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https://openalex.org/W2486281956	https://mts.intechopen.com/storage/books/213/authors_book/authors_book.pdf	English	null	Environmental Management in Practice	InTech eBooks	2,011	cc-by	209,848	Contributors Patricia Carla Giloni, Adriana Massaê Kataoka, Vanderlei Aparecido de Lima, Vanderlei Aparecido De Lima, Rosemary Caron, Michael Ericson Rezaee, Danielle Dionne, Hyunkee Bae, Richard C. Smardon, Hiroyuki Taguchi, Santosh Balkrishna Jaju, Tiberio Daddi, Maria Rosa De Giacomo, Marco Frey, Francesco Testa, Fabio Iraldo, Elzbieta Broniewicz, Masao Nakamura, Hitoshi Hayami, Elsa Mendonça, Ana Picado, Maria Ana Cunha, Justina Catarino, Sumiani Yusoff, Felipe Pombo, Alessandra Magrini, Alexandre Szklo, André Lermontov, Lidia Yokoyama, Mihail Lermontov, Maria Augusta Soares Machado, Jesús Martinez-Patino, Martín Picon-Nunez, Hui Lin Hai, Ana Luiza Spínola, Arlindo Philippi, Steven Odi-Owei, Itolima Ologhadien, Estibaliz Aranzabe, Arrate Marcaide, Marta Hernaiz, Nerea Uranga, Assed Naked Haddad, Fernando Beiriz, Iveta Čabalová, František Kačík, Danica Kačíková, Anton Geffert, Adebola Abosede Adeyi, Oladele Osibanjo, Zeynep Cetecioglu, Orhan Ince, Bahar Ince, Paulo Cesar Fernandes-Da-Silva, John Cripps Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html. ENVIRONMENTAL MANAGEMENT IN PRACTICE Edited by Elzbieta Broniewicz Environmental Management in Practice http://dx.doi.org/10.5772/738 Edited by Elzbieta Broniewicz © The Editor(s) and the Author(s) 2011 The moral rights of the and the author(s) have been asserted. g ( ) All rights to the book as a whole are reserved by INTECH. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECH’s written permission. Enquiries concerning the use of the book should be directed to INTECH rights and permissions department (permissions@intechopen.com). All rights to the book as a whole are reserved by INTECH. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECH’s written permission. Enquiries concerning the use of the book should be directed to INTECH rights and permissions department distributed or used for commercial or non-commercial purposes without INTECH’s written permission. Enquiries concerning the use of the book should be directed to INTECH rights and permissions department (permissions@intechopen.com). p p Violations are liable to prosecution under the governing Copyright Law. Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. First published in Croatia, 2011 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia Legal deposit, Croatia: National and University Library in Zagreb Additional hard and PDF copies can be obtained from orders@intechopen.com Environmental Management in Practice Edited by Elzbieta Broniewicz p. cm. ISBN 978-953-307-358-3 eBook (PDF) ISBN 978-953-51-6018-2 Environmental Management in Practice Edited by Elzbieta Broniewicz p. cm. ISBN 978-953-307-358-3 eBook (PDF) ISBN 978-953-51-6018-2 Meet the editor Dr. Elzbieta Broniewicz is a graduate of environmen- tal engineering on Technical University in Bialystok, Poland. In 1998 she received her PhD in the field of economics. In her teaching and scientific work she combines engineering with economics. The main fields of her interest are: environmental expenditure accounts, environmental goods and services sector, environmental management system and environmental impact assessment. In recent years the topic of environmental management has become very common. In sustainable development conditions, central and local gov- ernments much more often notice the need of acting in ways that diminish negative impact on environment. Contents Preface XIII Part 1 Environmental Management at the National and Regional Level 1 Curbing Climate Change through a Chapter 1 National Development of Climate Change Policy 3 Sumiani Yusoff Environmental Protection Chapter 2 Expenditure in European Union 21 Elzbieta Broniewicz Community Ecology and Capacity: Chapter 3 Advancing Environmental Communication Strategies among Diverse Stakeholders 37 Rosemary M. Caron, Michael E. Rezaee and Danielle Dionne Regional Issues in Environmental Management 67 Chapter 4 Hiroyuki Taguchi Geo-environmental Terrain Assessments Based on Chapter 5 Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 85 Paulo Cesar Fernandes da Silva and John Canning Cripps The Implementation of IPPC Directive Chapter 6 in the Mediterranean Area 119 Tiberio Daddi, Maria Rosa De Giacomo, Marco Frey, Francesco Testa and Fabio Iraldo Contaminated Sites and Public Policies Chapter 7 in São Paulo State, Brazil 145 Ana Luiza Silva Spínola and Arlindo Philippi Jr. X Contents X Contents Sustainable Management of Muddy Coastlines 159 Chapter 8 Steven Odi-Owei and Itolima Ologhadien Part 2 Environmental Management in Industry 175 Indicators of Sustainable Business Practices 177 Chapter 9 Hyunkee Bae and Richard S. Smardon Assessment of Industrial Pollution Load in Lagos, Chapter 10 Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 207 Adebola Oketola and Oladele Osibanjo Pollution Prevention in the Pulp and Paper Industries 223 Chapter 11 Bahar K. Ince, Zeynep Cetecioglu and Orhan Ince Retrofit Approach for the Reduction of Water and Energy Chapter 12 Consumption in Pulp and Paper Production Processes 247 Jesús Martínez Patiño and Martín Picón Núñez An Application Model for Chapter 13 Sustainability in the Construction Industry 267 Fernando Beiriz and Assed Haddad Assessing the SMEs’ Competitive Strategies Chapter 14 on the Impact of Environmental Factors: A Quantitative SWOT Analysis Application 285 Hui-Lin Hai Implementation of ISO 14000 in Chapter 15 Luggage Manufacturing Industry: A Case Study 297 S. B. Jaju Part 3 Technical Aspects of Environmental Management 311 The Statistical Distributions of Industrial Chapter 16 Wastes: an Analysis of the Japanese Establishment Linked Input-output Data 313 Hitoshi Hayami and Masao Nakamura The Effects of Paper Recycling Chapter 17 and its Environmental Impact 329 Iveta Čabalová, František Kačík, Anton Geffert and Danica Kačíková Overview Management Chemical Residues Chapter 18 of Laboratories in Academic Institutions in Brazil 351 Patrícia Carla Giloni-Lima, Vanderlei Aparecido de Lima and Adriana Massaê Kataoka Contents XI Contents Content Lengthening Biolubricants´ Lifetime Chapter 19 by Using Porous Materials 371 Estibaliz Aranzabe, Arrate Marcaide, Marta Hernaiz and Nerea Uranga A Fuzzy Water Quality Index for Chapter 20 Watershed Quality Analysis and Management 387 André Lermontov, Lidia Yokoyama, Mihail Lermontov and Maria Augusta Soares Machado Environmental Management of Wastewater Chapter 21 Treatment Plants – the Added Value of the Ecotoxicological Approach 411 Elsa Mendonça, Ana Picado, Maria Ana Cunha and Justina Catarino Technology Roadmap for Wastewater Chapter 22 Reuse in Petroleum Refineries in Brazil 425 Felipe Pombo, Alessandra Magrini and Alexandre Szklo Lengthening Biolubricants´ Lifetime Chapter 19 by Using Porous Materials 371 Estibaliz Aranzabe, Arrate Marcaide, Marta Hernaiz and Nerea Uranga A Fuzzy Water Quality Index for Chapter 20 Watershed Quality Analysis and Management 387 André Lermontov, Lidia Yokoyama, Mihail Lermontov and Maria Augusta Soares Machado Environmental Management of Wastewater Chapter 21 Treatment Plants – the Added Value of the Ecotoxicological Approach 411 Elsa Mendonça, Ana Picado, Maria Ana Cunha and Justina Catarino Technology Roadmap for Wastewater Chapter 22 Reuse in Petroleum Refineries in Brazil 425 Felipe Pombo, Alessandra Magrini and Alexandre Szklo Preface In recent years the topic of environmental management has become very common. In sustainable development conditions, central and local governments much more often notice the need of acting in ways that diminish negative impact on environment. Environmental management may take place on many different levels – starting from global level, e.g. climate changes, through national and regional level (environmental policy) and ending on micro level. This publication shows many examples of envi‐ ronmental management. In the chapters dealing with national and regional level of environmental manage‐ ment, authors have presented many different aspects: communication system, envi‐ ronmental costs, regional development indicators. Case studies from various world regions have also been included. The second section of the book deals with environmental management in various in‐ dustries. It presents sustainable business practices in construction industry, pulp and paper industry. Case studies in organizations have been a welcome addition to this section. The last section focuses on technical aspects of environmental management, mainly on water, waste and wastewater management. The diversity of presented aspects within environmental management and approach‐ ing the subject from the perspective of various countries contributes greatly to the de‐ velopment of environmental management field of research. I would like to thank all of the authors for presenting high quality chapters, Mr. Vidic for efficient project management and all InTech staff for making this publication pos‐ sible. PhD Elzbieta Broniewicz, Technical University of Bialystok Poland 1. Introduction In the last century, global development trends have tended to favour democratic systems and the capitalist ideology. In turn more equitable, economically secure, technologically advanced and intellectually progressive societies have developed around the world. However, at the same time, the side-effects of these trends have also been the creation of a materialistic society, high energy and resource consuming economy alongside inevitable, irreversible environmental damage and resource plundering. Economic analysis has a special role in contemporary national policy-making, as most of the important decisions fall within the economic domain. A country’s development level is principally measured by its GDP or monetary economic growth. Therefore, an economic model that is less materialistic and less energy demanding have to be introduced to achieve sustainable development, especially in the long-run. The development of a sustainable economic model has to recognise the environmental impact as part of the development agenda and finding a viable relationship between the two components. One common idea is to internalise the environmental impact into the economic scene with benefits to society and economy at large. To achieve the sustainable economic model, innovative policy instruments are essential in creating the necessary shifts in economic trends or patterns. A climate change policy plays a role in directing a country towards a sustainable economic development model by regulating the GHGs emission with appropriate sectoral policies in place. Curbing Climate Change through a National Development of Climate Change Policy Sumiani Yusoff University of Malaya Malaysia 1 Source: UNDP Human Development Report 2007/2008. < http://hdr.undp.org/en/media/HDR_20072008_Summary_English.pdf> p p / < http://hdr.undp.org/en/media/HDR_20072008_Summary_English.pdf> 2. Non-renewable energy and carbon emission With 0.4 per cent of the world’s population, Malaysia’s 27 million people accounted for 0.6 per cent of the global carbon emissions. As a developing country, Malaysia’s carbon emissions growth is one of the fastest; it grew by 221 per cent from 1990 to 2004 (UNDP Human Development Report 2007/2008).1 Malaysia’s rapid rise in its carbon emissions is the result of robust expansion in its industrial and automotive sectors, the over dependence on fossil fuel as its TPES (Total Primary Energy Supply), unsustainable waste management and forest and grassland conversion. With a CO2 emission intensity of GDP of 1.198 million metric tonne (MT) / USD million (IMF & CDIAC, 2006); Malaysia has one of the highest 4 Environmental Management in Practice carbon emission intensity of GDP in the world, indicated a low economy output to carbon emission. Malaysia had announced that it is taking a voluntary reduction of up to 40 per cent carbon emission intensity of GDP by the year 2020 compared to 2005 level at Copenhagen in 2009 (Theseira, 2010). To reach the carbon emission reduction, substantial action has to be taken. This requires first and foremost a viable policy on climate change to achieve this goal. Malaysia had announced that it is taking a voluntary reduction of up to 40 per cent carbon emission intensity of GDP by the year 2020 compared to 2005 level at Copenhagen in 2009 (Theseira, 2010). To reach the carbon emission reduction, substantial action has to be taken. This requires first and foremost a viable policy on climate change to achieve this goal. Country Annual CO2 emissions (in ‘000 MT) GDP (in billions of USD) CO2 intensity of GDP (million MT / USD billion) Malaysia 187,865 156.86 1.198 Thailand 272,521 206.99 1.317 Indonesia 333,483 364.35 0.915 Mexico 436,150 952.34 0.458 Argentina 173,536 212.71 0.816 Turkey 269,452 529.19 0.509 Sweden 50,875 393.76 0.129 UK 568,520 2,435.70 0.233 US 5,752,289 13,178.35 0.437 Japan 1,293,409 4,363.63 0.296 Table 1 Comparison of the carbon dioxide emissions intensity of GDP in 20062 A high carbon emission intensity of GDP would normally display the following results in the economy: The major sectors that drive the country’s economic growth have high carbon emissions with GDP by sector: Industrial: 42.3 per cent, Services: 47.6 per cent and Agricultural: 10.1 per cent (CIA, 2005). In 2000, the country’s total primary energy supply (TPES) was 49.47 million tons of oil equivalents (MTOE). 2 Sources: GDP data - IMF (International Monetary Fund), 2006 CO2 emission - CDIAC (Carbon Dioxide Information Analysis Center), 2006 Table 1. Comparison of the carbon dioxide emissions intensity of GDP in 20062 Fig. 1. Malaysia’s shares of TPES in 20073 Country MT of CO2 emission/ capita Energy use (kg of oil equivalent per capita) Energy use (kt of oil equivalent) Energy use / GDP (kt of oil equivalent / USD billion) Fossil fuel energy consumption (% of total) Malaysia 7.2 2733 72,589 462.76 95.5 Thailand 4.3 1553 103,991 502.40 81.2 Indonesia 1.5 849 190,647 523.25 68.8 Mexico 4.1 1750 184,262 193.48 89.3 Argentina 4.4 1850 73,065 343.50 89.5 Turkey 3.6 1370 100,005 188.98 90.5 Sweden 5.6 5512 50,422 128.05 32.9 UK 9.4 3464 211,308 86.76 89.6 US 19 7766 2,339,942 177.56 85.6 Japan 10.1 4019 513,519 117.68 83.2 Energy use refers to the TPES gy Fossil fuel refers to coal, oil and natural gas Table 2. Comparison of energy use and fossil fuel consumption, 20074 Table 2. Comparison of energy use and fossil fuel consumption, 20074 <http://www.iea.org/stats/pdf_graphs/MYTPESPI.pdf> 4 Source: CDIAC, 2006; IEA, 2007; IMF, 2006. 3 Source: IEA (International Energy Agency), 2008. 3 Source: IEA (International Energy Agency), 2008. <http://www.iea.org/stats/pdf_graphs/MYTPESPI.pdf> 4 Source: CDIAC, 2006; IEA, 2007; IMF, 2006. 2. Non-renewable energy and carbon emission The greatest percentage of the Malaysian fuel mix is petroleum products. In 2006, the TPES increase to 68.33 MTOE and it is projected to grow at a 3.5 per cent per year to 147 MTOE in 2030 because of the increase in demand for coal, oil and gas; with coal demand accounting for the highest growth rate at 9.7 per cent per year through 2030 (IEA, 2008). p p y g ( ) Higher energy use per GDP indicates a lower economy output per unit of energy use. Malaysia has one of the highest energy uses (oil equivalent) per unit GDP compared with the developed countries in the comparison lists. Although Malaysia shows a lower value compared with regional developing countries; the fossil fuel consumption in the total energy shares (95.5 per cent) is higher than Thailand (81.2 per cent) and Indonesia (68.8 per cent). This finding can deduce that Malaysia has the highest carbon emission intensity of GDP among the countries of comparison. Curbing Climate Change through a National Development of Climate Change Policy 5 48% 35% 12% 4% 1% Gas Oil Coal Comb. Renew. & Waste Hydro Fig. 1. Malaysia’s shares of TPES in 20073 Country MT of CO2 emission/ capita Energy use (kg of oil equivalent per capita) Energy use (kt of oil equivalent) Energy use / GDP (kt of oil equivalent / USD billion) Fossil fuel energy consumption (% of total) Malaysia 7.2 2733 72,589 462.76 95.5 Thailand 4.3 1553 103,991 502.40 81.2 Indonesia 1.5 849 190,647 523.25 68.8 Mexico 4.1 1750 184,262 193.48 89.3 Argentina 4.4 1850 73,065 343.50 89.5 Turkey 3.6 1370 100,005 188.98 90.5 Sweden 5.6 5512 50,422 128.05 32.9 UK 9.4 3464 211,308 86.76 89.6 US 19 7766 2,339,942 177.56 85.6 Japan 10.1 4019 513,519 117.68 83.2 Energy use refers to the TPES Fossil fuel refers to coal, oil and natural gas Table 2. Comparison of energy use and fossil fuel consumption, 20074 48% 35% 12% 4% 1% Gas Oil Coal Comb. Renew. & Waste Hydro Fig. 1. 2. Non-renewable energy and carbon emission Malaysia’s shares of TPES in 20073 Country MT of CO2 emission/ capita Energy use (kg of oil equivalent per capita) Energy use (kt of oil equivalent) Energy use / GDP (kt of oil equivalent / USD billion) Fossil fuel energy consumption (% of total) Malaysia 7.2 2733 72,589 462.76 95.5 Thailand 4.3 1553 103,991 502.40 81.2 Indonesia 1.5 849 190,647 523.25 68.8 Mexico 4.1 1750 184,262 193.48 89.3 Argentina 4.4 1850 73,065 343.50 89.5 Turkey 3.6 1370 100,005 188.98 90.5 Sweden 5.6 5512 50,422 128.05 32.9 UK 9.4 3464 211,308 86.76 89.6 US 19 7766 2,339,942 177.56 85.6 Japan 10.1 4019 513,519 117.68 83.2 Energy use refers to the TPES Fossil fuel refers to coal, oil and natural gas Table 2. Comparison of energy use and fossil fuel consumption, 20074 3. Unsustainable electricity production Energy in Malaysia is consumed mainly in the transportation and industrial sectors, 38.2 per cent and 37.8 per cent respectively in 2005, followed by commercial and residential sectors 48% 35% 12% 4% 1% Gas Oil Coal Comb. Renew. & Waste Hydro Fig. 1. Malaysia’s shares of TPES in 20073 3. Unsustainable electricity production Energy in Malaysia is consumed mainly in the transportation and industrial sectors, 38.2 per cent and 37.8 per cent respectively in 2005, followed by commercial and residential sectors at 12.5 per cent and the non-energy, which consumes 9.7 per cent of the total energy. Electrical energy production increased from 1,622 gigawatt per hour (GWh) in 1963 to 4,971 6 Environmental Management in Practice GWh in 1974 and 57,435 GWh in 1998. In 1996, 16 per cent of electrical production was hydro generated, and over 83 per cent was of thermal origin (National Energy Balance, PTM, 2006). In 2007, the country hit a staggering 101325 GWh of total electricity production with only about 6.4 per cent was hydro generated, (coal 29.5 per cent, natural gas 62 per cent and oil 2.1 per cent) according to the statistic shown by IEA in 2010. Country Electricity consumption* (TWh) Electricity Consumption /Population (kWh/capita) Electricity (production by source) % Fossil fuel* hydro nuclear others Malaysia 97.39 3668 93.6 6.4 - - Thailand 137.68 2157 91.4 5.7 - 2.9 Indonesia 127.17 564 87.1 7.9 - 5.0 Mexico 214.34 2028 81.4 10.6 4.0 4.0 Argentina 104.99 2658 65.8 26.7 6.3 1.2 Turkey 163.35 2210 80.9 18.7 - 0.4 Sweden 139.40 15238 2.4 44.5 45.0 8.1 UK 373.36 6142 77.6 2.3 15.9 4.2 US 4113.07 13616 71.6 6.3 19.2 2.9 Japan 1082.72 8475 66.8 7.4 23.3 2.5 Gross production + imports - exports - transmission/distribution losses Fossil fuel refers to oil, gas and coal Gross production + imports - exports - transmission/distribution losses Fossil fuel refers to oil, gas and coal Table 3. Electricity production by source in 20075 The country’s electricity consumption per capita is higher than the regional and other developing countries in the comparison list. Furthermore, the share of fossil fuel of the electricity production is the highest among all the countries in comparison. From the brief findings, it can be deduced that the factor contribute to the high carbon emission in any major sectors is the non-renewable energy supply. To reduce the carbon emission in any sector, a fundamental shift in the country’s TPES to a higher share of renewable energy is an imperative determinant. 5 Source: IEA, 2010. 4. Climate change related policies in Malaysia In general, Malaysia adopts a “precautionary principle” policy with actions to mitigate or adapt to climate change. A National Climate Committee was formed in 1995 with various government agencies, stakeholders from the business and civil society groups. The strategies adopted by the committee include to reduce the heavy reliance on fossil fuel in energy sector, promote renewable energy and energy efficiency, public awareness programme, sustainable forest management, ensure food sufficiency and undertaking coastal vulnerability index (CVI) study that serve as a basis for the development of adaptive 7 Curbing Climate Change through a National Development of Climate Change Policy measures to mitigate the impact of sea level rise. (Conservation and Environment Management Division, CEMD, 2007) measures to mitigate the impact of sea level rise. (Conservation and Environment Management Division, CEMD, 2007) Existing relevant policies in the country that will, directly or indirectly affect the development of an integrated and coherent climate change policy include: Existing relevant policies in the country that will, directly or indirectly affect the development of an integrated and coherent climate change policy include: 1. National Policy on the Environment, 2002 1. National Policy on the Environment, 2002 1. National Policy on the Environment, 2002 2. National Forestry Policy, 1978 3. National Policy on Biological Diversity, 1998 4. National Energy Policy, 1979 5. National Automotive Policy, 2009 6. Third National Agricultural Policy, 1998-2010 7. National Physical Plan, 2006 2. National Forestry Policy, 1978 2. National Forestry Policy, 1978 3. National Policy on Biological Diversity, 1998 5. National Automotive Policy, 2009 6. Third National Agricultural Policy, 1998-2010 7. National Physical Plan, 2006 6 Source: Abdul Rahim Nik, FRIM (Forest Reserve Institute of Malaysia), 2009. 5. National renewable energy policy 2011 Based on the data below (see Table 4), about 40-50 per cent of the carbon emissions originated from the energy and industrial sector. The emission from the industrial activities is mainly attributed to the energy sector as well. Therefore, the focus has to be on the energy sector in order to achieve any significant reduction goal. Rank Sub-sector GHGs Emission, CO2e (mil. MT) Percentage 1 Emission from energy industries CO2 58,486 28.2 2 Transportation CO2 35,587 17.2 3 Manufacturing and construction CO2 26,104 12.6 4 Landfills CH4 24,541 11.8 5 Forest and grassland conversion CO2 24,111 11.6 6 Fugitive emissions from fuel CH4 21,987 10.60 7 Mineral products CO2 9,776 4.7 8 Emission from soil CO2 4,638 2.2 9 Commercial CO2 2,122 1.0 207,352 99.9 Table 4. Key sources of GHGs emissions in Malaysia6 The key policies guiding energy-related activities in Malaysia consisted of:  National Petroleum Policy 1975  National Energy Policy 1979  National Depletion Policy 1980  Four Fuel Diversification Policy 1981  Fifth Fuel Diversification Policy (Eighth Malaysia Plan 2001-2005) In conjunction with these policies, a number of government supported projects to assist the National Energy Conservation plans, have been identified. Under the guidance and supervision of the Malaysia Energy Centre (PTM), some of the projects introduced are CDM (Clean Development Mechanisms), IRP (Integrated Resource Planning), MEDIS (Malaysia Rank Sub-sector GHGs Emission, CO2e (mil. MT) Percentage 1 Emission from energy industries CO2 58,486 28.2 2 Transportation CO2 35,587 17.2 3 Manufacturing and construction CO2 26,104 12.6 4 Landfills CH4 24,541 11.8 5 Forest and grassland conversion CO2 24,111 11.6 6 Fugitive emissions from fuel CH4 21,987 10.60 7 Mineral products CO2 9,776 4.7 8 Emission from soil CO2 4,638 2.2 9 Commercial CO2 2,122 1.0 207,352 99.9 T bl 4 K f GHG i i i M l i 6 The key policies guiding energy-related activities in Malaysia consisted of:  National Petroleum Policy 1975  National Energy Policy 1979  National Depletion Policy 1980  Four Fuel Diversification Policy 1981  Fifth Fuel Diversification Policy (Eighth Malaysia Plan 2001-2005) I j i i h h li i b f d j i h 8 Environmental Management in Practice Energy Database and Information System), MIEEIP (Malaysian Industrial Energy Efficiency Improvement Project), BioGen (biomass power generation and co-generation in palm oil industry), MBIPV (Malaysian Building Integrated Photovoltaic Technology Application Project) and Demand Side Management. 5. National renewable energy policy 2011 The SREP (Small Renewable Energy Programme) allows Renewable Energy (RE) projects with up to 10 megawatt (MW) of capacity only. The programme was introduced during 8th Malaysia Plan (2001-2005) under the fifth fuel diversification policy which targeted a 5 per cent renewable energy share of total electricity generation; however failed to achieve its target. In 9th Malaysia Plan or 9th MP, (2006-2010), targeted RE capacity to be connected to power utility grid is 300MW in Peninsula Malaysia and 50MW in Sabah with a 1.8 per cent of total power generation mix (65 per cent natural gas, 36 per cent coal, 6 per cent hydro & 0.2 per cent oil). However, RE capacity connected to power utility grid as of 31st December 2009 was 53MW which is barely 15 per cent of 9th MP target. The off grid RE (private palm oil millers and solar hybrids) is more than 430MW (Badriyah, 2010). y y The reasons for slow RE development are identified as market failure, absence of legal framework, lack of institutional measures and constraint in financial and technological aspects. A new ministry, Ministry of Energy, Water and Green Technology (KeTTHA) was formed in 2009 following the introduction of Green Technology Policy 2009. The ministry had formulated goals on sustainable use of energy and water. The ministry also provides incentive for the use of green technology. A new policy on renewable energy (National Renewable Energy Policy) will be introduced next year (Loo, 2010). With the new Act, a new feed-in tariff system will be introduced to stimulate the renewable energy sector. The policy statement is “Enhancing the utilization of indigenous renewable energy resources to contribute towards national electricity supply security and sustainable socio-economic development.” 7 Source: Loh, T., Yusoff, S., 2009. Source: Loh, T., Yusoff, S., 2009. Table 5. Comparison of currently installed and potential capacity of renewable energy7 urce: Loh, T., Yusoff, S., 2009. 6. Potential carbon emission reductions in energy sector The potential of carbon emissions reduction in energy sectors is discussed in this section. Comparison is made between the existing use of renewable energy and its potential in Malaysia. It is found that Malaysia has a vast potential in renewable energy as compared with the existing utilisation. Renewable energy Installed Capacity (MW) Potential Capacity (MW) Solar 6.2 6500 Wind 0.2 (low potential) Municipal Solid Waste - 400 Hydropower 2225 (year 2000) 22 000 Mini-Hydro 23.8 500 Biomass/Biogas 479 1300 (Palm Oil Waste) Table 5. Comparison of currently installed and potential capacity of renewable energy7 Curbing Climate Change through a National Development of Climate Change Policy 9 Year Cumulative RE Capacity RE Power Mix (vs Peak Demand) Cumulative CO2 avoided 2010 73 MW 0.5% 0.3 mt 2015 985 MW 6% 11.1 mt 2020 2080 MW 11% 42.2 mt 2030 4000 MW 17% 145.1 mt RE capacity achievements are dependent on the size of RE fund Assumptions: Feed-in Tariff (FiT) in place & 15.6 per cent compound annual growth rate (CAGR) of RE power capacity from 2011 to 2030 RE capacity achievements are dependent on the size of RE fund Assumptions: Feed-in Tariff (FiT) in place & 15.6 per cent compound annual growth rate (CAGR) of RE power capacity from 2011 to 2030 Table 6. National Renewable Energy Target8 The current power generation capacity connected to the Malaysia National Grid is 19,023 MW in 2007 (Energy Commission, 2007). Based on the data in Table 4, the potential power generation by renewable energy is 30,700 MW, which is more than the current power generation. Therefore, if the country can reach 40% of the potential renewable energy capacity, 60% of the existing power generation will be from renewable energy. However, less than 10% of the potential renewable energy is utilized currently. p gy y With the implementation of feed-in tariff, the RE power mix is projected to reach 11% in year 2020 (Badriyah, 2010) and the achievement of the 40% reduction of carbon intensity of GDP will be subjected to the country GDP growth. Therefore, the increase of the renewable energy share in the total power generation is a predominant agenda in the development of a climate change policy. 8 Source: Badriyah, KeTTHA, August 2010. 9 Source: Tan, C. T.; Pereira, J. J. & Koh, F. P. (2009). Stakeholder Consultation in the Development of Climate Change Policy: Malaysia’s Approach. Environmental Policy: A multinational conference on policy analysis and teaching methods, KDI School of Public Policy and Management, Seoul, Korea. 7. Draft national climate change policy The policy study on climate change was conducted by CEMD under the Ministry of Natural Resource Environment (MNRE) in collaboration with LESTARI (Institute for Environment and Development). The study adopted a three-pronged approach to support the national positions at the UNFCCC and Kyoto Protocol meetings, formulation of a national policy and action plan, and delineation of state level responses to climate change adaptation and mitigation (Figure 2). The first approach is the critical review of several international and local research papers and public documents that was related to post-2012 responses, decision documents of the UNFCCC and Kyoto Protocol, Malaysia’s Third Outline Perspective Plan (OPP3), Ninth Malaysia Plan (RMK9), relevant national policies, and Malaysia’s Initial National Communication (INC). The second approach involved the comparative studies of national policies or strategies on climate change from selected countries and the third approach focused on stakeholder consultation through national and regional workshops, interviews and surveys which were carried out in a four overlapping phases. The need for a national policy on climate change was articulated in the first and second phase; while in the third and fourth phase, the policy framework including its key actions was supported as a promising tool to mainstream climate change in national development (Tan, et al, 2009). 10 Environmental Management in Practice Fig. 2. Climate change policy study approach and expected outputs9 Fig. 2. Climate change policy study approach and expected outputs9 Fig. 2. Climate change policy study approach and expected outputs9 Fig. 3. Overall framework of a national climate change policy10 Fig. 3. Overall framework of a national climate change policy10 9 Source: Tan, C. T.; Pereira, J. J. & Koh, F. P. (2009). Stakeholder Consultation in the Development of Climate Change Policy: Malaysia’s Approach. Environmental Policy: A multinational conference on policy analysis and teaching methods, KDI School of Public Policy and Management, Seoul, Korea. Curbing Climate Change through a National Development of Climate Change Policy 11  Mainstreaming of measures to address climate change challenges through strengthened economic competitiveness, wise management of resources, environmental conservation and enhanced quality of life for sustainable development.  Mainstreaming of measures to address climate change challenges through strengthened economic competitiveness, wise management of resources, environmental conservation and enhanced quality of life for sustainable development. q y p  Integration of responses into national policies, plans and programmes to strengthen the resilience of development from arising impacts of climate change. 10 Op. cit. Tan, et al. (2009). 11 Source: Pereira, 2008 12 Based on the stakeholders discussion session at SLiM 2010 Roundtable: Creating an Institutional Framework for Implementing Sustainable Development (8th July) 7. Draft national climate change policy p g p g  Strengthening of institutional and implementation capacity to better harness opportunities in reducing negative impacts of climate change. Principles/Strategic Thrusts Principles/Strategic Thrusts Principle 1. Development on a Sustainable Path: Integrate climate change responses in national development plans to fulfil the country’s aspiration for sustainable development. y  Strategic Thrust 1. Facilitate the harmonisation of existing policies to address climate change adaptation and mitigation in a balanced manner.  Strategic Thrust 2. Institute measures to make development climate-resilient through low carbon economy to enhance global competitiveness and attain environmentally sustainable socio-economic growth. g  Strategic Thrust 3. Support climate-resilient industrial development and investmen pursuit of sustainable socio-economic growth. Principle 2. Sustainability of Environment and Natural Resources: Initiate actions on climate change issues that contribute to environmental conservation and sustainable use of natural resources while enhancing energy efficiency and sufficiency as well as water and food security.  Strategic Thrust 1. Adopt balanced adaptation and mitigation measures to climate- proof development, strengthen environmental conservation and promote sustainability of natural resources y Principle 3. Integrated Planning and Implementation: Integrate planning and implementation to climate-proof development.  Strategic Thrust 1. Institute measures to integrate cross-cutting issues in policies, plans, programmes and projects in order to increase resilience to and minimise negative impacts of climate change. g p g  Strategic Thrust 2. Support knowledge-based decision making through intensi climate related research and development and capacity building of human resources p p y g Principle 4. Effective Participation: Improve participation of stakeholders and majo groups for effective implementation of climate change responses.  Strategic Thrust 1. Improve collaboration through efficient communication and coordination among all stakeholders for effective implementation of climate change responses. p  Strategic Thrust 2. Increase awareness and public participation to promote behavioural responses to climate change. Principle 5. Common but Differentiated Responsibility: International involvement on climate change will be based on the principle of common but differentiated responsibility.  Strategic Thrust 1. Strengthen involvement in international activities on climate change based on the principle of common but differentiated responsibility. Table 7. The Draft National Policy on Climate Change – Objectives, Principles and Strategic Thrusts11 10 Op. cit. Tan, et al. (2009). 11 Source: Pereira, 2008 12 Environmental Management in Practice 8.2 Lack of expertise in climate change issue p g The R&D in climate change is relatively new in the country with concern on the matter is primarily driven by the signing and ratifying of Kyoto Protocol in carbon emission reduction and now more recently, the 40 per cent reduction of carbon emission intensity of GDP as pledged in COP 15. Local expertise on climate change issue especially the trend in the country is important and a proper data management plan needs to be implemented. The country need climate change experts that possess knowledge in climate change globally as well as have a strong understanding on the local socio economic development. Education from primary level is important to instil the understanding of climate change issue to the people since young. With proper education system, the topic can become more pertinent among the people and hopefully will produce more climate change experts. 8.1 Institutional reformation and policy restructuring p y g The formation of an institutional framework on climate change issue is the foundation to achieve any result in carbon emission. A clear cut government policy direction on sustainable development has to be established in the quest of achieving goal on carbon emission reduction. The agenda toward low carbon economy requires strong and persistent institutional reformation and political will and direction in a long term basis. A government’s primary concerns are the country’s economic growth and social welfare. Urgency and priority are the major considerations for the development of any public policy especially for a developing country. Tackling climate change issue or striving towards the sustainable development model is a long term planning process. A country abundant in natural resources may lack urgency in policy planning and implementation in climate change. Coordination and cooperation between various government agencies toward a achieving a common goal on carbon emission reduction is lacking at the moment. In the Malaysian context, political influence is always the prime mover in creating any trends as the decision making power is lacking among the civil servants. Another constraint in policy making process is the conflict in authority and power distribution between federal and state government.12 13 (Keizrul, 2010, Public Lecture at Faculty of Engineering, University of Malaya, on World Water Day, 22nd March). 14 Source: MPMA (Malaysian Plastics Manufacturers Association), 2010. 8.3 Economic status (lack of financial capacity) State of the art technology and solution to any environmental issue are readily available as solutions. The constraint is in its economic viability (i.e. pricing). The economic status does not allow solution for long term environmental issue which typically consume huge amount of money. For example, in waste management sector, the polluter pay principle (PPP) can directly encourage recycling. However it must be equipped with a proper pricing mechanism such as a variable unit based pricing scheme which will serve as a basis of the carrot and stick approach (Munasinghe, 2008). Another example is greywater or sullage that contributes to of 6 per cent of the total river point source pollution (DOE, 2001). The solution to sullage discharge is to retrofit the existing piping back to the sewer line for further treatment. However, the constraint is always related to the cost and priority. The DID (Department of Irrigation and Drainage) had introduced some measures in tackling river 13 Curbing Climate Change through a National Development of Climate Change Policy pollution such as gross pollutant trap (GPT) and FOG (fat, oil and grease) trap, but these have not been very effective. The most pragmatic solution is sullage pipe retrofitting which is costly and require strong political will to implement it.13 The same situation happens to renewable energy such as solar, biomass, biogas and the like. The example shows the inadequacy of financial capability in solving a pertinent local environmental issue. 8.4 Public understanding on climate change issue Public understanding on environmental issue is imperative in the development of the climate change policy as it directly affect the life of an individual. In Malaysia, the understanding of climate change by the public at large is rather insufficient. For example, most of the people are not aware that driving a car or petrol consumption can lead to climate change by carbon emission. Awareness campaigns by the government or NGOs does not take into account the bigger picture provide an understanding of the cause of environmental issues such as climate change. The publicity drive by the government produces only a superficial understanding on the part of the general public so it does not accurately reflect or actually lead to good environmental practices as such. That is to say, the rhetoric is not matched by the reality of an environmentally-friendly situation. y y y y One of the tools to analyse the entire environmental impacts of an issue is LCA (Life Cycle Assessment) which the public in general do not understand. One example is the issue of the banning on the use of plastic bags by certain states in the country. For most, plastic is something “negative” to the environment because of its non-biodegradable nature. Plastic which is beneficial as a “carrier” or for packaging may pose a problem when it comes to “inconsiderate” disposal, especially to any water bodies. However, banning the use of plastic bags will never solve the inconsiderate disposal problem and is myopic, impracticable and counter-productive. The solution has to be proper education and management since plastic bags are still a necessity as its use has yet to be outlasted by environmental concerns. The use of plastic packaging has lesser negative environmental impact if compared with other materials such as paper and metal because of the lower carbon emission in a life cycle perspective. In fact, plastic has reduced the consumption of fossil fuel on transportation and manufacturing. 14 22nd March). 8.5 Equity in socio-economic development y An important social criterion must be that climate change should not be a hindrance to the development of a more equitable society where no one is made worse off. Thus, environmental policies must leave room for the proper allocation and distribution of resources in the pursuit of socio-economic welfare of the least better off in society. Furthermore, without an equitable society, environmental policies are very difficult to be implemented. For example, solar panel is burdensome for most middle and lower income household without incentives. The total elimination of logging activities will cause the loss of employment among low income workers. However there are also counter-examples: An effective public transport system can bring benefit to the poor as well as reduce carbon emission. The development of a climate change policy has to include a concern of the poor as the policy may affect their livelihood. In a global perspective, the use of parameters like carbon 14 Environmental Management in Practice emission per GDP and per capita is questionable in relation to its applicability to all countries with differing levels of economic growth. Developed countries usually have a lower carbon emission per GDP but a higher carbon emission per capita; because of their high economy power. While developing countries usually have comparably higher carbon emission per GDP but lower carbon emission per capita. The measurement of emission with GDP (economy) correlation should be a short term parameter to provide incentive and opportunity for developing countries to boost their economy without much emission liability. However, for developed countries, the focus should be the emission per capita with the strong economy status. Kverndokk (1995)15 argued that conventional justice and moral principles should favour the equitable allocation of future GHG emission rights on the basis of population, consistent with the UN human rights declaration underlining the equality of all human beings. emission per GDP and per capita is questionable in relation to its applicability to all countries with differing levels of economic growth. Developed countries usually have a lower carbon emission per GDP but a higher carbon emission per capita; because of their high economy power. While developing countries usually have comparably higher carbon emission per GDP but lower carbon emission per capita. The measurement of emission with GDP (economy) correlation should be a short term parameter to provide incentive and opportunity for developing countries to boost their economy without much emission liability. 8.5 Equity in socio-economic development However, for developed countries, the focus should be the emission per capita with the strong economy status. Kverndokk (1995)15 argued that conventional justice and moral principles should favour the equitable allocation of future GHG emission rights on the basis of population, consistent with the UN human rights declaration underlining the equality of all human beings. 15 Source: Kverndokk, S., 1995. Tradeable CO2 Emission Permits: Initial Distribution as a Justice Problem, Environmental Value, 4(2), 129-48. <http://www.frisch.uio.no/sammendrag/14_eng.html> 9. Discussion The paper had introduced the existing carbon emission status in the country, two policies (National Renewable Energy and National Climate Change Policy) and identified the challenges of the development and implementation of these policies. To overcome the constraints arise in a viable approach, the climate change impacts have to be embedded into any policy development. Internalization of climate impacts in economic with mixed instruments, establishment of GHGs information centre, adaptation strategy and institutional redesigning and education at all levels are the keys to achieve the carbon reduction target. .1 Internalize climate change impact into the economy domain The approach is important to ensure fairness in the context of social equity. In this method, economic valuation is an important prerequisite. CBA (cost benefit analysis) can be applied to work out the valuation and quantify the damage in a monetary way. Multi-criteria analysis will be useful for environmental solution that can’t be evaluated with single criterion approach like CBA. MBI provides individual or company greater flexibility in their approaches to pollution management. Contrary to the command and control approach which is more prescriptive; MBI provides incentive to innovate and individual at large to make the correct decision. By having a clear and inclusive monetary structure in punishing or rewarding in the context of an environmental issue, the result will be more obvious. Multi-criteria analysis will be useful for environmental solution that can’t be evaluated with single criterion approach like CBA. MBI provides individual or company greater flexibility in their approaches to pollution management. Contrary to the command and control approach which is more prescriptive; MBI provides incentive to innovate and individual at large to make the correct decision. By having a clear and inclusive monetary structure in punishing or rewarding in the context of an environmental issue, the result will be more obvious. .1 Internalize climate change impact into the economy domain 9.1 Internalize climate change impact into the economy domain As an externality in the current economy trend, environmental issue always meets the problem of market failure. To achieve any goal and substantial result in environmental issue, it has to be internalized in the economy domain by putting a price tag on it. When there is a price to pay for any environmental damage caused, people will be able to feel it. For example, climate change issue is something either people can’t feel it or due to mere selfishness. Another example is landfill, which most people has never seen before and do not understand its negative impacts. The economic functions of the environment have to be valued for an internalization to happen. Three outlines for the evaluation of the functions are amenity services (natural beauty, recreational, etc), natural resources (minerals and non- minerals, forest, etc) and assimilation of waste products (land, water and air). With this perception of recognizing the economic value of environment, the traditional economic system and the environment will be dynamically interrelated (Munasinghe, 2008). The entire activity or process of an environmental issue has to be integrated with price mechanism to create a “market based” solution. Market based instrument (MBI) is an economic approach to influence people to include environmental matters in any decision making. Some of the examples are tradable permits, polluter pay principle, green levy, eco- labeling, landfill tax, etc. The polluter pay principle argues that those who create negative impact to the environment should pay the corresponding costs. The economic rationale is to provide alternatives and 15 Curbing Climate Change through a National Development of Climate Change Policy incentive for polluters to reduce their impact or emission to optimal level. This “carrot and stick” approach that incorporated in the principle can be further extended to principle of recompensing victim by using the revenues collected by polluters (Munasinghe, 2008). The approach is important to ensure fairness in the context of social equity. In this method, economic valuation is an important prerequisite. CBA (cost benefit analysis) can be applied to work out the valuation and quantify the damage in a monetary way. incentive for polluters to reduce their impact or emission to optimal level. This “carrot and stick” approach that incorporated in the principle can be further extended to principle of recompensing victim by using the revenues collected by polluters (Munasinghe, 2008). 9.2 Mixed instrument: A practical approach in the decoupling of economy growth and environmental impact (carbon emission) Besides, command and control, as a non-economic approach, may be beneficial as a starting point, when regulators are faced with a significant problem yet have too little information to support a MBI. In practice, the mixture of both command and control and MBI is more effective. The policy and economy instruments have to be implemented hand in hand to achieve any tangible target in carbon emission reduction. For the example, the readjustment of electricity tariff by TNB (Tenaga National Berhad), the main electricity distributor in Malaysia. With the differential tariff, users that consume less electricity are exempted from the levy which will be exerted on user that exceeds a certain levels of electricity consumption. (Rao, P.K., 2000) 9.2 Mixed instrument: A practical approach in the decoupling of economy growth and environmental impact (carbon emission) While MBI can be more cost-effective than regulatory instruments it has its own drawbacks. The major weaknesses of MBI are: (Munasinghe 2008) While MBI can be more cost-effective than regulatory instruments it has its own drawbacks. The major weaknesses of MBI are: (Munasinghe, 2008)  Their effects on environmental quality are not as predictable as those under a traditional regulatory approach as polluters may choose their own solutions.  Their effects on environmental quality are not as predictable as those under a traditional regulatory approach as polluters may choose their own solutions.  In the case of pollution charges, some polluters opt to pollute and to pay a charge if the charge is not set at the appropriate level.  In the case of pollution charges, some polluters opt to pollute and to pay a charge if the charge is not set at the appropriate level.  Require sophisticated institutions to implement and enforce them properly, particularly in the case of charges and tradable permits.  Require sophisticated institutions to implement and enforce them properly, particularly in the case of charges and tradable permits. Fi 4 D li f i h h d i l i Fig. 4. Decoupling of resource use with economy growth and environmental impact Fig. 4. Decoupling of resource use with economy growth and environmental impact Internalization of environmental impact in an economic perspective alone can’t bring out a comprehensive solution in carbon emission reduction. The most apparent drawback is the rich will pollute more while the poor will pollute less. It doesn’t solve the fundamental issue Internalization of environmental impact in an economic perspective alone can’t bring out a comprehensive solution in carbon emission reduction. The most apparent drawback is the rich will pollute more while the poor will pollute less. It doesn’t solve the fundamental issue 16 Environmental Management in Practice of sustainable consumption and production as the rich countries have the financial capacity to choose whether to increase or decrease their carbon emission. Therefore, both economic and non-economic approaches have to be considered in the internalization process with a flexible mode. Command and control policy such as placing standard, agreement, protocol, etc; is predominantly significance in ensuring a healthy development of MBI. 9.3 Establishment of GHGs information management system GHGs emission shouldn’t be merely calculated by the looking at the carbon source. The GHGs sequestration or carbon sink especially by natural sink such as forest has to be taken into account in figuring out the net carbon emission. The net emission data will represent a better overview and bigger picture of the issue in a country context. Fig. 5. Net carbon emission Malaysia with collaboration of the regional countries has to set up a world recognized independent body in the collection and management of data pertaining to carbon emission in the region. Data inconsistency can be an important factor that leads to the failure of the development of an environmental policy. For example, IEA reported that the CO2 emissions in Malaysia in 2005 was 5.45 MT/capita, UNEP revealed a value of 6.2 MT/capita in 2002, while CDIAC documented a 7.2 MT/capita in 2006; and on the other hand, WRI (World Resource Institute) published a value of 5.4 MT / capita in 2000. The questions are not only the consistency of the volume emissions but also the base year of those data. (Wee et al, 2008). O li bl GHG i i i t f th t d l d b M l i N ti l Net carbon emission Carbon sources (emission) Carbon sinks (sequestration) _ + Fig. 5. Net carbon emission Net carbon emission Carbon sources (emission) Carbon sinks (sequestration) _ + Net carbon emission Carbon sinks (sequestration) Carbon sources (emission) Fig. 5. Net carbon emission Malaysia with collaboration of the regional countries has to set up a world recognized independent body in the collection and management of data pertaining to carbon emission in the region. Data inconsistency can be an important factor that leads to the failure of the development of an environmental policy. For example, IEA reported that the CO2 emissions in Malaysia in 2005 was 5.45 MT/capita, UNEP revealed a value of 6.2 MT/capita in 2002, while CDIAC documented a 7.2 MT/capita in 2006; and on the other hand, WRI (World Resource Institute) published a value of 5.4 MT / capita in 2000. The questions are not only the consistency of the volume emissions but also the base year of those data. (Wee et al, 2008). 9.3 Establishment of GHGs information management system Malaysia with collaboration of the regional countries has to set up a world recognized independent body in the collection and management of data pertaining to carbon emission in the region. Data inconsistency can be an important factor that leads to the failure of the development of an environmental policy. For example, IEA reported that the CO2 emissions in Malaysia in 2005 was 5.45 MT/capita, UNEP revealed a value of 6.2 MT/capita in 2002, while CDIAC documented a 7.2 MT/capita in 2006; and on the other hand, WRI (World Resource Institute) published a value of 5.4 MT / capita in 2000. The questions are not only the consistency of the volume emissions but also the base year of those data. (Wee et al, 2008). One reliable GHGs emissions inventory of the country was developed by Malaysia National Steering Committee on Climate Change; which was established following the signing of Kyoto Protocol. The national GHGs emissions inventory was introduced during the preparation of the Initial National Communication (INC) for UNFCCC. In 2000, Malaysia submitted its INC comprising the national GHGs inventory and the assessment of possible Curbing Climate Change through a National Development of Climate Change Policy 17 impacts of climate change. It detailed the policies and plans in place that represent the national sustainable development agenda of the country. The preparation of Second National Communication (NC2) is a continual step towards further implementation of the UNFCCC at national level which aims to generate a comprehensive report on climate change related issues in Malaysia. (CEMD, 2007) Fig. 6. NC2 Institutional Arrangement (Source: DOE (Department of Environment), 2010 Fig. 6. NC2 Institutional Arrangement (Source: DOE (Department of Environment), 2010 9.5 Institutional framework redesigning Institution refers to a specific organization or a policy program. Organizations such as a specific government agency, departments, association are a manifestation of institution. For an institution to be changed, organizations are needed. But institutional change is harder to achieve than organizational change. For instance it is easier to restructure the Department of Environment than to transform Malaysia’s federal system which is constitutionally defined. Instructive guiding principles are found in Professor Dovers’ public lecture delivered in 2009 entitled Implementing Sustainable Development; six generic principles may be adopted and adapted by governments to suit relevant contexts: 1. Factoring in the long term 2. Integrating environment, society and economy in policy 3. Precautionary Principle 4. Global dimensions 5. Innovative policy approaches 6. Community participation y p p Institutionalizing sustainability by embedding these principles in their institutions is no easy feat for any government. A suggestion in institution reformation is the forming of “Green” ministries clusters typically share matters related to climate change; for example the alliance between Ministry of Natural Resource and Environment, Ministry of Energy, Green Technology and Water, Ministry of Housing and Local Government and Ministry of Science, Technology and Innovation. 9.6 Decision making at all levels Any decision making related to climate change is fundamentally affected by money and awareness. To look at the micro level in the implementation of the climate change policy, decision making process of all levels are the determining factor. For example, for an individual level, changing to energy saving light bulb can be costly for low income people; however the awareness can drive the individual to take the action. Same for the government level; for instance to embark in renewable energy technology will take tremendous efforts in various agencies; however, with proper planning and collaboration, the positive outcomes of the effort can outweigh the short term unfavorable financial constraint in an entire system outlook. Decision making is always affected by an individual’s knowledge, ethic, integrity and mindset. Education is the key toward correct decision making by all levels regarding matters related to climate change. Government had made the move to incorporate environmental issues in science and geography lessons at primary and secondary levels. The question remains is the comprehensiveness and sufficiency of the subject and the qualification and understanding of teachers on the subject. 9.4 Adaptation strategy to climate change Adaptation strategies to climate change are inevitable because of the limited available knowledge. As a global issue, the effects of climate change will happen everywhere around the world. Therefore, adaptation measures have to be taken as well besides the mitigation measures; to ensure the welfare of the society is under control. Adaptation is a shared responsibility between government, community and business entities; that have a stake and role in responding to the climate change impact. Government has to put efforts in studies and research in climate change vulnerability areas and develop the relevant policies. For example, the recent findings revealed in the national coastal vulnerability index (CVI) study conducted by the Drainage and Irrigation Department (DID) in 2006 shows that sea levels off the west coast of Peninsular Malaysia will rise by 10cm to 13cm in the next 100 years and 288.4km or 6% of the peninsula’s 4,809km-long coast is being eroded by sea water. As vulnerability to climate change is a new risk to a country, business and community need to assume the responsibility to manage the risk by factor climate change into everyday decision making. It takes time to adapt and as uncertainty exists in climate change effect, the reasons for taking the relevant action are flexibility and creativity. It is not cost effective for adaptation response measures to prevent all adverse impacts of current and future climate change. Adaptation actions will need to achieve a suitable balance between the risks of 18 Environmental Management in Practice acting too early or too late, and to balance the potential benefits of actions with the likely magnitude of impacts. In this context, the government plays a vital role in leading the action by providing information and setting the right conditions for business and community to adapt. 10. Conclusion Pending to the formulation of the climate change policy, the planning of the implementation part (enforcement, monitoring, measurement, improvement, etc) has to go concurrently. The introduction of a climate change policy is imperative for any country and in the right timing for the country to act as a strategic trajectory toward sustainable development. 10. Conclusion Mainstreaming sustainability has always proven difficult in any country either developed or developing. Nevertheless, the global challenge of climate change will serves as an impetus 19 Curbing Climate Change through a National Development of Climate Change Policy for the government to set new energy policy that based on clean and renewable energy. With a holistic policy and economy model in place, the achievement of sustainable development is not impossible. However implementation of the policy requires high quality governance and vast pools of expertise with high ethical and integrity. It is time to change the “Business As Usual” attitude and “NATO” (No Action Talk Only) syndrome. for the government to set new energy policy that based on clean and renewable energy. With a holistic policy and economy model in place, the achievement of sustainable development is not impossible. However implementation of the policy requires high quality governance and vast pools of expertise with high ethical and integrity. It is time to change the “Business As Usual” attitude and “NATO” (No Action Talk Only) syndrome. The country politicians, all business sectors and the community at large have to be involve and dwell on the environmental and especially climate change issue as it entails on numerous diverse topics and disciplines. Pending to the formulation of the climate change policy, the planning of the implementation part (enforcement, monitoring, measurement, improvement, etc) has to go concurrently. The introduction of a climate change policy is imperative for any country and in the right timing for the country to act as a strategic trajectory toward sustainable development. The country politicians, all business sectors and the community at large have to be involve and dwell on the environmental and especially climate change issue as it entails on numerous diverse topics and disciplines. Pending to the formulation of the climate change policy, the planning of the implementation part (enforcement, monitoring, measurement, improvement, etc) has to go concurrently. The introduction of a climate change policy is imperative for any country and in the right timing for the country to act as a strategic trajectory toward sustainable development. The country politicians, all business sectors and the community at large have to be involve and dwell on the environmental and especially climate change issue as it entails on numerous diverse topics and disciplines. 11. References Mohan Munasinghe, 2008. The sustainomics transdisciplinary meta-framework for marking development more sustainable: Applications to energy issues. In: Pushpam Kumar (eds): Economics of Environment & Development (pp. 11-87). Taylor & Francis Group: CRC Press. Rao, P.K. 2000. Sustainable development: Economics and policy. Blackwell Publisher. IEA Statistics, CO2 emissions from fuel combustion, 2009 edition, International Energy Agency. Mohan Munasinghe, 2008. Addressing the sustainable development and climate change challenges together: Applying the sustainomics framework. Procedia social and behavioral science 41 (2010); 6634-6640. Tan Ching Tiong, Joy Jacqueline Pereira and Koh Fui Pin, 2009. Stakeholder Consultation in the Development of Climate Change Policy: Malaysia’s Approach. Environmental Policy: a Multinational Conference on Policy Analysis and Teaching Methods, KDI School of Public Policy and Management - Seoul, South Korea. Badriyah Abdul Malek, 2010. Ministry of Energy, Green Technology & Water Malaysia. National Renewable Energy Policy & Action Plan. Energy Forum: Securing a Sustainable Energy for Malaysia. gy y PTM, 2007. Energy sector embracing climate change. National Conference on Climate Change Preparedness towards Policy Changes Al-Amin, Chamhuri Siwar, Abdul Hamid, Nurul Huda, 2005, Pollution implications of electricity generation in Malaysian economy: An input-output approach. APEC (Asia-Pacific Economic Cooperation). Energy Demand and Supply Outlook 2006: Malaysia. Gary W. Theseira, 2010. How can the 40% carbon (intensity) reductions can be achieved? Waste Management 2010 Conference, ENSEARCH. Loh, T., Yusoff, S., 2009, The clean development mechanism (CDM) as a driver for climate change adaptation in Malaysia. A. A. Hezri and Mohd. Nordin Hasan, 2006. Towards sustainable development? The evolution of environmental policy in Malaysia. Natural Resources Forum. A.A. Hezri, 2010. Sustainable Shift: Institutional Challenges for the Environment in Malaysia. EPSM SLiM Roundtable 2010, ‘Creating an Institutional Framework for Implementing Sustainable Development’. 20 Environmental Management in Practice National Hydraulic Research Institute Malaysia (NAHRIM). 2006. Study of the impact of climate change on the hydrologic regime and water resources of Peninsular Malaysia – Final Report. Ministry of Natural Resources and Environment. y p y Energy Commission of Malaysia, 2007. Statistics of interim on the performance of the electricity supply in Malaysia for the first half year of 2007. Universiti Kebangsaan Malaysia – Institute for Environment and Development (UKM- LESTARI), 2008a. Policy Framework on Climate Change: Stakeholder viewpoints. UKM-LESTARI. Universiti Kebangsaan Malaysia – Institute for Environment and Development (UKM- LESTARI), 2008b. National Policy on Climate Change (Draft 1: 10 September 2008): Stakeholder viewpoints. UKM-LESTARI. IPCC. 2005. Environmental Protection Expenditure in European Union Elzbieta Broniewicz Faculty of Management, Technical University of Bialystok Poland 2 Environmental Protection Expenditure in European Union Elzbieta Broniewicz Faculty of Management, Technical University of Bialystok Poland 2 11. References IPCC Special Report on Carbon Dioxide Capture and Storage Climate Change 2007. Geneva: Working Group III of the Intergovernmental Panel on Climate Change. IPCC. 2007. Climate Change 2007: The Physical Science Basis. Geneva: Intergovernmental Panel on Climate Change. Wee Kean Fong, Hiroshi Matsumoto, Chin Siong Ho, Yu Fat Lun, 2008. Energy consumption and carbon dioxide in the urban planning process in Malaysia. Joy Jacqueline Pereira, Ibrahim Komoo, Tan Ching Tiong, 2010. Climate change and disaster risk reduction, SEADPRI-UKM. UN. 2007. Sustainable Development Issues. United Nations Division for Sustainable Development. (http://www.un.org/esa/sustdev/documents/docs_sdissues.htm/, Retrieved: October 2007) Anwar Al-Mofleh, Soib Taib, M. Abdul Mujeebu, Wael Salah, 2007. Analysis of sectoral energy conservation in Malaysia. Energy Volume 34, Issue 6, June 2009. 1. Introduction Environmental protection expenditure should show the efforts being made to prevent, reduce and eliminate pollution resulting from the production or consumption of goods and services. The chapter presents the basic definitions and survey results of environmental protection expenditure in 25 European Union countries. p p p Environmental protection expenditure (EPE) is defined as the amount of money spent on all purposeful activities directly aimed at the prevention, reduction and elimination of pollution or nuisances resulting from the production processes (or consumption of goods and services). Data on environmental expenditure are collected from the European countries through the Joint OECD/Eurostat Questionnaire on Environmental Protection Expenditure and Revenues (EPER). The data covers five economic variables:  investments for environmental protection:  pollution treatment investments,  pollution prevention investments,  current expenditure for environmental protection,  subsidies/transfers given for environmental protection activities. subsidies/transfers given for environmental protection activities. The Questionnaire EPER contains also the data concerning household’s expenditure for environmental protection. The Questionnaire EPER contains also the data concerning household’s expenditure for environmental protection. The scope of Environmental Protection is defined according to the Classification of Environmental Protection Activities (CEPA, 2000), which distinguishes nine different environmental domains: protection of ambient air and climate, wastewater management, waste management, protection and remediation of soil, groundwater and surface water, noise and vibration abatement, protection of biodiversity and landscapes, protection against radiation, research and development and other environmental protection activities. The purpose of the chapter is to provide the information, how vary the environmental protection expenditure in European Union over the years and what are the trends in specific domains of environmental protection. The comparison between the amount of costs in different countries of European Union is very interesting. Environmental protection is an action or activity (which involves the use of equipment, labour, manufacturing techniques and practices, information networks or products) where the main purpose is to collect, treat, reduce, prevent, or eliminate pollutants and pollution or any other degradation of the environment resulting from the operating activity of the organization. 22 Environmental Management in Practice Environmental protection expenditure is the sum of capital and current expenditure for the undertaking of environmental protection activities. Environmental protection expenditure is the sum of capital and current expenditure for the undertaking of environmental protection activities. 1. Introduction Investment expenditure refers to financial or material costs, which aim at creating new permanent resources or improving (reconstruction, extension, restoration, adaptation or modernization) the existing objects of permanent property. It also means costs of so called first investment equipment. Presented division of investment costs is developed according to the rules of national accounting system, compliant with the “SNA 1993” recommendations. Investment expenditure can be divided into permanent resources and other costs. Environmental protection current expenditure includes costs of activity operation and maintenance (technology, process, equipment). Current expenditure is to prevent, reduce, dispose or eliminate pollution and other environmental losses caused by current activities of the entity. They include internal costs (including costs of operation and maintenance of environmental protection installations as well as environmental charges), costs of services provided by external entities, charges for sewage treatment and waste collection; costs of control systems, monitoring, laboratory research, management. Investment and current environmental expenditure have been divided, according to the property sectors, into: p p y public sector – government institutions (central public administration, regional and local governments as well as public organizations and institutions mainly classified in NACE, Rev. 1 as 75), public sector – government institutions (central public administration, regional and local governments as well as public organizations and institutions mainly classified in NACE, Rev. 1 as 75), - business sector – commercial enterprises, financial and insurance institutions as well as non-commercial institutions (all activities except NACE 75), - producers specialized in environmental protection (NACE 37 and 90) whose main activity is providing services for environment protection, mainly waste collection disposal and sewage treatment, g - household sector – there is no clear distribution into investment and current expenditure in this sector; the specificity of household activities combines all the types of expenditure together (SERIEE, 1994). p g ( ) The latest part of this chapter concerns Polish surveys of environmental protection expenditure in households. p g ( ) The latest part of this chapter concerns Polish surveys of environmental protection expenditure in households. 2 Household’s expenditure are excluded. 1 The latest available data. 2. Total environmental protection expenditure in UE Total environmental expenditure in 20071 costs European economy around 220 billion euro2. The biggest share was contributed by specialized producers – 41,2% of the total environmental expenditure, industry – 31,0% and public sector – 27,8% (Fig. 1 and Table 1). ) The basic indicators used to analyse the dynamics of environmental expenditure are:  contribution to Gross Domestic Product (GDP),  contribution to Gross Domestic Product (GDP),  the investment expenditure per inhabitant. Environmental expenditure in EU25 in 2007 accounted for 1,8% GDP and in 2002 for 1,7% GDP (except household expenditure) are presented in Fig. 2. Table 1. Environmental protection expenditure in European Union, million euro (Eurostat Data Navigation Tree) 2. Total environmental protection expenditure in UE Environmental Protection Expenditure in European Union 23 Specification Time 2007 2006 2005 2004 2003 2002 European Union (27 countries) 224 235e) 205 960 e) 192 387 e) 184 629 e) 179 409 e) 173 353 e) European Union (25 countries) 219 953 e) 202 686 e) 190 332 e) 182 792 e) 178 206 e) 172 052 e) European Union (15 countries) 205 186 e) 189 410 e) 179 624 e) 173 023 e) 169 671 e) 163 963 e) Belgium : : 133 6 245 5 963 5 752 Bulgaria 630 546 327 345 297 247 Czech Republic 2 613 2 309 1 449 1 410 1 050 675 Denmark 4 280 3 852 3 860 3 733 3 563 3 652 Estonia 424 399 265 209 121 134 Ireland : : : : : : Greece : : : : 12 15 Spain 21 410 e) 19 988 18 744 17 593 16 610 15 190 France 40 893 36 662 34 548 34 175 31 061 30 201 Italy 55 479 52 409 48 690 46 764 41 608 46 005 Cyprus 286 e) 173 128 166 124 37 Latvia 218 180 92 85 87 102 Lithuania 605 572 293 226 178 188 Luxembourg 279 294 280 262 242 239 Hungary 2 002 1 945 2 027 1 780 1 485 1 358 Malta : : : : : : Netherlands 11 493 e) 7 067 11 493 : 8 620 1 919 Austria 9 463 9 880 8 485 8 266 8 379 7 895 Poland 7 056 6 117 5 186 4 748 4 414 4 558 Portugal 1 773 e) 1 862 1 429 1 519 1 392 1 387 Romania 3 652 2 728 1 728 1 492 905 1 054 Slovenia 785 687 657 614 673 557 Slovakia 777 894 611 532 403 479 Finland 2 076 1 834 1 642 1 693 1 601 1 629 Sweden 2 169 1 989 2 055 1 807 1 776 1 677 United Kingdom 18 551 e) 15 903 14 456 13 224 12 454 11 802 : not available Table 1. Environmental protection expenditure in European Union, million euro (Eurostat Data Navigation Tree) Table 1. Environmental protection expenditure in European Union, million euro (Eurostat Data Navigation Tree) 24 Environmental Management in Practice Fig. 1. 2. Total environmental protection expenditure in UE During the period 2002-2007, the manufacturing sector in EU25, spent around 66% of total environmental protection expenditure, whilst electricity, gas and water supply sector and mining and quarrying sector 27% and 7% respectively. With reference to current expenditure this disproportion is bigger – 79%, 18% and 3% respectively (Georgescu, M.A. & Cabeca J. C., 2010). In 2007, the leading environmental domain in industry in 25 EU countries was waste management (25,7%). The other important area of environmental expenditure was the wastewater management and protection of ambient air and climate, which accounted for 25,7% and 25,4% of total expenditure. The structure of expenditure by the environmental domains in industry in selected countries in 2007 is shown in Fig. 5. Environmental expenditure, according to Classification of Environmental Protection Activities (CEPA), are divided into nine environmental domains: p g Activities (CEPA), are divided into nine environmental domains: 1. Protection of ambient air and climate 2. Wastewater management 3. Waste management 4. Protection and remediation of soil, groundwater and surface water 5. Noise and vibration abatement 6. Biodiversity and landscapes protection 7. Protection against radiation 8. Research and Development 9. Other environmental protection activities (mainly environmental administration and management, education, training and information, indivisible expenditure and other expenditure not classified elsewhere). The business sector consists of: 1. agriculture, hunting, fishing, forestry, 2 industry sector: 4. Protection and remediation of soil, groundwater and surface water p 9. Other environmental protection activities (mainly environmental administration and management, education, training and information, indivisible expenditure and other expenditure not classified elsewhere). 9. Other environmental protection activities (mainly environmental administration and management, education, training and information, indivisible expenditure and other expenditure not classified elsewhere). The business sector consists of: 1. agriculture, hunting, fishing, forestry, y - mining and quarrying, - manufacturing, - electricity, gas and water supply sector, 3. other business. However, the environmental protection expenditure occur mainly in the industry sector. During the period 2002-2007, the manufacturing sector in EU25, spent around 66% of total environmental protection expenditure, whilst electricity, gas and water supply sector and mining and quarrying sector 27% and 7% respectively. With reference to current expenditure this disproportion is bigger – 79%, 18% and 3% respectively (Georgescu, M.A. & Cabeca J. C., 2010). However, the environmental protection expenditure occur mainly in the industry sector. 2. Total environmental protection expenditure in UE The structure of environmental expenditure in 25 European Union countries in 2007 (Eurostat Data Navigation Tree) Fig. 1. The structure of environmental expenditure in 25 European Union countries in 2007 (Eurostat Data Navigation Tree) Fig. 2. Environmental protection expenditure in EU25 as % of GDP in 2002 and 2007 – by sectors (Eurostat Data Navigation Tree) Fig. 2. Environmental protection expenditure in EU25 as % of GDP in 2002 and 2007 – by sectors (Eurostat Data Navigation Tree) Comparing the share of environmental protection expenditures in GDP in particular countries, it could be noticed, that differences in environmental expenses are huge. Austria is one of the countries with the highest indicator in European Union (Fig. 3). Moreover, that expenditure per inhabitant in Austria is very high – in 2007 it was about 820 euro. In other EU countries this indicator came to 160 – 620 euro per inhabitant (Fig. 4). Fig. 3. Environmental protection expenditure in selected countries EU as % of GDP, data from the latest available survey (Eurostat Data Navigation Tree) Fig. 3. Environmental protection expenditure in selected countries EU as % of GDP, data from the latest available survey (Eurostat Data Navigation Tree) Environmental Protection Expenditure in European Union 25 Fig. 4. Environmental protection expenditure in selected countries EU – euro per inhabitant, data from the latest available survey (Eurostat Data Navigation Tree) Fig. 4. Environmental protection expenditure in selected countries EU – euro per inhabitant, data from the latest available survey (Eurostat Data Navigation Tree) Environmental expenditure, according to Classification of Environmental Protection Activities (CEPA), are divided into nine environmental domains: 1. Protection of ambient air and climate 2. Wastewater management 3. Waste management 4. Protection and remediation of soil, groundwater and surface water 5. Noise and vibration abatement 6. Biodiversity and landscapes protection 7. Protection against radiation 8. Research and Development 9. Other environmental protection activities (mainly environmental administration and management, education, training and information, indivisible expenditure and other expenditure not classified elsewhere). The business sector consists of: 1. agriculture, hunting, fishing, forestry, 2. industry sector: - mining and quarrying, - manufacturing, - electricity, gas and water supply sector, 3. other business. However, the environmental protection expenditure occur mainly in the industry sector. 2. Total environmental protection expenditure in UE During the period 2002-2007, the manufacturing sector in EU25, spent around 66% of total environmental protection expenditure, whilst electricity, gas and water supply sector and mining and quarrying sector 27% and 7% respectively. With reference to current expenditure this disproportion is bigger – 79%, 18% and 3% respectively (Georgescu, M.A. & Cabeca J. C., 2010). ) In 2007, the leading environmental domain in industry in 25 EU countries was waste management (25,7%). The other important area of environmental expenditure was the wastewater management and protection of ambient air and climate, which accounted for 25,7% and 25,4% of total expenditure. The structure of expenditure by the environmental domains in industry in selected countries in 2007 is shown in Fig. 5. In 2007, the leading environmental domain in industry in 25 EU countries was waste management (25,7%). The other important area of environmental expenditure was the wastewater management and protection of ambient air and climate, which accounted for 25,7% and 25,4% of total expenditure. The structure of expenditure by the environmental domains in industry in selected countries in 2007 is shown in Fig. 5. 26 Environmental Management in Practice 0% 20% 40% 60% 80% 100% Czech Republic Spain France Netherlands Poland United Kingdom Protection of ambient air and climate Wastewater management Waste management Non-core domains Fig. 5. Structure of environmental expenditure in industry of selected countries in 2007 (Eurostat Data Navigation Tree) Fig. 5. Structure of environmental expenditure in industry of selected countries in 2007 (Eurostat Data Navigation Tree) Current expenditure for environmental protection in 25 countries of European Union are higher than investments expenditure. In 2002-2007 current expenditure represented around 81% of total expenditure, whilst investment expenditure – 19% (Fig. 6). Fig. 6. Investment and current environmental protection expenditure in 25EU in 2002-2007, in million euro (Eurostat Data Navigation Tree) Fig. 6. Investment and current environmental protection expenditure in 25EU in 2002-2007, in million euro (Eurostat Data Navigation Tree) 2. Investment expenditure Following the methodology applied in European Union (SERIEE, 1994), the investment expenditure includes end-of-pipe and integrated investments: Following the methodology applied in European Union (SERIEE, 1994), the investment expenditure includes end-of-pipe and integrated investments:  the end-of-pipe investments (pollution treatment) – they do not affect in the production process itself (the production may be carried out without this kind of investment) but p p p g  the end-of-pipe investments (pollution treatment) – they do not affect in the production process itself (the production may be carried out without this kind of investment), but they reduce and dispose pollutants generated in the production process. The most  the end-of-pipe investments (pollution treatment) – they do not affect in the production process itself (the production may be carried out without this kind of investment), but they reduce and dispose pollutants generated in the production process. The most 27 Environmental Protection Expenditure in European Union investments in the public sector and in specialised producers – according to the methodology recommended by the Office of Statistics of the European Communities EUROSTAT – are entirely rated among end-of-pipe enterprises, investments in the public sector and in specialised producers – according to the methodology recommended by the Office of Statistics of the European Communities EUROSTAT – are entirely rated among end-of-pipe enterprises, y g  integrated technology (pollution prevention) – they lead to reduction of generated pollution through the modification of technological processes which makes the production cleaner and more environmentally friendly. When a new production process is introduced, the environmental expenditure refer to the expenditure that outstrip the costs of cheaper and in working order, but less environmentally friendly equipment.  integrated technology (pollution prevention) – they lead to reduction of generated pollution through the modification of technological processes which makes the production cleaner and more environmentally friendly. When a new production process is introduced, the environmental expenditure refer to the expenditure that outstrip the costs of cheaper and in working order, but less environmentally friendly equipment. q p The share of integrated technology in industry in EU25 exceeded the level of 35% in 2001 and in the year 2006 it increased to 43,0% (Georgescu, M.A. & Cabeca J. C., 2010). In 2007 it was 39% (Fig. 7). Companies adjust to the requirements of environmental protection by changing a production technology and implementing the best available productive and environmental solutions. 3 Connected products are products which are used directly and solely for environmental protection (for example septic tanks, filters, waste bags). Adapted products are products that are less polluting, at the time of their consumption and/or scrapping, than equivalent traditional products. In most cases, such products are more costly, and their production and consumption are usually encouraged by fiscal and other incentives. Products which are cleaner (and therefore more environmentally friendly) when used or disposed of. These products are sometimes also called (environmentally) cleaner products. Only the extra-cost is accounted for in the environmental protection expenditure (Glossary of Environment Statistics, 1997). Connected products are products which are used directly and solely for environmental protection (for example septic tanks, filters, waste bags). 3. Current environmental expenditure Total current expenditure is the sum of internal current expenditure and fees/purchases. Internal current expenditure includes the use of energy, material, maintenance and own personnel for measures made by the sector to protect the environment. A large part of internal expenditure is related to operating environmental protection equipment. There are also other internal expenditure such as general administration, education, information, environmental management and certification, research and development. Internal current expenditure includes purchases of connected and adapted non-capital goods3 such as extra cost for low sulphur fuels. These are sometimes not part of specific surveys but estimated based on existing information e.g. on number of units and unit costs. Fees/Purchases includes all purchases of environmental protection services, both from public and private producers. These payments are clearly linked with an environmental protection activity done outside the enterprise and should exclude e.g. fines and penalties. The payments include: - Payments to specialised producers (enterprises) for waste and wastewater collection and treatment and payments to environmental consultants linked e.g. with environmental management and education. - Payments to Public sector for waste and wastewater collection and treatment (whatever the name of the payments – fees, charges etc) as well as permits and surveillance fees. - Payments to Public sector for waste and wastewater collection and treatment (whatever the name of the payments – fees, charges etc) as well as permits and surveillance fees. Subsidies/Transfers (given or received) include all types of transfers financing Environmental Protection activities in other sectors, including transfers to or from other countries. These constitute expenditure for the paying sector (public sector), and revenue for the receiving sector (business sector and specialised producers sector). Payments of general environmental or green taxes (such as energy taxes) are excluded. Sometimes Environmental Protection activities produce by-products that have an economic value. These could either be sold and generate revenues, or be used internally and lead to reductions in costs. Examples include energy generated or material recovered, as a result of waste treatment. There should always be a specific Environmental Protection activity (and expenditure) that these receipts stem from. Receipts from by-products is the sum of the sales value and the value of the cost-saving (if used internally) related to these by-products. g ( y) y p Public sector and specialised producers receive the payments for environmental protection services. This is entered as revenues in the respective sector (EPER). 2. Investment expenditure Further changes in the structure of investment expenditure can be expected due to the implementation of a directive concerning integrated prevention and reduction of pollution (a Directive 96/62/EEC on integrated prevention and reduction of pollution – IPPC). Enforcement of the Directive requires establishing standards of pollution emission based on a concept of the Best Available Technique – BAT, that guarantees application of low-waste technologies, economical raw materials and energy use as well as application of the latest scientific and technical achievements. Fig. 7. Industry’s environmental protection investments in EU25 in 2002-2007, million euro (Eurostat Data Navigation Tree) Fig. 7. Industry’s environmental protection investments in EU25 in 2002-2007, million euro (Eurostat Data Navigation Tree) In the industry sector, the environmental domain, which attracted most of capital expenditure for both pollution treatment and pollution prevention investments, was protection of ambient air and climate. The second domain was wastewater management. This tendency is noticed since 2002 (Fig. 8, Fig. 9). The public sector and specialized producers sector were dominated by end-of-pipe investments, what resulted from the specificity of environmental protection activities. Major expenditure was allocated for building and modernization of wastewater plants, dumping sites and other waste disposal installations. 28 Environmental Management in Practice Fig. 8. The structure of industry’s pollution treatment investments in EU25 in 2002-2007 by the environmental domains (Eurostat Data Navigation Tree) Fig. 8. The structure of industry’s pollution treatment investments in EU25 in 2002-2007 by the environmental domains (Eurostat Data Navigation Tree) Fig. 9. The structure of industry’s integrated technlology in EU25 in 2002-2007 by the environmental domains (Eurostat Data Navigation Tree) Fig. 9. The structure of industry’s integrated technlology in EU25 in 2002-2007 by the environmental domains (Eurostat Data Navigation Tree) 29 Environmental Protection Expenditure in European Union Connected products are products which are used directly and solely for environmental protection (for example septic tanks, filters, waste bags). 3. Current environmental expenditure The main environmental domain of current costs in industry sector during the period 2002- 2007 was waste management (about 40%) and wastewater management (about 30%). Approximately, 10% concern other environmental protection activities, like general administration, education, information and environmental management – Fig 11. 30 Environmental Management in Practice Current expenditure in public and specialized producers sectors was directed largely towards ensuring a good provision of wastewater treatment and waste management services (Georgescu, M.A. & Cabeca J. C., 2010). Current expenditure in public and specialized producers sectors was directed largely towards ensuring a good provision of wastewater treatment and waste management services (Georgescu, M.A. & Cabeca J. C., 2010). Current expenditure in public and specialized producers sectors was directed largely towards ensuring a good provision of wastewater treatment and waste management services (Georgescu, M.A. & Cabeca J. C., 2010). Internal current expenditure Related to operating environmental protection equipment Protection of ambient air and climate Wastewater management Waste management Protection and remediation of soil, groundwater and surface water Noise and vibrations abatement Biodiversity and landscape protection Protection against radiation Research and development General administration, education, information, environmental management and certification (+) plus (-) minus Fees/purchases (+) plus or (-) minus Subsidies/Transfers (-) minus Receipts from by-products = (equals) Current expenditure Fig. 10. Classification of current expenditure on the environment in industry sector Fig. 11. The structure of industry’s current expenditure in EU25 in 2002-2007 by the environmental domains (Eurostat Data Navigation Tree) Internal current expenditure Related to operating environmental protection equipment Protection of ambient air and climate Wastewater management Waste management Protection and remediation of soil, groundwater and surface water Noise and vibrations abatement Biodiversity and landscape protection Protection against radiation Research and development General administration, education, information, environmental management and certification (+) plus (-) minus Fees/purchases (+) plus or (-) minus Subsidies/Transfers (-) minus Receipts from by-products = (equals) Current expenditure Fig. 10. Classification of current expenditure on the environment in industry sector Fig. 10. Classification of current expenditure on the environment in industry sector Fig. 10. Classification of current expenditure on the environment in industry sector g p y Fig. 11. The structure of industry’s current expenditure in EU25 in 2002-2007 by the environmental domains (Eurostat Data Navigation Tree) 31 Environmental Protection Expenditure in European Union 4. Environmental expenditure in households Environmental protection expenditure in households contains of 1) purchases of connected and adapted products and 2) payments and fees for environmental protection services – Fig. 12. Expenditure (investment and current) - purchases Protection of ambient air and climate Wastewater management Waste management Noise and vibrations abatement Biodiversity and landscape protection Other areas of environmental protection (-) minus Subsidies + (plus) Payments and fees collection and treatment of waste collection and treatment of wastewater = (equals) Environmental protection expenditure Fig. 12. Classification of households expenditure on the environment Based on Member Countries experience with the collection of data on private households there is no need to make a distinction between investments and current expenditure (EPER). Household purchases are viewed as current, in line with the national accounts. Examples are: p , p protection of ambient air and climate:  protection of ambient air and climate: - heat consumption meters and thermo regulators; - modernization of central heating systems for the entire building and for a single apartment; - installation of equipment for the treatment of fuel gases; - purchase, operation and maintenance of air pollution control devices for motor vehicles e.g. extra costs for use of more environmentally friendly goods such as unleaded petrol, or service costs for proper adjustments of engines, - purchase and installation of energy-saving windows; - additional insulation for the building protecting against cold; 32 Environmental Management in Practice  wastewater management: connection to the public sewer; - purchase of sewage treatment facilities such as septic tanks, - construction of individual wastewater treatment plants;  waste management: - purchase of goods used in connection with waste management such as bins, bags, composts etc.; - purchase of sewage treatment facilities such as septic tanks, - construction of individual wastewater treatment plants; g - purchase of goods used in connection with waste management such as bins, bags, composts etc.; g - purchase of goods used in connection with waste management such as bins, bags, composts etc.;  biodiversity and landscape protection: - tree and bush planting; - house facade repairs;  noise and vibrations abatement: - purchase and installation of noise reducing windows; - fences and live fences, noise and vibrations reducing screens. Household expenditure for environmental protection includes all payments and fees for services purchased from municipalities and specialised producers of environmental protection services. 4. Environmental expenditure in households These include mainly:  payments for the collection and treatment of waste,  payments for the collection and treatment of wastewater. Data of environmental protection expenditures in household is not available in Eurostat. Only a few EU countries conduct surveys in this sector (e.g. Austria, Hungary, Poland). In Poland, environmental protection expenditure in private households are examined from 1998. They are the biggest amount of environmental protection expenditures in Polish economy – during the period 1998-2009 it was approximately the same amount as the sum of expenditure in three sectors: public, business and specialized producers (Results of surveys of environmental protection expenditure conducting in 1998-2010. Ministry of the Environment in Poland). Data of environmental protection expenditures in household is not available in Eurostat. Only a few EU countries conduct surveys in this sector (e.g. Austria, Hungary, Poland). In Poland, environmental protection expenditure in private households are examined from 1998. They are the biggest amount of environmental protection expenditures in Polish economy – during the period 1998-2009 it was approximately the same amount as the sum of expenditure in three sectors: public, business and specialized producers (Results of surveys of environmental protection expenditure conducting in 1998-2010. Ministry of the Environment in Poland). ) The surveys are carried out on a representative sample of 1300 Polish households selected randomly by the Central Statistical Office for the purpose of examinating Polish households budgets. The survey covered 6 groups selected in accordance with their social and economic status, namely: - households of workers – 44,6% of the sample, - households of farmers with additional source of income – 4,3%, - households of farmers – 5,7%, - households of self-employed people – 6,1%, - households of the retired and pensioners – 35,2%; - households supported from non-profit sources – 4,1%. Environmental expenditure of households in 2009 amounted to 5,8 billion euro. The share of purchases, installations and constructions of appliances as well as connected goods accounted for 72,6%, while environmental services 27,4%. Environmental expenditure of households in 2009 amounted to 5,8 billion euro. The share of purchases, installations and constructions of appliances as well as connected goods accounted for 72,6%, while environmental services 27,4%. Costs of purchase, installation and construction of environmental devices and products referred mainly to air protection (77,6%), especially purchase and installation of energy- saving windows, houses heat-insulation and heating installation modernization. 4. Environmental expenditure in households The majority of expenditure concerning bio-diversity and landscape protection was allocated for renovations of building’s elevations and with regard to protection against noise and vibrations – purchase and installation of soundproof windows (Fig. 13). Costs of purchase, installation and construction of environmental devices and products referred mainly to air protection (77,6%), especially purchase and installation of energy- saving windows, houses heat-insulation and heating installation modernization. The majority of expenditure concerning bio-diversity and landscape protection was allocated for renovations of building’s elevations and with regard to protection against noise and vibrations – purchase and installation of soundproof windows (Fig. 13). 33 Environmental Protection Expenditure in European Union Among the costs of environmental services, the majority (68,3%) consisted of wastewater collection, treatment and discharge fees. The rest of 33,7% was constituted of waste collection charges. g It should be noted, however, that the rates of fees for the environmental services related to the environmental protection depended on the type of a building. For the purpose of the survey two main groups were defined: a multi-family apartment house (53% households in the sample) and a single-family house (43%). Moreover, in the case of 4% households the delivered information was the total cost of environmental protection products and services for their house (a single-family house), garage, summer house and bungalow. The average services fees for different types of buildings are presented in Fig. 14. Fig. 13. The structure of expenditure for purchasing connected goods to households in 2009 in Poland (Environment 2010. Statistical Information and Elaboration, 2010). Fig. 13. The structure of expenditure for purchasing connected goods to households in 2009 in Poland (Environment 2010. Statistical Information and Elaboration, 2010). Many owners of single-family houses, mainly in the country, most probably used to discharge their wastewater directly on the farmland and the most popular way of waste disposal was burning them or taking it to an unauthorized dumping ground to avoid the costs of utilization. The amount of charges for the environmental protection services was unrelated to the social and economic status of the members of the household. However, the highest expenditure on the purchase and installation of the equipment and products used directly for the purpose of environmental protection was recorded in households of self-employed people (excluding farmers) – 397 euro in 2009, whereas the lowest – 38 euro in households supported from nonprofit sources. 4. Environmental expenditure in households The average expenditure on the environment (services payments excluded) by source of income is presented in Fig. 15. 34 Environmental Management in Practice Fig. 14. Cost of environmental protection services for different types of building in Poland in 2009 (in euro). Fig. 14. Cost of environmental protection services for different types of building in Poland in 2009 (in euro). Fig. 15. The amount of expenditure on the purchase and installation of the equipment and products used directly for the purpose of environmental protection by source of income in all surveyed Polish households in 2009 (in euro). Fig. 15. The amount of expenditure on the purchase and installation of the equipment and products used directly for the purpose of environmental protection by source of income in all surveyed Polish households in 2009 (in euro). 35 Environmental Protection Expenditure in European Union 5. Conclusion Eurostat works towards systematically collecting environmental statistics for all economic sectors within the EU. These statistics are used to assess the effectiveness of new legislation and policies and to analyse the links between environmental pressures and the structure of the economy. For many years, European statistical services have collected data on air pollution, energy, water consumption, wastewater, solid waste, and their management. The links between these data and environmental data of an economic nature, such as environmental expenditure enable policymakers to consider the environmental impacts of economic activities, for example on resource consumption, air or water pollution, and waste production, and to assess actions (such as investment and current expenditure) that may be carried out to limit the causes and risks of pollution. The analysis of spending on environmental protection has a strategic interest and allows an evaluation of environmental policies already in place. A low level of expenditure does not necessarily mean that a country is not effectively protecting its environment. Indeed, information on expenditure tends to emphasise clean-up costs at the expense of cost reductions which may have resulted from lower emissions or more effective protection measures (Georgescu, M.A. & Cabeca J. C., 2010). Regulation NO 2056/2002 of 5 November 2002 amending Council Regulation NO58/97 concerning Structural Business Statistics Results of surveys of environmental protection expenditure conducting in 1998-2010. Ministry of the Environment in Poland Regulation NO 2056/2002 of 5 November 2002 amending Council Regulation NO58/97 concerning Structural Business Statistics Results of surveys of environmental protection expenditure conducting in 1998-2010. Ministry of the Environment in Poland SERIEE – European System for the Collection of Economic Information on the Environment, Manual, Luxemburg 1994 y SERIEE – European System for the Collection of Economic Information on the Environment, Manual, Luxemburg 1994 Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders Rosemary M. Caron, Michael E. Rezaee and Danielle Dionne University of New Hampshire 6. References Broniewicz, E. (2001). Expenditure on the Environment by Polish Households in the Year 2000, Economics and Environment, ed. by Poskrobko, B., pp. 117-132, Foundation of Environmental and Resource Economists, ISSN 0867-8898, Bialystok, Poland Broniewicz, E. (2004). Environmental protection expenditure in Poland in comparison with European Union countries, Proceedings of Business strategy and the environment, pp. 58-66, Leeds, UK, September 2004 p CEPA 2000 – Classification of Environmental Protection Activities and Expenditure Commission Recommendation of 30 May 2001 on the recognition, measurement and disclosure of environmental issues in the annual accounts and annual reports of companies (2001/453/EC). Official Journal of the European Communities g / g Environment 2010. Statistical Information and Elaboration (2010). Central Statistical Office, ISSN 0867-3217, Poland, 01-15.03.2011. Available from http://www.stat.gov.pl/gus p g p g EPER – Environmental Protection Expenditure And Revenues. Joint OECD/Eurostat Questionnaire, 2002-2010 Eurostat Data Navigation Tree, 02.01-20.03.2011. Available from http://ec.europa.eu/eurostat Georgescu, M.A. & Cabeca J. C. (2010). Environmental Protection Expenditure and Revenues in the EU, EFTA and candidate countries 2001-2006, In: Eurostat. Statistics in Focus, 31/2010, 12.01.2011. Available from http://ec.europa.eu/eurostat Glossary of Environment Statistics (1997). Studies in Methods, Series F, No. 67, United Nations, New York, 10.03.2011. Available from http://stats.oecd.org/glossary 36 Environmental Management in Practice 3 1. Introduction Many socioeconomically and geographically diverse communities in the United States have been challenged by occurrences of environmental contamination and the related complex public health issues. The investigations associated with such concerns have traditionally been the responsibility of governmental agencies. Communities facing potential environmental exposures often believe that government-based environmental agencies are not adequately addressing their concerns regarding risk, thus resulting in their misunderstanding and distrust of the regulatory process. A schism develops whereby the community perceives that government is either not doing enough to address their concerns and/or are being influenced by the relevant industry. The governmental agencies involved perceive that the community possesses an inaccurate or irrational perception of the potential risks. As a result, a stressful relationship often arises. p Recommendations for effective risk communication have been developed and published (Covello & Sandman, 2001; Hance et al., 1989; Sandman, 1989). Research has also demonstrated the importance of developing relationships among stakeholders and its impact on information delivery and reception (ATSDR, 2004). Given that stakeholder groups perceive risk differently, it is imperative for each group to appreciate the viewpoints of all involved to engage in effective dialog (Park et al., 2001; Tinker et al., 2001). g g g ( ) Cox (2006) defines environmental communication as “…the pragmatic and constitutive vehicle for our understanding of the environment as well as our relationships to the natural world; it is the symbolic medium that we use in constructing environmental problems and negotiating society’s different responses to them.” Although opportunities for public participation in environmental assessments have greatly increased, the environmental communication process among key stakeholders needs further evaluation (Charnley & Engelbert, 2005; McKinney & Harmon, 2002). The purpose of this chapter is to describe an evaluative process to develop and propose recommendations that could improve the environmental communication that occurs among diverse stakeholders, such as an environmental regulation and protection agency, waste disposal and energy producing facilities, community activists and the general public. Two case studies will be presented; the first describes the management of environmental permitting decisions in several disparate communities; and the second describes the management and perception of health risks from a single-owner waste-to-energy facility in two distinct communities. 1. Introduction To 38 Environmental Management in Practice accomplish this goal, this chapter will: 1.) examine how a state environmental agency and waste disposal and energy producing facilities describe their environmental communication experiences regarding various permitting operations and the risk perceptions of the impacted communities; 2.) identify effective communication methods; 3.) discuss the strengths and limitations of these activities; and 4.) propose recommendations for practitioners to advance environmental communication strategies among these key stakeholders. 1.1 Community ecology and capacity y gy p y Communities are important determinants in environmental health-related problems for populations. A community’s ecology (i.e., its social, cultural, economic, and political composition) can affect how a persistent and/or perceived environmental health problem is addressed. For example, the primary stakeholders in a refugee resettlement community’s childhood lead poisoning problem include the residents/resettled refugees in poor quality housing, refugee resettlement agencies, social service agencies, the local city health department, housing agencies, city building inspectors, realtors, property owners/managers, child care providers, health care community, etc. Some stakeholders view the childhood lead poisoning problem in the community as indicative of a larger issue, namely a community that is undergoing growth and diversification due to its refugee and immigrant resettlement status. Hence, others believe they are not able to solve the problem due to its enormity and complexity. As a result, this persistent environmental public health issue propagates in the community with varied efforts (Caron & Serrell, 2009; Wehrly, 2006). Childhood lead poisoning has been described as a wicked persistent environmental public health problem that is multi-factorial in nature and possesses no clear resolution due to the involvement of numerous stakeholders who define the problem differently and who pose uncoordinated solutions. Since wicked problems often possess no definitive solutions, remediation must focus on how to best manage them (Caron & Serrell, 2009). As part of a management practice for complex environmental public health issues, we propose that the community’s ecology – its political, ethnic and socioeconomic factors, including zoning laws, housing policies, cultural behavior, and language barriers - is a key determinant in shaping a population’s perception of risk and in developing effective communication strategies. In addition, understanding a community’s ecology can contribute to building the community’s capacity to affect the local management and communication of persistent and/or perceived environmental public health issues. 2. Case study: managing environmental permitting decisions in dissimilar communities The stakeholders considered in this work include a state environmental agency, facility managers of Title V operating facilities and community residents living near the facilities. Specifically, the New Hampshire Department of Environmental Services, Air Resources Division (NHDES ARD) is responsible for monitoring and regulating air quality that is protective of public health and the natural environment in the State of New Hampshire (ARD, 2010). NHDES ARD accomplishes this goal via numerous programs including a statewide permitting program to assure compliance with the Title V federal mandate. The purpose of the Title V permitting process is to ensure that facilities will not emit hazardous pollutants to a degree which could negatively affect human health. Specifically, the Title V mandate states that facilities which emit over 100 tons of any regulated pollutant, such as Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 39 carbon monoxide and sulfur oxides; or emit over 50 tons of nitrous oxides; or emit 10 tons of any of the federally regulated hazardous air pollutants need to apply to the state environmental agency for a Title V permit (ARD, 2008). Table 1 outlines the Title V operating facilities examined in this study: Turnkey Recycling and Environmental Enterprises, a solid waste management facility in operation since 1979 in Rochester, New Hampshire (NH); Mt. Carberry Landfill, historically used as a landfill for pulp and paper byproducts and a solid waste disposal site since 1989 in Berlin, NH; Four Hills Landfill, a solid waste disposal site since 1970 in Nashua, NH; Indeck Energy Services, Inc., a biomass electric generating facility in operation since 1987 in Alexandria, NH; Schiller Station, historically a coal burning facility from 1950 through 2006 and now a woodchip burning operation in Portsmouth, NH; and Wheelabrator Technologies, Inc., a solid waste energy plant in operation since 1987 in Claremont, NH. carbon monoxide and sulfur oxides; or emit over 50 tons of nitrous oxides; or emit 10 tons of any of the federally regulated hazardous air pollutants need to apply to the state environmental agency for a Title V permit (ARD, 2008). carbon monoxide and sulfur oxides; or emit over 50 tons of nitrous oxides; or emit 10 tons of any of the federally regulated hazardous air pollutants need to apply to the state environmental agency for a Title V permit (ARD, 2008). 1 U.S. Census Bureau. Population Finder. (http://www.census.gov/) 2. Case study: managing environmental permitting decisions in dissimilar communities g y p ( ) Table 1 outlines the Title V operating facilities examined in this study: Turnkey Recycling and Environmental Enterprises, a solid waste management facility in operation since 1979 in Rochester, New Hampshire (NH); Mt. Carberry Landfill, historically used as a landfill for pulp and paper byproducts and a solid waste disposal site since 1989 in Berlin, NH; Four Hills Landfill, a solid waste disposal site since 1970 in Nashua, NH; Indeck Energy Services, Inc., a biomass electric generating facility in operation since 1987 in Alexandria, NH; Schiller Station, historically a coal burning facility from 1950 through 2006 and now a woodchip burning operation in Portsmouth, NH; and Wheelabrator Technologies, Inc., a solid waste energy plant in operation since 1987 in Claremont, NH. Facility Name Type of Industry In Operation Since Location Population of Community1 Turnkey Recycling and Environmental Enterprises Landfill 1979 Rochester, NH 30,527 Mt. Carberry Landfill 1989 Berlin, NH 10,109 Four Hills Landfill 1970 Nashua, NH 86,837 Indeck Energy Services, Inc. Electricity 1987 Alexandria, NH 1,521 Schiller Station Electricity 1950 Portsmouth, NH 20,495 Wheelabrator Technologies, Inc. Incinerator 1987 Claremont, NH 13,097 Table 1. Facility stakeholders involved in the environmental communication of permitting decisions. The community members living in the midst of these Title V operating facilities represent the final stakeholder group. The demographics of these communities are diverse with three communities considered rural and the remaining considered urban. 3. Methods Data collection and analysis of the interactions among key stakeholders were conducted using collective case study methodology (Cottrell & McKenzie, 2005). Data was collected from publicly available New Hampshire Department of Environmental Services (NHDES) documents concerning specific Title V operating facilities in the State of New Hampshire. These documents were in the form of written or e-mail correspondence, phone logs and 40 Environmental Management in Practice public hearing audio tapes and written testimonies. A structured questionnaire was applied to each occurrence of communication. Each document was reviewed and information abstracted regarding the date and type of communication; origin of concern; responder; general summary of concern; action requested; response time; total number of complaints per facility; method of ongoing communication; whether feelings of distrust or doubt were expressed by the community with respect to facility operations; the type of organization(s) the community member contacted prior/following to communicating with the state agency or facility; and non-verbal communication (e.g., body language) at public hearings. Abstracted information was first organized in chronological order by facility; duplicate records were removed; and a search for potentially missed documents was conducted. A document summarizing record review information for each site was constructed. Additionally, public inquiries/concerns received about each facility were reviewed and classified into thematic areas. Semi-structured interviews were conducted, following Institutional Review Board approval from the University of New Hampshire, with NHDES employees involved in the Title V permitting process and Title V operating facility managers. Respondents were asked questions about the public’s perception of their work and whether the facility’s operations were considered to be contentious or non-contentious; the health and environmental concerns of the impacted community; and who they considered the major stakeholders. Respondents were asked if they had experience conducting and/or attending a public hearing about their facility. Information pertaining to the type and number of concerns communicated by the public was collected, as well as how these issues were addressed. With respect to the environmental management of concerns, the respondents were queried as to whether or not they believed they were proactive in involving the community and if there was a professional at their respective organizations who was responsible for handling the public’s concerns. 3. Methods The last series of questions posed to the respondents inquired about whether they thought improving environmental communication among all stakeholders would enhance working relationships; whether an appointed liaison would assist with environmental communication; and what specific recommendations they have to improve the communication of environmental permitting decisions among stakeholders. p g g The interviews were transcribed and a content analysis, using QSR NVivo (a computer- assisted qualitative data analysis program), was conducted of the structured interview responses to extract and code recurring themes. 4.1 Structured questionnaires q Tables 2A-F summarize the correspondence information among stakeholders regarding each facility. In general, public inquiries were fielded by NHDES ARD staff and/or the NHDES Complaint Manager. Inquiries were typically answered in two days or less. The concerns expressed ranged from health concerns (e.g., cancer, respiratory illness) to nuisance complaints (e.g., odor, noise, traffic). The actions most often requested involved scheduling a public hearing, extending the public comment period, conducting air and water quality testing, and initiating an independent investigation of NHDES’ administration. In some instances, the community members present at the public hearing called for the closure of the facility. Distrust of NHDES and/or the facility was expressed for the majority of sites. One exception to this sentiment was the Mt. Carberry Landfill. Community Ecology and Capacity: Advancing E i l C i i S i Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 41 Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders Common frustrations voiced by citizens included the inability to locate the appropriate representative, either at NHDES or the facility, to communicate their concern(s) and dissatisfaction with the response to their inquiry, thus leading them to contact the Environmental Protection Agency (EPA) or a local official to relay their concerns. Figures 1- 6 represent photographs of each facility examined. Common frustrations voiced by citizens included the inability to locate the appropriate representative, either at NHDES or the facility, to communicate their concern(s) and dissatisfaction with the response to their inquiry, thus leading them to contact the Environmental Protection Agency (EPA) or a local official to relay their concerns. Figures 1- 6 represent photographs of each facility examined. 4.1 Structured questionnaires Turnkey Recycling and Environmental Enterprises Correspondence Content Phone E-mail Public Hearing Written Time period of Correspondence 2004, 2005 2003, 2004, 2005 2004 2004 Total Number 59 7 7 7 Responder NHDES ARD; NHDES Complaint Manager NHDES ARD; Title V Program Manager NHDES ARD; Title V Permitting Engineer; Facility Manager None Summary of Concern Odor Odor Health (cancer); Odor; Air quality; Water quality Health (colitis); Odor; Air quality; Water quality Response Time Same day Same day Same day Not applicable Action Requested None Public hearing Air and water quality testing; Deny permit; Close facility Air and water quality testing; Deny permit; Close facility; Investigate NHDES Perception of Distrust Yes Yes Yes Yes Ongoing Communication None None None None Other Organizations Contacted None Director of Waste Management Services None Director of Waste Management Services Table 2A. Correspondence among stakeholders involved in the environmental communication of permitting decisions for a landfill facility. Table 2A. Correspondence among stakeholders involved in the environmental communication of permitting decisions for a landfill facility. 42 Environmental Management in Practice Mt. Carberry Landfill Correspondence Content Phone E-mail Public Hearing Written Time period of Correspondence 2006 No e-mail correspondence 2007 2007 Total Number 16 1 4 Responder NHDES ARD NHDES ARD and Facility Manager NHDES and Director of NHDES Summary of Concern Odor None – in support of facility Title V permitting process Response Time Same day Same day Two days Action Requested None Extension of public comment period Public hearing Perception of Distrust No No No Ongoing Communication NHDES Follow-up None None Other Organizations Contacted No No No Table 2B. Correspondence among stakeholders involved in the environmental communication of permitting decisions for a landfill facility. No No Table 2B. Correspondence among stakeholders involved in the environmental communication of permitting decisions for a landfill facility. Fig. 1. Turnkey Recycling and Environmental Enterprises, Rochester, New Hampshire. Source: http://www.greenrightnow.com/wabc/2009/05/19/unh-first-university-to-use- landfill-gas-as-primary-fuel-source/#more-3818 Fig. 1. Turnkey Recycling and Environmental Enterprises, Rochester, New Hampshire. Source: http://www.greenrightnow.com/wabc/2009/05/19/unh-first-university-to-use- landfill-gas-as-primary-fuel-source/#more-3818 Community Ecology and Capacity: Advancing E i t l C i ti St t i Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 43 Fig. 2A. Mt. Carberry Landfill, Berlin, NH. Fig. 2B. Flare at Mt. Carberry Landfill, Berlin, NH. Source for both photos: http://www.avrrdd.org/avrrdd-mt-carberry-landfill-berlin-nh.html Fig. 2B. Flare at Mt. Carberry Landfill, Berlin, NH. Fig. 2B. Flare at Mt. Carberry Landfill, Berlin, NH. Fig. 2A. Mt. Carberry Landfill, Berlin, NH. Source for both photos: http://www.avrrdd.org/avrrdd-mt-carberry-landfill-berlin-nh.html Fig. Fig. 3. Four Hills Landfill in Nashua, NH. 4.1 Structured questionnaires 3. Four Hills Landfill in Nashua, NH. Source:http://www.gonashua.com/CityGovernment/Departments/PublicWorks/SolidWa ste/tabid/135/Default.aspx Fig. 3. Four Hills Landfill in Nashua, NH. Fig. 3. Four Hills Landfill in Nashua, NH. Source:http://www.gonashua.com/CityGovernment/Departments/PublicWorks/SolidWa ste/tabid/135/Default.aspx Fig. 3. Four Hills Landfill in Nashua, NH. Source:http://www.gonashua.com/CityGovernment/Departments/PublicWorks/SolidWa ste/tabid/135/Default.aspx g Source:http://www.gonashua.com/CityGovernment/Departments/PublicWorks/SolidWa ste/tabid/135/Default.aspx 44 Environmental Management in Practice Environmental Management in Practice Environmental Management in Practice 44 Four Hills Landfill Correspondence Content Phone E-mail Public Hearing Written Time period of Correspondence 2007, 2008, 2009 2008 No public hearing No written correspondence Total Number 9 1 Responder NHDES ARD; NHDES Complaint Manager NHDES Complaint Manager Summary of Concern Odor; Noise Odor Response Time 1-2 days Same day Action Requested None None Perception of Distrust No No Ongoing Communication None Yes (via e-mail) Other Organizations Contacted EPA; Mayor’s office; local health department No Table 2C. Correspondence among stakeholders involved in the environmental communication of permitting decisions for a landfill facility. Fig. 4. Indeck Energy Services, Inc., Alexandria, NH. Source: http://www.indeckenergy.com/Alternative_Fuels.php Four Hills Landfill Correspondence Content Phone E-mail Public Hearing Written Time period of Correspondence 2007, 2008, 2009 2008 No public hearing No written correspondence Total Number 9 1 Responder NHDES ARD; NHDES Complaint Manager NHDES Complaint Manager Summary of Concern Odor; Noise Odor Response Time 1-2 days Same day Action Requested None None Perception of Distrust No No Ongoing Communication None Yes (via e-mail) Other Organizations Contacted EPA; Mayor’s office; local health department No Table 2C. Correspondence among stakeholders involved in the environmental communication of permitting decisions for a landfill facility. Yes (via e-mail) No Table 2C. Correspondence among stakeholders involved in the environmental communication of permitting decisions for a landfill facility. Fig. 4. Indeck Energy Services, Inc., Alexandria, NH. Source: http://www.indeckenergy.com/Alternative_Fuels.php Fig. 4. Indeck Energy Services, Inc., Alexandria, NH. Fig. 4. Indeck Energy Services, Inc., Alexandria, NH. Source: http://www.indeckenergy.com/Alternative_Fuels.php Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 45 Indeck Energy Services, Inc. 4.1 Structured questionnaires Correspondence Content Phone E-mail Public Hearing Written Time period of Correspondence 1986, 1991, 2008, 2009 No e-mail correspondence 2000, 2007 1986, 1999, 2007 Total Number 5 21 7 Responder NHDES Complaint Manager NHDES ARD and Facility Manager NHDES ARD and NHDES Director Summary of Concern Air quality; Noise Air quality, In support of permit for economic reasons Odor; Noise; Traffic; Air quality Response Time Same day Same day Two days Action Requested Air quality testing Air quality testing; more information on facility operations Information on facility operations and plans; Request a public hearing Perception of Distrust Yes Yes Yes Ongoing Communication None None None Other Organizations Contacted No No No Table 2D. Correspondence among stakeholders involved in the environmental communication of permitting decisions for an energy (electricity) facility. Air quality, In support of permit for economic reasons Odor; Noise; Traffic; Air quality Same day Two days No Table 2D. Correspondence among stakeholders involved in the environmental communication of permitting decisions for an energy (electricity) facility. 46 Environmental Management in Practice Schiller Station Correspondence Content Phone E-mail Public Hearing Written Time period of Correspondence 2002, 2004, 2007 2003, 2006, 2007 2004 2004 Total Number 5 3 3 3 Responder NHDES ARD; NHDES Complaint Manager NHDES ARD; NHDES Complaint Manager NHDES ARD and Facility Manager NHDES ARD Summary of Concern Coal dust damaged property; Air quality Health (cancer, allergies); Coal dust damaged property; Air quality Coal dust damaged property; Air quality Coal dust damaged property; Air quality Response Time Same day Same day Same day Two days Action Requested None None Air quality testing; One organization in support of the facility’s operation Air quality testing of ambient air in homes; Requested a public hearing Perception of Distrust No No Yes Yes Ongoing Communication None None None None Other Organizations Contacted No No No No Table 2E. Correspondence among stakeholders involved in the environmental communication of permitting decisions for an energy (electricity) facility. No No No Table 2E. Correspondence among stakeholders involved in the environmental communication of permitting decisions for an energy (electricity) facility. Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders Community Ecology and Capacity: Advancing E i t l C i ti St t i 47 Fig. 5. Schiller Station, Portsmouth, NH. Source:http://www.unhenergyclub.com/pastevents.php Fig. 5. Schiller Station, Portsmouth, NH. Source:http://www.unhenergyclub.com/pastevents.php Fig. 5. Schiller Station, Portsmouth, NH. Fig. 5. Schiller Station, Portsmouth, NH. g Source:http://www.unhenergyclub.com/pastevents.php Fig. 6. Fig. 5. Schiller Station, Portsmouth, NH. 4.1 Structured questionnaires Wheelabrator Technologies, Inc., Claremont, NH. Source: http://www.wheelabratortechnologies.com/index.cfm/our-clean-energy- plants/waste-to-energy-plants/wheelabrator-claremont-company-lp/ Fig. 6. Wheelabrator Technologies, Inc., Claremont, NH. Fig. 6. Wheelabrator Technologies, Inc., Claremont, NH. Source: http://www.wheelabratortechnologies.com/index.cfm/our-clean-energy- plants/waste-to-energy-plants/wheelabrator-claremont-company-lp/ Fig. 6. Wheelabrator Technologies, Inc., Claremont, NH. Source: http://www.wheelabratortechnologies.com/index.cfm/our-clean-energy- plants/waste-to-energy-plants/wheelabrator-claremont-company-lp/ 48 Environmental Management in Practice Wheelabrator Technologies, Inc. Correspondence Content Phone E-mail Public Hearing Written Time period of Correspondence 2005, 2006 2007 No public hearing 1995 Total Number 5 1 11 Responder NHDES ARD; NHDES Complaint Manager NHDES ARD; NHDES Complaint Manager NHDES ARD; NHDES Director Summary of Concern Health (respiratory illness); Odor; Air quality Health (respiratory illness); Air quality General health concerns; Air quality; Water quality; Failure of facility to comply with EPA’s emission standards Response Time Same day Same day Two days Action Requested Air quality testing Air quality testing Facility must engage in smoke stack emission testing; Development of more strict emission standards; Facility must become compliant with emission standards; Deny permit; Facility should communicate with the affected community Perception of Distrust Yes Yes Yes Ongoing Communication None None None Other Organizations Contacted EPA EPA No Table 2F. Correspondence among stakeholders involved in the environmental communication of permitting decisions for an incineration facility. 4 2 Structured interviews Table 2F. Correspondence among stakeholders involved in the environmental communication of permitting decisions for an incineration facility. 4.2 Structured interviews Carberry reported that “We told the public what was going on, how we were going to solve the problem, and we told them that we would keep them involved all along the way – and we did!” p g y When asked if NHDES and the facility were proactive in involving the public in the permitting process, there were varied responses including “…NHDES and my facility have been reactive instead of proactive” and “We [facility] weren’t that involved actually” and “I think it’s been a combination of both.” When asked if improving environmental communication would benefit the environmental permitting process, the responses varied. NHDES stated “Yes, hopefully, ideally. The more ongoing non-regulatory communication, the less issues are able to build up over time…There needs to be a continuous avenue for people to easily voice their concerns.” One facility manager stated “We feel that it isn’t very practical or efficient to reach out to the community before any kind of permitting decisions are started.” Another manager specifically noted that their “…filing for a Title V permit was completely voluntary…We don’t meet the guidelines to be considered a major polluting landfill. We applied for a Title V permit to be proactive.” The responses were also mixed about whether an appointed liaison would help improve environmental communication. NHDES stated “This depends on who they are affiliated with…If there was a person in this position, it would be helpful if each stakeholder had trust in this person. However, how this trust is built is unclear. It is quite possible that this person could be another barrier in the communication process and act as another layer of litigation.” One facility manager stated that “…one person, one contact would be very beneficial in improving environmental communication.” In contrast, another facility manager stated that “A person who has this position would get ‘beat up’ by all the stakeholders involved. I would have to say ‘No’.” When asked if improving environmental communication would benefit the environmental permitting process, the responses varied. NHDES stated “Yes, hopefully, ideally. The more ongoing non-regulatory communication, the less issues are able to build up over time…There needs to be a continuous avenue for people to easily voice their concerns.” Table 3 summarizes the recommendations of NHDES and the facility managers to improve the communication with impacted communities regarding environmental permitting decisions. 4.2 Structured interviews Both NHDES employees and Title V operating facility managers reported interacting with the public about environmental concerns and agreeing on who the stakeholders were in the environmental permitting process. All respondents believed that the respective facility was viewed positively by the public at the time of the interview. Initially, they may not have been viewed favorably but “Once there was some transparency developed, the public Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 49 welcomed the facility. They were happy that the facility was going to provide jobs in the area.” However, the incinerator was regarded by both NHDES and the facility manager as having a negative public perception. Interestingly, another incinerator, owned by the same parent company, located in a different part of the state is perceived positively by the surrounding community. The next case study examines the differences in environmental communication utilized by this facility in two distinct communities. One landfill experienced public outcry when it announced that it would be purchasing and re-opening a facility that had been closed for fifteen years. According to NHDES, this facility did not engage the surrounding community in their plans and the community attended the public hearing to obtain an update on the facility’s approach. Many of the issues presented at the public hearing could have been addressed beforehand but the facility was not proactive in involving the public. Another landfill facility manager reported that “Hearings have generally been a good experience, especially when the public doesn’t show up.” The facility manager from a similar site commented that “Our facility does a horrible job reaching out to the public…we are lacking in outreach.” In contrast, the Mt. Carberry Landfill held three public meetings. The first two meetings were sponsored by the facility owners and allowed “…the public to voice their concerns…” and served as informational sessions. When the official public hearing was held, all of the issues had been addressed and there was no conflict. The facility manager for Mt. 5. Managing environmental permitting decisions in dissimilar communities: discussion Effective environmental communication among all stakeholders is essential when addressing environmental health risks. Bennett (1999) and McComas (2003) describe how organizations will earn the trust of the community based on the content and delivery of their communication; the willingness for an inclusive, community-based participatory interaction; and their reputation for taking action. There is agreement that environmental communication among stakeholders be an integral component of the working relationship and that resources be allocated to develop public outreach plans that are tailored to the specific community (Brauer et al., 2004; Parkin, 2004). p y ( ) Given that stakeholder groups perceive risk differently, it is imperative for each group to appreciate the viewpoints of all involved to engage in effective dialog (Park et al, 2001; Tinker et al., 2001). Therefore, we propose that effective and proactive environmental communication that considers the community’s ecology (i.e., social, cultural, economic and political composition) among all stakeholders in all types of communities with a regulated industry is essential when addressing perceived health risks to the environmental and population. Based on our systematic examination of the environmental communication that occurred among a state environmental agency, six Title V operating facilities and the public concerning environmental permitting decisions perceived to impact human health, we developed recommendations to facilitate best practices in environmental communication. These recommendations for practitioners are presented in Section 10: Recommendations. 4.2 Structured interviews Key recommendations include conducting more informal “conversation” type meetings prior to the public hearing; presenting information at an appropriate educational level; and engaging in public outreach via the Internet, mailings, print media and/or a 50 Environmental Management in Practice community liaison; integrating a practice of transparency of information among stakeholders; and creating a uniform meeting setup.  Hold informal “conversational” type meetings prior to the public hearing for concerns and questions to be addressed (NHDES ARD)  Alter the meeting room setup for the public hearing so an “Us” versus “Them” scenario is not created (NHDES ARD)  Keep people informed via web sites, mailings, and newspapers (Landfill facility)  Community liaison who could share information among stakeholders (Incinerator facility)  Be transparent with information and the facility’s operations (Landfill facility)  Acknowledge differences in public perception (Electricity generating facility)  Explain the permitting process and emission standards to the public in an educationally appropriate manner (Landfill facility)  Facilities need to be more involved in the community (Landfill facility) Table 3. Summary of recommendations from state agency representatives and facility managers on how to improve environmental communication to the public. 6. Case study: managing perceived health risks from a single-owner waste- to-energy facility in two distinct communities The perceived health risks and environmental communication from two waste-to-energy facilities operated by the same parent company are examined in this work. Waste Community Ecology and Capacity: Advancing Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 51 Management, Inc., of Houston, Texas owns Wheelabrator Technologies, Inc. which operates several waste-to-energy facilities across the United States. Wheelabrator operates two such municipal solid waste incinerators in Claremont, New Hampshire (NH) and Concord, NH, respectively. The Claremont, NH facility began operation in 1987 and provides disposal of up to 200 tons of municipal solid waste daily for approximately 70,000 people. This facility can provide electricity to 5,600 homes. The Concord, NH facility began operation in 1989 and provides disposal of up to 500 tons of municipal solid waste daily for approximately 150,000 people. This facility can provide electricity to 17,000 homes (Wheelabrator, 2010). These facilities use the same waste-to-energy method and are considered Title V operating facilities by the New Hampshire Department of Environmental Services (NHDES). The purpose of the Title V permitting process is to ensure that facilities will not emit hazardous pollutants to a degree which could negatively affect human health. Specifically, facilities which emit over 100 tons of any regulated pollutant, such as carbon monoxide and sulfur oxides; emit over 50 tons of nitrous oxides; or emit 10 tons of any of the federally regulated hazardous air pollutants need to apply to the state environmental agency for a Title V permit (ARD, 2008). As required by current NHDES permits, the Wheelabrator sites continuously monitor carbon monoxide, sulfur dioxide, particulate matter, as well as other emission indicators such as steam flow and temperature. All monitoring and operational information are maintained in facility records, in accordance with state and federal requirements. “[NH]DES oversees and witnesses the performance of annual relative accuracy tests and audits facility records in order to ensure the accuracy of Wheelabrator’s continuous emissions monitoring system. [NH]DES also conducts full Compliance Evaluations at least every two years, witnesses annual compliance stack tests and reviews resultant stack test reports for accuracy” (ATSDR, 2009). 6.1 Two communities: home to the same environmental policy The demographics of the Claremont and Concord New Hampshire communities are similar with respect to age and sex. Both communities are also classified as cities. However, the demographic information for education, economic and housing characteristics are different. Table 4 outlines selected demographic characteristics of these two communities. Briefly, Claremont is a city in the western part of New Hampshire with a population of 12,968. It is situated along the Connecticut River in Sullivan County. It is the largest incorporated community in Sullivan County and ranks 22nd in population size among cities and towns in New Hampshire. The majority of the population (97.7%) is White and 78.7% of the population 25 years of age and older have completed high school while 12.8% have a Bachelor’s degree. The median household income in 1999 was $34,949 and the median value of a single-family owner-occupied home was $79,800 (Census, 2010). Concord is the state capital with a population of 42,255. It is situated along the Merrimack River in Merrimack County and ranks 3rd in population size among cities and town in New Hampshire. The majority of the population (95.5%) is White and 88.6% of the population 25 years of age and older have completed high school while 30.7% have a Bachelor’s degree. The median household income in 1999 was $42,447 and the median value of a single-family owner-occupied home was $112,300 (Census, 2010). 52 Environmental Management in Practice 3 Median household income for 1999, U.S. Census Bureau, Population Finder. (http://www.censu 2 Population estimate for 2008, U.S. Census Bureau, Population Finder. (http://www.census.gov/) 2 Population estimate for 2008, U.S. Census Bureau, Population Finder. (http://www.census.gov/) 3 Median household income for 1999, U.S. Census Bureau, Population Finder. (http://www.census.gov/) Table 4. Demographic characteristics of two communities that host a waste-to-energy facility4. 6.2 Stakeholders in environmental communication The stakeholders considered in this work include a state environmental agency, community activists living near the facilities and the general public. Specifically, the New Hampshire Department of Environmental Services, Air Resources Division (NHDES ARD) is responsible for monitoring and regulating air quality that is protective of public health and the natural environment in the State of New Hampshire (ARD, 2010). NHDES ARD accomplishes this goal via numerous programs including a statewide permitting program to assure compliance with the Title V federal mandate (ARD, 2008). p ( ) Citizens Leading for Environmental Action and Responsibility (CLEAR) is a community activist group that is primarily comprised of Claremont, NH residents. The mission of CLEAR is to “…respect and value the people, the environment, the public health, the political process, and the economics of our community and region;…encourage public participation in the decision-making process to promote the principles of environmental, political, social, and economic health;…commit to an organizational framework that is non- profit, open, democratic, and accountable” (CLEAR, 2010). The general public living or spending time in the communities that house these Title V operating facilities represents the final stakeholder group. Figure 7 represents photographs of the industry examined. Claremont, NH Concord, NH Total population2 12,968 42,255 Race: White 97.7% 95.5% High school graduate 78.7% 88.6% Bachelor’s degree 12.8% 30.7% Median household income3 $34,949 $42,447 Median value of a single- family owner-occupied home $79,800 $112,300 Table 4. Demographic characteristics of two communities that host a waste-to-energy facility4. 2 Population estimate for 2008, U.S. Census Bureau, Population Finder. (http://www.census.gov/) 3 Median household income for 1999, U.S. Census Bureau, Population Finder. (http://www.census.gov/) Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 53 Fig. 7. A and B. Wheelabrator Technologies, Inc. in Claremont and Concord, NH, respectively. Source: http://www.wheelabratortechnologies.com/index.cfm/our-clean-energy- plants/waste-to-energy-plants/wheelabrator-claremont-company-lp/ logies, Inc. in Claremont and Concord, NH, Fig. 7. A and B. Wheelabrator Technologies, Inc. in Claremont and Concord, NH, respectively. p y Source: http://www.wheelabratortechnologies.com/index.cfm/our-clean-energy- plants/waste-to-energy-plants/wheelabrator-claremont-company-lp/ 4 Source: U.S. Census Bureau. Population Finder. (http://www.census.gov/) 7.1 Survey instrument This question was followed by an inquiry regarding whether the respondents thought they were well-informed about environmental health issues in their community. Next, respondents were asked to indicate where they would rank their environmental issue of interest relative to other issues (e.g., property taxes) affecting their local community. and to choose what environmental health issue in their community concerned them the most from the following list: water quality, land conservation, air pollution, food security and other. This question was followed by an inquiry regarding whether the respondents thought they were well-informed about environmental health issues in their community. Next, respondents were asked to indicate where they would rank their environmental issue of interest relative to other issues (e.g., property taxes) affecting their local community. In order to determine sources of environmental health information, respondents were asked to choose from the following sources in the next section of the survey: federal agencies (e.g., Environmental Protection Agency, Agency of Toxic Substances and Disease Registry); state agencies (e.g., New Hampshire Department of Environmental Services, New Hampshire Department of Health and Human Services); local government (e.g., city councilor or Mayor); environmental groups (e.g., Greenpeace); academia (university presentations, studies, peer-reviewed literature); media sources (e.g., newspaper, television, radio, Internet); other. Respondents were instructed to circle all that applied to them. Respondents were then asked to rate their believability of the above-mentioned sources of information. Next, in order to determine which media sources were the most useful, respondents were asked to choose from the following sources: television programs, print resources (e.g., pamphlets), newspaper articles or editorials, community meetings, informational websites. The third series of questions pertained to the respondent’s attitude about public meetings. Respondents were asked if they had ever attended a public meeting and whether they believed public meetings were an effective means to communicate environmental health information. Next, respondents were asked if they believed whether their opinion, if voiced at a public meeting, would be taken seriously by officials. 7.1 Survey instrument ( g p p y ) g y In order to determine sources of environmental health information, respondents were asked to choose from the following sources in the next section of the survey: federal agencies (e.g., Environmental Protection Agency, Agency of Toxic Substances and Disease Registry); state agencies (e.g., New Hampshire Department of Environmental Services, New Hampshire Department of Health and Human Services); local government (e.g., city councilor or Mayor); environmental groups (e.g., Greenpeace); academia (university presentations, studies, peer-reviewed literature); media sources (e.g., newspaper, television, radio, Internet); other. Respondents were instructed to circle all that applied to them. Respondents were then asked to rate their believability of the above-mentioned sources of information. Next, in order to determine which media sources were the most useful, respondents were asked to choose from the following sources: television programs, print resources (e.g., pamphlets), newspaper articles or editorials, community meetings, informational websites. The third series of questions pertained to the respondent’s attitude about public meetings. Respondents were asked if they had ever attended a public meeting and whether they believed public meetings were an effective means to communicate environmental health information. Next, respondents were asked if they believed whether their opinion, if voiced at a public meeting, would be taken seriously by officials. g y y Finally, the last series of survey questions inquired whether or not the respondents believed the status of their personal health is related to the condition of the environment. Respondents were specifically asked if they were familiar with trash incineration and whether or not they believed it to be an effective form of waste disposal. y p All data were analyzed using the Statistical Package for Social Sciences. Descriptive analyses were done for each of the participant responses by determining frequencies and proportions. Comparisons of responses were made across both communities by utilizing the chi-square statistic, cross tabulations and independent sample t-tests to assess the statistical significance of these comparisons. For statistical tests, P-values less than 0.05 were considered to be statistically significant. Unknowns were accounted for in all variables. 7.1 Survey instrument Following Institutional Review Board approval from the University of New Hampshire, a cross-sectional study design was utilized to examine the sources, believability and utility of information and perceptions about environmental health issues among a relevant sample of residents and visitors of the two study communities. Self-report questionnaires utilizing a 4-point Likert scale and multiple choice questions were administered over a five month period at different times and locations (e.g., retail locations and churches of various denominations) in each community. These anonymous surveys were immediately collected from the participants upon completion. Alternatively, participants could choose to mail their completed survey to the University of New Hampshire via self-addressed and stamped envelopes. All questionnaires had a cover letter attached that explained the purpose of the study and emphasized the anonymity and confidentiality of the results. Participants were told to keep this letter for their records. There were no incentives for participating in this study. Additional open-ended comments from participants were recorded at the end of the survey. The 19-item questionnaire was designed to determine demographic information, self-reported knowledge about sources and believability of information and perceptions about environmental health issues in the community. Revisions were made during the pilot testing phase of the questionnaire. Ambiguities associated with the survey content were not identified during test trials that were conducted prior to official questionnaire administration. The survey questions were organized into four sections. First, respondents were asked for demographic information (e.g., length of residence in the community, education level, annual income) and questions pertaining to their interest and level of participation in community issues. Respondents were then asked how often they think about their physical environment 54 Environmental Management in Practice and to choose what environmental health issue in their community concerned them the most from the following list: water quality, land conservation, air pollution, food security and other. This question was followed by an inquiry regarding whether the respondents thought they were well-informed about environmental health issues in their community. Next, respondents were asked to indicate where they would rank their environmental issue of interest relative to other issues (e.g., property taxes) affecting their local community. and to choose what environmental health issue in their community concerned them the most from the following list: water quality, land conservation, air pollution, food security and other. 7.2 Structured interview instrument Structured interviews were conducted, following Institutional Review Board approval from the University of New Hampshire, with DES employees involved in Title V permitting and environmental health investigations and community activists from CLEAR to examine the experiences that shaped both parties’ perceptions of current environmental communication methods. Participants were asked semi-structured, open-ended questions about the public’s perception of their work, whether the facilities’ operations were considered to be contentious or non-contentious and the health and environmental concerns regarding the facilities. Participants were asked if they had experience conducting and/or attending a public hearing about the facility. Information pertaining to the type and number of concerns communicated by the public was collected, as well as how these issues were addressed. With respect to the environmental management of concerns, NHDES was Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 55 queried as to whether or not they believed they were proactive in involving the community and if they employed a professional who was responsible for handling the public’s concerns. CLEAR was queried as to their perception in regards of their inclusion, by NHDES, in health investigations concerning the facility and communication efforts from NHDES. The last series of questions posed to the participants inquired about whether they thought improving environmental communication among all stakeholders would enhance working relationships; the usefulness of having an appointed community liaison to assist with environmental communication; and what specific recommendations they have to improve the environmental communication among stakeholders. The interviews were transcribed and a content analysis, using QSR NVivo (a computer-assisted qualitative data analysis program), was conducted of the structured interview responses to extract and code recurring themes. 8.1 Two communities: sources, believability and utility of information and perceptions about environmental health issues Furthermore, 64.2% of Concord, NH respondents ranked the priority of environmental health issues higher than other community issues (e.g., property taxes) compared to 38.5% of Claremont, NH respondents. Lastly, 92.6% of Concord, NH respondents and 75.5% of Claremont, NH respondents were familiar with trash incineration as a waste disposal effort. p p As shown in Table 6, survey results demonstrate statistically significant differences and similarities between these two communities with respect to information sources, believability and usefulness. For instance, Concord, NH respondents were more likely to not only obtain information from state agencies (61.1%), but they were also more likely to believe it (67.3%) compared to Claremont, NH respondents. Also, Concord, NH respondents were more likely to obtain information from environmental groups (50.0%) compared to Claremont, NH respondents (18.5%). Interestingly, both Concord, NH (92.6%) and Claremont, NH (79.6%) respondents were very likely to obtain information from media sources such as newspapers, television, radio and the Internet. However, Claremont, NH respondents were more likely to believe media sources (46.0%) and use (56.6%) the information from the television compared to Concord, NH respondents. Yet, respondents from both the Concord, NH (55.6%) and the Claremont, NH (66.0%) communities reported newspapers to be the most useful source of information. In terms of having attended public meetings in the past and their effectiveness, both communities were similar in their responses. For example, respondents in the Concord, NH (70.9%) and Claremont, NH (56.6%) communities reported that they had attended a public meeting in the past. Respondents from Concord, NH (52.7%) and Claremont, NH (64.3%) reported that they found such a venue useful for communicating environmental health information. However, respondents from Concord, NH (31.5%) and Claremont, NH (24.5%) reported that if they voiced their opinion in a public meeting, they believed that their comments would not be taken seriously by officials in attendance. y y Furthermore, respondents from Concord, NH (63.6%) and Claremont, NH (58.5%) believed that the condition of the environment plays a role in their personal health. Respondents from Concord, NH (92.6%) and Claremont, NH (75.5%) reported that they were familiar with trash incineration but these same respondents did not believe it was an effective means of waste disposal (58.0% and 61.4%, respectively.) Cross-tabulation analyses indicated several statistically significant relationships (Table 7). 8.1 Two communities: sources, believability and utility of information and perceptions about environmental health issues One hundred and nine of 250 surveys (44% response rate) were completed and returned by community members and/or visitors to the Claremont and Concord, NH communities. Of the completed 109 surveys, 54 were from the Claremont community and 55 were from the Concord community. y As shown in Table 5, survey results indicate statistically significant differences between the Claremont, NH and Concord, NH survey respondents with respect to demographic Claremont, NH Concord, NH P-value College education 53.0% 92.2% 0.000 Annual income $25,000 or greater 55.5% 98.2% 0.000 Lived in the community for ten years or more 51.9% 76.4% 0.008 Active in community issues 42.6% 65.5% 0.017 Ranked the priority of environmental issues higher than other community issues (e.g., property taxes) 38.5% 64.2% 0.008 Familiar with trash incineration as a waste disposal method 75.5% 92.6% 0.015 Table 5. Demographic characteristics of two communities and survey respondents’ interest in environmental health issues in their community that hosts a waste-to-energy facility. characteristics and involvement in environmental health issues. For example, Concord, NH respondents reported higher annual incomes of $25,000 or more (98.2%) compared to Claremont, NH respondents (55.5%). In terms of education level, more Concord, NH respondents completed college education (92.2%) compared to Claremont, NH respondents (53.0%). In addition, Concord, NH respondents were more likely to have lived in their characteristics and involvement in environmental health issues. For example, Concord, NH respondents reported higher annual incomes of $25,000 or more (98.2%) compared to Claremont, NH respondents (55.5%). In terms of education level, more Concord, NH respondents completed college education (92.2%) compared to Claremont, NH respondents (53.0%). In addition, Concord, NH respondents were more likely to have lived in their characteristics and involvement in environmental health issues. For example, Concord, NH respondents reported higher annual incomes of $25,000 or more (98.2%) compared to Claremont, NH respondents (55.5%). In terms of education level, more Concord, NH respondents completed college education (92.2%) compared to Claremont, NH respondents (53.0%). In addition, Concord, NH respondents were more likely to have lived in their 56 Environmental Management in Practice community for more than ten years (76.4%) compared to Claremont, NH respondents (51.9%). Concord, NH respondents were also identified as being more active in community issues (65.5%) compared to Claremont, NH respondents (42.6%). 8.1 Two communities: sources, believability and utility of information and perceptions about environmental health issues For example, respondents with a college education were more likely to use environmental groups (43.4%) and the Internet (43.4%) as a source of environmental health information compared to respondents without a college education. Respondents who did not have a college education reported television (70.8%) as a useful media source for communicating environmental health information. In addition, respondents with a college education were more likely to report ever having attended a public meeting (70.2%), as well as being familiar with trash incineration as a disposal method (89.2%). Similarly, respondents who reported being more active in community issues were also more likely to report ever having attended a public meeting (81.0%), as well as being familiar with trash incineration as a disposal method (91.4%). Lastly, there were also significant relationships identified between living in the community for ten years or more and being well informed about community issues (62.3%). Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders Community Ecology and Capacity: Advancing E i l C i i S i 57 Claremont, NH Concord, NH P-value Sources of environmental health information State Agencies 24.1% 61.1% 0.000 Environmental Groups 18.5% 50.0% 0.001 Media Sources 79.6% 92.6% 0.051 Believability of sources of environmental health information State Agencies 42.3% 67.3% 0.030 Media Sources 46.0% 28.3% 0.042 Useful media sources for obtaining environmental health information Television 56.6% 18.5% 0.000 Newspapers 66.0% 55.6% 0.267 Table 6. Survey respondents’ sources, believability and usefulness of environmental health information from two communities that host a waste-to-energy facility. Table 6. Survey respondents’ sources, believability and usefulness of environmental health information from two communities that host a waste-to-energy facility. Table 6. Survey respondents’ sources, believability and usefulness of environmental health information from two communities that host a waste-to-energy facility. Table 6. Survey respondents’ sources, believability and usefulness of environmental health information from two communities that host a waste-to-energy facility. 8.1 Two communities: sources, believability and utility of information and perceptions about environmental health issues 58 Environmental Management in Practice Level of Education No College Education College Education P-value Environmental groups as source of environmental information 4.0% 43.4% 0.000 Television as useful media source for obtaining environmental information 70.8% 27.7% 0.000 Internet as useful media source for obtaining environmental information 20.8% 43.4% 0.045 Ever attended a public meeting 41.7% 70.2% 0.010 Familiar with trash incineration as a waste disposal method 66.7% 89.2% 0.008 Involvement in Community Issues Less Active More Active P-value Ever attended a public meeting 44.0% 81.0% 0.000 Familiar with trash incineration as a waste disposal method 75.5% 91.4% 0.025 Length of time lived in community Less than Ten Years More than Ten Years P-value Active in community issues 38.5% 62.9% 0.014 Well-informed about environmental health issues in the community 39.5% 62.3% 0.023 Table 7. Demographic characteristics and survey respondents’ practices about environmental health information and issues from two communities that host a waste-to- energy facility. Table 7. Demographic characteristics and survey respondents’ practices about Table 7. Demographic characteristics and survey respondents’ practices about environmental health information and issues from two communities that host a waste-to- energy facility. Table 7. Demographic characteristics and survey respondents practices about environmental health information and issues from two communities that host a waste-to- energy facility. Community Ecology and Capacity: Advancing E i t l C i ti St t i Community Ecology and Capacity: Advancing Environmental Communication Strategies among Dive Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 59 y gy p y g Environmental Communication Strategies among Diverse Stakeholders 8.2 State agency and community activists as stakeholders: perception of environmental communication Structured interviews with community activists (n=7) demonstrated that they “feel there is more that should be done regarding this issue (waste-to-energy).” All interviewees discussed this theme in their individual interviews. The activists recommended that state government should further restrict trash incineration. Several interviewees discussed the recent ban on construction and demolition material incineration and pointed out that if this material is outlawed, everything should be banned. y y p p Structured interviews with community activists (n=7) demonstrated that they “feel there is more that should be done regarding this issue (waste-to-energy).” All interviewees discussed this theme in their individual interviews. The activists recommended that state government should further restrict trash incineration. Several interviewees discussed the recent ban on construction and demolition material incineration and pointed out that if this material is outlawed, everything should be banned. y g Another theme that emerged was the activists’ perception that the state agency pays inadequate attention to the issue of waste incineration in their communities. The activists are also very distrustful of state and industry involvement because many believe the company that owns the two municipal waste incinerators of interest, discusses with NHDES when random emissions testing will occur in advance so the incinerator will burn “cleaner trash” on the testing days. They believe that this skews the data so any emission report released by NHDES is not accurate. y When asked about efforts to improve environmental communication, community activists had mixed reactions. The majority of activists reported that the state agency did a decent job at communicating environmental health information. Beyond typical communication venues, such as newspapers, Internet, and public meetings, activists were hard pressed to suggest anything new. Several community activists mentioned that there was discussion about creating a community panel to review environmental community issues. Decisions regarding the environment (and the incinerator) would go to this panel for review. This idea was met with opposition by the local government and never came to fruition. Community activists were asked about the effectiveness of having a community liaison located in their community. This individual would gather concerns and questions from the community, relay those concerns and questions to the appropriate state agency and then disseminate information back to the community. Unanimous support among the activists for such a position of this nature was expressed. 8.2 State agency and community activists as stakeholders: perception of environmental communication Twelve individual structured interviews with NHDES employees involved in Title V permitting and environmental health investigations and community activists from CLEAR were conducted to examine the experiences that shaped their perception of current environmental communication methods. Through structured interviews with NHDES and a review of publicly available documents (e.g., phone records, e-mail and written correspondence and public hearing recordings) housed at NHDES, it was determined that the public inquiries concerning the Wheelabrator companies were mainly for the facility in Claremont, NH and not Concord, NH, even though they have identical operations. The public inquiries were fielded by NHDES ARD staff and/or the NHDES Complaint Manager. The concerns expressed ranged from health issues (e.g., cancer, respiratory illness) to nuisance complaints (e.g., odor, noise) to environmental issues (e.g., poor air and water quality), all of which were perceived to be due to the operation of the incinerator. The actions most often requested by the public for the Claremont, NH facility included air and water quality testing, compliance evaluations with state and federal emission standards and communication from the facility with the affected community. In some instances, the community members called for the closure of the facility. Distrust of NHDES and/or the facility was expressed in the public documents. Through structured interviews with NHDES and a review of publicly available documents (e.g., phone records, e-mail and written correspondence and public hearing recordings) housed at NHDES, it was determined that the public inquiries concerning the Wheelabrator companies were mainly for the facility in Claremont, NH and not Concord, NH, even though they have identical operations. The public inquiries were fielded by NHDES ARD staff and/or the NHDES Complaint Manager. The concerns expressed ranged from health issues (e.g., cancer, respiratory illness) to nuisance complaints (e.g., odor, noise) to environmental issues (e.g., poor air and water quality), all of which were perceived to be due to the operation of the incinerator. The actions most often requested by the public for the Claremont, NH facility included air and water quality testing, compliance evaluations with state and federal emission standards and communication from the facility with the affected community. In some instances, the community members called for the closure of the facility. Distrust of NHDES and/or the facility was expressed in the public documents. 8.2 State agency and community activists as stakeholders: perception of environmental communication Interviews with NHDES regulators and investigators (n=5) revealed their belief that community activists do not acknowledge the state’s effort to respond to their concerns. On 60 Environmental Management in Practice multiple occasions, requests made by community activists were explored, such as the concern that the Claremont, NH facility was responsible for excessive cancer in that community. As a result, NHDES, in conjunction with the Agency for Toxic Substances and Disease Registry, conducted a community health investigation and analyzed twenty-four major cancer types from 1987-2001. It was determined, from the available data, the cancer rates for the specific types of cancer analyzed were within the expected range (ATSDR, 2006). This was a time-consuming endeavor and utilized many staff and budgetary resources. When results were presented to the community, activists were not pleased with the findings and discredited the initiative. The activists argued that the community health investigation was not done in a way that was inclusive of the community, and that the analysis was unacceptable and the results were inaccurate. As a result, state regulators believed that there was not much that could be done to remedy community activists concerns short of closing the Claremont, NH facility. g y Another major theme expressed by NHDES involved community activists’ communication with their organization. Direct questions and concerns were reported to be more effective than emotional propaganda from activists. An example expressed multiple times in NHDES interviews was that there were “two types of community activists.” There are the community activists that send emotional propaganda, such as hundreds of postcards with dead fish on them to NHDES claiming that the mercury emitted from the Claremont, NH facility is killing all the fish. Other types of emotional propaganda that have been used by this reported “type” of activist include the mailing of pictures of residents who have died from cancer with messages explaining that the negligence of NHDES to shut down the facility was the direct cause of their death. In contrast, the “other type” of community activist sends specific questions and concerns that NHDES can investigate and reply with factual data. This type of communication was preferred and was believed to be more effective. NHDES regulators and investigators were asked if it would be effective to have a community liaison position in New Hampshire communities where a contentious relationship exists between a community and an industry within the community. 8.2 State agency and community activists as stakeholders: perception of environmental communication The responses were mixed about whether an appointed community liaison would help improve environmental communication. NHDES stated “This depends on who they are affiliated with…If there was a person in this position, it would be helpful if each stakeholder had trust in this person. However, how this trust is built is unclear. It is quite possible that this person could be another barrier in the communication process and act as another layer of litigation.” 9. Managing perceived health risks from a single-owner waste-to-snergy facility in two distinct communities: discussion Our work suggests that ecological and demographic differences in communities need to be assessed, in order to identify the multidimensional components of the communities’ risk perception and to be able to determine the most effective means by which to communicate environmental information. Interestingly, a review of publicly available documents and structured interviews with community activists and agency stakeholders determined that although two NH communities host an identical municipal waste incinerator, the Claremont community, compared to the Concord community, was more vociferous in regards to their perception that the facility was a risk to the health of the population and their environment. In addition, the Claremont community was hesitant to believe the results of a health consultation and public health assessment conducted by NHDES and the federal Agency of Toxic Substances and Disease Registry that determined “…the Claremont area was in compliance with all National Ambient Air Quality Standards…” for the following criteria pollutants: sulfur dioxide, particulate matter less than 2.5 microns in diameter, ozone and nitrogen dioxide; and that “…cancer rates for 24 major cancer types were all within their expected ranges…” over the fifteen-year period studied (ATSDR, 2006; ATSDR, 2009). We suggest that demographic differences may contribute to the dissimilarity in risk perception of two communities for this industrial process, however, it is not the sole factor. We propose that effective and proactive environmental communicative structures that take into consideration the community’s ecology among all stakeholders in all types of communities with a regulated industry is essential when addressing perceived health risks to the environment and population. Such practices could result in improved relationships with communities and public perception and expectations of community health investigations. y Interestingly, a review of publicly available documents and structured interviews with community activists and agency stakeholders determined that although two NH communities host an identical municipal waste incinerator, the Claremont community, compared to the Concord community, was more vociferous in regards to their perception that the facility was a risk to the health of the population and their environment. 9. Managing perceived health risks from a single-owner waste-to-snergy facility in two distinct communities: discussion In addition, the Claremont community was hesitant to believe the results of a health consultation and public health assessment conducted by NHDES and the federal Agency of Toxic Substances and Disease Registry that determined “…the Claremont area was in compliance with all National Ambient Air Quality Standards…” for the following criteria pollutants: sulfur dioxide, particulate matter less than 2.5 microns in diameter, ozone and nitrogen dioxide; and that “…cancer rates for 24 major cancer types were all within their expected ranges…” over the fifteen-year period studied (ATSDR, 2006; ATSDR, 2009). We suggest that demographic differences may contribute to the dissimilarity in risk perception of two communities for this industrial process, however, it is not the sole factor. We propose that effective and proactive environmental communicative structures that take into consideration the community’s ecology among all stakeholders in all types of communities with a regulated industry is essential when addressing perceived health risks to the environment and population. Such practices could result in improved relationships with communities and public perception and expectations of community health investigations. 9. Managing perceived health risks from a single-owner waste-to-snergy facility in two distinct communities: discussion An ongoing, practical challenge for state agencies involved in investigating community concerns related to an industrial process perceived to impact the environment and human health is how to most effectively communicate with the community as a key stakeholder. We propose that investigators and regulators need to be able to 1.) identify the community’s ecology, that is the community’s social, cultural, economic and political composition and 2.) understand the community’s ecology to engage in effective environmental communication. State agencies frequently describe communities as groups of people living within a certain area, while communities may describe themselves on a Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 61 more detailed level, such as by their socioeconomic status, religious beliefs, race/ethnicity, etc. (Parkin, 2004). We present the relationships between the demographic characteristics of two communities that host an identical waste-to-energy facility owned by the same parent company, and various communicative structures, such as the sources, believability and utility of environmental health information accessed by these populations, as well as their level of knowledge about trash incineration, the industrial process of concern. We demonstrate that disparate populations that host a similar industry access and believe different sources of environmental health information rank the priority of environmental health issues compared to community health issues differently and have different levels of activity on community issues. Our work suggests that ecological and demographic differences in communities need to be assessed, in order to identify the multidimensional components of the communities’ risk perception and to be able to determine the most effective means by which to communicate environmental information. more detailed level, such as by their socioeconomic status, religious beliefs, race/ethnicity, etc. (Parkin, 2004). We present the relationships between the demographic characteristics of two communities that host an identical waste-to-energy facility owned by the same parent company, and various communicative structures, such as the sources, believability and utility of environmental health information accessed by these populations, as well as their level of knowledge about trash incineration, the industrial process of concern. We demonstrate that disparate populations that host a similar industry access and believe different sources of environmental health information rank the priority of environmental health issues compared to community health issues differently and have different levels of activity on community issues. 10. Recommendations for practitioners Both case studies utilized the cultural-experiential model of risk, which requests information regarding the experience and views of impacted populations and their assessment of risk (Cox, 2006). We propose that part of effective environmental communication on the part of practitioners involves not only understanding the community’s ecology but also the importance of engaging the public sphere to help build the community’s capacity to address the environmental health issue of concern. Cox (2006) defines the public sphere as “The realm of influence created when individuals engage others in communication – through conversation, argument, debate, questions and nonverbal acts – about subjects of shared concern of topics that affect a wider community.” The public sphere needs to be the common ground to communicate misunderstandings, knowledge deficits and environmental education. We utilized the cultural-experiential model to better understand 62 Environmental Management in Practice the public sphere experienced by dissimilar communities that host different regulated industries, and in one instance, an identical industry. Based on our systematic examination of the environmental communication preferences and practices among a state environmental agency, Title V operating facilities, community activists and the general public concerning environmental permitting decisions perceived to impact human health, we developed the below recommendations to facilitate best practices in environmental communication: 1. Initiate communication early with the community: Proactive communication to potentially affected communities by state agencies and neighboring facilities could facilitate the relationship among stakeholders and serve as the foundation for next steps. This recommendation arose from the experiences of two facilities which were completely different in their public outreach practices. One was not proactive in involving the community during the environmental permitting process and waited until the public hearing to address the community and explain the intent of their facility’s operations. In this case, the relationship between the facility and public was strained from the beginning of the permitting process and the situation became the facility versus the public, instead of the facility working with the public. In contrast, the other facility was proactive in involving the community and held public information sessions prior to the public hearing to address the community’s concerns. 2. Provide seminars to educate facility managers about public engagement: The state agency could offer seminars designed to educate facility managers on public outreach practices prior to the Title V permitting process. 10. Recommendations for practitioners To accommodate the high number of complaints the facility was receiving, one landfill utilized web-based technology for the public to express their concerns. However, it is important that this communication be “two-way.” For example, numerous entries stated that many inquiries had been filed online, yet the problems complained about were still in existence and the facility failed to respond to any concerns. Therefore, as part of the routine evaluation of their communication with the public, facilities need to establish processes to assure a timely response to the public’s comments. In addition, Claremont, NH respondents (56.6%) reported environmental health information obtained from the television more useful compared to Concord, NH respondents (18.5%). In addition, it is important that this communication be “two-way.” Therefore, as part of the routine evaluation of their communication with the public, state agencies and facilities need to establish processes to assure a timely response to the public’s comments. A community liaison could be proactive in this practice. p y p p 8. Increase state agency awareness: In several instances, the public contacted the EPA because they were unaware of who to contact at state government or the facility. Increasing awareness of the state agency as a stakeholder in the environmental permitting process would help the public understand who to contact concerning environmental issues and facilitate relationship-building between the state and the public. This may be accomplished through state agency and facility-sponsored community events or attendance at existing community events to raise awareness. 9. Use of appropriate information and meeting logistics: Information complexity as a communication barrier for the public was evident in the public hearing audiotapes and interviews with facility managers. For example, the public requested clarification by NHDES ARD concerning emissions and health effects and asked what “all the figures and tables meant.” Furthermore, facility managers expressed concern that the information presented by NHDES ARD to the public was too complex, thus leading the public to contact the facility. Taking the time to understand the community’s ecology will help state agencies develop appropriate information that is communicated in an effective forum for that community. Hence, this practice will be community-specific. y p y p In addition, the room for public hearings is traditionally organized in a polarized manner where the state agency and the facility are at one end and the community is at the other end. 10. Recommendations for practitioners These educational seminars would provide opportunities for facilities to develop an understanding of the concerns typically raised by communities and discuss how to be a “good neighbor” based on best practices. g g p 3. Require the permit application be accompanied by a public outreach plan: In order to maintain the neutrality of the official Title V permitting process, yet be proactive in communicating with stakeholders, the state agency could require the facility to include several objective public outreach activities that support public participation. An example could include engaging the community prior to the public hearing, via non-regulatory communication, which would ease the environmental permitting process by providing an opportunity for concerns to be addressed. 4. Advocate representatives from state government public health and environmental health bureaus be present at public hearings: The concerns expressed by the public are so varied that no one agency could address them. The inability to answer questions during public hearings led to the community’s frustration and increased stress on the communication among the stakeholders. Therefore, representatives from each public health and environmental health state bureau should be represented on the public hearing panel to address a broad array of questions and reduce the feelings of distrust. 5. Establish citizen advisory committees: This action could provide an opportunity for citizens to voice their concerns or ask questions about the facility operations on a regular basis. One facility manager explained that this has been a great way for the public to have direct communication with officials about the permitting process and their concerns. 6. Establishment of a community liaison position: The community liaison position is a neutral party who would be located in the community and have an established relationship with the community. He/she would gather concerns and questions from the community, relay those Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 63 concerns and questions to the appropriate state agency and then disseminate information back to the community. Similar to the community panel mentioned by community activists in Claremont, NH, this action could provide an opportunity for citizens to voice their concerns, ask questions about the facility operations on a regular basis and allow for the community to play a role in policy and program development. 7. Be accountable for communication among stakeholders: State agency(ies) and industry need to understand the best way to communicate with the community. 12. Acknowledgments The authors are grateful to the following: New Hampshire Department of Environmental Services, Air Resources Division; facility managers from Turnkey Recycling and Environmental Enterprises, Mt. Carberry Landfill, Four Hills Landfill, Indeck Energy Services, Inc., Schiller Station, and Wheelabrator Technologies, Inc.; CLEAR NH and the general public and businesses of Claremont, NH and Concord, NH for their participation in this study. In addition, the authors thank Robert J. McGrath, Assistant Professor in the Department of Health Management and Policy, University of New Hampshire for insightful discussions about survey design and analysis; and Holly Tutko, Clinical Assistant Professor, Department of Health Management and Policy, University of New Hampshire for her critical review of this work. 10. Recommendations for practitioners This creates an “us” versus “them” perception, which can inhibit positive communication among stakeholders. It would be optimal for the room to be organized so the stakeholders are interspersed at a roundtable. This format allows each participant to see each other and not feel as though any one viewpoint is valued over another. g y p 10. Provide routine updates to stakeholders: State agency and Title V facilities should provide concerned community members updates about progress made to address their concerns. These updates could be communicated via a list-serve; mass mailings of a newsletter; and updates posted to NHDES’ and the facility’s web page. This practice would keep the public informed about what the state agency and facilities are doing and can dissuade distrust or contention from developing. 64 Environmental Management in Practice Our recommendations provide a set of communicative structures to help advance effective environmental communication among stakeholders when dealing with regulated industry in different types of communities. Such practices may increase the community’s trust in government, as well as their belief in the credibility of community health investigations and their acceptance of the results (Charnley & Engelbert, 2005). 11. Future work Our future work involves examining how creative partnerships, such as those between academia and the community can further advance environmental communication strategies. Although academic institutions are rich resources for improving the health of the public and the environment, academic partnerships with community organizations can be challenging. Yet, such partnerships have been shown to translate science and best practices into social action and policy change at the local community level (Serrell et al., 2009). 13. References Agency for Toxic Substances and Disease Registry (ATSDR). (2004). Communicating results to community residents: Lessons from recent ATSDR health investigations. Journal of Exposure Analysis and Environmental Epidemiology, 14, 484-491. Agency for Toxic Substances and Disease Registry (ATSDR). (2006). Health Consultation for Cancer Incidence: Residents of Claremont, Sullivan County, New Hampshire (Wheelabrator – Claremont Site) Retrieved December 18, 2010, from: http://www.atsdr.cdc.gov/HAC/pha/WheelabratorClaremont/Wheelabrator- ClaremontHC09.30.06.pdf Agency for Toxic Substances and Disease Registry (ATSDR). (2009). Public Health Assessments and Health Consultations. Retrieved December 18, 2010 from http://www.atsdr.cdc.gov/HAC/PHA/index.asp p g p Agency for Toxic Substances and Disease Registry (ATSDR). (2009). Public Health Assessment for Ambient Air Quality in Claremont, Sullivan County, New Hampshire. Retrieved December 18, 2010, from: http://www.atsdr.cdc.gov/HAC/pha/AmbientAirQualityinClaremontNH/Clare montInitial-PublicCommentPHA03-02-2009a.pdf Community Ecology and Capacity: Advancing Environmental Communication Strategies amo Community Ecology and Capacity: Advancing Environmental Communication Strategies among Diverse Stakeholders 65 Air Resources Division (ARD), New Hampshire Department of Environmental Services. (n.d.). Overview. Retrieved December 15, 2010, from http://des.nh.gov/organization/divisions/air/overview.htm. Air Resources Division (ARD), New Hampshire Department of Environmental Services. (2008). Title V Operating Permit Reporting Guidance. Retrieved December 15, 2010, from: http://des.nh.gov/organization/commissioner/pip/forms/ard/documents/repo rting_guidance.pdf Bennett, P. (1999). Understanding responses to risk: Some basic findings. In Bennett, P. & Calman, K. (Eds.), Risk Communication and Public Health, (pp. 3-19). New York, New York: University Press. Brauer, M., Hakkinen, P.J., Gehan, B.M., Shirname-More, L. (2004). Communicating exposure and health effects results to study subjects, the community and the public: Strategies and challenges. Journal of Exposure Analysis and Environmental Epidemiology, 14, 479-483. Caron, R.M. & Serrell, N. (2009). Community ecology and capacity: Keys to progressing the environmental communication of wicked problems. Applied Environmental Education and Communication, 8(3-4), 195-203. ( ) Charnley, S. & Engelbert, B. (2005). Evaluating public participation in environmental decision-making: EPA’s superfund community involvement program. Journal of Environmental Management, 77, 165-182. g Citizens Leading for Environmental Action and Responsibility (CLEAR), Claremont, NH (n.d.). Overview. Retrieved December 18, 2010, from: http://www.clearnh.org/NewsAction.aspx Cottrell, R.R. & McKenzie, J.F. (2005). Qualitative research methods. In Health Promotion & Education Research Methods: Using the Five-Chapter Thesis/Dissertation Model, (pp. 217- 244). Massachusetts: Jones and Bartlett Publishers. Covello, V. & Sandman, P. (2001). Risk communication: Evolution and revolution. In Wolbarst, A. (Ed.), Solutions to an Environment in Peril, (pp.164-178). Baltimore: Johns Hopkins University Press. y Cox, R. (2006). Risk communication: Nonexpert publics and acceptable risk. In Environmental Communication and the Public Sphere, (pp.205-240). Thousand Oaks: Sage Publications, Inc. Hance B.J., Chess C., Sandman P.M. (1989). 1 The views expressed in this paper are those of the author and not those of the Ministry of Finance or the Policy Research Institute. 2 Kawai (2009) indicates, for example, that the ratio of intra-regional trade relative to world trade in East Asia has gone up from 35 percent in 1980 towards 54 percent in 2007, which is a little under 57 percent in EU and exceeding 43 percent in NAFTA. 3 The classification of income classes depends on World Development Indicators of World Bank. Regional Issues in Environmental Management Hiroyuki Taguchi Policy Research Institute, Ministry of Finance1 Japan 4 Regional Issues in Environmental Management Hiroyuki Taguchi Policy Research Institute, Ministry of Finance1 Japan 4 13. References Setting a context for explaining risk. Risk Analysis, 9,113-117. y McComas, K.A. (2003). Citizen Satisfaction with public meetings used for risk communication. Journal of Applied Communication Research, 31(2), 164-184. McKinney, M. & Harmon, W. (2002). Public participation in environmental decision making: Is it working? National Civic Review, 91(2), 149-170. Park, E., Scherer, C.W., Glynn, C.J. (2001). Community involvement and risk perception at personal and societal levels. Health, Risk & Society, 3(3), 281-292. Parkin, R.T. (2004). Communication with research participants and communities: Foundations for best practices. Journal of Exposure Analysis and Environmental Epidemiology, 14, 516-523. 66 Environmental Management in Practice Sandman, P. (1989). Hazard versus outrage in the public perception of risk. In Covello V, McCallum D, Pavlova M (Eds.), Effective risk communication (pp. 45-49). New York: Plenum Press. Serrell, N., Caron, R.M., Fleishman, B., Robbins, E.D. (2009). An academic-community outreach partnership: Building relationships and capacity to address childhood lead poisoning. Progress in Community Health Partnerships: Research, Education, and Action, 3.1, 53-59. Tinker, T.L., Zook, E., Chapel, T.J. (2001). Key challenges and concepts in health risk communication: Perspectives of agency practitioners. Journal of Public Health Management and Practice, 7, 67-75. g United States Census Bureau (Census). (n.d.). Population Finder. Retrieved December 18, 2010 from http://www.census.gov/ hrly, R. (2006). Constructing the problem of lead poisoning. Senior thesis (unpublished) Wheelabrator Technologies, Inc., A waste Management Company. (n.d.) Fact Sheet. Retrieved December 18, 2010, from: http://www.wheelabratortechnologies.com/index.cfm/our-clean-energy-plants/ 1. Introduction This chapter addresses regional issues in environment management. Economic integration beyond national boundaries has recently made great progress in regional levels as well as in global levels, with the formulations of Free Trade Agreement (FTA) and Economic Partnership Agreement (EPA) as typical examples. This trend in regional economic integration also refocuses attention on regional environmental issues including trans- boundary pollutions, and makes us rethink of what regional cooperation should be in environment management. Based on this context, we herein discuss regional environment issues, with a focus on East Asian region, from the following two perspectives. , g , g p p The first one is about which effects, i.e. technological spillovers or pollution haven damages, the regional latecomers have dominantly received in environment management under a growing trend in economic integration within East Asia. If the dominance of technological spillovers effect is identified for latecomer’s economies, we may have rather optimistic views on the future of environment quality as a whole region, because it implies that latecomers are absorbing the skills and technologies enough to leapfrog the mistakes made by developed economies in the past times. On the other hand, the dominance of pollution haven damages implies the mere relocation of polluters from developed economies towards latecomer’s economies, i.e. no decline in pollution as a whole region, thereby making us feel uneasy on regional prospect of environment. Thus, knowing the effects for latecomers seems to be linked with knowing the degree of demand for policy actions as a region. East Asia, in recent decades, has strengthened intra-economic integration in terms of trade and investment flows.2 At the same time, East Asian economies are still composed of a variety of countries with different stages of development: high-income countries like Japan and Korea, middle-income ones like Malaysia and Thailand, low-income ones such as Cambodia and Myanmar.3 Since the integration and diversification characterized by East Asian economies make East Asia a typical area with provability of technology spillovers or pollution haven damages, targeting East Asia seems to be meaningful in our analysis. g g g g y The second perspective is about what the regional framework of environmental cooperation should be in East Asia. 1. Introduction There have been intensive debates on the regional frameworks from 68 Environmental Management in Practice the viewpoints of commitment and compliance, especially in the cases of such trans- boundary issues as long-range air and water pollutions, freshwater resources in international rivers, migratory birds. The frameworks differ in the modality of cooperation: policy dialogue, cooperative environmental monitoring and assessment, implementation of project-based activities, and legal treaties and protocols. There seem to be some contrasts in the approaches towards regional cooperation between East Asia and Europe: Non-binding approaches in East Asia versus binding ones in Europe. The typical example is seen in the framework of the long-ranged trans-boundary air pollution control: East Asia is promoting non-binding agreements on pollution monitoring and other project-based activities, whereas Europe is controlling pollution based on binding agreement in terms of the Convention for the Long-Range Transmission of Air Pollutant in Europe (the LRTAP). Each approach appears to have advantages and disadvantages, and the choice of the approach seems to be linked with the region-specific properties in economical, political, and historical terms. The purpose of this section is, thus, to investigate the reason why East Asia has taken the non- binding approach, and to examine the justification of its choice considering the region- specific properties. The rest of the paper is structured as follows. Section 2 examines the effects for latecomers in East Asia: technological spillovers versus pollution haven, corresponding to the first perspective above. Section 3 discusses the regional frameworks of environmental cooperation in East Asia, corresponding to the second perspective above. 2. Effects for latecomers: technological spillovers versus pollution haven The purpose of this section is to examine whether the latecomer’s economies in East Asia enjoy technological spillover effects or suffer pollution haven damages in their environment management: in other words, which of latecomer’s advantage or latecomer’s disadvantage dominates for pollution control in East Asian economies. We focus on environmental indices with data availability: carbon dioxide emissions, consumption of ozone-depleting substances and industrial organic water pollutant (BOD) emissions. The analytical framework of the Environmental Kuznets curve (EK curve) is used to arrive at a conclusion. In the following subsections, we first review previous studies on the EK curve in brief and clarify this article’s contribution (Subsection 2.1), present our own empirical study of the effects for latecomers (Subsection 2.2), and end with brief summary (Subsection 2.3). 4 Borghesi (1999) criticized the cross-sectional approach by arguing that since environmental degradation is generally increasing in developing countries and decreasing in industrialized ones, the EK curve within the cross-sectional framework might reflect the mere juxtaposition of two opposite trends rather than describe the evolution of a single economy over time. 2.1 Previous studies and our contribution The environmental Kuznets curve (EK curve) provides an analytical framework to examine how economies deal with environmental issues. The EK curve postulates an inverted-U relationship between pollution and economic development; at early stages of development, environmental quality deteriorates with increases in per capita income, while at higher levels of development, environmental degradation is seen to decrease with further increases in per capita income. Kuznets's name was apparently attached to the curve by Grossman & Krueger (1993), who noted its resemblance to Kuznets inverted-U relationship between income inequality and development. Since the issue of the EK curve was first discussed in the World Bank’s 1992 World Development Report, there have been numerous empirical tests and theoretical debates on the EK curve. Until the mid of the 1990s, most of the empirical studies concentrated on validating the EK curve hypothesis and its requirements, using cross-sectional data. Some of evidences on specific pollutants supported the validity 69 Regional Issues in Environmental Management of the EK curve (e.g. Grossman & Krueger; 1995, Selden & Song; 1994), while some argue that the EK curve does not hold at all times and for all pollutants (e.g. Shafik; 1994). of the EK curve (e.g. Grossman & Krueger; 1995, Selden & Song; 1994), while some argue that the EK curve does not hold at all times and for all pollutants (e.g. Shafik; 1994). Since the late 1990s, the EK curve studies have shifted from cross-sectional analyses to time- series analyses, especially towards the analyses for comparing the EK curves of individual economies in terms of the height and the timing of their peaks, their shapes, etc (e.g. Panayotou; 1997, De Bruyn et al.; 1998).4 One of the frontiers in this direction of the EK curve studies is to put into empirical tests the two contrasting hypotheses presented by Dasgupta et al. (2002). One is the technological spillover hypothesis that developing societies, by utilizing progressive environmental management and the technologies of more advanced countries, might be able to experience an EK curve that is lower and flatter than what conventional wisdom would suggest. The other is the pollution haven hypothesis that the relatively high environmental standards in high-income economies impose high costs on polluters, and shareholders pressure firms to relocate to low-income countries. This pollution haven scenario may not shift the latecomer’s EK curves downward; on the contrary, it may even lift them up. 2.1 Previous studies and our contribution Taguchi & Murofushi (2009), by using the EK curve framework, examined whether developing countries enjoy the latecomer’s advantage or suffer the latecomer’s disadvantage in the environment management, focusing on sulfur emissions as local air pollutants and carbon emissions as global air pollutants, by using the world-wide samples for the 188 economies from 1960 to 1990 in sulfur emissions and from 1970 to 2003 in carbon emissions. It found contrasting result between sulfur and carbon emissions on the latecomer’s effects; sulfur emissions represent the dominance of the latecomer’s advantage (the downward shift of latecomer’s EK curve), while carbon emissions reveal that of the latecomer’s disadvantage (the upward shift of latecomer’s EK curve). It interpreted this contrast as the difference of maturity level in the know-how and technology to abate emissions: prevailing desulfurization technology and unrestricted “carbon leakage” (a kind of pollution haven in carbon emissions). that the EK curve does not hold at all times and for all pollutants (e.g. Shafik; 1994). Since the late 1990s, the EK curve studies have shifted from cross-sectional analyses to time- series analyses, especially towards the analyses for comparing the EK curves of individual economies in terms of the height and the timing of their peaks, their shapes, etc (e.g. Panayotou; 1997, De Bruyn et al.; 1998).4 One of the frontiers in this direction of the EK curve studies is to put into empirical tests the two contrasting hypotheses presented by Dasgupta et al. (2002). One is the technological spillover hypothesis that developing societies, by utilizing progressive environmental management and the technologies of more advanced countries, might be able to experience an EK curve that is lower and flatter than what conventional wisdom would suggest. The other is the pollution haven hypothesis that the relatively high environmental standards in high-income economies impose high costs on polluters, and shareholders pressure firms to relocate to low-income countries. This pollution haven scenario may not shift the latecomer’s EK curves downward; on the contrary, it may even lift them up. Taguchi & Murofushi (2009), by using the EK curve framework, examined whether developing countries enjoy the latecomer’s advantage or suffer the latecomer’s disadvantage in the environment management, focusing on sulfur emissions as local air pollutants and carbon emissions as global air pollutants, by using the world-wide samples for the 188 economies from 1960 to 1990 in sulfur emissions and from 1970 to 2003 in carbon emissions. 2.1 Previous studies and our contribution It found contrasting result between sulfur and carbon emissions on the latecomer’s effects; sulfur emissions represent the dominance of the latecomer’s advantage (the downward shift of latecomer’s EK curve), while carbon emissions reveal that of the latecomer’s disadvantage (the upward shift of latecomer’s EK curve). It interpreted this contrast as the difference of maturity level in the know-how and technology to abate emissions: prevailing desulfurization technology and unrestricted “carbon leakage” (a kind of pollution haven in carbon emissions). g ( p ) This study aims at testing the two contrasting hypothesis above in East Asia, – the dominance of the latecomer’s advantage (technological spillovers) or of the latecomer’s disadvantage (pollution haven). The main contribution is to extend the existing literature, mainly of Taguchi (2009), to the following directions. First, our study concentrates on East Asian economies (18 economies). The intra-area of East Asia with the characteristic of economic integration and diversification, as stated in Introduction, can be an experimental area suitable enough to put the hypotheses of technological spillovers and pollution haven into empirical tests. In addition, the evidence on the latecomer’s effects in East Asia has been extremely limited in the existing literature. Second, our analysis uses the latest data of the period for 1990-2007 on carbon dioxide emissions, consumption of ozone-depleting substances and industrial organic water pollutant (BOD) emissions. The usage of the latest data enables us to make the EK curve estimation reflect the recent trends of technological progress and policy responses to address environmental issues as well as growing economic interaction of East Asia. Third, our estimation for the EK curve adopts a dynamic panel model by a system of Generalized Method of Moments (GMM). It appears to take some periods for the current level of emissions to adjust toward their equilibrium level – a kind of inertia in the emission level. Most of previous studies for the EK curve have adopted a static 4 Borghesi (1999) criticized the cross-sectional approach by arguing that since environmental degradation is generally increasing in developing countries and decreasing in industrialized ones, the EK curve within the cross-sectional framework might reflect the mere juxtaposition of two opposite trends rather than describe the evolution of a single economy over time. 70 Environmental Management in Practice panel model in terms of ordinary fixed or random estimations. 5 See the website of http://www.unescap.org/stat/data/syb2008/syb2008_web/index.asp 2.1 Previous studies and our contribution When there is evidence of dynamics in the data, however, the validity of applying a static model might be questioned as being dynamically miss-specified. To our knowledge, it is only Halkos (2003) that constructed a dynamic panel model for the EK curve estimation. This paper adopts the method of Halkos (2003), which allows dynamic adjustments in the level of emissions. 2.2 Empirics We now turn to the empirical studies using the analytical framework of the EK curve. Our analysis consists of two steps. First, we simply overview the relationships between per capita real income and environmental indices. We then move to a dynamic panel analysis using cross-country panel data to examine the EK curve pattern and to see whether the latecomer’s advantage or its disadvantage dominates in the environmental management in East Asian economies. 2.2.1 Data We collect the data for three environmental indices per capita –carbon dioxide emissions, consumption of ozone-depleting substances and industrial organic water pollutant emissions– and real GDP per capita. All the data come from the Annual Core indicators online database developed by the Statistics Division of the United Nations Economic and Social Commission for Asia and the Pacific (ESCAP).5 The database covers data from 1990 to 2007, all of which we use as sample periods. The sample economies are the following 18 ones in East Asia: Brunei Darussalam, Cambodia, China, DPR Korea, Hong Kong, Indonesia, Japan, Lao PDR, Macao, Malaysia, Mongolia, Myanmar, Republic of Korea, Singapore, Thailand, the Philippines, Timor-Leste and Viet Nam. g p pp The indicator of “carbon dioxide emissions per capita” that we can obtain from the online database is defined as the quantity of estimated carbon dioxide emissions (tons of carbon dioxide) divided by total population, whose data sources are the United Nations Millennium Development Goals Indicators and the World Population Prospects: the 2006 Revision Population Database. The indicator of “consumption of ozone-depleting substances per capita” is defined as the sum of the national annual consumption in weighted tons of individual substances in the group of ozone-depleting substances multiplied by their ozone-depleting potential (Ozone-depleting substances are any substance containing chlorine or bromine that destroys the stratospheric ozone layer), expressed as ODP kilograms per 1,000 population. Its data sources are the same as those of carbon dioxide emissions per capita. The indicator of “industrial organic water pollutant emissions” is defined as the biochemical oxygen demand, which refers to the amount of oxygen that bacteria in water will consume in breaking down waste, expressed as kilograms per day. Its data source is the United Nations Environment Program, Emission Database for Global Atmospheric Research (EDGAR 3.2). This indicator shows total amount, thereby being divided by population. We can find the other emissions indicators in the online database: nitrous oxide emissions, sulfur dioxide emissions and PM10 concentration in urban area, but do not adopt them for the dynamic estimation later since their data cover only every five years. For the real GDP per capita, the indicator of “GDP per capita on 1990 US dollars base” is obtained from the online database. 2.2.1 Data 71 Regional Issues in Environmental Management To sum up, for conducting the dynamic panel estimation later on, we constructed a panel table of the annual data of the 18 economies from 1990 to 2007 on each of per capita environmental indices of carbon dioxide emissions, consumption of ozone-depleting substances and industrial organic water pollutant emissions, and on real GDP per capita. 2.2.2 Overview of the EK curves in sample economies in East Asia d h l h b l 2.2.2 Overview of the EK curves in sample economies in East Asia Fiure 1 indicates the time-series relationships between per capita real GDP and three kinds of environmental indices per capita in selected samples of East Asian economies. The rough Fiure 1 indicates the time-series relationships between per capita real GDP and three kinds of environmental indices per capita in selected samples of East Asian economies. The rough Carbon Dioxide Emissions (1990, 1995, 2000, and 2004) 0 2 4 6 8 10 100.00 1,000.00 10,000.00 100,000.00 GDP per capita: US dollar in 1990 emissions per capita: tons Japan China Republic of Korea Malaysia Thailand Philippines Indonesia Viet Nam Consumption of Ozone-depleting Substances (1990, 1995, 2000, and 2006) 0 100 200 300 400 500 600 700 800 900 1000 100.00 1,000.00 10,000.00 100,000.00 GDP per capita: US dollar in 1990 consumption per capita: grams Japan China Republic of Korea Malaysia Thailand Philippines Indonesia Viet Nam Industrial Organic Water Pollutant Emissions (1990, 1995, and 2000) 2 4 6 8 10 12 14 100.00 1,000.00 10,000.00 100,000.00 GDP per capita: US dollar in 1990 emissions per capita: grams Japan China Republic of Korea Malaysia Thailand Philippines Indonesia Fig. 1. Overview of the EK curves in selected sample economies Carbon Dioxide Emissions (1990, 1995, 2000, and 2004) 0 2 4 6 8 10 100.00 1,000.00 10,000.00 100,000.00 GDP per capita: US dollar in 1990 emissions per capita: tons Japan China Republic of Korea Malaysia Thailand Philippines Indonesia Viet Nam Consumption of Ozone-depleting Substances (1990, 1995, 2000, and 2006) 0 100 200 300 400 500 600 700 800 900 1000 100.00 1,000.00 10,000.00 100,000.00 GDP per capita: US dollar in 1990 consumption per capita: grams Carbon Dioxide Emissions (1990, 1995, 2000, and 2004) 0 2 4 6 8 10 100.00 1,000.00 10,000.00 100,000.00 GDP per capita: US dollar in 1990 emissions per capita: tons Japan China Republic of Korea Malaysia Thailand Philippines Indonesia Viet Nam Carbon Dioxide Emissions (1990, 1995, 2000, and 2004) Consumption of Ozone-depleting Substances (1990, 1995, 2000, and 2006) Industrial Organic Water Pollutant Emissions (1990, 1995, and 2000) Fig. 1. Overview of the EK curves in selected sample economies 72 Environmental Management in Practice findings are as follows. First, there appears to be no cases where the assembly of the economy’s trajectories clearly produces inverted-U shape patterns. 6 As Dasgupta et al. (2002) showed the revised EK curve that is actually dropping and shifting to the left as growth generates less pollution in the early stages of industrialization and pollution begins falling at lower income levels, the latecomer’s effects may not always be tantamount to a simple up- and downward shifts of the EK curve. However, we here simplify the analysis by focusing on up- and downward shift of the EK curve. 2.2.2 Overview of the EK curves in sample economies in East Asia d h l h b l The trajectories of carbon dioxide emissions represent an increasing trend whereas their slope seems to be flattened with higher real GDP per capita. The lines of consumption of ozone-depleting substances roughly represent declining slope. The cases of industrial organic water pollutant emissions have no clear trend of trajectories. We might speculate that the carbon dioxide emissions stay at the positively-sloping part of the EK curve, while the consumption of ozone-depleting substances stays at its negatively-sloping part. Second, the locations of the economy’s trajectories represent a clear contrast; the upward shifts of trajectories for latecomer’s economies are observed in the case of carbon dioxide emissions, while downward shifts are seen in the cases of consumption of ozone-depleting substances. The cases of industrial organic water pollutant emissions have no clear shift of trajectories. The GDP-emissions relationships described above may produce different implications among environmental indices. This point will be statistically tested through dynamic panel estimations in the following section. 2.2.3 Dynamic panel analysis y y We’ll now move to a dynamic panel analysis using cross-country panel data to examine the EK curve pattern and to see whether the latecomer’s advantage or its disadvantage dominates in the environmental management in East Asian economies. 2.2.3.1 Methodology We first clarify some methodological points related to our analysis. To study the relationship between pollution and growth, there are two possible approaches to model construction. One is to estimate a reduced-form equation that relates the level of pollution to the level of income. The other is to model the structural equations relating environmental regulations, technology, and industrial composition to GDP, and then to link the level of pollution to the regulations, technology, and industrial composition. We here take the reduced-form approach for the following reasons. First, the reduced-form estimates give us the net effect of a nation’s income on pollution. If the structural equations were to be estimated first, one would need to solve backward to find the net effect. Moreover, confidence in the implied estimates would depend on the precision and potential biases of the estimates at every stage. Second, the reduced-form approach spares us from having to collect data on pollution regulations and the state of the existent technology, which are not always available. Thus, we think that the reduced-form relationship between pollution and income is an important first step. p p We then specify the reduced-form equation by basically following the traditions of the literatures like Grossman and Krueger (1995) and Selden and Son (1994), and adding appropriate variables in accordance with our analytical interests. Our specific concern regarding the EK curve for the sample economies in East Asia is to see whether the EK- curve trajectories for the latecomer’s economies have shifted downward or upward, depending on the dominance of either the latecomer’s advantage or its disadvantage6; in 73 Regional Issues in Environmental Management other words, the levels of environmental pollution per capita have been affected not only by the level of per capita income following the EK curve, but also by the later degree of development among the economies. If a sample economy with later degree of development among the samples enjoys the lower level of environmental pollution (traces the downward course of the EK curve), we speculate that the economy, not repeating the EK-curve trajectories already experienced by the developed economies, should enjoy the latecomer’s advantage by absorbing the progress in environmental know-how, skills, and technology i.e. technological spillover. On the contrary, if the later development in a sample economy is linked with higher pollution, the economy may suffer from the latecomer’s disadvantage caused by the “pollution haven” scenario (see Figure 2). 2.2.3.1 Methodology Therefore, we will include a term representing the later degree of development among the economies into the equation for the EK curve. The later degree of development of a sample economy in a certain year is specified as the ratio of the GDP per capita of that economy relative to the maximum GDP per capita among sample economies (equivalent to the GDP per capita of Japan) in that year. Another methodological innovation in this study is to adopt a dynamic panel model. Halkos (2003), pointing out that a static model is justified either if adjustment processes are really very fast or if the static equation represents an equilibrium relationship, argued that since the assumption that the data are stationary is incorrect, and we are not expecting a very fast adjustment for estimating the EK curve, a statistically sound approach requires estimating a dynamic model. Following the argument of Halkos (2003), we construct a dynamic panel model by inserting a lagged dependent variable as a regressor into the EK curve equation for materializing a partial adjustment toward equilibrium emissions level. Per Capita Emissions Per Capita Emissions Per Capita Emissions Latecomer's Economy + Higher Pollution = Latecomer's Disadvantage (⇒Pollution Haven) Upward Shift Higher Income Economies Downward Shift Latecomer's Economy + Lower Pollution = Latecomer's Advantage (⇒Technological Spillover) Real GDP Per Capita Fig. 2. Latecomer’s advantage and disadvantage in the EK curves Based on analytical interests mentioned above, we specify the modified EK curve model as Latecomer's Economy + Higher Pollution = Latecomer's Disadvantage (⇒Pollution Haven) Upward Shift Higher Income Economies Downward Shift Latecomer's Economy + Lower Pollution = Latecomer's Advantage (⇒Technological Spillover) Real GDP Per Capita Real GDP Per Capita p Fig. 2. Latecomer’s advantage and disadvantage in the EK curves Fig. 2. Latecomer’s advantage and disadvantage in the EK curves Fig. 2. Latecomer’s advantage and disadvantage in the EK curves Fig. 2. 2.2.3.1 Methodology Latecomer’s advantage and disadvantage in the EK curves Based on analytical interests mentioned above, we specify the modified EK curve model as follows: Based on analytical interests mentioned above, we specify the modified EK curve model as follows: 2 it 0 1 it 2 it 3 it 4 it 1 5 i it EMS GDP GDP LAC EMS f e        (1) 2 it 0 1 it 2 it 3 it 4 it 1 5 i it EMS GDP GDP LAC EMS f e        (1) (1) 74 Environmental Management in Practice where i is the economy’s index (country), t is the time index, and e is the error term. The dependent variables EMS is measure of the per capita emissions: carbon dioxide emissions (CDE), consumption of ozone-depleting substances (ODS) and industrial organic water pollutant emissions (BOD). As for the independent variables, GDP is the real GDP per capita. LAC represents the later degree of development, specifically the ratio of the real GDP per capita of a certain economy relative to the maximum real GDP per capita among economies in a certain year (i.e. real GDP per capita of Japan) – the lower LAC means the later development of the economy. The fi denotes exogenously economy-specific factors that affect emissions; climate, geography, energy resources, etc. The equation does not include period dummy, because its inclusion was rejected significantly by statistical tests in the equation estimate. To verify the inverted-U shapes of the EK curves, the signs and magnitudes of α1 and α2 should be examined. Environmental emissions per capita can be said to exhibit a meaningful EK curve with the real GDP per capita, if α1>0 and α2<0, and if the turning point, –α1/2α2 is a reasonabe number. Of particular importance is the coefficient of LAC, α3, which is useful for identifying the dominance of the latecomer’s advantage or its disadvantage. The positive sign of α3, the lower pollution with the later development of the economy that creates the downward shift of the latecomers’ trajectories, indicates that the latecomer’s advantage surpasses its disadvantage. On the other hand, the negative sign of α3, the higher pollution with the later development of the economy equivalent to the upward shift of the latecomers’ curve, reveals the dominance of the latecomer’s disadvantage. 2.2.3.1 Methodology g Equation (1) contains the lagged dependent variable among the explanatory variables, thereby the ordinary OLS estimator being inconsistent. Obtaining consistent estimates requires the application of an instrumental variables estimator or Generalized Method of Moments (GMM). We here adopt the system GMM estimator developed by Arellano and Bond (1991) who argues that additional instruments can be obtained in a dynamic model from panel data if we utilize the orthogonality conditions between lagged values of the dependent and the disturbances. The GMM estimator eliminates country effects by first- differencing as well as controls for possible endogeneity of explanatory variables. The first- differenced endogenous variables of EMS with two lagged periods can be valid instruments provided there is no second-order autocorrelation in the idiosyncratic error terms. We also use the first differenced explanatory variables of GDP with one lagged period as an instrumental variable since GDP can possibly be correlated with the error term in case that environmental pollution might aggravates economic growth. We then conduct two step GMM iterations with updating weights once, and adopt White period as GMM weighting matrix. We present the tests for autocorrelations and the Sargan test of over-identifying restrictions in the table that follow. 2.2.3.2 Estimation results and interpretations Table 1 lists the results of the GMM estimation per capita on carbon dioxide emissions (CDE), consumption of ozone-depleting substances (ODS) and industrial organic water pollutant emissions (BOD). All the cases indicate that the inclusion of the lagged dependent variable of the emissions per capita proved to be positively discernable, thus imply inertia in the level of the emissions and justify forming the dynamic panel model. The Sargan tests do not suggest rejection of the instrumental validity at conventional levels for any cases estimated. As for the test results for autocorrelations, all the AR(2) test statistics reveal absence of second-order serial correlation in the first-differenced errors and thus that the instruments are valid. 75 Regional Issues in Environmental Management We first verify the shape of the EK curve of each emission index. There are no cases that reveal the meaningful EK curve with the inverted-U shape. The linear CDE estimation indicates upward sloping with real GDP per capita at significant level. The quadratic CDE estimation has the significant coefficients, α1 and α2 with correct signs of the inverted-U shape. Its turning point of 26,800 US dollars is, however, falling into the edge of the samples, i.e. only within the sample of Japan with the highest real GDP per capita. Almost all of the trajectories are within the monotonic increasing trend, i.e. the positively-sloping part of the EK curve. The ODS estimation indicates that the trajectories are in the monotonic decreasing trend regardless of the linear or quadratic equation forms. Although the quadratic estimation’s coefficients, α1 and α2, suggest not inverted-U but U shape, the turning point of 116,000 US dollars is far higher from the range of the samples. The BOD represents only monotonic downward sloping in its estimation, since the coefficient of the square of GDP, α2, is insignificant. We speculate that it is due to the shortage of sample data backward from 1990 that the ODS and BOD do not prove to form the inverted-U shape curve in their estimation. We next see if the latecomer’s EK trajectories show a downward shift or an upward shift, namely whether the latecomer’s advantage or its disadvantage dominate in the environmental management of latecomer’s economies. The CDE estimate has significantly negative α3, coefficient of LAC, thereby representing the upward shift of the latecomer’s trajectories and the dominance of the latecomer’s disadvantage. (Notes) i) The t-value are in parentheses. *, , and * indicate rejection at the 1 percent, 5 percent, and 10 percent significance levels significance levels. ii) "Sargan test" denotes the p-value of a Sargan-Hansen test of overidentifying restrictions. i) The t-value are in parentheses. *, , and * indicate rejection at the 1 percent, 5 percent, and 10 pe significance levels. Table 1. Results of dynamic panel estimation by GMM ii) Sargan test denotes the p-value of a Sargan-Hansen test of overidentifying restrictions. iii) AR(k) is the p-value of a test that the average autocovariance in residuals of order k is zero. 2.2.3.2 Estimation results and interpretations On the other hand, the ODS and BOD estimates have significantly positive α3, showing the downward shift of the latecomer’s trajectories, the dominance of the latecomer’s advantage. GDP 4.4310-4 ** 2.5710-3 ** -2.3310-2 ** -2.9810-2 ** -1.3810-4 ** -3.4110-4 * (978.87) (33.96) (-68204.89) (-6078.09) (-6.97) (-2.25) GDP2 -4.7810-8 ** 1.2810-7 ** 3.5310-9 (-21.42) (1150.38) (1.25) LAC -2.2110 *** -5.1810 ** 1.56102 ** 2.39102 ** 1.22 * 4.61 ** (-2980.99) (-291.37) (14700.19) (3125.34) (1.72) (2.12) (EMS) t-1 4.9610-1 ** 4.5310-1 ** 5.6610-1 ** 5.6510-1 ** 6.1110-1 ** 5.7610-1 ** (11958.02) (106.46) (517030.9) (171680.4) (19.48) (10.00) Tuning Point 2.68104 1.16105 4.83104 Sargan test 0.60 0.85 0.71 0.75 0.88 0.91 AR(1) 0.00 0.01 0.07 0.07 0.01 0.01 AR(2) 0.21 0.24 0.74 0.75 0.86 0.81 No. of obs. 222 222 192 192 93 93 (Notes) i) The t value are in parentheses and * indicate rejection at the 1 percent 5 percent and 10 percent CDE ODS BOD 76 Environmental Management in Practice There seem to be some contrasts of estimation results in terms of both the trajectory’s shape and location between CDE and the other indices of ODS and BOD. These contrasts appear to be interpreted as follows. The first contrast is concerned with the shape of the EK trajectories. The ODS and BOD mainly come from manufacturing production activities, thereby being subject to regulation due to their localized impact. In fact, the pollution controls on the ODS and BOD have intensively been promoted by East Asian countries. The ozone-depleting substances have been strictly regulated since the 1987’s signature of the Montreal Protocol, i.e. an international treaty designed to protect the ozone layer by phasing out the production of a number of substances believed to be responsible for ozone depletion. All of East Asian countries have had a commitment to the treaty or its amendments in terms of ratification, accession or acceptance. The issues of water pollution as well as air pollution have also been addressed with technological progress over a broad area of East Asia since the 1970-80s, when ASEAN countries formulated comprehensive environmental protection laws (the Philippines in 1977, Malaysia in 1974, Thailand in 1975, and Indonesia in 1982). These factual backgrounds seem to make the EK trajectories of ODS and BOD slope downward i.e. create downward sloping part of the inverted-U shaped EK curve. 7 See the website: http://www.theozonehole.com/montreal.htm. 2.3 Summary I thi ti In this section, we set out to examine, using the analytical framework of the environmental Kuznets curve, whether the latecomer’s economies in East Asia enjoy technological spillover effects or suffer pollution haven damages in their environmental pollution management, in other words, which of latecomer’s advantage or latecomer’s disadvantage for pollution control dominates in East Asian economies. For this purpose, we carried out dynamic panel estimation by a system of Generalized Method of Moments (GMM), using the panel data with 18 economies for the period from 1990 to 2007 on environmental indices of carbon dioxide emissions, consumption of ozone-depleting substances and industrial organic water pollutant emissions. p Through this analysis, we found two contrasting results among the environmental indices: 1) per capita consumption of ozone-depleting substances and industrial organic water pollutant emissions indicate monotonic decreasing trends with per capita real GDP while per capita carbon dioxide emissions show monotonic increasing trend, and 2) consumption of ozone-depleting substances and industrial organic water pollutant emissions represent the dominance of the latecomer’s advantage while carbon dioxide emissions reveal that of the latecomer’s disadvantage. We speculate that the contrast in the trends comes from the difference in the origin of emissions: consumption of ozone-depleting substances and industrial organic water pollutant emissions come mainly from production (easily regulated on the local level), and carbon dioxide emissions come from both production and consumption (easily externalized and not easily subject to regulation). We also presume that the contrast in the latecomer’s effects lies in the degree of maturity in regulatory framework and technology that offset pollution haven effect: good governance for controlling ozone- depleting substances and water pollutants, versus unrestricted “carbon leakage” for latecomer’s economies. The result implying “carbon leakage”, suggests the urgent necessity to facilitate the technological progress such as the development of technology on carbon dioxide capture and storage, and the internalization of external diseconomy through such methods as emissions charge and greenhouse taxes. For latecomer’s economies in East Asia, which appear to face a trade-off between environmental quality and productive activities and to strengthen regional economic integration, it can be expected that the spillover effects from technological progress and the consolidated regulatory framework should overcome “carbon leakage”. 2.2.3.2 Estimation results and interpretations On the other hands, the CDE is producing an opposite pattern of its trajectories, a positively- sloping part of the EK curve. It seems to be because carbon dioxide emissions arise from not only production but also from consumption such as automobile use and the burning of fossil fuels for the generation of electricity, thereby being easily externalized and thus not subject to regulation. The reality is that it is only after the Kyoto Protocol was approved in 1997 that regulatory frameworks on Greenhouse Gas have come to be set about domestically and internationally. The contrasting outcomes on the shape of the EK trajectories in this study appear to be consistent with those of previous works, which Nahman & Antrobus (2005) summarize by stating that the levels of the pollutants with local impacts fall with per capita income whilst the levels of easily externalized pollutants continue to rise with per capita income. The second contrast – downward shift of the latecomer’s trajectories on the ODS and BOD versus upward shift on the CDE – can be explained by the degree of maturity in the know- how and technology to abate those emissions in East Asia. More or less, the concentration of manufacturing industrial activities have tended to shift from advanced economies to developing economies since wealthy consumers in advanced economies demand a cleaner environment and stringent environmental regulations. Thus, the pollution haven effects can not help being avoided for latecomer’s economies. The question is, then, whether the technological spillover effects overcome the pollution haven effects for latecomer’s economies i.e. the dominance of latecomer’s advantage or disadvantage. The cases with downward shift of the latecomer’s trajectories on ODS and BOD can be interpreted in such a way that the policy efforts, know-how and technology to abate those emissions are mature and feasible enough to be transferred to latecomer’s economies and to exceed their suffering pollution haven effects in the area of East Asia. Especially, as Kofi Annan, the Former Secretary General of the United Nations, stated “perhaps the single most successful international agreement to date has been the Montreal Protocol”,7 the widespread adoption and implementation of the international framework to protect the ozone layer seems to be effective enough for developing economies in East Asia to enjoy the latecomer’s advantage. 2.2.3.2 Estimation results and interpretations On the contrary, the case with upward shift of the latecomer’s trajectories on CDE may be 77 Regional Issues in Environmental Management explained in such a way that the regulatory framework and technology to mitigate the emissions coming from both production and consumption are too immature to be transferred and disseminated to latecomer’s economies (Yaguchi et al. 2007). Thus, only the pollution haven effect seems to remain for latecomer’s economies. This phenomenon on carbon dioxide emissions might be regarded as what we call “carbon leakage” in the context of the Greenhouse Gas reduction at global level: the effect that there is an increase in carbon emissions in one country as a result of an emission reduction by a second country with a strict climate policy. 3.1 Trans-boundary issues in East Asia y We herein pick up major trans-boundary environmental issues in East Asia: acid deposition, marine pollution, haze pollution, and sand and dust storms. 3.1.1 Acid deposition8 Acid deposition originates from such pollutants as sulfur oxides (SOx) and nitrogen oxides (NOx), generated mainly by combustion of fossil fuels. Acid deposition appears in various forms of precipitation, such as rain, fog, mist, snow, etc. Its impacts are: affecting fishes due to the acidification of inland waters, threatening forests due to soil acidification, accelerating the decay of cultural monuments, and so forth. Since the substances causing acid deposition are transported over long distance, its influence diffuses to not only inside of the country but also to outside of the country. In East Asia, the rapid growth, accompanying the increasing energy consumption, has threatened to aggravate acid deposition since the 1990s. Some researches show that the trans-boundary acid rain in Northeast Asia is linked primarily to China’s coal consumption which accounts for two thirds of the country’s primary energy source (Yoon; 2007). Addressing the trans-boundary acid deposition in East Asia, was motivated by the Agenda 21 adopted by the United Nations Conference on Environment and Development in 1992, "the programs (in Europe and North America) need to be continued and enhanced, and their experience needs to be shared with other regions of the world". Since 2001, the Acid Deposition Monitoring Network in East Asia (EANET) under 13 countries participation has been running as a regional cooperative initiative for monitoring acid deposition. As a related cooperative framework, the Northeast Asian Sub-regional Programme of Environmental Cooperation (NEASPEC) is promoting the several projects for mitigation of trans-boundary air pollution from coal-fired power plants in North-east Asia. p http://www.env.go.jp/en/earth, http://www.eanet.cc/index.html, and http://www.neaspec.org. 9 Most of the description is based on http://www.nowpap.org/index.php. 8 Most of the description is based on 3. Regional framework of environmental cooperation In the previous section, we argued that the technological spillovers, offsetting the pollution haven damages, take an important role in environment management, especially in East Asia with the characteristic of economic integration and diversification. The significance of the 78 Environmental Management in Practice technological spillovers reminds us of the necessities of international cooperation in environment management in terms of regional framework as well as global and bilateral ones. The regional framework of environmental cooperation is, at the same time, crucial in addressing trans-boundary pollutions in specific region’s air and water. This section discusses the regional framework of environment cooperation, with a focus on the non- binding approach taken by East Asia. In the following subsections, we first review major trans-boundary environmental issues in East Asia (Subsection 3.1), represent the regional frameworks to address the trans-boundary issues (Subsection 3.2), and finally discuss the background and justification of the non-binding approach characterized by East Asia. http://www.env.go.jp/en/earth, http://www.eanet.cc/index.html, and 9 M f h d i i i b d h // /i d h 3.1.3 Haze pollution10 Haze is an atmospheric phenomenon where dust, smoke and other dry particles obscure the clarity of the sky. In the 1990s, haze pollution spreading across national boundaries was getting obvious in Southeast Asia. It originated from widespread land clearance through open forest burning, with the most well-known hotspots being in Sumatra, Borneo and the Malay Peninsula. Most of the smoke came from oil palm plantations which used burning instead of heavy equipment to clear land. The year 1997 was particularly noted for the raging forest fires in Indonesia which produced a pall of small particle pollution over the region for several weeks. Due to the prevalent monsoon winds, Malaysia, Singapore, Thailand and Brunei were seriously suffering from haze pollution. In light of the haze disaster, environmental ministers of the Association of Southeast Asian Nations (ASEAN) agreed on the Regional Haze Action Plan (RHAP) in 1997. As a further step, the ASEAN reached a legal agreement in 2002, which entered into force for the ratifying countries in 2003. The agreement contains provisions for monitoring, assessment and prevention, technical cooperation, scientific research, mechanisms for coordination and lines of communication, etc. for addressing trans-boundary haze pollution. Due to Indonesia’s current decision not to ratify and implement the agreement, however, the provisions of the agreement are not legally binding for the country which is perceived as being by far the greatest contributor to haze in Southeast Asia. 3.1.2 Marine pollution9 The Northwest Pacific sea region is specifically composed of the Yellow Sea, surrounded by China and the two Koreas, and the Sea of Japan/the East Sea, encircled by Japan, the two Koreas, and Russia. The region features coastal and island ecosystems with spectacular marine life and commercially important fishing resources. The region has, however, been getting enormous pressures and demands on its environment through coastal area 79 Regional Issues in Environmental Management development, river pollution flowing into the seas, and marine dumping. Marine contamination occurs also by such accidents as heavy oil spills from troubled tankers, e.g. the "Oil disaster of Nakhodka Accidents” in Japanese coastal sea by the Sea of Japan (the East Sea) in 1997. development, river pollution flowing into the seas, and marine dumping. Marine contamination occurs also by such accidents as heavy oil spills from troubled tankers, e.g. the "Oil disaster of Nakhodka Accidents” in Japanese coastal sea by the Sea of Japan (the East Sea) in 1997. As a regional framework for monitoring and assessing marine pollution, the Action Plan for the Protection, Management and Development of the Marine and Coastal Environment of the Northwest Pacific Region (NOWPAP) was adopted in September 1994 as a part of the Regional Seas Programme of the United Nations Environment Programme (UNEP). The participants of this plan are the countries bordering the sea region: China, Japan, Korea, and Russia (North Korea still reserves the option to become a regular member). p ( ) 11 Most of the description is based on http://www.env.go.jp/en/earth/dss/pamph/02.html, http://www.env.go.jp/earth/coop/temm/introduction_j.html, and http://www.neaspec.org. 10 Most of the description is based on UNEP (2010). 3.2 Modalities of regional frameworks in East Asia g The fore-mentioned trans-boundary environmental issues in East Asia have urged the countries in the region to make efforts to promote regional cooperation to address these issues. In this subsection, we pick up regional frameworks focusing on those for coping with trans-boundary environmental issues in East Asia, and examine their modalities in comparison with that in Europe. p p When we see the ongoing cases of regional frameworks for environmental cooperation in the world, we can find a variety of their modalities (e.g. IGES; 2001, Takahashi; 2003). We herein attempt to classify the modalities from the viewpoint of the consolidation of regional governance for cooperation as follows: a) Policy dialogue for sharing views and information on common environmental issues, b) Monitoring and assessment on trans-boundary environmental pollution by common methodologies, c) Project-based joint activities for mitigating pollution by utilizing permanent financial resources, d) Treaty and protocol for imposing common regulations on trans-boundary environmental pollution. g g y Table 2 reports the existing regional frameworks for environmental cooperation to address trans-boundary environmental issues. If we simply follow the modality classification above, the TEMM is classified into a)-type (policy dialogue); the EANET and the NOWPAP into b)- type (monitoring and assessment); the NEASPEC into c)-type (project-based joint activities); the ASEAN Agreement on Trans-boundary Haze Pollution into d)-type (treaty and protocol). It should be noted that the ASEAN Agreement on Trans-boundary Haze Pollution has problem in its implementation because of the lack of enforcement and liability clauses in the agreement in addition to Indonesia’s current decision not to ratify and implement the agreement as mentioned above (UNEP; 2010). g ( ) Europe, though having various modalities in regional frameworks for environmental cooperation, appears to depend more on legal frameworks backed by the EU organization than East Asia does. Some clear contrasts can be seen in the framework to address trans- boundary acid deposition and marine pollution. The acid deposition problem first came to trans-boundary attention in Europe in the 1970s. In the first place, the OECD responded to a request from Scandinavian countries to inaugurate a multilateral monitoring program of acid rain in 1972. And it was taken over by the United Nations Economic Commission for Europe in 1977. 3.1.4 Sand and dust storms11 The dust and sand storms (DSS) is a phenomenon of wind carrying dust, which originate in the arid and semi-arid regions of northern China and Mongolia, and arrive at Japan and Korea across national boundaries in the spring due to the region’s prevailing seasonal winds. DSS has recently been worsening in terms of their frequency and intensity because of China’s rapid desertification, soil degradation, and forest reduction. It causes problems in human health (e.g., sore eyes and respiratory infections), agricultural products, dust- sensitive industries (such as semi-conductor manufacturing), and transportation. Th T i i E i l Mi i M i (TEMM) Chi J d K ( g) p The Tripartite Environmental Ministers Meeting (TEMM) among China, Japan, and Korea, in its forth meeting in 2002, sharing their concern about the DSS problem, agreed to strengthen monitoring capacity to combat sandstorms, and stressed the importance of 80 Environmental Management in Practice extensive engagement of national environmental administrations in the region and international organizations in the efforts to cope with the DSS challenges. In 2003, the Global Environment Facility (GEF) launched the joint projects including those of improving monitoring and developing early warning network systems for DSS, in collaboration with the UNEP, the ADB, the UNESCAP, the United Nation Convention to Combat Desertification (UNCCD), and four countries (China, Japan, Korea and Mongolia). The NEASPEC is also promoting a demonstration project focusing on the prevention of dust and sandstorms at source areas in China and Mongolia. 3.2 Modalities of regional frameworks in East Asia Subsequently, the Convention for the Long-Range Transmission of Air Pollutant in Europe (the LRTAP) was agreed upon in 1979, and the protocols on 30% reduction of sulfur emission and on NOx emission control were adopted in the 1980s. For trans-boundary marine pollution, for instance, the Convention for the Protection of the Mediterranean Sea Against Pollution (the Barcelona Convention) was concluded in 1975, followed by the protocols on marine dumping, emergency oil pollution, land based pollution source, and so forth in 1975 and 1980. These conventions and protocols are definitely classified into d)-type in the classification above, whereas East Asian frameworks 81 Regional Issues in Environmental Management for trans-boundary acid deposition and marine pollution mainly fall into a), b), or c)-type of modalities. for trans-boundary acid deposition and marine pollution mainly fall into a), b), or c)-type of modalities. Acid Deposition Monitoring Network in East Asia (EANET) Staring year: 2001 Area: East Asia: 13 countries Issues: Acid Deposition Secretariat: UNEP Modality: Joint monitoring and assessment Northwest Pacific Action Plan (NOWPAP) Staring year: 1994 Area: Northeast Asia: China, Japan, Korea (South), and Russia Issues: Marine Pollution Secretariat: UNEP Modality: Joint monitoring and assessment ASEAN Agreement on Transboundary Haze Pollution Staring year: 2002 Area: ASEAN Issues: Haze Pollution Secretariat: ASEAN Modality: Legal agreement Tripartite Environmental Ministers Meeting (TEMM) Staring year: 1999 Area: China, Japan, and South Korea Issues: Comprehensive Secretariat: Rotation Modality: Policy dialogue North-east Asian Subregional Programme of Environmental Cooperation (NEASPEC) Staring year: 1993 Area: Northeast Asia: China, Japan, Korea (North), Korea (South), Mongolia, and Russia Issues: Comprehensive Secretariat: UN/ESCAP (Interim) Modality: Project-based activities Table 2. Regional frameworks for environmental cooperation 3.3 Discussion on non-binding approach in East Asia The typical contrast can be shown in the LRTAP Convention, which was created by homogenous advanced European nations and has well been maintained by strong links to EU policies and aid programs. Second, the environmental cooperation in East Asian region is too immature to lead to legal agreements. It was only after the Rio Earth Summit in 1992 that East Asian countries initiated environmental cooperation as an official diplomatic issue as shown in Table 2. We can also see a contrast in monitoring trans-boundary acid deposition: East Asian started its system in 2001 as the EANET, while Europe inaugurated it about thirty years earlier, in 1972. Finally, more importantly, political sentiments among East Asian nations are placing obstacles on the road toward binding agreements (see Yoon; 2007). The historical experiences of World War Two are making East Asian nations suspicious of Japanese initiatives on regional affairs. And China tends to prefer bilateral cooperation to supranational institutions, because bilateral negotiations do not place the country in the diplomatically unfavourable situation of being the main source of regional, trans-boundary pollution. The bilateral environmental cooperation promoted by Japan through official development assistant (ODA) may also have attenuated the need for binding agreements at multilateral level. To sum up, considering the region-specific properties in economical, political, and historical terms, non-binding approach as regional framework of environmental cooperation may be l h f E A h h d h “ ” “f ” d political reasons, and enables governments to formulate their commitments in a more precise and ambitious form than they would be possible in a binding treaty. Seeking deeper cooperation like a smaller club of “like-minded enthusiasts”, and facilitating learning processes or learning by doing, can be further benefits of nonbinding agreements. On the other hand, binding agreements strengthen the credibility of a commitment, increase compliance with the commitment, and reduce intergovernmental transaction costs. p , g Considering this theoretical viewpoint, we can interpret East Asian choice of non-binding approach in such a way that East Asia is getting or trying to get the non-binding advantages whereas facing the difficulties for getting the binding advantages. In fact, the progress in the trans-boundary on-going projects under the frameworks of EANET, NOWPAP, NEASPEC, etc., appears to be reflecting East Asian stances to pursue the “easier”, “faster” and “deeper” advantages of non-binding approach. Arellano, M. & Bond, S.R. (1991). Some tests of specification of panel data: Monte Carlo evidence and an application to employment equations. Review of Economic Studies, Vol.58, No.2, (April 1991), pp. 277–297, ISSN: 00346527 Borghesi, S. (1999). The Environmental Kuznets Curve: a survey of the literature. FEEM (Fondazione ENi Enrico Mattei) Working Paper, No. 85–99 3.3 Discussion on non-binding approach in East Asia On the other hand, the difficulties for binding approach in East Asia seem to come from the following economical, political and historical backgrounds. First, a lack of economic and political homogeneity is making it difficult for East Asia to reach binding agreements. As mentioned in Introduction, East Asian countries are composed of a variety of countries with different stages of development and with different political system. In addition, there is no regional organizations equivalent to the EU in East Asia except for ASEAN. The typical contrast can be shown in the LRTAP Convention, which was created by homogenous advanced European nations and has well been maintained by strong links to EU policies and aid programs. Second, the environmental cooperation in East Asian region is too immature to lead to legal agreements. It was only after the Rio Earth Summit in 1992 that East Asian countries initiated environmental cooperation as an official diplomatic issue as shown in Table 2. We can also see a contrast in monitoring trans-boundary acid deposition: East Asian started its system in 2001 as the EANET, while Europe inaugurated it about thirty years earlier, in 1972. Finally, more importantly, political sentiments among East Asian nations are placing obstacles on the road toward binding agreements (see Yoon; 2007). The historical experiences of World War Two are making East Asian nations suspicious of Japanese initiatives on regional affairs. And China tends to prefer bilateral cooperation to supranational institutions, because bilateral negotiations do not place the country in the diplomatically unfavourable situation of being the main source of regional, trans-boundary pollution. The bilateral environmental cooperation promoted by Japan through official development assistant (ODA) may also have attenuated the need for binding agreements at multilateral level. To sum up, considering the region-specific properties in economical, political, and historical terms, non-binding approach as regional framework of environmental cooperation may be To sum up, considering the region-specific properties in economical, political, and historical terms, non-binding approach as regional framework of environmental cooperation may be an optimal choice for East Asia, in the sense that it provides the “easier”, “faster” and “deeper” framework regardless of economical, political, and historical constraints. 3.3 Discussion on non-binding approach in East Asia The modality of regional frameworks for environmental cooperation has recently been discussed in terms of binding and non-binding approaches (e.g. Yoon; 2007, Köppel; 2009). Yoon (2007) argued that the environmental cooperation in Northeast Asia has evolved through non-binding agreements which do not contain official commitments on compliance or legal restrictions for non-compliance, whereas that in Europe has followed binding agreements by concluding with conventions and working through a series of protocols for solid compliance. This view is consistent with our comparative analysis on the modalities for environmental cooperation between in East Asia and Europe in the previous section. Then, why East Asia has taken the non-binding approach for environmental cooperation is the question in this section. Köppel (2009) explained theoretically the advantages of both binding and nonbinding agreements as follows. A nonbinding agreement is easier and faster to achieve, allows states to tackle a problem collectively at a time they otherwise might not due to economic or 82 Environmental Management in Practice political reasons, and enables governments to formulate their commitments in a more precise and ambitious form than they would be possible in a binding treaty. Seeking deeper cooperation like a smaller club of “like-minded enthusiasts”, and facilitating learning processes or learning by doing, can be further benefits of nonbinding agreements. On the other hand, binding agreements strengthen the credibility of a commitment, increase compliance with the commitment, and reduce intergovernmental transaction costs. Considering this theoretical viewpoint, we can interpret East Asian choice of non-binding approach in such a way that East Asia is getting or trying to get the non-binding advantages whereas facing the difficulties for getting the binding advantages. In fact, the progress in the trans-boundary on-going projects under the frameworks of EANET, NOWPAP, NEASPEC, etc., appears to be reflecting East Asian stances to pursue the “easier”, “faster” and “deeper” advantages of non-binding approach. On the other hand, the difficulties for binding approach in East Asia seem to come from the following economical, political and historical backgrounds. First, a lack of economic and political homogeneity is making it difficult for East Asia to reach binding agreements. As mentioned in Introduction, East Asian countries are composed of a variety of countries with different stages of development and with different political system. In addition, there is no regional organizations equivalent to the EU in East Asia except for ASEAN. 5. References 83 Regional Issues in Environmental Management Dasgupta, S.; Laplante, B.; Wang, H. & Wheeler, D. (2002). Confronting the Environmental Kuznets Curve. Journal of Economic Perspectives, Vol.16, No.1, (Winter 2002), pp. 147- 168, ISSN 08953309 De Bruyn, S.M.; Van den Bergh, J.C.J.M. & Opschoor, J.B. (1998). Economic Growth and Emissions: Reconsidering the Empirical Basis of 166 Journal of Economic Perspectives Environmental Kuznets Curves. Ecological Economics, Vol.25, No.2, (May 1998), pp. 161-175, ISSN 09218009 Grossman, G. & Krueger, A. (1993). Environmental Impacts of the North American Free Trade Agreement, In: The U.S.-Mexico Free Trade Agreement, P. Garber, (Ed.), 13-56, MIT Press, ISBN 0-262-07152-5, Cambridge g Grossman, G. & Krueger, A. (1995). Economic Growth and the Environment. Quarterly Journal of Economics, Vol.112, No.2, (May 1995), pp. 353–377, ISSN 00335533 Halkos, G.E. (2003). Environmental Kuznets Curve for sulfur: evidence using GMM estimation and random coefficient panel data models. Environment and Development Economics, Vol.8, No.4, (October 2003), pp. 581-601, ISSN 1355770X Institute for Global Environmental Strategies (IGES) (2001). Regional/Subr Environmental Cooperation in Asia, IGES, Japan Environmental Cooperation in Asia, IGES, Japan Kawai, M. (2009). International exchange and monetary system in East Asia. Financial Review, Vol 93, No 1, (March 2009), pp 176-194, ISBN 978-4-9904174-4-4 (Japanese) wai, M. (2009). International exchange and monetary system in East Asia. Financia Review, Vol.93, No.1, (March 2009), pp. 176-194, ISBN 978-4-9904174-4-4 (Japanese) Köppel, M. (2009). Explaining the Effectiveness of Binding and Nonbinding Agreements: Tentative Lessons from Transboundary Water Pollution. Paper prepared for the 2009 Amsterdam Conference on the Human Dimensions of Global Environmental Change, 2-4 Dec 2009, Available from http://www.earthsystemgovernance.org/ac2009/papers/AC2009-0283.pdf Panayotou, T. (1997). Demystifying the Environmental Kuznets Curve: Turning a Black Box into a Policy Tool. Environment and Development Economics, Vol.2, No.4, (October 1997), pp. 465-484, ISSN 1355770X Nahman, A. & Antrobus, G. (2005). The Environmental Kuznets Curve: A Literature Survey. South African Journal of Economics, Vol.73, No.1, (March 2005), pp.105–120, ISSN 00382280 Selden, T.M. & Song, D. (1994). Environmental Quality and Development: Is There a Kuznets Curve for Air Pollution Emissions? Journal of Environmental Economics and Management, Vol. 27, No.2, (September 1994), pp. 147-162, ISSN 00950696 Shafik, N. (1994). Economic Development and Environmental Quality: An Econometric Analysis. Oxford Economic Papers, Vol.46, (Supplement, October 1994), pp. 757-773, ISSN 00307653 Takahashi, W. (2003). Historical Development of Regional Cooperative Framworks on Environment of Europe. Paper Series of Utsunomiya University, No.17, (March 2003), pp.13-31, (Japanese) Taguchi, H. & Murofushi, H. (2009). Yoon, E. (2007). Cooperation for Transboundary Pollution in Northeast Asia: Non-binding Agreements and Regional Countries’ Policy Interests. Pacific Focus, Vol. XXII, No. 2 (Fall 2007), pp. 77-112, ISSN 1976-5118 Environment and Development Economics, Vol.12, No.3, (June 2007), pp. 445-470, ISSN 1355770X Environmental Management in Practice 5. References Environmental Latecomer’s Effects in Developing Countries – The Case of SO2 and CO2 Emissions. Journal of Developing Areas, Vol.44, No.2, (Spring 2011), pp. 143-164, ISSN 0022-037X , ( p g ), pp , UNEP (2010). Air Pollution : promoting regional cooperation, UNEP, ISBN 978-92-807-309 UNEP (2010). Air Pollution : promoting regional cooperation, UNEP, ISBN 978-92-807-3093-7 Yaguchi, Y. ; Sonobe, T. & Otsuka, K. (2007). Beyond the Environmental Kuznets Curve: A Comparative Study of SO2 and CO2 Emissions Between Japan and China. Yaguchi, Y. ; Sonobe, T. & Otsuka, K. (2007). Beyond the Environmental Kuznets Curve: A Comparative Study of SO2 and CO2 Emissions Between Japan and China. 84 Environmental Management in Practice 5 1. Introduction The responses of public authorities to natural or induced geological hazards, such as land instability and flooding, vary according to different factors including frequency of occurrence, severity of damage, magnitude of hazardous processes, awareness, predictability, political willingness and availability of financial and technological resources. The responses will also depend upon whether the hazard is 1) known to be already present thus giving rise to risk situations involving people and/or economic loss; or 2) there is a latent or potential hazard that is not yet present so that development and land uses need to be controlled in order to avoid creating risk situations. In this regard, geo-environmental management can take the form of either planning responses and mid- to long-term public policy based territorial zoning tools, or immediate interventions that may involve a number of approaches including preventative and mitigation works, civil defence actions such as hazard warnings, community preparedness, and implementation of contingency and emergency programmes. In most of cases, regional- and local-scale terrain assessments and classification accompanied by susceptibility and/or hazard maps delineating potential problem areas will be used as practical instruments in efforts to tackle problems and their consequences. In terms of planning, such assessments usually provide advice about the types of development that would be acceptable in certain areas but should be precluded in others. Standards for new construction and the upgrading of existing buildings may also be implemented through legally enforceable building codes based on the risks associated with the particular terrain assessment or classification. The response of public authorities also varies depending upon the information available to make decisions. In some areas sufficient geological information and knowledge about the causes of a hazard may be available to enable an area likely to be susceptible to hazardous processes to be predicted with reasonable certainty. In other places a lack of suitable data may result in considerable uncertainty. 86 Environmental Management in Practice In this chapter, a number of case studies are presented to demonstrate the methodological as well as the predictive and preventative aspects of geo-environmental management, with a particular view to regional- and semi-detailed scale, satellite image based terrain classification. If available, information on the geology, geomorphology, covering material characteristics and land uses may be used with remotely sensed data to enhance these terrain classification outputs. 1. Introduction In addition, examples provided in this chapter demonstrate the identification and delineation of zones or terrain units in terms of the likelihood and consequences of land instability and flooding hazards in different situations. Further applications of these methods include the ranking of abandoned and/or derelict mined sites and other despoiled areas in support of land reclamation and socio-economic regeneration policies. p The discussion extends into policy formulation, implementation of environmental management strategies and enforcement regulations. 2. Use of remote densing tools for terrain assessments and territorial zoning Engineering and geo-environmental terrain assessments began to play an important role in the planning process as a consequence of changing demands for larger urban areas and related infra-structure, especially housing, industrial development and the services network. In this regard, the inadequacy of conventional agriculturally-orientated land mapping methods prompted the development of terrain classification systems completely based on the properties and characteristics of the land that provide data useful to engineers and urban planners. Such schemes were then adopted and widely used to provide territorial zoning for general and specific purposes. The process of dividing a country or region into area parcels or zones, is generally called land or terrain classification. Such a scheme is illustrated in Table 1. The zones should possess a certain homogeneity of characteristics, properties, and in some cases, conditions and expected behaviour in response to human activities. What is meant by homogeneous will depend on the purpose of the exercise, but generally each zone will contain a mixture of environmental elements such as rocks, soils, relief, vegetation, and other features. The feasibility and practicability of delineating land areas with similar attributes have been demonstrated throughout the world over a long period of time (e.g. Bowman, 1911; Bourne, 1931; Christian, 1958; Mabbutt, 1968; amongst others), and encompass a wide range of specialisms such as earth, biological and agricultural sciences; hydrology and water resources management; military activities; urban and rural planning; civil engineering; nature and wildlife conservation; and even archaeology. gy According to Cendrero et al. (1979) and Bennett and Doyle (1997), there are two main approaches to geo-environmental terrain assessments and territorial zoning, as follows. 1) The analytical or parametric approach deals with environmental features or components individually. The terrain units usually result from the intersection or cartographic summation of several layers of information [thus expressing the probability limits of findings] and their extent may not corresponding directly with ground features. Examples of the parametric approach for urban planning, hazard mapping and engineering purposes are given by Kiefer (1967), Porcher & Guillope (1979), Alonso Herrero et al. (1990), and Dai et al. (2001). 2. Use of remote densing tools for terrain assessments and territorial zoning 2) In the synthetic approach, also termed integrated, landscape or physiographic approach, the form and spatial distribution of ground features are analysed in an integrated manner relating recurrent landscape patterns expressed by an interaction of Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 87 Terrain unit Definition Soil unit Vegetation unit Mapping scale (approx.) Remote sensing platform Land zone Major climatic region Order - < 1:50,000,000 Land division Gross continental structure Suborder Plant panformation Ecological zone 1:20,000,000 to 1:50,000,000 Meteorological satellites Land province Second-order structure or large lithological association Great group - 1:20,000,000 to 1:50,000,000 Land region Lithological unit or association having undergone comparable geomorphic evolution Subgroup Sub-province 1:1,000,000 to 1:5,000,000 Landsat SPOT ERS Land system * Recurrent pattern of genetically linked land facets Family Ecological region 1: 200,000 to 1:1,000,000 Landsat SPOT, ERS, and small scale aerial photographs Land catena Major repetitive component of a land system Association Ecological sector 1:80,000 to 1:200,000 Land facet Reasonably homogeneous tract of landscape distinct from surrounding areas and containing a practical grouping of land elements Series Sub- formation; Ecological station 1:10,000 to 1: 80,000 Medium scale aerial photographs, Landsat, and SPOT in some cases Land clump A patterned repetition of two or more land elements too contrasting to be a land facet Complex Sub- formation; Ecological station 1:10,000 to 1: 80,000 Land subfacet Constituent part of a land facet where the main formative processes give material or form subdivisions Type - Not mapped Large-scale aerial photographs Land element Simplest homogeneous part of the landscape, indivisible in form Pedon Ecological station element Table 1. Hierarchical classification of terrain, soil and ecological units [after Mitchell, 1991] Table 1. Hierarchical classification of terrain, soil and ecological units [after Mitchell, 1991] environmental components thus allowing the partitioning of the land into units. Since the advent of airborne and orbital sensors, the integrated analysis is based in the first instance, on the interpretation of remotely sensed images and/or aerial photography. In most cases, the content and spatial boundaries of terrain units would directly correspond with ground features. Assumptions that units possessing similar recurrent landscape patterns may be expected to be similar in character are required for valid predictions to be made by extrapolation from known areas. 2. Use of remote densing tools for terrain assessments and territorial zoning Thus, terrain classification schemes offer rational means of correlating known and unknown areas so that the ground conditions and potential uses environmental components thus allowing the partitioning of the land into units. Since the advent of airborne and orbital sensors, the integrated analysis is based in the first instance, on the interpretation of remotely sensed images and/or aerial photography. In most cases, the content and spatial boundaries of terrain units would directly correspond with ground features. Assumptions that units possessing similar recurrent landscape patterns may be expected to be similar in character are required for valid predictions to be made by extrapolation from known areas. Thus, terrain classification schemes offer rational means of correlating known and unknown areas so that the ground conditions and potential uses environmental components thus allowing the partitioning of the land into units. Since the advent of airborne and orbital sensors, the integrated analysis is based in the first instance, on the interpretation of remotely sensed images and/or aerial photography. In most cases, the content and spatial boundaries of terrain units would directly correspond with ground features. Assumptions that units possessing similar recurrent landscape patterns may be expected to be similar in character are required for valid predictions to be made by extrapolation from known areas. Thus, terrain classification schemes offer rational means of correlating known and unknown areas so that the ground conditions and potential uses 88 Environmental Management in Practice of unknown areas can be reasonably predicted (Finlayson, 1984; Bell, 1993). Examples of the applications of the landscape or physiographic approach include ones given by Christian & Stewart (1952, 1968), Vinogradov et al. (1962), Beckett & Webster (1969); Meijerink (1988), and Miliaresis (2001). of unknown areas can be reasonably predicted (Finlayson, 1984; Bell, 1993). Examples of the applications of the landscape or physiographic approach include ones given by Christian & Stewart (1952, 1968), Vinogradov et al. (1962), Beckett & Webster (1969); Meijerink (1988), and Miliaresis (2001). Griffiths and Edwards (2001) refer to Land Surface Evaluation as a procedure of providing data relevant to the assessment of the sites of proposed engineering work. The sources of data include remotely sensed data and data acquired by the mapping of geomorphological features. 2. Use of remote densing tools for terrain assessments and territorial zoning Although originally viewed as a process usually undertaken at the reconnaissance or feasibility stages of projects, the authors point out its utility at the constructional and post-construction stages of certain projects and also that it is commonly applied during the planning of engineering development. They also explain that although more reliance on this methodology for deriving the conceptual or predictive ground model on which engineering design and construction are based, was anticipated in the early 1980s, in fact the use of the methods has been more limited. Geo-environmental terrain assessments and territorial zoning generally involve three main stages (IG/SMA 2003; Fernandes da Silva et al. 2005b, 2010): 1) delimitation of terrain units; 2) characterisation of units (e.g. in bio-geographical, engineering geological or geotechnical terms); and 3) evaluation and classification of units. The delimitation stage consists of dividing the territory into zones according to a set of pre-determined physical and environmental characteristics and properties. Regions, zones or units are regarded as distinguishable entities depending upon their internal homogeneity or the internal interrelationships of their parts. The characterisation stage consists of attributing appropriate properties and characteristics to terrain components. Such properties and characterisitics are designed to reflect the ground conditions relevant to the particular application. The characterisation of the units can be achieved either directly or indirectly, for instance by means of: (a) ground observations and measurements, including in-situ tests (e.g. boring, sampling, infiltration tests etc); (b) laboratory tests (e.g. grain size, strength, porosity, permeability etc); (c) inferences derived from existing correlations between relevant parameters and other data such as those obtained from previous mapping, remote sensing, geophysical surveys and geochemical records. The final stage (evaluation and classification) consists of evaluating and classifying the terrain units in a manner relevant to the purposes of the particular application (e.g. regional planning, transportation, hazard mapping). This is based on the analysis and interpretation of properties and characteristics of terrain - identified as relevant - and their potential effects in terms of ground behaviour, particularly in response to human activities. p y p A key issue to be considered is sourcing suitable data on which to base the characterisation, as in many cases derivation by standard mapping techniques may not be feasible. The large size of areas and lack of accessibility, in particular, may pose major technical, operational, and economic constraints. 2. Use of remote densing tools for terrain assessments and territorial zoning Furthermore, as indicated by Nedovic-Budic (2000), data collection and integration into useful databases are liable to be costly and time-consuming operations. Such problems are particularly prevalent in developing countries in which suitably trained staff, and scarce organizational resources can inhibit public authorities from properly benefiting from geo-environmental terrain assessment outputs in planning and environmental management instruments. In this regard, consideration has been given to increased reliance on remote sensing tools, particularly satellite imagery. The advantages include: (a) the generation of new data in areas where existing data are sparse, discontinuous or non-existent, and (b) the economical coverage of large areas, availability of a variety of spatial resolutions, relatively frequent and periodic updating of images Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 89 (Lillesand and Kiefer 2000; Latifovic et al. 2005; Akiwumi and Butler 2008). It has also been proposed that developing countries should ensure that options for using low-cost technology, methods and products that fit their specific needs and capabilities are properly considered (Barton et al. 2002, Câmara and Fonseca 2007). Some examples are provided here to demonstrate the feasibility of a low-cost technique based on the analysis of texture of satellite imagery that can be used for delimitation of terrain units. The delimited units may be further analysed for different purposes such as regional and urban planning, hazard mapping, and land reclamation. The physiographic compartmentalisation technique (Vedovello 1993, 2000) utilises the spatial information contained in images and the principles of convergence of evidence (see Sabins 1987) in a systematic deductive process of image interpretation. The technique evolved from engineering applications of the synthetic land classification approach (e.g. Grant, 1968, 1974, 1975; TRRL 1978), by incorporating and advancing the logic and procedures of geological-geomorphological photo-interpretation (see Guy 1966, Howard 1967, Soares and Fiori 1976), which were then converted to monoscopic imagery (as elucidated by Beaumont and Beaven 1977; Verstappen 1977; Soares et al. 1981; Beaumont, 1985; and others). Image interpretation is performed by identifying and delineating textural zones on images according to properties that take into account coarseness, roughness, direction and regularity of texture elements (Table 2). 2. Use of remote densing tools for terrain assessments and territorial zoning The key assumption proposed by Vedovello (1993, 2000) is that zones with relatively homogeneous textural characteristics in satellite images (or air-photos) correspond with specific combinations of geo-environmental components (such as bedrock, topography and landforms, soils and covering materials) which share a common tectonic history and land surface evolution. The particular combinations of geo-environmental components are expected to be associated with specific ground responses to engineering and other land-use actions. The process of image interpretation (whether or not supported by additional information) leads to a cartographic product in which textural zones constitute comprehensive terrain units delimited by fixed spatial boundaries. The latter correspond with ground features. The units are referred to as physiographic compartments or basic compartmentalisation units (BCUs), which are the smallest units for analysis of geo-environmental components at the chosen cartographic scale (Vedovello and Mattos 1998). The spatial resolution of the satellite image or air-photos being used for the analysis and interpretation is assumed to govern the correlation between image texture and terrain characteristics. This correlation is expressed at different scales and levels of compartmentalisation. Figure 1 presents an example of the identification of basic compartmentalisation units (BCUs) based on textural differences on Landsat TM5 images. In this case the features on images are expressions of differences in the distribution and spatial organisation of textural elements related to drainage network and relief. The example shows the contrast between drainage networks of areas consisting of crystalline rocks with those formed on areas of sedimentary rocks, and the resulting BCUs. 3. Terrain susceptibility maps: applications to regional and urban planning Terrain susceptibility maps are designed to depict ground characteristics (e.g. slope steepness, landforms) and observed and potential geodynamic phenomena, such as erosion, instability and flooding, which may entail hazard and potential damage. These maps are useful for a number of applications including development and land use planning, environmental protection, watershed management as well as in initial stages of hazard mapping applications. 90 Environmental Management in Practice Textural entities and properties Description Image texture element The smallest continuous and uniform surface liable to be distinguishable in terms of shape and dimensions, and likely to be repetitive throughout an image. Usual types of image texture elements taken for analysis include: segments of drainage or relief (e.g. crestlines, slope breaks) and grey tones. Texture density The quantity of textural elements occurring within an area on image. Texture density is defined as the inverse of the mean distance between texture elements. Although it reflects a quantitative property, textural density is frequently described in qualitative and relative terms such as high, moderate, low etc. Size of texture elements combined with texture density determine features such as coarseness and roughness. Textural arrangement The form (ordered or not) by which textural elements occur and are spatially distributed on an image. Texture elements of similar characteristics may be contiguous thus defining alignments or linear features on the image. The spatial distribution may be repetitive and it is usually expressed by ‘patterns’ that tend to be recurrent (regularity). For example, forms defined by texture elements due to drainage expressed in rectangular, dendritic, or radial patterns. Structuring (Degree of spatial organisation) The greater or lesser organisation underlying the spatial distribution of textural elements and defined by repetition of texture elements within a certain rule of placement. Such organisation is usually expressed in terms of regular or systematic spatial relations, such as length, angularity, asymmetry, and especially prevailing orientations (tropy or directionality). Tropy reflects the anisotropic (existence of one, two, or three preferred directions), or the isotropic (multi-directional or no predominant direction) character of textural features. Asymmetry refers to length and angularity of linear features (rows of contiguous texture elements) in relation to a main feature identified on image. The degree of organisation can also be expressed by qualitative terms such as high, moderate, low, or yet as well- or poorly-defined. Structuring order Complexity in the organisation of textural elements, mainly reflecting superposition of image structuring. 3. Terrain susceptibility maps: applications to regional and urban planning For example, a regional directional trend of textural elements that can be extremely pervasive, distinctive and superimposed on other orientations also observed on imagery. Another example is drainage networks that display different orders with respect to main stream lines and tributaries (1st, 2nd, 3rd orders) Table 2. Description of elements and properties used for recognition and delineation of distinctive textural zones on satellite imagery [after Vedovello 1993, 2000]. Table 2. Description of elements and properties used for recognition and delineation o distinctive textural zones on satellite imagery [after Vedovello 1993, 2000]. Early multipurpose and comprehensive terrain susceptibility maps include examples by Dearman & Matula, (1977), Matula (1979), and Matula & Letko (1980). These authors described the application of engineering geology zoning methods to the urban planning process in the former Republic of Czechoslovakia. The studies in this and other countries focused on engineering geology problems related to geomorphology and geodynamic processes, seismicity, hydrogeology, and foundation conditions. Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 91 Culshaw and Price (2011) point out that in the UK, a major initiative on urban geology began in the mid-1970s with obtaining geological information relevant to aggregates and other industrial minerals together with investigations relating to the planning of the proposed 3rd London Airport. In the latter case, a very wide range of map types was produced, including one that could be viewed in 3D, using green and red anaglyph spectacles. Of particular interest was the summary ‘‘Engineering Planning Map which showed areas that were generally suitable for different types of construction and, also, detailed suggested site investigation procedures (Culshaw and Northmore 2002). gg g p ( ) As Griffiths and Hearn (2001) explain, subsequently about 50 experimental ‘environmental geological mapping, ‘thematic’geological mapping’ and ‘applied geological mapping’ projects were carried out between 1980 and 1996. Culshaw and Price (2011) explain that this was to investigate the best means of collecting, collating, interpreting and presenting geological data that would be of direct applicability in land-use planning (Brook and Marker 1987). Maps of a variety of geological and terrain types, including industrially despoiled and potentially unstable areas, with mapping at scales between 1:2500 and 1:25000 were produced. The derivation and potential applications of these sets of maps and reports are described by Culshaw et al. Environmental Geology in Land Use Planning: Guide to the sources of earth scienc information for planning and development (Ellsion and Smith, 1998) 3. Terrain susceptibility maps: applications to regional and urban planning (1990) who explain that they include basic data maps, derived maps and environmental potential maps. Typically such thematic map reports comprise a series of maps showing the bedrock and superficial geology, thickness of superficial deposits, groundwater conditions and areas of mining, fill, compressible, or other forms of potentially unstable ground. Maps showing factual information include the positions of boreholes or the positions of known mine workings. Derived maps include areas in which geological and / or environmental information has been deduced, and therefore is subject to some uncertainty. The thematic sets include planning advice maps showing the constraints on, and potential for, development and mineral extraction. Culshaw et al. (1990) also explained that these thematic maps were intended to assist with the formulation of both local (town or city), regional (metropolis or county) structure plans and policies, provide a context for the consideration of development proposals and facilitate access to relevant geological data by engineers and geologists. It was also recognised that these is a need for national (or state) policies and planning to be properly informed about geological conditions, not least to provide a sound basis for planning legislation and the issuing of advice and circulars. Examples of such advice include planning guidance notes concerning the granting of planning permission for development on potentially unstable land which were published (DOE, 1990, 1995) by the UK government. A further series of reports which were intended to assist planners and promote the consideration of geological information in land-use planning decision making were compiled between 1994 and 1998 by consultants on behalf of the UK government. Griffiths (2001) provides details of a selection of land evaluation techniques and relevant case studies. These covered the following themes:  Environmental Geology in Land Use Planning: Advice for planners and developers (Thompson et al., 1998a)  Environmental Geology in Land Use Planning: A guide to good practice (Thompson et al., 1998b)  Environmental Geology in Land Use Planning: Emerging issues (Thompson et al., 1998c)  Environmental Geology in Land Use Planning: Guide to the sources of earth science information for planning and development (Ellsion and Smith, 1998) 92 Environmental Management in Practice For an extensive review of world-wide examples of geological data outputs intended to assist with urban geology interpretation, land-use planning and utilisation and geological hazard avoidance, reference should be made to Culshaw and Price (2001). 3.1 Multipurpose planning p p p g The first example concerns the production of a geohazard prevention map for the City of São Sebastião (IG/SMA 1996), where urban and industrial expansion in the mountainous coastal zone of São Paulo State, Southeast Brazil (Figure 2) led to conflicts in land use as well as to high risks to life and property. Particular land use conflicts arose from the combinations of landscape and economic characteristics of the region, in which a large nature and wildlife park co-exists with popular tourist and leisure encroached bays and beaches, a busy harbour with major oil storage facilities and associated pipelines that cross the area. Physiographic compartmentalisation was utilised to provide a regional terrain classification of the area, and then interpretations were applied in two ways: (i) to provide a territorial zoning based on terrain susceptibility in order to enable mid- to long-term land use planning; and (ii) to identify areas for semi-detailed hazard mapping and risk assessment (Fernandes da Silva et al. 1997a, Vedovello et al., 1997; Cripps et al., 2002). Figure 2 presents the main stages of the study undertaken in response to regional and urban planning needs of local authorities. In the Land Susceptibility Map, the units were qualitatively ranked in terms of ground evidence and estimated susceptibility to geodynamic processes including gravitational mass movements, erosion, and flooding. Criteria for terrain unit classification in relation to erosion and mass movements (landslides, creep, slab failure, rock fall, block tilt and glide, mud and debris flow) were the following: a) soil weathering profile (thickness, textural and mineral constituency); b) hillslope profile; c) slope steepness; and d) bedrock structures (fracturing and discontinuities in general). Criteria in relation to flooding included: a) type of sediments; b) slope steepness; and c) hydrography (density and morphology of water courses). The resulting classes of terrain susceptibility can be summarised as follows: Low susceptibility: Areas where mass movements are unlikely. Low restrictions to excavations and man-made cuttings. Some units may not be suitable for deep foundations or other engineering works due to possible high soil compressibility and presence of geological structures. In flat areas, such as coastal plains, flooding and river erosion are unlikely. Moderate susceptibility: Areas of moderate to high steep slope (10 to 30%) with little evidence of land instability (small-scale erosional processes may be present) but with potential for occurrence of mass movements. 3. Terrain susceptibility maps: applications to regional and urban planning For an extensive review of world-wide examples of geological data outputs intended to assist with urban geology interpretation, land-use planning and utilisation and geological hazard avoidance, reference should be made to Culshaw and Price (2001). Three examples of terrain susceptibility mapping are briefly described and presented in this Section. The physiographic compartmentalisation technique for regional terrain evaluation was explored in these cases, and then terrain units were further characterised in geo- environmental terms. Fig. 1. Identification of basic compartmentalisation units (BCUs) based on textural differences on image. The image for crystalline rocks with rugged topography contrasts with sedimentary rocks with rolling topography. Top: Drainage network. Mid Row: Drainage network and delineated BCUs. Bottom: Composite Landsat TM5 image and delineated BCUs [after Fernandes da Silva et al. 2005b, 2010] Crystalline rocks + rugged topography Sedimentary rocks + rolling topography Crystalline rocks + rugged topography Crystalline rocks + rugged topography Sedimentary rocks + rolling topography Sedimentary rocks + rolling topography Fig. 1. Identification of basic compartmentalisation units (BCUs) based on textural differences on image. The image for crystalline rocks with rugged topography contrasts with sedimentary rocks with rolling topography. Top: Drainage network. Mid Row: Drainage network and delineated BCUs. Bottom: Composite Landsat TM5 image and delineated BCUs [after Fernandes da Silva et al. 2005b, 2010] Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 93 3.1 Multipurpose planning In lowland areas, reported flooding events were associated with the main drainage stream in relevant zones. Terrain units would possess moderate restrictions for land-use with minor engineering solutions and protection measures needed to reduce or avoid potential risks. High susceptibility: Areas of moderate (10 to 20%) and high steep slope (20 to 30%) situated in escarpment and footslope sectors, respectively, with evidence of one or more active land instability phenomena (e.g. erosion + rock falls + landslide) of moderate magnitude. Unfavourable zones for construction work wherein engineering projects would require accurate studies of structural stability, and consequently higher costs. In lowland sectors, recurrent flooding events were reported at intervals of 5 to 10 yrs, associated with main drainage streams and tributaries. Most zones then in use required immediate remedial action including major engineering solutions and protection measures. 94 Environmental Management in Practice Very high susceptibility: Areas of steeper slopes (> 30%) situated in the escarpment and footslope sectors that mainly comprised colluvium and talus deposits. There was evidence of one or more land instability phenomena of significant magnitude requiring full restriction on construction work. In lowland sectors, widespread and frequent flooding events at intervals of less than 5 years were reported and most land-used needed to be avoided in these zones. Very high susceptibility: Areas of steeper slopes (> 30%) situated in the escarpment and footslope sectors that mainly comprised colluvium and talus deposits. There was evidence of one or more land instability phenomena of significant magnitude requiring full restriction on construction work. In lowland sectors, widespread and frequent flooding events at intervals of less than 5 years were reported and most land-used needed to be avoided in these zones. Units or areas identified as having a moderate to high susceptibility to geodynamic phenomena, and potential conflicts in land use, were selected for detailed engineering geological mapping in a subsequent stage of the study. The outcomes of the further stage of hazard mapping are described and discussed in Section 4. Geological Information Geo morphological a nd Soil Information REGIONAL PHYSIOGRAPHIC CO MPARTMENTALISATION RS imagery  MAP LAND SUSCEPTIBILITY CLASS MAP REGIONAL RAINFALL EVALUATION 1:50.000 1:50.000 TIMESPACE ANALYSIS INVENTO RY 1:50.000 1:10.000 1:10.000 DETAILED SCALE GEOTECHNICAL CARTOGRAPHY REGIONAL EVALUATION LANDSLIDES MASS MOVEMENTS SELECTED AREAS LAND USE MAP LANDSLIDE EO CCURRENCE INVENTORY MINERAL EXPLOITATION INVENTORY HAZARD M APPING 1:10.000 Remotely sensed data A) B) Fig. 2. 3.1 Multipurpose planning A) Location map for the City of São Sebastião, north shore of São Paulo State, Southeast Brazil. B) Schematic flow diagram for the derivation of the geohazard prevention chart and structural plan (after IG/SMA, 1996). Location Map at South America Brazil Geological Information Location Map at South America Brazil Location Map at South America Brazil A) B) Fig. 2. A) Location map for the City of São Sebastião, north shore of São Paulo State, Southeast Brazil. B) Schematic flow diagram for the derivation of the geohazard prevention chart and structural plan (after IG/SMA, 1996). 3.2 Watershed planning and waste disposal The physiographic compartmentalisation technique was also applied in combination with GIS tools in support of watershed planning in the Metropolitan District of Campinas, central-eastern São Paulo State (Figure 3). This regional screening study was performed at 1:50,000 scale to indicate fragilities, restrictions and potentialities of the area for siting waste disposal facilities (IG/SMA, 1999). A set of common characteristics and properties (also referred to as attributes) facilitated the assessment of each BCU (or terrain unit) in terms of Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 95 susceptibility to the occurrence of geodynamic phenomena (soil erosion and land instability) and the potential for soil and groundwater contamination. As described by Brollo et al. (2000), the terrain units were mostly derived on the basis of qualitative and semi-quantitative inferences from satellite and air-photo images in conjunction with existing information (maps and well logs – digital and papers records) and field checks. The set of attributes included: (1) bedrock lithology; (2) density of lineaments (surrogate expression of underlying fractures and terrain discontinuities); (3) angular relation between rock structures and hillslope; (4) geometry and shape of hillslope (plan view and profile); (5) soil and covering material: type, thickness, profile; (6) water table depth; and (7) estimated permeability. These attributes were cross-referenced with other specific factors, including hydrogeological (groundwater production, number of wells per unit area), climatic (rainfall, prevailing winds), and socio-political data (land use, environmental restrictions). These data were considered to be significant in terms of the selection of potential sites for waste disposal. susceptibility to the occurrence of geodynamic phenomena (soil erosion and land instability) and the potential for soil and groundwater contamination. As described by Brollo et al. (2000), the terrain units were mostly derived on the basis of qualitative and semi-quantitative inferences from satellite and air-photo images in conjunction with existing information (maps and well logs – digital and papers records) and field checks. The set of attributes included: (1) bedrock lithology; (2) density of lineaments (surrogate expression of underlying fractures and terrain discontinuities); (3) angular relation between rock structures and hillslope; (4) geometry and shape of hillslope (plan view and profile); (5) soil and covering material: type, thickness, profile; (6) water table depth; and (7) estimated permeability. 3.2 Watershed planning and waste disposal These attributes were cross-referenced with other specific factors, including hydrogeological (groundwater production, number of wells per unit area), climatic (rainfall, prevailing winds), and socio-political data (land use, environmental restrictions). These data were considered to be significant in terms of the selection of potential sites for waste disposal. Fig. 3. Location map of the Metropolitan District of Campinas (MDC), central-eastern São Paulo State, Southeast Brazil (see Section 3.2). Detail map depicts Test Areas T1 and T2 within the MDC (see Section 3.3). Scale bar applies to detail map. Figure 4 displays the study area in detail together with BCUs, and an example of a pop-u window (text box) containing key attribute information, as follows: 1st row - BCU cod (COC1), 2nd - bedrock lithology, 3rd - relief (landforms), 4th – textural soil profil constituency, 5th - soil thickness, 6th - water table depth (not show in the example), 7th bedrock structures in terms of density of fracturing and directionality), 8th - morphometr (degree of dissection of terrain). The BCU coding scheme expresses three levels o 0 18 36 km  0 18 36 54 km 0 18 36 km  0 18 36 54 km Fig. 3. Location map of the Metropolitan District of Campinas (MDC), central-eastern São Paulo State, Southeast Brazil (see Section 3.2). Detail map depicts Test Areas T1 and T2 within the MDC (see Section 3.3). Scale bar applies to detail map. Figure 4 displays the study area in detail together with BCUs, and an example of a pop-up window (text box) containing key attribute information, as follows: 1st row - BCU code (COC1), 2nd - bedrock lithology, 3rd - relief (landforms), 4th – textural soil profile constituency, 5th - soil thickness, 6th - water table depth (not show in the example), 7th - bedrock structures in terms of density of fracturing and directionality), 8th - morphometry (degree of dissection of terrain). The BCU coding scheme expresses three levels of 96 Environmental Management in Practice compartmentalisation, as follows: 1st letter – major physiographic or landscape domain, 2nd– predominant bedrock lithology, 3rd - predominant landforms, 4th– differential characteristics of the unit such as estimated soil profile and underlying structures. 3.2 Watershed planning and waste disposal Using the example given in Figure 4, COC1 means: C = crystalline rock basement, O = equigranular gneiss, C = undulating and rolling hills, 1 = estimated soil profile (3 textural horizons and thickness of 5 to 10 m), underlying structures (low to moderate degree of fracturing, multi- directional). In terms of general interpretations for the intended purposes of the study, certain ground characteristics, such as broad valleys filled with alluvial sediments potentially indicate the presence water table level at less than 5 m below ground surface. Flood plains or concave hillside slopes that may indicate convergent surface water flows leading to potentially high susceptibility to erosion, were considered as restrictive factors for the siting of waste disposal facilities (Vedovello et al. 1998). Fig. 4. Basic compartmentalisation units (BCUs) and pop-up window showing key attribute information relevant to BCUs. See text for details. [Not to scale] [after IG/SMA, 1999] Fig. 4. Basic compartmentalisation units (BCUs) and pop-up window showing key attribute information relevant to BCUs. See text for details. [Not to scale] [after IG/SMA, 1999] 3.3 Regional development planning The third example is a territorial zoning exercise, in which terrain units delimited through physiographic compartmentalisation were further assessed in terms of susceptibility to land instability processes and groundwater vulnerability (Fernandes da Silva et al. 2005b). The study was conducted in two test areas situated in the Metropolitan District of Campinas (Figure 3) in order to assist State of São Paulo authorities in the formulation of regional development policies. It incorporated procedures for inferring the presence and characteristics of underlying geological structures, such as fractures and other discontinuities, then evaluating potential implications to ground stability and the flow of groundwater. Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 97 Details of image interpretation procedures for the delimitation of BCUs are described by Fernandes da Silva et al. (2010). The main image properties and image feature characteristics considered were as follows: (a) density of texture elements related to drainage and relief lines; (b) spatial arrangement of drainage and relief lines in terms of form and degree of organisation (direction, regularity and pattern); (c) length of lines and their angular relationships, (d) linearity of mainstream channel and asymmetry of tributaries, (e) density of interfluves, (f) hillside length, and (g) slope forms. These factors were mostly derived by visual interpretation of images, but external ancillary data were also used to assist with the determination of relief-related characteristics, such as slope forms and interfluve dimensions. The example given in Figure 1 shows sub-set images (Landsat TM5) and the basic compartmentalisation units (BCUs) delineated for Test Areas T1 and T2. p ( ) Based on the principle that image texture correlates with properties and characteristics of the imaged target, deductions can be made about geotechnical-engineering aspects of the terrain (Beaumont and Beaven 1977, Beaumont 1985). The following attributes were firstly considered in the geo-environmental characterisation of BCUs: (a) bedrock lithology and respective weathered materials, (b) tectonic discontinuities (generically referred to as fracturing), (c) soil profile (thickness, texture and mineralogy), (d) slope steepness (as an expression of local topography), and (e) water table depth (estimated). Terrain attributes such as degree of fracturing, bedrock lithology and presence and type of weathered materials were also investigated as indicators of ground properties. 3.3 Regional development planning For instance, the mineralogy, grain size and fabric of the bedrock and related weathered materials would control properties such as shear strength, pore water suction, infiltration capacity and natural attenuation of contaminants (Vrba and Civita 1994, Hudec 1998, Hill and Rosenbaum 1998, Thornton et al. 2001, Fernandes 2003). Geological structures, such as faults and joints within the rock mass, as well as relict structures in saprolitic soils, are also liable to exert significant influences on shear strength and hydraulic properties of geomaterials (Aydin 2002, Pine and Harrison 2003). In this particular case study, analysis of lineaments extracted from satellite images combined with tectonic modelling underpinned inferences about major and small-scale faults and joints. The approach followed studies by Fernandes and Rudolph (2001) and Fernandes da Silva et al. (2005b) who asserted that empirical models of tectonic history, based on outcrop scale palaeostress regime determinations, can be integrated with lineament analysis to identify areas: i) of greater density and interconnectivity of fractures; and ii) greater probability of open fractures; also to iii) deduce angular relationships between rock structures (strike and dip) and between these and hill slope directions. These procedures facilitated 3-dimensional interpretations and up-scaling from regional up to semi-detailed assessments which were particularly useful for assessments of local ground stability and groundwater flow. The BCUs were then classified into four classes (very high, high, moderate, and low) in terms of susceptibility to land instability and groundwater vulnerability according to qualitative and semi-quantitative rules devised from a mixture of empirical knowledge and statistical approaches. A spreadsheet-based approach that used nominal, interval and numerical average values assigned in attribute tables was used for this. A two-step procedure was adopted to produce the required estimates where, at stage one, selected attributes were analysed and grouped into three score categories (A - high, M - moderate, B - low B) according to their potential influence on groundwater vulnerability and land 98 Environmental Management in Practice instability processes. In the second step, all attributes were considered to have the same relative influence and the final classification for each BCU was the sum of the scores A, B, M. The possible combinations of these are illustrated in Table 3. Figure 5 shows overall terrain classifications for susceptibility to land instability. Combinations of scores Classification AAAA Very high AAAM, AAAB, AAMM High AAMB, AABB, AMMM, AMMB, MMMM Medium AMBB, ABBB, MMMB, MMBB, MBBB, BBBB Low Table 3. 3.3 Regional development planning Possible combinations of scores “A” (high), “M” (moderate), and “B” (low) respective to the four attributes (bedrock lithology and weathered materials, fracturing, soil type, and slope steepness) used for classification of units (BCUs) in terms of susceptibility to land instability and groundwater vulnerability. ( g ) ( ) ( ) respective to the four attributes (bedrock lithology and weathered materials, fracturing, soil type, and slope steepness) used for classification of units (BCUs) in terms of susceptibility to land instability and groundwater vulnerability. Fig. 5. Maps of susceptibility to land instability processes. Test Areas T1 and T2. UTM projection and coordinates [After Fernandes da Silva et al., 2010]. Fig. 5. Maps of susceptibility to land instability processes. Test Areas T1 and T2. UTM projection and coordinates [After Fernandes da Silva et al., 2010]. Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 99 4. Hazard mapping: Land instability and flooding In order to prevent damage to structures and facilities, disruption to production, injury and loss of life, public authorities have a responsibility to assess hazard mitigation and controls that may require remedial engineering work, or emergency and contingency actions. In order to accommodate these different demands, information about the nature of the hazard, and the consequences and likelihood of occurrence, are needed. Hazard maps aim to reduce adverse environmental impacts, prevent disasters, as well as to reconcile conflicting influences on land use. The examples given in this Section demonstrate the identification and zonation in terms of the likelihood and consequences of land instability and flooding hazards. There are several reasons for undertaking such work, for instance to provide public authorities with data on which to base structural plans and building codes as well as civil defence and emergency response programmes. 4.1 Application to local structural plans pp p As indicated in Section 3.1, the BCUs (terrain units) classified as having a moderate to high susceptibility to geodynamic processes (mass movements and flood) were selected for further detailed engineering geological mapping. This was to provide data and supporting information to the structure plan of the City of São Sebastião. The attributes of the selected units were cross-referenced with other data sets, such as regional rainfall distribution, land- use inventory, and mineral exploitation records to estimate the magnitude and frequency of hazards and adverse impacts. Risk assessment was based on the estimated probability of failure occurrence and the potential damage thus caused (security of life, destruction of property, disruption of production). Both the triggering and the predisposing factors were investigated, and, so far as was possible, identified. It is worth noting the great need to consider socio-economic factors in hazard mapping and risk analysis. For instance, areas of consolidated housing and building according to construction patterns and reasonable economic standards were distinguished from areas of unconsolidated/expanding urban occupation. Temporal analysis of imagery and aerial photos, such as densities of vegetation and exposed soil in non-built-up areas, were utilised to supplement the land use inventory. The mineral exploration inventory included the locations of active and abandoned mineral exploitation sites (quarries and open pit mining for aggregates) and certain geotechnical conditions. Besides slope steepness and inappropriate occupancy and land use, the presence of major and minor geological structures was considered to be one of the main predisposing factors to land instability in the region studied. Figure 6 depicts a detail of the hazard map for the City of São Sebastião. Zones of land instability were delimited and identified by code letters that correspond with geodynamic processes as follows: A - landslides, B - creep, C - block tilt/glide, and D - slab failure/rock fall. Within these zones, landsliding and other mass movement hazards were further differentiated according to structural geological predisposing factors as follows: r – occurrence of major tectonic features such as regional faults or brittle-ductile shear zones; f – coincidence of spatial orientations between rock foliation, hillslope, and man-made cuttings; t – high density of fracturing (particularly jointing) in combination with coincidence of spatial orientations between fracture and foliation planes, hillslope, and man- made cuttings (Moura-Fujimoto et al., 1996; Fernandes da Silva et al. 1997b). 100 Environmental Management in Practice Fig. 6. 4.1 Application to local structural plans Example of hazard map from the City of São Sebastião, north shore of São Paulo State, Southeast Brazil. Key for unit classification: Light red = very high susceptibility; Blue = high susceptibility; Light orange = moderate susceptibility; Yellow = low susceptibility. See Section 4.1 for code letters on geodynamic processes and predisposing factors. [after Fernandes da Silva et al. 1997b] (not to scale). Fig. 6. Example of hazard map from the City of São Sebastião, north shore of São Paulo State, Southeast Brazil. Key for unit classification: Light red = very high susceptibility; Blue = high susceptibility; Light orange = moderate susceptibility; Yellow = low susceptibility. See Section 4.1 for code letters on geodynamic processes and predisposing factors. [after Fernandes da Silva et al. 1997b] (not to scale). 4.2 Application to civil defence and emergency response programmes 4.2 Application to civil defence and emergency response programmes Methods of hazard mapping can be grouped into three main approaches: empirical, probabilistic, and deterministic (Savage et al. 2004, as cited in Tominaga, 2009b ). Empirical approaches are based on terrain characteristics and previous occurrence of geodynamic phenomena in order to estimate both the potential for, and the spatial and temporal distribution of, future phenomena and their effects. Probabilistic approaches employ statistical methods to reduce subjectivity of interpretations. However, the outcomes depend very much on measured patterns defined through site tests and observations, but it is not always feasible to perform this acquisition of data in developing regions and countries. Deterministic approaches focus on mathematical modelling that aims quantitatively to describe certain parameters and rules thought to control physical processes such as slope Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 101 stability and surface water flow. Their application tends to be restricted to small areas and detailed studies. stability and surface water flow. Their application tends to be restricted to small areas and detailed studies. In the State of Sao Paulo (Southeast Brazil), high rates of population influx and poorly planned land occupation have led to concentration of dwellings in unsuitable areas, thus leading to increasing exposure of the community to risk and impact of hazard events. In addition, over the last 20 years, landsliding and flooding events have been affecting an increasingly large geographical area, so bringing about damage to people and properties (Tominaga et al. 2009a). To deal with this situation, Civil Defence actions including preventive, mitigation, contingency (preparedness), and emergency response programmes have been implemented. The assessment of the potential for the occurrence of landslides, floods and other geodynamic processes, besides the identification and management of associated risks in urban areas has played a key role in Civil Defence programmes. To date, systematic hazard mapping has covered 61 cities in the State of São Paulo, and nine other cities are currently being mapped (Pressinotti et al., 2009). Examples that mix empirical and probabilistic approaches are briefly presented in this Section. The concepts of hazard mapping and risk analysis adopted for these studies followed definitions provided in Varnes (1984) and UN-ISDR (2004), who described risk as an interaction between natural or human induced hazards and vulnerable conditions. 4.2 Application to civil defence and emergency response programmes According to Tominaga (2009b), a semi-quantitative assessment of risk, R, can be derived from the product R = [H x (V x D)], where: H is the estimated hazard or likelihood of occurrence of a geodynamic and potentially hazardous phenomenon; V is the vulnerability determined by a number of physical, environmental, and socio-economic factors that expose a community and/or facilities to adverse impacts; and D is the potential damage that includes people, properties, and economic activities to be affected. The resulting risk, R, attempts to rate the damage to structures and facilities, injury and loss of lives, and disruption to production. The first example relates to hazard mapping and risk zoning applied to housing urban areas in the City of Diadema (Marchiori-Faria et al. 2006), a densely populated region (around 12,000 inhab. per km2) of only 31.8 km2, situated within the Metropolitan Region of the State Capital – São Paulo (Figure 7). The approach combined the use of high-resolution satellite imagery (Ikonos sensor) and ortho-rectified aerial photographs with ground checks. The aim was to provide civil defence authorities and decision-makers with information about land occupation and ground conditions as well as technical advice on the potential magnitude of instability and flooding, severity of damage, likelihood of hazard, and possible mitigating and remedial measures. Driving factors included the need to produce outcomes in an updateable and reliable manner, and in suitable formats to be conveyed to non-specialists. The outcomes needed to meet preventive and contingency requirements, including terrain accessibility, linear infrastructure conditions (roads and railways in particular), as well as estimations of the number of people who would need to be removed from risk areas and logistics for these actions. Risk zones were firstly identified through field work guided by local authorities. Site observations concentrated on relevant terrain characteristics and ground conditions that included: slope steepness and hillslope geometry, type of slope (natural, cut or fill), soil weathering profile, groundwater and surface water conditions, and land instability features (e.g. erosion rills, landslide scars, river 102 Environmental Management in Practice undercutting). In addition, information about periodicity, magnitude, and effects of previous landsliding and flooding events as well as perceptions of potential and future problems were gathered through interviewing of residents. Satellite images were further used to assist with the identification of buildings and houses liable to be affected and the delineation of risk zone boundaries. 4.2 Application to civil defence and emergency response programmes Risk assessment was based on a qualitative ranking scheme with four levels of risk: R1 (low); R2 (moderate); R3 (high); R4 (very-high). Low risk (R1) zones, for example, comprised only predisposing factors to instability (e.g. informal housing and cuttings in steep slope areas) or to flooding (e.g. informal housing in lowland areas and close to watercourses but no reported flood within the last 5 years). Very-high risk (R4) zones were characterized by significant evidence of land instability (e.g. presence of cracks in soil and walls, subsidence steps, leaning of trees and electricity poles, erosion rills and ravines, landslide scars) or flooding hazards (e.g. flooding height marks on walls, riverbank erosion, proximity of dwellings to river channel, severe floods reported within the last 5 years). The outcomes, including basic and derived data and interpretations, were integrated and then presented on a geo-referenced computational system designed to respond the needs of data displaying and information management of the State of São Paulo Civil Defence authorities (CEDEC). As described by Pressinotti et al. (2007), such system and database, called Map-Risk, includes cartographic data, interpretative maps (risk zoning), imagery, and layers of cadastral information (e.g. urban street network). The system also enabled generation and manipulation of outputs in a varied set of text (reports), tabular (tables), and graphic information including photographic inventories for risk zones. The system was fully conceived and implemented at low cost, utilizing commercial software available that were customized in this visualisation system through target-script programming designed to achieve user functionalities (e.g. ESRI/MapObjects, Delphi, Visual Basic, OCX MapObjects). Examples of delineated risk zones for the City of Diadema and a display of the Map-Risk functionalities are presented in Figure 7. The second example refers to a flooding hazard mapping performed at regional and local scales in the Paraiba do Sul River Watershed, Eastern São Paulo State (Figure 8), in order to provide a rapid and comprehensive understanding of hazard phenomena and their impacts, as well as to enable application of procedures of data integration and mapping in different socio-economic contexts (Andrade et al. 2010). The information was systematised and processed to allow the build-up of a geo-referenced database capable of providing information for both environmental regional planning (economic-ecological zoning) and local scale hazard mapping for civil defence purposes. 4.2 Application to civil defence and emergency response programmes The regional evaluation covered all the 34 municipalities located in the watershed, and comprised the following stages of work: 1) survey of previous flooding events reported in newspaper and historical archives; 2) data systematisation and consolidation to translate gathered news into useful pieces of technical information; 3) identification of flooding occurrence locations using Google Earth tools; 4) cartographic auditing, geo-referencing and spatial data analysis using a freeware GIS package called SPRING (see Section 5); 5) exploratory statistical analysis of data; 6) preliminary flooding hazard classification on the basis of statistical results. Such preliminary classification used geopolitical (municipality) and hydrographical sub-basin boundaries as units for the analysis. Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 103 A) B) . 7. A) Location of the City of Diadema in the Metropolitan District of São Paulo (Sta pital), Southeast Brazil and example of delineated risk zones over a high-resolution ellite image (Ikonos). B) Example of Map-Risk system display. See Section 4.2 for det ter Marchiori-Faria et al., 2006; Pressinotti et al., 2007] A) B) Fig. 7. A) Location of the City of Diadema in the Metropolitan District of São Paulo (State capital), Southeast Brazil and example of delineated risk zones over a high-resolution satellite image (Ikonos). B) Example of Map-Risk system display. See Section 4.2 for details. [after Marchiori-Faria et al., 2006; Pressinotti et al., 2007] B) B) Fig. 7. A) Location of the City of Diadema in the Metropolitan District of São Paulo (State capital), Southeast Brazil and example of delineated risk zones over a high-resolution satellite image (Ikonos). B) Example of Map-Risk system display. See Section 4.2 for details. [after Marchiori-Faria et al., 2006; Pressinotti et al., 2007] 104 Environmental Management in Practice Fig. 8. Location of Paraiba do Sul River Watershed in Eastern São Paulo State and distribution of flooding occurrences. Internal sub-divisions correspond to geopolitical boundaries (municipalities). [After Andrade et al., 2010] Fig. 8. Location of Paraiba do Sul River Watershed in Eastern São Paulo State and distribution of flooding occurrences. Internal sub-divisions correspond to geopolitical boundaries (municipalities). [After Andrade et al., 2010] Fig. 8. Location of Paraiba do Sul River Watershed in Eastern São Paulo State and distribution of flooding occurrences. Internal sub-divisions correspond to geopolitical boundaries (municipalities). 4.2 Application to civil defence and emergency response programmes [After Andrade et al., 2010] The regional evaluation was followed-up with detailed flooding hazard mapping (1:3,000 scale) in 7 municipalities, which included: a) ground observations - where previous occurrence was reported – to measure and record information on flooding height marks, land occupation, and local terrain, riverbank and water course characteristics; b) geo- referencing and spatial data analysis, with generation of interpolated numerical grids on flooding heights and local topography; c) data interpretation and delimitation of flooding hazard zones; d) cross-referencing of hazard zones with land use and economic information leading to delimitation of flooding risk zones. Numerical scoring schemes were devised for ranking hazard and risk zones, thus allowing relative comparisons between different areas. Hazard zone scores were based on intervals of flooding height (observed and interpolated) and temporal recurrence of flooding events. Flooding risk scores were quantified as follows: R = [H x (V x D)], in which potential damage and vulnerability were considered (housing areas, urban infrastructure, facilities and services to be affected) on the basis of image interpretation and cross-referencing with land use maps and information. A detail map (yet unpublished) showing the interpolated grid of flooding heights and delineated hazard zones is presented in Figure 9. Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 105 A) B) Fig. 9. A) Measurement of maximum flood height for recent flooding event. B) Numerical interpolated grid of flooding heights and delineated flooding hazard zones. Green = Low probability of occurrence, Estimated flooding heights (Efh) < 0.40 m. Yellow = Moderate probability, 0.40 < Efh < 0.80 m. Light Brown = High probability, 0.80 < Efh < 1.20 m. Red = Very high probability, Efh > 1.20 m. Ground observations and measurements: cross and rectangle. Continuous lines: black = topographic contour lines, blue = main river channel boundaries Not to scale A) A) B) B) Fig. 9. A) Measurement of maximum flood height for recent flooding event. B) Numerical interpolated grid of flooding heights and delineated flooding hazard zones. Green = Low probability of occurrence, Estimated flooding heights (Efh) < 0.40 m. Yellow = Moderate probability, 0.40 < Efh < 0.80 m. Light Brown = High probability, 0.80 < Efh < 1.20 m. Red = Very high probability, Efh > 1.20 m. Ground observations and measurements: cross and rectangle. 4.2 Application to civil defence and emergency response programmes Continuous lines: black = topographic contour lines, blue = main river channel boundaries. Not to scale. 5. Geo-environmental assessment: applications to land reclamation policies Land reclamation of sites of previous mineral exploitation frequently involve actions to minimize environmental damage and aim at re-establish conditions for natural balance and sustainability so reconciling former mined/quarried sites with their surroundings (Brollo et al., 2002). Strategies and programmes for land reclamation need to consider physical and 106 Environmental Management in Practice biological characteristics of the local environment as well as socio-economic factors. Socio- environmental regeneration, involves not only revegetation and land stabilisation engineering, but also rehabilitation or introduction of a new function for the area. A) B) tion map and satellite image of the Municipality of Ubatuba (North utheast Brazil). Dots on image represent quarried/mined sites. B) Sch ntegrated approach taken to reconcile mineral exploitation managem n. The scheme shows the three main issues to be addressed (centre) studied. [after Ferreira et al., 2006; Ferreira & Fernandes da Silva, 20 y concerns a GIS-based geo-environmental management scheme t A) A) B) B) Fig. 10. A) Location map and satellite image of the Municipality of Ubatuba (North Shore São Paulo State, Southeast Brazil). Dots on image represent quarried/mined sites. B) Schematic display of the integrated approach taken to reconcile mineral exploitation management and land reclamation. The scheme shows the three main issues to be addressed (centre) and topics of interest to be studied. [after Ferreira et al., 2006; Ferreira & Fernandes da Silva, 2008] The case study concerns a GIS-based geo-environmental management scheme to reconcile sustainable mineral exploration of aggregates and construction materials with regeneration Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 107 of abandoned and/or derelict mined sites in the municipality of Ubatuba (North Shore of São Paulo State, Brazil). Until the early 1990's intensive exploitation of residual soil and ornamental stone (for fill and civil construction) took place in an unplanned and unregulated manner. This led to highly adverse environmental impacts, including the creation of 114 derelict and abandoned sites which resulted in State and Federal authorities enforcing a virtual halt to mining activity in the region. Besides this, the municipality of Ubatuba is highly regarded for its attractive setting and landscape, including encroached coastline with sandy beaches and bays with growing leisure and tourism activities. 5. Geo-environmental assessment: applications to land reclamation policies The area encompasses the Serra do Mar Mountain Range covered by large remnants of Atlantic Forest so that approximately 80% of the municipal territory lies within a nature and wildlife reserve (Figure 10A). As described by Ferreira et al. (2005, 2006), the devised strategy required an integrated approach (Figure 10B) in order to address three key issues: 1) environmental recovery of a number of derelict (abandoned, unsightly) sites; 2) reduction of hazards (land instability, erosion, flooded areas etc), particularly at those sites informally occupied by low income populations; and 3) rational exploitation of materials for local building materials corresponding to local needs. The study was implemented using a freeware GIS and image processing package called SPRING (Câmara et al. 1996, INPE 2009) and ortho-rectified air photos (1-metre resolution, taken in 2001, leading to an approximate scale of 1:3,000). The key output of the land management strategy was a prioritisation ranking scheme based on a comprehensive site critical condition (ICR) score, which synthesised the significance of each factor or issue to addressed (IG/SMA, 2008; Ferreira et al., 2009). Accordingly, the score system consisted of three numerical indicators: 1) environmental degradation indicator (IDE), 2) mineral potential indicator (IPM), and 3) hazard/risk indicator (IRI). Each indicator was normalised to a scalar range (0 to 1), and the ICR was the sum of the three indicators. The ICR was then used to set up directives and recommendations to advise local and State authorities about the possible measures to be taken, through mid- and long-term policies and/or immediate remedial and mitigating actions. According to Ferreira et al. (2008), the IDE comprised four component criteria to estimate the degree of adverse environmental impact (or degradation) of the individual mineral extraction sites: erosional features, terrain irregularity, herbaceous and bushy vegetation, and exposed soil. Information on these factors was acquired from imagery and ground checks. Tracing of linear features on images was investigated as an indicator of the frequency and distribution of erosional processes (rills, ravines, piping scars) as well as for terrain irregularity. In the first case, the sum of linear features representative of erosional processes was ratioed by the area of each site to quantify the estimate. Similarly, linear features related to the contour of cutting berms, rill marks, and slope breaks caused by mining/quarrying activity, were also measured to quantify terrain irregularity. 5. Geo-environmental assessment: applications to land reclamation policies In summary, 47% of sites were classified as very low priority, 12% as low priority, 19% as moderate priority, 15% as high priority (18 sites), and 7% as very high priority (8 sites). The priorities represent a combination of availability of exploitable volumes of building materials and the need for measures to tackle adverse environmental impacts and high risk situations (Figure 12). Based on the application of the ICR scores and current land use, directives and recommendations for land reclamation and socio-regeneration of mined/quarried sites were consolidated into ten main groups (IG/SMA, 2008; Ferreira et al 2009) Such directives and recommendations ranged from simple measures such as Fig. 11. Example of the mineral potential indicator (IPM) for the Municipality of Ubatuba (North Shore São Paulo State, Southeast Brazil). A) Abandoned/quarried site delimited on image and numerical grid of topographic heights. B) Screen display from GIS-based computation of volumes of exploitable material [after Ferreira & Fernandes da Silva, 2008]. Fig. 11. Example of the mineral potential indicator (IPM) for the Municipality of Ubatuba (North Shore São Paulo State, Southeast Brazil). A) Abandoned/quarried site delimited on image and numerical grid of topographic heights. B) Screen display from GIS-based computation of volumes of exploitable material [after Ferreira & Fernandes da Silva, 2008]. Fig. 11. Example of the mineral potential indicator (IPM) for the Municipality of Ubatuba (North Shore São Paulo State, Southeast Brazil). A) Abandoned/quarried site delimited on image and numerical grid of topographic heights. B) Screen display from GIS-based computation of volumes of exploitable material [after Ferreira & Fernandes da Silva, 2008]. Figure 12 illustrates the application of the ICR scoring scheme to mined/quarried sites in Ubatuba. In summary, 47% of sites were classified as very low priority, 12% as low priority, 19% as moderate priority, 15% as high priority (18 sites), and 7% as very high priority (8 sites). The priorities represent a combination of availability of exploitable volumes of building materials and the need for measures to tackle adverse environmental impacts and high risk situations (Figure 12). Based on the application of the ICR scores and current land use, directives and recommendations for land reclamation and socio-regeneration of mined/quarried sites were consolidated into ten main groups (IG/SMA, 2008; Ferreira et al., 2009). Such directives and recommendations ranged from simple measures such as Figure 12 illustrates the application of the ICR scoring scheme to mined/quarried sites in Ubatuba. 5. Geo-environmental assessment: applications to land reclamation policies The areal extent of herbaceous and bushy vegetation as well as exposed soil were also delimited on images. The IPM, as described by Ferreira & Fernandes da Silva (2008), was achieved by means of the following procedures: 1) identification and delimitation of quarried/mined sites (polygons) on geo-referenced imagery; 2) derivation of local DEM (digital elevation model) from topographic contour lines to each delimited site; 3) calculation of local volume to material (V1) based on the original geometry of the quarried/mined sites; 4) calculation of 108 Environmental Management in Practice volume of material already taken (V2) and exploitable volume of material (V3), so that [V3] = [V1 – V2]; 5) application of classification rules based on legal environmental and land use restrictions. The calculation of volumes of material (residual soil and ornamental stone) was performed by means of GIS operations involving polygons (areas) and numerical grids of topographic heights generated with nearest neighbour interpolator in the SPRING package (Figure 11). The IRI was derived from R = [H x (V x D)] – see Section 4.2 – focussing on mass movement and flood hazards and their consequences to people, property and economic activity. According to Rossini-Penteado (2007) and Tominaga et al. (2008), Hazard, H, was quantified according to the spatial and temporal probability of occurrence of each phenomenon and then weighted in relation to areal distribution of such probabilities (percentage of sq. km). The vulnerability, V, was computed by means of scores assigned to socio-economic aspects such as nature of built structures, spatial regularity of land occupation, presence of urban infrastructure (e.g. water supply, sanitation, health services, refuse collection and disposal method), road/street network, educational and income patterns. Similarly, in order to estimate the extent of potential damage, D, numerical scores were devised and attributed to the estimated number of people and buildings per unit area, and to the proportion of built area in relation to total area of the site. Fig. 11. Example of the mineral potential indicator (IPM) for the Municipality of Ubatuba (North Shore São Paulo State, Southeast Brazil). A) Abandoned/quarried site delimited on image and numerical grid of topographic heights. B) Screen display from GIS-based computation of volumes of exploitable material [after Ferreira & Fernandes da Silva, 2008]. Figure 12 illustrates the application of the ICR scoring scheme to mined/quarried sites in Ubatuba. 5. Geo-environmental assessment: applications to land reclamation policies In summary, 47% of sites were classified as very low priority, 12% as low priority, 19% as moderate priority, 15% as high priority (18 sites), and 7% as very high priority (8 sites). The priorities represent a combination of availability of exploitable volumes of building materials and the need for measures to tackle adverse environmental impacts and high risk situations (Figure 12). Based on the application of the ICR scores and current land use, directives and recommendations for land reclamation and socio-regeneration of mined/quarried sites were consolidated into ten main groups (IG/SMA, 2008; Ferreira et al., 2009). Such directives and recommendations ranged from simple measures such as Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 109 routine maintenance and cleaning, revegetation with grass and control of water surface flow (including run-off) to the implementation of leisure and multi-purpose public facilities, major land stabilisation projects combined with mineral exploitation, and monitoring. Fig. 12. Spatial distribution of mined/quarried sites classified according to critical condition score scheme (ICR). Sites classified as High (triangles) and Very High (squares) priority are highlighted. Remarks: 1- Serra do Mar Nature and Wildlife State Park, 2- Environmentally sensitive protected areas. ICR-MB = Very Low priority, ICR-B = Low priority, ICR-M = Moderate priority, ICR-A = High priority, ICR-MA = Very high priority [after IG/SMA, 2008]. Fig. 12. Spatial distribution of mined/quarried sites classified according to critical condition score scheme (ICR). Sites classified as High (triangles) and Very High (squares) priority are highlighted. Remarks: 1- Serra do Mar Nature and Wildlife State Park, 2- Environmentally sensitive protected areas. ICR-MB = Very Low priority, ICR-B = Low priority, ICR-M = Moderate priority, ICR-A = High priority, ICR-MA = Very high priority [after IG/SMA, 2008]. 6. Conclusions Geo-environmental terrain classification may be used as part of the land-use planning decision making and may also provide the basis of responses to emergency situations. In most examples presented here, classification schemes were based on knowledge of the bedrock geology, topography, landforms, superficial geology (soil and weathered materials), groundwater conditions and land-uses. Information for the classification has been variously derived from remote sensing and fieldwork rather than specific site investigations. A framework for carrying out a terrain classification at different scales has been presented. In practice, the effectiveness of land zoning system requires the implementation of planning controls To do this the Local Authorit needs adequate resources and an appropriate legal y g p In practice, the effectiveness of land zoning system requires the implementation of planning controls. To do this the Local Authority needs adequate resources and an appropriate legal 110 Environmental Management in Practice or planning guidance policy framework. Preferably, the control process should be based on the principle that permission will be given unless there is a good reason for refusal. In granting permission, conditions may then be applied to ensure the safety of the development with regard to landsliding, flooding and other potential problems. However it must be recognised that where practical control over development cannot be exercised, other preventive, mitigative or advisory measures may be all that can be used. g y y Marker (1996) explains that the rate and style of development has a major impact on the information requirements. Without a rigorously enforced planning framework based on accurate information about the ground conditions very rapid urban development will generally lead to construction on areas of less stable land or land which may be subject to hazards such as flooding or pollution. This type of development may also result in the sterilisation of geological resources which it would be expedient to exploit as part of the development process. On the other hand restricting development to designated areas will generally require detailed information about the ground conditions and likelihood and potential impact of hazards to be available at the planning stage. It also assumes existence of the resources and will to enforce the plan. Such models can severely constrain the social and economic development of an area, lead to excessively high population densities and give rise to problems associated with the re-use of previously developed land. Policy formulation may incorporate incentives (e.g. 7. Acknowledgements The authors are very much indebted to the Sao Paulo State Geological Institute and its staff for most of the examples provided here. 6. Conclusions subsidies and reduced taxes) to be provided by local and state governments to encourage such measures and good practice, which can be viewed as kinds of voluntary or induced control. In some cases financial controls exerted by public funding as well as mortgage and insurance providers are the means by which some types of development may be curtailed but such controls may not prevent informal occupancy of hazardous areas. In this regard, some of the examples presented here, from a regional and local perspective, have also demonstrated environmental management regulations may have little meaning in some urban areas subject to rapid expansion where, because of population influx, informal housing is virtually an inevitable consequence. q Due to the latter hazard mapping updating and post-episode monitoring [failure episodes] are absolutely vital as these procedures facilitate a contemporary understanding of ground conditions and risk circumstances, which can be essential to provide timely and efficient advice for mitigation and control of hazards as well as to design effective contingency actions and engineering solutions. 8. References Akiwumi, F.A., Butler, D.R. (2008). Mining and environmental change in Sierra Leone, West Africa: a remote sensing and hydrogeomorphological study. Environmental Monitoring Assessment, 142: 309–318. ISSN 0167-6369 Akiwumi, F.A., Butler, D.R. (2008). 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Regional morpho-structural analysis on Radar and Landsat images at the Parana Basin. Proceedings of the 3rd South Brazilian Regional Symposium on Geology, Curitiba. p. 5-23. [In Portuguese] Thompson, A., Hine, P.D., Poole J.S. & Greig, J.R. 8. References (1998a) Environmental Geology in Land Use Planning: Advice for planners and developers. Published by Symonds Travers Morgan (East Grinstead, UK) on behalf of Department of Environment, Transport and the Regions, Great Britain. Thompson, A., Hine, P.D., Poole J.S. & Greig, J.R. (1998b) Environmental Geology in Land Use Planning: A guide to good practice. Published by Symonds Travers Morgan (East Grinstead, UK) on behalf of Department of Environment, Transport and the Regions, Great Britain. Thompson, A., Hine, P.D., Greig, J.R. & Poole J.S. (1998c) Environmental Geology in Land Use Planning: Emerging issues. Published by Symonds Travers Morgan (East Grinstead, UK) on behalf of Department of Environment, Transport and the Regions, Great Britain. Geo-environmental Terrain Assessments Based on Remote Sensing Tools: A Review of Applications to Hazard Mapping and Control 117 Thornton, S.F., Lerner, D.N., & Banwart, S.A. (2001) Assessing the natural attenuation of organic contaminants in aquifers using plume-scale electron and carbon balances: model development with analysis of uncertainty and parameter sensitivity. Journal of Contaminant Hydrology, 53(3-4): 199-232. ISSN 0169-7722. Tominaga, L.K., Rossini-Penteado, D., Ferreira, C.J., Vedovello, R. & Armani, G. (2008). Landsliding hazard assessment through the analysis of multiple geo-environmental factors. Proceedings of the 12th Brazilian Congress on Environmental and Engineering Geology. ABGE, ISBN 978-85-7270-052-8. Porto de Galinhas, November 2008. 15p. [In Portuguese] Tominaga, L.K. (2009a). Natural Disasters: Why they happen ? In: Disasters: Knowing to prevent. Tominaga, L.K., Santoro, J., & Amaral, R (eds). Pp. 147-160, Geological Institute, ISBN 978-85-87235-09-1, São Paulo. [In Portuguese] Tominaga, L.K. (2009b). Risk Analysis and Mapping. In: Disasters: Knowing to prevent. Tominaga, L.K., Santoro, J., & Amaral, R (eds). Pp. 13-23, Geological Institute, ISBN 978-85-87235-09-1, São Paulo. [In Portuguese] TRRL - Transport & Road Research Laboratory. (1978). Terrain evaluation for highway engineering and transport planning: a technique with particular value for developing countries. TRRL Supplementary Report No. 448, 21p. UN-ISDR – United Nations – International Strategy for Disaster Reduction. (2004). Living with Risk: A Global Review of Disaster Reduction Initiatives. In: Inter-Agency Secretariat International Strategy for Disaster Reduction. Geneve, 152p. Accessed in Aug, 2009. Available at http://www.unisddr.org. g p g Varnes, D.J. (1984). Landslide Hazard Zonation: Review of Principles and Practice. Unesco Press, Paris, 56p. Vedovello, R. (2000). Geotechnical Zoning for Environmental Management through Basic Physiographic Units. (unpublished PhD Thesis, Sao Paulo State University at Rio Claro). [In Portuguese] Vedovello, R. (1993). Geotechnical Zoning based on Remote Sensing Techniques for Urban and Regional Planning. Vrba J. & Civita, M. (1994) Assessment of groundwater vulnerability. In: Guidebook on Mapping Groundwater Vulnerability. Vrba, J. & Zaporozec, A. (eds.). 16: 31-48, International Association of Hydrogeologists (IAH). Vinogradov, B.V., Gerenchuk, K.I., Isachenko, A.G., Raman, K.G., & Teselchuk, Y.N. (1962). Basic principles of landscape mapping, Soviet Geography: Review and Translation, 3 (6), 15-20. Verstappen HTh (1977) Remote Sensing in Geomorphology. Elsevier Sci. Publ., Amsterdam. 214p. The Implementation of IPPC Directive in the Mediterranean Area Tiberio Daddi, Maria Rosa De Giacomo, Marco Frey, Francesco Testa and Fabio Iraldo Scuola Superiore di Studi Universitari e di Perfezionamento S. Anna, Pisa, Italy 1 In order to correct some failures in the application of the Directive, in 2008 the European Commission enacted a new IPPC Directive and many Countries are still implementing it. 8. References São José dos Campos, 186p. (unpublished M.Sc. Dissertation, National Institute for Space Research) [In Portuguese] Vedovello, R. & Mattos, J.T. (1998). The use of basic physiographic units for definition of geotechnical units: a remote sensing approach. Proceedings of the 3rd Brazilian Symposium on Geotechnical Cartography. São Paulo, November 1998, 11p. [In Portuguese] Vedovello, R., Brollo, M.J., & Fernandes da Silva, P.C. (1998). Assessment of erosion as a conditioning factor on the selection of sites for industrial waste disposal: an approach based on regional physiographic compartmentalisation obtained from satellite imagery. Proceedings of the 3rd Brazilian Symposium on Erosion Control. Presidente Prudente, March 1998. 9p. [In Portuguese] Vedovello, R., Tominaga, L.K., Moura-Fujimoto, N.S.V., Fernandes da Silva, P.C., Holl, M.C., & Maffra, C.Q.T. (1997). A mass movement hazard analysis approach for public authority responses. In: Proceedings of the 1st Latin American Forum on Applied Geography. Curitiba, September 1997. 9p. [In Portuguese] 118 Environmental Management in Practice Verstappen HTh (1977) Remote Sensing in Geomorphology. Elsevier Sci. Publ., Amsterdam. 214p. Vinogradov, B.V., Gerenchuk, K.I., Isachenko, A.G., Raman, K.G., & Teselchuk, Y.N. (1962). Basic principles of landscape mapping, Soviet Geography: Review and Translation, 3 (6), 15-20. Vrba J. & Civita, M. (1994) Assessment of groundwater vulnerability. In: Guidebook on Mapping Groundwater Vulnerability. Vrba, J. & Zaporozec, A. (eds.). 16: 31-48, International Association of Hydrogeologists (IAH). 6 The Implementation of IPPC Directive in the Mediterranean Area Tiberio Daddi, Maria Rosa De Giacomo, Marco Frey, Francesco Testa and Fabio Iraldo Scuola Superiore di Studi Universitari e di Perfezionamento S. Anna, Pisa, Italy 1. Introduction In Europe industrial activities are amongst the main causative factors of pollution. Until 1996 European Member States adopted separate regulations and multiple authorizations to address pollution control and prevention, and different laws separately dealt with air, water and soil issues, thus providing only partial solutions to the problem. The Council Directive 96/61/EC of 24 September 1996, on Integrated Pollution Prevention and Control (IPPC Directive1) aims at the integrated pollution prevention and control within European Member States (Schoenberger, 2009) starting from the activities listed in the annex I of the Directive (Honkasalo et al., 2005), which consider all environmental aspects (air, water, soil, waste, etc.) as a whole and unique integrated system. According to this approach, the Directive introduces a single authorization (Styles, et al., 2009) - the Integrated Environmental Authorization – the so-called “permit” to regulate the “environmental behaviour” of IPPC-related activities, to determine parameters of environmental aspects and establish measures to avoid or reduce environmental impact. Thanks to this Directive, European Member States shall correctly manage all aspects of industrial activity likely to generate environmental impacts, under the same administrative procedure in order to be granted the above mentioned permit. p g p The industrial activities listed in annex I of the law include six main topics: energy production, production and processing of metals, minerals, chemical, waste management and others activities – e.g. pulp and paper, pre-treatment or dyeing of textile fibres or textiles, tanning of hides and skins, intensive pig and poultry farming, surface treatments of substances, objects or products by means of organic solvents -. The Directive is addressed mostly at large installations, and indicates production capacity thresholds that exclude the smallest installations (Samarakoon & Gudmestad, 2011). This law lays down measures to prevent or, whereas not viable, to reduce emissions in air, water and land from the above-mentioned activities, as well as measures concerning waste, in order to achieve an overall high level of environmental protection (European Commission, 2008). The Directive thus provides an holistic approach to pollution prevention. 120 Environmental Management in Practice The IPPC Directive introduced some important improvements in the form of Best Available Techniques (BATs hereafter), i.e. 1. Introduction “the most effective and advanced stage in the development of activities and their methods of operation which indicate the practical suitability of particular techniques for providing in principle the basis for emission limit values designed to prevent and, where that is not practicable, generally to reduce emissions and the impact on the environment as a whole”. BATs concerns technologies and organizational measures expected to minimize overall environment pressures at acceptable private costs (Bréchet & Tulkens, 2009). Techniques should be available, so as to allow implementation in relevant industrial sectors, under economically and technically viable conditions, taking into consideration costs and advantages, whether or not the techniques are used or produced within the Member State in question, as long as they are reasonably accessible to the operator. Finally, techniques should be the most effective in achieving a high general degree of environmental protection. In view of that, BAT Reference Documents (BREF), published by the European IPPC Bureau, are the basic tools to implement the requirements of the Directive (Kocabas et al., 2009). The purpose of this chapter is to present some of the results of the European project MED IPPC NET (“Network for strengthening and improving the implementation of the IPPC Directive regarding the Integrated Pollution Prevention and Control in the Mediterranean”) whose main objective was the evaluation of the implementation of the IPPC Directive in seven European regions. g The chapter proceeds as follows. After a brief literature review about studies on IPPC topic, that will be included in paragraph 2, paragraph 3 illustrates the MED IPPC NET project. Paragraph 4 relates to the research question and the method applied to the study, while paragraphs 5 and 6 include some of the results achieved by the project. Finally, conclusions are included in paragraph 7, and reference list in paragraph 8. 2. The implementation of the IPPC directive By means of these data, the authors monitored the progress of BAT implementation through the comparison of indicators and after the normalization with benchmarks from BREF or granted environmental permits. Facilities have been ranked according to their BAT implementation so to provide clear indication about their environmental performance. The methodology proposed by the authors thus provides a useful evaluation of environmental performance in the pursue of IPPC targets. indicators and indices based on the reports of the European Polluting Emissions Register (EPER), and the application of environmental permits submitted by the Competent Authority for this matter. By means of these data, the authors monitored the progress of BAT implementation through the comparison of indicators and after the normalization with benchmarks from BREF or granted environmental permits. Facilities have been ranked according to their BAT implementation so to provide clear indication about their environmental performance. The methodology proposed by the authors thus provides a useful evaluation of environmental performance in the pursue of IPPC targets. Barros et. al. (Barros et al., 2009) in their work identified BAT in the seafood industry in the northwest of Spain. In particular, they carried out an analysis about the existing technologies in the mussel canning plant as well as a list of BAT both installed or not. Then BATs have been assessed in order to promote their implementation in a mussel canning facility. Barros et. al. (Barros et al., 2009) in their work identified BAT in the seafood industry in the northwest of Spain. In particular, they carried out an analysis about the existing technologies in the mussel canning plant as well as a list of BAT both installed or not. Then BATs have been assessed in order to promote their implementation in a mussel canning facility. The report “Assessment of the implementation of the IPPC Directive in the UK” of January 2008 – commissioned by the Air and Environment Quality Division of the Department for Environment, Food and Rural Affairs (Defra) - aimed to select ten UK-based installations with IPPC permits, and to assess the degree of implementation of IPPC requirements of the Directive by each case study. The survey has focused on the investigation of the procedures applied and the conditions set for selected permits, as also on the assessment of the current installation operation when compared to permit conditions and BAT. 2. The implementation of the IPPC directive Many studies deal with the evaluation of the IPPC Directive implementation, and most them refer to the application of BAT in the industrial field or in a localized nation or country. y The paper by Kobacas (Kobacas et. al., 2009) illustrates the results of the work derived by the first implementation of the IPPC Directive and the BREF Document within an industrial facility in Turkey (“Adoption of EU’s IPPC Directive to a Textile Mill in Turkey: BAT Applications”). In particular, the study focuses on water and energy consumption of a textile mill in Turkey, assessed further to the application of specific BAT aiming to reduce these consumptions. In their paper Bréchet & Tulkens (Bréchet & Tulkens, 2009) stated that Best Available Techniques should be best not only in term of private aims and interests, but also according to the society’s point of view. To this purpose, they present a modeling framework based on methodologies able to satisfy both these two purposes. They conclude that a fair combination of Best Available Techniques should be preferred to one single BAT. In their study they consider a lime factory. Karavanas et. al. (Karavanas et. al., 2009) presented an integrated methodological approach for the evaluation of the implementation of Best Available Techniques in facilities operating under the IPPC. For the application of the proposed methodology, the authors take into a account the Greek paper manufacturing sector and the relevant environmental performance 121 The Implementation of IPPC Directive in the Mediterranean Area indicators and indices based on the reports of the European Polluting Emissions Register (EPER), and the application of environmental permits submitted by the Competent Authority for this matter. By means of these data, the authors monitored the progress of BAT implementation through the comparison of indicators and after the normalization with benchmarks from BREF or granted environmental permits. Facilities have been ranked according to their BAT implementation so to provide clear indication about their environmental performance. The methodology proposed by the authors thus provides a useful evaluation of environmental performance in the pursue of IPPC targets. indicators and indices based on the reports of the European Polluting Emissions Register (EPER), and the application of environmental permits submitted by the Competent Authority for this matter. 2. The implementation of the IPPC directive As outlined in this paragraph, many studies exist in literature on the evaluation of IPPC and the effects that they have generated in some specific activities or sectors that also take into account the different experiences occurred in various different European Member States. p p Thanks to the MED IPPC NET project the study carried out represents an added value to the existent studies in literature on about the evaluation of the IPPC. We evaluated the implementation of the IPPC Directive in seven European regions analysing many of its aspects (laws that implemented the Directive, administrative procedure to issuing permits, control system in facilities envisaging permits, content of the permits and the analysis of requirements and prescriptions provided by them). The most important novelty of the study is that it allows a comparison among the regions of many European Member States. 3. The MED IPPC NET project The MED IPPC NET (“Network for strengthening and improving the implementation of the IPPC Directive regarding the Integrated Pollution Prevention and Control in the Mediterranean”) is a 30 month-project co-funded by the European Commission. Its main goal is to identify some crucial aspects in the implementation of the IPPC Directive 96/61/EC of 24 September 1996 concerning Integrated Pollution Prevention and Control within the Mediterranean area. In this area there are significant differences in how European Member States have perceived the importance of the IPPC, and on what kind of supporting or coercive mechanism they have implemented to improve its practical application. The MED IPPC NET project, by identifying these differences, aims to establish a set of common criteria that should be taken into account by all Mediterranean regions wishing to enhance their implementation. These common criteria will constitute the inputs to develop a common methodology in implementing the IPPC Directives within the Mediterranean area which will help it, in turn, to become a reference for the environmental behaviour of its industrial facilities. To achieve this goal the seven regions participating in the project, and belonging to four European Member States – Andalusia, Spain (Andalusian Institute of Technology), Piedmont, Italy (Arpa Piemonte), Sicily, Italy (Arpa Sicilia), Slovenia, whole national territory (Scientific Research Centre Bistra Ptuj), Tuscany, Italy (Scuola Superiore Sant’Anna and Eurobic Toscana Sud), Valencia, Spain (Environment, water, town planning and housing Department of Valencian Government), West Macedonia, Greece (Environmental Centre of Kozani) -, carried out in-depth studies in each region about the implementation modalities of the IPPC Directive. To this purpose the project provided an analytical phase (that constituted a specific task component of the project) in order to study how the seven regions involved in the project implemented the IPPC Directive. 2. The implementation of the IPPC directive In particular, the main objective of this study was to select and analyse some permits issued in UK in order to assess their compliance with the IPPC Directive. p A part of literature deals more specifically with the environmental performance or efficiency of IPPC industries. About this aspect another study (Honkasalo et. al., 2005) analyses some case studies of British, Finnish and Swedish industries and the corresponding regulatory bodies, to contribute to the discussion on the potentiality of the IPPC Directive as a driver of eco- efficiency in these industries. Styles et al. (2009) take into account the application of the Environmental Emissions Index (EEI) to reported emissions data about pharmaceutical-manufacturing installations and power stations holding IPPC in Ireland. Results on reported emissions demonstrated environmental performance improvements. Georgopoulou et al. (Georgopoulou et al., 2008) developed – within the framework of a research project - a decision-support tool for public and private administrators and managers (called “BEAsT”, BAT Economic Attractiveness Tool) in order to make possible an assessment of different BATs and their combinations in term of economic costs and environmental benefits deriving from their application. Since the development of this tool initiated by the necessity to provide an action plan for BAT promotion in a Greek region, where main environmental impacts of industrial activities derived from air pollution and liquid waste, the tool was mainly addressed to these two impacts. In practice, BEAsT was used to assess the environmental benefits to be expected from BATs, and to identify which BATs are attractive for end-users in the economic outlook. The main purpose of the study of Silvo et. al. (Silvo et. al., 2009) was to investigate the impacts of the IPPC Directive on environmental performance of pulp and paper mills in Finland in the period 2001-2006. To do this, the authors compared the Emission Limit Values of the IPPC permits with those of other permits not linked to IPPC. 122 Environmental Management in Practice As outlined in this paragraph, many studies exist in literature on the evaluation of IPPC and the effects that they have generated in some specific activities or sectors that also take into account the different experiences occurred in various different European Member States. 4. Research question and method In this framework, this chapter aims to answer to some research questions, such as: Has the Directive been implemented with the same approach in the Member States? Do permitting procedure, inspection and control system show differences among Member States? From June 2009 to May 2010 the seven regions involved in the project collected and analysed information and data on the implementation of the IPPC Directive applying a common research methodology. The results have been later compared building an Interregional Analysis report of the project. In this framework, this chapter aims to answer to some research questions, such as: Has the Directive been implemented with the same approach in the Member States? Do permitting procedure, inspection and control system show differences among Member States? From June 2009 to May 2010 the seven regions involved in the project collected and analysed information and data on the implementation of the IPPC Directive applying a common research methodology. The results have been later compared building an Interregional Analysis report of the project. 123 The Implementation of IPPC Directive in the Mediterranean Area The methodological approach, necessary to evaluate in each region the differences existing in the implementation of the IPPC, has been defined taking into account some existing studies to identify which aspects could be more interesting for the purpose of the project. On the basis of the final version of the methodology, some operational tools have been applied (e.g. questionnaires, guidelines, etc.) to carry out the Analysis through an homogeneous approach in each region participating to the project. g pp g p p g p j The methodological approach has included four typologies of Analysis: the Legislative Analysis, the Administrative Analysis, the Control and Inspection System Analysis and the Content of the Authorizations Analysis. The “Legislative Analysis” aimed to analyzing how the IPPC Directive has been implemented in national and local legislative frameworks. In particular, each region collected information and data through the study and the consultation of laws, reports and all documents about the IPPC. Moreover, interviews with Competent Authorities for permit issue have been carried out with the aim to collect information about the typologies of Competent Authorities involved in the issuing of the Integrated Environmental Authorization, the introduction of BAT Reference Documents (BREF) in the national, regional and local contexts, and some information concerning procedures and laws that guarantee the access to information and public participation in the permitting procedure. 4. Research question and method p p p p g p The “Administrative Analysis” aimed to acknowledge the procedure for the granting of permits in the several regions involved in the project. Some aspects collected and studied concerned data and documents requested by the procedure on issuing permits, the contents of these documents, the descriptions of simplifications in the permitting procedure for particular categories of enterprises/sectors, the number and the nature of the institutions involved in the permitting procedure, the description of the environmental assessment carried out in the permitting procedure, etc. The objective of the “Control and Inspection System Analysis” is to understand how that System has been implemented in the regions involved. To this purpose, some information has been collected about the nature and the role of Competent Authorities that carry out the inspections, the kinds of non-compliance identified by the Control Authorities, the public fares to pay, etc. For all above mentioned analyses, each region collected information also through interviews with the Competent Authorities for the granting of permits. Thanks to these interviews it was possible to collect opinions from these Authorities so as to identify strengths and weaknesses – but also best practices - on the IPPC implementation in the Mediterranean regions involved in the project. The objective of the “Control and Inspection System Analysis” is to understand how that System has been implemented in the regions involved. To this purpose, some information has been collected about the nature and the role of Competent Authorities that carry out the inspections, the kinds of non-compliance identified by the Control Authorities, the public fares to pay, etc. F ll b ti d l h i ll t d i f ti l th h i t i For all above mentioned analyses, each region collected information also through interviews with the Competent Authorities for the granting of permits. Thanks to these interviews it was possible to collect opinions from these Authorities so as to identify strengths and weaknesses – but also best practices - on the IPPC implementation in the Mediterranean regions involved in the project. 4. Research question and method The “Content of Authorizations Analysis” has been realized by consulting and analysing a sample of permits of some IPPC sectors selected for the project by the activities listed in the annex I of the Directive: - 1.1: Combustion installation with a rated thermal input exceeding 50 MW; - 3.5: Installation to manufacturing ceramic products; tallation to manufacturing ceramic products; - 3.5: Installation to manufacturing ceramic products; - 5.4: Landfills receiving more than 10 tons per day or with a total capacity exceeding 25.000 tons; - 2.6: installations for surface treatment of metals and plastic materials; - 6.1; Industrial plants for the production of pulp from timber or other fibrou - 6.1; Industrial plants for the production of pulp from timber or other fibrous materials and paper and board with a production capacity exceeding 20 tons per day. 6.1; Industrial plants for the production of pulp from timber or other fibrous material and paper and board with a production capacity exceeding 20 tons per day. p p p p and paper and board with a production capacity exceeding 20 tons per day. The objective of the Content of Authorization Analysis was to compare the content of permits for the industrial sectors mentioned, and to highlight the differences in the The objective of the Content of Authorization Analysis was to compare the content of permits for the industrial sectors mentioned, and to highlight the differences in the 124 Environmental Management in Practice environmental management prescriptions and requirements related to the several environmental aspects. This objective has been performed analyzing a representative sample of permits, as illustrated in the next paragraphs. 5. Result of the analysis phase of the MED IPPC NET project The Analysis carried out through the MED IPPC NET project has taken into account many aspects of the implementation of the IPPC Directive in the seven regions participating to the project. In this paragraph we highlight some interesting results derived from the Interregional Analysis (the Analysis that has allowed to compare results about the IPPC implementation among the seven regions). In particular, we included some results of the four Analyses which compose the Interregional Analysis report: Legislative, Administrative, Control and Inspection System and Content of Authorizations. 2 On 29th June 2010 the decree n. 59/2005 has been repealed by the legislative decree n. 128/2010. This latter integrates the IPPC within the legislative decree n. 152/2006. 5.1 Legislative analysis As outlined in paragraph 4, the Legislative Analysis aimed to analyze how the IPPC Directive has been implemented in the legislative framework. In the following paragraph, we consider the results obtained by implementing the Directive regulatory framework in the seven regions, by the Competent Authorities for issuing the Integrated Environmental Authorization, and the modalities to assure the access to information and public participation. 5.1.1 The implementation of the IPPC Directive in the seven regions participating to he project As regards regional laws, in Piedmont the resolution of the Regional Council (July 29, 2002) confirmed in the provinces the Competent Authorities to grant, renewal and review of IPPC permit3. p g p Also in Tuscany the Regional resolution n. 61 adopted in December 22nd 2003 identified as IPPC Competent Authorities the ten Tuscan Provinces (Firenze, Prato, Pistoia, Pisa, Massa Carrara, Livorno, Siena, Arezzo, Grosseto, Lucca), and one Circondario (Circondario Empolese Valdelsa). In respect to the ministerial decree 24/4/20084 on the fares to apply in connection with the preliminary inquires and controls provided by the national legislative decree, both in Piedmont and Tuscany there are more regional resolutions about this matter. They contain a general decrease of national rates, the administrative resolution on the advances of expenses for the preliminary examination on issuing the Integrated Environmental Authorization, the adaptation and the integration of fares to be applied according to the ministerial decree. In Tuscany other regional resolutions are applied such as, for example, the n. 151 of February 23rd 2004 that sets up the Coordination Technical Committee and the decree n. 1285 of March 10th 2004 concerning the appointment of the Coordination Technical Committee members. In Sicily a “Guideline” document to drafting, monitor and control a plan to set up IPPC tools has been prepared (reference document with the minimum information to be included into the Control and Monitoring Plan5), jointly with the ARTA Sicily Decree 12/08/2004 (GURS 36/04) approving the procedures for the application of the permit. In West Macedonia the IPPC Directive has been implemented at national level by law 3010/2002 that amended the basic Environmental Greek Law (L.1650/1986) to be harmonized with the European Directives 96/61 and 97/11. Moreover, two ministerial decisions have been adopted (CMD.15393/2332/2002 and CMD 11014/703/Φ104/2003). The subject of these two decisions is the adjustment of the environmental authorization procedure of activities included in the Annex I of the Directive. Besides, these activities are being categorized in relation of their impact on the environment. g g Finally, Slovenia has implemented the IPPC Directive with two acts: the Environmental Protection Act (ZVO-1; Official Gazette of the RS, no. 41/04), and the decree on activities and installations causing large-scale environmental pollution (IPPC Decree, Official Gazette of the RS, no. 97/04). 5.1.1 The implementation of the IPPC Directive in the seven regions participating to he project p j In the four European Member States involved in the MED IPPC NET project, the IPPC Directive has been implemented by specific national laws. In some regions also regional and local laws regarding specific aspects linked to the IPPC have been enacted. Directive has been implemented by specific national laws. In some regions also regional and local laws regarding specific aspects linked to the IPPC have been enacted. In Spain the IPPC Directive has been implemented through the national law 16/2002 on Integrated Pollution Prevention and Control. Besides, also two royal decrees have been enacted: the first one (the decree 508/2007) concerned the regulation for the information supply on emissions on Pollutant Release and Transfer Register (PRTR), and the integrated environmental authorizations; the second one (decree 509/2007) deals with the regulation of development and execution of national law 16/2002. The regional law 7/2007 implemented the Spanish national law about IPPC in Andalusia. p The Region of Valencia also applies a regional law (2/2006), enacted by the Generalitat Valenciana, on pollution prevention and environmental quality. In particular, the objective of this law is to define and regulate the instruments of environmental administrative intervention for those activities likely to affect security, health or environment. It established an Annex II for new categories of activities that must also obtain the Integrated Environmental Authorization. Additionally, the regional decree 127/2006, from the Valencia Council, concerns rules about the development and the execution of law 2/2006. p / The IPPC Directive was implemented in Italy in August 4th 1999 by the national legislative decree n° 372 that disciplined, for the first time in Italy, the issuing of the Integrated Environmental Authorization according to IPPC criteria. Consequently, the legislative decree 59/20052 replaced the first one. There are also other decrees in Italy that discipline 125 The Implementation of IPPC Directive in the Mediterranean Area some aspects of the IPPC: the redefinition of the National Competent Authorities on the issuing of the Integrated Environmental Authorization; the technical and administrative documents to submit for the permitting procedure about the issuing of permits; the institution of a national IPPC Commission with the function to supply support to the definition, the updating and the integration of BAT national guidelines; the modalities – also accounting – and the fares to apply in connection with the preliminary inquires and controls provided by the national legislative decree n. 59/05. 4 Since the national decree n. 59/05 has been repelled by the legislative decree n. 128/2010, a decree 24/4/2008 will be repelled in next months with a new one. 3 Once the operator of the installation presented an application to obtain the permit the Competent Authority issues the permit. 4 Since the national decree n. 59/05 has been repelled by the legislative decree n. 128/2010, also the decree 24/4/2008 will be repelled in next months with a new one. 5 This document should be filled in by the operator of the installation and should be presented jointly with the application to obtain the permit. 5 This document should be filled in by the operator of the installation and should be presented jointly with the application to obtain the permit. nce the operator of the installation presented an application to obtain the permit the Competen hority issues the permit. 5.1.2 Competent authorities in the granting of the integrated environmental authorization Another main aspect analysed is the type of Competent Authorities in charge of issuing the Integrated Environmental Authorization. In particular, the Analysis revealed that in some of the seven regions involved in the project these Authorities are provincial or regional, while in others they are ministerial or national ones. g p j p g , in others they are ministerial or national ones. In the case of the Region of Andalusia, the Competent Authorities are Provincial Delegations of the Department of Environment (Provincial Delegations are in Seville, Huelva, Cádiz, Córdoba, Málaga, Granada, Jaén y Almería). The territorial jurisdiction is determined by the location of the plant. When the plant serves more than one province, the competent Directorate General for Environmental Prevention and Control within the Department of Environment, will instruct and follow through with the proceedings, except when it delegates such competencies to one of the Provincial Delegations. In Tuscany and Piedmont the regional governments have delegated to provinces the competence for permit issue. The ten Tuscan Provinces are: Firenze, Prato, Pistoia, Pisa, Massa Carrara, Livorno, Siena, Arezzo, Grosseto and Lucca, besides the Circondario Empolese Valdelsa has also been appointed as Competent Authority. In Piedmont the eight provinces are: Alessandria, Asti, Biella, Cuneo, Novara, Torino, Verbano-Cusio-Ossola, Vercelli. For both regions (and also in Sicily), the Ministry of Environment is the Competent Authority in the place of provinces (or instead of the Region in the case of Sicily) when provided by the national law that implemented the IPPC Directive. In fact, in Sicily the whole process of permit release is under the responsibility of the Service II SEA-IEA (Regional Department of Territory and Environment). The Italian Regional Agencies for the Environmental Protection (ARPA) are involved in permit process, particularly in respect to the evaluation of the control and monitoring plan (PMC), included in every Integrated Environmental Authorization’s application. In Valencia, the Competent Authorities in charge of permit processing depend upon the y In the case of the Region of Andalusia, the Competent Authorities are Provincial Delegations of the Department of Environment (Provincial Delegations are in Seville, Huelva, Cádiz, Córdoba, Málaga, Granada, Jaén y Almería). The territorial jurisdiction is determined by the location of the plant. 5.1.1 The implementation of the IPPC Directive in the seven regions participating to he project We shall also consider the following regulations: the two Decrees amending the Decree on activities and installations causing large-scale environmental pollution (Official Gazette of the RS, n. 71/07 and n. 122/07), and two additional regulations on reporting to the European Pollutant Release and Transfer Register (PRTR). 126 Environmental Management in Practice The table below indicates the kind of legislation that implemented the IPPC Directive in each of the participant regions. The table below indicates the kind of legislation that implemented the IPPC Directive in each of the participant regions. IMPLEMENTATION OF THE IPPC DIRECTIVE IN THE SEVEN REGIONS State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany National laws and/or other national regulations/acts X X X X X X X 7 Regional law and/or other local laws X X - - X X X 5 Table 1. Implementation of the IPPC Directive in the seven regions. Table 1. Implementation of the IPPC Directive in the seven regions. 6 Regional law n. 2/2006 5.1.2 Competent authorities in the granting of the integrated environmental authorization When the plant serves more than one province, the competent Directorate General for Environmental Prevention and Control within the Department of Environment, will instruct and follow through with the proceedings, except when it delegates such competencies to one of the Provincial Delegations. g p g In Tuscany and Piedmont the regional governments have delegated to provinces the competence for permit issue. The ten Tuscan Provinces are: Firenze, Prato, Pistoia, Pisa, Massa Carrara, Livorno, Siena, Arezzo, Grosseto and Lucca, besides the Circondario Empolese Valdelsa has also been appointed as Competent Authority. In Piedmont the eight provinces are: Alessandria, Asti, Biella, Cuneo, Novara, Torino, Verbano-Cusio-Ossola, Vercelli. For both regions (and also in Sicily), the Ministry of Environment is the Competent Authority in the place of provinces (or instead of the Region in the case of Sicily) when provided by the national law that implemented the IPPC Directive. In fact, in Sicily the whole process of permit release is under the responsibility of the Service II SEA-IEA (Regional Department of Territory and Environment). The Italian Regional Agencies for the Environmental Protection (ARPA) are involved in permit process, particularly in respect to the evaluation of the control and monitoring plan (PMC), included in every Integrated Environmental Authorization’s application. pp In Valencia, the Competent Authorities in charge of permit processing depend upon the type of activities performed. For those activities included in the Annex I of the regional law6 (Annex I of the IPPC Directive) the CA is the Environment, Water, Town Planning and Housing Department of the Valencian Government (Conselleria de Medio Ambiente, Agua, 127 The Implementation of IPPC Directive in the Mediterranean Area Urbanismo y Vivienda de la Generalitat Valenciana), through its General Office of Climate Change (Dirección General para el Cambio Climático). For those activities included in the Annex II of the same regional law, the Competent Authorities are the provincial Offices (Direcciones Territoriales for the 3 provinces: Alicante, Castellón, Valencia) of Environment, Water, Town Planning and Housing Department of the Valencian Government. The activities included in this latter Annex II are similar to those included in the Annex I of the law, but with lower production capacity. 7 CMD 15393/2332/2002 and CMD11014/703/Φ104/2003. Table 2. Competent Authority/ies for the permitting procedure of the Integrated Environmental Authorization. o e ta ut o at o . 7 CMD 15393/2332/2002 and CMD11014/703/Φ104/2003. 5.1.3 The modalities to assure access to information and public participation in the permitting procedure for issuing the Integrated Environmental Authorization 5.1.3 The modalities to assure access to information and public participation in the permitting procedure for issuing the Integrated Environmental Authorization The primary method to ensure access to information and public participation in the permitting procedure in all regions is represented by the publication of some information (e.g. in newspapers, bulletins, etc.). g In Italy, according to the national decree that implemented the IPPC Directive, the Competent Authority identifies the offices where to record the proceedings for public consultation. Moreover, it provides that the operator publishes an advertisement for the public in a provincial, regional or national newspaper. Anyone can have access to a copy of the issued IPPC permits, and to any relating document in a public office, as determined by the Competent Authorities. The Competent Authority shall make available to the public the data provided by the operator relating to emission controls required by integrated environmental authorization. The results of the monitoring of emissions, required by permit conditions and held by the Competent Authority, should be available to the public. The legislative decree n. 195 adopted on 19th August 2005 – implementing the European Directive 2003/4/CE - disciplines public access to environmental information. On the one hand, this decree establishes terms, fundamental conditions and modalities to exert public access; on the other hand, it guarantees that the environmental information is at public disposal and it is disseminated accordingly. Moreover, when the Competent Authority informs a business company about the beginning date of the procedure, the operator should publish an announcement containing information on the plant at provincial, regional or national media outlets. In Valencia the Competent Authority submits the permit application along with the required documentation for public information procedure, within a minimum period of 30 days, by publishing it in the Official Diary of the Valencian Government, as well as in the relevant City Hall bulletin board. Its public dissemination (notification to neighbours, record of submitted documentation in CA offices) is therefore allowed, except for that data considered to be confidential. At the end of the procedure it also publishes the resolution of the permit, to which it is possible to make objections within a period of 30 days. In compliance with the principle of access to information relating to the environment, citizens can consult the emissions of specific pollutants of IPPC installations in the PRTR, and the content of the permit issued. 5.1.2 Competent authorities in the granting of the integrated environmental authorization p p y In the case of West Macedonia the law establishes that the Competent Authorities are the Ministry of Environment Energy and Climate Change and the Direction of Environment and Development, Department of Environment and Land-Planning of the Region of West Macedonia (Prefectures of Kozani, Kastoria, Grevena and Florina). In reality, the permits examined within the scope of the project have all been issued by the Ministry because the region did not authorise any IPPC plant at the time of the analysis. The jurisdiction between Ministry and Region is determined by the production ability of the installation7. y g y p y In Slovenia the Competent Authorities are national: the Ministry of Environment and Spatial Planning, and the Environmental Agency of the Republic of Slovenia (ARSO). ARSO performs professional, analytical, regulatory and administrative tasks in the field of environment nationally, it contributes to solving environmental problems as far as possible with the implementation of environmental legislation and keeps records of emissions, manages and monitors the implementation of remedial programs and seeks comprehensive solutions to the problems regarding climate change. Particularly, ARSO pays attention to raising public awareness on the environment and on environmental issues. Likewise, Slovenia set up a nation-wide special expert group established under the IPPC Directive, consisting of acting inspectors depending on the technological processes. The Environmental Agency of the Republic of Slovenia (ARSO) cooperates with the Inspectorate of the Republic of Slovenia for Environment and Spatial Planning (IRSOP) in the field of control of administrative decisions, since the IRSOP is responsible to supervise all environmental legislation adopted by the Parliament, the Government or the Ministry. W i d i h f ll i bl h C A h i i f h I d We summarized in the following table the Competent Authorities for the Integrated Environmental Authorizations: COMPETENT AUTHORITY/IES FOR THE PERMITTING PROCEDURE OF THE INTEGRATED ENVIRONMENTAL AUTHORIZATION State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany National Competent Authority X X X X X 5 Regional Competent Authority X X X 3 Provincial Competent Authority X X X X 4 Table 2. Competent Authority/ies for the permitting procedure of the Integrated Environmental Authorization. PETENT AUTHORITY/IES FOR THE PERMITTING PROCEDURE OF THE INTEGRATED ENVIRONMENTAL AUTHORIZATION 128 Environmental Management in Practice 5.1.3 The modalities to assure access to information and public participation in the permitting procedure for issuing the Integrated Environmental Authorization p In Andalusia the procedure of information and public participation for the Integrated Environmental Authorization falls at national level under the jurisdiction of the Autonomous Communities establishing only a minimum period of public information (30 days) - as in the case of Valencia -. Once the competent body verifies the project compatibility to the environmental regulations, it renders the Integrated Environmental Authorization available for public consultation and formulation of the relevant declarations, by including its advertisement in the Official Bulletin of the Government of Andalusia (Oficial de la Junta de Andalucía) (for 45 days), and through personal notification to the immediate neighbourhoods of the place where the activity is located (for 30 days). Following these terms, the competent body will remit all annexes and comments received to the requesting entity in charge of the Integrated Environmental Authorization, to the State body responsible for granting public permits in the field of maritime-terrestrial competences, and to the regional body responsible for granting the substantive permit, that can be declared within 15 days. g g p y In West Macedonia the competent Service of Environment of the Ministry of Environment Energy and Climate Change or the Region that has been granted the study, prior to approving the environmental procedure, conveys a copy to the Prefectural Council within a ten days period. Subsequently, the Prefectural Council has five days to publish the study in 129 The Implementation of IPPC Directive in the Mediterranean Area at least one local newspaper, and to disseminate it publicly (within 30 days) for information, to allow the public opinion to share objections on its content. at least one local newspaper, and to disseminate it publicly (within 30 days) for information, to allow the public opinion to share objections on its content. at least one local newspaper, and to disseminate it publicly (within 30 days) for information, to allow the public opinion to share objections on its content. In Slovenia the national legislation guarantees the access to information and public participation in the permitting procedure through many tools, as for example: an IPPC website, by organizing training cycles, seminars, workshops for operators of installations, specialized publications, public debates and round tables, public presentations to explaining the procedures for issuing the permit (application form, etc.). Table 3. Main modalities adopted by regions to assure the access to information and public participation in the permitting procedure. 9 The ATO is an institution of control and guidance, competent for the management of the water service, sewerage and waste. It was established by the national law n. 36/1994. 5.1.3 The modalities to assure access to information and public participation in the permitting procedure for issuing the Integrated Environmental Authorization The table below includes the main modalities adopted by each participating region to assure In Slovenia the national legislation guarantees the access to information and public participation in the permitting procedure through many tools, as for example: an IPPC website, by organizing training cycles, seminars, workshops for operators of installations, specialized publications, public debates and round tables, public presentations to explaining the procedures for issuing the permit (application form, etc.). p g p ( pp ) The table below includes the main modalities adopted by each participating region to assure the access to information and public participation in the permitting procedure. MAIN MODALITIES ADOPTED TO ASSURE THE ACCESS TO INFORMATION AND PUBLIC PARTICIPATION IN THE PERMITTING PROCEDURE State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany Record of document in specific offices X X X X 4 Advertisement publication in newspaper/ other publication about IPPC matter X X X X 4 Publication of permit and other documents in the Official Diary of Government and/or in City/Government bulletin X X 2 Publication of emissions of specific pollutants of IPPC installations in the Pollutant Release and Transfer Register X 1 Personal notification to neighbors X X 2 Publication of statement in the Table of Statements of the Prefecture X 1 IPPC portal on website X X 2 Training/ seminars/workshops for operators of installations and Public debates and round tables X X 2 Table 3. Main modalities adopted by regions to assure the access to information and public participation in the permitting procedure. AIN MODALITIES ADOPTED TO ASSURE THE ACCESS TO INFORMATION AND PUBLIC PARTICIPATION IN THE PERMITTING PROCEDURE Table 3. Main modalities adopted by regions to assure the access to information and public participation in the permitting procedure. 130 Environmental Management in Practice 5.2 Administrative analysis y The “Administrative Analysis” focuses essentially on the procedure for granting the permit in the seven regions of the project. As previously assessed for the legislative analysis, also in this case we identify some of the resulting elements, such as the nature of the institutions involved in the first issuing for new and existing installations, the content of documents to be submitted within the Integrated Environmental Authorizations, the time envisaged for the issuing of the Integrated Environmental Authorization, and the simplified rules and regulations within the permitting procedure for particular categories of enterprises. 8 The Basin Authority is an institution aiming to safeguard the whole catchment basins. It was established by the national law n. 183/1999. 5.2.1 Institutions involved in the first issue for new and existing installations The institutions involved in the permitting procedure for the permit issuing are more similar among the seven regions, in regards to content and object of the documents. However, in most of them are some institutions always participate in the permitting procedure, while some are present only in some cases. In Tuscany and in Piedmont, for example, the institutions involved in the permitting procedure are: the Municipality, the Local Health Authority and the Environmental Protection Regional Agency, while the Regional Administration, the waters managers, the Basin Authority8 and the fire department are sometimes present. In both regions the waste and sewage system competent authorities, the ATO9, the Basin Authority and, in the case of permit that should be issued for landfills, the superintendent can also be part of the process. In the case of Piedmont, public stakeholders can be part of it. In Tuscany the opinions of the above-mentioned institutions are generally not binding, but in the permitting procedure these latter tend to comply to them. In Piedmont the opinions of the municipality and the province are always binding, those of the regional administration is binding only for some sectors, while the opinions of the other institutions are not binding. p g In Sicily the opinion of the municipality, province, regional administration, local health authority, of the Provincial Committee for Environmental Protection (CPTA) and of the Ministry of Environment and Protection of territory (or in case of national Integrated Environmental Authorization process, the Ministry of Environment) are binding. On the contrary, for the following involved institutions the opinion is not binding: Regional Agencies for Environmental Protection, Regional Agencies for waste and water, the waste management ATO, Departments responsible for water, air, etc. In Andalusia the institutions participating in the permitting procedure are: the Municipality, the Regional Department of Environment, the State Environmental Body, the Water Basin Entity. Their opinions are binding. In Valencia the Environment, Water, Town Planning and Housing Department of the Valencia Government (EWTPH) has set up the Integrated Environmental Analysis Commission, a body whose representatives are one from each administration/institution involved in the permitting procedure. The institutions and organisations that are always involved in this Commission are: the IPPC Service of EWTPH, the Waste Service of EWTPH, the Environmental impact Service of EWTPH, the Air Service of EWTPH, the Water Service of EWTPH, the Basin authority, the Clean Technologies Centre of EWTPH. Table 4. Main institutions involved in the permitting procedure Table 4. Main institutions involved in the permitting procedure 5.2.2 Time forecast to issuing the Integrated Environmental Authorization 5.2.2 Time forecast to issuing the Integrated Environmental Authorization In Italy the time frame to issuing the permitting procedure for new and existing installations is established by the legislative decree 152/2006. In particular, within 30 days from the application for permit receipt, the Competent Authority communicates to the operator the starting date of the proceeding. Within 15 days from the receipt of communication, the operator publishes an announcement on the plant features. Further, within 30 days from the publication of the announcement, the interested parties can present observations. Subsequently, if there is compliance with the requirements of the decree 152/2006, the Competent Authorities may issue the permit within 150 days from the application; or in the case of no-compliance they deny the permit. However, in case of particular/relevant environmental impacts and of the complexity and/or national interest of a plant, specific agreements can be concluded. In this case, the 150 days timeframe is replaced with a 300 days timeframe. In Andalusia the deadline for granting the permit is of 10 months from the submission of the application. After this period without receiving the notification of any special resolution, the application can be rejected. In this case, the proceedings under the Integrated Environmental Authorization shall not become the subject neither of Municipal License, nor of substantive authorizations. In Valencia the permitting procedure begins with a public information phase, followed by a sector-based report to the concerned competent administrations and institutions requested by the Competent Authority that calls the IPPC operator for an audience. Closer to the end of the procedure, the Competent Authority carries out an environmental assessment of the IPPC activity which will take into account all factors involved in it, therefore completing the process by issuing a resolution containing all the constraints that the activity must comply for their exploitation. The resolution is then notified to the operator and published in the Official Diary of the Government of Valencia. As it happens in Andalusia, also in Valencia the maximum period for completing the permitting procedure is 10 months. This deadline is valid for the activities included in the Annex I of the regional law. For those activities included in the Annex II of the regional law, the deadline is 8 months. In Slovenia once a company has presented all the required documents, the Environmental Agency of the Republic of Slovenia may require further clarification. 5.2.1 Institutions involved in the first issue for new and existing installations This 131 The Implementation of IPPC Directive in the Mediterranean Area Commission is similar to the Italian one (called “Meeting of Public Services”) in its composition and activities. As in the case of Tuscany and Valencia also for West Macedonia there are some institutions that are always involved in the permitting procedure, and others that are present from time to time. The Special Service of Environment of the Ministry of Environment Energy and Climate Change, the Direction of Planning of the Ministry of Environment Energy and Climate Change, the Department of Environment of the Ministry of Environment Energy and Climate Change and the Region, the Prefectural Department of Environment, the Prefect and the Council for Public Information, belong to the first category. The Relative to the Investment Ministries, the Regional Department of Forests, the Regional Department of Waters, the Revenue of Antiquities, the Prefectural Departments of Agriculture and of Health belong to the second one. The opinions of all institutions are binding but not defined by law, the Competent Authority has the final decision but in almost all the cases it takes into account the remarks of the authorities involved. In Slovenia there is a sole institution involved in the permitting procedure is the Ministry of Environment and Spatial Planning, Environmental Agency of the Republic of Slovenia. Its opinion is binding. The table 4 indicates the institutions involved in the first issue of the permit for new and existing installations, in the seven regions of the project. MAIN INSTITUTIONS INVOLVED IN THE PERMITTING PROCEDURE State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany National institution X X X X X* X* 6 Regional institution X X X X X 5 Local institution X X X X X 5 Specific public institution (e.g. basin authority) X X X X X X 6 Other technical public departments (e.g. fireman) X X X X X 5 Public health and safety authority X X X X 4 Bearers of collective interests X X 2 * In Italian regions the national institution is involved when Ministry is the Competent Authority for the permit issue. Table 4. Main institutions involved in the permitting procedure MAIN INSTITUTIONS INVOLVED IN THE PERMITTING PROCEDURE 132 Environmental Management in Practice 5.2.2 Time forecast to issuing the Integrated Environmental Authorization 5.2.2 Time forecast to issuing the Integrated Environmental Authorization The presentation of them allows the Environmental Agency to prepare of a consensus and the public presentation of application, leading to the issuing of the permit. For the issuing of new permits, the deadline is set in 7 months, while for the existing installations the term of the permitting procedure is not determined. However, in the case of West Macedonia, once the Competent Authority judges that the documents presented are complete, it transmits them to the relevant consultative authorities within 10 days that can request additional data and clarifications of the investor in the following 15 days. Within 5 days from the interval of 15 days, the Competent Authority approves or denies the Pro-EIA. In West Macedonia first of all is realised an initially study called Pro-EIA where interested submits application to the responsible authority that is accompanied by file which contains 6 copies of the study with technical and administrative information. The approval and the Pro-EIA documents are transmitted to the Prefectural Council for public information, and within 30 days it is possible to formulate opinions and objections regarding the project. In order to receive the Environmental authorization, the investor submits a documented application accompanied by the 6 copies of EIA, including the approval of Pro-EIA. In case the competent authority judges the documents incomplete, it can ask for additional supporting information within 10 days, otherwise it transmits it within 10 days to the t t lt ti th iti j i tl ith th f t l b ti Th l tt 133 The Implementation of IPPC Directive in the Mediterranean Area should provide a final recommendation within 35 days, following this period of time, the Competent Authority has 15 days to approve or deny the EIA. Finally, the EIA approval is transmitted to the Prefectural Council for public information. If the entire documentation is complete the authorisation is granted within a maximum period of 90 days. This interval can be extended for another 90 days for peculiar cases. The table below indicates times for Integrated Environmental Authorization issuing. 10 Annex II of the Royal Decree 509/2007. 5.2.2 Time forecast to issuing the Integrated Environmental Authorization TIMES FOR THE FIRST ISSUING FOR INTEGRATED ENVIRONMENTAL AUTHORIZATION NEW AND EXISTING INSTALLATIONS State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany From 5 to 10 months X X X X 4 7 months X** 1 8 months X* 1 10 months X X* 2 * Times depend by activity typology for new installations Table 5. Times for the first Integrated Environmental Authorization issuing for new and existing installations TIMES FOR THE FIRST ISSUING FOR INTEGRATED ENVIRONMENTAL AUTHORIZATION NEW AND EXISTING INSTALLATIONS State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany From 5 to 10 months X X X X 4 7 months X 1 8 months X* 1 10 months X X* 2 MES FOR THE FIRST ISSUING FOR INTEGRATED ENVIRONMENTAL AUTHORIZATION NEW AND EXISTING INSTALLATIONS Table 5. Times for the first Integrated Environmental Authorization issuing for new and existing installations Table 5. Times for the first Integrated Environmental Authorization issuing for new an existing installations 5.2.3 The simplifications in the permitting procedure for particular categories of enterprises p In most regions (Andalusia, Valencia, Tuscany, Piedmont and Sicily), laws provide simplified permit procedures for particular categories of enterprises. p p p p g p In Spain the royal decree 509/2007 provided a simplified procedure to apply for an IPPC Permit for Farming Installations, as referred to in category 9.3 of the Law 16/2002 (paragraph 6.6 of the Directive 96/61/EC) for the documentation10 to be included in the basic project that accompanies the request of Integrated Environmental Authorization. Additionally, this decree grants Autonomous Regions the permission to establish measures simplifying the mechanisms used to verify the fulfilment of the conditions envisaged by the IPPC permit for those facilities that apply an Environmental Management System (as per the requirements established by International Standard UNE-EN ISO 14001 - International Organization for Standardization- and/or Regulation EMAS- Eco-Management and Audit Scheme -). In Valencia the regional decree 127/2006 establishes that for the renewal of the Integrated Environmental Authorization, jointly to the application the owner shall submit a certificate stating the environmental assessment adequacy of the facility to the existing environmental legislation. This adequacy will be certified by an Environmental Quality Collaborating Entity. Given that the EMAS register requires a yearly assessment/audit of compliance with the environmental legislation, if an IPPC company adhered to EMAS it is not necessary to submit the certificate for the renewal of the application. By consequence, the facility or activity will be in accordance to the current environmental constraints/legislation. 134 Environmental Management in Practice In Piedmont, in Sicily and in Tuscany some of the provided simplifications are laid out by the legislative decree n. 59/05: g / - If at the moment of issuing the permit a plant is registered according to the CE Regulation n. 1221/2009 (EMAS), the renewal of the Integrated Environmental Authorization is filled in every eight years (usually, it is a five years renewal). If the registration according to the CE Regulation n. 1221/2009 (EMAS) comply to the authorization, the renewal is carried out every eight years starting from the first following renewal. g - If at the moment of issuing the permit a plant is registered according to the CE Regulation n. 1221/2009 (EMAS), the renewal of the Integrated Environmental Authorization is filled in every eight years (usually, it is a five years renewal). If the registration according to the CE Regulation n. 5.3 Control and inspection system analysis The primary scope of this Analysis is to assess the system of Control and Inspection carried out in those facilities applying an Integrated Environmental Authorization. In the following paragraphs some of the elements resulting from this analysis are outlined. 5.2.3 The simplifications in the permitting procedure for particular categories of enterprises 1221/2009 (EMAS) comply to the authorization, the renewal is carried out every eight years starting from the first following renewal. g - If at the moment of issuing the permit a plant is certified according to the Regulation UNI EN ISO 14001, the renewal of the Integrated Environmental Authorization is filled in every six years. If the certification according to the Regulation UNI EN ISO 14001 comply to the authorization, the renewal is carried out every six years starting from the first following renewal. g - If at the moment of issuing the permit a plant is certified according to the Regulation UNI EN ISO 14001, the renewal of the Integrated Environmental Authorization is filled in every six years. If the certification according to the Regulation UNI EN ISO 14001 comply to the authorization, the renewal is carried out every six years starting from the first following renewal. MAIN SIMPLIFICATIONS IN THE PERMITTING PROCEDURE FOR PARTICULAR CATEGORIES OF ENTERPRISES State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany Any simplification X X 2 Simplification of documents to submit in order to obtain permit for farming installations X X 2 Simplification about documents to submit for installations registered EMAS X 1 Simplification in inspection control activities in facilities registered EMAS or certified ISO 14001 X X 2 Longer validity of permit enterprises registered EMAS or certified ISO 14001 X X X 3 Reduction of fares for enterprises registered EMAS or certified ISO 14001 X X X 3 Table 6. Main simplifications in the permitting procedure for particular categories of enterprises. MAIN SIMPLIFICATIONS IN THE PERMITTING PROCEDURE FOR PARTICULAR CATEGORIES OF ENTERPRISES According to article 5 of the decree n. 59/05, if the information and the descriptions provided comply to the Regulation UNI EN ISO 14001, or the data provided for the 135 The Implementation of IPPC Directive in the Mediterranean Area registered sites comply to the Regulation CE n. 1221/2009 (EMAS), and if any other information is in compliance with the overall rules, particularly with one or more of the requirements set in the Integrated Environmental Authorization application, these can be applied for the registration of the Integrated Environmental Authorization application. registered sites comply to the Regulation CE n. 5.2.3 The simplifications in the permitting procedure for particular categories of enterprises 1221/2009 (EMAS), and if any other information is in compliance with the overall rules, particularly with one or more of the requirements set in the Integrated Environmental Authorization application, these can be applied for the registration of the Integrated Environmental Authorization application. The Italian ministerial decree adopted in April 24th 2008 (decree about fares) provided other simplifications: one reads that the installations registered according to the CE Regulation n. 1221/2009 (EMAS), can be granted reduced fares between 1.000 up to 8.000 euro; whilst another provides that for the installations certified according to the Regulation UNI EN ISO 14001, the reduction is granted between 500 and 5.000 euro. Slovenia and West Macedonia do not provide for simplifications in the permitting procedure for particular categories of enterprises. The Table 6 summarizes main simplifications in the permitting procedure for particular categories of enterprises. 5.3.1 The Competent Authorities appointed for the inspections and control procedures in the regions The Competent Authorities for inspections and controls are regional for the most regions, while are national in few cases. In Andalusia the General Direction of Environmental Prevention and Quality of the Regional Government for Environment of Andalusia, is competent for drafting various Sector Plans on Environmental Inspections. In Valencia the Competent Authorities for control and inspection are: the IPPC Service - belonging to the Environment, Water, Town Planning and Housing Department of the Government of Valencia -, and the Environmental Quality Collaborating Entities (EQCE) duly authorized and recognized in the IPPC industry. Their technical competences are certified by the Spanish Accreditation Entity, and they are registered in the Valencia Register of EQCE (managed by the Clean Technologies Centre). There are 10 accredited EQCE in Valencia. In most of the installations, the inspections are being carried out by the EQCE. In Italy the legislative decree n. 59/05 states that the Agency for Environmental Protection and Technical Services (ISPRA), for facilities under State jurisdiction, or the regional and provincial environmental protection agencies, are the Competent Authorities for controls and inspections. In Italy for example there is a regional agency in each region (Environmental Protection Regional Agency), but in each province there is also a local department having a supervisory role. In this way the provincial departments guarantees the knowledge of the local reality although there is a risk to overlapping or of differing approaches. In Sicily, in the case of landfills, the Competent Authority for the release of permits (Service II SEA-IEA, Regional Department of Territory and Environment), let the agency ARPA make a testing visit coordinated with the Provinces, to value the compliance to the permit requirements. In West Macedonia and Slovenia the situation is different from the previous regions. In the former region there are several Authorities involved in controls, in the latter one the Control Authority is national. Specifically, in West Macedonia the Competent Authorities responsible for the inspection procedure can be 136 Environmental Management in Practice divided into those who inspect the enterprises during the permitting procedure, and those tasked of the ongoing renewal of permits. In Slovenia the inspections are carried out by the inspection service under the control program adopted for three years. The table-summary indicates the main Competent Authorities for the control and inspection procedure. divided into those who inspect the enterprises during the permitting procedure, and those tasked of the ongoing renewal of permits. 5.3.1 The Competent Authorities appointed for the inspections and control procedures in the regions In Slovenia the inspections are carried out by the inspection service under the control program adopted for three years. The table-summary indicates the main Competent Authorities for the control and inspection procedure. MAIN COMPETENT AUTHORITIES DESIGNATED FOR THE CONTROL AND INSPECTION PROCEDURE State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany National Competent Authorities X X X 3 Regional Competent Authorities X X - X X X X 6 Table 7. Main Competent Authorities designated for the control and inspection procedure. MAIN COMPETENT AUTHORITIES DESIGNATED FOR THE CONTROL AND INSPECTION PROCEDURE State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany National Competent Authorities X X X 3 Regional Competent Authorities X X - X X X X 6 Table 7. Main Competent Authorities designated for the control and inspection procedure. 5.3.2 Most frequently non-compliances identified The most non-compliances identified during the inspections in the seven regions are indicated below: THE MOST FREQUENTLY NON-COMPLIANCES IDENTIFIED State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany Non compliance ELVs X X X 3 Non regular data transmission X X X X 4 Non compliance with requirements contained in permit X X X X X X 6 Dissimilarity from the management of measuring instruments (incorrect positioning, operation, calibration, maintenance of instruments) X X X X 4 Table 8. The most frequently non-compliances identified N COMPETENT AUTHORITIES DESIGNATED FOR THE CONTROL AND INSPECTION PROCEDURE 5.3.2 Most frequently non-compliances identified 5.3.2 Most frequently non-compliances identified The most non-compliances identified during the inspections in the seven regions are indicated below: 5.3.2 Most frequently non compliances identified The most non-compliances identified during the inspections in the seven regions are indicated below: THE MOST FREQUENTLY NON-COMPLIANCES IDENTIFIED State Spain Slovenia Greece Italy Total Region Andalucía Valencia Slovenia West Macedonia Piedmont Sicily Tuscany Non compliance ELVs X X X 3 Non regular data transmission X X X X 4 Non compliance with requirements contained in permit X X X X X X 6 Dissimilarity from the management of measuring instruments (incorrect positioning, operation, calibration, maintenance of instruments) X X X X 4 Table 8. The most frequently non-compliances identified 137 The Implementation of IPPC Directive in the Mediterranean Area In most of the regions, the main non-compliance characteristics that have emerged when monitoring deal with the requirements contained in the permit, while the non conformity with Emission Limit Values has been indicated by a lower number of regions. Table 9. Sample of Integrated Environmental Authorizations analysed in the “Content of Authorizations Analysis” 5.4 Content of authorization analysis The Content of authorizations analysis was made possible by the study of a sample of IPPC Integrated Environmental Authorizations of four IPPC sectors, according to the activities listed in the annex I of the IPPC Directive:  1.1: Combustion installation with a rated thermal input exceeding 50 MW;  1.1: Combustion installation with a rated thermal input exceeding 50 MW;  3.5: Installation for the manufacture of ceramic products;  5.4: Landfills receiving more than 10 tonnes per day or with a total capacity exceeding 25.000 tonnes; 2.6: Installations for surface treatment of metals and plastic materials;  2.6: Installations for surface treatment of metals and plastic materials;  6.1: Industrial plants for the production of pulp from timber or other fibrous materials and paper and board with a production capacity exceeding 20 tonnes per day.  6.1: Industrial plants for the production of pulp from timber or other fibrous materials and paper and board with a production capacity exceeding 20 tonnes per day. The aim of the Content of Authorization analysis was the comparison of Integrated Environmental Authorizations within the industrial sectors of the seven regions involved in the project, to highlight differences as to the management of environmental prescriptions and requirements. The sample of Integrated Environmental Authorizations analysed for the MED IPPC NET project, with the indication of the number of permits analysed by each region for each IPPC sector, is indicated in the following table: Region Number of IPPC permits analyzed Combustion plants (1.1) Ceramics (3.5) Landfills (5.4) Surface treatment of metals and plastic materials (2.6) Paper production (6.1) TOTAL Andalusia 8 8 8 8 0 32 Valencia 4 8 7 8 0 27 Slovenia 7 8 1 8 0 24 West Macedonia 2 2 3 1 0 8 Piedmont 19 24 21 0 15 79 Sicily 1 0 6 1 0 8 Tuscany 5 13 16 0 13 47 TOTAL 46 63 62 26 28 225 Table 9. Sample of Integrated Environmental Authorizations analysed in the “Content of Authorizations Analysis”. Table 9. Sample of Integrated Environmental Authorizations analysed in the “Content of Authorizations Analysis”. Every partner has collected a high number of Integrated Environmental Authorizations. In any case it is important to highlight the work carried out by Piedmont, which collected 79 permits, i.e. about 80% of the total permits issued in its region in the four sectors covered by the project. 5.4 Content of authorization analysis 138 Environmental Management in Practice About 60% of the sample is represented by permits from Italy, followed by the Spanish regions with about 27% of analysed permits. About 60% of the sample is represented by permits from Italy, followed by the Spanish regions with about 27% of analysed permits. As to targets, the methodology envisages three fixed sectors: “combustion plants”, “ceramics”, “landfills”. All partners had to analyze these three sectors. For the fourth sector each partner was allowed to choose between “Surface treatment of metals and plastic materials” or “Paper production”. The partners from Tuscany and Piedmont chose the “paper production” sector due to its relevance in the two regions, while the other partners selected the sector “Surface treatment of metals and plastic materials”. Fig. 1. Percentage of Integrated Environmental Authorizations analysed for each IPPC sector of the project. Fig. 1. Percentage of Integrated Environmental Authorizations analysed for each IPPC sector of the project. Ceramics and landfills are the IPPC sectors most represented in the Analysis. The highest number of Authorisations for these two sectors was collected in Tuscany and Piedmont. The three fixed sectors (combustion plants, ceramics, landfills) are fully represented by permits of each region, which means that at least one permit was collected in each region involved in the project. Fig. 2. Percentage of Integrated Environmental Authorizations analysed by regions. Fig. 2. Percentage of Integrated Environmental Authorizations analysed by regions. 139 The Implementation of IPPC Directive in the Mediterranean Area 5.4.1 The requirements and conditions indicated in the permits to protect from contamination of soil and groundwater The permits require the highest possible frequency of controls and prescribe the protection of contamination of soil and groundwater, by means of measures for the storage of chemicals. In many cases the permits require a containment basin for the storage or prescribe a spillage kit. p p g Being an expensive activity, the requirement about monitoring the quality of groundwater is particularly significant, and as can be expected it is imposed most of all to landfills. Requirements about protection of contamination of soil and groundwater State Spain Slovenia Greece Italy Region Andalusia Valencia Slovenia West Macedonia Piedmont Sicily Tuscany Preliminary Report on the soil 43,8% 0,0% 58,3% 0,0% 0,0% 0,0% 0,0% Storage of chemical products 37,5% 92,6% 100,0% 100,0% 54,4% 12,5% 19,1% Spill walls 15,6% 3,7% 0,0% 100,0% 0,0% 0,0% 0,0% Draining and collection system 15,6% 0,0% 100,0% 100,0% 54,4% 12,5% 8,5% Proofs of leakage detection and watertight 12,5% 3,7% 100,0% 0,0% 5,1% 0,0% 0,0% Communication/information of some aspects 0,0% 14,8% 100,0% 0,0% 0,0% 0,0% 6,4% Control/analysis/monitoring of groundwater 3,1% 18,5% 0,0% 100,0% 25,3% 62,5% 14,9% Monitoring of ground-water level 3,1% 0,0% 0,0% 100,0% 25,3% 0,0% 10,6% Table 10. Requirements indicated in the permits about protection of contamination of soil and groundwater (disaggregate data for regions). Table 10. Requirements indicated in the permits about protection of contamination of soil and groundwater (disaggregate data for regions). Fig. 3. Requirements and conditions to protect from contamination of soil and groundwater (aggregate data for regions). Fig. 3. Requirements and conditions to protect from contamination of soil and groundwater (aggregate data for regions). 140 Environmental Management in Practice Another relevant requirement is related to the draining and collection system contained in 37,8% of permits. Another relevant requirement is related to the draining and collection system contained in 37,8% of permits. p In the table 10 the same requirements are classified according to an interregional perspective. The measures related to the monitoring of groundwater are mostly contained in the permits of Piedmont and West Macedonia. The need to submit a preliminary report on the soil is only required in Andalusia and Slovenia. The “measures related to the storage of chemical products” is the only requirement identified in at least 1 permit of each region. The other requirements show at least one region without frequency. As for emissions flowing in surface water, Slovenia presents a high variability in the limits imposed for COD. West Macedonia and Valencia have the same limit, lower than the limit required by the permits of the Italian Regions. For TSS the permits show one limit applied in each region. Slovenia and the Italian regions have the highest one (80 mg/l), while West Macedonia applies the strictest one. The permits of West Macedonia confirm the lowest 5.4.2 The emission limit values related to water emissions The table below indicates the Emission Limit Values in the four IPPC sectors analyzed (Ceramics -3.5-, Landfills -5.4-, Surface treatment of metals and plastic materials -2.6-, Paper production -6.1-) for which the destinations of industrial water emissions are the same (surface water and sewer), to compare limits among the seven regions of the project. Emission Limit Values related to industrial water emissions for Ceramics (3.5), Landfills (5.4), Surface treatment of metals and plastic materials (2.6), Paper production (6.1) (with data of number of permits) Destination Surface water Sewer Pollutants (mg/l) COD TSS Sulphates COD TSS Sulphates Andalusia n.a. (not available) n.a. n.a. 160 (2) 1550 (2) 30 (2) 35 (1) 42,5 (1) 800 (2) 400 (1) 800 (3) Valencia 125 (2) 60 (2) 250 (1) 400 (1) 1000 (1) 500 (2) n.a. Slovenia 100 (3) 120 (2) 150 (2) 500 (1) 268 (1) 80 (9) 3000 (3) 1900 (1) 1187 (1) n.a. 80 (2) 100 (1) 400 (1) 300 (1) 350 (1) 600 (2) 900 (1) 500 (1) West Macedonia 125 (3) 25 (3) 250 (3) n.a. n.a. n.a. Piedmont 160 (20) 80 (20) 1000 (20) 500 (7) 700 (1) 200 (7) 700 (1) 1000 (8) Sicily 160 (6) 80 (6) 1000 (6) 500 (6) 200 (6) 1000 (6) Tuscany 160 (14) 80 (14) 1000 (14) 500 (7) 3000 (1) 200 (8) 1000 (8) Table 11. Emission limit values related to industrial water emissions for all sectors Emission Limit Values related to industrial water emissions for Ceramics (3.5), Landfills (5.4), rface treatment of metals and plastic materials (2.6), Paper production (6.1) (with data of number of permits) As for emissions flowing in surface water, Slovenia presents a high variability in the limits imposed for COD. West Macedonia and Valencia have the same limit, lower than the limit required by the permits of the Italian Regions. For TSS the permits show one limit applied in each region. Slovenia and the Italian regions have the highest one (80 mg/l), while West Macedonia applies the strictest one. The permits of West Macedonia confirm the lowest As for emissions flowing in surface water, Slovenia presents a high variability in the limits imposed for COD. West Macedonia and Valencia have the same limit, lower than the limit required by the permits of the Italian Regions. For TSS the permits show one limit applied in each region. 6. Strengths and weaknesses identified in the analysis The Analysis carried out within the project, enhanced some strengths and some weaknesses of the IPPC matter, most of which reflecting the opinions of the Competent Authorities that issue the permits. One of the strengths of the Legislative Analysis, identified in Andalusia and Piedmont, is represented by the introduction - through national laws that implemented the IPPC Directive - of a single environmental permit which brings together all environmental-related permits. According to the Competent Authorities, in Piedmont the IPPC implementation has been a significant phase of reorganization and the introduction of the company’s monitoring and control plan is very important. In Tuscany a strength highlighted by the Legislative Analysis is that the environment is perceived as global and unique system that makes an integrated vision on enterprises activities possible. Another strength identified in Tuscany is that the IPPC Directive makes enterprises more motivated to achieve better work and activity levels also taking into account the environment preservation. The coordination among the Competent Authorities for issuing the permits is considered by West Macedonia, Sicily and Andalusia as a strength of the Legislative aspect of the IPPC Directive. As regards weaknesses, the Tuscan partners identified the considerable difference between the IPPC Directive principles and the real environment of enterprises applying for IPPC permit. So often the law adaptation to the firms and to the different situations is very difficult. Moreover, according to the Tuscan partners, the enforcement of the Directive does not take into account the complexity and the large number of environmental aspects to manage. One strength emerging in Piedmont from the Administrative Analysis is that the IPPC Directive enables companies to focus on the planning of future activities of environmental improvement. Tuscany identified as a strength the new and different conception of environmental authorizations that the Directive caused to companies and public administration. In West Macedonia and Sicily, the existence of one single authority responsible for administrative issues is considered a strength. Finally, simplifications in the authorization procedure for livestock categories and companies with environmental management systems are considered a positive factor in the Valencia region. As regards weaknesses linked to the administrative permit procedure, all regions see a problem in some delays in permit issue caused by several reasons. 5.4.2 The emission limit values related to water emissions Slovenia and the Italian regions have the highest one (80 mg/l), while West Macedonia applies the strictest one. The permits of West Macedonia confirm the lowest 141 The Implementation of IPPC Directive in the Mediterranean Area level also for the Sulphates with a value of 250 mg/l applied in three permits. The permits of companies from Slovenia include the highest limits for the same parameter achieving until a limit of 3000 mg/l imposed in three Authorisations. For the water emissions in sewer the limits change very much in the same region and among regions. One of the reasons could be the presence of a purification plant at the end of the industrial sewer. Often, the company responsible for the management of the purification plant can set the limits to be applied to companies that are connected to the sewerage. These limits are often decided by taking into account the characteristics of the purification plant and the number of connected companies. Thus each Management Body of purification plant could apply different limits. 6. Strengths and weaknesses identified in the analysis Delays are due to late application of regulations (Slovenia); to failure to meet deadlines of the Integrated Environmental Authorizations granting (West Macedonia and Andalusia); to the increased workload due to the need for the permit itself (Piedmont); to the absence of a deadline agenda for permit issuing (Tuscany); to the lack of human resources in the 142 Environmental Management in Practice Competent Authority organization, to the poor quality of the IPPC activity projects submitted by the operators; to the administrations and institutions that sometimes fail to meet deadlines in elaborating their reports (Valencia), and to the lack of authorities involved in the conference cycle (Sicily). Linked to the above aspect, the lack of preparation of the personnel of Competent Authorities, is a drawback for West Macedonia and Tuscany. There are no training activities and insufficient personnel in the Competent Authorities. Another weakness indicated by West Macedonia is the lack of exceptions or simplifications for enterprises certified according to EMAS or ISO 14001:04. Competent Authority organization, to the poor quality of the IPPC activity projects submitted by the operators; to the administrations and institutions that sometimes fail to meet deadlines in elaborating their reports (Valencia), and to the lack of authorities involved in the conference cycle (Sicily). Linked to the above aspect, the lack of preparation of the personnel of Competent Authorities, is a drawback for West Macedonia and Tuscany. There are no training activities and insufficient personnel in the Competent Authorities. Another weakness indicated by West Macedonia is the lack of exceptions or simplifications for enterprises certified according to EMAS or ISO 14001:04. g Some strengths and weaknesses have been identified for the Control and Inspection System Analysis as well. One positive aspect identified both by Andalusia and West Macedonia is the existence of clear and detailed definitions in the guidelines for operational and monitoring control and the measurement of environmental aspects in the Control and Monitoring Plans of the Integrated Environmental Authorization. Moreover, some aspects linked to the Competent Authorities in charge of controls and inspections are considered as strengths by some regions. Valencia indicates as a positive element the technical competence and the independence of the Environmental Quality Collaborating Entities, one of the Control Competent Authorities in Valencia, while Andalusia identifies as a positive aspect the appointment of a specific service to carry out the monitoring and inspection activities defined in the Environmental Control and Monitoring Plans. 6. Strengths and weaknesses identified in the analysis Still on this aspect, Sicily considers the existence of ARPA Provincial Department for each province a positive element. In Piedmont the punctual and systematic control of all environmental components is a positive aspect of the control system, but the difficulty in interpreting and understanding the rules about IPPC is considered by this region as a weakness. In Andalusia it is difficult to meet deadlines for the control and inspection activities listed in the Control and Monitoring Plans of the Integrated Environmental Authorizations. For West Macedonia a problem is the lack of specialized IPPC personnel and inspectors in control authorities. The latter is a weakness also indicated by Piedmont. 7. Conclusion According to European law, “the directive binds the Member State only about the results to obtain and leaves to its competence the way and the tools”. Also, according to the subsidiarity principle (art. 3 B Maastricht Treaty) the European Directives can bring some differences in the implementation among the Member States. This Analysis aimed at investigating how the IPPC Directive has been implemented and if the differences are able to affect cost-related competitiveness of firms subjected to the IPPC Directive and located in different Member States. Although this chapter only included a few results, it investigates the differences in permit procedures and contents, control and inspection systems included in the national and local legislative framework of the involved regions, in order to identify methodologies and approaches to reduce these differences as a top priority for the next phases of the MED IPPC NET Project. To this aim, the results obtained from the Analysis phase of the project were elaborated and assessed. As for the institutional analysis (legislative, administrative, control and inspection and content of authorisations analysis), significant differences emerged in the different regions. Among the most relevant issues, some concern the disparity of Competent Authorities for the permit issue (national, regional or provincial Authority) or the main modalities adopted by each region to assure the access to information and public 143 The Implementation of IPPC Directive in the Mediterranean Area participation in the permit issuing procedure. The deadline for issuing the permit, as well as the type of simplifications in the issuing procedure provided for specific categories of enterprises, also vary among the seven regions involved in the project, and time is a crucial factor for competitiveness. Most regions (and Countries) chose to enact some forms of simplifications to favour companies that developed and certified an environmental management system. One of the suggestions (that also relates to the duration of the permit), is to try and standardize at least the favourable conditions granted to EMAS-registered companies at the EU level. Many indications on the approach followed by the different Competent Authorities were provided also by the analysis of the permits. This also provided some insights on the differences among the investigated regions. For example, the detailed analysis of specific requirements of different environmental aspects outline a great distance between the approach chosen by the different Competent Authorities, especially as to the “typologies” of requirements. 7. Conclusion From the Analysis of all these aspects it is possible to specify some recommendations to improve the IPPC implementation within Europe. One of the most important is that the European Commission should promote national and regional actions concerning activities oriented to harmonize contents and approaches wherever there are many different Competent Authorities. It would be particularly useful to create a permanent forum for the monitoring and the comparison of different implementation modalities of the IPPC Directive, as it has been experimented within the MED IPPC NET project. In this way, it could be possible to provide timely feedback and suggestions to improve the whole system whenever these differences may cause excessive problems for some of the member states or a failure in achieving the Directive’s goals. Furthermore, to prevent disparities a “standard model” might be created at the European level, in order to coordinate contents among different Competent Authorities and Member States. Another recommendation could attain the setting up and the promotion of more specific and in-depth competences by training the personnel of competent and control authorities: the IPPC requires a holistic vision and wide qualification in many different environmental aspects to reach an integrated vision of environmental problems. 8. References Barros., M.C., Magán, A., Valiño, S., Bello, M.P., Casares, J.J. & Blanco, J.M. (2009). Identification of Best Available Techniques in the seafood industry: case study. Journal of Cleaner Production, Vol. 17, No. 3 , (February 2009), pp. 391-399. Bréchet, T. & Tulkens, H. (2009). Beyond BAT. Selecting optimal combinations of available techniques, with an example from the limestone industry. Journal of Environmental Management, Vol. 90, No. 5 , (April 2009), pp. 1790-1801. g ( p ) pp Entek UK Limites, (January 2008). Assessment of the implementation of the IPPC Directive in UK. Department for Environment, Food and Rural Affairs, Available from http://www.defra.gov.uk/environment/quality/pollution/ppc/background/doc uments/implementation-study.pdf European Commission (2008). Council Directive 2008/1/EC of 15 January 2008 Concerning Integrated Pollution Prevention and Control, Brussles. g Georgopoulou, E., Hontou, V., Gakis, N., Sarafidis, Y., Mirasgedis, S., Lalas, D.P., Loukatos, A., Gargoulas, Mentzis, A., Economidis, D., Triantafilopoulos, T. & Korizi, K. 144 Environmental Management in Practice (2008). BEAsT: a decision-support tool for assessing the environmental benets and the economic attractiveness of best available techniques in industry. Journal of Cleaner Production, Vol. 16, No.3 (February 2008), pp. 359-373. (2008). BEAsT: a decision-support tool for assessing the environmental benets and the economic attractiveness of best available techniques in industry. Journal of Cleaner Production, Vol. 16, No.3 (February 2008), pp. 359-373. Honkasalo, N., Rodhe, H., & Dalhammar, C. (2005). Environmental permitting as a driver for eco-efficiency in the dairy industry: A closer look at the IPPC directive. Journal of Cleaner Production, Vol. 13, No. 10-11, (August-September 2005), pp. 1049-1060. Karavanas, A., Chaloulakou, N. & Spyrellis, N. (2009). Journal of Cleaner Production, Vol. 17, No. 4 , (March 2009), pp. 480-486. Kocabas, A.M., Yukseler, H., Dilek, F.B. & Yetis, U. (2009). Adoption of European Union’s IPPD Directive to a textile mill: analysis of water and energy consumption. Journal of Environmental Management, Vol. 91, No. 1, (October 2009), pp. 102-113. f g ( ) pp Samarakoon, S.M.K. & Gudmestad, O.T. (2011). The IPPC directive and technique qualification at offshore oil and gas installations. Journal of cleaner production, Vol. 19 , No. 1 , (January 2011), pp. 13-20. Schoenberger, H. (2009). Integrated pollution prevention and control in large industrial installations on the basis of best available techniques – The Sevilla Process. Journal of Cleaner Production, Vol. 17, No. 16, (November 2009), pp. 1526-1529. f ( ) pp Silvo, K., Jouttijärvi, T. & Melanen, M. (2009). Contaminated Sites and Public Policies in São Paulo State, Brazil Ana Luiza Silva Spínola and Arlindo Philippi Jr. School of Public Health, University of São Paulo Brazil Brazil 1 CETESB is the institution that by law is in charge, among other assignments, of enforcing administrative policies for environmental issues throughout the entire State of São Paulo, as established by Laws no. 118/73 and no. 997/76. 8. References Implications of regulation based on the IPPC Directive – A review on the Finnish pulp and paper industry. Journal of cleaner production, Vol. 17, No. 8, (May 2009), pp. 713-723. Styles, D., O’Brien, K., & Jones, M. (2009). A quantitative integrated assessment of pollution prevention achieved by Integrated Pollution Prevention Control licensing. Environment International, Vol. 35, No. 8, (November 2009), pp. 1177-1187. 7 1. Introduction The existence of contaminated sites is an environmental issue that has been increasingly detected by environmental authorities in Brazil, most specifically in the State of São Paulo. The state government is in charge of the environmental management of contaminated sites through its environmental agency, the Environmental Company of the State of São Paulo [CETESB]1. Since the beginning of the 90’s the agency carries out the systematic management of these sites. g The environmental agency has pursued its own structuring to meet this growing demand, enabling its effective action and finding solutions for this serious environmental problem. Thus, since the beginning of the nineties, due to the German expertise and know-how regarding this subject, a technical cooperation was established with the German government, through its Technical Cooperation Society (Deutsche Gesellschaft für Technische Zusammenarbeit, GTZ), which encompasses both technical and financial support. This cooperation allowed the development of a specific project in order to build the capacity of the state agency for the management of contaminated sites (CETESB, 2011a). y g y g The origin of contaminated sites is related to a lack of knowledge, back in time, about safe procedures for the handling of hazardous substances, to the disrespect of such procedures and to accidents or spillings during the development of production processes, transportation or storage of raw materials and products (CETESB, 2011b). According to CETESB (2011a) “the soil and underground water contamination have been the focus of great concern for the last three decades in the industrialized countries, mainly in the USA and Europe. This environmental problem becomes more serious in urban industrial centres such as the Metropolitan Region of São Paulo”. The State of São Paulo has 3.675 contaminated confirmed sites (CETESB, 2011b). This inventory has been published since the year of 2002. Table 1 shows the distribution of contaminated sites registered in São Paulo by region and activity, regarding the last data published by CETESB on December 2010. Cunha (1997) considers that it is the inactive industries which more critically affect the environment and the population, among the contaminating sources in total. 1 CETESB is the institution that by law is in charge, among other assignments, of enforcing administrative policies for environmental issues throughout the entire State of São Paulo, as established by Laws no. 118/73 and no. 997/76. 2 From the conceptual point of view, it is important to highlight that the term “Brownfield” corresponds to a site degraded by previous use, which was abandoned or is under-utilized. It may or may not have real or perceptive contamination problems. Most are located in urbanized areas and require some type of intervention for a potential re-use as defined in the “Sustainable Brownfield Regeneration: CABERNET Network Report” (CABERNET, 2006, p. 26). Up until now the term “Brownfield” has not been translated into Portuguese. Sánchez (2001, p. 29) suggests the translation into Portuguese as “dysfunctional real estate/property”, for there is no possibility that the sites hold a new function without some type of treatment. Thus, the contaminated site (degraded site by contamination) is a type of Brownfield as described above. 1. Introduction 146 Environmental Management in Practice In addition, according to Sánchez (2001), it is possible to relate this issue to the life cycle of factories that had their doors shut for various motives: either because they had lost economic competitiveness or because their location became less advantageous. This means that there is the need for modernization and re-use, including potential interim uses up to the point of a potential industrial or other re-use. Cases such as these are part of urban dynamics and deserve special attention. Nowadays those are one of the main urban conflicts to be solved in cities worldwide. Region Activities Commercial Industrial Residues Gas Stations Accidents Total São Paulo 39 114 28 1.004 5 1.190 São Paulo Metropolitan Region - others 29 125 20 419 6 599 Country Side 60 158 40 1.105 12 1.375 Coastal Area 15 40 21 223 1 300 Paraíba Valey 4 34 1 171 1 211 Total 147 471 110 2.922 25 3.675 Table 1. Contaminated sites in São Paulo State, Brazil – December 2010 Table 1. Contaminated sites in São Paulo State, Brazil – December 2010 The existence of a contaminated site becomes the generator of many problems, such as the threat to superficial and underground water sources, the devaluation of properties, besides the threats to public health. Brazil does not have yet a national public policy, approved by law, about contaminated sites or brownfield redevelopment2. Therefore many contaminated sites have been reused for new commercial or housing purposes without any type of control from municipal public authorities – potentially ticking time bombs, entailing possibly serious health and environmental risks in the near future. As the appearance of contaminated sites is daily increasing, being a considerable problem mainly in the industrial cities, the State of São Paulo, breaking new ground, has approved on July 2009 a public policy, by which main regulatory aspects and legal framework on the issue are established, setting the tone for new legislation on this matter by the other states of the Brazilian Union. 147 Contaminated Sites and Public Policies in São Paulo State, Brazil A National Environmental Council Resolution has also been approved, on a federal basis, establishing guidelines values for soil and underground water, as well as environmental directions for contamination management. 2. Methodology The information and data collection methods combined literature and documental research. The literature research included bibliographies on contaminated sites management. The documental research included basically official documents published by CETESB, some federal legislation and the new legislation on contaminated sites management approved by the State of São Paulo (Act n. 13.577, of july 8th 2009). Given the newness of the law, there is no literature about it. 1. Introduction The objective of this paper is to comment the management of contaminated sites carried out by the State of São Paulo and to present its new legislation. 3.1 Environmental competences as determined by the constitution The State Act is composed of 6 chapters: i) general provisions (including the object, objectives, technical definitions and measures), ii) soil contamination prevention and 148 Environmental Management in Practice control, iii) contaminated site (divided in responsibilities, identification and remediation), iv) economical tools, v) infractions and penalties and vi) general provisions. The object is to protect the soil integrity against harmful contamination alterations, to establish liabilities, identify, register and remediate the contaminated sites in a way to make it safe for present and future use. As a general objective, it was established the sustainable soil use clause, protecting it from contaminations and preventing alterations on its natural features and functions by means of: i. preventive measures, including the protection of soil and underground water natural ti As a general objective, it was established the sustainable soil use clause, protecting it from contaminations and preventing alterations on its natural features and functions by means of: As a general objective, it was established the sustainable soil use clause, protecting it from contaminations and preventing alterations on its natural features and functions by means of: i. preventive measures, including the protection of soil and underground water natural properties; ii. corrective measures including procedures for contaminated sites identification, protection of the health and safety of the population exposed to the contamination; remediation ii. corrective measures including procedures for contaminated sites identification, protection of the health and safety of the population exposed to the contamination; remediation iii. incentive measures for the reuse of remediated sites; iv. promotion of exchange programs among institutions; v. guarantee to information and to participation by the population affected by decisions related with the contaminated areas. v. guarantee to information and to participation by the population affected by decisions related with the contaminated areas. 3 National Environmental Council 3.1 Environmental competences as determined by the constitution The Brazilian Federal Constitution establishes that “the protection of the environment and fighting pollution in any of its forms” is a common competence of the Union, the States, the Federal District and the Municipalities, according to article 24, I and IV. The competence to legislate upon the natural resources and soil protection, environment protection and pollution control is concurrent to the Union, the States and the Federal District, according to article 24, VI. With regard to Municipalities, the Brazilian Federal Constitution attributes competences to "legislate on matters of local interest", "complementation of federal and state laws, when applicable", and "promotion, when applicable, of adequate territorial order, through planning and control of urban land use, parceling and occupation", amongst others (art. 30, paragraphs I, II and VIII respectively). Although the environmental quality of urban sites depends on State control, the municipalities also have the duty to enforce this control, since their attribution to manage the use and occupation of the land is established by the Constitution. 3.2 Commentaries on the act n 13.577/2009 of the State of Sao Paulo 3.2 Commentaries on the act n 13.577/2009 of the State of Sao Paulo Even before the approval of the 2009 State Act CETESB had already developed specific procedures to manage contaminated sites based on “Executive Acts” n. 007 and n. 023 initially approved on the year 2000. 3.2 Commentaries on the act n 13.577/2009 of the State of Sao Paulo Even before the approval of the 2009 State Act CETESB had already developed specific procedures to manage contaminated sites based on “Executive Acts” n. 007 and n. 023 initially approved on the year 2000. On the year 2007 those statutes were revised and updated on a new Executive Act n. 103, which foresees a methodology to manage such sites aiming at their rehabilitation for future use, legal responsibilities for the contaminated site management, technical studies to be made in order to identify and delimit the contamination, emergency actions to be undertaken, remediation techniques, institutional and engineering control measures, monitoring deadlines and communication procedures towards governmental entities (such as the state health department and the municipalities, amongst others). a) Contaminated sites registration a) Contaminated sites registration The database will be composed by detailed information about: i) potentially pollutant activities and enterprises; ii) which, in the past, had promoted activities liable to provoke soil contamination; iii) which are under contamination suspicion iv) any other applicable cases; and will be published in the State of São Paulo Official Gazette and on the State of São Paulo Environment Department website. As mentioned the environmental agency of São Paulo is already keeping a database of contaminated and reabilitated areas which has been published in its website since 2002. b) Licenses and enforcement b) Licenses and enforcement 3.2.1 Definitions and tools As a mainly technical environmental statute, a whole section has being dedicated to the definition of mostly important terms for the soil quality management. Throughout this essay some technical and legal definitions shall be presented. A number of tools have been determined for the soil quality protection and contaminated sites management, among some of those that follow below: a) Contaminated sites registration b) Licenses and enforcement The licensing and the enforcement activities are important tools both in regards to the preventive and the corrective aspects in the management of contaminated sites. Environmental licensing is the administrative procedure under which the environmental agency licenses the location, installation, enlargement and operations of undertakings and activities using environmental resources, which are considered effectively or potentially pollutant, or those which, under whatever way, may cause environmental degradation (CONAMA3, 1997). ) procedure is divided into 3 phases, under which the following documents are issued: i. Preliminary License (granted in the undertaking’s planning phase, approving its location, conception and environmental viability); i. Preliminary License (granted in the undertaking’s planning phase, approving its location, conception and environmental viability); 149 Contaminated Sites and Public Policies in São Paulo State, Brazil ii. License for Installation (authorizes the undertaking’s installation according to the preliminarily approved specifications); and ii. License for Installation (authorizes the undertaking’s installation according to the preliminarily approved specifications); and ii. License for Installation (authorizes the undertaking’s installation according to the preliminarily approved specifications); and iii. License for Operations (authorizes the undertaking’s operation, after checking the compliance to previous licenses). iii. License for Operations (authorizes the undertaking’s operation, after checking the compliance to previous licenses). p p ) In the State of São Paulo the pollution sources, which depend on environmental licensing, are provided for in the Decree no. 8468/76. In the process of licensing an industry one should check if the activities performed may contaminate the soil or underground waters. Considering that the great majority of pollution sources depend on the license to operate, the occurrence of contaminated sites may be avoided in advance by including the suitable technical requirements in the undertaking’s installation or operation license, such as for example the obligation for adequate disposal of chemicals stored or of waste generated in the production process. Otherwise, from the correction stand point, it is possible that the undertaking’s operation license renewal be linked to the identification or remediation actions. c) Deactivation Plan The Act establishes that the entities legally responsible for undertakings, which are subject to environmental licensing and potential contamination generators, about to be totally or partially deactivated or disoccupied, must communicate the suspension or closing of activities to the competent state agency. At this moment a Deactivation Plan should be presented, contemplating the existing environmental condition, especially regarding the existence of contamination, and should contain, when applicable, information about the remediation measures to be taken. Sánchez (2005) highlights the deactivation planning importance. Shutting down is a stage of the industrial activities cycle of life in which the environmental damages accumulated throughout the previous enterprise stages should be repaired, to avoid conveying the reparation cost to either third parties or society. y Since year 2002, in the State of São Paulo, the obligation to present a deactivation plan for the termination of activities subject to environmental licensing is already in force (according to Decree n. 47.400/2002). d) City master plan and land use and occupation legislation This provision is timely to reinforce the need for municipalities to effectively take part in the prevention and management of contaminated sites. g Municipalities have a power-duty to act in the management of contaminated sites under two aspects: i) they hold the common competence with the Union, States and Federal District to protect the environment and fight pollution (since contamination, from the legal stand point, is a type of pollution), and ii) the hold the competence to legislate on matters of local interest and to promote territorial ordering through the planning and control of urban land use. The Master Plan is the basic instrument for urban development policies. The Act acknowledged the importance of the municipal action in the prevention and management of contaminated sites and has often summoned the municipality to take on its responsibility. The Master Plans and the legislation regarding land use and occupation should take into account the sites either potentially contaminated or under the suspicion of contamination, and the confirmed contaminated sites. The approval of land parceling and construction projects should guarantee the safe use of such sites. 150 Environmental Management in Practice Vis-à-vis the express determinations provided for in the Act, in practice, municipalities of the State of São Paulo should organize themselves administratively to tackle this issue by creating specific statutes and administrative procedures, providing equipment and technical capacity building of human resources, etc. c) Deactivation Plan In addition they are responsible for the land use restriction, in case the contaminated site poses health risks, so to avoid the receptors exposure to the existent contaminants. During the different phases of the contaminated sites management the local governments should be notified by the competent environmental agency when, e.g., the area is classified as contaminated under investigation, contaminated or remediated for the declared use (art. 18, clause II; art. 24, clause IV; and art. 27, clause III, respectively). p y) It is therefore not enough that local governments be notified about the presence of contaminated sites, they should know what to do with this information. Questions such as – “What should be done about a contaminated site?” “What are the consequences for urban planning in the presence of contaminated sites?” “Is there a risk to the extent that the land use should be restricted?” “How to restrict the land use?” “Which planning instruments can be created to attract investors to a contaminated site?” – need still to be discussed. Lazanha (2005) highlights that “the Municipality, as a federative entity holding the competence of territorial ordering, should be held accountable for corrective and pro-active measures in these sites, thus avoiding the spreading of further damages to society with the construction of housing and other buildings in contaminated sites” (p. 116). In this context, Marker (2003) highlights the importance of assessing – within the Brazilian context – which are the pre-requisites to “identify and mark the degraded sites in master plans and land use, to make diagnoses and develop strategies for their revitalization” (p.40). In São Paulo, despite the action of the state agency, there is urgent need to involve the municipalities on this issue. At the municipal level, however, there are neither instruments nor any kind of action to enforce the intervention on contaminated sites. In the State of São Paulo an exception is the municipality of São Paulo where urban planning provides the control of contaminated sites. e) “Bank collaterals” and “environmental insurance” Such tools, which still need to be regulated, were provided to ensure the fulfillment of the remediation plan and should reach the minimum value of 125% of the Plan’s estimated cost. In case of non-compliance there is a provision for the possibility that the environmental agency execute the collaterals aiming at covering the remediation measures complementary costs. 4 The Act defines “the holder of the right of surface use” as the “holder of the right to the surface of a land lot, for a determined or undetermined period of time, through a public deed registered in the Property Registry Office, in the terms of the Federal Law no. 10257, July 9th, 2001. According to this law of 2001 (named Statute of the City, that establishes the general guidelines of the urban policy) the right to the surface encompasses the right to use the ground, the underground or the land related aerial space, in the form established in the respective contract, meeting the urbanistic legislation (paragraph 1st of art. 21). h) Environmental compensation The environmental compensation was regulated by the State of Sao Paulo Decree no. 54.544/2009. In the environmental licensing of enterprises, potentially generating contaminated sites (to be defined by an executive act of the Environment Secretary), the entrepreneur will collect a value – to be determined by the competent agency – as a means of compensation to the Fund. c) Deactivation Plan f) Quality criteria for soil and underground waters In regards to the control of air and superficial waters pollution, the environmental legislation established standards of emission and quality, while the soil quality control is conducted through the use of “guideline values”. In regards to the control of air and superficial waters pollution, the environmental legislation established standards of emission and quality, while the soil quality control is conducted through the use of “guideline values”. “The adoption of guideline value lists has been the usual practice in countries with a tradition in soil and underground waters quality monitoring and in contaminated sites’ control” (CETESB, 2001, p. 10). “In a distinct manner there is no standardized international approach – regarding soil pollution – because of its complex and variable nature, and the soil is a privately owned economic asset” (CETESB, 2001, p.14). These “guideline values” are determining tools for the soil and underground waters quality management, since they are the fundamentals for making important decisions both These “guideline values” are determining tools for the soil and underground waters quality management, since they are the fundamentals for making important decisions both 151 Contaminated Sites and Public Policies in São Paulo State, Brazil preventive, and of pollution control in sites under the suspicion of contamination, as well as corrective, since they are indispensable for the area to be considered contaminated legally. This will eventually deploy a series of actions from the legally responsible agency and the Public Power. After specific studies which took into account the natural quality of the State of São Paulo soil, in 2001, CETESB published the “Report for the establishment of Guideline Values for Soils and Underground Waters in the State of São Paulo”. This report shows the approach of the USA, Germany and Holland in regards to the subject and it also reports the methodology adopted by the State of São Paulo for the establishment of its own values. The values published in 2001 were revised and nowadays those approved by the CETESB Executive Act n. 195/2005/E are ruling. g) Fund As it occurs in countries such as the USA, a specific Fund for contaminated sites can be used in cases where the site is considered “orphan”, i.e., where the responsible entity is not identified or localized. The Act provided for the creation of the State Fund for the Prevention and Remediation of Contaminated Sites, which is an investment fund linked to the Environment Department of the State of São Paulo and aimed at soil protection and contaminated sites’ identification and remediation. Different sources of revenue were considered, among which resources resulting from international aid and cooperation and from intergovernmental agreements; donations; environmental compensations resulting from potentially contaminating activities; 30% of the amount collected with fines applied based on the Act; reimbursement of State expenditures, etc. h) Environmental compensation 5 Article 264 of the Civil Code (Law no. 10.406/2002) establishes that “there is solidarity when in the same obligation more than one creditor or more than one debtor concur, each one with the right, or obliged to the whole debt”. 6 RESP n. 650.728 – SC 3.2.2 Legally responsible entities for the identification and remediation of contaminated sites The Act established the following subjects as legally, and solidarily, responsible for the prevention, identification and remediation of a contaminated site: the one that caused the contamination and its successors; the owner of the area; the “the holder of the right of surface use”4, the holder of the actual possession; whoever benefits directly or indirectly from it. 152 Environmental Management in Practice Solidarity is a legal concept provided for in the civil law5, and in the case of contaminated sites it is possible to demand total obligation (e.g. of remediation) from all the responsible listed above. The responsibility of the contaminated site’s owner is transmitted to the next owner in the case, e.g., of the sale of the contaminated land. The jurisprudence related to the matter has been understanding that a real estate environmental obligation (e.g. that of maintaining the green areas and the permanent preservation areas) is transmitted to the new acquirer. It is worth mentioning a recent decision held by the Brazilian Superior Court of Justice on an appeal reported by Justice Herman Benjamin: “the obligations derived from the illegal dumping of waste or residues on the land are of propter rem nature, meaning that they adhere to the title and are transferred to the future owner (…) regardless any discussion about the good or bad faith of the acquirer, for one is not in the condition of subjective responsibility based on the establishment of a personal guilt”6. The responsibility of the contaminated site’s owner is transmitted to the next owner in the case, e.g., of the sale of the contaminated land. The jurisprudence related to the matter has been understanding that a real estate environmental obligation (e.g. that of maintaining the green areas and the permanent preservation areas) is transmitted to the new acquirer. green areas and the permanent preservation areas) is transmitted to the new acquirer. It is worth mentioning a recent decision held by the Brazilian Superior Court of Justice on an appeal reported by Justice Herman Benjamin: “the obligations derived from the illegal dumping of waste or residues on the land are of propter rem nature, meaning that they adhere to the title and are transferred to the future owner (…) regardless any discussion about the good or bad faith of the acquirer, for one is not in the condition of subjective responsibility based on the establishment of a personal guilt”6. 3.2.3 Contaminated sites preventive actions As a general rule the Act provided that any individual or legal entity that may contaminate the soil should adopt the measures for not to allow the occurrence of changes that are significant and damaging to the functions of the soil. The functions of the soil were listed as: 1- sustainability of the life and “habitat” for humans, animals, plants and soil organisms; 2 – maintenance of the water cycle and its nutrients; 3 – protection of underground water; 4 – maintenance of the historical, natural and cultural heritage; 5 – conservation of the reserves of minerals and raw-materials; 6 – food production; 7 – means for the maintenance of social-economic activity. p y Environmental agencies were then obliged to act preventively and correctively with the purpose of preventing significant changes in the functions of the soil. As a parameter for actions, the use of the following guideline values (already cited above) was provided for: y Environmental agencies were then obliged to act preventively and correctively with the purpose of preventing significant changes in the functions of the soil. As a parameter for actions, the use of the following guideline values (already cited above) was provided for:  quality reference value is the “concentration of a determined substance in the soil and in the underground water that defines a soil as clean, or the natural quality of underground water”. These are to be used to guide the soil functions prevention and control policy.  prevention value is the “concentration of a determined substance above which harmful changes to the quality of soil and underground water may occur.” They shall be used to discipline the introduction of substances in the soil. In case they are surpassed there will be the need of monitoring resulting impacts.  intervention value is the “concentration of a determined substance in the soil and in the underground water, above which there are potential direct and indirect risks to human health, considering a generic exposure scenario”. These will be used to stop the continuous introduction of polluting loads in the soil. 3.2.4 Identification of contaminated sites 3.2.4 Identification of contaminated sites In the initial phase of contaminated sites identification, two concepts are important: i) “contamination potential area”: area, land, location, facility, construction or improvement 3.2.4 Identification of contaminated sites In the initial phase of contaminated sites identification, two concepts are important: i) “contamination potential area”: area, land, location, facility, construction or improvement 153 Contaminated Sites and Public Policies in São Paulo State, Brazil where activities, are or were developed and that due to their characteristics may accumulate amounts or concentrations of materials in such conditions which render it contaminated; and ii) “area under the suspicion of contamination”: site, land, location facility, construction or improvement with traces of being a contaminated site. where activities, are or were developed and that due to their characteristics may accumulate amounts or concentrations of materials in such conditions which render it contaminated; and ii) “area under the suspicion of contamination”: site, land, location facility, construction or improvement with traces of being a contaminated site. The Act foresees that when detecting traces or suspicions that a site is contaminated, the legally responsible entity should immediately communicate such fact to the competent environment and health agencies. The technical study that is to be conducted is the “preliminary assessment”, which has the purpose of clarifying if the contamination suspicion proceeds or not. This assessment was defined as an “initial assessment, based on available information, to fundament the suspicion of contamination in a site”. In case there is a fundamented suspicion of contamination one should start the “confirming investigation”, which “aims at proving the existence of a contaminated site”. This investigation is basically composed of the capturing and chemical analyses of samples and the interpretation of the results. The results are to be compared with the intervention values for soils and underground waters established by the CETESB. In case the concentrations observed in loco are above these values the site will be classified as “contaminated site under investigation”, which has been defined as the “contaminated site on which procedures are taking place to determine the extension of the contamination and the affected receptors”. In this management phase obligations were established for the environmental agency – such as, for example, carrying out the preliminary assessment at the site or request the entity responsible to adopt measures, demand confirmatory investigation – once significant changes harmful to the soil functions are detected. 3.2.4 Identification of contaminated sites In addition, the environmental agency must notify the State agencies involved (especially the organization in charge of granting the right of use of underground waters, the Municipalities, the Municipal Environmental Boards and other stakeholders), as well as determine the legally entity in charge that may start emergency action procedures. The environment and health agencies shall implement a program that guarantees the affected population, through its representatives, the access to available information and participation in the site assessment and remediation process. 3.2.5 Rehabilitation and remediation of contaminated sites One starts from the premise that “certain soil uses require it to have excellent quality – such as for housing and recreation – while other uses are less demanding, such as for industrial purposes or paved parking lots” (Sánchez, 2001, p. 131). Regarding the history of contaminated sites management in 16 European countries, Ferguson (1999, p. 33) mentions that “twenty or so years ago land contamination was usually perceived in terms of relatively rare incidents, with poorly known but possibly catastrophic consequences for human health and the environment. (…) As a result politicians responded by seeking maximum risk control: pollution should be removed or contained completely”. However it is now widely recognized that drastic risk control, for example cleaning up all sites to background concentrations or to levels suitable for the most sensitive land use, is neither technically nor economically feasible” (Ferguson, 1999, p. 33). However it is now widely recognized that drastic risk control, for example cleaning up all sites to background concentrations or to levels suitable for the most sensitive land use, is neither technically nor economically feasible” (Ferguson, 1999, p. 33). y y g To give an example, according to Ferguson (1999, p. 33) “in 1981 about 350 sites in the Netherlands were thought to be contaminated and possibly in need of remedial action. By 1995 the number had grown to 300,000 sites with and estimated clean-up cost of 13 billion ECU7. Similar circumstances exist in most other industrialized countries. Consequently, although the need for policies to protect soil and groundwater is recognized, strategies for managing contaminated land have moved towards fitness for use”. To give an example, according to Ferguson (1999, p. 33) “in 1981 about 350 sites in the Netherlands were thought to be contaminated and possibly in need of remedial action. By 1995 the number had grown to 300,000 sites with and estimated clean-up cost of 13 billion ECU7. Similar circumstances exist in most other industrialized countries. Consequently, although the need for policies to protect soil and groundwater is recognized, strategies for managing contaminated land have moved towards fitness for use”. The article written by Ferguson (1999) allows us to conclude that the management of contaminated sites in the 16 European countries studied is fundamentally based on the assessment of risk, be it to human health or to the environment. 3.2.5 Rehabilitation and remediation of contaminated sites The site rehabilitation process main objective is to “rehabilitate the site for a pre-established purpose”. The remediation goals to be achieved should be defined based on the scenario of the soil future occupation. p After the site is classified as “contaminated under investigation”, new studies are to be carried out to detail the amplitude of the contamination. p The legally responsible entity shall carry out a “detailed investigation” to establish a knowledge around the total extension of the contamination and the identification of all the receptors under risk. This investigation was legally defined as a “process of field data acquisition and interpretation which allows the understanding of the dynamics of the contamination plumes in each one of the physical means affected”. In case a water supply source is compromised, the entity responsible for the contamination shall provide an alternative drinking water source to supply the affected population. It is important to clarify that typically a contaminated site may be remediated focusing one between two possible future situations: that the soil be compatible with one previously 154 Environmental Management in Practice determined use (“fitness for use approach”), or with all possible uses (“multifunctionality of soil approach”):  Fitness for use clause: a health risk assessment is conducted and takes necessarily into account the scenario of future use of the land and ways through which people are (or may be) exposed to existing contaminants. The risk is minimized so it is possible to maintain the contaminants within the site, as long as this risk is manageable and maintained at an acceptable level. The site remediation is made as far as necessary to allow its use for the previously established purpose. Such decision is fundamented on “guideline values” for soils and underground waters (explained ahead) which vary according to each exposure scenario. In this case the site is rehabilitated for a determined purpose.  Multifunctionality of soil clause: the risk is eliminated by the removal or total destruction of the polluting substances. The site is recovered to its natural conditions (i.e., concentrations prior to contamination), and may be used for whichever purposes. In this case, remediation costs may be infinitely higher than in the previous hypothesis, even causing the non feasibility of site intervention. 7 European Currency Unit 3.2.5 Rehabilitation and remediation of contaminated sites Most of the countries adopt the “fitness for use approach” (or function oriented approach, suitable for use approach, cost- effective approach), i.e., the future use of the land is taken into account to define the remediation goals to be achieved. This is of great importance for remediation to be successful and efficient both from the technical and the economic point of view. p Furthermore, guideline values are adopted for different land use scenarios as a means to assess the risk of a determined area and based on which the objectives of remediation will be defined. Eventually, the need and tendency to take into account the economic aspect to 155 Contaminated Sites and Public Policies in São Paulo State, Brazil find solutions for a contaminated site stood out, and today managing sites aiming at the multifunctionality of soil approach is an exception. find solutions for a contaminated site stood out, and today managing sites aiming at the multifunctionality of soil approach is an exception. find solutions for a contaminated site stood out, and today managing sites aiming at the multifunctionality of soil approach is an exception. In this respect Cunha (1997) underlines that a point in common to all countries which elaborated contaminated sites management programs, was the high number of sites and the amount of resources necessary to their remedial. There is thus the need of evaluating the real need of remediating a contaminated site, considering the existing levels of contamination and the potential to cause harm to the health of the population. “Such procedure is generically known as health risk assessment and has been adopted in some countries as a way to determine the real need for a site remediation, as well as in the definition of the remediation system to be put in place” (p.09). In this respect Cunha (1997) underlines that a point in common to all countries which elaborated contaminated sites management programs, was the high number of sites and the amount of resources necessary to their remedial. There is thus the need of evaluating the real need of remediating a contaminated site, considering the existing levels of contamination and the potential to cause harm to the health of the population. 3.2.5 Rehabilitation and remediation of contaminated sites “Such procedure is generically known as health risk assessment and has been adopted in some countries as a way to determine the real need for a site remediation, as well as in the definition of the remediation system to be put in place” (p.09). Following the trend of countries that have a tradition in managing contaminated sites, the State of São Paulo adopted the “fitness for use” approach, finally putting an end to the issue related to how extensively a site should be remediated. The Act established the risk assessment as a subsidy for making decisions regarding interventions for remediation purposes to take place in a contaminated site. “Risk assessment” was legally defined as “the process through which risks to human health, to the environment and to other assets to be protected are identified, evaluated and quantified”. When the values defined for acceptable risk are surpassed, the site will be classified as “contaminated site” and its remediation is to be provided. The expression “contaminated site” was defined as “area, land, location, facility, building or improvement that contains amounts or concentrations of materials in such conditions that cause or may eventually cause harm to the human health, to the environment or to any other asset that is to be protected”8. Thus the legal concept of contaminated site is not precise and unequivocally limited, but it derives from the surpassing of acceptable risk values, that will be analyzed, in the concrete case, by means of a specific “risk assessment”. The site remediation will include the “adoption of measures for the elimination or reduction of risks to acceptable levels for the declared use”, according to definition established by the Act. After the site is classified as contaminated, the environmental agency shall inform the health agencies, when there is risk to human health, notify other public agencies involved, start the remediation procedures in tune with the ongoing emergency actions and demand the presentation of a remediation plan from the legally responsible entity. During this phase of the site management it is an attribution of the environmental agency to determine to the legally responsible entity the contaminated site status recording on the real state registration. Such imposition has been widely discussed about due to a possible conflict with article 22, clause XXV, of the Brazilian Constitution that says that “the Union has exclusive legislative competence on public recordings issues”. 8 The legal definition of contaminated site was provided for the first time in the Brazilian legislation, by the State of São Paulo Solid Residues Policy for (Law no. 12300, March 16th, 2006), and it is identical to the definition brought by the Act now discussed. 3.2.6 Infractions and penalties The actions or omissions contrary to the Act are considered administrative infractions and will be punished with written warnings, fines, embargo, demolition or suspension of funding and of tax benefits. g A limit was imposed to the fine penalty, ranging from 4 to 4,000,000 times the value of the Fiscal Unit of the State of São Paulo – UFESP (currently calculated at approximately R$ 17,45), and may not surpass the value of R$ 50,000,000 (fifty million reais). 3.2.5 Rehabilitation and remediation of contaminated sites However, even before the arrival of the State Act n. 13.577/2009, record on contaminated sites real state registration was already a procedure adopted by CETESB in respect to the Executive Act n. 167/2005 rendered by the Judiciary General Corrective Office, published on the State of Sao Paulo Official Gazzete on June 12th 2006. 8 The legal definition of contaminated site was provided for the first time in the Brazilian legislation, by the State of São Paulo Solid Residues Policy for (Law no. 12300, March 16th, 2006), and it is identical to the definition brought by the Act now discussed. 156 Environmental Management in Practice With all that, any possible acquirer could have available any information about past or present contamination related events on the real state. When an acceptable risk level is reestablished for the declared use, the site will be classified as “remediated site for declared use”, which is defined as “area, land, location, facility, building or improvement previously contaminated, which, after being submitted to remediation, has reestablished the level of risk acceptable to human health, considering the declared use”. Thus, besides information about the contamination (“Contaminated Site Term”) CETESB also emits the “Rehabilitation of the Site for Declared Use Term” which includes the kind of use the site is specifically rehabilitated for, that will also be recorded on the real state registration. 4. Conclusions The Act n. 13.577/2009 constitutes the first public policy issued in Brazil about contaminated sites management. It came to fulfill a great legal, as well as technical gap. It brought to the Brazilian Environmental Law important definitions on technical terms, it established the legal entities responsible for the contaminated site remediation and it determined the management stages to be followed. g g It also adopted the risk assessment as an important decision making tool as far as the site intervention will be developed on the terms of the “fitness for use” approach. The Fund created by the Act, following a tendency on countries with a established tradition on contaminated sites management will specially contribute to an effective management of so adopted the risk assessment as an important decision making tool as far as the site rvention will be developed on the terms of the “fitness for use” approach. The Fund created by the Act, following a tendency on countries with a established tradition on contaminated sites management, will specially contribute to an effective management of certain “orphan” and abandoned sites as it habilitates and financially enable the State`s action. Under the institutional aspect the Act established an “exchange among institutions” as one of many ways of reaching its goal. On the various management phases it will be necessary for the involved public bodies to communicate with each other imposing an effective and efficient information exchange in a way that each entity, in its particular area of competence, establishes the necessary procedures on it administrative routine. That was exactly the objective of the article 50 that states that “the State Environment Secretary and Health Secretary should establish common procedures and routines for any joint campaigns aiming the prevention or formation of contaminated sites as well as the identification and remediation of existing ones”. We point out that the municipalities also should be inserted in such a process in a way to specially consider the adoption of this issue in its urban and environmental planning. p g Under the important aspect of publicity the Act has institutionalized the contaminated sites registration database, imposing the publication of such information on the State 157 Contaminated Sites and Public Policies in São Paulo State, Brazil Environment Secretary website as well as on the State Official Gazzete. 4. Conclusions On an innovative fashion, meaning an important move to make the information available to the general public, it determined the record of contamination site status and of rehabilitated site status on the real state registration. g Some important issues still need to be regulated, for example: the way the participation of the affected population will take place on decisions regarding a site intervention; specification of “bank collaterals” and “environmental insurances” as financing tools to guarantee the site remediation; and effective creation of tax and crediting incentives. Some important issues still need to be regulated, for example: the way the participation of the affected population will take place on decisions regarding a site intervention; specification of “bank collaterals” and “environmental insurances” as financing tools to guarantee the site remediation; and effective creation of tax and crediting incentives. This innovative Act will probably set a national tendency in a way that other states of Brazil will probably create their own public policies on contaminated sites regarding regional particularities. A national public policy would be welcome as to establish directives and general clauses on the concurrent legislative competence of the Union, States and Federal District on environmental protection and pollution control issues. A national policy would also fulfill legal gaps for any state that hasn`t developed its own legislation, which can also be subject of future researches. This innovative Act will probably set a national tendency in a way that other states of Brazil will probably create their own public policies on contaminated sites regarding regional particularities. A national public policy would be welcome as to establish directives and general clauses on the concurrent legislative competence of the Union, States and Federal District on environmental protection and pollution control issues. A national policy would also fulfill legal gaps for any state that hasn`t developed its own legislation, which can also be subject of future researches. Sánchez, L. E. (2005). Danos e Passivo Ambiental, In: Curso Interdisciplinar de Direito Ambiental, Philippi Jr, A & Alves, A. C. (Ed.), pp. 261-293, Manole, ISBN: 85-204- 2187-3, São Paulo, Brazil. Environmental Management in Practice 1. Introduction The Coastal Zone is home to many heavy oil and gas industries, and a significant proportion of the population and wealth generating infrastructure. The coastal zone therefore, provides economic, transport, residential and recreational functions, all of which depend upon its physical characteristics, pleasant landscape, cultural heritage, natural resources and rich marine and terrestrial biodiversity. The United Nations estimated that by 2004, more than 75 percent of the world’s population would live within the coastal zone (Reeve et al., 2004). These regions are therefore of critical importance to a majority of humanity and affect an increasing percentage of our economic activities. The pressure on coastal environments is being exacerbated by rapid changes in global climate, overexploitation of fisheries, coastal and marine pollution, coastal erosion and flooding, physical modification and destruction of habitats, etc. For example, the Intergovernmental Panel on Climate Change (IPCC) has predicted a sea level rise of the order of 0.6m over the next century. For Nigeria, it is of the order of 0.83m (Nwaogazie & Ologhadien 2010) The value of the coastal zone to humanity, and the enormous pressure on it, provides strong incentives for a greater scientific understanding which can ensure effective coastal engineering practice and efficient and sustainable management of coastlines. 5. References CABERNET. (2006). Sustainable Brownfield Regeneration: CABERNET Network Report, University of Nottingham, ISBN: 0-9547474-5-3, Nottingham, United Kingdom. CETESB. (2001). Relatório de estabelecimento de valores orientadores para solos e águas subterrâneas no Estado de São Paulo, CETESB, São Paulo, Brazil. CETESB. (2011a). O que são áreas contaminadas, In: CETESB, 03.02.2011, Available from: <http://www.cetesb.sp.gov.br/areas-contaminadas/O-que-s%E3o-%E1%81reas- Contaminadas/1-> CETESB. (2011b). O gerenciamento de áreas contaminadas no Estado de São Paulo, In: CETESB, 03.02.2011, Available from: <http://www.cetesb.sp.gov.br/userfiles/file/areascontaminadas/texto_explicativ o_dez_10.pdf> Cunha, R. C. de A. (1997). Avaliação de risco em áreas contaminadas por fontes industriais desativadas – estudo de caso, Tese (Doutorado em Geociências), Instituto de Geociências, Universidade de São Paulo, São Paulo, Brazil. Ferguson, C. C. (1999). Assessing risks from contaminated sites: policy and practice in 16 European countries. In: Land Contamination and Reclamation, Rupert Hough, (Ed.), 33-54, EPP Publications, Retrieved from <http://epppublications.com/Documents/07-2-1.pdf> Lazanha, L. K. S (2005). Subsídios jurídico-sociais para formulação de políticas públicas: revitalização de áreas degradadas por contaminação no Estado de São Paulo. Dissertação (Mestrado em Saúde Pública), Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil. Marker, A. (2003). A revitalização de áreas urbanas degradadas: políticas, instrumentos e incentivos no cenário internacional (Relatório de Consultoria), GTZ, São Paulo, Brazil. Sánchez, L. E. (2001). Desengenharia: o passivo ambiental na desativação de empreendimentos industriais, Edusp, ISBN: 85-314-0599-8, São Paulo, Brazil. Sánchez, L. E. (2001). Desengenharia: o passivo ambiental na desativação de empreendimentos industriais, Edusp, ISBN: 85-314-0599-8, São Paulo, Brazil. 158 Environmental Management in Practice 8 2. Muddy coastline Coastal classification generally falls into two main categories; namely, genetic (nature) and descriptive (based on morphology). Within the descriptive classification, a sub classification in terms of particle size of the beach material have: muddy coasts, sand coast, gavel/shingle coasts and rock coast. Another sub-classification based on typical coastal features have the following: barrier island coasts, delta coasts, dune coasts, cliff coasts, coral reef coasts, mangrove coasts, marsh grass coasts, etc. While a vast majority of coastlines are made up of sediments ranging from coarse-grained fragments of rocks to fine-grained sand, only a few are muddy coasts. Sediment mixture with a fraction of clay particles (d < 4m, AGU scale), larger than about 10% have cohesive properties. Mud may be defined as a fluid-sediment mixture consist of (salt) water, sands, silt, clays and organic materials. Muddy coasts fall within the descriptive category of coasts in which classification are based on particle size of the beach material. In a coastal environment, there is a continuous cycle of mud flocs which consists of erosion, settling, deposition, consolidation and erosion. Since mud particles are denser than water and 160 Environmental Management in Practice unstable, the continuous agitation of the surf zone by breaking waves transport mud material cross-shore and equilibrium conditions are hardly attained. Thus muddy coastlines hardly form breaches, which offer natural coastal protection systems. Plate 1 shows the action of breaking waves on a muddy coastline. Plate 1. Wave breaking on a muddy coast at Aiyetoro, Nigeria Plate 1. Wave breaking on a muddy coast at Aiyetoro, Nigeria 3.1 Wave motion y wave celerity (c) and wave dispersion equations are : p y The wave celerity (c) and wave dispersion equations are : p y The wave celerity (c) and wave dispersion equations are : c = gω-1 tanh kd (3) c = gω-1 tanh kd (3) and c = gω-1 tanh kd (3) and ω2 = gk tanh kd (4) where k = 2 and ω = 2 c = gω-1 tanh kd (3 (3) and and ω2 = gk tanh kd (4 (4) ω2 = gk tanh kd (4) where k = 2 L  and ω = 2 T  ere k = 2 L  and ω = 2 T  where k = 2 L  and ω = 2 T  where k = L and ω = T Th i l l i i d i d f E i 2 i h d fi i i f l i i l L T The particle velocities are derived from Equation 2 using the definition of velocity potential: particle velocities are derived from Equation 2 using the definition of velocity potential: The particle velocities are derived from Equation 2 using the definition of velocity p u = HT-1 cosh ( ( ) sinh k y d kd  cos (kx – ωt) (5) v = HT-1 sinh ( ( ) sinh k y d kd  sin (kx – ωt) (6) (5) (6) where η is the height of the water surface above stillwater level, u is the horizontal water particle velocity, v is the vertical water particle velocity, d is the still water depth, H is the wave height, L is the wave length and T is the wave period. where η is the height of the water surface above stillwater level, u is the horizontal water particle velocity, v is the vertical water particle velocity, d is the still water depth, H is the wave height, L is the wave length and T is the wave period. For the computation of longshore sediment transport, coastline evolution, design of shore protection works and estimation of wave impact pressures on structures, historic wave data are required. The wave measurement facilities may be situated offshore in relatively deep water. By means of the wave dispersion equations (3 & 4), the wave conditions in the offshore station may be transferred to the coastal zone. 3. Coastal processes The hydraulic and morphological processes in the coastal zone are governed by two primary phenomena; namely, windwaves and astronomical tides. The wind stress on the water surface produces wind-generated waves which are of a relatively short period. The periodic rise and fall of water level is due to the astronomical tides produced by the gravitational field in the presence of the rotating earth, moon and sun. The timescale of tidal oscillations is very much larger than that of the wind-generated waves. Table 1 presents other free surface disturbances. Phenomena Generating force Time scale (period) Wind generated waves Shear and wind pressure on sea surface 0-15s Swell Long-distance wind wave 0-30s Surf beats Grouping of breaking waves 1-5 min Seiches Variations of wind speed and atmospheric pressure 1-60 min Basin resonance Tsunami, surf beats 1-60 min Tsunami Undersea earthquakes 5-60 min Tide Moon-sun influences on earth 12-24 hr Storm surge Wind shear and atmospheric pressure on sea 1-30 days Table 1. Free surface disturbances in the coast 161 Sustainable Management of Muddy Coastlines The most important hydraulic process in coastal engineering is the wave motion; the understanding of wave motion and of its interaction with structures and coastal hydrography is vital in the estimation of erosion and accretion, sediment transport and coastal morphology. These processes are also important in formulating sustainable management plans. 3.1 Wave motion The wave profile according to the linear wave theory is The wave profile according to the linear wave theory is (1) η a ︵ k x - ω t ︶ = c o s (1) where  is surface elevation, a is wave amplitude,  is circular frequency, k is wave number, t is time, and x is positive direction of wave travel. The solution of the velocity potential () for the wave profile of Equation 1, must satisfy the Laplace equation, boundary conditions at the sea bed and on the water surface. The resulting solution for  is given by:  = -gH 4 T        cosh ( ) cosh k d z kd  sin (kx – ωt) (2) (2) where g is acceleration due to gravity, H is wave height, T is wave period, k and are as previously defined. 3.2 Sediment transport p Coastal sediment transport consists of two aspects: sediment transport parallel to the shoreline (longshore) and sediment transport transverse to the shoreline (cross–shore). The imbalances in the longshore sediment transport are responsible for the long-term changes in the coastlines, whereas the cross-shore transport is responsible for the short-term variations. p Coastal sediment transport consists of two aspects: sediment transport parallel to the shoreline (longshore) and sediment transport transverse to the shoreline (cross–shore). The imbalances in the longshore sediment transport are responsible for the long-term changes in the coastlines, whereas the cross-shore transport is responsible for the short-term variations. The morphological consequences of shore protection works are assessed in terms of quantitative estimates of erosion and accretion. Waves and currents, along with the physical properties of the sediment materials, determine the rate of material transport in the coastal zone. The reliability of sediment transport predictions is strictly dependent upon the accuracy of the semi-empirical equations used to evaluate the sediment transport. Studies have been carried out to establish the validity and reliability of several solid transport formula (White et al. 1973; Gomez and Church 1989; Bathurst et al. 1987). These studies concluded that, there is no solid transport formula valid for all ranges of natural conditions and therefore, the more appropriate formula for each set of particular conditions can be chosen. A number of longshore transport models have been developed for a number of natural conditions; namely, 3.1 Wave motion Equations 5 and 6 are components of velocity used in estimating the wave forces exerted on structures. 162 Environmental Management in Practice 3.2.2 Bijker formula (1967 & 1968) formula (1967 & 1968) l i 3.2.1 Coastal erosion research council (CERC) formula (1963) In the CERC formula, S = A 2 2 sin cos o o rbr br br H C K   (7) S = A 2 2 sin cos o o rbr br br H C K   (7) where S is longshore transport due to breaking waves, A is a constant, Ho is deepwater wave height, Co is deepwater wave celerity, Krbr is wave refraction coefficient at the breaker line, and br is breaker angle. where S is longshore transport due to breaking waves, A is a constant, Ho is deepwater wave height, Co is deepwater wave celerity, Krbr is wave refraction coefficient at the breaker line, and br is breaker angle. The CERC formula does not account for differences in sediment materials often represented by d50 (mean size). The formula is often criticized for being only valid for relatively long and straight beaches, where the longshore differences in the breaking wave heights are small. Thirdly, the formula does not account for currents which are not generated by breaking waves, such as tidal currents. When tidal currents are important, another transport formula should be used. 4. Coastal morphology Morphological evolutions are a direct response to changes in sediment transport. The computation of longshore sediment transport rates preceeds prediction of coastal changes due to erosion and accretion. When the sediment transport rate reduces, accretion will occur; conversely, an increase in sediment transport will cause erosion. Consequently, morphological evolutions are indicative of changes in shoreline position, and these changes are often components of the decision making measures against coastal erosion. In conclusion, the coastline is in a state of dynamic equilibrium, characterized by the local wave climate, currents, and other water level fluctuations summarized in Table 1. In order to manage coasts sustainably, a good data gathering programme comprising: bathymetry/ topography, seabed characteristics/bedform, waterlevels/ waves, etc. is recommended. 3.2.3 Kamphius equation (1991) The Kamphius model was refined using a series of hydraulic model tests, giving Qk= 2.0 1.5 0.75 0.25 0.6 50 2.27 (tan ) (sin2 ) sb b H Tp D   (9) (9) where Hsb is breaker wave height, Tp is peak wave period,  is slope of the beach, D50 is medium sediment diameter, b is wave breaker angle. The Kamphius model does not take tidal currents along the coast in account. 3.2.2 Bijker formula (1967 & 1968) The Bijker formula is: jker formula (1967 & 1968) ker formula is: 2 50 50 2 2 0.27 exp 1 1 2 b b D C S bD g C u                                         (8) (8) where Sb is bed load transport, b is a constant (~5), D50 is mean grain diameter,  is current velocity, C is chezy coefficient = 18log 12h        , h is water depth, r is bed roughness, g is where Sb is bed load transport, b is a constant (~5), D50 is mean grain diameter,  is current velocity, C is chezy coefficient = 18log 12h        , h is water depth, r is bed roughness, g is 163 Sustainable Management of Muddy Coastlines acceleration due to gravity,  is specific density, 0.5 2 fw C g        with fw = exp     acceleration due to gravity,  is specific density, 0.5 2 fw C g        with fw = exp 6.0 5.2 0.19 oa                    , ao is the amplitude of orbital excursion at the bed, b is amplitude f bit l l it t th b d of orbital velocity at the bed. The Bijker longshore shore transport model takes into account the effect of tidal or other types of currents and may be coupled with other models. The Bijker model is unique, because it is adaptable to any current condition. 5.1 Mathematical modelling g Most coastal engineering models are non-linear equations, which do not have analytical solution. Therefore, they cannot be applied to problems involving complex boundaries and time-varying boundary conditions. Analytical solution of models of real world will be of little help and one has to resort to numerical techniques. Several types of numerical methods, such as finite differences, finite element, finite volume and boundary element methods have been widely used to coastal engineering problems. Such models are used in investigating coastal processes and the design of coastal engineering schemes. Experiments using physical models can also be undertaken using controlled conditions, thus allowing investigation of each controlling parameter independently. Physical models are normally smaller scale versions of the real situation. This requires a theoretical framework to relate model measurements to the real (prototype) situation. Unfortunately, the result of 164 Environmental Management in Practice this theoretical framework is that scaled physical models are unable to simultaneously replicate all of the physical processes present in the prototype in correct proportion. Thus, we return to nature, by way of field measurements. Such measurements obviously do contain all the real physics, if only we knew what to measure and the appropriate instruments to do so. Such measurement, as are possible, have to be taken in an often hostile environment, at considerable relative cost and under uncontrolled conditions. 5.2 Data gathering Field investigations are often carried out for major specific coastal defense projects. Basically, measurements are made on waves, tidal currents, water levels and beach profiles. Such measurements are often used to derive the local wave climate, current circulation patterns, extreme still-water levels and beach evolution through the use of numerical models which are calibrated and take their boundary conditions from the measurement. Field investigations are often carried out for major specific coastal defense projects. Basically, measurements are made on waves, tidal currents, water levels and beach profiles. Such measurements are often used to derive the local wave climate, current circulation patterns, extreme still-water levels and beach evolution through the use of numerical models which are calibrated and take their boundary conditions from the measurement. Mulder et al. (2000) described a set of measurement tools considered both comprehensive and informative, comprising descriptions of equipment to measure bathymetry/topography, seabed characteristics/bedforms, water levels/waves, velocities, suspended sediment concentrations, morphodynamics/sediment transport and instrument carrier/frames plat models which are calibrated and take their boundary conditions from the measurement. Mulder et al. (2000) described a set of measurement tools considered both comprehensive and informative, comprising descriptions of equipment to measure bathymetry/topography, seabed characteristics/bedforms, water levels/waves, velocities, suspended sediment concentrations, morphodynamics/sediment transport and instrument carrier/frames plat forms. Table 2 contains some recent tools in measurement equipment taken from Dominic et al. (2004). Interested readers are referred to the above texts for guidelines on how to use the tools and examples of results. In terms of the development of our understanding and the incorporation of that understanding in the management of coastlines, design process, field studies and physical model studies are required to improve both our knowledge of the physics and calibrate and verify our numerical models. These models are key component of the current state-of-the art tools. 5.3 Geographic information system (GIS) tools g p y ( ) Sustainable development and management of natural and economic resources depends on the ability to assess complex relationships between a variety of economic, environmental and social factors across space and time. Lack of Integrated data management tools among the Interrelated and Interwoven dimensions frequently Inhibit the quality of environmental and development planning. Consequently, information management systems are currently receiving growing attention. In this regard, GISs have emerged as a particularly promising approach, enabling users to collect, store, and analyze data that have been referenced to its geographic location. A Geographic Information System is a system of computer hardware, software, and procedures designed to support the capture, management, manipulation, analysis, and display of spatially referenced data for solving complex planning and management problems. The advantages of GIS capability can be categorized as long term or short term. The long- term category is where economic and environmental management on a national, regional or local level is called for , in other words, institutional or programmatic applications. The short-term category usually involve specific project situations, for example, Environmental Impact Assessment Studies. 165 Sustainable Management of Muddy Coastlines S/No Name of tool Brief description 1 Total station leveling for bathymetry/topography Method of surveying the coast and inter-tidal area, using laser leveling system. 2 Differential global positioning system (GPS) Method for fixing absolute position (three coordinates), based on calculated distance from at least four geo-stationary satellites. 3 Echo Sounder surveys Method of surveying the seabed using a standard maritime echo sounder. 4 Van Veen grab for seabed characteristics/bed forms A method of obtaining samples of subtidal seabed material either for visual analysis or for quantitative particle size distribution analysis. 5 Roxann system An acoustic system used to produce a map of the near shore and offshore zones of the study area. 6 Digital side-scan sonar An acoustic system designed to map the bedforms in the offshore and nearshore zones. 7 Pressure transducer (TP) for water levels/waves. A device for measuring total pressure, when installed underwater, analysis of instantaneous pressures gives measure of wave height/period. 8 Wave pole A pole or pile driven into the bed, and extending above the highest water level. 9 Directional wave Buoy A surface buoy for measuring offshore wave conditions, including wave height, period and direction. p Environmental Monitoring When monitoring environmental impacts during and after project completion, databases with multiple attributes must be integrated. GIS can help structure and integrate this diverse information ranging from water quality to soil productivity to habitat data. Specific GIS technologies that are useful in monitoring include remote sensing, which can be applied to monitor, for example, sewage disposal sites, effluent discharges and coastal areas for example. 5.3.1 Available GIS Geographic information systems are available both in PC/micro computers and mini and main frame computers. Table 3 lists a summary of some commercially available geographic information systems. 5.3 Geographic information system (GIS) tools The advantages of using GIS in environmental assessment include the followin g g  It encourages a more systematic approach to environmental data collection; It encourages a more systematic approach to environmental data collection;  It can reduce the overall costs and institutional overlap of environmental data collection and management;  It can reduce the overall costs and institutional overlap of environmental data collection and management; g  It increases comparability and compatibility of diverse data sets;  It makes data used in environmental assessment accessible to a wider range of decision- makers; and, encourages the spatial analysis of environmental impacts that would otherwise be ore easil ignored because of anal tical difficult or cost  It encourages the spatial analysis of environmental impacts that would otherwise be more easily ignored because of analytical difficulty or cost. more easily ignored because of analytical difficulty or cost. Besides Environmental Assessment, GIS provides a powerful set of tools for:  Supporting Resources Inventories and Baseline Surveys and land-use mapping;  Impact Assessment and Analysis of Alternatives; GIS modeling techniques allow complex interrelationships to be evaluated within comprehensive spatially referenced databases. Techniques such as network analysis, digital terrain modeling are routinely applied in coastal engineering to assess the vulnerability of climate change sea-level rise to coastal communities. g Decisions made in GIS application will be useful in designing mitigation measures. Risk assessment applications such as hazard identification, and risk minimization planning are other examples where GIS has been effective. Decisions made in GIS application will be useful in designing mitigation measures. Risk assessment applications such as hazard identification, and risk minimization planning are other examples where GIS has been effective. 5.3 Geographic information system (GIS) tools 10 Wave recording system (WRS) The wave recording system is an array of 6 pressure transducers used to derive the wave height, period and directional spectra in the nearshore zone. 11 Inshore Wave Climate Monitor (IWCM) The 5 wave staffs are driven into the beach in a triangular array and are connected to a central data storage/ battery power unit. Table 2. Names and brief description of measurement tools The 5 wave staffs are driven into the beach in a triangular array and are connected to a central data storage/ battery power unit. Table 2. Names and brief description of measurement tools The basic equipment, software and human resource skills required may be similar for both long-term and short-term, but the design, implementation and operation implications may be different. 166 Environmental Management in Practice GIS may be particularly useful in cross-sectoral and regional development, for example, in coastal zones, catchments, large urban areas, or multi-purpose development schemes within a given administrative region. g g Determining a region’s vulnerability to soil erosion for instance, requires the consideration of such factors as soil structure and chemistry, seasonal fluctuations in rainfall volume and intensity, geomorphology, and type of land management regime in practice. Assessing the feasibility of a soil conservation programme in an area requires additional information on the economic status of Inhabitants, the type of crops grown, and the responsiveness to incentives for soil conservation. Then, selecting the appropriate land rehabilitation models requires data on land capability and its suitability for different uses. GIS technologies handle both the spatial and non-spatial properties of data-sets, thus providing an extension to other statistical methods that disregard the spatial nature and variations of environmental data. The advantages of using GIS in environmental assessment include the following: g p data. 5.4 Salt intrusion/gravitational circulation Sediment-laden flowing water, other natural substances or pollutants move with the water, and therefore are transported by the flow. The flowing water is affected by density 167 Sustainable Management of Muddy Coastlines differences, causing density induced currents. These currents affect the direction of flow and transport, and may vary over the depth of water. Consequently, density currents are a factor to be considered when studying the sedimentation in estuaries, coast or the transport of pollutants through these systems. Another negative effect of gravitational circulation is the creation of “null points” causing shoaling and sedimentation which interferes with navigation. System name Hardware Geometric Storage Attribute storage ARC/INFO VAX, PRIME IBM, DG Vector Relational DELTAMAP HP, SUN Vector Relational INFORMAP VAX Vector Relational INTEGRAPH VAX Vector Network MAPS VAX, PRIME Vector Relational SICAD SIEMENS Vector Relational SYSSCAN VAX Vector Relational GEOBASED VAX Vector Relational SYSTEM 600 VAX Vector Relational ARC/INFO IBM PC/AT SYSTEM 2 Vector Relational SPANS IBM PC/AT SYSTEM 2 Quadtree vector Relational INFORMAP II IBM/AT Vector Relational ERDAS IBM/AT Vector Relational ILWIS IBM/AT Raster vector Relational PAMAP IBM/AT Raster Relational IDRISI IBM/AT Raster Relational able 3. GIS in mini and main-frame computers Table 3. GIS in mini and main-frame computers Management concerns frequently center on the concentration of waterborne indicators, including pollutants and plaktonic organisms. The need to consider the environmental and economic sustainability of present and future coastal management schemes on muddy coasts requires a good understanding of density currents and morpho-dynamics. Aquatic ecosystem sustainability is highly dependent on salinity concentration dynamics and must be studied for the particular environment. Both analytical and mathematical models are currently used to simulate salt intrusion. The models constitute a powerful tool for evaluation of salinity intrusion patterns and as supportive instruments for decision making in coast management. Table 4 contains some widely used coastal engineering models: 168 Environmental Management in Practice Designs Name Purpose 1 Genesis Simulation of coastal processes 2 SBEACH Coastal Engineering design 3 MODIFIED KRIEBEL Cross-shore simulation for berm dimensions and hurricane storm events. 4 CEQUALW2 Salinity Intrusion Table 4. Widely Used Coastal Engineering Models Salinity Intrusion Table 4. Widely Used Coastal Engineering Models Table 4. Widely Used Coastal Engineering Models 6. Sustainable management of coastlines Coastal management plans are designed to provide coastal zone resource development within the framework of: a. Technical: coastal processes and defense, etc a. Technical: coastal processes and defense, etc a. Technical: coastal processes and defense, etc b. Socio-economic: economic demography, regional planning and c. Environmental: water quality, biodiversity, etc. i. Coastal management is continually confronted with conflicting challenges. There are problems of jurisdiction involved in whether the responsibility for running the operation lies with the federal governments, a local government or some regulatory commission, and always there is application of priorities supposedly set by society as a whole. The basic tool is a legal framework to regulate the conflicting activities on the coast. These may include national laws made to meet specific requirements, e.g. National Environmental Policy Acts of 1969 which provides preparation of environmental impact statement, the Water Quality Act of 1970 which addresses oil pollution; international covenants and jurisdictional responsibility. p j p y ii. There is a problem of political process. The political process is such that technical standards will almost always yield to such things as austerity cases, emergency situations, or strong public sentiments. Consequently, decision on coastal environment must have a public input or else the decision will probably not be effective. The manager must be prepared to strike a compromise between the emotional public, individual agencies, both state and federal, often working at cross-purposes. ii. g g p p iii. Arising from (ii), is the need for coordinated approach such that environmental protection, fish and wildlife services, etc, may work together and adopt a consistent approach to survey, mitigation and monitoring. The coordinated approach achieves better results for the environment in terms of a more consolidated, integrated approach and saves on resources and repetition by stakeholders. g g p p iii. Arising from (ii), is the need for coordinated approach such that environmental protection, fish and wildlife services, etc, may work together and adopt a consistent approach to survey, mitigation and monitoring. The coordinated approach achieves better results for the environment in terms of a more consolidated, integrated approach and saves on resources and repetition by stakeholders. iv. The physical characteristics of coastal environment is dictated by the actions of breaking waves and currents on sediment materials. There is need for quality data gathering, both comprehensive and information, comprising bathymethry/topography, seabed characteristics/bedforms, water levels/waves, velocities, suspended sediment concentration. iv. 6. Sustainable management of coastlines The physical characteristics of coastal environment is dictated by the actions of breaking waves and currents on sediment materials. There is need for quality data gathering, both comprehensive and information, comprising bathymethry/topography, seabed characteristics/bedforms, water levels/waves, velocities, suspended sediment concentration. iv. The physical characteristics of coastal environment is dictated by the actions of breaking waves and currents on sediment materials. There is need for quality data gathering, both comprehensive and information, comprising bathymethry/topography, seabed characteristics/bedforms, water levels/waves, velocities, suspended sediment concentration. v. There is need to broaden the emphasis from assessment of physical environment aspects, to assessment of impacts on marine ecological resources, in particular benthic and epibenthic species, habitats. v. There is need to broaden the emphasis from assessment of physical environment aspects, to assessment of impacts on marine ecological resources, in particular benthic and epibenthic species, habitats. v. There is need to broaden the emphasis from assessment of physical environment aspects, to assessment of impacts on marine ecological resources, in particular benthic and epibenthic species, habitats. 169 Sustainable Management of Muddy Coastlines vi. Application of Hydroinformatics systems: Hydroinforamtics, the use of information and communication technology in hydraulics, encapsulates and integrates engineering methods in software systems. It provides powerful methods to engineers and rational solutions to policy makers. The application of hydroinformatics systems to problem solving in coastal environments requires the availability of databases for calibration and verification of engineered systems. It also calls for adequate instrumentation and experimental methods, and international cooperation for the acquisition and exchange of data. Hydroinformatics systems will have to be built up from proprietary codes and modeling systems that have been constructed, in most cases, for quite other purposes than those of hydroinformatics. Interested readers are referred to Abbot et al., (1988) for full description of hydroinformatics systems. p y y vii. The need for integrated coastal zone management (ICZM). Integrated coastal zone management has been widely accepted as an effective mechanism for addressing and resolving these types of issues throughout the developed and developing world. ICZM will enable the integration of all issues and emphasize the involvement of all key players in the planning process, coordination between sectoral agencies, and application of cooperative management strategies involving stakeholders. y y vii. The need for integrated coastal zone management (ICZM). 6. Sustainable management of coastlines Integrated coastal zone management has been widely accepted as an effective mechanism for addressing and resolving these types of issues throughout the developed and developing world. ICZM will enable the integration of all issues and emphasize the involvement of all key players in the planning process, coordination between sectoral agencies, and application of cooperative management strategies involving stakeholders. As a case study, the Nigerian coast and marine areas have been chosen under this heading: As a case study, the Nigerian coast and marine areas have been chosen under this heading: 7.1 Wave and tidal characteristics The Nigerian coast and marine areas are under the influence of moderate oceanographic forcing consisting of semi-diurnal tidal with spring tide ranging between 0.95m in the West to 3.25m in the East. The prevailing South-Westerly waves vary from spilling breaker to plunging waves. The persistence of significant wave height (hs) are in the order of 1.4m – 2.5m. Long shore currents and prevalent in the near shore while the West-East Guinea currents constitute the major ocean current. j The relative importance of diurnal and semidiurnal harmonics can be determined from the ratio, F, given by (10) F = K1 + O1 / M2 + S2 (10) where K1, O1, M2 and S2 denote the amplitudes of the respective tidal constituents. The form of tide (F) found in the Nigerian Atlantic Coast was calculated by substituting the amplitudes for K1, O1, M2 and S2 into Equation (10). The value of F calculated is 0.1601. Consequently, the tidal behaviour found along the Nigerian Atlantic coast is semi-diurnal, with two high and two low waters of approximately the same height. 7.4 Coastal erosion and flooding g The coastline has been subjected to erosion over the years in Nigeria. Scientist from the Nigerian Institute for Oceanography and Marine Research (NIOMR) have reported widespread erosion and flooding of the Barrier Islands and the Niger Delta (Ibe et al., 1984, Awosike 1993) created erosion resulting from deficit of sand due to natural and anthropogenic activities varies. Notable among the natural causes of coastal erosion are vulnerable soil characteristics, topography and occurrence of off-shore canyons. Anthropogenic causes include destruction of coastline dredging and river dams. The coastline has been subjected to erosion over the years in Nigeria. Scientist from the Nigerian Institute for Oceanography and Marine Research (NIOMR) have reported widespread erosion and flooding of the Barrier Islands and the Niger Delta (Ibe et al., 1984, Awosike 1993) created erosion resulting from deficit of sand due to natural and anthropogenic activities varies. Notable among the natural causes of coastal erosion are vulnerable soil characteristics, topography and occurrence of off-shore canyons. Anthropogenic causes include destruction of coastline dredging and river dams. The Victoria beach is fastest eroding beach in Nigeria with arrange erosion rate of 20 -30m yearly. Erosion rates range between 18-24m annually at Ugborodo/Escravos; Forcados, 20- 22m; 16-19m at Brass; Karamo, 15-20m; Bonny, 20-24m; and Opobo; 10-14m; as reported by Ibe in 1989. Coastal erosion with serious flooding has done widespread damage in many areas along the coastal zone. The beaches along the Nigerian coastline are very susceptible to flooding due to their very low topography. Flooding of the Victoria Island in Lagos State and other low-lying areas of the state are common during the rainy season (June-August). High rainfall in the Niger Delta coupled with poor drainage allow storm waters to collect in the hallows and eventually flood large areas within the Delta. The Victoria beach is fastest eroding beach in Nigeria with arrange erosion rate of 20 -30m yearly. Erosion rates range between 18-24m annually at Ugborodo/Escravos; Forcados, 20- 22m; 16-19m at Brass; Karamo, 15-20m; Bonny, 20-24m; and Opobo; 10-14m; as reported by Ibe in 1989. Coastal erosion with serious flooding has done widespread damage in many areas along the coastal zone. The beaches along the Nigerian coastline are very susceptible to flooding due to their very low topography. Flooding of the Victoria Island in Lagos State and other low-lying areas of the state are common during the rainy season (June-August). 7.3 Sea-break-through inlets g The major environmental concern of the Ondo Coastline in Nigeria is its susceptibility to sea-break through inlets (Odi-Owei and Ologhadien, 2009). The first sea break-through inlet occurred in 1983, when a canal was dredged oblique to the coastline, leaving a vertical head cut. The overhang, coupled with the poor geotechnical characteristics and mechanisms of sediment transport downstream in the channel, initiated an upstream migration at the head cut towards the sea. Over time, the combined actions of the breaking waves, tidal currents and sea wind migration of the head cut eventually opened up the inlet, discharging saline water into the fresh water forest. Consequently, over 20 hectares of fresh water forest resources were destroyed (Plate 2), impacting negatively on the local economy. It also reduced the volume of saw-logs supply to the timber markets in Lagos, Benin, etc. Freshwater resources are extensively exploited for cash or subsistence. The Ondo State coastline is fairly stable except in areas that are exposed to breaking waves. 7.2 Assessment of climate change The possible impacts of climate change include higher sea levels altered pattern of rainfall and air temperatures, and increased frequency and intensity of severe storms. Some industries could be directly affected by adverse impacts of climate change. The coastal tourism industry, for example, is vulnerable to both sea-level rise and greater weather extremes. Table 5 shows a comparison of sea-level rise indicator parameters with others (Nwaogazie and Ologhadien 2010). 170 Environmental Management in Practice Parameter Nigerian Coast IPCC Ghana England Remarks Temperature 1.8oC 1.5 – 4.5oC 0.11oC NA Per decade Rainfall 55.2mm NA 13mm NA Per decade Mean Sea Level 8.3cm 6cm 2.2cm 4cm-6cm Per decade NA - Not Available; IPCC – Intergovernmental Panel on Climatic Change Table 5. Comparison with IPCC and other Predictions Table 5. Comparison with IPCC and other Predictions 7.4 Coastal erosion and flooding High rainfall in the Niger Delta coupled with poor drainage allow storm waters to collect in the hallows and eventually flood large areas within the Delta. 171 Sustainable Management of Muddy Coastlines Plate 2. Dead vegetation around Awoye inlets Fig. 1. Map showing Canal and Sampling Stations Plate 2. Dead vegetation around Awoye inlets Plate 2. Dead vegetation around Awoye inlets Plate 2. Dead vegetation around Awoye inlets Fig. 1. Map showing Canal and Sampling Stations Fig. 1. Map showing Canal and Sampling Stations Fig. 1. Map showing Canal and Sampling Stations 172 Environmental Management in Practice 8. Conclusion In order to implement the Environmental Management Plan for Coastal Communities, guidelines for dealing with specific environmental issues identified should be developed. As part of the management plan, continuous data collection for bathymetry, topography, waves, tides, surges, wind and salinity need to be carried out. 7.6 Environmental management plan for coastal communities The key to effective environmental management plan is adequate monitoring of the projects implementation, predicted impacts and monitoring or implementation of predicted mitigation measures. The environmental issues that will be addressed are; i. Over exploitation of Fisheries resources, ii. Costal and Marine Pollution iii. Oil spills ii. Costal and Marine Pollution iv. Coastal Erosion and Flooding v. Physical modification and destruction of habitats vi. Climate change and sea-level rise vii. Invasive species (exotic species) viii. Storm surges. 7.5 Physical modification and festruction of habitats 7.5 Physical modification and festruction of habitats The coastal zones have undergone wide modifications in the last thirty years. Due to high pressures on coastal resources conflicting exploitation techniques and increasing population leading to loss of biodiversity, in the ecosystem, the value of coastlines has been diminished. The destruction of mangrove ecosystems has been on the increase since exploitation of oil and gas started in the Niger Delta resulting in replacement of mangrove vegetation by new vegetation species like nympa palms. The Kwale game reserve in the 1950s was rich in biodiversity but due to oil exploration, gas production and poaching elephants and many flora and fauna have disapproved in the Reserve several animal species of conservation interest including Scalter’s Guenon, Delta Red Columbus, the Crested Genet, the Pygmy Hippo, Chimpanzee and African Leopard have almost disappeared in the Niger Delta, many plants of medicinal, economic and cultural values such as Thaumatiococcus daniel (sweetener) Fegara sp. (for sickle cell anemia) and Rauvolfia vomitoria (for treatment of high blood pressure and now rare in the Niger Delta). ) The major socio-economic problems result from poverty ecosystem modification in the coastal zones include unemployment because the people depend on their tradition mean of livelihood. 9. References Abbot, M.B., (1991), Hydroinformatics; Information Technology and the Aquatic Environment, Avebury Technical, ISBN 1 85628 832 3. Adnitt, C. and Lewis J. (2004) “The Future of Environmental Impact Assessment for marine aggregate extraction-best practice and emerging issues”. Journal of marine Science and Environment, No. CI 2004, pp.36-44. 173 Sustainable Management of Muddy Coastlines Antonucci, J., GIS: A Guide to the Technology, New York: Von Nostrand Reinhold, 1991. Bathurst, J.C., Graf, W.H., and Cao, H.H. (1987). “Bed-load Discharge equation for steep mountain rivers” Sediment transport in Gravel-bed rivers, C.R. Thorne, J.C. Bathurst and R.D., Hey, eds., John Wiley and Sons ltd., new York, N.Y. y J y Bijker, E., “Sedimentation in Channels and Trenches”, Proc. 17th Conf. on Coastal Eng., Sydney, Australia, 1980, pp.299-300. y y pp Burrough, P.A., (1986), Principles of GISs for Earth Resources Assessment, Oxford: Clarendon Press. Christine, A. Coughonowr, Magnus N. Ngolie and Olof Linden (1995); “Coastal Zone Management on Eastern Africa Including the island States: A Review of issues and Initiatives” Ambio Vol.24, No.7-8, pp.448-457. pp Dominic Reeve, Andrew Chadwick and Christopher Fleming (2004). Coastal Engineer Processes, Theory and Design Practice”, Spon Press, OX14 4RN. y g p EA Source Book Update, GISs for Environmental Assessment and review, #3, April, 1993 Gomez, B., and Church, B. (1989) “An Assessment of bed load Sediment Transport Formulae for Gravel Bed Rivers”, J. Water Resources 25(6), 1161-1186. Hassan, H.M., and C, Hutchinson, Natural Resource and Environmental Information for Decisionmaking, World Bank, 1992. “Management of the Marine Environment” in Introduction to Marine Pollution Control, Jerome Williams, a Wiley-Interscience Publication. Chap 10. Mulder, J.P.M., Koningfield, M. Van Owen, M.W. and Rawson, J., 2001. Guidelines on the selection of CZM tools. Report RIKZ/2001.020, Rijkswanterstaat, April 2001. Nwaogazie I.L, and Ologhadien I. (2010). “Trend Analysis of Climate Change Indicators along the Nigerian Atlantic Coast”, Proceedings of the International Conf. on Climate Changes, Nigerian Society of Engineers”, Abuja 2010. g g y g j Nwilo, P.C. (1997). “Managing the Impacts of Storm Surge on Victoria Island, Lagos, Nigeria” IAHS, Publ. No. 239, pp. 325 – 330. Odi-Owei S. and Ologhadien I. (2009). “Environmental Aspect of Dredging Intra-coastal Navigation Channels in Muddy Coastline: The case of Awoye, Ondo State, Nigeria”. Journal of Food, Agr. & Environ, Vol. 7(2): 764-768. g J f g ( ) Paulson, B., Urban Applications of Satellite Remote Sensing and GIS Analysis, World Bank, 1992. Rijn, L.C. van Rijn, L.C. (1992) Morphological Processes, Lecture Note for Hydroinformatics Course at Unesco-IHE, Delft, The Wetherlands. White, W.R., Milli, H., and Crabbe, C. (1973). Sediment Transport: An Appraisal of Available Methods Hydr. Res. Station, Wallingford. Environmental Management in Practice 9. References van “Sediment Transport, Part II: Suspended Load Transport,” Journal of Hydraulic Engineering, vol. 110, No.11, 1984, pp.1613-1641. Rijn, L.C. van, “Initiation of Motion, Bed Forms, bed Roughness, Sediment Concentrations and Transport by Currents and Waves”, Report S 487-IV, Delft Hydraulics Laboratory, Delft, The Netherlands, 1985. y Rijn, L.C. van, “Model for Sedimentation Predictions”, Proc., 19th IAHR-Congress, vol.2, New Delhi, India, 1980, pp.321-329. Rijn, L.C. van, “Sediment Transport, Part I: Bed Load Transport”, Journal of Hydraulic Engineering, vol.110, No.10, 1984, pp.1431-1456. Rijn, L.C. van, “Sediment Transport, Part III: Bed Forms and Alluvial Roughness,” Journal of Hydraulic Engineering, vol.110, No.12, 1984, pp.1733-1754. y g g pp Van OS, A.G. (1990). Density currents and salt Intrusion, Lecture Note for the Hydraulic Engineering Course at Unesco-IHE, Delft, The Netherlands. 174 Environmental Management in Practice Environmental Management in Practice Environmental Management in Industry 9 1. Introduction Since the end of the 1990s, businesses have started to systematically consider environmental problems in terms of different positions and levels within a firm, such as design, purchase, sale, and disposal (Welford, 2000). The United Kingdom published BS 7750, a standardized specification for an environmental management system in 1994 and the International Organization for Standardization (ISO) published ISO 14001 - an environmental management standard in 1996. The main goal of these standards is to help all kinds of organizations to establish and implement environmental management systems by systematically setting up environmental policies, practices, objectives, and targets. The number of organizations with ISO 14001 certification around the world rapidly increased to 13,368 in December of 1999 to 129,031 in December of 2006 (Corporate Risk Management Company, 2000:2007). Welford (2000) insisted that Environmental Management Systems (EMSs), such as ISO 14001, are no longer options. However, there are some problems with EMSs. The ISO 14001 standard does not promote the flexibility needed to handle continuously changing environmental issues (Moxen & Strachan, 1998). The ISO 14001 mostly depends on action control and results based on environmental impacts, rather than social and ethical control. Thompson (2002) pointed out three areas of ISO 14001 that should be described: (i) social aspects and impacts and how to control them; (ii) guidelines for a set of widely recognized and accepted environmental performance principles; and (iii) a method to communicate environmental performance information to external stakeholders and decision makers. To address these areas, businesses should go even further than environmental management systems and completely integrate all the components of sustainable development into a new way of doing business (Welford, 2000). In addition, a variety of interested parties, such as governments, “green” consumers, and “green” investors, are also encouraging firms to incorporate their environmental management systems and sustainable development into their decision-making process for sustainable business practices and/or strategies. Companies could implement sustainable business practice to meet these demands for interested parties on sustainable business. To effectively implement sustainable business practices, firms need to know the kinds of indicators that meet the characteristics or concepts of sustainable business practices. Based on these needs, we aims to identify whether or not firms have applied sustainable business practices based on the Triple Bottom Line (Environmental, economic, and social 178 Environmental Management in Practice areas). To accomplish this goal, we conducted two surveys. 2.1 Sustainable business There is no single definition of sustainable business, as there is for sustainable development (Azapagic, 2003). A lack of a common accepted definition of sustainable business is the most critical problem because the definition is a fundamental tool to carry out new policies and actions. To overcome this, a few institutions have introduced the definition of sustainable business. The Evergreen Group (2008), a business brokerage dedicated to sustainable business, defines that a sustainable business is a business that carries out an environmentally friendly business processes without negative environmental impacts related to their activities, products, and services. Sustinable business.Com1 (2009) says that sustainable business is “a business that contributes to an equitable and ecologically sustainable economy.” Based on these examples of the definitions of sustainable business, sustainable business offers products and services that fulfill society's needs while contributing to the well-being of all earth's inhabitants. Sustainable business is a new, radical paradigm that considers the ecological, social, and economic impacts in a way that will not compromise the needs of future generations (Azapagic & Perdan, 2000; Welford, 2000). Azapagic and Perdan (2000) asserted that firms need a paradigm shift if firms want to integrate sustainable development into their business. Sustainable business requires effective harmonization of a Triple Bottom Line (TBL), which is the environmental, economic, and social areas. Since the TBL is the key element of sustainable development, firms that carry out sustainable business should not only understand the TBL, but also integrate it into their policies or strategies and decision- making processes (Desimone & Popoff, 1998; WBCSD, 2000). The environmental area consists of environmental impacts related to an organization’s diverse activities, products, and services. These environmental indicators should be identified in all stages of the organization’s full life cycle because they are used to track environmental progress, support environmental policy evaluation and inform the public. Examples of environmental indicators are energy and water consumption, air pollution, and solid and hazardous waste produced. The economic area includes an organization’s economic values and performance that are explained by economic indicators. The economy provides solutions and methods to invest in protecting the environment and conservation of natural resources as well as to sustain society. Examples are annual profits and sales, Research & Development investment, fines, capital investment, and share values or annual returns. 1. Introduction The first survey identified the trends of indicators in terms of the TBL used to describe sustainable business practices. The second survey assessed the degree to which firms have issued performance reports and what kinds of keywords were used in the titles of these reports. 1 Sustinablebusiness.Com: SustainableBusiness.com is an organization that “provides global news and networking services to help green business grow, covering all sectors: renewable energy, green building, sustainable investing, and organics” http://www.sustainablebusiness.com/ 2 Social Accountability 8000: Social Accountability 8000 was developed by the Council on Economic Priorities Accreditation Agency in 1997. “SA8000 is promoted as a voluntary, universal standard for companies interested in auditing and certifying labour practices in their facilities and those of their suppliers and vendors. It is designed for independent third party certification” http:// www.mallenbaker.net/csr/CSRfiles/SA8000.html 2.1 Sustainable business The social area is related to wider responsibilities that business has to communities within which it operates and to society in general, including both present and future generations. Since the importance of social and ethical responsibilities of a company is gradually 1 Sustinablebusiness.Com: SustainableBusiness.com is an organization that “provides global news and networking services to help green business grow, covering all sectors: renewable energy, green building, sustainable investing, and organics” http://www.sustainablebusiness.com/ 179 Indicators of Sustainable Business Practices increasing, its social responsibility has become a constituted element within what society expects from business. A few international organizations and institutions, such as the European Commission (EC), have developed and launched a variety of standards relevant to corporate social and ethical responsibility around the world. For instance, the Social Accountability 8000 (SA 8000)2 focused on social and ethical issues, and on Corporate Social Responsibility (CSR). It is not easy to define and quantify social indicators in terms of physical indicators like economic and environmental indicators. Nevertheless, many firms have set up a realistic goal to continuously measure these indicators in a comparable manner across organizations by using qualitative social indicators. These sets of qualitative social indicators are used to evaluate sustainable business embedded in the concept of sustainable development. Examples of social indicators are: (i) human development and welfare (e.g., education and training and health and safety); (ii) equity (e.g., wages, equal opportunity, and non-discrimination); and (iii) ethical considerations (e.g., human rights and child labor abolition) (Azapagic, 2003). 2.2 Voluntary communication to the public A firm that would like to apply sustainable business could voluntarily communicate diverse performance of their practices to the public because interested parties want to know information about the firms’ sustainable business practices (Adams, Houldrin & Slomp, 1999). Voluntary reporting information about firms’ environmental and social performance is becoming a powerful and popular tool to communicate with the public because interested parties can use such information to evaluate firms’ activities and performance (Feldman, Soyka, & Ameer, 1996; Sasseville, Willson, & Lawson, 1997). Internal or external reporting systems can have a significant effect on corporate culture for sustainable business because they are designed to support positive behaviors in terms of sustainable development. parties can use such information to evaluate firms activities and performance (Feldman, Soyka, & Ameer, 1996; Sasseville, Willson, & Lawson, 1997). Internal or external reporting systems can have a significant effect on corporate culture for sustainable business because they are designed to support positive behaviors in terms of sustainable development. Since the early 1990s, a few companies, such as Monsanto and Kodak, have disclosed outcomes of their environmental performance according to their own indicators. However, the lack of credibility and verifiability of the indicators and outcomes disclosed in these reports has become a significant problem (Lin & Wang, 2004; Thompson, 2002). To overcome these problems, in 2002, the Global Reporting Initiative (GRI) published the 2002 GRI Sustainability Reporting Guidelines based on the concepts of sustainable development (Lin & Wang, 2004 ; Thompson, 2002). The GRI guidelines propose principles and general indicators to report an organization’s performance in terms of the TBL: economic, environmental, and social dimensions. After publishing the GRI guidelines, many companies like 3M have integrated their own indicators into the GRI guidelines. SmiXXX (06) said that it used the Global Reporting Initiative’s 2002 Sustainability Reporting Guidelines to increase the credibility of its information and reports. In 2002, the European Commission (EC) published “Corporate Social Responsibility (CSR): A business contribution to Sustainable Development”. The EC formally defined corporate social responsibility: y g pp p p Since the early 1990s, a few companies, such as Monsanto and Kodak, have disclosed outcomes of their environmental performance according to their own indicators. However, the lack of credibility and verifiability of the indicators and outcomes disclosed in these reports has become a significant problem (Lin & Wang, 2004; Thompson, 2002). 2.3 Indicators for sustainable business practices An indicator is a measurement that shows the status of an environmental, economic, or social system over time (Redefining Progress, Sustainable Seattle, and Tyler Norris Associates, 1997). The goals of indicators are: ) g  to monitor and evaluate effectiveness and performance of goals and targets of sustainable business (Bennett & James,1999; Parris & Kates, 2003); ) g  to monitor and evaluate effectiveness and performance of goals and targets of sustainable business (Bennett & James,1999; Parris & Kates, 2003);  to communicate to diverse stakeholders (Thompson, 2002). Indicators can help stakeholders, including the pubic, decision makers, and managers, to assist in decision- making about sustainable business (Kuhndt & Geibler, 2002); and  to communicate to diverse stakeholders (Thompson, 2002). Indicators can help stakeholders, including the pubic, decision makers, and managers, to assist in decision- making about sustainable business (Kuhndt & Geibler, 2002); and g  to compare actions and performance of firms that may or may not be implementing sustainable business (Kuhndt & Geibler, 2002).  to compare actions and performance of firms that may or may not be implementing sustainable business (Kuhndt & Geibler, 2002). With these objectives in mind, numerous companies and international organizations, such as the International Organization for Standardization and the Global Reporting Initiatives, have developed a set of indicators to measure progress of environmental performance and sustainable business. Many organizations are using diverse indicators to integrate current environmental management systems into sustainable business. g y Indicators for sustainable business practices can be expressed in many different forms (e.g., qualitative or quantitative, general or specific, and absolute or relative), in accordance with objectives and applications of an indicator. Quantitative indicators are measured in terms of mass, volume or number of environmental pollutants or physical materials. Examples of quantitative indicators are total amount of air emissions like CO2, or total volume of hazardous waste. Not all indicators will be quantitative, and some will have to be expressed qualitatively because they cannot be defined in physical terms (Azapagic & Perdan, 2000). Qualitative indicators are expressed interpretively. Qualitative indicators include social dimensions of a firm’s activities, such as changes in cultural values or equity (Azapagic & Perdan, 2000). Sustainable business could be described by both qualitative and quantitative metrics because both are required to explain whether or not an organization’s diverse activities consider or meet human needs and social demands (Daly, 1990; Azapagic & Perdan, 2000). 2.2 Voluntary communication to the public To overcome these problems, in 2002, the Global Reporting Initiative (GRI) published the 2002 GRI Sustainability Reporting Guidelines based on the concepts of sustainable development (Lin & Wang, 2004 ; Thompson, 2002). The GRI guidelines propose principles and general indicators to report an organization’s performance in terms of the TBL: Since the early 1990s, a few companies, such as Monsanto and Kodak, have disclosed outcomes of their environmental performance according to their own indicators. However, the lack of credibility and verifiability of the indicators and outcomes disclosed in these reports has become a significant problem (Lin & Wang, 2004; Thompson, 2002). p g p ( g p ) To overcome these problems, in 2002, the Global Reporting Initiative (GRI) published the 2002 GRI Sustainability Reporting Guidelines based on the concepts of sustainable development (Lin & Wang, 2004 ; Thompson, 2002). The GRI guidelines propose principles and general indicators to report an organization’s performance in terms of the TBL: economic, environmental, and social dimensions. After publishing the GRI guidelines, many companies like 3M have integrated their own indicators into the GRI guidelines. SmiXXX (06) said that it used the Global Reporting Initiative’s 2002 Sustainability Reporting Guidelines to increase the credibility of its information and reports. In 2002, the European Commission (EC) published “Corporate Social Responsibility (CSR): A business contribution to Sustainable Development”. The EC formally defined corporate social responsibility: 180 Environmental Management in Practice CSR is a concept whereby companies integrate social and environmental concerns in their business operations and in their interaction with their stakeholders on a voluntary basis. (p. 7) CSR is a concept whereby companies integrate social and environmental concerns in their business operations and in their interaction with their stakeholders on a voluntary basis. (p. 7) The Corporate Social Responsibility (CSR) standard includes environmental, financial, and social performance information related to sustainable development. To meet the demands of the public for corporate social responsibility, many companies, such as Kodak and Ford, are annually disclosing the performance reports of their sustainable business practices with different titles, such as “Corporate Social report,” and “Sustainability Report” to the public. 2.3 Indicators for sustainable business practices Thus, many firms are setting up qualitative indicators as a substantial goal to measure the progress of the firms’ policies even though qualitative indicators are difficult to define in physical terms (Azapagic & Perdan, 2000). p y ( p g ) Indicators can also be divided into general and specific indicators (Verfaillie & Bidwell, 2000). General indicators are used by businesses across all industries in the world. These general indicators can be used to measure issues that have already been discussed globally, such as an international agreement or consensus: Agenda 21, Montreal Protocol, and Kyoto Protocol (global warming) (Verfaillie & Bidwell, 2000; Muller & Sturm, 2001). General indicators include energy, water and material consumption, greenhouse gas emissions, carbon dioxide, methane, and air emissions per unit product. These indicators can be used 181 Indicators of Sustainable Business Practices to compare one organization’s performance against another’s. Specific indicators are defined differently and measured in accordance to characteristics of each industry or firm (Verfaillie & Bidwell, 2000). For instance, Chemical Industries Association (2002) established the Responsible Care (RC) program for companies in the chemical industry. RC is the chemical industry's global voluntary initiative program. to compare one organization’s performance against another’s. Specific indicators are defined differently and measured in accordance to characteristics of each industry or firm (Verfaillie & Bidwell, 2000). For instance, Chemical Industries Association (2002) established the Responsible Care (RC) program for companies in the chemical industry. RC is the chemical industry's global voluntary initiative program. y g y p g Indicators for sustainable business practices can be expressed in absolute or relative forms. Absolute indicators are used to measure a firm’s quantitative environmental and social impact related to its activities, products, and services. Thompson (2002) said that absolute indicators are expressed in terms of measured quantities: total amount of energy consumed a year, total amount of water consumed, total amount of wastewater, and total amount of hazardous waste generated. These indicators can provide managers or the pubic with incomplete information relevant to operational levels because these indicators use a single value to represent how much a firm has accomplished towards its goals and targets over time (Bennett & James, 1999). For instance, a firm reduces the total energy consumed this year by 5% compared to last year’s total. 2.3 Indicators for sustainable business practices A manager cannot determine whether or not this reduction is an environmentally positive result since the reduction of energy could be the result of other factors, such as reduction of productivity, rather than actual improvements of environmental activities and technologies. Relative indicators were introduced to address this problem of absolute indicators. p Relative indicators are expressed in terms of a ratio or proportion that compares an absolute indicator with another absolute indicator (Thompson, 2002). Azapagic and Perdan (2000) argue that relative indicators enable firms and interested parties to evaluate improvement from year to year and figure out more sustainable opportunities and practices. Thus, relative indicators could help stakeholders understand whether or not a company truly increases efficiency of emissions by measuring levels of pollutant per unit of production (Bennett & James, 1999). Examples of relative indicators are eco-efficiency indicators, such as carbon dioxide emissions per unit of output, ratio of waste per unit of input material, ratio of total hazardous solid waste per unit of product, etc. These relative indicators can be used to measure the constant economic value of natural capital stocks. However, Bennett and James (1999) mentioned that relative indicators also have a problem because they do not show the total amount of pollutants in terms of absolute values, which could be used as firm to firm benchmarking. To resolve these problems of absolute and relative indicators, many companies choose to use both types of indicators to evaluate and report their performance. 3 Internet Archive Organization is “a 501(c)(3) non-profit that was founded to build an Internet library, with the purpose of offering permanent access for researchers, historians, and scholars to historical collections that exist in digital format” http://www.archive.org/index.php 3. Data collection We conducted two surveys. To conduct the first survey, we collected firms’ annual performance reports announced to the public through Internet media. There are two reasons why these performance reports were collected. The first reason is because the changes in the types of indicators for sustainable business practices were described in those performance reports. The second one is that the changes in the performance reports announced through Internet mass media can be used to investigate the extent to which firms have communicated their performance reports to the public. Sample performance reports for the first survey were collected from January 1999 to December 2006. Since the ISO published ISO 14031 Environmental Performance Evaluation - 182 Environmental Management in Practice guidelines in 1999, firms might have gained interest in reporting their environmental performance beginning in 1999. 2006 is the most current year that firms’ performance reports could be collected through firms’ Internet homepages. The announcements that were disclosed the performance reports were identified by using newswire databases; ABI/Inform, Global, Business & Industry, Business & Company Resource Center, and LexisNexis. The key words used to find the announcement events were “Environmental Performance,” “Reports,” “Sustainability,” “Corporate Social Responsibility,” and “Citizenship.” The following criteria were used to collect sample data: p y p g p  Only publicly traded firms on the New York Stock Exchange (NYSE) were consid y p g p nly publicly traded firms on the New York Stock Exchange (NYSE) were considered; y y g  Companies in the information, finance, and insurance industry were excluded b their businesses did not generate direct environmental pollution; and g p Firms that provide their performance reports (PDF file) were included.  Firms that provide their performance reports (PDF file) were included. Companies have created and continuously updated their Internet homepages to provide environmental and social performance reports. After identifying firms that announced their performance reports, the performance reports of sample firms were collected through each firm’s Internet homepage. The Internet Archive Organization3 was used to find the performance reports of companies that did not provide previous performance reports directly from the current homepage. The internet archive organization provides archive data of a firm’s Internet homepage according to the day that the firm updated the homepage. The North American Industry Classification System (NAICS) was used to classify types of industries A firm’s NAICS code categorized by the Wharton Research Data Service (WRDS) was used. 3. Data collection  Firms that provide their performance reports (PDF file) were included. Companies have created and continuously updated their Internet homepages to provide environmental and social performance reports. After identifying firms that announced their performance reports, the performance reports of sample firms were collected through each firm’s Internet homepage. The Internet Archive Organization3 was used to find the performance reports of companies that did not provide previous performance reports directly from the current homepage. The internet archive organization provides archive data of a firm’s Internet homepage according to the day that the firm updated the homepage. The North American Industry Classification System (NAICS) was used to classify types of industries A firm’s NAICS code categorized by the Wharton Research Data Service (WRDS) was used. The indicators for sustainable business practices were selected by reviewing diverse environmental and sustainable indicator guidelines, such ISO 14031, GRI guidelines, the Organisation for Economic Co-operation and Development (OECD), Social responsibility, and other researchers. The second survey was conducted to identify the current trend in the titles of firms’ performance reports. The terms used as key words in titles of firms’ performance reports could be used to identify the main themes or strategies of the reports (Bruemmer, 2000). Performance reports have been given diverse titles, such as “Environmental Reports,” “Environmental, Health, and Safety Report,” “Sustainable Reports,” “Corporate Social Reports,” “Citizenship Report,” etc. If a firm used “Environmental” as a key word in the titles of its performance report, it means that the firm did not set up social and economic indicators, which are the fundamental indicators of sustainable business. However, if a firm used the terms, “Social Responsibility,” “Corporate Social Responsibility,” “Sustainability,” and “Citizenship” as key words, it could indicate that the firm has likely incorporated the concepts of sustainable development into its business strategies, which is sustainable business. This is because these terms are evolved from the concept of sustainable development. For the second survey, we used S&P 500 firms as of December 2006 that reported their performance reports to the public in 2007. Since 2006 performance reports, disclosed in 2007, were the most current reports that could be collected through the Internet, they were chosen as the sample. Thus, the Internet homepages of S&P 500 sample companies were searched to identify annual sustainability or environmental reports for 2006. 3. Data collection Among S&P 500 firms, a 183 Indicators of Sustainable Business Practices few industries (e.g., Information; Finance and Insurance; Real Estate and Rental and Leasing; Educational Services; and Health Care and Social Assistance) were excluded from the sample because they neither generated environmental pollution nor had heavy environmental burdens. 4.1 Changes in indicators for sustainable business We found eighty-nine announcements eighty-nine announcements published by 40 companies through Internet media. Approximately eighty-eight percent (78 announcements) of the total sample was taken from the manufacturing industries (NAICS code 31, 32, and 33). The rest of the total samples (21 announcements) was disclosed by firms in other industries: the mining industry (NAICS code 21), the utilities industry (NAICS code 22), the miscellaneous store retailers (NAICS code 45), and the couriers and messengers industry (NAICS code 49). Table 1 presents the distribution of the sampled companies based on the NAICS. Table 2 lists the types of manufacturing industries. Of the NAICS Year Total Title (Two digit) Three digit '99 '00 '01 '02 '03 '04 '05 '06 Number % Mining (21) 212 1 1 1 3 3.4% Utilities (22) 221 2 2 4 4.5% Manufacturing (31,32,33) 311 1 3 4 87.6% 312 1 1 1 3 316 1 2 1 4 321 1 1 1 3 322 1 2 3 324 1 2 1 4 3 11 325 1 1 1 3 3 2 3 14 331 1 2 1 2 2 8 333 1 1 2 4 334 1 2 1 3 2 9 335 1 1 2 336 1 1 1 2 5 3 13 Miscellaneous Store Retailers(45) 453 1 1 1.1% Couriers and Messengers (49) 492 1 1 1 3 3.4% Total 2 1 5 4 11 12 26 28 89 Table 1. Distribution of Sampled Companies Based on the NAICS NAICS Year Total Title (Two digit) Three digit '99 '00 '01 '02 '03 '04 '05 '06 Number % Mining (21) 212 1 1 1 3 3.4% Utilities (22) 221 2 2 4 4.5% Manufacturing (31,32,33) 311 1 3 4 87.6% 312 1 1 1 3 316 1 2 1 4 321 1 1 1 3 322 1 2 3 324 1 2 1 4 3 11 325 1 1 1 3 3 2 3 14 331 1 2 1 2 2 8 333 1 1 2 4 334 1 2 1 3 2 9 335 1 1 2 336 1 1 1 2 5 3 13 Miscellaneous Store Retailers(45) 453 1 1 1.1% Couriers and Messengers (49) 492 1 1 1 3 3.4% Total 2 1 5 4 11 12 26 28 89 Table 1. Distribution of Sampled Companies Based on the NAICS Table 1. Distribution of Sampled Companies Based on the NAICS Table 1. Table 1. Distribution of Sampled Companies Based on the NAICS 4.1 Changes in indicators for sustainable business Distribution of Sampled Companies Based on the NAICS 184 Environmental Management in Practice NAICS Type of Manufacturing Number (%) 311 312 316 321 322 324 325 331 333 334 335 336 Food Manufacturing Beverage and Tobacco Product Manufacturing Leather and Allied Product Manufacturing Wood Product Manufacturing Paper Manufacturing Petroleum and Coal Products Manufacturing Chemical Manufacturing Primary Metal Manufacturing Machinery Manufacturing Computer and Electronic Product Manufacturing Electrical Equipment, Appliance, and Component Manufacturing Transportation Equipment Manufacturing 4 (5%) 3 (4%) 4 (5%) 3 (4%) 3 (4%) 11 (14%) 14 (18%) 8 (10%) 4 (5%) 9 (12%) 2 (3%) 13(17%) Total 78 Table 2. Types of Manufacturing in the Sample Announcements Table 2. Types of Manufacturing in the Sample Announcements 78 announcements in the manufacturing industries, 55 announcements (71%) are from firms in petroleum and coal products manufacturing, chemical manufacturing, primary metal manufacturing, computer and electronic products manufacturing, and transportation equipment manufacturing. The main reason why firms in these manufacturing industries have disclosed their performance reports more often than in other industries is that firms producing final consumer goods proactively meet needs and avoid potentially adverse stakeholders’ reactions (Anton, Deltas & Khanna, 2004). Anton et al. (2004) said that firms that produce consumer goods are pressured by environmental interests more than firms that produce industrial goods. To proactively respond to the increasing environmental pressures and social responsibilities, firms producing consumer goods have actively communicated their environmental and social information to their interested parties. We could not find many announcements in the mining sector related to the disclosure of environmental or sustainable performance reports during 1999 to 2006. Three announcements were reported by one firm, BXXX Ltd. Other firms in this industry have reported and provided their environmental performance reports on their Internet homepages. For instance, CXXX has reported the performance of a few environmental and social indicators relevant to sustainable development on its Internet homepage. It has monitored the performance of environmental and social indicators since 2005. p Since the utilities industry has to use natural capital to produce their products, such as electric power, natural gas, and fuel, it is one of the critical industries for sustaining society, doing business, and for activities such as the operation of factories and the routine activities of daily life. We found just four announcements in the utilities industry that were reported by. It does not seem that many firms in this industry proactively communicate their performance reports to the public. However, they have started disclosing their performance reports on Internet homepages since 2005 or 2006. For example, SXXX Company began providing its Corporate Responsibility Reports in 2006. To proactively respond to the increasing requirements of firms’ performance reports, they might realize that they should disclose their social and environmental performance reports. 185 Indicators of Sustainable Business Practices OXXX in the miscellaneous store retailers industry announced its performance reports based on the concept of sustainable development and business in 2006. Some firms in this industry have also reported their environmental or sustainability performance reports. For instance, StaXXX Inc. Table 2. Types of Manufacturing in the Sample Announcements has been reporting its corporate responsibility, which includes a few sustainable business indicators, on its Internet homepage since 2006. g There were three announcements of environmental or sustainable performance reports in the couriers and messengers industry. They were reported by UXXX. UXXX has disclosed its sustainability reports since 2003. Like the utilities industry and the miscellaneous store retailers industry, a few firms like FXXX had provided their environmental or sustainable performance reports on their Internet homepages. 4.1.1 Increasing announcements Figure 1 shows the trends of the announcements of the disclosure of firms’ performance reports during 1999 to 2006. We did not find many firms that announced their performance reports through diverse Internet media even though they began reporting their environmental performance in the early 2000s. This is consistent with previous studies. When Hamilton (1995) studied how media and stock market responded to the disclosure of the Toxic Release Inventory (TRI) data, he used 50 firms that reported TRI data through the media. This indicates that firms did not progressively communicate their environmental information to the public. Firms did not use various communication tools to inform the public about their environmental performance reports. According to Figure 1, the number of announcements of the disclosure of firms’ performance reports has been gradually increasing since 2003. Firms that announced performance reports before 2002 were in the manufacturing industry. From 2003, firms in other industries, such as the couriers and messengers, the mining, and the utilities industries, started announcing their performance reports through diverse Internet media. There are two reasons why the number of announcements of firms’ performance reports might have increased since 2003. 2 1 5 4 11 12 26 28 0 5 10 15 20 25 30 '99 '00 '01 '02 '03 '04 '05 '06 Year Number of Event Fig. 1. Trends of the Announcements from 1999 to 2006 The first reason is that after 2003 firms might have recognized that voluntarily announcing their performance reports by using various Internet media is a powerful tool to inform the public of their performance reports (Feldman et al, 1996; Sasseville et al., 1997). Firms can 2 1 5 4 11 12 26 28 0 5 10 15 20 25 30 '99 '00 '01 '02 '03 '04 '05 '06 Year Number of Event Fig. 1. Trends of the Announcements from 1999 to 2006 The first reason is that after 2003 firms might have recognized that voluntarily announcing their performance reports by using various Internet media is a powerful tool to inform the public of their performance reports (Feldman et al, 1996; Sasseville et al., 1997). Firms can The first reason is that after 2003 firms might have recognized that voluntarily announcing their performance reports by using various Internet media is a powerful tool to inform the public of their performance reports (Feldman et al, 1996; Sasseville et al., 1997). 4.1.1 Increasing announcements Firms can 186 Environmental Management in Practice use their socially and environmentally friendly management activities as key information in their marketing strategies because environmental and social information has been gaining significance as a marketing tool since the early 2000s. Another reason is that a few international guidelines relevant to the disclosure of environmental, social, and economic performance reports have been published since 2002, such as the 2002 GRI Sustainability Reporting Guidelines which is the fundamental guidelines of all GRI documents (GRI, 2004). The 2002 GRI guidelines included more detailed performance indicators of three sustainability dimensions (economic, environmental, and social) than the 2000 GRI guidelines first published by the GRI in 2000. Thus, many firms have actively adopted the 2002 GRI guidelines not only to voluntarily implement sustainable business, but also to voluntarily communicate the performance of sustainable business. After publishing the GRI guidelines, many global firms have integrated their own indictors into the GRI guidelines to meet the needs of their interested parties. For example, UXXX announced its first corporate social responsibility report with the title “Operating in Unison UXXX 2002 Corporate Sustainability Report” on Nov 14, 2003. In this report, they mentioned, “We used the Global Reporting Initiative (GRI) as the foundation for writing our first Corporate Sustainability Report.” 4.1.2 Identifying sustainable business indicators (SBIs) Firms in the mining industry started announcing their performance reports in 2004 and firms in the utility industries announced their performance reports in 2005 through Internet media. General indicators that were reported in over 60% of the samples of the pilot survey are defined as sustainable business indicators (SBIs) for this research. Table 3 shows the results of the pilot survey. significantly affect environmental and social impacts, such as the mining, utilities, and manufacturing industries. Firms in the mining industry started announcing their performance reports in 2004 and firms in the utility industries announced their performance reports in 2005 through Internet media. General indicators that were reported in over 60% of the samples of the pilot survey are defined as sustainable business indicators (SBIs) for this research. Table 3 shows the results of the pilot survey. Based on Table 3, the distribution of general indicators in each category is as follows: 9 environmental indicators; 5 economic indicators; 10 social indicators: and 5 integrated indicators. We did not find relative indicators, such as socio-environmental and socio- economic indicators that were reported in over 60% of the pilot sample. Based on the results p y Based on Table 3, the distribution of general indicators in each category is as follows: 9 environmental indicators; 5 economic indicators; 10 social indicators: and 5 integrated indicators. We did not find relative indicators, such as socio-environmental and socio- economic indicators that were reported in over 60% of the pilot sample. Based on the results p y Based on Table 3, the distribution of general indicators in each category is as follows: 9 environmental indicators; 5 economic indicators; 10 social indicators: and 5 integrated indicators. We did not find relative indicators, such as socio-environmental and socio- economic indicators that were reported in over 60% of the pilot sample. 4.1.2 Identifying sustainable business indicators (SBIs) After reviewing diverse environmental and sustainable indicator guidelines, such as the GRI guidelines, a total of 90 general indicators were selected. Table 3 shows the list of 90 general indicators. These general indicators were separated into seven categories in order to identify absolute and relative indicators types for sustainable business based on the TBL: 22 Environmental indicators; 14 economic indicators; 16 social indicators; 15 economic and environmental (eco-efficient) indicators; 7 social and environmental (socio-environmental) indicators; 6 social and economic (socio-economic) indicators; and 10 environmental, economic and social (integrated) indicators. Environmental, economic, and social indicators are absolute indicators. Eco-efficient, socio- environmental, socio-economic, and integrated indicators are relative indicators used to implement sustainable business practices. Socio-environmental indicators are focused on environmental impacts that affect social impacts, and vice versa. Azar, Holmberg, and Lindgren (1996) mentioned that the goal of the socio-environmental indicators is to serve as a tool in planning and decision-making processes at various managerial levels within society. Socio-economic indicators are related to the relationship between a firm’s economic activities and social effects. Socio-economic requires firms not only to consider one or more social impacts, but also one or more economic impacts (Etzioni, 2003). Unlike socio- environmental and socio-economic indicators, eco-efficient indicators are more easily understood and quantified than those of the socio-environmental and socio-economic indicators. Eco-efficient indicators incorporated with environmental and economic indicators mean business’s activities that increase economic values while decreasing ecological impacts and using natural capital stocks (Desimone & Popoff, 1998). Integrated indicators are comprehensively incorporated with economic, environmental, and social issues of the TBL. They are systematic and fundamental indicators that are built from the concepts of sustainable business as well as supporting the other indicators. p pp g To identify the general indicators for sustainable business, a pilot survey was conducted. This pilot survey was implemented by identifying whether or not each indicator of 90 general indicators was popularly reported in each pilot sample, which is a firm’s report. Of the 89 sample firms’ reports, 38 performance reports disclosed in 2004 and 2005 were selected as pilot samples in order to select a sample of firms in the industries that 187 Indicators of Sustainable Business Practices significantly affect environmental and social impacts, such as the mining, utilities, and manufacturing industries. 4.1.2 Identifying sustainable business indicators (SBIs) Based on the results Indicators Reporting (%) Not Reporting(%) Environmental 1)Total amount of water used 2)Total amount of materials used to package product 3)Total amount of materials used to produce products 4)Total amount of renewable resources used 5)Total amount of non-renewable resources used 6)Total amount of recycled or reused materials used 7)Total amount of energy used 8)Total amount of renewable energy used 9)Total amount of non-renewable energy used (oil) 10)Concentration of a specific contaminant in tissue of a specific plant species found in the local or regional area 11)Habitats protected or restored 12)Strategies, current actions, and future plans for managing impacts on biodiversity 13)Total amount of greenhouse gases generated (CO2) 14)Total amount of emissions of ozone-depleting substances 15)Total amount of Volatile Organic Compound (VOC) generated 16)Total amount of air emissions generated (SOx, NOx) 17)Total amount of waste recycled or reused 18)Total amount of solid waste generated 19)Total amount of hazardous waste generated 20)Total number and volume of significant spills and accidents 21)Total amount of wastewater 22)Total number of environmental violations 34(90%) 14(37%) 8(21%) 7(18%) 0(0%) 15(40%) 35(92%) 20(53%) 12(32%) 17(45%) 20(53%) 16 (42%) 38(100%) 18(47%) 24(63%) 28(74%) 26(68%) 32(84%) 31(82%) 21(55%) 16(42%) 30(79%) 4(10%) 24(63%) 30(79%) 31(82%) 38(100%) 23(60%) 3( 8%) 18(47%) 26(68%) 21 (55%) 18(47%) 22(58%) 0(0%) 20(53%) 14(37%) 10(26%) 12(32%) 6(16%) 7(18%) 17 (45%) 22(58%) 8(21%) Economic 1)Annual profits 2)Annual revenues 3)Annual sales 4)Annual operating costs (based on EHS) 5)Costs saving (based on EHS) 6)Capital expenditure (environmental) 7)Annual productivity 8)Fines 9)R & D investment (Based on EHS) 10)R & D investment (total) 11)Donations 12)Annual turnover 13)Value added 14)Stock price/dividends 23(61%) 18(47%) 30(79%) 14(37%) 8(21%) 11(29%) 15(40%) 28(74%) 8(21%) 24(63%) 37(97%) 3(8%) 0(0%) 19(50%) 15(39.5%) 20(52.6%) 8(21.1%) 24(63.2%) 30(78.9%) 27(71%) 23(60%) 10(26%) 30(79%) 14(37%) 1(3%) 35(92%) 38(100%) 19(50%) 188 Environmental Management in Practice Indicators Reporting (%) Not Reporting(%) Social 1)Female, disabled person’s rights 2)Abolition of all child labor 3)The recruitment of people from ethnic minorities, older workers, women 4)Empowerment of employees 5)Average hours of training per employee 6)Number of employees 7)Employment creation 8)Employment turn over 9)Recordable Illness rate (RIR) 10)Lost time Rate (LTR) 11)Total number of work-related fatalities 12)Whether or not firms implement a broad range of voluntary activities 13)Whether or not firms provide opportunities to communicate internally and externally to interested parties 14)Breakdown of employees in terms of gender, age, and minority group 15)Ratio of basic salary of men to women by employee category 16) Whether or not equity was mentioned 26(68%) 21(55%) 26(68%) 23(61%) 26(68%) 33(87%) 20(53%) 12(32%) 27(71%) 25(66%) 20(53%) 35(92%) 31(82%) 27(71%) 10(26%) 7(18%) 12(32%) 17 (45%) 12(32%) 15(40%) 12(32%) 5 (13%) 18 (47%) 26(68%) 11 (29%) 13(34%) 18(47%) 3( 8%) 7(18%) 11 (29%) 28(74%) 31(82%) Social- Environmental 1)Training time/total amount of solid waste generated 2)Employee’s training time /total amount of energy used 3)Total solid waste/employee 4)Total amount of energy used /employee 5)Voluntary activities/total amount of energy used 6)Recordable illness rate/total amount of energy used 7)Lost time rate/total amount of energy used 3(8%) 2(5%) 5(13%) 3(8%) 0(0%) 0(0%) 0(0%) 35(92%) 36(95%) 33(87%) 35(92%) 38(100%) 38 100%) 38(100%) Social – economic 1)Training time of employee per profit 2)Sales per employee 3)Lost time rate per profits 4)Donations per sales 5)Donations per profit 6)Donations per revenue 2(5%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 36(95%) 38 (100%) 38(100%) 38(100%) 38(100%) 38(100%) Eco-efficiency 1)Total amount of material used / sales 2)Total amount of material used /profits 3)Total amount of solid waste /revenue 4)Total amount of non-renewable energy used / sales 5)Total amount of non-renewable energy used / sales 6)Total amount of non-renewable energy used / revenues 7)Total amount of energy used / sales 8)Total amount of energy used /revenues 9)Total amount of toxic materials generated/sales 10)Total amount of toxic materials generated /profits 11)Total amount of material recycled and reused/ales 12)Total amount of material recycled and reused /revenue 13)Total amount of global warming materials generated/sales 14)Total amount of global warming materials generated/profits 15)Total amount of global warming materials generated/ revenue 1(3%) 1(3%) 1(3%) 0(0%) 0(0%) 0(0%) 21(55%) 1(3%) 2(5%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 37(97%) 37(97%) 37 (97%) 38 (100%) 38 (100%) 38(100%) 17(45%) 37(97%) 36 (95%) 38 (100%) 38(100%) 38(100%) 38(100%) 38(100%) 38(100%) Indicators of Sustainable Business Practices 189 Indicators Reporting (%) Not Reporting(%) Integrated 1)Whether or not firms implement voluntary environmental management systems (ISO 14001, LCA, etc) 2)Whether or not firms implement environmental accounting 3)Whether or not firms make decisions based on the concept of sustainable business and long-term objective 4)Whether or not firms enlighten consumers and suppliers for the concept of sustainable business 5)Whether or not firms deal with the impact on the Third World 6)Whether or not being verified their performance reports by third parties 7)Whether or not firms compare GRI 8)Whether or not firms mention culture 9)Whether or not firms survey in the reports (feedback) 10)Whether or not firms compare performance based on standard year (tota l values/relative values) 28(74%) 2(5%) 29(76%) 27(71%) 16(42%) 14(37%) 25(66%) 28(74%) 16(42%) 21(55%) 10(26%) 36(95%) 9(24%) 11(29%) 22 (58%) 24(63%) 13(34%) 10(26%) 22(58%) 17 (45%) Table 3. 4.1.2 Identifying sustainable business indicators (SBIs) The List of 90 General Indicators and the Results of Pilot Survey (Sustainable Business Indicators over 60% of the sample) (N=38) of the pilot survey, firms were not familiar with relative indicators. Since many firms had already measured and reported absolute indicators, absolute indicators made up a larger proportion of the SBIs than relative indicators such as socio-economic and socio- environmental indicators. With 29 SBIs identified from the pilot survey, a full survey was conducted to identify SBIs in the total sample of 89 firm’s reports. Table 4 shows the results of the full survey. of the pilot survey, firms were not familiar with relative indicators. Since many firms had already measured and reported absolute indicators, absolute indicators made up a larger proportion of the SBIs than relative indicators such as socio-economic and socio- environmental indicators. With 29 SBIs identified from the pilot survey, a full survey was conducted to identify SBIs in the total sample of 89 firm’s reports. Table 4 shows the results of the full survey. 4.1.3 Changes in sustainable business indicators disclosed in performance g Eighty-nine sample companies were separated into two categories, category I (1999 ~ 2002) and category II (2003~2006), to compare the trends of sustainable business indicators over a time period. These two categories were divided based on the year 2003 because the number of firms that announced their performance reports increased beginning in 2003. To compare the trends of sustainable business indicators, we chose firms in the manufacturing industries because all firms in category I were in the manufacturing industries. Among the 89 sample companies, the 78 announcements disclosed by the manufacturing industries were divided into category I (12 firms) and category II (66 firms). g y ( ) g y ( ) To identify the changes in SBIs used in manufacturing firms, we added four indicators to the previously defined 29 sustainable business indicators; total amount of renewable energy used (solar energy, clean energy); whether or not firms describe environmentally friendly product or process; abolition of all child labor; and whether or not firms use relative indicators (eco- efficiency). Although some of these four indicators were not reported at over 60% in the pilot survey, they are considered necessary by the authors as indicators to evaluate the characteristics of sustainable business. Total amount of renewable energy used and whether or not firms develop or describe environmentally friendly product or process are used to evaluate whether or not firms apply diverse technologies to implement sustainable business; whether or not firms use relative indicators, such as eco-efficiency, is used to identify the consistency of natural capital stocks; and abolition of all child labor is used to evaluate the social performance of sustainable business. Thus, we used a total of 33 SBIs to identify the trends of sustainable business indicators of firms in the manufacturing industries. The trends of sustainable business indicators used in category I and category II is shown in Table 5. 190 Environmental Management in Practice Indicators No. of Firms Reporting (%) No. of Firms Not Reporting (%) Quant. indicator Qual. Table 5. Changes in Sustainable Business Indicators Used in Category I and II 4.1.3 Changes in sustainable business indicators disclosed in performance indicator Sub-total Environmental Indictors 1)Total amount of water used 66 13 79(89%) 10(11%) 2)Total amount of energy used 69 16 85(96%) 4(4%) 3)Total amount of greenhouse gases generated (CO2) 70 17 87(98%) 2(2%) 4)Total amount of Volatile Organic Compound (VOC) generated 38 16 54(61%) 35(39%) 5)Total amount of air emissions generate (SOx, NOx) 65 14 79(89%) 10 (11%) 6)Total amount of waste recycled or reused 51 26 77(87%) 12(13%) 7)Total amount of solid waste generated 54 28 82(92%) 7(8%) 8)Total amount of hazardous waste generated 56 19 75(84%) 14 (16%) 9)Total number of environmental violations 44 24 68(76%) 21(24%) Economic Indicators 1)Annual profits 44 13 57(64%) 32(36%) 2)Annual sales 68 12 80(90%) 9(10%) 3)Fines 49 12 61(69%) 28(31%) 4)R & D investment (total) 30 22 52(58%) 37(42%) 5)Donations 52 3 85(96%) 4(4%) Social Indicators 1)Female, disabled person’s rights 0 59 59(66%) 30(34%) 2)The recruitment of people from ethnic minorities, older workers, women 0 56 56(63%) 33(37%) 3)Empowerment of employees 0 58 58(65%) 31(35%) 4)Average hours of training/ employee 4 71 75(84%) 14(16%) 5)Number of employees 61 20 81(91%) 8(9%) 6)Recordable illness rate (RIR) 68 2 70(79%) 19(21%) 7)Lost time rate (LTR) 64 1 65(73%) 24(27%) 8)Whether or not firms implement a broad range of voluntary activities 0 82 82(92%) 7(8%) 9)Whether or not firms provide opportunities to communicate internally and externally to interested parties 0 78 78(88%) 11(12%) 10)Breakdown of employees in terms of gender, age, and minority group 0 55 55(62%) 34 (38%) Integrated Indicators (reference) 1)Whether or not firms implement voluntary environmental management systems (ISO 14001, LCA, etc) 0 6 68(76%) 21(24%) 2)Whether or not firms make decisions based on the concept of sustainable business and long-term objective 0 74 74(83%) 15(17%) 3)Whether or not firms enlighten consumers and suppliers for the concept of sustainable business 0 71 71(80%) 18(20%) 4)Whether or not firms compare GRI 0 60 60(67%) 29(33%) 5)Whether or not firms mention Culture 0 71 71(80%) 18 (20%) Table 4. List of the Sustainable Business Indicators (SBIs) (1999 ~ 2006) (N=89) Indicators of Sustainable Business Practices 191 5. Changes in Sustainable Business Indicators Used in Category I and II 192 Environmental Management in Practice ble 5. 4.1.3 Changes in sustainable business indicators disclosed in performance Changes in Sustainable Business Indicators Used in Category I and II Indicators of Sustainable Business Practices 193 ble Business Indicators Used in Cat hanges in Sustainable Business Indicators Used in Cat ble Business Indicators Used in Category I and II Table 5. Changes in Sustainable Business Indicators Used in Category I and II 194 Environmental Management in Practice Key Words in the Titles in Category I (1999 ~ 2002) Key Words in the Titles in Category II (2003 ~ 2006) Environmental / Environmental, Health, and Safety Sustainability Total Environmental / Environmental, Health, and Safety Sustainability Total 5 (42%) 7 (58%) 12 11 (17%) 55 (83%) 66 Table 6. Changes in Key Words Used in the Title of Performance Reports in Category I and II Table 6. Changes in Key Words Used in the Title of Performance Reports in Category I d II Daly (1990) and Azapagic and Perdan (2005) said that sustainable development should be described by qualitative as well as quantitative measurement because it is required to explain whether or not an organization’s diverse activities consider or meet human needs and social demands. We also found that firms in category I and II used both qualitative and quantitative indicators in their sustainable business performance reports. q p p In category II, most social and integrated indicators except for four social indicators and one integrated indicator were qualitative indicators. Two quantitative social indicators, the Recordable Illness Rate (RIR) and the Lost Time Rate (LTR), are used to evaluate firms’ occupational safety and health. The recordable illness rate is the number of full-time employees suffering a recordable injury or illness during a given calendar year. The LTR is measured as the number of lost time claims per million hours worked and allows analysis of the number of lost time claims without the distorting effects of the size of the workforce. 4.1.3.1 Consistency of natural capital (2005) defined energy efficiency as cumulative % improvement in energy use per unit of production value and reported that energy efficiency increased from 12% in 2002 to 22% in 2004. Most firms in category I used absolute indicators, such as the total amount of energy consumed, rather than relative indicators, while firms in category II used absolute indicators as well as relative indicators, such as eco-efficient indicators based on their own firms’ characteristics. This is because firms in category I did not have diverse and sufficient guidelines for relative indicators. After a few international guidelines, such as the GRI guidelines, were published in 2002, firms had opportunities to use or consider relative indicators, such as various eco-efficient indicators. Those guidelines have introduced and proposed diverse relative indicators, such as eco-efficient indicators. By comparing firms that used eco-efficient indicators in category I and II, we found that firms in category II may have proactively monitored and improved the level of consistency of natural capital stocks by setting up and evaluating eco-efficiency more so than firms in category I. development into their business by applying constant economic values of the natural capital stocks, such as eco-efficient indicators. development into their business by applying constant economic values of the natural capital stocks, such as eco-efficient indicators. By providing the performance of various eco-efficient indicators, firms can help interested parties understand how effectively physical natural capital stocks, such as energy, have been used to retain an appropriate level of natural capital stock. For instance, AnhXXX Company (2006) measured and reported a few eco-efficient indicators, such as energy efficiency in 1,000 gig Joules (gJs) per million dollars Adjusted Net Sales (ANS), and Hazardous waste generated in kg per million dollars ANS. BaXXX Inc. (2005) defined energy efficiency as cumulative % improvement in energy use per unit of production value and reported that energy efficiency increased from 12% in 2002 to 22% in 2004. Most firms in category I used absolute indicators, such as the total amount of energy consumed, rather than relative indicators, while firms in category II used absolute indicators as well as relative indicators, such as eco-efficient indicators based on their own firms’ characteristics. This is because firms in category I did not have diverse and sufficient guidelines for relative indicators. 4.1.3.1 Consistency of natural capital After a few international guidelines, such as the GRI guidelines, were published in 2002, firms had opportunities to use or consider relative indicators, such as various eco-efficient indicators. Those guidelines have introduced and proposed diverse relative indicators, such as eco-efficient indicators. By comparing firms that used eco-efficient indicators in category I and II, we found that firms in category II may have proactively monitored and improved the level of consistency of natural capital stocks by setting up and evaluating eco-efficiency more so than firms in category I. 4.1.3.1 Consistency of natural capital The consistency of natural capital stocks can be measured by identifying the changes in the constant physical capital stocks, such as renewable energy and resources. This is because constant physical capital stock is one of the two concepts of the consistency of natural capital stock (Pearce, Barbier, & Markandya, 1990). Accordingly, the amount of renewable energy used in firms is a sustainable business indicator. Examples of renewable energy used in firms’ performance reports are wind, solar energy, hydrogen energy, and biogas. Based on Table 5, firms in category I reported the performance of this indicator by 25%, but firms in category II reported it by about 71%. Since 2003, many firms in category II had increased the use of renewable energy while they reduced the use of non-renewable energy. In the Corporate Responsibility Report 2005, STXXX electronics (2006) reported that they increased the use of wind and solar energy from 18.6GWh in 2003 and 30.5GWh in 2004. In the ‘2004 Sustainability Report’ published in 2005, POTXXX Corporate reported that it has started using renewable energy in 2004. The consistency of the natural capital stock can also be measured by identifying a constant economic value, which is another concept of the consistency of natural capital stock (Pearce et al., 1990). We found firms that disclosed different eco-efficient indicators in their performance reports, such as energy efficiency, the amount of pollution per dollar, etc. Based on Table 5, only 25% of the sample firms in category I disclosed eco-efficient indicators in their performance reports, while about 85% of the sample firms disclosed them in category II. Many firms in category II reported eco-efficient indicators, such as energy efficiency, in their performance reports. This is consistent with what WBCSD (2005) and Desimone and Popoff (1998) stated. They said that firms can integrate sustainable 195 Indicators of Sustainable Business Practices development into their business by applying constant economic values of the natural capital stocks, such as eco-efficient indicators. By providing the performance of various eco-efficient indicators, firms can help interested parties understand how effectively physical natural capital stocks, such as energy, have been used to retain an appropriate level of natural capital stock. For instance, AnhXXX Company (2006) measured and reported a few eco-efficient indicators, such as energy efficiency in 1,000 gig Joules (gJs) per million dollars Adjusted Net Sales (ANS), and Hazardous waste generated in kg per million dollars ANS. BaXXX Inc. 4.1.3.3 Harmonization of the triple bottom line Table 5 presents the trends of the Triple Bottom Line (TBL) used in firms’ performance reports in category I and category II. Sustainable business indicators that were reported in over 60% of sample firms in category I are: 9 environmental indicators (82% of total environmental indicators); 2 economic indicators (40% of total economic indicators); 5social indicators (45% of total social indicators); and one integrated indicator (20% of total integrated indicators). Firms in category I focused more on the environmental indicators, rather than social and economic indicators. On the other hand, many firms in category II described environmental indicators as well as economic and social indicators in their performance reports. Based on Table 5, all sustainable business indicators were reported in over 60% of all sample firms in category II. For example, 17% of firms in category I and 62% of firms in category II reported abolition of child labor. Only about 8% of firms in category I mentioned empowerment of employees in their performance reports, but firms in category II reported it by approximately 65%. Figure 2 shows the harmonization of the TBL used in firms in category I and II. It seems that most firms in category I focused more on environmental indicators and firms in category II tried to harmonize the TBL. Based on Table 6, about 42% of firms in category I used the term, “Environmental,” “Environmental, health, and safety” as key words in the titles of their performance reports. While approximately 58% of sample firms in category I used the term “Sustainability” or “Corporate social responsibility” as a key word in the title of their performance reports, about 83% of sample firms in category II used the term. These changes indicate that many firms have shifted the key words in the title of their performance reports from the concept of environmental performance to the concept of sustainable business practices, which is based on the concept of the TBL. For instance, STXXX electronics Company used “Environmental Report” as the title of its performance reports in 2001 and “Social and Environmental Report” in 2003. In 2004, STXXX electronics Company first used the term, “Corporate Responsibility Report 2004” as the title of its performance reports on sustainable business practices. 4.1.3.2 Culture for sustainable business However, in category II, it reported that it not only ensured diversity and equal opportunity, but also disclosed the changes in percentage of average employee age and seniority, number of nationalities by regions, and gender breakdown by regions in Corporate Responsibility Report 2005 and 2006. 4.1.3.2 Culture for sustainable business We found firms that had described their culture for sustainable business practices. This is consistent with what the International Institute for Sustainable Development (IISD) et al. (1992) and what Welford (1995) emphasized. They asserted that a firm should change its corporate culture to implement sustainable business practices. They also proposed some examples of corporate culture: employee participation in decision-making processes, the equitable treatment of women and minority groups, communication with the public, and the impact on the Third World and indigenous populations. We found these examples as SBIs. Table 5 shows the trends of these indicators in category I and II. g y We searched the terms, “Empower,” “Participation,” and “Decision,” to identify whether or not firms allow employee participation in the decision making process. Firms in category I and II reported that they involved their employees in their decision making process by empowering employees. While 62% of samples firms in category II reported that they involved their employees in their decision making process by empowering employees, only about 8% of sample firms in category I described the empowerment of employees. For instance, SXXXX Inc. in category II addressed, “Within this culture, employees are empowered and strongly encouraged to use their skills and experience to find better ways of doing business” (Corporate Social Responsibility Report 2005, p. 4). The CoXXX Company in category II also stated that its employees are empowered to keep the highest standards of quality in products, processes and relationships in 2006 Corporate Responsibility Review. STXXX electronics Company in category I did not mention employee empowerment in Corporate Environmental Report and Social Review 2001. However, STXXX electronics Company (2006) in category II stated that employee empowerment is one of the key principles for its sustainable business in their corporate responsibility report. 196 Environmental Management in Practice We found one indicator; breakdown of employees in terms of gender, age group, and minority group membership, as one of the SBIs. This indictor can be used to identify the equitable treatment of women and minority groups which is one aspect of corporate culture that the IISD et al. (1992) proposed. Firms in category II reported this indicator by 80% of the firms, and firms in category I described it 50% of the firms. STXXX electronics Company in category I did not mention diversity and equitable opportunity of women and minority in Corporate Environmental report and Social Review 2001. 4.1.3.4 Sustainability enlightenment Young (2000) insisted that sustainable business enlightens its interested parties, such as investors, customers, and employees, on the concept of sustainable business practices. This is because interested parties have significant roles in changing traditional consumption, purchasing, and investing behaviors toward sustainable business practices. We found firms behaviors that are consistent with Young’s (2000) findings. Based on Table 5, 83% of firms in category II and 33% of firms in category I reported that they enlightened their interested parties, such as customers, suppliers, investors, and employees about the concepts of sustainable business. Fig. 2. Trend of Sustainable Business Indicators Reported in Category I and II 4.1.3.4 Sustainability enlightenment It seems that firms in category II progressively enlightened their 197 Indicators of Sustainable Business Practices EN1:Total amount of water used;EN2:Total amount of energy used;EN3:Total amount of greenhouse gases generated (CO2);EN4:Total amount of Volatile Organic Compound (VOC) generated;EN5:Total amount of air emissions generated (SOx, NOx);EN6:Total amount of waste recycled or reused;EN7:Total amount of solid waste generated;EN8:Total amount of hazardous waste generated;EN9:Total number of environmental violations;EN10: EN1:Total amount of water used;EN2:Total amount of energy used;EN3:Total amount of greenhouse gases generated (CO2);EN4:Total amount of Volatile Organic Compound (VOC) generated;EN5:Total amount of air emissions generated (SOx, NOx);EN6:Total amount of waste recycled or reused;EN7:Total amount of solid waste generated;EN8:Total amount of hazardous waste generated;EN9:Total number of environmental violations;EN10: Total amount of renewable energy used (clean fuel, solar energy, clean energy);EN11:whether or not firms describe environmentally friendly product or process;EC1:Annual profits;EC2:Annual sales;EC3:Fines;EC4:R&D investment (total);EC5:Donations;SO1:Female, disabled person’s rights;SO2: Abolition of all child labor; SO3:The recruitment of people from ethnic minorities, older workers, women;SO4:Empowerment of employees;SO5:Average hours of training per employee;SO6:Number of employees; SO7:Recordable illness rate (RIR);SO8:Lost time rate (LTR);SO9:Whether or not firms implement a broad range of voluntary activities;SO10:Whether or not firms provide opportunities to communicate internally and externally to interested parties;SO11:Breakdown of employees in terms of gender, age, and minority group; I1:Whether or not firms implement voluntary environmental management systems (ISO 14001, LCA, etc);I2:Whether or not firms make decisions based on the concept of sustainable business and long-term objective;I3:Whether or not firms enlighten consumers and suppliers for the concept of sustainable business;I4:Whether or not firms compare GRI;I5:Whether or not firms mention culture;I6:Whether or not firms used relative indicators (eco-efficiency) EN1:Total amount of water used;EN2:Total amount of energy used;EN3:Total amount of greenhouse gases generated (CO2);EN4:Total amount of Volatile Organic Compound (VOC) generated;EN5:Total amount of air emissions generated (SOx, NOx);EN6:Total amount of waste recycled or reused;EN7:Total amount of solid waste generated;EN8:Total amount of hazardous waste generated;EN9:Total number of environmental violations;EN10: Total amount of renewable energy used (clean fuel, solar energy, clean energy);EN11:whether or not firms describe environmentally friendly product or process;EC1:Annual profits;EC2:Annual sales;EC3:Fines;EC4:R&D investment (total);EC5:Donations;SO1:Female, disabled person’s rights;SO2: Abolition of all child labor; SO3:The recruitment of people from ethnic minorities, older workers, women;SO4:Empowerment of employees;SO5:Average hours of training per employee;SO6:Number of employees; SO7:Recordable illness rate (RIR);SO8:Lost time rate (LTR);SO9:Whether or not firms implement a broad range of voluntary activities;SO10:Whether or not firms provide opportunities to communicate internally and externally to interested parties;SO11:Breakdown of employees in terms of gender, age, and minority group; I1:Whether or not firms implement voluntary environmental management systems (ISO 14001, LCA, etc);I2:Whether or not firms make decisions based on the concept of sustainable business and long-term objective;I3:Whether or not firms enlighten consumers and suppliers for the concept of sustainable business;I4:Whether or not firms compare GRI;I5:Whether or not firms mention culture;I6:Whether or not firms used relative indicators (eco-efficiency) EN1:Total amount of water used;EN2:Total amount of energy used;EN3:Total amount of greenhouse gases generated (CO2);EN4:Total amount of Volatile Organic Compound (VOC) generated;EN5:Total amount of air emissions generated (SOx, NOx);EN6:Total amount of waste recycled or reused;EN7:Total amount of solid waste generated;EN8:Total amount of hazardous waste generated;EN9:Total number of environmental violations;EN10: Total amount of renewable energy used (clean fuel, solar energy, clean energy);EN11:whether or not firms describe environmentally friendly product or process;EC1:Annual profits;EC2:Annual sales;EC3:Fines;EC4:R&D investment (total);EC5:Donations;SO1:Female, disabled person’s rights;SO2: Abolition of all child labor; SO3:The recruitment of people from ethnic minorities, older workers, women;SO4:Empowerment of employees;SO5:Average hours of training per employee;SO6:Number of employees; SO7:Recordable illness rate (RIR);SO8:Lost time rate (LTR);SO9:Whether or not firms implement a broad range of voluntary activities;SO10:Whether or not firms provide opportunities to communicate internally and externally to interested parties;SO11:Breakdown of employees in terms of gender, age, and minority group; I1:Whether or not firms implement voluntary environmental management systems (ISO 14001, LCA, etc);I2:Whether or not firms make decisions based on the concept of sustainable business and long-term objective;I3:Whether or not firms enlighten consumers and suppliers for the concept of sustainable business;I4:Whether or not firms compare GRI;I5:Whether or not firms mention culture;I6:Whether or not firms used relative indicators (eco-efficiency) Fig. 4.1.3.4 Sustainability enlightenment 2. Trend of Sustainable Business Indicators Reported in Category I and II 198 Environmental Management in Practice interested parties on sustainable business practices more so than firms in category I did. For instance, in their 2006 Citizenship Report, GeXXX Electronic Co. reported that it had required their suppliers to consider the concepts of sustainable business since 2002 by complying with laws and regulations governing minimum wage, hours of service, and overtime wages for employees. GeXXX Electronic Co. (2007) introduced “The Spirit & The Letter” polices so that GeXXX Electronic Co. could help its interested parties, such as employees, suppliers, and customers, understand the common standards of behaviors required to implement sustainable business practices of GeXXX Electronic Co. In KimXXX’s 2005 Sustainability Report, KimXXX Corporation reported that they enlightened and shared tools and technologies with suppliers to meet its social and environmental requirements that are sustainable business practices. 4.1.3.5 Voluntary programs and communication to the public We identified whether or not a firm implemented diverse voluntary programs as an indicator to evaluate a firm’s sustainable business, and found that most of the firms in category I and II have implemented and reported a variety of voluntary programs. This is consistent with Thompson (2002) and Scott (2001). They found that sustainable business should implement diverse voluntary programs to build strong relationships with stakeholders, increase a firm’s image and reputation, and consider ethical investment for individual investors and fund managers. In Table 5, about 83% of firms in category I and 97% of firms in category II reported their diverse voluntary programs. In their 2005 Sustainability Report, KimXXX Corporation disclosed that it voluntarily joined the U.S. Environmental Protection Agency’s Climate Leaders program in December 2005. GeXXX Company reported that it had implemented various voluntary greenhouse gas management initiatives to mitigate global climate change in their Corporate Responsibility Report of 2004/5. Many firms in category II implemented their voluntary programs especially for people employed in developing countries. AlXX X Inc. and KimXXX Corporation disclosed that they voluntarily implemented HIV/AIDS programs and management systems at places where they operate their facilities in South Africa. The goal of these programs is to help employees and their families undergo voluntary counseling and confidential testing for HIV/AIDS. The increasing number of voluntary HIV/AIDS programs implemented in developing countries is consistent with changes in corporate culture that the IISD et al. (1992) and Welford (1995) suggested in order to implement sustainable business. Many firms in category II have voluntarily applied to the GRI guidelines to report the performance of their sustainable business practices. AlcXXX Inc. reported that it voluntarily used the GRI guidelines to help its interested parties to understand its sustain able business practices in 2004 Sustainability Report. 4.2 Changes in the key words of the performance titles Data from a total of 287 firms were collected as sample data among all S&P 500 companies, as of December 2006. The following words were used to codify the results: Environmental; Report; Environmental, Health, and Safety Report; Sustainability; Corporate Social Responsibility; Corporate Report; and Citizenship Report. The results of these codes are separated into 3 categories; E (environmental report); EHS (environmental, health, and safety reports); Sustainability (sustainability, corporate social responsibility, and sustainable report). Sustainability, corporate social responsibility, corporate report, and sustainable report mean that the firm considered the concept of sustainable business because these words evolved from the concept of sustainable development. 4.2 Changes in the key words of the performance titles Table 7 presents the trends in key word usage Indicators of Sustainable Business Practices 199 NAICS Reporting Not Reporting Total Title(2 digit) Title(3 digit) E E, H, S Sustain ability Sub- total Agriculture, Forestry, Fishing and Hunting (11) Crop Production (111) 0(0%) 0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 Forestry and Logging (113) 1 (100%) 0 (0%) 0 (0%) 1 (100%) 0 (0%) 1 Sub-total 1 (50%) 0 (0%) 1 (50%) 2 (100%) 0 (0%) 2 Mining (21) Oil and Gas Extraction (211) 0 (0%) 2 (40%) 3 (60%) 5 (62%) 3 (38%) 8 Mining (except Oil and gas)(212) 1 (33%) 0 (0%) 2 (67%) 3 (75%) 1 (25%) 4 Support Activities for Mining (213) 0 (0%) 2 (50%) 2 (50%) 4 (57%) 3 (43%) 7 Sub-total 1 (8%) 4 (33%) 7 (58%) 12 (63%) 7 (37%) 19 Utilities(22) Utilities (221) 7 (28%) 6 (24%) 12 (48%) 25 (78%) 7 (22%) 32 Construction (23) Construction of Buildings (236) 0 (0%) 0 (0%) 1 (100%) 1 (20%) 4 (80%) 5 Heavy and Civil Engineering Construction (237) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (100%) 2 Sub-total 0 (0%) 0 (0%) 1 (100%) 1 (14%) 6 (86%) 7 Manufacturing (31,32,33) Food (311) 1 (14%) 2 (29%) 4 (57%) 7 (50%) 7 (50%) 14 Beverage and Tobacco Product (312) 3 (60%) 0 (0%) 2 (40%) 5 (50%) 5 (50%) 10 Apparel (315) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 4 (100%) 4 Leather and Allied Product (316) 0 (0%) 0 (0%) 1 (100%) 1 (50%) 1 (50%) 2 Wood Product (321) 0 (0%) 0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 Paper (322) 0 (0%) 1 (25) 3 (75%) 4 (57%) 3 (43%) 7 Printing and Related Support Activities (323) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (100%) 1 Petroleum and Coal Product (324) 0 (0%) 3 (43%) 4 (57%) 7 (88%) 1 (12%) 8 Chemical (325) 0 (0%) 5 (19%) 22 (81%) 27 (64%) 15 (36%) 42  Plastics and Rubber Product (326) 0 (0%) 1 (33%) 2 (67%) 3 (75%) 1 (25%) 4 Primary Metal (331) 1 (50%) 0 (0%) 1 (50%) 2 (40%) 3 (60%) 5 200 Environmental Management in Practice NAICS Reporting Not Reporting Total Title(2 digit) Title(3 digit) E E, H, S Sustain ability Sub- total Fabricated Metal Product (332) 0 (0%) 1 (33%) 2 (67%) 3 (60%) 2 (40%) 5 Machinery (333) 0 (0%) 1 (17%) 5 (83%) 6 (38%) 10 (62%) 16 Manufacturing (31,32,33) Computer and Electronic Product (334) 2 (9%) 3 (14%) 17 (77%) 22 (37%) 37 (63%) 59 Electrical Equipment, Appliance and Component (335) 0 (0 %) 0 (0%) 2 (100%) 2 (40%) 3 (60%) 5 Transportation Equipment (336) 1 (11%) 3 (33%) 5 (56%) 9 (60%) 6 (40%) 15 Furniture and Related Product (337) 0 (0%) 0 (0%) 1 (100%) 1 (50%) 1 (50%) 2 Miscellaneous (339) 0 (0%) 0 (0%) 3 (100%) 3 (33%) 6 (67%) 9 Sub-total 8 (8%) 20 (19%) 75 (73%) 103 (49%) 106 (51%) 209 Transportation and Warehousing (48,49) Air transportation (481) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (100%) 1 Rail Transportation (482) 1 (100 %) 0 (0%) 0 (0%) 1 (25%) 3 (75%) 4 Water Transportation (483) 1 (100%) 0 (0%) 0 (0%) 1 (100%) 0 (0%) 1 Pipeline Transportation (486) 1 (33%) 2 (67%) 0 (0%) 3 (100%) 0 (0%) 3 Couriers and Messengers(492) 0 (0%) 0 (0%) 2 (100%) 2 (100%) 0 (0%) 2 Sub-total 3 (43%) 2 (29%) 2 (28%) 7 (64%) 4 (36%) 11 Accommodati on and Food Service (72) Accommodation (721) 1 (100%) 0 (0%) 0 (0%) 1 (33%) 2 (67%) 3 Food Services and Drinking Places (722) 0 (0%) 0 (0%) 2 (100%) 2 (50%) 2 (50%) 4 Sub-total 1 (33%) 0 (0%) 2 (67%) 3 (43%) 4 (57%) 7 Total 21 (14%) 32 (21%) 100 (65%) 153 (53%) 134 (47%) 287 * E: Environment, H: Health, S: Safety Table 7. 4.2 Changes in the key words of the performance titles Changing the keywords used in the title of a firm’s performance reports means that the main strategies of the performance reports have likely changed and that the firm has informed the readers of what they have implemented and evaluated. 4.2 Changes in the key words of the performance titles Trends of the Key Words Used in the Titles of S&P 500 Firms’ Performance Reports in 2006 Table 7. Trends of the Key Words Used in the Titles of S&P 500 Firms’ Performance Repo n 2006 within the titles of performance reports based on the industry of all 287 sample firms. Of the 287 firms, approximately 53% of the firms (153 firms) reported their performance reports. Performance reports could not be found on the respective Internet homepages for the remaining firms. Of the 153 firms, 65.4% (100 firms) used “sustainability,” “sustainable,” or 201 Indicators of Sustainable Business Practices “corporate social” as (a) word(s) used in the titles of their performance reports; 20.9% (32 firms) used “environmental, health, and safety” as (a) word(s) for their performance reports; and 13.7% (21 firms) used “environmental” as (a) word(s) for their performance titles. This means that 65.4% of the 153 S&P 500 firms surveyed have reported the performance of sustainable business indicators; 20.9% have disclosed the performance of environmental, health, and safety indicators; and 13.7% have reported only environmental performance. Fifty-three firms, 18.5% of the total 287 S&P 500 firms surveyed reported that their environmental performance reports used the terms Environmental reports or environmental, health and safety reports in the title of their performance reports. This result is quite different from that of a previous study. In 1998, the Investor Responsibility Research Center (IRRC) conducted a survey to identify how many S&P 500 firms reported their performance reports to the public. They found that 61% of the 191 S&P 500 companies in 1998 used the term Environmental as a keyword in the title of their performance reports (Gozali et al., 2002). This indicates that 61% of the S&P 500 companies surveyed in 1998 focused on the performance of environmental indicators. The use of the term Environmental in the title of the performance reports swiftly dropped from 61% in 1998 to18.5% of the total 287 S&P 500 firms (53 firms) in 2006. On the other hand, the IRRC did not find firms that used the term Sustainability in the titles of their samples. However, we found 34.8% (100 firms) of 287 S&P 500 companies surveyed in 2006 used the term Sustainability as a keyword in the title of their performance reports. 4.2.1 Distribution of industries As of 2006, of the 287 S&P 500 companies surveyed, 19 firms were in the mining industry. 63.2% of these 19 firms (12 firms) provided their performance reports. Of the 12 firms, seven firms (58.3% of 12 firms) used the term, Sustainability and five firms (41.7% of 12 firms) used the term Environmental and EHS. In other words, 58.3% of firms described their performance in accordance with the concept of sustainable development. It could be said that firms in the mining industry have begun to progressively apply sustainable business strategies. g y g p g y pp y g Thirty-two firms in the utilities industry provided their performance reports. Among them, 48.0% of the firms used the term Sustainability, and 52% of the firms used the term Environmental and EHS in the title. Based on these numbers, it appears that many firms had still focused more on environmental management systems than on sustainable business even though international organizations had proposed guidelines, such as the Electric Utilities project proposed by the WBCSD in 2000, to help firms in the utilities industry implement sustainable business practices. p p Seventy-five firms (72.8% of 103 firms) in the manufacturing industry used the term Sustainability; 8 firms (7.8% of them) used the term Environmental; and 20 firms (19.4% of them) used the term EHS. It appears that firms in the manufacturing industry have proactively applied sustainable business practices or labels for such practices. Firms in the manufacturing industry have changed from environmental management strategies to sustainable business strategies. This shift was made possible in part because manufacturing firms could easily apply and implement sustainable business aided by the fact that most had already established and implemented several environmental management systems, such as ISO 14001. 202 Environmental Management in Practice The construction industry is a sector where sustainable business practices should be implemented as a business practice for two reasons: it is faced with indispensable challenges posed by “Sustainability”; and the construction industry is generally one of the largest industries in both developed and developing countries in terms of economic, social, and environmental impacts (Zhang, Shen, Love, & Treloar, 2000; Cole, 1998; Spence & Mulligan, 1995). However, we could not find many construction firms among S&P 500 companies in 2006 that reported their environmental or sustainable business performance. 4.2.1 Distribution of industries Of the seven S&P 500 companies in the construction industry, only one firm published its performance reports with a title that used the term Sustainability. Several international organizations, such as the WBCSD and the Institute of Sustainable Forestry (ISF), have encouraged firms in the agriculture, forestry, fishing, and hunting industry to apply sustainable development by proposing special programs, such as the Sustainable Forest Products Industry project and the Sustainable Forestry Initiatives. This is influenced by the fact that they deal with natural capital stocks. We found only two firms in the S&P 500, as of 2006, in Agriculture, Forestry, Fishing and Hunting. These two firms reported their performance reports and used the terms Sustainability and Environmental in the title of their performance reports. It is difficult to say whether firms in this industry have applied sustainable business practices because of the small sample. There are seven firms in the transportation and warehousing industry that published their performance reports. Of the seven firms, two firms (28.6%) used the term Sustainability and five firms (71.4%) used the term Environmental or EHS in their performance titles. It does not seem that firms in the transportation and warehousing industry have implemented sustainable business practices based on the key words used in the title of their performance reports. Of the seven firms, the main products of four firms are the transfer of water and gases through pipelines to their customers. Since transferring water and gases through pipelines has the potential for causing environmental accidents, such as spills and explosion incidents, the focus for these firms may be on the concept of environmental management strategies. g Three firms in the accommodation and food service industry disclosed environmental or sustainability performance reports even though this industry does not produce environmental impact directly. Of the three firms, two firms (66.7% of the 3 firms) used the term Sustainability and one report used Environmental. This implies that some firms in the accommodation and food service industry have begun to consider the concept of sustainable business. 5. Conclusions The objective of this research is to identify whether or not firms are applying sustainable business practice based on the Triple Bottom Line (Environmental, economic, and social areas). We found that more companies in the manufacturing industries have measured and disclosed diverse sustainable business indicators based on the Triple Bottom Line so that they have implemented sustainable business practices since 2003. In other words, firms in the manufacturing industries have integrated the concepts of sustainable business practices into their decision-making process and that some firms in other industries have begun incorporating the concepts of sustainable business practices into their business strategies since 2003. We conclude that since 2003 many companies have changed their strategies from environmental management to sustainable business. Although many firms have 203 Indicators of Sustainable Business Practices increasingly disclosed their performance reports to the public as one of their sustainable business practices, in many cases, they have not proactively announced the disclosure of their performance reports to the public through Internet mass media or newspapers. The results of this research, the distribution and types of sustainable business indicators, could contribute to the existing literature of firms’ sustainable business practices and activities. By providing empirical indicators that will be presented to the public, this research can help stakeholders, including “green” investors, “green” consumers, corporate firms, and others, recognize how the surveyed firms have implemented sustainable business practices. This research can also encourage scholars to actively study not only the theoretical methods for evaluating sustainable business practices, but also the theories or methods for the development of sustainable business strategies. Th l d i hi h d l ll d b f ll l d increasingly disclosed their performance reports to the public as one of their sustainable business practices, in many cases, they have not proactively announced the disclosure of their performance reports to the public through Internet mass media or newspapers. increasingly disclosed their performance reports to the public as one of their sustainable business practices, in many cases, they have not proactively announced the disclosure of their performance reports to the public through Internet mass media or newspapers. The results of this research, the distribution and types of sustainable business indicators, could contribute to the existing literature of firms’ sustainable business practices and activities. 5. Conclusions By providing empirical indicators that will be presented to the public, this research can help stakeholders, including “green” investors, “green” consumers, corporate firms, and others, recognize how the surveyed firms have implemented sustainable business practices. This research can also encourage scholars to actively study not only the theoretical methods for evaluating sustainable business practices, but also the theories or methods for the development of sustainable business strategies. p g The samples used in this research were not randomly collected, but purposefully sampled. Since the sample for this study is announcements that firms voluntarily disclosed their performance reports, it is not easy to randomly collect samples. Future researchers could conduct case studies to identify the changes in corporate culture and evaluate the benefits of those changes in corporate culture. 6. References Adams, R., Houldin, M. and Slomp, S. (1999). 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W., Barbier, E. and Markandya, A. (1990). Zhang, Z. H., Shen, L.Y., Love, P. E. D., & Treloar, G. (2000). A framework for implementing ISO 14001 in construction. Environmental Management and Health, Vol.11, No.2, pp.139-149 Environmental Management in Practice Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis Adebola Oketola and Oladele Osibanjo Department of Chemistry, University of Ibadan, Ibadan Nigeria Adebola Oketola and Oladele Osibanjo Department of Chemistry, University of Ibadan, Ibadan Nigeria 6. References Sustainable development: Economics and environment in the Third World, Edward Elgar Publishing, London, UK Redefining Progress, Sustainable Seattle, and Tyler Norris Associates. (1997). The Community indicators Handbook: Measuring progress toward healthy and sustainable communities, Redefining Progress, CA, USA g g Sasseville, D. R., Willson, G. W. and Lawson, R. W. (1997). 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Corporate environmental management 3: Toward sustainable development, Earthscan Publications Lt, London, UK Wharton Research Data Service. (n.d), 13.06.2008, Available from http://wrds.wharton.upenn.edu World Business Council for Sustainable Development (WBCSD). (2000). Sustainability report. 10.08.2008, Available from http://www.sustreport.org/background/definitions.html World Business Council for Sustainable Development (WBCSD). (2005).Eco-efficiency: Creating more value with less impact. 01.05.2007, Available from http://www.wbcsd.org Young, C .W. (2000). Towards sustainable production and consumption: From products to services, In: Corporate Environmental Management 3 Toward Sustainable Development, Welford, R, 79-108, Earthscan Publications Lt, London, UK 206 Environmental Management in Practice 10 1. Introduction Lagos is the economic capital of Nigeria with over 70% of industries in the country located there. It is also the fastest growing city in Nigeria in terms of development and industrial infrastructure, forecast to be one of the three megacities in the world with population of over 20 million by the year 2025. The rapid growth and haphazard urbanization have led to an increase in waste generation and environmental pollution. The industrial pollution problems faced by Lagos with over 7,000 medium and large scale manufacturing facilities are directly related to the rapid industrial growth and the haphazard industrialization without environmental consideration (Oketola and Osibanjo, 2009a). Pollution abatement technologies are largely absent and the consequence is a gross pollution of natural resources and environmental media. Since effective environmental protection cannot take place in a data vacuum, Industrial Pollution Projection System (IPPS), which is a rapid environmental management tool for pollution load assessment, has been employed in this study to estimate industrial pollution loads and to ascertain the agreement between IPPS models and conventional effluent analysis. y It has been recognized that the developing countries lack the necessary information to set priorities, strategies, and action plans on environmental issues. Plant-level monitoring of air, water and toxic emissions is at best imperfect, monitoring equipment is not available and where available is obsolete; data collection and measurement methodology are questionable, and there is usually lack of trained personnel on industrial sites (Oketola and Osibanjo, 2009b; Hettige et al., 1994). In the absence of reliable pollution monitoring data, the World Bank has created a series of datasets that have given the research community the opportunity to better understand levels of pollution in developing countries, and therefore issue policy advice with more clarity (Aguayo et al., 2001). Hence, the World Bank developed the Industrial Pollution Projection System (IPPS), which is a rapid assessment tool for pollution load estimation towards the development of appropriate policy formulation for industrial pollution control in the developing countries, where insufficient data on industrial pollution proved to be an impediment to setting-up pollution control strategies and prioritization of activities (Faisal, 1991; Arpad et al, 1995). g p ( p ) IPPS is a modeling system, which has been developed to exploit the fact that industrial pollution is heavily affected by the scale of industrial activity, by its sectoral composition, and by the type of process technology used in production. 1. Introduction IPPS combines data from 208 Environmental Management in Practice industrial activities (such as production and employment) with data on pollution emissions to calculate the pollution intensity factors based on the International Standard Industrial Classification (ISIC) (Hettige et al., 1994). The IPPS has been estimated from massive USA database. This database was created by merging manufacturing census data with USEPA data on air, water, and solid waste emissions. It draws on environmental, economic, and geographic information from about 200,000 US factories. The IPPS covers about 1,500 product categories, all operating technologies, and hundreds of pollutants. It can project air, water, or solid waste emissions, and it incorporates a range of risk factors for human toxic and ecotoxic effects (Hettige et al., 1995). ( g ) There are wide ranges of industries and the pollutants introduced largely depends on the type of industry, raw material characteristics, specific process methods, efficacy of facilities, operating techniques, product grades and climatic conditions (Onianwa, 1985). The industrial sectors in Lagos based on the Manufacturer’s Association of Nigeria (M.A.N) grouping are food, beverage and tobacco; textile, wearing apparel; pulp and paper products; chemical and pharmaceutical; wood and wood products; nonmetallic mineral products; basic metal; electrical and electronic; motor vehicle and miscellaneous; and domestic and industrial plastics (M.A.N., 1991).The Chemical and pharmaceutical sector is the most polluting industrial sector out of the ten major sectors based on the final ranking of IPPS pollution loads estimated with respect to employment and total value of output while basic metal, domestic and industrial plastics and textile wearing apparel sectors followed suit (Oketola and Osibanjo, 2009a). The chemical manufacturing facilities in the sector range from paint manufacturing industries, soap and detergents, pharmaceuticals, domestic insecticides and aerosol, petroleum products, toiletries and cosmetics, basic industrial chemicals while the basic metal manufacturing facilities are steel manufacturing, metal fabrication, aluminium extrusion etc. The magnitude of environmental pollution problem is related to the types and quantity of waste generated by industries and the methods of management of the waste. As indicated earlier, there are over 7,000 industries in Lagos state with less than 10% having installed treatment facilities (Onyekwelu et al., 2003). Majority of these industries discharge their partially treated or untreated effluents into the environment and the Lagos Lagoon has gradually become a sink for pollutants from these industries. 1. Introduction Industries utilize water for many purposes; these include processing, washing, cooling, boiler use, flushing sanitary/sewage use and general cleaning. Very large amount of water is required for these activities. Within a given industrial sector, water use correlates with the size of the industry, and also for predicting the rate of generation of wastewater. Water supply requirements of an industry vary from one sector to another. While some industries may only require smaller volumes for cooling and cleaning (as in metal fabrication, cement bagging, etc), some others due to the nature of their processes may require very large volumes of water. Among such industries are breweries, distilleries and soft drinks manufacturing industries where water forms the bulk of the products themselves as a solution. Total consumption is about 205,000 m3/day, with major users being Breweries, 22%; Textile, 18%; and Industrial chemicals, 16.6% (M.A.N., 2003). Industries utilize a vast array of input in the process of production of goods and services, and generate different forms of waste to varying degrees, which depends on the types and quantity of raw materials inputs, and the process technology employed (Ogungbuyi and Osho, 2005). This study estimated pollution loads of some industries among the top most polluting sectors in Lagos (i.e., chemical, basic metal, plastics and textile). The selection of the Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 209 industries was based on data availability and level of cooperation by industries studied. The industries selected are paint manufacturing, industrial gas manufacturing and lubricating oil production under the chemical and pharmaceutical sector while aluminium extrusion, steel manufacturing and glass bottle cap production industries were selected under the basic metal sector. Tyre manufacturing, foam and plastic production; and textile fabric and yarn production industries were selected under the domestic and industrial plastics and textile and wearing apparel sectors, respectively. IPPS pollution loads were estimated with respect to employment and total output, and the results of effluent pollution loads were compared statistically with IPPS pollution loads. 2.1 Description of the study area Lagos state has the largest population density of the four most industrialized states in Nigeria (Lagos, Rivers, Kano and Kaduna). It is also the state with the greatest concentration of industries, with well over seven thousand medium and large-scale industrial establishments. It is claimed that about 70-80% of the manufacturing facilities operating within the medium and large-scale industries are located there in. The major industrial estates in Lagos are: Ikeja, Agidingbi, Amuwo Odofin (industrial), Apapa, Gbagada, Iganmu, Ijora, Ilupeju, Matori, Ogba, Oregun, Oshodi/Isolo/Ilasamaja, Surulere (light industrial) and Yaba (Arikawe, 2002; Akinsanya, 2003; Ogungbuyi and Osho, 2005) as shown in Fig. 1. OGUN STATE AGBARA IBA OJO MOBA 166 AMUWO ISOLO (Proposed) 12 SURULERE 46 IGANMU 44 MATORI 56 ILUPEJU 25 OYINGBO YABA 46 AGIDINGBI OWORONSHOKI OJOTA 10 GBAGADA OREGUN OGBA IFAKO 56 IKEJA IJAIYE AKITAN 55 OTTA (Proposed) 446 IKORODU LAGOS LAGOS LAGOON OGUN STATE AKOWONJO IKOYI MAROKO SANGO-OTTA APAPA 700 PLOTS 400 PLOTS 300 200 100 ABESAN/IPAJA 665 (Proposed) Lagoon Fig. 1. Map of Industrial Estates in Lagos 2.2 Pollution data estimation methodology Economic considerations and lack of cooperation from the industries limited the selection of number of industries considered in this study and the number of samples analysed. Hence, two paint manufacturing industries represented as CAP and BGR, domestic insecticides and OTTA SANGO-OTTA LAGOS LAGOON LAGOS OJO MOBA MAROKO Fig. 1. Map of Industrial Estates in Lagos Fig. 1. Map of Industrial Estates in Lagos 2.2 Pollution data estimation methodology 2.3 Effluent sample analysis 2.3 Effluent sample analysis Treated and untreated effluent samples were collected from the industries at the point of discharge to the environment and production line, respectively. Effluent samples were analyzed for physico-chemical parameters and heavy metals using standard methods (APHA, 1992; Miroslav and Viadimir, 1999; Taras, 1950). The parameters determined were: temperature, pH, turbidity, conductivity, total suspended solids (TSS), total hardness, acidity, alkalinity, chloride, sulphate, nitrate, chemical oxygen demand (COD), biological oxygen demand (BOD), dissolved oxygen (DO), sodium chloride, calcium, magnesium, and heavy metals (e.g., Fe, Pb, Zn, Cd, Cr, Mn, Ni, Cu, and Co). 2.2 Pollution data estimation methodology The parameters determined were temperature, pH, turbidity, conductivity, total suspended solids (TSS), total hardness acidity, alkalinity, chloride, sulphate, nitrate, chemical oxygen demand (COD), biologica oxygen demand (BOD), dissolved oxygen (DO), sodium chloride, calcium, magnesium, and heavy metals (e.g., Fe, Pb, Zn, Cd, Cr, Mn, Ni, Cu, and Co). 2.4 Statistical analysis The data were validated statistically using t - test at 95% confidence interval (2- tailed) and analysis of variance (ANOVA) to ascertain if there is any significant difference between IPPS pollution loads with respect to employment and total output; and pollution loads from aerosol production (DIA), and basic industrial gas manufacturing (IGM) were considered under the chemical and pharmaceutical sector; steel manufacturing (UST), aluminium extrusion (AET), aluminium windows and doors production (AWD) and glass bottle cap production (CCP) were selected under the basic metal sector. Industries selected under the domestic and industrial plastics and textile and wearing apparels were tyre, foam and plastic manufacturing industries; and textile and yarn manufacturing industries, respectively. p y The total number of employees and average total output in CAP, BGR, LOP, UST, CCM, AWD, AET, FMI, TTP, CLP, WSY, RLT and APT were 225 and 3, 900 ton/yr; 250 and 8,000 ton/yr; 200 and 16.1 ton/yr; 120 and 1,170 ton/yr; 1,025 and 63,200 ton/yr; 370 while total output data was not available; 36 and 222 ton/yr; 200 and 1,800 ton/yr; 710 and 6,650 ton/yr; 1,000 and 9,560 ton/yr; 200 and 960,000 ton/yr; 350 and 12,000 ton/yr; 800 and 3,600 ton/yr; and 375 and 3,750 ton/yr, respectively. Lower Bound (LB) pollution intensities by medium with respect to total value of output and employment were obtained from the literature (Hettige, et al., 1994). The pollution intensities were used to estimate the pollution loads of these manufacturing industries based on the International Standard Industrial Classification (ISIC) code as found in the literature using the formulae: With respect to total output; Pollution intensity factor x Unit of Output Pollution load 2204.6  (1) With respect to employment; PI X TEM PL 1000 x 2204.6  (2) Pollution intensity factor x Unit of Output Pollution load 2204.6  (1) (1) (2) Where, PL = Pollution load of a sector in ton/year PI = Pollution intensity per thousand employees per year TEM = Total number of employees in that sector 2204.6 = Conversion factor from pounds to tonnes 2.3 Effluent sample analysis 2.2 Pollution data estimation methodology gy Economic considerations and lack of cooperation from the industries limited the selection of number of industries considered in this study and the number of samples analysed. Hence, two paint manufacturing industries represented as CAP and BGR, domestic insecticides and 210 Environmental Management in Practice aerosol production (DIA), and basic industrial gas manufacturin under the chemical and pharmaceutical sector; steel manufac extrusion (AET), aluminium windows and doors production (A production (CCP) were selected under the basic metal sector. Ind domestic and industrial plastics and textile and wearing appa plastic manufacturing industries; and textile and yarn m respectively. The total number of employees and average total output in CA AWD, AET, FMI, TTP, CLP, WSY, RLT and APT were 225 and 3, ton/yr; 200 and 16.1 ton/yr; 120 and 1,170 ton/yr; 1,025 and 63,2 output data was not available; 36 and 222 ton/yr; 200 and 1,8 ton/yr; 1,000 and 9,560 ton/yr; 200 and 960,000 ton/yr; 350 and 12 ton/yr; and 375 and 3,750 ton/yr, respectively. Lower Bound (L medium with respect to total value of output and employmen literature (Hettige, et al., 1994). The pollution intensities were use loads of these manufacturing industries based on the Internat Classification (ISIC) code as found in the literature using the formu With respect to total output; Pollution intensity factor x Unit Pollution load 2204.6  With respect to employment; PI X TEM PL 1000 x 2204.6  Where, PL = Pollution load of a sector in ton/year PI = Pollution intensity per thousand employees per TEM = Total number of employees in that sector 2204.6 = Conversion factor from pounds to tonnes 2.3 Effluent sample analysis Treated and untreated effluent samples were collected from the discharge to the environment and production line, respectivel analyzed for physico-chemical parameters and heavy metals (APHA, 1992; Miroslav and Viadimir, 1999; Taras, 1950). The par temperature, pH, turbidity, conductivity, total suspended sol acidity, alkalinity, chloride, sulphate, nitrate, chemical oxygen d oxygen demand (BOD), dissolved oxygen (DO), sodium chloride, heavy metals (e.g., Fe, Pb, Zn, Cd, Cr, Mn, Ni, Cu, and Co). 2.2 Pollution data estimation methodology 2.4 Statistical analysis The data were validated statistically using t - test at 95% confiden analysis of variance (ANOVA) to ascertain if there is any significan pollution loads with respect to employment and total output; i l ffl l i 0 05 aerosol production (DIA), and basic industrial gas manufacturing (IGM) were considered under the chemical and pharmaceutical sector; steel manufacturing (UST), aluminium extrusion (AET), aluminium windows and doors production (AWD) and glass bottle cap production (CCP) were selected under the basic metal sector. Industries selected under the domestic and industrial plastics and textile and wearing apparels were tyre, foam and plastic manufacturing industries; and textile and yarn manufacturing industries respectively. The total number of employees and average total output in CAP, BGR, LOP, UST, CCM AWD, AET, FMI, TTP, CLP, WSY, RLT and APT were 225 and 3, 900 ton/yr; 250 and 8,000 ton/yr; 200 and 16.1 ton/yr; 120 and 1,170 ton/yr; 1,025 and 63,200 ton/yr; 370 while tota output data was not available; 36 and 222 ton/yr; 200 and 1,800 ton/yr; 710 and 6,650 ton/yr; 1,000 and 9,560 ton/yr; 200 and 960,000 ton/yr; 350 and 12,000 ton/yr; 800 and 3,600 ton/yr; and 375 and 3,750 ton/yr, respectively. Lower Bound (LB) pollution intensities by medium with respect to total value of output and employment were obtained from the literature (Hettige, et al., 1994). The pollution intensities were used to estimate the pollution loads of these manufacturing industries based on the International Standard Industria Classification (ISIC) code as found in the literature using the formulae: With respect to total output; Pollution intensity factor x Unit of Output Pollution load 2204.6  (1 With respect to employment; PI X TEM PL 1000 x 2204.6  (2 Where, PL = Pollution load of a sector in ton/year PI = Pollution intensity per thousand employees per year TEM = Total number of employees in that sector 2204.6 = Conversion factor from pounds to tonnes 2.3 Effluent sample analysis Treated and untreated effluent samples were collected from the industries at the point of discharge to the environment and production line, respectively. Effluent samples were analyzed for physico-chemical parameters and heavy metals using standard methods (APHA, 1992; Miroslav and Viadimir, 1999; Taras, 1950). 2.4 Statistical analysis The data were validated statistically using t - test at 95% confidence interval (2- tailed) and analysis of variance (ANOVA) to ascertain if there is any significant difference between IPPS pollution loads with respect to employment and total output; and pollution loads from conventional effluent analysis at p > 0.05. Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 211 y j y ( ) y Industrial Sector Four ISIC Code Product Produced Major Raw Materials Types of Waste Generated Mode of Disposal Effluent Treatment Plant (ETP)/Constrain General Remarks CPH 3521 (CAP) Paints Pigment, resin, solvent and additives Effluent Waste solvent Discharge in drain By contractor off-site Operational Discharge treated effluent into the environment 3521 (BGR) Paints, wood preservative s, allied products Dyes, pigment, solvent, extender Effluent Sludge Discharge in drain By contractor off-site Operational Discharge treated effluent into the environment 3511 (IGM) Industrial gases e.g. O2, CO2, acetylene Caustic soda, soda ash, calcium carbide, ammonium nitrate. Effluent, Sludge Discharge in drain, Sludge is disposed by contractor off- site Not available, installing ETP Discharge effluent to the environment 3540 (LOP) Lubricants, aerosol insecticide etc Petroleum products Effluent Solid waste Sludge Used oil generated is discharged to cement kiln and solid/sludge by contractor off site Operational Treat effluent before discharge DIP 3551 (TTP) Tyres for cars, trucks and light trucks Natural and synthetic rubber, ZnO, cobalt stearate, carbon black, mineral oil Effluent Solid waste Discharge in drain, By contractor off-site Not available Uses effluent as cooling water 3513 (FMI) Flexible and rigid foams, adhesives Polyol, toluene- di-isocyanate (IDI), silicone oil, methylene chloride Solid waste Recycled Not available Emitting volatile organic compounds into the atmosphere 3560(CL P) Plastics Pigments and mastic batches Solid waste Waste oil discharged by contractor off- site Not Applicable Do not generate effluent at the production line TWA 3211 (RLT) Grey fabrics e.g. suiting, ankara Yarn, chemicals and dyes Effluent Solid waste In drain after treatment By contractor off-site Operational Discharge treated effluent into the environment 3211 (WSY) Textiles Dyes, pigment, caustic soda, acetic acid Effluent Solid waste Discharge in drain, by contractor off- site Operational Discharge treated effluent into the environment 3219 (APT) Yarn Cotton Solid waste By contractor off-site Not applicable Do not generate effluent. 2.4 Statistical analysis BML 3720 (AET) Aluminium profiles Aluminium billets, H2SO4, NaOH,Tin (II) Sulphate, Chromic acid Effluent, solid and sludge Effluent discharged in drain after treatment and sludge by contractor off- site. ETP operational Do not discharge effluent that contains hazardous substances into the environment. 3720 (AWD) Aluminium windows Aluminium profile from Solid waste Recycle waste Not applicable Do not generate effluent at all. 212 Environmental Management in Practice and doors aluminium ingot 3710 (UST) Steel bars, refractory bricks and enamelware Steel scrap, ferrous alloys (Fe-Mn, Fe-Si), NaOH, clay, silica. Effluent, Slag and Sludge Discharge in drain By contractor off-site Not available, installing ETP Reuse effluent as cooling water 3720 (CCM) Paint cans, crown caps and beverage cans Tin plate, copper wire etc Solid waste Molded together and sold off Not available Do not generate effluent during production Table 1. Major raw materials and types of waste generated by the selected industries in Lagos Table 1. Major raw materials and types of waste generated by the selected industries in Lagos 3. Results and discussion Emission to air was determined based on emission of total suspended particulate (TSP), fine particulate (FP, PM10), sulphur dioxide (SO2), nitrogen dioxide (NO2), carbon monoxide (CO), and volatile organic compounds (VOCs). Emission to water was estimated in terms of biological oxygen demand (BOD) and total suspended solid (TSS) while emission of toxic pollutants was estimated in terms of toxic chemicals and metals released into air, water and land, whose pollution intensities were available in the literature (Hettige, et al., 1994). The major raw materials and the type of waste generated by the selected industries are presented in Table 1 while the total number of employees and total value of output as well as the pollution loads are shown in Tables 2 and 3, respectively. UST have the highest number of employees and second highest total value of output while AWD have the lowest number of employees and LOP the lowest value of output. 3.1.1 Air pollution load Air pollution loads for all the selected industries are shown in Tables 2 and 3, respectively for pollution load estimated with respect to employment and total value of output. UST with 1025 employees and 63, 200 ton/yr of total output have the highest emission of all pollutants into environmental media (i.e., air, water, and land). The air pollution load with respect to employment and total value of output are 4,810 tons/yr and 1,860,000 tons/yr, respectively. This was followed by FMI,CCM, LOP, AET, TTP, IGM, RLT, APT, AWD, WSY, BGR, CAP, and CLP, respectively in decreasing order. p y g In most cases, the higher the number of employees and total output, the higher the air pollution loads. Basic metal, and domestic and industrial plastic (DIP) sectors are the most polluting sector in terms of air pollutant emission. UST ranked first while FMI and CCM ranked second and third, respectively. Total air pollution loads with respect to employment are 2,660 tons/yr and 2050 tons/yr in FMI and CCM, respectively. With respect to total output, air pollution loads are 94,500 ton/yr in FMI. Output data from CCM was not available thus; air pollution load with respect to total output cannot be estimated. Emission of CO and NO2 was the highest in UST and FMI when pollution load was estimated with respect to the two variables (i.e., employment and total output) while SO2 emission was the highest in CCM when pollution load was estimated with respect to employment. The trend in air pollution load by pollutant types in these industries are p yp UST: CO > SO2 > NO2 > FP > TSP > VOC UST: CO > SO2 > NO2 > FP > TSP > VOC Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 213 FMI: NO2 > VOC > SO2 > CO > TSP > FP CCM: SO2 > CO > TSP > VOC > NO2 > FP Pollution loads estimated with respect to employment and total output revealed that the most emitted air pollutant from UST was CO. This could be attributed to the fact that in steel making, oxygen reacts with several components in the bath, including Al, Si, Mn, P, C, and Fe, to produce metallic oxides which end up in the slag. Table 2. Pollution loads (ton/yr) with respect to employment NOTE: L = large scale, M = medium scale, S = small scale, NA = not available, NA* = not applicable Table 2. Pollution loads (ton/yr) with respect to employment Table 2. Pollution loads (ton/yr) with respect to employment ( /y ) p p y NOTE: L = large scale, M = medium scale, S = small scale, NA = not available, NA* = not applicable T bl 2 P ll i l d ( / ) i h l 3.1.1 Air pollution load It also generates carbon monoxide boil, a phenomenon common to all steel making processes and very important for mixing of the slag. Mixing enhances chemical reaction, purges hydrogen and nitrogen, and improves heat transfer. The CO supplies a less expensive form of energy to the bath, and performs several important refining reactions (Jeremy, 2003; and Bruce and Joseph, 2003). It is also important for foaming and help to bury the arc. INDUSTRIAL SECTOR/ SECTOR CODE CHEMICAL & PHARMACEUTICALS (CPH) BASIC METALS (BML) ISIC CODE 3521 (CAP) 3521 (BGR) 3540 (LOP) 3511 (IGM) 3710 (UST) 3720 (CCM) 3720 (AWD) 3720 (AET) EFFLUENT VOL. (L/day) 1,500 2,000 NA NA 1MILLON NA* NA 10 EFFLUENT TREATMENT PLANT (ETP) Operational Operational Operational NA NA NA* NA Operational NO OF EMPLOYEE 225 (M) 250 (M) 200 (M) 120 (M) 1025 (L) 370 (M) 36 (M) 200 (M) AIR POLLUTANTS SO2 5.88 6.53 565 200 1320 1,260 122 680 NO2 5.19 5.77 352 148 575 41.0 3.99 22.2 CO 0.73 0.81 266 115 2060 586 57.0 317 VOC 43.5 48.4 88.3 116 177 45.8 4.46 24.8 FP 1.78 1.98 17.4 6.77 366 11.6 1.13 6.25 TSP 3.49 3.88 217 32.1 307 106 10.3 57.2 TOTAL 60.6 67.3 1,510 617 4810 2,050 199 1,110 WATER POLLUTANTS BOD 0.01 0.07 0.59 68.3 0.89 96.5 9.39 52.2 TSS 0.03 0.03 0.73 105.6 14,400 1,400 136 754 TOTAL 0.04 0.10 1.32 174 14,400 1,490 145 806 TOXIC CHEMICALS TO AIR 38.8 43.1 10.8 101 73.0 97.3 9.47 52.6 TO LAND 93.1 103 3.17 353 418 258 25.1 140 TO WATER 0.10 0.11 0.32 51.3 25.9 3.78 0.38 2.04 TOTAL 132 147 14.7 505 517 359 35.0 194 TOXIC METALS TO AIR 0.33 0.37 0.02 0.50 12.5 6.73 0.66 3.64 TO LAND 2.54 2.82 0.30 15.9 276 223 21.7 121 TO WATER 0.002 0.002 0.01 0.47 1.89 0.13 0.01 0.07 TOTAL 2.89 3.18 0.33 16.9 291 230 22.4 124 NOTE: L = large scale M = medium scale S = small scale NA = not available NA* = not applicable 214 Environmental Management in Practice INDUSTRIAL SECTOR/SECTOR CODE DOMESTIC AND INDUSTRIAL PLASTICS (DIP) TEXTILE, WEARING APPAREL (TWA) ISIC CODE 3560 (CLP) 3513 (FMI) 3551 (TTP) 3219 (APT) 3211 (RLT) EFFLUENT VOL. NOTE: L = large scale, M = medium scale, S = small scale, NA = not available, NA* = not applicable Table 2. Contd. Pollution loads (ton/yr) with respect to employment 3.1.1 Air pollution load (L/day) NA* NA* 484,000 160 NA* 720 EFFLUENT TREATMENT PLANT (ETP) NA NA NA Operatio nal NA Operationa l NO OF EMPLOYEE 200 (M) 710 (L) 1,000 (L) 350 (M) 375 (M) 800 (L) AIR POLLUTANTS SO2 0.54 441 275 36.0 21.0 82.3 NO2 0.12 1,150 95.1 49.7 8.67 114 CO 0.04 169 11.7 6.67 1.58 15.3 VOC 6.48 838 278 13.6 166 31.2 FP 0.11 0.36 3.93 0.96 0.00 2.20 TSP 0.16 67.3 30.4 6.45 12.5 14.7 TOTAL 7.45 2,660 695 113 210 259 WATER POLLUTANTS BOD 4.97 1.89 0.002 1.46 0.00 3.34 TSS 0.11 58.2 0.68 2.27 0.09 5.18 TOTAL 5.08 60.0 0.68 3.73 0.09 8.52 TOXIC CHEMICALS TO AIR 18.2 484 9.98 5.22 147 11.9 TO LAND 5.38 401 17.2 4.85 33.2 11.1 TO WATER 0.04 35.4 0.21 2.66 0.01 6.08 TOTAL 23.6 920 27.4 12.7 180 29.1 TOXIC METALS TO AIR 0.004 0.13 0.39 0.04 0.03 0.10 TO LAND 0.16 20.9 15.1 0.09 0.01 0.20 TO WATER 0.01 0.44 0.02 0.003 - 0.01 TOTAL 0.18 21.5 15.5 0.13 0.04 0.31 Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 215 INDUSTRIAL SECTOR/ SECTOR CODE CHEMICAL & PHARMACEUTICALS (CPH) BASIC METALS (BML) ISIC CODE 3521 (CAP) 3521 (BGR) 3540 (LOP) 3511 (IGM) 3710 (UST) 3720(CCM) 3720 (AWD) 3720 (AET) EFFLUENT VOL. Table 3. Pollution loads (ton/yr) with respect to total value of output NOTE: NA = not available, NA* = not applicable 3.1.1 Air pollution load (L/day) 1,500 2,000 NA NA 1MILLION NA* NA 10 EFFLUENT TREATMENT PLANT (ETP) Operational Operational Operational NA NA NA* NA Operational TOTAL VALUE OF OUTPUT (ton/yr) 3,900 8,000 16.1 1,170 63,200 NA 222 1,800 AIR POLLUTANTS SO2 435 893 152 6,180 512,000 NA 3,890 31,600 NO2 384 787 94.7 4,590 222,000 NA 127 1,030 CO 54.8 112 71.7 3,550 798,000 NA 1,800 14,700 VOC 3,220 6,600 23.8 3,590 68,600 NA 141 1,150 FP 131 269 4.68 210 142,000 NA 35.7 290 TSP 258 530 58.4 994 119,000 NA 326 2,650 TOTAL 4,480 9,190 405 19,100 1,860,000 NA 6,320 51,300 WATER POLLUTANTS BOD 0.46 0.94 0.16 2,120 379 NA 298 2,410 TSS 1.91 0.26 0.20 3,270 5,580,000 NA 4,300 35,000 TOTAL 2.37 1.20 0.36 5,390 5,580,000 NA 4,600 37,400 TOXIC CHEMICALS NA TO AIR 2,870 5,880 2.90 3,140 28,000 NA 300 2,440 TO LAND 6,880 14,100 0.85 10,900 162,000 NA 796 6,470 TO WATER 7.47 15.3 0.09 1,590 10,000 NA 11.7 94.8 TOTAL 9,760 20,000 3.84 15,600 200,000 NA 1,110 9,000 TOXIC METALS TO AIR 24.3 49.9 0.01 15.6 4,850 NA 20.8 169 TO LAND 187 385 0.17 493 107,000 NA 689 5,590 TO WATER 0.15 0.32 0.002 14.5 732 NA 0.41 3.36 TOTAL 212 435 0.18 523 112,000 NA 710 5,760 NOTE: NA = not available, NA* = not applicable 216 Environmental Management in Practice INDUSTRIAL SECTOR/ SECTOR CODE DOMESTIC AND INDUSTRIAL PLASTICS (DIP) TEXTILE, WEARING APPAREL (TWA) ISIC CODE 3560 (CLP) 3513 (FMI) 3551 (TTP) 3211 (WSY) 3211 (RLT) 3219 (APT) EFFLUENT VOL. NOTE: NA = not available, NA* = not applicable 3.1.1 Air pollution load (L/day) NA* NA* 484,000 160 720 NA* EFFLUENT TREATMENT PLANT (ETP) NA NA NA Operational Operational NA TOTAL VALUE OF OUTPUT (ton/yr) 960,000 6,650 9,560 12,000 3,600 3,750 AIR POLLUTANTS SO2 24,400 15,600 16,500 13,200 3,950 1,270 NO2 5,230 40,600 5,690 18,300 5,460 526 CO 0.001 6,010 698 2,450 731 95.3 VOC 294,00 30,000 16,700 5,010 1,500 10,100 FP 5,230 12.1 234 355 106 0.00 TSP 7,400 2,390 1,820 2,360 707 757 TOTAL 337,000 94,400 41,600 41,700 12,400 12,800 WATER POLLUTANTS BOD 226,000 638 0.09 536 160 0.00 TSS 4,880 2,060 40.9 833 249 5.44 TOTAL 231,000 2,700 41.0 1,370 409 5.44 TOXIC CHEMICALS TO AIR 826,000 17.2 598 1,920 573 8,940 TO LAND 245,000 14.2 1,030 1,780 532 2,010 TO WATER 2,020 1.25 12.4 977 292 0.08 TOTAL 1,070,000 32.6 1,640 4,670 1,400 10,900 TOXIC METALS TO AIR 192 4.76 23.2 15.8 4.72 1.83 TO LAND 7,400 741.1 903 320 95.5 37.7 TO WATER 416 15.5 1.16 1.07 0.32 0.35 TOTAL 8,010 761 928 336 100.6 39.9 NOTE: NA = not available, NA* = not applicable Table 3. Contd. Pollution loads (ton/yr) with respect to total value of output Assessment of Industrial Pollution Load in Lagos, Nigeria b I d t i l P ll ti P j ti S t (IPPS) Eff Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 217 Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 3.1.2 Water pollution load p Of all the industries, UST ranked first in terms of total water pollution load while CCM and AET ranked second and third, respectively. This was due to the fact that emission of TSS from the two industries was more than BOD. Estimated TSS pollution load from these industries are 14,400 and 1,400 ton/yr, respectively while BOD pollution load are 0.89 and 96.5 ton/yr, respectively. The steel industry with the highest number of employees generated the highest water pollution load. Thus, the higher the number of employees, the higher the water pollution loads. Pollution load estimated with respect to total output showed that 5.6 million ton/yr of TSS was generated by UST. Water pollution load estimated with respect to employment and total output revealed that emission of TSS was more than BOD in all the manufacturing facilities under the basic metal sector with UST having the highest water pollution load with respect to the two variables (i.e., employment and total output). This is shown in Tables 2 and 3, respectively. APT and CAP have the lowest water pollution load thus, their contribution to water pollution is insignificant. 3.1.3 Toxic pollution load Toxic chemical and metal pollution load with respect to employment and total output are presented in Tables 2 and 3, respectively. Total chemical pollution load with respect to employment and total output is more than total metal pollution load in all the facilities. This may be attributed to the nature of the raw materials used by these facilities. Thus, raw material characteristics and product grades are some of the factors affecting pollution load (Oketola and Osibanjo, 2009b). 3.2 Pollution load assessment by effluent analysis The results of the composite untreated effluent samples collected from the production line of the facilities are presented in Tables 4 and 5, respectively. The result of effluents analysis showed varying concentration of some of the parameters such as heavy metals, COD etc., which are above the permissible limits of Federal Ministry of Environment, (FEPA, 1998) for effluent discharge thus indicating gross pollution. The values of some of the parameters obtained could be attributed to the production processes, raw material characteristics etc. 3.2 Pollution load assessment by effluent analysis Industrial Code /Parameters BGR CAP UST1 TTP1 WSY LOP IGM AET Sampling time (n) 4 5 2 2 3 2 2 5 Parameters Temp0C 30.3±1.7 29.2±1.8 45 33±1.4 46.3±7.8 36±1.4 29.5±0.7 30.5±0.7 pH 7.62±0.5 6.32±0.5 6.75±0.1 5.75±0.1 9.6±1.0 6.85±0.6 11.3±0.0 10.8±0.9 Turbidity (NTU) 4.15±0.3 3.53±0.5 ND ND 0.31±0.04 1,230±360 ND 0.72±0.1 Conductivity (µs/cm) 2210±410 810±85 104±5.7 260±14 0.31±0.04 305±78 2,700±28 0 3550±780 TSS (mg/L)* 9.65±2.8 1.40±0.8 0.28±0.3 0.05±0.01 0.14±0.1 301±66 1.55±1.3 2.33±1.4 218 Environmental Management in Practice Oil & Grease (mg/L) 3.42±8.8 6.30±1.5 104±5.7 260±14 2,400±400 91.2±30 0.34±0.4 34.3±30 Total Alkalinity (pH 4.3) (mg/L) 863±570 650±270 0.37±0.4 ND 1.0±0.4 32.6±46 505±710 3,730±2,400 Total Acidity (pH 8.3) (mg/L) 813±97 602±120 41.1±6.7 67.9±10 7931.0±61 40.5±31 ND 2,070±1,300 Methyl Orange Acidity (pH 3.7) (mg/L) 293±590 ND 34.9±32 ND 147±120 ND ND - Total Hardness (mg/L) 78.7±28 58.8±20 222.6±300 6.27±1.0 376±530 80.5±63 35.9±43 246±350 Cl- (mg/L) 82.2±38 33.6±10 8.57±4.1 1.79±0.1 36.7±18 9.06±0.5 2.44±1.0 21.1±38 SO42- (mg/L) 106±53 855±780 46.1±2.7 1.19±0.1 1,180±680 37.4±49 199±120 717±520 PO43- (mg/L) 94.5±20 46.2±17 ND ND 7 7.0±6.1 10.5±9.6 12.0±17 47.5±14 NO3- (mg/L) 2.12±1.4 ND ND ND 0.8±0.7 0.11±0.1 ND ND DO (mg/L) ND ND 7.50±1.4 6.80±0.1 ND ND ND 80±1.8 COD (mg/L) 1700±630 642±390 130±6.4 621±43 783±86 22,160±95 897±7.1 159±130 BOD5 (mg/L) * 23.4±2.9 20.3±7.7 10.5±3.0 0.48±0.04 4.56±0.4 54.5±18 ND 3.95±1.9 Ca (mg/L) 15.3±5.9 15.6±15 0.34±0.2 1.04±0.02 14.6±15 53.8±65 38.2±19 0.02±0.04 Mg (mg/L) 9.85±9.2 5.77±7.1 53.8±73 0.78±0.1 82.3±140 14.6±15 0.73±1.0 60.0±85 Pb (mg/L) 2.01±4.0 12.4±15 3.07±4.3 ND 9.07±16 0.22±0.3 ND 19.0±23 Ni (mg/L) 0.73±0.5 0.52±0.8 0.10±0.1 0.35±0.1 ND 0.1±0.1 0.6±0.8 0.48±0.8 Cd (mg/L) 0.78±1.1 1.77±1.3 0.11±0.2 ND 0.09±0.2 ND ND 0.44±0.6 Cr (mg/L) 0.53±0.4 0.41±0.3 0.18±0.2 0.05±0.01 0.18±0.1 ND 0.2±0.3 0.19±0.3 Fe (mg/L) 8.80±6.4 4.56±6.4 7.3±10 ND 8.27±7.2 1.40±2.0 4.9±6.9 8.96±12 Mn (mg/L) 2.71±2.2 1.02±0.9 ND 0.23±0.3 ND 0.06±0.1 0.27±0.4 0.98±1.5 Zn (mg/L) 0.15±0.1 0.02±0.04 1.00±1.4 ND 0.01±0.02 0.01±0.01 ND 0.06±0.1 Cu (mg/L) 20.7±14 8.48±7.0 2.70±2.2 0.30±0.1 2.54±0.6 7.8±7.8 4.98±7.0 14.3±6.5 Co (mg/L) 0.29±0.1 0.14±0.1 0.04±0.1 0.02±0.01 0.23±0.2 ND 0.14±0.2 0.25±0.1 TOTAL (mg/L) * 36.7 20.8 14.5 0.95 20.4 9.59 11.1 44.6 d h ll l d Note: * Parameters compared with IPPS pollution load Table 4. 3.3 Results of statistical analysis IPPS estimated pollution loads with respect to employment and total output in these industries were statistically analysed to ascertain the level of agreement between them. There is no significant difference between the pollution load estimated with respect to the two variables (i.e. employment and total output) at p > 0.05 in all the industries except in IGM, WSY, RLT, AWD, and AET. At the 0.05 level, the means are significantly different. IPPS pollution load was also compared with pollution load from conventional effluent analysis. There is no significant difference between them at p > 0.05 in CAP, BGR, UST, TTP and AET while there is significant different between IPPS pollution load and pollution load from conventional effluent analysis in WSY. Hence, IPPS compared favourably with effluent analysis in most of the industries. 3.2 Pollution load assessment by effluent analysis Mean concentration and standard deviation of physico-chemical parameters of untreated effluent from the selected industries Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 219 Industrial Code/Parameters BGR (n = 2) CAP (n = 2) WSY (n = 2) LOP (n = 2) IGM (n = 3) AET (n = 2) FMENV LIMIT Parameters Temp0C 30±2.8 28.8±3.2 47.8±1.8 30.8±0.4 35±2 29.5±0.7 pH 7.3±0.3 8.2±0.0 9.85±0.2 8.45±1.1 9.03±0.3 10.3±0.9 6.5 – 9.0 Turbidity (NTU) 0.05±0.01 0.06±0.01 0.44±0.2 137±52 ND 0.41±0.03 Conductivity (µs/cm) 545±92 2,300±140 4,500±710 289±150 5,670±610 3,400±57 0 TSS (mg/L) * 0.23±0.02 0.32±0.1 0.37±0.2 32.0±9.9 0.44±0.1 1.91±1.3 Oil & Grease (mg/L) 0.30±0.03 0.03±0.01 19.2±3.8 4.79±1.0 9.19±6.8 3.16±0.4 Total Alkalinity (pH 4.3) (mg/L) 293±57 572±97 1,350±440 131±56 2,880±170 1,720±1,1 00 Total Acidity (pH 8.3) (mg/L) 136±130 60±85 220±75 9.16±1.8 76.1±16 ND Total Hardness (mg/L) 118±67 44.5±20 32.1±25 22.0±8.5 207±330 1.57±2.2 Cl- (mg/L) 31.9±0.2 7.62±8.8 46.4±66 9.34±6.4 127±31 55.1±68 600 SO42- (mg/L) 103±16 471±83 303±84 36.4±36 111±32 1,100±89 0 PO43- (mg/L) 8. 85±5.2 ND 25.5±21 3.14±1.0 8.93±7.7 43.5±30 DO (mg/L) 3.75±3.5 ND ND 0.75±1.5 6.5±1.3 1.75±2.5 COD (mg/L) 1450±92 1,030±250 1,140±510 97.4±6.6 363±260 909±9.9 80.0 BOD5 (mg/L) * 27.0±1.1 16.1±2.7 60.1±11 21.8±8.5 10.2±11 6.55±1.0 30.0 Ca (mg/L) 16.3±16 2.34±0.1 5.31±6.5 10.9±13 2.4±0.4 ND Mg (mg/L) 18.7±6.6 9.38±4.8 4.55±2.1 5.38±3.7 48.8±79 0.38±0.5 Pb (mg/L) 3.27±4.6 4.7±6.7 6.35±9.0 7.0±9.9 0.28±0.4 ND < 1.0 Ni (mg/L) 2.8±0.6 1.20±0.3 0.90±0.1 ND 0.67±1.2 0.8±1.1 < 1.0 Cd (mg/L) 0.47±0.7 ND 0.97±1.4 ND 1.64±1.6 0.15±0.1 < 1.0 220 Environmental Management in Practice Cr (mg/L) 0.23±0.3 0.14±0.1 0.46±0.1 0.23±0.3 0.1±0.2 0.29±0.4 < 1.0 Fe (mg/L) 10.9±3.3 0.6±0.9 6.5±9.2 4.18±5.7 60.5±66 61.1±61 20.0 Mn (mg/L) ND ND 0.08±0.1 0.06±0.1 13±6.7 ND 5.0 Zn (mg/L) 0.11±0.6 0.20±0.3 0.01±0.01 0.12±0.2 0.1±0.1 0.07±0.1 < 1.0 Cu (mg/L) 9.21±7.4 8.03±4.6 3.18±4.5 1.81±2.4 11.5±10 6.85±2.0 < 1.0 Co (mg/L) 0.32±0.4 0.15±0.2 0.16±0.1 ND 0.11±0.2 0.14±0.03 < 1.0 TOTAL (mg/L)* 28.7 16.7 19.3 6.68 87.6 72.7 Note: * Parameters compared with IPPS pollution load T bl 5 M i d d d d i i f h i h i l f Table 5. Mean concentration and standard deviation of physico-chemical parameters of effluent discharged to the environment in the selected industries in Lagos Table 5. Mean concentration and standard deviation of physico-chemical parameters of effluent discharged to the environment in the selected industries in Lagos 4. Conclusion This study estimated pollution loads of some industries in Lagos using IPPS pollution intensities with respect to employment and total output. In most cases, the higher the total number of employees and total output, the higher the estimated pollution loads. There is no significant difference between the pollution loads estimated with respect to the two variables in all the industries except IGM where the two means are significantly different. IPPS pollution loads were also compared with pollution loads from conventional effluent analysis at p > 0.05. The two pollution loads compared favourably at this limit. Application of IPPS in Lagos and most developing countries will no doubt enable the regulatory and monitoring agencies in such countries to focus on the most polluting industries. This will on the long run increase the level of enforcement since more time can be spent on the few polluting industries. This will also enable the policy makers in the developing countries to tackle industrial pollution since IPPS is a cheap means of assessing industrial pollution when compared to running scientific monitoring data gathering, analysis and assessment which is time consuming, expensive and resource intensive. Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Efflu Assessment of Industrial Pollution Load in Lagos, Nigeria by Industrial Pollution Projection System (IPPS) versus Effluent Analysis 221 Detailed information on employment and total output obtained from the fourteen industries studied revealed that in most cases, the higher the total number of employees and output, the higher the pollution loads by pollutant types except in TTP where the higher the total number of employees and total output, the lower the estimated pollution loads. This variation can be attributed to other factors which affect pollution loads. These are types and quantity of raw materials, process technology, product grade, efficacy of facility, and source type etc. Also, pollution load of the fourteen industries estimated with respect to employment and total output were compared statistically using t-test at 95% confidence interval and analysis of variance (ANOVA). At this level, the two means are not significantly different in CAP, BGR, TTP, FMI, UST, LOP, CLP, and APT while there was significant different in AWD, WSY, RLT, IGM, and AET. These can be attributed to the information and data supplied by these industries including process efficiency and efficacy of installed pollution control technology if any. 4. Conclusion For example, IGM with only 120 employees produced 1,170 ton/yr of total output while LOP with 200 employees have a total production capacity of 16.1 tons/yr which is significantly less than that of IGM. The results of untreated effluent samples collected from these industries also revealed that most of the industries discharged untreated or partially treated effluent into the environment. Out of the 14 industries which data were available for this study, only 29% have effluent treatment plant which is operational, 36% have no effluent treatment plant while the remaining 36% operate dry process in which Effluent Treatment Plant (ETP) is not applicable. Unavailability of ETP in these industries could be attributed to the high cost of installing and maintaining an ETP, air pollution control devices, and weak enforcement of extant environmental regulations in Lagos. g g Pollution load from conventional effluent analysis were compared with IPPS pollution load in these industries. There is no significant difference between them at p > 0.05. IPPS pollution load of the selected industries compared favourably with pollution load from conventional effluent analysis in CAP, BGR, UST, TTP and AET. Enough data was not available from IGM and LOP. The exception was in WSY where there is significant difference between IPPS pollution load with respect to output and pollution load from conventional effluent analysis from effluent collected at the production line. Consequently, there was an agreement between effluent analysis or scientific monitoring and assessment and IPPS. Since IPPS compares favourably with scientific monitoring and analysis in these industries, IPPS therefore offers a cheap management tool for pollution load assessment in these industries; and directional basis for rapid policy intervention by government regulatory agencies in Lagos and other developing countries where pollution abatement technology is absent and level of enforcement is very low. 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Roya Society of Chemistry. Ogungbuyi, O.M. and Osho, Y.B. (2005). Study on Industrial Discharges to the Lagos Lagoon. Report Submitted by United Nations Industrial Development Organization (UNIDO), Country Service Framework Programme under the Ecological Sustainable Industrial Development Programme. g g Onianwa, P. C. (1985). Accumulation, exchange and retention of trace heavy metal in mosses from southwest Nigeria. Ph. D. thesis, University of Ibadan, Ibadan, Nigeria. Onyekwelu, I.U., Junaid, K.A., and Ogungbuyi, O.M. 2003. 5. References Recent trends in the pollution load on the Lagos Lagoon – A National perspective. Presented by Federal Ministryof Environment at the Ecological Sustainable Industrial Development Workshop. 2 - 20. g p p Oketola, A.A., and Osibanjo, O. (2009a). Estimating sectoral pollution load in Lagos by Industrial Pollution Projection System (IPPS): Employment versus Output. Toxicological & Environmental Chemistry. 91, (5), 799-818. g y ( ) Oketola, A.A., and Osibanjo, O. (2009b). Industrial pollution load assessment by Industrial Pollution Projection System (IPPS). Toxicological & Environmental Chemistry. 91, (5), 989-997. Taras J. Michael. (1950). Phenoldisulphonic acid method of determining nitrate in water. Anal Chem., 22, (8), 1020-102 11 Pollution Prevention in the Pulp and Paper Industries Bahar K. Ince1, Zeynep Cetecioglu2 and Orhan Ince2 1Bogazici University, Institute of Environmental Science, Istanbul, 2Istanbul Technical University, Environmental Engineering Department, Istanbul, Turkey Bahar K. Ince1, Zeynep Cetecioglu2 and Orhan Ince2 1Bogazici University, Institute of Environmental Science, Istanbul, 2Istanbul Technical University, Environmental Engineering Department, Istanbul, Turkey 1. Introduction Pulp and paper industry is considered as one of the most polluter industry in the world (Thompson et al., 2001; Sumathi & Hung, 2006). The production process consists two main steps: pulping and bleaching. Pulping is the initial stage and the source of the most pollutant of this industry. In this process, wood chips as raw material are treated to remove lignin and improve fibers for papermaking. Bleaching is the last step of the process, which aims to whiten and brighten the pulp. Whole processes of this industry are very energy and water intensive in terms of the fresh water utilization (Pokhrel & Viraraghavan, 2004). Water consumption changes depending on the production process and it can get as high as 60 m3/ton paper produced in spite of the most modern and best available technologies (Thompson et al., 2001). ( p ) The wastewaters generated from production processes of this industry include high concentration of chemicals such as sodium hydroxide, sodium carbonate, sodium sulfide, bisulfites, elemental chlorine or chlorine dioxide, calcium oxide, hydrochloric acid, etc (Sumathi & Hung, 2006). The major problems of the wastewaters are high organic content (20-110 kg COD/air dried ton paper), dark brown coloration, adsorbable organic halide (AOX), toxic pollutants, etc. The environmental problems of pulp and paper industry are not limited by the high water consumption. Wastewater generation, solid wastes including sludge generating from wastewater treatment plants and air emissions are other problems and effective disposal and treatment approaches are essential. The significant solid wastes such as lime mud, lime slaker grits, green liquor dregs, boiler and furnace ash, scrubber sludges, wood processing residuals and wastewater treatment sludges are generated from different mills. Disposal of these solid wastes cause environmental problems because of high organic content, partitioning of chlorinated organics, pathogens, ash and trace amount of heavy metal content (Monte et al., 2009). The major air emissions of the industry come from sulfite mills as recovery gurnaces and burnes, sulfur oxides (SOx), from Kraft operation as reduced sulfur gases and odor problems, from wood-chips digestion, spent liquor evaporation and bleaching as volatile organic carbons (VOCs), and from combustion process as nitrogen oxidies (NOx) and SOx. VOCs also include ketone, alcohol and solvents such as carbon disulfide methanol, acetone and chlorofom (Smook, 1992). 224 Environmental Management in Practice Many kinds of the wastes as summarized above are generated from different processes. 1. Introduction The amount, type and characteristics of these wastes are important to provide the best treatment technology. Physicochemical and biological treatment technologies are used extensively for the pulp and paper mills. The lab-scale and full-scale studies about sedimentation/floatation, coagulation and precipitation, adsorption, chemical oxidation and membrane filtration were carried out in the literature to examine physico-chemical approach (Pokhrel & Viraraghavan, 2004). Biological treatment both aerobic and anaerobic technologies are preferred for treatment of pulp and paper mills because of wastewater composition consisting of high organic compounds and economical aspects. Additionally, some fungi species are used to remove color and AOX from the effluents (Taseli and Gokcay, 1999). In some countries, tertiary treatment is applied to obtain discharge limits of regulations (Thompson et al., 2001). Finally, the wide application in the full-scale plants for treatment pulp and paper mills is hybrid systems, which is combined physico-chemical and biological treatment alternatives (Pokhrel & Viraraghavan, 2004). g ) Disposal strategy of solid wastes generated from pulp and paper industry is varied depends on the country and the regulations obeyed. After sorting and handling, dewatering, thermal application such as combustion and anaerobic digestion to obtain energy and deposit in landfills are general applications. However, the solid wastes should be monitored after landfill deposition because of toxic characteristics of the compounds (Monte et al., 2009). Also gaseous pollutants are other environmental problems generated from pulp and paper industry. To minimize these pollutants, physico-chemical methods such as adsorption to activated coal filters absorption, thermal oxidation, catalytic oxidation and condensation have been widely used (Eweis et al., 1998). In the last decade, low cost and effective trends have been developed to prevent the limitation of physico-chemical applications such as energy cost and generating secondary pollutants (Sumathi & Hung, 2006). Waste minimization, recycle, reuse, and innovative approaches developed in last 10 years become more than an issue. In this chapter, waste characterization of this industry in terms of type and source with management approaches was discussed. Exemplary applications were presented. Finally ‘state of the art’ approaches for the environmental problems of this industry were argued. 2. Waste characterization and source Pulp and paper industry is one of the most water and energy consuming industry in the world. This industry uses the fifth largest energy consumer processes; approximately 4% of total energy is used worldwide. Also during pulp and paper process, the important amount of waste is produced. It has been estimated that 500 million tons of paper and etc. per year will be produced in 2020. Three different raw materials are used in the pulp and paper industry as nonwood fibers and wood materials; soft and hard woods. Waste and wastewaters are generated from both of pulp and bleaching processes. Additionally, 100 million kg of toxic pollutants are released every year from this industry (Cheremisinoff & Rosenfeld, 2010). 2.1 Manufacturing technologies and process description Pulping process is the first step of the production. The main steps of this part are debarking, wood chipping, chip washing, chip digestion, pulp screening, thickening, and washing. Mechanical and chemical operation processes in pulping are used in the worldwide. While 225 Pollution Prevention in the Pulp and Paper Industries mechanical processes involve mechanical pressure, disc refiners, heating, and light chemical processes to increase pulping yield; wood chips are cooked in pulping liquors at high temperature and under pressure in the chemical pulping processes. (Sumathi & Hung, 2006). Additionally, mechanical and chemical processes can be combined in some applications. The yield of mechanical processes is higher (90-95%) compared to chemical processes (40-50%). However quality of the pulp obtained from mechanical processes is lower and also the pulp is highly coloured and includes short fibers (Pokhrel & Viraraghavan, 2004). Therefore, chemical pulping carrying out in alkaline or acidic media is mostly preferred. In alkaline media generally referred as Kraft Process, the woodchips are cooked in liquor including sodium hydroxide (NaOH) and sodium sulfide (NaS2). Mixture of sulphurous acid (H2SO3) and bisulfide ions (HSO3-) is used in acidic media named as sulfide process. ( ) p During the pulp processing, approximately 5-10% of the lignin comes from the raw materials cannot be removed and these are responsible from the dark colour of the end product. The production of white paper (pulp bleaching) includes five or optional six treatment steps with sequentially elemental chlorine (C1), alkali (E1), optional hypochlorite (H) stage, chlorine dioxide (D1), alkali (E2), and chlorine dioxide (D2). The general process steps are given in Figure 1. 2.2 Wastewater Different pulping processes utilize different amount of water and all of these processes are water intensive. The quality of wastewater generated from pulping and bleaching is significantly distinctive because of the process and chemical types (Billings and Dehaas, 1971). Approximately 200 m3 water are used for per ton of produced pulp and most of them are highly polluted, especially wastewater generated from chemical pulping process (Cecen et al., 1992). Wood preparation, pulping, pulp washing, screening, washing, bleaching, paper machine and coating operations are the most important pollution sources among various process stages. Wastewaters generated from pulping stage include mostly wood debris, soluble wood materials, and also some chemicals from chemical pulping process. Bleaching process wastewater has a different quality. These wastewaters are not higher strength than pulping process wastewater, however they include toxic components. p p g p y p Process steps and the generated wastewaters from these steps are given in Figure 2. The wastewater characteristics and their strengths changed depending upon the pulping processing. Kraft process is widely used worldwide approximately 60% within all pulp production includes both mechanical and chemical pulping (Holmberg & Gustavsson, 2007). The regional or geographical distribution of the pulping processes is given in Table 1. Region Process Type Pulp Production (million tons) 2004 2005 2006 2007 2008 2009 North America Chemical wood pulp 59.6 59.1 57.3 55.6 54.8 48.6 Mechanical wood pulp 16.3 16.2 15.3 14.4 13.6 11.7 Total Production 75.9 75.3 72.6 70.0 68.4 60.3 Europe Chemical wood pulp 26.8 25.9 27.5 27.3 32.4 29.5 Mechanical wood pulp 11.5 11.2 12.4 12.1 14.3 11.9 Total Production 38.3 37.1 39.9 39.4 46.7 41.4 Table 1. Pulp production in North America and Europe (Food and Agriculture Organization (FAO) Database, 2011) Table 1. Pulp production in North America and Europe (Food and Agriculture Organization (FAO) Database, 2011) 226 Environmental Management in Practice Environmental Management in Prac 6 Fig. 1. Process scheme of a conventional pulp and paper industry (Sumathi & Hung, 2006) Fig. 1. Process scheme of a conventional pulp and paper industry (Sumathi & Hung, 2006) 227 Pollution Prevention in the Pulp and Paper Industries Fig. 2. Wastewater producing steps from pulp and paper mill (Smook, 1992; EPA, 1995) Fig. 2. 2.2 Wastewater Wastewater producing steps from pulp and paper mill (Smook, 1992; EPA, 1995) The wastewaters generated from pulping process consist various wooden compounds such as lignin, carbohydrate and extractives and the treatment of these wastewaters by biologically is difficult. Addition of them, some toxic compounds such as resin acids, unsaturated fatty acids, diterpene alcohols, juvaniones, chlorinated resin acids, and others can exist in the wastewaters subjecting to the process (Pokhrel & Viraraghavan, 2004). The most important reaction in the bleaching step is oxidation of chlorine and the main problem about the wastewater content is chlorinated organic compounds or AOX (Sumathi & Hung, 2006). g p ( g ) The toxic effects of these by-products in the wastewaters on environment have been studied. Various studies reported that fish living in pulp and paper industry wastewaters have toxic/lethal effects on the daphnia, fish, planktons and other bioata in the receiving water bodies (Owens et al., 1994; Hickey and Martin, 1995; Yen et al., 1996; Vass et al., 1996; Liss et al., 1997; Lindstrom-Seppa et al., 1998; Leppanen and Oikari, 1999; Johnsen et al., 1998; Erisction and Larsson, 2000; Schnell et al., 2000b; Kovacs et al., 2002). 2.4 Gas emissions Air pollutants and gas emissions are the other concern about the pulp and paper industry. The most important gas emission is water vapours. Additionally, particulates, nitrogen oxides, volatile organic compounds (VOCs), sulfur oxides and total reduced sulfur compounds (TRS). The gas emissions sources and types are given Table 3. 2.3 Solid and hazardous wastes Wastewater and consequently solid wastes are the main environmental problem of the pulp and paper mills because this industry has a very water intensive production processes 228 Environmental Management in Practice (Cabral et al., 1998; Thompson et al., 2001). Solid wastes from pulp and paper industries are mainly treatment sludges, lime mud, lime slaker grits, green liquor dregs, boiler and furnace ash, scrubber sludges, and wood processing residuals. Wastewater treatment sludges have a significant concern for the environment because of including chlorinated compounds (EPA, 2002). The characteristics of all solid waste generated from the pulp and paper mills are organic exception of boiler and furnace ash. The chemicals of the solid wastes are varied depends on the process type. Solid wastes, sources and qualities are given in Table 2. Source Waste Type Waste Characteristic Wastewater Treatment Plant Sludge Organic fraction consistes wood fibers and biosludge. Inorganic fraction consists clay, calcium carbonate, and other materials 20-60% solid content pH≈7 Caustic Process Dregs, muds Green liquor dregs consisting of non- reactive metals and insoluble materials; lime mud Power Boiler Ash Inorganic compounds Paper Mill Sludge Colour waste and fibre clay including slowly biodegradable organics such as cellulose, wood fibres and lignin Table 2. Solid waste types and sources from pulp and paper mills (EPA, 2002; Nurmesniemi et al., 2007) Table 2. Solid waste types and sources from pulp and paper mills (EPA, 2002; Nurmesniemi et al., 2007) 3. Waste management During the pulp and paper production, high usage of water and energy results in large amount of waste generation like wastewater, solid waste and air emissions. Different types 229 Pollution Prevention in the Pulp and Paper Industries of waste are produced from different production steps and all these wastes pose important environmental problem. To solve this problem: of waste are produced from different production steps and all these wastes pose important environmental problem. To solve this problem: p p - Waste minimization can be done by using new and best available technologies. - End-of-pipe treatment technologies should be used before the discharge and/or disposal. p p Waste minimization can be done by using new and best available technologies. Source Major Pollutants Chemical Pulping Process VOCs (terpenes, alcohols, phenols, methanol, acetone, chloroform, methyl ethyl ketone [MEK]) Reduced sulfur compounds (TRS) Organo-chlorine compounds Bleaching VOCs (acetone, methylene chloride, chloroform, MEK, chloromethane, trichloroethane) Wastewater Treatment Plant VOCs (terpenes, alcohols, phenols, methanol, acetone, chloroform, MEK) Power Boiler SO2, Nox, fly ash, coarse particulates Evaporator Evaporator noncondensibles (TRS, volatile organic compounds: alcohols, terpenes, phenols) Recovery Furnace Fine particulates, TRS, SO2, Nox Calcining (Lime Kiln) Fine and coarse particulates Table 3. Air pollutants types and sources from pulp and paper mills (EPA, 2002) Table 3. Air pollutants types and sources from pulp and paper mills (EPA, 2002) 3.2 Treatment strategies Although the best approach is to minimize the waste generation from the pulp and paper mills and to recycle, the treatment applications are still necessary. In this section, up-to date treatment technologies are given. 3.1 Waste minimization Biopulping is preferred because: - To reduce the chemical and energy utilization To reduce the chemical and energy utilization - To reduce the pollutants h ld d h f l o educe t e c e ca a d e e gy ut at o - To reduce the pollutants T i th i ld d t th ti f l To reduce the pollutants - To reduce the pollutants - To increase the yield and strength properties of pulp. To increase the yield and strength properties of pulp. Elemental Chlorine Free (ECF) and Total Chlorine Free (TCF) Bleaching: Elemental chlorine has been used instead of chlorine dioxide and hypochlorite and oxygen, ozone, caustic soda, and hydrogen peroxide have been applied for TCF bleaching of Kraft pulps to reduce the chlorinated organic wastes (Sumathi & Hung, 2006). Biobleaching: Fungal cells and or their enzymes are used for pretreatment of pulp. A number of studies showed that application of white rod fungi reduces the chemical dosage of bleaching and enhances the brightness of paper (Kirkpatrick et al., 1990; Reid et al., 1990; Daneult et al., 1994). Extended Delignification: Enhanced removal of lignin before bleaching step is the main concern of this method (Gullichsen, 1991; McDonough, 1992). It may be achieved by extended cooking, oxygenation, ozonation, and addition of chemical catalysts. Extended delignification positively affect on the bleach effluent quality parameters such as COS, BOD, color and AOX. 3.2.1 Wastewater treatment End of the pipe pollution treatment strategies are necessary to provide the discharge limits. The general flow-chart of a typical wastewater treatment plant is given Figure 3. The main treatment application for wastewater generated from pulp and paper process is primary and secondary treatment. However, tertiary treatment can be an obligation in of the pipe pollution treatment strategies are necessary to provide the discharge limits general flow-chart of a typical wastewater treatment plant is given Figure 3. g y g g The main treatment application for wastewater generated from pulp and paper process is primary and secondary treatment. However, tertiary treatment can be an obligation in future due to possible new legislations. The physicochemical step is rare at present. 3.1 Waste minimization Modern waste minimization approach is by two means. This first way is chemical recovery and recycling. This system especially in chemical pulping process significantly reduces pollutants and additionally economical return is another important aspect. Chemical recovery is necessary because of the basic economic viability of the kraft process. According to EPA, all kraft pulp mills worldwide use chemical recovery systems. However, there is still no recovery system in some sulfite mills. Additionally, scrubber system particulate "baghouses" or electrostatic precipitators (ESPs) are often mill air pollution control components (EPA, 2002). p ( ) The second way to minimize waste production from pulp and paper mills is the application of best available techniques (BAT) according to the Integrated Pollution, Prevention and Control (IPPC) Regulation. An effective waste minimization method reduces cost, liability, regulatory burdens of hazardous waste management (Rouleau & Sasseville, 1996; Holland, 1997). Furthermore, hazardous waste generation can be reduced by waste management methods including: g - production, planning and sequencing - process adjustment and/or modification - raw material replacement - housekeepingwaste segregation ans seperation - housekeepingwaste segregation ans seperation - recycling - recycling 230 Environmental Management in Practice The industries have developed and applied new technologies instead of conventional pulping and bleaching processes. Some examples of these new technologies are given below: The industries have developed and applied new technologies instead of conventional pulping and bleaching processes. Some examples of these new technologies are given below: Organic Solvent Pulping: This process is more economical for small and medium scale plants for significant recovery and reuse of chemicals. In this process, organic solvent like ethanol, methanol, etc. are preferred. However, this process is more energy consumer than conventional ones (Sumathi & Hung, 2006). Acid Pulping: Acetic acid under the high pressure is used for treating of wood chips. The disadvantage of this process is to loss of acid, however recovery is possible (Sumathi & Hung, 2006). g ) Biopulping: Microorganism or microbial enzymes such as xylanases, pectinases, cellulases, hemicellulases, ligninases, and their combination are used in the pulping process to improve the properties of pulp (Kirk et al., 1996). Primary Treatment In this step, the aim is to remove suspended solid such as bark particles, fiber, fiber debris, filler and coating materials and consequently organic materials. Primary clarification can also be achieved without sedimentation and flotation. However Thompson et al. (2001) mentioned that sedimentation is generally preferred application for the pulp and paper mills in UK and approximately 80% of suspended solid was removed successfully. Also 231 Pollution Prevention in the Pulp and Paper Industries Rajvaid and Markandey (1998) reported 70-80% of removal in the sedimentation. Dissolved air flotation and filtration are the other option as primary treatment for pulp and paper mills Rajvaid and Markandey (1998) reported 70-80% of removal in the sedimentation. Dissolved air flotation and filtration are the other option as primary treatment for pulp and paper mills. Fig. 3. Flow scheme of general wastewater treatment plant of pulp and paper industry Fig. 3. Flow scheme of general wastewater treatment plant of pulp and paper industry Fig. 3. Flow scheme of general wastewater treatment plant of pulp and paper industry Secondary Treatment Aerobic lagoons, activated sludge systems, anaerobic treatment and sequential biological treatment (aerobic-anaerobic or anaerobic-aerobic) are the most common biological treatment application for pulp and paper mills. In this section, the details of these processes are given and they are discussed. Performances of various biological treatment processes are summarized in Tables 4. Aerated Lagoons (Stabilization Basins): Aerated Lagoons (Stabilization Basins): Aerated lagoons are the simple and economical biological systems and they have been studies very well as lab-scale and full-scale at the pulp and paper mills. These systems have been used for removal of BOD, low-molecular weight AOX and fatty acids at full-scale applications (Bajpai, 2001). Stuthridge and Macfarlane (1994) showed that 70% of AOX could be removed efficiently in a short residence time. Welander et al. (1997) reported that COD removal was achieved as 30-40% in a full-scale lagoon and 60-70% in a pilot-scale plant. Lab-scale treatability studies were conducted by Chernysh et al. (1992) to monitor the AOX and TOC removal of bleached kraft effluent. Slade et al. (1999) also reported three aerated stabilization basins, which treated elemental chlorine free (ECF) integrated bleached Kraft mill effluents. Anaerobic Treatment Processes: Anaerobic treatment processes are more suitable for treatment of high strength wastewater such as pulp and paper mills. In the literature, there are a variety of studies on the anaerobic treatability and microbial community of this type of effluents (Poggi-Varaldo et al., 1996; Bajpai, 2000; Ince et al., 2007). Also, anaerobic microorganisms are more efficient than aerobics in order to degrade chlorinated organic compounds. However, the sulphur content in the wastewaters is the main disadvantages for application of anaerobic systems, because one of the end products is hydrogen sulphide in the anaerobic biodegradation in the presence of sulphate (Lettinga et al., 1991). Although Hamm et al. (1991) reported that the toxic effect of H2S is less than high concentration of Ca2+ and SO42-. The other important issues for the application of anaerobic treatment in pulp and paper mills are toxicity of wastewater, anaerobic biodegradability characteristic of specific waste types such as lignin derivates, resin and fatty acids, loading capacity, response to loading fluctuation, and recovery of energy and chemicals (Sumathi and Hung, 2006). Several hundred tons of inorganic chemicals per day for delignification are used in a conventional pulp and paper mill. So, the recovery and reuse of these chemicals are one of the most economical and environmental concern. Addition of it, the black liquor is rich in lignin and a conventional pulp and paper mill produces 1.7-1.8 tons dry solid of black liquor per ton produced pulp and the potential energy of this liquor from anaerobic digestion is 250-500 MW (Stigsson, 1998; Larson et al., 2000). Activated Sludge Systems: This conventional treatment system is used in treatment of several industrial wastewater types in order to remove COD, BOD, SS, and AOX. There are a lot of studies in the literature to show the treatability of pulp and paper mills by activated sludge system. Some of them focused on the BOD, COD, AOX and other specific compound removal under different operation conditions. Schnell et al. (1997) showed that 74% of filtered COD, nearly %100 BOD5, resin and fatty acid removal were achieved in the full-scale plant. Saunamaki (1997) reported that 82% and 60% COD removal efficiency at paper mills and pulp mills, respectively in full-scale activated sludge systems of Finland. Knudsen et al. (1994) claimed high COD and BOD removal efficiency by two stage activated sludge process. Also Hansen et al. (1999) and Chandra (2001) showed similar results. The other part of these studies focused on the removal of AOX and other specific compounds such as chlorinated phenols, guaiacols, catechols, vanillins, 1,1- dichlorodimethyl sulfone (DSS), and chlorinated acetic acid (Mohamed et al., 1989; Demirbas et al., 1999; Bajpai, 2001; Chandra, 2001). The main operational problems of the pulp and paper mills are macro nutrient (N and P) limitation in the systems and growth of the filamentous microorganisms and bulking problems. The nutrient limitation problem is overcome by addition of nutrient. However, the dosage is important point on this step because the external addition causes adverse environmental effects such as eutrophication. 232 Environmental Management in Practice Cingolani et al. (1994) highlighted that the main causes of bulking in the pulp and paper mills treatment are poor oxygenation, low organic loading rates and also nutrient limitations. This problem can also be controlled by installation of a selector or addition of chemicals such as chlorine, ferrous salts, lime or talk powder. Selectors are mostly preferred application for bulking (Forster, 1996; Marten and Daigger, 1997; Prendle and Kroiss, 1998; Andreasen et al., 1999). Aerated Lagoons (Stabilization Basins): Anaerobic contact reactor, up-flow anaerobic sludge blanket (UASB) reactor, anaerobic filter, and fluidized bed reactor are mostly employed reactor types in pulp and paper mills. The anaerobic treatment efficiency of different plants from pulp and paper industry is given in Table 5. Fungal Treatment: Fungal Treatment: Fungal species have been used to remove colour and COD from pulp and paper mills (Eaton et al., 1980; Livernoche et al., 1983; Wang et al., 1992; Gokcay and Dilek, 1994; Duran et al., 233 Pollution Prevention in the Pulp and Paper Industries 1994; Sakurai et al., 2001). Pencillium sp., P. chrysosporium and white rod fungi are the most widely used species. Choudhury et al. (1998) reported that Pleurotus ostreatus was removed 77% of lignin, 76.8% of BOD, 60% of COD, and 80% of colour. 1994; Sakurai et al., 2001). Pencillium sp., P. chrysosporium and white rod fungi are the most widely used species. Choudhury et al. (1998) reported that Pleurotus ostreatus was removed 77% of lignin, 76.8% of BOD, 60% of COD, and 80% of colour. Coagulation/Precipitation: Addition of metal salts to generate larger flocs from small particles for removing the pollutants easily is the main principle of this method. There are some studies to find the most effective chemicals such as horseradish peroxide (chitosan), Al2(SO4)3, hexamethylene diamine epichlorohydrin polycondensate (HE), polyethyleneimine (PEI) to remove AOX, total organic carbon and colour (Tong et al., 1999; Ganjidoust et al., 1997). The authors reported that chitosan is more effective to remove these pollutants from others. Dilek and Gokcay (1994) stated that alum salts as coagulant were removed 96% of COD from the paper machine, 50% of COD from pulping, and %20 COD from bleaching effluents. The other study showed that polyelectrolytes were more effective than the conventional coagulant on the removal of turbidity, COD, and colour (Rohella et al., 2001). Tertiary Treatment Coagulation/Precipitation: Adsorption: This method relies on the addition of an adsorbant such as activated coke, fuller’s earth, coal ash, activated carbon, and activated charcoal to the wastewater to remove the pollutants. High removal of colour by activated charcoal, fuller’s earth, and coal ash was reported (Murthy et al., 1991). Also Shawwa et al (2001) showed that high removal of colour, COD, DOC, and AOX from bleaching wastewater by activated coke. Table 5. Selected anaerobic process performance at different pulp and paper industries (Bajpai, 2000) Chemical Oxidation: Advanced oxidation methods such as photocatalysis, photo-oxidation, Fenton type reactions, wet oxidation, ozonation are used to achieve the destruction of chromophoric and nonchromophoric pollutants in pulp and paper mills. The achievement of photocatalytic reaction in the removal of COD is depended on the concentration of COD and chloride, which are below a certain level (Balcioglu and Ferhan, 1999). Fenton and photo-fenton reactions are highly effective for the treatment of bleaching kraft mill effluent (Perez et al., 2002). Verenich et al. (2000) showed that wet-oxidation are increased the biodegradability of the pulp and paper mill effluent from 30% to 70%. Also ozonation is one of the most effective methods. Several author showed that the effectiveness of this method (Hostachy et al., 1997; Zhou and Smith, 1997; Yamamoto, 2001; Freire et al., 2000). Membrane filtration is a potential method to remove colour, COD, AOX, salts, heavy metals, and total dissolved solids from pulp and paper mills (Zaidi et al., 1992; Afonso and Pinho, 1991; Falth, 2000; Merrill et al., 2001). The effluent of membrane filtration can be used again in production process or discharge directly to the receiving water bodies. Dube et al. (2000) showed that 88% and 89% removal of BOD and COD, respectively was achieved by reverse osmosis (RO). ( ) The performance of physico-chemical process at the pulp and paper industry is summarized in Table 6 The performance of physico-chemical process at the pulp and paper industry is summarized in Table 6. 234 Environmental Management in Practice Pollution Prevention in the Pulp and Paper Industries 235 . Selected anaerobic process performance at different pulp and paper indus 2000) 236 Environmental Management in Practice 237 Pollution Prevention in the Pulp and Paper Industries 3.2.2 Management and disposal of solid wastes Steam reforming is a novel combustion technology, which carries out in a steam reforming reaction system (Durai- Swamy et al., 1991; Aghamohammadi and Durai-Swamy, 1995; Demirbas, 2007). Wet Oxidation: The principle of wet oxidation is that organic compound as solid or liquid form is firstly transferred to water where it contacts with an oxidant under high temperature and pressure. During wet oxidation, waste pulped with water is carbonized and its fuel value increases to the equivalent of medium-grade coal. The waste does not cause any air emission in order to combust without flame or smoke (Kay, 2002). This technology is also considered as an emerging technology like steam reforming. 3.2.2 Management and disposal of solid wastes g Integrated solid waste management of pulp and paper mills are through anaerobic digestion, composting, land applications, thermal processes such as incineration/combustion, pyrolysis, steam reforming, and wet oxidation. g Anaerobic Digestion: This process type is a cost effective way due to the high-energy recovery (Verstraete and Vandevivere, 1999; Mata-Alvarez et al., 2000). Industrial wastes, which have high organic content and digestable, are suitable for anaerobic digestion like paper sludge and wastewater treatment plant sludge (Kay, 2003; CANMET, 2005). g g y Composting: This method is suitable for the wastes and sludge, especially paper fibres and organic materials. The wastes are stabilized via microorganisms with minimal carbon loss. The end product of this process, humus-like material, can be used for houseplants, greenhouse and agriculture (Jokela et al., 1997; Hackett et al., 1999; Christmas, 2002; Gea et al., 2005). Land Application: This method has been preferred disposal method, especially for the acidic soil due to CaCO3 content of sludge. This application is widely used in the United Kingdom and Northern Europe. Before the application, dewatering and/or incineration treatment are done to the waste/sludge in order to reduce volume (Carr and Gay, 1997; Van Horn, 1997). and Northern Europe. Before the application, dewatering and/or incineration treatment are done to the waste/sludge in order to reduce volume (Carr and Gay, 1997; Van Horn, 1997). Incineration (Combustion): Combination of incineration with power and steam generation is one of the most applied methods in Europe, especially for wastewater treatment plant sludge. However, water and ash content of most sludges cause the energy deficiency. Fluidized bed boiler technology is becoming the one of the best solution for the final disposal of paper mill wastes in order to provide successful thermal oxidation of high ash, high moisture wastes (Busbin, 1995; Fitzpatrick and Seiler, 1995; Davis et al., 1995; Albertson, 1999; Porteous, 2005; Oral et al., 2005). ) Pyrolysis: In this process, organic wastes are converted to gaseous and liquid phase under high temperature and in the absence of oxygen. This is an alternative technology to incineration and landfill. This method is suitable for organic content high wastes such as wood, petroleum, plastic waste. However this technology is not sufficient for pulp and paper mill waste. Some investigations have been continue to adapt this technology to pulp and paper mills (Fio Rito, 1995; Frederik et al., 1996; Kay, 2002; Fytili and Zabaniotou, 2008). 3.2.2 Management and disposal of solid wastes Steam Reforming: This technology is used for sludge treatment, however it is still considered as an emerging technology for paper sludges. Steam reforming is a novel combustion technology, which carries out in a steam reforming reaction system (Durai- Swamy et al., 1991; Aghamohammadi and Durai-Swamy, 1995; Demirbas, 2007). Wet Oxidation: The principle of wet oxidation is that organic compound as solid or liquid form is firstly transferred to water where it contacts with an oxidant under high temperature and pressure. During wet oxidation, waste pulped with water is carbonized and its fuel value increases to the equivalent of medium-grade coal. The waste does not cause any air emission in order to combust without flame or smoke (Kay, 2002). This technology is also considered as an emerging technology like steam reforming. Pyrolysis: In this process, organic wastes are converted to gaseous and liquid phase under high temperature and in the absence of oxygen. This is an alternative technology to incineration and landfill. This method is suitable for organic content high wastes such as wood, petroleum, plastic waste. However this technology is not sufficient for pulp and paper mill waste. Some investigations have been continue to adapt this technology to pulp and paper mills (Fio Rito, 1995; Frederik et al., 1996; Kay, 2002; Fytili and Zabaniotou, 2008). Steam Reforming: This technology is used for sludge treatment, however it is still considered as an emerging technology for paper sludges. Steam reforming is a novel combustion technology, which carries out in a steam reforming reaction system (Durai- Swamy et al., 1991; Aghamohammadi and Durai-Swamy, 1995; Demirbas, 2007). W O id i Th i i l f id i i h i d lid li id Pyrolysis: In this process, organic wastes are converted to gaseous and liquid phase under high temperature and in the absence of oxygen. This is an alternative technology to incineration and landfill. This method is suitable for organic content high wastes such as wood, petroleum, plastic waste. However this technology is not sufficient for pulp and paper mill waste. Some investigations have been continue to adapt this technology to pulp and paper mills (Fio Rito, 1995; Frederik et al., 1996; Kay, 2002; Fytili and Zabaniotou, 2008). p p ( , ; , ; y, ; y , ) Steam Reforming: This technology is used for sludge treatment, however it is still considered as an emerging technology for paper sludges. 3.2.3 Treatment of gas emissions Air pollution control at pulp and paper mills has been important concern in the recent years. Especially VOCs produced form pulp and bleaching steps and steam are conventionally treated by physico-chemical methods such as adsorption to activated coal filters, absorption, thermal oxidation, catalytic oxidation, and condensation (Eweis et al., 1998). I spite of these 238 Environmental Management in Practice pollutants are removed from gaseous phase, they transferred another phase and they are also different pollutants for environment. More innovative approach to solve this problem is biofilters and bioscrubbers that have three steps to remove pollutants from gaseous phase; p p - The transfer of pollutants from air to liquid phase, - The transfer of pollutants from air to liquid phase, p q p - The transfer of pollutants from liquid phase to biofilm phase where microorganisms are located, and - The transfer of pollutants from liquid phase to biofilm phase where microorganisms are located, and - Mineralization of pollutants by microorganisms. - Mineralization of pollutants by microorganisms. 4. Conclusion The paper demand increases every day as a result of developed population and industrialisation. Water and energy utilization and in particularly waste generation are becoming more important concern ever worldwide. A major goal is to decrease damage to environment by waste minimization, reuse and recycle. To use best available techniques and innovative methods is becoming more an issue. However, end-of-pipe treatment is still the major approach to minimize the risk. To evaluate pollutants and to develop treatment technologies need a holistic approach. The major pollution load constitutes wastewaters from pulp and paper mills. A variety of wastewater is generated from diverse processes. Different technologies and their combinations have been used for their treatment. The most common applied systems are biological treatment, sequential anaerobic and aerobic systems, followed after primary treatment. Solid waste management and disposal are also another concern. During the final disposal step, the aim should be chemical compound and energy recovery because of environmental and economical aspects. However, the waste minimization has still the first and important approach. Biofilters and bioscrubbers are mostly used for removal of air pollutants and other applications are limited. 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Biological treatment of paper pulping effluents by using a fungal reactor. Water Sci Technol., Vol. 40, No. 11– 12, pp. 93–09. g pp Thompson, G.; Swain, J.; Kay, M. & Forster, C. (2001). The treatment of pulp and paper mill effluent: a review. Bioresource Technology, Vol. 77, pp. 275–286. Tong, Z.; Wada, S.; Takao, Y.; Yamagishi, T.; Hiroyasu, I. & Tamatsu, K. (1999). Treatment of bleaching wastewater from pulp-paper plants in China using enzymes and coagulants. J. Environ. Sci. Vol. 11, No. 4, pp. 480–484. Torrades, F.; Peral, J.; Perez, M.; Domenech, X.; Hortal, J.A.G. & Riva, M.C. (2001). Removal of organic contaminants in bleached kraft effluents using heterogeneous photocatalysis and ozone. Tappi J. Vol. 84, No. 6, pp. 63. p y pp pp US EPA. Zhou, H. & Smith, D.W. (1997). Process parameter development for ozonation of kraft pulp mill effluents. Water Sci. Technol., Vol. 35, No. 2–3, pp. 251– 259. Environmental Management in Practice Retrofit Approach for the Reduction of Water and Energy Consumption in Pulp and Paper Production Processes Jesús Martínez Patiño and Martín Picón Núñez University of Guanajuato México 5. References EPA office of compliance sector notebook project: profile of pulp and paper industry. Washington, DC 20460, USA: EPA/310-R-95-015; 1995. Vass, K.K.; Mukopadhyay, M.K.; Mistra, K. & Joshi, H.C. (1996). Respiratory stresses in fishes exposed to paper and pulp wastewater. Environ Ecol., Vol. 14, No. 4, pp. 895– 897. Verenich, S.; Laari, A. & Kallas, J. (2000). Wet oxidation of concentrated wastewater of paper mills for water cycle closing. Waste Manage (N.Y.), Vol. 20, No. 4, pp. 287– 293. Verstraete, W. & Vandevivere, P. (1999). New and broader applications of anaerobic digestion. Crit. Rev. Environ. Sci. Technol. Vol. 29, No. 2, pp. 151–173. Van Horn, J.T. (1997). Land Application of Solid Waste Stone Container Corporation. Environmental Conference and Exhibit. TAPPI Proceedings. Minneapolis Convention Center, pp. 845–848. Wang, S.H.; Ferguson, J.F. & McCarthy, J.L. (1992). The decolorization and dechlorination of Kraft bleach plant effluent solutes by use of three fungi: Ganderma lacidum, Coriolus versicolor and Hericium erinaceum. Holzforschung, Vol. 46, pp. 219–233 Welander, T.; Lofqvist, A. & Selmer, A. (1997). Upgrading aerated lagoons at pulp and paper mills. Water Sci Technol., Vol. 35, No. 2–3, pp. 117– 122. Yamamoto, S. (2001). Ozone treatment of bleached kraft pulp and waste paper. Japan Tappi J., Vol. 55, No. 4, pp. 90–97. Yen, N.T.; Oanh, N.T.K.; Reutergard, L.B.; Wise, D.L. & Lan, L.T.T. (1996). An integrated waste survey and environmental effects of COGIDO, a bleached pulp and paper mill in Vietnam on the receiving water body. Global Environ Biotechnol., Vol. 66, pp. 349– 364. Zaidi, A.; Buisson, H.; Sourirajan, S. & Wood, H. (1992). Ultra-and nano-filtration in advanced effluent treatment schemes for pollution control in the pulp and paper industry. Water Sci Technol., Vol. 25, No. 10, pp. 263– 276. 246 Environmental Management in Practice 12 2. Pulping process Pulp is obtained from different types of cellulosic material sources, e.g. wood and other fibrous plants. The procedure for obtaining pulp from these materials is called pulping and its purpose is the purification and separation of cellulosic. its purpose is the purification and separation of cellulosic. There are different categories of pulping processes: chemical and mechanical pulping. Chemical pulping methods rely on the effect of chemicals to separate fibers, whereas mechanical pulping methods rely completely on physical action. The two main chemical processes are: the Kraft process (alkaline) and the Sulfite process (acid). The mechanical process produces higher yields compared to the pulp process; Mechanical pulps are characterized by high yield, high bulk, high stiffness and low cost. They have low strength since the lignin interferes with hydrogen bonding between fibers when paper is made. Wood is debarked and chipped, and the chips screened to eliminate fine material and over- sized chips. The “accepted” chips are fed to a pressure vessel, the digester. The chips are steamed with direct steam to eliminate as much of the air as possible. The cooking temperature is maintained until the desired degree of delignification is reached, after which the digester contents go to a blow tank. The pulp from the blow tank is then washed and screened. Residual lignin is removed from pulp by bleaching with chemical reagents. All bleaching treatments have certain common steps. The consistency of the pulp suspension is set in a washer or de-watering device to a target level; temperature and pH may be adjusted by controlling the wash water temperature and pH on the washer of a preceding stage. The suspension is pumped via one or several mixers to a co-current tubular reactor, which may There are different categories of pulping processes: chemical and mechanical pulping. Chemical pulping methods rely on the effect of chemicals to separate fibers, whereas mechanical pulping methods rely completely on physical action. The two main chemical processes are: the Kraft process (alkaline) and the Sulfite process (acid). The mechanical process produces higher yields compared to the pulp process; Mechanical pulps are characterized by high yield, high bulk, high stiffness and low cost. They have low strength since the lignin interferes with hydrogen bonding between fibers when paper is made. Wood is debarked and chipped, and the chips screened to eliminate fine material and over- sized chips. 1. Introduction As it has been mentioned in the previous paragraphs, the application of technologies for the reduction of to reduce water and energy is performed independently, however, it has been 248 Environmental Management in Practice established that water and energy are directly related. First these papers works analyze the opportunities for water minimization and later, an energy study is realized in order to conclude that reducing water, energy consumption also reduces. established that water and energy are directly related. First these papers works analyze the opportunities for water minimization and later, an energy study is realized in order to conclude that reducing water, energy consumption also reduces. The methodologies mentioned in previous paragraphs do not discuss the main characteristics concerning the operation of pulp and paper process; in fact, process conditions (stream flow rate, temperature, concentration, etc.) give the information to identify the objectives of the minimal use of water and energy. Recently, Savulescu et al. (2008) published a work where heat is recovered through the mixing of streams and the dilutions that take place. However for this type of systems, they do not provide an integrated methodology where water and energy is reduced simultaneously. The central premise of this work is that internal aspects of the process must be analyzed in order to look into opportunities that will change the operating conditions to achieve a more efficient use of water and energy. The internal aspects of the process that must be analyzed are: separation processes, reaction processes and equipment performance. The chemical operations involved are those used for the separation of (lignin), unwanted material that accompanies the final product (cellulose). Depending on the level of conversion in the reaction, the next step (washing) will require more or less amount of water. Therefore, by increasing the conversion, a decrease in the water consumption can be expected. By modifying the water streams, the energy requirements for the bleaching operation are also modified. Any change in the operating conditions, will have an effect on the equipment performance, and this should be evaluated. This chapter presents a case study in a Kraft Pulp Mill (Fig. 1). The general process flowsheet is described in the following section. 1. Introduction This chapter describes a comprehensive approach that allows a water and energy reduction in industrial processes. This technique is based on the retrofit concept. An analysis of retrofit has the feature to perform in a systematic way, a series of steps that guides practices and help to identify opportunities for saving water and energy. p y pp g gy Methodologies and techniques have been implemented independently in (pulp) industries in order to reduce water and energy consumption. At industry level and particularly in real pulp and pulp processes, methodologies and techniques to reduce independently water consumption as well as energy consumption have been implemented. Pinch Technology began its application to this kind industry in 1990 (Calloway et at. 1990) to optimize energy using the traditional methodology introduced by Linnhoff et al. (1982). Subsequently, using the Pinch Analysis concept, Berglin et al. (1997) incorporated a mathematical programming work and an exergy analysis; they achieved the reduction of energy consumption in two pulp mills. Koufus et al. (2001), used sequentially Pinch Analysis and later on Water Pinch Analysis (WPA) methodology for these industries (Pulp and Paper), getting first of all an energy reduction and then a water reduction. In the paper of Rouzinuo et al. (2003) Pinch Technology proved to be a great tool for the integration of new equipment in processes for pulp and paper industry at an application in Albany (Oregon, USA) achieving the reduction of energy consumption significantly. Savulescu et al. (2005c), presented a processes integration technique based on Pinch Technology to reduce water (WPA) and energy (Pinch Analysis) in a Kraft process pulp mill; in the same way Towers (2007), applied Pinch Technology for water reduction. ( ) pp gy The concept of energy reduction through the water reduction in the pulp and paper industry was applied by Wising et al. (2005). With the same concept, Nordam et al. (2006), presented a design for water and energy systems reducing energy consumption by reducing water use. For water reduction (exclusively) in a pulp mill (Kraft process), Parthasarathy et al. (2001) used mass integration for effluent reuse and thereby reduce water consumption. Similarly Lovelady et. al (2007) reduced water consumption by optimizing the discharge effluent reuse water. Fig. 1. Simplified diagram of a pulping process. 3. Overall retrofit strategy for the reduction of water and energy in pulp and paper processes 3.1 Hierarchical methodology The guidelines set by Westerberg et al. (1979), the so-called strategy of the onion diagram (Linnhoff et al. 1982), (Shenoy, 1995) and the heuristic approach (Douglas, 1988), are examples of procedures for the design based on the decomposition of the process in stages. The philosophy behind each of these approaches is the basis for implementing the necessary strategies for the minimization of water and energy in real processes. In this work a hierarchical approach is developed for the retrofit of existing process aiming at the reduction of water and energy consumption. Basic to this approach is a profound knowledge of the process; it continues then with the extraction of information and then the implementation of the heuristic rules and methodologies for analysis. A graphical diagram of the hierarchical approach is shown in Fig. 2 by means of an “onion diagram”. The various steps are described below:  Analysis of chemical reaction route  Reaction system (reactors)  Reaction system (reactors)  Water use system  Water use system  Water use system  Water regeneration for reuse  Heat recovery system 3.2 Reaction The layer of reaction is subdivided into two levels: one is related to the analysis of the route of reaction and the other one is related to the system of reactors. In this stage, the type of chemical reaction, the kinetics and the reactor design are analyzed in detail. 2. Pulping process The “accepted” chips are fed to a pressure vessel, the digester. The chips are steamed with direct steam to eliminate as much of the air as possible. The cooking temperature is maintained until the desired degree of delignification is reached, after which the digester contents go to a blow tank. The pulp from the blow tank is then washed and screened. Residual lignin is removed from pulp by bleaching with chemical reagents. All bleaching treatments have certain common steps. The consistency of the pulp suspension is set in a washer or de-watering device to a target level; temperature and pH may be adjusted by controlling the wash water temperature and pH on the washer of a preceding stage. The suspension is pumped via one or several mixers to a co-current tubular reactor, which may Retrofit Approach for the Reduction of Water d E C ti i P l d P P Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 249 Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes be atmospheric or pressurised. The suspension is then transported to a washer for the removal of dissolved material. Finally, water is removed from the pulp through a drying process. be atmospheric or pressurised. The suspension is then transported to a washer for the removal of dissolved material. Finally, water is removed from the pulp through a drying process. Fig. 1. Simplified diagram of a pulping process. 3. Overall retrofit strategy for the reduction of water and energy in pulp and paper processes 3 1 Hi hi l th d l Fig. 1. Simplified diagram of a pulping process. Fig. 1. Simplified diagram of a pulping process. 3.2.1 Analysis of the chemical reaction route The stage of bleaching is a section of the process for pulp production where chemical reactions take place. The purpose of the bleaching process is to withdraw the maximum amount of lignin contained within the pulp. In this stage, the type of reaction that is carried out in each of the different stages of the bleaching process is analyzed. The chemical compounds that are used in the bleaching stages are identified. In the case of an existing plant, the analysis of the route of reaction may trigger a series of actions allowing the 250 Environmental Management in Practice implementation of technologies with greater reaction conversion while reducing the water consumption. implementation of technologies with greater reaction conversion while reducing the water consumption. Fig. 2. Retrofit approach based on the concept of the "Onion Diagram ". Fig. 2. Retrofit approach based on the concept of the "Onion Diagram ". 3.2.2 Reaction system (reactors) This Retrofit Approach for the Reduction of Water d E C ti i P l d P P Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 251 e o pp oac o e educ o o a e and Energy Consumptionin Pulp and Paper Production Processes condition results in less fresh water being needed for dilution and therefore water savings are obtained. In addition, warm water is added into the filter for furthering the removal of impurities from the cellulose. Fig. 3. Area of chemical reaction and filtering in the stage of bleaching. Fig. 3. Area of chemical reaction and filtering in the stage of bleaching. 3.2.2 Reaction system (reactors) Once the route of chemical transformation of the process is known, the reactor system design is then considered. This involves the examination of a three way trade-off between equipment, level of conversion and reduction in water consumption. In the case of the bleaching process, the lower the amount of solids product (pulp) at the outlet of the reactor; the lower is the amount of water that is needed to reach the required concentration in the filtering stage, as it is shown in the Fig. 3. Equation 1 (Walas, 1988) shows the relationship between mass flow rate, the volume of the reactor and concentrations. e F e x x V F r   (1) e F e x x V F r   (1) F = Flow rate V = Volume of the reactor xe = Final conversion xF = Initial conversion re = Conversion rate Knowledge of the characteristics of the bleaching reaction and reactor volume, the actual reaction rate can be determined. This information is then used to determine the additional reactor volume needed for more lignin to react. The flow diagram of Fig. 3 shows the way fresh water consumption is linked to the level of lignin conversion in the reactor. Since fresh water is used to dilute the reactor outlet stream for it to be filtered downstream, as amount of lignin that reacts increases, the lower the amount of solids at the reactor outlet. This re = Conversion rate Knowledge of the characteristics of the bleaching reaction and reactor volume, the actual reaction rate can be determined. This information is then used to determine the additional reactor volume needed for more lignin to react. The flow diagram of Fig. 3 shows the way fresh water consumption is linked to the level of lignin conversion in the reactor. Since fresh water is used to dilute the reactor outlet stream for it to be filtered downstream, as amount of lignin that reacts increases, the lower the amount of solids at the reactor outlet. 3.5 Heat recovery system The last stage in the hierarchical strategy is to identify the options for reducing energy consumption through the maximization of the heat recovery and the quantification of direct savings generated by the simple reduction of the water consumption. In some cases, when the economic scenario is favorable, the savings of steam can be channeled to the production of electrical power in cases where the process plant is integrated with a cogeneration system. 3.4 Water regeneration for reuse g Once exploited and completed all the options for the reduction of water consumption through the measures implemented in the first two layers of the onion diagram, the next step consists in the application of water regeneration techniques. At this level, different techniques for feasible decentralized regeneration of the effluents for water reuse should be evaluated (Fig. 5). Among the typical regeneration technologies are those of physical, chemical and biological nature. The selection of the regeneration system should be based on a series of considerations such as: equipment cost, operating costs, ease of implementation, availability, etc. Fig. 5. Regeneration system for water reuse. Fig. 5. Regeneration system for water reuse. 3.3 Water use system At this stage a water pinch analysis is carried out. Let us consider the washing section of the process that consists of a series of physical separations for the removal of impurities from the pulp coming out from the digester (Fig. 4). This pulp receives the name of raw flesh because it has not been bleached yet. At this stage, a large amount of water is used. It is therefore important the implementation of techniques that lead to the reduction of water. The large amounts of water used and the physical nature of the process are conducive for the implementation of the Water Pinch Analysis (WPA) technique which seeks to minimize the consumption of water. These conditions are also appropriate to pose an optimization problem by means of mathematical programming, seeking to reduce the total operation costs. Both techniques are effective for the analysis, synthesis and improvement of the water networks. Furthermore, they take into account the concepts of reuse and regeneration of water that have an impact on the generation of wastewater or effluents while minimizing the water consumption. Fig. 4. Washing system. Fig. 4. Washing system. 252 Environmental Management in Practice 4. Applications and case study The case study in this section uses information from a real pulp plant. The methodology described in the previous section is implemented step by step with the aim of reducing energy and water consumption. Knowledge of the various aspects of an existing plant allows us to identify particular situations that apart from theory lead us to incorporate certain considerations that make the Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 253 application practical. For instance, the plant layout, the economic environment, the time required for the delivery of the projects which will require modifications and investment, the plant production rate and its fluctuations throughout the year, etc. The application is shown below. 4.1 Process description The raw material used for the production of pulp is a short fiber wood from eucalyptus. The Kraft process is divided into four main stages, namely: cooking, screening and washing, bleaching and drying (see Fig. 1). 4.2.1 Analysis of the reaction stage in the bleaching process 4.2.1 Analysis of the reaction stage in the bleaching process As stated in the previous section, the first step consists in the analysis of the reaction route. In the case under consideration, chemical reactions take place during cooking and bleaching. The focus of the analysis will be around the former stage since it involves the consumption of fresh water. The process proceeds by means of an Elemental Chlorine Free reaction (ECF) (Gullichsen et al., 1999) and takes place in three stages with three reactors arranged in series. The reactions are: D0 (oxygen delignification), EOP (Alkaline extraction reinforced with oxygen and hydrogen peroxide) and D1 (Chlorine dioxide). It is important to mention that the plant under consideration does not have an oxidizing stage previous to bleaching for the removal of lignin which implies that pulp reaches the process with a large Kappa number (the Kappa number that determines the weight percentaje of lignin in the pulp. This is: % lignin in pulp = 0.15 x Kappa number). It has been identified that as the lignin conversion increases in the reactor, the consumption of fresh water needed for effluent dilution for the filtering stage is reduced. From equation 1 it is possible to calculate the rate of reaction (re ) since the design parameters of the installed reactors are known. In the case of the D0 reactor, with a volume of 183.084 m3, the volumetric feed (water and pulp mixture) is 216 m3/hr. For the calculation of the reaction conversion as the kappa number moves from 28 to 8, the density of the mixture is needed. To this end, the pulp concentration of the feed is known to be 9.4% by weight; the density of the pulp is 1250 kg/m3, so the overall density is determined below:     3 3 1250 0.094 1000 0.906 1023.5 DO DO kg m kg m                    ( 2 ) ( 2 ) Knowing the kappa number at the inlet and outlet of the reactor, the amount of lignin is calculated to be: Knowing the kappa number at the inlet and outlet of the reactor, the amount of lignin is calculated to be:  0.15 . 4.2.1 Analysis of the reaction stage in the bleaching process % 0.15 8 1.2 % final final final Lignin KappaNo Lignin      ( 4 ) ( 4 ) 254 Environmental Management in Practice The inlet and outlet lignin concentration is found to be: The inlet and outlet lignin concentration is found to be: The inlet and outlet lignin concentration is found to be:       3 3 100 4.2 1023.5 0.094 100 4.040778 initial o DO flow o o Lignin L Concentration kg L m kg L m                   (5) (5)       3 3 100 1.2 1023.5 0.094 100 1.154508 final f DO flow f f Lignin L Concentration kg L m kg L m                   (6)       3 3 100 1.2 1023.5 0.094 100 1.154508 final f DO flow f f Lignin L Concentration kg L m kg L m                   (6)    3 1.2 1023.5 0.094 100 1 154508 f kg L m kg L           (6) (6)   3 100 1.154508 f m kg L m    Once the lignin concentrations are known it is possible to determine the reaction conversion (XF) from: Once the lignin concentrations are known it is possible to determine the reaction conversion (XF) from:     - 4.040778 1.154508 4.040778 0.7143 o F F o F F L L X L X X     ( 7 ) ( 7 ) Then, the rate of reaction can be calculate from: Then, the rate of reaction can be calculate from: Then, the rate of reaction can be calculate from: Then, the rate of reaction can be calculate from: 1 - 183.084 0.7143 2163600 0.000234089 e F e e e x x V F r r r s    1 - 183.084 0.7143 2163600 0.000234089 e F e e e x x V F r r r s    Knowing the rate of reaction it is possible to determine the reactor volumen needed to take the kappa number form 8 to 4 (Gullichsen et al. 4.2.1 Analysis of the reaction stage in the bleaching process % 0.15 28 4.2 % initial initial initial Lignin KappaNo Lignin      ( 3 ) ( 3 )  0.15 . 4.2.1 Analysis of the reaction stage in the bleaching process 1999). This is, achiving higher conversion at the expense of investing in additional reaction volumne. Under the information so far obtained, it is determined that a volume of 219.7 m3 is needed. Fig. 6 shows the process information and the water consumption that are required for the two scenarios, namely: a conversion corresponding to a kappa number of 8 (original) and a conversion correponding to a kappa number of 4 (new). From the results it can be concluded that the increase of the reactor volume by 36.61 m3 allows more lignin to be removed from the pulp and consequently a mass reduction in the effluent is achieved; therefore, less fresh water is required to achieve a concentration of 1.2% which is required for an effective operation ofthe filter. In addition, the filter will consume less water for washing. g If the rector volumen was increased by 40 m3, for a total volume of 223.084 m3, calculations show that the fresh water consumption would be reduced by 11.511 m3/hr. For an Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 255 Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 255 Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes economical analysis, costs information is taken from Peters and Timmerhaus (1991). So, for the year 1990, the cost of a glass fiber linned reactor is approximately $ 190,000.00 USD. The cost is brought upto date by considering the the cost index according to: economical analysis, costs information is taken from Peters and Timmerhaus (1991). So, for the year 1990, the cost of a glass fiber linned reactor is approximately $ 190,000.00 USD. The cost is brought upto date by considering the the cost index according to: ( ) Cost Index (August 2008) f Cost Index Reference year  ( 8 ) ( 8 ) Taking the cost indexes from Chemical: Engineering Plant Cost Index-USA (1990) and Economic Indicator, Chemical Engineering (August 2008), the following factor is obtained: Taking the cost indexes from Chemical: Engineering Plant Cost Index-USA (1990) and Economic Indicator, Chemical Engineering (August 2008), the following factor is obtained: 389 1.6967 660 f  389 1.6967 660 f  So, the approximate up-to-date cost is: So, the approximate up-to-date cost is: 190000 1.6967 322365 $ US   190000 1.6967 322365 $ US   To determine the fresh water consumption, the cost of extraction per m3 of water is 1.2 US$ [Robin Smith, 2005]; considering a total of 8000 working hours, the water cost is To determine the fresh water consumption, the cost of extraction per m3 of water is 1.2 US$ [Robin Smith, 2005]; considering a total of 8000 working hours, the water cost is     3 3 $ 1.2 11.511 8000 Water US m Cost hrs hr m    110506 $ Water Cost US / year      3 3 $ 1.2 11.511 8000 Water US m Cost hrs hr m    110506 $ Water Cost US / year  110506 $ Water Cost US / year  From the information above, the payback period for the revamping of the bleaching reactor is approximately 3 years. For the second and third reaction stages, the lignin content is low enough to consider that the expected water saving would not justify the investment in reactor volume. Fig. 6. Fash water consumption for different reactor volume (D0). Fig. 6. Fash water consumption for different reactor volume (D0). 256 Environmental Management in Practice 4.2.2 Water using system Once the first hierarchical level has been covered, the second level is considered. This level   1 56.712 ton f hr  1, 0 ( ) in C ppm  1 1324 m   kg hr       4.2.2 Water using system Effluents reaching tanks 1 and 2 have different type of contaminant and different concentrations which imply that for water reuse, independent analysis must be conducted. Total water usage is given by stream 2, 4, 7, 11 y 13 (759.348 kg / hr). Streams 2 and 11 give the pulp the required consistency whereas streams 4, 7 and 11 are used for washing. Table 1 shows the mass flow rates, concentrations and mass content of these streams. Effluents reaching tanks 1 and 2 have different type of contaminant and different concentrations which imply that for water reuse, independent analysis must be conducted. Total water usage is given by stream 2, 4, 7, 11 y 13 (759.348 kg / hr). Streams 2 and 11 give the pulp the required consistency whereas streams 4, 7 and 11 are used for washing. Table 1 shows the mass flow rates, concentrations and mass content of these streams. y y Total water usage is given by stream 2, 4, 7, 11 y 13 (759.348 kg / hr). Streams 2 and 11 give the pulp the required consistency whereas streams 4, 7 and 11 are used for washing. Table 1 shows the mass flow rates, concentrations and mass content of these streams. In this part of the study, water pinch technology is applied (WPA). Some studies have been published on the application of this technology to total sites (Jacob et al., 2001; Koufus et al., Total water usage is given by stream 2, 4, 7, 11 y 13 (759.348 kg / hr). Streams 2 and 11 give the pulp the required consistency whereas streams 4, 7 and 11 are used for washing. Table 1 shows the mass flow rates, concentrations and mass content of these streams. In this part of the study, water pinch technology is applied (WPA). Some studies have been published on the application of this technology to total sites (Jacob et al., 2001; Koufus et al., 2001); however, in this work a local analysis is carried out. In a global study, one aspect that is ignored is the actual location of the water using operations; however, this aspect must be considered in a real plant application. Other aspects to be considered are: pulp recovery form water, the design of the piping network and the actual design and the operation of the equipment. 4.2.2 Water using system 4.2.2 Water using system O th fi t hi hi l l l h b d th d l l i id d Thi l l g y Once the first hierarchical level has been covered, the second level is considered. This level corresponds to the pulp washing stage. Once the first hierarchical level has been covered, the second level is considered. This level corresponds to the pulp washing stage. The purposes of the washing process are: a) the removal of un-reacted wood chips and non fibrous impurities from cellulose; b) the removal of soluble solids present in the fiber. The pulp washing step contains two filters that operate counter currently and a continuous rotary filter as shown in Fig. 4. Details of the operation of the equipment are shown in Fig. 7 and operating data are given in Table 1. The case is solved using a heuristic approach and the results are compared to those obtained a mathematical optimization. No. Flowrate (ton/hr) Concentration (%) Mass Load (Kg) No. Flowrate (ton/hr) Concentration (%) Mass Load (Kg) 1 72.250 12 8700 9 49.769 1.7 846 2 217.500 0 0 10 332.523 2.466 8223 3 290 3 8700 11 299.067 0 0 4 56.725 0 0 12 632.590 1.3 8223 5 62.971 2.1 1322.4 13 167.503 0 0 6 283.754 2.6 7377 14 732.134 0.009369 68.59 7 18.553 0 0 15 67.959 12 8155 8 31.755 1.5 476.3 Table 1. Operating data of the washing step. From Fig. 7 we see that the first filter removes the larger solids and its effluent is sent to filter 2 where smaller size solids are removed. The main stream from these two filters is sent to the rotary filter 3 where the pulp is finally washed for the bleaching step. y p p y g p The total fresh water consumption in this process is of 759.348 ton/hr. As mentioned before, the effluent from the first filter (62.97 ton / hr) is processed again for further pulp recovery. Effluents reaching tanks 1 and 2 have different type of contaminant and different concentrations which imply that for water reuse, independent analysis must be conducted. The total fresh water consumption in this process is of 759.348 ton/hr. As mentioned before, the effluent from the first filter (62.97 ton / hr) is processed again for further pulp recovery. 1 1324 m   kg hr       4.2.2 Water using system For the case of filter 1, the operating data is:   1 56.712 ton f hr  1, 0 ( ) in C ppm  1 1324 m   kg hr       1, 0 ( ) in C ppm  Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper P 257 ig. 7. Detail of streams and equipment in the washing process. Fig. 7. Detail of streams and equipment in the washing process. 258 Environmental Management in Practice The outlet concentration limit, C1,out, is obtained from equation (9): The outlet concentration limit, C1,out, is obtained from equation (9): The outlet concentration limit, C1,out, is obtained from equation (9): , lim 3 lim lim , , 10 [ ] i total i i out i in m f x C C    (9) (9) 3 lim , 1324( / ) 56.712( / ) 10 [ 0]( ) i out kg hr ton hr x C ppm   C1,out = 23346 (ppm) C1,out = 23346 (ppm) Table 2 shows the limit concentrations of the stream that will be used to remove the mass of contaminant (∆m) with the minimum amount of fresh water (56.712 ton/hr). The inlet and outlet concentrations of contaminant to the filters are given in Table 3. The information from Table 3 is used to construct the concentration-composite curve where the pinch point is obtained (424 ppm). This point represents the concentration limit of contaminant that can be used; it also shows the minimum fresh water consumption considering water reuse. Filter (no.) fi (ton/hr) Cin (ppm) Cout (ppm) ∆m (kg) 1 56.712 0 23346 1324 2 18.546 0 25666 476 3 167.520 0 424 71 Table 2. Fresh water data in filters. Filter (no.) fi (ton/hr) Cin (ppm) Cout (ppm) ∆m (kg) 1 56.712 0 23346 1324 2 18.546 0 25666 476 3 167.520 0 424 71 Table 2. Fresh water data in filters. The amount of contaminant removed from each process and the total removal (1871 kg/hr) are shown in Fig. 8. The analysis indicates that fresh water is fed to operation 3; part of its effluent is reused in operation 1 and 2 as shown in Fig. 9. 4.2.2 Water using system p g The data form Table 3 is used to design the water network structure by means of mathematical programming. The final design is shown in Fig. 10. Although using both approaches the same minimum water consumption is obtained; however, the network structure is different. Filter (no.) fi (ton/hr) Cin (ppm) Cout (ppm) ∆m (kg) 1 290.000 18780 23346 1324 2 62.952 18105 25666 476 3 632.400 311 424 71 Table 3. Process data of stream feeding the filters Table 3. Process data of stream feeding the filters Both the structures of Fig. 9 and 10 represent grass-root designs and they can be used to identify ways to improve o the existing structures. Now, there are some operating aspects that the former designs do not consider. For instance, none of the designs considers the start up and stabilization of the plant; besides, they do not allow residual water to be used to take the filter inlet to the required concentration. The actual operation of the various pieces of Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 259 equipment is difficult to incorporate in the design such as the case of the filters and its efficiency in connection to the concentration load and the required flow rate that will remove the contaminant. An important issue in this process consists in the removal undesirable material such as stones, plastic and other pulp residues. It is also true that the pulp still contains un-reacted wood chips that can be re-circulated back to the main reactor in order to increase the production of pulp. Taking all these elements into consideration, the series of practical implementations to the washing stage that allow for the reduction of water consumption are described below. Fig. 8. Concentration composite curve for the washing process. Fig. 8. Concentration composite curve for the washing process. Fig. 9. Design of the water network structure using WPA. Fig. 9. Design of the water network structure using WPA. 260 Environmental Management in Practice Fig. 10. Design of the water network structure using mathematical programming Fig. 10. Design of the water network structure using mathematical programming Fig. 11, shows the washing process where two intermediate stages have been included. The first one removes non usable contaminants such as stones, plastic, etc., and those that can be reused in the process (i.e. pulp and wood chips). Table 4. Process information for the start up of the washing process. Table 4. Process information for the start up of the washing process. 4.2.2 Water using system The effluent from this first stage passes through a second treatment where the pup is further cleaned prior to the bleaching stage. g p p p g g In order to incorporate these aspects such as the plant start up and the way the operating conditions change as the steady state is reached. Table 4 shows the process information during the start up, on the other hand Fig. 11 and Table 5 present the operating data once the process has been stabilized. No. Flowrate (ton/hr) Concentration (%) Mass Load (Kg) No. Flowrate (ton/hr) Concentration (%) Mass Load (Kg) 1 72.500 12 8700 14 732.480 0.009369 68.6 2 217.440 0 0 15 67.920 12 8151 3 289.920 3 8700 16 591.678 0.009369 55.4 4 56.712 0 0 17 140.322 0.009369 13.2 5 62.940 2.1 1322 18 31.746 1.5 476 6 283.680 2.6 7376 19 5.589 2.316 129.5 7 18.546 0 0 20 26.157 1.326 346.9 8 31.746 1.5 476 21 26.157 1.326 346.9 9 49.758 1.7 846 22 26.157 1.326 346.9 10 334.020 2.466 8237 23 1.260 0.238 3 11 298.980 0 0 24 26.157 1.314 343.9 12 632.400 1.3 8222 25 1.260 0 0 13 167.520 0 0 Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 261 Fig. 11. Representation of the pulp production process after reduction of fresh water consumption. Fig. 11. Representation of the pulp production process after reduction of fresh water consumption. 262 Environmental Management in Practice No. Flowrate (ton/hr) Concentration (%) Mass Load (Kg) No. Flowrate (ton/hr) Concentration (%) Mass Load (Kg) 1 72.500 12 8700 14 767.140 0.009369 71.87 2 202.731 0.009369 18.99 15 71.208 12 8545 3 302.600 3 9078 16 596.167 0.009369 55.85 4 54.412 0.009369 5.566 17 170.973 0.009369 16.02 5 65.748 2.1 1380.7 18 33.199 1.5 498 6 296.264 2.6 7702.9 19 5.831 2.321 135.4 7 19.483 0.009369 1.825 20 27.369 1.325 362.634 8 33.199 1.5 498 21 27.369 1.325 362.634 9 52.032 1.7 884.54 22 27.369 1.325 359.007 10 348.296 2.466 8547.4 23 1.260 0.2878 3.626 11 314.541 0.009369 29.47 24 27.369 1.312 359.007 12 662.837 1.3 8616.9 25 1.260 0 0 13 175.512 0 0 Table 5. Process information for the stabilized process. Table 5. Process information for the stabilized process. On the start up of the plant, 760.56 ton/hr of fresh water are needed. 4.2.2 Water using system Of these, 168.78 ton/hr are sent to the washing stage while the rest, 591.678 ton/hr are used for dilution purposes before entering filter 3. Once the regeneration processes enter into operation and the reuse of effluent 3 is established, the fresh water consumption is reduced to 176.772 ton/hr. From the ongoing discussion it can be seen that regeneration and reuse considerable reduce the fresh water consumption by reducing the need of using fresh water to feed the filter at a concentration of 1.2%, thus achieving a saving of 582.626 ton/hr. g g In the case under consideration there are various types of effluent stream with different contaminant concentrations, therefore it is important the adequate selection of the regeneration process for water reuse of recycling whatever the case. Regeneration processes are of the distributed type unlike the end of pipe treatment, which in the majority of cases is of centralized type. Fig. 12 shows the inlet and outlet process water flow rates. Fig. 12. Water effluent streams of the pulp production process. Fig. 12. Water effluent streams of the pulp production process. 4.2.3 Regeneration for water reuse Fig. 13 shows the application of a specific treatment to each of the effluent streams in the pulp production process. Appropriate selection of each of these treatments is critical since given the different contaminant composition. Retrofit Approach for the Reduction of Water d E C i i P l d P P Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 263 Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes The characteristic of the effluents of the cooking processes as given by Sumathi and Hung (2006) are: high oxygen demand (BOD), color, it may have sulfur and resin reduced compounds. The effluent of the washing process, on the other hand, contains large amounts of suspended solids (SS), BOD and color. The effluent from the bleaching process contains organochloride compounds, BOD and resin. Now, the level of regeneration can be total or partial. The main types of regeneration processes can be divided in to physical-chemical and biological. Amidst the physical-chemical are: membrane separation techniques (inverse osmosis, ultrafiltration, nanofiltration, etc.), chemical flotation and precipitation and advanced oxidation processes. The biological processes are: activated sludge, anaerobic treatment, sequential anaerobic-aerobic system and fungi system for color and organo- halogenated derivatives. g It is important to emphasize that in the majority of cases 100% regeneration is not targeted; however, what is sought is the minimization of the fresh water consumption and the flow rate o the discharged effluent. In this part, no numerical results are presented since this is outside the scope of this work. Fig. 13. Distributed treatment system for the effluents from each of the stages of the pulp production process. Fig. 13. Distributed treatment system for the effluents from each of the stages of the pulp production process. 4.2.4 Heat recovery system The reduction of fresh water brings about important changes in the need of energy consumption since the pulp production process requires water streams at different temperatures. This stage of the analysis seeks to clearly identify the situations where energy is reduced as a result of a reduction in water consumption through the application of pinch analysis. y Fig. 14 shows the case where water consumption is reduced after increasing the conversion in one of the reactors if the bleaching stage. If fresh water is available at 40ºC and it has to be heated up to 60 ºC before been fed to the filter as shown in Fig. 15, the amount of energy saved is 52.1 kW . So, in order to take the temperature from 20 ºC to 40 ºC, the water and energy saving is 10.391 ton/hr and 242.45 kW, respectively. Another type of sitations that arises is the one shown in Fig. 15, where stream 13 enters the process at 60 ºC and stream 12 reaches the filter at a temperature equal or larger than 35 ºC. After a water reuse scheme is applied, stream is reused 11 and since its temperature is above 35°C, an energy saving of 5,504 kW is achieved compared to the system where fresh water is used. 264 Environmental Management in Practice Fig. 14. Schematic of an energy saving application in the washing stage. Fig. 14. Schematic of an energy saving application in the washing stage. Fig. 15. Schematic of an energy saving process application. Fig. 15. Schematic of an energy saving process application. In summary and putting together the results of the reviewed operations (washing and bleaching), the total amount of water saved is 582.626 ton/hr and an energy saving of 5504 kW is achieved. En el blanqueo se obtiene un ahorro de agua fresca de 11.511 ton/hr y un ahorro de energía de 294.55 kW. It is important to mention that water and energy savings have been achieved simultaneously by applying the methodology to particular unit operations Fig. 15. Schematic of an energy saving process application. In summary and putting together the results of the reviewed operations (washing and bleaching), the total amount of water saved is 582.626 ton/hr and an energy saving of 5504 kW is achieved. En el blanqueo se obtiene un ahorro de agua fresca de 11.511 ton/hr y un ahorro de energía de 294.55 kW. 5. Conclusions This chapter has introduced a genera approach for the retrofit of existing processes for the reduction of water and energy consumption. The methodology introduced is based on a conceptual structured scheme with different hierarchical levels arranged in the following way: y Level 1. Analysis of the reaction system Level 2. Analysis of the water using network Level 3. Analysis and implementation of water regeneration schemes. Level 4. Analysis of the heat recovery system. This new approach direct us to determine the way changes to operating conditions affect the water and energy requirements in a process. In addition, these modifications can be viewed in the light of an economical analysis which shows the economical feasibility of the retrofit projects. 4.2.4 Heat recovery system It is important to mention that water and energy savings have been achieved simultaneously by applying the methodology to particular unit operations. In summary and putting together the results of the reviewed operations (washing and bleaching), the total amount of water saved is 582.626 ton/hr and an energy saving of 5504 kW is achieved. En el blanqueo se obtiene un ahorro de agua fresca de 11.511 ton/hr y un ahorro de energía de 294.55 kW. It is important to mention that water and energy savings have been achieved simultaneously by applying the methodology to particular unit operations. Retrofit Approach for the Reduction of Water d E C i i P l d P P Retrofit Approach for the Reduction of Water and Energy Consumptionin Pulp and Paper Production Processes 265 6. Acknowledgment Thanks to Haydee Morales Razo. This work was supported by SEP-PROMEP (México) through grant PROMEP/103.5/11/0140. 7. References Calloway, J., T. Retsina, et al. (1990). Pinch technology in practical kraft mill optimisation. Engineering Conference Proceedings. Linnhoff B. Townsend, D.W., Boland, D., Hewitt, D.F., Thomas, B.E.A., Guy, A.R. and Marsland RH. (1982)User Guide on Process Integration for the Efficient Use of Energy, Institution of Chemical Engineers. IchemE, Rugby-UK. gy g g y Berglin, N., J. Strömberg, et al. (1997). Using process integration to approach the minimum impact pulp mill. Environmental Conference Proceedings. Rouzinou, S., T. Retsina, et al. (2003). Pinch analysis: A powerful tool for the integration of new process equiment into existing pulp and paper. Fall Technical Conference. Savulescu, L., B. Poulin, et al. (September 2005 c). "Water and energy savings at a kraft paperboard mill using process integration." Pulp & Paper Canada 106(9): 29 -31 Towers, M. (March 2005). "Energy reduction at a kraft mill: Examining the effects of process integration, benchmarking, and water reduction,." Tappi Journal 4 (3): 15 - 21. Wising, U., T. Berntsson, et al. (2005). "The potencial for energy savings when reducing the water consumption in a Kraft Pulp Mill." Applied Thermal Engineering 25: 1057 - 1066. Nordman, R. and T. Berntsson (2006). "Design of kraft pulp mill hot and warm water systems- A new method that maximizes excess heat." Applied Thermal Engineering 26: 363 - 373. Parthasarathy, G. and G. Krishnagopalan (2001). "Sistematic reallocation of aqueous resources using mass integration in a typical pulp mill." Advances in Enviromental Research 5 61 - 79. Lovelady, E. M., M. El-Halwagi, et al. (2007). "An integrated approach to the optimisation of water usage and discharge in pulp and paper plants." International Journal of Environment and Pollution 2007 29(No. 1/2/3): 274 - 307. 266 Environmental Management in Practice Savulescu L. E., A. Alva-Argáez, Direct heat transfer considerations for improving energy efficiency in pulp and paper Kraft mills, Energy, 33(10) (2008), 1562-1571. y p p p p gy ( ) ( ) European Commission (2001). "BREF in the Pulp and Paper Industry". y Westerberg A. W., H.P. Hutchinson, R.L. Motard y P. Winter (1979). "Process Flowsheeting", Cambridge Univ. Press, Cambridge, England. g g g Shenoy U. V. (1995)."Heat Exchanger network Synthesis", Gulf Publishing Co. 1995 g g g Shenoy U. V. (1995)."Heat Exchanger network Synthesis" Douglas J. (1988), "Conceptual Design of Chemical Processes". Mc Graw-Hill Co. Walas S.M (1988) Chemical Process Equipment Selection and Design, Butterworths Gullichsen J., C.J. An Application Model for Sustainability in the Construction Industry Fernando Beiriz and Assed Haddad Federal Fluminense University and Federal University of Rio de Janeiro Brazil Fernando Beiriz and Assed Haddad Federal Fluminense University and Federal University of Rio de Janeiro Brazil Fernando Beiriz and Assed Haddad Federal Fluminense University and Federal University of Rio de Janeiro Brazil Brazil 7. References Fogelholm, Papermaking Science and Technology – Chemical Pulping, Fapet OY, Helsinki, 1999. Peters, M., Timmerhaus, K. 1991. Planta design and economics forchemical engineers. 4. ed. Mc.Graw Hill. Nueva York, NY. EEUU. R. Smith, Chemical Process Design and Integration, John Wiley & Sons Ltd., Chichester, 2005. Jacob, J., H. Kaipe, et al. (2002). "Water network analysis in pulp and paper processes by pinch and linear programming techniques." chemical Engineering Communications 189(2): 184 - 206. ( ) Koufos, D. and T. Retsina (2001). "Practical energy and water management through pinch analysis for the pulp and paper industry." Water Science Technology 43 (2): 327 - 332. Sumathi S., Yung-Tse Hung ,(2006). Treatment of Pulp and Paper Mill Wastes. Treatment in the Process Industries. (Editores: Wang, L.K., Hung Y., Lo H.H., Yapijakis, C.). Editado por Taylor and Francis. Pp 453-497. 13 1. Introduction 268 Environmental Management in Practice The important thing to be improved in this sector is the management process, with the decrease in solid waste generation and appropriate management of the same construction site, building awareness of the actors involved, creating the methodology. The important thing to be improved in this sector is the management process, with the decrease in solid waste generation and appropriate management of the same construction site, building awareness of the actors involved, creating the methodology. It is noteworthy that is necessary a change of culture among all those involved in the process of IC, indicating the importance of preserving the environment we live. Therefore, it is notorious the necessity of a mentality change in the aspect of environmental sustainability at the IC sector’s stakeholders, in order to fortify and develop a responsible conduct, aware of the relevance of preserving and extracting as better as possible the environment’s resources. The important thing to be improved in this sector is the management process, with the decrease in solid waste generation and appropriate management of the same construction site, building awareness of the actors involved, creating the methodology. It is noteworthy that is necessary a change of culture among all those involved in the process of IC, indicating the importance of preserving the environment we live. Therefore it is notorious the necessity of a mentality change in the aspect of environmental The important thing to be improved in this sector is the management process, with the decrease in solid waste generation and appropriate management of the same construction site, building awareness of the actors involved, creating the methodology. It is noteworthy that is necessary a change of culture among all those involved in the process of IC, indicating the importance of preserving the environment we live. Therefore, it is notorious the necessity of a mentality change in the aspect of environmental sustainability at the IC sector’s stakeholders, in order to fortify and develop a responsible conduct, aware of the relevance of preserving and extracting as better as possible the environment’s resources. 1. Introduction Over the years, mankind’s development of a large industrial capacity and its ability to create new technologies that turn easier society’s daily life has been a mark of innovation era. In many developing industries, technologies are incorporated into daily life by becoming indispensable to the modern lifestyle. Waste production has been increasingly alarming throughout the world, standing as a major problem to be solved.In order to achieve life quality and be able to provide favorable environmental conditions to future generations, it is indispensable to become conscious about environmental effects of all mankind’s production activities. It is vital to promote and encourage an environmental sustainability culture development: meeting society’s demand of industrial and technological products with the indispensable proper disposal of their products at the end of life, that is, discard minimizing environmental impacts on the completion of its life cycle. Some measures have been taken over recent years, with the intention of minimizing the generation of environmentally hazardous waste in the world, emphasizing the relevance of changes in production processes. In the specific case of construction, begins to be aroused interest from external factors. Among them, there is the availability of solutions to minimize negative environmental impacts identified and applicable management tools. Methods for evaluating environmental performance of the construction industry and increased competition in the industry and customer requirements are also seen as elements boosters, which come to be added to increase environmental awareness at the part of builders. Similarly, as many construction companies have implemented quality management systems that have brought them considerable benefits, it increases their interest in introducing environmental elements into existing systems. However, there are few builders that are committed to environmental issues. Still, environmental solutions have begun to be applied in enterprises, although this does not ensure continuous improvement and sustainable development of the sector. Despite its recognized economic impacts to the country such as: high job creation, income and viability of housing, infrastructure, roads and others; in the construction sector one still lacks a firm policy for disposal of solid waste, mainly in urban centers. The need to take the RCC not only results in a desire to economize. This is a fundamental attitude towards the preservation of our environment. 2. Construction industry sustainability In extreme ecocentric the authors emphasize 269 An Application Model for Sustainability in the Construction Industry the importance of natural capital and the need to preserve it, I value not only for financial but mainly for its substantive value. the importance of natural capital and the need to preserve it, I value not only for financial but mainly for its substantive value. Ecological sustainability means to expand the capacity of the planet by using the potential found in diverse ecosystems, while the continuing deterioration in a minimum level. Ecological sustainability means to expand the capacity of the planet by using the potential found in diverse ecosystems, while the continuing deterioration in a minimum level. It should reduce fossil fuel use and emission of pollutants, but also adopt policies for the g y p p y p y g p found in diverse ecosystems, while the continuing deterioration in a minimum level. It should reduce fossil fuel use and emission of pollutants, but also adopt policies for the conservation of energy and resources to replace. y g It should reduce fossil fuel use and emission of pollutants, but also adopt policies for the conservation of energy and resources to replace. The geographical sustainability can be achieved through a better distribution of human settlements and economic activities. It must seek a rural-urban setting most appropriate to protect biological diversity, while it improves the quality of life. Finally, cultural sustainability, the most difficult to bring the second SACHS (1997), is related to the path of modernization without the disruption of cultural identity within specific spatial contexts. To SACHS (1997), the concept of sustainable development refers to a conception of the limits and the recognition of the weaknesses of the planet; focuses on both the socioeconomic problem and satisfying the basic needs of populations. Although the starting point of the various approaches is different, there is a recognition that there is a space of interconnection or overlap between these different dimensions. p p Achieve progress toward sustainability is clearly a choice of society, organizations, communities and individuals. How covers different choices, change is only possible if there is greater involvement of society. In short, sustainable development requires the society to think in terms of long-term and recognize its place within the biosphere. 2. Construction industry sustainability The concept provides a new perspective of observing the world, which has proven to be the current state of human activity inadequate to meet existing needs, and seriously threaten the prospect of future generations. The goals of sustainable development challenge contemporary institutions. They have governed global changes reluctant to recognize that this process is actually occurring. The differences in the concept of sustainable development are so great that there is no consensus on how to measure sustainability. Unfortunately, for most authors cited earlier, does not have an operational definition of minimally acceptable. All definitions and tools related to sustainability must consider the fact that no one knows fully how the system operates; one can only discover environmental impacts of activities and interaction as human welfare, the economy and the environment. In general, it is known that the system interacts between different dimensions, but do not know specifically the impact of these interactions. All aspects presented show the diversity and complexity of the term sustainable development. 2. Construction industry sustainability The term sustainable development can be seen as a key word this time. As there are numerous definitions for this term, the two most common definitions known, cited and accepted are the Brundtland Report (WCED, 1987) and the document known as Agenda 21. The best known definition of the Brundtland Report, presents the question of future generations and its possibilities. It contains two key concepts: the necessity and the idea of limitation. The first refers particularly to the needs of developing countries and, second, the idea imposed by the state of technology and social organization to meet the needs of present and future. The question of emphasis on the social component of sustainable development is reflected in the debate taking place about the inclusion or not of social measures in the definition. This discussion appears in the variety of ideas about sustainability that contains components that are not usually measured, such as cultural and historical. Social indicators are considered particularly controversial, since they reflect political contexts and value judgments. The integration of mitigation measures is further complicated because of different and often conflicting dimensions. The definition of the Brundtland Report does not provide a static state, a more dynamic process that can continue to exist without self-defeating logic prevailing. The different forces acting on the system must be in balance for the system as a whole is maintained over time. According to Pearce (1993), there are different environmental ideologies that make environmentalism a complex and dynamic phenomenon. Inside of environmentalism, the author identifies two ideological extremes: on one hand the technocentrism, and the other the “ecocentrism”. Within this continuum one can identify four fields, with particular characteristic. Pearce uses four classifications: sustainability very weak (very weak sustentability), weak sustainability (weak sustentability), strong sustainability (strong sustentability) and sustainability very strong (very strong sustentability). You can also find a parallel Naess (1966) makes between Deep Ecology (deep ecology) and ecology superficial (shallow ecology). In ecology the central objective is superficial affluence and health, along with the fight against pollution and resource depletion. Focus on deep ecology focuses on biospheric egalitarianism and the principles of diversity, complexity and autonomy. Authors linked the trend technocentric believe that sustainability refers to the maintenance of total capital available on the planet and that it can be achieved by substituting natural capital for capital created by human ingenuity. 3. Reverse logistics and waste management The high competition among companies and constant increase in efficiency in the management processes of production, has characterized the current business environment. Among the many processes present in a company, there is the logistics business, which is geared to ensure the delivery of the product produced correctly in the right place at the moment and want the lowest possible cost. In many industries, logistics has received more attention, mainly due to the globalization of markets and consumer pressure to reduce distribution costs. The client, in turn, is embedded in consumer culture, which is driven by the cycle "buy-use- disposal", demonstrating that culture is unsustainable and inadequate to perishable 270 Environmental Management in Practice perpetuation of current conditions for survival in contemporary society, because it stimulates the increasing manufacture of new products to the detriment of reuse and recycling of byproducts or waste. perpetuation of current conditions for survival in contemporary society, because it stimulates the increasing manufacture of new products to the detriment of reuse and recycling of byproducts or waste. Thus it is observed that actions to boost consumption are not planned with a systemic view, since their products are not useless options, structured reuse, leaving only the landfilling as a solution to dispose of them. In this scenario, reverse logistics, or more precisely the deployment of reverse logistics gains importance in the supply chain. The structure of the reverse channel is a way to make new use of these products, through a new job or a transformation of industrial processing, in other useful products. Thus, reverse logistics has a great interface with sustainable development, since the mobilization of the chains allows the reuse of reverse obsolete products, byproducts and waste, reducing the volume of discarded into the environment and the extraction of new resources. It also presents another favorable feature, since the emergence of new business also promotes the social, financial returns and allows companies involved in chains reverses. Particularly in the construction industry, reverse logistics systems are designed to develop reverse chain for reuse of products and waste generated in production processes and establish the agents working in it, the census of responsibility throughout the product life cycle. This attitude is shared not only by builders but, especially, by supplying materials for these are in an industrial environment, where there is less variability of the process. 3. Reverse logistics and waste management Quantifying the generation of RCD is complex because it involves the collection of field data, since there are no precise data , nor indicators released. The generation relies heavily on project design and technologies used, the organization of the plot, containers for packaging of the various "bumps" of waste, and vary according to the stage of the work. Quantifying the generation of RCD is complex because it involves the collection of field data, since there are no precise data , nor indicators released. The generation relies heavily on project design and technologies used, the organization of the plot, containers for packaging of the various "bumps" of waste, and vary according to the stage of the work. Become evident throughout the academic, claims that the IC, as well as other industrial chains, must promote sustainable development, ie, it must develop in order to not compromise the ability of future generations to do it too. Among the many issues involved in policies for sustainable development of the productive chain of the IC in relation to environmental and social dimensions, are responsible for the use of natural resources and disposal of waste from industrial activities. p g g p y g g Become evident throughout the academic, claims that the IC, as well as other industrial chains, must promote sustainable development, ie, it must develop in order to not compromise the ability of future generations to do it too. Among the many issues involved in policies for sustainable development of the productive chain of the IC in relation to environmental and social dimensions, are responsible for the use of natural resources and disposal of waste from industrial activities. 4. Brazilian environmental legislation general requirements for construction companies There are several Brazilian environmental legislation aspects that affect the Construction Industry and construction companies operations in Brazil. The Waste Management Program for construction sites and Environmental Impact Assessment Program with respective Report and License are the most important items to be taken care. All construction sites demand waste management attention although this Environmental Impact Assessment Program and Report (Estudo de Impacto Ambiental - EIA/RIMA) are only mandatory in special cases. 3. Reverse logistics and waste management Thus, these companies can become drivers of implementation of this concept throughout the production chain construction. In the construction sector, it is assumed that interest is still incipient and demonstrated by a few industries, as are the Brazilian initiatives for the reuse of industrial waste. Applying the concept of reverse logistics in IC may occur in several ways. It can form themselves into organizational tool for the flow of aftermarket products, post-consumer waste from the production process of mobilization and demobilization of equipment used during construction of the project, and set yourself up as a new initiative or as an enhancement of existing reverse channel. g Specifically, with regard to flows, the amount and variability of waste composition of the construction industry generate flows of very different characteristics. IC flows in post-consumption and production (waste) are hardly distinguished, because they occur simultaneously, except when the demolition of a building, a notoriously product stream after consumption. Flows of products after sale are mainly for returns sent by mail-order and are usually intended for the secondary market, which, for example, donated to charity. Still others come from equipment and transportation as the return and withdrawal of lifts and cranes. q p p The biggest concern now rests on the post-consumer products or processes, generally named construction waste. Applying the concept of reverse logistics in IC may also have coverage from a company in isolation, this and its supply chain, as well as sectored organization, or the entire production chain (the reverse supply chain). When the reverse logistics systems of IC are shared by all actors in the chain and these are strategic objectives aligned on the reuse of reverse flow, consolidates the management of reverse supply chain (reverse supply chain management). 271 An Application Model for Sustainability in the Construction Industry The reverse logistics systems are formed by flows, distribution channels, reverse, or simply reverse channels. In this study of reverse logistics systems IC correspond to the flows of waste from construction and demolition-RCD and its reverse channels. Flows of post-consumer products industry, construction and demolition have their roots in construction sites. In this environment has recently developed some RCD management initiatives. The analysis of the requirements of laws versus the needs of the construction leads to the conclusion about the actions necessary for the establishment of a reverse logistics system for the RCD. 4.1 The waste management program This waste management program aims at the reduction of waste production and correct destination of what remains in activities involving in construction, retrofitting, remodeling, maintenance and demolition in all types of construction related activities and subsectors of the Construction Industry. Table 1. shows Brazilian Construction Waste Classification according to the legislation and its respective destinations. Reuse is the process of reapply some waste without transforming itself and Recycling is the process of reapply some waste after some transformation. These are possible final destination of A and B waste classes. Class A wastes, before reuse and recycle, can be stored in Building Construction landfills. In these sites special disposal storage techniques are used having in mind preservation of these segregated materials for its future use or the use of the land itself throughout application of some engineering principles to confine them. The Construction Industry Waste Management Integrated Plan (Plano Integrado de Gerenciamento de Resíduos da Construção Civil) structured as shown above. 272 Environmental Management in Practice Construction Waste Classification Classification Characteristics Examples Destination Class A Reusable waste or recyclable as aggregate Brick blocks and tiles, dirt, concrete, mortar Reused or recycled Class B Recyclable Plastics, paper/cardboard, metal, glass, wood pieces Class C Not existent technology for recycling Gypsum Stored, transported and disposed in conformity with specific standards. Class D Hazardous Waste Coats, solvents, oil Table 1. Brazilian Construction Waste Classification Fig. 1. Construction Industry Waste Management Integrated Plan Scheme. Construction Industry Waste Management Integrated Plan Construction Industry Waste Management Municipal Program Construction Industry Waste Management Projects Implemented and coordinated by citiy governments Establish technical standards and procedures for small waste generators responsabilities according to established rules Elaborated and implemented by generators Objectives the establishment of necessary environmental adequate procedures for handling and destinaton of waste Follow these steps:  caracterization;  segregation;  condittioning;  transport and  destination Construction Waste Classification Classification Characteristics Examples Destination Class A Reusable waste or recyclable as aggregate Brick blocks and tiles, dirt, concrete, mortar Reused or recycled Class B Recyclable Plastics, paper/cardboard, metal, glass, wood pieces Class C Not existent technology for recycling Gypsum Stored, transported and disposed in conformity with specific standards. Class D Hazardous Waste Coats, solvents, oil Table 1. Brazilian Construction Waste Classification Table 1. Brazilian Construction Waste Classification Table 1. Fig. 1. Construction Industry Waste Management Integrated Plan Scheme. 4.2 Environmental impact assessment program and report An Environmental Impact Assessment Program and Report is mandatory for CC seeking construction licenses for construction sites in which considerable environmental impacts will happen, such as: An Environmental Impact Assessment Program and Report is mandatory for CC seeking construction licenses for construction sites in which considerable environmental impacts will happen, such as:  Roads with two or more lanes;  Railroads;  Ports and terminals for oil and gas, chemicals products and mining;  Aeroports;  All types of pipelines including sewage, oil, gas, mining and others;  Power lines, beyond 230KV;  Water resources facilities including Hydro Plants beyond 10MW, irrigation works, sewers, navigation channels, etc;  Fossil fuels extraction;  Mining extraction;  Sanitary landfills, toxic or hazardous;  Power Plants generating more than 10MW;  Industrial and agricultural units and complexes;  Industrial districts and strictly industrial zones;  Wood exploration in large areas or in some subject to special environmental interest;  Urban Projects in large areas or in some subject to special environmental interest;  Any activity use coal from vegetal sources in excess to ten tons a day.  Canals and Harbour structures The Environmental Impact Assessment must: I - evaluate all technological and location alternatives for the project including the possibility of non development; II - identify and evaluate systematically all environmental impacts taken place during the implementation and operation phases of the project; III – determine the project directly and indirectly affected area geographic boundaries subject to environmental aspects and impacts of the project (denominated project influence area); IV - consider governmental proposed pans and programs for the project influence area and their compatibility.  Roads with two or more lanes;  Ports and terminals for oil and gas, chemicals products and mining;  Aeroports;  All types of pipelines including sewage, oil, gas, mining and others;  Water resources facilities including Hydro Plants beyond 10MW, irrigatio sewers, navigation channels, etc;  Sanitary landfills, toxic or hazardous; Power Plants generating more than 10MW;  Industrial and agricultural units and complexes;  Industrial districts and strictly industrial zones; y Wood exploration in large areas or in some subject to special environmental interest; Urban Projects in large areas or in some subject to special environmental interest;  Any activity use coal from vegetal sources in excess to ten tons a day. An Application Model for Sustainability in the Construction Industry This plan must have:  Technical standards and procedures for the Construction Industry Waste Management Municipal Program and the Construction Industry Waste Management Projects, elaborated by large waste generators, aiming the creation of a sense of responsibility by all generators;  Mapping of public or private areas, suitable for receiving, segregation a temporary storage of small waste volumes, according to urban municipal zoning. This allows further destination for waste management plants or recycling;  Mapping of public or private areas, suitable for receiving, segregation a temporary storage of small waste volumes, according to urban municipal zoning. This allows further destination for waste management plants or recycling;  Establishment of licensing procedures for areas of processing and final waste destination;  Determination of prohibition of disposal in non licensed areas; Incentives towards reuse or recycling;  Determination of parameters and criteria for registration of waste transportation companies;  Environmental education actions towards waste management of construction was 4.1 The waste management program Brazilian Construction Waste Classification Construction Industry Waste Management Integrated Plan Construction Industry Waste Management Municipal Program Construction Industry Waste Management Projects Fig. 1. Construction Industry Waste Management Integrated Plan Scheme. 273 An Application Model for Sustainability in the Construction Industry 4.2 Environmental impact assessment program and report The Environmental Impact Assessment must: I - evaluate all technological and location alternatives for the project including the possibility of non development; II - identify and evaluate systematically all environmental impacts taken place during the implementation and operation phases of the project; III – determine the project directly and indirectly affected area geographic boundaries subject to environmental aspects and impacts of the project (denominated project influence area); IV - consider governmental proposed pans and programs for the project influence area and their compatibility. 274 Environmental Management in Practice 4.3 Environmental licenses 4.3 Environmental licenses The Environmental License is an administrative act by which the environmental agency, establish conditions, restrictions and environmental control actions to be followed by companies and enterprises seeking construction, installation, addition and operation of projects and activities that which to use natural resources, with the potential to harm or affect the environment. The following construction activities demand licensing in Brazil:  Highways, railroads, subways and waterways;  Barrages and levees;  Drainage channels;  Water courses rectification;  Opening of channels, enlargement of rivers  Transposition of river basins;  Other special works.  Highways, railroads, subways and waterways;  Highways, railroads, subways and waterways;  Barrages and levees;  Drainage channels;  Water courses rectification;  Opening of channels, enlargement of rivers  Transposition of river basins;  Other special works.  Barrages and levees; ENVIRONMENTAL LICENSING License type Characteristics Validity Previous License Licença Prévia (LP) •Preliminary or in the planning phase of the project • Approves localization and concept • Ensures environmental availability • Basic requisites and conditions to fulfill in further phases towards project implementation Minimum: according to what was scheduled in the activity or project approved plans, programs and designs Maximum: 5 years Operation License “Licença de Operação (LO)” • Authorizes installation according to specifications from approved plans, programs and designs • Includes environmental control actions and conditions Minimum: according to what was scheduled in the activity or project Maximum: 6 year Installation License “Licença de Instalação (LI)” • Verifies effective accomplishment of previous licenses, conditions and environmental control plans for the operation • Authorizes the activity or project operation Should consider the environmental control plans Minimum: 4 years Maximum: 10 years Table. 2 Brazilian environmental Licenses. ENVIRONMENTAL LICENSING Table. 2 Brazilian environmental Licenses. The Brazilian Environmental Criminal Law (Brazilian Federal Law 9.605/98) was an important mark that determined higher attention in licensing activities. It determines that “Build, restore, addition, install or operate, in any part of the country, establishments, construction sites or services potentially hazardous without license or authorization from environmental agencies or against the rules and regulations ins unlawful and is subject to imprisonment from one to six months and fine”. 275 An Application Model for Sustainability in the Construction Industry 5.1 Principles of motion Any model to be functional and efficient it has to rely on a set of interdependent and harmonious elements, rules and procedures. In the proposal in focus, we list the main points and actions that should be considered: and actions that should be considered: and actions that should be considered:  Clear and comprehensive legislation - the recent National Policy on Solid Waste culminating in Brazil in August, 02 of 2010, is a great motivator to take seriously the treatment of waste from the construction industry. But, it is necessary that the state and local public authorities commensurate with their organic laws that policy, clearly and objectively, and promote a public-private partnership, to put into practice the recycling of construction debris in their areas of coverage ;  Effective supervision - one of the major problems faced by municipalities is the illegal dump sites and on public roads, including transport companies themselves accredited. It is essential to pursuing a proactive surveillance for the balance of the process, using modern technology, such as control by GPS;  Existence of incentives for products and services involved in the process - is important, for example, that recycled materials are treated with different taxes in relation to new products;  Existence of penalties for violating a law by service providers and generators of rubble - the penalties should be meaningful in order to promote greater accountability of individuals and companies in the process of disposal of construction waste, in favor of environmental control and panorama of cities; p  Encouraging the use of modern techniques and methodologies for building large projects in order to reduce the debris - debris is often generated by deficiencies in the construction process, such as failures or omissions in the preparation of projects and their implementation, poor quality of materials employees, for losses in transport and storage, improper handling by the workforce, as well as replacement components for the reform or reconstruction. 5. A model for sustainability in the construction industry The model presented here will apply for technical and economic issues, the major producing areas of waste. These regions represent large urban centers or may result from the formation of a conglomerate or consortium of adjacent municipalities, bringing together the legislation compatible. Importantly, the current stage of the construction industry in Brazil, already in itself justify the existence of a rigid model of treatment and recovery of debris from the construction industry in all regions of the country This scenario will be aggravated and may even be become untenable, with the advent of achievement, in Brazil, Football World Cup in 2014 and the Olympics in 2016, when major works and demolitions have to be made, generating an abnormal amount of debris. Moreover, the practice of waste treatment of IC is very incipient in the country, and even negligible in the State of Rio de Janeiro, where he will focus the Olympic Games of 2016. 5.1 Principles of motion Improved management and control of works, use of modulation techniques and also joint work with companies and construction workers can help to alleviate this waste;  The whole region should be provided with one or more treatment plants and waste processing, depending on the volume to be processed;  The whole region should be provided with one or more treatment plants and waste processing, depending on the volume to be processed;  Strategic location of collection points and disposal of debris (Ecopoints) for small and medium-sized generators of rubble; 276 Environmental Management in Practice  Area of Transshipment and Triage (ATT), which is the equivalent of an Eco Center for the receipt of large volumes of debris, from large generators;  Area of Transshipment and Triage (ATT), which is the equivalent of an Eco Center for the receipt of large volumes of debris, from large generators; g g  Location of areas of rubble landfill officials IC;  Implementation of policies for environmental management and waste treatment in large generators, such as construction and demolition;  Implementation of policies for environmental management and waste treatment in large generators, such as construction and demolition;  Specialized transportation network;  Educational campaign at all levels, including the population in general - is to clarify and encourage the integration of the self in the process.  Educational campaign at all levels, including the population in general - is to clarify and encourage the integration of the self in the process. 5.2 Operational architecture model The following Figure illustrates the components involved in the model for treatment of wastes from the construction industry, as well as the flow routing in each of the elements produced at each location. Fig. 2. Management architecture of the rubble IC 5.2.1 Small and medium generators of the debris of the IC and Ecopoints According to the definition given in Resolution No. 307 of CONAMA, generators are individuals or entities responsible for activities or enterprises that produce construction waste. Constitute small and medium-sized generators, for example, construction projects and reforms implemented in commercial or residential units of small or medium size. Fig. 2. Management architecture of the rubble IC Fig. 2. Management architecture of the rubble IC 5.2.1 Small and medium generators of the debris of the IC and Ecopoints According to the definition given in Resolution No. 307 of CONAMA, generators are individuals or entities responsible for activities or enterprises that produce construction waste. Constitute small and medium-sized generators, for example, construction projects and reforms implemented in commercial or residential units of small or medium size. 277 An Application Model for Sustainability in the Construction Industry The existence and management of eco-points are essential for efficient control of collection of debris from construction, to avoid dropping these, irregular, illegal or inappropriate points. p Ecopoint sites are provided, usually by public authorities, waste disposal in a voluntary and free basis. They can be made simply by buckets, properly prepared by a land or a house, always located near point of generation potential, and easy access. y p g p y Figure 3 shows some examples of existing Ecopoints in the State of São Paulo. Fig. 3. Ecopoint Bresser and Ecopoint Pinheiros Fig. 3. Ecopoint Bresser and Ecopoint Pinheiros The process for using these services is simple: just take the waste from construction such as cement, bricks, tiles, plaster, wood and other debris from construction, Ecopoints. It is anticipated with the disposal of debris up to 1m 3 cubic volume per user per day, equivalent to roughly 25% of a bucket. If the construction or renovation generates a tremendous amount to be disposed of more than 200 liters, it will be needed to hire a company specialized in the collection services and transportation of debris. 5.2 Operational architecture model As the debris in Ecopoints received, from small and medium generators are generally very impure, must be carefully separated, to be given the correct destination for each type of material found in them. Therefore, they should be compulsorily transferred to a triage area and Transshipment of Waste (TTA) for treatment. 5.2.2 Large generators of the debris of IC g g Large debris generators generate over 1m ³ of waste from construction or demolition. Are those that require buckets to carry your trash. Usually they are responsible for construction and remodeling of large, for example contractors, builders and technicians responsible for works. The big generators are responsible for disposal of rubbish they generate. In such cases it is necessary to hire a transport company of construction waste. y p p y In the case of buildings with more than 500m ², the generator must develop and deploy rubble in building site, a Waste Management Program of Construction and prove that the waste generated was disposed of in an environmentally correct. g p y The waste in construction site varies according to the execution phase of services. Much of the debris generated during the entire construction can be used as aggregate in various 278 Environmental Management in Practice stages of the building, considering, however, that the mineral fraction (cementitious material and ceramic) is the only one to be recycled and used on the construction, the other fractions as wood, metal, plaster, plastics and paper, should be directed to the appropriate local recycling or disposal of these materials, such as ATT's and Plants. Other components can even be sold or donated for reuse in the case of demolition, such as doors, windows, bathrooms and kitchens metals, etc. Therefore, it is necessary to be aware of the entire site, followed by pre-established procedures for the use and destination of the waste. First you must establish debris generated separation: ceramic and cement, wood, contaminants, metals, plastics and paper, for example. Each fraction will have its place of deposit in the quarry. This separation plot is not complex, because the debris is generated by separate activities, such as the use of mortars have will only cementitious material and other activities on the debris will only generate carpentry wood. The non-recyclable rubbish is disposed off on site, while recyclable rubbish is processed subsequently disposed. Fig. 4. Segregation of waste at the construction site In large enterprises, it is sometimes advantageous and desirable that the machining is done on the construction work. In this case, for crushing are generally used small equipment, with an average production of about 2m3 per hour, with power and manual removal of the products. Equipment is simple and easy to use, where: mortar-mill, hammer mill, grinder or plaster jaw crusher. 5.2.2 Large generators of the debris of IC B id i h i h f li i i Fig. 4. Segregation of waste at the construction site In large enterprises, it is sometimes advantageous and desirable that the machining is done on the construction work. In this case, for crushing are generally used small equipment, with an average production of about 2m3 per hour, with power and manual removal of the products. Equipment is simple and easy to use, where: mortar-mill, hammer mill, grinder or plaster jaw crusher. In large enterprises, it is sometimes advantageous and desirable that the machining is done on the construction work. In this case, for crushing are generally used small equipment, with an average production of about 2m3 per hour, with power and manual removal of the products. Equipment is simple and easy to use, where: mortar-mill, hammer mill, grinder or plaster jaw crusher. p j Besides improvements on the environment, the management of recycling at construction site brings good economic advantages, such as: 279 An Application Model for Sustainability in the Construction Industry  Reducing the volume of rubbish sent to ATT or plant, reducing the cost of removal; Reducing the volume of rubbish sent to ATT or plant, reducing the cost of removal;  Reducing the volume of rubbish sent to ATT or plant, reducing the cost of removal;  More organized and clean site;  Reducing the volume of rubbish sent to ATT or plant, reducing the cost of removal;  More organized and clean site;  More organized and clean site;  Reduced acquisition of aggregate material; The large main emphasis is to recycle the rubble in the works is the financial aspect: do not waste material already paid and still be able to produce products with low costs are rather compelling reasons. As an example we have that the projected cost per cubic meter of mortar with recycled material is around U$ 36, while a cubic meter of conventional mortar is US $ 62.00. 5.2.4 Landfills of waste places p In most cases, the debris is removed and disposed of the work clandestinely in places like vacant lots, riverbanks and streets of the suburbs. The social and environmental cost of this is beyond the control of the calculations, although its consequences are permanently noted. We can see the deterioration of quality of life in urban areas such as transport, flood, visual pollution, proliferation of disease vectors, among others. One way or another, the whole society suffers from the uneven deposition of debris. The debris is residue from a large volume, occupying so much space in landfills, transportation, depending not only on volume but the weight, it becomes expensive. Recycling and reuse of rubble are therefore of fundamental importance for the control and mitigation of environmental problems caused by the generation of waste. The existence of authorized local landfill dump in the context of this proposal is due to the fact that some debris from work, or certain residues after machining or segregation from the rubble at the ATT, is not be recovered. Have to be discarded, and this time it is important that they are received at licensed sites and specific for this purpose. The landfill is now the most widely used solution for its ease of implementation and others. But still has a very high environmental cost, and some administrators end up not respecting the rules or find other alternatives. When implementing the standards are not met, the sanitary landfill is no longer and begins to set up the so-called dump. A plausible alternative is the separation of waste into inert and non-polluting material (domestic waste, commercial, industrial and hospital) and material (waste derived from The landfill is now the most widely used solution for its ease of implementation and others. But still has a very high environmental cost, and some administrators end up not respecting the rules or find other alternatives. When implementing the standards are not met, the sanitary landfill is no longer and begins to set up the so-called dump. y g g p p A plausible alternative is the separation of waste into inert and non-polluting material (domestic waste, commercial, industrial and hospital) and material (waste derived from construction). This alternative, while reducing costs, since the landfill for inert material is cheaper than landfill, allows it to be used mainly in projects that address the reuse and recycling of such materials. 5.2.3 Area overflow and waste segregation (ATT) Transshipment Areas and Waste Sorting of Construction (ATT) are the premises for the receipt of bulky construction waste collected by private agents, which should be used for screening of incoming materials, processing and any subsequent removal for proper disposal. So are sites used for routing and segregation of waste for disposal. ATT is typically a business that belongs to the autonomous initiative of collecting small businesses or cooperatives, and are deployed and operated by observing the law of municipal land use and occupation, as well as federal and state legislation to control environmental pollution when appropriate. As these are areas, in the context of the proposed architecture, are administered by the private sector, generating jobs and revenues for receiving debris from the construction and sale of sorted waste, which could enable an effective overview for the efforts that are being developed in favor of a sustainable environment. This initiative is breaking old paradigms, showing that waste reduction can be combined with cost reduction, combining behavior change in various work to build partnerships with various vendors, abolishing the provision is irresponsible outlaws illegal boot through committed the allocation of each component of sorted waste, so that the responsibility to the environment that anchors the economic activity is exercised. The following picture illustrates an overview of the Transfer Area and Screening. Fig. 5. ATT in the outskirts of Guarulhos in Sao Paulo Fig. 5. ATT in the outskirts of Guarulhos in Sao Paulo 280 Environmental Management in Practice 5.2.4 Landfills of waste places This idea becomes valid once the aggregates are a major source of raw material at a relatively low cost. Fig. 6. Landfill construction debris (Hall BH) Fig. 6. Landfill construction debris (Hall BH) 281 An Application Model for Sustainability in the Construction Industry 5.2.5 Treatment plants and processing waste IC In the current global context is essential to improve the construction processes. However, recycling of rubbish comes as a solution to the materials that are inevitably lost. Recycling allows the reuse of raw materials, reducing the demand for more material, reducing energy consumption and protecting the environment more and more waste, which would take millions of years to be decomposed by nature. Recycling becomes disordered the mountains of building materials, piles of raw material, which serves both as building works for public works. There are two ways to turn losses into profits: one for the private sector and another for local governments. The process of recycling the rubble for obtaining aggregates basically involves the selection of recyclable materials from rubbish and grinding in their proper equipment. The screening phase of an ATT must proceed independently or integrated into the processing plant. The recycling of rubble can be done at facilities with different characteristics for equipment used which may be mobile or fixed. Fig. 7. Plant equipment: a set vibrating feeder jaw crusher + + belt Fig. 7. Plant equipment: a set vibrating feeder jaw crusher + + belt 5.2.6 Logistics of moving the rubble The debris generated by small works must necessarily be disposed in Ecopoints. Therefore, their transport should be done by an independent group of carriers, using vehicles intended for freight, cars and even wheelbarrows. Optionally, the service of withdrawal of small volumes of debris generated in small works may be offered by the city. In this case, the municipality may dismiss them Ecopoint a neighbor, or even carry an ATT. The debris generated by small works must necessarily be disposed in Ecopoints. Therefore, their transport should be done by an independent group of carriers, using vehicles intended for freight, cars and even wheelbarrows. Optionally, the service of withdrawal of small volumes of debris generated in small works may be offered by the city. In this case, the municipality may dismiss them Ecopoint a neighbor, or even carry an ATT. The transportation of debris from the ecopoint, should be done by the municipality or by an outside company for her. Should be discarded in an ATT, because it is a very heterogeneous rubble. The transportation of debris from the ecopoint, should be done by the municipality or by an outside company for her. Should be discarded in an ATT, because it is a very heterogeneous rubble. 282 Environmental Management in Practice For works of medium sized businesses that generate significant amounts of debris, transport must be hired by the generator, companies accredited by the Government, working with the transport of rubble multi cranes using trucks and dumpsters. In this case, the debris must be disposed directly in ATT, because it is a dump, as a rule, heterogeneous, with a disposal cost based on volume, the carrier paid to ATT. In large constructions and demolitions, debris intended for disposal should be conducted at a plant where have characteristics suitable for recycling at no cost by the receiving plant. When the debris is heterogeneous, an ATT should be discarded with the burden of the cost of disposal by the generator. The transfer of rubble in an ATT and screened for the disposal shall be transported to a recycling plant to the landfill or dump, depending on their character, on behalf of the Government, that you can do so directly or through third party service . The waste is not usable in a plant should be discarded in a landfill dump, under the responsibility of the plant. 6. Validation of the model The economic infeasibility of a CDR of recycling is often a logistical problem results mainly from the high cost of collection and transportation arising from the existence of many points of consumption and few points of waste recovery. The main objective of the recycling center regardless of whether a, private or public, is to diminish the distance between the product and its potential buyers. Consequently, this reduces the environmental and economic impacts arising from that transport. Efforts can be properly rewarded by the possibilities of raw material savings - as demonstrated in the analysis of economic viability, partnership with large generators, acting responsibly towards the environment and differential image in the marketplace. The simulations of economic viability analysis proved that, currently, there are conditions for the economic viability of waste recycling of ICC. The most appropriate model is the one that would establish co-responsibility, including environmental liabilities, among the managers of plants and ATT, waste generators and transporters. The manufacturer must develop, together with research institutions, appropriate disposal options and the developer must ensure that the flow of waste will be properly addressed to the appropriate places. 7. Conclusions Government agencies need to establish laws, which define: Government agencies need to establish laws, which define: Government agencies need to establish laws, which define:  Responsibilities and joint responsibilities of each agent on the management of RCD;  Forms of surveillance and punishment of its fulfillment;  A ban on the disposal of certain wastes at landfills, especially those based on plaster;  Tax on the disposal of certain wastes at landfills;  Tax on the purchase of certain products that generate waste disposal difficult to manage and / or high negative environmental impact;  Subsidies for the implementation and operation of recycling plants;  Minimum and maximum indices of recycled content in certain products;  The environmental certification of products. Responsibilities and joint responsibilities of each agent on the management of RCD; Subsidies for the implementation and operation of recycling plants; Minimum and maximum indices of recycled content in certain products;  The environmental certification of products. 283 An Application Model for Sustainability in the Construction Industry Trade unions should organize their members assisting in the dissemination studies and awareness of environmental responsibility and sustainable construction. The academy needs to develop knowledge about, especially the technical restrictions and new applications of the RCD. Consumers of recycled products shall also assist in establishing clear and objective specifications and minimum quality and performance required for their consumption. Despite growing concern for companies of ICC in relation to environmental sustainability, few initiatives have been taken. This finding is confirmed in the case of manufacturers who have a low involvement with respect to the disposal of waste solutions from the works, without taking into account that the generators and transporters do not show any commitment to proper disposal. It appears that manufacturers should encourage and support the research and take more active role in finding solutions and provision of recycling and take the RCD in a vertical process. This position might provide a differential in the relationship with customers, in particular the construction companies, with more active role in the searching of appropriate solutions for the allocation, since they are the manufacturers who have greater knowledge about the product. p Builders, distributors, installers and ATT should consolidate the processes of performing sorting and packaging accurately and hiring only suitable transport. They may also, through its purchasing power, pressuring suppliers to find solutions for allocation. 7. Conclusions g g Transport, in turn, must meet the stock of transport and disposal under the laws, and even agents of change in the behavior of generators. Finally, everyone needs to establish information system and effective communication between them. The establishment of reverse supply chain is subject to a strong cooperation between agents, which should be strategically aligned and have a shared vision and holistic environment in which they live, and census of all responsibilities on product life. We noted also that the consolidation of reverse logistics is a progressive and interdependent relationship between the suppliers and contractors. Efforts of a single side (agent) or scattered efforts tend to produce mediocre results and consequently no spread of its principles. 8. References Pinto, T. P. (1999). Metodologia para a Gestão Diferenciada de Resíduos Sólidos da Construção Urbana. São Paulo: EPUSP Manoliadis, O. G. (2007). The Role of Adaptive Environmental Management in Sustainable Development Case Study Assessing the Economical Benefits of Sustainable Construction in Greece, Environmental Technologies: New Developments, E. B. Ö. Güngör (Ed.), pp. 85-96, InTech, ISBN 978-3-902613-10-3, Democritus University of Thrace, Greece Chan, H. K., (2010). A Process Re-engineering Framework for Reverse Logistics based on a Case Study, University of East Anglia, Norwich, Norfolk, UK Naess, A. (1996). Ecology: The shallow and the deep. In: CAHN, MA. ; O’BRIEN, R. Thinking about the environment – readings on politics, property and the physical world. London: M. E. Sharpe p Reinhardt, F. L. (1999). Bring the environment Down to Earth Harvard Review. Nov. – Dec.. 284 Environmental Management in Practice RESOLUÇÃO CONAMA 307 DE 05 DE JULHO DE 2002. Dispõe sobre Gestão dos Resíduos da Construção Civil RESOLUÇÃO CONAMA 307 DE 05 DE JULHO DE 2002. Dispõe sobre Gestão dos Resíduos da Construção Civil Chattopadhyay, S. & Mo, John P.T. (2010). Modelling a Global EPCM (Engineering, Procurement and Construction Management) Enterprise, RMIT University, Australia Couto A. & Couto, J. P. (2010). Guidelines to Improve Construction and Demolition Waste Management in Portugal, Process Management, pp. 285-208, Intech, ISBN 978-953- 307-085-8, University of Minho, Portugal Rutherford, I. (1997). Use of models to link indicators of sustainable development. In: MOLDAN, B; BILHARDZ, S.; Sustainability indicators: report of the Project on indications of sustainable development. Chichester: John Willey & Sons ltd Sachs, I. (1997). Desenvolvimento sustentável, Bioindustrialização descentralizada e novas configurações rural-urbanas. Os casos da Índia e Brasil. In: VIEIRA, P. F.; WEBBER, J. Gestão de recursos naturais renováveis e desenvolvimento, Cortez (Ed.), São Paulo, Brasil Secretaria do Meio Ambiente do Estado de São Paulo. Governo do Estado Instituiu Selo Verde para Produtos que respeitem a Natureza. http://. www.ambiente.sp.gov.br, acess May 2010. Serviço de limpeza urbana da prefeitura municipal de Belo Horizonte – SLU/PMBH. Belo Horizonte, Feb. 2004 14 1. Introduction Most EMSs in Taiwanese SMEs are implemented in 286 Environmental Management in Practice accordance to specification in ISO 14001 or QC080000 standards, in which contain requirements that have to be fulfilled before third-party certification and /or registration can be achieved. accordance to specification in ISO 14001 or QC080000 standards, in which contain requirements that have to be fulfilled before third-party certification and /or registration can be achieved. Strength, Weakness, Opportunity and Threat (SWOT) analysis is an important support tool for decision-making, and is commonly used as a means to systematically analyze an organization’s internal management capability and its external environment. The purpose of the analysis on internal strengths and weaknesses is to assess how an enterprise carries out its internal work, such as R&D, day to day business operation, etc. On the other hand, the purpose of the analysis on the external opportunities and threats is to assess whether or not an enterprise can seize opportunities and avoid threats, whilst facing an uncontrollable external environment, such as fluctuating prices, political destabilization, etc. SWOT analysis has been successfully applied in EMS fields, such as the environmental impact assessment in India (Paliwal, 2006), the development of an environmental management system (Lozano and Vallés, 2007) and regional energy planning for renewable development (Terrados et al., 2007). ( ) For a quantitative SWOT, Kuttila et al. (2000) develop a hybrid method, the Analysis Hierarchy Process (AHP) in the SWOT analysis, to eliminate the weakness in the measurement and evaluation steps of the SWOT analysis. Examples in literature of studies that follow the method of Kuttila et al. include those by Kajanus et al. (2004), Leskinen et al. (2006) and Chang and Huang (2006). Yüksel and Dağdeviren (2007) demonstrate a process for quantitative SWOT analysis that can be performed even when there is dependence among strategic factors. They use the Analytic Network Process (ANP) that allows measurement of the dependency among the strategic factors as well as its AHP, which is based on independence between the factors. ANP is a more general form of its predecessor, the AHP, for ranking alternatives based on some set of criteria. Unlike AHP however, ANP is capable of handling feedbacks and interdependencies, which exist, in complex systems like a manufacturing system. ANP problem formulation starts by modeling the problem that depicts the dependence and influences of the factors involved to the goal or higher-level performance objectives. 1. Introduction In today’s highly competitive environment, strategic management has been widely used by all enterprises to withstand fierce competition. Environmental management has quickly emerged as an essential strategic factor in many industries. Environmental considerations are clearly becoming increasingly important and will be considered as one of the key factors in most companies’ success stories. For example, recently there are many firms in Asia that had already received ISO 14001 certification and adopted these Environmental Management Systems (EMS) standards as their state policy. No doubt that many firms have recognized the compatibility between environmental performance and profitability, as it witnessed by increasing interest in recycling programs and green marketing, in part due to realizing that the futility of running from such pressures. Melnyk et al. (2003) apply a survey of North American managers to demonstrate that firms having gone through EMS certification experience a greater impact on performance than do firms that have not certified their EMS. Pan (2003) applies questionnaires to the organizations within Taiwan, Japan, Hong Kong and Korea on regards of ISO9000 and ISO14000 issues. He uses statistical analysis results of the survey data to gain eight common points for ISO9000 and ISO14000 certified firm within these four countries. Tan et al. (2003) develop an e-commerce structure for sorting, selecting and utilizing information for the effect of ISO9000 system. The related studies of environmental issues will be listed in Environment Management (Ahsen and Funck, 2001; Rao et al., 2006; Gernuks et al., 2007), Environmental Management Accounting (Jasch, 2003), ISO14001 Certification (Fryxell and Szteo, 2002; Mbohwa and Fukada, 2002; Rennings et al., 2006) and Life Cycle Assessment for EMS (Zobel, 2002). In a country’s endeavor to implement EMS in both manufacturing and service sectors, the significance of Small and Medium Enterprises (SMEs) deserves special attention. In Taiwan, a SME is set under either two conditions. First, it is defined by the number of employees that they often refer to those with less than 200 employees involved in manufacturing, building and mining industries. Second, it is defined by its capital volume that is less than 80 million Taiwan dollars. The SMEs are typically much smaller in operation compared to the global and multinational enterprises, whereas most of the SEMs in Taiwan are positioned in the ending-role of the supply chain. 2. Vote-ranking methodology Data Envelopment Analysis (DEA) is an analytical procedure developed by Charnes et al. (1978) for measuring the relative efficiency of decision-making units (DMUs) that perform the same types of functions and have identical goals and objectives. The weights used for each DMU are those which maximize the ratio between the weighted output and weighted input. DEA is a mathematical programming technique that calculates the relative efficiencies of multiple DMUs, based on multiple inputs and outputs. A well-known method for ranking candidates in a ranked voting system is to compare the weighted sum of their votes after determining suitable weights. Cook and Kress (1990, 1992) present an approach to the problem of ranking candidates in a preferential election. They consider an alternative method which does not specify the sequence of weights by applying DEA. One would imagine that any reasonable person, voter, candidate or poll manager would agree that the first place votes should weigh at least as much as second place votes, and so on. They provide the following DEA model to obtain the total score for each candidate:       1 1 ( 1) max . . 1, 1,2,..., ; , , 1,2,..., 1; , . S rr rs rs s S rq rs qs s rs r s rS Z u x s t Z u x q R u u d s s S u d s                 (1)  1 . . 1, 1,2,..., ; S rq rs qs s s t Z u x q R      (1) Where, s: the number of places, s =1, … , S. p r: the number of candidates, r =1, … , R. r: the number of candidates, r =1, … , R. urs: the weights of the sth place with respect to the rth candidate. urs: the weights of the sth place with respect to the rth candidate. xrs: the total votes of the rth candidate for the sth place. d(s, ε): the given difference in weights between sth place with (s+1)th place; d(., ε), called the discrimination intensity function, is nonnegative and non-decreasing in ε. Parameter ε is nonnegative. 1. Introduction Dependence among the SWOT factors is observed to effect the strategic and sub-factor weights, as well as to change the strategy priorities. Dyson (2004) provides an SWOT and TOWS analysis to create strategy formation and its incorporation into the strategic development process at University of Warwick by scoring SWOT factors. A variation of SWOT analysis is the TOWS matrix. In the TOWS matrix the various factors are identified and these are then paired e.g. an opportunity with a strength, with the intention of stimulating a new strategic initiatives (Table 1). g g A “top-down” way of thinking could be used to guide the formulation of decision hierarchy. In this paper, a new quantitative SWOT analysis is provided that allows measurement of the strategic factors as well as its vote-ranking method. The first task is to invite eighteen certificated ISO9000 and ISO14000 auditors (or lead auditors) to organize a “Task Force (TF)”. The TF will discuss SWOT of Taiwanese SMEs within their EMS issues and assess the competitive strategies. The second task is to apply the internal competitive strengths to find external market opportunities. As a result, the strategy combination for max {strengths, opportunities} and min {weaknesses, threats} will be provided. The third task is to regard these SWOT indexes and their sub-criteria as the candidates voted by the task force. In conclusion, the different results of ranking will expose different weights among the votes of the candidates. This paper discusses the environmental issues of the SMEs not only by drawing insights from research conducted in different countries, but also look into the use of environmental Assessing the SMEs’ Competitive Strategies on the Impact of Environmental Factors:A Quantitative SWOT Analysis Application 287 factors of SWOT through their development, their context and adaptability to enhance the environment performance of SMEs. As for the medium, the vote-ranking method will be used to rank the different competitive strategies and priorities. This specific method provides a new quantitative SWOT methodology that will be extended to decision-making issues. The rest of this paper is organized as follows: Section 2 illustrates the vote-ranking method and the conceptual approach. Section 3 discloses the use of vote-ranking method to provide a quantitative SWOT method for assessing the SMEs’ competitive strategies in EMS by six-step procedure. Section 4 discusses the results of different strategies and suggestions. Section 5 highlights some conclusions and offers directions for further researches. 2. Vote-ranking methodology g The Cook and Kress’s ranked voting model (1) is assumed that in a voting system, each voter selects R candidates and ranks them from the 1st to the Sth places, S≤ R. The d(s, ε) is to ensure that first place votes are valued at least as highly as second place votes which are valued at least as highly as third place votes etc. The Zrq is the cross-efficiency which can be thought of here as candidate r’s evaluation of candidate q’s desirability. The constraints Zrq 288 Environmental Management in Practice are the usual DEA constraints i.e. that no candidate q should have a desirability greater than 1 under r’s weights. The Zrr has been used in the objective function to emphasize the candidate r’s evaluation of his/her own desirability. The rth candidate wishes to be assigned the weight urs so as to maximize the weighted sum of votes to candidate r, that is when the score Zrr becomes the largest. Notionally, each candidate was permitted to choose the most favorable weights to be applied to his/her standings in the normal DEA manner, with the additional ‘assurance region’ restriction, in which the weight for a sth place vote should be greater than the one for a (s+1)th place vote by some amount. Hashimoto and Ishikawa (1993) consider the candidates in ranked voting systems as the DMUs in DEA, and each is considered to have many outputs and only one input with unity. He also deems that it is fair to evaluate each candidate in terms of the weights optimal to himself/ herself. g p Green et al. (1996) further develop this model by setting certain constraints to the weights. They point out that the form of d(s, ε) affects the ranking results and does not allow DMUs to choose their own weights unreservedly. Therefore, they present an alternative procedure that involved using each candidate’s rating of him/herself along with each candidate’s rating of all the other candidates. They utilize the cross-efficiency model to DEA to obtain the best candidate. On the other hand, Hashimoto (1997) proposes a method to determine a total ordering of candidates specifying nothing arbitrary, but only assuming the condition of decreasing and convex sequence of weights. They incorporate the condition of decreasing and convex sequence of weights into DEA as the assurance region. Green et al. 2. Vote-ranking methodology and Hashimoto proposes these methods, whereabouts the existence of low preference candidates may change the ranks and DEA exclusion model, which seems to be unstable with respect to the inefficient candidates. Obata and Ishii (2003) consider that, the instability is caused by the fact mentioned above and that the inefficient candidates should not be used to discriminate efficient candidates. They also use this information only on efficient candidate while discriminating and realizing that the order of efficient candidates never changes even though the inefficient candidates are added or removed. Foroughi and Tamiz (2005) simplify the model of Obata and Ishii and extend it to rank the inefficient candidates as well as the efficient one. Noguchi et al. (2002) revise the application of Green’s method and show that different weights among objects gave rise to different ranking results. If one wants to set particular constraints to a weight can be employed, which is characterized by the following constraints: (a) ur1 2 ur23 ur3 … S urS , (b) urs  1/[(1+2+…+S)n] =2/(n S(S+1)), where n is the number of voters. In this multiple criteria case, the vote-ranking model is defined as follows:     1 1 ( 1) max . . 1 1,2,..., ; ( 1) , 1,...,( 1); 1 2 . * 1 1 2 ... S rr rs rs s S rq rs qs s rs r s rS Z u x s t Z u x q R su s u s S u n S S S n                        (2) (2) Where, these variables are the identical as model (1). Where, these variables are the identical as model (1). Where, these variables are the identical as model (1). Where, these variables are the identical as model (1). Assessing the SMEs’ Competitive Strategies on the Impact of Environmental Factors:A Quantitative SWOT Analysis Application 289 As for ranking of alternatives, one of the most popular methods compares the weighted sum of votes after determining suitable weights for each alternative. The different weights among objects resulted in different ranking results and propose a new method of ordering in order to solve the problem of weights ranking. 2. Vote-ranking methodology As a final point, the module solver imbedded in EXCEL of Microsoft Office [2003] will be applied to solve the above linear programming problems (Liu and Hai, 2005). 3. The competitive strategies of the Taiwanese SMEs for EMS This study proposes six-step procedure for selecting the competitive strategies of the Taiwanese SMEs. They are obtained from TF which will fall into four subjective criteria that discuss and analyze SWOT of Taiwan’s SMEs in the EMS. The first step is structuring the problem into a SWOT hierarchy. On the top level is the overall goal of selection competitive strategies. On the second level are the four SWOT criteria that contribute to the overall goal. The criteria (sub-criteria) for strengths (S1, S2, S3), opportunities (O1, O2, O3), weaknesses (W1, W2, W3) and threats (T1, T2, T3) are individually presented into Level 2 and 3. On the second level is that four criteria are decomposed into twelve sub-criteria under SWOT; additionally on the bottom (or fourth) level, there are five alternative competitive strategies that are to be evaluated in terms of the sub-criteria listed on the third level. These competitive strategies (OS-1, OS-2, OW-1, TS-1 and TW-1) are assessed in Level 4 and illustrated in Fig.1. Fig. 1. A SWOT hierarchy for selecting the competitive strategies Strengths Weaknesses Opportunities Threats S1, S2, S3 W1, W2, W3 O1, O2, O3 T1, T2, T3 Level-1 Level-2 Level-3 Level-4 Strategies Selection The competitive strategies: OS-1, OS-2, OW-1, TS-1, TW-1 Strategies Selection Level-1 The competitive strategies: OS-1, OS-2, OW-1, TS-1, TW-1 Fig. 1. A SWOT hierarchy for selecting the competitive strategies 3.1 Step 1: SWOT analysis Strengths: +S1: Capability to execute and develop EMS certification +S2: Synergy with commerce, environmental protection and education units +S3: Possessing high level of environmental education Weaknesses: -W1: SMEs respond slowly and difficultly for external customer requirements -W2: Some suppliers or manufacturers are unwilling to face higher environmental regulation required and seek other markets with lower quality consciousness -W3: The government’s regulations of environmental protection are too loose Opportunities: +O1: Change in customers’ preferences (increase in market demand for EMS or QMS certification) +O2: Increase value-added of product Maxi-maxi (O-S) Strategies OS-1: Extend EMS Certification effects to create high value-added markets OS-2: Involve in improving environment issues and promote company image and profits Maxi-mini (O-W) Strategies OW-1:Change directly in manufacture preferences to create products of high environmental requirement standard Strengths: +S1: Capability to execute and develop EMS certification +S2: Synergy with commerce, environmental protection and education units +S3: Possessing high level of environmental education Maxi-maxi (O-S) Strategies OS-1: Extend EMS Certification effects to create high value-added markets OS-2: Involve in improving environment issues and promote company image and profits Mini-maxi (T-S) Strategies TS-1: Increase strictly government or industry environmental regular Mini-mini (T-W) Strategies TW-1: Government impel environmental education and assistance for SMEs -T3: Negative corporation image if EMS certification is abandoned Table 1. SWOT and TOWS matrixes for EMS Table 1. SWOT and TOWS matrixes for EMS 3.1 Step 1: SWOT analysis p y First of all, the author invites the eighteen certificated ISO9000 or ISO14000 auditors (or lead auditors), consists of 14 part-time and 4 full-time auditors, to organize a TF in this particular study. They are first briefed about the overall objective of the study, then specifically on the SWOT and vote-ranking methodologies. The questionnaires are used for interviewing purposes; however they mainly use a board or group decision method to determine the criteria and sub-criteria for selecting the competitive strategies. The study is to apply the internal competitive strengths to find external market opportunities. This is followed by the analysis on the organization’s external competitive environment and internal operating environment. Consequently, the internal analysis is followed by the selection and 290 Environmental Management in Practice implementation of strategies. Due to highly global nature of the “Green House”, the requirements of EMS are also applicable for other countries in the European Union. For selecting the competitive strategies of SMEs for EMS, the TF has been mainly on the discussion of the SWOT method and problem defining after a series of revision. The strategy combination of EMS for max {strengths, opportunities} and min {weaknesses, threats}, OS-1, OS-2, OW-1, TS-1 and TW-1, is provided in Table 1. Mini-maxi (T-S) Strategies TS-1: Increase strictly government or industry environmental regular Mini-mini (T-W) Strategies TW-1: Government impel environmental education and assistance for SMEs Maxi-maxi (O-S) Strategies OS-1: Extend EMS Certification effects to create high value-added markets OS-2: Involve in improving environment issues and promote company image and profits 3.2 Step 2: priority votes of criteria and sub- criteria in SWOT 3.3 Step 3: calculate the weights of criteria and sub- criteria in SWOT The votes on Table 2 are used to calculate the weights of the four criteria by mod 3.2 Step 2: priority votes of criteria and sub- criteria in SWOT The second task is to regard these SWOT indexes as candidates that are voted by TF. The four criteria are the strength, weakness, opportunity and threat indexes and the twelve sub- criteria are S1-3, W1-3, O1-3 and T1-3 alternatively, within the SWOT. They are regarded as the selected items and expected to receive votes with respect to the related elements within the model, as shown in Table 1. The TF illustrate the order for the four criteria and the votes for each which are shown in Table 2. Every members will vote from 1 to S, (SR), where R is the number of criteria or sub-criteria. They are regarded as candidates whom are to be voted by different places. Afterward, TF will list its priority votes of sub-criteria in fixed first criterion within Table 3. They were only asked to determine the order of the criteria or sub-criteria, however not the weight of each criterion or sub-criterion. Assessing the SMEs’ Competitive Strategies on the Impact of Environmental Factors:A Quantitative SWOT Analysis Application 291 Criteria 1st 2nd 3rd 4th Weights Strengths 4 8 4 2 0.264 Opportunities 7 4 7 0 0.299 Weaknesses 2 0 7 9 0.174 Threats 5 6 0 7 0.263 * The weights are normalized and totally equal to one. Table 2. Priority votes of four criteria 3.3 Step 3: calculate the weights of criteria and sub- criteria in SWOT The votes on Table 2 are used to calculate the weights of the four criteria by model (2), R=4, S=4, n=18 and the lowest weights of the fourth place are 1 180 (ur4 2/ [nS(S+1)] =2/ [184(5) =0.0056]). The weights for strength, weakness, opportunity and threat at the second level are 0.884, 1.000, 0.581 and 0.882, respectively. After normalizing these data, the weights of outcome are 0.264, 0.299, 0.174 and 0.263, as it is illustrated in column 6 of Table 2, respectively. For “Strengths” in the Table 3, there are variables R=3, S=3, n=18 and the lowest weights of the third place are 1/108 (ur3 2/ [nS(S+1)] =2/ [183(4) =0.0093]). Similarly, the votes within Table 3 are using the same procedure in order to determine the weights of the sub-criteria. The results of the weight of sub-criteria are listed in columns 5 and 10 of Table 3. 3.3 Step 3: calculate the weights of criteria and sub- criteria in SWOT The votes on Table 2 are used to calculate the weights of the four criteria by =0.0056]). The weights for strength, weakness, opportunity and threat at the second level are 0.884, 1.000, 0.581 and 0.882, respectively. After normalizing these data, the weights of outcome are 0.264, 0.299, 0.174 and 0.263, as it is illustrated in column 6 of Table 2, respectively. For “Strengths” in the Table 3, there are variables R=3, S=3, n=18 and the lowest weights of the third place are 1/108 (ur3 2/ [nS(S+1)] =2/ [183(4) =0.0093]). Similarly, the votes within Table 3 are using the same procedure in order to determine the weights of the sub-criteria. The results of the weight of sub-criteria are listed in columns 5 and 10 of Table 3. Criteria Votes Weights Votes Weights 1st 2nd 3rd 1st 2nd 3rd Strengths Opportunities S1 13 4 1 0.465 O1 15 2 1 0.495 S2 0 9 9 0.227 O2 0 15 3 0.258 S3 5 5 8 0.308 O3 3 1 14 0.247 Total 18 18 18 Total 18 18 18 Weaknesses Threats W1 11 7 0 0.439 T1 5 5 8 0.304 W2 3 3 12 0.258 T2 5 9 4 0.336 W3 4 8 6 0.303 T3 8 4 6 0.360 Total 18 18 18 Total 18 18 18 * The weights are normalized and totally equal to one. Table 3 Priority votes and weights of twelve sub criteria 3.4 Step 4: scores of competitive strategies in SWOT The competitive strategies, OS-1, OS-2, OW-1, TS-1 and TW-1 are subjective indices that could be translated into numerical ratings using different methods, such as questionnaire, AHP or vote-ranking and so much more. TF may ask their colleagues to answer these questionnaires in order to rate the competitive strategies of sub-criteria of each SWOT. A major problem was thus, to ensure the consistency between managers and to avoid any bias creeping in. A set of standard guidelines was placed after discussions with the TF (voters). It is mainly agreed that all performance scores would be based on a nine points grade scale. 292 Environmental Management in Practice Each grade would have an adjective descriptor and an associated point score or range of point scores. The TF makes their judgment on the qualitative scale of adjectival descriptors. Table 4 lists the example for rating the strength and opportunity indices, where the lower and upper scores are predetermined from 1-9. Table 5. Grading different strategy scores in weakness and threat indexes The five competitive strategies, OS-1, OS-2, OW-1, TS-1 and TW-1, by means of the highest rating were regarded as the best competitive strategies, with the rest being ranked accordingly. The competitive strategies will earn the average scores of questionnaires within Table 4 and Table 5 by TF. The average of collected scores is listed in the columns 5-9 of Table 6. 3.3 Step 3: calculate the weights of criteria and sub- criteria in SWOT The votes on Table 2 are used to calculate the weights of the four criteria by The strength and opportunity indices should be maximized, the least-favorable candidate is assigned the smallest value and the most-favorable candidate is assigned the largest value. On the other hand, the weakness and threat indices need to be minimized, where the least-favorable candidate is assigned the largest value and the most-favorable candidate is assigned the smallest value. The overcoming range of subjective indices is set between 1 and 9 illustrated in Table 5. Therefore, each of the competitive strategy can be awarded a ‘score’ from 1 to 9 on each sub-criterion. Scores Rules 9 Greatly conforming to market and sub-criteria of requirement, successful probability more than 90% 7 Better conforming to market and sub-criteria of requirement, successful probability about 70% 5 Conforming to market and sub-criteria of requirement, successful probability about 50% 3 Slightly conforming to market and sub-criteria of requirement, successful probability about 30% 1 Not conforming to market and sub-criteria of requirement, successful probability about 10% Table 4. Grading different strategy scores in strength and opportunity indexes 3.5 Step 5: total weighted scores of competitive strategies This step requires the TF to assess the performance of all the competitive strategies within the twelve sub-criteria of SWOT identified as important for competitive strategies rating. Simple score sheets were provided to assist the manager to record the scores for each strategy on each of the twelve sub-criteria. An example of this strategy is shown in Table 6. In the first row of Table 6, the number 0.123 is equal to the product of the “Strength” criterion score 0.264 multiply with the S1 given value of “0.465”. Moreover, the same method is applied to obtain Assessing the SMEs’ Competitive Strategies on the Impact of Environmental Factors:A Quantitative SWOT Analysis Application 293 other results. Once the weights for sub-criteria have been determined, it is relatively easy to calculate the resulting competitive strategies rating scores. Mathematically, the rating is equivalent to the sum of the product of each sub-criterion weight and the competitive strategy performance score. The rating value of competitive strategies is obtained by summing the products of the respective elements. The competitive strategies rating value for strategy OS-1 is obtained by summing up the products of the respective elements in columns 4 and 5 for each row; given in the final column 10, the over all total weighted scores of the row is “6.859”. The rating method used in strategy OS-1, can be used to find the total scores of the other four strategies stated in columns 11-14 of Table 6. The rating value for each competitive strategy is obtained by summing the products of the respective elements in the matrix; given in the final score, the values of over all competitive strategies of OS-1, OS-2, OW-1, TS-1 and TW-1 respectively is, 6.859, 8.357, 7.532, 7.298 and 8.274 stated within the last row of Table 6. This gave a rating score for each competitive strategy, whereas the higher the rating, the better the overall performance for competitive strategy. other results. Once the weights for sub-criteria have been determined, it is relatively easy to calculate the resulting competitive strategies rating scores. other results. Once the weights for sub-criteria have been determined, it is relatively easy to calculate the resulting competitive strategies rating scores. Mathematically, the rating is equivalent to the sum of the product of each sub-criterion weight and the competitive strategy performance score. The rating value of competitive strategies is obtained by summing the products of the respective elements. 3.6 Step 6: assessment of competitive strategies In the last row of Table 6, the rating value for each strategy is obtained; the final score and the ranking of competitive strategies for OS-2, TW-1, OW-1, TS-1 and OS-1 is first, second, third, fourth and fifth respectively. Even though the score of OS-2 is only higher by 0.083 than TW-1 and the score of OW-1 is higher by 0.234 than TS-1, however for both of the competitive strategies, the difference of scores will definitely change the overall final rank. These results will be regarded as sensitivity analysis for five competitive strategies. Criteria (A) Sub-criteria (B) Weights (C= A×B) Grade Strategies Scores Weighted Strategies Scores OS-1 OS-2 OW-1 TS-1 TW-1 OS-1 OS-2 OW-1 TS-1 TW-1 Strengths S1 0.465 0.123 6.833 8.889 8.056 6.944 8.722 0.839 1.091 0.989 0.853 1.071 0.264 S2 0.227 0.060 6.944 8.944 7.611 7.278 8.500 0.416 0.536 0.456 0.436 0.509 S3 0.308 0.081 7.056 8.833 7.556 8.611 8.611 0.574 0.718 0.614 0.700 0.700 Opportunities O1 0.495 0.148 6.833 7.778 7.389 7.278 8.167 1.011 1.151 1.094 1.077 1.209 0.299 O2 0.258 0.077 6.944 8.000 7.611 7.389 8.000 0.536 0.617 0.587 0.570 0.617 O3 0.247 0.074 6.778 8.111 7.778 7.500 7.944 0.501 0.599 0.574 0.554 0.587 Weaknesses W1 0.439 0.076 6.611 7.778 7.222 6.944 7.833 0.505 0.594 0.552 0.530 0.598 0.174 W2 0.258 0.045 6.500 7.833 7.278 7.056 7.778 0.292 0.352 0.327 0.317 0.349 W3 0.303 0.053 6.667 7.944 7.500 7.278 7.722 0.351 0.419 0.395 0.384 0.407 Threats T1 0.304 0.080 7.056 8.611 7.278 7.000 8.444 0.564 0.688 0.582 0.560 0.675 0.263 T2 0.336 0.088 7.111 8.722 7.556 7.167 8.389 0.628 0.771 0.668 0.633 0.741 T3 0.360 0.095 6.778 8.667 7.333 7.222 8.556 0.642 0.821 0.694 0.684 0.810 Total Weighted Scores 6.859 8.357 7.532 7.298 8.274 Table 6. The SWOT analysis of different strategies Table 6. The SWOT analysis of different strategies 3.5 Step 5: total weighted scores of competitive strategies The competitive strategies rating value for strategy OS-1 is obtained by summing up the products of the respective elements in columns 4 and 5 for each row; given in the final column 10, the over all total weighted scores of the row is “6.859”. The rating method used in strategy OS-1, can be used to find the total scores of the other four strategies stated in columns 11-14 of Table 6. The rating value for each competitive strategy is obtained by summing the products of the respective elements in the matrix; given in the final score, the values of over all competitive strategies of OS-1, OS-2, OW-1, TS-1 and TW-1 respectively is, 6.859, 8.357, 7.532, 7.298 and 8.274 stated within the last row of Table 6. This gave a rating score for each competitive strategy, whereas the higher the rating, the better the overall performance for competitive strategy. 4. Discussion Which means that at the present moment, the demand on this particular product is lacking, furthermore, it might resulted in the incapability to agree on these certain analysis by some SMEs. p y g y y Moreover, from the strategy analysis OW-1and TS-1 point of view, direct changes in manufacture preferences to create products of high environmental requirement standard has a bigger risk toward the SMEs in term of direct investment. Generally, average companies do not have certain investment planning until it has reached a deal, order placement or customer’s promise in advance. Additionally, an increasingly strict government or industry environmental in carrying out this phase is facing difficulty, where presently the government mostly is using counseling method or fund assistance to encourage and urge the industry to increase its EMS ability in order to reach the low price product strategy and high level of product diversification. p gy g p Lastly, the strategy OS-2 imposes a similar way of thinking with strategy TW-1. Taiwanese SMEs apperceive the significance of EMS and also recognize the importance to survive within the diversified competing market environment, whereas they need to build up its environmental management that has to suit the EMS specification and attention. However, the investment within environmental protection for its resources and facilities requires a great amount of expenditures. Under this major investment, if the expected outcomes are unpredictable, therefore the willingness on investing within the environmental management will suffer an enormous drawback. These SMEs certainly would hope that government will work together with country resources, providing some assistance in procuring EMS needed facilities and equipments or even any related training within the environmental management scope, moreover guidance or counselling in obtaining different kinds of ISO authentic certificate will also be valuable resource. Obviously, most people are familiar with the conflicts between environmental protection and economic development. Those who are convinced of the consequences of global warming will remain convinced, while those suspicious will remain suspicious. After all, economic development means bread, while the mankind cannot immediately appreciate the deep implications of its damage to the great nature. Therefore, politicians should be aware of the environmental implications of legal provisions and regulations. 4. Discussion Likewise, the industry authorities, when developing new products, should consider the intangible social cost of pollution as a part of the overall cost and deal with the issue of pollution as a part of life cycle management, so that such considerations and practices will benefit our earth. In EMS, this will further our understanding of the potential poisonous substances to be produced in production, deployment and replacement stages, and will help us minimize pollution and thus contribute to environmental protection. 4. Discussion First of all, considering that the strategy OS-1 has the lowest score within the strategy analysis, most of the SMEs supposed that this strategy is quite acceptable even though there 294 Environmental Management in Practice are still have some doubts present, especially on whether or not by obtaining the EMS related international standard authentication, such as ISO14000, it will certainly create a high value-added market. From a present market condition which is quite unfeasible to reflect the practical demand, frequently as a final result it is invested in fund or modification. Even though most of the customers are quite optimistic and agreed to this way of doing, however when everything is fully involved within EMS in the future, it will certainly has some affect on its capital or product selling price. Which means that at the present moment, the demand on this particular product is lacking, furthermore, it might resulted in the incapability to agree on these certain analysis by some SMEs. Moreover, from the strategy analysis OW-1and TS-1 point of view, direct changes in manufacture preferences to create products of high environmental requirement standard has a bigger risk toward the SMEs in term of direct investment. Generally, average companies do not have certain investment planning until it has reached a deal, order placement or customer’s promise in advance. Additionally, an increasingly strict government or industry environmental in carrying out this phase is facing difficulty, where presently the government mostly is using counseling method or fund assistance to encourage and urge the industry to increase its EMS ability in order to reach the low price product strategy and high level of product diversification. L l h OS 2 i i il f hi ki i h TW 1 T i are still have some doubts present, especially on whether or not by obtaining the EMS related international standard authentication, such as ISO14000, it will certainly create a high value-added market. From a present market condition which is quite unfeasible to reflect the practical demand, frequently as a final result it is invested in fund or modification. Even though most of the customers are quite optimistic and agreed to this way of doing, however when everything is fully involved within EMS in the future, it will certainly has some affect on its capital or product selling price. 5. Conclusions With the continuing development of human civilization and technology, the life cycle of any products, from production, consumption to final waste, it is involving more and more external adverse factors which bring about direct or indirect impact on the environment. Assessing the SMEs’ Competitive Strategies on the Impact of Environmental Factors:A Quantitative SWOT Analysis Application 295 Economists said that we should stop aggravation of global warming now; and there is only one earth; therefore, be environmentally friendly. y y Lastly, the competitive strategies OS-2 and TW-1 will be provided to Taiwanese SME department and industry union. The main contributions of this study are as follows: tly, the competitive strategies OS 2 and TW 1 will be provided to Taiwanese SME artment and industry union. The main contributions of this study are as follows: p y y 1. The selection procedure of competitive strategies in SWOT can assist the audience to think in a very comprehensive and detailed manner, while allowing them to categorize various issues. p y y 1. The selection procedure of competitive strategies in SWOT can assist the audience to think in a very comprehensive and detailed manner, while allowing them to categorize various issues. 2. In this field, many researchers have sought to improve the different capabilities of quantitative SWOT, such as AHP, ANP or fully rank decision-making units. In this case, the vote-ranking methodology incorporated with SWOT is applied and as a result, it became the easiest and most convenient method compared to others. The vote-ranking is presented as an approach to the problem of ranking candidates in a preferential election. The future researches had suggested that the cross-evaluation method is better off to be applied to assess candidates through peer-group, whereas one can attain a more balanced view of the weight-setting. The cross evaluation can be used to overcome the problem of maverick decision-makers. The proposed methodology can be utilized to issues of SWOT, such as AHP or ANP within this study. 6. References Ahsen, A.V. ; Funck, D. (2001). Integrated management systems - opportunities and risks for corporate environmental protection. 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The influence of motivations for seeking ISO 14001 certification an empirical study of ISO 14001 certified facilities in Hong Kong. Journal of Environmental Management 65, pp. 223-238. Gernuks, M. ; Buchgeister, J. & Schebek, L. (2007). Assessment of environmental aspects and determination of environmental targets within environmental management systems (EMS) – development of a procedure for Volkswagen. Journal of Cleaner Production 15, pp. 1063-1075. Green, R.H. ; Doyle, J.R. & Cook, W.D. (1996). Preference voting and project ranking using DEA and cross-evaluation. European Journal of Operational Research 90, pp. 461-472. Green, R.H. ; Doyle, J.R. & Cook, W.D. (1996). Preference voting and project ranking using DEA and cross-evaluation. European Journal of Operational Research 90, pp. 461-472. Hashimoto, A. (1997). A ranked voting system using a DEA/ AR exclusion model: a note. , ; y , J , ( ) g p j g g DEA and cross-evaluation. European Journal of Operational Research 90, pp. 461-472. Hashimoto, A. (1997). A ranked voting system using a DEA/ AR exclusion model: a note. Journal of the Operational Research 97, 600-604. Hashimoto, A. (1997). A ranked voting system using a DEA/ AR exclusion model: a note. Journal of the Operational Research 97, 600-604. Hashimoto, A. & Ishikawa,H. (1993). Using DEA to evaluate the state of society as measured by multiple social indicators. Socio-Economic Planning Sciences 27(4), pp. 257-268. 296 Environmental Management in Practice Jasch, C. (2003). The use of environment management accounting (EMA) for identifying environmental costs. 6. References Journal of Cleaner Production 11, pp. 667-676. f Kajanus, M. ; Kangas, M. & Kuttila, M., (2004). The use of value focused thinking and the A’WOT hybrid method in tourism management. Tourism Management 25, pp. 499-506. Kuttila, M. ; Pesonen, M. ; Kangas, J. & Kajanus, M. (2000). Utilizing the analysis hierarchy process (AHP) in SWOT analysis – a hybrid method and its application to a forest- certification case. Forest Policy and Economics 1, pp. 41-45. y Leskinen, L.A. ; Leskinen, P. ; Kuttila, M. ; Kangas, M. & Kajanus, M. (2006). Adapting modern strategic decision support tools in the participatory strategy process- a case study of a forest research station. Forest Policy and Economics 8, pp. 206-216. y y pp Liu, F.H.F. & Hai, H.L. (2005). The voting analytic hierarchy process method for selecting suppliers. The International Journal of Production Economics 97, pp. 308-317. Lozano, M. & Vallés, J. (2007). An analysis of the implementation of an environmental management system in a local public administration. Journal of Environmental Management 82, 495-511. g Mbohwa, C. & Fukada, S. (2002). ISO 14001 certification in Zimbabwe experiences, problems and prospects. Corporate Environmental Strategy 9(4), pp. 427-436. Melnyk, S.A. ; Sroufe, R.P. & Calantone, R. (2003). Assessing the impact of environmental management systems on corporate and environmental performance. Journal of Operations Management 21, pp. 329-351. p g pp Noguchi, H. ; Ogawa, M. & Ishii, H. (2002). The appropriate total ranking method using DEA for multiple categorized purposes. Journal of Computational and Applied Mathematics 146, pp. 155-166. Obata, T. & Ishii, H. (2003). A method for discriminating efficient candidates with ranked voting data. European Journal of Operational Research 151, pp. 233-237. g p Paliwal, R. (2006). EIA practice in India and its evaluation using SWOT analysis. Environmental Impact Assessment Review 26, pp. 492-510. Pan, J.N. (2003). A comparative study on motivation for and experience with ISO9000 and ISO14000 certification among Far Eastern countries. Industrial Management & Data Systems 103(8), 564-578. Rao, P. ; Casttillo, O. ; Intal Tr, P.S. & Sajid, A. (2006). Environmental indicators for small and medium enterprises in the Philippines: An empirical research. Journal of Cleaner Production 14, pp. 505-515. pp Rennings, K. ; Ziegler, A. ; Ankele, K. & Hoffmann, E. (2006). The influence of different characteristics of the EU environmental management and auditing scheme on technical environmental innovations and economic performance. Ecological Economics 57, pp. 45-59. 6. References p g pp Tan, B. ; Lin, C. & Hung, H.C. (2003). An ISO 9001: 2000 quality information system in e- commerce environment. Industrial Management & Data Systems 103(9), pp. 666-676. Terrados, J. ; Almonacid, G. & Hontoria, L. (2007). Regional energy planning through SWOT analysis and strategic planning tools: impact on renewable development. Renewable & Sustainable Energy Reviews 11, pp. 1275-1287. gy pp Yüksel, Ì. & Dağdeviren, M. (2007). Using the analytic network process (ANP) in a SWOT analysis – a case study for a textile firm. Information Sciences 177, pp. 3364-3382. Zobel, T. ; Almroth, C. ; Bresky, J. & Burman, J-O. (2002). Identification and assessment of environmental aspects in an EMS context an approach to a new reproducible method based on LCA methodology. Journal of Cleaner Production 10, pp. 381-396. 15 Implementation of ISO 14000 in Luggage Manufacturing Industry: A Case Study S. B. Jaju G. H. Raisoni College of Engineering, Department of Mechanical Engineering, Nagpur India S. B. Jaju G. H. Raisoni College of Engineering, Department of Mechanical Engineering, Nagpur India 1. Introduction Definitions of EMS as provided by three separate documents on environmental management systems are as given below y g 001: "the organizational structure, responsibilities, practices, procedures, processes ources for implementing and maintaining environmental management" ISO 14001: "the organizational structure, responsibilities, practices, procedures, processes and resources for implementing and maintaining environmental management" BS 7750: "the organizational structure, responsibilities, procedures, processes and resources for implementing environmental management" p g g g 0: "the organizational structure, responsibilities, procedures, processes and resources lementing environmental management" Eco-Management and Audit Scheme (EMAS): "that part of the overall management system which includes the organizational structure, responsibilities, practices, procedures, processes and resources for determining and implementing the environmental policy" 4. Basic principles and methodology The fundamental principle and overall goal of the ISO 14001 standard, is the concept of continual improvement. ISO 14001 is based on the Plan-Do-Check-Act methodology, grouped into five phases that relate to Plan-Do-Check-Act; Environmental Policy, Planning, Implementation & Operation, Checking & Corrective Action and lastly Management Review. 2. Development of the ISO 14000 series The ISO 14000 family includes the ISO 14001 standard, which represents the set of standards used by various types of organizations for designing and implementing an effective environmental management system. The major objective of the ISO 14000 series of norms is "to promote more effective and efficient environmental management in organizations and to provide useful and usable tools - ones that are cost effective, system-based, and flexible and reflect the best organizations and the best organizational practices available for gathering, interpreting and communicating environmentally relevant information". Unlike previous environmental regulations, which began with command and control approaches, later replaced with ones based on market mechanisms, ISO 14000 was based on a voluntary approach to environmental regulation. The series includes the ISO 14001 standard, which provides guidelines for the establishment or improvement of an EMS. The standard shares many common traits with its predecessor ISO 9000, the international standard of quality management, which served as a model for its internal structure and both can be implemented side by side. As with ISO 9000, ISO 14000 acts both as an internal management tool and as a way of demonstrating a company’s environmental commitment to its customers and clients. Prior to the development of the ISO 14000 series, organizations voluntarily constructed their own EMS systems, but this made comparisons of environmental effects between companies difficult and therefore the universal ISO 14000 series was developed. An EMS is defined by 298 Environmental Management in Practice ISO as: “part of the overall management system, that includes organisational structure, planning activities, responsibilities, practices, procedures, processes and resources for developing, implementing, achieving and maintaining the environmental policy’. ISO as: “part of the overall management system, that includes organisational structure, planning activities, responsibilities, practices, procedures, processes and resources for developing, implementing, achieving and maintaining the environmental policy’. Customer pressure Lot of pressure is from customer end that is the basic aim of any organisation. Ultimately customer should have faith in the industry that the said industry is having compliance for environmental parameters. Legal obligations Over recent years, there have been considerable changes in environmental legislation. Every organisation wants to address its legal obligations, such as compliance with effluent discharge license parameters, or local authority planning requirements. Integrated Pollution Control licensing, for instance, will eventually oblige most industries to comply with stricter industry guidelines on pollution control, with the threat of heavy financial penalties resulting from non-compliance. Environmental concerns For a number of years preceding the introduction of a formal EMS, there was a genuine concern about its various environmental impacts. Some typical examples of impacts are:  energy and resource usage (electricity, gas , water)  raw material usage (paper, plates, inks, packaging, chemicals, film)  general waste (domestic)  recyclable waste (paper, timber, aluminum, silver, plastics)  hazardous waste (chemical wastes, liquid effluent, air emissions)  nuisances (noise, litter, dust, odors)  contracted activities (transport, subcontracted printing work)  product end use and disposal Plan – establish objectives and processes required Prior to implementing ISO 14001, an initial review or gap analysis of the organisation’s processes and products is recommended, to assist in identifying all elements of the current operation and if possible future operations, that may interact with the environment, termed environmental aspects. Environmental aspects can include both direct, such as those used during manufacturing and indirect, such as raw materials (Martin 1998). This review assists the organisation in establishing their environmental objectives, goals and targets, which should ideally be measurable; helps with the development of control and management Implementation of ISO 14000 in f Implementation of ISO 14000 in Luggage Manufacturing Industry: A Case Study 299 p Luggage Manufacturing Industry: A Case Study procedures and processes and serves to highlight any relevant legal requirements, which can then be built into the policy. procedures and processes and serves to highlight any relevant legal requirements, which can then be built into the policy. Do – implement the processes During this stage the organisation identifies the resources required and works out those members of the organisation responsible for the EMS’ implementation and control. This includes documentation of all procedures and processes; including operational and documentation control, the establishment of emergency procedures and responses, and the education of employees, to ensure they can competently implement the necessary processes and record results. Communication and participation across all levels of the organisation, especially top management is a vital part of the implementation phase, with the effectiveness of the EMS being dependant on active involvement from all employees. Check – measure and monitor the processes and report results During the check stage, performance is monitored and periodically measured to ensure that the organisation’s environmental targets and objectives are being met (Martin 1998). In addition, internal audits are regularly conducted to ascertain whether the EMS itself is being implemented properly and whether the processes and procedures are being adequately maintained and monitored. Act – take action to improve performance of EMS based on results After the checking stage, a regular planned management review is conducted to ensure that the objectives of the EMS are being met, the extent to which they are being met, that communications are being appropriately managed and to evaluate changing circumstances, such as legal requirements, in order to make recommendations for further improvement of the system. These recommendations are then fed back into the planning stage to be implemented into the EMS moving forward. 6.2 Corporate 1. Reduced financial costs through reduction in consumption of resources and through waste minimization. 1. Reduced financial costs through reduction in consumption of resources and through waste minimization. 2. Reduction and/or avoidance of potential emergency situations. 3. Avoidance of incidence of non-compliance with legislation and reduction in fines and cleanup costs. 4. Reduction in the cost of gaining capital, financial backing, insurance and valuation by becoming an "environmentally friendly" organization. 5. Improved marketing advantage as a "green" operation. 6. Increased staff morale and occupational safety and health standards. y 8. Increased documentation, communication and feedback of environmental policies and initiatives. 5. Role of EMS 1. An assessment of the existing practices and situation of an organization. 1. An assessment of the existing practices and situation of an organization. 2. A register of all environmental effects associated with the company's activities, established through an initial environmental review. 2. A register of all environmental effects associated with the company's activities, established through an initial environmental review. 3. A list of all legislation relevant and applicable to the environmental aspects of the activities, products and services of the organization. 3. A list of all legislation relevant and applicable to the environmental aspects of the activities, products and services of the organization. 4. Development of a corporate environmental policy and environmental management plan 4. Development of a corporate environmental policy and environmental management plan 5. The setting of environmental performance objectives and targets for both current and future activities. 6. Development of environmental performance evaluation procedures. 7. Establishment of an effective environmental training program for all employees within the organization, which will raise awareness, enhance skills for dealing with environmental issues and stress compliance with relevant legislation. 8. Implementation of a system, which reliably manages the performance of the organization, for both current and future activities. 8. Implementation of a system, which reliably manages the performance of the organization, for both current and future activities. 9. Documentation of the system communicated to all employees and distributed to all interested parties, especially to the public. 9. Documentation of the system communicated to all employees and distributed to all interested parties, especially to the public. p p y p 0. Establishment of non-conformance and corrective and preventive action procedures. 300 Environmental Management in Practice 11. Regular checking, reviewing and auditing of company practices and management commitment to reflect changing conditions with a focus on continual improvement. 6.1 Natural 2. Prevention and/or significant reduction of pollution and waste Generation. 3. Improved health and safety of interested parties. 4. Reduction in the use of non-renewable resources. 5. Improved conservation and efficient use of natural resources. 6. Benefits of EMS implementation 6.1 Natural 1. Clean Air, Water, Soil. 2. Prevention and/or significant reduction of pollution and waste Generation. 3. Improved health and safety of interested parties. 4. Reduction in the use of non-renewable resources. 5. Improved conservation and efficient use of natural resources. 7. Major requirements of ISO 14000 Following are some of the requirements of ISO 14000: g 1. Environmental protection as one of the highest corporate priorities with clear assignment of responsibilities and accountabilities to all employees. 1. Environmental protection as one of the highest corporate priorities with clear assignment of responsibilities and accountabilities to all employees. 2. Compliance with all environmental laws and regulations applicable to the company's activities, products and services. p 3. Ongoing communications on environmental commitment and performance with all shareholders. 4. Strategic planning that sets forth environmental performance objectives and targets, implemented through a disciplined management process. p g p g p 5. Periodic performance measurement (as well as systems audits and management reviews) to achieve continual improvement wherever possible. 6. Full integration with health and safety, quality, finance, business planning and other essential management processes. g p 7. Focus on EMS and looks for attributes that would sustain sound environmental decision making and performance. g 8. Top management commitment. 9. Third-party registration, through ISO 14001, or self-declaration for companies that meet the ISO 14000 standards. ISO 14001 includes discrete elements of environmental aspects, legal requirements, objectives and targets, environmental management program, communications, and emergency preparedness and response. Implementation of ISO 14000 in L M f t i I d t Implementation of ISO 14000 in Luggage Manufacturing Industry: A Case Study 301 8. Stage by stage implementation of ISO 14001 First stage: commitment and policy i. Environmental policy Second stage: Planning i. Environmental aspects ii. Legal and other requirements iii. Objectives and targets iv. Environmental management program Third stage: Implementation i. Structure and responsibility ii. Training, awareness and competence iii. Communication iv. Environmental documentation v. Document control Fourth stage: Operational control measurement and evaluation i. Monitoring and measurement ii. Non-conformance and corrective and preventive action iii. Records iv. Environmental management system audit Fifth stage: Review and improvement i. Management review 8. Stage by stage implementation of ISO 14001 First stage: commitment and policy i. Environmental policy Second stage: Planning i. Environmental aspects ii. Legal and other requirements iii. Objectives and targets iv. Environmental management program Third stage: Implementation i. Structure and responsibility ii. Training, awareness and competence iii. Communication iv. Environmental documentation v. Document control Fourth stage: Operational control measurement and evaluation i. Monitoring and measurement ii. Non-conformance and corrective and preventive action iii. Records iv. Environmental management system audit Fifth stage: Review and improvement i. Management review i. Management review 9. Benefits of developing ISO 14000 series . Having a single, global set of environmental management system guidance standard 2. The development of a common, global approach to voluntary and self-directed environmental management. 2. The development of a common, global approach to voluntary and self-directed environmental management. 2. The development of a common, global approach to voluntary and self-directed environmental management. 3. Enhancement of the ability to measure levels of sustainability and environmental performance through auditing methods. 3. Enhancement of the ability to measure levels of sustainability and environmental performance through auditing methods. 3. Enhancement of the ability to measure levels of sustainability and environmental performance through auditing methods. 4. Harmonization of non-uniform standards for a range of environmental impact iss 4. Harmonization of non-uniform standards for a range of environmental impact issues. 10. Implelementation OF ISO 14000 in luggage manufacturing industry: 7. Noise generation. 7. Noise generation. g The complete canteen as process showing input and output as well its effects on environment are tabulated in aspect register as shown in Table I. g The complete canteen as process showing input and output as well its effects on environment are tabulated in aspect register as shown in Table I. p g Each aspect is studied carefully for the following category of aspects: 1. Normal aspects: Effects of aspects are negligible. 2. Abnormal Aspects: Severe effect on environment. 3. Emergency Aspects: Nature of Aspect is emergency. Emergency prepared plan has to be there. Proper training to be given to the people for handling the emergency situations. They should be equipped with safety devices to tackle the emergency. For example: Suppose if there is a fire hazard. Whether the fire fighting equipment is there or not. If it is there then whether the people are trained to operate it and so on. 3. Emergency Aspects: Nature of Aspect is emergency. Emergency prepared plan has to be there. Proper training to be given to the people for handling the emergency situations. They should be equipped with safety devices to tackle the emergency. For example: Suppose if there is a fire hazard. Whether the fire fighting equipment is there or not. If it is there then whether the people are trained to operate it and so on. p p p ergency preparedness plan for every department, which will consist of the following few nts: p . Fire equipments are available or not that too in proper place. A: Occurrence 10. Implelementation OF ISO 14000 in luggage manufacturing industry: The study is done for Canteen and Tools and Mould repairing. y p g CANTEEN: Firstly the various input to canteen are identified viz. Vegetables, spices, oil, food grains, water, LPG gas, Electricity. Output of the canteen is Food products. The various effects of the canteen on the environment as a whole are identified CANTEEN: Firstly the various input to canteen are identified viz. Vegetables, spices, oil, food grains, water, LPG gas, Electricity. Output of the canteen is Food products. The various effects of the canteen on the environment as a whole are identified 1. Used water discharged to drainage. 1. Used water discharged to drainage. 1. Used water discharged to drainage. 2. Fire in L. P. G. (Emergency). 2. Fire in L. P. G. (Emergency). 3. Solid waste non biodegradable (Carboys, drums, containers, empty milk bags) 3. Solid waste non biodegradable (Carboys, drums, containers, empty milk bags). 4. Solid waste biodegradable (spent food and vegetables). 5. Fume generation. 5. Fume generation. 6. Heat loss to atmosphere. 6. Heat loss to atmosphere. 302 Environmental Management in Practice D: Controls F: Category of waste The factor rating of impacts for each aspect is shown in Table II. g p p After categorisation of aspects, their impact has to be ascertained. Then whether they can be measured or not. If measured, what is the present status? Then the frequency of occurrence is to be known. Further what is the treatment given to nullify the bad effects of aspects on environment? All these analysis is depicted in Table III. After rating and critical analysis of each aspect one should have the action plan for the aspect, which is affecting severely on environment. Meetings should be conducted to take reviews on the improvement after the implementation of action plan. The improved status to be maintained and periodic review are done to ascertain. In the similar way study is carried out for process of TOOL AND MOULD REPAIR and the reports are shown in Table IV, V and VI. A: Occurrence Continuous (8hours & above)/day 5 Less than 8 hours/day 4 Less than 8 hours/week 3 Less than 8 hours/month 2 Less than 8 hours/year 1 C: Significance of Impact C: Significance of Impact Causing death to human being on site/ offsite, damage to flora fauna, air, water, land (offsite). 5 Damage to flora fauna, air, water, land or hospitilisation to human being on site. 4 Damage to flora fauna, air, water, land or first aid to human being on shop 3 Damage to flora fauna, air, water, land and or first aid to human being on shop 2 Negligible impact 1 Implementation of ISO 14000 in L M f t i I d t Implementation of ISO 14000 in Luggage Manufacturing Industry: A Case Study 303 D: Controls Controls absent 5 Controls present 4 Controls present but needs human intervention 3 Controls present no human intervention 2 Closed loop control 1 E: Frequency of Detection Once in a year & above 5 Once in six months 4 Once in one to three months 3 Once in a week 2 Once in a day 1 F: Category of waste Hazardous waste 5 Non-hazardous waste (High) 4 Non-hazardous waste (Low) 3 100% recycled in house or recycled from outside party for inside use 2 Negligible waste generation 1 11. Conclusion With the implementation of EMS, communications concerning environmental practices were streamlined. It was possible to identify areas where utility savings existed. EMS defined roles and responsibilities towards each aspect of the process and their impacts on the environment. A systematic approach is understood to handle environmental issues in place and the overall plant cost savings coming from tracking resources and accounting for them. The EMS also provides the more intangible benefit of employee taking pride of working in the plant that is a good environmental neighbour. 304 Environmental Management in Practice Table No. I LUGGAGE INDUSTRIES LTD. ASPECT REGISTER DOC No. : ASP – 27 SHEET No. : 1 of 3 Dept. : Pers & Admn ISSUE DATE : 1-08-2003 REV No. : 00 Process :- Canteen REV. DATE : 00 Prepared by : Approved By : CANTEEN Noise Generation Heat loss to atmosphere Fume Generation Food products Solid waste biodegradable (spent food and Vegetable) Solid waste non biodegradable (carboys, drums, containers, empty milk bags) Fire in L.P.G. (Emergency) Used water discharged to drainage Vegetables, spices, Electricity Water Consumption L.P.G. Gas Fume Generation Food products Solid waste non biodegradable (carboys, drums, containers, empt Solid waste non biodegradable (carboys, drums, containers, empty milk bags) Prepared by : Approved By : Implementation of ISO 14000 in Luggage Manufacturing Industry: A Case Study 305 Table No. II LUGGAGE INDUSTRIES LTD. ASPECT REGISTER DOC No. : ASP – 27 SHEET No. : 3 of 3 Dept. : Pers & Admn ISSUE DATE : 1-08-2003 REV No. : 00 Process :- Canteen REV. DATE : 00 Sr. No. ASPECT FACTOR RATING TOTAL RATING IS IT SIGNIFICANT A B C D E F NORMAL ASPECTS 01 Fume Generation 5 1 1 2 5 1 15 No 02 Heat loss 5 1 1 2 5 1 15 No 03 Noise generation -- -- -- -- -- S T A T U T O R Y -- -- -- -- -- YES 04 Discharge of used water 5 3 1 3 3 3 18 YES 05 Solid waste biodegradable spent food vegetable 5 3 1 3 3 3 18 YES 06 Solid waste non biodegradable corboys drums containers etc. 2 1 1 3 3 3 13 No ABNORMAL ASPECTS 07 Nil Nil Nil Nil Nil Nil Nil Nil No EMERGENCY ASPECTS 08 Fire in L.P.G. Storage area YES Prepared by : Approved By : Table No. 11. Conclusion II 306 Environmental Management in Practice Table No. III LUGGAGE INDUSTRIES LTD. ASPECT REGISTER DOC No. : ASP – 27 SHEET No. : 2 of 3 Dept. : Pers & Admn ISSUE DATE : 1-08-2003 REV No. : 00 Process :- Canteen REV. DATE : 00 Sr. No. Aspect Impact Measur. Indicator Present status Frequency of Present treatment Type of control Remarks reference Occur Meas NORMAL ASPECTS 01 Fume generation Air Pollution Not measured Not measured Cont. Not Measured Nil Nil Nil 02 Heat loss Ambient warming Deg. Celsius Not measurable Cont. Not measurable Nil Nil Nil 03 Noise generation Noise pollution dB 72 dB Cont. Negligible Nil Statutory Noise level report 04 Solid waste biodegradable spent food and vegetable Land Contamination Kgs/Day 45 –50 kgs Cont. Once in a day Disposed to animal feeder Self EMP-P&A01 05 Discharge of used water to drainage Water pollution K Ltrs. Not measured Daily Not measured Disposed to sewage Self EMP-P&A02 06 Solid waste non- biodegradable carboys, drums, containers Land contamination Nos./month 18 Tins 20 Gunny bags 900 milk bags Cont. Once in a month Disposed to recyclers Self Nil ABNORMAL ASPECTS 07 Nil Nil Nil Nil Nil Nil Nil Nil Nil EMERGENCY ASPECTS 08 Fire in L.P.G. storage area Damage to flora fauna, air, land, human being & assets - No incident so far - - - - Emergency preparedness & response plan Prepared by : Approved By : Implementation of ISO 14000 in Luggage Manufacturing Industry: A Case Study 307 Table No. IV LUGGAGE INDUSTRIES LTD ASPECT REGISTER DOC No. : ASP – 13 SHEET No. : 1 of 3 Dept. : Tool Room ISSUE DATE : 1-08-2003 REV No. : 01 Process :- Tool / Mould Repairing REV. DATE : 18/01/2004 Prepared by : Approved By : TOOLS & MOULD REPAIRING Heat loss to atmosphere Dust generation Noise generation Finished / Semifinished Tools & Moulds Chips generation Coolant / Oil leakage Cotton waste M.S. Plate, Steel round, Copper plate / round, Graphite blocks, Water for machine coolant, machine oil LUGGAGE INDUSTRIES LTD M.S. Plate, Steel round, Copper plate / round, Graphite blocks, Water for machine coolant, machine oil Heat loss to atmosphere Finished / Semifinished Tools & Moulds TOOLS & MOULD REPAIRING Approved By : Approved By : Prepared by : Prepared by : 308 Environmental Management in Practice Table No. V Table No. V Table No. 11. Conclusion V LUGGAGE INDUSTRIES LTD ASPECT REGISTER DOC No. : ASP – 13 SHEET No. : 3 of 3 Dept. : Tool Room ISSUE DATE : 1-08-2003 REV No. : 01 Process :- Tool/ Mould Repairing REV. DATE : 18/01/2004 Sr. No. ASPECT FACTOR RATING TOTAL RATING IS IT SIGNIFICANT A B C D E F NORMAL ASPECTS 01 Noise generation -- -- -- -- -- S T A T U T O R Y -- -- -- -- -- YES 02 Heat loss to atmosphere 5 1 1 3 5 1 16 NO 03 Dust generation 3 1 1 5 5 1 16 NO 04 Cotton waste generation 5 1 1 3 3 3 16 NO 05 Metal scrap 5 1 1 3 3 1 14 NO 06 Spent coolant -- -- -- -- -- S T A T U T O R Y -- -- -- -- -- YES ABNORMAL ASPECTS 07 Oil leakage -- -- -- -- -- S T A T U T O R Y -- -- -- -- -- YES EMERGENCY ASPECTS 08 Nil Nil Nil Nil Nil Nil Nil Nil nil Prepared by : Approved By : Implementation of ISO 14000 in Luggage Manufacturing Industry: A Case Study 309 Table No. VI LUGGAGE INDUSTRIES LTD. ASPECT REGISTER DOC No. : ASP – 13 SHEET No. : 2 of 3 Dept. : Tool Room ISSUE DATE : 1-08-2003 REV No. : 01 Process :- Tool / Mould Repairing REV. DATE : 18/01/2004 Sr. No. Aspect Impact Measur. Indicator Present status Frequency of Present treatment Type of control Remarks reference Occur Meas NORMAL ASPECTS 01 Noise generation Noise pollution dB 80 Cont. Once in 6 months Nil Statutory Measurement of noise level report 02 Dust generation (By Grinding) Air Pollution Mg./Nm3 Negligible Cont. Not measured Nil Nil Nil 03 Heat loss to atmosphere Ambient warming  Cent. Max. 5C above ambient te perature Cont. Not Measured Nil Nil Nil 04 Cotton waste generation Land Contamination Kgs Approx. 60 Kgs/Month Cont. Monthly Disposed to recyclers Nil Monthly issue from store 05 Metal scrap Land Contamination Kgs. Appr x. 30 Kgs/Month Cont. Monthly Disposed to recyclers Nil Scrap ticket 06 Spent coolant Water pollution Ltrs. Approx. 30 Ltr./Month Cont. Monthly Effluent treatment plant Statutory Register for spent coolant to powder coating ABNORMAL ASPECTS 07 Oil leakage Land Contamination Ltrs. 11. Conclusion Negligible Rare Not Measured Secondary containment Statutory Nil EMERGENCY ASPECTS 08 Nil Nil Nil Nil Nil Nil Nil Nil nil Prepared by : Approved By : Table No. VI Table No. VI 310 Environmental Management in Practice 12. References [1] Ambika Zutshi and Amrik S. Sohal, (2000), Environmental management systems auditing: auditors’ experiences in Australia, Int. J. Environment and Sustainable Development, Vol. 1, No. 1, pp 73-87. [2] Subhash Babu, A., Madhu, K. and Sahani, N. (1998), Positioning ISO 14000 standards an investigative study covering selected Indian Industries, Proc. ISME Conference, Dec 1998, IIT Delhi, pp 286-291. [3] Fabio Orecchini, (2000), The ISO 14001 certification of a machine process, Journal of Cleaner Production, Vol. 8, Issue 1, February 2000, pp 61-68. [4] Martin, R 1998, ISO 14001 Guidance Manual, National Centre for environmental decision-making research: Technical report, viewed 23 August 2010 [5] Company manual. [5] Company manual. 1 See the Clean Japan Center (2005 and 2006) for details of this survey data. The Statistical Distributions of Industrial Wastes: an Analysis of the Japanese Establishment Linked Input-output Data Hitoshi Hayami1 and Masao Nakamura2 1Faculty of Business and Commerce, Keio University 2Sauder School of Business, University of British Columbia 1Japan 2Canada Hitoshi Hayami1 and Masao Nakamura2 1Faculty of Business and Commerce, Keio University 2Sauder School of Business, University of British Columbia 1Japan 2Canada Technical Aspects of Environmental Management 16 .1 Economic input-output-LCA: the theoretical background 2.1 Economic input-output-LCA: the theoretical background The input-output analysis is a powerful tool to evaluate environmental impacts within an interdependent economic system (Leontief 1970, Baumol and Wolff 1994). When production of a final product requires intermediate goods (e.g. parts), inter-industry effects along a supply chain generate various wastes in stages of the life cycle of the final product. The input-output (I-O) table is like a recipe of all economic activities for a national economy. Each column describes all the inputs used for an immediate economic activity, such as producing an automobile, supplying services such as education. It covers all economic activities and I-O relations are described in monetary terms. Recently publicly available I-O tables have been applied to the Economic Input-Output Life Cycle Assessment (EIO-LCA) (Hendrickson et al., 2006; Suh, 2010). Eiolca.net summarizes limitations of EIO-LCA compared to Process-Based LCA. 2.1 Economic input output LCA: the theoretical background The input-output analysis is a powerful tool to evaluate environmental impacts within an interdependent economic system (Leontief 1970, Baumol and Wolff 1994). When production of a final product requires intermediate goods (e.g. parts), inter-industry effects along a supply chain generate various wastes in stages of the life cycle of the final product. The input-output (I-O) table is like a recipe of all economic activities for a national economy. Each column describes all the inputs used for an immediate economic activity, such as producing an automobile, supplying services such as education. It covers all economic activities and I-O relations are described in monetary terms. Recently publicly available I-O tables have been applied to the Economic Input-Output Life Cycle Assessment (EIO-LCA) (Hendrickson et al., 2006; Suh, 2010). Eiolca.net summarizes limitations of EIO-LCA compared to Process-Based LCA. p One such limitation that EIO-LCA is difficult to apply to an open economy is overcome by using the methods given by us (Hayami & Nakamura, 2007). The most apparent disadvantage of EIO-LCA is that product assessments contain aggregate data containing uncertainty as Eiolca.net describes. Assume there are n commodities (including services) in an economy, each of which is an input for production of other commodities. A typical producer k produces output xj(k) of j-th commodity, which requires as inputs Xij(k) , where i=1,2,...,n. Governments provide the official I-O table with aggregate figures for all producers of j-th commodity xj=∑k=1mjxi(k), where mj is the number of producers of the j-th commodity. 1. Introduction Both waste management policies and the economic theories underlying them model the behaviour of a representative company or establishment using. For example, toxic wastes such as dioxin are regulated by the mean emission volume standard measured per Nm3, where the mean is estimated using data. As we will show, most establishments (particularly combustion plants) satisfy the required emission standard, while only a few exceed the regulation limit and must be checked by the authorities until regulation standards are met. But regulators must monitor all establishments incurring unnecessary costs. g g y Fullerton and Kinnaman 1995, among other theoretical contributions, show that taxing downstream establishments can achieve the second best policy. (See also Walls & Palmer 1998, who discuss more general market conditions.) Recent research shows that regulating downstream establishments promotes research and development by firms in upstream stages of a supply chain under certain market conditions (Calcott & Walls, 2000; Greaker & Rosendahl, 2006). These theoretical implications are important for policy making about how to design a tax system, but these theories also assume a typical producer and the regulation standard with respect to their mean emissions of waste materials. In practice, however, even though the coefficients of variation for the distributions of heavy metals in fly ash found in municipal solid waste are known to reach 50% (Nakamura et al., 1997), little statistical evidence in the published literature exists on the variation in industrial establishments’ waste generation and reuse-recycling per unit production, which is basic information required for economic and ecological design and general policy decisions. I hi fill hi i h li d h h di ib i f i g g g y In this paper we fill this gap in the literature and show the distributions of generation rates for various types of wastes and by-products in the production processes of establishments in Japanese manufacturing industries. We use the METI survey data (Survey on the Industrial Waste and By-Products, Japanese Ministry of Economy, Trade and Industry, 2005 and 2006). This survey gives the amounts of 37 types of industrial wastes generated for four different levels of the production processes (generation, intermediate reduction, reuse-recycle, and disposal to landfill) at 5048 establishments.1 314 Environmental Management in Practice We have linked the METI survey data with the Japanese Input-Output (I-O) table. 1. Introduction Using this linked data and the data on energy/CO2 requirements in industrial waste treatment, we are able to calculate the induced amounts of industrial wastes.2 For example, waste oil and waste plastic are generated in large quantities at 3080 and 3694 establishments, respectively. Estimated amounts of waste oil and waste plastic generated range, respectively, between 0 and 2.50 and between 0 and 2.11 (metric) tonnes per million yen of output. On the other hand, waste ferroalloy slag is produced at only 11 establishments, and its quantity ranges from 5.8 to 64.6 tonnes per million yen of output. We estimate that production of every car with a 2000cc engine or its equivalent induces, for example, 0.051 tonnes of all types of wastes combined in hot rolling processes and 0.677 tonnes of all types of wastes combined in iron steel making in upstream production activities. We estimate that a 2000cc equivalent automobile production generates 1.49 tonnes of all types of wastes combined. We believe that these averages and the distributions for waste generation rates along a production supply chain provide (currently unused) useful information for policy makers for further reductions in the generation of waste materials. 2 Induced amounts of output mean the amounts of output generated by upper (supplier) stages of a production supply chain in response to the production activities undertaken at downstream establishments. .1 Economic input-output-LCA: the theoretical background The Statistical Distributions of Industrial Wastes: A l i f th J E t bli h t Li k d The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data 315 ical Distributions of Industrial Wastes: s of theJapanese Establishment Linked Input-output Data 315 an Analysis of theJapanese Establishment Linked Input-output Data an Analysis of theJapanese Establishment Linked Input-output Data an Analysis of theJapanese Establishment Linked But these input coefficients Aij are different from producer k’s input coefficients Aij ( ) ( ) ( ) k k k i j i j j A X x  , 1,2, , i j n   and 1,2, , j k m   (2) (2) Similarly, by applying EIO-LCA to waste management with the same assumptions made above, we get the amount of waste i generated in producing output xj (we consider 37 waste materials as defined below): ( ) ( ) 1 1 m m k k i j i j j i j j k k j j W Waste x W x      1,2, ,37 i   1,2, , j n   (3) (3) Similarly, producer (k) generates i-th waste producing the j-th product: producer (k) generates i-th waste producing the j-th product: ( ) ( ) ( ) k k k i j i j j W Waste x  1,2, ,37 i   , 1,2, , j n   and 1,2, , j k m   (4) Japan Ministry of Economy, Trade and Industry (METI) conducts an annual survey that reports the amounts of 37 types of wastes observed in 4 stages: amounts generated by final production, Wij(k); amounts of reduction in intermediate steps of production, Vij(k); amounts recycled, Uij(k); and amounts sent for landfill, Tij(k).3 The most important assumption in our I- O analysis is that input coefficients and waste coefficient per output remain constant over time. If we can show empirically that these coefficients have narrow bell shape distributions, then the relative stability of these coefficients follows. In this paper, we will show using our data how the coefficients of waste generation Wij(k) distribute. g g j Using input coefficients, Aij, we can calculate the demand for goods made in stages of upstream sectors of a supply chain. 3 Vij(k) is defined as: Vij(k)=Intermediate Reduction/Waste Generated (Wasteij(k)); and Uij(k) and Tij(k) are similarly defined. The denominator is the amount of waste generated, rather than production output. The waste generated is measured at the gate of an industrial process. Generated wastes are reduced (sludge dewatering), recycled/reused, and finally disposed of (mainly by landfill). Waste reduction is often undertaken in production processes, for example, for reducing the failure rate (or increasing yields) for the processes. .1 Economic input-output-LCA: the theoretical background The same aggregation procedure is applied to inputs as follows: Xij=∑k=1mj Xij(k). EIO-LCA assumes that matrix of input coefficients Aij defined below is stable and represents a typical producer’s activity. ( ) ( ) 1 1 m m k k i j i j j i j j k k j j A X x X x      , 1,2, , i j n   (1) (1) 2 Induced amounts of output mean the amounts of output generated by upper (supplier) stages of a production supply chain in response to the production activities undertaken at downstream establishments. .1 Economic input-output-LCA: the theoretical background Unit production of j-th sector output induces production of i-th sector whose output is given by Aij. Similarly production of Aij induces production of Aki Aij in k-th sector. Repeating this, we can obtain output induced for any stage in upstream portions of a supply chain. Formally, multiplication of the I-O coefficients matrix A from left gives us induced output for all relevant goods and services in the immediate upstream stage of a supply chain. 2 , , , f A f A f  (5) 2 , , , f A f A f  (5) where f is a vector of demands for final goods and services where f is a vector of demands for final goods and services By multiplying production output for final production (downstream) stage and subsequent upstream stages (f, Af, ...) by waste generation matrix W, we obtain the amounts of waste generated in the corresponding stages of a supply chain: Wf, WAf, WA2f, .... By multiplying production output for final production (downstream) stage and subsequent upstream stages (f, Af, ...) by waste generation matrix W, we obtain the amounts of waste generated in the corresponding stages of a supply chain: Wf, WAf, WA2f, .... 2.2 Construction of a linked data set To properly allocate output of steel industry establishments in WBS among relevant I-O sectors, we have collected needed information by interviewing the Japan Iron and Steel Federation and the Nippon Slag Association. We then modified the allocation table to reflect our information. Secondly, in order to obtain the total amounts of industrial wastes in Japan, we multiplied the amounts derived from WBS by the proportionality constant since WBS is a survey and does not cover all Japanese establishments. The proportionality constant for each sector was obtained by comparing sales figures for the sector from the Census of Manufacturers data and WBS. Sectors of these two data sets are comparable since both use the JSIC system to define their sectors. Table 1 lists 37 types of industrial wastes discussed in this paper. Industrial wastes in Japan are classified into (1)37 types given in Table 1 and (2)especially regulated industrial wastes. Special industrial wastes in the latter category (2) are highly hazardous and include material contaminated with PCB, asbestos, strong acid with pH less than 2, strong alkali with pH higher than 12.5, highly inflammable waste oil and infectious wastes. WBS excludes wastes in category (2) that need to be treated separately. Industrial wastes other than those in category (2) include certain toxic substances (e.g. heavy metals, Pb, Cd) that must be treated properly. For each establishment and each type of waste, the following material balance equation must hold: ( ) ( ) ( ) k k k i j i j j Waste W x  and ( ) ( ) ( ) 1 k k k i j i j i j V U T    i=1,...,37 ; j=1,...,401, and k=1,...,5048 (6 (6) All wastes are measured by weight in metric tonnes, and output xj(k) is measured in monetary unit (in 1 million yen). All wastes are measured by weight in metric tonnes, and output xj(k) is measured in monetary unit (in 1 million yen). 2.2 Construction of a linked data set We briefly describe the procedure we used to link the Wastes and By-products Survey (WBS) data to the I-O table. We first note that the definition of a sector is different between the two data sets. WBS is based on the Japan Standard Industry Classification (JSIC) system, 316 Environmental Management in Practice but the I-O table uses its own more detailed classification system so that the stability of I-O coefficients over time is preserved. JSIC codes are divided into one or more of 401 I-O sectors, using the allocation matrix given in the appendix tables of the I-O table. This allocation method depends on the sales figures reported for different products of each establishment in WBS. One difficulty we encountered was for the steel industry sector. The steel industry in the I-O table is divided into 13 sectors and two related sectors (coal products and self power generation). Many of these I-O sectors belong to a single establishment in WBS because of their continuous casting production, and there are no sales figures reported on WBS for transactions for these I-O sector goods since these transactions occur within the same establishment. To properly allocate output of steel industry establishments in WBS among relevant I-O sectors, we have collected needed information by interviewing the Japan Iron and Steel Federation and the Nippon Slag Association. We then modified the allocation table to reflect our information. but the I-O table uses its own more detailed classification system so that the stability of I-O coefficients over time is preserved. JSIC codes are divided into one or more of 401 I-O sectors, using the allocation matrix given in the appendix tables of the I-O table. This allocation method depends on the sales figures reported for different products of each establishment in WBS. One difficulty we encountered was for the steel industry sector. The steel industry in the I-O table is divided into 13 sectors and two related sectors (coal products and self power generation). Many of these I-O sectors belong to a single establishment in WBS because of their continuous casting production, and there are no sales figures reported on WBS for transactions for these I-O sector goods since these transactions occur within the same establishment. 3.1 Distributions of unit waste generation rates The first objective of this paper is to estimate the statistical distributions of waste generation rates among establishments. Table 2 presents descriptive statistics for these waste generation rates, Wij(k)/xj(k), for waste of type i for establishment k in sector j. The number of observations (Nobs) denotes the number of establishment with non-zero production, xj(k)>0. Waste plastics other than synthetic rubber have the largest number of observations, which means waste plastics are the most common industrial waste. For all industrial wastes except waste animal-solidified, the sample mean is larger than the median, and the maximum value is far larger than the sample mean. This means that the distributions of unit waste generation rates W/x are asymmetric to left, with a few smaller values occur with very high frequencies and a long tail for large values. The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data 317 JSIC: 0110 Cinders other than coal 0111 Coal cinders 0210 Inorganic sludge other than polishing sand 0211 Inorganic sludge of polishing sand 022 Organic sludge 0230 Organic-inorganic mixed sludge other than polishing sand 0231 Organic-inorganic mixed sludge of polishing sand 031 Waste oil other than chlorinated solvent waste 032 Waste oil chlorinated solvent waste 040 Used acidic liquid 050 Waste alkali 061 Waste plastics other than synthetic rubber 062 Waste plastics synthetic rubber 070 Wastepaper 080 Chips and sawdust 090 Waste textile 100 Animal and vegetable remnants 101 Waste animal-solidified 110 Rubber waste 121 Scrap iron 122 Non-ferrous metal scrap 131 Scrap glass 132 Clay, porcelain, ceramic scrap 133 Scrap slab concrete 141 Waste moulding sands 1420 Slag other than steel, ferroalloy, and copper 1421 Iron-steel slag 1422 Ferroalloy slag 1423 Copper slag 1430 Slag other than aluminum dross 1431 Aluminum dross 150 Demolition debris 160 Animal manure 170 Animal carcasses 1800 Soot and dust other than coal ash 1810 Soot and dust flay ash 190 Processed material for disposal Table 1. 37 waste materials reported in the Wastes and By-Products Survey (WBS) Table 1. 37 waste materials reported in the Wastes and By-Products Survey (WBS) Waste Nobs Mean Median Max SD cinders other than coal 306 0.137 0.002 20.524 1.204 coal cinders 50 0.038 0.003 0.957 0.141 inorganic sludge excl. 3.1 Distributions of unit waste generation rates polishing sand 1815 0.091 0.006 25.744 0.905 inorganic sludge polishing sand 52 0.157 0.003 6.453 0.893 organic sludge 986 0.268 0.007 47.229 2.082 organic and inorganic mixed sludge 776 0.049 0.005 1.583 0.165 mixed sludge polishing sand 18 0.171 0.003 2.829 0.664 waste oil excl. chlorinated solvent waste 3080 0.019 0.002 2.495 0.090 waste oil chlorinated solvent waste 303 0.018 0.001 1.425 0.099 used acidic liquid 1242 0.153 0.002 50.131 1.738 waste alkali 1184 0.045 0.002 2.937 0.192 waste plastics excl. synthetic rubber 3694 0.027 0.005 2.114 0.080 waste plastics synthetic rubber 282 0.038 0.005 0.610 0.071 wastepaper 2612 0.069 0.005 2.631 0.244 chips and sawdust 2089 0.035 0.002 5.796 0.295 318 Environmental Management in Practice waste textile 261 0.033 0.001 5.898 0.366 animal and vegetable remnants 443 0.103 0.002 3.109 0.309 waste animal-solidified 2 1.174 1.174 2.230 1.494 lubber waste 61 0.017 0.000 0.280 0.051 scrap iron 3037 0.066 0.008 5.991 0.233 non-ferrous metal scrap 1464 0.017 0.002 0.561 0.050 scrap glass 1492 0.012 0.000 3.612 0.140 clay, porcelain, ceramic scrap 620 0.030 0.001 3.319 0.195 scrap slab concrete 8 0.687 0.008 4.733 1.653 waste moulding sands 214 0.493 0.195 4.326 0.708 slag excl. steel, ferroalloy, and copper 74 0.316 0.052 7.561 0.966 iron-steel slag 111 1.859 1.291 22.222 2.678 ferroalloy slag 11 11.191 3.956 64.592 19.683 copper slag 17 3.479 0.050 23.219 7.228 slag other than aluminum dross 192 0.193 0.038 4.485 0.527 aluminum dross 50 0.144 0.058 1.825 0.277 demolition debris 303 0.077 0.001 19.809 1.139 animal manure 6 0.001 0.000 0.003 0.001 animal carcasses 12 0.003 0.000 0.017 0.006 soot and dust excl. coal ash 434 0.160 0.008 3.462 0.354 soot and dust fly ash 46 0.067 0.032 0.534 0.109 processed material for disposal of industrial waste 119 0.007 0.001 0.170 0.020 Table 2. Descriptive Statistics for the unit waste generation rate W/x in 2006 Table 2. Descriptive Statistics for the unit waste generation rate W/x in 2006 Several typical shapes of statistical distributions are shown in Figures 1a and 1b. Figure 1a shows the distributions for waste moulding sands and iron and steel slag. Iron and steel slag does not have a large distance between the mean and the median, but it has a large maximum, 22.22 tonnes per 1 million yen, which is 12 times as large as the mean, 1.859 tonnes per 1 million yen. 3.1 Distributions of unit waste generation rates Standard deviation (SD) is larger than the mean, and the coefficient of variation is 1.44. Figure 1b shows two of common types of distributions for W/x for wastepaper and waste plastics, which concentrate around 0. Both have the median of 0.005 tonnes per 1 million yen of production. But the mean is 0.069 tonnes for wastepaper and 0.027 for waste plastics, with a maximum, 2.631 for wastepaper, and 2.114 for waste plastics. Extremely large maximum values may reflect irregular production and inventory practices at some establishments. p Figure 2 shows that the distributions for recycling rates for inorganic sludge and polishing sand. Both figures have concentrations around 0 and 1. This means that establishments face an all or nothing choice. Once a waste material is recycled, the establishment should choose recycling all wastes. This result follows because of the high initial cost of recycling equipment and the availability of outsourcing. But outsourcing is not available if the establishment location is far from the center of the recycling industry. As a result, the final disposal method (landfill here) is also highly concentrated around 0 and 1, as in Figure 3. We have tried statistical fitting of these empirical distributions derived here with only a partial success. First, we tried to use the Gamma distribution to fit observed distributions The Statistical Distributions of Industrial Wastes The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data 319 The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data for unit generation rate, W/x. But only 7 out of 37 distributions for industrial waste have been found not to be significantly different from the Gamma distribution. An appropriate theoretical distribution to fit the empirical distributions for recycling ratio, U/W, is the Beta distribution since recycling rations range between 0 and1. But our test of the goodness of fit rejected the Beta distribution for all cases. for unit generation rate, W/x. But only 7 out of 37 distributions for industrial waste have been found not to be significantly different from the Gamma distribution. An appropriate theoretical distribution to fit the empirical distributions for recycling ratio, U/W, is the Beta distribution since recycling rations range between 0 and1. But our test of the goodness of fit rejected the Beta distribution for all cases. a) b) Fig. 1. 3.1 Distributions of unit waste generation rates a)Typical distributions of waste generation coefficients: Waste Moulding Sands (214 establishments and Iron and Steel Slag (111 establishments), b) Typical distributions of waste generation coefficients: Wastepaper (2612 establishments), and Waste plastics other than synthetic rubber (3614 establishments) a) a) b) b) Fig. 1. a)Typical distributions of waste generation coefficients: Waste Moulding Sands (214 establishments and Iron and Steel Slag (111 establishments), b) Typical distributions of waste generation coefficients: Wastepaper (2612 establishments), and Waste plastics other than synthetic rubber (3614 establishments) 320 Environmental Management in Practice Fig. 2. Typical distributions of recycling rates: Inorganic sludge other than polishing sand (1815 establishments), and Wastepaper (2612 establishments) Fig. 2. Typical distributions of recycling rates: Inorganic sludge other than polishing sand (1815 establishments), and Wastepaper (2612 establishments) Fig. 3. Typical distributions of landfill rates: Inorganic sludge other than polishing sand of 1815 establishments (left), and wastepaper of 2612 establishments (right) Bootstrap resampling can calculate confidence intervals for the unit waste generation rate, W/x, from estimated empirical distributions. Table 3 shows simulated confidence intervals and the mean. The empirical distribution of Wij(k)/xj(k) used is based on observations from WBS 2005 and 2006. We used as re-sampling size 5000 for non-parametric estimation. The Fig. 3. Typical distributions of landfill rates: Inorganic sludge other than polishing sand of 1815 establishments (left), and wastepaper of 2612 establishments (right) Bootstrap resampling can calculate confidence intervals for the unit waste generation rate, W/x, from estimated empirical distributions. Table 3 shows simulated confidence intervals and the mean. The empirical distribution of Wij(k)/xj(k) used is based on observations from WBS 2005 and 2006. We used as re-sampling size 5000 for non-parametric estimation. The simulated mean uses weighs of output and our results correspond to unit waste generating The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data 321 rate W/x.4 We find that six out of seven wastes show the same statistical characteristics: (1)the median is smaller than the mean; and (2)the distributions have a long tail. But iron- steel slag (193 observations) has a nearly symmetric distribution as shown in Figure 4a. According to the central limit theorem, the distribution of a sample mean with a finite variance converges to the normal distribution. But our statistical test of the goodness of fit does not support gamma or normal distributions. 3.1 Distributions of unit waste generation rates The convergence in distribution to the normal distribution is not seen for distributions of other wastes either as shown in Figure 4b. The distribution for a positive random variable becomes exponential at the maximum entropy; in the present case a statistical test rejects the exponential distribution also. 2.50% 5% Median 95% 97.50% Mean Inorganic sludge 0.0073 0.0089 0.0343 0.2271 0.5347 0.0887 Sludge of polishing sand 0.0055 0.0071 0.0396 1.0490 1.1367 0.1888 Waste plastics 0.0062 0.0074 0.0227 0.0822 0.1100 0.0322 Waste paper 0.0024 0.0029 0.0156 0.4156 0.5044 0.0714 Scrap iron 0.0097 0.0119 0.0431 0.1666 0.2272 0.0623 Scrap glass 0.0001 0.0001 0.0007 0.0292 0.0822 0.0110 Iron-steel slag 1.4132 1.4787 1.9442 2.6046 2.7613 1.9810 Table 3. Simulated confidence intervals and the mean for unit waste generation rate W/x Table 3. Simulated confidence intervals and the mean for unit waste generation rate W Results for the distributions of the recycling rate using the same procedure as before are given in Table 4 and Figures 5a and 5b. Compared to distributions for the waste generation rates, distributions for the recycling rates are nearly symmetric. And the figures are clearly different from those given in Figure 2 for population the distributions (histograms) of the waste generation rate. This difference arises because, in case of distributions for recycling rates, there is the effect of aggregation of recycling rates. The sample mean is almost the same value as the sample median in Table 4. We can conclude that, for the distributions for recycling rates, U/W, for all sectors, observed values are close to both the mean and median of the simulated value and their confidence intervals are symmetric. y These results on the distributions of unit waste generation rate W/x and recycling rate U/W imply that the potential problems in policy making from assuming the representative (average) waste management activity come mostly from the distributions for unit waste generation rates W/x. The mean assumed in theory does not always reflect the typical intensity of waste generation. It also means that regulations based on the mean of a representative establishment does not always give effective regulations to the majority of establishments. Most of the establishments can clear the regulation standard, because the standard is based on the mean of the distribution. But as we have shown, the mean does not capture the essential property of the distributions underlying the waste generation rate. Table 4. Simulated confidence interval and mean of the recycling rate U/ W 3.1 Distributions of unit waste generation rates 4 This is because ( ) ( ) ( ) ( ) ( ) ( ) ( ) k k k k ij j ij j k k ij ij k k k k j j j j k Waste x Waste x W W x x x x                            , generating Wij(k) from the empirical distribution of Wij(k) and taking the weighted average gives Wij, which the Input-Output calculation uses. empirical distribution of Wij(k) and taking the weighted average gives Wij, which the Input-Output calculation uses. 322 Environmental Management in Practice a) b) Fig. 4. a) Distributions for unit waste generation rates, W/x, (bootstrapped weighted mean): Scrap Iron (left) and Iron-Steel Slag (right) b) Distributions for unit waste generation rates, W/x, (bootstrapped weighted mean): Waste plastics (left) and Wastepaper (right) a) b) b) Fig. 4. a) Distributions for unit waste generation rates, W/x, (bootstrapped weighted mean): Scrap Iron (left) and Iron-Steel Slag (right) b) Distributions for unit waste generation rates, W/x, (bootstrapped weighted mean): Waste plastics (left) and Wastepaper (right) 2.50% 5% Median 95% 97.50% Mean Inorganic sludge 0.398 0.414 0.513 0.609 0.626 0.513 Sludge of polishing sand 0.158 0.212 0.513 0.825 0.861 0.513 Waste plastics 0.546 0.552 0.584 0.616 0.622 0.584 Waste paper 0.730 0.741 0.791 0.831 0.837 0.789 Scrap iron 0.894 0.905 0.953 0.974 0.977 0.949 Scrap glass 0.436 0.480 0.677 0.858 0.886 0.679 Iron-steel slag 0.798 0.822 0.920 0.979 0.984 0.913 Table 4 Simulated confidence interval and mean of the recycling rate U/ W The Statistical Distributions of Industrial Wastes: A l i f th J E t bli h t Li k d I t t t D t The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data 323 The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data Iron-Steel Slag Frequency 0.5 0.6 0.7 0.8 0.9 1.0 0 50 100 150 200 a) Waste Plastics Frequency 0.5 0.6 0.7 0.8 0.9 1.0 0 20 40 60 80 100 Wastepaper Frequency 0.5 0.6 0.7 0.8 0.9 1.0 0 50 100 150 B) Fig. 5 a. 3.1 Distributions of unit waste generation rates Distribution of recycling rate U/W (bootstrapped weighted mean): Scrap Iron (left), and Iron-Steel Slag (right)b. Distribution of recycling rate U/W (bootstrapped weighted mean): Waste plastics (left), and Wastepaper (right) Iron-Steel Slag Frequency 0.5 0.6 0.7 0.8 0.9 1.0 0 50 100 150 200 a) a) Waste Plastics Frequency 0.5 0.6 0.7 0.8 0.9 1.0 0 20 40 60 80 100 Wastepaper Frequency 0.5 0.6 0.7 0.8 0.9 1.0 0 50 100 150 B) B) Fig. 5 a. Distribution of recycling rate U/W (bootstrapped weighted mean): Scrap Iron (left), and Iron-Steel Slag (right)b. Distribution of recycling rate U/W (bootstrapped weighted mean): Waste plastics (left), and Wastepaper (right) 3.2 Upstream waste generation: Calculation from the input-output analysis 3.2 Upstream waste generation: Calculation from the input-output analysis The second objective of this paper is to estimate the amounts of waste generated in various stages of production along a supply chain, starting from downstream production the final product to upstream production of supplies. We us the I-O table linked to the WBS data set explained in Section 2.1 above. Tables 5a and 5b, respectively, describe the total amounts of wastes generated average production supply chains for cellular phones and passenger car production in Japan in 2000. In both cases, pig iron is the most significant contributor of industrial waste. This is because production of pig iron generates heavy wastes such as iron-steel slag. The second most significant contributor is electricity for cell phones and passenger car final assembly for passenger cars. The total amounts of wastes generated are about 410 thousand tonnes for cellular phones and over 9 million tonnes for passenger car production. The cellular phone assembly sector generates relatively small amounts of wastes but the passenger car assembly sector generates large amounts of wastes. 324 Environmental Management in Practice One of the most important wastes generated in producing pig iron is iron-steel slag, whose unit generation rate distributes in a rather narrow range, has a symmetric distribution as shown in Figure 4a and its variance is smaller compared to other wastes generated in any other sectors. Unit waste generation rate for iron and steel slag lies between 1.4132 and 2.7613 at a 95% level (Table 3). 3.1 Distributions of unit waste generation rates One of the most important wastes generated in producing pig iron is iron-steel slag, whose unit generation rate distributes in a rather narrow range, has a symmetric distribution as shown in Figure 4a and its variance is smaller compared to other wastes generated in any other sectors. Unit waste generation rate for iron and steel slag lies between 1.4132 and 2.7613 at a 95% level (Table 3). Cellular phone production supply chain in Japan, 2000: final assembly and associated indirect (induced) stages of production by upstream suppliers Total amounts of wastes and by-products generated in stages of a supply chain (in tonnes) Pig iron 44,620 Electricity 42,440 Other electronic components 35,617 Copper 26,882 Plastic products 22,913 Crude steel (converters) 18,306 Paper 17,331 Cellular phone final assembly (direct stage) 13,434 Printing, plate making and book binding 13,367 Cyclic intermediates 12,002 Thermoplastics resins 9,258 Reuse and recycling 8,043 Aliphatic intermediates 7,925 Crude steel (electric furnaces) 7,782 Paperboard 6,832 Hot rolled steel 6,731 Cold-finished steel 6,151 Corrugated card board boxes 5,437 Petrochemical basic products 5,092 Lead and zinc (inc. regenerated lead) 4,490 Pulp 3,768 Other non-ferrous metals 3,696 Ferro alloys 3,621 Liquid crystal element 3,344 Integrated circuits 3,297 Other industrial inorganic chemicals 2,922 Iron and steel shearing and slitting 2,891 Electric wires and cables 2,802 Corrugated cardboard 2,791 Other metal products 2,601 Direct (final assembly of cell phones) 13,433 Total (all stages of production supply chain combined) 410,713 Table 5a. 3.1 Distributions of unit waste generation rates Generated wastes and by-products induced by cellular phone production The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data The Statistical Distributions of Industrial Wastes: A l i f h J E bli h Li k d 325 Passenger car production supply chain in Japan, 2000: final assembly and associated indirect (induced) stages of production by upstream suppliers Total amounts of wastes and by- products generated in stages of a supply chain (in tonnes) Pig iron 1,822,777 Passenger car final assembly (direct stage) 1,486,409 Crude steel (converters) 835,245 Motor vehicle parts and accessories 766,708 Electricity 422,843 Crude steel (electric furnaces) 365,982 Hot rolled steel 307,681 Internal combustion engines for motor vehicles and parts 276,001 Motor vehicle bodies 235,853 Cold-finished steel 200,976 Cast and forged materials (iron) 198,766 Ferro alloys 173,293 Coated steel 147,778 Reuse and recycling 132,619 Plastic products 120,871 Sheet glass and safety glass 108,440 Paper 89,860 Copper 84,560 Cyclic intermediates 81,456 Printing, plate making and book binding 70,531 Aliphatic intermediates 57,100 Thermoplastics resins 55,511 Synthetic rubber 46,305 Non-ferrous metal castings and forgings 44,044 Paperboard 39,111 Petrochemical basic products 37,843 Iron and steel shearing and slitting 37,178 Other metal products 37,135 Electrical equipment for internal combustion engines 36,679 Steel pipes and tubes 33,723 Direct (final assembly of passenger cars) 1,486,409 Total (all stages of production supply chain combined) 9,090,400 Table 5b. Generated wastes and by-products by Passenger Car production 326 Environmental Management in Practice Electricity sector also generates a significant amount of waste material, fly ash. The distribution for its unit waste generation rate is shown in Figure 6, with its 95% confidence interval (0.040, 0.110). Another waste, ferroalloy slag is generated by production supply chain stages for both cell phones and passenger cars. Its unit waste generation rate has a rather irregular distribution as shown in Figure 6, with its 95% confidence interval being very wide and given by (2.47, 34.96). This suggests that waste management policies based on point estimates for the unit waste generation rate for ferroalloy waste may lead to quite erroneous implications in practice. We have shown that unit waste generation rates for various wastes generated by production supply chains distribute in different manners, sometimes with large variances and asymmetric ways. 3.1 Distributions of unit waste generation rates This means serious limitations about the accuracy of policy decision making relying on point estimates for the waste generation by production supply chains as we do in EIO-LCA and other types of life cycle analyses. Given this limitation in mind, we may still be able to use information on waste generation in upstream production stages. Table 6 shows the total amounts of all wastes combined and amounts of CO2 emissions in the final (direct) assembly stage, a few upstream stages and all stages combined of the average production supply chain for passenger cars with 2000cc engines. Table 6 gives information about the stages which generate more waste than others. Generally waste materials tend to be generated evenly along stages of a supply chain while CO2 emissions tend to be generated more unevenly and fluctuate widely along stages of a supply chain. From policy perspectives, we conclude that application of production process LCA is more difficult for CO2 emissions than for generation of the 37 waste materials. Soot and Dust Fly Ash Frequency 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0 20 40 60 80 100 120 140 Ferroalloy Slag Frequency 0 10 20 30 40 50 0 50 100 150 200 Fig. 6. Distribution of the unit waste generation rate W/x (bootstrapped weighted mean): Fly ash (left) and ferroalloy slag (right) Soot and Dust Fly Ash Frequency 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0 20 40 60 80 100 120 140 Ferroalloy Slag Frequency 0 10 20 30 40 50 0 50 100 150 200 Fig. 6. Distribution of the unit waste generation rate W/x (bootstrapped weighted mean): Fly ash (left) and ferroalloy slag (right) The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data The Statistical Distributions of Industrial Wastes: an Analysis of theJapanese Establishment Linked Input-output Data 327 All wastes combined (summed in weight) Each Stage Cumulative (in tonnes) (in tonnes) Ratio direct stage (final assembly) 0.244 0.244 0.164 1st indirect stage 0.263 0.507 0.340 2nd indirect 0.226 0.733 0.491 3rd indirect 0.255 0.988 0.662 4th indirect 0.232 1.219 0.817 …… …… …… ….. Total (all stages combined) 1.493 1 CO2 emissions Each Stage Cumulative (in tonnes) (in tonnes) Ratio Direct 0.108 0.108 0.020 1st Indirect 0.707 0.814 0.155 2nd Indirect 1.206 2.020 0.384 3rd Indirect 1.152 3.172 0.602 4th Indirect 0.897 4.069 0.773 …… ….. ….. ….. Total 5.266 1 Table 6. 4. Conclusion Using the datasets that recently became available, we have obtained empirical distributions for generation, recycling and landfill rates for the 37 types of waste materials that are generated in the production processes of Japanese manufacturing establishments. Some of the statistics reported are for the total amounts of all the wastes combined to save space. Many empirical distributions obtained are not symmetric and have a long tail with the mean much larger than the median, making it inappropriate for policy decision making based on the mean generation rates. For example, if the regulation level is set at the industry mean, it is likely that most establishments satisfy the regulation level without efforts while a few large violators exceed the level by a big margin. In such a case it is more cost effective to set the regulation standard at a level much higher than the mean, thus saving the monitoring costs at most establishments while spending efforts to identify the few violators. In the second part of the paper we have shown how to estimate the amounts of wastes generated along stages of the average production supply chain and then given estimates for production processes of cellular phones and passenger cars. We have repeated this for emissions of carbon dioxide. In this supply chain analysis, we have shown that, given the large amounts of wastes generated in stages of upstream production supply chains, it is misleading to formulate waste management policies based only on the wastes generated in the final demand stages of supply chains. Our estimation results suggest that, in setting waste management policies, policy makers need to consider (1)not only the wastes generated from the final assembly stage but also the wastes generated from upstream stages of production supply chains and (2)such policies need to have different regulation standards for upstream stages depending on the final sector product and also the waste being considered to be Using the datasets that recently became available, we have obtained empirical distributions for generation, recycling and landfill rates for the 37 types of waste materials that are generated in the production processes of Japanese manufacturing establishments. Some of the statistics reported are for the total amounts of all the wastes combined to save space. 3.1 Distributions of unit waste generation rates Total wastes combines and CO2 generated by stages of the average production supply chain in Japan: passenger cars with 2000cc engines Table 6. Total wastes combines and CO2 generated by stages of the average production supply chain in Japan: passenger cars with 2000cc engines 4. Conclusion Many empirical distributions obtained are not symmetric and have a long tail with the mean much larger than the median, making it inappropriate for policy decision making based on the mean generation rates. For example, if the regulation level is set at the industry mean, it is likely that most establishments satisfy the regulation level without efforts while a few large violators exceed the level by a big margin. In such a case it is more cost effective to set the regulation standard at a level much higher than the mean, thus saving the monitoring costs at most establishments while spending efforts to identify the few violators. In the second part of the paper we have shown how to estimate the amounts of wastes generated along stages of the average production supply chain and then given estimates for production processes of cellular phones and passenger cars. We have repeated this for emissions of carbon dioxide. In this supply chain analysis, we have shown that, given the large amounts of wastes generated in stages of upstream production supply chains, it is misleading to formulate waste management policies based only on the wastes generated in the final demand stages of supply chains. Our estimation results suggest that, in setting waste management policies, policy makers need to consider (1)not only the wastes generated from the final assembly stage but also the wastes generated from upstream stages of production supply chains and (2)such policies need to have different regulation standards for upstream stages depending on the final sector product and also the waste being considered to be 328 Environmental Management in Practice regulated. For example, we have found that the amounts of CO2 emissions vary significantly from one stage to another of the Japanese production supply chain for passenger cars. 5. Acknowledgments This research was in part supported by the Keio University Fukuzawa Fund and the Social Sciences and Humanities Research Council of Canada. 6. Endnotes An earlier version of this paper was presented at the 18th International Input-Output Association Conference held at the University of Sydney in Australia, June 20-25, 2010. Preparation of the datasets used was done using Programming Language Pyhon 2.7 and statistical analyses were done using R 2.12.1. Further details are available by e-mailing: hayami@sanken.keio.ac.jp. 1. Introduction It is well known the paper production (likewise the other brands of industry) has enormous effects on the environment. The using and processing of raw materials has a variety of negative effects on the environment. g At the other hand there are technologies which can moderate the negative impacts on the environment and they also have a positive economical effect. One of these processes is the recycling, which is not only the next use of the wastes. The main benefit of the recycling is a double decrease of the environment loading, known as an environmental impact reducing. From the first view point, the natural resources conserves at side of the manufacturing process inputs, from the second view point, the harmful compounds amount leaking to the environment decreases at side of the manufacturing process outputs. The paper production from the recycled fibers consumes less energy; conserves the natural resources viz. wood and decreases the environmental pollution. The conflict between economic optimization and environmental protection has received wide attention in recent research programs for waste management system planning. This has also resulted in a set of new waste management goals in reverse logistics system planning. Pati et al. (2008) have proposed a mixed integer goal programming (MIGP) model to capture the inter- relationships among the paper recycling network system. Use of this model can bring indirectly benefit to the environment as well as improve the quality of waste paper reaching the recycling unit. In 2005, the total production of paper in Europe was 99.3 million tonnes which generated 11 million tonnes of waste, representing about 11% in relation to the total paper production. The production of recycled paper, during the same period, was 47.3 million tonnes generating 7.7 million tonnes of solid waste (about 70% of total generated waste in papermaking) which represents 16% of the total production from this raw material (CEPI 2006). The consumption of recovered paper has been in continuous growth during the past decades. According to the Confederation of European Paper Industries (CEPI), the use of recovered paper was almost even with the use of virgin fiber in 2005. This development has been boosted by technological progress and the good price competitiveness of recycled fiber, but also by environmental awareness – at both the producer and consumer ends – and regulation that has influenced the demand for recovered paper. The Effects of Paper Recycling and its Environmental Impact Iveta Čabalová, František Kačík, Anton Geffert and Danica Kačíková Technical University in Zvolen, Faculty of Wood Sciences and Technology Slovakia Iveta Čabalová, František Kačík, Anton Geffert and Danica Kačíková Technical University in Zvolen, Faculty of Wood Sciences and Technology Slovakia 17 The Effects of Paper Recycling and its Environmental Impact Iveta Čabalová, František Kačík, Anton Geffert and Danica Kačíková Technical University in Zvolen, Faculty of Wood Sciences and Technology Slovakia 17 7. References mol, W.J. & Wolff, E.N. (1994) A Key Role for Input-Output Analysis in Policy Design Vol.24, 93-113. cott, P. & Walls, M. (2000) Can Downstream Waste Disposal Policies Encourage Upstream „Design for Environment“? American Economic Review, Vol.90, 233-237 The Clean Japan Center (2005 and 2006) CJC-0708 and CJC-0809. Available from http://www.cjc.or.jp/. Eiocla.net www.eiocla.nt, Carnegie Mellon. Fullerton, D. & Kinnaman, T.C. (1995) Garbage, Recycling, and Illicit Burning or Dumping, Journal of Environmental Economics and Management, Vol.29, 78-91. f g Greaker, M. & Rosendahl, K.E. (2008) Environmental Policy with Upstream Pollution Abatement Technology Firms, Journal of Environmental Economics and Management, Vol.56,246-259. Hayami, H. & Nakamura, M. (2007) Greenhouse gas emissions in Canada and Japan: Sector- specific estimates and managerial and economic implications. Journal of Environmental Management, Vol. 85, 371-392. g Hendrickson, C. T.; Lave, L. B. & Matthews, H. S. (2006). Environmental Life Cycle Assessment of Goods and Services: An Input-Output Approach, Resources for the Future Press, ISBN-13 978-1933115245, Washington DC, USA. g Japan Ministry of Economy, Trade and Industry (METI) (2005 and 2006) the Waste and By- Products Surveys on Establishments („Haikibutsu-Fukusanbutsu Hasseijoukyo-tou no Chosa“). ) Japan Ministry of Internal Affairs and Communications (2000 and 2005) The Input Output Tables. Available from http://www.go.jp/ Leontief, W. (1970) Environmental Repercussions and the Economic Structure: An Input- Output Approach, Review of Economics and Statistics, Vol.52, No.3, 262-271. Nakamura, K.; Kinoshita, S. & Takatsuki, H. (1996) The Origin and Behavior of Lead, Cadmiun, and Antimony in MSW Incinerator, Waste Management, Vol.16 No.5/6, 509-517. Nakamura, K.; Kinoshita, S. & Takatsuki, H. (1996) The Origin and Behavior of Lead, Cadmiun, and Antimony in MSW Incinerator, Waste Management, Vol.16 No.5/6, 509-517. Suh, S., editor (2010) Handbook of Input-Output Economics in Industrial Ecology, Springer, ISBN-13 978-1402061547, New York, USA. Walls, M. & Palmer, K. (2001) Upstream Pollution, Downstream Waste Disposal, and the Design of Comprehensive Environmental Policies, Journal of Environmental Economics and Management, Vol. 41, 94-108. 1. Introduction The European paper industry suffered a very difficult year in 2009 during which the industry encountered more 330 Environmental Management in Practice down-time and capacity closures as a result of the weakened global economy. Recovered paper utilisation in Europe decreased in 2009, but exports of recovered paper to countries outside CEPI continued to rise, especially to Asian markets (96.3%). However, recycling rate expressed as “volume of paper recycling/volume of paper consumption” resulted in a record high 72.2% recycling rate after having reached 66.7% the year before (Fig. 1) (Hujala et al. 2010; CEPI 2006; European Declaration on Paper Recycling 2010; Huhtala & Samakovlis 2002; CEPI Annual Statistic 2010). Fig. 1. European paper recycling 1995-2009 in million tonnes (European Declaration on Paper Recycling 2006 – 2010, Monitoring Report 2009 (2010) (www.erpa.info) Fig. 1. European paper recycling 1995-2009 in million tonnes (European Declaration on Paper Recycling 2006 – 2010, Monitoring Report 2009 (2010) (www.erpa.info) Recycling is not a new technology. It has become a commercial proposition since Matthias Koops established the Neckinger mill, in 1826, which produced white paper from printed waste paper. However, there were very few investigations into the effect of recycling on sheet properties until late 1960's. From then until the late 1970's, a considerable amount of work was carried out to identify the effects of recycling on pulp properties and the cause of these effects (Nazhad 2005; Nazhad & Paszner 1994). In the late 1980's and early 1990's, recycling issues have emerged stronger than before due to the higher cost of landfills in developed countries and an evolution in human awareness. The findings of the early 70's on recycling effects have since been confirmed, although attempts to trace the cause of these effects are still not resolved (Howard & Bichard 1992). Recycling has been thought to reduce the fibre swelling capability, and thus the flexibility of fibres. The restricted swelling of recycled fibres has been ascribed to hornification, which has been introduced as a main cause of poor quality of recycled paper (Scallan & Tydeman 1992). Since 1950's, fibre flexibility among the papermakers has been recognized as a main source of paper strength. Therefore, it is not surprising to see that, for over half a century, papermakers have supported and rationalized hornification as a main source of tensile loss due to drying, even though it has never been fully understood (Sutjipto et al. 2008). 1. Introduction Recycled paper has been increasingly produced in various grades in the paper industry. However, there are still technical problems including reduction in mechanical strength for 331 The Effects of Paper Recycling and its Environmental Impact recycled paper. Especially, chemical pulp-origin paper, that is, fine paper requires a certain level of strength. Howard & Bichard (1992) reported that beaten bleached kraft pulp produced handsheets which were bulky and weak in tensile and burst strengths by handsheet recycling. This behaviour could be explained by the reduction in re-swelling capability or the reduction in flexibility of rewetted pulp fibers due to fiber hornification and, possibly, by fines loss during recycling processes, which decrease both total bonding area and the strength of paper (Howard 1995; Nazhad & Paszner 1994; Nazhad et al. 1995; Khantayanuwong et al. 2002; Kim et al. 2000). Paper recycling is increasingly important for the sustainable development of the paper industry as an environmentally friendly sound. The research related to paper recycling is therefore increasingly crucial for the need of the industry. Even though there are a number of researches ascertained the effect of recycling treatment on properties of softwood pulp fibres (Cao et al. 1999; Horn 1975; Howard & Bichard 1992; Jang et al. 1995), however, it is likely that hardwood pulp fibres have rarely been used in the research operated with recycling treatment. Changes in some morphological properties of hardwood pulp fibres, such as curl, kink, and length of fibre, due to recycling effects also have not been determined considerably. This is possibly because most of the researches were conducted in the countries where softwood pulp fibres are commercial extensively (Khantayanuwong 2003). Therefore, it is the purpose of the present research to crucially determine the effect of recycling treatment on some important properties of softwood pulp fibres. 2. Alterations of pulp fibres properties at recycling The goal of a recycled paper or board manufacturer is to make a product that meets customers΄ specification and requirements. At the present utilization rate, using recycled fibres in commodity grades such as newsprint and packaging paper and board has not caused noticeable deterioration in product quality and performance (Čabalová et al. 2009). The expected increase in recovery rates of used paper products will require a considerable consumption increase of recycled fibres in higher quality grades such as office paper and magazine paper. To promote expanded use of recovered paper, understanding the fundamental nature of recycled fibres and the differences from virgin fibres is necessary. y g y Essentially, recycled fibres are contaminated, used fibres. Recycled pulp quality is, therefore, directly affected by the history of the fibres, i.e. by the origins, processes and treatments which these fibres have experienced. McKinney (1995) classified the history into five periods: 1. fibre furnish and pulp history 2. paper making process history 3. printing and converting history 4. consumer and collection history 5. recycling process history. To identity changes in fibre properties, many recycling studies have occurred at laboratory. Realistically repeating all the stages of the recycling chain is difficult especially when including printing and deinking. Some insight into changes in fibre structure, cell wall properties, and bonding ability is possible from investigations using various recycling procedures, testing methods, and furnishes. p g Mechanical pulp is chemically and physically different from chemical pulp then recycling effect on those furnishes is also different. When chemical fibres undergo repeated drying 332 Environmental Management in Practice and rewetting, they are hornified and can significantly lose their originally high bonding potential (Somwand et al. 2002; Song & Law 2010; Kato & Cameron 1999; Bouchard & Douek 1994; Khantayanuwong et al. 2002; Zanuttini et al. 2007; da Silva et al. 2007). The degree of hornification can be measured by water retention value (WRW) (Kim et al. 2000). In contrast to the chemical pulps, originally weaker mechanical pulps do not deteriorate but somewhat even improve bonding potential during a corresponding treatment. Several studies (Maloney et al. 1998; Weise 1998; Ackerman et al. 2000) have shown good recyclability of mechanical fibres. y y Adámková a Milichovský (2002) present the dependence of beating degree (°SR – Schopper- Riegler degree) and WRV from the relative length of hardwood and softwood pulps. 2. Alterations of pulp fibres properties at recycling From their results we can see the WRV increase in dependence on the pulp length alteration is more rapid at hardwood pulp, but finally this value is higher at softwood pulps. Kim et al. (2000) determined the WRV decrease at softwood pulps with the higher number of recycling (at zero recycling about cca 1.5 g/g at fifth recycling about cca 1.1 g/g). Utilisation of the secondary fibres to furnish at paper production decrease of the initial need of woody raw (less of cutting tress) but the paper quality is not significantly worse. 2.1 Paper recycling The primary raw material for the paper production is pulps fibres obtaining by a complicated chemical process from natural materials, mainly from wood. This fibres production is very energy demanding and at the manufacturing process there are used many of the chemical matters which are very problematic from view point of the environment protection. The suitable alternative is obtaining of the pulp fibres from already made paper. This process is far less demanding on energy and chemicals utilisation. The paper recycling, simplified, means the repeated defibring, grinding and drying, when there are altered the mechanical properties of the secondary stock, the chemical properties of fibres, the polymerisation degree of pulp polysaccharidic components, mainly of cellulose, their supramolecular structure, the morphological structure of fibres, range and level of interfibres bonds e.g.. The cause of above mentioned alterations is the fibres ageing at the paper recycling and manufacturing, mainly the drying process. At the repeat use of the secondary fibres, it need deliberate the paper properties alter due to the fiber deterioration during the recycling, when many alteration are irreversible. The alteration depth depends on the cycle’s number and way to the fibres use. The main problem is the decrease of the secondary pulp mechanical properties with the continuing recycling, mainly the paper strength (Khantayanuwong et al. 2002; Jahan 2003; Hubbe & Zhang 2005; Garg & Singh 2006; Geffertová et al. 2008; Sutjipto et al. 2008). This decrease is an effect of many alterations, which can but need not arise in the secondary pulp during the recycling process. The recycling causes the hornification of the cell walls that result in the decline of some pulp properties. It is due to the irreversible alterations in the cells structure during the drying (Oksanen et al. 1997; Kim et al. 2000; Diniz et al. 2004). The worse properties of the recycled fibres in comparison with the primary fibres can be caused by hornification but also by the decrease of the hydrophilic properties of the fibres surface during the drying due to the redistribution or migration of resin and fat acids to the surface (Nazhad & Paszner 1994; Nazhad 2005). Okayama (2002) observed the enormous increase of the contact angle with water which is related to the fiber inactivation at the recycling. This process is known as „irreversible hornification“. 2.1 Paper recycling 333 The Effects of Paper Recycling and its Environmental Impact Paper recycling saves the natural wood raw stock, decreases the operation and capital costs to paper unit, decrease water consumption and last but not least this paper processing gives rise to the environment preservation (e.g. 1 t of waste paper can replace cca 2.5 m3 of wood). A key issue in paper recycling is the impact of energy use in manufacturing. Processing waste paper for paper and board manufacture requires energy that is usually derived from fossil fuels, such as oil and coal. In contrast to the production of virgin fibre-based chemical pulp, waste paper processing does not yield a thermal surplus and thus thermal energy must be supplied to dry the paper web. If, however, the waste paper was recovered for energy purposes the need for fossil fuel would be reduced and this reduction would have a favourable impact on the carbon dioxide balance and the greenhouse effect. Moreover, pulp production based on virgin fibres requires consumption of round wood and causes emissions of air-polluting compounds as does the collection of waste paper. For better paper utilization, an interactive model, the Optimal Fibre Flow Model, considers both a quality (age) and an environmental measure of waste paper recycling was developed (Byström & Lönnstedt 1997). 2.1.1 Influence of beating on pulp fibres the mechanical effect and the penetration into the interfibrilar spaces, it means to the amorphous region, there is the main portion of hemicelluloses. Češek & Milichovský (2005) showed that with the increase of pulp beating degree the standard rheosettling velocity of pulp decreases more at the fibres fibrillation than at the fibres shortening. Refining causes a variety of simultaneous changes in the fiber structure, such as internal fibrillation, external fibrillation and fines formation. Among these effects, swelling is commonly recognized as an important factor affecting the strength of recycled paper (Kang & Paulapuro 2006d). Scallan & Tigerstrom (1991) observed the elasticity modulus of the long fibres from kraft pulp during the recycling. Flexibility decrease was evident at the beating degree decrease (°SR), and also with the increase of draining velocity of low-yield pulp. 80 °C 100 °C 120 °C virgin pulp 0 1 2 3 4 5 6 7 8 number of recycling 0 1 2 3 4 5 6 7 8 9 10 11 breaking length [km] Fig. 2. Alteration of the breaking length of the paper sheet drying at the temperature of 80, 100 a 120 °C during eightfold recycling properties of paper sheets by drying temperature 80 °C number of recycling virgin pulp 0 1 2 3 4 5 6 7 8 Breaking length [km] 1.6 8.3 6.3 6.3 5.7 5.7 5.4 4.9 5.0 5.0 Tear index [mN.m2/g] 1.6 2.1 2.5 2.7 2.8 2.8 2.5 3.1 2.7 2.7 Brightnees [%MgO] 83.8 80.0 82.2 82.8 82.5 82.4 82.0 82.4 82.5 82.6 Opacity [%] 71.4 63.9 68.8 67.8 69.5 69.1 70.0 70.1 69.1 70.3 DP by viscometry 699 666 661 663 653 642 642 608 607 611 DP by SEC 1138 1128 1126 1136 1115 1106 1094 1069 1053 1076 80 °C 100 °C 120 °C virgin pulp 0 1 2 3 4 5 6 7 8 number of recycling 0 1 2 3 4 5 6 7 8 9 10 11 breaking length [km] 80 °C 100 °C 120 °C virgin pulp 0 1 2 3 4 5 6 7 8 number of recycling 0 1 2 3 4 5 6 7 8 9 10 11 breaking length [km] Fig. 2. Alteration of the breaking length of the paper sheet drying at the temperature of 80, 100 a 120 °C during eightfold recycling Fig. 2. 2.1.1 Influence of beating on pulp fibres Beating of chemical pulp is an essential step in improving the bonding ability of fibres. The knowledge complete about beating improves the present opinion of the fibres alteration at the beating. The main and extraneous influences of the beating device on pulps were defined. The main influences are these, each of them can be improve by the suitable beating mode, but only one alteration cannot be attained. Known are varieties of simultaneous changes in fibres, such as internal fibrilation, external fibrilation, fiber shortening or cutting, and fines formation (Page 1989; Kang & Paulapuro 2006a; Kang & Paulapuro 2006c).  Freeing and disintegration of a cell wall affiliated with strong swelling expressed as an internal fibrilation and delamination. The delamination is a coaxial cleavage in the middle layer of the secondary wall. It causes the increased water penetration to the cell wall and the fibre plasticizing.  External fibrillation and fibrils peeling from surface, which particularly or fully attacks primary wall and outside layers of secondary walls. Simultaneously from the outside layers there are cleavage fibrils, microfibrils, nanofibrils to the macromolecule of cellulose and hemicelluloses.  Fibres shortening in any place in any angle-wise across fibre in accordance with loading, most commonly in weak places.  Concurrently the main effects at the beating also the extraneous effects take place, e.g. fines making, compression along the fibres axis, fibres waving due to the compression. It has low bonding ability and it influences the paper porosity, stocks freeness (Sinke & Westenbroek 2004). The beating causes the fibres shortening, the external and internal fibrillation affiliated with delamination and the fibres plasticizing. The outside primary wall of the pulp fibre leaks water little, it has usually an intact primary layer and a tendency to prevent from the swelling of the secondary layer of the cell wall. At the beating beginning there are disintegrated the fibre outside layers (P and S1), the fibrilar structure of the fibre secondary layer is uncovering, the water approach is improving, the swelling is taking place and the fibrillation process is beginning. The fibrillation process is finished by the weaking and cleavaging of the bonds between the particular fibrils and microfibrils of cell walls during 334 Environmental Management in Practice the mechanical effect and the penetration into the interfibrilar spaces, it means to the amorphous region, there is the main portion of hemicelluloses. 2.1.1 Influence of beating on pulp fibres Alteration of the breaking length of the paper sheet drying at the temperature of 80, 100 a 120 °C during eightfold recycling properties of paper sheets by drying temperature 80 °C number of recycling virgin pulp 0 1 2 3 4 5 6 7 8 Breaking length [km] 1.6 8.3 6.3 6.3 5.7 5.7 5.4 4.9 5.0 5.0 Tear index [mN.m2/g] 1.6 2.1 2.5 2.7 2.8 2.8 2.5 3.1 2.7 2.7 Brightnees [%MgO] 83.8 80.0 82.2 82.8 82.5 82.4 82.0 82.4 82.5 82.6 Opacity [%] 71.4 63.9 68.8 67.8 69.5 69.1 70.0 70.1 69.1 70.3 DP by viscometry 699 666 661 663 653 642 642 608 607 611 DP by SEC 1138 1128 1126 1136 1115 1106 1094 1069 1053 1076 The Effects of Paper Recycling and its Environmental Impact 335 properties of paper sheets by drying temperature 100 °C number of recycling virgin pulp 0 1 2 3 4 5 6 7 8 Breaking length [km] 1.5 9.1 7.1 6.5 5.4 5.0 4.3 4.4 4.2 4.0 Tear index [mN.m2/g] 1.5 2.2 2.3 2.6 2.7 2.9 2.7 3.0 2.8 2.7 Brightnees [%MgO] 83.4 81.0 81.8 81.8 82.9 82.4 82.8 82.5 82.3 82.4 Opacity [%] 72.0 64.4 67.7 68.5 69.3 70.1 70.8 71.0 71.1 71.2 DP by viscometry 699 689 688 680 650 672 660 646 636 624 DP by SEC 1138 1012 1010 938 923 918 901 946 942 941 properties of paper sheets by drying temperature 120 °C number of recycling virgin pulp 0 1 2 3 4 5 6 7 8 Breaking length [km] 1.4 8.4 6.0 4.9 4.2 4.2 4.0 4.0 4.0 3.7 Tear index [mN.m2/g] 1.4 2.1 2.5 2.7 2.8 2.9 2.7 3.1 2.7 2.7 Brightnees [%MgO] 83.2 79.8 80.6 80.5 81.3 81.2 81.3 81.1 81.1 80.7 Opacity [%] 72.9 65.4 69.0 70.7 71.6 71.7 72.4 72.6 72.4 72.8 DP by viscometry 699 677 665 658 675 677 672 658 673 662 DP by SEC 1138 1030 1015 1059 1042 950 947 945 944 933 Table 1,2,3. The selected properties of the pulp fibres and the paper sheets during the process of eightfold recycling at three drying temperatures of 80 °C, 100 °C a 120 °C. From the result on Fig. 2 we can see the increase of the pulp fibres active surface takes place during the beating process, which results in the improve of the bonding and the paper strength after the first beating. 2.1.1 Influence of beating on pulp fibres It causes also the breaking length increase of the laboratory sheets. The secondary fibres wear by repeated beating, what causes the decrease of strength values (Tab. 1,2,3). The biggest alterations of tear index (Fig. 3) were observed after fifth recycling at the Table 1,2,3. The selected properties of the pulp fibres and the paper sheets during the process of eightfold recycling at three drying temperatures of 80 °C, 100 °C a 120 °C. From the result on Fig. 2 we can see the increase of the pulp fibres active surface takes place during the beating process, which results in the improve of the bonding and the paper strength after the first beating. It causes also the breaking length increase of the laboratory sheets. The secondary fibres wear by repeated beating, what causes the decrease of strength values (Tab. 1,2,3). The biggest alterations of tear index (Fig. 3) were observed after fifth recycling at the bleached softwood pulp fibres. The first beating causes the fibrillation of the outside layer of the cell wall, it results in the formation of the mechanical (felting) and the chemical bonds between the fibres. The repeated beating and drying dues, except the continuing fibrillation of the layer, the successive fibrils peeling until the peeling of the primary and outside 336 Environmental Management in Practice secondary layer of the cell wall. It discovers the next non-fibriled layer S2 (second, the biggest layer of the secondary wall) what can do the tear index decrease. The next beating causes also this layer fibrillation, which leads to the increase of the strength value (Fig. 3, Tab. 1,2,3). Paper strength properties such as tensile strength and Scott bond strength were strongly influenced by internal fibrillation; these could also be increased further by promoting mostly external fibrillation (Kang & Paulapuro 2006b). The course of the breaking length decrease and the tearing strength increase of the paper sheet is in accordance with the results of Sutjipto et al. (2008) at the threefold recycling of the bleached (88 % ISO) softwood pulps prepared at the laboratory conditions, beated on PFI mill to 25 °SR. 2.1.2 Drying influence on the recycled fibres Characteristic differences between recycled fibres and virgin fibres can by expected. Many of these can by attributed to drying. Drying is a process that is accompanied by partially irreversible closure of small pores in the fibre wall, as well as increased resistance to swelling during rewetting. Further differences between virgin and recycled fibres can be attributed to the effects of a wide range of contaminating substances (Hubbe et al. 2007). Drying, which has an anisotropic character, has a big influence on the properties of paper produced from the secondary fibres. During the drying the shear stress are formatted in the interfibrilar bonding area. The stresses formatted in the fibres and between them effect the mechanical properties in the drying paper. The additional effect dues the tensioning of the wet pulp stock on the paper machine. During the drying and recycling the fibres are destructed. It is important to understand the loss of the bonding strength of the drying chemical fibres. Dang (2007) characterized the destruction like a percentage reduction of ability of the water retention value (WRV) in pulp at dewatering. Hornification = [(WRV0-WRV1)/WRV0 ]. 100 [%], [%], 2.1.1 Influence of beating on pulp fibres 80 °C 100 °C 120 °C virgin pulp 0 1 2 3 4 5 6 7 8 number of recycling 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 tear index [mN.m2/g] 80 °C 100 °C 120 °C virgin pulp 0 1 2 3 4 5 6 7 8 number of recycling 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 tear index [mN.m2/g] Fig. 3. Tear index alteration of the paper sheets drying at the temperature of 80, 100 a 120 °C, during eightfold recycling number of recycling Fig. 3. Tear index alteration of the paper sheets drying at the temperature of 80, 100 a 120 °C, during eightfold recycling Song & Law (2010) observed kraft pulp oxidation and its influence on recycling characteristics of fibres, the found up the fibre oxidation influences negatively the tear index of paper sheets. Oxidation of virgin fibre prior to recycling minimized the loss of WRV and sheet density. The beating causes the fibres shortening and fines formation which is washed away in the large extent and it endeds in the paper sludges. This waste can be further processed and effective declined. Within the European Union several already issued and other foreseen directives have great influence on the waste management strategy of paper producing companies. Due to the large quantities of waste generated, the high moisture content of the waste and the changing composition, some recovery methods, for example, conversion to fuel components, are simply too expensive and their environmental impact uncertain. The thermal processes, gasification and pyrolysis, seem to be interesting emerging options, although it is still necessary to improve the technologies for sludge application. Other applications, such as the hydrolysis to obtain ethanol, have several advantages (use of wet sludge and applicable technology to sludges) but these are not well developed for pulp and paper sludges. 337 The Effects of Paper Recycling and its Environmental Impact Therefore, at this moment, the minimization of waste generation still has the highest priority (Monte et al. 2009). Therefore, at this moment, the minimization of waste generation still has the highest priority (Monte et al. 2009). WRV1 – the value of recycled pulp after drying and reslushing. According to the prevailing concept, hornification occurs in the cell wall matrix of chemical fibres. During drying, delaminated parts of the fiber wall, i.e., cellulose microfibrils become attached as Fig. 4 shows (Ackerman et al. 2000). Fig. 5. Shrinkage of a fiber cross section (Ackerman et al. 2000) Fig. 5. Shrinkage of a fiber cross section (Ackerman et al. 2000) Fig. 4. Changes in fiber wall structure (Weise & Paulapuro 1996) Fig. 4. Changes in fiber wall structure (Weise & Paulapuro 1996) Fig. 5. Shrinkage of a fiber cross section (Ackerman et al. 2000) Hydrogen bonds between those lamellae also form. Reorientation and better alignment of microfibrils also occur. All this causes an intensely bonded structure. In a subsequent Fig. 5. Shrinkage of a fiber cross section (Ackerman et al. 2000) Fig. 4. Changes in fiber wall structure (Weise & Paulapuro 1996) Fig. 5. Shrinkage of a fiber cross section (Ackerman et al. 2000) Hydrogen bonds between those lamellae also form. Reorientation and better alignment of microfibrils also occur. All this causes an intensely bonded structure. In a subsequent Hydrogen bonds between those lamellae also form. Reorientation and better alignment of microfibrils also occur. All this causes an intensely bonded structure. In a subsequent 338 Environmental Management in Practice reslushing in water, the fiber cell wall microstructure remains more resistant to delaminating forces because some hydrogen bonds do not reopen. The entire fiber is stiffer and more brittle (Howard 1991). According to some studies (Bouchard & Douek 1994; Maloney et al. 1998), hornification does not increase the crystallinity of cellulose or the degree of order in the hemicelluloses of the fiber wall. Fig. 6. The drying model of Scallan (Laivins & Scallan 1993) suggests that hornification prevents the dry structure in A from fully expanding to the wet structure in D. Instead, only partial expansion to B may be possible after initial drying creates hydrogen bonds between the microfibrils (Kato & Cameron 1999) Fig. 6. The drying model of Scallan (Laivins & Scallan 1993) suggests that hornification prevents the dry structure in A from fully expanding to the wet structure in D. Instead, only partial expansion to B may be possible after initial drying creates hydrogen bonds between the microfibrils (Kato & Cameron 1999) Weise & Paulapuro (1996) did very revealing work about the events during fiber drying. WRV1 – the value of recycled pulp after drying and reslushing. They studied fiber cross section of kraft fibers in various solids by Confocal Laser Scanning Microscope (CLSM) and simultaneously measured hornification with WRV tests. Irreversible hornification of fibers began on the degree of beating. It does not directly follow shrinkage since the greatest shrinkage of fibers occurs above 80 % solids content. In Figs. 4 and 5, stage A represented wet kraft fiber before drying. In stage B, the drainage has started to cause morphological changes in the fiber wall matrix at about 30 % solids content. The fiber wall lamellae start to approach each other because of capillary forces. During this stage, the lumen can collapse. With additional drying, spaces between lamellae continue shrinking to phase C where most free voids in the lamellar structure of the cell wall have already closed. Toward the end of drying in stage D, the water removal occurs in the fine structure of the fiber wall. Kraft fiber shrink strongly and uniformly during this final phase of drying, i.e., at solid contents above 75-80 %. The shrinkage of stage D is irreversible. At a repeated use of the dried fibres in paper making industry, the cell walls receive the water again. Then the opposite processes take place than in the Fig. 4 and 5. It show Scallan´s model of the drying in Fig. 6. The drying dues also macroscopic stress applied on paper and distributed in fibres system according a local structure. 2.1.3 Properties of fibres from recycled paper 2.1.3 Properties of fibres from recycled paper The basic properties of origin wet fibres change in the drying process of pulp and they are not fully regenerated in the process of slushing and beating. The basic properties of origin wet fibres change in the drying process of pulp and they are not fully regenerated in the process of slushing and beating. The same parameters are suitable for the description of the paper properties of secondary fibres and fibres at ageing as well as for description of primary fibres properties. The 339 The Effects of Paper Recycling and its Environmental Impact experiences obtained at the utilisation of waste paper showed the secondary fibres have very different properties from the origin fibres. Next recycling of fibres causes the formation of extreme nonhomogeneous mixture of various old fibres. At the optimum utilisation of the secondary fibres it need take into account their altered properties at the repeated use. With the increase number of use cycles the fibres change irreversible, perish and alter their properties. Slushing and beating causes water absorption, fibres swelling and a partial regeneration of properties of origin fibres. However the repeated beating and drying at the multiple production cycles dues the gradual decrease of swelling ability, what influences a bonding ability of fibres. With the increase of cycles number the fibres are shortened. These alterations express in paper properties. The decrease of bonding ability and mechanical properties bring the improving of some utility properties. Between them there is higher velocity of dewatering and drying, air permeability and blotting properties improve of light scattering, opacity and paper dimensional stability. g p y p p y The highest alterations of fibres properties are at the first and following three cycles. The size of strength properties depends on fibres type (Geffertová et al. 2008). Drying influences fibres length, width, shape factor, kinks which are the important factors to the strength of paper made from recycled fibres. The dimensional characteristics are measured by many methods, known is FQA (Fiber Quality Analyser), which is a prototype IFA (Imaging Fiber Analyser) and also Kajaani FS-200 fibre-length analyser. They measure fibres length, different kinks and their angles. Robertson et al. (1999) show correlation between methods FQA and Kajaani FS-200. A relatively new method of fibres width measurement is also SEM (Scanning Electron Microscope) (Bennis et al. 2010). 2.1.3 Properties of fibres from recycled paper Among devices for analyse of fibres different properties and characteristics, e.g. fibres length and width, fines, various deformations of fibres and percentage composition of pulp mixture is L&W Fiber Tester (Lorentzen & Wettre, Sweden). At every measurement the minimum of 20 000 fibres in a sample is evaluated. On Fig. 7 there is expressed the alteration of fibres average length of softwood pulps during the eightfold recycling at the different drying temperature of pulp fibres. Fig. 7. Influence of recycling number and drying temperature on length of softwood pulps 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 virgin pulp 0 1 2 3 4 5 6 7 8 80 °C 100°C 120 °C average length of fibres [mm] number of recycling 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 virgin pulp 0 1 2 3 4 5 6 7 8 80 °C 100°C 120 °C average length of fibres [mm] number of recycling number of recycling number of recycling Fig. 7. Influence of recycling number and drying temperature on length of softwood pulps Fig. 7. Influence of recycling number and drying temperature on length of softwood pulps 340 Environmental Management in Practice Fig. 8. Influence of recycling number and drying temperature on width of softwood pulps 25.0 25.5 26.0 26.5 27.0 27.5 28.0 virgin pulp 0 1 2 3 4 5 6 7 8 80 °C 100 °C 120 °C average width of fibres [μm] number of recycling Fig 8 Influence of recycling number and drying temperature on width of softwood pulps 25.0 25.5 26.0 26.5 27.0 27.5 28.0 virgin pulp 0 1 2 3 4 5 6 7 8 80 °C 100 °C 120 °C average width of fibres [μm] number of recycling number of recycling Fig. 8. Influence of recycling number and drying temperature on width of softwood pulps The biggest alteration were observed after first beating (zero recycling), when the fibres average length decrease at the sheet drying temperature of 80 °C about 17%, at the temperature of 100 °C about 15.6% and at the temperature of 120 °C about 14.6%. p p After the first beating the fibres average width was markedly increased at the all temperatures dues to the fibrillation influence. The fibres fibrillation causes the fibre surface increase. Following markedly alteration is observed after fifth recycling, when the fibres average width was decreased. 2.1.3 Properties of fibres from recycled paper Fi 9 I fl f li d d i t t l fi h 1.5 2.0 2.5 3.0 3.5 4.0 4.5 virgin pulp 0 1 2 3 4 5 6 7 8 80 °C 100 °C 120 °C pulp fines [%] number of recycling Fig 9 Influence of recycling process and drying temperature on pulp fines changes 1.5 2.0 2.5 3.0 3.5 4.0 4.5 virgin pulp 0 1 2 3 4 5 6 7 8 80 °C 100 °C 120 °C pulp fines [%] number of recycling number of recycling Fig. 9. Influence of recycling process and drying temperature on pulp fines changes The macroscopic level (density, volume, porosity, paper thickness) consists from the physical properties very important for the use of paper and paperboard. They indirectly characterize the three dimensional structure of paper (Niskanen 1998). A paper is a complex structure consisting mainly of a fibre network, filler pigment particles and air. Light is reflected at fibre and pigment surfaces in the surface layer and inside the paper structure. The light also penetrates into the cellulose fibres and pigments, and changes directions. Some light is absorbed, but the remainder passes into the air and is reflected and refracted again by new fibres and pigments. After a number of reflections and refractions, a certain proportion of the light reaches the paper surface again and is then reflected at all possible angles from the surface. We do not perceive all the reflections and refractions (the multiple reflections or refractions) which take place inside the paper structure, but we perceive that the paper has a matt white surface i.e. we perceive a diffuse surface reflection. Some of the incident light exists at the back of the paper as transmitted light, and the remainder has been absorbed by the cellulose and the pigments. Besides reflection, refraction and absorption, there is a fourth effect called diffraction. In other contexts, diffraction is usually the same thing as light scattering, but within the field of paper technology, diffraction is only one aspect of the light scattering phenomenon. Diffraction occurs when the light meets particles or pores which are as large as or smaller then the wavelength of the light, i.e. particles which are smaller than one micrometer (μm). These small elements oscillate with the light oscillation and thus function as sites for new light sources. 2.1.3 Properties of fibres from recycled paper We assume the separation of fibrils and microfibrils from the cell walls dues the separation of the cell walls outside layer, the inside nonfibriled wall S2 was discovered and the fibres average width decreased. After the fifth recycling the strength properties became worse, mainly tear index (Fig. 3). p p y ( g ) The softwood fibres are longer than hardwood fibres, they are not so straight. The high value of shape factor means fibres straightness. The biggest alterations of shape factor can be observed mainly at the high drying temperatures. The water molecules occurring on fibres surface quick evaporate at the high temperatures and fibre more shrinks. It can result in the formation of weaker bonds between fibres those surfaces are not enough near. At the beginning of wet paper sheet drying the hydrogen bond creates through water layer on the fibres surface, after the drying through monomolecular layer of water, finally the hydrogen bond results after the water removal and the surfaces approach. It results in destruction of paper and fibre at the drying. Chemical pulp fines are an important component in papermaking furnish. They can significantly affect the mechanical and optical properties of paper and the drainage properties of pulp (Retulainen et al. 1993). Characterizing the fines will therefore allow a better understanding of the role of fines and better control the papermaking process and the properties of paper. Chemical pulp fines retard dewatering of the pulp suspension due to the high water holding capacity of fines. In the conventional method for characterizing the role of fines in dewatering, a proportion of fines is added to the fiber furnish, and then only the drainage time. Fines suspension is composed of heterogeneous fines particles in water. The suspension exhibits different rheological characteristics depending on the degree of interaction between the fines particles and on their hydration (Kang & Paulapuro 2006b). From Fig. 9 we can see the highest formation of fines were after seventh and eight recycling, when the fibres were markedly weakened by the multiple using at the processes of paper 341 The Effects of Paper Recycling and its Environmental Impact making. They are easier and faster beating (the number of revolution decreased by the higher number of the recycling). making. They are easier and faster beating (the number of revolution decreased by the higher number of the recycling). 3. Paper ageing The recycled paper is increasingly used not only for the products of short term consumption (newspaper, sanitary paper, packaging materials e.g.), but also on the production of the higher quality papers, which can serve as a culture heritage medium. The study of the recycled papers alterations in the ageing process is therefore important, but the information in literature are missing. The recycling is also another form of the paper ageing. It causes the paper alterations, which results in the degradation of their physical and mechanical properties. The recycling causes a chemical, thermal, biological and mechanical destruction, or their combination (Milichovský 1994; Geffertová et al. 2008). The effect of the paper ageing is the degradation of cellulose, hemicelluloses and lignin macromolecules, the decrease of low molecular fractions, the degree of polymerisation (DP) decrease, but also the decline of the mechanical and optical properties (El Ashmawy et al. 1974; Valtasaari & Saarela 1975; Lauriol et al. 1987a,b,c; Bansa 2002; Havermans 2003; Dupont & Mortha 2004; Kučerová & Halajová, 2009; Čabalová et al. 2011). Cellulose as the most abundant natural polymer on the Earth is very important as a renewable organic material. The degradation of cellulose based paper is important especially in archives and museums where ageing in various conditions reduces the mechanical properties and deteriorates optical quality of stored papers, books and other artefacts. The low rate of paper degradation results in the necessity of using accelerating ageing tests. The ageing tests consist in increasing the observed changes of paper properties, usually by using different temperature, humidity, oxygen content and acidity, respectively. Ageing tests are used in studies of degradation rate and mechanism. During the first ageing stages—natural or accelerated—there are no significant variations in mechanical properties: degradation evidence is only provided by measuring chemical processes. Oxidation induced by environmental conditions, in fact, causes carbonyl and carboxyl groups formation, with great impact on paper permanence and durability, even if mechanical characteristics are not affected in the short term (Piantanida et al. 2005). During the degradation two main reactions prevail – hydrolysis of glycosidic bonds and oxidation of glucopyranose rings. As a result of some oxidation processes keto- and aldehyde groups are formed. These groups are highly reactive; they are prone to crosslinking, which is the third chemical process of cellulose decay (Bansa 2002, Calvini & Gorassini 2006). 2.1.3 Properties of fibres from recycled paper When the particles or pores are smaller than half of the light wavelength the diffraction decreases. It can be said that the light passes around the particle without being affected (Pauler 2002). g g The opacity, brightness, colouring and brilliance are important optical properties of papers and paperboards. For example the high value of opacity is need at the printing papers, but opacity of translucent paper must be lower. The paper producer must understand the physical principles of the paper structure and to determine their characteristics composition. It is possible to characterize nondirect the paper structure. The opacity characterizes the paper ability to hide a text or a figure on the opposite side of the paper sheet. The paper 342 Environmental Management in Practice brightness is a paper reflection at a blue light use. The blue light is used because the made fibers have yellowish colour and a human eye senses a blue tone like a white colour. The typical brightness of the printing papers is 70 – 95% and opacity is higher than 90% (Niskanen 1998). 3. Paper ageing At the accelerated paper ageing the decrease of DP is very rapid in the first stages of the ageing, later decelerates. During the longer time of the ageing there was determined the cellulose crosslinking by the method of size exclusion chromatography (SEC) (Kačík et al. 2009). The similar dependences were obtained at the photo-induced cellulose degradation (Malesic et al. 2005). An attention is pay to the kinetic of the cellulose degradation in several decades, this process was studied by Kuhn in 1930 and the first model of the kinetic of the cellulose chains cleavage was elaborated by Ekenstam in 1936. This model is based on the kinetic equation of first-order and it is used to this day in modifications for the watching of the cellulose degradation in different conditions. Hill et al. (1995) deduced a similar model with the The Effects of Paper Recycling and its Environmental Impact 343 Fig. 10. Alterations of DP (degree of polymerisation) of cellulose fibres due to recycling and ageing at the pulp fibres drying temperature of 80 °C, 100 °C a 120 °C. contribution of the zero order kinetic. 3. Paper ageing Experimental results are often controversial and new kinetic model for explanation of cellulose degradation at various conditions was proposed (C l i i t l 2008) Th fi t d ki ti d l d l d b th th t th t y = -2.1333x + 683.33 y = -10,22x + 603,3 450 500 550 600 650 700 750 virgin pulp 0 1 2 3 4 5 6 7 8 DP number of recycling before ageing 80 °C after ageing 80 °C y = -7,842x + 707,5 y = -7,521x + 595,0 450 500 550 600 650 700 750 virgin pulp 0 1 2 3 4 5 6 7 8 DP number of recycling before ageing 100 °C y = -9,357x + 696,6 y = -2,975x + 579,6 450 500 550 600 650 700 750 virgin pulp 0 1 2 3 4 5 6 7 8 DP number of recycling before ageing 120 °C after ageing 120 °C y = -2.1333x + 683.33 y = -10,22x + 603,3 450 500 550 600 650 700 750 virgin pulp 0 1 2 3 4 5 6 7 8 DP number of recycling before ageing 80 °C after ageing 80 °C y = -7,842x + 707,5 y = -7,521x + 595,0 450 500 550 600 650 700 750 virgin pulp 0 1 2 3 4 5 6 7 8 DP number of recycling before ageing 100 °C before ageing 80 °C after ageing 80 °C number of recycling before ageing 100 °C before ageing 120 °C after ageing 120 °C number of recycling Fig. 10. Alterations of DP (degree of polymerisation) of cellulose fibres due to recycling and ageing at the pulp fibres drying temperature of 80 °C, 100 °C a 120 °C. contribution of the zero order kinetic. Experimental results are often controversial and new kinetic model for explanation of cellulose degradation at various conditions was proposed (Calvini et al. 2008). The first-order kinetic model developed by these authors suggests that the kinetics of cellulose degradation depends upon the mode of ageing. An autoretardant path is followed during either acid hydrolysis in aqueous suspensions or oven ageing, while the production of volatile acid compounds trapped during the degradation in sealed contribution of the zero order kinetic. Experimental results are often controversial and new kinetic model for explanation of cellulose degradation at various conditions was proposed (Calvini et al. 2008). DP = LODP + DP01.e-k1.t + DP02.e-k2.t, where LODP is levelling-off degree of polymerisation. There is a first factor higher and quick decreasing during eight days and a second factor is lower and slow decreasing and dominant after eight days of the accelerating ageing in the equation. The number of cleavaged bonds can be well described by equation where LODP is levelling-off degree of polymerisation. There is a first factor higher and quick decreasing during eight days and a second factor is lower and slow decreasing and dominant after eight days of the accelerating ageing in the equation. The number of cleavaged bonds can be well described by equation DP0/DPt – 1 = n0.(1-e-k.t), where n0 is an initial number of bonds available for degradation. The equation of the regression function is in accordance with Calvini et al. (2007) proposal, the calculated value (4.4976) is in a good accordance with the experimentally obtained average values of DP0 a DP60 (4.5057). The DP decreased to cca 38% of the initial value and the polydispersity degree to 66% of the initial value. The decrease of the rate constant with the time of ageing was obtained also by next authors (Emsley et al. 1997; Zervos & Moropoulou 2005; Ding & Wang 2007). Čabalová et al. (2011) observed the influence of the accelerated ageing on the recycled pulp fibres, they determined the lowest decrease of DP at the fibres dried at the temperature of 120 °C (Fig. 10). The simultaneous influence of the recycling and ageing has the similar impact at the drying temperatures of 80 °C (decrease about 27,5 %) and 100 °C (decrease about 27.6%) in regard of virgin pulp, lower alterations were at the temperature of 120 °C (decrease about 21.5%). The ageing of the recycled paper causes the decrease of the pulp fiber DP, but the paper remains good properties. 3. Paper ageing The first-order kinetic model developed by these authors suggests that the kinetics of cellulose degradation depends upon the mode of ageing. An autoretardant path is followed during either acid hydrolysis in aqueous suspensions or oven ageing, while the production of volatile acid compounds trapped during the degradation in sealed 344 Environmental Management in Practice environments primes an autocatalytic mechanism. Both these mechanisms are depleted by the consumption of the glycosidic bonds in the amorphous regions of cellulose until the levelling-off DP (LODP) is reached. At the accelerated ageing of newspaper (Kačík et al. 2008), the cellulose degradation causes the decrease of the average degree of polymerisation (DP). The DP decrease is caused by two factors in accordance with equation DP = LODP + DP01.e-k1.t + DP02.e-k2.t, 5. Acknowledgment This work was financed by the Slovak Grant Agency VEGA (project number 1/0490/09). This work was financed by the Slovak Grant Agency VEGA (project number 1/0490/09). 4. Conclusion The recycling is a necessity of this civilisation. The paper manufacturing is from its beginning affiliated with the recycling, because the paper was primarily manufactured from the 100 % furnish of rag. It is increasingly assented the trend of the recycled fibers use from the European and world criterion. The present European papermaking industry is based on the recycling. The presence of the secondary fibres from the waste paper, their quality and amount is various in the time intervals, the seasons and the regional conditions. It depends on the manufacturing conditions in the paper making industry of the country. At present the recycling is understood in larger sense than the material recycling, which has a big importance from view point of the paper recycling. Repeatedly used fibres do not fully regenerate their properties, so they cannot be recycled ad anfinitum. It allows to use the alternative possibilities of the paper utilisation in the building industry, at the soil reclamation, it the agriculture, in the power industry. g p y The most important aim is, however, the recycled paper utilisation for the paper manufacturing. 345 The Effects of Paper Recycling and its Environmental Impact 6. References Rheosedimentation – a Tool for Evaluation of Pulp Fibre Behaviour in Wet State. Papír a celulóza 59 (7–8), 2005, pp. 224–229, ISSN 0031–1421 Češek, B. & Milichovský, M. (2005). Rheosedimentation – a Tool for Evaluation of Pulp Fibre Behaviour in Wet State. Papír a celulóza 59 (7–8), 2005, pp. 224–229, ISSN 0031–1421 El Ashmawy, A. E., Danhelka, J. & Kössler, I. (1974). Determination of molecular weight distribution of cellulosic pulps by conversion into tricarbanilate, elution fractionation and GPC. Svensk Papperstidning 16, 1974, pp. 603-608, ISSN 0283-6831 Emsley, A. M., Heywood, R. J., Ali, M. & Eley, C. (1997). 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Das Papier 50(6), pp. 328-333, ISSN 0031-1340 Environmental Management in Practice Overview Management Chemical Residues of Laboratories in Academic Institutions in Brazil Patrícia Carla Giloni-Lima, Vanderlei Aparecido de Lima and Adriana Massaê Kataoka Universidade Estadual do Centro Oeste (UNICENTRO) Brazil Carla Giloni-Lima, Vanderlei Aparecido de Lima and Adriana Massaê Kataoka Universidade Estadual do Centro Oeste (UNICENTRO) Brazil Brazil 6. References Measurement of fiber length, coarseness, and shape with the fiber quality analyser. Tappi Journal 82(10), pp. 93- 98, ISSN 0734-1415 Scallan, A. & Tigerstrom, A. C. (1991). Elasticity of fiber wall, effects of pulping and recycling. CPPA, 1st Research Forum on Recyclin, Montreal, pp. 149-154 Scallan, A.M. & Tydeman, A.C. (1992). Swelling and elasticity of the cell walls of pulp fibres, J. Pulp Paper Sci. 18(5), pp. 188-193, ISSN 0826-6220 Sinke, R.J. & Westenbroek, A.P.H. (2004). How to deal with the effects of recycling? Paper and Board. 8th Pira Paper Recycling Technology Conference, Prague, Czech Republic, 17-18 February 2004, 17.02. 2011. Available from: http://www.papierenkarton.nl/publications.htm#2004 Somwand, K., Enomae, T. & Onabe, F. (2002). Effect of Fiber Hornification in Recycling on Bonding Potential at Interfiber Crossings, Confocal Laser Scanning Microscopy. Japan TAPPI Journal, Vol.56, No.2, pp. 239 -245, ISSN 0022-815X Song, X. & Law, K.N. (2010). Kraft pulp oxidation and its inluence of recycling characteristics of fibres. Cellulose Chemistry and Technology 44(7-8), pp. 265-270, ISSN 0576-9787 Sutjipto, E.R., Li, K., Pongpattanasuegsa, S. & Nazhad, M.M. (2008). Effect of recycling on paper properties, TAPPSA (Technical articles), 07.02.2011. Available from: http://www.tappsa.co.za/archive3/index.html Valtasaari, L. & Saarela, K. (1975). Determination of chain lenght distribution of cellulose by gel permeation chromatography using the tricarbanilate derivate. Paper och Tra – Paperi ja Puu 1, pp. 5-10, ISSN 0031-1243 Zanuttini, M. A., McDonough, T. J., Courchene, C. E. & Mocchiutti, P. (2007). Upgrading OCC and recycled liner pulps by medium-consistency ozone treatment. Tappi Journal 6(2), pp. 3-8, ISSN 0734-1415 Zervos, S. & Moropoulov, A. (2005). Cotton cellulose ageing in sealed vessels. Kinetic model of autocatalytic depolymerization. Cellulose 12, 2005, pp. 485-496, ISSN 0969-0239 350 Environmental Management in Practice 18 1. Introduction In the last decades the discussion regarding the environmental theme has acquired concerning proportions for planetary order. Taking into account the civilization crisis in which we are immersed, the environment has been associated to a problem. p During the process of civilization, we perceive an accelerated growth of human population and the various wastes generated as byproducts of their activities surpass the resilience capacity of the environment, generating imbalances in their original cycles. Large discharges of artificial elements in high concentrations (many of them toxic and harmful to life) are constantly deposited in regions where its subsystem revolves around nature’s own dynamics. This flow of residues deposition returns to human beings life cycle as pollution, radiation, contamination, acid rain, among others (Jardim, 1993). In this sense, generation and fate of wastes have been one of the themes treated by environmental education means and the media. This outcome is a consequence of a capitalist society in which consumerism is required for its own maintenance. It is no surprise that the issue of waste generation and its fate are present in scientific discussions, as well as in common sense. It could not be different since each of us has its own direct contribution to this framework. The technical solution for the problem is fundamental and represents a challenge and an important research field for professionals in the area. But that technical knowledge alone is not adequate to solve the problem. Most waste management initiatives in universities emphasize the importance of environmental education, but merely quote them without exploring their full potential in addressing these issues. These works point out that one of the obstacles for the success of such management programs are the people, and more precisely, their understanding. Working with people in regards to environmental issues is exactly the field of action of environmental education in which, in general, universities employ professionals set in departments of biology, education, among others. It is worthwhile to ask ourselves: why do not these professionals working at universities communicate with each other? Another important inquiry: why cannot a university composed of professionals that comprise different fields of knowledge work in an interdisciplinary fashion on a problem generated by the university itself? Such discussion has been intensively investigated by universities and disseminated to society. 1. Introduction 352 Environmental Management in Practice Next we will try to answer these questions from reflections addressed by environmental education firstly contextualizing this matter in a broader perspective for we do not agree that a punctual approach could satisfactorily explain the complexity of this subject, incurring the risk of simplification, which would be insufficient to the quest for a more effective solution. 1.2 Overview of universities and institutions of higher education and research developed in Brazil Among them are: IQ/USP - Instituto de Química da Universidade de São Paulo (“Chemistry Institute of São Paulo University”); IQSC/USP - Instituto de Química da Universidade de São Paulo do Campus São Carlos (“Chemistry Institute of São Paulo University at São Carlos”); CENA/USP - Centro de Energia Nuclear na Agricultura da Universidade de São Paulo (“Center of Nuclear Energia in Agriculture of São Paulo University”); UNICAMP - Universidade de Campinas (“Campinas State University”); IQ/UERJ - Instituto de Química da Universidade do Estado do Rio de Janeiro (“Chemistry Institute of Rio de Janeiro State University”); DQ/UFPR – Departamento de Química da Universidade Federal do Paraná (“Chemistry Department of Paraná Federal University”); IQ/UFRGS - Instituto de Química da Universidade Federal do Rio Grande do Sul (“Chemistry Institute of Rio Grande do Sul Federal University”); UCB - Universidade Católica de Brasília (“Brasília Catholic University”); UFSCar - Universidade Federal de São Carlos (“São Carlos Federal University”); FURB – Universidade Regional de Blumenau (“Blumenau Regional University”); URI – Universidade Regional Integrada do Alto Uruguai e das Missões (“Alto Uruguai e das Missões Regional Integrate University”); UFRJ – Universidade Federal do Rio de Janeiro (“Rio de Janeiro Federal University”) (Afonso et al, 2004); UNIVATES – Centro Universitário Univates (“Univates University Center”). Usually in Brazilian universities, Chemistry departments, in addition to their practice classes in laboratories for their own students, they also attend to other undergraduate courses. A survey taken into account reagents used in basic experimental subjects within five major areas of Chemistry at Brazilian universities is presented. It includes about 180 substances among the ones applied and developed in laboratory activities. The curricular program for Chemistry undergraduates comprises five areas of experimental Chemistry: Experimental General Chemistry, Analytical Chemistry, Physical Chemistry, Organic Chemistry and Inorganic Chemistry. Experimental General Chemistry covers the initial concepts of Chemistry, when students start their learning process in experimental classes in laboratories. The subject program involves the following topics: laboratory safety, nomenclature and characteristics of glassware, measures of mass, volume, density and temperature, physical and chemical processing, standardization of substances, acid/base titrations and determination of levels of substances in our daily lives. Although this course is an introductory practice, in which students carry out their first experiments in Chemistry, a certain number of substances are required for the development of such laboratory experiments. The second area of experimental subjects is Qualitative and Quantitative Analytical Chemistry. 1.2 Overview of universities and institutions of higher education and research developed in Brazil Among them are: IQ/USP - Instituto de Química da Universidade de São Paulo (“Chemistry Institute of São Paulo University”); IQSC/USP - Instituto de Química da Universidade de São Paulo do Campus São Carlos (“Chemistry Institute of São Paulo University at São Carlos”); CENA/USP - Centro de Energia Nuclear na Agricultura da Universidade de São Paulo (“Center of Nuclear Energia in Agriculture of São Paulo University”); UNICAMP - Universidade de Campinas (“Campinas State University”); IQ/UERJ - Instituto de Química da Universidade do Estado do Rio de Janeiro (“Chemistry Institute of Rio de Janeiro State University”); DQ/UFPR – Departamento de Química da Universidade Federal do Paraná (“Chemistry Department of Paraná Federal University”); IQ/UFRGS - Instituto de Química da Universidade Federal do Rio Grande do Sul (“Chemistry Institute of Rio Grande do Sul Federal University”); UCB - Universidade Católica de Brasília (“Brasília Catholic University”); UFSCar - Universidade Federal de São Carlos (“São Carlos Federal University”); FURB – Universidade Regional de Blumenau (“Blumenau Regional University”); URI – Universidade Regional Integrada do Alto Uruguai e das Missões (“Alto Uruguai e das Missões Regional Integrate University”); UFRJ – Universidade Federal do Rio de Janeiro (“Rio de Janeiro Federal University”) (Afonso et al, 2004); UNIVATES – Centro Universitário Univates (“Univates University Center”). U ll i B ili i i i Ch i d i ddi i h i i g g y y In Brazil, only larger universities have programs for waste management and treatment. 1.2 Overview of universities and institutions of higher education and research developed in Brazil The 2009 census on Higher Education in Brazil, carried out by INEP (Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira; “National Institute of Educational Studies and Research Anísio Teixeira”, http://www.inep.gov.br/) showed that the number of Brazilian Higher Education Institutions (HEI) grows every year (Figure 1). In 2009, 2 314 HEI were registered, being 89.4  (p <0.001) private and only 10.6% public institutions. Colleges still account for most HEI’s, representing 85  of them. Nevertheless, the majority of courses are conglomerate on universities, with 49.8% of undergraduate The 2009 census on Higher Education in Brazil, carried out by INEP (Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira; “National Institute of Educational Studies and Research Anísio Teixeira”, http://www.inep.gov.br/) showed that the number of Brazilian Higher Education Institutions (HEI) grows every year (Figure 1). Estudos e Pesquisas Educacionais Anísio Teixeira; National Institute of Educational Studies and Research Anísio Teixeira”, http://www.inep.gov.br/) showed that the number of Brazilian Higher Education Institutions (HEI) grows every year (Figure 1). In 2009, 2 314 HEI were registered, being 89.4  (p <0.001) private and only 10.6% public institutions. Colleges still account for most HEI’s, representing 85  of them. Nevertheless, the majority of courses are conglomerate on universities, with 49.8% of undergraduate courses. In 2009, 2 314 HEI were registered, being 89.4  (p <0.001) private and only 10.6% public institutions. Colleges still account for most HEI’s, representing 85  of them. Nevertheless, the majority of courses are conglomerate on universities, with 49.8% of undergraduate courses. Fig. 1. Evolution the number of Institutions of Higher Education (HEI) - Brazil - 2000/2009. Source: INEP, http://www.inep.gov.br/. Fig 1 Evolution the number of Institutions of Higher Education (HEI) Brazil 2000/2009 Fig. 1. Evolution the number of Institutions of Higher Education (HEI) - Brazil - 2000/2009. Source: INEP, http://www.inep.gov.br/. In Brazilian HEI’s, the top ten courses in number of enrolled students are: administration, law, pedagogy, engineering, nursing, accountancy, communication, languages and literature, physical education and biology. These courses comprise 66.4% of students enrolled in Brazilian higher education institutions. g Modern Chemistry has revolutionized mankind for many years and is undoubtedly one of the basic sciences more present in people’s lives through closely related segments (e.g., textile, chemical, food and pharmaceutical industry). 1.2 Overview of universities and institutions of higher education and research developed in Brazil Chemistry has also contributed to Overview Management Chemical R id f L b i i A d Overview Management Chemical Residues of Laboratories in Academic Institutions in Brazil 353 develop human life’s quality for the last 40 years (Coelho, 2001). Institutes and departments of Chemistry at universities, as well as all segments that make use of chemicals in their daily work, have been confronting the issues linked to treatment and disposal of wastes brought forth in their teaching and research laboratories for many years (Gerbase et al., 2005). This is the reason why Chemistry courses are the target of discussions in this text, as it presents certain intrinsic features concerning the organization system of laboratory classes. develop human life’s quality for the last 40 years (Coelho, 2001). Institutes and departments of Chemistry at universities, as well as all segments that make use of chemicals in their daily work, have been confronting the issues linked to treatment and disposal of wastes brought forth in their teaching and research laboratories for many years (Gerbase et al., 2005). This is the reason why Chemistry courses are the target of discussions in this text, as it presents certain intrinsic features concerning the organization system of laboratory classes. develop human life’s quality for the last 40 years (Coelho, 2001). Institutes and departments of Chemistry at universities, as well as all segments that make use of chemicals in their daily work, have been confronting the issues linked to treatment and disposal of wastes brought forth in their teaching and research laboratories for many years (Gerbase et al., 2005). This is the reason why Chemistry courses are the target of discussions in this text, as it presents certain intrinsic features concerning the organization system of laboratory classes. In Brazil, only larger universities have programs for waste management and treatment. 1.2 Overview of universities and institutions of higher education and research developed in Brazil Chemical reactions experienced in these subjects aim to identify and isolate cations and anions by reactions of neutralization, precipitation, complexation, oxidation- reduction, release of gases, as well as volumetric and gravimetric determinations. Students are divided into groups of three or four for laboratory practices. A laboratory technician prepares and organizes the class so that students, under the professor’s 354 Environmental Management in Practice supervision, carry out experiments. In these practice classes, the volume of substances generated is relatively small when compared to industries; however, there is a wide range of chemical wastes generated. The list of chemicals produced in practice classes is considerable, so we listed some of the most important shown in Table 1. The third area of experimental subject in Chemistry is called Physical Chemistry (Table 1) and deals with concepts of energy associated with molecules and chemical reactions, electrochemistry, the laws of ideal gases, chemical kinetics and chemical and physical adsorption. p The fourth area is Organic Chemistry, which studies carbon compounds and their reactions. There are two experimental subjects, organic Chemistry I and II. In those subjects, most compounds that are required and generated in these classes are organic and are also presented in Table 1. Chemistry products Chemistry products 1. Experimental general Chemistry sodium hydroxide, oxalic acid, hydrochloric acid, benzoic acid, gasoline, solid iodine, phenolphthalein, methyl orange, thymol blue, magnesium, ammonium dichromate, phenol red sodium salt, alizarin yellow R sodium salt, methyl red sodium salt, bromo phenol, bromocresol green sultone, sodium bromide, sodium iodide, strontium chloride, copper(II) sulfate, chromium(III) chloride, potassium chloride, nickel(II) chloride, potassium iodate, potassium iodide, ethylene glycol, aniline, sodium sulfate, manganese(II) sulfate, iron(II) sulphate, aluminum sulfate, potassium nitrate, barium chloride, ferric chloride, butanol, ammonium carbonate, calcium hydroxide, cobalt(II) nitrate, lead II nitrate, sodium phosphate monobasic, ascorbic acid, lithium chloride, potassium permanganate, calcium cyanide, cobalt(II) sulphate. sodium hydroxide, oxalic acid, hydrochloric acid, benzoic acid, gasoline, solid iodine, phenolphthalein, methyl orange, thymol blue, magnesium, ammonium dichromate, phenol red sodium salt, alizarin yellow R sodium salt, methyl red sodium salt, bromo phenol, bromocresol green sultone, sodium bromide, sodium iodide, strontium chloride, copper(II) sulfate, chromium(III) chloride, potassium chloride, nickel(II) chloride, potassium iodate, potassium iodide, ethylene glycol, aniline, sodium sulfate, manganese(II) sulfate, iron(II) sulphate, aluminum sulfate, potassium nitrate, barium chloride, ferric chloride, butanol, ammonium carbonate, calcium hydroxide, cobalt(II) nitrate, lead II nitrate, sodium phosphate monobasic, ascorbic acid, lithium chloride, potassium permanganate, calcium cyanide, cobalt(II) sulphate. 2. Analytical Chemistry 4. Organic Chemistry 3. Physical Chemistry 2. Analytical Chemistry 1.2 Overview of universities and institutions of higher education and research developed in Brazil y sodium hydroxide, nitric acid, sulfuric acid, Hydrochloric acid, potassium thiocyanate, nickel salts, potassium ferrocyanide, and heavy metals in the form of their salts, as salts of silver, chromate and potassium dichromate, lead salts like lead II chloride, mercuric chloride, copper II salts and cadmium salts. chromate and potassium dichromate, ammonium thiocyanate, ethyl acetate, acetone, acetic acid, ethanol, naphthalene, diphenylamine, sodium dodecyl sulfate, copper sulfate, potassium chlorate, manganese(IV) oxide, phenol, zinc sulfate, nickel sulphate and silver chloride. 3. Physical Chemistry 4. Organic Chemistry 4. Organic Chemistry glycerin, benzoic acid, dinitrobenzene, glucose, copper(II) oxide, barium hydroxide, sodium ferrocyanide, aminobenzene, benzoic acid, ethoxy ethane, hydrochloric acid, ethanol, cyclohexanol, sulfuric acid, cyclohexene, potassium permanganate, nitrobenzene, acetanilide, 2-propanol, acetone, n-butyl ether, phenylamine, sodium nitrite, copper sulfate, adipic acid, carbon tetrachloride, liquid bromine, eugenol, urea, y p y pp adipic acid, carbon tetrachloride, liquid bromine, eugenol, urea, Overview Management Chemical Residues of Laboratories in Academic Institutions in Brazil 355 formaldehyde, salicylic acid, methyl salicylate, acetic anhydride, acetylsalicylic acid, 4-aminoazobenzene, caffeine, ethyl acetate, methanol, dichloromethane, benzanilide, 4- nitroacetanilide, methyl benzoate, hydroquinone diacetate, benzocaine, 1,3-dibenzoylacetone, butyraldehyde, benzaldehyde, dimethyl phthalate, phthalic anhydride, 2,4,6- tribromoaniline, methyl salicylate, isopropyl bromide, among others. 5. Inorganic Chemistry 5. Inorganic Chemistry potassium permanganate, sulfuric acid, hydrogen peroxide solution, sodium thiosulfate, sulfur, potassium iodate, sodium metabisulfite, potassium hydroxide, sodium hypochlorite, ferrous sulfate, nickel(II) nitrate, cobalt(II) chloride, calcium carbonate, magnesium carbonate, magnesium chloride, ammonium chloride, ammonium hydroxide solution, strontium chloride, ammonium sulfate, barium chloride, acetic acid, sodium tetraborate, aluminum sulfate, sodium carbonate, nickel(II) nitrate, lead(II) nitrate, barium sulfate, calcium sulfate, magnesium sulfate, strontium chloride, sodium acetate, ferric chloride, ammonium thiocyanate, sodium chloride, potassium chloride, sodium bromide, potassium iodide, ammonium chloride, calcium hydroxide, ammonium iron(II) sulfate hexahydrate, potassium fluoride, potassium thiocyanate, copper(II) sulfate pentahydrate, lithium chloride, methanol, potassium oxalate, ethanol, cobalt(II) sulfate heptahydrate, sodium silicate, potassium chromium(III) oxalate trihydrate, tris(ethylenediamine) nickel(II) chloride hydrate, cobalt (II) nitrate, tris (ethylenediamine) cobalt(III) nitrate, tris (ethylenediamine) nickel(II) chloride hydrate, nickel(II) acetate tetrahydrate, ammonium nitrate, tetra amin cobalt (III) carbonate, pentaamminechlorocobalt(III) chloride, tris (oxalate) chromate (III), potassium copper chloride (II), tartaric acid, formaldehyde solution. Table 1. A survey of reagents used in basic experimental subjects in five major areas of Chemistry in Brazilian universities. The last but not the least important area listed herein is Inorganic Chemistry. 1.2 Overview of universities and institutions of higher education and research developed in Brazil It is the branch of Chemistry that studies chemical elements and nature substances that do not display carbon in their structures, investigates structures, properties and explains the mechanism of their reactions and transformations. In Experimental Inorganic Chemistry, various chemicals are necessary for reactions (Table 1), as well as other several chemicals are formed after reactions. The great variety of substances used or generated during practice classes usually makes waste management in educational and research institutions or similar service providers more intricate than in industry. Unlike industries, these institutions generate small amounts of waste, most of which in laboratories. These wastes consist of a wide variety of substances, toxic or non-toxic, including new compounds of unknown toxicity. Besides, their compositions change on every new research project or experiment (Gerbase et al., 2005; Tavares & Bendassolli, 2005; Jardim, 1998; Ashbrook & Reinhardt, 1985). 356 Environmental Management in Practice Implantation of a Program Management of Chemical Residues (PMCR) should whenever possible accomplish the priority scales or hierarchy (Jardim, 1998):  Prevent waste generation (hazardous or not);  Minimize the amount of hazardous wastes that are inevitably generated;  Segregate and concentrate waste currents in order to make possible and economically viable a managing activity;  Segregate and concentrate waste currents in order to make possible and economically viable a managing activity;  Reuse internal or external wastes;  Recycle material or energetic components of the waste;  Keep all waste at its most treatable conformation;  Safely treat and dispose wastes. In this context, can we achieve this premise if we promote changes in daily activities of professionals: professors, researchers and laboratory technicians directly involved in the generation of chemical wastes (Nolasco et al., 2006; Tavares & Bendassolli, 2005; Singh, 2000): In this context, can we achieve this premise if we promote changes in daily activities of professionals: professors, researchers and laboratory technicians directly involved in the generation of chemical wastes (Nolasco et al., 2006; Tavares & Bendassolli, 2005; Singh, 2000):  a modification of a certain process (or analytical method), substitution of raw materials or inputs;  a modification of a certain process (or analytical method), substitution of raw materials or inputs;  minimization using microscale techniques;  segregation of waste into different classes of compatibility;  reapplication of waste inevitably produced by recycling or reuse; y y y g  treatment through acid/base neutralization and chemical precipitation of metals. 1.2 Overview of universities and institutions of higher education and research developed in Brazil In addition, practices of advanced oxidation processes (AOPs) (Pera-Titus et al., 2004; Perez et al., 2006), treatment and heavy metals recovery (Kurniawan et al., 2006), are already commonly used in several treatments of substances generated in laboratory. We can recommend other easily handling practices to reduce generated chemical wastes. One example is the preparation of the dye 1 - (p-nitrophenylazo)-2-naphthol from p- nitroacetanilide. Initially in experiments performed in Organic Chemistry I, where the p- nitroacetanilide is prepared, serving as a substrate for the next reaction, the production of 1 - (p-nitrophenylazo)-2-naphthol. This last reaction is performed in experimental classes of Organic Chemistry II. Therefore, the product generated in a practice class is applied in another, minimizing then generation of chemical wastes in class. One example is the preparation of the dye 1 - (p-nitrophenylazo)-2-naphthol from p- nitroacetanilide. Initially in experiments performed in Organic Chemistry I, where the p- nitroacetanilide is prepared, serving as a substrate for the next reaction, the production of 1 - (p-nitrophenylazo)-2-naphthol. This last reaction is performed in experimental classes of Organic Chemistry II. Therefore, the product generated in a practice class is applied in another, minimizing then generation of chemical wastes in class. We may also employ an alternative related to separation and identification of cations and anions in the subject Experimental Analytic Chemistry. Cations and anions not harmful to environment could be normally determined, but the identification of heavy metals, such as copper, silver, cadmium, lead, mercury and chromium, could be applied a single time. In experiments to be performed with these metals, procedures could be filmed and photographed step-by-step. Later, this material would be used to set a database with films and pictures containing all steps developed in the identification and determination of these elements. Then, the same experiments with these metals would not be conducted later, in the way that students of the following years would attend to audio-video classes, decreasing generation and supply of toxic residuals in laboratories. The final disposal of wastes may be achieved in industrial disposal sites or other adequate locations, avoiding the misinformation that incineration and co-processing are ways of disposal. In such processes residual material is burnt, gases that should be treated are produced and ashes are subsequently sent to landfills (Nolasco et al., 2006). 1.2 Overview of universities and institutions of higher education and research developed in Brazil The number of scientific publications coming from Brazil has grown steadily over the past 26 years, culminating in 26 482 in 2008 (Figure 2). In parallel, Brazil´s international Overview Management Chemical R id f L b i i A d 357 Overview Management Chemical Residues of Laboratories in Academic Institutions in Brazil contribution with articles has climbed from 0.8 % in 1992 to 2.7 % in 2008. There is a correlation between this increase and the growing number of PhDs awarded annually (UNESCO, 2010). Fig. 2. Evolution of number of scientific publications from Brazil in period 1992 to 2008. Source: UNESCO, 2010. Fig. 2. Evolution of number of scientific publications from Brazil in period 1992 to 2008. Source: UNESCO, 2010. We must be aware that benefits from our professional and scientific activities (publications, patents, scientific recognition, development of new products and technologies) may generate, on the other hand, chemical residues from varying degrees of dangerousness. They may require appropriate chemical treatment before being sent to final disposal (Afonso et al., 2003). Universities represent places where scientific knowledge is produced, from where usually their new products and technologies arise, building up its part in society and directly interfering in it. We know that training goes well beyond the execution of a specific undergraduate program, experiences through contact with professors, laboratory technicians and staff, apprenticeships, basic scientific research activities, tutoring and degree dissertations, among others. They surpass content or technical aspects of each area. Thus, we are concerned about the example being given to students when professionals apparently turn their backs to such problem. That is, students experience a total lack of accountability before an issue present in their own immediate environment. There is no denying that such experience will be part of this future professional education with possible repercussions in their professional future. p Universities, as opinion builders, should take advantage of all opportunities to create conditions for self-evaluation, seeking the formation of future professionals with environmental awareness, ethics and co-responsibility. In this sense is relevant to emphasize the fact that there is no sustainability in the social-environmental structure of universities, 358 Environmental Management in Practice either in the relationship among people, or the relationship among nature, people and their residuals. Another aspect to be taken into account is that, in the structure of the university, still prevail the issues created by knowledge fragmentation. 1.2 Overview of universities and institutions of higher education and research developed in Brazil This criticism is made by some parties in the university itself through scientific papers, books and lectures, though it does not resonate inside its own walls. We should not forget that universities are embedded in a social liberal context that seeks to adapt the principles of economic liberalism to the conditions of modern capitalism. Then, the entire academic community is suffocated by a "market" that requires high productivity and consequently hinders an integrated vision and a careful look to itself. In this context, environmental education plays a crucial role which can manage hazardous wastes in universities. In order to start wondering how could be this performance of environmental education, it is important to comprehend the significance of environmental education and what are its principles. 1.3 Brazilian legislation relevant to the theme It is also plausible to assume that waste production is not exclusive of industries, once laboratories in universities, colleges and research facilities also generate chemical residuals in high diversity and low volume, yet it may represent 1% of all hazardous wastes generated in a developed country (Ashbrook & Reinhardt, 1985). 2.9 million tons of industrial hazardous wastes annually produced in the country receive adequate treatment (ABETRE, 2002). It is also plausible to assume that waste production is not exclusive of industries, once laboratories in universities, colleges and research facilities also generate chemical residuals in high diversity and low volume, yet it may represent 1% of all hazardous wastes generated in a developed country (Ashbrook & Reinhardt, 1985). Such verification leads us to the fact that the matter of chemical residuals management, originated from research and teaching activities, should be taken as discussions and research themes that deserve more space in the Brazilian academic cycle. This should also be motivated by the important role that research and educational institutions play towards formation of human resources accustomed to environmental management practices (Afonso et al., 2003; Alberguini et al., 2003; Jardim, 1998). A survey regarding several management programs at institutions of higher education shows that management of their own wastes is an actual concern for most of them, and they are aware of the issues related to environmental degradation. However, between a spoken or written commitment and effective actions, there is a great distance. In this sense, we cannot just criticize universities for not complying with legislation, because, in most cases, the lack of infrastructure and resources, as well as public policies focused on the matter, limits the adequate disposal of wastes. As many industries do, colleges and universities encounter thorny problems dealing with hazardous wastes. Industry and academia alike are saddled with the rising cost of waste management and face sensitive liability for costs of waste cleanup (Ashbrook & Reinhardt, 1985). One institution experiencing problems with the dramatically rising costs of hazardous waste management is the University of Illinois. In 1977, the University spent $2,000 to dispose of 100 drums of chemical wastes. By 1982, the cost of disposing of 265 drums had risen to $46,000, an 87 % annual increase in the cost of storing, transportation and disposing of wastes in a landfill (Ashbrook & Reinhardt, 1985). Gerbase et al. 1.3 Brazilian legislation relevant to the theme There is a tendency in our society to consider harmful to the environment only those activities that generate large amounts of wastes. Consequently, these are great generators always under the supervision of state environmental protection agencies and subject to punishment. Small waste generators, such as educational and research institutions, biochemical and physicochemical laboratories, are usually considered not harmful to environment by inspection agencies and, therefore, rarely investigated as for discarding their chemicals wastes (Jardim, 1993). Solid, industrial, radioactive and health services wastes are under control of a specific legislation with standard rules and procedures for storing, transportation and final disposal. In these cases the national and state legislations indicate specific controlling agencies, and a valid principle is that polluter pays for its violations. This principle is part of the environmental law that forces the polluter to compensate all damages caused to the environment. In Brazil, Law 6.938/81 concerning the Environment National Policy establishes a "Objective Liability" which does not require proof of guilt in case of possible environment damage. That is, for a potential polluter to be punished it is solely necessary to demonstrate the link cause-effect between an activity developed by an organization and the given environmental damage. In summary, it means that a pollutant, even though being produced in acceptable concentrations established by current law, may cause environmental damage, subjecting responsible to compensation. Moreover, even if any indirect damage is detected, and since its connection to an organization is testified, the latter will be held responsible (Machado, 2002). Applying the “Objective Liability” Law is hindered by the difficulty in inspection procedures in several industry areas, research institutions and universities, inducing environmental risks and contributing to its degradation. This problem is almost always avoided until more serious threats, iniquities and environmental conflicts may reach people directly engaged to these contexts, such as inhabitants surrounding a degraded area where residuals present potential and effectively high levels of pollution and contamination (Penatti , 2008). ABETRE (Associação Brasileira de Empresas de Tratamento de Resíduos; “Brazilian Association of Waste Management Companies”) estimate that only 22% of approximately Overview Management Chemical R id f L b i i A d Overview Management Chemical Residues of Laboratories in Academic Institutions in Brazil 359 2.9 million tons of industrial hazardous wastes annually produced in the country receive adequate treatment (ABETRE, 2002). 2. Environmental education (EE) as a tool in the process of toxic waste management Several authors have addressed the lack of environmental awareness within academic communities as an obstacle to greening (Riera, 1996; Meyerson & Massey, 1995; Creighton, 1999). The consensus is that people must be educated before a change can take place. As for the sampled institutions, a double set of morals seems to exist. On the one hand, they all considered the lack of awareness to prevent a greening process within energy and waste management from taking place. However, practically nothing had been done to raise environmental awareness. The lack of environmental awareness was considered significant because people do not know how to act sustainable. In other words, investing in waste and energy reducing devices has no meaning unless people know how and why it should be carried out. Decision makers must be familiar with the benefits of greening to establish environmental policies and to invest in green devices, and academics must realize the necessity of being “green” role models to their students (Dahle & Neumayer, 2001). The importance of raising environmental awareness at high education institutions is now being recognized from various bodies; the UK Sustainable Development Education Panel (1999) notes that: p ( ) “All further and higher educational institutions should have staff fully trained and competent in sustainable development, and should be providing all students with relevant sustainable development learning opportunities.” g In Brazil, EE is guided by the Treaty on Environmental Education for Sustainable Societies, Environmental Education Policy and PRONEA (National Environmental Education Program) and has sought to build an interdisciplinary perspective to understand the issues that affect relationships between human groups and their environment and to intervene on them, activating several areas of knowledge (Carvalho, 1998). For the education to integrate in the process of Environmental Management, it is required that "conditions necessary for production and acquisition of knowledge and skills are provided and attitudes are developed attempting to individual and corporate participation in dealing with environmental resources and in conceiving and applying decisions that affect quality of physical-natural and sociocultural means" (Quintas, 2000). Thus, it is clear that we cannot expect a ready recipe for how environmental education deals with the management process. We may however start out a discussion on the principles of environmental education, having in mind that one of the aspects to be considered is the quest for the solution of a problem through a dialogue amongst actors involved in the problem. 1.3 Brazilian legislation relevant to the theme (2005) suggest some actions meant to funding, research and teaching regulation agencies in Brazil, in order to defeat financial difficulties inherent to the installation of programs for managing hazardous waste, such as: installation of programs for managing hazardous waste, such as:  resource allocation and specific convocations to Environmental and Hazardous Waste Management (chemical, biological and radioactive) in research and educational institutions;  resource allocation and specific convocations to Environmental and Hazardous Waste Management (chemical, biological and radioactive) in research and educational institutions;  establishment of working groups of experts in order to propose standard rules for Safety in Chemistry for research and educational institutions;  a quality criterion to be included as an item for evaluation by Ministério da Educação e Cultura (“Ministry for Education and Culture”; MEC) and Coordenadoria de Aperfeiçoamento de Profissional de Ensino Superior (“ Coordination for Improvement of Higher Education Professional”; CAPES), the existence, or project implementation, of a program for hazardous waste management in graduate and undergraduate institutions for education and research. Orloff and Falk (2003), discussing international perspectives on hazardous waste practices, suggest that an effective hazardous waste management program is a collaborative effort and must include input from all relevant parties: federal, state, and local government officials, citizens, academia, and representatives of industry and non-governmental organizations. Citizens are important stakeholders and their input about waste management is crucial to ensure acceptance of society. 360 Environmental Management in Practice 2. Environmental education (EE) as a tool in the process of toxic waste management Therefore, the construction of a management process will be conducted considering the several contributions by the actors involved, as well as its adequacy to local reality. The environmental educator must look for the meanings of human action that are in the roots of socio-environmental processes that seem to synthesize the core of interpretative making of environmental education. By demonstrating cultural and political meanings taking place in the interactive processes society-nature, the educator would be a translator of perceptions – which are also, on their turn, social and historical interpretations – that mobilize several interests and human interferences in the environment. In the opposite direction of an objective vision, in which interpreting the environment would mean conceive it in its factual reality, describe its laws, mechanisms and operation, one should demonstrate the horizons of historical-cultural sense that configure relationships with the environment for a certain human community and in a given time (Carvalho, 1998). Overview Management Chemical 361 g Residues of Laboratories in Academic Institutions in Brazil Our consideration on the contribution of environmental education is twofold. The first would be a broader approach, whose fundamental idea would be the accomplishment of an Environmental Education Policy for Sustainability at the University. The implementation of such policy, involving the entire academic community prioritizing the search for solutions for experienced environmental issues and often generated by the university itself, which would be extremely desirable, then, includes the toxic residues problem on a broader context, due to the complexity of the environmental problems nature. In this sense, the hazardous wastes problem would be treated like the solid wastes, sewage contamination, the rationalization of water and energy consumption, the quality of environment and interpersonal relationships at work, the institutional master plan, among others. Such an environmental policy would contribute in the sense of giving direction to where the toxic wastes matter resides in broader context. The acceptance of a systemic approach would be suggested, with the possibility to become clearer, as some problems may bear a common cause or a common solution. Hence, the possibility to combine financial resources would be eased, as well as the so much recommended interdisciplinary integration. y g The other approach would be associated to more technical aspects directly linked to management of self-generated wastes. 2. Environmental education (EE) as a tool in the process of toxic waste management In this sense, the interview methodologies, environment social representations, among others, would be applied at all times according to necessity, mainly during the development of work immediately with involved actors. 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) The system of environmental management is characterized by promoting sustainable development through a local procedure, aiming to develop a sense of global environmental responsibility. This system establishes in its execution general and specific processes for each section of activity in order to engage and involve all organization members. That is the reason why the institutional support is essential and the commitment of the highest hierarchies is an essential and ideal condition. Nevertheless, even when unconditional and unrestricted institutional support is lacking, is no reason for the process not to occur. A critical analysis on several programs of laboratory chemical wastes management at universities carried out by Nolasco et al. (2006) reveals a significant homogeneity regards adopted principles and emphasizes the opinion of professionals directly related to the execution of such programs. These professionals select as main difficulties the investment need in infrastructure and institutional support. Coelho (2001) states that within universities there is no lack of technical capability, yet political willingness of institutions in giving proper relevance to the matter, as well as the execution of internal policies, widely discussed and disseminated, involving the entire academic community and also support for scientific research by the responsible organs. Izzo (2000) acknowledges other aspects that complicates the implementation of PMCR in universities, HEI and research institutes and hinders the program coordination: (1) decentralization of these institutions in which different administration sections and departments work independently; (2) rotation of undergraduate and graduate students; (3) variation on research projects conducted in these institutions; (4) most of them do not present a centralized purchase and storing section. Variations in research projects and regular changes in lines of work reflect peculiarities in wastes generated in the universities when compared to industries. They present reduced 362 Environmental Management in Practice volume, though also a great diversity of compounds, which makes any establishment of a chemical treatment and or a standardized final disposal difficult (Gerbase et al., 2005; Jardim, 1998; Ashbrook & Reinhardt, 1985). ) The proposal to establish a Policy on Environmental Education for Sustainability in Universities (EEPSU) aims to unify planning, control and supervision of natural resources appliance that we use during our activities with awareness and always in search for sustainability. 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) It also has to do with us becoming responsible for the consequences of using these resources, trying to minimize our consumerism, avoiding waste of natural resources and promoting reduction of wastes generation of all kinds. All this tends to minimize any type of environmental impact. The policy established a pathway to best practice by requiring the establishment of an environmental management plan that would set out how the university would manage issues of environmental concern and interest “bearing in mind the commitment to principles of ecology-sustainable development”. The universities surveyed (Australian and New Zealand) have an environmental policy. The importance of developing an environmental plan is to establish concrete guidelines. Where a policy states that a university is committed to sustainable development, a plan will outline how sustainability will be included in university operations. Other aspects are that, by outlining future environmental development, plans facilitate accountability and leave the university open to criticism. Plans can foster participation and representation; committees and working groups may be established as representatives of the university community (Carpenter & Meehan, 2002). p y y ( p ) Dahle and Neumayer (2001) believed that the most important measure for reducing or overcoming established barriers to green university is to raise environmental awareness within campus communities, i.e. sustainable behavior cannot be expected to take place unless people understand the benefits and importance of doing so. Creighton (1999) suggests that, to achieve a “green” university that uses resources efficiently, creates little or no waste, and takes full responsibility for any waste that it does generate, a fundamental change in the thinking behind routine decisions of university administration, staff, faculty and students is needed. These alternatives can be achieved through the implementation of EEPSU increasing ethical conscience and co-responsible for the academic community (teachers, laboratory technicians and administrative and academic), which lead to formation of professionals aware of their citizenship and their professional role. p p Policies are necessary, it must be remembered that they are abstract statements of principles that, while creating a positive “environment”, do little to improve environmental performance; it is only when policies become operationalised through the establishment of a plan and subsequent programs that environmental performance can be improved (Carpenter & Meehan, 2002). 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) The Integrated Management for Chemical Residues in High Education Institutions (IMCRHEI) proposed by Giloni-Lima and Lima (2008) drew the scientific community’s interest, notwithstanding, as it was published in a Brazilian journal and written in Portuguese, its circulation was restrained. In this sense, we take the opportunity to spread it after revision and expansion, aiming to offer an integrated proposal, as its name implies, and in accordance with PEESU and other aspects of this chapter. It is worth mentioning that the MCRP is one of the problems experienced by HEI’s and will be addressed by EEPSU, and the former may happen even if the latter does not. On the other hand, if the policy is active, waste management would be part of a wider process. Overview Management Chemical R id f L b i i A d 363 g Residues of Laboratories in Academic Institutions in Brazil The participation of academics at all stages of the process would be of great value, because they would have the opportunity to demonstrate their interest in various working groups and act as multipliers, acting in the research for information and generating data applied for management of chemical residues. g The general structure of the flow chart proposed (Scheme 1) consists of four stages which The general structure of the flow chart proposed (Scheme 1) consists of four stages which Fig. 3. Basic flow chart to implantation the Integrated Management for Chemical Residues in High Education Institutions (IMCRHEI). Solid lines: direction to basic lines of work; dashed lines: feedback; dotted lines: direct application to results in the implantation of IMCRHEI. start from the constitution of a Manager Committee, which will coordinate the development process for deploying IMCRHEI, employing the formation of three Basic Work lines - 1st stage, with the formation of (1) Discussion Group, (2) Environmental Education Group and (3) of the Technical Chamber. The results of work developed in the first stage will define the main points to be worked out in stage 2, Pre-implantation of the Management Program. 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) It is worth noting that there is a consensus that environmental education is an important tool in the execution of waste management programs, though they are usually based on activities of passive and active environmental surveys (task proposed to discussion group) and application of hierarchy scales proposed by technical chamber, where EE is only mentioned in almost all programs. Activities of teaching and research for its peculiarities have a proper time to be concluded and execute the application of its results (dotted line in Scheme 1). The 4th stage, Process Evaluation, is the time to review achievements or failures from the Quality and Performance Indicators established by the basic lines of work, which can forward their findings to promote the necessary adjustments to the process as a whole. The learning process generate information to permit self-regulatory and self-correcting activities to occur, while involving monitoring, applying systems thinking, and performing self-assessment. Without the use of environmental indicators for monitoring, environmental audits and self-assessments, learning might not take place. These tools enhance learning process (Herremans & Allwright, 2000). Creation of the Manager Committee is based on a discussion process for the formation of groups of professionals from various fields of training and identified with the EEPSU. The form of constitution of the Manager Committee and groups which are willing to develop the lines of work can follow the own institution dynamics, or still can start from the creation of a discussion forum which may represent an open space for discussions about the theme, circulation of information about the institutional reality regarding the current management of self-generated waste, to exchange experiences, and especially will enable professionals to evaluate the profile that would be more suitable, aware and willing to engage in coordination of the process. p We suggest the formation of a multidisciplinary group, involving faculty members from areas of administration, chemistry and biology, education, and student body of the institution to coordinate the three basic lines of work (Stage 1) of IMCRHEI implementation, aiming to integrate the academic and scientific community in the process, being as follows: 1. Discussion Groups: These groups might work in survey information with two types of waste: the passive (which includes reaction from debris, passing through solid waste and bottles without labels) and active (continuously generated, the result of routines in the generating unit). 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) Once initiated this step, after gathering all basic requirements needed, i.e., setting goals and objectives of the management program, as well as a minimum training required for (1) Discussion Group Passive and active environmental survey, IMCRHEI goals (2) EE Group Academic community sensibilization and participation of all actors (3) Technical Chamber Priority scale application in IMCRHEI Management Committee IMCRHEI Goals Launching purposes for implantation of management program (2) Human resources Training (academic community or actors) (3) Teaching and Research Activities (Subjects and research lines towards management chemical residues) Application of results obtained in teaching, research and extension activities in IMCRHEI Popularization of goals and objectives established to management program Popularization to academic community or actors Scientific dissemination publication of data in monographs, degree words, other researches IMCRHEI implantation Quality and performance indicators Work lines (1st stage) Pre-implantation of IMCRHEI (2nd stage) Popularization and implantation of IMCRHEI (3rd stage) Process evaluation (4th stage) Management Committee Fig. 3. Basic flow chart to implantation the Integrated Management for Chemical Residues in High Education Institutions (IMCRHEI). Solid lines: direction to basic lines of work; dashed lines: feedback; dotted lines: direct application to results in the implantation of IMCRHEI. start from the constitution of a Manager Committee, which will coordinate the development process for deploying IMCRHEI, employing the formation of three Basic Work lines - 1st stage, with the formation of (1) Discussion Group, (2) Environmental Education Group and (3) of the Technical Chamber. The results of work developed in the first stage will define the main points to be worked out in stage 2, Pre-implantation of the Management Program. Once initiated this step, after gathering all basic requirements needed, i.e., setting goals and objectives of the management program, as well as a minimum training required for 364 Environmental Management in Practice compliance, from where stage 3 - Divulgation and Implantation IMCRHEI can be initiated, one should look for unambitious and real goals, as failure in a first attempt tends to discourage further attempts (Jardim, 1998). compliance, from where stage 3 - Divulgation and Implantation IMCRHEI can be initiated, one should look for unambitious and real goals, as failure in a first attempt tends to discourage further attempts (Jardim, 1998). 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) It can also be understood as a set of principles built interactively and shared by different groups that understand and transform reality through it (Moscovici, 2003). It is important to note that the information given here should be taken only as suggestions, because, as previously mentioned, the search for the paths to be followed will be built up by the group. Thus, the EE could help at times as in the situations listed below: g  assessment process through investigation of social representations of those involved: professors, students and laboratory technicians;  assessment process through investigation of social representations of those involved: professors, students and laboratory technicians;  in planning, involving results previously surveyed;  on the destination, by minimizing waste generation or disposal, and proper destination, developing in all groups involved an environmental and ethical conscience;  on the destination, by minimizing waste generation or disposal, and proper destination, developing in all groups involved an environmental and ethical conscience;  disseminating and instruction other laboratories that generate waste and chemicals which are not under the responsibility of professionals in the field;  internal and external divulgation of the management program implantation. The training program for the academic community may act in the formation of specialized human resources in the management and disposal of chemical waste, both undergraduates and graduates, which extends beyond technical skills, may further strengthen ethical and co-responsible conscience in regards of chemical safety in workplace and environmental liability. y Training courses could also involve practical actions that try to minimize environmental impact and risk to those involved with direct or indirect engagement with hazardous wastes, including chemical, biological and radioactive ones produced during teaching and research procedures (Gerbase at al., 2005). The acquiring funds could occur through projects submitted to funding agencies that support research through specific calls, or partnerships with public and private organizations that can be later profited with costless courses and supporting programs for social inclusion of people without qualifications. Such actions allow sharing with society the fundamental concepts of environmental wastes management through courses to the community (Bendassolli et al., 2003). The problems raised by the Technical Chamber in implementing a priority scale (Jardim, 1998), within the activities developed in the generator unit, may guide teaching, research and extension activities to be developed. 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) The characterization of passives shall be equated as well and using simple tests (Jardim, 1998; Armour, 1996). p ( ) 2. Group of Environmental Education aims to involve professionals in the field of Environmental Education (EE) and to promote a line of work to undertake a process to make the academic community aware of the relevance of the thematic, even before the establishment of plan goals. These professionals may also work in developing an Environmental Education Program based on information provided by discussion groups (1), in order to assist processes of dissemination and training of the academic community. y 3. Technical Chamber: this group may act to evaluate the practical activities carried out in laboratories (in conjunction with the teachers responsible for them), the implantation of the priority scale (Jardim, 1998) seeking the best alternatives within its institutional reality. This group can also act as a generator of theme for future research directed to Overview Management Chemical R id f L b i i A d Overview Management Chemical Residues of Laboratories in Academic Institutions in Brazil 365 monographs, works of completion and until the creation of lines of research in order to structure the management of hazardous chemicals or not, the proposition of safety standards chemistry, etc. monographs, works of completion and until the creation of lines of research in order to structure the management of hazardous chemicals or not, the proposition of safety standards chemistry, etc. The environmental education group, due to its interdisciplinary, contextualized, critical and emancipatory nature must somehow be involved with groups 1 and 3. This situation is justified by the need for this group to understand all relationships involved in the management process. Its performance may occur at all times that involve human relations. It is worth emphasizing that the participation of all stakeholders is crucial. In order to increase likelihood of successful management, actors must be heard and have opportunity of opinion for it generates commitment. A commonly used method for environmental education to achieve this practice is the research participant. The technique of social representations also brings great contributions. A social representation is the common sense that one has about a given topic (Reigota, 2010), through this technique, one can find out scientific concepts of how they were learned and perceived by people. 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) The creation of specific subjects or others related to the theme and the availabiliby of trainings (Alberguini et al., 2003; Tavares & Bendassolli, 366 Environmental Management in Practice 2005; Bendassolli et al., 2003) are able to promote an interdisciplinary approach, stimulating the formation of academic student within a more holistic perspective, working on his technical training and environmental responsibility, and to promote ethical attitudes improving the profile of the future professional. In regards to research procedures, there are innumerable possibilities to include researchers, technicians and undergraduate and graduate students in generating data and information that reach the basic precepts and its major aspects in order to achieve the requirements of current environmental legislation. 2005; Bendassolli et al., 2003) are able to promote an interdisciplinary approach, stimulating the formation of academic student within a more holistic perspective, working on his technical training and environmental responsibility, and to promote ethical attitudes improving the profile of the future professional. In regards to research procedures, there are innumerable possibilities to include researchers, technicians and undergraduate and graduate students in generating data and information that reach the basic precepts and its major aspects in order to achieve the requirements of current environmental legislation. Internal and external divulgation (Stage 3) of the Integrated Plan for Waste Management is essential for awareness and dissemination of ideas and attitudes that corroborate the process. Nolasco et al. (2006) posits that utilization of intercommunication ways in the unit eases maintenance and continuity of the program. The World Wide Web (Internet) has been extensively used for this purpose, facilitating communication and access to information relevant to the MCRP. Internal and external divulgation (Stage 3) of the Integrated Plan for Waste Management is essential for awareness and dissemination of ideas and attitudes that corroborate the process. Nolasco et al. (2006) posits that utilization of intercommunication ways in the unit eases maintenance and continuity of the program. The World Wide Web (Internet) has been extensively used for this purpose, facilitating communication and access to information relevant to the MCRP. 3. Overview of PMCR implanted and proposition integrated management of chemical residues (IMCR) based on a environmental education policy for sustainability in universities (EEPSU) The fourth stage of the process is extremely important because it will promote an assessment process as a whole, through the use of Indicators of Quality and Performance, which could provide an evaluation of the process efficiency depending on the products you want to achieve allowing the use of feedback mechanisms and recurrence of achievements and failures, redirecting them in order to reach the intended goals and objectives. Some elements that can assist in structuring indicators of quality and performance are: identifying the goals to be achieved, defining forms of measurement that may be used and for each raised indicator, how these are calculated, how often the assessment will occur and how to interpret results. These can provide information to raise the specific points in which objectives and goals are not yet reached and allowing to evaluate in which line (1, 2 or 3) adjustments are necessary and feedback subsequent actions (dashed lines in Scheme 1). 3. Conclusion a great deal of universities in Brazil and the world have expressed their concern on the issue, taking on their responsibility for the development of science and technology towards management of chemical waste. g Management of hazardous chemicals in universities is a problem that must be seriously considered and with responsibility, and face the consequences regarding the relevance of professional formation to academics with ability to fairly practice its profession committed to citizenship. The integrated management program for chemical residues in HEI’s, pervades legal, educational, scientific and environmental management aspects. It involves the entire academic community where the priority would be the establishment of an environmental policy in the institution. The process begins with the composition of a manager committee that coordinates and directs the formation of the three basic lines of work: discussion groups, environmental education and the technical chamber (1st stage). The second stage is the IMCRHEI pre-implantation that establishes goals and objectives and where the sensibilization process on academic community begins, both encompassing the participation of all actors involved. It is important to emphasize that every line of work has different times for its consolidation, in particular the activities to be undertaken by the technical chamber. The proposal anticipates the circulation of goals and objectives planned cooperatively and, after implantation (stage 3), performance and quality indicators would be used in the evaluation process (stage 4), a feedback mechanism supports good results and indicates alternatives in case of failures. Environmental education plays an extremely essential role in this process, since through the joint participation of the entire academic community (professors, administrative and laboratory technicians, students), shows where is the lack of sustainability in our activities (wastage, misuse of natural resources, in generating and disposing of solid and chemicals wastes) and, associated with institutional expertise, explains how to more sustainably execute them. Concerning more specific issues, such as the management of chemical wastes, EE presents an integrated proposal, where it is present since the conception of the politic- philosophical proposal, in sensibilization and training of actors involved. EE may also act to minimize problems caused by knowledge fragmentation, helping to sensibilize skillful professionals to work in an ethical, conscious, responsible, critical and contextualized manner. Professionals able to execute their tasks in a "green" form, knowing the basic procedures of safety and environmental protection, both within and outside the university. 3. Conclusion Chemistry is a basic science that has brought great benefits to humanity and is present in everyday life, in textile, food, pharmaceutical, agrochemical, petrochemical industries, etc., revolutionizing our lives in bringing comfort and technologies that translate into improved life quality and expectancy. This science, due to its own features, is linked to progress and development, representative characters of contemporary society, which, in search for life quality, generates concerning environmental impacts. We need to handle environmental impacts as political matters and to conceive the environment as a common public property, as well as its care as a political right, expanding its comprehension and citizenship practice. The proposal for a Policy on Environmental Education for Sustainability at the University is conceived in order to assess possible environmental impacts related to activities undertaken by universities and by means of technical and scientific competence, the pursuit of interdisciplinary solutions, and more sustainable alternatives for managing, treatment, storage and disposal of chemical wastes generated in their teaching and research activities. The establishment of a sustainability policy can also facilitate access to financial resources, scarce on this issue, but unexpendable. Chemistry is a basic science that has brought great benefits to humanity and is present in everyday life, in textile, food, pharmaceutical, agrochemical, petrochemical industries, etc., revolutionizing our lives in bringing comfort and technologies that translate into improved life quality and expectancy. This science, due to its own features, is linked to progress and development, representative characters of contemporary society, which, in search for life quality, generates concerning environmental impacts. We need to handle environmental impacts as political matters and to conceive the environment as a common public property, as well as its care as a political right, expanding its comprehension and citizenship practice. h l f l l d f b l h We regard that environmental legislation in Brazil and in the world is evolving and trying to adjust to technological, economical and social developments. Despite the lack of legislation specifically defined for peculiarities of waste generated in Institutions of Higher Education, Overview Management Chemical Residues of Laboratories in Academic Institutions in Brazil Overview Management Chemical R id f L b i i A d 367 a great deal of universities in Brazil and the world have expressed their concern on the issue, taking on their responsibility for the development of science and technology towards management of chemical waste. Alberguini, L. B. A.; Silva, L. C. & Rezende, M. O. O. (2003). Laboratório de Resíduos Químicos do Campus USP-São Carlos – Resultados da experiência pioneira em gestão e gerenciamento de resíduos em um campo universitário, Química Nova, Vol. 26, No.2, (Fevereiro/2003), pp. 291-295, ISSN 0100-4042 Associação Brasileira de Empresas de Tratamento de Resíduos, ABETRE. (July 2002). Brasil trata apenas 22% dos resíduos industriais perigosos, In: ABETRE, 25.04.2011, Available from http://www.abetre.org.br/noticia_completa.asp?NOT_COD=373 4. References Associação Brasileira de Empresas de Tratamento de Resíduos, ABETRE. (July 2002). Brasil trata apenas 22% dos resíduos industriais perigosos, In: ABETRE, 25.04.2011, Available from http://www.abetre.org.br/noticia_completa.asp?NOT_COD=373 Associação Brasileira de Empresas de Tratamento de Resíduos, ABETRE. (July 2002). Brasil trata apenas 22% dos resíduos industriais perigosos, In: ABETRE, 25.04.2011, Available from http://www.abetre.org.br/noticia_completa.asp?NOT_COD=373 Alberguini, L. B. A.; Silva, L. C. & Rezende, M. O. O. (2003). Laboratório de Resíduos Químicos do Campus USP-São Carlos – Resultados da experiência pioneira em gestão e gerenciamento de resíduos em um campo universitário, Química Nova, Vol. 26, No.2, (Fevereiro/2003), pp. 291-295, ISSN 0100-4042 Alberguini, L. B. A.; Silva, L. C. & Rezende, M. O. O. (2003). Laboratório de Resíduos Químicos do Campus USP-São Carlos – Resultados da experiência pioneira em gestão e gerenciamento de resíduos em um campo universitário, Química Nova, Vol. 26, No.2, (Fevereiro/2003), pp. 291-295, ISSN 0100-4042 368 Environmental Management in Practice Afonso, J. C.; Noronha, N. A.; Felipe, R. P. & Freidinger, N. (2003). Gerenciamento de Resíduos Laboratoriais: Recuperação de elementos e preparo para descarte final, Química Nova, Vol. 26, No.4, (Outubro/2002), pp. 602-611, ISSN 0100-4042 Armour, M. A. (1996). Hazardous Laboratory Chemicals Disposal Guide, Press Inc. Boca Raton, ISBN 1–56670–108–2, Florida Ashbrook, P. C. & Reinhardt, P. A. 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(2001). Segurança Química nas Instituições de Ensino Superior, Ciência Hoje, Vol.29, no.169, (Março/2001), pp. 63-65 ISSN 0101-8515 Creighton, S.H. (1999). Greening the Ivory Tower, Improving the Environmental Track Record of Universities, Colleges, and Other Institutions, MIT Press, Cambridge, MA ISBN 0-262- 53151-8 Dahle, M. & Neumayer, E. (2001). 4. References Overcoming barriers to campus greening: A survey among higher educational institutions in London, UK, International Journal of Sustainability in Higher Education, Vol.2, No.2, pp.139-160, MCB University Press 1467-6370 Gerbase, A. E.; Coelho, F. S.; Machado, P. F. L. & Ferreira, V. F. (2005). Gerenciamento de Resíduos Químicos em Instituições de Ensino e Pesquisa, Química Nova, Vol.28, No.1, (Fevereiro/2005), pp. 3-3, ISSN 0100-4042 Giloni-Lima, P. C. & Lima, V. A. (2008). 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Implantação de um programa de gerenciamento de resíduos e águas servidas nos laboratórios de ensino e pesquisa no CENA/USP, Química Nova, Vol.28, No.4, (Julho/Agosto/2005), pp. 732-738, ISSN 0100-4042 UK Sustainable Development Education Panel. (1999). First Annual Report 1998, Department of the Environment, Transport and the Regions, London. 370 Environmental Management in Practice Environmental Management in Practice 19 1. Introduction The ecological criteria for the product group “lubricants” shall be valid for 4 years from the date of notification of the Decision. On the occasion of the next revision particular attention will be paid to following issues: the possibility of including an additional test for toxicity to flora, the use of standardised performance tests, evaluating the criterion on biodegradability and bio-accumulative potential, the percentage&sourcing of the renewable raw materials, static or dynamic link to the OSPAR list and the Community list of priority substances in the field of the water policy, possible extension of the scope of the group, evaluating whether criteria need to be more ambitious, evaluating the consumer information. g The criteria are designed to reflect the philosophy of the new EU regulatory framework for chemicals (REACH - Registration, Evaluation and Authorization of Chemicals)4 and are in line with the Dangerous Substances Directive and Dangerous Preparations Directive. Besides that, substances appearing in the Community list of priority substances in the field of water policy and the OSPAR List of Chemicals for Priority Action, shall not be intentionally added as an ingredient in a product eligible for the European eco-label. According to the background document for the European Eco-label to lubricants, Several European countries regulations and policies exist in favour of biolubricants. In Germany, Austria and Switzerland is forbidden the use of mineral oil based lubricants around inland waterways and in forest areas. In Italy there is a tax for mineral oils. In Portugal there is a regulation that mandates the use of biolubricant two-stroke engine oils in outboard boat engines. In Belgium is required to use a biolubricant in all operations taking place near non- navigable waters. In the Netherlands there is an action programme in favour of biolubricants since 1996. The use of non-toxic lubricant will improve health and safety of all individuals that are in contact with products and materials during their whole lifecycle. At the end of the life of the lubricants, recycling or disposal will release lubricants to the environment and again to individuals, either directly or via intermediate steps such as animal food or drinking water. Such a release of solid and/or liquid wastes can affect for many years the quality of life. For instance, the very harmful PCB´s have been already been banned 20 years ago. 3 “Ecological Criteria for the award of the Community ecolabel to lubricants”- Regulatory Committee of the European Parliament and of the Council- 2005. 4 Regulation of the European Parliament and of the council concerning the Registration, Evaluation, Authorisation and Restrictions of Chemicals. 1. Introduction The most of the lubricants such as automotive lubricants, gear oils, automatic transmissions fluids, engine lubricants, compressors oils... have being disposed on the environment for years, without any special care. Waste disposal is becoming a major source of concern and the tolerance of unnecessary pollution by society decreases1. y p y y As it has been described in the European General Instructions, 75/439/CEE, 78/319/CEE and 87/101/CEE, used lubricants as toxic disposals are attached to several rules and standards. One of the main problems of lubricants is the proportion of them lost into the environment in the course of routine usage2. The spillage of lubricants to the environment is one of the main problems during their routine usage. About 600000 Tonnes of lubricating oils per year are lost without control which implies a hard damage to the environment because just 1 litre of lubricant over the water is needed to form a film of oil of 4000 m 2. In Europe the 50% of the lubricants are consumed for automotive purposes, and 35% for general industries (compressors, turbines, hydraulics, bearing, metal-working). More than 95% of the market is dominated by the mineral oil based lubricants which are polluting the environment but have in this moment a lower price and high availability. The market of biolubricants is still in a development stage and there is a priority to formulate high performance biodegradable lubricants. The growing importance of environmental awareness and regulations has leaded to new demands of lubricants based on biodegradable materials. Several national eco- labels/schemes and one international standard have been developed in the recent years setting requirements for the ecological and technical characteristics of lubricants: The main difference relating to the ecological criteria for lubricants is the use of renewable raw materials, a newly included concept that aids to meet the three pillars of sustainability. An example of requirements concerning renewable raw material are the Swedish Standard for greases (SS 155470 Greases) and the Nordic Swan for lubricating oils. 372 Environmental Management in Practice The final criteria of European Eco-label3 for lubricants was published in the Official Journal of 5 May 2005 and it will be valid until June 2011. It comprises hydraulic oils, greases, chainsaw oils, two stroke oils, concrete release agents and other total loss lubricants, for use by consumers and professional users. 5 Carnes K. “University Tests Biodegradable Soy-Based Railroad Lubricant”, Hart’s Lubricantes world 1998, Vol. September, pp 45-47. 6 Glancey J.L., Knowlton S., Benson E.R. “Development of a High-Oleic Soybean Oil-based Hydraulic Fluid”, Lubricants World 1999, Vol. January, pp 49-51. 7 Rajewski T.E., Fokens J.S., Watson M.C., “The development and Application of Syntetic Food Grade Lubricants”, Tribology, 2000, Vol 1, pp 83-89. 8 W. J. Bartz: “Comparison of Synthetic Fluids”, Lub. Eng., 1992, 48, (10), 765-774. 9 S.Z.Erhan: “Lubricant basestocks from vegetable oils”, Industrial Crops and Products 11 (2000) 277– 282 1. Introduction Notwithstanding that, they are still being released by dismantling of old electrical transformers containing these products as coolants. The use of biodegradable lubricants will reduce problems on disposal. In most of the countries in the European Union each consumer is responsible of its own lubricant. It means that disposal of lubricants must be done by each consumer following the rules of each country. The non fulfillment of these rules can be fined or even imprisoned. Then, mineral oil based lubricants can be critical when arrive to the environment (earth and water). They contain substance which are not compatible with the biosphere and can cause damage to soil organisms, plants, aquatic organism. The use of biodegradable lubricant means that in 28 days, the 98% of the lubricant will be biodegradable and no film of oil will remain on the 373 Lengthening Biolubricants´ Lifetime by Using Porous Materials water. The soft impact produced to the environment by biodegradable lubricants spill incidents due to their non toxic formulation makes it less worrying and preserves the environment from an irreversible deterioration. Try to restore the nature as well as difficult (sometimes impossible) is very expensive. Clearly, disposing of non-toxic and biodegradable lubricant will protect consumers. water. The soft impact produced to the environment by biodegradable lubricants spill incidents due to their non toxic formulation makes it less worrying and preserves the environment from an irreversible deterioration. Try to restore the nature as well as difficult (sometimes impossible) is very expensive. Clearly, disposing of non-toxic and biodegradable lubricant will protect consumers. Today, to deserve the name of “environmental friendly”, a lubricant must possess several characteristics in different aspects as biodegradability, toxicity, emissions and efficiency. Vegetable oils, carefully selected esters and polyglycolethers form the bulk of base fluids in this type of product, generally given the generic name of biodegradable lubricants5,6,7. Esters in general are known as good lubricants8, with low volatility, low pour points (-65 ºC compared with -15 ºC for mineral oils), good solubility for additives and high viscosity indexes. There are several types of biodegradable esters: dibasic esters or diesters, polyol esters (hindered esters), and phosphate esters. They find applications in engines, gears and compressors where cleanliness is important and the latter type as a fire-resistant fluid. Moderate corrosion behaviour is the major drawback. 1. Introduction It is recommended to use saturated natural esters (low iodine value) to formulate biodegradable oils, because they have better thermal and oxidation stability. Lubricants based on vegetable oils still comprise a narrow segment; however, they are finding their way into such applications as chainsaw bar lubricants, drilling muds and oils, straight metalworking fluids, food industry lubricants, open gear oils, biodegradable grease, hydraulic fluids, marine oils and outboard engine lubricants, oils for water and underground pumps, rail flange lubricants, shock absorber lubricants, tractor oils, agricultural equipment lubricants, elevator oils, mould release oils, two stroke engine lubricants and other. Volatility or viscosity index being cited the most often, vegetable oils clearly outperform mineral oils. Many of the other properties are similar between the fluids or may be manipulated with additives. However, low resistance to oxidative degradation and poor low temperature properties are major issues for vegetable oils. The lubricant industry’s inability to overcome these limitations ignited a rapid rise in demand of highly biodegradable synthetic basestocks as low molecular weight poly a- olefins (PAO 2 or PAO 4, essentially 20:1 and 10:1 mixtures of hydrogenated dimers:trimers of alpha-decene), di alkyl adipates (iso decyl, iso tridecyl) or polyol esters (mostly neopentyl glycol or trimethylol propane with fatty acids). The synthetic basestocks also have some imperfections, such as higher volatility of PAOs, seal swelling of adipates, questionable biodegradability of some polyols, and, frequently the major issue, costs of nearly three times higher than that of vegetable oils9. Polyalphaolefin fluids are enjoying a growing market share as synthetic base stocks. They are manufactured by the oligomerization of 1-decene, followed by hydrogenation and distillation into different viscosity grades. Applications range from hydraulic fluids to car 374 Environmental Management in Practice motor oils10,11,12.They have excellent thermal, oxidative and hydrolytic stabilities. Recently, it has been reported that 2cSt and 4cSt PAO fluids are easily biodegradable, so they can be used in environmentally sensitive applications13. From the Polyglycol side only Polyethyleneglycols (PEG) with mol-weights lower than 2000 are relatively good biodegradable, but water-soluble, means they migrate into the soil after an oil accident or via leakages. Polypropylenes (PPG) are not water-soluble but not easily biodegradable substance. Furthermore PAG´s are only partly or even not miscible with esters, mineral oils, etc. 10 C-X. Xiong: “The structure and Activity of Polyalphaolefins as Pour-Point Depressants”, Lub. Eng., 1993, 49, (3), 196-200. 11 G Kumar: “New Polyalphaolefin Fluids for specialty applications”, Lub. Eng., 1993, 49, (x), 723-725. 12 R. L. Shubkin: “Polyalphaolefins: Meeting the Challenge for High-Performance Lubrication”, Lub. Eng., 1994, 50, (x), 196-201. 13 J. F. Carpenter: “Biodegradability of Polyalphaolefin (PAO) Basestocks”, Lub. Eng., 1994, 50, (5), 359- 362. 14 M.K. Williamson “The emerging Role of Oil analysis in Enterprise-Wide decision making”. Practicig Oil analysis 2000. pp. 187-200. 15 “Lubricants and lubrication”. T. Mang, W. Dresel (Eds). Wiley-VCH. 2001 16 “Lubricating grease guide”. Fourth Edition. National Lubricating Grease Institute (NLGI) 17 A. Adhvaryu, “Oxidation kinetics studies of oils derived from unmodified and genetically modified vegetables using pressurized differential scanning calorimetry and nuclear magnetic resonance spectroscopy”. Thermochimica Acta, 364, 87-97. 2000 18 N.J. Fox, A.K. Simpson, G.W. Stachowiak, ”Sealed Capsule Differential Scanning Calorimetry-An Effective Method for Screening the oxidation Stability of vegetable oil formulations”. Lubrication Engineering, 57, 14-20. 2001 19 A. Adhvaryu, “Tribological studies of thermally and Chemically modified vegetable oils use as environmentally friendly lubricants”. Wear, 257, 359-367, 2004 20 F.Novotny-Farkas, P. Kotal, W. Bohme. “Condition monitoring of biodegradable lubricants”. World Tribology Congress. Vienna. 2001 15 “Lubricants and lubrication”. T. Mang, W. Dresel (Eds). Wiley-VCH. 2001 10 C-X. Xiong: “The structure and Activity of Polyalphaolefins as Pour-Point Depressants”, Lub. Eng., 1993, 49, (3), 196-200. 10 C-X. Xiong: “The structure and Activity of Polyalphaolefins as Pour-Point Depressants”, Lub. Eng., 1993, 49, (3), 196-200. 11 G Kumar: “New Polyalphaolefin Fluids for specialty applications”, Lub. Eng., 1993, 49, (x), 723-725. 12 R. L. Shubkin: “Polyalphaolefins: Meeting the Challenge for High-Performance Lubrication”, Lub. Eng., 1994, 50, (x), 196-201. 13 J. F. Carpenter: “Biodegradability of Polyalphaolefin (PAO) Basestocks”, Lub. Eng., 1994, 50, (5), 359- 362. 14 M.K. Williamson “The emerging Role of Oil analysis in Enterprise-Wide decision making”. Practicig Oil analysis 2000. pp. 187-200. Eng., 1994, 50, (x), 196-201. 13 J. F. Carpenter: “Biodegradability of Polyalphaolefin (PAO) Basestocks”, Lub. Eng., 1994, 50, (5), 359- 362. 14 M.K. Williamson “The emerging Role of Oil analysis in Enterprise-Wide decision making”. Practicig Oil analysis 2000. pp. 187-200. 15 “Lubricants and lubrication”. T. Mang, W. Dresel (Eds). Wiley-VCH. 2001 16 “Lubricating grease guide”. Fourth Edition. National Lubricating Grease Institute (NLGI) 17 A. Adhvaryu, “Oxidation kinetics studies of oils derived from unmodified and genetically modified vegetables using pressurized differential scanning calorimetry and nuclear magnetic resonance 6 “Lubricating grease guide”. Fourth Edition. National Lubricating Grease Institute (NLGI) ub cat g g ease gu de . ou t d t o . Nat o a ub cat g G ease st tute (N G ) 17 A. Adhvaryu, “Oxidation kinetics studies of oils derived from unmodified and genetically modified vegetables using pressurized differential scanning calorimetry and nuclear magnetic resonance spectroscopy”. Thermochimica Acta, 364, 87-97. 2000 1. Introduction But the compatibility of all the fluids is an absolutely neccesary item in case of re-filling or replacement of mineral oil by biodegradable fluids.The polyglycol used in this study (of about 320cSt of viscosity at 40ºC) is not biodegradable. y ( y ) g There is still a priority to formulate high performance biodegradable lubricants. The main limitation to introduce bio-degradable lubricants is the lack of knowledge of the performance of the biolubricants. A review of the state of the art has been developed to check the status of condition monitoring in mineral and biodegradable oils and greases14,15,16. Results indicated that there is hardly any documentation (application note, technical papers, standards) concerning how to tackle bio-lubricants. Also, documentation concerning how to perform a condition monitoring of greases is hardly non-existant. The procedures and test methods to detect contaminants has been developed for mineral based lubricants, but new procedures has to be developed for environmentally friendly lubricants. In order to assess this performance, it is important to understand how degradation processes occurs at biodegradable fluids17,18,19 and identify adequate control parameters, limits and sampling frequency (or re-greasing frequency). Apart from hydraulic fluids20, there is no information about how to efficiently handle the monitoring of biodegradable lubricants. In order to assess this performance, it is important to understand how degradation processes occurs at biodegradable fluids17,18,19 and identify adequate control parameters, limits and sampling frequency (or re-greasing frequency). Apart from hydraulic fluids20, there is no information about how to efficiently handle the monitoring of biodegradable lubricants. Table 1.1shows the status in the definition of parameters, limits and sample frequencies at different lubricant types (lubricating oils and greases and biodegradable lubricating oils and greases). The OK sign (√) indicates that the field (parameters, limits and sample frequencies) p py 18 N.J. Fox, A.K. Simpson, G.W. Stachowiak, ”Sealed Capsule Differential Scanning Calorimetry-An Effective Method for Screening the oxidation Stability of vegetable oil formulations”. Lubrication Engineering, 57, 14-20. 2001 g g 19 A. Adhvaryu, “Tribological studies of thermally and Chemically modified vegetable oils use as environmentally friendly lubricants”. Wear, 257, 359-367, 2004 y y 20 F.Novotny-Farkas, P. Kotal, W. Bohme. “Condition monitoring of biodegradable lubricants”. World Tribology Congress. Vienna. 2001 20 F.Novotny-Farkas, P. Kotal, W. Bohme. “Condition monitoring of biodegradable lubricants”. World Tribology Congress. Vienna. 1. Introduction 2001 375 Lengthening Biolubricants´ Lifetime by Using Porous Materials has already been properly defined and all aspects of these fields have been put into practice. The OK sign and plus mark (√/+) indicates that some studies have been carried out, however a improvement of the definition is necessary. The plus sign (+) indicates the field that we are trying to improve by means of this study. A high advance has been obtained in the work of obtaining a proper definition of each of fields. The question mark (?) indicates that there is not any adequate definition of the field and it is not going to obtain by means of this study. Table 1. Knowledge of parameters that have to be measured, limits and sample frequencies at lubricating oils, biodegradable lubricating oils, lubricating greases and biodegradable lubricating greases. Table 1. Knowledge of parameters that have to be measured, limits and sample frequencies at lubricating oils, biodegradable lubricating oils, lubricating greases and biodegradable lubricating greases. The development of the technology on different areas such as manufacturing, electronic and nanotechnology has allow us to develop new devices for improving the lubricants control and has opened an wide range of research areas. The main objectives of these developments are the following: to avoid the lubricant degradation by means of the use of filters or additives and to control “on-line” the condition of the lubricant by means of sensors21,22. This chapter includes a first proposal for the condition monitoring strategy developments of biolubricants (BIOMON Project contract NºCOOP-CT-2004-508208) and some results concerning the potential of porous materials for trapping oxidation molecules of the biolubricants during use for lengthening their lifetime instead of traditional antioxidant additives (SOILCYProject contract N515848). 21 Arnaiz, A., Aranzabe, A., Terradillos, J., Merino, S., Aramburu, I.: New micro-sensor systems to monitor on-line oil degradation, Comadem 2004. pp. 466-475 22 Kristiansen, P., Leeker, R.: U.S.Navy’s in-line oil analysis program, , lubr. Fluid powerj. 3, 3–12, aug 2001. 21 Arnaiz, A., Aranzabe, A., Terradillos, J., Merino, S., Aramburu, I.: New micro-sensor systems to monitor on-line oil degradation, Comadem 2004. pp. 466-475 g pp 22 Kristiansen, P., Leeker, R.: U.S.Navy’s in-line oil analysis program, , lubr. Fluid powerj. 3, 3–12, 2001. 2.1. Degradation mechanism g One of the important modes of lubricant degradation is oxidation. Oxidation products are the primary cause of metal corrosion, viscosity increase and sludge and varnish formation in lubrication systems. It is well-known that a lubricant when exposed to air at high temperatures undergoes a series of chain reactions that form peroxides, which upon further reaction produce low molecular weight products (products of the same or lower molecular weight than the original oil). The LMW products are a mixture of ketones, aldehydes, alcohols and acids. These primary oxidation products can polymerize to form higher molecular weight (HMW) viscous liquids, sludge and varnish. g ( ) q g Some studies of the oxidation mechanism of the vegetable oils reveal that the oxidation mechanism may be presented as a free radical chain reaction. It consists of four distinct reaction steps: initiation, propagation, branching and ending. These steps have been analyzed. The initiation step consists of forming free radical derived from R substance that undertake the peroxidation and it takes place due the high transformation energy, UV light of the mechanical shear stresses applied to a molecule. The appearance of the free radicals is due to one or more external factors (high temperature, moisture, pressure, oxygen presence) or internal factors (easily oxidable impurities, for instance: aromatic diphenol compounds). Under certain conditions the external factors produce the O2 activation and the development of some reactive elements and radicals. Although the radicals of the oxygen may accidentally appear, the reactions that release them are enough to initiate the formation of some free radicals from the initial organic substances. g The propagation step consists in the continuation of forming free radicals especially of the derived peroxides from the substance that bear the oxidation progress. The first propagation step involves an alkyl radical reacting with oxygen to form an alkyl peroxy radical (ROO.). The second propagation step is the hydrogen abstraction from a hydrocarbon molecule by an alkyl peroxy-radical to form a hydroperoxide and another alkyl radical.Generally the alkyl peroxy radicals are in a greater concentration than the alkyl radicals. This is due to the combination between high oxygen concentration with alkyl radical rather than the slow reaction rate of alkyl peroxy radical with hydrocarbon. The chain branching steps begin with the decomposition of hydroperoxide (ROOH) in alkoxy radicals and hydroxy radical. 2.1. Degradation mechanism This reaction has a great activation energy and the alkoxy and hydroxy radicals can react on with hydrocarbons to form more alkyl radicals, alcohols and water. Secondary and tertiary alkoxy radicals, will form aldehydes and ketones respectively. These condense via acid catalysed aldol reactions can lead to polymeric degradated products. The ending step may take place by the combination of the alkyl radicals. Two alkyl radicals can combine to form a hydrocarbon molecule. Alternatively, an alkyl radical may combine with an alkyl peroxy radical (ROO.) to form a peroxide or two alkyl peroxy radicals (2ROO.) (ROOR.). 2. Degradation mechanism of biolubricants and analytical techniques used for biolubricants monitoring A commercial hydraulic fluid (ISO VG 68/MP=Multi-purpose) which is currently in use for roller bearing purposes has been used in this study as fully formulated mineral oil. EP additives but no antioxidants have been included in its formulation. Secondly, a biodegradable nearly fully saturated ester has been developed in order to study its oxidation process. EP & antioxidant additives have been included in the formulation of the biodegradable oil. 376 Environmental Management in Practice 23Mang, T. y Dresel, W. (eds.), Lubricants and Lubrication, 2ª edición, Wiley-VCH, Weinheim (2007). 24 C.Duncan (2002), Lubrication Engineering, “Ashless Additives and New Polyol Ester Base Oils Formulated for Use in Biodegradable Hydraulic Fluid Applications” 2.2 Oxidation tests Oxidation tests can be differentiated into two groups: one kind of tests describe the condition of the lubricant after a defined test period by measuring several aging indicating parameters like acid number, viscosity change and sludge formation. The 1000-h TOST according to DIN 51587 and IP 280 oxidation test are examples for that kind of tests. Another group of oxidation tests measure the so-called induction time (time from the 377 Lengthening Biolubricants´ Lifetime by Using Porous Materials beginning of the oxidation to the autocatalytic phase of the autoxidation) by recording constantly or after defined periods of time the aging indicating parameters. Typical examples of this group are the lifetime TOST test according to ASTM-D 943 or the rotary bomb test (ASTM-D 2272) where the drop of oxygen pressure indicates the aging of the lubricant. More recently the high pressure differential scanning Calorimetry (HPDSC) as well as the sealed capsule differential scanning calorimetry (SCDSC) has been applied more and more to measure the stability of different antioxidants and formulations. These tests for lubricants are a function of temperature or other parameters. These aging tests are carried out on base oils as well as fully formulated products to test the efficiency of additives. There is a big variety of standardized test methods where we have duration of test between hours and months. The majority of these tests is based on exposing the test fluids to oxygen or air at relatively high temperatures in presence of catalyst metals to increase oxidation rates and to reduce the testing period. Oxidation stability is assessed by quantitative determination of oxidation products, oxygen absorption, viscosity changes, change in acidity or formation of sludge. The TOST (turbine oxidation stability test) is used to evaluate the oxidation stability of inhibited steam turbine oils in presence of oxygen, water, copper and iron catalyst at 95ºC. The test is continued until the total acid number (TAN) measured reaches at least 2.0 mg/g KOH. The number of test hours required for the oil to reach 2.0 mg/g KOH is the “oxidation lifetime” of an oil. The oxidation stability by rotary bomb test (ASTM D 2272) oxidizes the oil at 150ºC in presence of water, metallic copper catalyst and oxygen at 620 kPa pressure. The time is registered to reach a specific pressure drop and this is an indication of the oxidation stability. Fig. 1. Oxidation reactor 2.2 Oxidation tests The aging test according to Baader (DIN 51554) is an oxidation test of atmospheric air and intermittent immersion of a copper spiral at a test temperature of 95ºC. After a given time, the saponification number in mg/g KOH is measured. The oxidation stability by rotary bomb test (ASTM D 2272) oxidizes the oil at 150ºC in presence of water, metallic copper catalyst and oxygen at 620 kPa pressure. The time is registered to reach a specific pressure drop and this is an indication of the oxidation stability. The aging test according to Baader (DIN 51554) is an oxidation test of atmospheric air and g p p p y The aging test according to Baader (DIN 51554) is an oxidation test of atmospheric air and intermittent immersion of a copper spiral at a test temperature of 95ºC. After a given time, the saponification number in mg/g KOH is measured. For the oxidation stability of lubricating greases by oxygen bomb method grease is oxidized in a bomb heated to 99ºC and filled with oxygen at 110 psig. After a defined time, the drop of pressure is recorded. The degree of oxidation after a given period of time is determined by the corresponding decrease of oxygen pressure. y p g yg p In the table below equivalent standards are grouped together which are identical or technically equivalent respectively23. New biodegradable polyol ester base stocks formulated with the appropriate ashless additive technology outperform vegetable oils both hydrolytically and oxidatively and modified versions of the ASTM D 943 and D2274 tests, in which no water is employed have shown that they are very suitable for the evaluation of the long-term oxidative stability of biodegradable polyol esters24 . Tekniker proposes the development of a new oxidation procedure for biodegradable lubricants because this kind of oils needs higher temperatures for degradation. Based on Tekniker experience, it is proposed a new oxidation test running in a bath reactor (figure 1) at 120ºC with stirring, air flux and without presence of water and catalyst. 2.2 Oxidation tests Environmental Management in Practice 378 AFNOR ASTM DIN IP ISO Test method 51352t1 48 Determination of oxidation characteristics of lubricating oil 51554 Determination of oxidation characteristics of lubricating oil acc.to Baader 280 Determination of oxidation stability of inhibited mineral turbine oils T60-150 D943 51587 4263 Determination of oxidation stability of inhibited mineral oils D2893 51586 Determination of oxidation characteristics of extreme-pressure lubricating oils D4742 Oxidation stability of gasoline engine oils by thin film oxygen uptake (TFOUT) D2274 229 Determination of the relative oxidation stability by rotating bomb of mineral turbine oil D942 51808 142 Determination of oxidation stability of lubricating grease- oxygen bomb method Table 2. Equivalent oxidation standards Fig. 1. Oxidation reactor 378 Environmental Management in Practice Environmental Management in Practice AFNOR ASTM DIN IP ISO Test method 51352t1 48 Determination of oxidation characteristics of lubricating oil 51554 Determination of oxidation characteristics of lubricating oil acc.to Baader 280 Determination of oxidation stability of inhibited mineral turbine oils T60-150 D943 51587 4263 Determination of oxidation stability of inhibited mineral oils D2893 51586 Determination of oxidation characteristics of extreme-pressure lubricating oils D4742 Oxidation stability of gasoline engine oils by thin film oxygen uptake (TFOUT) D2274 229 Determination of the relative oxidation stability by rotating bomb of mineral turbine oil D942 51808 142 Determination of oxidation stability of lubricating grease- oxygen bomb method Table 2. Equivalent oxidation standards Table 2. Equivalent oxidation standards Table 2. Equivalent oxidation standards Fig. 1. Oxidation reactor Fig. 1. Oxidation reactor Fig. 1. Oxidation reactor 379 Lengthening Biolubricants´ Lifetime by Using Porous Materials The table 3 shows the advantages of the oxidation procedure proposed for biolubricants compared with the oxidation standard procedure for mineral lubricants. The table 3 shows the advantages of the oxidation procedure proposed for biolubricants compared with the oxidation standard procedure for mineral lubricants. Heterogeneous samples are taken due to the absence of stirring Homogeneous samples are taken due to the presence of stirring Long degradation time due to the temperature of the test. Shorter degradation time due to the temperature of the test Heterogeneous samples are taken due to the absence of stirring Homogeneous samples are taken due to the presence of stirring Table 3. Oxidation standards Table 4. Analytical techniques for biolubricants monitoring 2.3.1.1 Acid number (ASTM D974-04) Both in new lubricants and in-service lubricants, acidic constituents will appear, either in the form of additives or as a result of the oxidation of lubricant. The Acid Number test method is a measurement of the quantity of those acidic constituents. Measurement has been by titration which estimates the amount of KOH which is necessary to neutralize the acid compounds of the oil). 2.3.1.2 Viscosity at 40ºC (ASTM D445) The viscosity remains the most important property of the oil as part of the oil condition monitoring. Viscosity measurements have been done following ASTM D 445 standard. g y In general, the variation of oil viscosity can be caused by: In general, the variation of oil viscosity can be caused by: - The increase of viscosity results from the oxidation and/or polymerization of the oil, evaporation of the most light fractions even water presence or insoluble oxides formation. - The increase of viscosity results from the oxidation and/or polymerization of the oil, evaporation of the most light fractions even water presence or insoluble oxides formation. - The decrease of viscosity may be due to thermal cracking of the molecules of the lubricant, or shear stress from the viscosity modifiers.... - The decrease of viscosity may be due to thermal cracking of the molecules of the lubricant, or shear stress from the viscosity modifiers.... 2.3 Analytical techniques used for biolubricants monitoring 2.3 Analytical techniques used for biolubricants monitoring Oxidation is a major source for viscosity increase, acid number increase or corrosion, additive depletions, dispersant failures, base oil deterioration, varnish and sludge formation, filter plugging, oil darkening, as well as many of the wear root causes. For this reason, there have been many analytical techniques to evaluate the oxidation state of the lubricant. The table 4 shows the chemical, tribological and environmental analysis selected for monitoring biolubricants. The chemical analysis includes the following parameters: Acid Number (ASTM D 974-04), Viscosity at 40ºC (ASTM D 445), Density (PE-5053-AI), Emulsification Capacity (ASTM D 1401), DSC (PE-5035-AI), Fourier Transform Infrared Spectroscopy (FTIR) (PE-5008-AI), Remaining Useful Life Number (RULER) (PE-TA.090) and %Solids. Sliding tests has been selected in the tribological field. Finally, in accordance with the European Ecolabel for lubricants, the following environmental analyses have been selected: Ready Biodegradability according to OECD 301F Test, Inherent Biodegradability according to OECD 302B Test and Toxicity according to OECD 202 Test. Table 4. Analytical techniques for biolubricants monitoring Table 4. Analytical techniques for biolubricants monitoring 380 Environmental Management in Practice 2.3.1.3 Density (PE-5053-AI) Density is the mass of a unit volume of a substance. Its numerical value varies with the units used. A high level of oxidation and/or polymerization of the oil, causes an increase in density values. Density is the mass of a unit volume of a substance. Its numerical value varies with the units used. A high level of oxidation and/or polymerization of the oil, causes an increase in density values. 2.3.1.5 Differential scanning calorimetry (PE-5035-AI) Samples are tested at extreme conditions of temperature, pressure and in an oxidative environment. Remaining useful life can be determined concerning the oxidative stability. At constant temperature, the lubricant degradation takes place at a time called “onset time”, with its corresponding heat exchange. This value can give an idea of the oil stability. 2.3.1.6 Fourier transform infrared spectroscopy (PE-5008-AI) 2.3.1.4 Emulsification capacity (ASTM D1401) This test method measures the ability of petroleum oils or synthetic fluids to separate from water. This test provides a guide for determining the water separation characteristics of oils subject to water contamination and turbulence. It is used for specification of new oils and monitoring of in-service oils. 2.3.1.6 Fourier transform infrared spectroscopy (PE-5008-AI) The IR Spectroscopy is a well known technique for analysing the chemical properties of the oil such as oxidation products or additives. 2.3.1.7 Remaining useful life. RULER. (PE-TA.90) 2.3.3.1 Ready biodegradability (OECD 301F) If a chemical gives positive in this test will undergo rapid an ultimate biodegradation (CO2+H2O) in the environment and no further work on the biodegradability on the chemical, or on the possible environmental effects of biodegradation products, normally is required. Ultimate biodegradation within 28 days higher than 60% according to OECD 301 F. 2.3.3.2 Toxicity algae, daphnia (OECD 201, OECD 202). The level al which 50% of the test organisms show an adverse (lethal) effect. Exponentially-growing cultures of selected green algae or certain percentage of daphnia are exposed to various concentrations of the test substance under defined conditions. The inhibition of growth in relation to a control culture or the inhibition of the capability of swimming of daphnia is determined over a fixed period. g The 50% effect level (EC50) is chosen, the level at which 50% of the test organisms show an adverse (lethal) effect. 2.3.2.1 Sliding tests (DIN 51834-2) With the SRV tribometer reciprocating sliding tests in standard conditions using AISI 52100 steel standard balls and discs can be useful for finding any difference in the behavior of new and aged oils based on the results of friction (COF) and wear obtained during the tribological tests. 2.3.1.7 Remaining useful life. RULER. (PE-TA.90) The RULER (Remaining Useful Life) is a useful parameter to control the antioxidants consumption in biodegradable oils. The voltammetric test results are based on current, voltage and time relationships at the 3- electrode sensing system with a small, easily polarized microelectrode, and a large non- polarizable reference electrode. In performing a voltammetric analysis, the potential across the electrodes varies linearly with time (from 0 to 1.7 V at a rate of 0.1 V/second), and the resulting current is recorded as a function of the potential. With increased voltage to the sample in the cell, the various dissolved antioxidants oxidize electrochemically resulting in an oxidation reaction that can be used to predict the remaining useful life of the oil. 381 Lengthening Biolubricants´ Lifetime by Using Porous Materials 2.4 Identification of main condition monitoring patterns Regarding traditional lubricating oils, all condition monitoring parameters, limits and sample frequencies have been already established at different studies. However, there is not a clear rule of thumb, as small variations occurs in limits and sampling frequencies. Given this, the knowledge has been obtained through extensive usage occurred at WearCheck Ibérica Laboratories, which has helped to obtain enough expertise to study all condition monitoring fields. Regarding biodegradable lubricating oil parameters that have to be measured, an extensive tribological and physico-chemical comparison has been performed between normal and bio- degradable lubricants, in order to assess their conditions. The tests have demonstrated a superior working life-time for bio-degradable lubricants with respect to traditional ones that is mostly reflected in a much higher AN limit allowed for operation. As a result, similar parameters have been defined as of primary control. However, there are two important additions. The Ruler is a parameter for on site measurement of remaining useful lifetime of the oil. The analysis performed show that rules offer a quite reliable information on usage of the oil and can complement the information indicated by AN. g p y Also, the % of Solids parameter is a very useful parameter. However, it is very hard and expensive to measure and in the near future work the % of Solids parameter have to be eliminated to the monitoring routine and must be found a new parameter cheaper and easier to use it in the monitoring routine. g Of course, these are main parameters and limits. Depending on the type of lubricant and its application and the test cost, other parameters could be useful for mineral oils monitoring, 382 Environmental Management in Practice and biodegradable oils. For engine oils for example, it could be necessary to analyse Base Number (BN) parameter. The work should be completed with a complete identification of sample frequencies. Fig. 2. Parameters, monitor and warning limits, sample frequencies and analytical equipment for mineral oils. Fig. 2. Parameters, monitor and warning limits, sample frequencies and analytical equipment for mineral oils. Fig. 3. Limits and parameters for biodegradable oils. Fig. 3. Limits and parameters for biodegradable oils. Fig. 3. Limits and parameters for biodegradable oils. 3. New materials for enlarge biolubricants´lifetime One of the main concerns of lubricants is their performance which is improved using additives. The use of additives allows increasing the performance and physical properties of oil but they also increase the cost of lubricants and may even be harmful to health or environment. 383 Lengthening Biolubricants´ Lifetime by Using Porous Materials Adsorption in a porous material of oxidation products from a biodegradable lubricant is a promising approach to improve the performance of biolubricants in an environmentally friendly way. Antioxidant additives are commonly used to improve performance of biolubricants but they are expensive and even may be harmful. The development of a sieve able to trap oxidation products may be a way to reduce or avoid the use of additives. In our investigation, different oxidized samples of biolubricants obtained from the degradation process of TMP-trioleate have been characterized and the oxidation molecules to be trapped have been identified. The most suitable nanoporous material to trap the identified oxidation molecules has been selected. To do this the adsorption of biolubricant oxidation molecules in a nanoporous material has been examined by means of Monte Carlo (MC) and Molecular Dynamics (MD) computational methods and by means of Differential Scanning Calorimetry . Among the different framework types BEA, MFI, LTL and FAU zeolitic structures were selected due to their suitable pore size of molecular dimensions. All of them present an extensive channel network with elliptical or circular shape and cross section ranging from 0.5 and 0.8 nm. Besides, structural criteria, different compositions have been selected in order to analyze the effect of the physico-chemical properties of the solid surfaces (functional groups, acidity, hydrophilicity,…). It deserves to note that from the point of view of the composition, extremely hydrophobic materials with high silica content such as Silicalite-1, or highly hydrophilic materials with relatively low silica content such as zeolite x, have been considered. Prior to their use all the materials were dried and activated trough a thermal treatment using an exposure times of 2 h and temperatures of 150 or 300 ºC. Since crystal structure and grain size and morfology influences on total porosity of the material surface area of all the samples were measured after activation with a NOVA 1200e surface area and pore size analyzer from Quantachrome Instruments. Total surface area was computed according to BET Method. 3.3 Porous materials validation The hydrophilic and hydrophobic solids have showed the best performance trapping the oxidation molecules of TMP-trioleate. After testing the capacity of different nanoporous materials, the most suitable has been tested with the TMP-trioleate at 95ºC with stirring, air flux and without presence of water and catalyst. The chemical parameter which shows the effectiveness of the tested solid is Acid Number (AN). The following figure shows the trend of this parameter during the oxidation process. As it shows, both solids hydrophilic and hydrophobic one, delay the oxidation process of the oil due, these solids trap in their pores the acid compounds generated during the oxidation. Fig. 4. AN values trend in TMP-Trioleate oxidized with and without solid. Fig. 4. AN values trend in TMP-Trioleate oxidized with and without solid. 3.1 Oxidation conditions In order to analyze the capacity of the selective adsorption of oxidation by-products with nanoporous materials and predict the lubricating oil oxidation state, the Differential Scanning Calorimetry (DSC) analytical technique has been used. The experimental procedure consists on an analyzed sample heating it with a programmed temperature-time sequence: 3ºC/min heating from 100ºC at 600ºC at 20 bar of pressure. g p The oxidation method was described previously. The oxidation conditions were the following: 1.5 l of TMP-trioleate in a bath reactor at 95ºC with stirring, air flux and without presence of water and catalyst. The analytical parameters monitorized were the following: Acid Number (ASTM D 974-04), DSC (PE-5035-AI), Fourier Transform Infrarred Spectroscopy (FTIR) (PE-5008-AI) and Density (PE-5053-AI). Besides that, the oxidation molecules identification at different hours of oxidation has been made by GCMS and HPLC. The analytical parameters monitorized were the following: Acid Number (ASTM D 974-04), DSC (PE-5035-AI), Fourier Transform Infrarred Spectroscopy (FTIR) (PE-5008-AI) and Density (PE-5053-AI). Besides that, the oxidation molecules identification at different hours of oxidation has been made by GCMS and HPLC. After testing the capacity of different nanoporous materials, the most suitable has been tested with the TMP-trioleate at 95ºC with stirring, air flux and without presence of water and catalyst. 384 Environmental Management in Practice 3.2 MD simulations Molecular Dynamics (MD) has been used to study the interaction between the identified oxidation molecules and the selected nanoporous material. Results of the ability of the proposed material as absorbing media in terms of molecules per unit computational cell and preferred absorption sites are obtained. The simulations were performed at established conditions of pressure a temperature using the grand canonical and the NPT ensemble. Results of the ability of the proposed material as absorbing media in terms of molecules per unit computational cell and preferred adsorption sites are obtained. 4. Conclusions The second research work exposed is the use of nanoporous materials as tramp for oxidation compounds instead to use antioxidant additives in the bio oil formulation Antioxidant additives are commonly used to improve performance of biolubricants but they The second research work exposed is the use of nanoporous materials as tramp for oxidation compounds instead to use antioxidant additives in the bio oil formulation Antioxidant additives are commonly used to improve performance of biolubricants but they are expensive and even may be harmful. The development of a sieve able to trap oxidation oxidation compounds instead to use antioxidant additives in the bio oil formulation Antioxidant additives are commonly used to improve performance of biolubricants but they are expensive and even may be harmful. The development of a sieve able to trap oxidation products may be a way to reduce or avoid the use of additives. Adsorption in a porous material of oxidation products from a biodegradable lubricant is a promising approach to improve the performance of biolubricants in an environmentally friendly way. p p y y y  The use of biodegradable lubricants will reduce problems on disposal. The biodegradability in use must be tested in these types of friendly formulations.  The use of biodegradable lubricants will reduce problems on disposal. The biodegradability in use must be tested in these types of friendly formulations. g y yp y  The uses of hydrophilic solids delay oil oxidation, due the trap oxidation mole The uses of hydrophilic solids delay oil oxidation, due the trap oxidation molecules. y p y p  Acid Number (AN) seems to be a useful analytical technique for evaluate solid efficiency y p y p  Acid Number (AN) seems to be a useful analytical technique for evaluate solid efficiency y p y p  Acid Number (AN) seems to be a useful analytical technique for evaluate solid efficiency 4. Conclusions In this chapter it has been exposed two main research works; the first one is a proposal for the condition monitoring strategy for biolubricants. In this sense two oils, mineral and biodegrable; have been oxidized under a new oxidation procedure, based on Tekniker 385 Lengthening Biolubricants´ Lifetime by Using Porous Materials experience, which provide more advantage than traditional tests. Thanks to the chemical, tribological and environmental analyses monitor and warning limits can be proposed for bio-oil. experience, which provide more advantage than traditional tests. Thanks to the chemical, tribological and environmental analyses monitor and warning limits can be proposed for bio-oil. As it can be sawn these limits are different as traditional limits for mineral oils, what is mean that biodegradable oils shows different oxidations trends and traditional limits used As it can be sawn these limits are different as traditional limits for mineral oils, what is mean that biodegradable oils shows different oxidations trends and traditional limits used for mineral oils are nor accurate for these kind of biolubricants: for mineral oils are nor accurate for these kind of biolubricants:  Kinetic degradation reaction of biodegradable lubricants is differently than mineral oils and a specific maintenance approach is needed.  Kinetic degradation reaction of biodegradable lubricants is differently than mineral oils and a specific maintenance approach is needed.  DSC is a useful tool for studying the kinetic parameters of the new formulations.  Important research must be carried out to establish warning limits for biolubricants in order to develop condition monitoring strategies, assessment in mechanical components lubricated with biodegradable fluids.  Important research must be carried out to establish warning limits for biolubricants in order to develop condition monitoring strategies, assessment in mechanical components lubricated with biodegradable fluids.  In the standard tribological wear tests there is a direct relationship between aging hours and friction peaks. This test can be useful in the condition monitoring strategy.  In the standard tribological wear tests there is a direct relationship between aging hours and friction peaks. This test can be useful in the condition monitoring strategy. 5. References “Product Reviews: Liquid waste disposal and Recovery - Lubricant Recycling », Ind. Lub. Trib., 1994, 46, (4), 18-26. “Product Reviews: Liquid waste disposal and Recovery - Lubricant Recycling », Ind. Lub. Trib., 1994, 46, (4), 18-26. “The Need For Biodegradable Lubricants”, Ind. Lubr. and Trib., 1992, 44, (4), 6-7. ological Criteria for the award of the Community ecolabel to lubricants”- “Ecological Criteria for the award of the Community ecolabel to lubricants”- Regulatory committee of the European Parliament and of the Council- 2005 Regulation of the European Parliament and of the council concerning the Registration, Evaluation, Authorisation and Restrictions of Chemicals. Regulation of the European Parliament and of the council concerning the Registration, E l ti A th i ti d R t i ti f Ch i l Regulation of the European Parliament and of the council conce Evaluation, Authorisation and Restrictions of Chemicals. Carnes K. “University Tests Biodegradable Soy-Based Railroad Lubricant”, Hart’s Lubricantes world 1998, Vol. September, pp 45-47. Glancey J.L., Knowlton S., Benson E.R. “Development of a High-Oleic Soybean Oil-based Hydraulic Fluid”, Lubricants World 1999, Vol. January, pp 49-51. Rajewski T.E., Fokens J.S., Watson M.C., “The development and Application of Syntetic Food Grade Lubricants”, Tribology, 2000, Vol 1, pp 83-89. gy pp W. J. Bartz: “Comparison of Synthetic Fluids”, Lub. Eng., 1992, 48, (10), 765-774. S.Z.Erhan: “Lubricant basestocks from vegetable oils”, Industrial Crops and Products 11 (2000) 277–282 386 Environmental Management in Practice C-X. Xiong: “The structure and Activity of Polyalphaolefins as Pour-Point Depressants”, Lub. Eng., 1993, 49, (3), 196-200. G Kumar: “New Polyalphaolefin Fluids for specialty applications”, Lub. Eng., 1993, 49, (x), 723-725. R. L. Shubkin: “Polyalphaolefins: Meeting the Challenge for High-Performance Lubrication”, Lub. Eng., 1994, 50, (x), 196-201. g ( ) J. F. Carpenter: “Biodegradability of Polyalphaolefin (PAO) Basestocks”, Lub. Eng., 1994, 50, (5), 359-362. M.K. Williamson “The emerging Role of Oil analysis in Enterprise-Wide decision making”. Practicig Oil analysis 2000. pp. 187-200. g y pp Lubricants and lubrication”. T. Mang, W. Dresel (Eds). Wiley-VCH. 2001 “Lubricating grease guide”. Fourth Edition. National Lubricating Grease Institute (NLGI A. Adhvaryu, “Oxidation kinetics studies of oils derived from unmodified and genetically modified vegetables using pressurized differential scanning calorimetry and nuclear magnetic resonance spectroscopy”. Thermochimica Acta, 364, 87-97. 2000 N.J. Fox, A.K. Simpson, G.W. Stachowiak, ”Sealed Capsule Differential Scanning Calorimetry-An Effective Method for Screening the oxidation Stability of vegetable oil formulations”. Lubrication Engineering, 57, 14-20. 2001 A. A Fuzzy Water Quality Index for Watershed Quality Analysis and Management André Lermontov1,2, Lidia Yokoyama1, Mihail Lermontov3 and Maria Augusta Soares Machado4 1Universidade Federal do Rio de Janeiro 2Grupo Águas do Brasil S/A 3Universidade Federal Fluminense 4IBMEC-RJ Brazil A Fuzzy Water Quality Index for Watershed Quality Analysis and Management André Lermontov1,2, Lidia Yokoyama1, Mihail Lermontov3 and Maria Augusta Soares Machado4 1Universidade Federal do Rio de Janeiro 2Grupo Águas do Brasil S/A 3Universidade Federal Fluminense 4IBMEC-RJ Brazil Brazil 5. References Adhvaryu, “Tribological studies of thermally and Chemically modified vegetable oils use as environmentally friendly lubricants”. Wear, 257, 359-367, 2004 y y F.Novotny-Farkas, P. Kotal, W. Bohme. “Condition monitoring of biodegradable lubricants”. World Tribology Congress. Vienna. 2001 Arnaiz, A., Aranzabe, A., Terradillos, J., Merino, S., Aramburu, I.: New micro-sensor systems to monitor on-line oil degradation, Comadem 2004. pp. 466-475 Kristiansen, P., Leeker, R.: U.S.Navy’s in-line oil analysis program, , lubr. Fluid powerj. 3, 3– 12, aug 2001. C.Duncan (2002), Lubrication Engineering, “Ashless Additives and New Polyol Ester Base Oils Formulated for Use in Biodegradable Hydraulic Fluid Applications” 20 2.1 Water quality indices q y The purpose of an index is not to describe separately a pollutant's concentration or the changes in a certain parameter. To synthesize a complex reality in a single number is the biggest challenge in the development of a water quality index (IQA – Índice de Qualidade de Água, originally in Portuguese), since it is directly affected by a large number of environmental variables. Therefore, a clear definition of the goals to be attained by the use of such an index is needed. The formulation of a IQA may be simplified if one considers only the variables which are deemed critical for a certain water body. Among their advantages, indices facilitate communication with lay people. They are considered more trustful than isolated variables. They also integrate several variables in a single number, combining different units of measurement. g In a groundbreaking work, Horton (1965) developed general water quality indices, selecting and weighting several parameters. This methodology was then improved by the U.S. National Sanitation Foundation (NSF, 2007). The conventional way to obtain a IQA is to compute the weighted average of some predefined parameters, normalized in a scale from 0 to 100 and multiplied by their respective weights. y g Conesa (1995) modified the traditional method and created another index, called Subjective Water Quality Index (IQAsub), that includes a subjective constant, k. This constant assumes values between 0.25 and 1.00 at intervals of 0.25, with 0.25 representing polluted water and 1.00 a not polluted one. The parameters used to calculate this index (eq. 1) must be previously normalized using curves given by Conesa (1995). The Objective Water Quality Index (IQAobj) results from the elimination of the subjective constant k. IQAsub = x i i i i i C P k P   (1) (1) k is the subjective constant (0,25, 0,50, 0,75 and 1,00); j ( ) Ci the value of the ith normalized parameter (Conesa, 1995); j ( ) Ci the value of the ith normalized parameter (Conesa, 1995); p ( ) the relative weight of the ith parameter (Conesa, 1995). The Brazilian IQA is an adaptation from the NSF index. Nine variables, being the most relevant for water quality evaluation, are computed as the weighted product (eq. 1. Introduction The main purpose of this research is to propose a new water quality index, called Fuzzy Water Quality Index (INQA – Índice Nebuloso de Qualidade da Água, originally in Portuguese), to be computed using Fuzzy Logic and Fuzzy Inference tools. A second goal is to compare statistically the INQA with other indices suggested in the literature using data from hydrographic surveys of four different watersheds, in São Paulo State, Brazil, from 2004 to 2006 (CETESB, 2004, 2005, 2006). dealing with missing data. Fuzzy Logic can combine these different approaches. In this context new methodologies for the management of environmental variables are being developed (Silvert, 1997, 2000). p ( ) The main purpose of this research is to propose a new water quality index, called Fuzzy Water Quality Index (INQA – Índice Nebuloso de Qualidade da Água, originally in Portuguese), to be computed using Fuzzy Logic and Fuzzy Inference tools. A second goal is to compare statistically the INQA with other indices suggested in the literature using data from hydrographic surveys of four different watersheds, in São Paulo State, Brazil, from 2004 to 2006 (CETESB, 2004, 2005, 2006). 1. Introduction Climate change and hydric stress are limiting the availability of clean water. Overexploitation of natural resources has led to environmental unbalance. Present decisions relative to the management of hydric resources will deeply affect the economy and our future environment. The use of indicators is a good alternative for the evaluation of environmental behavior as well as a management instrument, as long as the conceptual and structural parameters of the indicators are respected. The use of fuzzy logic to study the influence and the consequences of environmental problems has increased significantly in recent years. According to Silvert (1997), most activities, either natural of anthropic, have multiple effects and any environmental index should offer a consistent meaning as well as a coherent quantitative and qualitative appraisal of all these effects. Among the several reasons for applying fuzzy logic to complex situations, the most important is probably the need to combine different indicators. Maybe the most significant advantage of the use of fuzzy logic for the development of environmental indicators is that it combines different aspects with much more flexibility than other methods, such as, for example, binary indices of the kind “acceptable vs. unacceptable.” y Methods to integrate several variables related to water quality in a specific index are increasingly needed in national and international scenarios. Several authors have integrated water quality variables into indices, technically called Water Quality Indices (WQIs) (Bolton et al., 1978; Bhargava, 1983; House, 1989; Mitchell, 1996; Pesce and Wunderlin, 1999; Cude, 2001; Liou et al., 2004; Said et al., 2004; Silva and Jardim, 2006; Nasiri et al., 2007). Most are based in a concept developed by the U. S. National Sanitation Foundation (NSF, 2007). There is an obvious need for more advanced techniques to assess the importance of water quality variables and to integrate the distinct parameters involved. In this context, new, alternative integration methods are being developed. Artificial Intelligence has thus become a tool for modeling water quality (Chau, 2006). Traditional methodologies cannot classify and quantify environmental effects of a subjective nature or even provide formalism for 388 Environmental Management in Practice dealing with missing data. Fuzzy Logic can combine these different approaches. In this context new methodologies for the management of environmental variables are being developed (Silvert, 1997, 2000). 2.1 Water quality indices 2) of the normalized values of these variables, ni: Temperature (TEMP), pH, Dissolved Oxygen (DO), Biochemical Oxygen Demand (BOD5), Thermotolerant Coliforms (TC), Dissolved Inorganic Nitrogen (DIN), Total Phosphorus (TP), Total Solids (TS) and Turbidity (T). Each parameter 389 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen is weighted by a value wi between 0 and 1 and the sum of all weights is 1. The result is expressed by a number between 0 and 100, divided in 5 quality ranges: (100 - 79) - Excellent Quality; (79 - 51) - Good Quality; (51 - 36) - Fair Quality; (36 - 19) - Poor Quality; [19 - 0] - Bad Quality, normalization curves for each variable, as well as the respective weights, are available in the São Paulo’s State Water Quality Reports (CETESB, 2004, 2005 and 2006). IQACETESB = 1 IQA q i n i i w   (2) (2) Silva and Jardim (2006) used the concept of minimum operator to develop their index, called Water Quality Index for protection of aquatic life (IQAPAL). The IQAPAL (eq. 3) is based on only two parameters, Total Ammonia (TA) and Dissolved Oxygen (DO): IQAPAL = min (TAn, DOn) (3) IQAPAL = min (TAn, DOn) (3) A fourth index, called IQAmin, proposed by Pesce and Wunderlin (2000), is the arithmetic mean (eq. 4) of three environmental parameters, Dissolved Oxygen (DO), Turbidity (T) and Total Phosphorus (TP), normalized using Conesa's curves (Conesa, 1995). IQAmin = DO+T+TP 3 (4) (4) Other indices are found in the literature and will not be considered in this study (Bordalo et al., 2001; SDD, 1976; Stambuk Giljanovic, 1999). Other indices are found in the literature and will not be considered in this study (Bordalo et al., 2001; SDD, 1976; Stambuk Giljanovic, 1999). 2.3 Development of the fuzzy water quality index (INQA) The fuzzy sets were defined in terms of a membership function that maps a domain of interest to the interval [0,1]. Curves are used to map the membership function of each set. They show to which degree a specific value belongs to the corresponding set (eq. 5): µA : X  [0,1] (5) µA : X  [0,1] (5) Trapezoidal and triangular membership functions (Figure 1) are used in this study, for the same nine parameters used by CETESB to calculate its IQA, so that this methodology can be statistically compared and validated. The data shown in Tables 1 and 2 are used according to Figure 1 to create the fuzzy sets: Fig. 1. Trapezoidal and triangular membership function. In a rule based fuzzy system, a linguistic description is attributed to each set. The sets are then named according to a perceived degree of quality, that ranges from very excellent to very bad (Tables 1 and 2). For the parameters temperature and pH, two sets for each linguistic variable are used. Temperature and pH sets have the same linguistic terms above and under the Very Excellent point while distancing from it. The sets under are marked with a (▼) symbol. The trapezoidal function is only used for the Very Excellent linguistic variable and the triangular for all others. This study uses the linguistic model of fuzzy inference, where the input data set, the water quality variables, called antecedents, are processed using linguistic if/then rules to yield an output data set, the so-called consequents. Fig. 1. Trapezoidal and triangular membership function. Fig. 1. Trapezoidal and triangular membership function. In a rule based fuzzy system, a linguistic description is attributed to each set. The sets are then named according to a perceived degree of quality, that ranges from very excellent to very bad (Tables 1 and 2). For the parameters temperature and pH, two sets for each linguistic variable are used. Temperature and pH sets have the same linguistic terms above and under the Very Excellent point while distancing from it. The sets under are marked with a (▼) symbol. The trapezoidal function is only used for the Very Excellent linguistic variable and the triangular for all others. 2.2 Fuzzy inference f h h f l allowing to avoid artificial precision and producing results that are more similar to the ecological complexity and real world problems in a more realistic panorama; and sixth, fuzzy logic can deal with and process missing data without compromising the final result. The way systems based on fuzzy rules have been successfully used to model dynamic y g p g p g The way systems based on fuzzy rules have been successfully used to model dynamic systems in other fields of science and engineering suggests that this approach may become an effective and efficient way to build a meaningful IQA. Fuzzy inference is the process that maps an input set into an output set using fuzzy logic. This mapping may be used for decision making or for pattern recognition. The fuzzy inference process involves four main steps: 1) fuzzy sets and membership functions; 2) fuzzy set operations; 3) fuzzy logic; and 4) inference rules. These concepts are discussed in depth in Bárdossy (1995), Yen e Langari (1999), Ross (2004), Cruz (2004) and Caldeira et al. (2007). ( ) The concept of fuzzy sets for modeling water quality was considered by Dahiya (2007), Nasiri et al. (2007) Chau (2006), Ocampo-Duque et al. (2006), Icaga (2007), and Chang et al. (2001), Lermontov et al. (2009), Ramesh et al. (2010), Taner et al. (2011). 2.2 Fuzzy inference One of the research fields involving Artificial Intelligence - AI is fuzzy logic, originally conceived as a way to represent intrinsically vague or linguistic knowledge. It is based on the mathematics of fuzzy sets (Zadeh, 1965). Fuzzy inference is the result of the combination of fuzzy logic with expert systems (Yager, 1994). The commonest models used to represent the process of classification of water bodies are called deterministic conceptual models. They are deterministic because they ignore the stochastic properties of the process and conceptual because they try to give a physical interpretation to the several subprocesses involved. These models often use a large number of parameters, making modeling a complex and time demanding task (Barreto, 2001). Models based on fuzzy rules are seen as adequate tools to represent uncertainties and inaccuracies in knowledge and data. These models can represent qualitative aspects of knowledge and human inference processes without a precise quantitative analysis. They are, therefore, less accurate than conventional numerical models. However, the gains in simplicity, computational speed and flexibility that result from the use of these models may compensate an eventual loss in precision (Bárdossy, 1995). There are at least six reasons why models based on fuzzy rules may be justified: first, they can be used to describe a large variety of nonlinear relations; second, they tend to be simple, since they are based on a set of local simple models; third, they can be interpreted verbally and this makes them analogous to AI models; fourth, they use information that other methods cannot include, such as individual knowledge and experience; fifth, the fuzzy approach has a big advantage over other indices, once they have the ability expand and combine quantitative and qualitative data that expresses the ecological status of a river, 390 Environmental Management in Practice allowing to avoid artificial precision and producing results that are more similar to the ecological complexity and real world problems in a more realistic panorama; and sixth, fuzzy logic can deal with and process missing data without compromising the final result. The way systems based on fuzzy rules have been successfully used to model dynamic systems in other fields of science and engineering suggests that this approach may become an effective and efficient way to build a meaningful IQA. 2.3 Development of the fuzzy water quality index (INQA) This study uses the linguistic model of fuzzy inference, where the input data set, the water quality variables, called antecedents, are processed using linguistic if/then rules to yield an output data set, the so-called consequents. In a rule based fuzzy system, a linguistic description is attributed to each set. The sets are then named according to a perceived degree of quality, that ranges from very excellent to very bad (Tables 1 and 2). For the parameters temperature and pH, two sets for each linguistic variable are used. Temperature and pH sets have the same linguistic terms above and under the Very Excellent point while distancing from it. The sets under are marked with a (▼) symbol. The trapezoidal function is only used for the Very Excellent linguistic variable and the triangular for all others. This study uses the linguistic model of fuzzy inference, where the input data set, the water quality variables, called antecedents, are processed using linguistic if/then rules to yield an output data set, the so-called consequents. 2.3 Development of the fuzzy water quality index (INQA) A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen 391 Gr01 Gr02 Gr03 Parameter Temperature pH Disolved Biochemical Thermotolerant Oxigen Oxigen Demand Coliforms Symbol Temp pH DO BOD Coli Unit oC mg/l mg/l Colonies/100ml Interval -6 - 45 1 - 14 0 - 9 0 - 30 0 - 18000 Linguistic Variable a b c d a b c d a b c d a b c d a b c d Very Excellent - VE 15 16 21 22 6.80 6.90 7.10 7.75 7.0 7.5 9.0 9.0 0 0 0.5 2 0 0 1 1 Excellent - E 14 15 16 7.10 7.75 8.25 6.5 7 7.5 0.5 2 3 1 2 3 Excellent - E▼ 21 22 24 6.60 6.80 6.90 Very Good - VG 13 14 15 7.75 8.25 8.50 6 6.5 7 2 3 4 2 3 8 Very Good - VG▼ 22 24 26 6.30 6.60 6.80 Good - G 10 13 14 8.25 8.50 8.75 5 6 6.5 3 4 5 3 8 16 Good - G▼ 24 26 28 6.10 6.30 6.60 Fair/Good - FG 5 10 13 8.50 8.75 9.00 4 5 6 4 5 6 8 16 40 Fair/Good - FG▼ 26 28 30 5.85 6.10 6.30 Fair - F 0 5 10 8.75 9.00 9.20 3.5 4 5 5 6 8 16 40 100 Fair - F▼ 28 30 32 5.60 5.85 6.10 Fair/Bad - FB -2 0 5 9.00 9.20 9.60 3 3.5 4 6 8 12 40 100 300 Fair/Bad - FB▼ 30 32 36 5.20 5.60 5.85 Bad - B -4 -2 0 9.20 9.60 10.00 2 3 3.5 8 12 15 100 300 1000 Bad - B▼ 32 36 40 4.75 5.20 5.60 Very Bad - VB -6 -4 -2 9.60 10.00 10.50 1 2 3 12 15 22 300 1000 6000 Very Bad - VB▼ 36 40 45 4.00 4.75 5.20 Poor - P -6 -6 -4 10.00 10.50 12.00 0 1 2 15 22 30 1000 6000 18000 Poor - P▼ 40 45 45 2.00 4.00 4.75 Very Poor - P -6 -6 -6 10.50 14.00 14.00 0 0 1 22 30 30 6000 18000 18000 Very Poor - P▼ 45 45 45 1.00 1.00 4.00 Table 1. 2.3 Development of the fuzzy water quality index (INQA) Fuzzy sets and linguistic terms for input parameters of Group 01, 02 and 03 Table 1. Fuzzy sets and linguistic terms for input parameters of Group 01, 02 and 03 Table 1. Fuzzy sets and linguistic terms for input parameters of Group 01, 02 and 03 Gr04 Gr05 Group Output Parameter Dissolved Total Total Solids Turbidity Output Inorg. Nitrogen Phosphorus Symbol DIN TP TS Turb Unit mg/l mg/l mg/l mg/l Interval 0 - 100 0 - 10 0 - 750 0 - 150 0 - 100 Linguistic Variable a b c d a b c d a b c d a b c d a b c d Very Excellent - VE 0 0 0.5 2 0 0 0.1 0.2 0 0 5 50 0 0 0.5 2.5 0 0 1 10 Excellent - E 0 2 4 0.1 0.2 0.3 0 50 150 0.5 2.5 7.5 0 10 20 Very Good - VG 2 4 6 0.2 0.3 0.4 50 150 250 2.5 7.5 12.5 10 20 30 Good - G 4 6 8 0.3 0.4 0.6 150 250 320 7.5 12.5 22.5 20 30 40 Fair/Good - FG 6 8 10 0.4 0.6 0.8 250 320 400 12.5 22.5 35 30 40 50 Fair - F 8 10 15 0.6 0.8 1 320 400 450 22.5 35 50 40 50 60 Fair/Bad - FB 10 15 25 0.8 1 1.5 400 450 550 35 50 70 50 60 70 Bad - B 15 25 35 1 1.5 3 450 550 600 50 70 95 60 70 80 Very Bad - VB 25 35 50 1.5 3 6 550 600 650 70 95 120 70 80 90 Poor - P 35 50 100 3 6 10 600 650 750 95 120 150 80 90 100 Very Poor - P 50 100 100 6 10 10 650 750 750 120 150 150 90 100 100 Table 2. Fuzzy sets and linguistic terms for input parameters of Group 04 and 05 and output parameters of all groups Table 2. Fuzzy sets and linguistic terms for input parameters of Group 04 and 05 and output parameters of all groups 392 Environmental Management in Practice Figure 2 shows the flow graph of the process, where the individual quality variables are processed by inference systems, yielding several groups normalized between 0 and 100. 2.3 Development of the fuzzy water quality index (INQA) The groups are then processed for a second time, using a new inference, and the end result is the Fuzzy Water Quality Index – INQA/FWQI. y y In the traditional methods used to obtain a IQA, parameters are normalized with the help of tables or curves and weight factors (Conesa, 1995; Mitchel, 1996; Pesce and Wunderlin, 1999; CETESB, 2004, 2005 and 2006; NSF, 2007) and then calculated by conventional mathematical methods, while in this work, parameters are normalized and grouped through a fuzzy inference system. Fig. 2. Flow Graph The NFS formulated the IQA as being a quantitative aggregation of various chosen and weighted water quality parameters to represent the best professional judgment of 142 expert respondants into one index (Mitchell, 1996). Working quantitatively with a mathematical equation, one uses a weight factor to differentiate the importance (weight - inferred and defined by experts) of each parameter for the outcoming result. NFS Brazilian CETESB Ocampo Duque et al (2006) Conessa (1997) and other authors who Fig. 2. Flow Graph Fig. 2. Flow Graph The NFS formulated the IQA as being a quantitative aggregation of various chosen and weighted water quality parameters to represent the best professional judgment of 142 expert respondants into one index (Mitchell, 1996). Working quantitatively with a mathematical equation, one uses a weight factor to differentiate the importance (weight - inferred and defined by experts) of each parameter for the outcoming result. The NFS formulated the IQA as being a quantitative aggregation of various chosen and weighted water quality parameters to represent the best professional judgment of 142 expert respondants into one index (Mitchell, 1996). Working quantitatively with a mathematical equation, one uses a weight factor to differentiate the importance (weight - inferred and defined by experts) of each parameter for the outcoming result. The NFS formulated the IQA as being a quantitative aggregation of various chosen and weighted water quality parameters to represent the best professional judgment of 142 expert respondants into one index (Mitchell, 1996). Working quantitatively with a mathematical equation, one uses a weight factor to differentiate the importance (weight - inferred and defined by experts) of each parameter for the outcoming result. y p ) p g NFS, Brazilian CETESB, Ocampo-Duque et al. (2006), Conessa (1997) and other authors who proposed IQA’s, used different weighting factors depending on the methodology and presence or absence of a specific monitoring parameter. 2.3 Development of the fuzzy water quality index (INQA) The rules for normalization and aggregation followed the logic described below and the consequent always obeyed the prescription of the minimum operator: If FP is VE and SP is VE then GR output is VE If FP is VE and SP is E then GR output is E If FP is E and SP is VE then GR output if E If FP is VE and SP is VP then GR output is VP If FP is VP and SP is VE then GR output is VP If FP is VE and SP is VE then GR output is VE If FP is VE and SP is E then GR output is E If FP is E and SP is VE then GR output if E If FP is VE and SP is VP then GR output is VP If FP is VP and SP is VE then GR output is VP If FP is VE and SP is VE then GR output is VE If FP is VE and SP is E then GR output is E If FP is E and SP is VE then GR output if E If FP is VE and SP is VP then GR output is VP If FP is VP and SP is VE then GR output is VP If FP is VE and SP is VE then GR output is VE If FP is VE and SP is E then GR output is E If FP is E and SP is VE then GR output if E If FP is VE and SP is VE then GR output is VE If FP is E and SP is VE then GR output if E If FP is VE and SP is VP then GR output is VP If FP is VP and SP is VE then GR output is VP where: FP - First Parameter / SP - Second Parameter / GR - Group / / p The INQA was developed from a fuzzy inference that had Groups 01 to 05 as input sets and a series or rules. The antecedent sets (Groups) and the consequent set (INQA) were created by trapezoid (Excellent and Poor sets) and triangular pertinence (all others) functions (Table 3, Figure 3); the INQA classes were the same as for the CETESB's IQA quality standards (Table 3). 2.3 Development of the fuzzy water quality index (INQA) Silva and Jardim (2006) and Pesce and Wunderlin (2000) did even not use weighting factors while developing respectively their IQAPAL and IQAmin. In a fuzzy inference system a quantitative numerical value is fuzzyfied into a qualitative state and processed by an inference engine, through rules, sets and operators in a qualitative sphere, allowing the use of information that other methods cannot include, such as individual knowledge and experience (Balas et al., 2004), permitting qualitative environmental parameters and factors to be integrated and processed (Silvert, 2000) producing similar to the real world results. A rule in the inference system is a mathematical formalism that translates expert judgment expressed in linguistic terms (as in NFS’s IQA formulation) and therefore is a subjective and qualitative weight factor in the inference engine. I.e.: Rule 1: if Thermotolerant Coliform is very high and pH is lower than average than index is very poor; Rule 2: if Thermotolerant Coliform is very high and pH is excellent than index is poor. One can notice that these rules have been designed as an expert system and a subjective and qualitative weight factor based on an 393 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen expert judgment has been introduced in the process scoop. In spite of the strong pH variation, the final score is not strongly affected. expert judgment has been introduced in the process scoop. In spite of the strong pH variation, the final score is not strongly affected. g y The physical parameters pH and Temp are normalized and aggregated into the first group (Gr01). DO and BOD comprise Gr02. Thermotolerant coliforms (Coli) were independently normalized as Gr03. The nutrients DIN and TP make up Gr04; TS and Turb are grouped in Gr05. The water analyses results used in this research were taken from the CETESB reports for the years of 2004, 2005 and 2006 (CETESB, 2004, 2005 and 2006). Curves to help in the creation and normalization of the fuzzy sets were taken these reports for the parameters pH, BOD, Coli, DIN, TP, TS and Turb and from Conesa (1995) for Temp and DO. y 2.4.1 Ribeira do Iguape river – environmental conservation area g p The watershed of Ribeira River and the Lagoone-Estuary Complex of Iguape, Cananéia and Paranaguá, called Ribeira Valley, comprises 32 counties and covers and area of 28,306 km2, with 9 cities and 12,238 km2 in Paraná State and 23 cities and 16,068 km2 in São Paulo State, Brasil. The economy of Ribeira Vally is based in livestock raising (200,421 hectares), fruticulture (49,942 hectares), silviculture (46,368 hectares), temporary cultures (15,965 hectares) and horticulture (2,773 hectares). Sand and turf extraction from low-lying areas are also significant. About 1% of the state population (396,684 people) live in this river basin, 68% of them in cities. About 56% of the effluents are collected and 49% are treated. It is estimated that approximately 8.8 tons of BOD5 (remaining pollutant charge) are launched in rivers for disposal within this watershed (CETESB, 2006). The sampling points are given in Table 4 and an illustrative map for this area is shown in Figure 4. Table 4. Sampling point locations in the Ribeira do Iguape river Table 4. Sampling point locations in the Ribeira do Iguape river Table 4. Sampling point locations in the Ribeira do Iguape river 2.3 Development of the fuzzy water quality index (INQA) For example, it was assumed that the boundary between Good and Excellent had a pertinence of 50% in the Excellent and Good fuzzy sets and so on, showing absence of a rigid boundary between classes. g y Fig. 3. Output Membership Function Gr 01, 02, 03, 04, 05 and INQAI IQA 0 - 100 CETESB a b c d Classes Excellent 65 90 100 100 79 < IQA ≤ 100 Good 44 65 90 51 < IQA ≤ 79 Fair 28 44 65 36 < IQA ≤ 51 Bad 0 28 44 19 < IQA ≤ 36 Poor 0 0 9 28 0 ≤ IQA ≤ 19 Table 3. Input and output fuzzy sets for inference IN06 and IQACETESB classes Fig. 3. Output Membership Function Fig. 3. Output Membership Function Gr 01, 02, 03, 04, 05 and INQAI IQA 0 - 100 CETESB a b c d Classes Excellent 65 90 100 100 79 < IQA ≤ 100 Good 44 65 90 51 < IQA ≤ 79 Fair 28 44 65 36 < IQA ≤ 51 Bad 0 28 44 19 < IQA ≤ 36 Poor 0 0 9 28 0 ≤ IQA ≤ 19 Table 3. Input and output fuzzy sets for inference IN06 and IQACETESB classes 394 Environmental Management in Practice The fuzzy inference system used to compute the INQA has 3125 rules. Being impossible to write them all in this paper, some examples are given below: R l 01 All the computations were processed using the “fuzzy logic toolbox” for MATLAB® (2006). 2.4.2 Paranapanema river – farming area Paranapanema River has a total extension of 929 km, with eight dams and barrages along its length. The area under study is about 29,114 km2. Soil use is predominantly rural and thus the region is considered a farming area, occupied mainly by pastures (1,781,625 ha) , followed by temporary cultures, such as sugar cane, soy and corn (764,476 ha) and silviculture (76,595 ha). Fruticulture occupies 40,917 ha and horticulture, 2,477 ha. The watershed comprises 63 counties, with a total population of 1,155,060, of which 88% is urban (CETESB, 2006). Approximately 95.5% of the effluents produced in this watershed are collected and about 79%of these are treated. It is estimated that approximately 20 tons of 395 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen BOD5 are dumped in reception bodies of this watershed for disposal (CETESB, 2006). The sampling points are given in Table 5 and an illustrative map for this area is shown in Figure 5. BOD5 are dumped in reception bodies of this watershed for disposal (CETESB, 2006). The sampling points are given in Table 5 and an illustrative map for this area is shown in Figure 5. Fig. 4. Map showing Ribeira do Iguape River in a conservation area. Fig. 5. Map showing Paranapanema River in a farming area. Fig. 4. Map showing Ribeira do Iguape River in a conservation area. Fig. 4. Map showing Ribeira do Iguape River in a conservation area. Fig. 5. Map showing Paranapanema River in a farming area. Fig. 5. Map showing Paranapanema River in a farming area. 396 Environmental Management in Practice Table 5. Sampling point locations in Paranapanema River Table 5. Sampling point locations in Paranapanema River 2.4.3 Pardo river – industrializing area Pardo River is born in a small spring in Minas Gerais state, crosses the northwest part of São Paulo state and, after running for 240 km with a watershed of 8,993 km2, empties in the estuary of Mogi-Guaçu river. The main uses of the soil in this watershed are urban- industrial and farming, with predominance of sugar cane (329,924 ha), followed by pastures (261,999 ha), fruticulture (83,611 ha) and silviculture (46,640 ha). About 3% of the state population live in this UGRHI (1,056,658 people) with 97% of the population in urban areas, scattered over 23 cities. More than 99% of the effluents are collected and 51% are treated. It is estimated that approximately 31 tons of BOD5 are dumped in reception bodies of this watershed for disposal (CETESB, 2006). The sampling points are given in Table 6 and an illustrative map for this area is shown in Figure 6. Table 6. Sampling point locations in Pardo River Fig. 6. Map showing Pardo River in an industrializing area. Table 6. Sampling point locations in Pardo River Table 6. Sampling point locations in Pardo River Table 6. Sampling point locations in Pardo River Fig. 6. Map showing Pardo River in an industrializing area. 397 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen Fig. 7. Map showing Paraíba do Sul River in an industrial area. 2.4.4 Paraíba do Sul river – industrial aea Paraíba do Sul River has an approximate length of 1,150 km (Jornal da ASEAC, 2001). Its watershed is located in the southwest region of Brazil and covers approximately 55,400 km2, including the states of São Paulo (13,500 km2), Rio de Janeiro (21,000 km2) and Minas Gerais (20,900 km2). The watershed comprises 180 counties, with a total population of 5,588,237, 88.8% in urban areas. The river is used predominantly for irrigation (49.73 m3/s), without taking into account the transposition of the Paraíba do Sul (160 m3/s) and Piraí (20 m3/s) rivers to the metropolitan region of Rio de Janeiro. The urban supply amounts to about 16.5 m3/s, while the industrial sector uses 13.6 m3/s, surpassing only the cattle-raising sector, with less than 4 m3/s. The main uses of the soil are urban-industrial and rural, the second with pastures (545,156 ha), temporary cultures (57,709 ha), fruticulture (2,996 ha), horticulture (438) and silviculture (83,667 ha). About 5% of the state population (1,944,638) live in this watershed, with 91% in urban areas, scattered throughout 34 counties. Of the total effluents produced in this watershed, 89% are collected and 33% of these are treated. It is estimated that about 72 tons of BOD are dumped in this river for disposal (CETESB, 2006). The sampling points are given in Table 7 and an illustrative map for this area is shown in Figure 7. Table 7. Sampling point locations in Paraíba do Sul River Table 7. Sampling point locations in Paraíba do Sul River Fig. 7. Map showing Paraíba do Sul River in an industrial area. Fig. 7. Map showing Paraíba do Sul River in an industrial area. 398 Environmental Management in Practice 3. Index results and discussion The IQACETESB was taken from the Relatórios de Qualidade das Águas Interiores do Estado de São Paulo (CETESB, 2004, 2005, 2006). The IQAsub was calculated with a weight factor k = 0.75 for good quality water. The IQAmin was calculated as described by Pesce and Wunderlin (2000) and the IQAPAL according to Silva e Jardim (2006), using the recommended technologies. The INQA was computed using the method previously outlined. In this work individual results will not be presented. The results will be graphically presented in the consolidated form of weighted averages. A statistical analysis of the results will then be performed. Factors or influences that lead to an increase or decrease of individual parameters will not be discussed, since this would take us too far afield. A discussion of the subject can be found in Lermontov (2009). 3.1 Ribeira do Iguape river indices – environmental conservation area The annual averages of the indices for 2004, 2005 and 2006 are shown in Figure 8 for all sampling points. The IQACETESB, IQAsub and INQA indices are strongly correlated. In most cases, the IQAsub index is the stricter and IQAmin is the less strict, attributing a better quality to the same water sample. Fig. 8. Annual averages of the indices for the Ribeira do Iguape River. Fig. 8. Annual averages of the indices for the Ribeira do Iguape River. 3.2 Paranapanema river indices – farming area 3.2 Paranapanema river indices farming area The results for the Parapanema River are shown in Figure 9. The IQAmin for 2004 is less strict than the other indices, while the IQAmin is the stricter. The other the indices are very close for sampling points SP 03, 04 and 05, but diverge somewhat for sampling points SP 01 and 02. In the case of 2005 data, the INQA stays close to the IQACETESB for all sampling points but the two indices are weakly correlated, specially at sampling point SP 02. The IQAsub is again the stricter index and the IQAmin the less strict. Data for 2006 confirm that the IQAsub is not the best indicator for the water quality of this river, since it diverges significantly from the other indices. The INQA is again very close to the IQACETESB, although slightly less strict. 3 a a apa e a e d ces a g a ea The results for the Parapanema River are shown in Figure 9. 3. Index results and discussion The IQAmin for 2004 is less strict than the other indices, while the IQAmin is the stricter. The other the indices are very close for sampling points SP 03, 04 and 05, but diverge somewhat for sampling points SP 01 and 02. In the case of 2005 data, the INQA stays close to the IQACETESB for all sampling points but the two indices are weakly correlated, specially at sampling point SP 02. The IQAsub is again p g The results for the Parapanema River are shown in Figure 9. The IQAmin for 2004 is less strict than the other indices, while the IQAmin is the stricter. The other the indices are very close for sampling points SP 03, 04 and 05, but diverge somewhat for sampling points SP 01 and 02. In the case of 2005 data, the INQA stays close to the IQACETESB for all sampling points but the two indices are weakly correlated, specially at sampling point SP 02. The IQAsub is again the stricter index and the IQAmin the less strict. Data for 2006 confirm that the IQAsub is not the best indicator for the water quality of this river, since it diverges significantly from the other indices. The INQA is again very close to the IQACETESB, although slightly less strict. In the case of 2005 data, the INQA stays close to the IQACETESB for all sampling points but the two indices are weakly correlated, specially at sampling point SP 02. The IQAsub is again the stricter index and the IQAmin the less strict. Data for 2006 confirm that the IQAsub is not the best indicator for the water quality of this river, since it diverges significantly from the other indices. The INQA is again very close to the IQACETESB, although slightly less strict. 399 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen Fig. 9. Annual averages of the indices for the Paranapanema River. Fig. 9. Annual averages of the indices for the Paranapanema River. 3.3 Pardo river indices – industrializing area g The results for the Pardo River are shown in Figure 10. For 2004, que IQACETESB, IQAsub e INQA índices are very close. A k = 0.75 value for the IQAsub index shows a less strict evaluation, while a k = 1.00 for the IQAobj shows a stricter evaluation. 3. Index results and discussion The INQA is in general close to the IQACETESB, albeit somewhat less strict for SP 04. The 2005 results show the INQA close to the IQACETESB for sampling points SP 01 e SP 02 but the indices diverge for SP 03 and SP 04. The IQAsub is again the stricter index. The results for 2006 are similar. Fig. 10. Annual averages of the indices for the Pardo River. 3.4 Paraíba do Sul indices – industrial area The results for the Paraíba do Sul River are shown in Figure 11. In the case, the IQAPAL is the stricter index, while the IQAobj and the IQAmin alternate as the less strict index, depending on the sampling point. The IQACETESB, IQAsub and INQA are closely related. Fig. 10. Annual averages of the indices for the Pardo River. 3.4 Paraíba do Sul indices – industrial area 3.4 Paraíba do Sul indices industrial area The results for the Paraíba do Sul River are shown in Figure 11. In the case, the IQAPAL is the stricter index, while the IQAobj and the IQAmin alternate as the less strict index, depending on the sampling point. The IQACETESB, IQAsub and INQA are closely related. The results for the Paraíba do Sul River are shown in Figure 11. In the case, the IQAPAL is the stricter index, while the IQAobj and the IQAmin alternate as the less strict index, depending on the sampling point. The IQACETESB, IQAsub and INQA are closely related. 400 Environmental Management in Practice Fig. 11. Annual averages of the indices for the Paraíba do Sul River. Fig. 11. Annual averages of the indices for the Paraíba do Sul River. 4.1 Statistical results The purpose of statistical analysis of the results for each watershed was to validate the use of fuzzy methodology to develop a fuzzy water quality index (INQA). In this process, the results for 2004, 2005 and 2006 were not separately studied, but were grouped in a single data set for each index. The results are shown in Table 8. Table 9. Relative differences between the means of the indices and IQACETESB and INQA. Table 8. Statistical Data The statistical data were computed using the StatSoft Statistica application and will be discussed in section 4.2. Figure 12 show the coefficient of variation of the indices. Table 9 shows the relative differences between the means of the indices and the official statistical data were computed using the StatSoft Statistica application and will b ussed in section 4.2. Figure 12 show the coefficient of variation of the indices. p g pp discussed in section 4.2. Figure 12 show the coefficient of variation of the indices. Table 9 shows the relative differences between the means of the indices and the official index (IQACETESB) and the proposed new index (INQA), calculated using Equation 6: g Table 9 shows the relative differences between the means of the indices and the official index (IQACETESB) and the proposed new index (INQA), calculated using Equation 6: 401 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen y y y y g % variation = (I1 - I2) / I1 x 100 (6) Where: I1 – First index I2 – Second index Fig. 12. Coefficients of variation of the indices. % variation = (I1 - I2) / I1 x 100 (6) Fig. 12. Coefficients of variation of the indices. Table 9. Relative differences between the means of the indices and IQACETESB and INQA. Table 9. Relative differences between the means of the indices and IQACETESB and INQA. 402 Environmental Management in Practice The frequency histograms of the indices for the four watersheds are shown in Figure 13 and correspond to a visual representation of the frequency distribution tables. For analysis and interpretation of these graphs, see Lermontov (2009). The frequency histograms of the indices for the four watersheds are shown in Figure 13 and correspond to a visual representation of the frequency distribution tables. For analysis and interpretation of these graphs, see Lermontov (2009). Ribeira do Iguape Paranapanema Pardo Paraíba do Sul Fig. 13. Frequency histograms for the four watersheds. Paranapanema Ribeira do Iguape Ribeira do Iguape Paraíba do Sul Pardo Paraíba do Sul Fig. 13. Frequency histograms for the four watersheds. Paraíba do Sul Fig. 13. Frequency histograms for the four watersheds. Fig. 13. Frequency histograms for the four watersheds. Table 10. Correlations between the INQA and the other indices for the four watersheds. Table 8. Statistical Data 403 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen Figures 14 and 15 show box & whiskers plots for all indices and watersheds. These plots are a convenient way to visualize the main trend and the data scatter and to show, in the same graph, the main results of a sampling. Figures 14 and 15 show box & whiskers plots for all indices and watersheds. These plots are a convenient way to visualize the main trend and the data scatter and to show, in the same graph, the main results of a sampling. Ribeira do Iguape Paranapanema Pardo Paraíba do Sul Fig. 14. Box & Whiskers plots of the mean, mean ± standard deviation and mean ± 1,96 times standard deviation for the four watersheds. Ribeira do Iguape Paranapanema Ribeira do Iguape Paranapanema Paranapanema Ribeira do Iguape Pardo Paraíba do Sul Fig. 14. Box & Whiskers plots of the mean, mean ± standard deviation and mean ± 1,96 times standard deviation for the four watersheds. Pardo Paraíba do Sul Paraíba do Sul Fig. 14. Box & Whiskers plots of the mean, mean ± standard deviation and mean ± 1,96 times standard deviation for the four watersheds. 404 Environmental Management in Practice Ribeira do Iguape Paranapanema Pardo Paraíba do Sul Fig. 15. Box & Whiskers plots of the median, upper and lower quartile and maximum and minimum value for the four watersheds. Rib i d I Paraíba do Sul Pardo Paraíba do Sul Fig. 15. Box & Whiskers plots of the median, upper and lower quartile and maximum and minimum value for the four watersheds. Table 10 shows the correlations between the fuzzy index (INQA) and the other indices. The best correlation, 0.8527 (a strong correlation), between the INQA and the IQACETESB for the Paranapanema River, is illustrated in Figure 16. The worst correlation, 0.3740, between the INQA and the IQAPAL for the Ribeira do Iguape River, is illustrated in Figure 17. Corelations - Pearson’s r Ribeira do Iguape Paranapanema Pardo Paraíba do Sul INQA x IQACETESB 0.79381 0.8527 0.8206 0.7943 INQA x IQAsub 0.57937 0.7710 0.7107 0.8127 INQA x IQAobj 0.57937 0.7710 0.7107 0.8742 INQA x IQAmin 0.59937 0.6444 0.6520 0.7483 INQA x IQAPAL 0.37406 0.3924 0.4025 0.5191 Table 10. Correlations between the INQA and the other indices for the four watersheds. 405 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen Fig. 16. 4.2 Statistical discussion The statistical data that were collected and presented in this work provide a rich field for discussion and analysis. However, our purpose here was only to validate the use of the fuzzy i d (INQA) A i lifi d t ti ti l l i i l t d d f lfill d it ndex (INQA). A simplified statistical analysis was implemented and fulfilled its purpose. index (INQA). A simplified statistical analysis was implemented and fulfilled its purpose. In the case of the Ribeira do Iguape River, we could compute all indices from the available data, except the IQACETESB, that was taken directly from reports. In the case of the Ribeira do Iguape River, we could compute all indices from the available data, except the IQACETESB, that was taken directly from reports. y In the case of the Paraíba do Sul River, since there was a minimum equal to zero, the geometric and harmonic means could not be computed. For all watersheds and all indices, the geometric mean was lower than the arithmetic mean and the harmonic mean was lower than the arithmetic mean. The geometric mean and the harmonic mean of the IQAPAL could not be computed for the Paraíba do Sul River because, in the case, the minimum value was 0. The coefficients of variation shown in the last column of Table 8 were plotted in Figure 12. In this kind of analysis, the statistical results are presented though a parameter that reflects the scattering of the data points. The worst coefficient of variation was that of the IQAPAL and the best were those of the IQAsub and the IQAobj. When the results for the INQA and the IQACETESB are compared, one notices that the coefficient of variation of the INQA was smaller than that of the IQACETESB in three watersheds: Ribeira do Iguape, Paranapanema and Pardo. Only in the industrial area of the Paraíba do Sul River the coefficient of variation of the IQACETESB was smaller than that of the INQA. This is probably due to the fact that the Paraíba do Sul watershed is more polluted than the others, with low quality water. The relative differences more relevant to our study, i.e. those between means of the other indices and the IQACETESB and the INQA means, were computed using Equation 6 and the results are shown in Table 9. Table 8. Statistical Data Best correlation – INQA x IQACETESB – r = 0.8527 – Paranapanema River Fig. 17. Worst correlation – INQA x IQApal – r = 0.3740 – Ribeira do Iguape River Fig. 16. Best correlation – INQA x IQACETESB – r = 0.8527 – Paranapanema River Fig. 16. Best correlation – INQA x IQACETESB – r = 0.8527 – Paranapanema River Fig. 16. Best correlation – INQA x IQACETESB – r = 0.8527 – Paranapanema River Fig. 17. Worst correlation – INQA x IQApal – r = 0.3740 – Ribeira do Iguape River Fig. 17. Worst correlation – INQA x IQApal – r = 0.3740 – Ribeira do Iguape River 406 Environmental Management in Practice 5. General conclusions The use of several water quality indices and the development, application and evaluation of a new indexing method to assess river water quality using fuzzy inference is discussed. A new index, called Fuzzy Water Quality Index (INQA) is developed to correct perceived deficiencies in environmental monitoring, water quality classification and management of water resources in cases where the conventional, deterministic methods can be inaccurate or conceptually limited. This methodology differs from other fuzzy water quality indexing methodologies by incorporating the weight factor in qualitative sphere throughout the rules in the inference engine. This is only possible due to a high variety of rules inserted in the inference system. The practical applications of the new index is tested in a realistic case study carried out in Ribeira do Iguape River in São Paulo State, Brazil, showing that the proposed index is reliable and consistent with the traditional qualitative methods. Most institutional players are not familiar with fuzzy logic concepts, therefore being unaware of the potential of this technique for the transfer of expert knowledge in a qualitative sphere into a formal system of environmental assessment. We think that this approach can and should be used as an alternate tool for the analysis of river water quality and for strategic planning and decision making in the context of integrated environmental management. The use of several water quality indices and the development, application and evaluation of a new indexing method to assess river water quality using fuzzy inference is discussed. A new index, called Fuzzy Water Quality Index (INQA) is developed to correct perceived deficiencies in environmental monitoring, water quality classification and management of water resources in cases where the conventional, deterministic methods can be inaccurate or conceptually limited. This methodology differs from other fuzzy water quality indexing methodologies by incorporating the weight factor in qualitative sphere throughout the rules in the inference engine. This is only possible due to a high variety of rules inserted in the inference system. The practical applications of the new index is tested in a realistic case study carried out in Ribeira do Iguape River in São Paulo State, Brazil, showing that the proposed index is reliable and consistent with the traditional qualitative methods. 4.3 Statistical conclusions The main conclusions of the statistical analysis are the following: The main conclusions of the statistical analysis are the following:  There is a strong correlation between the proposed fuzzy index (INQA) and a widely accepted, traditional index (IQACETESB);  The relative differences between the means of INQA and IQACETESB were less than 8% for all four watersheds;  The relative differences between the means of INQA and IQACETESB were less than 8% for all four watersheds;  The box and whiskers plots for the two indices are reasonably similar; y  The other statistical results for the two indices also were reasonably similar;  The coefficients of variation of the INQA were smaller than those of the IQACETESB for all four watersheds.  The coefficients of variation of the INQA were smaller than those of the IQACETESB for all four watersheds. Balas, C.E., Ergin, A., Williams, A.T., Koc, L. (2004). Marine litter prediction by artificial intelligence. Mar. Poll. Bull. 48, 449–457. 5. General conclusions Most institutional players are not familiar with fuzzy logic concepts, therefore being unaware of the potential of this technique for the transfer of expert knowledge in a qualitative sphere into a formal system of environmental assessment. We think that this approach can and should be used as an alternate tool for the analysis of river water quality and for strategic planning and decision making in the context of integrated environmental management. For this doctoral study, the same nine parameters used by CETESB State Organ to calculate its IQA were chosen for the methodology validation by statistical comparison. The authors also worked in the development of an index with additional parameters, such as heavy metals, organoleptic metals and toxic compounds, for a more realistic evaluation of the hydric bodies (Lermontov, 2009). 4.2 Statistical discussion This is probably due to the fact that this indicator is based on only two parameters;  The best correlation with INQA was that of IQAobj in the industrial region (Paraíba do Sul watershed), but the correlation of IQAobj with INQA was much weaker in the other regions;  The best correlation with INQA was that of IQAobj in the industrial region (Paraíba do Sul watershed), but the correlation of IQAobj with INQA was much weaker in the other regions; 407 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen  The best global correlation with INQA was that of IQACETESB, a widely accepted index; The best global correlation with INQA was that of IQACETESB, a widely accepted inde  The best global correlation with INQA was that of IQACETESB, a widely accepted index; Th b i di id l l i b INQA d IQA i h f i i g Q Q y p  The best individual correlation between INQA and IQACETESB was in the farming region (Paranapanema watershed).  The best individual correlation between INQA and IQACETESB was in the farming region (Paranapanema watershed). (Paranapanema watershed). 4.2 Statistical discussion In the case of the difference between the IQACETESB and the INQA, the main focus of our study, all the differences were smaller than 10%. The largest difference, 7.5%, was for the Paraíba do Sul watershed, an industrial area, and the smallest, 0.5%, was for the Paranapanema watershed, a farming area. Examining the box and whiskers plots of Figures 14 and 15 along with the data from Table 9, one can draw the following conclusions: Examining the box and whiskers plots of Figures 14 and 15 along with the data from Table 9, one can draw the following conclusions:  IQAobj and IQAmin are the indices that diverge more sharply from the others, especially from IQACETESB, calculated using a well accepted method;  INQA yielded satisfactory results when compared to a traditional method such as IQACETESB;  The results obtained using INQA and IQACETESB were closest for a farming region and were farthest for an industrial region. The correlation data are shown in Table 10. The correlation coefficient r, or “Pearson’s r”, as it is also called, is used in this study to measure the degree of correlation between INQA and the other indices for each watershed. Values between 0.7 and 1.0 (positive or negative) indicate a strong correlation between two parameters. Examining the correlation data, one can draw the following conclusions:  The worst correlation with INQA was that of IQAPAL in all four watersheds. 6. References 408 Environmental Management in Practice Bárdossy, A., Duckstein, (1995). Fuzzy rule-based modeling with applications to geophysical, biological and engineering systems. CRC Press, Boca Raton, New York, London, Tokyo. Barreto, J.M (2001). Inteligência Artificial no Limiar do Século XXI. 3ª Edição – Florianópolis; O Autor. 379p Bhargava, D. S. (1983). 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Journal of the Institution of Water and Environmental Management, 3, 1989, pp.336-344 Icaga, Y. (2007). Fuzzy evaluation of water classification. Ecological Indicators 7, pp.710-718. Jornal da ASEAC. (2001). Paraíba do Sul: um Rio no curso da morte. Informativo Mensal da Associação dos Empregados de Nível Universitário da CEDAE. 6. References Edição de Maio/Junho de 2001, Acessed on 15 dez 2008, Available from: <http://www.aseac.com.br/ jorn34_9.htm> Liou, S.; Lo S.; Wang, S. (2004). A generalized water quality index for Taiwan. Environmental Monitoring Assessment 96, pp. 35–52. 409 A Fuzzy Water Quality Index for Watershed Quality Analysis and Managemen Lermontov, A. (2009). Novo Índice de Qualidade das Águas com uso da Lógica e Inferência Nebulosa, Rio de Janeiro: Escola de Química/UFRJ, 2009. Doctoral Tesis. Lermontov, A., Yokoyama, L., Lermontov. M., Machado. M. A. S. (2009). River quality analysis using fuzzy water quality index: Ribeira do Iguape river watershed, Brazil. Ecological Indicators, Volume 9, p.1188-1197 Matlab® 6.0; 2006. Packaged software for technical computing, Release 14, The Math works, Inc. Mitchell, M.K., Stapp, W.B. (1996). Field Manual for Water Quality Monitoring: an Environmental Education Program for Schools, Thomson-Shore Inc., Dexter, Michigan, pp. 277. Nasiri, F., Maqsood, I., Huang, G. and Fuller, N. (2007). Water quality index: A fuzzy river- pollution decision support expert system, Journal of Water Resources Planning and Management, 133, pp. 95-105. [NSF] National Sanitation Foundation International. (2007). Acessed on October of 2007, Available from: <http://www.nsf.org> Ocampo-Duque W., Ferré-Huguet N., Domingo J. L., Schuhmacher M. (2006). Assessing water quality in rivers with fuzzy inference systems: A case study. Environment International 32, pp. 733–742. pp Pesce, S. F., Wunderlin, D. A. (2000). Use of water quality indices to verify the impact of Córdoba city (Argentina) on Suquía river. Water Research 34, pp.2915-2926. Ramesh, S., Sukumaran, N., Murugesan, A.G., Rajan, M.P. (2010). An innovative approach of Drinking Water Quality Index – A case study from Southern Tamil Nadu, India, Ecological Indicators 10, p.857-868. Ross, T. J., (2004). Fuzzy logic with engineering applications. New York: John Wiley & Sons. Said, A, Stevens, D, Selke, G. (2004). An innovative index for evaluating water quality in streams. Environ Manage, 34, pp.406–14. SDD (1976). Development of a Water Quality Index. Scottish Development Department, Report ARD3, Edinburgh, p.35. Silva, G. S.; Jardim, W. de F. (2006). Um novo índice de qualidade de águas para proteção de vida aquática aplicado ao rio Atibaia, região de Campinas/Paulínea – SP. Química Nova 29, No. 4, pp. 689-694. Silvert, W. (1997). Ecological impact classification with fuzzy sets. Ecological Modeling 96, pp.1–10. Silvert, W. (2000). Fuzzy indices of environmental conditions. Ecological Modeling 130, pp.111–119. Stambuk-Giljanovic N. (1999). Water quality evaluation by index in Dalmatia, Water Research 16, pp. 3423-3440. Taner, M. Ü, Üstün, B. Yen, J.; Langari, R. (1999). Fuzzy logic: intelligence, control, and information, Prentice-Hall, Inc. Zadeh, L.A. (1965). Fuzzy Sets, Information and Control 8, pp. 338-353. Environmental Management in Practice Environmental Management of Wastewater Treatment Plants – the Added Value of the Ecotoxicological Approach Elsa Mendonça1, Ana Picado1, Maria Ana Cunha2 and Justina Catarino1 1 Laboratório Nacional de Energia e Geologia (LNEG), Lisboa, 2 Agência Portuguesa do Ambiente (APA), Amadora, P t l Elsa Mendonça1, Ana Picado1, Maria Ana Cunha2 and Justina Catarino1 1 Laboratório Nacional de Energia e Geologia (LNEG), Lisboa, 2 Agência Portuguesa do Ambiente (APA), Amadora, P t l Portugal 6. References Erdinçler, A. (2011). A simple tool for the assessment of water quality in polluted lagoon systems: A case study for Küçükçekmece Lagoon, Turkey, Ecologigal Indicators, Volume 11, Issue 2, p.749-756 g g Yager, R. R.; Filvel, D.P. (1994). Essentials of Fuzzy Modeling and Control, New York: John Wiley & Sons. 410 Environmental Management in Practice 21 1. Introduction Pollution control has been changed by advances in scientific knowledge, because there is a connection of environmental contamination with the ability to measure it. With greater understanding of the impact of wastewater on the environment and more sophisticated analytical methods, advanced treatment is becoming more common (Lofrano & Brown, 2010). ) The assessment of biological effects of wastewater discharges in the ecosystems is today considered relevant and ecotoxicological tests identifying the ecological hazard are useful tools for the identification of environmental impacts. Direct toxicity assessment, making use of ecotoxicological tests, can play an important role in supporting decision-making, either regulatory driven or on a voluntary basis. Within the Integrated Pollution Prevention and Control Directive - IPPC, 2008/1/EC (European Commission [EC], 2008), the Direct Toxicity Assessment concept has been included as a suitable monitoring tool on effluent in several Best Available Techniques (BAT) Reference Documents. Also, in Water Framework Directive – WFD, 2000/60/EC (EC, 2000), direct toxicity assessment of Wastewater Treatment Plant (WWTP) effluents can contribute to attain or keep ecological quality objectives in water masses. So, for EU countries to comply with good ecological status, ecotoxicity evaluation of WWTP effluents is extremely relevant. y In many countries ecotoxicity tests are already in use for wastewater management (Power & Boumphrey, 2004; Tinsley et al., 2004; United States Environmental Protection Agency [USEPA], 2004; Vindimian et al., 1999). Bioassays are also used for wastewater surveillance and BAT compliance by authorities in Germany (Gartiser et al., 2010a). A global evaluation of wastewaters should include ecotoxicological tests to complement the chemical characterization, with advantages especially in the case of complex wastewaters (Mendonça et al., 2009). This approach has advantages particularly to protect biological treatment plants from toxic influents (Hongxia et al., 2004), to monitor the effectiveness of WWTP (Cēbere et al., 2009; Daniel et al., 2004; Emmanuel et al., 2005; Libralato et al., 2006; Metcalf & Eddy, 2003) and in the impact assessment of complex wastewaters. Bioassays are considered a suitable tool for assessing the ecotoxicological relevance of complex organic mixtures (Gartiser et al., 2010b). 412 Environmental Management in Practice As it is often referred (e.g. Metcalf & Eddy, 2003; Movahedian et al., 2005; Teodorović et al., 2009), physico-chemical parameters alone are not sufficient in obtaining reliable information on treated wastewater toxicity and toxicity tests must be performed in combination with routine chemical analysis. 1. Introduction The prediction of toxicity from chemical data is considered limited and the better coincidence between the toxicity and chemical-based assessments were achieved when information from all tests in a test-battery was assembled (Manusadžianas et al., 2003). ( ) In the framework of Life Cycle Assessment (LCA) comprehensive analysis of WWTP is evaluated for the physico-chemical characterization of the wastewaters as well as the inventory of inputs and outputs associated with the global process (Hospido et al., 2004). In a recent work Life Cycle Impact Assessment was done using emerging pollutants quantification to rank potential impacts in urban wastewater (Muñoz et al., 2008). A step forward in this approach would be to use ecotoxicological indicators. pp g In the last ten years and in the framework of European and National contracts developed in Lisbon area (Portugal) studies were conducted on the integrated evaluation of the ecotoxicological and physicochemical parameters of wastewaters from treatment plants receiving domestic and industrial effluents. The evaluation of ecotoxicological data from four of these WWTP was the main aim of this study. Data from acute tests with different species (bacteria, algae, crustaceans and plants) are discussed. 2.1 Wastewater treatment plants The characteristics of the four WWTP that receive domestic and industrial wastewaters are presented in Table 1. These systems differ from each other, namely in the magnitude of flows (the daily flow goes from 16 000 m3/day to 155 000 m3/day), the treatment level implemented (from preliminary treatment to tertiary treatment) and the site of discharge (river, estuary or coastal area). WWTP 1 WWTP 2 WWTP 3 WWTP 4 Population equivalent 130 000 700 000 800 000 250 000 Flow (m3/day) 16 000 70 000 155 000 54 500 Treatment type secondary tertiary preliminary tertiary Discharge River River Sea Estuary Table 1. General information on the Wastewater Treatment Plants (WWTP) 2.2 Wastewater sampling 2.2 Wastewater sampling p g Wastewater samples were collected with different strategies and periodicities in the different Treatment Plants: p g Wastewater samples were collected with different strategies and periodicities in the different Treatment Plants:  WWTP1 and WWTP2 – Influent and effluent 24h-composite samples collected seasonally in November, March, September and December 2003/2004; y p  WWTP3 – Effluent 24h-composite sample collected monthly from 2006 to 2009;  WWTP4 – Influent and effluent 1h-composite samples collected in different days of the week (Monday, Tuesday and Friday) at 10 h, 14h and 23h in April 2010. Environmental Management of Wastewater T t t Pl t th Add d V l f th E Environmental Management of Wastewater Treatment Plants – the Added Value of the Ecotoxicological Approach 413 As presented in Figure 1, sampling point for WWTP1 was after secondary treatment, for WWTP2 after tertiary treatment, for WWTP3 after preliminary treatment and for WWTP4 after primary treatment. As presented in Figure 1, sampling point for WWTP1 was after secondary treatment, for WWTP2 after tertiary treatment, for WWTP3 after preliminary treatment and for WWTP4 after primary treatment. p y Each sample was divided into subsamples, kept frozen (-20°C) for ecotoxicological analysis for no more than 1 month. Fig. 1. General Scheme of WWTP treatment process and identification of the level of treatment analyzed in each Treatment Plant. Preliminary Primary Advanced primary Secondary Secondary with nutrient removal Tertiary Advanced 2.3 Ecotoxicity tests y Ecotoxicological evaluation of the samples was performed using Vibrio fischeri, Pseudokirchneriella subcapitata, Thamnocephalus platyurus, Daphnia magna and Lemna minor as test organisms, to assess acute aquatic toxicity, according to the following methods: y Ecotoxicological evaluation of the samples was performed using Vibrio fischeri, Pseudokirchneriella subcapitata, Thamnocephalus platyurus, Daphnia magna and Lemna minor as test organisms, to assess acute aquatic toxicity, according to the following methods: Ecotoxicological evaluation of the samples was performed using Vibrio fischeri, Pseudokirchneriella subcapitata, Thamnocephalus platyurus, Daphnia magna and Lemna minor as test organisms, to assess acute aquatic toxicity, according to the following methods:  Microtox test: Bacterial toxicity was assessed by determining the inhibition of the luminescence of Vibrio fischeri (strain NRRL B-11177) exposed for 15 minutes (Microtox® Test, Microbics, Carlsbad, U.S.A.). The test was performed according to the basic test procedure (Microbics, 1992);  AlgalTox test: Algal toxicity was assessed by measuring the growth inhibition of Pseudokirchneriella subcapitata exposed for 72 hours, according to AlgalToxKit FTM test procedure (Microbiotests, 2004) that follow the OECD guideline 201 (Organisation for Economic Co-operation and Development [OECD], 1984). Optical density (OD 670 nm) of algae suspensions was determined;  ThamnoTox test: Crustacean toxicity was assessed by determining the mortality of Thamnocephalus platyurus exposed for 24 hours according to ThamnoToxKit FTM test procedure (Microbiotests, 2003);  Daphnia test: Crustacean toxicity was also assessed by determining the inhibition of the mobility of Daphnia magna (clone IRCHA-5) exposed for 48 hours, according to ISO 6341:1996 (International Organization for Standardization [ISO], 1996). Juveniles for testing were obtained from cultures maintained in the laboratory;  Lemna test: Plant toxicity was assessed by determining the growth inhibition of Lemna minor (clone ST) exposed for 7 days, according to ISO 20079: 2005 (ISO, 2005). Plants for testing were obtained from cultures maintained in the laboratory. Total frond area was used as growth parameter, quantified by an image analysis system – Scanalyzer (LemnaTec, Würselen, Germany). All samples were tested with Microtox, Daphnia and Lemna tests. For WWTP1, WWTP2 and WWTP4 samples, AlgalTox and ThamnoTox tests were also performed. Preliminary Secondary with nutrient removal Tertiary Advanced Fig. 1. General Scheme of WWTP treatment process and identification of the level of treatment analyzed in each Treatment Plant. 414 Environmental Management in Practice 2.4 Data analysis For each toxicity test EC50-t or LC50-t, the effective concentration (% v/v) responsible for the inhibition or lethality in 50% of tested population after the defined exposure period (t), was calculated: For each toxicity test EC50-t or LC50-t, the effective concentration (% v/v) responsible for the inhibition or lethality in 50% of tested population after the defined exposure period (t), was calculated:  EC50-72h for AlgalTox test, LC50-24h for ThamnoTox test and EC50-48 h for Daphnia test by using Tox-CalcTM software (version 5.0, Tidepool Scientific software, 2002); y g  EC50-7d for Lemna test by using Biostat 2.0 software (LemnaTec 2001);  EC50-15 min for Microtox test by using Microtox OmniTM software (Azur Environmental, 1999). ) To obtain a direct interpretation between values and toxicity, ecotoxicity test results are in this work presented in Toxic Units (TU), calculated as TU=1/ EC50100. Aiming to include all raw data for TU calculation and for statistical analysis, EC50 values not determined due to low effect levels were considered as 100%. For data analysis, values lower than 1 TU were considered as 0.5 TU. The tests sensitivity was assessed by Slooff’s index (Slooff, 1983): each single test result (expressed as EC50 or LC50) is divided by the arithmetic mean of all test results for each sample, and the geometric mean of these ratios for each test is calculated. The smaller value stands for the more sensitive test. The Slooff’s index was calculated for Microtox, AlgalTox, ThamnoTox, Daphnia and Lemna tests. Environmental Management of Wastewater Environmental Management of Wastewater Treatment Plants – the Added Value of the Ecotoxicological Approach 415 g Treatment Plants – the Added Value of the E Pearson correlations were determined for WWTP3 using statistical analysis software (JMP® 5.0.1) for the 48 samples on the following 4 variables:  Wastewater flow (pers. comm.);  Wastewater flow (pers. comm.);  Ecotoxicological data from Microtox, Daphnia and Lemna tests.  Ecotoxicological data from Microtox, Daphnia and Lemna tests. 3. Results and discussion Aiming to assess direct toxicity of samples from four WWTP we evaluated data from acute tests with different species: bacteria, algae, crustaceans and plants. The results are presented in Tables 2 to 5. Results obtained for WWTP1 (Table 2) show clearly that influent and effluent samples have different toxicity levels to the species tested, except for Lemna that shows no toxicity both for influent and effluent samples. Sample Microtox AlgalTox ThamnoTox Daphnia Lemna Influent Nov 03 27.0 3.8 <1 <1 Mar 04 19.2 5.0 2.3 1.4 Sep 04 5.6 <1 7.1 4.8 <1 Dec 04 11.5 1.8 1.7 2.4 <1 Effluent Nov 03 <1 <1 <1 <1 Mar 04 1.9 <1 2.2 <1 <1 Sep 04 <1 <1 <1 <1 <1 Dec 04 <1 <1 <1 <1 <1 Table 2. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP1 influent and effluent samples le 2. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP1 influent effluent samples For WWTP2 (Table 3), influent and effluent samples have also different toxicity levels to the species tested, except for AlgalTox that shows no toxicity both for influent and effluent samples. The effluent samples show in this case no toxicity in all the tests performed. Sample Microtox AlgalTox ThamnoTox Daphnia Lemna Influent Nov 03 17.2 3.7 1.2 1.1 Mar 04 62.5 <1 3.0 1.4 1.4 Sep 04 47.6 <1 2.0 1.8 1.1 Dec 04 83.3 <1 1.6 2.5 <1 Effluent Nov 03 <1 <1 <1 <1 Mar 04 <1 <1 <1 <1 <1 Sep 04 <1 <1 <1 <1 <1 Dec 04 <1 <1 <1 <1 <1 Table 3. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP2 influent and effluent samples Table 3. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP2 influent and effluent samples 416 Environmental Management in Practice For WWTP3 (Table 4), effluent samples have different toxicity levels to the species tested, with Microtox having the higher TU values along the four years. No significant correlations were obtained between toxicity test results and corresponding daily discharge flow. 3. Results and discussion Microtox Daphnia Lemna 2006 2007 2008 2009 2006 2007 2008 2009 2006 2007 2008 2009 Jan 16.3 14.5 5.9 33.3 3.2 1.4 2.4 1.5 1.6 <1 <1 <1 Feb 4.6 13.2 6.4 10.8 2.9 1.0 1.3 <1 1.2 <1 1.6 <1 Mar 2.2 10.4 15.6 11.6 1.4 2.0 2.9 1.8 <1 <1 1.3 1.0 Apr 8.1 8.4 14.9 10.9 1.4 1.9 2.6 2.5 1.4 <1 <1 <1 May 27.8 14.7 14.5 12.5 4.6 3.1 4.8 1.7 1.6 1.1 <1 1.0 Jun 32.3 16.4 13.2 10.3 7.1 2.6 2.1 1.2 <1 1.4 <1 1.0 Jul 13.5 25.0 19.2 22.2 6.6 2.2 1.2 <1 <1 1.1 1.1 1.0 Aug 14.5 12.2 19.2 4.1 3.2 3.1 3.6 2.2 <1 1.2 1.0 1.1 Sep 25.6 12.7 20.4 5.1 8.1 3.2 1.6 1.5 <1 1.4 <1 <1 Oct 17.5 7.8 31.3 10.0 2.6 1.5 1.5 1.3 1.1 <1 <1 <1 Nov 18.9 13.0 83.3 15.4 3.4 3.1 2.9 4.3 <1 1.3 1.0 1.1 Dec 16.7 4.7 71.4 9.4 3.2 1.4 3.2 <1 1.2 1.4 <1 <1 Table 4. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP3 effluent samples. Table 4. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP3 effluent samples. No time pattern for effluent toxicity was observed in WWTP3. Between October 2008 and January 2009, the effluent samples were particularly toxic to the bacteria, with 83.3 TU in November 2008 (Figure 2). For WWTP4 (Table 5), the difference in toxicity levels is not so clear between untreated and treated wastewater samples although for Microtox the range of values is higher for the untreated samples [5.8 TU - 93.5 TU] versus treated samples [2.3 TU – 35.8 TU]. During the week monitoring, the highest TU value was obtained on Friday night for Microtox. A peak in toxicity was obtained for Microtox in all samples collected at 23h. This is in line with Chapman (2007) that concludes that difficulties in obtaining representative samples arise in WWTP effluents, whose composition is highly variable, and repeated testing is required. Analyzing the mean TU values obtained in the different tests, Microtox test shows higher values in all WWTP, followed by the crustacean tests. Low toxicity values were obtained in the plant and algae tests (Figure 3). Table 5. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP4 influent and effluent samples 3. Results and discussion Environmental Management of Wastewater Treatment Plants – the Added Value of the Ecotoxicological Approach Environmental Management of Wastewater T t t Pl t th Add d V l f th E 417 Fig. 2. Distribution of sample toxicity in Toxic Units (TU) for WWTP3 monthly samples from 2006 to 2009. J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D Microtox Daphnia Lemna 2009 2006 2007 2008 Fig. 2. Distribution of sample toxicity in Toxic Units (TU) for WWTP3 monthly samples from 2006 to 2009. Sample Microtox AlgalTox ThamnoTox Daphnia Lemna Influent Mon-10h 5.8 <1 2.8 <1 1.3 Mon-14h 19.4 <1 3.0 <1 <1 Mon-23h 32.7 <1 3.6 1.5 1.1 Tues-10h 13.9 <1 2.7 <1 1.3 Tues-14h 12.6 <1 2.8 <1 <1 Tues-23h 46.5 <1 3.4 1.9 <1 Fri-10h 17.9 <1 2.6 3.6 <1 Fri-14h 43.9 <1 2.5 1.9 <1 Fri-23h 93.5 <1 2.4 1.4 <1 Effluent Mon-14h 2.3 <1 2.8 <1 <1 Mon-23h 11.1 <1 3.0 1.1 1.1 Tues-10h 2.9 1.1 1.8 <1 1.3 Tues-14h 4.8 <1 2.8 <1 1.3 Tues-23h 17.8 <1 2.4 1.5 <1 Fri-10h 16.6 <1 2.1 1.5 <1 Fri-14h 11.4 <1 2.2 1.1 <1 Fri-23h 35.8 <1 2.3 <1 <1 Table 5. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP4 influent and effluent samples Table 5. Values for ecotoxicological tests in Toxic Units (TU) obtained for WWTP4 influent and effluent samples 418 Environmental Management in Practice Fig. 3. Mean Toxic Units (TU) values for the tested species and for all effluent samples. Lemna Daphnia Microtox Thamnotox Algaltox WWTP1 WWTP2 WWTP3 WWTP4 1 10 100 Lemna 100 Daphnia Algaltox Microtox Thamnotox Fig. 3. Mean Toxic Units (TU) values for the tested species and for all effluent samples The acute toxicity is dependent on the treatment level of the studied WWTP and the species tested (Figure 3). TU values for Microtox and ThamnoTox are higher in the case of WWTP3 and 4, with preliminary and primary levels of treatment, respectively. The used tests are able to distinguish the different levels of treatment, with the exception of AlgalTox. The acute toxicity is dependent on the treatment level of the studied WWTP and the species tested (Figure 3). 3. Results and discussion TU values for Microtox and ThamnoTox are higher in the case of WWTP3 and 4, with preliminary and primary levels of treatment, respectively. The used tests are able to distinguish the different levels of treatment, with the exception of AlgalTox. The acute toxicity is dependent on the treatment level of the studied WWTP and the species tested (Figure 3). TU values for Microtox and ThamnoTox are higher in the case of WWTP3 and 4, with preliminary and primary levels of treatment, respectively. The used tests are able to distinguish the different levels of treatment, with the exception of AlgalTox. g p g From data presented in Figure 4, toxicity removal was obtained for all the WWTP where input and output wastewaters were monitored. For WWTP4 – primary treatment – removal values were in the range 15-60%. For the WWTP with secondary (WWTP1) and tertiary (WWTP2) levels of treatment toxicity removal evaluated by both crustaceans is similar, only the bacteria achieve to detect higher efficiency (100%) with the tertiary treatment. Tyagi et al. (2007) found that the mean percentage removal in toxicity for D. magna after primary, secondary and tertiary treatment were 29%, 76% and 100%, respectively. Also Movahedian et al. (2005) reinforces that toxicity removal increases with the level of treatment (e.g. 8% for preliminary treatment and 38% for primary treatment). p y p y ) A wastewater classification adapted from Tonkes et al. (1999) to the TU values, is as follows: samples with less than 1 TU are considered non toxic; between 1 and 10 TU are considered slightly toxic; with more than 10 TU are considered toxic. Values higher than 10 TU were obtained for Microtox test in 69% of the samples tested. Values between 1 and 10 TU were obtained for 79% of the samples for ThamnoTox and 74% of the samples for Daphnia. No toxicity to the alga and to the plant was registered for the majority of samples, respectively 90% and 65%. Slooff’s sensitivity index calculated for this group of acute test results shows that the bacterium Vibrio fischeri is the most sensitive species, and allows to establish the following gradient of test sensitivity, Microtox > ThamnoTox > Daphnia > AlgalTox > Lemna, from the corresponding Slooff’s index values 0.2 < 0.7 < 1.0 < 1.4 < 1.6. 3. Results and discussion bacterium Vibrio fischeri is the most sensitive species, and allows to establish the following gradient of test sensitivity, Microtox > ThamnoTox > Daphnia > AlgalTox > Lemna, from the corresponding Slooff’s index values 0.2 < 0.7 < 1.0 < 1.4 < 1.6. The sensitivity of Microtox test and the reliability of this test in monitoring toxicity of treatment plant wastewaters have also been observed by other authors (Araújo et al., 2005; Libralato et al., 2006; Lundström et al., 2010b). Related to the crustacean toxicity several authors concluded that Daphnia magna acute test can be a useful analytical tool for early The sensitivity of Microtox test and the reliability of this test in monitoring toxicity of treatment plant wastewaters have also been observed by other authors (Araújo et al., 2005; Libralato et al., 2006; Lundström et al., 2010b). Related to the crustacean toxicity several authors concluded that Daphnia magna acute test can be a useful analytical tool for early Environmental Management of Wastewater Treatment Plants – the Added Value of the Ecotoxicological Approach Environmental Management of Wastewater T t t Pl t th Add d V l f th E 419 warning system to monitor the different operational units of wastewater treatment plants (Movahedian et al., 2005; Tyagi et al., 2007) or to use in toxicity identification evaluation procedures (Hongxia et al., 2004). Also a study with a copepod as test organism showed that conventionally treated sewage effluent resulted in the most negative effects leading to the conclusion that additional treatments created effluents with less negative impacts (Lundström et al., 2010a). Fig. 4. Toxicity removal efficiency evaluated in WWTP 1, 2 and 4, for Microtox, Daphnia and ThamnoTox tests. 0 10 20 30 40 50 60 70 80 90 100 WWTP1 WWTP2 WWTP4 Toxicity removal efficiency (%) Microtox Daphnia Thamnotox Fig. 4. Toxicity removal efficiency evaluated in WWTP 1, 2 and 4, for Microtox, Daphnia and ThamnoTox tests. Though we found low sensitivity of Lemna minor in WWTP toxicity evaluation, the ecotoxicological assessment of pharmaceutical and food industries effluents using Lemna minor as a test organism was considered suitable by Radić et al. (2010) that demonstrated the relevance of Lemna as a sensitive indicator of water quality. 3. Results and discussion In nutrient rich wastewaters, although the algae test can be sensitive, it might not be the most appropriate test because of the complex relationship of inhibition and promotion of algae growth often observed (Gartiser et al., 2010a). When using the wastewater classification for the most sensitive species, in this study the bacteria V. fischeri used in the Microtox test, and considering all the WWTP under study, the distribution of toxicity level of treated samples in percentage is in accordance with the treatment process level implemented (Figure 5). For a tertiary treated effluent 100% samples are non toxic and for a preliminary treated effluent 75% are toxic. Concerning WWTP systems and considering the relative sensitivity of the organisms used in wastewater testing and the importance to consider effects at different trophic levels, the test battery proposed in a previous work (Mendonça et al., 2009) for characterization of WWTP discharges included tests with a bacterium, an alga and a crustacean to monitor this type of wastewaters. For a screening only one test with the most sensitive species, Microtox, was proposed. 420 Environmental Management in Practice Fig. 5. Distribution of treated samples according to toxicity level for the more sensitive species - Microtox, and Wastewater Treatment Plant process level. 0% 20% 40% 60% 80% 100% Preliminary Primary Secundary Tertiary Toxic Slightly toxic Non toxic 0% 20% 40% 60% 80% 100% Preliminary Primary Secundary Tertiary Toxic Slightly toxic Non toxic Fig. 5. Distribution of treated samples according to toxicity level for the more sensitive species - Microtox, and Wastewater Treatment Plant process level. Once secondary and tertiary treatment are employed, the prevention of eutrophication became the next goal for wastewater treatment, requiring the removal of nitrogen, phosphorous or both (Lofrano & Brown, 2010). On the other hand, little is known about the potential interactive effects of organic wastewater contaminants, namely steroids and hormones present in municipal effluents, when in complex mixtures that may occur in the environment and about their effect on human health (Filby et al., 2007). Chronic toxicity test and endocrine disruption assay of WWTP effluent samples indicated that, in a long term, potential population effects could arise in the receiving waters (Mendonça et al., 2009). Kontana et al. 3. Results and discussion (2008) in an ecotoxicological assessment of municipal wastewater using several test organisms including Vibrio fischeri and Daphnia magna, observed a decrease of ecotoxicological responses for all bioassays but also the induction of immune response after tertiary treatment, pointing to the need of using sensitive biomarkers if wastewaters are intended for reuse. g Considering ecotoxicity testing as an integral part of the toolbox to investigate the environmental impacts of effluents but knowing that it can be complex, time consuming and expensive, a tiered approach is recommended when defining a realistic assessment strategy (European Centre for Ecotoxicology and Toxicology of Chemicals [ECETOC], 2004; OSPAR Convention for the Protection of the marine Environment of the North-East Atlantic [OSPAR], 2007). The validity of the use of acute tests to drive environmental improvement has been demonstrated, but methodologies for chronic toxicity need further development. 5. Acknowledgment Research data were obtained under programs supported by the EU LIFE Environment Program (LIFE02 ENV/P/000416 and LIFE08 ENV/P/000237) and a contract with a public enterprise. 4. Conclusion This work shows that wastewater acute toxicity is dependent on the treatment level of the WWTP and the species tested. The bacterium Vibrio fischeri, the test organism in Microtox test, proved to be the most sensitive species in wastewater ecotoxicological evaluation. Environmental Management of Wastewater T Pl h Add d V l f h E Environmental Management of Wastewater Treatment Plants – the Added Value of the Ecotoxicological Approach 421 The distribution of treated samples according to the toxicity level to the most sensitive species clearly reveals the treatment process level implemented. All the used tests, with the exception of AlgalTox test, are able to distinguish the different levels of treatment and to assess toxicity removal efficiency. y y The ecotoxicological approach proves to have an added value to hazard and risk assessment of discharges to the receiving waters and environmental management of the Wastewater Treatment Plant can use this tool with advantages. Even if a preliminary treatment in the WWTP is associated with the discharge in a submarine outfall, environmental monitoring including toxicological parameters proves to be important. The inclusion of these ecological relevant data in the assessment of the grey water footprint for point sources of water pollution, like WWTP, can be the next step to have good indicators of the degree of water pollution. 6. References Araújo, C.V.M., Nascimento, R.B., Oliveira, C.A., Strotmann, U.J. & da Silva, E.M. (2005). The use of Microtox® to assess toxicity removal of industrial effluents from the industrial district of Camaçari (BA, Brazil), Chemosphere 58, pp. 1277-1281. Cēbere, B., Faltina, E., Zelčāns, N. & Kalnina, D. (2009). Toxicity tests for ensuring successful industrial wastewater treatment plant operation, Environmental and Climate Technologies 3, pp. 41-47. Chapman, P.M. (2007). Determining when contamination is pollution - Weight of evidence determinations for sediments and effluents, Environment International 33, pp. 492- 501. Daniel, M., Sharpe, A., Driver, J., Knight, A.W., Keenan, P.O., Walmsley, R.M., Robinson, A., Zhang, T. & Rawson, D. (2004). Results of a technology demonstration project to compare rapid aquatic toxicity screening tests in the analysis of industrial effluents, Journal of Environmental Monitoring 6, pp. 855-865. f g pp EC (2000). Water Framework Directive (WFD), Directive 2000/60/EC. (2008). Integrated Pollution Prevention and Control Directive (IPPC), Directive 2008/1/EC. ( ) g ( ), / / ECETOC (2004). Whole Effluent Assessment, Technical Report No. 94, European Centre for Ecotoxicology and Toxicology of Chemicals, Brussels, Belgium. Emmanuel, E., Perrodin, Y., Keck, G., Blanchard, J.-M. & Vermande, P. (2005). Ecotoxicological risk assessment of hospital wastewater: a proposed framework for raw effluents discharging into urban sewer network, Journal of Hazardous Materials A117, pp. 1-11. Filby, A.L., Neuparth, T., Thorpe, K.L., Owen, R., Galloway, T.S. & Tyler, C.R. (2007). Health Impacts of Estrogens in the Environment Considering Complex Mixture Effects. Environmental Health Perspectives, 115, pp. 1704-1710. 422 Environmental Management in Practice Gartiser, S., Hafner, C., Hercher, C., Kronenberger-Schäfer, K. & Paschke, A. (2010a). Whole effluent assessment of industrial wastewater for determination of BAT compliance. Part 1: paper manufacturing industry, Environmental Science and Pollution Research 17, pp. 856-865. Gartiser, S., Hafner, C., Hercher, C., Kronenberger-Schäfer, K. & Paschke, A. (2010b). Whole effluent assessment of industrial wastewater for determination of BAT compliance. Part 2: metal surface treatment industry, Environmental Science and Pollution Research 17, pp. 1149-1157. Hongxia, Y., Jing, C., Yuxia, C., Huihua, S., Zhonghai, D. & Hongjun, J. (2004). Application of toxicity identification evaluation procedures on wastewaters and sludge from a municipal sewage treatment works with industrial inputs, Ecotoxicology and Environmental Safety 57, pp.426-430. Hospido, A., Moreira, M.T., Fernández-Couto, M. & Feijoo, G. (2004). Environmental Performance of a municipal wastewater treatment plant, The International Journal of Life Cycle Assessment 9, pp. 261-271. ISO (1996). 6. References Water Quality – Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test, ISO 6341, International Standard Organization. ISO (2005). Water Quality – Determination of toxic effect of water constituents and wastewater to duckweed (Lemna minor) – Duckweed growth inhibition test, ISO 20079, International Standard Organization. Kontana, A., Yiangou, M., Papadimitriou, C.A., Samaras, P. & Zdragas, A. (2008). Bioassays and biomarkers for ecotoxicological assessment of reclaimed municipal wastewater, Water Science and Technology 57, pp. 947-953. Libralato, G., Losso, C., Arizzi Novelli, A., Avezzù, F., Scandella, A. & Volpi Ghirardini, A. (2006). Toxicity bioassays as effective tools for monitoring the performances of wastewater treatment plant technologies: SBR and UF-MBR as case studies, Proceedings of 4th MWWD and 2nd IEMES, Antalya, Turkey, 2006. Lofrano, G. & Brown, J. (2010). Wastewater management through the ages: A history of mankind, Science of the Total Environment 408, pp. 5254-5264. Lundström, E., Björlenius, B., Brinkmann, M., Hollert, H., Persson, J.-O. & Breitholtz, M. (2010a). Comparison of six sewage effluents treated with different treatment technologies – Population level responses in the harpacticoid copepod Nitroca spinipes, Aquatic Toxicology 96, pp. 298-307. Lundström, E., Adolfsson-Erici, M., Alsberg, T., Björlenius, B., Eklund, B., Lavén, M. & Breitholtz, M. (2010b). Characterization of additional sewage treatment technologies: Ecotoxicological effects and levels of selected pharmaceuticals, hormones and endocrine disruptors, Ecotoxicology and Environmental Safety 73, pp. 1612-1619. Manusadžianas, L., Balkelytė, L., Sadauskas, K., Blinova, I., Põllumaa, L. & Kahru, A. (2003). Ecotoxicological study of Lithuanian and Estonian wastewaters: selection of the biotests, and correspondence between toxicity and chemical-based indices, Aquatic Toxicology 63, pp. 27-41. Environmental Management of Wastewater Treatment Plants – the Added Value of the E Environmental Management of Wastewater Treatment Plants – the Added Value of the Ecotoxicological Approach 423 Mendonça, E., Picado, A., Paixão, S.M., Silva, L., Cunha, M.A., Leitão, S., Moura, I., Cortez, C. & Brito, F. (2009). Ecotoxicity tests in the environmental analysis of wastewater treatment plants: Case study in Portugal, Journal of Hazardous Materials 163: 665-670 Metcalf & Eddy (revised by Tchobanoglous, G., Burton, F.L. & Stensel, H.D.) (2003). Wastewater Engineering, Treatment and Reuse, 4th edition, McGraw-Hill, New York. Microbics (1992). Microtox Manual – A Toxicity Handbook, Vols.I-IV, Microbics Corporation Inc., Carlsbad, CA/USA. MicroBioTests (2003). ThamnoToxKit FTM – Freshwater Toxicity Screening Test. Standard Operational Procedure, MicroBioTests Inc., Nazareth, Belgium. MicroBioTests (2004). AlgalToxKit FTM - Freshwater Toxicity Test with Microalgae. Vindimian, E., Garric, J., Flammarion, P., Thybaud, E. & Babut, M. (1999). An index of effluent aquatic toxicity designed by partial least squares regression, using acute and chronic tests and expert judgments, Environmental Toxicology and Chemistry 18, pp. 2386-2391. USEPA (2004). NPDES Compliance Inspection Manual, Environmental Protection Agency, EPA 305-X-03-004. Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil Felipe Pombo, Alessandra Magrini and Alexandre Szklo Federal University of Rio de Janeiro, Energy Planning Program Brazil Felipe Pombo, Alessandra Magrini and Alexandre Szklo Federal University of Rio de Janeiro, Energy Planning Program Brazil 6. References Standard Operational Procedure, MicroBioTests Inc., Nazareth, Belgium. Movahedian, H., Bina, B. & Asghari, G.H. (2005). Toxicity Evaluation of Wastewater Treatment Plant Effluents Using Daphnia magna. Iranian Journal of Environmental Health, Science and Engineering 2, pp. 1-4. Muñoz, I., Gómez, M.J., Molina-Diáz, A., Huijbregts, M.A.J., Fernández-Alba, A.R. & García- Calvo, E. (2008). Ranking potential impacts of priority and emerging pollutants in urban wastewater through life cycle impact assessment, Chemosphere 74, pp. 37-44. g y p p pp OECD (1984). Alga Growth Inhibition Test. Guidelines for the testing of Chemicals, Test Guideline 201, OECD, Paris, France. OSPAR (2007). Practical Guidance Document on Whole Effluent Assessment. Ospar Comission, Publication Number 316/2007, ISBN 978-1-905859-55-9. http://www.ospar.org/documents/dbase/publications/p00316_WEA%20Guidan ce%20Document.pdf Power, E.A. & Boumphrey, R.S. (2004). International Trends in Bioassay Use for Effluent Management, Ecotoxicology 13, pp. 377-398. Radić, S., Stipaničev, D., Cvjetko, P., Mikelić, I.L., Rajčić, M.M., Širac, S., Pevalek-Kozlina, B. & Pavlica, M. (2010). Ecotoxicological assessment of industrial effluent using duckweed (Lemna minor L.) as a test organism, Ecotoxicology 19, pp. 216-222. Slooff, W. (1983). Benthic macroinvertebrates and water quality assessment: some toxicological considerations, Aquatic Toxicology 4, pp. 73-82. Teodorović, I., Bečelić, M., Planojević, I., Ivančev-Tumbas, I. & Dalmacija, B. (2009). The relationship between whole effluent toxicity (WET) and chemical-based effluent quality assessment in Vojvodina (Serbia), Environmental Monitoring and Assessment 158, pp. 381-392. Tinsley, D., Wharfe, J., Campbell, D., Chown, P., Taylor, D. & Upton, J. (2004). The use of Direct Toxicity Assessment in the assessment and control of complex effluents in the UK: a demonstration programme, Ecotoxicology 13, pp. 423-436. Tonkes, M., de Graaf, P.J.F. & Graansma, J. (1999). Assessment of complex industrial effluents in the Netherlands using a whole effluent toxicity (or WET) approach, Water Science and Technology 39, pp. 55-61. Tyagi, V.K., Chopra, A.K., Durgapal, N.C. & Kumar, A. (2007). Evaluation of Daphnia magna as an indicator of toxicity and treatment efficiency of municipal sewage treatment plant, Journal of Applied Sciences and Environmental Management 11, pp. 61-67. 424 Environmental Management in Practice USEPA (2004). NPDES Compliance Inspection Manual, Environmental Protection Agency, EPA 305-X-03-004. Vindimian, E., Garric, J., Flammarion, P., Thybaud, E. & Babut, M. (1999). An index of effluent aquatic toxicity designed by partial least squares regression, using acute and chronic tests and expert judgments, Environmental Toxicology and Chemistry 18, pp. 2386-2391. 22 1. Introduction Because of the planned expansion of Brazil’s refining capacity called for in the government’s energy policy and the scenario of stress on water resources, it is necessary to design the country’s new refineries so as to minimize water consumption and maximize reuse of effluents. Existing refineries are large water consumers. In 2009, Brazilian refineries consumed 254,093 m3 / day of water (estimated from the water consumption index of Petrobras refineries, of 0.9 m3 water/ m3 of oil) (Amorim, 2005). Empresa de Pesquisa Energética - EPE (“Energy Research Company”), a federally owned company that is part of the Ministry of Mines and Energy, forecasts an increase of 79% in Brazilian refining capacity with the construction of new refineries by 2030 (EPE, 2007). Some of them are planned for the Northeast region, which suffers from water shortage. While Brazil as a whole is blessed with water, having roughly 13% of the planet’s freshwater reserves (Mierzwa & Hespanhol, 2005), these resources are very unevenly distributed, with some regions plagued by shortages (arid and semi-arid regions) and others blessed with abundant water. Finally, although the country’s industrial heartland, the state of São Paulo, and the center of its oil industry, the state of Rio de Janeiro, both are in the country’s semi-tropical region, they still face problems of water shortages due to high demand, causing conflicts among watershed users. shortages due to high demand, causing conflicts among watershed users. The methods to reduce water consumption are conservation, recycling and reuse. Among the three, water conservation requires the least effort and investment costs. It involves the rational use of water by industry, incorporating measures to prevent physical losses and improve operations (Matsumura & Mierzwa, 2008). Recycling (with regeneration) refers to the use of treated wastewater at the place of origin. Finally, water reuse can occur in the following forms: a) direct reuse of wastewater in other processes, when the level of contamination does not interfere in the next process; and b) with regeneration, which is reuse of treated effluent in different processes than the original one (Wang & Smith, 1994). An important energy efficiency program was launched in 1992 by the U.S. Environmental Protection Agency, called Energy Star. As part of this program, a guide was issued focused on the refinery industry (Worrell & Galitsky, 2005). However, this document only covers energy use by refineries. 1. Introduction There is a need for a similar document on efficient water use by refineries. Therefore, against the backdrop depicted above of unevenly distributed and locally insufficient water resources, a technology roadmap for Brazilian refineries is important. g g g g The methods to reduce water consumption are conservation, recycling and reuse. Among the three, water conservation requires the least effort and investment costs. It involves the rational use of water by industry, incorporating measures to prevent physical losses and improve operations (Matsumura & Mierzwa, 2008). Recycling (with regeneration) refers to the use of treated wastewater at the place of origin. Finally, water reuse can occur in the following forms: a) direct reuse of wastewater in other processes, when the level of contamination does not interfere in the next process; and b) with regeneration, which is reuse of treated effluent in different processes than the original one (Wang & Smith, 1994). g g An important energy efficiency program was launched in 1992 by the U.S. Environmental Protection Agency, called Energy Star. As part of this program, a guide was issued focused on the refinery industry (Worrell & Galitsky, 2005). However, this document only covers energy use by refineries. There is a need for a similar document on efficient water use by refineries. Therefore, against the backdrop depicted above of unevenly distributed and locally insufficient water resources, a technology roadmap for Brazilian refineries is important. 426 Environmental Management in Practice There is a need to differentiate between treatment of refinery wastewater for discharge into water bodies and for reuse in other refinery units. The second case requires more advanced treatment systems, because the quality requirements are higher. Some examples of treatment techniques are reverse osmosis and reverse electrodialysis, with cost being the main barrier to widespread adoption of both (see Section 3). The first case requires more rudimentary treatment systems, as presented in Section 2. This chapter addresses the problem identified above by presenting a technology roadmap for reuse of the effluents produced by Brazilian oil refineries, which have a great need to minimize water use, a need that can be met through the significant recent technological advances. Section 2 presents the conventional treatment of refinery wastewater while Section 3 lays out the proposed technology roadmap for wastewater treatment for reuse by refineries, analyzing the following technologies: membranes, membrane bioreactors (MBRs), reverse osmosis, reverse electrodialysis, ion exchange and advanced oxidative processes. 1. Introduction Section 4 presents estimates of the costs and perspectives for application of these technologies in Brazil. Finally, Section 5 presents the conclusions of this chapter. 2. Conventional treatment of oil refinery effluents PPIs and CPIs improve the gravity separation because this process is based on the droplets reaching the continuous phase before leaving the separator, which is enhanced by increasing the specific surface area and reducing the height through which the oil droplets In general, refineries rely on the design standards of the manual entitled “Disposal of Refinery Wastes”, according to which API separators can be installed to work in series with parallel plate interceptors (PPI) or corrugated plate interceptors (CPI), the last of which are more modern and besides separating oil from the water, can also remove part of the solid material. With PPI or CPI separators, it is possible to remove oil droplets with sizes down to 75 m, representing an additional recovery of from 10 to 30 mg of oil per liter (Braile, 1979). In general, refineries rely on the design standards of the manual entitled “Disposal of Refinery Wastes”, according to which API separators can be installed to work in series with parallel plate interceptors (PPI) or corrugated plate interceptors (CPI), the last of which are more modern and besides separating oil from the water, can also remove part of the solid material. With PPI or CPI separators, it is possible to remove oil droplets with sizes down to 75 m, representing an additional recovery of from 10 to 30 mg of oil per liter (Braile, 1979). PPIs and CPIs improve the gravity separation because this process is based on the droplets reaching the continuous phase before leaving the separator which is enhanced by PPIs and CPIs improve the gravity separation because this process is based on the droplets reaching the continuous phase before leaving the separator, which is enhanced by increasing the specific surface area and reducing the height through which the oil droplets must rise before reaching the surface. Both of these devices accomplish this improvement (Jaworski, 2009). Flotation is a technique initially used in ore processing for selective separation of one type of solid from another, through the different specific gravities of the desired and undesired solids. But with recent advances, flotation devices are increasingly being used for treatment of industrial effluents. According to Rubio et al. (2002), these advances include the higher efficiency of modern equipment, new separation schemes, selective recovery of valuable ions (such as gold, palladium and silver) and lower generation of sludge. 2. Conventional treatment of oil refinery effluents To prevent the formation of very small particles that cannot be separated by this method, it is important that the wastewater in the outlet pipes and drainage systems be carefully conveyed, to avoid generating turbulence, such as that caused by pumps or sudden falls. It is also important to avoid the presence of emulsifiers (Braile, 1979). In general, refineries rely on the design standards of the manual entitled “Disposal of Refinery Wastes”, according to which API separators can be installed to work in series with parallel plate interceptors (PPI) or corrugated plate interceptors (CPI), the last of which are more modern and besides separating oil from the water, can also remove part of the solid material. With PPI or CPI separators, it is possible to remove oil droplets with sizes down to 75 m, representing an additional recovery of from 10 to 30 mg of oil per liter (Braile, 1979). PPIs and CPIs improve the gravity separation because this process is based on the droplets reaching the continuous phase before leaving the separator, which is enhanced by increasing the specific surface area and reducing the height through which the oil droplets must rise before reaching the surface. Both of these devices accomplish this improvement (Jaworski 2009) carefully conveyed, to avoid generating turbulence, such as that caused by pumps or sudden falls. It is also important to avoid the presence of emulsifiers (Braile, 1979). In general, refineries rely on the design standards of the manual entitled “Disposal of Refinery Wastes”, according to which API separators can be installed to work in series with parallel plate interceptors (PPI) or corrugated plate interceptors (CPI), the last of which are more modern and besides separating oil from the water, can also remove part of the solid material. With PPI or CPI separators, it is possible to remove oil droplets with sizes down to 75 m, representing an additional recovery of from 10 to 30 mg of oil per liter (Braile, 1979). 2. Conventional treatment of oil refinery effluents The main contaminants in wastewater from refineries are oils and greases, which can exist in three forms: free (droplets with diameters larger than 150 m), dispersed (droplets in the range of 20 to 150 m) and emulsified (droplets smaller than 20 m) (Cheryan & Rajagopalan, 1998). j g p ) The conventional methods to treat oily wastewater include (Cheryan & Rajagopalan, 1998  Gravity separation;  Dissolved air flotation;  Demulsification;  Coagulation;  Flocculation;  Biological treatment. Gravity separation is an efficient and low-cost method to remove free oil from wastewater (Cheryan & Rajagopalan, 1998). It is the first step of treatment of oily effluents at refineries. API (American Petroleum Institute) separators are the main such devices. This category also includes parallel and corrugated plate separators. The process for oil/water separation by gravity includes two mechanisms: decantation and coalescence. The first mechanism occurs according to Stokes’ Law (Equation (1)), while the second occurs through interactions at the interfaces of the dispersed oil droplets with the surrounding water (Jaworski, 2009). According to Equation (1), the smaller the oil droplet diameter, the more time it will take to separate the oil from the water. Vr = gDo2 (ρw – ρo) / 18µa (1) Vr = gDo2 (ρw – ρo) / 18µa (1) Here Vr is the velocity of rise, g is the acceleration of gravity, ρw and ρo are the density of water and oil, respectively, Do is the oil droplet diameter and µa is the absolute viscosity of water. In API separators, part of the oil accumulates at the liquid’s surface because of its lower specific gravity than water, but oil in emulsion and small oil droplets (with diameters under 150 m) are not separated. The part that rises is skimmed off, while another fraction, 427 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil consisting of oil-soaked solids, settles to the bottom of the separator, where it is also removed. To prevent the formation of very small particles that cannot be separated by this method, it is important that the wastewater in the outlet pipes and drainage systems be carefully conveyed, to avoid generating turbulence, such as that caused by pumps or sudden falls. It is also important to avoid the presence of emulsifiers (Braile, 1979). consisting of oil-soaked solids, settles to the bottom of the separator, where it is also removed. 2. Conventional treatment of oil refinery effluents Dissolved air flotation (DAF) is the technique most often used to treat industrial effluents, particularly oil refinery wastewater. It works through the formation of micro-bubbles, by pre-saturating the effluent with air at pressures of 3 to 6 atm and then rapidly reducing this pressure to 1 atm. By this process, the solution first becomes oversaturated under pressure and then when the pressure drops the air forms micro-bubbles with diameters of between 50 and 100 μm through nucleation/cavitation, rupturing the fluid’s structure (Rubio et al., 2002; Luz et al., 2002). Since oil is hydrophobic, with weak affinity for water, it tends to join with the air micro- bubbles and is carried to the top of the device. This process can be enhanced by the addition of surfactants, which work by controlling the surface properties of the oil droplets, making them more hydrophobic and easier to separate out selectively (Luz et al., 2002; Al-Shamrani et al., 2002). The most important factors in designing and dimensioning industrial DAF systems are the characteristics of the saturator, the air/solids ratio, the hydraulic discharge and the micro-bubble generation system (Luz et al., 2002). Conventional biological treatment is not able to remove all organic compounds to satisfy wastewater discharge standards. Therefore, pretreatment through biological purification is necessary. Among these processes, dissolved air flotation is the most common (Hami et al., 2007). These authors studied the effect of adding powdered activated carbon on the removal of pollutants in terms of BOD (biological oxygen demand) and COD (chemical oxygen demand) in a pilot-scale dissolved air flotation unit with a conical bottom, aiming to improve efficiency in adsorption of pollutants. They found that BOD and COD declined considerably with the addition of the activated carbon to the wastewater, and that increasing the quantity of activated carbon enhanced the pollutant removal efficiency (in %) for both BOD and COD. 428 Environmental Management in Practice Primary separators are used to break oil-water emulsions, allowing the demulsified oil to be separated from the water. Chemical methods (mainly addition of ferric and aluminum salts) are most commonly utilized. In this case, the process in general consists of rapid mixture of chemical coagulants with the wastewater, followed by flocculation and flotation/decantation. In turn, physical methods include heating, centrifugation, ultrafiltration and membrane processes (Yang, 2007). 2. Conventional treatment of oil refinery effluents There are various methods of breaking down emulsions and promoting coalescence of the oil droplets, after which they can be separated by gravity differential methods (Braile, 1979). Heating is used to reduce viscosity, accentuate density differences and weaken the interfacial films that stabilize the oil phase (Cheryan & Rajagopalan, 1998). Distillation is employed in some particularly resistant emulsions. Adjustment of the pH can destroy the protective colloid and permit sedimentation, which in some cases can also be achieved by aeration or chemical coagulation. Centrifugation increases the sedimentation force and can be used alone or together with heat or addition of chemical products. Filtration with diatomaceous earth or another element to assist filtration normally works well (Braile, 1979). In the case of chemical treatment, it is important to choose the right mixture of chemicals and optimize the process to reduce operating costs and enhance effectiveness (Cheryan & Rajagopalan, 1998). j g p ) Yang (2007) explained the mechanism of breaking down water-oil emulsions with electrochemical methods (electrochemical coagulation). This consists of an electrochemical reactor formed by iron electrodes (negative cathode and positive anode). During electrolysis, a DC voltage is applied to the electrodes, dissolving ferrous ions (Fe(II)) at the anode. These ions are in turn oxidized into ferric ions (Fe(III)), destabilizing the emulsion: Fe(s)  Fe(aq)2+ + 2e-  Fe(aq)3+ + 3e-, (2) 2H2O + 2e-  2H2(g) + 2OH(aq)-, (3) Fe(aq)3+ + 3OH(aq)-  Fe(OH)3(s). (4) (2) (3) (4) (2) (3) Fe(aq)3+ + 3OH(aq)-  Fe(OH)3(s). (4) (4) The coagulation is promoted by the addition of inorganic multivalent electrolytes, in general containing hydrosoluble cations, such as Al3+ and Fe3+ (Luz et al., 2002), and occurs when the attractive surface forces overcome the repulsive forces, allowing clots to form. The DLVO theory, named after the scientists who developed it independently (Derjaguin & Landau, 1941 and Verwey & Overbeek, 1948), explains the stability of colloid systems. This theory is based on the energy variations observed due to the aggregation of particles, considering only the Van der Waals attraction and electrostatic repulsion (Luz et al., 2002). The potential interaction energy (VT) is obtained by the balance of attractive (VA) and repulsive (VR) interactions, as shown in Equation (5), (VT = VA + VR) (5) (5) Aggregation occurs when VA>VR, while dispersion occurs when VA<VR (Luz et al., 2002). 2. Conventional treatment of oil refinery effluents In the case of two identical spherical particles, Equations (6) and (7) hold: Aggregation occurs when VA>VR, while dispersion occurs when VA<VR (Luz et al., 2002). In the case of two identical spherical particles, Equations (6) and (7) hold: VA = -Aa / 12d (6) VR = 2Пєaζ2exp (-κd), (7) VA = -Aa / 12d (6) (7) VR = 2Пєaζ2exp (-κd), 429 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil where A is the Hamaker constant, a is the radius of the particles, є is the permittivity of the solution, ζ is the zeta potential and κ is the Debye-Huckel parameter, or the inverse of the double electric layer thickness. where A is the Hamaker constant, a is the radius of the particles, є is the permittivity of the solution, ζ is the zeta potential and κ is the Debye-Huckel parameter, or the inverse of the double electric layer thickness. y Flocculation involves the addition of a polymer, called a flocculant, which promote the aggregation of fine particles to form flakes. The polymers can be classified in three ways: by origin (natural, modified or synthetic), molecular weight (low or high molecular weight) and electrical charge (neutral, anionic or cationic). The aggregates can be formed independently of the structural forces involved. The efficiency of the process depends, among other factors, on the choice of the proper flocculant, the way it is applied, the chemical environment, the system’s hydrodynamics and the sizes of the particles (Luz et al., 2002). ) Biological treatment is particularly useful to remove biodegradable organic matter from refinery wastewater. The main techniques are aerated lagoons, activated sludge and biodiscs. Aerated lagoons are artificial basins built to hold large volumes of effluents. They can be built above or below the original land surface. The aeration is not strictly natural; it is enhanced by the artificial introduction of oxygen, required by the organisms that decompose the soluble organic and fine particulate matter (Matos, 2005). In aerated lagoons, the aeration energy defines whether the liquid mass will be held in total or partial suspension. These lagoons can be classified as facultative aerated lagoons or suspension mixed lagoons. 2. Conventional treatment of oil refinery effluents Modeling these complex phenomena in advance is an important step in this sense (Piras, 1993). Fig. 1. Diagram of a conventional activated sludge process (Oever, 2005). Fig. 1. Diagram of a conventional activated sludge process (Oever, 2005). Biodiscs are cylindrical structures of plastic discs supported by a central axis. These structures are mounted horizontally above tanks so that about 30 to 40% of each disc is submersed in the liquid during rotation. The most common configuration is a disc diameter of 3.6 m (12 ft) by 8.2 m in length (27 ft). A typical biodisc system is in operation at the REFAP refinery in Brazil. It consists of four sets of discs. In the first set, the microorganisms, with adequate conditions in terms of oxygen, substrate, pH, ammonia and phosphate, attach themselves to the discs and start to grow, forming a biofilm. This biofilm uses the oxygen and organic carbon dissolved in the wastewater, removing the organic load by the action of heterotrophic bacteria. In the second set, the biofilm promotes nitrification by the action of nitrosomonas and nitrobacter bacteria. In the third set, the biofilm promotes denitrification by the action of specific bacteria. Finally, in the fourth set the process works as in the first set, to remove the residual organic load from the addition of methanol in the denitrification process (Ferreira et al., 2000). 2. Conventional treatment of oil refinery effluents In the first case, the formation and separation of biological flakes occurs in the lagoon itself, because the energy supplied to the aeration equipment is limited, ranging between 0.75 and 1.5 W/m³, which is insufficient to keep the sludge in suspension, so that solids settle in the lagoon. In the second case, the objective is to convert the soluble biodegradable organic material into biomass that can settle as sludge, which is done in secondary sedimentation ponds. The formation of biological flakes also occurs in the lagoon, but the aeration energy is greater than or equal to 3.0 W/m³, preventing the sedimentation of solids, which as stated, occurs in a secondary sedimentation pond. The removal rate in aerated lagoons is in general between 80 and 90% for total suspended solids (TSS), 65 and 80% for COD and 50 and 95% for BOD, depending on the type of setup (Matos, 2005). The activated sludge process is carried out in two main compartments: the aeration tank and the clarification tank (Figure 1). Microorganisms (specific types of bacteria) are used for biological degradation of the effluent. Some bacteria need an environment rich in oxygen (aerobic), while others need one poor in oxygen (anaerobic). The conversion products are water, carbon dioxide, nitrogen and dead microorganisms (called surplus sludge). In the aeration tank the wastewater and activated sludge are mixed so that the conversion reaction can occur. Then the activated sludge is separated out of the liquid in the other compartment by sedimentation. Most of the sludge precipitated out is returned to the aeration tank to repeat the process, but an excess portion is purged. Without this purge, the activated slugde concentration would increase too much, reducing the sedimentation efficiency in the clarification tank (Oever, 2005). In general treatment in a series of aerated lagoons is less expensive in terms of initial investment in equipment, but requires sufficient space. An activated sludge separation unit requires less area, but has higher operating cost and performs better. More demanding clean-up standards regarding removal of certain recalcitrant pollutants, mainly biomass, are only attained with units that enable extended residence times. Through knowledge of the 430 Environmental Management in Practice phenomena that govern the transformations of the biodegradable and oxidizable compounds is important to assure good performance of new biological treatment units or to improve the performance of existing ones. 3. Technological roadmap for wastewater treatment at oil refineries aiming euse The objective of this chapter is to study the best available techniques (BATs) for treatment of oil refinery wastewater for purposes of reuse. The expression “best available techniques” is defined in Section 5 of the U.S. Environmental Protection Agency Acts, 1992 and 2003, and Section 5(2) of the Waste Management Acts, 1996 to 2005, as the “most effective and advanced stage in the development of an activity and its methods of operation, which indicate the practical suitability of particular techniques for providing, in principle, the basis for emission limit values designed to prevent or eliminate or, where that is not practicable, generally to reduce an emission and its impact on the environment as a whole, where best in relation to techniques means the most effective in achieving a high general level of protection of the environment as a whole, available techniques means those techniques developed on a scale which allows implementation in the 431 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil relevant class of activity under economically the technically viable conditions, taking into consideration the costs and advantages, whether or not the techniques are used or produced within the State, as long as they are reasonably accessible to the person carrying out the activity, and techniques includes both the technology used and the way in which the installation is designed, built, managed, maintained, operated and decommissioned” (EPA, 2008). ) In designing an advanced treatment unit for secondary wastewater, the following aspects should be considered (Teodosiu et al., 1999):  complete characterization of the effluent;  the level of dissolved solids that can be reduced by coagulation-flocculation, sedimentation and/or filtration with sand, microfiltration or ultrafiltration;  the dissolved organic matter that can be removed by adsorption with activated carbon, chemical oxidation, reverse osmosis, and ultrafiltration for solid organic matter;  the dissolved solids that can be removed by reverse osmosis, ion exchange and electrodialysis;  the dissolved solids that can be removed by reverse osmosis, ion exchange and electrodialysis; y  the possibility of integrating the proposed unit with existing installations; and  the capital and operating costs. The wastewater treatment methods for reuse at refineries can be classified as primary, secondary and tertiary. The primary methods are the simplest, including techniques such as oil/water separation and dissolved air flotation. These are considered conventional treatment techniques (see Section 2). 3. Technological roadmap for wastewater treatment at oil refineries aiming euse Secondary treatment at refineries is used to remove a substantial part of the biodegradable organic matter. Tertiary treatment aims to remove ions (dissolved salts), to bring the quality up to the level required for reuse, mainly to feed cooling towers or boilers. 3.1 Membranes for micro, ultra and nanofiltration Membrane processes are used to treat stable oil/water emulsions, especially water-soluble oily wastes, rather than oil floating in unstable emulsions, for which other methods are more suitable (Cheryan, 1998, as cited in Cheryan & Rajagopalan, 1998). Membranes are effective in removing oil droplets with micrometric size, usually smaller than 10 m, and when the oil concentration is very low (Chakrabarty et al., 2008). These cases cannot be resolved by conventional techniques such as gravity separation, addition of chemical agents, thermal demulsification and biological treatment. The porous membrane matrix promotes coalescence of the micrometric and sub-micrometric oil droplets, which then can be easily removed by gravity (Hlavacek, 1995). y g y ( ) Membrane processes have several advantages, among them lower capital cost, no need to add chemicals and no subsequent generation of oily sludge (Ohya et al., 1998); operational simplicity, lower energy costs than for thermal treatment (Cheryan & Rajagopalan, 1998) and the capacity to produce a permeate with acceptable quality for discharge (Chakrabarty et al., 2008). A membrane is a barrier that serves to separate two phases by selectively restricting the passage of chemical agents. A membrane can be homogeneous or heterogeneous, with a symmetric or asymmetric structure, and solid or liquid. It can selectively conduct a positive or negative charge or be neutral or bipolar. The transport through the membrane can be by convection or diffusion of individual molecules, by induction by an electrical field or by pressure or temperature gradient. The membrane’s thickness can range from tens of microns to a few hundreds of micrometers (Ravanchi et al., 2009). 432 Environmental Management in Practice The pores of the membrane act as a physical barrier to impurities while permitting the passage of water molecules. A driving force must be applied to promote transport of the solution through the membrane. The main driving forces are pressure difference, concentration (or activity) difference – including difference in chemical potential () – or difference in electrical potential between the two sides of the membrane (Ravanchi et al., 2009). The permeate flux (Jp) and the selectivity of the membrane to a determined component of the feed solution are important properties for operation of membrane systems (Habert et al., 2006). The permeate flux in processes that use pressure difference as the driving force (microfiltration, ultrafiltration, nanofiltration and reverse osmosis) is given by Equation (8). 3.1 Membranes for micro, ultra and nanofiltration The membrane’s selective capacity can be calculated by the retention coefficient (R), defined as the fraction of the solution retained (retentate) by the membrane for a given feed concentration (Equation (9)): JP = LP (P - ), (8) (8) where LP is the hydraulic permeability (in L/m2h bar); P is the pressure difference (in bar); and  is the osmotic pressure difference (in bar). JP is therefore given in L/m2h. In turn, the retention coefficient is given by the following formula: where LP is the hydraulic permeability (in L/m2h bar); P is the pressure difference (in bar); and  is the osmotic pressure difference (in bar). JP is therefore given in L/m2h. In turn, the retention coefficient is given by the following formula: R (%) = [(Cf-Cp)/Ca] x 100, (9) (9) where Cf is the feed solute concentration (ppm) and Cp is the solute concentration of the permeate (ppm). where Cf is the feed solute concentration (ppm) and Cp is the solute concentration of the permeate (ppm). Figure 2 presents the range of pore diameters and retention efficiencies of micro, ultra and nanofiltration membranes and of the reverse osmosis process (Perry & Green, 2007). Note: RO - Reverse osmosis; NF - Nanofiltration; UF - Ultrafiltration; MF - Microfiltration. Fig. 2. Range of pore diameters and removal efficiencies of micro, ultra and nanofiltration membranes and the reverse osmosis process (Perry & Green, 2007). Note: RO - Reverse osmosis; NF - Nanofiltration; UF - Ultrafiltration; MF - Microfiltration. Fig. 2. Range of pore diameters and removal efficiencies of micro, ultra and nanofiltration membranes and the reverse osmosis process (Perry & Green, 2007). Fig. 2. Range of pore diameters and removal efficiencies of micro, ultra and nanofiltration membranes and the reverse osmosis process (Perry & Green, 2007). Fig. 2. Range of pore diameters and removal efficiencies of micro, ultra and nanofiltration membranes and the reverse osmosis process (Perry & Green, 2007). 3.1.1 Microfiltration Microfiltration and ultrafiltration membranes are used after conventional treatment techniques and as pretreatment just before reverse osmosis, to prolong the useful life of the 433 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil RO membrane and reduce incrustation (fouling) and operational costs of this process. They can also be used as part of a membrane bioreactor, to retain biomass, as will be discussed shortly. Microfiltration membranes are operated at a pressure of under 2.0 bar (Wagner, 2001). RO membrane and reduce incrustation (fouling) and operational costs of this process. They can also be used as part of a membrane bioreactor, to retain biomass, as will be discussed shortly. Microfiltration membranes are operated at a pressure of under 2.0 bar (Wagner, 2001). Fouling is one of the main problems reducing the efficiency of membrane filters. It is caused by various factors, such as clogging of the pores, adsorption of solute by the membrane and formation of a gel on the membrane surface, among others. Fouling causes a gradual decline in the permeate flux when all the other parameters are constant (pressure, flow, temperature and feed concentration). Fouling can be either reversible or irreversible. The distinction is a consequence of the characteristics of the deposit formed on the membrane surface (temporary or permanent) and the possibilities of restoring the initial flux by backwashing or chemical cleaning. It also raises operating costs because of the higher pressure required to maintain the retention rate and the need to clean the membrane or to replace it in cases of irreversible fouling (Teodosiu et al., 1999). 3.1.2 Ultrafiltration The permeate flux varied according to the chemical composition of the membrane studied, and was highest under normal pH 434 Environmental Management in Practice condition (of 6.12) in some cases, and at slightly alkaline pH (8.00) and slightly acid (5.00) conditions in other cases. condition (of 6.12) in some cases, and at slightly alkaline pH (8.00) and slightly acid (5.00) conditions in other cases. 3.1.3 Nanofiltration Nanofiltration membranes are operated at a pressure varying from 5 to 35 bar (Wagner, 2001). Nanofiltration membranes are generally used to separate multivalent ions and organic compounds with relatively low molecular weights (250 -1000 g/mol) from water. The treatment removes between 60 and 80% of the water hardness, over 90% of the color and all the turbidity (Bessarabov & Twardowski 2002). In aqueous solutions, the nanofiltration membranes become charged, permitting the separation of ionic species. It is believed that steric hindrance is the dominant retention mechanism in these membranes for colloids and large molecules, while physico-chemical interactions between the solute and membrane are more important for ions and organic materials with lower molecular weights. Figure 3 shows a hypothetical polymeric nanofiltration membrane with carboxyl groups linked at the membrane surface, which are produced in contact with an aqueous solution of an electrolyte. The presence of the carboxyl groups dissociated at the membrane surface (R-COO-) causes the occurrence of membrane charging (Bessarabov & Twardowski, 2002). Fig. 3. Hypothetical polymeric nanofiltration membrane containing carboxyl groups. The presence of these groups dissociated at the membrane surface (R-COO-) causes membrane charging. This charge repels large SO42- ions and permits the passage of smaller Cl- ions through the membrane (Bessarabov & Twardowski, 2002). Fig. 3. Hypothetical polymeric nanofiltration membrane containing carboxyl groups. The presence of these groups dissociated at the membrane surface (R-COO-) causes membrane charging. This charge repels large SO42- ions and permits the passage of smaller Cl- ions through the membrane (Bessarabov & Twardowski, 2002). Fig. 3. Hypothetical polymeric nanofiltration membrane containing carboxyl groups. The presence of these groups dissociated at the membrane surface (R-COO-) causes membrane charging. This charge repels large SO42- ions and permits the passage of smaller Cl- ions through the membrane (Bessarabov & Twardowski, 2002). 3.1.2 Ultrafiltration Ultrafiltration membranes are operated at a pressure range of 1.0 to 10.0 bar (Wagner, 2001). Teodosiu et al. (1999) studied the use of ultrafiltration (dual filtration with membranes made of polyethersulphone and polyvinylpirollidone) as pretreatment for reverse osmosis, for the purpose of recycling secondary refinery wastewater to feed cooling towers. An average efficiency of 98% was obtained for turbidity, meaning almost complete removal of suspended solids and colloids, along with efficiency of 30% for COD in the tests. In a system with ultrafiltration followed by reverse osmosis, the ultrafiltration can remove the suspended and colloidal material, bacteria, viruses and organic compounds, while the reverse osmosis removes dissolved salts, as will be discussed shortly. The quality requirements for cooling water are related to the limits established for substances that can cause scaling, corrosion, fouling and growth of microorganisms, all of which reduce the performance of cooling towers. Scaling is caused by the presence of carbonates and calcium and magnesium sulfates, which precipitate as scales in heat exchangers. Corrosion is related to the presence of large quantities of dissolved solids, including chloride and ammonia. Microorganisms grow because of the presence of high concentrations of nutrients or organic substances. And fouling occurs mainly due to the presence of high levels of suspended solids, although organic fouling via adsorption of dissolved organic compounds is also a problem (Teodosiu et al., 1999). Chakrabarty et al. (2008) used modified polysulfone membranes with the objective of attaining higher porosity and hydrophobicity through the use of additives such as polyvinylpirollidone and polyethylene glycol to remove oil from wastewater. The experiments were conducted in 12 different membranes in a semi-batch filtration cell made of Teflon. The authors evaluated the influence of feed properties such as initial oil concentration and pH of the solution on membrane performance. They concluded that these characteristics significantly affect the permeate flux and oil separation. With increasing concentration the flux diminished and the retention increased due to the formation of an oil layer on the membrane surface, leading to an increase in total resistance. With relation to pH, increased acidity or alkalinity of the feed solution caused greater oil retention for the four membranes selected in this analysis by the authors. 3.2 Membrane bioreactors (MBR) Membrane bioreactors (MBR) remove a large amount of biodegradable material (measured as BOD and COD) from oil refinery wastewater. MBR systems consist of a combination of the activated sludge biological process (see Section 2) with the membrane separation process. The reaction occurs like it does in the activated sludge process, with the added advantage of being able to operate without the need for clarification or steps like sand filtration (Melin et al., 2006). These systems use micro or ultrafiltration to separate the effluent from the activated sludge. The two main MBR configurations involve submerged or external separation membranes, as depicted in Figure 4. 435 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil Fig. 4. Configuration of MBR systems. (a) Submerged MBR. (b) Lateral flow MBR (Melin et al., 2006). Fig. 4. Configuration of MBR systems. (a) Submerged MBR. (b) Lateral flow MBR (Melin et al., 2006). The first option is more often applied to treat municipal wastewater (Melin et al., 2006), and can use both hollow fiber membranes (horizontal or vertical) and flat plate membranes (vertical). In side flow MBR systems, tubular membranes (horizontal or vertical) are placed outside the bioreactor and are fed by it. This system operates by cross-flow. Both systems are aerated at the lower part of the bioreactor, and the permeate is removed by suction (Oever, 2005). As in regular membrane processes, fouling is a problem of membrane bioreactors, by hindering the permeate flux during filtration. This problem is influenced by the characteristics of the biomass, the operating conditions and characteristics of the membrane (Chang et al., 2002). The cost of periodically replacing the membrane because of aging and fouling raises the operating costs and reduces the competitiveness of the MBR technology (Buetehorn et al., 2008). Fouling is also influenced by the hydrodynamic conditions, type of membrane and configuration of the unit, as well as by the presence of compounds with high molecular weight, which can be produced by microbial metabolism or introduced by the sludge growth process (Melin et al., 2006). Viero et al. (2008) evaluated the treatment of oil refinery wastewater using a submerged membrane bioreactor (SMBR), operating at a constant permeate flux. During the operation, high organic loading rates were applied to the unit by feeding mixtures of the effluent stream with another effluent having high phenolic strength, also generated by oil refineries. 3.3 Reverse osmosis Along with the reverse electrodialysis process, described next, reverse osmosis is used to remove ions (dissolved salts) from oil refinery wastewater, as part of the tertiary treatment cycle. Reverse osmosis is by far the most common membrane process used for desalination. It can reject nearly all the colloidal or dissolved material in an aqueous solution, producing concentrated salty water and a permeate of virtually pure fresh water. Reverse osmosis is based on the property of certain polymers called semi-permeability. While semi-permeable membranes are highly permeable to water, they have low permeability to dissolved substances. When a pressure difference is applied across the membrane, the water molecules contained in the feed stream are forced to permeate through the membrane. This pressure must be high enough to overcome the osmotic pressure working against the feed (Fritzmann et al., 2007). This pressure is generally in the range of 15 to 150 bar (Wagner, 2001). The osmosis process occurs when a semi-permeable membrane (permeable to water but not to the solute) separates the feed liquid into two aqueous solutions with different concentrations. At equal temperature and pressure on both sides of the membrane, the water will diffuse (permeate) through the membrane, resulting in an overall flow of the diluted solution to the more concentrated one until the concentrations on both sides of the membrane are equal. If the pressure differential (Δp) is greater than the osmotic pressure (Δ), the flow is reversed and the water flows from the concentrated to the diluted side. This process is called reverse osmosis. In water desalination, the feed side is operated under high pressure and the concentration of the solute on the permeate side (diluted) is negligible when compared to the feed concentration. In this case permeate flux occurs because the Δp exceeds the Δ of the feed solution (Fritzmann et al., 2007). ( ) According to Nazarov et al. (1979), reverse osmosis can be employed to desalt waste streams from crude oil electric desalting units, where the salt content of these streams is very high (above 5,000 mg/L, including 75-85% sodium chloride, 4-5% magnesium chloride and 10- 15% calcium chloride). They also mentioned the following aspects as advantages of reverse osmosis: the components (salts and water) are separated at ambient temperature without any phase conversion of the water (heating or cooling); the osmotic module is simple to design and operate; and the process can be fully automated. 3.2 Membrane bioreactors (MBR) The influence of the loading rate on the filtration was assessed, including the effects on the production of soluble microbial products (polysaccharides and proteins) and the retention of these compounds by the membrane. The membrane played a key role in the process, since it improved the COD and TOC (total organic carbon) removal efficiencies by 17 and 20%, respectively, in comparison with the results obtained with biomass alone. The authors observed that good efficiencies in removing organic matter, indicated by the COD and TOC results, were achieved considering the complexity of the wastewater stream processed. Additionally, the tested system was highly effective in removing phenols. 436 Environmental Management in Practice Scholz & Fuchsm (2000) reported tests of a MBR with high activated sludge concentration (above 48 g/L) and showed that oily wastewater also containing surfactants was biodegraded with high efficiency. During the different loading stages, a removal rate of 99.99% was attained for fuel oil as well as for lubricating oil, at a hydraulic retention time of 13.3 hours. The maximum biodegradation of the fuel oil was 0.82g of hydrocarbons degraded per day. The average removal of COD and TOC during the experiments was 94-96% for fuel oil and 98% for lubricating oil, respectively. Because of the high efficiency in removing oily pollutants and complete retention of suspended solids by the ultrafiltration system, the authors stated the MBR system has good potential for industrial applications aiming to recycle effluents. The MBR removed 93-98% of the COD and 95-98% of TOC in a hydraulic retention time of 7-14 h and oil loading rates of 3-5 g/L/day (Scholz & Fuchsm, 2000). 3.3 Reverse osmosis As disadvantages, they mentioned the difficulty of manufacturing reverse osmosis membranes, the low capacity of these membranes and the need to pre-treat the effluent to remove solid and emulsified contaminants or dissolved organic and inorganic substances. In reverse osmosis, a dynamic layer of water and solutes is formed on the membrane surface. Only molecules of a strictly determined size will penetrate through this layer and 437 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil through the membrane. Larger molecules do not pass through the membrane and are removed from the membrane surface under the influence of repulsive forces and longitudinal displacement of the liquid. Therefore, two streams are moving at the same time, a flow of liquid (water) and substances with low molecular weights through the layer of the membrane due to the difference in concentrations of these substances on either side of the membrane, and a flow of liquid and substances with high molecular weights from the surface of the membrane to within the solution, with subsequent carry-off (by purging) from the separation zone (Nazarov et al., 1979). 3.4 Reverse electrodialysis y The reverse electrodialysis process is also used to remove ions (dissolved salts) from oil refinery wastewater. Electrodialysis can be used to concentrate or remove charged species in aqueous solutions. The process is based on the movement of charged species in an electrical field. Dissolved anions, such as Cl- and NO3-, move towards the anode while cations, such as K+ and Na+, are attracted by the cathode. The movement of the ions is controlled by ion-selective membranes placed between the anode and cathode. Anion-exchange membranes (AEMs) are permeable to anions but not to cations, which are thus retained. Cation-exchange membranes (CEMs) work analogously but in the opposite way. The electrolysis device is divided into various cells by an alternating sequence of AEMs and CEMs. In this way, the concentration of ionic species is reduced in the diluted compartments and increased in the concentrate compartments. The basic unit of a device consists of a diluted compartment and a concentrate compartment in tandem (Fritzmann et al., 2007). Figure 5 shows a diagram of the reverse electrodialysis process. The electrodes’ polarity is Note: CEM - Cation Exchange Membrane; AEM - Anion Exchange Membrane. Fig. 5. The principle of reverse electrodialysis. Note: CEM - Cation Exchange Membrane; AEM - Anion Exchange Membrane. Note: CEM - Cation Exchange Membrane; AEM - Anion Exchange Membrane. Fig. 5. The principle of reverse electrodialysis. Fig. 5. The principle of reverse electrodialysis. 438 Environmental Management in Practice periodically reversed, so that the direction of the ion movement is also reversed. Therefore, the concentrate streams become diluted streams and vice versa. The periodic switching of polarity works as a self-cleaning mechanism, reducing the surface fouling of the ion- exchange membrane. Reverse electrodialysis systems are physically and chemically more durable than reverse osmosis systems and can support effluent flows with higher loads of organic matter, colloid particles and microorganisms than can reverse osmosis systems (Chao & Liang, 2008). 3.5 Ion exchange Ion exchange processes occur with the substitution of the undesirable ions of a liquid (such as wastewater) with ions like H+ and OH- from a solid material in which the ions are sufficiently mobile, usually a synthetic resin. Eventually the resin becomes exhausted and needs to be regenerated by contact with a small quantity of a solution with a high content of the desired ion. Resins can be tailored to have selective affinities for particular types of ions, such as mercury, boron, ferrous ions or copper in the presence of iron (Couper et al., 2010). An important property of these ion-exchange resins is their capacity to retain ions in their structure. There are specific resins for each ion species: cationic, with the capacity to retain cations, and anionic, specific for retention of anions. Within these two groups of resins there is a further subdivision, summarized below. Each of these is suitable to remove specific ions and has particularities in its regeneration processes (Mierzwa & Hespanhol, 2005).  Strongly acidic cation-exchange resin: This type of resin has a chemical structure formed by styrene and divinylbenzene. Its functional groups are sulfonic acid radicals (R-SO3- H+). It can operate in a broad pH range and can be conditioned to operate in a sodium or hydrogen cycle, depending on the application: water softening or demineralization.  Strongly acidic cation-exchange resin: This type of resin has a chemical structure formed by styrene and divinylbenzene. Its functional groups are sulfonic acid radicals (R-SO3- H+). It can operate in a broad pH range and can be conditioned to operate in a sodium or hydrogen cycle, depending on the application: water softening or demineralization.  Weakly acidic cation-exchange resin: Its functional groups are carboxylated (R-COOH), which are not ionized at low pH values. Therefore, this type of resin operates at pH values varying from neutral to alkaline. These resins are used to treat industrial water with high hardness, exclusively for calcium bicarbonate and carbonate.  Strongly basis anion-exchange resin: Its functional group is quaternary amine (R- N(CN3)3+). There are two sub-groups: Type I and Type II. The difference between these is basicity. Type I resins have a stronger basicity than Type II resins, and for this reason produce better output water, with less anion leakage principally of silica. 3.5 Ion exchange  Weakly basic anion-exchange resin: This type of resin is employed in water treatment systems designed mainly to remove anions from strong acids, among them chloride, sulfate and nitrate, because this type of resin is not able to remove weakly ionizable anions, among them bicarbonate and silica.  Weakly basic anion-exchange resin: This type of resin is employed in water treatment systems designed mainly to remove anions from strong acids, among them chloride, sulfate and nitrate, because this type of resin is not able to remove weakly ionizable anions, among them bicarbonate and silica. Except in very small-scale applications, ion exchangers are used in cyclical operations, involving sorption and desorption steps. A typical ion-exchange cycle used in water treatment applications involves: (a) backwashing: to remove accumulated solids and to fluidize the exchanger bed; (b) regeneration: a regenerant passes slowly through the exchanger to restore its original ionic form; (c) rinsing: water passes through the exchanger to remove the resin regenerant (in the case of porous exchangers, from the resin’s pores); and (d) loading: the solution to be treated passes through the exchanger until the leakage starts to occur. Water softening occurs in this way, with a cation-exchange column containing sodium. At the low ionic strength used in the loading step, calcium and magnesium are strongly preferred over sodium, permitting nearly all of it to be removed. 439 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil Since the selectivity for divalent cations diminishes sharply with ionic concentration, the regeneration is performed effectively with a concentrated sodium chloride solution. Removal of sulfates from boiler feed water is carried out by similar means with anion exchangers in chloride form (Perry & Green, 2007). g y Therefore, the ion-exchange process can treat effluents that contain dissolved ionic species, such as metals (Al3+, Pb2+, Sr2+, etc.), inorganic anions (F-, NO3-, SO42-, CN-, etc.) and organic acids (carboxylic, phenolic, etc.), among others. The advantages of using ion exchange are: (i) the generation of an outflow with higher quality than produced by other processes; (ii) the selective removal of undesired species; (iii) the fact that the process and equipment have been widely tested; (iv) the availability of automatic and manual systems in the market; and (v) the possibility of using them to treat small and large volumes of wastewater. 3.5 Ion exchange In contrast, the disadvantages are: (i) the chemical products involved in the regeneration process; (ii) the relatively high concentration of contaminants and other compounds in the outflow; (iii) the possibility that organic substances, microorganisms, suspended particles and other substances will degrade or reduce the treatment capacity of the resins; and (iv) the possible impairment of the process by small variations in the feed stream characteristics (Mierzwa & Hespanhol, 2005).  Termination (interaction of radicals): R· + R·  R-R (18) R· + R·  R-R (18) ·OH + ·OH  H2O2 (19) R· + R·  R-R (18) ·OH + ·OH  H2O2 (19) (19) Coelho et al. (2006) investigated the performances of various advanced oxidative processes to remove organic pollutants from sourwater from oil refineries. The preliminary experiments were conducted using the H2O2, H2O2/UV, UV, photocatalysis, ozonization, Fenton and photo-Fenton processes. Only the Fenton and photo-Fenton processes produced satisfactory results, considered to be reduction of the dissolved organic carbon (DOC) of the sourwater by 35%. For this reason, these two techniques were analyzed in more detail. Coelho et al. (2006) investigated the performances of various advanced oxidative processes to remove organic pollutants from sourwater from oil refineries. The preliminary experiments were conducted using the H2O2, H2O2/UV, UV, photocatalysis, ozonization, Fenton and photo-Fenton processes. Only the Fenton and photo-Fenton processes produced satisfactory results, considered to be reduction of the dissolved organic carbon (DOC) of the sourwater by 35%. For this reason, these two techniques were analyzed in more detail. p g p y Fenton and photo-Fenton processes. Only the Fenton and photo-Fenton processes produced satisfactory results, considered to be reduction of the dissolved organic carbon (DOC) of the sourwater by 35%. For this reason, these two techniques were analyzed in more detail. The authors observed that the photo-Fenton process was very rapid, taking only a few minutes to attain a final DOC removal of 13-27% due to the formation of iron complexes. Radiation for an additional period of 60 minutes was found to increase the DOC removal to above 87%. DOC removal rates above 75% were achieved when the reaction system was operated at hydraulic retention times longer than 85 min. In the batch experiments, the maximum DOC removal was 87%, using 4 and 0.4 g/L of ferrous sulfate and hydrogen peroxide, respectively. The Fenton reaction was also very fast, but lower DOC removal rates were attained, depending on the ferrous sulfate concentration used. The photo-Fenton process, meanwhile, was able to improve the DOC removal in a short interval (50 min), leading to high overall performance levels. The highest DOC removal (94%) was achieved with continuous operation of the combined processes (Fenton and photo-Fenton), when the system was operating with a hydraulic retention time of 1200 min.  Termination (interaction of radicals): However, appreciable DOC removal rates (greater than 75%) were also obtained with hydraulic retention times in the range of 60 to 120 min. (Coelho et al., 2006). 3.6 Advanced oxidative processes Advanced oxidative processes are characterized by producing OH radicals, an extraordinarily reactive chemical species that attacks the majority of organic molecules. These processes can be used in pre- or post-treatment in a biological process, contributing to the degradation of toxic or refractory substances (Coelho, 2004). Table 1 summarizes the existing advanced oxidative processes (Andreozzi et al., 1999). Advanced oxidative processes H2O2 / Fe2+ Fenton H2O2 / Fe3+ Fenton-like H2O2 / Fe2+ (Fe3+) / UV Photo / Fenton H2O2 / Fe3+ - Oxalate Photo / Fenton –like Mn2+ / Oxalic Acid / Ozone TiO2 / UV / O2 Photocatalysis O3 / H2O2 Peroxidation O3 / UV Oxidation by O3 / UV H2O2 / UV Oxidation by H2O2 / UV Table 1. Advanced oxidative processes (Andreozzi et al., 1999). Table 1. Advanced oxidative processes (Andreozzi et al., 1999). Some of the processes cited above are described in more detail below (Castro, 2004). The Fenton, photo-Fenton, ozone and H2O2/UV processes act in the following form (Reactions (10), (11), (12), (13), (14), respectively - Reactions (12) and (13) refer to the process with ozone): H2O2 + Fe2+  Fe3+ + OH- + ·OH (10) Fe3+ + H2O + hv  Fe2+ + ·OH + H+ (11) H2O2 + Fe2+  Fe3+ + OH- + ·OH (10) Fe3+ + H2O + hv  Fe2+ + ·OH + H+ (11) (10) (11) 440 Environmental Management in Practice O3 + OH-  ·O2 + O2 (12) O3 + H2O  2OH· + O2 (13) H2O2  (hv) 2 ·OH (14) Equations (15) to (19) represent the reactions between organic substances and the hydroxyl O3 + OH-  ·O2 + O2 (12) O3 + H2O  2OH· + O2 (13) H2O2  (hv) 2 ·OH (14) (12) (13) (14) Equations (15) to (19) represent the reactions between organic substances and the hydroxyl radical (Castro, 2004): Equations (15) to (19) represent the reactions between organic substances and the hydroxyl radical (Castro, 2004): Additi Ar-H + ·OH  Ar-OH + H· (15) (15)  Abstraction of hydrogen:  Abstraction of hydrogen: R-H + ·OH  H2O + R· (16) (16)  Electron transfer:  Electron transfer:  Electron transfer:  Electron transfer: R· + O2  ROO· (17) R· + O2  ROO· (17)  Termination (interaction of radicals):  Termination (interaction of radicals): 4. Assessment of the costs and applications in Brazil Wagner (2001) indicated the costs of membrane processes. According to him, the cost if installing a plant with spiral filters is between US$ 300 and 500 per m2 of membrane area, 441 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil while tubular systems are sold for more than US$ 1,000 per m2. The price of a flat plate system ranges from US$ 200 to 300 per m2. The cost of units with a fiber membrane configuration is US$ 1,700 per m2, while ceramic systems are the most expensive, at US$ 10,000 per m2. These costs refer to complete systems, that is, membranes, internal pipes, pumps and control equipment. Spiral membranes were originally made for water desalination, but their compact configuration and low cost make them attractive for industrial applications. Tubular systems are simple and have the advantage of tolerating suspended solids and fibers. Flat plate systems are robust and compact. Fiber membrane systems are mechanically more fragile and thus are used only for determined ultrafiltration applications and oil emulsions. Finally, ceramic systems can be very effective for microfiltration (Wagner, 2001). y y y g Depending on the capacity, a membrane filtration system can cost around US$ 1300- 5300/m3/day, while the operating costs are between US$ 0.79-3.96/m3 of treated wastewater (micro and ultrafiltration technologies) (Cheryan & Rajagopalan, 1998). g y j g Another source (CostWater, 2010) mentions the costs of membrane technology for microfiltration to remove particulate matter according to treatment capacity. According to this source, the costs are considerably lower than those mentioned above, ranging from US$ 400/m3/day for treatment capacity of 38,000 m3/day to around US$ 600/m3/day for a capacity of 3,800 m3/day. Membranes are a promising technology for treatment of refinery wastewater for reuse. They are generally used for pretreatment before reverse osmosis or reverse electrodialysis (micro and ultrafiltration technologies). Figure 6 shows a diagram of an advanced wastewater treatment system for reuse at oil refineries, considering ultra and microfiltration membranes and reverse osmosis or reverse electrodialysis as tertiary treatment. Fig. 6. Diagram of a wastewater treatment process for reuse at oil refineries (adapted from CENPES, 2004). Fig. 6. Diagram of a wastewater treatment process for reuse at oil refineries (adapted from CENPES 2004) Fig. 6. Diagram of a wastewater treatment process for reuse at oil refineries (adapted from CENPES, 2004). 4. Assessment of the costs and applications in Brazil For MBR systems, the costs of membrane bioreactors fell from US$ 400/m2 to around US$ 50/m2 in the 1990s. Figures from the EPA show an operating cost of US$ 0.47/m3 of treated wastewater, including electricity, membrane exchange and chemical inputs but not amortization of the initial outlay. Other estimates show the operating cost of MBR unit with capacity of 3,785 m3/day is around US$ 0.11-0.15 per m3 of treated effluent. These estimates are much lower than that of the EPA. The operating cost per volume treated appears not to very much with the system’s capacity (CostWater, 2010). 442 Environmental Management in Practice Oever (2005) mentioned the AirLift MBR system, which uses air to create turbulence and keep the membrane surface clean, with specific energy consumption of approximately 0.4 to 0.7 kWh/m3. This consumption would mean a cost of R$ 0.0965 to 0.1689/m3 at the industrial electricity tariff in the Southeast region of Brazil (where more than half of the country’s refining capacity is located), at the rate in effect in December 2009 (R$ 241.25/MWh) (ANEEL 2010). (US$ 1.00 = R$ 1.75228 on December 31, 2009.) Oever (2005) mentioned the AirLift MBR system, which uses air to create turbulence and keep the membrane surface clean, with specific energy consumption of approximately 0.4 to 0.7 kWh/m3. This consumption would mean a cost of R$ 0.0965 to 0.1689/m3 at the industrial electricity tariff in the Southeast region of Brazil (where more than half of the country’s refining capacity is located), at the rate in effect in December 2009 (R$ 241.25/MWh) (ANEEL 2010). (US$ 1.00 = R$ 1.75228 on December 31, 2009.) Figure 7 shows a diagram of an advanced refinery wastewater treatment system considering membrane bioreactors for secondary treatment and reverse osmosis or reverse electrodialysis as tertiary treatment (Torres at al., 2008). As can be observed, the effluent system passes through the primary treatment processes and then the oil polishing process, which can be by sand filtration, as shown in Figure 7, or nutshell filtration (for protection of the membrane) (CENPES, 2005). Nutshells are highly effective in absorbing free oil and suspended solids, due to their characteristic of promoting the coalescence of oil droplets. Activated carbon filters help remove refractory organic compounds from the biological process, allowing the treated effluent to be reused as industrial water. Note: GAC - Granulated Activated Carbon; RED - Reverse electrodialysis. Table 2. Membrane bioreactor units at refineries under construction (Santiago, 2009). Note: CENPES is a pilot project at the Petrobras Research Center. Note: * CENPES is a pilot project at the Petrobras Research Center. 4. Assessment of the costs and applications in Brazil Note: GAC - Granulated Activated Carbon; RED - Reverse electrodialysis. Fig. 7. Advanced oil refinery wastewater treatment (adapted from Torres et al., 200 Membrane bioreactors for wastewater reuse have been or are being installed at various existing Petrobras refineries or will be at new refineries. Table 2 presents these projects (Santiago, 2009). Refinery Description Capacity (m3/h) Start of Operation REVAP New Industrial Waste Treatment Station 300 2009 CENPES* New Industrial Waste Treatment Station 65 2010 REPAR New Industrial Waste Treatment Station 400 2011 COMPERJ New Refinery 1100 2012 RENEST New Refinery 600 2012 REGAP New Biological Treatment Unit 750 - LUBNOR New Industrial Waste Treatment Station 66 - 443 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil Fig. 8. Costs of wastewater treatment with ion exchange and competing technologies (Rautenbach & Melin, 2003, as cited in Fritzmann et al., 2007). Fig. 8. Costs of wastewater treatment with ion exchange and competing technologies (Rautenbach & Melin, 2003, as cited in Fritzmann et al., 2007). The capital cost of the reverse osmosis technology in Brazil is from US$1454 to 4483/m3/day, while the operating cost ranges from US$ 0.12 to 0.37/m3 (including amortization of the investment, operation and maintenance and membrane substitution) (OAS, 2010). According to Fritzmann et al. (2007), electricity consumption of the reverse osmosis process is between 0.4 and 7 kWh/m3 of treated effluent. That consumption range would mean a cost of R$ 0.0965 to 1.6888/m3, considering the industrial electricity tariff in the Southeast region of Brazil in December 2009 (R$ 241.25/MWh) (ANEEL, 2010). In treatment of refinery wastewater for reuse, reverse osmosis is used as tertiary treatment to remove ions (such as chlorides), as already mentioned. The competing technology is reverse electrodialysis. According to Fritzmann et al. (2007), electrodialysis consumes 1 kWh of electricity per m3 of treated wastewater. At the average industrial energy rate in the Southeast region in December 2009 (R$ 241.25/MWh), this would cost R$ 0.2413/m3 (ANEEL, 2010). Chao & Liang (2008) also estimated the operating cost of a mini reverse electrodialysis plant. According to their calculations, the electricity consumption by electrodes and pumps is 0.85 kWh/m3 of treated effluent, which works out to R$ 0.205/m3 at the same industrial rate applied above (ANEEL, 2010). They estimated the total operating costs, including electricity and chemicals, at US$ 0.146/m3. 4. Assessment of the costs and applications in Brazil The reverse electrodialysis process is promising for removal of ions (such as chlorides) from refinery wastewater, as mentioned. In a test program to analyze technologies to remove ions for reuse of wastewater (reverse osmosis and reverse electrodialysis) at pilot units at the REGAP refinery, reverse electrodialysis was the most promising. The advantage of this technology was its greater operational continuity. Both the reverse osmosis and reverse electrodialysis pilot plants were fed with effluent from the microfiltration unit, after filtration by activated charcoal. There was no fouling of the reverse electrodialysis 444 Environmental Management in Practice membranes, while this was a serious problem with the reverse osmosis membranes, causing increased pressure and frequent need for chemical cleaning (CENPES, 2004). With respect to the costs of the ion exchange process, Figure 8 presents these costs in comparison with those of competing technologies (Rautenbach & Melin, 2003, as cited in Fritzmann et al., 2007). ) It can be seen from the figure that for low salt concentrations, ion exchange is a competitive technology. This indicates that ion exchange can be used for the polishing step for reverse osmosis for treatment of wastewater for reuse at refineries in Brazil and the rest of the world. Table 3 shows the operating costs of treatment to remove nitrophenols with advanced oxidative processes, for 90% reduction (initial concentration of 0.04 mM) (Goi & Trapido, 2002). Treatment Process Compound [H2O2]o (mM) [Fe2+]o (mM) Energy required (kWh/m3) Energy cost (US$/m3) Total cost (US$/m3) UV 4,6-DN-0- CR 2,6-DNP 4-NP 0 0 0 0 0 0 901 658 872 63.10 46.10 61.10 63.10 46.10 61.10 UV / H2O2 4,6-DN-0- CR 2,6-DNP 4-NP 10 10 10 0 0 0 21.9 16.3 5.15 1.53 1.14 0.36 1.82 1.43 0.65 Fenton 4,6-DN-0- CR 2,6-DNP 4-NP 4 4 2 0.4 0.4 0.1 0 0 0 0 0 0 0.13 0.13 0.06 Photo- Fenton 4,6-DN-0- CR 2,6-DNP 4-NP 4 4 2 0.4 0.1 0.1 2.39 2.06 2.00 0.17 0.14 0.14 0.30 0.26 0.20 Note: DN - dinitro; CR - cresol; DNP - dinitrophenol; NP - nitrophenol. Table 3. Operating cost for treatment to remove nitrophenols with advanced oxidative Table 3. Operating cost for treatment to remove nitrophenols with advanced oxidative processes, for 90% reduction (initial concentration of 0.04 mM) (Goi & Trapido, 2002). 4. Assessment of the costs and applications in Brazil Advanced oxidative processes can be used to treat sourwater from oil refineries containing low biodegradability compounds and toxic substances that prevent use of biological treatment (Coelho et al., 2006). Sourwater is an industrial effluent that should be segregated and treated by combined processes because of its complex chemical composition (see Table 4), containing emulsified oil, phenols, sulfides, mercaptants, ammonia, cyanide and other micro-pollutants. Despite its complex composition, sourwater can be considered a candidate for industrial reuse when submitted to efficient treatment processes. It is produced when steam is injected in some refinery processing units to reduce the initial steam pressures of hydrocarbons, permitting operation at lower temperatures. After separation at the top of the tower, the sourwater can be 445 Technology Roadmap for Wastewater Reuse in Petroleum Refineries in Brazil fed into a rectification tower to remove ammonia and hydrogen sulfide. Besides being highly polluting, sourwater is very corrosive. The amount of sourwater generated by a refinery depends on various factors, including the process configuration and type and characteristics of the crude oil being processed. Typical production of sourwater at large refineries ranges from 0.2 to 0.5 m3/tonne (metric ton) of crude oil (Coelho et al., 2006). Parameter Range or average COD (mg / L) 850-1020 DOC(mg / L) 300-440 BOD5 570 Phenol (mg/L) 98-128 Ammonia (mg/L) 5.1-21.1 TSS (mg/L) n.da VSS (mg/L) n.da pH 8-8.2 Turbidity (NTU) 22-52 Sulfide (mg/L) 15-23 Toluene (μg/L) 1.1 Ethylbenzene (μg/L) 3.7 m,p-Xylene (μg/L) 15.4 o-Xylene (μg/L) 3.7 Oil and grease (mg/L) 12.7 Note: a - not detected; TSS - Total Suspended Solids; VSS – Volatile Suspended Solids. Table 4. Average characteristics of sourwater (Coelho et al., 2006). Parameter Range or average COD (mg / L) 850-1020 DOC(mg / L) 300-440 BOD5 570 Phenol (mg/L) 98-128 Ammonia (mg/L) 5.1-21.1 TSS (mg/L) n.da VSS (mg/L) n.da pH 8-8.2 Turbidity (NTU) 22-52 Sulfide (mg/L) 15-23 Toluene (μg/L) 1.1 Ethylbenzene (μg/L) 3.7 m,p-Xylene (μg/L) 15.4 o-Xylene (μg/L) 3.7 Oil and grease (mg/L) 12.7 Note: a - not detected; TSS - Total Suspended Solids; VSS – Volatile Suspended Solids. Table 4. Average characteristics of sourwater (Coelho et al., 2006). Note: a - not detected; TSS - Total Suspended Solids; VSS – Volatile Suspended Solids. Note: a - not detected; TSS - Total Suspended Solids; VSS – Volatile Suspended Solids. Table 4. Average characteristics of sourwater (Coelho et al., 2006). 5. Conclusion Despite the expensive investigations summarized here, further studies are necessary to 446 Environmental Management in Practice assess the feasibility of these technologies for large-scale application to treat oil refinery wastewater for reuse. assess the feasibility of these technologies for large-scale application to treat oil refinery wastewater for reuse. 6. Acknowledgments We thank the National Research Council (CNPq) and the Rio de Janeiro State Research Foundation (FAPERJ) for funding. We thank the National Research Council (CNPq) and the Rio de Janeiro State Research Foundation (FAPERJ) for funding. 5. Conclusion Brazil is currently expanding its refining capacity and faces problems of localized water shortage, both in semi-arid regions and intensely urbanized ones (such as the metropolitan areas of Rio de Janeiro and São Paulo), in the latter case because of high water demand. This chapter presented conventional wastewater treatment technologies and those aimed at improving the quality sufficiently for reuse. In the second case, the costs and perspectives for application in Brazil were presented. The conventional wastewater treatment methods at oil refineries include gravity separation (mainly in API separators), dissolved air flotation, demulsification, coagulation, flocculation and biological treatment (aerated lagoons, activated sludge systems and biodiscs). For treatment of refinery wastewater for reuse, the technologies include membranes (micro and ultrafiltration), membrane bioreactors (MBRs), reverse osmosis and reverse electrodialysis. These technologies work in series with conventional treatment techniques. Some technologies, not yet applied in large scale for treating refinery effluents aiming at reuse, are promising and should be tested further. Among these are nanofiltration, which presents removal efficiency levels between those of ultrafiltration and reverse osmosis; ion For treatment of refinery wastewater for reuse, the technologies include membranes (micro and ultrafiltration), membrane bioreactors (MBRs), reverse osmosis and reverse electrodialysis. These technologies work in series with conventional treatment techniques. Some technologies, not yet applied in large scale for treating refinery effluents aiming at reuse, are promising and should be tested further. Among these are nanofiltration, which presents removal efficiency levels between those of ultrafiltration and reverse osmosis; ion exchange, which as shown in this chapter would be useful for the polishing step of reverse osmosis; and advanced oxidative processes, which are effective for treating sourwater. Despite the expensive investigations summarized here, further studies are necessary to For treatment of refinery wastewater for reuse, the technologies include membranes (micro and ultrafiltration), membrane bioreactors (MBRs), reverse osmosis and reverse electrodialysis. These technologies work in series with conventional treatment techniques. Some technologies, not yet applied in large scale for treating refinery effluents aiming at reuse, are promising and should be tested further. Among these are nanofiltration, which presents removal efficiency levels between those of ultrafiltration and reverse osmosis; ion exchange, which as shown in this chapter would be useful for the polishing step of reverse osmosis; and advanced oxidative processes, which are effective for treating sourwater. 7. References Agência Nacional de Energia Elétrica - ANEEL. Retrieved on: August 26, 2010. Available from: <www.aneel.gov.br/arquivos/PDF/informacoes_gerenciais_novo.pdf>. 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( ) pp Torres, A.P.R.; Santiago, V.M.J.; Borges, C.P. (2008). Performance evaluation of submerged membrane bioreactor pilot units for refinery wastewater treatment. Environmental progress, Vol. 17, No. 2, (July 2008), pp.189-194, ISSN 1944-7442. p g ( y ) pp Viero, A.F.; Melo, T.M.; Torres, A.P.R.; Ferreira, N.R. Sant’Anna, G.L.; Borges, C.P.; Santiago, V.M.J. (2008). The effects of long-term feeding of high organic loading in a submerged membrane bioreactor treating oil renery wastewater. Journal of Membrane Science, Vol. 319, No. 1-2, (July 2008), pp. 223 – 230, ISSN 0376-7388. ( y ) pp Wagner, J. (2001). Membrane Filtration Handbook Practical tips and hints, 2nd ed., Osmonics Inc, Minnetonka. Wang, Y.P., Smith, R. (1994). Wastewater minimization. Chemical Engineering Science, Vol. 49, No. 7, (April 1994), pp. 981-1006, ISSN 0009-2509. Worrell, E., Galitsky, C. (2005). Energy Efficiency Improvement and Cost Saving Opportunities for Petroleum Refineries - An Energy Star Guide for Energy and Plant Managers, Ernest Orlando Lawrence. Berkeley National Laboratory (February 2005). Yang, C. (2007). Electrochemical coagulation for oily water demulsification. Separation Purification Technology, Vol. 54, No. 3, (May 2007), pp. 388-395, ISSN 1383-5866. Edited by Elzbieta Broniewicz Edited by Elzbieta Broniewicz In recent years the topic of environmental management has become very common. In sustainable development conditions, central and local governments much more often notice the need of acting in ways that diminish negative impact on environment. Environmental management may take place on many different levels - starting from global level, e.g. climate changes, through national and regional level (environmental policy) and ending on micro level. This publication shows many examples of environmental management. The diversity of presented aspects within environmental management and approaching the subject from the perspective of various countries contributes greatly to the development of environmental management field of research. ISBN 978-953-307-358-3 ISBN 978-953-51-6018-2 Photo by vitaga / iStock
https://openalex.org/W2131118581	https://hal.inria.fr/hal-01527643/document	English	null	Flexub: Dynamic Subscriptions for Publish/Subscribe Systems in MANETs	Lecture notes in computer science	2,012	cc-by	3,478	Flexub: Dynamic Subscriptions for Publish/Subscribe Systems in MANETs Engineer Bainomugisha1, Koosha Paridel2, Jorge Vallejos1, Yolande Berbers2, and Wolfgang De Meuter1 1Software Languages Lab, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Elsene , Brussels, Belgium. {ebainomu, jvallejo, wdmeuter}@vub.ac.be 1Software Languages Lab, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Elsene , Brussels, Belgium. {ebainomu, jvallejo, wdmeuter}@vub.ac.be 2Department of Computer Science, K.U.Leuven, Celestijnenlaan 200A, B-3001 Leuven, Belgium. {koosha.paridel, yolande.berbers}@cs.kuleuven.be Abstract. Current publish/subscribe systems provide very limited sup- port to modify subscriptions dynamically. Consequently, they cannot ef- ﬁciently control the ﬂow of events between publishers and subscribers, which may lead to unnecessary network traﬃc. In addition, it is not possible to automatically subscribe or unsubscribe to a service depend- ing on certain context of use. This implies for developers to manually manage subscriptions (e.g., taking care of when to cancel or re-issue a subscription), which may result in inappropriate subscription states (e.g., subscriptions that are cancelled too late). In this paper, we propose the concept of dynamic subscription mechanisms that improves the expres- siveness and ﬂexibility of subscriptions. We introduce a new dimension to a subscription that allows a subscriber to express the ﬂow of matched events, and when a new subscription can be (re)issued. We validate our claims for improved ﬂexibility and expressiveness by providing language abstractions and a prototype implementation of a dynamic subscription mechanism framework called Flexub that supports a variation of sub- scription mechanisms. When compared to existing subscription models, our experiment results show that the support for dynamic subscription mechanisms greatly reduces network traﬃc of events sent from publish- ers to the subscribers. In addition, our approach reduces the workload on the subscriber side. Keywords: Publish/Subscribe systems, subscription mechanisms, mo- bile ad hoc networks, context-aware systems 1 Introduction Event ﬁltering techniques and subscription models are two important areas of research for Publish/Subscribe systems [10, 3, 6, 9]. The works on event ﬁltering and subscription models propose various ways for specifying the interests of a subscriber and deciding which events the subscriber should receive. However, Bainomugisha et. al. 2 emerging mobile applications for MANETs such as context-aware applications, require more expressive subscription models, as it is desirable for subscribers to be able to dynamically modify subscriptions depending on some context condi- tions. For instance, a subscriber may need to specify that a subscription should be automatically adapted or cancelled after a certain number of matched events are received. Current solutions suﬀer from limited expressiveness and provide insuﬃcient support to modify subscriptions dynamically. While event ﬁlters [10] can be used to discard events that are of no interest to the subscriber, it is not possible to express when a subscription should be cancelled or modiﬁed. Consequently, subscribers have a limited control on the ﬂow of matched events, which may lead to unnecessary network traﬃc. Moreover, it is not possible to automatically subscribe or unsubscribe to a service depending on certain context conditions (e.g., battery level, number of required events, location, etc.). This implies for developers to manually manage subscriptions (e.g., taking care of when to cancel or re-issue a subscription), which may result in inappropriate subscription states (e.g., subscriptions that are cancelled too late). The main contributions of this paper are the following: (i) We propose a concept of dynamic subscription mechanisms that improves the expressiveness and ﬂexibility of subscriptions. To achieve this, we introduce a new dimension to a subscription that allows a subscriber to express the ﬂow of matched events, and a condition under which a new subscription should be issued or re-issued. (ii) We present a prototype extensible implementation of a framework called Flexub that supports a variation of subscription mechanisms for Publish/- Subscribe systems. (iii) We demonstrate that a variation of three subscription mechanisms can be easily expressed in Flexub: (a) continuous subscription, that allows the sub- scriber to receive all the events that match with the subscription, (b) sub- scribe once, that allows the subscriber to receive one and only one matched event, and (c) subscribe once and again, that allows the subscriber to re- subscribe, if needed, after each matching. 1 Introduction When compared to existing subscription models, our experiment results show that the support for dynamic subscription mechanisms greatly reduces network traﬃc of events sent from publishers to the subscribers. In addition, our approach reduces the workload on the subscriber side by ensuring that subscriptions that become irrelevant are cancelled as early as possible. 2 A Motivating Scenario Nowadays in public places such as an airport, there are many people who have mobile devices equipped with wireless communication capabilities. Such places create an opportunity for people to share information with each other without the need for ﬁxed infrastructure. For example, the passengers in an airport can Flexub: Dynamic Subscriptions for Publish/Subscribe Systems in MANETs 3 ﬁnd partners to share a cab with, exchange weather information, touristic tips, etc. Consider three passengers, Alice, Bob and Carol travelling through London Heathrow Airport. Alice is a passenger that is ﬂying indirectly and is waiting in the airport for transit. She has a few hours between her ﬂights and therefore, decides to ﬁnd other passengers that would like to join in for a game in the lounge. She would like to share this information and continuously get notiﬁed whenever there is an interested passenger. Bob wants to go for vacation to an island. He arrives at the airport and has two hours before his ﬂight. He is curious about the weather situation in the island. He would like to get notiﬁed only one time when there is a passenger who shares that information. Carol has just landed at the airport and wants to get a cab to her hotel. In order to minimise costs she would like to ﬁnd other passengers going to the same hotel to share a cab with. She would like to get notiﬁed whenever another person is interested in sharing until the cab’s capacity is reached. If the cab’s capacity is not yet reached, she would like to get notiﬁed once again about the next person who is interested. 2.1 Scenario Analysis The above scenario reveals interesting subscriptions that a subscriber may re- quire in order to control the ﬂow of information from the publisher to the sub- scriber. For instance, we can identify the following subscriptions: S.1 Alice needs to issue a subscription that continuously matches the publica- tions containing information about people interested in playing a game and notiﬁes her. S.2 Bob needs to issue a subscription that matches with only one publication containing weather information and notiﬁes him. S.3 Carol needs to issue a subscription that once and again matches with a publication containing information about people interested in sharing a cab and notiﬁes her. In addition, she speciﬁes a condition that the subscription should be reissued as long as the cab capacity is not yet reached. These sample subscriptions highlight the need for dynamic subscriptions i.e., subscriptions that allow the subscriber to specify the ﬂow of matched events as well as conditions under which a subscription may need to be adapted or cancelled. The current solutions suﬀer from limited expressiveness and ﬂexibility and require the programmer to formulate ad hoc implementations to manage subscriptions (e.g., taking care of when to cancel a subscription or re-issue a subscription). Managing subscriptions in an ad hoc manner is error-prone and may lead to subscriptions to be cancelled too early or too late. We further discuss existing solutions in Section 5. The next section presents our approach for expressing subscriptions with more ﬂexibility. Bainomugisha et. al. 4 3 Dynamic Subscription Mechanisms We propose the concept of dynamic subscription mechanisms that improves ex- pressiveness of subscriptions and allows the ﬂexibility to control the ﬂow of matched events. The model allows the subscriber not only to express the way events should be matched but also a means to control the ﬂow of events (e.g., a condition under which a subscription should be (re)issued or cancelled). We achieve this by introducing a new dimension of a subscription function Si to every subscription. In our model, subscription can be informally described as follows: (1) SUBSCRIBE(T, Si, u) (1) 4 Flexub In this section, we present a prototype implementation of a framework called Flexub that supports a variation of subscription mechanisms for Publish/Sub- scribe systems. 4.1 The Flexub Architecture The Flexub architecture is composed of two layers, namely, a communication middleware layer, and a language abstractions layer, that we explain below. Communication middleware layer. We chose Fadip [8] as our communica- tion middleware. Fadip is lightweight Publish/Subscribe middleware that is tailored to work in MANETs. Fadip supports a hybrid model for rout- ing by using bounded subscription propagation and message propagation at the same time. Moreover, it uses a fading gossip technique, which decreases the fanout of subscription and message propagation at each hop level. This technique ensures that there are less redundant message propagations in the network due to receiving the same message from diﬀerent routes. Language abstractions layer. The language abstractions layer provides high- level abstractions for expressing the dynamic subscription mechanisms sup- ported by Flexub. For each subscription mechanism, this layer provides the corresponding high-level language abstraction. Such high-level abstractions alleviate the programmer from the burden of manual management of sub- scription cancellation and event ﬁltering. Managing subscriptions in such an ad hoc manner is error-prone and may lead to subscriptions to be can- celled too early or too late. The details of using our language abstractions to implement the airport scenario can be found in [1]. Language abstractions layer. The language abstractions layer provides high- level abstractions for expressing the dynamic subscription mechanisms sup- ported by Flexub. For each subscription mechanism, this layer provides the corresponding high-level language abstraction. Such high-level abstractions alleviate the programmer from the burden of manual management of sub- scription cancellation and event ﬁltering. Managing subscriptions in such an ad hoc manner is error-prone and may lead to subscriptions to be can- celled too early or too late. The details of using our language abstractions to implement the airport scenario can be found in [1]. SUBSCRIBE(T, Si, u) The above deﬁnition shows a subscription to an event type T, with a subscrip- tion function Si, and an event ﬁltering function u. The subscription function (dimension) allows the subscriber to express extra speciﬁcation about the ﬂow of matched events, and a condition under which a new subscription can be is- sued or cancelled. We give a general description of the subscription dimension as follows: Si = (sm, p) (2) (2) Where sm denotes a subscription mechanism while p denotes a predicate func- tion that speciﬁes when to re-issue or cancel a subscription. A key observation here is that, introducing a new dimension to a subscription, makes it possible to express a subscription mechanism that speciﬁes the semantics of the ﬂow of matched events. So far, we have identiﬁed a variation of three subscription mechanisms: continuous subscription, subscribe once, and subscribe once and again that we explain below. Continuous subscription. In this mechanism, the subscriber continuously re- ceives all new matched events from the publishers. The current Publish/Sub- scribe systems support this kind of subscription mechanism. One drawback of this mechanism is that the publisher is always in control of the information ﬂow, which can result in the subscriber being ﬂooded with new information in case the publisher produces events at a higher rate than the subscriber can process. Subscribe once. The subscribe once mechanism enables the subscriber to reg- ister interest in receiving a single event from the publishers and the subscrip- tion is automatically cancelled as soon as the ﬁrst matched event is received. Therefore, the subscriber is in control of the information ﬂow. Subscribe once and again. This mechanism is a hybrid of the subscribe once and the continuous subscription mechanisms. The subscriber can receive one or multiple events depending on the need. Each subscription is speciﬁed with a predicate that is checked every time the subscriber receives an event to determine whether a subscription should be re-issued or automatically cancelled. The main advantage of this subscription mechanism is that the subscriber is in control and decides when to receive new events. 5 Flexub: Dynamic Subscriptions for Publish/Subscribe Systems in MANETs Flexub: Dynamic Subscriptions for Publish/Subscribe Systems in MANETs 5 4.2 Evaluation: Simulated Experiment We have performed a preliminary experiment in order to evaluate the beneﬁts of adding support for dynamic subscription mechanisms to a Publish/Subscribe system. The goal of this experiment was to investigate two properties: (i) the eﬀect of dynamic subscription mechanisms on the network traﬃc of matched events, and (ii) the workload on the subscriber side i.e., the amount of matched events that are received by a subscriber versus the amount of events that are required by the subscriber. 4.2.1 Experiment Setup We implemented Flexub on top of Fadip [8], to investigate the eﬀects of using a dynamic subscription mechanism to control the ﬂow of events. We performed the simulations using OMNeT++ [11], a component-based, open-architecture discrete event network simulator that is widely used in academia for the simu- lation of computer networks. We also used an OMNeT++ modelling framework called MiXiM to simulate wireless communication in the vehicular network. Bainomugisha et. al. 6 6 Bainomugisha et. al. Fig. 1. Network traﬃc usage by the number of nodes, with and without using Flexub Fig. 2. The number of desired events and the number of received events by sub- scribers in Fadip and in Fadip+Flexub Fig. 1. Network traﬃc usage by the number of nodes, with and without using Flexub Fig. 2. The number of desired events and the number of received events by sub- scribers in Fadip and in Fadip+Flexub We used 50 to 150 nodes in the simulations. We randomly chose 20% of these nodes as publishers and 5% of them as subscribers. The simulation runs for 50 seconds and nodes move randomly around with a speed of 2 meters per second in a playground with a size of 1500 meters by 1000 meters. With this setting, we tried to resemble the movement of people walking around with their computational devices in an airport terminal. Each subscribing node sends its subscription in the beginning of the simulation and each publishing node sends an event every 5 seconds. First, we performed the simulation without using Flexub. In this setting, the subscriptions are propagated without any ﬂow control, and they keep continuing to match and forward events. In the second setting, we use Flexub to add ﬂow control to the subscriptions. We assign a random number between 1 to 10, with a normal distribution, that indicates the number of matched events, after which the subscription is automatically cancelled. 5 Related Work In [4] the authors propose Cayuga that aims to extend the expressive power of Publish/Subscribe systems by allowing subscriptions that span multiple events and supporting parameterisation and aggregation of events. However, in Cayuga it is not possible to express dynamic subscriptions and it does not provide sup- port for specifying conditions under which a subscription can be automatically cancelled. Jayaram et. al. [7] propose parametric subscriptions in order to provide sup- port for dynamic subscription adaptations. They enrich content-based Publish/- Subscribe system with the ability for the subscriber to dynamically update its subscription without loosing any events that occur between the adaptations. Their work can be complementary to our approach in order to eﬃciently change from one subscription mechanism to another. Drosou et. al. [5] propose a preference-aware Publish/Subscribe system with a ranking mechanism based on the user preferences. Their system only delivers the top-ranked events to the subscribers, and ﬁlters out the rest. Therefore, they aim to increase the relevance of the events received by the users. In comparison with our system, they focus on the relevance of the matched events and not in controlling the ﬂow of the events. 4.2.3 Workload on the Subscribers Figure 2 illustrates the eﬀect of using Flexub on the number of events that subscribers receive, comparing to the total amount of desired matched events. The number of desired events is the sum of the required matched events for every subscriber in the network. Moreover, the number of received events is the sum of all events that a subscriber receives that can be more than what the subscribers initially asked for. As the results suggest, Flexub helps to the subscribers to receive fewer extra matching events. This reduces the workload on the subscribers. The reason is that Flexub automatically cancels the propagation of extra matched events in the early stages of their propagation. The beneﬁt of this eﬀect is reducing the processing eﬀort in the subscribers. 4.2.2 Network Traﬃc Figure 1 depicts the eﬀect of using Flexub on network traﬃc usage, with a network size of 50 nodes up to 150 nodes. We measure network traﬃc usage by counting the total number of messages that all nodes sent during the simulation time. This includes both subscription and publication messages. The results suggest that: – Flexub reduces the network traﬃc usage considerably. The reason is that in Flexub, subscriptions have a ﬂow control mechanism, and after the desired amount of matched events for a subscription has been reached, they prevents other unnecessary events to be further propagated in the network. – Flexub helps Fadip to scale better. Without Flexub, the network traﬃc usage increases exponentially by increasing the number of nodes. However, Flexub linearises this eﬀect on network traﬃc. This means that the support for dy- namic subscriptions makes a Publish/Subscribe system to be more scalable 7 Flexub: Dynamic Subscriptions for Publish/Subscribe Systems in MANETs and to work more eﬃciently with the increase in the number of nodes and messages on the network. Acknowledgements We would like to thank Pieter Mensalt for the initial experiments of subscription mechanisms in iScheme [2]. This work was partially funded by the SAFE-IS project of the Research Foundation - Flanders (FWO), the STADiUM project, and the VariBru project of the ICT Impulse Programme of the Institute for Research and Innovation. Bainomugisha et. al. For future work, we plan to investigate subscription mechanisms that reduce the workload at the publisher side as well as other intermediary nodes in a Publish/Subscribe system. 6 Conclusions and Future Work We proposed the dynamic subscription mechanisms concept that improves the expressiveness and ﬂexibility of subscriptions. Our preliminary experiments show that the adding support for dynamic subscriptions greatly reduces the network traﬃc of matched events received by subscribers. In addition, our approach re- duces the workload on the subscriber side by ensuring that subscriptions that become irrelevant are cancelled as early as possible. 8 References 1. E. Bainomugisha, K. Paridel, J. Vallejos, Y. Berbers, and W. D. Meuter. Flexub: Dynamic subscriptions for publish/subscribe systems in manets. Technical Report VUB-SOFT-TR, Vrije Universiteit Brussel, Belgium, April 2012. http://soft. vub.ac.be/~ebainomu/publications/flexub-tr.pdf. p p 2. E. Bainomugisha, J. Vallejos, E. G. Boix, P. Costanza, T. D’Hondt, and W. De Meuter. Bringing Scheme programming to the iPhone–Experience. Soft- ware: Practice and Experience, 2011. p , 3. S. Bittner and A. Hinze. Pruning subscriptions in distributed publish/subscribe systems. In Proceedings of the 29th Australasian Computer Science Conference - Volume 48, ACSC ’06, pages 197–206, Darlinghurst, Australia, 2006. Australian Computer Society, Inc. 4. A. Demers, J. Gehrke, M. Hong, M. Riedewald, and W. White. Towards expressive publish/subscribe systems. In Advances in Database Technology - EDBT 2006, volume 3896 of Lecture Notes in Computer Science, pages 627–644. Springer Berlin / Heidelberg, 2006. / 5. M. Drosou, K. Stefanidis, and E. Pitoura. Preference-aware publish/subscribe delivery with diversity. In Proceedings of the Third ACM International Conference on Distributed Event-Based Systems, page 6. ACM, 2009. 6. F. Fabret, H. A. Jacobsen, F. Llirbat, J. Pereira, K. A. Ross, and D. Shasha. Fil- tering algorithms and implementation for very fast publish/subscribe systems. In Proceedings of the 2001 ACM SIGMOD international conference on Management of data, SIGMOD ’01, pages 115–126, New York, NY, USA, 2001. ACM. f , , p g , , , , 7. K. R. Jayaram, C. Jayalath, and P. Eugster. Parametric subscriptions for content- based publish/subscribe networks. In Middleware, pages 128–147, 2010. / 8. K. Paridel, Y. Vanrompay, and Y. Berbers. Fadip: Lightweight publish/subscribe for mobile ad hoc networks. In R. Meersman, T. Dillon, and P. Herrero, editors, On the Move to Meaningful Internet Systems, OTM 2010, volume 6427 of Lecture Notes in Computer Science, pages 798–810. Springer Berlin / Heidelberg, 2010. 9. M. Petrovic, V. Muthusamy, and H. Jacobsen. Content-based routing in mobile ad hoc networks. In The Second Annual International Conference on Mobile and Ubiquitous Systems: Networking and Services, 2005., pages 45–55. IEEE, 2005. 10. S. Taherian and J. Bacon. State-ﬁlters for enhanced ﬁltering in sensor-based pub- lish/subscribe systems. In Proceedings of the International Workshop on Data In- tensive Sensor Networks (DISN’07), Mannheim, Germany, May 2007. IEEE Press. 11. A. Varga et al. The OMNeT++ discrete event simulation system. In Proceedings of the European Simulation Multiconference (ESM2001), pages 319–324, 2001.
https://openalex.org/W2031298822	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0104669&type=printable	English	null	Systemic CD8+ T Cell-Mediated Tumoricidal Effects by Intratumoral Treatment of Oncolytic Herpes Simplex Virus with the Agonistic Monoclonal Antibody for Murine Glucocorticoid-Induced Tumor Necrosis Factor Receptor	PloS one	2,014	cc-by	9,898	Abstract All relevant data a Supporting Information files. Funding: This work was supported by grants from Ministry of Education, Culture, Sports, Science and Technology of Japan (24390300 and 24800033). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by grants from Ministry of Education, Culture, Sports, Science and Technology of Japan (243 funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Specifically, the following co-authors: M. Tanaka and J. Mineno confirm that, in spite of them being employed by commercial companies Takara Bio Inc., this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials and that they are entitled and allowed to publish the results reported in this manuscript. H. Shiku is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria. * Email: seo-naohiro@clin.medic.mie-u.ac.jp (NS); shiku@clin.medic.mie-u.ac.jp (HS) oncolytic virus based on the JS-1 strain of herpes simplex virus type-1 (HSV-1) that has been engineered to express human GM- CSF [9–12]. The results of a phase III trial demonstrate that melanoma patients treated with this virus show statistically significant improvement with durable responses [12]. Mikiya Ishihara1, Naohiro Seo1, Jun Mitsui2, Daisuke Muraoka1, Maki Tanaka3, Junichi Mineno3, Hiroaki Ikeda1, Hiroshi Shiku1 Mikiya Ishihara1, Naohiro Seo1, Jun Mitsui2, Daisuke Muraoka1, Maki Tanaka3, Junichi Mineno3, Hiroaki Ikeda1, Hiroshi Shiku1 1 Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan, 2 Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Hokkaido, Japan, 3 Gene Medicine Business Unit, Takara Bio Inc., Shiga, Japan August 2014 \| Volume 9 \| Issue 8 \| e104669 Abstract Oncolytic virotherapy combined with immunomodulators is a novel noninvasive strategy for cancer treatment. In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor. Two murine tumor models were used to evaluate the therapeutic efficacies of HF10 virotherapy combined with DTA-1. The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry. Intratumoral administration of HF10 in combination with DTA-1 at a low dose resulted in a more vigorous attenuation of growth of the untreated contralateral as well as the treated tumors than treatment with either HF10 or DTA-1 alone. An accumulation of CD8+ T cells, including tumor- and herpes simplex virus type-1-specific populations, and a decrease in the number of CD4+ Foxp3+ T regulatory cells were seen in both HF10- and DTA-1-treated tumors. Studies using Fc-digested DTA-1 and Fcc receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages. These results indicated the potential therapeutic efficacy of a glucocorticoid-induced tumor necrosis factor receptor-specific monoclonal antibody in oncolytic virotherapy at local tumor sites. Citation: Ishihara M, Seo N, Mitsui J, Muraoka D, Tanaka M, et al. (2014) Systemic CD8+ T Cell-Mediated Tumoricidal Effects by Intratumoral Treatment of Oncolytic Herpes Simplex Virus with the Agonistic Monoclonal Antibody for Murine Glucocorticoid-Induced Tumor Necrosis Factor Receptor. PLoS ONE 9(8): e104669. doi:10.1371/journal.pone.0104669 Editor: Claude Krummenacher, University of Pennsylvania School of Veterinary Medicine, United States of America Received April 30, 2014; Accepted July 11, 2014; Published August 8, 2014 Copyright: 2014 Ishihara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Ishihara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Systemic CD8+ T Cell-Mediated Tumoricidal Effects by Intratumoral Treatment of Oncolytic Herpes Simplex Virus with the Agonistic Monoclonal Antibody for Murine Glucocorticoid-Induced Tumor Necrosis Factor Receptor Systemic CD8+ T Cell-Mediated Tumoricidal Effects by Intratumoral Treatment of Oncolytic Herpes Simplex Virus with the Agonistic Monoclonal Antibody for Murine Glucocorticoid-Induced Tumor Necrosis Factor Receptor HF10 Oncolytic Virotherapy with Murine GITR Targeting HF10 Oncolytic Virotherapy with Murine GITR Targeting Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a type I transmembrane protein of the tumor necrosis factor receptor family, and is involved in the regulation of T-cell receptor-mediated cell death [22]. GITR is similar to programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA- 4), both of which have been applied clinically as immune modifiers in tumor therapy [23]. GITR has been reported to be expressed at high levels on CD4+ CD25+ regulatory T (Treg) cells and to abrogate Treg cell-mediated immune suppression via intercellular signaling [24,25]. GITR has also been known to be expressed on activated CD8+ T cells and to act on the induction of tumor- specific CD8+ T cells [26]. In addition, GITR signaling via specific ligands seems to drive CD8+ T cell resistance to Treg cell- mediated inhibition [27]. Currently there is an ongoing clinical trial of a therapeutic anti-human GITR antibody [28]. Thus, GITR targeting is an attractive candidate method for use in HF10 virotherapy as it encourages tumoricidal cytotoxic T lymphocyte (CTL) activity and attenuates immune suppression. BioLegend), and anti-rat IgG2b monoclonal antibodies (mAbs) (MRG2b-85; BioLegend) as well as a FITC-conjugated rabbit anti-HSV-1 polyclonal antibody (Dako) were used in flow cytometric analysis and immunohistochemstry. An anti-mouse CD16/CD32 mAb (93; eBioscience, Inc) was used for Fc-blocking in all experiments. For in vivo administration, anti-mouse GITR mAb (DTA-1, rat IgG2b) and anti-mouse CD8a mAb were purified by protein G affinity column chromatography of ascites from BALB/c nude mice intraperitoneally inoculated with a 53- 6.7 hybridoma. Purifed rat serum IgG (Sigma) was used as the control antibody for all experiments with DTA-1. The Fab portion of DTA-1 was prepared by using a Pierce Fab Preparation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Mice To investigate DTA-1-mediated generation of tumor-specific CD8+ T cells, DTA-1 was injected i.t. into day 9 CT26/NY-ESO- 1 tumors at three doses (0.5, 2, or 10 mg). Tumor-regressed mice were selected from each DTA-1-treated group at 2 weeks after DTA-1 treatment, and the splenocytes from each group were pooled and incubated in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and 10 mg/mL of control peptide (9 m: QYIHSANVL) [32], CT26-specific AH-1 peptide (SPSYVYHQF) [33] or NY-ESO-181–88 peptide (RGPESRLL) [34] (all from MBL) for 5 hrs. The obtained cells were analyzed by flow cytometry to determine levels of intracellular IFN-c. Female BALB/c mice aged 6–8 weeks were obtained from SLC Japan. BALB/c mice deficient in the c subunit of the FccRI, FccRIII and FceRI receptors (FcRc KO mice) [29] were purchased from Taconic and bred at the Mie University Institute of Laboratory Animals. Experimental protocols were approved by the Animal Ethics Committee of Mie University, Tsu, Japan (Approval number: 23-8). Flow cytometric analysis of mAb-stained cells To confirm GITR expression on CMS5a/GITR cells, CMS5a/ GITR cells were incubated with DTA-1 (,26106 cells/mg in PBS supplemented with 0.2% BSA) for 15 min at 4uC. After washing 3 times with PBS containing 0.1% BSA, the cells were further treated with FITC-conjugated anti-rat IgG (H + L) Ab (Caltag Lab.) (,26106 cells/mg) for 15 min at 4uC. For staining of intracellular IFN-c in cultured splenic CD8+ T cells, GolgiPlug (BD Bioscience) protein transport inhibitor was added for the last 4 hrs of the incubation. The obtained cells were permeabilized using a Cytofix/Cytoperm Kit (BD Bioscience) and stained with a CD8a-specific mAb for 15 min at 4uC and followed by an IFN-c- specific mAb for 15 min at 4uC (,26106 cells/mg). For Foxp3- CD4 double labeling of TILs, TILs were first stained with a CD4- Cell preparations To collect tumor-infiltrating lymphocytes (TILs), a gentleMACS dissociator (Miltenyi Biotec K.K.) was used according to the manufacturer’s instructions with some modifications. Briefly, a CT26/NY-ESO-1 tumor cut into small pieces was incubated in 4.5 mL of RPMI-1640 medium supplemented with 1 mg/mL collagenase IA (Sigma) for 40 min at 37uC and then dissociated into single cells using the gentleMACS dissociator. DNase I was not used. The obtained cells were passed through a cell strainer (70 mm) to remove tissue aggregates. After washing 3 times with PBS containing 0.1% BSA, the TILs were evaluated by flow cytometric analysis for intracellular IFN-c as described below. When DTA-1-binding TIL populations were studied, collagenase I was not used so as to avoid the dissociation of DTA-1 bound to cells. In this study, we examined the anti-tumor effects of i.t. treatment of established murine tumors with HF10 in combination with the GITR-specific agonistic monoclonal antibody (mAb) DTA-1. Our results show that the combination therapy inhibited tumor growth at the contralateral as well as the injected tumor sites by promoting the accumulation of tumor-specific CD8+ T cells followed by DTA-1-mediated depletion of CD4+ Foxp3+ Treg cells. Thus, DTA-1 is an extremely effective partner for HF10 in oncolytic virotherapy. Cell lines CT26 is a colon tumor cell line derived from BALB/c mice [30]. A CT26 cell line transfected with the gene encoding the human cancer/testis antigen NY-ESO-1 (CT26/NY-ESO-1) was established as described previously [31]. CMS5a is a 3-methyl cholanthrene-induced fibrosarcoma cell line derived from BALB/c mice [32]. A CMS5a cell line transfected with the gene encoding GITR was established by retrovirus-mediated gene transfer. The retrovirus containing the murine GITR gene was purchased from Takara Bio Inc. Splenocytes from CT26/NY-ESO-1-bearing mice obtained at 5 days after i.t. treatment with both HF10 (days 7, 8 and 9) and DTA-1 (day 9) were cultured with CT26-specific AH-1 peptide (10 mg/mL) or HF10-infected CMS5a tumor cells [precultured with HF10 (MOI 1) for 12 hrs and irradiated with 50 Gy] at a ratio of 5 splenocytes to 1 HF10-infected CMS5a cell for 5 hrs. The obtained cells were then evaluated for intracellular IFN-c as described below. CT26/NY-ESO-1 and CMS5a cells were inoculated subcuta- neously (s.c.) into the hind flanks of mice (16106 cells/mouse and 26105 cells/mouse, respectively). HF10 or the vehicle was administered i.t. (16107 PFU/mouse) at 7, 8, and 9 days after tumor inoculation. DTA-1 was administered i.t. (10 mg/mouse) at 9 days after tumor inoculation. For the combination therapy, 10 mg of DTA-1 were mixed with the HF10 virus and administered to the mice at 9 days after tumor inoculation. Introduction Oncolytic virotherapy has existed for over 100 years and is a promising method for the treatment of cancer patients because of the strong cytolytic response of virus-infected tumor cells; however, complications may result from the use of oncolytic viruses including toxicity against normal cells [1–3]. Thus, artificially modified oncolytic viruses have been engineered to achieve low toxicity against normal tissues together with sufficient antitumor activity. Oncolytic viruses that have been modified to express human cytokines, such as granulocyte macrophage colony- stimulating factor (GM-CSF) have the potential for future therapeutic use in the treatment of solid tumors. JX-594 is a GM-CSF-armed oncolytic poxvirus that has shown promising outcomes when administered by either intratumoral (i.t.) injection or intravenous (i.v.) infusion [4–8]. OncoVEXGM-CSF is an HSV infection in wide ranges of cell populations results in degenerative change and death [13]. HF10 is a spontaneous mutant of HSV-1 strain HF [14] that lacks neuroinvasiveness and is at least 10,000-fold less virulent than wild-type HSV-1 in mice [15]. In several clinical studies of cancer patients, HF10 has been shown to have antitumor effects [16–19]. In murine studies, HF10 packaged with a GM-CSF-expressing amplicon has been reported to exhibit more tumoricidal activity than intact HF10 [20,21], supporting the hypothesis that HF10 exhibits maximal antitumor activity when used in combination with immunomodulators. August 2014 \| Volume 9 \| Issue 8 \| e104669 1 PLOS ONE \| www.plosone.org Immunohistochemistry Frozen CT26/NY-ESO-1 tumor specimens embedded in O.C.T compound (Sakura Finetechnical) were sectioned at a thickness of 3 mm, air dried for 2 hrs, fixed with cold acetone for 15 min, and then processed for immunohistochemistry. After washing 3 times with PBS, the slides were incubated at 4uC in blocking solution [PBS supplemented with 1% BSA, 5% Blocking One Histo (Nacalai Tesque, Inc.)] and 0.2 mg/mL anti-mouse CD16/CD32 mAb for 30 min. The tumor sections on the slides were then dual-labeled with PE-conjugated mAb and FITC- conjugated mAb diluted with PBS supplemented with 1% BSA and 5% Blocking One Histo for 1 hr at room temperature (r.t.) in a humidified chamber. After washing 3 times with PBS supplemented with 0.02% Tween-20, the slides were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen, Life Technologies, Inc.), and evaluated by fluorescence microscopy (BX53F; Olympus Co. Ltd.; Tokyo, Japan). The photographs from PE-, FITC-, and DAPI-stained tissue sections were merged and background fluorescence was deleted using Photoshop elements software (Adobe Systems Software Ltd.). HF10 Oncolytic Virotherapy with Murine GITR Targeting HF10 Oncolytic Virotherapy with Murine GITR Targeting Figure 1. Tumor growth following intratumoral administration of HF10 with/without DTA-1. Growth (mm2) of subcutaneously inoculated CT26/NY-ESO-1 (A) or CMS5a cells (B) following i.t. treatment with HF10 (open triangles) and/or DTA-1 (closed triangles) on indicated days. Purified rat IgG was used as a control for DTA-1. Data shown in Fig. 1 are representative of four independent experiments. By the Kruskal-Wallis ANOVA test, the CT26/NY-ESO-1 growth inhibition by combined treatment with HF10 and DTA-1 was significantly different from the HF10-treated or untreated control at day 42. CMS5a growth inhibition by the HF10 and DTA-1 combination was significantly different from the DTA-1-treated or untreated control at day 25. CMS5a growth inhibition was also significantly different in the HF10-treated group compared with the untreated control. doi:10.1371/journal.pone.0104669.g001 specific mAb [15 min, 4uC (,26106 cells/mg)], then fixed and permeabilized using a Foxp3 staining kit (eBioscience, Inc.), and then treated with a Foxp3-specific mAb [30 min, 4uC (,16106 cells/mg)]. The labeled cells were then analyzed by flow cytometry (FACSCanto II: BD Bioscience) with FlowJo software (Tomy Digital Biology). diacetate succinimidyl ester (CFSE) at 37uC for 6 min to be used as targets in the ADCC assay. After washing 3 times with RPMI- 1640 supplemented with 10% FCS, CFSE-labeled CMS5a or CMS5a/GITR cells were plated at various effector-to-target ratios with rat IgG or DTA-1 (2 mg/mL), incubated for 12 hrs, and analyzed by flow cytometry. For each sample, 20,000 non-CFSE labeled cells were collected, and the absolute number of CFSE- labeled surviving cells was counted. The survival percentage was calculated as the mean number of each of the three wells as follows: [(absolute number of surviving CFSE-labeled cells in control rat IgG-containing medium)2(absolute number of surviv- ing CFSE-labeled cells in DTA-1-containing medium)]6100/ (absolute number of surviving CFSE-labeled cells in control rat IgG-containing medium). Statistical analysis The Mann-Whitney U test was used to compare data from two groups. When equality of variance was proven by Levine’s test, data comparison between 2 groups was evaluated by Student’s t- test. The Kruskal-Wallis ANOVA test was used to compare data from four groups. p-values below 0.05 were considered statistically significant. Calculations were performed using SPSS Statistics v21.0 software (IBM). Effective inhibition of tumor growth by local treatment of HF10 combined with DTA-1 For hematoxylin and eosin (HE) staining, slides with acetone- fixed tissue sections were washed 3 times with PBS and incubated at r.t. in hematoxylin solution (New Hematoxylin Sol.: Muto Pure Chemicals Co., Ltd.) for 5 min. After washing with tap water, the cytoplasm was stained with eosin (r.t. for 2 min.; Pure Eosin Sol.: Muto Pure Chemicals). Samples were then dehydrated 3 times with xylene, and the slides were mounted with Malinol (Muto Pure Chemicals) and evaluated by microscopy. Since the therapeutic efficacy of the adjuvants included in immune-targeting Abs has been widely shown in the treatment of cancer, we hypothesized that the use of DTA-1, as an enhancer of tumor-specific CD8+ T cell responses [26,27] in HF10 virotherapy might produce a satisfactory treatment outcome. To investigate this hypothesis, we used human NY-ESO-1 gene-transfected CT26 tumor cells (CT26/NY-ESO-1) for in vitro and in vivo studies as an H-2Dd-restricted murine CTL epitope of NY-ESO-1 had been identified in our laboratory [31]. Subcutaneously inoculated CT26/NY-ESO-1 tumors were treated by i.t. admin- istration of HF10 with or without DTA-1 (Fig. 1A). Groups treated with either HF10 or DTA-1 showed weak suppression of tumor growth compared with the untreated group (control), and 2 out of 13 mice (15.4%) or 3 out of 19 mice (15.8%) showed Antibodies Fluorescein isothiocyanate (FITC)-conjugated and/or phycoer- ythrin (PE)-conjugated anti-mouse CD4 (RM4-5; eBioscience, Inc), anti-mouse CD8a mAb (53-6.7; BD Bioscience), anti-mouse/ rat Foxp3 mAb (FJK-16s; eBioscience, Inc), anti-mouse IFN-c mAb (XMG1.2; eBioscience, Inc), anti-mouse F4/80 mAb (BM8; PLOS ONE \| www.plosone.org August 2014 \| Volume 9 \| Issue 8 \| e104669 2 HF10 Oncolytic Virotherapy with Murine GITR Targeting y py g PLOS ONE \| www.plosone.org 3 August 2014 \| Volume 9 \| Issue 8 \| e10 PLOS ONE \| www.plosone.org 3 August 2014 \| Volume 9 \| Issue 8 \| e104669 August 2014 \| Volume 9 \| Issue 8 \| e104669 August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 HF10 Oncolytic Virotherapy with Murine GITR Targeting HF10 Oncolytic Virotherapy with Murine GITR Targeting Figure 2. Kinetics of CD8+ T cells after the combination therapy with HF10 and DTA-1. CT26/NY-ESO-1 tumor sections from untreated mice (control) or mice injected i.t. with DTA-1, HF10, or HF10 combined with DTA-1 were stained with hematoxylin and eosin (A), and phycoerythrin (PE)-conjugated anti-CD8a mAb and DAPI (B). (C) Frozen sections of CT26/NY-ESO-1 tumors from mice i.t. injected with HF10 combined with DTA-1 were stained with a PE-anti-CD8a monoclonal antibody, a fluorescein isothiocyanate (FITC)-anti-HSV-1 polyclonal antibody, and DAPI. (D) CT26/NY- ESO-1 growth (mm2) in both i.t. HF10- and DTA-1-treated control or CD8+ cell-depleted mice was measured. Seven mice per group were used. (E) Bilateral CT26/NY-ESO-1-bearing mice were treated with a combination of HF10 and DTA-1 in the tumors on the right flanks. Subsequent tumor growth (mm2) of the treated right and contralateral left sites was measured. Tumor growth in untreated mice was measured and used as a control. Fourteen mice per group were used. By the Kruskal-Wallis ANOVA test, CT26/NY-ESO-1 growth inhibition by the combined HF10 and DTA-1 treatment in contralateral as well as treated sites was significantly different from the untreated control group. (F) CT26/NY-ESO-1 tumors from one side of bilateral tumor-bearing mice were treated i.t. with HF10 combined with DTA-1. Frozen sections of contralateral CT26/NY-ESO-1 tumors were stained with a PE-anti-CD8a monoclonal antibody, a fluorescein isothiocyanate (FITC)-anti-HSV-1 polyclonal antibody, and DAPI. doi:10.1371/journal.pone.0104669.g002 a CT26-specific AH-1 peptide or an NY-ESO-1 81–88 peptide to expand each population of peptide-specific CD8+ T cells. As shown in Figure 3A, the response of CT26-specific IFN-c- producing CD8+ T cells was enhanced by DTA-1 in a dose- dependent manner. In addition, CD8+ T cells with NY-ESO-1 specificity were observed when DTA-1 was administered at a high dose (10 mg). In this experiment, we used tumor-regressed mice because we could not enhance the negligible CT26-specific IFN-c- producing CD8+ T cell responses seen in tumor-bearers in a DTA- 1 dose-dependent manner. Although CT26/NY-ESO-1 growth after i.t. treatment with DTA-1 was suppressed compared with the control group, tumor size was not different in each group of DTA- 1 (0.5, 2.0, or 10.0 mg/mouse). Furthermore, HF10-specific CD8+ T cells were found in addition to the AH-1-specific population (Fig. 3B left) when splenocytes from both HF10- and DTA-1- treated CT26/NY-ESO-1-bearing mice with HF10-infected CMS5a cells were cultured (Fig. 3B right). These results indicated that i.t. HF10 Oncolytic Virotherapy with Murine GITR Targeting treatment of HF10 and DTA-1 had the capacity to enhance tumor- and HF10-specific CD8+ T cell populations. complete tumor regression at 42 days after tumor inoculation, respectively (Table 1). In contrast, all mice in the group treated with both HF10 and DTA-1 showed statistically significant attenuation of tumor growth compared with the control group [p,0.001, Fig. 1A; complete tumor regression rate at 42 days: 60.0% (12 to 20 mice); Table 1]. In addition, CMS5a tumor growth in the group treated with both HF10 and DTA-1 was also suppressed significantly compared with the control or DTA-1- treated groups (Fig. 1B and Table 1). complete tumor regression at 42 days after tumor inoculation, respectively (Table 1). In contrast, all mice in the group treated with both HF10 and DTA-1 showed statistically significant attenuation of tumor growth compared with the control group [p,0.001, Fig. 1A; complete tumor regression rate at 42 days: 60.0% (12 to 20 mice); Table 1]. In addition, CMS5a tumor growth in the group treated with both HF10 and DTA-1 was also suppressed significantly compared with the control or DTA-1- treated groups (Fig. 1B and Table 1). We observed CT26/NY-ESO-1-regressed mice in another experiment for 2 months after HF10 and DTA-1 treatment. Tumor recurrence could not be seen in the tumor-regressed mice. In addition, these mice exhibited the resistance in tumor re- challenging. In addition, these mice exhibited the resistance in tumor re- challenging. Disappearance of tumor-infiltrating Foxp3+ cells after the treatment with DTA-1 in HF10 therapy We hypothesized that the increase in tumor-specific CD8+ T cell responses after DTA-1 treatment combined with HF10 therapy was involved in the attenuation and/or depletion of immune suppressors including Treg cells. To address this issue, CT26/NY-ESO-1 tumors obtained after DTA-1 treatment with or without HF10 were evaluated by immunohistological staining of tissue sections and flow cytometric analysis of infiltrating lymphocytes using a Foxp3-specific mAb. Foxp3+ cells accumu- lated abundantly in both untreated and HF10-treated tumors, whereas a vigorous decrease in the number of Treg cells was shown in tumors following treatment with DTA-1 alone or DTA-1 combined with HF10 (Fig. 4A). This result was confirmed by flow cytometric analysis of tumor-infiltrating Treg cells. The frequency of tumoral CD4+ Foxp3+ Treg cells from the HF10- and DTA-1- treated group was decreased significantly compared with that from the untreated (control) group but not from the HF10- or DTA-1- treated mice (Fig. 4B). The decrease in the frequency of Foxp3+ cells in the HF10-treated group (Fig. 4B) is possibly correlated with the decrease in tumor size due to HF10 treatment. This may be attributed to the lack of modulation of the absolute number of Foxp3+ cells in the HF10-treated group unlike in the control group (Fig. 4A). DTA-1 is a rat IgG2b class mAb. By visualizing DTA-1 with the FITC-conjugated anti-rat IgG2b mAb, it was demon- strated that the tumor-infiltrating CD4+ Foxp3+ Treg population bound predominantly with DTA-1 at 6 hrs after i.t. injection (Fig. 4C), in parallel with the disappearance of tumoral Treg cells after treatment with DTA-1. Taken together, these results strongly indicate that DTA-1 was essential to the decrease in the number of CD4+ Foxp3+ cells. CD8+ T cells act as tumoricidal effectors in the combination therapy of HF10 with DTA-1 py Intratumoral injection of HF10 resulted in the collapse of tumor structure with a decrease in the nuclear density of tumor cells, as shown at 7 days after the last HF10 treatment in both the group treated with HF10 and that treated with HF10 and DTA-1 (Fig. 2A). Tumor-infiltrating CD8+ T cells were shown to be the most frequent population after the administration of HF10 combined with DTA-1 at 3 days after the final treatment (Fig. 2B). Importantly, these cells appeared to accumulate near HF10- infected tumor areas (Fig. 2C and S1A), suggesting that HF10 infection is able to attract CD8+ T cells by leaking virus-associated proteins and tumor antigenic proteins from infected tumor cells and changing the tumor microenvironment after oncolysis. Inhibition of CT26/NY-ESO-1 growth by the combination therapy was completely negated by depleting CD8+ cells by intravenous treatment with a murine CD8a-specific mAb (Fig. 2D), indicating that the tumor-infiltrating CD8+ T cells shown in Figure 2B and 2C include tumoricidal effector popula- tions. In the study using bilateral tumor-bearing mice, tumor growth inhibition by HF10 combined with DTA-1 occurred not only in the treated tumors but also in the contralateral non-treated tumors (Fig. 2E and S1B). In addition, the sections of contralateral tumor showed infiltrating CD8+ T cells without HF10 infection (Fig. 2F and S1C). These results indicate that CD8+ T cells activated in a local tumor site under the influence of HF10 and DTA-1 participate in systemic surveillance and could attack distant tumors without tissue destruction due to HF10 infection. Antibody-dependent cellular cytotoxicity (ADCC) assay Antibody-dependent cellular cytotoxicity (ADCC) assay RAW264.7 cells were activated with 20 ng/mL murine IFN-c for 24 hrs in 24-well plates, after which the cells were gently washed with RPMI-1640 and the dish-adherent RAW264.7 cells were used as effectors in the ADCC assay. CMS5a or CMS5a/ GITR cells were labeled with 2.5 mM carboxyfluoresceine Table 1. Increase in the number of tumor-regressed mice by HF10 therapy combined with DTA-1. Table 1. Increase in the number of tumor-regressed mice by HF10 therapy combined with DTA-1. Treatment Number of complete tumor-regressed mice/Number of treated mice (%) CT26/NY-ESO-1 CMS5a Cont. IgG 0/10 (0.0%) 0/13 (0.0%) DTA-1 2/13 (15.4%) 0/13 (0.0%) HF10 3/19 (15.8%) 0/13 (0.0%) HF10+DTA-1 12/20 (60.0%) 3/13 (23.1%) Number of complete tumor-regressed mice was counted at 42 or 25 days after subcutaneous inoculation of CT26/NY-ESO-1 or CMS5a tumor cells, respectively. doi:10.1371/journal.pone.0104669.t001 PLOS ONE \| www.plosone.org 4 August 2014 \| Volume 9 \| Issue 8 \| e104669 4 HF10 Oncolytic Virotherapy with Murine GITR Targeting August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Augmentation of tumor-specific CD8+ T cell responses by DTA-1 treatment in HF10 therapy Next, we investigated whether the tumor- or HF10-specific CD8+ T cell response was enhanced in CT26/NY-ESO-1-bearing mice by i.t. treatment with DTA-1 alone or HF10 combined with DTA-1. To detect low proportions of CD8+ T cells with tumor specificity, spleen cells from tumor-regressed mice selected after i.t. treatment with DTA-1 at the indicated doses were stimulated with August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 6 HF10 Oncolytic Virotherapy with Murine GITR Targeting Figure 3. Generation of tumor- and HF10-specific CD8+ T cells by intratumoral treatment of DTA-1 and HF10 combined with (A) CT26- and NY-ESO-1-specific CD8+ T cell responses in CT26/NY-ESO-1-regressed mice by i.t. treatment of DTA-1 at indicated doses were by intracellular staining of IFN-c in splenocytes cultured with 10 mg/mL of the indicated peptides for 5 hrs. Splenocytes from two mice p were pooled and assessed. (B) Splenocytes from untreated and both i.t. HF10- and DTA-1-treated CT26/NY-ESO-1-bearing mice were obta days after final treatment, and cultured with AH-1 peptide (10 mg/mL) or HF10-infected CMS5a tumor cells for 5 hrs. Splenocytes from ten group were pooled and assessed. The obtained cells were immunohistologically stained for intracellular IFN-c. The 9 m peptide and u CMS5a cells were used as controls. doi:10.1371/journal.pone.0104669.g003 Figure 3. Generation of tumor- and HF10-specific CD8+ T cells by intratumoral treatment of DTA-1 and HF10 combined with DTA-1. (A) CT26- and NY-ESO-1-specific CD8+ T cell responses in CT26/NY-ESO-1-regressed mice by i.t. treatment of DTA-1 at indicated doses were assessed by intracellular staining of IFN-c in splenocytes cultured with 10 mg/mL of the indicated peptides for 5 hrs. Splenocytes from two mice per group were pooled and assessed. (B) Splenocytes from untreated and both i.t. HF10- and DTA-1-treated CT26/NY-ESO-1-bearing mice were obtained at 5 days after final treatment, and cultured with AH-1 peptide (10 mg/mL) or HF10-infected CMS5a tumor cells for 5 hrs. Splenocytes from ten mice per group were pooled and assessed. The obtained cells were immunohistologically stained for intracellular IFN-c. The 9 m peptide and uninfected CMS5a cells were used as controls. doi:10.1371/journal.pone.0104669.g003 Figure 3. Generation of tumor- and HF10-specific CD8+ T cells by intratumoral treatment of DTA-1 and HF10 combined with DTA-1. (A) CT26- and NY-ESO-1-specific CD8+ T cell responses in CT26/NY-ESO-1-regressed mice by i.t. Augmentation of tumor-specific CD8+ T cell responses by DTA-1 treatment in HF10 therapy (A) CT26/NY-ESO-1 tumor sections form untreated group (control) or group from mice injected i.t. with DTA-1, HF10, or HF10 combined with DTA-1 were stained with FITC-anti-Foxp3 mAb and DAPI. (B) The frequency of tumor-infiltrating CD4+ Foxp3+ Treg cells from mice injected i.t. with HF10, DTA-1, or HF10 combined with DTA-1 at 12 days after CT26/NY-ESO-1 inoculation was assessed by flow cytometry. Data from 4 individual experiments were analyzed statistically. Kruskal-Wallis ANOVA test was used to compare data from the 4 groups. The decrease in the frequency of tumoral CD4+ Foxp3+ Treg cells in the HF10 and DTA-1 combined treatment group was significantly different from untreated control, but not from the HF10- or DTA-1-treated group (N.S: Not significant). (C) At 6 hrs after DTA-1 injection into day 9 CT26/NY-ESO-1 tumors, tumor-infiltrating cells collected under collagenase-free conditions were analyzed by flow cytometry after staining with FITC-labeled anti-rat IgG2b mAb to detect DTA-1- bound cells. d i 10 1371/j l 0104669 004 doi:10.1371/journal.pone.0104669.g004 Augmentation of tumor-specific CD8+ T cell responses by DTA-1 treatment in HF10 therapy treatment of DTA-1 at indicated doses were assessed by intracellular staining of IFN-c in splenocytes cultured with 10 mg/mL of the indicated peptides for 5 hrs. Splenocytes from two mice per group were pooled and assessed. (B) Splenocytes from untreated and both i.t. HF10- and DTA-1-treated CT26/NY-ESO-1-bearing mice were obtained at 5 days after final treatment, and cultured with AH-1 peptide (10 mg/mL) or HF10-infected CMS5a tumor cells for 5 hrs. Splenocytes from ten mice per group were pooled and assessed. The obtained cells were immunohistologically stained for intracellular IFN-c. The 9 m peptide and uninfected CMS5a cells were used as controls. doi:10.1371/journal.pone.0104669.g003 Figure 3. Generation of tumor- and HF10-specific CD8+ T cells by intratumoral treatment of DTA-1 and HF10 combined with DTA-1. (A) CT26- and NY-ESO-1-specific CD8+ T cell responses in CT26/NY-ESO-1-regressed mice by i.t. treatment of DTA-1 at indicated doses were assessed by intracellular staining of IFN-c in splenocytes cultured with 10 mg/mL of the indicated peptides for 5 hrs. Splenocytes from two mice per group were pooled and assessed. (B) Splenocytes from untreated and both i.t. HF10- and DTA-1-treated CT26/NY-ESO-1-bearing mice were obtained at 5 days after final treatment, and cultured with AH-1 peptide (10 mg/mL) or HF10-infected CMS5a tumor cells for 5 hrs. Splenocytes from ten mice per group were pooled and assessed. The obtained cells were immunohistologically stained for intracellular IFN-c. The 9 m peptide and uninfected CMS5a cells were used as controls. doi:10.1371/journal.pone.0104669.g003 August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 7 HF10 Oncolytic Virotherapy with Murine GITR Targeting Figure 4. Disappearance of DTA-1-conjugated tumor-infiltrating CD4+ Foxp3+ cells after combined i.t. treatment with HF10 and DTA-1. (A) CT26/NY-ESO-1 tumor sections form untreated group (control) or group from mice injected i.t. with DTA-1, HF10, or HF10 combined with DTA-1 were stained with FITC-anti-Foxp3 mAb and DAPI. (B) The frequency of tumor-infiltrating CD4+ Foxp3+ Treg cells from mice injected i.t. with HF10, DTA-1, or HF10 combined with DTA-1 at 12 days after CT26/NY-ESO-1 inoculation was assessed by flow cytometry. Data from 4 individual experiments were analyzed statistically. Kruskal-Wallis ANOVA test was used to compare data from the 4 groups. The decrease in the frequency of tumoral CD4+ Foxp3+ Treg cells in the HF10 and DTA-1 combined treatment group was significantly different from untreated control, but not from the HF10- or DTA-1-treated group (N.S: Not significant). Augmentation of tumor-specific CD8+ T cell responses by DTA-1 treatment in HF10 therapy (C) At 6 hrs after DTA-1 injection into day 9 CT26/NY-ESO-1 tumors, tumor-infiltrating cells collected under collagenase-free conditions were analyzed by flow cytometry after staining with FITC-labeled anti-rat IgG2b mAb to detect DTA-1- bound cells. doi:10.1371/journal.pone.0104669.g004 HF10 Oncolytic Virotherapy with Murine GITR Targeting Figure 4. Disappearance of DTA-1-conjugated tumor-infiltrating CD4+ Foxp3+ cells after combined i.t. treatment with HF10 and DTA-1. (A) CT26/NY-ESO-1 tumor sections form untreated group (control) or group from mice injected i.t. with DTA-1, HF10, or HF10 combined with DTA-1 were stained with FITC-anti-Foxp3 mAb and DAPI. (B) The frequency of tumor-infiltrating CD4+ Foxp3+ Treg cells from mice injected i.t. with HF10, DTA-1, or HF10 combined with DTA-1 at 12 days after CT26/NY-ESO-1 inoculation was assessed by flow cytometry. Data from 4 individual experiments were analyzed statistically. Kruskal-Wallis ANOVA test was used to compare data from the 4 groups. The decrease in the frequency of tumoral CD4+ Foxp3+ Treg cells in the HF10 and DTA-1 combined treatment group was significantly different from untreated control, but not from the HF10- or DTA-1-treated group (N.S: Not significant). (C) At 6 hrs after DTA-1 injection into day 9 CT26/NY-ESO-1 tumors, tumor-infiltrating cells collected under collagenase-free conditions were analyzed by flow cytometry after staining with FITC-labeled anti-rat IgG2b mAb to detect DTA-1- bound cells. doi:10.1371/journal.pone.0104669.g004 Figure 4. Disappearance of DTA-1-conjugated tumor-infiltrating CD4+ Foxp3+ cells after combined i.t. treatment with HF10 and DTA-1. (A) CT26/NY-ESO-1 tumor sections form untreated group (control) or group from mice injected i.t. with DTA-1, HF10, or HF10 combined with DTA-1 were stained with FITC-anti-Foxp3 mAb and DAPI. (B) The frequency of tumor-infiltrating CD4+ Foxp3+ Treg cells from mice injected i.t. with HF10, DTA-1, or HF10 combined with DTA-1 at 12 days after CT26/NY-ESO-1 inoculation was assessed by flow cytometry. Data from 4 individual experiments were analyzed statistically. Kruskal-Wallis ANOVA test was used to compare data from the 4 groups. The decrease in the frequency of tumoral CD4+ Foxp3+ Treg cells in the HF10 and DTA-1 combined treatment group was significantly different from untreated control, but not from the HF10- or DTA-1-treated group (N.S: Not significant). (C) At 6 hrs after DTA-1 injection into day 9 CT26/NY-ESO-1 tumors, tumor-infiltrating cells collected under collagenase-free conditions were analyzed by flow cytometry after staining with FITC-labeled anti-rat IgG2b mAb to detect DTA-1- bound cells. Figure 4. Disappearance of DTA-1-conjugated tumor-infiltrating CD4+ Foxp3+ cells after combined i.t. treatment with HF10 and DTA-1. Depletion of tumor-infiltrating Treg cells by DTA-1- mediated cellular cytotoxicity fluorescence from DTA-1 or the control rat IgG merged with the green fluorescence from F4/80+ macrophages in nearby tumor stroma (Fig. 5B; C1, D1, and S2B) indicated that DTA-1 and rat IgG bound with macrophage-expressing FcRs. In addition, a large number of cells visualized in lymphocyte-like formation by staining with anti-rat IgG2b mAb were positive for Foxp3 (Fig. 5B; D2, D3; Fig. S3A and B) and were in contact with macrophages in various areas of DTA-1–treated tumors (Fig. 5B; D2; Fig. S3A). These results strongly support the hypothesis that DTA-1 Fluorescent immunohistological studies using double labeling with anti-rat IgG2b mAb (for DTA-1) and F4/80- (for macro- phages) or Foxp3- (for Tregs) specific mAbs were performed to determine the mechanisms of DTA-1-dependent depletion of CT26/NY-ESO-1 tumor-infiltrating Treg cells. At 6 hrs after DTA-1 treatment, Foxp3+ cells clustered at the DTA-1-stained peritumor sites, whereas Foxp3+ cells did not accumulate in the control rat IgG-treated case (Fig. 5A and S2A). Images of red August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 8 HF10 Oncolytic Virotherapy with Murine GITR Targeting Figure 5. Kinetics of Foxp3+ Treg cells and F4/80+ macrophages in DTA-1-treated tumors. Frozen sections of CT26/NY-ESO-1 tumors obtained at 6 hrs after DTA-1 i.t. injection were stained with FITC-conjugated anti-Foxp3 mAb, PE-conjugated anti-rat IgG2b mAbs and DAPI (A and B: D3), or FITC-anti-F4/80 mAb, PE-anti-rat IgG2b mAb, and DAPI (B: D1 and D2). Sections from untreated and control rat IgG-treated tumors were used as controls (A and B: N1, N2, C1, C2). Representative photos from three experiments are shown. doi:10.1371/journal.pone.0104669.g005 Figure 5. Kinetics of Foxp3+ Treg cells and F4/80+ macrophages in DTA-1-treated tumors. Frozen sections of CT26/NY-ESO-1 tumors obtained at 6 hrs after DTA-1 i.t. injection were stained with FITC-conjugated anti-Foxp3 mAb, PE-conjugated anti-rat IgG2b mAbs and DAPI (A and B: D3), or FITC-anti-F4/80 mAb, PE-anti-rat IgG2b mAb, and DAPI (B: D1 and D2). Sections from untreated and control rat IgG-treated tumors were used as controls (A and B: N1, N2, C1, C2). Representative photos from three experiments are shown. doi:10.1371/journal.pone.0104669.g005 Figure 5. Kinetics of Foxp3+ Treg cells and F4/80+ macrophages in DTA-1-treated tumors. Frozen sections of CT26/NY-ESO-1 tumors obtained at 6 hrs after DTA-1 i.t. injection were stained with FITC-conjugated anti-Foxp3 mAb, PE-conjugated anti-rat IgG2b mAbs and DAPI (A and B: D3), or FITC-anti-F4/80 mAb, PE-anti-rat IgG2b mAb, and DAPI (B: D1 and D2). Depletion of tumor-infiltrating Treg cells by DTA-1- mediated cellular cytotoxicity Sections from untreated and control rat IgG-treated tumors were used as controls (A and B: N1, N2, C1, C2). Representative photos from three experiments are shown. doi:10.1371/journal.pone.0104669.g005 August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 9 HF10 Oncolytic Virotherapy with Murine GITR Targeting Figure 6. DTA-1-mediated depletion of tumor-infiltrating CD4+ Foxp3+ Treg cells by ADCC. (A) DTA-1- (dotted line) or isotype control (solid line)-treated CMS5a and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells were stained with a FITC-conjugated anti-rat IgG (H+L) antibody and analyzed by flow cytometry. (B) CFSE-labeled CMS5a and CMS5a/GITR cells were used as targets. The mixture of IFN-c-activated RAW264.7 cells (effector cells) and target cells were incubated for 12 hrs with control IgG or DTA-1 at effector/target ratios of 5 and 15. (C) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after i.t. DTA-1 or Fc-digested DTA-1 (DTA-1 Fab) treatment was measured by flow cytometric analysis. (D) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after DTA-1 i.t. treatment in wild-type or FcRc KO mice was measured by flow cytometric analysis. By Student’s t-test, the decrease in the frequency of Foxp3+ cells in DTA-1-treated CT26/NY-ESO-1 tumors of wild type mice, but not FcRc KO mice (N.S.: Not significant), was significantly different from untreated control group. (E) Frozen sections of CT26/NY-ESO-1 tumors obtained at 3 days after DTA-1 i.t. treatment in wild type and FcRc KO mice were stained with FITC-anti-Foxp3 and PE-anti- CD8a mAbs, and DAPI. doi:10.1371/journal.pone.0104669.g006 y py g g Figure 6. DTA-1-mediated depletion of tumor-infiltrating CD4+ Foxp3+ Treg cells by ADCC. (A) DTA-1- (dotted line) or isotype control (solid line)-treated CMS5a and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells were stained with a FITC-conjugated anti-rat IgG (H+L) antibody and analyzed by flow cytometry. (B) CFSE-labeled CMS5a and CMS5a/GITR cells were used as targets. The mixture of IFN-c-activated RAW264.7 cells (effector cells) and target cells were incubated for 12 hrs with control IgG or DTA-1 at effector/target ratios of 5 and 15. (C) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after i.t. DTA-1 or Fc-digested DTA-1 (DTA-1 Fab) treatment was measured by flow cytometric analysis. (D) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after DTA-1 i.t. treatment in wild-type or FcRc KO mice was measured by flow cytometric analysis. Discussion Many studies involving oncolytic virus combined with systemic administration of cytotoxic agents have shown promising results in animal models. However, almost all of the studies have avoided the important issue of lymphocyte suppression caused by steroids as an antiemetic, implying the clinical inapplicability of such cytotoxic agents. Tumor therapy promises an era of safety in using noninvasive immunomodulatory agents including PD-1-, CTLA- 4- and GITR-specific mAbs. Unfortunately, all of them have produced slight immune-related slight adverse events such as diarrhea, rashes or pruritis [35–38]. In addition, systemic administration of immunomodulators can elicit serious autoim- mune diseases. A study from another group has shown that in a murine model, treatment with 50 mg/mouse DTA-1 induces antitumor activity and weak autoimmune reactions [39]. In this study, we also demonstrated that HF10 virotherapy combined with a GITR-targeting mAb in local tumor sites at more clinical appropriate lower and safer doses, elicits tumor lysis by augmented systemic tumor-specific CD8+ T cell activity with negligible toxicity. Therefore, local treatment of immunomodulators is a promising method for the future treatment of tumors. In conclusion, local HF10 therapy combined with DTA-1 should be suitable for the treatment of cancer patients without crucial side effects. The benefits of the combined treatment regimen include the vigorous expansion of tumoricidal CTLs associated with the early HF10-specific CTL responses, inhibition of tumor formation by HF10 infection, direct expansion of CD8+ T cells by DTA-1, and negation of immune suppressive Treg cell activities by DTA-1-mediated ADCC and/or DTA-1 signaling. The use of blocking Abs for suppressing immune signals has shown clinical benefits in the treatment of solid tumors [40–42]. Both PD-1 and CTLA-4, which are expressed on activated T cell surfaces, inhibit tumoricidal effector T cell responses by engage- ment via specific ligands that are expressed on various tumor cells [23]. However, high densities of tumor-infiltrating CD4+ CD25+ Foxp3+ Treg cells have been correlated with poor survival [43– 45]. Treg cells express both PD-1 and CTLA-4 in the steady state without activation. PD-1 and CTLA-4 signals result in Treg induction and maintenance, and subsequent outbreak of autoim- mune diseases [46]. Interestingly, it has been reported that an anti- CTLA-4 antibody augments tumoricidal effector T cells by downregulation of Treg cell functions, including ADCC-mediated depletion of Treg cells [47], which is similar to our GITR- targeting results. HF10 Oncolytic Virotherapy with Murine GITR Targeting participates in GITR+ Foxp3+ Treg depletion by ADCC at the treated tumor sites. The expression of GITR has been observed on CD4+ CD25+ Treg cells at relatively high levels [24,25], which is consistent with our results. In addition, the GITR-GITRL interaction has been known to attenuate Treg cell function via the loss of Foxp3 expression as well as enhance tumor-specific effector CD4+ and CD8+ T cell functions [27,39,48,49]. In this study, we demon- strated the use of DTA-1 as a depletion antibody because Fc- digested DTA-1 and intact DTA-1 in FcRc KO mice did not participate in the downregulation of Foxp3 expression. After i.t. DTA-1 injection, macrophages appeared to attract DTA-1- conjugated Treg cells via their FcR and migrate to peritumor sites, as shown in Fig. 5, and the results suggest that the peritumoral stroma is a crucial place for ADCC triggering. Since CCL22 secreted by macrophages is known to be a chemoattrac- tant for Treg cells [43,50], such chemokines might participate in DTA-1-mediated Treg cell depletion. Further studies are neces- sary to elucidate the molecular mechanisms of tumoral ADCC. To examine whether DTA-1 can mediate ADCC in a murine system, we performed an in vitro ADCC assay using IFN-c- activated RAW264.7 macrophage cells as an effector and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells (Fig. 6A) as a target. CMS5a/GITR cells were lysed in the presence of DTA-1 in a GITR-specific manner (Fig. 6B). We further investigated in vivo the ADCC effects of DTA-1 using Fc portion-digested DTA- 1 (DTA-1 Fab) and FcRc KO mice. Depletion of CD4+ Foxp3+ Treg cells in CT26/NY-ESO-1 tumors was not observed following i.t. DTA-1 Fab treatment (Fig. 6C). In addition, no significant decreases in the number of CD4+ Foxp3+ Treg cells and accumulation of CD8+ T cells were detected by DTA-1 treatment in FcRc KO mice, unlike the results from wild-type mice (Fig. 6D, 6E, and S4). These results clearly indicated the direct participation of DTA-1 in Treg cell depletion by ADCC. As indicated in Fig. 4C, a small proportion of tumor-infiltrating CD8+ T cells bound with i.t. treated DTA-1. In addition, DTA-1 enhanced tumor-specific CD8+ T cell responses in a dose- dependent manner in tumor-regressed mice as shown in Fig. 3. Discussion These reports indicate that the blockade of immune checkpoint molecules involves the activation of tumori- cidal effector T cells by preventing interactions with specific ligands on tumor cells and inhibiting Treg cell functions. HF10 Oncolytic Virotherapy with Murine GITR Targeting These results suggest that DTA-1 acts as a direct activator of CD8+ T cells, although we could not rule out the possibility that tumor-specific CD8+ T cell responses were increased by DTA-1 dose-dependent depletion of immune suppressive Treg cells. Indeed, it has been reported that the function and activity of CTLs are augmented by the signals through GITR [26,48,51]. In this study, HF10-specific CD8+ T cells were detected after both HF10 and DTA-1 injections, concomitant with vigorous tumor- specific CTL responses. La et al. have reported that DTA-1 elicits immediately explosive HSV-1-specific CD8+ CTL and CD4+ Th responses in HSV-1-infected mice [52]. In addition, we found in this study that DTA-1 was detected in tumor-draining lymph nodes soon after i.t. injection (Fig. S5), suggesting the relationship between DTA-1 and quick generation of tumor-specific CTLs. Thus, it is likely that HF10-specific CTL responses induced by DTA-1 change the tumor microenvironment to facilitate the expansion of CTLs in tumor-draining lymph nodes. Taken together, these results show that HF10 virotherapy combined with DTA-1 elicits a powerful therapeutic effect against tumors via the accumulation of CD8+ T cells, after tumor destruction by HF10 and the enhancement of tumor- and virus- specific CD8+ T cell responses directly or indirectly by depletion of immune-suppressive Treg cells at tumor sites by DTA-1. Depletion of tumor-infiltrating Treg cells by DTA-1- mediated cellular cytotoxicity By Student’s t-test, the decrease in the frequency of Foxp3+ cells in DTA-1-treated CT26/NY-ESO-1 tumors of wild type mice, but not FcRc KO mice (N.S.: Not significant), was significantly different from untreated control group. (E) Frozen sections of CT26/NY-ESO-1 tumors obtained at 3 days after DTA-1 i.t. treatment in wild type and FcRc KO mice were stained with FITC-anti-Foxp3 and PE-anti- CD8a mAbs, and DAPI. doi:10.1371/journal.pone.0104669.g006 August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 10 HF10 Oncolytic Virotherapy with Murine GITR Targeting HF10 Oncolytic Virotherapy with Murine GITR Targeting References 1. Vaha-Koskela MJ, Heikkila JE, Hinkkanen AE (2007) Oncolytic viruses in cancer therapy. Cancer Lett 254: 178–216. 19. Nakao A, Kasuya H, Sahin TT, Nomura N, Kanzaki A, et al. (2011) A phase I dose-escalation clinical trial of intraoperative direct intratumoral injection of HF10 oncolytic virus in non-resectable patients with advanced pancreatic cancer. Cancer Gene Ther 18: 167–175. 2. 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Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S (2002) Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self- tolerance. Nat Immunol 3: 135–142. 10. Kaufman HL, Kim DW, DeRaffele G, Mitcham J, Coffin RS, et al. (2010) Local and distant immunity induced by intralesional vaccination with an oncolytic herpes virus encoding GM-CSF in patients with stage IIIc and IV melanoma. Ann Surg Oncol 17: 718–730. 26. Cote AL, Zhang P, O’Sullivan JA, Jacobs VL, Clemis CR, et al. (2011) Stimulation of the glucocorticoid-induced TNF receptor family-related receptor on CD8 T cells induces protective and high-avidity T cell responses to tumor- specific antigens. J Immunol 186: 275–283. 11. Kaufman HL, Bines SD (2010) OPTIM trial: a Phase III trial of an oncolytic herpes virus encoding GM-CSF for unresectable stage III or IV melanoma. Future Oncol 6: 941–949. 27. Nishikawa H, Kato T, Hirayama M, Orito Y, Sato E, et al. (2008) Regulatory T cell-resistant CD8+ T cells induced by glucocorticoid-induced tumor necrosis factor receptor signaling. Cancer Res 68: 5948–5954. 12. Andtbacka RHI, Collichio FA, Amatruda T, Senzer NN, Chesney J, et al. (2013) OPTiM: A randomized phase III trial of talimogene laherparepvec (T-VEC) versus subcutaneous (SC) granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment (tx) of unresected stage IIIB/C and IV melanoma. J Clin Oncol 31: suppl; abstr LBA9008. 28. Rosenzweig M, Ponte J, Apostolou I, Doty D, Guild J, et al. (2010) Development of TRX518, an aglycosyl humanized monoclonal antibody (Mab) agonist of huGITR. J Clin Oncol 28: suppl; abstr e13028. 29. Takai T, Li M, Sylvestre D, Clynes R, Ravetch JV (1994) FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell 76: 519–529. pp 13. Campadelli-Fiume G, De Giovanni C, Gatta V, Nanni P, Lollini PL, et al. (2011) Rethinking herpes simplex virus: the way to oncolytic agents. Author Contributions Figure S4 The three fluorescence components of the merged images in Fig. 6E. The three separate images of red (PE), green (FITC), and blue (DAPI) fluorescence that were merged to produce Fig. 6E. (TIF) Conceived and designed the experiments: MI NS HS. Performed the experiments: MI NS. Analyzed the data: MI NS DM HI HS. Contributed reagents/materials/analysis tools: J. Mitsui MT J. Mineno. Contributed to the writing of the manuscript: MI NS. Figure S5 Drafting of i.t. treated DTA-1 into tumor- draining lymph nodes. Frozen sections of tumor-draining HF10 Oncolytic Virotherapy with Murine GITR Targeting HF10 Oncolytic Virotherapy with Murine GITR Targeting Three separate images of red (PE), green (FITC), and blue (DAPI) fluorescence that were merged to produce Fig. 5A (A) and N1, C1, and D1 of Fig. 5B (B). Three separate images of red (PE), green (FITC), and blue (DAPI) fluorescence that were merged to produce Fig. 5A (A) and N1, C1, and D1 of Fig. 5B (B). lymph nodes obtained at 6 hrs after intratumoral DTA-1 or DTA- 1 Fab treatment were stained with a FITC-conjugated anti-rat IgG2b antibody, a phycoerythrin (PE)-conjugated anti-F4/80 antibody, and DAPI. (TIF) Figure S3 The three fluorescence components of the merged images of N2, C2, D2, and D3 in Fig. 5B. The three separate images of red (PE), green (FITC), and blue (DAPI) fluorescence that were merged to produce N2, C2, D2, and D3 images in Fig. 5B. Acknowledgments We thank Drs. N. Harada, Y. Miyahara, T. Kato, and T. Takahashi for helpful discussions, and M. Yamane for technical assistance. We thank Drs. N. Harada, Y. Miyahara, T. Kato, and T. Takahashi for helpful discussions, and M. Yamane for technical assistance. Supporting Information Figure S1 Systemic surveillance of tumoricidal CTLs after HF10 combination therapy with DTA-1 at local tumor sites. (A) The images of red (PE), green (FITC), and blue (DAPI) fluorescence that were merged to produce Fig. 2C. (B) Bilateral CT26/NY-ESO-1-bearing mice were treated i.t. with a combination of HF10 and DTA-1 in tumors on the right flanks of mice. Tumor growth in the treated right and contralateral left sites was measured. Photos show representative mice at 25 days after CT26/NY-ESO-1 inoculation from the control and dual HF10- and DTA-1-treated groups. (C) The three images of red (PE), green (FITC), and blue (DAPI) fluorescence that were merged to produce Fig. 2F. p (TIF) Figure S2 The three fluorescence components of the merged images of Fig. 5A and Fig. 5B (N1, C1, and D1). Figure S2 The three fluorescence components of the merged images of Fig. 5A and Fig. 5B (N1, C1, and D1). August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 11 HF10 Oncolytic Virotherapy with Murine GITR Targeting 35. Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, et al. (2013) Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369: 134–144. FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res 11: 8326–8331. FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res 11: 8326–8331. 45. Wang W, Hodkinson P, McLaren F, MacKinnon A, Wallace W, et al. (2012) Small cell lung cancer tumour cells induce regulatory T lymphocytes, and patient survival correlates negatively with FOXP3+ cells in tumour infiltrate. Int J Cancer 131: E928–937. 36. Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, et al. (2013) Ipilimumab for patients with advanced mucosal melanoma. Oncologist 18: 726– 732. 37. Ribas A, Kefford R, Marshall MA, Punt CJ, Haanen JB, et al. (2013) Phase III randomized clinical trial comparing tremelimumab with standard-of-care chemotherapy in patients with advanced melanoma. J Clin Oncol 31: 616–622. 46. Francisco LM, Salinas VH, Brown KE, Vanguri VK, Freeman GJ, et al. (2009) PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. J Exp Med 206: 3015–3029. py p J 38. Weber JS, Dummer R, de Pril V, Lebbe C, Hodi FS (2013) Patterns of onset and resolution of immune-related adverse events of special interest with ipilimumab: detailed safety analysis from a phase 3 trial in patients with advanced melanoma. Cancer 119: 1675–1682. 47. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, et al. (2013) Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 210: 1695– 1710. 39. Ko K, Yamazaki S, Nakamura K, Nishioka T, Hirota K, et al. (2005) Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor- infiltrating Foxp3+CD25+CD4+ regulatory T cells. J Exp Med 202: 885–891. 48. Cohen AD, Diab A, Perales MA, Wolchok JD, Rizzuto G, et al. (2006) Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity. Cancer Res 66: 4904–4912. 40. Robert C, Thomas L, Bondarenko I, O’Day S, Weber J, et al. (2011) Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 364: 2517–2526. 49. Cohen AD, Schaer DA, Liu C, Li Y, Hirschhorn-Cymmerman D, et al. (2010) Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation. PLoS One 5: e10436. References Rev Med Virol 21: 213–226. 30. Lerner WA, Pearlstein E, Ambrogio C, Karpatkin S (1983) A new mechanism for tumor induced platelet aggregation. Comparison with mechanisms shared by other tumor with possible pharmacologic strategy toward prevention of metastases. Int J Cancer 31: 463–469. 14. Nishiyama Y, Kimura H, Daikoku T (1991) Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2. J Virol 65: 4520–4524. 31. Muraoka D, Kato T, Wang L, Maeda Y, Noguchi T, et al. (2010) Peptide vaccine induces enhanced tumor growth associated with apoptosis induction in CD8+ T cells. J Immunol 185: 3768–3776. 15. Mori I, Liu B, Goshima F, Ito H, Koide N, et al. (2005) HF10, an attenuated herpes simplex virus (HSV) type 1 clone, lacks neuroinvasiveness and protects mice against lethal challenge with HSV types 1 and 2. Microbes Infect 7: 1492– 1500. 32. Ikeda H, Ohta N, Furukawa K, Miyazaki H, Wang L, et al. (1997) Mutated mitogen-activated protein kinase: a tumor rejection antigen of mouse sarcoma. Proc Natl Acad Sci USA 94: 6375–6379. 16. Nakao A, Kimata H, Imai T, Kikumori T, Teshigahara O, et al. (2004) Intratumoral injection of herpes simplex virus HF10 in recurrent breast cancer. Ann Oncol 15: 988–989. 33. Huang AY, Gulden PH, Woods AS, Thomas MC, Tong CD, et al. (1996) The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product. Proc Natl Acad Sci USA 93: 9730–9735. 17. Fujimoto Y, Mizuno T, Sugiura S, Goshima F, Kohno S, et al. (2006) Intratumoral injection of herpes simplex virus HF10 in recurrent head and neck squamous cell carcinoma. Acta Otolaryngol 126: 1115–1117. 34. Mitsui J, Nishikawa H, Muraoka D, Wang L, Noguchi T, et al. (2010) Two distinct mechanisms of augmented antitumor activity by modulation of immunostimulatory/inhibitory signals. Clin Cancer Res 16: 2781–2791. 18. Kimata H, Imai T, Kikumori T, Teshigahara O, Nagasaka T, et al. (2006) Pilot study of oncolytic viral therapy using mutant herpes simplex virus (HF10) against recurrent metastatic breast cancer. Ann Surgical Oncol 13: 1078–1084. August 2014 \| Volume 9 \| Issue 8 \| e104669 August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 12 HF10 Oncolytic Virotherapy with Murine GITR Targeting g J 41. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, et al. (2012) Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366: 2455–2465. 50. Li YQ, Liu FF, Zhang XM, Guo XJ, Ren MJ, et al. (2013) Tumor secretion of CCL22 activates intratumoral Treg infiltration and is independent prognostic predictor of breast cancer. PLoS One 8: e76379. 42. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, et al. (2012) Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366: 2443–2454. p 51. Imai N, Ikeda H, Tawara I, Wang L, Wang L, et al. (2009) Glucocorticoid- induced tumor necrosis factor receptor stimulation enhances the multifunction- ality of adoptively transferred tumor antigen-specific CD8+ T cells with tumor regression. Cancer Sci 100: 1317–1325. g 43. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, et al. (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10: 942–949. 52. La S, Kim E, Kwon B (2005) In vivo ligation of glucocorticoid-induced TNF receptor enhances the T-cell immunity to herpes simplex virus type 1. Exp Mol Med 37: 193–198. p 44. Wolf D, Wolf AM, Rumpold H, Fiegl H, Zeimet AG, et al. (2005) The expression of the regulatory T cell-specific forkhead box transcription factor PLOS ONE \| www.plosone.org August 2014 \| Volume 9 \| Issue 8 \| e104669 PLOS ONE \| www.plosone.org 13
https://openalex.org/W2902726602	https://link.springer.com/content/pdf/10.1007/JHEP05(2019)208.pdf	English	null	Systematic construction of basis invariants in the 2HDM	The Journal of high energy physics/The journal of high energy physics	2,019	cc-by	18,184	Published for SISSA by Springer Published for SISSA by Springer Received: December 29, 2018 Accepted: May 23, 2019 Published: May 31, 2019 Received: December 29, 2018 Accepted: May 23, 2019 Published: May 31, 2019 Received: December 29, 2018 Accepted: May 23, 2019 Published: May 31, 2019 Systematic construction of basis invariants in the 2HDM JHEP05(2019)208 Andreas Trautner Max-Planck-Institut f¨ur Kernphysik, Saupfercheckweg 1, 69117 Heidelberg, Germany Bethe Center for Theoretical Physics und Physikalisches Institut der Universit¨at Bonn, Nussallee 12, 53115 Bonn, Germany E-mail: trautner@mpi-hd.mpg.de Andreas Trautner Max-Planck-Institut f¨ur Kernphysik, Saupfercheckweg 1, 69117 Heidelberg, Germany Bethe Center for Theoretical Physics und Physikalisches Institut der Universit¨at Bonn, Nussallee 12, 53115 Bonn, Germany E-mail: trautner@mpi-hd.mpg.de Keywords: Beyond Standard Model, CP violation, Global Symmetries, Renormalization Group Open Access, c⃝The Authors. Article funded by SCOAP3. Andreas Trautner https://doi.org/10.1007/JHEP05(2019)208 Contents 1 Introduction 1 2 Synopsis of jargon 4 3 Construction of the building blocks 5 4 CP properties of the building blocks 10 5 Construction of higher-order invariants 10 5.1 Number and structure of the invariants 10 5.2 Explicit construction of the invariants 13 5.2.1 Needed projection operators 13 5.2.2 Algebraically independent invariants 14 5.2.3 Completing the generating set 14 6 Systematic construction of Syzygies 15 7 Necessary and suﬃcient conditions for explicit CPV 16 8 Towards a Hironaka decomposition 18 9 Summary and discussion 19 A Algebraic (in)dependence of invariants 20 B Building blocks in conventional notation 21 C Relation to the invariants of Gunion and Haber 22 D Explicit statement of the invariants 22 D.1 Algebraically independent invariants 22 D.2 Invariants to complete the generating set 23 E Syzygies 26 Contents 1 Introduction 1 2 Synopsis of jargon 4 3 Construction of the building blocks 5 4 CP properties of the building blocks 10 5 Construction of higher-order invariants 10 5.1 Number and structure of the invariants 10 5.2 Explicit construction of the invariants 13 5.2.1 Needed projection operators 13 5.2.2 Algebraically independent invariants 14 5.2.3 Completing the generating set 14 6 Systematic construction of Syzygies 15 7 Necessary and suﬃcient conditions for explicit CPV 16 8 Towards a Hironaka decomposition 18 9 Summary and discussion 19 A Algebraic (in)dependence of invariants 20 B Building blocks in conventional notation 21 C Relation to the invariants of Gunion and Haber 22 D Explicit statement of the invariants 22 D.1 Algebraically independent invariants 22 D.2 Invariants to complete the generating set 23 E Syzygies 26 JHEP05(2019)208 Andreas Trautner E-mail: trautner@mpi-hd.mpg.de Abstract: A new systematic method for the explicit construction of (basis-)invariants is introduced and employed to construct the full ring of basis invariants of the Two-Higgs- Doublet-Model (2HDM) scalar sector. Co- and invariant quantities are obtained by the use of hermitian projection operators. These projection operators are constructed from Young tableaux via birdtrack diagrams and they are used in two steps. First, to extract basis- covariant quantities, and second, to combine the covariants in order to obtain the actual basis invariants. The Hilbert series and Plethystic logarithm are used to ﬁnd the number and structure of the complete set of generating invariants as well as their interrelations (syzygies). Having full control over the complete ring of (CP-even and CP-odd) basis invariants, we give a new and simple proof of the necessary and suﬃcient conditions for explicit CP conservation in the 2HDM, conﬁrming earlier results by Gunion and Haber. The method generalizes to other models, with the only foreseeable limitation being computing power. Keywords: Beyond Standard Model, CP violation, Global Symmetries, Renormalization Group ArXiv ePrint: 1812.02614 ArXiv ePrint: 1812.02614 ArXiv ePrint: 1812.02614 Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction Physical observables — i.e. measurable quantities like cross sections, branching ratios, CP asymmetries etc. — must not depend on arbitrary choices of basis and notation. Conse- quently, observables ultimately can only depend on basis invariant quantities. – 1 – Nevertheless, in order to formulate a theory and execute computations it arguably is necessary to pick a certain basis and parametrization. Given a theory formulated in an arbitrary basis, the following questions arise: • How does one obtain basis invariant quantities? • How many independent basis invariant quantities exist? • How are these basis invariant quantities related to physical observables? This paper will make contact with all of these questions. The exemplary case treated here is the scalar potential of the two-Higgs-doublet model (2HDM), see e.g. [1]. For this case, we will be able to give deﬁnite answers to questions one and two. Moreover, physical matters will be touched with respect to the violation of the combined charge-conjugation and parity (CP) symmetry. JHEP05(2019)208 Physical questions can be obscured by the presence of large basis redundancies, in which case an invariant formulation oﬀers a clear beneﬁt [2]. In particular — but certainly not limited to it — this is true for detecting the (non-)conservation of CP symmetry, which is commonly plagued by spurious phases [3]. Therefore, perhaps the most prominent example for a basis invariant measure is the well-known Jarlskog invariant, which detects CP non-conservation in the Standard Model (SM) [4]. While the non-vanishing of any CP-odd basis invariant unambiguously signals CP violation, suﬃcient conditions for CP conservation are much harder to ﬁnd. That is because in order to formulate suﬃcient conditions one has to know “when to stop looking” for new independent invariants, which is of course at the heart of our second question above. In this light, it is no surprise that basis invariant necessary conditions for CP conservation have been formulated for theories with extended fermion [5, 6] and/or scalar sectors [7, 8], while suﬃcient conditions for CP conservation, besides for the SM, are only known for the 2HDM [9] (see also [10–12]) and under limiting assumptions also for the three and N- HDM [12], as well as for certain supersymmetric models [13, 14]. Lately, also basis invariant necessary and suﬃcient conditions for the physically distinct order 4 CP transformation of the 3HDM [15] have been formulated [16, 17]. 1 Introduction Basis invariant methods have also been used to grasp physical aspects other than CP. For example, to investigate quark and lepton mixing in the SM and extensions [18–20], as well as to express physical observables of the 2HDM [21, 22], and more recently [23–25]. As an additional beneﬁt, a basis invariant parametrization simpliﬁes the analysis of renormal- ization group equations (RGE) and RGE running, both for SM fermions [26–28] as well as for extended scalar sectors [29–31], and so the question of how to construct basis invariants continues to be of interest [32, 33]. However, it remains in general an open question how a theory can be formulated solely in terms of basis invariant quantities. In a group theoretical sense, basis invariants are objects which do not transform under the action of the group of basis changes. This implies that basis invariants form a ring, – 2 – in the algebraic sense, and the question “when to stop looking” for new invariants turns out to be a mathematical exercise of invariant theory (see e.g. [34, 35]). The classical way of dealing with invariant rings is via their generating function, the so-called Hilbert- Poincar´e series (HS). A more modern tool in ring theory is the Plethystic logarithm (PL), introduced in [36] and further discussed in [37]. The methodology of the HS and PL has been put forward for physics applications in [38, 39], and has subsequently been applied to a plethora of formal questions, see e.g. [40–46]. Important phenomenological applications of the HS method are the characterization of quark and lepton invariants in and beyond the SM [19, 20], as well as the general construction of complete bases of gauge invariant operators in eﬀective ﬁeld theories [47–49]. We recommend [50] as a ﬁrst read on HS in the particle physics context. JHEP05(2019)208 In this paper, we will use the HS and PL to ﬁnd the number of independent basis invariants, a generating set of basis invariants, as well as the structure of interrelations between basis invariant (syzygies) — all for the 2HDM. The main original aspect of the present paper is our novel way of explicitly construct- ing the basis invariants in a systematic way. Of course, the construction of invariants from covariant objects is, in principle, a solved group theoretical problem for which even powerful computer codes exist (see e.g. [51]). 2 Synopsis of jargon Before starting the actual discussion of this paper let us introduce the technical terms used. All parameters of a theory do transform in some way or another under necessarily unphysical changes of basis. Thus, it is in general possible to form combinations of pa- rameters which are unaﬀected by all possible basis transformations: basis invariants (or short invariants in the following). Since any sum or product of invariants is an invariant itself, the invariants form a ring in the mathematical sense.1 An immediate question then is: how many invariant quantities are needed, in general, in order to be able to cover the space of all possible invariant quantities of a given model. JHEP05(2019)208 Some remarks are in order concerning the use of “generating set”, “generators”, “inde- pendent” and so forth since most of the physics literature lacks scrutiny in this point. By “independent” we always mean algebraically independent. To be clear, an invariant, say I1, is algebraically dependent on a set of invariants, say I2,3,.. , if and only if it is possible to ﬁnd a polynomial P such that P (I1, I2, I3, . . . ) = 0 . (2.1) (2.1) If such a polynomial does not exist, I1 is called algebraically independent of I2,3,.... The maximal number of algebraically independent invariants is equal to the number of physical parameters of a theory in the usual sense. Not surprisingly, this is the number of parameters which remains after all possible basis changes have been used to absorb parameters, i.e. set as many of them to zero as possible. Thus, having found a full set of algebraically independent invariants, the physical content of a theory is fully speciﬁed. Nevertheless, for many applications it makes sense to go beyond the set of algebraically independent invariants. This is the case, particularly because a relation of the kind (2.1) does not guarantee that we can solve for an arbitrary invariant. For this reason, it makes sense to discuss a generating set of a ring, which consists of all invariants that cannot be written as a polynomial of other invariants, Ii ̸= P (Ij, . . . ) . (2.2) (2.2) More intuitively perhaps, the generating set of invariants is a set such that all invariants in the ring can be written as polynomial in the generators, I = P (I1, I2 . . . ) . 2All ﬁnite groups and all semi-simple Lie groups are reductive. 1Most likely the invariants form even more than a ring, since one may also envisage more involv operations on them. 2 1 Introduction However, available methods quickly become unmanageable if it comes to construction of singlets from high-rank tensors or disentangling individual contributions of, in principle, independent singlets. Our approach is to use her- mitian projection operators [52] (see also [53–55]), which can conveniently be constructed from Young tableaux via birdtrack diagrams [56, 57]. As a pedagogical introduction to this we recommend [58]. These operators project arbitrary rank tensors onto their contained orthogonal trivial singlets, implying that they give rise to the shortest possible invariants by construction. Ultimately, it is the liaison of group theoretical and algebraic techniques that merges in a new and powerful systematic way for the construction of basis invariants. The thereby constructed invariants are short and their relations transparent, as demon- strated by our new and simple proof of necessary and suﬃcient conditions for explicit CP conservation in the 2HDM. The outline of the paper is as follows. In the subsequent section we will give a brief synopsis of indispensable terms and concepts. In section 3 we turn to the 2HDM, for which we construct the elementary building blocks of all invariants from the coupling tensors of the Lagrangian. The CP transformation behavior of the building blocks and higher- order invariants is derived in section 4. Using the building blocks as input, we proceed to construct the Hilbert series and Plethystic logarithm of the ring in section 5.2, thereby gaining information on the number and structure of the invariants and their syzygies. The explicit construction of the invariants is performed in section 5.2.2 and section 5.2.3, and a generally applicable strategy for the construction of syzygies is outlined in section 6. We then give a simple derivation of the necessary and suﬃcient conditions for explicit CP conservation in the 2HDM in section 7. Finally, section 8 contains some comments on the construction of a Hironaka decomposition of the 2HDM ring, after which we conclude. – 3 – nite groups and all semi-simple Lie groups are reductive. ope at o s o t e 2All ﬁnite groups and all semi-simple Lie groups are reductive. The most general 2HDM scalar potential can be written as The most general 2HDM scalar potential can be written as V = Φ† a Y a b Φb + Φ† a Φ† b Zab cd Φc Φd , (3.1) (3.1) where Φa are hyper charge-one scalar doublets of the SU(2)L gauge symmetry and a, b, c, d= 1, 2 are indices in the SU(2) space of Higgs-ﬂavor. We use upper and lower indices to distinguish ﬁelds transforming as 2 and 2 under SU(2) Higgs-ﬂavor basis changes. JHEP05(2019)208 Due to hermiticity of the Lagrangian, the coupling tensors satisfy Y a b = (Y b a )∗, Zab cd = (Zcd ab)∗. (3.2) (3.2) Because of SU(2)L invariance the quartic couplings fulﬁll in addition Zab cd = Zba dc . (3.3) (3.3) Due to these constraints, not all entries of the coupling tensors are independent. Y and Z have 4 and 10 independent real entries, respectively. Utilizing all possible basis changes to absorb parameters (i.e. set them to zero) one can show that the number of physical parameters is 11. We will, however, keep working in a general basis. Our ﬁrst goal is to construct linear combinations of the entries of Y and Z which transform in irreducible representations under basis changes. In particular we will see that Y and Z decompose into Y b= 1 ⊕3 , Z b= 1 ⊕1 ⊕3 ⊕5 . (3.4) ⊕ , Z b= 1 ⊕1 ⊕3 ⊕5 . (3.4) (3.4) There are three “trivial” basis invariants which arise as linear combinations of the potential parameters. Linearly independent of these, there are other linear combinations of entries of Y and Z which transform covariantly under basis changes, and they will be our building blocks for the construction of non-linear higher-order invariants below. There are three “trivial” basis invariants which arise as linear combinations of the potential parameters. Linearly independent of these, there are other linear combinations of entries of Y and Z which transform covariantly under basis changes, and they will be our building blocks for the construction of non-linear higher-order invariants below. Let us be very explicit in deriving (3.4). The undertaken steps may seem like an overkill to advanced readers, but it seems necessary to recall these details before entering the derivation of higher-order invariants below. As we want to make use of Young tableaux, it makes sense to convert all indices to one type (upper or lower). 2 Synopsis of jargon (2.3) (2.3) A classic result is that the generating set of a ring of invariants has ﬁnite size (“the ring is ﬁnitely generated”) if the underlying group of symmetry transformations (in our case the group of basis transformations) is reductive [59–61].2 General formulae for the number of invariants in the generating set are not available, but there exist bounds [62, 63] (see also [19] for applications). The full set of generators of a ring is, generally, algebraically dependent (otherwise the ring is called free). Algebraic relations among invariants are called syzygies. – 4 – 3 Construction of the building blocks The most general 2HDM scalar potential can be written as The most general 2HDM scalar potential can be written as To do this, we use the fully anti-symmetric Levi-Civita tensor with the convention ε12 = −ε21 = −ε12 = ε21 = 1 to raise or lower indices, thereby deﬁning the objects Y ab := εbc Y a c , and Zab,cd := εce εdf Zab ef . (3.5) (3.5) – 5 – Assigning a box to each index in the usual way, we can decompose Y and Z into their covariantly transforming irreducible components by the standard procedure,3 Y : a 2 ⊗b 2 = a b 1 ⊕a b 3 , (3.6) Z : a 2 ⊗b 2 ⊗c 2 ⊗d 2 = a b c d 1(1) ⊕a c b d 1(2) ⊕ a b c d 3(1) ⊕a b d c 3(2) ⊕a c d b 3(3) ⊕ a b c d 5 . (3.7) (3.6) JHEP05(2019)208 (3.7) Recall that writing boxes in a line of the Young tableaux means symmetrization of the respective indices, while boxes in the same column are to be anti-symmetrized. The order of indices has to be obeyed. The (anti-)symmetrization of indices can be performed by projection operators. Thus, in their very essence, Young tableaux correspond to projection operators. These projection operators can always be made hermitian [52]. Acting with these projection operators on a corresponding tensor projects out the respective covariantly transforming components of the tensor. Birdtracks [56, 57] can be used to construct the projection operators by hand. A pedagogical introduction to this can be found in [58]. We will only recall the very most relevant features along the way. The simplest projection operators are the total (anti-)symmetrizers of two indices denoted by P a b := a′ b′ a b ≡ 1 2! a′ a b′ b − a′ b b′ a ! , (3.8) P a b := a′ b′ a b ≡ 1 2! a′ a b′ b + a′ b b′ a ! . (3.9) (3.8) (3.9) Indices on incoming(outgoing) lines correspond to upper(lower) indices, and if two indices are directly connected by a line they are meant to be set equal by contraction with a Kro- necker delta. Using only these and larger (anti-)symmetrization operators consecutively, it is possible to diagrammatically construct projection operators for every Young tableaux. 3Due to the constraints (3.2) and (3.3), only one of the three triplets 3(1,2,3) in the decomposition of is independent, such that eq. (3.4) holds. We will show this explicitly around eq. (3.24). The most general 2HDM scalar potential can be written as Let us also introduce a diagrammatic representation of the Levi-Civita symbol Indices on incoming(outgoing) lines correspond to upper(lower) indices, and if two indices are directly connected by a line they are meant to be set equal by contraction with a Kro- necker delta. Using only these and larger (anti-)symmetrization operators consecutively, it is possible to diagrammatically construct projection operators for every Young tableaux. Let us also introduce a diagrammatic representation of the Levi-Civita symbol εab ≡a b = −a b , (3.10a) εab ≡a b = −a b , (3.10b) (3.10a) (3.10b) with the usual identities = = , etc. (3.11) (3.11) 3Due to the constraints (3.2) and (3.3), only one of the three triplets 3(1,2,3) in the decomposition of Z is independent, such that eq. (3.4) holds. We will show this explicitly around eq. (3.24). 3Due to the constraints (3.2) and (3.3), only one of the three triplets 3(1,2,3) in the decomposition of Z is independent, such that eq. (3.4) holds. We will show this explicitly around eq. (3.24). – 6 – It is very important to note that for indices in the fundamental of SU(N) we can anti- symmetrize at most N indices, otherwise the result is trivially zero. Diagrammatically, this gives rise to a factorization rule for anti-symmetrization operators with N indices. Here, for N = 2:4 P a b = a′ b′ a b = 1 2    a′ b′ a b   = −1 2εa′b′εab . (3.12) (3.12) Finally, let us also introduce a diagrammatic notation for Y and Z, Finally, let us also introduce a diagrammatic notation for Y and Z, JHEP05(2019)208 Y ab = a b Y =: a b Y , and (3.13) Y ab = a b Y =: a b Y , and (3.13) Zab,cd = a b c d Z =: a b c d Z . (3.14) (3.13) Zab,cd = a b c d Z =: a b c d Z . (3.14) (3.14) To ﬁnd the covariantly transforming components of the tensor Y , we simply act on it with the corresponding projection operators, Y a′b′ 1 = [P a b ]a′b′ ab Y ab , and Y a′b′ 3 = [P a b ]a′b′ ab Y ab . 5While the rules in [53–55] are derived with mathematical rigor, following them to construct projection operators in practice can be tedious. We remark here that there is a set of simple and very intuitive rules to construct these (and much larger) projection operators from scratch. We plan to communicate these rules in the future. The most general 2HDM scalar potential can be written as (3.15) (3.15) Diagrammatically the ﬁrst projection reads Y a′b′ 1 = 1 2 a′ b′ Y = 1 2 a′ b′ Y = −1 2εa′b′ Y a a . (3.16) (3.16) Note how due to factorization of the projection operator a bubble diagram is nucleated. Clearly, Y a′b′ 1 is a basis invariant because εa′b′ is invariant under any SU(2) rotation while Y a a is fully contracted. We are just interested in the non-trivial essence of the invariant. Hence, the global prefactors as well as the remaining ε-tensor are irrelevant and we will just drop them. The ﬁrst basis invariant hence is Y1 := Y = Y a a . (3.17) (3.17) Factorization does not take place for the second projection with the symmetrizer, which results in Factorization does not take place for the second projection with the symmetrizer, which results in Y a′b′ 3 := a′ b′ Y = 1 2 Y a′b′ + Y b′a′ . (3.18) (3.18) 4Spelled out as δa′ aδb′ b −δa′ bδb′ a = −εa′b′εab, this is nothing but the well-known Schouten identity for SU(2) with two indices lowered (see e.g. [64]). The crucial point here is really that this leads to (in some cases only partly) factorization of this and much larger projection operators (see below), also generalizing to SU(N). 4Spelled out as δa′ aδb′ b −δa′ bδb′ a = −εa′b′εab, this is nothing but the well-known Schouten identity for SU(2) with two indices lowered (see e.g. [64]). The crucial point here is really that this leads to (in some cases only partly) factorization of this and much larger projection operators (see below), also generalizing to SU(N). – 7 – – 7 – The hermitian projection operators needed to decompose Z are more involved. They can be constructed following the rules of [53–55].5 The projectors for the singlets read P a b c d = 4 3 , and P a c b d = 4 3 , (3.19) (3.19) while the triplet projection operators are given by while the triplet projection operators are given by P a b c d = 3 2 , P a c d b = 3 2 , P a b d c = 2 . (3.20) (3.20) JHEP05(2019)208 Here and in the following we will drop arrows and indices on birdtracks whenever restoring them works in the obvious way. The most general 2HDM scalar potential can be written as The last remaining projection operator is P5 to project on a b c d . This is simply the total symmetrizer of four indices, and so we do not display it. Here and in the following we will drop arrows and indices on birdtracks whenever restoring them works in the obvious way. The last remaining projection operator is P5 to project on a b c d . This is simply the total symmetrizer of four indices, and so we do not display it. Again, note how due to the factorization rule (3.12), both of the singlet projection operators in (3.19) factorize. Upon projection this nucleates a vacuum bubble diagram which transforms as a trivial singlet. Acting with the operators of eq. (3.19) on Z, the two singlets result as Z1(1) := Z = 1 2 Zab ab + Zab ba , and (3.21) Z1(2) := Z = εabεcdZab cd . (3.22) (3.21) Z1(2) := Z = εabεcdZab cd . (3.22) (3.22) Next we construct the triplet building blocks of Z. However, before applying projection operators on Z to extract the triplet irreps, observe that due to the symmetry in eq. (3.3), Z identically decomposes into a b c d Z = Z + Z . (3.23) a b c d Z = Z + Z . (3.23) a b c d Z = Z + Z . (3.23) (3.23) The second term is immediately identiﬁed as the trivial singlet Z1(2) by using the factoriza- tion rule (3.12). Hence, it will not contribute to the triplet irreps. Using this decomposition it is also easy to see that the triplet 3(2) vanishes identically: the corresponding projection operator (in the middle of (3.20)) anti-symmetrizes the ﬁrst two indices and symmetrizes the last two indices. Thus, acting with it on Z (using the decomposition (3.23)) it is – 8 – unavoidable that symmetrizers and anti-symmetrizers get mutually connected — which al- ways annihilates any contribution. The remaining two triplets, 3(1) and 3(3) of eq. (3.7), are degenerate. This can be shown via a straightforward direct computation, or alternatively diagrammatically. We ﬁnd 3 2 Zab 3(1) = Zab 3(3) = Z =: Zab 3 , (3.24) (3.24) JHEP05(2019)208 where we have deformed the box of Z to make the result more pleasant to the eye. The most general 2HDM scalar potential can be written as Spelling out Zab 3 explicitly would already be cumbersome, while diagrammatically it can be repre- sented in a compact way. where we have deformed the box of Z to make the result more pleasant to the eye. Spelling out Zab 3 explicitly would already be cumbersome, while diagrammatically it can be repre- sented in a compact way. Finally, the ﬁve-plet Z5 is simply given by the total symmetrization of all indices of and so we do not show this. To conclude their construction, we explicitly state the obtained building blocks in terms of the components of Y and Z. Denoting their components as [Y ]a b = yab and [Z]ab cd = zabcd, and dropping the irrelevant global prefactors of all covariants, one ﬁnds Y1 = y11 + y22 , (3.25a) Z1(1) = z1111 + z1212 + z1221 + z2222 , (3.25b) Z1(2) = z1212 −z1221 , (3.25c) Y ab 3 = y12 1 2 (y22 −y11) 1 2 (y22 −y11) −y∗ 12 ! , (3.25d) Y1 = y11 + y22 , (3.25a) Z1(1) = z1111 + z1212 + z1221 + z2222 , (3.25b) Z1(2) = z1212 −z1221 , (3.25c) Y ab 3 = y12 1 2 (y22 −y11) 1 2 (y22 −y11) −y∗ 12 ! , (3.25d) Zab 3 = z1112 + z1222 1 2 (z2222 −z1111) 1 2 (z2222 −z1111) −(z1112 + z1222)∗ ! , (3.25e) Zabcd 5 =       ζ1 ζ2 ζ2 ζ3 ! ζ2 ζ3 ζ3 −ζ∗ 2 ! ζ2 ζ3 ζ3 −ζ∗ 2 ! ζ3 −ζ∗ 2 −ζ∗ 2 ζ∗ 1 !       . (3.25f) 2 (y22 −y11) −y∗ 12 ! Zab 3 = z1112 + z1222 1 2 (z2222 −z1111) 1 2 (z2222 −z1111) −(z1112 + z1222)∗ ! , (3.25e) Zabcd 5 =       ζ1 ζ2 ζ2 ζ3 ! ζ2 ζ3 ζ3 −ζ∗ 2 ! ζ2 ζ3 ζ3 −ζ∗ 2 ! ζ3 −ζ∗ 2 −ζ∗ 2 ζ∗ 1 !       . (3.25f) Zab 3 = z1112 + z1222 1 2 (z2222 −z1111) 1 2 (z2222 −z1111) −(z1112 + z1222)∗ ! , (3.25e) Here, {y11, y22, z1111, z1212, z1221, z2222} ∈R, {y12, z1122, z1222, z1112} ∈C, and ζ1 := z1122 , (3.26a) ζ2 := 1 2 (z1222 −z1112) , (3.26b) ζ3 := 1 6 (z1111 −2z1212 −2z1221 + z2222) . The most general 2HDM scalar potential can be written as (3.26c) (3.26c) This explicitly shows how the 14 independent parameters of Y and Z combine to form the irreps of eq. (3.4). For completeness, we have also stated the building blocks in the conventional parametrization of the 2HDM scalar potential in appendix B. – 9 – 7This implies that we restrict ourselves here to CP transformation of order two, see e.g. [65]. F higher-order CP transformations one has to carry through the matrix U, but the result is the same. 4 CP properties of the building blocks Having the complete set of building blocks at hand, let us study their transformation behavior under general CP symmetries. Under a general CP transformation the scalars transform as6 Φa 7→Φ∗ b [U T]b a , Φ∗ a 7→[U ∗]a b Φb . (4.1) Φ∗ a 7→[U ∗]a b Φb . (4.1) Φa 7→Φ∗ b [U T]b a , (4.1) Applied to (3.1) we see that this transformation can equivalently be described by a mapping of parameters Applied to (3.1) we see that this transformation can equivalently be described by a mapping of parameters JHEP05(2019)208 Y a b 7→ U T Y T U ∗a b , (4.2) Y a b 7→ U T Y T U ∗a b , (4.2) Zab cd 7→[U T]a a′ [U T]b b′ [ZT]a′b′ c′d′ [U ∗]c′ c [U ∗]d′ d . (4.3) (4.2) b b Zab cd 7→[U T]a a′ [U T]b b′ [ZT]a′b′ c′d′ [U ∗]c′ c [U ∗]d′ d . (4.3) (4.3) Assuming U to be symmetric7 one can always choose a basis in ﬂavor space in which U ∝1. Assuming for a minute that we had transformed the theory to such a basis, the transformation of the coupling tensors simpliﬁes to Assuming U to be symmetric7 one can always choose a basis in ﬂavor space in which U ∝1. Assuming for a minute that we had transformed the theory to such a basis, the transformation of the coupling tensors simpliﬁes to Y a b 7→Y b a = (Y a b )∗, (4.4) Zab cd 7→Zcd ab = (Zab cd)∗, (4.5) (4.4) (4.4) (4.5) (4.4) (4 5) (4.5) where we have used hermiticity, cf. (3.2), for the last equalities. For the building blocks this implies the transformation where we have used hermiticity, cf. (3.2), for the last equalities. For the building blocks this implies the transformation Y1 7→Y1 , Z1(1) 7→Z1(1) , Z1(2) 7→Z1(2) , Y ab 3 7→−(Y3)ab , Zab 3 7→−(Z3)ab , Zabcd 5 7→(Z5)abcd . (4.6) (4.6) It follows that basis invariants (for which all indices have to be contracted) can at most transform with a sign under a CP transformation of order two. 4 CP properties of the building blocks Furthermore, we immedi- ately recognize a simple rule to distinguish CP-even and CP-odd basis invariants: It follows that basis invariants (for which all indices have to be contracted) can at most transform with a sign under a CP transformation of order two. Furthermore, we immedi- ately recognize a simple rule to distinguish CP-even and CP-odd basis invariants: A basis invariant is CP even odd iﬀit contains an even odd number of triplet building blocks (Y3, Z3). 6We focus here on the transformation behavior in the SU(2) Higgs-ﬂavor space, hence suppress t transformation with respect to spacetime and internal gauge symmetries. 5.1 Number and structure of the invariants We have identiﬁed all possible linear invariants. Our goal is now to construct non-linear (in the potential parameters) higher-order invariants out of the covariantly transforming 6We focus here on the transformation behavior in the SU(2) Higgs-ﬂavor space, hence suppress the ansformation with respect to spacetime and internal gauge symmetries. 7 6We focus here on the transformation behavior in the SU(2) Higgs-ﬂavor space, hence suppress the transformation with respect to spacetime and internal gauge symmetries. 7This implies that we restrict ourselves here to CP transformation of order two, see e.g. [65]. For higher-order CP transformations one has to carry through the matrix U, but the result is the same. – 10 – building blocks Y3, Z3, and Z5. To construct these invariants explicitly, we will once again make use of hermitian Young projection operators. However, before we do this we ﬁrst want to determine the size and structure (in terms of the building blocks) of the generating set of invariants. Even though it is conceivable that even this step can be performed diagrammatically, this seems like a rather tedious way to progress. We will ease this step by using the (multi-graded) Hilbert series [38, 39, 42] (see [50] for an accessible introduction and [19] for many examples in the particle physics context). The Hilbert series (HS) together with the Plethystic logarithm (PL) will allow us to fully characterize the ring of basis invariants and, furthermore, reveal the structure of all sought invariants. This input then will be merged with our diagrammatic approach to ﬁnally construct all required invariants explicitly. JHEP05(2019)208 The linear invariants are irrelevant for the construction of non-trivial higher-order invariants. We will, therefore, focus only on the non-trivially transforming building blocks in this section and add the linear invariants back in later. We will ﬁrst derive the (multi-graded) HS and PL and then discuss their informatio content. To ease the notation we deﬁne the symbols y b= Y3 , t b= Z3 , and q b= Z5 . We will need the character polynomials χr(z) for the relevant SU(2) irreps r, which are given by (see e.g. [50], appendix A.2) We will need the character polynomials χr(z) for the relevant SU(2) irreps r, which are given by (see e.g. 9The HS of the 2HDM scalar sector has only very recently appeared in the literature for the ﬁrst time, see eq. (A.5) of [30]. We do not include the linear invariants here, which explains the slight diﬀerence to [30]. Otherwise our results are in full agreement. 5.1 Number and structure of the invariants [50], appendix A.2) χ3(z) = z2 + 1 + 1 z2 , (5.1a) χ5(z) = z4 + z2 + 1 + 1 z2 + 1 z4 , (5.1b) (5.1a) (5.1b) as well as the plethystic exponential (PE), which is deﬁned as (see e.g. [38, 39]) PE [z, x, r] := exp ∞ X k=1 xk χr(zk) k ! . (5.2) (5.2) Using the token variables q, y, and t as deﬁned above, the multi-graded HS is computed as (see e.g. [44] for the integral measure) H(q, y, t) = 1 2πi I \|z\|=1 dz z 1 −z2 PE [z, q, 5] PE [z, y, 3] PE [z, t, 3] . (5.3) (5.3) Performing the integration via the residue theorem we ﬁnd Performing the integration via the residue theorem we ﬁnd H(q, y, t) = N (q, y, t) D (q, y, t) , (5.4) (5.4) with the numerator N (q, y, t) = 1 + qty + q2ty + qt2y + qty2 + q2t2y + q2ty2 + q3t3 + q3t2y + q3ty2 + q3y3 −q3t4y −q3t3y2 −q3t2y3 −q3ty4 −q4t3y2 −q4t2y3 −q5t3y2 −q5t2y3 −q4t3y3 −q5t3y3 −q6t4y4 , (5.5) (5.5) – 11 – and the denominator D (q, y, t) = 1 −t2 1 −y2 (1 −ty) 1 −q2 1 −q3 1 −qt2 1 −qy2 1 −q2t2 1 −q2y2 . (5.6) D (q, y, t) = 1 −t2 1 −y2 (1 −ty) 1 −q2 1 −q3 1 −qt2 1 −qy2 1 −q2t2 1 −q2y2 . (5.6) (5.6) We have expanded N and D of H(q, y, t) to a form in which the leading non-trivial term in N is positive. In this form, we observe that the numerator is anti-palindromic, N(q, y, t) = −q6y4t4 N(q−1, y−1, t−1). We have expanded N and D of H(q, y, t) to a form in which the leading non-trivial term in N is positive. In this form, we observe that the numerator is anti-palindromic, N(q, y, t) = −q6y4t4 N(q−1, y−1, t−1). Very important information is contained in the multi-graded PL, which is deﬁned a PL [H (q, y, t)] := ∞ X k=1 µ(k) ln H qk, yk, tk k , (5.7) (5.7) JHEP05(2019)208 here µ(k) is the M¨obius function. 8The original reference for this function seems to be [36] and it has also been discussed in [37]. It was introduced in a particle physics context in [38, 39]. 9 5.1 Number and structure of the invariants Expanding the PL around zero for all variables we ﬁnd PL [H (q, y, t)] = t2 + ty + y2 + q2 + qt2 + qty + qy2 + q3 + qt2y + q2t2 + qty2 + q2ty + q2y2 + q2t2y + q2ty2 + q3t3 + q3t2y + q3ty2 + q3y3 −q2t2y2 −q2t3y2 −q2t2y3 −q3t2y2 −q2t4y2 −q3t4y −q2t3y3 −3q3t3y2 −q2t2y4 −3q3t2y3 −q4t2y2 −q3ty4 −O [tyq]9 . (5.8) (5.8) The usual, ungraded, HS can directly be obtained from the multi-graded version by equat- ing all arguments9 h(z) ≡H(z, z, z) = 1 + z3 + 4 z4 + 2 z5 + 4 z6 + z7 + z10 (1 −z2)4 (1 −z3)3 (1 −z4) . (5.9) (5.9) As expected for a reductive group like SU(2), we can ﬁnd a form of h(z) in which all numerator coeﬃcients are positive. In this form the numerator of h(z) is palindromic, i.e. N(z) = z10N(z−1). Finally, the ungraded PL is given by PL [h(z)] = 4 z2 + 4 z3 + 5 z4 + 2 z5 + 3 z6 −3 z7 −O z8 . (5.10) (5.10) Now, let us point out the relevant information content of these functions for this study: Now, let us point out the relevant information content of these functions for this study: • The denominator of the HS in eq. (5.9) informs us about the smallest complete set of algebraically independent invariants. We read oﬀthat there are four algebraically independent invariants of order 2, three of order 3 and one of order 4. • The denominator of the HS in eq. (5.9) informs us about the smallest complete set of algebraically independent invariants. We read oﬀthat there are four algebraically independent invariants of order 2, three of order 3 and one of order 4. • The leading positive terms of the multi-graded PL in eq. (5.8) correspond to the number and structure of invariants in the generating set of the ring. To be clear, the leading 19 terms correspond to all invariants which are needed to express any other invariant as a polynomial of them. For example, the leading t2 term tells us that there will be one generating invariant originating from the tensor product Z3 ⊗Z3. The second term, ty, tells us that there will be one invariant from the tensor product Z3 ⊗Y3, etc. 5.1 Number and structure of the invariants – 12 – • The leading negative terms of the multi-graded PL in eq. (5.8) cut-oﬀthe set of generating invariants. Furthermore, these terms tell us the structure and number of relations between the invariants. For example, the leading negative term, −q2y2t2, of the total order 6 indicates that there is one relation between invariants of that structure. The term −3q3t3y2 tells us that there are three independent relations between invariants of that structure, etc. There is more useful information encoded in these functions but this is the most relevant for the sake of this study. There is more useful information encoded in these functions but this is the most relevant for the sake of this study. JHEP05(2019)208 5.2.1 Needed projection operators After having found the number and internal structure of the non-linear invariants, we proceed with their explicit construction. Due to their symmetry properties, we can directly represent the building blocks as symmetrized boxes Y3 ≡ , Z3 ≡ , Z5 ≡ . (5.11) (5.11) Here we have introduced a color coding to tell apart boxes of the diﬀerent building blocks. Arbitrary invariants of higher order are now obtained by taking tensor products of the building blocks and projecting out the invariants. It turns out that all required projection operators are very simple and always of the form 1 2 · · · n n+1n+2 · · · 2n =⇒P 1 2 · · · n n+1n+2 · · · 2n = a′ 1 a′ 2 . . . a′ n a′ n+1 . . . a′ 2n · · · a1 . . . an an+1 . . . a2n . (5.12) This follows from two facts: (i) We are only interested in invariants, and the operators that project onto invariants are precisely the ones which arise from Young tableaux of the “complete chocolate bar” shape (i.e. ··· ··· ). (ii) Other Young tableaux of the same shape but with a diﬀerent assignment of indices do exist (e.g. 1 2 3 5 4 6 7 8 instead of 1 2 3 4 5 6 7 8 ), but their corresponding projection operators in our case will not give rise to invariants which are independent of the ones obtained via the projection operators in (5.12). 1 2 · · · n n+1n+2 · · · 2n =⇒P 1 2 · · · n n+1n+2 · · · 2n = a′ 1 a′ 2 . . . a′ n a′ n+1 . . . a′ 2n · · · a1 . . . an an+1 . . . a2n . (5.12) This follows from two facts: (i) We are only interested in invariants, and the operators that project onto invariants are precisely the ones which arise from Young tableaux of the 1 2 · · · n n+1n+2 · · · 2n =⇒P 1 2 · · · n n+1n+2 · · · 2n = a′ 1 a′ 2 . . . a′ n a′ n+1 . . . a′ 2n · · · a1 . . . an an+1 . . . a2n . 5.2.2 Algebraically independent invariants (5.14) Here and in the following we suppress indices in the Young tableaux whenever they are meant to be assigned in the trivial way (incremental increase by one from left to right in each line, cf. (5.12)). The corresponding projection operators always have the form stated in (5.12), and acting with them on the respective tensor product of building blocks produces the corresponding invariant. Explicit expressions for the invariants obtained in this way are collected in appendix D and an explicit criterion to check the algebraic independence of arbitrary polynomials is given in appendix A. Here and in the following we suppress indices in the Young tableaux whenever they are meant to be assigned in the trivial way (incremental increase by one from left to right in each line, cf. (5.12)). The corresponding projection operators always have the form stated in (5.12), and acting with them on the respective tensor product of building blocks produces the corresponding invariant. Explicit expressions for the invariants obtained in this way are collected in appendix D and an explicit criterion to check the algebraic independence of arbitrary polynomials is given in appendix A. Including the linear invariants we have now constructed a total of eleven invariants. These form a maximal set of algebraically independent invariants for the 2HDM. This num- ber corresponds to the well-known number of 11 physical parameters of the 2HDM scalar sector. As always, the set of algebraically independent invariants is not unique. However, our choice certainly is the simplest in terms of the order of the individual invariants. We see that for the 2HDM scalar sector it is possible to ﬁnd a maximal set of alge- braically independent invariants which are all CP-even. Just as in the case of the SM [19], this indicates that one can express necessary and suﬃcient conditions for CP conservation solely in terms of CP-even quantities.10 11This number only by accident coincides with the numbers 11 above, i.e. it has nothing to do with the number of physical parameters. 5.2.2 Algebraically independent invariants 5.2.2 Algebraically independent invariants 10In the SM this would be the area of the CKM unitarity triangle reconstructed by the length of its sides. We thank Jo˜ao P. Silva for reminding us of this example. 10In the SM this would be the area of the CKM unitarity triangle reconstructed by the length of its sides. We thank Jo˜ao P. Silva for reminding us of this example. 11This number only by accident coincides with the numbers 11 above, i.e. it has nothing to do with the 5.2.2 Algebraically independent invariants We are now equipped to construct invariants of arbitrary order simply by projection. Let us introduce the following naming scheme for higher-order invariants: Ia,b,c b= Invariant containing powers qa, yb, and tc, that is Z⊗a 5 ⊗Y ⊗b 3 ⊗Z⊗c 3 . Ia,b,c b= Invariant containing powers qa, yb, and tc, that is Z⊗a 5 ⊗Y ⊗b 3 ⊗Z⊗c 3 . Ia,b,c b= Invariant containing powers qa, yb, and tc, that is Z⊗a 5 ⊗Y ⊗b 3 ⊗Z⊗c 3 . For completeness, we recall the three linear invariants, which are given by Y1 = 1 2 , Z1(1) = 1 2 3 4 , and Z1(2) = 1 3 2 4 . (5.13) (5.13) JHEP05(2019)208 We proceed with the construction of a set of non-linear independent invariants. The or- der of the sought invariants corresponds to the denominator factors of the HS (5.9), and their tensor product structure can be read oﬀfrom the graded PL (5.8). The resulting invariants are invariants are I2,0,0 := , I0,2,0 := , I0,1,1 := , I0,0,2 := , I3,0,0 := , I1,2,0 := , I1,0,2 := , and I2,1,1 := . (5.14) Here and in the following we suppress indices in the Young tableaux whenever they are meant to be assigned in the trivial way (incremental increase by one from left to right in each line, cf. (5.12)). The corresponding projection operators always have the form stated in (5.12), and acting with them on the respective tensor product of building blocks produces the corresponding invariant. Explicit expressions for the invariants obtained in this way are collected in appendix D and an explicit criterion to check the algebraic independence of arbitrary polynomials is given in appendix A. I2,0,0 := , I0,2,0 := , I0,1,1 := , I0,0,2 := , I3,0,0 := , I1,2,0 := , I1,0,2 := , and I2,1,1 := . (5.14) Here and in the following we suppress indices in the Young tableaux whenever they are meant to be assigned in the trivial way (incremental increase by one from left to right in I0,1,1 := , I0,0,2 := , I1,0,2 := , and (5.14) I0,2,0 := , I1,2,0 := , I2,0,0 := , I3,0,0 := , I2,1,1 := . I2,0,0 := , I3,0,0 := , I2,1,1 := . 5.2.1 Needed projection operators (5.12) This follows from two facts: (i) We are only interested in invariants, and the operators that project onto invariants are precisely the ones which arise from Young tableaux of the “complete chocolate bar” shape (i.e. ··· ··· ). (ii) Other Young tableaux of the same shape but with a diﬀerent assignment of indices do exist (e.g. 1 2 3 5 4 6 7 8 instead of 1 2 3 4 5 6 7 8 ), but their corresponding projection operators in our case will not give rise to invariants which are independent of the ones obtained via the projection operators in (5.12). This follows from two facts: (i) We are only interested in invariants, and the operators that project onto invariants are precisely the ones which arise from Young tableaux of the “complete chocolate bar” shape (i.e. ··· ··· ). (ii) Other Young tableaux of the same shape but with a diﬀerent assignment of indices do exist (e.g. 1 2 3 5 4 6 7 8 instead of 1 2 3 4 5 6 7 8 ), but their corresponding projection operators in our case will not give rise to invariants which are independent of the ones obtained via the projection operators in (5.12). Note that (ii) is not true in general, cf. the two independent singlets extracted from Z via 1 2 3 4 and 1 3 2 4 above. Nonetheless, for tensor products of only Y3, Z3, and Z5, the statement (ii) holds and we have conﬁrmed this explicitly. This can also be read oﬀdirectly from the PL (5.8), where we ﬁnd that no single invariant structure of the generating set appears with multiplicity higher than one — implying that each single tensor product Z⊗a 5 ⊗Y ⊗b 3 ⊗Z⊗c 3 can host at most one independent invariant. – 13 – 5.2.3 Completing the generating set The complete generating set of invariants contains eleven11 additional invariants and we collect them here. After reading oﬀtheir structure from the graded PL, eq. (5.8), the – 14 – construction proceeds in a straightforward way. We ﬁnd construction proceeds in a straightforward way. We ﬁnd I1,1,1 := , I2,2,0 := , J1,2,1 := , J2,2,1 := , J3,3,0 := , J3,2,1 := , I1,1,1 := , I2,2,0 := , I2,0,2 := , J1,2,1 := , J1,1,2 := , J2,2,1 := , J2,1,2 := , J3,3,0 := , J3,0,3 := , J3,2,1 := , J3,1,2 := . (5.15) I2,0,2 := , J1,1,2 := , J2,1,2 := , J3,0,3 := , J3,1,2 := . (5.15) JHEP05(2019)208 (5.15) Invariants that contain an odd total number of Y3 and Z3 are CP-odd, cf. section 4. and we denote them by the letter J instead of I. We give explicit expressions for all of these invariants in appendix D. All of our invariants group into permutation representations under a Y3(y) ↔Z3(t) exchange transformation, which is, of course, what one would expect because Y3 and Z3 behave identically under basis changes. This completes the construction of the generating set of invariants. Note that the invariants constructed in this section are not algebraically independent of the invariants in section 5.2.2 above. Therefore, each of these invariants fulﬁlls a polynomial relation with the other invariants and we will now proceed to construct some of these relations explicitly. 6 Systematic construction of Syzygies By deﬁnition of the generating set, it must be possible to express all higher-order invariants as polynomials in the invariants listed above. This requires relations between the higher- order invariants and the invariants of the generating set which are also called syzygies. We are not aware of any previously stated generally applicable strategy to system- atically construct syzygies. However, by explicit computation we ﬁnd that the following strategy works here: Any of the leading negative terms in the multi-graded PL, eq. (5.8), seems to corre- spond to a new, independent relation amongst the invariants. The structure of the cor- responding term corresponds to the structure of the relation(s), and the coeﬃcient of the term gives the number of independent relations of this structure. Simply making an ansatz of to-be related invariants in terms of suitable power products of lower order invariants, one just has to solve a linear system in order to obtain the desired syzygie(s). For example, the leading negative term in (5.8) is −q2y2t2. All possible power products of invariants that match this structure are I2 1,1,1 , I2,1,1 I0,1,1 , I2,2,0 I0,0,2 , I2,0,2 I0,2,0 , I1,2,0 I1,0,2 , I2,0,0 I0,2,0 I0,0,2 , I2,0,0 I2 0,1,1 . (6.1) I2,2,0 I0,0,2 , I2,0,2 I0,2,0 , I2,0,0 I2 0,1,1 . (6.1) I2 1,1,1 , I1,2,0 I1,0,2 , I2 1,1,1 , I2,2,0 I0,0,2 , I2,1,1 I0,1,1 , (6.1) – 15 – A simple linear ansatz then reveals the ﬁrst syzygy:12 A simple linear ansatz then reveals the ﬁrst syzygy:12 A simple linear ansatz then reveals the ﬁrst syzygy:12 3 I2 1,1,1 = 2 I2,1,1 I0,1,1 −I2,2,0 I0,0,2 −I2,0,2 I0,2,0 + 3 I1,2,0 I1,0,2 + I2,0,0 I0,2,0 I0,0,2 −I2,0,0 I2 0,1,1 . (6.2) (6.2) In principle it should be possible to ﬁnd all syzygies by this strategy and we have explicitly checked that this works for invariants up to a total order of 14, see table 1. There is a caveat: observe the diﬀerences between the (expanded) HS coeﬃcients and the PL coeﬃcients of the same term, for example There is a caveat: observe the diﬀerences between the (expanded) HS coeﬃcients an the PL coeﬃcients of the same term, for example H(q, y, t) = · · · + 6 q2t2y2 + · · · + 10 q4t2y2 + . . . 6 Systematic construction of Syzygies , PL [H (q, y, t)] = · · · −1 q2t2y2 + · · · −1 q4t2y2 + . . . . (6.3) JHEP05(2019)208 (6.3) For the above example — q2t2y2 — the diﬀerence between the HS and PL coeﬃcients 6 −(−1) = 7 matches the number 7 of possible power products of the generating invariants. In some cases, however, the number of possible power products exceeds this diﬀerence. For example, this happens for the invariants of order q4t2y2, for which according to the PL coeﬃcient there is one non-trivial relation. The diﬀerence in the coeﬃcients between HS and PL here is 11 — but we ﬁnd that there are 12 possible power products. This is indicative of an additional relation of the structure q4t2y2, in addition to the one counted by the “−1” in the PL, eq. (6.3). However, this additional relation turns out not to be independent of the other relations. Rather, it is the “old” q2t2y2 relation eq. (6.2) multiplied by the invariant q2, thus producing a relation of the order q4t2y2. While this happening does not give an obstacle for the explicit construction of independent syzygies, one should keep it in mind for explicit computations. The structure of all relations that we have explicitly constructed and checked in this way are shown in table 1 in appendix E. Altogether this gives some evidence to the suspicion that one can simply read oﬀthe total number of independent relations and their structure from the negative terms of the PL. 12Comparing this to the corresponding relation in the trace basis, e.g. eq. (A.4) in [30], gives a feeling f the simpliﬁcation arising from the use of orthogonal projectors. 7 Necessary and suﬃcient conditions for explicit CPV Let us now make use of our newly gained knowledge about the 2HDMs invariants. In a formidable explicit computation [9] it has been shown that an equivalent condition to explicit CP conservation in the 2HDM scalar sector is the vanishing of the four speciﬁc CP-odd basis invariants I2Y 2Z ∼J1,2,1 , IY 3Z ∼J1,1,2 , I3Y 3Z ∼J3,3,0 , I6Z ∼J3,0,3 . (7.1) IY 3Z ∼J1,1,2 , I6Z ∼J3,0,3 . (7.1) I2Y 2Z ∼J1,2,1 , I3Y 3Z ∼J3,3,0 , I2Y 2Z ∼J1,2,1 , (7.1) I3Y 3Z ∼J3,3,0 , We have also stated here the corresponding invariants in the notation of this paper. “∼” here means that the non-trivial parts of the invariants coincide while their exact expressions 12Comparing this to the corresponding relation in the trace basis, e.g. eq. (A.4) in [30], gives a feeling for the simpliﬁcation arising from the use of orthogonal projectors. – 16 – may diﬀer by a global numerical prefactor and an admixture of lower-order invariants of the correct structure. The exact relations are given in appendix C. We will now give a very simple proof of the necessary and suﬃcient conditions for explicit CPV by making use of the interrelation of the invariants. The ﬁrst step of this proof is the insight that instead of a potentially inﬁnite number of CP-odd invariants we only have to deal with the CP-odd invariants in the generating set of the ring. This is clear from the fact that any other invariant can be expressed as a polynomial in these. From eq. (5.15) we then ﬁnd that there are eight CP-odd invariants in the generating set of the ring. Thus, in order to prove Gunion and Haber right, there should be at least four independent relations amongst these invariants. In fact, we ﬁnd that there are many more relations among the CP-odd invariants, cf. table 1. JHEP05(2019)208 The ﬁrst two relations are arising at a total order 7 and they read J2,2,1I0,0,2 + J2,1,2I0,1,1 −J1,2,1I1,0,2 + J1,1,2I1,1,1 = 0 , and y ↔t . (7.2) (7.2) Note how this is a fully “CP-odd relation” which is actually sensitive to the signs of the CP-odd invariants and not only to their magnitude. This relation will not be used in the proof but we have stated it for completeness. 8 Towards a Hironaka decomposition Finally, we wish to comment on the possibility of representing the ring of invariants of the 2HDM in an even simpler way. Due to the fact that the SU(2) group of basis changes is reductive, it follows that the ring of invariants obeys the Cohen-Macaulay (CM) prop- erty implying the existence of a so-called Hironaka decomposition [60, 61] (cf. also [35] section 2.3, [49] section 5.4.1, [66] appendix A, or [67] section 3). Let us explain what that means. Writing the HS of eq. (5.9) as h(z) = sP i=1 zsi pQ j=1 (1 −zpj) , (8.1) JHEP05(2019)208 (8.1) deﬁnes the numbers p, pj, s, and si.13 The CM property then warrants that the ring can be generated in terms of a number of p primary invariants of the orders pj, commonly denoted as θj, together with a number of s secondary invariants of the orders si, commonly denoted as ηi. Together, the primary and secondary invariants form a Hironaka decomposition of the ring, implying that every invariant I can be written as I = s X i=1 ηi C [θ1, . . . , θp] . (8.2) (8.2) Here C [θ1, . . . , θp] denotes arbitrary polynomials in the primary invariants with potentially complex coeﬃcients and ηi are the secondary invariants. The astonishing feature of this decomposition is that the secondary invariants only enter linearly. Using the Hironaka decomposition can simplify the analysis of the ring and the related physical discussion. For example, imagine the situation that all primary invariants of a ring are CP-even, while CP-odd invariants only arise as secondary invariants. In order to ﬁnd necessary and suﬃcient conditions for CP conservation, one then would only have to investigate the linear span of the CP-odd secondary invariants (with coeﬃcients in the primary invariants). We stress that the power product invariants stated above in section 5.2.2 and sec- tion 5.2.3 do not directly correspond to the primary and secondary invariants of the Hiron- aka decomposition. Thus, given the generating set of a ring, the main task in obtaining a Hironaka decomposition is the identiﬁcation of primary and secondary invariants. This can be complicated by the fact that some of the primary and secondary invariants may only arise as combinations of invariants in the generating set. Whether this happens is dictated by the interrelations of invariants. 7 Necessary and suﬃcient conditions for explicit CPV Furthermore, we ﬁnd two CP-odd relations of total order 8 that read Note how this is a fully “CP-odd relation” which is actually sensitive to the signs of the CP-odd invariants and not only to their magnitude. This relation will not be used in the proof but we have stated it for completeness. Furthermore, we ﬁnd two CP-odd relations of total order 8 that read (7.3) 3 J2,2,1I1,2,0 −J3,2,1I0,2,0 + 3 J3,3,0I0,1,1 + J1,2,1I2,2,0 = 0 , and y ↔t . (7.3) nally, we also state the “CP-even” relations of the squared quintic CP-odd invariants 3 J 2 2,2,1 + 3 J3,3,0J1,1,2 −J3,2,1J1,2,1 −J 2 1,2,1I2,0,0 = 0 , and y ↔t . (7.4) (7.4) Remarkably, the squared quintics can be expressed almost entirely in terms of products of the other CP-odd invariants. Many more relations exist and have been derived, see table 1, but they are not needed for the proof and so we do not state them here explicitly. Let us now show that the vanishing of the invariants in (7.1), that is J1,2,1 = J1,1,2 = J3,3,0 = J3,0,3 = 0, (7.5) (7.5) is suﬃcient to conclude that all other CP-odd invariants are vanishing as well. This is readily conﬁrmed by inspection of eqs. (7.3) and (7.4). Using (7.5) it follows from (7.4) that also J2,2,1 = J2,1,2 = 0. Using this together with the condition (7.5) in (7.3) one ﬁnds that J3,2,1 = J3,1,2 = 0, but only under the assumption that I0,2,0 ̸= 0 ̸= I0,0,2. However, if I0,2,0 = I0,0,2 = 0 were to hold, this would itself imply that J3,2,1 = J3,1,2 = 0 to begin with, which is easy to show from the explicit form of the invariants given in appendix D. This completes the proof. The in total 6 novel relations (7.2), (7.3) and (7.4) are perhaps the main results of this paper. However, many more relations of this type exist, and so we are convinced that the main use of this work is the way of how we got there. – 17 – 13For the case of eq. (5.9) one ﬁnds p = 8, s = 14 as well as the corresponding orders of the invariants pj = {2, 2, 2, 2, 3, 3, 3, 4}, si = {0, 3, 4, 4, 4, 4, 5, 5, 6, 6, 6, 6, 7, 10}. 8 Towards a Hironaka decomposition In particular, any product of secondary invariants must either decompose in the form (8.2) or be a secondary invariant itself. The latter situation explains the appearance of secondary invariants of degree larger than any of the invariants in the generating set. These higher-order, so-called “reducible” secondary invariants can always be written as power products of a set of “irreducible” secondary invariants [68]. – 18 – We now comment on the construction of a Hironaka decomposition of the 2HDM ring. The primary invariants of order 2 and 3 are simply those listed in (5.14). The remaining cubic invariant I1,1,1 fulﬁlls the relation (6.2), rendering it a bona-ﬁde secondary invariant. The main obstacle, thus, is the identiﬁcation of the lone degree 4 primary invariant. It is clear that it must be a combination of the quartic invariants given in (5.14) and (5.15). Since there are no syzygies of the structure (q2y2)2, or (q2t2)2, and since there are no secondary invariants of order 8, both of the invariants I2,2,0, and I2,0,2 must be part of the sought primary invariant. In addition, the syzygy of the structure (q2yt)2 contains both, a term I2 2,1,1 as well as a term I2,2,0 × I2,0,2, thereby indicating that also the invariant I2,1,1 should appear in the sought quartic primary invariant. Other combinations of these invariants, together with the remaining quartic invariants I1,2,1 and I1,1,2 then should form the four quartic secondary invariants. Together, the sought primary and secondary invariants must obey the Hironaka decomposition (8.2). Trying to satisfy this requirement with an ansatz, however, leads to a complicated non-linear system of equations which we were unable to solve. The task of obtaining a Hironaka decomposition of the 2HDM ring, thus, remains to be solved in future work. JHEP05(2019)208 9 Summary and discussion We have presented a new algorithm to systematically construct basis invariants. Our method is based on algebraic invariant theory, with the powerful Hilbert series and Plethys- tic logarithm, as well as group theory, with Young tableaux and their corresponding her- mitian projection operators. Applied to the 2HDM, we have obtained a maximal set of algebraically independent invariants, as well as a complete generating set of invariants. We have also outlined a systematic approach for the construction of syzygies and showed some of them explicitly. Using the invariants of the generating set and their syzygies we have given a concise proof of the necessary and suﬃcient conditions for explicit CP conservation in the 2HDM. It should be remarked that the construction of building blocks and invariants in the 2HDM has also been performed by other means. For example, the building blocks of the present work correspond to the vectors and matrices obtained using the “bilinear formal- ism” [11, 69–77], and some of our CP-odd invariants are closely related to the ones obtained via graphical methods in [9, 21]. There are some advantages of our method as compared to previous approaches. First, our strategy does not require speciﬁc basis choices at any step. Then, by the use of the powerful invariant theory functions we can with certainty identify the full set of generating invariants, their structure, as well as their interrelations. This is the ﬁrst method where the answer to the question “when to stop constructing invariants” is given in a clear and quantitative fashion; it can be calculated in any model from our algorithm without the fear of miscounting. Furthermore, thanks to the use of orthogonal projection operators, our basis invariants are as short as possible by construction, and their CP properties are very transparent. Another attractive feature is the direct access to syzygies and their simple form of appearance. – 19 – An immediate further application of our method to the 2HDM would be the inclusion of vacuum expectation values to investigate possibly realistic models and the spontaneous violation of CP. Our method also includes the possibility of identifying how the invariants behave under the various additional Higgs-ﬂavor symmetries which are all subgroups of the group of basis changes. As a result, one would be able to identify how the number of independent invariants is reduced in models which are not the most general, but which have additional internal symmetries. 9 Summary and discussion Using our short invariants and relations, simpliﬁcations should also arise in the formulation of the renormalization group running [30, 31]. Another remaining open question in the 2HDM is the identiﬁcation of combinations of invariants to obtain a Hironaka decomposition of the ring. JHEP05(2019)208 Perhaps more important than our explicit results on the 2HDM is the method itself. In principle, our strategy generalizes to the fermion sector, three or even N-Higgs-doublet models, and also to completely diﬀerent models. A possible issue then could become computational power, since the construction of large Young projection operators easily ex- hausts memory limits of commercially available computing clusters. Also the computation of the Hilbert series and Plethystic logarithm straightforwardly extends to more compli- cated groups, but the corresponding integrals become more involved. Finally, the use of Young tableaux, at a ﬁrst glance, may seem to limit our explicit construction of invariants to SU(N) groups. However, all that is actually needed for the systematic construction of building blocks and invariants are the hermitian projection operators for the various tensor contractions. Since birdtracks can be used to construct these for all simple Lie groups [57], we are optimistic that our method for the construction of invariants can also be generalized in this direction. Acknowledgments I want to thank I. P. Ivanov, C. C. Nishi, and J. P. Silva for useful discussions and motiva- tion, and I. P. Ivanov and J. P. Silva for useful comments on the manuscript. Furthermore, I want to thank the Centre for Theoretical Particle Physics (CFTP) at T´ecnico Lisboa for hospitality at various stages of this work. This work has been supported by the German Science Foundation (DFG) within the SFB-Transregio TR33 “The Dark Universe”. The author is grateful to the authors of the LATEX packages ytableau and youngtab, and would also like to encourage the development of packages to typeset birdtracks and birdtrack pro- jection operators. 14Strictly speaking this works only for polynomials over ﬁelds of characteristic zero, see e.g. [78, 79]. A Algebraic (in)dependence of invariants The Jacobi criterion is an easy way to ﬁnd the number of algebraically independent polyno- mials.14 For a set of polynomials (here, invariants) Ii, depending on a number of variables xj (here, the components yab and zabcd of Y and Z), the number of algebraically indepen- – 20 – dent invariants is simply given by the rank of the Jacobian matrix: nt invariants is simply given by the rank of the Jacobian matrix: dent invariants is simply given by the rank of the Jacobian matrix: Number of algebraically independent invariants = rank ∂Ii ∂xj . (A.1) (A.1) Besides symbolic evaluation one can also use this criterion with all variables put to random numbers for a fast machine evaluation. Besides symbolic evaluation one can also use this criterion with all variables put to random numbers for a fast machine evaluation. B Building blocks in conventional notation JHEP05(2019)208 For completeness, we collect here the building blocks constructed in section 3 in the con- ventional parametrization of the 2HDM scalar potential in terms of m’s and λ’s. Following the replacement rules in ([9], eq. (21)) we ﬁnd (note that their Zabcd ≡2 Zac bd in our notation) y11 = m2 11 , y12 = −m2 12 , y22 = m2 22 , z1111 = λ1/2 , z2222 = λ2/2 , z1212 = λ3/2 , z1221 = λ4/2 , z1122 = λ5/2 , z1112 = λ6/2 z1222 = λ7/2 . (B.1) y11 = m2 11 , y12 = −m2 12 , y22 = m2 22 , z1111 = λ1/2 , z2222 = λ2/2 , z1212 = λ3/2 , z1221 = λ4/2 , z1122 = λ5/2 , z1112 = λ6/2 z1222 = λ7/2 . (B.1) (B.1) As usual m2 12, λ5, λ6, λ7 ∈C while all others are real. It is then straightforward to translate eq. (3.25) into this convention and we ﬁnd Y1 = m2 11 + m2 22 , (B.2a) Z1(1) = 1 2 (λ1 + λ2 + λ3 + λ4) , (B.2b) Z1(2) = 1 2 (λ3 −λ4) , (B.2c) 2 1 2 2 ! Y1 = m2 11 + m2 22 , (B.2a) Z1(1) = 1 2 (λ1 + λ2 + λ3 + λ4) , (B.2b) Z1(2) = 1 2 (λ3 −λ4) , (B.2c) Y ab 3 = −m2 12 1 2 m2 22 −m2 11 1 2 m2 22 −m2 11 (m2 12)∗ ! , (B.2d) Zab 3 = 1 2 λ6 + λ7 1 2 (λ2 −λ1) 1 2 (λ2 −λ1) −(λ6 + λ7)∗ ! , (B.2e) Zabcd 5 =       ξ1 ξ2 ξ2 ξ3 ! ξ2 ξ3 ξ3 −ξ∗ 2 ! ξ2 ξ3 ξ3 −ξ∗ 2 ! ξ3 −ξ∗ 2 −ξ∗ 2 ξ∗ 1 !       , (B.2f) Y1 = m2 11 + m2 22 , (B.2a) Z1(1) = 1 2 (λ1 + λ2 + λ3 + λ4) , (B.2b) Z1(2) = 1 2 (λ3 −λ4) , (B.2c) Y ab 3 = −m2 12 1 2 m2 22 −m2 11 1 2 m2 22 −m2 11 (m2 12)∗ ! , (B.2d) 1 λ + λ 1 (λ λ ) ! C Relation to the invariants of Gunion and Haber Here we state the original set of necessary and suﬃcient CP-odd invariants of Gunion and Haber ([9], eqs. (23)–(26)) in the notation of this paper, IY 3Z = Im h Zai icZej jbZbc edY d a i = −2i J1,1,2 , (C.1) I2Y 2Z = Im h Y a b Y c d Zbd af Zfi ic i = −2i J1,2,1 , (C.2) I6Z = Im h Zac bdZbℓ ℓf Zdp phZfj akZkm jnZnh mc i = −2i J3,0,3 , (C.3) I3Y 3Z = Im h Zab cdZcd egZef hqY g a Y h b Y q f i = 2i J3,3,0 + 2i J1,2,1 I0,1,1 + i Y 2 1 J1,1,2 . (C.4) (C.1) (C.2) (C.3) JHEP05(2019)208 (C.4) The ﬁrst three invariants only diﬀer by a global numerical factor, while the fourth in- variant of [9] actually contains admixtures of lower lying invariants. Note also that our invariants are pure complex numbers by construction. That is, they directly correspond to the imaginary, CP-odd part of a given contraction. The ﬁrst three invariants only diﬀer by a global numerical factor, while the fourth in- variant of [9] actually contains admixtures of lower lying invariants. Note also that our invariants are pure complex numbers by construction. That is, they directly correspond to the imaginary, CP-odd part of a given contraction. B Building blocks in conventional notation (B.2f) with with ξ1 := 1 2λ5 , ξ2 := 1 4 (λ7 −λ6) , ξ3 := 1 12 (λ1 −2λ3 −2λ4 + λ2) . (B.3) ξ1 := 1 2λ5 , ξ2 := 1 4 (λ7 −λ6) , ξ3 := 1 12 (λ1 −2λ3 −2λ4 + λ2) . (B.3) (B.3) – 21 – D.1 Algebraically independent invariants In this appendix we state explicitly our choice of a maximal set of algebraically independent basis invariants of the 2HDM. Note that these invariants are not written in any speciﬁc basis, i.e. they hold for all bases. To ease the notation we use redeﬁned parameters (conserving the total number of degrees of freedom, of course) which reﬂect the now known assignment of the Lagrangian parameters in Y and Z into the building blocks. Furthermore, it is convenient to split parameters into their real and imaginary parts. In our new conventions the building blocks of eq. (3.25) read Y1 : ys , (D.1a) Z1(1) : s1 , (D.1b) Z1(2) : s2 , (D.1c) Y ab 3 : i yi + yr y y i yi −yr ! , (D.1d) Zab 3 : i ti + tr t t i ti −tr ! , (D.1e) Zabcd 5 :       i qi1 + qr1 i qi2 + qr2 i qi2 + qr2 q3 ! i qi2 + qr2 q3 q3 i qi2 −qr2 ! i qi2 + qr2 q3 q3 i qi2 −qr2 ! q3 i qi2 −qr2 i qi2 −qr2 qr1 −i qi1 !       , (D.1f) – 22 – – 22 – – 22 – where now all fourteen parameters {ys, s1, s2, y, yr, yi, t, tr, ti, qr1, qi1, qr2, qi2, q3} are real. Their expressions in terms of the original parameters can easily be obtained by comparison with eq. (3.25) or eq. (B.2). The global prefactors of invariants are irrelevant for our purpose. However, the con- vention for the prefactors does, of course, matter in stating syzygies such as the ones in eq. (6.2), (7.2), (7.3), or (7.4). As a convention, we adjust the global prefactor of each invariant such as to render all internal coeﬃcients integer. This choice also makes the relative coeﬃcients in the syzygies very simple. D.1 Algebraically independent invariants Explicit expressions for the algebraically independent invariants of section 5.2.2 are JHEP05(2019)208 Y1 := 1 2 = (y11 + y22) ≡ys, Z1(1) := 1 2 3 4 = (z1111 + z1212 + z1221 + z2222) ≡s1, Z1(2) := 1 3 2 4 = (z1212 −z1221) ≡s2, (D.2a) := 1 2 = (y11 + y22) ≡ys, ) := 1 3 2 4 = (z1212 −z1221) ≡s2, (D.2a) Z1(2) := 1 3 2 4 = (z1212 −z1221) ≡s2, (D.2a) I0,2,0 := = y2 + y2 i + y2 r , I0,1,1 := = (ty + tiyi + tryr) , I0,0,2 := = t2 + t2 i + t2 r , I2,0,0 := = 3q2 3 + q2 i1 + 4q2 i2 + q2 r1 + 4q2 r2 , (D.2b) I0,0,2 := = t2 + t2 i + t2 r , I2,0,0 := = 3q2 3 + q2 i1 + 4q2 i2 + q2 r1 + 4q2 r2 , (D.2b) I2,0,0 := = 3q2 3 + q2 i1 + 4q2 i2 + q2 r1 + 4q2 r2 , (D.2b) (D.2b) (D.2b) I1,2,0 := = q3 2y2 −y2 i −y2 r + 4y (qi2yi + qr2yr) + 2qi1yiyr + qr1 y2 r −y2 i , I1,0,2 := = q3 2t2 −t2 i −t2 r + 4t (qi2ti + qr2tr) + 2qi1titr + qr1 t2 r −t2 i , I3,0,0 := = −q3 q2 i1 −2q2 i2 + q2 r1 −2q2 r2 + q3 3 −2q2 i2qr1 + 4qi1qi2qr2 + 2qr1q2 r2 , ( (D.2c) I2,1,1 := = {6q3 [−qi2(tyi + tiy) + qi1(tiyr + tryi) + qr1(tryr −tiyi)] −6qr2 [q3(tyr + try) + qi1(tyi + tiy) + 2qi2(tiyr + tryi) + qr1(tyr + try)] + 3q2 3(−2ty + tiyi + tryr) + 6qi2qr1(tyi + tiy) + q2 i1(2ty −tiyi −tryr) −4q2 i2(ty + tiyi −2tryr) −6qi1qi2(tyr + try) + q2 r1(2ty −tiyi −tryr) −4q2 r2(ty −2tiyi + tryr) . (D.2d) = {6q3 [−qi2(tyi + tiy) + qi1(tiyr + tryi) + qr1(tryr −tiyi)] −6qr2 [q3(tyr + try) + qi1(tyi + tiy) + 2qi2(tiyr + tryi) + qr1(tyr + try)] + 3q2 3(−2ty + tiyi + tryr) + 6qi2qr1(tyi + tiy) + q2 i1(2ty −tiyi −tryr) −4q2 i2(ty + tiyi −2tryr) −6qi1qi2(tyr + try) + q2 r1(2ty −tiyi −tryr) −4q2 r2(ty −2tiyi + tryr) . (D.2d) D.2 Invariants to complete the generating set Here we give explicit expressions for the invariants of section 5.2.3. The statement in text-form here is provided for completeness. For practical applications we also provide the invariants in an auxiliary Mathematica notebook. D.2 Invariants to complete the generating set = [q3 (2ty −tiyi −tryr) + 2qi2 (tyi + tiy) +qi1 (tiyr + tryi) + 2qr2 (tyr + try) + qr1 (tryr −tiyi)] , (D.3) = [q3 (2ty −tiyi −tryr) + 2qi2 (tyi + tiy) (D.3) – 23 – (D.4) (D.5) JHEP05(2019)208 = −24qi2qr2yiyr −12 (q3qi2yyi −q3qi1yiyr + q3qr2yyr −qi2qr1yyi + qi1qr2yyi + qi1qi2yyr + qr1qr2yyr) + 8 q2 i2y2 r + q2 r2y2 i −6 q3qr1y2 i −q3qr1y2 r + q2 3y2 −4 q2 i2y2 + q2 i2y2 i + q2 r2y2 + q2 r2y2 r + 3 q2 3y2 i + q2 3y2 r + 2 q2 i1y2 + q2 r1y2 −q2 i1y2 i −q2 i1y2 r −q2 r1y2 i −q2 r1y2 r (D.6) = −24qi2qr2titr −12 (q3qi2tti −q3qi1titr + q3qr2ttr −qi2qr1tti + qi1qr2tti + qi1qi2ttr + qr1qr2ttr) + 8 q2 i2t2 r + q2 r2t2 i −6 q3qr1t2 i −q3qr1t2 r + q2 3t2 −4 q2 i2t2 + q2 i2t2 i + q2 r2t2 + q2 r2t2 r + 3 q2 3t2 i + q2 3t2 r + 2 q2 i1t2 + q2 r1t2 −q2 i1t2 i −q2 i1t2 r −q2 r1t2 i −q2 r1t2 r (D.7) = i [ 4 (−q3qr1tyiyr + qi2qr2ty2 i −qi2qr2ty2 r −qi2qr2tiyyi + qi2qr2tryyr −q2 i2tyiyr + q2 i2tiyyr + q2 r2tyiyr −q2 r2tryyi + 3 q2 3tiyyr −q2 3tryyi + 2 (−q3qi2tyyr −q3qi1ty2 i + q3qi1ty2 r + q3qi1tiyyi + q3qi2tiyiyr + q3qi2try2 −q3qi1tryyr −q3qi2try2 i + q3qr2tyyi −q3qr2tiy2 + q3qr1tiyyr + q3qr2tiy2 r + q3qr1tryyi −q3qr2tryiyr −qi1qr2tyyr + qi2qr1tyyr −qi2qr1tiyiyr + qi1qr2tiyiyr −qi2qr1try2 + qi1qr2try2 −qi1qr2try2 i + qi2qr1try2 i + qi1qi2tyyi −qi1qi2tiy2 + qi1qi2tiy2 r −qi1qi2tryiyr + qr1qr2tyyi −qr1qr2tiy2 + qr1qr2tiy2 r −qr1qr2tryiyr) −q2 i1tiyyr + q2 i1tryyi −q2 r1tiyyr + q2 r1tryyi ] (D.8) = i [ −4 (q3qr1titry + qi2qr2ttiyi −qi2qr2ttryr −qi2qr2t2 i y + qi2qr2t2 r y −q2 i2ttryi + q2 i2titry + q2 r2ttiyr −q2 r2titry −3 q2 3ttiyr −q2 3ttryi −2 −q3qi2t2yr −q3qi1ttiyi + q3qi2ttry + q3qi1ttryr + q3qi1t2 i y + q3qi2t2 i yr −q3qi2titryi −q3qi1t2 r y + q3qr2t2yi −q3qr2ttiy −q3qr1ttiyr −q3qr1ttryi + q3qr2titryr −q3qr2t2 r yi −qi1qr2t2yr + qi2qr1t2yr −qi2qr1ttry + qi1qr2ttry −qi2qr1t2 i yr + qi1qr2t2 i yr −qi1qr2titryi + qi2qr1titryi + qi1qi2t2yi −qi1qi2ttiy + qi1qi2titryr −qi1qi2t2 r yi + qr1qr2t2yi −qr1qr2ttiy + qr1qr2titryr −qr1qr2t2 r yi + q2 i1ttiyr −q2 i1ttryi + q2 r1ttiyr −q2 r1ttryi ] (D.9) = i [ 18 yyiyrqr1q2 3 −16 y2 i yrqi2q2 r2 −yiy2 r q2 i2qr2 −12 yiq3qi1qi2y2 + yrq3qi2qr1y2 −yrq3qi1qr2y2 + yiq3qr1qr2y2 −yiyrq3q2 i2y + yiyrq3q2 r2y + y2 i q3qi2qr2y −y2 r q3qi2qr2y + y2 i qi2qr1qr2y + y2 r qi2qr1qr2y −y2 i yrq3qi2qr1 −yiy2 r q3qr1qr2 + 9 yy2 i q2 3qi1 −yy2 r q2 3qi1 + 8 qi2qr1qr2y3 −yrq3 i2y2 + yiq3 r2y2 −yrqi2q2 r2y2 + yiq2 i2qr2y2 −y2 r qi1q2 i2y + y2 i qi1q2 r2y + y2 i yrq3 i2 −yiy2 r q3 r2 + y3 r qi2q2 r2 −y3 i q2 i2qr2 + 6 q3qi1qi2y3 i + yrq3qi1qr2y2 i + yrqi1qr1qr2y2 i −y2 r q3qi1qi2yi + y2 r qi1qi2qr1yi −y3 r q3qi1qr2 + 4 qi1q2 i2y3 −qi1q2 r2y3 + yrqi2q2 r1y2 + yrq2 i1qi2y2 −yiq2 i1qr2y2 −yiq2 r1qr2y2 −y2 i qi1q2 i2y + y2 r qi1q2 r2y + yiyrqr1q2 r2y + yiyrq2 i2qr1y −y2 i yrqi2q2 r1 +yiy2 r q2 r1qr2 −2 qi1qi2qr1y3 i −q2 i1qr2y3 i −yrq2 i1qi2y2 i + yyrq3 r1yi + yyrq2 i1qr1yi + y2 r q2 i1qr2yi + y3 r q2 i1qi2 +y3 r qi1qr1qr2 −yy2 i q3 i1 + yy2 r q3 i1 −yy2 i q2 r1qi1 + yy2 r q2 r1qi1 ] (D.10) JHEP05(2019)208 JHEP05(2019)208 = i [ 4 (−q3qr1tyiyr + qi2qr2ty2 i −qi2qr2ty2 r −qi2qr2tiyyi + qi2qr2tryyr −q2 i2tyiyr + q2 i2tiyyr + q2 r2tyiyr −q2 r2tryyi + 3 q2 3tiyyr −q2 3tryyi + 2 (−q3qi2tyyr −q3qi1ty2 i + q3qi1ty2 r + q3qi1tiyyi + q3qi2tiyiyr + q3qi2try2 −q3qi1tryyr −q3qi2try2 i + q3qr2tyyi −q3qr2tiy2 + q3qr1tiyyr + q3qr2tiy2 r + q3qr1tryyi −q3qr2tryiyr −qi1qr2tyyr + qi2qr1tyyr −qi2qr1tiyiyr + qi1qr2tiyiyr −qi2qr1try2 + qi1qr2try2 −qi1qr2try2 i + qi2qr1try2 i + qi1qi2tyyi −qi1qi2tiy2 + qi1qi2tiy2 r −qi1qi2tryiyr + qr1qr2tyyi −qr1qr2tiy2 + qr1qr2tiy2 r −qr1qr2tryiyr) −q2 i1tiyyr + q2 i1tryyi −q2 r1tiyyr + q2 r1tryyi ] (D.8) = i [ 4 (−q3qr1tyiyr + qi2qr2ty2 i −qi2qr2ty2 r −qi2qr2tiyyi + qi2qr2tryyr −q2 i2tyiyr + q2 i2tiyyr + q2 r2tyiyr −q2 r2tryyi + 3 q2 3tiyyr −q2 3tryyi + 2 (−q3qi2tyyr −q3qi1ty2 i + q3qi1ty2 r + q3qi1tiyyi + q3qi2tiyiyr + q3qi2try2 −q3qi1tryyr −q3qi2try2 i + q3qr2tyyi −q3qr2tiy2 + q3qr1tiyyr + q3qr2tiy2 r + q3qr1tryyi −q3qr2tryiyr −qi1qr2tyyr + qi2qr1tyyr −qi2qr1tiyiyr + qi1qr2tiyiyr −qi2qr1try2 + qi1qr2try2 −qi1qr2try2 i + qi2qr1try2 i + qi1qi2tyyi −qi1qi2tiy2 + qi1qi2tiy2 r −qi1qi2tryiyr + qr1qr2tyyi −qr1qr2tiy2 + qr1qr2tiy2 r −qr1qr2tryiyr) −q2 i1tiyyr + q2 i1tryyi −q2 r1tiyyr + q2 r1tryyi ] (D.8) = i [ −4 (q3qr1titry + qi2qr2ttiyi −qi2qr2ttryr −qi2qr2t2 i y + qi2qr2t2 r y −q2 i2ttryi + q2 i2titry + q2 r2ttiyr −q2 r2titry −3 q2 3ttiyr −q2 3ttryi −2 −q3qi2t2yr −q3qi1ttiyi + q3qi2ttry + q3qi1ttryr + q3qi1t2 i y + q3qi2t2 i yr −q3qi2titryi −q3qi1t2 r y + q3qr2t2yi −q3qr2ttiy −q3qr1ttiyr −q3qr1ttryi + q3qr2titryr −q3qr2t2 r yi −qi1qr2t2yr + qi2qr1t2yr −qi2qr1ttry + qi1qr2ttry −qi2qr1t2 i yr + qi1qr2t2 i yr −qi1qr2titryi + qi2qr1titryi + qi1qi2t2yi −qi1qi2ttiy + qi1qi2titryr −qi1qi2t2 r yi + qr1qr2t2yi −qr1qr2ttiy + qr1qr2titryr −qr1qr2t2 r yi + q2 i1ttiyr −q2 i1ttryi + q2 r1ttiyr −q2 r1ttryi ] (D.9) = i [ −4 (q3qr1titry + qi2qr2ttiyi −qi2qr2ttryr −qi2qr2t2 i y + qi2qr2t2 r y −q2 i2ttryi + q2 i2titry + q2 r2ttiyr −q2 r2titry −3 q2 3ttiyr −q2 3ttryi −2 −q3qi2t2yr −q3qi1ttiyi + q3qi2ttry + q3qi1ttryr + q3qi1t2 i y + q3qi2t2 i yr −q3qi2titryi −q3qi1t2 r y + q3qr2t2yi −q3qr2ttiy −q3qr1ttiyr −q3qr1ttryi + q3qr2titryr −q3qr2t2 r yi −qi1qr2t2yr + qi2qr1t2yr −qi2qr1ttry + qi1qr2ttry −qi2qr1t2 i yr + qi1qr2t2 i yr −qi1qr2titryi + qi2qr1titryi + qi1qi2t2yi −qi1qi2ttiy + qi1qi2titryr −qi1qi2t2 r yi + qr1qr2t2yi −qr1qr2ttiy + qr1qr2titryr −qr1qr2t2 r yi + q2 i1ttiyr −q2 i1ttryi + q2 r1ttiyr −q2 r1ttryi ] (D.9) −yrq3qi1qr2y2 + yiq3qr1qr2y2 −yiyrq3q2 i2y + yiyrq3q2 r2y + y2 i q3qi2qr2y −y2 r q3qi2qr2y + y2 i qi2qr1qr2y + y2 r qi2qr1qr2y −y2 i yrq3qi2qr1 −yiy2 r q3qr1qr2 + 9 yy2 i q2 3qi1 −yy2 r q2 3qi1 + 8 qi2qr1qr2y3 −yrq3 i2y2 + yiq3 r2y2 −yrqi2q2 r2y2 + yiq2 i2qr2y2 −y2 r qi1q2 i2y + y2 i qi1q2 r2y + y2 i yrq3 i2 −yiy2 r q3 r2 + y3 r qi2q2 r2 −y3 i q2 i2qr2 + 6 q3qi1qi2y3 i + yrq3qi1qr2y2 i + yrqi1qr1qr2y2 i −y2 r q3qi1qi2yi + y2 r qi1qi2qr1yi −y3 r q3qi1qr2 + 4 qi1q2 i2y3 −qi1q2 r2y3 + yrqi2q2 r1y2 + yrq2 i1qi2y2 −yiq2 i1qr2y2 −yiq2 r1qr2y2 −y2 i qi1q2 i2y + y2 r qi1q2 r2y + yiyrqr1q2 r2y + yiyrq2 i2qr1y −y2 i yrqi2q2 r1 +yiy2 r q2 r1qr2 −2 qi1qi2qr1y3 i −q2 i1qr2y3 i −yrq2 i1qi2y2 i + yyrq3 r1yi + yyrq2 i1qr1yi + y2 r q2 i1qr2yi + y3 r q2 i1qi2 +y3 r qi1qr1qr2 −yy2 i q3 i1 + yy2 r q3 i1 −yy2 i q2 r1qi1 + yy2 r q2 r1qi1 ] (D.10) – 24 – = i [ 18 ttitrqr1q2 3 −16 t2 i trqi2q2 r2 −tit2 r q2 i2qr2 −12 tiq3qi1qi2t2 + trq3qi2qr1t2 −trq3qi1qr2t2 + tiq3qr1qr2t2 −titrq3q2 i2t + titrq3q2 r2t + t2 i q3qi2qr2t −t2 r q3qi2qr2t + t2 i qi2qr1qr2t + t2 r qi2qr1qr2t −t2 i trq3qi2qr1 −tit2 r q3qr1qr2 + 9 tt2 i q2 3qi1 −tt2 r q2 3qi1 + 8 qi2qr1qr2t3 −trq3 i2t2 + tiq3 r2t2 −trqi2q2 r2t2 + tiq2 i2qr2t2 −t2 r qi1q2 i2t + t2 i qi1q2 r2t + t2 i trq3 i2 −tit2 r q3 r2 + t3 r qi2q2 r2 −t3 i q2 i2qr2 + 6 q3qi1qi2t3 i + trq3qi1qr2t2 i + trqi1qr1qr2t2 i −t2 r q3qi1qi2ti + t2 r qi1qi2qr1ti −t3 r q3qi1qr2 + 4 qi1q2 i2t3 −qi1q2 r2t3 + trqi2q2 r1t2 + trq2 i1qi2t2 −tiq2 i1qr2t2 −tiq2 r1qr2t2 −t2 i qi1q2 i2t + t2 r qi1q2 r2t + titrqr1q2 r2t + titrq2 i2qr1t −t2 i trqi2q2 r1 +tit2 r q2 r1qr2 −2 qi1qi2qr1t3 i −q2 i1qr2t3 i −trq2 i1qi2t2 i + ttrq3 r1ti + ttrq2 i1qr1ti + t2 r q2 i1qr2ti + t3 r q2 i1qi2 +t3 r qi1qr1qr2 −tt2 i q3 i1 + tt2 r q3 i1 −tt2 i q2 r1qi1 + tt2 r q2 r1qi1 ] (D.11) = i [ 18 ttitrqr1q2 3 −16 t2 i trqi2q2 r2 −tit2 r q2 i2qr2 −12 tiq3qi1qi2t2 + trq3qi2qr1t2 −trq3qi1qr2t2 + tiq3qr1qr2t2 −titrq3q2 i2t + titrq3q2 r2t + t2 i q3qi2qr2t −t2 r q3qi2qr2t + t2 i qi2qr1qr2t + t2 r qi2qr1qr2t −t2 i trq3qi2qr1 −tit2 r q3qr1qr2 + 9 tt2 i q2 3qi1 −tt2 r q2 3qi1 + 8 qi2qr1qr2t3 −trq3 i2t2 + tiq3 r2t2 −trqi2q2 r2t2 + tiq2 i2qr2t2 −t2 r qi1q2 i2t + t2 i qi1q2 r2t + t2 i trq3 i2 −tit2 r q3 r2 + t3 r qi2q2 r2 −t3 i q2 i2qr2 + 6 q3qi1qi2t3 i + trq3qi1qr2t2 i + trqi1qr1qr2t2 i −t2 r q3qi1qi2ti + t2 r qi1qi2qr1ti −t3 r q3qi1qr2 + 4 qi1q2 i2t3 −qi1q2 r2t3 + trqi2q2 r1t2 + trq2 i1qi2t2 −tiq2 i1qr2t2 −tiq2 r1qr2t2 −t2 i qi1q2 i2t + t2 r qi1q2 r2t + titrqr1q2 r2t + titrq2 i2qr1t −t2 i trqi2q2 r1 +tit2 r q2 r1qr2 −2 qi1qi2qr1t3 i −q2 i1qr2t3 i −trq2 i1qi2t2 i + ttrq3 r1ti + ttrq2 i1qr1ti + t2 r q2 i1qr2ti + t3 r q2 i1qi2 +t3 r qi1qr1qr2 −tt2 i q3 i1 + tt2 r q3 i1 −tt2 i q2 r1qi1 + tt2 r q2 r1qi1 ] (D.11) JHEP05(2019)208 JHEP05(2019)208 = i [ −32 tiyiyrqi2q2 r2 −tryiyrq2 i2qr2 + 24 tqi2qr1qr2y2 −tyiq3qi1qi2y −tyrq3qi2qr1y = i [ −32 tiyiyrqi2q2 r2 −tryiyrq2 i2qr2 + 24 tqi2qr1qr2y2 −tyiq3qi1qi2y −tyrq3qi2 = i [ −32 tiyiyrqi2q2 r2 −tryiyrq2 i2qr2 + 24 tqi2qr1qr2y2 −tyiq3qi1qi2y −tyrq3qi2qr1y + tyrq3qi1qr2y −tiyiq3qi2qr2y + tryrq3qi2qr2y −tyiq3qr1qr2y −tiyiqi2qr1qr2y −tryrqi2qr1qr2y + try2 r qi2q2 r2 + tiyiyrq3qi2qr1 −tiy2 i q2 i2qr2 +tryiyrq3qr1qr2) + 18 tiq3qi1qi2y2 i + tiyq2 3qi1yi + tryq2 3qr1yi + tyrq2 3qr1yi −tryyrq2 3qi1 + tiyyrq2 3qr1 −try2 r q3qi1qr2 + 16 −tyyrq3 i2 + tiyiyrq3 i2 −tryyrqi1q2 i2 + tiy2 r qr2q2 i2 + tyyiqr2q2 i2 −try2 i q2 r2qi2 −tyyrq2 r2qi2 + tyyiq3 r2 −tryiyrq3 r2 +tiyyiqi1q2 r2 + 12 tqi1q2 i2y2 −tqi1q2 r2y2 −tiq3qi1qi2y2 −trq3qi2qr1y2 + trq3qi1qr2y2 −tiq3qr1qr2y2 + tryiq3q2 i2y + tiyrq3q2 i2y −tryiq3q2 r2y −tiyrq3q2 r2y + tyiyrq3q2 i2 −tyiyrq3q2 r2 −tryiyrq3qi1qi2 + try2 i q3qi2qr1 + tryiyrqi1qi2qr1 + tiyiyrq3qi1qr2 −ty2 i q3qi2qr2 + ty2 r q3qi2qr2 + tiy2 r q3qr1qr2 + tiyiyrqi1qr1qr2 −ty2 i qi2qr1qr2 −ty2 r qi2qr1qr2 + 9 ty2 i q2 3qi1 −ty2 r q2 3qi1 −8 try2q3 i2 −try2 i q3 i2 + ty2 r qi1q2 i2 + tiyyiqi1q2 i2 −tiy2qr2q2 i2 −tyyrq2 i1qi2 −tyyrq2 r1qi2 + tiyiyrq2 r1qi2 + try2q2 r2qi2 −tiy2q3 r2 + tiy2 r q3 r2 −ty2 i qi1q2 r2 −tryyrqi1q2 r2 + tyyiq2 i1qr2 + tyyiq2 r1qr2 −tryiyrq2 r1qr2 + 6 −tiqi1qi2qr1y2 i + tiq2 i1qr2y2 i + trq3qi1qr2y2 i + trqi1qr1qr2y2 i −try2 r q2 i1qi2 −tiy2 r q3qi1qi2 + tiy2 r qi1qi2qr1 −try2 r qi1qr1qr2 −4 −try2qi2q2 i1 −tiyiyrqi2q2 i1 + tiy2qr2q2 i1 + tryiyrqr2q2 i1 + ty2 i q2 i2qi1 −ty2 r q2 r2qi1 −try2qi2q2 r1 + try2 i qi2q2 r1 −tryyiqr1q2 r2 −tiyyrqr1q2 r2 −tyiyrqr1q2 r2 −tryyiq2 i2qr1 −tiyyrq2 i2qr1 −tyiyrq2 i2qr1 + tiy2q2 r1qr2 −tiy2 r q2 r1qr2 −2 tiyyiq3 i1 −tryyrq3 i1 −try2 i qi2q2 i1 + tryyiqr1q2 i1 + tiyyrqr1q2 i1 + tyiyrqr1q2 i1 + tiy2 r qr2q2 i1 + tiyyiq2 r1qi1 −tryyrq2 r1qi1 + tryyiq3 r1 + tiyyrq3 r1 +tyiyrq3 r1 −ty2 i q3 i1 + ty2 r q3 i1 −ty2 i q2 r1qi1 + ty2 r q2 r1qi1 ] (D.12) y q3qi1qr2y iyiq3qi2qr2y y q3qi2qr2y yiq3qr1qr2y iyiqi2qr1qr2y y qi2qr1qr2y yr qi2qr2 + tiyiyrq3qi2qr1 −tiy2 i q2 i2qr2 +tryiyrq3qr1qr2) + 18 tiq3qi1qi2y2 i + tiyq2 3qi1yi + tryq2 3qr1yi + tyrq2 3qr1yi −tryyrq2 3qi1 + tiyyrq2 3qr1 −try2 r q3qi1qr2 + 16 −tyyrq3 i2 + tiyiyrq3 i2 −tryyrqi1q2 i2 + tiy2 r qr2q2 i2 + tyyiqr2q2 i2 −try2 i q2 r2qi2 −tyyrq2 r2qi2 + tyyiq3 r2 −tryiyrq3 r2 +tiyyiqi1q2 r2 + 12 tqi1q2 i2y2 −tqi1q2 r2y2 −tiq3qi1qi2y2 −trq3qi2qr1y2 + trq3qi1qr2y2 −tiq3qr1qr2y2 + tryiq3q2 i2y + tiyrq3q2 i2y −tryiq3q2 r2y −tiyrq3q2 r2y + tyiyrq3q2 i2 −tyiyrq3q2 r2 −tryiyrq3qi1qi2 + try2 i q3qi2qr1 + tryiyrqi1qi2qr1 + tiyiyrq3qi1qr2 −ty2 i q3qi2qr2 + ty2 r q3qi2qr2 + tiy2 r q3qr1qr2 + tiyiyrqi1qr1qr2 −ty2 i qi2qr1qr2 −ty2 r qi2qr1qr2 + 9 ty2 i q2 3qi1 −ty2 r q2 3qi1 −8 try2q3 i2 −try2 i q3 i2 + ty2 r qi1q2 i2 + tiyyiqi1q2 i2 −tiy2qr2q2 i2 −tyyrq2 i1qi2 −tyyrq2 r1qi2 + tiyiyrq2 r1qi2 + try2q2 r2qi2 −tiy2q3 r2 + tiy2 r q3 r2 −ty2 i qi1q2 r2 −tryyrqi1q2 r2 + tyyiq2 i1qr2 + tyyiq2 r1qr2 −tryiyrq2 r1qr2 + 6 −tiqi1qi2qr1y2 i + tiq2 i1qr2y2 i + trq3qi1qr2y2 i + trqi1qr1qr2y2 i −try2 r q2 i1qi2 −tiy2 r q3qi1qi2 + tiy2 r qi1qi2qr1 −try2 r qi1qr1qr2 −4 −try2qi2q2 i1 −tiyiyrqi2q2 i1 + tiy2qr2q2 i1 + tryiyrqr2q2 i1 + ty2 i q2 i2qi1 −ty2 r q2 r2qi1 −try2qi2q2 r1 + try2 i qi2q2 r1 −tryyiqr1q2 r2 −tiyyrqr1q2 r2 −tyiyrqr1q2 r2 −tryyiq2 i2qr1 −tiyyrq2 i2qr1 −tyiyrq2 i2qr1 + tiy2q2 r1qr2 −tiy2 r q2 r1qr2 −2 tiyyiq3 i1 −tryyrq3 i1 −try2 i qi2q2 i1 + tryyiqr1q2 i1 + tiyyrqr1q2 i1 + tyiyrqr1q2 i1 + tiy2 r qr2q2 i1 + tiyyiq2 r1qi1 −tryyrq2 r1qi1 + tryyiq3 r1 + tiyyrq3 r1 +tyiyrq3 r1 −ty2 i q3 i1 + ty2 r q3 i1 −ty2 i q2 r1qi1 + ty2 r q2 r1qi1 ] (D.12) = i [ −32 titryiqi2q2 r2 −titryrq2 i2qr2 + 24 yqi2qr1qr2t2 −tiyq3qi1qi2t −tryq3qi2qr1t + tryq3qi1qr2t −tiyiq3qi2qr2t + tryrq3qi2qr2t −tiyq3qr1qr2t −tiyiqi2qr1qr2t −tryrqi2qr1qr2t + t2 r yrqi2q2 r2 + titryiq3qi2qr1 −t2 i yiq2 i2qr2 +titryrq3qr1qr2) + 18 yiq3qi1qi2t2 i + tyiq2 3qi1ti + tryq2 3qr1ti + tyrq2 3qr1ti −ttryrq2 3qi1 + ttryiq2 3qr1 −t2 r yrq3qi1qr2 + 16 −ttryq3 i2 + titryiq3 i2 −ttryrqi1q2 i2 + ttiyqr2q2 i2 + t2 r yiqr2q2 i2 −ttryq2 r2qi2 −t2 i yrq2 r2qi2 + ttiyq3 r2 −titryrq3 r2 +ttiyiqi1q2 r2 + 12 yqi1q2 i2t2 −yqi1q2 r2t2 −yiq3qi1qi2t2 −yrq3qi2qr1t2 + yrq3qi1qr2t2 −yiq3qr1qr2t2 + tryiq3q2 i2t + tiyrq3q2 i2t −tryiq3q2 r2t −tiyrq3q2 r2t + titryq3q2 i2 −titryq3q2 r2 −titryrq3qi1qi2 + t2 i yrq3qi2qr1 + titryrqi1qi2qr1 + titryiq3qi1qr2 −t2 i yq3qi2qr2 + t2 r yq3qi2qr2 + t2 r yiq3qr1qr2 + titryiqi1qr1qr2 −t2 i yqi2qr1qr2 −t2 r yqi2qr1qr2 + 9 t2 i yq2 3qi1 −t2 r yq2 3qi1 + 8 t2 i yrq3 i2 −t2yrq3 i2 −t2 r yqi1q2 i2 −ttiyiqi1q2 i2 + t2yiqr2q2 i2 + ttryq2 i1qi2 + ttryq2 r1qi2 −titryiq2 r1qi2 −t2yrq2 r2qi2 + t2yiq3 r2 −t2 r yiq3 r2 + t2 i yqi1q2 r2 + ttryrqi1q2 r2 −ttiyq2 i1qr2 −ttiyq2 r1qr2 +titryrq2 r1qr2 + 6 −yiqi1qi2qr1t2 i + yiq2 i1qr2t2 i + yrq3qi1qr2t2 i + yrqi1qr1qr2t2 i −t2 r yrq2 i1qi2 −t2 r yiq3qi1qi2 + t2 r yiqi1qi2qr1 −t2 r yrqi1qr1qr2 −4 −titryiqi2q2 i1 −t2yrqi2q2 i1 + t2yiqr2q2 i1 + titryrqr2q2 i1 + t2 i yq2 i2qi1 −t2 r yq2 r2qi1 −t2yrqi2q2 r1 + t2 i yrqi2q2 r1 −titryqr1q2 r2 −ttryiqr1q2 r2 −ttiyrqr1q2 r2 −titryq2 i2qr1 −ttryiq2 i2qr1 −ttiyrq2 i2qr1 + t2yiq2 r1qr2 −t2 r yiq2 r1qr2 −2 ttiyiq3 i1 −ttryrq3 i1 −t2 i yrqi2q2 i1 + titryqr1q2 i1 + ttryiqr1q2 i1 + ttiyrqr1q2 i1 + t2 r yiqr2q2 i1 + ttiyiq2 r1qi1 −ttryrq2 r1qi1 + titryq3 r1 + ttryiq3 r1 +ttiyrq3 r1 −t2 i yq3 i1 + t2 r yq3 i1 −t2 i yq2 r1qi1 + t2 r yq2 r1qi1 ] (D.13) = i [ −32 titryiqi2q2 r2 −titryrq2 i2qr2 + 24 yqi2qr1qr2t2 −tiyq3qi1qi2t −tryq3qi2qr1t + tryq3qi1qr2t −tiyiq3qi2qr2t + tryrq3qi2qr2t −tiyq3qr1qr2t −tiyiqi2qr1qr2t −tryrqi2qr1qr2t + t2 r yrqi2q2 r2 + titryiq3qi2qr1 −t2 i yiq2 i2qr2 +titryrq3qr1qr2) + 18 yiq3qi1qi2t2 i + tyiq2 3qi1ti + tryq2 3qr1ti + tyrq2 3qr1ti −ttryrq2 3qi1 + ttryiq2 3qr1 −t2 r yrq3qi1qr2 + 16 −ttryq3 i2 + titryiq3 i2 −ttryrqi1q2 i2 + ttiyqr2q2 i2 + t2 r yiqr2q2 i2 −ttryq2 r2qi2 −t2 i yrq2 r2qi2 + ttiyq3 r2 −titryrq3 r2 +ttiyiqi1q2 r2 + 12 yqi1q2 i2t2 −yqi1q2 r2t2 −yiq3qi1qi2t2 −yrq3qi2qr1t2 + yrq3qi1qr2t2 −yiq3qr1qr2t2 + tryiq3q2 i2t + tiyrq3q2 i2t −tryiq3q2 r2t −tiyrq3q2 r2t + titryq3q2 i2 −titryq3q2 r2 −titryrq3qi1qi2 + t2 i yrq3qi2qr1 + titryrqi1qi2qr1 + titryiq3qi1qr2 −t2 i yq3qi2qr2 + t2 r yq3qi2qr2 + t2 r yiq3qr1qr2 + titryiqi1qr1qr2 −t2 i yqi2qr1qr2 −t2 r yqi2qr1qr2 + 9 t2 i yq2 3qi1 −t2 r yq2 3qi1 + 8 t2 i yrq3 i2 −t2yrq3 i2 −t2 r yqi1q2 i2 −ttiyiqi1q2 i2 + t2yiqr2q2 i2 + ttryq2 i1qi2 + ttryq2 r1qi2 −titryiq2 r1qi2 −t2yrq2 r2qi2 + t2yiq3 r2 −t2 r yiq3 r2 + t2 i yqi1q2 r2 + ttryrqi1q2 r2 −ttiyq2 i1qr2 −ttiyq2 r1qr2 +titryrq2 r1qr2 + 6 −yiqi1qi2qr1t2 i + yiq2 i1qr2t2 i + yrq3qi1qr2t2 i + yrqi1qr1qr2t2 i −t2 r yrq2 i1qi2 −t2 r yiq3qi1qi2 + t2 r yiqi1qi2qr1 −t2 r yrqi1qr1qr2 −4 −titryiqi2q2 i1 −t2yrqi2q2 i1 + t2yiqr2q2 i1 + titryrqr2q2 i1 + t2 i yq2 i2qi1 −t2 r yq2 r2qi1 −t2yrqi2q2 r1 + t2 i yrqi2q2 r1 −titryqr1q2 r2 −ttryiqr1q2 r2 −ttiyrqr1q2 r2 −titryq2 i2qr1 −ttryiq2 i2qr1 −ttiyrq2 i2qr1 + t2yiq2 r1qr2 −t2 r yiq2 r1qr2 −2 ttiyiq3 i1 −ttryrq3 i1 −t2 i yrqi2q2 i1 + titryqr1q2 i1 + ttryiqr1q2 i1 + ttiyrqr1q2 i1 + t2 r yiqr2q2 i1 + ttiyiq2 r1qi1 −ttryrq2 r1qi1 + titryq3 r1 + ttryiq3 r1 +ttiyrq3 r1 −t2 i yq3 i1 + t2 r yq3 i1 −t2 i yq2 r1qi1 + t2 r yq2 r1qi1 ] (D.13) – 25 – E Syzygies Here we list syzygies that we have explicitly constructed in the course of this work. order structure CP HS PL PP new rels. old rels. comments 6 q2y2t2 + 6 −1 7 1 - I2 1,1,1, eq. (6.2) 7 q3y2t2 + 7 −1 8 1 - q2y3t2 / q2y2t3 − 3 −1 4 1 / 1 - eq. (7.2) 8 q2y4t2 / q2y2t4 + 9 −1 11 1 / 1 1 / 1 J 2 1,2,1 / J 2 1,1,2 q2y3t3 + 9 −1 10 1 - J1,2,1 × J1,1,2 q4y2t2 + 10 −1 12 1 1 I2 2,1,1 −(I2,2,0 × I2,0,2) q3y4t / q3yt4 − 4 −1 5 1 / 1 - I2,2,0J1,2,1 / I2,0,2J1,1,2, eq. (7.3) q3y3t2 /q3y2t3 − 6 −3 9 3 / 3 - I2,2,0J1,1,2 / I2,0,2J1,2,1 I2,1,1J1,2,1 / I2,1,1J1,1,2 9 q3y3t3 + 13 −2 17 2 2 q3y4t2 / q3y2t4 + 12 −1 15 1 / 1 2 / 2 q4y4t / q4yt4 − 5 −1 6 1 / 1 - q4y3t2 / q4y2t3 − 7 −3 11 3 / 3 1 / 1 ... ... ... ... 10 q4y4t2 / q4y2t4 + 18 −3 22 3 / 3 1 / 1 J 2 2,2,1 / J 2 2,1,2, eq. (7.4) q5yt4 / q5y4t − 7 −1 9 1 / 1 1 / 1 q5y3t2 / q5y2t3 − 10 −2 13 2 / 2 1 / 1 ... ... ... ... 12 q6y6 / q6t6 + 10 −1 11 1 / 1 - J 2 3,3,0 / J 2 3,0,3 q6y4t2 / q6y2t4 + 27 −2 38 2 / 2 9 / 9 J 2 3,2,1 / J 2 3,1,2 q6y3t3 + 28 −2 43 2 13 J3,3,0 × J3,0,3, J3,2,1 × J3,1,2 ... ... ... ... JHEP05(2019)208 Table 1. List of the syzygies of the 2HDM scalar sector. The ﬁrst columns give the order, structure and CP transformation behavior of the relation. The following columns summarize the corresponding coeﬃcients of the multi-graded Hilbert series (HS), Plethystic logarithm (PL) as well as the number of new relations that are arising (as evidence that the PL coeﬃcient indeed gives this number correctly). Furthermore, we list the total number of power products of generating invariants which give rise to the same structure (this is an indicator of how many previously found “old” relations appear in addition to possible new relations). E Syzygies The last column gives important structures of invariants that appear in the given syzygy. Table 1. List of the syzygies of the 2HDM scalar sector. The ﬁrst columns give the order, structure and CP transformation behavior of the relation. 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https://openalex.org/W4295048056	https://www.researchsquare.com/article/rs-2039272/latest.pdf	English	null	Histology and Ultrastructure of Olfactory and Nasal Respiratory Mucosae in Suckling and Adult African Grasscutters (Thryonomys swinderianus- Temminck, 1827)	Research Square (Research Square)	2,022	cc-by	9,338	Histology and Ultrastructure of Olfactory and Nasal Respiratory Mucosae in Suckling and Adult African Grasscutters (Thryonomys swinderianus- Temminck, 1827) Boniface M. Kavoi (  bmkavoi@uonbi.ac.ke ) University of Nairobi Shem J. Ochieng Kenyatta University Boniface M. Kavoi (  bmkavoi@uonbi.ac.ke ) Boniface M. Kavoi (  bmkavoi@uonbi.ac.ke ) University of Nairobi Shem J. Ochieng Kenyatta University Version of Record: A version of this preprint was published at Zoomorphology on January 5th, 2023. See the published version at https://doi.org/10.1007/s00435-022-00590-4. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Zoomorphology on January 5th, 2023. See the published version at https://doi.org/10.1007/s00435-022-00590-4. Page 1/21 Page 1/21 Page 1/21 Abstract Glasscutters (GRCs) are hystricognath rodents that predominate West African countries where they are captured and bred in captivity as “microlivestock” and for research. Consequently, research priority has, of late, shifted to aspects of GRC biology particularly with regard to morphofunctional aspects of its body systems. The olfactory system plays critical roles in regulating social, sexual, maternal and feeding behaviors. This study examines, histologically and ultrastructurally, the pattern and magnitude of remodeling of the GRC olfactory mucosa (OM) and nasal respiratory mucosa (NRM) between suckling and adult ages and compares these with what is documented for other mammals. In the adults, tubular- type Bowman’s glands, olfactory receptor neuron (ORN) axon bundles and blood vessels were uniformly distributed in the OM lamina propria contrary to sucklings where acinar-type Bowman’s glands lay superficially and the bundles relatively deeper. Apically in the adult NRM epithelium, ciliated and non- ciliated cells were uniformly distributed contrary to the sucklings where linearly arranged ciliated cell separated large numbers of non-ciliated cells. Quantitatively between the suckling and adult ages, respective increment values (%) were 28.2, 23.0, 28.1 and 52.9 for OM epithelial thickness, axon bundle diameter, ORN packing density and cilia number/ ORN dendritic knob. Age-related increment in volume density (%) was 53.9, 31.6, 19.4 and 46.3 for Bowman’s glands, axon bundles, OM vessels and NRM glands respectively. We conclude that microstructural refinement of the OM and NRM varies in qualitative and quantitative detail depending on age and species and that phenotypic plasticity in these structures suggests environmentally driven morphology. Introduction The Grasscutter (GRC) (Thryonomys swinderianus), also called great cane rat, is a tropical African cavy- like hystricomorph rodent that predominate the West African sub-region including Senegal, Ghana, Nigeria, Togo, Benin and Côte d’Ivoire (Ogunsanmi et al. 2002; Woods and Kilpatrick, 2005). In these countries, GRCs are aggressively hunted for their meat and also captured and raised in cages as “microlivestock” (home consumption and sale) and for use in research (Opara 2010; Jori et al., 2001; Fa et al. 2002; Ibe et al. 2017; Ingweye & Kalio, 2020). Indeed, the economic potential of GRCs is the reason why many organizations (including NGOs) interested in reducing poverty are promoting its production (Adu et al. 2017). Farming of GRCs is associated with good quality meat, inexpensive feeds, short gestation interval, large litter size and fast growth rate (Adu and Yeboah, 2003; Matthews, 2008; Owen and Dike, 2012). The meat of GRCs enjoys a higher premium price per kilogram weight than chicken, beef, pork and mutton among many West Africans and elsewhere (Adu et al. 2017). In the wild, GRCs inhabit dense and thick cane-like grasses growing in damp places where they live in small groups led by a single male (Woods and Kilpatrick, 2005; Aluko et al. 2015; Adu et al. 2017). These animals exhibit nocturnal and precocial behaviors and have an uncommon phenotype and life history (Mustapha et al. 2020). Despite their size and short limbs, GRCs are quick runner and skilled swimmers and their sense of sight is relatively poor, making communication to be highly dependent on hearing and well developed sense of Page 2/21 smell (Opara, 2010). They are mainly fossorial, nesting and living much of the time in burrows and feed on roots, shoots, and stems of various grasses (Wood, 1994; Williams et al., 2011). In mammals, the nasal cavity has important and diverse functions including filtering, warming and humidification of inhaled air as well as olfaction (Cole, 1993). Introduction Among the mammalian species, the nasal cavity varies, not only in gross structure, but also in the distribution of epithelial types as follows: (1) stratified squamous epithelium lining the nasal vestibule, (2) ciliated pseudostratified columnar epithelium (respiratory epithelium) in the main nasal chamber covering most of the nasal turbinates and parts of the nasal septum, (3) non-ciliated columnar epithelium (also called transitional epithelium) located between the squamous and respiratory epithelia, and (4) olfactory epithelium (OE) found in the dorsoposterior aspect of the nasal cavity mainly covering the ethmoidal concha (Harkema et al., 2006). Evolutionary pressure exerted on the olfactory cue defines two categories of mammals: (a) those with complex noses with the olfactory mucosa (OM) covering a great part of the nasal cavity having olfaction as the primary function (macrosmatic) e.g. rodents, rabbits and dogs and (b) those with simple noses (OM covers a small portion of nasal passage) and have breathing as the main function (microsmatic) e.g. humans and monkeys (Gross et al., 1982; Sorokin, 1988). In the main nasal chamber, the nasal respiratory mucosa (NRM) consists of a luminal surface epithelium and an underlying lamina propria containing glands, blood vessels and lymphatics and nerves embedded in a connective tissue matrix. The NRM epithelium has the following cell types: mucous (goblet) cells, ciliated cells, non-ciliated columnar cells and basal cells (Monteiro Rivier and Popp, 1984). The mucous cells are predominantly located in NRM epithelium lining the proximal part of the nasal septum whereas non-ciliated columnar cells (also called serous cells) are the primary secretory cells in the remainder of the NRM epithelium (Harkema et al., 1989). Goblet cells represent 5–15% of cells in the respiratory mucosa and produce secretions for the endonasal mucus together with the submucosal glands. Non- ciliated cells represent up to 70% of the epithelium and have 300–400 microvilli on their surface (Mygind et al. 1982). Another 20–50% of epithelial cells are ciliated cells possessing 200–300 cilia on their apical surfaces, which are the morphological substrate of the mucociliary clearance (Beule, 2010). In the main nasal chamber, the nasal respiratory mucosa (NRM) consists of a luminal surface epithelium and an underlying lamina propria containing glands, blood vessels and lymphatics and nerves embedded in a connective tissue matrix. The NRM epithelium has the following cell types: mucous (goblet) cells, ciliated cells, non-ciliated columnar cells and basal cells (Monteiro Rivier and Popp, 1984). Introduction The mucous cells are predominantly located in NRM epithelium lining the proximal part of the nasal septum whereas non-ciliated columnar cells (also called serous cells) are the primary secretory cells in the remainder of the NRM epithelium (Harkema et al., 1989). Goblet cells represent 5–15% of cells in the respiratory mucosa and produce secretions for the endonasal mucus together with the submucosal glands. Non- ciliated cells represent up to 70% of the epithelium and have 300–400 microvilli on their surface (Mygind et al. 1982). Another 20–50% of epithelial cells are ciliated cells possessing 200–300 cilia on their apical surfaces, which are the morphological substrate of the mucociliary clearance (Beule, 2010). The structure of the OM has comprehensively been described in our previous papers (Kavoi et al., 2010, 2012). In brief, the atypical OM epithelium has the basal (progenitor) cells residing at the bottom of the OE overlying the basement membrane. Above these cells is a layer of bipolar olfactory receptor neurons (ORN) whose dendrites extend to the apical surface of the epithelium to form knob-like structures from which several immotile cilia emanate. Axons of the ORN pierce the basement membrane to enter the lamina where they fasciculate before passing via the crevices of the cribriform plate to reach the olfactory bulb. The most apical zone of the OE is occupied by the nuclei and cytoplasm of the supporting cells, whose apices possess microvilli. On the apical surface of the OE, the ORN cilia are enmeshed with each other and with the supporting cell microvilli in the surface fluid thus providing an extensive surface area for reception of odorants (Menco, 1980; Menco et al., 1997). The lamina propria beneath the basement membrane accommodates the ORN axon bundles, Bowman’s glands and vasculature. The Bowman's glands have acini and narrow secretory ducts going out through the OE. The secretions Page 3/21 released by these glands on the epithelial surface provide an environment for dissolving odorants thus allowing their diffusion to the sensory receptor sites on the cilia (Getchell and Getchell, 1992). Structural remodeling of the OM takes place as animals transit from juvenile to adulthood and this has previously been demonstrated in the rat (Meisami 1989), dog (Kavoi et al. 2010) and rabbit (Kavoi et al. 2012). Experimental Animals Animals used for this study were healthy male captive-bred GRCs of two different age groups: ten sucklings aged 4–6 weeks (345- 460g) and ten adults aged 13–15 months (2.8-3.2kg). These animals were randomly selected from a colony raised in a conventional animal housing facility in the Department of Animal Science, University of Ghana. Animals selected for this study were transported in well ventilated cages to the School of Veterinary Medicine, University of Ghana, Anatomy Laboratory, where they were immediately euthanized with lethal doses of sodium pentobarbitone (140 mg/kg, intravenously). All procedures performed on the GRCs were approved by University’s Animal Care and Use Committee and strictly conformed to the guidelines provided in the Animals (Scientific Procedures) Act 1986. Animals used for this study were healthy male captive-bred GRCs of two different age groups: ten sucklings aged 4–6 weeks (345- 460g) and ten adults aged 13–15 months (2.8-3.2kg). These animals were randomly selected from a colony raised in a conventional animal housing facility in the Department of Animal Science, University of Ghana. Animals selected for this study were transported in well ventilated cages to the School of Veterinary Medicine, University of Ghana, Anatomy Laboratory, where they were immediately euthanized with lethal doses of sodium pentobarbitone (140 mg/kg, i t l ) All d f d th GRC d b U i it ’ A i l C d U ventilated cages to the School of Veterinary Medicine, University of Ghana, Anatomy Laboratory, where they were immediately euthanized with lethal doses of sodium pentobarbitone (140 mg/kg, intravenously). All procedures performed on the GRCs were approved by University’s Animal Care and Use Committee and strictly conformed to the guidelines provided in the Animals (Scientific Procedures) Act 1986. intravenously). All procedures performed on the GRCs were approved by University’s Animal Care and Use Committee and strictly conformed to the guidelines provided in the Animals (Scientific Procedures) Act 1986. Introduction Anatomical data on nervous structures are essential in understanding the adaptive physiology of an animal species including behavior in the natural habitat that would impact on performance when captured and bred in cages. In the literature, substantial amount of information is available on the GRC brain (Nzalak et al. 2008, Byanet et al., 2009; Ajayi et al., 2011; Byanet and Dzenda, 2014; Ibe 2016, Ibe et al., 2017, 2019), spinal cord (Mustapha et al. 2015, 2017) and the eye (Peter-Ajuzıe et al. 2019). The olfactory system plays a pivotal role in communication by regulating multiple and integrative functions including feeding, social behavior, reproductive events (mother-neonate interaction) and emotional responses (e.g. anxiety, fear and predator avoidance) (Lledo et al. 2005). In view of the importance attached to the olfactory system with respect to animal survival, adaptive behavior and performance during domestication, it is necessary that the anatomical design of GRC olfactory system is understood. To the best of our knowledge, the only study available on the GRC olfactory system is that by Kavoi et al. (2021) in which nasal and brain olfactory components were examined gross and histologically. To explore this further and to generate new information, we analyzed the OM and NRM in suckling and adult GRCs for qualitative and quantitative characteristics at light and electron microscopy in order to understand their morphofunctional dynamics. Additionally, data obtained in this study are compared with what is previously recorded for other mammalian species. Processing of tissues for light and scanning electron microscopy Tissues for histology (from animals perfused with formaldehyde) were decalcified in 5% EDTA (Alers et al., 1999), washed in distilled water and dehydration in increasing concentrations of ethanol (50%, 70%, 80%, 90% and twice in 100%). Dehydrated tissues were then transitioned through methyl benzoate and infiltrated and embedded in paraffin wax, sectioned in the transverse plane at 5 µm using a rotary microtome and stained in Masson’s trichrome. Glutaraldehyde-fixed tissues were post-fixed in 1% osmium tetroxide, dehydrated in graded dilutions of ethanol (70%, 80%, 95% and 100% ×2), cleared in propylene oxide and embedded in epoxy resin. The resin blocks were cut with glass knives using a Sorvall® ultramicrotome to obtain semi-thin sections which were picked on glass slides and stained with 0.5% toluidine blue. After dehydration, some sections were selected for SEM, critical point dried in liquid carbon dioxide, mounted on brass stubs and sputter coated with gold–palladium complex. The sample surfaces were then examined using a Jeol 330 SEM operating at 15 kV and images captured on Orion Saturn frame grabber system. Tissue fixation and harvesting Euthanized animals were immediately perfused transcardially through the left ventricle of the heart with 10% formaldehyde for histology (n = 5 animals/ age group) and 2.5% phosphate-buffered glutaraldehyde (pH 7.4) for scanning electron microscopy (SEM) and toluidine blue staining (n = 5 animals/ age group). Respectively, olfactory and nasal respiratory mucosae were obtained from nasal and ethmoidal conchae Page 4/21 Page 4/21 following mid-sagittal sectioning of the animal skull and removal of the nasal septum. In each case, the conchae were transected perpendicular to their long axes into posterior, middle and anterior portions (Kavoi et al., 2010) from which tissue sub-segments for microscopy were selected by systematic random sampling. Morphometric analysis Quantitative parameters were analyzed at both light microscopy and SEM levels. Tissue samples from each age group were obtained, randomly selected and processed from 10 animals (five for light microscopy and five for SEM) from which 10–15 histological and 8–10 SEM micrographs were prepared. In both cases, morphometric analysis was done on 30–35 test fields generated from randomly selected micrographs. The OM was analyzed for epithelial thickness, diameter of ORN axon bundles and volume densities for the axon bundles, Bowman’s glands and vessels at light microscopy and densities of ORN and cilia numbers/ORN knob at SEM. In the lamina propria of the NRM, volume density was worked out for both glands and vessels. The protocols followed in determining the aforementioned parameters are detailed in Kavoi et al. (2012). In brief, the thickness of OE was measured from the basement membrane to the apical surface while diameters ORN axon bundle were worked out from mean linear intercept lengths. Volume densities were estimated by point counting using an overlay of coherent test system of points. Packing densities of ORNs were estimated by counting the ORN knobs projecting from the OE in a millimeter square area while taking into account the forbidden line rule. In determining cilia counts/ ORN knob, the obscured ones, which are estimated at 25%, were incorporated in the final total value. Data on morphometry were analyzed using Student's t-test. In all cases, data were expressed as means ± standard deviation (SD). For differences in means between groups, statistical significance captured at p < 0.05. Page 5/21 Page 5/21 Page 5/21 Scanning electron microcopy The apical surfaces of the OM epithelium in suckling and adult animals are shown in Fig. 3. In both age groups, the apical surfaces of the OE showed ORN dendrites with cilia-bearing knobs, apices of supporting cells and tunnel-like openings of Bowman’s gland ducts. On the OE surface, individual ORN dendrites and supporting cell apices were almost equally distributed in the suckling animals (Fig. 3A1). This differed from the situation in the adults where the ORN dendrites appeared more numerous per unit area and were packaged in a manner that appeared to conceal the supporting cell apices (Fig. 3A2). Additionally, olfactory cilia in the adults emerged from around the ORN knobs to project in a radial manner as opposed to the suckling animals where the cilia arose from the knobs to run in parallel in form of a tuft (Fig. A2 & B2). Figure 4 shows the ultrastructural features of the NRM epithelium in suckling and adult GRCs. In both age groups, ciliated cells projected numerous cilia above the general NRM epithelial surface whereas apical surfaces of non-ciliated cells were covered with plenty of microvilli. Intercellular spaces and tunnel-like openings of subepithelial gland ducts were well discerned in both postnatal age groups. The NRM epithelium in the suckling animals was characterized by linearly arranged ciliated cells separating relatively large numbers of non-ciliated cells (Fig. 4A). Conversely in the adults, individual ciliated and non-ciliated cells appeared uniform in terms of number per field and distribution (Fig. 4B). Results The GRC OM is shown in Fig. 1. The OM epithelial zones (nuclear, non-nuclear and free zones) were well established in both suckling and adult animals, with basement membranes of almost the same thickness. In the lamina propria of both age groups were ORN axon bundles, Bowman’s glands and vessels. These three structures were distributed uniformly in the lamina propria of the suckling animals in contrast to the situation in the adults where the bundles were placed deeper than the glands. The glands in the suckling animals were of the acinar type while those in adults were tubular. In Fig. 2A1, semi-thin sections showed the cellular components of the NRM epithelium in both suckling and adult GRCs to be: (1) ciliated cells whose apical surfaces possess numerous cilia, (2) non-ciliated columnar cells whose nuclei are positioned deeper than those of the ciliated cells and, (3) basal cells that are restricted to the lowermost region of the epithelium superjacent to the basement membrane. The NRM lamina accommodates glands and blood vessels (Fig. 2A & B). The glands in both age groups were of the acinar-type and in the adult the glands appeared more closely packed and the vessels were of greater diameters. Morphometry Table 1 presents data on thicknesses of OM epithelia and diameters of ORN axon bundles in the suckling and adult GRCs (current study) and in rabbits, dog and sheep (previous studies). Between the suckling Page 6/21 and adult stages of development, increment values were 28.2% (59.5 ± 4.1–76.3 ± 5.4 µm) for OE thickness and 23.0% (57.4 ± 6.0- 70.6 ± 8.6 µm) for axon bundle diameters. These values exceed, for both structures, those obtained in dogs and sheep but were less than those for the rabbit. and adult stages of development, increment values were 28.2% (59.5 ± 4.1–76.3 ± 5.4 µm) for OE thickness and 23.0% (57.4 ± 6.0- 70.6 ± 8.6 µm) for axon bundle diameters. These values exceed, for both structures, those obtained in dogs and sheep but were less than those for the rabbit. Table 1 Mean values (± SD given in parentheses) for OM epithelial thickness and ORN axon bundles (in µm) and their percentage increment between suckling and adult ages in the grasscutter (current study) and in previous studies in the rabbit, dog and sheep. Table 1 Table 1 Mean values (± SD given in parentheses) for OM epithelial thickness and ORN axon bundles (in µm) and their percentage increment between suckling and adult ages in the grasscutter (current study) and in previous studies in the rabbit, dog and sheep. Thickness of OE (µm) Diameter of axon bundles (µm) Suckling Adult Increase (%) Suckling Adult Increase (%) GRC (Current study) 59.5 (4.1) 76.3 (5.4) 28.2 57.4 (6.0) 70.6 (8.6) 23.0 Rabbit (Kavoi et al, 2012) 69.8 (5.3) 92.9 (2.3) 33.1 54.6 (8.3) 149.5 (9.4) 173.8 Dog (Kavoi et al., 2010) 64.7 (1.7) 72.5 (2.9) 12.1 68.2 (9.9) 73.3 (10.3) 7.5 Sheep (Kavoi et al., 2010) 47.8 (3.8) 56.8 (3.1) 18.8 43.8 (5.7) 50.6 (6.8) 15.5 Regarding the OM lamina propria, volume density values for Bowman’s glands, axonal bundles and blood vessels in the GRC are compared with those earlier recorded in rabbits (Table 2). In the GRC between suckling and adult ages, volume density increment was 53.9% (32.3 ± 6.0-49.7 ± 5.1) for the glands and 19.4% (7.2 ± 1.1–8.6 ± 3.3) for the vessels. These percentage increments exceeded those previously reported in rabbits (Table 2). Morphometry For the axon bundles, GRCs recorded a volume density increment of 31.6% (23.7 ± 5.2–31.2 ± 4.4), a value which is less than that documented for the rabbit (Table 2). Diameter of axon bundles (µm) vessels in the GRC are compared with those earlier recorded in rabbits (Table 2). In the GRC between suckling and adult ages, volume density increment was 53.9% (32.3 ± 6.0-49.7 ± 5.1) for the glands and 19.4% (7.2 ± 1.1–8.6 ± 3.3) for the vessels. These percentage increments exceeded those previously reported in rabbits (Table 2). For the axon bundles, GRCs recorded a volume density increment of 31.6% (23.7 ± 5.2–31.2 ± 4.4), a value which is less than that documented for the rabbit (Table 2). vessels in the GRC are compared with those earlier recorded in rabbits (Table 2). In the GRC between suckling and adult ages, volume density increment was 53.9% (32.3 ± 6.0-49.7 ± 5.1) for the glands and 19.4% (7.2 ± 1.1–8.6 ± 3.3) for the vessels. These percentage increments exceeded those previously reported in rabbits (Table 2). For the axon bundles, GRCs recorded a volume density increment of 31.6% (23.7 ± 5.2–31.2 ± 4.4), a value which is less than that documented for the rabbit (Table 2). Page 7/21 Page 7/21 Page 7/21 Table 2 Table 2 Mean volume densities (% ±SD given in parenthesis) for Bowman’s glands, axonal bundles and blood vessels in the lamina propria of the OM in suckling and adult GRC (current study) and in the rabbit (Kavoi et al., (2012). Mean volume densities (% ±SD given in parenthesis) for Bowman’s glands, axonal bundles and blood vessels in the lamina propria of the OM in suckling and adult GRC (current study) and in the rabbit (Kavoi et al., (2012). Morphometry ( ( ) GRC (current study) Rabbit (Kavoi et al., 2012) Suckling Adult Increase (%) Suckling Adult Increase (%) Axon bundles 23.7 (5.2) 31.2 (4.4) 31.6 14.6 (2.5) 24.1 (3.3) 65.1 Bowman’s glands 32.3 (6.0) 49.7 (5.1) 53.9 31.3 (2.9) 44.3 (3.7) 41.5 Blood vessels 7.2 (1.1) 8.6 (3.3) 19.4 11.5 (1.4) 12.5 (1.5) 8.7 t ki d iti f ORC ( illi t ) d ili b ORN k b i kli Data on packing densities of ORCs (per square millimeter) and cilia numbers per ORN knob in suckling Data on packing densities of ORCs (per square millimeter) and cilia numbers per ORN knob in suckling Data on packing densities of ORCs (per square millimeter) and cilia numbers per ORN knob in suckling and adult GRC (current study), rabbit, dog and sheep (earlier studies) are shown in Table 3. Respective values for the GRC ORN packing densities and cilia number/ ORN knob increased by 28.1% (61.2 ± 4.3– 78.4 ± 6.1 mm− 3 x 103) and 52.9% (17 ± 4–26 ± 5) between suckling and adult ages. Compared to the GRC, values for the above parameters (cilia counts and ORN densities) were greater in the rabbit but lesser in the dog and sheep. and adult GRC (current study), rabbit, dog and sheep (earlier studies) are shown in Table 3. Respective values for the GRC ORN packing densities and cilia number/ ORN knob increased by 28.1% (61.2 ± 4.3– 78.4 ± 6.1 mm− 3 x 103) and 52.9% (17 ± 4–26 ± 5) between suckling and adult ages. Compared to the GRC, values for the above parameters (cilia counts and ORN densities) were greater in the rabbit but lesser in the dog and sheep. Table 3 Discussion In the current study, the anatomical design of the OM and NRM are analyzed using histological, ultrastructural and morphometric methods in order to unravel salient features of the mucosae that are specific to this species and those that accompany growth to adulthood. Additionally, a comparison is made to clarify how and to what extent, between suckling and adult ages, structural remodeling of the OM and NRM in the GRC (order Rodentia) varies from other taxonomically different mammals already studied including dogs (order Carnivora), sheep (order Artiodactyla) and rabbits (order Langomorpha). The increasing trend in GRC domestication coupled with the paucity of data on its biology calls for rigorous search of information particularly on the functional morphology of the GRC’s nervous structures, which is essential in understanding its physiological adaptability and performance in captivity. Anatomical modification levels and patterns, particularly for nervous system structures, are never similar across mammalian taxa (orders, species etc.) because of the following facts: (i) that structures of the nervous system evolve in strict conformity to the functional needs of a given species (Kaas & Collins, 2001) and (ii) that evolution involves diversification of animals’ lifestyles including aspects of feeding and habitat (Wiens et al., 2010) and (iii) that the structure of the olfactory system and therefore the olfactory cue determines much of the behavior and ecology of a myriad of animal species (Rinaldi, 2007). At histological level, the OM in both suckling and adult GRCs presents features that typify the mammalian OM. As the animals grew to attain adulthood, OE thickness and ORN axon bundle diameters are noted to increase. We attribute this to age-related increment in ORN densities and the associated increase in axon numbers arising from the newly forming ORNs. Furthermore, optimization of olfactory function during juvenile development would require greater numbers of ORN axons to converge upon central relay neurons in the brain. This enhances the physiological capacities of the olfactory afferent pathway by increasing the opportunity for spatial summation and facilitation (Meisami, 1989). Extensive work on mammalian OM structure (Yamagishi et al. 1989; Ferrari et al. 2000; Kumar et al. 2000; Kavoi et al. 2010, 2012; Onyono et al. 2017; Kavoi, 2018) has shown the distribution of ORN axon bundles and Bowman’s glands and the structural form of the glands to vary among species and postnatal age groups. Table 3 Mean values (± SD given in parentheses) for cilia counts per ORN knob, densities of ORCs (mm− 2 x 103) and their percentage increase between suckling and adult ages of the grasscutter (current study) and in earlier studies in the rabbit, dog and sheep. Mean values (± SD given in parentheses) for cilia counts per ORN knob, densities of ORCs (mm− 2 x 103) and their percentage increase between suckling and adult ages of the grasscutter (current study) and in earlier studies in the rabbit, dog and sheep. Number of cilia/ORC knob Density of ORN (mm− 2x103) Suckling Adult Increase (%) Suckling Adult Increase (%) GRC (Current study) 17 (4) 26 (5) 52.9 61.2 (4.3) 78.4 (6.1) 28.1 Rabbit (Kavoi et al, 2012) 13 (3) 23 (4) 76.9 56.2 (4.3) 87.6 (4.8) 55.9 Dog (Kavoi et al., 2010) 18 (3) 19 (3) 5.6 66.7 (4.9) 81.7 (7.2) 22.5 Sheep (Kavoi et al., 2010) 9 (1) 7 (2) -2.2 63.3 (4.9) 46.7 (4.9) -26.2 (2) In the NRM lamina propria, volume densities of glands and blood vessels in suckling and adult GRCs are shown in Fig. 5. For both the glands and vessels, an age-related increase in volume density was noted, with the increment being 46.3% (32.4 ± 5.1–47.4 ± 5.5) for the glands and 32.5% (19.1 ± 3.5–25.3 ± 4.8) for the vessels. In the NRM lamina propria, volume densities of glands and blood vessels in suckling and adult GRCs are shown in Fig. 5. For both the glands and vessels, an age-related increase in volume density was noted, with the increment being 46.3% (32.4 ± 5.1–47.4 ± 5.5) for the glands and 32.5% (19.1 ± 3.5–25.3 ± 4.8) for the vessels. Discussion Similar differences are noted in the GRC where the Bowman’s are of the tubular-type and acinar-type in the adults and suckling animals respectively. In the suckling animals, the glands and the bundles are uniformly distributed as opposed to the adults where the glands are located superficially while the axon bundles are placed deeper in the lamina propria. Prenatally, Bowman’s glands develop as buds of epithelial tissue in the lamina propria (Sangari et al. 1992). Arguably therefore, age-related differences in distribution pattern, structural forms and volume densities of the Bowman’s glands may be linked to the anatomical transformation that happen as the glands develop from primitive to definitive forms. Page 9/21 In the NRM epithelium, ciliated cells play role in mucociliary clearance whereas non-ciliated columnar cells have microvilli that increase their apical surface area for retention of moisture and to prevent drying of the epithelial surface (Mygind et al. 1982; Beule 2010). Basal cells, on the other hand, have progenitor properties and can self-renew and differentiate into other epithelial cell types (Rock et al., 2010). In suckling GRCs, the ciliated cells are linearly arranged leaving wide spaces occupied by the non-ciliated cells whereas adult GRCs showed ciliated and non-ciliated cells that are fairly uniformly distributed. In a study by Maina et al. (1992) in which the ultrastructure of the trachea was compared in two species of East African mole rats, differences in the number and distribution pattern of ciliated and non-ciliated cells was observed, a feature that was believed to signify differences in structural modulation of the airway and to have a bearing on phylogeny, functional requirements and behavior. Notably also, age-related increase in vascularization in the GRC NRM and OM may, according to Sangari et al. (2000), be associated with increasing metabolic demands of replicating mucosal cells toward completion of their functional maturation. The ultrastructural appearance of the apical surface of the OE is influence by both the animal’s age and species. The radial pattern of cilia projection, which has also been demonstrated in humans (Lenz, 1977), horses (Kumar et al., 2000), dogs (Kavoi et al., 2010) and Sengis (Kavoi, 2018) is associated with higher cilia numbers when compared with the parallel pattern commonly seen in bovids (Menco, 1978; Kavoi et al., 2010). In rabbits (Kavoi et al. 2012), the transition from suckling to adulthood showed OE surface changes similar to those currently noted in GRCs. CONFLICT OF INTEREST The authors declare no conflict of interest. The authors declare no conflict of interest. Discussion Such refinement in OE structure are function-related and an important prerequisite to self-dependency (change in lifestyle including feeding from sucking to gnawing) at adulthood. Furthermore, the aforementioned differences in OE cytoarchitecture reflects the great variability in the number of ORN receptor genes among species that are reminiscent of the species’ lifestyles and which is caused by frequent gene gains and losses during evolution (Niimura et al. 2014). Besides, Dubied et al. (2021) compared ontogenetic trajectories of mandible shape in rodents (suborders Hystricomorpha and Myomorpha) to reveal that epigenetic interactions between the skull bones and muscles influence spatialization of bone formation and remodeling in response to biomechanical strain, a mechanism that was ascribed to developmental plasticity emanating from evolutionary divergences between species and clades. This study compares GRC data on packing densities of ORNs and cilia number/ per ORN knob between suckling and adult ages with what is documented for the rabbit (Kavoi et al. 2012) whose altricial young rely heavily on olfaction for nipple attachment (Coureaud et al. 2008), the dog (Kavoi et al. 2010), a relatively generalized carnivore with well-developed olfactory cue for tracking and catching prey (Walker, 1975) and the sheep, a specialized grazer whose olfactory cue is comparatively less important (Levy et al. 2004). We note that the above mentioned parameters are greatest in rabbits, followed by GRCs, dogs and then sheep, a pattern that appears to match with olfactory function demand levels for each species. ORN cilia are the principal sites for the initial events of olfactory transduction and signaling (Menco et al. 1997) and therefore the higher the ORN density and cilia number/ ORN knob, the greater the area of the cilia plasmalemma is made available for odor binding. Page 10/21 Page 10/21 Overall, results of this work show that the GRC OM and NRM undergo, during juvenile development, histological and ultrastructural modifications at levels that result to a functionally better specialized mucosae than is the case (for the OM) in the dog and sheep (Kavoi et al. 2010) but generally less so when compared with the rabbit (Kavoi et al. 2012). Behaviorally, GRCs are nocturnal herbivores that travel at night through trails in reeds and grass and that exhibit fossorial, precocial and solitary lifestyles (NRC 1991; Mustapha et al. 2020; Williams et al. 2011; Adu et al. 2017). Discussion Adaptation to these behavioral characteristics requires a sophisticated olfactory system whose structural details are presented here and in our earlier paper (Kavoi et al. 2021). We assert that different selective pressures, which are undoubtedly liked to dynamics of evolution, have acted upon the olfactory systems of the above species to produce the observed outcomes. These data form an important basis for understanding GRC biology and physiological adaptation and may find application in the broader areas of comparative anatomy, wildlife behavior and mammalian evolution. Future studies on the olfactory system in hystricognaths should incorporate more representative species. ACKNOWLEDGEMENTS We thank the Department of Animal Science, University of Ghana, for providing the GRCs for this work. Laboratory space, equipment and chemicals were made available by the School of Veterinary Medicine, University of Ghana, and also the Departments of Veterinary Anatomy & Physiology and Human Anatomy, University of Nairobi. Messrs. Derrick Asare, Kingsley Amoako and Ephraim Yawson of the School of Veterinary Medicine, University of Ghana, helped in tissue harvesting. SEM work was guided by Mr. Shem Ochieng’ of Kenyatta University at International Centre for Insect Physiology & Ecology (ICIPE), Nairobi, Kenya. 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Rock JR, Randell SH, Hogan BL (2010) Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling. Dis Model Mech 3:545–556 55. Sangari SK, Sengupta P, Pradhan S (1992) Histogenesis of human olfactory mucosa. J Anat Soc India 41:111–115. 56. Sangari SK, Sengupta P, Pradhan S, Khatri K (2000) Vascularization of developing human olfactory neuroepithelium—a morphometric study. Cells Tissues Organs 166:349–353 57. Sorokin SP (1988) A Textbook of Histology. In: Weiss L (ed) Cell and tissue biology: The respiratory system. Urban and Schwarzenberg, Baltimore, pp 751–814. 58. Walker EP (1975) Mammals of the world, 3rd edn. The Johns Hopkins University Press, Baltimore and London. 59. Wiens J, Ackerly DD, Allen AP et al (2010) Niche conservatism as an emerging principle in ecology and conservation biology. Ecol Lett 13: 1310–1324. 60. Willams OS, Ola SI, Boladuro BA, Badmus RT (2011) Diurnal variation in ambient temperature and humidity in a pit pen grasscutter (Thryonomys swinderianus) house in IleIfe. Proceedings of the 36th annual conference of the Nigerian Society of Animal Production. Abuja, Nigeria, pp 111–113. 61. Wood B.J (1994) Rodents in agriculture and forestry. In: Buckle AP, Smith RH (eds) Rodent pests and their control. CAB International, Wallingford, pp 45–83. 61. Wood B.J (1994) Rodents in agriculture and forestry. In: Buckle AP, Smith RH (eds) Rodent pests and their control. CAB International, Wallingford, pp 45–83. 62. Woods CA, Kilpatrick CW (2005) Infraorder Hystricognathi Brandt, 1855. In: Wilson DE, Reeder DM (eds) Mammal species of the world: a taxonomic and geographic reference, 3rd edn. Figures Page 15/21 Figure 1 Light micrographs showing the OM in suckling (A1) and adult ages (B) of the GRC. A1 & B: the OM Page 16/21 Figure 1 Light micrographs showing the OM in suckling (A1) and adult ages (B) of the GRC. A1 & B: the OM presents a distinctly formed nuclear (Nu) and non-nuclear (Nn) zones and an equally thick basement membrane (asterisks). In the lamina propria, ORN axon bundles (B), Bowman’s glands (G) and blood vessels (V) present a uniform pattern of distribution in suckling animals in contrast to the adults where the bundles take a deeper position than the glands. The glands are acinar type in suckling and tubular Figure 1 Light micrographs showing the OM in suckling (A1) and adult ages (B) of the GRC. A1 & B: the OM presents a distinctly formed nuclear (Nu) and non-nuclear (Nn) zones and an equally thick basement membrane (asterisks). In the lamina propria, ORN axon bundles (B), Bowman’s glands (G) and blood vessels (V) present a uniform pattern of distribution in suckling animals in contrast to the adults where the bundles take a deeper position than the glands. The glands are acinar type in suckling and tubular in Page 16/21 Page 16/21 the adults A2: shows the conspicuous free zone (double arrow heads) and terminal bar (arrow) that characterizes the OE in both age groups. A1 & A2- Masson’s trichrome stain, B- toluidine blue stain. Scale bar= 40µm in A1 & B, 20µm in A2. Page 17/21 Figure 2 Figure 2 Figure 2 Page 17/21 Page 17/21 Page 17/21 Semi-thin sections of GRC nasal respiratory mucosa in suckling (A1 & A2) and adult (B) age groups. A1: the epithelium in both groups present three cell types namely, ciliated cells (C) projecting tufts of cilia (arrows) on the epithelial surface, non-ciliated columnar cells (N) with nuclei positioned below those of the ciliated cells and basal cells (B) that are confined to the basal part of the epithelium where they overlie the basement membrane (asterisks). A2 & B: the lamina propria is observed to have a greater number (per unit area) of acinar-type glands (G) and larger diameter blood vessels (V) in the adult than in the suckling animals. Toluidine blue stain. Scale bar= 10µm in A1, 20µm in A2 & B. Page 18/21 Page 18/21 Figure 3 Scanning electron micrographs illustrating the apical surface appearance of OE in suckling (A1 & A2) and adult (B1 & B2) GRCs. Observed on the epithelial surface in each age group are dendrites of ORNs (O), apices of supporting cells (S) and tunnel-like openings (dotted arrows) of Bowman’s gland ducts. A1 & A2: supporting cells and ORNs appear nearly equal in number (per area) and cilia project in a parallel pattern from the ORN dendritic knobs (Dk) in form of a tuft B1 & B2: ORNs predominate the epithelial surface to almost obscure the supporting cell apices and cilia emerge from around the bases of the ORN dendritic knobs in a radial pattern. Scale bar= 5µm in A1 & B1, 1µm in A2 & B2. Scanning electron micrographs illustrating the apical surface appearance of OE in suckling (A1 & A2) and adult (B1 & B2) GRCs. Observed on the epithelial surface in each age group are dendrites of ORNs (O), apices of supporting cells (S) and tunnel-like openings (dotted arrows) of Bowman’s gland ducts. A1 & A2: supporting cells and ORNs appear nearly equal in number (per area) and cilia project in a parallel pattern from the ORN dendritic knobs (Dk) in form of a tuft B1 & B2: ORNs predominate the epithelial surface to almost obscure the supporting cell apices and cilia emerge from around the bases of the ORN dendritic knobs in a radial pattern. Scale bar= 5µm in A1 & B1, 1µm in A2 & B2. Page 19/21 4 Page 20/21 Figure 4 SEM features of NRM epithelial surface in weanling (A) and adult (B) GRCs. In both age groups, the epithelium is characterized by ciliated (C) cells that project hundreds of cilia (arrow heads) above the epithelial surface, and non-ciliated cells (N) with a numerous microvilli on their apical surfaces. Distinct intercellular boundaries (arrows) and tunnel-like openings of subepithelial gland ducts (asterisks) also characterize the epithelia of both age groups A: the epithelium contains a preponderance of non-ciliated Figure 4 SEM features of NRM epithelial surface in weanling (A) and adult (B) GRCs. In both age grou epithelium is characterized by ciliated (C) cells that project hundreds of cilia (arrow heads) a epithelial surface, and non-ciliated cells (N) with a numerous microvilli on their apical surfac intercellular boundaries (arrows) and tunnel like openings of subepithelial gland ducts (aster Figure 4 Figure 4 SEM features of NRM epithelial surface in weanling (A) and adult (B) GRCs. In both age groups, the epithelium is characterized by ciliated (C) cells that project hundreds of cilia (arrow heads) above the epithelial surface, and non-ciliated cells (N) with a numerous microvilli on their apical surfaces. Distinct intercellular boundaries (arrows) and tunnel-like openings of subepithelial gland ducts (asterisks) also characterize the epithelia of both age groups A: the epithelium contains a preponderance of non-ciliated SEM features of NRM epithelial surface in weanling (A) and adult (B) GRCs. In both age groups, the epithelium is characterized by ciliated (C) cells that project hundreds of cilia (arrow heads) above the epithelial surface, and non-ciliated cells (N) with a numerous microvilli on their apical surfaces. Distinct intercellular boundaries (arrows) and tunnel-like openings of subepithelial gland ducts (asterisks) also characterize the epithelia of both age groups A: the epithelium contains a preponderance of non-ciliated SEM features of NRM epithelial surface in weanling (A) and adult (B) GRCs. In both age groups, the epithelium is characterized by ciliated (C) cells that project hundreds of cilia (arrow heads) above the epithelial surface, and non-ciliated cells (N) with a numerous microvilli on their apical surfaces. Distinct intercellular boundaries (arrows) and tunnel-like openings of subepithelial gland ducts (asterisks) also characterize the epithelia of both age groups A: the epithelium contains a preponderance of non-ciliated Page 20/21 Page 20/21 cells forming a carpet punctuated by linearly arranged ciliated cells (see dotted arrow) B: ciliated and non- ciliated cells occur in nearly equal numbers and their distribution pattern is fairly uniform. Scale bar= 10 µm in A & B. cells forming a carpet punctuated by linearly arranged ciliated cells (see dotted arrow) B: ciliated and non- ciliated cells occur in nearly equal numbers and their distribution pattern is fairly uniform. Scale bar= 10 µm in A & B. Figure 5 Mean volume densities (%) for glands and blood vessels in the NRM of suckling and adult GRCs. Volume density increment is age-related and quite significant in the case of the glands, p< 0.05. Figure 5 Mean volume densities (%) for glands and blood vessels in the NRM of suckling and adult GRCs. Volume density increment is age-related and quite significant in the case of the glands, p< 0.05. Mean volume densities (%) for glands and blood vessels in the NRM of suckling and adult GRCs. Volume density increment is age-related and quite significant in the case of the glands, p< 0.05. Page 21/21
https://openalex.org/W3170615192	https://zenodo.org/records/5348092/files/Rausp%20volume%2056%20revista%203%20artigo%204.pdf	English	null	The case for social support in social marketing	RAUSP Management Journal	2,021	cc-by	12,033	The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/2531-0488.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/2531-0488.htm Abstract Purpose – This paper aims to reinforce the arguments for applying the social support concept in social marketing. Design/methodology/approach – This paper aims to conceptually outline the potential positiv contribution of social support for social marketing practice as a tool to induce behavior change. Findings – This paper focuses on the philosophical principle of social exchange, highlights the consumer- centered perspective of social marketing, which implies the natural evaluation of the social networks of inﬂuence and support and presents social support as a mechanism to induce long-term behavior change. Research limitations/implications – No empirical (qualitative or quantitative) investigations wer used to test the application of the concept in practical interventions. Research limitations/implications – No empirical (qualitative or quantitative) investigations were used to test the application of the concept in practical interventions. Practical implications – This paper provides signiﬁcant insights for intervention developers that can be used to program and theoretically justify future social marketing interventions applying the social support concept. Practical implications – This paper provides signiﬁcant insights for intervention developers that can b used to program and theoretically justify future social marketing interventions applying the social suppor concept. Social implications – Empirical research concluded for a positive relation between social support and human health and well-being. Thus, increasing the use of the concept in social marketing can serve to attain these social goals. Originality/value – The concept of social support has gained considerable interest in the areas of behavioral medicine and health psychology. Despite such interest, it is still not clear how it can be approached in social marketing as there is a lack of conceptual literature discussing social support from a social marketing perspective, the number of social marketing interventions operationalizing the concept is limited and, till date, no research has focused in comprehensively establishing a theoretical rationale to operationalize the concept in social marketing. Keywords Social marketing, Social support, Behavior change Keywords Social marketing, Social support, Behavior change Paper type Research paper RAUSP Management Journal Vol. 56 No. 3, 2021 pp. 295-313 EmeraldPublishingLimited 2531-0488 DOI 10.1108/RAUSP-08-2020-0193 © Nuno Baptista, Helena Alves and José Pinho. Published in RAUSP Management Journal. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. 295 Helena Alves University of Beira Interior and NECE, Covilhã, Portugal, and Received 30 August 2020 Revised 1 November 2020 7 February 2021 Accepted 2 March 2021 José Pinho University of Minho and NIPE, Braga, Portugal The authors contributed in following ways: Baptista, Nuno, Investigation (Equal), Alves, Helena, Conceptualization (Lead), Investigation (Equal), Methodology (Lead), Pinho, José, Conceptualization (Lead), Investigation (Equal), Methodology (Lead). The case for social support in social marketing Social support in social marketing Social support in social marketing Nuno Baptista Instituto Politécnico de Lisboa, Escola Superior de Comunicação Social and NECE, Covilhã, Lisboa, Portugal © Nuno Baptista, Helena Alves and José Pinho. Published in RAUSP Management Journal. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence maybe seen at http://creativecommons.org/ licences/by/4.0/legalcode RAUSP Management Journal Vol. 56 No. 3, 2021 pp. 295-313 EmeraldPublishingLimited 2531-0488 DOI 10.1108/RAUSP-08-2020-0193 Abstract The full terms of this licence maybe seen at http://creativecommons.org/ licences/by/4.0/legalcode RAUSP Management Journal Vol. 56 No. 3, 2021 pp. 295-313 EmeraldPublishingLimited 2531-0488 DOI 10.1108/RAUSP-08-2020-0193 The authors contributed in following ways: Baptista, Nuno, Investigation (Equal), Alves, Helena, Conceptualization (Lead), Investigation (Equal), Methodology (Lead), Pinho, José, Conceptualization (Lead), Investigation (Equal), Methodology (Lead). 1. Introduction Outside the domain of social marketing, a mechanism that has been extensively used in the health area consists in enhancing the social support networks of individuals (Baptista, Alves, & Pinho, 2020a). The overwhelming evidence about the positive impact of social support on individuals’ physical, emotional, and social well-being (Pan, Feng, & Skye Wingate, 2018) has fueled the development of many public health interventions designed to improve social support (Uchino, 2009; Baptista et al., 2020a). RAUSP 56,3 296 296 ) Social marketing literature exploring social support is very limited. Albrecht (1995) proposed that social marketers, when promoting health services, should take in consideration the capacity of social support to reduce the recipients’ uncertainty under stressful conditions and the increased sense of personal control. This paper reinforces Albrecht’s (1995) arguments, highlighting the utility of social support as a mechanism to attain long-term behavior change in social marketing. Although several mechanisms have been suggested for social marketing to be effective, there is some level of agreement in the literature that three main elements are central to social marketing, namely, the principle of exchange, a consumer-centered perspective; and a long-term focus (Glenane-Antoniadis, Whitwell, Bell, & Menguc, 2003). This paper analyzes each of these key principles that constitute the basis of the authors’ argument for promoting social support within social marketing. The paper proceeds as follows. After this brief introduction, the concept of social support is presented, followed by a narrative review of social marketing literature. The argument for social support in social marketing is then presented, focusing on the three core issues of social exchange, consumer-centered perspective and long-term focus. 2. Concept of social support 2. Concept of social support Despite being Caplan (1974) who ﬁrst introduced the notion of social support, the scientiﬁc interest in social support was triggered largely from independent research by two physician epidemiologists, presented in the same year of 1976: John Cassel and Sidney Cobb (Barrera, 1986; House, Landis, & Umberson, 1988; House, Umberson, & Landis, 1988; Uchino, Cacioppo, & Kiecolt-Glaser, 1996; Berkman, 2000; Sarason and Sarason, 2009). Curiously, Cassel’s (1976) and Cobb’s (1976) methods and ﬁndings were very similar. Both authors reviewed extant empirical evidence and concluded about the importance of social relationships and support to the maintenance and promotion of good health. Cassel (1976) and Cobb (1976) found that people with more limited or negative social relationships appeared to get health problems more frequently than those with vaster and more abiding relationships (Baptista et al., 2020a). The authors advanced several possible reasons for this link, but they both emphasized the role of social relationships in buffering the negative health consequences of psychosocial stress (Baptista et al., 2020a). Cassel focused on the physiological processes mediating the effects of social relationships in health outcomes. Drawing from numerous animal and human studies, Cassel theorized social conditions relevant to health from a functionalist perspective, assigning one category to the factors that promote health, and other to the elements that produce disease. In the ﬁrst category, Cassel included social support, as a factor that protects people from the physiologic or psychological consequences of stress exposure. Cobb on the other side, applied a communication perspective and synthetized evidence showing supportive communication to be protective against the health consequences of various life stresses, conceiving social support as “information leading the subject to believe that he is cared for and loved, esteemed, and a member of a network of mutual obligations” (1976, p. 1). believe that he is cared for and loved, esteemed, and a member of a network of mutual obligations” (1976, p. 1). 2. Concept of social support Cassel’s and Cobb’s studies were fundamental in stimulating new research on the impact Social support in social k i Cassel’s and Cobb’s studies were fundamental in stimulating new research on the impact of social support on all aspects of health (House et al., 1988) and while the concept of social support started to be extensively studied in the years that followed, there was little agreement among researchers as to its theoretical deﬁnition and, as a result, the concept was initially vague, with anything that inferred a social interaction or relationship being classiﬁed as indicative of social support (Hupcey, 1998; Baptista et al., 2020a). Discussions about the conceptual problems of social support have been provided by several academics, most notably Thoits (1982), Bruhn and Philips (1984); Barrera (1986), Hupcey (1998), and more recently, Williams Barclay and Schmied (2004) and Sarason and Sarason (2009). On the whole, these authors consider social support as a multi-faceted concept that cannot be deﬁned by a few attributes (Baptista et al., 2020a). Rather, it is the process as a whole that differentiates social support from similar concepts and constructs such as caring, social integration, social participation, social ties, social anchorage, social control, social inﬂuence, social modeling, social norms or social identity (House, 1987; Due, Holstein, Lund, Modvig, & Avlund, 1999; Berkman, Glass, Brissette, & Seeman, 2000; Finfgeld-Connett, 2005; Baptista et al., 2020a). Further problems identiﬁed by the previously mentioned authors include the lack of a conceptual deﬁnition of social support, a certain bias in the literature favoring the stress buffering explanation for the effects of social support in health, as opposed to direct effects, and difﬁculties in social support measurement (Williams et al., 2004; Sarason and Sarason, 2009). 297 ; , ) While a substantial body of work on the concept of social support has resulted in a large number of deﬁnitions, used across disciples and research ﬁelds, three basic dimensions of social support are often identiﬁed in the literature as follows: social embeddedness, referring to the network relations that individuals develop, and that allow support ﬂow; enacted support, or supporting behavior, meaning the actual supportive actions that individuals perform; and perceived social support, consisting in the subjective perceptions or evaluations of the support that is given or accessible (Barrera, 1986; Vaux, 1988; Hupcey, 1998; Baptista et al., 2020a). 2. Concept of social support Documented positive health outcomes of social support include psychological adjustment, improved adherence and increased efﬁcacy of treatments, better coping with illness uncertainty, reduced stress and depressive symptoms, increased resistance and recovery to disease, improved immune function, mitigation of the risks of suicide and reduced mortality (Cassel, 1976; Cobb, 1976; Barrera, 1986; Thoits, 1986; Heitzmann & Kaplan, 1988; Mattson & Hall, 2011; Nolan, Hendricks, Ferguson, & Towell, 2017; Li, He, Wang, & Wang, 2019; Spence, March, & Donovan, 2019; Schrock, Snodgrass, & Sugiyama, 2020; Baptista et al., 2020a). Slack, Da Silva, & Sinclair, 2000), sociology (Wellman & Wortley, 1990; Turner & Marino, 1994), education (Wall, Covell, & MacIntyre, 1999; Wilcox, Winn, & Fyvie-Gauld, 2005) and social work (Letourneau, Stewart, & Barnfather, 2004; Msengi, Arthur-Okor, Killion, & Schoer, 2015). Nonetheless, the main interest in social support is motivated by its relationship to physical and mental health (House, 1987; Hobfoll, Freedy, Lane, & Geller, 1990), hence the concept has gain considerable traction in health psychology and behavioral medicine (DiMatteo, 2004). Several reviews of empirical research concluded for a positive relation between social support and human health and well-being (Hogan, Linden, & Najarian, 2002; Uchino, 2006; Mazzoni & Cicognani, 2011; Taylor, 2011; Nurullah, 2012; Paterson, Jones, Rattray, & Lauder, 2013; Silva & Loureiro, 2014; Barton, Efﬁng, & Cafarella, 2015; Gariépy, Honkaniemi, & Quesnel-Vallée, 2016; Brunelli, Murphy, & Athanasou, 2017; Smith, Banting, Eime, O’Sullivan, & van Uffelen, 2017; Ali, Kokorelias, MacDermid, & Kloseck, 2018; Teoh & Hilmert, 2018; Wang, Mann, Lloyd-Evans, Ma, & Johnson, 2018). Documented positive health outcomes of social support include psychological adjustment, improved adherence and increased efﬁcacy of treatments, better coping with illness uncertainty, reduced stress and depressive symptoms, increased resistance and recovery to disease, improved immune function, mitigation of the risks of suicide and reduced mortality (Cassel, 1976; Cobb, 1976; Barrera, 1986; Thoits, 1986; Heitzmann & Kaplan, 1988; Mattson & Hall, 2011; Nolan, Hendricks, Ferguson, & Towell, 2017; Li, He, Wang, & Wang, 2019; Spence, March, & Donovan, 2019; Schrock, Snodgrass, & Sugiyama, 2020; Baptista et al., 2020a). 298 The theoretical background explaining the positive effects of social support in health consists in two basic theories: the relational regulation theory and the stress and coping theory. 2. Concept of social support Since the 1980s, researchers have moved away from considering social support a unitary concept, and attempted to increase the speciﬁcity of the term by identifying its core components (Wortman, 1984; Baptista, Pinho, & Alves, 2020b). Various taxonomies of social support have been proposed, with considerable similarity among the various typologies. Since the 1980s, researchers have moved away from considering social support a unitary concept, and attempted to increase the speciﬁcity of the term by identifying its core components (Wortman, 1984; Baptista, Pinho, & Alves, 2020b). Various taxonomies of social support have been proposed, with considerable similarity among the various typologies. Considering the multidimensional nature of the concept, and drawing from previous frameworks, Cutrona and Suhr (1992) proposed a social support behavior code that models social support into ﬁve basic dimensions. Informational support offers facts, advice, guidance or feedback, whereas emotional support consists in expressions of affection, concern, empathy or sympathy. Esteem support shows a positive regard for the recipient’s skills, abilities and intrinsic value. Network support consists of promoting a sense of membership or belonging and tangible support is conceived as providing goods and services to the recipient. As noted by Baptista et al. (2020a), since the introduction of the concept in health studies, several other disciplines have found value in better understanding the effects of social support in human well-being, including, for example, psychology (Chay, 1993; Tetrick, RAUSP 56,3 Slack, Da Silva, & Sinclair, 2000), sociology (Wellman & Wortley, 1990; Turner & Marino, 1994), education (Wall, Covell, & MacIntyre, 1999; Wilcox, Winn, & Fyvie-Gauld, 2005) and social work (Letourneau, Stewart, & Barnfather, 2004; Msengi, Arthur-Okor, Killion, & Schoer, 2015). Nonetheless, the main interest in social support is motivated by its relationship to physical and mental health (House, 1987; Hobfoll, Freedy, Lane, & Geller, 1990), hence the concept has gain considerable traction in health psychology and behavioral medicine (DiMatteo, 2004). Several reviews of empirical research concluded for a positive relation between social support and human health and well-being (Hogan, Linden, & Najarian, 2002; Uchino, 2006; Mazzoni & Cicognani, 2011; Taylor, 2011; Nurullah, 2012; Paterson, Jones, Rattray, & Lauder, 2013; Silva & Loureiro, 2014; Barton, Efﬁng, & Cafarella, 2015; Gariépy, Honkaniemi, & Quesnel-Vallée, 2016; Brunelli, Murphy, & Athanasou, 2017; Smith, Banting, Eime, O’Sullivan, & van Uffelen, 2017; Ali, Kokorelias, MacDermid, & Kloseck, 2018; Teoh & Hilmert, 2018; Wang, Mann, Lloyd-Evans, Ma, & Johnson, 2018). RAUSP 56,3 3. Social marketing 299 Drawing on its parent discipline of commercial marketing, which largely drew on economics and psychology, social marketing has become an important research ﬁeld and evolved over the years into a discipline in its own right (Buyucek, Kubacki, Rundle-Thiele, & Pang, 2016). The salient characteristic of social marketing is that it takes learning from commercial marketing and applies it to the resolution of social problems (Stead, Gordon, Angus, & McDermott, 2007). Andreasen (1994) deﬁned the scope of social marketing by proposing the following deﬁnition: “Social marketing is the application of commercial marketing technologies to the analysis, planning, execution and evaluation of programs designed to inﬂuence the voluntary behavior of target audiences in order to improve their personal welfare and that of society” (p. 110). Two key attributes in this deﬁnition shaped the current understanding of social marketing. First, Andreasen observed that social marketing should inﬂuence not only “social ideas” but also attitudes and behaviors and introduced the notion of voluntary behavior change, implying that social marketing is not about coercion or enforcement; second, there is the implied principle that, contrary to commercial marketing, social marketing is not intended to beneﬁt the organization that promotes the marketing actions, but rather to beneﬁt individuals’ welfare or promote societal good (Baptista et al., 2020a). Furthermore, the emphasis on society as well as on the individual, implies another fundamental aspect about social marketing: it can apply not only to the behavior of individuals but also to that of professionals, organizations and policymakers, meaning the contextual actors that can inﬂuence individuals’ behavior change (Gordon, Mcdermott, Martine, & Angus, 2006; Baptista, Pinho, & Alves, 2021). , ) Traditional downstream social marketing focuses on the micro-level and places the responsibility for change in the individual (Russell-Bennett, Wood, & Previte, 2013). Midstream social marketing refers to community-based interventions, focused at the meso level and involving collaborations with community actors (people and organizations) and personal networks such as family and friends as means to achieve change (Luca, Hibbert, & McDonald, 2016). Upstream social marketing focuses on the macro system, by concentrating efforts on decision-making groups or individuals who have an inﬂuence over a target audience such as governments, politicians, regulators and other decision-makers (Cherrier & Gurrieri, 2014). 2. Concept of social support Research suggests these systems inﬂuence each other as they all share central nervous system mechanisms as their basis and regulator (Ditzen & Heinrichs, 2014). effects of social support in the neural system (Ditzen & Heinrichs, 2014; Eisenberger, Taylor, Gable, Hilmert & Lieberman, 2007; Morese, Lamm, Bosco, Valentini, & Silani, 2019) and immunologic responses (Pressman et al., 2005; Schrock et al., 2020). Research suggests these systems inﬂuence each other as they all share central nervous system mechanisms as their basis and regulator (Ditzen & Heinrichs, 2014). 2. Concept of social support In 1985 Cohen and Wills established a distinction between social support main effects and stress buffering effects in health, and this distinction has since played an essential role in shaping research and theory (Lakey & Orehek, 2011). The stress and coping theory explores social support as a response tool for dampening or protect against stress (Cohen & Wills, 1985; Barrera, 1986; Thoits, 1986). The relational regulation theory reports a main-effect model, in which social support is considered to improve health, irrespective of whether people are under stress, through relational inﬂuences, relationship stability or socially rewarded roles (Lakey & Orehek, 2011).The stress and coping theory, also referred to as the “stress-buffering” model, has been thoroughly developed and become dominant in social support research (Barrera, 1986; Lakey & Orehek, 2011; Baptista et al., 2020a). Social support theorists such as Cassel (1976) and Cobb (1976) were particularly concerned with the role of social support in the prevention of disease. They suggested that social support facilitates coping and adaptation by acting as a moderator of the pathogenic inﬂuence of stressful events. Social support is said to play a role at two different stages of the causal chain relating stress to illness: ﬁrst, the perception that others will provide support redeﬁnes the potential for harm posed by a stressful situation and increases one’s self-conﬁdence to cope with imposed demands, preventing an event from being appraised as stressful; second, social support intervenes between the experience of stress and the onset of the pathological outcome by providing a solution to the problem or by reducing the perceived relevance of the event (Cohen & Wills, 1985). Despite existing theories, the precise psychobiological pathways by which social support leads to health beneﬁts and the factors that moderate and mediate this relationship are still not completely understood (DiMatteo, 2004; Gale, Kenyon, MacArthur, Jolly, & Hope, 2018; Szkody & McKinney, 2019; Baptista et al., 2020b; Wilson, Weiss, & Shook, 2020). To solve this gap research has mostly focused on the effects of social support in cardiovascular response to stress (Teoh & Hilmert, 2018), Social support in social marketing effects of social support in the neural system (Ditzen & Heinrichs, 2014; Eisenberger, Taylor, Gable, Hilmert & Lieberman, 2007; Morese, Lamm, Bosco, Valentini, & Silani, 2019) and immunologic responses (Pressman et al., 2005; Schrock et al., 2020). 3. Social marketing Recent appeals of social marketing to address complex social problems and sustainable change in face of complex and wicked societal ills emphasize the importance of adopting a macro system-thinking perspective and facilitate supportive joint actions by a variety of stakeholders across and between sectors, communities and social networks (Brennan, Previte, & Fry, 2016; Domegan et al., 2016; Kennedy, Kapitan, Bajaj, Bakonyi, & Sands, 2017; Carvalho & Mazzon, 2019; Truong, Saunders, & Dong, 2019). For example, Brennan et al. (2016) argue for the value of a relational logic perspective to attain social change and the importance of collaboration and social support relations across a network of actors to overcome barriers and identify solutions to social problems, classifying social support for affected community members as a “strategic toolkit” when applying a systems approach to societal change. Discussing social change coalitions, Gurrieri, Gordon, Barraket, Joyce, and Green (2018) highlight the importance of supportive relations and the need to create common unity through emotional investment and returns. Furthermore, for addressing grand challenges social marketing scholars argue for the need to acknowledge socio-cultural factors to a signiﬁcant level, to adopt a cultural market orientation and to consider the cultural perspective of behavior change, including group inﬂuences, traditions and accepted narratives in supportive networks (Brennan et al., 2016; Gordon, Russell-Bennett, & Lefebvre, 2016; Martín-Santana, Cabrera-Suarez, & Déniz-Déniz, 2020). 300 , , ) Despite the several deﬁnitions of social marketing offered in seminal literature, one difﬁculty was deﬁning what constitutes a social marketing program (Stead, Mcdermott, Gordon, Angus, & Hastings, 2006; Firestone, Rowe, Modi, & Sievers, 2017) and what elements could potentially improve the impact of an intervention (French & Blair- Stevens, 2005). Considering this difﬁculty, and to establish a distinction between social marketing and alternative social approaches, such as social advertising, social media marketing, social policy or education (Russell-Bennett, Fisk, Rosenbaum, & Zainuddin, 2019), Andreasen (2002) proposed a scheme of six social marketing benchmarks that provides a basic structure for social marketing interventions. The six benchmark criteria proposed by this author include behavioral change, formative research, segmentation, the use of the marketing mix, the principle of exchange and competition. Alternative frameworks of social marketing benchmarks were proposed by other authors, including French and Blair-Stevens (2005). A relevant criteria not included in Andreasen’s (2002) benchmark scheme and contained in French and Blair-Stevens’s (2005) framework is the use of theory to understand behavior and inform social marketing interventions. 3. Social marketing Despite the several deﬁnitions of social marketing offered in seminal literature, one difﬁculty was deﬁning what constitutes a social marketing program (Stead, Mcdermott, Gordon, Angus, & Hastings, 2006; Firestone, Rowe, Modi, & Sievers, 2017) and what elements could potentially improve the impact of an intervention (French & Blair- Stevens, 2005). Considering this difﬁculty, and to establish a distinction between social marketing and alternative social approaches, such as social advertising, social media marketing, social policy or education (Russell-Bennett, Fisk, Rosenbaum, & Zainuddin, 2019), Andreasen (2002) proposed a scheme of six social marketing benchmarks that provides a basic structure for social marketing interventions. The six benchmark criteria proposed by this author include behavioral change, formative research, segmentation, the use of the marketing mix, the principle of exchange and competition. Alternative frameworks of social marketing benchmarks were proposed by other authors, including French and Blair-Stevens (2005). A relevant criteria not included in Andreasen’s (2002) benchmark scheme and contained in French and Blair-Stevens’s (2005) framework is the use of theory to understand behavior and inform social marketing interventions. Theory can help social marketing developers to understand the social system that inﬂuence behavior change, to better interpret relationships between different factors that affect behavior change, to improve the efﬁcacy of social marketing interventions and to make predictions and generalizations for similar events by tracing the pathways by which social marketing achieves behavior change outcomes (Luca & Suggs, 2013; Manikam & Russell-Bennett, 2016; Luecking et al., 2017; Firestone et al., 2017; Basil, 2019; Baptista et al., 2020a). The focus on a multilevel approach and the importance of theory to sustain social marketing practice is present in current deﬁnitions of social marketing. For example, in October 2013, the International Social Marketing Association (ISMA), the European Social Marketing Association (ESMA) and the Australian Association of Social Marketing RAUSP 56,3 300 Many issues that afﬂict human societies, such as poverty, unsustainable food systems, have a complex nature that is not compatible with traditional social marketing approaches (Carvalho & Mazzon, 2019). 3. Social marketing The turn from traditional downstream and micro-marketing approaches toward ecological perspectives, which account for change at the micro, meso and macro level has been pivotal in social marketing development (Cherrier & Gurrieri, 2014; Baptista et al., 2020a). The call for more ecological approaches is present in critical social marketing, which has been deﬁned by Gordon (2011, p. 89) as “critical research from a marketing perspective on the impact commercial marketing has upon society, to build the evidence base, inform upstream efforts such as advocacy, policy and regulation, and inform the development of downstream social marketing interventions.” The objective of critical social marketing is not simply to promote a critical analysis of social marketing but to change the discipline to become more reﬂexive, inclusive, transtheoretical and multidisciplinary in its approach to behavior and social change. Many issues that afﬂict human societies, such as poverty, unsustainable food systems, have a complex nature that is not compatible with traditional social marketing approaches (Carvalho & Mazzon, 2019). Recent appeals of social marketing to address complex social problems and sustainable change in face of complex and wicked societal ills emphasize the importance of adopting a macro system-thinking perspective and facilitate supportive joint actions by a variety of stakeholders across and between sectors, communities and social networks (Brennan, Previte, & Fry, 2016; Domegan et al., 2016; Kennedy, Kapitan, Bajaj, Bakonyi, & Sands, 2017; Carvalho & Mazzon, 2019; Truong, Saunders, & Dong, 2019). For example, Brennan et al. (2016) argue for the value of a relational logic perspective to attain social change and the importance of collaboration and social support relations across a network of actors to overcome barriers and identify solutions to social problems, classifying social support for affected community members as a “strategic toolkit” when applying a systems approach to societal change. Discussing social change coalitions, Gurrieri, Gordon, Barraket, Joyce, and Green (2018) highlight the importance of supportive relations and the need to create common unity through emotional investment and returns. Furthermore, for addressing grand challenges social marketing scholars argue for the need to acknowledge socio-cultural factors to a signiﬁcant level, to adopt a cultural market orientation and to consider the cultural perspective of behavior change, including group inﬂuences, traditions and accepted narratives in supportive networks (Brennan et al., 2016; Gordon, Russell-Bennett, & Lefebvre, 2016; Martín-Santana, Cabrera-Suarez, & Déniz-Déniz, 2020). 3. Social marketing Theory can help social marketing developers to understand the social system that inﬂuence behavior change, to better interpret relationships between different factors that affect behavior change, to improve the efﬁcacy of social marketing interventions and to make predictions and generalizations for similar events by tracing the pathways by which social marketing achieves behavior change outcomes (Luca & Suggs, 2013; Manikam & Russell-Bennett, 2016; Luecking et al., 2017; Firestone et al., 2017; Basil, 2019; Baptista et al., 2020a). The focus on a multilevel approach and the importance of theory to sustain social marketing practice is present in current deﬁnitions of social marketing. For example, in October 2013, the International Social Marketing Association (ISMA), the European Social Marketing Association (ESMA) and the Australian Association of Social Marketing (AASM) (ISMA, ESMA, & AASM, 2013) endorsed a comprehensive deﬁnition of social marketing, which states that: (AASM) (ISMA, ESMA, & AASM, 2013) endorsed a comprehensive deﬁnition of social marketing, which states that: Social support in social (AASM) (ISMA, ESMA, & AASM, 2013) endorsed a comprehensive deﬁnition of social marketing, which states that: Social Marketing seeks to develop and integrate marketing concepts with other approaches to Social support in social marketing Social Marketing seeks to develop and integrate marketing concepts with other approaches to inﬂuence behaviors that beneﬁt individuals and communities for the greater social good. Social Marketing practice is guided by ethical principles. It seeks to integrate research, best practice, theory, audience and partnership insight, to inform the delivery of competition sensitive and segmented social change programmes that are eﬀective, eﬃcient, equitable and sustainable (p. 1). 301 The principle of exchange has been identiﬁed by authors such as Andreasen (2002) as a suitable framework for conceptualizing social marketing, notwithstanding, some authors resist to accept exchange as relevant, arguing that the social issues social marketing seeks to address make exchange difﬁcult to occur and the reciprocal nature or balanced mutual beneﬁt underpinning the logic of exchange is not always evident (Duane, Domegan, McHugh, & Devaney, 2016). g , y, ) While social support is under-investigated in social marketing, in the close ﬁeld of transformative service research (TSR) the concept of social support has been intensively explored in the study of vulnerable consumers, with interesting results. TSR is an emerging ﬁeld of service marketing research that is focused on the relationships between services and consumer well-being to relieve or minorize forms of consumer suffering. 3. Social marketing The term “transformative service research” was ﬁrst used by Rosenbaum (2006) in a study of the role of third places (places such as diners, coffee shops and taverns) in providing support to consumers. In this study, Rosenbaum explored how third places can serve to satisfy not only consumption needs but also consumer needs for social support, and concluded for a positive relationship between received social support and loyalty to these commercial places. Under a TSR framework, and studying two online communities for people with Parkinson’s disease and motor neuron disease, Loane, Webster, and D’Alessandro (2015) demonstrated that members experience the co-creation of different types of consumer value through the exchange of social support in these consumer-dominated online environments. More recently and also studying an online support group for weight management, Parkinson, Schuster, Mulcahy, and Taiminen (2017) showed how vulnerable consumers experience transformative services which are consumer-dominant to overcome their vulnerability, by creating safe third places, to receive social support and concluded that transformative services have the capacity to optimize their services to enable vulnerable consumers to co-create social support. 4. Framing social support within social marketing 4 1 P i i l f i l h Similarly to social marketing, exchange models also provide methods for addressing the interdependency of relationships involved in social support exchanges (Shumaker & Brownell, 1984; Baptista et al., 2020a). Social support is basically an exchange of resources between individuals (Gottlieb & Bergen, 2010) and there are potential costs and beneﬁts associated with the exchange for the participants (Baptista et al., 2020a). The beneﬁts may include enhanced coping, self-efﬁcacy, improved decision- making and reduced stress (Mattson & Hall, 2011). The costs of social support may assume the form of time and effort (spent in support interactions), inadequate or misguided information, unwanted control and attention, social aggression, reduced self-efﬁcacy and stress (Taylor, 2011; Gale et al., 2018; Reijnders, Schalk, & Steen, 2018; Gray et al., 2019; Palant & Himmel, 2019; Zee & Kumashiro, 2019; Baptista et al., 2021). According to Baptista et al. (2021) the norm of generalized reciprocity, involves the intention to provide social support without a calculation of immediate value or dyad repayment, and can be motivated by altruistic reasons, a desire to help others and see the community ﬂourish or based on the hope that support exchanges will eventually be reciprocal, in the long run, or that payback is in a different currency such as network status, honor or others’ approval (Baker & Bulkley, 2014). Reciprocity refers to a form of conditional gains in which people’s present actions are expected to generate future beneﬁts (Hung, Durcikova, Lai, & Lin, 2011) contributing to present well-being by anticipating future positive rewards (Baptista et al., 2021). According to Mathwick, Wiertz, & De Ruyter (2008), the expectation of long-term repayment imposes an informal social control system that obviates the need for more formal, institutionalized hierarchical control, resulting in a highly efﬁcient social system that requires less formal control. This stability of the social system facilitates the accumulation of trust and further fosters social support provision (Baptista et al., 2021). RAUSP 56,3 302 (Duane et al., 2016). Social exchange involves actions that are contingent on rewarding reactions from others. The content of social exchange can be utilitarian (e.g. tangible, network or informational types of social support) or symbolic (e.g. emotional and esteem social support) or mixed (involving a combination of both utilitarian and symbolic). 4. Framing social support within social marketing 4 1 P i i l f i l h Traditionally, the content of exchange under a social marketing framework has tended to be utilitarian, with social marketers mostly focusing their efforts on marketing tangible offers, for example, food and medicine (Andreasen, 2002) and in restricted types of exchanges, involving two party direct relationships (Duane et al., 2016), were the intervention promoter offers tangible assistance to the targeted individuals. However, as the discipline evolved, the content of exchange has become more symbolic as its application has broadened to wider social causes (Glenane-Antoniadis et al., 2003) and restricted exchanges has since been criticized for neglecting to examine the broader structural, behavioral and exchange contexts (Duane et al., 2016). Generalized exchanges involve a system of mutual relationships between three or more actors, whereas complex exchanges involve mutual relationships between at least three actors but are reciprocal and have an extended timeframe, unlike generalized exchanges (Glenane-Antoniadis et al., 2003). According to Duane et al. (2016) when social marketing wants to induce social change, the problematic nature of exchange becomes more salient because the social marketer is faced, not with a singular exchange but with coordinating numerous exchanges, that is, one web of complex exchanges. (Duane et al., 2016). Social exchange involves actions that are contingent on rewarding reactions from others. The content of social exchange can be utilitarian (e.g. tangible, network or informational types of social support) or symbolic (e.g. emotional and esteem social support) or mixed (involving a combination of both utilitarian and symbolic). Traditionally, the content of exchange under a social marketing framework has tended to be utilitarian, with social marketers mostly focusing their efforts on marketing tangible offers, for example, food and medicine (Andreasen, 2002) and in restricted types of exchanges, involving two party direct relationships (Duane et al., 2016), were the intervention promoter offers tangible assistance to the targeted individuals. However, as the discipline evolved, the content of exchange has become more symbolic as its application has broadened to wider social causes (Glenane-Antoniadis et al., 2003) and restricted exchanges has since been criticized for neglecting to examine the broader structural, behavioral and exchange contexts (Duane et al., 2016). Generalized exchanges involve a system of mutual relationships between three or more actors, whereas complex exchanges involve mutual relationships between at least three actors but are reciprocal and have an extended timeframe, unlike generalized exchanges (Glenane-Antoniadis et al., 2003). According to Duane et al. 4. Framing social support within social marketing 4 1 P i i l f i l h p f g The social exchange-based view of human behavior was introduced by authors such as Thibaut and Kelley (1959), Homans (1958); and Blau (1964) in the ﬁelds of psychology and sociology. According to social exchange theory, people’s actions result from subjective cost– beneﬁt analysis and the comparison of alternatives, thus social marketers must provide strong incentives by emphasizing how the beneﬁts of their offerings outweigh the respective costs (Luca & Suggs, 2013; Baptista et al., 2020a). The exchange element of social marketing is considered a key component in social marketing interventions and is one of Andreasen’s (2002) benchmarks. ( ) Social marketing interventions consider what will motivate the target individuals to engage voluntarily in behavior change and offers them something beneﬁcial in return RAUSP 56,3 (Duane et al., 2016). Social exchange involves actions that are contingent on rewarding reactions from others. The content of social exchange can be utilitarian (e.g. tangible, network or informational types of social support) or symbolic (e.g. emotional and esteem social support) or mixed (involving a combination of both utilitarian and symbolic). Traditionally, the content of exchange under a social marketing framework has tended to be utilitarian, with social marketers mostly focusing their efforts on marketing tangible offers, for example, food and medicine (Andreasen, 2002) and in restricted types of exchanges, involving two party direct relationships (Duane et al., 2016), were the intervention promoter offers tangible assistance to the targeted individuals. However, as the discipline evolved, the content of exchange has become more symbolic as its application has broadened to wider social causes (Glenane-Antoniadis et al., 2003) and restricted exchanges has since been criticized for neglecting to examine the broader structural, behavioral and exchange contexts (Duane et al., 2016). Generalized exchanges involve a system of mutual relationships between three or more actors, whereas complex exchanges involve mutual relationships between at least three actors but are reciprocal and have an extended timeframe, unlike generalized exchanges (Glenane-Antoniadis et al., 2003). According to Duane et al. (2016) when social marketing wants to induce social change, the problematic nature of exchange becomes more salient because the social marketer is faced, not with a singular exchange but with coordinating numerous exchanges, that is, one web of complex exchanges. Social marketing is in a good position to conceptually interpret and explore the interdependent and reciprocal nature of social support. RAUSP 56,3 4.2 Consumer-centered perspective of social marketing An additional element in making the case for this social support in social marketing rests in the consumer-centered perspective of social marketing. As in mainstream marketing, customer orientation is fundamental for social marketing (Glenane- Antoniadis et al., 2003). For Grier and Bryant (2005), social marketing is a consumer- centered, research-driven approach to promote voluntary behavior change in a priority population, meaning that peoples’ needs, values and preferences are evaluated and pondered before a certain behavior change strategy is selected, planned and implemented (Baptista et al., 2020a). This approach makes social marketing more competitively minded and audience centered than alternative social intervention approaches, whose projects are usually designed based on political as well as expert assessments of risk and solutions, minimizing the importance of consumer insights (French & Apfel, 2014). Essential to the social marketing process of consumer research is the harnessing of deep, meaningful insight of consumers’ behaviors, roles and networks of inﬂuences in order to develop a deep understanding of the social issues under investigation (Brennan, Fry, & Previte, 2015). In practice, the evaluation of existing networks of personal and supportive relationships is a natural process of social marketing that comes with its consumer centered approach (Baptista et al., 2020a). 303 Traditional downstream social marketing focuses attention on the individual to understand how behaviors can be modiﬁed to achieve the desired change, placing the responsibility for change in the individual (Russell-Bennett et al., 2013; Brennam, Binney, Parker, Aleti, & Nguyen, 2014). The downstream approach still dominates social marketing practice, however in recent years there is a turn from traditional downstream and micro-marketing approaches toward ecological perspectives, that concentrate efforts in upstream audiences (Cherrier & Gurrieri, 2014; David & Rundle- Thiele, 2018). Contrary to alternative approaches to social and individual well-being, such as law or policy development, that use coercion or the threat of punishment to change behavior, social marketing is supposed to inﬂuence behavior by offering alternative choices that invite voluntary exchange (Grier & Bryant, 2005). The new trend toward “upstream” social marketing is focused on the social or environmental determinants of behavioral change and tries to act upon the social system within which the individual operates. In practice many social marketing interventions using upstream approaches violate the basic principle of “voluntary behavior change” in their attempts to inﬂuence regulators or legislators to improve policies or impose new laws. 4. Framing social support within social marketing 4 1 P i i l f i l h (2016) when social marketing wants to induce social change, the problematic nature of exchange becomes more salient because the social marketer is faced, not with a singular exchange but with coordinating numerous exchanges, that is, one web of complex exchanges. 302 Social marketing is in a good position to conceptually interpret and explore the interdependent and reciprocal nature of social support. Similarly to social marketing, exchange models also provide methods for addressing the interdependency of relationships involved in social support exchanges (Shumaker & Brownell, 1984; Baptista et al., 2020a). Social support is basically an exchange of resources between individuals (Gottlieb & Bergen, 2010) and there are potential costs and beneﬁts associated with the exchange for the participants (Baptista et al., 2020a). The beneﬁts may include enhanced coping, self-efﬁcacy, improved decision- making and reduced stress (Mattson & Hall, 2011). The costs of social support may assume the form of time and effort (spent in support interactions), inadequate or misguided information, unwanted control and attention, social aggression, reduced self-efﬁcacy and stress (Taylor, 2011; Gale et al., 2018; Reijnders, Schalk, & Steen, 2018; Gray et al., 2019; Palant & Himmel, 2019; Zee & Kumashiro, 2019; Baptista et al., 2021). , ) According to Baptista et al. (2021) the norm of generalized reciprocity, involves the intention to provide social support without a calculation of immediate value or dyad repayment, and can be motivated by altruistic reasons, a desire to help others and see the community ﬂourish or based on the hope that support exchanges will eventually be reciprocal, in the long run, or that payback is in a different currency such as network status, honor or others’ approval (Baker & Bulkley, 2014). Reciprocity refers to a form of conditional gains in which people’s present actions are expected to generate future beneﬁts (Hung, Durcikova, Lai, & Lin, 2011) contributing to present well-being by anticipating future positive rewards (Baptista et al., 2021). According to Mathwick, Wiertz, & De Ruyter (2008), the expectation of long-term repayment imposes an informal social control system that obviates the need for more formal, institutionalized hierarchical control, resulting in a highly efﬁcient social system that requires less formal control. This stability of the social system facilitates the accumulation of trust and further fosters social support provision (Baptista et al., 2021). Social support in social marketing 4.2 Consumer-centered perspective of social marketing 4.2 Consumer-centered perspective of social marketing Critics of upstream social marketing approaches argue in favor of a return to self- regulation as opposed to outright policy interventions or bans that impinge upon consumers’ freedoms and choices (Cherrier & Gurrieri, 2014) and point the fact that social marketing may have oversimpliﬁed the problem of bringing societal change by considering that upstream actors can be inﬂuenced in the same way as downstream actors (Carvalho & Mazzon, 2019). Furthermore, when discussing the need to create inclusive social change coalitions, authors such as Gurrieri et al. (2018) highlight the frequent disconnection between upstream actors and the voices of those they are meant to be representing and argue for social marketing to promote support relations between actors. Social support can be used in social marketing as a mechanism to involve midstream and upstream audiences in voluntary behavior change efforts (Baptista et al., 2020a). This approach to social change can move social marketing toward systems thinking, and market- place solutions that focus on supportive collaboration amongst actors. Besides close ties, RAUSP 56,3 social support can be provided to focal individuals by community actors and the diverse stakeholders that constitute the involving social systems without coercion and upstream actors can create the conditions that favor the development of supportive networks. As noted by Brennan et al. (2016, p. 6), behavior change depends ultimately on the target audience “laws can be passed, environments altered, and communication campaigns established; yet, if individuals choose not to act, social change will not happen.” Social support provision is based on voluntary acceptance. Social support is not a function that can be imposed on providers and recipients, it is an expression of mutuality and requires consent. 304 4.3 Long-term focus of social marketing To solve complex social problems there is the need to involve diverse stakeholders from government, business and civil society in nonlinear, dynamic and involving solution that must be sustained over long periods (Addy & Dubé, 2018). In social marketing, a long-term focus implies that interventions are strategic and that behavior change can be sustained in the long run (Glenane-Antoniadis et al., 2003). This implies a shift in the exchange context from singular transactions to the development of long-term relationships (Glenane-Antoniadis et al., 2003). 4.2 Consumer-centered perspective of social marketing The relational regulation theory of social support (also known as the “main-effect” or “direct-effect model”) was developed based on statistical evidence that social support beneﬁts health and longevity independently of stress (Cohen & Wills, 1985; Thoits, 1985; Procidano, 1992; Finch, Okun, Pool, & Ruehlman, 1999). According to this theoretical view, social supportive networks can provide individuals with long-term positive emotional experiences and a set of stable and socially rewarded roles, reducing role ambiguity, leading to a sense of predictability and stability in one’s life situation and a recognition of self-worth, social integration, relational rewards or status (Cohen & Wills, 1985). For example, family living has been proved to be associated with a broad spectrum of healthy behaviors, including reduced probability of drugs use, alcohol abuse and smoking and an increased likelihood of a balanced diet and good sleep habits, while social isolation has been related with unhealthy responses to stress, including smoking and alcohol consumption (Holahan & Moos, 1981; Broadhead et al., 1983; Taylor, 2011). Social marketing research has examined how individuals implement behavior change, but there has been less emphasis on how people can cope with the long-term cognitive and emotional aspects of maintaining change (Logie-Maciver & Piacentini, 2011; Baptista et al., 2020a). The role of social support as a coping resource in stressful situations has long been emphasized in social support theory (Kim, Han, & Shaw, 2010; Mattson & Hall, 2011). Under conditions of pressure related with behavior change, social support can potentially act as a buffer by reducing stress (Barrera, 1986; Cohen & Wills, 1985; Thoits, 1986). 4.4 Madres para la salud In this section, we provide an example of a cultural sensitive, community-based, social marketing intervention that operationalized the social support concept for behavior change, named Madres para la Salud (Keller et al., 2011; Keller et al., 2012; Keller et al., 2014). The intervention in case was developed in the United States and targeted overweight postpartum Hispanic women. The behavior change goal consisted in leading the targeted individuals to increase physical activity to attain improvements in body composition, inﬂammatory markers and postpartum depression symptoms. 4.2 Consumer-centered perspective of social marketing Social support in social marketing Social support in social marketing The main component of the intervention involved the promotion of social support as a resource from family, friends and intervention walking groups within local neighborhoods, led by health advisors or peer counselors (named Promotoras) to whom other community members could turn for care, advice, information and support. During the consumer research phase Hispanic women were invited to join the intervention team in discussing strategy development to strengthen the cultural relevance of the intervention, to report their personal experiences with physical activity, the ways in which group support could facilitate walking and share with the program developers existing networks of social support for walking, which included family, friends and neighbors, highlighting the consumer-centered perspective of this intervention. 305 The critical inputs of the Madres para la Salud included the following typologies of social support, which were offered in exchange for the adoption of walking behaviors: emotional support provided by the Promotoras to initiate and sustain walking; emotional support provided by the Promotoras to initiate and sustain walking instrumental support, including a set of activities that provided tangible aid or services, such as a stroller loaner program, childcare, walking shoes and teaching partners/spouses how and why to encourage walking; informational support, consisting in facts and advice covering topics such as strategies for walking with family members, safe walking routes, health beneﬁts of walking, time management strategies to create opportunities for walking; and appraisal support feedback consisting in self-monitoring activities, through accelerometer, pedometers, weekly diaries, Promotora review of physical activity and goal setting and attainment. A total of 177 postpartum Hispanic women were recruited. Participants were randomly assigned to the intervention or attention-control group. Evaluation data was gathered at baseline, 3, 6, 9 and 12 months using questionnaires assessing the availability of social support, body scans and self-report measures of walking adherence. The results showed increases in social support for exercise following delivery of the 48-week social support intervention, positive achievements in aerobic and total steps across the 12 months of the intervention and declines in body adiposity and fat tissue inﬂammation. Although this intervention study presented some ﬂaws, including the use of very high cut scores for measurement of depressive symptoms, resulting in inconclusive results in this respect, the intervention demonstrated the potential of social support to attain consistent behavior change in social marketing interventions. 5. Conclusions l ki h f In looking to the future and the continuing development of social marketing research, there is a need to further explore the utility of concepts borrowed from other disciplines, such as social support, to improve the efﬁcacy of social marketing practice. This paper reinforces Albrecht’s (1995) arguments for social support in social marketing. First, the paper highlights the principle of exchange in social marketing. Social support when involves reciprocal supportive networks where network members are engaged in direct and indirect, restrict and generalized, as well as utilitarian and symbolic exchanges at the same time, can be identiﬁed as a complex social system which has the potential to induce sustainable systemic change. A second argument is based on the consumer-centered perspective of social marketing, which implies the necessity to evaluate the networks of inﬂuence of targeted individuals. 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Social support in social marketing 313 For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com Wortman, C. B. (1984). Social support and the cancer patient. Cancer, 53(10), 2339–2362, doi: https://doi. org/10.1002/cncr.1984.53.s10.2339. marketing Corresponding author Nuno Baptista can be contacted at: nunotiagobaptista@gmail.com Nuno Baptista can be contacted at: nunotiagobaptista@gmail.com For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com
https://openalex.org/W2973017325	https://pure.eur.nl/ws/files/48232050/Repub_120290_O-A.pdf	English	null	Cell Heterogeneity and Phenotypic Plasticity in Metastasis Formation: The Case of Colon Cancer	Cancers	2,019	cc-by	14,671	Received: 25 July 2019; Accepted: 11 September 2019; Published: 14 September 2019 Received: 25 July 2019; Accepted: 11 September 2019; Published: 14 September 2019 Abstract: The adenoma-to-carcinoma progression in colon cancer is driven by a sequential accumulation of genetic alterations at speciﬁc tumor suppressors and oncogenes. In contrast, the multistage route from the primary site to metastasis formation is underlined by phenotypic plasticity, i.e., the capacity of disseminated tumor cells to undergo transiently and reversible transformations in order to adapt to the ever-changing environmental contexts. Notwithstanding the considerable body of evidence in support of the role played by epithelial-to-mesenchymal transition (EMT)/mesenchymal-to-epithelial transition (MET) in metastasis, its rate-limiting function, the detailed underlying cellular and molecular mechanisms, and the extension of the necessary morphologic and epigenetic changes are still a matter of debate. Rather than leading to a complete epithelial or mesenchymal state, the EMT/MET-program generates migrating cancer cells displaying intermediate phenotypes featuring both epithelial and mesenchymal characteristics. In this review, we will address the role of colon cancer heterogeneity and phenotypic plasticity in metastasis formation and the contribution of EMT to these processes. The alleged role of hybrid epithelial/mesenchymal (E/M) in collective and/or single-cell migration during local dissemination at the primary site and more systemic spreading will also be highlighted. Keywords: colon cancer; Wnt signaling; tumor heterogeneity; phenotypic plasticity; EMT; hybrid E/M; collective and single-cell migration; beta-catenin paradox Cell Heterogeneity and Phenotypic Plasticity in Metastasis Formation: The Case of Colon Cancer Miriam Teeuwssen and Riccardo Fodde * Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center, 3015 GD Rotterdam, The Netherlands; m.teeuwssen@erasmusmc.nl * Correspondence: r.fodde@erasmusmc.nl; Tel.: +31-010-7043896 www.mdpi.com/journal/cancers Cancers 2019, 11, 1368; doi:10.3390/cancers11091368 cancers cancers cancers cancers cancers 1. Introduction—Tumor Heterogeneity in Colon Cancer Colon cancer is the third most commonly diagnosed malignancy and the second leading cause of cancer-related death worldwide. It is predicted that its mortality burden will increase by 75% by 2040 [1]. Apart from its clinical impact, colon cancer also represents a unique study model to elucidate the cellular and molecular mechanisms underlying tumor onset, progression towards malignancy, and metastasis formation at distant organ sites [2]. It is generally accepted that primary colon carcinomas are heterotypic, i.e., they feature a heterogeneous composition of epithelial cancer cells intermingled with lymphocytes, stromal ﬁbroblasts, endothelial, and other cell types from the micro- and macro-environment [3]. This heterogeneity is matched by the diversity of parenchymal cancer cells encompassing a broad spectrum of morphologies, gene expression proﬁles, and functional characteristics [4–6]. Likewise, heterogeneity within the stromal compartment, i.e., the tumor microenvironment, has also been demonstrated [5,7]. Intrinsic, i.e., (epi)genetic, as well as extrinsic factors, such as spatial location within the tumor (e.g., at the invasive front vs. tumor center), inﬂammation, and treatment history underlie the observed intra-tumor heterogeneity. Consequently, diﬀerent cellular subpopulations within the primary tumor mass and its metastatic lesions are observed [8,9]. Next to ‘spatial’ heterogeneity, ‘temporal’ heterogeneity has also been demonstrated relative to changes in the (epi)genetic landscape Cancers 2019, 11, 1368; doi:10.3390/cancers11091368 www.mdpi.com/journal/cancers Cancers 2019, 11, 1368 2 of 19 of colon cancer within individual tumors over time [10]. Of note, tumor heterogeneity is thought to underlie the disappointing results of many currently employed anti-cancer therapies as it not only supports tumor progression and metastatic dissemination but it also lies at the basis of the development of therapy resistance and of overall poor clinical prognosis [11]. Metastasis formation is a process encompassing multiple steps: (1) Local tumor invasion across the basement membrane into the surrounding stroma, (2) intravasation into the vasculature, (3) survival in the circulatory system, (4) extravasation into the parenchyma of the distant organ, (5) colonization into a distal organ, and (6) re-initiation of proliferation to form macroscopic metastases [12]. In order to successfully complete this challenging series of events, the most important feature of the metastasizing cancer cell is the capacity to adapt to the ever-changing environmental contexts by undergoing reversible changes in its cellular identity. This ‘Dr. Jekyll and Mr. 1. Introduction—Tumor Heterogeneity in Colon Cancer Hide’ feature of migrating cancer cells is often referred to as phenotypic plasticity [13] and is controlled by epigenetic mechanisms which regulate, among other processes, epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) [14]. A variety of chromatin remodeling complexes such as Polycomb and NuRD, play a central role in the transcriptional regulation of EMT-related transcription factors (EMT-TFs) and micro RNAs (miRs) by determining the accessibility of regulatory DNA elements and positioning of nucleosomes [15,16]. In addition, post-translational histone modiﬁcations which modulate chromatin folding and inﬂuence recruitment of regulatory proteins and control gene expression [17]. Accordingly, contextual EMT-promoting signals epigenetically modify the repression of epithelial genes and consequently drive the transition of cells into more mesenchymal-like states. These are epigenetically sustained unless the presence of EMT-promoting signals is discontinued leading to the reversion to more epithelial phenotypes [15]. Notwithstanding the considerable body of evidence in support of the role played by EMT/MET in metastasis, its rate-limiting function, and the detailed underlying cellular and molecular mechanisms, and the extension of the necessary morphologic and epigenetic changes are still a matter of debate [14,18,19]. Rather than leading to a complete epithelial or mesenchymal state, the EMT/MET programs generate migrating cancer cells displaying intermediate phenotypes featuring both epithelial and mesenchymal characteristics. These hybrid E/M cancer cells have been the focus of much attention in the most recent scientiﬁc literature as they are likely to be metastable and as such very eﬃcient in causing metastasis [20]. Here, we will address the role of tumor cell heterogeneity and phenotypic plasticity in colon cancer metastasis formation and the contribution of EMT to these processes. The alleged role of hybrid E/M in collective and/or single-cell migration during local dissemination at the primary site and more systemic spreading will be highlighted. 2. The Adenoma-Carcinoma Sequence in Colon Cancer: The β-Catenin Paradox Here, β-catenin interacts with members of the TCF/LEF family of transcription factors and modulates the expression of a broad spectrum of Wnt downstream target genes with cellular functions ranging from stemness to proliferation [23] (Figure 1a). Consequently, loss- and gain-of-function genetic alterations in APC and β-catenin respectively, result in the constitutive signaling of β-catenin to the nucleus [2]. crypt of Lieberkühn where it regulates the homeostatic equilibrium between stemness, proliferation, and differentiation [22]. In the bottom third of the crypt, where stem cells reside, Wnt signaling is particularly active due to signals from the surrounding stromal environment. Moving along the crypt-villus axis however, Wnt is progressively less active in a decreasing gradient inversely proportional to the grade of differentiation of the epithelial lining [23]. Here, in the absence of canonical Wnt ligands such as Wnt3a, intracellular β-catenin levels are controlled by the formation of a multiprotein “destruction complex” encompassing protein phosphatase 2A (PP2a), glycogen synthase kinase 3 (GSK3β) and casein kinase 1α (CK1α), and the scaffold proteins adenomatous polyposis coli (APC), and Axin1/2. This complex binds and phosphorylates β-catenin at specific serine and threonine residues, thereby targeting it for ubiquitination and proteolytic degradation by the proteasome [23] (Figure 1a). In the presence of Wnt ligands instead, i.e., in the stem cell compartment, co-activation of the Frizzled and LRP5/6 (low-density lipoprotein receptor-related proteins) receptors prevents the formation of the destruction complex thus resulting in the stabilization and consequent translocation of β-catenin from the cytoplasm to the nucleus. Here, β-catenin interacts with members of the TCF/LEF family of transcription factors and modulates the expression of a broad spectrum of Wnt downstream target genes with cellular functions ranging from stemness to proliferation [23] (Figure 1a). Consequently, loss- and gain-of-function genetic alterations in APC and β-catenin respectively, result in the constitutive signaling of β-catenin to the nucleus [2]. Figure 1. The (a) Wnt/β-catenin signal transduction pathway and the (b) β-catenin paradox in colon cancer. (a) Illustration of the canonical Wnt signaling in homeostasis. Left panel: In the absence of Wnt ligands, intracellular β-catenin levels are controlled by a destruction complex encompassing protein phosphatase 2A (PP2a), glycogen synthase kinase 3 (GSK3β) and casein kinase 1α (CK1α), adenomatous polyposis coli (APC), and Axin1/2. This complex binds and phosphorylates β-catenin at serine and threonine residues, thereby targeting it for ubiquitination and proteolytic degradation by the proteasome. 2. The Adenoma-Carcinoma Sequence in Colon Cancer: The β-Catenin Paradox Colon cancer arises and progresses through a well-deﬁned series of histologic stages along which normal colonic epithelial cells transform in stepwise fashion into precursor lesions which eventually evolve to increasingly more invasive and malignant stages. This sequence, often referred to as ‘the adenoma-carcinoma sequence’, features a gradual accumulation of genetic alterations in speciﬁc tumor suppressors and oncogenes generally regarded as the main underlying and driving forces in the progression of colonic adenomas towards malignancy [21]. The initiating and rate-limiting event in the vast majority of sporadic colon cancer cases is represented by the constitutive activation of canonical Wnt signaling through loss of function mutations at the APC (adenomatous polyposis coli) tumor suppressor gene. Alternatively, gain of function or ‘activating’ mutations in Wnt agonists such as the β-catenin (CTNNB1) oncogene have functionally equivalent consequences, i.e., the ligand-independent and constitutive signaling activation of the pathway [2]. The reason for the pivotal role of the Wnt/β-catenin signal transduction pathway in colon cancer onset mainly resides in its functional role in the intestinal crypt of Lieberkühn where 3 of 19 f 20 Cancers 2019, 11, 1368 Cancers 2019, 11, x it regulates the homeostatic equilibrium between stemness, proliferation, and diﬀerentiation [22]. In the bottom third of the crypt, where stem cells reside, Wnt signaling is particularly active due to signals from the surrounding stromal environment. Moving along the crypt-villus axis however, Wnt is progressively less active in a decreasing gradient inversely proportional to the grade of diﬀerentiation of the epithelial lining [23]. Here, in the absence of canonical Wnt ligands such as Wnt3a, intracellular β-catenin levels are controlled by the formation of a multiprotein “destruction complex” encompassing protein phosphatase 2A (PP2a), glycogen synthase kinase 3 (GSK3β) and casein kinase 1α (CK1α), and the scaﬀold proteins adenomatous polyposis coli (APC), and Axin1/2. This complex binds and phosphorylates β-catenin at speciﬁc serine and threonine residues, thereby targeting it for ubiquitination and proteolytic degradation by the proteasome [23] (Figure 1a). In the presence of Wnt ligands instead, i.e., in the stem cell compartment, co-activation of the Frizzled and LRP5/6 (low-density lipoprotein receptor-related proteins) receptors prevents the formation of the destruction complex thus resulting in the stabilization and consequent translocation of β-catenin from the cytoplasm to the nucleus. 2. The Adenoma-Carcinoma Sequence in Colon Cancer: The β-Catenin Paradox Right panel: In presence of Wnt, co-activation of the Frizzled and LRP5/6 (low-density lipoprotein receptor-related proteins) receptors prevents the formation of the destruction complex leading to the stabilization and consequent translocation of β-catenin from the cytoplasm to the nucleus. Here, β-catenin interacts with members of the TCF/LEF family of transcription factors and modulates the expression of a broad spectrum of Wnt downstream target genes. Adapted from [24]. (b) The β-catenin paradox in colon cancer. β-catenin IHC analysis of the invasive front of a colon carcinoma show marked nuclear β-catenin accumulation in the proximity of the stromal microenvironment. In contrast, the majority of tumor cells localized inside the tumor mass are characterized by membrane-bound and cytoplasmic β-catenin staining. Scale bar: 50 µm. h d l d h h f l d b h Figure 1. The (a) Wnt/β-catenin signal transduction pathway and the (b) β-catenin paradox in colon cancer. (a) Illustration of the canonical Wnt signaling in homeostasis. Left panel: In the absence of Wnt ligands, intracellular β-catenin levels are controlled by a destruction complex encompassing protein phosphatase 2A (PP2a), glycogen synthase kinase 3 (GSK3β) and casein kinase 1α (CK1α), adenomatous polyposis coli (APC), and Axin1/2. This complex binds and phosphorylates β-catenin at serine and threonine residues, thereby targeting it for ubiquitination and proteolytic degradation by the proteasome. Right panel: In presence of Wnt, co-activation of the Frizzled and LRP5/6 (low-density lipoprotein receptor-related proteins) receptors prevents the formation of the destruction complex leading to the stabilization and consequent translocation of β-catenin from the cytoplasm to the nucleus. Here, β-catenin interacts with members of the TCF/LEF family of transcription factors and modulates the expression of a broad spectrum of Wnt downstream target genes. Adapted from [24]. (b) The β-catenin paradox in colon cancer. β-catenin IHC analysis of the invasive front of a colon carcinoma show marked nuclear β-catenin accumulation in the proximity of the stromal microenvironment. In contrast, the majority of tumor cells localized inside the tumor mass are characterized by membrane-bound and cytoplasmic β-catenin staining. Scale bar: 50 µm. Figure 1. The (a) Wnt/β-catenin signal transduction pathway and the (b) β-catenin paradox in colon cancer. (a) Illustration of the canonical Wnt signaling in homeostasis. 2. The Adenoma-Carcinoma Sequence in Colon Cancer: The β-Catenin Paradox Left panel: In the absence of Wnt ligands, intracellular β-catenin levels are controlled by a destruction complex encompassing protein phosphatase 2A (PP2a), glycogen synthase kinase 3 (GSK3β) and casein kinase 1α (CK1α), adenomatous polyposis coli (APC), and Axin1/2. This complex binds and phosphorylates β-catenin at serine and threonine residues, thereby targeting it for ubiquitination and proteolytic degradation by the proteasome. Right panel: In presence of Wnt, co-activation of the Frizzled and LRP5/6 (low-density lipoprotein receptor-related proteins) receptors prevents the formation of the destruction complex leading to the stabilization and consequent translocation of β-catenin from the cytoplasm to the nucleus. Here, β-catenin interacts with members of the TCF/LEF family of transcription factors and modulates the expression of a broad spectrum of Wnt downstream target genes. Adapted from [24]. (b) The β-catenin paradox in colon cancer. β-catenin IHC analysis of the invasive front of a colon carcinoma show marked nuclear β-catenin accumulation in the proximity of the stromal microenvironment. In contrast, the majority of tumor cells localized inside the tumor mass are characterized by membrane-bound and cytoplasmic β-catenin staining. Scale bar: 50 µm. Thi ti d l di t th t th t j it f l i iti t d b th tit ti Figure 1. The (a) Wnt/β-catenin signal transduction pathway and the (b) β-catenin paradox in colon cancer. (a) Illustration of the canonical Wnt signaling in homeostasis. Left panel: In the absence of Wnt ligands, intracellular β-catenin levels are controlled by a destruction complex encompassing protein phosphatase 2A (PP2a), glycogen synthase kinase 3 (GSK3β) and casein kinase 1α (CK1α), adenomatous polyposis coli (APC), and Axin1/2. This complex binds and phosphorylates β-catenin at serine and threonine residues, thereby targeting it for ubiquitination and proteolytic degradation by the proteasome. Right panel: In presence of Wnt, co-activation of the Frizzled and LRP5/6 (low-density lipoprotein receptor-related proteins) receptors prevents the formation of the destruction complex leading to the stabilization and consequent translocation of β-catenin from the cytoplasm to the nucleus. Here, β-catenin interacts with members of the TCF/LEF family of transcription factors and modulates the expression of a broad spectrum of Wnt downstream target genes. Adapted from [24]. (b) The β-catenin paradox in colon cancer. β-catenin IHC analysis of the invasive front of a colon carcinoma show marked nuclear β-catenin accumulation in the proximity of the stromal microenvironment. 2. The Adenoma-Carcinoma Sequence in Colon Cancer: The β-Catenin Paradox In contrast, the majority of tumor cells localized inside the tumor mass are characterized by membrane-bound and cytoplasmic β-catenin staining. Scale bar: 50 µm. g p j y , y activation of Wnt signaling, should feature nuclear β-catenin localization throughout the entire This genetic model predicts that the vast majority of colon cancers, initiated by the constitutive activation of Wnt signaling, should feature nuclear β-catenin localization throughout the entire tumor mass. However, extensive immunohistochemical analysis of sporadic colon cancers has contradicted 4 of 19 Cancers 2019, 11, 1368 this prediction. In fact, only a minority of colon cancer cells, non-randomly distributed along the invasive front of the primary mass and of quasi-mesenchymal morphology, show nuclear β-catenin accumulation. In contrast, the majority of more diﬀerentiated (epithelial-like) tumor cells localized inside the tumor mass are characterized by an apparently normal (membrane-bound) subcellular distribution of β-catenin together with increased cytoplasmic staining [25] (Figure 1b). This “β-catenin paradox” is generally explained by the fact that the APC and β-catenin mutations are necessary for the constitutive activation of the pathway though insuﬃcient for nuclear β-catenin accumulation and full-blown Wnt signaling [24] (Figure 1b). The latter is only achieved in colon cancer cells located at the invasive front where they are exposed to stromal cues capable of further promoting the nuclear translocation of β-catenin from the cytoplasm [26]. Of note, the same heterogeneous β-catenin distribution, with nuclear staining in less diﬀerentiated cells located in closer proximity to the microenvironment and membranous staining in more diﬀerentiated cells in the center of the lesion, has also been observed in colon cancer metastases [27]. The reacquisition of epithelial features at the metastatic sites is required for cancer cell proliferation, as mesenchymal-like cells are generally hindered in their proliferative activity and are therefore not able to underlie the expansion of the metastasis. Hence, diﬀerent levels of Wnt signaling activity between the tumor center and the invasive front are likely to account for the ‘spatial’ intra-tumor heterogeneity and to underlie distinct Wnt downstream cellular eﬀectors such as proliferation and EMT leading to tumor growth and invasion, respectively [28]. 2. The Adenoma-Carcinoma Sequence in Colon Cancer: The β-Catenin Paradox These observations have led to the hypothesis according to which, apart from its role in colon cancer initiation, Wnt signaling and the consequent downstream EMT activation, also underlies the onset of migrating cancer stem cells (mCSC) at the invasive front of the primary lesion which locally invade the tumor microenvironment and eventually form distant metastases [29]. This paracrine—and presumably epigenetic—control of local invasion and metastasis also oﬀers an explanation to the so-called “progression puzzle” [30], i.e., the lack of main genetic and expression diﬀerences between matched primary tumors and metastases as reported in colon cancer and other tumor types [31–33]. This suggests that although the adenoma-carcinoma progression at the primary site is clearly driven by the sequential accumulation of genetic mutations at key genes, the multistage route from dissemination into the tumor microenvironment to metastasis formation is underlined by phenotypic plasticity, i.e., the capacity of circulating tumor cells (CTCs) to undergo transient phenotypic changes to adapt to the ever-changing cellular contexts en route to distant organ sites. As previously and eloquently proposed by Thomas Brabletz and collaborators, EMT and its reverse program MET play pivotal roles in regulating phenotypic plasticity of CTCs [29]. In the next section, we will discuss the current understanding of the role of EMT in local invasion and metastasis. 3. Epithelial to Mesenchymal Transition in Local Invasion and Metastasis As pointed out in the previous section, tumor cells within primary and metastatic tumor masses, as well as CTCs, display substantial phenotypic heterogeneity representing various intermediate stages of the EMT program [34,35]. EMT is a developmental program exploited by carcinoma cells to switch from their epithelial state, featuring cell–cell contacts and apical–basal polarity, to more motile and invasive quasi-mesenchymal phenotypes with spindle-like morphology and front-back-end polarity. During cancer invasion, EMT provides cells with the ability to produce, interact with, and digest the surrounding extracellular matrix (ECM), detach from the primary tumor, and invade into the surrounding tissue [14]. In addition to promoting cellular migration and invasion, the transient phenotypic changes associated with formation of the mesenchymal state during EMT have been associated with the acquisition of stem-like properties, resistance to therapy, and immune suppression [36–39]. The epigenetic, and as such reversible nature of EMT is crucial as the reverse mesenchymal-to-epithelial (MET) process allows migrating cancer (stem-like) cells to regain proliferative and epithelial characteristics to colonize distant organ sites [14]. The initiation and 5 of 19 Cancers 2019, 11, 1368 execution of EMT are orchestrated by a set of transcription factors (i.e., ZEB1/2, SNAIL1/SLUG, and TWIST1/2) and miRNAs (e.g., the miR200 family) [40]. Hallmarks of EMT include the silenced expression of integral members of epithelial cell adhesion structures such as adherens- and tight-junctions, and desmosomes, and/or proteins involved in cytoskeleton (re)organization and in cell-matrix adhesion. Next, EMT-TFs can also activate the expression of mesenchymal cell markers resulting in changes in cell morphology, enhanced migratory properties, and ECM remodeling. EMT is induced by cytokines and growth factors secreted from the tumor microenvironment in response to metabolic changes, hypoxia, innate and adaptive immune responses, and treatment by cytotoxic drugs [40]. In addition, the mechanical composition and properties of the ECM also play an important role in EMT regulation. Both shear stress of cancer cells and increasing matrix stiﬀness in the microenvironment activate EMT, tumor invasion and metastasis [41–43]. In turn, as noted before, EMT also stimulates the composition and mechanics of the ECM, thereby forming a tightly controlled feedback loop that is often dysregulated in cancer. As mentioned above, colon carcinomas display nuclear β-catenin accumulation at the invasive front simultaneously with the acquisition of mesenchymal-like morphologic features [24]. 3. Epithelial to Mesenchymal Transition in Local Invasion and Metastasis In this respect, it has been shown that EMT can be activated downstream of canonical Wnt/β-catenin signaling as GSK3β kinase activity inhibition stabilizes SLUG, thereby initiating EMT [44]. Alternatively, active Wnt signaling also inhibits SNAIL1 phosphorylation, leading to increased protein levels of this transcriptional repressor of E-cadherin, EMT initiation, and local invasion [45]. In colon cancer, overexpression of the Wnt ligand Wnt3a is associated with EMT and cancer progression. Accordingly, Wnt3a overexpression in both in vitro and in vivo models was shown to induce SNAIL expression thus promoting EMT, an eﬀect that is abrogated by the Wnt antagonist Dickkopf1 (Dkk1) [28]. More recently, the intestinal microbiome has also been shown to contribute to EMT. A variety of enterotoxins secreted by microbes, including Bacteriodes fragilis, Fusobacterium nucleatum, and Enterococcus faecalis have been demonstrated to alter normal cell–cell adhesion by interfering with E-cadherin function [46–48]. F. nucleatum adheres through FadA (Fusobacterium adhesion A), an adhesion protein, to E-cadherin in colon cancer cells. The FadA/E-cadherin interaction leads to activation of β-catenin signaling and of oncogenic and inﬂammatory responses [48]. Interestingly, Fusobacterium and its associated microbiome (including Bacteroides, Selenomonas, and Prevotella) are sustained in distal metastases and mouse xenografts of primary colorectal tumors. Treating tumor-bearing mice with the antibiotic metronidazole reduced the amount Fusobacterium and abrogated cancer cell proliferation and growth [49]. 3.2. Hybrid E/M Phenotypes and Partial EMT: Many Shades of Gray y yp y f y As mentioned above, the transient and reversible nature of As mentioned above, the transient and reversible nature of EMT represents an essential feature for a metastatic lesion to develop [52,54,55]. Recent experimental evidence indicates that EMT, rather than acting as a binary switch where cells transit between fully epithelial and mesenchymal states, generates a broad spectrum of intermediate E/M stages where cells co-express both types of markers [14] (Figure 2). These partial EMT states are metastable and as such confer to the cancer cell enhanced phenotypic plasticity, an essential hallmark of the migrating/metastatic cancer (stem) cell [14]. , p for a metastatic lesion to develop [52,54,55]. Recent experimental evidence indicates that EMT, rather than acting as a binary switch where cells transit between fully epithelial and mesenchymal states, generates a broad spectrum of intermediate E/M stages where cells co-express both types of markers [14] (Figure 2). These partial EMT states are metastable and as such confer to the cancer cell enhanced phenotypic plasticity, an essential hallmark of the migrating/metastatic cancer (stem) cell [14]. Figure 2. Epithelial to mesenchymal transition (EMT). Schematic overview of epithelial (E) cells transitioning to mesenchymal (M) phenotypes through an intermediate E/M state, and vice versa. EMT can be induced by various stimuli and is dependent on the environmental context. Figure 2. Epithelial to mesenchymal transition (EMT). Schematic overview of epithelial (E) cells transitioning to mesenchymal (M) phenotypes through an intermediate E/M state, and vice versa. EMT can be induced by various stimuli and is dependent on the environmental context. Figure 2. Epithelial to mesenchymal transition (EMT). Schematic overview of epithelial (E) cells transitioning to mesenchymal (M) phenotypes through an intermediate E/M state, and vice versa. EMT can be induced by various stimuli and is dependent on the environmental context. Figure 2. Epithelial to mesenchymal transition (EMT). Schematic overview of epithelial (E) cells transitioning to mesenchymal (M) phenotypes through an intermediate E/M state, and vice versa. EMT can be induced by various stimuli and is dependent on the environmental context. Two recent studies, in particular, have highlighted the relevance of partial EMT in metastasis. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), Aiello et al. (2018) sorted primary tumor cells according to their membranous expression of E-cadherin (Cdh1). Additional RNAseq and protein analysis of Cdh1-negative cancer cells revealed the presence of two distinct groups of Two recent studies, in particular, have highlighted the relevance of partial EMT in metastasis. 3.1. Role of EMT in Metastasis under Debate 3 2 H b id E/M Ph t d P ti l EMT M Sh d f G Although compelling, the Fischer et al. (2015) and Zheng et al. (2015) studies are mainly based on the analysis of individual transcription factors and downstream targets in speciﬁc tumor models [39,40] and cannot as such be used to discard EMT’s role in metastasis against an overwhelming body of experimental evidence from the scientiﬁc literature. Several TFs are known to cooperate in eliciting EMT and in controlling the extension of the execution of the trans-diﬀerentiation program. Also, EMT-TFs are known to act in a cooperative and context-dependent fashion, and loss of individual factors in speciﬁc organ sites may well not suﬃce to initiate EMT and facilitate metastasis formation. The same is true for the employed mesenchymal markers the expression of which cannot be employed as universal readouts of EMT activation [14,50]. on the analysis of individual transcription factors and downstream targets in specific tumor models [39,40] and cannot as such be used to discard EMT’s role in metastasis against an overwhelming body of experimental evidence from the scientific literature. Several TFs are known to cooperate in eliciting EMT and in controlling the extension of the execution of the trans-differentiation program. Also, EMT-TFs are known to act in a cooperative and context-dependent fashion, and loss of individual factors in specific organ sites may well not suffice to initiate EMT and facilitate metastasis formation. The same is true for the employed mesenchymal markers the expression of which cannot be employed as universal readouts of EMT activation [14,50]. 3 2 H b id E/M Ph t d P ti l EMT M Sh d f G 3.1. Role of EMT in Metastasis under Debate EMT is often deﬁned by the respective down- and up-regulation of epithelial (e.g., E-cadherin, catenins, and cytokeratins) and mesenchymal markers (e.g., vimentin, ﬁbronectin, and N-cadherin) by the above-mentioned ZEB1/2, SNAIL1/SLUG, and TWIST1/2 transcription factors (EMT-TFs). However, no single transcription factor (TF) or downstream target can universally deﬁne EMT throughout diﬀerent cancer types and cellular contexts. Distinct EMT-TFs are likely to act in a tumor- and dosage-speciﬁc manner and as such diﬀerentially repress or enhance the transcription of speciﬁc downstream target genes. From this perspective, the recent debate on whether EMT is an essential requirement for metastasis to occur [14,50] reﬂects the complexity of the network of transcription factors and their downstream targets in the activation of the EMT program. Two provocative studies, in particular, have raised questions on the relative importance of the role played by EMT along the multistep sequence of events leading to metastasis. Fischer et al. (2015) employed in vivo mesenchymal GFP reporters to study EMT onset in the MMTV-PyMT mammary cancer model. Notwithstanding the observed mesenchymal expression within the primary lesions, albeit in low proportion, and its enrichment in CTCs, GFP-positive tumor cells did not contribute to distant metastases [18]. Moreover, Zeb1/2 inhibition by miR-200 overexpression did not reduce lung metastasis incidence. In a second study by Zheng et al. (2015) it was shown, by taking advantage of a pancreatic ductal carcinoma Cancers 2019, 11, 1368 pancreatic ductal 6 of 19 t affect mouse model, that genetic ablation of Snail1 or Twist1 did not aﬀect dissemination and lung metastasis development [19]. The latter is in contrast with a later study by Krebs et al. (2017), showing that Zeb1 downregulation in the same pancreatic cancer models negatively aﬀects the formation of precursor lesions, tumor grading, invasion, and metastasis [51]. Additionally, other studies using diﬀerent cancer mouse models point to a key role of Snail1- and Twist1-driven EMT in metastatic colonization [52–54]. g p y y Krebs et al. (2017), showing that Zeb1 downregulation in the same pancreatic cancer models negatively affects the formation of precursor lesions, tumor grading, invasion, and metastasis [51]. Additionally, other studies using different cancer mouse models point to a key role of Snail1- and Twist1-driven EMT in metastatic colonization [52–54]. Although compelling, the Fischer et al. (2015) and Zheng et al. 3.1. Role of EMT in Metastasis under Debate (2015) studies are mainly based mouse model, that genetic ablation of Snail1 or Twist1 did not aﬀect dissemination and lung metastasis development [19]. The latter is in contrast with a later study by Krebs et al. (2017), showing that Zeb1 downregulation in the same pancreatic cancer models negatively aﬀects the formation of precursor lesions, tumor grading, invasion, and metastasis [51]. Additionally, other studies using diﬀerent cancer mouse models point to a key role of Snail1- and Twist1-driven EMT in metastatic colonization [52–54]. Although compelling, the Fischer et al. (2015) and Zheng et al. (2015) studies are mainly based on the analysis of individual transcription factors and downstream targets in speciﬁc tumor models [39,40] and cannot as such be used to discard EMT’s role in metastasis against an overwhelming body of experimental evidence from the scientiﬁc literature. Several TFs are known to cooperate in eliciting EMT and in controlling the extension of the execution of the trans-diﬀerentiation program. Also, EMT-TFs are known to act in a cooperative and context-dependent fashion, and loss of individual factors in speciﬁc organ sites may well not suﬃce to initiate EMT and facilitate metastasis formation. The same is true for the employed mesenchymal markers the expression of which cannot be employed as universal readouts of EMT activation [14,50]. Krebs et al. (2017), showing that Zeb1 downregulation in the same pancreatic cancer models negatively affects the formation of precursor lesions, tumor grading, invasion, and metastasis [51]. Additionally, other studies using different cancer mouse models point to a key role of Snail1- and Twist1-driven EMT in metastatic colonization [52–54]. Although compelling, the Fischer et al. (2015) and Zheng et al. (2015) studies are mainly based on the analysis of individual transcription factors and downstream targets in specific tumor models [39,40] and cannot as such be used to discard EMT’s role in metastasis against an overwhelming body of experimental evidence from the scientific literature. Several TFs are known to cooperate in eliciting EMT and in controlling the extension of the execution of the trans-differentiation program. Also, EMT-TFs are known to act in a cooperative and context-dependent fashion, and loss of individual factors in specific organ sites may well not suffice to initiate EMT and facilitate metastasis formation. The same is true for the employed mesenchymal markers the expression of which cannot be employed as universal readouts of EMT activation [14,50]. 3.2. Hybrid E/M Phenotypes and Partial EMT: Many Shades of Gray y yp y f y As mentioned above, the transient and reversible nature of In a mouse model of pancreatic ductal adenocarcinoma (PDAC), Aiello et al. (2018) sorted primary tumor cells according to their membranous expression of E-cadherin (Cdh1). Additional RNAseq and protein analysis of Cdh1-negative cancer cells revealed the presence of two distinct groups of tumors: while the ﬁrst resulted from the transcriptional downregulation of E-cadherin (and of other epithelial markers), the second and major group showed E-cadherin expression both at the mRNA and 7 of 19 Cancers 2019, 11, 1368 protein levels. However, rather than being presented at the membrane, E-cadherin was internalized in recycling endosomes [34]. These two distinct E-cadherin negative and EMT-competent subpopulations of tumor cells were also earmarked by diﬀerent invasive and metastatic behavior. Whereas cancer cells featuring a complete EMT (i.e., E-cadherin downregulated at the transcriptional level) invaded the tumor microenvironment mostly as single cells, cells with internalized E-cadherin in a partial EMT state (E/M) migrate collectively as multicellular clusters which are also found in the blood of the pancreatic cancer mouse model [34]. Of note, it has also been shown that the diﬀerent degrees of epithelial-mesenchymal plasticity aﬀect the tumor cells’ metastatic organotropism, i.e., their capacity to metastasize a spectrum of diﬀerent organ sites [56]. p g In a second study, Pastushenko et al. (2018) employed a mouse model of squamous cell carcinoma (SCC) and, by taking advantage of the diﬀerent expression levels of the CD106, CD61, and CD51 cell-surface markers, identiﬁed six distinct EpCAM-negative tumor cell subpopulations, each characterized by a diﬀerent degree of EMT. The diﬀerent SCC subpopulations, encompassing both fully mesenchymal (complete EMT) and hybrid E/M subtypes (partial EMT), were characterized by distinct chromatin landscapes and gene expression proﬁles. Similar EMT-heterogeneity was also found in mouse models for metaplastic and luminal breast cancer [35]. Although the tumor-propagating capacity of hybrid E/M EpCAM-negative SCC cells was found to be comparable with that of their fully mesenchymal equivalents, those with a partial EMT phenotype showed increased CTC multiplicities and metastasis formation at distant organs [35]. Overall, partial EMT seems to confer increased phenotypic plasticity to the cancer cells especially when it comes to regaining epithelial characteristic (by MET), an essential requirement for metastasis formation at speciﬁc organ sites [14]. 3.2. Hybrid E/M Phenotypes and Partial EMT: Many Shades of Gray y yp y f y As mentioned above, the transient and reversible nature of Of note, HNSCC (head and neck squamous cell carcinoma) cells with partial EMT are preferentially localized at the invasive front of the primary tumors in close proximity to CAFs (cancer-associated ﬁbroblasts) [57], reminiscent of the “β-catenin paradox” in colon cancer [24]. The elucidation of the molecular mechanisms underlying partial EMT is still in its early days. Nonetheless, the diﬀerent intermediate E/M phenotypes are likely to be driven by speciﬁc epigenetic and transcriptional modiﬁcations. Kröger et al. (2019) isolated subpopulation tumor cells stably residing in a hybrid E/M state from both in vitro and in vivo models using a human immortalized and transformed mammary epithelial cell line. These E/M tumor cells were characterized by upregulation of the SNAIL EMT-TF and of canonical Wnt-signaling. Ectopic ZEB1 expression resulted in a fully mesenchymal transformation of the E/M cells accompanied by a reduction of their tumorigenic potential and a switch from canonical to non-canonical Wnt signaling [58]. Apart from SNAIL, other transcription factors including NUMB, GRLH2, and OVOL have been proposed to act as ‘phenotypic stability factors’ which promote, control, and stabilize the hybrid E/M state, possibly by interfering with the core EMT decision-making circuit [59,60]. As mentioned above, the cancer cell’s ability to revert back from EMT-induced phenotypes is critical for metastasis formation in distant organs and full mesenchymal transformation may result in the irreversible loss of MET capacity [58,61,62]. For example, activation of TGF-β signaling triggers EMT in carcinoma cells in a dosage-dependent fashion. Upon short-term treatment, the induced EMT is reversible. However, prolonged exposure of cancer cells to TGF-β result in more stable and irreversible transitions even upon ligand withdrawal [62]. Next to the speciﬁc expression signatures of EMT-related transcription factors and their downstream signaling pathways driving hybrid E/M and fully mesenchymal states in cancer cells, the existence of other alternative EMT-programs with distinct outcomes has been proposed [34,56]. Overall, it is still unclear whether hybrid E/M cells represent a metastable population or are just captured in a time frame transitioning from the epithelial to mesenchymal phenotype. Also, it remains uncertain which context-dependent environmental factors and downstream signaling paths are responsible for driving heterogeneous phenotypic fates during tumor progression. Nonetheless, as mentioned earlier, ample experimental evidence clearly indicates that the hybrid E/M cells state is involved in the collective invasion, migration, and dissemination of tumor cells en route to form Cancers 2019, 11, 1368 8 of 19 distant metastases. 4.1. Single Cell Migration Cancer cells lacking interactions with neighboring tumor cells can detach from the primary mass and migrate individually into the microenvironment. There are two diﬀerent mechanisms of single-cell invasion, namely amoeboid and mesenchymal migration [64]. The involvement of one of these two modes is dependent on the rigidity of the cell-matrix adhesions, the tumor cell’s capacity to remodel the extracellular matrix, and the contractility of the cytoskeleton [65]. In amoeboid invasion, an EMT-independent mechanism, cancer cells have a characteristic rounded cell shape. Here, migration relies on the contractility of cortical actomyosin, promoted by the Rho/ROCK signaling pathway [66]. The proteolysis-independent actomyosin contractility results in membrane blebbing, i.e., the formation of membrane protrusions that enable cancer cells to squeeze through gaps within the ECM [66,67]. In contrast, during mesenchymal single-cell invasion, cells adopt an elongated spindle-like phenotype with front-back polarity as a result of EMT [68,69]. Additionally, cells that engage the mesenchymal mode are dependent on the activity of enzymes such as matrix metalloproteinases (MMPs) and serine protease seprase that degrade the ECM and, as tumor cells invade, progressively create channels which can be used for the cells lagging behind the leading ones [70]. Interestingly, inhibition of ECM remodeling leads to amoeboid migration with cancer cells squeezing through pre-existing pores by actomyosin contractility [67]. Of note, MMPs are generally regarded as integral members of the EMT program. In hepatocellular carcinoma (HCC), upregulation of the EMT-TF Snail not only repressed E-cadherin transcription but also increased expression of MMP-1, MMP-2, MMP-7, and MT1-MMP leading to accelerated invasion [71,72]. Alternatively, several ECM components and even MMPs can, in some cases, act as EMT initiators [73,74]. Induction of MMP-3, also known as stromelysin-1 (SL-1), in the mammary epithelium resulted in cleavage of E-cadherin leading to removal of E-cadherin and catenins from adherens junctions, downregulation of cytokeratins, upregulation of vimentin and of endogenous MMPs [73]. Although single-cell invasion is linked to tumor cells undergoing the full EMT-program leading to suppression of E-cadherin and induction of vimentin [68,69], there is evidence that partial EMT, i.e., the retention of epithelial features, can also feature single-cell migration [75,76]. Additionally, cancer cells can switch between amoeboid and mesenchymal states spontaneously or through changes in ECM composition [67]. 3.2. Hybrid E/M Phenotypes and Partial EMT: Many Shades of Gray y yp y f y As mentioned above, the transient and reversible nature of In the next sections, we will portray the role of EMT in collective cell invasion into the local tumor stroma and dissemination as CTC-clusters, when compared with single migrating cancer cells that complete the full EMT-program. 4. Single versus Collective Cell Migration The initial detachment of the cancer cell from the primary mass and its invasion in the surrounding stromal microenvironment represent critical and rate-limiting steps in the metastatic cascade responsible for 90% of deaths in patients with malignancies [12,63]. In order to invade, cancer cells employ distinct invasion modalities: single (amoeboid or mesenchymal invasion) and collective cell migration. Of note, cancer cells can switch between these invasion modes, an important feature when it comes to the development of anti-invasive and anti-metastatic therapies [64]. 4.2. Collective Cell Migration and the Role of EMT In collective cell migration, cancer cells retain intact cell–cell adhesions while invading the tumor microenvironment, the vasculature, and distant organ sites [77]. A variety of migration modalities feature collective cell migration, ranging from narrow linear connected cell strands to broad sheets or compact cluster/budding of cells [77]. Unlike single-cell migration resulting from fully mesenchymal cells, the role of EMT in collective migration is subtler. Recently, using a Drosophila melanogaster model of colon cancer, it was shown that the Snail homolog Sna can activate partial EMT in tumor cells leading to their collective invasion through the basement membrane and muscle ﬁbers [78]. Additional 9 of 19 Cancers 2019, 11, 1368 evidence pointing at the correlation between hybrid E/M and collective cell migration lies in the onset of ‘leader’ cells at the invasive margin that are selected to guide other ‘following’ cancer cells [79]. These leader cells show a bi-phenotypic state with mesenchymal features as altered polarity and development of protrusions at their front. Yet, they also maintain attachments to their follower cells at their rear end. The follower cells, on the other hand, retain apical–basal polarity and migrate taking advantage of the pulling force generated by leader cells [80]. Knockdown of the epithelial marker cytokeratin 14 in leader cells is suﬃcient to block collective migration suggesting that the hybrid E/M state is mandatory for establishment of the leader cells [79]. The onset, activity, and maintenance of leader cells are coordinated by environmental stimuli, i.e., the local increase of compression [81], soluble factors, and chemokines [82], but is also controlled within the collective tumor group by autocrine or juxtacrine fashion. Of note, also in this case several MMPs are expressed at the leading edge to facilitate ECM degradation and to create a migration path for the cell clusters [83]. Notably, non-cancer cells can also contribute to collective cell migration. The movement of cancer cells can be conducted by migratory stromal cells such as ﬁbroblasts [84,85]) or macrophages [86,87]. Labernadie et al. (2017) demonstrated that cancer-associated ﬁbroblasts (CAFs) exert a physical force on cancer cells that leads to their collective migration. This intercellular force transduction is achieved by the formation of heterophilic adhesion complexes between N-cadherin on the CAF membrane and E-cadherin on the cancer cell membrane [85]. Moreover, CAFs are also a source of ECM-degrading proteases such as MMPs thereby creating micro tracks used by cancer cells to migrate through [84]. 4.2. Collective Cell Migration and the Role of EMT In addition to degrading the ECM, CAFs also secrete growth factors and chemokines that generate chemotactic gradients to direct cell migration [88]. Last, cancer cells can ingest exosomes secreted by CAFs thereby activating intracellular pathways known to trigger EMT [89]. In colon cancer, CAFs release exosomes containing miR-92a-3p and promote invasion and chemotherapy resistance. miR-92a-3p directly binds to FBXW7 and MOAP1 thereby activating Wnt-induced EMT and mitochondrial apoptosis [89]. Overall, single and collective cell migration share some of the underlying mechanisms (e.g., cell–cell and cell-matrix communication, and the establishment of a migratory polarity). Moreover, during invasion tumor cells can switch between diﬀerent modes of migration depending on intrinsic (cell adhesion) and extrinsic cues (ECM composition and density). In general terms, a complete EMT is associated with single-cell migration, whereas collective cell migration seems to result from partial EMT. Nonetheless, the mechanisms underlying the role of EMT in determining the invasion modalities, the intercellular communication among invading cells, and the tumor microenvironmental cues leading to collective migration are yet poorly deﬁned. This is further complicated by the fact that invasion modalities are likely to be cell type-, tissue-, and time-dependent. The plasticity of cancer cells to switch between diﬀerent invasion modes is a key feature and a putative target for the development of novel therapeutic strategies [90]. 5.1. Single CTCs versus CTC Clusters Single CTCs disseminate into distant organs upon EMT [14]. However, the discovery of CTC clusters has raised questions on the relative role of EMT in local invasion and systemic dissemination from the primary tumor mass. CTC clusters are deﬁned as a group of 2–3 or more tumor cells that travel as a group through the bloodstream [91]. In 1954, Watanabe showed that, by injecting bronchogenic carcinoma cells in the jugular vein of recipient mice, tumor clumps, in contrast to single cells, were able to form metastasis [102]. Accordingly, aggregated colon cancer cells also showed increased metastatic eﬃciency in the liver when compared with single cells after intra-portal injection in rat [103]. These initial observations, however, did not explain how and where CTC clusters are formed. More recently, it has been demonstrated that CTC clusters do not derive from the intravascular aggregation of single CTCs or from proliferating single CTCs, but rather from clumps of primary tumor cells that collectively detach from the primary mass and enter the vasculature as CTC clusters [104–106]. Moreover, it was also shown that the metastatic capacity of CTC clusters was up to ﬁfty-fold higher when compared with single CTCs [104]. Genome-wide single-cell DNA methylation analysis demonstrated distinct methylomes between CTC clusters and single CTCs in human breast cancer patients. CTC clusters were shown to be hypo-methylated at stemness- and proliferation-associated transcription regulators including OCT4, NANOG, SOX2, and SIN3A, and hyper-methylated at Polycomb target genes [107]. Lastly, the presence of circulating tumor micro emboli in peripheral blood of patients with cancer arising from colon, breast, and lung was predictive of poor survival [104,108,109]. 5.2. CTC Cluster Heterogeneity The heterogeneous composition of CTC clusters encompassing parenchymal cancer cells together with immune cells, cancer-associated ﬁbroblasts, tumor stroma, and platelets, seems to reﬂect the heterogeneity of the primary tumors they originate from [94–98]. The presence of non-malignant cells within CTC clusters contributes to their improved survival and metastatic capacity. Normal epithelial cells undergo cell anoikis upon the detachment from the extracellular matrix (ECM), which establishes an important defense mechanism to prevent abnormal growth in inappropriate places. However, EMT can circumvent anoikis in individual cells during dissemination and metastasis [110]. The transition of single CTCs to a mesenchymal phenotype results in the expression of adherence-independent survival signals that compensate for the loss of attachment to the ECM [111]. Alternatively, CTC clusters may prevent tumor cell anoikis by retaining epithelial cell–cell interactions and thus contributing to the activation of survival stimuli [104,105]. Next, the non-malignant cell microenvironment can protect CTC cells from immune cells [112,113], shield cells from mechanical stress, and promotes adhesion to the endothelium [114,115]. It also has been shown that platelets can induce EMT in CTCs via TGF-β and NF-κB signaling while enhancing their metastatic potential [97]. Thus, secretion of growth factors and cytokines by the non-cancer cells may represent an additional survival advantage for the CTC clusters in the vasculature. Last, yet another advantage of the CTC clusters when compared to single CTCs is the capacity of remodeling the microenvironment at the metastatic site, thereby facilitating colonization [95]. 5. Circulating Tumor Cells Circulating tumor cells (CTCs) are deﬁned as those cancer cells disseminated from the primary tumor mass and intravasated into blood vessels which are thought to underlie metastasis at distant organ sites [91]. CTCs have been identiﬁed at diﬀerent multiplicities in many carcinomas including colon, breast, prostate, lung, bladder, and gastric cancer, while they are extremely rare in healthy individuals or in patients with non-malignant disease [91]. However, even in cancer patients, CTCs are extremely rare and, accordingly, their prospective isolation and characterization have proven to be a challenge [91]. Heterogeneity also exists among CTCs, possibly reﬂecting the above discussed intra-tumor heterogeneity. Likewise, the existence of both single CTCs, as well as CTC clusters comprising multiple (from few to hundreds) cells, has been well established in the scientiﬁc literature [92,93]. Of note, CTC clusters are not exclusively composed of epithelial cancer cells but are often intermingled with immune cells, cancer-associated ﬁbroblasts, tumor stroma, and platelets [94–98]. 10 of 19 Cancers 2019, 11, 1368 In addition to this heterogeneity, CTCs and CTC clusters have been captured that express both epithelial and mesenchymal features [93,99–101]. 5.3. EMT in CTC Clusters Next to the heterogeneity of CTC clusters in terms of cell lineage composition, the degree of EMT activation among the parenchymal cancer cells within CTCs can also vary considerably. CTC clusters display epithelial cell–cell interactions as shown by the retention of expression of several epithelial-speciﬁc genes such as K5, K8, K14, E-cadherin, P-cadherin, and plakoglobin in metastatic breast CTC clusters [116]. Accordingly, knockdown of plakoglobin, a member of the catenin protein family and homologous to β-catenin, led to disaggregation of the CTC clusters, thereby compromising 11 of 19 tastatic protein Cancers 2019, 11, 1368 epithelial-specific b CTC l metastasis formation [104]. Also, disruption of K14 expression negatively aﬀected the expression of key downstream eﬀectors in metastatic niche remodeling and metastasis survival, leading to compromised eﬃciency in metastasis formation [105]. However, CTC clusters with predominant hybrid E/M or fully mesenchymal features have been observed in human colon, prostate, lung, and breast cancer patients [93,99–101]. At least one-third of cancer cells from within CTC clusters derived from colon cancer patients were negative for cytokeratin expression [100]. In breast cancer, CTC clusters show shifts in their EMT status according to treatment modalities with predominant mesenchymal expression patterns during cancer progression and/or in refractory disease [93] (Figure 3). This dynamic EMT proﬁle allows for cellular plasticity and adaptation to the diverse cellular contexts encountered by CTCs during dissemination and metastasis formation, and to diﬀerent treatments regimes. The latter is also of relevance for the use of prognostic epithelial markers of CTCs likely to fail to detect cancer cells that have undergone EMT. Additional mesenchymal CTCs markers are needed for more accurate prognostic studies [117]. family and homologous to β catenin, led to disaggregation of the CTC clusters, thereby compromising metastasis formation [104]. Also, disruption of K14 expression negatively affected the expression of key downstream effectors in metastatic niche remodeling and metastasis survival, leading to compromised efficiency in metastasis formation [105]. However, CTC clusters with predominant hybrid E/M or fully mesenchymal features have been observed in human colon, prostate, lung, and breast cancer patients [93,99–101]. At least one-third of cancer cells from within CTC clusters derived from colon cancer patients were negative for cytokeratin expression [100]. In breast cancer, CTC clusters show shifts in their EMT status according to treatment modalities with predominant mesenchymal expression patterns during cancer progression and/or in refractory disease [93] (Figure 3). 5.3. EMT in CTC Clusters This dynamic EMT profile allows for cellular plasticity and adaptation to the diverse cellular contexts encountered by CTCs during dissemination and metastasis formation, and to different treatments regimes. The latter is also of relevance for the use of prognostic epithelial markers of CTCs likely to fail to detect cancer cells that have undergone EMT. Additional mesenchymal CTCs markers are needed for more accurate prognostic studies [117]. Figure 3. EMT features in single circulating tumor cells (CTCs) and CTC clusters from a metastatic breast cancer patient. (a) Longitudinal monitoring of EMT features in CTCs. The Y-axis indicates the number of CTCs per 3 mL of blood. The patient was monitored over time (X-axis) during treatment with inhibitors targeting the PI3K and MEK pathways (months 1–8), followed by chemotherapy with Adriamycin (8–12). The color-coded quantification bars indicate the EMT status of the CTCs based on RNA-ISH (in situ hybridization) analysis at each indicated time point. P = disease progression; R = tumor response. (b) RNA-ISH analysis of EMT markers in CTCs derived from patients with metastatic breast cancer. Green dots represent epithelial (E) and red marks mesenchymal (M) markers. Scale bar: 5 µm. Adapted from [93]. Figure 3. EMT features in single circulating tumor cells (CTCs) and CTC clusters from a metastatic breast cancer patient. (a) Longitudinal monitoring of EMT features in CTCs. The Y-axis indicates the number of CTCs per 3 mL of blood. The patient was monitored over time (X-axis) during treatment with inhibitors targeting the PI3K and MEK pathways (months 1–8), followed by chemotherapy with Adriamycin (8–12). The color-coded quantiﬁcation bars indicate the EMT status of the CTCs based on RNA-ISH (in situ hybridization) analysis at each indicated time point. P = disease progression; R = tumor response. (b) RNA-ISH analysis of EMT markers in CTCs derived from patients with metastatic breast cancer. Green dots represent epithelial (E) and red marks mesenchymal (M) markers. Scale bar: 5 µm. Adapted from [93]. Figure 3. EMT features in single circulating tumor cells (CTCs) and CTC clusters from a metastatic breast cancer patient. (a) Longitudinal monitoring of EMT features in CTCs. The Y-axis indicates the number of CTCs per 3 mL of blood. The patient was monitored over time (X-axis) during treatment with inhibitors targeting the PI3K and MEK pathways (months 1–8), followed by chemotherapy with Adriamycin (8–12). 6. Partial EMT, Collective Cell Migration, and Metastasis: Therapeutic strategies Metastasis formation involves the successful completion of a sequential series of challenging steps. Phenotypic plasticity refers to the key feature of the metastasizing cancer cell to adapt to the environment where it resides through reversible changes of its cellular identity [13]. This ‘Dr. Jekyll and Mr. Hide’ feature of migrating cancer cells is controlled by epigenetic mechanisms which regulate E-to-M and M-to-E transitions (EMT and MET) [14]. However, EMT cannot be regarded as a binary process as it generates hybrid E/M cancer cells encompassing a range of intermediate stages. Partial EMT has been correlated with collective cell migration and with the presence of CTC clusters with enhanced metastatic potential in the peripheral blood of cancer patients [119]. Moreover, the unaﬀected CTC clusters multiplicity upon chemotherapy is indicative of treatment failure in colorectal cancer [120]. From this perspective, the elucidation of the underlying intrinsic and extrinsic mechanisms is bound to lead to the development of novel therapeutic and even preventive strategies based on the targeting of cell–cell and/or cell-matrix interactions and the disruption of CTC clusters. Gkountela et al. (2019) tested a library of approximately 2500 FDA-approved compounds and identiﬁed Na+/K+ ATPase inhibitors able to disaggregate derived CTC clusters derived from breast cancer patients into single cells. Mechanistically, Na+/K+ ATPase inhibition in tumor cells leads to an increase of intercellular Ca2+ concentration and to the consequent inhibition of formation of cell–cell junctions. In an in vivo xenograft model using NSG mice injected with patient-derived breast cancer cells in their fat pad, treatment with the Na+/K+ ATPase inhibitor ouabain resulted in a marked reduction of CTC cluster formation together with the increase of single CTC multiplicity. Although the size of the primary tumor was unaﬀected upon ouabain treatment, the overall number of metastatic lesions, corresponding to the number of CTC clusters, was reduced [107]. An alternative approach towards the development of therapeutic strategies based on CTC clusters may be represented by inhibition of platelet function. Platelets make integral part of CTC clusters where they are thought to protect the cancer cells from shear stress and immune attacks [121]. Acetylsalicylic acid (i.e., aspirin) inhibits platelet function by acetylation of cyclooxygenase (COX) thereby preventing arachidonic acid (and prostaglandin) production and consequently resulting in irreversible inhibition of platelet-dependent thromboxane formation. Based on this, aspirin has been employed as an anticoagulant for the prevention of thrombosis [122]. 6. Partial EMT, Collective Cell Migration, and Metastasis: Therapeutic strategies In experimental cancer models and clinical trials, inhibiting the interaction between cancer cells and platelets have been shown to hamper tumor cell survival, growth and metastasis formation [123,124]. Notwithstanding the above promising and innovative therapeutic strategies based on CTC clusters, their allegedly high degree of plasticity—as a mechanism to escape targeted treatment—is also likely to result in therapy resistance. Nonetheless, future research towards the identiﬁcation of novel therapeutic targets to lower the risk of CTC cluster formation is expected to improve the eﬃcacy of cancer treatment in the long run. 5.3. EMT in CTC Clusters The color-coded quantification bars indicate the EMT status of the CTCs based on RNA-ISH (in situ hybridization) analysis at each indicated time point. P = disease progression; R = tumor response. (b) RNA-ISH analysis of EMT markers in CTCs derived from patients with metastatic breast cancer. Green dots represent epithelial (E) and red marks mesenchymal (M) markers. Scale bar: 5 µm. Adapted from [93]. Figure 3. EMT features in single circulating tumor cells (CTCs) and CTC clusters from a metastatic breast cancer patient. (a) Longitudinal monitoring of EMT features in CTCs. The Y-axis indicates the number of CTCs per 3 mL of blood. The patient was monitored over time (X-axis) during treatment with inhibitors targeting the PI3K and MEK pathways (months 1–8), followed by chemotherapy with Adriamycin (8–12). The color-coded quantiﬁcation bars indicate the EMT status of the CTCs based on RNA-ISH (in situ hybridization) analysis at each indicated time point. P = disease progression; R = tumor response. (b) RNA-ISH analysis of EMT markers in CTCs derived from patients with metastatic breast cancer. Green dots represent epithelial (E) and red marks mesenchymal (M) markers. Scale bar: 5 µm. Adapted from [93]. As mentioned above, alternative EMT-programs accounts for different CTC phenotypes. Aiello et al. (2018) suggested that single CTCs arise from cancer cells that have completed a full As mentioned above, alternative EMT-programs accounts for diﬀerent CTC phenotypes. Aiello et al. (2018) suggested that single CTCs arise from cancer cells that have completed a full EMT-program, whereas tumor cells characterized by partial EMT tend to present as clusters resulting from collective migration [34]. However, it has been demonstrated that, next to those mainly composed by CTCs, cell clusters have been isolated from colon cancer patients which consist of endothelial cells 12 of 19 Cancers 2019, 11, 1368 12 of 19 without any genetic aberrations found in their matched primary tumor of origin. These cell clusters were positive for both epithelial and mesenchymal markers and are thought to result from the direct release of clusters from the tumor vasculature due to impaired neo-angiogenesis [118]. 7. Final Remarks and Conclusions Acknowledgments: The authors would like to thank Mathijs Verhagen for his assistance with the artwo Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 7. Final Remarks and Conclusions EMT contributes to a considerable degree of cellular heterogeneity in both primary tumors and metastatic lesions as it aﬀects a broad spectrum of cellular functions beyond the transitions between epithelial and mesenchymal states associated with enhanced invasive and metastatic abilities. Changes in stem cell behavior, escape from apoptosis and senescence, ECM and tumor-microenvironment remodeling, and resistance to cytotoxic treatments are only a few among the broad spectrum of downstream EMT eﬀectors which contribute to intra-tumor cell heterogeneity with profound implications for cancer therapeutics, especially in the decade of personalized treatments [11]. Cancers 2019, 11, 1368 13 of 19 13 of 19 In fact, EMT is thought to play key roles in each and every step of the metastatic cascade including intra- and extravasation [125], and the colonization of distant organ sites [126,127]. For the sake of brevity, these latter aspects are not discussed in this review. The observed broad spectrum of EMT eﬀectors may well reﬂect the pleiotropic functional roles of the EMT-TFs such as ZEB1 [128] that go well beyond the E to M (and vice versa) trans-diﬀerentiation, and include angiogenesis, remodeling of the tumor microenvironment, immune escape, mechanotransduction, and possibly many more. The identiﬁcation and elucidation of the complex network of intrinsic and extrinsic mechanisms driving EMT at “just-right” (E/M) levels to trigger collective migration, generate CTC clusters and successfully metastasize distant organ sites represent the major future research challenge in the translation of our fundamental understanding of metastasis into therapy. From this perspective, single-cell epigenetic and transcriptomic analysis will provide powerful approaches to address this challenge. These high-resolution techniques will be key to elucidate the heterogeneous composition of malignancies including the identiﬁcation of distinct and rare cell types arising transiently in time and at speciﬁc locations within tumors. Moreover, single-cell proﬁles will help to investigate the variability among individuals, disease states, microenvironments, and treatment history. Overall, the realization of the importance of epigenetics and the elucidation of the mechanisms underlying transient changes in the cellular identity of individual circulating and metastasizing tumor cells will lay the basis for the development of novel treatment modalities. These will complement the current ‘personalized cancer medicine’ mainly directed at somatic gene mutations arisen at the primary site and unlikely to be rate-limiting in the clinical management of a more advanced malignant disease. Funding: This research was funded by the Dutch Cancer Society (KWF), grant number EMCR 2015-7588. References 1. 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https://openalex.org/W4393393377	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0301197&type=printable	English	null	Improving the efficiency of adaptive management methods in multiple fishways using environmental DNA	PloS one	2,024	cc-by	16,903	PLOS ONE PLOS ONE RESEARCH ARTICLE Masahiko Nakai1, Taku MasumotoID2, Takashi Asaeda3, Mizanur Rahman3 Masahiko Nakai1, Taku MasumotoID2, Takashi Asaeda3, Mizanur Rahman3 Masahiko Nakai1, Taku MasumotoID2, Takashi Asaeda3, Mizanur Rahman3 1 Japan International Consultants for Transportation Co., Ltd, Tokyo, Japan, 2 Energy Planning Department, East Japan Railway Company, Tokyo, Japan, 3 Saitama University, Saitama, Japan 1 Japan International Consultants for Transportation Co., Ltd, Tokyo, Japan, 2 Energy Planning Department, East Japan Railway Company, Tokyo, Japan, 3 Saitama University, Saitama, Japan 1 Japan International Consultants for Transportation Co., Ltd, Tokyo, Japan, 2 Energy Planning Department, East Japan Railway Company, Tokyo, Japan, 3 Saitama University, Saitama, Japan t-masumoto@jreast.co.jp * t-masumoto@jreast.co.jp * t-masumoto@jreast.co.jp OPEN ACCESS Citation: Nakai M, Masumoto T, Asaeda T, Rahman M (2024) Improving the efficiency of adaptive management methods in multiple fishways using environmental DNA. PLoS ONE 19(4): e0301197. https://doi.org/10.1371/journal. pone.0301197 Editor: Petr Heneberg, Charles University, CZECH REPUBLIC Editor: Petr Heneberg, Charles University, CZECH REPUBLIC Received: July 8, 2023 Accepted: March 12, 2024 Published: April 1, 2024 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0301197 Copyright: © 2024 Nakai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Dams and weirs impede the continuity of rivers and transit of migratory fish. To overcome this obstacle, fishways are installed worldwide; however, management after installation is important. The Miyanaka Intake Dam has three fish ladders with different flow velocities and discharges and has been under adaptive management since 2012. Fish catch surveys, con- ducted as an adaptive management strategy, place a heavy burden on fish. Furthermore, a large number of investigators must be mobilized during the 30-day investigation period. Thus, a monitoring method using environmental DNA that exerts no burden on fish and requires only a few surveyors (to obtain water samples) and an in-house analyst was devised; however, its implementation in a fishway away from the point of analysis and with limited flow space and its effective water sampling frequency have not been reported. Therefore, in 2019, we started a trial aiming to evaluate the methods and application condi- tions of environmental DNA surveys for the continuous and long-term monitoring of various fish fauna upstream and downstream of the Miyanaka Intake Dam. To evaluate the fish fauna, the results of an environmental DNA survey (metabarcoding method) for 2019 to 2022 were compared to those of a catch survey in the fishway from 2012 to 2022. The results confirmed the use of environmental DNA surveys in evaluating the contribution of fishways to biodiversity under certain conditions and introduced a novel method for sample collection. Introduction Dams and weirs disrupt river continuity and impede migratory fish movement [1,2]. The risks from habitat degradation due to dams are possibly much higher than those from climate change [3–10]. The focus of conservation has gradually shifted from only fish, considered as economic resources in Europe and the United States, to overall biodiversity, which includes a variety of fish [11]. Thus, the protection of biodiversity upstream and downstream of dams is critical, and such work is based on assessments of factors that include river gradients, tributary confluence, riverbank environments, and downstream conditions [12,13]. Structures that Data Availability Statement: All relevant data are within the manuscript. Data Availability Statement: All relevant data are within the manuscript. Funding: The authors received no specific funding for this work. 1 / 29 PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. cross rivers interrupt the continuity of habitats for organisms that must travel upstream and downstream. Although fishways have been installed worldwide to remove these obstacles, management following fishway installation is critical and should consider distribution patterns and extinction risks [14–16]. The monitoring required for conventional management is performed using net capture and expert analysis; however, these methods are costly and require considerable labor [17,18]. In areas where local survey data are lacking, complementary biological assessments may be conducted using data from species distribution models that are readily available elsewhere [19]. Segments originating from various types of vertebrates, such as fish and amphibians, sequenced via normal PCR amplification contain sufficient nucleotide differences to distin- guish between animal species [20]. Surveys using environmental DNA present high cost effec- tiveness and biodiversity assessment standardization [21]; thus, they are considered suitable for understanding the diversity of fish and reduce the required time and expenses [22–30]. Moreover, because of species extinctions, ecosystems must be assessed before and after struc- ture formation. Therefore, environmental DNA could represent a sensitive and powerful tool for monitoring such interruptions in river continuity [31–33]. In 2008, the first successful identification of organisms using environmental DNA occurred when the presence and density of the alien species Rana catesbeiana were inferred by extract- ing environmental DNA from pond water [34]. Subsequently, the development of metabar- coding technology, which detects multiple types of DNA simultaneously, progressed in Japan. Attempts were made to obtain a more complete picture of fish species by applying noninvasive methods to detect target alien species. In 2011, "all types of DNA from aquarium water" and "four types of DNA from wild water" were successfully detected using environmental DNA for the first time [35]. This method is more sensitive than capture surveys [36,37]; therefore, it is more effective when targeting rare organisms [38–42]. Environmental DNA has been success- fully used in ocean studies and is becoming a standard method in this field to enhance the study of ecosystems. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Data Availability Statement: All relevant data are within the manuscript. Acoustic sonar, which has an established track record in sea environ- ments, can also be applied in rivers to detect deep rivers and salmon, which are strong swim- mers [43]. However, because of the uncertainty of recognition by acoustic sonar, the environmental DNA technology approach, which has been validated in the ocean, may also become the standard for rivers [44–46]. Optimal filtration contributes to ensuring optimal environmental DNA to maximize the detection probability in rare species habitats [47]. More- over, compared to aqueous environmental DNA, sedimentary environmental DNA useful for benthic inference may be confounded due to resuspension and transport [48]. Analyses using environmental DNA have been performed for surveys of a wide range of target areas, including remote regions, irrespective of the classification of organisms. In many inaccessible locations, this method requires multiple data sources to establish the potential presence or absence of species [49]. The potential of environmental DNA has been strength- ened by its ability to reveal unrecorded biodiversity components and human influences [50]. This method is widely used for analyses of organisms in water and soil [51]. Discussions about advances in environmental DNA indicate that this method will have wide-ranging applications for information processed based on conventional morphological classifications [52]. Using a non-idealistic environmental DNA barcode data approach facilitates access to new taxa [53]. Metabarcoding for biodiversity assessment provides a rapid and inexpensive alternative that does not require taxonomic expertise; in addition, instead of routine identification, researchers can focus on important aspects, such as species habitats [54]. The development of metabarcod- ing alleviates some of the challenges in morphological identification of bioindicator taxa [55]. The remarkable advances and developments in environmental DNA sequencing have elimi- nated the need for DNA amplification, and collections of DNA with standardized barcodes PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 2 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways have created a comprehensive taxonomic reference [56]. Therefore, prior to its practical use, environmental DNA underwent a number of experimental verifications [57]. Even in the absence of a complete database, elusive patterns of biodiversity may be revealed in environ- mentally heterogeneous and biologically diverse regions using environmental DNA metabar- coding [58]. Seasonally different activity patterns in different species of the same genus affect the probability of environmental DNA detection. Natural history information can therefore guide monitoring plans [59]. Data Availability Statement: All relevant data are within the manuscript. Organisms that cannot be directly observed are also detected using this method because environmental DNA carries the molecular signatures of a species [29], which has been con- firmed in several studies [60,61]. However, an understanding of the history of environmental DNA analysis by investigators and researchers is required [62]. Species can be clearly detected in areas where they are not actually present [63]. Controlling false positives remains a major challenge in environmental DNA analysis [64]. Misidentification hinders the reliability of DNA-based assessments of biodiversity [65]. A balanced discussion with consistent communi- cation, controls, and limits of detection to clarify false positives is important for resolving mis- conceptions about false positives [66]. Analyzing more filters can reduce the risk of false negatives when individuals live in low densities [67]. When used appropriately, this method can be used to formulate plans for managing ecosystem conservation [68–70]. Moreover, this method allows for a better understanding of the relationship between changes due to natural or artificial factors and fish populations [71]; therefore, this approach is recommended [72]. p p [ ] pp [ ] The diversity of fish based on environmental DNA in a space that presents limited flow and occurs a certain distance from the point of analysis, such as in a fishway, is poorly understood because of the limited number of reported surveys [73]. Therefore, in this study, we focused on developing a continuous, long-term, and efficient method for understanding the diverse fish fauna within fishway groups and elucidating the optimal water sampling frequency for environmental DNA research that can corroborate the results of real fish capture research. The Miyanaka Intake Dam has three fishways (ice-harbor-, stair-, and rock-ramp-type), each of which maintains its own independent environment. The most significant feature of the Miyanaka Dam is the difference in its flow velocity settings [74]. Specifically, the flow velocity in the ice-harbor-type fishway is two-fold that of the rock-ramp-type fishway [75]. During the fishway improvement project, which targeted 16 types of fish species, the fish targeted for each type of fishway were determined. The ice-harbor-type fishway is intended for use by Oncor- hynchus keta and Oncorhynchus masou, the stair-type fishway is intended for Plecoglossus alti- velis and Tribolodon hakonensis, and the rock-ramp-type fishway is intended for bottom- dwelling type fish and small fish with poor swimming ability. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Data Availability Statement: All relevant data are within the manuscript. A correlation was confirmed between individual density and biomass estimated by the capture survey in the upstream section from the water sampling point and environmental DNA concentration [80]. However, in other studies, environmental DNA concentrations were not correlated with bio- mass or population [81]. One of the reasons for this could be that all measured samples were below the quantification limit for the target DNA sequence, such as in the case of American crayfish [82]. In addition, in an experiment in which the dilution of environmental DNA released in a river was measured as it flowed downstream, the concentration of DNA decreased in the downstream direction when the flow velocity was low. However, when the flow rate was high, the same DNA concentration was observed both downstream and upstream [83]. In addition, the following points were evaluated in order to develop a new research method: 1) effects when the number of migratory individuals increased or decreased significantly, 2) types of fish that migrated upstream in different fishways with different cur- rent velocities and discharges, and 3) presence or absence of changes in the fish fauna over a long period of time. Based on our findings, a detailed analysis of the population numbers of P. altivelis and T. hakonensis was performed in another study that drew from these survey results. Furthermore, a modification in a survey policy that focuses on changes in fish fauna was con- sidered. We hypothesized that surveys using environmental DNA would not only reduce the burden on fish and surveyors but also contribute to the efficient and economical realization of medium- to long-term surveys by appropriately setting the number of places and times to obtain water in a day. Therefore, in 2019, a study was conducted to identify fish fauna using environmental DNA analysis. We compared the results of the metabarcoding method with those of capture surveys for fish fauna around the Miyanaka Intake Dam. Masumoto et al. [76] highlighted the most distinctive feature of the Miyanaka Intake Dam: the fish fauna varies across its three fishways. This offers a fine-grained system to benchmark environmental DNA against capture surveys. It was crucial to clarify the fish species that did not overlap between the two surveys. Next, a study was conducted using environmental DNA analysis to understand the utilization of these fish passages by different fish species. Data Availability Statement: All relevant data are within the manuscript. Considering studies determining the overflow depth of the bulkhead suitable for the target fish, an upstream environment with different current speeds was created [76]. Subsequently, it was confirmed that the flow velocity within each fishway remained below the rush speed of the targeted fish species. Monitoring studies conducted since 2012 have shown that fish preferentially select certain fishways based on their biological characteristics [75]. Therefore, the fish fauna varies among fishways. We conducted fish catch surveys since 2012 by setting traps at the upstream end of the fishway and identified 37 species of fish. Monitoring surveys based on captures have confirmed that the improved fishway is fully functioning [76]. However, it became clear that catching surveys are a heavy burden on fish [77,78] and that the number of individuals caught in fishways fluctuates with various factors, such as river water temperature, turbidity, and flow conditions due to flooding. A total of 180 investigators surveyed the fishway for one month. Specifically, six investigators each day retrieved catch baskets from the traps in the fishway once every hour and checked the types of fish eight times a day. This analysis has been conducted since 2012, and it is performed in June, when P. altivelis and T. hakonensis, which attract particular PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 3 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways attention in this river and represent small fish, swim upstream. Surveys have focused not only on the number of species but also on the number of individuals. However, the populations of migratory fish such as Plecoglossus altivelis and Oncorhynchus keta fluctuate owing to not only downstream environmental conditions during their upstream migration but also autumn floods during the spawning season and changes in seawater temperature. In another survey on salmon conducted by the Ministry of Land, Infrastructure, Transport and Tourism in the same fishway, the impact of natural fluctuations, such as the number of salmon migrating to Niigata Prefecture, was evaluated as one of the factors behind the changes in salmon popula- tions. Regarding the relationship between the number of individuals counted and environ- mental DNA, a study showed that the environmental DNA concentration was higher in ponds with a larger number of individuals estimated via existing methods [79]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Data Availability Statement: All relevant data are within the manuscript. Environmental DNA analysis (metabarcoding method) was performed for each fishway, and the findings were compared with the results of the corresponding fish catch surveys [84– 88]. The metabarcoding results for 2021 and 2022 from the three different types of fishways (ice-harbor-type, stair-type, and rock-ramp-type fishways) were compared to evaluate whether the status of fish can be understood based on fish passages through an environmental DNA survey. Specifically, the effectiveness of determining the fishway usage status using an environ- mental DNA survey was examined by focusing on reproducibility and comparing the charac- teristics of the fish fauna in terms of the amount of environmental DNA between fish passages. In addition, changes in the number of fish caught were compared with changes in environ- mental DNA to understand the use of fishways through environmental DNA surveys. The PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 4 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways amount of DNA corresponding to fish that showed migration status changes between 2021 and 2022 was evaluated under the assumption that if the amount of environmental DNA in a fishway reflects the use of the fishway by fish, the amount of DNA for species with changes in the number of catches from 2021 to 2022 will also show corresponding changes. The effort required for water sampling was less than that for the fish-catching survey, and the cost of the environmental DNA survey was lower, even after accounting for the cost of reagents and analysis [89]. Here, we aimed to obtain results that could be substituted for con- tinuous, long-term, and efficient evaluation of various fish fauna in the fishway without impos- ing a burden on the fish. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Survey methods using environmental DNA Collection and preparation of samples for environmental DNA. Water sampling and analysis were carried out according to the Environmental DNA Sampling and Experiment Manual Version 2.1 (published on April 25, 2019, environmental DNA Methods Standardiza- tion Committee). A bucket with an attached string, water sampler with a handle, and measur- ing cup were used according to the sampling environment. These instruments were decontaminated with chlorine bleach before and after use to prevent false positives. New gloves were used when handling water sampling equipment and water sampling pads and per- forming filtration work. In st1 where the height difference of the water surface and water sam- pling point was approximately 2 m, a bucket with a string was used to sample surface water. In st2 and st3 where surface water was sampled across a rock-ramp-type fishway, a water sampler with a handle was used. Surface water was used as the sample because the bottom water and surface water in a 2 m deep pool mix as they flow down more than 200 m. In st4, where the environment was normal, a measuring cup was used to collect water samples near the center. One liter of water was collected for each sample (Fig 2). Environmental DNA research workers avoided contact with fish capture research workers who were working in the same fishway management yard to prevent false positives. The collected samples were injected into a disposable water collection bag. To protect against DNA degradation, which can cause false negatives, 1 ml of benzalkonium chloride 10% w/v aqueous solution (commercial product chloride: OSVAN) was added for every 1 L of water. Following the temporary storage under cold conditions, the samples were filtered through glass fiber filters (mean pore size, 0.7 μm) on site within a few hours [90,91]. A filtra- tion device developed for this study that is capable of filtering 10 samples at the same time was temporarily installed in a car parked at the management yard next to the fishway where the water was sampled. The filtration device used a 100 V power source and thus could be oper- ated using car batteries. The time required for one filtration was approximately 15 to 30 min. Because DNA degrades over time, short-term filtration is desirable. However, increasing the pump capacity may cause the filter paper to tear. Survey points, timing, and number of water samples The target fishways are located on the right bank of the Miyanaka Intake Dam (37˚3’58.445”N, 138˚41’50.321”E) at 134 km from the mouth of the Shinano River [76]. There were four collec- tion points for environmental DNA analysis in 2022 (Fig 1): st1 was located at the exit of the fishways (upstream) and st2–4 were located at the entrance (downstream) of each of the three fishways (ice-harbor-type, stair-type, and rock-ramp-type). st1 was set as the control point for assessing the environmental DNA flowing into the fishway, and st2–4 were used to assess the effect of fish existing in the fishway. To prevent differences in the environmental DNA flowing into each fishway, st1 was set at a point before the river was divided into the three fishways. st2–4 were set slightly upstream of the submerged area and established to evaluate fish Fig 1. Survey station in the fishways of Miyanaka Intake Dam. Three types of fishways with different flow velocities and water depths are installed on the right bank side. hways of Miyanaka Intake Dam. Three types of fishways with different flow velocities and water depths are installed on the Fig 1. Survey station in the fishways of Miyanaka Intake Dam. Three types of fishways with different flow velocities and water depths are installed on the right bank side. ey station in the fishways of Miyanaka Intake Dam. Three types of fishways with different flow velocities and water depths a id https://doi.org/10.1371/journal.pone.0301197.g001 https://doi.org/10.1371/journal.pone.0301197.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 5 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways swimming up the fishway or staying in the fishway based on the difference in concentration of environmental DNA between st1 and st2–4. There were two water sampling points in 2019 and 2020 (st1 and st2) and four in 2021 and 2022 (st1 and st2–4). Samples used for environmental DNA analysis were collected during the same period as that of the comparative capture survey (29-day period from June 6 to July 4). Water sampling was excluded on days when fish were not expected to migrate due to flooding. The capture sur- vey was carried out every hour from 9:00 to 17:00 each day; water sampling was carried out at the same times. Survey points, timing, and number of water samples As a result, 360 water samples for environmental DNA analysis were collected in 2019 (two sites × 9 times/day × 20 days), 408 were collected in 2020 (two sites × 9 times/day × 20 days + two sites × 2 times/day × 12 days), 972 were collected in 2021 (four sites × 9 times/day × 27 days), and 936 were collected in 2022 (four sites × 9 times/day × 26 days). To determine the frequency (time interval and date interval) sufficient to replace the capture survey with an environmental DNA survey, samples were selected for analysis based on the results of the cap- ture survey. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Survey methods using environmental DNA A time of 15–30 min is appropriate because the samples were collected once every hour and a pump type was used instead of free-fall. The filtered sample (filter paper) was folded in half, with the filter side toward the interior; it was shielded from light using aluminum foil, placed in a small plastic bag, and stored in a frozen PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 6 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 2. Water sampling, developed in-vehicle unique filtration system, and storage of filter paper. https://doi.org/10.1371/journal.pone.0301197.g002 Fig 2. Water sampling, developed in-vehicle unique filtration system, and storage of filter paper. https://doi.org/10.1371/journal.pone.0301197.g002 Fig 2. Water sampling, developed in-vehicle unique filtration system, and storage of filter paper. https://doi.org/10.1371/journal.pone.0301197.g002 https://doi.org/10.1371/journal.pone.0301197.g002 state (below −20˚C) [92]. Specifically, the filter paper was stored in a portable freezer on site, and after one day of research was complete, it was stored in a stationary freezer indoors. The survey required more than 900 water samples over 29 days for analysis based on the results of the capture survey. Filtration performed as soon as possible after sampling and cold storage contribute to the longer retention of analyzable concentrations of environmental DNA, as the concentration of environmental DNA in water samples declines rapidly over time [93,94]. Environmental DNA was successfully stored at room temperature (20˚C) for 2 weeks [95–98]. However, the Niigata Prefecture, where the water was sampled, and the Kyoto Prefec- ture, where the analysis was performed, are approximately 500 km apart. Completing the transport before the rapid DNA degradation in all water samples was difficult; therefore, a unique filtration system was built into the car taken to the water sampling point [99]. On-site filtration and cold storage (−20˚C) enabled efficient collection of a large number of samples. Selection of sample for analysis. The water sampling frequency was adequately verified to ensure the development of environmental DNA surveys as an alternative to capture surveys. Catch surveys were conducted hourly from 9:00 to 17:00 during the survey period (June 6 to July 4) [76]. To improve the efficiency of the alternative survey, the sampling frequency (period and time) was determined based on the results of a trial conducted since 2019. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Survey methods using environmental DNA In this exhaustive analysis, samples for environmental DNA analysis were selected based on the results of fish catch surveys (the number of species and individuals confirmed at each time point), taking into consideration the migration status of P. altivelis and T. hakonensis repre- senting the ice-harbor-type fishway, Opsariichthys platypus representing the stair-type fishway, and Rhinogobius kurodai representing the rock-ramp-type fishway. Six specimens (two survey points at st1 and st2) were analyzed in 2019, the first year of the survey, which covered 3 days. 7 / 29 PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Twelve specimens (similarly, two survey points) were analyzed in the fiscal year 2020, the sec- ond year, which covered 3 days (11:00 and 15:00, respectively). To verify whether it is possible to understand the difference in the migration situation among the three types of fishways (ice- harbor-type, stair-type, and rock-ramp-type), the number of specimens obtained in 2021 and 2022 was 16 (four survey points for st1 and st2–4). The number of species and individuals of fish caught tended to increase in the afternoon. Furthermore, there was no clear difference between the two fish fauna analysis results at 11:00 and 15:00. Therefore, from 2021, fish fauna analysis was performed using the results at 15:00. Water sampling was still done hourly in case the trend of variety and population of fish caught changed. Specimens for analysis were selected based on the capture results. Based on the results of the advanced capture survey, the number of fish species was not expected to change significantly over time. Therefore, the results obtained at 15:00 each day, when the number of the confirmed types was high, were used as the sample. Samples were not collected immediately after a flood, when the water is often cloudy and DNA is washed out, because of the potential to induce false negatives. To further prevent false negatives, the water was not sampled during the spawning season of the dominant species P. altivelis, and the water sampling point was not a spawning site. Analysis of environmental DNA. The analysis procedure was performed according to Environmental DNA Sampling and Experiment Manual Version 2.2 (published April 3, 2020, environmental DNA Methods Standardization Committee). An outline of each analysis method is presented below [100]. DNA metabarcoding is a next-generation sequencing-based technique for the analysis of environmental samples. Table 1. Sequences of primers used for PCR. Primer name Sequence information MiFish U-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTAAAACTCGTGCCAGC MiFish E-v2-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNRGTTGGTAAATCTCGTGCCAGC MiFish U2-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGCCGGTAAAACTCGTGCCAGC Ayu-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTTAATCTCGTGCCAGC Yatsume-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTTAATCTCGTGCCAGC MiFish U-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNCATAGTGGGGTATCTAATCCCAGTTTG MiFish E-v2-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNGCATAGTGGGGTATCTAATCCTAG MiFish U2-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNCATAGGAGGGTGTCTAATCCCCGT Table 1. Sequences of primers used for PCR. Primer name Sequence information MiFish U-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTAAAACTCGTGCCAGC MiFish E-v2-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNRGTTGGTAAATCTCGTGCCAGC MiFish U2-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGCCGGTAAAACTCGTGCCAGC Ayu-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTTAATCTCGTGCCAGC Yatsume-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTTAATCTCGTGCCAGC MiFish U-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNCATAGTGGGGTATCTAATCCCAGTTTG MiFish E-v2-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNGCATAGTGGGGTATCTAATCCTAG MiFish U2-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNCATAGGAGGGTGTCTAATCCCCGT Table 1. Sequences of primers used for PCR. U is a primer suitable for fish in general, U2 is applicable to Pseudoblennius percoides Gu¨nther, and E-v2 is specific to cartilaginous fish. These primer sequences all refer to the Environmental DNA Sampling and Experiment Manual Version 2.2. Ayu is the primer specific for P. altivelis, and Yatsume is the primer specific to Lethenteron spp. Each primer was designed with reference to the sequence stored on the NCBI public DNA database (https://www.ncbi.nlm.nih.gov/genbank/). The forward primer (F) was designed to anneal to the 3’ end of the region to be amplified, while the reverse primer (R) was designed to anneal to the 5’ end of the region to be amplified. Note that the primer sequence of RNA synthesis proceeds in the 5’!3’ direction. https://doi.org/10.1371/journal.pone.0301197.t001 conditions for the 2nd PCR are as follows: one cycle at 95˚C for 180 s, 10–12 cycles at 98˚C for 20 s, 72˚C for 15 s, and one cycle at 72˚C for 300 s. MiFish metabarcoding uses 2-step PCR, with 35 cycles as the standard for the first PCR (1st PCR). However, if the fish are less active, it should be increased to a maximum of 40 cycles to recover a sufficient amount of DNA accord- ing to the Environmental DNA Sampling and Experiment Manual Version 2.2. No more than 40 cycles should be run, as excessive cycles can cause false positives. PCR cycles were carefully set to avoid inappropriate amplification. In this study, MiFish primers that specifically amplify fish DNA and a SimpliAmp thermal cycler (Thermo Fisher Scientific) were used. Table 1 lists the sequences of primers used for PCR. 2) NGS NGS was performed for the 2nd PCR product (library). The prepared libraries were sequenced on the MiSeq platform using the MiSeq Reagent v2 Kit (Illumina, CA, USA) for 2 × 150 bp PE or 2 × 250 bp PE following the manufacturer’s protocol. Sequence information and read counts for each operational taxonomic unit (OTU) were obtained by collating the DNA sequences into OTUs (sequence-based taxon units). An OTU was obtained by classifying the base sequences of multiple amplified DNA fragments using similarity as an index. Survey methods using environmental DNA It uses marker genes to characterize the species composition of whole communities [101,102]. During analysis to prevent false positives, the DNA extraction room was sufficiently spa- tially separated from PCR-related rooms in accordance with the manual. Two primers were designed to flank the gene region of interest. The MiFish primers [103] that can amplify the DNA of all Osteichthyes species and P. altivelis-specific and Lethenteron spp.-specific primers were mixed for the metabarcoding in fish fauna analysis. P. altivelis and Lethenteron spp. are difficult to detect using the MiFish method; therefore, specific primers were added to prevent false negatives. MiFish primers are a set of primers developed for fish fauna analysis; they amplify the DNA of all ichthyes. However, amplifying DNA from some species is difficult. Therefore, the basic set U, Pseudoblennius percoides Gu¨nther-specific U2, and Chon- drichthyes-specific E-v2 were mixed and used. DNA was eluted (56˚C, 30 min) from selected samples (filter paper with environmental DNA attached) using a buffer containing proteinase K. DNA was extracted and purified from the filter using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The extracted DNA was stored frozen at −30˚C until analysis. DNA was purified using a filter with selective affinity to DNA. Comprehensive DNA analysis. Metabarcoding (simultaneous detection of multiple species) to understand the fish fauna was carried out in three steps: 1) library creation, 2) next-genera- tion sequencing (NGS), and 3) data analysis (matching with database). The library was constructed using a two-step PCR: (1) 1st PCR to amplify the target DNA sequence, which was repeated eight times according to the Environmental DNA Sampling and Experiment Manual Version 2.2, and (2) 2nd PCR to add a tag (marker) that allows the target sequence to be identified during the sequencing. The reaction mix consisted of KAPA HiFi HS ready mix (6.0 μL), Primer Mix (MiFish U, U2, E v2)–F/R (2.0 μL), sample DNA (2.0 μL), and water (2.0 μL). The cycling conditions for the 1st PCR are as follows: one cycle at 95˚C for 180 s, 35 cycles at 98˚C for 20 s, 65˚C for 15 s, 72˚C for 15 s, and one cycle at 72˚C for 300 s. The 8 / 29 PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Environmental DNA in adaptive management methods in multiple fishways PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 3) Data analysis (matching with database) Candidate species names for each OTU were obtained by comparing the sequence of each OTU obtained from the NGS analysis with sequences in the MitoFish database (MitoFish http://mitofish.aori.u-tokyo.ac.jp/). The species names were determined according to the guid- ance for freshwater fish survey methods using environmental DNA analysis technology [104]. Specifically, it was determined based on the candidate species names of each OTU and the existing fish fauna around the Miyanaka Intake Dam. When multiple candidate species existed and could not be distinguished from the existing fish fauna, the possible species were listed together (e.g., Misgurnus anguillicaudatus and Paramisgurnus dabryanus) and treated as one species for convenience. Conditions for analysis. A quantitative comparison of the number of reads obtained using metabarcoding within the same sample for each fish was possible. However, a compari- son between samples was not possible because 1) the amount of PCR inhibitors varied between samples; 2) the dilution ratios at the time of analysis varied between samples; and 3) the num- ber of amplifications varied between the samples because the threshold would be reached for samples with high DNA concentrations before the predetermined number of amplifications. However, the water flowing into the three types of fishways in the Miyanaka Intake Dam is the same water from the dam reservoir upstream of the fishway exit (st1); therefore, the water quality was assumed to be the same. These problems were interpreted as follows in this study: 1) the concentrations of PCR inhibitors could be considered approximately equal; 2) the meta- barcoding results could be converted to the number of reads per unit volume based on the dilution ratio of each sample; and 3) differences in the number of amplifications were difficult to correct for. However, the differences were not large because the DNA concentrations in the samples were adjusted to a certain extent by the dilution during the analysis. Considering the conditions of the analysis in this light, the number of reads for each fish species obtained through metabarcoding was assumed to reflect the differences between the samples to some extent. Therefore, the metabarcoding results were converted to per unit volumes and used for comparison. Handling of data in comparison studies. The environmental DNA obtained at the st1 water sampling points was influenced by both the environmental DNA originating from the fish in the fishway and the environmental DNA flowing from upstream of the fishway. Table 1. Sequences of primers used for PCR. Primer name Sequence information MiFish U-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTAAAACTCGTGCCAGC MiFish E-v2-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNRGTTGGTAAATCTCGTGCCAGC MiFish U2-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGCCGGTAAAACTCGTGCCAGC Ayu-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTTAATCTCGTGCCAGC Yatsume-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGTCGGTTAATCTCGTGCCAGC MiFish U-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNCATAGTGGGGTATCTAATCCCAGTTTG MiFish E-v2-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNGCATAGTGGGGTATCTAATCCTAG MiFish U2-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNCATAGGAGGGTGTCTAATCCCCGT The name of the genus was identified by matching the base sequence of each OTU with those in the database. Processing was performed by paired-end sequencing. The target had an average length of 170 bp. All data preprocessing and analyses of MiSeq raw reads were performed using PMiFish version 2.4.1 (MIT license Copyright 2020 rogotoh, https://github.com/ rogotoh/PMiFish; Miya et al., 2015) and USEARCH v11 (Edgar 2010) based on the following steps. Both forward and reverse reads were merged using the fastq_mergepairs command. In this process, low-quality tail reads with a cutoff threshold set to a quality (Phred) score of 2, reads that were too short (<50 bp) after tail trimming, and paired reads with too many differ- ences (>5 positions) in the alignment region were discarded. The fastx_truncate command was used to remove primer sequences from merged reads, and the fastq_filter command was used to quality-filter reads without primer sequences, remove low-quality reads with expected error rates >1%, and remove reads that were too short (<50 bp). The preprocessed reads were dereplicated using the fastx_uniques command, and all singletons, doubletons, and tripletons PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 9 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways were removed from the subsequent analysis following the recommendation of Edgar (2010). The dereplicated reads were denoised using the unoise3 command, and all putatively chimeric and erroneous sequences were removed from the subsequent amplicon sequence variant (ASV) assignment. Finally, the usearch_global command was used to align all processed reads with sequences in a database prepared from GenBank, MitoFish (http://mitofishaoriu- tokyoacjp/), and an in-house database and then to assign sequences with >80% identity to fish sequences (Miya et al., 2015). PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 the final value represented the environmental DNA derived from fish in the fishway. When the difference was negative, it was treated as 0. the final value represented the environmental DNA derived from fish in the fishway. When the difference was negative, it was treated as 0. Method used for the fish catch survey Comparative fish catch surveys have been conducted annually since 2012, and the rock-ramp- type fishway was constructed in 2012. A capture survey was conducted for 29 days from June 6 to July 4 using the method described by Masumoto et al. [76], with a temporarily installed trap as shown in Fig 3 in the fishways. The Governor of Niigata Prefecture granted a “Special Cap- turing Permit” (permit number: special No. 31) necessary for conducting the survey. The per- mit consisted of taking all animals for a specified period of time and reporting the results. A temporary net cage caught all the fish at the upstream end of each fishway. Catch baskets set at 9:00 caught fish every hour from 10:00 to 17:00. A dam upstream of the fishway received all fish after their length was recorded and photographed. Fig 4 presents the results of the capture surveys. To continuously confirm that P. altivelis, which represents small fish with a relatively weak swimming ability, reached upstream via the fishway, surveys continued to be carried out in June, during their migration season. Moreover, in order to verify the appropriate operation of the dam and fishway, their upstream status must be confirmed in the future, mainly in June. 3) Data analysis (matching with database) A com- bination of cell sedimentation, turbulence, and dilution effects showed that detectable levels of environmental DNA extended by a distance of 239.5 m regardless of the current [83]. The length of the ice-harbor-type fishway and stair-type fishway of the Miyanaka Intake Dam is approximately 210 m, the length of the stair-type fishway is approximately 240 m, and the length of the rock-ramp-type fishway is 260 m. Therefore, the environmental DNA at St2–4 in the large and small fishways was not assumed to reflect the presence of fish in the fishway. To understand the contribution of environmental DNA derived from fish that migrate up the fishway in this study, the amount of environmental DNA at the upstream point (st1) was sub- tracted from the amount of environmental DNA (number of reads) at each point (st2–4), and PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 10 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 3. Position and state of the temporary trap for catching fish. Three traps were temporarily installed at the upstream ends of all fishways for one month each. Fish were caught in underwater net cages. The blue arrow indicates the flow direction. https://doi.org/10.1371/journal.pone.0301197.g003 of the temporary trap for catching fish. Three traps were temporarily installed at the upstream ends of all fishways for one ught in underwater net cages. The blue arrow indicates the flow direction. Fig 3. Position and state of the temporary trap for catching fish. Three traps were temporarily installed at the upstream e month each. Fish were caught in underwater net cages. The blue arrow indicates the flow direction. Fig 3. Position and state of the temporary trap for catching fish. Three traps were temporarily installed at the upstream ends of all fishways for one month each. Fish were caught in underwater net cages. The blue arrow indicates the flow direction. https://doi.org/10.1371/journal.pone.0301197.g003 https://doi.org/10.1371/journal.pone.0301197.g003 https://doi.org/10.1371/journal.pone.0301197.g003 the final value represented the environmental DNA derived from fish in the fishway. When the difference was negative, it was treated as 0. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Narrowing down of samples for analysis In this study, we considered reducing the frequency of water sampling to ensure continuous and efficient long-term monitoring of the fish fauna. For this reason, water was sampled at the same frequency as in the capture survey, although the samples to be analyzed were narrowed 11 / 29 PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 4. Results obtained from catch surveys in fish passages (number of species, number of P. altivelis populations, and other populations). https://doi.org/10.1371/journal.pone.0301197.g004 Fig 4. Results obtained from catch surveys in fish passages (number of species, number of P. altivelis populations, and other populations). https://doi org/10 1371/journal pone 0301197 g004 Fig 4. Results obtained from catch surveys in fish passages (number of species, number of P. altivelis populations, and other populations). https://doi.org/10.1371/journal.pone.0301197.g004 https://doi.org/10.1371/journal.pone.0301197.g004 down based on the following considerations. The estimated results were evaluated by analyz- ing the samples at 15:00 on the target day (one sample per day), which was either a day when the number and species of fish migrating upstream on ice-harbor-type, stair-type, and rock- ramp-type fishways were high; a day with a large population of fish representative of each fish- way (ice-harbor-type fishway: P. altivelis, T. hakonensis, stair-type fishway: O. platypus, and rock-ramp-type fishway: R. kurodai); or a day during the time period when the number of spe- cies and individuals was large on the days selected above. Rarely identified fish that did not affect the results of environmental DNA fish fauna analysis were not considered in the selec- tion of days for analysis. Environmental DNA surveys record fish species that inhabit the area because environmen- tal DNA is measured in a range that includes the upstream side of the survey point for a short time. The DNA detection rate was negatively correlated with time, and the probability of unde- tectable DNA after 17 days was greater than 95% [105]. Studies have indicated that the effec- tiveness of biomonitoring using environmental DNA analysis requires preliminary verification of the DNA detection rate in an environment close to nature before conducting the analysis [106]. Therefore, we verified the period of time in which the number of confirma- tions by environmental DNA at a given time reflected the situation. verification of the DNA detection rate in an environment close to nature before conducting the analysis [106]. Comparison of the metabarcoding and capture survey results Table 2 shows the results of the metabarcoding from 2019 to 2022. The number of days of cap- ture surveys and the number of specimens analyzed differed with the year of the survey; how- ever, the number of species recorded in 2022 was the largest. The species observed in all samples also remained the same, and Squalidus biwae was newly recorded in all samples in 2022. S. biwae was also frequently caught in the fishway catch survey carried out in 2022. Therefore, this result was considered to reflect an increase in S. biwae population. Fig 5 shows the results for two common locations (st1 and st2) from 2019 to 2022. The number of species confirmed by both the environmental DNA survey and the capture survey was 16–24. When including the number of species identified only by environmental DNA sur- vey, the number of species was 19–35. Thus, the proportion of species confirmed by both envi- ronmental DNA survey and capture survey was 72.4% (65.7–84.2%) of the number of species confirmed using the environmental DNA survey (df = 6, t = 2.447, t<0.05). Species such as Anguillidae (one individual), Cyprinus carpio (one individual), Carassius cuvieri (zero individ- uals), Lefua echigonia (zero individuals), and Gymnogobius urotaenia (one individual) were identified only in the environmental DNA analysis; they were rare in the 4-year catch survey. However, species such as Nipponocypris temminckii (three individuals) and Sarcocheilichthys variegatus microoculus (two individuals) were confirmed only in the capture survey. A factor contributing to this false negative value was the low number of these fish that were caught. A total of 37 species were identified in the fish catch surveys from 2012 to 2022, which was approximately the same as that recorded using environmental DNA surveys (Fig 6). Among the 37 species of fish identified using environmental DNA, C. cuvieri was the only fish species that had never been caught. Narrowing down of samples for analysis Therefore, we verified the period of time in which the number of confirma- tions by environmental DNA at a given time reflected the situation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 12 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways In the fishway fish catch survey, the species and number of fish caught in temporary traps at the upstream end of the fishway were investigated on an hourly basis. Therefore, the number of fish species recorded using environmental DNA may reflect the fish species upstream of the dam at a certain time. However, the number of fish species obtained from catch surveys reflects the situation in the fishway over a short period. Survey results for each fishway Effectiveness of evaluating fishway usage status through environmental DNA survey. The composition of fish in each fishway, as indicated by the number of environmental DNA reads, differed with the survey year and date. However, two species, M. anguillicaudatus and R. kurodai, dominated the rock-ramp-type fishway; three species, O. platypus, T. hakonensis, and P. altivelis, dominated the ice-harbor-type fishway; and two species, O. platypus and P. altivelis, dominated the stair-type fishway. Table 2. Number of species detected by metabarcoding and captured by survey from 2019 to 2022. Year Specimens Number of species detected by eDNA Captured species Fish confirmed in all samples 2019 6 20 21 4 2020 12 35 21 18 2021 16 31 (31) 24 14 2022 16 36 (32) 22 15 The numbers shown in parentheses (2021: 31 species, 2022: 32 species) are the number of species limited to st1 and st2 to match the conditions for 2019 and 2020. Table 2. Number of species detected by metabarcoding and captured by survey from 2019 to 2022. Year Specimens Number of species detected by eDNA Captured species Fish confirmed in all samples 2019 6 20 21 4 2020 12 35 21 18 2021 16 31 (31) 24 14 2022 16 36 (32) 22 15 The numbers shown in parentheses (2021: 31 species, 2022: 32 species) are the number of species limited to st1 and st2 to match the conditions for 2019 and 2020. Table 2. Number of species detected by metabarcoding and captured by survey from 2019 to 2022. species, 2022: 32 species) are the number of species limited to st1 and st2 to match the conditions for 2019 and 2020. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 13 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 5. Comparison of fish catch survey results and environmental DNA analysis results (2019–2022). https //doi org/10 1371/jo rnal pone 0301197 g005 Fig 5. Comparison of fish catch survey results and environmental DNA analysis results (2019–2022). https://doi.org/10.1371/journal.pone.0301197.g005 In addition, the numbers of Pelteobagrus nudiceps and S. biwae, which ranked high in the stair-type fishway in 2022, increased significantly since 2021 (P. nudiceps increased from 9 individuals in 2021 to 125 individuals in 2022; S. biwae increased from 1 individual in 2021 to 122 individuals in 2022). The results of these environmental DNA surveys revealed a signifi- cant change in the number of individuals that migrated upstream. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Survey results for each fishway Effectiveness of understanding fishway usage status through environmental DNA sur- vey. Based on results of the catch surveys conducted in 2021 and 2022, the fish identified in the environmental DNA survey were grouped according to the fish representative of each fish- way and their utilization. In Case 1, the percentage of read numbers for each fish species was examined. Fish that were predominantly caught in each fish passage, such as O. platypus, T. hakonensis, P. altivelis, Cobitis biwae, and R. kurodai, were distinguished as individual indica- tors. Fish other than these five species were prevalent in the ice-harbor-type, stair-type, and rock-ramp-type fishways. They were classified into groups 1 to 4 (Group 1: Fish species in which many individuals mainly use the ice-harbor-type fishway; Group 2: Fish species that mainly used the ice-harbor-type and stair-type fishways; Group 3: Fish species in which many individuals mainly used the rock-ramp-type fishway; Group 4: Other fish species with no clear difference in usage by fishway type). The ratio of DNA amount represented the average value for 4 days obtained through metabarcoding, and the ratio of capture survey results was calcu- lated from the total number of captures during the survey period (29 days). In Case 2, the fish of Group 2 and Group 3 were divided by lifestyle type (middle-level swimming type and bottom-dwelling type). In Group 2, Squalidus chankaensis biwae, Sarco- cheilichthys variegatus microoculus, and O. masou were classified as middle-level swimming PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 14 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 6. Comparison of the number of fish species confirmed using environmental DNA surveys and capture surveys. Environmental DNA survey results are the total for 2021 and 2022, and capture survey results are the total for 2012–2022. One species that was confirmed only through the environmental DNA survey is C. cuvieri, and the three species confirmed only through the capture survey are Tanakia lanceolata (past one individual), Rhodeus ocellatus (past five individuals), and Tribolodon nakamurai (past two individuals). The numbers of all of these fish were too small for confirmation. Fig 6. Comparison of the number of fish species confirmed using environmental DNA surveys and capture surveys. Environmental DNA survey results are the total for 2021 and 2022, and capture survey results are the total for 2012–2022. One species that was confirmed only through the environmental DNA survey is C. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 https://doi.org/10.1371/journal.pone.0301197.g006 Division of analysis period to reflect capture results The period during which the capture survey results were reflected in the environmental DNA results was identified because water sampling for environmental DNA and trap surveys was conducted hourly. To efficiently monitor the fish fauna over time and facilitate comparison with previous surveys, improvements were sought to enhance long-term survey efficiency. Evaluating the possibility of transitioning from a schedule of 30 days × 8 times/day to several days × 1 time/day, it was decided to solely use the samples collected at 15:00 as the environ- mental DNA sample. First, the results of the capture survey were divided into four survey peri- ods: (1) that time, (2) that day, (3) 1 week before and after, and (4) 15 days before and after (targeting eight survey time slots: June 17, 21, 28, and 30 in 2021 and June 20, 22, 30, and July 2 in 2022, at 15:00 on every day). The relationship between the number of species confirmed by environmental DNA at 15:00 and the number of species captured at the four times shown above was arranged for each fishway. Fig 8 shows the results of verifying the natural state indi- cated by the environmental DNA at a specific time. As a result of verifying four periods (that time, that day, 1 week before and after (±3 days), and 15 days before and after (±7 days)) and the number of fish species caught in three fishways, the correlation coefficient (r) and decision coefficient (R2) were large on that day (r = -0.587, R2 = 0.345, p <0.05) and 1 week before and after (±3 days) (r = -0.442, R2 = 0.196, p<0.05). Furthermore, when the sampling window was increased to 15 days before and after (±7 days), the r and R2 values became smaller (r = -0.277, R2 = 0.077, and p = 0.11). To investigate this factor, the correlation coefficient was verified in each fishway. The r values between the ice-harbor-type fishway and the stair-type fishway showed the same trend as those for the whole fishway (r>0.74, R2>0.54, p<0.05), while the r values of the rock-ramp-type fishway showed a tendency different from those of the whole fishway (r<0.5, R2<0.25, p<0.05). P. altivelis, T. hakonensis, and O. platypus were mainly caught owing to the fast current and deep water of the ice-harbor-type fishway and stair-type fishway. However, C. biwae and R. Survey results for each fishway Thus, the differences between environmen- tal DNA surveys and capture surveys may have reflected the behavior of these fish. Fig 7(b) shows the results for Case 2. A clear difference was not observed in the proportion of environmental DNA reads (metabarcoding method), even when separated into middle- level swimming type and bottom-dwelling type, because the composition ratio of Group 2 and Group 3 was originally small in the ice-harbor-type fishway and stair-type fishway. In Group 3, the bottom-dwelling type was approximately twice as common as the middle-level swim- ming type in the rock-ramp-type fishway in 2021. In addition, the bottom-dwelling type accounted for the majority in both Group 2 and Group 3 in 2022. The results of environmental DNA surveys captured such changes in the flow regime of the fishway. Therefore, identification of fish species in each fishway was possible by evaluating the read numbers of the major fish and the other groups of fish in the environmental DNA survey in each fishway. Survey results for each fishway cuvieri, and the three species confirmed only through the capture survey are Tanakia lanceolata (past one individual), Rhodeus ocellatus (past five individuals), and Tribolodon nakamurai (past two individuals). The numbers of all of these fish were too small for confirmation https://doi.org/10.1371/journal.pone.0301197.g006 https://doi.org/10.1371/journal.pone.0301197.g006 types and Cottus pollux, Pseudogobio esocinus esocinus, and Pseudobagrus nudiceps were classi- fied as bottom-dwelling types. In Group 3, Rhynchocypris lagowskii steindachneri, Rhodeus ocellatus ocellatus, Pseudorasbora parva, and Gnathopogon elongatus elongatus were classified as middle-level swimming types and Liobagrus reini, Lethenteron reissneri, Misgurnus anguilli- caudatus, Lefua echigonia, and Gymnogobius urotaenia were classified as bottom-dwelling types. Furthermore, there was no bottom-dwelling type in Group 1, where many individuals mainly used the large fishway. Fig 7(a) shows a comparison of the results of the environmental DNA survey and capture survey for each fishway. The fish fauna determined from the percentage of environmental DNA reads for each fishway was similar to the results of the capture survey. T. hakonensis, P. altivelis, and Group 1 fish constituted the main species in the ice-harbor-type fishway. O. platy- pus, P. altivelis, and Group 1 fish were the main species recorded in the stair-type fishways. In contrast, the ratios of R. kurodai, C. biwae, and Group 3 fish were high in the 2021 rock-ramp- type fishway. In addition, the number of swimming fish, such as O. platypus and Group 2 fish, increased in 2022. The discharge from the rock-ramp-type fishway during the 2022 catch sur- vey was higher than that in 2021 owing to the operation of the fishway; many swimming fish were confirmed owing to this. Among the fish that migrated upstream through the rock- ramp-type fishway, bottom-dwelling type fish take time to reach the upstream end of the fish- way. Therefore, their behavior was different from that of fish that migrated upstream through ramp-type fishway, bottom-dwelling type fish take time to reach the upstream end of the fish- way. Therefore, their behavior was different from that of fish that migrated upstream through PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 15 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 7. Comparison of environmental DNA survey results and capture survey results for each fishway. (a) Case 1 and (b) Case 2. https://doi.org/10.1371/journal.pone.0301197.g007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 16 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways the ice-harbor-type fishway and stair-type fishway. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Division of analysis period to reflect capture results kurodai were mainly caught owing to the slow current and shallow depth of the rock-ramp-type fishway. Vegetation studies are being conducted on the rock-ramp-type fishway of the Miyanaka Intake Dam to enhance fish migration and habitat [107]. Therefore, C. biwae and R. kurodai migrate slowly and inhabit the rock-ramp-type fish- way; accordingly, the results for the other two fishways are different [75]. As the number of species actually caught increased, many of them included fish with extremely low population numbers. Negative correlations occurred because of increased types not detected in environ- mental DNA (false negatives). Therefore, we focused on the five species of fish typically caught in the fishway of the Miyanaka Intake Dam and continued our verification. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 17 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 8. Verification of natural conditions indicated by environmental DNA at a specific time targeting four periods and number of fish species caught in each fishway. (Ice: ice-harbor-type fishway; Stair: stair-type fishway; and Rock: rock-ramp-type fishway). https://doi.org/10.1371/journal.pone.0301197.g008 Fig 8. Verification of natural conditions indicated by environmental DNA at a specific time targeting four periods and number of fish species caught in each fishway. (Ice: ice-harbor-type fishway; Stair: stair-type fishway; and Rock: rock-ramp-type fishway). https://doi.org/10.1371/journal.pone.0301197.g008 Subsequently, a more detailed analysis was performed. The most frequently caught species, O. platypus, T. hakonensis, P. altivelis, C. biwae, and R. kurodai, were sorted. Fig 9 shows the correlation between the number of fish and environmental DNA reads for each survey period segment. Most O. platypus (approximately 82%) individuals used the low-flow stair-type fish- ways, and approximately 16% utilized the high-flow ice-harbor-type fishways. O. platypus has a short body length and low charging speed. Therefore, it is possible that a large amount of environmental DNA from O. platypus remained in the fishway because it required a long time to swim through the fishway. O. platypus was confirmed using environmental DNA in all sur- vey period divisions; it was used as an indicator. Therefore, there was a significant correlation (r>0.4, especially a strong correlation for 15 days before and after; r>0.7, p<0.05, for 1 week before and after) in all survey periods. Most (approximately 70%) of the T. hakonensis individ- uals utilized ice-harbor-type fishways with high flow, and approximately 29% utilized stair- type fishways with low flow. Many T. hakonensis individuals migrated in groups, and large T. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Division of analysis period to reflect capture results hakonensis individuals were strongly motivated to migrate. These factors contribute to short- term mass migration; this tendency reduces the retention of T. hakonensis environmental DNA in the fishway during non-migratory periods. Therefore, T. hakonensis, used as an indi- cator, was not recorded in the environmental DNA survey during four of the eight survey peri- ods. Therefore, there was a weak correlation (r<0.4, p>0.05) in all survey period divisions. Most of the P. altivelis (approximately 63%) individuals used the ice-harbor-type fishways with high flow, and approximately 37% utilized stair-type fishways with low flow. The body length of P. altivelis was approximately 0.1 m, and all those that migrated upstream were juveniles in search of a habitat to establish a territory. Therefore, the number of captured P. altivelis indi- viduals was large, indicating that their motivation to move upstream was high. However, few PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 18 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Fig 9. Verification of natural conditions indicated by environmental DNA at a specific time targeting four periods and the five main types of fish caught. Fig 9. Verification of natural conditions indicated by environmental DNA at a specific time targeting four periods and the five main types of fish caught. https://doi.org/10.1371/journal.pone.0301197.g009 https://doi.org/10.1371/journal.pone.0301197.g009 P. altivelis individuals (1%) were caught in the rock-ramp-type fishways with lower flows. P. altivelis was confirmed using environmental DNA analysis during all eight index sampling periods. Therefore, there was a significant correlation (r>0.4, p>0.05) in the survey period divisions in addition to that during the full survey period. C. biwae, a bottom-dwelling fish, was caught in rock-ramp-type fishways with low flow and still water. C. biwae was confirmed using environmental DNA analysis during all eight index survey periods. The total number of catches was as low as 76 individuals; thus, there was a weak correlation (0.4>r>0.2, p<0.05) in all survey period segments. R. kurodai not only migrated using the rock-ramp-type fishways but also lived in these fishways [72], and 637 individuals were captured. R. kurodai was con- firmed using environmental DNA at all index times, indicating a significant correlation (r>0.4, especially r>0.7, for 1 week before and after, p<0.05) in all survey period segments. P. altivelis individuals (1%) were caught in the rock-ramp-type fishways with lower flows. P. altivelis was confirmed using environmental DNA analysis during all eight index sampling periods. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Discussion To restore the continuity of movement for river fish hampered by dams and weirs, adaptive management of fishways is emphasized worldwide. Adaptive management was first described in 1976 under the term “adaptive control” [108]. Originally used in areas related to fisheries resource management, it has also been used in forest and river ecosystem management in Aus- tralia and North America [109,110]. The long-term sustainability of natural ecosystems and ecological links is of paramount importance. In scenarios where scientific forecasts are unreli- able, adaptive management strategies are employed, aiming to mitigate various natural uncer- tainties by reducing them over time [111]. To navigate these uncertainties, the integration of adaptive learning and feedback mechanisms is crucial [112]. Adaptive learning consists of active investigation to obtain information efficiently and passive investigation to obtain infor- mation from normal investigation [113]. These management techniques, which carefully monitor nature’s response to projects and subsequently integrate these observations into project plans, are combined with traditional Japanese knowledge and techniques [114,115]. Management to ensure biodiversity is also underway in Japan, with the enactment of the Nature Restoration Promotion Act (a law enacted by legislators in December 2002). The fishway at the Miyanaka Intake Dam has been appropriately maintained as a civil engineering structure. However, management that focuses on fish in consideration of biodiversity was not implemented. Following the completion of fishway improvements in 2012, a five-year monitoring survey up to 2016 showed that the improvements to the fishway structure were having the desired effect. However, because the habitat and migratory environment of fish may change in the future, monitoring to under- stand the status of fish migration is ongoing. Continuous monitoring verified that the fishway was functioning as intended and was effective. To adapt without changing the goal [116], it is crucial to develop new hypotheses and implement corresponding measures. The process of repeating this series of processes through trial and error is part of the ongoing adaptive man- agement strategy being implemented at the fishway at the Miyanaka Intake Dam. A rock-ramp-type fishway was installed at the Miyanaka Intake Dam in 2012, and adaptive management has continued since then, which involved fish catch surveys for monitoring [117]. The burden of this method on the fish and facility managers is heavy; therefore, a survey method utilizing environmental DNA, established in recent years, was adopted. Division of analysis period to reflect capture results Therefore, there was a significant correlation (r>0.4, p>0.05) in the survey period divisions in addition to that during the full survey period. C. biwae, a bottom-dwelling fish, was caught in rock-ramp-type fishways with low flow and still water. C. biwae was confirmed using environmental DNA analysis during all eight index survey periods. The total number of catches was as low as 76 individuals; thus, there was a weak correlation (0.4>r>0.2, p<0.05) in all survey period segments. R. kurodai not only migrated using the rock-ramp-type fishways but also lived in these fishways [72], and 637 individuals were captured. R. kurodai was con- firmed using environmental DNA at all index times, indicating a significant correlation (r>0.4, especially r>0.7, for 1 week before and after, p<0.05) in all survey period segments. The results of the capture surveys 1 week before and after and 15 days before and after were well reflected in the environmental DNA results. The ice-harbor-type fishway had a high dis- charge of approximately 1.637 m3/s and a high velocity of 1.27–2.43 m/s. This suggests that the environmental DNA is probably diluted depending on the size of the fish species and speed of migration. The stair-type fishway had a flow rate of 0.133 m3/s and a flow velocity of 0.87–1.05 m/s; therefore, fish with a strong swimming ability and bottom-dwelling fish do not use this fishway. The environmental DNA and capture survey results were relatively consistent because fish with a low swimming ability did not stay and took an appropriate amount of time to swim upstream. The rock-ramp-type fishway has a low flow rate (0.071 m3/s) and low velocity (0.64 m/s). Small fish with a weak swimming ability took time to swim upstream. Many bottom- dwelling fish move slowly or live alone. The continuous release of environmental DNA from these fish suggests that the results of the environmental DNA and capture surveys did not PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 19 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways match depending on the type of fish. C. biwae was caught entirely from the rock-ramp-type fishways, and R. kurodai was partially caught from the ice-harbor- and stair-type fishways. Some species of R. kurodai migrate between the sea and upstream rivers; therefore, more migrating individuals were observed for R. kurodai than for C. biwae. This could explain the capture of a larger number of R. kurodai. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Discussion Environmen- tal DNA studies have limitations, one of which is the survival time of the samples. Considering the challenges in sample transportation while ensuring the preservation of samples, we devel- oped an on-site filtration system and a mechanism for freezing and preserving the samples at −20˚C. Environmental DNA surveys (metabarcoding) conducted in 2021 and 2022 identified 30 and 31 species of fish, respectively, of which 24 and 22 species of fish were identified in common from the capture surveys, respectively, based on the sum of those from the three fish- ways (ice-harbor-type, stair-type, and rock-ramp-type). A comparison of the results of 2021 and 2022 revealed an increasing trend in the environmental DNA results for P. nudiceps and S. biwae, which increased significantly in the capture survey. Therefore, when the number of individuals that migrated upstream increased, the change was reflected in the environmental DNA survey. Determining the fishway usage status of each fish species in each fishway is PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 20 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways possible by calculating the ratio of the number of environmental DNA reads of the main fish in each fishway and the total fish classified into groups. A comparison of the results of previous environmental DNA surveys with fish catch surveys showed that the number of species recorded using the environmental DNA surveys tended to be higher than that recorded through fish catches during the same survey period. This discrep- ancy can arise if only a small number of fish inhabit a specific area, with their detection influ- enced by factors, such as the flow rate of the river, strength of the current, and behavior of the fish. This pattern is consistent with observations made in numerous studies across various communities and ecosystems [118,119]. The fish recorded at the fishway exit (upstream end of the fishway) through the catch survey were those that had migrated up the fishway. In contrast, the fish confirmed through the environmental DNA survey may have lived upstream of the Miyanaka Intake Dam or potentially migrated upstream along the fishway. Therefore, a com- parative study with the results of surveys conducted over the past few years is necessary. Discussion This method used for understanding and monitoring biodiversity presents certain limita- tions and challenges, such as incomplete recovery of species with very low abundance [120], reliance on methods that need to be further developed in terms of technological advancement and standardization of procedures [121], and issues with abundance quantification, taxonomic assignment, and environmental DNA spatial and temporal dynamics [122]; thus, further opti- mization is required [123,124]. The results of the catch survey in the fishway included only the types of fish that temporarily reached the trap installed at the upstream end of the fishway. The environmental DNA survey uses the difference between the fishway entrance (downstream) and exit (upstream); therefore, only the types of fish remaining in the fishway were included. However, it is assumed that the samples collected for environmental DNA analysis contain the environmental DNA of fish that lived upstream, in addition to the fish that stayed in the fish- way. Therefore, we aimed to confirm whether the frequency of water sampling and the results of the environmental DNA analysis reflected the actual situation. Environmental DNA is a widespread survey method used to understand the status of fish habitat distribution and reduce the load on fish, and it was used in this study to evaluate the adaptive management of the fishways in the Miyanaka Intake Dam. Environmental DNA anal- ysis was incorporated into the plan as a research method. The surveys in this study are still in the experimental stage of determining the number of fish that have migrated upstream. How- ever, by determining the types of fish using environmental DNA over a long period, it is possi- ble to continuously confirm the presence or absence of changes in fish fauna. The appropriate frequency of water sampling and analysis is 1 week before and after or 15 days before and after. While continuing the survey, which mainly analyzed environmental DNA, the applica- bility of the analysis was verified by capture surveys conducted every 5 years. This research presented certain limitations. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 Discussion With river flows being continuous and the life history of fish varying, these considerations are cru- cial for future research on fish descending downstream from dams [127]. Therefore, identify- ing the target fish and the optimal timing for surveys is essential. To compare the environmental DNA survey results with the fish capture survey results, the samples in this study were collected at times when the number of captured individuals and species was high based on the results of the capture survey. Environmental DNA analysis was also conducted on samples taken at times when many representative species, such as P. altive- lis, T. hakonensis, O. platypus, C. pollux, and R. kurodai, were captured. Normally, the number of species and populations of fish cannot be determined unless a capture survey is conducted. Therefore, future challenges lie in clarifying the frequency and time of water sampling that will enable both improved accuracy of environmental DNA survey results and improved efficiency of long-term monitoring by analyzing the relationship among various environmental factors, including water temperature and fish migration timing. In addition, given the special situation of monitoring within a fishway rather than within a river, water sampling locations within the fishway should be identified that can more accurately reproduce the local situation. The impact of fish caught in net cages must also be examined to resolve this issue by collect- ing water samples, setting times when net cages are not set up, or setting specific days during the survey to capture fish. To compare the results of environmental DNA surveys and fish cap- ture surveys and verify the differences, careful and continuous research must be conducted over a certain number of years. Conclusion We verified whether environmental DNA analysis could be used as a substitute for traditional surveying methods by performing a comparison with the capture survey method using five representative species (O. platypus, T. hakonensis, P. altivelis, C. biwae, and R. kurodai) around the Miyanaka Intake Dam. The results of environmental DNA analysis for 4 years (2019– 2022) and capture surveys for 11 years (2012–2022) were compared. The substitutability of environmental DNA analysis was confirmed based on a comparison period of 1 week to 15 days and according to its tolerance of certain errors, and the proposed method achieved both reduction in the burden on fish and facility managers and a semi-permanent adaptive manage- ment strategy for fishways. Thus, we developed a survey method using a new environmental DNA analysis method for fish passages far from the analysis point. However, environmental DNA research methods are continuously evolving, and trial-and-error methods are applied worldwide. Therefore, we will continue to confirm the applicability of this survey method, which mainly analyzes environ- mental DNA based on capture surveys and findings obtained every 5 years. Acknowledgments We thank Editage (www.editage.com) for their assistance with English language editing. Discussion For example, the water used for analysis in the environmental DNA study was sampled at hourly intervals at the downstream end of the fish- way; therefore, the water samples may have contained environmental DNA originating from the following situations: fish living upstream of the Miyanaka Intake Dam, fish staying in the fishway without reaching the net cage at the upstream end of the fishway, and fish caught in net cages. Environmental DNA research methods differ from those used to confirm the biology of fish, and environmental DNA survey results cannot specifically reveal the behavior of the fish, such as whether identified fish were individuals that previously migrated upstream through the fish- way, individuals that are currently migrating upstream, or individuals that reside in the fishway. In this study, the survey compared with the environmental DNA survey targeted fish migrating upstream through the fishway. The hole of the trap for catching fish faced down- stream. Considering the viewpoint of the fish, the water sampling point on the downstream side where fish enter the fishway was determined to be the entrance point of the fishway. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 21 / 29 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways However, from a river-based perspective, where environmental DNA flows from upstream, the upstream side of the fishway may be considered as the entry point of the fishway. No sur- vey method observed fish moving down the adjacent fishway when maintenance flow is being released from the spillway gate. Owing to the increased concentration of environmental DNA after spawning [125,126], environmental DNA perturbs the detected fish population. With river flows being continuous and the life history of fish varying, these considerations are cru- cial for future research on fish descending downstream from dams [127]. Therefore, identify- ing the target fish and the optimal timing for surveys is essential. However, from a river-based perspective, where environmental DNA flows from upstream, the upstream side of the fishway may be considered as the entry point of the fishway. No sur- vey method observed fish moving down the adjacent fishway when maintenance flow is being released from the spillway gate. Owing to the increased concentration of environmental DNA after spawning [125,126], environmental DNA perturbs the detected fish population. Author Contributions Conceptualization: Masahiko Nakai, Taku Masumoto, Takashi Asaeda. 22 / 29 PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 PLOS ONE Environmental DNA in adaptive management methods in multiple fishways Data curation: Taku Masumoto, Takashi Asaeda, Mizanur Rahman. Data curation: Taku Masumoto, Takashi Asaeda, Mizanur Rahman. Formal analysis: Masahiko Nakai, Taku Masumoto, Takashi Asaeda. Funding acquisition: Masahiko Nakai, Taku Masumoto. Investigation: Masahiko Nakai, Taku Masumoto, Takashi Asaeda. Methodology: Masahiko Nakai, Taku Masumoto, Takashi Asaeda, Mizanur Rahman. Project administration: Masahiko Nakai, Taku Masumoto. Resources: Taku Masumoto. Software: Taku Masumoto. Supervision: Masahiko Nakai. Validation: Masahiko Nakai, Taku Masumoto, Takashi Asaeda, Mizanur Rahman. Validation: Masahiko Nakai, Taku Masumoto, Takashi Asaeda, Mizanur Rahman. Visualization: Taku Masumoto, Takashi Asaeda, Mizanur Rahman. Writing – original draft: Masahiko Nakai. Writing – review & editing: Masahiko Nakai, Taku Masumoto, Takashi Asaeda, Mizanur Rahman. PLOS ONE \| https://doi.org/10.1371/journal.pone.0301197 April 1, 2024 References He F, Zarfl C, Bremerich V, Henshaw A, Darwall W, Tockner K, et al. Disappearing giants: A review of threats to freshwater megafauna. Wiley Interdiscip Rev Water. 2017; 4(3): e1208. 15. 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https://openalex.org/W2126950311	https://repository.helmholtz-hzi.de/bitstream/10033/558827/1/Xiong%20et%20al_final.pdf	English	null	The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions	Open biology	2,015	cc-by	14,252	The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy- dependent and -independent functions. Item Type Article Authors Xiong, Qiuhong; Ünal, Can; Matthias, Jan; Steinert, Michael; Eichinger, Ludwig Citation The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions. 2015, 5 (4):150008 Open Biol DOI 10.1098/rsob.150008 Journal Open biology Download date 24/10/2024 05:25:00 Link to Item http://hdl.handle.net/10033/558827 This is an Open Access-journal’s PDF published in Xiong, Q., Ünal, C., Matthias, J., Steinert, M., Eichinger, L. The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions. (2015) Open Biology; 5 (4); art.-no: 150008. This is an Open Access-journal’s PDF published in Xiong, Q., Ünal, C., Matthias, J., Steinert, M., Eichinger, L. The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions. (2015) Open Biology; 5 (4); art.-no: 150008. on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from Qiuhong Xiong1, Can U¨nal2,3, Jan Matthias1, Michael Steinert2,4 and Ludwig Eichinger1 Author for correspondence: Ludwig Eichinger e-mail: ludwig.eichinger@uni-koeln.de Author for correspondence: Ludwig Eichinger e-mail: ludwig.eichinger@uni-koeln.de Research Cite this article: Xiong Q, U¨nal C, Matthias J, Steinert M, Eichinger L. 2015 The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions. Open Biol. 5: 150008. http://dx.doi.org/10.1098/rsob.150008 1Zentrum fu¨r Biochemie, Medizinische Fakulta¨t, Universita¨t zu Ko¨ln, Joseph-Stelzmann-Strasse 52, Ko¨ln 50931, Germany 2Institut fu¨r Mikrobiologie, Technische Universita¨t Braunschweig, Spielmannstrasse 7, Braunschweig 38106, Germany 3Fen Faku¨ltesi, Tu¨rk-Alman-U¨niversitesi, Istanbul 34820, Turkey 4Helmholtz Centre for Infection Research, Braunschweig 38124, Germany 3Fen Faku¨ltesi, Tu¨rk-Alman-U¨niversitesi, Istanbul 34820, Turkey 4Helmholtz Centre for Infection Research, Braunschweig 38124, Germany 4Helmholtz Centre for Infection Research, Braunschweig 38124, Germany The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions Keywords: Dictyostelium, autophagy, development, phagocytosis, proteasome, protein aggregate Dictyostelium, autophagy, development, phagocytosis, proteasome, protein aggregate rsob.royalsocietypublishing.org Research Cite this article: Xiong Q, U¨nal C, Matthias J, Steinert M, Eichinger L. 2015 The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions. Open Biol. 5: 150008. http://dx.doi.org/10.1098/rsob.150008 1. Summary Received: 13 January 2015 Accepted: 26 March 2015 Subject Area: biochemistry/cellular biology/ molecular biology/microbiology Keywords: Dictyostelium, autophagy, development, phagocytosis, proteasome, protein aggregate Author for correspondence: Ludwig Eichinger e-mail: ludwig.eichinger@uni-koeln.de Electronic supplementary material is available at http://dx.doi.org/10.1098/rsob.150008. Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG162 and ATG92/162 cells and compare them to the previously reported ATG92 mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 para- logues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phago- cytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG92 and ATG162 cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG92/162 double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiqui- tin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG92/162 cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG92/162 cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes. Subject Area: biochemistry/cellular biology/ molecular biology/microbiology 2. Background Domain structure of ATG16 and verification of generated strains by immunoblotting. (a) Domain structure of the 612 amino acid ATG16 protein (http:// smart.embl-heidelberg.de/). The ATG5 binding site (light grey) and the coiled-coil domain responsible for homodimerization (hatched box) are located in the N-terminal half of the protein. The seven WD40 repeats make up the C-terminal half and are depicted as black rectangles. (b) Verification of mutant strains. Western blot of total cell lysates of wild-type AX2, the ATG162, ATG92 and ATG92/162 mutants and two mutants that express GFP fused C-terminally to ATG16 in the ATG162 mutant (ATG162/ATG16-GFP) or, respectively, in the ATG92/162 double mutant (ATG92/162/ATG16-GFP). Top row: anti-GFP antibody; second row from top: anti-ATG9 antibody; third row from top: anti-ATG16 antibody; bottom row: Act1-7 anti-actin antibody. this domain is crucial for its role in xenophagy [20–24]. The Dictyostelium orthologue has a similar size and structure as mammalian ATG16L1 (figure 1a). such as macromolecules and organelles, until its edges are fused with each other to form a double-membrane structure called the autophagosome. Finally, fusion of the outer membrane of the autophagosome with the lysosome (or vacuole in yeast) leads to the formation of the autophagolysosome in which the sequestered cytoplasmic components together with the inner membrane of the autophagosome are degraded by the resident hydrolases [7–9]. ATG16L1 deficiency in mouse embryonic fibroblasts dis- rupted the recruitment of the ATG12–ATG5 conjugate to the isolation membrane and resulted in a loss of ATG8 (LC3) link- age to PE [25]. Recently, a non-synonymous single-nucleotide polymorphism (A to G; T300A in the protein) was identified in the ATG16L1 gene as a risk factor for the development of Crohn’s disease (CD) [26,27]. The T300A mutation had no effect on ATG16L1 binding to ATG5 and on basal autophagy. However, the T300A variant showed impaired xenophagy against Salmonella typhimurium suggesting that the increased risk of CD is due to less-efficient bacterial capture by auto- phagy in cells expressing the mutant variant [28]. ATG9 is so far the only known integral membrane protein of the core autophagy machinery and is thought to deliver membrane lipids to the site of autophagosome formation [29–32]. The core autophagy protein ATG16 has been shown to be involved in the expansion of the isolation membrane [10,11]. The protein is part of one of the two ubiquitin-like protein conjugation systems of the autophagy machinery [9]. 2. Background Macroautophagy (hereafter autophagy) is the major lysosomal route for the turnover of cytoplasmic components. It serves as a housekeeping mechanism and as a cellular response to different stresses such as starvation, the presence of protein aggregates or intracellular bacteria and is essential for temporary cell survival [1]. In addition, it also plays a key role in a range of normal develop- mental processes such as sporulation in Saccharomyces cerevisiae, fruiting body formation in Dictyostelium discoideum, dauer development of Caenorhabditis elegans and pupa formation in Drosophila melanogaster [2,3]. Furthermore, it may promote type II programmed cell death, a type of programmed cell death distinct from apoptosis [4,5]. The autophagymachinery ishighlycomplex and conserved inall eukaryotes. It is composed of many core and accessory autophagy-related (ATG) proteins, many of which have been initially characterized in yeast [6]. Autophagy can be subdi- vided into three main stages: initiation, expansion and lysosomal degradation. Initially, upon appropriate signals, the phagophore or isolation membrane is formed. This structure is elongated and thereby enwraps cytoplasmic constituents Electronic supplementary material is available at http://dx.doi.org/10.1098/rsob.150008. & 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from 1 156 198 313 612 100 aa AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG16-GFP ATG9 ATG16 actin (a) (b) ATG9–/16– ATG9–/16–/ATG16-GFP Figure 1. Domain structure of ATG16 and verification of generated strains by immunoblotting. (a) Domain structure of the 612 amino acid ATG16 protein (http:// smart.embl-heidelberg.de/). The ATG5 binding site (light grey) and the coiled-coil domain responsible for homodimerization (hatched box) are located in the N-terminal half of the protein. The seven WD40 repeats make up the C-terminal half and are depicted as black rectangles. (b) Verification of mutant strains. Western blot of total cell lysates of wild-type AX2, the ATG162, ATG92 and ATG92/162 mutants and two mutants that express GFP fused C-terminally to ATG16 in the ATG162 mutant (ATG162/ATG16-GFP) or, respectively, in the ATG92/162 double mutant (ATG92/162/ATG16-GFP). Top row: anti-GFP antibody; second row from top: anti-ATG9 antibody; third row from top: anti-ATG16 antibody; bottom row: Act1-7 anti-actin antibody. (a) 612 AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG16-GFP ATG9 ATG16 actin (b) ATG9–/16– ATG9–/16–/ATG16-GFP (b) Figure 1. rsob.royalsocietypublishing.org Open Biol. 5: 150008 D. discoideum proteins show high sequence similarities with their human orthologues [38–40]. Thus, in recent years, the organism has become also an increasingly important model for the investigation of biological problems that are relevant to human health, such as for example autophagy, protein aggrega- tion diseases or, as the organism can be infected with several medically relevant pathogens, host–pathogen interactions [41–43]. strain. As expected, no bands were detected in the parental strains for these primer combinations (electronic supple- mentary material, figure S2A,B). RT-PCR of purified total cytoplasmic RNA confirmed loss of the atg16 mRNA in the gen- erated mutants. atg16 cDNA of the expected size was detected in the parental strains and pdi (protein disulfide isomerase) cDNA as positive control was amplified in all analysed strains (electronic supplementary material, figure S2C). The two inde- pendent atg162 strains displayed a comparable phenotype in development (data not shown). Therefore, we performed all other experiments with the ATG162 #1 strain (hereafter referred to as ATG162 strain). Furthermore, we generated two strains that express ATG16 C-terminally tagged with GFP in the ATG162 and the ATG92/ATG162 background. All strains were verified by western blotting of total cell lysates and immunological detection of the corresponding proteins with specific antibodies (figure 1b). Table 1 [32] provides an overview of all strains used in this study. Here, we describe the consequences of deletion of atg16 in AX2 wild-type and ATG92 cells for development, growth, the ability to survive starvation, pinocytosis, phagocytosis and protein homoeostasis. We find that deletion of atg16 resulted in a complex phenotype with significant defects in all of the above-stated cellular processes. Expression of GFP-tagged ATG16 largely rescued the mutant phenotype. We also find that the cell survival after starvation, and the developmental and proteasomal phenotypes of the ATG9/16 double mutant were much more severe than for either of the single mutants, while pinocytosis and phagocytosis defects were similar in all three mutant strains, suggesting that ATG9 and ATG16 probably have cellular functions in addition to their role in autophagy. Our results support links between autophagy and the uptake of nutrients and particles as well as between autophagy and the ubiquitin–proteasome system (UPS). 3.2. Knock-out of atg9 or atg16 caused similar developmental and cell survival defects which were much more severe in the double mutant It has been previously shown that autophagy is required for a number of developmental processes in Dictyostelium [32,44–46]. We investigated the development of AX2, ATG162, ATG92/162 and the ATG162/ATG16-GFP strains by starving cells on phosphate agar plates. Development was severely impaired in the ATG162 mutant and the developmen- tal phenotype was very similar to the previously reported ATG92 mutant [32]. In contrast to the AX2 wild-type strain, the ATG162 mutant generally generated three tips per mound in the tipped mound stage, the mutant produced longer and thinner slugs in the slug stage which frequently broke and its fruiting bodies were extremely misshapen (figure 2). The few fruiting bodies that were produced in the ATG162 mutant possessed a thickened stalk and were much smaller than wild-type fruiting bodies. Re-expression of ATG16 fused C-terminally with GFP in the mutant back- ground rescued the developmental phenotype (data not shown). The developmental defect of the ATG92/162 double mutant was much more severe. In contrast to the single mutants, development of the double mutant was completely arrested at the tipped mound stage and we could not detect 3. Results 3.1. Generation of ATG162 and ATG16-GFP expressing strains 2. Background It associ- ates non-covalently with the ATG12–ATG5 conjugate and thereby forms a tetrameric complex consisting of two ATG12–ATG5 conjugates bound to an ATG16 dimer [12,13]. In this tetrameric complex, ATG16 determines the binding site at the preautophagosomal structure (PAS) in yeast or the isolation membrane in higher eukaryotes [10,14], while ATG12–ATG5 catalyses the lipidation of ATG8 with phos- phatidylethanolamine (PE) through its E3-ligase activity [15]. Mammals possess two ATG16 paralogues, ATG16L1 and ATG16L2, and D. discoideum ATG16 is the orthologue of mam- malian ATG16L1 [3]. The ATG16L1 protein is composed of an N-terminal half which harbours the ATG5 binding site, a coiled-coil domain responsible for homodimerization, and binding sites for clathrin, FIP200 and Rab33B [12,13,16–19]. The C-terminal half is composed of seven WD-repeats, which fold into a b-propeller structure, that contains binding sites for NOD 1 and 2, TMEM59 and ubiquitin. It is assumed that Dictyostelium discoideum amoebae grow as separate, inde- pendent cells but develop into a multicellular organism upon starvation. In this developmental programme up to 100 000 cells aggregate by chemotaxis towards cAMP. The aggregate transforms via distinct morphological states into a mature fruit- ing body, composed of a ball of spores supported bya thin, long stalk made of vacuolized dead cells [33]. The social amoeba is a well-established model organism for the study of several basic biological processes such as signal transduction, cell motility, cytokinesis and phagocytosis [34–37]. Despite the large evol- utionary distance of around 1 billion years, many of the on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from Table 1. Dictyostelium discoideum mutant strains used in this study. 3 Table 1. Dictyostelium discoideum mutant strains used in this study. strains summary references ATG92 ATG9 null mutant [32] ATG162 ATG16 null mutant this study ATG92/162 ATG9/16 double null mutant this study ATG162/[act15]:ATG16-GFP expression of ATG16-GFP in ATG162 this study ATG92/162/[act15]:ATG16-GFP expression of ATG16-GFP in ATG92/162 this study AX2/[act15]:RFP-GFP-ATG8a expression of RFP-GFP-ATG8a in AX2 this study ATG92/[act15]:RFP-GFP-ATG8a expression of RFP-GFP-ATG8a in ATG92 this study ATG162/[act15]:RFP-GFP-ATG8a expression of RFP-GFP-ATG8a in ATG162 this study ATG92/162/[act15]:RFP-GFP-ATG8a expression of RFP-GFP-ATG8a in ATG92/162 this study 3.4. Upregulation of the autophagosomal membrane elongation machinery in ATG162 cells The ATG12–ATG5/ATG16 complex acts as an E3 ligase in the lipidation of ATG8 (LC3). This is the last step in the ATG8 ubi- quitin-like conjugation system and promotes the elongation of the autophagosomal membrane. D. discoideum encodes two ATG8 paralogues, ATG8a (DDB_G0286191) and ATG8b (DDB_G0290491), and another crucial component of the autop- hagosomal membrane elongation machinery is the transmembrane protein ATG9 (DDB_G0285323), which is thought to deliver membrane lipids to the growing auto- phagosome. To investigate whether the absence of ATG16 influences the abundance of these proteins, we analysed western blots of total cell lysates of AX2 cells, the ATG162 mutant and of ATG162/ATG16-GFP cells with antibodies specific for ATG8a, ATG8b or ATG9. Comparable loading of total cellular protein was confirmed by detection of actin. We observed a significant increase in protein levels for all three proteins in the ATG162 strain (figure 3a). Quantification of three independent experiments revealed approximately twice as much ATG8a, three times as much ATG9 and four times as much ATG8b in the ATG162 strain in comparison with AX2 (figure 3b). The levels of these proteins were approxima- tely back to normal after expression of ATG16-GFP in the ATG162 mutant (figure 3a,b). The observed increase in ATG8a, ATG8b and ATG9 protein levels could be due either to the defect in autophagosome maturation (table 2; electronic supplementary material, figure S3B), which could be respon- sible for their reduced degradation, or to an upregulation of gene expression. To address this issue, we performed real- time PCR analysis of the atg8a, atg8b and atg9 genes and of atg5, atg7 and atg12 whose gene products are also part of the ubiquitin-like conjugation mechanism. The results showed a 3.5- to 17-fold increase in mRNA levels for all of these genes in the ATG162 strain (figure 3c). Rescue of the mutant pheno- type through expression of ATG16-GFP reduced the expression of atg5, atg7, atg8b and atg9 to approximately AX2 wild-type levels. The mRNA levels of atg8a and atg12 were also strongly further morphological states even after prolonged starvation of several days on phosphate agar plates (figure 2). Next, we measured the ability of wild-type and mutant cells to survive starvation, which is more specifically reliant on autophagy than development. Cell survival was measured after starving cells in amino acid-free medium for the indicated times. 3. Results The ATG162 cells generated considerably smaller fruiting bodies (please note the different scale) that had thickened stalks in comparison to AX2 fruiting bodies. The ATG92/162double mutant wasstill arrested at the tipped mound stage. Developmentand generation of fruiting bodies were rescued in the ATG162/ATG16-GFP strain (not shown). Scale bar, 0.1 mm. 3.4. Upregulation of the autophagosomal membrane elongation machinery in ATG162 cells We found that wild-type AX2 cells retained high viability over several days, while the survival of ATG92 and ATG162 strains was significantly reduced. Consistent with the most severe developmental defect, the ATG92/162 strain showed also the largest reduction in cell viability (electronic supplemen- tary material, figure S3A). Thus, the ability to survive starvation confirmed the results of the developmental assay with respect to the severity of the phenotypes of the different strains. 3. Results 3.1. Generation of ATG162 and ATG16-GFP expressing strains Dictyostelium atg16 encodes a protein of 612 amino acids and is composed of an N-terminal half which mediates homodimeri- zation and binding to ATG5 and a C-terminal half with seven WD40 repeats which form a b-propeller (figure 1a). To investi- gate the cellular function of ATG16, we generated atg16 gene replacement mutants in AX2 wild-type cells and in the ATG92 strain which had previously been characterized [32]. The atg16 gene locus was inactivated by the integration of the replacement construct containing a blasticidin resistance (bsr) cassette (electronic supplementary material, figure S1). Success- ful targeted integration was confirmed by PCR of genomic DNA with gene-specific primers in combination with bsr- specific primers for two independent ATG16 knock-out strains in the AX2 background and for the atg9/atg16 double knock-out on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from AX2 (a) (b) (c) ATG9–/16– ATG16– Figure 2. Development of the ATG162 and ATG92/162 mutants is severely impaired. Cells were developed on phosphate agar plates and pictures were taken every 6 min. Shown are three developmental stages. (a) Tipped mound stage. Wild-type AX2 cells produced only one tip per mound (black arrows), while mutant cells generally produced three tips per mound (black arrows). (b) Slug stage. In contrast to AX2 slugs, ATG162 mutant slugs were generally longer and thinner and frequently broke (black arrow). In the ATG92/162 double mutant, development stopped at the tipped mound stage and the mutant never proceeded to the slug stage. (c) Side view of terminally differen- tiated fruiting bodies after 48 h of development. The ATG162 cells generated considerably smaller fruiting bodies (please note the different scale) that had thickened stalks in comparison to AX2 fruiting bodies. The ATG92/162double mutant wasstill arrested at the tipped mound stage. Developmentand generation of fruiting bodies were rescued in the ATG162/ATG16-GFP strain (not shown). Scale bar, 0.1 mm. of AX2, ATG92, ATG162 and ATG92/162 cells revealed that in AX2 cells approximately 41% of the fluorescent punctae were red indicating maturation into autophagolysosomes. In contrast, in ATG92, ATG162 and ATG92/162 cells only 8.0, 2.3 and 2.2%, respectively, of the fluorescent punctae were red (table 2; see electronic supplementary material, figure S3B, for exemplary images). This corresponds to an approxima- tely fivefold decrease of autophagolysomes in ATG92 cells, an 18-fold decrease in ATG162 and a 19-fold decrease in ATG92/162 cells. 3. Results In contrast to AX2 slugs, ATG162 mutant slugs were generally longer and thinner and frequently broke (black arrow). In the ATG92/162 double mutant, development stopped at the tipped mound stage and the mutant never proceeded to the slug stage. (c) Side view of terminally differen- tiated fruiting bodies after 48 h of development. The ATG162 cells generated considerably smaller fruiting bodies (please note the different scale) that had thickened stalks in comparison to AX2 fruiting bodies. The ATG92/162double mutant wasstill arrested at the tipped mound stage. Developmentand generation of fruiting bodies were rescued in the ATG162/ATG16-GFP strain (not shown). Scale bar, 0.1 mm. Figure 2. Development of the ATG162 and ATG92/162 mutants is severely impaired. Cells were developed on phosphate agar plates and pictures were taken every 6 min. Shown are three developmental stages. (a) Tipped mound stage. Wild-type AX2 cells produced only one tip per mound (black arrows), while mutant cells generally produced three tips per mound (black arrows). (b) Slug stage. In contrast to AX2 slugs, ATG162 mutant slugs were generally longer and thinner and frequently broke (black arrow). In the ATG92/162 double mutant, development stopped at the tipped mound stage and the mutant never proceeded to the slug stage. (c) Side view of terminally differen- tiated fruiting bodies after 48 h of development. The ATG162 cells generated considerably smaller fruiting bodies (please note the different scale) that had thickened stalks in comparison to AX2 fruiting bodies. The ATG92/162double mutant wasstill arrested at the tipped mound stage. Developmentand generation of fruiting bodies were rescued in the ATG162/ATG16-GFP strain (not shown). Scale bar, 0.1 mm. Figure 2. Development of the ATG162 and ATG92/162 mutants is severely impaired. Cells were developed on phosphate agar plates and pictures were taken every 6 min. Shown are three developmental stages. (a) Tipped mound stage. Wild-type AX2 cells produced only one tip per mound (black arrows), while mutant cells generally produced three tips per mound (black arrows). (b) Slug stage. In contrast to AX2 slugs, ATG162 mutant slugs were generally longer and thinner and frequently broke (black arrow). In the ATG92/162 double mutant, development stopped at the tipped mound stage and the mutant never proceeded to the slug stage. (c) Side view of terminally differen- tiated fruiting bodies after 48 h of development. 3. Results Interestingly, the phenotype of the ATG162 and ATG92/162 cells was comparable and much more severe than for ATG92 cells. This indicates that ATG9 is less important for the completion of autophagosomal membrane expansion, which appears to be a prerequisite for fusion with the lysosome. The result is consistent with a significant reduction in auto- phagic flux in all three mutants which is probably caused by inefficient membrane expansion. It is also noteworthy that at least some of the large punctae in the mutant strains probably represent protein aggregates. In addition, we observed a slight decrease in the average number of autophagosomes per cell, from 1.40 for AX2 wild-type cells to 1.13 for ATG92/162 cells (table 2). AX2 (a) (b) (c) ATG9–/16– ATG16– AX2 (a) (b) (c) ATG9–/16– ATG16– of AX2, ATG92, ATG162 and ATG92/162 cells revealed that in AX2 cells approximately 41% of the fluorescent punctae were red indicating maturation into autophagolysosomes. In contrast, in ATG92, ATG162 and ATG92/162 cells only 8.0, 2.3 and 2.2%, respectively, of the fluorescent punctae were red (table 2; see electronic supplementary material, figure S3B, for exemplary images). This corresponds to an approxima- tely fivefold decrease of autophagolysomes in ATG92 cells, an 18-fold decrease in ATG162 and a 19-fold decrease in ATG92/162 cells. Interestingly, the phenotype of the ATG162 and ATG92/162 cells was comparable and much more severe than for ATG92 cells. This indicates that ATG9 is less important for the completion of autophagosomal membrane expansion, which appears to be a prerequisite for fusion with the lysosome. The result is consistent with a significant reduction in auto- phagic flux in all three mutants which is probably caused by inefficient membrane expansion. It is also noteworthy that at least some of the large punctae in the mutant strains probably represent protein aggregates. In addition, we observed a slight decrease in the average number of autophagosomes per cell, from 1.40 for AX2 wild-type cells to 1.13 for ATG92/162 cells (table 2). AX2 (c) rsob.royalsocietypublishing.org Open Biol. 5: 150008 4 4 (b) (c) Figure 2. Development of the ATG162 and ATG92/162 mutants is severely impaired. Cells were developed on phosphate agar plates and pictures were taken every 6 min. Shown are three developmental stages. (a) Tipped mound stage. Wild-type AX2 cells produced only one tip per mound (black arrows), while mutant cells generally produced three tips per mound (black arrows). (b) Slug stage. 3.3. Impaired autophagosome maturation in ATG92, ATG162 and ATG92/162 cells ATG162 and ATG92/162 cells We next assessed autophagosome number and maturation by expressing the RFP-GFP-ATG8a fusion protein in AX2 and the three mutant strains. Autophagosomes containing RFP- GFP-ATG8a emit red and green light and thus appear yellow in fluorescence microscopy, while autophagolysomes only emit red light because the fluorescence of GFP is very sensitive to the acidic environment of the lysosome. Thus, the presence of red punctae lacking the green fluorescence is an indication of the acidification of the autophagosome through fusion with the lysosome [47]. This way, the maturation of autophagosomes to autophagolysosomes can be analysed. Quantitative analysis on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from Table 2. Autophagosome maturation is impaired in ATG92, ATG162 and ATG92/162 cells. Table 2. Autophagosome maturation is impaired in ATG92, ATG162 and ATG92/162 cells. Table 2. Autophagosome maturation is impaired in ATG92, ATG162 and ATG92/162 cells. 5 strain cells (no.) punctae (no.) punctae/cell green and red punctae (no.) red punctae (no.) (%) AX2 173 242 1.40 + 0.44 141 101 41.0 + 9.5 ATG92 143 193 1.38 + 0.37 178 15 8.0 + 6.2 ATG162 257 323 1.24 + 0.15 315 8 2.3 + 1.6 ATG92/162 277 313 1.13 + 0.03 306 7 2.2 + 0.5 reduced in the rescued strain but were still significantly higher than in AX2 wild-type cells. However, the remaining increase in transcript level of atg8a in the rescued strain was not reflec- ted in the protein level (figure 3b,c). The results are consistent with the existence of a sensing system for autophagosome maturation with a positive feedback regulation of genes in this pathway. reduced in the rescued strain but were still significantly higher than in AX2 wild-type cells. However, the remaining increase in transcript level of atg8a in the rescued strain was not reflec- ted in the protein level (figure 3b,c). The results are consistent with the existence of a sensing system for autophagosome maturation with a positive feedback regulation of genes in this pathway. ATG92/162 strains differed with respect to growth in shaking culture and uptake of TRITC-labelled dextran. The growth defect of ATG162 cells was much more severe than that of ATG92 and ATG92/162 cells. However, the pinocytic activity of these strains was similarly reduced. Next, we performed phagocytosis assays to further investi- gate the mutant strains. First, we analysed the uptake of TRITC-labelled yeast cells. 3.5. ATG9 and ATG16 deficiency resulted in impaired pinocytosis and phagocytosis pinocytosis and phagocytosis The D. discoideum AX2 strain is able to grow axenically in liquid medium, a mode of growth that is supported by macropino- cytic uptake of nutrients from the broth. We first analysed the growth of ATG92, ATG162, ATG92/162 and ATG162/ ATG16-GFP cells in comparison with AX2 in shaking culture. Log phase cells were adjusted to a density of 2 104 cells ml21 and cell proliferation was followed over 11 days in shaking cul- ture. The ATG162 strain displayed a severe growth defect over the whole time period. For example, after 72 h of growth, its cell titre was about 14 times lower than that of AX2 cells. Expression of ATG16-GFP in the ATG162 strain rescued the growth defect. Unexpectedly, the growth defect of the ATG92/162 double mutant was similar to the ATG92 strain and less severe than for the ATG162 strain (figure 4a). Calcu- lation of the generation times in the logarithmic growth phase revealed a dramatic increase to 22 h in the case of ATG162 cells in comparison with a 12 h generation time for AX2 and the ATG162/ATG16-GFP cells. The generation time of the ATG92/162double knock-out strain was 15 h, similar to the ATG92 mutant [32]. Due to the longer generation time, the ATG92 and ATG92/162 strains reached stationary phase between day 6 and 7, which was about 2 days later than AX2 and ATG162/ATG16-GFP cells. Final cell densities were similar for these strains. The ATG162 mutant reached stationary phase only after 11 days at a cell density of approxi- mately 8 106 cell ml21, which is approximately 1.5-fold lower than the stationary cell density of AX2 (data not shown). The growth defect of the mutant strains pointed to a pino- cytosis defect and/or a defect in the intracellular utilization of nutrients. We therefore analysed the uptake of tetra- methylrhodamineisothiocyanate (TRITC)-labelled dextran by AX2, ATG92, ATG162, ATG162/ATG16-GFP and ATG92/ 162 cells. We found a significant decrease in the uptake of TRITC-labelled dextran in ATG162, ATG92 and ATG92/162 cells. The reduced pinocytosis of ATG16 deficient cells was res- cued through expression of ATG16-GFP (figure 4b). The result suggests that autophagy has a positive function in macropino- cytosis. Notably, the phenotypes of the ATG162, ATG92 and The D. discoideum AX2 strain is able to grow axenically in liquid medium, a mode of growth that is supported by macropino- cytic uptake of nutrients from the broth. 3.3. Impaired autophagosome maturation in ATG92, ATG162 and ATG92/162 cells AX2 cells showed a strong increase in fluorescence in the first hour of incubation with TRITC- labelled yeast. In the case of the ATG92, ATG162 and ATG92/162 cells, the increase in fluorescence was only moderate and final fluorescence values after 2 h were approxi- mately 60% compared with AX2. Re-expression of ATG16-GFP rescued the phagocytic activity of the ATG162 mutant (figure 5a). We then analysed the uptake of pHrodoTM Escherichia coli particles. The fluorescence of pHrodoTM E. coli strongly increases as the pH of its surrounding becomes acidic. This is the case when phagosomes fuse with lysosomes, therefore phagolysosomal fusion of ingested particles can be followed. We found that the fluorescence in ATG162, ATG92 and ATG92/162 strains was decreased to approximately 60% as compared with AX2 cells. ATG162 cells that expressed ATG16-GFP were similar to AX2 (figure 5b). Both results indi- cate a phagocytosis defect in autophagy compromised strains. Because of the reduced pinocytotic and phagocytotic activities of the mutant strains, we next wanted to know whether the uptake of Legionella pneumophila would also be affected. We measured the uptake of L. pneumophila by determining the colony-forming units (cfu) 3 h post-infection. At this stage, intracellular bacteria have not yet started to replicate. For all mutant strains, we observed a significant decrease in the uptake of L. pneumophila that was approximately 75% in com- parison to AX2 wild-type cells. Re-expression of ATG16-GFP rescued this defect (figure 5c). 3.5. ATG9 and ATG16 deficiency resulted in impaired pinocytosis and phagocytosis We first analysed the growth of ATG92, ATG162, ATG92/162 and ATG162/ ATG16-GFP cells in comparison with AX2 in shaking culture. Log phase cells were adjusted to a density of 2 104 cells ml21 and cell proliferation was followed over 11 days in shaking cul- ture. The ATG162 strain displayed a severe growth defect over the whole time period. For example, after 72 h of growth, its cell titre was about 14 times lower than that of AX2 cells. Expression of ATG16-GFP in the ATG162 strain rescued the growth defect. Unexpectedly, the growth defect of the ATG92/162 double mutant was similar to the ATG92 strain and less severe than for the ATG162 strain (figure 4a). Calcu- lation of the generation times in the logarithmic growth phase revealed a dramatic increase to 22 h in the case of ATG162 cells in comparison with a 12 h generation time for AX2 and the ATG162/ATG16-GFP cells. The generation time of the ATG92/162double knock-out strain was 15 h, similar to the ATG92 mutant [32]. Due to the longer generation time, the ATG92 and ATG92/162 strains reached stationary phase between day 6 and 7, which was about 2 days later than AX2 and ATG162/ATG16-GFP cells. Final cell densities were similar for these strains. The ATG162 mutant reached stationary phase only after 11 days at a cell density of approxi- mately 8 106 cell ml21, which is approximately 1.5-fold lower than the stationary cell density of AX2 (data not shown). 3.6. Increase in poly-ubiquitinated proteins, appearance of protein aggregates and decrease in proteasomal activity in ATG9 and ATG16 deficient cells 3.6. Increase in poly-ubiquitinated proteins, appearance of protein aggregates and decrease in proteasomal activity in ATG9 and ATG16 deficient cells The growth defect of the mutant strains pointed to a pino- cytosis defect and/or a defect in the intracellular utilization of nutrients. We therefore analysed the uptake of tetra- methylrhodamineisothiocyanate (TRITC)-labelled dextran by AX2, ATG92, ATG162, ATG162/ATG16-GFP and ATG92/ 162 cells. We found a significant decrease in the uptake of TRITC-labelled dextran in ATG162, ATG92 and ATG92/162 cells. The reduced pinocytosis of ATG16 deficient cells was res- cued through expression of ATG16-GFP (figure 4b). The result suggests that autophagy has a positive function in macropino- cytosis. Notably, the phenotypes of the ATG162, ATG92 and The autophagolysosomal and ubiquitin–proteasome path- ways are responsible for cellular homoeostasis and are the major routes for protein and organelle clearance in eukaryotic cells. They were originally considered as independent degra- dative pathways, however recent evidence suggests that autophagy and the UPS are interrelated [49,50]. We first ana- lysed the presence of poly-ubiquitinated proteins in whole cell lysates of AX2, ATG162, ATG162/ATG16-GFP and ATG92/162cells using the monoclonal P4D1 antibody, which on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from AX2 ATG16– ATG16–/ ATG16-GFP ATG8a actin ATG8b actin ATG9 actin AX2 ATG16– ATG16–/ATG16-GFP 5 4 3 2 1 0 18 16 14 12 10 8 6 4 2 0 ATG8a atg8a atg8b atg9 atg5 atg7 atg12 ATG8b ATG9 rel. protein content fold change (a) (b) (c) Figure 3. Upregulation of core autophagy genes and proteins in ATG162 cel (a) Representative western blots of total cell lysates of AX2, ATG162 an ATG162/ATG16-GFP cells. ATG8a, ATG8b and ATG9 were detected with polycl nal antibodies described previously [32,48]. Actin was used as loading cont and detected with the monoclonal Act1-7 antibody. (b) Quantification ATG8a, ATG8b and ATG9 protein levels in AX2, ATG162 and ATG162/ATG1 GFP strains. ATG8a, ATG8b and ATG9 signals were quantified by densitomet analysis and normalized based on the actin signal. Bars represent me values and standard errors of three independent experiments. (c) Real-tim PCR analysis of selected core autophagy genes. RNA was isolated from AX ATG162 and ATG162/ATG16-GFP cells and reverse transcribed in cDNA. Rea time PCR was carried out with primers specific for atg8a, atg8b, atg9, atg atg7 and atg12 (electronic supplementary material, table S1). Bars represe similar increase in ubiquitinated proteins was previously observed in the ATG92 strain [48]. Expression of ATG16 fused to GFP in the knock-out strain reduced ubiquitination to similar levels as in AX2 (figure 6a). 3.6. Increase in poly-ubiquitinated proteins, appearance of protein aggregates and decrease in proteasomal activity in ATG9 and ATG16 deficient cells protein content (b) 18 16 14 12 10 8 6 4 2 0 atg8a atg8b atg9 atg5 atg7 atg12 fold change (c) We observed a significant decrease of proteasomal activity to 40% of wild-type levels in ATG162 cells and a partial rescue to about 80% in the ATG162/ATG16-GFP strain. Reduction of proteasomal activity in comparison with AX2 was even higher in lysates of ATG92 and ATG92/162 cells, where we measured 32% and 5% proteasomal activity, respectively (figure 6e). The further reduction of proteasomal activity in the double knock-out mutant suggests that ATG9 and ATG16 act in parallel pathways that are independently of each other important for full proteasomal activity. atg8a atg8b atg5 atg9 Figure 3. Upregulation of core autophagy genes and proteins in ATG162 cells. (a) Representative western blots of total cell lysates of AX2, ATG162 and ATG162/ATG16-GFP cells. ATG8a, ATG8b and ATG9 were detected with polyclo- nal antibodies described previously [32,48]. Actin was used as loading control and detected with the monoclonal Act1-7 antibody. (b) Quantification of ATG8a, ATG8b and ATG9 protein levels in AX2, ATG162 and ATG162/ATG16- GFP strains. ATG8a, ATG8b and ATG9 signals were quantified by densitometric analysis and normalized based on the actin signal. Bars represent mean values and standard errors of three independent experiments. (c) Real-time PCR analysis of selected core autophagy genes. RNA was isolated from AX2, ATG162 and ATG162/ATG16-GFP cells and reverse transcribed in cDNA. Real- time PCR was carried out with primers specific for atg8a, atg8b, atg9, atg5, atg7 and atg12 (electronic supplementary material, table S1). Bars represent mean values and standard errors of three independent experiments. Figure 3. Upregulation of core autophagy genes and proteins in ATG162 cells. 3.6. Increase in poly-ubiquitinated proteins, appearance of protein aggregates and decrease in proteasomal activity in ATG9 and ATG16 deficient cells Concomitant with the increase in poly-ubiquitinated proteins we frequently observed in immunofluorescence studies the appearance of large protein aggregates in ATG162 and ATG92/162 cells, which stained positive for ubiquitin (figure 6b). Large protein aggregates positive for ubiquitin were previously also docu- mented in ATG92 cells [48]. The presence of ubiquitin- positive protein aggregates in ATG162 cells prompted us to investigate a possible co-localization of ATG16 with ubiquitin. For this purpose, we expressed ATG16-GFP in the ATG92/ 162 mutant. The resulting strain showed a similar increase in protein aggregates as the ATG92 mutant [48]. In confocal microscopy of fixed cells which had been stained with the P4D1 monoclonal antibody, we detected co-localization of ATG16 and ubiquitin for many protein aggregates. It appears that ATG16-GFP localized to the surface of and partially cov- ered ubiquitin-positive aggregates (figure 6c). rsob.royalsocietypublishing.org Open Biol. 5: 150008 6 6 AX2 ATG16– ATG16–/ ATG16-GFP ATG8a actin ATG8b actin ATG9 actin (a) (a) The deficiency in autophagy in ATG92, ATG162 and ATG92/162 cells may be counteracted by the other major protein degradation pathway, the UPS. This could be achieved by an increase in proteasome number or by upregulation of proteasomal activity. Thus, we first assessed proteasome number by analysing the expression level of the proteasomal subunit 5 (SU5) in AX2, ATG162, ATG162/ATG16-GFP, ATG92 and ATG92/162 cells by western blot analysis (elec- tronic supplementary material, figure S4). Quantification of three independent experiments revealed no significant differ- ences in the amount of SU5 in the analysed strains, indicating that expression of the proteasome is unchanged in the mutant strains (figure 6d). We next measured the specific pro- teasomal activity and normalized it to the proteasomal content. We observed a significant decrease of proteasomal activity to 40% of wild-type levels in ATG162 cells and a partial rescue to about 80% in the ATG162/ATG16-GFP strain. Reduction of proteasomal activity in comparison with AX2 was even higher in lysates of ATG92 and ATG92/162 cells, where we measured 32% and 5% proteasomal activity, respectively (figure 6e). The further reduction of proteasomal activity in the double knock-out mutant suggests that ATG9 and ATG16 act in parallel pathways that are independently of each other important for full proteasomal activity. AX2 ATG16– ATG16–/ATG16-GFP 5 4 3 2 1 0 ATG8a ATG8b ATG9 rel. 4. Discussion An alternative explanation for the strong upreglation of ATG8a, ATG8b and ATG9 in the ATG16 mutant would be a sensing system for autophagosome completion which would regulate expression of the involved proteins. Therefore, we analysed the transcription of these genes and of atg5, atg7 and atg12, whose gene products are also important for autophagosomal maturation, by real-time PCR. The results showed that the expression of all of these genes was significantly increased in the ATG16 knock-out strain. This indicates that the increase in protein levels of ATG8a, ATG8b and ATG9 was mainly caused by an upregulation of their expression in ATG162 cells. We predict that also atg3, atg4 and atg10, as being also part of the two ubiquitin-like protein conjugation systems which are essential for elongation of the autophago- somal membrane, are upregulated in ATG162 cells. How the cell senses the status of the autophagosome and elicits the corresponding response needs to be addressed in future work. It is well established that autophagy plays an important role in differentiation and development in multicellular eukar- yotes [56]. The same is true for Dictyostelium, and previous studies showed that deletion of atg1, atg5, atg6, atg7, atg8, atg9 or vmp1 caused aberrant development of varying severity [32,44–46]. We found that also deletion of atg16 resulted in a strong developmental phenotype which was very similar to the ATG92 strain. Loss of either protein led to severe impair- ments in the tipped aggregate stage, the slug stage and in fruiting body generation (figure 2) [32]. The observed pheno- types were also similar to the ones reported for the knock-out mutants of ATG5 and ATG7 [45]. The similar developmental phenotypes for these knock-out strains make perfect sense, because all four proteins are essential components of the two ubiquitin-like protein conjugation systems involved in the elongation of the autophagosomal membrane. The develop- mental phenotype of the ATG9/16 double knock-out mutant was therefore of considerable interest. Unexpectedly, loss of both proteins led to a much more severe phenotype with an early developmental arrest at the tipped mound stage. As both proteins function in membrane elongation of the pha- gophore, we assessed autophagosome maturation in the mutant strains by monitoring RFP-GFP-ATG8a (table 2; electronic supplementary material, figure S3B). We found that autophagosome maturation was similarly impaired in ATG162 and ATG92/162 cells, but to a lesser extent in ATG92 cells. 4. Discussion Macroautophagy is a very complex and evolutionarily highly conserved process which involves more than 20 ‘core’ ATGs, most of which have originally been described in the yeast S. cerevisiae [51]. Autophagosome formation is governed by the ULK1 complex, the PI3K complex, two ubiquitin-like protein conjugation systems and several additional proteins. ATG16 and ATG8 (LC3) are central components of the two ubiquitin-like protein conjugation systems, which include eight core ATG proteins and are crucial for the expansion of the membrane of the growing PAS in yeast or the phago- phore in, for example, mammals and Dictyostelium [3]. ATG16 associates non-covalently with the ATG12–ATG5 conjugate and forms a tetrameric complex which catalyses ATG8 lipidation as an E3 ligase [12,13]. In S. cerevisiae, the ATG12–ATG5/ATG16 complex resides on the PAS, but is recognizes mono-ubiquitin, poly-ubiquitin and ubiquitinated proteins and cross-reacts with Dictyostelium ubiquitin. We detected a clear increase of ubiquitinated proteins in ATG162 and ATG92/162cells as compared with AX2. Immu- nodetection of actin served as loading control (figure 6a). A recognizes mono-ubiquitin, poly-ubiquitin and ubiquitinated proteins and cross-reacts with Dictyostelium ubiquitin. We detected a clear increase of ubiquitinated proteins in ATG162 and ATG92/162cells as compared with AX2. Immu- nodetection of actin served as loading control (figure 6a). A on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from 107 106 105 104 24 48 72 96 120 144 time (h) time (min) 0 0.2 0.4 0.6 0.8 1.0 1.2 AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– 30 60 90 120 cell titre (cells ml–1) relative fluorescence (a) (b) Figure 4. Growth in shaking culture and macropinocytosis are impaired in mutant strains. (a) Growth in shaking culture of AX2, ATG162, ATG162/ATG16-GFP, ATG92 and ATG92/162 cells. The strains were inoculated at a starting density of 2 104 cells ml21 and the cell titre was determined every 24 h. Shown are cell titres for the first 6 days of the growth experiment. Please note the logarithmic scale of the y-axis. Mean values and standard errors of three independent experiments were calculated. (b) Macropinocytosis of TRITC-labelled dextran. The analysed Dictyostelium strains were adjusted to 4 106 cells ml21 and TRITC- dextran was added at a final concentration of 2 mg ml21. Fluorescence with excitation at 544 nm and emission at 574 nm was determined at the indicated time points. 4. Discussion As autophagosomes cannot be completed in this mutant, a subfraction of ATG8a and ATG8b would no longer be degraded and hence would accumulate. However, the observed block in autophagosome maturation in the ATG16 mutant (table 2) cannot satisfactorily explain the increased level of ATG9 because it has been shown that ATG9 is recycled upon autophagosome completion [55]. An alternative explanation for the strong upreglation of ATG8a, ATG8b and ATG9 in the ATG16 mutant would be a sensing system for autophagosome completion which would regulate expression of the involved proteins. Therefore, we analysed the transcription of these genes and of atg5, atg7 and atg12, whose gene products are also important for autophagosomal maturation, by real-time PCR. The results showed that the expression of all of these genes was significantly increased in the ATG16 knock-out strain. This indicates that the increase in protein levels of ATG8a, ATG8b and ATG9 was mainly caused by an upregulation of their expression in ATG162 cells. We predict that also atg3, atg4 and atg10, as being also part of the two ubiquitin-like protein conjugation no longer present on the completed autophagosome [14]. Similarly, ATG12–ATG5/ATG16L1 predominantly localizes on the outer surface of the phagophore in mammals and dis- sociates from the membrane immediately before or after completion of the autophagosome [7]. Thus, it is assumed that the ATG16 complex delivers ATG8 through lipidation to the membrane of the forming autophagosome and that ATG8 then functions in the closing of the autophagosome [11]. Another important player in autophagosomal mem- brane elongation is the transmembrane protein ATG9, which is thought to deliver membrane lipids [52–54]. We reasoned that the abundance of ATG proteins involved in membrane expansion must be coordinately regulated and that deletion of an essential protein in this process such as ATG16 could influence the concentration of the other pro- teins. Indeed, we found that the protein levels of ATG8a, ATG8b and ATG9 were strongly increased in the ATG16 knock-out strain. The accumulation of ATG8a and ATG8b could be caused by autophagy deficiency in the ATG16 mutant. As autophagosomes cannot be completed in this mutant, a subfraction of ATG8a and ATG8b would no longer be degraded and hence would accumulate. However, the observed block in autophagosome maturation in the ATG16 mutant (table 2) cannot satisfactorily explain the increased level of ATG9 because it has been shown that ATG9 is recycled upon autophagosome completion [55]. 4. Discussion The fluorescence of AX2 at 120 min was set to 1. Mean values and standard errors of three independent experiments were calculated. time (min) 0 0.2 0.4 0.6 0.8 1.0 1.2 AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– 30 60 90 120 relative fluorescence (b) 107 106 105 104 24 48 72 96 120 144 time (h) AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– cell titre (cells ml–1) (a) (b) 7 Figure 4. Growth in shaking culture and macropinocytosis are impaired in mutant strains. (a) Growth in shaking culture of AX2, ATG162, ATG162/ATG16-GFP, ATG92 and ATG92/162 cells. The strains were inoculated at a starting density of 2 104 cells ml21 and the cell titre was determined every 24 h. Shown are cell titres for the first 6 days of the growth experiment. Please note the logarithmic scale of the y-axis. Mean values and standard errors of three independent experiments were calculated. (b) Macropinocytosis of TRITC-labelled dextran. The analysed Dictyostelium strains were adjusted to 4 106 cells ml21 and TRITC- dextran was added at a final concentration of 2 mg ml21. Fluorescence with excitation at 544 nm and emission at 574 nm was determined at the indicated time points. The fluorescence of AX2 at 120 min was set to 1. Mean values and standard errors of three independent experiments were calculated. no longer present on the completed autophagosome [14]. Similarly, ATG12–ATG5/ATG16L1 predominantly localizes on the outer surface of the phagophore in mammals and dis- sociates from the membrane immediately before or after completion of the autophagosome [7]. Thus, it is assumed that the ATG16 complex delivers ATG8 through lipidation to the membrane of the forming autophagosome and that ATG8 then functions in the closing of the autophagosome [11]. Another important player in autophagosomal mem- brane elongation is the transmembrane protein ATG9, which is thought to deliver membrane lipids [52–54]. We reasoned that the abundance of ATG proteins involved in membrane expansion must be coordinately regulated and that deletion of an essential protein in this process such as ATG16 could influence the concentration of the other pro- teins. Indeed, we found that the protein levels of ATG8a, ATG8b and ATG9 were strongly increased in the ATG16 knock-out strain. The accumulation of ATG8a and ATG8b could be caused by autophagy deficiency in the ATG16 mutant. 4. Discussion Next, we measured the ability of the cells to sur- vive starvation, which is more specifically reliant on autophagy, and found a similar decrease in survival in the single knock- out mutants, while the ATG92/162 cells were much more severely impaired (electronic supplementary material, figure S3A). The results suggest that there is still some albeit strongly reduced autophagic activity in the single mutants which is suf- ficient for development to proceed beyond the tipped mound on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from AX2 AX2 ATG16– ATG16– ATG16–/ATG16-GFP ATG16–/ATG16-GFP ATG9– ATG9– ATG9–/16– ATG9–/16– AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– time (min) time (h) 0 0 0 1 2 3 4 5 6 7 8 30 60 90 120 relative fluorescence relative uptake relative fluorescence 1.2 1.0 0.8 0.6 0.4 0.2 1.2 1.0 0.8 0.6 0.4 0.2 1.2 1.0 0.8 0.6 0.4 0.2 (a) (b) (c) AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– time (min) time (h) 0 0 1 2 3 4 5 6 7 8 30 60 90 120 relative fluorescence relative fluorescence 1.2 1.0 0.8 0.6 0.4 0.2 1.2 1.0 0.8 0.6 0.4 0.2 (a) (b) AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– time (min) 0 30 60 90 120 relative fluorescence 1.2 1.0 0.8 0.6 0.4 0.2 (a) human pathogen L. pneumophila (figures 4 and 5). The link between autophagy and phagocytosis is not clear. One possi- bility is that disruption of cellular recycling processes may lead to a shortage of membranes and other components that are needed for efficient phagocytosis. Alternatively, there may be a direct link between autophagy and phagocy- tosis. The defect in pinocytosis might be explained by the fact that ATG9 and ATG16 are localized to recycling endosomes and may have important functions in endocytosis [53]. Thus, knock-out of either protein could lead to the observed pinocytic defect (figure 4). 8 The complex phenotypes of the ATG162 and ATG92/162 strains were similar to the ATG9 knock-out strain with the exceptions of growth in shaking culture, survival upon star- vation, development and proteasomal activity. This suggests that both proteins may, at least partially, act in par- allel in these cellular processes. Surprisingly, the growth phenotype of the ATG9/16 double mutant was similar to the ATG92 strain and milder than for the ATG162 strain, while development and proteasomal activity were much more severly impaired in the ATG92/162 strain (figures 2, 4 and 6). 4. Discussion We currently do not have an explanation for this opposing result. The strong decrease in proteasomal activity in the ATG92, ATG162 and ATG92/162 strains was mir- rored by an increase in total poly-ubiquitinated proteins and the appearance of large protein aggregates which stained positive for ubiquitin (figure 6). Autophagy and the UPS are the two major intracellular pathways for protein degrada- tion and are crucial for normal protein homoeostasis. Until recently, they were thought to act independently, but evi- dence is accumulating that there is crosstalk between the two pathways [49,57,58]. It is well established that there is compensatory upregulation of autophagy upon inhibition of proteasomal activity [49,59–61]. The picture with respect to a possible compensatory regulation of the UPS in the case of autophagy deficiency is less clear. Wang et al. [62] recently reported an upregulation of proteasomal subunits and an increase in proteasomal activity upon pharmacologi- cal inhibition of autophagy and also upon downregulation of autophagy genes by RNAi in colon cancer cells. By con- trast, autophagy inhibition through treatment with the autophagy inhibitor bafilomycin or siRNA knock-down of atg7 or atg12 in HeLa cells resulted in impaired clearance of UPS clients. The authors concluded that autophagy inhibition may impact the flux through the UPS and that this deficiency is predominantly mediated by the adaptor molecule p62/ SQSTM1 [63]. Of note, also a decrease of proteasomal activity in the case of autophagy reduction has been observed. Inhibi- tion of lysosomal activities by chloroquine in neuroblastoma cells resulted in an accumulation of ubiquitinated proteins and reduced proteasomal activities. Furthermore, in mice deficient for the lysosomal enzyme cathepsin D proteasomal activities were also significantly reduced [64,65]. We found an increase of poly-ubiquitinated proteins and the appearance of large ubiquitin-positive protein aggregates in ATG92, ATG162 and ATG92/162 cells, which indicated a strong imbalance in protein homoeostasis in these cells. We hypo- thesized that the disturbance of protein degradation might be compensated through either an upregulation of the protea- some number or proteasomal activity. 4. Discussion However, we found no significant difference in the expression level of proteasomal SU5 in these strains and, to our surprise, proteasomal activity of ATG92 and ATG162 cells was strongly reduced and the AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– time (min) time (h) 0 1 2 3 4 5 6 7 8 relative fluorescence 1.2 1.0 0.8 0.6 0.4 0.2 (b) AX2 ATG16– ATG16–/ATG16-GFP ATG9– ATG9–/16– time (h) 0 relative uptake 1.2 1.0 0.8 0.6 0.4 0.2 (c) Figure 5. Phagocytosis is less efficient in the mutant strains. (a) Quantitative analysis of the phagocytosis of TRITC-labelled yeast. Cells were resuspended at 2 106 cells ml21 in Soerensen buffer, and an up to fivefold excess of flu- orescent yeast was added. Data are presented as relative fluorescence and the final fluorescence of AX2 was set to 1. Mean values and standard errors of three independent experiments are shown. (b) Uptake of pHrodoTM E. coli. Fluorescence of the internalized bacteria was measured at the indicated time points. Data are presented as under (a) and represent the mean values and standard errors of three independent experiments. (c) Uptake of L. pneumophila. Intracellular bacteria in Dictyostelium cells were deter- mined 3 h post-infection with an MOI of 10. Graphs represent the mean values and standard errors of four independent experiments. Figure 5. Phagocytosis is less efficient in the mutant strains. (a) Quantitative analysis of the phagocytosis of TRITC-labelled yeast. Cells were resuspended at 2 106 cells ml21 in Soerensen buffer, and an up to fivefold excess of flu- orescent yeast was added. Data are presented as relative fluorescence and the final fluorescence of AX2 was set to 1. Mean values and standard errors of three independent experiments are shown. (b) Uptake of pHrodoTM E. coli. Fluorescence of the internalized bacteria was measured at the indicated time points. Data are presented as under (a) and represent the mean values and standard errors of three independent experiments. (c) Uptake of L. pneumophila. Intracellular bacteria in Dictyostelium cells were deter- mined 3 h post-infection with an MOI of 10. Graphs represent the mean values and standard errors of four independent experiments. Figure 5. Phagocytosis is less efficient in the mutant strains. (a) Quantitative analysis of the phagocytosis of TRITC-labelled yeast. Cells were resuspended at 2 106 cells ml21 in Soerensen buffer, and an up to fivefold excess of flu- orescent yeast was added. 4. Discussion Data are presented as relative fluorescence and the final fluorescence of AX2 was set to 1. Mean values and standard errors of three independent experiments are shown. (b) Uptake of pHrodoTM E. coli. Fluorescence of the internalized bacteria was measured at the indicated time points. Data are presented as under (a) and represent the mean values and standard errors of three independent experiments. (c) Uptake of L. pneumophila. Intracellular bacteria in Dictyostelium cells were deter- mined 3 h post-infection with an MOI of 10. Graphs represent the mean values and standard errors of four independent experiments. stage. The much more severe developmental phenotype of the double mutant in conjunction with a similar deficiency in the ability to survive starvation suggests that ATG9 and ATG16 act in parallel in autophagosome formation. The ATG92 and ATG92/162 mutants displayed also defects in pinocytosis, as evidenced by the reduced uptake of TRITC-labelled dextran, and phagocytosis, as evidenced by phagocytosis of TRITC-labelled yeast, pHrodoTM E. coli BioParticlesw and the uptake of the clinically relevant on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from AX2 ATG16– ATG16–/ATG16-GFP ATG9–/16– AX2 ATG16– ATG16–/ ATG16-GFP ATG9– ATG9–/16– AX2 ATG16– ATG16–/ ATG16-GFP ATG9– ATG9–/16– AX2 ATG16– ATG16–/ATG16-GFP Ab: ubiquitin actin 1.2 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 ATG9–/16– relative SU5 content relative proteasomal activity (a) (b) (c) (d) (e) merge ATG16-GFP Ubiquitin Ubiquitin e 6. Deranged protein homoeostasis in ATG92, ATG162 and ATG92/162 cells. (a) Total ubiquitinylated proteins in AX2 and mutant strains. Total cell lysat 2, ATG162, ATG162/ATG16-GFP and ATG92/162 cells were separated by SDS-PAGE, followed by western blotting. Ubiquitinylated proteins were detect he monoclonal P4D1 antibody against ubiquitin. Actin was used as loading control and detected with the monoclonal Act1-7 antibody. (b) Immunofluo e microscopy of AX2, ATG162, ATG162/ATG16-GFP and ATG92/162 cells. Cells were fixed and stained with the P4D1 anti-ubiquitin antibody. ATG162 a 2/162 cells frequently contained large ubiquitin-positive protein aggregates. (c) Immunofluorescence microscopy of ATG92/162/ATG16-GFP cells. ATG16-G xpressed in ATG92/162 double knock-out cells. Cells were fixed and stained with the anti-ubiquitin P4D1 monoclonal antibody. Nuclei are visualized by DA g. In many instances and as exemplified by the enlargement of the inset, we detected partial co-localization of ATG16-GFP and ubiquitin in protein agg Scale bar, 2 mm. (d) Expression of the proteasomal SU5 in AX2 wild-type and mutant strains. 6.1. Dictyostelium strains, growth and development Dictyostelium discoideum AX2 was used as wild-type strain. The ATG162 and ATG92/162 strains were generated by repla- cing part of the atg16 gene with the Bsr resistance cassette in AX2 and ATG92 cells. ATG162/ATG16-GFP and ATG92/ 162/ATG16-GFP mutants were generated by transformation of ATG162 and ATG92/162 cells, respectively, after transient expression of the Cre recombinase with an expression construct encoding GFP fused C-terminally to ATG16. Strains used in this study are listed in table 1. AX2 and mutant cells were grown at 218C in Ax2 medium (for 1 l: 14.3 g bacteriological peptone, 7.15 g yeast extract, 18 g maltose, 0.62 g Na2HPO2 2H2O, 0.49 g KH2PO4, pH 6.7) that contained in the case of mutant strains in addition 5 mg ml21 Blasticidin S (ICN Bio- chemicals) or 6 mg ml21 G418 (Sigma) with shaking at 160 r.p.m. [66] or on SM agar plates with Klebsiella aerogenes [67]. For growth experiments, log phase cells with a cell titre of 2–4 106 cells ml21 were inoculated at a density of 2 104 cells ml21 in 30 ml Ax2 medium and growth was monitored by measuring the cell titre every 24 h. For the calculation of gen- eration times in the logarithmic growth phase, cell titres of AX2 and the ATG162/ATG16-GFP strain between days 2 and 3, of ATG92/162 and ATG92 between days 3 and 4 and of ATG162 between days 6 and 8 were used. Generation times were calculated using n ¼ [logNt 2 logN0]/log2 where n is the number of generations, N0 is the cell titre at time 0 and Nt is the cell titre at time t. For the measurement of cell survival upon starvation, cells were resuspended in SIH medium with- out amino acids (Formedium, UK) and cell survival was determined at the indicated times by counting the number of cfu on lawns of K. aerogenes. Two independent experiments 5. Conclusion In summary, our results clearly demonstrate a crosstalk between autophagy and the UPS with the unexpected out- come that a functioning autophagy is essential for optimal proteasomal activity. They also suggest that the core ATGs, ATG9 and ATG16, have likely additional autophagy- independent functions. This could explain why the pheno- types of the ATG92, ATG162 and ATG92/162 strains are similar for some cellular processes but different for others. on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from proteasomal activity of the double mutant was even more compromised (figure 6e; electronic supplementary material, figure S4). This result clearly shows that, opposite to the expectation, there is no compensatory increase of the UPS in these strains. Accordingly, an intact autophagy pathway is needed for full proteasomal activity in Dictyostelium cells. The further significant reduction of proteasomal activity in the ATG92/162 strain suggests that both proteins, ATG9 and ATG16, have overlapping as well as independent func- tions that are needed for full proteasomal activity. Thus, it appears that at least some human or mouse cell types as well as Dictyostelium cannot compensate an imbalance in protein degradation because of an autophagic block through an upregulation of the UPS. Rather, the imbalance is exacer- bated because the second arm of protein degradation gets also severly compromised. with two parallel cultures each were performed, day 1 was set to 1 for each strain and standard errors of the mean were calculated. 10 rsob.royalsocietypublishing.org Open Biol. 5: 150008 10 For monitoring development on phosphate agar plates, log phase cells from a shaking culture were washed twice with Soerensen buffer (2.0 mM Na2PO4, 14.6 mM KH2PO4, pH 6.0). A total of 1 108 cells were then resuspended in 1 ml of Soerensen buffer and 500 ml of this solution corresponding to 5 107 cells was evenly distributed onto a phosphate-buffered agar plate (9 cm in diameter) and incubated at 218C. Different stages of development were observed and the images were captured either manually at defined time points or automati- cally at 6 min intervals using a stereomicroscope (MZFIII, Leica, Germany). 6.2. Vector construction and transformation The atg16 gene replacement construct was generated in the pLPBLP vector where the Bsr resistance cassette is flanked by loxP sites [68]. A PCR-amplified fragment of 521 bp (bases 23–543 of the coding sequence) was cloned into the BamHI and PstI sites and a 646 bp fragment (bases 1630–2276) into the HindIII and SalI sites of the pLPBLP vector. The plasmid was introduced into AX2 cells and the ATG92 mutant by elec- troporation, and transformants were selected in the presence of 5 mg ml21 Blasticidin S. Gene replacement mutants were ident- ified by PCR screening of blasticidin resistant clones followed by western blotting. For ectopic expression of full-length ATG16 fused N-terminally to GFP, the pBsr-C-GFP C1 vector was used [69]. The full-length ATG16-coding sequence was amplified by PCR and cloned into the pBsr-C-GFP C1 vector via the BamHI and XmaI restriction sites, and the final expression construct was verified by sequencing. In the ATG16-GFP fusion protein, a linker of six amino acids with the sequence GGSGGS, which was introduced through the PCR primer, separates the GFP moiety from the full-length ATG16. The plasmid was introduced into ATG162 and ATG92/162 mutants by electroporation and transformants were selected in the presence of 5 mg ml21 Blasticidin S. Blasticidin resistant clones expressing ATG16-GFP were identified by visual inspection under a fluorescence micro- scope and verified by western blotting. 6. Material and methods 6.1. Dictyostelium strains, growth and development 4. Discussion SU5 signals were quantified in AX2, ATG162, ATG162/ATG1 ATG92 and ATG92/162 strains by densitometric analysis and normalized based on the actin signal. The SU5 value of AX2 has been set to 1. Bars represe AX2 ATG16– ATG16–/ATG16-GFP ATG9–/16– AX2 ATG16– ATG16–/ATG16-GFP Ab: ubiquitin actin ATG9–/16– (a) (b) (c) merge ATG16-GFP Ubiquitin Ubiquitin (a) AX2 ATG16– ATG16–/ATG16-G ATG9–/16– Ab: ubiquitin actin AX2 ATG16– ATG16–/ ATG16-GFP ATG9– ATG9–/16– AX2 ATG16– ATG16–/ ATG16-GFP ATG9– ATG9–/16– 1.2 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 relative SU5 content relative proteasomal activity (d) (e) Figure 6. Deranged protein homoeostasis in ATG92, ATG162 and ATG92/162 cells. (a) Total ubiquitinylated proteins in AX2 and mutant strains. Total cell lysates of AX2, ATG162, ATG162/ATG16-GFP and ATG92/162 cells were separated by SDS-PAGE, followed by western blotting. Ubiquitinylated proteins were detected with the monoclonal P4D1 antibody against ubiquitin. Actin was used as loading control and detected with the monoclonal Act1-7 antibody. (b) Immunofluor- escence microscopy of AX2, ATG162, ATG162/ATG16-GFP and ATG92/162 cells. Cells were fixed and stained with the P4D1 anti-ubiquitin antibody. ATG162 and ATG92/162 cells frequently contained large ubiquitin-positive protein aggregates. (c) Immunofluorescence microscopy of ATG92/162/ATG16-GFP cells. ATG16-GFP was expressed in ATG92/162 double knock-out cells. Cells were fixed and stained with the anti-ubiquitin P4D1 monoclonal antibody. Nuclei are visualized by DAPI staining. In many instances and as exemplified by the enlargement of the inset, we detected partial co-localization of ATG16-GFP and ubiquitin in protein aggre- gates. Scale bar, 2 mm. (d) Expression of the proteasomal SU5 in AX2 wild-type and mutant strains. SU5 signals were quantified in AX2, ATG162, ATG162/ATG16- GFP, ATG92 and ATG92/162 strains by densitometric analysis and normalized based on the actin signal. The SU5 value of AX2 has been set to 1. Bars represent mean values and standard errors of four independent experiments. A representative western blot of SU5 expression is shown in the electronic supplementary material, figure S4. (e) Proteasomal activity of AX2 and mutant strains. The proteasomal activity assay was performed as described in Material and methods. Pro- teasomal activity was normalized to proteasome content, and the chymotrypsin-like activity of AX2 was set to 1. Bars represent mean values and standard errors of three independent experiments. on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from 6.4. Phagocytosis and pinocytosis analysis Quantitative phagocytosis of TRITC-labelled heat-killed yeast cells was performed as described [75]. Briefly, D. discoideum cells at a density of 2–4 106 cells ml21 were washed twice with Soerensen buffer and resuspended at a density of 2 106 cells ml21 in Soerensen buffer. One millilitre of the cell suspension was centrifuged for 4 min at 500g in an Eppen- dorf tube, the supernatant was carefully removed and the pellet frozen at 2208C. This sample was used for the determi- nation of the protein content. Ten microlitres of the cell suspension was dispensed in a 30-ml Erlenmeyer flask and incubated for 15 min on a rotary shaker to allow cells to recup- erate. Then TRITC-labelled yeast cells were added at a fivefold excess relative to Dictyostelium cells. A 1-ml sample was removed immediately (time point 0) and transferred to an Eppendorf tube containing 100 ml 0.4% Trypan Blue solution to quench external fluorescence, washed twice with Soerensen buffer, the cell pellet resuspended in 500 ml Soerensen buffer and fluorescence measured immediately using an Infinite M1000 plate reader (Tecan Group Ltd., Switzerland) at 544 nm excitation and 574 nm emission. The other time points (30, 60, 90 and 120 min) were processed as described for the 0 time point. The measured fluorescence values were corrected with the background fluorescence of cells at time point 0 and for differences in protein content. Mean values and standard errors of three independent experiments were calculated. Quantitative phagocytosis of pHrodoTM E.coli Bio- particles (Invitrogen) was performed as described [32]. Mean values and standard errors of four independent experiments were calculated. The uptake of L. pneumophila Corby by Dictyostelium AX2 cells and mutant strains was measured after 3 h of infection. The infection assay and the determination of intracellular bacteria were performed as described [32]. on July 3, 2015 http://rsob.royalsocietypublishing.org/ Downloaded from rsob.royalsocietypublishing.org Open Biol. 5: 150008 11 11 ATG9, ATG8a and ATG8b were detected with specific pAbs at a 1 : 10 000 dilution as described [32,48]. Secondary antibodies used were anti-mouse and anti-rabbit lgG conjugated with per- oxidase (Sigma) followed by chemiluminescence detection. Images were recorded and analysed using the Fluorchem SP imaging system (Alpha Innotech, USA). Relative protein amounts were determined densitometrically using the Spot Denso tool of the ALPHAEASEFC software (Alpha Innotech). Background values were subtracted and the resulting intensi- ties normalized based on actin staining. Mean values and standard errors of three independent experiments were calculated. fluorescence values were corrected by the background fluor- escence of cells at time point 0 and for differences in protein content. Mean values and standard errors of three indepen- dent experiments were calculated. 6.6. Proteasomal activity analysis Proteasomal activity assays of the different D. discoideum strains were performed using the established protocol from skeletal muscle tissue [77] with minor changes as described [48]. Mean values and standard errors of three independent experiments were calculated. The chymotrypsin-like activity of AX2 wild-type cells was set to 1. 6.5. Fluorescence microscopy Immunofluorescence microscopy was essentially done as described [32]. Fixed cells were incubated with the monoclonal P4D1 anti-ubiquitin antibody (Cell Signaling Technology) at a 1 : 100 dilution in PTB (1 PBS, 0.1% Triton X-100, 0.1% BSA). Secondary antibody was Alexa-fluor 568 conjugated goat anti- mouse IgG at a 1 : 2000 dilution (Invitrogen). Nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI, Sigma). Confocal images of fixed cells were recorded in sequential mode with an inverted TCS SP5 laser scanning microscope (Leica) with a 100 HCX PL APO NA 1.40 oil immersion objec- tive. The RFP-GFP-ATG8a autophagosome maturation assay was essentially done as described [47]. Briefly, incorporation of RFP-GFP-ATG8a into the autophagolysosome can be mon- itored because the acidic environment of the lysosome quenches the more sensitive fluorescence of GFP while the fluorescence of RFP is preserved for longer. Therefore, yellow punctae (green and red) indicate autophagosomes, while red punctae (green fluorescence is quenched) indicate autophago- lysosomes. To allow monitoring of RFP-positive punctae, lysosomal degradation was slowed down by treatment of the cells with 100 mM NH4Cl twice for 2 h. In vivo imaging was done with an inverted TCS SP5 laser scanning microscope (Leica). Three independent experiments were performed and the number of RFP-GFP-ATG8a punctae were counted from single plane. Excitation of GFP was at 488 nm, emission 500– 550 nm; of Alexa-fluor 568 at 568 nm, emission 600–630 nm; and of DAPI at 405 nm, emission 412–464 nm. Images were processed using the accompanying LEICA APPLICATION SUITE (LAS) software. 6.3. Antibody generation, SDS-PAGE, western blotting and protein quantification For generation of ATG16-specific polyclonal antibodies (pAbs), the cDNA sequence encoding the coiled-coil domain (aa 160–210) was amplified by PCR and cloned into the pGEX-6p-1 GST expression vector. The fusion protein was expressed in E. coli XL1 blue, purified using GST-Sepharose beads, released through cleavage with PreScission protease and used for the immunization of rabbits (BioGenes GmbH, Germany). SDS-PAGE and western blotting were essentially performed as described [70,71]. The proteins of 2 105 cells per lane were separated by SDS gel electrophoresis of total cell lysates. The generated ATG16 pAbs were affinity purified and used for western blotting at a 1 : 1000 dilution. GFP was detected with monoclonal antibody K3-184-2 at a 1 : 15 dilution [72]. The proteasomal SU5 was detected with monoclonal anti- body 171-337-2 at a 1 : 100 dilution [73]. 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The authors declare that they have no competing financial interests. and coordinated it and wrote the manuscript. All authors gave final approval for publication. and coordinated it and wrote the manuscript. All authors gave final approval for publication. 12 6.7. Miscellaneous methods p Fluid phase macropinocytosis was determined using TRITC-labelled dextran [76]. Dictyostelium cells at a density of 2–4 106 cells ml21 were pelleted and resuspended at 4 106 cells ml21 in fresh nutrient medium. One millilitre of the cell suspension was centrifuged for 4 min at 500g in an Eppendorf tube, the supernatant was carefully removed and the pellet was frozen at 2208C. This sample was used for the determination of the protein content. Five millilitres of the cell suspension was dispensed in a 30-ml Erlenmeyer flask and incubated for 15 min on a rotary shaker to allow cells to recuperate. Then TRITC-labelled dextran (Sigma) was added to a final concentration of 2 mg ml21. Five hun- dred microlitre samples were removed immediately (time point 0) and after 30, 60, 90 and 120 min and processed and fluorescence measured as described for the phagocytosis of TRITC-labelled yeast cells (see above). The measured RNA isolation, cDNA generation and real-time PCR were essentially done as described [78]. Table S1 of the electronic supplementary material lists the primers that were used in this study. Acknowledgements. We thank Dr Ricardo Escalante for providing the plasmid for expression of RFP-GFP-ATG8a, Dr Sze Man Tung for the ATG92 strain, Natalie Heuer for expert technical assistance and Rosemarie Blau-Wasser for help with immunofluorescence studies. Funding statement. L.E. and M.S. acknowledge support of their work by the German Research Foundation (Deutsche Forschungsgemeinschaft: CRC670, TP01; STE838/8-1) and L.E. by Ko¨ln Fortune. This work was supported by the China scholarship council to Q.X. Acknowledgements. 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https://openalex.org/W3046236294	https://bmcemergmed.biomedcentral.com/counter/pdf/10.1186/s12873-020-00354-7	English	null	A descriptive study of orthopaedic injuries due to parachute jumping in soldiers	BMC emergency medicine	2,020	cc-by	9,182	Sahin and Batın BMC Emergency Medicine (2020) 20:58 https://doi.org/10.1186/s12873-020-00354-7 Sahin and Batın BMC Emergency Medicine (2020) 20:58 https://doi.org/10.1186/s12873-020-00354-7 (2020) 20:58 Open Access © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. A descriptive study of orthopaedic injuries due to parachute jumping in soldiers Taner Sahin1* and Sabri Batın2 Abstract Background: While parachute jumping, soldiers may suffer minor or life-threatening injuries in various parts of the body. Several trauma scoring systems assess the severity of such injuries. The primary goal of this study was to assess clinical characteristics and the severity of orthopaedic, musculoskeletal, and other injuries from military training-related parachute jumping using two trauma scoring systems (AIS and ISS). Our secondary goal was to assess whether there was an increase in injury rates with age. Methods: In total, 185 military personnel between 19 and 51 years old who were injured as a result of daytime static parachute jumping during 44 months between January 2016 and August 2019 were included in the study. Demographic data; vital signs; the level of consciousness; the Glasgow Coma Scale; ISS trauma region classifications; anatomical injury sites; AIS and ISS scores; diagnoses; treatment methods; hospitalization status; and duration of hospital stay were examined descriptively. Results: There were 184 male and one female participant. The most commonly injured body site was the foot (33.5%), and the most common diagnosis was soft tissue trauma (64.3%). The most commonly injured body site was the foot (33.5%), and the most common diagnosis was soft tissue trauma (64.3%). Regarding treatment methods, 51.4% received analgesic pills and cold pack application, 42.7% received a plaster splint, and 5.9% had orthopaedic surgery. The mean ISS score was 5.16 ± 3.92. The hospitalization rate was significantly higher for patients with a critical AIS score than those with a severe AIS score (p < 0.001). Conclusions: The use of trauma scoring systems to assess injury severity among patients admitted to an ED due to a parachute jumping injury may facilitate treatment selection. We found that AIS and ISS were useful in determining injury severity. Therefore, we recommend the use of trauma scoring systems for assessing the injury severity and the therapeutic approach, and we advocate for the use of the 17 anatomical regions we used in this study. We also found that with increasing age, the severity of injury could increase. Keywords: Parachuting injuries, Abbreviated injury scale, Injury severity score balloon, took place in 1797; subsequently, modern para- chuting developed [2]. The word parachute derived from the Greek word ‘para,’ indicating protection, and the French word ‘chute,’ indicating fall [1, 2]. * Correspondence: drmtsahin@gmail.com 1Emergency Medicine Clinic Kayseri City Education and Research Hospital affiliated with University of Health Sciences Turkey, 38090 Kayseri, Turkey Full list of author information is available at the end of the article Background The idea of jumping from a high place without injury first appeared in China about 2000 years ago [1]. As a re- sult of trials conducted throughout history, the first suc- cessful parachute jump, which was a jump from a With the Second World War, parachuting was used and developed for military purposes. Today, many coun- try armies still contain parachute troops. Military or hobby parachute jumps are widely used today [2, 3]. * Correspondence: drmtsahin@gmail.com 1Emergency Medicine Clinic Kayseri City Education and Research Hospital affiliated with University of Health Sciences Turkey, 38090 Kayseri, Turkey Full list of author information is available at the end of the article Study design and setting This study was conducted at Kayseri City Training and Research Hospital, a tertiary center in Kayseri, Turkey. Ethical approval was obtained through the Kayseri City Training and Research Hospital Research Committee (16.11.2016/09) and the Erciyes University Ethics Com- mittee (96,681,246/340). The study adhered to the Dec- laration of Helsinki [21]. pp The AIS anatomical scoring system first published in 1969; it was last updated in 2015 and is still in use today. AIS can be used to reveal the epidemiological features of trauma patients. AIS also allows wound severity to com- pared through the use of standard terminology for the identification of wounds and simple numerical sequen- cing methods. According to the first AIS systems, the body anatomically divided into six regions: head (includ- ing neck), face, chest, abdomen or pelvic contents, ex- tremities, and external. In later versions of AIS, this anatomical classification slightly expanded. AIS uses a six-point scale to classify injury severity (minor = 1, moderate = 2, serious = 3, severe = 4, critical = 5 and un- survivable = 6) [20]. In this descriptive study, we examined the orthopaedic, musculoskeletal, and other injuries of patients brought to the ED after parachute jumping. All patients who were injured while the emergency medicine specialist ex- amined parachuting, and consultations were requested from relevant departments as deemed necessary. Special- ists or assistant doctors performed consultations. Importance ISS is an AIS-based scoring system developed in 1974 by Baker et al. [14]. ISS provides numerical calculations and identifies the total injury severity of people with in- juries to more than one body area. Like AIS, ISS divides the body into six anatomical regions, too. AIS scores for each of the three most severely injured body areas. If the ISS score is higher than 16, there is major trauma. The maximum score is 75 points, which indicates a deadly level of injury [14]. Parachuting requires high levels of concentration and physical performance. Various injuries may occur in the air and during landing; in some cases, fatal accidents can occur. According to the literature, the rate of injuries as a result of parachute jumping is 3–24 per 1000 persons [4, 5]. The factors affecting the injury rate include alti- tude; whether the jump performed at night; the equip- ment and technique used; plane-related factors; the parachute type; the weather and other environmental factors; the ground is adequate and personal factors, such as age, weight, height, experience, aircraft departure technique and loss of control [3, 6–10]. Most parachut- ing accidents and fatal injuries occur at low altitudes and are due to sudden turns [3, 4, 8]. Page 2 of 11 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Goal of this investigation Due to its geographical structure, air sports, such as paragliding; military training; hobby parachuting; and balloon tours, are performed frequently in Central Ana- tolia and its surroundings. Different scoring systems used to determine the sever- ity of trauma patients before and in the hospital. These scoring systems generally based on consciousness, vital signs, and trauma mechanisms and can be classified as physiological, anatomical, triage, or combined scoring systems. Physiological scoring systems consider con- sciousness and vital signs; anatomical scoring systems focus on the anatomical regions of the body, and triage scoring systems consider consciousness, vital signs, and other scoring systems. Specific scoring systems can be classified as triage scoring systems, e.g., the prehospital index (PHI) and the trauma triage rules (TTR). The ana- tomical scoring systems, e.g., the abbreviated injury scale (AIS), the injury severity score (ISS), the new injury se- verity score (NISS), the anatomic profile score (APS) and the anatomic profile. The physiological scoring sys- tems, e.g., the revised trauma score (RTS), the Glasgow coma scale (GCS), and the acute physiology and chronic health evaluation (APACHE). Or the combined scoring systems, e.g., the trauma and injury severity score (TRIS S), the organ injury scale (OIS), the international classifi- cation of diseases-based ISS (ICISS), a severity characterization of trauma (ASCOT), the trauma and in- jury severity score comorbidity (TRISSCOM) and the advanced trauma life support (ATLS) [11-19]. In Kayseri, a city of Central Anatolia, paragliding is done for hobby and parachuting for military training. We observe an increase in the number of emergency de- partment (ED) admissions as a result of parachute jumps has increased in recent years. In the present study, we aimed to assess the orthopaedic, musculoskeletal, and other organ-related injuries among patients admitted at the Kayseri City Training and Research Hospital Emer- gency Medicine Clinic, which injured as a result of jumping from parachute for military training. The pri- mary goal of this study was to assess clinical characteris- tics and the severity of orthopaedic, musculoskeletal, and other injuries from military training-related para- chute jumping using two trauma scoring systems (AIS and ISS) in 44 months between January 2016 and August 2019. Our secondary goal was to assess whether there was an increase in injury rates with age. Selection of participants Total 371.324 patients admitted to our hospital ED be- cause of all the complaints between January 2016 and August 2019. A total of 86.787 trauma patients admitted Sahin and Batın BMC Emergency Medicine (2020) 20:58 Page 3 of 11 Page 3 of 11 to the hospital, ED, in 2019. Of these, 200 military flight school personnel initially selected for the study. These patients were males and females between 19 and 51 years old who injured as a result of daytime static para- chute jumping for military training purposes. for continuous variables—were used to summarize the data. Categorical variables summarized as numbers and percentages. The normality of the numerical variables checked with the Kolmogorov-Smirnov test. The Mann- Whitney U test used to compare two independent groups when the numerical variables did not show nor- mal distribution. The Pearson chi-square test or the Fisher Freeman Halton test used to compare the cat- egorical variables. Descriptive and frequency analyses used in the analysis of intergroup distributions. The ISS score averages compared by age group using one-way ANOVA and Spearman correlation tests. A total of 15 patients were excluded because they were not recorded in the hospital information management system (HIMS), their AIS and ISS trauma scores were not calculated, they did not provide consent to partici- pate in the study and/or parts of their patient observa- tion forms were missing. Verbal or written consent to participate in the study obtained from the remaining 185 patients. The statistical analysis conducted by using the Jamovi version 1.0.7 software program in this study. And the significance level set at a p-value of 0.05 Microsoft Of- fice Excel 2016 software used to create the graphics. Outcomes AIS scores calculated. ISS scores calculated as the sum of the squares of the three sites with the most severe in- juries (ISS = a2 + b2 + c2); the minimum score is 1 point, and the maximum is 75 points. In the trauma regions where we thought the anatomical classification of AIS and ISS were inadequate, we divided the body into 17 different anatomical regions (head, cervical, thorax, shoulder, arm, elbow, forearm, wrist, hand, spine, abdo- men, pelvic region, thigh, knee, leg, ankle, and foot) and calculated the injury rates separately. The patients who presented to the ED with an injury complaint due to a parachute jump in 2016 (7%, n = 13), 2017 (39.5%, n = 73), 2018 (24.3%, n = 45) and the first 8 months of 2019 (29.2%, n = 54) were evaluated. The total of the patients for all conditions of ED patients was 460.716 in 2016, 464.637 in 2017, 468.223 in 2018 and 371.324 in the first 8 months of 2019. The ratio of ED patients with injury complaints due to parachute jump to other patients in 2016 was 0.003%; the ratios were 0.016% in 2017, 0.009% in 2018, and 0.015% in 2019 (Table 1). The measurements of systolic blood pressure (SBP), which taken upon each patient’s arrival to the ED, were divided into four groups: > 100 mmHg, 86–100 mmHg, 75–84 mmHg and 0–74 mmHg. The patients’ pulse rates (P.R.s) divided into three groups: > 120/min, 51–119/ min and < 50 mmHg, and their respiratory rates (R.R.s) divided into three groups: > 20/min, 10–12/min and < 10/min. The patients’ consciousness states were classi- fied as normal, confused and meaningless speech, while the GCS scores recorded numerically. The most common injury site was the foot (33.5%, n = 62), followed by the ankle (29.1%, n = 54), the spine (18.3%, n = 34) and the head (12.4%, n = 23). The ISS anatomical classifications of the patients’ in- jury sites were the abdomen/pelvis (75.7%), the ex- tremity (67.6%), the head/neck (13.5%), and the chest (13.5%). When the injury sites were examined, 33.5% of the foot injuries were assessed as intensive, along with 29.2% of the ankle injuries, 18.4% of the spine injuries, 14.1% of the pelvis injuries, 13.5% of the head injuries, 13% of the leg injuries, 7.6% of the Results After considering exclusion criteria, a total of 185 pa- tients included in the study, which focused on the 44 months from January 2016 to August 2019. Among the 185 participants, 99.5% (n = 184) were male and 0.5% (n = 1) were female. The mean age of the patients was 26.7 ± 6.2 years old (youngest: 19, oldest: 51). In total, 43.8% were 19–23 years old, 31.4% were 24–29 years old, and 24.8% were 30 years old and older. Among the par- ticipants, 43.7% were private soldiers, and 56.3% were ranked soldiers. It learned from the commanders that private soldiers were working on average 7 ± 2.3 months, while ranked soldiers were working on average 6 ± 1.4 years of military service. Besides, it also learned that the ranked soldiers had more parachute jumping experience. We selected AIS and ISS trauma scoring since these systems allowed us to make fast and easy calculations. Measurements For the 185 participants, demographic data, such as age and gender; ISS trauma region classification; anatomical injury sites; AIS and ISS scores; diagnoses; treatment methods; hospitalization status; and duration of hospitalization stay obtained from face-to-face interviews with the patients, HIMS and the patient registration forms. The injuries classified according to age and the trauma scoring scores. The injury rate stated as a per- centage. The patients divided into three age groups: 19– 23 years old (n = 81), 24–29 years old (n = 58), and 30 years old and older (n = 46). Data analysis One patient (0.5%) had a GCS of 13, 10 ti t (5 5%) h d GCS f 14 d 174 ti t Table 1 Distribution of patients by age group, gender and year Age Mean ± SS 26,7 ± 6,2 Age n (%) between ages 19–23 81 (43,8) between ages 24–29 yaş 58 (31,4) 30 years and over 46 (24,9) Rank n (%) Private soldier 81 (43,7) Rank soldier 104 (56,3) Gender n (%) Male 184 (99,5) Female 1 (0,5) Patient application rates due to parachuting injury by years n (%) 2016 13 (7) 2017 73 (39,5) 2018 45 (24,3) 2019 54 (29,2) Proportion of parachuting injury to all other emergency patients by years n (%) 2016 13 (0,003) 2017 73 (0,016) 2018 45 (0.009) 2019 54 (0.015) Descriptive statistics are given as number (%) for categorical variables and mean ± standard deviation for numerical variables Table 2 Distribution of trauma site, injury site, diagnosis and treatment method applied according to ISS n (%) Rates of injury sites by anatomical classification of the ISS Pelvic, yes 140 (75,7) Extremities, yes 125 (67,6) Head neck, yes 25 (13,5) Chest, yes 25 (13,5) Subcutaneous superficial, yes 8 (4,3) Face, yes 4 (2,2) West, yes 3 (1,6) Injury Site Foot, yes 62 (33,5) Ankle, yes 54 (29,2) Spine, yes 34 (18,4) Pelvic region, yes 26 (14,1) Head yes 25 (13,5) Leg, yes 24 (13) Knee, yes 14 (7,6) Thigh, yes 12 (6,5) Shoulder, yes 11 (5,9) Thorax, yes 6 (3,2) Cervical, yes 5 (2,7) Wrist, yes 4 (2,2) Abdomen, yes 3 (1,6) Hand, yes 3 (1,6) Elbow, yes 2 (1,1) Arm, yes 2 (1,1) Forearm, yes 1 (0,5) Diagnosis STT, yes 119 (64,3) Lower limb fracture, dislocation, yes 28 (15,1) Head injury, yes 19 (10,3) Spinal injury, yes 10 (5,4) Upper limb fracture, dislocation, yes 4 (2,2) Cervical injury, yes 4 (2,2) Facial trauma, yes 4 (2,2) Abdominal injury, yes 2 (1,1) Thoracic injury, yes 3 (1,6) Pelvis fracture, yes 1 (0,5) Treatment method applied Medication and cold application 95 (51,4) Splint plaster 79 (42,7) treatment method applied according to ISS n (%) Rates of injury sites by anatomical classification of the ISS Pelvic, yes 140 (75,7) Extremities, yes 125 (67,6) Head neck, yes 25 (13,5) Chest, yes 25 (13,5) Subcutaneous superficial, yes 8 (4,3) Face, yes 4 (2,2) West, yes 3 (1,6) Injury Site Foot, yes 62 (33,5) Ankle, yes 54 (29,2) Spine, yes 34 (18,4) Pelvic region, yes 26 (14,1) Head yes 25 (13,5) Leg, yes 24 (13) Knee, yes 14 (7,6) Thigh, yes 12 (6,5) Shoulder, yes 11 (5,9) Thorax, yes 6 (3,2) Cervical, yes 5 (2,7) Wrist, yes 4 (2,2) Abdomen, yes 3 (1,6) Hand, yes 3 (1,6) Elbow, yes 2 (1,1) Arm, yes 2 (1,1) Forearm, yes 1 (0,5) Diagnosis STT, yes 119 (64,3) Lower limb fracture, dislocation, yes 28 (15,1) Head injury, yes 19 (10,3) Spinal injury, yes 10 (5,4) Upper limb fracture, dislocation, yes 4 (2,2) Cervical injury, yes 4 (2,2) Facial trauma, yes 4 (2,2) Abdominal injury, yes 2 (1,1) Thoracic injury, yes 3 (1,6) Pelvis fracture, yes 1 (0,5) Treatment method applied Medication and cold application 95 (51,4) Splint plaster 79 (42,7) Surgery 11 (5,9) Descriptive statistics are given as numbers (%). Data analysis Descriptive statistics—mean ± standard deviation, me- dian (width between quarters), and minimum-maximum Sahin and Batın BMC Emergency Medicine (2020) 20:58 Page 4 of 11 Table 1 Distribution of patients by age group, gender and year Age Mean ± SS 26,7 ± 6,2 Age n (%) between ages 19–23 81 (43,8) between ages 24–29 yaş 58 (31,4) 30 years and over 46 (24,9) Rank n (%) Private soldier 81 (43,7) Rank soldier 104 (56,3) Gender n (%) Male 184 (99,5) Female 1 (0,5) Patient application rates due to parachuting injury by years n (%) 2016 13 (7) 2017 73 (39,5) 2018 45 (24,3) 2019 54 (29,2) Proportion of parachuting injury to all other emergency patients by years n (%) 2016 13 (0,003) 2017 73 (0,016) 2018 45 (0.009) 2019 54 (0.015) Descriptive statistics are given as number (%) for categorical variables and mean ± standard deviation for numerical variables knee injuries, 6.5% of the thigh injuries and 5.9% of the shoulder injuries. In total, 64.3% of the injuries were diagnosed as soft tissue trauma (strain and stretching), followed by lower limb fracture- dislocation (15.1%), head trauma (10.3%), and spinal injury (5.4%). Regarding the treatment methods, 51.4% (n = 95) were given medication (Nonsteroidal anti- inflammatory drugs (NSAID), paracetamol pills and topical analgesic gel) and cold pack application, 42.7% (n = 79) received a plaster splint and 5.9% (n = 11) had surgery (Table 2, Figs. 1, 2 and 3). Thirteen of the participants (7%) had SBPs between 75 and 84 mmHg, 144 (77.8%) had SBPs between 86 and 100 mmHg and 28 (15.1%) had SBPs > 100 mmHg. PR was > 120/min in 32 patients (17.3%) and 51–119/min in 153 patients (82.7%). RR was < 10/min in 1 patient (0.5%), > 20/min in 17 patients (9.2%) and 10–12/min in 167 patients (90.3%). Regarding level of consciousness, 1 patient (0.5%) was confused, and the others were assessed as normal. Data analysis STT Soft Tissue Trauma Descriptive statistics are given as number (%) for categorical variables and mean ± standard deviation for numerical variables Descriptive statistics are given as number (%) for categorical variables and mean ± standard deviation for numerical variables knee injuries, 6.5% of the thigh injuries and 5.9% of the shoulder injuries. In total, 64.3% of the injuries were diagnosed as soft tissue trauma (strain and stretching), followed by lower limb fracture- dislocation (15.1%), head trauma (10.3%), and spinal injury (5.4%). Regarding the treatment methods, 51.4% (n = 95) were given medication (Nonsteroidal anti- inflammatory drugs (NSAID), paracetamol pills and topical analgesic gel) and cold pack application, 42.7% (n = 79) received a plaster splint and 5.9% (n = 11) had surgery (Table 2, Figs. 1, 2 and 3). g y g Thirteen of the participants (7%) had SBPs between 75 and 84 mmHg, 144 (77.8%) had SBPs between 86 and 100 mmHg and 28 (15.1%) had SBPs > 100 mmHg. PR was > 120/min in 32 patients (17.3%) and 51–119/min in 153 patients (82.7%). RR was < 10/min in 1 patient (0.5%), > 20/min in 17 patients (9.2%) and 10–12/min in 167 patients (90.3%). Regarding level of consciousness, 1 patient (0.5%) was confused, and the others were assessed as normal. One patient (0.5%) had a GCS of 13, 10 patients (5.5%) had a GCS of 14 and 174 patients (94%) had a GCS of 15. The mean ISS score of the patients was 5.16 ± 3.92 (minimum = 1, maximum = 25). The most frequent ISS trauma region was the extremity, followed by the pelvis Sahin and Batın BMC Emergency Medicine (2020) 20:58 Page 5 of 11 Fig. 1 Injury rates by site, according to the ISS anatomical classifications Fig. 1 Injury rates by site, according to the ISS anatomical classifications (67.6%, n = 125), the head/neck (8.6%, n = 16) and the chest (7.0%, n = 13). Injuries were rarely observed in the abdomen (1.6%, n = 3). in the body group were thoracic (66.7%, n = 6), abdom- inal (33.3%, n = 3) and cutaneous-subcutaneous superfi- cial (11.1%, n = 1) injuries (Table 3). Using the AIS classifications, 22.2% (n = 41) of the pa- tients’ injuries were mild, 62.2% (n = 115) were moder- ate, 15.1% (n = 28) were severe and 0.5% (n = 1) were critical; no deadly injuries were observed. Data analysis The median ISS score was 4, and the mean was 5.16 ± 3.92 (mini- mum = 1, maximum = 25) (Table 4). According to the ISS anatomical classification, shoul- der injuries (50%, n = 9) were the most commonly ob- served upper extremity injuries, followed by injuries to the whole upper extremity, the arm, the elbow, the hand and the wrist (the whole upper extremity n = 18). The most common lower extremity injuries were foot injuries (24.1%, n = 32), followed by ankle (13.5%, n = 18), leg (7.5%, n = 10), knee (5.3%, n = 7), thigh (3.8%, n = 5), whole lower extremity (31.6%, n = 42) and pelvis (14.3%, n = 19) injuries (Table 3). Although the average ISS scores (minimum = 1, max- imum = 25) increased with age, no statistically significant difference was observed when examining these scores by age group using one-way ANOVA. There was also no statistically significant relationship found between pa- tients’ age values and ISS values when using Spearman correlation (p = 0.108) (Table 5). Similarly, the most common head, neck, and spinal in- juries were to the spine (18.4%, n = 34), the head (13.5%), and the neck (1.1%). The most common injuries Fig. 2 Injury rates by sites, according to the our anatomical classifications Fig. 2 Injury rates by sites, according to the our anatomical classifications Sahin and Batın BMC Emergency Medicine (2020) 20:58 Page 6 of 11 Fig. 3 Diagnosis frequency of patients Meanwhile, regarding injury sites, the rate of leg injury in patients 30 years and older were found to be statisti- cally more significant than in patients between 24 and 29 years old (p = 0.043). Also, in patients between 19 and 23 years old, the rate of knee injury was statistically more significant than for those between 24 and 29 years old and those over 30 years old (p = 0.006). Furthermore, there were no statistical differences between the foot, ankle, thigh, pelvis, spine, abdomen, thorax, shoulder, hand, ankle, forearm, elbow, arm, cervical and head in- jury rates when compared by age group (p > 0.05 for each) (Table 6). There was a statistically significant difference in the hospital admission rates based on the AIS scores (p < 0.001); the hospitalization rate of patients with a critical AIS score was significantly higher than those with a se- vere AIS score. Data analysis Similarly, the difference between the me- dian ISS scores was statistically significant based on their hospitalization status (p < 0.001). Accordingly, the median ISS score was significantly higher among Table 3 Injury rates of upper-lower extremity, head/neck, spinal and body group according to the ISS anatomical classifications n (%) ISS Upper Extremity group Hand 1 (5,6) Wrist 1 (5,6) Elbow 2 (11,1) Arm 2 (11,1) Whole upper extremity 3 (16,7) Shoulder 9 (50) ISS Lower Extremity group Foot 32 (24,1) Ankle 18 (13,5) Leg 10 (7,5) Knee 7 (5,3) Thigh 5 (3,8) Whole lower extremity 42 (31,6) Pelvis 19 (14,3) ISS head and neck spinal group Head yes 25 (13,5) Neck, yes 2 (1,1) Spine, yes 34 (18,4) ISS Body group Thorax, yes 6 (66,7) Abdomen, yes 3 (33,3) Skin, subcutaneous superficial, yes 1 (11,1) Descriptive statistics are given as numbers (%) Table 4 AIS classifications and ISS scores n (%) AIS score Mild 41 (22,2) Moderate 115 (62,2) Severe 28 (15,1) Critical 1 (0,5) ISS score Median [IQR] 4 [4–8] Min.-Max 1–25 Descriptive statistics were given as number (%) for categorical variables and mean ± standard deviation and minimum-maximum for numerical variables Meanwhile, regarding injury sites, the rate of leg injury in patients 30 years and older were found to be statisti- cally more significant than in patients between 24 and 29 years old (p = 0.043). Also, in patients between 19 and 23 years old, the rate of knee injury was statistically more significant than for those between 24 and 29 years old and those over 30 years old (p = 0.006). Furthermore, there were no statistical differences between the foot, ankle, thigh, pelvis, spine, abdomen, thorax, shoulder, hand, ankle, forearm, elbow, arm, cervical and head in- jury rates when compared by age group (p > 0.05 for each) (Table 6). Discussion Various injuries can occur during parachute training and jumping. Due to advances in developing para- chute equipment and landing techniques, injuries tend to decrease day by day [5, 7–9]. Leggat and Smith stated that musculoskeletal disorders (MSD) are a common occurrence among soldiers and represent a valuable source of morbidity for the military as a whole. Intrinsic risk factors linked to military training injuries include a diverse range of inherent variables, such as the level of prior physical conditioning, psy- chological makeup, age, height, weight, and gender. Extrinsic risk factors for military MSDs include train- ing surface, exercising when fatigued, progressive training in place of cyclical training, and the type of footwear usually worn. Other military-specific vari- ables may include drill methods, the arrangement of platoons, training techniques, and the actual training distance [22]. In our study, we think that the rate of injury as a result of jumping from a parachute varies depending on the age, experience, landing technique, and the ground descended. Table 6 Comparison of injury sites by age group Table 6 Comparison of injury sites by age group Between ages 19–23 Between ages 24–29 30 and older p Injury site Foot, yes 31 (38,3) 21 (36,2) 10 (21,7) 0,144* Ankle, yes 27 (33,3) 19 (32,8) 8 (17,4) 0,127* Leg, yes 11 (13,6)a,b 3 (5,2)b 10 (21,7)a 0,043* Knee, yes 12 (14,8)a 1 (1,7)b 1 (2,2)b 0,006 Thigh, yes 7 (8,6) 2 (3,4) 3 (6,5) 0,504 Pelvis, yes 8 (9,9) 11 (19) 7 (15,2) 0,304* Spine, yes 13 (16) 14 (24,1) 7 (15,2) 0,390* Abdomen, yes 2 (2,5) 1 (1,7) 0 (0) 0,789 Thorax, yes 4 (4,9) 2 (3,4) 0 (0) 0,428 Shoulder, yes 3 (3,7) 6 (10,3) 2 (4,3) 0,266 Hand, yes 1 (1,2) 1 (1,7) 1 (2,2) 0,999 Wrist, yes 1 (1,2) 1 (1,7) 2 (4,3) 0,463 Forearm, yes 1 (1,2) 0 (0) 0 (0) 0,999 Elbow, yes 2 (2,5) 0 (0) 0 (0) 0,500 Arm, yes 0 (0) 1 (1,7) 1 (2,2) 0,318 Cervical, yes 4 (4,9) 1 (1,7) 0 (0) 0,316** Head, yes 8 (9,9) 6 (10,3) 11 (23,9) 0,059* Descriptive statistics were given as numbers (%) . Pearson Chi-Square test was used . Fisher Freeman Halton test was used The average age of the patients included in the study was 26.70 ± 6.24 years old. Data analysis Table 3 Injury rates of upper-lower extremity, head/neck, spinal and body group according to the ISS anatomical classifications n (%) ISS Upper Extremity group Hand 1 (5,6) Wrist 1 (5,6) Elbow 2 (11,1) Arm 2 (11,1) Whole upper extremity 3 (16,7) Shoulder 9 (50) ISS Lower Extremity group Foot 32 (24,1) Ankle 18 (13,5) Leg 10 (7,5) Knee 7 (5,3) Thigh 5 (3,8) Whole lower extremity 42 (31,6) Pelvis 19 (14,3) ISS head and neck spinal group Head yes 25 (13,5) Neck, yes 2 (1,1) Spine, yes 34 (18,4) ISS Body group Thorax, yes 6 (66,7) Abdomen, yes 3 (33,3) Skin, subcutaneous superficial, yes 1 (11,1) Descriptive statistics are given as numbers (%) There was a statistically significant difference in the hospital admission rates based on the AIS scores (p < 0.001); the hospitalization rate of patients with a critical AIS score was significantly higher than those with a se- vere AIS score. Similarly, the difference between the me- dian ISS scores was statistically significant based on their hospitalization status (p < 0.001). Accordingly, the median ISS score was significantly higher among Sahin and Batın BMC Emergency Medicine (2020) 20:58 Page 7 of 11 Table 5 Average ISS score by age group Tukey HSDa,b Age groups N Subset for alpha = 0.05 (ISS mean score) 1 Between 19 and 23 years old 81 4‚6667 Between24–29 years old 58 5‚0862 30 years old and above 46 6‚1304 Sig. ,108 Means for groups in homogeneous subsets are displayed aUses Harmonic Mean Sample Size = 58,450 bThe group sizes are unequal. The harmonic mean of the group sizes is used. Type I error levels are not guaranteed Table 7 Comparison of hospitalization rates based on AIS scores and ISS score medians and hospitalization duration, according to the AIS score Hospitalization Status p Yes No AIS score Mild 0 (0) 41 (100) < 0,001 Moderate 0 (0) 115 (100) Severe 10 (35,7) 18 (64,3) Critical 1 (100) 0 (0) ISS score 9 [9–18] 4 [4–6] < 0,001** Hospitalization days AIS score Mild – Moderate – Severe 3 [1–4] Critical 6 [6–6] . Fisher Freeman Halton test was used. Descriptive statistics are given as numbers (%) . Mann-Whitney U test was used. Data analysis Descriptive statistics are given as median [IQR] Table 7 Comparison of hospitalization rates based on AIS scores and ISS score medians and hospitalization duration, according to the AIS score hospitalized patients than those who were not hospital- ized (Table 7). While 5.9% (n = 11) of the patients received inpatient treatment, 94.1% (n = 174) discharged with outpatient treatment (NSAID or paracetamol pills, topical analgesic gel, and cold pack application). Furthermore, 54.5% (n = 6) of the inpatients had lower extremity injuries, 27.3% (n = 3) had upper extremity injuries and 18.2% (n = 2) had shoulder surgery, which was performed by ortho- paedist. Of these patients, 18.1% (n = 2) were hospital- ized in the ICU, and 81.9% (n = 9) were hospitalized in the ED. None of the hospitalized patients died. The aver- age length of hospitalization was 3.45 ± 2.20 days . Fisher Freeman Halton test was used. Descriptive statistics are given as numbers (%) *. Mann-Whitney U test was used. Descriptive statistics are given as median [IQR] (minimum = 1, maximum = 7). Also, in Table 7, the me- dian day of hospitalization of patients with a severe AIS score was 3 days, while the median day of hospitalization of critical patients was 6 days. . Pearson Chi-Square test was used **. Fisher Freeman Halton test was used Discussion In Ekeland’s study, the aver- age age was 24.5 ± 3.6 years old, and the age ranges in that study, and our study were similar [4]. The rate of injury increased with age in our study. This rate was similar to the literature but, severe and critical patient Sahin and Batın BMC Emergency Medicine (2020) 20:58 Page 8 of 11 Page 8 of 11 Page 8 of 11 rates were higher (15.1 and 0.5%, respectively) [4, 5]. This difference may be because more people participated in our study, and the average age of the participants was higher. landing techniques and the fact that most of the partici- pants were private soldiers, and the parachute experi- ence was low. Besides, the equipment of all of the jumpers was complete and suitable for jumping. Static, free fall and high altitude-low opening (HALO) jumps specified by the United States (US) Army as jumping techniques used by special forces [3]. In litera- ture studies, it was reported that 141 injuries detected during 134 HALO jumps [3, 8]. Meanwhile, in two US and British army studies, approximately 2.5% of para- chute jumps resulted in injuries, the majority of which were minor [1, 8]. The injury rate for static jumps has been specified as 8.1/1.000 [3]. The vitals of the majority of the participants in our study was generally stable, which was similar to the re- sults found in Ekeland’s study [4]. There are differences in the frequency of age and diag- nosis of injuries caused by parachuting in literature stud- ies. In our study, the patients were most frequently diagnosed with soft tissue trauma (sprain and stretching) (64.3%, n = 119) and lower limb fracture-dislocation (15.1%, n = 28). The percentage of these diagnoses is 64.3 and 15.1%, respectively. These rates were higher than those in a study by Craig et al. [3]. The reasons for this high rate in our study may be because most soldiers (43.7%) were between the ages of 19–23 and inexperi- enced of the soldiers, the landing technique problems, and the inappropriate landing area selection. In our study, patients aged 30 years old and older had a statisti- cally significantly higher leg injury rate than patients aged 24–29 years old (p = 0.043). Discussion In patients between 19 and 23 years old, the knee injury rate was statistically significantly higher than those between 24 and 29 years old and those over 30 years old (p = 0.006). These find- ings are similar to the literature, which we believe was due to increases in parachuting experience with age. However, proper techniques used during descent, age, and joint, bone, muscle, and ligament degeneration may also be factors in the development of leg injuries. Mean- while, we think that the reason for the higher rate of knee injuries among younger patients may have been due to not using the five-point technique. The patients in our study typically engaged in self- opening and low-altitude jumping. Since the total num- ber of people who jumped was not known, the injury rate could not be calculated. However, we did determine that, during the four-year study period, and an average of 0.011% of the ED patients admitted for parachute jumping-related trauma injuries. In our study, 22.2% of the 185 patients suffered mild injuries, while 62.2% had moderate, 15.1% had severe, and 0.5% had critical injur- ies. Besides, 43.7% of the participants were private sol- diers, and 56.3% were ranked soldiers. The ranks of those who jumped in detail in the literature studies were not fully specified, like our study. Scoring systems, such as GCS, AIS, ISS, TRISS, and RTS, are used to assess the severity of trauma patients in EDs [12, 13, 17, 18, 20]. We chose to use AIS and ISS scores, which can be easily calculated to evaluate the se- verity of injuries of patients and which we think is ap- propriate for crowded EDs such as ours. Since we had difficulties calculating the ISS score for multiple injuries in the same region, we assessed injury severity by divid- ing the body into 17 different anatomical areas, noting the injury areas and evaluating them using AIS and ISS. The mean of the participants ISS was 5.16 ± 3.92 in our study. This mean was lower than the mean in a study by Sozuer et al. (8.15 ± 4.29) [24]. We think that the reason for this difference is due to the more significant number of patients included in our study and the more severe in- juries of those patients. The rate of foot injury of the participants in our study was higher than in previous studies [4, 7, 10]. Discussion In our study, as the AIS and ISS scores increased, the rate of hospitalization in- creased. Therefore, the use of trauma scoring systems can be interpreted as facilitating the work of emergency staff in determining the severity of trauma, hospitalization decisions, and possible mortality risks. p p Previous studies in the literature on parachute injuries are usually in the form of a case report or a literature summary [1, 7, 9, 25, 26]. In our study, 28 patients had lower extremity fractures, four patients had upper ex- tremity fractures, and one patient had pelvic fractures. The fractures of the upper extremity related to para- chute jumping are generally reported as a humeral surgi- cal neck fracture or stable humerus fracture in Kirby’s study [27]. Also, in our study, the most common shoul- der injury was observed in the upper extremity, and the fracture was found in 4 people. Kirby was observed that the most common foot and ankle injuries were similar to our study [27]. In one study, the ankle injury rate was calculated as 0,45% [10]; in our study, the ankle injury rate was 29.1%. This injury rate difference may be due to the larger number of patients included in our study. In one study, the ankle injury rate was calculated as 4.5/ 1000 [10]; in our study, the ankle injury rate was 29.1%. This injury rate difference may be due to the larger number of patients included in our study. In a retro- spective study by Zakowski et al., the injury rate was 1.48–3.76/1000 jumps [26]. In our study, 11 people were operated on by orthopaedists, and 54.5% (n = 6) had lower extremity surgery, 27.3% (n = 3) had upper ex- tremity surgery and 18.2% (n = 2) had shoulder surgery. Of these patients, 18.1% (n = 2) were hospitalized in the ICU, and 81.9% (n = 9) were hospitalized in the ED. p p y In the literature, it has been stated that musculoskel- etal disorders (MSD) are a common occurrence for sol- diers and an essential source of morbidity for the army as a whole [22]. Regarding the average age of the partici- pants in our study, 43.8% were 19–23 years old, 31.4% were 24–29 years old, and 24.8% were 30 years old and older. Although there was an increase in ISS score with age in our study, there was no statistically significant dif- ference. Strengths and limitations g Our study has some limitations. Since we wanted to evaluate and compare cases of isolated parachute injur- ies, our study did not include a control group. We did not consider the jumpers how many times they had jumped before. And, how many soldiers had been in- jured on the same day were not questioned. We learned from the soldiers’ commander that all of the jumps oc- curred in the daytime. Since only soldiers who fall from the parachute during the day are included in our study, we do not know the injury characteristics that occur as a result of parachuting at night. In later studies, day and night parachute jumps can be compared. p In a study by Farrow, 63 of 8886 jumps resulted in death, and the injury rate was 7.1/1000 [5]. In our study, there were no fatal cases. Westman et al. examined deadly events due to free fall building-antenna-span- earth (BASE) jumping between 1981 and 2006 and found that the deaths were related to human, environ- ment and equipment problems, reverse airflow, loss of vision due to weather and acrobatic movements [6]. An- other study found that similar factors affected the injury rate and also found a high injury rate for night-time jumps [9]. Since the patients in our study did not per- form night-time jumps, the injury rate of night-time jumps could not be calculated. In the literature, an in- correct landing technique was mentioned as the most common cause of injury [4, 23]. Since the night jump was not performed in our study, the rate of injury in the night jump could not be calculated. It is pleasing that no fatal case was encountered in our study. It may be be- cause the soldiers did not prefer to jump out of the para- chute while the weather was windy and badly, and a lower altitude was preferred for training. We also think that all of the jumping during the daytime could be an effective factor on the absence of fatal injury. After the emergency treatments, only hospitalized pa- tients continued to be followed, and outpatients did not follow after they discharged from the ED For this reason, it did not know whether any of the patients had any complications after discharge. Besides, only parachute and related injuries were included in the study; para- gliding and balloon accidents did not included. Strengths and limitations Since all jumps were made in the form of a static line, injury rates due to free jumps were not studied. In terms of the strengths of the study, we conducted this prospective study over a 44-week observation period, included many cases, detailed the anatomical lo- cations of the parachute injuries, used two different scoring systems to assess the severity of the trauma, col- lected data on treatment measures and recorded the pa- tients’ hospital stay duration. Also, as the scoring systems were considered insufficient, we used more de- tailed classifications of the injury sites. Discussion In a com- prehensive study conducted by Ekeland, the most com- mon injury site was the ankle (36%) [4]. In our study, when the foot and ankle sites were evaluated together, the injury rate reached 62.6%. When the two injuries evaluated together, similar to this study, the rate of foot and ankle injury is most common. Similarly to our study, in a retrospective study by Ball et al., which examined injuries due to static parachute jumps, the lower extrem- ity was the most frequently injured body region [23]. The most commonly observed ISS anatomically trauma region was the extremity and the pelvis (67.6%, n = 125), followed by the head/neck (8.6%, n = 16) and the chest (7.0%, n = 13). Injuries rarely observed in the abdomen (1.6%, n = 3). The rates of injury site separation compared to ISS were higher in our study than in the lit- eratüre. Compared to the ISS anatomically trauma re- gion exposed, in our study, the most frequent was extremity and pelvis, with a rate of 67.6% (n = 125). Also, the least number of injuries observed in the abdomen (n = 3) with 1.6%. We have found that injury site separ- ation rates are higher than in previous literature studies Bricknell reported in his study that the injury rate in- creases when wind speed exceeds nine knots and during high altitude jumps [7]. In our study, we learned that from patients commanders, they each jumped with a self-opening mushroom-shaped static parachute from a plane at an altitude of approximately 400 m (1200 ft), using a five-point technique at 1–2 knots wind speed, 5.9–7.1 km/h landing speed, and they landed on flat ground. Although the wind speed was not high, we think that the injury rate was high, maybe due to improper Sahin and Batın BMC Emergency Medicine (2020) 20:58 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Page 9 of 11 Page 9 of 11 [3–5, 7, 24]. It may be because 43.7% of the injured are private soldiers who do not have sufficient experience. ISS scores [14, 16, 28, 29]. In our study, as the AIS and ISS scores increased, the rate of hospitalization in- creased. Therefore, the use of trauma scoring systems can be interpreted as facilitating the work of emergency staff in determining the severity of trauma, hospitalization decisions, and possible mortality risks. ISS scores [14, 16, 28, 29]. Discussion We think that the reason for this is that, as age increases, attention may decrease due to an increase in self-confidence as the person gains experience, which, in turn, may cause an increase in injury rates. Funding h h 16. Roy N, Gerdin M, Schneider E, Veetil DKK, Khajanchi M, Kumar V, et al. Validation of international trauma scoring systems in urban trauma centres in India. Injury. 2016;47(11):2459–64. The authors received no specific funding for this work. The authors covered all expenses of the study. 17. Greenspan L, McLELLAN BA, Greig H. Abbreviated injury scale and injury severity score: a scoring chart. J Trauma. 1985;25(1):60–4. 17. Greenspan L, McLELLAN BA, Greig H. Abbreviated injury scale and injury severity score: a scoring chart. J Trauma. 1985;25(1):60–4. Presentations d l h 11. Wisner DH. History and current status of trauma scoring systems. Arch Surg. 1992;127(1):111–7. 11. Wisner DH. History and current status of trauma scoring systems. Arch Surg. 1992;127(1):111–7. We declare that this study has not been previously published in another journal and not currently evaluated in another journal. 12. Maslanka AM. Scoring systems and triage from the field. Emerg Med Clin North Am. 1993;11(1):15–27. 12. Maslanka AM. Scoring systems and triage from the field. Emerg Med Clin North Am. 1993;11(1):15–27. Abbreviations AIS Abb i t d AIS: Abbreviated Injury Scale; APS: Anatomic Profile Score; ASCOT: A Severity Characterization of Trauma; GCS: Glasgow Coma Score; HALO: High Altitude- Low Opening; HBYS: Hospital Information Management System; ICU: Intensive Care Unit; ISS: Injury Severity Score; NSAID: Nonsteroidal anti- inflammatory drugs; NISS: New Injury Severity Score; OIS: Organ Injury Scale (OIS),; PR: Pulse Rate; RR: Respiratory Rate; RTS: Revised- Trauma Score; SBP: Systolic Blood Pressure; STT: Soft Tissue Trauma; TRISS: Trauma and Injury Severity Score; TRISSCOM: Trauma and Injury Severity Score Comorbidity 5. Farrow GB. Military static line parachute injuries. Aust N Z J Surg. 1992;62(3): 209–14. 6. Westman A, Rosén M, Berggren P, Björnstig U. Parachuting from fixed objects: descriptive study of 106 fatal events in BASE jumping 1981–2006. Br J Sports Med. 2008;42(6):431–6. 7. Bricknell MCM, Craig SC. Military parachuting injuries: a literature review. Occup Med (Chic Ill). 1999;49(1):17–26. 8. Glorioso JE Jr, Batts KB, Ward WS. Military free fall training injuries. Mil Med. 1999;164(7):526–30. 9. Knapik J, Steelman R. Risk factors for injuries during military static-line airborne operations: a systematic review and meta-analysis. J Athl Train. 2016;51(11):962–80. Conflict of interest disclosure 13. Senkowski CK, McKenney MG. Trauma scoring systems: a review. J Am Coll Surg. 1999;189(5):491–503. 13. Senkowski CK, McKenney MG. Trauma scoring systems: a review. J Am Coll Surg. 1999;189(5):491–503. TS and SB declared that there is no conflict of interest. 14. Baker SP, O’Neill B, Haddon WJ, Long WB. The injury severity score: a method for describing patients with multiple injuries and evaluating emergency care. J Trauma. 1974;14(3):187–96. 14. Baker SP, O’Neill B, Haddon WJ, Long WB. The injury severity score: a method for describing patients with multiple injuries and evaluating emergency care. J Trauma. 1974;14(3):187–96. Consent for publication Not Applicable. Consent for publication Not Applicable. Consent for publication Not Applicable. Not Applicable. Authors’ contributions Meeting and TS performed follow-up of patients in the emergency room. SB performed confirmation of diagnosis of patients. TS and SB determined to- gether what would be the treatment of patients. TS and SB did the literatüre review together. TS made data collection and analysis. TS, and SB read and approved the final format of the manuscript. TS drafted the manuscript. 15. Galvagno SM Jr, Massey M, Bouzat P, Vesselinov R, Levy MJ, Millin MG, et al. Correlation Between the Revised Trauma Score and Injury Severity Score: Implications for Prehospital Trauma Triage. Prehospital emergency care: official journal of the National Association of EMS Physicians and the National Association of State EMS Directors. 2019;23(2):263–270. https://doi. org/10.1080/10903127.2018.1489019. Acknowledgements We would like to thank the Kayseri City Hospital Trauma Unit Healtcare workers who assisted incollecting the cases. 10. Schumacher JTJ, Creedon JF, Pope RW. The effectiveness of the parachutist ankle brace in reducing ankle injuries in an airborne ranger battalion. Mil Med. 2000;165(12):944–8. References D Davison DJ. A review of parachuting injuries. Injury. 1990;21(5):314– 1. Davison DJ. A review of parachuting injuries. Injury. 1990;21(5):314–6. 2. Lucas J. The silken canopy: a history of the parachute. Airlife; 1997. 2. Lucas J. The silken canopy: a history of the parachute. Airlife; 1997. 3. Craig SC, Lee T. Attention to detail: injuries at altitude among U.S. Army Military Static Line Parachutists. Mil Med. 2000;165(4):268–71. Available from https://doi.org/10.1093/milmed/165.4.268. 4. Ekeland A. Injuries in military parachuting: a prospective study of 4499 jumps. Injury. 1997;28(3):219–22. Author details 1 1Emergency Medicine Clinic Kayseri City Education and Research Hospital affiliated with University of Health Sciences Turkey, 38090 Kayseri, Turkey. 2Orthopedist, Orthopedic Clinic Kayseri City Education and Research Hospital affiliated with University of Health Sciences Turkey, 38090 Kayseri, Turkey. As a result, parachuting and air sports are becoming more popular day by day. For the asses injury severity of the trauma caused by these jumps made for military or sports purposes and the planning of the treatment, we recommend that the emergency departments use a clas- sification of trauma scores systems alone, combined, or divide the body into more anatomical regions as we use. Received: 16 March 2020 Accepted: 23 July 2020 Received: 16 March 2020 Accepted: 23 July 2020 AIS: Abbreviated Injury Scale; APS: Anatomic Profile Score; ASCOT: A Severity Characterization of Trauma; GCS: Glasgow Coma Score; HALO: High Altitude- Low Opening; HBYS: Hospital Information Management System; ICU: Intensive Care Unit; ISS: Injury Severity Score; NSAID: Nonsteroidal anti- inflammatory drugs; NISS: New Injury Severity Score; OIS: Organ Injury Scale (OIS),; PR: Pulse Rate; RR: Respiratory Rate; RTS: Revised- Trauma Score; SBP: Systolic Blood Pressure; STT: Soft Tissue Trauma; TRISS: Trauma and Injury Severity Score; TRISSCOM: Trauma and Injury Severity Score Comorbidity Ethics approval and consent to participate pp p p Ethical approval was obtained for this study from the Kayseri City Education and Resources Hospital Research Committee by ID 16.11.2016/09 and Erciyes University Ethics Committee by ID 96681246/340 and adhered to the Declaration of Helsinki. Individual informed consent was taken verbally or writing from the participants in this study. Verbal consent was obtained from some participants by informing the ethics committee due to the disruptions in moving the hospital to its new location. The researchers recorded the emergency workers as witnesses from the participants who gave verbal consent and recorded them on the patient observation paper. On the other hand, emergency workers should be kept in mind that injuries due to air sports are more common in inexperienced people and that more severe injuries may occur in people with advanced age. Therefore, we suggest that military personnel over the age of 30 behave more cautiously when jumping from a parachute and es- pecially during a landing. We also believe that injury rates may decreased by correcting risk factors linked with the person and the environment. Conclusions According to the literature, hospitalization and mortal- ity are associated with increases in AIS and especially Various injuries can occur during parachute training and jumping. Due to advances in developing parachute Page 10 of 11 Page 10 of 11 Page 10 of 11 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Sahin and Batın BMC Emergency Medicine (2020) 20:58 equipment and landing techniques, injuries tend to de- crease day by day. We believe that parachute jumping- related injury rates will be reduced by increasing the first aid training given to parachute instructors and students and by further improving parachute jumping techniques. However, although new jumping techniques and equip- ment developed, we think that the most critical factors on the parachute-related injury are the human factors. Ethics approval and consent to participate Availability of data and materials 18. Baker SP, O’neill B. The injury severity score: an update. J Trauma Acute Care Surg. 1976;16(11):882–5. The datasets analyzed during the current study are available from the corresponding author on reasonable request. The datasets analyzed during the current study are available from the corresponding author on reasonable request. Page 11 of 11 Sahin and Batın BMC Emergency Medicine (2020) 20:58 Sahin and Batın BMC Emergency Medicine (2020) 20:58 19. Copes WS, Champion HR, Sacco WJ, Lawnick MM, Gann DS, Gennarelli T, et al. Progress in characterizing anatomic injury. J Trauma. 1990;30(10):1200–7. 20. Loftis KL, Price J, Gillich PJ. Evolution of the abbreviated injury scale: 1990– 2015. Traffic Inj Prev. 2018;19(sup2):S109–13. 2015. Traffic Inj Prev. 2018;19(sup2):S109–13. 21. Williams JR. The declaration of Helsinki and public health. Bull World Health Organ. 2008;86:650–2. 22. Leggat PA, Smith DR. Military training and musculoskeletal disorders. J Musculoskelet Pain. 2007;15(2):25–32. Available from. https://doi.org/10. 1300/J094v15n02_06. 23. Ball VL, Sutton JA, Hull A, Sinnott BA. Traumatic injury patterns associated with static line parachuting. Wilderness Environ Med. 2014;25(1):89–93. 24. Sozuer EM, Ozkan S, Akdur O, Durukan P, Ikizceli I, Avsarogullari L. Injuries due to parachute jumping. Ulus Travma Acil Cerrahi Derg. 2008;14(3):201–4. 25. Bourghli A, Fabre A. Proximal end clavicle fracture from a parachute jumping injury. Orthop Traumatol Surg Res. 2017;98(2):238–41. Available from:. https://doi.org/10.1016/j.otsr.2011.09.021. 25. Bourghli A, Fabre A. Proximal end clavicle fracture from a parachute jumping injury. Orthop Traumatol Surg Res. 2017;98(2):238–41. Available from:. https://doi.org/10.1016/j.otsr.2011.09.021. 26. Zakowski B, Wagner I, Domzalski M. Analysis of a military parachutist injury– a retrospective review of over 37,000 landings. Mil Med. 2019;184(1–2): e261–5. 26. Zakowski B, Wagner I, Domzalski M. Analysis of a military parachutist injury– a retrospective review of over 37,000 landings. Mil Med. 2019;184(1–2): e261–5. 27. Kirby CN. Parachuting injuries. Proc R Soc Med. 1974;67(1):17–21 28. Narcı A, Solak O, Turhan-Haktanır N, Ayçiçek A, Demir Y, Ela Y, et al. The prognostic importance of trauma scoring systems in pediatric patients. Pediatr Surg Int. 2009;25(1):25. 29. Mullins RJ, Veum-Stone J, Helfand M, Zimmer-Gembeck M, Hedges JR, Southard PA, et al. Outcome of hospitalized injured patients after institution of a trauma system in an urban area. Jama. 1994;271(24):1919–24. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2914003466	https://dergipark.org.tr/en/download/article-file/642205	English	null	Efficiency of a Herbal Liquid Extract Mixture for the Prevention of Salmonella Growth in Whipped Cream	Natural and engineering sciences	2,019	cc-by	4,686	* Corresponding Author: Özüm Özoğlu, e-mail: ozoglu@uludag.edu.tr Article history: y Received 07 September 2018, Accepted 26 November 2018, Available online 31 January 2019 Özüm Özoğlu1* and Evrim Güneş Altuntaş2 Özüm Özoğlu1* and Evrim Güneş Altuntaş2 1Department of Food Engineering, Faculty of Agriculture, Uludağ University, 16059 Bursa, Turkey 2Ankara University, Biotechnology Institute, System Biotechnology Advanced Research Unit Tandoğan Campus, 06560, Ankara, Turkey 1Department of Food Engineering, Faculty of Agriculture, Uludağ University, 16059 Bursa, Turkey 2Ankara University, Biotechnology Institute, System Biotechnology Advanced Research Unit Tandoğan Campus, 06560, Ankara, Turkey NESciences, 2019, 4(1): 65-75 NESciences, 2019, 4(1): 65-75 Abstract In this study, a herbal liquid extract mixture (ASATİM® ST 1412; recommended for milk-based product and whipped cream by company, Kayseri, Turkey) was used to exhibit the inhibitory effect on Salmonella Enteritidis growth in whipped cream. For this aim, 8 different groups (K1, K2, K3, K4, K5, 0.1%, 1%, 10%) were designed and the samples were left 1 to 3 hours at room temperature. Then, the bacterial counts were obtained. The experiment was performed in duplicate. In the first experiment number of the bacteria of whipped cream (K1) showed roughly 1 logarithmic unit rising as from 4.6 to 5.5 in 3 hours. Calculated results were at the end of the 3rd hour for cream added Salmonella and the herbal liquid extract mixture of 0.1%, 1%, 10% to the samples; 3x107cfu/g, 2.05x108cfu/g and 1.3x105cfu/g respectively for the first experiment. Then, in the results of second experiment these values were found as <108cfu/g, 4.5x108cfu/g and 3.5x104cfu/g respectively. Thus, 10% ratio herbal liquid extract mixture was much more effective on Salmonella growth than the other ratios. According to the results of this study, the commercial herbal liquid extract mixture is an alternative and natural method for precluding Salmonella growth in whipped cream. Keywords: Salmonella, whipped cream, herbal liquid, extract mixture Article history: Article history: Introduction Pastry products with cream are commonly used in the food and confectionery industries (Sharifzadeh, Hajsharifi-Shahreza, & Ghasemi-Dehkordi, 2016). Some foods like muscle meat Natural and Engineering Sciences 66 have natural barriers which prevent or delay to the attack of microorganisms on the inner parts, while desserts and cakes containing milk or cream which are milk-based products are suitable media with high nutrient content, water activity (aw) and pH values for microbial growth (Sharifzadeh et al., 2016; Alişarlı et al., 2002). Besides, pastry creams tend to contamination with several pathogenic microorganisms because of their ingredients and production methods like using raw cream (Sharifzadeh et al., 2016; Alişarlı et al., 2002; Ray, 2004; Pajohi-Alamoti et al., 2016; Kotzekidou, 2013). Therefore; pastry cream is the main cause of food poisoning among humans that is an important and common problem all around the world and Salmonella is one of the major pathogenic microorganisms in the pastry cream (Sharifzadeh et al., 2016; Ray, 2004; Pajohi- Alamoti et al., 2016). Salmonella is a member of Enterobacteriaceae family, Gram-negative, nonsporulating, mesophile, facultative anaerobic microorganism and has more than 2000 serovars (Ray, 2004; Mahmoud, 2012). Although Salmonella is sensitive to low pH, some strains can survive in low pH due to fact that they have acid-resistance (Ray, 2004). Salmonella is a heat-sensitive pathogen and it can be eliminated with heat processes (Mahmoud, 2012; Kavaz Yüksel & Yüksel, 2015). This pathogen is found in the environment such as water, soil, sewage and gastrointestinal tract of animals and humans (Ray, 2004; Mahmoud, 2012). Meat, poultry, fish, egg, milk, dairy products, fruits and vegetables are transmission vector of Salmonella (Ray, 2004; Kotzekidou, 2013; Mahmoud, 2012; Kavaz Yüksel & Yüksel, 2015; Paião et al., 2013; Newell et al., 2010; Holliday et al., 2003). The most common recorded cause of diarrheal illnesses is Salmonella contagion in industrialized countries and all over the world (Ray, 2004; Kotzekidou, 2013; Mahmoud, 2012). In addition to that, the Ministry of Health in Italy was reported that Salmonella was the most important reason of the outbreaks (Argenio et al., 1999). Salmonella causes to Salmonellosis (Salmonella infection), which is infected by consuming Salmonella-contaminated foods and it can be fatal (Ray, 2004; Kotzekidou, 2013; Mahmoud, 2012; Kavaz Yüksel & Yüksel, 2015; Paião et al., 2013; Newell et al., 2010; Holliday et al., 2003). Introduction Therefore, it is essential to apply microbial control to foods such as pastry cream, which are suitable media for Salmonella growth based on both health and industrial prospects (Pajohi-Alamoti et al., 2016; Mahmoud, 2012). While Salmonella infection is prevented with lots of different methods, including adding antibiotic or antimicrobial compounds such as spice or essential oil (Mahmoud, 2012; Kavaz Yüksel & Yüksel, 2015; Paião et al., 2013), last studies have been focused on using spice liquid extract for preventing Salmonella growth, because of increasing antibiotic resistance of some food borne Salmonella strains (Mahmoud, 2012; Amrutha et al., 2017; Bernbom et al., 2009; Mahgoub et al., 2013; Perumalla & Hettiarachchy, 2011; Sanchez et al., 2006; Kelati et al., 2017). A chemical compound of the plant extract or essential oils has various important antimicrobial ingredients which are terpenoides, phenols, alkaloids and flavanoids (Mahmoud, 2012; Botsoglou et al., 2003; Exarchou et al., 2002; Burt et al., 2007). These components have a hydrophobicity which provides them to divide in the lipids of the cell membrane and mitochondria making them more permeable (Mahmoud, 2012; Burt et al., 2007). An example of these plants is a garlic (Allium sativum) that is used against to pathogenic bacteria, including Salmonella, especially in fish products (Bernbom et al., 2009; Kumar & Berwal, 1998). The other examples to benefit from antibacterial characteristics of medicinal and aromatic plants’ extracts as Rumex acetosella, Achillea millefolium, Plantago Lanceolata (Burt et al., 2007; Kokoska et al., 2002; Macit & Köse, 2015; Shale et al., 1999; Stewart, 1996; Yaldız et al., 2010). Rumex acetosella is used as digestive, nutritive and cure of Natural and Engineering Sciences 67 wounds and bruises. But also its leave extract has antibacterial activity on both of Gram-negative and Gram-positive bacteria such as Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae (Hızlısoy et al., 2010; Shale et al., 1999). Achillea millefolium is consumed as painkiller, diuretic, antiviral, vermifural, antifebrile, neuroleptic, cure of prostate and as many similar treatment (Hızlısoy et al., 2010; Yaldız et al., 2010). In addition to these, extract of Achillea millefolium is effective on bacteria such as Bacillus cereus and Staphylococcus aureus (Kokoska et al., 2002). Plantago Lanceolata is used for cure of wound, eczema, callus, ambustion and for some other statements (Hızlısoy et al., 2010; Macit & Köse, 2015). Introduction Phenylpropanoid glycoside verbascoside which is found Plantago Lanceolata has an antimicrobial activity. Besides, antimicrobial compounds of Plantago Lanceolata affect to inhibit rumen fermentation and volatile acid composition of it. Furthermore, Plantago Lanceolata can change milk composition (Stewart, 1996). In the light of this information, this study aimed using a commercial herbal liquid extract mixture which is composed of sorrel extract (Rumex acetosella), millfoil extract (Achillea millefolium), ribwort plantain extract (Plantago Lanceolata) (ASATİM® ST 1412;Kayseri, Turkey) for the preventing of Salmonella growth in whipped cream which is used commonly in the food industry. The Herbal Liquid Extract Mixture and Bacteria In the current study, a herbal liquid extract mixture containing sorrel extract (Rumex acetosella), millfoil extract (Achillea millefolium), ribwort plantain extract (Plantago Lanceolata) (ASATİM® ST 1412; recommended for milk-based product and whipped cream by company, Kayseri, Turkey) was used to exhibit the inhibitory effect on Salmonella Enteritidis ATCC 13076 growth in whipped cream (prepared under sterile conditions with milk as the company proposed; 75g powder whipped cream is mixed with 200 mL UHT milk). Dropping Plate Technique Eight different groups which were designed as total concentration of each one at 10 mL like that only pastry cream as K1, cream with Salmonella as K2, cream added only the herbal liquid extract mixture at 0.1%, 1% and 10% ratio as K3, K4, K5 respectively, cream added Salmonella and the herbal liquid extract mixture of 0.1%, 1%, 10% to the samples were called with their herbal liquid extract mixture. The samples were left 1, 2 and 3 hours at room temperature. Furthermore, there were three samples for each 8 groups by each hour and diluted them seven-fold serial dilution for enumeration. The test bacteria, Salmonella, was adjusted to 0.4 (O.D.600) which was calculated equal to 106cfu/mL in order to add to the samples. After that, dropping plate technique was performed by adding 0.01 mL proportion of each samples on to the Tryptic Soy Agar plates in duplicate. After inoculation, incubation temperature and time was chosen as 37 ̊C for 24 hours in accordance with the optimum growth condition of the bacterium. Natural and Engineering Sciences 68 Counting and Calculation of the Colonies After 37 ̊C for 24 hour-incubation process, occured colonies were counted according to seen clearly and denumerability. Thus, colony forming units were calculated with regard to the following formula (Halkman & Ayhan, 2000): Results The experiment was performed in two recurrences and the data obtained in this study are given in Table 1 and Table 2. The results of bacterial counts belong to only whipped cream as K1, cream with Salmonella as K2, cream added herbal liquid extract mixture at 0.1%, 1% and 10% ratio as K3, K4, K5 respectively, cream added Salmonella and herbal liquid extract mixture of 0.1%, 1%, 10% to the samples at the end of 1st, 2nd and 3rd hour for first experiment (Table 1). Table 1. The results of bacterial counts belong to only whipped cream. 1st Hour 2nd Hour 3rd Hour N(cfu/g) Log N(cfu/g) Log N(cfu/g) Log K1 4.0x104 4.6 1.9x105 5.3 3.4x105 5.5 K2 1.5x108 8.2 1.0x108 8.0 2.5x108 8.4 K3 1.6x105 5.2 2.7x105 5.4 3.0x105 5.5 K4 1.7x105 5.2 3.1x105 5.5 8.6x104 4.9 Table 1. The results of bacterial counts belong to only whipped cream. Natural and Engineering Sciences 69 K5 4.5x104 4.7 7x104 4.8 1.4x105 5.2 0.1% 3.0x108 8.5 2.8x107 7.4 3.0x107 7.5 1% 1.5x108 8.2 8.6x106 6.9 2.1x108 8.3 10% 3.8x104 4.6 2.7x105 5.4 1.3x105 5.1 As far as the Table 1, number of the bacteria of K1 which is non-added bacteria to whipped cream sample was calculated as 4x104cfu/g at the end of the 1st hour and then almost 1 logarithmic unit increasing was observed and the colony number has reached to 3.4x105cfu/g at the end of the 3rd hour. This means that, whipped cream has microbial risk when it is waited in room temperature. The other remarkable result was observed in the sample containing 10% herbal liquid extract mixture and 106cfu/g Salmonella as well. The amount of bacteria was calculated as 3.8x104cfu/g, 2.7x105cfu/g and 1.3x105cfu/g at the end of the 1st, 2nd and 3rd hours respectively. The calculated number of the bacteria of K5 which is 10% herbal liquid extract and the bacteria-free whipped cream mixture was similar to the sample containing 10% herbal liquid extract mixture and 106cfu/g Salmonella except for 2 hours due to the fact that there was not a fluctuation. Furthermore, there was a slight increasing in the following hours as 4.5x104cfu/g, 7x104cfu/g and 1.4x105cfu/g at the end of the 1st, 2nd and 3rd hours respectively. Results Likewise, the amount of bacteria of K3 sample increased proportionally from 1 to 3 hours, but it had higher amounts bacteria than K5 because of containing 0.1% ratio the herbal liquid extract mixture. When the number of the bacteria of K2 was investigated; it was calculated as 1.5x108cfu/g at the end of the 1st hour. Then, it decreased at 1x108cfu/g surprisingly at the end of the 2nd hour and it reached as 2.5x108cfu/g at the end of the 3rd hour. The very similar result was observed in the sample containing 1% herbal liquid extract mixture and 106cfu/g Salmonella, it decreased and then rose over 3-hour period. The number of bacteria of K4 and added 0.1% herbal liquid extract mixture and 106cfu/g Salmonella in whipped cream were shown a decrease from the end of the 1st hour to end of the 3rd hour. To sum up, none of the samples which were treated with the herbal liquid extract mixture changed as much as K1 (containing only whipped cream) that was shown 1 logarithmic unit increasing. The results of the first experiment was proved that the herbal liquid extract mixture with different ratio is effective in Salmonella growth in whipped cream. The results of bacterial counts belong to only whipped cream as K1, cream with Salmonella as K2, cream added herbal liquid extract mixture at 0.1%, 1% and 10% ratio as K3, K4, K5 respectively, cream added Salmonella and herbal liquid extract mixture of 0.1%, 1%, 10% to the samples at the end of 1st, 2nd and 3rd hour for second experiment (Table 2). Natural and Engineering Sciences 70 Table 2. The results of bacterial counts belong to only whipped cream. 1st Hour 2nd Hour 3rd Hour N(cfu/g) Log N(cfu/g) Log N(cfu/g) Log K1 <103 <3.0 1.0x104 4.0 <103 <3.0 K2 4.5x109 9.7 2.0x108 8.3 2.5x109 9.4 K3 2.5x105 5.4 3.0x105 5.5 3.4x105 5.5 K4 <103 <3.0 1.0x105 5.0 1.8x106 6.3 K5 <103 <3.0 8.6x105 5.9 <103 <3.0 0.1% 4.5x108 8.7 2.0x109 9.3 <108 <8.0 1% 1.5x108 8.2 <107 <7.0 4.5x108 8.7 10% <103 <3.0 3.5x105 5.5 3.5x104 4.5 Table 2. The results of bacterial counts belong to only whipped cream. Results With reference to the Table 2, number of the bacteria of the samples which were entreated with the herbal liquid extract mixture (0.1%, 1% and 10 %) have not never reached too high amount like K2 which was bacteria and whipped cream mixed. It was observed that, somehow number of the bacteria did not change too much such as nearly 1 logarithmic unit, the others were roughly 2 logarithmic unit, increasing between 1st and 2nd hours like K4, K5 and the sample containing 10% herbal liquid extract mixture and the bacteria. When considered K1, the number of the bacteria was almost stable in 3-hour period, which were calculated as <103, 1x104cfu/g, <103 respectively. The result was shown a little alteration by comparing with results of first experiments. It can be said that, the lowest number of the bacteria is in the sample containing 10% herbal liquid extract mixture and 106cfu/g Salmonella according to the table and also it is seen in Figure 1. In other words, the most effective amount of the herbal liquid extract mixture was 10% portion for holding down the number of bacteria. Which is a very similar result with the first trial. When compared the data obtained in the study, it can be said clearly the bacterial numbers were not calculated same for both two experiments. The reason of it should be that Salmonella has a quick and different growth rate at the suitable media. However, it is also obvious that the sample containing herbal extract at 10% has a significant inhibitory effect on Salmonella. Natural and Engineering Sciences 71 Figure 1. Counted colonies of the second experiment, after 37 ̊C for 24 hours incubation of the sample containing 10%, 1% and 0.1% herbal liquid extract mixture and 106cfu/g Salmonella at top- down which were seen clearly and denumerability. A, D and G: The Petri dishes incubated for 1 hour. B, E and H: The Petri dishes incubated for 2 hours. C,F and I: The Petri dishes incubated for 3 hours. Figure 1. Counted colonies of the second experiment, after 37 ̊C for 24 hours incubation of the sample containing 10%, 1% and 0.1% herbal liquid extract mixture and 106cfu/g Salmonella at top- down which were seen clearly and denumerability. A, D and G: The Petri dishes incubated for 1 hour. B, E and H: The Petri dishes incubated for 2 hours. Results C,F and I: The Petri dishes incubated for 3 hours. Discussion The results of that study are shown similarity with the other studies which are used the same herbal extract in the studies. For example; in 2010, Hızlısoy H. et al. demonstrated that 5% diluted of the herbal extract is an effective cleaning of chicken carcasses from Campylobacter jejuni (Hızlısoy et al., 2010). According to another study which was done by Tornuk F. et al. (2011) was indicated that washing treatment of raw meat surface with the herbal extract reduced the number of Listeria monocytogenes and Escherichia coli O157:H7 almost 3 log cfu/cm2 (Tornuk et al., 2011). Also, in the literature some experiments are seen remarkable when compared to our study. One of them was performed by A. Sharifzadeh et al. (2016) and they showed evaluation of microbial contamination (for coliforms, E. coli, Staphylococcus aureus, and yeast) of cream-filled pastries in confectioneries of Chaharmahal Va Bakhtiari (Iran) with their study that the number of Natural and Engineering Sciences 72 bacteria (total) was 8.21 logarithmic units (Sharifzadeh et al., 2016). Also, in the study of P. Kotzekidou (2013); samples of desserts with dairy cream were analysed and according to the results; aerobic colony counts ranged from 103 to <109 cfu/g and the majority of samples (76.7%) contained Enterobacteriaceae with population dissipation in the range of 103 to <107 cfu/g; some samples had enterococci and Staphylococcus spp. which was counted as >105 cfu/g (Kotzekidou, 2013). Similarly, M. Alişarlı et al. (2002) searched about the determination of the microbiological quality of some dairy desserts. They calculated the number of microorganisms in cream, pastries higher than other milk-based desserts (Alişarlı et al., 2002). Thus, it can be deduced that pastry creams are suitable media for microorganism growth and they can cause food poisoning. According to the study of S.L. Holiday et al. (2003) in which sweet cream whipped salted butter, sweet cream whipped unsalted butter, salted light butter, yellow fat spread and light margarine were used as designing products to determine the survival of pathogens, five serotypes of Salmonella which were Anatum, Enteritidis, Montevideo, Muenchen, and Typhimurium were inoculated two different types. These were high humidity and temperature abuse studies consisted of 20 mL of mixed-serotype or mixed-strain suspensions deposited in each indentation on the surface of test products followed by exposure to 85% relative humidity at 37 ̊C for 1 h and temperature. Discussion And physical abuse studies consisted of 2 mL of mixed-serotype or mixed strain suspensions added to 200 g of warm (37 ̊C) test products and pummeled for 3 min. The bacterial numbers were 7.22, 7.82, 7.26, 7.32, 7.11 7.27 logarithmic units for 1 hour incubation and 3.91, 4.26, 4.47, 4.41, 4.37, 4.22 logarithmic units for 3 minutes incubation respectively for S.Anatum, S.Enteritidis, S.Montevideo, S.Muenchen, S.Typhimurium and mixture type (Holliday et al., 2003). When these results compared to ours the results of K2 samples were more higher, due to the higher first inoculation amount of the bacteria. On the other hand, the data of these studies are close and they show that dairy or milk-based products, such as butter and whipped cream are convenient to Salmonella growth. M. Kumar and J.S. Berwal (1998) calculated minimum inhibitory concentration (MIC) of garlic (Allium sativum) against Staphylococcus aureus, Salmonella Typhi, Escherichia coli and Listeria monocytogenes. Then the results were found like that E. coli and Staph. aureus, inhibition occurred quickly and up to the 5% level of garlic, there was an almost 80% inhibition and then, a significant inhibition was seen, for L. monocytogenes, an important fall in growth was observed with rising garlic level and finally for Salmonella Typhi, a rapid reduction in growth was seen, with 50% inhibition at 1% garlic and 90% inhibition at 10% (Kumar & Berwal, 1998). Besides, Z. Kalaycıoğlu et al. (2017) demonstrated that, turmeric extract incorporation increased the physical and antibacterial characteristics of chitosan films due to the fact that, turmeric incorporated chitosan film decreased by 2 logarithmic units to number of Salmonella over 3 hour periods, although only chitosan film decreased that roughly 1 logarithmic unit (Kalaycıoğlu et al., 2017). E. Kakouri et al. (2017) indicated that minimum 1.5 mg of Crocus sativus L. tepals hydrolysed extract has antimicrobial effect on Enterococcus faecium, Listeria monocytogenes, Staphylococcus xylosus, St. simulans, Salmonella enterica and Escherichia coli by using Well Diffusion Assay (Kakouri et al., 2016). Y. Shahbazi (2016) found that Ziziphora clinopodioides essential oil (ZEO) in 2% portion and nisin at 500 IU mL-1 had an important antibacterial effect on Salmonella Typhimurium and Staphylococcus aureus growth in milk (Shahbazi, 2016). These studies and the current study indicated that certain amounts of herbal extracts are significant effects on pathogen Natural and Engineering Sciences 73 microorganisms which contaminate food. Discussion Therefore, using herbal extracts should be one of the important solutions for preventing microorganism growth in foods. microorganisms which contaminate food. Therefore, using herbal extracts should be one of the important solutions for preventing microorganism growth in foods. As far as the results of the present study and other studies in the literature, milk products such as whipped creams are pretty suitable growth media for pathogen microorganisms especially Salmonella. Thus, some treatments are necessary for like as these food products. Then, using of herbal mix extracts on the degenerative foods is a novel and effective method. According to our study, it can be reported that 10% ratio of herbal liquid extract mixture in whipped cream has the most inhibitory effect on the bacteria when the result compared with the control sample. The producing company of the herbal liquid extract mixture proposes that the herbal liquid extract mixture should be added from 0.2% to 1% ratio to milk-based product and whipped cream. This approach is acceptable, so that in this study, high level of Salmonella was inoculated to the samples at the beginning which is an unusual situation. In conclusion, in the daily use of whipped cream sample we can offer that in case of any risk, adding this herbal extract will be helpful to avoid food poisonings. Acknowledgments This study was supported by ASATİM® Kayseri, Turkey and carried out at Ankara University Biotechnology Institute System Biotechnology Advanced Research Unit Laboratory. References Alişarlı, M., Sancak, Y. C., Akkaya, L., & Elibol, C. (2002). 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https://openalex.org/W2035311489	https://www.scielo.br/j/rbpv/a/TkQFRLdr6MJb35KsXTsHkXs/?lang=en&format=pdf	English	null	Effects of destruxin A on Rhipicephalus (Boophilus) microplus ticks (Acari: Ixodidae)	Revista Brasileira de Parasitologia Veterinária/Brazilian Journal of Veterinary Parasitology	2,011	cc-by	3,261	Resumo Este estudo avaliou os efeitos da destruxina A em fêmeas de Rhipicephalus (Boophilus) microplus, uma vez que essa toxina é uma das prováveis causas da alta mortalidade induzida pelo fungo entomopatogênico Metarhizium anisopliae em artrópodes. Os carrapatos foram imersos ou inoculados com diferentes concentrações de destruxina A. Apesar das doses aplicadas, não houve mortes ou alterações significativas de postura entre os grupos tratados com destruxinas A e os grupos controle. Nenhum outro efeito externo provocado pela destruxina A, tal como paralisia tetânica, foi observado nas fêmeas ingurgitadas de carrapato após o tratamento. Palavras-chave: Metarhizium anisopliae, metabólitos fúngicos, controle biológico. Efeitos da destruxina A no carrapato Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Patrícia Silva Gôlo1; Isabele da Costa Angelo2; Mariana Guedes Camargo1; Wendell Marcelo de Souza Perinotto1; Vânia Rita Elias Pinheiro Bittencourt1* 1Postgraduate Program on Veterinary Sciences, Department of Animal Parasitology, Veterinary Institute, Federal Rural University of Rio de Janeiro – UFRRJ rtment of Epidemiology and Public Health, Veterinary Institute, Federal Rural University of Rio de Janeiro – UFRRJ Received February 18, 2011 Accept March 4, 2011 Abstract This study evaluated the effects of destruxin A on Rhipicephalus (Boophilus) microplus females, since this toxin is one of the likely causes of high mortality induced by the entomopathogenic fungus Metarhizium anisopliae in arthropods. Ticks were immersed or inoculated with different concentrations of destruxin A. Despite the doses applied, there were no deaths or significant alterations in oviposition between the groups treated with destruxin A and the control groups. No other external effect caused by destruxin, such as tetanic paralysis, was observed in these engorged female ticks after the treatment. Keywords: Metarhizium anisopliae, fungal metabolites, biological control. Research Note Research Note Research Note Rev. Bras. Parasitol. Vet., Jaboticabal, v. 20, n. 4, p. 338-341, out.-dez. 2011 ISSN 0103-846X (impresso) / ISSN 1984-2961 (eletrônico) Rev. Bras. Parasitol. Vet., Jaboticabal, v. 20, n. 4, p. 338-341, out.-dez. 2011 ISSN 0103-846X (impresso) / ISSN 1984-2961 (eletrônico) Corresponding author: Vânia Rita Elias Pinheiro Bittencourt Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro – UFRRJ, Rod. BR 465, Km 7, CEP 23890-000, Seropédica, RJ, Brasil e-mail: vaniabit@ufrrj.br Corresponding author: Vânia Rita Elias Pinheiro Bittencourt Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro – UFRRJ, Rod. BR 465, Km 7, CEP 23890-000, Seropédica, RJ, Brasil e-mail: vaniabit@ufrrj.br www.cbpv.com.br/rbpv Introduction The target organs of these cyclic peptides in insects generally include the gut, Malpighian tubules and circulating hemocytes (DUMAS et al., 1996). The reports on insects also include different modes of action, such as contact, antifeedant and growth regulation modes. Nevertheless, there are no reports on the effects of destruxin on ticks. Moreover, clarification of the mechanisms involved in the process of tick infection by fungi is a crucial step towards developing new biological agents that can be used against these ectoparasites. hf VAINSTEIN, 2010). They differ in the R-group of the hydroxyl acid residue and appear to have overlapping but different biological effects. These effects include insecticidal, herbicidal and antiviral ones (HU; REN, 2004). However, the mechanism through which destruxin production increases fungal pathogenicity is not totally understood. Studies have reported that destruxins may be involved with insect-cuticle dissolution, immune system suppression and interference in host ion channels (causing tetanic paralysis), as well as other cell dysfunctions (DUMAS et al., 1996; VILCINSKAS et al., 1997). The target organs of these cyclic peptides in insects generally include the gut, Malpighian tubules and circulating hemocytes (DUMAS et al., 1996). The reports on insects also include different modes of action, such as contact, antifeedant and growth regulation modes. Nevertheless, there are no reports on the effects of destruxin on ticks. Moreover, clarification of the mechanisms involved in the process of tick infection by fungi is a crucial step towards developing new biological agents that can be used against these ectoparasites. After treatment, the engorged females were placed in Petri dishes, labeled and incubated at 27 ± 1 °C and RH ≥ 80%. Some biological parameters of the females were analyzed to determine the effects of destruxin A on R. (B.) microplus. The egg mass laid by each female was weighed daily and placed into individual test tubes. The eggs were then incubated at the same temperature and RH, to allow the larvae to hatch. The main parameters studied were: oviposition period (OP), hatching percentage (HP), egg production index (EPI) and nutrient index (NI). The EPI was calculated as the ratio between the total weight of eggs and the initial weight of engorged females and the NI was calculated as the ratio between the total weight of eggs and the subtraction between the initial and residual female weights (BENNETT, 1974). Introduction The parametric data (OP) were assessed using analysis of variance followed by the Student-Newman-Keuls test. The nonparametric data (NI, EPI and HP) were assessed using the Kruskal-Wallis test followed by Student’s t test. P values less than 0.05 were considered to be significant. i To detect whether there was any tetanic paralysis, the movement of the engorged females’ legs was observed. The purpose of this study was to evaluate the effects of destruxin A from M. anisopliae on engorged R. (B.) microplus females, using immersion and inoculation methods for infection. This communication reports the first comprehensive bioassays on destruxins acting against ticks. To test the viability and effectiveness of the destruxin A used in these bioassays, larvae of Galleria mellonella, a species known to be sensitive to this toxin (DUMAS et al., 1996; ROBERTS, 1966; VILCINSKAS et al., 1997), were also subjected to treatment. The larvae were provided by the Brazilian Agriculture and Livestock Research Company (Empresa Brasileira de Pesquisa Agropecuária; Embrapa), from the Embrapa Dairy Cattle Research Unit, Juiz de Fora, MG, Brazil. Twelve final-instar G. mellonella larvae (around 250 mg) were distributed into two groups. In the first group, 50 mL of sterile distilled water was applied to the body surface of each larva and the same volume of destruxin solution, at 10 ppm, was applied to each larva in the second group. The bioassays were performed twice. Material and Methods Rhipicephalus (Boophilus) microplus colony was maintained at the Federal Rural University of Rio de Janeiro, which is located in Seropédica, RJ, Brazil. To prepare the destruxin solution, one milligram of destruxin A from M. anisopliae (Sigma®; D4921 Destruxin A) was diluted in 1 mL of sterile distilled water. Firstly, 200 mL of acetone PA (Merck®) was added to optimize the dilution, followed by 500 mL of sterile distilled water. After stirring, the solution was placed in a water bath for acetone evaporation. The final volume was then made up to 1 mL by adding 500 mL of water. Results There was no statistically significant difference (P ≥ 0.05) between the groups treated with destruxin A and the control groups. In other words, not only was there no contact virulence, but also the females’ biological parameters that were analyzed did not differ from those of the control groups, even when the toxin was injected. The data are shown in Tables 1 and 2. The treated engorged females did not present any paralysis just after infection. For the immersion bioassays on engorged females, four groups containing six engorged female ticks of similar weights were tested: a control group and three treatment groups using destruxin A at concentrations of 5, 10 or 20 parts per million (ppm). The destruxin solutions were diluted, according to each concentration, in sterile distilled water plus 0.05% Tween 80. In the control group, the engorged females were immersed only in sterile distilled water with 0.05% Tween 80. Each engorged female was immersed in the solution for five minutes, as described by Drummond et al. (1973), with modifications. Galleria mellonella larvae treated with destruxin A presented immediate tetanic paralysis, while the water-treated group did not suffer any paralysis. This result indicates that the destruxin A solutions were viable. i In the inoculation bioassay, a perforation was made at the body insertion of the fourth leg, using a hypodermic needle. The inoculum was individually calculated based on the weight of each female, as 0.075, 0.15 and 0.3 mg of destruxin per gram of tick. Two control groups were established for the inoculation bioassay: one composed of engorged females injured by a needle, with no inoculation, and the other composed of females inoculated with 5 mL of physiological solution. Each inoculated group was composed of six engorged female ticks, of similar weights. The immersion and inoculation bioassays were performed twice. Introduction The infection pathway of M. anisopliae consists of attachment of the spore to the cuticle, germination and formation of appressoria, thereby leading to penetration through the cuticle. After overcoming the host’s response and immune defense reactions, this fungus spreads within the host by forming hyphal bodies, outgrowing the dead host and producing new conidia (ZIMMERMANN, 2007). While spreading within the host, the fungus produces secondary metabolites, which have a toxic effect on the host. Thus, the efficiency of this fungus is due not only to its physical proliferation, but also to its chemical action. Studies on insects have shown that there is a direct relationship between M. anisopliae virulence and destruxin production (PAL et al., 2007). However, the mechanisms for this effectiveness against ticks are unclear. The cattle tick Rhipicephalus (Boophilus) microplus causes significant economic losses to cattle farmers due to reduced milk and meat production, slower growth of infested animals and disease transmission. In Brazil, these economic losses have been estimated as two billion dollars a year (GRISI et al., 2002). Tick control is generally based on the use of chemical acaricides, but their continual application and improper use have many negative side effects, including the development of chemical resistance in tick populations and food and environmental contamination. Biological control is an alternative to the use of chemical acaricides. Of all the entomopathogenic fungal genera and species that have been tested, Metarhizium anisopliae is among the most often investigated, because of its potential for controlling tick species worldwide (FERNANDES; BITTENCOURT, 2008). Destruxins are toxic secondary metabolites produced by entomopathogenic fungi, including M. anisopliae, and are considered to have insecticidal properties. This group of cyclic depsipeptides is composed of five amino acids and one a-hydroxy acid. Thirty‑eight destruxin analogues have been reported to date (SCHRANK; www.cbpv.com.br/rbpv Effects of destruxin A on ticks v. 20, n. 4, out.-dez. 2011 339 VAINSTEIN, 2010). They differ in the R-group of the hydroxyl acid residue and appear to have overlapping but different biological effects. These effects include insecticidal, herbicidal and antiviral ones (HU; REN, 2004). However, the mechanism through which destruxin production increases fungal pathogenicity is not totally understood. Studies have reported that destruxins may be involved with insect-cuticle dissolution, immune system suppression and interference in host ion channels (causing tetanic paralysis), as well as other cell dysfunctions (DUMAS et al., 1996; VILCINSKAS et al., 1997). Discussion Although in this study the doses applied to the female ticks were above the LD50 for insects (AMIRI et al., 1999; THOMSEN; EILENBERG, 2000; SREE et al., 2008), the females also remained alive and did not show any change during the oviposition process. Similarly, Hu et al. (2009) reported there was no significant difference in oviposition shown by the whitefly Bemisia tabaci Gôlo, P.S. et al. Rev. Bras. Parasitol. Vet. 340 Table 1. Biological parameters of engorged Rhipicephalus (Boophilus) microplus females immersed in destruxin A and incubated at 27 ± 1 °C and RH ≥ 80%. Group OP (days) EPI (%) HP (%) NI (%) control 17.5 ± 2.72ª 62.84 ± 8.79ª 98.63 ± 0.74ª 81.14 ± 9.69ª 5 ppm 16.88 ± 2.53ª 61.94 ± 7.09ª 98.25 ± 0.71ª 79.9 ± 10.32ª 10 ppm 17.63 ± 2.06ª 62.84 ± 8.21ª 96.25 ± 6.6ª 81.93 ± 9.83ª 20 ppm 15.88 ± 4.36ª 49.82 ± 20.44ª 85.25 ± 29.08ª 79.23 ± 10.24ª OP: oviposition period; HP: hatching percentage; EPI: egg production index; NI: nutrient index; ppm: parts per million. The data are expressed as mean ± stand ard deviation. Means followed by the same letter in the same column do not differ statistically (P ≥ 0.05). ephalus (Boophilus) microplus females immersed in destruxin A and incubated at 27 ± 1 °C of engorged Rhipicephalus (Boophilus) microplus females immersed in destruxin A and incubated at 27 ± 1 °C Table 1. Biological parameters of engorged Rhipicephalus (Boophilus) microplus females immersed in destruxi and RH ≥ 80%. ogical parameters of engorged Rhipicephalus (Boophilus) microplus females immersed in destruxin A and incub % Table 2. Biological parameters of engorged Rhipicephalus (Boophilus) microplus females inoculated with destruxin A (mg per gram of tick) and incubated at 27 ± 1 °C and RH ≥ 80%. References AMIRI, B.; IBRAHIM, L.; BUTT, T. M. Antifeedant properties of destruxins and their potential use with the entomogenous fungus Metarhizium anisopliae for improved control of crucifer pests. Biocontrol Science and Technology, v. 9, n. 4, p. 487-498, 1999. http://dx.doi. org/10.1080/09583159929451 AMIRI, B.; IBRAHIM, L.; BUTT, T. M. Antifeedant properties of destruxins and their potential use with the entomogenous fungus Metarhizium anisopliae for improved control of crucifer pests. Biocontrol AMIRI, B.; IBRAHIM, L.; BUTT, T. M. Antifeedant properties of destruxins and their potential use with the entomogenous fungus Metarhizium anisopliae for improved control of crucifer pests. Biocontrol p p p Science and Technology, v. 9, n. 4, p. 487-498, 1999. http://dx.doi. org/10.1080/09583159929451 p p p Science and Technology, v. 9, n. 4, p. 487-498, 1999. http://dx.doi. org/10.1080/09583159929451 BENNETT, G. F. Oviposition of Boophilus microplus (CANESTRINI) (ACARIDA:IXODIDAE) I. Influence of tick size on egg production. Acarologia, v. 16, n. 1, p. 52-61, 1974. PMid:4463680. Bioassays using other destruxin analogues are required in order to determine the real role of production of fungal secondary metabolites and their effects on ticks. DRUMMOND, R. O. et al. Boophilus annulatus and B. microplus: laboratory tests of insecticides. Journal of Economic Entomology, v. 66, n. 1, p. 130-133, 1973. PMid:4690254. DUMAS, C. et al. Comparative study of the cytological aspects of the mode of action of destruxins and other peptidic fungal metabolites on target epithelial cells. Journal of Invertebrate Pathology, v. 67, n. 2, p. 137-146, 1996. http://dx.doi.org/10.1006/jipa.1996.0021 DUMAS, C. et al. Comparative study of the cytological aspects of the mode of action of destruxins and other peptidic fungal metabolites on Discussion Group OP (days) EPI (%) HP (%) NI (%) IJ 17 ± 1.77ª 61.98 ± 6.27ª 96.0 ± 3.93ª 79.92 ± 7.35ª INps 16.5 ± 2.88ª 54.84 ± 6.82ª 96.0 ± 3.93ª 75.28 ± 5.32ª INd 0.075 16.25 ± 1.28ª 59.37 ± 8ª 96.88 ± 1.55ª 79.22 ± 10.33ª INd 0.15 16.13 ± 1.81ª 57.24 ± 10.98ª 97.25 ± 3.45ª 77.55 ± 11.85ª INd 0.3 16.38 ± 1.69ª 56.91 ± 12.21ª 98.0 ± 1.31ª 77.86 ± 11.81ª OP: oviposition period; HP: hatching percentage; EPI: egg production index; NI: nutrient index; IJ: females only injured by the needle; INd: females inoculated with destruxin; INps: females inoculated with physiological solution. The data are expressed as mean ± standard deviation. Means followed by the same letter in the same column do not differ statistically (P ≥ 0.05). f engorged Rhipicephalus (Boophilus) microplus females inoculated with destruxin A (mg per gram of tick) and ≥80% Table 2. Biological parameters of engorged Rhipicephalus (Boophilus) microplus females inoculated with destruxin A (mg per gram of tick) and incubated at 27 ± 1 °C and RH ≥ 80%. phalus (Boophilus) microplus females inoculated with destruxin A (mg per gram of tick) and OP: oviposition period; HP: hatching percentage; EPI: egg production index; NI: nutrient index; IJ: females only injured by the needle; INd: females inoculated with destruxin; INps: females inoculated with physiological solution. The data are expressed as mean ± standard deviation. Means followed by the same letter in the same column do not differ statistically (P ≥ 0.05). between a group treated with destruxins and the control group. Since the toxicity of molecules is usually attributed to the interaction between the substance and its target protein (HU et al., 2009), the results suggest that target proteins were absent from the organs or tissues relating to the tick oviposition process. Furthermore, based on these results, destruxin A production was not a determining factor for entomopathogenic fungal virulence towards R. (B.) microplus females, since the hosts’ biological parameters did not change. This is an important observation with regard to clarifying the process of fungal infection in tick biological control. Acknowledgements target epithelial cells. Journal of Invertebrate Pathology, v. 67, n. 2, p. 137-146, 1996. http://dx.doi.org/10.1006/jipa.1996.0021 We are grateful to the Embrapa Dairy Cattle Research Unit (Embrapa Gado de Leite, Juiz de Fora, MG, Brazil), and especially to Dr. Marcia Prata and the PhD student Caio de Oliveira Monteiro, for providing the G. mellonella larvae used in this study. This research was supported by grants from the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico; CNPq) and the Carlos Chagas Filho Research Support Foundation of the State of Rio de Janeiro (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro; FAPERJ). We also thank the Coordination Office for Advancement of University-level Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; CAPES). FERNANDES, E. K. K.; BITTENCOURT, V. R. E. P. Entomopathogenic fungi against South American tick species. Experimental and Applied Acarology, v. 46, n. 1-4, p. 71-93, 2008. PMid:18563593. http://dx.doi. org/10.1007/s10493-008-9161-y GRISI, L. et al. Impacto econômico das principais ectoparasitoses em bovinos no Brasil. A Hora Veterinária, v. 21, n. 125 p. 8-10, 2002. HU, Q.; REN, S. Review of destruxins of Metarhizium anisopliae Sorokin. Chinese Journal of Biological Control, v. 20, n. 4, p. 234‑242, 2004. HU, Q. et al. Toxicities of destruxins against Bemisia tabaci and its natural enemy Serangium japonicum. Toxicon, v. 53, n. 1, p. 115-121, 2009. PMid:19000916. http://dx.doi.org/10.1016/j.toxicon.2008.10.019 Effects of destruxin A on ticks v. 20, n. 4, out.-dez. 2011 341 PAL, S.; LEGER, R. J. S.; WU, L. P. Fungal peptide destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster. Journal of Biological Chemistry, v. 282, n. 12, p. 8969-8977, 2007. PMid:17227774. http://dx.doi.org/10.1074/jbc. M605927200 Management Science, v. 64, n. 2, p. 119-125, 2008. PMid:17935266. http://dx.doi.org/10.1002/ps.1480 THOMSEN, L.; EILENBERG, J. Time-concentration mortality of Pieris brassicae (Lepidoptera: Pieridae) and Agrotis segetum (Lepidoptera: Noctuidae) larvae from different destruxins. Environmental THOMSEN, L.; EILENBERG, J. Time-concentration mortality of Pieris brassicae (Lepidoptera: Pieridae) and Agrotis segetum (Lepidoptera: Noctuidae) larvae from different destruxins. Environmental Entomology, v. 29, n. 5, p. 1041-1047, 2000. http://dx.doi. org/10.1603/0046-225X-29.5.1041 p p g g p p Noctuidae) larvae from different destruxins. Environmental Entomology, v. 29, n. 5, p. 1041-1047, 2000. http://dx.doi. org/10.1603/0046-225X-29.5.1041 ROBERTS, D. W. Toxins from entomogenous fungus Metarhizium anisopliae: II. Symptoms and detection in moribund hosts. Journal of Invertebrate Pathology, v. 8, n. 2, p. 222-227, 1966. http://dx.doi. Acknowledgements org/10.1016/0022-2011(66)90132-7 ROBERTS, D. W. Toxins from entomogenous fungus Metarhizium anisopliae: II. Symptoms and detection in moribund hosts. Journal of p y p Invertebrate Pathology, v. 8, n. 2, p. 222-227, 1966. http://dx.doi. org/10.1016/0022-2011(66)90132-7 VILCINSKAS, A.; MATHA, V.; GÖTZ, P. Inhibition of phagocytic activity of plasmatocytes isolated from Galleria mellonella by entomogenous fungi and their secondary metabolites. Journal of Insect Physiology, v. 43, n. 5, p. 475-483, 1997. http://dx.doi.org/10.1016/ S0022-1910(96)00120-5 SCHRANK, A.; VAINSTEIN, M. H. Metarhizium anisopliae enzymes and toxins. Toxicon, v. 56, n. 7, p. 1267-1274, 2010. PMid:20298710. http://dx.doi.org/10.1016/j.toxicon.2010.03.008 SREE, K. S.; PADMAJA, V.; MURTHY, Y. L. N. Insecticidal activity of destruxin, a mycotoxin from Metarhizium anisopliae (Hypocreales), against Spodoptera litura (Lepidoptera: Noctuidae) larval stages. Pest ZIMMERMANN, G. Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Science and Technology, v. 17, n. 9, p. 879-920, 2007. http://dx.doi.org/10.1080/09583150701593963
https://openalex.org/W2466183535	https://vestnikramn.spr-journal.ru/jour/article/download/492/463	Russian	null	Intramedullary Elastic Transphyseal Tibial Osteosynthesis and Its Effect on Segmental Growth	Vestnik Rossijskoj akademii medicinskih nauk	2,015	cc-by	6,422	АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ DOI: 10.15690/vramn.v70.i4.1410 Российский научный центр «Восстановительная травматология и ортопедия» им. акад. Г.А. Илизарова, Курган, Российская Федерация Интрамедуллярный эластичный трансфизарный остеосинтез большеберцовой кости и его влияние на рост сегмента Интрамедуллярный трансфизарный эластичный остеосинтез применяется у детей при заболеваниях, сопровождающихся сниженны- ми прочностными свойствами костной ткани, в первую очередь при несовершенном остеогенезе. Цель исследования: изучить рост боль- шеберцовой кости в условиях трансфизарного встречного проведения эластичных стержней без нарушения целостности кости, при моделировании поперечного перелома, а также сочетании трансфизарного армирования и поднадкостничного расположения титано- вой сетки с вплетенными в нее эластичными стержнями. Методы: проведено нерандомизированное контролируемое исследование на 3 группах (сериях) щенков (по 6 животных в каждой серии). В серии I производили встречное трансфизарное армирование большеберцовой кости, в серии II — трансфизарное армирование в сочетании с поперечной остеотомией костей голени, в серии III трансфизарный эла- стичный остеосинтез выполнялся вместе с поднадкостничным введением сетки из никелида титана с вплетенными в нее эластичными стержнями при поперечной остеотомии костей голени. Результаты: трансфизарное армирование вызывало замедление роста опери- рованной большеберцовой кости с потерей длины на 3,8 мм (p =0,078) в I, на 7,8 мм (p =0,032) во II и на 7,7 мм (p =0,042) в III серии. Эксцентричное проведение трансфизарных стержней формировало угловую деформацию в процессе последующего роста, в среднем на 7° (p =0,023) на уровне дистального эпифиза во всех сериях. Пери- и эндостальные реакции способствовали увеличению диаметра диа- физа большеберцовой кости в серии II на 3,9 мм (p =0,037), в серии III — на 3,8 мм (p =0,041) по сравнению с контрольной конечностью. В первой серии разницы диаметров между оперированной и интактной большеберцовой костью не наблюдалось. Заключение: интра- медуллярное трансфизарное армирование замедляет рост кости в длину. Для предотвращения угловых деформаций расположение телескопических систем должно быть максимально близким к центру ростковых зон. Поднадкостничное армирование не замедляет консолидацию фрагментов и может сочетаться с интрамедуллярным трансфизарным остеосинтезом. К ф й й й 441 Ключевые слова: трансфизарный эластичный остеосинтез, несовершенный остеогенез. (Для цитирования: Попков Д.А., Кононович Н.А., Мингазов Э.Р., Шутов Р.Б., Барбье Д. Интрамедуллярный эластичный трансфизарный остеосинтез большеберцовой кости и его влияние на рост сегмента. Вестник РАМН. 2015; 70 (4): 441–449. Doi: 10.15690/vramn.v70.i4.1410) D.A. Popkov, N.A. Kononovich, E.R.Mingazov, R.B. Shutov, D. Barbier Russian Ilizarov Scientiﬁ c Center «Restorative Traumatology and Orthopaedics», Kurgan, Russian Federation D.A. Popkov, N.A. Kononovich, E.R.Mingazov, R.B. Shutov, D. Barbier Russian Ilizarov Scientiﬁ c Center «Restorative Traumatology and Orthopaedics», Kurgan, Russian Federation Intramedullary Elastic Transphyseal Tibial Osteosynthesis and Its Effect on Segmental Growth Background: Intramedullary transphyseal elastic osteosynthesis is used in children for the diseases accompanied by the reduced strength proper- ties of bone tissue, and primarily for osteogenesis imperfecta. Objective: The purpose of the experimental study was to investigate tibial growth under the conditions of transphyseal counter-directed insertion of elastic rods without bone integrity breaking, under transverse fracture model- ing, as well as under combining transphyseal reinforcement and subperiosteal positioning the titanium mesh with the elastic rods intervolved in it. Methods: Non-randomized controlled trial was performed. Three series of experiments performed in 18 puppies. Counter-directed transphyseal reinforcement of tibia performed in Series I,, transphyseal reinforcement combined with transverse osteotomy of leg bones — in Series II, trans- physeal elastic osteosynthesis and subperiosteal positioning the titanium nickelide mesh with intervolved in it elastic rods during transverse leg bone osteotomy performed in Series III. Results: Transphyseal reinforcement resulted in growth retardation of the operated tibia. The loss of residual growth was 3.8 mm (p =0.078) in series I; 7.8 mm (p =0.032) — in series II; 7.7 mm (p =0.042) — in series III. Eccentric insertion of transphyseal rods formed an angular deformity (mean value 7°; p =0.023) of the distal tibial epiphysis in the process of residual growth. Periosteal and endosteal reactions contributed to enlargement of diaphyseal diameter of 3.9 mm (series II; p =0.037) and 3.8 mm (series III; p =0.041). Any difference of diameter between operated and intact tibia was not observed in series I. Conclusion: Intramedullary transphyseal reinforcement retards longitudinal bone growth. The positioning of the telescopic systems should be as close as possible to the center of growth plates in order to prevent angular deformities. Subperiosteal reinforcement doesn’t retard consolidation of fragments, and it can be combined with intramedullary transphyseal osteosynthesis. Key words: transphyseal elastic osteosynthesis, osteogenesis imperfecta. (For citation: Popkov D.A., Kononovich N.A., Shutov R.B., Barbier D. Intramedullary Elastic Transphyseal Tibial Osteosynthesis and Its Effect on Segmental Growth. Vestnik Rossiiskoi Akademii Meditsinskikh Nauk = Annals of the Russian Academy of Medical Sciences. 2015; 70 (4): 441–449. Doi: 10.15690/vramn.v70.i4.1410) (For citation: Popkov D.A., Kononovich N.A., Shutov R.B., Barbier D. Intramedullary Elastic Transphyseal Tibial Osteosynthesis and Its Effect on Segmental Growth. Vestnik Rossiiskoi Akademii Meditsinskikh Nauk = Annals of the Russian Academy of Medical Sciences. 2015; 70 (4): 441–449. Обоснование ничного расположения титановой сетки с вплетенными в нее эластичными стержнями. ничного расположения титановой сетки с вплетенными в нее эластичными стержнями. Системные заболевания скелета, сопровождающиеся cниженными прочностными свойствами костной ткани, особенно несовершенный остеогенез, крайне часто ос- ложняются патологическими переломами, деформаци- ями и неравенством длины конечностей [1–3]. Sofield и Millar первыми предложили для коррекции деформаций при несовершенном остеогенезе выполнять трансфи- зарный интрамедуллярный остеосинтез [1]. При этом интрамедуллярно введенный материал остается на весь период роста, выполняя роль внутреннего тутора на время сращения костных фрагментов, а также предотвращая по- явление новых деформаций. Данный принцип хирургиче- ской профилактики и лечения деформаций конечностей остается актуальным [2]. Intramedullary Elastic Transphyseal Tibial Osteosynthesis and Its Effect on Segmental Growth Doi: 10.15690/vramn.v70.i4.1410) 28.09.2015 12:09:15 28.09.2015 12:09:15 flat Vestnik 4-2015.indd 441 flat Vestnik 4-2015.indd 441 flat Vestnik 4-2015.indd 441 flat Vestnik 4-2015.indd 441 ВЕСТНИК РАМН /2015/ 70 (4) Дизайн исследования Проведено нерандомизированное контролируемое исследование на 18 щенках. Животных параллельно раз- делили на 3 группы (серии) в зависимости от вида опера- тивного вмешательства. Критерии соответствия Основным критерием включения являлись возраст и общее состояние животных. Условия проведения В 1987 г. Métaizeau для лечения обычных переломов трубчатых костей у детей, а также для предотвращения формирования деформаций при системных заболева- ниях скелета предложил использовать интрамедулляр- ное встречное трансфизарное эластичное армирование (flexible intramedullary nailing) [3]. Преимуществом эла- стичных стержней является возможность их использо- вания при любом диаметре костномозгового канала, что особенно важно у детей в возрасте до 5–6 лет. Трансфизарное расположение стержней предполагает их прохождение через зоны роста. Следовательно, вы- бор оптимальных участков введения интрамедуллярных фиксаторов имеет важное клиническое значение в ор- топедии детского возраста. Однако экспериментальные работы в области влияния дозированного повреждения ростковых пластинок немногочисленны и не позволяют прийти к единому мнению в вопросе определения оп- тимального пространственного расположения трансфи- зарных стержней [4–6]. Такая ситуация требует экспе- риментального обоснования методики трансфизарного армирования. Эксперименты выполнены на базе вивария РНЦ «Восстановительная травматология и ортопедия» им. акад. Г.А. Илизарова (Курган). Животных содержали в индивидуальных боксах (по одному). Получали одина- ковые стандартные, сбалансированные по питательным веществам, корма и чистую питьевую воду. Каждое жи- вотное в группе было идентифицировано индивидуаль- ным четырехзначным номером. Все хирургические мани- пуляции осуществлены в условиях операционной одной хирургической бригадой. 442 Продолжительность исследования Период наблюдения составил 180 сут после вы- полнения оперативного вмешательства, по окончанию которого биологический возраст животных соответ- ствовал 12 мес. Анализировали результаты исследова- ния, полученные непосредственно перед выполнением экспериментов (контрольная точка Д0, соответствую- щая предоперационному периоду) и через 180 сут по- сле его начала (контрольная точка М6, т.е. 6 мес после операции). Другим нерешенным вопросом остается сохранение функций тутора трансфизарно проведенных спиц по мере их расхождения в средней части кости в процессе естественного роста сегмента. Единственным решением в настоящее время остается замена спиц на более длинные для предотвращения деформаций и/или патологических переломов [3, 6]. Однако до сих пор отсутствует предло- женный и тем более экспериментально апробированный способ пролонгации функции тутора интрамедуллярной (или комбинированной) системы. flat Vestnik 4-2015.indd 442 flat Vestnik 4-2015.indd 442 р р р д р р Методы статистического анализа данных • длину большеберцовых костей (рис. 2а); Полученные количественные данные подвергали статистической обработке с использованием програм- мы AtteStat 13.1 (Россия). Статистическое исследование включало в себя описательную статистику: средние зна- чения (М) и стандартное отклонение (SD). Сравнитель- ные исследования производили с применением критерия Стьюдента и Вилкоксона для независимых (для проверки равенства средних между показателями правой и левой конечности) и парных (для проверки различий между двумя выборками парных изменений, в т.ч. проверки ра- венства средних, между показателями одной конечности в начале и конце эксперимента) выборок. Разницу изме- рений значений между исследователями оценивали с по- мощью коэффициента вариации. Различия показателей считали статистически значимыми при р < 0,05. • величину перекреста проксимальной и дистальной спицы в костномозговом канале (рис. 2б); • поперечный размер диафиза большеберцовой кости в средней трети (см. рис. 2а); • механический медиальный проксимальный угол (см. рис. 2а) большеберцовой кости (mecanical Medial а б Рис. 2. Схема расположения изучаемых параметров. Примечание. а — длина большеберцовой кости, поперечный размер диафиза большеберцовой кости в средней трети, угол mMPTA, угол mLDTA, б — величина перекреста проксимальной и дистальной спицы в костномозговом канале. а Методы регистрации исходов Примечание. а — превентивный интрамедуллярный остеосинтез, б — при поперечной остеотомии диафиза, в — при поперечной остеотомии диафиза и в сочетании с поднадкостничным арми- рованием. Примечание. а — превентивный интрамедуллярный остеосинтез, б — при поперечной остеотомии диафиза, в — при поперечной остеотомии диафиза и в сочетании с поднадкостничным арми- рованием. Основные исходы исследования были получены путем выполнения остеометрии большеберцовых костей по их рентгеновским изображениям. Измерения производил каждый соавтор работы независимо друг от друга. Допол- нительные исходы выявляли рентгенологическим и кли- ническим методом. Рентгенологические исследования правых и левых голеней выполняли в прямой и боковой проекции с использованием рентгеновского аппарата VEP X Technology Premium VET (Испания). остеосинтеза титановыми стержнями поднадкостнично располагали сетку из никелида титана марки TN-10 (тол- щина нити 150 мкм, диаметр ячеек 2–2,5 мм) с титано- выми стержнями. Сетка перекрывала зону остеотомии большеберцовой кости по всей длине окружности на про- тяжении 2 см проксимальнее и 2 см дистальнее от концов отломков. Прямые титановые стержни (n =3) диаметром 2 мм были заранее равномерно распределены в сетке и ориентированы параллельно оси сегмента. Надкостницу отдельными узловыми швами фиксировали к сетке. 443 Описание оперативного вмешательства Всем животным удаляли шерстный покров на правой тазовой конечности. Животных наркотизировали путем внутривенного введения тиопентала натрия в рекомен- дуемых дозах. В серии I (рис. 1а) для выполнения встречного бипо- лярного трансфизарного эластичного интрамедуллярно- го остеосинтеза неповрежденной правой большеберцо- вой кости использовали по 2 предварительно изогнутых стержня (титановые спицы) диаметром 1,8 мм и длиной 10 см. Радиус изгиба спиц составлял 40°. Одну из спиц наркотизированным животным вводили через прокси- мальный паратендинозный доступ. Эту спицу проводили антеградно через центр проксимального эпифиза больше- берцовой кости. Вторую спицу вводили навстречу преды- дущей ретроградно эксцентрично на уровне медиальной лодыжки. Свободные концы спиц U-образно изгибали и инпактировали в медиальном направлении в эпифизы с целью предотвращения миграции стержней внутрь кости по мере роста сегмента. Таким образом, вопрос совершенствования транс- физарных систем остается актуальным. Кроме того, при клиническом использовании отсутствуют количествен- ные данные влияния трансфизарно проведенных эле- ментов на продольный рост конечности, формирование эпифизов армированных сегментов или развитие угловых деформаций. Наше экспериментальное исследование было на- правлено на изучение следующих вопросов: оптималь- ное расположение трансфизарных стержней, влияние их на продольный рост и развитие суставных концов оперированного сегмента, а также разработка принци- пиально нового способа продления свойств внутрен- него тутора добавлением поднадкостничного армиро- вания. В серии II (рис. 1б) выполняли поднадкостничную по- перечную остеотомию середины диафиза большеберцо- вой кости вибропилой и остеотомию малоберцовой кости долотом. После этого осуществляли биполярный транс- физарный эластичный интрамедуллярный остеосинтез (аналогично серии I). Целью исследования было изучить рост большебер- цовой кости в условиях трансфизарного встречного про- ведения эластичных стержней неповрежденной кости и при моделировании поперечного перелома, а также в сочетании трансфизарного армирования и поднадкост- В серии III (рис. 1в) после остеотомий берцовых костей и интрамедуллярного эластичного стабильного 28.09.2015 12:09:15 28.09.2015 12:09:15 Этическая экспертиза До начала экспериментальных исследований было получено одобрение Комитета по этике РНЦ «ВТО» им. акад. Г.А. Илизарова на их выполнение (протокол № 5 (30) заседания от 14.11.2012 г.). Дополнительные исходы исследования Дополнительные исходы исследования Во всех сериях дополнительно оценивали функцио- нальное состояние конечности. В сериях II и III отмечали сроки консолидации костных отломков и возможные де- формации сегментов в результате возможных вторичных смещений. б Рис. 1. Рентгенограммы костей голени экспериментальных животных, интрамедуллярный эластичный трансфизарный осте- осинтез. flat Vestnik 4-2015.indd 443 flat Vestnik 4-2015.indd 443 Принципы расчета размера выборки Для достижения поставленной цели в периоды экс- перимента, обозначенные контрольными точками, опре- деляли: АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ а б в Рис. 1. Рентгенограммы костей голени экспериментальных животных, интрамедуллярный эластичный трансфизарный осте- осинтез. Примечание. а — превентивный интрамедуллярный остеосинтез, б — при поперечной остеотомии диафиза, в — при поперечной остеотомии диафиза и в сочетании с поднадкостничным арми- рованием б Proximal Tibial Angle, mMPTA), измеряемый между механической осью сегмента и линией, соединяющей 2 точки (вершины) вогнутых суставных поверхностей мыщелков большеберцовой кости [7]; Proximal Tibial Angle, mMPTA), измеряемый между механической осью сегмента и линией, соединяющей 2 точки (вершины) вогнутых суставных поверхностей мыщелков большеберцовой кости [7]; Proximal Tibial Angle, mMPTA), измеряемый между механической осью сегмента и линией, соединяющей 2 точки (вершины) вогнутых суставных поверхностей мыщелков большеберцовой кости [7]; Proximal Tibial Angle, mMPTA), измеряемый между механической осью сегмента и линией, соединяющей 2 точки (вершины) вогнутых суставных поверхностей мыщелков большеберцовой кости [7]; • механический латеральный дистальный угол (см. рис. 2а) большеберцовой кости (mecanical Lateral Distal Tibial Angle m, LDTA), образуемый механиче- ской осью сегмента и суставной линией [7]; • расположение эластичных стержней на уровне физи- сов относительно их срединных осей в сагиттальной и фронтальной плоскости. Размер выборки предварительно не рассчитывали. Методы статистического анализа данных Размер выборки предварительно не рассчитывали. Методы статистического анализа данных АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ Таблица 1. Значения показателей продольного роста большеберцовых костей (M ± SD) Серия Общая длина большеберцовой кости, мм Потеря общей длины «П» от длины «Л» в период М6 Величина прироста в длину за период Д0–М6 Д0 М6 От общей длины сегмента М6, % «П» от «Л», % «П» «Л» «П» «Л» мм % «П» «Л» I 136,5±39,5 136,7±39,4 152,7±36,71 156,5±37,31 3,8±2,9 2,5±2,1 11,5±5,0 13,6±4,6 17±12,3 II 139,2±9,5 139,2±9,5 172,5±9,81 180,3±8,51 7,8±8,0 4,3±4,3 19,3±3,6 22,8±3,7 14,9±14,0 III 146,8±7,9 146,4±7,8 174±10,51 181,7±11,31 7,7±6,6 4,2±3,6 15,5±3,6 19,1±3,6 18,2±15,2 Примечание. 1 — статистически значимые различия с исходными значениями данной серии; «П» — правая большеберцовая кость, «Л» — левая большеберцовая кость. Таблица 1. Значения показателей продольного роста большеберцовых костей (M ± SD) При непосредственной оценке полученной величины прироста опытного сегмента по отношению к контроль- ным значениям в период Д0–М6 во всех сериях определя- ли их уменьшение на 15–18%, при этом различий между сериями не зарегистрировано. внутреннюю лодыжку. В этой области стержень занимал 0,63±0,03% площади метафиза. На уровне проксималь- ного физиса расположение стержней было более цен- тральное, но также в большинстве случаев с тенденцией к смещению в медиальном направлении относительно продольной оси кости на 1–5 мм. От площади прокси- мального метафиза стержень занимал 0,32±0,03%. внутреннюю лодыжку. В этой области стержень занимал 0,63±0,03% площади метафиза. На уровне проксималь- ного физиса расположение стержней было более цен- тральное, но также в большинстве случаев с тенденцией к смещению в медиальном направлении относительно продольной оси кости на 1–5 мм. От площади прокси- мального метафиза стержень занимал 0,32±0,03%. При измерении длины перекреста спиц в костномоз- говом канале отмечено его уменьшение по мере роста большеберцовой кости. Прекращение уменьшения длины перекреста соответствовало закрытию зон роста больше- берцовой кости. К концу эксперимента в первой серии уменьшение перекреста по отношению к послеопераци- онному состоянию в среднем составило 13,4±0,9, во вто- рой — 15,6±5,7, в третьей — 11,2±3,4 мм. Разница этого показателя между сериями не достигла статистической значимости. В серии II в 2 случаях произошла миграция проксимального стержня внутрь кости на 19 и 25 мм, в се- рии III — в 1 случае на 24 мм. В серии I мы не отметили слу- чаев миграции стержней внутрь в процессе роста сегмента. Расположение стержней диаметром 1,8 мм относи- тельно центров физисов в каждой серии представлено на рис. 4. Результаты исследования длины сегментов представ- лены в табл. 1. 445 Как видно из представленных данных, рост больше- берцовых костей продолжался у собак всех эксперимен- тальных серий. Участники исследования Для выполнения экспериментов были отобраны кли- нически здоровые беспородные собаки (щенки) обоего пола в возрасте 6 мес. При этом в каждой отдельной груп- пе использовали щенков из одного помета. У всех живот- ных правая голень являлась опытным сегментом, а левая служила контролем (рис. 3). Рис. 2. Схема расположения изучаемых параметров. Примечание. а — длина большеберцовой кости, поперечный размер диафиза большеберцовой кости в средней трети, угол mMPTA, угол mLDTA, б — величина перекреста проксимальной и дистальной спицы в костномозговом канале. В серии I при трансфизарном армировании не- поврежденной большеберцовой кости имитирова- 28.09.2015 12:09:15 28.09.2015 12:09:15 flat Vestnik 4-2015.indd 443 flat Vestnik 4-2015.indd 443 ВЕСТНИК РАМН /2015/ 70 (4) Рис. 3. Схема проведения экспериментального исследования. ис. 3. Схема проведения экспериментального исследования. Основные результаты исследования ли превентивный интрамедуллярный трансфизарный остеосинтез, осуществляемый при несовершенном ос- теогенезе. При выполнении остеометрических исследований разница в измерениях изучаемых показателей между ис- следователями не превышала 2,5%, что позволяет под- твердить воспроизводимость и точность измерений. В сериях II и III в определенном приближении ими- тировали интрамедуллярный трансфизарный эластичный остеосинтез и его сочетание с поднадкостничным арми- рованием при переломе костей голени на фоне несовер- шенного остеогенеза. Анализ полученных результатов показал, что на уровне дистальной зоны роста прохождение стержней было медиальнее, что обусловлено проведением их через Рис. 4. Расположение стержней диаметром 1,8 мм относительно центров физисов. Примечание. а, в, д — проксимальный метафиз, б, г, е — дистальный метафиз. а в д б г е а б 444 г в е д Рис. 4. Расположение стержней диаметром 1,8 мм относительно центров физисов. Примечание. а, в, д — проксимальный метафиз, б, г, е — дистальный метафиз. Рис. 4. Расположение стержней диаметром 1,8 мм относительно центров физисов. Примечание. а, в, д — проксимальный метафиз, б, г, е — дистальный метафиз. flat Vestnik 4-2015.indd 444 flat Vestnik 4-2015.indd 444 Примечание. p1 — значения p при сравнении правой и левой стороны Д0; p2 — значения p при сравнении правой и левой стороны M6; p3— значения p правой стороны Д0 по отношению к M6; p4— значения p левой стороны Д0 по отношению к M6; pt — p-значения теста Стьюдента. 1 — значения p при сравнении правой и левой стороны Д0; p2 — значения p при сравнении правой и левой стороны M6; p правой стороны Д0 по отношению к M6; p4— значения p левой стороны Д0 по отношению к M6; pt — p-значения теста АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ Исходные значения длины сегментов значимо отличались от длины, достигнутой по окончании эксперимента. При этом между собой в каждый отдель- ный период времени продольные размеры опытного и контрольного сегмента достоверных отличий не имели. В серии I величина потери продольного роста за время экс- перимента была наименьшей и составила от 1 до 7 мм (1– 5,4%), в серии II — от 5 до 17 мм (2,8–9,5%), но у одной собаки отмечено ускорение роста оперированной голени, и разница в длине к концу эксперимента составила 5 мм (2,7%). В серии III потеря продольного роста составила от 2 до 16 мм (1,1–9,3%). На разных этапах эксперимента во всех сериях у опытного сегмента не обнаружено достоверных отличий значений mMPTA между собой и в сравнении с доопе- рационным значением (табл. 2). У левого (контрольного) сегмента имелись незначительно выраженные изменения этого показателя. Однако эти изменения в 11 (61,1%) слу- чаях из 18 были не значимы по отношению к опытному сегменту. Таблица 2. Значения проксимального медиального большеберцового угла (mMPTA) № серии Конечность Д0 M6 M6–Д0 (правая сторона) M6–Д0 (левая сторона) М ± SD p1 M ± SD p2 p3 p4 (min-max) (min-max) Серия I Правая 91,7±2,4 pt=0,914 90,8±2,1 pt=0,642 pt=0,306 pt=0,101 (87–96) (88–94) Левая 90,9±2,8 93,4±2,3 (84–95) (90–99) Серия II Правая 92,3±2,1 pt=0,481 91,8±1,9 pt=0,079 pt=0,675 pt=0,128 (90–95) (90–95) Левая 91,5±1,9 90,0±1,1 (89–94) (89–92) Серия III Правая 90,7±5,5 pt=0,905 90,5±2,0 pt=0,453 pt=0,946 pt=0,541 (83–96) (87–92) Левая 90,3±3,8 91,5±2,4 (85–95) (87–94) Примечание. p1 — значения p при сравнении правой и левой стороны Д0; p2 — значения p при сравнении правой и левой стороны M6; p3— значения p правой стороны Д0 по отношению к M6; p4— значения p левой стороны Д0 по отношению к M6; pt — p-значения теста Стьюдента. 28.09.2015 12:09:16 28.09.2015 12:09:16 flat Vestnik 4-2015.indd 445 flat Vestnik 4-2015.indd 445 ВЕСТНИК РАМН /2015/ 70 (4) Таблица 3. Значения латерального дистального большеберцового угла (mLDTA) № серии Конечность Д0 M6 M6–Д0 (правая конечность) M6–Д0 (левая конечность) М ± SD p1 M ± SD p2 p3 p4 (min-max) (min-max) Серия I Правая 85,2±3,1 pt=0,563 88,2±5,3 pt=0,023 pw=0,045 pt=0,267 pt=0,088 (81–89) (84-96) Левая 84,2±2,7 81,2±2,8 (79–87) (76–84) Серия II Правая 87,7±2,1 pt=0,718 84,2±6,4 pt=0,691 pt=0,251 pt=0,016 pw=0,026 (86–91) (78–96) Левая 87,2±2,6 83±2,5 (84–91) (80–85) Серия III Правая 83,3±4,3 pt=0,607 80,3±2,7 pt=0,190 pt=0,183 pt=0,040 pw=0,058 (77–89) (78–85) Левая 82,2±3,3 78,5±1,5 (77–86) (76–80) Примечание. АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ p1 — значения p при сравнении правой и левой стороны Д0; p2— значения p при сравнении правой и левой стороны M6; p3— значения p правой стороны Д0 по отношению к M6; p4 — значения p левой стороны Д0 по отношению к M6; pt — значения теста Стьюдента; pw — значения непараметрического теста Вилкоксона. Таблица 3. Значения латерального дистального большеберцового угла (mLDTA) Примечание. p1 — значения p при сравнении правой и левой стороны Д0; p2— значения p при сравнении правой и левой стороны M6; p3— значения p правой стороны Д0 по отношению к M6; p4 — значения p левой стороны Д0 по отношению к M6; pt — значения теста Стьюдента; pw — значения непараметрического теста Вилкоксона. 446 Таблица 4. Поперечные размеры большеберцовой кости в средней трети (мм) Серия Д0 (прямая проекция) М6 (прямая проекция) Д0 (боковая проекция) М6 (боковая проекция) Правая Левая Правая Левая Правая Левая Правая Левая I 11,3±0,82 11,3±0,82 13,2±0,841 13,2±0,841 10±1,55 10±1,55 11,8±1,72 11,2±1,47 II 12,2±1,17 12,2±1,17 17,2±1,941 14,3±1,211,2 11,2±1,17 11,2±0,75 17,2±1,831 13,3±1,212 III 11,7±1,03 11,5±0,84 14,8±0,411 13,5±1,221,2 10±0,63 10,2±0,41 16,1±1,911 12,3±1,372 Примечание. 1 — значимые различия Д0 по отношению к М6 для данной серии (p <0,05); 2 — значимые различия между правой и левой стороной М6 (p <0,05). Таблица 4. Поперечные размеры большеберцовой кости в средней трети (мм) зарегистрированы во всех сериях. В серии I, согласно данным табл. 4, увеличение диаметра диафиза было одинаковым для оперированной и интактной конеч- ности. Значения mLDTA опытного (правого) сегмента во всех сериях к концу эксперимента между собой и доопе- рационным уровнем значимо не изменились. У контроль- ного сегмента во всех сериях было установлено значимое (p =0,088 для серии I, р =0,016 для серии II и р =0,04 для серии III) снижение угла mLDTA к моменту заверше- ния роста в сравнении с дооперационными значениями (табл. 3). В сериях II и III к окончанию эксперимента значимое увеличение поперечных размеров кости отмечали как по сравнению с исходными данными, так и между контроль- ной и опытной конечностью (см. табл. 4). Интересно от- метить, что наиболее выраженная периостальная реакция и, соответственно, утолщение кортикальной пластинки происходили по задней поверхности большеберцовой кости. Изменение значений внешнего диаметра больше- берцовой кости в средней трети в сторону увеличения происходило у всех животных на правой и левой ко- нечности. Значимые (p <0,05) различия между Д0 и М6 г а б в г Рис. 5. Фрагменты рентгенограмм: серия II (а, б), серия III (в, г). Примечание. Нежелательные явления На протяжении эксперимента не было зарегистри- ровано нежелательных последствий, в т.ч. осложнений инфекционного и неврологического характера. Резюме основного результата исследования нта укорочение сегмента будет выражено значительнее Интрамедуллярное трансфизарное армирование за- медляло рост кости в длину. Проведение эластичных стержней, близкое к центру ростковой зоны кости, зна- чимо не влияло на формирование угла наклона суставной поверхности. Эксцентричное проведение трансфизарных стержней формировало угловую деформацию в процессе последующего роста. Поднадкостничное армирование в сочетании с трансфизарным остеосинтезом не замедляло консолидацию фрагментов. При трансфизарном армиро- вании неповрежденной кости влияние интрамедуллярных эластичных стержней на функциональную активность эн- достальной выстилки и надкостницы было несуществен- ным, а остеотомия и отслаивание надкостницы являлись условием для развития периостальной реакции. Наше исследование впервые показало, что интраме- дуллярный трансфизарный остеосинтез влияет на фор- мирование угла наклона дистальной суставной поверхно- сти большеберцовой кости. Эксцентричное проведение интрамедуллярных стержней через внутреннюю лодыжку привело либо к варизации голеностопного сустава (в се- рии I) со значимым отличием между контрольной и опыт- ной конечностью к моменту завершения роста, либо, как в сериях II и III, к торможению физиологической валь- гизации (уменьшение aLDTA). Результаты исследований других авторов подтверждают факт угловой девиации при эксцентричном повреждении зоны роста [17, 18]. Центральное трансфизарное проведение стержней не вызывало угловой девиации ни в одной серии исследова- ния. R. Seil и соавт. при выполнении центрально распо- ложенного канала у 18 ягнят также не наблюдали угловой девиации в процессе последующего роста сегмента [19]. АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ Дополнительные результаты исследования и соавт. описывают частичный эпифизеодез и развитие угловой деформации [14]. В свою очередь, P.S. Yung и соавт. R. Langenhan и соавт. и другие ученые не находят влияния трансфизарно проведенных прямых спиц Кирш- нера на последующий рост оперированной конечности [15, 16]. Во всех случаях животные начинали опираться на ко- нечность через 2–3 сут после операции. Функция конеч- ности была сохранна до окончания эксперимента. В сериях II и III рентгенологические признаки консо- лидации костных фрагментов (исчезновение линии пере- лома, непрерывность кортикальных пластинок) были отмечены во всех случаях к окончанию 2-го мес экспери- мента (рис. 5). Вторичное смещение фрагментов наблю- дали лишь у 1 животного в серии II, оно характеризова- лось антекурвационной деформацией на 7˚. В нашем исследовании повреждение площади физиса составило менее 6% при нахождении имплантов в тече- ние 25 нед, что не привело к необратимому эпифизеоде- зу. Во всех трех сериях было зафиксировано замедление роста, однако статистически значимой разницы длины оперированного сегмента по отношению к интактному мы не наблюдали. Трансфизарное армирование при вы- полнении остеотомии (серия II) или остеотомии в соче- тании с интрамедуллярным и поднадкостничным остео- синтезом (серия III) приводило к большему укорочению (до 9,5%) в сравнении с армированием неповрежденной кости (до 5,4%). flat Vestnik 4-2015.indd 447 flat Vestnik 4-2015.indd 447 АКТУАЛЬНЫЕ ВОПРОСЫ ТРАВМАТОЛОГИИ а, в — срок эксперимента 60 сут; б, г — срок эксперимента 180 сут (М6). а б в Рис. 5. Фрагменты рентгенограмм: серия II (а, б), серия III (в, г). Примечание. а, в — срок эксперимента 60 сут; б, г — срок эксперимента 180 сут (М6). в г Рис. 5. Фрагменты рентгенограмм: серия II (а, б), серия III (в, г). Примечание. а, в — срок эксперимента 60 сут; б, г — срок эксперимента 180 сут (М6). flat Vestnik 4-2015.indd 446 flat Vestnik 4-2015.indd 446 Обсуждение Возможно, при подобных исследованиях на животных меньшего возраста и более длительном периоде экспери- мента укорочение сегмента будет выражено значительнее. 1. Sofield H.A., Millar E.A. Fragmentation, realignment, and intra- medullary rod fixation of deformities of the long bones in children. J. Bone Joint Surg. Am. 1959; 41: 1371–1391. Конфликт интересов Авторы данной статьи подтвердили отсутствие финан- совой поддержки исследования / конфликта интересов, о которых необходимо сообщить. Выражение признательности Авторы выражают признательность Н.В. Попковой за помощь п ри подготовке данной статьи. Обнадеживающие результаты применения бисфосфо- натов у пациентов с несовершенным остеогенезом [8, 9] Ограничения исследования Несомненно, сложно экстраполировать результаты данного исследования для применения в клинической практике при несовершенном остеогенезе, когда проч- ностные свойства костной ткани снижены. Ограничени- ем служит и сравнительно небольшое число животных, что, к примеру, не позволяет установить корреляцию между топографией проведения трансфизарных стержней и величиной угловой деформации. Интрамедуллярный эластичный остеосинтез при ле- чении диафизарных переломов у детей, в т.ч. и патологи- ческих, является наиболее распространенным и надеж- ным хирургическим методом, используемым детскими ортопедами многих стран [23, 24]. Вполне естественно, что консолидация костных отломков в серии II нашего исследования произошла во всех случаях и в обычные сроки. Тем интереснее констатировать, что отслаивание надкостницы на уровне остеотомии и поднадкостничное введение сетки и эластичных спиц (серия III) также со- провождались консолидацией костных фрагментов в тот же срок. При этом наблюдали выраженную периосталь- ную реакцию, способствовавшую значимому увеличению поперечных размеров диафиза кости на данном уровне. Таким образом, можно полагать, что поднадкостничное введение титановой сетки и поднадкостничное армиро- вание эластичными стержнями не нарушают процессов костеобразования и ведут к консолидации в обычные сроки. Аспектом новизны полученных данных является не только возможность создания условия для продления механического увеличения свойств кости за счет интра- медуллярного и поднадкостничного перекрытия зоны перелома, но и получение локального увеличения кост- ной массы за счет периостальной реакции, сопровожда- ющейся утолщением кортикальных пластинок. Это очень важно при лечении патологических переломов у пациен- тов с заболеваниями, сопровождающимися снижением прочностных свойств костной ткани. Заключение Исследование показало, что трансфизарный эластич- ный остеосинтез влияет на продольный рост сегмента. Наименьшие потери длины отмечены при изолирован- ном интрамедуллярном трансфизарном остеосинтезе, имитирующем превентивное армирование. 448 Эксцентричное проведение трансфизарных стерж- ней способствует формированию угловой деформа- ции либо замедляет физиологическую ориентацию су- ставной поверхности. Таким образом, трансфизарные телескопические системы должны предусматривать расположение элементов импланта только в центре ростковых зон. При трансфизарном интрамедуллярном остеосинтезе или при его сочетании с поднадкостничным армирова- нием консолидация костных отломков наступает в оди- наковые сроки. При трансфизарном эластичном остеосинтезе в про- цессе роста сегмента может происходить затруднение расхождения интрамедуллярных стержней в результате формирования эндостального регенерата в области кон- такта импланта с костью. Энд- и периостальные реакции на введение интраме- дуллярных и поднадкостничных имплантов ведут к уве- личению толщины кортикальных пластинок и диаметра кости, что может иметь значение при комбинированном лечении несовершенного остеогенеза фармакологически- ми и хирургическими методами. Поднадкостничное армирование тонкими эластичны- ми стержнями, чрескожное введение спиц при тяжелых формах остеогенеза применяют в тех случаях, когда от- сутствует костномозговой канал кости, и ввиду чрезвы- чайно тонкого диаметра диафиза рассверливание его для проведения спицы невозможно [8, 12]. Серия III экспери- мента показала, что поднадкостничное эластичное арми- рование стрежнями, закрепленными в сетке из никелида титана, в сочетании с интрамедуллярным эластичным остеосинтезом, способствует стабильности остеосинте- за, не нарушает консолидации фрагментов. Кроме того, такой остеосинтез предотвращает ситуацию, когда из-за расхождения интрамедуллярных стержней средняя треть диафиза остается без внутреннего тутора. 2. Laron D., Pandya N.K. Advances in the orthopedic management of osteogenesis imperfecta. Orthop. Clin. North Am. 2013; 44 (4): 565–573. Обсуждение основного результата исследования Horn 28.09.2015 12:09:16 28.09.2015 12:09:16 ВЕСТНИК РАМН /2015/ 70 (4) чередование повторных эпифизеодезов и спонтанных дезэпифизеодезов в процессе роста служит объяснением локального торможения функции физиса в нашем экс- перименте. При этом наблюдается замедление роста, но не закрытие зон роста сразу после операции. и/или имплантов с биоактивным покрытием позволяют надеяться, что комбинирование терапевтических и хирур- гических методов лечения позволит улучшить исходы как с точки зрения анатомического (продольный рост сегмен- тов и отсутствие деформаций), так и функционального результата лечения. чередование повторных эпифизеодезов и спонтанных дезэпифизеодезов в процессе роста служит объяснением локального торможения функции физиса в нашем экс- перименте. При этом наблюдается замедление роста, но не закрытие зон роста сразу после операции. Следует отметить, что мы не наблюдали нарушений расхождения спиц в процессе роста в костномозговом канале в серии I, имитирующей превентивное армиро- вание сегмента. Эти данные демонстрируют преимуще- ства интрамедуллярного эластичного армирования перед телескопическими стержнями. Однако в 3 случаях, когда производилось нарушение целостности кости (серии II и III), было отмечено блокирование скольжения эластич- ных стержней. Препятствием расхождению стержней, на наш взгляд, может служить их фиксация эндостальной мозолью на уровне остеотомии. Обсуждение основного результата исследования Реконструктивные ортопедические вмешательства на конечностях при системных заболеваниях, сопрово- ждающихся хрупкостью костной ткани, направлены на предотвращение деформаций или их коррекцию. Это, не- сомненно, оказывает положительное влияние на функци- ональное состояние пациентов, связанное с увеличением их двигательной активности [8, 9]. Однако в клинической практике при применении трансфизарного армирования по-прежнему отсутствуют данные о количественном влиянии данного способа ле- чения на рост и развитие сегмента; эта важная функция ростковых зон никак не оценивается в условиях транс- физарного расположения имплантов [20]. B. Boutaud и J.M. Laville считают, что использование интрамедуллярного трансфизарного эластичного остео- синтеза предпочтительнее, чем телескопических стерж- ней, т.к. общее число осложнений ниже, чем при при- менении других систем [10]. Общие сложности и/или недостатки телескопических систем чаще связаны с необ- ходимостью их замены по мере роста ребенка; миграцией стержней или частей (например, резьбовых), осуществля- ющих фиксацию к кости (от 10,5 до 23,7%); деформацией телескопического стержня (до 18,8%); нерасхождением частей стержней (до 2,1%); переломами стержней (до 6,9%) [11, 12]. Наше исследование трансфизарного армирования показало, что центральное длительное нахождение им- планта не вызывает угловых деформаций, но может спо- собствовать формированию укорочения сегмента, а пери- ферическое расположение эластичного стержня ведет к развитию угловой деформации. При этом получены ранее не известные количественные характеристики потери роста во время нахождения импланта in situ — 14,9–18,2%. Важно отметить, что помимо топографии и величины повреждения ростковой зоны постоянное трансфизарное присутствие импланта имеет значение для изменения функции роста. Имеется ряд исследований, в которых установлено, что одномоментное формирование транс- физарного канала без расположения в нем стержней или сухожилий не ведет к образованию эпифизеодеза и не вызывает каких-либо нарушений продольного роста кости [18, 21], в то время как образование узкой зоны эпифизеодеза, окружающей постоянно присутствующий имплант, является необратимой причиной торможения роста в данной части физиса [5, 22]. В то же время именно относительно небольшие размеры эпифизеодеза (менее 7%) позволяют происходить спонтанному дезэпи- физеодезу в процессе активного роста. Мы полагаем, что В некоторых экспериментальных работах показано, что трансфизарное нахождение прямых спиц диаметром 1,3–2 мм менее 12 нед не вызвало развития нарушения функции роста, а в период 12–24 нед наблюдали эпифи- зеодез и укорочение сегмента. Использование спиц диа- метром 3,2–3,5 мм, занимающих 7% площади зоны роста, приводило к эпифизеодезу и укорочению [4, 13, 14]. 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Илизарова Попков Дмитрий Арнольдович, доктор медицинских наук, заведующий лабораторией коррекции деформаций и удли- нения конечностей РНЦ «ВТО» им. акад. Г.А. Илизарова Ц р Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: dpopkov@mail.ru р 014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: dpopkov@mail.ru Кононович Наталья Андреевна, кандидат ветеринарных наук, ведущий научный сотрудник лаборатории коррекции деформаций и удлинения конечностей РНЦ «ВТО» им. акад. Г.А. Илизарова Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: n.a.kononovich@mail.ru Кононович Наталья Андреевна, кандидат ветеринарных наук, ведущий научный сотрудник лаборатории коррекции деформаций и удлинения конечностей РНЦ «ВТО» им. акад. Г.А. Илизарова Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: n.a.kononovich@mail.ru Мингазов Эдуард Рифович, аспирант очной аспирантуры РНЦ «ВТО» им. акад. Г.А. Илизарова Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: edikmed@mail.ru Шутов Роман Борисович, врач травматолог-ортопед, травматолого-ортопедического отделения № 11 РНЦ «ВТО» им. акад. Г.А. Илизарова Мингазов Эдуард Рифович, аспирант очной аспирантуры РНЦ «ВТО» им. акад. Г.А. Илизарова Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: edikmed@mail.ru Шутов Роман Борисович, врач травматолог-ортопед, травматолого-ортопедического отделения № 11 РНЦ «ВТО» им. акад. Г.А. Илизарова д р Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: shrb78@mail.ru р рес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: shrb78@mail.ru Барбье Доминик, интерн РНЦ «ВТО» им. акад. Г.А. Илизарова Барбье Доминик, интерн РНЦ «ВТО» им. акад. Г.А. Илизарова Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43 Адрес: 640014, Курган, ул. М. Ульяновой, д. 6., тел.: +7 (3522) 43-05-37, e-mail: d.barbier.mail@gmail.com flat Vestnik 4-2015.indd 449 flat Vestnik 4-2015.indd 449 28.09.2015 12:09:16 28.09.2015 12:09:16 flat Vestnik 4-2015.indd 449 flat Vestnik 4-2015.indd 449
https://openalex.org/W3144814345	https://eprints.gla.ac.uk/235415/2/235415.pdf	English	null	Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank	PLOS genetics	2,021	cc-by	16,588	RESEARCH ARTICLE Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank Keira J. A. JohnstonID1,2,3, Joey WardID1, Pradipta R. Ray4, Mark J. AdamsID2, Andrew M. McIntoshID2, Blair H. SmithID5, Rona J. StrawbridgeID1,6, Theodore J. PriceID4, Daniel J. SmithID1, Barbara I. NichollID1, Mark E. S. BaileyID3 Keira J. A. JohnstonID1,2,3, Joey WardID1, Pradipta R. Ray4, Mark J. AdamsID2, Andrew M. McIntoshID2, Blair H. SmithID5, Rona J. StrawbridgeID1,6, Theodore J. PriceID4, Daniel J. SmithID1, Barbara I. NichollID1, Mark E. S. BaileyID3 1 Institute of Health and Wellbeing, University of Glasgow, Glasgow, Scotland, United Kingdom, 2 Division of Psychiatry, University of Edinburgh, Edinburgh, Scotland, United Kingdom, 3 School of Life Sciences, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom, 4 School of Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, Texas, United States of America, 5 Division of Population Health Sciences, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, United Kingdom, 6 Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * k.johnston.2@research.gla.ac.uk OPEN ACCESS Chronic pain is highly prevalent worldwide and imparts a significant socioeconomic and pub- lic health burden. Factors influencing susceptibility to, and mechanisms of, chronic pain development, are not fully understood, but sex is thought to play a significant role, and chronic pain is more prevalent in women than in men. To investigate sex differences in chronic pain, we carried out a sex-stratified genome-wide association study of Multisite Chronic Pain (MCP), a derived chronic pain phenotype, in UK Biobank on 178,556 men and 209,093 women, as well as investigating sex-specific genetic correlations with a range of psychiatric, autoimmune and anthropometric phenotypes and the relationship between sex- specific polygenic risk scores for MCP and chronic widespread pain. We also assessed whether MCP-associated genes showed expression pattern enrichment across tissues. A total of 123 SNPs at five independent loci were significantly associated with MCP in men. In women, a total of 286 genome-wide significant SNPs at ten independent loci were discov- ered. Meta-analysis of sex-stratified GWAS outputs revealed a further 87 independent asso- ciated SNPs. Gene-level analyses revealed sex-specific MCP associations, with 31 genes significantly associated in females, 37 genes associated in males, and a single gene, DCC, associated in both sexes. We found evidence for sex-specific pleiotropy and risk for MCP was found to be associated with chronic widespread pain in a sex-differential manner. Male and female MCP were highly genetically correlated, but at an rg of significantly less than 1 (0.92). All 37 male MCP-associated genes and all but one of 31 female MCP-associated genes were found to be expressed in the dorsal root ganglion, and there was a degree of enrichment for expression in sex-specific tissues. Overall, the findings indicate that sex dif- ferences in chronic pain exist at the SNP, gene and transcript abundance level, and highlight possible sex-specific pleiotropy for MCP. Results support the proposition of a strong central nervous-system component to chronic pain in both sexes, additionally highlighting a poten- tial role for the DRG and nociception. Citation: Johnston KJA, Ward J, Ray PR, Adams MJ, McIntosh AM, Smith BH, et al. (2021) Sex- stratified genome-wide association study of multisite chronic pain in UK Biobank. PLoS Genet 17(4): e1009428. https://doi.org/10.1371/journal. pgen.1009428 Editor: Michael P. Epstein, Emory University, UNITED STATES * k.johnston.2@research.gla.ac.uk Editor: Michael P. Epstein, Emory University, UNITED STATES Received: August 20, 2020 Accepted: February 16, 2021 Published: April 8, 2021 Copyright: © 2021 Johnston et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: August 20, 2020 Accepted: February 16, 2021 Published: April 8, 2021 Received: August 20, 2020 Accepted: February 16, 2021 Published: April 8, 2021 Copyright: © 2021 Johnston et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: UK Biobank Data is available upon application to UK Biobank. Summary statistics of the sex-stratified and GWAS meta-analyses are available for download from the University of Glasgow Enlighten system http://dx. doi.org/10.5525/gla.researchdata.1050. p pp Summary statistics of the sex-stratified and GWAS meta-analyses are available for download from the University of Glasgow Enlighten system http://dx. doi.org/10.5525/gla.researchdata.1050. Funding: RJS is supported by a UKRI Innovation- HDR-UK Fellowship (MR/S003061/1). JW is supported by the JMAS Sim Fellowship for depression research from the Royal College of Physicians of Edinburgh (173558). KJAJ is 1 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS supported by an MRC Doctoral Training Programme Studentship at the Universities of Glasgow and Edinburgh (MR/N013166/1). DJS acknowledges the support of a Lister Prize Fellowship (173096) and the MRC Mental Health Data Pathfinder Award (MC_PC_17217). M.J.A. and A.M.M. are supported by Pathfinder Award MC_PC_17209 and by Wellcome Trust Grant 104036/Z/14/Z. TJP and PRR are supported by a National Institute of Neurological Disorders and Stroke (Bethesda) grant (R01NS102161). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author summary Chronic pain is a highly prevalent and debilitating condition, which is more common in women than in men. Sex differences in this condition may be a result of several factors, including differences between the sexes in genetic variation related to chronic pain and gene expression differences related to sex. To explore sex differences in chronic pain from a genetic perspective, we looked for genetic variants associated with chronic pain in men and women separately in a large general-population cohort, and compared the variants we identified between the sexes. We assessed the degree of overlap between genetic vari- ants associated with chronic pain in each sex and those associated with a wide range of other traits, including major depression, body-mass index and suicidality. We also investi- gated gene expression patterns across a range of tissues for genes associated with chronic pain in each sex, in particular examining expression in neural and non-neural human and mouse tissues and assessing the degree of Dorsal Root Ganglion (DRG) enrichment, an important peripheral nervous system component involved in chronic pain. This work contributes to understanding of chronic pain as a trait and of sex differences in chronic pain at the levels of genetics and gene expression. Competing interests: The authors have declared that no competing interests exist. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Introduction Chronic pain is widely defined as pain persisting beyond 3 months [1,2], and can be a primary disorder [3] or secondarily associated with injury, surgery or a range of medical conditions. Chronic pain is highly prevalent worldwide [4–9] and imparts a significant socioeconomic and public health burden [10]. Factors influencing susceptibility to chronic pain, and the mechanisms underlying its development and maintenance, are not fully understood. Several aspects of chronic pain including Chronic Pain Grade [11], severe chronic pain and low back pain have been studied from a genetic perspective and found to be complex traits. Heritability estimates vary from ~30–46% in twin, pedigree and factor analysis studies [12– 15], while single nucleotide polymorphism (SNP) heritability has been estimated from genome-wide association studies (GWAS) to be ~7–10% [16,17]. It is increasingly recognised that sex differences in many complex human traits are biologi- cally important, with genetic architecture for many traits being to some extent sex-specific [18], and a ‘sex-aware’ approach to genetic analysis has been widely advocated [19]. Sex as a biological variable has wide-ranging effects on the functioning of the genome and on resultant phenotypes. These effects can be mediated via sex-differential gene expression [20,21], sex differences in methylation [22–26] and expression quantitative trait locus (eQTL) effects [27,28], or differing levels and actions of hormones [29,30]. Sex can also influence traits through environmental fac- tors strongly correlated with sex [23,24] and sex-specific pleiotropy [25,31]. Chronic pain exhibits sex-related prevalence differences, and is more common in women than in men [32–34]. There are also potential sex differences in the impact of pain on functioning in daily life, and in the suc- cess of specific coping strategies [35]. In addition to differences in prevalence between the sexes, sex differences in underlying pain mechanisms and their modulation by immune cells have been recently reported [36–38], and immune responses in general can differ by sex [39]. Multisite Chronic Pain (MCP) is a derived chronic pain phenotype, defined as the sum of the number of sites of chronic pain on the body, here expressed on a scale from 0–7 [17]. GWAS of MCP in males and females separately To detect sex-differential genetic influences on multisite chronic pain, GWASs were run sepa- rately for males and females in UK Biobank. In men, a total of 123 SNPs at five independent loci were associated with MCP at a genome-wide significance threshold of p < 5 x 10−8 (Table 1, Fig 1). In women, a total of 286 genome-wide significant SNPs at ten independent loci were discovered (Table 1, Fig 1). All 15 of these loci were differentially associated with sex —none of the genome-wide significant SNPs at these loci had p < 5 x 10−8 in the GWAS con- ducted in the opposite sex. However, a total of 257 SNPs were found to have suggestive levels of association with MCP (p < 5 x 10−5) in both men and women. Two SNPs had suggestive evidence in men and were genome-wide significant in women, and eight SNPs had suggestive evidence in women and were genome-wide significant in men. In addition, the genome-wide significant loci on chromosome 6 in each sex were separated by less than 1 Mbp and may potentially exert their influence via differential effects on the same gene. lead SNPs associated with MCP (p < 5 x 10−8) in male and female sex-stratified GWAS. CHR, chromosome; BP, here A1 is the effect allele; BETA/SE, coefficient and its standard error for the effect allele; Gene = ANNOVAR annota- i i ANNOVAR) Table 1. Genome-wide significant independent lead SNPs associated with MCP (p < 5 x 10−8) in male and female sex-stratified GWAS. CHR, chromosome; BP, chromosome coordinate; A1/A2, alleles 1 and 2, where A1 is the effect allele; BETA/SE, coefficient and its standard error for the effect allele; Gene = ANNOVAR annota- tion gene symbol for variant (N/A, no gene annotation in ANNOVAR). Introduction We have previously shown in UK Biobank [17] that genetic predisposition to MCP (as captured by a polygenic risk score; PRS) was associated with Chronic Widespread Pain (CWP), a separate PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 2 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS but related chronic pain phenotype, in women but not men [17]. Additional unpublished find- ings using the Generation Scotland study [40] demonstrated that the PRS-MCP was associated with both chronic pain grade and an MCP-like phenotype in both men and women, but that the magnitude of effect was roughly twice as great in women as it was in men. There was also a significant PRS-by-sex interaction. These findings suggest that sets of variants contributing to chronic pain in males and females may act differently, or have different genetic effect sizes, in the two sexes. Here we report on an exploration of this preliminary evidence for the existence of sex-specific loci associated with MCP using a sex-stratified GWAS analysis approach in UK Biobank, and identify several sex-specific MCP loci. A meta-analysis of the female- and male- specific GWASs also revealed novel MCP loci not identified in the original MCP GWAS. We have also investigated possible functional effects associated with sex-specific MCP-associated genes as revealed by gene expression data in multiple relevant tissues, including dorsal root ganglion (DRG) samples, in both human and mouse. GWAS of MCP in males and females separately SNP rsID CHR BP A1 A2 BETA SE P Sex Gene rs35072907 1 51189556 G C 0.020 0.004 2.40E-08 Female FAF1 rs59898460 1 150493004 T C 0.025 0.004 4.90E-12 Female LINC00568; RP11-54A4.2 rs147903676 2 5835352 C CT -0.031 0.006 2.00E-08 Female SOX11 rs13135092 4 103198082 A G -0.038 0.006 2.30E-09 Female SLC39A8 rs3080367 5 57576558 TACAC T 0.024 0.004 2.90E-08 Female PGAM1P1; PLK2 rs62381120 5 120176330 T C -0.021 0.004 3.50E-08 Female CTD-2334D19.1; AC008565.1 rs74274428 5 170842428 CA C 0.020 0.004 2.80E-08 Female NPM1; FGF18 rs151060048 6 34633069 CA C -0.035 0.006 5.40E-09 Female C6orf106 rs34003284 13 53902876 C A -0.024 0.004 3.20E-10 Female RN7SL618P; AL450423.1 rs11079993 17 50301552 G T -0.021 0.004 4.50E-09 Female CA10; snoZ178 rs10660361 6 33741371 C CG 0.020 0.004 1.80E-08 Male LEMD2 9:140251458_G_A 9 140251458 G A -0.030 0.005 3.00E-09 Male EXD3 rs16909443 11 6192462 T C -0.040 0.007 4.40E-08 Male RP11-290F24.3 18:50442591_TTTC_T 18 50442591 TTTC T -0.020 0.004 1.60E-08 Male N/A 20:19709268_AAAAT_A 20 19709268 AAAAT A 0.030 0.005 1.20E-08 Male SLC24A3; AL121761.1 htt //d i /10 1371/j l 1009428 t001 Table 1. Genome-wide significant independent lead SNPs associated with MCP (p < 5 x 10−8) in male and female sex-stratified GWAS. CHR, chromosome; BP, chromosome coordinate; A1/A2, alleles 1 and 2, where A1 is the effect allele; BETA/SE, coefficient and its standard error for the effect allele; Gene = ANNOVAR annota- tion gene symbol for variant (N/A, no gene annotation in ANNOVAR). PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 3 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fig 1. Manhattan plots for the stratified GWAS analyses. Upper panel: female, lower panel: male. Red dotted line indicates genome-wide significant p-value threshold. https://doi.org/10.1371/journal.pgen.1009428.g001 Fig 1. Manhattan plots for the stratified GWAS analyses. Upper panel: female, lower panel: male. Red dotted line indicates genome-wide significant p-value threshold. https://doi.org/10.1371/journal.pgen.1009428.g001 https://doi.org/10.1371/journal.pgen.1009428.g001 LDSR analysis demonstrated that inflation of test statistics in each GWAS was due to poly- genicity (Table 2; LDSR intercept). SNP heritability was moderate, estimated as 0.125 and 0.106 in females and males, respectively (Table 2). The genetic correlation between male and female MCP was high (rg = 0.92, SE 0.03; p = 3.32 x 10−213), but significantly less than 100% (based on confidence intervals calculated as +/- 2 x SE). Meta-Analysis of Sex-Stratified MCP GWAS Outputs 87 independent SNPs were found to be associated with MCP at genome-wide significance in total, 11 of which were novel (not found in the unstratified or in each sex-stratified GWAS analysis). Each of the 87 independent significant SNPs showed consistent direction of effect between males and females (Table 3), but seven showed significant heterogeneity in effect size (I2 p < 0.05). In total, 49 lead SNPs across 46 genomic risk loci were found to be associated with MCP in meta-analysis of sex-stratified outputs. Genetic correlations between sex-stratified MCP and other disorders and i A range of complex trait phenotypes were selected for LDSR analysis with male and female MCP based on previous evidence for phenotypic correlation [17], with the addition of newly available trait data such as GWAS outputs on suicide and self-harm [41] and mood instability [42]. Suicidality and self-harm are important comorbidities of chronic pain, an issue Table 2. Trait genetic attributes from the male and female MCP GWASs. SNP-heritability, BOLT-LMM pseudo- heritability estimate; λGC1000, λGC value adjusted for sample size; LDSR_intercept (SE), LD-score regression intercept value and its standard error. Attribute female male SNP-heritability 0.125 0.106 λGC1000 1.002 1.001 LDSR_intercept (SE) 1.03 (0.006) 1.025 (0.005) https://doi.org/10.1371/journal.pgen.1009428.t002 4 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 3. Independent, genome-wide significant (GWS) SNPs from meta-analysis of sex-stratified MCP GWASs. Genomic Locus = genomic risk locus context of SNP with multiple independent GWS SNPs present at some of the 46 loci, rsID = SNP rsID identifier, Position = genomic position (chromosome: base-pair start position), Meta p = p value for association from the GWAS meta-analysis, Direction = direction of effect in female MCP and male MCP GWASs, respectively (+ if association beta value for effect allele > 0,—if < 0), I2 = effect size heterogeneity estimate, I2 p = p value for heterogeneity estimate, Symbol = ANNOVAR gene annotation for variant (N/ A, no gene annotation in ANNOVAR). Significant (unadjusted) heterogeneity I2 p values (I2 p < 0.05) are marked with . Table 3. Independent, genome-wide significant (GWS) SNPs from meta-analysis of sex-stratified MCP GWASs. Genomic Locus = genomic risk locus context of SNP with multiple independent GWS SNPs present at some of the 46 loci, rsID = SNP rsID identifier, Position = genomic position (chromosome: base-pair start position), Meta p = p value for association from the GWAS meta-analysis, Direction = direction of effect in female MCP and male MCP GWASs, respectively (+ if association beta value for effect allele > 0,—if < 0), I2 = effect size heterogeneity estimate, I2 p = p value for heterogeneity estimate, Symbol = ANNOVAR gene annotation for variant (N/ A, no gene annotation in ANNOVAR). Significant (unadjusted) heterogeneity I2 p values (I2 p < 0.05) are marked with . Genetic correlations between sex-stratified MCP and other disorders and i value for effect allele > 0,—if < 0), I2 = effect size heterogeneity estimate, I2 p = p value for heterogeneity estimate, Symbol = ANNOVAR gene annotation for variant (N/ A, no gene annotation in ANNOVAR). Significant (unadjusted) heterogeneity I2 p values (I2 p < 0.05) are marked with . compounded by the common co-occurrence of mental health traits, such as major depressive disorder (MDD), with chronic pain, and by the fact that use of certain medication in chronic pain contributes to increased risk for self-harm, suicidal ideation and suicide attempt [43–48]. Genetic correlations between sex-stratified MCP and other disorders and i rsID Genomic Locus Position Meta p Direction I2 I2 p Symbol rs909001 1 1:32196647 2.80E-08 – 7.6 0.298 BAI2 1:51042504_CT_C 2 1:51042504 2.35E-08 – 59.1 0.118 FAF1 rs197441 3 1:112283655 4.33E-10 – 0 0.644 FAM212B-AS1 rs12033257 3 1:112318484 2.79E-08 ++ 0 0.824 KCND3 rs509345 4 1:150276022 1.42E-10 – 71.3 0.062 MRPS21 rs367563576 4 1:150495378 2.24E-11 ++ 89.6 0.002 LINC00568; RP11-54A4.2 rs9700909 5 1:243255124 4.07E-09 – 30 0.232 RP11-261C10.3 1:243461350_CT_C 5 1:243461350 9.48E-09 – 53 0.145 SDCCAG8 rs6721975 6 2:5832667 2.86E-08 – 67 0.082 SOX11 rs4852567 7 2:80703379 3.51E-08 ++ 25 0.248 CTNNA2 rs5832889 8 2:100503396 2.06E-08 ++ 74.9 0.046 AFF3 rs112908707 9 3:49865628 4.35E-10 – 0 0.866 TRAIP rs62260755 9 3:49898318 5.36E-09 – 0 0.619 CAMKV 3:50098024_CAA_C 9 3:50098024 4.79E-09 ++ 0 0.75 RBM6 rs13067082 9 3:50221715 3.66E-08 ++ 0 0.534 SEMA3F rs144433312 10 3:84591507 1.07E-08 ++ 26.7 0.243 AC107025.1; LINC00971 rs62263345 11 3:107252190 5.01E-10 – 0 0.848 BBX rs28750366 12 3:136361055 3.55E-08 – 0 0.498 STAG1 rs56203712 13 4:25342606 1.37E-11 ++ 0 0.783 ZCCHC4 rs201081507 14 4:102681041 8.93E-09 – 51.9 0.149 BANK1 rs13109404 14 4:102896591 1.17E-09 – 0 0.768 BANK1 rs13135092 14 4:103198082 2.61E-14 – 3.4 0.309 SLC39A8 rs6869446 15 5:65570607 3.23E-08 – 3.4 0.309 snoU13; RP11-305P14.1 rs10076888 16 5:103786487 1.06E-08 ++ 0 0.872 RP11-6N13.1 rs147831713 16 5:103787168 9.96E-09 – 0 0.66 RP11-6N13.1 rs325485 16 5:103995368 3.15E-08 ++ 0 0.576 RP11-6N13.1 rs1976423 16 5:104042643 1.48E-08 – 58.3 0.121 RP11-6N13.1 rs137863733 17 5:160890323 8.13E-10 ++ 23.3 0.254 GABRB2 rs6915136 18 6:33651322 1.09E-08 – 37.2 0.207 ITPR3 rs482786 18 6:33707599 1.51E-09 – 29.8 0.233 IP6K3 6:33709752_CA_C 18 6:33709752 1.08E-08 ++ 0 0.5 IP6K3 rs28651968 18 6:33717424 2.16E-08 ++ 0 0.945 IP6K3; LEMD2 rs17529077 18 6:33793332 1.27E-09 ++ 0 0.997 MLN; LINC01016 rs17600945 18 6:33802263 1.21E-09 ++ 0 0.365 MLN; LINC01016 rs6907508 19 6:34592090 2.81E-08 – 50.3 0.156 C6orf106 rs151060048 19 6:34633069 6.92E-09 – 84.4 0.011 C6orf106 rs142415291 19 6:34755312 9.43E-09 – 81.6 0.020 SNRPC; UHRF1BP1 rs6926377 20 6:145105354 7.17E-09 – 49.6 0.159 UTRN rs148148187 21 7:3602520 1.29E-08 ++ 0 0.839 SDK1 rs7798894 22 7:21552995 3.44E-08 ++ 17 0.273 SP4 rs6966540 23 7:95727967 1.09E-08 – 0 0.67 DYNC1I1 rs10156143 23 7:95844896 6.43E-09 – 0 0.349 SLC25A13 rs1450833 24 7:113865735 1.16E-08 – 0 0.715 FOXP2 7:113945981_CCACTTATAAATACTGTCCCTTGGGCA_C 24 7:113945981 1.24E-08 ++ 0 0.657 FOXP2 ( d) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 5 / 27 https://doi.org/10.1371/journal.pgen.1009428.t003 PLOS GENETICS PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 3. (Continued) rsID Genomic Locus Position Meta p Direction I2 I2 p Symbol rs1527146 24 7:113987281 4.56E-08 ++ 27.4 0.241 FOXP2 7:114058731_CA_C 24 7:114058731 5.08E-10 ++ 23.2 0.254 AC073626.2 rs55671932 25 7:150556803 4.07E-08 ++ 68.2 0.076 AOC1 rs6997840 26 8:141658361 2.97E-08 – 0 0.37 AGO2; PTK2 9:96168164_CT_C 27 9:96168164 1.83E-08 ++ 0 0.87 RNU6-829P; Y_RNA rs7869969 27 9:96217447 9.87E-10 ++ 0 0.797 FAM120A rs6478241 28 9:119252629 6.61E-09 ++ 5.6 0.303 ASTN2 9:140247497_A_C 29 9:140247497 5.65E-12 – 0 0.567 EXD3 9:140249861_A_C 29 9:140249861 6.70E-09 ++ 23.9 0.252 EXD3 9:140260266_T_G 29 9:140260266 3.38E-14 – 0 0.571 EXD3 rs2183271 30 10:21957229 4.47E-08 – 4.3 0.307 MLLT10 10:99784552_CCA_C 31 10:99784552 1.05E-08 – 40.4 0.195 CRTAC1 rs11599236 32 10:106454672 3.33E-08 ++ 0 0.351 SORCS3 rs17553733 33 11:16362089 1.70E-10 – 0 0.392 SOX6 rs2118362 33 11:16373083 1.32E-08 – 31.9 0.226 SOX6 rs55670730 34 11:43620008 4.18E-08 – 0 0.925 N/A rs7303462 35 12:23974911 4.61E-08 – 32.5 0.224 SOX5 rs184483429 36 12:107620106 4.87E-08 ++ 0 0.58 RP11-797M17.1; SETP7 rs2759694 37 13:53695378 3.17E-08 ++ 76.8 0.038 OLFM4; LINC01065 rs67128127 37 13:53889000 7.48E-09 ++ 3.4 0.309 RN7SL618P; AL450423.1 rs1443914 37 13:53917230 4.73E-10 ++ 76.7 0.038 RN7SL618P; AL450423.1 rs7335163 37 13:53989975 7.23E-09 ++ 8.9 0.295 AL450423.1; LINC00558 rs17574479 37 13:54049489 2.97E-09 – 88.3 0.003 AL450423.1; LINC00558 rs34521521 38 14:73832318 4.99E-08 – 0 0.512 NUMB rs4886649 39 15:75328595 2.25E-08 – 0 0.892 PPCDC 15:75348905_CAACA_C 39 15:75348905 1.62E-08 – 0 0.924 PPCDC rs2386584 40 15:91539572 6.68E-12 – 0 0.578 PRC1 rs285027 41 16:77100932 9.39E-09 – 0 0.748 CTD-2336H13.2; MON1B rs11871043 42 17:43172849 1.49E-09 ++ 0 0.945 NMT1 rs967823 43 17:50317276 1.98E-11 – 58.8 0.119 snoZ178; RP11-429O1.1 rs35518690 44 18:42136963 2.15E-08 ++ 41.5 0.191 CTC-782O7.1; RP11-456K23.1 rs2043187 45 18:50394405 3.58E-10 – 0 0.668 DCC rs72922230 45 18:50394407 4.12E-10 – 29.7 0.233 DCC rs767443167 45 18:50622162 1.43E-08 – 0 0.469 DCC rs8089828 45 18:50669725 9.63E-10 ++ 0 0.783 DCC rs8099145 45 18:50743672 2.15E-12 – 0 0.871 DCC rs12968428 45 18:50750225 1.77E-09 ++ 0 0.413 DCC rs17410557 45 18:50776391 1.03E-11 – 0 0.561 DCC rs773737322 45 18:50846440 9.82E-11 ++ 0 0.529 DCC rs1367635 45 18:50861409 5.25E-09 – 0 0.659 DCC rs766498304 45 18:50871256 7.87E-09 – 35.6 0.213 DCC rs10164055 45 18:50919600 2.53E-08 – 0 0.659 DCC rs16980973 46 20:19648493 1.51E-10 ++ 0 0.322 SLC24A3 https://doi.org/10.1371/journal.pgen.1009428.t003 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 6 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fig 2. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS https://doi.org/10.1371/journal.pgen.1009428.g004 Fig 4. MCP-associated gene expression tissue enrichment analysis. Significant (-log10 p > 3) results in female analyses and their corresponding tissue results in male analyses are shown for brevity. No significant enrichment for expression in any tissue was found for MCP-associated genes in males. Dashed line = significance threshold. ACC_BA24 = anterior cingulate cortex BA24, Caudate_BG = caudate basal ganglia, NAc_BG = Basal ganglia— Nucleus accumbens region, Putamen_BG = Basal ganglia—putamen region. Full results for all 53 tissues can be found in S1 and S2 Figs. htt //d i /10 1371/j l 1009428 004 Fig 4. MCP-associated gene expression tissue enrichment analysis. Significant (-log10 p > 3) results in female analyses and their corresponding tissue results in male analyses are shown for brevity. No significant enrichment for expression in any tissue was found for MCP-associated genes in males. Dashed line = significance threshold. ACC_BA24 = anterior cingulate cortex BA24, Caudate_BG = caudate basal ganglia, NAc_BG = Basal ganglia— Nucleus accumbens region, Putamen_BG = Basal ganglia—putamen region. Full results for all 53 tissues can be found in S1 and S2 Figs. https://doi.org/10.1371/journal.pgen.1009428.g004 https://doi.org/10.1371/journal.pgen.1009428.g004 PLOS GENETICS Genetic correlations (rg) between male (M) and female (F) MCP and multiple traits (rg values with 95% CI error bars). Phenotypes differentially correlated with MCP between the sexes are plotted in orange. https://doi.org/10.1371/journal.pgen.1009428.g002 Fig 2. Genetic correlations (rg) between male (M) and female (F) MCP and multiple traits (rg values with 95% CI error bars). Phenotypes differentially correlated with MCP between the sexes are plotted in orange. https://doi.org/10.1371/journal.pgen.1009428.g002 MCP in both men and women was found to be significantly genetically correlated with a range of traits and disorders, including psychiatric and mood phenotypes such as anhedonia, mood instability, depressive symptoms, MDD, anxiety, suicidality and subjective wellbeing (Fig 2, S1 Table). In addition, as expected, genetic correlations between both male and female MCP and unstratified MCP from our previous analysis [17]were essentially perfect (rg = 1.00, p < 1 x 10−120 for both sexes). PTSD, schizophrenia, autism spectrum disorder, anorexia ner- vosa, PGC cross-disorder phenotype and primary biliary cirrhosis were found to be signifi- cantly correlated with MCP in one sex and not the other. Several phenotypes were found not to be genetically correlated with MCP in either sex (pfdr > 0.05), including inflammatory bowel diseases, Parkinson’s disease, bipolar disorder, rheumatoid arthritis, and low relative amplitude (a circadian rhythmicity-related phenotype). Fig 3. Venn diagram of number of genes found significantly associated with MCP in MAGMA gene-based test analyses. Sectors: Original = non-sex-stratified MCP GWAS, Meta = meta-analysed female and male GWAS output, Female = female MCP GWAS, Male = male MCP GWAS. https://doi.org/10.1371/journal.pgen.1009428.g003 Fig 3. Venn diagram of number of genes found significantly associated with MCP in MAGMA gene-based test analyses. Sectors: Original = non-sex-stratified MCP GWAS, Meta = meta-analysed female and male GWAS output, Female = female MCP GWAS, Male = male MCP GWAS. https://doi.org/10.1371/journal.pgen.1009428.g003 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 7 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fig 4. MCP-associated gene expression tissue enrichment analysis. Significant (-log10 p > 3) results in female analyses and their corresponding tissue results in male analyses are shown for brevity. No significant enrichment for expression in any tissue was found for MCP-associated genes in males. Dashed line = significance threshold. ACC_BA24 = anterior cingulate cortex BA24, Caudate_BG = caudate basal ganglia, NAc_BG = Basal ganglia— Nucleus accumbens region, Putamen_BG = Basal ganglia—putamen region. Full results for all 53 tissues can be found in S1 and S2 Figs. Gene-level analysis Genes enriched for variants associated with MCP were identified using a gene-level association analysis (gene-based test) [49] approach implemented by MAGMA as part of the FUMA suite that tests 19,012 separate genes. The results are summarised in Fig 3 and S2 Table. In females and males, 31 and 37 genes, respectively, were found to be significantly (Bonferroni-adjusted significance criterion: p < 2.63 x 10−6) associated with MCP. The only gene found to be signifi- cantly associated with both male and female MCP was DCC. 24 out of the 31 genes signifi- cantly associated with MCP in females in the sex-stratified analyses, and 31 out of the 37 genes significantly associated in males, were also significantly associated in our previous non-strati- fied analysis [17]. Six genes were significantly associated with MCP only in females (NCAN, SPATS2L, TBC1D9, CAMK1D, SOX11, GON4L), while 4 genes were associated only in males (CENPW, MTCH2, NICN1, DNAJA4). Twenty-four genes were identified as significantly asso- ciated with MCP only in the meta-analysis of the sex-stratified GWAS outputs (Fig 3 and S3 Table). Nineteen genes found in previous sex-combined GWAS analyses [17] were not found to be associated with MCP in either sex-stratified GWAS, or in GWAS meta-analysis (Fig 3 and S2 Table). None of the six and four genes associated with MCP in females and males only respectively had a mouse orthologue listed in the Pain Genes Database [50], an interactive web browser listing mouse genes associated with pain-related phenotypes when knocked out. None of the six and four genes associated with MCP in females and males only respectively had a mouse ortholog listed in the Pain Genes Database [50], an interactive web browser of pain-related transgenic knockout studies. Of the twenty-four novel genes found to be associated with MCP in GWAS meta-analyses and associated downstream analyses, one had an ortholog listed in the Pain Genes Database (Gnaq)[51]. Gene expression analysis Tissue-enrichment of MCP-associated gene expression was analysed using FUMA, which implements a MAGMA gene-property analysis [52] using GTEx [53] gene expression datasets to determine association between trait-associated genes and expression in a range of bodily 8 / 27 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Fi M ll ifi i f diff i d MCP i d Th b Sex stratified multisite chronic pain G Fig 5. Mouse cell-type-specific expression patterns for sex-differentiated MCP-associated genes. The mousebrain. org database was used to identify cell types of expression in the mouse nervous system (brain neurons and glia have been collapsed to two lines at the base of the plot for clarity) for orthologues of genes identified in this study. Trinarization score (posterior probability of detection in a particular cell type) was used, rather than expression levels. Fig 5. Mouse cell-type-specific expression patterns for sex-differentiated MCP-associated genes. The mousebrain. org database was used to identify cell types of expression in the mouse nervous system (brain neurons and glia have been collapsed to two lines at the base of the plot for clarity) for orthologues of genes identified in this study. Trinarization score (posterior probability of detection in a particular cell type) was used, rather than expression levels. The analysis was carried out for 25 genes selected from the male-specific (n = 14) and female-specific (n = 11; note that Dcc appears in both lists) MCP-associated sets as being enriched for neural tissue expression (neural proportion score > 0.5, S4 and S5 Tables). While most of these show pan-neuronal expression, a few are expressed in mouse glial subpopulations (Cdhr4), primarily in CNS neurons (Gabrb2), primarily in DRG neurons (Amigo3), or in limited subsets of neurons (Dcc, Slc4a10, Camkv). https://doi.org/10.1371/journal.pgen.1009428.g005 https://doi.org/10.1371/journal.pgen.1009428.g005 https://doi.org/10.1371/journal.pgen.1009428.g005 tissues. MCP-associated genes in females were found to be enriched for expression in the brain, particularly the cerebellum, and frontal cortex (Fig 4). There was no significant tissue enrichment for expression of MCP genes found in males (Fig 4). tissues. MCP-associated genes in females were found to be enriched for expression in the brain, particularly the cerebellum, and frontal cortex (Fig 4). There was no significant tissue enrichment for expression of MCP genes found in males (Fig 4). Gene expression analysis PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 9 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS For genes identified (using MAGMA, as reported above) as significantly associated with MCP in either the male-only or female-only GWAS, we carried out further analyses of gene expression at tissue and cell-type level by querying existing transcriptomic data- sets, focusing on neural tissues and tissues specific to each sex. For most tissues GTEx data were used, but because GTEx does not contain data for dorsal root ganglion (DRG) neu- rons, which are key for the generation of the nociceptive signals that initiate pain in chronic pain patients, we assessed expression in this tissue using other comparable data- sets [54], which additionally contained measures of gene expression enrichment in human neural tissues (neural proportion score) and in the DRG (DRG enrichment score) (details in Methods). We also used single cell RNA-seq datasets to estimate whether genes of inter- est are likely to be expressed in neurons in the peripheral or central nervous systems (CNS). Full results of this analysis are given in S4 and S5 Tables, while those for a subset of genes (those enriched for neural tissue expression) are shown in Fig 5. Most of the 37 genes identified from the male-only GWAS were observed to be expressed in the nervous system. Two of the 37 genes, IP6K3 and FAM129A, had low neural proportion scores, sug- gesting that they are non-neuronal and non-glial. All 37 genes, however, showed DRG expression, although expression level was very low for DCC and IP6K3, which were more highly expressed in the CNS. One gene, AMIGO3, was found to be enriched solely in the DRG; its orthologue has also been found to be primarily expressed only in mouse DRG neurons in the www.mousebrain.org dataset (Fig 5). Of the 30 female-specific MCP-asso- ciated genes, all showed high neural expression except CPS1, which was not expressed in neural tissue at all. Again, we noted that all 30 genes except GABRB2 (whose mouse ortho- logue is primarily expressed in CNS neurons, Fig 5) did show expression in DRG (S5 Table), though none of them showed enriched expression in the DRG compared to the CNS. Several genes in either the male-only and female-only lists (e.g. Gene expression analysis CPS1, SEMA3F, MST1, MST1R, SDK1, ECM1; S4 and S5 Tables) that were expressed in neural tissues but with low (< 0.5) neural proportion scores were found to be involved in immune function. Among the genes with high (> 0.5) neural proportion scores, mouse orthologues of many are pan-neuro- nal in expression based on the mousebrain.org dataset (Fig 5), with a few genes having ubiqui- tous but neural-enriched expression (Mrps21, Ip6k1), solely glial expression (Cdhr4), or being expressed in limited neuronal subpopulations including CNS, sensory and enteric neuronal subtypes (Dcc, Camkv, Slc4a10 respectively). Several of the neuronally expressed genes are known to be involved in axon pathfinding and neurite outgrowth. Many of these genes have elevated expression levels in sex-specific tissues like testis and ovary, and / or are somewhat differentially expressed (10% or more difference in median TPMs across sexes) between male and female cohorts in brain sub-regions in the GTEx database (S4 and S5 Tables), suggesting they may be androgen- or estrogen-regulated. Comparing male and female multisite chronic pain Prevalence, coping strategies, and, potentially, mechanisms of development and maintenance of chronic pain vary between the sexes. To explore underlying genetic differences that may contribute to these sex differences in chronic pain, we carried out a large-scale sex-stratified GWAS of a quantitative chronic pain phenotype, MCP. We found both male and female MCP to be moderately heritable. Although the estimated female SNP heritability was higher than that in males (12.5% versus 10.6% respectively), this difference was not significant. Pathway analyses No significant results were found for male MCP-associated genes using FUMA GENE2FUNC [55]. There was no overrepresentation of male MCP-associated genes within any of the Molec- ular Signatures Database (MSigDB) gene sets (h, c1-c7) [56–58]. Two of the male MCP-associ- ated genes were associated with disease-related entries in OMIM (Online Mendelian Inheritance in Man) [59], neither of which mention a pain-related phenotype, and none of the genes were listed as previously identified drug targets in DrugBank [60] (S6 Table). Amongst the female MCP-associated genes, overrepresentation was only found for two of the positional PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 10 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS gene sets listed in MsigDB (S3 Fig). Nineteen female MCP genes were associated with an OMIM entry, and five were listed as drug targets in DrugBank (S7 Table). Polygenic risk score analysis Polygenic risk scores were used to assess whether MCP and chronic widespread pain (CWP), a related but distinct chronic pain phenotype, are likely to be related biologically. For both men and women, the sex-specific PRS-MCP was significantly associated with CWP (p < 2 x 10−17; O.R. = 1.0034 (female) and 1.0026 (male); S8 and S9 Tables), indicating that increased genetic risk for MCP is significantly associated with having chronic widespread pain. MCP-associated SNP loci Twice as many genomic risk loci were identified in GWAS analyses in females as in males (10 versus 5 respectively), with no risk loci shared between the sexes. This may be due to lower sample size in the male MCP GWAS, both in comparison to previous non-stratified analyses and to sample size in the female MCP GWAS analysis (roughly 30,000 more participants are included in the female MCP GWAS than in the male GWAS). Loci were found across the genome (Fig 1 and Table 3) in both males and females, with genomic location varying by sex. We note also, however, that significant loci were found in both men and women on chromo- some 6, only 0.89 Mbp apart (Table 3). Additional trait-associated SNPs were discovered when the sex-stratified GWAS outputs were meta-analysed, likely due to increased power. Twenty-four loci that had not reached genome-wide significance in our previous sex-combined analysis were found to be associated with MCP after meta-analysis. The fact that these loci were not identified in the non-stratified GWAS could be due to effect heterogeneity, in terms either of direction or magnitude, between the sexes reducing the overall signal at these loci. Previous studies have highlighted sex-specific or sex-differentiated loci in a range of disor- ders and traits, such as ASD, anthropometric traits and asthma [31,61–63]. Four of the loci found to be associated with MCP in our meta-analysis of sex-stratified GWAS outputs, includ- ing 7 SNPs in total, showed signs of heterogeneity of effect size between the sexes (I2 p < 0.05), though the evidence was not significant after FDR-adjustment. Nevertheless, if these results are replicated in future studies, it may be that these loci are found to contribute to sex differ- ences in chronic pain. Genes of interest Genes associated with MCP in males. The sex-stratified gene-level analysis discovered a number of genes with significant evidence for a genetic contribution to MCP. Significant genes in males included CENPW, MTCH2, NICN1, AMIGO3, DNAJA4, CTBP2 and NOP14, with the latter two also being significant in the meta-analysis gene-level testing (S2 and S3 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 11 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Tables). CENPW encodes centromere protein W, involved in kinetochore assembly and func- tion, and associated with diseases such as type 1 diabetes [64–66]. MTCH2 (mitochondrial car- rier 2) encodes a member of the SLC25 family, a family of transporters localised to the inner membrane of mitochondria and involved in a wide range of cell metabolism functions [67]. SNPs in this locus have previously been associated with obesity [68–70], and this gene may be involved in regulation of development of adipocytes. NICN1 (nicolin 1) encodes a nuclear pro- tein of unknown function expressed in a variety of tissues [71]. AMIGO3 (adhesion molecule with Ig-like domain 3), the only male-specific MCP-associated gene whose expression was enriched in DRG, is a member of a small family of cell-surface immunoglobulin domain- and leucine-rich repeat-containing adhesion molecules. Its function is not well understood, but it is expressed in a range of DRG neuronal subtypes and may play a specialized role in nocicep- tion or other sensory modalities. Interestingly, AMIGO3 is located almost next to and within 40 kbp of IP6K1, which was also found to be associated in the male gene-level analysis (S2 Table). It may be that there is coordinate regulation of these two genes in tissues relevant to MCP, or that a number of separate functional variants are distributed across this genomic locus but in fact only influence expression of one of these two genes. DNAJA4 (DnaJ Heat Shock Protein Family (Hsp40) Member A4) encodes a heat shock protein [72] previously shown to be involved in melanoma metastasis and angiogenesis regulation, but is generally poorly characterised [73]. Genes associated with MCP in females. Genes found to be associated with MCP in females included NCAN, SPATS2L, TBC1D9, CAMK1D, SOX11, GON4L, and DAGLB, the last of which was also significantly associated in the meta-analysis. NCAN (neurocan) encodes a chondroitin sulfate proteoglycan [74] potentially involved in the modulation of cell adhesion and migration, and previously linked to bipolar disorder in GWAS and mouse model studies [75,76]. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Genes of interest SPATS2L (spermatogenesis associated serine rich 2 like) encodes a protein that may be involved in ribosome biogenesis and translational control as a response to oxidative cellular stress [77]. TBC1D9 (TBC1 domain family member 9) encodes a potential GTPase and was found to be overexpressed in mantle cell lymphoma [78]. TBC1D9 was also recently found to be involved in a Ca2+-dependent cellular response to infection [79]. CAMK1D (calcium/cal- modulin dependent protein kinase ID) encodes a member of the calcium/calmodulin-depen- dent protein kinase 1 family involved in granulocyte regulation, activating CREB-dependent gene transcription, the activation and differentiation of neutrophils, promotion of basal den- dritic growth of hippocampal neurons, and apoptosis in erythroleukemia cells [80]. SOX11 (SRY-box transcription factor 11) encodes a member of the SOX (SRY-related HMG-box) family of transcription factors, with potential roles both in nervous system development and in neurogenesis during adulthood [81–85]. De novo mutations in this gene have also been associated with Coffin-Siris syndrome [86]. GON4L (Gon-4 like) encodes a protein involved in transcriptional repression [87,88]. DAGLB (diacylglycerol lipase beta) encodes an enzyme that participates in the endocannabinoid synthesis pathway and is required for axonal growth during development and for retrograde synaptic signalling in mature synapses [89]. Overall, it was notable that genes found to be significantly associated with male and female MCP were largely different. Only one gene, DCC (DCC netrin 1 receptor; a.k.a. deleted in colorectal carcinoma), was associated with both male and female MCP. DCC encodes a recep- tor for the guidance cue netrin 1, and is important for development of the nervous system, par- ticularly the dopaminergic system [90]. Mutations in the DCC gene have been found in those with congenital mirror movement disorder (MRMV-1; [91] and it has also been previously associated with a range of complex brain-related traits, including suicidality, mood instability, intelligence and putamen volume [41,42,92–94]. DCC has also been highlighted as a risk gene PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 12 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS for major depression, and may be involved in the pathology of depression through effects on axon guidance in the developing and adult CNS [95,96]. Gene expression differences in male and female MCP. Genes of interest Gene expression analyses in GTEx (which does not have DRG samples) carried out using FUMA indicated that expression of the female-specific MCP-associated genes was enriched primarily in brain tissue, and this pattern was also seen when meta-analysed sex-stratified GWAS outputs were analysed simi- larly (not shown). Almost all of these genes were also expressed in the human DRG—it is inter- esting to speculate as to whether the role of these genes in initiation or maintenance of chronic pain phenotypes is mediated through roles in the brain or via effects on cells located within the DRG. No significant enrichment for specific tissues was seen in analyses of male MCP-associ- ated genes using GTEx and the human DRG expression profiles. The lack of tissue-enrichment findings for the male-specific genes may, as with the lower number of MCP-associated loci, be due to reduced power resulting from lower sample size in the male GWAS. However, these patterns of tissue-level gene expression in GTEx may also indicate differing gene expression between the sexes, with more ubiquitous expression across all tissues for genes associated with MCP in males, while genes conferring risk in females may tend to have more tissue-specific expression patterns. These expression patterns may also be associated with the fact that the GTEx resource is enriched for male tissue samples (V8 release; 67.1% male)–sex-differential enrichment of certain genes may be conflated with tissue-differential gene enrichment. It is still notable that almost all the male or female MCP-associated genes were found to be expressed in human CNS tissues and in DRG. A subset of these are enriched in human neural tissues and additionally are expressed in mouse neuronal subpopulations when examining sin- gle cell sequencing databases (Fig 4). All of these lines of evidence, together, suggest putative central and peripheral neuronal roles for some of these genes, many of which have not been historically well studied in the field of chronic pain. Genetic correlations. For both males and females, MCP was genetically correlated with non-stratified MCP [17], at rg = 1. In contrast, the genetic correlation between male MCP and female MCP was found to be high but significantly less than 1 (rg = 0.92), with around 8% of common SNP-tagged trait variation therefore not shared between the two traits. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Genes of interest However, it could be argued that although this figure suggests that a small subset of genetic variation linked to MCP is unique to each sex, this difference is not large enough to consider MCP in the two sexes as biologically distinct to a considerable extent—traits correlated at lower rg are routinely used as proxies for one another in GWAS settings e.g. educational attainment as proxy for intelligence (rg ~ 70%) [92], or current age as a proxy for life span (rg ~40–70%) [97]. g g g y g Genetic correlations with a range of psychiatric disorders, psychological traits and somatic traits and disorders were explored and notable sex-related commonalities and differences were observed. Significant genetic correlations of similar magnitude for both sexes were found between MCP and a range of psychiatric, autoimmune and anthropometric traits. Some phe- notypes were significantly genetically correlated only with female MCP (schizophrenia, rg = 0.13; PGC cross-disorder phenotype, rg = 0.14; PTSD, rg = 0.44; anorexia nervosa, rg = -0.08), while others were significantly genetically correlated only with male MCP (autism spectrum disorder, rg = -0.16; primary biliary cholangitis, rg = 0.14). In most cases, where one sex was significant and the other was not, the rg values for both sexes were not very different, suggest- ing that the underlying biology is only subtly, quantitatively different. The sex differences that were observed may reflect sex-differential patterns of pleiotropy, with some genetic factors contributing differentially to multiple phenotypes in males and females. It would be of interest to investigate the relative effect size of loci contributing to MCP and to schizophrenia and PTSD in the two sexes and the biological mechanisms underlying this difference. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 13 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Observed genetic correlation differences may also, however, be due to differences in sample size between male and female MCP GWASs (with lower sample size for men compared to women). Differences may also be a result of either men or women being over-represented in the comparison GWAS. For example, in the GWAS meta-analysis of autism spectrum disorder [98] contributing cohorts had M:F ratios from 1.2:1 to as high as 8.6:1, PBC GWASs contribut- ing to the discovery set in the PBC GWAS meta-analysis [99] contained > 90% female cases, and the anorexia nervosa GWAS [100] contained only female cases. Genes of interest Analysis of the cohorts used in the schizophrenia and PTSD GWAS meta-analyses [101–106], suggests that the sex dif- ference in genetic correlation with MCP for these phenotypes is not primarily driven by an overrepresentation of one sex. Comparing the relationship between chronic widespread pain and MCP in males and females Each sex-specific PRS was significantly associated with CWP in the corresponding sex, but the magnitude of association was much lower in comparison to the sex-combined PRS analysis reported previously [17], which may reflect the smaller sample sizes in the sex-stratified analy- ses. As previously found in sex combined analyses, results indicated a moderate degree of shared genetic basis for MCP and chronic widespread pain in both sexes, with degree of shar- ing potentially slightly stronger in females than in males. It is possible that this difference is driven at least in part by the overrepresentation of females in CWP cases (M:F ratio 1:1.74). Clinical perspective on findings and potential impact on treatment Overall, our findings suggest that MCP shows genetic (and therefore biological) differences between men and women. If women with MCP are more likely to be at risk of PTSD and schizophrenia (and vice versa) specific screening for these potential comorbidities could be appropriate, with a view to instigating additional appropriate management or referrals. Except for PTSD, however, these sex-differential genetic correlations were relatively small in compari- son with those that affected both sexes, particularly a range of mood phenotypes. Nevertheless, the shared biology evident in the significant genetic correlations should stimulate investigation of whether screening for these potential clinically important co-morbidities can be used to improve management of chronic pain patients. Enhanced attention to the sex differences in manifestation and underlying biology of chronic pain is also merited, and may lead to improvements in clinical assessment, awareness of risks and choice of medical treatment. For example, if MCP in women is more strongly associated with immune function (based on evidence from MCP-associated genes), efficacy and side-effect profiles for drugs targeting immune system function may be different in women and men. Inappropriate treatments, such as chronic opioid prescribing, might also have sex-differential consequences—opioids are known to adversely affect immune function [107,108]. These sex differences in MCP biology may also inform the search for new or re-pur- posed drugs that can be prescribed in a sex specific manner. It is already known that specific proteins play sex-specific roles in pain processing and that certain drugs have been found to have sex-specific analgesic effects (reviewed by [109]). Limitations These analyses were carried out using UK Biobank, which was used in our previous sex-com- bined MCP GWAS [17]. In comparison to our previous analysis, sample size in each individ- ual sex-stratified GWAS was lower, which leads to somewhat reduced power. However, sample sizes are still larger than many sex-combined GWAS analyses of chronic pain PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 14 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS phenotypes, and meta-analysis of the sex-stratified GWAS outputs resulted in an increase in power to find MCP-associated SNPs overall. True replication is difficult due to heterogeneity in chronic pain phenotyping and available sample sizes of potential independent cohorts, but in an independent subset of the UK Biobank, a PRS constructed from each sex-stratified GWAS output was found to be significantly associated with CWP, a related but distinct chronic pain phenotype of interest. Although this work was focused on sex differences in the genetics of MCP we examined only autosomal variation. An important extension of this work would be to assess genetic asso- ciations with X chromosome loci, which is likely to provide an additional heritability contribu- tion and give a fuller picture of sex differences in MCP at the genetic level. Inclusion of the X chromosome in GWAS analyses is associated with specific methodological and statistical issues including lower quality genotyping array coverage of the X chromosome compared to autosomes, differences in how imputation needs to be implemented, differences in X chromo- some dosage between the sexes leading to differences in population genetics/demographic his- tory of the X chromosome relative to the autosomes, and changes to quality control required (and differing QC protocols between sexes) [110,111]. We aim to address this in future work as we adapt our BOLT-LMM pipeline and downstream analysis pathway. These GWAS analyses were carried out on a white British subset of UK Biobank, and there- fore may not generalise to admixed or non-white populations. GTEx donors are also primarily white (v8 release: 84.6% white, 12.9% African American, 0.2% American Indian, 1.3% Asian, 1.1% Unknown, [https://www.gtexportal.org/home/tissueSummaryPage]), as are donors who provided DRG tissue (white females [54]), again potentially limiting generalisability of our findings to non-white populations. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Chronic pain phenotyping UK Biobank participants were asked via a touchscreen questionnaire about “pain type(s) expe- rienced in the last month” (field ID 6159), with possible answers: ‘None of the above’; ‘Prefer not to answer’; pain at seven different body sites (head, face, neck/shoulder, back, stomach/ abdomen, hip, knee); or ‘all over the body’. The seven individual body-site pain options were not mutually exclusive, but those who chose ‘all over the body’ could not also select from the seven individual body sites. Where patients reported recent pain at one or more body sites, or all over the body, they were additionally asked (category ID 100048) whether this pain had lasted for 3 months or longer. Chronic Widespread Pain (CWP) was defined as reported [112], and the ‘case’ group included only those participants who answered that they had pain ‘all over the body’ that was longer than 3 months in duration in the touchscreen questionnaire. These individuals were excluded from analyses of other chronic pain phenotypes as there is some evidence that this phenotype can be substantially different from more localised chronic pain [113]. Multisite Chronic Pain (MCP) was a quasi-quantitative variable defined as previously reported [17]; briefly, this variable captures the number of body sites at which chronic pain (at least 3 months duration) was recorded (excluding those with CWP): phenotypic values therefore ranged from 0 to 7. 10,000 randomly selected individuals reporting no chronic pain were excluded from the GWAS to use as controls in subsequent polygenic risk score (PRS) analyses. Conclusions We also observed that several MCP-associated loci in both sexes have been linked to immune function. Overall, our findings indicate the existence of potential sex differences in chronic pain at multiple levels, from SNP-level to transcript abundance and the results support theories of strong nervous system and immune involvement in chronic pain in both sexes. These findings may inform development of novel treatment approaches in future, as well as adding to our understanding of the physiology of chronic pain. Conclusions Sex differences in chronic pain likely have, at least in part, a genetic basis and the study of com- plex traits such as chronic pain is likely to benefit from “sex-aware” analytical approaches. This study comprises one of the largest sex-stratified genetic analyses of a chronic pain phenotype, and highlights sex-differential MCP-associated loci, genes, genetic correlations, and patterns of tissue expression. We also examined transcriptome abundance of key sex-differential MCP- associated genes in a range of neural and non-neural tissues, including DRG, an important nervous system component in chronic pain which is not part of the GTEx resource and so may be understudied in GWASs with follow-up conducted solely using FUMA. Sex-stratified GWASs can provide an increase in power if heterogeneity in effects of trait- associated variants is seen between the sexes. Here, 24 novel genes and 11 novel independent lead SNPs were associated with MCP, in addition to the findings from previous non-sex-strati- fied work, further contributing to understanding of genetic variation predisposing to chronic pain. Genetic correlation results indicated possible sex-differential pleiotropy, including differing genetic correlations between certain psychiatric disorders and traits and chronic pain in women compared to men. However, it is of note that genetic correlations between the sexes are largely similar, particularly with respect to psychiatric disorders and traits including MDD, anhedonia and depressive symptoms. This is the first study to use novel GWAS outputs from studies of suicidality and of RDoC mental health traits such as mood instability and anhedonia in genetic correlation analyses with chronic pain and it provides an important insight into shared genetic factors between these comorbidities of chronic pain and MCP. The patterns of gene expression enrichment associated with the identified predisposing genes supports and enhances our previous conclusion that MCP derives more strongly from PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 15 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS brain and/or CNS-based mechanisms than from other organ systems and functions. We did observe, however, that almost all the sex-specific MCP-associated genes identified are expressed in the DRG as well as in the brain, with one sex differentiated MCP gene, AMIGO3, being DRG-specific amongst the tissues assessed, raising the possibility that cells in the DRG that play a specialised role in nociception or other sensory function might be involved in chronic pain mechanisms. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 BOLT-LMM GWAS and gene-level analysis in FUMA Sex-stratified GWASs of MCP, modelled as a quantitative trait, were carried out using BOLT-LMM [115], adjusting for age and chip (genotyping array), under the infinitesimal model of genetic risk, as previously described for our unstratified MCP GWAS [17]. BOLT-LMM uses a genetic relatedness matrix in the model to adjust for population stratifica- tion and familial relationships. We therefore did not include genetic principal components (GPCs) in the model. The SNP-level GWAS summary statistics were then analysed using FUMA [55] to obtain genome-wide plots and carry out MAGMA [49] gene-set and gene- based test analyses and gene expression analysis using GTEx [53] for male-enriched and female-enriched MCP genes. Significant independent lead SNPs were determined according to FUMA. Briefly, FUMA defines lead SNPs as the subset of independent significant SNPs (SNPs associated with the trait at p < 5 x 10−8 and having LD r2 < 0.6 with any other signifi- cant SNP) that are not in LD (r2 > 0.1) with any other lead SNP [55]. In addition, when these LD blocks of independent significant SNPs are in close proximity (< 250kbp apart), separate loci based on LD thresholds are merged into a single genomic locus, and thus each genomic risk locus can contain multiple lead and independent significant SNPs [55]. Meta-analysis of male and female GWAS Summary statistics. Meta-analysis of the two sex-specific GWAS summary statistics datasets was carried out using METAL [116], deploying a fixed-effects model and weighted by standard error (‘SCHEME STDERR’) with default options aside from selecting ‘heterogeneity’ in order to analyse heterogeneity (‘ANALYZE HETEROGENEITY’). A meta-analysis p-value of < 5 x 10−8 was selected as the significance threshold for association. Gene-level analysis (MAGMA) was also carried out using meta-anal- ysis output. Genetic quality control For the GWAS analyses, SNPs with an imputation quality score of less than 0.3, minor allele frequency (MAF) < 0.01 and/or Hardy-Weinberg equilibrium (HWE) test p < 10−6 were excluded. Participants whose self-reported sex did not match their genetically-determined sex, those who had putative sex-chromosome aneuploidy, those considered outliers in UK Biobank QC in terms of missingness or heterozygosity [114], and those who were not of self-reported white British ancestry were excluded from analyses. A list of “poor quality” samples (due to missingness, putative genetic and reported sex mismatch and/or unexpectedly high heterozy- gosity) was derived by Bycroft et al [114] and is available to all researchers using UK Biobank, and was used here as part of genetic quality control. Briefly, putative sex chromosome aneu- ploidy was defined by visual inspection of scatterplots of mean log2 ratio (L2R) on X and Y chromosomes, and 652 UKB participants meet these criteria for putative sex-chromosome aneuploidy (Supplemental Information S 3.6 [114]). Samples with a heterozygosity value, adjusted for both ancestry and genetic principal components (GPCs), above the mean PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 16 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 4. Number of participants per MCP phenotype level group included in each GWAS (male or female sex- stratified analysis). MCP N female % female N male % male 0 113148 54.11 105474 59.07 1 49984 23.91 42734 23.93 2 26000 12.43 18612 10.42 3 12376 5.92 7771 4.35 4 5319 2.54 2970 1.66 5 1723 0.82 780 0.44 6 471 0.23 181 0.10 7 72 0.03 34 0.02 total n in each GWAS 209093 NA 178556 NA https://doi.org/10.1371/journal.pgen.1009428.t004 Table 4. Number of participants per MCP phenotype level group included in each GWAS (male or female sex- stratified analysis). ber of participants per MCP phenotype level group included in each GWAS (male or female sex- ysis) heterozygosity value (0.1903) and missing rate greater than 0.05 as computed using PLINK ‘— miss’ command were also flagged as potentially poor quality ([114]; 968 such samples are listed in this paper’s Supplemental Information S 3.5.3). heterozygosity value (0.1903) and missing rate greater than 0.05 as computed using PLINK ‘— miss’ command were also flagged as potentially poor quality ([114]; 968 such samples are listed in this paper’s Supplemental Information S 3.5.3). A summary of participant MCP phenotypic information for those included in each GWAS is shown in Table 4. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Transcriptome analysis of sex-specific association gene lists We further analysed the tissue and cell type of expression of male-specific and female-specific genes identified from the MAGMA gene-based analysis of the sex-stratified GWAS results. Specifically, we characterized gene expression in mammalian nervous system tissues and cell PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 17 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS types as a potential starting point for identifying the functions of these genes with respect to pain. We also characterised expression for the one gene found to be associated with both male and female MCP in the gene-based analyses (DCC). types as a potential starting point for identifying the functions of these genes with respect to pain. We also characterised expression for the one gene found to be associated with both male and female MCP in the gene-based analyses (DCC). RNA-seq-derived gene expression values have been previously reported [54] as relative abundances in TPMs (standardised Transcripts per Million mapped reads) for 12 adult human tissues (6 neural and 6 non-neural). Additionally, the study notes 3 metrics on a scale of 0 to 1 for each gene based on expression in these 12 tissues: normalized Shannon’s entropy as a mea- sure of tissue specificity (0 for highly tissue-specific and 1 for tissue-agnostic gene expression in the quantified tissues), neural proportion score as a measure of enriched expression (possi- bly neuronal and/or glial) in the nervous system (0 for genes not expressed in the nervous sys- tem and 1 for genes expressed solely in neural tissues with respect to these 12 tissues), and DRG enrichment score for identifying specificity of gene expression in the DRG with respect to the other 11 profiled tissues (0 for no expression in the DRG or for tissue-agnostic gene expression, and 1 for DRG-specific gene expression in the context of the set of profiled tissues). The mathematical formulations for these scores are provided in detail in Ray et al. 2018 [54]. The corresponding tables are presented in S4 Table (male) and S5 Table (female). Genes with neural proportion scores > 0.5 (with overall more neural than non-neural tissue expression), were further characterized by the putative cell type(s) of gene expression in the mammalian nervous system (Fig 4). While human single cell resolution RNA-seq datasets are not publicly available for the peripheral nervous system, a comprehensive database of gene expression in the mouse nervous system exists: the www.mousebrain.org repository [117]. Transcriptome analysis of sex-specific association gene lists While it is true that nervous system expression patterns may be different between human and mouse between nervous system subpopulations due to regulatory evolution or due to differ- ences in nervous system cell types [118], it is unlikely that expression would change categories across the 4 broad nervous system cell type categories: neurons, glia, immune and vascular cells) between human and mouse, given overall conservation of tissue gene expression profiles across humans and mice [54]. Trinarization scores, defined as the posterior probability (using a Bayesian framework) of detecting reads from a particular gene in a cell type subpopulation (details are given in ref. [117]), were used to characterise gene expression by cell-type. Fig 4 visualizes trinarization scores for a range of peripheral nervous system cell types (sensory neu- rons and glia, enteric neurons and glia, and sympathetic neurons). While peripheral nervous system vascular and immune cells have not been profiled so far in www.mousebrain.org, we depict CNS vascular immune cells and vascular cells as surrogate cell types for their PNS coun- terparts. Both CNS neurons and glia play a critical role in chronic pain, but due to high diver- sity of cell types were not ideal for summarizing expression patterns in each subpopulation of these categories succinctly in a single figure. Instead, summary rows for CNS neurons and glia expression for relevant genes are provided at the bottom of the figure. For further details of expression in these subtypes, www.mousebrain.org can be queried for mouse gene expression profiles. Finally, for genes with neural proportion scores > 0.5, the GTEx database was queried for sex differential gene expression in profiled CNS regions, and for high expression in sex-specific tissues, such as testis and ovary, noted in the comments section of S4 and S5 Tables. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Polygenic risk score analysis Previous analyses showed that a polygenic risk score (PRS) for MCP was associated with the phenotype of chronic widespread pain (CWP) in women but not in men, and that this PRS was associated more strongly with chronic pain phenotypes in women than in men in an inde- pendent cohort (Johnston K.J.A. et al., unpublished). To further explore the relationship between MCP and CWP, we assessed how separate male and female PRSs for MCP were asso- ciated with CWP. Separate sex-specific PRSs for MCP, based on the sex-specific GWAS results reported here, were calculated for a ‘case’ group consisting of participants who reported pain all over the body that lasted for three months or longer (a proxy phenotype for Chronic Wide- spread Pain; CWP; N = 6, 813)), and for a ‘control’ group consisting of 10,000 randomly selected UKB controls, both of which had been excluded from the GWAS analyses (demo- graphic data for this subsample are given in Table 5). SNPs associated with MCP at p < 0.01 in the original sex-specific GWAS were selected and LD-pruned (at a threshold of r2 < 0.1 within a 250kbp window using the PLINK ‘—clump’ command). Sex-specific PRS-MCPs were calculated for each individual in the analysis as the sum of risk alleles at each SNP, weighted by effect size (beta value) in the GWAS [122]. PRS values were standardised and z-scores were used in the analysis. Association between stan- dardised sex-specific PRS-MCP and CWP status was investigated separately in males and in females in the target case-control subsample using logistic regression, adjusted for chip (geno- typing array), age and the first eight genetic principal components. Genetic correlations Genetic correlations with a range of neuropsychiatric disorders and traits were assessed by LD-score regression (LDSR) for the male and female MCP GWAS outputs separately. Sum- mary statistics datasets employed were publicly available or available via LD Hub [119,120], or were results from published and unpublished in-house GWASs. Pre-computed LD scores PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 18 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Table 5. Summary of sample sizes of participants used in each of the two PRS analyses (male or female). Female Male Combined Totals N mean age (years) N mean age N mean age Control 5135 56.68 4865 56.96 10000 56.81 CWP 4328 57.00 2485 57.27 6813 57.10 Total 9463 56.83 7350 57.07 16813 56.93 https://doi.org/10.1371/journal.pgen.1009428.t005 Summary of sample sizes of participants used in each of the two PRS analyses (male or female). were used, along with HapMap3 SNPs as a reference. By default the munging part of LDSR fil- ters out SNPs with an info score of < 0.9, a MAF of < 0.01, a GWAS p value outside the range 0–1, strand-ambiguous SNPs, non-SNP (e.g. indel) variants, and SNPs with low sample size. The default option of no constraint on the LDSR intercept was used. LDSR p-values for genetic correlation were FDR-corrected within each sex. LDSR was also used to assess trait polygeni- city and to calculate a SNP-heritability estimate. Note that although some of the GWAS results used in this analysis came from studies that included UK Biobank data, genetic correlations estimated using LDSR are not subject to bias caused by sample overlap [121]. were used, along with HapMap3 SNPs as a reference. By default the munging part of LDSR fil- ters out SNPs with an info score of < 0.9, a MAF of < 0.01, a GWAS p value outside the range 0–1, strand-ambiguous SNPs, non-SNP (e.g. indel) variants, and SNPs with low sample size. The default option of no constraint on the LDSR intercept was used. LDSR p-values for genetic correlation were FDR-corrected within each sex. LDSR was also used to assess trait polygeni- city and to calculate a SNP-heritability estimate. Note that although some of the GWAS results used in this analysis came from studies that included UK Biobank data, genetic correlations estimated using LDSR are not subject to bias caused by sample overlap [121]. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 S6 Table. OMIM and DrugBank derived information (male MCP). (PDF) S6 Table. OMIM and DrugBank derived information (male MCP). (PDF) S7 Table. OMIM and DrugBank derived information (female MCP). (PDF) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 Supporting information S1 Table. Genetic correlations between MCP and other disorders and traits. Results of LDSR analysis using summary statistics from the sex-stratified GWASs of MCP versus a range of potentially related disorders and traits. Genetic correlations are given as rg values (and FDR-corrected p-values) sorted in order of numerically decreasing rg for female MCP vs other traits. f_rg and m_rg = genetic correlation value for female and male MCP versus trait, respec- tively, f_p_fdr and m_p_fdr = FDR-corrected p value for genetic correlation, source = source of trait GWAS data, PMID = PubMed ID of associated publication for GWAS of trait. Signifi- cant genetic correlations (FDR-corrected p value < 0.05) within each sex are highlighted PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 19 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS orange, non-significant in blue. (PDF) S2 Table. Table accompanying Venn diagram of genes associated with multisite chronic pain in MAGMA gene-level analyses. ‘Found in’ refers to the GWAS and corresponding MAGMA gene-level analyses where genes were found to be significantly associated with MCP: Female = sex-stratified GWAS (female), Male = sex-stratified GWAS (male), Meta = GWAS meta-analysis of male and female sex-stratified GWAS outputs, Original = sex-combined GWAS analysis described previously [17]. Total = total number of genes in category. Elements = gene names. (PDF) S3 Table. Genes associated with MCP in MAGMA gene-level analyses of GWAS meta-anal- ysis output. (PDF) S4 Table Expression of genes associated with male MCP in MAGMA analyses across neural and non-neural tissues TPM = transcripts per million, DRG = dorsal root ganglion h_DRG_enrich = DRG enrichment score h_entropy = normalized Shannon’s entropy h_neural_propn = neural proportion score. (PDF) S5 Table. Expression of genes associated with female MCP in MAGMA analyses across neural and non-neural tissues. TPM = transcripts per million, DRG = dorsal root ganglion. h_DRG_enrich = DRG enrichment score. h_entropy = normalized Shannon’s entropy. h_neural_propn = neural proportion score. (PDF) S6 Table. OMIM and DrugBank derived information (male MCP). (PDF) S7 Table. OMIM and DrugBank derived information (female MCP). (PDF) S8 Table. Association between male MCP PRS and CWP in men. Full results (chip, PCs) not shown for brevity. SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z- standardised PRS value. (PDF) S9 Table. Association between female MCP PRS and CWP in women. Full results (chip, PCs) not shown for brevity. Supporting information SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z-standardised PRS value. (PDF) S1 Fig. Tissue Expression of MCP-associated Genes (male) (53 tissues). (PDF) S2 Fig. Tissue Expression of MCP-associated Genes (female) (53 tissues). (PDF) S3 Fig. FUMA gene set analysis results for female MCP genes from the gene-level analysis. (TIFF) ( ) S2 Table. Table accompanying Venn diagram of genes associated with multisite chronic pain in MAGMA gene-level analyses. ‘Found in’ refers to the GWAS and corresponding MAGMA gene-level analyses where genes were found to be significantly associated with MCP: Female = sex-stratified GWAS (female), Male = sex-stratified GWAS (male), Meta = GWAS meta-analysis of male and female sex-stratified GWAS outputs, Original = sex-combined GWAS analysis described previously [17]. Total = total number of genes in category. Elements = gene names. (PDF) S3 Table. Genes associated with MCP in MAGMA gene-level analyses of GWAS meta-anal- ysis output. (PDF) S3 Table. Genes associated with MCP in MAGMA gene-level analyses of GWAS meta-anal- ysis output. (PDF) S4 Table Expression of genes associated with male MCP in MAGMA analyses across neural and non-neural tissues TPM = transcripts per million, DRG = dorsal root ganglion h_DRG_enrich = DRG enrichment score h_entropy = normalized Shannon’s entropy h_neural_propn = neural proportion score. (PDF) S4 Table Expression of genes associated with male MCP in MAGMA analyses across neural and non-neural tissues TPM = transcripts per million, DRG = dorsal root ganglion h_DRG_enrich = DRG enrichment score h_entropy = normalized Shannon’s entropy h_neural_propn = neural proportion score. (PDF) S5 Table. Expression of genes associated with female MCP in MAGMA analyses across neural and non-neural tissues. TPM = transcripts per million, DRG = dorsal root ganglion. h_DRG_enrich = DRG enrichment score. h_entropy = normalized Shannon’s entropy. h_neural_propn = neural proportion score. (PDF) S7 Table. OMIM and DrugBank derived information (female MCP). (PDF) S8 Table. Association between male MCP PRS and CWP in men. Full results (chip, PCs) not shown for brevity. SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z- standardised PRS value. (PDF) S9 Table. Association between female MCP PRS and CWP in women. Full results (chip, PCs) not shown for brevity. SE = standard error, Z = Z value, P = p value, OR = odds ratio, PRS = z-standardised PRS value. (PDF) S1 Fig. Tissue Expression of MCP-associated Genes (male) (53 tissues). (PDF) S2 Fig. Tissue Expression of MCP-associated Genes (female) (53 tissues). (PDF) S3 Fig. FUMA gene set analysis results for female MCP genes from the gene-level analysis. (TIFF) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1009428 April 8, 2021 20 / 27 PLOS GENETICS Sex-stratified multisite chronic pain GWAS Author Contributions Conceptualization: Keira J. A. Johnston, Daniel J. Smith, Mark E. S. Bailey. Conceptualization: Keira J. A. Johnston, Daniel J. Smith, Mark E. S. Bailey. Data curation: Joey Ward, Pradipta R. Ray. Data curation: Joey Ward, Pradipta R. Ray. Formal analysis: Keira J. A. Johnston, Joey Ward, Pradipta R. Ray, Theodore J. Price. Supervision: Mark J. Adams, Andrew M. McIntosh, Daniel J. Smith, Barbara I. Nicholl, Mark E. S. Bailey. Writing – original draft: Keira J. A. Johnston, Pradipta R. Ray, Blair H. Smith, Theodore J. Price. Writing – review & editing: Keira J. A. Johnston, Joey Ward, Pradipta R. Ray, Mark J. Adams, Andrew M. McIntosh, Blair H. Smith, Rona J. Strawbridge, Theodore J. Price, Daniel J. Smith, Barbara I. Nicholl, Mark E. S. Bailey. Acknowledgments We thank participants in the UK Biobank study. UK Biobank was established by the Wellcome Trust, Medical Research Council, Department of Health, Scottish Government and Northwest Regional Development Agency. This work was carried out under approved UK Biobank appli- cations 6553 and 7155. 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https://openalex.org/W3180834865	https://backend.orbit.dtu.dk/ws/files/266756569/Laqua_2021_Nucl._Fusion_61_106005.pdf	English	null	High-performance ECRH at W7-X: experience and perspectives	Nuclear fusion	2,021	cc-by	4,450	Citation (APA): Laqua, H. P., Baldzuhn, J., Braune, H., Bozhenkov, S., Brunner, K., Hirsch, M., Hoefel, U., Knauer, J., Langenberg, A., Marsen, S., Moseev, D., Pasch, E., Rahbarnia, K., Stange, T., Wolf, R. C., Pablant, N., & Grulke, O. (2021). High-performance ECRH at W7-X: Experience and perspectives. Nuclear Fusion, 61(10), Article 106005. https://doi.org/10.1088/1741-4326/ac1a1b Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. You may also like Validation of theory-based models for the control of plasma currents in W7-X divertor plasmas A. Dinklage, G. Fuchert, R.C. Wolf et al. - (Expected difficulties with) density-profile control in W7-X high-performance plasmas C D Beidler, Y Feng, J Geiger et al. - Plasma–surface interaction in the stellarator W7-X: conclusions drawn from operation with graphite plasma-facing components S. Breznsek, C.P. Dhard, M. Jakubowski et al. - This content was downloaded from IP address 192.38.67.116 on 03/01/2022 at 10:19 High-performance ECRH at W7-X Experience and perspectives Laqua, H. P.; Baldzuhn, J.; Braune, H.; Bozhenkov, S.; Brunner, K.; Hirsch, M.; Hoefel, U.; Knauer, J.; Langenberg, A.; Marsen, S. Total number of authors: 17 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Laqua, H. P., Baldzuhn, J., Braune, H., Bozhenkov, S., Brunner, K., Hirsch, M., Hoefel, U., Knauer, J., Langenberg, A., Marsen, S., Moseev, D., Pasch, E., Rahbarnia, K., Stange, T., Wolf, R. C., Pablant, N., & Grulke, O. (2021). High-performance ECRH at W7-X: Experience and perspectives. Nuclear Fusion, 61(10), Article 106005. https://doi.org/10.1088/1741-4326/ac1a1b this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim. ∗Author to whom any correspondence should be addressed. a See Klinger et al 2019 (https://doi.org/10.1088/1741-4326/ab03a7) for the W7-X Team. Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. High-performance ECRH at W7-X: experience and perspectives Validation of theory-based models for the control of plasma currents in W7-X divertor plasmas A. Dinklage, G. Fuchert, R.C. Wolf et al. - (Expected difficulties with) density-profile control in W7-X high-performance plasmas C D Beidler, Y Feng, J Geiger et al. - Plasma–surface interaction in the stellarator W7-X: conclusions drawn from operation with graphite plasma-facing components S. Breznsek, C.P. Dhard, M. Jakubowski et al. - Validation of theory-based models for the control of plasma currents in W7-X divertor plasmas - p A. Dinklage, G. Fuchert, R.C. Wolf et al. (Expected difficulties with) density-profile control in W7-X high-performance plasmas C D Beidler, Y Feng, J Geiger et al. - To cite this article: H.P. Laqua et al 2021 Nucl. Fusion 61 106005 To cite this article: H.P. Laqua et al 2021 Nucl. Fusion 61 106005 Plasma–surface interaction in the stellarator W7-X: conclusions drawn from operation with graphite plasma-facing components - View the article online for updates and enhancements. View the article online for updates and enhancements. This content was downloaded from IP address 192.38.67.116 on 03/01/2022 at 10:19 International Atomic Energy Agency Nuclear Fusion https://doi.org/10.1088/1741-4326/ac1a1b Nucl. Fusion 61 (2021) 106005 (6pp) Abstract The second operation phase of W7-X (OP1.2) showed the potential of exclusively electron cyclotron resonance heating (ECRH)-sustained plasma operations in stellarators. Employing multi-pass ECRH scenario in the second harmonic O-mode (O2-ECRH), stationary densities of up to 1.4 × 1020 m−3 could be achieved. This scenario also made stationary divertor detachment possible, which is a reactor-relevant scenario for power and particle exhaust. At high densities and with sufficiently high density gradients for an improved ion confinement, the coupling between the electrons and ions was strong enough to bring the ion temperature to values above 3 keV and to the neoclassical limit for some magnetic configurations, thus enabling to test the W7-X neoclassical optimization. The planned enhancement of the ECRH performance will enable to advance towards reactor-relevant beta values and to investigate their stability and confinement of fast particles, which is a priority goal of W7-X. Keywords: ECRH, W7-X, stellarator, high performance, high density, detachment (Some figures may appear in colour only in the online journal) similar to that expected in a fusion reactor, where in a reac- tor it is the fusion alpha particles that transfer their energy to the electrons by slowing down collisions. This scenario can be simulated in W7-X with microwave heating of the electrons. However, it should be noted that although the fast alpha par- ticles do not significantly interact with the thermic ions, they can have an influence on the ion confinement [1]. High-performance ecrh at w7-x: experience and perspectives H.P. Laqua1,∗J. Baldzuhn1 , H. Braune1, S. Bozhenkov1, K. Brunner1, M. Hirsch1, U. Hoefel1, J. Knauer1, A. Langenberg1, S. Marsen1, D. Moseev1, E. Pasch1, K. Rahbarnia1, T. Stange1, R.C. Wolf1, N. Pablant2 , O. Grulke1,3 and the W7-X Team1,a 1 Max-Planck-Institute for Plasma Physics, Greifswald, Germany 2 Princeton Plasma Physics Laboratory, 08543 Princeton, United States of America 3 Technical University of Denmark, Kongens Lyngby, Denmark 1 Max-Planck-Institute for Plasma Physics, Greifswald, Germany 2 Princeton Plasma Physics Laboratory, 08543 Princeton, United States of America 3 Technical University of Denmark, Kongens Lyngby, Denmark E-mail: laqua@ipp.mpg.de Received 31 May 2021 Accepted for publication 3 August 2021 Published 30 August 2021 1. Introduction W7-X is an optimized stellarator in which the otherwise strong neoclassical transport is reduced so that high confinement time values can be achieved.In particular this can be done with elec- tron cyclotron resonance heating (ECRH) only, whereby only the electrons are heated directly, while the ion temperature is increased by electron ion collisions. Although it is expected that high ion temperatures will also be achieved by direct ion heating with NBI or ICRH, pure electron heating is a situation The superconductingcoil system of W7-X generates a mag- netic field strength of 2.5 T. This requires an ECRH frequency of 140 GHz, which corresponds to the second harmonic res- onance of the electrons. Unfortunately, only the X2 mode is completely absorbed in the plasma at the resonance sur- face. However, the X2-mode can only propagate up to 1.2 × 1020 m−3. The second harmonic O-mode, which has a cut- off density of 2.4 × 1020 m−3, is incompletely absorbed in the single pass. On the other hand, the highest performance is expected at densities above 1 × 1020 m−3, because of the © EURATOM 2021 Printed in the UK 1 1741-4326/21/106005+6$33.00 Nucl. Fusion 61 (2021) 106005 H.P. Laqua et al Figure 1. W7-X ECRH system. Here, one out of two models with five gyrotrons/beams is shown. The individual gyrotron beams, marked by different colours, are combined into a bundle and sent over a long distance with large common mirrors, the so called multi-beam section. They are divided into individual beams close to the vacuum vessel. Inside the vacuum vessel the beams are launched into the plasma by steerable mirrors. Figure 1. W7-X ECRH system. Here, one out of two models with five gyrotrons/beams is shown. The individual gyrotron beams, marked by different colours, are combined into a bundle and sent over a long distance with large common mirrors, the so called multi-beam section. They are divided into individual beams close to the vacuum vessel. Inside the vacuum vessel the beams are launched into the plasma by steerable mirrors. an efficiency of 95% as shown in figure 1. The transmission takes place with cooled mirrors and at atmospheric pressure. A diamond disc forms the interface to the plasma vessel. Inside the plasma vessel, the individual rays are radiated flexibly in both toroidal and poloidal direction into the plasma with the help of movable mirrors. 1. Introduction The 140 GHz EC-wave with ordinary (O2) polarization gives access to densities beyond the cut-off limit (1.2 × 1020 m−3) of the commonly used extra-ordinary (X2) polarized waves, but to the expense of incomplete single pass absorption for the W7-X plasma parameters. This dis- advantage could be compensated by a special multi-pass sce- nario, where the partially (60%–70%) absorbed ECRH-beams were reflected by specially shaped tiles and passed three times through the plasma core with an overall absorption of up to 90%. The O2-operation scenario has been also routinely used for plasma operation below the X2 cutoff at densities above 0.8 × 1020 m−3 prohibiting the otherwise high risk of uncon- trolled beam deflection in the X2-modeECRH case. In order to keep the absorption high, the density was feed-back controlled in such a way that a central Te was always kept above 2 keV. It should be noted, however, that the O2 process comes at the price of flexibility. Ray trajectories are fixed by the reflector tile positions. positive confinement scaling and the increasing coupling between the electrons and ions with density. Therefore, a multi-pass ECRH scenario was developed so that an absorp- tion of the O2 mode of more than 90% could be achieved. It should be noted that in a stellarator reactor a magnetic field strength of above 5T is expected. In that case the well absorbed first harmonic O-mode (O1) will be used. Here the full flexibil- ity of the ECRH with respect of current drive and off-axis heat- ing will be achieved and further optimization of the electron temperature profile as well as the iota profile will be possible. The high performance high density scenario must also be compatible with a technically reasonable particle and energy exhaust. For this purpose, W7-X is equipped with a so-called island divertor, which separates the core plasma from the plasma wall interaction. 3. Results An outstanding result is the stationary operation with pure O2-ECRH at plasma densities of 1.4 × 1020 m−3, which is clearly above the X2 cutoff density as shown in figure 2. Here the electron-ion coupling was so high that the ion tempera- ture approached the values of the electrons. Unfortunately, the error bars of the x-ray spectroscopy were too high to carry out a precise profile analysis. The high density operation with the multi-pass O2-ECRH scenario showed an excellent plasma performance. However the maximum achieved temperature was limited the available heating power (6 MW for 15 s) and the respective transport parameters of the plasma scenarios. This scenario could be kept stationary and was only limited in time by of the maximum heating energy applicable to this W7-X operation phase with uncooled first wall elements.i The achievable large density range enabled a combined operation with pellet injection without the risk of approach- ing the cut-off condition for the here pellet-induced peaked density profiles. After the pellet injection phase, the trans- port properties were improved for both ions and electrons and thus high plasma performance with high triple product values has been achieved [5]. Here the ion power flux approached the neoclassical value enabling to test the neoclassical trans- port optimization of W7-X [6]. In figure 3, the time traces of the pellet-fueled plasma discharge is shown. First of all, the plasma start-up had to be performed with the well-absorbed X2 mode. While the plasma density was then built up with gas fuelling, the polarization of the ECRH beams was changed from X-mode to O-mode. It should be noted that the polar- ization change can be performed during gyrotron operation. As long as the plasma density is below the X2 cutoff den- sity both ECRH modes will be absorbed. Then the density was further increased with the aid of pellet injection and fur- ther ECRH beams were switched on. After the pellet injection, a peaked density profile was established which improved the ion confinement and increased both the ion temperature and the plasma energy (diamagnetic energy). There reason for the confinement improvement is the simultaneous stabilization of the ion temperature gradient (ITG) turbulence and the trapped electron mode (TEM) turbulence. This is a unique feature of A further benefit of the O2-ECRH scenario was its com- patibility with high neutral pressure at the plasma edge. 2. Experimetal set-up W7-X has the world’s largest microwave heating system for plasma generation at the moment [2]. The ECRH works at a frequency of 140 GHz. That is twice the resonance fre- quency of the electrons at a magnetic field strength of 2.5 T, the nominal magnetic field of the superconducting coil sys- tem. The ECRH consists of 10 microwave sources, the so- called gyrotrons, with a unit power of up to 1 MW and an operating time of 30 min, which is also the targeted W7- X plasma duration of 30 min in the final completion phase. The maximum total port through power was about 8 MW, but for higher reliability in longer discharges the gyrotrons were detuned to a total port through power of 6 MW. The main reason for power limitation was arcing in the transmis- sion line due to a high air humidity during the experimental campaign in summer 2018.The 10 microwavebeams are trans- mitted with a purely quasi-optical transmission line from the gyrotrons into the plasma over a distance of about 40 m with Three high performance scenarios are reported here. In the first scenario, a stationary plasma at a density of 1.4 × 1020 m−3 with only 6 MW O2-ECRH was achieved. The elec- tron and ion temperatures almost equalized each other. In the second scenario, the density was built up with the help of pel- let injection, which lead to a temporarily peaked density profile with a strongly improved ion heat confinement. This scenario could not be explored towards steady state due to the limited number of available hydrogen ice pellets of the existing blower gun pellet injector. In the third scenario, the so-called detach- ment was demonstrated,in which the power flux to the divertor is strongly reduced. 2 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 Figure 2. Left: time traces of a gas fuelled high density plasma with O2-ECRH only. Right: density and temperature profiles of the high density discharge. Figure 2. Left: time traces of a gas fuelled high density plasma with O2-ECRH only. Right: density and temperature profiles of the high density discharge. neoclassical confinement times of 2–10 s [4]. Even more in cases, where impurity accumulation has been found, like in the exclusively NBI-heated high density plasmas, additional O2- ECRH significantly flattened the otherwise peaked impurity profiles and pushed the impurities towards the plasma edge. 4. Outlook For the next operation campaign an upgrade of the available ECRH power to 10 MW and more efficient multi-pass reflector tiles are planned. In particular a new more powerful (1.5 MW) gyrotron is being developed now and an increase of the num- ber of gyrotrons and beamlines from 10 to 12 is envisaged. The development strategy for the new gyrotron is to largely retain the successful basic W7-X gyrotron design and only to increase the operating mode from TE28.8 to TE28.10 in order to increase the cavity diameter and thus to keep the power load density at the cavity surface below the critical level of 20 MW m−2. Keeping the azimuthal mode unchanged allowed to use the same electron optics as for the 1 MW class W7-X gyrotrons. Therefore the new 1.5 MW class gyrotron is fully compatible with the W7-X installation, in particular with the gyrotron magnet, and outdated gyrotrons can easily be replaced by new more powerful gyrotrons. Inside the new gyrotron only cavity and the microwave optics are adjusted accordingly. The beam current is increased from 40 to 60 A. In experiments at KIT with an uncooled short-pulse gyrotron with the same TE28.10 microwave system, 1.5 MW output power was achieved and thus the gyrotron concept has been successfully confirmed [10]. The 1.5 MW power cw gyrotron is currently being built by the Thales company and expected for November 2021 [11]. A general challenge of nuclear fusion with magnetic con- finement is the power and particle exhaust. The advantage of the particle motion guided by field lines in magnetic confine- ment turns into a challenge when the power and the particles come into contact with the walls. Here the flux concentrated on the magnetic field lines leads to power densities that are technically difficult to control. The low shear concept of the W7-X has an advantage here, because it has long (>100 m) connection lengths between the last closed flux surface and the divertor target. Due to the different magnetic geometry and the much longer connection length compared to tokamak diver- tors, the W7-X stellerator detachment differs fundamentally from that in tokamaks. A detailed description can be found in [8]. Nevertheless, the detachment at the W7-X is also charac- terized by a strong reduction of the power flux on the divertor surface. 3. Results Even though after boronization of the W7-X wall no glow discharge cleaning has been performed, the density control was never lost and the high density operation was very robust. But on the other hand in the presence of high neutral fluxes, plasma radiation and charge exchange losses pushed down the edge temperatures as shown in figure 2. The neoclassical impu- rity transport in stellarators predicts an inward pinch and thus an impurity accumulation. Temperature gradient driven turbu- lence is counteracting here and thus in ECRH-plasmas with gas fuelling no accumulation has been found [3]. In particu- lar the laser blow off experiments estimated impurity confine- ment time of the order of 70–80 ms which is far below the 3 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 Figure 3. O2 ECRH scenario in combination with pellet injection (#20170904.015). For the plasma start-up three ECRH beams are polarized in X2-mode. During the density ramp their polarization is changed into O2-mode. The gray shaded area is the over-dense phase. Figure 3. O2 ECRH scenario in combination with pellet injection (#20170904.015). For the plasma start-up three ECRH beams are polarized in X2-mode. During the density ramp their polarization is changed into O2-mode. The gray shaded area is the over-dense phase. plasma was terminated regularly and no components had been at their power load limit. the W7-X configuration with a ‘stability valley’ for both ITGs and TEMs [7]. Unfortunately, in this experimental campaign W7-X was insufficiently equipped to control the density gradient. This requires a controlled pellet injection for the central fuelling and a high neutral gas pumping capability for a low neutral gas pressure at the edge. 5. Summary Figure 5. Tungsten covered TZM reflector tile with prolarization grating on top. In the W7-X operation phase 1.2, a routine high-density opera- tion with O2 ECRH could be established. The O2-ECRH made it possible to achieve high performance plasma scenarios in various aspects. Stationary gas puff fuelled discharges of high densities were achieved, where the electron and ion tempera- tures were equalized due to the high collision frequency. The good heating efficiency at high densities also made it possi- ble to use hydrogen ice pellet injection with which the density gradients were increased with a strongly beneficial effect on the confinement. Finally, the stationary high-density operation also enabled to reach the detachmentat the plasma edge, which strongly reduced the thermal load on the divertor plates and thus represents a possible scenario for safe long-pulse oper- ation. For the next phase of operation for W7-X (OP2), in addition to the installation of water-cooled first wall and diver- tor components, further upgrades of the active components are also planned. The ECRH power will be increased by two more gyrotron positions and new, more powerful gyrotrons. The continuous-wave pellet injector and the high-performance cryo-pumps should, among other things, enable the density profile control for scenario optimization. humidity below 20% at 20◦c. Here a high microwave power transmission test has been performed in 2020 where the suc- cessful transmission of a 0.9 MW beam for 2 min through the whole in air section of the transmission line has been demon- strated. Since here the maximum power was limited by the maximum gyrotron out-put power only, we expect a further margin enabling the transmission of the 1.5 MW beam. A detailed analyses of the absorption and losses with the O2-mode for all 10 ECRH beams in the experiments enabled further optimization of the reflector tiles. The new reflec- tor will also correct the polarization of the reflected beam, such that the O-mode polarization is maintained as shown in figure 5. Thus in the next campaign an overall absorption of 95% is envisaged. This is at first glance a small improvement. 5. Summary But it will reduce the none-absorbed ECRH stray radiation by 50%, which is a remarkable step forward for prospective future steady state operation, since the microwave stray radia- tion gives an additional load to all W7-X components even if they are outside a line of sight to the plasma [12].li The multi-reflection system can also be used for efficient plasma heating with the third harmonic X-mode at the mag- netic field strength of 1.7T, thus opening a new operation point for W7-X. In particular with reduced magnetic field a higher relative plasma pressure (beta) can be achieved with same 4. Outlook With its the help, steady state conditions could also be achieved for the uncooled test divertor of W7-X. Although the peak densities in the detachment experiments were just below the X2 cutoff density, the plasma had to be heated with the O2 mode in order to avoid the risk of uncontrolled ECRH X2 beam deflection so close to the X2-cutoff. In the experiment shown in figure 4 the transition to detachment was reached at 3 s and the radiated power approached the heating power. The power flux on the divertor was reduced from 3.5 MW m−2 to 0.4 MW m−2. This state could be kept stable for 26 s [9]. The In addition the maximum power capability of the in- air quasi-optical transmission line is being enhanced with a powerful air drying system which brings down the relative 4 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 Figure 4. Left: time traces of the maximum heat flux at the divertor tile. Right: IR-picture of the divertor for attachment and detachment. Reproduced from [9]. Figure 4. Left: time traces of the maximum heat flux at the divertor tile. Right: IR-picture of the divertor for attachment and detachment. Reproduced from [9] Figure 4. Left: time traces of the maximum heat flux at the divertor tile. Right: IR-picture of the divertor for attachment and detachment. Reproduced from [9]. Figure 5. Tungsten covered TZM reflector tile with prolarization grating on top. heating power compared to the nominal operation at 2.5 T. Plasma performance enhancement is also expected by an improved edge neutral density control with powerful cryo pumps in the divertor pumping gap and gas valves in the diver- tor region. In addition a steady state pellet injector will be installed as a US contribution to W7-X. It will enable contin- uous core fuelling at low neutral edge density. This injector is a prototype of the US ITER pellet injector. Four microwave pickup holes are connected to waveguides the back of the tile, which measure the position, power and polarization of the incoming beam. ORCID iDs y [8] Reinbold F. et al 2021 IAEA-CN-123/45 (private communica- tion) J. Baldzuhn https://orcid.org/0000-0001-5667-351X N. Pablant https://orcid.org/0000-0001-6617-8459 [9] Jakubowski M. et al 2021 Overview of the results from the diver- tor experiments at Wendelstein 7-X and their implications for steady state operation Preprint: 2020 IAEA Fusion Energy Conf. [EX/7-4] Acknowledgments This work has been carried out within the framework of the EUROfusion Consortium and has received funding from 5 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 the Euratom research and training program 2014–2018 and 2019–2020 under Grant Agreement No. 633053. The views and opinions expressed herein do not necessarily reflect those of the European Commission. the Euratom research and training program 2014–2018 and 2019–2020 under Grant Agreement No. 633053. The views and opinions expressed herein do not necessarily reflect those of the European Commission. [4] Wegner T. et al 2018 Rev. Sci. Instrum. 89 073505 g [5] Sunn Pederson T. et al 2019 Plasma Phys. Control. Fusion 61 014035 [6] Beidler C. et al 2021 Nature 596 221–6 [7] Alcus´on J.A., Xanthopoulos P., Plunk G.G., Helander P., Wilms F., Turkin Y., von Stechow A. and Grulke O. 2020 Plasma Phys. Control. Fusion 62 035005 [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev. Lett. 125 105002 [2] Erckmann V. et al 2007 Fusion Sci. Technol. 52 291–312 [3] Langenberg A. et al 2019 Plasma Phys. Control. Fusion 61 014030 References 0] Avramidis K.A. et al 2021 Fusion Eng. Des. 164 112173 [11] Gantenbein G. et al 2021 High power gyrotron development for advanced fusion devices Preprint: 2020 IAEA Fusion Energy Conf. [TECH/P7-19] [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev. Lett. 125 105002 [2] Erckmann V. et al 2007 Fusion Sci. Technol. 52 291–312 [3] Langenberg A. et al 2019 Plasma Phys. Control. Fusion 61 014030 [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev. Lett. 125 105002 [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev. Lett. 125 105002 [2] Erckmann V. et al 2007 Fusion Sci. Technol. 52 291–312 [3] Langenberg A. et al 2019 Plasma Phys. Control. Fusion 61 014030 [12] Laqua H.P. et al 2001 Proc. of the 28th EPS Conf. Control. Fusion and Plasma Phys., Funchal 2001, ECA 25A (Geneva: European Physical Society) pp 1277–80 6
https://openalex.org/W1022329560	https://europepmc.org/articles/pmc4511751?pdf=render	English	null	Rapid Expansion of Phenylthiocarbamide Non-Tasters among Japanese Macaques	PloS one	2,015	cc-by	11,947	RESEARCH ARTICLE Rapid Expansion of Phenylthiocarbamide Non-Tasters among Japanese Macaques Nami Suzuki-Hashido1,2, Takashi Hayakawa1,2¤a¤b, Atsushi Matsui1¤c, Yasuhiro Go1¤d, Yoshiro Ishimaru3, Takumi Misaka3, Keiko Abe3, Hirohisa Hirai1, Yoko Satta4, Hiroo Imai1 1 Molecular Biology Section, Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan, 2 Japan Society for the Promotion of Science, Tokyo, Japan, 3 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 4 Department of Evolutionary Studies of Biosystems, The Graduate University for Advanced Studies (Sokendai), Hayama, Kanagawa, Japan ¤a Current Address: Department of Wildlife Science (Nagoya Railroad Co., Ltd.), Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan. ¤a Current Address: Department of Wildlife Science (Nagoya Railroad Co., Ltd.), Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan. y y y p ¤b Current Address: Japan Monkey Centre, Inuyama, Aichi, Japan. ¤b Current Address: Japan Monkey Centre, Inuyama, Aichi, Japan. ¤c Current Address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. ¤c Current Address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. p ¤d Current Address: Center for Novel Science Initiatives, National Institute of Natural Sciences, Okazaki, Aichi, Japan. p ¤d Current Address: Center for Novel Science Initiatives, National Institute of Natural Sciences, Okazaki, Aichi, Japan. * satta@soken.ac.jp (YS); imai.hiroo.5m@kyoto-u.ac.jp (HI) Abstract Citation: Suzuki-Hashido N, Hayakawa T, Matsui A, Go Y, Ishimaru Y, Misaka T, et al. (2015) Rapid Expansion of Phenylthiocarbamide Non-Tasters among Japanese Macaques. PLoS ONE 10(7): e0132016. doi:10.1371/journal.pone.0132016 , , , ( ) p Expansion of Phenylthiocarbamide Non-Tasters among Japanese Macaques. PLoS ONE 10(7): e0132016. doi:10.1371/journal.pone.0132016 Editor: John I. Glendinning, Barnard College, Columbia University, UNITED STATES Received: April 11, 2015 Accepted: June 9, 2015 Published: July 22, 2015 Bitter taste receptors (TAS2R proteins) allow mammals to detect and avoid ingestion of tox- ins in food. Thus, TAS2Rs play an important role in food choice and are subject to complex natural selection pressures. In our previous study, we examined nucleotide variation in TAS2R38, a gene expressing bitter taste receptor for phenylthiocarbamide (PTC), in 333 Japanese macaques (Macaca fuscata) from 9 local populations in Japan. We identified a PTC “non-taster” TAS2R38 allele in Japanese macaques that was caused by a loss of the start codon. This PTC non-taster allele was only found in a limited local population (the Kii area), at a frequency of 29%. In this study, we confirmed that this allele was present in only the Kii population by analyzing an additional 264 individuals from eight new populations. Using cellular and behavioral experiments, we found that this allele lost its receptor function for perceiving PTC. The nucleotide sequences of the allele including flanking regions (of about 10 kb) from 23 chromosomes were identical, suggesting that a non-taster allele arose and expanded in the Kii population during the last 13,000 years. Genetic analyses of non- coding regions in Kii individuals and neighboring populations indicated that the high allele frequency in the Kii population could not be explained by demographic history, suggesting that positive selection resulted in a rapid increase in PTC non-tasters in the Kii population. The loss-of-function that occurred at the TAS2R38 locus presumably provided a fitness advantage to Japanese macaques in the Kii population. Because TAS2R38 ligands are often found in plants, this functional change in fitness is perhaps related to feeding habit specificity. These findings should provide valuable insights for elucidating adaptive evolu- tionary changes with respect to various environments in wild mammals. Editor: John I. Glendinning, Barnard College, Columbia University, UNITED STATES Editor: John I. Glendinning, Barnard College, Columbia University, UNITED STATES Editor: John I. Abstract Glendinning, Barnard College, Columbia University, UNITED STATES Received: April 11, 2015 Accepted: June 9, 2015 Published: July 22, 2015 Received: April 11, 2015 Accepted: June 9, 2015 Published: July 22, 2015 Received: April 11, 2015 Accepted: June 9, 2015 Published: July 22, 2015 Copyright: © 2015 Suzuki-Hashido et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Japanese macaques (Macaca fuscata) are widely distributed from the northern part of the main island of Japan (Shimokita Peninsula) to the southern island of Japan (Yakushima Island) (Fig 1), and thus occupy the northernmost habitats of all non-human primates. Japanese macaques adapted to the Japanese environment after they diverged from rhesus macaques (M. mulatta) approximately 0.31–0.88 million years ago (mya) [1] and can survive even in the snowy environments of mountainous areas or in northern areas where the temperatures reach below −20°C during the winter. Therefore, they have suitable food habits for survival in vari- able environments [2]. The genetic variability within and among various local populations of Japanese macaques has been well studied [3–5]. A phylogenetic study of mitochondrial DNA (mtDNA) showed that Japanese macaques that live in eastern Japan have lower genetic diversity than those living in western Japan. This was attributed to an ancient population expansion that occurred in the eastern habitats after the last glacial period approximately 15,000 years ago [6]. A population genetic study of blood protein polymorphisms showed that genetic variation in Japanese macaques is not uniformly distributed and that variants are shared only in limited areas [4]. The characteristics are maintained by frequent exchanges of reproductive males between local populations. A previous study showed that different selection pressures have acted on the gene encoding Toll-like receptor 2, which plays an important role in the recognition of a variety of pathogenic microbes, between Japanese macaques and rhesus macaques, reflecting adaptation of the species to different habitats [7]. Thus, other functional genes, particularly those involved in perceiving environmental signals or stimuli, may also vary among habitats within Japanese macaques, assuming that they are adapted to particular environments in Japan. However, such variation in functional genes has not been fully characterized with respect to environmental variation. The sense of taste allows mammals to evaluate consumed food. Among the five recognized taste sensations (sweet, bitter, sour, salty, and umami), bitter taste plays an important role in avoiding ingestion of toxins. In mammals, bitter taste is mediated by a family of seven-trans- membrane G protein-coupled receptor genes, TAS2Rs [8,9]. Humans and mice have 26 and approximately 40 functional TAS2R genes, respectively, and some loci exhibit intraspecific var- iation including nonfunctional alleles [10–14]. Because TAS2Rs are directly involved in food choice, the number of TAS2R genes in the genome has changed frequently during mammalian evolution [13–17]. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from MEXTand JSPS KAKENHI to NS (12J04300) and to HI (21370109, 24370096, and 26117512). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Rapid Expansion of PTC Non-Tasters among Japanese Macaques PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Introduction Some studies have suggested that TAS2R function varies, even within spe- cies, owing to different selection pressures depending on the habitat [18,19]. TAS2R38 is the best-studied bitter taste receptor in terms of intraspecific phenotypic varia- tion [20,21]. It is a receptor for the synthetic bitter compounds phenylthiocarbamide (PTC) and propylthiouracil (PROP). Among natural bitter compounds, TAS2R38 recognizes glucosi- nolates and limonin, which are found in cruciferous and citrus plants, respectively [22]. Indi- vidual variation in human ability to taste PTC was first recognized in 1931 [23]. Such phenotypic variation has subsequently been found in many other primates, including chim- panzees (Pan troglodytes) [24,25]. Most phenotypic variation among humans is caused by three amino acid substitutions in TAS2R38, whereas for chimpanzees the observed variation results from a start codon mutation (ATG>AGG) in TAS2R38 [20,21]. Premature stop codons have been identified in brown wooly monkeys (Lagothrix lagotricha) and black-handed spider monkeys (Ateles geoffroyi) [26]. These mutations are predicted to disrupt receptor function. Although it is not clear whether these disruptive mutations in the wooly monkey and spider monkey are polymorphic or fixed, these findings indicate that multiple primate species have phenotypic variation in PTC sensitivity. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 2 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Fig 1. Sampling sites for Japanese macaques. The 17 sampling sites are indicated by black circles with their abbreviated locality name (Table 1). Fig 1. Sampling sites for Japanese macaques. The 17 sampling sites are indicated by black circles with their abbreviated locality name (Table 1). Fig 1. Sampling sites for Japanese macaques. The 17 sampling sites are indicated by black circles with their abbreviated locality name (Table 1). doi:10.1371/journal.pone.0132016.g001 doi:10.1371/journal.pone.0132016.g001 Furthermore, it has been suggested that different types of natural selection have acted on TAS2R38 in humans and chimpanzees. In humans, balancing selection may have maintained phenotypic variation resulting from “taster” and “non-taster” alleles at the TAS2R38 locus [27]. In contrast, in chimpanzees, the non-taster allele was found in only western chimpanzees (P. troglodytes verus) where it was present in 76% of individuals, while it was not found in eastern chimpanzees (P. troglodytes schweinfurthii) [19]. In addition, an FST analysis suggested that dif- ferent types of selective pressures have acted on western chimpanzees and eastern chimpan- zees. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 TAS2R38 genetic variation among Japanese macaques We previously identified a non-taster allele, designated Mf-K, with a start codon mutation (ATG>ACG) that occurred in only the Kii population [28]. In this study, to investigate whether other local populations also had the Mf-K allele, we analyzed many more samples and determined sequence variation in TAS2R38 (1002 bp, including 239 synonymous sites and 760 nonsynonymous sites). We analyzed a total of 597 Japanese macaques including 264 new and 333 previously identified individuals from 17 local populations (Table 1). Single nucleotide var- iants (SNVs) found among these 597 Japanese macaques included 3 synonymous and 12 non- synonymous substitutions, for a total of 15 variable sites. We identified 20 alleles based on combinations of these 15 SNVs and designated them Mf- A–T (S1 Table). This included 7 new alleles that were not identified in our previous study. These 20 alleles were categorized into three groups (Fig 2, S2 Table). The first group included only the Mf-A and Mf-B haplotypes; it comprised many populations that were widely distrib- uted and the haplotypes retained a high frequency in each local population. The second group included the Mf-G, Mf-K, Mf-M, Mf-N, Mf-R, Mf-S, and Mf-T alleles. These alleles were identi- fied as population-specific. Eleven residual alleles were included in the third group; they were shared by several populations with a low frequency in each local population. Because Japanese macaques diverged from rhesus macaques 0.31–0.88 mya [1], their nucle- otide sequences are very similar and any phylogenic relationships among alleles from both spe- cies are expected to be intermingled. To elucidate the phylogenetic relationships among these TAS2R38 alleles, we constructed a network of 20 TAS2R38 alleles, including 10 rhesus macaque alleles identified in our previous study (Fig 2). This network showed that the Japanese and rhe- sus macaque alleles were not completely differentiated; rather, the Japanese macaque sequences formed 2 clusters that diverged from Mm-a, a rhesus macaque allele. However, the non-taster Mf-K allele clustered separately from rhesus macaque alleles and arose independently in Japa- nese macaques after the species divergence. Although we investigated 8 additional local populations in this study, we did not detect the Mf-K allele in any of the other local populations. Thus, we confirmed that Mf-K occurred in only the Kii population. TAS2R38 genetic variation among Japanese macaques Interestingly, the frequency of the Mf-K allele was 29% in the Kii pop- ulation, whereas the other alleles of the second group (Mf-G, Mf-M, Mf-N, Mf-R, Mf-S, and Mf-T) had frequencies of <13% in each of the local populations (S2 Table). Introduction Therefore, the evolutionary mechanisms underlying the phenotypic variation associated with TAS2R38 in multiple primate species may be related to their diets and habitats. In our previous study, we determined TAS2R38 sequences for 333 Japanese macaques and 55 rhesus macaques. We identified a PTC “non-taster” allele in Japanese macaques that was caused by a start codon mutation (ATG>ACG) using nucleotide sequence comparisons and qualitative behavioral tests [28]. Interestingly, the non-taster allele was restricted to the Kii population and had a frequency of 29% in this population. However, the evolutionary history of this non-taster allele remains uncertain. In the present study, we confirmed that individuals PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 3 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques with the PTC non-taster phenotype were only in the Kii population using many more samples, and quantitatively evaluated the causal mutation leading to a nonfunctional TAS2R38 receptor. Furthermore, we obtained evidence of positive selection on the non-taster TAS2R38 allele in the Kii population. This positive selection in a restricted region of Japan may ultimately reveal that adaptive evolution in Japanese macaques depended on their habitat. Rapid Expansion of PTC Non-Tasters among Japanese Macaques Table 1. TAS2R38 variants in Japanese macaques and rhesus macaques. Population Name Abbreviation Population Typea nb Sc hd πe θf Dg Japanese macaques Shimokita SMK (3) 83 3 4 0.083 0.053 0.94 Kinkazan KKZ (3) 9 0 1 0.000 0.000 NA Numata NMT (3) 20 3 4 0.038 0.070 -1.01 Takahamah THM (1) 27 5 6 0.168 0.110 1.26 Jigokudani JGD (2) 40 4 4 0.072 0.081 -0.22 Hagachih HGC (2) 15 3 3 0.065 0.076 -0.34 Okazaki OKZ (3) 4 2 3 0.100 0.077 1.10 Mie MIE (3) 78 9 10 0.174 0.160 0.21 Shigah SHG (3) 37 6 8 0.167 0.123 0.86 Arashiyamah ARY (1) 29 4 3 0.065 0.086 -0.54 Minooh MNO (2) 41 7 5 0.088 0.140 -0.90 Kiih KII (2) 40 8 6 0.118 0.161 -0.67 Wakasah WKA (1) 41 8 7 0.224 0.160 0.99 Shodoshimah SHD (1) 11 1 2 0.017 0.027 -0.64 Kami KAM (3) 12 6 8 0.247 0.160 1.64 Takaoka TKO (3) 29 3 2 0.072 0.065 0.24 Koshimah KOS (2) 81 4 4 0.113 0.071 1.09 All 597 15 20 0.142 0.195 -0.60 rhesus macaques Chinah CHN (1) 27 7 7 0.265 0.153 1.89 Indiah IND (1) 27 5 5 0.156 0.110 1.02 All 54 8 10 0.236 0.152 1.33 aPopulation types: (1) captive born populations that retained their locality attribution (2) wild born populations and (3) wild populations Table 1. TAS2R38 variants in Japanese macaques and rhesus macaques. gTajima’s D. Two-sided Tajima’s D test was performed using coalescent simulations with 10,000 replicates, assuming no recombination and a Poisson distribution of mutations along lineages. All Tajima’s D values were not signiﬁcant (P 0.05). hReported in our previous study [28]. doi:10.1371/journal.pone.0132016.t001 MfTAS2R38RC-K variant that was engineered to have a start codon upstream of the native start site detected PTC, suggesting a complete rescue of receptor function. These results showed that MfTAS2R38-A,-B,-C, and MmTAS2R38-a produced a functional receptor, but the MfTAS2R38-K allele did not. To determine the effects of amino acid variation on receptor sensitivity, we compared the responsiveness of receptor variants, MfTAS2R38WT-A,-B,-C, and MmTAS2R38WT-a, by cal- culating their EC50 values (half maximal effective concentration values; see Materials and methods). The EC50 value of MfTAS2R38WT-B was 3.2–5.0 times higher than those of MfTAS2R38WT-A,-C, and MmTAS2R38WT-a (Fig 3). There were no differences among the latter three variants. aPopulation types: (1) captive-born populations that retained their locality attribution, (2) wild-born populations, and (3) wild populations. Functional analysis of TAS2R38 variants using cultured cells In our previous study, we showed that Mf-K homozygous individuals had a lower sensitivity to PTC than those individuals lacking the Mf-K allele. In this study, we quantitatively assessed the receptor activity of the high-frequency alleles, including Mf-K. We performed functional assays using PTC as the ligand and a calcium imaging method. We first tested whether a mutation in the start codon altered the receptor activity. Four receptor variants—MfTAS2R38WT-A,-B,-C, and MmTAS2R38WT-a, all of which were predicted to be full-length receptors—responded to PTC (Fig 3). In contrast, MfTAS2R38TR-K, which lacked 96 amino acids in the TAS2R38WT receptor owing to a missense mutation in the start codon, failed to respond to PTC. An PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 4 / 21 doi:10.1371/journal.pone.0132016.t001 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 aPopulation types: (1) captive-born populations that retained their locality attribution, (2) wild-born populations, and (3) wild populations. bNumber of individuals. Watterson s θ per site (%). gTajima’s D. Two-sided Tajima’s D test was performed using coalescent simulations with 10,000 replicates, assuming no recombination and a Poisson distribution of mutations along lineages. All Tajima’s D values were not signiﬁcant (P 0.05). hReported in our previous study [28]. which was the only difference between MfTAS2R38WT-A and-C, did not influence sensitivity to PTC. which was the only difference between MfTAS2R38WT-A and-C, did not influence sensitivity to PTC. which was the only difference between MfTAS2R38WT-A and-C, did not influence sensitivity to PTC. A modified MfTAS2R38RC-K that was engineered to have an upstream start codon had an EC50 value comparable to that of MfTAS2R38WT-B. Because both MfTAS2R38WT-B and MfTAS2R38RC-K had a common amino acid substitution at position 271 from isoleucine to threonine (S1 Fig), this amino acid substitution was responsible for the slight decrease in TAS2R38 receptor sensitivity. An amino acid substitution at position 203, 5 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Fig 2. Median-joining network for TAS2R38 alleles of Japanese and rhesus macaques. Each circle represents a different haplotype and is shown along with the allele name. Mf and Mm indicate Japanese and rhesus macaque haplotypes, respectively. Colors within a circle indicate each local population (Table 1), and the size of a circle is proportional to the number of chromosomes. Nucleotide positions of mutations that differentiate alleles are indicated on the network branches. Line styles of branches indicate mutation types. Fig 2. Median-joining network for TAS2R38 alleles of Japanese and rhesus macaques. Each circle represents a different haplotype and is shown along with the allele name. Mf and Mm indicate Japanese and rhesus macaque haplotypes, respectively. Colors within a circle indicate each local population (Table 1), and the size of a circle is proportional to the number of chromosomes. Nucleotide positions of mutations that differentiate alleles are indicated on the network branches. Line styles of branches indicate mutation types. doi:10.1371/journal.pone.0132016.g002 Behavioral experiments In our previous study, we found qualitative differences in sensitivity between 4 and 3 individu- als with ATG/ATG and ACG/ACG homozygous TAS2R38 start codons, respectively [28]. We used a two-bottle preference test with PTC solutions of various concentrations to quantify the differences in sensitivity thresholds between these 2 homozygotes (Fig 4, S2 Fig). We calculated the EC50 values that indicate the PTC concentration at which the preference ratio becomes 25%, half of the level due to chance, and compared these values between the 2 homozygotes. The average EC50 value for ATG/ATG individuals was 18.9 μM with a large standard error (±22.1). This result was consistent with results obtained for humans, for which PTC thresholds showed considerable variability within the “taster” group [29]. In contrast, the average EC50 value for ACG/ACG individuals was 1542.1 μM (±8.9), which was significantly higher than that of ATG/ATG individuals (80-fold). We also used this test for an individual that was an ATG/ACG heterozygote at the TAS2R38 start codon and obtained an EC50 value of 179.2 μM (Fig 4). This was intermediate between the values of each homozygote and was consistent with human studies showing that taster/non- 6 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Rapid Expansion of PTC Non-Tasters among Japanese Macaques 2 Fig 3. Dose-response curves for intracellular Ca2+ levels in cells that express TAS2R38 variants against PTC concentrations. Each point represents the mean ± standard error determined from 3 independent measurements. Points on the horizontal axis indicate the EC50 values for alleles. doi:10.1371/journal.pone.0132016.g003 Fig 3. Dose-response curves for intracellular Ca2+ levels in cells that express TAS2R38 variants against PTC concentrations. Each point represents the mean ± standard error determined from 3 independent measurements. Points on the horizontal axis indicate the EC50 values for alleles. doi:10.1371/journal.pone.0132016.g003 taster heterozygote individuals (PAV/AVI genotype) had intermediate sensitivity between taster and non-taster groups [30]. taster heterozygote individuals (PAV/AVI genotype) had intermediate sensitivity between taster and non-taster groups [30]. Analysis of TAS2R38 flanking regions In our genetic analysis of TAS2R38, population-specific alleles, except for the Mf-K allele, had frequencies of <1% overall in Japanese macaques and frequencies of <13% in each local popu- lation (S1 and S2 Tables). It is expected that population-specific alleles would be maintained at a low frequency in each population because they probably arose after population differentia- tion. However, the Mf-K allele had a frequency of 29% in the Kii population and its distribution was distinctly different from that of the other alleles. To clarify the evolutionary history of TAS2R38 alleles found in the Kii population, we sequenced TAS2R38 flanking regions and determined non-coding SNVs linked to each TAS2R38 allele. We determined the nucleotide sequences for the TAS2R38 flanking region (10,231 bp) in all 40 individuals sampled from the Kii population. The TAS2R38 allele distributions for these sequenced samples were 32 Mf-A, 17 Mf-B, 1 Mf-F, 6 Mf-J, 23 Mf-K, and 1 Mf-Q. The overall nucleotide diversity (π) values for the 50 (4229 bp), coding (1,002 bp), and 30 (5000 bp) regions were 0.49%, 0.12%, and 0.06%, respectively. These alleles comprised 13 long-range haplotypes and were named for the TAS2R38 coding region with distinguishing numbers, such as A-1, A- PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 7 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Fig 4. Gustatory responsiveness of Japanese macaques to various PTC solution concentrations. The vertical axis indicates the preference ratio for PTC, which is the ratio of PTC solution consumed to the total liquid consumed. Each point represents the mean of 6 trials. Point and line colors indicate the genotype of the start codon for TAS2R38. Points on the horizontal axis indicate the EC50 values for individuals. The responsiveness of each individual is shown in the (S2 Fig). doi:10.1371/journal.pone.0132016.g004 Fig 4. Gustatory responsiveness of Japanese macaques to various PTC solution concentrations. The vertical axis indicates the preference ratio for PTC, which is the ratio of PTC solution consumed to the total liquid consumed. Each point represents the mean of 6 trials. Point and line colors indicate the genotype of the start codon for TAS2R38. Points on the horizontal axis indicate the EC50 values for individuals. The responsiveness of each individual is shown in the (S2 Fig). doi:10.1371/journal.pone.0132016.g004 2, B-1, and B-2 (S3 Fig). There were 7 and 2 different haplotypes for Mf-A (A-1–A-7) and Mf-B (B-1 and B-2) alleles, respectively. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Analysis of TAS2R38 flanking regions Median-joining network for TAS2R38 flanking region haplotypes in the Kii population. Gray and white circles represent the flanking haplotypes and median-vectors, respectively. The sizes of gray circles are proportional to the numbers of chromosomes. The numbers along branches indicate nucleotide positions of mutations that distinguish haplotypes and correspond to the last 4 digits of the positions in the rheMac2 genome sequences (S3 Fig). Bold numbers indicate mutations in the TAS2R38 coding region and the others indicate mutations in non-coding regions. Dashed lines indicate indel mutations. A substitution numbered 8166 indicates a mutation at the start codon. doi:10.1371/journal.pone.0132016.g005 doi:10.1371/journal.pone.0132016.g005 mutation at the start codon (Fig 5). However, the preK-1 haplotype was not found in the sam- pled Kii population. The K-1 haplotype was presumably derived from preK-1 by acquiring a mutation at the start codon in the limited 6411-bp region that excluded regions of recombination. It is expected to take 160,000 years to acquire a single mutation in this 6411-bp sequence assuming a muta- tion rate of 10−9/site/year. This may be an overestimation because our data were limited to these 6411-bp. If we subsequently find no differences between K-1 and preK-1 in a longer sequence, this would indicate an earlier estimated divergence time between these 2 haplotypes. Furthermore, the K-1 haplotype expanded to 23 chromosomes without acquiring any muta- tions in this 10-kbp sequence. When the K-1 haplotype expanded to 23 chromosomes for t years under a star phylogeny, the total branch length was 23t years. For a mutation rate of 10−9/site/year, the mutation rate λ for a 10-kbp region for 23t years is 23t × 10−5. The assump- tion of a star phylogeny is conservative for this time estimation. Based on our data, no muta- tions occurred for 23t years. If the probability of this was set to >5%, then based on a Poisson distribution for the number of substitutions in this region, the upper limit for the time would be approximately 13,000 years (t < 13,024). Analysis of TAS2R38 flanking regions All Mf-K haplotype sequences for the 23 chromosomes were identical. For other alleles, only 1 haplotype was detected for each allele (F-1, J-1, and Q-1). We compared the informative sites of these 13 haplotypes and investigated their phyloge- netic relationships. There were numerous incompatible sites in the 50- and 30-flanking regions (S3 Fig). This strongly suggested that genetic recombination occurred at a site located between 2 polymorphic sites: positions 179,408,639 and 179,408,483 of chromosome 3. Indeed, the topology and branch lengths of the tree for the 50 region, including position 179,408,639, were different from those of the 30 region, including position 179,408,483. Therefore, we excluded a possible 50-recombinant region when we conducted a haplotype network analysis. Then, we determined the phylogenetic relationships based on TAS2R38 using the 6411 bp sequence, including the TAS2R38 coding region (S3 Fig). We constructed a haplotype network for TAS2R38 flanking regions to determine when and how the Mf-K allele arose (Fig 5). The presumable most recent common ancestral haplotype of K-1 and A-4 designated median-vector-1 (mv-1) was derived from the A-5 haplotype by obtaining 2 insertion-deletion (indel) mutations and 1 substitution (Fig 5). K-1 then uniquely acquired 4 substitutions, including a nonsense mutation at the start codon. Of the 4 unique substitution sites, 2 were located in the 50 region and the others were located in the coding region. We analyzed whether these 4 substitutions occurred in 6 other neighboring local popu- lations and identified the most similar haplotype, designated “preK-1”, in the JGD and HGC populations. The haplotype sequence was identical to that of the K-1 haplotype, except for a PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 8 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Fig 5. Median-joining network for TAS2R38 flanking region haplotypes in the Kii population. Gray and white circles represent the flanking haplotypes and median-vectors, respectively. The sizes of gray circles are proportional to the numbers of chromosomes. The numbers along branches indicate nucleotide positions of mutations that distinguish haplotypes and correspond to the last 4 digits of the positions in the rheMac2 genome sequences (S3 Fig). Bold numbers indicate mutations in the TAS2R38 coding region and the others indicate mutations in non-coding regions. Dashed lines indicate indel mutations. A substitution numbered 8166 indicates a mutation at the start codon. d i 10 1371/j l 0132016 005 Fig 5. General genetic characteristics of the Kii population Japanese macaques generally have high migration rates between neighboring troops, and the breeding structure of this species follows the two-dimensional stepping-stone model developed by Kimura and Weiss [31]; genetic diversity within groups has been maintained by gene flow [4]. However, the Mf-K allele was not found in other local populations despite the frequency of PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 9 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Mf-K being 29% in the Kii population. This may be explained by the genetic specificity (i.e., a small effective population size or a low migration rate between neighboring populations) of the Kii population or natural selection for the Mf-K allele of TAS2R38. Therefore, we analyzed the sequences of other non-coding regions in the Kii and 7 neighboring populations to evaluate their genetic diversity and migration level. We determined the sequences of 9 loci in non-coding regions (average length, 725 bp/locus) in the Kii and 7 additional neighboring populations and compared these sequences within each population (Table 2 and S3 Table). The average nucleotide diversity (π) over 9 loci in the Kii population was 0.076% and was similar to that of the other 7 populations, 0.087% (SD: 0.017%). Additionally, to investigate the demography of the Kii population, we estimated Taji- ma’s D, which provides an index of an effect of population dynamics including demography and selection (S3 Table). Although most of these values were not significantly different from zero, the D value at the IGS09 locus was significantly positive in the Kii population. However, this tendency was also found in the other populations, except for the HGC population. This showed that this tendency, observed in all 8 populations including the Kii population, was inherited from an ancestral population. To evaluate the effects of gene flow between these local populations, we calculated the FST value for each of the 8 populations in a pairwise manner (Table 3). The average differentiation of 9 loci in the Kii population was 0.08 and was similar to the average of the other 7 popula- tions, 0.11. We also calculated the Nm values, where N is the effective population size and m is the migration rate. If Nm > 1, then local differentiation is less pronounced. In particular, if Nm 4, then the entire population tends to behave as a single panmictic population, assuming the two-dimensional stepping-stone model [32]. bAverage nucleotide diversity per site (%), with standard deviation in parentheses. aAverage number of polymorphic sites per locus. cAverage number of segregating sites per site (%), with standard deviation in parentheses. doi:10.1371/journal.pone.0132016.t002 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Table 3. Pairwise migration levels for Kii and 7 neighboring populations. Nmb THM JGD HGC MIE SHG ARY MNO KII average FST a Takahama 　 1.50 0.98 2.41 4.34 1.38 1.86 3.27 2.25 Jigokudani 0.14 3.88 2.46 1.95 0.90 1.38 2.19 2.04 Hagachi 0.20 0.06 2.00 1.65 1.05 1.14 1.75 1.78 Mie 0.09 0.09 0.11 6.06 1.78 1.53 12.02 4.04 Shiga 0.05 0.11 0.13 0.04 3.57 4.17 15.49 5.32 Arashiyama 0.15 0.22 0.19 0.12 0.07 2.63 2.20 1.93 Minoo 0.12 0.15 0.18 0.14 0.06 0.09 2.09 2.11 Kii 0.07 0.10 0.12 0.02 0.02 0.10 0.11 5.57 average 0.12 0.13 0.14 0.09 0.07 0.13 0.12 0.08 　 aPairwise FST values. bPairwise Nm values were calculated as follows: FST = 1/(1 + 4Nm). Table 3. Pairwise migration levels for Kii and 7 neighboring populations. doi:10.1371/journal.pone.0132016.t003 Although such population-specific TAS2R38 haplotypes were found in other populations, these proportions were relatively low (0.6%–12%). It would be very rare for a population-spe- cific haplotype to have expanded to almost one-third of a particular population, and this char- acteristic appeared to be unique to the TAS2R38 locus in the Kii population. g y these proportions were relatively low (0.6%–12%). It would be very rare for a population-spe- cific haplotype to have expanded to almost one-third of a particular population, and this char- acteristic appeared to be unique to the TAS2R38 locus in the Kii population. General genetic characteristics of the Kii population The Nm value for the Kii population was 5.6, which suggested high migration between the Kii and neighboring populations. We also separately calculated the proportions of shared haplotypes at 9 non-coding regions and the TAS2R38 coding region (Fig 6). In non-coding regions, the average proportion of specific haplotypes in the Kii population was 5.15%, and 94.85% of haplotypes were shared with other populations. This indicated that the Kii population was not genetically isolated from these other populations or that this population was not sufficiently differentiated from the other populations. In contrast, the proportion of shared haplotypes in the TAS2R38 coding region was 71%, and that of region-specific haplotypes was 29%, which corresponded to the Mf-K frequency. Table 2. Average genetic diversity of 9 loci in non-coding regions. Population Name Sa πb θc Takahama 1.2 0.059 (0.09) 0.050 (0.07) Jigokudani 2.3 0.105 (0.09) 0.100 (0.08) Hagachi 2.9 0.101 (0.10) 0.119 (0.10) Mie 2.0 0.082 (0.13) 0.082 (0.10) Shiga 2.7 0.101 (0.12) 0.112 (0.10) Arashiyama 1.6 0.073 (0.09) 0.066 (0.06) Minoo 1.8 0.088 (0.11) 0.074 (0.09) Kii 1.7 0.076 (0.10) 0.069 (0.07) Average 2.0 0.085 0.084 Table 2. Average genetic diversity of 9 loci in non-coding regions. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 10 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Computer simulations for allele expansion The Mf-K allele, a non-taster allele, was found in only the Kii population at a frequency of 29%. Because this allele was associated with the loss of receptor function owing to a missense muta- tion at the start codon, the Mf-K allele could have expanded neutrally in the Kii population. We examined whether a neutral allele could increase in frequency to 29% without migration by computer simulations using various Nm values (Fig 7). The number of replicates used in these simulations was 1000. The estimated time for a neutral allele to increase in frequency to 29% without migration was 0.69N generations, where N is the effective population size. We con- firmed that this value was consistent with the expected value using the formula in Kimura and Ohta [33]. Fig 6. Proportion of shared and non-shared haplotypes among 8 Japanese macaque populations. Proportions of shared and non-shared haplotypes (a) in 9 loci in non-coding regions and (b) in TAS2R38 coding regions. Gray and white bars indicate the rates of non-shared and shared haplotypes, respectively. doi:10.1371/journal.pone.0132016.g006 Fig 6. Proportion of shared and non-shared haplotypes among 8 Japanese macaque populations. Proportions of shared and non-shared haplotypes (a) in 9 loci in non-coding regions and (b) in TAS2R38 coding regions. Gray and white bars indicate the rates of non-shared and shared haplotypes, respectively. doi:10.1371/journal.pone.0132016.g006 doi:10.1371/journal.pone.0132016.g006 11 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Fig 7. Computer simulations for migration among populations. The horizontal axis indicates the Nm value used as an index of the migration level. Graphs indicate the probability that all alleles remain in each population during expansion to each frequency with 1000 replications. A value of 0 indicated that all alleles migrated to other populations. Straight and dashed lines indicate Mf-K alleles in the Kii population and Mf-M alleles in the Minoo population, respectively, with each Nm value estimated from a non-coding region (Table 3) indicated by arrows. Fig 7. Computer simulations for migration among populations. The horizontal axis indicates the Nm value used as an index of the migration level. Graphs indicate the probability that all alleles remain in each population during expansion to each frequency with 1000 replications. A value of 0 indicated that all alleles migrated to other populations. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Discussion In the present study, we found that positive selection operated on a loss-of-function TAS2R38 mutation in Japanese macaques. Loss-of-function mutations that provide a selective advantage have been reported in humans, such as the delta-32 (Δ32) mutation in the chemokine receptor 5 (CCR5) gene. Because CCR5 is related to human immunodeficiency virus (HIV) infection and this 32-bp deletion results in a non-functional protein, individuals with this deletion have a selective advantage with respect to HIV resistance. It was estimated that this deletion arose and expanded within the past few thousand years [35]. A loss-of-function that has a selective advantage rather than a deleterious effect is curious, and it is of interest to determine the timing of this mutation in TAS2R38 and to evaluate the evolutionary history. Our results suggested that the non-taster allele Mf-K arose and expanded in the Kii population approximately 13,000 years ago. Mutations that caused phenotypic varia- tion for tasting PTC have been identified in other primate species as well as in Japanese macaques [20,21]. A non-taster TAS2R38 allele in humans arose 0.3–1.6 mya and has been maintained by balancing selection [27,36]. For chimpanzees, a non-taster allele was found only in western chimpanzees, and this allele has expanded after the diversification between eastern and western chimpanzees approximately 0.5 mya [19]. The times for the emergence of non-taster alleles differed among these 3 species, including the Holocene epoch for Japanese macaques, the Pleistocene epoch for humans, and after the Pleistocene epoch for chimpanzees. Therefore, ecological factors related to the expansion of the non-taster allele by positive selection in Japanese macaques may also be different from those of the other 2 species. For Japanese macaques, the specific characteristics of the feeding habits and habitat of the Kii population may have affected this expansion. The evolutionary and eco- logical factors that are related to non-taster TAS2R38 variation in other primate species should be examined carefully by focusing on species-specific properties, as well as common factors and processes of expansion beyond species. TAS2R38 enables the recognition of glucosinolates in cruciferous plants and limonin in cit- rus plants as natural bitter compounds [22], and Japanese macaques often eat some parts of these plants [2]. Computer simulations for allele expansion Straight and dashed lines indicate Mf-K alleles in the Kii population and Mf-M alleles in the Minoo population, respectively, with each Nm value estimated from a non-coding region (Table 3) indicated by arrows. Fig 7. Computer simulations for migration among populations. The horizontal axis indicates the Nm value used as an index of the migration level. Graphs indicate the probability that all alleles remain in each population during expansion to each frequency with 1000 replications. A value of 0 indicated that all alleles migrated to other populations. Straight and dashed lines indicate Mf-K alleles in the Kii population and Mf-M alleles in the Minoo population, respectively, with each Nm value estimated from a non-coding region (Table 3) indicated by arrows. doi:10.1371/journal.pone.0132016.g007 doi:10.1371/journal.pone.0132016.g007 The nucleotide sequence analyses of 9 neutral regions showed that the average Nm value was 5.6 (SD: 5.7) with a range of 1.8 < Nm < 15.5 between the Kii and neighboring populations (Table 3). These simulation results showed that a lack of migrants of a particular allele during 0.69N generations for all migration rates of >1.8 was unlikely (P < 0.005). This suggested that the absence of Mf-K alleles in the neighboring populations could not be explained by neutral processes (Fig 7). We also conducted these simulations for the Minoo population, which had a population-specific allele, Mf-M, at a frequency of 12%; this was the highest frequency for a population-specific allele, except for Mf-K, among our samples. The expected time for a neutral allele to expand to 12% was 0.27N generations, and the average Nm value between the Minoo and other populations was 2.1 (SD: 1.0). Based on these values, the occurrence of Mf-M at 12% without any migration under neutrality was not rejected (Fig 7). Thus, only the frequency of the Mf-K allele could not be explained by neutral processes, which suggested that the Mf-K allele had expanded rapidly in a much shorter time than expected under neutrality. The time required for expanding to 29% in a neutral case, 0.69N generations, is 84,000 years when applying an effective population size and a generation time of Japanese macaques of 20,000 [34] and 6 years, respectively. Computer simulations for allele expansion The lack of mutations among the 23 Mf-K alleles showed that it expanded in the Kii population for 13,000 years at most, 12 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Rapid Expansion of PTC Non-Tasters among Japanese Macaques much shorter than 84,000 years. The most reasonable explanation for these data is that positive selection on the non-taster Mf-K phenotype promoted rapid expansion of the Mf-K allele. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Discussion A previous study on humans showed that people who are homozygous for a sensitive allele (PAV/PAV) have a higher sensitivity to the bitterness of glucosinolate-produc- ing vegetables than those who are homozygous for insensitive alleles (AVI/AVI), as well as their sensitivity to PTC. In addition, people who are heterozygous for these alleles have inter- mediate sensitivity [37]. In the present study, we revealed that Japanese macaques are divided into 3 types with regard to PTC sensitivity based on TAS2R38 genotypes, as in humans. There- fore, Japanese macaques could have a similar tendency as humans with respect to sensitivity to bitterness in cruciferous plants. Although the contribution of different TAS2R38 genotypes to sensitivity to limonin bitterness remains unclear, it is also possible that TAS2R38 genotypes in Japanese macaques affect the citrus feeding behavior. The first citrus species in Japan was Cit- rus tachibana (After its Japanese common name, “tachibana”), which has grown wild for 2800 years. It is thought that the original site of C. tachibana growth was the Kii peninsula, which includes the habitat of the Kii population of Japanese macaques analyzed in the present study [38,39]. Agriculture over the past several hundred years has rapidly expanded the distribution of most cruciferous (e.g., cabbage and radish) and citrus plants, which are ingested by wild Jap- anese macaques [2]. Such expansion of these plants may be related to the rapid expansion of the non-taster allele among Japanese macaques. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 13 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Other geological factors should also be taken into consideration. The Kii peninsula has been struck by tsunamis at 400–600 year intervals [40]. One study reported that there was no signifi- cant change in the number of long-tailed macaques (M. fascicularis) after the earthquake in the Indian Ocean off Sumatra in 2004, where coastal forests were washed away by the ensuing tsu- nami [41]. Therefore, a decrease and transition in available food with dramatically changing vegetation due to a tsunami in the Kii area may have also favored individuals with low sensitiv- ity to a particular bitterness. Monkeys in the Kii population with the non-taster allele are housed in open enclosures at the Primate Research Institute, Kyoto University; accordingly, in future studies we can observe changes in behavior and fitness in response to various candidate plants (e.g., cruciferous plants and citrus fruits). Discussion In preliminary experiments, we provided citrus fruits including mandarin oranges and C. tachibana to Japanese macaques in individual cages. Monkeys often peeled, removed the seeds, and ate relatively large fruits, but did not peel or eat whole fruits when we provided small fruits (2–4 cm) such as C. tachibana. These results suggest that non-taster indi- viduals have an advantage in terms of eating small fruits, presumably because they contain higher concentrations of limonin, which is a ligand for TAS2R38, in their peels and seeds. Although we need to account for changes in vegetation other factors, these preliminary results provide insight into the fitness advantage of PTC non-tasters in the Kii population of Japanese macaques. A previous study that examined blood protein polymorphism in Japanese macaques showed that major alleles are distributed throughout Japan and that specific mutation types are con- fined to limited areas [4]. The TAS2R38 allele distributions investigated in the present study were mostly consistent with the results of this previous study. However, a study by Nozawa [4] indicated that some mutants are not restricted to a certain local population, but are shared among some adjacent regions owing to migration. This pattern was not consistent with the dis- tribution of the non-taster Mf-K TAS2R38 allele, which was found only in the Kii population, despite its high frequency in this population. The Kii population is located in the western Kii peninsula. Thus, we also investigated TAS2R38 polymorphisms in the Mie population, which is located in the eastern Kii peninsula, to compare the genetic variation among these adjacent populations (Fig 1). The pairwise FST value for these 2 populations in the non-coding region was only 0.02 (SD: 0.045), which sug- gested that Japanese macaques in this area migrate frequently between these 2 populations. This was also supported by TAS2R38 allele distributions in these 2 neighboring populations; 5 of 6 alleles in the Kii population were shared with the Mie population. However, the Mf-K allele was not found in the Mie population despite its high frequency in the Kii population, which suggested that the Mf-K allele had expanded more rapidly in the Kii population than it had spread to the Mie population by migration. Although the population structure of TAS2R38 was generally similar to those of blood proteins in a previous study, only the non-taster allele Mf-K exhibited specific characteristics in terms of its distribution and frequency in the Kii population. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Ethics statement This study was carried out in strict accordance with recommendations in the Guidelines for Care and Use of Nonhuman Primates Version 2 and 3 of the Primate Research Institute, Kyoto University (2002, 2010). This guideline was prepared based on the provisions of the Guidelines for Proper Conduct of Animal Experiments (June 1, 2006; Science Council of Japan), Basic Pol- icies for the Conduct of Animal Experiments in Research Institutions under the Jurisdiction of the Ministry of Health, Labor and Welfare (effective on June 1, 2006; Ministry of Health, Labor and Welfare (MHLW)), Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions (Notice No. 71 of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) dated June 1, 2006), and Stan- dards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Notice No. 88 of the Ministry of the Environment dated April 28, 2006). The experiments were approved by the Animal Welfare and Animal Care Committee (Monkey Committee) of the Primate Research Institute (2010-066, 2011-093, 2012-047). PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Discussion In TAS2R38 flanking regions, the nucleotide diversity (π) of the excluded region within the 50 region (3,820 bp) was 0.50% and this was approximately 10 times higher than that of the non-coding region (0.076%) and the 30-flanking region (0.055%) of TAS2R38. Additionally, Tajima’s D for this region was significantly positive, 2.36 (P < 0.01), which indicated that there were an excess of haplotypes with intermediate frequencies. Furthermore, the divergence time (2.5 mya) between these haplotypes was much longer than the time since speciation between Japanese and rhesus macaques, 0.5 my. Shared ancestral polymorphisms may have been main- tained among these macaque species rather than within Japanese macaques. Perhaps this 50 region affects TAS2R38 expression by providing diversity to the TAS2R38 control region. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 14 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques Accordingly, we only focused on the start codon mutation that occurred in a restricted manner in the Kii population. Other factors likely to affect TAS2R38 receptor function are mentioned above. Thus, we must investigate other possibilities. We can postulate that wild mammals adapted to various environments by altered molecular mechanisms as well as learning. This is supported by our results showing that a loss-of-func- tion mutation for specific bitter tastes was acquired by Japanese macaques in a specific popula- tion with region-specific vegetation and that this mutation expanded rapidly under positive selection. Samples A total of 597 Japanese macaques from 17 local populations in Japan were studied, including 333 samples analyzed for the TAS2R38 locus in our previous study [28] and 264 additional individuals from 8 populations. These 17 local populations were divided into 3 categories: (1) captive-born populations that retained their locality attribution, (2) wild-born populations, and (3) wild populations (Table 1). Because both (1) and (2) populations were kept in the Pri- mate Research Institute, Kyoto University, blood samples were obtained when these macaques underwent health status check-ups. Monkeys were housed in open enclosures at the Primate Research Institute, Kyoto University, and lived with their mothers and group members. Mon- keys received water ad libitum and received leaves, seeds, fruits, and insects in addition to pel- lets. Tissue, blood, or DNA samples of individuals belonging to populations in category (3) were provided during the course of government population management or population cen- suses. No animals were sacrificed for the purposes of the present study; however, monkeys in some populations were culled previously as a pest-control measure against crop damage, with the permission of the local government. Genetic and morphological data from these monkeys are utilized for population census used by various institutes (universities, museums, non-prof- its, etc.). Tissue sample from this group were used with the cooperation of the respective orga- nizations. For monkeys in the government census, body measurement, blood collection, and application of a GPS telemetry device, and release were performed by a veterinarian belonging to a non-profit organization aimed at Japanese macaque conservation and management. Blood samples were used with the cooperation of this organization. PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 15 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques TAS2R38 sequencing and haplotype inferences Genomic DNA was extracted from blood or tissue samples using the Quick Gene DNA Whole Blood Kit S (Fujifilm, Tokyo, Japan) or the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Ger- many). To amplify and sequence the entire coding region of the Japanese macaque TAS2R38 gene, primers were designed based on the whole-genome sequences of rhesus macaques (MGSC Merged 1.0/rheMac2) from the University of California, Santa Cruz website (http:// genome.ucsc.edu/) using software available on the Primer3Plus website (http://www. bioinformatics.nl/primer3plus/). The primer sequences and the PCR conditions used are shown in S4 Table. PCR mixtures (25 μl total) contained 0.625 U of ExTaq DNA polymerase (Takara Bio Inc., Shiga, Japan), 2 mM reaction buffer and 0.2 mM deoxynucleoside triphos- phates provided by the DNA polymerase manufacturer (Takara), primers (0.2 μM each), and an adequate amount of genomic DNA as the template. Amplification was performed using the following conditions: initial denaturation at 94°C for 10 min; 35–40 cycles of denaturation at 94°C for 10 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. PCR products were checked by electrophoresis in 1% aga- rose gels and purified by isopropanol precipitation and/or ExoSAP-IT (Affymetrix Inc., Santa Clara, CA, USA). Using the PCR primers and internal primers, the purified PCR products were directly sequenced in both strand orientations with a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Chromatograms were imported to the ATGC software (Genetyx Corporation, Tokyo, Japan) and analyzed. Haplotype sequences were reconstructed from diploid sequence sets using PHASE v2.1 [42,43]. During reconstruction, sequences with sites inferred to have probabilities of <0.95 were excluded. Sequences of these unphased haplotypes were determined by cloning using a TOPO TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA, USA). The TAS2R38 coding sequences determined in the present study were deposited in GenBank under the accession numbers AB907224–AB907230. TAS2R38 population and sequence analyses Common population parameters were estimated using DnaSP v5.1 [44]. To summarize diver- sity levels, pairwise nucleotide diversity (π) and Watterson's θ based on the number of segregat- ing sites were calculated [45,46]. Tajima’s D was also calculated to assess TAS2R38 neutrality [47]. Median-joining networks of evolutionary relationships among the alleles were con- structed, rooted with the TAS2R38 sequence of the hamadryas baboon (Papio hamadryas; accession number AY724835.1) using NETWORK v4.6 [48]. Functional analysis using cultured cells To investigate the PTC responsiveness of each TAS2R38 variant, a functional assay with the major alleles found in Japanese and rhesus macaques, Mf-A, Mf-B, Mf-C, Mf-K, and Mm-a, was used with a calcium imaging method [49]. The PCR products for these alleles were tagged at the last 8 amino acids of bovine rhodopsin (bRh) at the C-terminal end. Subsequently, the tagged TAS2R38 fragments and the first 45 amino acids of rat somatostatin receptor type 3 (ssr3) were ligated into a pEAK10 expression vector (Edge Biosystems, Gaithersburg, MD, USA) using Geneart (Invitrogen) to enhance cell-surface expression (S1 Fig). ) g ( g ) p ( g) Chimpanzees also have a start codon mutation (ATG>AGG) in TAS2R38, and the receptor activity of this allele was tested using a truncated polypeptide resulting from translation initia- tion at a downstream ATG codon (M97) described by Wooding et al. [21]. Thus, MfTAS2R38-K that produced MfTAS2R38TR-K (truncated type) with 96 amino-terminal Chimpanzees also have a start codon mutation (ATG>AGG) in TAS2R38, and the receptor activity of this allele was tested using a truncated polypeptide resulting from translation initia- tion at a downstream ATG codon (M97) described by Wooding et al. [21]. Thus, MfTAS2R38-K that produced MfTAS2R38TR-K (truncated type) with 96 amino-terminal PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 16 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques residues missing compared with wild-type MfTAS2R38WT was constructed (S1 Fig). As a con- trol, MfTAS2R38-K (rescued) that produced MfTAS2R38RC-K (rescued type) resulting from translation initiation at an upstream ATG codon in ssr3 was constructed (S1 Fig). All plasmids were sequenced to verify mutations and to exclude plasmids with amplification errors during PCR. Expression plasmids were used for the chimeric G-protein subunit Gα16gust44, which was subcloned into pcDNA 3.1 (Invitrogen), to be co-expressed with TAS2R38 [50]. Human embryonic kidney 293T (HEK293T) cells were cultured at 37°C in Dulbecco’s mod- ified Eagle’s medium (DMEM; Sigma-Aldrich, Tokyo, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY). HEK293T cells were kindly provided by Dr. Hiroaki Matsunami (Department of Neurobiology and Duke Institute for Brain Sciences, Duke University Medical Center). For transfection, cells were seeded in 35-mm dishes and transiently transfected with plasmids that expressed each recombinant TAS2R38 along with Gα16gust44 using Lipofectamine 2000 (Invitrogen). Functional analysis using cultured cells Transfected cells were transferred to a 96-well lumox multiwell plate (SARSTEDT AG & Co., Nümbrecht, Germany) 6 h after trans- fection and then incubated for an additional 18–20 h. Wells were rinsed with an assay buffer (130 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES; pH 7.4) and loaded with 5 μM fluo-4 AM (Dojindo Laboratories, Kumamoto, Japan). Plates were then incubated for 45 min at 27°C in the dark. Subsequently, a plate was placed on a Flex- Station 3 (Molecular Devices, Inc., Sunnyvale, CA, USA) for fluorescence detection. Changes in fluorescence intensity were monitored at 2-s intervals. After baseline readings for 20 s, PTC (Sigma-Aldrich) dissolved in assay buffer was added and scanning resumed for an additional 100 s. Fluorescence signals were evaluated as F values. A response was expressed as the ΔF/F value [ΔF/F = (F −F0)/F0], which was the normalized peak response relative to background fluores- cence (F0). Based on the mean ΔF/F value at each concentration across 3 independent experi- ments, these data were fit to the equation: f(x) = Imin + [Imax −Imin/(1 + (x/EC50)h], where x, EC50, and h represent the ligand concentration, the half maximal effective concentration value, and the Hill coefficient, respectively. Curve fitting and parameter estimations were done using the non-linear least squares method (Levenberg–Marquardt algorithm) implemented in R v2.14.0 (http://www.r-project.org/). PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 Analyses of TAS2R38 coding and flanking regions in the Kii population To elucidate the evolutionary background of Mf-K the non-taster TAS2R38 allele, sequence variation in the TAS2R38 gene (1,002 bp) and additional non-coding regions that flanked either side of this gene was examined. For this analysis, 40 DNA samples derived from the Kii population were used, including all individuals that had the Mf-K allele. We designed the primer sets shown in S4 Table to amplify the TAS2R38 flanking region of Japanese macaques based on the genome sequence of rhesus macaques in 2 parts: the 50-flanking region plus the TAS2R38 coding region and the 30-flanking region plus the TAS2R38 coding region. The PCR extension time was 5 min and the other conditions were the same as those used for TAS2R38 sequencing and haplotype inferences described above. Haplotype names were assigned on the basis of the TAS2R38 coding region. These flanking sequences were deposited in GenBank under the accession numbers AB907288–AB907300. Recombination in this flanking region was identified using GENECONV [51] and the recombi- nation boundary in the 50 region of TAS2R38 was estimated. This region was excluded from analysis, and only a 6411 bp sequence (rheMac2, chr3: 179,402,166–179,408,638) that included the TAS2R38 coding region was used for further analyses. A median-joining network of evolutionary relationships among available haplotypes was constructed using NETWORK v4.6 [48]. Indel polymorphisms were weighted twice as much as substitution polymorphisms. Sequencing analysis of non-coding regions in the Kii population To characterize the genetic diversity and migration rate of genes in the Kii population, the non-coding intergenic regions were analyzed in the Kii and 7 neighboring populations, Taka- hama, Jigokudani, Hagachi, Mie, Shiga, Arashiyama, and Minoo (Fig 1). Eight individuals from each population were investigated. Nine non-coding loci were randomly selected from the 27 regions described by Osada et al. [52], and they are listed in S4 Table along with the primers used for amplification and sequencing. The methods used for amplifying and sequenc- ing a target region were the same as those described above. If a sample had more than 1 hetero- zygous indel in the target region, the sequences were not used for further analyses. Haplotypes were reconstructed from diploid sequences sets using PHASE v2.1 [42,43]. The non-coding sequences were deposited in GenBank under the accession numbers AB907231–AB907287. Summary statistics, including π, θ, D, and pairwise FST, were determined using DnaSP v5.1 [44]. Nm values were calculated from the formula: FST = 1/(1 + 4Nm), where N and m represent the effective population size and migration rate, respectively [53]. Two-bottle preference test Behavioral tests were performed for 7 Japanese macaques from the Kii population, which were housed in individual cages at the Primate Research Institute, Kyoto University. Of these 7 macaques, 4 had the ATG/ATG genotype, 1 had the ATG/ACG genotype, and 2 had the ACG/ ACG genotype at the start codon of the TAS2R38 gene. Monkeys were kept in an air-condi- tioned room in individual cages (90 cm wide, 76 cm length, 85 cm height). For environmental enrichment, monkeys were able to play with toys (wood blocks, chains, and cords) and inter- acted with other monkeys visually and vocally. All monkeys were given fruits and sweet pota- toes in addition to pellets. Monkeys received water ad libitum during the two-bottle preference test; the automatic water supply was disabled and distilled water and PTC solutions were instead provided in distinctive bottles (500 cc) placed in an individual macaque’s cage for 4 h. PTC solutions were prepared at concentrations of 1, 10, 100, 250, and 1000 μM in distilled water. PTC solution concentrations and positions were randomly changed to prevent position preferences. During the test, we monitored monkey behavior by video and checked for liquid spillage caused by accidents due to monkey interference. To prevent monkey interference, we covered the bottles, exposing only the feed-water nozzle as needed. The health of animals was carefully checked daily by the experimenter and keeper. This trial was conducted once a day PLOS ONE \| DOI:10.1371/journal.pone.0132016 July 22, 2015 17 / 21 Rapid Expansion of PTC Non-Tasters among Japanese Macaques every weekday and repeated 6 times for each concentration. A preference ratio was calculated as the ratio of PTC solution volume consumed to the total liquid volume consumed. Based on the mean preference ratio at each concentration, we fit the data to the equation: f(x) = 0.5/[1 + (x/EC50)h], where x was the PTC concentration, with the Levenberg–Marquardt algorithm using R v2.14.0. Author Contributions Conceived and designed the experiments: NSH TH YI TM YS HI. Performed the experiments: NSH. Analyzed the data: NSH TH AM YG YS. Contributed reagents/materials/analysis tools: YI TM KA HH HI. Wrote the paper: NSH TH HH YS HI. Acknowledgments We thank Dr. Naoyuki Takahata and Dr. Yukako Katsura for valuable discussions; Ms. Miho Hakukawa for experimental support; Dr. Takashi Ueda for providing the G16/gust44 expres- sion vector; Mr. Yoshiro Kamanaka and Ms. Mayumi Morimoto for support with behavior experiments; the Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University; the NPO Saru-Doko-Net; Dr. Tomoko Anezaki (Gunma Museum of Natural History); Dr. Shuji Yachimori (Shikoku Institute of Natural History); local governments in Shi- mokita (Mutsu city, Oma town, Sai village, Kazamaura village); and Mr. Hideharu Yamazaki, Dr. Shin-ichi Hayama, Dr. Toshinori Omi, Dr. Yoshi Kawamoto, Dr. Nobuko Kazahari, Dr. Miho Murayama, Dr. Eiji Inoue, and Dr. Naofumi Nakagawa for providing DNA samples. A Japanese macaque that was a heterozygote for the non-taster allele at TAS2R38 was provided by NBRP “Japanese Monkeys” through the National BioResource Project of MEXT, Japan. 1. 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The seven Japanese macaques have 3 geno- types at the TAS2R38 start codon: (A-D) ATG/ATG, (E-F) ACG/ACG and (G): ATG/ACG. (PDF) S2 Fig. Gustatory responsiveness of each individual to various PTC solution concentra- tions. These Figs show the individual data summarized in Fig 4. Each point represents the mean ± standard error determined from 6 trials. The seven Japanese macaques have 3 geno- types at the TAS2R38 start codon: (A-D) ATG/ATG, (E-F) ACG/ACG and (G): ATG/ACG. (PDF) S3 Fig. Segregating sites in 13 haplotypes of the TAS2R38 flanking region. Segregating sites are divided into 3 parts: 50-flanking, TAS2R38 coding, and 30-flanking regions. Numbers above the segregating sites indicate the nucleotide position of each site and correspond to the last 5 positional digits in the rheMac2 genome (chr3;179,4xx,xxx). Dots indicate nucleotides identical to those in the top line sequence (A-1). Pound signs and asterisks indicate indel polymor- phisms. 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https://openalex.org/W3097360571	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0233941&type=printable	English	null	Deviation from Mendelian transmission of autosomal SNPs can be used to estimate germline mutations in humans exposed to ionizing radiation	PloS one	2,020	cc-by	8,871	PLOS ONE PLOS ONE RESEARCH ARTICLE Deviation from Mendelian transmission of autosomal SNPs can be used to estimate germline mutations in humans exposed to ionizing radiation Hugo Pereira Leite FilhoID1,2, Irene Plaza PintoID3, Lorraynne Guimarães OliveiraID3,4, Emı´lia Oliveira Alves Costa3, Alex Silva da CruzID3, Daniela de Melo e Silva3,4, Claudio Carlos da Silva1,2,3,5, Alexandre Rodrigues CaetanoID6, Aparecido Divino da Cruz1,3,4,5* 1 Programa de Po´s-Graduac¸ão em Biotecnologia e Biodiversidade, Universidade Federal de Goia´s, Goiaˆnia, Goia´s, Brazil, 2 Universidade Estadual de Goia´s, Ana´polis, Goia´s, Brazil, 3 Nu´cleo de Pesquisa Replicon, Mestrado em Gene´tica, Escola de Ciências Agra´rias e Biolo´gicas, Pontı´ficia Universidade Cato´lica de Goia´s, Goiaˆnia, Goia´s, Brazil, 4 Programa de Po´s-Graduac¸ão em Gene´tica e Biologia Molecular, Universidade Federal de Goia´s, Goiaˆnia, Goia´s, Brazil, 5 Laborato´rio de Gene´tica Molecular e Citogene´tica Humana, Laborato´rio Estadual de Sau´de Pu´blica Dr. Giovanni Cysneiros, Secretaria de Sau´de Pu´blica do Estado de Goia´s, Goiaˆnia, Goia´s, Brazil, 6 Embrapa Recursos Gene´ticos e Biotecnologia, Brası´lia, Distrito Federal, Brazil * acruz@pucgoias.edu.br Abstract Citation: Leite Filho HP, Pinto IP, Oliveira LG, Costa EOA, da Cruz AS, e Silva DdM, et al. (2020) Deviation from Mendelian transmission of autosomal SNPs can be used to estimate germline mutations in humans exposed to ionizing radiation. PLoS ONE 15(10): e0233941. https://doi.org/ 10.1371/journal.pone.0233941 We aimed to estimate the rate of germline mutations in the offspring of individuals acciden- tally exposed to Cesium-137 ionizing radiation. The study included two distinct groups: one of cases, consisting of males and females accidentally exposed to low doses of ionizing radi- ation of Cs137, and a control group of non-exposed participants. The cases included 37 peo- ple representing 11 families and 15 children conceived after the accident. Exposed families incurred radiation absorbed doses in the range of 0.2 to 0.5 Gray. The control group included 15 families and 15 children also conceived after 1987 in Goiaˆnia with no history of radiation exposure. DNA samples from peripheral blood were analyzed with the Affymetrix GeneChip® CytoScanHD™to estimate point mutations in autosomal SNPs. A set of scripts previously developed was used to detect de novo mutations by comparing parent and off- spring genotypes at the level of each SNP marker. Overall numbers of observed Mendelian deviations were statistically significant between the exposed and control groups. Our retro- spective transgenerational DNA analysis showed a 44.0% increase in the burden of SNP mutations in the offspring of cases when compared to controls, based on the average of MFMD for the two groups. Parent-of-origin and type of nucleotide substitution were also inferred. This proved useful in a retrospective estimation of the rate of de novo germline mutations in a human population accidentally exposed to low doses of radiation from Cesium-137. Our results suggested that observed burden of germline mutations identified in offspring was a potentially useful biomarker of effect to estimate parental exposure to low doses of IR and could become an important marker suitable for biomonitoring human popu- lation exposed to environmental mutagens. Editor: Roberto Amendola, ENEA Centro Ricerche Casaccia, ITALY * acruz@pucgoias.edu.br Introduction In 1987, a series of unexpected events resulted in a major radiological accident in Goiaˆnia, Goia´s, Brazil, causing human, animal, plant and environmental exposure to gamma ray ioniz- ing radiation (IR) of Cesium-137 and contamination by the radionuclide [1]. For some people, individual exposure resulted from internal and external contamination by the radioactive salt, while others were exposed to radiation emitted by the decay of Cesium-137. In some cases, people were both exposed to radiation and contaminated by the radionuclide. In the aftermath, 249 people were exposed to IR from Cesium-137, leading to individual absorbed doses of IR ranging from 0 to 7 Gy, resulting in four fatalities during the acute phase of the accident [2, 3]. Following the accident, the exposed population has been extensively monitored using genetic biomarkers, as they have been shown to be efficient biomarkers of exposure to gamma rays [4]. However, each biomarker tends to reveal a distinct biological phenomenon in the exposed cells, mostly associated with DNA repair and how cells physiologically coped to sur- vive a specific insult. In this context, our group and others have established somatic mutation frequencies using data from glycophorin A [5] and HPRT assays [6], chromosomal aberrations [7–9], BCL2/J(H) translocation [10], and micronucleus frequencies [11] in T-lymphocytes of the cohort accidently exposed to Cesium-137 IR. Moreover, in order to understand the effect of IR on the induction of germ line mutations, STR markers [12] were initially used to estimate the rate of germline mutations in the offspring of the exposed cohort. More recently, CNVs have been used as biomarkers for parental exposure to demonstrate the effect of low absorbed doses of IR on germline mutations in the cohort’s offspring conceived after the accident [13]. Radiation-absorbed dose relates to the estimated quantity of energy deposited in the mater per unit of mass. Thus, it can be used as an indirect measurement of the harmful biological effect of the radioactive energy on the cellular system. It is calculated by estimating the concen- tration of energy from radiation exposure deposited in each organ, using a reference value, the type of radiation and the potential for radiation-related mutagenic changes in each organ or tissue [14]. The exposure of cells to IR delays the normal progression of the cell cycle [15–17], initially observed as a passive cellular response resulting from of the induction of DNA damage in the exposed cells. Editor: Roberto Amendola, ENEA Centro Ricerche Casaccia, ITALY Copyright: © 2020 Leite Filho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data included in the article are available at https://github.com/ hugofilho/sipo/tree/master/data. Funding: The author received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. 1 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 Sample collection, processing, and genotyping The experiment was designed as a case-control observational study. The group of cases con- sisted of 11 families, of whom at least one of the parents was accidentally exposed to IR during the Cesiu-137 accident, totaling 37 participants (11 fathers, 11 mothers, and 15 children con- ceived after the accident). The radiation absorbed doses for the exposed parents ranged from 0.2 to 0.5 Gy [3, 13]. As controls, biological samples were obtained from 15 families living in Goiaˆnia since the time of the accident with no prior history of exposure to IR. Thus, the con- trol group was comprised of 15 fathers, 15 mothers, and 15 children also conceived after 1987. A total of 82 subjects were used in the study whose DNA samples were analyzed using the SNP-array GeneChip1 CytoScanHD™(Thermo Fisher Scientific). Cases and controls participated voluntarily in the study, which was approved by the ethics committee on research with humans from the Pontifical Catholic University of Goia´s (PUC-- Goia´s)–CAAE number 49338615.2.0000.0037. At the time of blood collection, participants answered a lifestyle questionnaire and signed an informed consent form. A total of 10 mL of peripheral blood in EDTA was voluntarily donated by all participants. Total genomic DNA was isolated from whole blood using Illustra blood genomicPrep Mini Spin Kit1 (GE Health- care, Milwaukee, WI, USA) and stored at −20˚C. The remaining biological material was stored according to CNS Resolution 441/11. 1 Chromosomal microarray analyses were carried out in GeneChip CytoScanHD1 arrays (Thermo Fisher Scientific) in order to collect individual genotypes from polymorphic autoso- mal markers. SNP genotypes were generated using ChAS© (Thermo Fisher Scientific). Every array met the quality controls recommended in the manufacturer’s guidelines. SNP genotypes were filtered based on individual call confidence levels for each marker, thus calls with confi- dence levels <5x10-2 and invalid (no call or null) in one or more samples were removed from the dataset. Therefore, only markers with quality-controlled genotypes in all samples were con- sidered for the analysis. Genotyping was based on the hg19 version of the human genome hosted on the UCSC Genome Browser (University of California, Santa Clara, CA, USA). We also applied the CpG island track from UCS browser in order to stablish the rule out C>T mutations at CpG sites. As the array genotypes didn’t allow the discrimination from which strand the damage was derived, all substitutions were included in the data sets. Introduction The irradiated cell must adapt to the insult and facilitate DNA repair processes, especially fixing double-strand breaks, the most common damage after DNA exposure to IR [18–22]. The mutagenic effects of IR on the human germ line cells are of concern, as they lead to the accumulation of mutations in the offspring of irradiated parents, amounting to an increase in the mutational burden [23]. Despite numerous studies, little is known about the genetic effects of low doses of radiation from low linear energy transfer gamma radiation exposure in humans. Most of the consolidated evidence comes from the extrapolation of the induction of germline mutations in mammals, often rat and mouse models [24, 25]. Advances in the methodologies of genomic analysis have greatly increased the volume of nucleotide sequence data, enabling the identification of thousands of SNPs (single-nucleotide polymorphisms). Variations in SNPs are important to determine genotypic and phenotypic relationships, within and between species and populations, and also to identify variants related to genetic diseases in humans and animals [26]. In this context, genomic analysis can be a use- ful tool to study and understand the effects of IR exposure on animals and humans [13, 27]. In recent decades, several genotyping technologies have been developed to characterize SNPs all producing genotype matrices with hundreds of thousands of datapoints. Algorithms based on parametric and nonparametric statistical models have been used to determine the genotype of each SNP from the fluorescence signal intensity of marked probes, which are PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 2 / 17 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation scanned, captured, and arranged in a matrix format [28, 29]. One commercially available SNP array, the GeneChip1 CytoScanHD™(Thermo Fisher Scientific, Waltham, MA, USA), is con- sidered to be a high-density matrix, including about 750,000 polymorphic markers with an average genotyping accuracy of >99% [30]. In the aforementioned context, the general objective of the current study was to quantify Mendelian deviations (MD) in genome-wide autosomal SNP data from a cohort of people conceived after parental exposure to Cesium-137 IR, and a group of non-exposed people from the same geographical area. The rate of MD was applied to evaluate if the observed burden of germline mutations identified in the offspring could be a potentially useful biomarker of parental exposure to low doses of IR. Principle component analysis Principle Component Analysis (PCA) methods were used to assess whether participants in the case and control groups came from the same genetic population, the dataset contained about 522K SNPs. This step was also included to assess whether individual sample quality effects may have generated spurious results. SNPs with minor allele frequencies (MAF) below 0.01 3 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation were removed from the dataset, including all mendelian errors in the samples. Data pruning of the final dataset was performed using the PLINK (2.0) package [31] to generate a subset of markers for PCA analysis using the following parameters: window size of 500 SNP with a step size of 5 SNP, using an r2 threshold of 0.1. Pruning resulted in a subset of 2.789 SNPs that were used to estimate principal components and to generate plots for each test group. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 Analysis and phasing of genotyping data to identify mendelian deviations MDs were inferred with a set of previously developed Perl scripts and R libraries [32] termed SIPO (Scripts for Inference of Parental Origin) to mine SNP data in MySql© format. The SIPO pipeline was listed in (Fig 1) and supporting information files are accessible in a GitHub under the accession URL: https://github.com/hugofilho/sipo. Parent genotypes were compared with respective offspring genotypes for each individual marker. Sex chromosome data were excluded from the analysis, as X-linked data showed elevated noise and Y-specific regions had low marker coverage. Table 1 shows all data variables considered by SIPO. First, SIPO validated the.CYCHP file generated by ChAS1, then SIPO identified trio vari- ables and started to generate inferences for de novo mutations, corresponding to MDs in the child. Parental origin of observed mendelian deviations were inferred using basic expected mendelian inheritance rules applied over family trio data. For instance, if parent 1 had a geno- type "AA" and parent 2 had a genotype "CC", and their child had genotype "GC", the germline mutation was inferred to parent 1. Executed steps allowed to determine nucleotide substitution type in addition to inferring the parent of origin of the MD observed in the offspring. Derived information was loaded into a MySQL database and R scripts were used to perform linear regression, clustering and PCA with the resulting data (Fig 2). In some situations, SIPO was not able to identify the parental origin of a SNP based on Men- delian transmissions. To solve this challenge, two deductions were incorporated into the Fig 1. Steps performed by SIPO to infer de novo mutations. Deducting the parental origin of the MD, indicating the type of substitution and generating the estimated rate of Mendelian deviation in the offspring of people accidentally exposed to Cs-137 ionizing radiation. https://doi.org/10.1371/journal.pone.0233941.g001 Fig 1. Steps performed by SIPO to infer de novo mutations. Deducting the parental origin of the MD, indicating the type of substitution and generating the estimated rate of Mendelian deviation in the offspring of people accidentally exposed to Cs-137 ionizing radiation. https://doi.org/10.1371/journal.pone.0233941.g001 https://doi.org/10.1371/journal.pone.0233941.g001 4 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Table 1. Variables generated by ChAS1 and considered by SIPO to identify MDs. Marker SNP marker identifier Genotyping Genotyping call. Biallelic information. Analysis and phasing of genotyping data to identify mendelian deviations Three possible combinations: AA/BB/AB Trust Value Confidence value for each genotyping call Sign A Gross sign value for sign A on the marker Sign B Gross sign value for sign B on the marker Nitrogen Base Call from the nitrogen base. Biallelic information. Ten possible combinations: AA\|AC\|AG\| AT\|CC\|CG\|CT\|GG\|TT dbSNP SNP identifier record in the NCBI dbSNP database Chromosome Autosome associated with the marker Chromosome Position SNP locus on the chromosome https://doi.org/10.1371/journal.pone.0233941.t001 Table 1. Variables generated by ChAS1 and considered by SIPO to identify MDs. pipeline. The first deduction was coded into SIPO to identify confidence interval values of indi- vidual SNPs using ChAS1 data from the parents of a family trio. The second consisted of identi- fying the nearest mutated SNP, based on Euclidean distance using tools from Microsoft Excel1 (version 365), which had the parent of origin previously inferred following mendelian transmis- sions rules. Thus, at the end of the pipeline, the deductions aided to attribute the origin of a mutation to the parent who had both the lowest confidence interval value for that particular mutated SNP and who transmitted that chromosomal segment to the child based on the nearest variant SNP. The total count of MD was used to estimate the germline mutation frequency (MFMD) in the offspring, using Eq 1: [13, 33] MFMD ¼ P TMD b x nvp ð1Þ ð1Þ Where STMD = Total MD; b is a biallelic locus (2); nvp is the number of valid SNPs in the array according to the assembly of the human reference sequence (GRCh37/hg19) as indicated in S1 Table. Where STMD = Total MD; b is a biallelic locus (2); nvp is the number of valid SNPs in the array according to the assembly of the human reference sequence (GRCh37/hg19) as indicated in S1 Table. In the present study, all statistical tests were performed considering a 95% confidence inter- val and 5% significance level. The statistical tests used were the Shapiro-Wilk test, Student’s T test, regression analysis, clustering [34], and principal component analysis [35, 36]. The R sta- tistical package [32] was used in all analyses. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 Results and discussion The current study used SNP genotypes from a cohort of offspring born to parents accidentally exposed to Cs-137 to estimate the induction of germline mutations in humans exposed to low doses of ionizing radiation. As a cautionary note, in the current work, deviation of a Mende- lian transmission implies that a point mutation observed in a child wasn’t observed in his/her parents, thus it was herein interpreted as a de novo mutation. However, we are aware that SNP variants can rise somatically due to DNA repair failure in the first cell divisions of the embryo, a variable common to both cases and controls and expected to be equally represented in the study datasets, bearing little bias to the dataset if any. Before disclosing the results of the study, we also wish to note the limitation regarding the small size of the study cohort, which could render meaningful conclusions at first glance. In this context, two important rationales support the value of considering follow-up studies of human populations exposed to IR. First, considering the global effort with respect to radiopro- tection and regulation, it’s very unlikely that large accidentally exposed cohorts will be 5 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Fig 2. Workflow of the SIPO. Steps performed to infer de novo mutations from SNPs obtained from a high-density genotyping array. Fig 2. Workflow of the SIPO. Steps performed to infer de novo mutations from SNPs obtained from a high-density genotyping array. https://doi.org/10.1371/journal.pone.0233941.g002 available world-wide to be investigated with the newest methodologies. Second, a high-density SNP array was used to call thousands of SNPs, covering a very large proportion of the genome. Thus, increasing the chances of identifying genomic variation in small populations that could be potentially useful to establish new biomarkers of effect to be applied in future studies inves- tigating genotoxic and mutagenic responses to environmental stressors. The current available technologies applied to the study of genomes have that intrinsic characteristic, making them tools of first-tier choice in a variety of investigations, particularly when assessing small cohorts. available world-wide to be investigated with the newest methodologies. Second, a high-density SNP array was used to call thousands of SNPs, covering a very large proportion of the genome. Results and discussion Thus, increasing the chances of identifying genomic variation in small populations that could be potentially useful to establish new biomarkers of effect to be applied in future studies inves- tigating genotoxic and mutagenic responses to environmental stressors. The current available technologies applied to the study of genomes have that intrinsic characteristic, making them tools of first-tier choice in a variety of investigations, particularly when assessing small cohorts. CytoScan HD Suite had an intrinsic algorithm, which allowed the analysis of a chromosome segment given the presence of polymorphic markers within that region. In the current study, the challenge was to establish the parent-of-origin for a point mutation based solely on PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 6 / 17 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Fig 3. PCA with 2.7K SNP. The variables contained in the PCA represented the standardized relationship matrix by variance, multidimensional sizing (MDS) based on Hamming distances. https://doi.org/10.1371/journal.pone.0233941.g003 Fig 3. PCA with 2.7K SNP. The variables contained in the PCA represented the standardized relationship matrix by variance, multidimensional sizing (MDS) based on Hamming distances. Fig 3. PCA with 2.7K SNP. The variables contained in the PCA represented the standardized relationship matrix by variance, multidimensional sizing (MDS) based on Hamming distances. https://doi.org/10.1371/journal.pone.0233941.g003 https://doi.org/10.1371/journal.pone.0233941.g003 Mendelian transmissions. In order to infer that origin, two deductions were incorporate into our pipeline, which allowed the inclusion of 9,522 and 4,821 MDs for case and control groups, respectively, into the dataset. In this context, the current pipeline could be used as an addi- tional tool to define the parental origin of polymorphic variants obtained from SNP array genotypes. PCA results using a subset of LD-pruned data (522 Kb SNPs) indicated subjects included in both case and control groups belonged to the same population and there were no recognizable additional confounding factors associated with the test groups other than exposure to Cs-137 (Fig 3). Therefore, the MFMD could be compared between groups, even with a reduced sample size. Observed MDs followed a normal distribution (p = 0.5592) and were all included in sub- sequent statistical analyses. The lowest individual numbers of MDs were 972 and 682, and the highest were 2,875 and 1,635 for the case and control groups, respectively (Table 2). Observed MDs were randomly distributed on the SNPs in the array. Results and discussion When performing family trio com- parisons, most MDs (60%) were observed only once with no repetition, while 27%, 9% and 4% of the same MDs were respectively observed twice, three and four times in the family trios, confirming both the random effect of DNA damage induced by IR and spontaneous replica- tion errors. Moreover, this observation also favors the quality of the array avoiding artefactual genotyping errors to be included in the dataset. In the current study, mutation burden was defined by the number of de novo base substitu- tions in an assayed SNP of a child born to a parent exposed to IR. Thus, a total of 18,429 and 26,533 SNPs showed MD for control and cases, respectively. Thus, the overall frequencies of PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 7 / 17 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation germline mutations observed in the different trios were, on average, 1.3 x10-3 and 0.9 x10-3 mutations per polymorphic marker. The Student’s T test showed the difference in the means was statistically significant assuming equal variances for both groups (p = 0.002). Tables 2 and 3 contain the summary of the data used in this study. Our retrospective transgenerational DNA analysis showed about a 44.0% increase in the burden of SNP mutations in the offspring Table 2. Overall data from both control and exposed groups regarding the study of germline mutation in the offspring of people accidentally exposed to low absorbed doses of Cesium-137 ionizing radiation in Goiania (Brazil). https://doi.org/10.1371/journal.pone.0233941.t002 g p All ages are in years old. Results and discussion Group Family Exposed Progenitor Absorbed Dose (Gy) Paternal Age2, Maternal Age2 Age of offspring Sex of offspring MDs1 Total of valid SNPs Frequency of MDs Father Mather Unknown Total Control Ct001 None 0 40 36 9 Female 783 694 10 1487 702,304 1,06E-03 Ct25 None 0 47 36 9 Male 545 631 3 1179 683,381 8,62E-04 Ct27 None 0 26 26 23 Male 594 729 9 1332 692,311 9,62E-04 Ct39 None 0 24 24 3 Female 679 679 11 1369 674,320 1,02E-03 Ct40 None 0 45 37 3 Male 710 711 30 1451 696,544 1,04E-03 Ct45 None 0 37 31 15 Male 643 663 8 1314 683,243 9,62E-04 Ct51 None 0 35 34 2 Female 384 479 8 871 697,737 6,24E-04 Ct52 None 0 55 41 1 Male 1015 588 32 1635 710,477 1,15E-03 Ct53 None 0 35 27 8 Male 315 361 6 682 713,372 4,78E-04 Ct60 None 0 40 38 1 Female 501 482 6 989 712,261 6,94E-04 Ct66 None 0 31 20 26 Female 543 594 2 1139 707,798 8,04E-04 Ct68 None 0 33 20 10 Male 758 654 11 1423 712,191 1,00E-03 Ct70 None 0 20 24 8 Female 448 545 3 996 714,892 6,96E-04 Ct72 None 0 31 20 14 Female 521 614 4 1139 710,259 8,02E-04 CtF09 None 0 19 21 25 Male 718 696 9 1423 712,352 9,98E-04 Exposed Ex04 Father 0.1 27 27 20 Male 900 966 7 1873 698,305 1,34E-03 Ex06 Father 0.3 35 26 9 Male 1045 1082 9 2136 693,858 1,54E-03 Ex07- 1F Mother 0.2 54 24 19 Male 976 850 8 1834 692,375 1,32E-03 Ex07- 4F Mother 0.2 56 26 17 Female 1509 1085 18 2612 689,467 1,89E-03 Ex08 Mother 0.2 18 20 8 Male 538 718 4 1260 706,759 8,92E-04 Ex10 Mother 0.2 21 24 2 Female 1187 1054 21 2262 705,993 1,60E-03 Ex12 Mother 0.3 31 30 3 Male 1361 1486 28 2875 693,463 2,08E-03 Ex15 Father 0.2 18 27 16 Male 560 645 7 1212 706,780 8,58E-04 Ex18 Father 0.2 47 30 18 Male 819 800 12 1631 707,366 1,15E-03 Ex21 Mother 0.2 38 27 20 Female 457 513 2 972 707,827 6,86E-04 Ex22- 2F Mother 0.2 29 31 20 Female 1006 664 3 1673 707,075 1,18E-03 Ex22- 3F Mother 0.2 32 34 17 Female 845 566 3 1414 708,278 9,98E-04 Ex22- 4F Mother 0.2 33 35 16 Male 781 518 5 1304 708,885 9,20E-04 Ex24 Father 0.5 21 19 12 Female 1010 1102 47 2159 703,635 1,53E-03 Ex25 Father 0.5 18 16 15 Female 598 708 10 1316 706,598 9,32E-04 1Medelian deviations 2A l and exposed groups regarding the study of germline mutation in the offspring of people accidentally exposed to low radiation in Goiania (Brazil). PLOS ONE PLOS ONE Table 3. Summary of the descriptive data of the case and control groups for parental and F1 generations in the study of the effect of IR exposure on the induction of germline mutations in humans. Generation Variables Cases Control Parental N 15 15 Age range (years) 16 −56 19–55 Mean age at conception (years ± SD) Paternal 31.9 (12.5) 34.4 (9.9) Maternal 26.4 (5.3) 28.9 (7.4) Absorbed dose (Gy) 0.2 −0.5 0 F1 N 15 15 Age range (years) 2 −20 1–26 Mean age (years ± SD) 14.0 (5.9) 10.5 (8.5) Sex ratio (Male:Female) 8:7 8:7 Mean MFMD (± SD) 1.3 x 10−3 (± 0.4 x 10−3) 0.9 x 10−3 (± 0.2 x 10−3) SD = Standard Deviation. https://doi org/10 1371/journal pone 0233941 t003 Table 3. Summary of the descriptive data of the case and control groups for parental and F1 generations in the study of the effect of IR exposure on the induction of germline mutations in humans. Generation Variables Cases Control Parental N 15 15 Age range (years) 16 −56 19–55 Mean age at conception (years ± SD) Paternal 31.9 (12.5) 34.4 (9.9) Maternal 26.4 (5.3) 28.9 (7.4) Absorbed dose (Gy) 0.2 −0.5 0 F1 N 15 15 Age range (years) 2 −20 1–26 Mean age (years ± SD) 14.0 (5.9) 10.5 (8.5) Sex ratio (Male:Female) 8:7 8:7 Mean MFMD (± SD) 1.3 x 10−3 (± 0.4 x 10−3) 0.9 x 10−3 (± 0.2 x 10−3) SD = Standard Deviation. https://doi.org/10.1371/journal.pone.0233941.t003 Table 3. Summary of the descriptive data of the case and control groups for parental and F1 generations in the study of the effect of IR exposure on the induction of germline mutations in humans. Generation Variables Cases Control f the case and control groups for parental and F1 generations in the study of the effect of IR exposure on the induction https://doi.org/10.1371/journal.pone.0233941.t003 of cases when compared to controls, based on the average of MFMD for the two groups. The current study pioneered the application of SNP data analysis to identify MD and estimate germline mutations in the offspring of humans accidentally exposed to low absorbed doses of IR. Current findings corroborated our first study reporting the usefulness of small CNVs to estimate de novo human germline mutation rates in a similar cohort [13]. Results and discussion ta from both control and exposed groups regarding the study of germline mutation in the offspring of people accidentall esium-137 ionizing radiation in Goiania (Brazil). Table 2. Overall data from both control and exposed groups regarding the study of germline mutation in the offspring o absorbed doses of Cesium-137 ionizing radiation in Goiania (Brazil). PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 8 / 17 Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation PLOS ONE A previous study by [23] also described the usefulness of the mutation frequencies of de novo CNV and SNVs as biomarkers of effect for paternal exposure to IR in mice. Moreover, a recent study using whole genome sequencing data from an offspring of radar soldiers potentially exposed to IR found the differences in the frequency of de novo SNVs might be suited for the assessment of DNA damage from IR in humans [37]. We also carried out a liner regression in order to evaluate the relationship between the radi- ation-absorbed doses and the MFMD in our cohorts. Our results were statistically significant (p = 0.004; R2 = 0,257), suggesting that low absorbed doses of IR could predict an increase of the mendelian deviation in the exposed group, which could be linearly fitted (Fig 4) following Fig 4. Representation the relationship between the radiation-absorbed doses and the means frequency of Mendelian deviations in a cohort of people conceived after parental exposure to ionizing radiation. https://doi.org/10.1371/journal.pone.0233941.g004 Fig 4. Representation the relationship between the radiation-absorbed doses and the means frequency of Mendelian deviations in a cohort of people conceived after parental exposure to ionizing radiation. htt //d i /10 1371/j l 0233941 004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 9 / 17 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Fig 5. Potential parental age effect. (A) The fraction of paternal mutations as a function of paternal age at conception. Each point represents the data for one child (proband) with similar parental ages. The x-axis position is parental age, the y-axis position is fraction among paternal and maternal. Show the broadcast of data regarding the parental origin that transmitted the mutation to the child in the case group (B) and control group (C). Fig 5. Potential parental age effect. (A) The fraction of paternal mutations as a function of paternal age at conception. Each point represents the data for one child (proband) with similar parental ages. The x-axis position is parental age, the y-axis position is fraction among paternal and maternal. Show the broadcast of data regarding the parental origin that transmitted the mutation to the child in the case group (B) and control group (C). PLOS ONE https://doi.org/10.1371/journal.pone.0233941.g005 the equation below: the equation below: MFMD ¼ 0:001 þ 0:001ðdoseÞ MFMD ¼ 0:001 þ 0:001ðdoseÞ To date, there is extensive evidence supporting sex differences in mutation rates, with older fertile males expected to contribute more to the burden of a mutational health hazard than older females. A greater number of continuous cell divisions in the male germ line has been implicated as one reasonable explanation for such difference on paternal age effect [38, 39]. However, although this have been consistently reported, a clear and definite conclusion on the subject remains to be reached [38, 39]. In our study, the sex of the progenitors had no effect on the MFMD of autosomal SNPs as for both case and control groups mothers and fathers contrib- ute equal numbers of de novo MD to their offspring. When taken into consideration the sex of the exposed parent, the average of the frequencies of germline mutations of children born to exposed fathers was 1.2 x10-3 (±0.3 x10-3) and for exposed mothers was 1.3 x10-3 (±0.5 x10-3), with no statistical differences (p = 0.195) intragroup. With respect to the potential parental age effect, our control group revealed older fathers contributed more MDs to their offspring (Fig 5A–5C), which could be modeled by the number of mitotic spermatogonia divisions as a function of age, reinforcing previous findings regarded as male-mutation bias [39, 40]. However, our study failed to detect the maternal age effect on the number of MDs (Fig 6). Although there has been increasing evidence of maternal contri- butions to the de novo point mutations in the offspring [41, 42], others have argued that females contribute less MD to their offspring based on sex differences in gametogenesis and development [43]. To date, there is an ongoing debate about the maternal and paternal contri- butions to the germline mutation burden in the offspring [44]. New genomic and statistic tools applied to large and diverse populational datasets will soon help bring forth a resolution for this biological conundrum. Although larger number of family trios might be needed to assess the female contribution on the germline point mutations in their offspring, our results sug- gested that strength of male-mutation bias could be observed even in small family cohorts. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 PLOS ONE Single base substitutions have been a common and frequent mutational event subjacent to cell divisions spontaneously that rise as a consequence of DNA replication errors or induced by environmental stressors, such as IR. Some previously published studies on the types of DNA spontaneous base substitutions indicated all possible substitutions are well represented in germline cells [45]. Such studies suggested that transition rates tend to be higher [46] than transversion rates [47]. The findings in the current study supported these observations, since a higher proportion of transitions was observed in the children from both cases and controls. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 10 / 17 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Fig 6. Representation maternal age effect on the number of Mendelian deviations. https://doi.org/10.1371/journal.pone.0233941.g006 Fig 6. Representation maternal age effect on the number of Mendelian deviations. https://doi.org/10.1371/journal.pone.0233941.g006 It has been generally assumed that in groups of small sample sizes, it would be very difficult to detect the maternal age effect on the burden of point mutations in the offspring. Neverthe- less, in order to test the hypothesis that in our exposed cohort germline mutations in both sexes were damage-induced by exposure to low doses of IR, we stratified our set of phased de novo mutations in 6 classes based on parental and derived alleles (Table 4). All the SNPs harboring C>T transitions in the data sets were not located in CpG islands and were all included in the analyses. IR is known to cause double stand brakes and all types of base substitutions. Although all transitions and transversions were observed in our data set (Fig 7), C>T and T>C were overrepresented, for both cases and controls, favoring the well- known hypothesis that human genome harbor a mutational bias toward A/T composition in the DNA stand [48]. In our study, although the base line of the MFMD in SNPs were different, the mutational spectra of cases and controls, considering all base substitutions, were Table 4. Summary of the descriptive data of the case and control groups for the six classes of base substitution in the genome of children conceived after parental exposure to low doses of ionizing radiation and their controls. https://doi.org/10.1371/journal.pone.0233941.t004 PLOS ONE lGroup Class Minimum Maximum Mean SD Total Control C>A 38 137 100.93 29.058 1,514 C>G 45 141 111.47 28.538 3,344 C>T 238 558 435.93 92.669 2,798 T>A 45 84 64.67 14.034 6,539 T>C 252 635 418.93 99.427 970 T>G 61 141 96.67 21.091 6,284 Exposed C>A 72 209 148.27 45.325 2,224 C>G 90 291 168.60 56.616 2,529 C>T 376 1.166 672.67 234.248 10,090 T>A 48 147 85.27 27.825 1279 T>C 307 847 561.60 161.834 8,424 T>G 75 219 132.47 42.797 1,987 Table 4. Summary of the descriptive data of the case and control groups for the six classes of base substitution in the genome of children conceived after parental exposure to low doses of ionizing radiation and their controls. case and control groups for the six classes of base substitution in the genome of children conceived after parental their controls. Table 4. Summary of the descriptive data of the case and control groups for the six classes of base substitution in the gen exposure to low doses of ionizing radiation and their controls. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 11 / 17 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Fig 7. Phased de novo mutations based on parental and derived alleles distributed in classes of base substitutions for cases and controls from the Goiaˆnia population accidentally exposed to low doses of ionizing radiation. https://doi.org/10.1371/journal.pone.0233941.g007 Fig 7. Phased de novo mutations based on parental and derived alleles distributed in classes of base substitutions for cases and controls from the Goiaˆnia population accidentally exposed to low doses of ionizing radiation. https://doi.org/10.1371/journal.pone.0233941.g007 https://doi.org/10.1371/journal.pone.0233941.g007 remarkably similar. This observation supports previous claims regarding the random effect of the deposition of radiation energy on biological systems [49]. In the context described before, MFMD of polymorphic markers was a quantifiable and use- ful variable to estimate the parental contribution to the mutational burden of their children, as a consequence of transmitting non-deleterious point mutations induced by IR above the threshold expected from the control population. DNA damage in the parental germ lines could have gone uncorrected by the DNA repair system, fixed in the cells and then transmitted to the offspring. The F test, to evaluate MD frequencies in the test groups, showed the number of observed MDs were significantly different (F = 4.47; p = 8 x10-3). PLOS ONE The arithmetic mean of the MD in the offspring of case and control groups are shown in Fig 8A, whereas Fig 8B shows the representation of the total of MDs observed in each family trio in both groups. To validate the findings of the current study, which analyzed the MFMD of a small cohort of children conceived after their parents were accidentally exposed to ionizing radiation from Cs- 137, we suggest the application of the current study design to larger cohorts. It might be advis- able to include a wider range of absorbed doses, resulting from either therapeutic or occupa- tional exposures, to assess the potential of Mendelian deviations as retrospective biomarkers for IR exposure in human populations. In the present study, the case and control groups belonged to the same population and, therefore, were subjected to similar general environ- mental effects. Thus, it was safe to conclude that the average MFMD was higher in the exposed group as a result of higher germline base substitutions than in the control group, which could be reasonably assumed as a consequence of parental exposure to low doses of IR. In this con- text, low doses of low-LET radiation induced MD in autosomal SNPs that could be identified, quantified and, therefore, used as a biomarker of effect to study human populations according to their history of exposure to environmental mutagenic insults. Conclusions This study pioneered the analysis of MDs using autosomal SNP data observed in parent-off- spring trios as biomarkers of effect to low doses of ionizing radiation. We succeeded estimating retrospectively the germline mutation frequency of SNPs in a human population accidentally exposed to low doses of radiation from Cs-137 and estimated the burden of germline muta- tions in the offspring. 12 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Fig 8. Representation of the variability of Mendelian deviations. (A) Mean numbers of Mendelian deviations in the offspring of the case and control groups. (B) Representation of the number of Mendelian deviations for each trio in the case and control groups. https://doi.org/10.1371/journal.pone.0233941.g008 Fig 8. Representation of the variability of Mendelian deviations. (A) Mean numbers of Mendelian deviations in th offspring of the case and control groups. (B) Representation of the number of Mendelian deviations for each trio in t Fig 8. Representation of the variability of Mendelian deviations. (A) Mean numbers of Mendelian deviations in the offspring of the case and control groups. (B) Representation of the number of Mendelian deviations for each trio in the case and control groups. https://doi.org/10.1371/journal.pone.0233941.g008 https://doi.org/10.1371/journal.pone.0233941.g008 We found the sex of the progenitors had no effect on the MFMD of autosomal SNPs, for both case and control groups, mother and fathers contributed equal numbers of de novo MD to their offspring. After accounting for age, our control group revealed older fathers contrib- uted more MD to their offspring, which could be modeled by number of mitotic spermatogo- nia divisions as a function of age, supporting previous findings of male-mutation bias. However, our study failed to detect the maternal age effect on the frequency of MDs. In summary, there was a 44.0% increase in the MFMD of the offspring of those accidentally exposed to low doses of IR, from a radiological accident in Goiaˆnia. Low absorbed doses of IR could predict the increase of the mendelian deviation in the exposed group. Therefore, we con- cluded that MFMD is a potentially useful biomarker to estimate parental exposure to IR and suitable for human population biomonitoring. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 Acknowledgments At first, the authors wish to express their gratitude to the volunteers who selfishly agreed to participate in the study. We also thank CARA (Centro de Assistência ao Radioacidentado da SES-GO) for helping with contacting the group of exposed parents. Moreover, we thank Dr. Fernando Nodari and his team for assisting with the issues regarding the commercial genotyp- ing array. Lastly, we would like to thank Mr. Sean Quail for proofreading the manuscript. A.D. C., A.R.C., and D.M.S are CNPq research fellows. Conclusions In this context, future studies involving the behavior of MDs following diverse genomic and mutagenic hazards, caused by exposure to environmental agents, may provide important knowledge of the biological effects, mecha- nisms, and risks resulting from human exposure to such agents. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233941 October 27, 2020 13 / 17 PLOS ONE Deviation from Mendelian transmission in SNPs of humans exposed to ionizing radiation Finally, we are confident SNP array data can be used to estimate ionizing radiation-induced mutagenesis in human populations, provided the appropriate bioinformatics and statistical tools are used to extract the necessary information for biological inferences and to validate the scientific hypotheses underlying each investigation. References 1. da Cruz ASP, de Melo e Silva D, Godoy FR, de Melo AV, Costa EOA, Pedrosa, et al. 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Author Contributions Conceptualization: Hugo Pereira Leite Filho, Irene Plaza Pinto, Emı´lia Oliveira Alves Costa, Alex Silva da Cruz, Daniela de Melo e Silva, Claudio Carlos da Silva, Alexandre Rodrigues Caetano, Aparecido Divino da Cruz. Conceptualization: Hugo Pereira Leite Filho, Irene Plaza Pinto, Emı´lia Oliveira Alves Costa, Alex Silva da Cruz, Daniela de Melo e Silva, Claudio Carlos da Silva, Alexandre Rodrigues Caetano, Aparecido Divino da Cruz. Data curation: Hugo Pereira Leite Filho, Irene Plaza Pinto, Lorraynne Guimarães Oliveira, Emı´lia Oliveira Alves Costa, Claudio Carlos da Silva, Alexandre Rodrigues Caetano, Apare- cido Divino da Cruz. Formal analysis: Hugo Pereira Leite Filho, Claudio Carlos da Silva, Alexandre Rodrigues Cae- tano, Aparecido Divino da Cruz. Funding acquisition: Hugo Pereira Leite Filho, Alexandre Rodrigues Caetano, Aparecido Divino da Cruz. Investigation: Hugo Pereira Leite Filho, Irene Plaza Pinto, Emı´lia Oliveira Alves Costa, Alex Silva da Cruz, Daniela de Melo e Silva, Claudio Carlos da Silva, Alexandre Rodrigues Cae- tano, Aparecido Divino da Cruz. Methodology: Hugo Pereira Leite Filho, Alex Silva da Cruz, Claudio Carlos da Silva, Alex- andre Rodrigues Caetano, Aparecido Divino da Cruz. Project administration: Hugo Pereira Leite Filho, Claudio Carlos da Silva, Alexandre Rodri- gues Caetano, Aparecido Divino da Cruz. Resources: Hugo Pereira Leite Filho, Alexandre Rodrigues Caetano, Aparecido Divino da Cruz. Software: Hugo Pereira Leite Filho, Alexandre Rodrigues Caetano. Supervision: Hugo Pereira Leite Filho, Claudio Carlos da Silva, Alexandre Rodrigues Caetano, Aparecido Divino da Cruz. Validation: Hugo Pereira Leite Filho, Alexandre Rodrigues Caetano, Aparecido Divino da Cruz. Visualization: Hugo Pereira Leite Filho, Alexandre Rodrigues Caetano, Aparecido Divino da Cruz. 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W2011817570.txt	https://bmcplantbiol.biomedcentral.com/counter/pdf/10.1186/1471-2229-13-162	en		Combined linkage and association mapping reveals candidates for Scmv1, a major locus involved in resistance to sugarcane mosaic virus (SCMV) in maize	BMC plant biology	2,013	cc-by	8,822	Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 RESEARCH ARTICLE Open Access Combined linkage and association mapping reveals candidates for Scmv1, a major locus involved in resistance to sugarcane mosaic virus (SCMV) in maize Yongfu Tao1†, Lu Jiang1†, Qingqing Liu1†, Yan Zhang1, Rui Zhang1, Christina Roenn Ingvardsen3, Ursula Karoline Frei2, Baobao Wang1, Jinsheng Lai1, Thomas Lübberstedt2 and Mingliang Xu1* Abstract Background: Sugarcane mosaic virus (SCMV) disease causes substantial losses of grain yield and forage biomass in susceptible maize cultivars. Maize resistance to SCMV is associated with two dominant genes, Scmv1 and Scmv2, which are located on the short arm of chromosome 6 and near the centromere region of chromosome 3, respectively. We combined both linkage and association mapping to identify positional candidate genes for Scmv1. Results: Scmv1 was fine-mapped in a segregating population derived from near-isogenic lines and further validated and fine-mapped using two recombinant inbred line populations. The combined results assigned the Scmv1 locus to a 59.21-kb interval, and candidate genes within this region were predicted based on the publicly available B73 sequence. None of three predicted genes that are possibly involved in the disease resistance response are similar to receptor-like resistance genes. Candidate gene–based association mapping was conducted using a panel of 94 inbred lines with variable resistance to SCMV. A presence/absence variation (PAV) in the Scmv1 region and two polymorphic sites around the Zmtrx-h gene were significantly associated with SCMV resistance. Conclusion: Combined linkage and association mapping pinpoints Zmtrx-h as the most likely positional candidate gene for Scmv1. These results pave the way towards cloning of Scmv1 and facilitate marker-assisted selection for potyvirus resistance in maize. Keywords: Maize, SCMV, QTL, Fine-mapping, Association mapping Background Sugarcane mosaic virus is one of the most prevalent viral pathogens, causing significant global losses in maize grain and forage yields [1], especially in China and Germany [1,2]. A recent report of SCMV in Poland and Central France revealed that the incidence of SCMV continues to increase [3,4]. The diagnostic symptoms of infection with SCMV include stunting, chlorosis, and reduction in plant weight and grain yield [1,5]. SCMV, formerly denoted as maize dwarf mosaic virus (MDMV) strain B, and belongs to the family Potyviridae [5]. Other * Correspondence: mxu@cau.edu.cn † Equal contributors 1 National Maize Improvement Center, China Agricultural University, 2 West Yuanmingyuan Road, Beijing 100193, People’s Republic of China Full list of author information is available at the end of the article members of the family Potyviridae that also infect maize include MDMV, wheat streak mosaic virus (WSMV), Johnson grass mosaic virus (JGMV), sorghum mosaic virus (SrMV), and Zea mosaic virus (ZeMV) [6,7]. Control of SCMV by agronomic and chemical means is ineffective owing to the non-persistent transmission mode of the virus by aphid vectors. Thus, deployment of resistant varieties is the most important way to prevent yield losses, which is based on screening and identification of resistant germplasm [8]. U.S. inbred line Pa405 shows complete resistance to MDMV and SCMV under both field and greenhouse conditions after artificial inoculation [9]. In Europe, a collection of 122 early maturing maize inbred lines was screened for resistance against SCMV, resulting in the © 2013 Tao et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 identification of three lines, D21, D32, and FAP1360A, that displayed complete resistance to SCMV [6]. In China, many elite inbred lines display complete resistance when challenged with SCMV inoculum [2,10]. Analysis of quantitative trait loci (QTL) in a cross between the resistant line D32 and susceptible line D145 revealed two major dominant genes on chromosomes 3 and 6, and three minor QTL on chromosomes 1, 5, and 10 [11,12]. Highresolution mapping using progeny from the cross between FAP1360A (completely resistant to SCMV) and F7 (highly susceptible to SCMV) confirmed that Scmv1 (on the short arm of chromosome 6) and Scmv2 (near the centromere of chromosome 3) are crucial for maize resistance to SCMV [13]. Similar mapping results have been reported in several independent experiments [8,14,15]. The Scmv1 locus has also been detected in a resistant Chinese line, Huangzao4, together with additional QTL on chromosomes 1, 3, 5, and 10 [14]. In the progeny of Siyi×Mo17, two major genes were mapped on chromosome 3 (bin 3.04/05) and chromosome 6 (bin 6.00/01), respectively [8]. By using an F9-derived immortalized RIL population obtained from the cross of Huangzao4×Mo17, a major QTL was detected between chromosome bins 6.00 and 6.01, accounting for 50.0% of the total phenotypic variation and decreasing the disease index by 27.9% [16]. The loci on chromosomes 3 and 6 have also been confirmed in tropical maize germplasm from Brazil [15]. With the aid of the whole-genome B73 sequence, the Scmv2 region was fine-mapped to a region of 0.28 cM, covering a physical distance of 1,342.6 kb. Four predicted genes possibly involved in virus movement are likely candidates for Scmv2 [17]. Existence of two closely linked resistance genes within the Scmv1 region has been postulated [18,19]. Interestingly, several QTL that confer resistance to various viruses all map to the same region on chromosome 6 [20]. It is currently uncertain, whether a single pleitropic Scmv1 gene confers resistance to different viruses, or distinct resistance genes are clustered in this region. Even though diverse mapping populations have been used, all studies consistently identified the Scmv1 and Scmv2 genomic regions as being critical to confer resistance to SCMV. In this study, we first conducted the fine-mapping of Scmv1 using a large isogenic mapping population previously shown to segregate solely for Scmv1 [13,18,19,21,22]. Second, we employed two RIL populations from elite parental lines with contrasting resistance to SCMV to confirm and further fine map the Scmv1 locus. Third, we identified a limited number of positional Scmv1 candidate genes based on the combined fine-mapping results. Finally, we conducted candidate gene-based association mapping to characterize the QTL region and candidate genes using a panel of 94 maize inbred lines with variable resistance to SCMV. Page 2 of 13 Results Fine-mapping of Scmv1 in a near-isogenic F2 population Of 177 unselected F2 plants, 50 exhibited typical symptoms of SCMV infection within 2–3 weeks after the first inoculation. The numbers of resistant and susceptible plants segregated in a 3:1 ratio (χ2 = 0.83 < χ20.01, 1), in agreement with inheritance of a single dominant gene. Using the 17 polymorphic simple sequence repeat (SSR) markers available from the Maize Genetics and Genomics Database (http://www.maizegdb.org/) (Additional file 1), the Scmv1 locus was mapped between umc1018 and umc1753, with a physical distance of 18.80 Mb according to the B73 physical map (Figure 1A). Owing to the lack of high-density markers, 28 recombinants between these two markers could not be resolved. Accordingly, we developed 11 new markers (Table 1) based on the public maize Bacterial Artificial Chromosome (BAC) sequences accessible in GenBank at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). We used these 11 new markers to resolve the 28 recombinants. Subsequent marker-phenotype analysis enabled us to progressively refine the Scmv1 region into a 710.97-kb interval between STS-5 and STS-15 (Figure 1B), with still two recombinants in between those two markers. Thereafter, the markers STS-5 and STS-15 were used to genotype 510 F2 susceptible plants, resulting in 13 new recombinants. Within the STS-5/STS-15 interval, single/low copy sequences were exploited to develop seven additional markers (Table 1). These markers were used to genotype 15 recombinants, which allowed us to delimit the Scmv1 locus into a 112.39-kb interval flanked by the two closest markers, R1-2 and STS-11 (Figures 1C and 1D). There were still six recombinants to the left and eight recombinants to the right side of the Scmv1 locus (Figure 1D). Comparison with the B73 physical map indicated that the two closest markers, R1-2 and the STS-11, reside on the adjacent BACs in GenBank: b0129G15 and b0598N23, respectively. Validation of Scmv1 in RILs Given that the Scmv1 region could not be narrowed further using the near-isogenic F2 population, we used two RIL populations with parental lines contrasting in resistance to SCMV to confirm and fine-map the Scmv1 region. As both of the Scmv1 and Scmv2 loci are involved in maize resistance to SCMV, each RIL was investigated for its genotype at the Scmv2 locus using the co-segregating marker DJF004 developed by Ingvardsen et al. [17]. Given that one of the Scmv1 flanking markers, STS-11, failed to reveal any polymorphism between parents of each of the two RIL populations, we searched for an alternative marker around STS-11 and finally developed a new marker, B4, which is located approximately 103.75-kb downstream of STS11. The B4 Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 Page 3 of 13 A D B R1-2 0.1 0.3 1.8 4.5 C bnlg1043 umc2309 umc2208 umc2068 umc1018 12.7 6 B6-MseI STS-5 umc1018 2.7 18.8 6002I 260A16 0.5 3.6 umc1753 bnlg1165 umc2312 bnlg1432 umc1600 bnlg1867 umc1229 umc1196 1.0 1.4 1.7 0.3 2.8 2.2 STS-5 STS-8 STS-15 0.6 0.1 2.2 59.6 0 6003B 4.3 14.2 261A11 261B1 0.6 umc1825 48.0 1.9 6004F 186564A 262M1 umc1753 0.3 0.3 1.7 0.2 1.3 6 0.4 0.5 2.7 0.4 11.7 G7-2 R1-2 B6-MseI R18-2 R7B-2 STS-8 STS-12 STS-11 STS-15 21.8 4.0 0 4.7 0 27.0 0 R18-2 R7B-2 STS-8 10 kb phi077 3.7 8 STS-12 STS-11 1Mb 1Mb 1 kb Figure 1 Diagram of the sequential fine-mapping process. (A) The physical map of 17 polymorphic SSR markers available from MaizeGDB (http://www.maizegdb.org/) in the Scmv1 genome region, 2 of the 17 markers with ambiguous location were deleted. By assaying 177 unselected F2 plants, Scmv1 was narrowed to an 18.8 Mb interval flanked by SSRs umc1018 and umc1573. (B) The physical map of the Scmv1 region after fine-mapping with 28 recombinants and 13 new developed markers. (C), (D) The final physical maps of Scmv1, estimated by genotyping 15 recombinants between STS-15 and STS-5 using eight newly developed markers. The number on the left side of the chromosome segments correspond to the physical distance of AGPv2, the units of A, B, C and D were 1 Mb, 1 Mb, 10 kb, 1 kb, respectively. The numerals on the right side of the chromosome segments represent the number of recombinants between two flanking markers. marker, together with the R1-2 marker, were then used to jointly genotype all RILs at the Scmv1 locus. For each RIL population, four sets of 30 RILs, which differed at either the Scmv1 or Scmv2 or both loci, were separately selected for phenotypic evaluation. In the RIL population of Zheng58×Chang7-2, 28 of 30 RILs that share the same haplotype as Chang7-2 at both Scmv1 and Scmv2 loci (RR/RR) were highly resistant to SCMV. Of 30 RILs with the Chang7-2 haplotype at Scmv1, but the Zheng58 haplotype at Scmv2 (SS/RR), only one RIL was resistant to SCMV. Another two sets of 30 RILs sharing the same haplotype as Zheng58 at the Scmv1 locus (SS/SS and RR/SS) were all susceptible to SCMV. A similar result was observed in the RIL population of X178×HuangC, all 30 RILs sharing the same haplotype as X178 at both Scmv1 and Scmv2 loci (RR/RR) displayed high resistance to SCMV. Of 30 RILs with the X178 haplotype at Scmv1 and HuangC haplotype at Scmv2 (SS/RR), only four RILs were resistant to SCMV. Another two sets of 30 RILs which share the same haplotype as HuangC at the Scmv1 locus (SS/SS or RR/SS) were all susceptible to SCMV. These findings indicate that the presence of both Scmv1 and Scmv2 loci are prerequisite for SCMV resistance as described in previous reports [13]. The significant difference (P < 0.01) in resistance percentages between RILs differing in haplotypes at both Scmv1 and Scmv2 loci suggested the presence of the same resistance loci in both RIL populations (Table 2). Further fine-mapping of Scmv1 in RILs Given the validation result, the flanking markers B4 and R1-2 were used to screen for recombinants within the 215.47-kb Scmv1 interval. Eight and nine recombinants were detected in the RIL populations Zheng58×Chang7-2 and X178×HuangC, respectively. For the Scmv2 locus, a co-segregating marker DFJ004 was used to determine the genotype for each RIL. All recombinants were repeatedly investigated for their resistance to SCMV under both greenhouse and field conditions. On the other hand, Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 Page 4 of 13 Table 1 New markers developed for fine mapping of the Scmv1 locus Name BAC Forward primer Reverse primer Type 6002I c0288C01 GTTAGAACGTCTGCGCCTGT TCCGTGCTGCGTAGTTACCT STS STS-5 c0149g11 GCAGGAGAAGAATCGGAGG ACAAGAACAAGACAGCAGCG STS G7-2 b0129G15 AGTGTTCGTACCATGAGGTC AATCCAGCCTCAGTAGAGTC STS R1-2 b0129G15 GCGTAGCAATATCCGTGTCA TTCTGGTGTGAGCTTGAGCC STS R18-2 b0598N23 GATCTCGCAATCGTTGTAGC CTCTCAATGCGGTAACCATC STS STS-8 b0129G15 ACCGATTATGTTCCATTGGC GGTCCGACGCTCACTTCC STS STS-11 b0598N23 CAATAGACGTCGGACGAATG ATGACATGCAACTACAGGCG STS STS-12 b0598N23 CTAACAAGCATGACGATCCG AATTGCATCGATAAGCCACC STS STS-15 b0606n15 CTATTGGGCTTCCTTGTTCG TATTCCTGTAGGCGACCTGG STS 261B1 c0042B13 AGCTTCCAAGTGTCCTTAGC GCATCCTGGCTCAGCAATAC STS 6003B b0191N03 CTGATGACTGCCACCATAGC AACACGCTCGTGAGCAATAC STS 186564A b009O12 GTTCACGTTTCCCATGCTG GCCACGTCGAACAACCTTAT SSR 261A11 c0361N19 GAGAAGGCGAGAACGCATTG AAGAGGCTACCGTAGGCGTC SSR Restriction enzymes 6004F b0390N08 CGAGCTACTCAGCGTTGTCC AAGAGGCTACCGTAGGCGTC SSR R7B-2 b0598N23 CACCGGAATAGTAGACGCAC AGCTCTACTCCACCGGAACA SSR 260A16 c0115G19 ATGTCTGCTGCCGTGAGTTC CCTCCTGTCTGGTTGTCGTC CAPS HaeIII B6-MseI b0129G15 GGATGAGAGGACTCTTGCAC CTGCAAGCATGTCACAACA CAPS MseI 262M1 c0043A01 CAACTTCCATGACAGTGTCC GTGTGATTCGGTGGCATAAC CAPS MfeI RL3* 207B1-77 GGATACAGCACCAAGGTTGA AAGAGCTGTACACAGACGCC SNP SNP1* 207B1-77 GTAAGTGATAGGCGGAGTGG AGGCTATCGTCGTGGATTGA SNP SNP2* 207B1-77 CCACTCTTGCTTCATCCTCA AGTGTGTACGTGACCTTGATCT SNP SNP3* 207B1-77 AGATGGTGGTGAAGTGAAGG CCTAGCAGGCTAGCACTGAT SNP SNP4* 207B1-77 GATGGAGTGCCGATTGCTAG GGTCAACGAAGCCGATATTG SNP R6* 207B1-77 CACACACCACTTGCGATGTT TCATGCGATGCCAGTGATAG SNP 2562F* 207B1-77 GCCGTATACAGGTCGAGCTT GGAAGGAAGCCTAACTCGGA SNP Trx-1* 207B1-77 CGATGCCGCCTAATATTCTC GTAGCGGATCACGGATAACG STS 579P4* 207B1-77 AACGCGAGTGCAGTAAGTCT TGTTCCACAATGCTTTATCC CAPS TC-4* 579B1-75 CAGAGGAACAACAACCACCA GCACATTACGGTTGAGTTGG SNP B-4* 579B1-75 TACAAGTCAGGAGGTCCGGT CTGGATGGTGTTGTGTCGAG SSR AccII : Markers used for validation and further mapping in RILs, and these markers were developed based on the BAC sequences from 1145 inbred line. through multiple comparisons of three resistant inbred lines, namely 1145, FAP1360A, and Huangzao4, and one susceptible B73 sequences within the B4/R1-2 interval, ten markers were developed to resolve the 17 recombinants. Of the eight recombinants screened from the RIL population of Zheng58×Chang7-2, four RILs were as resistant to SCMV as the resistant parent Chang7-2, whereas another four RILs were as susceptible to SCMV as the susceptible parent Zheng58. The Scmv2 locus in three of the susceptible recombinants, Nos.70, 683 and 1045, was identical to that in the susceptible parent Zheng58, and thus they could not be used to define the Scmv1 locus. The four resistant RILs, Nos. 413, 966, 246, and 841, together with one susceptible RIL No. 1012 had the resistance allele as Chang7-2 at the Scmv2 locus and thus could be used to fine-map the Scmv1 locus. The four resistant RILs had the Chang7-2 donor region upstream of the marker B4, and one susceptible RIL No. 1012 had the Chang7-2 donor region downstream of the marker SNP4. These data support that the Scmv1 locus is located upstream of the marker B4 (Figure 2A). The mapped Scmv1 region in the Zheng58×Chang7-2 RIL population spanned the 112.39-kb Scmv1 region revealed by the near-isogenic F2 population and was thus of no value to further narrow down the Scmv1 locus. Among the nine recombinants screened from the RIL population X178×HuangC, two displayed the same high resistance to SCMV as seen in the resistant parent X178, whereas another seven were as susceptible to SCMV as the parent HuangC. Three highly susceptible RILs, Nos. 1779, 1553, and 1746, carry the HuangC susceptibility allele at Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 Page 5 of 13 Table 2 Confirmation of the Scmv1 and Scmv2 regions in two RIL populations P-value Genotype SS/SS SS/RR RR/SS RR/RR Zheng58×Chang7-2 Resistant RILs 0 1 0 28 Susceptible RILs 30 29 30 2 Resistant RILs 0 4 0 30 Susceptible RILs 30 26 30 0 <0.01 HuangC×X178 <0.01 The haplotype at the Scmv1 locus was detected by its flanked SSR markers R1-2 and B4; while the haplotype at the Scmv2 locus was identified by its co-segregating marker DJF004. The genotype SS/SS means both of the Scmv1 and Scmv2 loci are the same as Zheng58 or HuangC; RR/RR means both loci are the same as Chang7-2 or X178; RR/SS means Scmv2 and Scmv1are the same as Chang7-2/X178 and Zheng58/HuangC, respectively; while SS/RR means Scmv2 and Scmv1 are the same as Zheng58/HuangC and Chang7-2/X178, respectively. P-value and χ2 were calculated by SAS 9.1 using two-way ANOVA. the Scmv2 locus. They were thus excluded from finemapping of the Scmv1 locus. Four RILs, Nos. 1900, 1876, 2041, and 2062, had the X178 resistance allele at the Scmv2 locus. However, they were all susceptible to SCMV. This finding indicates the absence of the X178 resistance allele at the Scmv1 locus for these four RILs. Among them, Nos. 1876 and 1900 RILs had the donor HuangC region B4 SNP4 Trx-1 2562F 276P2 R1-2 A downstream of the marker R1-2, and Nos. 2041 and 2062 RILs had the donor HuangC region upstream of the marker SNP3, suggesting the Scmv1 locus is confined by the markers R1-2 and SNP3. Two highly resistant RILs, Nos. 2009 and 1523, carried the X178 resistance allele at the Scmv2 locus and their Scmv1 regions were flanked by the markers 579P4 and B4. Thus, the Scmv1 locus can be narrowed Percentage susceptibility Zheng58 Chang7-2 413 966 246 841 683 1012 70 B 1045 B4 SNP2 SNP3 TC4 SNP1 Trx-1 R6 579P4 R1-2 HuangC X178 1523 2009 1779 2062 1553 2041 1746 1876 1900 0% 20% 40% 60% 80% 100% Figure 2 Further fine-mapping of Scmv1 in two RIL populations. The genetic structure for each recombinant type is depicted as green and red rectangles, (green symbolizes the fragment from resistant parents, chang7-2 or X178; red symbolizes the fragment from susceptible parents, Zheng58 or HuangC). The star represents allele at the Scmv2 locus on chromosome 3 (green symbolizes resistance allele from Chang7-2 or X178; red symbolizes susceptibility allele from Zheng58 or HuangC). The light-green bars on the right represent the average percentage of plants with SCMV symptoms based on three independent experiments in greenhouse. A) The Scmv1 locus can be delimited to a 215.47-kb region in RIL population Zheng58×Chang7-2. B) The Scmv1 locus can be delimited to a 59.21-kb region flanked by markers 579P4 and SNP3 (in red dash-dot lines) in RIL population HuangC×X178. Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 down into a 59.21-kb region flanked by the markers 579P4 and SNP3 (Figure 2B), located within the 112.39kb interval defined by the near-isogenic F2 population. Predicting putative genes within and around the mapped Scmv1 region A 215.47-kb sequence flanked by the markers B4 and R1-2 was retrieved from www.maizesequence.org to identify candidate Scmv1 genes. There are still seven gaps left in this region, indicating some missing sequences. Transposable elements account for 55.27% of this region, of which 82.28% are retrotransposons, and 99.87% of the retrotransposons are long terminal repeat elements (data not show). We used Fgenesh software (http://linux1. softberry.com/berry.phtml, version 2.6) to predict ten putative genes from the masked sequence, including seven genes with putative functions, two hypothetical genes, and one gene without significant similarity to known genes (Figure 3C). No typical resistance (R) gene was found in the Scmv1 region. Analysis of possible functions for the seven predicted genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation indicated three of the genes were likely to confer resistance to SCMV (Table 3). Two of the predicted genes are cycloartenol synthase1like (CAS1-like) genes with high sequence similarity to genes from Arabidopsis thaliana. The first had the coverage of 76% and E-value of 1.00E-129, whereas the second had the coverage of 60% and E-value of 1.00E-74. These two CAS1-like genes have 74.18% similarity (204/275), with an E value of 2.00E-117, although they differ in the lengths of their amino-acid sequences. We designated the larger gene (403 residues) as CAS1-like-1, and the smaller one (360 residues) as CAS1-like-2. The other candidate gene, Zmtrx-h, encodes a putative 119-residue protein that is highly similar to thioredoxin (100% coverage; E = 4.00E-64), an oxidoreductase that acts on sulfur groups of donors, using disulfide as an acceptor. Integration of the mapping results indicated that Scmv1 localizes to a 59.21-kb region flanked by 579P4 and SNP3 (Figure 3B). Of the five predicted genes located in this region, CAS1-like-2 and Zmtrx-h are more likely than the other three genes to confer resistance to SCMV. Candidate gene–based association mapping of the Scmv1 locus To verify the candidate region and possibly refine the analysis by identifying quantitative trait nucleotide polymorphisms, we conducted an association mapping study using a panel of 94 inbred lines (Additional file 2), which were divided into six subgroups based on 70 SSR markers (Additional file 3). Multiple sequence alignment analysis identified 30 polymorphisms in six informative amplicons. Linkage disequilibrium (LD) analysis revealed Page 6 of 13 lower r2 between these polymorphisms (Figure 4), with 91.03% of the r2 < 0.20. Two (single nucleotide polymorphism) SNP sites, S454 and S126, separately located on the 3′-end and intron of Zmtrx-h were significantly associated with SCMV resistance (P < 0.05; Table 4 and Additional file 4). Nonetheless, a low r2 value of 0.01 indicated two independent functional variants. Given that 19 of the 94 maize inbred lines, including Mo17, could not be amplified by polymerase chain reaction (PCR) using any of the 17 primer pairs scattered throughout the Scmv1 region, we were able to identify a large PAV covering the Scmv1 region. This PAV was verified by Springer et al. [23], who reported a 2.6-Mb region on chromosome 6 including the Scmv1 region present in the B73 genome but not in Mo17. Strikingly, all of the 19 inbred lines were susceptible to SCMV, resulting in a strong association with SCMV resistance (P = 9.96E-4). Discussion Precise phenotypic evaluation is crucial for marker-trait association analysis [24-27], especially for those traits with incomplete penetrance and phenotypic plasticity. Apart from being influenced by two major genes, resistance of maize to SCMV is also affected by genetic background and various environmental factors [13,17,18]. To obtain reliable phenotypic data, we developed a F2 mapping population from a near-isogenic pair where the resistance allele was fixed at the Scmv2 locus, and only Scmv1 was segregating [28]. Furthermore, selection of only susceptible individuals from the F2 population for fine-mapping of the Scmv1 locus reduced the possibility of misclassification, as heterozygotes tend to be more plastic in their response to SCMV inoculation. Advances in multiple genomic platforms and analytical methods allow fine-mapping and cloning of QTL responsible for important agronomic traits [29]. In the current study, development of polymorphic markers in the 112.39-kb Scmv1 region was a challenge due to highly repetitive sequences. We thus sequenced the positive BAC clones from the 1145, FAP1360A, and Huangzao4 BAC libraries to mask the repetitive sequences at the Scmv1 locus, in order to reveal single/low-copy sequences for marker development. Generally, re-sequencing of more maize inbred lines [30] and the availability of additional publicly accessible sequence information (such as for Mo17: http://www.phytozome.net/maize. php; and Palomero: http://www.palomerotoluqueno. org/index.php) will facilitate the development of markers in targeted genome regions. During the process of finemapping, we found that the published order of BAC contigs (http://www.maizesequence.org) for the Scmv1 region is incorrect. The BAC-based B73 whole-genome physical map suggests that the adjacent BAC contigs #260 and #261 should be physically close to one another. However, the ctg260 6003B 262M1 6004F 186564A 261A11 A Page 7 of 13 261B1 6002I 260A6 Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 B73 ctg263 ctg261 ctg262 ctg263 c0430B07 b0129G15 579P4 B 1 2 3 b0606N15 B4 59.21kb 4 5 6 c0078M12 SNP-3 R1-2 STS-11 b0598N23 7 9 8 10 215.47Kb Candidate genes Genes with putative function Hypothetical protein No significant similarity Figure 3 Integration of the mapping results from three populations. (A) The combined mapping of Scmv1. The revised order of contigs on the Scmv1 region is #260-#263-#261-#262-#264 (ctg263 in red is where Scmv1 located ). Scmv1 was finally assigned to a 59.21-kb region flanked by markers 579P4 and SNP3. (B) Gene prediction of a 215.47-kb B73 sequence covering the targeted region: each arrow stands for a predicted gene, the head represents gene orientation and the length of each arrow corresponds to gene length (detailed information listed in Table 3). genetic distance is so large that none of the recombinants within the umc1018/umc1753 interval could be further resolved by the newly developed markers based on the contig #261. Only the markers developed from the contig #263 could resolve the recombinants. This indicates that contig #263 is located between contigs #260 and #261, and that the correct contig order must be #260-#263-#261-#262#264 (Figure 3A). This correct contig order has been released (www.maizesequence.org). Searching for recombinants with an unambiguous phenotype is crucial for fine-mapping. Thus, we used only highly susceptible individuals in the F2 mapping population for fine-mapping, which allowed us to fine-map the Scmv1 locus into a 112.39-kb region flanked by R1-2 and STS-11. From the fact that the Scmv1 locus has been repeatedly detected [8,12,14,21], we inferred that Scmv1 is likely present in various resistant inbred lines. Based on this hypothesis, two RIL populations were used to fine-map the Scmv1 locus into a 59.21-kb region flanked by the markers 579P4 and SNP3. The public B73 sequence suggested that two predicted genes, the CAS1-like homolog and Zmtrx-h gene, are the most likely candidates for Scmv1 owing to their potential roles in disease defense response [31,32], although the potential influence of hypothetical proteins in the Scmv1 region on SCMV resistance cannot be excluded. Comparison with the complete CAS1 gene from Arabidopsis thaliana, which encodes 759 residues, Table 3 Analysis of genes predicted from the repeat-masked sequence between the 215.47-kb interval of the markers B4/R1-2 Predicted gene Length of gene First hit E-value Query coverage KEGG entry 1 403aa putative cycloartenol synthase 1.00E-129 76% AT2G07050 2 137aa 60S ribosomal protein L24 7.00E-04 16% AT3G53020 3 119aa thioredoxin H-type 4.00E-64 100% AT5G39950 4 239aa putative transposable element 2.00E-29 89% AT3G22220 5* 360aa putative cycloartenol synthase 1.00E-74 60% AT2G07050 6 618aa sucrose synthase2 4.00E-52 36% AT4G02280 7 342aa salT gene product 3.00E-20 100% AT1G19715 8 93aa no significant similarity NA NA NA 9 330aa hypothetical protein cauri_1913 3.30E+00 53% NA 10 455aa hypothetical protein 2.00E-107 98% NA stands for candidates. First hit, E-value, and Query coverage were BLASTP results of predicted genes from NCBI. KEGG entry is the search word for pathway information about the predicted genes based on GO blast search results. NA means not available. Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 Page 8 of 13 Zmtrx-h CAS1-like-1 A6 R1-2 5 end 3 end 1 O1 5 6 13 14 7-5 3 end 20 21 22 23 2652F IDP11 5 end 25 26 30 r2 1.0 0.8 0.6 0.4 0.1 0.0 Figure 4 Gene structure and LD pattern of the Scmv1 region across 94 maize inbred lines. All polymorphic sites with MAF ≥ 0.05 were used. In the gene diagram, filled black boxes represent exons, and open boxes indicate the UTRs. Dash-dot boxes mark the regions sequenced in this study. Polymorphic sties 1 to 5 are from the sequenced PCR product R1-2, 6 to 13 from A6, 14 to 20 from O1, 21 to 22 from 7–5, 23 to 25 from 2652F, and 26 to 30 from IDP11. The polymorphic sites and their locations on the gene diagram are connected by Dash-dot lines. suggests that the putative CAS1-like gene may be partial gene containing the TERPENE_SYNTHASES motif. CAS1 is involved in biosynthesis of steroids and nonsteroidal secondary metabolites, such as campesterol and stigmasterol [31]. Campesterol is the precursor of brassinosteroids, an important class of plant hormones that functions in cellular signal transduction, whereas the accumulation of stigmasterol stimulates an important plant metabolic process that occurs following bacterial infection of leaves [32]. Given that phenotypic variance associated with changes in the Scmv1 region are influenced by the stage of plant development [19], it remains to be established whether the situation with Scmv1 closely resembles that of Hm2. a truncated duplicate of Hm1. Compared to Hm1, Hm2 preferentially functions in adult plants, conferring resistance against the leaf spot and ear mold disease caused by Cochliobolus carbonumrace 1 [33,34]. Thioredoxin is a master regulator of cellular redox status [35], with h-type thioredoxin reported to function in defense responses to viruses [36] and fungi [37]. Given that the Zmtrx-h protein in the Scmv1 region lacks the conserved WC(G/P)PC motif, which is essential for redox protein activities, it seems unlikely that Zmtrx-h affects cellular redox status. Hence, additional investigation of the roles of the three candidate genes is required to uncover the mechanism of SCMV resistance. Linkage and association analysis are two prevalent approaches to map genes or QTL. Both can be used in a complementary manner to dissect the genetic basis of traits of interest [38,39], as well as to fine-map causative variants of targeted QTL [40]. Six pairs of primers were used to test the association of specific stretches of sequence in the Scmv1 region with SCMV resistance. General linear model (GLM) analysis highlighted three polymorphisms associated with SCMV resistance. The significant association between the PAV and SCMV resistance validates the role of the Scmv1 region with disease resistance, whereas the association between two Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 Page 9 of 13 Table 4 Associations between SCMV resistance and polymorphic sites in the Scmv1 genome region Sites Location Allelesa Frequency P-value FAP1360A/ F7d GLMb SMAc 0.0145 0.0039 G/T S454 3,-end of Zmtrx-h T/G S126 Intron of Zmtrx-h T/C 5/68 0.0371 0.0281 C/C PAV Bin 6.01 0/1 19/75 9.56E-4 2.07E-5 1/1 15/50 S454 was identified from the marker O1, while S126 was identified from the marker 7–5. a Letters (or number) in bold indicate the favourable alleles. b P-value was calculated using GLM in TASSEL 2.0. c P-value was calculated using single marker analysis. d The alleles of inbred lines FAP1360 and F7. other SNPs and SCMV resistance provides statistical support for Zmtrx-h as the primary candidate for Scmv1. Moreover, these two SNP located in the intron and 3′-end of Zmtrx-h, respectively, seemingly having an influence on the gene’s expression. The CAS1-like-2 gene cannot yet be excluded, as there was no primer pair available in the vicinity of it. Therefore, a complementary functional test via transformation is still needed to identify which candidate gene, if any, defines Scmv1. These transgenic experiments are underway in our lab. Broad-spectrum resistance (BSR), resistance against two or more types of pathogen species or the majority of races of the same pathogen species [41], is essential to improve the resistance of crops to various diseases. The quantitative resistance gene Lr34 cloned recently confers resistance to leaf rust, stripe rust, powdery mildew and various other diseases of wheat [42]. The evidence for the presence of multiple disease resistance genes in a maize association population [43] revealed that quantitative BSR can be conferred by a single gene. The Scmv1 region has been reported to associate with resistance to other members of the Potyviridae family, including WSMV and MDMV [44,45]. The near isogenic line F7RR/RR, which was used as a resistant parental line in this study, was also found to be resistant to MDMV and ZeMV [28]. According to a model proposed by Kou [46], a single gene conferring BSR may function in either basal-resistance pathways, in overlapping pathways that confer race-specific resistances, or at sites of cross-talk between different defense pathways. Given that both candidate genes have putative roles in basal resistance, Scmv1 likely confers BSR, which would be advantageous in environments where plants are threatened by multiple pathogens. However, this speculation remains to be verified by artificial inoculation of transgenic plants and recombinants with the appropriate range of pathogens. Conclusion We have fine-mapped Scmv1 to a 59.21-kb region through integrating mapping data from different populations. This provides indispensable data for initiating molecular breeding to improve SCMV resistance in maize, a crop that is critical to world food security. We identified two candidates, of which the Zmtrx-h gene seems the most promising. Map-based cloning of Scmv1 and transgenic analyses will shed light on the molecular mechanism of this important plant-virus interaction. Methods Populations used for fine-mapping The F7 near-isogenic lines, F7RR/RR , F7RR/SS, and F7SS/RR , derived from two early-maturing European maize inbred lines, FAP1360A (completely resistant to SCMV) and F7 (highly susceptible to SCMV), have been developed as described by Ingvardsen [17] (Additional file 5). A total of 2,200 F2 plants derived from the cross of F7RR/RR×F7RR/SS were collected as a mapping population for fine-mapping of Scmv1. Initially, a panel of 177 plants, randomly selected from the F2 mapping population, was used as an unselected mapping population to delimit the Scmv1 region. Each plant was mechanically inoculated with SCMV twice and scored at a weekly interval to evaluate resistance to SCMV. Additional markers in the Scmv1 region were developed to genotype the unselected F2 population. In parallel, the remaining 2,023 F2 plants were investigated for their resistance to SCMV, resulting in the identification of 510 plants with typical SCMV symptoms indistinguishable from their susceptible parent F7. These 510 susceptible F2 plants, together with 28 recombinants from the unselected F2 mapping population, formed the F2 sub-population for fine-mapping of Scmv1. Validation of the Scmv1 locus in two RIL populations Two single-seed descent F6 RIL populations were employed to validate and further fine map the Scmv1 locus. One was developed from the cross between Zheng58 (susceptible to SCMV) and Chang7-2 (resistant to SCMV), a commercial hybrid (ZD958) that is currently widely cultivated in China. The other RIL population was derived from the cross between X178 (completely resistant to SCMV) and HuangC (highly susceptible to SCMV), another commercial hybrid (ND108) widely grown in China. A total of 2,206 F6 RILs (1,285 from Zheng58×Chang7-2 and 921 from X178×HangC) were planted in the experimental station at Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 Shangzhuang, Beijing, during the summer of 2010. All RILs were genotyped using the markers R1-2 and B4 flanking the Scmv1 locus, as well as a co-segregating marker DJF004 at the Scmv2 locus [17]. For each RIL population, four sets of 30 RILs, which differed at the Scmv1 or Scmv2 locus or both loci, were separately selected for phenotypic evaluation, whereas all recombinants between R1-2 and B4 were investigated for their SCMV resistance. Artificial inoculation Seeds were sown in a soil/vermiculite mixture in 0.80-L pots maintained under greenhouse conditions. Virus inocula (Chinese isolate) were prepared from the SCMV-infected susceptible F7 plants. Young leaf tissue with typical mosaic symptoms was homogenized in five volumes of 0.01 M sodium phosphate buffer, pH 7.0. Carborundum was added to the sap to facilitate rubinoculation. All leaves of each plant were rub-inoculated at the three- to four-leaf stage. One week after inoculation, all the symptomless plants were re-inoculated to avoid any escape. During sap preparation and mechanical inoculation, the inocula were put on ice to maintain low-temperature conditions. For phenotypic evaluation of inbred lines and selected RILs, 12 kernels of each line were planted in four 0.80-L seeding pots. Whereas 10 plants were rub-inoculated with virus inoculum, two plants were not inoculated. These RILs were evaluated along with their parent lines as positive and negative controls for all tests. RILs with the same symptoms (P > 0.05, t-test) as the susceptible parent or the resistant parent were regarded as susceptible (S) or resistant (R). Three independent experiments were performed during September 2010 to July 2011 to ensure accurate phenotyping. DNA extraction and molecular analyses Immature leaf samples were harvested from each individual and immediately frozen in liquid nitrogen. DNA was extracted using the SDS method described by Murray and Thompson [47]. Apart from new markers developed from public BAC sequences, all SSR markers were retrieved from the Maize Genetics and Genomics Database (www.maizegdb.org) (Additional file 1) and synthesized by Invitrogen (Beijing, China). To design specific markers for fine-mapping, we first anchored the QTL in bin 6.01 to the B73 BAC-based whole-genome physical map (http://www.maizesequence. org/) using flanking SSR markers. We next extracted BACs, expressed sequence tags, and Indel polymorphic sequences (http://www.ncbi.nlm.nih.gov/; http://magi.plantgenomics. iastate.edu/browseMarkers.do) available in the targeted region to develop new polymorphic markers (SSR, STS, SNP, and CAPS). The retrieved sequences were analyzed Page 10 of 13 with RepeatMasker (http://www.repeatmasker.org/cgi-bin/ WEBRepeatMasker) followed by BLAST analysis against maize high-throughput genome sequence and genome sequence survey to remove redundant sequences [48]. All primers for the single/low-copy sequences were designed using the software PRIMER 5.0 with the criteria that the primer length should be 20 nucleotides, with a 40–60% GC content, no secondary structure, and no consecutive tracts of the same nucleotide. The microsatellite search tool SSRHunter1.3 [49] was used to mine SSR sequences of 4–6 tandem copies of >2 base pairs. For PCR-mediated detection of SSR and STS markers, reactions were prepared in a 10-μL reaction volume containing 100 ng of genomic DNA, 1 μL of 10× PCR buffer, 0.2 mM of each dNTP, 0.2 μM of each forward and reverse primer, and 1 U of Taq polymerase. To detect SNP and CAPS markers, 50 μL reactions contained 300 ng of template DNA, 5 μL of 10× PCR buffer, 1 mM of each dNTP, 1 μM of each forward and reverse primer, and 5 U Taq polymerase (Beijing Transgen Biotech Co.LTD). All PCR reactions were performed using a PTC-200 Peltier Thermal Cycler (MJ Research Inc., Canada) with particular annealing temperature and elongation duration adapted to each reaction. The PCR products were subjected to electrophoresis using either 1% agarose gels (visualized using a gel imaging system from BioRad Laboratories Inc., USA) or 6% polyacrylamide gels (amplification products were visualized by silver staining). SNP markers were sequenced at the Beijing Genomic Institute. Analysis of candidate genes To predict candidate genes, we retrieved the DNA sequence of the Scmv1 region from the B73 wholegenome sequence (http://www.maizesequence.org) for candidate gene prediction. The repetitive sequence in the Scmv1 region was analyzed by RepeatMasker version 3.3.0 (http://www.repeatmasker.org). We used the Fgenesh program (http://linux1.softberry.com/berry.phtml, version 2.6) to predict genes present in the single/lowcopy Scmv1 region. For functional analysis, the deduced amino-acid sequence of each putative gene was compared against the “nr” database via BLASTP on National Center of Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/). Detailed GO and KEGG annotation was applied to the gene sequences predicted by BLAST searches (http://www.geneontology.org/ and http://www.genome.jp/kegg/). Association mapping of the Scmv1 region A total of 94 maize inbred lines were phenotyped in two distinct experiments (October 2007, May 2010) under greenhouse conditions using the same artificial inoculation Tao et al. BMC Plant Biology 2013, 13:162 http://www.biomedcentral.com/1471-2229/13/162 procedure as RIL populations. The same 94 inbred lines were genotyped using a set of 70 SSR markers that were evenly distributed throughout the maize genome (Additional file 6). Based on the B73 sequence and using Primer 3 (http://frodo.wi.mit.edu/) or Premier 5.00 (PREMIER Biosoft International, USA), we designed 17 pairs of PCR primers (Additional file 7) flanking the three candidate genes. We used these primers to first screen for polymorphisms between F7, FAP1360A, Zheng58, Chang7-2, HuangC, and X178. Most primer pairs were discarded because of unreliable amplification. Six primer pairs with polymorphic products were used to amplify these 94 inbred lines to identify potential polymorphic sites. Of these six polymorphic amplicons, two and three are located in the vicinity of CAS1-like-1 and Zmtrx-h, respectively, whereas no primer pair around CAS1-like-2 was available. Nucleotide sequences alignments were done using the multiple sequence alignment software MUSCLE [50] and subsequently refined manually. Q was inferred using STRUCTURE 2.2 [51,52] with 70 SSR markers. Five independent runs were performed setting the number of populations (k) from 1 to 10, burn in time and MCMC (Markov Chain Monte Carlo) replication number both to 500,000, and a model for admixture and correlated allele frequencies. The k value was determined by LnP(D) in STRUCTURE output and an ad hoc statistic Δk based on the rate of change in LnP(D) between successive k. Lines with membership probabilities ≥ 0.75 were assigned to corresponding clusters; lines with membership probabilities < 0.75 were assigned to a mixed group. Groups were further subdivided into subgroups using a similar methodology. The runs most consistent with breeder’s knowledge about pedigree were used to assign lines into clusters. The levels of LD between two sites were calculated using TASSEL2.0 [53]. The associations between the extracted polymorphic sites with Minor Allele Frequency (MAF) ≥ 0.05 and SCMV resistance were carried out using GLM in TASSEL2.0. Additional files Additional files 1: The list of public SSR markers for mapping of Scmv1. Additional files 2: The 94 maize inbred lines and their resistance performance to SCMVR means resistance to SCMV, while S means susceptible to SCMV. Additional files 3: Population structure of 94 maize inbred lines estimated using 70 SSRs. Population structure was assessed by STRUCTURE. Each individual is represented by a vertical bar, partitioned into colored segments with the length of each segment representing the proportion of the individual’s genome from k = 6 groups. For all classes, a given group is represented: red, REID; Blue, P; green, LAN; Yellow, TSPT; pink, Z330; turquoise, OTHER. Additional files 4: Associations between SCMV resistance and polymorphic sites in the Scmv1 genome region. a: P-value calculated using GLM. #: polymorphic sites associated with SCMV resistance. Page 11 of 13 Additional files 5: The procedure of developing the Scmv1 mapping population. Additional files 6: List of SSR markers for structure and kinship analysis. Additional files 7: Primers designed for association mapping. : The PCR products amplified by these primers contained polymorphic sites that could be used for association mapping. Abbreviations BAC: Bacterial artificial chromosome; BSR: Broad-spectrum resistance; CAS: Cycloartenol synthase1-like; GO: Gene ontology; K: Kinship; KEGG: Kyoto encyclopedia of genes and genomes; LD: Linkage disequilibrium; MAF: Minor Allele Frequency; JGMV: Johnson grass mosaic virus; GLM: General linear model; PCR: Polymerase chain reaction; Q: Population structure; PAV: Presence/absence variations; QTL: Quantitative trait loci; RIL: Recombinant inbred line; SCMV: Sugarcane mosaic virus; SNP: Single nucleotide polymorphism; SrMV: Sorghum mosaic virus; SSR: Simple sequence repeat; WSMV: Wheat streak mosaic virus; ZeMV: Zea mosaic virus. Competing interests The authors declare that they have no competing interests. Authors’ contributions LJ, QL, CRI and UKF performed mapping experiment in segregating population. BW and JL provided two RIL populations. YT, QL, carried out fine-mapping in these two RIL populations. YZ, RZ and YT completed the association mapping work. MX and TL supervised the research, designed the experiments and were involved in data analysis. YT wrote the manuscript draft, MX and TL edited and revised the manuscript. All authors read and approved the final manuscript. Acknowledgements This study was financially supported by the NSFC (31371625), Ministry of Agriculture of China (2011ZX08009-003-001), National High-tech and Development Program of China (2012AA10A306 and 2012AA101104). We thank Dr. Xiaohong Yang at the China Agricultural University for critical guidance of association analysis in this study. Author details 1 National Maize Improvement Center, China Agricultural University, 2 West Yuanmingyuan Road, Beijing 100193, People’s Republic of China. 2 Department of Agronomy, Iowa State University, 1204 Agronomy Hall, Ames, Iowa 50011, USA. 3Research Center Flakkebjerg, University of Aarhus, 4200, Slagelse, Denmark. Received: 1 August 2013 Accepted: 9 October 2013 Published: 18 October 2013 References 1. 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https://openalex.org/W2750999242	https://hal-obspm.ccsd.cnrs.fr/obspm-04001840/file/atoms-05-00027.pdf	Latin	null	Semiclassical Stark Broadening Parameters of Ar VII Spectral Lines	Atoms	2,017	cc-by	2,976	y Received: 16 August 2017; Accepted: 6 September 2017; Published: 8 September 2017 Received: 16 August 2017; Accepted: 6 September 2017; Published: 8 September 2017 Abstract: Using the semi-classical perturbation approach in the impact approximation, full width at half maximum and shift have been determined for eight spectral lines of Ar VII, for broadening by electron-, proton-, and He III-impacts. The results are provided for temperatures from 20,000 K to 500,000 K, and for an electron density of 1018 cm−3. The obtained results will be included in the STARK-B database, which is also in the virtual atomic and molecular data center (VAMDC). Keywords: stark broadening; atomic data; atomic processes; line proﬁles; Ar VII 1. Introduction With the development of space astronomy and satellite-born spectroscopy, trace elements—which have been without importance for astrophysics—now become increasingly important, and the corresponding data of interest for the analysis of stellar spectra. For example, spectral lines of Ar VII have been observed in the spectrum of extremely hot and massive galactic O3 If supergiant HD 93129A [1]. Additionally, Werner et al. [2] have found Ar VII lines in some of the hottest known central stars of planetary nebulae, with the effective temperatures of 95,000–110,000 K, and in (pre-) white dwarfs by analyzing high-resolution spectra from the Far Ultraviolet Spectroscopic Explorer (FUSE). We note that Stark broadening is the principal pressure broadening mechanism in such hot stars, and without the corresponding Stark broadening data, reliable analysis and modelling of high-resolution spectra are not possible. Concerning Stark broadening parameters for Ar VII spectral lines, there is only one article where Stark broadening parameters are provided for three transitions [3]. Here, full widths at half intensity maximum (FWHM) W and shifts d for eight additional transitions have been calculated by using semiclassical perturbation method (SCP, [4,5]) for collisions of Ar VII ions with electrons, protons, and He III ions, since hydrogen and helium are the main constituents of stellar atmospheres. Article Milan S. Dimitrijevi´c 1,2,† ID , Aleksandar Valjarevi´c 3,† ID and Sylvie Sahal-Bréchot 2,,† ID 1 Astronomical Observatory, Volgina 7, 11060 Belgrade 38, Serbia; mdimitrijevic@aob.rs 2 LERMA, Observatoire de Paris, PSL Research University, CNRS, Sorbonne Universités, UPMC (Univ. Pierre & Marie Curie) Paris 06, 5 Place Jules Janssen, 92190 Meudon, France 3 Department of Geography, Faculty of Natural Sciences and Mathematics, University of Kosovska Mitrovica, Ive Lole Ribara 29, 38220 Kosovska Mitrovica, Serbia; aleksandar.valjarevic@pr.ac.rs Correspondence: sylvie.sahal-brechot@obspm.fr; Tel.: +381-64-297-8021 † These authors contributed equally to this work. Academic Editor: Robert C Forrey Milan S. Dimitrijevi´c 1,2,† ID , Aleksandar Valjarevi´c 3,† ID and Sylvie Sahal-Bréchot 2,,† ID LERMA, Observatoire de Paris, PSL Research University, CNRS, Sorbonne Universités, UPMC (Univ. Pierre & Marie Curie) Paris 06, 5 Place Jules Janssen, 92190 Meudon, France 3 Department of Geography, Faculty of Natural Sciences and Mathematics, University of Kosovska Mitrovica, Ive Lole Ribara 29, 38220 Kosovska Mitrovica, Serbia; aleksandar.valjarevic@pr.ac.rs C d l i h l b h @ b f T l 381 64 297 8021 Academic Editor: Robert C. Forrey 2. The Impact Semiclassical Perturbation Method The semiclassical perturbation formalism (SCP) applied here for the calculations of Stark broadening parameters, full width at half intensity maximum (FWHM-W), and shift of spectral line (d) has been formulated in [4,5], and later updates, optimisations, and innovations are presented in Sahal-Bréchot [6], Sahal-Bréchot [7], Dimitrijevi´c et al. [8], Dimitrijevi´c and Sahal-Bréchot [9], and Sahal-Bréchot et al. [10]. Within the frame of this method, FWHM (W) and shift (d) may be expressed by the following relation: Atoms 2017, 5, 27; doi:10.3390/atoms5030027 www.mdpi.com/journal/atoms 2 of 5 Atoms 2017, 5, 27 Atoms 2017, 5, 27 W = N Z v f (v)dv  ∑ i′̸=i σii′(v) + ∑ f ′̸= f σf f ′(v) + σel   d = N Z v f (v)dv Z RD R3 2πρdρ sin(2ϕp). (1) (1) Here, N is electron density, f (υ) the Maxwellian velocity distribution function for electrons, ρ is the impact parameter of the incoming electron, and with i′, f ′ are denoted the perturbing levels of the initial (i) and ﬁnal (f) state. The inelastic cross-section σjj′(υ), j = i, f is expressed as: ∑ i′̸=i σii′(υ) = 1 2πR2 1 + Z RD R1 2πρdρ ∑ i′̸=i Pii′(ρ, υ), (2) (2) where Pjj′(ρ, υ), j = i, f; j′ = i′, f ′ is the transition probability. The elastic cross-section is where Pjj′(ρ, υ), j = i, f; j′ = i′, f ′ is the transition probability. The elastic cross-section is σel = 2πR2 2 + Z RD R2 2πρdρ sin2 δ + σr, σel = 2πR2 2 + Z RD R2 2πρdρ sin2 δ + σr, δ = (ϕ2 p + ϕ2 q) 1 2 . (3) (3) The phase shift due to the polarization potential is ϕp (r−4), and due to the quadrupolar potential ϕq (r−3) (see Section 3 of Chapter 2 in Sahal-Bréchot [4]). R1, R2, R3, and RD are cut-offs, deﬁned and described in Section 1 of Chapter 3 in Sahal-Bréchot [5]. σr denotes the contribution of Feshbach resonances, explained in detail in [11]. A review of the theory, all approximations and details of applications is given in Sahal-Bréchot et al. [10]. 3. Stark Broadening Parameter Calculations By using Equations (1)–(3) we have calculated widths (FWHM) and shifts for eight transitions (triplets) in Ar VII spectrum. The necessary atomic energy levels have been taken from Saloman [12]. The oscillator strengths—needed for calculations—have been obtained by using the method of Bates and Damgaard [13] and the tables of Oertel and Shomo [14]. When there was no corresponding data in Oertel and Shomo [14] (for some higher levels), the needed oscillator strengths have been calculated using the method of Van Regemorter et al. [15]. In Table 1 are shown widths (FWHM) and shifts of Ar VII spectral lines broadened by electron-, proton-, and He III-impacts, for a perturber density of 1018 cm−3 and for a set of temperatures from 20,000 K to 500,000 K. The temperature range is of interest for astrophysics, laboratory plasmas, fusion research, various plasmas in technology and laser-produced plasmas. If we want to use these data for higher perturber densities, the inﬂuence of Debye screening should be checked and taken into account if needed (e.g., [16]). g The accuracy of the semiclassical perturbation method is estimated by comparison with numerous experimental data for different elements and spectral lines, and is estimated to be 20–30% (see discussion in [10]). Since Ar VII is a member of the magnesium isoelectronic sequence with relatively simple spectrum, we suppose that the error of results shown in Table 1 is not much higher than 20%. 3 of 5 Atoms 2017, 5, 27 Table 1. Electron-, proton-, and doubly charged helium-impact broadening parameters for Ar VII spectral lines, for a perturber density of 1018 cm−3 and temperatures from 20,000 to 500,000 K. The calculated wavelength of the transitions (in Å) and parameter C are also given. When divided by the corresponding Stark width, this parameter gives an estimate for the maximal perturber density for which the line may be treated as isolated. We: electron-impact full width at half maximum of intensity; de: electron-impact shift; Wp: proton-impact full width at half maximum of intensity; dp: proton-impact shift; WHe++: doubly charged helium ion-impact full width at half maximum of intensity; dHe++: doubly charged helium ion-impact shift. 3. Stark Broadening Parameter Calculations Transition T(K) We (Å) de (Å) WH+ (Å) dH+ (Å) WHe++ (Å) dHe++ (Å) 4s 3S −5p 3Po 20,000 0.510E−01 0.174E−03 0.298E−03 0.197E−04 0.492E−03 0.272E−04 443.2 Å 50,000 0.328E−01 0.217E−03 0.898E−03 0.636E−04 0.173E−02 0.117E−03 C = 0.47E+20 100,000 0.243E−01 0.158E−03 0.148E−02 0.129E−03 0.291E−02 0.249E−03 200,000 0.186E−01 0.215E−03 0.203E−02 0.223E−03 0.403E−02 0.444E−03 300,000 0.162E−01 0.211E−03 0.220E−02 0.275E−03 0.439E−02 0.550E−03 500,000 0.139E−01 0.227E−03 0.242E−02 0.352E−03 0.482E−02 0.709E−03 3p 3Po −4s 3S 20,000 0.490E−02 −0.779E−03 0.744E−06 0.135E−04 0.129E−05 0.186E−04 250.4 Å 50,000 0.270E−02 0.615E−04 0.642E−05 0.432E−04 0.124E−04 0.792E−04 C = 0.32E+20 100,000 0.191E−02 0.113E−03 0.295E−04 0.835E−04 0.579E−04 0.161E−03 200,000 0.142E−02 0.131E−03 0.819E−04 0.133E−03 0.163E−03 0.265E−03 300,000 0.121E−02 0.143E−03 0.113E−03 0.162E−03 0.227E−03 0.323E−03 500,000 0.101E−02 0.136E−03 0.173E−03 0.196E−03 0.347E−03 0.396E−03 3p 3Po −3d 3D 20,000 0.911E−02 −0.564E−03 0.361E−05 −0.107E−05 0.598E−05 −0.148E−05 477.5 Å 50,000 0.585E−02 −0.436E−04 0.148E−04 −0.348E−05 0.282E−04 −0.639E−05 C = 0.48E+21 100,000 0.417E−02 −0.288E−04 0.396E−04 −0.740E−05 0.767E−04 −0.143E−04 200,000 0.297E−02 −0.165E−04 0.900E−04 −0.150E−04 0.176E−03 −0.298E−04 300,000 0.245E−02 −0.134E−04 0.129E−03 −0.220E−04 0.253E−03 −0.438E−04 500,000 0.195E−02 −0.237E−04 0.177E−03 −0.333E−04 0.349E−03 −0.669E−04 3p 3Po −4d 3D 20,000 0.421E−02 −0.445E−03 0.671E−05 0.621E−05 0.111E−04 0.858E−05 192.3 Å 50,000 0.249E−02 0.896E−05 0.263E−04 0.200E−04 0.506E−04 0.366E−04 C = 0.94E+19 100,000 0.181E−02 0.259E−04 0.589E−04 0.394E−04 0.115E−03 0.760E−04 200,000 0.135E−02 0.428E−04 0.100E−03 0.646E−04 0.199E−03 0.128E−03 300,000 0.115E−02 0.345E−04 0.127E−03 0.782E−04 0.252E−03 0.156E−03 500,000 0.951E−03 0.379E−04 0.155E−03 0.971E−04 0.308E−03 0.196E−03 4p 3Po −4d 3D 20,000 0.309 −0.849E−02 0.811E−03 0.262E−03 0.134E−02 0.362E−03 1425.9 Å 50,000 0.199 0.324E−03 0.292E−02 0.845E−03 0.562E−02 0.155E−02 C = 0.52E+21 100,000 0.145 0.336E−03 0.577E−02 0.169E−02 0.113E−01 0.327E−02 200,000 0.108 0.126E−02 0.877E−02 0.286E−02 0.173E−01 0.569E−02 300,000 0.927E−01 0.595E−03 0.106E−01 0.346E−02 0.211E−01 0.691E−02 500,000 0.777E−01 0.645E−03 0.120E−01 0.440E−02 0.237E−01 0.886E−02 3d 3D −4p 3Po 20,000 0.184E−01 −0.111E−02 0.352E−04 0.757E−05 0.584E−04 0.105E−04 416.0 Å 50,000 0.116E−01 0.474E−04 0.133E−03 0.245E−04 0.256E−03 0.450E−04 C = 0.87E+20 100,000 0.835E−02 0.115E−03 0.286E−03 0.513E−04 0.560E−03 0.991E−04 200,000 0.612E−02 0.105E−03 0.469E−03 0.946E−04 0.926E−03 0.188E−03 300,000 0.518E−02 0.121E−03 0.577E−03 0.126E−03 0.114E−02 0.251E−03 500,000 0.427E−02 0.140E−03 0.679E−03 0.161E−03 0.135E−02 0.323E−03 3d 3D −5p 3Po 20,000 0.139E−01 −0.555E−03 0.900E−04 0.185E−04 0.148E−03 0.256E−04 240.6 Å 50,000 0.868E−02 0.137E−03 0.271E−03 0.587E−04 0.523E−03 0.108E−03 C = 0.14E+20 100,000 0.642E−02 0.162E−03 0.445E−03 0.109E−03 0.877E−03 0.211E−03 200,000 0.490E−02 0.190E−03 0.616E−03 0.166E−03 0.122E−02 0.331E−03 300,000 0.425E−02 0.199E−03 0.669E−03 0.204E−03 0.133E−02 0.406E−03 500,000 0.362E−02 0.199E−03 0.739E−03 0.237E−03 0.147E−02 0.479E−03 4d 3D −5p 3Po 20000 0.243 −0.224E−03 0.163E−02 0.134E−03 0.269E−02 0.185E−03 952.0 Å 50,000 0.158 0.166E−02 0.477E−02 0.431E−03 0.923E−02 0.790E−03 C = 0.22E+21 100,000 0.118 0.174E−02 0.767E−02 0.856E−03 0.151E−01 0.165E−02 200,000 0.902E−01 0.184E−02 0.104E−01 0.142E−02 0.207E−01 0.283E−02 300,000 0.786E−01 0.221E−02 0.113E−01 0.172E−02 0.224E−01 0.343E−02 500,000 0.672E−01 0.208E−02 0.124E−01 0.217E−02 0.246E−01 0.437E−02 Si th l th i T bl 1 l l t d th diff t f i t Since the wavelengths in Table 1 are calculated ones, they are different from experimental wavelengths. Since the wavelengths in Table 1 are calculated ones, they are different from experimental wavelengths. However, we notice that they are correct in angular frequency units, because in such 3. Stark Broadening Parameter Calculations However, we notice that they are correct in angular frequency units, because in such 17, 5, 27 4 of 5 4 of 5 Atoms 2017, 5, 27 a case, relative and not absolute positions of energy levels are important for calculations. For the transformation of the Stark widths from Å-units to angular frequency units, the following formula can be used: a case, relative and not absolute positions of energy levels are important for calculations. For the transformation of the Stark widths from Å-units to angular frequency units, the following formula can be used: W( ˚A) = λ2 2πcW(s−1) (4) (4) where c is the speed of light. For the correction of widths and/or shifts for the difference between calculated and experimental wavelength, one can use the expression: where c is the speed of light. For the correction of widths and/or shifts for the difference between calculated and experimental wavelength, one can use the expression: Wcor = λexp λ 2 W. (5) (5) The corresponding expressions for the shifts are analogous to Equations (4) and (5). Here, Wcor denotes the corrected width, while λexp is the experimental wavelength, λ the calculated wavelength, and W the width from Table 1. Dividing the parameter C [17] from Table 1 by the corresponding full width at half maximum, one obtains the maximal perturber density for which the line may be treated as isolated. The obtained Ar VII Stark broadening parameters shown in Table 1 will be implemented in the STARK-B database [18,19]. This database contains Stark widths and shifts needed ﬁrst of all for the investigations, modelling, and diagnostics of the plasma of stellar atmospheres, but also for diagnostics of laboratory plasmas and investigation of laser produced, inertial fusion plasma, and for plasma technologies. The STARK-B database is one of the databases included in the virtual atomic and molecular data center—VAMDC [20,21], created in order to enable more effective search and mining of atomic and molecular data which are in different databases. Databases with atomic and molecular data which are in VAMDC—including STARK-B—can be accessed and searched through the VAMDC portal: http://portal.vamdc.org/. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Taresch, G.; Kudritzki, R.P.; Hurwitz, M.; Bowyer, S.; Pauldrach, A.W.A.; Puls, J.; Butler, K.; Lennon, D.J.; Haser, S.M. Quantitative analysis of the FUV, UV and optical spectrum of the O3 star HD 93129A. Astron. Astrophys. 1997, 321, 531–548. 2. Werner, K.; Rauch, T.; Kruk, J.W. Discovery of photospheric argon in very hot central stars of planetary nebulae and white dwarfs. Astron. Astrophys. 2007, 466, 317–322. 3. Dimitrijevi´c, M.S.; Sahal-Bréchot, S. On the Stark broadening of Ar VII spectral lines. Univ. Thought 2017, 7, 46–50. 4. Sahal-Bréchot, S. Impact theory of the broadening and shift of spectral lines due to electrons and ions in a plasma. Astron. Astrophys. 1969, 1, 91–123. 5. Sahal-Bréchot, S. Impact theory of the broadening and shift of spectral lines due to electrons and ions in a plasma (continued). Astron. Astrophys. 1969, 2, 322–354. 6. Sahal-Bréchot, S. Stark broadening of isolated lines in the impact approximation. Astron. Astrophys. 1974, 35, 319–321. 7. Sahal-Bréchot, S. Broadening of ionic isolated lines by interactions with positively charged perturbers in the quasistatic limit. Astron. Astrophys. 1991, 245, 322–330. 8. Dimitrijevi´c, M.S.; Sahal-Bréchot, S.; Bommier, V. Stark broadening of spectral lines of multicharged ions of astrophysical interest. I-C IV lines. Astron. Astrophys. 1991, 89, 581–590. 9. Dimitrijevi´c, M.S.; Sahal-Bréchot, S. Stark broadening of Li II spectral lines. Phys. Scr. 1996, 54, 50–5 10. Sahal-Bréchot, S.; Dimitrijevi´c, M.S.; Ben Nessib, N. Widths and Shifts of Isolated Lines of Neutral and Ionized Atoms Perturbed by Collisions with Electrons and Ions: An Outline of the Semiclassical Perturbation (SCP) Method and of the Approximations Used for the Calculations. Atoms 2014, 2, 225–252. 1. Fleurier, C.; Sahal-Bréchot, S.; Chapelle, J. Stark proﬁles of some ion lines of alkaline earth elements. J. Qu Spectrosc. Radiat. Transf. 1977, 17, 595–603. 12. Saloman, E.B. Energy Levels and Observed Spectral Lines of Ionized Argon, Ar II through Ar XVIII. J. Phys. Chem. Ref. Data 2010, 39, 033101. 13. Bates, D.R.; Damgaard, A. The Calculation of the Absolute Strengths of Spectral Lines. Philos. Trans. R. Soc. Lond. Ser. A Math. Phys. Sci. 1949, 242, 101–122. 14. Oertel, G.K.; Shomo, L.P. Tables for the Calculation of Radial Multipole Matrix Elements by the Coulomb Approximation. Astrophys. J. Suppl. Ser. 1968, 16, 175–218. pp p y pp 15. Van Regemorter, H.; Hoang Binh, D.; Prud’homme, M. Radial transition integrals involving low or high effective quantum numbers in the Coulomb approximation. J. Phys. 4. Conclusions The semiclassical perturbation calculation of Stark broadening parameters, widths, and shifts for spectral lines broadened by collisions of Ar VII ions with electrons, protons, and doubly charged helium ions have been performed for eight multiplets of Ar VII. The obtained values of Stark broadening parameters will be implemented in the STARK-B database—one of the databases included in the virtual atomic and molecular data center (VAMDC). Since Stark broadening data for Ar VII spectral lines considered here do not exist in the literature, we hope that they will be of interest for the relevant problems in astrophysical, laboratory, laser-produced, inertial fusion, and technological plasmas. Acknowledgments: The support of the Ministry of Education, Science and Technological Development of the Republic of Serbia through project 176002 is gratefully acknowledged. This work has also been supported by the Paris Observatory, the PADC (Paris Data Center_OV), and the CNRS which are greatly acknowledged. We also acknowledge ﬁnancial support from “Programme National de Physique Stellaire” (PNPS) of CNRS/INSU, CEA and CNES, France. This work has also been supported by the VAMDC (Virtual Atomic and Molecular Data Centre). VAMDC is funded under the Combination of Collaborative Projects and Coordination and Support Actions Funding Scheme of The Seventh Framework Program. Call topic: INFRA-2008-1.2.2 Scientiﬁc Data Infrastructure, Grant Agreement number: 239108. A part of this work has also been supported by the LABEX Plas@par project and received ﬁnancial state aid managed by the Agence Nationale de la Recherche, as part of the programme “Investissements d’Avenir” under the reference, ANR11IDEX000402. Author Contributions: These authors contributed equally to this work. 5 of 5 Atoms 2017, 5, 27 c⃝2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References B 1979, 12, 1053–1061. 16. Griem, H.R. Spectral Line Broadening by Plasmas; Academic Press, Inc.: New York, NY, USA, 1974. 17. Dimitrijevi´c, M.S.; Sahal-Bréchot, S. Stark broadening of neutral helium lines. J. Quant. S Radiat. Transf. 1984, 31, 301–313. 18. Sahal-Bréchot, S.; Dimitrijevi´c, M.S.; Moreau, N. STARK-B Database, Observatory of Paris, LERMA and Astronomical Observatory of Belgrade, 2017. Available online: http://stark-b.obspm.fr (accessed on 24 August 2017). 19. Sahal-Bréchot, S.; Dimitrijevi´c, M.S.; Moreau, N.; Ben Nessib, N. The STARK-B database VAMDC node: A repository for spectral line broadening and shifts due to collisions with charged particles. Phys. Scr. 2015, 50, 054008. 20. Dubernet, M.L.; Boudon, V.; Culhane, J.L.; Dimitrijevic, M.S.; Fazliev, A.Z.; Joblin, C.; Kupka, F.; Leto, G.; Le Sidaner, P.; Loboda, P.A.; et al. Virtual atomic and molecular data centre. J. Quant. Spectrosc. Radiat. Transf. 2010, 111, 2151–2159. 21. Dubernet, M.L.; Antony, B.K.; Ba, Y.A.; Babikov, Y.L.; Bartschat, K.; Boudon, V.; Braams, B.J.; Chung, H.K.; Daniel, F.; Delahaye, F.; et al. The virtual atomic and molecular data centre (VAMDC) consortium. J. Phys. B 2016, 49, 074003. c⃝2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W1969087669	https://www.scielo.br/j/rbz/a/6YS5ShSYwLSFjfzLDrXzsxw/?lang=pt&format=pdf	Portuguese	null	Repetibilidade de caracteres agronômicos em Panicum maximum Jacq.	Revista Brasileira de Zootecnia	2,007	cc-by	5,026	Janaina Azevedo Martuscello2, Liana Jank3, Dilermando Miranda da Fonseca4, Cosme Damião Cruz5, Daniel de Noronha Figueiredo Vieira da Cunha2 1 Parte da tese da primeira autora. Financiamento: Unipasto e CNPq. 2 Doutorando do Departamento de Zootecnia – Universidade Federal de Viçosa. Bolsista do CNPq. 3 Embrapa Gado de Corte. Bolsista de Produtividade em Desenvolvimento Tecnológico e Extensão Inovadora do CNPq. 4 Departamento de Zootecnia – Universidade Federal de Viçosa. 5 Departamento de Biologia Geral – Universidade Federal de Viçosa. 1 Parte da tese da primeira autora. Financiamento: Unipasto e CNPq. 2 Doutorando do Departamento de Zootecnia – Universidade Federal de Viçosa. Bolsista do CNPq. 3 Embrapa Gado de Corte. Bolsista de Produtividade em Desenvolvimento Tecnológico e Extensão Inovadora do CNPq. 4 Departamento de Zootecnia – Universidade Federal de Viçosa. 5 Departamento de Biologia Geral – Universidade Federal de Viçosa. RESUMO - O experimento foi conduzido para avaliar o coeficiente de repetibilidade de algumas características agronômicas em híbridos de Panicum maximum. Dez parcelas de plantas sexuais da espécie foram aleatoriamente distribuídas entre as 230 parcelas de acessos apomíticos. Após a fecundação natural, sementes de cada planta sexual constituíram-se uma família de meios-irmãos. Trinta plantas de cada progenitora foram avaliadas em delineamento de blocos ao acaso, com cinco plantas por parcela e seis repetições. A partir de cinco cortes, estimou-se o coeficiente de repetibilidade pelos métodos da Análise de Variância, Componentes principais (matriz de correlação e covariância) e Análise estrutural. Os coeficientes de repetibilidade (r) nos diferentes métodos, para todas as características avaliadas, oscilaram entre 0,51 e 0,86 e podem ser considerados altos, assim como os coeficientes de determinação. As estimativas do coeficiente de repetibilidade obtidas para as quatro características avaliadas pelo método da análise de variância foram quase sempre menores que as obtidas pelos demais métodos. Pelo método dos componentes principais (baseado na matriz de covariância), as estimativas foram sempre maiores em relação aos demais métodos. Considerando satisfatórios níveis de 80 ou 90% de confiabilidade para avaliação da superioridade relativa dos híbridos para todas as características avaliadas, as cinco medições realizadas são suficientes para escolha da melhor planta. A exclusão dos cortes 1 e 2 promove aumento nos coeficientes de repetibilidade e determinação. Palavras-chave: análise estrutural, análise de variância, componentes principais, produção de matéria seca Agronomic characters repeatability in Panicum maximum Jacq. ABSTRACT - The experiment was carried out to evaluate the coefficient of repeatability of some agronomic characteristics in Panicum maximum hybrids. Ten plots of sexual plants of this species were randomly distributed among 230 plots of apomitic accessions. After natural hybridization, seeds of each sexual plant constituted a half-sib family. Thirty plants of each female progenitor were evaluated in a randomized blocks design, with five plants per plot and six replications. Based on five evaluation cuts, the coefficient of repeatability was estimated by the methods analysis of variance, principal components (correlation and covariance matrices) and structural analyses. The coefficient of repeatability (r) estimated by the different methods, for all the characteristics evaluated, was high and varied from 0.51 to 0.86. The estimates of the coefficient of determination were also high. It was verified that the estimates of the repeatability coefficient for the four characteristics evaluated by the analyses of variance method were almost always smaller than the estimates obtained by the other methods. By the principal component method (based on the covariance matrix), the estimates were always the highest. Considering a level of 80 or 90% as satisfactory for the confidence in the decision of the relative superiority of the hybrids based on green matter yield, these five evaluation cuts would be sufficient for the choice of the best plant. The exclusion of the first two cuts will increase the repeatability and determination coefficients. Key Words: analysis of variance, dry matter production, principal components, structural analysis Correspondências devem ser enviadas para: jazevedom@bol.com.br Revista Brasileira de Zootecnia © 2007 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br Revista Brasileira de Zootecnia © 2007 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br R. Bras. Zootec., v.36, n.6, p.1975-1981, 2007 (supl.) Material e Métodos O experimento foi conduzido na Embrapa Gado de Corte no município de Campo Grande, Mato Grosso do Sul, a 20°27' de latitude e 54°57' de longitude.O clima, segundo a classificação de Köppen, é do tipo tropical chuvoso de savana, subtipo Aw, caracterizado por distribuição anual irregular das chuvas e pela ocorrência bem definida do período seco durante os meses frios e do período chuvoso durante o verão. Os 250 acessos do banco de germoplasma de Panicum maximum estavam bem conservados em campo e cultivados em parcelas de 4 × 4 m2 com 2 m entre elas. Distribuídos aleatoriamente entre os acessos apomíticos (tetraplóides), encontravam-se dez parcelas com plantas sexuais (tetraplóides) que tiveram o número de cromossomos duplicados pela aplicação de colchicina. Na época de florescimento (março a maio), as plantas sexuais foram fecundadas naturalmente pelos acessos vizinhos, fazendo com que suas sementes gerassem famílias de meios-irmãos. Sementes híbridas de dez plantas sexuais foram colhidas em maio e junho de 2004 e semeadas em outubro em bandejas de isopor com substrato vermiculita:areia 1:1. Após dois meses, 30 plântulas de cada progenitora sexual foram transplantadas para terra em sacos plásticos e mantidas até condições adequadas para plantio no campo. Durante o processo de seleção de plantas visando ao lançamento de novos cultivares ou à escolha de genitores superiores para recombinação, é importante certificar-se de sua superioridade genética. A análise de sucessivas medi- ções de uma característica em um grupo de indivíduos é desejável no melhoramento genético de plantas perenes, uma vez que, espera-se, a superioridade ou a inferioridade inicial de um indivíduo mantém-se ao longo das medições. Portanto, coeficiente de repetibilidade valida essa expecta- tiva (Cruz et al., 2004). Altos valores de repetibilidade para quaisquer caracte- rísticas indicam que é possível predizer o valor real dos indivíduos com base em determinado número de medições. Assim, a medida de consistência da posição em relação à classificação dos indivíduos durante sucessivas medições de determinado caráter é denominada repetibilidade (Turner & Young, 1969). Segundo alguns autores (Lush, 1964; Abeywardena, 1972; Kempthorne, 1973; Cruz et al., 2004), a repetibilidade pode ser definida como a correlação entre sucessivas medidas, obtidas de um mesmo indivíduo, e avaliações tomadas ao longo do tempo ou do espaço. Quando uma característica possui alta repetibilidade, há indicativo de pouco ganho em acurácia com o aumento do número de medidas repetidas. Introdução dos sistemas de produção de bovinos no Brasil, a espécie P. maximum, pelo seu potencial produtivo e sua qualidade, é uma das mais indicadas para esse tipo de exploração. A disponibilidade de cultivares melhorados, como Tanzânia, Mombaça, Massai e Milênio, tem sido responsável pela ampliação da área de cultivo desta forrageira. A continuação dos trabalhos de melhoramento genético dessa forrageira A área ocupada pela espécie Panicum maximum Jacq. corresponde a aproximadamente 20% de toda a área de pastagens cultivadas no Brasil (em torno de 100 milhões de hectares) suprindo 30% do mercado de sementes forrageiras (Abrasem, 2004). Considerando a crescente intensificação Martuscello et al. 1976 contribuirão significativamente para o aumento da produção de carne, leite, couro e lã no País. verde (MV), produção de matéria seca (MS), produção de matéria seca foliar (MSF) e porcentagem de folhas (%F) em híbridos de P. maximum a partir de dados obtidos durante dois anos (cinco cortes). Estimou-se ainda o número mínimo de medições para a seleção com maior eficiência e confiabilidade e para a estabilização fenotípica da pro gênie. A maioria dos acessos de P. maximum tem reprodução do tipo apomítica, ou seja, a planta gerada possui as mesmas características da planta-mãe, portanto, não possui varia- bilidade genética. Neste caso, então, verifica-se a necessi- dade de plantas sexuais para intercâmbio gênico (Savidan, 1989). Assim, o cruzamento entre plantas sexuais e apomíticas de P. maximum constitui alternativa viável para obtenção de forrageiras superiores quanto à produção vegetal e, conseqüentemente, à produção animal. © 2007 Sociedade Brasileira de Zootecnia Material e Métodos )1 ( ^ 2 2 ^ − − = n r y y σ σ λ em que: 2 y σ = σ2 + σ2g em que: 2 y σ = σ2 + σ2g em que: 2 y σ = σ2 + σ2g O método da análise estrutural apresenta apenas dife- renças conceituais em relação ao método dos componentes principais. Obtiveram-se também as estimativas da repetibilidade por meio da matriz de correlações entre genótipos em cada par de avaliação. Assim,o estimador do coeficiente de repetibilidade foi determinado por: O coeficiente de repetibilidade (r) foi estimado por quatro procedimentos estatísticos, que possibilitaram ava- liar a consistência da estimativa e permitiram conclusões mais confiáveis sobre as características estudadas. Os estimadores dos coeficientes de repetibilidade foram obti- dos por meio do método da análise de variância, do método dos componentes principais (baseado na matriz de covariância ou correlação) e da análise estrutural (baseado na matriz de correlação). 1 1 ˆ' ˆ − − = n a R a r em que: a’ = autovetor com elementos paramétricos associa- dos ao maior autovalor de R; R = matriz paramétrica de correlação entre genótipos em cada par de avaliação; ^ R = estimador da matriz R; e n = número de períodos avaliados. Para estimação do coeficiente de repetibilidade pelo método da Análise de Variância (ANOVA), utilizou-se o modelo estatístico com dois fatores de variação: Após a obtenção do coeficiente de repetibilidade, esti- mou-se o número mínimo de avaliações necessárias para predizer o valor real dos indivíduos, com base em um coefi- ciente de determinação (R2) preestabelecido. A predição desse valor foi realizada com base na seguinte expressão: Yij = µ + gi + aj + eij, em que µ = média geral; gi = efeito aleatório do i-ésimo genótipo sob a influência do ambiente permanente; aj = efeito fixo do ambiente temporário na j-ésima medição; eij = efeito do ambiente temporário associado à j-ésima medição no i-ésimo genótipo. n0 = R2 (1- ^ r )/(1-R2) r Para esse modelo, o coeficiente de repetibilidade foi determinado por: em que: R2 = nr / 1 + r (n-1) ^ 2 ^ 2 ^ 2 ' , ' ) ( ) ( ) , cov( ˆ g g ij ij ij ij V V r σ σ σ + = Υ Υ Υ Υ = A estabilização fenotípica das quatro características estudadas nos híbridos de P. Material e Métodos maximum foi avaliada utilizando-se os métodos dos componentes principais, obtidos a partir da matriz de correlações para as sucessivas medições, considerando-se 2,3... até todas as n avaliações efetuadas. Portanto, foram realizadas n-1 análises e duas medições sucessivas; n-2, três avaliações sucessivas até chegar às cinco medições. A metodologia dos componentes principais se baseou na matriz de correlação ou na matriz de covariância entre os genótipos, obtidas em cada par de medições, que estimam de forma mais eficiente o coeficiente de repetibilidade em casos no quais os genótipos apresentam comportamento cíclico em relação ao caráter estudado. Todas as análises estatísticas foram realizadas por meio do programa computacional GENES (Programa Computacional em Genética e Estatística) (Cruz, 2001). Quando se baseou na matriz de correlações, o coefi- ciente de repetibilidade foi determinado por: 1 1 1 ^ − − = n r λ © 2007 Sociedade Brasileira de Zootecnia Material e Métodos No entanto, quando a repetibilidade apresenta baixos valores, é necessário grande número de medições para se alcançar valor de determinação satisfatório. Assim, ao aumentar o número de medidas tomadas para determinada característica, diminui-se a variância temporária causada pelo ambiente e, evidente- mente, reduz-se também a variância fenotípica, melhorando a acurácia do coeficiente de repetibilidade. Ressalta-se que, quando a variância ambiental temporária é baixa e a repetibilidade é alta, o aumento no número de tomada de dados pouco acrescentará na melhoria da inferência do valor genotípico do indivíduo. No entanto, se o valor da repetibilidade é baixo, aumentando o número de avaliações haverá aumento de ganho. Em dezembro de 2005, as mudas por progênies foram transplantadas para a área experimental em um delineamento de blocos ao acaso, com cinco plantas por parcela e seis repetições, totalizando 30 plantas por família. O espaçamento foi de 1 m entre parcelas e entre plantas na parcela e de 2 m entre repetições. O solo da área experimental, um Latossolo Vermelho Escuro, apresentou as seguintes características: pH CaCl2 = 4,53; P = 1,29 mg/dm3; K = 48,6 mg/dm3; Ca²+ = 1.64 cmolc/dm3; Mg2+ = 0,93 cmolc/dm3; H + Al = 8,31 cmolc/dm3). No início de novembro de 2005, efetuou-se a aplicação de calcário dolomítico (3 t/ha) incorporado. As adubações fosfatada e potássica consistiram, respectivamente, de 100 kg de P205/ha (superfosfato simples) e 100 kg de K2O/ha (cloreto de potássio). Procedeu-se também à aplica- ção de FTE BR16 (50 kg/ha) e à adubação nitrogenada com 100 kg de N/ha (uréia) no transplantio. Outra adubação Neste trabalho objetivou-se determinar os coeficientes de repetibilidade das características produção de matéria © 2007 Sociedade Brasileira de Zootecnia Repetibilidade de caracteres agronômicos em Panicum maximum Jacq. 1977 idêntica à anterior, à exceção dos 50 kg de FTE BR16/ha, foi realizada em dezembro de 2005. Quando se baseou na matriz de covariâncias, o coefi- ciente de repetibilidade foi estimado considerando-se a matriz paramétrica de variâncias e covariância fenotípicas: Foram feitos cinco cortes no período das águas em todas as plantas (3/3/2005, 13/4/2005, 8/12/2005, 24/1/2006 e 8/3/2006). Imediatamente após o corte, o material foi pesado para obtenção da produção de matéria verde (MV). Amostras foram separadas em lâmina, colmo + bainha e material morto para estimativa da produção de MS total e da MSF. Em seguida, determinou-se a % de folhas de cada genótipo. Resultados e Discussão Observou-se que os coeficientes de repetibilidade (r) oscilaram entre 0,51 e 0,86 (Tabela 1). Os maiores valores foram registrados para a MSF e os menores para produção de MS. No processo de seleção de novas forrageiras, a em que: λ1 = autovalor obtido da matriz ^ R , associado ao autovetor, cujos elementos tiveram o mesmo sinal e magnitudes semelhantes; e n = número de períodos avaliados. Martuscello et al. 1978 característica produção de MSF, fundamental na alimenta- ção animal, é imprescindível, pois as folhas apresentam maior digestibilidade em relação ao colmo. A seleção com base nessa característica tende a tornar o processo de melhoramento genético mais eficiente, principalmente na etapa final, quando a avaliação do ganho de peso é o principal indicador do potencial de utilização da planta. Segundo Abeywardena (1972), o método dos compo- nentes principais permite estimar de forma mais eficiente o coeficiente de repetibilidade em situações em que os genótipos apresentam comportamento cíclico em relação ao caráter estudado, por basear-se na matriz de covariância entre genótipos obtidas em cada par de medições. De acordo com Cruz et al. (2004), o método da análise de variância pode, em alguns casos, levar a estimativas subdimensionadas do coeficiente de repetibilidade. As estimativas dos coeficientes de repetibilidade para avaliação das características MV, MS, MSF e % F, de modo geral, podem ser consideradas altas e indicam a confiabilidade na identificação dos melhores genótipos, considerando as cinco avaliações utilizadas. De modo geral, a diferença entre as estimativas do coefi- ciente de repetibilidade nos quatro métodos indica a necessi- dade de utilização de vários métodos disponíveis para se obter um intervalo preciso dentro do qual, com maior probabilidade, seja encontrado o valor real para esse parâmetro. Independentemente do método utilizado para estima- tiva do coeficiente de repetibilidade, os coeficientes de determinação variaram de 83,96 a 96,88% (Tabela 1) e, portanto, podem ser classificados como altos. Como o coeficiente de determinação expressa a acurácia na predi- ção do valor real do indivíduo, a confiabilidade na seleção das melhores progênies de P. maximum, baseada no valor fenotípico de MSF, é de 96,88% (Método dos componen- tes principais baseado na matriz de covariância), portanto, as cinco colheitas foram suficientes para proporcionar alta confiabilidade na predição do comportamento de diferen- tes genótipos. Resultados e Discussão O aumento da precisão em relação à predição do valor real das avaliações, em todos os métodos utilizados, impli- cou aumento considerável do número de medições para todas as características (Tabela 2). Esses dados sugerem que a tentativa de aumento de precisão além de 95% exigiria aumento significativo do número de medições, o que pouco acrescentaria em precisão, tornando seu uso injustificável. Na seleção de híbridos de P. maximum, Resende et al. (2004) obtiveram 80% de precisão para MSF em quatro anos de avaliação sob cortes, porém com um baixo au- mento na eficiência de seleção após três anos de avali- ação (15 cortes). Para Brachiaria brizantha, no entanto, Basso (2005) obteve 80% de precisão para esta variável com 6 a 14 cortes, dependendo dos genótipos avaliados em diferentes campos. Alguns estudos de repetibilidade para outras plantas forrageiras baseados em dados obti- dos no Brasil estão disponíveis na literatura (Shimoya et al., 2002; Pereira et al., 2002; Daher et al., 2004; Souza Sobrinho et al., 2004). As estimativas do coeficiente de repetibilidade obtidas para as quatro características avaliadas pelo método da análise de variância foram quase sempre menores que as obtidas pelos demais métodos avaliados. Pelo método dos componentes principais (baseado na matriz de covariância), essas estimativas foram sempre maiores que as obtidas pelos demais métodos. Ainda assim, para todas as características avaliadas, as estimativas obti- das pelas quatro metodologias utilizadas comprovaram acurácia nas medições realizadas e alta regularidade da superioridade dos indivíduos. Resultados e Discussão O número de cortes necessários para caracterizar as produções de MV e MS com 80% de probabilidade de seu Tabela 1 - Estimativas do coeficiente de repetibilidade e do coeficiente de determinação (entre parênteses) das características produção de matéria verde (MV), produção de MS, produção de MS foliar (MSF) e porcentagem total de folhas (% F) avaliados por quatro métodos Table 1 - Estimates of the coefficients of repeatability and determination (between brackets) for the characteristics green mass yield (MV), DM yield, leaf DM yield (MSF) and leaf percentage (% F), evaluated by four methods Método Característica Method Characteristic MV MS MSF % F Análise de variância 0,5114(83,96) 0,5165(84,23) 0,7038(92,24) 0,6386(89,83) Analysis of variance Componentes principais (covariância) 0,7491(93,72) 0,6182(88,99) 0,8611(96,88) 0,7067(92,33) Principal component (covariance) Componentes principais (correlação) 0,6087(88,61) 0,5594(86,39) 0,7929(95,03) 0,6555(90,49) Principal component (correlation) Análise estrutural (correlação) 0,6063(88,51) 0,5571(86,28) 0,7825(94,73) 0,6503(90,29) Strutural analysis (correlation) Tabela 1 - Estimativas do coeficiente de repetibilidade e do coeficiente de determinação (entre parênteses) das características produção de matéria verde (MV), produção de MS, produção de MS foliar (MSF) e porcentagem total de folhas (% F) avaliados por quatro ét d e coefficients of repeatability and determination (between brackets) for the characteristics green mass yield (MV), DM yield, leaf and leaf percentage (% F), evaluated by four methods © 2007 Sociedade Brasileira de Zootecnia 1979 Repetibilidade de caracteres agronômicos em Panicum maximum Jacq. Resultados e Discussão Tabela 2 - Número de medições das características produções de massa verde (MV), matéria seca (MST) e matéria seca foliar (MSF) e porcentagem de folhas (% folha) em híbridos de Panicum maximum em quatro métodos de estimação do coeficiente de repetibilidade Table 2 - Number of measurements of the characteristics green mass yield (MV), dry matter yield (MS), leaf dry matter yield (MSF) and leaf percentage (% F) in Panicum maximum hybrids, associated to different coefficients of determination of the genotypic value (R2), in four methods of estimation of the coefficient of repeatability R² Anova Componentes principais (covariância) Componentes principais (correlação) Análise estrutural (Correlação) Analysis of variance Principal components (covariance) Principal components (correlation) Strutural analysis (correlation) Matéria verde (Green matter) 0,8 4 1 2 3 0,85 5 2 3 4 0,9 9 3 6 6 0,95 18 6 12 12 0,99 94 33 64 64 Matéria seca (Dry matter) 0,8 4 2 3 3 0,85 5 3 4 4 0,9 8 6 7 7 0,95 18 12 15 15 0,99 93 61 78 79 Matéria seca foliar (Leaf dry matter) 0,8 2 1 1 1 0,85 2 1 1 2 0,9 4 2 2 3 0,95 8 3 5 5 0,99 42 16 26 27 % de folhas (Leaf %) 0,8 2 2 2 2 0,85 3,2 2 3 3 0,9 5 4 5 5 0,95 11 8 10 10 0,99 56 41 52 53 casos, é possível um caráter ser regido por conjuntos gênicos distintos e os genes estarem mais ou menos ativos de acordo com o estado de desenvolvimento dos indivíduos. Em alguns casos, a inclusão de avaliações em estádio precoce onde não há plena manifestação do potencial genético da planta, ou em avaliações tardias, quando a forrageira apresenta determinado grau de senescência, a repetibilidade pode ser subestimada. Assim, se o genótipo no qual as medidas são tomadas não estiver estabilizado, a variação dentro de indivíduos incluirá parte considerável da variância da interação genótipo × ambiente temporário e o aumento do número de medições como forma de reduzir a variação dentro de indivíduos pode não ser vantajoso, pois a variância adicional poderá ser suficiente para neutra- lizar a redução do componente. © 2007 Sociedade Brasileira de Zootecnia Resultados e Discussão Tabela 3 - Números de medições (n) e obtenção de R2 por meio da estabilização fenotípica utilizando-se o método dos componentes principais a partir da matriz de correlação de quatro características agronômicas avaliadas em híbridos de Panicum maximum em cinco sucessivas colheitas Table 3 - Estimate of the number of measurements (n) and obtention of the coefficients of determination of the genotypic value (R2), by the phenotypic stabilization using the principal components method, based on the correlation matrix of four agronomic characteristics evaluated in hybrids of Panicum maximum in five successive harvests Colheita No de medições Coeficiente de repetibilidade R2 Colheita No de medições Coeficiente de repetibilidade R2 Harvest Number of harvests Coefficient of repeatability Harvest Number of harvests Coefficient of repeatability Matéria verde Matéria seca foliar Green yield Leaf dry matter yield 1-2 2 0,92 95 1-2 2 0,52 69 2-3 2 0,35 52 2-3 2 0,61 76 3-4 2 0,90 95 3-4 2 0,89 94 4-5 2 0,69 82 4-5 2 0,92 96 1, 2 e 3 3 0,62 83 1, 2 e 3 3 0,68 87 2, 3 e 4 3 0,57 80 2, 3 e 4 3 0,71 88 3, 4, e 5 3 0,75 90 3, 4, e 5 3 0,92 97 1, 2, 3 e 4 4 0,60 86 1, 2, 3 e 4 4 0,75 92 2, 3, 4 e 5 4 0,59 85 2, 3, 4 e 5 4 0,77 93 1, 2, 3, 4 e 5 5 0,61 89 1, 2, 3, 4 e 5 5 0,79 95 Matéria seca % folhas Dry matter Leaf % 1-2 2 0,85 92 1-2 2 0,86 93 2-3 2 0,36 54 2-3 2 0,51 67 3-4 2 0,86 92 3-4 2 0,97 98 4-5 2 0,58 73 4-5 2 0,71 83 1, 2 e 3 3 0,58 81 1, 2 e 3 3 0,68 87 2, 3 e 4 3 0,57 80 2, 3 e 4 3 0,66 86 3, 4, e 5 3 0,64 84 3, 4, e 5 3 0,77 91 1, 2, 3 e 4 4 0,58 85 1, 2, 3 e 4 4 0,70 90 2, 3, 4 e 5 4 0,53 82 2, 3, 4 e 5 4 0,62 87 1, 2, 3, 4 e 5 5 0,56 87 1, 2, 3, 4 e 5 5 0,65 90 Tabela 3 - Números de medições (n) e obtenção de R2 por meio da estabilização fenotípica utilizando-se o método dos componentes principais a partir da matriz de correlação de quatro características agronômicas avaliadas em híbridos de Panicum maximum em cinco sucessivas colheitas 2 verde foi a de maior instabilidade. Resultados e Discussão valor real foi de no máximo quatro (paro o método da análise de variância) e apenas um e dois, respectivamente, para o método de componentes principais (baseado na matriz de covariância), o que indica não serem necessários cinco cortes para essas características, pois com apenas quatro (para o método que estima o maior número de medições) obtém-se boa confiabilidade, além de redução da mão-de- obra e do tempo. Essa observação é válida também para as demais carac- terísticas avaliadas. Para a produção de MSF e a % folhas, o número de medições necessárias para os mesmos 80% de probabilidade é de duas medições, o que indica novamente que os cinco cortes foram suficientes para estimativa confiável do coeficiente de repetibilidade. Para se obter maior confiabilidade, pode-se utilizar o coeficiente de deter- minação de 90% para a seleção dos melhores genótipos para todas as características avaliadas. Neste caso, para maior acurácia, são necessários 3 a 9 cortes, de acordo com a metodologia. Para a variável produção de MV, o maior coeficiente de repetibilidade foi obtido quando se correlacionaram os cortes 1 e 2 (Tabela 3) e o menor, na correlação dos cortes 2 e 3, o que provavelmente foi ocasionado pelo fato de que, nos dois primeiros cortes, os genótipos ainda não estavam estabilizados. De fato, a produção de matéria A utilização de genótipos estabilizados na obtenção de estimativas de repetibilidade é fundamental. Em alguns © 2007 Sociedade Brasileira de Zootecnia 1980 Martuscello et al. Resultados e Discussão Esse resultado era previsível, uma vez que a teor de água em cada planta tem reflexo direto nessa estimativa. Não só para MV, mas também para MS, a baixa correlação entre os cortes 2 e 3 pode ser explicada pela época do corte, pois o terceiro corte foi realizado em dezembro e as plantas se desenvolveram após rebrotação de corte na seca. Para MS o maior coeficiente de repetibilidade e o maior coeficiente de determinação estive- ram correlacionados entre os cortes 3 e 4, provavelmente em razão das condições ambientais, que foram bastante homo- gêneas quanto à temperatura e à precipitação. produção de MS, apresentou maiores coeficientes de repetibilidade e determinação. Apesar da regularidade no comportamento dos indiví- duos nos cortes 1 e 2 (Tabela 3), em geral, a inclusão dos dados a partir desses cortes tende a diminuir o coeficiente de repetibilidade, evidenciando que a estabilização de genótipos de P. maximum ocorre provavelmente após o segundo corte, o que sugere a exclusão dos dados dos dois primeiros cortes em futuras avaliações. Conclusões A correlação entre os cortes e a estabilização do genótipo para a produção de MSF e a porcentagem de folhas provavelmente sofreu efeito direto da época de florescimento de cada progênie, uma vez que em florescimento ocorre maior acúmulo de colmo e, conse- qüentemente, efeito direto desse acúmulo sobre a produção de MSF e sobre a porcentagem de folhas em cada genótipo. Para MSF, o maior coeficiente de repetibilidade foi na correlação entre os cortes 4 e 5 e o menor nas correlações entre os cortes 2 e 3 (Tabela 3). A correlação entre os cortes 3 e 4 para a porcentagem de folhas, assim como para Os coeficientes de repetibilidade, assim como os coefici- entes de determinação obtidos para as características agronô- micas produção de matéria verde, produção de MS, produção de MSF e porcentagem de folhas em genótipos de P. maximum, podem ser considerados altos para todas as características em todos os métodos avaliados. Para maior acurácia (90% de confiabilidade) na seleção dos melhores híbridos, são necessárias três (método dos componentes principais, baseado na matriz de correlação) a nove (método da análise de variância) medições. © 2007 Sociedade Brasileira de Zootecnia Repetibilidade de caracteres agronômicos em Panicum maximum Jacq. 1981 A exclusão dos cortes 1 e 2 promove aumento nos coeficientes de repetibilidade e determinação. É necessário utilizar os vários métodos de análise disponíveis para se obter intervalo preciso para cada parâmetro visando encon- trar o valor real com maior probabilidade. KEMPTHORNE, O. The design and analysis of experiments. New York: Robert E. Krieger Publishing Company, 1973. 631p. LUSH, J.L. Melhoramento dos animais domésticos. Tradução de CARNEIRO, G.G.; MEMORIA, J.M.P.; DRUNMOND, G. (Eds.) Rio de Janeiro: CEDEGRA, 1964. 566p. PEREIRA, A.V.; CRUZ, C.D; FERREIRA, R.P. et al. Influência da estabilização de genótipos sobre a estimativa de repetibilidade em caracteres produtivos de capim-elefante. C i ê n c i a e Agrotecnologia, v.26, n.4, p.762-767, 2002. © 2007 Sociedade Brasileira de Zootecnia Literatura Citada RESENDE, R.M.S.; JANK, L.; VALLE, C.B. et al. Biometrical analysis and selection of tetraploid progenies of “Panicum maximum” Jacq. using mixed model methodology. Pesquisa ABEYWARDENA, V. An application of principal component analysis in genetics. Journal Genetics, v.16, p.27, 1972. Agropecuária Brasileira, v.39, n.4, p.335-341, 2004. Agropecuária Brasileira, v.39, n.4, p.335-341, 2004. SAVIDAN, Y.H.; JANK, L.; COSTA, J.C.G. et al. Breeding Panicum maximum in Brazil: 1. Genetic resourses, modes of reproduction SAVIDAN, Y.H.; JANK, L.; COSTA, J.C.G. et al. Breeding Panicum maximum in Brazil: 1. Genetic resourses, modes of reproduction ASSOCIAÇÃO BRASILEIRA DE SEMENTES - ABRASEM. Disponível em: http://<www.abrasem.br.> Acesso em 1/12/2004. and breeding procedures. Euphytica, v.41, p.107-112, 1989. SHIMOYA A PEREIRA A V FERREIRA R P l BASSO, K.C. Estimação de parâmetros genéticos e índice de seleção em genótipos de Brachiaria brizantha. Dourados: Universidade Federal de Mato Grosso do Sul, 2005. 63p. Dissertação (Mestrado em Produção Vegetal) - Universidade Federal de Mato Grosso do Sul, 2005. g p p y , , p , 9 9 SHIMOYA, A.; PEREIRA, A.V.; FERREIRA, R.P. et al. Repetibilidade de características forrageiras de capim-elefante. Scientia Agricola, v.59, n.2, p.227-234, 2002. SOUZA SOBRINHO, F.; LEDO, F.J.S.; PEREIRA, A.V. et al. Estimativas de repetibilidade para produção de matéria seca em lf f Ciê i R l 34 2 531 537 2004 CRUZ, C.D. Programa Genes Versão Windons: aplicativo computacional em genética e estatística. Viçosa, MG: Universidade Federal de Viçosa, 2001. 684p. alfafa. Ciência Rural, v.34, n.2, p.531 537, 2004. TURNER, H.N.; YOUNG, S.Y. Quantitative gentics insheep breeding. Ithaca: Cormell University Press, 1969. 331p. ç , p CRUZ, C.D.; REGAZZI, A.J.; CARNEIRO, P.C.S. Modelos biométricos aplicados ao melhoramento genético. 3.ed. CRUZ, C.D.; REGAZZI, A.J.; CARNEIRO, P.C.S. Modelos biométricos aplicados ao melhoramento genético. 3.ed. Viçosa, MG: Universidade Federal de Viçosa, 2004. v.1. 480p. Viçosa, MG: Universidade Federal de Viçosa, 2004. v.1. 480p. DAHER, R.F.; VASQUEZ, H.M.; PEREIRA, A.V. et al. Estimativas de parâmetros genéticos e de coeficientes de repetibilidade de caracteres forrageiros em clones de capim- elefante ( Pennisetum purpureum Schum). Acta Scientiarum, v.26, n.4, p.483-490, 2004. Recebido: 9/11/2006 Aprovado: 5/7/2007
https://openalex.org/W2752311711	https://europepmc.org/articles/pmc6151834?pdf=render	English	null	Mechanochemical Synthesis and Biological Evaluation of Novel Isoniazid Derivatives with Potent Antitubercular Activity	Molecules/Molecules online/Molecules annual	2,017	cc-by	38,839	Article Mechanochemical Synthesis and Biological Evaluation of Novel Isoniazid Derivatives with Potent Antitubercular Activity Paulo F. M. Oliveira 1,2,3,† ID , Brigitte Guidetti 2,3, Alain Chamayou 1, Christiane André-Barrès 2,3, Jan Madacki 4, Jana Korduláková 4,, Giorgia Mori 5, Beatrice Silvia Orena 5, Laurent Roberto Chiarelli 5 ID , Maria Rosalia Pasca 5, ID , Christian Lherbet 2,3, Chantal Carayon 2,3, Stéphane Massou 2, Michel Baron 1 and Michel Baltas 2,3,* 1 Department of Process Engineering, Université de Toulouse, Mines-Albi, CNRS UMR 5302, Centre RAPSODEE, Campus Jarlard, 81013 Albi, France; paul_marqs@hotmail.com (P.F.M.O.); alain.chamayou@mines-albi.fr (A.C.); baron@mines-albi.fr (M.B.) 2 Department of Chemistry, Université de Toulouse, UPS, CNRS UMR 5068, LSPCMIB, 118 Route de Narbonne, 31062 Toulouse, France; guidetti@chimie.ups-tlse.fr (B.G.); candre@chimie.ups-tlse.fr (C.A.-B.) christian.lherbet@itav.fr (C.L.); andre@chimie.ups-tlse.fr (C.C.); massou@chimie.ups-tlse.fr (S.M.) 3 Narbonne, 31062 Toulouse, France; guidetti@chimie.ups-tlse.fr (B.G.); candre@chimie.ups-tlse.fr (C.A.-B.); christian.lherbet@itav.fr (C.L.); andre@chimie.ups-tlse.fr (C.C.); massou@chimie.ups-tlse.fr (S.M.) 3 CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, LSPCMIB, UMR-5068, 118 Route de Narbonne, 31062 Toulouse, France p p 3 CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, LSPCMIB, UMR-5 118 Route de Narbonne, 31062 Toulouse, France 4 Department of Biochemistry, Comenius University in Bratislava, Faculty of Natural Science Mlynská Dolina, Ilkoviˇcova 6, 84215 Bratislava, Slovakia; jan.madacki@gmail.com 5 Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia; via Ferrata 1, 27100 Pavia, Italy; giorgia.mori@unipv.it (G.M.); beatricesilvia.orena01@universitadipavia.it (B.S.O.); laurent.chiarelli@unipv.it (L.R.C.) * Correspondence: kordulakova@fns.uniba.sk (J.K.); mariarosalia.pasca@unipv.it (M.R.P.); baltas@chimie.ups-tlse.fr (M.B.); Tel.: +421-(0)260296547 (J.K.); +39-0382-985576 (M.R.P.); +33-(0)561556289 (M.B.) † Current address: Université de Lille, UMET, Unité Matériaux et Transformations, CNRS UMR 8207, F-59000 Lille, France. Received: 2 July 2017; Accepted: 24 July 2017; Published: 1 September 2017 Received: 2 July 2017; Accepted: 24 July 2017; Published: 1 September 2017 Abstract: A series of isoniazid derivatives bearing a phenolic or heteroaromatic coupled frame were obtained by mechanochemical means. Their pH stability and their structural (conformer/isomer) analysis were checked. The activity of prepared derivatives against Mycobacterium tuberculosis cell growth was evaluated. Some compounds such as phenolic hydrazine 1a and almost all heteroaromatic ones, especially 2, 5 and 7, are more active than isoniazid, and their activity against some M. tuberculosis MDR clinical isolates was determined. Compounds 1a and 7 present a selectivity index >1400 evaluated on MRC5 human ﬁbroblast cells. The mechanism of action of selected hydrazones was demonstrated to block mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell. Keywords: Mycobacterium tuberculosis; mechanochemistry; hydrazone Molecules 2017, 22, 1457; doi:10.3390/molecules22091457 molecules molecules molecules 1. Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), represents an enduring, deadly infectious disease worldwide. According to the World Health Organization (WHO), one third of the global population is infected with M.tb. In comparison with other diseases caused by a single infectious agent, TB is the second leading cause of mortality. It is estimated that in 2015 TB killed 1.8 million people, mainly in underdeveloped countries [1]. New effective drugs for the treatment www.mdpi.com/journal/molecules Molecules 2017, 22, 1457; doi:10.3390/molecules22091457 2 of 27 Molecules 2017, 22, 1457 of TB are necessary ﬁrstly to reduce the duration of TB treatment and, secondly, for the treatment of M.tb multidrug-resistant (MDR) [2,3], extensively-drug resistant (XDR) [4], and totally-drug resistant (TDR) strains [5]. In the recent years, two molecules bedaquiline and delamanid (Figure 1) have been approved for the treatment of MDR-TB when an effective treatment regimen is not otherwise available [6,7]. Molecules 2017, 22, 1457 2 of 27 (TDR) strains [5]. In the recent years, two molecules bedaquiline and delamanid (Figure 1) have been approved for the treatment of MDR-TB when an effective treatment regimen is not otherwise available [6,7]. Figure 1. Antitubercular drug Isoniazid (1952), and Bedaquiline and Delamanid, two new compounds approved for the treatment of MDR-TB. Figure 1. Antitubercular drug Isoniazid (1952), and Bedaquiline and Delamanid, two new compounds approved for the treatment of MDR-TB. Figure 1. Antitubercular drug Isoniazid (1952), and Bedaquiline and Delamanid, two new compounds approved for the treatment of MDR-TB. Figure 1. Antitubercular drug Isoniazid (1952), and Bedaquiline and Delamanid, two new compounds approved for the treatment of MDR-TB. One of the main known drug targets to fight M.tb growth is the enoyl-ACP reductase or InhA. This well-known enzyme is already the indirect target of Isoniazid (INH), a front-line prodrug used clinically to treat TB. INH remains a key component in all multiple drug treatment regiments recommended by the WHO even if M.tb resistant isolates have been rapidly generated during monotherapy or inappropriate treatment. Hence, improvement of INH molecule by introducing chemical modifications in its core structure in order to enhance biological response (prodrug, increase of bioavailability, and membrane permeability) continues to be an interesting scientific challenge. Recently, for example, compounds containing isonicotinoyl moiety with potential dual inhibition targeting FabG4 and HtdX, were successfully characterized [8]. 1. Introduction Hydrazide-hydrazone derivatives have long attracted attention because of their wide range of applications in medicinal chemistry [9–12] Hydrazide and hydrazone derivatives showed strong One of the main known drug targets to ﬁght M.tb growth is the enoyl-ACP reductase or InhA. This well-known enzyme is already the indirect target of Isoniazid (INH), a front-line prodrug used clinically to treat TB. INH remains a key component in all multiple drug treatment regiments recommended by the WHO even if M.tb resistant isolates have been rapidly generated during monotherapy or inappropriate treatment. Hence, improvement of INH molecule by introducing chemical modiﬁcations in its core structure in order to enhance biological response (prodrug, increase of bioavailability, and membrane permeability) continues to be an interesting scientiﬁc challenge. Recently, for example, compounds containing isonicotinoyl moiety with potential dual inhibition targeting FabG4 and HtdX, were successfully characterized [8]. applications in medicinal chemistry [9–12]. Hydrazide and hydrazone derivatives showed strong antioxidant and radical scavenging properties, while others ones displayed potent anticancer, antimicrobial, anticonvulsant or anti-inflammatory activities in vitro [9]. Recently, interesting anti- mycobacterial activities were reported about the following derivatives: guanylhydrazones [13], trans- cinnamic acid hydrazides derivatives [14], fluorine containing hydrazones [15], sulfonyl-hydrazones [16], and L-proline derived hydrazones [17]. Moreover, isoniazid-related hydrazones showed similar or better efficiency than the INH [18]. Isonicotinoylhydrazone derivatives were also synthesized and evaluated as anti-mycobacterial agents [19 20] Interestingly vanillic acylhydrazones were reported as potential β keto acyl carrier g g y Hydrazide-hydrazone derivatives have long attracted attention because of their wide range of applications in medicinal chemistry [9–12]. Hydrazide and hydrazone derivatives showed strong antioxidant and radical scavenging properties, while others ones displayed potent anticancer, antimicrobial, anticonvulsant or anti-inflammatory activities in vitro [9]. Recently, interesting anti-mycobacterial activities were reported about the following derivatives: guanylhydrazones [13], trans-cinnamic acid hydrazides derivatives [14], fluorine containing hydrazones [15], sulfonyl-hydrazones [16], and L-proline derived hydrazones [17]. agents [19,20]. Interestingly, vanillic acylhydrazones were reported as potential β-keto acyl carrier protein synthase III (FabH) inhibitors [21]. Schiff bases of isoniazid, considered as chemical modification that can block N-acetylation of INH, showed good activity in vitro and in vivo and in some cases low toxicity [22,23]. In 2008, an extended study reported a quantitative structure activity relationships (QSAR) of a large hydrazide family for the developing of antitubercular compounds [24]. Finally, some researchers focused on establishing a predictive QSAR model for different INH derivatives including isonicotinoylhydrazones [25]. 1. Introduction Our group is involved both in synthesizing new antitubercular compounds, including cinnamic acid derivatives [26–29], triazoles [30–33], pyrrolidines [34–36], semicarbazones and hydrazine/hydrazones Moreover, isoniazid-related hydrazones showed similar or better efﬁciency than the INH [18]. Isonicotinoylhydrazone derivatives were also synthesized and evaluated as anti-mycobacterial agents [19,20]. Interestingly, vanillic acylhydrazones were reported as potential β-keto acyl carrier protein synthase III (FabH) inhibitors [21]. Schiff bases of isoniazid, considered as chemical modiﬁcation that can block N-acetylation of INH, showed good activity in vitro and in vivo and in some cases low toxicity [22,23]. In 2008, an extended study reported a quantitative structure activity relationships (QSAR) of a large hydrazide family for the developing of antitubercular compounds [24]. Finally, some researchers focused on establishing a predictive QSAR model for different INH derivatives including isonicotinoylhydrazones [25]. Molecules 2017, 22, 1457 3 of 27 Our group is involved both in synthesizing new antitubercular compounds, including cinnamic acid derivatives [26–29], triazoles [30–33], pyrrolidines [34–36], semicarbazones and hydrazine/hydrazones [37], and in searching for new and innovative synthetic reactions. We have recently reported the solvent-free mechanosynthesis of a series of hydrazones [38,39]. Mechanochemistry has been used for a long time for the chemical and physicochemical transformations of inorganic materials to generate all states of aggregation produced by the effect of mechanical energy [40]. More recently, the mechanical energy has been used to synthesize organic molecules in milling devices [41–52]. Molecules 2017, 22, 1457 3 of 27 [37], and in searching for new and innovative synthetic reactions. We have recently reported the solvent- free mechanosynthesis of a series of hydrazones [38,39]. Mechanochemistry has been used for a long time for the chemical and physicochemical transformations of inorganic materials to generate all states of Considering the pharmaceutical area [53], the mechanical action was used in particular to develop nitrogen-containing heterocycles, well represented in many therapeutic classes. For example, phthalazoles [54], phenazines [55], pyrazoles, pyridazinones [56], and pyrroles [57] were obtained by mechanosynthesis. Non-heterocyclic nitrogen-containing molecules were also synthesized under mechanical solid-state and solvent-free conditions, including imines, azomethines [58,59], azines [56], enamines and hydrazones [60–62]. for the chemical and physicochemical transformations of inorganic materials to generate all states of aggregation produced by the effect of mechanical energy [40]. More recently, the mechanical energy has been used to synthesize organic molecules in milling devices [41–52]. 1. Introduction Considering the pharmaceutical area [53], the mechanical action was used in particular to develop nitrogen-containing heterocycles, well represented in many therapeutic classes. For example, phthalazoles [54], phenazines [55], pyrazoles, pyridazinones [56], and pyrroles [57] were obtained by mechanosynthesis. Non-heterocyclic nitrogen-containing molecules were also synthesized under h i l lid t t d l t f diti i l di i i thi [58 59] i [56] In this work, this methodology was used in order to synthesize hydrazones under solvent-free conditions, in particular the isonicotinoyl ones. Thus, herein we report the synthesis of a series of phenol and hetero aryl isonicotinoylhydrazones through mechanochemistry and the evaluation of their anti-tuberculosis activities. mechanical solid-state and solvent-free conditions, including imines, azomethines [58,59], azines [56], enamines and hydrazones [60–62]. In this work, this methodology was used in order to synthesize hydrazones under solvent-free conditions, in particular the isonicotinoyl ones. Thus, herein we report the synthesis of a series of phenol and hetero aryl isonicotinoylhydrazones through mechanochemistry and the evaluation of 2. Results and Discussion their anti tuberculosis ac 2.1. Chemistry 2. Results 2 1 Ch i Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones Table 1 Hydra ones 2 11 produced mechanochemically by reacting INH and imida olic inda olic y g g g times of 2 h. Table 1 Hydra ones 2 11 produced mechanochemically by reacting INH and imida olic inda olic The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding times of 2 h. The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding times of 2 h. The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding times of 2 h. The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding times of 2 h. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives or indolic aldehydes. The reaction was catalyzed by p TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. It is still important to mention that, differently from the phenolic hydrazones, a melting was or indolic aldehydes. The reaction was catalyzed by p TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. It is still important to mention that, differently from the phenolic hydrazones, a melting was Indazole derivatives or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones It is still important to mention that, differently from the phenolic hydrazones, a melting was or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. It is still important to mention that, differently from the phenolic hydrazones, a melting was Indole derivatives Table 1. Hydrazones 2 11 produced mechanochemically by reacting INH and imidazolic, indazolic or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. I i ill i i h diff l f h h li h d l i Table 1. Hydrazones 2 11 produced mechanochemically by reacting INH and imidazolic, indazolic or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. I i ill i i h diff l f h h li h d l i Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. Table 1. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic or indolic aldehydes. The reaction was catalyzed by p-TSA. Aldehydes Heterocyclic Hydrazones (6) Derived From Isoniazid Imidazole derivatives 2 (80%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] 5 (99%) [a] Indole derivatives 6 (99%) [a] 7 (94%) [a] 8 (99%) [a] 9 (99%) [a] 10 (91%) [a] 11 (99%) [a] [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H-NMR and MS, the conversions were quantitative. Heterocyclic Hydrazones (6) Derived From Isoniazid Heterocyclic Hydrazones (6) Derived From Isoniazid Heterocyclic Hydrazones (6) Derived From Isoniazi y y p Heterocyclic Hydrazones (6) Derived From Isoniazid y y p Heterocyclic Hydrazones (6) Derived From Isoniazi was catalyzed by p-TSA. n was catalyzed by p-TSA. y y g was catalyzed by p-TSA. n was catalyzed by p-TSA. ed mechanochemically by reacting INH and imidazolic, was catalyzed by p-TSA. ced mechanochemically by reacting INH and imidazoli n was catalyzed by p-TSA. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydrazones The classical methods to synthesize hydrazones are generally carried out at low concentration, and require times from 3 to 24 h or even 48 h under reﬂux of toluene or ethanol in order to obtain good yields. We previously employed the vibratory mill Pulverisette 0 (P0) (Fritsch, Germany) to synthesize phenolic hydrazones mechanochemically [38]. A comparative study has been therein reported with various hydrazides, among them the isoniazid and phenolic aldehydes leading to compounds already described 1a–d (Figure 2). Mechanosynthesis of Isonicotinoyl Hydrazones The classical methods to synthesize hydrazones are generally carried out at low concentration, and require times from 3 to 24 h or even 48 h under reflux of toluene or ethanol in order to obtain good yields. We previously employed the vibratory mill Pulverisette 0 (P0) (Fritsch, Germany) to synthesize phenolic hydrazones mechanochemically [38]. A comparative study has been therein reported with various hydrazides, among them the isoniazid and phenolic aldehydes leading to compounds already described 1a–d (Figure 2). Figure 2. Isonicotinoyl hydrazones synthesized by co-grinding of isoniazide and an aldehyde. Figure 2. Isonicotinoyl hydrazones synthesized by co-grinding of isoniazide and an aldehyde. Figure 2. Isonicotinoyl hydrazones synthesized by co-grinding of isoniazide and an aldehyde. Figure 2. Isonicotinoyl hydrazones synthesized by co-grinding of isoniazide and an aldehyde. We decided to synthesize a series of isonicotinoylhydrazones derivatives bearing various N- heterocyclic indole, indazole or imidazole moieties using the mechanochemical approach. The corresponding aldehydes were selected because of the importance of these N-heterocyclic fragments in a large number of natural or synthetic biologically active molecules. Indeed, compounds bearing We decided to synthesize a series of isonicotinoylhydrazones derivatives bearing various N-heterocyclic indole, indazole or imidazole moieties using the mechanochemical approach. The corresponding aldehydes were selected because of the importance of these N-heterocyclic 4 of 27 Molecules 2017, 22, 1457 fragments in a large number of natural or synthetic biologically active molecules. Indeed, compounds bearing these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti-inﬂammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones Molecules 2017, 22, 1457 4 of 27 these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- Molecules 2017, 22, 1457 4 of 27 these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- Molecules 2017, 22, 1457 4 of 27 these frames may exhibit various activities i e antibacterial anticancer antioxidant anti- Molecules 2017, 22, 1457 4 of 27 these frames may exhibit various activities i e antibacterial anticancer antioxidant anti- Molecules 2017, 22, 1457 4 of 27 Molecules 2017, 22, 1457 4 of 27 Molecules 2017, 22, 1457 4 of 27 Molecules 2017, 22, 1457 4 of 27 Molecules 2017, 22, 1457 4 of 27 Molecules 2017, 22, 1457 4 of 27 The reaction described for phenolic compounds was ﬁrstly used with the 4-methyl-5-imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efﬁciency of the reaction in acidic media. inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours TLC and NMR analysis showed incomplete conversion In that inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours TLC and NMR analysis showed incomplete conversion In that these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was ese a es ay e ibi a ious ac i i ies, i e , a ibac e ia , a ica ce , a io ida , a i inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- imidazolecarboxaldehyde as model of heterocyclic aldehyde. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones However, in spite of the milling was these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- i id l b ld h d d l f h t li ld h d H i it f th illi these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- i id l b ld h d d l f h t li ld h d H i it f th illi these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- these frames may exhibit various activities, i.e., antibacterial, anticancer, antioxidant, anti- inflammatory, anti-diabetic, antiviral, anti-proliferative, antituberculosis, antispermatogenic or antipsychotic activities [63–67]. The reaction described for phenolic compounds was firstly used with the 4-methyl-5- The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone, which was conﬁrmed by 1H- and 13C-NMR analysis. carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones TLC showed the consumption of the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone which was confirmed by 1H- and 13C- carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone which was confirmed by 1H- and 13C- imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones 3 (98%) [a] 3 (98%) [a] 3 (98%) [a] 3 (98%) [a] ) Derived From Isoniazid 6) Derived From Isoniazid ) Derived From Isoniazid 6) Derived From Isoniazid ) Derived From Isoniazid 6) Derived From Isoniazid 2 (80%) [a] 2 (80%) [a] y y 2 (80%) [a] y y 2 (80%) [a] Heterocyclic Hydrazone (80%) [ ] Heterocyclic Hydrazon (80%) [ ] Heterocyclic Hydrazone Heterocyclic Hydrazon Heterocyclic Hydrazone Heterocyclic Hydrazon 2 (80%) [a] 2 (80%) [a] 2 (80%) [a] 2 (80%) [a (98%) [a] 3 (98%) [a] (98%) [a] 3 (98%) [a] 5 (99%) [a] 5 (99%) [a] 5 (99%) [a] 5 (99%) [a] 3 (98%) [a] 5 (99%) [a] 3 (98%) [a] 5 (99%) [a] 3 (98%) [a] (99%) [ ] 3 (98%) [a] 5 (99%) [ ] 3 (98%) [a] 3 (98%) [a] Indazole derivatives 4 (98%) [a] Indazole derivatives 4 (98%) [a] Indazole derivatives Indazole derivatives 4 (98%) [a] Indazole derivatives 4 (98%) [a] ( ) Indazole derivatives 4 (98%) [a] ( ) Indazole derivatives 4 (98%) [a] 2 (80%) [a] Indazole derivatives 2 (80%) [a] Indazole derivatives 2 (80%) [a] Indazole derivatives 2 (80%) [a] Indazole derivatives atives 4 (98%) [a] vatives 4 (98%) [a] satives 4 (98%) [a] vatives 4 (98%) [a] ( ) tives 4 (98%) [a] ( ) vatives 4 (98%) [a] 2 (80%) [a] tives 2 (80%) [a] vatives 2 (80%) [a] atives 2 (80%) [a] vatives Indazole derivativ Indazole derivat Indazole derivatives Indazole derivativ Indazole deriva Indazole derivativ Indazole derivat Indazole derivativ Indazole derivat 5 (99%) [a] 5 (99%) [a] 5 (99%) [a] 5 (99%) [a] 5 (99%) [a] 5 (99%) [a] 4 (98%) [a] 4 (98%) [a] 4 (98%) [a] 4 (98%) [a] 4 (98%) [a] 4 (98%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 5 (99%) [ ] 7 (94%) [ ] 5 (99%) 7 (94%) [a] 6 (99%) [a] 6 (99%) [a] 6 (99%) [a] 6 (99%) [a] 6 (99%) [a] 6 (99%) [a] ( ) 6 (99%) [a] ( ) 6 (99%) [a] 4 (98%) [a] 6 (99%) [ ] 4 (98%) [a] 6 (99%) [a] Indole derivatives 7 (94%) 7 (94%) 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 7 (94%) [a] 6 (99%) 6 (99%) 6 (99%) [a] 6 (99%) [a 6 (99%) [a] 6 (99%) [a] 6 (99%) [a] 6 (99%) [a] 6 (99%) [ ] 6 (99%) [a] 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 7 (94%) (9 %) 7 (94%) [a] 7 (94%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 6 (99%) [ ] 8 (99%) [a] 6 (99%) 8 (99%) [a] 8 (99%) [ ] 8 (99%) 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 8 (99%) [a] 11 (99%) [a] nate p-TSA. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones TLC showed the consumption of imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p TSA TLC showed the consumption of imidazolecarboxaldehyde as model of heterocyclic aldehyde. However, in spite of the milling was carried out by up to eight hours, TLC and NMR analysis showed incomplete conversion. In that respect, we evaluated the efficiency of the reaction in acidic media. The reaction was thus carried out in the presence of AlCl3 or of p-toluenesulfonic acid (p-TSA); the latter one showed the best result when using 50% mol of p TSA TLC showed the consumption of The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding times of 2 h. NMR analysis. The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding i f 2 h NMR analysis. The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding i f 2 h the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- NMR analysis. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding the reagents and the consequent appearance of the hydrazone, which was confirmed by H and C NMR analysis. The hydrazones listed in Table 1 were thus obtained with a high transformation ratio in grinding the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- NMR analysis. Th h d li t d i T bl 1 th bt i d ith hi h t f ti ti i i di the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- NMR analysis. Th h d li t d i T bl 1 th bt i d ith hi h t f ti ti i i di the latter one showed the best result when using 50% mol of p TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- NMR analysis. the latter one showed the best result when using 50% mol of p TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- NMR analysis. the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- NMR analysis. the latter one showed the best result when using 50% mol of p-TSA. TLC showed the consumption of the reagents and the consequent appearance of the hydrazone, which was confirmed by 1H- and 13C- NMR analysis. Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic or indolic aldehydes. The reaction was catalyzed by p-TSA. Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic i d li ld h d Th ti t l d b TSA Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic i d li ld h d Th ti t l d b TSA times of 2 h. Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic times of 2 h. Table 1. Hydrazones 2–11 produced mechanochemically by reacting INH and imidazolic, indazolic y g g g times of 2 h. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones According to TLC, 1H- 11 (99%) [a] minate p-TSA. According to TLC, 1H- 9 (99%) 11 (99%) [a] nate p-TSA. According to TLC, 1H- 9 (99%) 11 (99%) [a] minate p-TSA. According to TLC, 1H- 9 (99%) [a] 11 (99%) [a] nate p TSA According to TLC 1H 9 (99%) [a] 11 (99%) [a] minate p TSA According to TLC 1H 9 (99%) [a] 11 (99%) [a] 9 (99%) [a] 11 (99%) [a] 9 (99%) [a] 11 (99%) [a] 9 (99%) [a] 11 (99%) [a] 9 (99%) 9 (99%) 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 9 (99%) [a] 10 (91%) [a] ing with NaHCO3 aqueous solution to el onversions were quantitative 10 (91%) [a] shing with NaHCO3 aqueous solution to conversions were quantitative 10 (91%) [a] ing with NaHCO3 aqueous solution to el 10 (91%) [a] shing with NaHCO3 aqueous solution to 10 (91%) [a] ing with NaHCO3 aqueous solution to el 10 (91%) [a] shing with NaHCO3 aqueous solution to 8 (99%) [ ] 10 (91%) [a] h O l l 8 (99%) 10 (91%) [a] h h CO l 8 (99%) [a] 10 (91%) [a] 8 (99%) [a] 10 (91%) [a] According to According t According to . According t 11 (99%) [a] According to 11 (99%) [a] According to 11 (99%) [a] 11 (99%) [a] 11 (99%) [a] 11 (99%) [a] CO3 aqueous re quantitativ HCO3 aqueou were quantitat CO3 aqueous HCO3 aqueou 10 (91%) [a] CO3 aqueous 10 (91%) [a] HCO3 aqueou 10 (91%) [a] 10 (91%) [a] 10 (91%) [a] 10 (91%) [a] NM a MS, e co e sio s e e qua i a i e It is still important to mention that, differently from the phenolic hydrazones, a melting was a , e o e io e e qua i a i e It is still important to mention that, differently from the phenolic hydrazones, a melting wa NMR and MS, the conversions were quantitative. It is still important to mention that differently from the phenolic hydrazones a melting was NMR and MS, the conversions were quantitative. It is still important to mention that differently from the phenolic hydrazones a melting w g q p g , NMR and MS, the conversions were quantitative. g q p g , NMR and MS, the conversions were quantitative. Molecules 2017, 22, 1457 5 of 27 Molecules 2017, 22, 1457 It is still important to mention that, differently from the phenolic hydrazones, a melting was produced when the p-TSA was added, and, therefore, the reaction was not fully in solid-state. The formation of a ﬂuid phase is vastly found for solid mixtures (eutectic melting) [68] and surely contributed to reach high conversions in short times of grinding for these hydrazones. Molecules 2017, 22, 1457 5 of 27 formation of a fluid phase is vastly found for solid mixtures (eutectic melting) [68] and surely contributed to reach high conversions in short times of grinding for these hydrazones. 2.2. Structural Analysis of Isonicotinoyl Hydrazones by DFT and NMR: Determination of the Free Activation Energy (∆G̸=) between Conformers of Selected Compounds 1a and 5 2.2. Structural Analysis of Isonicotinoyl Hydrazones by DFT and NMR: Determination of the Free Activation Energy (G≠) between Conformers of Selected Compounds 1a and 5 The structures of all the hydrazones were identiﬁed and fully characterized by 1H- and 13C-NMR, MS and HRMS, FTIR and UV-vis (see Supplementary Materials). The structures of all the hydrazones were identified and fully characterized by 1H- and 13C-NMR, MS and HRMS, FTIR and UV-vis (see Supplementary Materials). C h d ll l h d h d h f f Concerning the NMR data, all isonicotinoyl hydrazones showed the presence of conformers in dimethylsulfoxide (DMSO) solution. NMR spectral and theoretical studies previously demonstrated that acylhydrazones generally exist predominantly or solely as a mixture of isomers [69–71]. In theory, N-acylhydrazones may exist with four possible arrangements in respect to (E/Z)-conﬁgurational isomers relative to the C=N bond and (E’/Z’)-rotamers caused by inversion of the amide bonds C(O)NH, here named cis/trans amide conformers (Figure 3) [69,72]. Concerning the NMR data, all isonicotinoyl hydrazones showed the presence of conformers in dimethylsulfoxide (DMSO) solution. NMR spectral and theoretical studies previously demonstrated that acylhydrazones generally exist predominantly or solely as a mixture of isomers [69–71]. In theory, N-acylhydrazones may exist with four possible arrangements in respect to (E/Z)- configurational isomers relative to the C=N bond and (E’/Z’)-rotamers caused by inversion of the amide bonds C(O)NH, here named cis/trans amide conformers (Figure 3) [69,72]. Figure 3. E/Z-configurational isomers and cis/trans amide conformers for N-acylhydrazones. Figure 3. E/Z-conﬁgurational isomers and cis/trans amide conformers for N-acylhydrazones. Figure 3. E/Z-configurational isomers and cis/trans amide conformers for N-acylhydrazones. Figure 3. E/Z-conﬁgurational isomers and cis/trans amide conformers for N-acylhydrazones. Molecules 2017, 22, 1457 Although the four forms were considered, E/Z isomerization is generally not observed and the Z geometric isomers are absent or present only in poor part. An exception is for R’ = 2-pyridyl, in which strong intramolecular hydrogen bonds are present in the (Z)-form [70] mainly in less polar Although the four forms were considered, E/Z isomerization is generally not observed and the Z geometric isomers are absent or present only in poor part. An exception is for R’ = 2-pyridyl, in which strong intramolecular hydrogen bonds are present in the (Z)-form [70] mainly in less polar solvents. solvents. While all isonicotinoyl hydrazones showed the presence of conformers in DMSO solution, for study purposes, two derivatives were chosen to be further investigated: phenolic hydrazone (1a) and indazole derivative (5). Both of them showed two sets of signals indicating the possibility of equilibrium between rotamers in solution. Theoretical assessment of the existence of the isomers was While all isonicotinoyl hydrazones showed the presence of conformers in DMSO solution, for study purposes, two derivatives were chosen to be further investigated: phenolic hydrazone (1a) and indazole derivative (5). Both of them showed two sets of signals indicating the possibility of equilibrium between rotamers in solution. Theoretical assessment of the existence of the isomers was carried out. carried out. The four structures of Z/E geometrical isomers and cis/trans amide conformers of 1a were modeled by Density Functional theory (DFT), using Gaussian 09, firstly at HF/STO-3G level. The Z conformers were found higher in energy than the E ones, around 6 kcal/mol (see Table S1 in the Supplementary Materials). Thus, only the cisE and transE-isomers were then modeled at the B3LYP/6- 31+G(d,p) level and the frequencies calculations were performed on the optimized geometries at 298 The four structures of Z/E geometrical isomers and cis/trans amide conformers of 1a were modeled by Density Functional theory (DFT), using Gaussian 09, ﬁrstly at HF/STO-3G level. The Z conformers were found higher in energy than the E ones, around 6 kcal/mol (see Table S1 in the Supplementary Materials). Thus, only the cisE and transE-isomers were then modeled at the B3LYP/6-31+G(d,p) level and the frequencies calculations were performed on the optimized geometries at 298 K, showing all positive frequencies and allowing evaluation of the Gibbs free energy. K, showing all positive frequencies and allowing evaluation of the Gibbs free energy. Mechanosynthesis of Isonicotinoyl Hydrazones 2.1. Chemistry Mechanosynthesis of Isonicotinoyl Hydra ones [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H- NMR and MS, the conversions were quantitative. [a] Yields after washing with NaHCO3 aqueous solution to eliminate p-TSA. According to TLC, 1H-NMR and MS, the conversions were quantitative. Molecules 2017, 22, 1457 p At the coalescence temperature Tc At the coalescence temperature Tc kexch = π/√2 = 2.22· (Hz) kexch = (kB·Tc/h)exp(−G≠/RTc) (Eyringss equation G≠ = −RTcLn(kexch·h/kB·Tc) with kB = 1.38 × 10−23 kJ/K, h = 6.626 × 10−34 J·s, and R kexch = π∆ν/√2 = 2.22·∆ν (Hz) kexch = (kB·Tc/h)exp(−∆G̸=/RTc) (Eyringss equation) ∆G̸= = −RTcLn(kexch·h/kB·Tc) J J J The 1H-NMR study at increasing temperatures (from 298 K to 388 K) allo with kB = 1.38 × 10−23 kJ/K, h = 6.626 × 10−34 J·s, and R = 8.314 J·mol−1·K−1. The 1H-NMR study at increasing temperatures (from 298 K to 388 K) allo with kB = 1.38 × 10−23 kJ/K, h = 6.626 × 10−34 J·s, and R = 8.314 J·mol−1·K−1. of coalescence temperatures of the same signals of each isomer (Figure S1). Correlation of the coalescence temperatures Tc with the difference in chemical shift of the signals led to G≠ following the precedent equation described (Supplementary Materials, Table S3). The mean value obtained on several signals led to G≠ = 17.58 kcal·mol−1. This experimental value is in agreement with the calculated one of 17.63 kcal·mol−1 at the B3LYP/6-31+G(d,p) level. The same study was performed on compound 5, which possesses eight isomers. As previously d b d h f h h h h / O h d h h The 1H-NMR study at increasing temperatures (from 298 K to 388 K) allowed the determination of coalescence temperatures of the same signals of each isomer (Figure S1). Correlation of the coalescence temperatures Tc with the difference in chemical shift of the signals led to ∆G̸= following the precedent equation described (Supplementary Materials, Table S3). The mean value obtained on several signals led to ∆G̸= = 17.58 kcal·mol−1. This experimental value is in agreement with the calculated one of 17.63 kcal·mol−1 at the B3LYP/6-31+G(d,p) level. described, the optimization of geometries on the height isomers at the HF/STO-3G showed that the Z isomers are higher in energy (6–7.5 kcal/mol) (Supplementary Materials, Table S4). Tsshus, the calculations were carried out at the B3LYP/6-31+G(d,p) level on the four isomers: cis and trans E- isomers and the two rotamers, in the gas phase and in the DMSO modeled by the SMD polarizable continuum model. Boltzmann analysis was used to determine the relative distribution of each conformer both in the gas phase and in the DMSO continuum model. Molecules 2017, 22, 1457 Following Boltzmann distribution, (Pi/Pj = exp((Gj − Gi)/kBT), cisE-isomer was present at 92% and transE at 8% in the gas phase, whereas in DMSO, using the polarizable solvent continuum model (SMD) th ti i t d i /t 6/94 (T bl 2) Following Boltzmann distribution, (Pi/Pj = exp((Gj −Gi)/kBT), cisE-isomer was present at 92% and transE at 8% in the gas phase, whereas in DMSO, using the polarizable solvent continuum model (SMD), the ratio was inverted: cis/trans: 6/94 (Table 2). 6 of 27 6 of 27 6 of 27 6 of 27 Molecules 2017, 22, 1457 Molecules 2017, 22, 1457 Table 2. Geometries and energies of minima and transition state for cis and trans E-isomers of 1a obtained at B3LYP/6-31+G(d,p) level in the gas phase and using the DMSO polarizable continuum model (SMD). Table 2. Geometries and energies of minima and transition state for cis and trans E-isomers of 1a obtained at B3LYP/6-31+G(d,p) level in the gas phase and using the DMSO polarizable continuum model (SMD). Table 2. Geometries and energies of minima and transition state for cis and trans E-isomers of 1a obtained at B3LYP/6-31+G(d,p) level in the gas phase and using the DMSO polarizable continuum model (SMD). Table 2. Geometries and energies of minima and transition state for cis and trans E-isomers of 1a obtained at B3LYP/6-31+G(d,p) level in the gas phase and using the DMSO polarizable continuum model (SMD). transE-1a cisE-1a TS-E-1a In the Gas Phase E (ua) G (ua) G (kcal/mol) % transE_1a −816.730569 −816.553707 1.47 8 cisE_1a −816.733307 −816.55605 0 92 In DMSO E (ua) G (ua) G (kcal/mol) % transE_1a −816.763427 −816.585816 −1.68 94 cisE_1a −816.761415 −816.583145 0 6 TS_1a −816.765271 −816.557724 17.63 - In the Gas Phase E (ua) G (ua) ∆G (kcal/mol) % transE_1a −816.730569 −816.553707 1.47 8 cisE_1a −816.733307 −816.55605 0 92 In DMSO E (ua) G (ua) ∆G (kcal/mol) % transE_1a −816.763427 −816.585816 −1.68 94 cisE_1a −816.761415 −816.583145 0 6 TS_1a −816.765271 −816.557724 17.63 - A transition state (TS-1a) was found between transE and cisE, characterized by its imaginary frequency at −115.79 cm−1. The difference between Gibbs free energy of the transition state and the energy of the cis-E-isomer (G≠) was equal to 17.63 kcal·mol−1. A transition state (TS-1a) was found between transE and cisE, characterized by its imaginary frequency at −115.79 cm−1. Molecules 2017, 22, 1457 The difference between Gibbs free energy of the transition state and the energy of the cis-E-isomer (∆G̸=) was equal to 17.63 kcal·mol−1. e e gy o e cis E iso e ( G ) as equa o 63 a o The NMR chemical shift calculations were then performed at B3LYP/6-311+ (2d,p) level, using the DMSO polarizable continuum model (PCM). Isotropic shielding constants () for 1H and 13C nuclei were transformed in chemical shift (δ) using linear regression procedure proposed by Lodewyk [73]. Calculated values are presented in the Supplementary Materials (Table S2). By comparing calculated values and experimental ones, we unambiguously concluded that the transE- isomer is the major one. This result is also in agreement with the calculated Gibbs free energy values in DMSO, at 298 K, as transE-isomer is −1.68 kcal/mol lower than the cis (ratio cis/trans around 6/94). gy q The NMR chemical shift calculations were then performed at B3LYP/6-311+ (2d,p) level, using the DMSO polarizable continuum model (PCM). Isotropic shielding constants (σ) for 1H and 13C nuclei were transformed in chemical shift (δ) using linear regression procedure proposed by Lodewyk [73]. Calculated values are presented in the Supplementary Materials (Table S2). By comparing calculated values and experimental ones, we unambiguously concluded that the transE-isomer is the major one. This result is also in agreement with the calculated Gibbs free energy values in DMSO, at 298 K, as transE-isomer is −1.68 kcal/mol lower than the cis (ratio cis/trans around 6/94). gy ( ) q The NMR chemical shift calculations were then performed at B3LYP/6-311+ (2d,p) level, using the DMSO polarizable continuum model (PCM). Isotropic shielding constants () for 1H and 13C nuclei were transformed in chemical shift (δ) using linear regression procedure proposed by The NMR chemical shift calculations were then performed at B3LYP/6-311+ (2d,p) level, using the DMSO polarizable continuum model (PCM). Isotropic shielding constants (σ) for 1H and 13C nuclei were transformed in chemical shift (δ) using linear regression procedure proposed by Lodewyk [73]. , , / ( / / ) This activation energy can also be measured by 1H-NMR analysis, by determining the coalescence temperature Tc. This activation energy can also be measured by 1H-NMR analysis, by determining the coalescence temperature Tc. 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability Stability studies were carried out in order to confirm that biological activities evidenced for isonicotinoyl hydrazones arise from the tested compounds, and not from the hydrolysis of the imine bond. UV-vis spectrophotometry at the λmax of absorbance of the related molecule was used to monitor the stability of the most prominent synthesized compounds. The study was carried out for all compounds; here, we present the results for some of them while all other data are in the Supplementary Materials. Table 4 summarizes the conditions used and the stability results observed for 1a–1d, 5 and 7 compounds. Stability studies were carried out in order to confirm that biological activities evidenced for isonicotinoyl hydrazones arise from the tested compounds, and not from the hydrolysis of the imine bond. UV-vis spectrophotometry at the λmax of absorbance of the related molecule was used to monitor the stability of the most prominent synthesized compounds. The study was carried out for all compounds; here, we present the results for some of them while all other data are in the Supplementary Materials. Table 4 summarizes the conditions used and the stability results observed for 1a–1d, 5 and 7 compounds. Stability studies were carried out in order to confirm that biological activities evidenced for isonicotinoyl hydrazones arise from the tested compounds, and not from the hydrolysis of the imine bond. UV-vis spectrophotometry at the λmax of absorbance of the related molecule was used to monitor the stability of the most prominent synthesized compounds. The study was carried out for all compounds; here, we present the results for some of them while all other data are in the Supplementary Materials. Table 4 summarizes the conditions used and the stability results observed for 1a–1d, 5 and 7 compounds. Stability studies were carried out in order to confirm that biological activities evidenced for isonicotinoyl hydrazones arise from the tested compounds, and not from the hydrolysis of the imine bond. UV-vis spectrophotometry at the λmax of absorbance of the related molecule was used to monitor the stability of the most prominent synthesized compounds. The study was carried out for all compounds; here, we present the results for some of them while all other data are in the Supplementary Materials. Table 4 summarizes the conditions used and the stability results observed for 1a–1d, 5 and 7 compounds. Molecules 2017, 22, 1457 Isomer Geometry (In Gas Phase) G (In Gas Phase) % G (In DMSO) % transE_5_1 −888.90736 0.34 −888.94471 28.22 transE_5_2 −888.909926 5.09 −888.94528 51.50 cisE_5_1 −888.910465 9.0 −888.941968 1.54 cisE_5_2 −888.912591 85.6 −888.944326 18.72 −888.90736 0.34 −888.94471 28.22 transE_5_2 ( ,p) g p ( ) Isomer Geometry (In Gas Phase) G (In Gas Phase) % G (In DMSO) % transE_5_1 −888.90736 0.34 −888.94471 28.22 transE_5_2 −888.909926 5.09 −888.94528 51.50 cisE_5_1 −888.910465 9.0 −888.941968 1.54 cisE_5_2 −888.912591 85.6 −888.944326 18.72 −888.909926 5.09 −888.94528 51.50 cisE_5_1 5 at the B3LYP/6 31 G(d,p) in the gas phase and in the DMSO continuum solvent model (SMD). Isomer Geometry (In Gas Phase) G (In Gas Phase) % G (In DMSO) % transE_5_1 −888.90736 0.34 −888.94471 28.22 transE_5_2 −888.909926 5.09 −888.94528 51.50 cisE_5_1 −888.910465 9.0 −888.941968 1.54 cisE_5_2 −888.912591 85.6 −888.944326 18.72 −888.910465 9.0 −888.941968 1.54 cisE_5_2 5 at the B3LYP/6 31 G(d,p) in the gas phase and in the DMSO continuum solvent model (SMD). Isomer Geometry (In Gas Phase) G (In Gas Phase) % G (In DMSO) % transE_5_1 −888.90736 0.34 −888.94471 28.22 transE_5_2 −888.909926 5.09 −888.94528 51.50 cisE_5_1 −888.910465 9.0 −888.941968 1.54 cisE_5_2 −888.912591 85.6 −888.944326 18.72 −888.912591 85.6 −888.944326 18.72 1H- and 13C-NMR chemical shifts were calculated as previously described at the B3LYP/6- 311+G(2d,p) using DMSO continuum model (PCM) and taking into account the Boltzmann distribution of the two conformers for each isomer, which means transE-1/transE-2: 35%/65%, and cisE-1/cisE-2: 8%/92%. The comparison of calculated chemical shifts and experimental ones of both 1H and 13C (Supplementary Materials, Figure S2 and Table S5), showed that trans isomer is the major compound in DMSO. 1H- and 13C-NMR chemical shifts were calculated as previously described at the B3LYP/6- 311+G(2d,p) using DMSO continuum model (PCM) and taking into account the Boltzmann distribution of the two conformers for each isomer, which means transE-1/transE-2: 35%/65%, and cisE-1/cisE-2: 8%/92%. The comparison of calculated chemical shifts and experimental ones of both 1H and 13C (Supplementary Materials, Figure S2 and Table S5), showed that trans isomer is the major compound in DMSO. Molecules 2017, 22, 1457 The a ti atio e e y a e aluated by 1H NMR a aly i at i ea i te e atu e (f o 298 1H- and 13C-NMR chemical shifts were calculated as previously described at the B3LYP/6- 311+G(2d,p) using DMSO continuum model (PCM) and taking into account the Boltzmann distribution of the two conformers for each isomer, which means transE-1/transE-2: 35%/65%, and cisE-1/cisE-2: 8%/92%. The comparison of calculated chemical shifts and experimental ones of both 1H and 13C (Supplementary Materials, Figure S2 and Table S5), showed that trans isomer is the major compound in DMSO. Th i i l d b 1H NMR l i i i (f 298 1H- and 13C-NMR chemical shifts were calculated as previously described at the B3LYP/6- 311+G(2d,p) using DMSO continuum model (PCM) and taking into account the Boltzmann distribution of the two conformers for each isomer, which means transE-1/transE-2: 35%/65%, and cisE-1/cisE-2: 8%/92%. The comparison of calculated chemical shifts and experimental ones of both 1H and 13C (Supplementary Materials, Figure S2 and Table S5), showed that trans isomer is the major compound in DMSO. Th i i l d b 1H NMR l i i i (f 298 1H- and 13C-NMR chemical shifts were calculated as previously described at the B3LYP/6-311+G(2d,p) using DMSO continuum model (PCM) and taking into account the Boltzmann distribution of the two conformers for each isomer, which means transE-1/transE-2: 35%/65%, and cisE-1/cisE-2: 8%/92%. The comparison of calculated chemical shifts and experimental ones of both 1H and 13C (Supplementary Materials, Figure S2 and Table S5), showed that trans isomer is the major compound in DMSO. The activation energy was evaluated by 1H-NMR analysis at increasing temperatures (from 298 K to 388 K) and was evaluated at 17.8 kcal/mol (Supplementary Materials, Table S6) in the same order as for compound 1a. The activation energy was evaluated by 1H-NMR analysis at increasing temperatures (from 298 K to 388 K) and was evaluated at 17.8 kcal/mol (Supplementary Materials, Table S6) in the same order as for compound 1a. Th b ti i t t t i t d t ti i ht h i t The activation energy was evaluated by 1H-NMR analysis at increasing temperatures (from 298 K to 388 K) and was evaluated at 17.8 kcal/mol (Supplementary Materials, Table S6) in the same order as for compound 1a. Molecules 2017, 22, 1457 Th b i i i d i i h h i The activation energy was evaluated by 1H-NMR analysis at increasing temperatures (from 298 K to 388 K) and was evaluated at 17.8 kcal/mol (Supplementary Materials, Table S6) in the same order as for compound 1a. Th b i i i d i i h h i The activation energy was evaluated by 1H-NMR analysis at increasing temperatures (from 298 K to 388 K) and was evaluated at 17.8 kcal/mol (Supplementary Materials, Table S6) in the same order as for compound 1a. These observations are important as restricted rotations might have an impact on pharmacological properties. These observations are important as restricted rotations might have an impact on pharmacological properties. These observations are important as restricted rotations might have an impact on pharmacological properties. These observations are important as restricted rotations might have an impact on pharmacological properties. p These observations are important as restricted rotations might have an impact on pharmacological properties. 2.3. Physicochemical Studies of Some Isonicotinoyl Hydrazones 2.3. Physicochemical Studies of Some Isonicotinoyl Hydrazones 2.3. Physicochemical Studies of Some Isonicotinoyl Hydrazones 2.3. Physicochemical Studies of Some Isonicotinoyl Hydrazones 2.3. Physicochemical Studies of Some Isonicotinoyl Hydrazones Molecules 2017, 22, 1457 Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of 5 at the B3LYP/6 31+G(d p) in the gas phase and in the DMSO continuum solvent model (SMD) Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of 5 at the B3LYP/6-31+G(d,p) in the gas phase and in the DMSO continuum solvent model (SMD). Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of 5 at the B3LYP/6-31+G(d p) in the gas phase and in the DMSO continuum solvent model (SMD) Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of 5 at the B3LYP/6-31+G(d p) in the gas phase and in the DMSO continuum solvent model (SMD) at the B3LYP/6-31+G(d,p) in the gas phase and in the DMSO continuum solvent model (SMD). Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers o 5 at the B3LYP/6 31+G(d p) in the gas phase and in the DMSO continuum solvent model (SMD) Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers o 5 at the B3LYP/6-31+G(d,p) in the gas phase and in the DMSO continuum solvent model (SMD). Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers o 5 at the B3LYP/6-31+G(d p) in the gas phase and in the DMSO continuum solvent model (SMD) Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers o 5 at the B3LYP/6-31+G(d p) in the gas phase and in the DMSO continuum solvent model (SMD) Isomer Geometry (In Gas Phase) G (In Gas Phase) % G (In DMSO) % transE_5_1 5 at the B3LYP/6 31+G(d,p) in the gas phase and in the DMSO continuum solvent model (SMD). Molecules 2017, 22, 1457 In the gas phase, cis compounds are the major ones, although, in the DMSO continuum, trans h E % i E % ( bl ) The same study was performed on compound 5, which possesses eight isomers. As previously described, the optimization of geometries on the height isomers at the HF/STO-3G showed that the Z isomers are higher in energy (6–7.5 kcal/mol) (Supplementary Materials, Table S4). Tsshus, the calculations were carried out at the B3LYP/6-31+G(d,p) level on the four isomers: cis and trans E-isomers and the two rotamers, in the gas phase and in the DMSO modeled by the SMD polarizable continuum model. Boltzmann analysis was used to determine the relative distribution of each conformer both in the gas phase and in the DMSO continuum model. 7 of 27 Molecules 2017, 22, 1457 In the gas phase, cis compounds are the major ones, although, in the DMSO continuum, trans isomers are the major ones: transE: 80%, cisE: 20% (Table 3). Molecules 2017, 22, 1457 7 of 27 Molecules 2017, 22, 1457 7 of 27 Molecules 2017, 22, 1457 7 of 27 Molecules 2017, 22, 1457 7 of 27 Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of 5 Table 3 Geo et ies Gibbs f ee e e gies a d Bolt a dist ibutio of the fou ajo co fo e s of Table 3 Geometries Gibbs free energies and Boltzmann distribution of the four major conformers o Table 3 Geometries Gibbs free energies and Boltzmann distribution of the four major conformers o Table 3 Geometries Gibbs free energies and Boltzmann distribution of the four major conformers of Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of 5 at the B3LYP/6-31+G(d p) in the gas phase and in the DMSO continuum solvent model (SMD) Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of Table 3. Geometries, Gibbs free energies and Boltzmann distribution of the four major conformers of 5 at the B3LYP/6-31+G(d,p) in the gas phase and in the DMSO continuum solvent model (SMD). Table 3. 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability Stability studies were carried out in order to conﬁrm that biological activities evidenced for isonicotinoyl hydrazones arise from the tested compounds, and not from the hydrolysis of the imine bond. UV-vis spectrophotometry at the λmax of absorbance of the related molecule was used to monitor the stability of the most prominent synthesized compounds. The study was carried out for all compounds; here, we present the results for some of them while all other data are in the Supplementary Materials. Table 4 summarizes the conditions used and the stability results observed for 1a–1d, 5 and 7 compounds. p As expected, UV-vis spectroscopy demonstrated that all the tested compounds are stable for a prolonged time (up to 22 h–7 days) and no signiﬁcant decomposition was observed. Only 5 showed a very small reduction (3%) of the absorbance after 20 h. It is known that hydrazones possess greater 8 of 27 8 of 27 Molecules 2017, 22, 1457 Molecules 2017, 22, 1457 intrinsic hydrolytic stability than that of imines. In addition to the contribution of the NH-N=C in electron delocalization, the resonance forms in acylhydrazones increase the negative-charge on the C=N and thus, reduce its electrophilicity and the afﬁnity to the nucleophile attack from water [74]. Furthermore, the repulsion of the lone pairs of the NH-N can be relieved in the conjugates [75]. Table 4. Stability study of hydrazones. Compound Medium Conc. (mol/L) pH Time Stability 1a 28% EtOH/H2O 6.0 × 10−5 6.1 22 h stable 1b 6.3 × 10−5 6.5 22 h stable 1c 6.9 × 10−5 6.3 15 h stable Table 4. Stability study of hydrazones. Compound Medium Conc. (mol/L) pH Time Stability 1a 28% EtOH/H2O 6.0 × 10−5 6.1 22 h stable 1b 6.3 × 10−5 6.5 22 h stable 1c 6.9 × 10−5 6.3 15 h stable 1d 5.9 × 10−5 6.2 21 h stable 5 5% EtOH/PIPES buffer (50 mM) 4.1 × 10−5 6.8 20 h 3% Abs. reduction 7 3.1 × 10−5 6.8 20 h stable 7 days 5% Abs. reduction 1d 5.9 × 10−5 6.2 21 h stable 5 5% EtOH/PIPES buffer (50 mM) 4.1 × 10−5 6.8 20 h 3% Abs. reduction 7 3.1 × 10−5 6.8 20 h stable 7 days 5% Abs. reduction As expected, UV-vis spectroscopy demonstrated that all the tested compounds are stable for a prolonged time (up to 22 h–7 days) and no significant decomposition was observed. 2.3.2. pKa Determination 2.3.2. pKa Determination The acid dissociation constant (Ka, or more commonly expressed by pKa) is a very important physicochemical parameter in a wide range of research areas, including the development of active molecules due to solubility issues. The pKa of the molecules studied for stability could also be determined by using UV-vis spectrometry based on the variation of the absorbance as function of the pH due to the presence of chromophores close to the ionization site of the molecules. The molar absorptivity varies according to the conjugation forms that dynamically change when and where the molecule is charged by the effect of pH and protonation/dissociation. As an example, the different protonation states for the hydrazone 1d are shown in Figure 4. The acid dissociation constant (Ka, or more commonly expressed by pKa) is a very important physicochemical parameter in a wide range of research areas, including the development of active molecules due to solubility issues. The pKa of the molecules studied for stability could also be determined by using UV-vis spectrometry based on the variation of the absorbance as function of the pH due to the presence of chromophores close to the ionization site of the molecules. The molar absorptivity varies according to the conjugation forms that dynamically change when and where the molecule is charged by the effect of pH and protonation/dissociation. As an example, the different protonation states for the hydrazone 1d are shown in Figure 4. Figure 4. Protonation states of the hydrazone 1d as function of pH. Figure 4. Protonation states of the hydrazone 1d as function of pH. Figure 4. Protonation states of the hydrazone 1d as function of pH. Figure 4. Protonation states of the hydrazone 1d as function of pH. For experimental pKa determination, the molecules were solubilized in hydroalcoholic solutions at ethanol 28% and the ionic strength was maintained by the addition of KCl (0.1 M). The pH was adjusted with concentrated solutions of KOH and HCl. The UV spectra were recorded at each pH value and, at least three wavelengths were monitored. The absorbance variation was plotted as function of the pH. As an example, the variations on the UV-vis spectra for compound 1d and the resulting plot for six wavelength values are shown in Figure 5a,b, respectively (all other spectra are present in Figures S4–S8). 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability 2.3.1. Hydrolytic Stability Only 5 showed a very small reduction (3%) of the absorbance after 20 h. It is known that hydrazones possess greater intrinsic hydrolytic stability than that of imines. In addition to the contribution of the NH-N=C in electron delocalization, the resonance forms in acylhydrazones increase the negative-charge on the C=N and thus, reduce its electrophilicity and the affinity to the nucleophile attack from water [74]. Furthermore the repulsion of the lone pairs of the NH N can be relieved in the conjugates [75] Table 4. Stability study of hydrazones. 5.9 × 10 5 6.2 4.1 × 10−5 6.8 Moreover, the stability of compound 1d was further assessed upon incubation in DMSO, the solvent used for biological assays, by mass spectrometry analysis; in addition, no relevant peaks corresponding to possibly released INH have been detected (Figure S3). , p p j g [ ] Moreover, the stability of compound 1d was further assessed upon incubation in DMSO, the solvent used for biological assays, by mass spectrometry analysis; in addition, no relevant peaks corresponding to possibly released INH have been detected (Figure S3). Moreover, the stability of compound 1d was further assessed upon incubation in DMSO, the solvent used for biological assays, by mass spectrometry analysis; in addition, no relevant peaks corresponding to possibly released INH have been detected (Figure S3). p p j g [ ] Moreover, the stability of compound 1d was further assessed upon incubation in DMSO, the solvent used for biological assays, by mass spectrometry analysis; in addition, no relevant peaks corresponding to possibly released INH have been detected (Figure S3). 2.3.2. pKa Determination 2.3.2. pKa Determination The protonation/dissociation of the molecule is accompanied by a variation For experimental pKa determination, the molecules were solubilized in hydroalcoholic solutions at ethanol 28% and the ionic strength was maintained by the addition of KCl (0.1 M). The pH was adjusted with concentrated solutions of KOH and HCl. The UV spectra were recorded at each pH value and, at least three wavelengths were monitored. The absorbance variation was plotted as function of the pH. As an example, the variations on the UV-vis spectra for compound 1d and the resulting plot for six wavelength values are shown in Figure 5a,b, respectively (all other spectra are present in Figures S4–S8). The protonation/dissociation of the molecule is accompanied by a variation in the 9 of 27 7 Molecules 2017, 22, 1457 absorbance (Figure 5a) and is graphically represented for six wavelength values as function of pH in Figure 5b. in the absorbance (Figure 5a) and is graphically represented for six wavelength values as function of pH in Figure 5b. (a) (b) Figure 5. The variations on the UV-vis spectra for compound 1d and the resulting plot for six wavelength values. (a) UV-vis spectra obtained for compound 1d as function of pH variation; (b) Plots of absorbance for six wavelength values (λ/nm) as function of pH for compound 1d. Th i fl ti i t d t th h i th t t t d/di i t d t t f th Wavelength (nm) 250 300 350 400 450 500 Absorbance (AU) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 221 334 Figure 5. The variations on the UV-vis spectra for compound 1d and the resulting plot for six wavelength values. (a) UV-vis spectra obtained for compound 1d as function of pH variation; (b) Plots of absorbance for six wavelength values (λ/nm) as function of pH for compound 1d. (a) Wavelength (nm) 250 300 350 400 450 500 Absorbance (AU) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 221 334 (a) (a) (a) (b) (b) Figure 5. The variations on the UV-vis spectra for compound 1d and the resulting plot for six wavelength values. (a) UV-vis spectra obtained for compound 1d as function of pH variation; (b) Plots of absorbance for six wavelength values (λ/nm) as function of pH for compound 1d. Figure 5. The variations on the UV-vis spectra for compound 1d and the resulting plot for six wavelength values. 2.3.2. pKa Determination 2.3.2. pKa Determination (a) UV-vis spectra obtained for compound 1d as function of pH variation; (b) Plots of absorbance for six wavelength values (λ/nm) as function of pH for compound 1d. molecule, and, therefore, the pH at that point amounts to the pKa. Thus, by using this method, the pKa values of 1d were determined [76]. The same protocol was applied for the other molecules and the resulted pKa are presented in Table 5, as an average value from three different λ. Table 5. pKa values determined for the isonicotinoylhydrazones. The inﬂection points correspond to the change in that protonated/dissociated state of the molecule, and, therefore, the pH at that point amounts to the pKa. Thus, by using this method, the pKa values of 1d were determined [76]. The same protocol was applied for the other molecules and the resulted pKa are presented in Table 5, as an average value from three different λ. Compound 1a 1b 1c 1d 5 7 pKa1 nd a nd a 3.4 3.0 3.4 3.6 pKa2 9.2 ± 0.1 7.4 ± 0.1 9.1 ± 0.1 9.5 ± 0.1 10.4 ± 0.1 11 pKa3 - 8.9 ± 0.1 - >11 - - a nd for not determined. Table 5. pKa values determined for the isonicotinoylhydrazones. Compound 1a 1b 1c 1d 5 7 pKa1 nd a nd a 3.4 3.0 3.4 3.6 pKa2 9.2 ± 0.1 7.4 ± 0.1 9.1 ± 0.1 9.5 ± 0.1 10.4 ± 0.1 11 pKa3 - 8.9 ± 0.1 - >11 - - a nd for not determined. 1a 1b 1c 1d d d 3 4 3 0 3 Table 5. pKa values determined for the isonicotinoylhydrazones. a nd for not determined. An inﬂection point was always observed close to pH 3, which is related with the protonation of 4-pyridinic nitrogen of isoniazid moiety (pKa2 of isoniazid = 3.5), and corresponds to pKa1 of the 10 of 27 Molecules 2017, 22, 1457 hydrazones. The pKa2 of the molecules ranges from 7.7 to 11. It means that they are in dissociated form from milder (1b) to stronger (7) basic conditions. hydrazones. The pKa2 of the molecules ranges from 7.7 to 11. It means that they are in dissociated form from milder (1b) to stronger (7) basic conditions. 2.4. Biology of Mechanochemically Synthesized Hydrazones 2.4. Biology of Mechanochemically Synthesized Hydrazones 2.4.1. InhA Inhibition Assay InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoylreductase from M. tuberculosis, has emerged as a promising drug target due to its vital role in synthesis of mycolic acids. InhA is the main target of Isoniazid [77–80]. Recombinant M.tb InhA was expressed in E. coli and subsequently puriﬁed. The selected synthetic compounds, corresponding to the phenolic isonicotinoyl hydrazones 1a–d and the corresponding heterocyclic ones 2–11 series, were evaluated in vitro for the inhibition of M.tb InhA activity at 50 µM by applying a previously described method [81] (Table 6). Table 6. Enzyme inhibition values for the INH derivatives. Results are expressed as a percentage of InhA inhibition. Compound % Inhibition at 50 µM (Inhibitor) Compound % Inhibition at 50 µM (Inhibitor) 1a 45 5 19 1b 54 6 43 1c 48 7 39 1d 64 8 42 2 54 9 32 3 3 10 79 4 33 11 not soluble TCL >99 Considering the four phenolic derivatives 1a–d, the tri-substituted one (1d) presents the better InhA inhibition activity with 64% value at 50 µM. Derivatives 1a–c are less potent with 45–54% InhA inhibition range at 50 µM. Under these conditions, 99% inhibition is obtained for Triclosan (TCL). Concerning the heterocyclic isonicotinoyl hydrazones, compounds 2, 3 and 11 were difﬁcult to evaluate due to solubility issues at high concentrations. Compound 5 is a very poor inhibitor (19% InhA inhibition), while compounds 4, 6 and 7 may be considered as poor inhibitors with 32%, 43% and 39% values, respectively. Compound 10 is the derivative exhibiting the highest inhibition of InhA enzyme with 79% at 50 µM. In comparing isonicotinoyl derivatives derived from indoles (compounds 6–10), we can notice that those possessing an indol-3-yl frame have the same activities with inhibition values between 32% and 43%. Compounds 6 and 8 differing by one methyl group (position 2 of the indol-3-yl frame) have the same inhibition values. On the contrary, compound 10 possessing an indole-2-yl frame is two-fold more active (79% of inhibition). Concerning the imidazole and indazole derivatives, we might hypothesize that the striking differences in activities could arise by a better positioning of the compounds 2 and 4 guided by their nitrogen atoms on their heterocyclic parts. Molecules 2017, 22, 1457 Molecules 2017, 22, 1457 11 of 27 It is noteworthy that a series of phenolic hydrazones bearing the four phenolic frames (a–d), previously synthetized by mechanochemical means were already tested against M.tb. cell growth [38]. Among the different hydrazines (isoniazid, hydralazine, 2-hydrazino-benzothiazole, 3-aminorhodanine, benzyl carbazate and benzhydrazide), used to form the corresponding hydrazones, only the isonicotinoyl derivatives were active against M.tb growth, whilst the other compounds were not effective (MIC > 30 µM; data not shown, but reported in Reference [39]). p Isonicotinoyl derivatives 1a–d showed good anti-mycobacterial activity with 0.0125 or 0.125 µg/mL MIC values. Among these four, those bearing two or more substituents ortho to the phenolic function presented potent activities with a MIC value of 0.125 µg/mL, which is ﬁve times higher than that of INH. Compound 1a, synthesized from p-hydroxybenzaldehyde and INH, is the most potent derivative with a MIC value of 0.0125 µg/mL (0.05 µM), which is 2–4 times lower than that of INH (0.025 µg/mL, 0.18 µM). 2.4.3. M.tb H37Rv Growth Inhibition Assays of Nitrogen Heterocyclic Hydrazones (2–11) 2.4.3. M.tb H37Rv Growth Inhibition Assays of Nitrogen Heterocyclic Hydrazones (2–11) The MIC values of the series of hydrazones, bearing the INH moiety coupled with different N-heterocyclic aldehydes, were also determined (Table 8). The MIC values of the series of hydrazones, bearing the INH moiety coupled with different N-heterocyclic aldehydes, were also determined (Table 8). Table 8. MICs of Isoniazid-Nitrogen heterocyclic hydrazones against M.tb H37Rv. Table 8. MICs of Isoniazid-Nitrogen heterocyclic hydrazones against M.tb H37Rv. Compound MW (g/mol) MIC (µg/mL)/(µM) LogP Cpd MW (g/mol) MIC (µg/mL)/(µM) LogP 2 215.21 0.03/0.14 −1.00 7 265.27 0.015/0.056 0.24 3 229.24 0.03/0.13 −1.37 8 264.28 0.06/0.23 0.86 4 265.27 0.06/0.23 1.38 9 343.18 0.125/0.36 1.69 5 265.27 0.03/0.11 −0.52 10 264.28 0.06/0.23 0.52 6 278.31 0.25/0.90 0.49 11 309.28 0.25/0.81 1.39 INH 137.14 0.05/0.36 −0.64 All four imidazole and indazole derivatives (2–5) were 1.5–3 times more effective than isoniazid, while their InhA inhibition activities were much lower to inexistent with activities against M.tb growth, in the range of 0.11–0.23 µM. For indole derivatives, nitro substitution on the aromatic ring (compound 11) or methyl substitution on the indole ring (compound 6), compromised the anti-TB activity. While the bromo derivative 9 had the same activity as INH (0.36 µM), compounds 7, 8 and 10 presented better MIC values than INH. 2.4.2. Activity of Phenolic Isonicotinoyl Hydrazones (1a–d) against M.tb Cell Growth 2.4.2. Activity of Phenolic Isonicotinoyl Hydrazones (1a–d) against M.tb Cell Growth The determination of the minimal inhibitory concentration (MIC) was performed using M.tb H37Rv strain and INH as control (Table 7). Table 7. Phenolic isonicotinoyl hydrazones tested as inhibitory agents against M.tb growth (H37Rv strain). Compound MW (g/mol) MIC (µg/mL)/(µM) LogP Cpd MW (g/mol) MIC (µg/mL)/(µM) LogP 1a 241.25 0.0125/0.05 1.64 1c 271.27 0.125/0.46 1.51 1b 257.24 0.125/0.49 1.25 1d 301.30 0.125/0.41 1.38 INH 137.14 0.025/0.18 −0.64 Table 7. Phenolic isonicotinoyl hydrazones tested as inhibitory agents against M.tb growth (H37Rv strain). Molecules 2017, 22, 1457 Interestingly, the azaindole derivative 7 showed the highest anti-TB activity in this series and was found as active as the phenolic compound 1a (MIC = 0.015 µg/mL/0.056 µM). Finally, we can notice that all active compounds here presented, can show different lipophilic values as given by their LogP values, probably supporting the inference that there is no clear relationship between lipophilicity and in vitro activity as pointed also by others [25]. The resistance to the current tested drugs (ﬁrst- and second-line) remains a very serious problem, mostly resulting from inhA and katG mutations [82] and culminates in the occurrence of M.tb multidrug-resistant (MDR) strains. Owing the good results obtained for the herein studied INH-derived hydrazones, we tested them against a M.tb multidrug-resistant clinical isolate (IC2; resistant to streptomycin, INH, rifampicin, ethambutol, pyrazinamide, ethionamide, and capreomicin) (Table 9). The indazole and indole derivatives were not active against IC2 isolate (MICs > 10 µg/mL), except for the nitro derivative 11. The imidazole containing derivatives 2 and 3 were poor active (MIC = 5–10 µg/mL). By comparing results for compounds 10 and 11, it appears that the nitro substituent improves the activity against IC2 clinical isolate. Interestingly, the phenolic derivatives 1a and 1b presented the best activities against the MDR isolate, with MIC of 2.5 µg/mL (10.36 µM) and 1 µg/mL (3.89 µM), respectively. 12 of 27 Molecules 2017, 22, 1457 Table 9. MIC of isoniazid derivatives against M.tb MDR isolate IC2. Table 9. MIC of isoniazid derivatives against M.tb MDR isolate IC2. Table 9. MIC of isoniazid derivatives against M.tb MDR isolate IC2. Compound MIC (µg/mL)/(µM) H37Rv IC2 1a 0.0125/0.05 2.5/10.36 1b 0.125/0.49 1/3.89 1c 0.125/0.46 >2.5/>9.22 1d 0.125/0.41 >2.5/>8.30 2 0.03/0.14 5/23.2 3 0.03/0.13 5–10/21.8–43.6 4 0.06/0.23 >10 5 0.03/0.11 >10 6 0.25/0.90 >10 7 0.015/0.056 >10 8 0.06/0.23 >10 9 0.125/0.36 >10 10 0.06/0.23 >10 11 0.25/0.81 5–10/18.9–37.8 INH 0.025/0.18 >2/>14.58 2.4.4. Cytotoxicity and Selectivity Index Determination 2.4.4. Cytotoxicity and Selectivity Index Determination Cytotoxicities of all compounds bearing the INH moiety were also evaluated on MRC5 human ﬁbroblast cells. Almost all compounds tested presented LD50 values above 80 µM (Table 10), with the exception of 1b (LD50 = 36.3 µM). The LD50 evaluation is essential to determine the selectivity index (SI), which indicates the best candidates in terms of high biological activity against the target and low cytotoxicity. The SI presented in Table 10 are the ratio between LD50 and the in vitro MIC value against M.tb H37Rv previously obtained. Table 10. Cytotoxicity (LD50) and selectivity index (SI) for the most active hydrazones against H37Rv M.tb. Compound LD50 (µM) SI Compound LD50 (µM) SI 1a >80 >1600 5 >80 >727 1b 36.3 74 6 129 143 1c >80 >173 7 >80 >1429 1d >80 >195 8 >80 >364 2 >80 >571 9 >80 >222 3 >80 >615 10 71.4 310 4 >80 >364 11 156 193 INH - - Apart from compound 1b, due to its high toxicity, the phenolic hydrazones presented good selectivities higher than 170. A great result was obtained for 1a which has the lowest MIC value (0.0125 µg/mL), LD50 > 80 µM and the highest SI (>1600). The N-heterocyclic INH derivatives 2–11 presented good SI values, with the exception of 6 and 11, which possessed the highest MIC values. Compounds 4, 8, 9 and 10 conducted to comparable SI. Compound 10 must be considered (MIC = 0.06 µg/mL, LD50 = 71.4 µM and SI = 310), mainly due to its InhA inhibition of 78%. Great SI values closer to 600 are found for 2 and 3 but, nonetheless, some reservation must be taken due to the poor solubility of these molecules. Finally, the SI of compound 5 is higher than 727 and 7 is highlighted with MIC = 0.015 µg/mL and an SI higher than 1469. 2.4.5. In Search for the Molecular Target of Prepared Hydrazones in Mycobacteria 2.4.5. In Search for the Molecular Target of Prepared Hydrazones in Mycobacteria In order to conﬁrm the enoylreductase InhA as a target of synthesized hydrazones, we analyzed the effect of selected compounds with the best MIC values and cytotoxicity scores on synthesis of 13 of 27 13 f 27 Molecules 2017, 22, 1457 mycolic acids in avirulent strain M.tb H37Ra. Tested hydrazones, speciﬁcally 1a, 3, 5, 7 and 10, as well as INH as control InhA inhibitor, were added to M.tb H37Ra culture when it reached early mid-log phase of growth and, after subsequent 24 h cultivation, 14C acetate was added as a metabolic tracer. TLC analysis of lipid fractions extracted from harvested 14C labeled cells revealed that, similar to INH, all of the tested hydrazones abolish the synthesis of trehalose monomycolates and trehalose dimycolates (Figure 6). mycolic acids in avirulent strain M.tb H37Ra. Tested hydrazones, specifically 1a, 3, 5, 7 and 10, as well as INH as control InhA inhibitor, were added to M.tb H37Ra culture when it reached early mid-log phase of growth and, after subsequent 24 h cultivation, 14C acetate was added as a metabolic tracer. TLC analysis of lipid fractions extracted from harvested 14C labeled cells revealed that, similar to INH, all of the tested hydrazones abolish the synthesis of trehalose monomycolates and trehalose dimycolates (Figure 6). mycolic acids in avirulent strain M.tb H37Ra. Tested hydrazones, specifically 1a, 3, 5, 7 and 10, as well as INH as control InhA inhibitor, were added to M.tb H37Ra culture when it reached early mid-log phase of growth and, after subsequent 24 h cultivation, 14C acetate was added as a metabolic tracer. TLC analysis of lipid fractions extracted from harvested 14C labeled cells revealed that, similar to INH, all of the tested hydrazones abolish the synthesis of trehalose monomycolates and trehalose dimycolates (Figure 6). Figure 6. TLC analysis of lipids extracted from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Lipids were separated in chloroform:methanol:water (20:4:0.5) and detected by autoradiography (TDM: trehalose dimycolates; TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin). Figure 6. TLC analysis of lipids extracted from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. 2.4.5. In Search for the Molecular Target of Prepared Hydrazones in Mycobacteria Lipids were separated in chloroform:methanol:water (20:4:0.5) and detected by autoradiography (TDM: trehalose dimycolates; TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin). Figure 6. TLC analysis of lipids extracted from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Lipids were separated in chloroform:methanol:water (20:4:0.5) and detected by autoradiography (TDM: trehalose dimycolates; TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin). Figure 6. TLC analysis of lipids extracted from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Lipids were separated in chloroform:methanol:water (20:4:0.5) and detected by autoradiography (TDM: trehalose dimycolates; TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin). Figure 6. TLC analysis of lipids extracted from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Lipids were separated in chloroform:methanol:water (20:4:0.5) and detected by autoradiography (TDM: trehalose dimycolates; TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin). Figure 6. TLC analysis of lipids extracted from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Lipids were separated in chloroform:methanol:water (20:4:0.5) and detected by autoradiography (TDM: trehalose dimycolates; TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin) Analysis of fatty/mycolic acids isolated from whole 14C labeled cells proved that these compounds specifically inhibit synthesis of mycolic acids (Figure 7). Analysis of fatty/mycolic acids isolated from whole 14C labeled cells proved that these compounds speciﬁcally inhibit synthesis of mycolic acids (Figure 7). Analysis of fatty/mycolic acids isolated from whole 14C labeled cells proved that these compounds specifically inhibit synthesis of mycolic acids (Figure 7). Analysis of fatty/mycolic acids isolated from whole 14C labeled cells proved that these compounds specifically inhibit synthesis of mycolic acids (Figure 7). Analysis of fatty/mycolic acids isolated from whole 14C labeled cells proved that these compounds speciﬁcally inhibit synthesis of mycolic acids (Figure 7). Analysis of fatty/mycolic acids isolated from whole 14C labeled cells proved that these compounds specifically inhibit synthesis of mycolic acids (Figure 7). Figure 7. TLC analysis of methyl esters of fatty (FAME) and mycolic (MAME) acids isolated from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. 2.4.5. In Search for the Molecular Target of Prepared Hydrazones in Mycobacteria Different forms of methyl esters were separated in n-hexane:ethyl acetate (95:5; 3×) and detected by autoradiography. (, methoxy, and keto refer to forms of MAMEs). 0.1 0.5 0 5 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5 g/mL 1a INH 3 5 7 10 FAME methoxy keto M A M E y y y ( g ) compounds specifically inhibit synthesis of mycolic acids (Figure 7). Figure 7. TLC analysis of methyl esters of fatty (FAME) and mycolic (MAME) acids isolated from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Different forms of methyl esters were separated in n-hexane:ethyl acetate (95:5; 3×) and detected by autoradiography. (, methoxy, and keto refer to forms of MAMEs). 0.1 0.5 0 5 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5 g/mL 1a INH 3 5 7 10 FAME methoxy keto M A M E Figure 7. TLC analysis of methyl esters of fatty (FAME) and mycolic (MAME) acids isolated from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Different forms of methyl esters were separated in n-hexane:ethyl acetate (95:5; 3×) and detected by autoradiography. (α, methoxy, and keto refer to forms of MAMEs). Figure 7. TLC analysis of methyl esters of fatty (FAME) and mycolic (MAME) acids isolated from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Different forms of methyl esters were separated in n-hexane:ethyl acetate (95:5; 3×) and detected by autoradiography. (, methoxy, and keto refer to forms of MAMEs). Figure 7. TLC analysis of methyl esters of fatty (FAME) and mycolic (MAME) acids isolated from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Different forms of methyl esters were separated in n-hexane:ethyl acetate (95:5; 3×) and detected by autoradiography. (, methoxy, and keto refer to forms of MAMEs). Figure 7. TLC analysis of methyl esters of fatty (FAME) and mycolic (MAME) acids isolated from 14C acetate labeled M.tb H37Ra cells treated with compounds 1a, 3, 5, 7 and 10, INH and DMSO as a control. Different forms of methyl esters were separated in n-hexane:ethyl acetate (95:5; 3×) and detected by autoradiography. A series 3.1. Material ability to inhibit M.tb H37Rv strain growth, the most active being 1a and 7. Imidazole derivative 5 and indazole one 10 are also active against H37Rv with their nitro substituent. Compounds 1a and 1b were the most effective against both M.tb H37Rv strain and drug-resistant IC2 isolate. The mechanism of anti-mycobacterial activities of selected hydrazones with the best scores regarding the MICs and toxicities was confirmed to be through the cessation of mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell. 4. Materials and Methods 4.1. Material All chemicals were obtained from Maybridge, TCI, Aldrich or Alfa Aesar, 97–99% and used without further purification. Nuclear magnetic resonance spectra (1H- and 13C-NMR) were recorded on Bruker AC 300, Avance-400 MHz and Avance-500 spectrometers with DMSO-d6 as solvent. Chemical shifts δ were expressed in parts per million (ppm) relative to TMS. Solvent residue signals were used for calibration of spectral data. Mass spectrometry (MS) data were obtained from the “Service Commun de Spectrométrie de masse” of the Plateforme Technique, Institut de Chimie de Toulouse (Toulouse France) MS were performed using a Waters Quadrupole Time of flight mass All chemicals were obtained from Maybridge, TCI, Aldrich or Alfa Aesar, 97–99% and used without further puriﬁcation. Nuclear magnetic resonance spectra (1H- and 13C-NMR) were recorded on Bruker AC 300, Avance-400 MHz and Avance-500 spectrometers with DMSO-d6 as solvent. Chemical shifts δ were expressed in parts per million (ppm) relative to TMS. Solvent residue signals were used for calibration of spectral data. Mass spectrometry (MS) data were obtained from the “Service Commun de Spectrométrie de masse” of the Plateforme Technique, Institut de Chimie de Toulouse (Toulouse, France). MS were performed using a Waters Quadrupole Time-of-ﬂight mass spectrometer XEVO G2-S QTof. The samples were dissolved in methanol and Electrospray ionization method was used. High-resolution mass spectra (HRMS) were recorded on a ThermoFinnigan MAT 95 XL spectrometer using electrospray ionization (ESI) methods. Melting points were measured using a Koﬂer heating bench system Heizbank Type WME (Wagner &Munz GmbH, Munich, Germany), with measuring accuracy of ±1 ◦C in the range of 50–260 ◦C. If the melting point was higher than 260 ◦C or if it could not be exactly determined because of an apparent degradation, the DSC analysis was employed. The analysis was performed in a ATG-DSC 111 (Sertaram). Molecules 2017, 22, 1457 Molecules 2017, 22, 1457 Molecules 2017, 22, 1457 Molecules 2017, 22, 1457 Next, we overproduced InhA protein in M.tb H37Ra and tested sensitivity of overproducer, as well as control strain carrying empty vector against synthesized hydrazones by drop dilution method. Clearly, this testing showed, that MICs of all of tested compounds against M.tb H37Ra pMV261-InhA are 5–10× higher comparing to control strain conﬁrming InhA as molecular target of these inhibitors inside mycobacterial cells (Figure 8). e , e o e p o u e I A p o ei i a a e e e i i i y o o e p o u e , a well as control strain carrying empty vector against synthesized hydrazones by drop dilution method. Clearly, this testing showed, that MICs of all of tested compounds against M.tb H37Ra pMV261-InhA are 5–10× higher comparing to control strain confirming InhA as molecular target of these inhibitors inside mycobacterial cells (Figure 8). Figure 8. Determination of sensitivity of M.tb H37Ra pMV261 and M.tb H37Ra pMV261-InhA against 1a (Left panel) and 3, 5, 7 and 10 (Right panel) by drop dilution method. Figure 8. Determination of sensitivity of M.tb H37Ra pMV261 and M.tb H37Ra pMV261-InhA against 1a (Left panel) and 3, 5, 7 and 10 (Right panel) by drop dilution method. Figure 8. Determination of sensitivity of M.tb H37Ra pMV261 and M.tb H37Ra pMV261-InhA against 1a (Left panel) and 3, 5, 7 and 10 (Right panel) by drop dilution method. Figure 8. Determination of sensitivity of M.tb H37Ra pMV261 and M.tb H37Ra pMV261-InhA against 1a (Left panel) and 3, 5, 7 and 10 (Right panel) by drop dilution method. 3. Conclusions 3. Materials and Methods A series 3.1. Material 2.4.5. In Search for the Molecular Target of Prepared Hydrazones in Mycobacteria (α, methoxy, and keto refer to forms of MAMEs). 14 of 27 14 of 27 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis The derivatives 1a–d were synthesized as previously described by us [38]. Compounds 1a–1d have also a CAS number: 1a (840-81-3); 1b (13838-18-1); 1c (149-17-7); 1d (315230-80-9). The derivatives 1a–d were synthesized as previously described by us [38]. Compounds 1a–1d have also a CAS number: 1a (840-81-3); 1b (13838-18-1); 1c (149-17-7); 1d (315230-80-9). The derivatives 1a–d were synthesized as previously described by us [38]. Compounds 1a–1d have also a CAS number: 1a (840-81-3); 1b (13838-18-1); 1c (149-17-7); 1d (315230-80-9). The derivatives 1a–d were synthesized as previously described by us [38]. Compounds 1a–1d have also a CAS number: 1a (840-81-3); 1b (13838-18-1); 1c (149-17-7); 1d (315230-80-9). 3.2.2. General Procedure for Isoniazid Nitrogen-Containing Heterocycles Derivatives 2–11 4.2.2. General Procedure for Isoniazid Nitrogen-Containing Heterocycles Derivatives 2–11 A mixture the solid reactants, INH (1 equivalent), the aldehyde (1 equivalent) and the catalyst (p-TSA, 0.5 equivalent) were placed in milling device and the reaction proceeded between 1 h–2 h, depending on the aldehyde. The Cryomill (Restch) was used for the screening of catalysts (milling started at the room temperature) at 25 Hz during 1 h. After the choice of the catalyst (p-TSA), all the reactions were carried out in the vibratory ball-mill Pulverisette 0 (Fritsch, Germany) equipped with a single stainless steel ball of 50 mm of diameter and 500 g, in a semi-spherical vessel of 9.5 cm of diameter. The plate vibrates with a frequency of 50 Hz and amplitude of 2.0 mm. The amounts of reactant powder were stoichiometric conditions for reactants totalizing 1 g + the amount of catalyst. The transformation was monitored by TLC. After the reaction time, the powder mixture was washed with a NaHCO3 solution to eliminate the catalyst and the powder was dried under vacuum. 1H-, 13C-NMR spectra and Mass Spectra for all new compounds are included in Supplementary Materials; NMR data show both conformers). NMR data reported below correspond to the major conformer. A mixture the solid reactants, INH (1 equivalent), the aldehyde (1 equivalent) and the catalyst (p-TSA, 0.5 equivalent) were placed in milling device and the reaction proceeded between 1 h–2 h, depending on the aldehyde. The Cryomill (Restch) was used for the screening of catalysts (milling started at the room temperature) at 25 Hz during 1 h. A series 3.1. Material The temperature programming was from 20 ◦C to 200 or 260 ◦C according to the sample with a constant rate of 5 ◦C/min under nitrogen atmosphere. Fourier Transformed Infrared Spectroscopy (FTIR) analysis for identiﬁcation was performed using KB ll t Th Ni l t 5700 t t (Th Ni l t M di WI USA) Th i “Service Commun de Spectrométrie de masse” of the Plateforme Technique, Institut de Chimie de Toulouse (Toulouse, France). MS were performed using a Waters Quadrupole Time-of-flight mass spectrometer XEVO G2-S QTof. The samples were dissolved in methanol and Electrospray ionization Fourier Transformed Infrared Spectroscopy (FTIR) analysis for identiﬁcation was performed using KBr pellets on a Thermo Nicolet 5700 spectrometer (Thermo-Nicolet, Madison, WI, USA). The main peaks/bands were identiﬁed, especially the –C=N– that is attributed to the hydrazone. FTIR studies “Service Commun de Spectrométrie de masse” of the Plateforme Technique, Institut de Chimie de Toulouse (Toulouse, France). MS were performed using a Waters Quadrupole Time-of-flight mass spectrometer XEVO G2-S QTof. The samples were dissolved in methanol and Electrospray ionization Fourier Transformed Infrared Spectroscopy (FTIR) analysis for identiﬁcation was performed using KBr pellets on a Thermo Nicolet 5700 spectrometer (Thermo-Nicolet, Madison, WI, USA). The main peaks/bands were identiﬁed, especially the –C=N– that is attributed to the hydrazone. FTIR studies 15 of 27 formed A). The 15 of 27 formed A). The Molecules 2017, 22, 1457 Fourier Transfo using KBr pellets on 22, 1457 with the solid hydrazines as function of temperature were recorded in IN10MX Thermo Scientiﬁc FTIR microscope equipped with THMS600 (Linkam Scientiﬁc Instruments, Tadworth, Surrey, UK) heating and freezing stage. studies with the solid hydrazines as function of temperature were recorded in IN10MX Thermo Scientific FTIR microscope equipped with THMS600 (Linkam Scientific Instruments, Tadworth, Surrey, UK) heating and freezing stage. 2C, C2,6), 134,36 (1C, C5′), 143.08 (2C, C3,5). KBr) ν cm−1: 3193.59 (N-H), 3038.08 (C-Har), 1648.96 (C=O), 1626.02 (C=N-N), 1596.86 1551.04 (Car N), 1506.46 (C=N). OH 182 μM 25 °C): λ = 309 nm ε = 5495 05 dm3·mol−1·cm−1 (very poorly soluble) g g UV-vis spectroscopy was performed using a HP (Hewlett Packard, Palo Alto, CA, USA) 8452A diode array spectrophotometer from 200 to 400 nm, with ethanol as a solvent at 20 ◦C and using quartz cells. A series 3.1. Material The molar absorptivity was determined for the wavelength with the highest absorbance through Lambert–Beer’s law with the molar absorptivity ε in (dm3·mol−1·cm−1) expressed for the λmax of the molecule. UV-vis spectroscopy was performed using a HP (Hewlett Packard, Palo Alto, CA, USA) 8452A diode array spectrophotometer from 200 to 400 nm, with ethanol as a solvent at 20 °C and using quartz cells. The molar absorptivity was determined for the wavelength with the highest absorbance through Lambert–Beer’s law with the molar absorptivity ε in (dm3·mol−1·cm−1) expressed for the λmax of the molecule. OH, 182 μM, 25 C): λ = 309 nm, ε = 5495.05 dm3·mol 1·cm 1 (very poorly soluble). , TOF, MeOH) m/z: 238.0708 [M + Na+]; 216.0887 [M + H+]. (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 216.0887. ethyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis After the choice of the catalyst (p-TSA), all the reactions were carried out in the vibratory ball-mill Pulverisette 0 (Fritsch, Germany) equipped with a single stainless steel ball of 50 mm of diameter and 500 g, in a semi-spherical vessel of 9.5 cm of diameter. The plate vibrates with a frequency of 50 Hz and amplitude of 2.0 mm. The amounts of reactant powder were stoichiometric conditions for reactants totalizing 1 g + the amount of catalyst. The transformation was monitored by TLC. After the reaction time, the powder mixture was washed with a NaHCO3 solution to eliminate the catalyst and the powder was dried under vacuum. 1H-, 13C-NMR spectra and Mass Spectra for all new compounds are included in Supplementary Materials; NMR data show both conformers). NMR data reported below correspond to the major conformer. 9 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). R (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). R (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 , 1551.49 (Car N). OH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 230.1049. -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) (E)-N′-((1H-imidazol-4-yl)methylene)isonicotinohydrazide (2) (E)-N′-((1H-imidazol-4-yl)methylene)isonicotinohydrazide (2) (E)-N′-((1H-imidazol-4-yl)methylene)isonicotinohydrazide (2) (E) N′ ((1H imidazol 4 yl)methylene)isonicotinohydrazide (2) m.p.: 296.7 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). 1H NMR (300 MH DMSO d ) δ 8 15 (d J 1 4 H 1H H ) 8 36 (dd J 6 6 1 5 H 2H H ) .: 296.7 ◦C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 0 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, m.p.: 296.7 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v H NMR ( MH DMSO d ) δ 8 1 (d J 1 m.p.: 296.7 ◦C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). 2.5 C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H 1H-NMR (300 MHz, DMSO-d6) δ ppm: 8.15 (d, J = 1.4 Hz, 1H, H5′), 8.36 (dd, J = 6.6, 1.5 Hz, 2H, H2,6), 8.52 (s, 1H, H-C=N), 9.10 (dd, J = 6.6, 1.5 Hz, 2H, H3,5), 9.21 (d, J = 1.3 Hz, 1H, H3′), 15.67 (s, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 122.02 (1C, C5′), 125.10 (2C, C2,6), 128.25 (1C, C1′), 138.37 (1C, C=N), 136,80 (1C, C3′), 144.77 (2C, C3,5), 146.91 (1C, C1), 160.14 (1C, C=O). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 8.15 (d, J = 1.4 Hz, 1H, H5′), 8.36 (dd, J = 6.6, 1.5 Hz, 2H, H2,6), 8.52 (s, 1H, H-C=N), 9.10 (dd, J = 6.6, 1.5 Hz, 2H, H3,5), 9.21 (d, J = 1.3 Hz, 1H, H3′), 15.67 (s, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 122.02 (1C, C5′), 125.10 (2C, C2,6), 128.25 (1C, C1′), 138.37 (1C, C=N), 136,80 (1C, C3′), 144.77 (2C, C3,5), 146.91 (1C, C1), 160.14 (1C, C=O). 126.78 (2C, C2,6), 134,36 (1C, C5′), 143.08 (2C, C3,5). R (300 MHz, DMSO d6) δ ppm: 7.63 (d, J 8.7 Hz, 1H, H3), 7.84 (dd, J 4.4, 1.6 Hz, 2H, 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, =N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KBr) ν cm−1: 3193.59 (N-H), 3038.08 (C-Har), 1648.96 (C=O), 1626.02 (C=N-N), 1596.86 (C=Car), 1551.04 (Car KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 1549.40 (Car N). N), 1506.46 (C=N). ( ) ( ) OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF MeOH) m/z: 266 1046 [M + H+] ) ( ) UV (EtOH, 182 µM, 25 ◦C): λ = 309 nm, ε = 5495.05 dm3·mol−1·cm−1 (very poorly soluble). MS (ES TOF M OH) / 238 0708 [M N +] 216 0887 [M H+] (( ) OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. 3.2. Chemistry 4.2. Chemistry 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KB ) 1 3188 96 (N H) 3027 37 (C H) 1652 (C O) 1622 47 (C N N) 1607 84 ) ( 3,5) ( ) ( ind) 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). C N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N H), 13.31 (s, 1H, N Hind). R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KB ) 1 3188 96 (N H) 3027 37 (C H) 1652 (C O) 1622 47 (C N N) 1607 84 , 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). ), ( , , , ), ( , , ), ( , , ) R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 9 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). R (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). R (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), 1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). FTIR (KB ) 3194 75 (N H) 3097 19 (C H) 1660 58 (C O) 1621 45 (C N N) 1602 24 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). pp ( ) ( ) ( ) ( 19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), 138 44 (1C C N) 142 84 (1C C ) 144 03 (2C C ) 147 78 (1C C ) 159 52 (1C C O) pp ( ) ( J ) H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). R (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 (Car=Car), 1551.49 (Car N). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) y ) y ) y ( ) yl)methylene)isonicotinohydrazide (4) p ( ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H NMR (300 MH DMSO d ) δ 7 63 (d J 8 7 H 1H H ) 7 84 (dd J 4 4 1 6 H 2H .: 302.5 ◦C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). p ( ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′) m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H NMR (300 MH DMSO d ) δ 7 63 (d J 8 7 H 1H H ) m.p.: 302.5 ◦C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). .5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). p 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8. m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). H NMR (300 MH DMSO d ) δ 7 63 (d J 8 m.p.: 302.5 ◦C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 2.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 13C NMR (75 MH DMSO) δ 111 39 (1C C ) 121 98 (2C C ) 122 73 (1C C ) 123 35 (1C 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 3 , FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 (Car=Car), 1551.49 (Car ( , ), ( , ) KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 , 1549.40 (Car N). N). 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 (C C ) 1551 49 (C N) , pp ( ) ( ) ( ) ( ) 1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 UV (EtOH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. HRMS (ES TOF) m/z: [M + H+] calc for C11H12N5O: 230 1042 Found: 230 1049 OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. , TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 161.58 µM, 25 ◦C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. (Car=Car), 1551.49 (Car N). UV (EtOH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). MS (ES, TOF, MeOH) m/z: 252 0866 [M + Na+]; 230 1049 [M + H+] , 1551.49 (Car N). OH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 230.1049. ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 230.1049. MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 230.1049. TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis ES TOF) / [M + H+] al fo C H N O 230 1042 Fou d 230 1049 (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) , [ ] dih d 1H i d l 5 l) th l )i i ti h d id (4) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 ( 1H H C N) 11 ( 1H H ) 14 (dd J 1 H H H ) 1 (b H N H) 9 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). R (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 (Car=Car), 1551.49 (Car N). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 (Car=Car), 1551.49 (Car C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, .19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 , 1549.40 (Car N). N). m.p.: 299 C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) / / ( / / ) R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, =N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). C NMR ( MH DMSO) δ 111 3 (1C C ) 1 1 ( C C ) 1 3 (1C C ) 1 3 3 (1C 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). ( ) R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, =N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KB ) 1 3188 96 (N H) 3027 37 (C H) 1652 (C O) 1622 47 (C N N) 1607 84 , 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). ), ( , , , ), ( , , ), ( , , ) R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). (Car=Car), 1549.40 (Car N). UV (EtOH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. MS (ES TOF M OH) / 266 1046 [M H ] FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 (Car=Car), 1549.40 (Car N). UV (EtOH 38 37 μM 25 °C): λ = 234 nm ε = 22 666 92 dm3·mol−1·cm−1 , FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 (Car=Car), 1549.40 (Car ( , ), ( , ) KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 , 1549.40 (Car N). N). MS (ES, TOF, MeOH) m/z: 266.1046 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. UV (EtOH, 38.37 μM, 25 C): λ = 234 nm, ε = 22,666.92 dm3·mol 1·cm 1. MS (ES, TOF, MeOH) m/z: 266.1046 [M + H+]. HRMS (ES TOF) m/z: [M + H+] calc for C14H12N5O: 266 1042 Found: 266 1046 OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. , TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 38.37 µM, 25 ◦C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), / / m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v m.p.: 299 ◦C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 299 °C (d ) R 0 1 E OA /M OH (4 1 / 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). ( , J , , , ), ( , , ), ( , J , , ), ( , =N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 dd b 9 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). R (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). ( , J , , , ), ( , , ), ( , J , , ), ( , =N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 3.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis 4.2.1. General Procedure for Phenolic Isonicotinoyl Hydrazones Synthesis ) ( ) UV (EtOH, 182 µM, 25 ◦C): λ = 309 nm, ε = 5495.05 dm3·mol−1·cm−1 ( MS (ES, TOF, MeOH) m/z: 238.0708 [M + Na+]; 216.0887 [M + H+]. OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. UV (EtOH, 182 µM, 25 ◦C): λ = 309 nm, ε = 5495.05 dm3·mol−1·cm−1 (very poorly so MS (ES, TOF, MeOH) m/z: 238.0708 [M + Na+]; 216.0887 [M + H+]. HRMS (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 216.0887. (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. HRMS (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 216.0887. 16 of 27 16 of 27 Molecules 2017, 22, 1457 MS (ES, TOF, (ES, TOF) m/z: [M + H UV (EtOH, 182 MS (ES TOF M , 1551.04 (Car N), OH, 182 μM, 25 °C): λ (E)-N′-((4-methyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) (E)-N′-((4-methyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) y y y y HRMS (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 2 , , [ ]; [ ] (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 216.0887. (E)-N′-((4-methyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) (E)-N′-((4-methyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) HRMS (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 2 , , [ ] [ ] (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 216.0887. E)-N′-((4-methyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) E)-N′-((4-methyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) HRMS (ES, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 2 [ ] [ ] S, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 216.0887. eOH (5:5:3 v/v/v). y y y hylene)isonicotinohydrazide (3) 5:5:3 m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). m.p.: 299 ◦C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, 299 °C (d ) R 0 1 E OA /M OH (4 1 / ) m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v m.p.: 299 ◦C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz DMSO-d6) δ ppm: 2 54 (s 3H CH3) 7 12 (ddt J = 24 4 9 3 1 9 1 9 Hz 2H m.p.: 281.1 ◦C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, C H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 m.p.: 281.1 °C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v) 1H NMR (300 MH DMSO d ) δ 2 54 ( 3H C m.p.: 281.1 ◦C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, C H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 m.p.: 281.1 °C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v) 1H NMR (300 MH DMSO d ) δ 2 54 ( 3H C m.p.: 281.1 ◦C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, C H5′ H6′) 7 35 (tt J = 2 4 1 7 0 9 0 8 Hz 1H H4′) 7 85 m.p.: 281.1 °C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v) 1H NMR (300 MH DMSO d ) δ 2 54 ( 3H C m.p.: 281.1 ◦C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). H5, H6), 7.35 (tt, J 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4), 7.85 (dd, J 4.5, 1.8 Hz, 2H, H2,6), 8.16 8.32 (m, 1H, H7′), 8.71 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3,5), 11.55 (d, J = 7.4 Hz, 1H, N-H), 11.67 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), 121 63(1C C5′) 121 87 (2C C2 6) 122 34 (1C C6′) 125 85 (1C C9′) 136 19 (1C C8′) 141 05 (1C C2′) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, 9.3, 1.9, 1.9 Hz, 2H, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H, H2,6), 8.16–8.32 (m, 1H, H7′), 8.71 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3,5), 11.55 (d, J = 7.4 Hz, 1H, N-H), 11.67 (s, 1H, N-Hind). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 13C NMR (75 MH DMSO) δ 110 75 (1C C ) 119 03 (1C C ) 121 51 (2C C ) 122 01 (1C C ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.61 (dd, J = 8.5, 1.3 Hz, 1H, H7′), 7.84 (m, 4H, H2,6, H2′, H6′), 8.13 (d, J = 1.3 Hz, 1H, H5′), 8.60 (s, 1H, H-C=N), 8.80 (br, 2H, H3,5), 12.12 (s, 1H, N-H), 13.28 (s, 1H, N-Hind). / / ( / / ) R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). m.p.: 295.2 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.61 (dd, J = 8.5, 1.3 Hz, 1H, H7′), 7.84 (m, 4H, H2,6, H2′, H6′), 8.13 (d, J = 1.3 Hz, 1H, H5′), 8.60 (s, 1H, H-C=N), 8.80 (br, 2H, H3,5), 12.12 (s, 1H, N-H), 13.28 13C-NMR (75 MHz, DMSO) δ ppm: 110.75 (1C, C2′), 119.03 (1C, C7′), 121.51 (2C, C2,6), 122.01 (1C, C6′), 124.39 (1C, C8′), 132.43 (1C, C1′), 134.21 (1C, C5′), 140.33 (1C, C1), 140.95 (1C, C9′), 150.03 (1C, C=N), 150.79 (2C, C3,5), 162.08 (1C, C=O). FTIR (KB ) 1 3193 59 (N H) 3038 08 (C H ) 1648 96 (C O) 1626 02 (C N N) 1596 86 13C-NMR (75 MHz, DMSO) δ ppm: 110.75 (1C, C2′), 119.03 (1C, C7′), 121.51 (2C, C2,6), 122.01 (1C, C6′), 124.39 (1C, C8′), 132.43 (1C, C1′), 134.21 (1C, C5′), 140.33 (1C, C1), 140.95 (1C, C9′), 150.03 (1C, C=N), 150.79 (2C, C3,5), 162.08 (1C, C=O). ), ( , , ), ( , , ), ( , , ) R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 110.75 (1C, C2′), 119.03 (1C, C7′), 121.51 (2C, C2,6), 122.01 (1C, C6′), 124.39 (1C, C8′), 132.43 (1C, C1′), 134.21 (1C, C5′), 140.33 (1C, C1), 140.95 (1C, C9′), 150.03 (1C, C=N), m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N- N), 1599.50 (C=Car), 1550.60 (Car N), 1506.46 (C=N). UV (EtOH, 57.29 μM, 25 °C): λ = 224 nm, ε = 21,909.58 dm3·mol−1·cm−1. 141.56 (1C, C1), 146.10 (1C, C=N), 150.69 (2C, C3,5), 161.00 (1C, C=O). FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N-N), 1599.50 (C=Car), 1550.60 (Car C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.8 (Car=Car), 1549.40 (Car N). UV (EtOH 38 37 M 25 °C) λ 234 22 666 92 d 3 ol 1 1 N), 1506.46 (C=N). UV (EtOH 57 29 M 25 ◦C) λ 224 21 909 58 d 3 l 1 1 N), 1599.50 (C Car), 1550.60 (Car N), 1506.46 (C N). UV (EtOH, 57.29 μM, 25 °C): λ = 224 nm, ε = 21,909.58 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 279.1246 [M + H+]. FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N- N), 1599.50 (C=Car), 1550.60 (Car N), 1506.46 (C=N). UV (EtOH 57 29 μM 25 °C): λ = 224 nm ε = 21 909 58 dm3 mol−1 cm−1 , FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N-N), 1599.50 (C=Car), 1550.60 (Car ) ( ) ( ) FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.8 (Car=Car), 1549.40 (Car N). N), 1506.46 (C=N). ( , , ) [ ] HRMS (ES, TOF) m/z: [M + H+] calc. for C16H15N4O: 279.1246. Found: UV (EtOH, 57.29 μM, 25 C): λ = 224 nm, ε = 21,909.58 dm3·mol 1·cm 1 MS (ES TOF MeOH) m/z: 279 1246 [M + H+] ( ) ( ) UV (EtOH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm UV (EtOH, 57.29 µM, 25 ◦C): λ = 224 nm, ε = 21,909.58 dm3·mol−1·cm−1. , , [ ] HRMS (ES, TOF) m/z: [M + H+] calc. for C16H15N4O: 279.1246. Found: 279.12 UV (EtOH, 57.29 μM, 25 C): λ = 224 nm, ε = 21,909.58 dm mol cm . MS (ES TOF MeOH) m/z: 279 1246 [M + H+] UV (EtOH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. UV (EtOH, 57.29 µM, 25 ◦C): λ = 224 nm, ε = 21,909.58 dm3·mol−1·cm−1. E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, 9.3, 1.9, 1.9 Hz, 2H, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H, H2,6), 8.16–8.32 (m, 1H, H7′), 8.71 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3,5), 11.55 (d, J = 7.4 Hz, 1H, N-H), 11.67 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), m.p.: 281.1 ◦C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, 9.3, 1.9, 1.9 Hz, 2H, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H, H2,6), 8.16–8.32 (m, 1H, H7′), 8.71 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3,5), 11.55 (d, J = 7.4 Hz, 1H, N-H), 11.67 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na ]; 230.1049 [M + H ]. HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 230.1049. (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 ( 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 13C-NMR (75 MHz DMSO) δ ppm: 111 39 (1C C3′) 121 98 (2C C2 6) 122 73 (1C C7′) 123 35 (1 (E) N ((2 methyl 1H indol 3 yl)methylene)isonicotinohydrazide (6) (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na ]; 230.1049 [M + HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. 55 PE/EtOAc/MeOH (5:5:3 v/v/v). o-1H-indazol-5-yl)methylene)isonicotinohydrazide ( EtO 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, 9.3, 1.9, 1.9 Hz, 2H, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H, H2,6), 8.16–8.32 (m, m.p.: 281.1 °C. E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H 1H, H7′), 8.71 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3,5), 11.55 (d, J (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) m.p.: 281.1 °C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H m.p.: 281.1 ◦C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, 9.3, H ) 7 35 (tt J 2 4 1 7 0 9 0 8 H 1H H ) 7 85 (dd J 4 5 1 8 H 2H H ) 8 MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na ]; 230.1049 [M + H ]. HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 230 (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). ( d ) d (E) N ((2 methyl 1H indol 3 yl)methylene)isonicotinohydrazide (6) (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na ]; 230.1049 [M + HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. (E) N ((2 methyl 1H indol 3 yl)methylene)isonicotinohydrazide (6) m.p.: 281.1 °C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, 9.3, 1.9, 1.9 Hz, 2H, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H, H2,6), 8.16–8.32 (m, 1H, H7′), 8.71 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3,5), 11.55 (d, J = 7.4 Hz, 1H, N-H), 11.67 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), 121.63(1C, C5′), 121.87 (2C, C2,6), 122.34 (1C, C6′), 125.85 (1C, C9′), 136.19 (1C, C8′), 141.05 (1C, C2′), 141 56 (1C C1) 146 10 (1C C=N) 150 69 (2C C3 5) 161 00 (1C C=O) (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) (E)-N′-((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) m.p.: 281.1 °C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) MS (ES, TOF, MeOH) m/z: 266.1046 [M + H+]. MS (ES, TOF, MeOH) m/z: 266.1046 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.104 UV (EtOH, 38.37 μM, 25 C): λ = 234 nm, ε = 22,666.92 dm3·mol 1·cm 1. MS (ES, TOF, MeOH) m/z: 266.1046 [M + H+]. OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF MeOH) m/z: 266 1046 [M + H+] UV (EtOH, 38.37 µM, 25 ◦C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. MS (ES TOF M OH) / 266 1046 [M H+] HRMS (ES, TOF) m/z: [M + H ] calc. for C14H12N5O: 266.1042. Found: 266.1046 ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. HRMS (ES, TOF) m/z: [M + H ] calc. for C14H12N5O: 266.1042. Found: 266.1046 (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. 17 of 27 16 of 2 17 of 27 16 of 2 Molecules 2017, 22, 1457 OH, 161.58 μM, 25 °C ) / Molecules 2017, 22, 14 (E)-N′-((2H-indazol-6-yl)methylene)isonicotinohydrazide (5) , , (E)-N′-((2H-indazol-6-yl)methylene)isonicotinohydrazide (5) ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 23 Molecules 2017, 22, 1457 FTIR (KBr) ν cm−1: 3193.59 (N-H), 3038.08 (C-Har), 1 (C=Car), 1551.04 (Car N), 1506.46 (C=N). (E)-N′-((2H-indazol-6-yl)methylene)isonicotinohydrazide (5) (E)-N′-((2H-indazol-6-yl)methylene)isonicotinohydrazide (5) (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 23 Molecules 2017, 22, 1457 FTIR (KBr) ν cm−1: 3193.59 (N-H), 3038.08 (C-Har), 1 (C=Car), 1551 04 (Car N), 1506 46 (C=N) y ) y ) y ( ) zol-6-yl)methylene)isonicotinohydrazide (5) H, 182 μM, 25 C): λ = 309 nm, ε = 5495.05 dm3·mol 1·cm 1 (very poorly soluble). TOF, MeOH) m/z: 238.0708 [M + Na+]; 216.0887 [M + H+]. S, TOF) m/z: M + H+ calc. for C10H10N5O: 216.0885. Found: 216.0887. hyl-1H-imidazol-5-yl)methylene)isonicotinohydrazide (3) m.p.: 295.2 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H NMR (300 MH DMSO d ) δ 7 61 (dd J 8 5 1 3 H 1H H ) 7 84 ( 4H H H .: 295.2 ◦C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 295.2 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 1H NMR (300 MH DMSO d ) δ 7 61 (dd J m.p.: 295.2 ◦C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 2.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 295.2 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H NMR (300 MH DMSO d ) δ 7 61 (dd J m.p.: 295.2 ◦C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 2.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.61 (dd, J = 8.5, 1.3 Hz, 1H, H7′), 7.84 (m, 4H, H2,6, H2′, H6′), 8.13 (d, J = 1.3 Hz, 1H, H5′), 8.60 (s, 1H, H-C=N), 8.80 (br, 2H, H3,5), 12.12 (s, 1H, N-H), 13.28 (s, 1H, N-Hind). 13C NMR (75 MH DMSO) δ 110 75 (1C C ) 119 03 (1C C ) 121 51 (2C C ) 122 01 (1C C ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.61 (dd, J = 8.5, 1.3 Hz, 1H, H7′), 7.84 (m, 4H, H2,6, H2′, H6′), 8.13 (d, J = 1.3 Hz, 1H, H5′), 8.60 (s, 1H, H-C=N), 8.80 (br, 2H, H3,5), 12.12 (s, 1H, N-H), 13.28 (s, 1H, N-Hind). / / ( / / ) R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). m.p.: 295.2 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.61 (dd, J = 8.5, 1.3 Hz, 1H, H7′), 7.84 (m, 4H, H2,6, H2′, H6′), 8.13 (d, J = 1.3 Hz, 1H, H5′), 8.60 (s, 1H, H-C=N), 8.80 (br, 2H, H3,5), 12.12 (s, 1H, N-H), 13.28 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.61 (dd, J = 8.5, 1.3 Hz, 1H, H7′), 7.84 (m, 4H, H2,6, H2′, H6′), 8.13 (d, J = 1.3 Hz, 1H, H5′), 8.60 (s, 1H, H-C=N), 8.80 (br, 2H, H3,5), 12.12 (s, 1H, N-H), 13.28 (s, 1H, N-Hind). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.5 (s 1H H C=N) 9 11 (s 1H H3′) 9 14 (dd J = 5 5 1 8 Hz 2H H3 5) 15 98 (br 2H N H) FTIR (KBr) ν cm−1: 3193.59 (N-H), 3038.08 (C-Har), 1648.96 (C=O), 1626.02 (C=N-N), 1596.86 (C=Car), 1551.04 (Car N), 1506.46 (C=N). UV (EtOH 37 39 μM 25 °C): λ 313 nm ε 25 055 1 dm3 mol−1 cm−1 ( , 3,5), ( , ) FTIR (KBr) ν cm−1: 3193.59 (N-H), 3038.08 (C-Har), 1648.96 (C=O), 1626.02 (C=N-N), 1596.86 (C=Car), 1551.04 (Car 50.76 (2C, C3,5), 161.87 (1C, C O). KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 , 1549.40 (Car N). N), 1506.46 (C=N). 150.79 (2C, C3,5), 162.08 (1C, C=O). FTIR (KBr) ν cm−1: 3193.59 (N-H), 3038.08 (C-Har), 1648.96 (C=O), 1626.02 (C=N-N), 1596.86 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′ 138 44 (1C C=N) 142 84 (1C C3′) 144 03 (2C C3 5) 147 78 (1C C1) 159 52 (1C C=O) UV (EtOH, 37.39 μM, 25 °C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1 MS (ES, TOF, MeOH) m/z: 266.1047 [M + H+]. HRMS (ES TOF) m/z: [M + H+] calc for C14H12N5O: 266 1042 Found 1551.04 (Car , 1549.40 (Car N). OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. N), 1506.46 (C N). UV (EtOH, 37.39 µM, 25 ◦C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 266.1047 [M + H+]. (C=Car), 1551.04 (Car N), 1506.46 (C=N). UV (EtOH, 37.39 μM, 25 °C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1 MS (ES TOF M OH) / 266 1047 [M H ] 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C= (Car=Car), 1551.49 (Car N). UV (EtOH, 37.39 μM, 25 °C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 266.1047 [M + H+]. E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N-N), 1599 50 (C=C ) 1550 60 (C , ), ( , , ), ( , , ), ( , , ) 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 (C =C ) 1549 40 (C N) N) 1506 46 (C=N) 121.63(1C, C5), 121.87 (2C, C2,6), 122.34 (1C, C6), 125.85 (1C, C9), 136.19 (1C, C8), 141.05 (1C, C2), 141.56 (1C, C1), 146.10 (1C, C=N), 150.69 (2C, C3,5), 161.00 (1C, C=O). FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N- N), 1599.50 (C=Car), 1550.60 (Car N), 1506.46 (C=N). 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), 121.63(1C, C5′), 121.87 (2C, C2,6), 122.34 (1C, C6′), 125.85 (1C, C9′), 136.19 (1C, C8′), 141.05 (1C, C2′), 141.56 (1C, C1), 146.10 (1C, C=N), 150.69 (2C, C3,5), 161.00 (1C, C=O). FTIR (KBr) ν cm−1: 3385 07 (N H) 3209 03 (N H) 3049 08 (C H) 1655 09 (C=O) 1626 02 (C=N 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), 121.63(1C, C5′), 121.87 (2C, C2,6), 122.34 (1C, C6′), 125.85 (1C, C9′), 136.19 (1C, C8′), 141.05 (1C, C2′), 141.56 (1C, C1), 146.10 (1C, C=N), 150.69 (2C, C3,5), 161.00 (1C, C=O). ) ( ) ( ) ( ) 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). N), 1599.50 (C=Car), 1550.60 (Car N), 1506.46 (C=N). UV (EtOH, 57.29 μM, 25 °C): λ = 224 nm, ε = 21,909.58 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 279.1246 [M + H+]. HRMS (ES TOF) / [M H ] l f C H N O 279 1246 F d 279 1246 141.56 (1C, C1), 146.10 (1C, C N), 150.69 (2C, C3,5), 161.00 (1C, C O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) HRMS (ES TOF) m/z: [M + H+] calc for C14H12N5O: 266 1042 Found: 26 ( ar , ( ) OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. ), ( ) UV (EtOH, 37.39 µM, 25 ◦C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 266.1047 [M + H+]. (C=Car), 1551.04 (Car N), 1506.46 (C=N). UV (EtOH, 37.39 μM, 25 °C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1. (E O O ) / 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159 FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), (Car=Car) 1551 49 (Car N) UV (EtOH, 37.39 μM, 25 °C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 266.1047 [M + H+]. HRMS (ES TOF) m/z: [M + H+] calc for C14H12N5O: 266 1042 Found: 266 1047 ( ar , ( ) OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. ), ( ) UV (EtOH, 37.39 µM, 25 ◦C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 266.1047 [M + H+]. (C=Car), 1551.04 (Car N), 1506.46 (C=N). UV (EtOH, 37.39 μM, 25 °C): λ = 313 nm, ε = 25,055.1 dm3·mol−1·cm−1. MS (ES TOF M OH) / 266 1047 [M H+] 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.2 (Car=Car), 1551.49 (Car N). HRMS (ES, TOF) m/z: [M + H ] calc. for C14H12N5O: 266.1042. Found: 266.1047 ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1047. MS (ES, TOF, MeOH) m/z: 266.1047 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1047 ( ), ( ) UV (EtOH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very (E)-N -((2-methyl-1H-indol-3-yl)methylene)isonicotinohydrazide (6) m.p.: 281.1 °C. Rf: 0.55 PE/EtOAc/MeOH (5:5:3 v/v/v). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 13C NMR (75 MH DMSO) δ 11 97 (1C CH ) 107 84 (1C C ) 111 34 (1C C ) 120 82 (1C C ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.54 (s, 3H, CH3), 7.12 (ddt, J = 24.4, 9.3, 1.9, 1.9 Hz, 2H, H5′, H6′), 7.35 (tt, J = 2.4, 1.7, 0.9, 0.8 Hz, 1H, H4′), 7.85 (dd, J = 4.5, 1.8 Hz, 2H, H2,6), 8.16–8.32 (m, 1H, H7′), 8.71 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3,5), 11.55 (d, J = 7.4 Hz, 1H, N-H), 11.67 (s, 1H, N-Hind). m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 121.63(1C, C5), 121.87 (2C, C2,6), 122.34 (1C, C6), 125.85 (1C, C9), 136.19 (1C, C8), 141.05 (1C, C2), 141.56 (1C, C1), 146.10 (1C, C=N), 150.69 (2C, C3,5), 161.00 (1C, C=O). FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N- N), 1599.50 (C=Car), 1550.60 (Car N), 1506.46 (C=N). UV (EtOH, 57.29 μM, 25 °C): λ = 224 nm, ε = 21,909.58 dm3·mol−1·cm−1. MS (ES TOF M OH) / 279 1246 [M H+] 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), 121.63(1C, C5′), 121.87 (2C, C2,6), 122.34 (1C, C6′), 125.85 (1C, C9′), 136.19 (1C, C8′), 141.05 (1C, C2′), 141.56 (1C, C1), 146.10 (1C, C=N), 150.69 (2C, C3,5), 161.00 (1C, C=O). FTIR (KBr) ν cm−1: 3385.07 (N-H), 3209.03 (N-H), 3049.08 (Car-H), 1655.09 (C=O), 1626.02 (C=N- N), 1599.50 (C=Car), 1550.60 (Car N), 1506.46 (C=N). 13C-NMR (75 MHz, DMSO) δ ppm: 11.97 (1C, CH3), 107.84 (1C, C1′), 111.34 (1C, C4′), 120.82 (1C, C7′), 121.63(1C, C5′), 121.87 (2C, C2,6), 122.34 (1C, C6′), 125.85 (1C, C9′), 136.19 (1C, C8′), 141.05 (1C, C2′), 141.56 (1C, C1), 146.10 (1C, C=N), 150.69 (2C, C3,5), 161.00 (1C, C=O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) HRMS (ES, TOF) m/z: [M H ] calc. for C16H15N4O: 279.1246. Found: 279.1246 Compound 6 can also be found (commercial source; CAS No.: 113143-57-0). HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.10 HRMS (ES, TOF) m/z: [M + H+] calc. for C16H15N4O: 279.1246. Found: 279.1246. p ( Compound 6 can also be found (commercial source; CAS No.: 113143-57-0). 18 of 27 N), 1596.8 18 of 27 N), 1596.8 Molecules 2017, 22, 1457 MS (ES, TO HRMS (ES FTIR (KBr) (C=Car), 155 (E)-N′-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)isonicotinohydrazide (7) , , (E)-N′-((1H-pyrrolo[2 3-b]pyridin-3-yl)methylene)isonicotinohydrazide (7) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide ( Molecules 2017, 22, 1457 μ ( MS (ES, TOF, MeOH) m/z: 238.0708 [M + Na+]; 216.0887 [M + H+]. HRMS (ES TOF) m/z: M + H+ calc for C10H10N5O: 216 0885 Found: 2 (E)-N′-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)isonicotinohydrazide (7) (E)-N′-((1H-pyrrolo[2 3-b]pyridin-3-yl)methylene)isonicotinohydrazide (7) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide ( Molecules 2017, 22, 1457 μ ( MS (ES, TOF, MeOH) m/z: 238.0708 [M + Na+]; 216.0887 [M + H+]. HRMS (ES TOF) m/z: M + H+ calc for C10H10N5O: 216 0885 Found: 2 ]pyridin-3-yl)methylene)isonicotinohydrazide (7) midazol-5-yl)methylene)isonicotinohydrazide (3) 323.2 °C (dec.). Rf: 0.34 PE/EtAc/MeOH (5:5:3 v/v). 2 ◦C (dec.). Rf: 0.34 PE/EtAc/MeOH (5:5:3 v/v). p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 323.2 °C (dec.). Rf: 0.34 PE/EtAc/MeOH (5:5:3 v/v m.p.: 323.2 ◦C (dec.). Rf: 0.34 PE/EtAc/MeOH (5:5:3 v/v). m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v 1H-NMR (300 MHz, DMSO-d6) δ ppm: 77.24 (tt, J = 7.8, 4.7, 4.7 Hz, 1H, H6′), 7.84 (dd, J = 4.2, 1.7 Hz, 2H, H2,6), 8.03 (d, J = 2.3 Hz, 1H, H2′), 8.33 (dd, J = 4.7, 1.7 Hz, 1H, H7′), 8.58 (d, J = 1.6 Hz, 1H, H5′), 8.62 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 11.86 (s, 1H, N-H), 12.17 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO)δ ppm: 110.85 (1C, C1), 117,13 (1C, C9′), 117.44 (1C, C6′), 121.95 (2C, C2,6), 130.56 (1C, C5′), 131.51 (1C, C2′), 141.39 (1C, C1), 144.56 (1C, C7′), 146.09 (1C, C=N), 149.84 (1C, C8′), 150.71 (2C, C3,5), 161.49 (1C, C=O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) ( ) O p ( ) f ( ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 77.24 (tt, J = 7.8, 4.7, 4.7 Hz, 1H, H6′), 7.84 (dd, J = 4.2, 1.7 Hz, 2H, H2,6), 8.03 (d, J = 2.3 Hz, 1H, H2′), 8.33 (dd, J = 4.7, 1.7 Hz, 1H, H7′), 8.58 (d, J = 1.6 Hz, 1H, H5′), 8.62 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 11.86 (s, 1H, N-H), 12.17 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO)δ ppm: 110.85 (1C, C1), 117,13 (1C, C9′), 117.44 (1C, C6′), 121.95 (2C, C2,6), 130.56 (1C, C5′), 131.51 (1C, C2′), 141.39 (1C, C1), 144.56 (1C, C7′), 146.09 (1C, C=N), 149.84 (1C, C8′), 150.71 (2C, C3,5), 161.49 (1C, C=O). m.p.: 302.5 C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). m.p.: 323.2 °C (dec.). Rf: 0.34 PE/EtAc/MeOH (5:5:3 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 77.24 (tt, J = 7.8, 4.7, 4.7 Hz, 1H, H6′), 7.84 (dd, J = 4.2, 1.7 Hz, 2H, H2,6), 8.03 (d, J = 2.3 Hz, 1H, H2′), 8.33 (dd, J = 4.7, 1.7 Hz, 1H, H7′), 8.58 (d, J = 1.6 Hz, 1H, H5′), 8.62 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 11.86 (s, 1H, N-H), 12.17 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO)δ ppm: 110.85 (1C, C1), 117,13 (1C, C9′), 117.44 (1C, C6′), 121.95 (2C, C2,6), 130.56 (1C, C5′), 131.51 (1C, C2′), 141.39 (1C, C1), 144.56 (1C, C7′), 146.09 (1C, C=N), 149.84 m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) Hz, 2H, H2,6), 8.03 (d, J = 2.3 Hz, 1H, H2′), 8.33 (dd, J = 4.7, 1.7 Hz, 1H, H7′), 8.58 (d, J = 1.6 Hz, 1H, H5′), 8.62 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 11.86 (s, 1H, N-H), 12.17 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO)δ ppm: 110.85 (1C, C1), 117,13 (1C, C9′), 117.44 (1C, C6′), 121.95 (2C, C2,6), 130.56 (1C, C5′), 131.51 (1C, C2′), 141.39 (1C, C1), 144.56 (1C, C7′), 146.09 (1C, C=N), 149.84 m.p.: 299 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.5 (s, 1H, H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′) FTIR (KBr) ν cm−1: 3454.03 (N-H), 3199.51 (N-H), 3031.09 (Car-H), 1662.68 (C=O), 1611.72 (C=N- N), 1600.48 (C=Car), 1551.26 (Car N), 1284.78 (C-N). UV (EtOH 58 08 M 25 °C) λ 200 18 839 92 d 3 l 1 1 λ 218 17 446 62 FTIR (KBr) ν cm−1: 3454.03 (N-H), 3199.51 (N-H), 3031.09 (Car-H), 1662.68 (C=O), 1611.72 (C=N-N), 1600.48 (C=Car), 1551.26 (Car ) ( ) ( ) FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.8 (Car=Car), 1549.40 (Car N). N), 1284.78 (C-N). (1C, C8′), 150.71 (2C, C3,5), 161.49 (1C, C=O). FTIR (KBr) ν cm−1: 3454.03 (N-H), 3199.51 (N-H), 3031.09 (Car-H), 1662.68 (C=O), 1611.72 (C=N- N) 1600 48 (C C ) 1551 26 (C N) 1284 78 (C N) 138.44 (1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). FTIR (KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.2 UV (EtOH, 58.08 μM, 25 °C): λ = 200 nm, ε = 18,839.92 dm3·mol−1·cm−1, λ = 218 nm, ε = 17,446.62 dm3·mol−1·cm−1, λ = 322 nm, ε = 17,193.69 dm3·mol−1·cm−1. MS (ES TOF MeOH) / 266 1045 [M + H+] ( ) ( ) UV (EtOH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 58.08 µM, 25 ◦C): λ = 200 nm, ε = 18,839.92 dm3·mol−1·cm−1, λ = 218 nm, ε = 17,446.62 dm3·mol−1·cm−1, λ = 322 nm, ε = 17,193.69 dm3·mol−1·cm−1. E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 13C-NMR (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 77.24 (tt, J = 7.8, 4.7, 4.7 Hz, 1H, H6′), 7.84 (dd, J = Hz, 2H, H2,6), 8.03 (d, J = 2.3 Hz, 1H, H2′), 8.33 (dd, J = 4.7, 1.7 Hz, 1H, H7′), 8.58 (d, J = 1.6 H dd 1H-NMR (300 MHz, DMSO-d6) δ ppm: 77.24 (tt, J = 7.8, 4.7, 4.7 Hz, 1H, H6′), 7.84 (dd, J = 4.2, 2H, H2,6), 8.03 (d, J = 2.3 Hz, 1H, H2′), 8.33 (dd, J = 4.7, 1.7 Hz, 1H, H7′), 8.58 (d, J = 1.6 Hz, 1H p / / ( / / ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1. H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H m.p.: 323.2 °C (dec.). Rf: 0.34 PE/EtAc/MeOH (5:5:3 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 77.24 (tt, J = 7.8, 4.7, 4.7 Hz, 1H, H6′), 7.84 (dd, J = H5′), 8.62 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 11.86 (s, 1H, N-H), 12.17 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO)δ ppm: 110.85 (1C, C1), 117,13 (1C, C9′), 117.44 (1C, C6′), 121.95 (2C, C2,6), 130.56 (1C, C5′), 131.51 (1C, C2′), 141.39 (1C, C1), 144.56 (1C, C7′), 146.09 (1C, C=N), 149.84 (1C, C8′), 150.71 (2C, C3,5), 161.49 (1C, C=O). 8.62 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 11.86 (s, 1H, N-H), 12.17 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO)δ ppm: 110.85 (1C, C1), 117,13 (1C, C9′), 117.44 (1C, C6′), 121.95 (2C, C2,6), 130.56 (1C, C5′), 131.51 (1C, C2′), 141.39 (1C, C1), 144.56 (1C, C7′), 146.09 (1C, C=N), 149.84 (1C, C8′), 150.71 (2C, C3,5), 161.49 (1C, C=O). 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) Rf: 0.45 PE/EtOAc/MeOH (5:5:3 1H-NMR (300 MHz, DMSO-d6) δ ppm: δ 7.11–7.28 (m, 2H, H5′,6′), 7.47 (dt, J = 7.9, 0.9 Hz, 1H, H4′), 7 85 (dd J 4 4 1 6 H 2H H ) 7 88 (d J 2 8 H 1H H ) 8 31 (dd J 6 8 1 5 H 1H H ) m.p.: 242 °C. Rf: 0.55 PE/EtAc/MeOH (5:5:3 v/v/v). ( O d ) d m.p.: 242 ◦C. Rf: 0.55 PE/EtAc/MeOH (5:5:3 v/v/v). m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 7.85 (dd, J = 4.4, 1.6 Hz, 2H, H2,6), 7.88 (d, J = 2.8 Hz, 1H, H2′), 8.31 (dd, J = 6.8, 1.5 Hz, 1H, H9′), 8.65 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3, 5), 11.65 (s, 1H, N-H), 11.76 (s, 1H, N-Hind). 13C-NMR (75 MHz, DMSO) δ ppm: 111.96 (1C, C1′), 112.34 (1C, C4′), 121.00 (1C, C5′), 121.95 (2C, 1H-NMR (300 MHz, DMSO-d6) δ ppm: δ 7.11–7.28 (m, 2H, H5′,6′), 7.47 (dt, J = 7.9, 0.9 Hz, 1H, H4′), 7.85 (dd, J = 4.4, 1.6 Hz, 2H, H2,6), 7.88 (d, J = 2.8 Hz, 1H, H2′), 8.31 (dd, J = 6.8, 1.5 Hz, 1H, H9′), 8.65 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3, 5), 11.65 (s, 1H, N-H), 11.76 (s, 1H, N-Hind). 1H-NMR (300 MHz, DMSO-d6) δ ppm: δ 7.11–7.28 (m, 2H, H5′,6′), 7.47 (dt, J = 7.9, 0.9 Hz, 1H, H4′), 7.85 (dd, J = 4.4, 1.6 Hz, 2H, H2,6), 7.88 (d, J = 2.8 Hz, 1H, H2′), 8.31 (dd, J = 6.8, 1.5 Hz, 1H, H9′), 8.65 (s, 1H, H-C=N), 8.78 (dd, J = 4.4, 1.6 Hz, 2H, H3, 5), 11.65 (s, 1H, N-H), 11.76 (s, 1H, N-Hind). p / / ( / / ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). C2,6), 122.44 (1C, C7′), 123.17 (1C, C6′), 123.35 (1C, C7′), 124.77 (1C, C9′), 131.36 (1C, C2′), 137.53 (1C, C8′), 141.54 (1C, C1), 146.63 (1C, C=N), 150.69 (2C, C3,5), 161.33 (1C, C=O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) N), 1600.48 (C=Car), 1551.26 (Car N), 1284.78 (C-N). UV (EtOH, 58.08 μM, 25 °C): λ = 200 nm, ε = 18,839.92 dm3·mol−1·cm−1, λ = 218 nm, ε = 17,446.62 d 3 l 1 1 λ 322 17 193 69 d 3 l 1 1 (Car=Car), 1551.49 (Car N). UV (EtOH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). MS (ES, TOF, MeOH) m/z: 266.1045 [M + H HRMS (ES TOF) m/z: [M + H+] calc for C14 HRMS (ES, TOF) m/z: [M + H+] calc. for C MS (ES, TOF, MeOH) m/z: 266.1045 [M + H+]. dm3·mol−1·cm−1, λ = 322 nm, ε = 17,193.69 dm MS (ES TOF MeOH) m/z: 266 1045 [M + H MS (ES, TOF, MeOH) m/z: 252.0866 [M + HRMS (ES TOF) / [M H ] l f C MS (ES, TOF, MeOH) m/z: 266.1045 [M + H+]. HRMS (ES TOF) m/z: [M + H+] calc for C14H12N5O: 266 1042 Found: 266 1045 HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. MS (ES, TOF, MeOH) m/z: 266.1045 [M + H+]. dm3·mol−1·cm−1, λ = 322 nm, ε = 17,193.69 dm3·mol−1·cm−1. MS (ES TOF MeOH) m/z: 266 1045 [M + H+] MS (ES, TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. HRMS (ES TOF) / [M H+] l f C H N O 230 1042 F d 230 1049 HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1045 HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1045. MS (ES, TOF, MeOH) m/z: 266.1045 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1045 HRMS (ES, TOF) m/z: [M + H+] calc. for C11H12N5O: 230.1042. Found: 230.10 (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) m p : 242 °C Rf: 0 55 PE/EtAc/MeOH (5:5:3 v/v/v) (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonico (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((1H-indol-3-yl)methylene)isonicotinohydrazide (8) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonic 5 PE/EtAc/MeOH (5:5:3 v/v/v) A / p ( ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: δ 7.11–7.2 m.p.: 242 °C. Rf: 0.55 PE/EtAc/MeOH (5:5:3 v/v/v) m.p.: 242 ◦C. Rf: 0.55 PE/EtAc/MeOH (5:5:3 v/v/v). m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 1H-NMR (300 MHz, DMSO-d6) δ ppm: δ 7.11–7.2 m.p.: 242 °C. Rf: 0.55 PE/EtAc/MeOH (5:5:3 v/v/v) m.p.: 242 ◦C. Rf: 0.55 PE/EtAc/MeOH (5:5:3 v/v/v). m.p.: 302.5 °C. E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) FTIR (KBr) ν cm−1: 3543.66 (N-H), 3395.82 (N-H), 2886.55 (C-Har), 1656.52 (C=O), 1626.02 (C=N- N) 1598 83 (C C ) 1550 54 (C N) 1496 83 (C N) 13C-NMR (75 MHz, DMSO) δ ppm: 111.96 (1C, C1′), 112.34 (1C, C4′), 121.00 (1C, C5′), 121.95 (2C, C2,6), 122.44 (1C, C7′), 123.17 (1C, C6′), 123.35 (1C, C7′), 124.77 (1C, C9′), 131.36 (1C, C2′), 137.53 (1C, C8′), 141.54 (1C, C1), 146.63 (1C, C=N), 150.69 (2C, C3,5), 161.33 (1C, C=O). TIR ( B ) 4 66 ( H) ( H) 6 (C H ) 16 6 (C O) 16 6 (C 13C-NMR (75 MHz, DMSO) δ ppm: 111.96 (1C, C1′), 112.34 (1C, C4′), 121.00 (1C, C5′), 121.95 (2C, C2,6), 122.44 (1C, C7′), 123.17 (1C, C6′), 123.35 (1C, C7′), 124.77 (1C, C9′), 131.36 (1C, C2′), 137.53 (1C, C8′), 141.54 (1C, C1), 146.63 (1C, C=N), 150.69 (2C, C3,5), 161.33 (1C, C=O). , ), ( , , ), ( , , ), ( , , ) 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). C2,6), 122.44 (1C, C7′), 123.17 (1C, C6′), 123.35 (1C, C7′), 124.77 (1C, C9′), 131.36 (1C, C2′), 137.53 (1C, C8′), 141.54 (1C, C1), 146.63 (1C, C=N), 150.69 (2C, C3,5), 161.33 (1C, C=O). FTIR (KBr) ν cm−1: 3543.66 (N-H), 3395.82 (N-H), 2886.55 (C-Har), 1656.52 (C=O), 1626.02 (C=N- ) (C C ) (C ) (C ) 13C-NMR (75 MHz, DMSO) δ ppm: 111.96 (1C, C1′), 112.34 (1C, C4′), 121.00 (1C, C5′), 121.95 (2C, C2,6), 122.44 (1C, C7′), 123.17 (1C, C6′), 123.35 (1C, C7′), 124.77 (1C, C9′), 131.36 (1C, C2′), 137.53 (1C, C8′), 141.54 (1C, C1), 146.63 (1C, C=N), 150.69 (2C, C3,5), 161.33 (1C, C=O). 13C-NMR (75 MHz, DMSO) δ ppm: 111.96 (1C, C1′), 112.34 (1C, C4′), 121.00 (1C, C5′), 121.95 (2C, C2,6), 122.44 (1C, C7′), 123.17 (1C, C6′), 123.35 (1C, C7′), 124.77 (1C, C9′), 131.36 (1C, C2′), 137.53 (1C, C8′), 141.54 (1C, C1), 146.63 (1C, C=N), 150.69 (2C, C3,5), 161.33 (1C, C=O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) , ), ( , , ), ( , , ), ( , , ) 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). N), 1598.83 (C=Car), 1550.54 (Car N), 1496.83 (C=N). UV (EtOH, 40.01 μM, 25 °C): λ = 221 nm, ε = 22,519 dm3·mol−1·cm−1. MS (ES TOF M OH) / 265 1092 [M H+] FTIR (KBr) ν cm−1: 3543.66 (N-H), 3395.82 (N-H), 2886.55 (C-Har), 1656.52 (C=O), 1626.02 (C=N- N), 1598.83 (C=Car), 1550.54 (Car N), 1496.83 (C=N). UV (EtOH 40 01 M 25 °C) λ 221 22 519 d 3 l 1 1 , FTIR (KBr) ν cm−1: 3543.66 (N-H), 3395.82 (N-H), 2886.55 (C-Har), 1656.52 (C=O), 1626.02 (C=N-N), 1598.83 (C=Car), 1550.54 (Car ), ( , ), ( , ) FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.8 (Car=Car), 1549.40 (Car N). N), 1496.83 (C=N). N), 1598.83 (C=Car), 1550.54 (Car N), 1496.83 (C=N). UV (EtOH, 40.01 μM, 25 °C): λ = 221 nm, ε = 22,519 dm3·mol−1·cm−1. MS (ES TOF M OH) / 265 1092 [M H+] FTIR (KBr) ν cm−1: 3543.66 (N-H), 3395.82 (N-H), 2886.55 (C-Har), 1656.52 (C=O), 1626.02 (C=N- N), 1598.83 (C=Car), 1550.54 (Car N), 1496.83 (C=N). UV (EtOH 40 01 M 25 °C) λ 221 22 519 d 3 l 1 1 , FTIR (KBr) ν cm−1: 3543.66 (N-H), 3395.82 (N-H), 2886.55 (C-Har), 1656.52 (C=O), 1626.02 (C=N-N), 1598.83 (C=Car), 1550.54 (Car ) ( ) ( ) FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.8 (Car=Car), 1549.40 (Car N). N), 1496.83 (C=N). MS (ES, TOF, MeOH) m/z: 265.1092 [M + H+]. HRMS (ES TOF) m/z: [M + H+] calc for C H N O: 265 1089 Found UV (EtOH, 40.01 μM, 25 °C): λ = 221 nm, ε = 22,519 dm3·mol−1·cm−1. MS (ES TOF MeOH) m/z 265 1092 [M + H+] ( ) ( ( ) ( ) UV (EtOH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·c ) ( ) UV (EtOH, 40.01 µM, 25 ◦C): λ = 221 nm, ε = 22,519 dm3·mol−1·cm−1. Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1092. E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.35 (dd, J = 8.7, 2.1 Hz, 1H5′), 7.44 (d, J = 8.5 Hz, 1H, H4′), 7.84 (dd, J = 4.5, 1.9 Hz, 2H, H2,6), 7.95 (d, J = 2.7 Hz, 1H, H9′), 8.48 (s, 1H, H2′), 8.62 (s, 1H, H-C=N), 8.73–8.89 (m, 2H, H3,5), 11.83 (d, J = 6.0 Hz, 2H, N-H). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.35 (dd, J = 8.7, 2.1 Hz, 1H5′), 7.44 (d, J = 8.5 Hz, 1H, H4′), 7.84 (dd, J = 4.5, 1.9 Hz, 2H, H2,6), 7.95 (d, J = 2.7 Hz, 1H, H9′), 8.48 (s, 1H, H2′), 8.62 (s, 1H, H-C=N), 8.73–8.89 (m, 2H, H3,5), 11.83 (d, J = 6.0 Hz, 2H, N-H). / / ( / / ) R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). m.p.: 309.3 °C (dec.). Rf: 0.61 PE/EtAc/MeOH (5:5:3 v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.35 (dd, J = 8.7, 2.1 Hz, 1H5′), 7.44 (d, J = 8.5 Hz, 1H, H4′), 7.84 (dd, J = 4.5, 1.9 Hz, 2H, H2,6), 7.95 (d, J = 2.7 Hz, 1H, H9′), 8.48 (s, 1H, H2′), 8.62 (s, 1H, H-C=N), 13C-NMR (75 MHz, DMSO) δ ppm: 111.59 (1C, C1′), 113,70 (1C, C6′), 114.42 (1C, C4′), 121.93 (2C, C2,6), 124.60 (1C, C2′), 125.71 (1C, C5′), 126.42 (1C, C9′), 132.71 (1C, C7′), 136.27 (1C, C8′), 141.38 (1C, C1), 146.13 (1C, C=N), 150.72 (2C, C3,5), 161.40 (1C, C=O). FTIR (KB ) 31 (N H ) 1 3 (C H ) 166 6 (C O) 161 34 (C N N) 1 3 13C-NMR (75 MHz, DMSO) δ ppm: 111.59 (1C, C1′), 113,70 (1C, C6′), 114.42 (1C, C4′), 121.93 (2C, C2,6), 124.60 (1C, C2′), 125.71 (1C, C5′), 126.42 (1C, C9′), 132.71 (1C, C7′), 136.27 (1C, C8′), 141.38 (1C, C1), 146.13 (1C, C=N), 150.72 (2C, C3,5), 161.40 (1C, C=O). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. [ ] HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1092. Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1092 Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.10 HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1092. Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1092. Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.104 HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1092. Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). Compound 8 can also be found (commercial source; CAS No.: 10245 44 Compound 8 can also be found (commercial source; CAS No.: 10245-44-0). 19 of 27 Molecules 2017, 22, 1457 , ) (ES, TOF) m/z: [M + H 2C, C2,6), 134,36 (1C, (E)-N′-((5-bromo-1H-indol-3-yl)methylene)isonicotinohydrazide (9) (E)-N′-((5-bromo-1H-indol-3-yl)methylene)isonicotinohydrazide (9) a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) Molecules 2017, 22, 1457 , 1551.04 (Car N), 1506.46 (C=N). OH 182 μM 25 °C): λ = 309 nm ε = 5495 05 dm3·mol−1·cm−1 (very poorly (E)-N′-((5-bromo-1H-indol-3-yl)methylene)isonicotinohydrazide (9) (E)-N′-((5-bromo-1H-indol-3-yl)methylene)isonicotinohydrazide (9) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) Molecules 2017, 22, 1457 1551.04 (Car N), 1506.46 (C=N). OH, 182 μM, 25 °C): λ = 309 nm ε = 5495 05 dm3·mol−1·cm−1 (very poorly 3 yl)methylene)isonicotinohydrazide (9) 8 [M + Na+]; 216.0887 [M + H+]. for C10H10N5O: 216.0885. Found: 216.0887. hylene)isonicotinohydrazide (3) p.: 309.3 °C (dec.). Rf: 0.61 PE/EtAc/MeOH (5:5:3 v/v). NMR (300 MH DMSO d ) δ 7 35 (dd J 8 7 2 1 H 1H ) 7 44 (d J 8 5 H 1H H ) 09.3 ◦C (dec.). Rf: 0.61 PE/EtAc/MeOH (5:5:3 v/v). f: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 309.3 °C (dec.). Rf: 0.61 PE/EtAc/MeOH (5:5:3 v/v m.p.: 309.3 ◦C (dec.). Rf: 0.61 PE/EtAc/MeOH (5:5:3 v/v). 2.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3, 5), 161.84 (1C, C=O). FTIR (KBr) ν cm−1: 3250.19 (N-H), 3032.16 (C-Har), 1689.09 (C=O), 1621.50 (C=N-N), 1599.80 (C C ) 1548 50 (C N) Hz, 1H), 7.18 (ddd, J 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J 8.2, 0.9 Hz, 1H), 7.58 (dd, J 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3, 5), 161.84 (1C, C=O). 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3, 5), 161.84 (1C, C=O). 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, .19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3, 5), 161.84 (1C, C=O). FTIR (KBr) ν cm−1: 3250.19 (N-H), 3032.16 (C-Har), 1689.09 (C=O), 1621.50 (C=N-N), 1599.80 (C C ) (C ) 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3, 5), 161.84 (1C, C=O). (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 / / ( / / ) R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, =N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2.1, 0.9 Hz, 1H), 7.02 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2.1, 0.9 Hz, 1H), 7.02 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). ( ) R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, =N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). 7.86 (dd, J 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2 6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3 5), 2.5 C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). R (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H, 0 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 (s, C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, .19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2.1, 0.9 Hz, 1H), 7.02 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2.1, 0.9 Hz, 1H), 7.02 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3, 5), 161.84 (1C, C=O). ), ( , , , ), ( , , ), ( , , ) R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, .19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). (C=Car), 1548.50 (Car N). UV (EtOH, 52.65 μM, 25 °C): λ = 206 nm, ε = 27,480 dm3·mol−1·cm−1, λ = 350 nm, ε = 33,825 d 3 l 1 1 FTIR (KBr) ν cm−1: 3250.19 (N-H), 3032.16 (C-Har), 1689.09 (C=O), 1621.50 (C=N-N), 1599.80 (C=Car), 1548.50 (Car N). U (E O °C) 6 d l FTIR (KBr) ν cm−1: 3250.19 (N-H), 3032.16 (C-Har), 1689.09 (C=O), 1621.50 (C=N-N), 1599.80 (C=Car), 1548.50 (Car ( ) ( ) KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 , 1549.40 (Car N). N). (C=Car), 1548.50 (Car N). UV (EtOH, 52.65 μM, 25 °C): λ = 206 nm, ε = 27,480 dm3·mol−1·cm−1, λ = 350 nm, ε = 33,825 d l FTIR (KBr) ν cm−1: 3250.19 (N-H), 3032.16 (C-Har), 1689.09 (C=O), 1621.50 (C=N-N), 1599.80 (C=Car), 1548.50 (Car N). FTIR (KBr) ν cm−1: 3250.19 (N-H), 3032.16 (C-Har), 1689.09 (C=O), 1621.50 (C=N-N), 1599.80 (C=Car), 1548.50 (Car KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 , 1549.40 (Car N). N). dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 265.1090 [M + H+]. HRMS (ES TOF) / [M H ] l f C H N O 265 1089 F d 265 1090 UV (EtOH, 52.65 μM, 25 °C): λ = 206 nm, ε = 27,480 dm3·mol−1·cm−1, λ = 350 nm, ε = 33,825 dm3·mol−1·cm−1. S (ES O O ) / OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 52.65 µM, 25 ◦C): λ = 206 nm, ε = 27,480 dm3·mol−1·cm−1, λ = 350 nm, ε = 33,825 dm3·mol−1·cm−1. dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 265.1090 [M + H+]. (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 HRMS (ES TOF) / [M H+] l f C H N O 265 1089 F d 265 1090 UV (EtOH, 52.65 μM, 25 °C): λ = 206 nm, ε = 27,480 dm3·mol−1·cm−1, λ = 350 nm, ε = 33,825 dm3·mol−1·cm−1. MS (ES TOF M OH) / 265 1090 [M H ] OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 52.65 µM, 25 ◦C): λ = 206 nm, ε = 27,480 dm3·mol−1·cm−1, λ = 350 nm, ε = 33,825 dm3·mol−1·cm−1. HRMS (ES, TOF) m/z: [M + H+] calc. for C1 X Ray structure of this compound has been MS (ES, TOF, MeOH) m/z: 265.1090 [M + H HRMS (ES TOF) m/z: [M + H+] calc for C15 ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1 MS (ES, TOF, MeOH) m/z: 265.1090 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C1 X Ray structure of this compound has been MS (ES, TOF, MeOH) m/z: 265.1090 [M + H HRMS (ES TOF) m/z: [M + H+] calc for C ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1 MS (ES, TOF, MeOH) m/z: 265.1090 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090. X-Ray structure of this compound has been recently reported [83]. MS (ES, TOF, MeOH) m/z: 265.1090 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090. X-Ray structure of this compound has been recently reported [83]. (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. MS (ES, TOF, MeOH) m/z: 265.1090 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090. X Ray structure of this compound has been recently reported [83] , [ ] X-Ray structure of this compound has been recently reported [83]. ( , , ) [ ] HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090. X-Ray structure of this compound has been recently reported [83]. (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. Found: 266.1046. MS (ES, TOF, MeOH) m/z: 265.1090 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090. X-Ray structure of this compound has been recently reported [83]. HRMS (ES, TOF) m/z: [M + H+] calc. (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 0 82 PE/EtA /M OH (5 5 3 / / ) p ( ) / / ( / / ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2.1, 0.9 Hz, 1H), 7.02 (ddd, J = 8.0, 7.0, 1.1 m.p.: 231 °C (dec.). Rf: 0.82 PE/EtAc/MeOH (5:5:3 v/v/v). m.p.: 231 ◦C (dec.). Rf: 0.82 PE/EtAc/MeOH (5:5:3 v/v/v). 2.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). p ( ) ( ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2 m.p.: 231 °C (dec.). Rf: 0.82 PE/EtAc/MeOH (5:5:3 v/v/v) m.p.: 231 ◦C (dec.). Rf: 0.82 PE/EtAc/MeOH (5:5:3 v/v/v). .5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 (2C, C3, 5), 161.84 (1C, C=O). p ( ) ( ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2.1, 0.9 Hz, 1H), 7.02 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). 13C-NMR (75 MHz, DMSO) δ ppm: 107.98 (1C, C2′), 112.57 (1C, C3′), 120.06 (1C, C4′), 121.31 (1C, C6′), 122.01 (2C, C2,6), 128.05 (1C, C9′), 133.28 (1C, C1′), 138.47 (1C, C8′), 142.16 (1C, C=N), 150.79 p ( ) f ( ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 76.90 (dd, J = 2.1, 0.9 Hz, 1H), 7.02 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.18 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.47 (dq, J = 8.2, 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 1.1 Hz, 1H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 8.51 (s, 1H), 8.81 (dd, J = 4.4, 1.7 Hz, 2H), 11.65 (s, 1H), 12.05 (s, 1H). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) ), ( , , ), ( , , ), ( , , ) R (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C, 19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C, 50.76 (2C, C3,5), 161.87 (1C, C=O). 8.73–8.89 (m, 2H, H3,5), 11.83 (d, J = 6.0 Hz, 2H, N-H). 13C-NMR (75 MHz, DMSO) δ ppm: 111.59 (1C, C1′), 113,70 (1C, C6′), 114.42 (1C, C4′), 121.93 (2C, C2,6), 124.60 (1C, C2′), 125.71 (1C, C5′), 126.42 (1C, C9′), 132.71 (1C, C7′), 136.27 (1C, C8′), 141.38 (1C, C1), 9 °C (dec.). Rf: 0.1 EtOAc/MeOH (4:1 v/v). R (300 MHz, DMSO-d6) δ ppm: 2.43 (s, 3H, CH3), 8.42 (dd, J = 5.5, 1.8 Hz, 2H, H2,6), 8.53 FTIR (KBr) ν cm−1: 3127.58 (N-Hind), 2891.39 (C-Har), 1662.69 (C=O), 1618.34 (C=N-N), 1538 (C=Car), 1552.13 (Car N), 1041.12 (Car-Br). UV (EtOH 52 65 M 25 °C) λ 201 29 890 d 3 l 1 1 λ 226 25 981 , FTIR (KBr) ν cm−1: 3127.58 (N-Hind), 2891.39 (C-Har), 1662.69 (C=O), 1618.34 (C=N-N), 1538 (C=Car), 1552.13 (Car ( , ), ( , ) KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.84 , 1549.40 (Car N). N), 1041.12 (Car-Br). 146.13 (1C, C=N), 150.72 (2C, C3,5), 161.40 (1C, C=O). FTIR (KBr) ν cm−1: 3127.58 (N-Hind), 2891.39 (C-Har), 1662.69 (C=O), 1618.34 (C=N-N), 1538 (C C ) 1552 13 (C N) 1041 12 (C B ) pp ( ) ( J ) H-C=N), 9.11 (s, 1H, H3′), 9.14 (dd, J = 5.5, 1.8 Hz, 2H, H3,5), 15.98 (br, 2H, N-H). R (75 MHz, DMSO) δ ppm: 9.38 (1C, CH3), 123.60 (1C, C1′) 125.38 (2C, C2,6), 131.78 (1C, C5′), UV (EtOH, 52.65 μM, 25 °C): λ = 201 nm, ε = 29,890 dm3·mol−1·cm−1, λ = 226 nm, ε = 25,981 dm3·mol−1·cm−1, λ = 330 nm, ε = 18,196 dm3·mol−1·cm−1. MS (ES TOF MeOH) m/z: 343 0194 [M + H+] ( ) OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 52.65 µM, 25 ◦C): λ = 201 nm, ε = 29,890 dm3·mol−1·cm−1, λ = 226 nm, ε = 25,981 dm3·mol−1·cm−1, λ = 330 nm, ε = 18,196 dm3·mol−1·cm−1. (C=Car), 1552.13 (Car N), 1041.12 (Car-Br). E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) Compound 9 can also be found (CAS No.: 113143-44-5). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). OH, 161.58 μM, 25 C): λ 316 nm, ε 5551.73 dm mol cm (very poorly soluble). TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. (ES TOF) m/z: [M + H+] calc for C11H12N5O: 230 1042 Found: 230 1049 Compound 9 can also be found (CAS No.: 113143-44-5). Compound 9 can also be found (CAS No.: 113143-44-5). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. ES TOF) m/z: [M + H+] calc for C11H12N5O: 230 1042 Found: 230 1049 (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) UV (EtOH, 52.65 μM, 25 °C): λ = 201 nm, ε = 29,890 dm3·mol−1·cm−1, λ = 226 nm, ε = 25,981 dm3 mol 1 cm 1 λ 330 nm ε 18 196 dm3 mol 1 cm 1 1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 UV (EtOH, 52.65 μM, 25 °C): λ = 201 nm, ε = 29,890 dm3·mol−1·cm−1, λ = 226 nm, ε = 25,981 dm3·mol−1·cm−1, λ = 330 nm, ε = 18,196 dm3·mol−1·cm−1. MS (ES TOF MeOH) m/z: 343 0194 [M + H+] OH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 52.65 µM, 25 ◦C): λ = 201 nm, ε = 29,890 dm3·mol−1·cm−1, λ = 226 nm, ε = 25,981 dm3·mol−1·cm−1, λ = 330 nm, ε = 18,196 dm3·mol−1·cm−1. (C=Car), 1552.13 (Car N), 1041.12 (Car-Br). UV (EtOH, 52.65 μM, 25 °C): λ = 201 nm, ε = 29,890 dm3·mol−1·cm−1, λ = 226 nm, ε = 25,981 d 3 ol 1 1 λ 330 18 196 d 3 ol 1 1 1C, C=N), 142.84 (1C, C3′), 144.03 (2C, C3,5), 147.78 (1C, C1), 159.52 (1C, C=O). KBr) ν cm−1: 3194.75 (N-H), 3097.19 (Car-H), 1660.58 (C=O), 1621.45 (C=N-N), 1602.24 MS (ES, TOF, MeOH) m/z: 343.0194 [M + H HRMS (ES TOF) m/z: [M + H+] calc for C1 OF) m/z: [M + H+] calc. for C14H12N5O: 266.1 ES, TOF, MeOH) m/z: 343.0194 [M + H+]. dm3·mol−1·cm−1, λ = 330 nm, ε = 18,196 dm3·m MS (ES TOF MeOH) m/z: 343 0194 [M + H 49 (Car N). MS (ES, TOF, MeOH) m/z: 343.0194 [M + H HRMS (ES TOF) m/z: [M + H+] calc for C15 ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1 MS (ES, TOF, MeOH) m/z: 343.0194 [M + H+]. dm3·mol−1·cm−1, λ = 330 nm, ε = 18,196 dm3·m MS (ES TOF MeOH) m/z: 343 0194 [M + H 1551.49 (Car N). H 161 8 M 2 °C) λ 316 5551 73 d MS (ES, TOF, MeOH) m/z: 343.0194 [M + H HRMS (ES TOF) m/z: [M + H+] calc for C1 ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1 MS (ES, TOF, MeOH) m/z: 343.0194 [M + H+]. E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4) dih d 1H i d l 5 l) th l )i i ti h d id (4) dm3·mol−1·cm−1, λ = 330 nm, ε = 18,196 dm3· MS (ES TOF MeOH) m/z: 343 0194 [M + H 1551.49 (Car N). H 161 58 M 25 °C) λ 316 5551 73 d HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). ( , , ) [ ] HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). MS (ES, TOF, MeOH) m/z: 343.0194 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). OH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. (ES TOF) m/z: [M + H+] calc for C11H12N5O: 230 1042 Found: 230 1049 HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). MS (ES, TOF, MeOH) m/z: 343.0194 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No : 113143-44-5) OH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. ES TOF) / [M H ] l f C H N O 230 1042 F d 230 1049 HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No.: 113143-44-5). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. Compound 9 can also be found (CAS No : 113143 44 5) MS (ES, TOF, MeOH) m/z: 343.0194 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. OH, 161.58 μM, 25 °C): λ = 316 nm, ε = 5551.73 dm3·mol−1·cm−1 (very poorly soluble). , TOF, MeOH) m/z: 252.0866 [M + Na+]; 230.1049 [M + H+]. Compound 9 can also be found (CAS No.: 113143-44-5). HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12BrN4O: 343.0193. Found: 343.0194. (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 for C15H13N4O: 265.1089. Found: 265.1090. X Ray structure of this compound has been recently reported [83] HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090. X-Ray structure of this compound has been recently reported [83]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090 X Ray structure of this compound has been recently reported [83] HRMS (ES, TOF) m/z: [M + H+] calc. for C15H13N4O: 265.1089. Found: 265.1090. X-Ray structure of this compound has been recently reported [8 X-Ray structure of this compound has been recently reported [83]. X-Ray structure of this compound has been recently reported [8 X-Ray structure of this compound has been recently reported [83]. 20 of 27 20 of 27 Molecules 2017, 22, 1457 HRMS (ES, (E)-N′-((4-nitro-1H-indol-3-yl)methylene)isonicotinohydrazide (11) (E)-N′-((4-nitro-1H-indol-3-yl)methylene)isonicotinohydrazide (11) (E)-N′-((3a,7a-dihydro-1H-indazol-5-yl)methylene)isonicotinohyd m.p.: 317.1 °C (dec.). Rf: 0.70 PE/EtOAc/MeOH (5:5:3 v/v/v) m.p.: 317.1 ◦C (dec.). Rf: 0.70 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 317.1 °C (dec.). Rf: 0.70 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 317.1 ◦C (dec.). Rf: 0.70 PE/EtOAc/MeOH (5:5:3 v/v/v). m.p.: 302.5 °C. Rf: 0.45 PE/EtOAc/MeOH (5:5:3 v/v/v). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.36 (t, J = 8.0 Hz, 1H, H5′), 7.84 (dd, J = 4.4, 1.7 Hz, 2H, H2,6), 7.94 (dd, J = 11.8, 7.9 Hz, 2H, H4′,6′), 8.28 (d, J = 2.9 Hz, 1H, H2′), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 8.91 (s, 1H, H-C=N), 12.00 (s, 1H, N-H), 12.55 (s, 1H, N-H). 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.36 (t, J = 8.0 Hz, 1H, H5′), 7.84 (dd, J = 4.4, 1.7 Hz, 2H, H2,6), 7.94 (dd, J = 11.8, 7.9 Hz, 2H, H4′,6′), 8.28 (d, J = 2.9 Hz, 1H, H2′), 8.78 (dd, J = 4.4, 1.7 Hz, 2H, H3,5), 8.91 (s, 1H, H-C=N), 12.00 (s, 1H, N-H), 12.55 (s, 1H, N-H). p / / ( / / ) 1H-NMR (300 MHz, DMSO-d6) δ ppm: 7.63 (d, J = 8.7 Hz, 1H, H3′), 7.84 (dd, J = 4.4, 1.6 Hz, 2H H2,6), 7.90 (dd, J = 8.8, 1.5 Hz, 1H, H2′), 8.03–8.10 (m, 1H, H7′), 8.17 (t, J = 1.2 Hz, 1H, H6′), 8.57 ( 1H, H-C=N), 8.79 (s, 2H, H3,5), 12.01 (s, 1H, N-H), 13.31 (s, 1H, N-Hind). Physicochemical studies: hydrolytic stability and pKa determination: UV spectra were recorded with HP8453 (Agilent) temperature controll Physicochemical studies: hydrolytic stability and pKa determination: The pHs of the solutions were measured at room temperature (temperature probe) with a combined pH electrode with Seven Multi (Mettler Toledo) pHmeter. The pHs of the solutions were measured at room temperature (temperature probe) with a combined pH electrode with Seven Multi (Mettler Toledo) pHmeter. The pHs of the solutions were measured at room temperature (temperature probe) with a combined pH electrode with Seven Multi (Mettler Toledo) pHmeter. The pHs of the solutions were measured at room temperature (temperature probe) with a combined pH electrode with Seven Multi (Mettler Toledo) pHmeter. 4.3.1. Hydrolytic Stability 3.3.1. Hydrolytic Stability In the case of compounds 1a, 1b, 1c and 1d, a small quantity (respectively, 1.45 mg, 1.63 mg, 1.87 mg, and 1.79 mg) of each product was weighted and dissolved in 1 mL of EtOH. The aliquots were then placed in 100 mL standard flask with a concentration of 28% EtOH/H2O. Final concentrations of compounds were respectively 1a: 6.0 × 10−5 mol/L, 1b: 6.3 × 10−5 mol/L, 1c: 6.0 × 10−5 mol/L and 1d: 5.9 × 10−5 mol/L. In the case of compounds 5 and 7, a small quantity (respectively, 1.65 mg and 1.20 mg) of each d h d d d l d 4 L f E OH A f f h l ( l 1 30 In the case of compounds 1a, 1b, 1c and 1d, a small quantity (respectively, 1.45 mg, 1.63 mg, 1.87 mg, and 1.79 mg) of each product was weighted and dissolved in 1 mL of EtOH. The aliquots were then placed in 100 mL standard ﬂask with a concentration of 28% EtOH/H2O. Final concentrations of compounds were respectively 1a: 6.0 × 10−5 mol/L, 1b: 6.3 × 10−5 mol/L, 1c: 6.0 × 10−5 mol/L and 1d: 5.9 × 10−5 mol/L. product was weighted and dissolved in 4 mL of EtOH. A fraction of this solution (respectively 1.30 mL and 1.36 mL) were placed in 50 mL standard flask and mixed with PIPES buffer (50 mM) with a final concentration of 5% EtOH/PIPES. The solution was stirred at room temperature; pH values and λ max of absorbance of the related d d f l h 15 h In the case of compounds 5 and 7, a small quantity (respectively, 1.65 mg and 1.20 mg) of each product was weighted and dissolved in 4 mL of EtOH. A fraction of this solution (respectively 1.30 mL and 1.36 mL) were placed in 50 mL standard ﬂask and mixed with PIPES buffer (50 mM) with a ﬁnal concentration of 5% EtOH/PIPES. compounds were measured for longer than 15 h. 4 3 2 pKa Determination [84] The solution was stirred at room temperature; pH values and λ max of absorbance of the related compounds were measured for longer than 15 h. (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 Found: 266.10 MS (ES, TOF, MeOH) m/z: 332.0760 [M + Na+]; 310.0940 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12N5O3: 310.0937. Found: 310.0940. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12N5O3: 310.0937. Found: 310.0940 HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12N5O3: 310.0937. Found: 310.0940. (E)-N -((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) (E)-N′-((1H-indol-2-yl)methylene)isonicotinohydrazide (10) -dihydro-1H-indazol-5-yl)methylene)isonicotinohydrazide (4 13C-NMR (75 MHz, DMSO) δ ppm: 110.29 (1C, C9′), 117.59 (1C, C1′), 118.94 (1C, C4′), 119.92 (1C, C6′), 121.50 (1C, C5′), 122.01 (2C, C2, 6), 131.31 (1C, C2′), 139.55 (1C, C8′), 141.22 (1C, C1), 141.90 (1C, C-NO2), 146.43 (1C, C=N), 150.66 (2C, C3,5), 161.56 (1C, C=O). 13C-NMR (75 MHz, DMSO) δ ppm: 110.29 (1C, C9′), 117.59 (1C, C1′), 118.94 (1C, C4′), 119.92 (1C, C6′), 121.50 (1C, C5′), 122.01 (2C, C2, 6), 131.31 (1C, C2′), 139.55 (1C, C8′), 141.22 (1C, C1), 141.90 (1C, C-NO2), 146.43 (1C, C=N), 150.66 (2C, C3,5), 161.56 (1C, C=O). , ), ( , , , ), ( , , ), ( , , ) 13C-NMR (75 MHz, DMSO) δ ppm: 111.39 (1C, C3′), 121.98 (2C, C2,6), 122.73 (1C, C7′), 123.35 (1C C9′), 124.19 (1C, C2′), 127.23 (1C, C1′), 134.99 (1C, C6′), 141.07 (1C, C1), 141.14 (1C, C8′), 150.31 (1C C=N), 150.76 (2C, C3,5), 161.87 (1C, C=O). FTIR (KBr) ν cm−1: 3156.34 (N-H), 3137.68 (N-H), 3053.25 (C-Har), 1664.06 (C=O), 1628.34 (C=N-N), 1590.99 (C=Car), 1554.10 (Car N), 1513.15 (C=N-NO2). UV (EtOH 52 64 M 25 °C) λ 214 26 971 88 d 3 l 1 1 λ 331 17 046 35 , FTIR (KBr) ν cm−1: 3156.34 (N-H), 3137.68 (N-H), 3053.25 (C-Har), 1664.06 (C=O), 1628.34 (C=N-N), 1590.99 (C=Car), 1554.10 (Car ), ( , ), ( , ) FTIR (KBr) ν cm−1: 3188.96 (N-H), 3027.37 (Car-H), 1652 (C=O), 1622.47 (C=N-N), 1607.8 (Car=Car), 1549.40 (Car N). N), 1513.15 (C=N-NO2). UV (EtOH, 52.64 μM, 25 °C): λ = 214 nm, ε = 26,971.88 dm3·mol−1·cm−1, λ = 331 nm, ε = 17,046.35 dm3·mol−1·cm−1. UV (EtOH, 38.37 μM, 25 °C): λ = 234 nm, ε = 22,666.92 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 266.1046 [M + H+]. UV (EtOH, 52.64 µM, 25 ◦C): λ = 214 nm, ε = 26,971.88 dm3·mol−1·cm−1, λ = 331 nm, ε = 17,046.35 dm3·mol−1·cm−1. MS (ES, TOF, MeOH) m/z: 332.0760 [M + Na+]; 310.0940 [M + H HRMS (ES TOF) m/z: [M + H+] calc for C15H12N5O3: 310 0937 F HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042 MS (ES, TOF, MeOH) m/z: 332.0760 [M + Na+]; 310.0940 [M + H+]. MS (ES, TOF, MeOH) m/z: 332.0760 [M + Na+]; 310.0940 [M + H+]. HRMS (ES, TOF) m/z: [M + H+] calc. for C15H12N5O3: 310.0937. Found: 310.0940 HRMS (ES, TOF) m/z: [M + H+] calc. for C14H12N5O: 266.1042. 4.3. Physicochemical Studies: Hydrolytic Stability and pKa Determination 3.3. Physicochemical Studies: Hydrolytic Stability and pKa Determination 4.3. Physicochemical Studies: Hydrolytic Stability and pKa Determination 3.3. Physicochemical Studies: Hydrolytic Stability and pKa Determination Physicochemical studies: hydrolytic stability and pKa determination: UV spectra were recorded with HP8453 (Agilent) temperature controll Physicochemical studies: hydrolytic stability and pKa determination: A small quantity (1–2 3.3.2. pKa Determination aliquots were then placed in 100 mL standard flask with a final concentration of 28% EtOH/H2O. The ionic strength was fixed at 0.1 M with potassium chloride. The pH was adjusted to the required value by adding concentrated 0.1 M KOH or 0.1 M HCl. The solution was stirred at room temperature. The absorbance at three different wavelengths was measured. Absorbance vs. pH curves were plotted and the pKa values were evaluated graphically by geometric method. A small quantity (1–2 mg) of each product was weighted and dissolved in a 1 mL of EtOH. The aliquots were then placed in 100 mL standard ﬂask with a ﬁnal concentration of 28% EtOH/H2O. The ionic strength was ﬁxed at 0.1 M with potassium chloride. The pH was adjusted to the required value by adding concentrated 0.1 M KOH or 0.1 M HCl. The solution was stirred at room temperature. The absorbance at three different wavelengths was measured. Absorbance vs. pH curves were plotted and the pKa values were evaluated graphically by geometric method [84]. 21 of 27 21 of 27 Molecules 2017, 22, 1457 3.4.2. MIC Determination in M.tb H37Rv strain was used as the reference strain. M.tb H37Rv and IC2 clinical isolate [31] were grown at 37 ◦C in Middlebrook 7H9 broth (Difco), supplemented with 0.05% Tween 80, or on solid Middlebrook 7H11 medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC). MICs for the compounds were determined by means of the micro-broth dilution method. Dilutions of M.tb wild-type or mutant strain (about 105–106 cfu/mL) were streaked onto 7H11 solid medium containing a range of drug concentrations (0.125 µg/mL to 40 µg/mL). Plates were incubated at 37 ◦C for about 21 days and the growth was visually evaluated. The lowest drug dilution at which visible growth failed to occur was taken as the MIC value. Results were expressed as the average of at least three independent determinations. 3.4.3. Determination of LC50 Human primary ﬁbroblast (MRC5 ATCC CCL171) were seeded at 5 k/well in a corning Cell bind 96-well plate and treated with growing concentration of compounds. Seventy-two hours post-treatment, cells were directly stained with Hoechst 33342 and imaged under a Cellomics Array scan HCS microscope using the cell cycle algorithm. Total number of cells were assessed in each condition and normalized over untreated wells. LC50 were determined as concentration inducing 50% reduction in cell number. 3.3.3. DMSO stability of Compound 1d The stability of compound 1a in DMSO was assessed at 25 ◦C. To this purpose, the compound was dissolved in DMSO, at a ﬁnal concentration of 10 mM, and incubated for 16 h at 25 ◦C. The compound was then diluted in methanol, and analysed. The mass spectra were recorded in negative ESI resolution mode with a Thermo LTQ-XL mass spectrometer, and compared with those of compound 1a and of INH freshly dissolved in DMSO (10 mM) and diluted in methanol. 3.4. Biological Assays 3.4. Biological Assays 3.4.1. Inhibition Kinetics in the Presence of InhA Inhibition kinetic was performed as described [81,85]. Inhibition kinetic was performed as described [81,85]. Supplementary Materials: Supplementary Materials are available online. Acknowledgments: We thank the CNRS and University Paul Sabatier for ﬁnancial support. We thank the Minister of Industry for Ph.D. grant (P.F.M.O.); William R. Jacobs for pMV261-InhA construct and grant VEGA 1/0284/15. We thank F. Gaillardo (NeoVirTech, ITAV, Centre P. Potier, Toulouse) for the cytotoxicity evaluation of the hydrazone compounds. Author Contributions: P.F.M.O.: Ph.D. student synthetic experiments; B.G., A.C. and M.Baron (co-director of P.F.M.O. thesis): supervised the synthetic part; C.A.B.: NMR and DFT studies; G.M. and B.S.O. performed the MIC experiments; L.R.C. performed MS; M.R.P. directed this part; J.M., J.K.: analysis on lipids and mycolic acids, J.K. directed this part; C.L.: InhA inhibition experiments; C.C.: hydrolytic stability and pKa determination; S.M.: NMR experiments; M.Baltas: conceive and direct the project, co-direct the P.F.M.O. thesis. All authors contributed in writing the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. 3.4.5. Determination of Sensitivity of M.tb H37Ra Strain Overproducing InhA to Hydrazones InhA protein was overproduced in M.tb H37Ra using pMV261-InhA construct as already described [88]. Sensitivity of InhA overproducing strain, as well as control strain carrying empty vector to compounds 1a, 3, 5, 7 and 10 was analysed by drop dilution methods. Both cultures grown in 7H9 broth supplemented with albumin-dextrose-catalase and 0.05% Tween 80 were adjusted to OD600 0.5 and 4 µL aliquots of 100, 10−1, 10−2 and 10−3 dilutions were dropped on 7H11 agar supplemented with oleic acid-albumin-dextrose-catalase and incubated 25 days at 37 ◦C. 4. Conclusions A series of hydrazones were synthesized through mechanochemistry and evaluated for their ability to inhibit M.tb H37Rv strain growth, the most active being 1a and 7. Imidazole derivative 5 and indazole one 10 are also active against H37Rv with their nitro substituent. Compounds 1a and 1b were the most effective against both M.tb H37Rv strain and drug-resistant IC2 isolate. The mechanism of anti-mycobacterial activities of selected hydrazones with the best scores regarding the MICs and toxicities was conﬁrmed to be through the cessation of mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell. 3.4.4. Analysis of the Effect of Hydrazones on Lipids and Mycolic Acids of M.tb H37Ra The modes of actions of selected hydrazones were analyzed by metabolic labeling of M.tb H37Ra strain with 14C acetate as already described [34]. Brieﬂy, M.tb H37Ra culture was grown in Middlebrook 7H9 broth (Difco) supplemented with albumin-dextrose-catalase (Difco) and 0.05% Tween 80 (MP Biomedicals) at 37 ◦C till OD600 reached 0.24. The culture was then divided into 20 mL aliquots and tested compounds dissolved in DMSO were added in 0.41 µM (0.1 µg/mL) and 2.05 µM (0.5 µg/mL) ﬁnal concentrations for 1a, 0.44 µM (0.1 µg/mL) and 2.2 µM (0.5 µg/mL) for 3, 0.38 µM (0.1 µg/mL) and 1.9 µM (0.5 µg/mL) for 5, 0.38 µM (0.1 µg/mL) and 1.9 µM (0.5 µg/mL) for 7, 0.38 µM (0.1 µg/mL) and 1.9 µM (0.5 µg/mL) for 10 and 36.5 µM (5 µg/mL) for INH. The ﬁnal concentration of DMSO was kept at 1% in each experiment. 14C acetate (speciﬁc activity 106 mCi/mmol, ARC) in the ﬁnal concentration of 0.5 µCi/mL was added to each of the cultures after 24 h of cultivation with shaking (120 rpm) and the cells were cultivated for next 24 h. Lipids were extracted from whole cells harvested from 10 mL culture aliquots as described earlier [34,86], dissolved in chloroform:methanol (2:1)—350 µL per 1 unit of OD600 of harvested cells. 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Chollet, A.; Mori, G.; Menendez, C.; Rodriguez, F.; Fabing, I.; Pasca, M.R.; Madacki, J.; Korduláková, J.; Constant, P.; Quémard, A.; et al. Design, synthesis and evaluation of new GEQ derivatives as inhibitors of InhA enzyme and Mycobacterium tuberculosis growth. Eur. J. Med. Chem. 2015, 101, 218–235. [CrossRef] [PubMed] 27 of 27 27 of 27 Molecules 2017, 22, 1457 86. Stadthagen, G.; Korduláková, J.; Grifﬁn, R.; Constant, P.; Bottová, I.; Barilone, N.; Gicquel, B.; Daffé, M.; Jackson, M. p-Hydroxybenzoic acid synthesis in Mycobacterium tuberculosis. J. Biol. Chem. 2005, 280, 40699–40706. [CrossRef] [PubMed] 87. Phetsuksiri, B.; Baulard, A.R.; Cooper, A.M.; Minnikin, D.E.; Douglas, J.D.; Besra, G.S.; Brennan, P.J. Antimycobacterial activities of isoxyl and new derivatives through the inhibition of mycolic acid synthesis. Antimicrob. Agents Chemother. 1999, 43, 1042–1051. [PubMed] 88. Larsen, M.H.; Vilchèze, C.; Kremer, L.; Besra, G.S.; Parsons, L.; Salﬁnger, M.; Heifets, L.; Hazbon, M.H.; Alland, D.; Sacchettini, J.C.; et al. Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis. Mol. Microbiol. 2002, 46, 453–466. [CrossRef] [PubMed] Sample Availability: Samples of the compounds 1a–d, 2–11 are available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2891977167	https://advances.umw.edu.pl/pdf/2018/27/9/1253.pdf	English	null	Anti-CCP antibodies and rheumatoid factor in systemic sclerosis: Prevalence and relationships with joint manifestations	Advances in Clinical and Experimental Medicine	2,018	cc-by	4,908	Original papers Original papers Advances in Clinical and Experimental Medicine, ISSN 1899-5276 (print), ISSN 2451-2680 (online) Advances in Clinical and Experimental Medicine, ISSN 1899-5276 (print), ISSN 2451-2680 (online) Adv Clin Exp Med. 2018;27(9):1253–1257 Adv Clin Exp Med. 2018;27(9):1253–1257 Address for correspondence Ewa Wielosz E-mail: ewa.wielosz@wp.pl Funding sources None declared Conflict of interest None declared Copyright Copyright by Author(s) This is an article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc-nd/4.0/) DOI 10.17219/acem/69921 Copyright Copyright by Author(s) This is an article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc-nd/4.0/) Anti-CCP antibodies and rheumatoid factor in systemic sclerosis: Prevalence and relationships with joint manifestations Ewa WieloszA–F, Maria MajdanA,C,E,F, Magdalena DryglewskaB,C, Robert ZwolakB,C,E Department of Rheumatology and Connective Tissue Diseases, Medical University of Lublin, Poland Introduction IgM RF were determined using the enzyme-linked im- munosorbent assay (ELISA) commercial test : EUROIM- MUN for IgM RF (EUROIMMUN AG, Lübeck, Germany) and INOVA for a-CCP antibodies (INOVA Diagnostics, San Diego, USA). The test kit was used according to the manufacturer’s suggested procedures. The samples were classified as negative for a-CCP antibodies at <20 units and negative for IgM RF at <30 RU/mL. Two markers of in- flammation, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), were also determined. The sam- ples were classified as negative for ESR at <16 mm/h for women and at <11 mm/h for men, and at <10 mg/L for CRP. Joint involvement was assessed according to clinical manifestations, such as arthralgia or arthritis. Arthral- gia was assessed by involving 2 or more peripheral joints characterized by tenderness. Non-erosive arthritis was determined by involving 2 or more peripheral joints char- acterized by tenderness and swelling or confirmed by ultra- sonography (USG). Moreover, flexion contractures, tendon friction rubs (TFRs) and finger-to-palm distance in flexion (FTP) were assessed. Finger-to-palm distance in flexion was determined by measuring the minimal distance (mm) between the nail tip of the middle finger and the transverse palmar creases in both hands (normally no distance can be measured).12 Rheumatoid arthritis was diagnosed accord- ing to the ACR criteria.13 Anti-topoisomerase I (a-Scl-70) and anti-centromere antibodies (ACA) were determined using a commercial test, the EUROLINE Systemic Sclerosis Profile (EUROIMMUN AG, Lübeck, Germany). Systemic sclerosis (SSc) is a multisystem disorder char- acterized by vascular damage, immune activation and fi- broblast activation, changes which lead to a progressive thickening of the skin with structural and functional ab- normalities of different organs. Musculoskeletal involve- ment is present in 24–97% of SSc patients.1–3 A more com- mon manifestation is arthralgia, whereas arthritis is rare.4,5 Hand involvement is often the first clinical manifestation of SSc.6,7 Symmetrical polyarthritis similar to rheumatoid arthritis (RA) can also be found in SSc, but it particularly characterizes a systemic sclerosis-rheumatoid arthritis (SSc-RA) overlap syndrome.2,8–10 In some cases, arthritis in the course of SSc and SSc-RA overlap syndrome can be very difficult to distinguish. It is known that the main se- rological markers for diagnosing RA are anti-citrullinated protein (a-CCP) antibodies and rheumatoid factor (RF). Abstract Background. It is known that anti-citrullinated protein (a-CCP) antibodies and rheumatoid factor (RF) can be present in systemic sclerosis (SSc) patients, particularly with joint involvement. Objectives. The aim of the study was to assess the prevalence of a-CCP antibodies and immunoglobulin M class (IgM) RF, and the relationships between their presence and joint manifestations in patients with SSc. Material and methods. The study included 100 European Caucasian SSc patients hospitalized consecutively in the Department of Rheumatology and Connective Tissue Diseases (Lublin, Poland). Anti-citrullinated protein antibodies and IgM RF were determined using a commercial enzyme-linked immunosorbent assay (ELISA) test. Received on October 30, 2016 Reviewed on December 14, 2016 Accepted on March 28, 2017 Results. Anti-citrullinated protein antibodies were found in 10 out of 100 (10%) SSc patients and IgM RF in 71 out of 100 (71%) SSc patients. In the study, 90/100 (90%) SSc patients had joint manifestations (arthralgia or arthritis), 34/100 (34%) had arthritis and 6/100 (6%) had a systemic sclerosis-rheumatoid arthritis (SSc-RA) overlap syndrome. Significantly higher a-CCP antibody levels (p = 0.012), erythrocyte sedimentation rate (ESR) (p = 0.029) and C-reactive protein (CRP) levels (p = 0.020) were observed in the SSc group with arthritis. A significant correlation was found between the group with arthritis and the presence of a-CCP antibodies, and between the arthralgia group and the presence of IgM RF. Conclusions. The prevalence of RF and a-CCP antibodies is relatively high in SSc, and joint involvement occurs frequently. There was a significantly higher prevalence of IgM RF in the group with joint manifesta- tions. About 1/3 of SSc patients had symptoms of arthritis. Arthritis is connected with the presence of a-CCP antibodies, while arthralgia is connected with the presence of IgM RF. Key words: systemic sclerosis, arthritis, rheumatoid factor, anti-citrullinated protein antibodies, arthralgia DOI 10.17219/acem/69921 Copyright Copyright by Author(s) This is an article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc-nd/4.0/) E. Wielosz, et al. A-CCP antibodies and rheumatoid factor in SSc 1254 Introduction These parameters can be present in SSc patients, particu- larly with joint involvement.1,2,8 The aim of the study was to assess the prevalence of a-CCP antibodies and immuno- globulin M class (IgM) RF, and the relationships between their presence and joint manifestations in patients with SSc. Material and methods The study included 100 European Caucasian SSc pa- tients (82 female and 18 male) hospitalized consecutively in the Department of Rheumatology and Connective Tis- sue Diseases (Lublin, Poland). The patients fulfilled the American College of Rheumatology (ACR) classification criteria of SSc (Table 1).11 Serum samples were obtained from 100 patients and a joint examination was performed at the same time. Anti-citrullinated protein antibodies and All calculations were performed with STATISTICA v. 10.0 software (StatSoft, Kraków, Poland). Data was analyzed using the non-parametric χ2 test for comparisons be- tween the groups. Quantitative data was assessed using the Mann-Whitney U test, whereas qualitative data was de- termined by the Yule correlation. Probability value p < 0.05 was considered statistically significant. Table 1. Characteristics of the SSc study group Characteristics Patients’ data Number of patients 100 SSc subtype dcSSc – 50 lcSSc – 50 Age [years] 52.68 ±13.85 (range 18–81) Duration of disease [years] 6.46 ±6.05 (range 0.1–23) ACA 19/100 (19%) Anti-Scl-70 46/100 (46%) Overlap syndrome 21/100 (21%) SSc/RA 6/100 (6%) SSc/SS 8/100 (8%) SSc/DM/PM 7/100 (7%) SSc – systemic sclerosis; dcSSc – diffuse cutaneous SSc; lcSSc – limited cutaneous SSc; ACA – anti-centromere antibodies; anti-Scl-70 – anti- topoisomerase I antibodies; RA – rheumatoid arthritis; SS – Sjögren's syndrome; DM/PM – dermatomyositis/polymyositis. Data is presented as mean ± standard deviation (SD), number and percentage. Results 0 arthritis (–) 1 arthritis (+) arthritis –10 0 10 20 30 40 50 60 70 80 a-CCP antibodies titers 0 arthritis (–) 1 arthritis (+) arthritis 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 ESR [mm/h] IgM RF – immunoglobulin M class rheumatoid factor; a-CCP – anti-citrullinated protein; SSc – systemic sclerosis; NS – nonsignificant. Data is presented as number and percentage; p-value of <0.05 was considered statistically significant. t. Fig. 3. Statistically significant differences in the titers of CRP in groups with and without arthritis CRP – C-reactive protein; probability value: p = 0.0201. 0 arthritis (–) 1 arthritis (+) arthritis 4 6 8 10 12 14 16 18 20 22 CRP [mg/L] Fig. 3. Statistically significant differences in the titers of CRP in groups with and without arthritis CRP – C-reactive protein; probability value: p = 0.0201. 0 arthritis (–) 1 arthritis (+) arthritis 4 6 8 10 12 14 16 18 20 22 CRP [mg/L] 0 arthritis (–) 1 arthritis (+) arthritis –10 0 10 20 30 40 50 60 70 80 a-CCP antibodies titers CRP [mg/L] Fig. 1. Statistically significant differences in the titers of a-CCP antibodies in groups with and without arthritis Fig. 3. Statistically significant differences in the titers of CRP in groups with and without arthritis a-CCP – anti-citrullinated protein; probability value: p = 0.0119. a-CCP – anti-citrullinated protein; probability value: p = 0.0119. a-CCP – anti-citrullinated protein; probability value: p = 0.0119. Fig. 2. Statistically significant differences in ESR in groups with and without arthritis ESR – erythrocyte sedimentation rate; probability value: p = 0.0294. 0 arthritis (–) 1 arthritis (+) arthritis 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 ESR [mm/h] CRP – C-reactive protein; probability value: p = 0.0201. at a medium value and 6 (9%) at a low value. Anti-citrulli- nated protein antibodies were found in 3 (5%) SSc patients without arthritis, 1 (2%) with a high value and 2 (3%) with low. However, in the group of 46 patients with positive IgM RF without overlapped RA and without arthritis, 42 (91%) patients had arthralgia. Our findings indicated that the prevalence of IgM RF was significantly higher in the group with joint manifestations compared to the group without it (p = 0.0230). Results According to our observations, 90 out of the 100 (90%) SSc patients had arthralgia, 34 (34%) SSc patients had arthritis and 24 (24%) SSc patients developed flexion contractures. Finger-to-palm distance in flexion was decreased in 26 out of 100 (26%) SSc patients, while 5 (5%) SSc patients had TFRs. An SSc-RA overlap syndrome was found in 6 (6%) SSc patients. The mean disease activity score 28 (DAS 28) in the group with an SSc-RA overlap syndrome was 4.46 ±1.29. The prevalence of IgM RF and a-CCP antibod- ies in SSc patients with and without joint manifestations is presented in Table 2. SSc – systemic sclerosis; dcSSc – diffuse cutaneous SSc; lcSSc – limited cutaneous SSc; ACA – anti-centromere antibodies; anti-Scl-70 – anti- topoisomerase I antibodies; RA – rheumatoid arthritis; SS – Sjögren's syndrome; DM/PM – dermatomyositis/polymyositis. Data is presented as mean ± standard deviation (SD), number and percentage. In the SSc study group without overlapped RA, 28 pa- tients had arthritis, 65 patients were positive for IgM RF and 7 patients were positive for a-CCP antibodies. In the group of 28 SSc patients with arthritis, IgM RF was present in 19 patients (68%), at a high value in 15 (54%) patients and Adv Clin Exp Med. 2018;27(9):1253–1257 1255 Table 2. The prevalence of IgM RF and a-CCP antibodies in SSc patients with and without joint manifestations Prevalence of IgM RF and a-CCP antibodies Total Group with joint manifestations Group without joint manifestations p-value arthralgia arthritis Number of patients 100 90/100 (90%) 34/100 (34%) 10/100 (10%) – IgM RF 71/100 (71%) 67/90 (74.4%) 25/34 (73.5%) 4/10 (40%) 0.023 p = 0.049 a-CCP antibodies 10/100 (10%) 9/90 (10%) 7/34 (20.6%) 1/10 (10%) NS NS IgM RF – immunoglobulin M class rheumatoid factor; a-CCP – anti-citrullinated protein; SSc – systemic sclerosis; NS – nonsignificant. Data is presented as number and percentage; p-value of <0.05 was considered statistically significant. Fig. 1. Statistically significant differences in the titers of a-CCP antibodies in groups with and without arthritis a-CCP – anti-citrullinated protein; probability value: p = 0.0119. 0 arthritis (–) 1 arthritis (+) arthritis –10 0 10 20 30 40 50 60 70 80 a-CCP antibodies titers 44 Fig. 3. Statistically significant differences in the titers of CRP in groups with and without arthritis CRP – C-reactive protein; probability value: p = 0.0201. Results 0 arthritis (–) 1 arthritis (+) arthritis 4 6 8 10 12 14 16 18 20 22 CRP [mg/L] Table 2. The prevalence of IgM RF and a-CCP antibodies in SSc patients with and without joint manifestations Prevalence of IgM RF and a-CCP antibodies Total Group with joint manifestations Group without joint manifestations p-value arthralgia arthritis Number of patients 100 90/100 (90%) 34/100 (34%) 10/100 (10%) – IgM RF 71/100 (71%) 67/90 (74.4%) 25/34 (73.5%) 4/10 (40%) 0.023 p = 0.049 a-CCP antibodies 10/100 (10%) 9/90 (10%) 7/34 (20.6%) 1/10 (10%) NS NS IgM RF – immunoglobulin M class rheumatoid factor; a-CCP – anti-citrullinated protein; SSc – systemic sclerosis; NS – nonsignificant. Data is presented as number and percentage; p-value of <0.05 was considered statistically significant. Table 2. The prevalence of IgM RF and a-CCP antibodies in SSc patients with and without joint manifestations Prevalence of IgM RF and a-CCP antibodies Total Group with joint manifestations Group without joint manifestations p-value arthralgia arthritis Number of patients 100 90/100 (90%) 34/100 (34%) 10/100 (10%) – IgM RF 71/100 (71%) 67/90 (74.4%) 25/34 (73.5%) 4/10 (40%) 0.023 p = 0.049 a-CCP antibodies 10/100 (10%) 9/90 (10%) 7/34 (20.6%) 1/10 (10%) NS NS IgM RF – immunoglobulin M class rheumatoid factor; a-CCP – anti-citrullinated protein; SSc – systemic sclerosis; NS – nonsignificant. Data is presented as number and percentage; p-value of <0.05 was considered statistically significant. Table 2. The prevalence of IgM RF and a-CCP antibodies in SSc patients with and without joint manifestations at a low value in 4 (14%) patients. Moreover, a-CCP anti- a-CCP antibodies 10/100 (10%) 9/90 (10%) IgM RF – immunoglobulin M class rheumatoid factor; a-CCP – anti-citrullinate number and percentage; p-value of <0.05 was considered statistically signific Fig. 1. Statistically significant differences in the titers of a-CCP antibodies in groups with and without arthritis a-CCP – anti-citrullinated protein; probability value: p = 0.0119. Fig. 2. Statistically significant differences in ESR in groups with and without arthritis ESR – erythrocyte sedimentation rate; probability value: p = 0.0294. Discussion Ac- cording to our study, 90% of SSc patients had joint mani- festations and 71% were positive for IgM RF, but only 6% developed an SSc-RA overlap syndrome. The above re- sults suggest that the presence of RF or a-CCP antibodies in SSc patients does not lead to a diagnosis of an SSc-RA overlap syndrome. According to literature data, the search for anti-CCP antibodies might be of great help in some infrequent cases of an SSc-RA overlap syndrome, but not in all situations.19 Destructive joint disease in patients with SSc may suggest an overlap syndrome with RA.6,10 Numerous studies have reported such results. According to Arslan Tas et al., such radiographic findings as erosions, joint space narrowing and arthritis were less frequent in the SSc group, but acro-osteolysis, flexion contracture and calcinosis were more frequent than in the RA group.6 The abovementioned radiological findings can worsen the prognosis. Apart from hand radiography, the potential of USG in the assessment of joint and tendon involvement in the course of SSc was evaluated. Some studies revealed that SSc patients with a history of arthralgia without ar- thritis developed synovial inflammation, which could be observed during an USG examination, particularly in the wrist and small hand joints.20,21 Another important finding was that TFRs were part of functional impairment in SSc.22 In the present study, we investigated the presence of serological parameters of joint involvement: a-CCP antibodies and IgM RF in SSc. According to our obser- vations, a-CCP antibodies were found in 10% of SSc pa- tients. Our findings are similar to those reported in the literature. Polimeni et al. Results However, no significant intergroup differ- ences in the presence of a-CCP antibodies were determined (Table 2). Significantly higher concentrations of a-CCP antibodies (p = 0.0119) and CRP (p = 0.0201), as well as ESR (p = 0.0294) were observed in the SSc group with ar- thritis compared to the group without arthritis (Fig. 1–3). The titer of IgM RF was not significantly higher in the SSc group with arthritis (p = 0.0713) than in the group with- out arthritis. Interestingly, a significant correlation was determined between the SSc group with arthritis and the presence of a-CCP antibodies (p = 0.013; Φ = 0.263) and between the SSc group with arthralgia and the presence of IgM RF (p = 0.025; Φ = 0.238). Moreover, a significant correlation was noted between the SSc group with arthritis and the concentrations of inflammatory parameters (ESR and CRP) (Table 3). ESR – erythrocyte sedimentation rate; probability value: p = 0.0294. at a low value in 4 (14%) patients. Moreover, a-CCP anti- bodies were present in 4 out of 28 patients (14%) with ar- thritis, 3 (11%) with a high value and 1 (3%) with low. In the group of 66 SSc patients without arthritis, 46 patients (70%) were positive for IgM RF, 32 (49%) at a high value, 8 (12%) E. Wielosz, et al. A-CCP antibodies and rheumatoid factor in SSc 1256 Table 3. Correlations between IgM RF, a-CCP antibodies, inflammation parameters and joint involvement Joint manifestations ESR CRP IgM RF a-CCP antibodies Arthritis p = 0.0158 Φ = 0.234 p = 0.050 Φ = 0.192 p = 0.481 Φ = 0.073 p = 0.013 Φ = 0.263 Arthralgia p = 0.467 Φ = 0.070 p = 0.844 Φ = 0.025 p = 0.025 Φ = 0.238 p = 0.895 Φ = 0.012 ESR – erythrocyte sedimentation rate; CRP – C-reactive protein; IgM RF – immunoglobulin M class rheumatoid factor; a-CCP – anti-citrullinated protein; p-value of <0.05 was considered statistically significant. Discussion patients with joint manifestations in our study group. Ac- cording to our study, 90% of SSc patients had joint mani- festations and 71% were positive for IgM RF, but only 6% developed an SSc-RA overlap syndrome. The above re- sults suggest that the presence of RF or a-CCP antibodies in SSc patients does not lead to a diagnosis of an SSc-RA overlap syndrome. According to literature data, the search for anti-CCP antibodies might be of great help in some infrequent cases of an SSc-RA overlap syndrome, but not in all situations.19 Destructive joint disease in patients with SSc may suggest an overlap syndrome with RA.6,10 Numerous studies have reported such results. According to Arslan Tas et al., such radiographic findings as erosions, joint space narrowing and arthritis were less frequent in the SSc group, but acro-osteolysis, flexion contracture and calcinosis were more frequent than in the RA group.6 The abovementioned radiological findings can worsen the prognosis. Apart from hand radiography, the potential of USG in the assessment of joint and tendon involvement in the course of SSc was evaluated. Some studies revealed that SSc patients with a history of arthralgia without ar- thritis developed synovial inflammation, which could be observed during an USG examination, particularly in the wrist and small hand joints.20,21 Another important finding was that TFRs were part of functional impairment in SSc.22 Moreover, according to the largest worldwide database, joint and tendon involvement predict disease progression in SSc.23 Using the latest data from the European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) cohort, this prospective study inves- tigating 1,301 patients with SSc demonstrated that joint synovitis and TFRs were strong independent predictors of skin progression and that positivity for a-Scl-70 anti- bodies and a history of digital ulcers were also predic- tive markers. Furthermore, joint synovitis was predictive of the occurrence of decreased left ventricular ejection fraction and the occurrence of new digital ulcers.23 To sum up, according to literature data, hand arthropathy in SSc should alert one to functional damage, severe skin pro- gression and serious internal organ involvement, particu- larly cardiopulmonary complications, which was the main cause of death in SSc.24–27 In conclusion, serological pa- rameters which could predict severe articular involvement are needed. According to Generini et al., antibodies such as heterogeneous nuclear ribonucleoprotein isoform A1 patients with joint manifestations in our study group. Discussion demonstrated that in a SSc group, 7 of 78 (9%) patients were a-CCP positive.2 According to Ingegnoli et al., the presence of a-CCP antibodies was detected in 8 of 75 (10.7%) patients with SSc.1 Other stud- ies showed that a-CCP antibodies were revealed in 18 out of 146 (12.3%), 3 out of 28 (10.7%), 11 out of 82 (13.4%), and 3 out of 114 (2.6%) SSc patients.6,14–17 Moreover, Horimoto and Costa found a-CCP antibodies in 4 out of 24 patients (16.7%), but all patients had an SSc-RA overlap syndrome.9 Based on literature data, we can suppose that a-CCP an- tibodies are a useful marker in identifying patients diag- nosed with an SSc-RA overlap syndrome; however, these antibodies may also be positive in 7% of SSc patients with- out arthritis.15 In our study, we also detected a-CCP anti- bodies in 10% of SSc patients without joint involvement. There are many data indicating a significant association between positive a-CCP antibodies and arthritis and bone erosions in SSc.1,6,16,17 According to our findings, there was a significant correlation between the group of SSc patients with arthritis and the presence of a-CCP anti- bodies; higher titers of a-CCP antibodies were observed in the SSc group with arthritis compared to the group without arthritis. Additionally, the prevalence of IgM RF in SSc was evaluated; IgM RF was detected in 71% of pa- tients, which seems to be higher than in other studies. According to our research, the prevalence of RF in SSc ranged from 12% to 35% and may be positive in SSc pa- tients without joint manifestations.1,6,9,15,18,19 Avouac et al. was that TFRs were part of functional impairment in SSc. Moreover, according to the largest worldwide database, joint and tendon involvement predict disease progression in SSc.23 Using the latest data from the European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) cohort, this prospective study inves- tigating 1,301 patients with SSc demonstrated that joint synovitis and TFRs were strong independent predictors of skin progression and that positivity for a-Scl-70 anti- bodies and a history of digital ulcers were also predic- tive markers. Discussion demonstrated that in a SSc group, 7 of 78 (9%) patients were a-CCP positive.2 According to Ingegnoli et al., the presence of a-CCP antibodies was detected in 8 of 75 (10.7%) patients with SSc.1 Other stud- ies showed that a-CCP antibodies were revealed in 18 out of 146 (12.3%), 3 out of 28 (10.7%), 11 out of 82 (13.4%), and 3 out of 114 (2.6%) SSc patients.6,14–17 Moreover, Horimoto and Costa found a-CCP antibodies in 4 out of 24 patients (16.7%), but all patients had an SSc-RA overlap syndrome.9 Based on literature data, we can suppose that a-CCP an- tibodies are a useful marker in identifying patients diag- nosed with an SSc-RA overlap syndrome; however, these antibodies may also be positive in 7% of SSc patients with- out arthritis.15 In our study, we also detected a-CCP anti- bodies in 10% of SSc patients without joint involvement. There are many data indicating a significant association between positive a-CCP antibodies and arthritis and bone erosions in SSc.1,6,16,17 According to our findings, there was a significant correlation between the group of SSc patients with arthritis and the presence of a-CCP anti- bodies; higher titers of a-CCP antibodies were observed in the SSc group with arthritis compared to the group without arthritis. Additionally, the prevalence of IgM RF in SSc was evaluated; IgM RF was detected in 71% of pa- tients, which seems to be higher than in other studies. According to our research, the prevalence of RF in SSc ranged from 12% to 35% and may be positive in SSc pa- tients without joint manifestations.1,6,9,15,18,19 Avouac et al. suggested that the RF test seems non-specific and does not distinguish SSc patients with musculoskeletal mani- festations from those unaffected by them.19 Rheumatoid factor may also be seen in patients with SSc associated with secondary Sjögren’s syndrome, which is not uncom- mon in SSc patients.19 However, our results revealed that the prevalence of IgM RF was significantly higher in the group with joint manifestations as compared to the group In the present study, we investigated the presence of serological parameters of joint involvement: a-CCP antibodies and IgM RF in SSc. According to our obser- vations, a-CCP antibodies were found in 10% of SSc pa- tients. Our findings are similar to those reported in the literature. Polimeni et al. References 19. Avouac J, Clements PJ, Khanna D, Furst DE, Allanore Y. Articular involvement in systemic sclerosis. Rheumatology (Oxford). 2012;51: 1347–1356. 1. Ingegnoli F, Galbiati V, Zeni S, et al. Use of antibodies recognizing cyclic citrullinated peptide in the differential diagnosis of joint involvement in systemic sclerosis. Clin Rheumatol. 2007;26:510–514. 20. Iagnocco A, Vavala C, Vasile M, Stefanantoni K, Valesini G, Riccieri V. Power Doppler ultrasound of the hand and wrist joints in systemic sclerosis. Clin Exp Rheumatol. 2013;31:89–95. 2. Polimeni M, Feniman D, Skare TS, Nisihara RM. Anti-cyclic citrulli- nated peptide antibodies in scleroderma patients. Clin Rheumatol. 2012;31:877–880. 21. Gutierrez M, Pineda C, Cazenave T, et al. Ultrasound in systemic scle- rosis. A multi-target approach from joint to lung. Clin Rheumatol. 2014;33:1039–1047. 3. Schmeiser T, Pons-Kühnemann J, Özden F, Müller-Ladner U, Dinser R. Arthritis in patients with systemic sclerosis. Eur J Intern Med. 2012; 23:25–29. 22. Stoenoiu MS, Houssiau FA, Lecouvet FE. Tendon friction rubs in sys- temic sclerosis: A possible explanation – an ultrasound and magnetic resonance imaging study. Rheumatology (Oxford). 2013;52:529–533. 4. Lima TR, Guimarães FS, Silva LA, Silva DP, Menezes SL, Lopes AJ. Relationship between functional capacity, joint mobility and pul- monary function in patients with systemic sclerosis. J Bodyw Mov Ther. 2015;19:17–24. 23. Avouac J, Walker UA, Hachulla E, et al; EUSTAR collaborators. Joint and tendon involvement predict disease progression in system- ic sclerosis: A EUSTAR prospective study. Ann Rheum Dis. 2016;75: 103–109. 5. Chevreul K, Brigham KB, Gandré C, Mouthon L; BURQOL-RD Research Network. The economic burden and health-related quality of life associated with systemic sclerosis in France. Scand J Rheumatol. 2014; 18:1–9. 24. Bálint Z, Farkas H, Farkas N, et al. A three-year follow-up study of the development of joint contractures in 131 patients with systemic scle- rosis. Clin Exp Rheumatol. 2014;32:68–74. 6. Arslan Tas D, Erken E, Sakalli H, Yucel AE. Evaluating hand in system- ic sclerosis. Rheumatol Int. 2012;32:3581–3586. 25. Ingegnoli F, Boracchi P, Ambrogi F, Gualtierotti R, Galbiati V, Meroni PL. Hand impairment in systemic sclerosis: Association of different hand indices with organ involvement. Scand J Rheumatol. 2010;39: 393–397. 7. Mouthon L. Hand involvement in systemic sclerosis. Presse Med. 2013; 42:1616–1626. 8. Marrone M, Chialà A, Tampoia M, et al. Prevalence of anti-CCP anti- bodies in systemic sclerosis. Reumatismo. 2007;59:20–24. 26. Fett N. Scleroderma: Nomenclature, etiology, pathogenesis, progno- sis, and treatments: Facts and controversies. Clin Dermatol. 2013;31: 432–437. 9. Discussion Furthermore, joint synovitis was predictive of the occurrence of decreased left ventricular ejection fraction and the occurrence of new digital ulcers.23 To sum up, according to literature data, hand arthropathy in SSc should alert one to functional damage, severe skin pro- gression and serious internal organ involvement, particu- larly cardiopulmonary complications, which was the main cause of death in SSc.24–27 In conclusion, serological pa- rameters which could predict severe articular involvement are needed. According to Generini et al., antibodies such as heterogeneous nuclear ribonucleoprotein isoform A1 suggested that the RF test seems non-specific and does not distinguish SSc patients with musculoskeletal mani- festations from those unaffected by them.19 Rheumatoid factor may also be seen in patients with SSc associated with secondary Sjögren’s syndrome, which is not uncom- mon in SSc patients.19 However, our results revealed that the prevalence of IgM RF was significantly higher in the group with joint manifestations as compared to the group without joint manifestations. This observation seemed to be associated with the presence of a large number of SSc Adv Clin Exp Med. 2018;27(9):1253–1257 1257 and A2 antibodies (anti-hnRNP-A1 and anti-hnRNP-A2) may have some diagnostic value for joint involvement and the risk of developing erosive arthritis in SSc.28 11. Arslan Tas D; Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Com- mittee. Preliminary criteria for the classification of systemic sclero- sis (scleroderma). Arthritis Rheum. 1980;23:581–590. sis (scleroderma). Arthritis Rheum. 1980;23:581–590. 12. McHugh NJ, Distler O, Giacomelli R, Riemekasten G. Non organ based laboratory markers in systemic sclerosis. Clin Exp Rheumatol. 2003;21: 32–38. Conclusions 13. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31:315–324. Immunoglobulin M class rheumatoid factor and a-CCP antibodies seem to be a helpful tool to differentiate cases with joint manifestations, although not in all patients. In our study, arthralgia was a common manifestation in SSc patients and the prevalence of RF was high in this group. Furthermore, RF correlated with arthralgia and a-CCP antibodies correlated with arthritis in SSc pa- tients. Our study requires further analysis with the re- sults based on clinical picture, USG and radiological joint examinations. 14. Payet J, Goulvestre C, Bialé L, et al. Anticyclic citrullinated peptide antibodies in rheumatoid and nonrheumatoid rheumatic disorders: Experience with 1162 patients. J Rheumatol. 2014;41:2395–2402. 15. Ueda-Hayakawa I, Hasegawa M, Kumada S, et al. Usefulness of anti- cyclic citrullinated peptide antibody and rheumatoid factor to detect rheumatoid arthritis in patients with systemic sclerosis. Rheumatol- ogy (Oxford). 2010;49:2135–2139. 16. Stamenković B, Stanković A, Dimić A, et al. The clinical significance of antibody determination to cyclic citrullinated peptides in system- ic sclerosis. Srp Arh Celok Lek. 2012;140:350–354. 17. Morita Y, Muro Y, Sugiura K, Tomita Y. Anti-cyclic citrullinated peptide antibody in systemic sclerosis. Clin Exp Rheumatol. 2008;26:542–547. 18. Allali F, Tahiri L, Senjari A, Abouqal R, Hajjaj-Hassouni N. Erosive arthropathy in systemic sclerosis. BMC Public Health. 2007;7:260–266. References Horimoto AM, Costa IP. Overlap between systemic sclerosis and rheu- matoid arthritis: A distinct clinical entity? Rev Bras Reumatol. 2015;4: 482–504. 27. Grant-Kels JM, Rothe MJ, Mease P. Rheumatology and dermatology: Part 1. Clin Dermatol. 2006;24:347. 10. Chung L, Lin J, Furst DE, Fiorentino D. Systemic and localized sclero- derma. Clin Dermatol. 2006;24:374–392. 28. Generini S, Steiner G, Miniati I, et al. Anti-hnRNP and other autoan- tibodies in systemic sclerosis with joint involvement. Rheumatology (Oxford). 2009;48:920–925.
https://openalex.org/W4310577483	https://zenodo.org/records/7340631/files/Agroprocessing-2022-7-10.pdf	Russian	null	ЭГАТ ТУБИНИ ЎЗГАРУВЧАН ЗИЧЛОВЧИ ТЕХНИК ВОСИТА ВА СУҒОРИШ ТЕХНОЛОГИЯСИ БЎЙИЧА ТАЖРИБАЛАР ТАҲЛИЛИ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	5,433	SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 SVcabcaiXdd[`VYfc`S^[ Y[^W!da` Yfc`S^SVcabcaiXdd[`V ea_`a_Xc! ɮɮɹ ! ISSN 2181-9904 Doi Journal 10.26739/2181-9904 ISSN 2181-9904 Doi Journal 10.26739/2181-9904 Хамидов Мухаммадхон Хамидович Хамидов Мухаммадхон Хамидович доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Хамидов Мухаммадхон Хамидович қишлоқ хўжалиги фанлар доктори, “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети профессори Хамидов Мухаммадхон Хамидович доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” eSȗc[c[\_Sd^ShSe]X`VSk[ Исаев С.Х., қишлоқ хўжалиги фанлар доктори, “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети профессори; Муродов Ш.М., иқтисодиёт фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Худайев И.Ж., техника фанлари доктори (DSc)номзоди, “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети Бухоро филиали; Матякубов Б.Ш, қишлоқ хўжалиги фанлар доктори, “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети профессори; Ахмедов Д.Х., биология фанлари доктори, Пахта селекцияси, уруғчилиги ва етиштириш Ахмедов Д.Х., биология фанлари доктори, Пахта селекцияси, уруғчилиги ва етиштириш агротехнологиялари илмий-тадқиқот институти, катта илмий ҳодими; Мирхасилова З.Қ., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; агротехнологиялари илмий-тадқиқот институти, катта илмий ҳодими; Равшанов А.Э., қишлоқ хўжалиги фанлари доктори, Пахта селекцияси, уруғчилиги ва етиштириш агротехнологиялари илмий-тадқиқот институти директори; Атажанов А., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Нурматов Ш.Н., қишлоқ хўжалик фанлари доктори, Қишлоқ хўжалик экинлари навларини синаш маркази директори; Аманов Б.Т., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Авлияқулов М.А., қишлоқ хужалиги фанлари доктори (DSc), Пахта селекцияси, уруғчилиги ва етиштириш агротехнологиялари илмий-тадқиқот институти, катта илмий ходими; Улжаев Ф.Б., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Каримов Ш.А., қишлоқ хўжалиги фанлари фалсафа доктори, Пахта селекцияси, уруғчилиги ва етиштириш агротехнологиялари илмий-тадқиқот институти, катта илмий ходим; Гадаев Н.Н., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Муратов А.Р., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Гуломов С.Б., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Касымбетова С.А., техника фанлари номзоди (PhD), “Тошкент ирригация ва қишлоқ хўжалиги Уразбаев И.К., “Тошкент ирригация ва қишлоқ хўжалиги механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; механизациялаш муҳандислар институти” миллий тадқиқот университети доценти; Khamidov Mukhammadkhan Doctor of Agricultural Sciences, Professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University Tak_fȗScc[c¬V^SU`n\cXWS]eac¬ɹɖ Khamidov Mukhammadkhan Doctor of Agricultural Sciences, Professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University cXWS]i[a``n\daUXe Исаев С.Х., доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Матякубов Б.Ш., доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Исаев С.Х., доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Матякубов Б.Ш., доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” у Худайев И.Ж., доктор техничких наук, доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Бухарского филиала Мирхасилова З.Қ., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” у Худайев И.Ж., доктор техничких наук, доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Бухарского филиала Ахмедов Д.Х., доктор биологических наук, НИИ хлопководства, семеноводства и агротехнологии, старший научный сотрудник; Ахмедов Д.Х., доктор биологических наук, НИИ хлопководства, семеноводства и агротехнологии, старший научный сотрудник; 2 2 ɹ №7 \| 2022 Атажанов А., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Аманов Б.Т., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Улжаев Ф.Б., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Гадаев Н.Н., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Гуломов С.Б., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Уразбаев И.К., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” ɹ №7 \| 2022 Атажанов А., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Аманов Б.Т., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Улжаев Ф.Б., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Гадаев Н.Н., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Гуломов С.Б., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Уразбаев И К кандидат технических наук (PhD) доцент VcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮS Равшанов А.Э., доктор сельскохозяйственных наук, директор научно-исследовательского института селекции, семеноводства и агротехнологии выращивания хлопка; VcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮS Равшанов А.Э., доктор сельскохозяйственных наук, директор научно-исследовательского института селекции, семеноводства и агротехнологии выращивания хлопка; Нурматов Ш.Н., доктор сельскохозяйственных наук, директор Центра сортоиспытаний сельскохозяйственных культур; ɹ №7 \| 2022 Атажанов А., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” VcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮS Равшанов А.Э., доктор сельскохозяйственных наук, директор научно-исследовательского института селекции, семеноводства и агротехнологии выращивания хлопка; Нурматов Ш.Н., доктор сельскохозяйственных наук, директор Центра сортоиспытаний сельскохозяйственных культур; Авлияқулов М.А., доктор сельскохозяйственных наук, НИИ хлопководства, семеноводства и агротехнологии, старший научный сотрудник; Каримов Ш.А., доктор сельскохозяйственных наук (DSc), старший-научный сотрудник научно-исследовательского института селекции, семеноводства и агротехнологии выращивания хлопка; Муратов А.Р., к.т.н., (PhD), доцент Национального исследовательского университета "Ташкентский институт инженеров ирригации и механизации сельского хозяйства"; Касымбетова С.А., кандидат технических наук, (PhD), доцент Национального исследовательского университета "Ташкентский институт инженеров ирригации и механизации сельского хозяйства"; р р ; Нурматов Ш.Н., доктор сельскохозяйственных наук, директор Центра сортоиспытаний сельскохозяйственных культур; Аманов Б.Т., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Авлияқулов М.А., доктор сельскохозяйственных наук, НИИ хлопководства, семеноводства и агротехнологии, старший научный сотрудник; Улжаев Ф.Б., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Каримов Ш.А., доктор сельскохозяйственных наук (DSc), старший-научный сотрудник научно-исследовательского института селекции, семеноводства и агротехнологии выращивания хлопка; Гадаев Н.Н., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Муратов А.Р., к.т.н., (PhD), доцент Национального исследовательского университета "Ташкентский институт инженеров ирригации и механизации сельского хозяйства"; Муратов А.Р., к.т.н., (PhD), доцент Национального исследовательского университета "Ташкентский институт инженеров ирригации и механизации сельского хозяйства"; Касымбетова С.А., кандидат технических наук, (PhD), доцент Национального исследовательского университета "Ташкентский институт инженеров ирригации и механизации сельского хозяйства"; Гуломов С.Б., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Касымбетова С.А., кандидат технических наук, (PhD), доцент Национального исследовательского университета "Ташкентский институт инженеров ирригации и механизации сельского хозяйства"; Уразбаев И.К., кандидат технических наук (PhD), доцент национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” ɖɮɸɮɖ Doctor of Agricultural Sciences, Professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; g y Akhmedov D., doctor of Biological Sciences, Research Institute of Cotton Breeding, Seed Production and Agrotechnology, Senior Research Fellow; Khudoev I.J., Bukhara Institute of Natural Resources Management of the National Research University of Tashkent Institute of Irrigation and Agricultural Mechanization Engineers Rabshanov A., Doctor of Agricultural Sciences, Director of the Research Cotton Breeding, Seed Production and Agrotechnologies Research Institute; Nurmatov Sh., Doctor of Agricultural Sciences, Director of the Center for Variety Testing of Agricultural Crops; Rabshanov A., Doctor of Agricultural Sciences, Director of the Research Cotton Breeding, Seed Production and Agrotechnologies Research Institute; Mirkhasilova Z., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Nurmatov Sh., Doctor of Agricultural Sciences, Director of the Center for Variety Testing of Agricultural Crops; Atadjanov A., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Avliyakulov M., Doctor of Agricultural Sciences (DSc), Research Institute of Cotton Breeding, Seed Production and Agrotechnology, Senior Research Fellow; g gy Karimov Sh., Doctor of Agricultural Sciences (DSc), Senior Researcher, Research Institute of Cotton Breeding, Seed Production and Agrotechnology; Muratov A.R., doctor of philosophy (PhD) technics, associate- professor, National Research University "Tashkent Institute of Irrigation and Agricultural Mechanization Engineers"; Karimov Sh., Doctor of Agricultural Sciences (DSc), Senior Researcher, Research Institute of Cotton Breeding, Seed Production and Agrotechnology; Amanov B.T., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Muratov A.R., doctor of philosophy (PhD) technics, associate- professor, National Research University "Tashkent Institute of Irrigation and Agricultural Mechanization Engineers"; Uljayev F.B., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Uljayev F.B., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Kasimbetova S.A., doctor of philosophy (PhD) technics, associate-professor, National Research University "Tashkent Institute of Irrigation and Agricultural Mechanization Engineers"; Kasimbetova S.A., doctor of philosophy (PhD) technics, associate-professor, National Research University "Tashkent Institute of Irrigation and Agricultural Mechanization Engineers"; Gadayev N.N., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Urazbayev I.K., “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Agricultural Mechanization Engineers” National Research University; y Guamov S.B., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; y Guamov S.B., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Page Maker \| Верстка \| Саҳифаловчи: Хуршид Мирзахмедов Контакт редакций журналов. ɖɮɸɮɖ Murodov Sh.M., doctor of philosophy of economic sciences(PhD), associate-professor, National Research University "Tashkent Institute of Irrigation and Agricultural Mechanization Engineers".Botirov Sh., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Isaev S, Doctor of Agricultural Sciences, Professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; ( ) p University "Tashkent Institute of Irrigation and Agricultural Mechanization Engineers".Botirov Sh., candidate of technical sciences, associate professor of the “Tashken Institute of Irrigation and Agricultural Mechanization Engineers” National Research University; Matyakubov B. cXWS]i[a``n\daUXe Исаев С.Х., доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Матякубов Б.Ш., доктор сельскохозяйственных наук, профессор национального исследовательского университета “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” Муродов Ш.М., к.э.н., (PhD), доцент "Ташкентского институтга инженеров ирригации и механизации сельского хозяйства" Национальный исследовательский институт. Муродов Ш.М., к.э.н., (PhD), доцент "Ташкентского институтга инженеров ирригации и механизации сельского хозяйства" Национальный исследовательский институт. ɖɮɸɮɖ www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000 Editorial staff of the journals of www.tadqiqot.uz Tadqiqot LLC The city of Tashkent, Amir Temur Street pr.1, House 2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Phone: (+998-94) 404-0000 Контакт редакций журналов. www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000 Editorial staff of the journals of www.tadqiqot.uz Tadqiqot LLC The city of Tashkent, Amir Temur Street pr.1, House 2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Phone: (+998-94) 404-0000 3 3 АННОТАЦИЯ Суғоришда эгатнинг узунлиги ва чуқурлиги бўйича тупроқ намлигининг бир хиллигини таъминлаш ҳамда ҳосилдорликнинг ижобий натижаларига эришиш талаб этилади. Мазкур мақола ер устидан эгат олиб суғоришда мавжуд сув ресурсларидан самарали фойдаланиш мақсадида яратилган технология ва ишлаб чиқарилган техник воситани тажриба даласида қўллашда ғўзанинг ўсиши ва ривожланишига суғориш технологиясининг, техник воситани ишлатишнинг суғориш меъёри ва ғўза ҳосилдорлигига таъсири ҳамда тажрибадан олинган натижалар таҳлили масаласига бағишланган. Калит сўзлар: вегетация, зичлик, қатлам, майдон, намланиш, нишаб, суғориш, суғорма сув, технология, техник восита, тажриба, текислаш, қумоқ тупроқ, эгат, ғўза. Атажанов А. У. Национальный исследовательский университет “Ташкентский институт инженеров ирригации и механизации сельского хозяйства” доцент, PhD. http://dx.doi.org/10.5281/zenodo.7340631 http://dx.doi.org/10.5281/zenodo.7340631 SVcabcaiXdd[`V¬ ɮSɹ _f`WSc[YS¬daWXcYS`[X¬ɹ 1. Базаров Дилшод, Шодиев Бобур, Назарова Шохида РАЗРАБОТКА МЕТОДИКИ ГАШЕНИЯ ЭНЕРГИИ ПОТОКА НА СРЕДНЕНАПОРНЫХ И НИЗКОНАПОРНЫХ ГИДРОУЗЛАХ………………………………...5 2. Уралов Бахтиёр, Муталов Шухрат, Сирожов Бурхон, Вохидов Ойбек, Арзиева Диловар ВЛИЯНИЕ ГИДРОАБРАЗИВНОГО ИЗНОСА ЛОПАСТЕЙ РАБОЧЕГО КОЛЕСА НА НАПОР ЦЕНТРОБЕЖНОГО НАСОСА…………………………………………………………13 3. Bekchanov A. Faxriddin THEORY QUESTIONS OF THE REPLACEMENT OF THE MAIN EQUIPMENT OF PUMPING STATIONS...............................................................................................................20 4. Шаазизов Фаррух, Вохидов Ойбек ОЦЕНКА УЩЕРБА ПРИ ОБРАЗОВАНИИ И ПРОХОЖДЕНИИ СЕЛЕЙ ПО ТАШКЕНТСКОЙ ОБЛАСТИ…………………………………………………………………….27 5. Norov Kh. Begmat, Kholmatova N. Khusnora TECHNOLOGY OF ELECTROMECHANICAL HARDENING OF SURFACES OF WORKING BODIES OF EARTH-MOVING MACHINES.......................................................37 6. Азимов Азам, Хидиров Санъат, Шодиев Бобур, Шомуродов Абдулазиз НАСОС СТАНЦИЯЛАРДАГИ СЎРИШ ҚУВУРЛАРИНИНГ ИШЛАШ РЕЖИМИ………...46 7. Усманов Шавкат, Рахимов Нурбек, Мирхасилова Зулфия, Якубова Хуршида ИРРИГАЦИОННАЯ ОЦЕНКА КАЧЕСТВА КОЛЛЕКТОРНО-ДРЕНАЖНЫХ ВОД ПО ВОЗМОЖНОСТИ ИСПОЛЬЗОВАНИЯ ИХ НА ОРОШЕНИЕ………………………………...52 8. Бекмуродов Хумойиддин, Хаитов Эргаш, Ҳайдаров Туйгун, Ражабов Нурмамат УНУМДОРЛИГИ ПАСТ ТУПРОҚЛАРДА ҒЎЗАГА ҲАМКОР ЭКИН СИФАТИДА МОШ ВА СОЯ ЭКИЛГАНДА ТУПРОҚ УНУМДОРЛИГИГА ТАЪСИРИ………………………….58 9. Алланазаров Олимжон, Хикматуллаев Санжар МАВЖУД ДАВЛАТ КАДАСТРЛАРИНИ БОШҚАРИШ ТИЗИМИ ВА ЖАҲОН ТАЖРИБАЛАРИ…………………………………………………………………………………..63 10. Атажанов Адилжан ЭГАТ ТУБИНИ ЎЗГАРУВЧАН ЗИЧЛОВЧИ ТЕХНИК ВОСИТА ВА СУҒОРИШ ТЕХНОЛОГИЯСИ БЎЙИЧА ТАЖРИБАЛАР ТАҲЛИЛИ…………………………………….70 4 SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 Ў УЎК: 631.675.2 УЎК: 631.675.2 Атажанов Адилжан “Тошкент ирригация ва қишлоқ хўжалигини механизациялаш мухандислари институти” Миллий тадқиқот университети доцент, PhD. Е-mail: a.atajanov@tiiame.uz ANNOTATION ANNOTATION When irrigating, it is required to ensure uniform soil moisture along the length and depth of the furrows, as well as to achieve positive yield results. This article is devoted to the issues of the impact of technology on the growth and development of cotton, the influence of the use of technical means on the irrigation rate and cotton yields by using the created technology on the experimental field and developed technical means for the rational use of existing water resources in surface irrigation along furrows, as well as analysis of the obtained experimental results. g g , y p Keywords: vegetation, density, layer, area, moisture, slope, irrigation, irrigation water, technology, technical means, experience, layout, loam, furrow, cotton. Кириш. Республикамизда суғориладиган майдонлардан фойдаланиш ва самарадорлигини оширишга ёрдам берадиган янги технологияларни яратиш бўйича тадқиқотлар ўтказиш ва уларни амалда қўллаш бўйича чора- тадбирлар амалга оширилмоқда. 2017-2021 йилларга мўлжалланган Ўзбекистон Республикасини ривожлантиришнинг бешта устувор йўналиши бўйича Ҳаракатлар стратегиясида “Суғориладиган ерларнинг мелиоратив ҳолатини янада яхшилаш, мелиоратив ва ирригация объектлари тармоқларини ривожлантириш, қишлоқ хўжалиги ишлаб чиқариш соҳасига интенсив усулларни, энг аввало, сув ва энергетик ресурсларни тежайдиган замонавий технологияларни кенг жорий этиш” бўйича вазифалар белгиланган. Мазкур вазифаларни амалга оширишда, жумладан инновацион технологиялар ва техник воситаларни қўллаш орқали янги замонавий сувтежамкор технологияларни ишлаб чиқиш муҳим ҳисобланади. Ўзбекистон Республикаси Президентининг 2020 йил 10 июлдаги ПФ-6024 “Ўзбекистон Республикаси сув хўжалигини ривожлантиришнинг 2020 -2030 йилларга мўлжалланган концепциясини тасдиқлаш тўғрисида”ги фармони, 2019 йил 9 октябрдаги ПҚ-4486 сонли “Сув ресурсларини бошқариш тизимини янада такомиллаштириш бўйича чора-тадбирлари тўғрисида” ги қарори, 2021 йил 24 февралдаги ПҚ-5005-сонли “Ўзбекистон Республикасида сув ресурсларини бошқариш ва ирригация секторини ривожлантиришнинг 2021-2023 йилларга мўлжалланган СТРАТЕГИЯСИ” қарорида ҳамда мазкур фаолиятга тегишли бошқа меъёрий-ҳуқуқий ҳужжатларда белгиланган вазифаларни амалга оширишга ушбу тадқиқот муайян даражада хизмат қилади [1,2]. Тадқиқот методикаси. Эгат тубини ўзгарувчан зичловчи техник восита ва суғориш технологиясини қўллаш мақсадида тажриба далалари тупроқларининг генетик қатламлари таснифи ва механик таркибини аниқлаш учун тупроқ кесмалари қазилиб, генетик қатламлар бўйича тупроқ морфологияси аниқланди ҳамда тупроқнинг сув-физик хоссалари аниқланди. Шу тариқа ҳар хил механик таркибли тажриба далалари танлаб олинди ва илмий тадқиқот ишлари амалга оширилди. Ғўза экилган тажриба даласи тупроғининг механик таркиби тадқиқотлар бошида ковланган тупроқ кесмасидан генетик қатламлар бўйича олинган тупроқ намуналарида аниқланди. Лаборатория таҳлилининг маьлумотларига кўра тажриба даласи тупроғининг механик таркиби Н.Качинский тавсифномасига кўра, 0-39 см чуқурликдаги қатлами оғир қумоқ, 39-75 см чуқурликдаги қатлами оғир қумоқ 75-92 см чуқурликдаги қатлами ўрта қумоқ тупроққа, 92-118 см чуқурликдаги қатлами ўрта қумоқ тупроқлар таснифига киради. АННОТАЦИЯ Ц При орошении требуется обеспечить равномерность увлажнение почвы по длине и по глубине борозд, а также достижение положительных результатов урожайности. Данная статья посвящена вопросам влияние технологии на рост и развитие хлопчатника, влияние использование технического средства на поливную норму и урожайности хлопчатника применением на опытном поле созданной тенологии и разработанного технического средства с целью рационального использования существующих водных ресурсов при поверхностном орошении по бороздам, а также анализу полученных опытных результатов. Ключевые слова: вегетация, плотность, слой, площадь, увлажнение, уклон, полив, поливная вода, технология, техническое средство, опыт, планировка, суглинок, борозда, хлопчатник. 70 SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 National Research University “Tashkent Institute of Irrigation and Agricultural Mechanization Engineers” PhD, associate professor, e-mail: a.atajanov@tiiame.uz ANNOTATION Тажриба даласи тупроғининг механик таркиби пастга томон енгиллашиб бориши кузатилди, бу эса ўсимликлар ер ости сувларидан фойдаланишида муҳим аҳамиятга эга. риба даласи тупроғининг механик таркиби Н.Качинский тавсифномасига кўра, 0- 2-тажриба даласи тупроғининг механик таркиби Н.Качинский тавсифномасига кўра, 0- 71 SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 №7 \| 2022 65 сантиметр чуқурликдаги қатлами ўрта қумоқ тупроққа ва 85-150 сантиметр чуқурликдаги қатлами енгил қумоқ тупроқлар тавсифига киради. Тупроқнинг сув-физик хоссалари унинг тури, механик таркиби, тузилиши, органик ва маданли моддалар миқдори, тузилмаси, экини, алмашлаб экиш ва етиштиришига қараб ўзгариб бориши билан бир қаторда тажриба далаларининг тупроқдаги бошқа сув-физик хоссалари, жумладан тупроқнинг ҳажм оғирлиги, тупроқнинг чекланган дала намлик сиғими ва сув ўтказувчанлиги ҳам аниқланди. Тупроқнинг сув-физик хоссалари унинг тури, механик таркиби, тузилиши, органик ва минерал моддаларнинг миқдори, структураси, маданийлашганлиги, экинларни алмашлаб экиш ва ерга ишлов бериш даражаларига боғлиқ ҳолда турлича бўлади. Ўтлоқи тупроқлар Республикада тарқалган бошқа тупроқларга нисбатан қулай сув-физик хусусиятларга эгалиги билан ажралиб туради. Тупроқ ҳайдов қатламининг тузилишини белгиловчи асосий кўрсаткичлардан бири унинг ҳажмий оғирлиги ҳисобланади. Тажриба олиб борилган йилларда 1-тажрибада вегетация бошида тупроқнинг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,36-1,38 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,42-1,43 г/см3 ва 0-100 см қатламда 1,41-1,42 г/см3 ни ташкил этиб, вегетация охирига бориб, ишлаб чиқариш назоратида тупроқнинг ҳажмий оғирлиги ҳайдаладиган (0-30 см) қатламда 1,41-1,42 г/см3, ҳайдаладиган қатлам остидаги (30- 50 см) 1,46-1,48 г/см3 ва 0-100 см қатламда 1,44-1,45 г/см3 ни ташкил қилди ҳамда тупроқнинг ҳажмий оғирлиги 0,03-0,05 г/см3 га ортганлиги аниқланди. Тупроқнинг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,39-1,40 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,44-1,46 г/см3 ва 0-100 см қатламда 1,43 г/см3 ни ташкил этди, тупроқнинг ҳажмий оғирлиги 0,01-0,02 г/см3 га, яъни назоратга нисбатан кам миқдорда ортди. Олинган маьлумотларга асосан 2-тажрибада вегетация бошида тупроқнинг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,35-1,36 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,37-1,38 г/см3 ва 0-100 см қатламда 1,37-1,38 г/см3 ташкил этиб, вегетация охирида ишлаб чиқариш назоратида тупроқнинг ҳажмий оғирлиги ҳайдаладиган (0-30 см) қатламда 1,38-1,40 г/см3, ҳайдаладиган қатлам остида (30-50 см) 1,42 г/см3 ва 0-100 см қатламда 1,41-1,42 г/см3 ни ташкил қилди ҳамда тупроқнинг ҳажмий оғирлиги 0,04-0,05 г/см3 га ортди. Тупроқнинг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,38-1,40 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,42 г/см3 ва 0-100 см қатламда 1,41-1,42 г/см3 ни ташкил қилди ҳамда тупроқнинг ҳажмий оғирлиги 0,03-0,004 г/см3 га, яъни назоратга нисбатан кам миқдорда ортди. ANNOTATION 3-тажрибада вегетация бошида тупроқнинг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,31-1,32 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,34-1,35 г/см3 ва 0-100 см қатламда 1,32-1,33 г/см3 ташкил этди ва вегетация охирида ишлаб чиқариш назоратида тупроқнинг ҳажмий оғирлиги ҳайдаладиган (0-30 см) қатламда 1,35-1,36 г/см3, ҳайдаладиган қатлам остида (30-50 см) 1,39-1,40 г/см3 ни ва 0-100 см қатламда 1,37 г/см3 ни ташкил қилди ҳамда тупроқнинг ҳажмий оғирлиги 0,04-0,05 г/см3 га ортди Тупроғининг ҳажмий оғирлиги ҳайдаладиган 0 30 см қатламда 1 36 1 38 г/см3 3-тажрибада вегетация бошида тупроқнинг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,31-1,32 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,34-1,35 г/см3 ва 0-100 см қатламда 1,32-1,33 г/см3 ташкил этди ва вегетация охирида ишлаб чиқариш назоратида тупроқнинг ҳажмий оғирлиги ҳайдаладиган (0-30 см) қатламда 1,35-1,36 г/см3, ҳайдаладиган қатлам остида (30-50 см) 1,39-1,40 г/см3 ни ва 0-100 см қатламда 1,37 г/см3 ни ташкил қилди ҳамда тупроқнинг ҳажмий оғирлиги 0,04-0,05 г/см3 га ортди. Тупроғининг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,36-1,38 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,39-1,41 г/см3 ва 0-100 см қатламда 1,38-1,39 г/см3 ни ташкил қилди ҳамда тупроқнинг ҳажмий оғирлиги 0,04-0,06 г/см3 га, яъни назоратга нисбатан кам миқдорда ортди. 3-тажрибада вегетация бошида тупроқнинг ҳажмий оғирлиги ҳайдаладиган 0-30 см қатламда 1,31-1,32 г/см3 ни, ҳайдов ости (30-50 см) қатламда 1,34-1,35 г/см3 ва 0-100 см қатламда 1,32-1,33 г/см3 ташкил этди ва б й й Тажриба далаларини экишга тайёрлаш, экиш, культивация қилиш, эгат олиш, эгатни зичлаш қурилмасини ишлатиш ва ундан кейин бажариладиган агротехника тадбирларини амалга ошириш, суғориш тартибларининг ҳамда трактор-механизмларни кириши тупроқнинг зичланишига таъсир этиши натижасида тупроқнинг ҳажмий оғирлигининг ортиши кузатилди. р р р у зичлаш қурилмасини ишлатиш ва ундан кейин бажариладиган агротехника тадбирларини амалга ошириш, суғориш тартибларининг ҳамда трактор-механизмларни кириши тупроқнинг зичланишига таъсир этиши натижасида тупроқнинг ҳажмий оғирлигининг ортиши кузатилди. Ғўза экилган тажриба далаларида тупроқнинг чекланган дала нам сиғими бўйича олинган натижаларига кўра, 1-тажриба дала бўйича тупроқнинг чекланган дала нам сиғими ҳайдалма 0-50 см қатламида қуруқ тупроқ оғирлигига нисбатан 22,0 % ни, тупроқнинг 0-100 см қатламида чекланган дала нам сиғими қуруқ тупроқ оғирлигига нисбатан 22,3 % ни, 2- тажрибада 0-50 см қатламда 21,0 % ни, 0-100 см қатламда 21,3 % ни, 3-тажрибада 0-50 см қатламда 19,5 % ни, 0-100 см қатламда 19,2 % ни ташкил этди. ANNOTATION Ғўза экилган тажриба далаларида тупроқнинг чекланган дала нам сиғими бўйича олинган натижаларига кўра, 1-тажриба дала бўйича тупроқнинг чекланган дала нам сиғими ҳайдалма 0-50 см қатламида қуруқ тупроқ оғирлигига нисбатан 22,0 % ни, тупроқнинг 0-100 см қатламида чекланган дала нам сиғими қуруқ тупроқ оғирлигига нисбатан 22,3 % ни, 2- тажрибада 0-50 см қатламда 21,0 % ни, 0-100 см қатламда 21,3 % ни, 3-тажрибада 0-50 см қатламда 19,5 % ни, 0-100 см қатламда 19,2 % ни ташкил этди. Тажриба далалари тупроғининг сув ўтказувчанлиги тупроқнинг механик таркиби, 72 SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 №7 \| 2022 структураси, чиринди миқдори ва шўрхоклик даражасига боғлиқ ҳолда турлича бўлади. Тадқиқот олиб борилган ўтлоқи тупроқлар С.В.Астапов бўйича ўртача сув ўтказувчанликка эга ҳисобланади. Тупроқ сув ўтказувчанлиги тупроқнинг механик таркибига ва сув-физик хоссасига, унинг структура ҳолатига, зичлиги, ғоваклиги, намлик ҳамда намланиш давомийлигига ҳам боғлиқ бўлади. Сув ўтказувчанлик оғир қумоқ механик таркибли тупроқларда ҳар доим енгил тупроқларга нисбатан паст бўлади. Тажриба далаларида тупроқнинг сув ўтказувчанлиги бўйича олинган натижалари бўйича 1-тажрибада вегетация даврининг бошида тупроқ сув ўтказувчанлиги 6 соат давомида 980 м3/га ёки 0,272 мм/мин ни ташкил қилди (2018 й.). Вегетация даврининг охирига бориб, сув ўтказувчанлик 6 соат давомида ишлаб чиқариш назоратида 739 м3/га ни ёки 0,205 мм/мин ни ташкил қилган бўлса тажриба далада 764 м3/га ни ёки 0,212 мм/мин бўлди. Бу кўрсатгич 1- назоратга нисбатан 25 м3/га ва 0,007 мм/мин га юқоридир. 2-тажриба участкасида вегетация даврининг бошида тупроқ сув ўтказувчанлиги 6 соат давомида 1300 м3/га ёки 0,361 мм/мин га тенг бўлди. Вегетация даврининг охирига бориб, 2 тажриба далада сув ўтказувчанлик 6 соат давомида ишлаб чиқариш назоратида 836 м3/га ни ёки 0,232 мм/мин ни ташкил қилган бўлса тажриба далада 920 м3/га ни ёки 0,256 мм/мин бўлди. Бу кўрсатгич назоратга нисбатан 84 м3/га ва 0,024 мм/мин га юқори бўлганлиги кузатилди. 3-тажриба участкасида ҳам шундай қонуниятлар кузатилди. Вегетация даврининг бошида тупроқ сув ўтказувчанлиги 6 соат давомида 1490 м3/га ёки 0,417 мм/мин ни ташкил қилган бўлса, вегетация даврининг оҳирига бориб, 6 соат давомида 946 м3/га ни ёки 0,263 мм/мин. ни ташкил этди. Бу кўрсатгич назоратга нисбатан 40 м3/га ва 0,011 мм/мин га пастдир. Демак, вегетация давридаги суғориш сонининг ортиши ҳамда эгат тубини ўзгарувчан зичлаш қурилмасини ишлатиш орқали тупроқнинг зичлашуви ортади ва унинг сув ўтказувчанлиги камайиб боради. Умуман олганда, далани экишга тайёрлаш, агротехника тадбирлари, суғориш усуллари, мақбул суғориш меъёрлари ва тартибини тўғри белгилаш ҳамда қўллаш тупроқнинг сув ўтказувчанлик хусусиятини бошқариш имкониятини беради. ANNOTATION Ғўзанинг ўсиши ва ривожланиши бўйича фенологик кузатувлар шуни кўрсатадики, шўрланган ёки шўрланишга мойил ерларда ўсимликнинг фаол қатламларида мақбул сув режимини сақлаб туриш, ўсимликлар таналаридаги физиологик жараёнларнинг йўналишини белгилайдиган тупроқдаги сувда эрувчан тузларнинг таркиби ва миқдорига боғлиқдир. Ҳудди шундай майдонларда пахта етиштиришнинг асосий даври бўлиб, ғўзанинг гуллаш ва ҳосил тугиш фазаси бўлиб ҳисобланади (1-жадвал). у ф ў ( ) 1-жадвал маълумотларига биноан 1-тажриба далада вегетация бошида ғўза кўчатнинг қалинлиги гектар бошига 97,6 минг тупни, ҳосил шохи 6,7 донани, 2-тажрибада, вегетация бошида ғўза кўчатнинг қалинлиги гектар бошига 98,4 минг тупни, ҳосил шохи 6,4 донани, 3- тажрибада, вегетация бошида ғўза кўчатнинг қалинлиги гектар бошига 96,5 минг тупни, ҳосил шохи 5,7 донани ташкил этди [5; 10]. 1 жадвал. Ғўзанинг ўсиши ва ривожланиши (2018-2020 йй.) Вариантлар Кўчат қалинлиги, минг дона Чин барги, см Ғўзанинг бўйи, см Ҳосил шохлар сони, дона 1.06 1.06 1.06 1.07 1.07 1-тажриба ИЧН 96,8 3,7 9,5 47,1 6,27 ТД 97,0 3,8 9,8 47,1 7,02 2-тажриба ИЧН 99,9 3,4 9,6 35,8 6,0 ТД 100,0 3,7 10,1 35,0 6,5 3-тажриба ИЧН 96,7 3,5 8,3 35,7 6,43 ТД 98,2 3,8 8,6 36,4 7,14 1 жадвал. Ғўзанинг ўсиши ва ривожланиши (2018-2020 йй.) 1 жадвал. Ғўзанинг ўсиши ва ривожланиши (2018-2020 йй.) Кўчат қалинлиги, минг Чин барги, Ғўзанинг бўйи см Ҳосил шохлар 73 SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 №7 \| 2022 1-расм. 1 тажриба даладаги ғўзанинг ўсиши ва ривожланишининг ўртача кўрсаткичлари. 1-расм. 1 тажриба даладаги ғўзанинг ўсиши ва ривожланишининг ўртача кўрсаткичлари. Ғўзанинг ўсиши ва ривожланишига суғориш технологиясининг таъсирини аниқлаш бўйича фенологик кузатувлар шуни кўрсатадики, шўрланган ёки шўрланишга мойил ерларда ўсимликнинг илдизи тарқаладиган қатламларида мақбул сув режимини сақлаб туриш, ўсимликлар таналаридаги физиологик жараёнларнинг йўналишини белгилайдиган тупроқдаги сувда эрувчан тузларнинг таркиби ва миқдорига боғлиқдир. Ҳудди шундай майдонларда пахта етиштиришнинг асосий даври бўлиб, ғўзанинг гуллаш ва ҳосил тугиш фазаси бўлиб ҳисобланади. Ғўзанинг ўсиши ва ривожланишига суғориш тартибларининг таъсири Ғўзанинг ўсиши ва ривожланишига суғориш тартибларининг таъсири маълумотларига кўра 1-тажриба даланинг вегетация бошида ғўза кўчатнинг қалинлиги гектар бошига 95,6-97,6 минг тупни ташкил этган бўлса, вегетация охирига бориб кўчатининг қалинлиги гектарига 94,6-96,9 минг туп бўлиб, камайиши кузатилди. 1- сентябр ҳолати кўра ғўзанинг бўйи 91,6-92,9 см ни, ҳосил шохлари 11,8-11,9 донани, кўсакларининг сони 10,4-11,1 донани ва очилган кўсаклар сони 4,4-4,9 донани ташкил қилди. Ўсиши ва ривожланиши ишлаб чиқариш назоратига нисбатан ҳосил шохлари 1,2-1,7 донага, кўсакларининг сони 0,4-0,8 донага ва очилган кўсаклар сони 0,4-0,7 донага кўп бўлди. ANNOTATION 1- сентябр ҳолати кўра ғўзанинг бўйи 91,6-92,9 см ни, ҳосил шохлари 11,8-11,9 донани, кўсакларининг сони 10,4-11,1 донани ва очилган кўсаклар сони 4,4-4,9 донани ташкил қилди. Ўсиши ва ривожланиши ишлаб чиқариш назоратига нисбатан ҳосил шохлари 1,2-1,7 донага, кўсакларининг сони 0,4-0,8 донага ва очилган кўсаклар сони 0,4-0,7 донага кўп бўлди. 2-тажрибада ҳам шундай кўрсатгичлар бўлиб, 1- сентябр ҳолатига ғўзанинг бўйи 87,8- 88,9 см ни, ҳосил шохлари 10,9-11,5 донани, кўсакларининг сони 10,5-11,5 донани ва очилган кўсаклар сони 2,6-3,7 донани ташкил қилди ҳамда, назоратга нисбатан ҳосил шохлари 0,8-0,9 донага, кўсакларининг сони 0,8-1,3 донага ва 1-сентябрда очилган кўсаклар сони 0,1-0,7 донага кўп бўлди. 3-тажрибада вегетация бошида ғўза кўчатининг қалинлиги гектар бошига 96,5-98,7 минг тупни ташкил этган бўлса, вегетация охирига бориб кўчатининг қалинлиги гектарига 95,0-98,2 минг тупни ташкил қилди ёки 0,5-1,5 минг тупга камайиши кузатилди. 1- сентябр холатига ғўзанинг бўйи 85,3-88,6 см ни, ҳосил шохлари 10,5-11,6 донани, кўсакларининг сони 10,4-10,8 донани ва очилган кўсаклар сони 3,1-4,2 донани ташкил қилиб, назоратга нисбатан ҳосил шохлари 0,5-0,9 донага, кўсакларининг сони 0,5-0,8 донага ва 1-сентябрда очилган кўсаклар сони 0,9 донага кўп бўлди. Ғўза экилган тажриба далаларида бир кўсакдаги пахта оғирлиги кузатувларга кўра 1- тажрибанинг ишлаб чиқариш назоратида теримлар бўйича ўртача 5,3-5,4 граммга тенг бўлди. Тажриба далада теримлар бўйича бир кўсакдаги пахта оғирлиги ўртача 5,5-5,7 граммга тенг бўлди. 2- тажрибада ишлаб чиқариш назоратида теримлар бўйича бир кўсакдаги пахта оғирлиги 6,0-6,2 граммга, тажриба далада эса, ўртача 6,3-6,5 граммга тенг бўлди. 2- тажрибада ишлаб чиқариш назоратида теримлар бўйича бир кўсакдаги пахта оғирлиги 6,0-6,2 граммга, тажриба далада эса, ўртача 6,3-6,5 граммга тенг бўлди. 3- тажрибада ишлаб чиқариш назоратида теримлар бўйича бир кўсакдаги пахта 3- тажрибада ишлаб чиқариш назоратида теримлар бўйича бир кўсакдаги пахта 74 SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 №7 \| 2022 оғирлиги ўртача 5,1-5,2 граммга, тажриба далада эса, теримлар бўйича ўртача 5,4-5,5 граммга бў оғирлиги ўртача 5,1-5,2 граммга, тажриба далада эса, теримлар бўйича ўртача 5,4-5,5 граммга тенг бўлди. 2-расм. Бир кўсакдаги пахта оғирлиги. Техник воситани ишлатишнинг суғориш меъёри ва ғўза ҳосилдорлигига таъсирини аниқлаш бўйича олиб борилган 1-тажрибанинг ишлаб чиқариш назоратида 31,6-33,4 ц/га пахта ҳосили олинди ва 1 центнер пахта етиштириш учун -115,4-116,6 м3 дарё суви сарфланди. Тажриба далада - 37,9-38,5 ц/га пахта ҳосили олиниб, 1 центнер пахта етиштириш учун энг кам: 57,7-58,2 м3 дарё суви сарфланди. 2-тажрибанинг ишлаб чиқариш назоратида 31,9-32,8 ц/га пахта ҳосили олинди ва 1 центнер пахта етиштириш учун - 143,8-146,3 м3 дарё суви сарфланди. Тажриба далада - 35,8- 36,3 ц/га пахта ҳосили олиниб, 1 центнер пахта етиштириш учун энг кам: 77,6-79,8 м3 дарё суви сарфланди. 3-тажрибада ишлаб чиқариш назоратида 31,5-31,6 ц/га пахта ҳосили олинди ва 1 центнер пахта етиштириш учун -165,4-171,1 м3 дарё суви сарфланди. Тажриба далада эса - 35,5-37,5 ц/га пахта ҳосили олиниб, 1 центнер пахта етиштириш учун 102,7-105,9 м3 дарё суви сарфланди. 3-тажрибада ишлаб чиқариш назоратида 31,5-31,6 ц/га пахта ҳосили олинди ва 1 центнер пахта етиштириш учун -165,4-171,1 м3 дарё суви сарфланди. Тажриба далада эса - 35,5-37,5 ц/га пахта ҳосили олиниб, 1 центнер пахта етиштириш учун 102,7-105,9 м3 дарё суви сарфланди. Тадқиқот натижалари шуни кўрсатдики, суғоришда мазкур ускунани қўллаш хамда суғориш технологияни ишлатиш натижасида ғўзадан юқори ҳосил олиб билан бир қаторда, мавсум давомида бериладиган дарё суви миқдорини иқтисод қилиш имконияти яратилиши исботланди. Хулоса. Янги техник восита асосидаги сув тежамкор суғориш технологиясининг ғўзанинг ўсиши, ривожланиши ва ҳосилдорлигига таъсири ўрганилди: Хулоса. Янги техник восита асосидаги сув тежамкор суғориш технологиясининг ғўзанинг ўсиши, ривожланиши ва ҳосилдорлигига таъсири ўрганилди: -енгил механик таркибли тупроқларда ғўзанинг асосий поясининг баландлиги 1 сентябр ҳолатига 92,2 см. ни, кўсаклар сони 10,7 донани, шундан очилганлари 4,6 донани ташкил этиб, ҳосилдорлик 38,5 ц/га ни; -ўрта механик таркибли тупроқларда ғўзанинг асосий поясининг баландлиги 1 сентябр ҳолатига 88,4 см. ни, кўсаклар сони 11,2 донани, шундан очилганлари 3,1 донани ташкил этиб, ҳосилдорлик 36,0 ц/га ни; -оғир механик таркибли тупроқларда ғўзани асосий поясининг баландлиги 1 сентябр ҳолатига 87,1 см. ни кўсаклар сони 10,6 донани, шундан очилганлари 3,7 донани ташкил этиб, ҳосилдорлик 36,5 ц/га ни ташкил этиши аниқланди. Ғўза ҳосилдорлиги ишлаб чиқариш назоратига нисбатан мос равишда 6,3 ц/га, 3,2 ц/га ва 5,0 ц/га га юқори бўлди. -оғир механик таркибли тупроқларда ғўзани асосий поясининг баландлиги 1 сентябр ҳолатига 87,1 см. ни кўсаклар сони 10,6 донани, шундан очилганлари 3,7 донани ташкил этиб, ҳосилдорлик 36,5 ц/га ни ташкил этиши аниқланди. Ғўза ҳосилдорлиги ишлаб чиқариш назоратига нисбатан мос равишда 6,3 ц/га, 3,2 ц/га ва 5,0 ц/га га юқори бўлди. 2-расм. Бир кўсакдаги пахта оғирлиги. 75 SVcabcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 №7 \| 2022 Адабиётлар рўйхати 1. Ўзбекистон Республикаси Президентининг 2020 йил 10 июлдаги ПФ -6024 сонли “Ўзбекистон Республикаси сув хўжалигини ривожлантиришнинг 2020-2030 йилларга мўлжалланган концеспцияси” фармони. Ў 1. Ўзбекистон Республикаси Президентининг 2020 йил 10 июлдаги ПФ -6024 сонли “Ўзбекистон Республикаси сув хўжалигини ривожлантиришнинг 2020-2030 йилларга мўлжалланган концеспцияси” фармони. 2. Ўзбекистон Республикаси Президентининг 2021 йил 24 февралдаги ПҚ-5005-сонли “Ўзбекистон Республикасида сув ресурсларини бошқариш ва ирригация секторини ривожлантиришнинг 2021-2023 йилларга мўлжалланган СТРАТЕГИЯСИ” қарори. 2. Ўзбекистон Республикаси Президентининг 2021 йил 24 февралдаги ПҚ-5005-сонли “Ўзбекистон Республикасида сув ресурсларини бошқариш ва ирригация секторини ривожлантиришнинг 2021-2023 йилларга мўлжалланган СТРАТЕГИЯСИ” қарори. р р р ў р р 3. Атажанов А.У. Суғориш эгатларининг тубини зичлаш қурилмаси. (Илмий ишланма- патент). IP CONSULTING CENTER. GUVOHNOMA № 002635. 12.11.2020. р р р ў р р 3. Атажанов А.У. Суғориш эгатларининг тубини зичлаш қурилмаси. (Илмий ишланма- патент). IP CONSULTING CENTER. GUVOHNOMA № 002635. 12.11.2020. 4. Атажанов А.У., Ирмухамедова Л.Х., Атажанов А.А. “Технология планировки орошаемого поля, обеспечивающая равномерность увлажнение почвы” // Международный научный журнал «Молодой ученый». Г.Казан. № 8 (142)/2017. стр. 43- 46 4. Атажанов А.У., Ирмухамедова Л.Х., Атажанов А.А. “Технология планировки орошаемого поля, обеспечивающая равномерность увлажнение почвы” // Международный научный журнал «Молодой ученый». Г.Казан. № 8 (142)/2017. стр. 43- 46 5. Атажанов А.У., Матякубов Б.Ш. “Совершенствование технологии, обеспечивающей равномерность увлажнения корнеобитаемого слоя почвы, орошаемой по бороздам” //Международная научно-практическая конференция «Вода для устойчивого развития Центральной Азии».23-24 марта 2018г. г. Душанбе, Таджикистан. cтр. 237-241. 5. Атажанов А.У., Матякубов Б.Ш. “Совершенствование технологии, обеспечивающей равномерность увлажнения корнеобитаемого слоя почвы, орошаемой по бороздам” //Международная научно-практическая конференция «Вода для устойчивого развития Центральной Азии».23-24 марта 2018г. г. Душанбе, Таджикистан. cтр. 237-241. 6. Атажанов А.У. Суғориш эгатлари тубини зичлаш қурилмаси. Ихтиролар.Расмий ахборотнома, №12. 31.12.2020. (21) IAP 2019 0274. (51) А01В 13/00, А01G 25/00. 7. Атажанов А.У. “Новая технология и техническое средство создания устойчивого профиля и проектного уклона поливных борозд”. Монография. Типография ТИИИМСХ. 2019 г. 126 стр. р 8. Atajanov A.U. Sug’orish egatlarining turg’un profili va loyihaviy nishabini yaratuvchi texnologiya va texnik vosita. Монография. ТИҚХММИ босмахонаси, 2021йил, 158 бет. 76 bcaiXdd[`VYfc`S^[¬Yfc`S^SVcabcaiXdd[`V¬ ɮSɹ №7 \| 2022 ISSN 2181-9904 Doi Journal 10.26739/2181-9904 SVcabcaiXdd[`VYfc`S^[ Y[^W!da` Yfc`S^SVcabcaiXdd[`V ea_`a_Xc! ɮɮɹ ! 77 Контакт редакций журналов. www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000 Editorial staff of the journals of www.tadqiqot.uz Tadqiqot LLC the city of Tashkent, Amir Temur Street pr.1, House 2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Phone: (+998-94) 404-0000 Контакт редакций журналов. www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000 Editorial staff of the journals of www.tadqiqot.uz Tadqiqot LLC the city of Tashkent, Amir Temur Street pr.1, House 2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Phone: (+998-94) 404-0000
https://openalex.org/W2588681194	https://napier-repository.worktribe.com/preview/428733/ckw169.049.full.pdf	English	null	Cultural reflections on the Scottish HPV vaccination programme	European journal of public health	2,016	cc-by	468	191 191 9th European Public Health Conference: Parallel Sessions E Carnegie1, A Whittaker1, C Gray Brunton1, N Hanif1, S Harding2, S Hilton3, R Hogg4, C Kennedy5, K Pollock6, J Pow1 1School of Nursing, Midwifery and Social Care, Edinburgh Napier University, Edinburgh, UK 2Diabetes & Nutritional Sciences Division, King’s College, London, UK 3MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow, UK 4Applied Research and Development, NHS Greater Glasgow and Clyde, Glasgow, UK 5School of Nursing and Midwifery, Robert Gordon University, Aberdeen, UK 6Vaccine Preventable Diseases, Health Protection Scotland, Glasgow, UK Contact: e.carnegie@napier.ac.uk Background g The Scottish HPV vaccination programme serves young women aged 11 to 18 years and reports consistently high uptake rates, yet these figures may conceal levels of under- standing and antecedents to decision-making. Evidence from other European countries indicates that ethnicity may influence decision-making regarding vaccination. The aim of the study was to identify understandings and explanations for HPV-related health behaviours within differing cultural contexts by examining accounts of young people from Black, Asian and Minority Ethnic (BAME) communities. h d Methods A critical qualitative exploratory study utilising Foucauldian discursive analysis was conducted. Seven focus groups and four paired interviews were conducted with 40 young people aged 16-26, from BAME communities: South Asian/Black African/ Arab, Muslim/Sikh/Christian. Stimulus material was utilised to explore understandings of HPV, experiences of vaccine programme, views on universal vaccination. Results 1School of Nursing, Midwifery and Social Care, Edinburgh Napier University, Edinburgh, UK g y S Hilton3, R Hogg4, C Kennedy5, K Pollock6, J Pow1 1 g 2Diabetes & Nutritional Sciences Division, King’s College, London, UK 3 p g Results Contrasting attitudes and perceptions across ethnicity and gender were observed: openness of Black African participants to information and partnership working; barriers to inter- generational dialogue expressed by Asian men; intracultural and intercultural opportunities for information-sharing pro- posed by Asian women. Participants identified solutions for sensitising formative public health interventions - how they are to be delivered and in which contexts including a flexible approach to offering information and the vaccine. by guest on November 21, 2016 http://eurpub.oxfordjournals.org/ Downloaded from Public health strategies should consider including: a staged and tailored approach to information-giving throughout school and beyond; extending the age of vaccination and including boys being offered the vaccine at a culturally acceptable stage and age; developing neutral and destigmatised messages in partnership with communities/elders; employing multi-media information campaigns for young men and women. Key messages: Embracing a flexible intracultural and cross-cultural approach will include utilising general and specific strategies that embrace diversity and increase public access to information and vaccine Working in partnership with BAME communities is essential for the development of destigmatising messages and intercultural communications Cultural reflections on the Scottish HPV vaccination programme Elaine Carnegie E Carnegie1, A Whittaker1, C Gray Brunton1, N Hanif1, S Harding2,
https://openalex.org/W3102057651	https://link.springer.com/content/pdf/10.1140/epjc/s10052-015-3736-7.pdf	English	null	Corpuscular consideration of eternal inflation	European physical journal. C, Particles and fields	2,015	cc-by	5,190	Corpuscular consideration of eternal inﬂation Florian Kühnel1,a, Marit Sandstad2,b 1 Department of Physics, The Oskar Klein Centre for Cosmoparticle Physics, Stockholm University, AlbaNova, 106 91 Stockholm, Sweden 2 Institute of Theoretical Astrophysics, University of Oslo, P.O. Box 1029, Blindern, 0315 Oslo, Norway Florian Kühnel1,a, Marit Sandstad2,b Received: 14 August 2015 / Accepted: 16 October 2015 / Published online: 26 October 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com Abstract We review the paradigm of eternal inﬂation in the light of the recently proposed corpuscular picture of space- time. Comparing the strength of the average ﬂuctuation of the ﬁeld up its potential with that of quantum depletion, we show that the latter can be dominant. We then study the full respec- tive distributions in order to show that the fraction of the space-time which has an increasing potential is always below the eternal-inﬂation threshold. We prove that for monomial potentials eternal inﬂaton is excluded. This is likely to hold for other models as well. into account. In fact, some phenomena, like Hawking radi- ation, the Bekenstein entropy, or, the information paradox, can only be fully understood in this quantum picture [9–15] (cf. also [16–27] for recent progress). The mentioned attempt leads to regard space-time, such as black holes, de Sitter spaces, etc., as gravitationally bound states in the form of weakly/marginally bound states, or Bose–Einstein condensates, of gravitons with a mean wave- length equal to the curvature radius of that space-time. Due to the weak binding, quantum ﬂuctuations are responsible for emptying the ground state of the condensate. This depletion is an intrinsically quantum effect, which is entirely missed in any (semi-)classical treatment. In inﬂationary spaces, it acts like a quantum clock which works against the semiclas- sical one and, as we will investigate below, also against the ﬂuctuations of the scalar ﬁeld up the potential. Eur. Phys. J. C (2015) 75:505 DOI 10.1140/epjc/s10052-015-3736-7 Regular Article - Theoretical Physics Regular Article - Theoretical Physics Regular Article - Theoretical Physics a e-mail: ﬂorian.kuhnel@fysik.su.se b e-mail: marit.sandstad@astro.uio.no a e-mail: ﬂorian.kuhnel@fysik.su.se b e-mail: marit.sandstad@astro.uio.no 1 Introduction C (2015) 75 :505 be deﬁned in terms of a scale, which is also the characteristic length scale of the system. The self-interactions of the gravi- tons make this critical phase stable. This means that removal or addition of gravitons to the critical condensate happens self-similarly—changing the deﬁning length scale of the sys- tem while remaining at criticality. The deﬁning length scale is proportional to the square root of the number of gravitons in the critical condensate. A prime example of such a state is a Schwarzschild black hole, where the deﬁning length scale is the Schwarzschild radius RS and the graviton number is N = (RS/LP)2. where nφ is the number density of inﬂatons in the condensate. The number of coherent gravitons is given by where nφ is the number density of inﬂatons in the condensate. The number of coherent gravitons is given by N = R2 H = 1 H2 . (2) (2) When considering eternal inﬂation in view of the cor- puscular description of gravity, we ﬁnd that two competing quantum effects are active: the quantum ﬂuctuations of the inﬂaton ﬁeld due to the uncertainty principle, and the quan- tum depletion of the inﬂaton and graviton condensates due to graviton–inﬂaton scattering. The de Sitter or inﬂationary patch is another such con- densate at criticality, where the deﬁning length scale is the Hubble radius RH and the graviton number N = (RH/LP)2. This inﬂationary case is somewhat more complicated than the black hole case, as the inﬂationary state is a compos- ite, comprising a critical graviton Bose–Einstein condensate interacting with a much higher-occupied inﬂaton condensate. The typical quantum ﬂuctuation due to the uncertainty principle reads (cf. [6]) \| ˙φ\| = H2 2π . (3) \| ˙φ\| = H2 2π . (3) With an interaction strength of α = 1 N and with combina- toric factors of N Nφ and N(N −1) for graviton–inﬂaton and graviton–graviton scattering, respectively, the quantum depletion of the coherent gravitons is, to leading order in 1/N, (cf. [9]) Though in the large-N limit the emergent geometries are classical, the above outlined corpuscular description is fully quantum at heart. Belonging to the quantum critical conden- sate, the ground state of the gravitons has nearby, tightly spaced Bogoliubov states, accessible through graviton-to- graviton or graviton-to-inﬂaton scattering. 1 Introduction Cosmological inﬂation [1,2] is one of the central building blocks of our current understanding of the Universe. One of its simplest realizations, which is still compatible with obser- vations, is via a single scalar ﬁeld, called inﬂaton. Today’s structure in the Universe is seeded by the quantum ﬂuctua- tions of this ﬁeld and of the space-time, and it is in remark- able agreement with measurements (cf. [3–5]). Depending on the value of the inﬂaton, it might experience large quantum ﬂuctuations, also and in particular, up its potential, therefore inducing ever expanding inﬂationary patches of the Universe. This is the idea behind eternal inﬂation [6,7] (cf. for a recent review [8]). Recently, in [15] it has been argued that the corpuscular picture is incompatible with a positive cosmological con- stant. Also, in Ref. [9] the authors argue that quantum deple- tion sets a limit on the total number of e-foldings. Here, we investigate those qualitative considerations in more detail by quantitatively comparing the strengths of the relevant effects and considering, via the full respective probability distribu- tions, the fraction of the space-time which has an increasing potential. The mentioned corpuscular picture of quantum gravity, as introduced in [9] (cf. also [10–15]), assumes gravitons on a Minkowski background. Since they are bosons, and given their peculiar attractive derivative self-coupling, they gener- ically form Bose–Einstein condensates. In the limit of very high graviton ground-state occupation number N, these con- densates yield the emergent geometry which is observed at the classical level. These considerations are, however, only valid if the semi- classical description of space-time is a faithful approxi- mation. If gravity, like all other fundamental interactions, possesses a quantum description, inevitably the question arises when such a corpuscular picture of space-time starts to become relevant. Recent progress by Dvali and Gomez (cf. [9–15]), elaborating precisely on this topic, suggests that in certain situations one necessarily needs to take the graviton nature of what is classically regarded as space-time geometry Certain instances of these Bose–Einstein condensates are very special as their particular densities put them at a point of quantum criticality. This criticality occurs when the inter- action strength α is inversely proportional to the number of gravitons present in the condensate. In turn, these states can 12 3 3 505 Page 2 of 6 505 Page 2 of 6 Eur. Phys. J. 1 Introduction In the inﬂationary case, inﬂatons vastly outnumber gravitons, but inﬂaton self- interactions cannot excite the inﬂatons from the ground state, and hence graviton-to-inﬂaton scattering is the dominant quantum processes which deteriorates the classical geom- etry, and thereby also the anchor point of all semiclassical computations. ˙N ≃−1 √ N Nφ N − 1 √ N Nls. (4) (4) Here Nls is the number of species that are lighter than the energy of the gravitons in the condensate, which then present a possible decay channel. If no such lighter species exist the second term in Eq. (4) will just be −1/ √ N and represents graviton–graviton scattering. In [9] it was argued qualitatively that this quantum deple- tion of the condensates excludes eternal inﬂation. Here we will expand on that argument to consider eternal inﬂation for this corpuscular description of quantum gravity to do a quan- titative exploration of the subject. We include all monomial potentials, not only the m2 φ2-version that was considered in [9]. The quantum ﬂuctuations in the inﬂaton medium is the only source that may increase the energy of the inﬂaton medium, pushing the inﬂaton upward in the potential. For eternal inﬂation to be realized, this effect has to be larger than the effect of depletion in a large enough fraction of the space, so that combined with the continued inﬂation of this fraction of space it increases the volume of the inﬂating part of space-time. Since for each Hubble time this increase in volume is e3, this fraction must be < e−3 ≈1/20 = 0.05. 123 2 Competing ﬂuctuations Taking the condition given for the vacuum ﬂuctuations to dominate over depletion (10) and requiring that it holds above the bound yielding eternal inﬂa- tion classically (10), we ﬁnd To have eternal inﬂation, this bound must be fulﬁlled while the quantum-depletion effects are still smaller than the reg- ular quantum ﬂuctuations. Taking the condition given for the vacuum ﬂuctuations to dominate over depletion (10) and requiring that it holds above the bound yielding eternal inﬂa- tion classically (10), we ﬁnd (7c) (7d) \|n −1\| 2500π2n ≳N. (12) where we assume that n ̸= 1. where we assume that n ̸= 1. where we assume that n ̸= 1. (12) For quantum ﬂuctuations in Eq. (3) to move the inﬂaton up the potential, the ﬂuctuations have to be larger than the average depletion (4), which deteriorates the state away from semiclassicality. Using Eq. (7b) for Nφ in Eq. (4), we realize that the magnitude of depletion is Since N can never go below 1, naïvely, eternal inﬂation can never take place, because regardless of the value of n, this boundwillalwaysimplythat N ismuchsmallerthan1,unless n is extremely close to zero, which means that the potential is extremely ﬂat. In fact, formally \| ˙Ndep\| ≃ n 3n! N λn 3 n\|n −1\| N = 3 meff N . (8) (8) n ≲4 × 10−5. (13) (13) We then insert Eqs. (7c) and (7d) into Eq. (3) to ﬁnd the contribution to ˙N stemming from a typical quantum ﬂuctua- tion: in order for eternal inﬂation to have a chance of dominating before depletion takes over, hence, naïvely we could already exclude the occurrence of eternal inﬂation for all monomial potentials. \| ˙Nqf\| ≃n 2π n N λn 3n! = 1 2π n 3\|n −1\| meff √ N. (9) (9) Irrespective of the value of n, the above calculation was just done by comparing the typical quantum ﬂuctuation and depletion. In order to get a more reﬁned exclusion of eternal inﬂation, we must consider the two distributions properly. The quantum ﬂuctuations approximately follow a Gaussian distribution centered around zero. The depletion process is given by a Poisson distribution [18–21]. For eternal inﬂation to make any sense, the quantum- ﬂuctuating effects that drive it must be dominating the effects of the quantum depletion of the condensates. 2 Competing ﬂuctuations To compare the two effects we look for simplicity at an inﬂaton in the monomial potential We consider a Universe ﬁlled with inﬂaton and graviton condensates. The number of coherent inﬂatons in the inﬂa- ton condensate is Nφ and the number of coherent gravitons in the graviton condensate is N.1 Working in Planck units (c = ℏ= MPl = 1), the number of coherent inﬂatons can easily be deﬁned as V (φ) = 1 n! λn φn, (5) (5) where n > 0, and we deﬁne the effective mass scale meff(φ) ≡ V ′′(φ) , (6) Nφ := nφ R3 H = nφ H3 , (1) (6) (1) which for the case of V (φ) = 1/2m2 φ2 yields meff(φ) ≡ meff = m. We shall work in the slow-roll regime, \| ˙φ2\| ≪\|V \| and \| ¨φ2\| ≪3 H ˙φ ∼\|V,φ\|, and use Eq. (4), the Friedmann 1 We will sometimes refer to the number of coherent inﬂatons/gravitons in the condensates only as the number of inﬂatons/gravitons, as this is the only numbers of particles we will be interested in. 1 We will sometimes refer to the number of coherent inﬂatons/gravitons in the condensates only as the number of inﬂatons/gravitons, as this is the only numbers of particles we will be interested in. 123 Eur. Phys. J. C (2015) 75 :505 Page 3 of 6 505 equation H2 = 1/3 V , and Nφ ≃(V/meff) R3 H, to obtain the useful relations down the potential. This gives the criterion: H2/\| ˙φcl\| ≳3.8 (see [8]). Inserting for the corpuscular variables and our potential (5), this translates to the demand: φ ≃ n 3n! N λn , (7a) Nφ ≃ n 3n! N λn 3 n\|n −1\| N, (7b) ˙φ ≃−1 n n 3n! N λn ˙N N , (7c) meff ≃ n N λn 3n! 3n\|n −1\| N , (7d) (7a) N meff ≲ 3\|n −1\| n 1 3.8. (11) (11) To have eternal inﬂation, this bound must be fulﬁlled while the quantum-depletion effects are still smaller than the reg- ular quantum ﬂuctuations. 2 Competing ﬂuctuations Note that the true value of Vup will be lower in practice because the classical ﬂow will also pull the inﬂaton down its potential. Below we will include this effect in the convolution integral. Vup ≃ ∞ 0 dt λt e−λ t! −(t+tcl) −∞ dx 1 σ √ 2π e−x2 2σ2 , (16) (16) with λ = ˙Ndep, σ = ˙Nqf and tcl = ˙Ncl as given in Eq. (15a- c). The maximum N, which corresponds to standard/non- corpuscular eternal inﬂation, reads with λ = ˙Ndep, σ = ˙Nqf and tcl = ˙Ncl as given in Eq. (15a- c). The maximum N, which corresponds to standard/non- corpuscular eternal inﬂation, reads When investigating the effect of the classical ﬂow down the potential along with the two quantum effects, we need to consider the convoluted volume for decreasing values of N, from the classical onset of eternal inﬂation, and down toward Planckian values, N ≈1. The magnitudes of the three effects in this regime can be written Nmax ≃ 3n! λn 2 n+2 1 3.8n 2n n+2 . (17) (17) \| ˙Nqf\| ≃n 2π n λn 3n! N 1 n , (15a) \| ˙Ndep\| ≃ 3 n\|n −1\| n 3n! λn N −2+n 2n , (15b) \| ˙Ncl\| ≃ V ′ V 2 H N ≃n2 λn 3n! 2 n N 4+n 2n . (15c) (15a) For instance, λn = 10−12 yields Nmax(n = 2) ∼O(106), Nmax(n = 3) ∼O(105), and Nmax(n = 4) ∼O(104). As long as none of the resulting fractions (16) exceed 0.05, eternal inﬂation will not occur. For instance, λn = 10−12 yields Nmax(n = 2) ∼O(106), Nmax(n = 3) ∼O(105), and Nmax(n = 4) ∼O(104). As long as none of the resulting fractions (16) exceed 0.05, eternal inﬂation will not occur. (15b) In Fig. 2 we depict results of the general convolution integral (16) for various values of the self-coupling λn (10−12, 10−10, and 10−8) as well as for two values of N— once for a 1/10 of the maximum allowed value as given by (17), and once for 1/20 of it. For the values consid- ered eternal inﬂation is dwarfed out by many orders of magnitude. As n or λn increases, Nmax decreases, so for values much larger than the ones considered here, either for n or λn, even considering eternal inﬂation becomes nonsensical. 2 Competing ﬂuctuations The two effects might seem, in the case when the quantum ﬂuctuations drive the inﬂaton up its potential, to both push the conden- sate toward lower values of N. However, the way in which this happens is different. The depletion drives the conden- sate away from its classical inﬂating description toward an intrinsically quantum state. For this the coherent graviton- condensate behavior which constitutes the inﬂating geome- try no longer exists, and the description of this as inﬂation (eternal or otherwise) disappears. Comparing (8) and (9) we ﬁnd that the condition for the quantum ﬂuctuations being dominant reads For safe bounds on eternal inﬂation, we can compare the two distributions at the lower bound for the potential, there- fore, at the value of N meff where standard (non-corpuscular) eternal inﬂation would occur [cf. Eq. (10)]. Inserting this into Eq. (9) we ﬁnd that here the Gaussian distribution has a stan- dard deviation σ ≃(n/2π)(3.8n)−2/(2+n)(λn/3n!)1/(2+n), whereas Eq. (8) implies that the Poisson distribution has an expectation value of λ ≃3.8√3n/\|n −1\| . In order to compare the two competing effects we must then convolute the two probability distribution functions to ﬁnd the fractional convoluted area that gives an increase in inﬂaton energy. That is, for each possible value for depletion, we multiply its probability with the probability of all quan- tum ﬂuctuations that are large enough to dominate over it. In practice this is done by integrating the Gaussian up to where its contribution is the negative of each point on the Poisson curve along the Poisson distribution: N 3/4meff ≳ 6π 3\|n −1\| n . (10) (10) In order for eternal inﬂation to proceed when no corpuscu- lar effects are present, the standard deviation of the Gaussian- distributed quantum ﬂuctuations must be large enough for 1/e3 ≈1/20 of the ﬂuctuations to exceed the classical roll 12 3 123 Eur. Phys. J. C (2015) 75 :505 505 Page 4 of 6 505 Page 4 of 6 1 2 3 4 5 0.000 0.002 0.004 0.006 0.008 n Vup 1 2 3 4 5 0.000 0.002 0.004 0.006 0.008 n Vup Fig. 1 The convolution integral Vup (cf. Eq. (14)) as a function of the exponent n in V (φ) = 1/n! λn φn, for various values of the self- coupling λn: 10−8 (blue dotted), 10−10 (red dashed), 10−12 (black solid) makes inﬂation (eternal or not) meaningless. 2 Competing ﬂuctuations Thus it is in fact classical ﬂow and the depletion that together both pull the physical state away from the eternally inﬂating state, but in radically different ways, one by ﬂowing toward the bottom of the potential, and one by destroying the coherence of the underlaying quantum state. The naïve absolute maximum value for λn, which is remotely sensible to consider, is the value for which eter- nal inﬂation can begin only at Planck scales N ≃1 in Eq. (10), that is, λn, max ≃3n!(3.8n)−n. In practice λn would be much smaller than this as terms of higher order in 1/N become important when N approaches 1. The minimum value of λn is harder to obtain. However, as λn decreases, the width of the Gaussian function for the quantum ﬂuctuations decreases, making the quantum-depletion effects more dom- inant. n Fig. 1 The convolution integral Vup (cf. Eq. (14)) as a function of the exponent n in V (φ) = 1/n! λn φn, for various values of the self- coupling λn: 10−8 (blue dotted), 10−10 (red dashed), 10−12 (black solid) For a given value of n and the self-coupling λn we can cal- culate the fraction of the Universe undergoing eternal inﬂa- tion by evaluating the convolution integral for a given value of N, which is formulated in such a way as to account for the classical ﬂow and the depletion both driving the state away from the potentially eternally inﬂating state, Vup ≃ ∞ 0 dt λt e−λ t! −t −∞ dx 1 σ √ 2π e−x2 2σ2 ; (14) (14) Vup is the fraction of the space-time that has an increasing potential. Since the space-time volume in the inﬂating parts of the Universe is multiplied by 20, eternal inﬂation can only occur when Vup ≳0.05. Figure 1 shows that, at least for small n, eternal inﬂation is excluded. Note that the true value of Vup will be lower in practice because the classical ﬂow will also pull the inﬂaton down its potential. Below we will include this effect in the convolution integral. Vup is the fraction of the space-time that has an increasing potential. Since the space-time volume in the inﬂating parts of the Universe is multiplied by 20, eternal inﬂation can only occur when Vup ≳0.05. Figure 1 shows that, at least for small n, eternal inﬂation is excluded. 123 3 Discussion and outlook In this work we investigated the paradigm of eternal inﬂation in view of the corpuscular picture of space-time for single- ﬁeld inﬂation models with monomial potentials. We have compared the strength of the average ﬂuctuation of the ﬁeld up its potential with that of quantum depletion, and showed that the latter is dominant at least for small n. Note that the mentioned physical mechanism investigated in this work is very different from all the (semi-)classical ones discussed previously in the literature, which investigate bounds on the total number of e-foldings, originating from extra-dimensions (cf. e.g. [28]), by assigning ﬁnite entropy to de Sitter space [29–31], or, via thoroughly incorporating the null-energy condition [32], for instance. Instead, here, the limited duration of inﬂation originates from the quantum resolution of the inﬂation as well as of the graviton conden- sate, which constitute the classical backgrounds in the limit of inﬁnite N. In this limit no quantum depletion is present, which, as we quantiﬁed, turns out to be crucial for prop- erly deriving the criterion for the occurrence of eternal inﬂa- tion. In order to make a more reﬁned statement, we then studied the fraction of space-time which has an increasing potential both with and without the effects of the classical roll present. For the case where we only considered quantum ﬂuctuations versus quantum depletion, we could already prove the non- existence of eternal inﬂation for the observationally relevant small-n potential. Including the classical effects we could show that the frac- tion of space-time moving up the potential is always, i.e. for any n, way below the eternal-inﬂation threshold. In summary, we have proven that eternal inﬂation does strictly not occur for all canonical single-ﬁeld inﬂation models with monomial self-interactions. This is a quantitative substantiation of the claim made in [9,15] that corpuscular gravity prohibits eter- nal inﬂation. As the de Sitter solution might be approximated by the extreme slow-roll version of inﬂation, the fact that inﬂation- ary theories in the near-Planckian range are strongly domi- nated by the depletion effect also strengthens the argument found in [15] that the corpuscular view of gravity may have bearing on the cosmological constant problem. We believe that these ﬁndings are rather generic. Eur. Phys. J. C (2015) 75 :505 Fig. 2 The general convolution integral Vup including the classical drift (cf. Eq. (16)) as a function of the exponent n in V (φ) = 1/n! λn φn for various values of the self-coupling λn: 10−8 (blue dotted), 10−10 (red dashed), 10−12 (black solid); upper panel: N = 0.05 Nmax; lower panel: N = 0.1 Nmax 3.0 3.2 3.4 3.6 3.8 4.0 0 2. 10 8 4. 10 8 6. 10 8 8. 10 8 n Vup N 0.05 Nmax 3.0 3.2 3.4 3.6 3.8 4.0 0 5. 10 9 1. 10 8 1.5 10 8 2. 10 8 2.5 10 8 3. 10 8 3.5 10 8 n Vup N 0.1 Nmax 3 Discussion and outlook In this work we investigated the paradigm of eternal inﬂation in view of the corpuscular picture of space-time for single- cal General Relativity and it will be impossible to reliably say that eternal inﬂation occurs. These statements are com- pletely generic for any corpuscular treatment of inﬂation, and in a forthcoming publication we will further elaborate 3.0 3.2 3.4 3.6 3.8 4.0 0 10 8 10 8 10 8 10 8 n N 0.05 Nmax 3.0 3.2 3.4 3.6 3.8 4.0 0 5. 10 9 1. 10 8 1.5 10 8 2. 10 8 2.5 10 8 3. 10 8 3.5 10 8 n Vup N 0.1 Nmax eternal inﬂation time for single- ntials. We have tion of the ﬁeld on, and showed cal General Relativity and it will be impossible to reliably say that eternal inﬂation occurs. These statements are com- pletely generic for any corpuscular treatment of inﬂation, and, in a forthcoming publication, we will further elaborate on this (non-eternal) inﬂation for generic single-ﬁeld inﬂa- tion models, using the quantitative methods developed in this work. N t th t th ti d h i l h i i ti t d 3.0 3.2 3.4 3.6 3.8 4.0 0 2. 10 8 4. 10 8 6. 10 8 8. 10 8 n Vup N 0.05 Nmax 3.0 3.2 3.4 3.6 3.8 4.0 0 5. 10 9 1. 10 8 1.5 10 8 2. 10 8 2.5 10 8 3. 10 8 3.5 10 8 n p N 0.1 Nmax Fig. 2 The general convolution integral Vup including the classical drift (cf. Eq. (16)) as a function of the exponent n in V (φ) = 1/n! Eur. Phys. J. C (2015) 75 :505 λn φn for various values of the self-coupling λn: 10−8 (blue dotted), 10−10 (red dashed), 10−12 (black solid); upper panel: N = 0.05 Nmax; lower panel: N = 0.1 Nmax n n cal General Relativity and it will be impossible to reliably say that eternal inﬂation occurs. These statements are com- pletely generic for any corpuscular treatment of inﬂation, and, in a forthcoming publication, we will further elaborate on this (non-eternal) inﬂation for generic single-ﬁeld inﬂa- tion models, using the quantitative methods developed in this work. 2 Competing ﬂuctuations Hence eternal inﬂation does not occur for canonical single-ﬁeld inﬂation models with monomial self- interactions. (15c) The classical effect is always positive, whereas the depletion effect is always negative. The quantum ﬂuctuations can take either positive or negative values, but the interesting ones that may lead to eternal inﬂation are the negative ones. However, as discussed above, this does not mean that the depletion and the quantum ﬂuctuations pull together toward an eternally inﬂating state. The quantum-depletion effect on the contrary drives the entire state away from its semiclassical descrip- tion taking the gravitons out of their coherent state, which 123 Eur. Phys. J. 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Mukhanov, Black Hole M 3 Discussion and outlook In the case of more general potentials, such as for instance hill- top inﬂation, we still need the quantum ﬂuctuations to be comparable to the classical evolution in order to drive eter- nal inﬂation. In these situations we also expect the deple- tion effects to become large, and more importantly domi- nate with respect to the usual quantum ﬂuctuations, more or less regardless of the detailed shape of the potential. Also for many non-monomial potentials, the shape of the poten- tial as seen in the case of an inﬂaton high enough up in the potential for quantum ﬂuctuations to be comparable to clas- sical ﬂow may be well approximated by a monomial poten- tial. Acknowledgments F. K. acknowledges support from the Swedish Research Council (VR) through the Oskar Klein Centre and thanks the Institute of Theoretical Astrophysics at the University of Oslo, where part of this work as been performed. M.S. thanks David F. Mota for motivation to discover and explore the ﬁeld of corpuscular gravity and is also grateful to Nordita for hospitality during the ”Extended theories of Gravity” workshop. We are indebted to Gia Dvali and Douglas Spolyar for valuable comments. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecomm ons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Funded by SCOAP3. We should stress that, in any case, at some ﬁnite point in time the quantum-depletion effects will accumulate to an extent that invalidates any (semi-)classical treatment, and hence constitutes a radical shift away from the ”standard” non-corpuscular description of eternal inﬂation. 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https://openalex.org/W2120129393	https://journals.plos.org/plosmedicine/article/file?id=10.1371/journal.pmed.1001192&type=printable	English	null	Planned Vaginal Birth or Elective Repeat Caesarean: Patient Preference Restricted Cohort with Nested Randomised Trial	PLoS medicine	2,012	cc-by	9,176	Planned Vaginal Birth or Elective Repeat Caesarean: Patient Preference Restricted Cohort with Nested Randomised Trial Caroline A. Crowther1, Jodie M. Dodd1, Janet E. Hiller2, Ross R. Haslam3, Jeffrey S. Robinson1, on behalf of the Birth After Caesarean Study Group" 1 Australian Research Centre for Health of Women and Babies (ARCH), The Discipline of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, South Australia, Australia, 2 Faculty of Health Sciences, Australian Catholic University, Melbourne, Victoria, Australia, 3 Department of Neonatal Medicine, The Women’s and Children’s Hospital, Adelaide, South Australia, Australia Abstract Background: Uncertainty exists about benefits and harms of a planned vaginal birth after caesarean (VBAC) compared with elective repeat caesarean (ERC). We conducted a prospective restricted cohort study consisting of a patient preference cohort study, and a small nested randomised trial to compare benefits and risks of a planned ERC with planned VBAC. Methods and findings: 2,345 women with one prior caesarean, eligible for VBAC at term, were recruited from 14 Australian maternity hospitals. Women were assigned by patient preference (n = 2,323) or randomisation (n = 22) to planned VBAC (1,225 patient preference, 12 randomised) or planned ERC (1,098 patient preference, ten randomised). The primary outcome was risk of fetal death or death of liveborn infant before discharge or serious infant outcome. Data were analysed for the 2,345 women (100%) and infants enrolled. The risk of fetal death or liveborn infant death prior to discharge or serious infant outcome was significantly lower for infants born in the planned ERC group compared with infants in the planned VBAC group (0.9% versus 2.4%; relative risk [RR] 0.39; 95% CI 0.19–0.80; number needed to treat to benefit 66; 95% CI 40– 200). Fewer women in the planned ERC group compared with women in the planned VBAC had a major haemorrhage (blood loss $1,500 ml and/or blood transfusion), (0.8% [9/1,108] versus 2.3% [29/1,237]; RR 0.37; 95% CI 0.17–0.80). Conclusions: Among women with one prior caesarean, planned ERC compared with planned VBAC was associated with a lower risk of fetal and infant death or serious infant outcome. The risk of major maternal haemorrhage was reduced with no increase in maternal or perinatal complications to time of hospital discharge. Women, clinicians, and policy makers can use this information to develop health advice and make decisions about care for women who have had a previous caesarean. Trial registration: Current Controlled Trials ISRCTN53974531 Please see later in the article for the Editors’ Summary. Citation: Crowther CA, Dodd JM, Hiller JE, Haslam RR, Robinson JS, et al. (2012) Planned Vaginal Birth or Elective Repeat Caesarean: Patient Preference Restricted Cohort with Nested Randomised Trial. PLoS Med 9(3): e1001192. doi:10.1371/journal.pmed.1001192 Academic Editor: Gordon C. Smith, Cambridge University, United Kingdom Received July 25, 2011; Accepted February 2, 2012; Published March 13, 2012 Received July 25, 2011; Accepted February 2, 2012; Published March 13, 2012 Copyright: 2012 Crowther et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was funded by a three-year project grant from the National Health and Medical Research Council, Australia; and a grant from the Women’s and Children’s Hospital Foundation, South Australia. JD was supported through a National Health and Medical Research Council Neil Hamilton Fairley Clinical Fellowship (ID 399244) and an NHMRC Practitioner Fellowship (ID 627005). Infrastructure support was provided by the Australian Research Centre for Health of Women and Babies (ARCH), The University of Adelaide. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: BMI, body mass index; ERC, elective repeat caesarean; SES, socioeconomic status; RR, relative risk; VBAC, vaginal birth after caesarean E-mail: caroline.crowther@adelaide.edu.au Abbreviations: BMI, body mass index; ERC, elective repeat caesarean; SES, socioeconomic status; RR, relative risk; VBAC, vaginal birth after caesarean * E-mail: caroline.crowther@adelaide.edu.au " Membership of the Birth After Caesarean Study Group is provided in the Acknowledgments. PLoS Medicine \| www.plosmedicine.org 1 March 2012 \| Volume 9 \| Issue 3 \| e1001192 PLoS Medicine \| www.plosmedicine.org Planned VBAC or ERC: Health Outcomes Planned VBAC or ERC: Health Outcomes Interventions l d Planned VBAC group. Participating clinicians agreed to follow the study protocol for intrapartum care for women in the planned VBAC group on the basis of clinical practice guidelines [12,14–17,20–22]. After study entry, women who planned to have a vaginal birth awaited spontaneous onset of labour. The attending obstetrician made assessment of the woman’s on-going suitability for a planned VBAC. Planned VBAC group. Participating clinicians agreed to follow the study protocol for intrapartum care for women in the planned VBAC group on the basis of clinical practice guidelines [12,14–17,20–22]. After study entry, women who planned to have a vaginal birth awaited spontaneous onset of labour. The attending obstetrician made assessment of the woman’s on-going suitability for a planned VBAC. Planned ERC group: Women who planned to have an ERC had this scheduled between 38 wk and 40 wk, preferably at 39 wk. If a woman in the planned ERC group entered labour prior to the scheduled elective surgical procedure, a caesarean was considered as an emergency. Randomisation, Masking, and Group Allocation Randomisation, Masking, and Group Allocation Women who gave written informed consent to the randomised trial, were randomly assigned to either planned VBAC or planned ERC, using a central telephone randomisation service. The randomisation schedule was prepared by an investigator not involved with clinical care, with stratification by centre and previous successful vaginal birth, using balanced variable blocks. Participants, staff, and investigators were not masked to treatment allocation. Women who gave written informed consent to the preference study were asked their preference for either planned VBAC or planned ERC, and were assigned to their preferred study group. Baseline information, including age, parity, marital status, body mass index (BMI), socioeconomic status (SES), psychological wellbeing, quality of life, and ethnicity self-reported by the participant was collected to compare the study groups. Both ERC and VBAC have benefits and harms. Risks of planned VBAC when compared with planned ERC include haemorrhage, need for blood transfusion, endometritis, uterine rupture, perinatal death, and hypoxic ischaemic encephalopathy [8–12]. By comparison, women planning ERC are at increased risk of surgical complications, placenta accreta, and risks of multiple caesareans [11] and their infants are at risk of respiratory morbidity [10,11]. Couples may experience subsequent infertility. [9–12]. There have been no randomised controlled trials comparing health outcomes after VBAC and ERC although the difficulty of conducting such trials has been recognised [9,11]. A comprehen- sive systematic review of the nonrandomised literature comparing ERC with VBAC concluded that the current literature was ‘‘significantly flawed,’’ and that future research ‘‘should focus on comparability of the groups, specificity of the intervention, and standard outcome measures.’’ [10]. The need for evidence to inform women, clinicians, and policy makers about health outcomes of intended planned mode of birth rather than actual has been highlighted as critical [11]. To address these research gaps we conducted a prospective restricted cohort study that became effectively a patient preference study, with a smaller randomised trial (Text S1) [13] to compare the benefits and risks of a planned ERC with planned VBAC. Outcomes Ethics approval was granted by the Children’s Youth and Women’s Health Services Human Research Ethics Committee at the Women’s and Children’s Hospital, and by the local institutional review boards for each centre. Study outcomes were important established measures of term infant morbidity and maternal morbidity, up to the time of primary hospital discharge after birth collected by trained research personnel. The primary prespecified outcome for this study was a composite of death or serious outcome for the infant defined as: death (any fetal death after study entry or death of a liveborn infant before hospital discharge [excluding lethal congenital anomalies]); or serious morbidity (defined as one or more of: birth trauma [subdural or intracerebral haemorrhage, spinal cord injury, basal skull fracture, other fracture, peripheral nerve injury present at discharge from hospital]); seizures at ,24 h age or requiring two or more drugs to control; Apgar score ,4 at 5 min; cord pH,7.0 (arterial or venous cord blood) and/or cord blood base deficit $12; neonatal encephalopathy stage 3; admission to the neonatal intensive care unit (NICU).4 d; severe neonatal lung disease (defined as mean airway pressure .10 and or fraction of inspired oxygen .0.80 with need for ventilation); proven PLoS Medicine \| www.plosmedicine.org Introduction perforation; multiple pregnancy; any contraindication to vaginal birth (including placenta praevia, transverse lie, active genital herpes infection); cephalo-pelvic disproportion as judged by the clinician; lethal congenital anomaly; or fetal anomaly associated with mechanical difficulties at birth. The inclusion and exclusion criteria were based on clinical practice guidelines for eligibility for a VBAC [12,14–17]. Caesarean section is one of the commonest operations performed on childbearing women, with rates continuing to rise worldwide. For women who have had a previous caesarean, choices for mode of birth in their next pregnancy are either a trial of vaginal birth after caesarean (VBAC) or an elective repeat caesarean (ERC). For women who attempt a VBAC, the chance of achieving vaginal birth has been variably reported between 56% [1] and 80% [2]. The proportion of women attempting a VBAC has been declining in many countries [3], fuelled by negative reports of an increase in the risk of maternal and infant complications related to VBAC [4], including uterine rupture [5] and perinatal death [6]. The rates of repeat caesarean birth following a previous caesarean have risen commensurately, reaching 83% in Australia [7] and almost 90% in the US [3]. Repeat caesarean now accounts for 28% of all births in the United Kingdom [8]. In the antenatal clinic eligible women were provided with written information about the randomised trial and patient preference study, pamphlets on VBAC and caesarean [18,19], and asked if they would participate by the research officer. Recruitment started in November 2002 and was completed in May 2007. PLoS Medicine \| www.plosmedicine.org Study Design and Participants We conducted a multicentre study at 14 Australian maternity hospitals, all staffed and equipped to provide recommended care for VBAC or caesarean [12–17]. Women were eligible who had a single prior caesarean presenting in their next pregnancy with a live singleton in cephalic presentation, at 37 wk gestation or more, and who were considered eligible to attempt planned VBAC by their obstetrician (Text S1) [13]. Women were ineligible with more than one prior caesarean birth; a vertical, inverted T or unknown uterine incision; previous uterine rupture; previous uterine surgery (including hysterotomy or myomectomy involving entry of the uterine cavity or excessive myometrial dissection); previous uterine March 2012 \| Volume 9 \| Issue 3 \| e1001192 2 Planned VBAC or ERC: Health Outcomes Planned VBAC or ERC: Health Outcomes necrotising enterocolitis; and proven systemic infection in first 48 h of life treated with antibiotics. incision; occurrence of a fistula involving the genital tract; bowel obstruction or paralytic ileus; pulmonary oedema; stroke (defined as acute neurological deficit .24 h); cardiac arrest or respiratory arrest; any other serious maternal complication related to birth (as judged by the adverse events committee, while remaining masked to group allocation and mode of birth). Secondary study outcomes related to serious outcomes for the woman defined as one or more of: maternal death; uterine rupture (defined as a clinically significant rupture involving the full thickness of the uterine wall and requiring surgical repair); severe haemor- rhage (blood loss of $1,500 ml and/or requiring blood transfusion); hysterectomy for any complications resulting from birth; vulvar or perineal haematoma requiring evacuation; deep vein thrombosis or thrombophlebitis requiring anticoagulant therapy; pulmonary embolus requiring anticoagulant therapy; pneumonia due to infection, aspiration or other causes; adult respiratory distress syndrome; wound infection (requiring prolongation of hospital stay or readmission) or wound dehiscence; damage to the bladder, ureter, or bowel requiring repair, or cervical laceration extending to the lower uterine segment, or abnormal extension of the uterine Statistical Methods Analyses were performed using intention-to-treat principles, according to the woman’s assigned mode of birth at study entry, with the use of SAS software, version 9.1. Initial analyses were unadjusted. We prespecified that analyses would be adjusted for key prognostic variables with imbalance. We therefore adjusted analyses for maternal SES, BMI, and indication for previous caesarean birth where possible. Binary variables were analysed with log-binomial regression to give relative risks and 95% CIs. Figure 1. Flow of participants in the study. Data are numbers (%). doi:10.1371/journal.pmed.1001192.g001 Figure 1. Flow of participants in the study. Data are numbers (%). doi:10.1371/journal.pmed.1001192.g001 March 2012 \| Volume 9 \| Issue 3 \| e1001192 PLoS Medicine \| www.plosmedicine.org 3 Planned VBAC or ERC: Health Outcomes Table 1. Baseline maternal characteristics. Statistical Methods Outcome n (%), Mean ± SD, or Median and Interquartile Range RCT Patient Preference Planned ERC Group (n = 1,108) Planned VBAC Group (n = 1,237) Planned ERC (n = 10) Planned VBAC (n = 12) Planned ERC (n = 1,098) Planned VBAC (n = 1,225) Age (y) 29.064.2 32.666.2 31.165.2 30.765.1 31.165.2 30.765.1 Gestational age entry (wk) 37.6 (37.0–38.0) 38.1 (37.3–39.1) 37.4 (37.1–38.2) 37.5 (37.1–38.4) 37.4 (37.1–38.2) 37.5 (37.1–38.4) Previous vaginal birth 1 (10.0) 5 (41.7) 143 (13.0) 188 (15.3) 144 (13.0) 193 (15.6) Smoker 1 (10.0) 3 (25.0) 177 (16.1) 194 (15.8) 178 (16.1) 197 (15.9) Body mass index 23.1 (21.6–26.9) 28.5 (27.1–33.4) 26.7 (22.2–32.0) 25.9 (22.3–30.8) 27.9 (23.9–32.9) 26.8 (23.5–31.4) Ethnicitya Caucasian 7 (70.0) 9 (75.0) 957 (87.2) 1,038 (84.7) 964 (87.0) 1,047 (84.6) Asian 3 (30.0) 3 (25.0) 95 (8.7) 116 (9.5) 98 (8.8) 119 (9.6) Other 0 (0.0) 0 (0.0) 46 (4.2) 71 (5.8) 46 (4.2) 71 (5.8) Married/de facto 9 (90.0) 10 (83.3) 908 (82.7) 989 (80.7) 917 (82.8) 999 (80.8) Socioeconomic indexb Low SE index (disadvantaged) 1 (10.0) 1 (8.3) 256 (23.3) 341 (27.8) 257 (23.2) 342 (27.6) Low-mid SE index 2 (20.0) 3 (25.0) 265 (24.1) 314 (25.6) 268 (24.2) 318 (25.7) Mid-high SE index 7 (70.0) 2 (16.7) 210 (19.1) 240 (19.6) 218 (19.7) 241 (19.5) High SE index 0 (0.0) 6 (50.0) 367 (33.4) 330 (26.9) 365 (32.9) 336 (27.2) Emotional wellbeing EPDS Score 10.0 (3.0–13.0) 6.0 (5.0–14.0) 6.0 (3.0–9.0) 6.0 (3.0–10.0) 6.0 (3.0–9.0) 6.0 (3.0–10.0) Depressed (EPDS.12) 3 (33.3) 4 (36.4) 111 (11.4) 121 (11.4) 114 (11.6) 125 (11.6) Anxiety (STAI) 12.063.6 12.564.6 10.863.6 10.963.4 10.963.6 10.963.5 Physical functioning (SF36) 59.4629 51.1625 61.5625 62.5624 61.5625.5 62.3624.1 Role physical (SF36) 58.3645 38.6645 41.8642 43.7641 41.9641.6 43.6641.2 Bodily pain (SF36) 51.9620 52.7629 58.8622 60.4622 58.7622.4 60.3621.9 General health (SF36) 72.9623 70.4620 78.6617 79.0616 78.5616.7 78.9615.8 Vitality (SF36) 43.9619 58.2616 50.4620 51.5619 50.3619.6 51.5618.7 Social functioning (SF36) 73.6618 78.4626 76.7622 76.8622 76.7621.9 76.8621.9 Role emotional (SF36) 63.0642 75.8642 83.8632 82.1633 83.6632.2 82.0633.1 Mental health (SF36) 77.8613 69.5616 77.7615 77.0615 77.7615.0 76.9615.0 Physical component (SF36) 40.069.3 36.669.7 39.1610 40.069.6 39.169.9 39.969.6 Mental Component (SF36) 48.769.5 51.869.6 52.569.0 52.169.0 52.569.0 52.169.0 Brazier health utility (SF36) 0.760.2 0.7160.1 0.7460.1 0.7460.1 0.760.1 0.760.1 Main reasons for previous caesarean Failure to progress 1 (10.0) 5 (41.7) 469 (42.7) 394 (32.2) 470 (42.4) 399 (32.3) Fetal distress 6 (60.0) 8 (66.7) 423 (38.5) 467 (38.1) 429 (38.7) 475 (38.4) Breech 2 (20.0) 1 (8.3) 217 (19.8) 411 (33.6) 219 (19.8) 412 (33.3) Cephalopelvic disproportion 1 (10.0) 1 (8.3) 81 (7.4) 64 (5.2) 82 (7.4) 65 (5.3) Failed induction 2 (20.0) 1 (8.3) 46 (4.2) 59 (4.8) 48 (4.3) 60 (4.9) Antepartum haemorrhage 1 (10.0) 1 (8.3) 42 (3.8) 60 (4.9) 43 (3.9) 61 (4.9) Maternal choice 0 (0.0) 0 (0.0) 73 (6.6) 32 (2.6) 73 (6.6) 32 (2.6) Maternal medical disorder 0 (0.0) 2 (16.7) 31 (2.8) 24 (2.0) 31 (2.8) 26 (2.1) Pre-eclampsia/eclampsia 1 (10.0) 0 (0.0) 66 (6.0) 63 (5.1) 67 (6.0) 63 (5.1) Other 1 (10.0) 1 (8.3) 65 (5.9) 63 (5.1) 66 (6.0) 64 (5.2) aEthnicity as reported by the participant. y g g g EPDS, Edinburgh Postnatal Depression Scale [32]; SD, standard deviation; SE, socioeconomic; STAI, Spielberger State-Trait Anxiety Inventory [33]; SF3, SF36-Health Survey Questionnaire [34]. y p y p p bSocioeconomic index as measured by SEIFA where high index scores indicate increasing levels of social disadvantage. PDS di b h P l D i S l [ ] SD d d d i i S i i S S i lb S i participant. sured by SEIFA where high index scores indicate increasing levels of social disadvantage. y g g g EPDS, Edinburgh Postnatal Depression Scale [32]; SD, standard deviation; SE, socioeconomic; STAI, Spielberger State-Trait Anxiety Inventory [33]; SF3, SF36-Health S Q i i [34] y p y p p bSocioeconomic index as measured by SEIFA where high index scores indicate increasing levels of social disadvantage. EPDS, Edinburgh Postnatal Depression Scale [32]; SD, standard deviation; SE, socioeconomic; STAI, Spielberger State-Trait A nicity as reported by the participant. ioeconomic index as measured by SEIFA where high index scores indicate increasing levels of social disadvantage. y y bSocioeconomic index as measured by SEIFA where high index scores indicate increasing levels of social disadvantage. EPDS, Edinburgh Postnatal Depression Scale [32]; SD, standard deviation; SE, socioeconomic; STAI, Spielberger State-Tra Survey Questionnaire [34]. y g g g gh Postnatal Depression Scale [32]; SD, standard deviation; SE, socioeconomic; STAI, Spielberger State-Trait Anxiety Inventory [33]; S onnaire [34]. Socioeconomic index as measured by SEIFA where high index scores indicate increasing levels of social disadvantage. EPDS, Edinburgh Postnatal Depression Scale [32]; SD, standard deviation; SE, socioeconomic; STAI, Spielberger State-Trait Anxiety Inventory [33]; SF3, SF36-Health Survey Questionnaire [34]. doi:10 1371/journal pmed 1001192 t001 Primary Outcomes The risk of fetal or liveborn infant death prior to discharge or serious outcome was significantly reduced for infants born to women in the planned ERC group compared with infants of women in the planned VBAC group (planned ERC 10/1,108 [0.9%] versus planned VBAC 30/1,237 [2.4%], relative risk [RR] 0.39, 95% CI 0.19–0.80, p = 0.011) (Table 2). The number needed to treat to benefit with planned ERC to prevent fetal death or Results Of the 2,345 women enrolled, 1,108 (47.2%) were in the planned ERC group (ten randomised; 1,098 patient preference) and 1,237 (52.8%) in the planned VBAC group (12 randomised; 1,225 patient preference) (Figure 1). Clinical outcomes to primary hospital discharge after birth were available for all women and their infants. The two treatment groups were similar at the time of study entry apart from women in the planned ERC group compared with women in the VBAC group being of slightly higher BMI, and SES, and there were some differences in the reasons for the previous caesarean section (Table 1). Statistical Methods bSocioeconomic index as measured by SEIFA where high index scores indicate increasing levels of social disadvantage. EPDS, Edinburgh Postnatal Depression Scale [32]; SD, standard deviation; SE, socioeconomic; STAI, Spielberger State-Trait Anxiety Inventory [33]; SF3, SF36-Health Survey Questionnaire [34]. n (%), Mean ± SD, or Median and Interquartile Range PLoS Medicine \| www.plosmedicine.org March 2012 \| Volume 9 \| Issue 3 \| e1001192 Planned VBAC or ERC: Health Outcomes Continuous variables, if normally distributed, were analysed with a Student’s t-test, and nonparametric tests were used for skewed data. We tested for interaction between treatment groups (VBAC and ERC) by study arm (randomised and patient preference) by calculating 95% CIs on the basis of the score method for the difference in treatment of the outcome proportions for binary data [23]. For all outcomes, apart from emergency caesarean where results are presented separately, no interactions were detected and the results of the preference and randomised arms were combined. The difference of proportions metric was used, as the number of women in the randomised arm was small generating zero cells. Consequently the relative risk was undefined for some outcomes. A p-value of less than 0.05 was considered to indicate statistical significance (2-sided). An interim analysis was not performed. aValues adjusted for maternal SES, BMI, and indication for previous caesarean birth. bDeath or serious outcome includes: death (any fetal death after study entry or death of a liveborn infant prior to hospital discharge [excluding lethal congenital anomalies]); or serious neonatal morbidity (defined as one or more of: birth trauma [subdural or intracerebral haemorrhage, spinal cord injury, basal skull fracture, other fracture, peripheral nerve injury present at discharge from hospital]; seizures at ,24 h age or requiring two or more drugs to control; Apgar score ,4 at 5 min; cord pH,7?0; cord blood base deficit $12 mmol/l; neonatal encephalopathy stage 3; admission to NICU.4 d; severe neonatal lung disease [defined as MAP.10 and or FiO2.0?80 with need for ventilation]; proven necrotising enterocolitis; or proven systemic infection in first 48 h of life treated with antibiotics). NICU, neonatal intensive care unit. Sample Size We estimated that a sample size of 2,314 women would have statistical power of 80% (two-tailed alpha level 0.05) to detect an increase of 2% in the risk of fetal death or liveborn infant death prior to discharge or serious infant outcome from 1.6% for planned ERC [24] to 3.6% for planned VBAC (Text S1) [13]. Table 2. Primary outcomes. Table 2. Primary outcomes. Sample Size y Outcome RCT Patient Preference Planned ERC Group (n = 1,108) Planned VBAC Group (n = 1,237) Adjusted RR (95% CI)a p- Value Planned ERC (n = 10) Planned VBAC (n = 12) Planned ERC (n = 1,098) Planned VBAC (n = 1,225) n Percent n Percent n Percent n Percent n Percent n Percent CI Range Death or serious infant outcomeb 0 (0.0) 0 (0.0) 10 (0.9) 30 (2.4) 10 (0.9) 30 (2.4) 0.39 (0.19– 0.80) 0.011 Perinatal death 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.2) 0 (0.0) 2 (0.2) 0.50 Stillbirth 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.2) 0 (0.0) 2 (0.2) Neonatal death 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Serious neonatal morbidity 0 (0.0) 0 (0.0) 10 (0.9) 28 (2.3) 10 (0.9) 28 (2.3) 0.41 (0.20– 0.83) 0.014 Birth trauma 0 (0.0) 0 (0.0) 0 (0.0) 3 (0.2) 0 (0.0) 3 (0.2) 0.25 Seizures 0 (0.0) 0 (0.0) 1 (0.1) 1 (0.1) 1 (0.1) 1 (0.1) 1.11 (0.07– 17.8) 1.00 Apgar #4 at 5 min 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.1) 0 (0.0) 1 (0.1) 1.00 Cord pH,7.0 0 (0.0) 0 (0.0) 1 (0.1) 6 (0.5) 1 (0.1) 6 (0.5) 0.19 (0.02– 1.54) 0.13 Cord blood base deficit $12 mmol/l 0 (0.0) 0 (0.0) 1 (0.1) 9 (0.7) 1 (0.1) 9 (0.7) 0.13 (0.02– 1.06) 0.06 Stage 3 encephalopathy 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) NICU admission .4 d 0 (0.0) 0 (0.0) 4 (0.4) 7 (0.6) 4 (0.4) 7 (0.6) 0.64 (0.18– 2.25) 0.48 Severe lung disease 0 (0.0) 0 (0.0) 2 (0.2) 1 (0.1) 2 (0.2) 1 (0.1) 2.23 (0.20– 24.6) 0.61 Necrotising enterocolitis 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Proven systemic infection 0 (0.0) 0 (0.0) 1 (0.1) 4 (0.3) 1 (0.1) 4 (0.3) 0.28 (0.03– 2.49) 0.38 Planned VBAC Group (n = 1,237) Adjusted RR (95% CI)a aValues adjusted for maternal SES, BMI, and indication for previous caesarean birth. Values adjusted for maternal SES, BMI, and indication for previous caesarean birth. Secondary Outcomes There were no maternal deaths in this study. The risk of maternal death or serious morbidity for women in the planned ERC group was 3.1% (34/1,108) and 4.5% (56/1,237) for women in the planned VBAC group; this was not a statistically significant difference (RR 0.69, 95% CI 0.46–1.05, p = 0.08). When the individual components of the composite outcome were examined, significantly fewer women in the planned ERC group had a major haemorrhage (defined as blood loss .1, 500 ml and/or need for blood transfusion) when compared with planned VBAC (planned No perinatal deaths occurred among infants of mothers in the planned ERC group. There were two stillbirths in the planned Table 3. Secondary maternal outcomes by planned mode of birth. Table 3. Secondary maternal outcomes by planned mode of birth. Table 3. Secondary maternal outcomes by planned mode of birth. Sample Size bDeath or serious outcome includes: death (any fetal death after study entry or death of a liveborn infant prior to hospital discharge [excluding lethal congenital anomalies]); or serious neonatal morbidity (defined as one or more of: birth trauma [subdural or intracerebral haemorrhage, spinal cord injury, basal skull fracture, other fracture, peripheral nerve injury present at discharge from hospital]; seizures at ,24 h age or requiring two or more drugs to control; Apgar score ,4 at 5 min; cord pH,7?0; cord blood base deficit $12 mmol/l; neonatal encephalopathy stage 3; admission to NICU.4 d; severe neonatal lung disease [defined as MAP.10 and or FiO2.0?80 with need for ventilation]; proven necrotising enterocolitis; or proven systemic infection in first 48 h of life treated with antibiotics). NICU neonatal intensive care unit PLoS Medicine \| www.plosmedicine.org March 2012 \| Volume 9 \| Issue 3 \| e1001192 5 Planned VBAC or ERC: Health Outcomes VBAC group. Both infants were born at 39 wk. The cause of death after autopsy for both infants was unexplained stillbirth. liveborn infant death prior to discharge or serious outcomes in one infant was 66 (95% CI 40–200). When the individual components of the primary endpoint were examined there was a statistically significant reduction in the risk of serious morbidity for infants born to women in the planned ERC group, compared with infants born to women in the planned VBAC group (planned ERC 10/1,108 [0.9%] versus planned VBAC 28/1,235 [2.3%], RR 0.41, 95% CI 0.20–0.83, p = 0.014). There were no statistically significant differences identified for any of the other individual components of the primary outcome between the two treatment groups. Secondary Outcomes Outcome RCT Patient Preference Planned ERC Group (n = 1,108) Planned VBAC Group (n = 1,237) Adjusted RR (95% CI)a p- Value Planned ERC (n = 10) Planned VBAC (n = 12) Planned ERC (n = 1,098) Planned VBAC (n = 1,225) n Percent n Percent n Percent n Percent n Percent n Percent CI Range Serious maternal outcomeb 0 (0.0) 0 (0.0) 34 (3.1) 56 (4.6) 34 (3.1) 56 (4.5) 0.69 (0.46– 1.05) 0.08 Maternal death 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Uterine rupture 0 (0.0) 0 (0.0) 1 (0.1) 3 (0.2) 1 (0.1) 3 (0.2) 0.37 (0.04– 3.57) 0.63 Major haemorrhage .1500 ml 0 (0.0) 0 (0.0) 9 (0.8) 29 (2.4) 9 (0.8) 29 (2.3) 0.37 (0.17– 0.80) 0.011 Hysterectomy 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.1) 0 (0.0) 1 (0.1) 1.0 Vulval/perineal haematoma 0 (0.0) 0 (0.0) 1 (0.1) 2 (0.2) 1 (0.1) 2 (0.2) 0.56 (0.05– 6.15) 1.0 DVT requiring anticoagulation 0 (0.0) 0 (0.0) 1 (0.1) 0 (0.0) 1 (0.1) 0 (0.0) 0.47 PE requiring anticoagulation 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Pneumonia 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Adult respiratory distress 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Wound infection/ dehiscence 0 (0.0) 0 (0.0) 18 (1.6) 13 (1.1) 18 (1.6) 13 (1.1) 1.62 (0.77– 3.40) 0.20 Organ damage requiring repair 0 (0.0) 0 (0.0) 6 (0.5) 15 (1.2) 6 (0.5) 15 (1.2) 0.46 (0.17– 1.20) 0.11 Genital tract fistula 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.1) 0 (0.0) 1 (0.1) 1.0 Bowel obstruction/ileus 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Pulmonary oedema 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.1) 0 (0.0) 1 (0.1) 1.0 Stroke 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Cardiac arrest 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Respiratory arrest 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) aValues adjusted for maternal SES, BMI, and indication for previous caesarean birth. j , , p bDeath or serious maternal outcome includes: one or more of maternal death; uterine rupture (defined as a clinically significant rupture involving the full thickness of the uterine wall and requiring surgical repair); severe haemorrhage (blood loss of $1,500 ml and/or requiring blood transfusion); hysterectomy for any complications resulting from birth; vulval or perineal haematoma requiring evacuation; deep vein thrombosis (DVT) or thrombophlebitis requiring anticoagulant therapy; pulmonary embolus (PE) requiring anticoagulant therapy; pneumonia due to infection, aspiration or other causes; adult respiratory distress syndrome; wound infection (requiring prolongation of hospital stay or readmission) or wound dehiscence; damage to the bladder, ureter, or bowel requiring repair, or cervical laceration extending to the lower uterine segment, or abnormal extension of the uterine incision; occurrence of a fistula involving the genital tract; bowel obstruction or paralytic ileus; pulmonary oedema; stroke (defined as acute neurological deficit .24 h); cardiac arrest; respiratory arrest. doi:10.1371/journal.pmed.1001192.t003 Secondary Outcomes j p bDeath or serious maternal outcome includes: one or more of maternal death; uterine rupture (defined as a clinically significant rupture involving the full thickness of the uterine wall and requiring surgical repair); severe haemorrhage (blood loss of $1,500 ml and/or requiring blood transfusion); hysterectomy for any complications resulting from birth; vulval or perineal haematoma requiring evacuation; deep vein thrombosis (DVT) or thrombophlebitis requiring anticoagulant therapy; pulmonary embolus (PE) requiring anticoagulant therapy; pneumonia due to infection, aspiration or other causes; adult respiratory distress syndrome; wound infection (requiring prolongation of hospital stay or readmission) or wound dehiscence; damage to the bladder, ureter, or bowel requiring repair, or cervical laceration extending to the lower uterine segment, or abnormal extension of the uterine incision; occurrence of a fistula involving the genital tract; bowel obstruction or paralytic ileus; pulmonary oedema; stroke (defined as acute neurological deficit .24 h); cardiac arrest; respiratory arrest. doi:10.1371/journal.pmed.1001192.t003 PLoS Medicine \| www.plosmedicine.org March 2012 \| Volume 9 \| Issue 3 \| e1001192 6 Planned VBAC or ERC: Health Outcomes Table 4. Labour and birth outcomes. Secondary Outcomes Outcome n (%), Mean ± SD, or Median and Interquartile Range Adjusted RR (95% CI)a p-Value RCT Patient Preference Planned ERC Group (n = 1,108) Planned VBAC Group (n = 1,237) Planned ERC (n = 10) Planned VBAC (n = 12) Planned ERC (n = 1,098) Planned VBAC (n = 1,225) CI Range Gestational age at birth (wk) 39.260.7 40.061.0 38.860.7 40.061.1 38.860.7 40.061.1 21.1 (21.2 to 1.01) ,0.001 Induction of labour 0 (0.0) 2 (16.7) 4 (0.4) 151 (12.3) 4 (0.4) 153 (12.4) 0.03 (0.01–0.08) ,0.001 Mode of birth Vaginal birth 2 (20.0) 7 (58.3) 23 (2.1) 528 (43.1) 25 (2.3) 535 (43.2) 0.06 (0.04–0.09) ,0.001 Caesarean section 8 (80.0) 5 (41.7) 1,075 (97.9) 697 (56.9) 1,083 (97.7) 702 (56.8) 1.67 (1.59–1.75) ,0.001 Elective caesarean section 5 (50.0) 4 (33.3) 966 (88.0) 330 (26.9) 971 (87.6) 334 (27.0) 3.10 (2.82–3.42) ,0.001 Emergency caesarean section Preference arm 109 (9.9) 367 (30.0) 109 (9.9) 367 (30.0) 0.32 (0.27–0.40) ,0.001 Randomised arm 3 (30.0) 1 (8.3) 3 (30) 1 (8.3) 3.60 (0.44–29.5) 0.19 Analgesia/anaesthesia 9 (90.0) 12 (100) 1,080 (98.4) 1,148 (93.7) 1,089 (98.3) 1,160 (93.8) 1.04 (1.02–1.06) ,0.001 Epidural 3 (30.0) 4 (33.3) 183 (16.7) 425 (34.7) 186 (16.8) 429 (34.7) 0.47 (0.41–0.55) ,0.001 Postpartum haemorrhage .500 ml 2 (20.0) 2 (16.7) 184 (16.8) 214 (17.5) 186 (16.8) 216 (17.5) 0.91 (0.76–1.09) 0.30 Postpartum infection 0 (0.0) 0 (0.0) 4 (0.4) 3 (0.2) 4 (0.4) 3 (0.2) 1.49 (0.33–6.64) 0.71 Length of stay (d) 5 (3–5) 4 (3–5) 5 (4–6) 4 (3–5) 5 (4–6) 4 (3–5) ,0.001 Length of stay .7 d 0 (0.0) 0 (0.0) 60 (5.5) 62 (5.1) 60 (5.4) 62 (5.0) 1.08 (0.76–1.54) 0.67 Infant birthweight (g) 3,4016475 3,5346425 3,4626451 3,5716495 3,4626451 3,5716494 256 (2252 to 139.4) 0.57 Apgar score ,7 at 5 min 0 (0.0) 0 (0.0) 3 (0.3) 8 (0.7) 3 (0.3) 8 (0.6) 0.48 (0.12–1.86) 0.29 aValues adjusted for maternal SES, BMI, and indication for previous caesarean birth. RCT, randomised controlled trial. doi:10.1371/journal.pmed.1001192.t004 n (%), Mean ± SD, or Median and Interquartile Range ERC 9/1,108 [0.8%] versus planned VBAC 29/1,237 [2.3%], RR 0.37, 95% CI 0.17–0.80, p = 0.011, NNTB 66, 95% CI 40– 187). There were no statistically significant differences between the treatment groups for any of the other secondary outcomes (Table 3). serious outcome for the infant, when compared with women who planned VBAC. Secondary Outcomes For women planning a VBAC our 2.4% risk of death or serious outcome for the infant is similar to that reported from previous cohort studies that compared actual rather than planned mode of birth where risks ranged between 0.13% and 2.4% [4,9]. In the planned ERC group 1,083 (97.7%) women gave birth by caesarean section, with the majority (87.6%) as an elective procedure. In the planned VBAC group 535 (43.2%) women had a vaginal birth and 702 (56.8%) had a caesarean section; 334 (27.0%) as an elective and 368 (29.7%) as an emergency procedure (Table 4). The main indications for caesarean section in the planned VBAC group were previous caesarean section, failure to progress, and fetal distress. Women in the planned ERC group compared with women in the planned VBAC group gave birth at an earlier gestational age and although their median length of postnatal hospital stay was longer they were not more likely to stay more than 7 d (Table 4). There are a number of strengths to our study design, enhancing the validity of our results. To our knowledge, this is the first randomised trial to report on health outcomes in this setting. However, few women consented to the randomised trial, as was suggested likely by our pre-trial survey of women’s views [25]. Although the randomised controlled trial is regarded as the ‘‘gold standard’’ research methodology for assessing the effects of health care interventions, some research questions cannot be fully answered using this design, particularly where patients have strong treatment preferences, and decline randomisation as in our setting. Given our experience here and the recognised difficulty of recruitment to randomised trials related to VBAC [9,11], it seems unlikely that large randomised trials will be conducted, although these may still be possible in other health care settings. PLoS Medicine \| www.plosmedicine.org Birth After Caesarean Study Group: Birth After Caesarean Study Group: Study authors: CA Crowther, JM Dodd, JE Hiller, RR Haslam, JS Robinson; Study authors: CA Crowther, JM Dodd, JE Hiller, RR Haslam, JS Robinson; There is a well-documented increase in the risk of both perinatal mortality and infant morbidity with increasing gestational age beyond term [27,28]. The differences in the risk of death or serious outcome for infants born to women in the planned VBAC group could be related to the difference in gestational age at birth observed between the two groups, rather than planned mode of birth. The relationship between advancing gestational age and morbidity and mortality, and the optimal time of birth for women at term therefore warrants further prospective evaluation. Birth After Caesarean Study Group: Serious Adverse Events Committee Collaborators: TY Khong, JE Hiller; Writing Group: CA Crowther, JM Dodd, JE Hiller, RR Haslam, JS Robinson; Serious Adverse Events Committee Collaborators: TY Khong, JE Hiller; Writing Group: CA Crowther, JM Dodd, JE Hiller, RR Haslam, JS Robinson; Collaborators (by hospital; total number of women recruited from each site in parentheses), indicates named associate investigator for the NHMRC grant: Caboolture Hospital (Queensland) (117): R Alexander, L Cochrane, M Ratnapala, S Rehman; Lyell McEwin Hospital (South Australia) (107): G Dekker, S Kennedy-Andrews, C Pawley; Modbury Hospital (South Australia) (5): J Hillen, G Matthews, J Sieben; Nepean Hospital (New South Wales) (416): C Dunn, M Mongelli, M Peek, S Sellar; Redcliffe Hospital (Queensland) (73): A McCartney, M Shallcross, A Stamatiou; Royal North Shore Hospital (New South Wales) (312): J Milligan, J Morris, S Nettleton, K Rickard; Royal Prince Alfred Hospital (New South Wales) (143): A Child, H Phipps, J Ludlow, E Olive; The Mater Hospital, Brisbane (Queensland) (173): P Devenish-Meares, V Flenady, M Jell, S Jenkins-Manning, J MacPhail; The Royal Women’s Hospital, Brisbane (Queensland) (25): Y Chadha, R Heath, M Pritchard; The Royal Women’s Hospital, Melbourne (Victoria) (91): M Cram, L Kornman, S Oldfield; The Townsville Hospital (Queensland) (99): C Boniface, S Campbell, A Contarino, E Green, A Lawrence, D Watson; Toowoomba Base Hospital (Queensland) (66): M Cybulski, K Mahomed, J Thomae, V Volobuev; Westmead Hospital (New South Wales) (209): C Compton, D Fleming, T McGee, D Wilton; Women’s and Children’s Hospital (South Australia) (509): S Agett, B Antonas, P Ashwood, V Ball, H Campbell, CA Crowther, H Dent, A Deussen, JM Dodd, L Evele, M Ewens, K Henderson, JE Hiller, C Holst, Y Khong, D McCormack, P Although statistically significant, overall the absolute risk difference in adverse health outcomes between the two forms of care remains small. Nevertheless, these small differences in the risk of short-term adverse health outcomes, either for the women or for their infants, are likely to be of considerable importance to the women, and therefore influence their choice of preferred mode of birth [29]. Absolute risk differences (related to mode of birth) may vary by factor, such as previous successful vaginal birth, where planned VBAC is more likely to result in vaginal birth. Discussion In this study involving women with a single prior caesarean who had reached 37 wk gestation in their next pregnancy, and who did not have a contraindication to a planned VBAC, a plan to birth by ERC was associated with a beneficial reduction in the risk of fetal death or liveborn infant death prior to discharge or This is the first study designed around women’s planned preferences for birth after caesarean, among women who were eligible for a VBAC, and therefore provides a high quality estimate of the benefits and harms associated with the two planned or March 2012 \| Volume 9 \| Issue 3 \| e1001192 March 2012 \| Volume 9 \| Issue 3 \| e1001192 7 Planned VBAC or ERC: Health Outcomes either VBAC or ERC should be included in the counselling provided to women to assist with their decision making. either VBAC or ERC should be included in the counselling provided to women to assist with their decision making. intended treatment choices for birth, not previously reported. Most of the previous evidence has been based on comparison of actual mode of birth where groups were less comparable [11]. Information relating to health outcomes for women and their infants is known for all 2,345 women enrolled in the study, and our sample size was sufficiently large to allow us to detect important, small differences between the two policies for care. There is a need to establish whether the identified short-term benefits in health and wellbeing persist or are balanced by later risks. Therefore the evaluation of longer-term health, for both the women and children in this study, will be important. Our planned longer-term follow-up at early school age will assess maternal and child health as well as outcome in subsequent pregnancies including the risk from multiple caesareans, such as placenta praevia and accreta, and fertility [11,12]. Flaws identified in the literature related to the risks and benefits of planned ERC and planned VBAC have included a lack of comparability of groups, specifically being unclear whether women included in the ERC group were truly eligible to attempt VBAC [10]. Our study methodology ensured that all women recruited were considered eligible to attempt VBAC, on the basis of current clinical practice guidelines, as assessed by qualified staff at the time of study entry. Discussion We made a comprehensive assessment of known confounders with statistical adjustments for minor imbalances found between treatment groups for BMI, SES, and indication for previous caesarean. Nevertheless, unmeasured confounding may still account in part for the study findings. Our intention-to-treat analysis ensured that the study evaluated a policy of choice around planned ERC and planned VBAC. Our ‘‘restricted’’ prospective cohort study design used method- ological features of high quality randomised trials, which included identification of a ‘‘zero time’’ for determining eligibility, study entry, and baseline characteristics; use of inclusion and exclusion criteria; treatment protocols derived from evidence-based clinical practice guidelines; standardised definitions for clinical outcomes; adjustment for imbalance in confounders at study entry; and the use of intention-to-treat analyses [30,31]. Our results, whilst not generalisable for other populations, indicate that for women who have had one previous caesarean birth and are considered eligible at term to attempt a planned VBAC in their next pregnancy, an ERC as planned mode of birth is significantly associated with a lower risk of both fetal death or liveborn infant death prior to discharge or serious infant morbidity and major maternal haemorrhage without increasing other maternal and perinatal complications. Women, clinicians, and policy makers can use this information to develop health advice to assist in making evidence-based decisions about care for women who have had a previous caesarean and their infants. We identified no increase in the risk of short-term maternal morbidity related to planned ERC, but rather a beneficial reduction in the risk of major maternal haemorrhage and/or the need for blood transfusion compared with planned VBAC. This finding is in sharp contrast to other reports where ERC has been associated with an increase in maternal blood loss [4,26] and justifies further study. The risk of symptomatic uterine scar rupture was low for both treatment groups being 0.1% for women in the planned ERC group and 0.2% for women in the planned VBAC group. This risk of uterine rupture related to VBAC is lower than that reported from the NICHD cohort study of 0.7% [4] and lower than the rate of symptomatic uterine scar rupture of 1.2/100 to 3.9/1,000 among women having a VBAC reported in systematic reviews of other cohort studies [9–11]. The standardised treatment schedules for VBAC and ERC, based on relevant evidence-based clinical practice guidelines used by all participating hospitals, may account for the low rates observed. PLoS Medicine \| www.plosmedicine.org References Dodd JM, Crowther CA, Huertas E, Guise JM, Horey D (2004) Planned elective repeat caesarean section versus planned vaginal birth for women with a previous caesarean birth. Cochrane Database Syst Rev 4: CD004224. 25. Dodd J, Pearce E, Crowther C (2004) Women’s experiences and preferences following Caesarean birth. Aust N Z J Obstet Gynaecol 44: 521–524. 26. Mozurkewich EL, Hutton EK (2000) Elective repeat cesarean delivery versus trial of labor: a meta-analysis of the literature from 1989 to 1999. Am J Obstet Gynecol 183: 1187–1197. y 10. Guise JM, McDonagh MS, Hashima J, Kraemer DF, Eden KB, et al. (2003 Mar) Vaginal birth after cesarean (VBAC). Evid Rep Technol Assess (Summ) 71: 1–8. 27. Gulmezoglu AM, Crowther CA, Middleton P (2006) Induction of labour for improving birth outcomes for women at or beyond term. Cochrane Database Syst Rev 4: CD004945. 11. Guise JM, Eden K, Emeis C, Denman MA, Marshall N, et al. (2010) Vaginal birth after cesarean: new insights. Evid Rep Technol Assess (Full Rep) 191: 1–397. 28. Heimstad R, Romundstad PR, Eik-Nes SH, Salvesen KA (2006) Outcomes of pregnancy beyond 37 weeks of gestation. Obstet Gynecol 108: 500–508. 12. Royal College Of Obstetricians and Gynaecologists (RCOG) (2007) Birth after caesarean birth: Green-top guideline no 45. London: RCOG Press. caesarean birth: Green-top guideline no 45. London: RCOG Press 29. Walker SP, McCarthy EA, Ugoni A, Lee A, Lim S, et al. (2007) Cesarean delivery or vaginal birth: a survey of patient and clinician thresholds. Obstet Gynecol 109: 67–72. 13. Dodd JM, Crowther CA, Hiller JE, Haslam RR, Robinson JS (2007) Birth after caesarean study–planned vaginal birth or planned elective repeat caesarean for women at term with a single previous caesarean birth: protocol for a patient preference study and randomised trial. BMC Pregnancy Childbirth 7: 17. 30. Solomon MJ, McLeod RS (1998) Surgery and the randomised controlled trial: past, present and future. Med J Aust 169: 380–383. 14. American College of Obstetricians and Gynecologists (1999) ACOG Practice Bulletin: vaginal birth after previous cesarean delivery. Number 5, July 1999 (replaces practice bulletin number 2, October 1998). Clinical management guidelines for obstetrician-gynecologists. American College of Obstetricians and Gynecologists. Int J Gynaecol Obstet 66: 197–204. 31. Horwitz RI, Viscoli CM, Clemens JD, Sadock RT (1990) Developing improved observational methods for evaluating therapeutic effectiveness. Am J Med 89: 630–638. 32. References 18. Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) (2002) Caesarean section - a guide for women. Camberwell, Australia: Mi-tec Publishing. 1. Stone C, Halliday J, Lumley J, Brennecke S (2000) Vaginal births after Caesarean (VBAC): a population study. Paediatr Perinat Epidemiol 14: 340–348. 2. Cowan RK, Kinch RAH, Ellis B, Anderson R (1994) Trial of labor following cesarean delivery. Obstet Gynecol 83: 933–936. 19. Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) (2002) Vaginal birth after caesarean section – a guide for women. Camberwell, Australia: Mi-tec Publishing. 3. Hamilton BE, Martin JA, J. VS (2009) Births: preliminary data for 2007. Natl Vital Stat Rep 57: 1–23. 20. American College of Obstetricians and Gynecologists (2002) Committee opinion: induction of labor for vaginal birth after cesarean delivery. Obstet Gynecol 99: 679–680. 4. Landon MB, Hauth JC, Leveno KJ, Spong CY, Leindecker S, et al. (2004) Maternal and perinatal outcomes associated with a trial of labor after prior cesarean delivery. N Engl J Med 351: 2581–2589. 21. Royal College Of Obstetricians and Gynecologists (RCOG) (2001) Evidence- based clinical guideline number 8: the use of electronic fetal monitoring. London: RCOG Press. 5. Lydon-Rochelle M, Holt VL, Easterling TR, Martin DP (2001) Risk of uterine rupture during labor among women with a prior cesarean delivery. N Engl J Med 345: 3–8. 22. Royal College Of Obstetricians and Gyanecologists (RCOG) (2001) Evidence- based clinical guideline number 9: induction of labour. London: RCOG Press. 6. Smith GCS, Pell JP, Cameron AD, Dobbie R (2002) Risk of perinatal death associated with labor after previous cesarean delivery in uncomplicated term pregnancies. JAMA 287: 2684–2690. g 23. Newcombe RG (1998) Interval estimation for the difference between independent proportions: Comparison of eleven methods. Stat Med 17: 873–890. p g J 7. Laws P, Li Z, Sullivan EA (2010) Australia’s mothers and babies 2008. Perinatal statistics series no. 24. Cat. no. PER 50. Canberra: AIHW. 24. Hannah ME, Hannah WJ, Hewson SA, Hodnett ED, Saigal S, et al. (2000) Planned caesarean section versus planned vaginal birth for breech presentation at term: a randomised multicentre trial. Lancet 356: 1375–1383. l College Of Obstetricians and Gynaecologists (RCOG) (2001) 8. Royal College Of Obstetricians and Gynaecologists (RCOG) (2001) The National Sentinel Caesarean Section Audit Report. London: RCOG Press. onal Sentinel Caesarean Section Audit Report. London: RCOG Pres 9. Birth After Caesarean Study Group: Similar proportions of women in each treatment group had achieved a previous vaginal birth prior to the caesarean (13% in the planned ERC group and 15.6% in the planned VBAC group). There have been no studies comparing the risks and benefits of VBAC with ERC that have reported on the health outcomes beyond the neonatal period. This lack of information on long-term health of March 2012 \| Volume 9 \| Issue 3 \| e1001192 8 Planned VBAC or ERC: Health Outcomes Planned VBAC or ERC: Health Outcomes JMD JEH RRH JSR. Contributed reagents/materials/analysis tools: CAC JMD JEH RRH JSR. Wrote the first draft of the manuscript: CAC. Contributed to the writing of the manuscript: CAC JMD JEH RRH JSR. ICMJE criteria for authorship read and met: CAC JMD JEH RRH JSR. Agree with manuscript results and conclusions: CAC JMD JEH RRH JSR. Obtained funding: CAC JMD JEH RRH JSR. Muller, H Oakey, E Pearce, B Peat, JS Robinson, K Robinson, S Russell, J Svigos, R Sweet, J Thomas, N Thomas, C Wilkinson, K Willson. Muller, H Oakey, E Pearce, B Peat, JS Robinson, K Robinson, S Russell, J Svigos, R Sweet, J Thomas, N Thomas, C Wilkinson, K Willson. We are indebted to the 2,345 women who participated in this research study. We are indebted to the 2,345 women who participated in this research study. Author Contributions Conceived and designed the experiments: CAC JMD JEH RRH JSR. P f d h i CAC JMD RRH A l d h d CAC Conceived and designed the experiments: CAC JMD JEH RRH JSR. Performed the experiments: CAC JMD RRH. Analyzed the data: CAC Conceived and designed the experiments: CAC JMD JEH RRH JSR. Performed the experiments: CAC JMD RRH. Analyzed the data: CAC g p J J J Performed the experiments: CAC JMD RRH. Analyzed the data: CAC 34. Ware JE, Sherbourne CD (1992) The MOS 36-Item Short-Form Health Survey (Sf-36). Conceptual-Framework and Item Selection. Med Care 30: 473–483. Editors’ Summary Most obviously, having a surgical procedure puts the woman having the repeat caesarean section at risk of surgical complications, especially if performed under a general anesthetic, and her baby may be at risk of respiratory complications. However, subsequent vaginal delivery following a previous caesarean section may put the mother at risk of bleeding severely enough to need a blood transfusion (more than 1,500 ml blood loss) and she may also be at increased risk of rupturing her uterus; and her baby may have an increased risk of dying or of becoming brain damaged due to lack of oxygen. What Do These Findings Mean? These findings show that in women who had delivered by a previous caesarean section delivering their next baby by planned caesarean section was associated with less infant death and better health outcomes for the mother before she was discharged from the hospital compared to women who had a subsequent vaginal delivery. This information can be used by women, clinicians, and policy makers in helping to make decisions about the mode of subsequent deliveries and best care for women who have had a previous caesarean section. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10. 1371/journal.pmed.1001192. However, to date there have been no randomized controlled trials of elective repeat caesarean section versus vaginal delivery following a previous caesarean section to compare the health outcomes of mother and baby and a recent systematic review could draw no conclusions. So the researchers conducted this prospective cohort study based on patient preference (with a few women agreeing to be randomized to mode of delivery), to compare the health outcomes for mother and baby for elective repeat caesarean section versus vaginal delivery in women following a previous caesarean section. However, to date there have been no randomized controlled trials of elective repeat caesarean section versus vaginal delivery following a previous caesarean section to compare the health outcomes of mother and baby and a recent systematic review could draw no conclusions. So the researchers conducted this prospective cohort study based on patient preference (with a few women agreeing to be randomized to mode of delivery), to compare the health outcomes for mother and baby for elective repeat caesarean section versus vaginal delivery in women following a previous caesarean section. Editors’ Summary on the woman’s preferences for mode of birth, participating obstetricians either scheduled a date for an elective caesarean section (1,098 women) or assessed on-going suitability for the woman to have a planned vaginal delivery (1,225 women). However only 535 (43.2%) women who chose to have a vaginal birth were able to deliver this way because of failure to progress in labor or fetal distress: 334 of these women (27.0%) had to have an elective caesarean section and 368 women had to have an emergency caesarean section. Background. Rates of caesarean section are rising around the world, particularly in high- and middle-income countries, where most women have a choice of how their baby is delivered. Historically, the obstetrician in charge of the woman’s care made the decision on whether to perform an elective (planned) caesarean section based on medical criteria. For women who have had a previous caesarean section, typically, their options for mode of childbirth are either a trial of vaginal birth or an elective repeat caesarean section. The proportion of women attempting a vaginal birth after a previous caesarean section has been declining in many countries partly due to the variable chance of achieving a successful vaginal birth (reported between 56% and 80%) and partly because of negative reports of the risk of complications, both to the mother and the baby, of a having a vaginal delivery following a caesarean section. Consequently, the rates of repeat caesarean section have risen sharply, for example, currently 83% in Australia and almost 90% in the US. Although no women died, women who had a planned caesarean section experienced less severe bleeding than women who delivered vaginally. There were no infant deaths in those born by elective caesarean section but two unexplained stillbirths in the planned vaginal delivery group. There was also a reduced risk of nonfatal serious outcome before discharge from hospital for infants delivered by in the elective caesarean section. The researchers calculated that one infant death or near death would be prevented for every 66 elective caesarean sections performed in women who had a previous caesarean section. Why Was This Study Done? Both elective repeat caesarean section and subsequent vaginal delivery after a previous caesarean section have clinical risks and benefits. References Cox JL, Holden JM, Sagovsky R (1987) Detection of postnatal depression - development of the 10-item Edinburgh postnatal depression scale. Br J Psychiatry 150: 782–786. y g J y 15. Institute for Clinical Systems Improvement (ICSI) (2001) Vaginal birth after cesarean. Bloomington (Minnesota): ICSI. cesarean. Bloomington (Minnesota): ICSI. 33. Marteau TM, Bekker H (1992) The development of a six-item short-form of the state scale of the Spielberger State-Trait Anxiety Inventory (STAI). Br J Clin Psychol 31(Pt 3): 301–306. 16. National Institute for Clinical Effectiveness (NICE) (2004) Caesarean section. London: RCOG Press. 17. Society of Obstetricians and Gynecologists of Canada (2005) Clinical Practice Guidelines: Guidelines for vaginal birth after previous caesarean birth. Int J Gynaecol Obstet 89: 319–331. 34. Ware JE, Sherbourne CD (1992) The MOS 36-Item Short-Form Health Survey (Sf-36). Conceptual-Framework and Item Selection. Med Care 30: 473–483. PLoS Medicine \| www.plosmedicine.org March 2012 \| Volume 9 \| Issue 3 \| e1001192 9 Planned VBAC or ERC: Health Outcomes Editors’ Summary Editors’ Summary N This study is linked to a PLoS Medicine Research Article by Fitzpatrick and colleagues and a PLoS Medicine Perspective by Catherine Spong N The American Congress of Obstetricians and Gynecologists has information sheets for patients on caesarean sections and on vaginal birth after caesarean delivery N Childbirth Connection, a US-based not-for-profit organiza- tion, provides information about caesarean sections and about vaginal birth after caesarean What Did the Researchers Do and Find? Between 2002 and 2007, the researchers recruited 2,345 suitable women (that is, women who had one previous caesarean section, were currently 37 weeks pregnant with a single baby, and who were clinically able to have a vaginal delivery) from 14 maternity hospitals throughout Australia. A few women (22) agreed to be randomized to either mode of delivery but most women chose her preferred option. Then, depending N The National Childbirth Trust, a UK charity, provides information for parents on all aspects of pregnancy and birth, including caesarean sections and vaginal birth after caesarean delivery N The UK charity Healthtalkonline has personal stories from women making decisions about birth after a caesarean section PLoS Medicine \| www.plosmedicine.org March 2012 \| Volume 9 \| Issue 3 \| e1001192 PLoS Medicine \| www.plosmedicine.org 10
https://openalex.org/W3173353230	https://www.researchsquare.com/article/rs-596604/latest.pdf	English	null	Chinese Aid Guyana Medical Team Member HAMA Under the COVID-19 Epidemic Correlation Study with TCSQ	Research Square (Research Square)	2,021	cc-by	4,222	Chinese Aid Guyana Medical Team Member HAMA Under the COVID-19 Epidemic Correlation Study with TCSQ Jin Zhengxi (  550807853@qq.com ) Nanjing Medical University Zhang Xiangxin Nanjing Medical University Qiao Zhiming Nanjing Medical University Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Chinese aid Guyana medical team member HAMA under the COVID-19 epidemic correlation study with TCSQ Jin Zhengxi1 Zhang Xiangxin2 Qiao Zhiming3 1. The first author and corresponding author, Department of Nephrology, The Affiliated Suzhou Hospital of Nanjing Medical University(Suzhou Municipal Hospital),242 Guangji Road, Suzhou City, Jiangsu Province, China. 550807853@qq.com 2. Co-corresponding author, Department of Orthopedics, The Affiliated Suzhou Hospital of Nanjing Medical University(Suzhou Municipal Hospital), Email: lovezxx2003@qq.com 3.Department of Surgery, The Affiliated Suzhou Hospital of Nanjing Medical University(Suzhou Municipal Hospital) , Email: zhiming_qiao@163.com [Summary] [Purpose]: To understand the mental health level (HAMA) and trait coping style (TCSQ) of the Chinese aid Guyana medical team members (CAGMTM) under the new coronavirus (COVID-19) epidemic, in order to take effective measures to improve the CAGMTM Mental health provides a theoretical basis. [Method]: From October 2020 to March 2021, the team members have worked and lived in Guyana for 6 months. The mental health level (Hamilton Anxiety Scale, HAMA) and trait coping styles were investigated for 16 team members who aided Guyana. [Results]: The HAMA score of the CAGMTM was (14.75±5.12); 14 doctors had a score of (15.57+6.01); 2 chefs had a score of (17.5+7.78). The results of multivariate regression analysis showed that among the CAGMTM. Whether there are team members in isolation (t=3.383, p<0.05), and whether any medical staff in the hospital where the CAGMTM are located have tested positive for COVID-19 (t=-2.831, p<0.05) are important factors affecting the mental health of the team members during the epidemic. factor. The score of active coping in the coping style is (29.38±6.28) points, which is lower than the Chinese conventional model (t=-0.392, p<0.05); the negative coping score is (30.37±5.05) points, which is higher than the Chinese conventional model (t=4.908) , p<0.05); the HAMA score of the CAGMTM in the COVID-19 epidemic was negatively correlated with the positive response in TCSQ (r=-0.654, p<0.05); the HAMA score was positively correlated with the negative response in TCSQ (r=0.654) ,p<0.05). [Conclusion]: The mental health level of the CAGMTM under the COVID-19 epidemic is closely related to their idiosyncratic coping styles. For foreign aid medical team members working in countries with severe epidemics, attention should be paid to the mental health of foreign aid medical team members, timely intervention should be given, and psychological counseling should be carried out regularly, so that the aid team members can work and live better. [Keywords] COVID-19; foreign aid medical treatment; Chinese aid Guyana medical team members; mental health; coping style. The WHO declared the outbreak of COVID-19[1] infection in China as a "Public Health Emergency of International Concern"[2] on January 30, 2020. After China has actively mobilized and organized the people across the country to fight COVID-19 scientifically, dynamically and effectively, the epidemic has been brought under control. However, due to different levels of attention and prevention and control measures in other countries, the COVID-19 pandemic has resulted. (2)Past or current mental illness. A total of 16 CAGMTM were included in this study, with an age distribution ranging from 34 to 49 (42.50±4.44) years; education level: 2 junior college students, 14 undergraduates and above; Aid hospital level: 11 people are at national level and 5 people are at the regional level; Isolation of team members: 11 people are isolated, 5 people are not isolated; whether the hospital is admitted to treat COVID-19 patients: yes 11 people, no 5 people; whether the doctor's department has a colleague who has confirmed COVID-19 positive status: Yes 9 people , No 5 people. [Summary] In this context, The 16th Chinese Medical Team Aid to Guyana arrived in Guyana on October 1, 2020 for medical assistance. At the same time, it imparted China's COVID-19 prevention and anti-epidemic experience and donated medicine equipment and anti-epidemic materials. Due to the limited capacity of local nucleic acid testing, national nucleic acid testing cannot be performed. Medical staff are at greater risk of contacting COVID-19 patients and infection during diagnosis and treatment, and they are extremely prone to cross-infection and occupational exposure. The infection of COVID-19 among medical staff in the department puts medical team members under tremendous psychological pressure in their daily stressful and arduous work, which will affect their physical and mental health and the quality of clinical work, and even affect their ability to cope with work. This study aims to explore the correlation between the mental health level of the aid team members in the COVID-19 epidemic and their idiosyncratic coping styles. Provide theoretical basis for CAGMTM to carry out psychological intervention and improve work quality. 1.1 Survey object In March 2021, this study selected 16 members (14 doctors and 2 chefs) of the 16th China-aided Guyana Medical Team for investigation. Inclusion criteria: (1)Selected by the Jiangsu Provincial Office of International Cooperation and Exchange, the doctor has a doctor's qualification certificate, the chef has a cook certificate, and has no criminal record, and the doctor has passed the medical council assessment in Guyana and obtained the local practicing qualification certificate. (2)No mental illness or major physical illness. (3)Informed consent to this research, voluntarily join the research project. 1.2 Survey method 1.2.1 General information questionnaire items include gender, age, marital status, education level, job title, hospital level, health status, whether there are team members isolated, whether it is a national designated hospital for COVID-19, and whether colleagues in the department have COVID- 19 positive cases. Exclusion criteria: (1)Severe physical illness; (2)Past or current mental illness. 1.2.3 Trait Coping Style Questionnaire Trait coping style questionnaire (TCSQ) consists of 20 items, including 10 items for positive response and 10 items for negative response. The questionnaire adopts a five-level scoring method, with scores of 5, 4, 3, 2, and 1 respectively indicating complete agreement, agreement, neutrality, disagreement, and complete disagreement. It is used to reflect the subjects' positive and negative attitudes and behavior characteristics when facing difficulties and frustrations.A high score for active coping reflects that the active coping characteristic is obvious, and a high score for negative coping reflects that the negative coping characteristic is obvious. The scale for positive response to Cronbach’s α is 0.89, and negative response to Cronbach’s α is 0.78, which has good reliability. 1.2.2 Hamilton Anxiety Scale Hamilton Anxiety Scale (HAMA)[3] is often used clinically to diagnose and classify anxiety disorders. All items on the scale adopt a 5-level scoring method from 0 to 4 points. The scoring standards at all levels are: 0 points: asymptomatic; 1 point: mild; 2 points: moderate; 3 points: severe; 4 points: extremely severe. The higher the score, the more severe the anxiety symptoms. According to the information provided by the Chinese Scale Cooperation Group[4], The total score of the scale is ≥29 points, which may be severe anxiety; the total score ≥21 points, there must be obvious anxiety; the total score ≥14 points, there must be anxiety; >7 points, there may be anxiety; <7 points, there is no anxiety symptoms. The Cronbach’s α coefficient of the scale is 0.835, which has good reliability. 1.3 Statistical methods SPSS 26.0 statistical software was used for data analysis, general data was described by frequency, quantitative data was described by ( x±S), and the comparison between the sample and the domestic regular model was constructed by t-test. The correlation between mental health level and trait coping style was Pearson correlation For sex analysis, the test level α is set to 0.05 unless otherwise specified, with a two-sided probability. 2 Results 2 Results The total HAMA score of the CAGMTM is (14.75±5.12) points. Items 1 to 6 and 14 are mental anxiety with a score of (12.88±4.06); items 7 to 13 are physical anxiety with a score of (1.88±2.80). 0 were severely anxious, 2 were obviously anxious, scored (25.50±3.54), and 9 were mildly anxious, scored (15.11±1.27). CAGMTM A multi-factor linear regression analysis of the mental health status of the CAGMTM, taking the anxiety of the CAGMTM as the dependent variable, and whether the CAGMTM are isolated and whether a colleague in the department is diagnosed with COVID-19 as the independent variables. Regression analysis showed that the difference was statistically significant (p<0.05). The adjusted R-square, ∆R2=0.665 (Table 2). Table 2 Regression analysis of HAMA influencing factors of the CAGMTM Table 2 Regression analysis of HAMA influencing factors of the CAGMTM Influencing factors B SE Beta t p R square ∆R square Teammates isolated or not? 6.286 1.858 0.549 3.383* 0.005 0.709 0.665 Department colleagues positive or not? -4.914 1.736 -0.460 -2.831* 0.014 Note: p<0.05，∆R2=0.665 2.2 Single factor analysis of mental health status scores of CAGMTM Whether the CAGMTM are isolated, whether there are colleagues in the department where the COVID-19 is diagnosed and HAMA scores are compared, the difference is statistically significant (p<0.05). The other team members who are quarantined around the team tend to have a higher tendency to score (13.45±2.91), and the department has a colleague with a positive score of COVID-19 (16.88±4.67) (Table 1, t-test). Table 1 Single factor analysis of the scores of mental health status of the CAGMTM Project n HAMA F p Age 34-40 7 16.14±5.58 1.368 0.323 41-49 9 13.67±4.77 Hospital level National level 10 13.6±3.03 7.722 0.0042 Regional level 4 16.25±8.42 Table 1 Single factor analysis of the scores of mental health status of the CAGMTM Note: p<0.05 2.3 Regression analysis of influencing factors of mental health status of the Education background Junior college 2 17.5±7.78 2.501 0.1179 Bachelor or above 14 14.36±4.92 Teammate isolated or not? Yes 11 13.45±2.91 7.351 0.0030* No 5 17.6±7.89 Department colleagues covid positive or not? Have 9 15.11±5.9 6.303 0.0283* No 5 13.0±2.35 2.4 TCSQ scores of the CAGMTM (x±S) The TCSQ scores of the CAGMTM were compared with the Chinese norm. Their positive response (29.38±6.28) score was lower than the Chinese norm (30.26±8.74); the negative response (30.37±5.05) score was higher than the Chinese norm ( 21.25±7.41) points, the difference was statistically significant (p<0.05) (Table 3). Table 3 TCQS scores ofthe CAGMTM (x±s) Project n Positive response Negative response CAGMTM 16 29.38±6.28 30.37±5.05 Domestic norm 1305 30.26±8.74 21.25±7.41 t -0.392 4.908 p 0.0457* 0.0364* Note: p<0.05 Table 3 TCQS scores ofthe CAGMTM (x±s) 3 Discussion The Cooperative Republic of Guyana is located in the northern part of South America. South America currently has a large number of people infected with COVID-19, and the exchange of people between local residents and surrounding countries affects epidemic control; at the same time, Guyana is one of the countries with the highest immigration rate in the world, with an immigration rate of more than 55 %, due to the epidemic, many Guyanese who have immigrated abroad returned to the country to avoid the epidemic, further aggravating the epidemic in their country. A total of 16 members of the Chinese medical aid team to Guyana, 11 of them assisted the Georgetown Hospital, Guyana's largest national public hospital, and 5 assisted the Linden Hospital, the second largest public hospital in Guyana. Research[5] confirms that most people will have varying degrees of physical and mental stress response when facing work pressure. If they do not adjust in time, they may experience boredom and tiredness at work. Difficulty in exerting subjective initiative, leading to reduced work quality. Different coping styles can affect the development results of things, and even change the emotional state of people, and play an important role in regulating physical and mental health[6]. 2.5 Pearson's correlation analysis between the total scores of HAMA and the total scores of coping styles 2.5 Pearson's correlation analysis between the total scores of HAMA and the total scores of coping styles The HAMA total score of the CAGMTM was significantly correlated with the coping style, the positive coping style was negatively correlated with the anxiety total score (r=-0.654, p<0.05), and the negative coping style was positively correlated with the anxiety total score (r= 0.654, p<0.05), in the face of COVID-19, between the anxiety and coping styles of the CAGMTM, there is a negative correlation between active coping and mental anxiety (p<0.05),Negative coping and mental anxiety were positively correlated, and the difference was statistically significant (p<0.05) (Table 4). 4). Table 4 Correlation between total scores of the CAGMTM' HAMA and total scores of coping styles Project Positive response Negative response Mental anxiety Physical anxiety HAMA total score Positive response 1 -0.480 -0.627* -0.285 -0.654 p 0 0.06 0.009 0.285 0.006 Negative response -0.480 1 0.754 0.103 0.654 p 0.06 0 0.001 0.705 0.006 Table 4 Correlation between total scores of the CAGMTM' HAMA and total scores of coping styles Mental anxiety -0.627 0.754 1 0.081 0.838 p 0.009 0.001 0 0.767 0 Physical anxiety -0.285 0.103 0.081 1 0.611 p 0.285 0.705 0.767 0 0.012 HAMA total score -0.654 0.654 0.838 0.611* 1 p 0.006 0.006 0 0.012 0 Note: *p<0.01, p<0.05. Note: *p<0.01, p<0.05. 3.1 Mental health status of the CAGMTM The results of this study showed that the total HAMA score of the CAGMTM was (14.75±5.12), of which 68.8% were anxious team members, and those without severe anxiety accounted for 12.5% of obvious anxiety and 56.3% of mild anxiety, which was higher than Liu Liying[7] research and report the data of foreign aid team members facing the Ebola virus epidemic. Anxiety is a negative emotional state[8] subjectively felt by an individual, and it is one of the common psychological obstacles faced by medical staff in the face of COVID-19[9]. Each member of the CAGMTM is located in a different department. In addition to contacting patients, they have close contact with local doctors and nurses at work. Nurses take buses to and from get off work. Local buses are also called minibuses. They are small in size and have many passengers. Both increase the probability of potential infection, and also increase the risk of cross-infection and occupational exposure. These risk factors will directly or indirectly affect the physical and mental health of the CAGMTM. Due to the special nature of the work of medical staff, the CAGMTM in the hospital have high risks and heavy responsibilities. They face high-intensity work during the epidemic.Latent anxiety can have a negative psychological impact. In assisting Guyana, team members should strengthen contact and share difficult cases and unsolvable problems encountered in work; teammates discuss and multi-disciplinary consultations to face and solve problems together, and relieve pressure and work in the work. anxiety. In life, the emotional communication between team members should be strengthened, and mutual care should be taken. Help each other, discover and eliminate the bad emotions of teammates in time, and protect the physical and mental health of the team members. 3.2 The influencing factors of the mental health of the CAGMTM The results of multivariate regression analysis showed that whether the CAGMTM are isolated and whether there are COVID-19-positive medical staff in the doctor's department are important factors affecting the mental health of the CAGMTM in the epidemic. The reason for the analysis may be ①Quarantine teammates increase other teammates’ self-infection concerns; ②The isolation of team members increases teammates’ thoughts of their family members and worries about their future work; ③For team members whose doctors have infections from medical staff in the same department in the hospital, they will consider that the risk of infection is significantly increased, and they are afraid of work; ④The infection of medical staff in the team's department will lead to a shortage of department staff, workload and work pressure; the above reasons will all lead to the psychological pressure of medical team members increased significantly, and negative emotions such as anxiety were extremely easy to appear. In the special period of the global epidemic of COVID-19, attention should be paid to the mental health of the CAGMTM, coordinated with the hospital to conduct nucleic acid tests in a timely manner, reasonably adjust the diagnosis and treatment shifts, and regularly invite psychologists to perform decompression and psychological counseling, and analyze the reasons for their existence. Look for positive ways to cope, and actively respond to reduce work stress by directly and indirectly affecting negative coping[10]. Play an effective intervention for the mental health of the CAGMTM, strengthen the psychological endurance and adaptability, keep a good and positive attitude, and improve the CAGMTM' psychological rescue ability in the face of the outbreak of public health incidents. 3.3 The situation of the response methods of the CAGMTM Coping style is the cognitive or behavioral effort that everyone makes to reduce stress or injury when facing a stressful environment. It is the result of the interaction between individual stability factors and situational factors. The results of this study show that the active response score (29.38±6.28) of the Chinese aided medical team in the COVID-19 epidemic is lower than the Chinese norm[11] (30.26±8.74); the passive response score (30.37±5.05) is higher than that of China Normal mode (21.25±7.41) points, which are consistent with the results of Zhou Zhenhua[12] in the face of SARS. Research[9]shows that in the COVID-19 epidemic, the length of working hours of clinical front-line staff and the amount of contact with patients during treatment are clearly related to the severity of anxiety. Under the heavy work and special environment of this investigation, the CAGMTM gradually became anxious with the prolonged working hours of foreign aid, the active response gradually weakened, and the passive coping style gradually increased. The results of Valizadeh[13] showed that negative coping can bring more negative emotional experiences, such as pessimism, anxiety, fatigue and so on. This reminds us that in the face of the COVID-19 epidemic, we should strengthen the prevention and control of hospital infections, the rescue of critically ill patients, the learning of professional knowledge and skills, establish a safe and efficient work process, and improve work efficiency under the premise of ensuring the safety of self-protection, and provide rich diagnosis and treatment. Experience and strong professional skills are applied to practice; strengthen teamwork spirit, actively provide psychological counseling, relieve work pressure, increase work enthusiasm, and respond to public health incidents with a positive attitude. 3.4 Correlation between mental health level and coping styles of the CAGMTM 3.4 Correlation between mental health level and coping styles of the CAGMTM CAGMTM Coping styles play a mediating role in stressful events and mental health, and there is a correlation with mental health[14]. The results of this study showed that the total HAMA score of the CAGMTM was significantly correlated with TCQS, and the positive coping score was negatively correlated with HAMA (p<0.05); the negative coping score was positively correlated with HAMA (p<0.05), and it was positively correlated with Jiang Ganjin[15]. The research results are consistent.When the CAGMTM first invested in the recipient country for medical assistance, they were able to actively respond to the COVID-19 epidemic and make self-psychological adjustments, but as time passed, their ability and level to withstand stress events at work decreased, and the department A colleague who tested positive for COVID-19 may make him more sensitive and vulnerable psychologically. The more concerned about the epidemic situation, the more anxious he will be. Worrying about being infected by a patient or colleague, or even worrying about whether he can return home safely or whether he will infect family members through himself, exceeds his normal psychological load, which makes the team members gradually generate anxious negative emotions from the active response. The CAGMTM have been in a high-intensity and high-pressure environment for a long time, resulting in a decline in their self-regulation ability, which seriously affects work and life. Supportive therapy and stress intervention[15] achieve the purpose of treatment by providing methods to eliminate or reduce the negative effects of multiple stress factors. During the period of foreign aid, the CAGMTM should strengthen the systematic training of hospital infection prevention and control knowledge. Do a good job in hospital hand hygiene management, strictly implement the standard prevention system in the diagnosis and treatment process, actively participate in online training and learning organized by domestic experts through the Internet, strictly control the quality of hospital infection management, and reduce the risk of infection for the CAGMTM. The current situation of the COVID-19 epidemic in Guyana is still severe, and the team members should adopt a positive attitude and deal with the pressure correctly. With the help of domestic psychologists, establish a positive psychological response model, improve stress coping skills, improve mental health, reduce anxiety, and enhance psychological adaptability. CAGMTM In summary, the mental health level and idiosyncratic response methods of the CAGMTM show that in the face of COVID-19 global pandemic stress events, positive psychological interventions should be taken to reduce negative responses and improve the response ability of the CAGMTM to maintain good physical and mental health; establish a complete emergency response plan, comprehensively improve the overall quality of the CAGMTM. In this study, only 16 team members who aided Guyana in the 16th phase were selected. The sample size is small. When facing public health incidents in foreign aid medical treatment, the mental health status and coping methods of Chinese foreign aid medical team members still need to be further explored. [Declarations:] (1)This study was approved and approved by the Ethical Committee of Suzhou Municipal Hospital. (2) Ethics approval and consent to participate: The human questionnaire for this study was conducted in accordance with relevant guidelines and regulations. This study was certified by the ethics committee of Suzhou Municipal hospital. All enrolled patients volunteered to participate in the study. Informed consent was obtained from all subjects prior to the study. (3)Consent for publication: All authors agree to the publication of this study. (3)Consent for publication: All authors agree to the publication of this study. (4)Availability of data and material: All data and materials in this study belong to the author and consent to be published and used. The supplementary material contains all the original data. (5)Conflict of Interest: All authors of the article have no conflict of interest. (6)Funding：This fund belongs to the subject applied by the corresponding author. 2017 Suzhou Key Industrial Technology Innovation Project (People's Livelihood Science and Technology -- Basic Research of Medical and Health Application) （SYSD2017112）. (7) Author's contribution statement: This research is designed by Jin Zhengxi and Zhang Xiangxin, research implementation and data collection are Qiao Zhiming, Zhang Xiangxin, Jin Zhengxi, and the paper is written by Jin Zhengxi. (8)Acknowledgements: This article was written with the help of two doctors (Wu Zhengyan and Yang Xuna) from Suzhou Guangji Hospital (Suzhou Mental Health Center). After the article was written, they reviewed the article and would like to send my sincere thanks. 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https://openalex.org/W3087315063	https://discovery.dundee.ac.uk/files/51971330/Li2020_Article_FungalBioremediationOfSoilCo_c.pdf	English	null	Fungal bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals	Applied microbiology and biotechnology	2,020	cc-by	7,555	University of Dundee Citation for published version (APA): Li, Q., Liu, J., & Gadd , G. M. (2020). Fungal bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals. Applied Microbiology and Biotechnology, 104, 8999-9008. https://doi.org/10.1007/s00253-020-10854-y Fungal bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals Li, Qianwei; Liu, Jicheng; Gadd , Geoffrey Michael Li, Qianwei; Liu, Jicheng; Gadd , Geoffrey Michael Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Li, Q., Liu, J., & Gadd , G. M. (2020). Fungal bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals. Applied Microbiology and Biotechnology, 104, 8999-9008. https://doi.org/10.1007/s00253-020-10854-y General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-020-10854-y MINI-REVIEW MINI-REVIEW Key points y p • Fungi play important roles in soil co-contaminated with TPH and toxic metals. • Soil characteristics, enzymes, and metabolites are major factors in bioremediation. • DGT and metabolomics can be applied to overcome current bottlenecks. Keywords Fungi . Bioremediation . Petroleum hydrocarbons . Toxic metals Keywords Fungi . Bioremediation . Petroleum hydrocarbons . Toxic metals Abstract Much research has been carried out on the bacterial bioremediation of soil contaminated with petroleum hydrocarbons and toxic metals but much less is known about the potential of fungi in sites that are co-contaminated with both classes of pollutants. This article documents the roles of fungi in soil polluted with both petroleum hydrocarbons and toxic metals as well as the mechanisms involved in the biotransformation of such substances. Soil characteristics (e.g., structural components, pH, and temperature) and intracellular or excreted extracellular enzymes and metabolites are crucial factors which affect the efficiency of combined pollutant transformations. At present, bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals is mostly focused on the removal, detoxification, or degradation efficiency of single or composite pollutants of each type. Little research has been carried out on the metabolism of fungi in response to complex pollutant stress. To overcome current bottle- necks in understanding fungal bioremediation, the potential of new approaches, e.g., gradient diffusion film technology (DGT) and metabolomics, is also discussed. Fungal bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals Qianwei Li1 & Jicheng Liu1 & Geoffrey Michael Gadd1,2 Received: 2 June 2020 /Revised: 11 August 2020 /Accepted: 23 August 2020 # The Author(s) 2020 Received: 2 June 2020 /Revised: 11 August 2020 /Accepted: 23 August 2020 # The Author(s) 2020 1 State Key Laboratory of Heavy Oil Processing, State Key Laboratory of Petroleum Pollution Control, China University of Petroleum-Beijing, Beijing 102249, China Co-contamination in the soil environment major challenging problems in petroleum-producing coun- tries, such as Qatar (Freije 2015), China (Cheng et al. 2019; Dong et al. 2013), and Russia (Kuyukina et al. 2018). Petroleum hydrocarbons and associated by-products found in soil are usually generated from accidental spills of crude oil, fuel contamination, refining processes, and subsequent problems associated with distribution and utilization. Spilled contaminants penetrate into soil pores and adsorb onto soil particles, moving vertically with capillary and gravitational forces which alters chemical, physical and biological proper- ties, and composition (Czarny et al. 2020; dos Santos and Maranho 2018). Alkanes, aromatic compounds, nitrogen- sulfur-oxygen-containing compounds, and asphaltene are the major constituents of total petroleum hydrocarbons (TPH). The aromatic fraction refers to those compounds with benzene rings including polycyclic aromatic hydrocarbons (PAHs), which contain multiple fused aromatic rings, and are listed as priority pollutants due to their carcinogenic, mutagenic, and toxic properties as well as environmental recalcitrance (Czarny et al. 2020; Khan et al. 2018). With the accelerating pace of industrialization and urbaniza- tion, soil contamination has become a critical worldwide con- cern because of the threat to natural ecosystems and human health and much research has been carried out on innovative and cost-effective remediation technologies (Dong et al. 2013; Khan et al. 2018; Song et al. 2017). Soils co-contaminated with petroleum hydrocarbons and toxic metals are one of the * Qianwei Li qianweili@cup.edu.cn * Geoffrey Michael Gadd g.m.gadd@dundee.ac.uk 1 State Key Laboratory of Heavy Oil Processing, State Key Laboratory of Petroleum Pollution Control, China University of Petroleum-Beijing, Beijing 102249, China 2 Geomicrobiology Group, School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK Appl Microbiol Biotechnol Toxic metals found in petroleum-contaminated soils in- clude As, Ba, Cd, Cr, Pb, Hg, Ni, V, and Zn, and these are mainly associated with petroleum extraction and refining, and combustion of fuel for heat and transport (Adeniyi and Afolabi 2002; Klimek et al. 2016; Kuyukina et al. 2018; Muniz et al. 2004). The heavy oils in Russia are enriched with V and Ni, and trace amounts of Cd, Pb, and Zn (Kuyukina et al. 2018). More than 20 soil samples collected from petroleum-producing sites in China contained Cd, Ni, Cr, and Zn at concentrations ranging from 0.08–8.18, 21.6–40.7, 25.9–71.5, and 36.7–226.0 mg/kg dry weight, respectively (Cheng et al. 2019). (Harms et al. 2011). Co-contamination in the soil environment Compared with bacteria, filamentous fungi show some advantages in the transport or translocation of essential substances, including nutrients and water, and the pollutant itself, over significant distances (Boswell et al. 2003; Furuno et al. 2012; Boswell et al. 2002; Harms et al. 2011; Jacobs et al. 2002; Worrich et al. 2018). It is also significant that fungal mycelia can act as “highways” in facilitating the transport of pollutant-degrading bacteria over distance in soil which can enhance bioremediation (Banitz et al. 2013; Kohlmeier et al. 2005; Wick et al. 2007). ) Many fungi can survive and grow in the presence of toxic metals and this depends on intrinsic biochemical and structur- al properties, physiological and/or genetic adaptation, includ- ing morphological changes, and environmental modification of metal speciation, bioavailability, and toxicity (Gadd 1993; Gadd 2010; Glasauer et al. 2004; Sullivan and Gadd 2019). Filamentous fungi, e.g., Aspergillus and Penicillium spp., have been investigated for the degradation of aliphatic hydro- carbons, chlorophenols, and polycyclic aromatic hydrocar- bons, with the organic pollutants serving as carbon and energy sources (Harms et al. 2011; Hofrichter et al. 1994; Pinedo- Rivilla et al. 2009) (Table 1). The ability of ureolytic fungi, such as Neurospora crassa, to immobilize metals has been investigated because, when incubated in urea-supplemented media, toxic metals are precipitated as carbonates and/or ox- ides (Li et al. 2015; Li and Gadd 2017a; Li and Gadd 2017b; Li et al. 2019; Li et al. 2016; Li et al. 2014). When grown in urea-containing media supplemented with heavy oil and Ca2+, such mineral precipitation tended to aggregate along the edge of the heavy oil which may provide an additional carbon or energy source during the biomineralization process (Fig. 1). Moreover, fungi are primary decomposers of organic matter and plant biomass in soil with the chemical structure of lignin polymers of wood being comparable with the aromatic struc- ture of PAHs (Haritash and Kaushik 2009; Vanholme et al. 2010) (Fig. 2). Because of this, many lignin-degrading fungi, e.g., Phanerochaete chrysosporium, have been investigated for degradation of PAHs and other aromatic compounds be- cause of the wide range of substances that can be attacked by such organisms (Gadd 2004; Gadd 2001). Some fungi convert high-molecular-mass PAHs such as the highly carcinogenic benzo[α]pyrene into water-soluble products using non- specific detoxification mechanisms (Harms et al. 2011). Co-contamination in the soil environment Fusarium solani and Hypocrea lixii isolated from petrol sta- tion soil were investigated for the degradation of pyrene and tolerance to copper and zinc. These organisms degraded more than 60% of the supplied pyrene and could also accumulate Cu and Zn (Hong et al. 2010). In co-contaminated soil, Fe(III) coordinating fungal siderophores could play an important role not only by binding metals other than Fe(III), e.g., Cd, Cu, Ni, Pb, Zn, Th (IV), U(IV), and Pu (IV) (Ahmed and Holmström 2014) but also by facilitating the biodegradation of petroleum hydrocarbons by satisfying the Fe requirement for the ( g ) Remediation of soil co-contaminated with organic and in- organic pollutants is a complex problem as these two pollutant classes need to be treated differently (Gadd 2004). The pres- ence of toxic metals in co-contaminated soil can inhibit petro- leum biodegrading microorganisms, affecting growth and me- tabolism, nitrogen and sulfur conversions, and dehalogenation (Biswas et al. 2015; El-Azeem et al. 2013; Sandrin and Maier 2003). Toxic metal species may exert a plethora of toxic ef- fects depending on metal concentration and speciation, phys- icochemical factors, and the organism’s ability to respond to metal stress through intrinsic or induced mechanisms (Gadd 1993; Gadd 2007; Rangel et al. 2018). Metals can exert tox- icity in many ways, e.g., inhibition of enzymes, displacement or substitution of essential metals, disruption of cell and organellar membranes, and interaction with normal cellular homeostatic and stress response systems (Gadd 1993; Gadd 2007; Sullivan and Gadd 2019). For example, toxic metal cations may substitute for essential metal co-factors within an enzyme (e.g., Cd2+ may substitute for Zn2+) resulting in enzyme dysfunction (Sandrin and Maier 2003). Petroleum hydrocarbons in toxic metal-contaminated soils are hydropho- bic materials with low water solubility and preferentially at- tach to the soil matrix which reduces the bioavailability of toxic metals to potential bioremediating microorganisms (Lai et al. 2009). Although research has been carried out on the bioremediation of co-contaminated soil with bacterial sys- tems, much less attention has been paid to the potential roles of fungi in soils contaminated with petroleum hydrocarbons and toxic metals. Fungi in soil co-contaminated with petroleum hydrocarbons and toxic metals Fungi are ubiquitous chemoorganotrophic (heterotrophic) or- ganisms (Gadd 2008; Gadd 2017), and are one of the three major clades of eukaryotic life that independently evolved multicellular organization (Stajich et al. 2010). The coloniza- tion of soil by fungal mycelium results in enmeshment and aggregation of soil particles and improvement of soil struc- ture, sometimes facilitating contaminant bioavailability Appl Microbiol Biotechnol Table 1 Some examples of degradation of petroleum hydrocarbons by different fungal species Species Hydrocarbons Formula Structure Removal efficiency (%) Treatment length (d) Reference Penicillium sp. Decane C10H22 49.0 28 Govarthanan et al. (2017) Aspergillus sp. N-hexadecane C16H34 86.3 10 Al-Hawash et al. (2018) Fusarium sp. N-octadecane C18H38 89 60 Hidayat and Tachibana (2013) Phomopsis liquidambari Phenanthrene C14H10 77.4 10 Fu et al. (2018) Irpex lacteus Anthracene C14H10 60 25 Drevinskas et al. (2016) Pleurotus ostreatus Anthracene C14H10 56 23 Drevinskas et al. (2016) Ganoderma lucidum Pyrene C16H10 99.6 30 Agrawal et al. (2018) Polyporus sp. Chrysene C18H12 65 30 Hadibarata et al. (2009) Table 1 Some examples of degradation of petroleum hydrocarbons by different fungal species speciation, bioavailability, and toxicity of the metal and the organic pollutant (Ceci et al. 2019). degrading microorganisms in Fe-limited habitats. In co- contaminated soil, PAHs may interact with lipophilic compo- nents of the fungal cytoplasmic membrane, changing the per- meability, which can result in penetration of toxic metals into cells and resulting effects/on cellular functions. Shen et al. (2005) investigated the effects of Cd and phenanthrene (Phe) on the growth of certain soil fungi and showed that growth was strongly inhibited in soil containing Cd and Phe com- pared with soil containing only Cd. Examples of selected fun- gal species interacting with PAHs and toxic metals are pre- sented in Table 2. The pH is another crucial factor in determining the bio- degradation of petroleum hydrocarbons and biotransforma- tion of toxic metals. Changes in pH can alter fungal, and bacterial, community structure and enzyme activities as well as affecting metal speciation. Such effects of pH on the speciation of metal ions can be simulated using geo- chemical modeling software, e.g., Geochemists’ work- bench (GWB) (Carrillo-Chávez et al. 2014; Li et al. 2019), MINEQL+ (Cloutier-Hurteau et al. 2007; Kocaoba 2020), and PHREEQC (Ceci et al. 2015; Liang et al. 2016b). Fungi in soil co-contaminated with petroleum hydrocarbons and toxic metals For example, the speciation and solubility of Zn2+ in a simulated fungal system for metal remediation calculated using GWB showed that smithsonite (ZnCO3) (pH = 1.7–3.2) and Zn3(PO4)2∙4H2O (pH = 3.2–14) were the main mineral phases over different pH ranges (Fig. 3). Importance of metabolites and enzymes Many transforming interactions between fungi and different pollutants depend on a variety of extracellular excreted sub- stances and metabolites (Gadd 2004; Kirtzel et al. 2020). Fungi are capable of degrading petroleum hydrocarbons by secreting enzymes (e.g., laccases, tyrosinases, manganese per- oxidases, cytochrome P450 monooxygenases, reductive dehalogenases), and affecting metal speciation by excretion of a variety of other metabolites (e.g., organic acids, amino acids, siderophores, extracellular proteins, etc.) (Fig. 4). , p , p , ) ( g ) Petroleum hydrocarbons can be used by several fungal species as a carbon and energy source and assimilated into fungal biomass. Fungal taxa including Amorphoteca, Neosartorya, Talaromyces, Aspergillus, Fusarium, Paecilomyces, Sporobolomyces, Cephalosporium, Penicillium, and Graphium have all been reported to include potential degraders of petroleum hydrocarbons (Chulalaksananukul et al. 2006; Das and Chandran 2011; Varjani 2017). Some species can oxidize pollutants (e.g., phe- nols and aromatic amines) extracellularly by the production of laccases (Martínková et al. 2016), manganese peroxidases (Zhang et al. 2016), or lignin peroxidases (Falade et al. 2017; Grossart and Rojas-Jimenez 2016). Moreover, fungal cell membranes are permeable to many organic pollutants and these can be degraded by intracellular enzymes, e.g., cyto- chrome P450 (Ostrem Loss and Yu 2018), reductive dehalogenases (Stella et al. 2017), and nitroreductases (Tripathi et al. 2017; Xu and Zhou 2016), to simpler organic compounds, followed by further metabolism such as β- oxidation and entry into the tricarboxylic acid (TCA) cycle (Varjani 2017). Degradation of petroleum hydrocarbons con- sists of several different enzymatic steps with biodegradability depending on the chemical structure and other factors that affect fungal growth and metabolism, and pollutant chemistry and speciation. In general, biodegradation efficiency can be ranked as linear alkanes > branched alkanes > small aromatics > cyclic alkanes (Das and Chandran 2011; Guermouche M’rassi et al. 2015; Varjani 2017). In co-contaminated soil, petroleum hydrocarbons may pro- vide a carbon and energy source for certain fungal species while toxic metals can also exert significant effects on fungal popula- tions and activity. Despite the potential toxicity of many metal species, many fungi can flourish in contaminated conditions although there may be shifts in species composition (Fomina et al. 2005; Gadd 2005; Gadd 2007). The major survival mech- anisms involved can be explained as changes in toxic metal mobility resulting from various tolerance and resistance mech- anisms (Gadd 2007; Gadd 2010). Soil characteristics Temperature can influence the bioremediation of co- contaminated soil by affecting the chemistry of pollutants and fungal biodiversity (Rangel et al. 2018). The viscosity of petroleum increases at low temperatures and volatility is reduced which results in retardation of biodegradation. The highest degradation rates for hydrocarbon pollutants generally occur around 30–40 °C in the soil environment (Das and Chandran 2011). At higher temperatures, the solubility of PAHs and toxic metal ions increases which improves their bioavailability, although such high temperatures will also af- fect microbial community structure and activity. Compared with effects at 20 °C and 40 °C, 30 °C was found to be the optimum temperature for metal removal by Beauveria bassiana due to increased biomass production which provided more metal-binding sites (Gola et al. 2016). Soil components, pH, and temperature are key factors in fun- gal bioremediation and metal speciation and affect the trans- portation and bioavailability of contaminants (Liu et al. 2017; Rangel et al. 2018). Organic matter and clay minerals signif- icantly reduce the solution-phase concentration of metal ions. It was reported that in mineral-dominated soil, 0.01-mg L−1 Cd2+ inhibited the dechlorination of trichloroaniline (TCA) while 0.2-mg L−1 Cd2+ was necessary in an organic- dominated soil, which correlated with the metal-binding ca- pacity of the organic material (Zhang et al. 2016). Clay min- erals, e.g., montmorillonite, possess high cation exchange ca- pacities (CECs), and can efficiently reduce metal bioavailabil- ity and toxicity (Sandrin and Maier 2003). Moreover, metals in soil may react with the organic pollutants to affect the Appl Microbiol Biotechnol Table 2 Some applications of fungal species for the bioremediation of PAHs and toxic metals Fungus PAHs Initial content Removal efficiency Toxic metals Initial content Bioremediation efficiency Data source Reference Acremonium sp. Naphthalene, fluorine, phenanthrene, anthracene, fluoranthene 25 mg L−1 64.9–96.9% Mn, Fe, Zn, Cu, Al, Pb 50 mg L−1 – Lab Ma et al. (2014) Pleurotus ostreatus Pyrene, benzo[a]anthracene, chrysene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene 10 ppm 0.5–52.2% Cd, Hg 10–500 ppm – Lab Baldrian et al. (2000) Fusarium flocciferum, Trichoderma sp., Trametes versicolor, Pleurotus sp. Benzo[a]anthracene, benzo[a]fluoranthene, benzo[a]pyrene, chrysene, phenanthrene 150– 250 mg kg−1 21–93% Cd, Ni 50–500 mg kg−1 – Lab Al-Turki (2009) Pleurotus ostreatus Crude oil – – Pb, Cu, Mn, Cd, Ni 0.79–52.07 mg kg−1 28.2–75.9% Lab Anacletus et al. (2017) Importance of metabolites and enzymes Fungi possess many mecha- nisms or properties that influence metal toxicity and mobility, including the production of metal-binding proteins, organic and inorganic precipitation, active transport, and intracellular com- partmentalization, while cell walls and associated pigments and Appl Microbiol Biotechnol polysaccharides have significant metal-binding abilities (Gadd 1993; Gadd 2007). The secretion of fungal metabolites (e.g., metal-binding peptides, polysaccharides, amino acids, organic acids) is particularly important for metal and mineral transfor- mations playing roles in both mobilization and immobilization of metal species (Gadd 2007; Gadd et al. 2014). Moreover, fungal phenolic polymers and melanin possess many potential metal-binding sites with oxygen-containing groups, such as carboxyl, phenolic and alcoholic hydroxyl, carbonyl, and methoxyl groups (Fomina and Gadd 2014). Fungal surface Fig. 1 Fungal biomineralization of carbonates in media amended with heavy oil. Neurospora crassa was incubated on solid agar medium, supplemented with 40-mM urea and 50-mM CaCl2, at 25 °C in the dark for 5 days. Four wells (5-mm diameter) were made in the agar medium using a sterile cork borer and filled with heavy oil prior to fungal inoculation. Scale bars = 200 μm (Li et al., unpublished data) Fig. 1 Fungal biomineralization of carbonates in media amended with heavy oil. Neurospora crassa was incubated on solid agar medium, supplemented with 40-mM urea and 50-mM CaCl2, at 25 °C in the dark for 5 days. Four wells (5-mm diameter) were made in the agar medium using a sterile cork borer and filled with heavy oil prior to fungal inoculation. Scale bars = 200 μm (Li et al., unpublished data) of metal species (Gadd 2007; Gadd et al. 2014). Moreover, fungal phenolic polymers and melanin possess many potential metal-binding sites with oxygen-containing groups, such as carboxyl, phenolic and alcoholic hydroxyl, carbonyl, and methoxyl groups (Fomina and Gadd 2014). Fungal surface of metal species (Gadd 2007; Gadd et al. 2014). Moreover, fungal phenolic polymers and melanin possess many potential metal-binding sites with oxygen-containing groups, such as carboxyl, phenolic and alcoholic hydroxyl, carbonyl, and methoxyl groups (Fomina and Gadd 2014). Fungal surface polysaccharides have significant metal-binding abilities (Gadd 1993; Gadd 2007). The secretion of fungal metabolites (e.g., metal-binding peptides, polysaccharides, amino acids, organic acids) is particularly important for metal and mineral transfor- mations playing roles in both mobilization and immobilization Appl Microbiol Biotechnol Fig. 3 Geochemical simulation of Zn2+ versus pH in a simulated fungal system at 25 °C. Importance of metabolites and enzymes The chemical parameters were set at 0.33-M CO3 2−, 6.1- mM Cl−, 0.83-mM SO4 2−, 0.66-M NH4 +, 4-mM K+, 0.8-mM Mg2+, 1.7- mM Na+, 0.2-mM Ca2+, 0.02-mM Mn2+, and 9-μm Fe3+. The letter a on the y-axis represents the effective concentration of a given chemical species in the mixture (Li et al., unpublished data) complex formation may be related to the coordination of metal ions with oxygen donor atoms and proton release (Gadd 2009): S‐OH þ Cu2þ⇌S ‐ OCuþ þ Hþ Bidentate surface complexation may also result: Bidentate surface complexation may also result: Metal immobilization appears particularly relevant to bio- remediation approaches and fungi are capable of mediating precipitation of metals as insoluble oxalates, oxides, carbon- ates, and phosphates (Fomina et al. 2008; Gadd et al. 2014; Liang and Gadd 2017; Suyamud et al. 2020). For example, the liberation of phosphate from organic or inorganic phosphate hydrolysis proved to be an efficient method for metal immo- bilization, including Zn, Pb, La, and U, which were precipi- tated on and around hyphal surfaces (Ezawa and Saito 2017; Fomina et al. 2008; Liang et al. 2016a; Liang and Gadd 2017; Liang et al. 2015; Suyamud et al. 2020). Urease-positive fungi (e.g., Neurospora crassa, Pestalotiopsis sp., and Myrothecium gramineum) are promising candidates for the immobilization of toxic metals because the mechanism in- volved is associated with urea degradation. Such fungi grown Fig. 3 Geochemical simulation of Zn2+ versus pH in a simulated fungal system at 25 °C. The chemical parameters were set at 0.33-M CO3 2−, 6.1- mM Cl−, 0.83-mM SO4 2−, 0.66-M NH4 +, 4-mM K+, 0.8-mM Mg2+, 1.7- mM Na+, 0.2-mM Ca2+, 0.02-mM Mn2+, and 9-μm Fe3+. The letter a on the y-axis represents the effective concentration of a given chemical species in the mixture (Li et al., unpublished data) g g g Fig. 4 Simplified diagram of fungal interactions with petroleum hydrocarbons and toxic metals. Fungal cell membranes are permeable to petroleum hydrocarbons or simpler organic compounds oxidized by extracellular enzymes, which can undergo further metabolism including hydrolysis, dehalogenation, β-oxidation, and entry into the tricarboxylic acid cycle. Toxic metals can accumulate on fungal cell surfaces through biosorption, which can result in nucleation and subsequent precipitation of biominerals. Importance of metabolites and enzymes Some metals can be intracellularly accumulated by active transport or diffusion through the cell membrane, and localized within vacuolar or other organellar compartments and/or be sequestered by sulfide, metal-binding proteins/peptides, and other macromolecules Fig. 4 Simplified diagram of fungal interactions with petroleum hydrocarbons and toxic metals. Fungal cell membranes are permeable to petroleum hydrocarbons or simpler organic compounds oxidized by extracellular enzymes, which can undergo further metabolism including hydrolysis, dehalogenation, β-oxidation, and entry into the tricarboxylic acid cycle. Toxic metals can accumulate on fungal cell surfaces through biosorption, which can result in nucleation and subsequent precipitation of biominerals. Some metals can be intracellularly accumulated by active transport or diffusion through the cell membrane, and localized within vacuolar or other organellar compartments and/or be sequestered by sulfide, metal-binding proteins/peptides, and other macromolecules Appl Microbiol Biotechnol in urea-containing medium hydrolyze urea producing ammo- nia and free carbonate which results in the precipitation of metals as carbonates, e.g., BaCO3, CdCO3, CoCO3, Cu2(OH)2CO3, La2(CO3)3, and NiCO3 (Li and Gadd 2017b; Li et al. 2019; Liang and Gadd 2017; Liu et al. 2019; Rautaray et al. 2004). Fungi can produce a variety of metal oxalates on interacting with metals and metal-bearing minerals including those of Ca, Cd, Co, Cu, Mg, Mn, Sr, Zn, Ni, and Pb (Gadd et al. 2014). Extracellular proteins, amino acids, and polysac- charides also play an important role in toxic metal immobili- zation. Extracellular nickel precipitation was associated with the removal of extracellular protein (Li et al. 2019), and it has been demonstrated that extracellular protein may act as a tem- plate for mineral formation, influencing the size of the resul- tant biominerals (Li and Gadd 2017a; Li et al. 2019; Liu et al. 2019). resolution spatial distribution characteristics of toxic metals and reveal interface reaction processes of soil-metals-fungal interactions, new methods such as gradient diffusion film technology (DGT) combined with laser ablation-inductively coupled plasma mass spectrometry could be applied to ana- lyze dynamic changes in toxic metal speciation at soil- microbe interfaces (Challis et al. 2018; Feng et al. 2016; Koppel et al. 2020; Wang et al. 2018). Such approaches will further clarify fungal transformation mechanisms in soil con- taminated with petroleum hydrocarbons and toxic metals and may contribute to more effective strategies for fungal bioremediation. Funding This work was supported by the National Natural Science Foundation of China (Grant No. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, pro- vide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Importance of metabolites and enzymes 41701352), the Open Project Program of the State Key Laboratory of Petroleum Pollution Control (Grant No. PPC2019012), and the National Key R&D Program of China (No. 2018YFC1801903-01). GMG gratefully acknowledges the support of the Geomicrobiology Group from the Natural Environment Research Council, UK (NE/M010910/1 (TeaSe); NE/M011275/1 (COG3)). Future perspectives and conclusions At present, bioremediation of soil co-contaminated with pe- troleum hydrocarbons and toxic metals is mostly focused on the removal, detoxification, or degradation efficiency of single or composite pollutants of each type. Little research has been carried out on the metabolism of fungi in response to complex pollutant stress. Fungal responses to petroleum hydrocarbons can be reflected by differences in metabolic responses, en- zyme induction and synthesis, and extracellular metabolite production, which will also affect the migration and transfor- mation of toxic metals. This is clearly a complex problem, affected by many variables, and sometimes limited by the availability of appropriate analytical techniques. For example, understanding the spatial distribution of toxic metals in the soil and/or the migration and transformation processes medi- ated by different fungal species depends on the sampling and analysis methods used. Sampling technology can be the main bottleneck that limits understanding due to heterogeneity of the soil in vertical and horizontal dimensions. In fact, toxic metals may show different gradient distributions over very small interfaces. Conventional techniques are also based on sampling and subsequent transport to the laboratory for anal- ysis, but there may be subsequent changes during collection and storage due to, e.g., contamination and changes in envi- ronmental conditions (e.g., metabolic activity, pH, dissolved oxygen, Eh), which conceal the dynamic changes in biodiver- sity or chemical speciation that may occur in contaminated soil. To overcome these difficulties, new technology, includ- ing microbial metabolomics (Dombrowski et al. 2016; Tian et al. 2018) and proteomics, needs to be applied to the study of fungal bioremediation in co-contaminated soil, which could inform about metabolic responses under multiple pollutant conditions. This could provide scientific explanations for fun- gal responses to multiple contaminants at the molecular level (Aydin et al. 2017; Wang et al. 2017). 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https://openalex.org/W2806548634	https://europepmc.org/articles/pmc5975581?pdf=render	English	null	Paraquat induced acute kidney injury and lung fibrosis: a case report from Bangladesh	BMC research notes	2,018	cc-by	2,668	Background Unfortunately, this compound has no effective antidote and rapidly causes multi-organ failure [3]. It has high mortality even with standard care and early management [3]. Diagnostic limitations and unavailable sample at presentation have resulted in under-reporting and lack of awareness among the treating physicians, making Para- quat poisoning one of the most neglected toxicological emergencies in Bangladesh. Herein this article, we pre- sent a case of Paraquat poisoning complicated by renal failure and lung fibrosis and emphasis on pitfalls in the management. Since Bangladesh government issued a ban on WHO class I pesticides in 2000 [1], the use of herbicides have been increasing in our agriculture and most toxic her- bicides like Paraquat entered into our market [2]. The annual consumption of Paraquat is sharply increasing in Bangladesh [2], so is the incidence of Paraquat poisoning, posing a major threat to public health. While the organo- phosphate (OP) poisonings still account for the majority of hospital admissions, the fatality of Paraquat poisoning cases has been an emerging concern. © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Since Bangladesh government issued a ban on the use of highly toxic WHO Class I pesticides, annual consumption of herbicides like Paraquat have been sharply increasing in the markets. Paraquat poisoning is an emerging public health threat and its high mortality rate is responsible for a significant number of deaths. Diagnostic limitations and unavailable sample at presentation have resulted in under-reporting and lack of awareness among the treating physicians, making Paraquat poisoning one of the most neglected toxicological emergencies. Herein, we present a case of Paraquat induced multi-organ failure and emphasis on pitfalls in the management. Case presentation: An 18-years-old healthy male was admitted in Sylhet M.A.G Osmani Medical College Hospital with history of attempted suicide by Paraquat ingestion. On admission, he had high serum creatinine but otherwise asymptomatic. He was discharged on day 10 when his renal functions returned to normal. But On day 15, he started having respiratory symptoms—unresponsive to any of the local treatments he received, and by day 30, he developed overt lung fibrosis. We present sequential blood picture, radiographs and CT scans demonstrating Paraquat induced kidney and lung injury over the course of 30 days. Conclusion: Paraquat poisoning can lead to death and fatal long-term consequences. All cases of Paraquat poison- ing, regardless of symptoms, must be hospitalized and observed for early detection of complications. Distribution of Paraquat should be restricted and/or banned as 38 other countries have done so, which we believe will greatly reduce poisoning related mortality. Keywords: Paraquat, Poisoning, Bangladesh, Acute kidney injury, Lung fibrosis Paraquat induced acute kidney injury and lung fibrosis: a case report from Bangladesh Ishrat Tahsin Isha1, Z. H. M. Nazmul Alam1, Bidur Kanti Shaha1, Md. Shafiqul Bari1, Mohammad Zabed Jillul Bari1 and Fazle Rabbi Chowdhury2,3 Isha et al. BMC Res Notes (2018) 11:344 https://doi.org/10.1186/s13104-018-3425-3 Isha et al. BMC Res Notes (2018) 11:344 https://doi.org/10.1186/s13104-018-3425-3 BMC Research Notes Open Access Case presentation An 18-year-old healthy male was brought to the emer- gency room, Sylhet M.A.G Osmani Medical College Hospital with a history of attempted suicide by ingestion Correspondence: isha5@live.com 1 Department of Medicine, Sylhet M.A.G Osmani Medical College Hospital, Medical College Road, Sylhet 3100, Bangladesh Full list of author information is available at the end of the article *Correspondence: isha5@live.com 1 Department of Medicine, Sylhet M.A.G Osmani Medical College Hospital, Medical College Road, Sylhet 3100, Bangladesh Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Isha et al. BMC Res Notes (2018) 11:344 Page 2 of 4 Table 1 Laboratory profile after Paraquat poisoning Investigation Day 1 Day 10 Day 30 Haemoglobin 13.6 11.5 13.7 WBC 10,600 7200 8100 Platelets 276,000 379,000 332,000 Serum creatinine 4.32 2.1 0.84 Bilirubin total 2.0 – – ALT 36 13.7 48 Na+/K+ 138.9/3.71 136.7/4.12 132.3/4.71 HCO3 − 25.8 – – SpO2 98% – 97% Others – – Sputum for AFB: nega- tive Table 1 Laboratory profile after Paraquat poisoning of about 30 ml of an unknown poison, later revealed to be Paraquat 20 SL. He was initially managed at a local health complex with gastric lavage, intravenous fluids, antiemetic, and H2 blocker, and referred to this tertiary hospital for further management. On admission, he had vomiting, difficulty in opening his mouth and inability to drink or swallow. He was conscious and oriented and had mucosal erosion of tongue (Fig. 1), palate, and lips with some mucosal bleeding having poisoning severity score (PSS) grade one. His heart rate was 78/min and regular, blood pressure was 100/60 mm Hg, respiratory rate was 20/min and temperature 98°K. Pupils were normal and reacting to light. Oxygen saturation was 98% on room air. Both lung fields were clear on auscultation. Other sys- temic examinations were normal. Laboratory investigations revealed high serum creati- nine (PSS grade 2). Complete blood count, serum ALT and electrolytes were within normal limits (Table 1). Case presentation Chest radiograph was also normal (Fig. 2a). a high-resolution CT scan of chest was obtained, which revealed bilateral pulmonary fibrosis (Fig. 3). i He was started on cyclophosphamide and methylpred- nisolone and his condition was static. There was no fur- ther deterioration during his stay. (Additional file 1) To rule out OP poisoning, atropine challenge test was performed which was negative. He was managed con- servatively and was soon able to swallow liquids. He was discharged on day 10 when his renal function settled (Table 1). Discussion and conclusion On day 15, he developed irregular fever, shortness of breath and non-productive cough and as these symptoms progressed, he consulted with a local physician. Chest radiograph revealed diffuse consolidation (Fig. 2b) and he was prescribed a 14-day course of antibiotics. But his condition deteriorated and he had to get admitted to the hospital on day 30. The lesions on his chest radiograph showed bilateral diffuse alveolar shadowing (Fig. 2c) and Paraquat (1,1′-Dimethyl-4,4′-bipyridinium dichloride) has the ability to generate highly reactive oxygen and nitrite species which cause cellular damage and apoptosis in many organs [3]. The clinical manifestations depend upon the quantity ingested. Ingestion of large amounts of liquid concentrate (> 50–100 ml of 20% w/v) results in fulminant multi-organ failure and death within several hours to a few days [3]. Ingestion of smaller quantities i h h f Fig. 1 Paraquat tongue, within 24 h of ingestion Isha et al. BMC Res Notes (2018) 11:344 Page 3 of 4 Fig. 2 Serial chest radiographs after Paraquat poisoning. a Day 1—Normal. b Day 15—Diffuse alveolar shadowing predominantly involving left mid and lower zone. c Day 30—Diffuse alveolar shadowing extending to right apical and mid zone Fig. 2 Serial chest radiographs after Paraquat poisoning. a Day 1—Normal. b Day 15—Diffuse alveolar shadowing predominantly involving left mid and lower zone. c Day 30—Diffuse alveolar shadowing extending to right apical and mid zone Fig. 2 Serial chest radiographs after Paraquat poisoning. a Day 1—Normal. b Day 15—Diffuse alv and lower zone. c Day 30—Diffuse alveolar shadowing extending to right apical and mid zone Fig. 3 High resolution CT scan of chest demonstrating bilateral pulmonary fibrosis 30 days after Paraquat poisoning Fig. 3 High resolution CT scan of chest demonstrating bilateral pulmonary fibrosis 30 days after Paraquat poisoning to erosion and irritation. Activated charcoal or Fuller’s earth was not used due to unavailability. Irrespective of its route of administration, it is rapidly distributed in most tissues, with the highest concentra- tion found in the lungs and kidneys [3, 4]. It is actively taken up by the type II pneumocytes against a concen- tration gradient. Lung damage occurs in two phases, ini- tially from destructive alveolitis over one to three days followed by proliferative phase leading to fibrosis [3, 4]. usually leads to toxicity to two key target organs, kidneys and lungs, developing over days to weeks [3, 4]. to erosion and irritation. References Four-year study on acute poisoning cases admitted to a tertiary hos- pital in Bangladesh: emerging trend of poisoning in commuters. Asia Pac J Med Toxicol. 2014;3(4):152–6. https://doi.org/10.22038/apjmt.2014.3485. 7. Bari MS, Chakraborty SR, Alam MMJ, Qayyum JA, Hassan N, Chowdhury FR. Four-year study on acute poisoning cases admitted to a tertiary hos- pital in Bangladesh: emerging trend of poisoning in commuters. Asia Pac J Med Toxicol. 2014;3(4):152–6. https://doi.org/10.22038/apjmt.2014.3485. Funding Funding Not applicable. References 1. Chowdhury FR, Dewan G, Verma VR, Knipe DW, Isha IT, Faiz MA, et al. Bans of WHO class I pesticides in Bangladesh—suicide prevention without hampering agricultural output. Int J Epidemiol. 2018;47(1):175–84. https ://doi.org/10.1093/ije/dyx157. 1. Chowdhury FR, Dewan G, Verma VR, Knipe DW, Isha IT, Faiz MA, et al. Bans of WHO class I pesticides in Bangladesh—suicide prevention without hampering agricultural output. Int J Epidemiol. 2018;47(1):175–84. https ://doi.org/10.1093/ije/dyx157. Up to date, 38 countries have issued a ban on Paraquat including the European Union, Sri Lanka, Vietnam and South Korea [8–11]. Bans of W.H.O class I pesticides is proved to significantly lower the suicide rates in Bangla- desh [1]. Therefore it is the high time to implement such regulation on Paraquat also. 2. Pesticides use. http://www.fao.org/faostat/en/#data/RP. Accessed 15 Apr 2018. 3. Gawarammana IB, Buckley NA. Medical management of paraquat ingestion. Br J Clin Pharmacol. 2011;72(5):745–57. https://doi.org/10.111 1/j.1365-2125.2011.04026.x. Paraquat poisoning can lead to death and fatal long- term consequences. Unfortunately, there is no avail- able antidote, which makes it more hazardous. All cases, regardless of symptoms, must be hospitalized and observed for early detection of complications. We recom- mend the government should look into the problem at large and issue a ban on Paraquat which will effectively lower the poisoning death-rates. j 4. Wunnapuk K, Mohammed F, Gawarammana I, Liu X, Verbeeck RK, Buckley NA, et al. Prediction of paraquat exposure and toxicity in clinically ill poisoned patients: a model based approach. Br J Clin Pharmacol. 2014;78(4):855–66. https://doi.org/10.1111/bcp.12389. 5. Houze P, Baud FJ, Mouy R, Bismuth C, Bourdon R, Scherrmann JM. Toxi- cokinetics of paraquat in humans. Hum Exp Toxicol. 1990;9:5–12. 6. Lee SH, Lee KS, Ahn JM, Kim SH, Hong SY. Paraquat poisoning of the lung: thin-section CT findings. Radiology. 1995;195:271–4. 4. Wunnapuk K, Mohammed F, Gawarammana I, Liu X, Verbeeck RK, Buckley NA, et al. Prediction of paraquat exposure and toxicity in clinically ill poisoned patients: a model based approach. Br J Clin Pharmacol. 2014;78(4):855–66. https://doi.org/10.1111/bcp.12389. 5. Houze P, Baud FJ, Mouy R, Bismuth C, Bourdon R, Scherrmann JM. Toxi- cokinetics of paraquat in humans. Hum Exp Toxicol. 1990;9:5–12. 6. Lee SH, Lee KS, Ahn JM, Kim SH, Hong SY. Paraquat poisoning of the lung: thin-section CT findings. Radiology. 1995;195:271–4. 7. Bari MS, Chakraborty SR, Alam MMJ, Qayyum JA, Hassan N, Chowdhury FR. Authors’ contributions 11. PAN Vietnam Welcomes the Ban of Paraquat and 2,4-d. http://panap .net/2017/02/pan-vietnam-welcomes-the-ban-of-paraquat-and-24-d/. Accessed 12 Oct 2017. ITI, ZNA, BKS, MZJB and MSB diagnosed the case, collected data, supervised the management and followed-up the patient. ITI and FRC drafted the manu- script. All authors read and approved the final manuscript. Competing interests Availability of data and materials Not applicable. Availability of data and materials Not applicable. i This case posed a number of diagnostic challenges to the medical team. Paraquat poisoning is an emerging problem of Bangladesh and never been reported from this region [7]. Therefore, there was considerable confusion regard- ing the identity of the pesticide ingested. This patient got gastric lavage (which made his condition worse) and test doses of atropine initially and might have looked upon as OP poisoning. Later he was confirmed as a case of Para- quat poisoning after examining the container. Secondly, the initial clinical features were nonspecific. Initial symp- toms of vomiting and mucosal ulceration mislead to other corrosive agents. Thirdly, failure to anticipate the com- plications led to an early discharge of the patient. Subse- quently, he developed overt lung fibrosis within 30 days. Methylprednisolone and cyclophosphamide therapy was not given initially (started later), which was a major pitfall in the management of our patient. Early initiation of these therapies might reduce the accumulation into lung [3]. Abbreviations PSS: poisoning severity score; AFB: acid fast bacilli; ALT: alanine transaminase; OP: organophosphate. PSS: poisoning severity score; AFB: acid fast bacilli; ALT: alanine transaminase; OP: organophosphate. 10. Cha ES, Chang SS, Gunnell D, Eddleston M, Khang Y-H, Lee WJ. Impact of paraquat regulation on suicide in South Korea. Int J Epidemiol. 2016;45:470–9. https://doi.org/10.1093/ije/dyv304. Additional files 8. EU Court Reimposes Ban on Paraquat Weed killer. http://www.reute rs.com/article/environment-eu-paraquat-dc/eu-court-reimposes-ban- on-paraquat-weedkiller-idUSL1166680020070711. Accessed 12 Oct 2017. 8. EU Court Reimposes Ban on Paraquat Weed killer. http://www.reute rs.com/article/environment-eu-paraquat-dc/eu-court-reimposes-ban- on-paraquat-weedkiller-idUSL1166680020070711. Accessed 12 Oct 2017. Additional file 1. Timeline of clinical events. 9. Pearson M, Zwi AB, Buckley NA, Manuweera G, Fernando R, Dawson AH, et al. Policymaking ‘under the radar’: a case study of pesticide regula- tion to prevent intentional poisoning in Sri Lanka. Health Policy Plan. 2015;30(1):56–67. https://doi.org/10.1093/heapol/czt096. 11. PAN Vietnam Welcomes the Ban of Paraquat and 2,4-d. http://panap .net/2017/02/pan-vietnam-welcomes-the-ban-of-paraquat-and-24-d/. Accessed 12 Oct 2017. Discussion and conclusion Activated charcoal or Fuller’s earth was not used due to unavailability. An ingestion of 10–15 ml of 20% w/v Paraquat solution is considered lethal [4]. The estimated amount taken by our patient was 30 ml of 20 SL solution, which was quite high. Following ingestion, the herbicide induces a burn- ing sensation of the mouth and throat, gastrointestinal irritation, abdominal pain, nausea, vomiting, and diar- rhea [3]. In our patient, all the initial symptoms were due Irrespective of its route of administration, it is rapidly distributed in most tissues, with the highest concentra- tion found in the lungs and kidneys [3, 4]. It is actively taken up by the type II pneumocytes against a concen- tration gradient. Lung damage occurs in two phases, ini- tially from destructive alveolitis over one to three days followed by proliferative phase leading to fibrosis [3, 4]. Isha et al. BMC Res Notes (2018) 11:344 Page 4 of 4 Page 4 of 4 Excretion of Paraquat is biphasic, owing to lung accumu- lation and occurs largely in the urine [4, 5]. In our patient, renal failure was evident by a rise of serum creatinine to 4.32 mg/dl on day 1, which subsequently normalized by day 30 with conservative treatment. Respiratory symp- toms appeared at the end of 2nd week and features of lung fibrosis became evident within 1 month, consistent with hallmark lung findings of Paraquat poisoning [6].h Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh. 3 Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK. Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh. 3 Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK. Acknowledgements Not applicable. Acknowledgements Not applicable. Consent for publication W i i f d Written informed consent was obtained from the patient for publication of this case report and any accompanying images. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Received: 26 October 2017 Accepted: 10 May 2018 Author details 1 1 Department of Medicine, Sylhet M.A.G Osmani Medical College Hospital, Medical College Road, Sylhet 3100, Bangladesh. 2 Department of Medicine,
https://openalex.org/W4385570211	https://aclanthology.org/2023.acl-industry.59.pdf	English	null	Context-Aware Query Rewriting for Improving Users’ Search Experience on E-commerce Websites	null	2,023	cc-by	9,008	Abstract From the query alone, it is implausible to tell if the user’s intent is for costumes of actual bumblebee (i.e., the animal) or the character from the movie fran- chise. This type of ambiguity is common in e- commerce search, where queries are usually short (only 2-3 terms) and insufficiently informative (He et al., 2016b). Therefore, it is not possible to dis- ambiguate queries using only the instant search. A common solution is to use statistical rules to differentiate the possible choices. Specifically, in our example, suppose a total of 100 users entered the “bumblebee costumes” query, and 70 of them eventually purchased the movie character costume. When a new user searches for the same query, the recommended products will consist of 70% movie character costumes and 30% animal costumes. This procedure is problematic because each user has a specific intent, i.e., either the movie character cos- tume or the animal costume, but rarely both, which the aforementioned method fails to address. Context-Aware Query Rewriting for Improving Users’ Search Experience on E-commerce Websites Simiao Zuo⋄, Qingyu Yin∗†, Haoming Jiang†, Shaohui Xi†, Bing Yin†, Chao Zhang⋄and Tuo Zhao⋄ {simiaozuo, chaozhang, tourzhao}@gatech.edu {qingyy, jhaoming, shaohux, alexbyin}@amazon.com ⋄Georgia Institute of Technology †Amazon Figure 1: Searching for “bumblebee costumes” with (right) and without (left) history searches. Abstract E-commerce queries are often short and am- biguous. Consequently, query understanding often uses query rewriting to disambiguate user- input queries. While using e-commerce search tools, users tend to enter multiple searches, which we call context, before purchasing. These history searches contain contextual in- sights about users’ true shopping intents. There- fore, modeling such contextual information is critical to a better query rewriting model. How- ever, existing query rewriting models ignore users’ history behaviors and consider only the instant search query, which is often a short string offering limited information about the true shopping intent. We propose an end-to-end context-aware query rewriting model to bridge this gap, which takes the search context into account. Specifically, our model builds a ses- sion graph using the history search queries and their contained words. We then employ a graph attention mechanism that models cross-query relations and computes contextual information of the session. The model subsequently calcu- lates session representations by combining the contextual information with the instant search query using an aggregation network. The ses- sion representations are then decoded to gener- ate rewritten queries. Empirically, we demon- strate the superiority of our method to state-of- the-art approaches under various metrics. Figure 1: Searching for “bumblebee costumes” with (right) and without (left) history searches. (left) demonstrates searching for “bumblebee cos- tumes” without considering search context. From the query alone, it is implausible to tell if the user’s intent is for costumes of actual bumblebee (i.e., the animal) or the character from the movie fran- chise. This type of ambiguity is common in e- commerce search, where queries are usually short (only 2-3 terms) and insufficiently informative (He et al., 2016b). Therefore, it is not possible to dis- ambiguate queries using only the instant search. A common solution is to use statistical rules to differentiate the possible choices. Specifically, in our example, suppose a total of 100 users entered the “bumblebee costumes” query, and 70 of them eventually purchased the movie character costume. When a new user searches for the same query, the recommended products will consist of 70% movie character costumes and 30% animal costumes. This procedure is problematic because each user has a specific intent, i.e., either the movie character cos- tume or the animal costume, but rarely both, which the aforementioned method fails to address. (left) demonstrates searching for “bumblebee cos- tumes” without considering search context. ∗Correspondence to Qingyu Yin (qingyy@amazon.com). oceedings of the 61st Annual Meeting of the Association for Computational Linguistics Volume 5: Industry Track, pages 616–628 July 10-12, 2023 ©2023 Association for Computational Linguistics 2 Related Works To overcome the drawbacks of existing meth- ods, we propose an end-to-end context-aware query rewriting algorithm. Our model’s backbone is the Transformer (Vaswani et al., 2017). In our context- aware model, the Transformer encoder learns rep- resentations for individual history queries. The rep- resentations are further transformed to carry cross- query relational information using a graph atten- tion mechanism (GAT, Velickovic et al. 2018). The GAT computes contextual information of a session based on a session graph, where its nodes contain the history queries and the tokens contained in the history queries. After obtaining the contextual in- formation from the GAT, it is aggregated with the instant search using an aggregation network. The augmented information is subsequently fed into the Transformer decoder to generate rewritten queries. One line of work uses statistical methods. For ex- ample, Cui et al. (2002, 2003) extract probabilistic correlations between the search queries and the product descriptions. Other works extract features that are related to the user’s current search (Huang et al., 2003; Huang and Efthimiadis, 2009), or from relational information among the user’s his- tory searches (Billerbeck et al., 2003; Baeza-Yates and Tiberi, 2007; Wang and Zhai, 2007; Cao et al., 2008; Wang and Zhai, 2008). There are also sta- tistical machine translation-based models (Riezler et al., 2007; Riezler and Liu, 2010) that employ sequence-to-sequence approaches. The aforemen- tioned statistical methods suffer from unreliable extracted features, such that the rewritten queries differ from the original one in intent. Another line of work focuses on neural query rewriting models (He et al., 2016b; Xiao et al., 2019; Yang et al., 2019). These models adopt re- current neural networks (RNNs, Hochreiter and Schmidhuber 1997; Sutskever et al. 2014) to learn a vectorized representation for the user’s search query, after which KNN-based methods are used to find queries that yield similar representations. One major limitation is that the rewritten queries are limited to the previously presented ones. Also, these methods often involve complicated and un- grounded feature function designs, e.g., He et al. (2016b) and Xiao et al. (2019) hand-crafted 18 feature functions, or require excessive labeled data (Yang et al., 2019). Other works (Sordoni et al., 2015; Dehghani et al., 2017; Jiang and Wang, 2018) use RNNs for generative query suggestion, Our proposed method improves upon existing works from three aspects. 1 Introduction Query rewriting is a task where a user inputs a potentially problematic query (e.g., typos or insuf- ficient information), and we rewrite it to a new one that better matches the user’s real shopping intent. This task plays an important role in e-commerce query understanding, where without proper rewrit- ing, search engines often return undesired items, rendering the search experience unsatisfactory. We propose to explore contextual information from users’ history searches to resolve the query ambiguity issue. Taking the “bumblebee costumes” example again, in Figure 1 (right), suppose a rewrit- ing model recognizes that the user searched for “Transformers movie” earlier, then it could infer One major issue that impedes query rewriting is the ambiguity of queries. For example, Figure 1 ∗Correspondence to Qingyu Yin (qingyy@amazon.com). 616 Proceedings of the 61st Annual Meeting of the Association for Computational Linguistics Volume 5: Industry Track, pages 616–628 July 10-12, 2023 ©2023 Association for Computational Linguistics that the user’s purchase intent is the movie char- acter costume, and hence can remove the input ambiguity. There have been existing works that utilize search logs for query rewriting. For ex- ample, Wang and Zhai (2007, 2008) use tradi- tional TF-IDF-based similarity metrics to capture relational information among the user’s history searches. These approaches are too restrictive to handle the increasingly complex corpus nowadays. As such, the rewritten queries significantly differ from the original one in intent. More recently, neu- ral network-based query rewriting algorithms (He et al., 2016b; Xiao et al., 2019; Yang et al., 2019) are proposed. Most of such approaches employ a multi-stage training approach. Consequently, they involve complicated hand-crafted features or re- quire excessive human annotations for the interme- diate features (sometimes both). age powerful pre-trained language models, such as BART (Lewis et al., 2020). Such models contain rich semantic information and are successful in nu- merous natural language processing tasks (Devlin et al., 2019; Liu et al., 2019; Radford et al., 2019). We demonstrate the effectiveness of our method on in-house data from an online shopping plat- form. Our context-aware query rewriting model outperforms various baselines by large margins. Notably, comparing with the best baseline method (Transformer-based model), our model achieves 11.6% improvement under the MRR (Mean Recip- rocal Rank) metric and 20.1% improvement under the HIT@16 metric (a hit rate metric). We further verify the effectiveness of our approach by conduct- ing online A/B tests. History: {dodge banners; mopar poster} Source (Instance): dodger posters Target: dodge posters We collect about 3 million (M) sessions, where each session consists of at least 3 history queries, a source query (i.e., the one we need to rewrite), and a target query (i.e., the ground-truth query that is associated with the purchase). We have roughly 18.7M queries, and on average, each session con- tains 4 history queries. Query rewriting is con- sequently formulated as a sequence-to-sequence learning problem. We highlight that per our formu- lation, we do not need human annotations, unlike existing approaches. 4.1 Transformer Encoder For a given source query, we first pad it with a <boq> (begin-of-query) token. Then, we pass the padded query through a Transformer encoder, after which we have its hidden representation Hs ∈ RLs×d. Here Ls is the length of the padded query, and d is the hidden dimension. We also pass all the history queries corresponding to this source query through the encoder, and we have the history query representation Uh ∈RNh×Lh×d, where Nh is the number of history queries and Lh is the padded length. More details are presented in Appendix A. Below is an example of a search session. From the history queries, the user is interested in car related banners/posters. The source query contains a typo (“doger” is a baseball team) and we should rewrite it to the target query (“dodge posters”). 3 Problem Setup The session data are collected from search logs. First, we collect all the searches from a specific user within a time window, and we call the searches a “session”. After the user purchases a product, the session ends, i.e., we do not consider subsequent queries and behaviors after a purchase happens. This is because after a purchase, the user’s intent often change. Note that different sessions may be collected from different users. Each session contains multiple searches from the same user. We call the last query in the session the “target” query, the second to the last query the “source” (or the “instance) query, and the others the “history” queries. The intuition behind this is that because sessions always end with a purchase, the last search (i.e., the target) reflects the user’s real intent. When the user enters the second to the last search (i.e., the source), if we can rewrite it to the target query, the user’s intent will be fulfilled. Figure 2: Overview of model. 2 Related Works First, our model does not involve recursion, unlike conventional recur- rent neural network-based approaches (He et al., 2016b; Yang et al., 2019; Xiao et al., 2019). This facilitates training deep models containing dozens of layers capable of capturing high-order informa- tion. Second, our end-to-end sequence-to-sequence learning formulation eliminates the necessity of ex- cessive labeled data. Previous approaches (Yang et al., 2019; Xiao et al., 2019) require the judg- ment of “semantic similarity”, and thus crave for human annotations, which are expensive to obtain. In contrast, our method uses search logs as super- vision, which does not involve human effort, and are cheap to acquire. Third, our method can lever- 617 but they inherit the weaknesses of RNNs and yield unsatisfactory performance in practice. 4 Method Figure 2 illustrates our context-aware query rewrit- ing model. The model contains four parts: a con- ventional Transformer (Vaswani et al., 2017) en- coder, a graph attention mechanism (Velickovic et al., 2018) that captures the user’s purchase intent, an aggregation network that encodes the history searches, and a conventional Transformer decoder that generates the rewritten query candidates. Note that Grbovic et al. (2015) construct context- aware query embeddings using word2vec (Mikolov et al., 2013). In their approach, an embedding is learned for each distinct query in the dataset. As such, the quality of the learned embeddings rely heavily on the number of occurrences of each query. This method is not applicable to our case because in our dataset, almost all the queries are distinct. Figure 2: Overview of model. 4.2.2 Node Representations The next step is to refine the node representations. Each of the nodes in the session graph has its own representation. The token representations are sim- ply the corresponding representations of the tokens, extracted from the token embedding matrix. The query representations are the representations of the <boq> token in each padded history query, i.e., the representation of the Q1 query node in Figure 3 is found by Uh[0, 0, :] ∈Rd. Denote Gq = {qi}Nq i=1 and Gt = {ti}Nt i=1 the sets of representations for the query and token nodes, respectively. Here Nq is the number of query nodes and Nt is the number of token nodes. Note that all the node representations have the same size, i.e., qi, ti ∈Rd. Eventually, we obtain the updated vectorized rep- resentations {hi}Ng i=1 for all the nodes, and we treat them as the contextual information of the session. We remark that the GAT mechanism explicitly models cross-query relations by associating query representations with word representations. Such an approach is fundamentally different from exist- ing methods, where the relations are either ignored (e.g., conventional Transformer attention) or cap- tured via recursion (e.g., RNN-based approaches). 4.2 Contextual Information from Session Graphs After we obtain the history query representations Uh, the next step is to refine them. Such refinement is necessary because the Transformer encoder con- siders the history queries separately, such that their interactions are not taken into account. However, since each search depends on its previous searches in the same session, modeling cross-query relations are imperative for determining the user’s purchase 618 Figure 3: Left: Illustration of a session graph, where “T” stands for tokens and “Q” stands for queries. Right: One-step update based on the session graph. attention mechanism is defined as hi = gi + ELU  X j∈Ni αijWvgj  , where αij = exp(zij) P ℓ∈Ni exp(ziℓ), (2) (2) Figure 3: Left: Illustration of a session graph, where “T” stands for tokens and “Q” stands for queries. Right: One-step update based on the session graph. Figure 3: Left: Illustration of a session graph, where “T” stands for tokens and “Q” stands for queries. Right: One-step update based on the session graph. Figure 3: Left: Illustration of a session graph, where “T” stands for tokens and “Q” stands for queries. Right: One-step update based on the session graph. zij = LeakyReLU (Wa[Wqgi; Wkgj]) . zij = LeakyReLU (Wa[Wqgi; Wkgj]) . Here ELU(x) = x · 1{x > 0} + (exp(x) −1) · 1{x ≤0} is the exponential linear unit, Ni de- notes the neighbor of the i-th node, and Wa, Wq, Wk, Wv are trainable weights. Note that a residual connection (He et al., 2016a) is added to the last equation in Eq. 2. This has proven to be an effec- tive technique to prevent gradient vanishing, and hence, to stabilize training. intent. To this end, we use a graph attention mech- anism (Velickovic et al., 2018; Wang et al., 2020) to capture contextual information from Uh. 4.2.1 Session Graph Construction First we specify how to build a graph for each ses- sion, which we call the session graph. Suppose we have a session that contains three history queries: The session graph only induces attention be- tween nodes that are connected. For example, in Figure 3 (right), the model updates Q1 and Q2 using T3, while Q3 is unchanged, i.e., NT3 = {Q1, Q2}. A multi-head graph attention mech- anism is then defined as the concatenation of [h1 i , h2 i , · · · , hK i ], where K is the number of heads, and each of the hi is calculated via Eq. 2. Q1 : Search query : T1, T3}, Q1 : Search query : T1, T3}, Q2 : Search query : T1, T2, T3 , (1) Q3 : Search query : T1, T2, T4, T5 , where Q1, Q2, Q3 are the three queries, and T1, · · · , T5 are the five tokens that appear in the three queries. Recall Section 3 for the problem setup. Figure 3 (left) illustrates the session graph. The token node representations and the query node representations are updated iteratively. First, we update the token representations (Gt) using the query representations (Gq), in order that the tokens acknowledge to which queries they belong. Then, Gq is re-computed using the updated version of Gt, which essentially evaluates cross-query relations, using the token nodes as intermediaries. Note that the graph attention mechanism (GAT) used in each of the two steps are distinct, i.e., there are two different sets of weights [Wa, Wq, Wk, Wv]. 4.2.3 Update Node Representations We use a multi-head graph attention mechanism to update the node representations. For simplicity, denote Ng = Nq +Nt the number of distinct nodes in the session graph, and G = Gq ∪Gt = {gi}Ng i=1 the set of all the node representations. 5.2 Evaluation Metrics We use BLEU, MRR (Mean Reciprocal Rank), HIT@1, and HIT@16 to evaluate the query rewrit- ing models. For all metrics except BLEU, we report the gains over the the results calculated by using only source queries. We remark that MRR, HIT@1, and HIT@16 (the percentage that the actual prod- uct is ranked within the first 16 products i.e., the first page when we search the rewritten query) are more important than BLEU, because MRR and HIT are directly linked to user experience. Please refer to Appendix B.2 for details about these metrics. Hsess = Hs + PNg i=1 αiWvhi, (3) Hsess = Hs + PNg i=1 αiWvhi, (3) where αi = exp(zi) PNg j=1 exp(zj), zi = (Wkhi)⊤hs, Wk and Wv are trainable weights. The summation in the last equation in Eq. 3 is conducted row-wise, wherein Hsess, Hs ∈RLs×d, and v ∈Rd. The matrix Hsess serves as the representation of the ses- sion. Intuitively, by incorporating the aggregation network, we can filter out redundant information from the session history and only keep the ones pertinent to the source query. (3) where αi = exp(zi) PNg j=1 exp(zj), zi = (Wkhi)⊤hs, Wk j 1 and Wv are trainable weights. The summation in the last equation in Eq. 3 is conducted row-wise, wherein Hsess, Hs ∈RLs×d, and v ∈Rd. The matrix Hsess serves as the representation of the ses- sion. Intuitively, by incorporating the aggregation network, we can filter out redundant information from the session history and only keep the ones pertinent to the source query. j and Wv are trainable weights. The summation in the last equation in Eq. 3 is conducted row-wise, wherein Hsess, Hs ∈RLs×d, and v ∈Rd. The matrix Hsess serves as the representation of the ses- sion. Intuitively, by incorporating the aggregation network, we can filter out redundant information from the session history and only keep the ones pertinent to the source query. 5.3 Experimental Results Table 1 summarizes experimental results. Recall that in our formulation, we rewrite a source query to a target query. The “target query” entry in Ta- ble 1 is the performance gain of the ground truth target query, i.e., this entry signifies upper bounds of performance gain that any model can achieve. After the Transformer encoder, the graph atten- tion mechanism, and the aggregation network, we obtain Hsess, the session representation that con- tains information on both the source query and its history searches. Subsequently, Hsess is fed into the Transformer decoder to generate rewrit- ten query candidates. The algorithm is detailed in Algorithm 1 in Appendix D. We can see that the attention-based models (i.e., BART, MeshBART, Transformer and MeshTrans- former) outperforms the recurrent neural network- based approach (i.e., LQRW and HRED). This is because RNNs suffer from forgetting and training issues. In contrast, Transformer-based models use the attention mechanism instead of recursion to cap- ture dependencies, which has proven to be more ef- fective. Moreover, by aggregating history searches, BART+Aggregation and Transformer+Aggregation consistently outperform their vanilla alternatives. Essentially performance of these two methods indi- cate that integrating history queries into training is critical. The performance is further enhanced by in- corporating the session graphs. Specifically, Trans- former+Aggregation+Graph achieves the best per- formance under almost all the metrics. Notice that the HIT@16 metric gain improves from +15.9 to 5.1 Baselines For baselines with pre-training, we use MeshBART (Chen and Lee, 2020) and BART (Lewis et al., 2020). For baselines without pre-training, we use LQRW (He et al., 2016b), HRED (Sordoni et al., 2015) and MeshTransformer (Chen and Lee, 2020) (a variant of MeshBART where we train the model from scratch). We also compare our algorithm with two model variants: Transformer+Aggregation and BART+Aggregation, where we use the aggregation network but not the GAT mechanism. Please refer to Appendix B.1 for details. Figure 4: Aggregation network. prepended <boq> token as the representation of the source query, which is denoted hs ∈Rd. We adopt an aggregation network to extract useful in- formation with respect to hs from the contextual information {hi}Nh+Nt i=1 . The network employs an attention mechanism that determines to what extent each vector hi contributes to the source query hs. Figure 4 illustrates the architecture of the aggrega- tion network. Concretely, 4.3 Session Representations Recall that we pass the source query through a Transformer encoder and obtain Hs ∈RLs×d. The matrix Hs contains representations for all the tokens in the source query. We use that of the With the above notations, a single-head graph 619 Figure 4: Aggregation network. Figure 4: Aggregation network. 5 Experiments We conduct experiments on some in-house data. We implement two methods with different model architectures: Transformer+Aggregation+Graph and BART+Aggregation+Graph. The first one is constructed in the previous section, and the sec- ond one employs a fine-tuning approach instead of training-from-scratch. The training details are de- ferred to Appendix B.3. More experimental results are shown in Appendix C. 620 Table 1: Experimental results. The results of MRR, HIT@1, and HIT@16 are shown as gain over the source query. The best results are shown in bold. Number of candidates #Candidates=5 #Candidates=10 BLEU Metric MRR HIT@1 HIT@16 MRR HIT@1 HIT@16 Source Query 0 0 0 0 0 0 — Target Query +16.1 +10.6 +29.0 +16.1 +10.6 +29.0 — Baseline methods LQRW +3.5 +2.5 +6.4 +6.8 +4.9 +12.6 29.4 HRED +4.7 +3.2 +8.4 +8.1 +5.7 +14.2 25.7 BART +4.6 +3.1 +8.2 +8.2 +5.5 +14.8 30.9 Transformer +4.3 +2.6 +9.2 +8.5 +5.6 +15.9 25.3 MeshBART +5.0 +3.8 +8.7 +8.3 +5.8 +14.3 31.7 MeshTransformer +4.0 +2.7 +8.4 +8.3 +5.6 +15.7 25.9 Our methods BART+Aggregation +6.3 +3.9 +10.9 +9.7 +6.4 +17.1 31.9 Transformer+Aggregation +5.2 +2.9 +10.8 +10.2 +7.0 +17.3 27.2 BART+Aggregation+Graph +6.9 +4.6 +11.8 +10.5 +7.5 +17.6 32.9 Transformer+Aggregation+Graph +6.6 +4.6 +12.0 +11.6 +8.3 +20.1 28.2 Table 1: Experimental results. The results of MRR, HIT@1, and HIT@16 are shown as gain over the source query. The best results are shown in bold. Table 2: Two examples of context-aware query rewriting with and without context. +20.1 when employing both the aggregation net- work and the session graph formulation for the Transformer-based models. We highlight that the graph attention mechanism can directly captures cross-query relations, which is implausible for all the baselines. We can see that this property indeed contributes to model performance, i.e., HIT@16 in- creases from +17.3 to +20.1 when we equip Trans- former+Aggregation with the GAT mechanism. +20.1 when employing both the aggregation net- work and the session graph formulation for the Transformer-based models. We highlight that the graph attention mechanism can directly captures cross-query relations, which is implausible for all the baselines. We can see that this property indeed contributes to model performance, i.e., HIT@16 in- creases from +17.3 to +20.1 when we equip Trans- former+Aggregation with the GAT mechanism. 5 Experiments Example 1 dodge led sign; History dodge banners; mopar banner; mopar poster Source dodger posters Target dodge posters Rewritten w/o context dodger flag Rewritten w/ context dodge poster Example 2 samsung galaxy case; History samsung galaxy a11 case; samsung a11 case Source samsung galaxy a7 Target samsung galaxy a7 case Rewritten w/o context samsung galaxy a7 charger Rewritten w/ context samsung galaxy a7 case Notice that BLEU is not a definitive metric. For example, the MRR and HIT metrics of HRED are consistently higher than those of LQRW, even though the BLEU score of the former is signifi- cantly lower than the latter. Also, compared with Transformer-based models, the BLEU score is con- sistently higher when using the BART model as the backbone. This is because a pre-trained lan- guage model contains more semantic information. However, the MRR and HIT metrics of the BART- based models are slightly worse than those of the Transformer-based models. search system. Experiments are conducted over five days, during which our system processed over 30 million sessions. The proposed method im- proves business metrics in terms of revenue; and also significantly decreases the number of refor- mulated searches. This indicates that the rewrit- ten queries better meet customers’ shopping intent since customers are able to find their desired prod- ucts with less number of searches. However, the BLEU score is comparable for models with the same backbone. For example, for Transformer vs. Transformer+Aggregation vs. Transformer+Aggregation+Graph, the BLEU scores are 25.3 vs. 27.2 vs. 28.2. Such a tendency coincides with the online metrics. We observe the same results from BART-based models. 5.4 Online A/B Test ⋄Advantages of leveraging history information Two examples are shown in Table 2. The first exam- ple is error correction. In the example, the customer wishes to purchase dodge (a car brand) posters, but she mistakenly searches for dodger (a baseball team) posters. Without history information, it is impossible to determine the customer’s true intent. We conduct online A/B experiments on a large- scale e-commerce shopping platform with our query rewriting models. For a given search query within a session, we generate one reformulated query using the proposed model, and we feed both the original query and rewritten query into the 621 Table 3: Two examples of generated queries and their associated likelihood. Type Query Likelihood Query Likelihood History iphone 11 pro case pokemon; colorado 2005 tail lights; iphone 11 pro case eevee; — colorado 2005 door — iphone 11 pro case hetalia; colorado 2005 accessories iphone 11 pro case sailor moon Source iphone 11 pro case snow leopard — colorado headlights — Target iphone 11 pro case tiger — colorado 2005 headlights — Rewritten iphone 11 pro case disney 0.497 2005 colorado headlights 0.566 iphone 11 pro case sailor moon 0.492 colorado headlights 2005 0.458 iphone 11 pro case harry potter 0.445 colorado headlights led 0.357 iphone 11 pro case 0.440 colorado headlights assembly 0.301 iphone 11 pro case cute 0.419 colorado tail lights 0.289 iphone 11 pro case leopard 0.391 colorado headlights housing 0.237 iphone 11 pro case clear 0.379 colorado led headlights 0.234 iphone 11 pro case disney princess 0.372 2004 colorado headlights 0.230 iphone 11 pro case pink 0.364 colorado 2004 headlights 0.214 iphone 11 pro case totoro 0.353 colorado headlights 2004 0.208 acters. Finally, notice that the likelihood of all the suggested queries is similar, which means our model cannot single out a significantly better query than the others. Therefore our model generated a diverse group of queries. However, by looking at session histories, we find that all the previous searches are related to auto- mobiles (e.g., dodge and mopar), and therefore the query should be rewritten to “dodge posters”. Our model successfully captures this pattern. Notice that the rewritten query without leveraging context does not match the user’s intent. In the second example (the right two columns), the generated query successfully matches the target query. 5.4 Online A/B Test Note that the top two generated queries have high likelihood, and the likelihood decreases dras- tically as the suggested queries become more and more implausible. In this example, the first query is 172% more likely than the tenth query, whereas this number is only 41% in the previous example. This suggests that our model can differentiate be- tween good quality suggestions and poor quality alternatives. The second example is keyword refinement. In the example, by looking at the history searches, it is obvious that the customer wishes to find phone cases, instead of phones. However, this intent is im- possible to capture by using only the source query. Our model automatically adds the keyword “case” to the source query and matches the target query. On the other hand, without the context information, the rewritten result is not satisfactory. References Kaiming He, Xiangyu Zhang, Shaoqing Ren, and Jian Sun. 2016a. Deep residual learning for image recog- nition. In 2016 IEEE Conference on Computer Vision and Pattern Recognition, CVPR 2016, Las Vegas, NV, USA, June 27-30, 2016, pages 770–778. IEEE Com- puter Society. Ricardo Baeza-Yates and Alessandro Tiberi. 2007. Ex- tracting semantic relations from query logs. 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In Proceedings of the Eleventh International World Wide Web Conference, WWW 2002, May 7- 11, 2002, Honolulu, Hawaii, USA, pages 325–332. ACM. Jyun-Yu Jiang and Wei Wang. 2018. RIN: reformula- tion inference network for context-aware query sug- gestion. In Proceedings of the 27th ACM Interna- tional Conference on Information and Knowledge Management, CIKM 2018, Torino, Italy, October 22- 26, 2018, pages 197–206. ACM. Hang Cui, Ji-Rong Wen, Jian-Yun Nie, and Wei-Ying Ma. 2003. 6 Conclusion and Discussion ⋄Diversity of query generation Table 3 demon- strates two examples. In the first example (the left three columns), notice that our model can grep in- formation from history queries, e.g., “iphone 11 case sailor moon”, and can delete keywords that are deemed insignificant or too restrictive, e.g., “iphone 11 case leopard” instead of “snow leop- ard”. Also, our model can effectively capture do- main information. For example, some of the history query keywords (e.g., pokemon, eevee) are often described as “cute”, and our model recommends this keyword. All the history keywords are from Japanese anime series, therefore our model sug- gests another popular character, “totoro”. Addition- ally, the “disney” and “disney princess” keywords are generated based on the interest to virtual char- We propose an end-to-end context-aware query rewriting model that can efficiently leverage user’s history behavior. Our model infers a user’s pur- chase intent by modeling her history searches as a graph, on which a graph attention mechanism is applied to generate informative session repre- sentations. The representations are subsequently decoded into rewritten queries. Our proposed ses- sion graph can be extended to incorporate more information. Here, we present a bipartite graph, which contains words and queries. Additional com- ponents can be added as extra layers. For example, we can add product information such as categories to the session graph, which will create 3-partite session graphs (word, query and product). 622 References 2019. fairseq: A fast, extensible toolkit for sequence modeling. In Proceedings of the 2019 Con- ference of the North American Chapter of the Associa- tion for Computational Linguistics (Demonstrations), pages 48–53, Minneapolis, Minnesota. Association for Computational Linguistics. Petar Velickovic, Guillem Cucurull, Arantxa Casanova, Adriana Romero, Pietro Liò, and Yoshua Bengio. 2018. Graph attention networks. In 6th International Conference on Learning Representations, ICLR 2018, Vancouver, BC, Canada, April 30 - May 3, 2018, Conference Track Proceedings. OpenReview.net. 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In Proceedings of the Twelfth ACM International Con- ference on Web Search and Data Mining, WSDM 2019, Melbourne, VIC, Australia, February 11-15, 2019, pages 402–410. ACM. Stefan Riezler and Yi Liu. 2010. Query rewriting using monolingual statistical machine translation. Compu- tational Linguistics, 36(3):569–582. References Query expansion by mining user logs. IEEE Transactions on knowledge and data engineer- ing, 15(4):829–839. Diederik P. Kingma and Jimmy Ba. 2015. Adam: A method for stochastic optimization. In 3rd Inter- national Conference on Learning Representations, ICLR 2015, San Diego, CA, USA, May 7-9, 2015, Conference Track Proceedings. Mostafa Dehghani, Sascha Rothe, Enrique Alfonseca, and Pascal Fleury. 2017. Learning to attend, copy, and generate for session-based query suggestion. In Proceedings of the 2017 ACM on Conference on In- formation and Knowledge Management, CIKM 2017, Singapore, November 06 - 10, 2017, pages 1747– 1756. ACM. Mike Lewis, Yinhan Liu, Naman Goyal, Marjan Ghazvininejad, Abdelrahman Mohamed, Omer Levy, Veselin Stoyanov, and Luke Zettlemoyer. 2020. BART: Denoising sequence-to-sequence pre-training for natural language generation, translation, and com- prehension. In Proceedings of the 58th Annual Meet- ing of the Association for Computational Linguistics, pages 7871–7880, Online. Association for Computa- tional Linguistics. Jacob Devlin, Ming-Wei Chang, Kenton Lee, and Kristina Toutanova. 2019. BERT: Pre-training of deep bidirectional transformers for language under- standing. In Proceedings of the 2019 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Tech- nologies, Volume 1 (Long and Short Papers), pages 4171–4186, Minneapolis, Minnesota. Association for Computational Linguistics. Yinhan Liu, Myle Ott, Naman Goyal, Jingfei Du, Man- dar Joshi, Danqi Chen, Omer Levy, Mike Lewis, Luke Zettlemoyer, and Veselin Stoyanov. 2019. Roberta: A robustly optimized bert pretraining ap- proach. arXiv preprint arXiv:1907.11692. Mihajlo Grbovic, Nemanja Djuric, Vladan Radosavl- jevic, Fabrizio Silvestri, and Narayan Bhamidipati. 2015. Context- and content-aware embeddings for query rewriting in sponsored search. In Proceedings of the 38th International ACM SIGIR Conference on Research and Development in Information Retrieval, Santiago, Chile, August 9-13, 2015, pages 383–392. ACM. Ilya Loshchilov and Frank Hutter. 2019. Decoupled weight decay regularization. In 7th International Conference on Learning Representations, ICLR 2019, New Orleans, LA, USA, May 6-9, 2019. OpenRe- view.net. 623 Tomas Mikolov, Kai Chen, Greg Corrado, and Jef- frey Dean. 2013. Efficient estimation of word representations in vector space. arXiv preprint arXiv:1301.3781. Ashish Vaswani, Noam Shazeer, Niki Parmar, Jakob Uszkoreit, Llion Jones, Aidan N. Gomez, Lukasz Kaiser, and Illia Polosukhin. 2017. Attention is all you need. In Advances in Neural Information Pro- cessing Systems 30: Annual Conference on Neural Information Processing Systems 2017, December 4-9, 2017, Long Beach, CA, USA, pages 5998–6008. Myle Ott, Sergey Edunov, Alexei Baevski, Angela Fan, Sam Gross, Nathan Ng, David Grangier, and Michael Auli. B.1 Baselines The baselines are split into two groups: without pre-training and with pre-training. For the w/o pre-training group, we build the following models: ⋄Learning to Rewrite Queries (LQRW) (He et al., 2016b) is one of the first methods that applies deep learning techniques to query rewriting. Specif- ically, the LQRW model combines a sequence- to-sequence LSTM (Hochreiter and Schmidhuber, 1997; Sutskever et al., 2014) model with statisti- cal machine translation (Riezler and Liu, 2010) techniques to generate queries. The candidates are subsequently ranked using hand-crafted feature functions. After the initial embedding layers, we pass Ys through the self-attention module. Specifically, we compute attention output S by S = Softmax QKT √dk V , where Q = YsWq, K = YsWk, V = YsWv. (4) ⋄ Hierarchical Recurrent Encoder-Decoder (HRED) (Sordoni et al., 2015) employs a hierarchi- cal recurrent neural network for generative query suggestion. The model is a step forward from its predecessors in that HERD is sensitive to the order of queries and the method is able to suggest rare and long-tail queries. ⋄ Hierarchical Recurrent Encoder-Decoder (HRED) (Sordoni et al., 2015) employs a hierarchi- cal recurrent neural network for generative query suggestion. The model is a step forward from its predecessors in that HERD is sensitive to the order of queries and the method is able to suggest rare and long-tail queries. Here Wq, Wk ∈Rd×dK, Wv ∈Rd×dV are learn- able weights. In practice we use multi-head self- attention to increase model flexibility. To facil- itate this, different attention outputs S1, · · · , SH are computed using different sets of weights {W h q , W h k , W h v }H h=1. The final attention output is ⋄Transformer (Vaswani et al., 2017) has achieved superior performance in various sequence-to- sequence (seq2seq) learning tasks. To adopt Trans- former to query rewriting, we treat the source query as the source-side input, and the target query as the target-side input. Then we train a model using only these constructed inputs, similar to machine trans- lation. Note that this setting resembles most of the existing works. We adopt the Transformer-base ar- chitecture, which contains about 72M parameters. S = [S1, S2, · · · , SH] Wo, (5) S = [S1, S2, · · · , SH] Wo, (5) where Wo ∈RHdV ×d is a learnable aggregation matrix. References Stefan Riezler, Alexander Vasserman, Ioannis Tsochan- taridis, Vibhu Mittal, and Yi Liu. 2007. Statistical machine translation for query expansion in answer re- trieval. In Proceedings of the 45th Annual Meeting of the Association of Computational Linguistics, pages 464–471, Prague, Czech Republic. Association for Computational Linguistics. Ya Yatao Yang, Jun Tan, Hongbo Deng, Zibin Zheng, Yu- tong Lu, and Xiangke Liao. 2019. An active and deep semantic matching framework for query rewrite in e-commercial search engine. In Proceedings of the 28th ACM International Conference on Information and Knowledge Management, CIKM 2019, Beijing, China, November 3-7, 2019, pages 309–318. ACM. Alessandro Sordoni, Yoshua Bengio, Hossein Vahabi, Christina Lioma, Jakob Grue Simonsen, and Jian-Yun Nie. 2015. A hierarchical recurrent encoder-decoder for generative context-aware query suggestion. In Proceedings of the 24th ACM International Confer- ence on Information and Knowledge Management, CIKM 2015, Melbourne, VIC, Australia, October 19 - 23, 2015, pages 553–562. ACM. Ilya Sutskever, Oriol Vinyals, and Quoc V. Le. 2014. Sequence to sequence learning with neural networks. In Advances in Neural Information Processing Sys- tems 27: Annual Conference on Neural Information Processing Systems 2014, December 8-13 2014, Mon- treal, Quebec, Canada, pages 3104–3112. 624 B Experiments Details For a given source query, we first pad it with a <boq> (begin-of-query) token. Then, we pass the padded query through a token embedding layer and a position embedding layer, and we obtain Ys ∈RLs×d. Here Ls is the length of the padded source query, and d is the embedding dimension. Note that the position embedding can either be a sinusoidal function or a learned matrix. B.1 Baselines The attention output is then fed through a position-wise feed-forward neural network to gen- erate encoded representation Hs ∈RLs×d for the source query: Hs = ReLU S W 1 FFN + b1 W 2 FFN + b2. (6) (6) ⋄MeshTransformer (Chen and Lee, 2020) is a variant of MeshBART, where the pre-trained BART module is replaced by a Transformer and the model is trained from scratch. The method concatenates history queries to the source query in order to inte- grate contextual information. See the MeshBART method below for details. Here {W 1 FFN, W 2 FFN, b1, b2} are weights of the neu- ral network. Equations 4, 5, and 6 constitute as an encoder block. In practice we stack multiple encoder blocks to build the Transformer encoder, as demonstrated in Figure 2. For the history queries in this session, we also pad them with <boq> tokens. Suppose that we have Nh padded history queries (recall a session contains multiple history queries), and their respec- tive length is denoted by L1 h, · · · , LNh h . We pad the history queries to the same length, and we obtain the history query matrix Xh ∈RNh×Lh, where Lh = max{L1 h, · · · , LNh h }. Then, following the same procedures as encoding the source query, we pass Xh through the embedding layers and the en- coder blocks, after which we obtain the history query representations Uh ∈RNh×Lh×d. ⋄Transformer+Aggregation is the model where we use the aggregation network to encode history search queries, i.e., without the graph attention mechanism. Specifically, we first obtain the rep- resentations of the source query and the history queries from the Transformer encoder. Then, we extract information related to the source query from the history representations using an aggregation network. Such information is added to the source representation, and we follow a standard decoding 625 procedure using these two factors. See Section 4.3 for details. for r1 is score1 = 1/2 = 0.5. The score of the rewritten queries r1, · · · , r10 is then defined as max{scorei}10 i=1. Finally, the score for the query rewriting model is the average over all the source query scores. The second group of methods adopt pre-trained language models for query rewriting. ⋄BART (Lewis et al., 2020) is a pre-trained seq2seq model. 1https://github.com/pytorch/fairseq/blob/ master/examples/translation/README.md B.3 Training Details We use the Fairseq (Ott et al., 2019) code-base with PyTorch (Paszke et al., 2019) as the back-end to implement all the methods. All the experiments are conducted using 8 NVIDIA V100 (32GB) GPUs. For training a Transformer model from scratch, we adopt the Transformer-base (Vaswani et al., 2017) architecture. We use Adam (Kingma and Ba, 2015) as the optimizer, and the learning rate is chosen from {3 × 10−4, 5 × 10−4, 1 × 10−3}. We use 4 heads for the multi-head graph attention mechanism, where the head dimension is set to be 128 (note that the Transformer-base architecture has embedding dimension 512). ⋄MeshBART (Chen and Lee, 2020) is a BART- based model that first concatenates the history queries to the source query, and then feeds the concatenated input to a pre-trained BART model for query generation. Note that the original method requires click information. We remove this com- ponent as the proposed method do not need such data. ⋄ BART+Aggregation is similar to Trans- former+Aggregation, except we replace the Trans- former backbone with the pre-trained seq2seq BART model. For fine-tuning a BART model, we adopt the BART-base (Lewis et al., 2020) architecture. We use AdamW (Loshchilov and Hutter, 2019) as the optimizer, and the learning rate is chosen from {3 × 10−5, 5 × 10−5, 1 × 10−4}. Similar to the training from scratch scheme, we adopt 4 heads, each with dimension 192, for the graph attention mechanism. B.1 Baselines We adopt this particular model instead of, for example, BERT (Devlin et al., 2019) or GPT-2 (Radford et al., 2019), because we treat query rewriting as a seq2seq task. And the aforementioned architectures have either the Transformer encoder (e.g., BERT) or the Trans- former decoder (e.g., GPT-2), but not both. In our experiments, BART is fine-tuned in a setting sim- ilar to training the Transformer model. We adopt the BART-base architecture in all the experiments, which contains about 140M parameters. We also use HIT@1 and HIT@16 as evaluation metrics. The HIT@16 metric is the percentage that the actual product is ranked within the first 16 products (the first page) when we search the rewritten query. And the HIT@1 metric is similarly defined. B.2 Evaluation Metrics We use the BLEU score (Post, 2018) as an eval- uation metric. This metric is constantly used to evaluate the quality of translation. We adopt it here because similar to machine translation, we formu- late query rewriting as a seq2seq learning task. The correlation between the rewritten query and the tar- get query reflects the model’s ability to capture the user’s purchase intent. For both training-from-scratch and fine-tuning, please refer to1 Ott et al. (2019) for more de- tails such as pre-processing steps and other hyper- parameters. C More Experimental Results The MRR metric describes the accuracy of the rewritten queries. For each source query in the test set, we generate 10 candidate queries r1, · · · , r10. Then we search each of these candidates using our production search engine, and we obtain the re- turned products, of which we only keep the top 32. Recap that we know the actual product that the customer purchased. The next step is to cal- culate the reciprocal of the actual product’s rank for each of r1, · · · , r10. For example, suppose for r1, the actual purchased product is the sec- ond within the 32 returned products, then the score C.1 Analysis ⋄BART vs. Transformer Even though BART contains twice the number of parameters compared with Transformer (140M vs. 70M), models fine- tuned on BART yield lower MRR and HIT metrics (Table 1). One reason is that publicly available pre- trained models are pre-trained on natural language corpus, but queries are usually short and have dis- tinct structures. This raises doubts on whether cur- 626 the relation between model size and performance, where we decrease the embedding dimension (cor- respondingly the FFNs’ hidden dimensions) and the number of layers. We can see that even with 1/8 of the parameters, model performance does not decrease much. Moreover, our model is more than 20% smaller than a BERT-base model (85M vs. 110M), rendering online deployment more than possible. rent pre-trained models are suitable for the query domain. Indeed, the rich semantic information en- ables a much better BLEU score (32.9 vs. 28.2), but the MRR and HIT metrics suggest the fine-tuned models’ unsatisfactory performance. Another reason is that in a conventional fine- tuning task, a task-specific head is appended to the pre-trained model, and the head usually con- tains only a small number of parameters. But in the query rewriting task, both the aggregation net- work and the graph attention mechanism contain a significant amount of parameters (about 10% of BART). This is problematic because in fine- tuning, the learning rate is usually small since nearly all the weights are supposed to be mean- ingful and should not change much. Yet, in our case, we need to properly train a large amount of randomly initialized parameters. Moreover, the aggregation network and the GAT are added in- side the pre-trained model (more specifically, they are added to the BART encoder) instead of ap- pended after BART. Essentially this nullifies the pre-trained parameters on the decoder side, impos- ing additional challenges to the fine-tuning task. Nevertheless, the BART+Aggregation model still outperforms the vanilla BART model, and the per- formance is further improved by adding the GAT (i.e., BART+Aggregation+Graph). ⋄Query length vs. performance Figure 7 demon- strates model performance regarding length of the instant query. We can see that the BLEU score grad- ually decreases when the length increases. This is because long queries are often very specific (e.g., down to specific models or makes), making the rewriting task harder. D Detailed Algorithm D Detailed Algorithm ⋄Training from scratch vs. fine-tuning Figure 5 plots the training and validation perplexity (ppl) of the training-from-scratch approach and the fine- tuning approach. From Figure 5a and Figure 5b, we can see that by employing the aggregation network, Transformer+Aggregation fits the data better and exhibits enhanced generalization. The training and validation ppls are further significantly improved by incorporating the graph attention mechanism, i.e., by using Transformer+Aggregation+Graph, we achieve even better performance. Notice that in Figure 5c, BART+Aggregation out- performs BART+Aggregation+Graph in terms of training ppl, which is different from the training- from-scratch approach. As indicated by Figure 5d, BART+Aggregation shows clear sign of over-fitting. This is because even though pre-trained language models contain rich semantic information, much of it is considered “noisy” for query rewriting. Thus feature enhancement initiated by the graph atten- tion mechanism is needed. ⋄Model size vs. performance Figure 6 illustrates ⋄Model size vs. performance Figure 6 illustrates 627 (a) Transformer (train). (b) Transformer (valid). (c) BART (train). (d) BART (valid). Figure 5: Training and validation perplexity using Transformer and BART as backbone. Figure 5: Training and validation perplexity using Transformer and BART as backbone. Figure 6: Model performance (in BLEU scores) vs. model size. The model size (in millions of parameters) are shown above the bars. Figure 7: Query length vs. rewrit- ing quality. Figure 6: Model performance (in BLEU scores) vs. model size. The model size (in millions of parameters) are shown above the bars. Figure 7: Query length vs. rewrit- ing quality. Algorithm 1: Context-aware query rewriting. Input: D: dataset containing sessions; Initial parameters for the Transformer encoder and the Transformer decoder; Initial parameters for two graph attention mechanism (Eq. 2): GA GA i i l f h i k K h b Figure 6: Model performance (in BLEU scores) vs. model size. The model size (in millions of parameters) are shown above the bars. Figure 7: Query length vs. rewrit- ing quality. Figure 7: Query length vs. rewrit- ing quality. Figure 6: Model performance (in BLEU scores) vs. model size. The model size (in millions of parameters) are shown above the bars. Algorithm 1: Context-aware query rewriting. Input: D: dataset containing sessions; Initial parameters for the Transformer encoder and the Transformer decoder; Initial parameters for two graph attention mechanism (Eq. 2): GATt→q, GATq→t; Initial parameters for the aggregation network (Eq. 3); K: the number of updates on the session graph; N: the number of rewritten queries for each session. O t t A li t th t t i N t d i f h i i th d t t Input: D: dataset containing sessions; Initial parameters for the Transformer encoder and the Transformer decoder; Initial parameters for two graph attention mechanism (Eq. 2): GATt→q, GATq→t; Initial parameters for the aggregation network (Eq. 3); K: the number of updates on the session graph; N: the number of rewritten queries for each session. g Output: A list that contains N generated queries for each session in the dataset. Rewritten results: rewritten = {}; Output: A list that contains N generated queries for each session in the dataset. Rewritten results: rewritten = {}; for each session in D do /* Encode input data. / Compute source representation Hs and history representation Uh using the Transformer encoder; Compute source representation Hs and history representation Uh using the Transformer encoder; / Apply graph attention. / / Apply graph attention. / / Apply graph attention. / Obtain initial representations G0 t , G0 q; Obtain initial representations G0 t , G0 q; for k = 1 · · · K do Gk t = GATq→t(Gk−1 q , Gk−1 t ); Gk q = GATt→q(Gk t , Gk−1/2 q ); end Set history representation {hi}Nt+Nh i=1 = GK t ∪GK h ; Set history representation {hi}Nt+Nh i=1 = GK t ∪GK h ; Set history representation {hi}Nt+Nh i=1 = GK t ∪GK h ; i 1 / Apply aggregation network. / i / Apply aggregation network. / Compute session representation Hsess from Hs and {hi}Nt+Nh i=1 using Eq. 3; / Generate rewritten queries. / Compute session representation Hsess from Hs and {hi}Nt+Nh i=1 using Eq. 3; Compute session representation Hsess from Hs and {hi}Nt+Nh i=1 using Eq. 3; / Generate rewritten queries. */ Generate N rewritten queries {qi}N i=1 using the Transformer decoder and a beam search procedure; Generate N rewritten queries {qi}N i=1 using the Transformer decoder and a beam search procedure; N p rewritten ←rewritten ∪{qi}N i=1; end rewritten ←rewritten ∪{qi}N i=1; end end Output: The rewritten queries. 628
https://openalex.org/W4288357278	https://zenodo.org/records/2801294/files/124.project%20165.pdf	English	null	ANALYSIS OF SALIVARY PROFILE IN TYPE 2 DIABETES MELLITUS ADULT PATIENTS	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	2,096	Ahmad Hassan et al Ahmad Hassan et al IAJPS 2019, 06 (05), 9552-9554 ISSN 2349-7750 ISSN: 2349-7750 Research Article Research Article Available online at: http://www.iajps.com ANALYSIS OF SALIVARY PROFILE IN TYPE 2 DIABETES MELLITUS ADULT PATIENTS 1Dr Ahmad Hassan, 2Dr Haseeb ur Rehman, 3Dr Ramna Chahal 1House Officer at Holy Family Hospital, Rawalpindi, 2Medical Officer at BHU Dhoke Budhal, Rawalpindi, 3House Officer at Benazir Bhutto Hospital, Rawalpindi. Article Received: March 2019 Accepted: April 2019 Published: May 2019 Abstract: Introduction: Diabetes mellitus is a chronic condition with severe long-term, disabling complications, and unknown remedy that is characterized by disorders in carbohydrate, fat, and protein metabolism. Aims and objectives: The basic aim of the study is to analyze the salivary profile in type 2 diabetes mellitus adult patients. Introduction: Diabetes mellitus is a chronic condition with severe long-term, disabling complications, and unknown remedy that is characterized by disorders in carbohydrate, fat, and protein metabolism. unknown remedy that is characterized by disorders in carbohydrate, fat, and protein metabolism. Aims and objectives: The basic aim of the study is to analyze the salivary profile in type 2 diabetes mellitus adult patients. Material and methods: This case control study was conducted in Holy Family Hospital, Rawalpindi during April 2018 to November 2018. The data was collected from 100 type-2 DM patients. We divided the data into two groups. One was control group and one was diseased group. The enrolled cases had a history of T2DM which had a minimum duration of 2 years. Samples were centrifuged at 5000 rpm, the supernatants were collected and they were stored at – 8000C until further analysis. Results: The data were collected from 100 patients. The mean age of the participants in the study was 46.5 years. No significant differences were observed between the sexes in both groups in pH, salivary flow rates and biochemical parameters. Salivary pH was observed to be significantly lower in diabetics as compared to that to non-diabetics. Flow rate was significantly diminished in diabetics. Biochemical determinations showed significant differences between non– diabetics and diabetics. Conclusion: It is concluded that there is a significant variations in both physical and biochemical parameters of saliva in T2DM, thereby emphasizing the fact that the salivary composition was not just a reflection of the oral health state of a subject, but also of one’s systemic state. Corresponding author: Dr. Ahmad Hassan, House Officer at Holy Family Hospital, Rawalpindi. Ahmad Hassan et al Please cite this article in press Ahmad Hassan et al., Analysis Of Salivary Profile In Type 2 Diabetes Mellitus Adult Patients., Indo Am. J. P. Sci, 2019; 06(05). QR code MATERIAL AND METHODS: MATERIAL AND METHODS: This case control study was conducted in Holy Family Hospital, Rawalpindi during April 2018 to November 2018. The data was collected from 100 type-2 DM patients. We divided the data into two groups. One was control group and one was diseased group. The enrolled cases had a history of T2DM which had a minimum duration of 2 years. Data collection: People who were on treatment with antidepressants, antihistaminics and anti hypertensives, those who were edentulous or had any systemic illnesses and those who were undergoing radiotherapy to head and neck region were excluded from the study. Questionnaires which requested details of demographic data and medical history were administered to the subjects, following which saliva was collected from them in the morning between 7 am–8.30 am in the fasting state. Unstimulated whole saliva was collected by means of the standardised spitting technique, for 5 minutes. Samples were centrifuged at 5000 rpm, the supernatants were collected and they were stored at – 8000C until further analysis. This disease is a public health problem with important social repercussions. It is a relatively common ill-health condition in Iran with a prevalence of 7.7% in the age range of 25-64 years and with a rather high burden [3]. Diabetes can be divided into types 1 and 2. In type 1 diabetes, the - cells of the pancreas are destroyed by the autoimmune mechanism. In type 2 diabetes mellitus (T2DM), a resistance to insulin is developed. A common oral problem associated with DM is xerostomia, whose intensity is significantly correlated with salivary level of glucose, increased infections, salivary dysfunction, dental caries and lichenoid reaction caused by certain anti-diabetic drugs in DM patients [4]. Differences in saliva production and composition have been observed previously between diabetic and non-diabetic subjects. Approximately 5% of all patients visiting dental clinics are reported to have diabetes. Therefore, examination of the composition of saliva in patients with diabetes [5]. INTRODUCTION: imbalances or a combination of these are responsible for salivary hypo function and dehydration in diabetics [6]. Diabetes mellitus is a chronic condition with severe long-term, disabling complications, and unknown remedy that is characterized by disorders in carbohydrate, fat, and protein metabolism. Prevalence of diabetes in the adult population is currently considered to be 6.4% in the world. Type 2 diabetes (T2D) is caused primarily by insulin resistance in the liver, muscle, and adipose tissue as peripheral target organs [1]. This disease strongly impacts the production and composition of saliva because it is connected with autonomic neuropathies, microvascular alternations, and hormonal imbalances, or a combination of all these. Saliva is a fluid with complex compound and specific roles as well as the principal defensive factor in the mouth which contains informative components that can be used as diagnostic markers for human diseases. There are specific antibody systems in saliva [2]. Statistical analysis The data was collected and analyzed using SPSS version 21.0. Bi-variate analysis was conducted using Chi-square and t-test to investigate associations. Aims and objectives The basic aim of the study is to analyze the salivary profile in type 2 diabetes mellitus adult patients. Corresponding author: Corresponding author: Dr. Ahmad Hassan, Dr. Ahmad Hassan, House Officer at Holy Family Hospital, Rawalpindi. article in press Ahmad Hassan et al., Analysis Of Salivary Profile In Type 2 Diabetes Mellitus Adult Patients., Indo Am. J. P. Sci, 2019; 06(05). se cite this article in press Ahmad Hassan et al., Analysis Of Salivary Profile In Type 2 Diabetes Mellitu Adult Patients., Indo Am. J. P. Sci, 2019; 06(05). Page 9552 w w w . i a j p s . c o m Ahmad Hassan et al Ahmad Hassan et al ISSN 2349-7750 IAJPS 2019, 06 (05), 9552-9554 INTRODUCTION: DISCUSSION: 1. Lasisi TJ, Fasanmade AA. Salivary flow and composition in diabetic and non-diabetic subjects. Niger J Physiol Sci. 2012;27:79– 82. [PubMed] [Google Scholar] Several studies which were done on resting salivary pH estimated a range of 5.5 – 7.9 in normal individuals. The pH of saliva is maintained by carbonic acid and bicarbonate system, phosphate system and protein system of buffers [7]. This study showed a significant decrease in pH in diabetics in comparison with that in non diabetic subjects. Acidic pH was also observed in diabetic subjects by M E Lopez et al., in their study and this was attributed to either the microbial activity or a decrease in bicarbonate, which had occurred along with the flow rate. Nevertheless, not much literature which pertains to salivary pH changes in T2DM is available [8]. Several studies which were done on resting salivary pH estimated a range of 5.5 – 7.9 in normal individuals. The pH of saliva is maintained by carbonic acid and bicarbonate system, phosphate system and protein system of buffers [7]. This study showed a significant decrease in pH in diabetics in comparison with that in non diabetic subjects. 2. 32. Chorzewski M, Orywal K, Sierpinska T, Golebiewska M. Salivary protective factors in patients suffering from decompensated type 2 diabetes. Adv Med Sci. 2017;62:211– 5. [PubMed] [Google Scholar] 3. 33. Wang B, Du J, Zhu Z, Ma Z, Wang S, Shan Z, et al. Evaluation of parotid salivary glucose level for clinical diagnosis and monitoring type 2 diabetes mellitus patients. Biomed Res Int 2017. 2017:2569707.[PMC free article] [PubMed] [Google Scholar] 4. 34. Tabesh M, Noroozi A, Amini M, Feizi A, Saraf-Bank S, Zare M, et al. Association of retinol-binding protein 4 with metabolic syndrome in first-degree relatives of type 2 diabetic patients. J Res Med Sci. 2017;22:28. [PMC free article] [PubMed] [Google Scholar] Salivary parameters are altered by metabolic, nutritional and neurological abnormalities, the hydration status of a person and by drugs like anticholinergics, diuretics, antihistaminics, anti hypertensives, etc. Diabetes is associated with microvascular complications and hence, autonomic neuropathy, both of which may affect the salivary secretions. But the knowledge on the effect of T2DM on salivary functions remains equivocal, in spite of several studies which have been conducted in this regards [9]. IAJPS 2019, 06 (05), 9552-9554 ISSN 2349-7750 Ahmad Hassan et al Table 01: Salivary Biochemical Alterations In Non-Diabetic & Diabetic Subjects Parameter Studied Non-Diabetic Subjects (Mean ± SD) Diabetic Subjects (Mean ± SD) p Value Glucose (mg/dl) 4.33 ± 0.29 17.31 ± 2.05* p=0.000 Salivary α amylase (nm maltose liberated /mt/mg protein) 92.51 ± 13.74 19.20 ± 1.8* p=0.005 Total proteins(g/l) 424.46 ± 237.34 877.29 ± 603.84* p=0.000 Sodium(mEq/l) 4.31 ± 0.65 14.42 ± 1.83* p=0.000 Potassium(mEq/l) 20.84 ± 0.71 25.95 ± 1.56* p=0.000 Calcium(mEq/l) 6.39 ± 0.5 4.22 ± 0.12* p=0.000 Table 01: Salivary Biochemical Alterations In Non-Diabetic & Diabetic Subjects cal Alterations In Non-Diabetic & Diabetic Subjects RESULTS: The data were collected from 100 patients. The mean age of the participants in the study was 46.5 years. No significant differences were observed between the sexes in both groups in pH, salivary flow rates and biochemical parameters. Salivary pH was observed to be significantly lower in diabetics as compared to that to non-diabetics. Flow rate was significantly diminished in diabetics. Biochemical determinations showed significant differences between non– diabetics and diabetics. A statistically significant decrease was noticed in the salivary amylase concentrations in diabetics (p<0.0001). In addition, it has been noted that the salivary glands are also affected directly or indirectly. Reported oral health complications which are associated with T2DM, which are usually encountered by practitioners include xerostomia, tooth loss, gingivitis, periodontitis, odontogenic abscesses and soft tissue lesions of the tongue and the oral mucosa. Multiple physiologic factors contribute to compromised salivary function in poorly controlled T2DM. Diabetes-associated autonomic neuropathies, microvascular changes, hormonal Page 9553 w w w . i a j p s . c o m IAJPS 2019, 06 (05), 9552-9554 Ahmad Hassan et al DISCUSSION: Hence, we planned to do this study on a south Indian diabetic population, to investigate whether the salivary physical and biochemical characteristics would be altered in diabetics and to compare them with those of non diabetic controls. Further, it was intended to propose the possibility of using saliva as an alternative to blood in the diagnosis/monitoring of diabetes mellitus [10]. 5. 35. Andjelski-Radicevic B, Dozic R, Todrovic T, Dozic I. Biochemical markers in saliva of patients with diabetes mellitus. Serbian Dent J. 2012;59:198–201. [Google Scholar] 6. 36. Agrawal RP, Sharma N, Rathore MS, Gupta VB, Jain S, Agarwal V, et al. Noninvasive method for glucose level estimation by saliva. J Diabetes Metab. 2013;4:266. [Google Scholar] 7. 37. Jurysta C, Bulur N, Oguzhan B, Satman I, Yilmaz TM, Malaisse WJ, et al. Salivary glucose concentration and excretion in normal and diabetic subjects. J Biomed Biotechnol 2009. 2009:430426. 8. Ben-Aryeh H, Serouya R, Kanter Y, Szargel R, Laufer D. Oral health and salivary composition in diabetic patients. Journal of diabetes and its complications. 1993;7(1):57– 62. [PubMed] [Google Scholar] CONCLUSION: It is concluded that there is a significant variations in both physical and biochemical parameters of saliva in T2DM, thereby emphasizing the fact that the salivary composition was not just a reflection of the oral health state of a subject, but also of one’s systemic state. Future studies can be conducted on a larger scale, taking into account the various limitations. 9. [5] Chavez EM, Taylor GW, Borrell LN, Ship JA. Salivary function and glycemic control in older persons with diabetes. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2000;89(3):305–11. 10. Adler AI, Stevens RJ, Manley SE, Bilous RW, Cull CA, Holman RR. Development and REFERENCES: Page 9554 w w w . i a j p s . c o m IAJPS 2019, 06 (05), 9552-9554 Ahmad Hassan et al ISSN 2349 progression of nephropathy in type 2 diabetes: the United Kingdom Prospective Diabetes Study (UKPDS 64) Kidney international. 2003;63(1):225–32. Ahmad Hassan et al ISSN 2349-7750 ISSN 2349-7750 Ahmad Hassan et al IAJPS 2019, 06 (05), 9552-9554 progression of nephropathy in type 2 diabetes: the United Kingdom Prospective Diabetes Study (UKPDS 64) Kidney international. 2003;63(1):225–32. Page 9555 w w w . i a j p s . c o m
https://openalex.org/W4225164663	https://www.imrpress.com/journal/RCM/23/5/10.31083/j.rcm2305162/pdf	English	null	Clinical Features of Aortitis with Gastrointestinal Involvement	Reviews in cardiovascular medicine	2,022	cc-by	7,296	1. Introduction Gastrointestinal manifestations are rarely the predom- inating features of systemic vasculitides but can rapidly be- come life-threatening. Aortitis with small vessel involve- ment can cause various gastrointestinal manifestations, in- cluding mucosal purpura (risk of hemorrhage), patchy gran- ulomatous or ischemic ulcerations that can mimic inflam- matory bowel disease (IBD) and can cause intestinal perfo- ration. The most common vascultides that involve the aorta are Takayasu arteritis, Behcet’s disease, giant cell arteritis, and infectious arteritis. Vasculitides is a group of diseases that present with inflammation of blood vessel walls and earn their classifi- cations based on the size and type of the vessels involved, influencing the type and area of the ischemic injury [1]. Aortitis is vasculitis of the aortic wall and it can be a feature of systemic rheumatological, infectious or neoplas- tic disorders, and it can also be idiopathic [2]. Diagnosis of aortitis requires a high index of suspicion since clinical fea- tures are atypical and nonspecific [3]. While histopathology is the gold standard for diagnosing aortitis, tissue biopsy is not usually feasible, and correlating clinical findings with imaging and laboratory tests helps with the final diagnosis. As the clinical manifestation is nonspecific, aortitis could be easily overlooked if not suspected as part of the initial differential diagnosis. Abstract Few vascultides have a predilection for the aorta. Among those are Takayasu arteritis, Behcet’s disease, giant cell arteritis, and infec- tious aortitis. Diagnosis of aortitis requires a high index of suspicion since clinical features are atypical and nonspecific. However, many patients present with gastrointestinal manifestations owing to mesenteric involvement, intestinal infarction, and hepatitis. The most common vascultides that involve the aorta are Takayasu arteritis, Behcet’s disease, giant cell arteritis, and infectious arteritis. Here- with, we review the literature on epidemiology, gastrointestinal manifestations, and management of each form of aortitis that affects the gastrointestinal tract. Keywords: vasculitis; aortitis; gastrointestinal manifestations Mansour Altuwaijri1,†, Abdulmajeed Altoijry2,,† 1Division of Gastroenterology, Department of Medicine, College of Medicine, King Khalid University Hospital, King Saud Univer Saudi Arabia 2Division of Vascular Surgery, Department of Surgery, College of Medicine, King Saud University, 11472 Riyadh, Saudi Arabia Correspondence: maltuwaijri@ksu.edu.sa (Mansour Altuwaijri) †These authors contributed equally. Academic Editors: Brian Tomlinson and Takatoshi Kasai 2Division of Vascular Surgery, Department of Surgery, College of Medicine, King Saud University, 11472 Riyadh, Saudi Arabia *Correspondence: maltuwaijri@ksu.edu.sa (Mansour Altuwaijri) †These authors contributed equally. Academic Editors: Brian Tomlinson and Takatoshi Kasai Academic Editors: Brian Tomlinson and Takatoshi Kasai Submitted: 4 January 2022 Revised: 3 March 2022 Accepted: 25 March 2022 Published: 28 April 2022 Copyright: © 2022 The Author(s). Published by IMR Press. This is an open access article under the CC BY 4.0 license. Publisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. MR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rev. Cardiovasc. Med. 2022; 23(5): 162 https://doi.org/10.31083/j.rcm2305162 Review 2. Methods Records removed before screening: Duplicate records removed (n = 4,371) Records removed for other reasons (n = 4,154) Records excluded (n = 1,378) Full text excluded: Beyond scope (n = 141) Insufficient details (n = 116) Not in English (n = 21) Studies included in review (n = 87) Fig. 1. PRIMSA flow diagram for clinical features of aortitis with gastrointestinal involvement. 3.1 Epidemiology The most common non-infectious aortitis (NIA) is takayasu arteritis (TKA). This is a rare obliterative and necrotizing idiopathic large vessel, segmental panarteritis [3]. It is most commonly found in women between the ages of 20 and 40 years in Southeast Asia, India and Mexico with Japan holding the highest prevalence [5,6]. Due to its rarity, epidemiological data for incidence rates of TKA are lim- ited. However, recent studies have put the incidence rate at 1–2 per million in Japan and 2.2 per million Kuwait [7,8]. Recent European studies have put their specific incident rates between 0.4 and 1.3 per million, recognizing an in- crease in the recent years compared to older estimates from the European countries [9–14]. Although its etiology is un- known, the frequency in specific populations and familial aggregation of TKA and its association with HLA alleles suggest involvement of genetic factors in the etiopathogen- esis of TKA [15]. Arterial stenoses manifests with a bruit which is usu- ally audible over the subclavian, brachial, and carotid ar- teries. Stenosis also manifests with discrepancies in limb blood pressure of 10 mmHg or more. Therefore, patients with suspected TKA should have their blood pressure mea- sured in all four limbs. When stenosis involves coronary vessels, the most common feature is angina. Aortitis and coronary arteritis have been described in patients with TKA. In such cases, Myocardial infarction and death may occur. 3. Takayasu Arteritis progress and become symptomatic. In patients with TKA, constitutional symptoms such as weight loss, low grade fever and fatigability are common especially in the early period. Additionally, arthralgias and myalgias are occur in about one-half of cases. Tenderness of the carotid artery is also observed in 10–30% patients at presentation [16]. Peripheral pulses may be weak or absent, especially at the level of the radial arteries [17]. Ischemic ulceration and gangrene of the extremities my occur, but this is rare due to the fact that these complications are preceded by formation of collateral vessels. In all cases, limb claudication is com- mon and involvement of the subclavian artery may be asso- ciated with subclavian steal syndrome, which gives rise to neurological symptoms and syncope during exercise [18]. 2. Methods We systematically searched MEDLINE (from 1940) and EMBASE (from 1972) up to the end of December 2021 using a comprehensive search strategy that combined MeSH terms and free text for “Aortitis”, “Gastrointestinal”, and “Takayasu”, “Behcet’s disease”, “giant cell arteritis”, “infectious”, and “mycotic”. Reference lists of all relevant studies, reviews, and letters were also searched to identify additional studies. The searches were limited to humans and adults. In cases of aortitis presenting with gastrointesti- nal involvement, the large-vessel vasculitis could lead to widespread intestinal infarction and even involve other or- gans. Aortitis has a variable presentation ranging from mild abdominal pain to more severe and life-threatening bowel perforation and peritonitis. These manifestations could happen during diagnosis or could present later at a relapse time and are often isolated. The Five Factor Score (FFS) of 1996 described gastrointestinal manifestations as a major predictor of mortality in microscopic polyangiitis, polyarteritis nodosa and eosinophilic granulomatosis with polyangiitis (EGPA) with involvement of the central ner- vous system, kidneys and the heart [4]. Despite advances in diagnostics and management of aortitis, gastrointestinal manifestations remain, till this day, a serious problem. Our inclusion criteria were broad and included prior systematic reviews and meta-analysis, clinical trials, cohort studies, case series, and case reports. Both authors independently screened all titles and ab- stracts to identify potentially relevant articles. Disagree- ments were resolved by repeated review and discussion. They independently extracted data from the full-text arti- cles using structured review forms that included epidemi- ology, diagnosis, gastrointestinal manifestations, and man- agement. Articles that did not fulfill any of the review form items were excluded (Fig. 1). Records identified from MEDLINE, EMBASE and reference lists: n = 8,525 Records removed before screening: Duplicate records removed (n = 4,371) Records removed for other reasons (n = 4,154) Title/Abstract screened (n = 1,743) Records excluded (n = 1,378) Full text assessed (n = 365) Full text excluded: Beyond scope (n = 141) Insufficient details (n = 116) Not in English (n = 21) Studies included in review (n = 87) Identification Screening Included Fig. 1. PRIMSA flow diagram for clinical features of aortitis with gastrointestinal involvement. 3.2 Diagnosis Usually, TKA has a sabacute course lasting months to years. During this period, vascular involvement may 2 Table 1. Gastrointestinal manifestations of the most common etiologies of aortitis. Disease Gastrointestinal manifestation Clinical features suggesting GI involvement Takayasu arteritis Splenic infarction and hepatic ischemia Abdominal pain, abdominal bruits, jaundice Mesenteric ischemia Occlusive or stenotic lesions in the celiac or superior mesenteric arteries Elevated liver enzymes Behçet’s disease Mesenteric ischemia Nausea and vomiting, dyspepsia, anorexia, melena, diarrhea, abdominal pain Mucosal ulcers Esophageal ulcers and varices Aphthous, geographic and volcano ulcers in colon Giant cell arteritis Aortic aneurysm Abdominal pain, elevated liver enzymes, nonspecific fever Mesenteric ischemia Infective aortitis Mesenteric ischemia High CRP and ESG Abnormal echocardiography Nausea and vomiting, dyspepsia, anorexia, melena, diarrhea, abdominal pain Table 1. Gastrointestinal manifestations of the most common etiologies of aortitis. Clinical features suggesting GI involvement Clinical features suggesting GI Laboratory findings in TKA are nonspecific. Erythro- cyte sedimentation rate (ESR) and C-reactive protein (CRP) may be elevated, and anemia of chronic disease may be ob- served. ESR and CRP do not reflect disease progression and can be normal in active TKA. At present, there are no diag- nostic tests for TKA. Nevertheless, the American College of Rheumatology criteria demonstrated a sensitivity and a specificity of 90.5% and 97.8%. The presence of at least 3 of the following factors is considered suggestive of TKA: onset at age less than or equal to 40 years, claudication of an extremity, decreased brachial artery pulse, greater than 10 mmHg difference in systolic blood pressure between arms, a bruit over the subclavian arteries or the aorta, and arteri- ographic evidence of narrowing or occlusion of the entire aorta, its primary branches, or large arteries in the proximal upper or lower extremities [19]. with significant side effects. Therefore, patients may be prescribed an immunosuppressive agent to maintain long- term remission [24]. Surgery and endovascular procedures may be indicated in cases of significant stenosis, critical is- chemia, or large aneurysms. Patients who develop new-onset arterial stenosis or major vessel inflammation (e.g., aortitis) should receive oral prednisone at a dose of 1 mg/kg per day, up to a max- imum daily dose of 60 to 80 mg. This regimen should be continued for two to four weeks. 4.1 Epidemiology Another NIA is Behçet’s disease, most commonly found in the Mediterranean and Asia where 80–420 cases in 100,000 are found in Turkey alone compared to 0.12– 0.64 cases per 100,000 in Western countries [28]. It com- monly manifests in males in Mediterranean and Asian coun- tries and females in Western ones [29]. The gastrointestinal manifestations of Behçet’s disease vary greatly by region with presentations in 2.8% of patients from a Turkish se- ries, 37–43% in the US and 50–60% in Japan [29,30]. 3.2 Diagnosis High-dose intravenous steroids can be used to initiate treatment for up to three days in order to prevent impending organ failure (e.g., se- vere carotid or vertebral artery stenosis) [25]. As for non-steroid immunosuppressant, methotrexate and azathioprine were found to reduce the need for gluco- corticoids while maintaining adequate disease control [26]. Restenosis after percutaneous angioplasty or surgical bypass is not uncommon. The rate of restenosis after open surgery reaches up to 30% at 5–20 years postop with some estimates reaching 70% [27]. 3.3 Gastrointestinal Manifestation of TKA The gastrointestinal manifestations of TKA mainly in- volve the ileum and the colon (Table 1). Additionally, splenic infarction and hepatic ischemia have been observed in TKA due to occlusion of medium and large gastrointesti- nal arteries [20]. In a study of 126 subjects with TKA, 16% had abdominal pain, 14% had abdominal bruits, while 4% had mesenteric ischemia. One in four patients in the study had occlusive or stenotic lesions in the celiac or superior mesenteric arteries [21]. Interestingly, another study in- volving 40 subjects with TKA reported elevated levels of alkaline phosphatase in three-quarters of patients, suggest- ing hepatic involvement [22]. Additionally, inflammatory bowel disease (IBD) has been reported to coexist with TKA. In a study with 160 subjects with TKA, 5% had IBD, and almost 70% of those patients presented with IBD 4 years before being diagnosed with to TKA [23]. 5.2 Diagnosis GCA has a subacute course with abrupt flareups [45]. It is often associated with constitutional symptoms including low-grade fever, fatigability, and weight loss. Headache is also a common symptom that occurs in two- thirds of patients. Headache is classically associated with scalp tenderness, but it often has no defining characteris- tics [46,47]. Jaw claudication is present in about half of GCA patients. In some cases, patients notice a trismus-like symptom with restriction in the movement of the temporo- mandibular joint. Claudication symptoms occasionally af- fect the tongue during eating or with repeated swallowing [48]. The American College of Rheumatology established diagnosis criteria for GCA based on clinical and laboratory assessments in 1990. The criteria include Age at disease on- set ≥50 years, New headache, Temporal artery abnormal- ity (such as blood vessel occlusion or weakening and sub- sequent rupture), elevated erythrocyte sedimentation rate, and abnormal artery biopsy, i.e., non-caseating granuloma- tous inflammatory process along the internal elastic lamina [49]. In 2016, some authors suggested revising the criteria to a point-based system where scoring 3 or more points sug- gested GCA. The additional criteria included sudden onset of visual disturbances, polymyalgia rheumatica, jaw clau- dication, unexplained fever and/or anemia, and compatible pathology [50]. 5. Giant Cell Arteritis (GCA) 5.1 Epidemiology GCA affects the aorta and branches of large arteries with a predilection for the vertebral and carotid branches [1]. A systematic review by Gonzalez-Gay and colleagues found that 10–25% of patients with GCA develop aortitis. Additionally, the systematic review found that GCA usually occurs in patients older than 50 years with a peak incidence in 70 and 80 years. GCA is associated with polymyalgia rheumatica, and is more common in Western countries and Caucasians [44]. Behçet syndrome affects venous and arterial vessels of all sizes, and most clinical features of Behçet syndrome are secondary to vasculitis. 4.3 Gastrointestinal Manifestations Gastrointestinal manifestations of Behçet’s disease in- clude vomiting, dyspepsia, anorexia, melena, diarrhea and abdominal pain. Behçet’s disease is also associated with intestinal perforation requiring emergency surgical inter- vention [29]. A distinction of the intestinal Behçet’s dis- ease can be made between its two forms: Large-vessel vas- culitis (including aortitis) causing intestinal infraction and ischemia, and mucosal ulcers from neutrophilic infiltrates mimicking IBD [35,36]. Although the involvement of any part of the gastrointestinal tract is possible, the ileocaecal junction and terminal ileum are the most common [37]. Esophageal ulcers frequently occur in inferior esophagus, and varices have been reported in association with occlu- sion of the vena cava [38]. Furthermore, pyloric stenosis and ulcers present as part of the gastric manifestations of Behçet’s disease [39]. Additionally, aphthous, geographic and volcano ulcers may be found in the colon [40], and they have the highest risk of perforation in those 25 years and older [41]. In people with Behçet’s disease, 1.3–3.2% suffer from Budd-Chiari syndrome with risks increasing in young males [28]. The main determinant of survival is in this case the extent of the thrombus in the inferior vena cava. If dif- fuse occlusion is complete the mean survival becomes only 10 months [42]. 3.4 Management Behçet syndrome commonly presents with recurrent, painful mucocutaneous ulcers. Oral ulcers usually heal spontaneously within three weeks, while recurrent lesions The mainstay of therapy for TKA is systemic gluco- corticoids. However, long-term use of steroid is associated 3 mour necrosis factor-alpha inhibitors or apremilast may be considered in select cases. In patients with eye involve- ment, an ophthalmologist should be involved. may persist. The most specific lesions associated with Behçet syndrome are painful genital ulcers, which occur in more than three-quarters of patients [31]. Cutaneous lesions are also common and include acneiform lesions, papulo-vesiculo-pustular eruptions, pseudofolliculitis, nod- ules, erythema nodosum (septal panniculitis), superficial thrombophlebitis, and palpable purpura [32]. Behçet syn- drome may also present with arthritis; in which case ac- neiform lesions are commonly found [33,34]. 4.4 Management The goal of treatment is to suppress exacerbations and relapses in order to prevent end organ damage. Multidisci- plinary management is necessary to ensure good outcomes. The European League against Rheumatism (EULAR) pub- lished guidelines on the management of Behcet Disease [43]. The recommendations can be summarized as follows: Transient monocular (and rarely binocular) visual dis- turbances may be an early manifestation of GCA. In tran- sient monocular vision loss (TMVL), affected patients typ- ically notice a sudden partial visual field loss or a transient curtain effect in the visual field of one eye. Even in the era of effective therapies, the incidence of permanent partial or complete loss of vision in one or both eyes due to GCA, as described by several centers, is between 15 and 20 percent of patients [51–56]. Permanent vision loss may be preceded by single or multiple episodes of transient vision loss, but it may also occur with devastating rapidity. Once vision loss has occurred, it is rarely reversible [57]. In addition, High-dose glucocorticoids can be used for rapid sup- pression of inflammation during acute attacks, while reg- ular doses can be used for gastrointestinal manifestations. Additionally, colchicine is used to prevent mucocutaneous lesion recurrence, especially if oral and genital ulcers are present. Treatment of leg ulcers, however, should involve a dermatologist and a vascular surgeon since the ulcers are usually caused by venous stasis or obliterative vasculitis. Moreover, azathioprine, thalidomide, interferon-alpha, tu- 4 it is estimated that 25 to 50 percent of untreated patients will experience further loss of vision in the unaffected eye within one week. Nevertheless, prompt initiation of appro- priate steroid treatment virtually eliminates the risk of sub- sequent vision loss. If vision loss is already present, such treatment significantly reduces the risk of further deteriora- tion but does not improve the existing vision loss [58]. currence of cranial symptoms. Some cases of large bowel infarction infraction of the large bowel were described. In such cases, patients usually present with in the literature and present with nonspecific fever, acute abdomen or ab- dominal pain. Some extremely rare occurrences are that of granulomatous inflammation of the liver and the portal tract hepatic arteritis that can induce gastrointestinal symptoms and fever before the cranial symptoms that are suggestive of GCA [70,71]. Large vessel (LV) involvement in GCA causes aneurysms and dissections especially in the thoracic aorta. 4.4 Management Stenosis, occlusion, and ectasia of large arteries have also been described [59]. Authors studied 40 patients with con- firmed GCA using computed tomographic (CT) angiogra- phy and found evidence of large-vessel vasculitis (includ- ing the aorta and/or its tributaries arteritis in two-thirds of patients. Authors defined aortitis as circumferential aortic wall thickness ≥2 mm with or without contrast enhance- ment of the vessel wall observed in zones without adja- cent atheroma. The aortic tributaries including the bra- chiocephalic trunk, carotid, subclavian, axillary, splanch- nic (coeliac and mesenteric), renal, iliac and femoral arter- ies were also evaluated. Radiological findings considered included circumferential wall thickness, contrast enhance- ment of the artery wall, arterial diameter and the presence of stenoses. Arteritis was considered to be present when the thickness of the artery wall was >1 mm. Sixty-five percent of those patients in the study had aortitis, 47 per- cent had brachiocephalic trunk involvement, 42% subcla- vian arteries and 30% had femoral arteries vasculitis. [60]. Clinical recognition of aortic aneurysms/dilatation has been described in 10 to 20 percent of cases [61–64]. The tho- racic aorta, especially the ascending aorta, is affected more often than the abdominal aorta. Nevertheless, major com- plications such as aortic dissection and rupture occur less frequently [61,62]. 5.4 Management Glucocorticoids are the treatment of choice for GCA. In patients with a positive biopsy, high-dose systemic glu- cocorticoids are the mainstay of therapy and should be in- stituted promptly once the diagnosis of giant cell arteritis (GCA) is strongly suspected, especially in patients with re- cent or threatened visual loss. A temporal artery biopsy or other diagnostic procedure should be obtained as soon as possible, but treatment should not be withheld while await- ing the performance or results. In cases where the clinical scenario for GCA is compelling but the diagnostic workup is negative, the diagnosis of GCA may be arrived at on clin- ical grounds. GCA is treated with daily glucocorticoids [72]. Ad- juvant treatment with tocilizumab or methotrexate may be used to avoid steroid side effects [73]. These options are in- dicated in patients with significant co-morbidities, in those with significant corticosteroid side effects, and when a re- lapse necessitates prolonged immunosuppression. In case of severe gastrointestinal manifestations, an immunosuppressant is usually used with a steroid. Surgery and endovascular procedures are used in an as-needed basis. Surgical treatment should be considered in patients who de- velop an aortic aneurysm, ideally in the dormant phase of the disease. Owing to the morbidity risk associated with surgical repair of GCA-related aneurysms, we recommend performing it only in specialized, experienced tertiary care centers. Endovascular repair has also been reported for aortic aneurysms. Endovascular repair can be considered for particularly ill patients and provides them with superior short-term outcomes compared to those undergoing open surgery [74]. 6.2 Diagnosis Patients usually present with a fever, back, chest, or abdominal pain, pulsatile abdominal mass, leukocytosis, and a positive blood culture. Diagnosing infectious aorti- tis requires a high index of suspicion since symptoms and signs are nonspecific. Blood cultures can help identify bacterial causes of aortitis. However, signs on CT scan can help guide a di- agnosis. CT scan is rapidly indicated for patients with sus- pected abdominal aortic aneurysm, which frequently ac- companies aortitis. Features that can be identified periaor- tic soft tissue or fluid accumulation, aneurysmal dilatation, and vertebral body osteomyelitis. Other diagnostic options include magnetic resonance imaging and nuclear medicine scintigraphy. Additionally, transesophageal echocardiog- raphy may provide insight into the thoracic aorta. The goals of surgical therapy are removal of infected tissue, often including an aneurysm resection, and restora- tion of distal arterial flow [86]. This should be followed by long-term systemic antibiotic therapy. Overall, the surgical mortality rates range from 40% to 45%, much of which is influenced by the presence of vessel rupture prior to surgery, whether the infection involves an exist- ing prosthetic aortic graft, and the suprarenal extent of the aneurysm. 6.1 Epidemiology IA is managed by adequate antibiotic therapy depend- ing on the infectious agent in question. Broad-spectrum antibiotics may be used while waiting for blood culture re- sults [80]. Hospitalization with extensive workup is indi- cated for any adult, especially over the age of 50 years, who presents with fever, chest or abdominal pain, and posi- tive blood cultures in which the diagnosis of infectious aor- titis is suspected [81]. Any patient with fever associated with a palpable aneurysm should also be hospitalized, be- cause rapid evaluation and diagnosis are required to avoid aneurysm rupture. Aneurysms due to gram-negative infec- tions are associated with a greater tendency toward early rupture than those associated with gram-positive infections (84% vs 10%) [82]. The aorta is normally resistant to infection. Risk fac- tors for infective aortitis include atherosclerosis, syphilis, cystic medial necrosis, and aortic prosthesis. IA is more frequent in med and elderly patients. It usually presents with aneurysmal disease or infective endocarditis [78]. In- fectious aortitis is an uncommon finding, representing only 2.6% of all abdominal aortic aneurysm. Infection may follow septic embolization of the aorta (“embolomycotic”), hematogenous seeding (“micro- bial aortitis or infected aneurysm”), or spread from a con- tiguous focus of infection. The mortality associated with in- fectious aortitis usually ranges from 21% to 44%, higher if managed with antibiotics alone. Increased mortality is asso- ciated with uncontrolled infection or sepsis, infection with more virulent microorganisms, suprarenal extension of the aneurysm, and perhaps aneurysm rupture, whereas 30-day mortality may be decreased in patients who are revascular- ized using cryopreserved arterial homografts [79]. If surgical intervention is immediately planned, an- tibiotics should be initiated after intraoperative cultures are obtained. Because gram-negative bacteria, like Salmonella species, and gram-positive organisms, like S. aureus, are the most commonly isolated bacterial pathogens, initial antibi- otic selection should be active against these bacteria. The duration of antimicrobial therapy is usually 6 to 12 weeks, possibly 1 year or indefinitely in the immunocompromised patient; however, controlled trials are lacking [83,84]. Ri- fampin impregnated grafts have been used successfully in a limited number of patients [85]. However, it must be em- phasized that treatment of infectious aortitis requires a com- bined medical and surgical approach. 5.3 Gastrointestinal Manifestations In patients with GCA, abdominal pain can result from abdominal aortic dissection or aneurysm. A cohort study from a clinic in Minnesota followed 96 patients who devel- oped GCA between 1950 and 1985. Authors reported aortic artery aneurysms in 11.5% of patients. Most of those pa- tients developed aortic aneurysms after a median of 6 years from diagnosis [65]. Thus, patients diagnosed with GCA should have regular screening for aortic aneurysms at the time of diagnosis and throughout follow-up [66]. It is noteworthy that patients suffering from GCA tend to be older than those suffering from Takayasu arteritis, which is why the morbidity and mortality of GCA is higher [75]. Adjunctive methotrexate could reduce relapse as well as reliance on steroids [76]. The use of tocilizumab has also been studied in clinical trials; It was found that 85% of pa- tients with GCA experience sustained remission within one year, and 80% of patients are able to discontinue glucocor- ticoids [77]. GCA has also been shown to affect the liver. Twelve of 56 patients with GCA who were followed in Jerusalem had elevated liver enzymes including alkaline phosphatase and transaminase levels [67]. These elevated levels could be a result from bile duct epithelial cells being injured due to neighboring arteritis [68]. GCA rarely affects the mesenteric vessels. A litera- ture review in 2008 found 12 cases of GCA with mesen- teric involvement [69]. Fifty percent of these cases had pre- dominating abdominal symptoms with a less common oc- 5 5 6.3 Gastrointestinal Manifestations According to the International Chapel Hill Consen- sus Conference on the Nomenclature of Systemic Vasculi- tides [1], this condition can be classified into large, medium, and small vasculitis. However, small- and medium-vessel vasculitis can also affect the aorta, although this is rare. For instance, Veraldi and colleagues reported the case of a 46-year-old man who was admitted for investigation of an abdominal aortic aneurysm with the presence of solid fi- brous inflammatory tissue surrounding the aortic wall. Au- thors suspected infective or autoimmune etiology. They performed a laparotomy during which they noted extensive solid fibrous tissue surrounding the aorta was found without any cleavage planes between anatomical structures. For this reason, they performed aneurysmectomy, in-situ revascu- larization with an arterial homograft, and obtained periaor- tic specimens for histopathologic examination. The histo- Diagnosis often occurs after suspicion from the pa- tient’s symptoms and history supported by peculiarly high c-reactive protein and erythrocyte sedimentation rate. The presentation is usually non-specific and for this reason a high index of suspicion must be maintained. Symptoms in- clude pyrexia of unknown origin, abdominal and/or back pain, palpable pulsatile abdominal mass, and signs of rup- ture abdominal aortic aneurysm rupture. Hemorrhage into the gastrointestinal tract, which manifests in hemateme- sis, coffee ground vomitus, and/or melena occurs in pa- tients with bowel erosion or an aorto-enteric fistula. Imag- ing such as echocardiography also guides the diagnosis [2]. Salmonella spp. are the most common bacteria causing ab- dominal aortitis. However, two-thirds of cases of aortitis in developing countries are due to Mycobacterium tuberculo- sis. 6 logical specimens confirmed the presence of vasculitis le- sions, associated with eosinophilic and plasma cellular in- filtration. The patient was diagnosed with Anti-neutrophil cytoplasmic antibody (ANCA) vasculitis complicated by symptomatic infrarenal aortic aneurysm was concluded. He responded well to therapy with a glucocorticoid in addition to methotrexate and was discharged 3 weeks after surgery [87]. [10] Reinhold-Keller E, Herlyn K, Wagner-Bastmeyer R, Gross WL. Stable incidence of primary systemic vasculitides over five years: results from the German vasculitis register. Arthritis and Rheumatism. 2005; 53: 93–99. [11] Dadoniene J, Kirdaite G, Mackiewicz Z, Rimkevicius A, Hauge- berg G. Incidence of primary systemic vasculitides in Vilnius: a university hospital population based study. Annals of the Rheumatic Diseases. 2005; 64: 335–336. Rheumatic Diseases. 2005; 64: 335–336. [12] Romero-Gómez C, Aguilar-García JA, García-de-Lucas MD, Cotos-Canca R, Olalla-Sierra J, García-Alegría JJ, et al. 6.3 Gastrointestinal Manifestations Epi- demiological study of primary systemic vasculitides among adults in southern Spain and review of the main epidemiologi- cal studies. Clinical and Experimental Rheumatology. 2015; 33: S–11–8. 8. Conclusions Aortitis may present with gastrointestinal manifesta- tions. While this is rare, it could quickly become life- threatening and physicians must therefore maintain a high index of suspicion. A multidisciplinary protocol must be put in place to improve patient prognosis. [13] Watts R, Al-Taiar A, Mooney J, Scott D, MacGregor A. The epi- demiology of Takayasu arteritis in the UK. Rheumatology. 2009; 48: 1008–1011. [14] Mohammad AJ, Mandl T. Takayasu arteritis in southern Sweden. The Journal of Rheumatology. 2015; 42: 853–858. [15] Onen F, Akkoc N. Epidemiology of Takayasu arteritis. La Presse MéDicale. 2017; 46: e197–e203. The authors declare no conflict of interest. [21] Schmidt J, Kermani TA, Bacani AK, Crowson CS, Cooper LT, Matteson EL, et al. Diagnostic features, treatment, and outcomes of Takayasu arteritis in a us cohort of 126 patients. Mayo Clinic Proceedings. 2013; 88: 822–830. Author Contributions MA and AA—designed the study, screened literature, wrote manuscript and approved the final version. [16] Mason JC. Takayasu arteritis–advances in diagnosis and man- agement. Nature Reviews. Rheumatology. 2010; 6: 406–415. [17] Serra R, Butrico L, Fugetto F, Chibireva MD, Malva A, De Caridi G, et al. Updates in Pathophysiology, Diagnosis and Management of Takayasu Arteritis. Annals of Vascular Surgery. 2016; 35: 210–225. Acknowledgment Not applicable. [19] Arend WP, Michel BA, Bloch DA, Hunder GG, Calabrese LH, Edworthy SM, et al. The American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis. 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https://openalex.org/W1911369697	https://europepmc.org/articles/pmc4423447?pdf=render	English	null	Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle formation in zebrafish	Frontiers in cell and developmental biology	2,015	cc-by	6,541	REVIEW published: 07 May 2015 doi: 10.3389/fcell.2015.00027 Introduction Individual cells have the potential to migrate randomly. When chemo-attractants are distributed in an environment, the cells can migrate toward the attractants. In some situations, cell aggregates can move directionally while maintaining cell adhesions in a process called collective cell migration. Collective cell migration is essential for the generation of basic organ structures such as sheets, clusters, spheres, sprouts, and vesicles in the morphogenetic processes of animal development (Rorth, 2009, 2012; Weijer, 2009; Reig et al., 2014), and is also observed in wound closure and cancer invasion (Friedl and Gilmour, 2009; Friedl et al., 2012). Collective cell migration shares some features with individual cell migration but also has unique ones. These features have been described in several in-depth reviews cited in this review (Friedl and Gilmour, 2009; Rorth, 2009, 2012; Weijer, 2009; Friedl et al., 2012; Reig et al., 2014). Specialty section: This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology Received: 16 March 2015 Accepted: 22 April 2015 Published: 07 May 2015 Specialty section: This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology Loss-of-function of chemo-attractants, their receptors, signal mediators, and cell adhesion molecules blocks or delays the directed migration of the cell groups in model systems of collective cell migration such as zebraﬁsh lateral lines and Drosophila border cells (Niewiadomska et al., 1999; Kerstetter et al., 2004; Wilson et al., 2007; Friedl and Gilmour, 2009; Rorth, 2009; Reig et al., 2014). However, these manipulations unexpectedly does not dissociate the cell groups, suggesting that how “cell collectivity,” a feature in which cells stay assembled as a single cell cluster, is produced and maintained within a migrating cell cluster remains largely unknown. Cell collectivity regulation within migrating cell cluster during Kupffer’s vesicle formation in zebraﬁsh Takaaki Matsui, Hiroshi Ishikawa and Yasumasa Bessho Gene Regulation Research, Nara Institute of Science and Technology, Nara, Japan Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called “collective cell migration,” is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as “cell collectivity,” remain largely unknown. During the formation of Kupffer’s vesicle (KV, an organ of laterality in zebraﬁsh), KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent ﬁndings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration. Edited by: Edited by: Mitsugu Fujita, Kindai University Faculty of Medicine, Japan Reviewed by: Takaki Miyata, Nagoya University, Japan Siu Ping Ngok, Stanford University School of Medicine, USA Keywords: collective cell migration, cell adhesion, Kupffer’s vesicle, cell signaling, zebraﬁsh Correspondence: Takaaki Matsui, Gene Regulation Research, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Nara, Japan matsui@bs.naist.jp Keywords: collective cell migration, cell adhesion, Kupffer’s vesicle, cell signaling, zebraﬁsh KV Formation and Function When DFCs appear as a single cluster at the mid-gastrulation stage (6 hpf), cadherin1 and cadherin2 (E-cadherin and N-cadherin in zebraﬁsh, respectively) are expressed in DFCs (Babb et al., 2001; Babb and Marrs, 2004; Esguerra et al., 2007; Harrington et al., 2007; Warga and Kane, 2007; Matsui et al., 2011), suggesting that cadherin-based adherens junction mediates DFC-DFC interaction (Figure 1). Although, in general, formation of adherens junction leads to generation of tight junction (Hartsock and Nelson, 2008), tight junction is not formed between DFCs at 6 hpf. Instead, tight junction is formed between a subset of DFCs and overlying enveloping layer In zebraﬁsh, DFCs ﬁrst appear in the dorsal site adjacent to the embryonic margin at 6 h post-fertilization (hpf) (Figure 1) (Essner et al., 2002, 2005; Oteiza et al., 2008; Matsui and Bessho, 2012). About 20–30 DFCs form a single cluster that migrates toward the vegetal pole accompanied by epiboly movement by 10 hpf (Cooper and D’Amico, 1996; Melby et al., 1996). During migration, the DFC cluster undergoes compaction and changes into a bottled shaped cluster. Around the late-gastrulation stage (8 hpf), DFCs start to polarize so that multiple focal points are generated within the cluster (Cooper and D’Amico, 1996; Melby et al., 1996; Amack et al., 2007). These points are then FIGURE 1 \| Cell adhesion status during KV organogenesis. KV organogenesis is divided into four steps. First, 20–30 DFCs form a cluster through the formation of adherens junction. A subset of DFCs binds to EVL through the formation of tight junction. Second, the cluster becomes compact. Multiple focal points that enrich tight junction components are generated between some DFCs. Third, these points are then rearranged into a single focal point. Fourth, an internal lumen is generated. During collective DFC migration, adherens junction, tight junction, and cell-ECM interaction are generated in a migrating DFC clusters. junction components are generated between some DFCs. Third, these points are then rearranged into a single focal point. Fourth, an internal lumen is generated. During collective DFC migration, adherens junction, tight junction, and cell-ECM interaction are generated in a migrating DFC clusters. FIGURE 1 \| Cell adhesion status during KV organogenesis. KV organogenesis is divided into four steps. First, 20–30 DFCs form a cluster through the formation of adherens junction. A subset of DFCs binds to EVL through the formation of tight junction. Second, the cluster becomes compact. Citation: Matsui T, Ishikawa H and Bessho Y (2015) Cell collectivity regulation within migrating cell cluster during Kupffer’s vesicle formation in zebraﬁsh. Front. Cell Dev. Biol. 3:27. doi: 10.3389/fcell.2015.00027 Kupﬀer’s vesicle (KV) is a key organ required for the left-right asymmetric patterning in zebraﬁsh (Amack and Yost, 2004; Essner et al., 2005; Matsui and Bessho, 2012). During KV May 2015 \| Volume 3 \| Article 27 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 1 Collectivity regulation within cell cluster Matsui et al. rearranged into a single focal point that will expand and become a vesicle lumen by 12 hpf. At the same time, motile monocilia are generated on the apical membrane facing the lumen. In the KV, rotation of motile cilia generates a counterclockwise ﬂow of ﬂuid, called nodal ﬂow, and leads to the establishment of left- right asymmetry in the body (Essner et al., 2005; Amack et al., 2007; Oteiza et al., 2008). Several excellent reviews have already described the molecular and cellular mechanisms of left-right patterning and ciliogenesis (Hirokawa et al., 2006, 2012; Ishikawa and Marshall, 2011; Nakamura and Hamada, 2012; Blum et al., 2014; Choksi et al., 2014), so we do not go into those here. Instead, we focus on cell collectivity regulation occurring within a migrating cell cluster. organogenesis, 20–30 KV progenitors called dorsal forerunner cells (DFCs) make a single cluster and migrate together, so KV organogenesis is represented as one of the model systems of collective cell migration. In our and other’s studies, loss-of- functional situations of genes/signals have led to a breaking up of the cluster of KV progenitors (DFCs) without aﬀecting their directed migration (Matsui and Bessho, 2012). We therefore feel that KV organogenesis is a good model for investigating the regulatory mechanisms of cell collectivity formation. In this review, we describe our recent understanding of how cell collectivity is generated and maintained during collective DFC migration. KV Formation and Function Multiple focal points that enrich tight May 2015 \| Volume 3 \| Article 27 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 2 Collectivity regulation within cell cluster Matsui et al. (EVL) cells (Figure 1) (Oteiza et al., 2008). In addition, it has been reported that integrinα5 and integrinβ1b are expressed in migrating DFC cluster at 6 hpf (Ablooglu et al., 2010). These ﬁndings suggest that multiple types of cell adhesions including adherens junctions, tight junctions, and cell-extracellular matrix (ECM) interactions are generated in DFCs at the onset of DFC clustering. chaperones enhances fgfr1 protein folding within endoplasmic reticulum and increases mature fgfr1 at the membrane of DFCs, resulting in much higher FGF/Erk signal activity in DFCs. The potentiated FGF/Erk signal induces the expression of cadherin1 via transcriptional activation of tbx16 (Table 1). Furthermore, it has been reported that Nodal signaling regulates the expression of cadherin1 in DFCs via repression of snail1 expression (Table 1) (Esguerra et al., 2007), which is known as a transcriptional repressor of E-cadherin expression in several animals (Batlle et al., 2000; Cano et al., 2000; Hajra et al., 2002). During epiboly (6–10 hpf), cadherin1 expression increases in migrating DFCs (Figure 1) (Babb et al., 2001; Babb and Marrs, 2004; Esguerra et al., 2007; Matsui et al., 2011), suggesting that a cadherin1-based adherens junction contributes to the compaction and shape change of the cluster. Within the bottled shaped cluster seen in 8–10 hpf, focal points that enrich the tight junction components are generated, and then multiple focal points are rearranged into a single focal point that will be an internal vesicle lumen (Figure 1) (Amack et al., 2007; Oteiza et al., 2008, 2010; Siddiqui et al., 2010). On the other hand, DFC-EVL tight junction is maintained by the end of epiboly (10 hpf) (Figure 1) (Oteiza et al., 2008, 2010; Siddiqui et al., 2010). This means that there is a transition of tight junction formation from DFC-EVL to DFC-DFC pairs during collective DFC migration (Figure 1). However, how the tight junction formation is regulated and how this transition occurs remain unexplored. j In addition to these transcriptional regulations of cadherin1, the post-translational regulations of cadherin1 contribute to the ﬁne-tuning of cell collectivity. The lateral giant larvae 2 (lgl2), one of the lateral giant larvae genes initially identiﬁed as tumor suppressor genes in Drosophila, is expressed in DFCs (Tay et al., 2013). Regulatory Mechanisms of Cell Collectivity Formation in the DFC Cluster When cadherin1 is knocked down speciﬁcally in DFCs, the DFC cluster is broken up into multiple groups of cells at 60–80% epiboly stages (6.5–8.5 hpf) (Matsui et al., 2011); the “broken- up DFC cluster phenotype” is represented as a sign of failures of cell collectivity (Table 1). This phenotype eventually results in formation of small KV and randomization of left-right patterning (this means the loss of KV function), indicating that cell collectivity regulation is required for formation of a functional KV. We also ﬁnd that strengthening a cadherin1-based adherens junction by cadherin1 overexpression induces the formation of a highly compacted oval-shaped DFC cluster, leading to abnormal formation of KV and loss of KV function (Matsui et al., 2011). These observations suggest that KV formation becomes abnormal in either weak or strong cadherin1-based adherens junctions. This may be a common feature of collective migration because it has been reported that both up- and down-regulation of DE-cadherin induce the delayed collective migration of the border cell cluster during Drosophila oogenesis (Niewiadomska et al., 1999; Schober et al., 2005). The DFC-speciﬁc knockdown of integrinα5 or integrinβ1b results in the breaking up of the DFC cluster (Ablooglu et al., 2010), demonstrating that cell-ECM interaction contributes to DFC collectivity (Figure 1 and Table 1). integrinα5/β1b is known to recognize RGD-peptide containing ligands (e.g., ﬁbronectin, vitronectin, and osteopontin). Although overexpression of a dominant negative form of ﬁbronectin and ﬁbronectin mutations, including natter, leads to the randomization of left-right asymmetric body plan and cardia biﬁda, respectively (Trinh and Stainier, 2004; Compagnon et al., 2014), it has not been reported that DFC collectivity is compromised in embryos injected with the dominant negative form of ﬁbronectin and ﬁbronectin mutants. Although laminin-α1β1γ1 accumulate around KV and DFC-speciﬁc knockdown of laminin-γ1 results in ciliogenesis failures and the randomization of a left-right asymmetric body plan (Compagnon et al., 2014), this manipulation does not lead to the breaking up of the DFC cluster. Therefore, it is unlikely that ﬁbronectin and laminins are a ligand for integrinα5/β1b in migrating DFCs. Additional experiments are required to identify the ligand(s) for integrinα5/β1b during collective DFC migration in the near future. These ﬁndings suggest that a ﬁne-tuning of cadherin1 expression and function into a proper range is required for generating proper DFC collectivity. KV Formation and Function lgl2 in cooperation with rab11b GTPase enhances the traﬃcking of cadherin1 proteins into a lateral membrane to generate KV lumen, suggesting that regulation of the membrane traﬃcking of cadherin1 protein is required for ensuring cell collectivity (Table 1). Heterotrimeric G proteins of the Gα12 family (Gα12 and Gα13) bind to the intercellular domain of cadherin1 and interfere with the link between cadherin1 and β-catenin, leading to the inhibition of cell-cell adhesion (Lin et al., 2009). Importantly, loss- and gain-of-function of the Gα12 family in zebraﬁsh result in a breaking up of the DFC cluster into small groups of cells (Table 1) (Lin et al., 2009), suggesting again that ﬁne-tuning of the cadherin1 expression is required for generating cell collectivity. Taken together, these results suggest that the cadherin1-based adherens junction is regulated by both transcriptional and post-translational mechanisms and that these ﬁne-tuning systems are essential for generating proper DFC collectivity during collective DFC migration. Regulatory Mechanisms of Cell Collectivity Formation in the DFC Cluster Our recent study has revealed a part of this mechanism: speciﬁcally, we characterized a ﬁbroblast growth factor (FGF) positive feedback regulator canopy1 in zebraﬁsh and found that FGF-signal dependent regulation of cadherin1 expression is required for generating proper cell collectivity in the DFC cluster (Matsui et al., 2011). fgf8a secreted from DFCs binds to and activates FGF receptor 1 (fgfr1) in DFCs. The intracellular signal pathway (Ras-Erk pathway) induces canopy1 expression. canopy1 with As stated above, loss-of-function of cadherin1-based adherens junction or integrinα5/β1b-ECM interaction in DFCs leads to a broken up DFC cluster (Table 1) (Ablooglu et al., 2010; Matsui et al., 2011). However, some DFCs bind to each other even Frontiers in Cell and Developmental Biology \| www.frontiersin.org May 2015 \| Volume 3 \| Article 27 3 Collectivity regulation within cell cluster Matsui et al. TABLE 1 \| Genes essencial for cell collectivity regulation during collective DFC migration. Loss/Gain of function Gene Protein Adherens junction Tight junction Cell-ECM interaction DFC clustering KV structure References DFC-KD cadherin1 Adhesion molecule Weaken n.d. n.d. Broken-up Small/disrupted Esguerra et al., 2007; Oteiza et al., 2010; Matsui et al., 2011 Mutation, KD ace/fgf8a FGF ligand Weaken (Reduced cdh1 expression) n.d. n.d. Broken-up Small/disrupted Matsui et al., 2011 DFC-KD; KD canopy1 FGF positive regulator Weaken (Reduced cdh1 expression) n.d. n.d. Broken-up Small/disrupted Matsui et al., 2011 DFC-KD; KD tbx16 TF induced by FGF Weaken (Reduced cdh1 expression) Weaken (wide aPKC focal points) n.d. Broken-up Small/disrupted Amack et al., 2007; Matsui et al., 2011 KD ier2 FGF mediator n.d. n.d. n.d. Broken-up Small/disrupted Hong and Dawid, 2009 KD ﬁbp1 FGF mediator n.d. n.d. n.d. Broken-up Small/disrupted Hong and Dawid, 2009 KD fgf2 FGF ligand n.d. Normal n.d. Broken-up Small Arrington et al., 2013 DFC-KD; KD syndecan2 Heparan sulfate proteoglycan n.d. Normal n.d. Broken-up Small Arrington et al., 2013 OE gna13a Cadherin1 binding G protein Weaken (Reduced cdh1 binding activity) n.d. n.d. Broken-up n.d. Lin et al., 2009 KD prickle1a nc-Wnt mediator Weaken (Reduced adhesion forces) Weaken (wide aPKC focal points) n.d. Failed compaction Fragmented lumens Oteiza et al., 2010 DFC-KD; KD lpar3 LPA receptor n.d. Weaken (Reduced aPKC expression) n.d. Broken-up Small/disrupted Lai et al., 2012 DFC-KD; KD autotaxin Lysophospholipase D n.d. Weaken (Reduced aPKC expression) n.d. Broken-up Small/disrupted Lai et al., 2012 DFC-KD; KD ttrap Nodal antagonist Weaken (Reduced cdh1 expression) n.d. n.d. Spread n.d. Genes Essential for DFC Collectivity Many genes have been reported to be involved in the regulation of DFC collectivity, as listed in Table 1. As stated above, genes related to FGF and to Nodal signaling can function as regulators of DFC collectivity. In addition, knockdown of prickle1a, a non- canonical Wnt (nc-Wnt) regulator, leads to failure to compact the DFC cluster during late-gastrulation stages (9–10 hpf) (Table 1) (Oteiza et al., 2010), suggesting that nc-Wnt signaling also regulates cell collectivity. This idea is supported by data from the direct single-cell force spectroscopy measurement of adhesion properties between a pair of DFCs, which showed that the adhesion force between DFCs isolated from embryo depleted of prickle1a is reduced as compared to wildtype DFCs (Oteiza et al., 2010). This method is very good for measuring cell adhesion forces at the single cell level, while physiological interactions are disrupted during cell preparation. It would be of great interest to develop a method to measure physiological cell adhesion forces within living zebraﬁsh embryos in the near future. Because the DFC-speciﬁc gene knockdown approach is absolutely indispensable to provide new insights into collective DFC migration, KV formation, KV ciliogenesis, and left- right patterning in the near future, a guideline for DFC- speciﬁc gene knockdown is needed. We therefore propose one based on guidelines reported by Eisen and Smith (2008) and Kok et al. (2015). First, as with standard morpholino- based knockdown approaches, validate morpholino-induced phenotypes and compare them to those of the mutant. If the mutant is not available, generate one for the gene of interest and make sure of the morpholino-induced phenotypes by comparison. Second, inject the morpholino tagged with ﬂuorescein or lissamine into the yolk of embryos at 256–512 cell stages (2.5–2.75 hpf), and select embryos in which morpholino has been correctly delivered into the DFCs. As an important control, also inject the same morpholino into the yolk of embryos at the sphere-dome stages (4–4.3 hpf) (Amack and Yost, 2004; Amack et al., 2007). Third, perform a rescue experiment by co- injecting morpholino and mRNA into the yolk of embryos at 256–512 cell stages (Matsui and Bessho, 2012). Regulatory Mechanisms of Cell Collectivity Formation in the DFC Cluster Esguerra et al., 2007 OE smad3b Nodal mediator Weaken (Reduced cdh1 expression) n.d. n.d. Broken-up n.d. Esguerra et al., 2007 KD wdr18 WD-repeat protein n.d. Normal Weaken Broken-up Small Gao et al., 2011 DFC-KD; KD integrinα5, β1b Adhesion molecule to ECM n.d. Normal Weaken Broken-up Small Ablooglu et al., 2010; Gao et al., 2011 KD lgl2 A homolog of Tumor suppressor protein in Drosophila Weaken (Reduced cdh1 accumulation at membrane) Normal n.d. n.d. Small Tay et al., 2013 DFC-KD; KD cateninβ1, β2 Wnt mediator n.d. Normal n.d. Small (Reduced proliferation) Small Zhang et al., 2012 Inhibition – Ca2+ -ATPase Weaken n.d. n.d. Broken-up Small/disrupted Schneider et al., 2008 DFC KD DFC speciﬁc knockdown; KD knockdown; OE overexpression; n d not determined May 2015 \| Volume 3 \| Article 27 Collectivity regulation within cell cluster Matsui et al. in the near future, we feel that it would be diﬃcult to apply it to understand the roles of genes in DFC/KV formation. in these situations. These ﬁndings suggest that cooperation of several adhesive interactions is required to regulate DFC collectivity. Therefore, it will be important to characterize cooperative eﬀects of adhesive interactions on cell collectivity regulation during collective DFC migration. In general, conditional gene knockout is carried out by using a Cre/loxP system. The expression of Cre recombinase is induced by a tissue/organ-speciﬁc promoter and Cre protein modulates the genome at the speciﬁc insertion site of loxP. Thus, it takes time to observe the signs of Cre/loxP recombination. For instance, in the case of conditional knockout mice, the signs of Cre/loxP recombination are detected an average of 24 h after the induction (Hayashi and McMahon, 2002; Chen et al., 2007). In the case of DFCs, however, collective DFC migration starts at almost the same time as the DFC speciﬁcation (evident by the expression of early DFC marker genes, sox32 and sox17) and ﬁnishes just 3 h (Oteiza et al., 2008; Matsui and Bessho, 2012). It has been suggested that Cre/loxP recombination may not occur simultaneously with collective DFC migration without applying much faster recombination methods. Identiﬁcation of Genes Required for DFC Collectivity Formation in Future y A DFC-speciﬁc gene knockdown approach in which morpholino is injected into the yolk of embryos at the 256–512 cell stages has greatly contributed to our understanding of how DFC collectivity is regulated (this review), how DFC clustering occurs and whether DFC/KV morphogenesis is required for the establishment of left-right patterning (Amack and Yost, 2004; Amack et al., 2007; Matsui and Bessho, 2012). We therefore believe that this approach will be important to identify new genes required for DFC collectivity. However, very recently, Kok et al. have reported a problem for morpholino-induced knockdown approach (Kok et al., 2015). Approximately 80% of phenotypes induced by morpholinos are not observed in mutant embryos, indicating that morpholinos highly induce oﬀ-target eﬀects; moreover, these problems are shared with other antisense technologies. It is thus recommended that only morpholinos that recapitulate the respective phenotypes seen in mutant embryos should be applied for ancillary analyses. As the DFC-speciﬁc gene knockdown approach is based on morpholino-induced knockdown, here is a good opportunity to consider whether we can apply this approach to investigate how DFC collectivity is regulated in future. Frontiers in Cell and Developmental Biology \| www.frontiersin.org Dynamics of Collective DFC Migration y g During epiboly (6–10 hpf), the EVL cells tightly bind to the yolk syncytial layer (YSL) and purse string contraction of the actin in YSL drives the migration of EVL toward the vegetal pole (Lepage and Bruce, 2010; Lee, 2014). Because a subset of DFCs is linked to EVL through the formation of tight junction, it has been suggested that DFC-EVL tight junction is important to mediate the vegetal migration of DFCs during epiboly (Figures 1, 2). However, live imaging has revealed that DFCs located at the leading edge side of the cluster frequently generate cell protrusions such as ﬁlopodia and lamellipodia toward the vegetal pole (Ablooglu et al., 2010 and see also Figure 2). These ﬁndings suggest that, in addition to passive migration mediated by DFC-EVL tight junction, DFCs have the potential to migrate themselves toward the vegetal pole. As this approach has similarities to the conditional knockout strategy, it is best to evaluate whether DFC-speciﬁc gene knockdown recapitulates the phenotypes caused in the DFC- speciﬁc conditional knockout zebraﬁsh. Although insertion of loxP site into a zebraﬁsh genome by genome editing technologies such as TALEN and CRISPER/Cas9 systems has recently been successful (Bedell et al., 2012; Chang et al., 2013), a method generating conditional knockout zebraﬁsh has not yet been developed. Even though such a method is likely to be developed In collective cell migration of endothelial cells, the cells behave dynamically. For instance, a follower cell sometimes moves to the leading edge and becomes a leader cell (Jakobsson et al., 2010; Arima et al., 2011; Rorth, 2012), suggesting that cell behaviors are highly variable in individuals and positions, and Frontiers in Cell and Developmental Biology \| www.frontiersin.org May 2015 \| Volume 3 \| Article 27 5 Matsui et al. Collectivity regulation within cell cluster FIGURE 2 \| Cell dynamics during collective DFC migration. Depending on the formation of tight junction between DFC and EVL, directed migration of the DFC cluster toward the vegetal pole passively occurs (blue arrows). EVL, enveloping layer; DC, deep cells; YSL, yolk syncytial layer; DFC, dorsal forerunner cells. Time-lapse images from 0 to 16 min (4 min intervals) in the control Tg[sox17:GFP] embryo (left panels). Dorsal view, anterior to the top. Scale bar: 20 μm. As shown previously (Ablooglu et al., 2010), GFP-positive DFCs at the leading edge frequently generate ﬁlopodium (arrow) and lamellipodium (bracket) (right panels). Dynamics of Collective DFC Migration These ﬁndings suggest that, in addition to the passive migration through the formation of DFC-EVL tight junction, DFCs have a potential to migrate toward the vegetal pole. Dorsal view, anterior to the top. Scale bar: 20 μm. As shown previously (Ablooglu et al., 2010), GFP-positive DFCs at the leading edge frequently generate ﬁlopodium (arrow) and lamellipodium (bracket) (right panels). These ﬁndings suggest that, in addition to the passive migration through the formation of DFC-EVL tight junction, DFCs have a potential to migrate toward the vegetal pole. FIGURE 2 \| Cell dynamics during collective DFC migration. Depending on the formation of tight junction between DFC and EVL, directed migration of the DFC cluster toward the vegetal pole passively occurs (blue arrows). EVL, enveloping layer; DC, deep cells; YSL, yolk syncytial layer; DFC, dorsal forerunner cells. Time-lapse images from 0 to 16 min (4 min intervals) in the control Tg[sox17:GFP] embryo (left panels). Dorsal (Abloog generat These the form toward Dorsal view, anterior to the top. Scale bar: 20 μm. As shown previously (Ablooglu et al., 2010), GFP-positive DFCs at the leading edge frequently generate ﬁlopodium (arrow) and lamellipodium (bracket) (right panels). These ﬁndings suggest that, in addition to the passive migration through the formation of DFC-EVL tight junction, DFCs have a potential to migrate toward the vegetal pole. FIGURE 2 \| Cell dynamics during collective DFC migration. Depending on the formation of tight junction between DFC and EVL, directed migration of the DFC cluster toward the vegetal pole passively occurs (blue arrows). EVL, enveloping layer; DC, deep cells; YSL, yolk syncytial layer; DFC, dorsal forerunner cells. Time-lapse images from 0 to 16 min (4 min intervals) in the control Tg[sox17:GFP] embryo (left panels). dynamic than previously thought. Despite this substantial progress, many important questions remain. For instance, how do collective cell dynamics contribute to generating functional organs? How does the pairing of tight junction change? Are adherens junctions, tight junctions, and cell-ECM interaction coordinated? Does mechanical force contribute to collective DFC migration? Does collective DFC migration have analogy with other collective cell migrations seen in normal development, wound repair, and cancer invasion? It is of great interest to ﬁll in these gaps to further clarify the regulatory mechanisms and importance of collective cell migration during organogenesis. that cell identities as leaders and followers can be changeable during collective cell migration. Dynamics of Collective DFC Migration If individual DFCs behave dynamically during collective DFC migration, signal activities, which regulate cell collectivity and/or migration, are diﬀerent among individual DFCs and change from time to time even in an individual DFC. Therefore, precise observation of individual DFC behaviors, adhesive properties, and signal activities in living zebraﬁsh embryos will be important to understand mechanisms of collective DFC migration. Acknowledgments We are grateful to the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Uehara Memorial Foundation, the Nakajima Foundation and the Mochida Memorial Foundation for past and current support. Conclusion and Outlook Many studies using zebraﬁsh as a model system of vertebrate development have provided us with new insights into how the laterality organ (KV) breaks left-right symmetry. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Warga, R. M., and Kane, D. A. (2007). A role for N-cadherin in mesodermal morphogenesis during gastrulation. Dev. Biol. 310, 211–225. doi: 10.1016/j.ydbio.2007.06.023 May 2015 \| Volume 3 \| Article 27 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 8
https://openalex.org/W4229801816	https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1007639&type=printable	English	null	Correction: Maternal Diet and Insulin-Like Signaling Control Intergenerational Plasticity of Progeny Size and Starvation Resistance	PLOS genetics	2,018	cc-by	529	1. Hibshman JD, Hung A, Baugh LR (2016) Maternal Diet and Insulin-Like Signaling Control Intergenera- tional Plasticity of Progeny Size and Starvation Resistance. PLoS Genet 12(10): e1006396. https://doi. org/10.1371/journal.pgen.1006396 PMID: 27783623 The fifth and sixth sentences in the second paragraph under the subheading “Liquid culture system for dietary restriction” in the Materials and Methods should read as follows: The fifth and sixth sentences in the second paragraph under the subheading “Liquid culture system for dietary restriction” in the Materials and Methods should read as follows: “Subsequently OD600 was measured in 1:20 dilutions to ensure measurements were in the linear range of measurements. At this dilution, absorbance readings were consistently around 0.7–0.8 for AL and 0.09–0.1 for DR (S1 Fig).” OPEN ACCESS Citation: Hibshman JD, Hung A, Baugh LR (2018) Correction: Maternal Diet and Insulin-Like Signaling Control Intergenerational Plasticity of Progeny Size and Starvation Resistance. PLoS Genet 14(8): e1007639. https://doi.org/10.1371/journal. pgen.1007639 Supporting information S1 Fig. A liquid culture system for DR. A) Schematic of DR by food dilution in liquid culture. Worms are grown in standard conditions on plates with OP50 and then bleached to obtain embryos. Embryos are hatched in buffer so they enter L1 arrest for synchronization. Arrested L1 larvae are added to culture flasks at a very low density of 10 worms/mL so that they do not reduce bacterial density during culture. E. coli HB101 is used for liquid culture to avoid floccu- lation. Worms are cultured at 20˚C with shaking and typically harvested at 96 hr to collect their embryos for phenotypic analysis. B) Optical density at 600 nm (OD600) is plotted for 1:5 dilutions of different densities of HB101 over time in S-complete, showing that density is roughly constant. C) OD600 is plotted for 1:20 dilutions of AL and DR cultures with worms at 0 and 96 hr of culture. There is not a significant change in bacterial density in either AL or DR (p = 0.10, p = 0.19 respectively, paired t-test, n = 3). The data points obscure SEM bars. (TIF) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 CORRECTION Correction: Maternal Diet and Insulin-Like Signaling Control Intergenerational Plasticity of Progeny Size and Starvation Resistance a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Jonathan D. Hibshman, Anthony Hung, L. Ryan Baugh Jonathan D. Hibshman, Anthony Hung, L. Ryan Baugh S1 Fig reports OD600 values for ad libitum (25 mg/mL E. coli HB101) and dietary restriction (3.1 mg/mL E. coli HB101) with incorrect dilution factors. The measurements reported in S1B Fig were from a 5-fold dilution and those in S1C were from a 20-fold dilution. The ad libitum and dietary restriction conditions corresponded to approximately 16 and 2 OD600 units, respectively. The authors are issuing a correction to rectify this mistake. Please view the cor- rected version of S1 Fig and its legend below. These changes also affect two sentences in the Materials and Methods section. Published: August 30, 2018 Copyright: © 2018 Hibshman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 / 1 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007639 August 30, 2018
https://openalex.org/W3127691301	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/12/e3sconf_icersd2020_01015.pdf	English	null	Research on Investment Trend of Distribution Network Based on Support Vector Machine	E3S web of conferences	2,021	cc-by	2,799	1 Introduction investment prediction model. Literature [5] established a power grid infrastructure investment calculation model using gray theory, and verified the validity and rationality of the model through an example of power grid infrastructure investment calculation in a certain area. As a basic power facility, the distribution network plays an extremely important role in the development of the economy and the improvement of people's living standards. Therefore, strengthening the investment forecast management of the distribution network is of vital importance to improving the operating efficiency of power grid enterprises. To sum up, the current grid investment forecasts are mostly the application of gray models and linear regression models. Due to the lack of grid-related data and insufficient analysis, the final investment forecast results are relatively one-sided and not accurate enough. Therefore, this paper first applies the fishbone diagram theory to systematically analyze the main factor indicators that affect the distribution network investment; then combines the relevant expert opinions and the measurability of the factor indicator data to select the key factor indicators to construct a support vector machine-based distribution network investment forecast Model; Finally, the relevant data of a certain area distribution network is selected as a sample to verify the validity and feasibility of the model. Literature [1] uses five mathematical models to predict the power consumption and maximum load of Wuhai City on the premise of analyzing the development status and problems of Wuhai City to support the investment and construction of the power grid. Literature [2] based on a comprehensive analysis of the current status of distribution network management, combined with historical data, obtained some problems in the distribution network investment planning of county-level power supply enterprises, and then used the gray prediction method and the multiple linear regression prediction model to carry out Load forecasting avoids the shortcomings of a single forecasting method, and achieves the purpose of reducing forecasting risks and increasing forecasting accuracy. Literature [3] built a medium- and long-term investment scale prediction model for provincial power grids based on support vector machines based on analyzing the new environment, new directions, and new requirements of power grid investment. Literature [4] firstly analyzes the main factors affecting power grid investment and its impact mechanism from the macroeconomic and microeconomic aspects, and then builds an ARMA-based power grid Research on Investment Trend of Distribution Network Based on Support Vector Machine Chao Wang1, Jiyuan Zhang1, Yaling Jian1, Jie Jiao1, Xi Yang1, Guangxiu Yu1, Le Du2ˈ* 1 State Grid Sichuan Economic Research Institute, Chengdu, Sichuan ,China 2 School of Economics and Management, North China Electric Power University, Beijing, China Abstract: Under the new situation, with the continuous development of my country's economy and the implementation of power system reforms, higher development requirements have been put forward for the distribution network investment plan. Through the scientific and reasonable calculation of the investment scale of the distribution network, optimizing the investment scale of the distribution network and rationally arranging the investment planning of the distribution network project have become one of the key concerns of the current power grid enterprises. This paper uses fishbone diagram theory to analyze the factors that affect the investment scale of the distribution network, and selects the key factor indicators to construct a distribution network investment trend prediction model based on support vector machines. By selecting a certain region's distribution network investment for empirical forecasting analysis, and comparing with the planned investment of the distribution network in the region, the validity of the model is verified. Corresponding author’s e-mail: 1182306191@ncepu.edu.cn © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/202123601015 https://doi.org/10.1051/e3sconf/202123601015 E3S Web of Conferences 236, 01015 (2021) ICERSD 2020 Research on Investment Trend of Distribution Netwo Support Vector Machine Chao Wang1, Jiyuan Zhang1, Yaling Jian1, Jie Jiao1, Xi Yang1, Guangxiu Yu1, Le Du2ˈ 1 State Grid Sichuan Economic Research Institute, Chengdu, Sichuan ,China 2 School of Economics and Management, North China Electric Power University, Beijing, China DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2 Analysis of influencing factors of distribution network investment Figure 1 stribution network investmentInfluencing factors g If there is a classification hyperplane: If there is a classification hyperplane: 0 WX b ˄1˅ Meet the constraints: 1 0, 1,2, , iy WX b i l ˄2˅ If there is a classification hyperplane: 0 WX b ˄1˅ Meet the constraints: 1 0, 1,2, , iy WX b i l ˄2˅ assification hyperplane: 0 WX b ˄1˅ ˄1˅ Meet the constraints: 1 0, 1,2, , iy WX b i l ˄2˅ ˄2˅ It is said that the training set is linearly separable. Where W is the weight vector, X is the input vector, and b R is the bias. It is said that the training set is linearly separable. Where W is the weight vector, X is the input vector, and b R is the bias. Use p to represent the distance between the hyperplane and the nearest sample. The purpose of the support vector machine is to find a hyperplane with the largest separation edge. This hyperplane is the optimal hyperplane (OptimalHyperplane). Therefore, w and b when p is the largest must be determined. The resulting prediction function: Figure 1.stribution network investmentInfluencing factors Figure 1.stribution network investmentInfluencing factors. Comprehensively consider the complexity of the forecast model, the quantitative analysis of the influencing factor index data, and the availability of data, combined with the suggestions of relevant experts, select four areas: total electricity consumption, proportion of electricity, reliability of power supply, and coverage of distribution network automation Key indicators are the basic elements of model construction. The basic element index system of the forecasting model is shown in Table 1 below: ( ) Y f x sgn WX b ˄3˅ ˄3˅ Its promotion ability is the best. Among them, sgn(·) is a symbolic function. That is, the classification error rate of the original sample should be minimized. Its promotion ability is the best. Among them, sgn(·) is a symbolic function. That is, the classification error rate of the original sample should be minimized. From analytic geometry, the distance from any point in the sample space to the optimal hyperplane is: Table 1. Predictive model building elements. S i Table 1. Predictive model building elements. 2 Analysis of influencing factors of distribution network investment Serial number Indicator type Index 1 Electricity demand Regional electricity consumption 2 The grid is safe and reliable Power supply reliability rate 3 Promote energy consumption Electricity ratio 4 Distribution network technology innovation Distribution network automation coverage rate 0 0 0 r \|\| \|\| W X b W ˄4˅ ˄4˅ mplification: 0 0 0 g( ) \|\| \|\| x r W W X b ˄5˅ ˄5˅ Normalize the function so that 0 \|g( ) \| 1 x , which is the support vector, is the closest to the classification decision plane, and plays a leading role in the SVM. Normalize the function so that A, which is the support vector, is the closest to the classification decision plane, and plays a leading role in the SVM. So the algebraic distance from the support vector to the optimal hyperplane is: 2 Analysis of influencing factors of distribution network investment Under the new situation, the influence factors of power distribution network investment of power grid enterprises are becoming more diversified and complicated. From the perspective of power grid companies, when making distribution network investment planning, in addition to regional economic development plans, industrial development policies, and the company’s own *Corresponding author’s e-mail: 1182306191@ncepu.edu.cn https://doi.org/10.1051/e3sconf/202123601015 E3S Web of Conferences 236, 01015 (2021) ICERSD 2020 problem that the low-dimensional space cannot use linear regression. The basic principles of the period are as follows: development strategies, it is also necessary to comprehensively consider the optimization of the distribution network structure and the reliable and safe power supply quality. , Electric energy substitution, and promotion of electric energy consumption. This article combines the current situation of distribution network investment planning and management and relevant expert suggestions, from the four dimensions of power demand, grid safety and reliability, promotion of energy consumption, and distribution network technology innovation, combined with fishbone diagram theory, a systematic analysis of the impact on distribution network investment Factors, as shown in Figure 1 below. The given sample set is , , 1,2, , i i x y i l , where n ix R ˈ 1, 1 iy . The principle of support vector machine for solving nonlinear function optimization is to solve the high-dimensional feature space by linear programming by mapping the nonlinearity to the high-dimensional space. First, we must construct a decision function (classification hyperplane) to classify the sample data. The classification hyperplane can maximize the blank area on both sides of the hyperplane while ensuring the classification accuracy, so as to realize the optimal classification of linear separable problems. Linear separability refers to the use of one or several straight lines to separate sample points belonging to different categories. 4 Empirical analysis This paper selects 2016-2019 distribution network investment data in a certain region for simulation analysis. The original data of distribution network investment and electricity consumption levels in the region from 2016 to 2019 are shown in Table 2. The classification machine is an algorithm that converts the optimization problem of the maximum interval method to solve the optimal classification surface into its dual problem, so as to solve the original classification problem by solving the relatively simple dual problem. Table 2. Original data table of distribution network investment and electricity consumption in a certain region from 2016 to 2019 Table 2. Original data table of distribution network investment and electricity consumption in a certain region from 2016 to 2019. Table 2. Original data table of distribution network investment and electricity consumption in a certain region from 2016 to 2019. year s Distributi on network investmen t (100 million yuan) Regional electricity consumpti on (100 million kWh) Power supply reliabilit y rate (%)˅ Electrici ty ratio (%) Distributi on network automatio n coverage rate (%) 2016 37.6 365.4 99.936 16.63 25.8 2017 31.2 378.9 99.965 18.98 61.2 2018 28.3 420.7 99.964 25.12 63.7 2019 34.5 543.6 99.968 32.68 88.5 Using Lagrange optimization method and Wofle duality theory, the above problem can be transformed into a dual problem, that is, to maximize the functional function: 1 , 1 1 1 ( ) 2 0 . . 0 1, . n n i i j i j i j i i j n i i i i Q y y x x y s t i n ˄7˅ ˄7˅ Among them, i is the Lagrange multiplier corresponding to sample i . According to the support vector machine algorithm, the predicted value of the distribution network investment scale in the region from 2020 to 2022 in the next three years is as follows: The optimal classification function obtained by solving the above problems is: 1 ( ) sgn sgn , nsv i i i i f x WX b a y x x 8˅ Table 3. Comparative analysis of model predicted value and planned investment value Table 3. Comparative analysis of model predicted value and planned investment value. Table 3. Comparative analysis of model predicted value and planned investment value. 3 Construction of distribution network investment prediction model based on regression tree theory 0 0 0 g(x ) 1 r \|\| \|\| \|\| \|\| W W ˄6˅ ˄6˅ The basic idea of the support vector machine is that the function of reducing the dimensional space is transformed into a high-dimensional linear mapping through non-linear mapping, so as to better solve the Therefore, it can be considered that maximizing the separation edge is equivalent to minimizing the norm W 2 E3S Web of Conferences 236, 01015 (2021) ICERSD 2020 https://doi.org/10.1051/e3sconf/202123601015 of the weight vector. Therefore, the classification hyperplane that minimizes W is the optimal hyperplane. 4 Empirical analysis years Forecast value of distribution network investment (100 million yuan) Planned investment value (100 million yuan) Relative error 2020 34.9 35 -0.29% 2021 35.8 35.5 0.85% 2022 35.2 36 -2.22% 8˅ Among them, nsv is the number of support vectors, and b is the classification threshold, which can be obtained by taking the median of any pair of support vectors in the two categories. By introducing the relaxation term i , a generalized classification surface can be realized, and the situation that the training samples are linearly inseparable can be solved, that is, the minimum error classification sample and the maximum classification interval are considered as a compromise. For nonlinear problems, it can be transformed into a linear problem in a high-dimensional space through nonlinear transformation, and then the optimal classification surface is sought in this high-dimensional space. Only the inner product operation i j x x between samples is involved, so only the inner product operation needs to be performed in the high-dimensional space, and the inner product operation can be realized by the function in the original space. According to the Hilbert-schmidt principle, as long as the kernel function i j K x x satisfies the Mercer condition, it corresponds to the inner product in a certain exchange space. As can be seen from the above table, the application model predicts that the investment scale of the distribution network in the region in the next three years will be 3.49 billion yuan, 3.58 billion yuan and 3.52 billion yuan respectively. According to the distribution network investment development plan in the region, the planned investment scale of the distribution network in the region from 2020 to 2022 will be 3.5 billion yuan, 3.55 billion yuan and 3.6 billion yuan, respectively. The relative errors of the investment prediction model are -0.29%, 0.85%, and -2.22% respectively, indicating the model Has good prediction accuracy. 5 Conclusion In view of the importance of improving the research technology of distribution network investment forecasting, this paper constructs a distribution network investment forecasting model based on support vector machines based on the analysis of the influence factors of distribution network investment, and applies the model to predict 2020-2022 in a certain area The annual distribution network investment scale, through comparison with the planned investment in the region, verifies that the model has good prediction accuracy, and can provide reference and reference for the prediction of the distribution network investment scale of power grid Therefore, by replacing the inner product in the formula with the kernel function i j K x x that satisfies the Mercer condition, a certain linear classification after nonlinear transformation can be realized, and the optimal classification function becomes: ˄ ˅ ˄9˅ 1 ( ) sgn nsv i i i i f x a y K x x b 3 https://doi.org/10.1051/e3sconf/202123601015 E3S Web of Conferences 236, 01015 (2021) ICERSD 2020 3. Xie Wei, Pan Wenming, Wang Chenghua, Chen Li, Wang Yang, Ma Jing, Li Zhiwei, Du Haihong. Research on the medium and long-term investment scale prediction model of provincial power grid based on support vector machine[J]. Industrial Technology Economy, 2019, 38 (08):154-160. companies. companies. Acknowledgments This research was supported by the Science and Technology Project of the State Grid Sichuan Economic Research Institute "Multi-scale Evaluation of Distribution Network Investment and Comprehensive Optimization Technology Evaluation under the New Reform Situation". 4. Ji Liwei, Yang Liping, Fei Gaiying. Analysis and prediction of influencing factors of power grid project investment [J]. China Electric Power Enterprise Management, 2016(06): 85-92. 5. Hu Baichu, Hu Gang, Hu Chaohua, Qing Song, Li Mingwei, Peng Chao. Power grid infrastructure investment calculation model based on gray prediction[J]. Journal of University of Electronic Science and Technology of China, 2013, 42(06): 890-894 References 1. Wu Rong. Research on Wuhai Distribution Network Planning Scheme[D]. North China Electric Power University, 2018. 1. Wu Rong. Research on Wuhai Distribution Network Planning Scheme[D]. North China Electric Power University, 2018. 2. Liao Junjie. Research on Pingnan County Distribution Network Planning [D]. Guangxi University, 2016. 2. Liao Junjie. Research on Pingnan County Distribution Network Planning [D]. Guangxi University, 2016. 4
https://openalex.org/W3016943283	https://www.research.ed.ac.uk/files/156107705/s40814_020_00606_0.pdf	English	null	Increasing uptake of structured self-management education programmes for type 2 diabetes in a primary care setting: a feasibility study	Pilot and feasibility studies	2,020	cc-by	15,340	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Increasing uptake of structured self-management education programmes for type 2 diabetes in a primary care setting: A feasibility study Citation for published version: Davies, M, Kristunas, C, Huddlestone, L, Alshreef, A, Bodicoat, D, Dixon, S, Eborall, H, Glab, A, Hudson, N, Khunti, K, Martin, G, Northern, A, Patterson, M, Pritchard, R, Schreder, S, Stribling, B, Turner, J & Gray, L 2020, 'Increasing uptake of structured self-management education programmes for type 2 diabetes in a primary care setting: A feasibility study', Pilot and Feasibility Studies. https://doi.org/10.1186/s40814-020- 00606-0 Citation for published version: Digital Object Identifier (DOI): 10.1186/s40814-020-00606-0 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Pilot and Feasibility Studies RESEARCH Open Access © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Increasing uptake of structured self- management education programmes for type 2 diabetes in a primary care setting: a feasibility study Melanie Davies1, Caroline A. Kristunas1,2 , Lisa Huddlestone2, Abualbishr Alshreef3, Danielle Bodicoat1, Simon Dixon3, Helen Eborall2, Agnieszka Glab4, Nicky Hudson5, Kamlesh Khunti1, Graham Martin6, Alison Northern4, Mike Patterson4, Rebecca Pritchard4, Sally Schreder4, Bernie Stribling4, Jessica Turner5 and Laura J. Gray2 Abstract Background: Structured self-management education (SSME) for people with type 2 diabetes mellitus (T2DM) improves biomedical and psychological outcomes, whilst being cost-effective. Yet uptake in the UK remains low. An ‘Embedding Package’ addressing barriers and enablers to uptake at patient, health care professional and organisational levels has been developed. The aim of this study was to test the feasibility of conducting a subsequent randomised controlled trial (RCT) to evaluate the Embedding Package in primary care, using a mixed methods approach. Methods: A concurrent mixed methods approach was adopted. Six general practices in the UK were recruited and received the intervention (the Embedding Package). Pseudonymised demographic, biomedical and SSME data were extracted from primary care medical records for patients recorded as having a diagnosis of T2DM. Descriptive statistics assessed quantitative data completeness and accuracy. Quantitative data were supplemented and validated by a patient questionnaire, for which two recruitment methods were trialled. Where consent was given, the questionnaire and primary care data were linked and compared. The cost of the intervention was estimated. An integrated qualitative study comprising ethnography and stakeholder and patient interviews explored the process of implementation, sustainability of change and ‘fit’ of the intervention. Qualitative data were analysed using a thematic framework guided by the Normalisation Process Theory (NPT). (Continued on next page) Correspondence: melanie.davies@uhl-tr.nhs.uk * Correspondence: melanie.davies@uhl-tr.nhs.uk 1Diabetes Research Centre, University of Leicester, Leicester, UK Full list of author information is available at the end of the article Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Davies et al. Pilot and Feasibility Studies (2020) 6:71 https://doi.org/10.1186/s40814-020-00606-0 * Correspondence: melanie.davies@uhl-tr.nhs.uk 1Diabetes Research Centre, University of Leicester, Leicester, UK Full list of author information is available at the end of the article (Continued from previous page) (Continued from previous page) Results: Primary care data were extracted for 2877 patients. The primary outcome for the RCT, HbA1c, was over 90% complete. Questionnaires were received from 423 (14.7%) participants, with postal invitations yielding more participants than general practitioner (GP) prompts. Ninety-one percent of questionnaire participants consented to data linkage. The mean cost per patient for the Embedding Package was £8.94, over a median follow-up of 162.5 days. Removing the development cost, this reduces to £5.47 per patient. Adoption of ethnographic and interview methods in the collection of data was appropriate, and the use of NPT, whilst challenging, enhanced the understanding of the implementation process. The need to delay the collection of patient interview data to enable the intervention to inform patient care was highlighted. Conclusions: It is feasible to collect data with reasonable completeness and accuracy for the subsequent RCT, although refinement to improve the quality of the data collected will be undertaken. Based on resource use data collected, it was feasible to produce cost estimates for each individual component of the Embedding Package. The methods chosen to generate, collect and analyse qualitative data were satisfactory, keeping participant burden low and providing insight into potential refinements of the Embedding Package and customisation of the methods for the RCT. Trial registration: ISRCTN, ISRCTN21321635, Registered 07/07/2017—retrospectively registered. Keywords: Type 2 diabetes Structured self-management education Feasibility study Key messages regarding feasibility result in improved biomedical and psychological out- comes [5, 7, 8]. Improvements have been seen in HbA1c, lipids, weight and blood pressure, as well as in depres- sion and quality of life [5, 7, 8]. It is therefore unsurpris- ing that NICE recommends SSME to be made available to patients “at and around the time of diagnosis, with annual reinforcement and review” [9]. However, despite the demonstrated benefits of SSME and it being made a national priority by NICE, rates of uptake to SSME for those with T2DM have remained low. Latest figures show that although the number of patients with T2DM- offered SSME has increased substantially in the last 5 years, and as of 2015, this figure was in excess of 90%, in the same year, less than 10% of those diagnosed with T2DM were recorded as having attended [10]. A system- atic review of the reasons why patients referred to dia- betes education programmes choose not to attend found two broad categories of non-attenders [11]. The authors found the first category to comprise patients who were unable to attend due to social or logistical reasons, whilst the second category comprised those patients who chose not to attend or perceived no benefit in doing so [11]. The review concluded that the referrers had a responsibility to ensure that “those who are referred are appropriate, ready and fully informed”, whilst the courses that they are referred to should be ‘accessible in time and place wherever possible’ [11]. The feasibility of data collection procedures and utility of the chosen theoretical framework were uncertain. The study identified that it was feasible to collect quantitative data with reasonable completeness and accuracy directly from primary care. The collection of qualitative data was also found to be feasible, and the appropriateness of the selected theoretical framework was confirmed. Feasibility and potential benefits of the intervention were indicated. Refinement of quantitative data collection methods was recommended to enhance quality. Further development of the Embedding Package was highlighted to improve clinician engagement. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 19 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies Background Type 2 diabetes mellitus (T2DM) is a progressive chronic disease which can lead to a reduced quality of life and increased prevalence of long-term health com- plications. Diabetes affects almost 3.7 million people in the UK [1], a figure that continues to rise [2] despite ef- forts to promote healthier lifestyle changes and develop- ments in pharmacological interventions. It is predicted that by 2035, diabetes will account for 17% of the National Health Service (NHS) expenditure [3]. There is now a burgeoning literature on the ways in which health care interventions are implemented in a variety of organisational settings, yet authors have highlighted the absence of studies which focus on the longer-term sustainability of such interventions [12–14]. The question of how improvements in health care are retained and become embedded or ‘routinised’ in every- day practice remains poorly understood, and is as much A substantial body of evidence demonstrates the bene- fits of structured self-management education (SSME) in T2DM [4, 5]. Here, the term SSME is used to encompass any education programme meeting the recommended National Institute for Health and Care Excellence (NICE) criteria [6], regardless of the mode of delivery. SSME has been shown to be both cost-effective [4] and Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies Page 3 of 19 The appointment of an ‘Embedder’ was part of the new/amended professional roles. The role of the Embed- der is to liaise between all relevant stakeholders to pro- mote SSME and use of the resources. A local champion, identified in each CCG, will promote SSME and the Em- bedding Package at both practice and CCG level. a matter of networks of influence and knowledge of pri- orities and incentive frameworks than as of clinical or cost-effectiveness in themselves [13]. It is important, therefore, to explore how to ensure that SSME can be- come part of routine care in the new organisational structures of the NHS in ways that are feasible for all stakeholders. This requires a better understanding of the contextual factors and processes that encourage the adoption of structured education interventions and what the barriers might be to longer term sustainable change and how to overcome these barriers. Background Promotional resources targeting different stakeholders will include (a) patient-facing resources such as promo- tional posters, SSME invitation letters and self-referral forms; (b) resources for health care professionals including document templates, guidance on recruiting staff, refer- ring patients and increasing staff engagement; and (c) re- sources for education providers and commissioners that will consist of electronic administration and referral systems, sample referral pathways and evidence summar- ies. In addition, the toolkit will also include guidance on communication strategies, auditing, conducting local needs assessments, as well as details of how to ensure pa- tient accessibility and tailoring of SSME courses and sessions. To address this need, an intervention to increase uptake to SSME was developed. In accordance with guidance from the UK Medical Research Council [15], multiple phases of work were undertaken using a multiple-method approach, informed by the Normalisation Process Theory (NPT) [16]. Three stages of work were undertaken over a 12-month period. The process was iterative, with each stage informing subsequent stages. Stage one [an evidence synthesis comprising a review of 23 published articles and a secondary analysis of five qualitative data sets (n = 74 in- terviews) concerning referral to and uptake of SSME for T2DM] intended to understand how and why stake- holders engage with and participate in SSME for T2DM and identify barriers and enablers to SSME referral and uptake. Next, a guided discussion (Stage two) was facili- tated among the research team, primary care stakeholders and providers of SSME to understand which characteris- tics of an SSME programme are prioritised by stake- holders, as well as the resources required to address barriers and deliver solutions. Finally, in Stage three, a purposive sample of 16 individuals with a professional interest in SSME, management of chronic health condi- tions, or the implementation and adoption of interven- tions in primary care were recruited to participate in a consultation to rank and select the key functions of an intervention to increase referral to and engagement with SSME for T2DM. The overall aim of the study presented here was to test feasibility of conducting a large-scale evaluation of the Embedding Package in primary care using a mixed methods approach. This was achieved through the fol- lowing objectives: Quantitative: 1. To assess the feasibility of two recruitment and consent approaches 2. Background To assess the feasibility of extracting demographic and biomedical data and information on SSME referral and attendance from primary care medical records with sufficient accuracy and completeness for use in an RCT, particularly HbA1c, the primary outcome for the RCT. 3. To assess patient willingness to provide consent for accessing and extracting identifiable data from their medical records 4. To assess the willingness of patients to provide consent and complete a questionnaire asking for demographic data and information on the diagnosis and management of their diabetes, their history of being invited to or attending SSME and their preferences around the method of delivery of SSME The Embedding Package A theory-and-evidence-based intervention (the Embed- ding Package) was developed to overcome the previously identified barriers to the uptake of SSME. The Embed- ding Package incorporates four key components: (1) a clear marketing strategy for SSME, (2) a user friendly and effective referral pathway, (3) new/amended profes- sional roles, including local clinical champions and an ‘Embedder’, and (4) a toolkit of resources, complemen- ted by a website. In the operation of the intervention, the Embedder guides practices and SSME providers and educators through the implementation process. Tailor- ing of the intervention to the local context is achieved through a series of meetings between stakeholders and the Embedder. 5. To assess the feasibility of capturing cost data for embedding activities at participating practices and clinical commissioning groups (CCGs) Qualitative: 1. To assess the feasibility of using ethnographic methods in a range of primary care settings g p y g 2. To identify and collect context-specific data on the processes of implementation, sustainability of Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies Page 4 of 19 Page 4 of 19 (2020) 6:71 change, and the fit of the Embedding Package with routine practice undertaken, incorporating observations, interviews and document analysis. The use of qualitative methods en- abled an understanding of the implementation and adop- tion of the Embedding Package within the social, environmental and organisational context and facilitated theoretical evaluation of intervention components [17]. In addition, data relating to the cost of implementing the intervention were obtained from the practice managers and an appropriate member of staff in the CCG, using a questionnaire and interview. 3. To evaluate the application of the Normalisation Process Theory (NPT) as a way of analysing data and understanding factors contributing to the embedding of SSME g 4. To explore methods for providing formative feedback to aid in the refinement of the Embedding Package and development of RCT study procedures Sample For the integrated ethnographic component, a purpos- ive approach to sampling aimed to achieve a range of staff and patients, in terms of roles demography and ex- perience, within the size and time limits of the wider study. The aim was to recruit between 12 and 15 patient participants and conduct between six and ten interviews with participants representing practices and providers. Participant recruitment continued until data saturation was reached. Concurrent qualitative studies Informed written consent was obtained from all partici- pants undertaking interviews. Informed verbal consent was sought and obtained prior to and during all observa- tions. No incentives or disbursements were provided to participants. Eligibility criteria General practices using either EMIS Web or TPP Sys- temOne electronic health record systems and located within the two participating CCGs were invited to participate in the study. Practices had to be referring pa- tients with T2DM to an SSME programme and be will- ing to sign a data sharing and remote data collection agreement with PRIMIS (a third party company provid- ing the data extraction service for the study). This allowed the collection of anonymised patient data, and where consent had been provided by patients’ identifi- able data. Since this study did not aim to assess the ef- fectiveness of the intervention, a comparator was not required, and all general practices (and patients with T2DM within them) received the intervention. Patient recruitment and consent Quantitative study Participants were recruited in two ways. Firstly, through the use of a prompt installed on the electronic health record sys- tem, which notified the clinician during a consultation of a patient’s potential eligibility for inclusion in the study. This encouraged the clinician to make the patient aware of the study and provide them with a participant information pack and a copy of the data collection questionnaire for self- completion. The second method involved postal invitations to participate. All potentially eligible patients who had not been invited in person received an information pack and self-completion questionnaire. Informed consent was implied by the return of a completed questionnaire directly to the study team using a pre-paid, self-addressed envelope. As we did not intend to generate inference from our qualitative data, there was no optimum number of partici- pants. Rather, we invited participation from individuals who could provide the most productive information, based on a framework of variables [maximum variation (patients) and key informants (and where appropriate de- viant cases)], our practical knowledge of the research area, and existing literature on primary care implementation re- search. In addition to relying on saturation of data [19], we were also guided by principles of adequacy, appropri- ateness and analytical redundancy—in that one or many more interviews would not have made additional contri- butions or provide further insight [20]. The questionnaire could be completed anonymously as questions asking for sex, year of birth and ethnicity were optional. For patients opting to share and link their health record data to their questionnaire responses, additional written informed consent was sought and obtained. Sample For the integrated ethnographic component, a purpos- ive approach to sampling aimed to achieve a range of staff and patients, in terms of roles demography and ex- perience, within the size and time limits of the wider study. The aim was to recruit between 12 and 15 patient participants and conduct between six and ten interviews with participants representing practices and providers. Participant recruitment continued until data saturation was reached. Patients from participating practices were eligible to have their anonymised data extracted if they were aged 18 years or over and coded in their electronic medical record as diagnosed with T2DM. Patients with a record of a terminal illness and life expectancy of < 12 months, housebound or in residential care, or with a dissent code to data sharing as part of a research study, were ex- cluded. If the above criteria were met, and the patient was able to provide informed consent and able to speak, understand, and read English to a sufficient level to par- ticipate in the study, they were invited to complete a questionnaire and an additional consent form, allowing their data extracted by PRIMIS to be linked to their questionnaire response. practices were invited to participate in the questionnaire. It was assumed that 30% of the patients on the dia- betes register would attend their surgery during the recruitment window and be given study information by a health care professional. It was estimated that there would be a 20% questionnaire return rate for this group, so this would collect data from 250 pa- tients. All remaining eligible patients with T2DM would receive a postal questionnaire; it was estimated that 10% of these would be returned. This would re- sult in a total of ~ 540 patient participants returning a questionnaire with implied consent. This sample would then be large enough that a sufficient number of patients would have returned a completed ques- tionnaire and agreed to data linkage, to allow assess- ment of the agreement between the two data sources. Health care professionals were eligible for inclusion if they were employed by a participating practice or an or- ganisation referring to, providing, or commissioning SSME, and willing and able to provide informed written or verbal consent. Sample Three groups of participants were included in this research: general practice staff, patients and staff in orga- nisations providing SSME. As this was a feasibility study, there was no formal sample size calculation. The num- ber of practices was based on a balance between practi- calities and having sufficient data to assess its quality and to give adequate variation in urban and rural prac- tices. It was anticipated that six practices would provide data extracted from around 4200 patients. This was based on an average diabetes register size of 700, in- formed by figures from a Quality and Outcomes Frame- work report [18]. All eligible patients in these included The feasibility study involved collecting data from six general practices and two SSME providers within two CCGs in the East Midlands, UK, between May and Au- gust 2017. Figure 1 provides an overview of the quantita- tive data collection process. Quantitative demographic and biomedical data and in- formation relating to SSME referral and attendance and diabetes diagnosis and management were gathered from primary care electronic medical records (extracted data), SSME providers (health economic data) and question- naires completed by patients with T2DM (self-reported data). An integrated concurrent ethnographic study was Fig. 1 Flow diagram of the quantitative data collection process Fig. 1 Flow diagram of the quantitative data collection process Fig. 1 Flow diagram of the quantitative data collection process Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Page 5 of 19 practices were invited to participate in the questionnaire. It was assumed that 30% of the patients on the dia- betes register would attend their surgery during the recruitment window and be given study information by a health care professional. It was estimated that there would be a 20% questionnaire return rate for this group, so this would collect data from 250 pa- tients. All remaining eligible patients with T2DM would receive a postal questionnaire; it was estimated that 10% of these would be returned. This would re- sult in a total of ~ 540 patient participants returning a questionnaire with implied consent. This sample would then be large enough that a sufficient number of patients would have returned a completed ques- tionnaire and agreed to data linkage, to allow assess- ment of the agreement between the two data sources. Quantitative data collection Pseudonymised patient data were extracted from pri- mary care medical records for eligible patients by a third party (PRIMIS). Data were extracted from each practice at a single time point in September or October 2017. Page 6 of 19 Page 6 of 19 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies Data extraction procedures were based on those used by a similar study [21] and extracted data recorded over the previous 12 months. Information was extracted on age, sex, ethnicity, date of T2DM diagnosis, HbA1c (mmol/ mol), body mass index (BMI), weight, lipids (total and HDL), blood pressure (SBP and DBP), medications, atrial fibrillation, smoking status, hospital admissions and car- diovascular risk (QRisk2) score. The last value recorded within the last 12 months was extracted for all biomed- ical/anthropometric data. Any Read coded data relating to offering, referring, uptake or did not attend (DNA) of SSME at any time since diagnosis, with dates, whether the patient had been approached to take part in the questionnaire, and whether they declined participation in the questionnaire study was also collected. interview discussions. The observational guide aided our understanding of the intervention implementation process, and a schedule of topics for discussion (Add- itional File 1) explored the variety of experiences of each participant group in relation to intervention implemen- tation, outcomes and barriers and enablers to adopting the toolkit. Observations were undertaken totalling approximately 25 h of practice and provider meetings and activities, taking field notes in situ, which were then transcribed to provide a full and rich account. Ethnography team de- brief meetings were held to reflect on the observations and interviews and identify salient points for future ex- ploration in interviews. Tensions, inconsistencies, or am- biguities in the data were also identified. Individual and group interviews with stakeholders (pa- tients, practice staff and SSME providers) were held and the Embedder was interviewed at three-month intervals to ex- plore perceptions of the implementation process, including delivery and use of the toolkit and outcomes. All interviews were audio-recorded with participant consent and tran- scribed verbatim. Demographic data on participant charac- teristics were collected using a brief questionnaire. Qualitative data collection A questionnaire and face-to-face interview were used to obtain data on the cost of all the embedding activity from the practice manager and an appropriate member of staff in the CCG. A simple tick box tracker covered the type of embedding activity implemented, the dur- ation over which it was applied and whether it was still ongoing. This tracker was completed by the practice and the ‘Embedder’. This provided a census of what activities were attempted as well as providing a measure of re- source use in terms of time spent by the Embedder and staff from each provider and practice, against which unit costs could be applied. The unit cost was generated by the practice manager interviews which asked for details of staff time, consumables and other costs that have been devoted to each individual activity over the dur- ation that the activity was undertaken. The resource use data for developing the web-based toolkit (a key compo- nent of the Embedding Package) were obtained from the designer. Traditional ethnographic methods were used to collect data (key-informant and semi-structured interviews, nat- uralistic observation and collection of relevant docu- ments and publically available information). The aim was to identify methodological barriers and facilitators, develop feedback processes between the qualitative and wider study team, provide insight into quantitative out- comes and contribute to the refinement of intervention content, processes and definitive trial procedures—doing so in a manner that was both rigorous and acceptable to primary care stakeholders and patients. To gain insight into clinical and organisational culture and context, we collected relevant information and documentation as appropriate. Practice websites, Care Quality Commission reports and local authority public health Joint Strategic Needs Assessment reports were reviewed to provide contextual information in relation to practice size, services and local area demographic. Diabetes- and diabetes education-related patient infor- mation materials were also collated. Meeting notes and task plans developed between the Embedder and prac- tices or Educators were also reviewed and contributed to the final analysis. Action planning and action planning review documents were analysed to assess the develop- ment of embedding activity, the delegation of roles and responsibilities and outputs. Quantitative data collection The questionnaire gathered data on registered practice, diagnosis and management of diabetes (time since T2DM diagnosis, HbA1c and diabetes medication), history of be- ing invited to SSME and of attendance at SSME, including delivery method. The questionnaire also asked for patient preferences around the delivery of SSME, i.e. web-based or face-to-face. Basic demographic information was also sought on year of birth, sex and ethnicity; however, re- sponses to these items were optional. Statistical analysis Quantitative data The data were analysed using de- scriptive summary statistics to assess data completeness and accuracy using mean, standard deviation (SD) and range for continuous, normally distributed variables, me- dian, interquartile range and range for continuous, non- normally distributed variables and number (percentage) NPT informed data collection instruments [16]. Guides were designed to facilitate observations and Page 7 of 19 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies for categorical variables. Extracted primary care data were summarised overall, by CCG and by practice to identify whether there were CCG or practice-level varia- tions in recording. Data from the questionnaire were summarised using the same descriptive statistics. For the participants who had consented to data linkage, their questionnaire responses were linked to their practice data using the pseudonymised NHS number. Where the same categorical measure was available for both the questionnaire and primary care data, these were cross- tabulated and the percentage agreement, calculated. In this comparison, missing values were ignored. For con- tinuous variables, mean difference and limits of agree- ment were calculated. All analyses were performed in Stata 14.1. organised by the four domains of NPT. Different approaches to grouping and defining different stakeholder data were considered. IV. Application of the analytical framework: Using the descriptors of the NPT domains [16] as guidance, narrative data summaries were produced that incorporated narrative and documentary data. After making several attempts to logically organise data, data were categorised and presented according to stakeholder category (patient, practice, provider and the Embedder). V. Data interpretation: Throughout the analysis process, the entire ethnography team met in a series of analysis meetings to review findings and procedures. These discussions formed the basis of an agreed coding framework and where there was uncertainty or disagreements concerning coding, these were resolved through further discussion and consensus between the research team. V. Data interpretation: Throughout the analysis process, the entire ethnography team met in a series of analysis meetings to review findings and procedures. These discussions formed the basis of an agreed coding framework and where there was uncertainty or disagreements concerning coding, these were resolved through further discussion and consensus between the research team. For the practice activity tracker, a cross-tabulation of activity by practice was undertaken, together with the calculation of the mean duration of each activity across practices. Assessment of the feasibility of two recruitment and consent approaches Two CCGs were approached; four expressions of inter- est were received from one CCG and six from the other. Six of these practices were then selected to participate in the study and agreed to the extraction of their primary care data. Eligible patients were then approached to par- ticipate in the questionnaire, either by GP prompt or postal invite. After the removal of duplicates, 423 unique questionnaires (response rate 14.7%) were received across the six practices (66 patients per practice on aver- age; range = 8 to 102). Twenty eight patients did not have a practice recorded (i.e. they returned the question- naire anonymously). More participants were recruited via the postal invite (85%) than the GP prompt (15%). Statistical analysis For the practice manager interviews, central estimates of resource use for each activity are reported, together with upper and lower bounds where there was uncertainty relating to the best estimate. Estimates of re- source use for each activity in each practice were multi- plied by relevant unit costs and used to produce an average cost per activity over time; this was then divided by the number of patients with T2DM at the practice to get a per-patient cost. Resource use data were valued by applying unit costs using the University of Kent’s Per- sonal Social Services Research Unit (PSSRU) unit costs 2016 and other sources where relevant (Additional File 2) [22, 23]. Qualitative data analysis A thematic framework approach to the analysis of the complete data corpus was adopted, to enable compari- son within and between cases [24]. Management of data were undertaken in NVivo. Analysis followed the pro- cedure described by Gale and colleagues [24]: Of those returning a questionnaire, 384 (90.8%) con- sented to have their questionnaire responses and pri- mary care data linked. Of these, 20 (5.2%) could not be linked because they did not provide their NHS number and eight (2.1%) because they did not have their primary care record extracted (three of these were recruited via their GP and five via postal invitation). I. Familiarisation with the transcript data: This stage involved the reading, re-reading and open coding of interview and debrief transcripts, supplemented by the additional documentation to become familiar with the data. Additionally, notes were made on the transcripts to reflect any first impressions or thoughts. Missing data Regarding completeness of demographic and biomedical data, age, sex and smoking status had no missing values. HbA1c and blood pressure had less than 10% missing data, but there were more missing data for total (12%) and HDL (17%) cholesterol, ethnicity (17%), weight (22%) and QRisk score (51%). BMI and hospital admis- sions were missing for all patients. Regarding SSME attendance data, 46% of patients were not recorded as being referred to SSME. This is lower than the 63% estimate based on the referral data. The dis- crepancy arises because where patients were recorded as attending or declining SSME, it was assumed that they had been referred to SSME (n = 492 patients). Only 14% of patients were recorded as having attended SSME, 26% did not attend, and 14% were referred but had no GP rec- ord as to whether or not they subsequently attended. There were some fairly large differences in the per- centages of missing data between CCGs. For example, weight was missing for 9% of patients in CCG 1 and for 29% in CCG 2. Similarly, cholesterol was missing for a higher percentage of patients in CCG 2 than in CCG 1, whereas the higher percentage of missing QRisk scores was in CCG 1. Examining the summary by practice sug- gests that these differences are driven by one or two practices with particularly high percentages of missing values. For example, Practice 3 had only 24% missing QRisk scores, whereas Practice 2 had 71% missing QRisk scores. CCG 1 had more referrals recorded than CCG 2 (47% vs 31%, respectively) but lower attendance recorded (ab- solute values: 6% vs 19%, respectively, indicating that 13% of referrals in CCG1 resulted in attendance com- pared with 60% in CCG 2). This difference may be as a result of differing “quality” of the offer of SSME. Assessment of the feasibility of extracting demographic and biomedical data, and information on SSME referral and attendance from primary care medical records Assessment of the feasibility of extracting demographic and biomedical data, and information on SSME referral and attendance from primary care medical records Primary care data were extracted for all six practices and for 2877 patients [mean = 479.5 patients per practice (SD = 262.3); median = 459 (range = 118 to 824)]. All pa- tients had a T2DM code and were aged 18 years or older as required, but it was not possible to verify the other eligibility criteria from the extracted data. The extracted II. Coding: Open (inductive) coding was conducted independently by two researchers and aimed to classify all of the data; preliminary themes were then organised and charted to identify accounts that differed from the rest, or that which challenged or reconciled anomalies in accounts. Primary care data were extracted for all six practices and for 2877 patients [mean = 479.5 patients per practice (SD = 262.3); median = 459 (range = 118 to 824)]. All pa- tients had a T2DM code and were aged 18 years or older as required, but it was not possible to verify the other eligibility criteria from the extracted data. The extracted III. Development of an analytical framework: After coding and organising the data, initial themes were Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies Page 8 of 19 Page 8 of 19 data are summarised overall and by CCG in Tables 1 and 2 and by practice in Additional File 3. spurious values identified were for weight which had two likely outliers (< 0.001% of values) of 1.5 kg and 8.8 kg. Regarding SSME referral data, 63% of patients had no record of ever being referred to an SSME programme, 20% had been referred to DESMOND, and 16% did not have a specific type of course entered (i.e. the generic SSME Read code had been used which is the recom- mended code). The majority of patients’ last referral had been over a year previously. There were 18 spurious re- ferrals to SSME where the patients were referred to DAFNE, which is a course for patients with type 1 dia- betes, not T2DM. Accuracy of variables Regarding accuracy of these variables, mean and SD esti- mates were similar for all continuous variables when summarised overall, by CCG and by practice, suggesting that there were no consistent reporting errors in any of the practices or CCGs. The only exception to this was mean QRisk score which ranged from 17.4% to 28.3% between the six practices. On the range checks, the only Assessment of patient willingness to provide consent and complete the questionnaire The questionnaire had a 14.7% response rate across the six practices, receiving 423 unique responses, which are summarised in Table 3. An average of 66 patients per practice (range 8 to 102) completed the questionnaire. moving outliers minimum weight was 39.7 kg L high-density lipoproteins, CCG clinical commissioning group, SD standard deviation Removing outliers minimum weight was 39.7 kg HDL high-density lipoproteins, CCG clinical commissioning group, SD standard deviation g y p p Removing outliers minimum weight was 39.7 kg lipoproteins, CCG clinical commissioning group, SD standard deviation rs minimum weight was 39.7 kg Demographics The questionnaire participants were representative of the eligible population (i.e. those for whom primary care data were extracted) in terms of mean age [68 (SD = Table 1 Summary of extracted continuous primary care data overall and by CCG All (N = 2877) CCG 1 (N = 1036) CCG 2 (N = 1841) Variable N (%) missing Mean (SD) Median (range) N (%) missing Mean (SD) N (%) missing Mean (SD) Age, years 0 (0) 66.0 (13.3) 67 (23, 101) 0 (0) 68.6 (12.5) 0 (0) 64.6 (13.5) HbA1c, mmol/mol 189 (7) 56.1 (15.5) 52 (29, 179) 22 (2) 55.0 (13.4) 167 (9) 56.8 (16.6) HbA1c, % 189 (7) 7.3 (1.4) 6.9 (4.8, 18.5) 22 (2) 7.2 (1.2) 167 (9) 7.3 (1.5) Weight, kg 622 (22) 88.1 (21.9) 85.7 (1.5, 215.0) 89 (9) 85.9 (20.4) 533 (29) 89.7 (22.8) Total cholesterol, mmol/L 349 (12) 4.3 (1.1) 4.2 (1.9, 13.4) 67 (6) 4.3 (1.1) 282 (15) 4.3 (1.1) HDL cholesterol, mmol/L 487 (17) 1.3 (0.4) 1.2 (0.2, 4.4) 73 (7) 1.3 (0.3) 414 (22) 1.3 (0.4) Systolic blood pressure, mmHg 204 (7) 132.5 (13.7) 132 (86, 240) 41 (4) 133.7 (13.8) 163 (9) 131.8 (13.6) Diastolic blood pressure, mmHg 204 (7) 75.1 (9.5) 76 (45, 134) 41 (4) 75.2 (9.6) 163 (9) 75.1 (9.4) QRisk score, % 1468 (51) 23.1 (15.4) 19.9 (0, 98.7) 669 (65) 25.4 (15.4) 799 (43) 22.3 (15.3) HDL high-density lipoproteins, CCG clinical commissioning group, SD standard deviation Removing outliers minimum weight was 39.7 kg Table 1 Summary of extracted continuous primary care data overall and by CCG All (N = 2877) CCG 1 ( Table 1 Summary of extracted continuous primary care data overall and by CCG Davies et al. Pilot and Feasibility Studies (2020) 6:71 Page 9 of 19 Davies et al. Demographics Pilot and Feasibility Studies Table 2 Summary of extracted categorical primary care data overall and by CCG Variable N (%) All (N = 2877) CCG 1 (N = 1036) CCG 2 (N = 1841 Sex Male 1588 (55.2) 575 (55.5) 1013 (55.0) Female 1289 (44.8) 461 (44.5) 828 (45.0) Ethnicity White European 1948 (67.7) 618 (59.7) 1330 (72.2) South Asian 219 (7.6) 56 (5.4) 163 (8.9) Black 57 (2.0) 5 (0.5) 52 (2.8) Other 32 (1.1) 12 (1.2) 20 (1.1) Not otherwise stated 121 (4.2) 102 (9.9) 19 (1.0) Missing 500 (17.4) 243 (23.5) 257 (14.0) Smoking status Never smoker 1425 (49.5) 508 (49.0) 917 (49.8) Ex-smoker 1102 (38.3) 435 (42.0) 667 (36.2) Current smoker 350 (12.2) 93 (9.0) 257 (14.0) HbA1c, mmol/mol (%) ≤53 (≤7%) 1465 (50.9) 585 (56.5) 880 (47.8) 54–58 (7.1–7.5%) 385 (13.4) 148 (14.3) 237 (12.9) 59–64 (7.6–8.0%) 279 (9.7) 100 (9.7) 179 (9.7) 65–69 (8.1–8.5%) 152 (5.3) 58 (5.6) 94 (5.1) 70–86 (8.6–10.0%) 266 (9.3) 83 (8.0) 183 (9.9) ≥87 (≥10.1%) 141 (4.9) 40 (3.9) 101 (5.5) Missing 189 (6.6) 22 (2.1) 167 (9.1) Diabetes medication in the last 12 months None recorded 639 (22.2) 215 (20.8) 424 (23.0) DPP-IV 101 (3.5) 22 (2.1) 79 (4.3) GLP-I 20 (0.7) 10 (1.0) 10 (0.5) Insulin 263 (9.1) 80 (7.7) 183 (9.9) Metformin 1425 (49.5) 559 (54.0) 866 (47.0) SGLT-2 21 (0.7) 5 (0.5) 16 (0.9) Sulphonylurea 346 (12.0) 136 (13.1) 210 (11.4) Multiple diabetes medications 38 (1.3) 0 (0.0) 38 (2.1) Other 24 (0.8) 9 (0.9) 15 (0.8) Previous atrial fibrillation diagnosis Yes 246 (8.6) 106 (10.2) 140 (7.6) No 2631 (91.5) 930 (89.8) 1701 (92.4) SSME referral No record 1809 (62.9) 546 (52.7) 1263 (68.6) DESMOND 586 (20.4) 106 (10.2) 480 (26.1) X-PERT 2 (0.1) 0 (0.0) 2 (0.1) DAFNE 18 (0.6) 2 (0.2) 16 (0.9) Generic SSME code 462 (16.1) 382 (36.9) 80 (4.4) Date of SSME referral ≤1 year 270 (9.4) 138 (13.3) 132 (7.2) Table 2 Summary of extracted categorical primary care data overall and by CCG Davies et al. Pilot and Feasibility Studies (2020) 6:71 Page 10 of 19 Davies et al. Management of diabetes For HbA1c, the percentage reporting that they had had it measured in the last 12 months (93%) was the same as in the primary care data (93%). Self-reported HbA1c was missing for 42% of participants. However, when missing data were excluded, the percentages of patients with HbA1c ≤53 mmol/mol were very similar in the self- report (56%) and primary care (55%) data, suggesting that self-reported HbA1c data were incomplete but accurate. The majority of the participants (70%) reported that they would attend SSME if invited. The preferred formats were one-to-one (29%) or group (24%). Fewer participants preferred a format including an online com- ponent (online, group/online or one-to-one/online; 19%) than face-to-face only formats (group, one-to-one or group/one-to-one; 62%). Another notable difference between the primary care and self-report data is that 29% self-reported being pre- scribed with multiple diabetes medications, whereas only 2% were recorded as such in the primary care data. Met- formin was the most common medication (47% primary care and 34% self-reported). A similar proportion re- ported not being on any medication in the primary care and self-reported data (23% and 22.9% respectively). Demographics Pilot and Feasibility Studies Table 2 Summary of extracted categorical primary care data overall and by CCG (Continued) Variable N (%) All (N = 2877) CCG 1 (N = 1036) CCG 2 (N = 1841) > 1 year 798 (27.7) 352 (34.0) 446 (24.2) Missing 1809 (62.9) 546 (52.7) 1263 (68.6) SSME attendance Not referred 1317 (45.8) 311 (30.0) 1006 (54.6) Attendance not recorded 403 (14.0) 266 (25.7) 137 (7.4) Did not attend 744 (25.9) 395 (38.1) 349 (19.0) Attended 413 (14.4) 64 (6.2) 349 (19.0) Date of SSME attendance ≤1 year 121 (4.2) 12 (1.2) 108 (5.9) > 1 year 291 (10.1) 52 (5.0) 239 (13.0) Missing 2465 (85.7) 972 (93.8) 1493 (81.1) CCG clinical commissioning goup, SSME structured self-management education, DPP-IV dipeptidyl peptidase 4, GLP-I glucagon-like peptide-1, SGLT-2 sodium- glucose transport protein 2 Table 2 Summary of extracted categorical primary care data overall and by CCG (Continued) attendance at group SSME (45%) was much higher than that recorded in the extracted primary care data (14%). 11.1) and 66 (SD = 13.3) years, respectively) and sex dis- tribution (58% and 55% male, respectively). White par- ticipants were however over-represented in the questionnaire population compared to those for whom primary care data were extracted (91% and 68%, respect- ively). Over half (52%) of the questionnaire participants had long-standing T2DM (> 10 years). Time since diag- nosis was not available in the primary care data for comparison. In relation to the question, ‘Have you ever attended a one-to-one session with an educator to teach you about your diabetes (this would be outside of the normal care you receive at your GP practice)?’, 17 of the 21 partici- pants who responded positively reported that this had happened as part of their normal care at their practice, and so had incorrectly answered the question. Among the eight participants who reported attending an online SSME programme, four completed a Diabetes UK online programme. Assessment of patient willingness to provide consent for accessing and extracting identifiable data from their medical records In total, 356 participants were included in the linked analyses, which are summarised in Table 4. High levels of agreement were demonstrated for the demographic variables: age (mean difference = 0.3 years), sex (~ 100.0% agreement) and ethnicity (93% agree- ment). The large discrepancy between multiple diabetes medications described above is also present in the linked data with 97% of those who self-reported taking multiple diabetes medications having only one medication listed in their extracted data over the previous 12 months. Referrals and attendance at SSME Regarding SSME, 37% of patients reported never having been referred compared with 63% in the extracted pri- mary care data. The percentage who self-reported Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Referrals and attendance at SSME Pilot and Feasibility Studies Page 11 of 19 Table 3 Summary of patient questionnaire data (n = 423) Characteristic N missing values Mean (SD) Age, years 6 68.3 (11.1) Categories N (%) Recruitment source General practitioner 64 (15.1) Postal invite 359 (84.9) Sex Male 243 (57.5) Female 179 (42.3) Missing 1 (0.2) Ethnicity White 385 (91.0) South Asian 21 (5.0) Black 8 (1.9) Other 6 (1.4) Missing 3 (0.7) Time since type 2 diabetes diagnosis < 12 months 19 (4.5) 1 to 3 years 29 (6.9) 4 to 10 years 146 (34.5) > 10 years 220 (52.0) Missing 9 (2.1) HbA1c measured in the last 12 months Yes 392 (92.7) No 13 (3.1) Do not know 16 (3.8) Missing 2 (0.5) HbA1c result, mmol/mol ≤53 (≤7%) 136 (32.2) 54–69 (7.1–8.5%) 82 (19.4) 70–86 (8.6–10.0%) 19 (4.5) ≥87 (≥10.1%) 8 (1.9) Missing 178 (42.1) Diabetes medication None (diet and lifestyle) 97 (22.9) DPP-IV 7 (1.7) GLP-I 1 (0.2) Insulin 24 (5.7) Metformin 145 (34.3) SGLT-2 1 (0.2) Sulphonylurea 10 (2.4) Multiple diabetes medications 121 (28.6) Other 0 (0.0) Missing 17 (4.0) Ever referred to SSME No 158 (37.4) Yes 241 (57.0) Do not know 14 (3.3) Missing 10 (2.4) Date of SSME referral ≤1 year 55 (13.0) > 1 year 166 (39.2) Table 3 Summary of patient questionnaire data (n = 423) (Continued) Do not know 5 (1.2) Missing 197 (46.6) Ever attended group SSME No 211 (49.9) Yes 190 (44.9) Missing 22 (5.2) Type of SSME attended N/A 233 (55.1) Unknown 20 (4.7) DESMOND 72 (17.0) Juggle 53 (12.5) Tonic 1 (0.2) Other 21 (5.0) Missing 22 (5.2) Date of SSME attendance ≤1 year 29 (6.9) > 1 year 116 (27.4) Missing 278 (65.7) Re-invited if did not attend No 128 (30.3) Yes 11 (2.6) Missing 284 (67.1) Reason for not attending SSME Lack of information 2 (0.5) Lack of perceived benefit 16 (3.8) Unsuitable time 5 (1.2) Suitable transport unavailable 3 (0.7) Othera 33 (7.8) Rather not say 1 (0.2) Missing 363 (85.8) Ever attended one-to-one SSME No 372 (87.9) Yes 21 (5.0) Do not know 15 (3.6) Missing 15 (3.6) Ever attended online SSME No 403 (95.3) Yes 8 (1.9) Do not know 2 (0.5) Missing 10 (2.4) Would you attend SSME if invited? Interviews One GP practice agreed to take part in the face-to-face interview with the Health Economist to discuss resource use and costs incurred to allow the Embedding interven- tion to take place. The interview was undertaken with the Champion (a senior health care professional) and practice administrator who were both involved in implementing the feasibility study at practice level. Since no intervention activity tracker or proforma was completed in advance of the meeting, the interview was based on the information from the Embedder’s tracker record for the practice (e.g. staff time spent in providing each activity). The interview provided the information required for estimating unit costs for valuation of resource use at the practice level (e.g. diabetes awareness event, providing resources in dif- ferent languages to patients, photocopying). SD standard deviation, SSME structured self-management education aThese reasons are listed in Additional File 4 bThese reasons are listed in Additional File 5 Referrals and attendance at SSME No 99 (23.4) Yes 299 (70.7) Missing 25 (5.9) Reason for not wanting to attend if invited Lack of information 1 (0.2) Lack of perceived benefit 24 (5.7) Unsuitable time 3 (0.7) Suitable transport unavailable 9 (2.1) Otherb 44 (10.4) Rather not say 1 (0.2) Missing 341 (80.6) Table 3 Summary of patient questionnaire data (n = 423) (Continued) Page 12 of 19 Page 12 of 19 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies Table 3 Summary of patient questionnaire data (n = 423) (Continued) Preferred format Group 103 (24.4) One-to-one 122 (28.8) Online 64 (15.1) Group/one-to-one 39 (9.2) Group/online 9 (2.1) One-to-one/online 7 (1.7) Any 12 (2.8) Missing 67 (15.8) SD standard deviation, SSME structured self-management education aThese reasons are listed in Additional File 4 bThese reasons are listed in Additional File 5 Table 3 Summary of patient questionnaire data (n = 423) (Continued) Table 3 Summary of patient questionnaire data (n = 423) (Continued) Preferred format Group 103 (24.4) One-to-one 122 (28.8) Online 64 (15.1) Group/one-to-one 39 (9.2) Group/online 9 (2.1) One-to-one/online 7 (1.7) Any 12 (2.8) Missing 67 (15.8) SD standard deviation, SSME structured self-management education aThese reasons are listed in Additional File 4 bThese reasons are listed in Additional File 5 collection, champion, administrator, and web-based tool- kit) was attempted by at least one practice over the study follow-up. However, none of the CCG designates and practice managers completed the intervention tracker or proforma. Management of diabetes g Most participants were aware whether they had had HbA1c measured in the last 12 months (98% agreement), but self-reporting of HbA1c values was fairly poor. For example, the mean difference was small (−0.6 mmol/ mol), but the 95% limits of agreement were large ranging from −13.1 mmol/mol (−3.4%) to 12.0 mmol/mol (3.2%). There seemed to be a particular problem with participants reporting high HbA1c values (≥70 mmol/ mol; 8.6%) that were not reflected in their primary care record. This may be due to participants confusing glu- cose measurements with measurements of HbA1c. Cost estimation For the six practices, the median period over which re- source use data was recorded was 162.5 days (range: 144 to 191). For CCG and SSME providers, the median follow-up was 152 days (range: 150 to 154). The mean total cost per practice over the study period was able to be estimated as £3363 (SD = 2140), with an average cost per patient of £8.94 across all practices. However, if the cost for developing the Embedding Package toolkit is re- moved from this calculation, the mean total cost per practice is reduced. The Embedding intervention will be tested across 66 general practices in the full RCT (sam- ple size informed by the average practice size for this feasibility study), and if shown to be effective, the toolkit will be provided across the whole NHS [25]. Therefore, it is felt the development cost per practice for this feasi- bility study provides an unrealistic real-world cost per practice. Referrals and attendance at SSME For SSME referrals, agreement was moderate (60%). Of those who self-reported that they had not been re- ferred, 77% also had no record of referral in their pri- mary care data. However, of those who self-reported that they had been referred, only 54% had a record of this in their primary care data suggesting that not all of the discrepancy between the two sources is due to the representativeness of the questionnaire partici- pants. For SSME attendance, there was high agree- ment between the two sources when the participant self-reported that they had not attended (97%). How- ever, when the participant reported that they had attended SSME, there was only a primary care record of this for 46% of the participants. Where both sources had a record of referral and attendance, there was high agreement as to when this had occurred (87% and 86% respectively). The breakdown of costs for each individual activity by CCG/SSME provider and practice and the mean cost per patient (weighted by the number of patients with T2DM in each CCG and practice) were also able to be estimated. Initiative tracker and proforma completion It was concluded that the methods chosen to generate and collect qualitative data kept the participant burden low and would be feasible in a larger scale RCT. The fol- lowing sub-sections describe the feasibility of collecting documentary, observational and interview data. Resource use and cost data were collected for all partici- pating practices, CCGs and SSME providers based on the Embedder’s tracker. Each of the pre-specified inter- vention components (marketing, referral and data Davies et al. Pilot and Feasibility Studies (2020) 6:71 Page 13 of 19 Davies et al. Pilot and Feasibility Studies Table 4 Agreement between primary care and patient questionnaire data (n = 356) Characteristic N Mean difference 95% Limits of agreement Age, years 352 0.32 −2.17, 2.81 HbA1c, mmol/mol 40 −0.66 −11.33, 10.00 Characteristic Categories N (%) agree N (%) disagree % Agreement Sex Male 212 (99.5) 1 (0.5) Female 142 (100.0) 0 (0.0) 99.7 Ethnicity White 256 (93.8) 17 (6.2) South Asian 13 (81.3) 3 (18.7) Black 5 (83.3) 1 (16.7) Other 1 (50.0) 1 (50.0) 92.6 HbA1c measured in the last 12 months Yes 323 (97.9) 7 (2.1) No 9 (81.8) 2 (18.2) 94.7 HbA1c result, mmol/mol ≤53 (≤7%) 108 (93.1) 8 (6.9) 54–69 (7.1–8.5%) 43 (61.4) 27 (38.6) 70–86 (8.6–10.0%) 5 (33.3) 10 (66.7) ≥87 (≥10.1%) 2 (28.6) 5 (71.4) 76.0 Diabetes management Diet and lifestyle 71 (88.9) 9 (11.3) DPP-IV 5 (71.4) 2 (28.6) GLP-I 1 (100.0) 0 (0.0) Insulin 15 (75.0) 5 (25.0) Metformin 118 (96.7) 4 (3.3) SGLT-2 1 (100.0) 0 (0.0) Sulphonylurea 8 (100.0) 0 (0.0) Multiple diabetes medications 3 (2.9) 101 (97.1) Other 0 (0.0) 0 (0.0) 55.9 SSME referral No 101 (76.5) 31 (23.5) Yes 111 (54.2) 94 (45.9) 59.6 Date of SSME referral ≤1 year 26 (76.5) 8 (23.5) > 1 year 67 (91.8) 6 (8.2) 86.9 SSME attendance No 169 (96.6) 6 (3.4) Yes 78 (46.4) 90 (54.6) 72.0 Date of SSME attendance ≤1 year 13 (100.0) 0 (0.0) > 1 year 37 (82.2) 8 (17.8) 86.2 SSME structured self-management education Table 4 Agreement between primary care and patient questionnaire data (n = 356) SSME referral Feasibility of collection and review of documents understand the wider context of practices, SSME pro- viders and commissioners. Of note, documents revealed the financial, workforce capacity and policy constraints facing practices and providers, which impacted on their ability to improve and innovate patient care. Both publicly available information and internal commu- nications and documents arising from the intervention delivery were collected. This included emails between the Embedder and stakeholders about local information, planning, refining and evaluating the impact of interven- tion features; the development of promotional and pa- tient information material; SSME attendance rates; presentations given by the Embedder and providers; meeting notes; and practice workflows. For some of these, further explanation was sought during interviews with the Embedder. These documents helped to Identifying and collecting contextual data on implementation processes, sustainability of change and fit of the intervention with routine practice It was anticipated that it would be possible to arrange face-to-face interviews with stakeholders during lunch breaks or allotted Continuing Professional Development (CPD) or administrative times. However, given constraints on stakeholder availability, more success was had conduct- ing these by telephone. It was helpful to start by briefly re- capping their involvement in the study, which enabled us to focus on the most relevant issues. The questions were refined based on the content of the discussion. Once agreeing to participate, no participant withdrew. However, considerable efforts were made by the research team to re- tain participants including flexibility in the timings, loca- tions and methods of interviewing. Data identified a number of contextual factors likely to impact on the implementation, adoption and sustainabil- ity of the intervention. Leadership, workforce focus and motivation to change were the three main factors identi- fied as impacting intervention implementation and adoption. During the interviews and observations and through document analysis, it became evident that prac- tice leaders had to align innovation work with national and local priorities and areas of focus. Example quota- tions are referred to in text and are presented in Table 6. In relation to leadership, practice managers and lead clinicians regarded it as their responsibility to be both familiar with and committed to the research agenda and improving patient outcomes. Nothing explicit regarding leadership and SSME for T2DM was identified through document analysis of the practices strategy. However, leadership was implicit when organisational issues were described, both at a practice and provider level, as well as wider levels of service commissioning (Table 6; Quotation A). Patient interviews conducted over the telephone at a pre-arranged time were logistically easy, but did not gen- erate useful data, owing to the limited time since the intervention had been implemented, resulting in little impact and change to usual care being reported by pa- tients. This has led to refinement of the plans for the RCT; patients will be recruited and interviewed much later in the implementation process, to allow for the intervention to filter through to patient care. Interviews with the Embedder transpired to be crucial in facilitating understanding of the embedding process and stakeholder engagement. It became apparent that practice and provider stakeholders were excessively reli- ant on the Embedder due to resource and capacity limi- tations, and the need to facilitate low-resource, high- impact strategies was identified. Evaluation of applying Normalisation Process Theory Evaluation of applying Normalisation Process Theory Overall, NPT was useful in structuring the approach to the ethnographic data collection and analysis. It was a helpful device to sensitise and focus the researchers’ consideration on structural, individual and cognitive fac- tors related to the implementation of the Embedding Package, as well as to uncover challenges and enablers, practical issues and the collection of data (Table 5). Structuring the findings into the NPT domains enabled pragmatic solutions and recommendations to be devel- oped and fed back to the wider team and, in turn, in- form the RCT design. However, it was not possible to apply some of the findings to just one particular domain construct and encountered a good deal of crossover be- tween concepts, particularly within the domain of coher- ence. To enhance the analysis for the RCT, some of the constructs and domain descriptors have been modified to fit the context. Feasibility of collecting observational data Over the course of the study, the two qualitative re- searchers became familiar to practice staff and other stakeholders from regular attendance at meetings and events accompanying the Embedder. It was found to be helpful to spend time engaging in informal conversations Davies et al. Pilot and Feasibility Studies (2020) 6:71 Page 14 of 19 Davies et al. Pilot and Feasibility Studies Davies et al. Pilot and Feasibility Studies (2020) 6:71 making process changes; this directly informed the methods and strategies for the RCT. with practice staff and observing workflows, use (or non-use) of the intervention tools, as well as interaction with the Embedder. These interactions, and researcher interpretations of them, were documented in detailed narrative field notes. Through more structured observa- tions (for example, shadowing staff during a half-day pa- tient engagement and screening event) further workflow were able to be observed, featuring patient engagement and the availability of education materials, as well as the clinic environment. This approach helped to achieve the goal of gaining a concrete understanding of how a prac- tice works, in order to ground the analyses of the Em- bedding Package’s adoption within the day-to-day work of practice staff. Observation identified issues that could be fed back to inform refinements to the Embedding Package; for instance, a community engagement event uncovered a previously unidentified potential for patient stigmatisation and highlighted the need to consider the acceptability of locations for public engagement. Exploring methods to refine the intervention and study procedures As indicated, a key objective was to develop and opti- mise methods for feeding back findings from the ethno- graphic study to the wider team to inform the refinement of the Embedding Package. Bi-monthly meet- ings were held between two members of the ethno- graphic team (LH and JT), the Embedder and the trial manager to explore the findings, study challenges and enablers, and any intervention content, potential issues, or study procedures that required further exploration or refinement. Primary care data were extracted for all practices, but from fewer patients than anticipated, as practice sizes were lower than predicted. The proportion of patients returning a completed questionnaire was higher than ex- pected, and over 90% of these then consented to the linking of their data. These recruitment figures will be used to calculate the sample size for the RCT. Generally, there was good completeness of the extracted data, par- ticularly for HbA1c, the primary outcome for the RCT; however, the same cannot be said for the self-reporting of HbA1c. This was over 40% missing; although, where it was reported, it was found to be accurate. This is in line with the findings of research assessing the preva- lence of HbA1c self-knowledge among T2DM patients [26]. Due to the poor reporting of HbA1c in the ques- tionnaire, this will be removed from the questionnaire for the RCT. Findings were shared with the feasibility study team and trial management group on a monthly basis. In brief, findings were structured into three categories (Fig. 2): (a) organisational factors that are specific to the local setting (including support, leadership, culture, values and resources); (b) intervention features that may influ- ence implementation and adoption (e.g. documents, clarity of aims/objectives, systems and procedures, team capacity and feedback); and (c) characteristics of local research teams (including staff roles, communication and information sharing). Health professionals believed the intervention in- creased their awareness of SSME, which in turn meant that greater encouragement and more options for acces- sing SSME could be offered to patients newly diagnosed with T2DM. Case examples demonstrating impact can be found in Additional File 6. Other changes will also be made to the questionnaire, including the method of invitation, which will now be solely via postal invite, due to the GP prompts yielding fewer participants and being unpopular with the prac- tices. Identifying and collecting contextual data on implementation processes, sustainability of change and fit of the intervention with routine practice The Embedder’s insights identified the need to build trust and enhance communi- cation with stakeholders to develop their confidence in The focus of the workforce, infrastructure and re- source availability were contextual factors that emerged during observations and interviews with practice stake- holders. Challenges in relation to implementation and integration were identified through observations and document analysis and were directly related to the lack of readiness, both in terms of research and the organisa- tion, local infrastructure and limited guidance from commissioners (Table 6; Quotation B). Page 15 of 19 Page 15 of 19 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies Davies et al. Pilot and Feasibility Studies (2020) 6:71 study, in relation to each of the feasibility objectives, is provided in Additional File 7. Motivation to change emerged from the interviews with both practice and provider stakeholders, where partici- pants talked about the potential benefits that could arise from the intervention. Analysis of interviews revealed that practice stakeholders were motivated to use the Embed- ding Package to inform and refine their practice, if there was a practical and observable benefit for it (Table 6; Quotation C). However, unlike provider participants who reported leadership support to refine existing practices and procedures, practice stakeholders did not address leadership as a focal point when describing how the pack- age was integrated (Table 6; Quotation D). Leadership did, however, emerge as a factor in observational data, but re- lated to a lack of dissemination with respect to the adop- tion of the intervention in certain practices. Data were successfully extracted from primary care health records. Patients were willing to be recruited to the study and to consent to their questionnaire data be- ing linked to their extracted primary care data. It was feasible to capture cost data for Embedding activities and produce a cost estimate for each individual initiative attempted as part of the Embedding Package. Stake- holders from practices that engaged fully reported in- creased understanding of the content of SSME, which was used to inform the information delivered to patients during consultations. However, there was limited en- gagement from the majority of the participating prac- tices. Focussing the intervention implementation on practice staff, who are ultimately not responsible for de- livering SSME, was not feasible given the capacity con- straints in practices. Identifying and collecting contextual data on implementation processes, sustainability of change and fit of the intervention with routine practice The RCT will therefore focus intervention implementation at the provider-level where possible, whilst maintaining relationships with practices and CCG personnel. This includes action planning meetings taking place with local SSME providers and then cascading the information down to practices when/ where appropriate. Exploring methods to refine the intervention and study procedures Biomedical and SSME information and preferences will also be removed from the questionnaire, to better complement the data available from primary care and other studies. Whether or not a participant has been re- ferred to and attended SSME will still be collected, as this has been shown to be a useful tool for validating primary care records. However, there was only moderate agreement between the questionnaire and primary care records in terms of referral and attendance at SSME, Reflexive monitoring Reflexive monitoring (systemisation, communal appraisal, individual appraisal, reconfiguration) ‘In one of the pharmacies that we visited, there was on particular gentleman that has, I don’t know a couple of Pharmacies in the local area, and was really enthusiastic about it, and was giving us feedback as well about people using the Pharmacy, and has given them feedback about [Structured Education] and it was positive’ (systemisation, communal appraisal, individual appraisal, reconfiguration) p (Educator, Provider 2). suggesting that not all of the discrepancy between the two sources is due to the representativeness of the ques- tionnaire participants. This discrepancy may be due to participants misunderstanding what was meant by SSME; a clearer explanation of what SSME is will there- fore be included. There is also the possibility that since referral rates appeared to differ from national figures [10], that some referrals may not have been recorded in the primary care records or that the practices recruited were not as representative as we hoped. The full RCT will include a larger number of practices, so will be more representative. Printed on each questionnaire booklet will be a preassigned participant identification number which can be linked to the individual’s NHS number; this will reduce the quantity of missing NHS numbers suggesting that not all of the discrepancy between the two sources is due to the representativeness of the ques- tionnaire participants. This discrepancy may be due to participants misunderstanding what was meant by SSME; a clearer explanation of what SSME is will there- fore be included. There is also the possibility that since referral rates appeared to differ from national figures [10], that some referrals may not have been recorded in the primary care records or that the practices recruited were not as representative as we hoped. The full RCT will include a larger number of practices, so will be more representative. Printed on each questionnaire booklet will be a preassigned participant identification number which can be linked to the individual’s NHS number; this will reduce the quantity of missing NHS numbers which prevented data linkage despite the participant providing consent. Reflexive monitoring Although it was found to be feasible to collect data for costing the Embedding Package initiatives in the full RCT, it was not feasible to categorise initiative costs based on ‘development’ and ‘steady’ state phases, as these data were planned to be collected by a practice and CCG proforma, but these were not completed. In- stead, costs were estimated as an average cost per initia- tive over the study period. Completing the initiative tracker and proforma by the CCG designates and prac- tice managers proved challenging and unfeasible. It is anticipated that the majority of the work in the RCT will occur at the ‘provider’ level, although most of this may be down to the Embedder. Accordingly, the education The appraisal work that people undertake to assess and understand the ways that a new set of practices affects them and others around them A It has been massively beneficial from a personal point of view in terms of helping us get things [monitoring and referral strategies] in place that will hopefully, help longer term. It’s also helped us as an organisation think about things that perhaps we’ve not considered before like working a bit more up closely with Pharmacies and so forth. (Educator Lead) Cognitive participation ‘No I didn’t use any [Embedding Package Resources] because the doctors don’t actually do the direct referral to [Structured Education] because it is an administrative task’ (GP, Practice 5). g p p (initiation, enrolment, legitimation, activation) The operational work that people undertake to enact a set of practices Collective action (interactional workability, relational integration, skillset workability, contextual integration) The operational work that people undertake to enact a set of practices ‘We sourced a lot of brochures and leaflets from the Diabetes websites and we made contact with a little bit of help from [The Embedder] with the local diabetes support group which has been fantastic they’re really, really, useful’ (Practice Nurse, Practice 5). The appraisal work that people undertake to assess and understand the ways that a new set of practices affects them and others around them A It has been massively beneficial from a personal point of view in terms of helping us get things [monitoring and referral strategies] in place that will hopefully, help longer term. It’s also helped us as an organisation think about things that perhaps we’ve not considered before like working a bit more up closely with Pharmacies and so forth. (Educator Lead) B There is a read code that comes in so we can capture the people that have been [to SSME] but really what we want to capture more is the people that have not… And not the ones who have cancelled their appointment, but the ones who didn’t bother to make one even in the first place. (Practice Manager) C I have seen plenty of patients walking out with them in various languages and I think sometimes, it is surprising that somebody might have had diabetes for the last 10 years but never really had a good conversation about it. I have had at least one consultation where somebody had read the leaflet and actually, it prompted him or her [patient] to re-engage. I have seen it as a positive thing. (Practice Nurse) D We’ve never done anything for World Diabetes Day, Diabetes Awareness anything like that before. Obviously having done several diabetes trials and working with [The Embedder] this year as well, we decided that we would promote it and try and raise awareness. (Practice Nurse) A It has been massively beneficial from a personal point of view in terms of helping us get things [monitoring and referral strategies] in place that will hopefully, help longer term. It’s also helped us as an organisation think about things that perhaps we’ve not considered before like working a bit more up closely with Pharmacies and so forth. (Educator Lead) B There is a read code that comes in so we can capture the people that have been [to SSME] but really what we want to capture more is the people that have not… And not the ones who have cancelled their appointment, but the ones who didn’t bother to make one even in the first place. (Practice Manager) C I h l t f ti t lki t ith th i i l d I thi k ti it i i i th t b d i ht h h d B There is a read code that comes in so we can capture the people that have been [to SSME] but really what we want to capture more is the people that have not… And not the ones who have cancelled their appointment, but the ones who didn’t bother to make one even in the first place. (Practice Manager) C I have seen plenty of patients walking out with them in various languages and I think sometimes, it is surprising that somebody might have had diabetes for the last 10 years but never really had a good conversation about it. I have had at least one consultation where somebody had read the leaflet and actually, it prompted him or her [patient] to re-engage. I have seen it as a positive thing. (Practice Nurse) A It has been massively beneficial from a personal point of view in terms of helping us get things [monitoring and referral strategies] in place that will hopefully, help longer term. It’s also helped us as an organisation think about things that perhaps we’ve not considered before like working a bit more up closely with Pharmacies and so forth. (Educator Lead) B There is a read code that comes in so we can capture the people that have been [to SSME] but really what we want to capture more is the l th t h t A d t th h h ll d th i i t t b t th h did ’t b th t k i th fi t Discussion The feasibility of conducting a large-scale evaluation of the Embedding Package in primary care was tested, using a concurrent mixed methods approach. Although no formal feasibility thresholds were set, which is a limi- tation of this study, we have been able to demonstrate the feasibility of a future RCT based on nine predefined feasibility objectives. A summary of the findings of this Page 16 of 19 Davies et al. Pilot and Feasibility Studies Davies et al. Pilot and Feasibility Studies (2020) 6:71 Table 5 Normalisation Process Theory constructs, domains and coding examples Domain (Components) Description Example Coherence (differentiation, communal specification, individual specification, internalisation) The sense-making work that people undertake indi- vidually and collectively ‘One of the practices responded to a very specific question which was around me going and putting the display board up for World Diabetes Day. But when asked to review the Action Plan and things like that have not had any response, and I have followed things up two or three times’ (The Embedder). Table 5 Normalisation Process Theory constructs, domains and coding examples Domain (Components) Description Example Coherence (differentiation, communal specification, individual specification, internalisation) The sense-making work that people undertake indi- vidually and collectively ‘One of the practices responded to a very specific question which was around me going and putting the display board up for World Diabetes Day. But when asked to review the Action Plan and things like that h h d d I h f ll d The relational work undertaken by people to build and sustain a community of practice around a new intervention Cognitive participation (initiation, enrolment, legitimation, activation) Table 6 Example quotations C I have seen plenty of patients walking out with them in various languages and I think sometimes, it is surprising that somebody might have had diabetes for the last 10 years but never really had a good conversation about it. I have had at least one consultation where somebody had read the leaflet and actually, it prompted him or her [patient] to re-engage. I have seen it as a positive thing. (Practice Nurse) D We’ve never done anything for World Diabetes Day, Diabetes Awareness anything like that before. Obviously having done several diabetes trials and working with [The Embedder] this year as well, we decided that we would promote it and try and raise awareness. (Practice Nurse) D We’ve never done anything for World Diabetes Day, Diabetes Awareness anything like that before. Obviously having done several diabetes trials and working with [The Embedder] this year as well, we decided that we would promote it and try and raise awareness. (Practice Nurse) Davies et al. Pilot and Feasibility Studies (2020) 6:71 Page 17 of 19 Davies et al. Pilot and Feasibility Studies Fig. 2 Findings from the ethnographic study, grouped by organisational, intervention and team factors Funding g This project is funded by the National Institute for Health Research (NIHR) Programme Grants for Applied Research (Increasing uptake of effective self- management education programmes for type 2 diabetes in multi-ethnic pri- mary care settings RP-PG-1212-20004). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. The Trial Sponsor is the University of Leicester uolsponsor@le.ac.uk. Ethics approval and consent to participate Ethics approval and consent to participate This study has been approved by the Yorkshire & The Humber—Leeds West Research Ethics Committee (03/05/2017, ref. 17/YH/0135). All participants provided informed consent to the questionnaire, observations, interviews and data linkage, respectively. Consent was implied by the completion and return of the questionnaire if it was returned anonymously. Abbreviations BMI: Body mass index; CCG: Clinical commissioning group; CPD: Continuing Professional Development; DBP: Diastolic blood pressure; DNA: Did not attend; GP: General practitioner; HDL: High-density lipoproteins; NHS: National Health Service; NICE: National Institute for Health and Care Excellence; NPT: Normalisation Process Theory; RCT: Randomised controlled trial; SBP: Systolic blood pressure; SD: Standard deviation; SSME: Structured self-management education; T2DM: Type 2 diabetes mellitus; UK: United Kingdom Authors’ contributions All authors contributed to the conception or design of the study, the acquisition, analysis or interpretation of data or drafting of the work. CK and LH prepared and drafted the final manuscript. All authors have read and approved the submitted version. Conclusion It is feasible to collect demographic, biomedical and SSME referral and attendance data with reasonable com- pleteness and accuracy for the subsequent RCT; how- ever, measures can and will be taken to further improve the quality of the data collected. Based on this feasibility study, it is also feasible to collect data for costing the Embedding Package initiatives for the full RCT; however, changes are required to some elements of the resource use data collection strategy. Based on data collected, it was feasible to produce cost estimates for each individ- ual initiative attempted as part of the Embedding Pack- age. The methods chosen to generate, collect and analyse qualitative data were satisfactory; they kept par- ticipant burden low and provided insight into potential refinements of the Embedding Package. Some further development of the Embedding Package is required to improve engagement from practices. Fig. 2 Findings from the ethnographic study, grouped by organisational, intervention and team factors addressed in the RCT in order to optimise the likelihood for successful implementation and adoption. In order to scale the Embedding Package across many organisations, the work of the Embedder will be imperative to local en- gagement with and adoption of the intervention. With knowledge of the local context gained at the launch of the intervention and continued contact and develop- ment, implementation issues can be identified and miti- gated. Furthermore, with the RCT planning to target intervention implementation at the provider level where possible, the challenges identified with practice engage- ment with the Embedder will be overcome, since one or- ganisation provides SSME for numerous practices [25]. Low levels of uptake and use may be explained in part by a lack of organisational readiness at a local (practice) level and different focus of various stakeholders; thus, provider will be added as a key source for collecting re- source use data and the role of the Embedder will be maintained and strengthened in the full RCT. Both CCG designates and practice managers will be removed as sources for collecting resource use data because it has proven unfeasible. Consequently, the resource use data collection tools for the RCT will be amended to reflect this change. Education providers will be supported to ensure that all relevant resource use and cost data are captured over the full RCT follow-up. The qualitative work highlights that the success of the intervention depends on the organisational context. There were challenges directly related to a lack of readi- ness, both in terms of research and the organisation, local infrastructure and limited guidance from commis- sioners. These various contextual factors need to be Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Page 18 of 19 Davies et al. Pilot and Feasibility Studies aligning interests across multiple stakeholders and orga- nisations remains a challenge when planning an inter- vention in primary care. Another challenge lies in the implementation process and in the integration of SSME in usual practice. For practices, there is uncertainty about SSME in general, and concerted and ongoing ef- forts will be required in the RCT to integrate referral and engagement practices as a routine and sustained part of primary care provision. Despite the challenges that were present, a number of low-capacity high-return activities were identified. These include patient informa- tion leaflets tailored to the cultural and demographic needs of the practice, promotional videos, waiting room or reception area displays, self-referral forms, and en- gagement with existing initiatives and partnership work- ing. However, constraints on the timings of this feasibility study meant that the Embedding Package was not utilised sufficiently in practice to have impacted on patient’s awareness and/or experience, particularly at the time patients were recruited to participate in interviews. Patient interviews will be conducted later in the RCT, to allow time for the intervention to have filtered through to patient care. NPT was found to be useful in structur- ing our approach to ethnographic data collection and analysis. However, it was difficult to apply some of the findings to a single domain construct, and so, to enhance the analysis in the RCT, some of the constructs and do- main descriptors will be customised. Additional file 3. Summary of extracted continuous primary care data by practice. Additional file 4. Other reasons given for not attending SSME. Additional file 5. Other reasons given for not wanting to attend SSME if invited. Additional file 6. Case examples demonstrating impact. Additional file 7. Summary of the findings for each feasibility objective. Additional file 4. Other reasons given for not attending SSME. Additional file 5. Other reasons given for not wanting to attend SSME if invited. Additional file 4. Other reasons given for not attending SSME. Additional file 5. Other reasons given for not wanting to attend SSME if invited. Additional file 6. Case examples demonstrating impact. Additional file 7. Summary of the findings for each feasibility objective. Acknowledgements The authors also acknowledge support from the National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care—East Midlands (NIHR CLAHRC—EM) and the NIHR Leicester Biomedical Research Centre, which is a partnership between University Hospitals of Leicester NHS Trust, Loughborough University and the University of Leicester. Availability of data and materials The dataset used and analysed during the current study is available from the corresponding author on reasonable request. References 25. Davies MJ, Kristunas CA, Alshreef A, Dixon S, Eborall H, Glab A, Huddlestone L, Hudson N, Khunti K, Martin G, Northern A, Patterson M, Pritchard R, Schreder S, Stribling B, Turner J, Gray L. The impact of an intervention to increase uptake to structured self-management education for people with type 2 diabetes mellitus in primary care (the embedding package), compared to usual care, on glycaemic control: study protocol for a mixed methods study incorporating a wait-list cluster randomised controlled trial. BMC Fam Pract. 2019;20(1):152. 1. DIABETES UK, Diabetes prevalence 2017, https://www.diabetes.org.uk/ professionals/position-statements-reports/statistics/diabetes-prevalence-201 7, accessed 08 Jan 2019. 2. International Diabetes Foundation. Diabetes Atlas 8th Edition, 2017. 2. International Diabetes Foundation. Diabetes Atlas 8th Edition, 2017. l h l h d f f 3. Hex N, Bartlett C, Wright D, et al. Estimating the current and future costs of type 1 and type 2 diabetes in the UK, including direct health costs and indirect societal and productivity costs. Diabet Med. 2012;29(7):855–62. 3. Hex N, Bartlett C, Wright D, et al. Estimating the current and future costs of type 1 and type 2 diabetes in the UK, including direct health costs and indirect societal and productivity costs. Diabet Med. 2012;29(7):855–62. 4. Gillett M, Dallosso H, Dixon S, et al. Delivering the diabetes education and self management for ongoing and newly diagnosed (DESMOND) programme for people with newly diagnosed type 2 diabetes: cost effectiveness analysis. BMJ. 2010;341:c4093. 26. Trivedi H, Gray L, Seidu S, et al. Self-knowledge of HbA1c in people with type 2 diabetes mellitus and its association with glycaemic control. Primary Care Diabet. 2017;11(5):414–20. 5. Davies MJ, Heller S, Skinner T, et al. Effectiveness of the diabetes education and self management for ongoing and newly diagnosed (DESMOND) programme for people with newly diagnosed type 2 diabetes: cluster randomised controlled trial. BMJ. 2008;336(7642):491–5. Received: 20 November 2019 Accepted: 22 April 2020 24. Gale NK, Heath G, Cameron E, Rashid S, Redwood S. Using the framework method for the analysis of qualitative data in multi-disciplinary health research. BMC Med Res Methodol. 2013;13(1):117. Competing interests There are no conflicts of interest; however, for transparency, the following are provided: MD is the Principal Investigator (PI) on the DESMOND programme (Diabetes education and self-management for people with newly diagnosed Type 2 diabetes). BS, SS and AN are employed by the Uni- versity Hospitals of Leicester NHS Trust, which receives not-for-profit income for DESMOND. MD, KK, HE, LG, AN, SS and BS are actively engaged in re- search and have previously received grants for DESMOND from NIHR, MRC and Diabetes UK to develop and test Diabetes Self-Management Education and Support programmes like DESMOND. The University Hospitals of Leices- ter (UHL) NHS Trust (for which BS, SS and AN have a contract) receives li- censing fees to support implementation of the DESMOND programme in CCGs in the UK, Ireland and Australia. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s40814-020-00606-0. Additional file 1. Ethnographic interview topic guides. Additional file 2. Unit costs applied for valuation of resource use. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s40814-020-00606-0. Additional file 1. Ethnographic interview topic guides. Additional file 2. Unit costs applied for valuation of resource use. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s40814-020-00606-0. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s40814-020-00606-0. Additional file 1. Ethnographic interview topic guides. Additional file 2. Unit costs applied for valuation of resource use. All other authors have no competing interests. Page 19 of 19 Page 19 of 19 Page 19 of 19 Page 19 of 19 Davies et al. Pilot and Feasibility Studies (2020) 6:71 Davies et al. 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UK Annual Survey of Hours and Earnings (ASHE) 2015. URL: https://www. wcva.org.uk/media/5625220/10_volunteer_time_as_match_funding.pdf (accessed 11 Dec 2017). Received: 20 November 2019 Accepted: 22 April 2020 Received: 20 November 2019 Accepted: 22 April 2020 Received: 20 November 2019 Accepted: 22 April 2020 Publisher’s Note Qualitative research sample design and sample size: resolving and unresolved issues and inferential imperatives. Prev Med. 2012;55(5):398–400. 20. Sobal J. Sample extensiveness in qualitative nutrition education research. J Nutr Educ. 2001;33(4):184–92. 20. Sobal J. Sample extensiveness in qualitative nutrition education research. J Nutr Educ. 2001;33(4):184–92.
https://openalex.org/W2008356963	https://www.scielo.br/j/paz/a/W948hc9gDMCMxrTGMJcg6RF/?lang=en&format=pdf	English	null	A new genus and species of cavernicolous Pomatiopsidae (Mollusca, Caenogastropoda) in Bahia, Brazil	Papéis Avulsos de Zoologia	2,012	cc-by	7,570	Volume 52(40):515‑524, 2012 Volume 52(40):515‑524, 2012 Abstract Spiripockia punctata is a new genus and species of Pomatiopsidae found in a cave from Serra Ramalho, SW Bahia, Brazil. The taxon is troglobiont (restricted to subterranean realm), and is characterized by the shell weakly elongated, fragile, translucent, normally sculptured by pus‑ tules with periostracum hair on tip of pustules; peristome highly expanded; umbilicus opened; radular rachidian with 6 apical and 3 pairs of lateral cusps; osphradium short, arched; gill filaments with rounded tip; prostate flattened, with vas deferens inserting subterminally; penis duct narrow and weakly sinuous; pallial oviduct simple anteriorly, possessing convoluted by‑ pass connecting base of bulged portion of transition between visceral and pallial oviducts with base of seminal receptacle; spermathecal duct complete, originated from albumen gland. The description of this endemic species may raise protective environmental actions to that cave and to the Serra Ramalho Karst area. Key-Words: Pomatiopsidae; Spiripockia punctata gen. nov. et sp. nov.; Brazil; Cave; Tro- globiont; Anatomy. A new genus and species of cavernicolous Pomatiopsidae (Mollusca, Caenogastropoda) in Bahia, Brazil Luiz Ricardo L. Simone 1 1. Museu de Zoologia, Universidade de São Paulo. Caixa Postal 42.494, 04218‑970, São Paulo, SP, Brasil. E‑mails: lrsimone@usp.br; lrlsimone@gmail.com List of included taxa: S. punctata new species. List of included taxa: S. punctata new species. Etymology: The generic epithet is a contraction of Latin words spiri, from spira, and pocillum a little cup. Meaning something like a spiral small cup. This name is also used by my father, Egydio do Carmo de Sim- one, to designate any female pet in which he does not know the name. Pronunciation: in English terms the genus must be vo- calized as “speereepowqueeah”. Introduction An enigmatic tiny gastropod has been collected in caves from the Serra Ramalho Kars area, southwestern Bahia state, Brazil. It has a pretty, fragile, translucent shell in such preliminary gross anatomy, which already reveals troglobiont adaptations, i.e., depigmentation, lack of eyes and small size. The sample has been brought by Maria Elina Bichuette, who is specialized in subter- ranean fauna. Complementary analysis revealed a new pomatiopsid for which no known genus can be attrib- uted. Both genus and species are hereby formally in- troduced, including a detailed anatomical investigation. The description of this new taxon is not only academi- cally urgent, but the cave is also threatened for econom- ic reasons. The description of an endemic species helps in the argumentation for preserving this fragile location. The family Pomatiopsidae is represented in the Brazilian region by only two species of the genus Id‑ iopyrgus Pilsbry, 1911 (Simone, 2006: 94). However, the taxon is much richer in remaining mainland ar- eas, with both freshwater and semi-terrestrial habits (Ponder & Keyzer, 1998; Kameda & Kato, 2011). Common features of the pomatiopsids are small size (smaller than 15 mm), elongated to short spired, and smooth shell with paucispiral protoconch. The main distinction from other rissooideans is the diaulic pal- lial oviducts of the females; i.e., the typical pallial ovi- duct has two separate openings to the pallial cavity (Davis, 1967, 1979; Ponder, 1988). Simone, L.R.L.: A new Pomatiopsidae from Bahia 516 Spiripockia puncata new species (Figs. 1‑33) Types: Holotype MZSP 105000. Paratypes: MZSP 104435, 19 specimens, MNRJ 30503, 1 specimen, USNM 2060411, 1 specimen, all from type locality. Type locality: BRAZIL. Bahia; Serra Ramalho Karst area, Middle São Francisco River basin, Lapa dos Peixes cave, 13°49’21.78”S, 43°57’24.39”W (M.E. Bichuette col., 29/vii/2005). Abbreviatons of institutions: MNRJ, Museu Na- cional da Universidade Federal do Rio de Janeiro; MZSP, Museu de Zoologia da Universidade de São Paulo; USNM, National Museum of Natural History, Smithsonian Institution. Diagnosis: Shell weakly elongated; walls thin, fragile, translucent, unpigmented. Sculptured by pustules somewhat spirally aligned, possessing periostracum hair on tip of pustules (sometimes absent). Peristome expanded in plane ~90° in relation to last quarter whorl. Umbilicus opened. Strong pair of ventral snout retractor muscles passing through nerve ring. Radular rachidian with 6 apical and 3 pairs of lateral cusps, ~10 terminal cusps in radular lateral and mar- ginal teeth. Eyes absent. Head-foot unpigmented. Os- phradium short, arched. Gill filaments with rounded Type species: S. punctata new species. Type species: S. punctata new species. List of included taxa: S. punctata new species. Material and Methods pustules containing hair-like projections of periostra- cum. Freshwater environment. All specimens were fixed in 70% Ethanol. The dissections were performed under a stereomicro- scope by standard techniques, with the specimens immersed under fixative. Digital photos of each step of the dissection were obtained, as well as drawings aided by a camera lucida. A scanning electron mi- croscope (SEM) was employed to view details of the shell and radula in the Laboratório de Microscopia Eletrônica of Museu de Zoologia da Universidade de São Paulo. Description: Outline turbiform to slightly turriform. Protoconch paucispiral, of single whorl. Outer sur- face simple, except for small pustules distributed in somewhat spiral alignment, absent in some specimens (surface totally smooth). Peristome highly expanded and sprayed except for implantation on columellar surface. Umbilicus somewhat opened. Operculum thin. Pair of strong ventral retractor muscles of snout. Salivary gland reduced. Gonoducts closed (tubular); oviduct with bypass in posterior region. Spermathecal duct originated from albumen gland. Anatomical abbreviations in the figures: ag, albumen gland; an, anus; au, auricle; bm, buccal mass; br, sub- radular membrane; ce, cerebral ganglion; cg, capsule gland; cm, columellar muscle; co, bulged region of oviduct; cv, ctenidial vein; dd, duct to digestive gland; df, dorsal folds of buccal mass; dg, digestive gland; es, esophagus; fe, fecal pellets; fp, female pores; ft, foot; gi, gill; in, intestine; ir, insertion of m4 in subradular cartilage; is, insertion of m5 in subradular cartilage; jw, jaws; ki, kidney; m2‑m12, odontophore muscles; mb, mantle border; mj, jaw and peribuccal muscles; mo, mouth; ne, nephrostome; oc, odonto- phore cartilage; od, odontophore; of, Spermathecal oviduct; op, opercular pad; os, osphradium; ov, pal- lial oviduct; oy, ovary; pa, penis aperture; pc, peri- cardium; pd, penis duct; pe, penis; pg, anterior fur- row of pedal glands; pl, pleural ganglion; pn, pedal glanglion; pt, prostate; ra, radula; rm, snout ventral retractor muscle; rn, radular nucleus; rs, radular sac; rt, rectum; sa, salivary gland aperture; sc, subradular cartilage; sd, sperm duct; sg, salivary gland; sn, snout; st, stomach; su, subesophageal ganglion; sv, seminal vesicle; sy, statocyst; te, cephalic tentacle; tg, integu- ment; tn, tentacular nerve; ts, testis; ve, ventricle; vd, vas deferens; vo, visceral oviduct. Family Pomatiopsidae Genus Spiripockia new genus Family Pomatiopsidae Genus Spiripockia new genus Diagnosis: Turbiform shell with highly expand- ed peristome. Walls translucent, smooth except for Papéis Avulsos de Zoologia, 52(40), 2012 517 Description 8‑9); gradually after 2‑3 whorls sculpture changing to adult pattern. Peristome expanding in plane ~90° in relation to terminal region of body whorl (Figs. 1‑5, 10‑11) in mature specimens; expan- sion edges extremely fragile and somewhat irregular (apparently to fit in substrate), particularly wider in anterior region (Fig. 1) and narrower in columel- lar implantation; expansion extending ~15 % shell width. Aperture oval, superior angulation blunt; re- maining somewhat rounded. Umbilicus opened and relatively wide (Figs. 2, 4, 11), occupying ~15% of shell width (excluding peristome); no callus, except for short and narrow portion of peristome attached to preceding whorl (Figs. 2‑4, 10‑11). located approximately in opposed side from aper- ture (Figs. 2, 5, 11) (more visible in apical or ante- rior views); projection absent in younger specimens (Figs. 8‑9); being clearly derived from previous ap- erture. Sculpture minute pustules aligned in spiral lines (Figs. 12, 14), ~6 doted spiral lines, equidis- tantly spaced, in penultimate whorl; each line with ~40 pustules in penultimate whorl disposed some- what alternately each line in relation to neighbor lines (Figs. 1‑3, 10‑14); each pustule in form of small, cir- cular convexity, separated from neighbor pustules by smooth distance equivalent to twice pustules width (Fig. 12); pustules somewhat uniformly distributed, and sometimes forming wide angulation on shell sur- face (Fig. 14); this dotted sculpture absent in ~15% of specimens, possessing completely smooth outer sur- face (Figs. 4‑5); periostracum possessing 5‑6 axially aligned hair on pustules tip (Figs. 3, 10‑11), looking like flexible scales. Sculpture on younger specimens similar to this above described, but with pustule lo- cated closer to each other and with 4 inferior lines located closer to each other and far from subsutural Head-foot (Figs. 19‑21): Relatively small, stubby, un- pigmented. Foot thick, as wide as shell aperture. Mes- opodium thick, flanked dorsally by shallow lateral fur- rows (Fig. 21: ft). Anterior furrow of pedal glands (pg) deep, restricted to anterior edge (Figs. 19‑20). Oper- cular pad (Fig. 21: op) elliptic, terminal, occupying half of posterior foot dorsal surface. Description tip. Salivary glands short, small. Odontophore lacking pair of ventral tensor muscles (m11). Genital pallial gonoducts closed (tubular). Prostate flattened, with vas deferens inserting subterminally. Penis duct nar- row and weakly sinuous. Pallial oviduct simple ante- riorly; posterior region possessing convoluted bypass connecting base of bulged portion of transition be- tween visceral and pallial oviducts with base of semi- nal receptacle; spermathecal duct complete, opening close to anus level. Nerve ring with well delimited ganglia. Shell (Figs. 1‑5, 8‑14): Up to 5 mm; about as wide as long, turbiform to slightly turriform. Spire angle ~50°. Color pale beige to white, translucent. Protoconch occupying ~20% of shell width and ~7% of length; globose, smooth, transparent (Figs. 3, 5, 10, 13). Transition protoconch-teleoconch weak, orthocline. Teleoconch up to 4 convex whorls; suture deep, with ~90°; whorls somewhat uniform, except for last whorl in adult specimens, with bluntly pointed projection Figures 1‑11: Spiripockia punctata shell and operculum: 1) holotype, dorsal view (H 4.6 mm); 2) same, apertural-slightly anterior view; 3) same, apertural view; 4) paratype MZSP 104435 2♀ with specimens extracted, apertural view (H 3.3 mm); 5) same, apical-slightly apertural view; 6) holotype operculum, outer view (H 1.2 mm); 7) same, inner view; 8) young paratype, apertural view; 9) same, another specimen, left view; 10) paratype 6♀, apertural-slightly left view (H 3.4 mm); 11) same, anterior-slightly apertural view. Figures 1‑11: Spiripockia punctata shell and operculum: 1) holotype, dorsal view (H 4.6 mm); 2) same, apertural-slightly anterior view; 3) same, apertural view; 4) paratype MZSP 104435 2♀ with specimens extracted, apertural view (H 3.3 mm); 5) same, apical-slightly apertural view; 6) holotype operculum, outer view (H 1.2 mm); 7) same, inner view; 8) young paratype, apertural view; 9) same, another specimen, left view; 10) paratype 6♀, apertural-slightly left view (H 3.4 mm); 11) same, anterior-slightly apertural view. Figures 1‑11: Spiripockia punctata shell and operculum: 1) holotype, dorsal view (H 4.6 mm); 2) same, apertural-slightly anterior view; 3) same, apertural view; 4) paratype MZSP 104435 2♀ with specimens extracted, apertural view (H 3.3 mm); 5) same, apical-slightly apertural view; 6) holotype operculum, outer view (H 1.2 mm); 7) same, inner view; 8) young paratype, apertural view; 9) same, another specimen, left view; 10) paratype 6♀, apertural-slightly left view (H 3.4 mm); 11) same, anterior-slightly apertural view. Simone, L.R.L.: A new Pomatiopsidae from Bahia 518 line (Figs. Description 23: mo). Pair of strong ventral retractor muscles of snout and mouth (Figs. 23‑24: rm); originating in middle level of hae- mocoelic ventral floor; running close to median line towards anterior, flanking ventral surface of buccal mass, passing through nerve ring (Figs. 23, 33); insert- ing along ventral wall of snout, close to ventral border of mouth. Buccal mass bulged, occupying entire snout inner surface and ~1/3 of haemocoelic volume. Pair of jaw plates (Fig. 25: jw) somewhat separated from each other; each jaw oval and very thin, translucent. Odon- tophore spherical, with ~half of buccal mass volume. Odontophore muscles (Figs. 24‑26, 28): mj, pair of jaw and peribuccal muscles, working also as odonto- phore protractors, originating in lateral and ventral region of mouth, running towards posterior as part of oral tube along ~half odontophore length (Fig. 24), inserting in latero-ventral mid region of odontophore cartilages (Fig. 26); m2, pair of posterior retractor muscles of odontophore (Figs. 24‑25), narrow, origi- nating in lateral region of haemocoel inner surface, in its middle level, running towards anterior a distance slightly longer than buccal mass length, inserting in postero-lateral region of odontophore cartilages (Fig. 28); m4, pair of main dorsal tensor muscle of radula (Fig. 28), broad and thick, surrounding outer lateral surface of odontophore cartilages, originating from their ventro-lateral surface, inserting in lateral edges of subradular cartilage in its region in buccal cavity, and also in radular sac region preceding buccal cavity (Fig. 28: ir); m5, pair of secondary dorsal ten- sor muscles of radula (Figs. 26, 28), narrow and thick, originating in posterior edge of odontophore carti- lages, running towards medial and anterior, between cartilages, inserting in ventral side of radular ribbon in its region exposed in buccal cavity (Figs. 28: is); m6, horizontal muscle, thin and wide, connecting ventral edge of both odontophore cartilages along ~75% of their length (Fig. 28); m10, pair of ventral Mantle organs (Figs. 22, 27): Somewhat narrow, with ~1 whorl in length. Mantle edge simple, weakly thick; unpigmented. Osphradium short (os), curved (con- cavity left), simple; length ~6% pallial cavity length; located in anterior-left corner of cavity, close to man- tle border. Gill elongated, narrow, with ~85% of pal- lial cavity length and ~25% of cavity width; anterior end bluntly pointed, close to mantle border; posterior region narrowing gradually, filaments ending at some distance from pericardium; gill filaments somewhat triangular, with distal tip rounded (Fig. Description Head bulbous, Figures 12‑18: Spiripockia punctata hard parts in SEM: 12) paratype MZSP 104435 3♂, detail of penultimate whorl, dorsal view, scale = 200 µm; 13) same, detail of apical region in profile, scale = 200 µm; 14) same, whole dorsal view (H 3.9 mm); 15) radula, partial whole view, scale = 20 µm; 16) same, detail of central region, scale = 10 µm; 17) same, wider magnification, scale = 10 µm; 18) same, detail of more lateral region, scale = 10 µm. Figures 12‑18: Spiripockia punctata hard parts in SEM: 12) paratype MZSP 104435 3♂, detail of penultimate whorl, dorsal view, scale = 200 µm; 13) same, detail of apical region in profile, scale = 200 µm; 14) same, whole dorsal view (H 3.9 mm); 15) radula, partial whole view, scale = 20 µm; 16) same, detail of central region, scale = 10 µm; 17) same, wider magnification, scale = 10 µm; 18) same, detail of more lateral region, scale = 10 µm. Papéis Avulsos de Zoologia, 52(40), 2012 519 digestive gland. Other genital and digestive details below. with ~85% of food width; pair of cephalic tentacles lateral positioned (Figs. 19‑20, 23: te), each tentacle simple, thick, with ~twice foot length; no detectable eye. Snout with ~twice tentacles width and ~half ten- tacles length (sn); anterior end weakly bilobed, mouth oval, wide, subterminal, ventral (Figs. 23: mo). Colu- mellar muscle thick, ~3/4 whorl. Haemocoel ellipti- cal, on central region of head-foot (Fig. 23). Circulatory and excretory systems (Fig. 31): Pericar- dium narrow, located transversely between stomach and pallial cavity (Fig. 22: pc), volume ~1/20 of that of visceral mass; auricle anterior, small, just posterior to posterior end of ctenidial vein; ventricle posterior, simple. Kidney minute, weakly smaller than pericar- dium, located on right side of pallial cavity posterior end; inner tissue totally solid, white. Nephrostome (ne) small, transverse, located close to pericardium. Operculum (Figs. 6‑7): Corneous, thin, translucent, yellow-beige, flexible, paucispiral. Outline elliptical- almost circular. Edges thin. Nucleus located in middle region of interior half, weakly dislocated internally (Fig. 6); from nucleus, ~2 whorls uniformly growing, appearing; outer surface also sculptured by weak un- dulations parallel to outer edge. Inner surface glossy, scar elliptic, occupying ~half of inner surface; location dislocated to internal edge, but not touching internal edge. Occupies entire shell aperture (Figs. 3, 10‑11). Digestive system (Figs. 22‑26, 28): Mouth in antero- ventral end of snout (Fig. Description 27: gi). Be- tween gill and rectum a narrow distance equivalent to 10% of pallial cavity width. Hypobranchial gland inconspicuous. Rectum wide, reaching up to half of pallial cavity width in some points; bearing aligned series of elliptical fecal pellets (fe) disposed transver- sally or weakly oblique, easily seen by translucence (Figs. 1‑3, 10‑11); rectum displaced from right man- tle cavity edge by pallial genital ducts. Anus simple, shortly siphoned, distant from mantle border ~10% of cavity length, in anterior-right corner of pallial roof. Genital ducts running along right edge, rela- tively massive, described below. Visceral mass (Figs. 22, 31): Length ~3 whorls, keep- ing ~2 first shell whorls empty in adult specimens (Figs. 2‑3, 10). Color of most structures pale beige to white. Stomach as anterior structure, compressing ventrally small reno-pericardial structures; stomach of ~0.5 whorl and with almost entire adjacent whorl width. Digestive gland of ~2 whorls, mostly posterior to stomach. Gonad running along columellar surface of each whorl when mature, weakly distinct from Visceral mass (Figs. 22, 31): Length ~3 whorls, keep- ing ~2 first shell whorls empty in adult specimens (Figs. 2‑3, 10). Color of most structures pale beige to white. Stomach as anterior structure, compressing ventrally small reno-pericardial structures; stomach of ~0.5 whorl and with almost entire adjacent whorl width. Digestive gland of ~2 whorls, mostly posterior to stomach. Gonad running along columellar surface of each whorl when mature, weakly distinct from Simone, L.R.L.: A new Pomatiopsidae from Bahia 520 protractor muscle of buccal mass (Figs. 24, 26), rela- tively wide and thick, originating in ventral edge of mouth, just ventral to retractor muscle (mr) origin, running towards posterior a distance equivalent to odontophore length, inserting in posterior-ventral edge of odontophore cartilages. Subradular cartilage relatively wide in region of buccal cavity (Fig. 25: sc). Odontophore cartilages elliptic, somewhat flattened laterally, anterior edge weakly narrower than posterior edge (Fig. 28: oc). Description Radular sac with length ~3 times p g g p g g g Figures 19‑27: Spiripockia punctata anatomy: 19) head-foot, male, dorsal view; 20) same, female; 21) same, right view; 22) pallial cavity roof and partially uncoiled visceral mass, female, ventral view, some genital structures displaced, transverse section in indicated region of pallial oviduct also shown; 23) head and haemocoel, ventral view, foot and columellar muscle removed; 24) foregut, right-slightly dorsal view; 25) same, ventral view, odontophore partially removed and deflected, esophagus partially sectioned longitudinally; 26) odontophore, ventral view, radula partially deflected upwards; 27) pallial cavity hoof, transverse section in middle level of gill. Scales = 0.5 mm. Figures 19‑27: Spiripockia punctata anatomy: 19) head-foot, male, dorsal view; 20) same, female; 21) same, right view; 22) pallial cavity roof and partially uncoiled visceral mass, female, ventral view, some genital structures displaced, transverse section in indicated region of pallial oviduct also shown; 23) head and haemocoel, ventral view, foot and columellar muscle removed; 24) foregut, right-slightly dorsal view; 25) same, ventral view, odontophore partially removed and deflected, esophagus partially sectioned longitudinally; 26) odontophore, ventral view, radula partially deflected upwards; 27) pallial cavity hoof, transverse section in middle level of gill. Scales = 0.5 mm. Papéis Avulsos de Zoologia, 52(40), 2012 521 that of odontophore (Figs. 24‑26: rs), and ~1/4 odon- tophore width; radular nucleus (rn) weakly broader. edge; lateral tooth with main region rectangular, with ~double rachidian height and its same width, ~15 cusps similar to those of rachidian, cusps smaller in both ends, being gradually larger towards middle- weakly lateral region (Figs. 16‑17), basal region wid- ening obliquely externally (Figs. 15, 17) along dis- tance equivalent to ~30% radula width, being covered by marginal teeth (Figs. 17‑18); inner and outer lat‑ eral tooth similarly shaped, being outer tooth ~30% narrower than inner marginal tooth, ~double length Radular tooth (Figs. 15‑18): rachidian tooth triangu- lar, distal tip curved inwards, with ~16% of radular ribbon width, about as tall as wide, base simple, weak- ly arched, 3 pairs of latero-basal cusps (Figs. Papéis Avulsos de Zoologia, 52(40), 2012 Description Sperm duct, or bypass (sd) as convoluted duct connecting anterior region of bulged region of oviduct (co) with base of seminal receptacle (rs); posterior half clearly narrower, gradually broadening in anterior half. Capsule gland (cg) occupying ~80% of pallial oviduct length; walls thick glandular, white, lumen flattened (Fig. 22); short terminal atrium with walls weakly thick, taper- ing up to female pore. Female pore shortly siphoned, papilla-like, turned anteriorly, located close to and at right of anus. No special female genital structure in head-foot (Figs. 20‑21). Salivary gland small (Figs. 23‑24: sg), white, with maximum length ~half buccal mass length, and sometimes smaller; about 3 times longer than wide, tip rounded; salivary aperture in postero-lateral re- gion of oral cavity (Fig. 25: sa). Dorsal folds of buc- cal mass (Fig. 25: df) smooth, simple, wide, located close to each other. Esophagus simple and sinuous along haemocoel (Figs. 23‑24: es), generally with an- terior glandular enlargement (Fig. 24), but sometimes (~30%) with same width along length; most remain- ing esophageal inner surface simple, lacking folds; posterior esophagus (Fig. 22: es) narrow, inserting in ventral-mid region of stomach. Stomach dimensions and positions above described (visceral mass); pos- sessing main gastric chamber posterior, with rounded posterior surface, and narrow style sac (Figs. 22: ss), with ~half remaining gastric dimensions. Style sac to- tally separated from intestine; esophagus inserting just ventrally to origins of style sac and intestine, on ante- rior stomach side; duct to digestive gland as wide as posterior esophagus, originating at short distance pos- terior to esophageal insertion (Fig. 22: dd), running towards posterior. Intestine originating dorsally and at left from esophageal insertion (Fig. 22: in), with ~double esophagus width, and at left from style sac; running surrounding externally style sac up to dorsal region of kidney, broadening in region preceding pal- lial cavity. Rectum, fecal pellets and anus above de- scribed (pallial cavity). Fecal pellets formed only after renal portion of intestine (Figs. 11, 22). Central nervous system (Figs. 30, 33): Nerve ring lo- cated just posterior to buccal mass, with pedal ganglia weakly more anterior (Fig. 23: nr) than remaining ganglia. Each cerebral ganglion (ce) oval, with size equivalent to esophageal section; cerebral commissure narrow, with ~half-length as each ganglion. Pleural ganglia (pl) with ~half size of cerebral ganglia, located just ventral to them. Description 16‑18), each cusp small, triangular, pointed tip; 5‑7 terminal triangular cusps, central cusp larger, cusps weakly di- minishing towards lateral, restricted to distal, curved Figures 28‑33: Spiripockia punctata anatomy: 28) odontophore, dorsal view, radula, outer layer of muscles and membranes removed, both cartilages deflected; 29) penis, ventral view, adjacent region of integument also shown, penis duct seen by translucence; 30) nerve ring, right view; 31) part of pallial cavity roof and partially uncoiled visceral mass, male, ventral view; 32) posterior end of pallial oviduct, ventral view; 33) nerve ring, dorsal view, topology of adjacent retractor muscle also shown. Scales = 0.5 mm. Figures 28‑33: Spiripockia punctata anatomy: 28) odontophore, dorsal view, radula, outer layer of muscles and membranes removed, both cartilages deflected; 29) penis, ventral view, adjacent region of integument also shown, penis duct seen by translucence; 30) nerve ring, right view; 31) part of pallial cavity roof and partially uncoiled visceral mass, male, ventral view; 32) posterior end of pallial oviduct, ventral view; 33) nerve ring, dorsal view, topology of adjacent retractor muscle also shown. Scales = 0.5 mm. Simone, L.R.L.: A new Pomatiopsidae from Bahia 522 weak zigzag coils along outer, almost central penial region, up to penis distal tip (Fig. 29: pd); aperture distal, simple, small (pa). as lateral tooth and ~40% narrower, general form as rod curved inwards at tip, tip flattened, bearing ~13 (inner) and ~10 (outer) terminal cusps of similar characters as lateral tooth cusps. Female (Figs. 22, 32): Visceral structures similar to those of males; ovary (oy) somewhat broader than testis. Visceral oviduct very narrow, running along middle level of columellar surface of visceral mass ~half whorl (vo). Visceral oviduct inserting terminally in complex posterior region of pallial oviduct (Fig. 32) in bulged region of oviduct (co) with simple glandular walls and hollow inside. This bulged region continu- ing to albumen gland (ag) strongly curved in ~180°. Spermathecal duct (of) originating in ventral-left side of albumen gland, very narrow but with thick walls, running along right edge of pallial cavity ventral to re- maining pallial oviduct ~90% of pallial cavity length; opening simple by side and ventral to main female pore (Figs. 22: fp). Seminal receptacle (rs) bulging subterminally in posterior region of pallial oviduct, possessing two successive bulbs or sometimes horse- shoe-shaped; inserting by narrow duct in adjacent left side of posterior end of capsule gland. Discussion Spiripockia punctata is the second troglobiont gastropod described in Brazilian territory. The first one is a relative taxon, Potamolithus troglobius Simone & Moracchioli, 1994, a Hydrobiidae from Ribeira Valley, southern São Paulo state. Both species share the characters to consider them as troglobiont – the total lack of pigmentation and eyes, and small size – and the freshwater environment. In light of the enor- mous quantity of caves in the Brazilian region, it is difficult to believe that only 2 troglobiont gastropod species exist. Bichuette & Trajano (2003) cited at least five more troglobiont species, all belonging to Hydro- biidae and probably to the genus Potamolithus Pilsbry, 1911, occurring in Ribeira Velley, SE Brazil. Though, certainly many more studies on this issue and urgent preservation of cave environments are necessary. I can affirm that the MZSP collection already has some samples of troglobiont rissooideans and even pulmo- nates waiting for study. With respect to the radula, Spiripockia punctata possesses the normal pattern of the family (Ponder & Keyzer, 1998), which includes the lateral cusps in the rachidian and the basal lateral expansion of the lateral toot base. It differs in number and form of the cusps, as normally found amongst closely related species. g y Spiripockia punctata has no South American species with which it can be confused. The set of conchological features are so far exclusive and suf- ficiently distinctive to erect a new genus. From the family Pomatiopsidae, the only two species hitherto described belong to the genus Idiopyrgus – I. rudolphi Haas, 1838 and I. souleyetianus Pilsbry, 1911 (Sim- one, 2006), from which S. punctata differs in having a shorter and wider shell, fewer whorls, peristome more expanded, umbilicus wider, and the sculpture of most specimens of pustules with periostracum hair in them (Figs. 1‑3, 10‑11). The radula and genital part of the anatomy of I. souleyetianus has been investigated (Rey, 1959; Malek, 1983), which permits some extra-shell comparison. The operculum of S. punctata has a more central nucleus. Its osphradium is much shorter and curved. Spiripockia punctata also has the insertion and pallial origin of the vas deferens in the prostate gland in a subterminal manner (Fig. 31: vd), however, that of I. Description Each pedal ganglion (pn) weakly larger than cerebral ganglion, somewhat spherical, located very close to each other (pedal commissure extremely short). Cerebro-pedal and pleuro-pedal connectives about as long as pedal ganglia (Fig. 30). Subesophageal ganglion (su) with ~half pleural gan- glion size, located a distance from right pleural gan- glion equivalent to 1.5 times each cerebral ganglion length. Pair of statocysts (sy) located in ventro-poste- rior side of pedal ganglia; with large, single statolith inside. Single pair of tentacular nerve (tn), lacking optical nerve. Genital system: Male (Figs. 19, 29, 31): Testis restrict- ed to posterior half of digestive gland (Fig. 31: ts). Seminal vesicle (sv) weakly convolute, with 3‑4 zig- zag loops in columelar region of visceral mass preced- ing stomach, color pale grey-iridescent; vas deferens relatively straight in gastric and renal region, insert- ing in prostate subterminally (Fig. 31: vd). Prostate with ~1/4 pallial cavity width, somewhat dorso- ventrally flattened, white, totally closed (tubular); inner lumen simple, flat (Fig. 27: pt). Vas deferens originating subterminally from prostate anterior re- gion (Fig. 31: vd) in level between anterior and mid thirds of pallial cavity; in short distance penetrating in floor of pallial cavity by side of penis base, running immersed in integument (Figs. 19: vd, 29: pd). Penis (Figs. 19‑29: pe) simple, curved, base broad, gradually tapering up to sharp pointed tip. Penis duct seen by translucence, color pale-green-iridescent, performing Papéis Avulsos de Zoologia, 52(40), 2012 523 Measurements (in mm): Holotype: 4.6 by 4.3; para- types: 2♀: 3.3 by 3.1; 4♂: 3.3 by 3.5; 5♀: 3.6 by 3.4. Measurements (in mm): Holotype: 4.6 by 4.3; para- types: 2♀: 3.3 by 3.1; 4♂: 3.3 by 3.5; 5♀: 3.6 by 3.4. mid-level of that gland. The penis is somewhat simi- lar, with totally closed (tubular) duct, but the penis duct of S. punctata appears to be more convolute. The contrary happens in the seminal vesicle, such as in the case of I. souleyetianus whose seminal vesicle is appar- ently much more coiled. Related to the pallial oviduct, S. punctata lacks a clear separation between albumen and capsule glands (Fig. 32), as happens in I. souleye‑ tianus; the bypass duct (sd) is much more coiled in S. punctata, and its seminal receptacle is connected directly to the pallial oviduct, while that of I. souleye‑ tianus is connected to the visceral oviduct; S. Description punctata lacks a small secondary receptacle, which is present in I. souleyetianus; the spermathecal duct (of) has its origin in the end of the albumen gland, close to inser- tion of the visceral oviduct in S. punctata, while that of I. souleyetianus is originated from the receptacle (also known, in rissooideans, as spermatheca or bursa copulatrix). Actually, the arrangement of the posterior region of the pallial oviduct of I. souleyetianus looks more like the pattern of the family (Davis, 1967, 1979; Ponder, 1988; Davis et al., 1992) than that of S. punctata, with the spermathecal duct connected to the pallial oviduct instead to the receptacle. In this point of view, the name “spermathecal duct” is not totally accurate; however the name was maintained in order to permit further comparisons and to suggest homology. This singularity of S. punctata is another indication of generic separation, beyond those fea- tures of the shell. Distribution: Only known in the type locality (Lapa dos Peixes cave). Distribution: Only known in the type locality (Lapa dos Peixes cave). Habitat: In a cave surrounded by Caatinga semi-arid environment, attached to rocks inside a small stream. Material examined: Types. Material examined: Types. Material examined: Types. Discussion souleyetianus is inserted and originated at It is expected that with the description of this new endemic taxon, further argumentation for the conservation of the cave type locality for the present species and others can be instituted. Some pomatiop- sids have been considered as endangered species on the IUCN Red Lists. One example is Tomichia caw‑ stoni Connolly, 1939, from South Africa (Kristensen et al., 2010). According to the IUCN terms, S. punc‑ tata is already considered threatened based on its en- demicity, as well as on its troglomorphic features, such as depigmentation and being eyeless. Resumo Spiripockia punctata é gênero e espécie novos de Poma‑ tiopsidae encontrado em uma caverna da Serra Ramalho, Simone, L.R.L.: A new Pomatiopsidae from Bahia 524 SW Bahia, Brasil. O táxon é troglóbio (restrito à vida subterrânea), caracterizado pela concha levemente alon‑ gada, frágil, translúcida, normalmente esculturada por pústulas contendo pelos do perióstraco no topo das pús‑ tulas; perístoma altamente expandido; umbílico aberto; dente raquidiano da rádula com 6 cúspides apicais e 3 pares de basais; osfrádio curto e curvo; filamentos bran‑ quiais com topo arredondado; próstata achatada, com vaso deferente inserindo subterminalmente; ducto do pênis fracamente sinuoso; oviduto palial anteriormen‑ te simples, possuindo um by-pass convoluto da base da porção bulbosa da porção entre oviduto visceral e palial com a base do receptáculo seminal; ducto da espermateca completo, originado da glândula de albume. A descrição dessa espécie endêmica deve contribuir com ações proteti‑ vas do ambiente da caverna e à área da Serra Ramalho. References Bichuette, M.E. & Trajano, E. 2003. A population study of epigean and subterranean Potamolithus snails from southeast Brazil. Hydrobiologia, 505:107‑117. Davis, G.M. 1967. The systematic relationship of Pomatiopsis lapidaria and Oncomelania hupensis formosana (Prosobranchia: Hydrobiidae). Malacologia, 6(1‑2):1‑143. Davis, G.M. 1979. The origin and evolution of the gastropod family Pomatiopsidae, with emphasis on the Mekong River Triculinae. Monograph of the Academy of Natural Sciences of Philadelphia, 20:1‑120. Davis, G.M.; Chen, C.E.; Wu, C.; Kuang, T.F.; Xing, G.; Li, L.; Liu, W.J. & Yan, Y.L. 1992. The Pomatiopsidae of Hunan, China (Gastropoda: Rissoacea). Malacologia, 34(1‑2):143‑342. Kameda, Y. & Kato, M. 2011. Terrestrial invasion of pomatiopsid gastropods in the heavy-snow region of the Japanese Archipelago. BioMed Central Evolutionary Biology, 11(118):1‑14. Kristensen, T.K.; Stensgaard, A.S. & Appleton, C. 2010. Tomichia cawstoni. The IUCN Red List of Threatened Species. www.iucnredlist.org/apps/redlist/details/63444/0 (extracted in 26/iv/2011). Kristensen, T.K.; Stensgaard, A.S. & Appleton, C. 2010. Tomichia cawstoni. The IUCN Red List of Threatened Species. Palavras-Chave: Pomatiopsidae; Spiripockia punc‑ tata gen. nov. et sp. nov.; Brasil; Caverna; Troglóbio; Anatomia. p www.iucnredlist.org/apps/redlist/details/63444/0 (extracted in 26/iv/2011). Malek, E.A. 1983. The South American hydrobioid genus Idiopyrgus Pilsbry, 1911. Nautilus, 97(1):16‑20. Ponder, W.F. 1988. The truncatelloidean (= rissoacean) radiation l h l d d b h Ponder, W.F. 1988. The truncatelloidean (= rissoacean) radiation – a preliminary phylogeny. In: Ponder, W.F. (Ed.). Prosobranch Phylogeny. Malacological Review supplement, 4:129‑166. Acknowledgments Ponder, W.F. & Keyzer, R.G. 1998. Superfamily Rissooidea. In: Beesley, P.L.; Ross, G.J.B. & Wells, A. (Eds.). Mollusca: the southern synthesis. CSIRO Publishing, Melbourne. p. 745‑766. (Fauna of Australia v. 5, part B, p. 565‑1234). The material was collected under IBAMA li- cense 027/00‑DIFAS/DIREC by Maria Elina Bi- chuette, Universidade Federal de São Carlos. I am deeply grateful to her for donation of the specimens, location description, and for reviewing the paper. I am also grateful to Winston Ponder, Australian Muse- um, for suggestions on systematics in the early phase of this study. I further acknowledge FAPESP (Funda- ção de Amparo à Pesquisa do Estado de São Paulo) for financial support in the field activities of E. Trajano’s project (process 2003/0794‑5). Rey, L. 1959. Molluscs of the genus Oncomelania, in Brazil, and their possible epidemiological significance. Revista do Instituto de Medicina Tropical de São Paulo, 1(2):144‑149. Simone, L.R.L. 2006. Land and freshwater molluscs of Brazil. EGB. FAPESP. São Paulo, 390 pp. Simone, L.R.L. & Moracchioli, N. 1994. Hydrobiidae (Gastropoda: Hydrobioidea) from the Ribeira Valley, S.E. Brazil, with descriptions of two new cavernicolous species. Journal of Molluscan Studies, 60:445‑459. 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https://openalex.org/W2796771843	https://www.duo.uio.no/bitstream/10852/67688/2/journal.pone.0193677.pdf	English	null	Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells	PloS one	2,018	cc-by	9,314	RESEARCH ARTICLE Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells Halh Al-Serori1, Franziska Ferk1, Michael Kundi2, Andrea Bileck3, Christopher Gerner3, Miroslav Misˇı´k1, Armen Nersesyan1, Monika Waldherr1, Manuel Murbach4, Tamara T. Lah5, Christel Herold-Mende6, Andrew R. Collins7, Siegfried Knasmu¨ller1* 1 Institute of Cancer Research, Department of Internal Medicine 1, Medical University of Vienna, Vienna, Austria, 2 Center for Public Health, Department of Environmental Health, Medical University of Vienna, Vienna, Austria, 3 Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria, 4 IT ‘IS Foundation, Zurich, Switzerland, 5 Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia, 6 Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany, 7 Department of Nutrition, University of Oslo, Oslo, Norway OPEN ACCESS Citation: Al-Serori H, Ferk F, Kundi M, Bileck A, Gerner C, Misˇı´k M, et al. (2018) Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells. PLoS ONE 13(4): e0193677. https://doi.org/10.1371/ journal.pone.0193677 Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malig- nant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is sup- ported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activa- tion of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems. Editor: Maria Rosaria Scarfi, Consiglio Nazionale delle Ricerche, ITALY Received: September 25, 2017 Accepted: February 13, 2018 Published: April 12, 2018 Copyright: © 2018 Al-Serori et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the Austrian Workers Compensation Board (Allgemeine Unfallversicherungsanstalt, AUVA). * siegfried.knasmueller@meduniwien.ac.at Mobile phone specific EMF and DNA damage carcinogenic for humans”[1]. This decision was based on results of epidemiological studies which indicated that the RF signals from mobile phones may cause glioblastomas and other malignant brain tumors as well as schwannomas (for reviews see [2, 3, 4]). Abbreviations: BER, base excision repair; DSB, double strand breaks; GSM, global system for mobile communications; H2O2, hydrogen peroxide; LC, liquid chromatography; MS, mass spectrometry; NER, nucleotide excision repair; γH2AX, phosphorylated histone H2AX; RF, radiofrequency; RF-EMF, radiofrequency electromagnetic fields; SAR, specific absorption rate; SCGE, single cell gel electrophoresis; SSB, single strand break; UMTS, universal mobile telecommunication system. It is known that damage of the genetic material plays a key role in the etiology of cancer [5, 6, 7], therefore, we investigated for the first time the effects of the universal mobile telecommu- nication system (UMTS) signal on DNA stability in human glioblastoma cell lines (U87, U251 and U373). Additionally, we included further human nerve tissue derived cell lines i.e. primary astrocytes, a neuroblastoma line (SH-SY5Y) and a human stem cell like glioblastoma line (NCH421k). We conducted also experiments with cells from organs other than the brain, i.e. liver derived cells (HepG2), buccal mucosa derived and fibroblast cells (TR-146 and ES-1) as well as lymphocytes. All experiments were conducted under conditions relevant for humans (i.e. with specific absorption rate (SAR) values 1 W/kg) and with a RF-frequency of 1950 MHz. This signal is currently widely used for 3rd generation (“smart”) phones. The impact of RF on DNA stability was studied in the present investigation in single cell gel electrophoresis (SCGE) assays, which are based on the measurements of DNA migration in an electric field [8, 9]. This approach is currently widely used in genetic toxicology [10]. The experiments were conducted under alkaline conditions, which allow the detection of single and double strand breaks (SSBs and DSBs) and apurinic sites [11]. The cells were treated in all experiments additionally with hydrogen peroxide as some earlier studies indicated that the effects of EMF-fields are due to formation of ROS, therefore we wanted to know if they increase the sensitivity of the different cell types towards oxidative damage. Furthermore, we performed γH2AX experiments which reflect DSBs under identical conditions [12]. This method is based on the measurement of phosphorylation of the histone protein H2AX [13]. It was postulated that RF effects are cell cycle dependent, and it was hypothesized that alter- ations of DNA repair processes may play a causal role [14], but no results from experiments are available which concern the impact of the UMTS signal on DNA stability in non-dividing cells and on DNA repair functions. Therefore, we studied the effects of RF exposure in two selected glioblastoma lines (U87 and U251, which appeared to be more sensitive towards RF- fields as the other cell types) after cultivation under serum free conditions, which leads to cell cycle arrest and reflects the in vivo situation which is characterized by low mitotic activity. Fur- thermore we investigated the impact of the UMTS signal on the activities of nucleotide exci- sion repair (NER) and base excision repair (BER), which are major repair pathways in mammalian cells [15]. To provide a mechanistic explanation of our results a proteome analysis was conducted to investigate if proteins which are upregulated as a consequence of DNA dam- age are affected by the signal. About 4000 individual proteins were quantified before and after treatment of the cells. According to our knowledge, the impact of the UMTS signal on protein expression has not been studied in high-throughput experiments. Introduction About 6.8 billion mobile phone subscriptions are active at present (www.itu.int). The adverse health effects of telecommunication radiofrequencies (RF) are controversially discussed since the development of this technology. In 2011, the IARC classified mobile phone RF as “possibly Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 1 / 17 Collection and cultivation of human cell lines The cell lines were collected from different sources and were stored deep frozen. Prior to the experiments they were checked for contaminations with mycoplasmas, bacteria and fungi. Human derived glioma lines U87, U251 and U373 were provided by the National Institute of Biology (Ljubljana, Slovenia). The cells were cultivated under standard conditions (37˚C, humidified atmosphere, 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM) which was supplemented with 10% FCS. The human neuroblastoma cell line (SH-SY5Y) was grown in complete DMEM: Ham’s F12 (1:1 mixture) supplemented with 2.0 mM L-glutamine and 10% FCS. The cells were obtained from S. Bo¨hm (Medical University of Vienna, Austria). The glioblastoma stem-like cell line NCH421k was generated in the laboratory of the Department of Neurosurgery (University of Heidelberg, Germany) and was cultured as non-adherent spheres in DMEM-F12 medium containing 1x BIT100, 2.0 mM L-glutamine, 30.0 U/ml Pen- Strep, 1 U/ml Heparin, 20.0 ng/ml bFGF and 20.0 ng/ml EGF. The human diploid fibroblast line (ES-1 cells) was grown in DMEM with 10% FCS. The cells were obtained from A. Pilger (Department of Occupational Medicine, General Hospital of Vienna, Austria). The human buccal cell line (TR-146) came from J. G. Rheinwald (Dermatology Institute of Boston, USA) and was cultured in Dulbecco’s medium with 10% FCS. The human hepatocellular carcinoma cell line HepG2 was provided by F. Darroudi (University of Leiden, Netherlands). The cells were cultured in Eagle’s minimal essential medium (MEM) supplemented with 1.0 mM sodium pyruvate and 10% FCS. All cell lines were stored in liquid nitrogen. After re-cultivation, experiments were per- formed up to the 5th to 7th passage. Media were changed every 2–3 days. When the cultures reached 80% confluency, the cells were washed with DPBS, detached with Trypsin-EDTA, cen- trifuged and sub-cultured. Chemicals Low melting point agarose (LMPA) and normal melting point agarose (NMPA) were pur- chased from Gibco (Paisley, UK). Propidium iodide, hydrogen peroxide (H2O2), Triton X- 100, Trizma base, trypan blue, fetal calf serum (FCS), Dulbecco’s Phosphate Buffered Saline (DPBS), Ham’s F12, L-glutamine, Pen-Strep, formamidopyrimidine DNA glycosylase (FPG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA), Histopaque 1077, bovine serum albumin (BSA), heparin, Trypsin-EDTA, sodium pyruvate, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), potassium chloride (KCl), ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA) and all PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 2 / 17 Mobile phone specific EMF and DNA damage media for the cultivation of the cell lines were from Sigma-Aldrich (Steinheim, Germany). Cancer stem cell medium (BIT100) came from Provitro (Berlin, Germany); T4 endonuclease V was purchased from Biozym Scientific (Vienna, Austria). The photosensitizer Ro 19–8022, was from Chiron AS (Trondheim, Norway) and Biofreeze came from Biochrom AG (Berlin, Germany). PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Treatment of the cells 5 x 105 cells were seeded into the Petri dishes and allowed to attach to the bottom overnight before exposure. Aliquots of lymphocyte suspensions were immediately exposed after isolation. Six dishes were exposed simultaneously in a waveguide chamber; in a second chamber, the cells were sham exposed. The cells were treated intermittently (cycles of 5 min on, 10 min off) as it was postulated that intermittent RF-EMF (radiofrequency electromagnetic fields) may be more effective than continuous exposure [17]. In all experiments, RF-exposed as well as unex- posed cells were treated with H2O2. For investigations of the SAR dependency, the cells were exposed to intermittent SAR levels of 0.25, 0.5 and 1.0 W/kg for 16 hours. In a second series of experiments, the cells were grown overnight in FCS medium; subse- quently, they were kept in serum free medium for 104 hrs and during the exposure phase (16 hrs); all other experimental conditions were identical as in the first series. Collection and cultivation of primary cells Normal human primary astrocytes were purchased from iXCells Biotechnologies (Cat#10HU- 035, San Diego, USA). Cells arrived as passage 4 and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS and EGF. The cell line was stored and re-cultivated as described above. Peripheral blood samples were collected from three healthy, non-smoking male volunteers without any history of recent disease or exposure to toxic chemical agents. The samples were collected by venipuncture in heparinized tubes (BD, Heidelberg, Germany). Blood samples were centrifuged (650 g, 10 min, 4˚C) immediately after collection. Subse- quently, the plasma was removed, the cell suspensions diluted with RPMI 1640 and the lym- phocytes isolated by gradient centrifugation (800 g, 16˚C min, 16˚C) with Histopaque 1077 in Accuspin tubes (Sigma Aldrich, Steinheim, Germany). The cells were collected and washed twice in RPMI 1640 (332 g, 10 min 16˚C). The Ethical Committee of the Medical University of Vienna specifically approved this study. 3 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Mobile phone specific EMF and DNA damage Exposure system The waveguide-based and computer-controlled exposure system was built and provided by the IT’IS Foundation (Foundation for Research on Information Technologies Society, Zurich, Switzerland, www.itis.ethz.ch). A detailed description of the experimental set-up and of the dosimetry can be found in an article of Schuderer et al. [16]. The exposure unit consists of two rectangular waveguides operating at a frequency of 1950 MHz. Both waveguides were placed in a commercial incubator (HeraCell 240 CO2 incubator, Kendro Laboratory, Germany), which provided the environmental conditions for the cell cultures (37˚C ± 0.1˚C, 5% CO2, 95% humidity). A commercial broadband coax-to-waveguide coupler was used to excite the waveguides, each one containing up to six 35 mm ø Nunc Petri dishes (Nunc, Roskilde, Den- mark). The detailed electrothermal analysis of the waveguide exposures included both, numer- ical estimation and experimental validation. The field level was controlled via monopole antennas in the waveguides and linked to the corresponding SAR and temperature values from the dosimetric assessment. The Petri dishes were placed in the H-field maxima of the standing-wave within the waveguide, such that the highest SAR level occurred at the bottom- layer of the dishes, where the monolayer cells are located. The dishes were filled with 4.0 ml medium. A SAR value of 1.0 W/kg, (the maximum intensity which was used in the present investigation) causes under these conditions an increase of the temperature 0.03˚C [16]. The pH values of the cultures were identical in treated and untreated cultures. The system was fully computer controlled and logs the status of the field-level, environmental temperature, and ventilators every 10 seconds for a fully reproducible experiment. Exposure to the different intensities followed the same UMTS protocol at a 5 minutes on/10 minutes off schedule. Measurement of DNA repair Preparation of the DNA substrate cells. BER and NER activities were analyzed by use of a modified protocol of the SCGE assay according to A Azqueta et al. [18]. To prepare DNA for BER measurements, HepG2 cells were used as an indicator line and were treated with 0.1 μM of a photosensitizer (Ro 19–8022) for 7 min, and irradiated subsequently on ice with a 400 W halogen lamp (SON-TAGRO, Philips, Vienna, Austria) at a distance of 33 cm to induce forma- tion of 8-oxoguanine. For the measurement of NER, HepG2 cells were irradiated with 2 Jm−2 UVC (Stratalinker UV Crosslinker, Model 1800, Agilent Technologies, Vienna, Austria) to generate cyclobutane pyrimidine dimers and 6–4 photoproducts. In parallel, also untreated HepG2 were processed. After two washes with DPBS, the cells were detached by trypsinization and centrifuged at 200 g at 4˚C for 3 min. The pellet was resuspended in freezing medium, divided into aliquots and frozen slowly to −80˚C. Preparation of the cytosolic extracts from exposed cells. After exposure to RF, the cells were washed twice with cold DPBS, trypsinized and resuspended in medium. Subsequently, they were centrifuged at 800 g (4˚C) for 5 min and resuspended in cold DPBS at a concentra- tion of 1.5 × 10⁶/ml and again centrifuged (14.000 g, 4˚C) for 5 min. Subsequently, the super- natants were gently removed, then the pellets were snap frozen by dropping into liquid nitrogen and stored at -80˚C. Determination of DNA repair. Prior to the repair assay, the cytosolic extracts of RF- exposed cells were thawed and the suspensions were diluted with four volumes of buffer B (45 mM HEPES, 0.25 mM EDTA, 0.3 mg/mL BSA, 2% glycerol, pH 7.8) before use. Extracts were kept on ice until use. Substrate cells were placed on pre-coated agarose slides and lysed in the dark at 4˚C overnight. Slides were washed with reaction buffer (0.1 M KCl, 0.5 mM Na2EDTA, 40 mM HEPES, 0.2 mg/mL BSA, pH 8.0) for 3 × 5 min. For NER measurements additionally MgCl2 solution (final concentration 1 mM) was added. Subsequently, 30 μl of the cytosolic extracts were incubated with substrate DNA for BER and NER measurements for 30 min. Reaction buffer was used as a negative control; for BER assays formamidopyrimidine DNA glycosylase (FPG) served as a positive control while T4 endonuclease V was used in NER experiments. Measurement of DNA repair After incubation, the slides were processed immediately according to the protocol for con- ventional comet assays [19] to measure DNA breaks introduced by the initial incision events of repair. For each experimental point, three gels were prepared and 50 randomly distributed cells were evaluated per gel. All experiments were performed in triplicate. Single cell gel electrophoresis (SCGE) assays SCGE experiments were conducted under standard alkaline conditions as described by Tice et al. [8]. Briefly, the cells were collected by trypsinization after exposure, their vitality and numbers were determined with the trypan blue dye exclusion test with a Neubauer chamber (LO-Laboroptik GmbH, Germany). Subsequently, the pellets were resuspended in low-melting agarose (LMA, 0.5%) and spread on pre-coated agarose slides (1.5% normal melting agarose). H2O2 was used as a positive con- trol. In all series, also non-RF exposed cultures were treated with H2O2. The slides were immersed in H2O2 solution (30 μM) on ice for 10 min. After the treatment, the slides were washed with DPBS (2 x 8 min) and lysed in the dark at 4˚C for at least 60 min. After 30 min PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 4 / 17 Mobile phone specific EMF and DNA damage unwinding under alkaline conditions (pH > 13), electrophoresis was carried out for 30 min (300 mA, 25 V). Neutralization was performed twice for 8 min; then the air-dried slides were stained with propidium iodide (20.0 μg/mL). The percentage of DNA in tail was measured by use of a computer-aided image analysis sys- tem (Comet IV, Perceptive Instruments Ltd., Haverhill, UK). Three independent experiments were conducted per experimental point, and three cultures were set up per experiment. From each culture one slide was prepared and 50 randomly distributed cells were evaluated per slide. Hedgehogs were identified visually and excluded from the evaluation. However, the frequen- cies were recorded and did not exceed 2%. Proteome analysis Subcellular fractionation. The analysis was performed as described previously by Bileck et al. [20]. Briefly, the cells were washed and lysed in isotonic lysis buffer (10 mM HEPES/ NaOH, pH 7.4, 0.25 M sucrose, 10 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100) supplemented with protease inhibitors (pepstatin, leupeptin and aprotinin, each at 1.0 μg/ml; 1.0 mM PMSF) and exposed to mechanical shear stress as described previously after removal of the superna- tants. [21] Cytoplasmic proteins were separated from the nuclei by centrifugation at 2300 g and 4˚C for 5 min and precipitated overnight with ice-cold ethanol at -20˚C. To obtain nuclear proteins, pellets were swelled up for 10 min in 500 mM NaCl and 1:10 diluted with NP-40 buffer for 15 min. For the separation of nuclear proteins, a centrifugation step at 2300 g and 4˚C was performed for 5 min. The extracted proteins were again precipitated overnight with ice-cold ethanol at -20˚C. All samples were dissolved in sample buffer (7.5 M urea, 1.5 M thio- urea, 4% CHAPS, 0.05% SDS, 100 mM DDT) and protein concentrations were determined with the Bradford assay (Bio-Rad-Laboratories, Germany). MS-sample preparation. All protein samples were enzymatically digested using a modifi- cation of the FASP protocol as described previously [21, 22]. Briefly, 3 kD MWCO filters (Pall Austria Filter GmbH) were pre-washed using LC-MS grade water (Millipore GesmbH). 20 μg of each protein sample were pre-concentrated onto the filter by centrifugation at 15,000 g for 15 min. Protein reduction using DTT (5.0 mg/ml dissolved in 8.0 M guanidinium hydrochlo- ride in 50 mM ammonium bicarbonate buffer (ABC buffer), pH 8) and protein alkylation using IAA (10 mg/ml in 8 M guanidinium hydrochloride in 50 mM ABC buffer) were per- formed. Afterwards, 0.1 μg/μl trypsin was added and incubated at 37˚C for 18 hrs. Subse- quently, trypsin was added again for another 4 hrs. Finally, digested peptide samples were dried and stored at -20˚C until further MS analyses. LC-MS analysis. Peptide samples were reconstituted in 5.0 μl 30% formic acid (FA) con- taining 10 fmol each of 4 synthetic standard peptides and diluted with 40 μl mobile phase A (98% H2O, 2% ACN, 0.1% FA), as described previously [22, 23]. LC-MS/MS analyses were conducted using a Dionex Ultimate 3000 nano LC-system coupled to a QExactive orbitrap mass spectrometer (Thermo Fisher Scientific, Austria). Fluorescence-activated cell sorting (FACS, analysis of the cell cycle and γH2AX analysis) Cell-cycle analyses were performed with a propidium iodide flow cytometry kit (ABCam, 139418, Cambridge, UK) according to the instructions of the manufacturer. A H2AX 5 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Mobile phone specific EMF and DNA damage phosphorylation assay kit (Millipore, 17–344, Vienna, Austria) was used to analyze DNA dam- age by flow cytometry. At least 10,000 stained cells were analyzed per experimental point in a FACS BD LSR II (Becton Dickinson, Schwechat, Austria) with FACS Diva Software (Becton Dickinson Biosciences, Schwechat, Austria); the flow cytometry gates remained "fixed" in each experimental series according to the controls for each experimental series. All experiments were performed in triplicate. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Mobile phone specific EMF and DNA damage peptide tolerance of 25 ppm, a minimum of two peptide identifications per protein, at least one of them unique as well as a maximum of 2 missed cleavages were included as search criteria. Furthermore, carbamidomethylation on cysteines was set as fixed modification and methi- onine oxidation as well as N-terminal protein acetylation as variable modifications. Match between runs was performed using a 5 min match time window and a 15 min alignment time window. All identified peptides and proteins are meeting an FDR < 0.01. Statistical analysis was per- formed using Perseus version 1.3.0.4 [25]. Therefore, proteins were first filtered for contami- nants, reversed sequences and a minimum of three independent identifications per protein. A two-sided t-test with a significance threshold of p < 0.05 was applied to determine significantly up- and down-regulated proteins between control and treated samples. Evaluation of the samples To ensure blinded evaluation a computer program was used, which generated randomized radiation of the cells. The information concerning the exposure of the cultures was only avail- able to an operator located at the IT’IS Foundation in Zurich. The slides of comet assays were evaluated by an experienced scorer and were cross-checked by a second scorer. The raw data were collected by a scientist who supervised the study and who received also information concerning the treatment of the cultures (via e-mail). To ensure adequate handling, the mails were opened in presence of an external controller (not involved in the project), then both data sets (scoring results and information concerning radiation) were immediately forwarded to a statistician. An identical strategy was used in the proteomics and in the γH2AX experiments. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Proteome analysis 5.0 μl of the peptide samples were pre- concentrated on a 2 cm x 75 μm C18 Pepmap100 pre-column (Thermo Fisher Scientific, Aus- tria) at a flow rate of 10 μl/min using mobile phase A. Afterwards, peptides were eluted from the pre-column to a 50 cm x 75 μm Pepmap100 analytical column (Thermo Fisher Scientific, Austria) at a flow rate of 300 nl/min. Chromatographic separation was accomplished using a gradient from 8% to 40% mobile phase B (80% ACN, 20% H2O, 0.1% FA) over 95 min. All samples were analyzed in technical duplicates. MS scans were achieved in the range from m/z 400–1400 at a resolution of 70,000 (at m/z = 200), whereas MS/MS scans of the 8 most abundant ions were performed using HCD fragmentation at 30% normalized collision energy and analyzed in the orbitrap at a resolution of 17,500 (at m/z = 200). LC-MS data analysis. MaxQuant 1.3.0.5 running the Andromeda search engine and searching against the SwissProt Database (Version 240314 with 16 717 entries) was used for protein identification as well as label-free quantitative (LFQ) data analysis [24]. An allowed PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 6 / 17 γH2AX assays γH2AX assays were performed to study the induction of DSBs. It can be seen in Table 1 that no significant increases were obtained. Genotoxic responses of different cell lines The results of screening experiments with the different cell lines exposed to a SAR of 1.0 W/kg in presence or absence of hydrogen peroxide (H2O2) are summarized in Fig 1A and 1B. Exper- iments which were performed with lower doses with selected lines are listed in S1 Table. No significant induction of DNA migration was observed after exposure to RF under all experi- mental conditions. However, an increased level of DNA damage was seen with all neural tis- sue-derived cell lines after exposure to RF fields; the most pronounced increase in% tail DNA was found with two glioblastoma cell lines U251 and U87 (Fig 1A). Combined exposure to RF fields and H2O2 was also assessed in all cell lines; the results are summarized in Fig 1B. Expo- sure to the peroxide caused significant DNA damage in all cell types, but no further increase of DNA damage was detected in RF-exposed cells. The effect of RF-exposure on the vitality of the cells was monitored in all experiments with trypan blue and no cytotoxic effects were detected. Statistical evaluation Each SAR intensity was tested in three independent experiments. In each experiment, six cul- tures were set up. Three of them were treated with RF and three were sham exposed. From each culture, one slide was prepared. Sample size determination was based on the requirement that a 50% increase of tail intensity should be detected at a two-sided significance level of 5% with a power of 80%. Three experiments with three cultures per experiment are necessary to fulfill this requirement. From each culture one slide was made and from each slide 50 cells were evaluated in comet assays by use of a computer-aided image analysis system (Comet IV, Perceptive Instruments Ltd., Haverhill, UK). The percentage of DNA in tail (tail intensity) was determined and stored in a computer file separately for each slide. Hedgehogs were excluded from the evaluation. The data from each file were then automatically extracted and the medians of the tail intensities computed for the 50 cells of each slide. The percentage values of DNA in tail were arcsine transformed (arcsin ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ð% DNA in tail=100Þ p ) to remove correlations between means and variances. It is well known that means and vari- ances of tail intensities are correlated [11] which violates a requirement of analysis of variance (ANOVA). The arcsine transformation is efficient in removing this correlation [26]. These data were then evaluated by ANOVA using Statistica 12.0 (StatSoft Inc, Tulsa, OK, USA) including a random factor (experimental repetition) and factors for intensity (different levels of SAR) and exposure (sham/real exposure). Comparisons between real and sham exposed slides were done by linear contrasts. Assumptions of homogeneity of variances were tested by Levene’s tests and assumptions of normality of residuals by Kolmogorov-Smirnov tests with Lilliefors’ corrected p-values. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 7 / 17 Mobile phone specific EMF and DNA damage Data from the cell cycle and γH2AX experiments were analyzed using ANOVA and linear contrasts. For all statistical tests p-values below 0.05 were considered significant. Impact of serum deprivation on the sensitivity of selected cell lines The most pronounced (although statistically non-significant) increase in% tail DNA was observed after RF-exposure in lines U87 and U251. These cell types were studied in further experiments. It is known that the sensitivity of cells towards ionizing radiation can be increased by serum deprivation [27]. Therefore, we monitored in subsequent experiments the impact of different RF doses on induction of DNA damage in U87 and U251 cells cultivated in absence of serum. Fig 2 shows that serum deprivation had a strong impact on the cell cycle; i.e. when the cells were cultivated with serum, 60–70% were in G0/G1 and 20–40% in S-phase, without serum for 120 hrs, the majority (80%) was in G0/G1 and only 8% were in S-phase (Fig 2). RF exposure for 16 hrs had no effect on the cell cycle kinetics of both cell lines (see Fig 2). When the cells were exposed after serum deprivation to RF-EMF, significant induction of% tail DNA was observed in line U87 while no such effect was detected in U251 (Fig 3A and 3B). The vitality of the cells was in all experimental series above 78% (see S1 Fig); therefore, it can be excluded that cytotoxic effects had an impact on the results of the SCGE experiments. Fig 3C shows that the “comets” induced in cell line U87 after serum deprivation disap- peared after one or two hours, when the cells were re-cultivated in serum supplemented medium. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Induction of BER and NER In further experiments which were conducted under identical conditions as the standard SCGE experiments, the impact of RF-exposure on DNA-repair was investigated with a modi- fied version of the comet assay enabling to monitor the activities of NER and BER. The results are shown in Fig 4A–4D. In U87, induction of NER was detected with all SAR intensities (Fig 4A) while U251 was less sensitive (only at the highest dose, a moderate effect was detected). PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 8 / 17 Mobile phone specific EMF and DNA damage ps://doi.org/10.1371/journal.pone.0193677 April 12, 2018 9 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 9 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 9 / 17 Mobile phone specific EMF and DNA damage Fig 1. A and B Impact of RF-exposure on% tail DNA in different human derived cell lines. The cells were exposed to RF (Specific Absorption Rate 1.0 W/kg) for 16 hrs. Subsequently, some of the cultures were treated with H2O2 (30 μM) for 10 min as a positive control (Fig 1B). % Tail DNA was monitored in SCGE experiments under standard conditions as described in materials and methods. Bars represent means ± SEM of results obtained in three independent experiments. (Six cultures were prepared per experimental point, three were treated with RF and three were sham exposed. From each culture, one slide was made and 50 cells were evaluated per slide). White bars represent sham exposed cells (controls), grey bars exposed cells. Statistical comparisons were performed by linear contrast after analysis of variance. https://doi.org/10.1371/journal.pone.0193677.g001 https://doi.org/10.1371/journal.pone.0193677.g001 No effects were observed with the latter cell line U251 in BER measurements, but a marginal effect was found in U87 cells with the highest dose. Mobile phone specific EMF and DNA damage Fig 3. A-C Impact of RF exposure on DNA stability and vitality of human derived glioblastoma cell lines. The cells were cultivated in presence or absence of serum and exposed to different RF intensities (0.25, 0.5 and 1.0 W/kg) over a period of 16 hrs. Subsequently, DNA damage was monitored in SCGE experiments under standard conditions (Fig 3A and 3B). Fig 3C shows the disappearance of% tail DNA in U87 cells after RF exposure under serum free conditions and recultivation in serum supplemented medium. The cells were treated with 1.0 W/kg; subsequently, they were cultivated in medium with serum up to two hours. Bars represent means ± SEM of three independent experiments (Six cultures were prepared per experimental point, three were treated with RF and three were sham exposed. From each culture, one slide was made and 50 cells were evaluated per slide). Stars indicate statistical significance (p < 0.05). White bars represent sham exposed cells (controls), grey bars exposed cells. Statistical comparisons were performed by linear contrast after analysis of variance. https://doi.org/10.1371/journal.pone.0193677.g003 Fig 3. A-C Impact of RF exposure on DNA stability and vitality of human derived glioblastoma cell lines. The cells were cultivated in presence or absence of serum and exposed to different RF intensities (0.25, 0.5 and 1.0 W/kg) over a period of 16 hrs. Subsequently, DNA damage was monitored in SCGE experiments under standard conditions (Fig 3A and 3B). Fig 3C shows the disappearance of% tail DNA in U87 cells after RF exposure under serum free conditions and recultivation in serum supplemented medium. The cells were treated with 1.0 W/kg; subsequently, they were cultivated in medium with serum up to two hours. Bars represent means ± SEM of three independent experiments (Six cultures were prepared per experimental point, three were treated with RF and three were sham exposed. From each culture, one slide was made and 50 cells were evaluated per slide). Stars indicate statistical significance (p < 0.05). White bars represent sham exposed cells (controls), grey bars exposed cells. Statistical comparisons were performed by linear contrast after analysis of variance. https://doi.org/10.1371/journal.pone.0193677.g003 expression levels of 72 proteins were altered. 16 proteins were up-regulated and 56 were down-regulated, respectively (Fig 5A, S2 Table). Fig 5B shows the significant induction of sev- eral proteins related to NER. Furthermore, several proteins associated with the γ-interferon pathway were up-regulated (one of them significant) after RF-exposure (Fig 5C). Proteome analysis To investigate the impact of RF-exposure on the expression of proteins associated with DNA stability, a mass spectrometry-based proteomic analysis was conducted with U87 cells exposed at a SAR of 1.0 W/kg for 16 hrs. The results are summarized in Fig 5A. In total, 3955 proteins were analyzed. A two-sided t-test between control and RF-exposed cells revealed that the Fig 2. A-D Impact of the cultivation conditions and RF-exposure on the cell cycle. The cells were grown in presence and absence of serum and exposed to different RF intensities (Specific Absorption Rates 0.25, 0.5 and 1.0 W/kg) over a period of 16 hrs before they were analyzed by FACS. Cells in G0/G1, S and G2/M phase were sorted on the basis of their DNA contents. 10 000 cells were analyzed per experimental point and all experiments were performed in triplicate. https://doi org/10 1371/journal pone 0193677 g002 Fig 2. A-D Impact of the cultivation conditions and RF-exposure on the cell cycle. The cells were grown in presence and absence of serum and exposed to different RF intensities (Specific Absorption Rates 0.25, 0.5 and 1.0 W/kg) over a period of 16 hrs before they were analyzed by FACS. Cells in G0/G1, S and G2/M phase were sorted on the basis of their DNA contents. 10 000 cells were analyzed per experimental point and all experiments were performed in triplicate. https://doi.org/10.1371/journal.pone.0193677.g002 Fig 2. A-D Impact of the cultivation conditions and RF-exposure on the cell cycle. The cells were grown in presence and absence of serum and exposed to different RF intensities (Specific Absorption Rates 0.25, 0.5 and 1.0 W/kg) over a period of 16 hrs before they were analyzed by FACS. Cells in G0/G1, S and G2/M phase were sorted on the basis of their DNA contents. 10 000 cells were analyzed per experimental point and all experiments were performed in triplicate. https://doi.org/10.1371/journal.pone.0193677.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 10 / 17 Mobile phone specific EMF and DNA damage Table 1. Impact of RF on γH2AX phosphorylation in human glioblastoma cell lines1. Cell line SAR (W/kg) with serum without serum (mean ± SEM) (mean ± SEM) Sham exposed2 Exposed p-value Sham exposed2 Exposed p-value U87 0.25 1.69±0.28 2.14±0.13 0.452 2.49±0.18 2.61±0.15 0.834 0.5 1.38±0.41 1.39±0.52 0.982 1.83±0.49 1.95±0.41 0.838 1 2.19±0.13 2.13±0.12 0.914 1.87±0.54 2.12±0.33 0.675 U251 0.25 0.83±0.34 1.03±0.11 0.742 1.37±0.63 1.72±0.49 0.556 0.5 1.31±0.44 1.06±0.20 0.704 2.19±0.41 1.99±0.55 0.742 1 1.42±0.37 1.54±0.77 0.847 1.31±0.59 1.98±0.39 0.257 Table 1. Impact of RF on γH2AX phosphorylation in human glioblastoma cell lines1. 1 The cells were grown either in serum supplemented medium or in absence of serum. Induction of DNA breaks was monitored in γH2AX experiments (which reflect double strand breaks) after exposure to different specific absorption rates (SAR) for 16 hrs. Values indicate means ± SEM of results obtained in three independent experiments (four cultures were set per experimental point, two were treated with RF and two were sham exposed). Data were analyzed using ANOVA and linear contrasts. For all statistical tests p-values below 0.05 were considered significant. 2 Sham exposed cells (controls). 1 The cells were grown either in serum supplemented medium or in absence of serum. Induction of DNA breaks was monitored in γH2AX experiments (which reflect double strand breaks) after exposure to different specific absorption rates (SAR) for 16 hrs. Values indicate means ± SEM of results obtained in three independent experiments (four cultures were set per experimental point, two were treated with RF and two were sham exposed). Data were analyzed using ANOVA and linear contrasts. For all statistical tests p-values below 0.05 were considered significant. 2 Sham exposed cells (controls). https://doi.org/10.1371/journal.pone.0193677.t001 https://doi.org/10.1371/journal.pone.0193677.t001 DNA damage under “normal” cultivation conditions in any of the ten cell types investigated. However, evidence for formation of comets was found in a glioblastoma cell line under specific experimental conditions (i.e. when cells were cultivated in absence of serum); furthermore, we found clear evidence for induction of a specific form of DNA repair (NER) in this cell line. As mentioned above, only few studies have been published so far about the effects of the UMTS signal on DNA stability. In three former investigations, SCGE experiments were con- ducted with lymphocytes from healthy volunteers with consistently negative results (for review see IARC [1] and D Manna, R Ghosh [28]). Discussion The present investigation concerns the impact of the UMTS-signal on genomic stability in human derived cells from different organs. Overall, our findings show that this signal presently widely used for mobile telecommunications worldwide does not cause significant induction of PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 11 / 17 1 The cells were grown either in serum supplemented medium or in absence of serum. Induction of DNA breaks was monitored in γH2AX experiments (which reflect double strand breaks) after exposure to different specific absorption rates (SAR) for 16 hrs. Values indicate means ± SEM of results obtained in three independent experiments (four cultures were set per experimental point, two were treated with RF and two were sham exposed). Data were analyzed using ANOVA and linear contrasts. For all statistical tests p-values below 0.05 were considered significant. 2 Sham exposed cells (controls). PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Mobile phone specific EMF and DNA damage hi h b it f RNA l II [38] ll DNA li ti f t [39 40] d Fig 4. A-D Measurement of the NER and BER activity in glioblastoma cells after exposure to different RF intensities (Specific Absorption Rate 0.25 W/kg—1 W/ kg) for 16 hrs. Bars indicate means ± SEM of results obtained in the three independent experiments. (Six cultures were prepared per experimental point, three were treated with RF and three were sham exposed. From each culture, one slide was made and 50 cells were evaluated per slide). Left bars show results of control experiments, which were performed in parallel (for details see also Collins and Dusinska [48]). Striped bars show results obtained with isolated nuclei which were exposed to buffer only, hatched bars show results with nuclei which were treated either with UV (for determination of NER) or with Ro-19-8022 (determination of BER). Subsequently, the nuclei of the cells were incubated with T4 endonuclease V (for NER measurements) or FPG (for BER measurements). Other bars show results of experiments in which cells were treated either with UV (determination NER) or with Ro-19-8022 (determination BER) and subsequently with cytosols of sham exposed (white bars) or RF treated (grey bars) glioblastoma cells. Stars indicate significance (p < 0.05). Statistical comparisons were performed by linear contrasts after analysis of variance. https://doi.org/10.1371/journal.pone.0193677.g004 Fi 4 A D M f h NER d BER i i i li bl ll f diff RF i i i (S ifi Ab i R 0 25 W/k 1 W/ Fig 4. A-D Measurement of the NER and BER activity in glioblastoma cells after exposure to different RF intensities (Specific Absorption Rate 0.25 W/kg—1 W/ kg) for 16 hrs. Bars indicate means ± SEM of results obtained in the three independent experiments. (Six cultures were prepared per experimental point, three were treated with RF and three were sham exposed. From each culture, one slide was made and 50 cells were evaluated per slide). Left bars show results of control experiments, which were performed in parallel (for details see also Collins and Dusinska [48]). Striped bars show results obtained with isolated nuclei which were exposed to buffer only, hatched bars show results with nuclei which were treated either with UV (for determination of NER) or with Ro-19-8022 (determination of BER). Furthermore, one article with the human primary fibroblast line ES-1 was published; the experiments were conducted under the same experi- mental conditions used in the present study [29]. The reliability of these findings was contro- versially discussed and criticized [30]; furthermore, a German group failed to reproduce these results [31]. Although we found significant but less pronounced increases than Schwarz et al. [29] in a previous experiment applying the same cell line ES-1, in the present experiments three years later the cell line showed no responsiveness to the UMTS signal in accordance with Speit et al. [31]. Induction of DNA damage was observed in the present study only in the U87 glioblastoma line when the cells were cultivated in absence of serum, which leads to cell cycle arrest. In this context, it is notable that earlier experiments with ionizing radiation found that human cells are more sensitive when arrested in G0 phase [32]. This phenomenon was explained by lower levels of protective sulfhydryl compounds in the cytosol at this stage of the cell cycle [27]. The induction of DNA damage which was seen under these conditions may be related to the p53 status of the cells as significant increase of DNA damage was only detected in the p53 profi- cient line U87. In this context it is notable, that dose dependent induction of NER studied applying a modified protocol of the SCGE assay was only observed in this line. It is well docu- mented that the expression of wild type p53 is involved in the activation of NER [33, 34, 35, 36]. The results of the proteome analysis support the assumption that NER is induced by the UMTS signal as they show that several proteins involved in this repair system are up-regulated as a consequence of RF exposure under serum-free conditions in U87 cells (Fig 5B), namely, ERCC2 a protein belonging the excision-repair cross-complementing family, which is required for transcription initiation by RNA polymerase II [37], two proteins (RPB11-a and RPB-3), PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 12 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 Mobile phone specific EMF and DNA damage Fig 5. A-C Results of proteomic analysis of RF exposed glioblastoma cell line U87. The cells were exposed to RF for 16 hrs (Specific Absorption Rate 1.0 W/kg). The distribution of up- and down-regulated proteins is shown in a volcano plot (Fig 5A). For each identified protein, the fold-change on a logarithmic scale to the basis of 2 (ln2Δt-test) and the corresponding p-value (-log p-value) are indicated. Label-free quantification intensities of proteins related to NER activity are shown in Fig 5B and of proteins related to the γ-interferon pathway in Fig 5C. White bars represent sham exposed cells, and grey bars the exposed cells. Stars indicate statistical significance (p < 0.05). htt //d i /10 1371/j l 0193677 005 Fig 5. A-C Results of proteomic analysis of RF exposed glioblastoma cell line U87. The cells were exposed to RF for 16 hrs (Specific Absorption Rate 1.0 W/kg). The distribution of up- and down-regulated proteins is shown in a volcano plot (Fig 5A). For each identified protein, the fold-change on a logarithmic scale to the basis of 2 (ln2Δt-test) and the corresponding p-value (-log p-value) are indicated. Label-free quantification intensities of proteins related to NER activity are shown in Fig 5B and of proteins related to the γ-interferon pathway in Fig 5C. White bars represent sham exposed cells, and grey bars the exposed cells. Stars indicate statistical significance (p < 0.05). https://doi.org/10.1371/journal.pone.0193677.g005 Taken together, the present findings show that UMTS-modulated RF fields did not cause induction of DNA damage in 10 cell lines in either the presence or absence of H2O2. Only the U87 cell line under serum-free conditions demonstrated increased DNA damage in response to RF field exposure, where induction of NER appears to be involved. Experiments are in prog- ress to clarify if the activation of NER plays a causal role in regard to synergistic/antagonistic effects which were seen in combined treatment experiments with mobile phone specific radia- tion and chemicals [45, 46, 47]. Subsequently, the nuclei of the cells were incubated with T4 endonuclease V (for NER measurements) or FPG (for BER measurements). Other bars show results of experiments in which cells were treated either with UV (determination NER) or with Ro-19-8022 (determination BER) and subsequently with cytosols of sham exposed (white bars) or RF treated (grey bars) glioblastoma cells. Stars indicate significance (p < 0.05). Statistical comparisons were performed by linear contrasts after analysis of variance. https://doi.org/10.1371/journal.pone.0193677.g004 which are subunits of RNA polymerase II [38], as well as a DNA replication factor [39, 40] and a subunit of a transcription factor, which are both involved in NER [41, 42]. As shown in Fig 5C we found also limited evidence for activation of the interferon-γ-pathway, i.e. we detected significant up-regulation of the HLA class II histocompatibility antigen (HLA-DRB1), which may mediate cytokine production [43], additionally also some other proteins (B2M, IRF 3, Yes1) were activated which are involved in this pathway, but these latter effects were statisti- cally not significant. The findings of the γH2AX experiments (Table 1) and also the results of our former MN study with the glioblastoma lines U87 and U251 indicate that the UMTS signal does not cause DSBs and MN which reflect (persisting) chromosomal damage [44]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193677 April 12, 2018 13 / 17 S2 Table. List of proteins which were altered significantly after exposure to RF in addition to NER-associated proteins. (DOCX) S2 Table. List of proteins which were altered significantly after exposure to RF in addition to NER-associated proteins. (DOCX) S1 Fig. Impact of radiation exposure on the vitality of the cells which was analyzed with trypan blue exclusion technique. Bars show means ±SD. The experimental setup is described in detail in the legend of Fig 3. (TIF) Author Contributions Conceptualization: Halh Al-Serori, Michael Kundi, Siegfried Knasmu¨ller. Data curation: Manuel Murbach. Formal analysis: Michael Kundi, Miroslav Misˇı´k. Investigation: Halh Al-Serori, Franziska Ferk, Andrea Bileck, Christopher Gerner, Armen Nersesyan, Monika Waldherr. Methodology: Halh Al-Serori, Franziska Ferk, Michael Kundi, Tamara T. Lah, Andrew R. Collins, Siegfried Knasmu¨ller. Project administration: Siegfried Knasmu¨ller. Resources: Tamara T. Lah, Christel Herold-Mende. Supervision: Siegfried Knasmu¨ller. Visualization: Michael Kundi, Miroslav Misˇı´k. Writing – original draft: Halh Al-Serori, Siegfried Knasmu¨ller. Conceptualization: Halh Al-Serori, Michael Kundi, Siegfried Knasmu¨ller. Conceptualization: Halh Al-Serori, Michael Kundi, Siegfried Knasmu¨ller. Data curation: Manuel Murbach. Formal analysis: Michael Kundi, Miroslav Misˇı´k. Investigation: Halh Al-Serori, Franziska Ferk, Andrea Bileck, Christopher Gerner, Armen Nersesyan, Monika Waldherr. Methodology: Halh Al-Serori, Franziska Ferk, Michael Kundi, Tamara T. Lah, Andrew R. Collins, Siegfried Knasmu¨ller. Project administration: Siegfried Knasmu¨ller. Resources: Tamara T. Lah, Christel Herold-Mende. Supervision: Siegfried Knasmu¨ller. Visualization: Michael Kundi, Miroslav Misˇı´k. Writing – original draft: Halh Al-Serori, Siegfried Knasmu¨ller. Writing – review & editing: Michael Kundi, Tamara T. Lah, Andrew R. Collins, Siegfried Knasmu¨ller. Supporting information S1 Table. Impact of RF-exposure on comet formation in different human glioblastoma cell lines. (DOCX) S1 Table. Impact of RF-exposure on comet formation in different human glioblastoma cell lines S1 Table. 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https://openalex.org/W2989813375	https://europepmc.org/articles/pmc6869181?pdf=render	English	null	The preliminary results of proton and carbon ion therapy for chordoma and chondrosarcoma of the skull base and cervical spine	Radiation oncology	2,019	cc-by	6,171	© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Keywords: Chordoma, Chondrosarcoma, Proton, Carbon ion, Radiotherapy Keywords: Chordoma, Chondrosarcoma, Proton, Carbon ion, Radiotherapy bone tumors with 5–12% occurring in the head and neck, mostly at the skull base [3]. The preliminary results of proton and carbon ion therapy for chordoma and chondrosarcoma of the skull base and cervical spine Xiyin Guan1,2, Jing Gao1,2, Jiyi Hu1,2, Weixu Hu1,2, Jing Yang1,2, Xianxin Qiu1,2, Chaosu Hu2,3, Lin Kong2,3* and Jiade J. Lu1,2* Abstract Purpose: To evaluate the short-term outcomes in terms of tumor control and toxicity of patients with skull base or cervical spine chordoma and chondrosarcoma treated with intensity-modulated proton or carbon-ion radiation therapy. Methods: Between 6/2014 and 7/2018, a total of 91 patients were treated in our Center. The median age was 38 (range, 4–70) years. Forty-six (50.5%) patients were treated definitively for their conditions as initial diagnosis, 45 (49.5%) patients had recurrent tumors including 14 had prior radiotherapy. The median gross tumor volume was 37.0 (range, 1.6–231.7) cc. Eight patients received proton therapy alone, 28 patients received combined proton and carbon ion therapy, 55 patients received carbon-ion therapy alone. Results: With a median follow-up time of 28 (range, 8–59) months, the 2-year local control (LC), progression free (PFS) and overall survival (OS) rates was 86.2, 76.8, and 87.2%, respectively. Those rates for patients received definitive proton or carbon-ion therapy were 86.7, 82.8, and 93.8%, respectively. On multivariate analyses, tumor volume of > 60 cc was the only significant factor for predicting PFS (p = 0.045), while re-irradiation (p = 0.012) and tumor volume (> vs < 60 cc) (p = 0.005) were significant prognosticators for OS. Grade 1–2 late toxicities were observed in 11 patients, and one patient developed Grade 3 acute mucositis. Conclusions: Larger tumor volume and re-irradiation were related to inferior survival for this group of patients. Further follow-up is needed for long-term efficacy and safety. Guan et al. Radiation Oncology (2019) 14:206 https://doi.org/10.1186/s13014-019-1407-9 Guan et al. Radiation Oncology (2019) 14:206 https://doi.org/10.1186/s13014-019-1407-9 Introduction Chordomas originate from transformed undifferentiated notochordal remnants that exist throughout the skull base and axial skeleton. The incidence of clival chordo- mas is approximately 0.8–1/106 [1, 2]. Chondrosarcomas arise from cartilage and represent 10–20% of malignant Both chordomas and chondrosarcomas are locally in- vasive, and surgery is their mainstay treatment. However, complete resection is nearly universally constrained for the skull base lesions by their proximity to critical neural or vascular structures [4]. Adjuvant radiation therapy (RT) can improve local control (LC) and overall survival (OS). A systematic review reported that the 5-year mor- tality rate decreased from 25 to 9% with the addition of any form of RT [3]. Nevertheless, high dose RT is usu- ally not feasible due to the dose constrain of the critical organs at risk (OARs) particularly the optic nerve/ * Correspondence: jiade.lu@sphic.org.cn; lin.kong@sphic.org.cn 2 * Correspondence: jiade.lu@sphic.org.cn; lin.kong@sphic.org.cn 2Department of Radiation Oncology, Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, 4365 Kangxin Road, Shanghai 201315, China 1 Correspondence: jiade.lu@sphic.org.cn; lin.kong@sphic.org.cn 2Department of Radiation Oncology, Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, 4365 Kangxin Road, Shanghai 201315, China 1 1Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, 4365 Kangxin Road, Shanghai 201315, China Full list of author information is available at the end of the article Intensity modulated particle radiation therapy Treatment planning for IMPT or MICT was performed using the Siemens Syngo® treatment planning system (version VC11). The beam arrangement varied depend- ing on the target volume geometry and dose limits to neighboring organs at risk, such as those with prior radi- ation exposure. Treatments typically consisted of 2–5 beams with a median of 3 fields. Individual factors such as patient positioning reproducibility and/or beam angles were chosen for optimal dosimetry. Immobilization and registration All patients were registered and immobilized in the su- pine position with individualized thermoplastic masks. Planning CT scans without intravenous contrast from the vertex to the inferior margin of the clavicular heads were performed at 1 mm slice thickness. MRI-CT fusion was performed for all patients prior to target volume de- lineation. The gross tumor volume (GTV) consisted of the gross tumor discovered on clinical examination or imaging studies. We defined CTV-boost as a GTV with a 1–3 mm margin to deliver the prescribed dose to the tumor. The clinical target volume (CTV) included the GTV and suspected subclinical disease based on clinical risk estimation. A maximum of a 5 mm margin was typ- ically added to the CTV for the planning target volume Guan et al. Radiation Oncology (2019) 14:206 Page 2 of 9 (PTV) to mitigate potential setup errors and uncertain- ties in the geometric precision of the dose distribution. chiasm, brain stem, spinal cord, and brain, if conven- tional photon-based RT technology is used. As such, long-term LC and OS remained suboptimal with the addition of adjuvant RT despite of the relatively indolent behavior of the conditions. Patients and methods Pretreatment evaluation Patient evaluation included a complete history and phys- ical examination (H&P), complete blood count and elec- trolyte panel, renal and liver function tests, magnetic resonance imaging (MRI) of the head and neck region (CT was allowed when MRI was contraindicated), posi- tron emission tomography (PET-CT) when appropriate. All cases were discussed in our institutional multidiscip- linary tumor clinic for their indication for particle RT prior to registration and inclusion to the tumor registry. Setup accuracy was confirmed with daily orthogonal X-ray using bony landmarks as a reference. Verification CT scans were typically performed on a weekly basis after the second week of the IMCT course to assess any changes in anatomy. Recalculation was required if the coverage of the CTV is substantially altered, based on the assessment of the attending radiation oncologist. Dose prescription and delivery p p y Doses were measured by Gy-equivalents (GyE) to ac- count for the RBE differences of particle RT as com- pared to photon-based RT. IMPT and IMCT were delivered with PBS technology. For IMPT plan, as we using conventional fractionation,the dose constraints of the OARs were based on the TD5/5 described by Emami et al. [8] However, hypo-perfraction of 2.5–3.0GyE was used in IMCT plan. Due to the lack of experience, extra caution must be applied. The critical organs including the optic nerve (D20 < 30 GyE), brain stem (Dmax< 45 GyE), spinal cord (Dmax< 30 GyE), and temporal lobes (V40 < 7.66 cc; V50 < 4.66 cc) were based on previous ex- perience from the National Institute of Radiation Science (NIRS) of Japan [9]. For patients who were irradiated previously, recovery from previous radiation therapy dose was set at 70% regardless of the latent time be- tween the two courses of RT [10]. Proton and carbon ion beams have the physical advan- tage of a finite range in depth with a steep dose fall-off after the Bragg peak. In addition, compared to photon and pro- ton beams, relative biological effectiveness (RBE) calcula- tion results of about 3 for carbon ion beams within the target volume,which may theoretically has the enhanced advantage of biological advantages [5]. The effectiveness of particle radiation therapy for both chordoma and chondro- sarcoma have been reported [4, 6]. Furthermore, dosimetry studies has confirmed that the use intensity-modulated pro- ton (IMPT) and carbon ion radiotherapy (IMCT) with pen- cil beam scanning (PBS) technology could improve target volume coverage while minimizing the dose to the sur- rounding OARs thereby improving the therapeutic ratio for base of skull tumors [7]. In this paper, we try to bolster the literature with the results of a group of patients with skull base/cervical spine chordoma and chondrosarcoma treated with IMPT and IMCT at the Shanghai Proton and Heavy Ion Center (SPHIC). Follow-up All patients were required to adhere to the standardized institutional follow-up protocol. The first follow-up was provided at 4–6 weeks after the completion of particle RT. Patients were then required to be follow-up every 3 months in the first 2 years, every 6 months in the follow- ing 3 years, and annually thereafter. A complete H&P examination focused to the head and neck regions, and an enhanced MRI scan of the head and neck area are re- quired at each follow-up. PET-CT, other laboratory, or imaging studies were ordered based on any evidence of metastasis or other concurrent diseases. Page 3 of 9 Guan et al. Radiation Oncology (2019) 14:206 Guan et al. Radiation Oncology (2019) 14:206 Table 1 Patient characteristics Parameter Total N = 91(%) Age (years)/ Median (range) 38(14–76) Gender Female 40(44.0) Male 51(56.0) Histology Chordoma 77(84.6) Chondrosarcoma 14(15.4) Tumor site Skull base 85(93.4) Cervical spinal 6(6.6) KPS scores 100 26(19.8) 90 47(51.6) 80 18(19.8) GTV/Median (range, cc) 37.0(1.6–231.7) ≤60 cc 62(68.1) >60 cc 29(31.9) Brainstem compression No 32(35.2) Abutment 14(15.4) Yes 45(49.5) Optic apparatus compression No 51(56) Yes 40(44) Any compression No 22(24.2) Yes 69(75.8) Symptom headache 45(49.5) cranial nerve injury 68(74.7) endocrine dyscrasia 8(8.8) Recurrent disease No 46(50.5) Yes 45(49.5) Re-radiotherapy No 77(84.6) Yes 14(15.4) Number of surgeries 1 49(53.8) 2 31(34.1) ≥3 11(12.1) Data analysis Table 1 Patient characteristics y The duration of survival was calculated from the diagnosis of the current condition (first diagnosis or recurrence), until death or the date of the last follow-up. The time to local, regional, distant failure or progression was measured from the date of any treatment for the current diagnosis until documented treatment failure or progression. Free- dom from failure and OS rates were calculated using the Kaplan-Meier method. Tumor response was determined as a maximum response on follow-up MRI according to the RECIST criteria. Log-rank test was used for univariate analysis to compare the differences of the survival prob- abilities. Multivariate analysis using a Cox regression model was performed to define significant prognostic fac- tors. All analyses were performed in SPSS statistics version 24.0 software package (Chicago, IL USA). Acute adverse events were scored using the Common Terminology Criteria for Adverse Events (CTC AE) ver- sion 4.03, and included the adverse events that occurred during or within 3 months after the initiation of particle RT. Late toxicities were scored using the Radiation Therapy Oncology Group (RTOG) late radiation mor- bidity scoring system for toxicities observed beginning at 90 days after completion of particle RT. Patients and particle RT Ninety-one consecutive and non-selected patients with cervical and skull base chordomas or chondrosarcomas treated at SPHIC with IMPT or IMCT between 6/2014 and 7/2018 were included in the current analyses. Four- teen (15.4%) patients who had previous photon-based RT (gamma-knife = 7, cyber-knife = 2, or conventional RT = 5) received salvage particle treatment. The remaining 77 pa- tients received particle RT with definitive indication, and most were treated in phase 1 (dose escalation) or phase 2 clinical trials or according to our institutional treatment protocol. The characteristics of patients, their conditions, and treatment were detailed in Table 1. Adverse effects A total of 25 (27.5%) patients developed Grade 1–2 oral mucositis, including 10 (11.9%) Grade 2 mucositis. Only one patient developed Grade 3 acute mucositis. Eight (20.2%) patients experienced grade 1 skin reaction and 9 (9.9%) with focal hair loss. Other acute toxicities included grade 1 myelosuppression in 6 patients, and grade 1 hear- ing loss in 5 patients. Late toxicities of grade 1 or 2 were observed in 19 pa- tients: 9 with hearing loss, 5 with xerostomia, 1 with dys- phagia, another with reduced prolactin, and 3 with unilateral white matter changes in the temporal lobe (1 also experienced memory loss). No patient experienced severe toxicities of grade 3 or above. Fig. 1 Kaplan Meier survival curves for local control (LC), progression free survival (PFS), and overall survival (OS) rates of the 91 patients with chordoma and chondrosarcoma of the skull base and cervical spine proton and/or carbon ion therapy at the Shanghai Pro- ton and Heavy Ion Center. A substantial portion of pa- tients presented with locally recurrent disease after surgery or surgery followed by photon radiotherapy. We have discovered that the 2-year os for radiation naïve Prognostic factors Significant predictive factors for OS and PFS (progres- sion free survival) previously reported in the literature for chordoma and chondrosarcoma were assessed in both uni- and multi-variate analyses. Potential prognos- tic factors included age, gender, KPS, histology, presence of cranial nerve injury, radiological critical organ com- pression including brainstem or optic apparatus (with vs. without), GTV volume, as well as technology of particle radiotherapy (IMPT, IMCT, and IMPT + IMCT boost), recurrence status (initial vs. recurrent diagnosis), RT sta- tus (RT-naïve vs. re-RT), and surgery stutus (1 vs. > 1 surgery excluding biopsy). Univariate analysis showed that KPS (p = 0.029), the presence of cranial nerve injury (p = 0.011), re-irradiation (p = 0.015), GTV volume of > 60 cc (p = 0.004), as well as brainstem or optic apparatus compression (p = 0.012) were significant negative prognosticators for PFS. And KPS (p = 0.008), the presence of cranial nerve injury (p = 0.043), re-irradiation (p<0.001), GTV volume of > 60 cc (p<0.001), as well as brainstem or optic apparatus com- pression (p = 0.048) were also significant negative prog- nosticators for OS. (Table 2, Fig. 2). On multivariate analyses, GTV volume of > 60 cc (p = 0.045) was a significant prognosticator for PFS (Table 4), while GTV volume of > 60 cc (p = 0.012) and re- irradiation (p = 0.005) were significant prognosticator for OS (Table 3 and Table 4). Disease control and survival Eight patients received IMPT alone to the standard 70 GyE/35 fractions according to the protocol of the registra- tion trial required by the Chinese FDA. The remaining 69 patients were treated according to our institutional dose escalation schemes: 22 patients received IMRT to 50 GyE/ 25 fractions to CTV 2 plus IMCT boost to the CT-boost to 15~21 GyE/5~8 fractions; 47 patients received 45 GyE/ 15 fractions to the CTV followed to a boost to CTV-boost to the total of 63~69GyE/21~23 fractions. With a median follow-up time of 28 (range 8–59) months, 28 developed progression/recurrence and 14 of them had deceased. Among the 14 patients deceased at the time of this analysis: 12 (7 re-irradiated and 5 RT-naïve) patients died of uncontrolled local disease including 9 without reaching the RECIST criteria of disease progression, and 2 additional RT-naïve patients died of liver metastasis and stroke, respectively. The 2-year local control, progress free, and overall survival rates were 86.2, 76.8, and 87.2%, re- spectively for the entire cohort. The 2-year OS rates for patients after first-time radiation vs. re-irradiation were 93.8 50.3%, respectively (p<0.001). Twenty-eight (28) pa- tients developed progression after the completion of par- ticle RT: 7 experienced local recurrence, 8 had local progression, 1 had metastases to cervical spine, 1 had both For the 14 patients who received re-irradiation, 6 re- ceived proton RT (50GyE/25 fractions) followed by IMCT boost to 15~18GyE/5~6 fractions, and 8 received 57~69 GyE in 19~23 fractions. The dose/fraction scheme was decided based on the previous RT regimen and the limitation of the OARs. Guan et al. Radiation Oncology (2019) 14:206 Guan et al. Radiation Oncology (2019) 14:206 Page 4 of 9 Fig. 1 Kaplan Meier survival curves for local control (LC), progression free survival (PFS), and overall survival (OS) rates of the 91 patients with chordoma and chondrosarcoma of the skull base and cervical spine local progression and metastasis to cervical spine, 1 had liver metastasis, and 9 had uncontrolled local disease (and died) without reaching the RECIST criteria of disease pro- gression. The median time to progression/recurrence was 20.5 (range, 5–48) months. (Fig. 1). Discussion The present study reported the first 91 consecutive and non-selected patients with skull base or cervical spine chordoma and chondrosarcoma patients treated with Guan et al. Radiation Oncology (2019) 14:206 Page 5 of 9 Guan et al. Radiation Oncology Table 2 Univariate analysis of risk factors for progression free survival (PFS) and overall survival (OS) in 91 patients Prognostic factors 2-year PFS (%) [95% CI] P value 2-year OS (%) [95% CI] P value Age (yeaars) ≤38 64.1(7.5) 0.142 81.8(6.3) 0.130 >38 89.6(5.0) 92.5(4.3) Gender Female 82.8(6.6) 0.513 88.9(5.3) 0.686 Male 72.7(6.5) 86.5(5.2) KPS ≥90 80.1(4.8) 0.029 90.8(3.6) 0.008 80 53.0(15.5) 59.7(15.9) Critical organ compression Yes 71.1(5.8) 0.012 83.4(4.9) 0.048 No 95.7(4.3) 100 Cranial nerve injury Yes 71.4(5.8) 0.011 83.5(4.8) 0.043 No 95.7(4.3) 100 Histology Chordoma 73.5(5.5) 0.132 85.9(4.4) 0.340 Chondrosarcoma 92.9(6.9) 92.9(6.9) GTV ≤60 cc 84.9(5.0) 0.004 93.9(3.4) <0.001 >60 cc 59.7(9.5) 73.2(8.8) Recurrent disease Yes 73.8(6.9) 0.828 82.7(6.0) 0.260 No 80.4(6.4) 92.5(4.2) Re-radiotherapy Yes 41.3(15.7) 0.015 50.3(15.5) <0.001 No 82.8(4.6) 93.8(3.0) Surgery times 1 81.1(6.1) 0.609 87.7(5.2) 0.654 ≥2 71.5(7.3) 86.3(5.7) RT Proton 100 0.69 100 0.489 Proton+carbon 74.2(8.4) 84.6(7.1) Carbon 75.6(6.3) 87.3(5.1) RT patients was 93.8%, significantly better than 50.3% of those who failed prior photon radiation. Furthermore, tumor volume (> vs. < 60 cc) was a significant prognosti- cator for PFS, and re-irradiation and tumor volume were significant prognosticators for os. Therefore, adjuvant RT is considered necessary for most if not all patients, and could improve local control espe- cially for patients with residual disease after surgery [3]. cially for patients with residual disease after surgery [3]. Because of their radio-resistance, high doses are needed for successful local control for chordomas and chondrosarcomas. However, the doses/fractions at 70– 74 GyE (1.8-2GyE) or 60.8–67.6 GyE (hypo-fractionation at 3–4.2 GyE) that is considered minimum efficacious doses for local control are difficult to achieve with photon-based RT [6, 13, 14]. Particle RT is featured with its far superior physical properties and enables more fo- cused dose delivery to the tumor volumes thereby im- proves therapeutic ratio especially for tumors positioned near critical OARs. In addition, carbon-ion beam has higher linear-energy transfer (LET) and may inflict higher relative biological effectiveness [5]. As such, pro- ton or carbon-ion RT is more superior RT technique for skull base tumors that requires high-dose irradiation. The outcomes after proton RT had been reported in nu- merous retrospective studies. Discussion The 5-year OS rates ranged between 67%~ 81%, and those of LC rates ranged between 46%~ 81% [15]. Schulz-Ertner et al. of the Heidelberg Ion Therapy Center (HIT) reported their ex- perience of skull base chordomas using the median Chordomas and chondrosarcomas are rare malignan- cies that present multiple management challenges be- cause of their proximity to vital structures and relative resistance to chemotherapy and radiotherapy. Both con- ditions are highly locally invasive and usually occur near critical organs especially the central nervous system (CNS), thus those occurs in the head and neck are often amalgamated because of similar clinical presentation, radiologic finding, anatomic location, and treatment. Maximal safe resection combined with adjuvant RT is a key component of successful management of cervical and skull base chordoma and chondrosarcoma. How- ever, even by experienced teams, the complete resection rate in published studies has ranged from 0 to 60%. In a study of 159 skull base chondrosarcoma patients re- ported by Feuvret et al. [11], the complete resection rate was only 8.2%. Gay et al. [12] attempted radical surgery for this condition. However, only 60% could undergo surgery and 30% experienced cerebrospinal fluid leakage. Guan et al. Radiation Oncology (2019) 14:206 Page 6 of 9 Fig. 2 Overall survival (OS) and progression free survival (PFS) for all patients based on Karnofsky performance status (80 vs. ≥90), the presence of cranial nerve injury, nature of radiotherapy (RT vs. re-RT), tumor volume (> vs. < 60 cc), and the presence of critical organ compression ents based on Karnofsky performance status (80 vs. ≥90), the presence of > vs < 60 cc) and the presence of critical organ compression Fig. 2 Overall survival (OS) and progression free survival (PFS) for all patients based on Karnofsky performance status (80 vs. ≥90), the presence of cranial nerve injury, nature of radiotherapy (RT vs. re-RT), tumor volume (> vs. < 60 cc), and the presence of critical organ compression Guan et al. Discussion Radiation Oncology (2019) 14:206 Page 7 of 9 Table 3 Multivariate analysis of risk factors for progression free survival (PFS) B SE Wald df significance Exp(B) KPS −.031 .058 .273 1 .601 .970 Critical organ compression 1.522 1.058 2.070 1 .150 4.582 Cranial nerve injury 1.312 1.064 1.520 1 .218 3.714 Histology −.380 .762 .249 1 .618 .684 Tumor volume 1.037 .517 4.030 1 .045 2.822 Recurrent disease −.927 .631 2.162 1 .141 .396 Re-radiotherapy .774 .553 1.956 1 .162 2.168 Surgery −.076 .574 .018 1 .895 .927 carbon ion dose of 60 GyE in 20 fractions [16, 17]. The 3-year OS and LC rates were 80.6 and 91.8%, respect- ively. In a phase I/II dose escalation study from the NIRS of Japan with a total dose of 48.0, 52.8, 57.6, and 60.8 GyE in 16 fractions, Mizoe et al. reported that 60.8 GyE/ 16 fractions of carbon ion radiotherapy provided the best local control (at 100%) at 5 years [18]. Cautions must be applied when comparing the outcomes using different beam types or carbon ion therapy using differ- ent biological models. In the current study, we analyzed our patients treated at different dosage schemes of pro- ton and or carbon ion therapy mostly according to our trial protocols. Findings at 2-years for RT-naïve patients were similar to data reported in previous literatures: the OS and LPFS rates were ~ 94% and ~ 83%, respectively, which are significantly better than those after salvage particle radiotherapy. In addition, we didn’t find any sig- nificant differences between any kinds of particle therapy or the dosage levels for local control or overall survival due to, at least in part, relatively high RBE doses used and relatively short follow-up. prior to adjuvant RT. Unfortunately, the extent of surgery is usually limited by risk of severe neurological morbidities. GTV with cut points from 20 to 110 cc were used to report its association with the outcomes [4, 6, 19, 20] [[21, 22], and GTV > 25 mL was mostly reported as the negative fac- tor for local control [4, 6]. As the majority of patients in our series presented with locally advanced diseases, a higher tumor volume (i.e., > 60 CC) was found to be the cut off for predicting both OS and PFS. Discussion The high proportion of pa- tients with more advanced disease in our cohort could be the reason, at least in part, for the slightly inferior LC rate in our study. For tumors with large volumes, especially when compression of CNS is evident, a portion of the tumor may be under dosed in order to meet constraints to critical OARs such as optic nerves or brainstem. Optic ap- paratus or brainstem compression was identified as a prognostic factor for patient’s outcome in the univariate analyses of our study. Our finding echoed the series previ- ously reported. Weber et al. [23] reported the 7-year OS rates of 73.7 and 90.3% for patient with or without optic apparatus or brainstem compression, respectively (P = 0.025). Hug et al. [4] reported the local control rate for pa- tients with or without brainstem involvement were 53 and 94%, respectively (p = 0.04). Our results also demonstrated that the presence of cranial nerve injury was a significant negative prognosticator in the univariate analysis. How- ever, compression of the critical structures of the central nervous system was not shown to be significant in our The size of GTV is the most frequently reported prog- nostic factor for local control or OS in patients with skull base chordomas and chondrosarcomas [6, 19]. McDonald et al. reported postoperative GTV had a statistically signifi- cant association with LC on multivariate analysis, whereas preoperative GTV had no apparent association with out- comes [20]. This supports a strategy of maximal resection Table 4 Multivariate analysis of risk factors for overall survival (OS) B SE Wald df significance Exp(B) KPS .003 .077 .002 1 .967 1.003 Critical organ compression 11.162 195.729 .003 1 .955 70,401.455 Cranial nerve injury 11.376 321.728 .001 1 .972 87,178.423 Histology .500 1.105 .205 1 .651 1.649 Tumor volume 2.081 .824 6.372 1 .012 8.013 Recurrent disease −1.116 1.034 1.166 1 .280 .327 Re-radiotherapy 2.376 .840 7.993 1 .005 10.759 Surgery −.896 .811 1.220 1 .269 .408 Table 4 Multivariate analysis of risk factors for overall survival (OS) B SE Guan et al. Radiation Oncology (2019) 14:206 Page 8 of 9 Page 8 of 9 Page 8 of 9 multi-various as significant factors, because most of the compressions were presented in patients with larger GTV. durability of disease control and to monitor for additional particle RT-induced late toxicities. Discussion In addition, chordoma seemed to have a suboptimal outcome compared to chon- drosarcoma in reported literatures [6, 23]; however, there was no significant survival difference in our study due to, at least in part, the limited patient number for chondro- sarcomas and short follow-up time. Third, most of our pa- tients were treated in prospective phase 1 and 2 trials. For those received dose-escalating schemes, different dose and combination of beams were used. Nevertheless, the bio- logical equivalent doses are considered sufficient even at the lower levels in phase 1 trials based on historical data. p p p g One feature of our study was that close to 50% of pa- tients presented with recurrent disease after surgery with or without prior adjuvant photon RT. Patients with locally recurrent chordoma and chondrosarcoma represent a sig- nificant clinical challenge, with few promising treatment options and few articles reported the results of these dis- ease [24]. Surgical re-resection provides a transient benefit only for most patients. Fagundes et al. reported on 49 pa- tients who received salvage therapy for local relapse of chordomas of the base of skull or cervical spine [25]. The 2-year OS rates after salvage therapy was 63%. In our study, 45 of the 91 patients (49.5%) presented as recurrent tumors, 44 (46.2%) had 2 or more surgeries, and 14 (15.4%) failed previous photon-based RT. In addition, all patients received R2 resection. The 2-year OS was 80.7% for this sub-group of patients. Although we didn’t find sig- nificant differences in PFS and OS between recurrent and primary tumors, patients who failed previous course of RT had significantly worse outcomes. Similar findings were demonstrated in the experience from M.D. Anderson Cancer Center. Holliday et al. reported that in 19 patients with spinal chordomas and chondrosarcomas, those who received proton RT at initial presentation had a local con- trol rate of 80%, as compared with 46% in those who were treated at the time of recurrence [26]. Indelicato et al. [27] reported the similar results of 51 patients with spinal chordoma, and the 4-year LC rate after initial radiotherapy was 71% versus 19% for those irradiated for recurrence. McDonald et al. [28] reported 16 chordoma patients treated with a median re-irradiation dose of 75.6 GyE pro- ton therapy, and the 2-year OS rate was 80%. Funding d f g Funding from Science and Technology Commission of Shanghai Municipality (Project No. 19411951000), Join Breakthrough Project for New Frontier Technologies of the Shanghai Hospital Development Center (project no. SHDC12016120) and Shanghai Sailing Program (project no.15YF1411300) are gratefully acknowledged. Although we presented the largest series of skull base/ cervical chordoma and chondrosarcoma treated mainly with IMCT component, and most patients were treated in a prospective manor, a number of pitfalls need to be dis- cussed. First, the patient composition of our series is rela- tively heterogenic, and recurrent disease consisted close to 50% of patients. Treatments received prior to our proton and carbon-ion RT were also mixed. Second, the follow- up time of 2 years for the entire cohort was short, al- though such a time frame maybe sufficient for recurrent diseases received salvage treatment for short-term out- comes. Further follow-up is surely necessary to assess the Abbreviations CNS: central nervous system; CTC AE: the Common Terminology Criteria for Adverse Events; CTV: clinical target volume; GTV: gross tumor volume; HIT: Heidelberg Ion Therapy Center; IMCT: intensity-modulated carbon ion radiotherapy; IMPT: intensity-modulated proton therapy; LC: local control; LET: linear-energy transfer; MRI: magnetic resonance imaging; OARs: organs at risk; OS: overall survival; PBS: pencil beam scanning; PET-CT: positron emission tomography; PFS: progression free survival; PTV: planning target volume; RBE: relative biological effectiveness; RTOG: the Radiation Therapy Oncology Group; SPHIC: Shanghai Proton and Heavy Ion Center Discussion However, higher dosage of re-irradiation led to more toxicity, and the 2-year estimate grade 3 and 4 late toxicity was as high as 19%. Re-irradiation would certainly increase the risk to OARs for radiation-induced toxicities. The concerns of overdosing brainstem, spinal cord, or optic apparatus con- strained the doses of second course of RT. It is difficult to derive clear guidelines for normal tissue dose constraints in the setting of re-irradiation, given the numerous factors involved that may influence organ tolerance. Acknowledgements W k l d h We acknowledge the work of colleagues in the department of Radiation Oncology, Shanghai Proton and Heavy Ion Center related to this article. Conclusions Proton and/or carbon ion therapy was effective and safe for patients with skull base or cervical spine chordoma and chondrosarcoma. Larger tumor volume (> 60 cc) and re-irradiation were significant prognostic factors and predicts inferior survival. Further follow-up is needed for long-term efficacy and safety. Role of the funding source All the grant sponsors had no involvement in the study design, data analysis, preparation, or writing of this manuscript. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Authors’ contributions Lin Kong, Jiade J. Lu developed the study design and coordination, and helped to review the manuscript, Xiyin Guan carried out the data collection and the statistical analysis, and drafted the manuscript, Jing Gao, Jiyin Hu, Weixu Hu, Jing Yang, Xianxin Qiu and Chaosu Hu recruit patients and provide patients care. All authors read and approved the final manuscript. Ethics approval and consent to participate The retrospective study was approved by the Ethical Review Board of Shanghai Proton and Heavy Ion Center, and written informed consent could be obtained from all patients prior to inclusion into the study. References 1. Chambers KJ, Lin DT, Meier J, Remenschneider A, Herr M, Gray ST. Incidence and survival patterns of cranial chordoma in the United States. 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https://openalex.org/W4307567565	https://insu.hal.science/insu-04155759/document	English	null	Crustal Structure Constraints From the Detection of the SsPp Phase on Mars	Earth and space science	2,023	cc-by	15,406	To cite this version: Jiaqi Li, Caroline Beghein, Paul Davis, Mark A. Wieczorek, Scott M. Mclennan, et al.. Crustal Structure Constraints From the Detection of the SsPp Phase on Mars. Earth and Space Science, 2023, 10, 10.1029/2022EA002416. insu-04155759 Crustal Structure Constraints From the Detection of the SsPp Phase on Mars Jiaqi Li, Caroline Beghein, Paul Davis, Mark A. Wieczorek, Scott M. Mclennan, Doyeon Kim, Ved Lekić, Matthew Golombek, Martin Schimmel, Eleonore Stutzmann, et al. To cite this version: Jiaqi Li, Caroline Beghein, Paul Davis, Mark A. Wieczorek, Scott M. Mclennan, et al.. Crustal Structure Constraints From the Detection of the SsPp Phase on Mars. Earth and Space Science, 2023, 10, 10.1029/2022EA002416. insu-04155759 SsPp Phase on Mars Jiaqi Li, Caroline Beghein, Paul Davis, Mark A. Wieczorek, Scott M. Mclennan, Doyeon Kim, Ved Lekić, Matthew Golombek, Martin Schimmel, Eleonore Stutzmann, et al. Distributed under a Creative Commons Attribution 4.0 International License Key Points: Jiaqi Li1 , Caroline Beghein1 , Paul Davis1, Mark. A. Wieczorek2 , Scott M. McLennan3 , Doyeon Kim4,5, Ved Lekić5 , Matthew Golombek6, Martin Schimmel7 , Eleonore Stutzmann8 , Philippe Lognonné8 , and William Bruce Banerdt6 Jiaqi Li1 , Caroline Beghein1 , Paul Davis1, Mark. A. Wieczorek2 , Scott M. McLennan3 , Doyeon Kim4,5, Ved Lekić5 , Matthew Golombek6, Martin Schimmel7 , Eleonore Stutzmann8 , Philippe Lognonné8 , and William Bruce Banerdt6 • We analyzed marsquakes and made the first coherent detection of the SsPp phase (an SV- to P-wave reflection on the receiver side) 1Department of Earth, Planetary, and Space Sciences, University of California, Los Angeles, CA, USA, 2Université Côte d’Azur, Observatoire de la Côte d’Azur, CNRS, Laboratoire Lagrange, Nice, France, 3Department of Geosciences, Stony Brook University, Stony Brook, NY, USA, 4Department of Earth Sciences, Institute of Geophysics, ETH Zürich, Zürich, Switzerland, 5Department of Geology, University of Maryland, College Park, MD, USA, 6Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA, 7Geosciences Barcelona – CSIC, Barcelona, Spain, 8Université de Paris, Institut de physique du globe de Paris, CNRS, Paris, France • We determined that the average P-wave speed in the top crustal layer (Layer 1, above 8 km) is between 2.5 and 3.2 km/s • The average P-wave speed in Layer 1 is consistent with the current understanding of the upper crustal stratigraphy beneath InSight • The average P-wave speed in Layer 1 is consistent with the current understanding of the upper crustal stratigraphy beneath InSight Abstract The shallowest intracrustal layer (extending to 8 ± 2 km depth) beneath the Mars InSight Lander site exhibits low seismic wave velocity, which is likely related to a combination of high porosity and other lithological factors. The SsPp phase, an SV- to P-wave reflection on the receiver side, is naturally suited for constraining the seismic structure of this top crustal layer since its prominent signal makes it observable with a single station without the need for stacking. We have analyzed six broadband and low-frequency seismic events recorded on Mars and made the first coherent detection of the SsPp phase on the red planet. The timing and amplitude of SsPp confirm the existence of the ∼8 km interface in the crust and the large wave speed (or impedance) contrast across it. HAL Id: insu-04155759 https://insu.hal.science/insu-04155759v1 Submitted on 7 Jul 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Author Contributions: Author Contributions: Conceptualization: Jiaqi Li, Caroline Beghein Data curation: Jiaqi Li, Paul Davis, Doyeon Kim, Philippe Lognonné Formal analysis: Jiaqi Li, Caroline Beghein, Paul Davis, Mark. A. Wieczorek, Scott M. McLennan, Doyeon Kim, Ved Lekić, Martin Schimmel, Eleonore Stutzmann Funding acquisition: Caroline Beghein Investigation: Jiaqi Li, Caroline Beghein, Scott M. McLennan, Matthew Golombek Author Contributions: Conceptualization: Jiaqi Li, Caroline Beghein Data curation: Jiaqi Li, Paul Davis, Doyeon Kim, Philippe Lognonné Formal analysis: Jiaqi Li, Caroline Beghein, Paul Davis, Mark. A. Wieczorek, Scott M. McLennan, Doyeon Kim, Ved Lekić, Martin Schimmel, Eleonore Stutzmann Funding acquisition: Caroline Beghein Investigation: Jiaqi Li, Caroline Beghein, Scott M. McLennan, Matthew Golombek Crustal Structure Constraints From the Detection of the SsPp Phase on Mars Crustal Structure Constraints From the Detection of the SsPp Phase on Mars RESEARCH ARTICLE Key Points: Key Points: With our new constraints from the SsPp phase, we determined that the average P-wave speed in the top crustal layer is between 2.5 and 3.2 km/s, which is a more precise and robust estimate than the previous range of 2.0–3.5 km/s obtained by receiver function analysis. The low velocity of Layer 1 likely results from the presence of relatively low-density lithified sedimentary rocks and/or aqueously altered igneous rocks that also have a significant amount of porosity, possibly as much as 22%–30% by volume (assuming an aspect ratio of 0.1 for the pore space). These porosities and average P-wave speeds are compatible with our current understanding of the upper crustal stratigraphy beneath the InSight Lander site. Supporting Information: Supporting Information may be found in the online version of this article. Correspondence to: J. Li, jli@epss.ucla.edu Citation: Li, J., Beghein, C., Davis, P., Wieczorek, M. A., McLennan, S. M., Kim, D., et al. (2023). Crustal structure constraints from the detection of the SsPp phase on Mars. Earth and Space Science, 10, e2022EA002416. https://doi. org/10.1029/2022EA002416 Received 6 MAY 2022 Accepted 18 OCT 2022 Crustal Structure Constraints From the Detection of the SsPp Phase on Mars Crustal Structure Constraints From the Detection of the SsPp Phase on Mars Citation: Li, J., Beghein, C., Davis, P., Wieczorek, M. A., McLennan, S. M., Kim, D., et al. (2023). Crustal structure constraints from the detection of the SsPp phase on Mars. Earth and Space Science, 10, e2022EA002416. https://doi. org/10.1029/2022EA002416 Plain Language Summary The NASA InSight mission sent a seismometer to Mars in 2018. One of the science goals of the mission is to better understand how rocky planets form and evolve by investigating the interior structure of Mars. Previous seismological studies with InSight data have revealed a shallow crustal layer (i.e., Layer 1, extending to 8 ± 2 km depth) with low seismic wave speed under the instrument. In this study, we have identified a new seismic signal on the seismograms recorded on Mars. The existence of this seismic phase confirmed the low speed of compressional (P) waves in Layer 1 and provided additional constraints on the average P-wave speed, that is, between 2.5 and 3.2 km/s. Based on these low speeds, we found that the seismic properties of Layer 1 likely result primarily from the presence of sedimentary rocks and/or aqueously altered igneous rocks that also have a significant amount of porosity, possibly as much as ∼30% by volume. These porosities and average P-wave speeds are compatible with our current understanding of the upper crustal stratigraphy beneath the InSight Lander site. Received 6 MAY 2022 Accepted 18 OCT 2022 Received 6 MAY 2022 Accepted 18 OCT 2022 © 2022 The Authors. Earth and Space Science published by Wiley Periodicals LLC on behalf of American Geophysical Union. © 2022 The Authors. Earth and Space Science published by Wiley Periodicals LLC on behalf of American Geophysical Union. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Author Contributions: Conceptualization: Jiaqi Li, Caroline Beghein Data curation: Jiaqi Li, Paul Davis, Doyeon Kim, Philippe Lognonné Formal analysis: Jiaqi Li, Caroline Beghein, Paul Davis, Mark. A. Wieczorek, Scott M. McLennan, Doyeon Kim, Ved Lekić, Martin Schimmel, Eleonore Stutzmann Funding acquisition: Caroline Beghein Investigation: Jiaqi Li, Caroline Beghein, Scott M. McLennan, Matthew Golombek 1. Introduction (2021) subsequently found that both S-to-p receiver functions and receiver functions constructed from free-surface P-wave multiples (PPs) favor the 3-layer model. Durán et al. (2022) also supported the 3-layer model using a more complete marsquake cata- log and phase picks, though with slightly different average interface depths of 10 km, 20 km, and 45 km. Using ambient noise auto-correlation, Deng and Levander (2020), Kim, Davis, et al. (2021), and Schimmel et al. (2021) observed the strongest signal at a lag time of 10.6 s, which corresponds to a discontinuity at about 21 km depth, in agreement with the observed receiver function amplitudes. Methodology: Jiaqi Li, Caroline Beghein, Paul Davis Project Administration: Caroline Beghein Resources: Jiaqi Li, Paul Davis, Philippe Lognonné Software: Jiaqi Li, Paul Davis, Doyeon Kim, Ved Lekić Supervision: Caroline Beghein Validation: Jiaqi Li, Paul Davis, Mark. A. Wieczorek, Doyeon Kim, Ved Lekić, Martin Schimmel, Eleonore Stutzmann, Philippe Lognonné Visualization: Jiaqi Li Writing – original draft: Jiaqi Li, Caroline Beghein, Mark. A. Wieczorek, Scott M. McLennan Writing – review & editing: Jiaqi Li, Caroline Beghein, Paul Davis, Mark. A. Wieczorek, Scott M. McLennan, Doyeon Kim, Ved Lekić, Matthew Golombek, Martin Schimmel, Eleonore Stutzmann, Philippe Lognonné, William Bruce Banerdt Methodology: Jiaqi Li, Caroline Beghein, Paul Davis Project Administration: Caroline Beghein Resources: Jiaqi Li, Paul Davis, Philippe Lognonné Software: Jiaqi Li, Paul Davis, Doyeon Kim, Ved Lekić Supervision: Caroline Beghein Validation: Jiaqi Li, Paul Davis, Mark. A. Wieczorek, Doyeon Kim, Ved Lekić, Martin Schimmel, Eleonore Stutzmann, Philippe Lognonné Visualization: Jiaqi Li Writing – original draft: Jiaqi Li, Caroline Beghein, Mark. A. Wieczorek, Scott M. McLennan Writing – review & editing: Jiaqi Li, Caroline Beghein, Paul Davis, Mark. A. Wieczorek, Scott M. McLennan, Doyeon Kim, Ved Lekić, Matthew Golombek, Martin Schimmel, Eleonore Stutzmann, Philippe Lognonné, William Bruce Banerdt Methodology: Jiaqi Li, Caroline Beghein, Paul Davis Resources: Jiaqi Li, Paul Davis, Philippe Lognonné Another prominent teleseismic signal well-recorded on Earth and often used to constrain the depth of the Mohor- ovičić discontinuity (hereafter referred to as Moho) is the SsPp phase, an SV- to P-wave reflection off the free surface on the receiver side. For incoming S-waves (i.e., SV-wave) polarized in the P-SV plane containing the event and the receiver, phase conversion occurs at the free surface and the converted P-waves reflect at the Moho (or any other intracrustal discontinuity) before being recorded by the seismometer (Figure 1b). 1. Introduction On Earth, this SsPp phase has been analyzed in data from isolated stations (e.g., Owens & Zandt, 1997; Zandt & Randall, 1985; Zhou et al., 2000) and seismic arrays (Chen & Jiang, 2020; Tseng et al., 2009; Yu et al., 2012) to constrain crustal thickness. Cunningham and Lekic (2019) and Liu et al. (2019) additionally showed that SsPp phases provide complementary constraints that remove the trade-off between velocity and thickness inherent in receiver func- tion analysis. Because there is a near-critical (sometimes post-critical) reflection within the top layer, the SsPp phase is usually stronger than an SV- to p-wave transmission in the conventional S-wave receiver function (Chen & Chen, 2020), and has been observed in several regions on Earth. For example, SsPp phases arrive at 4–11 s after the direct SV-phase in the western United States (Yu et al., 2016), 7–12 s across the North China craton (Yu et al., 2012), and 12–18 s across the Himalayan-Tibetan orogeny (Tseng et al., 2009). Writing – original draft: Jiaqi Li, Caroline Beghein, Mark. A. Wieczorek, Scott M. McLennan In continental regions on Earth, the Moho lies between depths of about 15 and 75 km (e.g., Brown & Mussett, 1993; Chen et al., 2013; Laske et al., 2013). When there are no sedimentary basins, the Moho is usually where the most significant jump in seismic wave speed occurs within the lithosphere of the average Earth model (e.g., Kennett & Engdahl, 1991). At the Mars InSight lander site, the situation is different, and the interface with the largest wave speed change corresponds to the shallowest intracrustal layer, hereafter referred to as Layer 1, at 8 ± 2 km (Knapmeyer-Endrun et al., 2021). The velocity contrast is estimated to be up to +40% due to the relatively low wave speed within Layer 1. The low velocity and recently discovered seismic anisotropy of Layer 1 (Li et al., 2022), make it an important region to study since both features are likely related to high porosity in the Martian crust (Knapmeyer-Endrun et al., 2021; Li et al., 2022). The low observed velocities could potentially be a result of sedimentary or volcanic ash and pyroclastic deposits that have intrinsically high porosity, or a high density of fractures in the upper crust generated by impact cratering events, such as is observed on the Moon (e.g., Milbury et al., 2015; Soderblom et al., 2015; Wieczorek et al., 2013). 1. Introduction Alternatively, the low velocities could be the result of a high quantity of aqueously altered materials (Lognonné et al., 2022). Understanding the origin of the low seismic velocity in this layer would not only provide clues to the origin of this layer but would also provide useful information for future studies of the deeper crustal layers (e.g., Wieczorek et al., 2022). The large amplitude characteristics of SsPp and the previously observed large wave speed jump across the base of Layer 1 make the SsPp an ideal phase to further constrain the properties of Layer 1. In addition, because it can be observed with a single station without the need for stacking, this phase is naturally suitable for seismic studies on Mars where we only have one instrument at a single location on the planet. 1. Introduction One of the science goals of the NASA InSight mission is to better understand how rocky planets form and evolve by investigating the interior structure of Mars (Banerdt et al., 2020). Since the landing in November 2018, the Seismic Experiment for Interior Structure (SEIS, Lognonné, et al., 2019) Very Broadband (VBB) seismometer has recorded more than one thousand events (InSight Marsquake Service, 2020, 2021a, 2021b, 2022a, 2022b). Preliminary models of the crust and mantle structure, as well as core size, have been obtained with receiver func- tion analysis (Kim, Lekić, et al., 2021; Knapmeyer-Endrun et al., 2021; Lognonné et al., 2020), P- and S-wave differential travel-times and surface-reflected body-wave phases (Khan et al., 2021), and ScS waves (Stähler et al., 2021), respectively. © 2022 The Authors. Earth and Space Science published by Wiley Periodicals LLC on behalf of American Geophysical Union. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 of 16 1 of 16 LI ET AL. Earth and Space Science 10.1029/2022EA002416 Using P-to-s receiver functions, Knapmeyer-Endrun et al. (2021) found two possible sets of crustal models at the lander site: a 2-layer model with a crustal thickness of 20 ± 5 km and a 3-layer model with a thickness of 39 ± 8 km depth (with a weaker impedance contrast across it). Kim, Lekić, et al. (2021) subsequently found that both S-to-p receiver functions and receiver functions constructed from free-surface P-wave multiples (PPs) favor the 3-layer model. Durán et al. (2022) also supported the 3-layer model using a more complete marsquake cata- log and phase picks, though with slightly different average interface depths of 10 km, 20 km, and 45 km. Using ambient noise auto-correlation, Deng and Levander (2020), Kim, Davis, et al. (2021), and Schimmel et al. (2021) observed the strongest signal at a lag time of 10.6 s, which corresponds to a discontinuity at about 21 km depth, in agreement with the observed receiver function amplitudes. Using P-to-s receiver functions, Knapmeyer-Endrun et al. (2021) found two possible sets of crustal models at the lander site: a 2-layer model with a crustal thickness of 20 ± 5 km and a 3-layer model with a thickness of 39 ± 8 km depth (with a weaker impedance contrast across it). Kim, Lekić, et al. Methodology: Jiaqi Li, Caroline Beghein, Paul Davis Project Administration: Caroline Beghein Resources: Jiaqi Li, Paul Davis, Philippe Lognonné Software: Jiaqi Li, Paul Davis, Doyeon Kim, Ved Lekić Supervision: Caroline Beghein Validation: Jiaqi Li, Paul Davis, Mark. A. Wieczorek, Doyeon Kim, Ved Lekić, Martin Schimmel, Eleonore Stutzmann, Philippe Lognonné Visualization: Jiaqi Li Writing – original draft: Jiaqi Li, Caroline Beghein, Mark. A. Wieczorek, Scott M. McLennan Writing – review & editing: Jiaqi Li, Caroline Beghein, Paul Davis, Mark. A. Wieczorek, Scott M. McLennan, Doyeon Kim, Ved Lekić, Matthew Golombek, Martin Schimmel, Eleonore Stutzmann, Philippe Lognonné, William Bruce Banerdt 2. Data and Methods Colorscales correspond to the arrival time of the traces. (d) Particle motion analysis for part of the waveforms (−1 to 5 s) on the radial and vertical components shown in (c). Colorscales correspond to the arrival time of the traces, and are the same as in (c). Oblique lines indicate the estimated direction for the pseudo-P (in blue) and pseudo-S (in red) components. (e) Synthetic waveforms on the pseudo-P (in blue) and pseudo-S (in red) components, derived from the particle motion analysis in (d). arrive later, do not interfere with SsPp. This criterion could have been a problem to detect SsPp on Mars since most of the events detected so far likely originate from depths shallower than 40 km (Drilleau et al., 2021; Durán et al., 2022). Nevertheless, Yu et al. (2013) showed that this problem could be mitigated by removing the source wavelet complexity resulting from source-side scattering. Specifically, analyses of particle motion provide clues for deriving a "pseudo-S" wave train, which contains information about both the source time function and depth phases. After the deconvolution of this "pseudo-S” wave train, the authors showed that shallow events with complex source wavelets display signals in the seismic data of similar clarity to those from deep earthquakes (Yu et al., 2013). This method thus greatly increases the number of earthquakes that can be used to study SsPp phases and makes it possible to look for them on Mars. In addition to these two criteria, we found that the duration of the source wavelet, which is measured based on the direct SV phase (Ss), plays a crucial role in reliably detecting SsPp phases on Mars, and that it needs to be rela- tively short. On Earth, the SsPp phase is mostly used to study the Moho and arrives at relatively large differential travel times to the Ss phase (e.g., 4—11 s in the western United States (Yu et al., 2016)). This implies that even a relatively long source wavelet duration does not affect the SsPp detection. On Mars, however, the base of Layer 1 is located at about 8 km depth and synthetic waveforms (where the source time function has a short duration of 1 s) predict differential arrival times of only about 4 s (Figure 1c). 2. Data and Methods There are two main criteria for SsPp data selection on Earth. First, the epicentral distance should be larger than 30° to avoid mantle triplications generated by the 410-km and 660-km discontinuities (Kang et al., 2016). On Mars, the ideal epicentral distance to detect SsPp should be smaller than 60°, since the phase transformation of olivine to its higher-pressure polymorphs occurs at around 1,000 km depth (Huang et al., 2022). Second, the source wavelet should be simple. Deep earthquakes are therefore usually preferred (e.g., Tseng et al., 2009) since their source time function is often simple, and the depth phases from deep earthquakes, which 2 of 16 Earth and Space Science 10.1029/2022EA002416 Figure 1. (a) Topographic map from the Mars Orbiter Laser Altimeter (MOLA, Smith et al., 2001) near Seismic Experiment for Interior Structure (SEIS) (black triangle). The red stars mark the six events used in this study. The red and black error bars indicate the uncertainties in epicentral distance and back azimuth, respectively (see Supporting Information S1, Section 1.1). (b) Ray paths of the SsPp phase from a planar, incident teleseismic SV-wave near the SEIS instrument. The S-waves are shown as dashed red lines and the converted P-waves are in blue. (c) Synthetic waveforms on the radial and vertical components. Colorscales correspond to the arrival time of the traces. (d) Particle motion analysis for part of the waveforms (−1 to 5 s) on the radial and vertical components shown in (c). Colorscales correspond to the arrival time of the traces, and are the same as in (c). Oblique lines indicate the estimated direction for the pseudo-P (in blue) and pseudo-S (in red) components. (e) Synthetic waveforms on the pseudo-P (in blue) and pseudo-S (in red) components, derived from the particle motion analysis in (d). Figure 1. (a) Topographic map from the Mars Orbiter Laser Altimeter (MOLA, Smith et al., 2001) near Seismic Experiment for Interior Structure (SEIS) (black triangle). The red stars mark the six events used in this study. The red and black error bars indicate the uncertainties in epicentral distance and back azimuth, respectively (see Supporting Information S1, Section 1.1). (b) Ray paths of the SsPp phase from a planar, incident teleseismic SV-wave near the SEIS instrument. The S-waves are shown as dashed red lines and the converted P-waves are in blue. (c) Synthetic waveforms on the radial and vertical components. 2. Data and Methods This means that if the source time function has a relatively long duration, the SsPp phase will likely be buried in the Ss phase source wavelet, making it undetect- able directly (Figure S1 in the Supporting Information S1). We verified that as long as the duration of the source wavelet is less than 𝐴𝐴 𝐴𝐴𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆−𝑇𝑇𝑆𝑆𝑆𝑆 , the particle motions show linear trends and the source-normalization technique (Yu et al., 2013) can effectively obtain the corresponding SV- to P-phase (e.g., Figure S1f in the Supporting Information S1). On the contrary, if the duration of the source wavelet is larger than 𝐴𝐴 𝐴𝐴𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆−𝑇𝑇𝑆𝑆𝑆𝑆 , the particle motions no longer exhibit linear trends (Figures S1d and S1e in the Supporting Information S1), and the short time function approximation cannot be applied, in which case the derived SsPp phase is unreliable (Figure S1c in the Supporting Information S1). 3 of 16 LI ET AL. Earth and Space Science Earth and Space Science 10.1029/2022EA002416 Locating marsquakes using a single station is challenging because both the arrival times of P- and S-waves and P-wave polarization information are needed to determine the epicentral distance and the back azimuth, respec- tively. During 1,133 Mars solar days (i.e., over three Earth years), SEIS has recorded 32 broadband (with energy up to 2.4 Hz) and 52 low-frequency (with energy below 1 Hz) marsquakes, and only 11 of them are labeled “quality-A” by the InSight Marsquake Service (MQS) 2020, 2021a, 2021b, 2022a, 2022b. To be designated quality A, an event needs to have both clear back azimuth and epicentral distance. Quality-A marsquakes, in most cases, are ideal candidates for many seismological studies due to their strong seismic energy, high signal-to-noise ratio, and well-constrained event location. However, most of the quality-A events happen to have relatively long source wavelets (Figure S2 in the Supporting Information S1), therefore, are not necessarily ideal for analyzing SsPp phases. To date, nine quality-A events have an epicentral distance smaller than 60° (Figure S2 in the Supporting Infor- mation S1). Four of them (S0235b, S1015f, S1022a, and S1048d) exhibit very long (e.g., larger than 5 s) and complex source wavelets, indicating that they are not suitable for our SsPp study. There is less low-frequency content in the source time function of the other five quality-A events (S0173a, S0809a, S0820a, S0864a, and S1133c) and although multiple peaks or oscillations are observed, they are potential candidates for this study. 2. Data and Methods Compared with quality-A events, marsquakes of quality B, in general, have shorter and simpler source wavelets. There are 18 marsquakes of quality B (eight broadband and 10 low-frequency), and the epicentral distances have been measured to be within 60° (with an uncertainty smaller than 10°) for 12 of them. However, due to their rela- tively low signal-to-noise ratio, the MQS has not determined the back azimuth for the quality-B events. Recent studies by Drilleau et al. (2021) and Zenhäusern et al. (2022) with detailed analyses of the waveforms provided back azimuth estimates for 10 of these 12 events. After excluding event S0325a whose estimated back azimuth shows large discrepancies between the two studies, we are left with a total of nine quality-B events with both epicentral distance and back azimuth information. In this study, we focused on seven of these nine quality-B events with back azimuths between 0 and 180° (Table S1 in the Supporting Information S1) and which are in the same direction (to the east of the InSight lander) as the events used in the receiver function study (Knapmeyer-Endrun et al., 2021). Besides these seven quality-B events, we also included one quality-A event S1133c, which has a relatively short source time function of ∼4 s. Comparisons with the other eight quality-A events can be found in the Supplementary Material (i.e., Figures S2 and S3 in the Supporting Information S1). 2.1. Data Processing The waveform data (InSight Mars SEIS data service, 2019) were processed by first applying a pre-filtering from 0.01 to 8 Hz (zero-phase, second-order Butterworth filter) to the deglitched data set (Scholz et al., 2020, with a sampling rate of 20 samples per second), and then removing the instrument response to get the ground motion records. Finally, we filtered (zero-phase, second-order Butterworth filter) the data into periods from 1.5 to 5 s. We prefer working with the displacement record because there are fewer oscillations compared with the velocity record. To analyze the SsPp, we need to use data from the radial (R) and vertical (Z) components. We thus converted the waveforms from the original UVW to NEZ channels using ObsPy (Beyreuther et al., 2010) and rotated the coor- dinates from NEZ to RTZ using the back azimuth information provided by previous studies (Drilleau et al., 2021; Zenhausern et al., 2022) and listed in Table S1 in the Supporting Information S1. 2.2. SsPp Phase We first calculated synthetic seismograms for the radial (R) and vertical (Z) components using a MATLAB package (Yu et al., 2017) based on the propagator method (Kennett, 2009) with a planar incident SV-wave (with a delta source wavelet, filtered between 1.5 and 5 s). Using one of the models from the receiver function study (Knapmeyer-Endrun et al., 2021) with a Layer 1 thickness of 8 km and an average P-wave velocity of 3.0 km/s, we found that the simulated SsPp phase arrives about 4 s after the direct SV phase for marsquakes with an epicentral distance of 30° (i.e., a ray parameter of 13.3 s/deg). This SsPp phase is observable on both the radial and vertical components (Figure 1c). Particle motion analysis of the radial and vertical components of the synthetics shows that the first signal (at −1 to 2 s) and the second signal (at 3–5 s) have distinctive particle motions. Specifically, the first signal follows a linear trend in the second and fourth quadrants, and the second signal is polarized in the first and third quadrants (Figure 1d). We can define pseudo-S and pseudo-P components according to the direction of these two sub-linear particle motions (Yu et al., 2013). On the pseudo-S component, the main phase is the direct SV phase at 0 s (Figure 1e). On the pseudo-P component, there is no direct SV phase at 0 s, but a strong SsPp phase is visible at around 4 s (Figure 1e). We applied the same particle motion analysis and the pseudo-P and S separation technique to the real data. Figure 2 shows examples of quality-A event S1133c, quality-B events S0105a, S0407a, S0484b, S0784a, and S0918a. In Figure 2, the start time (0 s) is selected based on the arrival time of the S-wave measured by MQS (Table S2 in the Supporting Information S1), where the signal envelope is analyzed in multiple narrowband filters and the coherent start time in the largest possible bandwidth is selected (Clinton et al., 2021). In all cases, there are two linear trends in the particle motion analysis and there is a strong signal at around 4 s on the pseudo-P components. 2.2. SsPp Phase Figure 1b illustrates the ray path of the SsPp phase, where the Ss-leg of the ray path (the dashed line in red to the right of the lander) is almost parallel to that of the direct SV phase (the dashed line in red below the lander). The major difference between the ray paths is the near- or post-critical Pp reflection at the base of Layer 1. Therefore, the travel-time difference between the direct Ss phase and SsPp phase can provide constraints on the average P-wave speed and thickness of Layer 1: 𝑇𝑇𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆−𝑆𝑆𝑆𝑆= 2𝐻𝐻𝐻𝐻𝛽𝛽= 2𝐻𝐻 √ 1 𝑉𝑉2 𝑃𝑃 −𝑝𝑝2 𝛽𝛽, (1) (1) 4 of 16 LI ET AL. Earth and Space Science 10.1029/2022EA002416 Figure 2. (a1) Raw displacement waveforms on the radial and vertical components for event S0407a. The layout is the same as Figure 1c. (a2) Particle motion analysis for event S0407a. The layout is the same as Figure 1d. (a3) Separated waveforms on the pseudo-P and pseudo-S components for event S0407a. The peak on the pseudo-S component (at around 0 s) and the trough on the pseudo-P component (at around 4 s) are indicated by black dots. Same analysis for event S1133c (b1-b3), S0105a (c1–c3), S0484b (d1–d3), S0784a (e1–e3), and S0918a (f1–f3). Note that the waveforms are flipped if needed to make the amplitude at 0 s on the pseudo-S component to be positive for better illustration. Figure 2. (a1) Raw displacement waveforms on the radial and vertical components for event S0407a. The layout is the same as Figure 1c. (a2) Particle motion analysis for event S0407a. The layout is the same as Figure 1d. (a3) Separated waveforms on the pseudo-P and pseudo-S components for event S0407a. The peak on the pseudo-S component (at around 0 s) and the trough on the pseudo-P component (at around 4 s) are indicated by black dots. Same analysis for event S1133c (b1-b3), S0105a (c1–c3), S0484b (d1–d3), S0784a (e1–e3), and S0918a (f1–f3). Note that the waveforms are flipped if needed to make the amplitude at 0 s on the pseudo-S component to be positive for better illustration. where 𝐴𝐴 𝐴𝐴𝛽𝛽 and 𝐴𝐴 𝐴𝐴𝛽𝛽 are the vertical and horizontal slowness (i.e., ray parameter) of the incident SV wave, respectively. 𝐴𝐴𝑃𝑃 is the average P-wave speed in the layer, and H is its overall thickness. 2.2. SsPp Phase The following analysis is based on these six events, and we excluded the other two quality-B events due to no clear pseudo-P and S separation (S0409d, in Figure S4a in the Supporting Information S1), and no clear direct-SV arrival (S0802a, in Figure S4b in the Supporting Information S1). 5 of 16 Earth and Space Science 10.1029/2022EA002416 Figure 3. (a) L2-norm misfit map for event S0918a. The red star marks one of the acceptable models within the strict threshold of Figure 4b, and the P-wave speed of the second layer is shown in the lower right corner. The corresponding synthetic waveforms are shown in (d) in the same color. (b) L2-norm misfit map for event S0105a. (c) L2-norm misfit map for event S0407a. (d) Comparison between data (in black) and synthetics (in red) on the pseudo-P components. (e) L2-norm misfit map for event S0484b. (f) L2-norm misfit map for event S1133c. (g) L2-norm misfit map for event S0784a. Figure 3. (a) L2-norm misfit map for event S0918a. The red star marks one of the acceptable models within the strict threshold of Figure 4b, and the P-wave speed of the second layer is shown in the lower right corner. The corresponding synthetic waveforms are shown in (d) in the same color. (b) L2-norm misfit map for event S0105a. (c) L2-norm misfit map for event S0407a. (d) Comparison between data (in black) and synthetics (in red) on the pseudo-P components. (e) L2-norm misfit map for event S0484b. (f) L2-norm misfit map for event S1133c. (g) L2-norm misfit map for event S0784a. Although the arrival times of the signals on the pseudo-P components are coherent, their waveforms vary between different events. This is mainly due to the different source time functions, which are indicated by the phase at 0 s on the pseudo-S components. To consider the effect of different kinds of source wavelets, we followed Yu et al. (2013) to assume the waveforms on the pseudo-S component to be an approximation of the source wavelet and then convolved the assumed source wavelet (from −5.0 s to +2.5 s) with the synthetic Green's function to simulate the pseudo-P component (Figure 3). Although the arrival times of the signals on the pseudo-P components are coherent, their waveforms vary between different events. This is mainly due to the different source time functions, which are indicated by the phase at 0 s on the pseudo-S components. 2.2. SsPp Phase To consider the effect of different kinds of source wavelets, we followed Yu et al. (2013) to assume the waveforms on the pseudo-S component to be an approximation of the source wavelet and then convolved the assumed source wavelet (from −5.0 s to +2.5 s) with the synthetic Green's function to simulate the pseudo-P component (Figure 3). 3. Results 3.1. Waveform Comparison and Misfit Map 3.1. Waveform Comparison and Misfit Map Therefore, although we simultaneously searched for three parameters to better fit the waveforms, we only aimed to constrain the thickness and average P-wave speed of Layer 1, whereas the velocity of the second layer is more difficult to constrain given the relatively low signal-to-noise ratio on Mars compared with that on Earth (e.g., Liu et al., 2019). map for the SsPp data. (b) Acceptable ray) and strict (in blue) thresholds Figure 4. (a) Summed L2-norm misfit map for the SsPp data. (b) Acceptable model regions defined by the loose (in gray) and strict (in blue) thresholds extracted from (a). At each grid cell, we calculated synthetic Green's functions, performed the particle motion analysis, separated the synthetic pseudo-P and pseudo-S components, convolved the assumed source wavelet (i.e., S wave, from −5.0 s to +2.5 s, on the pseudo-S or the tangential component), and then compared the simulated waveforms with the data. To quantify the waveform similarity between the data and the synthetics, we selected a misfit window from −5.0 to 6.0 s. We chose the L2-norm of the waveform differences (after normalization) in the time domain as the misfit function. The misfit maps are shown in Figure 3. Models in the dark regions have smaller misfits and are thus more acceptable than models in the bright area. We found that the average L2-norm misfit along the approx- imate diagonal (i.e., regions close to the predictions from the ray theory with Equation 1) is systematically lower, confirming our identification of the SsPp phase. We note that there is another signal at around −2.5 s in the data which is also fitted by the synthetics. This phase is likely to be the S-to-p transmission at the base of Layer 1 (i.e., the S-to-p receiver function), and could provide additional constraints on the properties of Layer 1 (e.g., Chen & Chen, 2020). However, due to its relatively smaller amplitude compared with the SsPp, this phase has a limited contribution to the total misfit. To suppress the data noise, we averaged the L2-norm misfit maps (with the same weight) to get the final misfit map in Figure 4a. We first found the best-fitting model with the smallest misfit, then defined the range of accept- able models using a misfit threshold (i.e., strict and loose thresholds were set for misfits within 130% and 150% of the minimum misfit, respectively). 3.1. Waveform Comparison and Misfit Map In the SsPp phase, there is a near- or post-critical Pp reflection at the base of Layer 1, and thus a phase shift might occur. When such a phase shift happens, it prevents us from accurately picking the arrival time of SsPp (e.g., Figure S6 in the Supporting Information S1). Therefore, we prefer to perform a waveform comparison (between the data and the synthetics) rather than refer to Equation 1, to constrain the model parameters (i.e., the average P-wave speed and thickness of Layer 1). 6 of 16 Earth and Space Science 10.1029/2022EA002416 Figure 4. (a) Summed L2-norm misfit map for the SsPp data. (b) Acceptable model regions defined by the loose (in gray) and strict (in blue) thresholds extracted from (a). To compare the synthetic SsPp waveforms with the real data, we first applied the same particle motion analysis to the synthetic vertical and radial Green's functions to separate the synthetic pseudo-P and pseudo-S components, then we convolved the synthetic pseudo-P waveforms with the source wavelet (derived from the real data) to simulate the observations. We found that the simulated waveforms (i.e., the red waveforms in Figure 3d), generated by the acceptable models (i.e., corresponding to the red stars in Figure 3), match the pulse at around 4 s in the data well. Although these models predict waveforms that are similar to the data, trade-offs between model parameters (the average P-wave speed and layer thickness) exist as shown in Equation 1. To find all acceptable models, we performed forward modeling and sampled the average P-wave speed (from 1.3 km/s to 4.2 km/s with an interval of 0.02 km/s) and layer thickness (from 4 to 14 km with an interval of 0.05 km) of Layer 1. Since the velocity of the second layer will affect both the amplitude and the phase of the SsPp signal (Figure S6 in the Supporting Information S1), we also varied the velocity of the second layer (from 1.1 to 1.8 times the velocity of Layer 1 in 0.1 intervals). Of these three parameters, the Layer 1 thickness and average P-wave speed are more directly related to the differential arrival time (e.g., Equation 1), and the velocity of the second layer affects the possible phase shift of the SsPp (i.e., the waveform of SsPp, also see Figure S6 in the Supporting Information S1). 3.1. Waveform Comparison and Misfit Map (d) Same as (b) for the 3-layer crustal case. Figure 5. (a) Colored dots represent the original 20,000 models from the receiver function study (Knapmeyer-Endrun et al., 2021) for the 2-layer crustal case. The color scale indicates the number of models at each grid point. The acceptable model regions with the loose (in gray) and strict (in blue) thresholds derived from the SsPp data in this study are superimposed. (b) Reduced models for the 2-layer crustal case with the constraint from the SsPp phase in this study. (c) Same as (a) for the 3-layer crustal case. (d) Same as (b) for the 3-layer crustal case. We note, however, that there are discrepancies between our derived acceptable regions and the ray-theory-based predictions (Figure S7 in the Supporting Information S1). These arise because, when the velocity of the second layer is large enough, the SsPp is a post-critical reflection and a phase shift occurs. In such a case, the location of the negative pulse deviates from the actual arrival time of the SsPp phase (Figure S6 in the Supporting Infor- mation S1). Therefore we trust the results from the misfit map since the possible phase shift (for the critical P-p reflection off Layer 1) is included in the synthetics calculations. In addition, multiple sources of uncertainties are automatically included in the final misfit map such as the noise in the data, the duration of the pulse, and even finite-frequency effects. 3.1. Waveform Comparison and Misfit Map Those strict and loose thresholds were set to extract the acceptable region of the model space (i.e., Figure 4b). We also see that there are other sets of solutions (e.g., near the lower left corner and near the top in Figure 4b) in addition to the one along the approximate diagonal. Nevertheless, those solutions are of relatively larger misfits and are only observed with the loose misfit threshold. Although the choice of the L2-misfit threshold is somewhat subjective, we are confident our analysis is robust because we tested several thresholds and compared them with predictions from ray theory. For example, if we consider an even smaller threshold value of 110% of the minimum misfit, there are very few acceptable regions, indicating that this threshold is too strict to be used in practice (in Figure S7a in the Supporting Information S1). We also compared the acceptable regions (derived from the L2-norm misfit maps) with the ray-theory-based calcu- lation using formula 1. The first-order trends are similar between these two approaches (Figure S7 in the Support- ing Information S1). In addition, the choice of the misfit function (e.g., L2-norm, L1-norm, or cross-correlation coefficient) does not affect the pattern of the misfit map (Figure S8 in the Supporting Information S1). 7 of 16 Earth and Space Science 10.1029/2022EA002416 Figure 5. (a) Colored dots represent the original 20,000 models from the receiver function study (Knapmeyer-Endrun et al., 2021) for the 2-layer crustal case. The color scale indicates the number of models at each grid point. The acceptable model regions with the loose (in gray) and strict (in blue) thresholds derived from the SsPp data in this study are superimposed. (b) Reduced models for the 2-layer crustal case with the constraint from the SsPp phase in this study. (c) Same as (a) for the 3-layer crustal case. (d) Same as (b) for the 3-layer crustal case. Figure 5. (a) Colored dots represent the original 20,000 models from the receiver function study (Knapmeyer-Endrun et al., 2021) for the 2-layer crustal case. The color scale indicates the number of models at each grid point. The acceptable model regions with the loose (in gray) and strict (in blue) thresholds derived from the SsPp data in this study are superimposed. (b) Reduced models for the 2-layer crustal case with the constraint from the SsPp phase in this study. (c) Same as (a) for the 3-layer crustal case. 3.2. Constraints From SsPp Our study provides constraints on the average P-wave velocity in Layer 1 using the SsPp phase, which can be compared to the acceptable models in the receiver function study (Knapmeyer-Endrun et al., 2021). In Figures 5a and 5c, we first plotted the distribution of the average P-wave speed and thickness of Layer 1 from the receiver function study (Knapmeyer-Endrun et al., 2021), for the 2-layer and 3-layer crust cases, respectively. In each case, there are 20,000 acceptable models, and most of those models are located along a sub-linear trend reflecting the trade-offs between the wave speed and layer thickness. Then, we superimposed the acceptable model space regions determined from our SsPp analysis for the strict and loose thresholds (blue and gray regions, respectively). For both the 2-layer (Figure 5a) and 3-layer (Figure 5c) cases, acceptable regions derived from the SsPp analyses intersect with the models from Knapmeyer-Endrun et al. (2021). Because the acceptable ensemble of models derived from the SsPp analysis has a different slope compared to the models obtained with receiver functions, the trade-offs between model parameters and the number of possible models can be reduced. That is, models located at the intersection of the two regions are accepted by both the receiver function and the SsPp data. When they are outside the loose threshold contours, 8 of 16 Earth and Space Science 10.1029/2022EA002416 Figure 6. (a) Histograms for the thickness of Layer 1 in the original 2-layer crustal case (in gray) of Knapmeyer-Endrun et al. (2021) and reduced model ensemble (in red). (b) Histograms of the average P-wave speed in Layer 1 for the 2-layer crustal case. (c) Same as (a) for the 3-layer crustal case. (d) Same as (b) for the 3-layer crustal case. Figure 6. (a) Histograms for the thickness of Layer 1 in the original 2-layer crustal case (in gray) of Knapmeyer-Endrun et al. (2021) and reduced model ensemble (in red). (b) Histograms of the average P-wave speed in Layer 1 for the 2-layer crustal case. (c) Same as (a) for the 3-layer crustal case. (d) Same as (b) for the 3-layer crustal case. those models are rejected. Models lying in between the two thresholds have a certain chance of being accepted, according to their misfit (e.g., smaller misfits correspond to larger acceptance possibilities). 3.2. Constraints From SsPp With this new constraint, the total number of possible models is reduced from 20,000 to 8,100 for the 2-layer crustal case, and from 20,000 to 9,982 for the 3-layer crustal case (Figures 5b and 5d). The most prominent feature of these smaller model sets is a cut-off P-wave speed of about 3.2 km/s. 4.1. Method Validation The separation of the pseudo-SV and pseudo-P wave trains is key to removing the source-side scatterings and enhancing the signals from the structure. In this study, we estimated those based on a particle motion analysis (Yu et al., 2012). This approach is fully based on the data and does not rely on a priori knowledge of the near-surface. However, the results could be affected by the presence of noise. To test the influence of the possible noise, we also applied a free-surface transform matrix (Kennett, 1991), constructed from prior information about the P- and S-wave speeds of the near-surface from Kim, Lekić, et al. (2021), to estimate the P- and SV-waveforms. Results show that the derived pseudo-S and pseudo-P wave trains from this free-surface transformation are consistent with those from the particle motion analysis (see Supporting Information S1, Section 1.2). To search for acceptable models, we performed grid searches for three parameters: the average P-wave velocity and the thickness of Layer 1, and the P-wave velocity of the second layer. An alternative way to assess our results is to directly use the 40,000 receiver-function-derived models (Knapmeyer-Endrun et al., 2021) to calculate the synthetic SsPp waveforms and compare them with the SsPp data. Results show that both approaches exhibit the same first-order pattern: models with an average P-wave velocity larger than 3.2 km/s are rejected. This implies that the upper limit of the average P-wave velocity (at around 3.2 km/s) is required by the data and is not depend- ent on the inversion approach (see Supporting Information S1, Section 1.3). We further analyzed the model uncertainties resulting from location error (i.e., epicentral distance and back azimuth) and glitches (see Supporting Information S1, Section 1.1). We also investigated the possible interfer- ences with other signals (see Supporting Information S1, Section 1.4), the sharpness and dip of the interface (see Supporting Information S1, Section 1.5), the effects of different data types (i.e., displacement or velocity records, see Supporting Information S1, Section 1.6), and compared our models with auto-correlation results (see Supporting Information S1, Section 1.7). We concluded that our derived average P-wave speed in Layer 1 (between 2.5 and 3.2 km/s) is robust. 4.2. Origin of Layer 1 The low average P-wave speed in Layer 1 indicates the presence of materials with low seismic velocity in the upper crust at the InSight landing site. Low seismic velocities, in turn, imply materials with low density (compared to the middle or lower crust), that could result from elevated porosity (e.g., Lognonné et al., 2020), low-density lithologies (including chemically altered lithologies), or a combination of intrinsically low-velocity materials and porosity (e.g., Wieczorek et al., 2022). The near-surface geology and stratigraphy in the vicinity of the InSight landing site are now reasonably well understood although the constitution of the deeper crust (i.e., >0.2 km) is less well constrained (Golombek et al., 2020; Pan et al., 2017, 2020; Warner et al., 2022). The subsurface at the InSight landing site includes a shal- low impact-generated regolith (several meters thick) that grades into ∼170 m of Early Amazonian to Hesperian basalt lava flows that are underlain by sedimentary rocks of the Noachian age (Golombek et al., 2017, 2018; Pan et al., 2020; Warner et al., 2022). Orbital imaging and spectral evidence from lithologies thought to be excavated in nearby craters suggest that rocks at greater depth are characterized by Fe/Mg-bearing phyllosilicates and are interpreted to be either Noachian sedimentary rocks (Warner et al., 2022; also see Pan et al., 2020) or aqueously altered Noachian igneous rocks (Pan et al., 2017), or presumably some combination, and that could extend to depths up to 5 km—possibly the entire thickness of Layer 1. 3.3. Average P-Wave Speed and Thickness of Layer 1 In Knapmeyer-Endrun et al. (2021), two ensembles of crustal models were shown to be compatible with the receiver function data: a 2-layer model and a 3-layer model. However, discrepancies were found between the two sets of models, for both the average P-wave speed and the thickness of Layer 1 (i.e., gray histograms in Figure 6): the preferred thickness for Layer 1 is 8.5–9.0 km for the 2-layer case and 7.0–7.5 km for the 3-layer case, and the preferred average P-wave speed for Layer 1 is 3.0–3.5 km/s for the 2-layer case and 2.5–3.0 km/s for the 3-layer case. These discrepancies might be because the properties of Layer 1 (e.g., velocity and thickness) have to be able to explain both the Ps phase and its multiple PpPs phase for the 2-layer crustal case (Knapmeyer-Endrun et al., 2021). The analyses we performed in the present work allow us to obtain new distributions (i.e., red histograms in Figure 6) of possible average P-wave speed and thickness for Layer 1 (Cunningham & Lekic, 2019; Liu et al., 2019) in both the 2-layer case and the 3-layer case. Using the ensemble of models obtained with our additional constraints from the SsPp data, we found that the preferred thickness of Layer 1 for the 3-layer crust case remains unchanged (7.0–7.5 km). However, the preferred thickness for the 2-layer crust case is shallower (7.5–9.0 km) than in the original receiver function study (8.5–9.0 km, from Knapmeyer-Endrun et al., 2021). The preferred average P-wave speeds in both cases are centered between 2.5—3.2 km/s, which is also seen in Figure 5. In addition, the estimated average P-wave speed and thickness of Layer 1 are more consistent between 9 of 16 Earth and Space Science Earth and Space Science 10.1029/2022EA002416 the two cases when adding our SsPp constraints to the original receiver function study. However, based solely on our SsPp analysis and the receiver function analysis of Knapmeyer-Endrun et al. (2021), we cannot distinguish between a 2-layer and a 3-layer crust. 4. Discussion 4.1. Method Validation 4.2.1. Porosity Effects A seismic velocity of 6.8 km/s was assumed for non-porous basaltic materials (Christensen, 1972), a pore aspect ratio of 0.1 was assumed (Heap, 2019), and properties of the void filling materials were also taken from Heap (2019). We see that as the porosity increases, the P-wave speed in the layer decreases, being reduced by a factor of two for porosities close to 20%–25%. The P-wave velocity is somewhat larger when the pores are filled by liquid water than by atmos- pheric gas, but the difference is only moderate for the majority of the range of porosities that we consider. Our average P-wave speeds for the upper 8 km of Mars from InSight data (from 2.5 to 3.3 km/s) can be accounted for by a porosity of 25%–30% when the pores are filled by liquid water, or 22%–26% when the pores are filled by atmospheric gas. If the pores were more spherical than our assumed aspect ratio of 0.1, the amount of required porosity would be greater (see Heap, 2019). In contrast, if the seismic velocity of the matrix materials was lower than assumed, the amount of required porosity would be reduced. t with intrusions of carbon dioxide function of porosity (with the aspect ks the range of the average P-wave er and upper limits for the porosity Figure 7. P-wave speed of porous basalt with intrusions of carbon dioxide (in black) and liquid water (in gray) as a function of porosity (with the aspect ratio of 0.1). The shaded red region marks the range of the average P-wave speeds from this study. The derived lower and upper limits for the porosity estimations are also indicated. Our computed porosities are consistent with the range of values found for a variety of typical extrusive rocks at volcanoes on Earth, which can approach 30% (see data tabulated in Lesage et al., 2018) and for clastic sediments that often exceed 30% (e.g., Boggs, 2009). On the other hand, these porosities are somewhat higher than those directly measured in Martian meteorites, with porosities mostly in the range of 2%–12% (Coulson et al., 2007). Though near-surface volcanic deposits can form with high porosities over a range of length scales (from gas bubbles in magmas to evacuated lava tubes), impact cratering is an additional mechanism that can fracture and generate significant porosity in crustal materials. 4.2.1. Porosity Effects 4.2.1. Porosity Effects Since all of the possible Layer 1 lithologies (sedimentary, volcanic, altered Noachian basement) in the vicinity of the landing site could contain significant porosity, we first consider the influence of porosity alone on seismic wave speed. 10 of 16 Earth and Space Science 10.1029/2022EA002416 Figure 7. P-wave speed of porous basalt with intrusions of carbon dioxide (in black) and liquid water (in gray) as a function of porosity (with the aspect ratio of 0.1). The shaded red region marks the range of the average P-wave speeds from this study. The derived lower and upper limits for the porosity estimations are also indicated. Here, we assess how porosity affects the wave speed of typical Martian basal- tic materials. This will provide us with a maximum allowable porosity, given that other materials (e.g., sedimentary rocks) have intrinsically lower wave speeds. Here, we assess how porosity affects the wave speed of typical Martian basal- tic materials. This will provide us with a maximum allowable porosity, given that other materials (e.g., sedimentary rocks) have intrinsically lower wave speeds. We make use of the scattering theory of Toksöz et al. (1976) to estimate the P-wave speeds of a given material as a function of porosity. As demon- strated by Heap (2019), the bulk seismic velocity depends upon the matrix composition, the amount of porosity, the composition of the material filling the pores, and the pore aspect ratio. We have performed similar calculations as in that study and compared the predicted wave speeds with our average P-wave speed results for Layer 1 beneath the InSight lander. For the model setup, we assumed a basaltic composition for the matrix, given that basalts are the dominant rock type found near the surface of Mars (e.g., McSween et al., 2009). Such a composition could be representative of either basaltic lavas or the detrital grains of unaltered basaltic sediment (e.g., McLennan et al., 2019). After including a specified porosity, the pore space was filled with either atmospheric gas (e.g., carbon dioxide) or liquid water. Manga and Wright (2021) demonstrated that the observed low S-wave speeds in the upper 8 km beneath the InSight lander preclude the existence of water ice in this layer, so we did not consider this case further in our analysis. We plotted our predicted P-wave speeds in Figure 7. 4.2.2. Lithological Effects When considering lithological effects, it is also convenient to think in terms of rock density since seismic veloc- ities for several different possible lithologies are more difficult to directly predict in any systematic manner. Although it is well known that there is a relationship between seismic velocities and rock density, the detailed nature of that relationship is less certain (e.g., Brocher, 2005). Nevertheless, values of the average P-wave speeds in the range of 2.5–3.3 km/s likely correspond to rock densities in the range of about 2,000 to 2,300 kg m −3 (Brocher, 2005; Christensen & Salisbury, 1975; Gardner et al., 1974), compared with an average crustal grain density of <3,100 kg m −3 (Wieczorek et al., 2022; also see Taylor & McLennan, 2009). In Figure 7, we assumed that porosity-free upper crustal basaltic rocks have a P-wave speed of 6.8 km/s which is broadly consistent with the average crustal bulk density that is constrained to be <3,100 kg m −3 (Wieczorek et al., 2022). However, recent findings from rover activities in Gale crater and lithologies preserved in the Martian meteorite breccia NWA7533 (and its numerous pairs) indicate that the early crust of Mars is lithologically diverse (e.g., Cousin et al., 2017; Humayun et al., 2013) with compositions ranging from picrobasalt (SiO2 < 45%) through to alkali-rich intermediate-felsic compositions (SiO2 > 60%). Wieczorek et al. (2022) estimated the grain densities for known igneous lithologies and found them to be in the range of 2,680–3,420 kg m −3, thus varying by over 25% relatively. Accordingly, there is a possibility that at least some of the igneous materials making up the upper crust in the vicinity of InSight have porosity-free P-wave velocities that are lower than the 6.8 km/s assumed here. The favored interpretation of the presence of several kilometers of Noachian sedimentary rocks beneath the landing site is consistent with our current understanding of the scale of the Martian sedimentary record. Based on geochemical mass balance, McLennan (2012) estimated the minimum size of the Martian sedimentary mass to be between 5e10 22 and 5e10 23 g, which, assuming an average density of 2,000 kg m −3 (see below), corresponds to a global average thickness of 0.17–1.7 km. In several locations, sedimentary rock thicknesses are known to be very much greater. 4.2.2. Lithological Effects For example, the sedimentary sequence in Gale crater is measured to be 5 km (Grotzinger et al., 2015), the sedimentary sequence in Juventae Chasma (Valles Marineris) may be on the order of 3–6 km thick (Grotzinger & Milliken, 2012), and the Medusae Fossae Formation in places is up to 3 km thick (Bradley et al., 2002). Globally, the Martian sedimentary rock record is lithologically and mineralogically complex and influenced by a variety of sedimentary processes (e.g., chemical weathering, mineral sorting) and diagenetic processes (e.g., cementation, compaction, secondary porosity formation) (McLennan & Grotzinger, 2008; McLennan et al., 2019). Although sedimentary rocks can contain large amounts of primary intergranular porosity, in many cases that porosity may be lost during compaction and/or filled by diagenetic cements during the lithification process (Boggs, 2009), and for Mars, such cements can be highly variable with respect to mineralogy (phyllosilicates, sulfates and chlorides of variable hydration state, amorphous silica and other amorphous, commonly hydrated, phases) (McLennan & Grotzinger, 2008; McLennan et al., 2019). Although such cements eliminate porosity, their densities can be significantly lower than the grain density of the clastic particles and so the overall effects on both bulk density and seismic velocities would be to lower them but it is not possible to make quantitative predictions. There have been some attempts to independently constrain the densities of Martian sedimentary rocks. Using combined gravity and topography signatures, Ojha and Lewis (2018) estimated a bulk density of 1,765 ± 105 kg m −3 for the Medusae Fossae Formation (also see Watters et al., 2007), a notably low value that was attributed to the result from either high contents of water ice (Watters et al., 2007) or, more likely, elevated porosity (>35% averaged over 1.5 km depth) (Ojha & Lewis, 2018). Using the Curiosity rover accelerometer to measure the gravity field (Lewis et al., 2019), and correcting for the gravity field resulting from the Gale impact, Johnson et al. (2021) estimated the mean bulk density of the sedimentary rocks in Gale crater to be 2,300 ± 130 kg m −3. This density was considered consistent with a porosity of 18 ± 6%, a value in turn consistent with lithified sedimentary rocks that have undergone about 4–5 km of burial compaction (Johnson et al., 2021). 4.2.1. Porosity Effects As an example, combined gravity and remote sensing data imply that the average porosity of the crust of the Moon is about 12% (Wieczorek et al., 2013) and that the poros- ity could be even higher for the uppermost crust (Besserer et al., 2014). Analyses of feldspathic samples from the lunar highlands reveal impact-generated porosities that range from about 2% to 20% (Kiefer et al., 2012). Drill core samples from the central peak ring of the Chixulub impact basin have similar average porosities as the lunar samples, near 12% at depths near a kilometer, with values that reach as high as 20% at shallower depths (Rae et al., 2019). Drill cores from within the Reis impact crater on Earth also show the presence of up to about 30% porosity in the upper few hundred meters (Förstner, 1967). Given the ancient age of the surface volcanic materi- als at the Insight landing site (from Hesperian to Early Amazonian, see Warner et al., 2022), combined with the presence of an about 10 m thick impact generated regolith at the surface in the vicinity of the landing site, impact processes could have plausibly contributed to high levels of porosity in the upper 8 km of the crust beneath the InSight lander. 11 of 16 Earth and Space Science 10.1029/2022EA002416 4.2.3. Synthesis From the above analysis, it is clear that porosity alone—in both sedimentary and volcanic rocks—could poten- tially explain the low average P-wave speed observed in Layer 1. On the other hand, it is less likely that litho- logical factors alone, such as the presence of cemented sedimentary rocks, aqueously altered igneous rocks, or more felsic rocks, could do so. Nevertheless, our current understanding suggests that the upper crustal stratig- raphy beneath the landing site is dominated by lithologies that have reduced densities related to the presence of secondary materials such as sedimentary cements and other hydrous alteration phases. Accordingly, our favored hypothesis is that a combination of low-density lithologies (cemented sedimentary rocks, intermediate-felsic igneous rocks, and aqueously altered Noachian igneous rocks) almost certainly played a significant role in reduc- ing P-wave speed. However, in addition to that, it is also necessary that significant primary porosity (and for sedimentary rocks, possibly secondary porosity) remained in many of these rocks. A final issue is what is the origin of the seismic discontinuity at the base of Layer 1. Given the known thicknesses of sedimentary rocks on Mars, a Noachian sedimentary succession on the order of ≥7 km thickness is plausible and so one possibility is that the base of Layer 1 is essentially the base of a sedimentary rock sequence. If, on the other hand, aqueously altered igneous rocks dominate at these depths, then the boundary could also correspond to the maximum depth of aqueous alteration and therefore fluid flow. In either case, it is likely that porosity also plays a significant role. Both compaction and viscous deformation will result in porosity reduction with depth. Gyalay et al. (2020) showed that the closure of pore space should occur over a narrow depth range of a few kilometers. Above this transition zone, the rocks retain their initial porosity, whereas below this transition zone all porosity is removed. The absolute depth of the transition zone depends upon the heat flow at the time when the porosity was created. If most porosities were created by impacts before 3.9 Ga, based on reasonable estimates of the surface heat flow at that time, all porosity would have since been removed for depths greater than about 12–23 km (Wieczorek et al., 2022). Thus, if Layer 1 initially contained high porosities near 22%–30% at 3.9 Ga, this porosity would remain to the present day. 4.2.3. Synthesis If Layer 1 instead formed at a later date (such as from sedimentary processes), these materials would also retain their initial porosity to the present day. Accordingly, a combination of lithological change and pore reduction (or elimination) is a plausible mechanism to explain the seismic discontinuity at the base of Layer 1 and is also consistent with the known geological rela- tionships in the vicinity of the InSight lander site. 4.2.2. Lithological Effects Although considered less likely, another deep upper crustal lithology that may underlie the InSight landing site is aqueously altered Noachian igneous rocks, possibly similar to the rocks in the ancient highlands ∼500 km to the southwest. Pan et al. (2017) examined the mineralogy of deeper crustal materials exposed in craters throughout the northern lowland and found a variable mixture of primary volcanic mafic minerals and a variety of hydrous minerals, dominated by Fe/Mg phyllosilicates, interpreted to have formed by aqueous alteration processes in 12 of 16 Earth and Space Science Earth and Space Science 10.1029/2022EA002416 the Noachian crust. The density of Fe/Mg phyllosilicates (e.g., nontronite, saponite) is mostly in the range of 2,200–2,300 kg m −3 (Anthony et al., 2002) and thus are also likely to lower the density of the primary crustal igneous materials. the Noachian crust. The density of Fe/Mg phyllosilicates (e.g., nontronite, saponite) is mostly in the range of 2,200–2,300 kg m −3 (Anthony et al., 2002) and thus are also likely to lower the density of the primary crustal igneous materials. 5. Conclusions We have analyzed one quality-A and five quality-B broadband and low-frequency events and made the first coherent detection of the SsPp phase on Mars, which helps us constrain the crustal structure at the lander site. We found that quality-B marsquakes, with simpler source wavelets, behave better than quality-A events when constraining the structure of the uppermost crustal layer (at about 8 km depth) when using the SsPp phase. We found coherent signals that are consistent with wave reflections off the first crustal interface, and this new phase confirms the existence of the ∼8 km interface in the crust and the large wave speed (or impedance) contrast across it. The detected SsPp phase helped reduce the number of acceptable models used in the previous receiver function analysis of Knapmeyer-Endrun et al. (2021) from 20,000 models to about 10,000 models. Using our new constraint from the SsPp phase, we determined that the average P-wave speed in Layer 1 is between 2.5 km/s and 3.2 km/s, compared to the previous range of 2.0–3.5 km/s obtained by receiver function analysis. Based on these low average P-wave speeds, the seismic properties of Layer 1 likely result primarily from the presence of relatively low-density lithified sedimentary rocks and/or aqueously altered igneous rocks that also have a significant amount of porosity, possibly as much as ∼30% by volume. 13 of 16 Earth and Space Science 10.1029/2022EA002416 Data Availability Statement Data sets (both the raw data and the deglitched data in SAC format, after removing the instrument response) for this research are available on the Zenodo repository: https://doi.org/10.5281/zenodo.6784826. References Constraining crustal structure in the presence of sediment: A multiple converted wave approach. Geop ical Journal International, 219(1), 313–327. https://doi.org/10.1093/gji/ggz298 Cunningham, E., & Lekic, V. (2019). Constraining crustal structure in the presence of sediment: A ical Journal International, 219(1), 313–327. https://doi.org/10.1093/gji/ggz298 Deng, S., & Levander, A. (2020). 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Compressional and shear wave velocities at pressures to 10 kilobars for basalts from the east Pacific rise. Geophysical Journal International, 28(5), 425–429. https://doi.org/10.1111/j.1365-246x.1972.tb06140.x Christensen, N. I. (1972). Compressional and shear wave velocities at pressures to 10 kilobars for b Journal International, 28(5), 425–429. https://doi.org/10.1111/j.1365-246x.1972.tb06140.x ristensen, N. I., & Salisbury, M. W. (1975). Structure and constitution of the lower oceanic crust. Reviews of Geophysics and Space Physics, 13, 57–86. https://doi.org/10.1029/rg013i001p00057 Christensen, N. I., & Salisbury, M. W. (1975). Structure and constitution of the lower oceanic cru 13, 57–86. https://doi.org/10.1029/rg013i001p00057 This is InSight Contribution Number ICN 245. InSight seismic data presented here (http://dx.doi.org/10.18715/SEIS. INSIGHT.XB_2016) are publicly avail- able through the Planetary Data System (PDS) Geosciences node (InSight SEIS Data Bundle 2021), the Incorporated Research Institutions for Seismology (IRIS) Data Management Center under network code XB and through the Data center of Institut de Physique du Globe, Paris. The authors acknowledge NASA, CNES, their partner agencies and Institutions (UKSA, SSO, DLR, JPL, IPGP-CNRS, ETHZ, IC, MPS-MPG) and the flight operations team at JPL, SISMOC, MSDS, IRIS-DMC and PDS for providing SEED SEIS data. p g g p Clinton, J. F., Ceylan, S., Van Driel, M., Giardini, D., Stähler, S. C., Böse, M., et al. (2021). The marsquake catalogue from InSight, sols 0–478. Physics of the Earth and Planetary Interiors, 310, 106595. https://doi.org/10.1016/j.pepi.2020.106595 y f y p g j p p Coulson, I. M., Beech, M., & Nie, W. (2007). Physical properties of Martian meteorites: Porosity and density measurements. 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The misfit maps are plotted with the Scientific color maps (Crameri, 2018). Acknowledgments J. Li thank Tiezhao Bao and Weiliang Yin for sharing codes for the particle motion analysis, Ross Maguire for providing the Marsquakes downloading script, and Brigitte Knapmeyer-Endrun for discuss- ing the models from the receiver function study. The authors thank three anonymous reviewers and editor David Baratoux for their constructive comments. The misfit maps are plotted with the Scientific color maps (Crameri, 2018). J. Li and C. Beghein were supported by NASA InSight PSP grant #80NSSC18K1679 and S. M. McLennan by NASA InSight PSP grant #80NSSC18K1622. P. Lognonné, E. Stutzmann, and M. Wieczorek are supported by Agence Nation- ale de la Recherche (MAGIS, ANR-19-CE31-0008-08; IdEx Université Paris Cité, ANR-18-IDEX-0001) and by CNES for SEIS science support. M. Schimmel thanks SANIMS (RTI2018-095594-B-I00). This is InSight Contribution Number ICN 245. 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https://openalex.org/W2043601120	https://europepmc.org/articles/pmc4113732?pdf=render	English	null	Novel Preclinical and Radiopharmaceutical Aspects of [68Ga]Ga-PSMA-HBED-CC: A New PET Tracer for Imaging of Prostate Cancer	Pharmaceuticals	2,014	cc-by	8,475	Pharmaceuticals 2014, 7, 779-796; doi:10.3390/ph7070779 pharmaceuticals ISSN 1424-8247 www.mdpi.com/journal/pharmaceuticals Article Novel Preclinical and Radiopharmaceutical Aspects of [68Ga]Ga-PSMA-HBED-CC: A New PET Tracer for Imaging of Prostate Cancer Matthias Eder 1,2,†,, Oliver Neels 1,2,†, Miriam Müller 1, Ulrike Bauder-Wüst 1, Yvonne Remde 1, Martin Schäfer 1, Ute Hennrich 1,2, Michael Eisenhut 1, Ali Afshar-Oromieh 3, Uwe Haberkorn 3 and Klaus Kopka 1,2 1 German Cancer Research Center (dkfz), Division of Radiopharmaceutical Chemistry, Im Neuenheimer Feld 280, Heidelberg 69120, Germany; E-Mails: o.neels@dkfz.de (O.N.); miriam.mueller@wak-gmbh.de (M.M.); u.bauder-wuest@dkfz.de (U.B.-W.); y.remde@dkfz.de (Y.R.); martin.schaefer@dkfz.de (M.S.); u.hennrich@dkfz.de (U.He.); m.eisenhut@dkfz.de (M.E.); k.kopka@dkfz.de (K.K.) 2 German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, Heidelberg 69120, Germany 3 Department of Nuclear Medicine, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany; E-Mails: Ali.Afshar@med.uni-heidelberg.de (A.A.-O.); Uwe.Haberkorn@med.uni-heidelberg.de (U.Ha.) † The authors contributed equally to this work. Author to whom correspondence should be addressed; E-Mail: m.eder@dkfz.de; Tel.: +49-622-142-2423; Fax: +49-622-142-2434. Received: 20 February 2014; in revised form: 11 June 2014 / Accepted: 12 June 2014 / Published: 30 June 2014 OPEN ACCESS Pharmaceuticals 2014, 7, 779-796; doi:10.3390/ph7070779 1. Introduction Early detection of metastases or recurrent prostate cancer (PC) lesions is of utmost clinical relevance in terms of clinical staging, prognosis and therapy management [1,2]. The high clinical impact of targeting the prostate-specific membrane antigen (PSMA) was recently demonstrated in a series of first-in-man examinations with either 68Ga- [3–5] or 123I-labelled [6] PSMA inhibitors. PSMA is a membrane-type zinc protease, also called glutamate carboxypeptidase II (GCPII), which is expressed by nearly all prostate cancers. Enhanced expression levels were found in poorly differentiated, metastatic and hormone-refractory carcinomas [7,8]. Since urea-based inhibitors of PSMA clear rapidly from the circulation and since only low levels of physiological PSMA expression were detected in a few organs like the brain, kidney, salivary gland and small intestine [7,9–11] PSMA represents an ideal biological target for high quality PET imaging of prostate cancer [12–17]. Initial clinical experiences with the 68Ga-labelled PET tracer Glu-NH-CO-NH-Lys-(Ahx)-[[68Ga] Ga(HBED-CC)] ([68Ga]Ga-PSMA-HBED-CC) suggest that this novel tracer detects PC relapses and metastases with higher contrast as compared to 18F-choline. In a retrospective study, the images of 37 patients who received both 18F-choline PET/CT and 68Ga-PSMA PET/CT were analyzed [3]. Especially at low PSA values, PET/CT images obtained with [68Ga]Ga-PSMA-HBED-CC showed more PC lesions as compared to 18F-choline. It was concluded that [68Ga]Ga-PSMA-HBED-CC represents an attractive new imaging agent for the detection of recurrent prostate cancer and metastatic spread. The chelator HBED-CC (N,N'-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid), represents a hitherto rarely used acyclic complexing agent especially allowing efficient radiolabelling with 68Ga even at ambient temperature [18,19]. By combining HBED-CC with the PSMA inhibitor Glu-urea-Lys, a favourable aromatic part is introduced into the radiotracer which was found to be a necessary requirement for a sustainable interaction with the PSMA receptor, putatively with the accessory hydrophobic pocket of the PSMA S1 binding site [15,20,21]. We have indeed shown in a preclinical study that the replacement of HBED-CC by DOTA (1,4,7,10- tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid) resulted in a molecule not able to image the tumour at all [20]. Moreover, besides these biological advantages, HBED-CC represents a highly effective chelator. Extraordinary high thermodynamic stability constants of >1039 were determined for the complexation of Ga with HBED [22]. As mentioned before the structure is acyclic and demands rather low energy for complex formation which allows fast labelling at ambient temperature [19,20]. Novel Preclinical and Radiopharmaceutical Aspects of [68Ga]Ga-PSMA-HBED-CC: A New PET Tracer for Imaging of Prostate Cancer Matthias Eder 1,2,†,, Oliver Neels 1,2,†, Miriam Müller 1, Ulrike Bauder-Wüst 1, Yvonne Remde 1, Martin Schäfer 1, Ute Hennrich 1,2, Michael Eisenhut 1, Ali Afshar-Oromieh 3, Uwe Haberkorn 3 and Klaus Kopka 1,2 1 German Cancer Research Center (dkfz), Division of Radiopharmaceutical Chemistry, Im Neuenheimer Feld 280, Heidelberg 69120, Germany; E-Mails: o.neels@dkfz.de (O.N.); miriam.mueller@wak-gmbh.de (M.M.); u.bauder-wuest@dkfz.de (U.B.-W.); y.remde@dkfz.de (Y.R.); martin.schaefer@dkfz.de (M.S.); u.hennrich@dkfz.de (U.He.); m.eisenhut@dkfz.de (M.E.); k.kopka@dkfz.de (K.K.) German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, Heidelberg 69120, Germany 3 Department of Nuclear Medicine, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany; E-Mails: Ali.Afshar@med.uni-heidelberg.de (A.A.-O.); Uwe.Haberkorn@med.uni-heidelberg.de (U.Ha.) † The authors contributed equally to this work. † The authors contributed equally to this work. Author to whom correspondence should be addressed; E-Mail: m.eder@dkfz.de; Tel.: +49-622-142-2423; Fax: +49-622-142-2434. Received: 20 February 2014; in revised form: 11 June 2014 / Accepted: 12 June 2014 / Published: 30 June 2014 Abstract: The detection of prostate cancer lesions by PET imaging of the prostate-specific membrane antigen (PSMA) has gained highest clinical impact during the last years. 68Ga-labelled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]Ga-PSMA-HBED-CC) represents a successful novel PSMA inhibitor radiotracer which has recently demonstrated its suitability in individual first-in-man studies. The radiometal chelator HBED-CC used in this molecule represents a rather rarely used acyclic complexing agent with chemical characteristics favourably influencing the biological functionality of the PSMA inhibitor. The simple replacement of HBED-CC by the prominent radiometal chelator DOTA was shown to dramatically reduce the in vivo imaging quality of the respective 68Ga-labelled PSMA-targeted tracer proving that HBED-CC contributes intrinsically to the PSMA binding of the 780 Pharmaceuticals 2014, 7 Glu-urea-Lys(Ahx) pharmacophore. Owing to the obvious growing clinical impact, this work aims to reflect the properties of HBED-CC as acyclic radiometal chelator and presents novel preclinical data and relevant aspects of the radiopharmaceutical production process of [68Ga]Ga-PSMA-HBED-CC. Keywords: 68Ga-PET imaging; PSMA; HBED-CC; prostate cancer; radiopharmaceutical production; good manufacturing practice; GMP Keywords: 68Ga-PET imaging; PSMA; HBED-CC; prostate cancer; radiopharmaceutical production; good manufacturing practice; GMP Pharmaceuticals 2014, 7 represents a highly attractive radiogallium chelator for high-stability labelling of radiopharmaceuticals. However, in contrast to other clinically well-established radiometal chelators, HBED-CC forms three NMR-distinguishable diastereomers (RR, RS and SS configurations at the amine nitrogens) during gallium complexation, whereas presumably the RR configuration is thermodynamically favoured [24]. Besides the influence of the temperature the formation of the diastereomers was reported to be pH- and concentration-dependent as well [23]. In a standard labelling protocol, [68Ga]Ga-PSMA-HBED-CC is incubated at a pH of ~4 and heated at 95 °C. The thermodynamically favoured diastereomer is formed; however, a small fraction of one of the other two diastereomers is still present in the labelling reaction and would be part of the final formulation prepared for the patient. Thus, it is of utmost importance to analyze the two major diastereomers according to their biological activity in cell-based assays. The aim of this study is to summarize hitherto existing radiochemical experiences with HBED-CC conjugated low-molecular weight compounds and to confirm that the configuration of Ga-HBED-CC does not influence the cell binding properties of the resulting PSMA-targeted radioligand [68Ga]Ga- PSMA-HBED-CC, especially since the chelator has high impact on the interaction with the aforementioned accessory hydrophobic pocket of PSMA. Owing to the high and growing clinical impact, this work aims to present novel important preclinical data of [68Ga]Ga-PSMA-HBED-CC and relevant aspects of its radiopharmaceutical production. The fully automated radiosynthesis as well as the essential quality control parameters according to current EU-GMP regulations for radiopharmaceuticals are given. 1. Introduction Moreover, a high kinetic stability of the Ga-HBED-CC complex at physiological pH was reported [23] resulting in a stable complex in vivo [24] and in human serum for at least 72 h [25]. Thus, HBED-CC 781 Pharmaceuticals 2014, 7 Pharmaceuticals 2014, 7 782 The peptide c(RGDyK) was synthesised as described previously [26]. HBED-CC was conjugated by reacting with 1.2 equivalents of HBED-CC-TFP-ester which was synthesised as previously described [19]. The reaction mixture was supplemented with two equivalents of DIPEA in N,N-dimethylformamide (DMF). After HPLC purification (vide supra) the product was treated with TFA at room temperature for one hour resulting in the final compound. NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation was carried out by reacting the peptide with 3 equivalents of SCN-Bn-NOTA (purchased from Macrocyclics, Dallas, TX, USA) in 0.1 M sodium carbonate buffer (pH 9.5) for 20 h at room temperature. The reaction mixture was purified by semipreparative HPLC. Mass spectrometry was used to confirm the identity of the synthesised compounds (m/z (NOTA-c(RGDyK) = 1071.3 (calc. for [M+H]+ = 1071.2); m/z (HBED-CC-c(RGDyK) = 1035.2 (calc. for [M+H]+ = 1035.2)). 2.2.1. 68Ga-Radiolabelling 68Ga (half-life 68 min; β+ 89%; Eβ+ max. 1.9 MeV) was obtained from a 68Ge/68Ga-generator based on a pyrogallol resin support [27]. For radiolabelling, the conjugates (0.1–1 nmol in 0.1 M HEPES buffer, pH = 7.5, 100 µL) were added to a mixture of 10 µL HEPES (2.1 M in H2O) and 40 µL [68Ga]Ga3+ eluate. The pH of the labelling solution was adjusted using NaOH. The reaction mixture was incubated at room temperature or 95 °C, respectively. The radiochemical yield (RCY) was determined via analytical RP-HPLC and thin layer chromatography (ITLC, Pall, Crailsheim, Germany) with a mixture of 0.9% NaCl and methanol (5:1) as solvent. Free activity was complexed by supplementing with 10 µL 0.1 M EDTA. In case of non-conjugated chelators, 0.9% NaCl was used as solvent. 2.2.2. natGa-Complexes A 10 times molar excess of Ga(III)-nitrate (Sigma Aldrich, Munich, Germany) in 10 µL 0.1 N HCl was reacted with the conjugates (1 mM in 0.1 M HEPES buffer pH 7.5, 40 µL) in a mixture of 10 µL 2.1 M HEPES solution and 2 µL 1 N HCl for 2 min at room temperature or 95 °C, respectively. 2.1. Reagents and Chemical Syntheses The chemicals were of analytical grade and were used without further purification. Analysis of the synthesised molecules was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Chromolith RP-18e, 100 mm × 4.6 mm; Merck, Darmstadt, Germany) with a linear A–B gradient (0% B to 100% B in 6 min) at a flow rate of 4 mL/min (analysis) or 6 mL/min (purification). Solvent A consisted of 0.1% aqueous trifluoroacetic acid (TFA) and solvent B was 0.1% TFA in acetonitrile. The HPLC system (L6200 A; Merck-Hitachi, Darmstadt, Germany) was equipped with a UV and a gamma detector (Bioscan, Washington, DC, USA). UV absorbance was measured at 214 nm and 254 nm. Mass spectrometry was performed with a MALDI-MS Daltonics Microflex system (Bruker Daltonics, Bremen, Germany). The natGa-labelled reference Glu-urea-Lys(Ahx)-[Ga(HBED-CC)] (DKFZ-GaPSMA-11) was purchased from ABX Advanced Biochemical Compounds (Radeberg, Germany) and dissolved in 40 µL CH3CN/H2O (1:1). 2.6 µg were characterised at RT by mass spectrometry using an Agilent 1200 HPLC-MS system connected to an Orbitrap Mass Spectrometer (Exactive, Thermo Fisher Scientific) on a Hypersil Gold C18 column (2.1 mm × 200 mm, 1.9 µm; Thermo Scientific, Bremen, Germany) eluted with a linear gradient (eluent A: 0.05% TFA in water; eluent B: 0.05% TFA in acetonitrile; 0%–30% B in 30 min at RT, flow rate: 0.2 mL/min, absorbance: λ = 214 nm). Full scan single mass spectra (positive mode) were obtained by scanning from m/z = 200–4000. 2.3.2. Cell Binding and Internalisation 2.3.2. Cell Binding and Internalisation The competitive cell binding assay and internalisation experiments were performed as described previously [20]. Briefly, LNCaP cells (105 per well) were incubated with the radioligand [68Ga] Ga-PSMA-HBED-CC in the presence of 12 different concentrations of analyte (0–5000 nM, 100 µL/well, natGa-labelled Glu-urea-Lys(Ahx)-HBED-CC prepared either by reaction at room temperature (RT) or by reaction at 95 °C). After incubation, washing was performed using a multiscreen vacuum manifold from Millipore (Billerica, MA, USA). Cell-bound radioactivity was determined using a gamma counter (Packard Cobra II, GMI, Ramsey, MN, USA). The 50% inhibitory concentration (IC50) was calculated using a nonlinear regression algorithm (GraphPad Software, version 5.01, La Jolla, CA, USA). Experiments were performed in triplicate. The specific cell uptake and internalisation was determined in another cell-based assay. Briefly, 105 cells per well were seeded in poly-L-lysine coated 24-well cell culture plates 24 h before incubation. After washing, the cells were incubated with 25 nM of the radiolabelled compounds (either labelled at RT or 95 °C) for 45 min at 37 °C and at 4°C, respectively. Specific cellular uptake was determined by competitive blocking with the PSMA inhibitor 2-(phosphonomethyl)pentanedioic acid (500 µM final concentration, 2-PMPA, Axxora, Loerrach, Germany). Cellular uptake was terminated by washing 4 times with 1 mL of ice-cold PBS. Cells were subsequently incubated twice with 0.5 mL glycine-HCl in PBS (50 mM, pH = 2.8) for 5 min to remove the surface-bound fraction. The cells were washed with 1 mL of ice-cold PBS and lysed using 0.5 mL 0.3 N NaOH. The radioactivity of the probes was measured in a gamma counter. The cell uptake was calculated as per cent of the initially added radioactivity bound to 106 cells [%IA/106 cells]. 2.3. In Vitro Testing 2.3. In Vitro Testing 2.3. In Vitro Testing 2.2.3. Radiochemical Stability The radiochemical stability of the 68Ga-labelled compounds was determined by incubating in both phosphate buffered saline (PBS) and human serum at 37 °C. An equal volume of acetonitrile was added to the samples to precipitate serum proteins. Subsequently, the samples were centrifuged for 5 min at 13,000 rpm (Heraeus Picofuge fresco, Thermo Fisher Scientific Germany, Schwerte, Germany). An aliquot of the supernatant and the PBS sample was analysed by RP-HPLC. In addition, serum samples were run on a Superdex 75 5/150 GL gel filtration column (GE Healthcare, Munich, Germany) in order to analyse protein binding. To investigate the complex stability against human transferrin, a 400 µL aliquot of the 68Ga-labelled peptide was added to 250 µg apo-transferrin and incubated at 37 °C in PBS at pH 7. The complex stability was determined using a Superdex 75 GL 5/150 short column with PBS (pH 7) as eluent. 783 2.3.1. Cell Culture LNCaP cells (metastatic lesion of human prostatic adenocarcinoma, ATCC CRL-1740) were cultured in RPMI medium supplemented with 10% fetal calf serum and Glutamax (PAA, Pasching, Austria). The cells were grown at 37 °C in an incubator with humidified air equilibrated with 5 % CO2. The cells were harvested using trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; 0.25% trypsin, 0.02% EDTA, all from PAA, Austria) and washed with PBS. 2.4. Automated Synthesis A fully automated synthesis module (Scintomics GRP, Fürstenfeldbruck, Germany) and its ControlCenter and GRP-Interface software were used to transfer the radiosynthesis of [68Ga] Ga-PSMA-HBED-CC into an environment suitable for clinical application. The 68Ge/68Ga-generator used for radiopharmaceutical production was purchased from IDB-Holland BV (Baarle-Nassau, The Netherlands). Disposable cassette kits and chemicals including the precursor PSMA-HBED-CC (DKFZ-PSMA-11) in GMP-compliant grade used for the radiosynthesis were obtained from ABX advanced biochemical compounds. A Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Dreieich, Germany) equipped with a Chromolith Performance RP-18e column (100 mm × 4.6 mm, 784 Pharmaceuticals 2014, 7 Merck) and a NaI radiodetector (Raytest, Straubenhardt, Germany) was used to determine the radiochemical purity. The mobile phase consisted of gradient mixtures of acetonitrile (A) and 0.1% aqueous TFA (B); 0–0.5 min: 95% B; 0.5 to 10 min linear gradient to 80% A; flow rate 2 mL/min. Residual solvents were determined using a 6850 Series gas chromatograph (Agilent Technologies, Böblingen, Germany). Bacterial endotoxin testing was performed using the LAL test with an Endosafe®-PTS device (Charles River Laboratories, Wilmington, DE, USA). The radionuclide was identified by determination of the half-life (67.9 min) using a CRC-15R dose calibrator (Capintec, Ramsey, NJ, USA). Radionuclidic purity of the final product solution and separation cartridges was analysed using gamma spectrometry (HPGe Canberra GC 5020, Meriden, CT, USA). Sterility testing was realised at the Department for Infectiology of the Heidelberg University Hospital. 1.9 µg (2 nmol) of PSMA-HBED-CC were dissolved in a mixture of 1.5 M acetate buffer pH 4.5 (1 mL) and 1 M ascorbic acid (10 µL) and the mixture was transferred into the reaction vessel. The 68Ge/68Ga-generator was eluted with 10 mL of 0.6 M HCl and the eluate diluted with 9 mL of ultrapure water. The mixture was then transferred to a cation exchange cartridge (Macherey-Nagel PS-H+, Size M, Düren, Germany) and eluted with 5 M NaCl solution (1.2 mL) into the preheated reaction vessel (100 °C). The reaction mixture was heated for 10 min. The crude reaction mixture was then removed from the reaction vessel and transferred to a pre-conditioned (1. 10 mL EtOH/ 2. 10 mL ultrapure water) C18 cartridge (Waters Sep-Pak light, Eschborn, Germany). 9 mL ultrapure water was used to rinse the reaction vessel and passed over the C18 cartridge. The C18 cartridge was washed with another 5 mL of ultrapure water. The final product was eluted from the C18 cartridge with 2 mL of EtOH/H2O (1:1 v:v), sterile filtered (Millipore Cathivex-GV, 0.22 µm) and diluted with 8 mL of PBS solution (according to Ph. Eur. 8.0 (4005000)). All quality control tests except those for sterility and radionuclidic purity were determined prior release of the final product. 3.1. HBED-CC as Chelator for Highly Efficient Radiolabelling of Peptides A B 0 5 10 15 20 25 30 35 40 45 50 0 20 40 60 80 100 HBED-CC-Peptide NOTA-Peptide c [µM] RCY [%] 0 5 10 15 20 0 20 40 60 80 100 1.7 µM NOTA-Peptide 1.7 µM HBED-CC-Peptide reaction time [min] RCY [%] h diff l b lli ki i f CC d O A b d i h i l A B 0 5 10 15 20 25 30 35 40 45 50 0 20 40 60 80 100 HBED-CC-Peptide NOTA-Peptide c [µM] RCY [%] 0 5 10 15 20 0 20 40 60 80 100 1.7 µM NOTA-Peptide 1.7 µM HBED-CC-Peptide reaction time [min] RCY [%] A B 0 5 10 15 20 25 30 35 40 45 50 0 20 40 60 80 100 c [µM] RCY [%] RCY [%] B 0 0 5 10 15 20 0 20 40 60 80 100 reaction time [min] RCY [%] B The different labelling kinetics of HBED-CC and NOTA observed in these experiments were also confirmed in a competition-for-chelation assay performed with the HBED-CC- and NOTA-conjugated RGD-peptides. Equal concentrations of HBED-CC-c(RGDyK) and NOTA-c(RGDyK) were incubated with [68Ga]Ga3+ at room temperature. Also under these conditions, HBED-CC turned out to be the more potent chelator in direct challenging with NOTA, as 16% of the activity was complexed by NOTA-c(RGDyK) whereas 84% of the activity was incorporated in HBED-CC-c(RGDyK). Labelling the free acid of HBED-CC was effective at pH values between 3.5 and 5 with no significant reduction of RCY between pH 4 and 5 (Figure 2). This fact ensures a high reproducibility and highly reliable radiopharmaceutical production process as a broad range of pH and an efficient radiolabelling at low concentrations of precursor enhance the robustness of the synthesis. Another aspect which determines the quality and the clinical impact of a radiometal chelator is its serum stability. As metal complexes of open chain ligands generally show a lower degree of kinetic stability compared to cyclic chelators it is of importance to prove the long term stability of Ga-HBED-CC as active pharmaceutical ingredient of a radiopharmaceutical before its clinical use. The long term stability of radiogallium labelled HBED-CC-c(RGDyK) and NOTA-c(RGDyK) in human serum was investigated using the longer living radionuclide 67Ga. Neither the NOTA-conjugate nor the HBED-CC-conjugate showed non-complexed 67Ga in ionic form after 48 h-incubation in human serum at 37 °C. 3.1. HBED-CC as Chelator for Highly Efficient Radiolabelling of Peptides The acyclic radiometal chelator HBED-CC was reported to represent a highly effective 68Ga complexing agent mainly suitable for gentle room temperature-radiolabelling of antibodies and proteins [18,19,24]. However, less is known about labelling kinetics and stability issues of 68Ga-labelled HBED-CC-conjugated peptides. First insights in the quality of HBED-CC as labelling moiety of low-molecular weight compounds were given in the context of HBED-CC-conjugated PSMA inhibitors. We have recently reported that Glu-urea-Lys(Ahx)-HBED-CC can be labelled in less than 2 min with 99% radiochemical yield (RCY) at room temperature [20,28]. Here we summarise our PSMA-independent radiochemical experiences with the HBED-CC-conjugated peptide c(RGDyK) compared to the NOTA-labelled counterpart. Figure 1A shows a typical dependency of the radiochemical yield (RCY) over time demonstrating the fast labelling kinetics of the examined HBED-CC-conjugated peptides at room temperature. A direct comparison of the labelling kinetics of a NOTA-conjugate and a HBED-CC-conjugate on the basis of proteins was already published previously [18] and is in good agreement with the herein presented data. In comparison to NOTA, HBED-CC complexes [68Ga]Ga3+ more efficiently at low 785 Pharmaceuticals 2014, 7 concentrations and low temperatures. A concentration of 1.7 µM was found to be sufficient to form the 68Ga-labelled peptide with high radiochemical yields in less than one minute at room temperature. Clear differences were still observed after 20 min incubation of the reaction mixtures (Figure 1B). Figure 1. The radiochemical yields (RCY) of the HBED-CC- and NOTA-conjugated model-peptide c(RGDyK) as a function of (A) peptide concentration (2 min reaction time); and (B) reaction time (1.7 µM peptide concentration), respectively. The compounds were incubated with the generator eluate at room temperature in HEPES buffer (pH 4.2). The reaction was stopped by adding 10 µL of a 0.1 M EDTA solution and the reaction mixture was subsequently analysed via radio-HPLC (n = 6). The different labelling kinetics of HBED-CC and NOTA observed in these experiments were also confirmed in a competition-for-chelation assay performed with the HBED-CC- and NOTA-conjugated RGD-peptides. Equal concentrations of HBED-CC-c(RGDyK) and NOTA-c(RGDyK) were incubated with [68Ga]Ga3+ at room temperature. Also under these conditions, HBED-CC turned out to be the more potent chelator in direct challenging with NOTA, as 16% of the activity was complexed by NOTA-c(RGDyK) whereas 84% of the activity was incorporated in HBED-CC-c(RGDyK). 3.1. HBED-CC as Chelator for Highly Efficient Radiolabelling of Peptides Remarkably, ~3% of the radioactivity was released from the NOTA-conjugate after 1 week in PBS at room temperature while the HBED-CC conjugate remained unchanged (>99% radiochemical purity (RCP)). This might be explained by a slight instability of the NOTA complex which was also demonstrated by analysing the serum samples on gel filtration (Figure 3). About 13% of the original 786 Pharmaceuticals 2014, 7 peptide-bound radioactivity was eluted at about the same time as the serum proteins. In order to specify the transfer of 67Ga to the serum proteins, the stability was further investigated by incubating the 68Ga-labelled peptides in the presence of an excess of apo-transferrin for 2 h at 37 °C. However, no radiogallium was transchelated to apo-transferrin in both cases. Figure 2. The pH dependence of the complexation reaction was determined by incubating the free acid of (A) HBED-CC (10 µM) and (B) NOTA (10 µM), respectively, at RT for 10 min. The radiochemical yield was determined by ITLC at the indicated time points (n = 6). Figure 2. The pH dependence of the complexation reaction was determined by incubating the free acid of (A) HBED-CC (10 µM) and (B) NOTA (10 µM), respectively, at RT for 10 min. The radiochemical yield was determined by ITLC at the indicated time points (n = 6). B 0 1 2 3 4 5 6 7 8 9 10 0 20 40 60 80 100 pH 3 pH 3.5 pH 5 pH 4 reaction time [min] RCY [% ] A 0 1 2 3 4 5 6 7 8 9 10 0 20 40 60 80 100 reaction time [min] RCY [% ] Figure 3. Superdex 75 5/150 GL runs of (A) 67Ga-labelled HBED-CC-c(RGDyK); and (B) 67Ga-labelled NOTA-c(RGDyK) after 48 h incubation in human serum at 37 °C. Only the radiometric signal is shown. The UV trace showed a major peak at ~3.9 min p.i.; (C) which corresponds with the elution time of serum proteins. B A 0 1 2 3 4 5 6 7 8 9 10 0 20 40 60 80 100 reaction time [min] RCY [% ] 0 1 2 3 4 5 6 7 8 9 10 0 20 40 60 80 100 pH 3 pH 3.5 pH 5 pH 4 reaction time [min] RCY [% ] A B reaction time [min] Figure 3. 3.2. The Influence of Diastereomers of HBED-CC on the Binding Properties The active site of a PSMA inhibitor consists of two independent main binding sites, a zinc-containing rigid site and an efferent tunnel with rather lipophilic characteristics [29,30]. The classical binding motif of urea-based PSMA inhibitors typically interacts with its carboxylic groups and the carbonylic oxygen. For efficient internalisation of a PSMA-directed radiotracer, however, the interaction of the linker region of the molecule with the aforementioned rather hydrophobic tunnel region seems of crucial importance. HBED-CC turned out to presumably interact with this region as the replacement of DOTA by HBED-CC has shown a significant influence on the internalisation efficiency of [68Ga]Ga-PSMA-HBED-CC [20]. Due to this specific interaction with the hydrophobic binding pocket, slight chemical differences caused by the known formation of different diastereomers of HBED-CC after Ga-complexation might influence the binding properties of the whole molecule. The reaction temperature influences the proportional distribution of the three diastereomers. Typically, the GMP-compliant synthesis is realised at 100 °C resulting in the predominant formation of the thermodynamically more stable configuration of Ga-HBED-CC whereby the composition proved to be stable for at least 3 h in the injection buffer (Figure 4C). HPLC-MS measurements confirmed the identity of both diastereomers of the natGa-labelled complex (supporting information). If the reaction is carried out at RT, a fraction of about 50% of another diastereomer is formed (Figure 4A). This diastereomer converts into the thermodynamically more stable one quite rapidly in a few hours at pH 4 (Figure 4D). However, neutralising the reaction mixture resulted in a much slower interconversion. As shown in Figure 4E the percentage composition remains nearly unchanged for hours which is in agreement with the observation of Schuhmacher et al., who reported that the interconversion into the most stable configuration at pH 7 took several days [23]. From the in vivo point of view, the distribution of the diastereomers might be of high impact if one configuration of Ga-HBED-CC would not interact with the binding site and therefore would hamper the overall functionality of the PSMA inhibitor. Since the quality of the PET/CT images would be affected by the presence of a potentially non-functional diastereomer, it is important to elucidate their influence on the PSMA binding characteristics. Therefore, the cell binding properties of [68Ga] Ga-PSMA-HBED-CC labelled at ambient temperature and 95 °C, respectively, were evaluated in cell-based assays. 3.1. HBED-CC as Chelator for Highly Efficient Radiolabelling of Peptides Superdex 75 5/150 GL runs of (A) 67Ga-labelled HBED-CC-c(RGDyK); and (B) 67Ga-labelled NOTA-c(RGDyK) after 48 h incubation in human serum at 37 °C. Only the radiometric signal is shown. The UV trace showed a major peak at ~3.9 min p.i.; (C) which corresponds with the elution time of serum proteins. B C 0.0 2.0 4.0 6.0 8.0 10.0 0.0 2.0 4.0 6.0 8.0 0.0 2.0 4.0 6.0 8.0 3.943 7.014 7.691 3.878 1.283 6.160 A B C 0.0 2.0 4.0 6.0 8.0 10.0 0.0 2.0 4.0 6.0 8.0 0.0 2.0 4.0 6.0 8.0 3.943 7.014 7.691 3.878 1.283 6.160 A B C 0.0 2.0 4.0 6.0 8.0 10.0 0.0 2.0 4.0 6.0 8.0 3.943 7.014 6.160 A A B 787 Pharmaceuticals 2014, 7 Taken together, HBED-CC represents an attractive acyclic alternative chelator for room temperature radiolabelling of proteins and peptides. Due to its different chemical characteristics compared to NOTA or DOTA such as the higher lipophilicity and the presence of aromatic residues, HBED-CC might have a positive impact on the pharmacokinetics or binding properties of a molecule. For [68Ga]Ga-PSMA-HBED-CC these chemical characteristics, obviously being a part of the pharmacophore, were shown to be advantageous for the PSMA binding behaviour of the molecule, presumably by interacting with the known accessory hydrophobic pocket of the PSMA S1 site [20]. 3.2. The Influence of Diastereomers of HBED-CC on the Binding Properties Figure 5A shows that the PSMA-specific cell surface binding and internalisation of the mixture of diastereomers (RT labelling) and the thermodynamically most stable diastereomer (95 °C labelling) were comparable. In addition, the affinity related IC50-values of the reaction mixtures of both labelling conditions were determined on the PSMA expressing cell line LNCaP (Figure 5B). Indeed, both the RT-labelled fraction and the 95 °C-labelled fraction bound PSMA with identical Pharmaceuticals 2014, 7 788 affinities (IC50 values: 27.4 ± 1.3 nm and 24.8 ± 1.2 nm, respectively). It has to be concluded that the presence of a thermodynamically less stable diastereomer does not have any negative influence on the PSMA-binding properties indicating a robust stereochemical independence of the pharmacophore Glu-NH-CO-NH-Lys sidechain. Figure 4. Radio-HPLC traces of RT (A) and 95 °C (B) labelled Glu-urea-Lys(Ahx)- HBED-CC. The peaks correspond to (1), the thermodynamically more stable, and (2), the thermodynamically less stable diastereomer, respectively. Graphs C–E show the radiochromatograms after various storage and labelling conditions: 95 °C labelled Glu-urea-Lys(Ahx)-HBED-CC stored for 3 h in injection buffer (C); RT labelled Glu-urea-Lys(Ahx)-HBED-CC after 3 h incubation in labelling reaction buffer at (D) pH 4, and (E) pH 7. ( ) p 789 Pharmaceuticals 2014, 7 Figure 5. (A) LNCaP-cell binding and internalisation of [68Ga]Ga-PSMA-HBED-CC labelled at RT or 95 °C, respectively. Specific cell uptake was determined by competitive blockade with 500 µM of the PSMA inhibitor 2-PMPA. Values are expressed as percentage of applied radioactivity bound to 106 cells [%IA/106 cells]. Data are expressed as mean ± SD (n = 3); (B) Determination of binding affinity of [natGa]Ga-PSMA-HBED-CC to LNCaP cells as a function of the labelling temperature. The cells (105 per well) were incubated with the radioligand (68Ga-labelled Glu-urea-Lys(Ahx)-HBED-CC) in the presence of different concentrations of natGa-analyte (0–5000 nM, 100 µL/well). (A) (B) This information is important with regard to the radiopharmaceutical production process as a small fraction of one of the thermodynamically less stable diastereomers is still present in the labelling reaction mixture even at 95 °C labelling condition (Figure 4B) and would be part of the final formulation prepared for the patient. According to our results, a fraction of approx. 50% of the thermodynamically less stable diastereomer does not reduce the PSMA-specific cellular uptake and should therefore not have any negative influence on the quality of the PET images. 3.2. The Influence of Diastereomers of HBED-CC on the Binding Properties However, it has to be considered that our conclusions are based on an indirect reasoning as the two major diastereomers were not separated chemically by HPLC for these experiments. A chemical separation via HPLC would result in an unknown concentration of unlabelled compound. As the cell binding directly depends on the used concentration it is crucial to know exactly the amount of analyte given to the cells. Since the proportional distribution of the observed two diastereomers can be easily controlled by pH and temperature of the labelling reaction mixture, the influence of the thermodynamically less stable diastereomer on the cell binding properties can be reliably determined by comparing RT-labelled and 95°C-labelled Glu-urea-Lys(Ahx)-HBED-CC. If the thermodynamically less stable diastereomer would not bind PSMA-presenting cell lines at all it would reduce the cell uptake considerably in the RT-labelled approach 37°C 4°C blocked blocked + 4°C 37°C 4°C blocked blocked + 4°C 0 1 2 3 4 5 Lysate Cell surface bound RT 95°C %IA / 106 cells 10-1 100 101 102 103 104 0 500 1000 1500 2000 Glu-urea-Lys(Ahx)-HBED-CC labelled with natGa at RT Glu-urea-Lys(Ahx)-HBED-CC labelled with natGa at 95°C c [nM] cpm (A) (B) This information is important with regard to the radiopharmaceutical production process as a small 37°C 4°C blocked blocked + 4°C 37°C 4°C blocked blocked + 4°C 0 1 2 3 4 5 Lysate Cell surface bound RT 95°C %IA / 106 cells 10-1 100 101 102 103 104 0 500 1000 1500 2000 Glu-urea-Lys(Ahx)-HBED-CC labelled with natGa at RT Glu-urea-Lys(Ahx)-HBED-CC labelled with natGa at 95°C c [nM] cpm (A) 37°C 4°C blocked blocked + 4°C 37°C 4°C blocked blocked + 4°C 0 1 2 3 4 5 Lysate Cell surface bound RT 95°C %IA / 106 cells (B) 10-1 100 101 102 103 104 0 500 1000 1500 2000 Glu-urea-Lys(Ahx)-HBED-CC labelled with natGa at RT Glu-urea-Lys(Ahx)-HBED-CC labelled with natGa at 95°C c [nM] cpm (A) (B) This information is important with regard to the radiopharmaceutical production process as a small fraction of one of the thermodynamically less stable diastereomers is still present in the labelling reaction mixture even at 95 °C labelling condition (Figure 4B) and would be part of the final formulation prepared for the patient. According to our results, a fraction of approx. 3.3. Automated Synthesis The radiosynthesis of [68Ga]Ga-PSMA-HBED-CC as described earlier [20] was reproduced reliably and slightly modified on a fully automated synthesis module in 80% ± 5% decay corrected radiochemical yield within 35 min applying single-use cassette-based kits. An audit trail was recorded for each radiosynthesis including all performed steps and courses of radioactivity on three gamma detectors, gas flow and temperature of the heating unit. The steps involved include conditioning of the purification cartridge, elution of the 68Ge/68Ga-generator, purification of the generator eluate, radiosynthesis and purification and sterile filtration of the final product. [68Ga]Ga-PSMA-HBED-CC was obtained in >99% radiochemical purity as two diastereomers (Figure 4B) with a pH ranging between 6 and 8. Stability of [68Ga]Ga-PSMA-HBED-CC in the final product solution was tested for up to 2 h after the end of radiosynthesis and yielded identical results for the radiochemical purity. The only residual solvent found in the final product solution was ethanol in less than 10% v/v taking the density at 20°C to be 0.79 g/mL. Bacterial endotoxin testing showed <2 IU/mL and all samples tested were sterile. Filter integrity of the sterile filter was tested using the bubble-point test. Gamma spectrometry showed characteristic peaks at 0.511 and 1.077 MeV. For the production of radiopharmaceuticals using gallium-68 obtained from a generator system it is crucial that possible metal impurities washed off from the 68Ge/68Ga-generator as well as 68Ge- breakthrough have to be eliminated prior the radiolabelling. Examples of methods for purification of the eluate are fractionated elution of the 68Ge/68Ga-generator or the trapping of the generator eluate on either a cationic or anionic exchange cartridge [31–33]. One of the most prominent methods has been described earlier by Zhernosekov et al. [33]. The drawback of this method for the purification of 68Ga is the use of acetone, which has to be removed after synthesis and bares the risk of additional impurities [34]. Recently, a method based on the use of a strong NaCl solution to purify 68Ga on a cation exchange cartridge has been reported [35,36]. For the radiosynthesis of [68Ga]Ga-PSMA-HBED-CC we adapted the method from Martin et al. [36]. A volume of 1.2 mL of 5 M NaCl solution was confirmed to elute up to 85% of the trapped 68Ga activity from the PS-H+ separation cartridge. Pharmaceuticals 2014, 7 Pharmaceuticals 2014, 7 790 3.2. The Influence of Diastereomers of HBED-CC on the Binding Properties 50% of the thermodynamically less stable diastereomer does not reduce the PSMA-specific cellular uptake and should therefore not have any negative influence on the quality of the PET images. However, it has to be considered that our conclusions are based on an indirect reasoning as the two major diastereomers were not separated chemically by HPLC for these experiments. A chemical separation via HPLC would result in an unknown concentration of unlabelled compound. As the cell binding directly depends on the used concentration it is crucial to know exactly the amount of analyte given to the cells. Since the proportional distribution of the observed two diastereomers can be easily controlled by pH and temperature of the labelling reaction mixture, the influence of the thermodynamically less stable diastereomer on the cell binding properties can be reliably determined by comparing RT-labelled and 95°C-labelled Glu-urea-Lys(Ahx)-HBED-CC. If the thermodynamically less stable diastereomer would not bind PSMA-presenting cell lines at all it would reduce the cell uptake considerably in the RT-labelled approach. 3.3. Automated Synthesis As the majority of the 68Ga activity was eluted with the last 200 µL of NaCl solution, a smaller volume than 1.2 mL could not be applied. Larger volumes did not lead to higher elution efficiency. Remaining 68Ge was neither observed on the PS-H+ separation cartridge nor in the reaction vessel after elution with 5 M NaCl solution and neither in the final product preparation 48 h after purification of the generator eluate. This confirms the efficacy of this method to remove possible 68Ge-breakthrough. We modified the reaction parameters due to the larger geometry of the reaction vessel on the synthesis module compared to the method described by Eder et al. [20]. To allow good mixing of 68Ga and PSMA-HBED-CC in the reaction vessel, a minimum volume of 1 mL was necessary. Additionally, pH of the reaction mixture is seen to be a critical factor to obtain high radiochemical yields. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a well-known buffer in radiolabelling procedures using 68Ga, but as we consider HEPES as a critical impurity for radiopharmaceutical applications we were looking for alternative buffer solutions. Firstly, citrate buffer was applied at different concentrations and at different pH but this was unsuccessful resulting in radiochemical yields <5%. We used different sodium acetate buffer solutions (pH 3.2–5.5) in concentrations ranging from 20 mM to 1.5 M and volumes ranging from 1 to 3 mL. The optimal reaction conditions with an 791 Pharmaceuticals 2014, 7 average radiochemical yield of 80% were found using 1 mL of 1.5 M sodium acetate buffer solution with a pH of the reaction mixture ranging from 3.6 to 4.2. Lower or higher pH of the reaction mixture lead to a significant decrease in radiochemical yields. Although the 68Ga-eluate was purified and concentrated using 5 M NaCl solution, sodium acetate buffer solutions with volumes smaller than 1 mL were not able to stabilise the pH of the reaction mixture sufficiently. Additionally, 10 µL of 1 M ascorbic acid was added to avoid the forming of side products when using a 68Ge/68Ga-generator with high starting activities [37]. We prolonged the reaction time to 10 min and heated to 100 °C because the resulting volume of the reaction mixture was by a factor 20 higher than previously reported [20]. 3.3. Automated Synthesis High radiochemical yields were obtained reliably with only 1.9 µg (2 nmol) of PSMA-HBED-CC in our setup, different setups due to the properties of other synthesis modules might require slightly higher amounts (5–10 µg) of radiolabelling precursor. The crude reaction mixture was purified using a C18 cartridge which was then rinsed with ultrapure water to remove unreacted 68Ga. After sterile filtration 10 mL of the final product solution were obtained and 1.5–2 mL taken for quality control and keeping of a retain sample. Batches of [68Ga]Ga-PSMA-HBED-CC produced following this fully automated procedure are being released after passing quality control requirements according to a defined product specification (Table 1). The whole automated synthesis procedure was also successfully applied for the radiopharmaceutical production of [68Ga]Ga-DOTA-TOC and is a useful tool for a quick clinical implementation of other novel 68Ga-labelled compounds. efined product specification of the final preparation of [68Ga]Ga-PSMA-HBED-CC. Table 1. Defined product specification of the final preparation of [68Ga]Ga-PSMA-HBED-CC Appearance Clear and Colourless pH 4–8 Radioactivity concentration 10–200 MBq/mL Radiochemical purity (HPLC) ≥95% Chemical impurities (HPLC) ≤5 µg/mL PSMA-HBED-CC Concentration ethanol (GC) <10% v/v Approximate half-life 68 ± 6 min Bacterial endotoxins <17.5 IU/mL Filter integrity (bubble-point test) >3.5 bar Radionuclidic purity (γ-spectrometry) 68Ga > 99.9% (γ-lines at 0.511 MeV and 1.077 MeV) 68 Ge: ≤0.001% Sterility Sterile Author Contributions Matthias Eder contributed in writing of the manuscript and data interpretation and analysis. Oliver Neels contributed in writing of the manuscript and the development of the GMP-compliant automated synthesis and quality control procedures. Miriam Müller contributed in the synthesis of peptides and chemicals, data interpretation and analysis and carried out the complex stability and labelling kinetics experiments. Ulrike Bauder-Wüst contributed in the data interpretation and analysis and carried out in vitro experiments. Yvonne Remde contributed to the development of the GMP-compliant automated synthesis. Martin Schäfer carried out and evaluated the synthesis of peptides and chemicals. Ute Hennrich contributed to the development of quality control procedures for the GMP-compliant automated synthesis. Michael Eisenhut contributed to the design of experiments for HBED-CC- and NOTA-conjugated model-peptide c(RGDyK). Ali Afshar-Oromieh gave scientific advice for the development of the GMP-compliant automated synthesis. Uwe Haberkorn gave scientific advice for the design of preclinical experiments. Klaus Kopka gave scientific advice, contributed to the design of experiments as well as in writing of the manuscript. All authors approved the manuscript. 4. Conclusions We have recently introduced HBED-CC in the PSMA inhibitor motif Glu-urea-Lys as radiometal chelator in order to optimise the interactions of the pharmacophore with the accessory hydrophobic pocket of the PSMA S1 binding site. In addition, HBED-CC represents a very effective and stable radiometal chelator allowing fast radiolabelling at room temperature while exhibiting an exceptionally high complex stability similar to the clinically used DOTA chelator. The formation of diastereomers can be controlled via distinct temperature conditions during the radiolabelling reaction and directed predominantly to the formation of the thermodynamically more stable one. In case of small fractions of other diastereomers, the biological functionality of the radiotracer [68Ga]Ga-PSMA-HBED-CC is 792 Pharmaceuticals 2014, 7 proven to be not affected. Taken together, the HBED-CC-conjugated PSMA inhibitor [68Ga]Ga- PSMA-HBED-CC is suitable for being used in typical kit like radiochemical production processes for clinical use with high reproducibility and robustness. Acknowledgments There is no other financial relationship of the authors than a grant from the DFG (Deutsche Forschungsgemeinschaft) for M. Eder (ED234/2-1), which is gratefully acknowledged. HPLC-MS measurements were kindly performed and evaluated by Susanne Krämer (Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany). Conflicts of Interest The authors declare no conflict of interest. 1. Andriole, G.L.; Crawford, E.D.; Grubb, R.L., 3rd; Buys, S.S.; Chia, D.; Church, T.R.; Fouad, M.N.; Gelmann, E.P.; Kvale, P.A.; Reding, D.J.; et al. Mortality results from a randomized prostate-cancer screening trial. N. Engl. J. Med. 2009, 360, 1310–1319. References 1. Andriole, G.L.; Crawford, E.D.; Grubb, R.L., 3rd; Buys, S.S.; Chia, D.; Church, T.R.; Fouad, M.N.; Gelmann, E.P.; Kvale, P.A.; Reding, D.J.; et al. Mortality results from a randomized prostate-cancer screening trial. N. Engl. J. Med. 2009, 360, 1310–1319. 2. Beheshti, M.; Langsteger, W.; Fogelman, I. Prostate cancer: Role of SPECT and PET in imaging bone metastases. Semin. Nucl. Med. 2009, 39, 396–407. 3. Afshar-Oromieh, A.; Zechmann, C.M.; Malcher, A.; Eder, M.; Eisenhut, M.; Linhart, H.G.; Holland-Letz, T.; Hadaschik, B.A.; Giesel, F.L.; Debus, J.; et al. Comparison of PET imaging with a 68Ga-labelled PSMA ligand and 18F-choline-based PET/CT for the diagnosis of recurrent prostate cancer. Eur. J. Nucl. Med. Mol. Imaging 2014, 41, 11–20. Pharmaceuticals 2014, 7 793 4. Afshar-Oromieh, A.; Malcher, A.; Eder, M.; Eisenhut, M.; Linhart, H.G.; Hadaschik, B.A.; Holland-Letz, T.; Giesel, F.L.; Kratochwil, C.; Haufe, S.; et al. PET imaging with a [68Ga]Gallium-labelled PSMA ligand for the diagnosis of prostate cancer: Biodistribution in humans and first evaluation of tumour lesions. Eur. J. Nucl. Med. Mol. Imaging 2013, 40, 486–495. 5. Afshar-Oromieh, A.; Haberkorn, U.; Eder, M.; Eisenhut, M.; Zechmann, C.M. [68Ga]Gallium-labelled PSMA ligand as superior PET tracer for the diagnosis of prostate cancer: Comparison with 18F-FECh. Eur. J. Nucl. Med. Mol. Imaging 2012, 39, 1085–1086. 6. Barrett, J.A.; Coleman, R.E.; Goldsmith, S.J.; Vallabhajosula, S.; Petry, N.A.; Cho, S.; Armor, T.; Stubbs, J.B.; Maresca, K.P.; Stabin, M.G., et al. First-in-man evaluation of 2 high-affinity psma- avid small molecules for imaging prostate cancer. J. Nucl. Med. 2013, 54, 380–387. 7. Silver, D.A.; Pellicer, I.; Fair, W.R.; Heston, W.D.; Cordon-Cardo, C. 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Identification, characterization and suppression of side-products formed during the synthesis of high dose 68Ga-DOTA-TATE. Appl. Radiat. Isot. 2013, 76, 63–69. Appendix HPLC-MS analysis HPLC-MS analysis The natGa-labelled reference Glu-urea-Lys(Ahx)-[Ga(HBED-CC)] (DKFZ-GaPSMA-11) was purchased from ABX advanced biochemical compounds (Radeberg, Germany) and dissolved in 40 µL CH3CN/H2O (1:1). 2.6 µg were characterised at RT by mass spectrometry using a Agilent 1200 HPLC-MS system connected to an Orbitrap Mass Spectrometer (Exactive, Thermo Fisher Scientific) on a Hypersil Gold C18 column (2.1 × 200 mm, 1.9 µm; Thermo Scientific, Bremen, Germany) eluted with a linear gradient (eluent A: 0.05% TFA in water; eluent B: 0.05% TFA in acetonitrile; 0%–30% B in 30 min at RT, flow rate: 0.2 mL/min, absorbance: λ=214 nm). Full scan single mass spectra (positive mode) were obtained by scanning from m/z = 200−4000. 796 Pharmaceuticals 2014, 7 Figure S1. MS analysis of HPLC fractions of the mixture of diastereomers (fraction 23.84–27.00 min), of the thermodynamically more stable (fraction 23.95-25.04 min) and of the less stable diastereomer (fraction 26.58-26.88 min) of Ga-PSMA-HBED-CC, respectively. Figure S1. MS analysis of HPLC fractions of the mixture of diastereomers (fraction 23.84–27.00 min), of the thermodynamically more stable (fraction 23.95-25.04 min) and of the less stable diastereomer (fraction 26.58-26.88 min) of Ga-PSMA-HBED-CC, respectively. the less stable diastereomer (fraction 26.58-26.88 min) of Ga-PSMA-HBED-CC, respectively. 14 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access a buted under the terms and conditions of the Creative Commons Attribution li //creativecommons.org/licenses/by/3.0/). © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W2072669442	https://europepmc.org/articles/pmc3022638?pdf=render	English	null	Plant identification credibility in ethnobotany: a closer look at Polish ethnographic studies	Journal of ethnobiology and ethnomedicine	2,010	cc-by	12,525	Open Access Open Access Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 JOURNAL OF ETHNOBIOLOGY AND ETHNOMEDICINE © 2010 Łuczaj; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Łukasz J Łuczaj Łukasz J Łuczaj Abstract p g y , g Not all of the invented credibility codes were used in the list of wild food plants, but they may be useful for other researchers. The most often used codes were the ones signifying identification by: voucher specimen, botanical expert and by a common name used throughout the country. Conclusions: The results of this study support the rigorous use of voucher specimens in ethnobotany, although they also reveal a relatively high percentage of misidentified taxa in the specimens studied. Conclusions: The results of this study support the rigorous use of voucher specimens in ethnobotany, although they also reveal a relatively high percentage of misidentified taxa in the specimens studied. The invented credibility coding system may become a useful tool for communication between historical ethnobotanists, particularly in creating larger databases. they also reveal a relatively high percentage of misidentified taxa in the specimens studied. The invented credibility coding system may become a useful tool for communication between historical ethnobotanists, particularly in creating larger databases. Abstract Background: This paper is an attempt to estimate the percentage of erroneously identified taxa in ethnographic studies concerning the use of plants and to propose a code for recording credibility of identification in historical ethnobotany publications. Methods: A sample of Polish-language ethnobotanical literature (45 published sources from 1874-2005) and four collections of voucher specimens (from 1894-1975) were analyzed. Errors were detected in the publications by comparing the data with existing knowledge on the distribution of plant names and species ranges. The voucher specimens were re-examined. A one-letter code was invented for quick identification of the credibility of data published in lists of species compiled from historical or ethnographic sources, according to the source of identification: voucher specimen, Latin binominal, botanical expert, obvious widespread name, folk name, mode of use, range, physical description or photograph. To test the use of the code an up-to-date list of wild food plants used in Poland was made. Results: A significant difference between the ratio of mistakes in the voucher specimen collections and the ratio of detectable mistakes in the studies without herbarium documentation was found. At least 2.3% of taxa in the publications were identified erroneously (mean rate was 6.2% per publication), and in half of these mistakes even the genus was not correct. As many as 10.0% of voucher specimens (on average 9.2% per collection) were originally erroneously identified, but three quarters of the identification mistakes remained within-genus. Results: A significant difference between the ratio of mistakes in the voucher specimen collections and the ratio of detectable mistakes in the studies without herbarium documentation was found. At least 2.3% of taxa in the publications were identified erroneously (mean rate was 6.2% per publication), and in half of these mistakes even the genus was not correct. As many as 10.0% of voucher specimens (on average 9.2% per collection) were originally erroneously identified, but three quarters of the identification mistakes remained within-genus. The species of the genera Thymus, Rumex and Rubus were most often confused within the genus. Not all of the invented credibility codes were used in the list of wild food plants, but they may be useful for other researchers. The most often used codes were the ones signifying identification by: voucher specimen, botanical expert and by a common name used throughout the country. Correspondence: lukasz.luczaj@interia.pl The Wild Garden; Pietrusza Wola 86; 38-471 Wojaszówka; Poland Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 2 of 16 It seems that no one has ever endeavored to estimate the possible percentage of mistakes in ethnobotanical publications. One of the very few authors who has dealt with the credibility of data in historical ethnobotany is Svanberg [8,9]. He presented a few examples of some so called “ghost data” - old and erroneous information, which has been repeated by subsequent authors. The importance of identification credibility in historical eth- nobotany can be clearly shown by the study of Kufer et al. [10], who compared present use of plants by the Ch’orti’ Maya from Guatemala with data gathered in the same population in the 1930s by Charles Wisdom. It turned out that some mistakes occurred in the former study, where a taxon was misidentified as belonging to a different family. containing the same or similar folk names as used in the studied population. 2.1.3 The local name is identical or similar to an official ‘scientific’ name of a species and the plant was (often erroneously) identified by assuming that the local name referred to the same taxon. 2.2 The plant was named by the informant using its scientific name (and a local name). 2.3 The plant was identified by the ethnobotanist from a verbal description. Obviously the ideal situation is 1.2.2.2, particularly if voucher specimens were shown/brought by more than one informant. However, different scenarios happen for a variety of reasons, of which the major three are: The quality of ethnobotanical information is increas- ingly discussed in a variety of contexts [11-13], for instance ethnobotanical databases [14,15]. For example in a database of ethnobotanical data on the Campania region in Italy [14], levels of certainty of identification were introduced (sure, unsure, etc.). Generally, the like- lihood of a mistake in identification probably increases with the age of the studied publication/information. This happens for a variety of reasons, e.g. changing folk names or uses in time. Background particularly in the field of historical ethnobotany, where not only is there a lack of voucher specimens, but often we have to hypothesize about the taxonomic position of certain species known only by their extinct folk/local names [6]. Ethnobotanists may include sources in their databases, which contain Latin binominals that come from reliable authors (preferably from professional bota- nists), but which are not confirmed by voucher speci- mens. This situation comes about because historical data are often too important to be discarded just on the basis of insufficient documentation [7]. One of the main problems ethnobotanists face when publishing their results is the possibility of a mistake in the identification of the studied taxa. Therefore securing voucher specimens is now standard procedure in ethno- botany [1-3], required by major journals and discussed in ethnobotany method manuals [e.g. [4,5]]. On the other hand the results of studies not documented by voucher specimens are still sometimes published, Correspondence: lukasz.luczaj@interia.pl The Wild Garden; Pietrusza Wola 86; 38-471 Wojaszówka; Poland Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 1 the ignorance of the researcher, 1 the ignorance of the researcher, 1 the ignorance of the researcher, 2 the fact that the information may be published/ recorded even if securing of a voucher specimen is not possible, because of the importance of studying the use of a taxon for the researcher, 2 the fact that the information may be published/ recorded even if securing of a voucher specimen is not possible, because of the importance of studying the use of a taxon for the researcher, 3 the use of a plant is extinct and we have only histor- ical records without voucher specimens. 3 the use of a plant is extinct and we have only histor- ical records without voucher specimens. In this study I would like to consider the problem of the credibility of ethnobotanical data in one country - Poland. Poland, like a few other European countries, has a rich 19 and 20th century ethnographic literature con- cerning the traditional use of plants - for a bibliography see Klepacki’s review [16]. As the Polish flora is rela- tively poor in plant species (it has approximately three thousand species), the concept of voucher specimens was difficult to understand, not only for ethnographers studying the traditional use of plants, but also for bota- nists, who were relatively sure of their identifications. In order to analyze the issue of mistakes in plant iden- tification we should look at the whole process of plant identification. With ethnobotanical data a few scenarios are most likely: 1. The plant was shown to the ethnobotanist by an informant. Methods Kielak’s book contains colour photographs of around half of the voucher speci- mens in the archive (129 specimens out of 280). Plants were re-identified using photographs from this book. 2. The documentation of Orzeszkowa’s ethnobotani- cal study from the river Niemen region (now wes- tern Belarus) published in a few parts in the periodical Wisła between 1888 and 1891 [e.g. [44,45]] stored in the archive of the Poznańskie Towarzystwo Przyjaciół Nauk society in Poznań. The detailed description of this herbarium was pub- lished by Kielak [77]. Kielak’s book contains colour photographs of around half of the voucher speci- mens in the archive (129 specimens out of 280). Plants were re-identified using photographs from this book. The total number of identified plant taxa was recorded for each publication, as well as the number of taxa which were presumably identified erroneously. A refer- ence to a species from one publication and each herbar- ium specimen were later referred to as a use-report, a term, which, although mainly applied to indicate a plant-use mentioned by a given informant [72], in this case can be used with a publication as a unit. This way of treating a literature citation as one use-report is used in ethnobotanical studies, which review earlier publica- tions, where the number of informants and informant consensus is not given. For example this approach was used by Leonti et al. [73] to analyze the influence of the 16th century herbal of Matthioli on present day ethno- botanical knowledge in Campania (Italy), and in reviews of edible plants of Spain [74]. 3. The archives of the Polish Ethnographic Atlas study of wild edible plants from 1948-49 and medic- inal plants from 1949-50 [78]. The herbarium (as a part of the field questionnaires) is stored in the office of the Polish Ethnographic Atlas in Cieszyn (University of Silesia) but formally belongs to the Polish Folklore Society in Wrocław. For this study 196 questionnaires (concerning edible plants) con- taining herbarium specimens, identified with Latin names, were used. The person who identified them is not recorded, the name of the Department of Plant Systematics and Geography of the University of Curie-Skłodowska in Lublin is printed as the identifying institution. 1. The plant was shown to the ethnobotanist by an informant. 1. The plant was shown to the ethnobotanist by an informant. 1.1 The informant showed the wrong plant. 1.2 The informant showed the right plant. 1.2.1 The plant was not taken from the field and the identification was performed “from memory”. 1.2.2 The plant was picked and used in the identification process. 1.2.2.1 The plant was not preserved. 1.2.2.1.1 A voucher specimen was not made. 1.2 The informant showed the right plant. The first person who tried to verify the credibility of older ethnobotanical studies in Poland was Köhler in 1996 [17], who checked the identification of plants in Udziela’s herbarium from the turn of the 19th and 20th century. A few years earlier Radwańska-Paryska [18] re- examined the herbarium of an 18th century monk, Brother Cyprian, containing Slovak and Polish plant names from the Pieniny and Tatra mountain ranges bordering the two countries. Later, the author of this paper (ŁŁ) published an article on the taxonomic issues concerning the quality of the data and mistakes in the identification of taxa in ethnobotanical studies in Poland [19]. 1.2.2.1.1 A voucher specimen was not made. 1.2.2.1.2 A voucher specimen was made later. from a plant, which according to the ethnobotanist’s knowledge belongs to the same taxon. 1.2.2.2 The plant was preserved as a vou- cher specimen. The aim of this article is to extend the investigations of the previous work [19], in particular: p 2 The plant was not shown to the ethnobotanist. 2.1 The plant was named by the informant using a local name. 2.1.1 A scientific name was not assigned. 2.1.2 The scientific name was found/hypothe- sized using other ethnobotanical literature 2 The plant was not shown to the ethnobotanist. 2.1 The plant was named by the informant using a local name. 2.1.1 A scientific name was not assigned. 2.1.2 The scientific name was found/hypothe- sized using other ethnobotanical literature 1. To quantify a possible percentage of taxonomic errors in publications from this field. 2.1.1 A scientific name was not assigned. 2.1.2 The scientific name was found/hypothe- sized using other ethnobotanical literature 2. To propose a standard of coding the credibility of identification of scientific names in ethnobotanical Page 3 of 16 Page 3 of 16 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 publications, and test its usefulness by making a list of edible plants used in Poland. 1. The plant was shown to the ethnobotanist by an informant. publications, and test its usefulness by making a list of edible plants used in Poland. - Some taxa were widely used under one name and their ‘identity’ is obvious but a different Latin name had been erroneously assigned to this folk taxon. For example in one publication szałwia - Salvia officina- lis was named S. pratensis, although the description of the plant without doubt refers to the former. Methods A sample of Polish-language ethnobotanical literature consisting of 45 published sources [20-64] (Table 1) and four voucher specimen collections were analyzed (Table 2). The analyzed publications consisted of a large pro- portion of Polish-language ethnographic publications with ethnobotanical content, which contained lists of regionally used plants including at least one Latin name. All such papers available to the author were taken into account. Most of the analyzed sources deal with either wild food plants (reviewed in the Journal of Ethnobiol- ogy and Ethnomedicine in 2007 [65]) or medicinal plants. Papers without Latin binominals or monographs on the use of single species were not included. Mauri- zio’s [66] and Moszyński’s [67,68] major works were not taken into account, as they are syntheses concerning the whole of northern Eurasia (the former author) or all Slavs (the latter). Lists of plant names and databases compiled mainly on the basis of other published sources were not included either [e.g. [69-71]]. The analyzed publications usually concern studies from the present area of Poland and in a few cases - western Belarus [44,45,47], western Ukraine [37] and Lithuania [56]. The publications from these countries were included in the analyses as they were written by Polish ethnographers working close to the present area of Poland, within its former, broader territory from before World War II. The second part of the study dealt with the re-exami- nation of voucher specimens (Table 2). The voucher specimen collections for ethnobotanical data are extre- mely rare in Poland and so far only four such herbar- iums have been found: 1. The documentation of Udziela’s study [61] of medicinal and ritual plants of the Kraków area, stored in the Herbarium of the Institute of Botany of the Polish Academy of Sciences in Kraków (KRAM). The whole collection (119 specimens) was already previously checked by Köhler [17] but in 2010 I reexamined the collection. The specimens probably come from 1894-99 when Udziela collected his field data [17]. 2. The documentation of Orzeszkowa’s ethnobotani- cal study from the river Niemen region (now wes- tern Belarus) published in a few parts in the periodical Wisła between 1888 and 1891 [e.g. [44,45]] stored in the archive of the Poznańskie Towarzystwo Przyjaciół Nauk society in Poznań. The detailed description of this herbarium was pub- lished by Kielak [77]. Methods The content of these ques- tionnaires was published in 2008 with identifications already corrected by Łuczaj [79] - however in this study the original identifications were analyzed with reference to the kinds of errors that were made. The archive contains a few hundred more voucher speci- mens but they were not included in this study as The following methods of identifying errors were used: The following methods of identifying errors were used: - For wild taxa the distribution was checked in the atlas of the distribution of Polish vascular plants [75] - if the species did not occur in the geobotanical region (kraina geobotaniczna as mapped by Matusz- kiewicz [76]) of the publication, an error was assumed. - For wild taxa the distribution was checked in the atlas of the distribution of Polish vascular plants [75] - if the species did not occur in the geobotanical region (kraina geobotaniczna as mapped by Matusz- kiewicz [76]) of the publication, an error was assumed. - For wild taxa the distribution was checked in the atlas of the distribution of Polish vascular plants [75] - if the species did not occur in the geobotanical region (kraina geobotaniczna as mapped by Matusz- kiewicz [76]) of the publication, an error was assumed. Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 4 of 16 Page 4 of 16 they were only recently rediscovered and have not been analyzed in detail. 4. The herbarium of Szychowska-Boebel, stored in the archive of the Ethnographic Museum in Toruń. It is a documentation of her studies of ethnomedic- inal plants in the village of Wiele in Eastern Pomera- nia in 1975 [59]. It contains 43 specimens, including 21 identified taxa. Table 1 Literature sources [20-64] where the level of botanical mistakes was assessed using comparative methods (using the present knowledge of species ranges and the distribution of folk names) Author’s name Reference Number Year Main topic No. Methods The author set up a code of credibility for presenta- tion of historical ethnobotanical data in tables: H - confirmed by (a) voucher specimen(s), A - confirmed by authority (expert botanist), O - obvious common name universally used in a large area, L - highly probable Latin name or a binominal scienti- fic name used in the language of a given country corre- sponding to a Latin name, given by non-botanist, The mistakes concerned a variety of taxa but only a few taxa were mistaken more than twice: Thymus, ten times (e.g Thymus serpyllum confused with Thymus pulegioides or T. vulgaris), Rubus (six), Rumex (six), Cir- sium, Trifolium (both four), Chenopodium/Atriplex, Malva and Mentha (three each). When the taxa from two families were confused this usually happened because of two similar folk/scientific names (e.g. Cheno- podium - ’lebioda’, Origanum vulgare - ‘lebiodka’; Hip- pophae rhamnoides -’ rokitnik’, narrow leaved Salix spp. - ‘rokita’, etc.), which suggests that the author looked up Latin names in a scientific key without illustrations. This kind of error was the commonest type of mistake (eighteen out of thirty-six errors where a possible reason for the error was identified). The second commonest type (twelve cases) were simplifications and inaccuracies - such as reporting the use of only one species when more species from the same genus were used at least as frequently (Table 3). N - identified using comparative analysis of folk names, N - identified using comparative analysis of folk names, M - identified using data on the species’ mode of use (in case of unusual species/uses), M - identified using data on the species’ mode of use (in case of unusual species/uses), D - identified using physical description of species, R - identified with the help of the data of a species range or/and habitat, U - highly uncertain (should be combined with another code), P - identified using pictures (photographs or drawings). The usefulness of such a code was tested by compiling an up-to-date list of wild food plants used in Poland from the 19th to 21st century (within the present terri- tory, excluding the German population pre-1939). The list was based on the review of edible plants of Poland [65] and amended by recent publications by Łuczaj [79-81] and Pirożnikow [82,83] bringing more data on the subject. Methods In the list of edible plants of Poland (Table 6) 39% of 192 use-reports are confirmed by voucher specimens (code H), 30% by scholars with reliable botanical exper- tise (code A), 13% using folk names known widely throughout the country and 11% by scientific names Methods Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 5 of 16 Table 2 Voucher specimen collections analyzed Author’s name Publication place of original names Publication place of corrected names No. of voucher specimens analyzed No. of errors Udziela [61] [17] 119 8 Orzeszkowa [44,45] partly in [77] 129 8 Gajek unpublished [79] 196 28 Szychowska- Boebel [59] - 21 2 Table 2 Voucher specimen collections analyzed (Tables 1 and 3) and in the voucher specimens (Tables 2, 4 and 5). This constitutes 2.3% of the analyzed use- reports for the former set of data and 10.0% of voucher specimens. The mean mistakes rates per publication dif- fer significantly between the two sets of data (Mann- Whitney U test, U = 35.5, P (exact version) = 0.032, P (Monte Carlo version) = 0.022), they were 6.2% and 9.2% respectively. Both in publications and voucher specimen collections, only taxonomic errors were taken into account. Spelling mistakes were not included, nor were cases where the author was cautious and identified only the genus (for example Equisetum sp. instead of Equisetum hyemale). However the cases when only one species was reported in the literature as used in the area, though we have firm evidence that a larger number of closely related species was/is utilized were also treated as errors (inaccuracies), for example, a passage like: “blackberries (Rubus caesius) are used as food”, as “Rubus caesius” should be replaced by “Rubus subgenus Rubus” or “Rubus spp.” The comparative method revealed a relatively large number of mistakes in a few publications, both older [26,32,52,53] and new ones [49,54,60], however no or single mistakes were found in most sources. The author set up a code of credibility for presenta- tion of historical ethnobotanical data in tables: There was no correlation between the year of publica- tion and the percentage of errors in the species list (Pearson correlation coefficient, r = -0.004, P = 0.98, Kolberg’s postmortem publications were assigned to his death date of 1890). Longer lists of plants had slightly lower error rates (the correlation between the number of Latin binominals in a list and the percentage of errors in it was r = -0.28, P = 0.060). Methods of taxa with Latin names Errors Bohdanowicz [20] 1996 food 10 2 Chętnik [21] 1936 food 14 0 Dekowski [22] 1968 food 35 0 Dekowski [23] 1973 foraging 38 0 Dydowiczowa [24] 1964 foraging 44 0 Eljasz-Radzikowski [25] 1897 general ethnographic 18 0 Gajkowa [26] 1947 general ethnographic 4 3 Gawełek [27] 1910 ethnomedicine 36 0 Gustawicz [28] 1904 general ethnographic 18 0 Henslowa [29] 1962 selected edible taxa 12 0 Janicka-Krzywda [30] 2004 food 5 1 Jostowa [31] 1954 food 1 0 Kantor [32] 1907 general ethnogr. 46 5 Kolberg [33] 1962 (1874) general ethnogr. 54 1 Kolberg [34] 1962 (1882) general ethnogr. 35 1 Kolberg [35] 1962 (1891) general ethnogr. 75 0 Kolberg [36] 1973 general ethnogr. 6 0 Kolberg [37] 1963 (1888) general ethnogr. 22 0 Kolberg [38] 1968 general ethnogr. 29 0 Libera, Paluch [39] 1993 ethnomedicine 98 0 Łęga [40] 1961 general ethnogr. 2 0 Malicki [41] 1971 foraging 20 0 Ochrymowicz [42] 1900 beliefs about herbs 52 1 Oczykowski [43] 1896 ethnomedicine 10 1 Orzeszkowa [44] 1888 ethnomedicine and beliefs 69 0 Orzeszkowa [45] 1891 ethnomedicine and beliefs 51 0 Paluch [46] 1984 ethnomedicine 176 0 Pietkiewicz [47] 1928 material culture 23 0 Plichta [48] 1891 ethnomedicine 8 0 Ruszel [49] 2004 ethnographic dictionary 85 8 Siarkowski [50] 1890 ethnomedicine 17 0 Siarkowski [51] 1891 ethnomedicine 5 1 Sulisz [52] 1906 general ethnogr. 11 3 Sulisz [53] 1906 general 8 4 Szot-Radziszewska [54] 2005 ethnomedicine 129 4 Szromba-Rysowa [55] 1966 foraging 63 2 Szukiewicz [56] 1903 folk beliefs 5 0 Szulczewski [57] 1996 general ethnogr. >100 0 Szychowska-Boebel [58] 1972 ethnomedicine 95 1 Szychowska-Boebel [59] 1978 ethnomedicine 49 1 Tylkowa [60] 1988 ethnomedicine 81 4 Wawrzeniecki [61] 1916 ritual plants 14 2 Weryho [62] 1888 ethnomedicine 31 1 Wysłouchowa [63] 1896 general ethnogr. 38 0 Udziela [64] 1931 ethnomedicine and beliefs 141 0 Table 1 Literature sources [20-64] where the level of botanical mistakes was assessed using c (using the present knowledge of species ranges and the distribution of folk names) they were only recently rediscovered and have not been analyzed in detail. It is a documentation of her studies of ethnomedic- inal plants in the village of Wiele in Eastern Pomera- nia in 1975 [59]. It contains 43 specimens, including 21 identified taxa. 4. The herbarium of Szychowska-Boebel, stored in the archive of the Ethnographic Museum in Toruń. Results Forty-six identification mistakes were detected both in the published material using comparative methods Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 6 of 16 Page 6 of 16 Table 3 Errors detected in the studied publications, assessed using comparative methods Author Name in the publication (with original spelling) The correct name Type of mistake/inaccuracy Bohdanowicz [20] Origanum vulgare Chenopodiumsp. L Bohdanowicz [20] Carduus Cirsium sp. L Gajkowa [26] cuminum cyminum Carum carvi L. L Gajkowa [26] panicum miliaceum Echinochloa crus-galli (L.)P.Beauv. L Gajkowa [26] atriplex hortense Chenopodium & Atriplex spp. L Janicka- Krzywda [30] Carlina vulgaris Carlina acaulis L. ? Kantor [32] Geranium Dahlia sp. ? Kantor [32] Iris Lilium sp.? ? Kantor [32] Salsola ? ? Kantor [32] Selinum carvifolia Carum carvi L. I Kantor [32] Sesleria coerulea Sesleria sadlerana Janka ssp. tatrae (Degen) Deyl? I Kolberg [33] Hippophae rhamnoides Salix sp. L Kolberg [34] Helleborus albus Veratrum sp.? Vincetoxicum hirundinaria Medik.? L Ochrymowicz [42] Iris germanica Iris sp. or Eupatorium cannabinum L. ? Oczykowski [43] Rumex hydrolapathum some other Rumex spp. I Ruszel [49] Plantago major Plantago spp. S (the name refers to all the Plantago species) Ruszel [49] Helleborus viridis Veratrum lobelianum Bernh.? L Ruszel [49] Carum carvi Glechoma hederacea L. s.l. ? Ruszel [49] Thymus serpyllum Thymus serpyllum L. em. Fr. &Th. pulegioides L. S (both species are used) Ruszel [49] Tilia cordata Tilia cordata Mill. &T. platyphyllos Scop. S (both species are used equally frequently) Ruszel [49] Rubus plicatus Rubus subgenus Rubus spp. S (there are a few dozen species of Rubus in this area, R. plicatus is not the most frequent [84] Ruszel [49] Carduus Cirsium & Carduus spp. L Ruszel [49] Rumex hydrolapathum Rumex spp. mainly R. obtusifolius L. I Siarkowski [51] Thymus serpyllum Thymus spp. S Sulisz [52] Origanum vulgare Chenopodium sp. L Sulisz [52] Thymus vulgaris Thymus serpyllum L. em. Fr. or Th. pulegioides L. I Sulisz [52] Matricaria Chamomilla Tanacetum parthenium (L.) Sch.Bip. I Sulisz [53] Acorus calamus Calamagrostis epigejos (L.)Roth ? Sulisz [53] Rhamnus cathartica Staphylea pinnata L. ? Sulisz [53] Galium cruciata Euonymus europaeus L./Rhamnus cathartica L.? ? (L?) Sulisz [53] Ledum palustre the term bagnięta was used erroneously as it refers to any wooden branches L Szot- Radziszewska [54] Cicuta virosa Solanaceae, probably Hyoscyamus niger L. L Szot- Radziszewska [54] Thymus serpyllum Thymus spp. Results L - wrong Latin name given by a researcher who looked the plant up in a guide using the local name as if it was an official name; I - other kind of wrong identification of a species; S - oversimplification/inaccuracy - one name given because more than one species from the genus is known under the same folk name, and the names are used with at least equal frequency, and in the same way, in the local area. with unknown reliability (L). Only ten out of 192 were identified using folk names (N) and four by comparing species ranges (R; with help of other data, e.g. folk names). None of the species were identified by only using a physical description from literature (D), pictures (P) or mode of use (M). In ten cases the code U (uncer- tain) was used. Rosa, Rumex and in the subgenus Rubus. The identifica- tion may be particularly difficult in apomictic taxa (like Rubus - [84]). Intergeneric mistakes occur either between two closely related genera not distinguished by folk taxonomy (Carduus and Cirsium) or less related (or unrelated) genera, if one of them has a folk name identi- cal or similar to the scientific or folk name of another genus (in Poland, Origanum and Chenopodium, Sonchus and Taraxacum). Results S Szot- Radziszewska [54] Salvia pratensis Salvia officinalis L. L Szot- Radziszewska [54] Papaver rhoeas Papaver officinalis L. L Table 3 Errors detected in the studied publications, assessed using comparative methods Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 7 of 16 Table 3 Errors detected in the studied publications, assessed using comparative methods (Continued) Szromba- Rysowa [55] Rubus caesius Rubus subgenus Rubus spp. S (other Rubus spp. are used more frequently) Szromba- Rysowa [55] Carduus sp. Cirsium & Carduus spp. L Szychowska- Boebel [59] Crataegus oxyacantha L. Crataegus spp. S (Crataegus monogyna is more frequent) Szychowska- Boebel [59] Crataegus oxyacantha L. Crataegus spp. S (as above) Tylkowa [60] Sonchus olearceus L. Taraxacum sp. L Tylkowa [60] Rubus plicatus L. Rubus spp. S (there are a few dozen species of Rubus in this area, R. plicatus is not the most frequent [84] Tylkowa [60] Malva alcea L. Alcea rosea L. L Tylkowa [60] Thymus serpyllum L. Thymus pulegioides L. S - T. serpyllum does not occur in the region, the other species is commonly used Wawrzeniecki [61] Thymus vulgaris Thymus spp. I Wawrzeniecki [61] Urtica urens Urtica dioica L. & U. urens L. S Weryho [62] Vinca major Vinca minor L. ? L - wrong Latin name given by a researcher who looked the plant up in a guide using the local name as if it was an official name; I - other kind of wrong identification of a species; S - oversimplification/inaccuracy - one name given because more than one species from the genus is known under the same folk name, and the names are used with at least equal frequency, and in the same way, in the local area. Table 3 Errors detected in the studied publications, assessed using comparative methods (Continued) L - wrong Latin name given by a researcher who looked the plant up in a guide using the local name as if it was an official name; I - other kind of wrong identification of a species; S - oversimplification/inaccuracy - one name given because more than one species from the genus is known under the same folk name, and the names are used with at least equal frequency, and in the same way, in the local area. Discussion in the genera Mentha, Thymus, Tilia, Crataegus, Page 8 of 16 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Table 4 Errors detected in the voucher specimen collections Collector Name in the publication The correct name Orzeszkowa Anchusa arvensis ? Orzeszkowa Thymus serpyllum Thymus pulegioides L. Orzeszkowa Anchusa arvensis Anchusa officinalis L. Orzeszkowa Asarum europaeum Hepatica nobilis L. Orzeszkowa Sium latifolium Cicuta virosa L. Orzeszkowa Ranunculus sceleratus Ranunculus flammula L. Orzeszkowa Ranunculus flammula Ranunculus sceleratus L. Orzeszkowa Lamium maculatum Lamiaceae but not Lamium Udziela Arabis arenosa Epilobium adenocaulon Hasskn. Udziela Daucus carota Pimpinella saxifraga L. Udziela Inula germanica Inula britannica L. Udziela Lappa maior (=Arctium lappa) Arctium tomentosum Mill. Udziela Marrubium vulgare Nepeta cataria L. Udziela Mentha piperita Mentha cfr verticillata L. Udziela Thymus serpyllum Thymus pulegioides L. Udziela Tilia grandiflora Tilia cordata Mill. PAE Betula alba Betula pubescens Ehrh. PAE Carlina vulgaris Cirsium arvense (L.) Scop. PAE Echium vulgare Symphytum officinale L. PAE Hypericum perforatum Vaccinium uliginosum L. PAE Malva neglecta (x3) Malva sylvestris L. PAE Mentha cfr. aquatica Mentha longifolia (L.)Huds. PAE Mentha piperita Mentha longifolia (L.)Huds. PAE Polygonum bistorta Rumex acetosa L. PAE Polygonum convolvulus Convolvulus arvensis L. PAE Polygonum mite (x2) Polygonum lapathifolium L. s.l. (including P. tomentosum Schrank) PAE Ribes rubrum (x2) Ribes spicatum Robson PAE Rosa canina (x3) Rosa sp. PAE Rubus hirtus Rubus sp. PAE Rubus hirtus Rubus caesius L. PAE Rubus saxatilis Rubus caesius L. PAE Rumex acetosella R. thyrsiforus Fing. PAE Rumex acetosella L (x 2) Rumex acetosa L. PAE Thymus serpyllum (x2) Thymus pulegioides L. PAE Trifolium medium Trifolium repens L. and T. pratense L. PAE Trifolium medium Trifolium pratense L. Szychowska-Boebel Trifolium arvense L. Trifolium pratense L. Szychowska-Boebel Trifolium arvense L. Trifolium repens L. PAE - Polish Ethnographic Atlas (specimens collected by numerous researchers). Table 4 Errors detected in the voucher specimen collections Collector Name in the publication The Table 4 Errors detected in the voucher specimen collections Trifolium repens L. and T. pratense L. Trifolium pratense L. Trifolium pratense L. PAE - Polish Ethnographic Atlas (specimens collected by numerous researchers). Lamium spp., Symphytum officinale and Pulonaria obscura [65,79-81]. other Cirsium species [85]. Discussion Nearly all of the quoted studies were performed by eth- nographers, not botanists, so it is impossible to quantita- tively compare the quality of their work with that of the few people with a biological background who have con- tributed to ethnobotany in Poland (e.g. Pirożnikow, Rosta- fiński, Maurizio, Moszyński, Szulczewski, though the latter two were known mainly as ethnographers). This compari- son is particularly difficult given that most of these expert botanists supplied us with relatively large synthetic studies [66-69], and only Szulczewski and Pirożnikow contributed local monographs with ethnobotanical data [57,82]. Each of these studies [57,66-69,82] contains well over a hundred species. In five out of six of these works I have not encountered any identification mistakes. On the other hand in the work of Maurizio two mistakes can be sus- pected, which probably arose from the misidentification of folk taxa. Both concern famine plants used in Poland, quoted by the author. One of them is Cichorium, suppo- sedly used as famine food in the Tatra mountains. Mauri- zio got this information from an ethnographic paper [25]. However the original source does not mention Cichorium but only a folk name - szczerbak. This folk name was used both for Cichorium intybus, Cirsium rivulare, as well as The lack of voucher specimens is obviously a problem in determining the real level of mistakes in older Eur- opean ethnobotanical studies. A considerable number of errors was detected in the studied herbariums. The comparative analysis of species ranges and names did not reveal these mistakes. One may wonder to what extent other works can be trusted? Some ethnographers probably avoided making taxonomic mistakes by writing only about more common and widely known taxa and identifying taxa only to the genus level. Some authors mentioned in their publications that their study was documented by voucher specimens identified by a pro- fessional biologist (e.g. Orzeszkowa [77] and Wysłou- chowa [64]) or that living/dried specimens were at least shown to professional botanists [30,46] or that voucher specimens from the Polish Ethnographic Atlas were used [46]. Thus in the above mentioned cases the possi- bility of mistakes is much lower. Some taxa are more likely to be confused than others. Unsurprisingly, errors often occur in genera with more than one species, in which the species are similar to each other and are poorly recognized in folk taxonomy, e.g. Discussion H 1, 2, 3, 5, 6 leaves and flowering stalks soup Pastinaca sativa L. O 1, 2, 6 roots cooked foods Acorus calamus L. Araceae H 1, 2, 5 inner parts of stems raw H 1, 5 leaves under baking bread Achillea millefolium L. Asteraceae A 4, 5, 6 leaves raw and as spice Arctium sp. A 3 leaf stalks lacto-fermented A 6 roots boiled Artemisia absinthium L. O 6 leaves spice for meat Bellis perennis L. N 2 unspecified unspecified A 5, 6 flowers raw Carlina acaulis L. H 1, 2, 3 receptacles, roots unknown Carlina vulgaris L. U 1 unspecified parts unkown Centaurea cyanus L. H 1 petals fermented drink Chamomilla recutita (L.)Rauschert L 2 shoots infusion Cichorium intybus L. U 1, 6 leaves boiled (ff), raw L 1, 5, 6 roots coffee surrogate Cirsium oleraceum Scop. A 1, 3, 4 leaves, roots boiled, ff Cirsium rivulare All. H 1, 2, 3, 4 leaves boiled, ff Table 5 Comparison of error rates in the studied sources Type of study Literature Voucher specimens Number of publications/herbariums 45 4 No. of use-reports/specimens 1983 459 No. of errors detected 46 46 Average rate of mistakes per publication/source 6.2 9.2 Percentage of errors detected 2.3 10.0 Types of errors: Number of taxa (Percentage given in parentheses) wrong genus 22 (48%) 11 (24%) wrong species within the same genus 7 (15%) 29 (63%) more species from the same genus are actually used in the area 16 (35%) - the identification is too detailed (the voucher specimen is in bad condition - it should have been identified only to the genus level) - 5 (11%) Table 6 The list of wild food plants used in Poland since the 19th century Species Family Method of Identification Source Parts Used Mode of Use Acer platanoides L. Aceraceae H 1, 2, 5 sap raw and fermented A 1 cambium raw A 1 fruits raw A 1 opening leaf buds fermented A 1, 5 leaves under baking bread Acer pseudoplatanus L. A 1, 2 sap raw A 1 leaf buds raw, ff Aegopodium podagraria L. Apiaceae A 3, 5, 6 young leaves soup Carum carvi L. H 1, 2, 5 seeds spice A 1 young plants soup Daucus carota L. A 5, 6 roots, leaves, fruits soup, spice Heracleum sphondylium L. H 1, 2, 3, 5, 6 leaves and flowering stalks soup Pastinaca sativa L. Discussion Cirsium rivulare was a very important famine plant in the Tatras [85], whereas Cichor- ium intybus was never mentioned as famine food by any other source listed in this article. Another possible mistake concerns the use of Mellitis melisophyllum. Maurizio mentioned that this plant (he also cited the folk name miodnik) was used during famine in Poland. However this is a relatively rare plant. On the other hand there are a few species of plants, which were used as famine food under similar names (miodunka, medunka, miodownik), i.e. Even if these two mistakes were confirmed, the total ratio of mistakes in the works of the five professional botanists would remain well below half a percent. How- ever, due to the different character of these studies, I restrained from deeper statistical comparisons. It is worth pointing out that the quality of the ethno- graphers’ work is highly variable. More than half of the publications contained no detectable mistakes, in Page 9 of 16 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Table 5 Comparison of error rates in the studied sources Type of study Literature Voucher specimens Number of publications/herbariums 45 4 No. of use-reports/specimens 1983 459 No. of errors detected 46 46 Average rate of mistakes per publication/source 6.2 9.2 Percentage of errors detected 2.3 10.0 Types of errors: Number of taxa (Percentage given in parentheses) wrong genus 22 (48%) 11 (24%) wrong species within the same genus 7 (15%) 29 (63%) more species from the same genus are actually used in the area 16 (35%) - the identification is too detailed (the voucher specimen is in bad condition - it should have been identified only to the genus level) - 5 (11%) Table 6 The list of wild food plants used in Poland since the 19th century Species Family Method of Identification Source Parts Used Mode of Use Acer platanoides L. Aceraceae H 1, 2, 5 sap raw and fermented A 1 cambium raw A 1 fruits raw A 1 opening leaf buds fermented A 1, 5 leaves under baking bread Acer pseudoplatanus L. A 1, 2 sap raw A 1 leaf buds raw, ff Aegopodium podagraria L. Apiaceae A 3, 5, 6 young leaves soup Carum carvi L. H 1, 2, 5 seeds spice A 1 young plants soup Daucus carota L. A 5, 6 roots, leaves, fruits soup, spice Heracleum sphondylium L. Discussion O 1, 2, 6 roots cooked foods Acorus calamus L. Araceae H 1, 2, 5 inner parts of stems raw H 1, 5 leaves under baking bread Achillea millefolium L. Asteraceae A 4, 5, 6 leaves raw and as spice Arctium sp. A 3 leaf stalks lacto-fermented A 6 roots boiled Artemisia absinthium L. O 6 leaves spice for meat Bellis perennis L. N 2 unspecified unspecified A 5, 6 flowers raw Carlina acaulis L. H 1, 2, 3 receptacles, roots unknown Carlina vulgaris L. U 1 unspecified parts unkown Centaurea cyanus L. H 1 petals fermented drink Chamomilla recutita (L.)Rauschert L 2 shoots infusion Cichorium intybus L. U 1, 6 leaves boiled (ff), raw L 1, 5, 6 roots coffee surrogate Cirsium oleraceum Scop. A 1, 3, 4 leaves, roots boiled, ff Cirsium rivulare All. H 1, 2, 3, 4 leaves boiled, ff Cirsium arvense (L.) Scop. H 2, 3, 4 leaves, stalks boiled, ff Sonchus arvensis L. LU 1, 3 green parts raw Taraxacum sp. pl. A 1, 5 inflorescences syrup, wine Table 6 The list of wild food plants used in Poland since the 19th century Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 10 of 16 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 10 of 16 Table 6 The list of wild food plants used in Poland since the 19th century (Continued) H 1, 2, 5 leaves raw, boiled Tragopogon pratensis L. s.l. H 2 stalks raw Tussilago farfara L. A 1 leaves boiled, ff Berberis vulgaris L. Berberidaceae H 1, 2, 3 fruits raw, preserves Alnus sp. Betulaceae O 2 Betula pendula Roth & Betula pubescens Ehrh. H 1, 2, 5 sap raw or fermented H 1 leaf buds fermented H 1, 2 cambium flour, ff Anchusa arvensis (L.) M.Bieb. Boraginaceae A 3 shoots boiled, ff Echium vulgare L. A 6 flowers nectar sucked Pulmonaria obscura L. AR 1 leaves boiled, ff Symphytum officinale L. H 1, 2, 3 leaves boiled, ff H 2, 6 flowers nectar sucked A 6 roots boiled (ff?) Armoracia rusticana P.Gaertn., B.Mey, &Scherb. Brassicaceae H 1, 2, 5 roots spice A 1 leaves under baking bread or as spice Capsella bursa-pastoris (L.)Medik. A 1, 2 fruits raw A 5, 6 whole plant? boiled Cardamine amara L. RU 3 leaves raw Cardamine pratensis L. Discussion A 3 leaves ff Raphanus raphanistrum L. H 1, 2, 3, 4 leaves boiled, ff Sinapis arvensis L. H 1, 2, 3, 4 leaves boiled, ff Campanula persicifolia L. Campanulaceae L 1, 6 flowers raw Phyteuma spicatum L. L 1 roots unspecified Humulus lupulus L. Cannabaceae O 1, 2, 5 inflorescences and fruits beer, mead, bread O 1 probably shoots ff Sambucus nigra L. Caprifoliaceae H 1, 2, 3, 5, 6 fruits boiled: wine, jam, soup, rarely raw O 3, 5 flowers fried in batter or preserves Viburnum opulus L. A 1, 2, 5 fruits boiled: wine, juice, jam Silene vulgaris (Moench) Garcke Caryophyllaceae A 3 shoots boiled, ff Stellaria media (L.) Vill. N 3 shoots boiled, ff Euonymus verrucosus Scop. Celastraceae A 5 fruits added to wine? Atriplex patula L. Chenopodiaceae L? 1 leaves boiled, fried Chenopodium album L. H 1, 2, 3, 5 leaves boiled, fried Chenopodium bonus-henricus L. A 1, 3 leaves boiled, fried Chenopodium hybridum L. A 3 leaves boiled, fried Chenopodium polyspermum L. A 3 leaves boiled, fried Chenopodium polyspermum L. A 3 leaves boiled, fried Convolvulus arvensis L. Convolvulaceae H 1, 2, 3 shoots boiled, ff Carpinus betulus L. Corylaceae H 1 sap raw Corylus avellana L. H 1, 3 inflorescences, leaves ff, mainly for flour H 1, 2, 3, 5 fruits raw and in cakes Juniperus communis L. Cupressaceae O 1, 2, 5 pseudofruits spice, beer, snack Scirpus sylvaticus L. Cyperaceae A 1, 6 inner parts of young shoots raw Pteridium aquilinum L. Dennstaedtiaceae LU 1 rhizomes unspecified, ff Table 6 The list of wild food plants used in Poland since the 19th century (Continued) Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 11 of 16 Table 6 The list of wild food plants used in Poland since the 19th century (Continued) Empetrum nigrum L. Empetraceae A 1 fruits unspecified Equisetum arvense L. Equisetaceae H 1, 2, 6 strobils raw, cooked A 2, 6 bulbils raw Calluna vulgaris (L.) Hull ON Ericaceae L 1 seeds bread, ff Vaccinium myrtillus L. H 1, 2, 3 fruit raw, boiled Vaccinium oxycoccos L. H 1, 2, 3, 5 fruit raw or in preserves Vaccinium vitis-idaea L. H 1, 2, 3, 5 fruit raw or in preserves Vaccinium uliginosum L. Discussion Oleaceae O 5, 6 fruits boiled, ff Table 6 The list of wild food plants used in Poland since the 19th century (Continued) Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 12 of 16 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 12 of 16 Table 6 The list of wild food plants used in Poland since the 19th century (Continued) Oenothera sp. Onagraceae A 6 roots boiled Oxalis acetosella L. Oxalidaceae H 1, 2, 3, 5, 6 leaves raw, cooked Oxalis stricta L. s.l. H 1, 2 leaves raw, cooked Papaver rhoeas L. Papaveraceae N 2, 5 seeds unspecified Abies alba Mill. Pinaceae O 1 young shoots syrup Picea abies (L.) H. Karst. O 1 young shoots raw, syrup O 1 male inflorescences raw O 1 young cones raw O 2 cambium ff Pinus cembra L. O 1 male inflorescences raw O 1 seeds raw Pinus sylvestris L. O 5 young vegetative and generative shoots raw, syrup Plantago lanceolata L. Plantaginaceae L 1, 2, 6 leaves boiled, raw Bromus secalinus L. Poaceae A 1, 2, 3 seeds ground for flour, ff Dactylis glomerata L. A 1 stem base raw Elymus repens (L.) Gould H 1 rhizomes ground for flour Festuca pratensis L. LU 1 seeds unspecified Glyceria fluitans (L.) R. Br. A 1 seeds seeds, boiled or for flour Glyceria plicata Fries L 1 seeds seeds, boiled or for flour Phleum pratense L. N 2 seeds for flour, ff Setaria pumila (Poir.) Schult. or/and Setaria viridis (L.) P.Beauv. NR 3 seeds boiled Fallopia convolvulus (L.) AA.Löve Polygonaceae H 3 shoots boiled, ff Polygonum hydropiper L. A 5 leaves raw Polygonum lapathifolium L. ssp. pallidum H 1 shoots fried, ff Rumex acetosa L. H 1, 3, 5 leaves raw, cooked Rumex acetosella L. H 1, 3 leaves raw, cooked Rumex crispus L. A 6 leaves cooked Rumex crispus L. or R. obtusifolius L. A 5, 6 fruit boiled, ff Rumex hydrolapathum Huds. A 6 leaves cooked Rumex obtusifolius L. HU 2 leaves for compotes Rumex thyrsiflorus Fing. H 2 leaves raw, cooked Rumex crispus L. L 1 leaves, seeds flour, ff Polypodium vulgare L. Polypodiaceae H 1,2,3 rhizomes raw or cooked as sweetener Ranunculus ficaria L. Ranunculaceae A 1, 3, 6 leaves boiled, raw Nigella sp. L 1 seeds spice Nigella arvensis L. Discussion H 1, 2, 3, 5 Euphorbia peplus L. Euphorbiaceae L 1 whole plant boiled, ff Astragalus glycyphyllos L. Fabaceae A 5, 6 stalks raw Medicago lupulina L. A 6 thickened parts of the roots raw Robinia pseudoacacia L. H 2 flowers raw, jams Trifolium pratense L., T. repens L., T. montanum L. H 1, 2 inflorescences nectar sucked or dried for baking bread Vicia sp. pl. L 1, 2 seeds flour for bread, ff Fagus sylvatica L. Fagaceae H 1, 3 fruits raw or baked, oil Quercus robur L. & Q. petraea Mattuschka (Liebl.) H 1 fruits flour (ff), coffee surrogate Ribes alpinum L. Grossulariaceae A 5 fruits raw (rarely) Ribes alpinum L. or R. petraeum Wulfen NR 1 fruits raw Ribes nigrum L. H 1, 2, 6 fruits raw, jams H 1, 6 leaves spice Ribes spicatum Robson H 2, 6 fruits raw, jams Ribes uva-crispa L. O 2, 3 fruits raw Stratiotes aloides L. Hydrocharitaceae H 2 leaves, roots boiled, ff Dracocephalum ruyschiana L. Lamiaceae A 5 flowers nectar sucked Galeopsis sp. A 1 leaves boiled, ff Glechoma hederacea L. s.l. H 1, 2, 3 leaves spice Lamium sp pl. (mainly L. album L.) A 3 shoots boiled Lamium purpureum L. A 5 shoots boiled Melittis melisophyllum L. LU 1 leaves unspecified, ff; liquors A 6 flowers nectar sucked Mentha arvensis L. H 1, 6 leaves spice, infusions, raw Mentha longifolia (L.)Hudson H 1 leaves spice Origanum vulgare L. A 1 flowering tops beer condiment Prunella vulgaris L. N 4 shoots boiled, ff Stachys palustris L. H 2, 3 rhizomes raw, boiled Thymus pulegioides L. H l flowering tops spice, teas Thymus serpyllum L. H 1, 5 flowering tops spice, teas Lemna minor L. L 1 leaves fried, ff Allium ursinum L. Liliaceae LU 1 roots spice O 1, 5, 6 leaves raw Allium sp. N 2 ? ? Maianthemum bifolium (L.) F. W. Schmidt H 1, 3 fruits raw, wine Viscum album L. Loranthaceae H 2 fruits raw Malva neglecta Wallr. Malvaceae H 1, 2 leaves boiled H 1, 2, 5 immature fruits raw Malva sylvestris L. H 1, 2 leaves boiled H 1, 2 immature fruits raw Fraxinus excelsior L. Discussion A 6 seeds spice Frangula alnus Miller Rhamnaceae NU 2 fruits jams Alchemilla sp. Rosaceae L 1 leaves boiled, ff Crataegus sp. pl. H 1, 2, 3, 5 fruit raw, wine, jams A 6 flowers raw Fragaria vesca L. H 1, 2, 3, 5 fruit raw, wine, jams Malus domestica Borkh. (feral plants) O 1, 2, 3, 5 fruit raw, compotes or in sauerkraut Potentilla anserina L. L 1 young shoots raw Prunus avium L. O 1 fruits raw, compotes O 1 solidified sap raw Prunus padus L. H 1, 2, 3, 5 fruits mainly raw Table 6 The list of wild food plants used in Poland since the 19th century (Continued) Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 13 of 16 Page 13 of 16 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Table 6 The list of wild food plants used in Poland since the 19th century (Continued) Prunus spinosa L. H 1, 2, 3 fruits raw, compotes, jams, wine Pyrus pyraster (L.) Burgsd. & Pyrus communis L. em. Gaertner O 1, 2, 3, 5 fruits raw, dried, pickled Rosa sp. pl. (mainly Rosa canina L.) H 1, 2, 5 fruits raw, wine, jams, infusions Rubus caesius L. H 1, 2, 3, 5 fruits raw, wine, jams, infusions Rubus idaeus L. H 1, 2, 3, 5 fruits raw, wine, jams, infusions Rubus L. sect. Rubus sp. pl. H 1, 2, 3, 5 fruits raw, wine, jams, infusions Rubus saxatilis L. A 1, 2, 5 fruits raw, juice Sedum maximum (L.) Hoffm. H 2 thick roots unspecified Sorbus aucuparia L. emend. Hedl. H 1, 2, 3, 5, 6 fruits wine, jams, liqueur, rarely as spice Galium odoratum (L.) Scop. Rubiaceae H 2 flowering shoots spice, infusions V. beccabunga L. or V. anagallis-aquatica L. Scrophulariaceae A 3 shoots raw Tilia cordata Miller & Tilia platyphyllos Scop. Tiliaceae H 1, 2 flowers infusion O 2 cambium raw O 3 seeds oil O 3, 4 leaves boiled or into flour, ff Trapa natans L. Trapaceae L 1 leaves raw, boiled, flour Typha latifolia L. Typhaceae A 5, 6 shoots and rhizomes boiled, roasted Ulmus sp. Ulmaceae N 2 fruits, leaves unspecified Urtica dioica L. & Urtica urens L. Urticaceae H 1, 2, 3 shoot tops boiled, infusions Parthenocissus sp. Vitaceae H 2 fruits fermented drink Viola arvensis Murr. Discussion Violaceae A 5 flowers raw The credibility of identification: H - confirmed by voucher specimen; A - confirmed by a reliable professional or amateur botanist; O - obvious common name universally used in a large area; L - probable Latin name or scientific name used in the language of a given country, given by a non-botanist; N - determined using comparative analysis of folk names; R - determined with the help of the data of a species range or/and habitat; U - highly uncertain; ff - used only as famine food. Source: 1 - [65], 2 - [79], 3 - [80], 4 - [81], 5 - [82], 6 - [83]. Table 6 The list of wild food plants used in Poland since the 19th century (Continued) The credibility of identification: H - confirmed by voucher specimen; A - confirmed by a reliable professional or amateur botanist; O - obvious common name universally used in a large area; L - probable Latin name or scientific name used in the language of a given country, given by a non-botanist; N - determined using comparative analysis of folk names; R - determined with the help of the data of a species range or/and habitat; U - highly uncertain; ff - used only as famine food. Source: 1 - [65], 2 - [79], 3 - [80], 4 - [81], 5 - [82], 6 - [83]. contrast to a few authors who frequently misnamed the plants they had studied. names with Polish official names (scenario 2.1.3 in the Background chapter). This probably occurred by the researcher looking up a particular local name in a plant guide and then automatically assigning to it the Latin binominal of a different genus whose official name was identical to the local name of the studied species. It must be noted that there is a significant difference between the mean percentage of mistakes detected in the studied works without voucher specimens (6.2%) and the level of errors found in the works documented by voucher specimens (9.2%). This also illustrates that even in works documented by a herbarium, gross mis- takes can occur when the specimens are not verified by a good taxonomist. Single voucher specimens impose yet another threat: although the plant was correctly identified in the field or due to a widely known folk name a specimen of the wrong plant may be collected. Conclusions Fortunately the errors made in the presented publica- tions were rarely repeated later. The only case of erro- neous “ghost information” in Polish studies is a list of plant names compiled in two ethnographic works [70,71]. This situation contrasts with Swedish publica- tions, which according to Svanberg [8,9] contain numer- ous ghost data. Ethnographic papers without herbarium documentation contain on average at least 6.2% of mistakes. The verifi- cation of voucher specimens can increase this ratio to 9.2%. These mistakes most often arise by looking up plants using a local name in a botanical guide, and by the lack of cooperation between ethnographers and botanists. The large difference between the ratio of mis- takes in the voucher specimen collections and the ratio of detectable mistakes in the literature is an argument for the rigorous use of voucher specimens, which are identified by a specialist, and for creating a service of online depositories of photographs of voucher specimens. In all the studied cases the ethnobotanical herbaria contained species which had been reported in the given region or cultivated there, which supports the idea of using detailed atlases of plant distribution to verify eth- nobotanical data. Such biogeographical data could be coupled with estimates of species abundance and distri- bution in local habitat spectra. The presented code of credibility may become a useful tool for historical ethnobotany. Not all the codes presented in the methodology sec- tion were used in the real-life list of edible plants of Poland. Most taxa were identified using the codes H, A and L, more rarely O, N or R. The codes D, M and P were not used. However this list was compiled using numerous voucher specimens (hence code H predomi- nates) and data from ‘reliable’ researchers (like Rosta- fiński and Pirożnikow, hence code A). If a similar list were to be compiled for countries where voucher spe- cimens were not collected, or for earlier periods, the proportion of codes in the list may have been reversed. Competing interests h h d l h The author declares that they have no competing interests. The author declares that they have no competing interests. Received: 15 October 2010 Accepted: 17 December 2010 Received: 15 October 2010 Accepted: 17 December 2010 Published: 17 December 2010 Received: 15 October 2010 Accepted: 17 December 2010 Published: 17 December 2010 Published: 17 December 2010 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 14 of 16 At the end of this paper the author must confess his own error. While preparing a table for the article on the taxonomic issues in Polish ethnobotanical studies [19], a mistaken name for Veronica chamaedrys was published, i.e. wole oczy instead of żabie oczka. (’ox-eyes’ instead of ‘frogs’ eyes’). This mistake happened when transferring hand-written records to the computer. That is another example of the way errors and “ghost information” can enter ethnobotany, even via a botanist. in the field and for those identified by a non-specialist (e.g. a separate code E could be used for a taxonomic expert). This could be useful, but on the other hand it is very difficult to draw a line between these two cate- gories. As a rule, all voucher specimens should be iden- tified/verified by a specialist - in case of easily identified taxa - a botanist, and in the case of critical taxa (in the Polish flora: Rubus, Rosa, Hieracium, Oenothera, Alchemilla) - a specialized taxonomist, or the specimens should be identified only to the genus, subgenus or sec- tion level [2-5,19]. in the field and for those identified by a non-specialist (e.g. a separate code E could be used for a taxonomic expert). This could be useful, but on the other hand it is very difficult to draw a line between these two cate- gories. As a rule, all voucher specimens should be iden- tified/verified by a specialist - in case of easily identified taxa - a botanist, and in the case of critical taxa (in the Polish flora: Rubus, Rosa, Hieracium, Oenothera, Alchemilla) - a specialized taxonomist, or the specimens should be identified only to the genus, subgenus or sec- tion level [2-5,19]. Acknowledgements I f l I am very grateful to Dr Ingvar Svanberg (Uppsala) for an inspiring discussion on the topic of mistakes in ethnobotanical publications. I am very grateful to Dr Ingvar Svanberg (Uppsala) for an inspiring discussion on the topic of mistakes in ethnobotanical publications. Discussion This may have happened to Orzeszkowa. Her herbarium contains a specimen of Hepatica nobilis identified as ‘kopytnik’ Asarum europaeum. The name kopytnik is universally used throughout Poland to name Asarum, so Orzeszkowa may have collected a wrong specimen as both species have evergreen leaves of similar size and grow in the same habitat (identification scenario 1.1 or 1.2.2.1.2 in the Background chapter). The studied papers usually contained little or no methodological information, so in most cases we cannot be sure if mistakes arose with or without seeing the actual studied plants in the field. Most errors in the identification of voucher specimens occurred within the same genus, and only 24% of genera were misidentified. In contrast with this, 48% of mis- takes detected in the publications without voucher spe- cimens concern incorrectly identified genera. As the total number of detectable mistakes in the studies with- out voucher specimens is roughly four times lower than that of the studies with voucher specimens (2.3% versus 10%), we can assume that three quarters of all the errors and half of the misidentified genera remained unde- tected in the studies without voucher specimens. It is a matter of dispute whether there should be sepa- rate codes for voucher specimens identified by an expert It must be emphasized that the main source of errors in the analyzed literature was the confusion of local Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Łuczaj Journal of Ethnobiology and Ethnomedicine 2010, 6:36 http://www.ethnobiomed.com/content/6/1/36 Page 15 of 16 9. Svanberg I: The use of lichens for dyeing candles: ethnobotanical documentation of a local Swedish practice? 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https://openalex.org/W2802266914	https://thescipub.com/pdf/jcssp.2018.562.573.pdf	English	null	Development of Web-Based Electronic Learning System (WELS) in Improving the Effectiveness of the Study at Vocational High School “Dharma Nusantara”	Journal of computer sciences/Journal of computer science	2,018	cc-by	6,842	1Deni Darmawan, 2Handy Kartawinata and 3Widya Astorina 1Educational Technology, Graduate School of Universitas Pendidikan Indonesia and Educational Technology of Postgraduate of Educational Institute of Indonesia, Indonesia 2Program Studies, Educational Technology, Instructional Technology Concentration, Graduate Study Educational Institute of Indonesia, Indonesia 3Program Studies, Computer Engineering and Networks, SMK Dharma Nusantara, Indonesia Abstract: The background of this research question comes from teachers of the vocational high school in Indonesia who are difficulties and not sure apply WEB-Based learning to students of Computer and Network engineering program. To answer that question, researchers are developing a learning model based on Electronic Learning Systems (WELS) web-based. The development process is done through Research and Development Approach. From this WELS model, then used in learning on computer technique and network technique and the result is quite effective. Then tested the level of relationship between WELS with the level of effectiveness obtained, the results showed a positive and significant relationship on vocational school students in Indonesia, especially in vocational high school "Dharma Nusantara". In order to produce better future research, we recommend that further research is undertaken by examining other factors that may contribute to and influence the improvement of WELS for all vocational high schools in Indonesia. Article history Received: 03-09-2017 Revised: 16-03-2018 Accepted: 21-04-2018 Corresponding Author: Deni Darmawan Educational Technology, Graduate School of Universitas Pendidikan Indonesia and Educational Technology of Postgraduate of Educational Institute of Indonesia, Indonesia Corresponding Author: Deni Darmawan Educational Technology, Graduate School of Universitas Pendidikan Indonesia and Educational Technology of Postgraduate of Educational Institute of Indonesia, Indonesia Email: itikurih71@gmail.com deni_darmawan@upi.edu Keywords: e-Learning, Learning Website, WELS namely computer technique and network with the overall number of students were 414 students. Teachers and students in Vocational High School “Dharma Nusantara” currently requires the presence of the application of new learning methods that are effective and fun in accordance with the development of information and communication technologies, one of which is the existence of a medium of instruction. Learning media can be created and used in accordance with needs in the process of implementation of the study at Vocational High School “Dharma Nusantara”, interactive learning media is media that is needed in the implementation of learning, either directly in the class or indirect learning or distance learning are often called or also called online learning (e-learning). Distance education has become a popular term to describe learned through telecommunications. 1Deni Darmawan, 2Handy Kartawinata and 3Widya Astorina As described by Keegan (1986) on the books of Instructional Technology and Media for Learning that distance education has a definition and characteristics of © 2018 Deni Darmawan, Handy Kartawinata and Widya Astorina. This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license. Journal of Computer Science Journal of Computer Science Original Research Paper Introduction Technological developments have brought such great impact on the quality of learning and the quality of education, including the process of teaching and learning activities in the classroom, is no longer centered against the teacher. Students can study in accordance with what he wants, according to what he is thinking, so it inspires students in creative thinking and innovative. As is the case when the students learned one of the subjects, when in the process of learning, teachers, as well as students, don't find the right solution in practice his studies, computer technology can be one of the best solutions as a medium of instruction. Vocational High School “Dharma Nusantara” is a school that has a Program of Information and Communication Technology Skills. Where teachers and students are required to follow the development of technology and information. One of his competence, Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 the physical separation of the learner from the teacher, the managed instruction program, communication technology and two-way communication. Smaldino et al. (2012) in Instructional Technology and Media for Learning found that "online learning (also known as learning electronic learning or E-Learning) is the result of teaching being delivered electronically using a computer-based media". From the observations, while conducted by a research team at Vocational High School “Dharma Nusantara”, researchers found the problem about the delay of learning activities directly in class due to a variety of factors over which requires the existence of a solution to overcome these problems. Thus this research is focused on questions about (1) How does Electronic Learning System development in improving the effectiveness of Web-based study at Vocational High School “Dharma Nusantara”?; (2) How is the effectiveness of the application of the Web-based Learning Activities on computer network engineering student in Vocational High School “Dharma Nusantara”?; (3) How is the relationship between the application of Web-based Electronic Learning Systems that have been developed with the effectiveness of student learning at Vocational High School “Dharma Nusantara”? p Learning activities in a way that makes it easy for users in online. Because the students or teachers no longer have difficulty in terms of distance or time when the learning activity directly in the class or can be said face to face directly cannot be done. Literature Review gy E-learning applied in Vocational High School “Dharma Nusantara” motivated by difficulties of learning material, assignments or quiz when the teacher was unable to attend to implement learning directly. Other problems are also often experienced by teachers when they have tasks outside school, but the task of teaching is required to be implemented. From these conditions then e-learning based learning is needed as a solution. Not less important is when students are carrying out the activities of the work practices of the industry, where students carry out work directly in the learning industry for a little over two months. Teaching and learning activities are no longer carried out effectively in the classroom. Meanwhile, the activities of the midterm and final exams of the semester must remain they follow. So the students lack get lessons at school. In General, teachers have yet to understand the technology of learning online learning system, on the other hand, the current students are enjoying activities in the form of online communication, ranging from socializing using social media online, search learning materials online, to the activities of entertainment such as game playing. This phenomenon gave rise to the impression students more prepared to face the development of information and communication technology than the teacher. Therefore, teachers need to compensate by following the development of information and communication technologies especially in the field of online learning, so that students can learn in a way that they enjoy doing. In addition, along with the development of technology, students and teachers will also need to know online learning that demands the ability to be able to do a variety of activities-based internet technologies. Whereby indirectly, e-learning can improve the quality of learning in a fun learning method in accordance with their interest will be matters relating to the development of technologies that are currently available. Introduction Darmawan (2014a) in his book entitled the development of E- Learning Design theory and argued that: "The system of electronic learning or e-learning is the new way of teaching and learning. E-learning is a basic and logical consequence of the development of information and communication technology". Learning system Learning in effective teaching and learning activities, in fact, is the learning that is implemented systematically. Shrode and Voich (1974) in Zakapedia (2015) defines that "the System is a set of interrelated parts, working independently and jointly, in pursuit of common objectives of the whole, within a complex environment". Learning activities in it there is the activity of teaching educators and student learning activities, teaching activities among teachers and learning activities of students this is what is often called the learning interaction. Learning depends very much on the ability of teachers in implementing or pack the learning process. The study was carried out in a good and right will provide the dominant contribution in order for students, instead of learning that is implemented in a way that is not good will lead to potential student hard developed or empowered. Hamalik in Sanjaya (2013) suggested that the “learning system is an organized combination that includes elements of human, material, facilities, equipment and procedures that interact to accomplish a goal”. The elements in the system of learning should be integrated with each other so that the learning objectives to be achieved can be realized. As well as a system of online learning element of humane is items must exist in the learning system, which consists of the elements of the system developers, teachers and students as a user. Element material consisting of materials and supporting equipment in systems such as online learning, the computer as a medium, the user interface as the media liaison between students and teachers in learning online, access to the internet as a transmission medium in the process of implementation of distance learning. 563 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 competency skills to the needs of the industrial world in the future when the students have finished school and work in the industrial environment. Methods Research and development in terms of education, producing specific products that can be used to achieve the goal of effective learning. Borg et al. (2003) in Sugiyono (2015), The steps of this process are usually referred to as the R and D cycle, which consist of Wadi research findings pertinent to the product to be developed, developing the product based on these findings, field testing it in the testing where it will be used eventually and revising to correct the deficiencies found in the field-testing stage. In more rigorous programs of R and D, this cycle is repeated until the field test data indicate that the product meets its behaviorally defined objectives. To analyze the impact of the research product, the quantitative data is analyzed through the result of experimental data of the product at trial (Darmawan, 2014b). Sugiyono (2015) explained the steps the research and development level 4 as follows: Website The website or often known as the current website often used by the user in the exchange of information that consists of a few pages use technologies that are connected to the internet. Yuhefizar (2016) in the Carapedia page.com, that: the Web is a method for displaying information on the internet, either in the form of text, images, sound or interactive videos and merit to linking (link) a document with other documents (hypertext) that can be accessed through a browser. How to make use of information technology for learning activities according to Warsita (2008), as follows: (1) Web Course, namely the use of information technology for the purposes of education, where all learning materials, discussions, consultation, training, assignments and exams are delivered entirely over the internet. Students and teachers are entirely separate and not needed the presence of face-to-face, (2) Web-Centric Course, where most materials, discussions, consultation, assignments and exercises are delivered via the internet; While the exam and some consultations, discussions and exercises done in face-to-face and (3) Web-Enhanced Course, namely the utilization of the internet for education, to support the improvement of the quality of face-to-face learning activities in the classroom. Based on the above, some sense of the Web according to researchers are information and communication technology-based wherein it has the features of multimedia (text, images, sound and video) in the form of information that can be accessed interactively through internet network. Electronic According to large Indonesian Language Dictionary (KBBI) online, an electronic device that is "based on the principles of electronics; things or objects using tools created or worked on the basis of Electronics". An example of an electronic device is the driver electronics (electronic control), electronic equipment, electronic devices and musical instruments that use electrical tools. Based on the above understanding, electronic learning in context can be understood as a medium that was created with the intention to accommodate the learning needs was formed with the basic principles of electronics such as televisions, radios, computers and more. Moodle in the Development of Learning Management System (LMS) Moodle is an acronym for Modular Object-Oriented Dynamic Learning Environment (Xing, 2013). Darmawan and Bariyah (2014), "Moodle is an open source application program in developing e-learning systems". Using Moodle in building web-based learning system, can create an effective teaching and learning activities without limits of space and time. This means the learning system can be done electronically (e-learning) or also called remote learning system (distance learning). Moodle can be used freely as open source products (open source) under the GNU license. Moodle can be installed on any computer and operating system that can run PHP and SQL (Alshraideh et al., 2013) database support. The features are presented in a Learning Management System is quite comprehensive, (Lourdes et al., 2013), including Assignment Submission, discussion forums, download archives, rankings, chat, online calendar, news, online quiz, wikis and Video Conference, (McKnight and McKnight, 2012). How to develop a Learning Management System is divided into two ways, i.e., offline and Online. To construct a Learning Management System by way of offline-only required supporting software, such as Xampp and Moodle's course to the next can be installed and configured. As for building a Learning Management System by means of online just by visiting a website on https://gnomio.com. Collection of Information At this stage, the researcher collects a variety of information from the administrative data of the associated students, teachers, list of subjects correspond to the structure of the curriculum, lessons schedule, materials, articles, news of the school as well as other information that supports the completeness of data in a web-based electronic learning systems. This flowchart from Fig. 2 described the process of operating WELS (left side). It starts from data input until Finish. Then right side the system display and respon the whole system of prototype WELS. It begins with controling based on database until system respons and display. Both must show smooth communication. Validation of the Design At this stage product design was validated by media experts to obtain a judgment that the products developed are worthy to be used in the stage of research in the field. Literature Study At this stage the researcher is collecting and studying some literature relating to research and development (Borg et al., 2003) that is based on the results of research to determine what products need to be developed in order to overcome the problems, especially the development of web-based electronic learning system using Learning Management System Moodle as an attempt to increase the effectiveness of learning in the Vocational High School “Dharma Nusantara”. Identify Potential and Problems (https://smkgdn.gnomio.com). Researchers develop online LMS with consideration in terms of time, cost and ease of build. This phase consists of (1) the pre-production stage, (2) production stage and (3) post production stage. The following flowchart of the use of web-based electronic learning system based on the access rights of the user who performed the activity in the pre-production. This is intended to help the legibility of the system of information for the admin in using this product as described by Paydar and Kahani (2011) which explains that Information Sources: This item indicates the types of information sources that are utilized by the pre-test process Information Sources: This item indicates the types of information sources that are utilized by the framework to automate the test process. At this stage, researchers conducted a series of structured and unstructured interviews with several Parties at the school, including interviews with the principal, some teachers and students. In addition, researchers doing research in the field, researching the teaching and learning activities when the teacher was unable to attend class. Based on the identification process, obtained the impression that the problems that appear in the activity of learning when teachers and students cannot face to face directly, learning becomes not effective though the task is already given. Design Revision This stage is carried out after the first product design validation is performed. The revision is carried out based on some revisions of media experts. The revision is done including a banner on the initial display (homepage), user permissions and category rendering course which is the core content of web- based electronic learning system. Training Materials Training Materials stands for education and training materials. Unlike adaptive and normative subjects, subjects in high school vocational training or productive subject there is often referred to by the term vocational subjects. It's called a training subject because it contains knowledge, theory and practice. More than that, on the subject of productive training exercise activity there is in accordance with the Based on the steps in Fig. 1, here are the steps of WELS development, which are as follows: 564 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 Fig. 1: Steps of Level 4 research and development here are the stages of research with reference to the flow chart above R&D Potential and problems Literature study Information gathering Product design Design validation Product design Product-1 revision Limited trial Product development Design revision Product-2 Revision Operational field trial Product-3 Revision Disemination and Implementation Literature study Product-1 revision Product development Fig. 1: Steps of Level 4 research and development here are the stages of research with reference to the flow chart above R&D Fig. 1: Steps of Level 4 research and development here are the stages of research with reference Identify Potential and Problems Designing Products On this stage researchers design products using Learning Management System (LMS) Moodle online 565 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 DOI: 10.3844/jcssp.2018.562.573 Fig. 2: Administrator access Flowchart for Administrator User: Administrator System Start Operating WELS Display Features MENU Explore available Features Processing data? Login Data Validation No Yes Update and delete user No Yes Valid? Mode: Administrator Administrator can processing data (Input, Update and Delete) Processing Data Setting site Display Setting Language Database Autentification and user Registration Append Categories, Lessons and Teachers Upload data user Registering Student to the lessons Any Activity? No Yes Logout Finish Note : Advanced workflow from user to system : Flow back from system to user Paralel Mode: System Respons and Display Flowchart for Administrator User: Administrator System Display Features Processing data? Valid? Administrator can processing data (Input, Update and Delete) Fig. 2: Administrator access access. This test was performed twice to make sure that the features tested was understandable by the students. Tests performed include: (1) A test user login (access rights), (2) testing the menu, (3) applying the test profile settings, (4) a test display, (5) access trials per session Limited Trials The Main Field Trials On the stage of this main field trials, researchers are testing the feasibility of the system with the approach of the application of the material on the training subject to assemble Personal computers in class X-A competency expertise of computer technique and network. Limited Trials Limited trials carried out to some students to know the quality of the electronic web-based learning system that was built, the introduction of the system and ease of 566 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 material, (6) trials of Deuteronomy/quiz and (7) trial of value processing. Revision of the Product 1 Product 1 revision is carried out based on the evaluation of the limited trials. At this stage, the product only at the extent of the revision is the default setting for the language setting, i.e., the language of Indonesia. Fig. 3: Teacher access User: Teacher Start Operating WELS Surfing available Features System Processing data/teaching Flowchart for Teacher MENU Display feature Login Yes No No Date validation Valid? Teaching and learning activity (Lessons, schedule, attendance, quiz, value and participation of students in ‘class’) Yes Mode: user- teacher Database Data stored Data lesson Doing teacher activity Data processing/ teaching Teaching Prepare materials (lesson, exercise, deuteronomy)- weekly Data stored Data processing Seeing the student’ activity Managing value Print out/update? Updated Student data Create a schedule, classes, assignment, add chatting activity Print out Student score report Logout Finish Note : Advanced workflow from user to system : Flow back from system to user Display feature Date validation Valid? Data stored Fig. 3: Teacher access Fig. 3: Teacher access 567 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 The Operational Field Trial The stage of operational field trials researchers are testing the feasibility of the system with a two-stage approach to the material on the training subject of Assembling Personal Computers in class X-A competency skills as computer and network improvements from previous trials. Product revisions at this stage are only done on technical features. The Dissemination and Implementation of the Results of Product Development This stage is a stage where students and teachers can use the web-based electronic learning systems in everyday teaching and learning activities. WELS can already be used in teaching and learning activities in accordance with the conditions and needs. After learning the system developed further do test process Improvement the effectiveness of Learning in the application of WELS with the acquisition of the data as it appears below. Revision of the Product 2 the suitability of the schedule and (3) that features cannot be used because at that session has not yet been activated. Product 2 revision is done based on the evaluation of field trials. At this stage of revision, products include: (1) Clarify the title Subject, where previously the title subject matter not listed. The information shown is date per session, so students are hard-pressed to choose the material, (2) features "Attendance system" could not be used because of This flowchart from Fig. 3 described smoothness of teacher communication as user of WELS. It important, that WELS prototype in this case how the teacher activity during teaching. Teacher should be able to generate a number of learning resources which will be stored in the WELS program database for teaching and learning activity. Fig. 4: Student access User: Student Start Operating WELS System Explore the features available Flowchart for Student MENU Display Features Login Yes No Date Validation Valid? Mode: User- student No Learning activity? Explore the other features? No Yes Logout Finish Note : Advanced workflow from user to system : Flow back from system to user Paralel Mode: System Response and Display Database Seeing the schedule and lesson activity Seeing and taking an available lesson Following lesson and exercise Seeing and giving task Answer the quiz Using chat facility Seeing the result Fig. 4: Student access Fig. 4: Student access 568 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 Research and information Collecting. -include needs assessment, review of literature, small-scale study, research and preparation of report on state of the art of tea, (2) Planning-include defining the skills to be learned, stating objectives, identifying and sequencing learning activities and small-scale business feasibility testing, (3) Develop a Preliminary Form of a Product- Includes preparation of instructional materials, procedures and evaluation instrument, (4) a Preliminary Field Testing-Conducted in form 1 to 3 schools, using 6 to 12 subjects. Interview, observational and questionnaire data are collected and analyzed, (5) Main Product Revision-Revision of the product suggested by the preliminary field-test result, (6) Main Field Testing- Conducted in 5 to 15 schools with 30 to 100 subjects. Quantitative and pre-course and the post course subject are on performance are collected. Revision of the Product 2 Results are evaluated with respect to course objective and are compared with a control group of data, when appropriate, (7) Operational Product Revision-Revision of the product suggested by main field-test result, (8) Operational Field Testing-Conducted in 10 to 30 schools, involving 40-400 subjects. Interview, questionnaire and observational data collected and analyzed (9) Final Product Revision-Revision of the product suggested by operational field-test result, (10) Dissemination and Implementation-Report on the product at professional meetings and in journals. This flowchart from Fig. 4 described the process of smooth communication between students as users with WELS prototypes. The workflow of the system is expected to provide facilities for learning students interactively by utilizing the learning resources of the WELS program Database through Paralel Mode: Respons and Display. Revision of Product 3 This stage is actually no revisions, problems at the stage of field trials only on operational activities of the chat. So that repairs can be made at that time as well. The Dissemination and Implementation of the Results of Product Development Result and Discussion Development of WELS to Enhance the Effectiveness of the Study at Vocational High School “Dharma Nusantara” The data in the Table 1 shows the increase in the average value on every test run. This suggests that the development of WELS can increase the effectiveness of the study at Vocational High School “Dharma Nusantara”. Through WELS all data and learning information that students need can be easily obtained from the existing cloud base in the system, as explained by Molamoganyi Gorata et al. (2017) which states that Cloud scenarios face a huge amount of data which is known as big data, as such, the replication method affect the storage and communication cost directly. Based on the table presented above, the increased effectiveness can be seen in the Fig. 5. The researchers do development process using the steps put forth by Sugiyono (2015:48), namely research and development of level 4. These steps comprise the potential and problems, the study of literature, information collection, product design, design validation, revision of design, product manufacturing, limited trials, product revision 1, main field trials, product revision 2, operational field trial, product revision 3, as well as dissemination and implementation. As for the measures put forward by the Borg et al. (2003) in Sugiyono (2015) are as follows: Table 1: Improvement of learning effectiveness No Kind of trial Value Average 1 Actual Condition 1644 46.97 2 Limited Trial 690 69.00 3 Main Field Trial 2333 66.66 4 Operational Field Trial 3977 87.91 5 Effectiveness Test 3082 88.06 569 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 Fig. 5: Increase the effectiveness of Learning 46,97 69,00 72,09 87,89 88,06 90,00 80,00 70,00 60,00 50,00 40,00 30,00 20,00 10,00 0,00 Improvement of learning effectiveness Actual condition Limited trial Main field trail Operational field trail Effectiveness test Improvement of learning effectiveness Fig. 5: Increase the effectiveness of Learning The Application of WELS Effective in Learning Activities The Application of WELS Effective in Learning Activities Development of web-based electronic learning system was successfully applied in order to achieve effective learning without worrying about the problems of distance learning, (Heshmatpanah and Neyestanak, 2011). The time as well as to educate students and teachers to be more prepared for the technological developments in the field of learning. This kind of learning can be said also as distance learning or system known as e-learning. Result and Discussion This result support to Al-Hasani and Elgazzar (2015) An Initial list of instructional design standards of the two e- Learning Multimedia Educational Computer program Instructional designs were derived. Changes in the concept of learning not an easy thing to be implemented. Need for education to teachers and students to learn, adapt and utilize this system of distance learning, In addition, the required presence of conditioning in order to make this web-based learning activity can be implemented in Dharma Nusantara. Husna and Wahyuni (2008) explain the importance of Human Resources, that "through the efforts of the Human Resources education quality can be improved. While the quality of education depends very much on learning model developed. Therefore various models of learning need to be continuously developed because the learning process is still being an influential factor in education efforts. One of the models of learning that is now needed is information technology-based learning (E-Learning), a more open, easy and quick to be accessed by anyone, such as distance education. The statement confirms that e-learning has contributions to the teacher in teaching to their students in a dynamic, interactive and nonlinear access to the text, graphics and animations in a new environment so that students and teachers have new knowledge especially in the field of information and communication technology. The application of web-based electronic learning system developed in this study has the following advantages: (1) For students, with the presence of e- learning activities, allow the growing flexibility of the higher learning activities, i.e., students not only able to access learning materials when in class but can also do so repeatedly according to their needs by downloading it via the internet. Students also can learn themselves faster to enhance knowledge and expand their insights, learn interactively through communication with other speakers that can motivate the spirit of student learning, as well as develop ability in the field of research at the same time can also increase sensitivity to the various problems that exist and (2) for teachers, with the utilization of the internet and teacher have a chance developing the professional ability of them namely increasing knowledge, share resources among fellow teachers with the same subjects, publishing and publication, in collaboration with teachers in foreign countries as well as participate in forums and colleagues both locally and internationally. Result and Discussion The application of web-based electronic learning system has functioned as (1) Additional (Supplement) like asynchronous system (Oluwaniyi et al., 2015). The web-based asynchronous e-learning system will reduce the constraints of the classical learning system and save time and resources. It is said to function as an additional (supplement), if the student has freedom of choice, whether to make use of electronic learning materials or not. In this case, there is no obligation/necessity for students to access learning materials electronically. 570 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 H0: t < 0 H1: t > 0 Though their nature optional, students who use it will have additional knowledge or insight, (2) Complementary (complement). It is said to function as a complement of electronic learning materials when added to supplement accepted student learning material in the classroom (Lewis and Goodison, 2004). As a complementary means of electronic learning materials are to be materials, (3) Enrichment (reinforcement) or repair (remedial) for learners in the following activities of conventional learning, (4) a substitute (substitution). Some schools in developed countries provide some alternative models of learning activities to students. For testing the hypothesis, then in the methodology used, will test the correlation included Kendall Tau (t) whose results can be seen in the Table 2. So there is a positive relationship as big as These findings suggest that the model designed for the general didactic tutor Lourdes et al. (2013), is required in online learning such as WELS 0.764 between the development of web-based electronic learning system with increased effectiveness study at Vocational High School “Dharma Nusantara”. In order to prove whether the coefficients that can be imposed on the population where the sample is taken then it needs to be tested for their significance by using the formula z. In this case the extent of his error of 5%. These findings suggest that the model designed for the general didactic tutor Lourdes et al. (2013), is required in online learning such as WELS. From this point of view, a reasonable design helps to improve the extensibility and flexibility of the framework, (Paydar and Kahani, 2011). Result and Discussion The flow of the application of web-based electronic learning system was initiated by the work undertaken by the management systems that involve: (1) An administrator; working on various activities administering learning as a preparation phase of teaching and learning activities between teachers and students, (2) teachers; working on a wide range of activities to teach students online and (3) students; see a list of the subjects that must be followed. Thus it can be concluded that the problems of the system of learning in a classroom that had occurred in the Vocational High School of “Dharma Nusantara” can be resolved. ( ) ( ) 0,74 0,74 0,74 6,19 0,12 2(2 5) 2(2.35 5) 2 75 9 ( 1 9.35(35 1) 315 34 z z N N N τ = = = = = + + − − The Relationship between the Applications of WELS that has been Developed with Increasing Effectiveness Study at Vocational High School “Dharma Nusantara” The Relationship between the Applications of WELS that has been Developed with Increasing Effectiveness Study at Vocational High School “Dharma Nusantara” A z-count value above then compared to the value of z-table. To test the two-party, then 1% error level is divided into two, becomes 0.5%. Next, the value of z can be seen on the normal curve with z = 0.495 (0.495 retrieved from 0.5-0.005). On normal curve table, 495 figure does not exist, but the most figures approach is a number 4951. Based on these figures, then z = 2.58 price. To be able to give an interpretation of whether the price is significant or not then be able to use a provision that if the count is greater than the z-table, then the coefficient of correlation being found is significant. It turns out that calculating z 6.19 is greater than the z table 2.58. Thus it can be concluded that the correlation between the development of web- based electronic learning system with increased effectiveness study of 0.74 was significant. It can also be described by a normal curve as follows. Researchers test the relationship between the development of web-based electronic learning system by improving the effectiveness of learning. To that end, researchers are formulating a hypothesis as follows: H0: There is no relationship between the development of web-based electronic learning a system with increased effectiveness study at Vocational High School “Dharma Nusantara”. Result and Discussion H1: There is a significant and positive relationship between the development of web-based electronic learning system with increased effectiveness study at Vocational High School “Dharma Nusantara”. Correlation is significant at the 0.01 level (2-tailed) Acknowledgement This work is a collaboration between Postgraduate of Educational Technology of STKIP/Indonesia Institute of Education and Vocational High school of Dharma Nusantara. It has been proven by the presence of a positive relationship between the development of WELS with the improvement of study effectiveness in Vocational High School “Dharma Nusantara”, based on the curve in Fig. 6 that calculating z = 0.74 6.19 is located in the rejection of H0, H1 then accepted. Conclusion From this research can be concluded that the development of electronic learning system which is then produced with WELS has been done by using the stages of research and development. The development research process undertaken through the revision test stage on prototypes generated in the trial is limited to a wider trial. The results of the revision test model that has obtained information learning through WELS can improve the quality of learning in vocational school level Or it can be written with the test hypothesis as follows: Or it can be written with the test hypothesis as follows: Table 2: Correlation of Kendall Tau (t) Developing Improvement of WELS Learning Activities Kendal’tau_b Development of WELS Correlation Coefficient 0.764 0,764 Sig, (2tailed) 0,000 N 35 35 Improvement of Correlation Coefficient 0,764 1,000 Learning Effectiveness Sig. (2 tailed) 0,000 N 35 35 Correlation is significant at the 0.01 level (2-tailed) **Correlation is significant at the 0.01 level (2-tailed) 571 Deni Darmawan et al. / Journal of Computer Science 2018, 14 (4): 562.573 DOI: 10.3844/jcssp.2018.562.573 Fig. 6: The applicability of test 2 party -4.25 -2.58 2.58 4.25 0 0,74 Z-Table Z-Count Denial Area H0 Denial Area H0 Denial Area H0 Denial Area H0 Fig. 6: The applicability of test 2 party Testing is done to prove the existence of a relationship (Correlation) between the application of web-based electronic learning system with increased effectiveness of study at Vocational High School “Dharma Nusantara”. In the draft of correlational research, research using correlational statistic test to describe and measure the degree of interrelationship or relation between two variables. In this draft, the researcher does not attempt to control or manipulate variables as in the experiments. Creswell (2015:664), "correlation is a statistical test to determine the inclination or pattern for two or more variables or two data sets to very consistently." "Dharma Nusantara". Further research is able to prove that the application of WELS-based learning has resulted in improved learning outcomes are quite good by learners who learn about computer engineering and network at the vocational school level. Likewise from this study obtained information that WELS learning is able to produce a fairly positive relationship with improving the quality of learning vocational high school students at Indonesia like "Dharma Nusantara". Ethics This article is original and contains unpublished material. Participants are made known that their feedbacks will be contributing to a research project. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved. Author’s Contributions Based on the calculation of z-count above, then it can be inferred that the hypothesis that states there is no positive relationship between development of web-based electronic learning system with increased effectiveness study at Vocational High School “Dharma Nusantara” at the level of 0.01 rejected, meaning that in this study it was proved that there was a significant positive relationship. This result it’s was relevant with the result of research from Elabaid and Elobaid (2017) that However, students are convinced of ICT and e-learning utility and applicability. This opens new perspectives to find and adopt more effective tools to improve implementation of e-learning in teaching and learning. Deni Darmawan: Design the research plan, Data analysis, contribute to the writing of the manuscript and revised. Handy Kartawinata: Data analysis and writing the manuscript, Edit and revised. Widya Astorina: Data collection, Edit figure. 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https://openalex.org/W4318821177	https://www.frontiersin.org/articles/10.3389/fpsyg.2023.1096516/pdf	English	null	Knowledge, attitudes, and practices of parents toward sexuality education for primary school children in China	Frontiers in psychology	2,023	cc-by	8,294	OPEN ACCESS Wenjing Zhang1 and Yuzhi Yuan2* Wenjing Zhang1 and Yuzhi Yuan2* 1Beijing Academy of Educational Sciences, Beijing, China, 2School of Education, Renmin University of China, Beijing, China This cross-sectional study provides insight into the perceptions of Chinese parents of primary school children with respect to sexuality education. A sample of 19,745 parents was surveyed using an online questionnaire in Beijing, Tianjin, and Hebei, China. SPSS version 23.0 was used for data analysis. In this study, nearly 90% of parents had positive attitudes toward the sexuality education of children in primary schools. However, Chinese parents had limited knowledge of sexuality education. More than 60% of parents were unaware of the sexuality questions that children may encounter at diferent ages and did not have any accurate information on child sexual abuse prevention education. Although ∼70% of parents (both fathers and mothers) reported that they had used television and Internet resources to talk with their children about sexuality and 63% reported that they had read books with their children about sexuality, < 30% reported using appropriate terminology with their children on sexuality education and only 40% reported talking to their children about sexuality comfortably. The multivariate linear regression equation showed some factors associated with the practices of parents in sexuality education, including parental knowledge, attitudes, their experience in receiving sex education in childhood, and their educational level. The ﬁndings from this study suggest that it is important to develop culturally relevant training programs for parents of primary schools in Chinese society. The implications and limitations of these ﬁndings are discussed. Zhang W and Yuan Y (2023) Knowledge, attitudes, and practices of parents toward sexuality education for primary school children in China. Front. Psychol. 14:1096516. doi: 10.3389/fpsyg.2023.1096516 COPYRIGHT © 2023 Zhang and Yuan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS sexuality education, inﬂuencing factors, primary schools, parents, China COPYRIGHT © 2023 Zhang and Yuan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 01 February 2023 DOI 10.3389/fpsyg.2023.1096516 TYPE Original Research PUBLISHED 01 February 2023 DOI 10.3389/fpsyg.2023.1096516 TYPE Original Research PUBLISHED 01 February 2023 DOI 10.3389/fpsyg.2023.1096516 Zhang W and Yuan Y (2023) Knowledge, attitudes, and practices of parents toward sexuality education for primary school children in China. Front. Psychol. 14:1096516. doi: 10.3389/fpsyg.2023.1096516 Knowledge, attitudes, and practices of parents toward sexuality education for primary school children in China OPEN ACCESS EDITED BY Nelly Lagos San Martín, University of the Bío Bío, Chile REVIEWED BY Oana D˘anil˘a, Alexandru Ioan Cuza University, Romania Donna-Maria Maynard, The University of the West Indies, Cave Hill, Barbados *CORRESPONDENCE Yuzhi Yuan yuanbnu@163.com SPECIALTY SECTION This article was submitted to Educational Psychology, a section of the journal Frontiers in Psychology RECEIVED 12 November 2022 ACCEPTED 04 January 2023 PUBLISHED 01 February 2023 sexuality education, inﬂuencing factors, primary schools, parents, China Parental knowledge, attitudes, and practices in the sexuality education of children Previous studies showed that a majority of parents have positive attitudes toward sexuality education at school and have general sexuality knowledge (Mckay et al., 2014; Morawska et al., 2015; Shin et al., 2019). For example, one study in Australia, with a sample of 557 parents of children between the ages of 3 and 10 years, demonstrated that parents felt knowledgeable about sexuality education (Morawska et al., 2015). Another survey of 337 primary school parents conducted in Korea revealed that more than 70% of respondents have the knowledge of the structure and function of sexual organs, pregnancy, and childbirth (Shin et al., 2019). Although the education policy of China supports primary and middle schools to carry out sexuality education, very few schools oﬃcially teach sexuality education courses for children (Liu and Su, 2014). Chinese children, especially primary school students, have limited sexuality knowledge and skills. A survey with 773 primary students (aged 6–14 years) conducted in 12 schools in Zhejiang, Hunan, Shanxi, Sichuan, Ningxia, and Liaoning provinces in China found that primary school students lack sexuality knowledge (Hu et al., 2015). Some studies in China on child sexual abuse prevention education revealed that primary school children lack the knowledge of sexual abuse prevention and self-protection skills (Chen, 2012; Jin et al., 2016). Research has also shown that few parents discuss and communicate about sexuality with their children. Some studies indicated that few parents could provide adequate and good-quality sexuality education for their children (Jerman and Constantine, 2010; Morawska et al., 2015; Shin et al., 2019). Morawska et al. (2015) found that more than half of the Australian parents did not start up a conversation about sexuality with their children (aged 3–10 years). A recent study in South Korea also showed that two-thirds of parents had never provided sexuality education to their elementary school children (Shin et al., 2019). Scholars and researchers have proposed several reasons for the reluctance of parents to talk about sexuality with their children. Introduction Sexuality education is one of the most important aspects of education for children. The international technical guidance of the United Nations Educational, Scientiﬁc, and Cultural Organization (UNESCO) on sexuality education emphasizes the need for comprehensive sexuality education (CSE) programs that aim to equip children and young people with the knowledge, skills, attitudes, and values to make responsible choices about their sexual and social relationships in the world (UNESCO, 2018). CSE programs are scientiﬁcally accurate, culturally and age-appropriate, gender-sensitive, and life skills-based. Sexuality education should not be assumed to begin in adolescence. Sexuality begins at birth, and talking about sex and sexuality with children should be a continuous process. Sexuality education if started early in childhood may help children develop a sense of themselves and their bodies while strengthening their self-conﬁdence and helping them take charge of their lives (UNESCO, 2018). Sexuality education is critical in the development of a healthy life; it is both formal (e.g., school-based curricula and health professionals) and informal (e.g., parents /caregivers, Internet, and peers). Frontiers in Psychology Frontiers in Psychology frontiersin.org 01 10.3389/fpsyg.2023.1096516 Zhang and Yuan Sexuality education of children in China China has long recognized the signiﬁcance of sexuality education for children. In March 1963, Premier Zhou Enlai stressed the need to promote sexuality education for adolescents as an important element for the healthy growth of the Chinese population on the occasion of the national conference on the hygiene science and technology program (Liu, 1994). However, for a long time, there are neither national guidelines nor a national curriculum for sexuality education in China. In 2008, the Ministry of Education issued a set of health education guidelines for primary and middle schools that contained some reference to sexuality education (Ministry of Education of People’s Republic of China, 2008). Recently, child sexual abuse has become a major concern in Chinese society. A meta-analysis found that child sexual abuse prevalence rates are 8.9% for women and 9.1% for men in China (Ma, 2018). Therefore, in 2013 and 2018, the Ministry of Education issued opinions on sexual abuse education prevention for children and adolescents (Ministry of Education of People’s Republic of China, 2013, 2018). In the present time, part from sexual abuse, children in China are frequently exposed to sexual messages through ﬁlms, television, the media, and the Internet. Thus, the importance of quality sexuality education at an early age has been increasingly emphasized in modern Chinese society (Zhang et al., 2013; Liu and Su, 2014). Parents play an important part in the sexuality education of children feeling embarrassment (Turnbulla et al., 2008; Morawska et al., 2015); and parents with the fear of destroying the “innocence” of their children (Alldred et al., 2016; Robinson and Davies, 2017; Rudolph and Zimmer-Gembeck, 2018). Sexuality education is eﬀective when parents and their children discuss sex-related issues together. Furthermore, parent-child communication, parental monitoring, and involvement are all beneﬁcial to protecting children from sexual abuse (Wurtele and Kenny, 2010; Rudolph and Zimmer-Gembeck, 2018). Parent-child communication about sexuality in the family has been linked to reductions in risky sexual behaviors in adolescence (Huebner and Howell, 2003; Zhao et al., 2022). Parents play a key role in educating their children about sexuality. They could oﬀer sexuality education sequentially and provide time-sensitive information as they receive questions from their children at home (Krauss and Miller, 2012). CSE programs at school can help children and young people maintain sexuality and relationship health in physical, emotional, spiritual, and social adaptation. The impact of CSE lessons at school depends on the support of parents at home, such as clarifying concepts and helping children apply their new knowledge and skills in daily life. Moreover, one of the most important extended environments aﬀecting the learning activities of their children in sexuality is the teaching of parents at home (Morawska et al., 2015). Children and young people often want to learn about sexuality matters from their parents (Turnbulla et al., 2008). Young children are curious about the issues related to sexuality and ask their parents questions about body diﬀerences between boys and girls, where babies come from, how babies are made, and so on (Brilleslijper-Kater and Baartman, 2000; Martin and Torres, 2014). When parents talk about sexuality issues with their children at an early age, they have the opportunity to foster safe and healthy attitudes toward sexuality (Morawska et al., 2015). Therefore, the knowledge, attitudes, and practices of parents would have very strong inﬂuences on the formation of sound sex-related values in their children. Frontiers in Psychology frontiersin.org Parental knowledge, attitudes, and practices in the sexuality education of children These include parents having an inability or unwillingness to discuss topics of a sensitive nature, especially on sexuality (Morawska et al., 2015; Alldred et al., 2016); parents lacking sexuality knowledge and teaching skills to provide education (Morawska et al., 2015; Shin et al., 2019); parents with low self-eﬃcacy or Although more than 70% of primary school students identify their parents as the main source of sexuality education in China (Hu et al., 2015), there is less research on the sexuality education of the children of primary school parents. In Fuzhou, a city in the southeast of China, one study investigated 209 parents in four primary schools. It was found that <40% of parents reported their children needed to receive sexuality education at school and that nearly one-half of parents were reluctant to answer questions about the sexuality Frontiers in Psychology 02 frontiersin.org Zhang and Yuan 10.3389/fpsyg.2023.1096516 Of the 28,155 enrolled primary students, 21,679 adults responded (77.0%), with only one adult permitted to respond per family. Among the 21,679 online questionnaires returned, 277 questionnaires were rejected due to the fact that the respondents missed over one-third of the questions (199) or gave ﬁctitious or inconsistent (e.g., The answer for more than two-thirds of the items in the questionnaire was the same option, such as option A, option B, and so on; and more than one-third of the items had been given two answers, but two answers were inconsistent) answers (78). The data analyses in the current study were restricted to respondents of mothers and fathers only. The ﬁnal participants were 19,745 (92.3%) parents, including 15,217 mothers (77.1%, M age = 35.96, SD = 4.42) and 4,528 fathers (22.9%, M age = 37.37, SD = 4.87). Their children were aged 6–13 years (M age = 8.77, SD = 1.78). development of their children (Hu, 2017). Diao surveyed a sample of 179 primary school parents in Chengdu, a city in southwest China. It was reported that more than 90% of parents had positive attitudes toward school-based sexuality education, but only one-third of parents could answer questions about the sexuality development of their children (Diao, 2019). In the last three decades, it has been found that a series of qualitative or review studies involves the concept of sexuality education, teaching modes, education lessons, resources, and practices in China (Liu and Yuan, 2017; Wu and Zeng, 2020; Zhao et al., 2020). Parent questionnaire The parental questionnaire was developed based on a series of published studies (Chen and Chen, 2005; Dake et al., 2014; Morawska et al., 2015; Robinson et al., 2017; Depauli and Plaute, 2018; Rudolph and Zimmer-Gembeck, 2018; UNESCO, 2018; Shin et al., 2019) and included 15 items on knowledge, attitudes, and practices regarding sexuality education of children. Parental knowledge, attitudes, and practices in the sexuality education of children However, there is less empirical and quantitative research on sexuality education. Previous quantitative studies in China that included primary school parents only had small sample sizes, mainly in the south of China. In addition, previous studies showed that there are some impact factors related to parental communication about sexuality with their children. The main related factors include parental characteristics, such as parental gender, educational level, experience in receiving sexuality education in childhood, sexuality knowledge, and attitudes (Mckay et al., 2014; Flores and Barroso, 2017; Robinson and Davies, 2017; Shin et al., 2019), and child characteristics, such as child gender, age, and grade (Flores and Barroso, 2017; Shin et al., 2019). Procedures and participants A cross-sectional survey was conducted among a convenience sample of primary school parents in Beijing, Tianjin, and Hebei. Participating parents were from six primary schools located in Beijing, four schools in Tianjin, and ﬁve schools in Hebei. An online questionnaire was used to collect data from participating parents. First, a link to the questionnaire was sent to the heads of participating schools. Second, school head teachers were asked to forward the link to parents to promote participation. Third, parents accessed the survey online and were presented with an information page about the study as well as a consent page, such as the voluntary nature of their participation, the anonymity and privacy of their responses, and so on. Participants could exit the online survey at any time. The questionnaire was available online for 4 months, from September to December 2018. The current study The knowledge subscale included ﬁve items, including knowledge of correct terminology for genitalia, daily sexual healthcare, child sexual development, and sexuality questions, that may be encountered at diﬀerent ages of development of children, and accurate information on child sexual abuse prevention education, “e.g., that do you know the correct terminology for genitalia”. Response options were “yes,” “no,” or “unsure” for each item. “Yes” responses scored 1, while “no” and unsure responses scored 0. The scores for each item were summed for a total knowledge score (range = 0–5). A brief attitudes subscale consists of three items that ask whether parents agree or disagree with the aspects of sexuality education for children in primary school (0–3). The practices subscale included seven items. These seven questions were asked about parental communication with their child about sexuality. Examples of questions in this section included the following: “Did you use appropriate terminology with your child in the process of sexuality education?” “Did you encourage your child to share their thoughts and feelings about sexuality?” and “Did you give brochures or other materials to your child to help them learn about sexuality?” Response options were “yes,” “no,” or “unsure” for each item. “Yes” responses scored 1, while “no” and unsure responses scored 0. The scores for each item were summed for a total practice score (range = 0–7). Internal consistency analyses of subscales of knowledge, attitudes, and practices produced alpha levels of 0.83, 0.90, and 0.80, respectively. Therefore, the present study had two aims. First, this study aimed to examine knowledge, attitudes, and educative practices toward sexuality education among parents of primary school students in Beijing, Tianjin, and Hebei in China. Second, the study sought to explore whether sexuality communication of Chinese parents with their children is associated with certain “facilitators” such as the parental experience of receiving sexuality education in childhood, parental sexuality knowledge, parental attitudes toward sexuality education, parental educational level, child gender, child grade, and other demographic factors. The ﬁndings of this study are expected to be used as baseline data to develop an education program to assist parents in discussing sexuality with their children in China. Frontiers in Psychology frontiersin.org Data analyses All data were analyzed using the SPSS 23.0 software. First, descriptive statistics were used to report the demographic characteristics of parents and their knowledge, attitudes, and practices toward the sexuality education of children in primary schools. Second, a series of variance analyses were conducted to investigate the associations between scores of parents on sexuality knowledge, attitudes, and educative practices and their sociodemographic characteristics variables. Finally, a stepwise multivariate linear regression was conducted to ﬁnd the correlated factors of parental practices toward the sexuality education of children. TABLE 1 Characteristics of the parents involved in the study (N = 19,745). TABLE 1 Characteristics of the parents involved in the study (N = 19,745). own experience of receiving sexuality education in childhood: “In your childhood, did you ever get the information about sexuality education from your parents or other family members?” and “In your childhood, did you ever get the information about sexuality education at school?” (response options: yes, no, or unsure). Other four items were used to gather the characteristics of their children, including child gender (boys/girls), child grade, child age, and only one child or not (yes/no). Characteristics n (%) Living region Beijing 5,639 (28.6%) Tianjin 2,681 (13.6%) Hebei 11,425 (57.9%) Gender Mothers 15,217 (77.1%) Fathers 4,528 (22.9%) Mothers’ educational level Junior high school or less 4,480 (22.7%) Senior high school 5,526 (28.0%) College degree 4,710 (28.6%) Bachelor degree or above 5,029 (25.5%) Fathers’ educational level Junior high school or less 4,338 (22.0%) Senior high school 6,041 (30.6%) College degree 4,609 (23.3%) Bachelor degree or above 4,757 (24.1%) Only-one child Yes 9,218 (46.7%) No 10,527 (53.3%) Child gender Girls 9,408 (47.6%) Boys 10,337 (52.4%) Child grade Grade 1 3,763 (19.1%) Grade 2 3,005 (15.2%) Grade 3 3,562 (18.0%) Grade 4 3,407 (17.3%) Grade 5 2,896 (14.7%) Grade 6 3,112 (15.8%) The whole questionnaire was reviewed by two psychologists, two sexuality education professionals, and two educational researchers. These interviews and their evaluation were discussed. Then, their suggestions and proposals for improvement were taken into account in the design of the ﬁnal questionnaire. The ﬁnal questionnaire was piloted with ten parents of primary students, including four fathers and six mothers, to determine whether they were able to comprehend the instructions and items of the questionnaire. No changes were made to the ﬁnal questionnaire following the current study. Demographic characteristics of parents Correct responses of parents to sexuality knowledge questions of children are summarized in Table 2. More than 80% of the parents responded that they have the knowledge of the correct terminology for genitalia and the knowledge about daily sexual healthcare. In other respects, the sexuality knowledge of parents is lacking. Nearly half of the participating parents did not know about sexual development and behavior across childhood, while almost three-ﬁfths did not know about sexuality questions that children may encounter at diﬀerent ages and did not have any accurate information on child sexual abuse prevention education. On average, parents of primary school students had 60% of sexuality knowledge (M = 3.02, SD = 1.54). The present sample included 19,745 parents. Table 1 shows the frequency distribution of parental demographic variables. Frontiers in Psychology frontiersin.org Demographic form A demographic form was designed to gather background information about parents. Four items gathered demographic information about themselves, including gender (fathers/mothers), age, education qualiﬁcations (junior high school or less/senior high school/college degree/bachelor degree or high), and their childhood sexuality education history. Parents were asked about their Frontiers in Psychology 03 frontiersin.org Zhang and Yuan 10.3389/fpsyg.2023.1096516 History of sexuality education for parents Further analysis demonstrated that parents in Beijing and Tianjin had higher scores of sexuality knowledge and practices than that in Hebei (p < 0.05), while parents in Beijing and Tianjin had similar levels of knowledge and practices. The results also found that parents in Beijing had more positive attitudes toward the sexuality education of children than that in Tianjin and Hebei (p < 0.05), while parents in Tianjin and Hebei had no living region diﬀerences in scores of attitudes. The level of education of parents was positively associated with their sexuality knowledge, attitudes, and educative practices. As shown in Table 5, with the increase in the level of education of parents, their scores on sexuality education knowledge, attitudes, and practices of children were also increasing. Parents who had a bachelor’s degree or above scored highest on the knowledge, attitudes, and practice toward sexuality education of children. The experiences of parents receiving sexuality education in childhood were also analyzed. Parents who had experiences receiving sexuality education in childhood at home or school showed signiﬁcantly higher knowledge scores, more positive attitudes, and more active education practices with their children on sexuality than those who had not (see Table 5). History of sexuality education for parents Questions about sexuality that may be encountered at diﬀerent ages of children development 39.1 38.6 39 5. Accurate information on child sexual abuse prevention education 40.5 37.1 39.7 TABLE 3 Parental attitudes toward sexuality education of children (N = 19,745). TABLE 3 Parental attitudes toward sexuality education of children (N = 19,745). Items Answered “agree” (%) Mothers Fathers Total 1. Primary school students should receive sexuality education? 88 84 87.1 2. Primary school teachers should provide their students with sexuality education 87.5 85.4 87.0 3. Parents should join in school sexuality education programs? 87.9 84.6 87.1 education was signiﬁcantly higher than that of the father (mothers: 2.63 ± 0.84 vs. fathers: 2.54 ± 0.65; F = 40.64, p < 0.01). However, the score of practices of the father in the sexuality education of children was signiﬁcantly higher than that of the mother (fathers: 3.52 ± 2.38 vs. mothers: 3.63 ± 2.23; F = 17.98, p < 0.01). Further analysis showed that mothers had a higher percentage than fathers in two items “read books with my child about sexuality” (mothers: 64% vs. fathers: 60.8%, χ2 = 15.03, p < 0.001) and “made use of the television and the Internet resources to my child about sexuality appropriately” (mothers: 70.3% vs. fathers: 66.9%, χ2 = 19.22, p < 0.001). answered “yes” to all seven items of sexuality education practices. Approximately 70% of parents reported that they had used television and Internet resources to talk with their children about sexuality appropriately, and nearly two-thirds of parents reported that they had read books with their children about sexuality. More than half of the parents had encouraged their children to share thoughts and feelings about sexuality and had given brochures or other materials to their children to help them learn about sexuality. Using appropriate terminology with their children in the process of sexuality education and talking to their children about sexuality comfortably were noticeably less common. Table 4 shows that the great majority of parents did not respond conﬁdently to questions from their children about sexuality topics. On average, parents of primary school students had limited practices of sexuality education of children at home (M = 3.39, SD = 2.27). The living region of the parents was associated with knowledge, attitudes, and practices toward the sexuality education of children. History of sexuality education for parents Parents were asked about their own experiences of receiving sexuality education in childhood. Less than 7% (6.6%; fathers: 5.7% and mothers: 6.8%) of respondents reported that they did receive sexuality education from their family in childhood, and ∼10% (10.3%; fathers: 10.8% and mothers: 10.1%) of respondents reported that they received sexuality education from the school in childhood. The proportion of parents who received sexuality education in childhood at school was signiﬁcantly higher than that at home (χ2 = 3,524.45, p < 0.001). More mothers than fathers received family sexuality education in childhood (χ2 = 7.13, p < 0.01). There was no gender diﬀerence in the parental history of receiving sexuality education at school (p > 0.05). As shown in Table 3, ∼90% of parents agreed that sexuality education should be oﬀered to primary school students, and primary school teachers should provide sexuality education to their students. Moreover, the great majority of parents were willing to join school sexuality education programs. In short, parents had positive attitudes toward sexuality education in primary schools (M = 2.61, SD = 0.87). As a whole, 87.1% of parents had provided at least one form of sexuality education at home. However, only 13.6% of the parents Frontiers in Psychology 04 frontiersin.org Zhang and Yuan 10.3389/fpsyg.2023.1096516 TABLE 4 Parental practices on sexuality education of children (N = 19,745). TABLE 2 Parental knowledge of children sexuality education (N = 19,745). TABLE 2 Parental knowledge of children sexuality education (N = 19,745). TABLE 4 Parental practices on sexuality education of children (N = 19,745). Items Answered “yes” (%) Mothers Fathers Total 1. Responded conﬁdently to a question about a sexuality topic 38.1 46.1 40 2. Used appropriate terminology with my child in the process of sexuality education 27.7 37.2 29.8 3. Talked to my child about sexuality comfortably 30.2 34.6 31.2 4. Encouraged my child to share their thoughts and feelings about sexuality 54.9 53.0 54.5 5. Read books with my child about sexuality 64.0 60.8 63.3 6. Made use of the television and the internet resources to my child about sexuality appropriately 70.3 66.9 69.5 7. Gave brochures or other materials to my child to help them learn about sexuality 50.5 53.4 51.1 Items Answered correctly (%) Mothers Fathers Total 1. Correct terminology for genitalia 84.9 90.0 86.1 2. Daily sexual health care 86.7 82.2 85.7 3. Sexual development and behavior across childhood 52.3 49.6 51.7 4. Frontiers in Psychology frontiersin.org Associations between demographic factors and knowledge, attitudes, and educative practices toward sexuality education of children Compared to parents of boys, parents of girls scored higher on Frontiers in Psychology 05 frontiersin.org frontiersin.org Zhang and Yuan 10.3389/fpsyg.2023.1096516 Variables Categories Sexuality knowledge (0–5) Sexuality attitudes (0–3) Educative practices (0–7) M (SD) F M (SD) F M (SD) F Living region Beijing 3.05 (1.54) 7.35∗∗∗ 2.67 (0.80) 16.46∗∗ 3.49 (2.30) 10.43∗∗∗ Tianjin 3.10 (1.57) 2.62 (0.87) 3.47 (2.32) Hebei 2.99 (1.54) 2.58 (0.90) 3.33 (2.23) Gender Mothers 3.04 (1.54) 5.20∗ 2.63 (0.84) 40.64∗∗∗ 3.36 (2.23) 17.98∗∗∗ Fathers 2.98 (1.54) 2.54 (0.95) 3.52 (2.38) Mothers’ educational level Junior high school or less 2.64 (1.63) 171.10∗∗∗ 2.46 (1.02) 96.26∗∗∗ 2.91 (2.20) 179.78∗∗∗ Senior high school 2.98 (1.54) 2.57 (0.91) 3.22 (2.21) College degree 3.09 (1.48) 2.67 (0.80) 3.49 (2.25) Bachelor degree or above 3.34 (1.44) 2.74 (0.69) 3.93 (2.28) Fathers’ educational level Junior high school or less 2.63 (1.63) 162.13∗∗∗ 2.50 (0.99) 64.85∗∗∗ 2.93 (2.19) 169.09∗∗∗ Senior high school 3.00 (1.54) 2.56 (0.93) 3.22 (2.24) College degree 3.11 (1.48) 2.67 (0.79) 3.52 (2.25) Bachelor degree or above 3.33 (1.54) 2.72 (0.72) 3.92 (2.27) Only-one child Yes 3.11 (1.51) 53.82∗∗∗ 2.66 (0.81) 52.43∗∗∗ 3.56 (2.27) 94.25∗∗∗ No 2.95 (1.56) 2.57 (0.92) 3.25 (2.25) Child gender Girls 3.11 (1.53) 64.81∗∗∗ 2.65 (0.82) 34.35∗∗∗ 3.42 (2.24) 3.03 Boys 2.94 (1.55) 2.58 (0.91) 3.37 (2.29) Child grade Grade 1 2.86 (1.48) 19.08∗∗∗ 2.58 (0.92) 1.32 3.32 (2.26) 3.29∗∗ Grade 2 3.01 (1.54) 2.62 (0.88) 3.45 (2.25) Grade 3 2.95 (1.55) 2.61 (0.87) 3.32 (2.24) Grade 4 3.05 (1.56) 2.62 (0.85) 3.37 (2.27) Grade 5 3.15 (1.54) 2.62 (0.84) 3.46 (2.28) Grade 6 3.16 (1.56) 2.61 (0.87) 3.48 (2.30) Received sexuality education Yes 4.21 (1.27) 859.80∗∗∗ 2.84 (0.55) 100.02∗∗∗ 5.11 (2.05) 833.45∗∗∗ as a child at home No 2.94 (1.52) 2.60 (0.88) 3.27 (2.23) Received sexuality education Yes 3.87 (1.39) 711.36∗∗∗ 2.79 (0.63) 100.40∗∗∗ 4.59 (2.20) 656.54∗∗∗ as a child at school No 2.92 (1.53) 2.59 (0.89) 3.26 (2.23) ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. sexuality knowledge and attitudes. Regarding the educative practices of parents, there was no signiﬁcant diﬀerence between boys and girls. Moreover, the ﬁndings showed that parents who had only one child scored signiﬁcantly higher scores on knowledge, attitudes, and practices toward sexuality education than those who had not. Child grade was related to the scores of parental sexuality knowledge and practices. Associations between demographic factors and knowledge, attitudes, and educative practices toward sexuality education of children Associations between demographic factors of parents and their knowledge, attitudes, and practices toward sexuality education of children are summarized in Table 5. The results showed that the gender of parents was associated with diﬀerences in the scores of sexuality knowledge, attitudes, and educative practices. The total score of knowledge of the mother was signiﬁcantly higher than that of the father (mothers: 3.04 ± 1.54 vs. fathers: 2.98 ± 1.55; F = 5.21, p < 0.05). Similarly, the score of attitudes of the mother toward sexuality It is also noteworthy that child gender was associated with parental knowledge and attitudes toward sexuality education. Associations between demographic factors and knowledge, attitudes, and educative practices toward sexuality education of children Further analysis showed that parents whose children were in grades 5 and 6 had signiﬁcantly higher sexuality knowledge scores and had more active practices than those whose children were in other grades (see Table 5). Regarding the attitudes of parents toward sexuality education, there was no signiﬁcant diﬀerence by child grade. on sexuality education practices and nine independent variables, including parental knowledge, parental attitudes, parental history of receiving sexuality education at home in childhood, parental history of receiving sexuality education at school in childhood, parental educational level, parental living region, child grade, and only one child. The multivariate linear regression showed that, among all these variables, the knowledge of the father (B = 0.50, SE B = 0.02, p < 0.00), the attitude of the father (B = 0.63, SE B = 0.03, p < 0.001), history of the father receiving sexuality education at home in childhood (B = 0.96, SE B = 0.15, p < 0.001), history of the father receiving sexuality education at school in childhood (B = 0.43, SE B = 0.11, p < 0.001), and educational level of the father (B = 0.16, SE B = 0.03, p < 0.01) were the signiﬁcant factors for education practices of the father. The ﬁve variables together accounted for 24.4% of the variance in scores on practices of the father toward sexuality education of children (F = 183.48, p < 0.001, R2 = 0.24). Frontiers in Psychology Inﬂuencing factors for practices of parents on sexuality education of children y p y Most parents (87%) agreed about the importance of sexuality education lessons in primary schools. This ﬁnding is consistent with observations in previous studies conducted on US parents of primary school children (∼90% in Fisher et al., 2015; 80% in Dake et al., 2014), on Canadian parents (87% in Mckay et al., 2014), and on Australian parents (71% in Robinson et al., 2017) but much higher than the study in Shenzhen province in China 15 years ago (∼60% in Wu, 2005). It is suggested that Chinese parents have more positive attitudes toward sexuality education than before. Scholars and researchers advocate that better communication between parents and school teachers could enhance the eﬀect of sexuality education (Robinson et al., 2017). Approximately 90% of parents of primary school students expressed their willingness to join in school-based sexuality education in the current study. This ﬁnding suggests that there is substantial support from the majority of parents for implementing sexuality education in primary schools. In future, it is supposed to be a collaborative approach between families and schools on sexuality education in China. Our study provided a factual basis that parental knowledge and attitudes are inﬂuencing factors for their practices on the sexuality education of children. With the increase in parental scores on knowledge and attitudes, their scores on educative practices about sexuality were also increasing. Hence, sexuality education needs to target not just children but also their parents (Robinson et al., 2017). In view of this, scholars and researchers should develop parent- focused sexuality education programs that include more accurate and speciﬁc sexuality information to help Chinese parents improve their family communication about sexuality. Consistent with previous studies (Lee and Kweon, 2013; Flores and Barroso, 2017), the results of our study showed that mother respondents had more knowledge and positive attitudes toward sexuality education of children. However, unlike the previous studies in the United States (Flores and Barroso, 2017) and Korea (Lee and Kweon, 2013), which found that mothers were predominant providers of sexuality education at home, this study showed that fathers were more likely to provide educative practices to their children in China. Fathers also play an important role in sexuality education at home, like mothers. Compared to mothers, fathers may discuss male issues more eﬀectively with their boys (Shin et al., 2019; Zhang et al., 2019). Inﬂuencing factors for practices of parents on sexuality education of children The results of this study suggest that both mothers and fathers of young children should be the target population for sexuality education training programs in China in future. Discussion Chinese parents rarely used correct terminology for genitalia (30%), which is lower than the previous studies with parents of primary school students in Australia (∼80% in Morawska et al., 2015) and the United States (∼40% in Dake et al., 2014) but much higher than the recent Muslim research in Pakistan (∼6% in Nadeem et al., 2021) and the Korean research (∼6% in Shin et al., 2019). Parents commonly feel embarrassed or uncomfortable talking to their children about sexuality (Turnbulla et al., 2008). Our study found that less than one-third of the parents had talked to their children comfortably about sexuality, and only two-ﬁfths responded conﬁdently to a question about a sexuality topic. It is much lower than the previous studies in Australia (∼75% in Morawska et al., 2015) and Korea (∼70% in Shin et al., 2019). The Chinese tradition of sexual conservatism may make it diﬃcult for parents to discuss sexuality topics with their children openly and comfortably (Xie et al., 2015). Knowledge and attitudes of parents toward sexuality education of children In this study, parents of primary students had gaps in their sexuality knowledge. Although the average sexuality knowledge score was 3.02, the correct rate for some items was very low. Only 39% of parents reported that they had knowledge of the sexuality questions that children may encounter at diﬀerent ages. Although children commonly ask their parents some sexuality questions (Krauss and Miller, 2012; Martin and Torres, 2014; Hu et al., 2015), parents who lack sexuality knowledge feel embarrassed (Xie et al., 2015; Shin et al., 2019) and may be unable to answer questions from their children in time. Similar to the previous studies done in China (Chen and Chen, 2005; Chen et al., 2007; Xie et al., 2015; Jin et al., 2019), more than 60% of the parents in this study reported that they lacked knowledge about child sexual abuse prevention education. Thus, children may be unable to be protected well and taught accurate information about sexual abuse by their parents (Wurtele and Kenny, 2010). Regression analyses The stepwise multivariate linear regression was used to explore the associations between the scores of the father (or mother) Frontiers in Psychology 06 frontiersin.org 10.3389/fpsyg.2023.1096516 Zhang and Yuan There are also ﬁve signiﬁcant factors for the educative practices of the mother. The multivariate linear regression showed that these signiﬁcant factors included knowledge of the mother (B = 0.46, SE B = 0.01, p < 0.001), the attitude of the mother (B = 0.63, SE B = 0.02, p < 0.001), history of the mother receiving sexuality education at home in childhood (B = 0.89, SE B = 0.07, p < 0.001), history of the mother receiving sexuality education at school in childhood (B = 0.50, SE B = 0.06, p < 0.001), and educational level of the mother (B = 0.20, SE B = 0.02, p < 0.001). The ﬁve variables together accounted for 24.9% of the variance in scores on practices of the mother toward sexuality education of children (F = 630.38, p < 0.001, R2 = 0.25). to provide sexuality education to their children. The percentage is higher than the previous studies in Australia (Morawska et al., 2015), the United States (Dake et al., 2014), and Korea (Shin et al., 2019). In addition, more than 60% of the parents reported reading books with their children about sexuality, which is a substantial rise from the Australian study which found that <50% of parents had read books with their children about sexuality (Morawska et al., 2015). At present, the media has become one of the most important sources of sexuality health information in China. Access to media resources could increase motivation and enhance the quality of sexuality education (Lou et al., 2006). Therefore, it is helpful and convenient for parents to discuss sexuality with their children by using the Internet and television. Frontiers in Psychology Zhang and Yuan Ethics statement The study involving human participants was reviewed and approved by Beijing Academy of Educational Sciences. The participants provided their written informed consent to participate in this study. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Acknowledgments The authors wish to thank the parents who participated in this study. Conclusion This study concluded that the majority of parents of primary school students had positive attitudes toward sexuality education in China. However, they had limited sexuality knowledge. Less than 40% of the parents knew that there were sexuality questions that children may encounter at diﬀerent ages and had accurate information on child sexual abuse prevention education. One major beneﬁt of this study lies in the novel investigation that most fathers and mothers had communication speciﬁcally with their children about sexuality, with the help of the Internet, television, and books. Moreover, this study documented that parental practices are positively associated with their knowledge and attitudes toward sexuality education of children. Based on these ﬁndings, it is important to develop culturally relevant training programs for parents in Chinese society in future. Sexuality education training programs are supposed to help parents learn more accurate knowledge and improve their communication with their young children at home. Funding This study was supported by the Fundamental Research Funds for the Central Universities and the Research Funds of Renmin University of China (22XNC003). Study limitations This study had several limitations. The ﬁrst limitation relates to the purpose-built questionnaire. The scale of knowledge, attitudes, and practices in sexuality education for children was designed to be simple and relatively superﬁcial. While the scales appeared to demonstrate good internal consistency, the results need to be interpreted with caution. To obtain a deeper understanding of the awareness of parents about sexuality education, a more detailed assessment, focus groups, and qualitative interviews need to be conducted in future research. Second, this study used an Internet-based survey that relied on self-reporting. Therefore, the reliability of the data may depend on the sincerity of the answers of respondents. Third, although this study had a suﬃcient sample size, it was limited to self-reports from a sample of parents only in Beijing, Tianjin, and Hebei in China. Thus, the ﬁndings cannot be generalized to the whole of China. Finally, the information collected was cross-sectional; thus, causation between associated factors and parental educative practices in sexuality education cannot be inferred from this study. Data availability statement Despite the vast majority of parents (90%) in the sample having not had received any experience of sexuality education either from their own families or in their school settings, this study still demonstrated that the experience of parents of sexuality education in childhood was associated with better communication about sex-related issues with their child. Consistent with the previous studies (Wight et al., 2006; Jerman and Constantine, 2010; Zhang et al., 2013; Dake et al., 2014), this study also found that parents with higher educational levels were more likely to talk with their children about sexuality. Therefore, attention should be paid to parents with low academic qualiﬁcations in future training programs. The datasets presented in this article are not readily available because the data have not been made available on a permanent third- party archive because Beijing academy of educational sciences ruled that we could not post the data. Requests to access the datasets should be directed to the corresponding author (yuanbnu@163.com). Practices of parents on sexuality education of children at home With the help of media (e.g., television and the Internet), parents actively carry out sexuality education at home in China. Almost 70% of parents reported using television and Internet resources Frontiers in Psychology 07 frontiersin.org Zhang and Yuan Zhang and Yuan 10.3389/fpsyg.2023.1096516 10.3389/fpsyg.2023.1096516 Author contributions WZ: acquisition of data, conception and design of study, conducting a research and investigation process, and drafting the manuscript. YY: data curation, revising the manuscript critically for important intellectual content, and development or design of methodology. All authors contributed to the article and approved the submitted version. Zhang and Yuan References Alldred, P., Fox, N., and Kulpa, R. (2016). 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https://openalex.org/W4376645892	https://www.frontiersin.org/articles/10.3389/fmed.2023.1075672/pdf	English	null	A network analysis of psychological flexibility, coping, and stigma in dermatology patients	Frontiers in medicine	2,023	cc-by	11,178	TYPE Original Research PUBLISHED 16 May 2023 DOI 10.3389/fmed.2023.1075672 TYPE Original Research PUBLISHED 16 May 2023 DOI 10.3389/fmed.2023.1075672 TYPE Original Research PUBLISHED 16 May 2023 DOI 10.3389/fmed.2023.1075672 OPEN ACCESS OPEN ACCESS EDITED BY Robert Gniadecki, University of Alberta, Canada REVIEWED BY Karolina Chilicka-Hebel, Opole University, Poland Hanlin Zhang, Peking Union Medical College Hospital (CAMS), China Adam Reich, University of Rzeszow, Poland Piotr K. Krajewski, Wroclaw Medical University, Poland REVIEWED BY Karolina Chilicka-Hebel, Opole University, Poland Hanlin Zhang, Peking Union Medical College Hospital (CAMS), China Adam Reich, University of Rzeszow, Poland Piotr K. Krajewski, Wroclaw Medical University, Poland Vasilis S. Vasiliou 1,2, Hellen Russell 3, Sarah Cockayne 4, Gabriel Lins de Holanda Coelho 5 and Andrew R. Thompson 1,3 1 School of Psychology, South Wales Clinical Psychology Doctorate, Cardiff and Vale University Health Board, Cardiff University, Cardiff, United Kingdom, 2 Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Oxford, United Kingdom, 3 Department of Psychology, University of Sheffield, Sheffield, United Kingdom, 4 Department of Dermatology, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom, 5 School of Applied Psychology, University College Cork, Cork, Ireland Introduction: Despite the negative effects of stigma in individuals with skin conditions, interventions to address its effects are rare. This might be in part due to a continued lack of understanding as to how individuals respond to stigma. Methods: In this study, we employed a step-case analytic method, using traditional regression, moderation, and network analyses, to examine the role of psychological flexibility (PF) with stigmatized experiences, and stigma-related outcomes. We run a cross-sectional study (n = 105 individuals with various skin conditions) and analyzed stigma-related variables. We included variables examining perceived stigmatization (PSQ), anxiety (GAD-7), depression (PHQ-9), well-being (EQ5D5L), and variables stemming from the PF model (CompACT), presented as three coping with stigma responses, namely “open,” “aware,” and “active.”. © 2023 Vasiliou, Russell, Cockayne, Coelho and Thompson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: Using network analysis, the most influential or central variables that contributed to stigma were generalized anxiety, perceived stigmatization, and valued actions. OPEN ACCESS In relation to PF, being open to the experience of stigma (as opposed to avoidance), keeping a distance from stigmatized thoughts (as opposed to self-stigmatizing), and bringing attention to value-based committed actions (as opposed to passivity) were all found to contribute to less stigmatized experiences. Discussion: The results indicate that two of the three skills of the PF model (“open” and “active”) may be important targets for interventions targeting stigma in people living with skin conditions. stigma, psychodermatology, process-based therapy, psychological flexibility, coping A network analysis of psychological flexibility, coping, and stigma in dermatology patients OPEN ACCESS EDITED BY Robert Gniadecki, University of Alberta, Canada REVIEWED BY Karolina Chilicka-Hebel, Opole University, Poland Hanlin Zhang, Peking Union Medical College Hospital (CAMS), China Adam Reich, University of Rzeszow, Poland Piotr K. Krajewski, Wroclaw Medical University, Poland CORRESPONDENCE Andrew R. Thompson thompsona18@cardiff.ac.uk Vasilis S. Vasiliou vasiliou.v1@cardiff.ac.uk; vasilis.vasiliou@ndorms.ox.ac.uk RECEIVED 20 October 2022 ACCEPTED 25 April 2023 PUBLISHED 16 May 2023 CITATION Vasiliou VS, Russell H, Cockayne S, Coelho GLH and Thompson AR (2023) A network analysis of psychological flexibility, coping, and stigma in dermatology patients. Front. Med. 10:1075672. doi: 10.3389/fmed.2023.1075672 1. Introduction Stigma is characterized by a proneness to either devaluate and discredit a person/group considered to possess a negative attribute (1), or an individual’s/group’s tendency to come to believe what others attribute to them (2, 3). Given the highly visible nature of skin conditions, it is unsurprising that stigma is commonly experienced (4–7). The visible marks on the skin can Frontiers in Medicine 01 frontiersin.org Vasiliou et al. Vasiliou et al. 10.3389/fmed.2023.1075672 10.3389/fmed.2023.1075672 10.3389/fmed.2023.1075672 be perceived as “deviant” from what is considered the norm in appearance, making it easier for people in society to stigmatize individuals with skin conditions, compared to other conditions that show no visible differences in appearance (e.g., individuals with diabetes) (8, 9). Overall, traditional moderation and mediation methods limit practical applications for intervention development targeting stigma (41, 42). This is because they may generate a wide range of skills (20), potentially increasing uncertainty about which skills to select and target (20, 43). Further, these approaches do not allow the dynamic and simultaneous bi-directional interaction of stigma-related thoughts, emotions, and behaviors (responses to stigma) to be studied. Given that stigma is a multi-dimensional construct (41, 42), new innovative data-driven methods that can address these complexities, such as network analyses, are needed. Existing research shows that stigma in various skin conditions, including acne, atopic dermatitis, vitiligo, and psoriasis, is associated with poorer quality of life and increased distress (7, 10–18). For example, individuals with psoriasis often feel “different” from others. This increases stigma-related stress, consequently, impacting individuals’ daily functioning (19). Further, studies in patients living with acne show that stigma is the largest contributor in predicting poorer quality of life, over and above disease and demographic variables (11). These findings are concerning, highlighting that individuals with skin conditions have to deal with the diagnosis/ management of the condition in addition to the potential negative effects of feeling stigmatized. Promoting approaches that focus on managing stigma and distress, is required, yet, this has proven to be difficult thus far to achieve (20, 21). Unlike traditional mediation and moderation analyses, network analysis explores relations between variables through partial correlations, which are visually illustrated with links (e.g., lines connecting different variables) that show the connection between the variables. 1 https://www.psychodermatology.net/ 2.1. Design The study was nested in a multi-center European study conducted by the European Society for Dermatology and Psychiatry (ESDaP)1. The ESDaP multi-country study collected data on the association between stigmatization and the psychosocial burden of individuals living with a skin condition in 17 European countries (ESDaP, 2016). In addition to the variables examined across all countries, some countries also investigated other variables. In the UK, the survey was expanded to include variables related to psychological flexibility so that the aims of this study could be addressed. The study had ethical approval from the NHS Health Research Authority (18/LO/0639). To date, the existing studies attempting to identify parameters of coping with stigma in this population are rare (6, 20). Further, existing studies have employed traditional methods to examine variables, such as mediation and moderation analyses that only present a static picture of how stigma, coping with stigma, and stigma-related outcomes interact. For example, McCleary-Gaddy and James (36) found mediating effects of stigma consciousness between skin tone, life satisfaction, and psychological distress among African Americans, highlighting the potential role of increased awareness of stigmatization in reducing distress. Further, Bohm et al. (37), and Schmid-Ott et al. (38) both found mediating effects of reduced self-esteem and rejection as stigmatization parameters in skin condition severity and quality of life, indicating the potential role of defusion from stigma related experiences as a coping response. Likewise, Krüger and Schallreuter (39) found behavioral avoidance as the main coping with stigma response in patients with vitiligo, and Lu et al. (40) found helplessness as an illness cognition response to stigmatization in patients with psoriasis and atopic dermatitis. Frontiers in Medicine 1. Introduction Adopting such an approach would allow the conceptualization of stigma as a network of interactional patterns, centred around defining variables of interest, such as coping with stigma responses, and stigma- related outcomes, rather than artificially assigning variables into static dependent and independent variables (24). A network analytic approach was taken in this study that tested the importance of variables and identified an empirically dynamic network of skills focusing on stigma alleviation. Stigma-related variables, including perceived stigmatization, anxiety, depression, well-being, and psychological variables, such as PF, were examined. In short, this study aimed to identify the most influential or central parameters contributing to stigma alleviation by attempting to determine (a) the relationships among all variables of interest, (b) the variance of stigma and PF skills in explaining individuals’ well-being; (c) the potential role of certain or all the three PF skills in buffering the effects of stigma; and (d) the bidirectional relations among the PF processes, stigma, and stigma related outcomes. One approach that helps researchers and clinicians to identify effective responses to stigma is the process-based approach (22–25). This approach attempts to identify common responses to stigma that can be flexible enough so that they can concurrently target the contextual (e.g., stigmatization) and psychological (e.g., how individuals cope with thoughts and emotions) elements of stigma (20). A therapeutic approach that can target both the context of stigma and the way individuals respond to it is psychological flexibility (PF) (26–28). PF includes three trainable psychological skills, named “openness to experience” (defusion and acceptance), “behavioral awareness” (contacting the present moment and self-as-context), and “valued action” (values and committed action) that can be presented as “coping with stigma” responses. Research examining these PF-related skills on other conditions, such as stigma in relation to chronic pain or weight self stigma shows that the PF skills can buffer the effects of stigma (29–32). These sets of psychological skills are amenable to interventions (e.g., can be employed as coping with stigma responses outside of a therapy room) and can be delivered in different forms (e.g., digitally, in-group, one-to-one, etc.) (33–35). Yet, no research so far has examined how these skills can help individuals with skin conditions, experiencing stigma. 2.3. Recruitment and study procedures Eligible participants were recruited using convenience and purposive approaches. During clinic appointments, Dermatologists invited consecutive patients who met the study criteria to participate. Upon consent, participants completed the package of questionnaires with the assistance of a research team member, and study Dermatologists recorded their skin condition and severity. Dermatologists used the International Classification of Diseases (ICD-10) criteria to rate the participating individuals’ severity of their skin disease as mild, moderate or severe. Data collection occurred between July and September 2018. Figure 1 presents the flow chart with all the study procedures. The EuroQOL 5-Dimensions (EQ5D5L) (48) is a visual analog scale (VAS) assessing self-reported health. Participants are asked to rate their health on the day of reporting (“today”), using a zero (“the worst health you can imagine”) to 100 (“the best health you can imagine”) metric. The validity of the EQ5D5L in skin populations shows good psychometric characteristics (49), showing moderate-to- strong correlations with other health-related quality-of-life measures (e.g., SF-12) and can detect significant changes in health status over time. The VAS is a subtest within this measure, and validation is not available for this subscale alone. Thus, the psychometric assessments refer to the whole EQ5D5L. 2.2. Inclusion and exclusion criteria Eligible participants were recruited from patients attending outpatient appointments with a dermatology department within a large teaching hospital in the UK. Inclusion criteria consisted of individuals 02 frontiersin.org frontiersin.org Vasiliou et al. 10.3389/fmed.2023.1075672 and test–retest reliability (r = 0.84) (46), as well as an acceptance construct validity, as assessed by functional status (46). The Cronbach’s alpha for this study was a = 0.98. over 18 years of age with a sufficient English capacity to complete questionnaires and provide consent, and a diagnosis of a chronic skin condition. The exclusion criteria consisted of a non-primary diagnosis of chronic skin conditions, the presence of a primary psychiatric condition relevant to skin distress (e.g., trichotillomania, delusional parasitosis etc.), a benign skin lesion (e.g., a noncancerous related skin lesion), and/or a suspected/diagnosed skin cancer. The Generalized Anxiety Disorder Assessment (GAD-7) (47) is a self-administered 7 items measure of symptoms of a generalized anxiety disorder (GAD). The questionnaire asks participants to rate the frequency of nine symptoms of GAD within the last 2 weeks on a scale from “not at all” to “nearly every day” (scored 0–3 with a total score ranging from 0 to 21). Total scores can be interpreted of “minimal” (0–4), “mild” (5–9), “moderate” (10–14), or “severe” (15– 21) anxiety. Research shows that the GAD-7 has excellent reliability (test–retest correlation of 0.83) and construct validity, as presented with correlations measuring functional impairment (47). The Cronbach’s alpha for this study was a = 0.98. 2.4.3. PF related variablesh Participants completed a series of measures, including demographics, such as age, gender, education level, and employment status, clinically relevant questions about their skin conditions (disease severity and intensity), and a set of five standardized self-reported questionnaires, measuring stigma, depression, anxiety, quality of life, and a measure assessing the skills stemming from the PF, presented as three dyads or coping responses: “open,” “aware,” and “active.” In sum, the following measures were completed by the participants. The Comprehensive Assessment of Acceptance and Commitment Therapy Processes (CompACT) (50) is a 23-item measure of psychological flexibility with three subscales: openness to experience, behavioral awareness, and valued actions. The three factors represent latent constructs of PF skills, merged as dyads, reflecting acceptance and defusion (“open” being present; CompOE), present moment awareness and self-as-context (“aware” behavioral awareness; CompBA), and values and committed actions (“active”; doing what matters”; CompVA) (51). Participants respond to a series of items (e.g., I behave in line with my personal values) on a 7-point Likert scale from “strongly disagree” to “strongly agree.” Higher scores in each subscale or the total score indicate greater psychological flexibility (e.g., greater openness to experience, mindful attention to current activities and engagement in valued actions). The measure demonstrates excellent internal consistency in its subscales (α = 0.90, 0.87, and 0.90, respectively) and acceptable criterion validity with existing ACT measures, such as the Acceptance and Action Questionnaire (50). The Cronbach’s alpha for this study was for the CompOE a = 0.88, for the CompBA a = 0.91, and for the CompVA a = 0.93, correspondingly. Frontiers in Medicine 2.4.1. Stigma-related variables Perceived Stigmatization Questionnaire (PSQ) (44) consists of 21 items, assessing perceived stigmatization in social experiences (e.g., people avoid looking at me or people do not know how to act around me) in individuals with visible differences in appearance. Higher scores indicate a greater perception of stigmatized behaviors. The measure assesses stigmatized behaviors on a 5-point Likert scale (never, almost never, sometimes, often, always) and has good internal consistency and criterion validity with other related psychosocial constructs (e.g., good convergent and discriminant validity within a sample of adult burn survivors) (44). The Cronbach’s alpha for this study was a = 0.90. 2.5. Statistical analyses The Patient Health Questionnaire (PHQ-9) (45) is a self- administered questionnaire with 9 items, measuring the presence and severity of depressive symptoms (e.g., feeling down, depressed, hopeless or having little interest or pleasure in doing things). Participants are required to rate the frequency of nine symptoms of depression on a scale from “not at all (0)” to “nearly every day (3)” for the past 2 weeks. Total scores can range from 0 to 27. Depression is indicative of “mild” (scores 5–9), “moderate” (scores 10–14), “moderately severe” (scores 15–19), or “severe” depression (>20). The measure presents excellent internal reliability (Cronbach’s alpha = 0.89) As part of the preliminary analysis, we examined the parametric assumptions and tested the normality of distribution by visually inspecting the histograms, P–P residual plots, and missing cases. We did not detect a serious violation of the normality assumptions (linearity, homoscedasticity, collinearity, and multicollinearity). Also, univariate and multivariate outliers and missing cases were negligible. We examined the histograms and plots for any issues with the skewness and kurtosis. There were no values below or above the −/+3. 03 frontiersin.org Vasiliou et al. 10.3389/fmed.2023.1075672 FIGURE 1 Flow chart with study procedures. FIGURE 1 Flow chart with study procedures. The measures of psychological distress (anxiety and depression) were positively skewed. Ratings of self-reported health were negatively skewed. There was a notable outlier in the stigma data, with one participant scoring very high on the Perceived Stigmatisation Questionnaire (49, z = 3.15). We rerun all the reported analyses without the outlier and conclusions drawn were the same. Therefore, the reported results include the outlier as it was deemed a genuine, although extreme score. Hence we left the data intact. To corroborate with the visual inspection of the dataset, we run a missing data analysis to assess any pattern of non-identifiable missingness (52). Little’s MCAR test indicated that the data were missing completely at random. relationships of the variables using network analyses. We used IBM SPSS Statistics 27 to test and compare the variables’ importance. We then used the packages of JASP2 and R studio (53) to run the network analysis. Firstly, we run a series of Pearson’s correlation coefficient analyses (54) and a series of hierarchical multiple linear regression analyses (simultaneous forced entry method using R2 and adjusted R2), to examine the prediction of stigma on well-being, controlling for any effects of age, gender, and clinician-rated severity of the skin condition. Frontiers in Medicine 2 https://jasp-stats.org/ 2.5. Statistical analyses Other skin diseases diagnoses that occurred in >3% of the sample, included acne (n = 7), rosacea (n = 3), and urticaria (n = 2). Skin disease diagnoses given in >1% are presented in the Supplementary material S4 where we also present the comprehensive list of participants’ skin diagnoses. Clinicians’ ratings of the severity of participants’ skin disease were most commonly moderate (45%, n = 44) or severe (34%, n = 36). There were no differences between males and females in the employment status and clinicians’ rated severity of skin diseases (both ps < 0.05). Table 1 presents more detailed characteristics of participants’ demographic information. outcomes. We examined the partial correlations network (total scale/ subscale scores, rather than individual items) of the PSQ (stigma), GAD (generalized anxiety), PHQ9 (depression), EQ5D5L (perceived health), and the three sub-scales of the CompACT, CompOE (open to experience), CompVA (values), and CompBA (Behavioral awareness). Using the glasso R package (55) embedded in the JASP, we depicted graphically the edge weights connecting the nodes (e.g., the variables included in the model) and examined the nodes’ strengths. We also used the Fruchterman-Reingold positioning algorithm (56)- a forced- directed method- to visualize the network model variables and examine which variables are posed in the center of the graph. For the interpretation of the outcomes, we applied the graphic LASSO [Least Absolute Shrinkage and Selection Operator (57)] estimator [a stunning parameter set to 0.5-using the EBIC; Extended Bayesian Information Criterium (58)], to counterbalance the relevant small sample size of the study (model regularization) (59). The technique estimates the variance–covariance matrix and removes less relevant edges from the model, returning a parsimonious network of partial correlation coefficient which is more conservative and easily interpretable (e.g., only a reasonably small number of edges are used to explain the covariation structure of the model). We also examined the stability of centrality indices using a parametrization technique called bootent (59) in the R software (53). We estimated the Coefficient Intervals (CIs), to examine if the order of centrality indices remains the same after bootstrapping (re-estimating) the network with fewer cases (e.g., dropping cases from the original dataset) and without replacing them. To assess this stability, we used the correlation stability coefficient, or CS-coefficient (quantification of stability). CS-coefficient defines the percentage of cases that can be dropped, with a 95% probability of maintaining ~0.70 correlation, compared to the completed data (59). 3.3. Multivariate analyses The hierarchical multiple regression models consisted of seven predictors. We firstly entered (forced entry) demographics and clinical characteristics (step 1), followed by stigma (step 2), and finally, the three PF dyads of response processes (step 3). Before running the models, we log-transformed anxiety, depression, and self-reported health variables as they did not meet the criteria for normality due to skewness. Screening criteria showed no multicollinearity or the presence of multivariate outliers, and the variables met the criteria for normality, linearity, and homoscedasticity. For all the models, the variance inflation factor (VIF) was less than 3.3, and tolerance statistics were all 0.296 or above. 3.2. Correlation analyses The stigma experience scale (PSQ) score demonstrated medium negative correlations with the openness to experience subscale score (CompACT_OE, r > −0.33), the behavioral awareness (CompACT_ BA, r > −0.27) scores of the PF processes, and the perceived health (VAS, r > −0.24) scores. Further, stigma showed a positive correlation with the study outcomes, such as higher levels of stigma experiences being associated with higher levels of depression (PHQ-9, r > 0.34) and generalized anxiety (GAD-7; r > 0.29). As Table 2 shows, these findings support the first study hypothesis, indicating a significant relationship between stigma, PF processes, and stigma-related outcomes, consequently, allowing us to build the predictive models. 2.5. Statistical analyses The edge-weight accuracy is estimated when values are over 0.50 but not lower than 0.25. Finally, we examined the edge weights CIs to assess the precision with which PF processes are strongly interconnected within the network. Narrower CIs indicate better accuracy (59). 2.6. Statistical power and sample size The proposed analysis included a maximum of seven variables to detect medium effect sizes in the first round of analyses which included multiple regression and moderation analyses. Following suggestions from Cohen and Field (60, 61) a G power analysis (62) suggested a sample size of 105 participants, for p < 0.05. For the second round of network analysis, the number of observations in our tested model (e.g., n ~ 100) seemed appropriate for estimating the partial correlation network analysis. That is, we expect 20 nodes to occur on the network model, allowing us to examine the validation and robustness of the model even when the highly conservative Lasso penalty estimator is applied (59). As Table 3 shows, the seven predictors, after controlling for demographics and clinical characteristics accounted for 57% of the variance explained in generalized anxiety (adj. R2 = 0.53). The equation was highly significant [F (7,95) = 16.53, p < 0.001], representing a large effect size, f2 = 1.14. Age, skin condition severity, stigma, and the three PF response styles were all significant predictors in the final model, with behavioral awareness (CompACT_BA) showing the highest contribution (b = −0.451) when compared with the other six predictors. In predicting depression, the seven predictors accounted for 38% of the variance (Adj. R2 = 0.379). The equation was highly significant [F 2.5. Statistical analyses Then, we performed a series of moderation analyses, to test whether PF moderated the relationship between feelings of stigma and well- being. We then run a network analysis to simulate a hypothesized stigma model and identify the most central, therefore, most influential, PF skills that correlated with stigmatization and stigma-related The main analytic plan followed an exploratory step-wise approach. We first explored a “static” or pre-defined model of variables, using traditional regression and moderation regression analyses, to examine predictive relationships among the variables (e.g., stigmatized experiences as predictors of distress and low perceived health). We then examined “dynamic” and “bidirectional” 04 frontiersin.org Vasiliou et al. 10.3389/fmed.2023.1075672 10.3389/fmed.2023.1075672 90). Most of the participants had completed the highest level of education (GCSE equivalent or below; 63%, n = 66), with more females (n = 46) than males (n = 20), achieving the highest level of education. Most of the participants were, at the time of the study, employed (41%, n = 43) or retired (40%, n = 42). As for the participants clinical characteristics, among the 29 reported primary skin conditions, the most common diagnoses were: psoriasis (n = 23), eczema (n = 16), and alopecia (n = 11). Other skin diseases diagnoses that occurred in >3% of the sample, included acne (n = 7), rosacea (n = 3), and urticaria (n = 2). Skin disease diagnoses given in >1% are presented in the Supplementary material S4 where we also present the comprehensive list of participants’ skin diagnoses. Clinicians’ ratings of the severity of participants’ skin disease were most commonly moderate (45%, n = 44) or severe (34%, n = 36). There were no differences between males and females in the employment status and clinicians’ rated severity of skin diseases (both ps < 0.05). Table 1 presents more detailed characteristics of participants’ demographic information. 90). Most of the participants had completed the highest level of education (GCSE equivalent or below; 63%, n = 66), with more females (n = 46) than males (n = 20), achieving the highest level of education. Most of the participants were, at the time of the study, employed (41%, n = 43) or retired (40%, n = 42). As for the participants clinical characteristics, among the 29 reported primary skin conditions, the most common diagnoses were: psoriasis (n = 23), eczema (n = 16), and alopecia (n = 11). Frontiers in Medicine frontiersin.org 3.4. Moderation mediation analysis (7,95) = 9.28, p < 0.001], representing a large effect size f2 = 0.85. Examining the individual prediction (criterion) of the seven variables, one can see that stigma and valued-based actions approached significance (p = 0.07). In contrast, the two other PF dyads, openness to experience and behavioral awareness were significant. The variable with the highest prediction was behavioral awareness (b = −0.34, p < 0.01) compared to the other six variables. Finally, as for the perceived health, the overall model accounted for 22% of the variance explained (Adj. R2 = 0.218). This finding was also highly significant [F (7,95) = 1.69, p < 0.01], representing a large effect size f2 = 0.52. Behavioral awareness and value-based actions were the only significant predictors in the final model, with an almost equal prediction of perceived health (b = −0.281 and b = −0.241). The regression analyses supported the second study hypotheses, where perceived stigmatization predicts higher anxiety, depression, and lower self-reported health. Notably, PF processes might revert the negative effects of stigma on individuals’ well- being, particularly the process of behavioral awareness (being present). We tested which PF processes of change exert effects in the following analyses. (7,95) = 9.28, p < 0.001], representing a large effect size f2 = 0.85. Examining the individual prediction (criterion) of the seven variables, one can see that stigma and valued-based actions approached significance (p = 0.07). In contrast, the two other PF dyads, openness to experience and behavioral awareness were significant. The variable with the highest prediction was behavioral awareness (b = −0.34, p < 0.01) compared to the other six variables. Finally, as for the perceived health, the overall model accounted for 22% of the variance explained (Adj. R2 = 0.218). This finding was also highly significant [F (7,95) = 1.69, p < 0.01], representing a large effect size f2 = 0.52. Behavioral awareness and value-based actions were the only significant predictors in the final model, with an almost equal prediction of perceived health (b = −0.281 and b = −0.241). The regression analyses supported the second study hypotheses, where perceived stigmatization predicts higher anxiety, depression, and lower self-reported health. Notably, PF processes might revert the negative effects of stigma on individuals’ well- being, particularly the process of behavioral awareness (being present). We tested which PF processes of change exert effects in the following analyses. 3.1. Sample and descriptive characteristics One hundred five participants filled out the questionnaires, and 57% (n = 59) were women, with a mean age of 54 (ranging from 19 to 05 frontiersin.org 10.3389/fmed.2023.1075672 Vasiliou et al. Characteristic1 Sex Total Male (n = 44) (Mean, N or %) Female (n = 59) T r x2 (p/df)* n = 105 n = 105 (Mean, N or %) Age 52.50 (16.96) 54.88 (18.71) 0.26 53.86 (17.94) Educational level (% years completed) 0.002 (2) GCSE or below 20 46 62.9% (n = 66) A Level or equivalent 7 3 9.5% (n = 10) Degree or above 18 11 27.6% (n = 29) Employment Status 0.11 (5) Unemployed 6 9 14.3% (n = 15) Retired 13 29 40% (n = 42) Sick leave 1 0 1% (n = 1) In education 1 1 1.9% (n = 2) Employed 24 19 41% (n = 43) Clinician rated severity of skin disease 0.21 (3) Mild 7 13 19% (n = 20) Moderate 15 32 44.8% (n = 47) Severe 21 15 34.3% (n = 36) Descriptive Characteristics2 Stigma (PSQ score range 0–27) 16.64 (11.18) 12.61 (10.68) 0.99 (100) 14.42 (11.04) Anxiety (GAD 7 score range: 0–21) 6.31 (6.04) 6.95 (6.23) 0.37 (100) 6.67 (6.12) Depression (PHQ 9 score range: 5–27) 7.43 (6.96) 8.03 (7.85) 0.20 (100) 7.77 (7.45) Self-rated health (EQ5D5L score range, 0–100) 67.45 (17.73) 66.04 (22.92) 0.06 (99) 66.65 (20.73) CompOE (Open; open to experience) 31.02 (9.40) 30.75 (9.89) 0.53 (97) 30.87 (9.63) CompBA (Aware; Behavioral awareness) 16.64 (6.49) 17.04 (7.49) 0.22 (97) 16.86 (7.03) CompVA (Active; Doing what matters) 35.11 (9.16) 34.38 (9.95) 0.61 (97) 34.71 (9.57) 1Mean comparisons between groups were executed with independent t-tests for continuous variables and ×2 fisher tests for categorical variables. Due to missing, the overall sum up does not equate n = 105 in all variables examined. 2Descriptive characteristics present means and standard deviation of the total scores for the study variables, split into males and females. Frontiers in Medicine 4. Discussion p < 0.001, but again the interaction was not significant. Finally, the same results were observed for perceived health where stigma and PF explained 16% of the variance in log self-reported health R2 = 0.16, F (3,94) = 7.2, p < 0.001, yet the interaction was not significant. In sum, the third aim was not supported, indicating that the relationship between stigma, PF processes, and stigma-related outcomes appears to be more complex and dynamic than static, as these predictive models indicate. To examine the dynamic role of the PF processes, we finally run a network analysis. Stigmatization is a common problem associated with living with a skin condition, yet relatively little is known about how this is influenced by psychological variables associated with distress. Network analysis has the potential to examine the multifaceted and bidirectional interactions associated with several variables potentially relevant to stigmatization in skin conditions (1, 59). In this study we specifically examined the relationship between psychological flexibility (PF), quality of life, stigmatization, and distress. Findings showed that stigma was negatively related to the three skills PF associated with depression and anxiety. Behavioral awareness accounted for the largest portion of variance explained among the three skills of PF response styles (open, aware, and active), predicting lower anxiety, depression, and higher perceived health. Moderation analyses showed no effect of the three PF response styles between stigma and outcomes. This finding suggests that PF responses may not be considered as static-not amenable to direct change variables, but as dynamic, sharing some potentially therapeutic role in buffering the effects of stigma in individuals with skin conditions. To further examine our hypothesis, we run a network analysis. Findings indicated generalized anxiety, depression, perceived stigmatization, and value- based actions as the most highly interconnected variables within the network. Stigma was most strongly negatively associated with avoidance (as opposed to being open) and value-based actions (as opposed to being active), and positively with anxiety and depression. These findings are congruent with existing research demonstrating the negative role of stigma in increasing psychological distress to individuals with skin conditions. However, our study provides support for the role of the PF responses as trainable skills that may play central role in tackling stigma. As such, these responses may be foci for interventions, designed that can lower stigma-related distress. 3.4. Moderation mediation analysis We conducted a moderated regression analysis to assess whether PF (total score on the CompACT questionnaire) moderates the relationship between stigma and well-being. We hypothesized that higher levels of PF would indirectly buffer the negative effects of stigma and stigma-related outcomes. To test for moderation, stigma, PF, and their interaction was entered together in a single block to three models, predicting generalized anxiety, depression, and perceived health. Variables were mean-centered prior to computing the interaction terms to minimize multicollinearity problems. A significant interaction term would indicate the presence of moderating effects. As Supplementary material S1 shows, none of the moderation analyses were significant. For example, when stigma and PF were entered together, they explained 50% of the variance in log anxiety R2 = 0.50, F (3, 91) = 28.92, p < 0.001, but the interaction term was not a significant predictor of anxiety. For depression, when the same variables were entered together (stigma and PF), they explained 38% of variance in log depression scores R2 = 0.38, F (3,93) = 20.63, 06 frontiersin.org Vasiliou et al. 10.3389/fmed.2023.1075672 E 2 Correlations between predictor variables (stigma), mediators (PF processes), and outcome variables (stigma-related impact). 1 2 3 4 5 6 7 1. PSQ −0.33 −0.27 −0.15 0.34 0.29 −0.24* 2. CompACT_OE −0.33 0.68 0.08 0.58 −0.65 0.34 3. CompACT_BA −0.27 0.68 0.22 −0.58 −0.63 0.41 4. CompACT_VA −0.15 0.08 0.02 −0.23* −0.28 0.30 5. PHQ-9 0.34 −0.58 −0.58** −0.23* 0.84 −0.62 6. GAD-7 0.29 −0.64 −0.63 −0.28 0.84 −0.61 7. VAS −0.24* 0.34 0.41 0.30 −0.62 −61 All correlations are Pearson’s r; n = 107; PSQ, perceived stigmatization questionnaire; CompACT_OE, openness to experience; CompACT_BA, behavioral awareness; CompACT_VA, valued actions; PHQ-9, patient health questionnaire; GAD-7, generalised anxiety disorder; VAS, EQ5D5L. p < 0.01. p<0 05 All correlations are Pearson’s r; n = 107; PSQ, perceived stigmatization questionnaire; CompACT_OE, openness to experience; CompACT_BA, behavioral awareness; CompACT_VA, valued actions; PHQ-9, patient health questionnaire; GAD-7, generalised anxiety disorder; VAS, EQ5D5L. p < 0.01. p<0 05 Frontiers in Medicine frontiersin.org 3.5. Network analysis TABLE 3 Linear regression for the prediction of anxiety, depression, and perceived health. Independent variables (Predictors) Steps (blocks)1 Β2 t p R2 Adj. R2 F (Df) p Dependent variable: anxiety Age 1 −0.151 −2.210 0.030 0.051 0.020 1.66 (3,95) 0.18 Gender 0.036 0.417 0.677 Severity −0.024 −0.024 −0.024 Stigma (PSQ) 2 0.005 2.48 0.015 0.11 0.072 2.85 (4,95) 0.03* Openness to experience (CompACT_OE) 3 −300 −3.07 0.003 0.57 0.534 16.53 (7,95) <0.001 Behaviorsal Awareness (CompACT_BA) −0.451 −4.71 <0.001 Valued-based actions (CompACT_VA −0.180 −2.52 0.013 Dependent variable: depression Age 1 0.027 0.326 0.745 0.004 −0.028 0.13 (3,95) 0.93 Gender 0.018 0.209 0.835 Severity −0.012 −0.148 0.883 Stigma (PSQ) 2 0.196 1.885 0.063 0.132 0.094 3.46 (4,95) 0.011 Openness to experience (CompACT_OE) 3 −0.258 −2.285 0.025 0.425 0.379 9.28 (7,95) <0.001 Behaviorsal Awareness (CompACT_BA) −0.343 −3.107 0.003 Valued-based actions (CompACT_VA) −0.149 −1.816 0.073 Dependent variable: perceived health Age 1 0.085 0.881 0.381 0.013 −0.019 0.41 (3,95) 0.742 Gender 0.021 0.213 0.832 Severity 0.105 1.075 0.285 Stigma (PSQ) 2 0.140 1.338 0.184 0.079 0.038 1.95 (4,95) 0.109 Openness to experience (CompACT_OE) 3 −0.032 −0.243 0.808 0.218 0.156 1.69 (7,95) 0.002 Behaviorsal Awareness (CompACT_BA) −0.281 −2.185 0.032 Valued-based actions (CompACT_VA −0.241 −2.520 0.014 1Variables were entered simultaneously in blocks (steps) and each independent variable was assessed in terms of what it adds to the prediction of the dependent, when the previous variables were controlled for. 1Variables were entered simultaneously in blocks (steps) and each independent variable was assessed in terms of what it adds to the prediction of the dependent, when the previous variables were controlled for. 2Beta represents standardized coefficients to the equation to allow for comparisons. represents standardized coefficients to the equation to allow for comparisons. population (66, 67). In this case, a measure assessing generalized anxiety disorder (GAD-7) might not entirely capture the distress individuals with skin problems experience. stigma (e.g., when others impose stigmatized attitudes) can reduce stigma-related distress and improves daily functioning (34, 51, 68). In our case, such finding suggests that being more open and engaged allows individuals with skin conditions first to acknowledge more willingly that their visible difference in appearance may trigger stigmatized reactions and correspondingly respond to stigma more openly by minimizing avoidance (e.g., attempting to control one’s stigmatized behaviors) and by abstaining from attaching rigidly to stigmatized thoughts (e.g., seen stigmatized thoughts as true literal entities that can define behaviors) (29, 30, 69). Frontiers in Medicine frontiersin.org 3.5. Network analysis The final network is illustrated in Figure 2. Based on the strength centrality indices, the node with the highest centrality, and therefore the most influential within the model, was generalized anxiety (GAD-7), followed by perceived stigmatization (PSQ), valued actions (CompACT_VA), and depression (PHQ-9). As expected, the model’s strongest (more meaningful) positive relations, excluding the PF, were observed between depression and anxiety, and stigma and depression (see Supplementary Table S2 for all the variables examined weights partial correlations). The strongest negative relationships were observed between anxiety and perceived health, and depression and perceived health. We found strong positive relationships between open and aware, and active and perceived health. The strongest negative relationships of PF with stigma were observed between open and anxiety, aware and anxiety, and open and stigma. Table 4 presents the edge weights partial network correlations of PF processes when LASSO regularization was applied. Stigma had the strongest negative relationships (edge) with openness to experience (weight matrix), followed by valued actions and behavioral awareness. Further, generalized anxiety was also found to exert a large negative relationship with openness to experience, followed by behavioral awareness, and valued actions. Depression was only found to be negatively related to behavioral awareness and positively to stigma. The Supplementary Table S2 presents all the relevant partial correlations among the examined variables. When percentages of cases were dropped off, stability assessment showed that the order of node strength was interpretable with some care. The edge weight accuracy (CIs) was found narrow for most PF processes when interconnecting with other nodes (see Supplementary material S3). The role of depression and anxiety is consistent with studies on stigma (7, 63, 64). In our study, we observed depression as the only variable associated with stigma in the network model. Concerning stigma, depression in individuals with skin conditions might be seen as a form of avoidance and passivity behaviors (39). These behaviors can lead individuals with skin conditions to avoid seeking support as a result of of stigma and shame (65). On the other hand, anxiety can be seen as a form of social anxiety related to the visible difference in appearance, further supporting some studies, showing that social anxiety is the most common form of distress for this 07 frontiersin.org 10.3389/fmed.2023.1075672 Vasiliou et al. TABLE 3 Linear regression for the prediction of anxiety, depression, and perceived health. 3.5. Network analysis Findings from research in the area of social psychology resonate with this approach of The findings in this study point towards the role of PF skills as effective responses to stigmatization. More specifically, findings from the network analysis indicated the influential central role of the “open” response style (comprising the PF skills of acceptance and defusion) as a promising intervention target, to reduce the effects of stigmatized behaviors in people with skin conditions. Existing research shows that being willing to experience both internalized stigma (e.g., when individuals come to believe the stigmatized thoughts) and enacted 08 frontiersin.org Vasiliou et al. 10.3389/fmed.2023.1075672 FIGURE 2 Network model of Stigma, PF responses skills (dyads), and stigma-related outcomes. Red edges indicate negative partial correlations; blue edges indicate positive partial correlations; PSQ: Perceived Stigmatisation Questionnaire; PHQ9: Patient Health Questionnaire; GAD7: Generalised Anxiety Disorder Assessment; VAS: EuroQOL 5- Dimensions- EQ5D5L Visual Analogue Scale; CompactOE: openness to experience; CompactBA: behavioral awareness; CompactVA; valued actions. FIGURE 2 Network model of Stigma, PF responses skills (dyads), and stigma-related outcomes. Red edges indicate negative partial correlations; blue edges indicate positive partial correlations; PSQ: Perceived Stigmatisation Questionnaire; PHQ9: Patient Health Questionnaire; GAD7: Generalised Anxiety Disorder Assessment; VAS: EuroQOL 5- Dimensions- EQ5D5L Visual Analogue Scale; CompactOE: openness to experience; CompactBA: behavioral awareness; CompactVA; valued actions. TABLE 4 LASSO regularized partial correlation coefficients for PF processes. Psychological flexibility processes Stigma Emotional functioning Daily functioning Anxiety Depression Perceived healthy (QoL) Openness (Compact OE) −0.142 −0.218 −0.010 0.000 Awareness (Compact BA) −0.022 −0.177 −0.060 0.170 Active (Compact VA) −0.062 −0.102 0.000 0.055 LASSO estimator was applied to controls for spurious (non-reliable) relations and to return a sparse (conservative) network model where only a relatively small number of edges are used to explain the covariation structure in the data. Therefore, edge (nodes) relations estimated as 0.00 reflect negligible relations within the model. TABLE 4 LASSO regularized partial correlation coefficients for PF processes. LASSO estimator was applied to controls for spurious (non-reliable) relations and to return a sparse (conservative) network model where only a relatively small number of edges are used to explain the covariation structure in the data. Therefore, edge (nodes) relations estimated as 0.00 reflect negligible relations within the model. Additionally, the study showed the important role of the “active” response style (comprising of the PF skills of values and committed actions), indicating foci for intervention development. Frontiers in Medicine 3.5. Network analysis Findings showed value-based actions as one of the most influential nodes in the awareness vs. control of stigmatized behaviors (70, 71) or changing self-stigmatized thoughts (72), yet further research is warranted, especially, to indicate how being open to stigmatized experiences, is an effective practice for individuals with skin problems. 09 frontiersin.org frontiersin.org Vasiliou et al. 10.3389/fmed.2023.1075672 10.3389/fmed.2023.1075672 intervention to tackle the effects of stigma, is likely to be suboptimal. One should move beyond skills and attempt to understand stigma as a context-specific problem, including biophysiological and sociocultural levels of stigma. Consistent with the network intervention approach, these skills should not be seen as snapshots that can be delivered across skin conditions. Rather, they ought to be seen as dynamic and interconnected systems of an intervention that are likely to modify person-specific coping with stigma responses, including broader sociocultural parameters that feed into the stigma. This requires a deliberate shift to models that organize different intervention strategies into a more coherent network (25). Such a model is the new Extended Evolutionary Meta-Model [see further here (81)]. It is based on evolutionary science and allows interventions to expand targeted PF skills, including conceptions about adaptation and resilience (84–86). network, exerting a negative association with stigma, anxiety, and a positive one with perceived health. Other studies indicates that values can lower distress and increase daily functioning (73–75). Value-based interventions help individuals identify and clarify their values, shift attention toward value-based actions in moments of distress, and guide them to resonate with those choices (76). Because stigma can promote a disconnect and a dissonance from one’s values (77, 78), the process of increased attention to value-based actions may not directly impact the cognitive or emotional content related to stigma but cultivate engagement of individuals’ to more healthy behaviors, such as adhering to medical prescription or taking care of ongoing flares due to the disease’s progress (79). Consequently, this can increase the frequency where healthy behaviors are chosen in different contexts where stigma occurs (e.g., “I can see others frown their eyes when they spot my pale white patches on my face, but this does not stop me from enjoying shopping in the mall or attending a social event”). 3.5. Network analysis Research indicates values as the process that increases motivation towards health behavioral changes (31, 80), yet, future research will shed more light on how individuals with skin conditions, in particular, can use values in this way, even in the presence of stigma. The present study had several limitations. First, the study used self-reported subjective measures known for their source bias and shared method variance. Secondly, the study was part of a larger cross- sectional epidemiological study that employed only a few psychosocial parameters involved in stigma. While we present new knowledge using variables that indicated the “central” role of PF, other variables that were not included, should be measured for a more integrated interpretation of stigma, such as contextual, interpersonal, and functional (6). Likewise, we made use of the UK-only self-selected sample, and this narrows the interpretation of the findings to predominantly white Caucasian populations. Equally, the sub-sample that measured stigma and PF parameters was underpowered for network analysis. Although the network model stability was found to be within acceptable ranges, interpretation should be cautious as the interpretation of CIs in analyses such as LASSO regularization is problematic because the initial estimates of network analyses are biased towards zero (59). Therefore, further replication of the study findings is warranted. The present study findings are noteworthy, suggesting both theoretical and clinical implications. From a theoretical point of view, our findings indicate two of the three PF response of psychological flexibility as being essential to tackle the effects of stigma and related psychological distress. This contains a set of trainable behavioral responses that allow individuals to address concurrently core psychological, behavioral, and contextual parameters of perceived stress (27, 81), such as stigma. Because these skills reflect common responses to perceived threats (e.g., stigmatized behaviors), we can more directly specify what are the core functionally important pathways that we can focus on and change. Theoretically, for this to occur, we first need to link how individuals respond to stigma. Findings from this study indicated the use of the “open” and “active” response styles of the PF as skills that hold the potential to reduce the effects of stigma. Secondly, we need to find approaches that incorporate all the relevant past, present and contextual factors (e.g., demographic, disease severity, health care professionals’ behaviors) that seem to contribute to the psychological reaction involved in stigma (e.g., social anxiety and avoidance). 3.5. Network analysis Notably, we can achieve this level of analysis by employing methods, such as momentary ecological assessments that can collect high temporal personalized density data at the context of individuals’ lives (82). As a first step towards this approach, our findings indicated foundational knowledge about the nuances of unidirectional and bidirectional relationships of stigma-related associations within a nomological network that goes beyond static correlational, regression, and moderation analyses. Such a level of analysis can propose future directions and indicates clinical progress (83). Future research should attempt to collect multiple and large-scale data, using measures that examine the experience of stigma holistically, with samples from different countries and with more heterogeneous skin conditions. This will allow researchers to use network comparison analyses and explore coping with stigma-related outcomes interconnections, including several contextual characteristics (e.g., demographics, race/ethnicity, disease onset or progress, etc.). Further, as the affective component of body image (e.g., anxiety, distress, shame, etc.) may be related to specific aspects of physical appearance (21), future studies should use disease-specific measures to assess affection. Likewise, future studies should attempt to examine stigma and coping responses, employing more idiographic and personalized methods, such as ecological momentary assessments (EMA). These methods can longitudinally collect behavioral and self- reported highly temporal data to assess the impact of targeted skills on stigma in the context of within-person variability, indicating personalized interventions. From a clinical perspective, focusing on functionally important skills, clinicians can develop scalable interventions for stigma that can meet the needs of a heterogeneous group of individuals with skin conditions (21). For example, the open response style should be employed when the problem is a narrow response to self- stigmatization where individuals attempt to reduce the stigmatized thoughts or replace them with more neutral or informative ones. On the contrary, when individuals respond to stigma with avoidance or passivity, values and commitment to health behaviors (as opposed to avoidance) should be employed. As stigma is a multidimensional phenomenon, focusing solely on individuals’ responses as the main This study applied step-wise analytic approaches to individuals with skin problems. Among the examined variables, stigma, depression, and two of the three response styles of the PF model, namely “open” and “active” skills, appeared important. The role of PF in the network analyses indicate certain functionally important pathways that may have clinical utility in psychosocial programs, attempting to reduce the effects of stigma in skin populations. Frontiers in Medicine Data availability statement The data analyzed in this study is subject to the following licenses/ restrictions: data are part of a larger European epidemiological research and can be available upon request. Requests to access these datasets should be directed to AT, thompsona18@cardiff.ac.uk. Supplementary material This manuscript was prepared as part of the first co-author Post- doctoral Research Associate Position in Clinical Health Psychology which is funded by the Health Education and Improvement Wales (HEIW). The data collection was fulfilled as part of the partial completion The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed.2023.1075672/ full#supplementary-material 3.5. Network analysis Tailoring personalized approaches may increase the likelihood of a truly good outcome for individuals with skin 10 frontiersin.org Vasiliou et al. 10.3389/fmed.2023.1075672 10.3389/fmed.2023.1075672 of the second co-author’s doctoral in Clinical Psychology program (D.Clin.Psy) which was conducted at the University of Sheffield and funded by the Health Education England, United Kingdom. problems, experiencing stigma. For this to occur, researchers and implementation scientists should employ newest approaches, such as the process-based intervention approach (25) and the Extended Evolutionary Meta-Model (EEMM) (81) as guides to develop a coherent network of intervention strategies that will tap across the multiple nature of stigma. Acknowledgments The present study was nested within a multi-center European study coordinated by the European Society for Dermatology and Psychiatry (ESDAP). The ESDAP study aimed to gather epidemiological data regarding the psychosocial burden of skin disease. The measures and hypotheses can be found at: https:// bmjopen.bmj.com/content/8/12/e024877. The current study utilized only data collected in the United Kingdom (UK), does not use the full data set collected, and includes specific measures only administered in the UK for the purpose of this study. We would also like to thank all the individuals who helped us to collect the data. Ethics statement The studies involving human participants were reviewed and approved by NHS Health Research Authority (18/LO/0639). The patients/participants provided their written informed consent to participate in this study. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note VV: writing – original draft (lead), methodology (equal), formal analyses (equal), and writing – review & editing (equal). HR: conceptualization (equal), formal analysis (equal), project administration (lead), and resources (equal). SC: investigation (lead) and resources (equal) GC: software (lead). AT: methodology (equal), conceptualization (equal), writing – review & editing (equal), and supervision (lead). All authors contributed to the article and approved the submitted version. 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https://openalex.org/W3177657142	https://journal.uniga.ac.id/index.php/MJA/article/download/1235/961	Indonesian	null	ANALISIS KEPUASAN PELANGGAN TERHADAP JASA PENGGILINGAN PADI (Studi Kasus Penggilingan Padi Tunas Karya Desa Persil Raya, Kecamatan Seyuran Hilir, Kabupaten Seruyan)	Mahatani	2,021	cc-by	5,554	ANALISIS KEPUASAN PELANGGAN TERHADAP JASA PENGGILINGAN PADI (Studi Kasus Penggilingan Padi Tunas Karya Desa Persil Raya, Kecamatan Seyuran Hilir, Kabupaten Seruyan) ANALISIS KEPUASAN PELANGGAN TERHADAP JASA PENGGILINGAN PADI (Studi Kasus Penggilingan Padi Tunas Karya Desa Persil Raya, Kecamatan Seyuran Hilir, Kabupaten Seruyan) Analysis of Customer Satisfaction on Rice Milling Services (Case Study of Rice Mill Milling Tunas Karya in Persil Raya Village, Seyuran Hilir District, Seruyan Regency) Anggun Erli Santika1, Tirsa Neyatri Bandrang1* 1Politeknik Seruyan, Jl A.Yani, KualaPembuang, Seruyan Hilir Kabupaten Seruyan, Kalimatan Tengah, Indonesia. *Email: tneyatri.poltes@gmail.com MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X terdapat 4 atribut, Pada kuadran IV terdapat 3 atribut. Berdasakan metode Customer Satisfaction Index (CSI) posisi pada tingkat kepuasan pelanggan berada pada tingkat sangat puas dengan hasil 0,85 yang didasarkan pada tabel indeks kepuasan pelanggan untuk atribut-atribut yang telah diuji. Kata Kunci: Penggilingan Padi, Analisis Deskriptif, Performance Analysis (IPA), Customer Satisfaction Index (CSI) Kata Kunci: Penggilingan Padi, Analisis Deskriptif, Performance Analysis (IPA), Customer Satisfaction Index (CSI) Keywords: Rice Milling, Descriptive Analysis, Performance Analysis (IPA), Customer Satisfaction Index (CSI) ABSTRAK Penelitian ini bertujuan menganalisis karakteristik pelanggan dan menganalisis kepuasan pelanggan Jasa Penggilingan Padi Tunas Karya, karena pelanggan yang merasa puas merupakan asset penting bagi tempat usaha Penggilingan Padi Tunas Karya. Dalam melakukan penelitian ini, untuk tujuan pertama menggunakan metode analisis deskriptif yakni untuk melihat karakteristik pengunjung dan untuk tingkat kepuasan konsumen di Penggilingan Padi Tunas Karya menggunakan dua metode, yakni Customer Satisfaction Index (CSI) digunakan untuk menentukan kepuasan pelanggan secara menyeluruh dengan pendekatan yang mempertimbangkan tingkat kepentingan dari variabel-variabel yang diukur, dan Importance Perfoemance Analysis (IPA) digunakan untuk mengetahui keadaan masing-masing variabel dari faktor-faktor kepuasan ditinjau dari segi tingkat kepentingan dan tingkat kinerja atribut, serta menggunakan diagram kartesius dan yang terdapat pada empat kuadrat. Hasil uji realibilitas untuk butir soal evaluasi kepentingan dan evaluasi kinerja menunjukan nilai yang lebih besar dari nilai kritis yang ditoleransi yaitu 0,81 untuk butir soal evaluasi kepentingan dan 0,86 untuk butir soal evaluasi kinerja. Hasil penelitian berdasarkan metode Importance Performance Analysis (IPA) pada kuadran I terdapat 1 atribut, Pada kuadran II terdapat 11 atribut, Kuadran III 299 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X P-ISSN : 2622- 2896 E-ISSN : 2721-513X ABSTRACT This study aims to analyze customer characteristics and analyze customer satisfaction in Tunas Karya Rice Milling Services. Because a satisfied customer is an important asset for the Tunas Karya Rice Mill business. In conducting this research, for the first purpose using descriptive analysis method, namely to see the characteristics of visitors and for the level of customer satisfaction at Tunas Karya Rice Mill using two methods, namely the Customer Satisfaction Index (CSI) used to determine overall customer satisfaction with an approach that considers the level the importance of the measured variables, and Importance Performance Analysis (IPA) is used to determine the state of each variable from the satisfaction factors in terms of importance level and attribute performance level, as well as using Cartesian diagrams and those contained in four squares. The results of the reliability test for the interest evaluation and performance evaluation items showed a value greater than the tolerable critical value, namely 0.81 for the evaluation of interest items and 0.86 for the performance evaluation items. The results of the study are based on the Importance Performance Analysis (IPA) method in quadrant I there is 1 attribute, in quadrant II there are 11 attributes, Quadrant III there are 4 attributes, in quadrant IV there are 3 attributes. Based on the Customer Satisfaction Index (CSI) method, the position on the level of customer satisfaction is at a very satisfied level with a result of 0.85 which is based on the customer satisfaction index table for the attributes that have been tested. Keywords: Rice Milling, Descriptive Analysis, Performance Analysis (IPA), Customer Satisfaction Index (CSI) 300 MAHATANI Vol. 4, No. 1, Juni 2021 PENDAHULUAN Beras merupakan kebutuhan dasar bagi kehidupan manusia baik ditinjau dari segi fisiologis, phisikologis, sosial maupun antropologis. Bagi masyarakat indonesia, beras menjadi komoditas yang sangat penting, tidak saja dilihat dari sisi produsen tetapi juga dari sisi konsumen. Sebelum menjadi beras, padi akan melalui beberapa tahap pascapanen yaitu: pemanenan padi, penyimpanan padi, perontokan padi, pengeringan padi, pengilingan gabah hingga menjadi beras. Masalah utama yang dihadapi petani dari proses pascapanen padi tersebut adalah kehilangan hasil yang cukup tinggi yang dikarenakan ada beras yang menjadi remuk ketika digiling. Besarnya jumlah penggilingan padi yang menyebar hampir merata di seluruh daerah sentra produksi padi di Indonesia ini menggambarkan bahwa Penggilingan padi memiliki peran yang sangat penting, dibidang Pertanian. Supply beras nasional dituntut untuk dapat memberikan kontribusi dalam penyediaan beras, baik dari segi kuantitas maupun kualitas yang mendukung ketahanan pangan nasional, maka Penggilingan padi yang merupakan tempat pertemuan antara produksi, pasca panen, pengolahan dan pemasaran gabah/beras sehingga merupakan mata rantai penting untuk menghasilkan beras/gabah secara maksimal dan baik (Arsyad, dkk., 2015). Besarnya jumlah penggilingan padi yang tersebar di sejumlah daerah tidak menjamin kualitas beras yang dihasilkan (Asmawati, 2009). Proses pengolahan gabah menjadi beras merupakan suatu proses yang terjadi pada penggilingan padi dengan batas kadar air 13-14%. Umumnya proses penggilingan padi dapat dipisahkan antara pengolahan gabah sampai menjadi beras pecah kulit (BPK) dan proses penyosohan yakni pengolahan beras pecah kulit menjadi beras sosoh. Pemisahan proses ini menggunakan alat yang terpisah yakni husker (pemecah kulit) dan whitener (pemutih/penyosoh). Berdasarkan penggunaan alat pada penggilingan secara umum, penggilingan padi cenderung untuk meningkatkan mutu, terutama pada penggilingan yang berskala kecil (Sudirman, 2011). Penggilingan padi merupakan salah satu unsur dalam penanganan pascapanen yang memerlukan proses penanganan dan pengolahan. Ketersediaan panen yang melimpah menimbulkan ketidakseimbangan dengan 301 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 output beras yang dihasilkan dikarenakan banyak tingkat kehilangan pada proses penanganan pascapanen yang masih menggunakan alat tradisional. Dengan adanya permasalahan tersebut maka sebaiknya pelaku usaha mengembangkan penggunaan teknologi dalam proses penggilingan padi, yaitu dengan cara menggunakan mesin dengan teknologi yang lebih efisien. Dalam hal ini untuk membuat pelaku usaha agar lebih baik dalam menggunakan mesin penggilingan padi, sebaiknya pemerintah menunjang itu memberikan pelatihan penggunaan mesin padi tersebut agar pelaku usaha dapat dengan mahir untuk menggunakannya, sehingga pengolahan beras yang dihasilkan lebih optimal. output beras yang dihasilkan dikarenakan banyak tingkat kehilangan pada proses penanganan pascapanen yang masih menggunakan alat tradisional. Dengan adanya permasalahan tersebut maka sebaiknya pelaku usaha mengembangkan penggunaan teknologi dalam proses penggilingan padi, yaitu dengan cara menggunakan mesin dengan teknologi yang lebih efisien. Dalam hal ini untuk membuat pelaku usaha agar lebih baik dalam menggunakan mesin penggilingan padi, sebaiknya pemerintah menunjang itu memberikan pelatihan penggunaan mesin padi tersebut agar pelaku usaha dapat dengan mahir untuk menggunakannya, sehingga pengolahan beras yang dihasilkan lebih optimal. Beras yang dijual di tempat penggilingan padi adalah beras yang diolah secara alami oleh para petani mulai dari peprosesan benih menjadi padi hingga dari padi menjadi beras, yang mana beras yang dihasilkan tersebut benar - benar alami, tidak menggunakan bahan kimia untuk mendapatkan hasil yang maksimal. Karena hal itu pula yang menjadikan masyarakat memiliki minat untuk membeli langsung beras yang dijual dipenggilingan padi ataupun mereka menggiling sendiri padi yang mereka miliki selain hal itu kenapa masyarakat lebih meminati beras hasil yang digiling secara langsung dikarenakan harga jual yang lebih murah. Pembangunan sektor pertanian yang memiliki nilai strategis, antara lain dalam memenuhi kebutuhan pangan yang terus meningkat akibat bertambahnya jumlah penduduk, peningkatan rata-rata pendapatan penduduk, luasnya penciptaan lapangan pekerjaan dan Usaha Jasa Penggilingan Padi yang bergerak disektor pertanian merupakan bagian dari salah satu pembangunan sektor pertanian tersebut serta semakin meningkatkan kesadaran gizi masyarakyat. Bertempat di Kabupaten Seruyan, Kecamatan Seruyan Hilir khususnya di Desa Persil Raya usaha Jasa Penggilingan Padi merupakan salah satu cabang dari sektor pertanian yang sedang terus berjalan usahanya. Banyak terdapat usaha dibidang pertanian di Kecamatan Seruyan Hilir, salah satunya adalah tempat usaha Jasa Penggilingan Padi. Adapun yang berdiri di Kecamatan Seruyan Hilir khususnya di Desa Persil Raya juga sangat pesat untuk perkembangan usaha jasa tempat penggilingan padi ini. 302 MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X Tabel 1. MAHATANI Vol. 4, No. 1, Juni 2021 pelanggan dengan tujuan pelanggan tetap setia dan senang membeli atau memakai jasa dari, untuk membuat usaha akan terus berkembang dan pelangganpun akan terus berdatangan serta menggunakan ulang jasa yang dijual. Pengukuran kepuasan pelanggan dapat mengetahui cara yang bisa membuat pelanggan merasa puas dan akan terus menjadi pelanggan tetap, penelitian mengukur kepuasan pelanggan dapat digunakan untuk membantu melakukan perbaikan dari segi kualitas pelayanan dan hasil produk, agar membuat usaha tetap berjalan dan lebih maju lagi Berdasarkan hasil survey di lapangan, diperoleh tempat penggilingan padi yang aktif produksi 2 ton perhari yaitu penggilingan padi Tunas Karya. Pengambilan tempat penggilingan padi yang akan dijadikan objek penelitian ini didasarkan melalui beberapa faktor, seperti penggilingan padi Tunas Karya mampu bersaing dengan penggilingan padi yang lebih dahulu berdiri seperti tempat Penggilingan Padi Karya Keluarga yang sudah memiliki pelanggan tetap. Pelanggan yang menggiling padinya di penggilingan padi Tunas Karya tidak hanya yang berada disekitar wilayah tempat usaha berdiri saja, tetapi yang dari luar wilayah desa persil raya pun juga ada, bahkan informasi yang penulis dapat dari narasumber jika ada pelanggan penggilingan padi Karya Keluarga yang berpindah menjadi pelanggan penggilingan padi Tunas Karya. Pentingnya melakukan penelitian pengukur kepuasan konsumen terhadap penggilingan padi Tunas Karya karena untuk mengetahui sejauh mana para pelanggan penggilingan padi Tunas Karya merasakan puas terhadap jasa yang dijual oleh penggilingan padi. Berdasarkan latar belakang itulah maka perlu dilakukan penelitian mengenai “Analisis Kepuasan Pelanggan Terhadap Jasa Penggilingan Padi (Studi Kasus Penggilingan Padi Tunas Karya Desa Persil Raya, Kecamatan Seyuran Hilir, Kabupaten Seruyan)”. MAHATANI Vol. 4, No. 1, Juni 2021 Data Penggilingan Padi di Desa Persil Raya Nama Penggilingan Padi Kapasitas Produksi Perhari (ton) Alamat Penggilingan Padi ATI 1 ton Sungai Mitak Penggilingan Padi Tunas Karya 2 ton Rt 001 Penggilingan Padi Dolah 2 ton Rt 003 Penggilingan Padi Karya Keluarga 2,5 ton Rt 001 Penggilingan Padi Karya Mandiri 1 ton Rt 003 Tabel 1. Data Penggilingan Padi di Desa Persil Raya Berdasarkan Tabel 1 dapat diketahui kapasitas produksi perhari masing – masing tempat usaha penggilingan padi, dimana untuk penggilingan padi ATI memiliki kapasitas satu hari produksi 1 ton begitu juga untuk penggilingan padi Karya Mandiri, penggilingan padi Dolah memiliki kapasitas perharinya 2 ton perproduksi, tetapi untuk ke tiga tempat usaha jasa penggilingan padi ini tidak buka setiap tiap hari kecuali padi yang akan mereka giling sudah sampai kapasitas yang mereka tentukan maka mereka akan memproduksi, berbeda dengan diwaktu musim panen dimana disaat musim panen produksi mereka dalam satu hari dapat dipastikan satu sampai dua ton. Untuk penggilingan padi Tunas Karya dalam satu hari produksi sebanyak dua ton yang mana penggilingan padi tunas karya ini buka setiap hari dan jarang libur terkecuali untuk hari – hari yang memang diliburkan karena ada kepentingan atau acara lain, penggilingan padi ini juga memiliki pelanggan yang tetap. Tempat penggilingan padi yang terakhir adalah tempat penggilingan padi Karya Keluarga dimana penggilingan padi ini adalah tempat penggilingan padi yang paling lama berdiri diantara yang lain, yang memiliki kapasitas produksi dua setengah ton perharinya, untuk jam operasi usaha bejalan beliau membatasi dari jam 8 pagi sampai jam 12 siang dan masuk lagi dari jam 2 siang sampai jam 4 sore, dimana beliau juga memiliki pelanggan tetap. Mengukur kepuasan pelanggan, dapat mengetahui cara membuat pelanggan puas terhadap suatu jasa atau produk yang mereka pakai, serta yang menyebabkan pelanggan puas terhadap jasa atau produk yang mereka pakai dan untuk tetap terus membuat pelanggan bertahan melakukan pembelian atau pemakaian jasa, maka perlu mengetahui cara untuk tetap mempertahankan 303 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X sengaja dengan pertimbangan bahwa Penggilingan Padi Tunas Karya merupakan salah satu penggilingan padi yang menerima jasa penggilingan Padi dan menyediakan beras untuk konsumen yang ada di Kecamatan Seruyan Hilir, tidak hanya itu juga didasarkan melalui beberapa faktor, seperti penggilingan padi mampu bersaing dengan penggilingan padi yang lebih dahulu berdiri, waktu beroperasi tempat usaha setiap hari, dalam satu tahun hampir tidak pernah tutup atau libur. Waktu penelitian ini dilakukan pada Bulan April sampai dengan Bulan Mei 2020. sengaja dengan pertimbangan bahwa Penggilingan Padi Tunas Karya merupakan salah satu penggilingan padi yang menerima jasa penggilingan Padi dan menyediakan beras untuk konsumen yang ada di Kecamatan Seruyan Hilir, tidak hanya itu juga didasarkan melalui beberapa faktor, seperti penggilingan padi mampu bersaing dengan penggilingan padi yang lebih dahulu berdiri, waktu beroperasi tempat usaha setiap hari, dalam satu tahun hampir tidak pernah tutup atau libur. Waktu penelitian ini dilakukan pada Bulan April sampai dengan Bulan Mei 2020. Lokasi dan Waktu Penelitian Lokasi penelitian dilakukan di Penggilingan Padi Tunas Karya, Desa Persil Raya, Kecamatan Seruyan Hilir, Kabupaten Seruyan. Pemilihan tempat penggilingan padi yang akan dijadikan objek penelitian ini dilakukan secara 304 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X Metode Analisis Menganalisis kepuasan pelanggan Penggilingan Padi Tunas Karya menggunakan 2 metode analisis data, yaitu metode analisis Impotance Performance Analysis (IPA) dan Customer Satisfaction Index (CSI). Analisis Importance Performance Analysis (IPA) dimaksudkan untuk mengetahui keadaan masing-masing variabel dari faktor-faktor kepuasan ditinjau dari segi kepentingan dan kinerja. Dimana pengukuran variabel dari masing-masing tingkat kepentingan dan tingkat kinerja menggunakan skala likert, adapun panduan pengukuran menggunakan skala likert yaitu: Tingkat kepentingan, sangat penting= 5, penting= 4, netral= 3, tidak penting= 2, sangat tidak penting = 1, dan tingkat kinerja, sangat baik= 5, baik= 4, netral= 3, tidak baik= 2, sangat tidak baik= 1. Analisis tingkat kepentingan dan tingkat kinerja Importance Performance Analysis (IPA) menggunakan diagram kartesius dimana penilaian kepentingan pelanggan ditunjukan dengan huruf Y, sementara penilaian kinerja perusahaan ditunjukan oleh huruf X. Untuk memenuhi maksud maka langkah-langkah yang ditempuh adalah sebagai berikut. a. Sebagai indikator skala ukuran kuantitatif untuk tingkat kepentingan menurut persepsi pelanggan dan tingkat kinerja secara nyata dinyatakan dalam bentuk tanggapan pelanggan terhadap kepuasan berupa skala likert. Skala ini memungkinkan responden untuk dapat mengekspresikan intensitas perasaan mereka terhadap karakteristik suatu produk dengan menentukan jumlah skor dari setiap variabel X dan Y. Skor ini diperoleh dengan mengalikan seluruh frekuensi data dengan bobotnya (Simamora, 305 MAHATANI Vol. 4, No. 1, Juni 2021 2002). Total penilaian tingkat kepentingan dan tingkat kinerja masing-masing atribut diperoleh dengan cara menjumlahkan hasil perkalian bobot masing-masing skala dengan jumlah responden yang memilih skala tersebut. 2002). Total penilaian tingkat kepentingan dan tingkat kinerja masing-masing atribut diperoleh dengan cara menjumlahkan hasil perkalian bobot masing-masing skala dengan jumlah responden yang memilih skala tersebut. b. Selanjutnya dilakukan pembagian jumlah skor dengan banyaknya responden, hasilnya berupa skor rata-rata tingkat kepentingan (Y) dan skor rata-rata tingkat kinerja (X). Dalam penyederhanaan rumus, maka untuk setiap faktor yang mempengaruhi kepuasan pelanggan adalah sebagai berikut: b. Selanjutnya dilakukan pembagian jumlah skor dengan banyaknya responden, hasilnya berupa skor rata-rata tingkat kepentingan (Y) dan skor rata-rata tingkat kinerja (X). Dalam penyederhanaan rumus, maka untuk setiap faktor yang mempengaruhi kepuasan pelanggan adalah sebagai berikut: Keterangan: Keterangan: X = Skor rata-rata tingkat kinerja atribut pada setiap variabel Penggilingan Padi Tunas Karya Y =Skor rata-rata tingkat kepentingan atribut pada setiap variabel Penggilingan Padi Tunas Karya =Total skor tingkat kinerja dari seluruh responden =Total skor tingkat kepentingan dari seluruh responden n =Jumlah responden =Total skor tingkat kepentingan dari seluruh responden 306 MAHATANI Vol. 4, No. Metode Analisis 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X c. Selanjutnya skor rata-rata tingkat kepentingan dan kinerja dianalisis pada diagram kartesius. Gambar 1. Diagram Cartesius Importance Performance Analysis Kurang Penting Kepentingan (Y) Penting Kurang Baik Kinerja (X) Baik I Prioritas Utama II Pertahankan Prestasi III Prioritas Rendah IV Berlebihan Gambar 1. Diagram Cartesius Importance Performance Analysis Kurang Penting Kepentingan (Y) Penting Kurang Baik Kinerja (X) Baik I Prioritas Utama II Pertahankan Prestasi III Prioritas Rendah IV Berlebihan Kurang Penting Gambar 1. Diagram Cartesius Importance Performance Analysis Dapat dilihat dari diagram tersebut, dapat dirumuskan strategi yang dilakukan berkaitan dengan posisi masing-masing atribut pada ke empat kuadran tersebut yang dapat dijelaskan sebagai berikut: a) Kuadran I (Prioritas Utama), posisi ini memuat atribut-atribut pada setiap variabel yang dianggap penting oleh pelanggan namun pada kenyataannya kinerja dari variabel ini belum sesuai dengan yang diharapkan. Artinya tingkat kepuasan yang diperoleh pelanggan masih sangat rendah dan perlu dilakukan perbaikan terus-menerus. b) Kuadran II (Pertahankan Prestasi), posisi ini memuat atribut-atribut pada setiap variabel yang dianggap penting oleh pelanggan telah dilaksanakan dengan baik dan dapat memuaskan pelanggan. c) Kuadran III (Prioritas Rendah), posisi ini memuat atribut-atribut pada setiap variabel yang dianggap kurang penting pengaruhnya bagi pelanggan, dan pada kenyataannya juga kinerjanya kurang istimewa. Perbaikan dan peningkatan kinerja variabel-variabel ini dapat dipertimbangkan kembali karena pengaruhnya kecil terhadap pelanggan. 307 MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 d) Kuadran IV (Berlebihan), posisi ini memuat atribut-atribut pada setiap variabel yang dianggap kurang penting pengaruhnya bagi pelanggan, namun pelaksanaannya telah dilaksanakan dengan sangat baik. Kinerja variabel-variabel ini dapat dihemat karena dapat menghemat biaya. d) Kuadran IV (Berlebihan), posisi ini memuat atribut-atribut pada setiap variabel yang dianggap kurang penting pengaruhnya bagi pelanggan, namun pelaksanaannya telah dilaksanakan dengan sangat baik. Kinerja variabel-variabel ini dapat dihemat karena dapat menghemat biaya. Customer Satisfaction Index (CSI) digunakan untuk menentukan tingkat kepuasan pelanggan secara menyeluruh dengan pendekatan yang mempertimbangkan tingkat kepentingan atribut-atribut pada setiap variabel yang diukur. Menurut Aritonang dan Lebrin (2005) tahap-tahap dalam pengukuran CSI adalah sebagai berikut: a. Menentukan Mean Importance Score (MIS). Nilai ini berasal dari rata-rata kepentingan tiap pelanggan. Dimana: n = Jumlah pelanggan Yi = Nilai kepentingan produk layanan Y ke-i b. Membuat Weight Factors (WF). Bobot ini merupakan persentase nilai MIS per atribut pelayanan terhadap total MIS seluruh atribut pelayanan. Dimana: p Dimana: p = Atribut pelayanan kepentingan ke-p Dimana: p = Atribut pelayanan kepentingan ke-p c. Membuat Weight Score (WS). Bobot ini merupakan perkalian antara WF dengan rata-rata tingkat kepuasan (x) (Mean Satisfaction Score = MSS). c. Membuat Weight Score (WS). Bobot ini merupakan perkalian antara WF dengan rata-rata tingkat kepuasan (x) (Mean Satisfaction Score = MSS). 308 HATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E ISSN 2721 513X P-ISSN : 2622- 2896 E-ISSN : 2721-513X E-ISSN : 2721-513X d. Menentukan Customer Satisfaction Index (CSI). d. Menentukan Customer Satisfaction Index (CSI). d. Menentukan Customer Satisfaction Index (CSI). Dimana: p = Atribut pelayanan kepentingan ke-p HS = (Highest Scale) Skala maksimum yang dig Pada umumnya, apabila nilai CSI di atas 50% dapat dikatakan jika pelanggan sudah merasa puas terhadap kinerja Penggilingan Padi Tunas Karya namun sebaliknya jika nilai CSI di bawah 50% pelanggan dikatakan belum puas terhadap kinerja Penggilingan Padi Tunas Karya. Nilai CSI dalam penelitian ini dibagi ke dalam 5 kriteria mulai dari tidak puas sampai dengan sangat puas. Kriteria ini mengikuti modifikasi kriteria yang dilakukan oleh PT. Sucofindo dalam melakukan Survei Kepuasan Pelanggan, adapun kriteria nilai CSI Survei Kepuasan Pelanggan PT. HASIL DAN PEMBAHASAN 1. Importance Performance Analysis (IPA) MAHATANI Vol. 4, No. 1, Juni 2021 Sucofindo dapat dilihat pada Tabel 2 berikut: Tabel 2 Kriteria Nilai Customer Satisfaction Index Nilai CSI Kriteria CSI 0,81 - 1,00 Sangat Puas 0,66 - 0,80 Puas 0,51 - 0,65 Cukup Puas 0,35 - 0,50 Kurang Puas 0,00 - 0,34 Tidak Puas Sumber: Riandinia dkk (2006). Tabel 2 Kriteria Nilai Customer Satisfaction Index Sumber: Riandinia dkk (2006). 1. Importance Performance Analysis (IPA) Importance Performance Analysis (IPA) digunakan untuk mengetahui tingkat kepuasan pelanggan Penggilingan padi Tunas Karya. Kepuasan pelanggan tercapai apabila kinerja perusahaan sesuai dengan tingkat kepentingan pelanggan. Tabel 3 menunjukkan data tingkat kepentingan dan kinerja serta nilai rata-rata masing-masing atribut. 309 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X Tabel 3. Tingkat Kepentingan dan Kinerja Serta Nilai Rata-Rata Atribut Atribut/ variabel Rata-Rata Kepentingan Kinerja 1. (V1) Harga Jual Jasa Penggilingan Padi 4.2 4.6 2. (V2) Harga Jual Sesuai Kualitas 4.6 4.7 3. Kualitas Pelayanan (V3) Kecepatan Respon (sigap) 4.1 3.9 (V4) Diskon (diatas 1 ton) 3.8 4.0 (V5) Kesopanan Merespon 3.7 3.9 (V6) Kemahiran 4.5 4.3 (V7) Keterampilan 4.5 4.6 (V8) Keramahan 4.5 4.6 (V9) Kecepatan Bekerja 4.5 4.6 (V16) Kejujuran 4.7 4.6 (V12) Lokasi 4.2 3.9 (V13) Pelayanan via telpon 4.6 3.9 (V14) Berperilaku baik 3.6 4.4 (V15) Mendengar masukkan 4.3 4.5 (V18) Penyediaan tempat penjemuran padi 4.6 4.5 (V19) Tingkat kebersihan lingkungan Penggilingan Padi 2.9 3.8 4. Kualitas Produk (V11) Tekstur 3.4 3.5 (V10) Warna 4.2 4.6 (V17) Kebersihan beras yang dihasilkan 4.7 3.5 Rata – Rata 4.19 4.23 Sumber : Referensi ( 2020), diolah. Tabel 3. Tingkat Kepentingan dan Kinerja Serta Nilai Rata-Rata Atribut 3. Tingkat Kepentingan dan Kinerja Serta Nilai Rata-Rata Atribut Importance Performance Analysis (IPA) bertujuan untuk mengetahui tingkat kepuasan pelanggan, kepuasan pelanggan tercapai apabila kinerja perusahaan sesuai dengan tingkat kepentingan pelanggan. Untuk mengetahui tingkat kepuasan pelanggan terhadap atribut Penggilingan Padi Tunas Karya maka akan dikelompokkan atribut-atribut berdasarkan masing-masing kuadran tingkat perbaikan kinerja. Dari analisis data tingkat kepentingan dan kinerja serta nilai rata-rata masing-masing atribut, diperoleh empat kuadran pada diagram kartesius Importance 310 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X Performance Analysis dengan garis pembagi adalah rata-rata tingkat kepentingan atribut yang diharapkan oleh pelanggan dan rata-rata tingkat kinerja atribut yang diterima dan dirasakan oleh pelanggan. Berdasarkan perhitungan rata-rata tingkat kepentingan dan kinerja masing-masing atribut, maka ke 19 atribut telah dikelompokkan ke dalam 4 Kuadran. Pada kuadran I terdapat 1 atribut yaitu: berperilaku baik pelayan penggilingan padi. Pada kuadran II terdapat 11 atribut yaitu: harga jual jasa penggilingan padi, Harga Jual Sesuai Kualitas, Kemahiran, Keterampilan, Keramahan, Kecepatan Bekerja, Kejujuran, Mendengar masukkan, Penyediaan tempat penjemuran padi, Tekstur, dan Warna. Pada Kuadran III terdapat 4 atribut yaitu: Kecepatan Respon, Diskon, Kesopanan Merespon, Tingkat kebersihan lingkungan Penggilingan Padi. Pada kuadran IV terdapat 3 atribut yaitu: Lokasi, Kecepatan pelayanan (via telpon), Kebersihan beras yang dihasilkan. Pengelompokan masing-masing atribut pada ke 4 kuadran digunakan untuk memperbaiki kinerja atribut yang dapat mempengaruhi kepuasan pelanggan. Tingkat kepentingan tergantung pada persepsi masing-masing pelanggan sehingga tiap-tiap pelanggan berbeda tingkat kepentingannya. Jadi untuk meningkatkan kepuasan pelanggan dilakukan melalui peningkatan kinerja, dengan pengelompokan Importance Performance Analysis sehingga dapat diketahui atribut mana yang harus menjadi prioritas peningkatan kinerja. Berdasarkan data rata-rata tingkat kepentingan dan kinerja maka ke 19 atribut dikelompokkan ke dalam 4 kuadran, yaitu sebagai berikut: 311 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X Kinerja Penggilingan Padi Tunas Karya Gambar 2. Diagram Cartesius Penggilingan Padi Tunas Karya S b R f i (2020) di l h Kinerja Penggilingan Padi Tunas Karya Kinerja Penggilingan Padi Tunas Karya Gambar 2. Diagram Cartesius Penggilingan Padi Tunas Karya Gambar 2. Diagram Cartesius Penggilingan Padi Tunas Karya Keterangan: (V1) Harga Jual Jasa Penggilingan Padi (V2) Harga Jual Sesuai Kualitas (V3) Kecepatan Respon (sigap) (V4) Diskon (V5) Kesopanan Merespon (V6) Kemahiran (V7) Keterampilan (V8) Keramahan (V9) Kecepatan Bekerja (V10) Warna (V11) Tekstur (V12) Lokasi (V13) Kecepatan pelayanan (via telpon) (V14) Berperilaku baik (V15) Mendengar masukkan (V16) Kejujuran (V17) Kebersihan Beras Yang dihasilkan (V18) Penyediaan tempat penjemuran padi (V19) Tingkat kebersihan lingkungan Penggilingan Padi Kuadran I MAHATANI Vol. 4, No. 1, Juni 2021 pelanggan menganggap atribut berperilaku baik pelayan penggilingan padi penting namun kinerjanya tidak sesuai dengan apa yang diharapkan pelanggan. pelanggan menganggap atribut berperilaku baik pelayan penggilingan padi penting namun kinerjanya tidak sesuai dengan apa yang diharapkan pelanggan. Berdasarkan hasil pengamatan di lapangan, pelanggan penggilingan padi melakukan penggilingan dalam waktu yang berbeda – beda yang mana ketika mereka menggiling padi akan berinteraksi dengan petugas yang menggiling padi dan dari sini para pelanggan dapat menilai sikap dan perilaku yang diberikan oleh petugas penggilingan padi agar mereka merasa nyaman untuk melakukan penggunaan jasa dan membuat mereka terus akan memakai jasa penggilingan padi tersebut karena itulah menurut para pelanggan berperilaku baik dalam melayani pengguna jasa itu penting. Maka hendaknya pihak manajemen Penggilingan Padi Tunas Karya harus melakukan perbaikan kinerja terhadap atribut berperilaku baik dalam melayani pelanggan penggilingan, dengan cara menyesuaikan sikap dalam merespon pelanggan agar pelanggan merasa nyaman dalam melakukan penggunaan jasa dan sesuai dengan kepentingan serta harapan pelanggan sehingga pada akhirnya dapat meningkatkan kepuasan pelanggan. Kuadran I Kuadran I terdapat 1 atribut yaitu: berperilaku baik pelayan penggilingan padi. Atribut ini menjadi prioritas utama bagi manajemen Penggilingan Padi Tunas Karya dalam peningkatan kinerja, karena memiliki nilai kepentingan yang tinggi sementara nilai kinerjanya rendah. Artinya 312 312 Kuadran III Kuadran III terdapat 4 atribut yaitu: Kecepatan Respon, Diskon, Kesopanan Merespon, Tingkat kebersihan lingkungan Penggilingan Padi, atribut yang berada diposisi ini dinilai oleh pelanggan tidak terlalu penting, dikarenakan pelanggan tidak terlalu mengharapkan kinerja atribut tersebut dan nyatanya kinerjanya biasa-biasa saja. Dapat dikatakan bahwa atribut kerapian pelayanan memiliki pengaruh yang tidak terlalu besar apabila dilakukan perbaikan kinerjanya. Berdasarkan hasil pengamatan di lapangan, tenaga kerja Penggilingan Padi Tunas Karya di hadapkan langsung dalam proses menggiling padi. Pada proses tersebut tenaga kerja menggunakan mesin penggilingan untuk menggiling padi menjadi beras dalam proses penggilingan padi, terjadi pemecahan terhadap kulit padi, dilanjutkan pemisahan bulir beras dari kulit, setelah itu dimasukkan kedalam mesin penyosoh yang digunakan untuk membuat beras bersih, selanjutnya memasukkan kedalam mesin pemisah beras yang utuh dan beras yang patah, dan tahap terakhir menggunakan mesin shining yang berfungsi sebagai pencuci dan pembersih beras dan membersihkan lokasi setelah proses penggilingan padi selesai. Untuk perbaikan kinerja atribut pada kuadran ini tidak terlalu penting dilakukan, artinya lebih baik dilakukan perbaikan pada kuadran I (prioritas). MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X P-ISSN : 2622- 2896 E-ISSN : 2721-513X Kuadran II Kuadran II menunjukan bahwa antara tingkat kepentingan dan harapan pelanggan sudah sesuai dengan kinerja masing-masing atribut. Menyatakan pelanggan sudah merasa puas terhadap kinerja atribut-atribut yang mereka terima sehingga usaha Penggilingan Padi Tunas Karya hanya perlu mempertahankan kualitas kinerja yang ada pada kuadran ini, atribut yang ada di kuadrat ini sebanyak 10. Atribut-atribut yang terdapat pada kuadran ini adalah harga jual jasa penggilingan padi, Harga Jual Sesuai Kualitas, Kemahiran, Keterampilan, Keramahan, Kecepatan Bekerja, Kejujuran, Mendengar masukkan, Penyediaan tempat penjemuran padi, Tekstur, dan Warna. Namun untuk jangka panjang tetap akan diperlukan berbagai peningkatan seiring bermunculannya pesaing-pesaing baru yang turut bersaing dalam memenuhi kepuasan pelanggan. 313 MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X tunas karya juga sudah terjamin kebersihannya dan tidak akan mempengaruhi hasil giling. Pelanggan menganggap atribut Lokasi, Kecepatan pelayanan (via telpon), Kebersihan Padi yang dihasilkan, tidak terlalu penting karena pelanggan merasa meskipun lokasi penggilingan padi terletak dibelakang dari jalan raya, tetapi penggilingan padi menyediakan tempat untuk menjemur padi pelanggan yang belum kering, sehingga pelanggan dapat dengan mudah untuk melakukan penjemuran padi mereka sebelum digiling. Kuadran IV Pada kuadran IV terdapat 3 atribut dinilai oleh pelanggan memiliki kepentingan yang rendah tetapi kinerjanya bagus. Atribut pada kuadran ini adalah yaitu: Lokasi, Kecepatan pelayanan (via telpon), Kebersihan Padi yang dihasilkan. Berdasarkan hasil pengamatan di lapangan masing-masing pelanggan yang menggiling padi di Tunas Karya memiliki tempat tinggal yang berbeda beda lokasinya, sehingga terkadang ada pelanggan yang menggunakan pelayanan via telpon dan ada juga pelanggan yang memilih datang langsung untuk mengetahui padinya sudah selasai digiling atau belum. Lokasi penggilingan padi Tunas Karya yang terletak didalam gang tidak menjadi masalah bagi para pelanggan, dan kebersihan padi yang dihasilkan penggilingan 314 MAHATANI Vol. 4, No. 1, Juni 2021 MAHATANI Vol. 4, No. 1, Juni 2021 Sumber: referensi (2020), diolah. Sumber: referensi (2020), diolah. MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X Nilai Customer Satisfaction Index (CSI) kepuasan pelanggan Penggilingan Padi Tunas Karya untuk keseluruhan dilihat berdasarkan penilaian kinerja dan kepentingan atribut-atribut yang diterima oleh pelanggan menunjukan angka Customer Satisfaction Index (%) sebesar 0,85. Dengan hasil ini menunjukan jika pelanggan penggilingan padi sangat puas terhadap pelayanan dan produk yang dihasilkan dan diberikan penggilingan padi Tunas Karya, dan membuat mereka akan terus bertahan menggunakan jasa penggilingan padi Tunas Karya. Perhitungan analisis CSI kepuasan pelanggan Penggilingan Padi Tumas Karya yang berada dikriteria “sangat puas”, untuk kepuasan pelanggan baiknya terus ditingkatkan hingga mencapai taraf maksimal yang diindikasikan dengan nilai CSI mendekati angka 1,00 akan lebih baik, pelanggan akan benar-benar merasakan kinerja atribut-atribut Penggilingan Padi Tugas Karya sama dengan tingkat kepentingan yang pelanggan harapkan. Kepuasan sangat berhubungan dengan loyalitas, karena itu ketika pelanggan merasa puas maka mereka akan loyal terhadap usaha jasa Penggilingan Padi Tunas Karya. 2. Customer Satisfaction Index (CSI) Customer Satisfaction Index (CSI) digunakan untuk mengalisis kepuasan pelanggan secara menyeluruh dari semua atribut-atribut yang diukur. Nilai CSI diperoleh dengan cara membagi Weigthted Average (penjumlahan seluruh Weighted Score) dengan skala maksimum (skala 5) yang digunakan dalam penelitian ini. Nilai CSI pada penelitian ini adalah 0,85, menurut Panduan Survei Kepuasan Pelanggan yang berada direntang 0,81 - 1,00 artinya pelanggan Penggilingan Padi Tunas Karya secara keseluruhan berdasarkan kinerja dan kepentingan atribut-atribut produk berada pada kriteria “sangat puas” yang ditunjukkan dengan angka hasil 0,85. Customer Satisfaction Index (CSI) digunakan untuk tujuan menganalisis kepuasan pelanggan secara menyeluruh dari semua atribut-atribut yang diukur. Hasil dari analisis Customer Satisfaction Index (CSI) secara rinci dapat dilihat pada Tabel 4 berikut: 315 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X Tabel 4. Analisis Customer Satisfaction Index (CSI) Penggilingan Penggilingan Padi Tunas Karya No Atribut Kepentin gan Importance Weighting Factors (%) Kinerja Weighted Score 1 Harga Jasa Penggilingan Padi 4.2 5.28 4.6 0.24 2 Harga Seusai Kualitas 4.6 5.78 4.7 0.27 3 Kecepatan Pelayanan Merespon (sigap) 4.1 5.15 3.9 0.20 4 Memberikan Potongan Harga 3.8 4.77 4.0 0.19 5 Memberikan Respon Sopan 3.7 4.65 3.9 0.18 6 Kemahiran Petugas Penggilingan 4.5 5.65 4.3 0.24 7 Keterampilan dalam pengerjaan 4.5 5.65 4.6 0.26 8 Keramahan dalam Melayani 4.5 5.65 4.6 0.26 9 Kecepatan dalam Pengerjakan 4.5 5.65 4.6 0.26 10 Warna Beras yang dihasilkan 4.7 5.90 4.6 0.27 11 Tekstur Beras yang dihasilkan 3.4 4.27 3.5 0.15 12 Lokasi Penggilingan Padi 4.2 5.28 3.9 0.21 13 Pelayanan Via Telpon 4.6 5.78 3.9 0.23 14 Memberikan respon tutur kata yang baik 3.6 4.52 4.4 0.20 15 Mendengarkan masukkan 4.3 5.40 4.5 0.24 16 Kejujuran dalam pelayanan 4.2 5.28 4.6 0.24 17 kebersihan beras yang dihasilkan 4.7 5.90 3.5 0.21 18 Penyediaan tempat penjemuran 4.6 5.78 4.5 0.26 19 Kebersihan lingkungan penggilingan 2.9 3.64 3.8 0.14 Total 79.6 100 80.4 4.25 Customer satisfaction Index (%) 0.85 Sumber: referensi (2020) diolah Tabel 4. Analisis Customer Satisfaction Index (CSI) Penggilingan Penggilingan Padi nalisis Customer Satisfaction Index (CSI) Penggilingan Penggilingan Padi 316 316 UCAPAN TERIMAKASIH Ucapan terimakasih kepada semua pihak yang sudah berpartisipasidalam penelitian ini serta para petani penggilingan padi Tunas Karya Desa persil Raya Kecamatan Seuryan Hilir Kabupaten Kuala Pembuang Kalimantan Tengah. penelitian ini serta para petani penggilingan padi Tunas Karya Desa persil Raya Kecamatan Seuryan Hilir Kabupaten Kuala Pembuang Kalimantan Tengah. Kecamatan Seuryan Hilir Kabupaten Kuala Pembuang Kalimantan Tengah. Kesimpulan Berdasarkan Importance Performance Analysis (IPA) atribut yang diteliti masuk ke dalam empat kuadran. kuadran I terdapat 1 atribut yaitu: berperilaku baik pelayan penggilingan padi. Pada kuadran II terdapat 11 atribut yaitu: harga jual jasa penggilingan padi, harga jual sesuai kualitas, kemahiran, keterampilan, keramahan, kecepatan bekerja, kejujuran, mendengar masukkan, penyediaan tempat penjemuran padi, tekstur, dan warna. Pada Kuadran III terdapat 4 atribut yaitu: kecepatan respon, diskon, kesopanan merespon, tingkat kebersihan lingkungan penggilingan padi. Pada kuadran IV terdapat 3 atribut yaitu: lokasi, kecepatan pelayanan (via telpon), kebersihan beras yang dihasilkan. Berdasarkan Customer Satisfaction Index (CSI) secara keseluruhan pelanggan sangat puas terhadap kinerja Penggilingan Padi Tunas Karya hal ini terlihat dari nilai Customer Satisfaction Index (CSI) yaitu sebesar 0,85 yang berada pada rentang 0,81 - 1,00. 317 MAHATANI Vol. 4, No. 1, Juni 2021 P-ISSN : 2622- 2896 E-ISSN : 2721-513X P-ISSN : 2622- 2896 E-ISSN : 2721-513X Rekomonedasi Kebijakan 1. Atribut perilaku penggilingan padi hendaknya terus meningkatkan kinerjanya,. 1. Atribut perilaku penggilingan padi hendaknya terus meningkatkan kinerjanya,. 2. Pertahankan kinerja terutama harga jual jasa penggilingan padi, harga jual sesuai kualitas, kemahiran, keterampilan, keramahan, kecepatan bekerja, kejujuran, mendengar masukkan, penyediaan tempat penjemuran padi, tekstur, dan warna. 3. Terus melakukan evaluasi kepuasan terhadap pelanggan secara bertahap untuk menindak lanjuti keluhan-keluhan yang didapat dari pelanggan terhadap kualitas pelayanan Penggilingan Padi DAFTAR PUSTAKA Arsyad, M., Junaedi, M., Mulyati, dan Tahir, M. 2015. Evaluasi Kualitas beras dan Unit Cost Penggiling Padi untuk Varietas dan Ciliwung. Journal Ilmiah Indonesia. 10(1). Ariani, D. Wahyu. 2009. Manajemen Operasi Jasa. Yogyakarta : Graha Ilmu. Limbong Ismail., Mozart B Darus, dan Emalisa. (2015). Analisis Kelayakan Usaha Penggilingan Padi Skala Kecil (Studi Kasus: Kecamatan Tanjung Morawa, Kabupaten Deli Serdang, Provinsi Sumatera Utara). Journal on social economic of agriculture and agribusiness, 4(12). Umar, Sudirman. 2011. Pengaruh Sistim Penggilingan Padi Terhadap Kualitas Giling di Sentra Produksi Beras Lahan Pasang Surut. Jurnal Teknologi Pertanian. Universitas Mulawarman. Volume 7 No 1. Warisno Wowon, Tamrin, dan Budianto Lanya. (2014). Analisis Mutu Beras Pada Mesin Penggilingan Padi Berjalan di Kabupaten Pringsewu. Yuliarmi, Ni Nyoman dan Putu Riyasa. 2007. Analisis Faktor-Faktor Yang Mempengaruhi Kepuasan Pelanggan Terhadap Pelayanan PDAM Kota Denpasar. Denpasar: Buletin Studi Ekonomi Volume 12 Nomor 1 Tahun 2007. 318
W1563218668.txt	https://sjer.ch/article/download/4900/7180	de		Der Begriff Heterogenität in empirischen Studien	Schweizerische Zeitschrift für Bildungswissenschaften	2,018	cc-by	5,390	Thema Der Begriff Heterogenität in empirischen Studien Sandra Zulliger und Sabine Tanner Welche Themen und Heterogenitätskategorien werden erforscht, wenn im Titel einer empirischen Studie der Begriff „Heterogenität“ verwendet wird? Anhand einer systematischen Datenbankrecherche und Inhaltsanalyse von empirischen Studien wird dieser Frage nachgegangen. 21 Studien entsprachen den definierten Kriterien und lassen sich in die folgenden drei Themenschwerpunkte aufteilen: 1. Die Heterogenität der Schülerschaft in Bezug auf unterschiedliche Heterogenitätskategorien und Analyseebenen. Die Leistungsheterogenität ist in diesem Zusammenhang die am häufigsten untersuchte Heterogenitätskategorie. 2. Die Anwendung, Evaluierung und Entwicklung von integrativen Unterrichtsmethoden. 3. Die subjektiven Theorien der Lehrpersonen zur Heterogenität ihrer Schülerschaft. Einleitung Der Begriff Heterogenität ist in den letzten Jahren zu einem wichtigen Begriff der deutschsprachigen Erziehungswissenschaft avanciert. International vergleichende Schulleistungsstudien wie bspw. PISA, TIMSS oder IGLU tragen dazu bei, dass Heterogenität als Sammelbegriff eines mit vielerlei Herausforderungen behafteten Themenfeldes in einer zunehmend breiteren Öffentlichkeit diskutiert wird (vgl. z.B. Wenning, 2007). Wie sich in den folgenden Ausführungen zur Begriffsbestimmung zeigen wird, handelt es sich bei Heterogenität um einen sehr vielschichtigen Begriff, der viele Unklarheiten offen lässt. Zur Begriffsbestimmung werden u.a. Listendefinitionen ausgeführt, welche die heterogenitätsstiftenden Schülermerkmale nennen (z.B. Wischer, 2009). Schülerinnen und Schüler können sich diesem Verständnis zufolge theoretisch in unendlich vielen verschiedenen Merkmalen unterscheiden. Im Schulkontext wird der Fokus dabei häufig auf Merkmale gerichtet, die in Zusammenhang mit dem Lern- und Leistungsverhalten resp. dem resultierenden Bildungserfolg von Interesse sind (Stöger & Ziegler, 2012). Eigenschaften des Heterogenitätsbegriffs fassen Lang, Grittner, Rehle und Revue suisse des sciences de l’éducation, 35 (1) 2013, 37-52 ISSN 1424-3946, Academic Press Fribourg 37 Thema Hartinger (2010) in vier Punkten zusammen: - Relativität: Heterogen ist keine absolute Eigenschaft. Was als heterogen bezeichnet wird, ist das Ergebnis eines Vergleichs eines bestimmten Kriteriums zu einem bestimmten Massstab. - Partialität: Mit Heterogenität werden nur zeitlich begrenzte Zustände beschrieben, da sich die Unterschiede wieder vergrössern oder verringern können. Zudem gilt die Bezeichnung nur partiell und kann nicht verwendet werden, um Schülerinnen und Schüler oder eine Gruppe von Schülern als Ganzes oder deren zukünftige Entwicklung zu beschreiben. - Konstruiertheit: Erst durch das Anlegen eines bestimmten Massstabs kann Heterogenität bzw. Homogenität bestimmt werden. Es handelt sich somit um ein Konstrukt, das vom jeweiligen Massstab abhängig ist. - Wertneutralität: Die Konstruktion von Heterogenität bzw. Homogenität ist nicht grundsätzlich gut oder schlecht. Sie erfolgt jedoch immer aus einem bestimmten Interesse und in bestimmten Zusammenhängen. Lernrelevante Unterschiede der Schülerinnen und Schüler werden z.B. häufig normativ interpretiert. Zur Bezeichnung der Unterschiedlichkeit von Personen findet neben dem Begriff der Heterogenität häufig der Begriff Diversity / Diversität Verwendung. Die beiden Begriffe erzeugen nach Krüger-Potratz (2011) je unterschiedliche Vorstellungen. Diversity / Diversität ist überwiegend im ökonomischen Bereich im Sinne von «Diversity Management» in Gebrauch. Diversity Management bezeichnet die Berücksichtigung der Vielfalt und der Verschiedenheit des Personals zur Effizienzsteigerung. Im Gegensatz zu Diversität / Diversity ist der Begriff Heterogenität binär codiert. Der Gegenbegriff Homogenität ist eher positiv besetzt und bezeichnet Einheitlichkeit. Der hauptsächlich im schulischen Kontext verwendete Begriff Heterogenität ist hingegen vielfach mit Schwierigkeiten assoziiert. Diesen Schwierigkeiten gilt es im Schulalltag entgegenzuwirken (Krüger-Potratz, 2011). Das System Schule ist in seiner Grundstruktur darum bemüht, Homogenität herzustellen. In diesem Sinne werden Schülerinnen und Schüler nach Alter und Leistung gruppiert (Leiprecht & Lutz, 2006). Es ist unbestritten, dass die schulische Leistung das zentrale Ordnungskriterium des heutigen Schulsystems repräsentiert (Lindemann, Schwanenberg & Bos, 2011). Angesichts der Aktualität und Brisanz von Schulleistungsunterschieden wird angenommen, dass die Leistungsheterogenität in empirischen Studien einen zentralen Untersuchungsgegenstand darstellt. Eine in jüngster Zeit aufgekommene Forderung an die Forschung ist dabei, nach Möglichkeit mehrere Heterogenitätsdimensionen miteinander zu betrachten, um z.B. die Ursachen von Schulerfolg oder -misserfolg aufschlussreicher zu ergründen (Prengel, 2007). Die aufgezeigten Begriffsannäherungen lassen im Zusammenhang mit der Verwendung des Begriffs Heterogenität in empirischen Studien vielerlei Fragen 38 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013 Thema aufkommen. Welche Schülermerkmale werden herangezogen, um Heterogenität festzustellen? Welche Massstäbe werden angelegt? Für diesen Beitrag ist die Forschungsfrage wegleitend, was genau erforscht wird, wenn im Titel eines Beitrags von Heterogenität die Rede ist. Folgende Fragestellungen werden ausformuliert: 1. In welchem thematischen Kontext wird der Begriff ‚Heterogenität‘ in empirischen Studien aus dem Schulumfeld eingebracht? 2. Welche Heterogenitätskategorien stehen im Mittelpunkt des Forschungsinteresses? Mit der Analyse dieser Fragestellungen sollen die übergeordneten Themengebiete der verschiedenen Beiträge und die verwendeten Heterogenitätskategorien aufgezeichnet werden. Die Studie leistet dabei keinen Beitrag zur genaueren Begriffsbestimmung, sondern zeigt lediglich die Bandbreite der inhaltlichen Anwendungen auf. Im Folgenden wird das methodische Vorgehen erläutert. Im Ergebnisteil und der Zusammenschau werden die zentralen Erscheinungsformen herausgearbeitet, die den Begriff Heterogenität im schulischen Kontext kennzeichnen. Das Forschungsvorgehen bezweckt insofern das Rekonstruieren und Aufschlüsseln von Realität (Diaz-Bone, 2006). Dies ist für den pädagogischen Heterogenitätsdiskurs der Gegenwart wünschenswert, da der Umgang mit Heterogenität als grosse zukünftige Herausforderung der Schule gilt (Wischer, 2007). Methodisches Vorgehen Die Zusammenstellung der wissenschaftlichen Veröffentlichungen folgt dem Interesse, die empirische Sättigung des Begriffs Heterogenität in Forschungsstudien zu erfassen. Bestandteil und Grundlage der Suche ist der Begriff Heterogenität, verschränkt mit der Suche nach empirischen Studien. Konkret wird nach Einzelbeiträgen gesucht, die sich mit Heterogenität befassen und diesen Begriff im (Unter-)Titel aufführen. Beim (Unter-)Titel kann davon ausgegangen werden, dass dieser von der Autorenschaft gewählt wurde und somit bewusst ein Einbringen in den Heterogenitätsdiskurs angestrebt wird. Im Gegensatz dazu verfügt der Heterogenitätsbegriff im Abstract oder in der Verschlagwortung des Beitrags über eine geringere Priorität, da beide nicht zwingend auf die Autorenschaft zurückgehen und somit nicht mit einer entsprechenden Absicht in Zusammenhang stehen müssen. Im Rahmen dieser Untersuchung wird eine systematische Forschungsreview angestrebt. Systematische Reviews zeichnen sich gemäss Petticrex und Roberts (2006, zitiert nach Steinke & Badura, 2011) durch folgende Merkmale aus: Revue suisse des sciences de l’éducation 35 (1) 2013 39 Thema - präzise, operationalisierte Fragestellung - definierte Ein- und Ausschlusskriterien, die zu Beginn der Recherche festgelegt werden - eine systematische und umfassende Recherche, die sicherstellt, dass alle relevanten Studien zu der untersuchten Fragestellung berücksichtigt werden - strukturierte und transparente Darstellung der Ergebnisse und insbesondere der Vorgehensweise Um die Fragestellung beantworten zu können, anerbietet sich eine Eingrenzung der Recherche auf den deutschsprachigen Raum. Somit werden Ergebnisse hervorgebracht, die für den Gebrauch des Heterogenitätsbegriffs im schweizerischen, im deutschen und im österreichischen Forschungskontext aussagekräftig sind. Als Datengrundlage wird die deutschsprachige FIS Bildung Literaturdatenbank ausgewählt. Sie repräsentiert die für den deutschsprachigen Raum wichtigste und umfangreichste Metadatenbank. In die Datenbank werden Artikel aus ca. 430 Fachzeitschriften, Monografien, Beiträge aus Sammelwerken und elektronische Dokumente aus Datenbankbeständen von ca. 30 Dokumentationseinrichtungen in Deutschland, der Schweiz und Österreich einbezogen (Fachportal Pädagogik, 2012). Die Recherchestrategie charakterisiert sich dadurch, dass Beiträge ausgewählt werden, die im Titel oder Untertitel den Begriff ‚Heterogenität‘ mitführen und zudem eine empirische Studie darstellen, die von der Urheberschaft selbst publiziert wurde. Um sicherzugehen, dass es sich beim ausgewählten Dokument auch wirklich um eine empirische Studie handelt, wurden aus dem Schlagwortregister alle ähnlichen oder abgewandelten Begriffe zu ‚Empirie‘, ‚Untersuchung‘, ‚Forschen‘ und ‚Studie‘ einbezogen und mit der Suche nach dem Begriff ‚Heterogenität‘ im Titel und im Untertitel verknüpft. Thematisch berücksichtigt wurden Beiträge aus dem Schulumfeld, in welchen sich der Begriff Heterogenität auf die Schülerinnen und Schüler bezieht. Bezüglich der Schulstufen wurden Studien der Primarstufe, der Sekundarstufe I und der Sekundarstufe II (Gymnasium, Berufsbildung) aufgenommen. Empirische Studien zur Heterogenität von Studierenden und Erwachsenen werden nicht einbezogen. Themen, die Kinder und Jugendliche betreffen, jedoch nicht klar erkennbar mit dem schulischen Kontext in Verbindung stehen (z.B. Mädchenarbeit, Medienkonsum) werden ausgeschlossen. Nebst den inhaltlichen Merkmalen zeichnet sich die durchgeführte Recherche durch folgende Formalitäten aus: - Einbezug aller Dokumentationstypen: Monografien, Sammelwerksbeiträge und Zeitschriftenaufsätze. Das ursprüngliche Vorhaben, nur Zeitschriftenartikel aufzunehmen, die ein Peer-Review-Verfahren durchlaufen haben, wurde u.a. aus dem Grund wieder fallen gelassen, als pädagogische Forschungsbeiträge nicht ausschliesslich in Zeitschriften publiziert werden, sondern zu 40 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013 Thema einem grossen Teil in Sammelwerken Aufnahme finden. - Ausgeschlossen werden publizierte Vorträge und Publikationen aus nicht von einem Fachpublikum rezipierten Zeitschriften wie z.B. dem ‚Schulblatt Aargau‘. - Bei mehreren Beiträgen aus dem gleichen Forschungsprojekt wurde die jeweils aktuellste Publikation in die Analyse aufgenommen. - Es fand eine Eingrenzung auf die Sprache Deutsch statt. - Zeitlich wurde keine Beschränkung vorgenommen. Insgesamt ergab die Recherche 162 Treffer (Stichtag der Suche: 15. Juni 2012). Sämtliche Beiträge wurden auf alle genannten Selektionskriterien hin überprüft. Viele Beiträge wurden ausgegrenzt, da sie nicht von der Urheberschaft der Studie selber publiziert wurden1. Übrig blieben 21 Beiträge, die allen Kriterien entsprachen und somit als Datengrundlage für die vorzunehmende Analyse herangezogen wurden. Die ausgewählten Studien werden dabei keiner Qualitätsprüfung unterzogen, sondern lediglich hinsichtlich der interessierenden Inhaltsdimensionen analysiert. Ergebnisse Die 21 Beiträge wurden zwischen 2001 und 2011 publiziert. Es handelt sich um 3 Dissertationen aus den Jahren 2009, 2010 und 2011, 11 Sammelwerksbeiträge, von denen 2 im gleichen Werk erschienen sind, und um 7 Zeitschriftenaufsätze. 2 Beiträge stammen aus Österreich, 6 aus der (Deutsch-)Schweiz und 13 aus Deutschland. Auffallend ist hier, dass eine relativ geringe Zahl empirischer Studien den Begriff Heterogenität im Titel bringt. Es fehlen z.B., ausser der IGLU-Studie, die wichtigen internationalen Studien wie die PISA-Studie oder Studien, die verschiedene Aspekte von Heterogenität ausdrücklich thematisieren (z.B. EDK-ost 4bis8). Dennoch ist die Datenbasis breit genug, um in einem explorativen Verfahren Aussagen zur Verwendung des Begriffs Heterogenität in empirischen Studien zu generieren. Allgemein fällt die unterschiedlich häufige Verwendung des Begriffs Heterogenität innerhalb der Beiträge auf. Neben der Verwendung im Titel reicht die Spannweite von einer zweimaligen Nennung des Begriffs im Text (z.B. Allemann-Ghionda, Auernheimer & Grabbe, 2006) bis zu einem Versuch der Begriffsbestimmung (Grunder, 2009) und näheren Erläuterungen des Begriffs (Schründer-Lenzen, 2008). Der (Unter-)Titel des Texts gibt dabei teilweise schon Auskunft über die behandelten Heterogenitätskategorien – z.B. «Lernentwicklung in leistungshomogenen und -heterogenen Schulklassen» (Bos & Scharenberg, 2010). Bei anderen Beiträgen sind die verwendeten Heterogenitätskategorien aus dem Titel nicht ersichtlich wie z.B. in «Homogene oder heterogene Schulen oder Klassen?» (Wirszing & Weichert, 2008). Revue suisse des sciences de l’éducation 35 (1) 2013 41 Thema Thematisch eingeführt werden die Beiträge häufig, wie dies Budde (2012) auch bereits angemerkt hat, mit dem Hinweis auf die PISA-Studien (z.B. Schneider, 2009), das stark selektive Schulsystem im deutschsprachigen Raum (z.B. Schratz, Schwarz & Westfall-Greiter, 2011) oder mit dem Hinweis auf die grosse und stetig steigende Heterogenität der Schülerschaft (z.B. Schenz, 2011). Aus den einzelnen Beiträgen geht die grobe Kategorisierung in die folgenden Themengebiete hervor: 1. Schülerinnen und Schüler: Heterogenität in der Schule. 2. Unterricht: Forschungsstudien (Interventionsstudien, Evaluationen) zu didaktischen Methoden/pädagogischen Handlungsweisen. 3. Lehrpersonen: Unterrichten in heterogenen Klassen; Einstellungen der Lehrpersonen. Die drei Themengebiete werden im Folgenden mit Hilfe der analysierten Beiträge genauer vorgestellt und konkretisiert. Schülerinnen und Schüler Unterschiede zwischen den Schülerinnen und Schülern an der Schule stehen hier im Vordergrund. Die acht Beiträge zu diesem Themengebiet lassen sich verschiedenen Ebenen zuordnen. Eine Zuordnung kann hinsichtlich der jeweils obersten Analyse- und Vergleichsebene unternommen werden, nämlich auf Ebene des Individuums, der Schulklasse, der Schule oder auf Ebene eines Ländervergleichs. Individuum: Zwei Studien untersuchen die Lernprozesse einzelner Individuen. Die Mikroebene von Lernprozessen und die interindividuellen Unterschiede stehen hier im Vordergrund. Die qualitativ ausgerichtete Studie von Schratz et al. (2011) untersucht die Bedeutungen von Lernerfahrungen für die Lernenden. Schneider (2009) ermittelt mit einer Längsschnittstudie die literale Resilienz von Kindern mit ungünstigen Lernbedingungen. Die Studie kontrolliert bei den Schülerinnen und Schülern die Merkmale Geschlecht, Sozialstatus, kognitive Grundfähigkeiten, Migrationshintergrund und untersucht im Besonderen deren Leseleistung. Der Begriff Heterogenität wird in beiden Studien im Titel erwähnt, ohne dass im Text darauf Bezug genommen wird. Schratz et al. (2011) zeigen am Beispiel eines Mädchens mit Migrationshintergrund die Erfassung personaler Bildungsprozesse auf. Insgesamt wurden 48 Schülerinnen und Schüler untersucht, von denen angenommen wird, dass sie einer heterogenen Gruppe entstammen, da es «aufgrund der demographischen, wirtschaftlichen und gesellschaftlichen Entwicklungen zu einer starken Durchmischung der Schülerinnen und Schüler an allen Schularten» (Schratz et al., 2011, S. 26) kommt. Die Heterogenität der Schülerschaft wird hier implizit vorausgesetzt, ohne ein genaueres Augenmerk auf deren Ausprägungen zu werfen. 42 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013 Thema Schulklasse: Die Schulklasse ist eine durch das Schulsystem gegebene Gruppierung von Schülerinnen und Schülern, die gemeinsam beschult werden. Dementsprechend häufig wird die Schulklasse als Bezugssystem und Referenzpunkt für Vergleiche herangezogen. Die Schülerinnen und Schüler werden innerhalb ihrer Schulklassen untereinander verglichen oder es finden Vergleiche zwischen den Schulklassen statt. Die Altersheterogenität ist Thema der einen Studie, welche die Zusammensetzung von in Berufsschulen ‚natürlich‘ vorkommenden altersheterogenen Lerngruppen untersucht (Reinhardt & Rosen, 2004). Der Begriff Altersheterogenität wird hier in Abgrenzung zum Begriff Altershomogenität verwendet. Eine einzelne Studie aus der Integrationsforschung befasst sich mit der Ausgrenzung von Schülerinnen und Schülern mit Behinderungsmerkmalen und dem Zusammenhang zu verschiedenen Schülermerkmalen (Huber, 2009). Die Untersuchung findet in Klassen statt, in welche ein Kind mit besonderem Förderbedarf integriert ist. In diesen Klassen werden alle Kinder gemeinsam unterrichtet. Insgesamt 11 Schülermerkmale (Intelligenz, Schulleistung, Konzentration, Motivation, Selbstständigkeit, Sozialkompetenz, Alter, sozialer Rückzug, Aggressivität, Belastbarkeit und Selbstreflexion) werden erfasst und normiert. Die Varianz dieser Merkmale bildet die Lerngruppenheterogenität in den einzelnen Klassen ab und wird mit anderen Schulklassen (insgesamt 30 Schulklassen) verglichen. Auf den Begriff Heterogenität wird explizit Bezug genommen. «Heterogenität ist ein abstrakter Begriff, der hier als möglichst vielfältige und häufige Abweichung von einer Gruppennorm betrachtet wird» (Huber, 2009, S. 181). Der Begriff der Norm wird hier im Sinne der häufigsten Ausprägung verwendet. Eine weitere Untersuchung, die einzelne Schulklassen miteinander vergleicht, ist die KILIA-Studie von Martschinke und Kammermeyer (2003). Fragestellungen sind: Wie sehen Klassen im Anfangsunterricht bezüglich der leistungsund persönlichkeitsspezifischen Lernvoraussetzungen Leistung (phonologisches Bewusstsein, Selbstkonzept im Schriftspracherwerb, Aspekte der Persönlichkeit wie z.B. leistungsbezogene Angst, Selbstkonzept usw.) und Kontextvariablen wie z.B. der Klassengrösse und der Elternunterstützung aus? Welche Unterrichtsmassnahmen (Differenzierung, Freiheitsspielräume) werden von den Lehrkräften ergriffen und wie hängen diese mit der Heterogenität der vormals aufgezeigten Merkmale der jeweiligen Klasse zusammen? Die Studie gilt es hervorzuheben, da sie die Ebene der Schulklasse mit derjenigen der Lehrperson verknüpft und aufzuzeigen versucht, wie die Lehrperson auf die im Anfangsunterricht vorgefundenen Bedingungen, d.h. die Heterogenität der Schülerschaft, reagiert. Die Komplexität des Unterrichts wird mit verschiedenen Heterogenitätskategorien in Zusammenhang gebracht. Eine weitere Studie, die diese beiden Ebenen miteinander verknüpft, ist die explorative Studie von Eckhart (2002), welche die Unterrichtsformen fokussiert (genauer erläutert und eingeordnet wird die Studie unter dem Thema Unterricht, s. unten). Revue suisse des sciences de l’éducation 35 (1) 2013 43 Thema Die weiteren Beiträge auf der Ebene Schulklasse behandeln die Klassenunterschiede zwischen verschiedenen Schulformen. Anders als in den vorangehenden Beiträgen wird nicht mit Absolutwerten gerechnet, sondern mit den Unterschieden zwischen vermeintlich homogeneren und heterogeneren Schulklassen. Im Forschungsbeitrag von Amberg und Posch (2004) werden die nach dem herkömmlichen Schulmodell gebildeten altershomogenen Schulklassen bezüglich ihrer Leistung und dem Sozialverhalten mit altersgemischen Modellen wie der flexiblen Schuleingangsphase und den Mehrstufenklassen verglichen. Schule/Schulform: Wirszing und Weichert (2008) vergleichen zwei Schulen miteinander bezüglich der Persönlichkeitsentwicklung ihrer Abgängerinnen und Abgänger. Die «sehr heterogene» Schule unterscheidet sich von der «normal heterogen und homogenisierenden» (Wirszing & Weichert, 2008, S. 11) Schule durch ein multikulturelles Einzugsgebiet und «baut zudem auf ein Schulkonzept, welches darüber hinaus Heterogenität über das natürliche Mass hinaus ‚herstellt’» (ebd., S. 11), dies mit jahrgangsübergreifenden Integrationsklassen und gezieltem sozialen Bezugsgruppenwechsel. Die homogene Schule weist die «klassische Heterogenität einer Jahrgangsschule» (ebd., S. 11) auf. Ländervergleich: Aus einer soziologischen Perspektive wird im letzten Beitrag die Verschiedenheit zwischen den Schülerinnen und Schülern betrachtet. Im Beitrag zur IGLU-Studie von Schwippert, Bos und Lankes (2004) wird anhand von Schüler- und Elternbefragungen der Zusammenhang zwischen den Leistungen in Lesen, Mathematik und den Naturwissenschaften mit der Sozialschicht, dem Migrationshintergrund und dem Geschlecht ermittelt. Die Ergebnisse der Studie können zwar auch für den Vergleich von einzelnen Schulklassen verwendet werden, hier steht jedoch der Vergleich mit anderen Ländern im Vordergrund. Unterricht Die folgenden sieben Beiträge fokussieren den Unterricht und untersuchen die Anwendung von einzelnen Unterrichtsmethoden, welche besonders für eine heterogene Schülerschaft gedacht sind. Anhand von Interventionsstudien und/ oder Evaluationen werden didaktische Methoden und pädagogische Handlungsweisen überprüft. Kontext dieser Untersuchungen ist die Kritik an der äusseren Differenzierung und der damit verbundenen Aussonderung von Kindern, die besonderer Förderung bedürfen (siehe z.B. Eckhart, 2002). In diesem Zusammenhang werden Unterrichtsmethoden vorgestellt, die einen integrativen Unterricht erlauben und insbesondere auch Schulleistungsschwachen und/oder Kindern mit Migrationshintergrund zugutekommen sollen. Eine Studie, die das Unterrichtsgeschehen aufnimmt, ist diejenige von Hugener, Krammer und Pauli (2008). Anhand von gefilmten Mathematiklektionen wird das Differenzierungsrepertoire von einzelnen Lehrpersonen erfasst. Eckhart (2002) beschreibt die Ergebnisse einer explorativen Studie und 44 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013 Thema inwieweit sich integrative didaktisch-methodische Vorgehensweisen auf den Lernerfolg sowie auf die soziale Integration von in- und ausländischen Kindern mit Schulleistungsschwächen auswirken. Hierzu wurden bei den Schülerinnen und Schülern die soziale Integration und die Intelligenz erfasst. Bei den Lehrpersonen wurden Angaben zur Verwirklichung von integrativen Unterrichtsformen (innere Differenzierung, zieldifferenziertes Lernen, fächerübergreifendes Lernen, offene Lernformen) eingeholt. Wie bereits im vorangehenden Kapitel erwähnt, wird hier die Ebene der Schülerinnen und Schüler mit derjenigen der Lehrpersonen verknüpft. Drei Interventionsstudien mit anschliessender Evaluation untersuchen erstens kooperative Lernformen hinsichtlich Leistung und ethischen Hintergrunds von heterogenen Lerngruppen und deren integrationsfördernde Wirkung (Lanphen, 2001), zweitens eine für Schülerinnen und Schüler mit ungünstigen Lernvoraussetzungen2 gestaltete adaptive Unterrichtseinheit (kognitive Strukturierung, Peer Learning und formatives Assessment) (Warwas & Kunter, 2011) und drittens Fördermassnahmen (FörMig), welche insbesondere für Schülerinnen und Schüler mit Migrationshintergrund gedacht sind (Schründer-Lenzen, 2008). Eine weitere Studie ist diejenige von Wischer (2001), welche die Absolventinnen und Absolventen der Laborschule Bielefeld analysiert. An der Laborschule werden leistungsheterogene Gruppen bis zum Abschluss des 9. Schuljahres unterrichtet. Es wird untersucht, wie erfolgreich die innere Differenzierung des Unterrichts aus Sicht der Schülerinnen und Schüler durchgeführt wird, und ob, insbesondere bei den Leistungsschwächeren, ein Effekt auf das Selbstkonzept und das Wohlbefinden nachweisbar ist. Ein Dissertationsprojekt befasst sich mit der Herausarbeitung von Best-Practice für die Basisstufe. Mittels teilnehmender Beobachtung werden günstige pädagogische Handlungsweisen abgeleitet, welche insbesondere auch benachteiligten Kindern, d.h. Kindern, deren Erstsprache nicht Deutsch ist und/oder die aus einem bildungsfernen Milieu stammen, gerecht werden (Urech, 2010). Auffallend an den zum Thema Unterricht eingeordneten Studien ist die Verwendung der Heterogenitätskategorien. Bei der Studie von Hugener et al. (2008), welche das Unterrichtsgeschehen bezüglich der Methoden der inneren Differenzierung thematisiert, werden Heterogenitätskategorien nicht explizit erwähnt. Bei den übrigen Studien zum Unterricht stehen die Merkmale Migrationshintergrund und Leistungsschwäche in Kombination mit einer für alle Schülerinnen und Schüler gedachten Unterrichtsmethode im Fokus des Interesses. Die letztgenannte Untersuchung von Urech (2010) ist als Ausnahme zu bezeichnen, da es um die Entwicklung von pädagogischem Handlungswissen (Best-Practice) für eine neu organisierte, ‚heterogenitätsfördernde‘ Schulform geht. Charakteristiken, die einen gelingenden Unterricht in der Basisstufe ausmachen, werden herausgearbeitet. Revue suisse des sciences de l’éducation 35 (1) 2013 45 Thema Lehrpersonen Bei Beiträgen, die dieser Thematik zugeteilt wurden, liegt der Fokus auf der Lehrperson und deren Umgang mit Heterogenität in Schule und Unterricht. Leitend ist jeweils die Frage, wie Lehrpersonen mit Heterogenität oder der schulischen Selektionsfunktion umgehen. Die Heterogenitätskategorien werden hierbei nicht genauer fokussiert, sondern es wird davon ausgegangen, dass die Schülerschaft per se heterogen ist. Die subjektiven Theorien der Lehrpersonen stehen im Vordergrund. In sämtlichen Beiträgen wurden Lehrpersoneninterviews als Erhebungsmethode eingesetzt. Den Lehrpersonen obliegt die Aufgabe der Förderung wie auch der Auslese. Der Aspekt der Auslese steht in einer soziologisch und einer pädagogisch ausgerichteten Studie im Vordergrund. In der soziologisch ausgerichteten Studie werden vor dem Hintergrund der Reproduktion von Ungleichheit nach Bourdieu Lehrpersonen nach dem Spannungsfeld zwischen Förderung und Selektion sowie ihrem Heterogenitätsverständnis befragt (Streckeisen, Hänzi & Hungerbühler, 2009). In der pädagogisch ausgerichteten Studie von Allemann-Ghionda et al. (2006) wird mit explorativen Interviews die diagnostische Kompetenz von Lehrpersonen im Hinblick auf Kinder mit Migrationshintergrund untersucht. Das professionelle Verständnis der Lehrpersonen im Umgang mit einzelnen Heterogenitätskategorien wird in drei Studien erfasst. Dies mit Blick auf (hoch) begabte Kinder und Jugendliche bei Gymnasiallehrpersonen (Schenz, 2011), in Bezug auf kulturelle Heterogenität (Edelmann, 2006) und in einem Dissertationsprojekt zur vergleichenden Betrachtung von deutschen und französischen Lehrpersonen zu ihrem Umgang mit soziokultureller Vielfalt (Sievers, 2009). Zusammenschau Mit den angewendeten Methoden konnten empirische Studien identifiziert und analysiert werden, welche sich explizit mit Heterogenität befassen und sich bewusst in den Heterogenitätsdiskurs einbringen. Die Themenbereiche und aufgegriffenen Heterogenitätskategorien wurden aufgeschlüsselt und werden im Folgenden zusammenfassend dargestellt. Eine wichtige Erkenntnis ist, dass je nach Themenbereich andere Heterogenitätskategorien aufgegriffen werden. Allerdings ist die Ausdifferenzierung in verschiedene Heterogenitätskategorien nicht in allen Themenbereichen von gleicher Relevanz. Relativierend wird angemerkt, dass eine (weit grösser gefasste) Analyse unter Einbezug aller empirischen Studien, welche sich mit Heterogenität(skategorien) befassen, ev. zu anderen Ergebnissen gekommen wäre. Die Datenbasis von 21 Studien erlaubt im Sinne einer explorativen Auswertung, Aussagen zur thematischen Breite und den verwendeten Kategorien zu machen. Des Weiteren sind die konkreten Ergebnisse der untersuchten Studien und insbesondere das Ensemble der in den einzelnen Beiträgen erforschten Heterogenitätskategorien 46 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013 Thema nicht Bestandteil des vorliegenden Beitrags, könnten jedoch Anlass für an diese Studie anschliessende Forschungsfragen sein. In geraffter Form werden die ermittelten Themenbereiche der empirischen Studien wie folgt dargestellt: Tabelle 1: Themenbereiche der 21 untersuchten empirischen Studien Schülerinnen und Schüler (9) Unterricht (7) Lehrpersonen (5) Analyseebene: Unterrichtsmethoden anwenden, evaluieren, entwickeln (5) Subjektive Theorien der Lehrpersonen zu Selektionsfunktion; Umgang mit und Einstellung zu (einzelnen) Heterogenitätskategorien Individuum (2) Klasse (5) Schule (1) Ländervergleich (1) Häufigkeit/Anwendung der inneren Differenzierung durch Lehrpersonen (2) In Klammern steht die Anzahl der Beiträge. Die aus der systematischen Recherche hervorgegangenen Beiträge liessen sich eindeutig einem Themengebiet zuordnen (Ausnahmen: die Studien von Martschinke und Kammermeyer, 2003 und Eckhart, 2002). Die in der Einleitung skizzierte, inhaltliche Vielschichtigkeit des Begriffs Heterogenität kommt in den ausgewählten empirischen Studien zum Tragen. Die Konstruiertheit und Relativität des Begriffs Heterogenität (vgl. Lang et al., 2010) zeigt sich insbesondere im Themenbereich Schülerinnen und Schüler, und zwar anhand der verschiedenen Analyseebenen, die vom Individuum bis hin zum Ländervergleich reichen. Die Unterschiede der Schülerinnen und Schüler resp. das Ausmass der Heterogenität wird in Relation zur jeweiligen Analyseebene beurteilt. Es sei an dieser Stelle an den Beitrag von Wirszing und Weichert (2008) erinnert, die eine «sehr heterogene» Schule mit einer «normal» heterogenen Schule vergleichen. In anderen Studien (z.B. Huber, 2009; Martschinke & Kammermeyer, 2003) wurden in als ‚normal‘ heterogen geltenden Klassen/ Schulen Unterscheidungsmerkmale der einzelnen Schülerinnen und Schüler untersucht. Dabei wird auf den Umstand aufmerksam gemacht, dass scheinbar homogenisierte Gruppen auch heterogen sind. Bezüglich der untersuchten Heterogenitätskategorien bestätigte sich die in der theoretischen Einleitung aufgezeigte, zentrale Rolle der Leistungsheterogenität. Die Leistung bildete in den meisten Studien zusammen mit weiteren Aspekten, z.B. Persönlichkeit (Martschinke & Kammermeyer, 2003) oder Sozialverhalten (Amberg & Posch, 2004) die interessierende Zielgrösse oder erwies sich, wie in der Studie von Huber (2009), als zentrale Variable zur Aufklärung von Lerngruppenheterogenität. Ausnahmen bildeten die Studien von Reinhardt und Rosen (2004) zur Altersheterogenität, sowie Wirszing und Weichert (2008), welche die Persönlichkeitsentwicklung in Abhängigkeit verschiedener Schulformen analysierten. Das in der Einleitung von Krüger-Potratz (2011) im Heterogenitätsbegriff erwähnte Mitschwingen von Schwierigkeiten und Konflikten lässt sich in den Beiträgen zum Themenbereich Unterricht und Lehrpersonen am deutlichsten Revue suisse des sciences de l’éducation 35 (1) 2013 47 Thema erkennen. Bei Beiträgen, die diesen beiden Bereichen zugeordnet wurden, blieben Heterogenitätskategorien und die genaue Bestimmung, worin die Unterschiedlichkeiten der Schülerinnen und Schüler bestehen, im Hintergrund. Dass die Schülerschaft heterogen ist, wurde vorausgesetzt und dahingehend die Umsetzung von Unterricht sowie die Sichtweise der Lehrpersonen fokussiert. Insbesondere wurden von der jeweiligen Norm abweichende Heterogenitätsausprägungen wie Migrationshintergrund und die schulische Leistungsschwäche thematisiert sowie der Umgang mit entsprechenden Normabweichungen im Rahmen selektiver, aussondernder Schulsysteme kritisiert (z.B. Eckhart, 2002). Eine Ausnahme bildete die Studie von Schenz (2011), die Vorstellungen von Lehrpersonen über (Hoch-)Begabte thematisiert. Hervorgehoben seien zum Schluss die KILIA-Studie von Matschinke und Kammermeyer (2003) und die explorative Studie von Eckhart (2002), welche die Ebene der Schülerinnen und Schüler und diejenige der Lehrpersonen miteinander verschränken. Beide Studien verbinden die Heterogenitätskategorien auf Ebene der Schülerinnen und Schüler mit der Ebene des Unterrichts und der Lehrperson. Unterschiedliche Heterogenitätskategorien werden dadurch kombiniert und zudem mit der Komplexität des Unterrichts in Zusammenhang gebracht. Studien mit entsprechend kombinierten Analyseebenen sind gegenüber Studien, die einzelne Ebenen isoliert untersuchen, aufgrund der dargestellten Ergebnisse klar in der Minderheit, jedoch für zukünftige Forschungsprojekte erwünscht. Anmerkungen Bspw. wird eine Publikation, die einen Überblick über verschiedene Studien leistet (und den Begriff Heterogenität im Titel führt), in der Analyse nicht berücksichtigt. 2 Der Beitrag ist sehr kurz gehalten, woraus keine Angaben zu einer genaueren Bestimmung dieser ungünstigen Lernvoraussetzungen entnommen werden können. 1 Literatur Allemann-Ghionda, C., Auernheimer, G. & Grabbe, H. (2006). Beobachtung und Beurteilung in soziokulturell und sprachlich heterogenen Klassen. Die Kompetenzen der Lehrpersonen. In C. Allemann-Ghionda (Hrsg.), Kompetenzen und Kompetenzentwicklung von Lehrerinnen und Lehrern. (Zeitschrift für Pädagogik, Beiheft 51, S. 250-266). Weinheim: Beltz. Amberg, I. & Posch, V. (2004). Schulleistungen und Sozialverhalten in altersheterogenen und altershomogenen Lerngruppen auf der Grundstufe. Eine empirische Untersuchung auf der 2. Schulstufe an Wiener Volksschulen. Erziehung & Unterricht, 154 (1-2), 88-114. Bos, W. & Scharenberg, K. (2010). Lernentwicklung in leistungshomogenen und -heterogenen Schulklassen. In W. Bos, E. Klieme & O. Koller (Hrsg.), Schulische Lerngelegenheiten und Kompetenzentwicklung (S. 173-194). Münster u.a.: Waxmann. Budde, J. (2012). Die Rede von der Heterogenität in der Schulpädagogik. Diskursanalytische Perspektiven. Forum Qualitative Sozialforschung/Forum: Qualitative Social ResearchOnline, 13 (2), Art. 16. Zugriff am 28.08.2012 unter http://www.qualitative-research.net/ index.php/fqs/article/view/1761/3358 48 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013 Thema Diaz-Bone, R. (2006). Zur Methodologisierung der Foucaultschen Diskursanalyse. Historical Social Research, 31 (2), 243-274. Eckhart, M. (2002). Unterricht in heterogenen Schulklassen. Theoretische Überlegungen und explorative Untersuchung unter besonderer Berücksichtigung von Kindern mit Schulleistungsschwächen. Vierteljahresschrift für Heilpädagogik und ihre Nachbargebiete, 71 (2), 152-172. Edelmann, D. (2006). Pädagogische Professionalität im transnationalen sozialen Raum. Eine Studie über Sichtweisen und Erfahrungen von Primarlehrpersonen in Bezug auf die kulturelle Heterogenität ihrer Schulklassen. In C. Allemann-Ghionda (Hrsg.), Kompetenzen und Kompetenzentwicklung von Lehrerinnen und Lehrern (Zeitschrift für Pädagogik, Beiheft 51, S. 235-249). Weinheim u.a.: Beltz. Fachportal Pädagogik (2012). Policy der FIS Bildung Literaturdatenbank. Zugriff am 24.04.2012 unter http://www.fachportal-paedagogik.de/fis_bildung/fis_policy.html Huber, C. (2009). Soziale Ausgrenzung in der Integration von Schülern mit sonderpädagogischem Förderbedarf. Zusammenhang von Persönlichkeit, Gruppenheterogenität und sozialer Ausgrenzung. Empirische Pädagogik, 23 (2), 170-190. Hugener, I., Krammer, K. & Pauli, C. (2008). Kompetenzen der Lehrpersonen im Umgang mit Heterogenität: Differenzierungsmassnahmen im Mathematikunterricht. In M. Gläser-Zikuda & J. Seifried (Hrsg.), Lehrerexpertise. Analyse und Bedeutung unterrichtlichen Handelns (S. 47-66). Münster u.a.: Waxmann. Krüger-Potratz, M. (2011). Intersektionalität. In H. Faulstich-Wieland (Hrsg.), Umgang mit Heterogenität und Differenz (S. 183-200). Baltmannsweiler: Schneider Verlag. Lang, E., Grittner, F., Rehle, C. & Hartinger, A. (2010). Das Heterogenitätsverständnis von Lehrkräften im jahrgangsgemischten Unterricht der Grundschule. In J. Hagedorn, V. Schurt, C. Steber & W. Waburg (Hrsg.), Ethnizität, Geschlecht, Familie und Schule (S. 315-331). Wiesbaden: VS Verlag für Sozialwissenschaften. Lanphen, J. (2011). Kooperatives Lernen und Integrationsförderung. Eine theoriegeleitete Intervention in ethnisch heterogenen Schulklassen. Münster u.a.: Waxmann. Dissertation, Philipps-Universität Marburg. Leiprecht, R. & Lutz, H. (2006). Intersektionalität im Klassenzimmer: Ethnizität, Klasse, Geschlecht. In R. Leiprecht & A. Kerber (Hrsg.), Schule in der Einwanderungsgesellschaft. Ein Handbuch (S. 218-234). Schwalbach: Wochenschau Verlag. Lindemann, S., Schwanenberg, J. & Bos, W. (2011). Heterogenität im Lichte rezenter Schulleistungsstudien. In R. Hinz & R. Walthes (Hrsg.), Verschiedenheit im Diskurs (S. 74-96). Tübingen: Narr Francke Attempto Verlag. Martschinke, S. & Kammermeyer, G. (2003). Jedes Kind ist anders. Jede Klasse ist anders. Ergebnisse aus dem KILIA-Projekt zur Heterogenität im Anfangsunterricht. Zeitschrift für Erziehungswissenschaft , 6 (2), 257-275. Prengel, A. (2007). Im Schwebezustand: Schulen und transgressive Lebenswelten. Ansätze in Forschung und Lehre. Schweizerische Zeitschrift für Bildungswissenschaften, 29 (3), 363-378. Reinhardt, C. & Rosen, E. (2004). Einfluss der Altersheterogenität auf Kleingruppenarbeit. In W. Bos (Hrsg.), Heterogenität (S. 245-252). Münster, Westfalen u.a.: Waxmann. Schenz, C. (2011). Lehrerinnenexpertise im Umgang mit Heterogenität. In C. Schenz, S. Rosebrock & M. Soff (Hrsg.), Von der Begabtenförderung zur Begabungsgestaltung. Vom kreativen Umgang mit Begabungen in der Mathematik (S. 11-25). Berlin u.a.: LIT. Schneider, H. (2009). Soziale Heterogenität und Leseleistung. Wenn Schriftaneignung trotzdem gelingt. In H.-U. Grunder & A. Gut (Hrsg.), Zum Umgang mit Heterogenität in der Schule (Bd. 1, S. 67-81). Baltmannsweiler: Schneider-Verlag Hohengehren. Schratz, M., Schwarz, J. F. & Westfall-Greiter, T. (2011). Personale Bildungsprozesse in heterogenen Gruppen. Zeitschrift für Bildungsforschung, 1 (1), 25-39. Revue suisse des sciences de l’éducation 35 (1) 2013 49 Thema Schründer-Lenzen, A. (2008). Bedingungen guten Unterrichts für heterogene Lerngruppen – Befunde der empirischen Forschung. In H. Kiper, S. Miller, C. Palentien. & C. Rohlfs (Hrsg.), Lernarrangements für heterogene Gruppen – Lernprozesse professionell gestalten (S. 106-126). Bad Heilbrunn: Klinkhardt. Schwippert, K., Bos, W. & Lankes, E.-M. (2004). Heterogenität und Chancengleichheit am Ende der vierten Jahrgangsstufe in den Ländern der Bundesrepublik Deutschland und im internationalen Vergleich. In W. Bos, E.-M. Lankes, M. Prenzel, K. Schwippert, R. Valtin & G. Walther (Hrsg.), IGLU. Einige Länder der Bundesrepublik Deutschland im nationalen und internationalen Vergleich (S. 165-190). Münster, Westfalen: Waxmann. Sievers, I. (2009). Individuelle Wahrnehmung, nationale Denkmuster. Einstellungen deutscher und französischer Lehrkräfte zu Heterogenität im Unterricht (1. Auflage). Frankfurt, Main: Brandes & Apsel. Dissertation, Universität Hannover. Steinke, M. & Badura, B. (2011). Präsentismus. Ein Review zum Stand der Forschung. Zugriff am 25.04.2012 unter http://www.gesundheitsfoerderung.ch/pdf_doc_xls/d/betriebliche_ gesundheitsfoerderung/grundlagen_wissen/Praesentismus_2011.pdf Stöger, H. & Ziegler, A. (2012). Heterogenität und Inklusion im Unterricht. In L. Haag & S. Rahm (Hrsg.), Studienbuch Schulpädagogik. Bad Heilbrunn: Klinkhardt/UTB. Streckeisen, U., Hänzi, D. & Hungerbühler, A. (2009). Fördern, Selektion und der Umgang mit Heterogenität. Deutungsmuster von Lehrpersonen in soziologischer Hinsicht. In H.-U. Grunder & A. Gut (Hrsg.), Zum Umgang mit Heterogenität in der Schule (Bd. 1, S. 128-147). Baltmannsweiler: Schneider-Verlag Hohengehren. Urech, C. (2010). Die heterogene Schulklasse. Fallstudien zum pädagogischen Handeln in Basisstufen. Zürich: Rüegger. Dissertation, Universität Freiburg. Warwas, J. & Kunter, M. (2011). Individuelle Förderung in heterogenen Klassen. Im Projekt ,Igel’ werden adaptive Lernumgebungen in der Grundschule evaluiert. Forschung Frankfurt, 29 (1), 14-15. Wenning, N. (2007). Heterogenität als Dilemma für Bildungseinrichtungen. In S. Boller, E. Rosowski & T. Stroot (Hrsg.), Heterogenität in Schule und Unterricht. Handlungsansätze zum pädagogischen Umgang mit Vielfalt (S. 21-31). Weinheim und Basel: Beltz. Wirszing, D. & Weichert, W. (2008). Homogene oder heterogene Schulen oder Klassen? Grundschulmagazin, 76 (3), 11-12. Wischer, B. (2001). Lernen in heterogenen Gruppen aus Schülersicht. Ausgewählte Ergebnisse aus der Absolventenstudie. In I. Demmer-Dieckmann & B. Struck (Hrsg.), Gemeinsamkeit und Vielfalt. Pädagogik und Didaktik einer Schule ohne Aussonderung (S. 227-246). Weinheim; München: Juventa. Wischer, B. (2007). Wie sollen LehrerInnen mit Heterogenität umgehen? Über «programmatische Fallen» im aktuellen Reformdiskurs. Die deutsche Schule, 99 (4), 422-433. Wischer, B. (2009). Der Diskurs um Heterogenität und Differenzierung. Beobachtungen zu einem schulpädagogischen «Dauerbrenner». In B. Wischer & K.-J. Tillmann (Hrsg.), Erziehungswissenschaft auf dem Prüfstand. Schulbezogene Forschung und Theoriebildung von 1970 bis heute (S. 69-93). Weinheim, München: Juventa. Schlagworte: Heterogenität, Heterogenitätskategorien, empirische Studien 50 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013 Thema La notion d’hétérogénéité dans des études empiriques Résumé Lorsque le titre d’une étude empirique comprend la notion d’hétérogénéité, quels objets et quelles catégories d’hétérogénéité sont examinés? La recherche rapportée dans cet article contribue à répondre à cette question en s’appuyant sur une revue systématique et sur l’analyse de contenu d’un corpus d’études empiriques. À partir d’un ensemble de critères, 21 travaux ont été catégorisés dans l’une des trois thématiques génériques suivantes: 1. L’hétérogénéité d’une population d’élèves selon différentes catégories d’hétérogénéité et niveaux d’analyse. Il ressort que la catégorie d’hétérogénéité la plus fréquemment étudiée concerne celle liée à la performance des élèves. 2. L’implémentation, l’évaluation et le développement de méthodes d’enseignement intégratives. 3. Les théories subjectives des enseignants à propos de l’hétérogénéité des élèves. Mots-clés: Hétérogénéité, catégories d’hétérogénéité, revue d’études empiriques, analyse de contenu Il concetto di eterogeneità in studi empirici Riassunto Su quali argomenti e quali categorie di eterogeneità si concentrano le ricerche quando nel titolo di uno studio empirico viene utilizzato il termine «eterogeneità»? Questa domanda viene approfondita sulla base di una ricerca sistematica su banche dati e un’analisi dei contenuti di studi di ricerca. 21 studi, che possono essere suddivisi nei tre punti tematici cruciali di seguito riportati, hanno mostrato una corrispondenza con i parametri definiti. 1. L’eterogeneità degli studenti rispetto a differenti categorie di eterogeneità e livelli di analisi. In tale contesto, l’eterogeneità del rendimento è la categoria esaminata più spesso. 2. L’applicazione, la valutazione e l’evoluzione di metodi d’insegnamento integrativi. 3. Le teorie soggettive dei docenti rispetto all’eterogeneità dei propri studenti. Parole chiave: Eterogeneità, categorie di eterogeneità, studi empirici Revue suisse des sciences de l’éducation 35 (1) 2013 51 Thema The Notion of «Heterogeneity» in Empirical Studies Summary When the term «heterogeneity» occurs in the title of an empirical study, wich topics and wich heterogeneity categories are being studied? This question is explored by means of a systematic database research and content analysis of empirical studies. With respect to defined criteria, 21 studies are classified into the following three key topics: 1. Student heterogeneity with respect to different heterogeneity categories and levels of analysis. It appears that performance-related heterogeneity is the most frequently investigated category. 2. The implementation, evaluation and development of inclusive teaching methods. 3. Teachers’ subjective theories about students’ heterogeneity. Keywords: Heterogeneity, heterogeneity categories, empirical studies review 52 Schweizerische Zeitschrift für Bildungswissenschaften 35 (1) 2013
https://openalex.org/W3209444632	https://revistas.ucn.cl/index.php/estudios-atacamenos/article/download/4353/3921, http://www.scielo.cl/pdf/eatacam/v67/0718-1043-eatacam-67-23.pdf	es		Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu	Estudios atacameños	2,021	cc-by	16,145	Estudios Atacameños vol. 67 (2021) ARQUEOLOGÍA e4353 10.22199/issn.0718-1043-2021-0023 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu Appropiation and State control on high-altitude environments in Southern border of Tawantinsuyu Víctor Alberto Durán1 https://orcid.org/0000-0002-0534-0739 Sol Zárate Bernardi1 https://orcid.org/0000-0002-9124-1090 Lucía Yebra1 https://orcid.org/0000-0002-2049-199X Cecilia Frigolé1 https://orcid.org/0000-0002-2163-792X Gustavo Lucero1,2 https://orcid.org/0000-0002-4687-3929 Diego Andreoni3 https://orcid.org/0000-0002-9975-240X Diego Winocur4 https://orcid.org/0000-0001-8354-7818 Alejandra Gasco1 https://orcid.org/0000-0003-2670-1377 Erik Marsh1 https://orcid.org/0000-0003-2355-5415 Silvina Castro1 https://orcid.org/0000-0002-0868-1715 María Inés Zonana1 https://orcid.org/0000-0002-3318-5668 Ramiro Barberena1 https://orcid.org/0000-0002-4323-1197 Valeria Cortegoso1 https://orcid.org/0000-0003-3361-815X Laboratorio de Paleoecología Humana, Instituto Interdisciplinario de Ciencias Básicas, CONICET-Universidad Nacional de Cuyo. Mendoza, ARGENTINA. Email: duranvic2@gmail.com; zaratebernardisol@gmail.com; yebralucia@hotmail.com; cfrigole@gmail.com; soljandra@gmail.com; erik.marsh@gmail.com; silcastro24@hotmail.com; inezonana@gmail.com; ramidus28@gmail.com; vcortegoso@gmail.com 2 Departamento de Antropología, Facultad de Ciencias Sociales y Humanidades, Universidad Católica de Temuco, CHILE. Email: glucero18@gmail.com 3 División Arqueología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, CONICET. La Plata, Buenos Aires, ARGENTINA. Email: andreondieg@hotmail.com 4 Instituto de Estudios Andinos. Departamento de Ciencias Geológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires-CONICET. CABA, ARGENTINA. Email: diegowinocur@hotmail.com 1 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Resumen Se presentan resultados sobre técnicas constructivas y patrones de ocupación del espacio, cronología, tecnología lítica, tipología y tecnología cerámica, zooarqueología de camélidos, antracología y arte rupestre, correspondientes a un conjunto de sitios arqueológicos del Área Natural Protegida Laguna del Diamante (Mendoza, Argentina). Esta área se ubica en la cordillera Principal y vincula las cuencas altas de los ríos Diamante y Maipo. Los registros obtenidos corresponden al período de consolidación de la ocupación inca de la región y prueban que el Tawantinsuyu controló este sector de la cordillera para explotar una fuente de obsidiana y mantener en funcionamiento una red de tráfico especializado que vinculaba al valle central chileno con los valles orientales de Uco y Jaurúa. Por otra parte, las manifestaciones de arte rupestre halladas permiten inferir un proceso de sacralización y apropiación de este espacio dominado por el volcán Maipo y la laguna del Diamante. Palabras clave: arqueología, control inca, ambientes de altura, frontera meridional del Tawantinsuyu. Abstract This paper presents the results of constructive techniques, space occupation patterns, chronology, lithic technology, ceramic technology and typology, camelid zooarchaeology, anthracology and studies of rock art from a group of archaeological sites in the Laguna del Diamante Natural Protected Area (Mendoza, Argentina). This area is located in the Cordillera Principal of the Andes and connects the upper Diamante and Maipo River Basins. Chronologically, the archaeological data are from the consolidation of the Inca occupation in the region. The studies demonstrate that the Inca state controlled this sector of the Andes in order to exploit an obsidian source and maintain a specialized trade network that linked the Central Valley in Chile with the eastern valleys of Uco and Jaurúa. Additionally, the rock art motifs allow us to infer a process of sacralization and appropriation of this landscape, which is dominated by the Maipo Volcano and the Diamante Lake. Keywords: archaeology, Inca control, high altitude environments, Southern border of Tawantinsuyu. Recibido: 18 julio 2020. Aceptado: 10 diciembre 2020 2 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu El dominio incaico del valle central chileno y noroeste de la provincia de Mendoza, iniciado en las primeras décadas del siglo XV DC (Cornejo, 2014; Marsh, Kidd, Ogburn y Durán, 2017), generó cambios importantes en los modos de vida de las sociedades locales. Estudios históricos y arqueológicos muestran que, en ambas regiones vinculadas por la cordillera de los Andes, aumentó durante este período la producción agrícola mediante obras hidráulicas que permitieron un mejor manejo del agua (Michieli, 1994; Stehberg y Sotomayor, 2012), y posiblemente también la ganadera (Durán, Gasco, Paiva, Zonana y Barberena, 2020). Se hicieron explotaciones mineras de oro, plata y cobre (Cornejo, Miranda y Saavedra, 1999; Stehberg y Sotomayor, 2012) y mejoró sensiblemente la capacidad de transportar bienes e información a través de un sistema vial complejo (Stehberg, 1995; Bárcena, 1998). Para mantener el control sobre las sociedades locales, el Estado inca no solo utilizó la fuerza, sino que impuso también nuevas formas de organización social y creencias religiosas (Sánchez, 2004), destacándose el culto a los cerros o apus que incluyó, en ocasiones, sacrificios humanos (Schobinger, 2001). En lo referido al límite meridional de la expansión incaica, para la vertiente occidental de la cordillera de los Andes existe consenso acerca de que el control estatal directo/efectivo (político y económico) llegó hasta el río Maipo/Cachapoal, con una frontera fluctuante entre estos ríos y el Maule, que perduró hasta la conquista hispana (Silva, 1985; Stehberg, 1995). Para la vertiente oriental, en cambio, se mantienen dos posiciones diferentes. Una considera que el control efectivo de los incas solo llegó hasta el río Mendoza, fundamentándose en la ausencia de arquitectura y obras viales de origen incaico al sur de este río (Hyslop, 1984; Bárcena, 1992; Cahiza y Ots, 2005; Ots, 2009). Para explicar la presencia de otros restos materiales de clara filiación incaica en los valles de Uco y Jaurúa (cuenca pedemontana del río Tunuyán), se ha propuesto que los incas explotaron estos espacios a través de enclaves o colonias que tenían propósitos económicos específicos (Bárcena 1992; Cahiza y Ots 2005; Ots 2009). En contraposición, García (2011) asume que los incas mantuvieron un control económico y político de esos valles, que puede percibirse en la cultura material (la cerámica específicamente) y en las formas de organización sociopolítica de las sociedades que describen las fuentes históricas del primer siglo de contacto hispano-indígena. Al considerar más sólida la propuesta de García (2011), e incorporando los resultados arqueológicos publicados para las cuencas altas de los ríos Aconcagua, Mapocho y Maipo (Stehberg, 1995; Cornejo, Saavedra y Vera, 2006; Cornejo, 2008; Cornejo y Sanhueza, 2011a; Stehberg y Sotomayor, 2012; Pavlovic et al., 2019), se plantea aquí como hipótesis de trabajo que el Estado inca estableció un control directo de la franja oriental cordillerana comprendida entre los ríos Mendoza y Diamante, con el propósito de mantener en funcionamiento vías de comunicación trans e intercordilleranas y sus redes de intercambio vinculadas, como también para aprovechar ambientes de cordillera ricos en recursos (vegetales, animales y minerales) que solo podían obtenerse en el estío por condicionantes climáticos. Para contrastar esta hipótesis se desarrolló un proyecto de investigación centrado en la cuenca alta septentrional del río Diamante. Se presentan en este trabajo resultados de distintas líneas de estudio (técnicas constructivas y patrón de ocupación del espacio, cronología, tecnología lítica, tipología y tecnología cerámica, zooarqueología de camélidos, antracología y arte rupestre) desarrolladas en el Área Natural Protegida Laguna del Diamante. También se hace una revisión de los resultados alcanzados en la región por otros proyectos de investigación, con el objeto de discutir modelos de poblamiento vigentes que, por su rigidez, han obstaculizado la búsqueda de explicaciones alternativas para los registros del Período Inca obtenidos en lugares donde no se esperaba encontrarlos. estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 3 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Descripción geográfica del área El Área Natural Protegida Laguna del Diamante (en adelante, ANPLD) está emplazada sobre una caldera volcánica de forma elíptica de (20 x 16 km) ubicada en la cordillera del Límite (Sruoga, Etcheverría, Folguera, Repol y Zanettini, 2004) (Figura 1). Las cotas bajas del interior de la caldera (˂3300 msnm) han sido ocupadas, en su mayor parte, por la laguna del Diamante y otros cuerpos de agua dulce menores. El agua de derretimiento de las crestas montañosas que rodean la laguna discurre hacia ella a través de arroyos y por infiltración dentro de las coladas volcánicas que se extienden desde el cono del volcán Maipo hacia el este y sudeste. Del extremo meridional de la laguna nace el río homónimo que en su curso inicial fluye sobre un valle con poca pendiente hasta vencer la pared sur de la caldera. Figura 1. Mapa de la macrorregión. En las superficies planas libres de agua domina una estepa arbustiva baja y muy abierta, en donde se destacan elementos altoandinos y patagónicos (Dalmasso, Martínez Carretero, Videla y De Lugan, Dalmaso et al., 1996). Extensas zonas con saturación de agua permiten el crecimiento de vegas o humedales. Dentro de los ambientes de cordillera, las vegas son los ecosistemas con mayor productividad (Scotti, Durán y Lecitra, 2012). Eso hace que sean aprovechadas estacionalmente por distintas clases de herbívoros (especialmente Lama guanicoe) y sus depredadores. Vegas, estepas, lagunas y cursos de agua, en los que se concentra una gran cantidad de aves, ocupan una parte importante de las cotas bajas de Caldera del Diamante. Por ello, en la estación cálida, la oferta de recursos bióticos de estos ambientes es elevada, relativamente constante y, en consecuencia, predecible (Figura 2). 4 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu En cuanto a los recursos abióticos, el área ofrece agua dulce en abundancia y con una distribución amplia. Hay disponibilidad de rocas silíceas y de dos variedades de obsidiana (Cortegoso, Barberena, Durán y Lucero, 2016; Durán et al., 2018; Cortegoso et al., 2020). También se han localizado manifestaciones de material cuprífero diseminado y óxidos de hierro y arcillas (Durán, Neme, Gil y Cortegoso, 2006). Figura 2. Fotografía de la laguna del Diamante y del volcán Maipo. Las condiciones ambientales son favorables para su ocupación estacional y es el clima1 la limitación más importante para su uso humano. Las vías de acceso también son factores limitantes, pero no todas presentan igual grado de dificultad. Para ingresar desde el oeste solo es necesario atravesar el portezuelo del Maipo, ubicado a 3470 msnm. A través del mismo, ANPLD se vincula al Cajón del Maipo y por este último al valle central chileno. Ingresar desde el este es, en cambio, una tarea más compleja que obliga a cruzar por valles muy encajonados como el del río Diamante o portezuelos emplazados por encima de los 3700 msnm. Antecedentes En los últimos 20 años, las cuencas cordilleranas altas de los ríos Maipo y Diamante han sido intensamente estudiadas por distintos equipos de investigación (Lagiglia, 1999; Durán et al., 1 La temperatura media anual es en la laguna de 2 ºC y el promedio de precipitaciones, 600 mm, con un régimen invernal. De acuerdo con la clasificación de Koeppen, es un clima polar ETH –de tundra con alta heliofanía– (Dalmasso et al., 1996). estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 5 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso 2006; Neme, 2007; Cornejo, 2008). La cuenca del Maipo es la que tiene los registros más antiguos, con fechas comprendidas entre los años 6000 y 4000 AP (Cornejo y Sanhueza, 2011a y b). Dado que los sitios con ocupaciones de cazadores-recolectores se encuentran a no más de 25 km de la Caldera del Diamante o en sus límites, se entiende que grupos de cazadoresrecolectores debieron transitarla o aprovechar sus recursos durante ese período y posterior. Todavía esta presunción no ha podido contrastarse, ya que los registros arqueológicos obtenidos en las cotas más bajas de la Caldera (˂3300 msnm) se inician con fechas próximas a los 2100 años AP y mantienen características que perduran hasta el Período Inca. Se trata de sitios con estructuras circulares pircadas que aparecen en agrupaciones variables (de algunas unidades a varias decenas) y se ubican próximos a fuentes de agua y vegas. Sus registros incluyen elementos de molienda, artefactos líticos confeccionados, en general, sobre obsidiana y fragmentos de cerámica con frecuencias predominantes de tipos propios de los períodos Alfarero Temprano (PAT), Intermedio Tardío (PIT) y Tardío (PT) de la periodización del valle central chileno (Durán et al., 2006, 2018). Para explicar la existencia de este tipo de sitios, ampliamente distribuidos en la región, están vigentes tres propuestas, dos de las cuales tienen una clara base etnohistórica.2 La primera fue publicada por Lagiglia (1999) –siguiendo a Madrid (1977)– y la mantuvo Neme posteriormente (2007, 2016; Neme et al., 2016). Ambos autores entienden que estos ambientes de altura fueron ocupados casi exclusivamente por cazadores-recolectores de las planicies orientales. Neme considera que su ocupación se dio tardíamente (segunda mitad del Holoceno Tardío) como consecuencia de un proceso de intensificación iniciado por crecimiento demográfico en los ambientes más bajos de la vertiente oriental. Este proceso habría llevado a aquellos cazadores-recolectores orientales a hacer uso de ambientes altos que no habían sido atractivos, hasta entonces, por presentar una oferta menor e incierta de recursos (Neme, 2007, 2016; Morgan et al., 2017). La segunda propuesta tiene por autores a Cornejo y Sanhueza (2011a y b), quienes también retoman lo expuesto por Madrid (1977) y consideran que en el valle del Maipo se dio, desde alrededor del año 2000 AP hasta la llegada de los españoles, una coexistencia de cazadoresrecolectores y agricultores, correspondiéndoles a los primeros el control de las tierras altas de la cuenca del Maipo y de los valles intercordilleranos orientales. Difieren del modelo anterior al entender que estos cazadores-recolectores eran dos sociedades distintas: una que provenía de las planicies orientales ubicadas entre los ríos Diamante y Atuel y otra que permanecía en los ambientes cordilleranos occidentales intermedios durante las estaciones frías. El tercer modelo propone, para los últimos 2000 años, un uso recurrente de la cuencas altas de los ríos Diamante y Maipo por sociedades con economías agropastoriles provenientes del oeste –valle del Maipo y valle central de Chile–,3 sin negar la posibilidad de que hayan sido ambientes compartidos con cazadores-recolectores orientales (Durán et al., 2006, 2018; Barberena et al., 2019; Andreoni y Durán, 2021). 2 Madrid (1977) y Lagiglia (1999) utilizan la descripción de Bibar (siglo XVI DC) sobre los puelches –cazadores recolectores orientales– y sus actividades de intercambio con agricultores occidentales para inferir que eran ellos, específicamente los chiquillames, los que ocupaban las cuencas cordilleranas altas de los ríos Maipo y Diamante. 3 La posibilidad de ocupaciones estacionales de agricultores occidentales había sido considerada para el sitio El Indígeno por Falabella y Sanhueza (Falabella, Sanhueza, Neme y Lagiglia, 2001; Sanhueza et al., 2004), pero la abandonan posteriormente (Falabella et al., 2016). 6 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu El Período Inca, en las cuencas cordilleranas de los ríos Maipo, Diamante y Tunuyán, cuenta con una cantidad destacable de registros arqueológicos. La primera es la que manifiesta mayor densidad y diversidad, seguramente motivada por su proximidad al centro administrativo mayor o capital provincial que los incas establecieron entre los ríos Mapocho y Maipo (Stehberg y Sotomayor, 2012) y que aprovecharon los españoles posteriormente para fundar la ciudad de Santiago. En la cuenca cordillerana del Maipo se han encontrado sitios arqueológicos vinculados a vías de comunicación transcordilleranas (Cornejo et al., 2006), explotación minera (Cornejo et al., 1999), ceremonias religiosas en montañas (Ibacache y Cantarutti, 2003) y también prácticas funerarias (Cornejo, 2008). Para el sitio más meridional, Puente de Tierra, se ha propuesto que tuvo la función de marcar el límite de la expansión del Estado (Cornejo, 2008). Este sitio incluye dos estructuras pircadas, rectilíneas, con muros dobles orientados siguiendo los puntos cardinales, un bloque con un petroglifo serpentiforme y un topu. En las proximidades de estos, además, se ubicaron alrededor de una decena de recintos circulares y un alero. Si bien Cornejo menciona que pudo haber un interés del Estado inca por controlar el paso del Maipo (distante unos 30 km) y los recursos que por allí fluían, considera que el sitio funcionó como un demarcador del límite de la expansión estatal. En un trabajo posterior, Cornejo y Sanhueza (2011a) profundizan esta propuesta al concluir que este paso perdió durante el Período Inca el protagonismo que había tenido hasta entonces, y que los espacios que estaban fuera del límite establecido por el Estado (en Puente de Tierra específicamente) se mantuvieron bajo el control de cazadores-recolectores cordilleranos. En esos espacios se encuentra la mayor densidad de sitios de la cuenca alta del Maipo, los cuales se destacan por presentar estructuras pircadas adosadas a grandes bloques y cerámica inca en sus superficies y estratigrafías. En la cuenca cordillerana alta del río Diamante, fuera de ANPLD, se encuentran dos sitios con registros que pueden atribuirse al Período Inca: Risco de Los Indios (Neme et al., 2016; Morgan et al., 2017; Sugrañes, 2019) y El Indígeno (Lagiglia, 1999; Neme, 2007). El primero se ubica aproximadamente a 20 km al sur-sureste del Paso del Maipo y a menor distancia de los pasos del río Bayo y de La Cruz de Piedra, que conducen al valle del río Maipo y al sitio Puente de Tierra. Lo conforman un conjunto de 19 recintos circulares, en los que fueron registradas cerámicas de estilos Diaguita inca y/o Inca local del valle central chileno. Esta asignación tipológica se basa en la observación de fotografías de fragmentos cerámicos considerados de estilo Aconcagua en el trabajo de Morgan y colaboradores (2017, p. 219, Fig. 5B, D y F). También se incluyen en este contexto vegetales domesticados (Phaseolus sp.), una estructura amojonada demarcatoria (tipo topu o apacheta) y tres fechas radiocarbónicas correspondientes al Período Inca (se analizan posteriormente). Varios autores sostienen que este conjunto de recintos fue la base residencial de un grupo de cazadores-recolectores con alta movilidad y que obtenían parte de su cerámica y vegetales domesticados a través de intercambio (Neme et al., 2016, 2020; Morgan et al., 2017; Sugrañes, 2019). Por su parte, El Indígeno está ubicado por encima de los 3400 msnm y tiene 136 estructuras pircadas y registros similares a los descriptos para ANPLD, asignados también a cazadores-recolectores de las planicies orientales (Lagiglia, 1999; Neme, 2016). Se ha propuesto que su ocupación se inicia alrededor del año 1500 AP y termina en el 800 AP (Neme, 2016; Morgan et al., 2017). Con referencia a esta última fecha, resulta necesario destacar que estos autores no tuvieron en cuenta registros de cerámica Diaguita inca y posiblemente otras del mismo período, que aparecen en las recolecciones de superficie y que describe Lagiglia (1999). Aproximadamente 25 km al noroeste de la laguna del Diamante, se ubica la localidad arqueológica Arroyo Campos Borbarán (Bárcena, 2003, 2010), situada en la cuenca alta meridional del río Tunuyán. Comprende dos refugios naturales ubicados a 2800 msnm, en un depósito de estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 7 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso ignimbritas con petroglifos distribuidos a lo largo de varias decenas de metros. Uno de los aleros (Alero Ernesto) tiene un contexto estratificado con cerámica de tipología incaica (Diaguita inca e Inca local de la vertiente oriental-Viluco) y una fecha radiocarbónica correspondiente al Período Inca (se analiza posteriormente). Esta localidad está ubicada en un nodo de sendas pre y poshispánicas que permiten acceder desde el valle de Jaurúa al del Maipo. A pesar de las características de este registro y de su contexto, Bárcena (2003, 2010) lo asocia a puelches, huarpes o pehuenches (sociedades locales de la vertiente oriental). Es claro que el modelo de poblamiento de base etnohistórica ha influido en la interpretación de los registros arqueológicos del Período Inca de la región. Pero este modelo se afirma en un supuesto erróneo que lo debilita. La documentación histórica señala que los chiquillames (cazadores-recolectores orientales) se establecieron en la franja pedemontana de la cuenca del río Diamante recién a principios del siglo XVIII, por imposición de los españoles (Prieto, 1989). Antes su territorio estaba en la Planicie Oriental, al sur de los ríos Diamante y Atuel (Durán, 2000). No hay información en la documentación histórica que permita ubicarlos en las cuencas altas de los ríos Diamante y Maipo durante el período inicial de contacto hispanoindígena. Registros arqueológicos del Período Inca en ANPLD Características de los sitios En ANPLD se han localizado diez sitios arqueológicos que pueden asociarse al Período Inca (ver Figura 1), cuatro de ellos con recintos pircados (LD-S2, LD-S4, LD-S13 y LD-S21), cinco con petroglifos (LD-S7C, LD-S15, LD-S19, LD-S22 y LD-S23) y una cantera de obsidiana (LD-S24). Los cuatro sitios con recintos, caracterizados en este punto, tienen material en superficie y estratificado asignables al período indicado a partir de criterios tipológicos; dos de ellos cuentan con fechados radiocarbónicos que lo corroboran (LD-S4 y LD-S13). LD-S2 incluye 29 recintos de planta circular con diámetros de tres metros promedio (Figura 3A). Se ubica a más de 250 m de la costa occidental de la laguna del Diamante, en el extremo de una colada volcánica. Los muros de los recintos se han construido superponiendo, en forma irregular, bloques extraídos de la colada. Presentan distintas condiciones de conservación y alturas variables. Las alturas de los que han sido refuncionalizados, en su último período de ocupación o recientemente, superan en algunos casos el metro, mientras que otros solo están definidos por una línea de rocas. Se realizaron excavaciones de 1 m2 en tres recintos, siguiendo niveles artificiales de 5 cm hasta alcanzar profundidades próximas a los 20 cm. Cuatro fechados radiocarbónicos (tres sobre carbón y uno sobre hueso) arrojaron edades comprendidas entre 2100 y 1400 años AP –uno de los fechados de 1400 años AP corresponde a un esqueleto humano localizado en la estructura 6– (Yebra et al., 2021). Aunque no se cuenta con fechados absolutos, por las características tipológicas del registro cerámico, se asignó al Período Inca el primer nivel por debajo de la superficie de las estructuras 1 y 3 (Durán et al., 2006, 2018; Yebra et al., 2021). En superficie, tanto en el interior de las estructuras como en el espacio abierto que se define hacia el este, se distribuyen molinos planos, moletas, productos de talla y artefactos líticos formatizados, mayoritariamente de obsidiana, y fragmentos cerámicos. Entre estos últimos también aparecen tipos asignables al Período Inca, en especial en las proximidades de la estructura 7, la de mayor tamaño (Figura 3A). 8 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu LD-S4 está conformado por cuatro estructuras pircadas, tres recintos de planta circular/irregular y una estructura cuadrangular que encierra un espacio relativamente plano (Figura 3B). Se ubica sobre la margen izquierda del río Diamante, en el extremo de una colada volcánica y a 120 m de la llanura de inundación del río y de las vegas que allí se desarrollan. Se excavó en uno de los recintos una superficie próxima a los 3 m2, alcanzándose una profundidad de 55 cm –11 extracciones de 5 cm–. La secuencia se inicia con una fecha de alrededor de 1100 años AP y termina con registros del siglo XX (Durán et al., 2006, 2018; Yebra et al., 2021). En la extracción 5 (cuadrícula B) se obtuvo una fecha radiocarbónica de 490±40 años AP (Yebra et al., 2021). Al tener en cuenta este fechado y otras características del registro, puede proponerse que este sitio fue abandonado después de la ocupación incaica y que recién volvió a ocuparse a principios del siglo XX, produciéndose como consecuencia de esta nueva instalación el desplazamiento vertical de material moderno. Se ha agrupado por ello dentro del Período Inca el material procedente de las extracciones 2 a 5. LD-S13 incluye una cavidad natural situada entre dos grandes bloques rocosos. Dos recintos pircados adosados a dichos bloques, uno circular y otro de planta irregular (Figura 3C). El sitio se ubica a 50 m de la margen izquierda del río Diamante, en una zona con un amplio desarrollo de vegas. Los trabajos arqueológicos incluyeron tres sondeos de 0,50 por 0,50 m: uno en el recinto circular (sondeo 1) y dos en el interior de la cueva (sondeos 2 y 3). Los sedimentos se retiraron siguiendo niveles artificiales de 5 cm; en el sondeo 1 se extrajeron nueve niveles y doce en los sondeos 2 y 3. Se obtuvieron fechados radiocarbónicos que ubican la ocupación inicial del sitio (tanto en la cueva como en el exterior) alrededor de 1500 AP (Durán et al., 2018). A esta ocupación inicial corresponden dos entierros humanos. Estudios isotópicos realizados sobre huesos y dientes de estos dos individuos permiten asignar su procedencia a la vertiente occidental y definir que uno de ellos tuvo una dieta rica en maíz en su infancia (Durán et al., 2018; Barberena et al., 2019). Una fecha radiocarbónica de 420±40 años AP (Yebra et al., 2021), obtenida sobre carbón vegetal del nivel 5 del sondeo 1, permitió generar para este sondeo un componente que reúne los niveles 2 a 5 y se asocia al Período Inca. LD-S21 está conformado por 11 recintos pircados: tres de forma circular o semicircular, tres de planta rectilínea yuxtapuestos y seis irregulares adosados a bloques (Figura 3D). Incluye además un área despedrada de alrededor de 200 m2. Desde esta plataforma se observa en plenitud el volcán Maipo y la confluencia de los arroyos Paramillos y de Las Vacas, que se ubica a 100 m al oeste. Los recintos se ubican en una zona con una oferta importante de agua y de recursos vegetales. En 2020, se realizó una excavación de 1 m2 en el recinto 6 y una Figura 3. Planimetrías de sitios con componente inca y recintos pircados. estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 9 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso recolección sistemática de material superficial sobre un área de 300 m2. Dentro del material cerámico recuperado aparecen tipos propios del Período Inca. Por otra parte, este sitio cobra una importancia particular por estar emplazado a alrededor de 600 m de una cantera de obsidiana y tener una clara vinculación con ella. Análisis espacial SIG Este análisis integra múltiples caminos de menor costo (LCP, Least Cost Paths) para predecir conectividad geográfica en paisajes topográficamente heterogéneos. A través de un DTM (Digital Terrain Model), se generaron maquetas del terreno con las posibles vías de circulación y los costos de desplazamiento entre sitios. Fueron objetivos específicos de este trabajo: a) modelar la conectividad de todos los sitios con asociación inca (de cualquier tipo) y la posible existencia de una red de corredores; y b) evaluar el grado de conectividad del paso Maipo y discutir la relevancia de ANPLD en la red de corredores establecida. Se utilizó información georreferenciada obtenida en terreno e insumos ALOS-PALSAR de la fuente Alaska Satellite Facility. Los datos fueron procesados mediante SIG con los programas Q-Gis, ArcGis y Linkage Mapper (Núñez et al., 2013; Lucero, Castro, Marsh y Cortegoso, 2016). Se empleó el algoritmo de Tobler Off-Path modificado para calcular movimientos humanos en ambientes de altura (Lucero et al., 2016). De acuerdo a este modelo y a información etnográfica procedente de ambientes andinos comparables, los desplazamientos se miden en jornadas con rangos de 15 a 20 km de distancia (Nielsen, 2006; Yacobaccio, 2012). Los costos de desplazamiento se evaluaron a partir de la información resultante de la superficie de fricción y se aplicó, además, un modelo generado para ambientes de altura que pondera los movimientos en función de jornadas (Lucero et al., 2016). A partir de la ubicación de sitios con registros del Período Inca, se modelaron corredores que siguen cuencas fluviales y sectores de menor resistencia. Los análisis de múltiples LCP muestran que se da una alta conectividad entre todos los sitios, dentro de ANPLD y en su periferia. Existen dos corredores de alta conectividad y varios de menor jerarquía que vinculan la cuenca cordillerana alta del río Maipo, a través de las cuencas cordilleranas altas de los ríos Diamante y Tunuyán con el Piedemonte Oriental (Figura 4). Por otra parte, el análisis LCP señala a ANPLD como un área con una alta conectividad, ya que es allí donde muchos flujos convergen. Dado que esta zona presenta la mayor biomasa asociada a la subsistencia, podría considerársela como un nodo de conectividad (sensu Nielsen, 1997). En ANPLD la conexión de las dos vertientes cordilleranas se produce a través de varios pasos (Alvarado, Maipo, Río Bayo y Cruz de Piedra), siendo el del Maipo el de mayor amplitud y menor altura (Figura 4). En la Tabla 1, se han incorporado los valores obtenidos y promediados desde LD-S2/19 hacia los otros sitios, para demostrar la importancia de ANPLD en la articulación de espacios entre vertientes y su conectividad y transitabilidad. Los caminos delineados son producto de un modelo que traza rutas óptimas de menor costo en función de la topografía. En este tipo de modelo no se tienen en cuenta aspectos sociales o simbólicos de la conducta humana. Esto implica que las rutas trazadas pudieron no ser necesariamente la única alternativa, por ejemplo, la conectividad entre el sitio LD-S13 y Risco de los Indios se proyecta por el río Bayo y no por el río Diamante que parecería ser la ruta más viable. 10 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu Tabla 1. Transitabilidad y conectividad entre sitios desde LD-S2/19. Hacia Distancia Km Horas Jornadas 10 hs Km/h Conectividad LD-S4 2 0.98 0.1 2 Alta LD-S22 3 1.73 0.2 1.6 Alta LD-S15 3 1.93 0.2 1.6 Alta LD-S13 6 3.17 0.3 1.9 Alta LD-S21 6 3.62 0.4 1.7 Alta LD-S23 6 3.93 0.4 1.6 Alta LD-S24 6 4.17 0.4 1.4 Alta LD-S7 10 5.72 0.6 1.7 Alta Vega Linda 15 9.00 0.9 1.6 Alta Alero 16 9.97 1.0 1.6 Alta A. Ernesto 30 20.67 2.1 1.5 Alta Puente de Tierra 38 22.83 2.3 1.7 Alta Risco de Los Indios 33 23.17 2.3 1.4 El Indígeno A 51 36.17 3.6 1.4 Alta AR-Petros 55 36.97 3.7 1.5 Alta Alta Figura 4. Corredores de conectividad. estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 11 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Cronología Se calibraron y agruparon fechados radiocarbónicos de dos sitios de ANPLD, LD-S4 y LDS13 (Yebra et al., 2021), y de otros dos con registros contemporáneos y comparables, Alero Ernesto (Bárcena, 2010) y Risco de Los Indios (Morgan et al., 2017) (Tabla 2). Estos sitios se seleccionaron por su proximidad a ANPLD y por estimarse que formaban parte de la misma red de sendas de circulación. Tabla 2. Fechados modelados. Sitio Contexto Profundidad (cm) Código de laboratorio Edad 14 C ± Mediana calibrada (DC) 95% rango de probabilidad Material LD-S13 Sondeo externo, ext. 5 25 LP3539 420 40 1510 1440-1630 carbón LD-S4 Estructura 1, cuadrícula B, sector NE-SE, ext. 5 25 LP3658 490 40 1450 1400-1600 carbón Alero Ernesto Nivel superior Beta162400 460 60 1480 1400-1630 carbón Risco de Los Indios Estructura 22, Estrato III, Nivel 10 75-90 AA102653 478 38 1450 1410-1620 carbón disperso Risco de Los Indios Estructura 22, Estrato III, Nivel 7, lente de carbón 72 UGAMS13578 500 20 1440 1420-1460 carbón Risco de Los Indios Estructura, 22, Estrato II, Nivel 6, lente de ceniza 55 UGAMS13579 480 20 1450 1420-1480 carbón δ13C fue estimado en -24±2‰ para las fechas de LD-S4 y LD-S13; para las demás, no se informó. Los fechados radiocarbónicos se calibraron y modelaron con la curva actual del hemisferio sur, SHCal (Hogg et al., 2013) en OxCal 4.3 (Bronk Ramsey, 2009). Sus calibraciones tienen rangos de probabilidad superpuestas con medianas de un rango restringido de 1450-1500 años DC (ver Figura 3). Luego se refinó la cronología con un modelo bayesiano simple. Son supuestos de este modelo que las fechas corresponden a una sola fase y que las de Risco de Los Indios se depositaron en orden estratigráfico, según los detalles publicados de la estructura excavada (Morgan et al., 2017). El modelo tiene un índice de acuerdo alto (146) y estima que la fase se dio entre 1440 y 1460 años DC. Todas las fechas tienen medianas de 1450 DC y rangos de error reducidos de 5 a 10 años cada uno. Sin embargo, se toma en cuenta la posibilidad que 12 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu las fechas reales de estos eventos no se acerquen tanto en el tiempo. Este acercamiento puede ser el efecto del supuesto de que todas las fechas pertenecen a la misma fase, en donde la mitad correspondiente a Risco de Los Indios cae dentro de un lapso muy acotado. Figura 5. Rangos de probabilidad de los fechados de LD-S4, LD-S13, Alero Ernesto y Risco de los Indios. Las curvas en gris claro muestran las distribuciones de probabilidad calibradas, sin modelar. Las partes de las curvas de probabilidad menor de LP-3539 y Beta-162400, posterior a 1550 DC, se extienden fuera del gráfico. Las curvas en gris oscuro muestran las distribuciones de probabilidad luego de modelarse. Las líneas verticales bajo cada curva indican las medianas modeladas. La curva “KDE” (Kernel Density Estimate) es una síntesis de la densidad temporal de las seis fechas dentro de la fase (Bronk Ramsey, 2017). Si se consideran los rangos calibrados o modelados, el patrón de ocupaciones ocurre cerca del año 1450 DC o en las décadas posteriores. Estas fechas tienen una correspondencia clara con la presencia incaica registrada para el norte de Mendoza (Marsh et al., 2017) y Chile central (Cornejo, 2014). Estudios recientes de las fechas de contextos incas de ambas regiones sugieren una presencia inicial del Imperio próxima al año 1400 DC o algo anterior. También sitúan la densidad más alta de fechados en Chile central durante el lapso comprendido entre los años 1450 y 1500 DC (Cornejo, 2014). Por lo expuesto, puede concluirse que las ocupaciones registradas en ANPLD corresponden al inicio del período de consolidación de la ocupación inca de Chile central. Tecnología lítica En esta sección se presenta un estudio de intensidad de ocupación a través de la cuantificación del volumen de lascas proximales, una discusión sobre los resultados del análisis químico de artefactos de obsidiana y una descripción breve de la cantera/taller ubicada en ANPLD. estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 13 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Intensidad de ocupación en el rango temporal 450-500 años cal. AP Para realizar una estimación sobre la intensidad de ocupación, se analizó el registro lítico de los sitios LD-S2 (Estructura 1 y 3, Ext.1 y Superficie), LD-S4 (Cuad. A y B, Ext. 2 a 6 y Cuad. C, Ext. 2 a 5) y LD-S13 (Sondeo 1, Ext. 2 a 5) (ver Figura 3). Se cuantificó el volumen de lascas proximales, ya que los productos de talla que conservan el remanente de plataforma de percusión representan un golpe real practicado en la talla. De esta manera, el conteo de lascas con plataforma permite calcular el NMG (Número Mínimo de Golpes), que puede ser considerado como un indicador de intensidad de ocupación (Andrefsky, 1998). El NMG está representado en este caso por las lascas proximales, LP, e instrumentos confeccionados en lascas, IL (Hiscock, 2002). Se registró para el Período Inca un n de 252 LP e IL, cuya división por el volumen de metros cúbicos excavados (0,6055) arroja un total de 416,18 LP e IL por metro cúbico. Este período registra el índice más elevado de LP e IL, duplicando el del anterior más alto (1200-1280 años cal. AP; n=203 por m3) (Yebra et al. 2021) (Figura 6). Los resultados indican que la ocupación tardía del área habría registrado una intensidad sensiblemente superior a todas las anteriores. Esto podría ser consecuencia de estadías más largas o recurrentes y/o un mayor número de personas. Figura 6. Índice de Lascas Proximales por Componentes temporales (fases de ocupación de ANPLD). 14 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu Análisis químicos de obsidianas de fuentes y arqueológicas Se aplicaron estudios geoquímicos mediante XRF (Cortegoso et al., 2020) a muestras de artefactos de obsidiana procedentes del registro asignado al Período Inca (n=343). Los resultados muestran un uso mayoritario de las fuentes locales y la mayor diversidad de fuentes no locales de obsidiana en los cerca de dos mil años de ocupación de ANPLD. La fuente empleada de manera mayoritaria fue Laguna del Diamante –64,43%– (n=221, fuente local), seguida por Arroyo Paramillos –29,73%– (n=102, fuente local), Las Cargas –2,91%– (n=10), Laguna del Maule 1 y Cerro Huenul, ambas con 0,87% (n=3 para cada fuente) y Laguna del Maule 2 y Desconocida B, ambas con 0,29% (n=1 para cada fuente). Finalmente, hay artefactos de obsidiana que no han sido asignados a ninguna de las fuentes regionales, 0,58% (n=2). La variabilidad de fuentes cordilleranas (LD, AP, LC, LM-1) y extracordilleranas (HU y LM-2) sugiere un cambio en relación a las ocupaciones previas. Desde ca. 1210 años cal. AP se registró la presencia de la fuente LC y Desconocida B. Las fuentes de obsidiana identificadas para el Período Inca indican desplazamientos de sur a norte que alcanzan, en línea recta, 300 km para HU, 290 km para LM-2, 220 km para LM-1 y 140 km para LC. Cantera/taller de obsidiana (LD-S24) En la ladera septentrional del cerro El Relincho, en un depósito morénico ubicado a 3700 msnm, se detectó un sector con disponibilidad natural de obsidiana concentrada en forma de nódulos con tamaños comprendidos entre 5 y 15 cm (Figura 7). Su dispersión a lo largo de 700 m sobre un talud de detritos alcanza a 3400 msnm la margen izquierda de los arroyos de Las Vacas y Paramillos. La calidad para la talla de esta fuente de obsidiana es de muy buena a excelente. Sus características macroscópicas son similares a la obsidiana LD, que también se ha detectado y caracterizado por XRF en otro flanco del mismo cerro (Cortegoso et al., 2020). En la pendiente abundan nódulos y fragmentos con negativos de lascados de diversos tamaños. Por las evidencias de su explotación, LD-S24 puede ser considerado como una cantera/ taller (Nami, 1992). Pero resulta conveniente destacar que, dadas las condiciones naturales de fragilidad de la obsidiana y la dinámica del terreno, las evidencias de actividad de talla podrían estar sobrerrepresentadas debido a procesos tafonómicos. Existe una clara vinculación entre esta cantera/taller y el sitio LD-S21 localizado a alrededor de 600 m. Se registró allí un número importante de productos de talla y artefactos de obsidiana, tanto en superficie como en la excavación realizada. La presencia de percutores y productos de obsidiana en distintas etapas de la secuencia de talla son indicadores de su asociación a actividades de extracción y procesamiento de esa materia prima. Por otra parte, los dos sitios están relacionados con un bloque con grandes serpentiformes (LD-S23) que se ubica dentro de la misma cantera/taller (ver Figura 7). La asociación de estos serpentiformes a prácticas rituales incaicas, la presencia de cerámica inca en LD-S21 y su arquitectura similar a la del sitio Puente de Tierra (Cornejo, 2008) sugieren el interés de ese Estado por controlar la explotación de esta fuente de obsidiana. estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 15 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Figura 7. Sitio LD-S24, cantera/taller. Arriba, laguna del Diamante y volcán Maipo vistos desde la cantera. Abajo, detalle de la cantera y ejemplos de nódulos hallados. Tecnología y tipología cerámica En esta sección se retoman trabajos previos que consistieron en clasificar en categorías de vasijas y tipologías los conjuntos cerámicos recuperados de los sitios LD-S2 y LD-S4 (Durán et al., 2006, 2018). Se actualizaron dichos resultados y se reagrupó la muestra en categorías de acuerdo a tratamientos de superficie y decoración; además se tuvieron en cuenta estados de atributos y rasgos técnicos considerados diagnósticos de tipos incaicos que pueden reconocerse en conjuntos fragmentados mediante observación macroscópica (Pavlovic, Troncoso, Sánchez y Pascual, 2012; Stehberg, Prado y Rivas, 2017). Con la finalidad de evaluar en términos relativos la cantidad de cerámica inca de los sitios de ANPLD, se estimaron frecuencias y densidades por categoría (Tabla 3) y se compararon los valores con densidades y frecuencias obtenidas para otros sitios incas de Chile central. 16 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu Tabla 3. Frecuencias y densidades de materiales cerámicos del componente inca en los sitios LD-S2 y LD-S4. LD-S4 LD-S2 TratamientoDecoración Frecuencia absoluta Frecuencia relativa Densidad Frecuencia absoluta Frecuencia relativa Densidad Pintura roja y negro s/rojo (Inca) 19 18% 0,0150 1 5% 0,0100 Pintura roja 15 14% 0,0119 0 0% 0,0000 Alisado-inciso 2 2% 0,0016 0 0% 0,0000 Bruñido 2 2% 0,0016 0 0% 0,0000 Pulido 37 35% 0,0293 8 38% 0,0800 Alisado 32 30% 0,0253 12 57% 0,1200 Total 107 100% 0,0847 21 100% 0,2100 La muestra incluye el total de fragmentos cerámicos pertenecientes al Período Inca de los sitios LD-S4 (n=107) y LD-S2 (n=21). El 35,1% (n=45) corresponde a cerámica pulida y el 34,3% a cerámica alisada (n=44). Ambos grupos presentan pastas y superficies grises y marrones con distintas tonalidades. No poseen atributos diagnósticos que permitan asociarlos claramente a una tradición cerámica. A pesar de ello, cerámica que presenta estas características ha sido asignada en Risco de Los Indios a tipos alfareros locales de cazadores-recolectores orientales (Neme et al., 2016; Morgan et al., 2017; Sugrañes, 2019). Este tipo de asociaciones tipológicas debe realizarse con cautela, en especial para el caso de la cerámica Overo (Lagiglia, 1999), cuyos atributos externos y patrones de pasta (estilos tecnológicos) guardan estrechas similitudes con la cerámica Llolleo (Sanhueza, Falabella, Fonseca y Andonie, 2004). Atribuir a cazadoresrecolectores esta cerámica es discutible, dado que podría tratarse de cerámica de estilo/tradición Llolleo replicada a nivel local, o bien haber sido producida en la vertiente occidental y trasladada hacia las tierras altas cordilleranas. Trabajos recientes de mineralogía automatizada de pastas por QEMSCAN, Quantitative Evaluation of Minerals by Scaning Electron Microscopy (Frigolé et al., 2018), aplicados a materiales cerámicos del área de estudio, Chile central y norte semiárido, muestran similitudes en la composición mineralógica de Overo con Llolleo. Por otra parte, resultados de Análisis de Activación de Neutrones (Sugrañes, 2019), aplicados a muestras halladas en distintos ambientes de la cuenca del río Diamante y Atuel, evidencian que los tipos cordilleranos de la vertiente oriental (Overo y Nihuil) y los de la vertiente occidental (Llolleo, Aconcagua y posibles tipos Incas) poseen una composición química similar y conforman un mismo grupo que se diferencia de los de las tierras bajas de la vertiente oriental. Estos resultados sugieren una misma área de procedencia para todos los tipos mencionados, lo que apoya la propuesta del traslado de alfarería de los valles occidentales por sociedades que explotaban recursos de las tierras altas cordilleranas durante el verano (Durán et al., 2006, 2018). Los fragmentos del conjunto clasificado como inca representan el 15,6% (n=20) del total de la muestra. Poseen superficies externas pintadas monocromas (rojo) y bicromas (negro sobre rojo) y pulidas. En las superficies internas presentan un tipo de escobillado (Figura 8) que no se halla en la cerámica de los contextos del PIT y es característico de la alfarería incaica en Chile central y norte semiárido chileno (Pavlovic et al., 2012; Stehberg et al., 2017; Cornejo y estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 17 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Saavedra, 2018). La densidad de dicho conjunto expresada según la razón fragmentos/litros es de 0,01 en el sitio LD-S2, y de 0,0150 en el sitio LD-S4. En términos generales, estos valores de densidad se encuentran dentro de los rangos estimados para fragmentos diagnósticos incas en contextos incaicos de Chile central, incluso podrían considerarse más altos que algunos de ellos (Cornejo y Saavedra, 2018: Tabla 2, p. 139). La categoría de cerámica pintada roja representa el 11,7% (n=15). Incluye fragmentos que poseen superficies externas engobadas rojas y pulidas y podrían corresponder tanto a tipos Incas como Aconcagua (Durán et al., 2006, 2018). Los conjuntos menos representados corresponden a cerámica de superficies externas bruñidas y pastas finas de color negro (1,5%, n=2) y gris incisa (1,5%, n=2). El primer tipo se asemeja a otros del norte semiárido chileno (Durán et al., 2006; Cornejo y Sanhueza, 2011b) y el segundo a tipos frecuentes en los períodos Temprano/Medio y Tardío del noroeste y centro-oeste de Mendoza (García, 2004). En la mayor parte de los sitios cordilleranos de la cuenca del Maipo aparecen tipos bruñidos en muy bajas frecuencias (Cornejo y Sanhueza, 2011b) y también en el área cordillerana y precordillerana del norte de Mendoza y sur de San Juan (Michieli y Gambier, 1998). Análisis de pastas por QEMSCAN asignan este tipo de cerámica a la vertiente occidental y la gris incisa, a la oriental (Frigolé et al., 2018). Figura 8. Superficies externas e internas de cerámica Inca en el sitio LD-S4. Zooarqueología: camélidos silvestres y domésticos Este estudio retomó el conjunto de Camelidae del registro arqueofaunístico del sitio LDS4. Los especímenes muestran una alta fragmentación producto de la meteorización. Solo el 4,3% (NSP=15.202) pudo ser determinado a algún nivel taxonómico. En toda la secuencia de ocupación los camélidos predominan (más del 75%), seguidos por aves de diferentes tamaños (7,7%) y Dasipódidos (3,8%) (Durán et al., 2006: Tabla 8). En los primeros están representados individuos neonatos, juveniles y adultos y no se registra selección de partes anatómicas, lo que podría indicar que ingresaron completos al sitio. El análisis de las marcas de origen cultural permitió inferir un aprovechamiento intensivo de las presas, por medio de la cocción y para el consumo de médula ósea. En trabajos anteriores 18 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu referidos a este conjunto arqueofaunístico (Durán et al., 2006; Neme et al., 2020) no se observó una tendencia clara que marcara cambios en el aprovechamiento de Camelidae a través del tiempo. No obstante, al reagrupar los datos, se advirtió un aumento de su presencia durante el Período Inca, en el que se aprecia un NISP de 107, mientras que en los dos períodos anteriores es de 79 y 69 (Durán et al., 2006). Si se suma a estos últimos los especímenes de Artiodactyla, esa tendencia se robustece (NISP=115, 89 y 75, respectivamente). A pesar del bajo tamaño de la muestra, es importante considerar esta tendencia, ya que resulta poco frecuente hallar en sitios de altura, a cielo abierto, registros faunísticos numerosos y con buenas condiciones de preservación. También se aplicaron análisis osteométricos y estadística multivariada (análisis de componentes principales y análisis de conglomerados) sobre 12 especímenes que cumplieron los requisitos para la aplicación de técnicas métricas. Todos fueron comparados con la Colección de Referencia de Camélidos, con una base analítica comparativa más robusta (Gasco, 2014), y se los discriminó taxonómicamente. Nueve fueron identificados como Lama guanicoe y tres como Lama glama (ver detalles en Gasco, 2013), estos últimos corresponden al Período Inca. Al tener en cuenta que es complejo diferenciar llama y guanaco a nivel osteológico (Yacobaccio y Vilá, 2013), se prefirió utilizar osteometría (Cartajena, Núñez y Grosjean, 2007; Izeta, 2009; Gasco, 2013, y citas en ellos), técnica que ofrece numerosas ventajas analíticas como la replicabilidad y la sencillez. Solo precisa una base de datos actual numerosa, representativa y controlada de cada especie de camélidos, que actúe como referencia comparativa (Gasco, 2014 y citas allí). Además, al no establecer grupos de tamaño/forma a priori y trabajar con los datos métricos crudos, las pruebas estadísticas que se aplican evidencian la variabilidad de las relaciones entre los datos arqueológicos y actuales, evitando sesgos en el análisis, lo que no sucede cuando se aplica anatomía comparada, ya que generalmente utiliza pocos esqueletos de referencia. Por otra parte, para la región de estudio, otros análisis arqueofaunísticos han partido de un sesgo actualístico, al aceptar que todo el registro identificado macroscópicamente como Camelidae pertenece a Lama guanicoe porque es la única especie de camélidos que vive actualmente en estos ambientes (Neme et al., 2020). La presencia de animales domésticos identificada con una base metodológica fuerte invita a replantear hipótesis sobre estrategias de subsistencia y patrones de ocupación del espacio de las sociedades que habitaron ANPLD y disgrega en, al menos, dos especies la preponderancia de camélidos registrada en la secuencia de ocupación del sitio. En este contexto, se asume que la evidencia de especímenes de Lama glama da cuenta de la ganadería como estrategia combinada con la caza y del uso del área como un ámbito de pastoreo para rebaños, implicando una posible práctica de trashumancia (Gasco, 2013). Asimismo, abre el juego sobre su utilización como animales de carga en circuitos de intercambio. Antracología El conjunto antracológico analizado procede de los registros del Período Inca de LD-S2, LDS4 y LD-S13. La metodología de trabajo consistió en la comparación de los carbones arqueológicos con referencias de maderas actuales. Con anterioridad a la determinación, los carbones se pesaron, contabilizaron y se separaron en tres tamaños distintos (a- 2-5 mm; b- 5 a 10 mm y c- > a 10 mm) para evitar sesgar la muestra en favor de aquellos taxones que dejan carbones de mayor tamaño. La submuestra analizada se definió por medio de la construcción de una curva de riqueza específica (Andreoni y Capparelli, 2012). estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 19 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Para la identificación taxonómica se utilizó la colección de referencia de maderas del área de estudio (Andreoni, 2015; Andreoni y Durán, 2021) y descripciones anatómicas de maderas de Chile (Rancusi, Nishida y Nishida, 1987). Respecto del nivel de determinación, en algunos casos fue posible arribar a familia (p.e., Asteraceas), en otros se llegó a género (p.e.,Adesmia sp.) y en otros a nivel especie (p.e., Gomortega keule). En algunos casos, debido al solapamiento de rasgos anatómicos, fue imposible diferenciar entre dos géneros (p.e., Acacia/Prosopis), mientras que otros se definieron como afines (aff.) a una especie (p.e., Acacia aff. caven). La categoría indeterminable incluye los carbones en los que no fue posible reconocer la estructura de la madera (Andreoni y Durán, 2021). Se analizaron 179 carbones, de los cuales se identificó el 94,97%. Se pudieron diferenciar 10 taxones vegetales distintos: Familia Asteracea, Acacia aff. caven, Gomortega keule, Austrocedrus chilensis, Persea lingue, Quillaja saponaria, Ochetophila trinervis, Prosopis aff. alpataco (raíz), Adesmia sp. y Acacia/Prosopis (Tabla 4). En términos de frecuencia, Adesmia sp. fue la madera mayormente colectada para leña, representando el 75%, que son junto con Ochetophila trinervis los dos únicos taxones identificados en los tres sitios. En términos de riqueza taxonómica, en LD-S4 se identificaron seis taxones, en LD-S13, siete y en LD-S2, dos. Un aspecto relevante del registro antracológico es la presencia de maderas de distintos ambientes: pudieron diferenciarse maderas locales, como Adesmia sp. y Familia Asteraceae, y otras procedentes de pisos de vegetación más bajos de la vertiente occidental. Entre estos se incluyen Acacia aff. caven que actualmente crece en el Bosque Espinoso Mediterráneo y se distribuye en todo el valle central chileno y en el piedemonte occidental de la cordillera de los Andes. De este mismo piso procede, probablemente, Acacia sp./Prosopis sp. Mientras que en el Bosque Esclerófilo Andino, ubicado entre los 1400 y 2200 msnm, se debió colectar Austrocedrus chilensis, Persea lingue, Quillaja saponaria y probablemente Gomortega keule (Luebert y Pliscoff, 2017). Por otra parte, del piedemonte oriental de la cordillera Frontal se debió colectar Prosopis aff. alpataco, especie nativa de las provincias fitogeográficas de Monte y Patagónica. La procedencia de Ochetophila trinervis es más compleja de establecer, ya que esta especie crece tanto en la cuenca del río Maipo como en arroyos cordilleranos orientales. El estudio del registro antracológico de los períodos previos en ANPLD también muestra que la recolección de maderas locales fue predominante (p.e., Adesmia sp.) y que se introducen, aunque en menor proporción, especies de pisos altitudinales inferiores (p.e., Ochetophila trinervis). Para estas primeras ocupaciones la riqueza taxonómica es baja identificándose solo cuatro taxones: dos locales –Adesmia sp. y Asteraceae– y dos foráneos –Quillaja saponaria y Ochetophila trinervis– (Andreoni y Durán, 2021). Esta tendencia en el uso de especies locales se mantiene durante el Período Inca y se incrementa la riqueza taxonómica de los conjuntos a nueve taxones. Se incluyen maderas de los valles de Chile central (p.e., Acacia aff. caven, Quillaja saponaria, Austrocedrus chilensis, entre otras) y otras del piedemonte oriental de la cordillera Frontal (p.e., Prosopis aff. alpataco), ausentes en las primeras ocupaciones del área. Esta ampliación en los rangos de aprovisionamiento de leña refleja el aprovechamiento de recursos de distintos pisos ecológicos y la existencia de una estrategia de aprovisionamiento previa al ascenso a las tierras altas. 20 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu Tabla 4. Resultados antracológicos para ANPLD. Taxones LD-S2 Nº LD-S4 % LD-S13 Nº % Nº 8 7,77 - Total ANPLD % Nº % - 8 4,47 Familia Asteraceae - - Austrocedrus chilensis - - 0,00 3 8,33 3 1,68 Gomortega keule - - 0,00 1 2,78 1 0,56 Ochetophila trinervis 5 12,5 1 0,97 2 5,56 8 4,47 Persea lingue - - 1 0,97 - - 1 0,56 Prosopis aff. alpataco (raíz) - - 3 2,91 - - 3 1,68 Quillaja saponaria - - 3 2,91 1 2,78 4 2,23 Acacia sp./ Prosopis sp. - - - - 2 5,56 2 1,12 Adesmia sp. 34 85 79 76,70 23 63,89 136 75,98 Indeterminables 1 2,5 6 5,83 2 5,56 9 5,03 40 - 103 - 36 - 179 - Arte rupestre En ANPLD se descubrieron cinco sitios con arte rupestre, en cuatro de los cuales (LD-S15, LD-S19, LD-S22 y LD-S23) aparecen motivos serpentiformes de gran tamaño. Estos motivos han sido registrados en otras regiones del Tawantinsuyu y se los ha caracterizado como incas (Hernández Llosas, 2006; Cornejo, 2008; Cruz, 2015). Es preciso señalar que fue necesario diferenciarlos de las fulguritas, improntas que los rayos dejan en las rocas y que también están presentes en ANPLD. Las fulguritas son vidrios formados por el rápido calentamiento que genera el impacto de un rayo sobre roca, arena o suelo. A medida que pasa, esta energía derrite, vaporiza y reduce químicamente el material expuesto, lo que resulta en un rápido cambio físico, químico y/o morfológico (Jones et al., 2005). Es común que en la zona de impacto se forme un pequeño orificio desde donde la energía se transmite a través de la superficie de la roca (Pasek, Block y Pasek, 2012), también puede generarse una aureola de contacto o un halo con presencia de minerales de cuarzo o vidrio generado por las altas temperaturas que superan los 1700 °C (Elmi, Chen, Goldsby y Gieré, 2017). La fulgurita identificada (Figura 9) comienza en un orificio de aproximadamente 3 cm de diámetro (con una pequeña aureola alrededor compuesta presumiblemente por óxidos o vidrio de aproximadamente 2 cm de ancho) ubicado en el sector más alto del bloque, a partir del cual se desarrolla la impronta hacia abajo de manera vertical. Presenta una forma serpentiforme, con bordes angulosos y es cuasisimétrica a lo largo de su eje vertical. Su impronta es blanquecina y forma una cobertura continua e intensa que impide la identificación de los minerales afectados. Finalmente, hacia la parte basal del bloque va angostando progresivamente su traza, concluyendo casi en un punto en cercanía del suelo. estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 21 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso Figura 9. Fulgurita registrada en ANPLD. A la derecha, detalles de orificio de impacto del rayo y del angostamiento y finalización en un punto. Fotografía: Gentileza del guardaparque Javier López. A diferencia de la fulgurita, los motivos serpentiformes identificados en ANPLD tienen orientaciones disímiles, improntas más claras y en algunos casos se presentan acompañados por otros grabados. La técnica de ejecución varía de acuerdo a las características de superficie del panel: en los sectores donde es lisa y sin afectaciones se percibe un abradido suave, sin profundidad de surco, pero con marcado contraste con la coloración de la pátina; en los sectores donde la superficie del panel está meteorizada, se registran piqueteados que forman el surco. La técnica utilizada es particular, pues aunque se trata de abrasión, la sutileza de su ejecución hace que sea difícil de identificar. Para petroglifos similares se ha planteado su ejecución mediante termoalteración/rubificación (Cruz, 2015), aunque queda por experimentar otras opciones como abrasión con agua y arena o pumicitas. LD-S15 es un bloque de coloración oscura y aspecto suave, típico del barniz o pátina del desierto, que proviene de una colada del volcán Maipo de composición andesítica. Tiene adosada a su cara oriental una estructura parcialmente pircada, semicircular, de alrededor de 1 m2 y contiene un único panel grabado que se orienta hacia el noreste, de 120 cm de altura y 70 cm de ancho (Figura 10). Se trata de una superficie plana vertical afectada por agrietamientos y meteorización en su extremo inferior. El serpentiforme se desarrolla de forma vertical y alcanza los 100 cm de largo, comienza en el extremo superior derecho y finaliza en el sector inferior medio; la impronta oscila entre los 3 y los 5 cm de ancho. Se observa piqueteado en el sector inferior, que coincide con la parte meteorizada de la roca. Se ubica a 130 m de una laguna menor y a 260 m al oeste de la margen derecha del río Diamante, sobre una meseta abierta que une el paso del Maipo con el valle del río mencionado. LD-S22 es una gran roca andesítica con un único panel grabado que se orienta hacia el noreste (Figura 10). Como en el caso anterior, se trata de una superficie plana vertical, pero 22 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu con dimensiones mayores, ya que el panel alcanza 140 cm de alto por casi 170 cm de ancho. Presenta una pátina negra en la que resaltan dos serpentiformes que se yuxtaponen y que parecen formar una única figura altamente visible. El primero supera los 100 cm de largo y se desarrolla desde el extremo superior del panel hasta el sector izquierdo del mismo, mientras que el segundo recorre el panel desde el sector izquierdo hacia la parte inferior. Se registraron también dos figuras más pequeñas, una línea recta vertical de 27 cm y un tridígito de 20 cm en el sector superior del bloque donde se inicia el primer serpentiforme (existe una yuxtaposición parcial de las figuras, quedando el tridígito por debajo). Ambas figuras, la línea y el tridígito, fueron manufacturadas a partir de piqueteado lineal cuyo surco oscila entre los 7 y 10 mm de ancho. Este bloque se ubica a 70 m de la margen derecha del río Diamante y tiene una vinculación clara con LD-S15. Los dos bloques grabados parecen marcar un segmento de una senda, hoy inexistente, que debió unir el río Diamante con el paso del Maipo. Figura 10. Serpentiformes de ANPLD y similares de otras regiones del Tawantinsuyu. A y B) Petroglifos de LD-S15 y LD-S22, en ANPLD; C) Serpentiforme ubicado en la cumbre del cerro Cuzco (cordillera de los Frailes, Potosí, Bolivia), asociado con santuario de altura inca (Fotografía: Gentileza Pablo Cruz); D) Serpentiforme del sitio Puente de Tierra (Fotografía: Gentileza Luis Cornejo). estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 23 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso LD-S19 es un bloque de andesita con varios sectores de su pátina meteorizados. Este proceso ha afectado incluso al panel grabado, de 80 cm de alto por 75 cm de ancho. Contiene un serpentiforme en el costado derecho del panel, de 60 cm de longitud, efectuado mediante abrasión. Es el serpentiforme menos notorio de ANPLD debido a su meteorización. Además, se registró un círculo pequeño realizado mediante piqueteado areal, con un diámetro de 5 cm. Este bloque está ubicado a unos metros de una línea de costa pasada la laguna del Diamante y, aproximadamente, 70 m al norte del recinto más septentrional de LD-S2. LD-S23 es un bloque de una roca intrusiva, de coloración gris oscura y de textura microgranosa. Tiene un único panel vertical grabado de gran tamaño (sobrepasa los 2 m tanto de alto como de ancho), orientado hacia el sur-sureste. En la pátina rojiza resaltan dos serpentiformes horizontales que superan los 2 m de extensión y se desarrollan casi en paralelo (Figura 11). La figura superior es más visible, alcanza los 5 cm de ancho y se yuxtapone con la inferior (menos notoria debido a que su impronta promedia 1 cm de ancho) en el extremo derecho de ambas. Se ubica en la ladera norte del cerro Paramillos, a 400 m al sureste del sitio LD-S21, dentro del área de dispersión de material tallado de la cantera (LD-S24). Al emplazarse en el sector medio del cerro tiene un dominio visual de la mayor parte de la cantera, de la laguna del Diamante y de los arroyos de Las Vacas y Paramillos. Figura 11. Petroglifo de LD-S23. Calco obtenido mediante Adobe Photoshop. En la esquina inferior, fotografía original. 24 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu Para hacer una propuesta sobre la adscripción cronocultural de las figuras serpentiformes y su funcionalidad, se consideraron los registros rupestres de una región distante y dos próximas. En el altiplano surandino boliviano (departamento de Potosí), Cruz (2015) identificó varios sitios con motivos serpentiformes, que presentan una llamativa semejanza con los de ANPLD, tanto en lo figurativo como en lo técnico. Cruz (2015) relaciona estas grafías con la imagen de Illapa, deidad asociada al rayo, y destaca su asociación con adoratorios incaicos de altura y/o establecimientos mineros del mismo período. En la cuenca cordillerana alta del río Mendoza, a más de 3000 msnm, se ubica el sitio Portezuelo Colorado, donde varios serpentiformes más pequeños que los de ANPLD están en asociación directa con figuras vinculadas a los incas: cruz inscrita, motivos cuatripartitos y fitomorfos semejantes a la planta de maíz (Zárate Bernardi, Puerto y Marsh, 2020). El emplazamiento del sitio permite postular que su funcionalidad se relacionaría a marcación de rutas de movilidad intracordillerana y al acceso a espacios sacralizados (al cerro Aconcagua, específicamente). Por su parte, en la cuenca cordillerana alta del río Maipo, Cornejo (2008) registra un serpentiforme en el sitio Puente de Tierra. La presencia de este motivo a ambos lados de la cordillera y prácticamente en la misma latitud permite asumir que estas grafías podrían estar marcando rutas de movilidad y/o servir como una forma de apropiación del paisaje. El quinto sitio con grabados es LD-S7, formado por cuatro sectores: dos con recintos pircados circulares agrupados, uno con un recinto pircado aislado mayor (6 x 4 m), y el sector con los grabados (LD-S7C). Uno de los bloques grabados presenta cuatro figuras: una línea con ángulo recto hecha mediante un piqueteado lineal profundo; dos areales –una triangular pequeña semejante a un tridígito y otra rectangular no figurativa, muy degradada–; y una ancoriforme (cuadrangular de lados curvos). Esta última, vertical y de 10 cm de alto, ha sido manufacturada a partir de piqueteado areal; ocupa el área central del panel y está cerca de la rectangular, aunque no se puede establecer una asociación directa entre ambas (Figura 12). En las cercanías al recinto mayor, existe otro soporte menor cuyo panel con orientación cenit también presenta grafías: una línea recta de 15 cm, efectuada mediante abrasión, y un mortero pequeño o cupulita de 8 cm de diámetro. La visibilidad de estas marcas está dada por su profundidad, no por generar contraste entre fondo y figura. LD-S7 se emplaza a metros de la línea de costa antigua de una laguna de agua dulce menor, ubicada a 1500 m de la ladera septentrional del volcán Maipo y a 10 km al este de los pasos Alvarado. Figura 12. Soportes del sitio LD-S7C. A la izquierda, soporte con figura ancoriforme (rodeada por un círculo); a la derecha, soporte con cupulita y línea recta. estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 25 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso La figura ancoriforme de LD-S7C sirve también para ubicar cronológicamente las manifestaciones de arte rupestre de ANPLD, ya que figuras semejantes han sido asignadas al arte rupestre del Período Tardío en centro-norte de Chile y en el centro-oeste argentino (García, 2014; Zárate et al., 2020). Se los asocia a tumis, y se los vincula a símbolos de poder incorporados en épocas del dominio incaico, tanto en el norte de Chile (Montt y Pimentel, 2003) como en el Noroeste Argentino (Hernández Llosas, 2006; Martel y Giraudo, 2014). Por último, se considera el arte rupestre de la localidad Arroyo Campos Borbarán (Bárcena, 2003, 2010). Aunque la descripción publicada no permite un acercamiento preciso a las características de sus petroglifos, da cuenta de figuras cuadrangulares y “signos escudo” –motivos estándar dentro del arte rupestre inca del centro de Chile (Troncoso, 2001)–. Discusión Para entender la forma en que las sociedades prehispánicas hicieron uso de las cuencas cordilleranas altas de los ríos Maipo y Diamante, se ha adherido a un modelo de complementariedad ecológica4 derivado del de Murra (1975), que propone que, en ambientes andinos, la supervivencia de sociedades con economías de caza y recolección y agropastoriles se basó en la complementación anual de recursos provenientes de distintos pisos altitudinales (Durán y García, 1989). Dentro de un marco con estas características, resulta inadecuado categorizar cualquiera de esos pisos como marginal/pobre o central/rico,5 ya que es la oferta del conjunto total la que importa. Teniendo en cuenta lo expuesto, se ha considerado que los ambientes de altura de la cordillera Principal, en la franja latitudinal tomada, fueron ocupados estacionalmente durante todo el Holoceno, primero por cazadores-recolectores y luego, a partir del 2000 AP, por grupos agropastoriles del PAT, PIT y PT de la cuenca cordillerana del río Maipo. Se entiende que estas sociedades pudieron crecer demográficamente y pasar por un proceso de intensificación que las llevó a hacer un uso cada vez más prolongado de las tierras altas, con el objeto de asegurar el rendimiento de sus actividades pastoriles y de caza y recolección. Esta cordillera ocupada por pastores pudo favorecer la circulación de bienes a través de mecanismos de tráfico de tipo incorporado y/o especializado. El primero, desarrollado por pastores individuales y el segundo, por individuos especializados dentro de las comunidades pastoriles (Nielsen, 2006; Yacobaccio, 2012). Esta manera de ver el proceso de poblamiento humano de la región se contrapone a otras que entienden que los ambientes de altura considerados se mantuvieron siempre ocupados por cazadores-recolectores, aun durante el período en el que el valle central chileno y el centro-oeste y noroeste de Mendoza se anexaron al Tawantinsuyu (Cornejo y Sanhueza 2011a; Neme et al., 2016; Morgan et al., 2017). También se contrapone a una propuesta muy arraigada en la arqueología chilena que considera que el pastoreo de camélidos se inicia en el valle central con la conquista incaica6 (Falabella, Pavlovic, Planella y Sanhueza, 2016). 4 El modelo de complementariedad de pisos ecológicos elegido tiene mayor correspondencia con el inicial de base claramente funcionalista/adaptacionista (Murra, 1975). Pero también se han tenido en cuenta propuestas de arqueología internodal realizadas en ambientes puneños (Nielsen, 2006). 5 Se asume una posición crítica a la propuesta de Neme (2007, 2016) que considera las tierras altas de cordillera un ambiente marginal y, por serlo, que fue ocupado en forma tardía. 6 Al aplicar el método osteológico/anatómico de Becker a registros arqueofaunísticos preincas de Chile central, no se ha podido determinar la presencia de Lama glama. Pero hay otros indicadores en esos registros que permitirían proponer prácticas de pastoralismo. Por ejemplo, para el PIT se ha registrado un incremento en la frecuencia de camélidos y que algunos fueron alimentados con maíz (Falabella et al., 2016; Pavlovic et al., 2019). 26 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu El objetivo principal de este trabajo es probar que las cuencas cordilleranas altas de los ríos Diamante y Maipo fueron controladas por el Estado inca. Para ello se llevaron adelante distintas líneas de investigación en ANPLD. Los resultados obtenidos muestran que las cronologías, tanto de los sitios del área como de otros próximos, corresponden al inicio del período de consolidación del dominio incaico del valle central chileno y el noroeste y centro-oeste de Mendoza –1450-1500 años DC– (Cornejo, 2014; Marsh et al., 2017). También ha podido demostrarse que este período muestra una mayor intensidad de uso de los sitios, que se percibe claramente al cuantificar el volumen de lascas proximales y al comparar los valores obtenidos con los de ocupaciones previas. Esta mayor intensidad de ocupación tiene un correlato en los índices de densidad de cerámica de tipos incaicos que resultan similares a los de contextos incas del valle central chileno (Cornejo y Saavedra, 2018). Se da en ANPLD, al mismo tiempo, un incremento de la presencia de Camelidae. Si se asocia a esta información el aumento considerable de taxones de especies vegetales provenientes de pisos de vegetación más bajos (predominantes de la vertiente occidental), puede considerarse que la ejecución de esta estrategia preascenso de aprovisionamiento de leña fue una consecuencia de la disminución de la oferta de leñas locales, provocada por ese incremento en la intensidad de uso. La identificación de Lama glama en el registro arqueológico de LD-S4 permitiría proponer la utilización de animales de carga para el traslado de distintos recursos desde y hacia la vertiente occidental. El aprovisionamiento de leña en ambientes más bajos para equipar los emplazamientos de verano y su transporte en llamas puede considerarse un comportamiento más viable que el acarreo humano, en lo que hace a energía invertida. Por otra parte, el registro de estos animales es un claro argumento a favor de la presencia de pastores. El interrogante que surge es si estos pastores ingresaron a ANPLD siguiendo una actividad trashumante tradicional, sin ser controlados por el Estado inca, o si, por el contrario, fue una actividad supervisada y controlada por ese Estado para favorecer, por ejemplo, el funcionamiento de mecanismos de tráfico caravanero especializado. Al incorporar en este escenario el caravaneo, cobran otro sentido las características arquitectónicas y la distribución en el espacio de los recintos pircados presentes en ANPLD y regiones vecinas. Su forma de construcción y diseño (plantas circulares, semicirculares o irregulares, próximas o adosadas a grandes bloques, con muros simples de pirca seca y alturas bajas) es semejante a los que han dejado los caravaneros en la puna chilena, boliviana y argentina (Nielsen, 2006; Pimentel, Montt, Blanco y Reyes, 2007; Yacobaccio, 2012). El tránsito con llamas de estos caravaneros actuales requiere lugares para pasar una noche (jara), después de hacer movimientos diarios de entre 15 y 25 km, y de otros mejor equipados (jarana de ocupación prolongada7) que se emplazan en lugares con recursos vitales (leña, agua y forraje) en donde se permanece uno o más días con el objeto de recuperar la energía de los animales o para prepararlos para un esfuerzo mayor, como el paso por un portezuelo (Nielsen, 2006; Martel, Zamora y Lépori, 2017). Si se observa con esta perspectiva la distribución, características y conectividad de los sitios de ANPLD y de localidades próximas de la vertiente oriental (Risco de Indios, El Indígeno y Arroyo Campos Borbarán) y occidental (Vega Linda, Buena Vista, Cruz de Piedra y Puente de Tierra), puede reconocerse que forman parte de una red vial amplia y compleja. En esa red estos sitios pudieron funcionar como lugares de permanencia corta o prolongada de acuerdo a sus características contextuales y/o a las decisiones y necesidades de quienes circulaban. 7 En las jaranas de ocupación prolongada suelen aparecer estructuras mayores en forma de U que se utilizan para controlar y cargar los animales (Nielsen, 2006). Una estructura con estas características ha sido registrada en El Indígeno (Neme, 2016, Fig. 6, p. 104). estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 27 Durán, Zárate, Yebra, Frigolé, Lucero, Andreoni, Winocur, Gasco, Marsh, Castro, Zonana, Barberena y Cortegoso En su conjunto, ANPLD tiene características extraordinarias: extensas mesetas, con alturas comprendidas entre los 3300 y 3400 msnm, con la mayor laguna de agua dulce de la región y varias menores, además de arroyos y surgentes y una oferta destacable de recursos vegetales y animales. Debe sumarse a estas características ventajosas su proximidad al paso del Maipo, el más bajo y amplio de la región (Cornejo y Sanhueza, 2011a). Se entiende que fue, por ello, un lugar obligatorio de tránsito para aquellos que se movían sobre la red de sendas que surcaban la cordillera en sentidos norte-sur y este-oeste y, al mismo tiempo, un lugar óptimo para establecimientos estacionales de pastores. También el registro intrasitio muestra evidencias que podrían asociarse a tráfico especializado. Para el caso específico de la obsidiana, la variabilidad de fuentes marca un punto de inflexión en lo referido a estrategias de interacción interregional. Se halla presente la mayor parte de las fuentes externas cordilleranas y extracordilleranas registradas (cinco), mientras que en períodos anteriores solo aparecen dos. Ya que el aumento en la circulación de obsidiana para el Período de Desarrollos Regionales ha sido propuesto como una prueba de tráfico especializado en el NOA (Yacobaccio, 2012), se entiende que puede hacerse una inferencia similar para ANPLD. Para este supuesto sistema de tráfico, se destaca un flujo sur-norte de obsidiana desde fuentes ubicadas a cientos de kilómetros y totalmente vinculadas a la cuenca del río Grande. Este valle es predominantemente latitudinal y conforma con las nacientes de los ríos Atuel y Diamante un corredor natural que conecta la cordillera sur de Mendoza con la central. Al considerar el funcionamiento de esa red, resulta sugerente no haber encontrado en los registros de ANPLD obsidiana de la fuente El Peceño, que se ubica en las planicies orientales. Ya que es de allí de donde procederían los cazadores-recolectores que, según el modelo de base etnohistórica, ocupaban la cuenca cordillerana alta del río Diamante y manejaban el tráfico de bienes entre ambas vertientes durante el período considerado. Por otra parte, el flujo de bienes con dirección norte-sur y este-oeste también podría inferirse a través de la cerámica negra pulida y la gris incisa, que procederían del norte semiárido chileno y el noroeste y centro-oeste de Mendoza, respectivamente. Se ha señalado que fue una política del Estado inca controlar las rutas de circulación de bienes, entre ellos la obsidiana (Bray, 1992; Stehberg 1995; Yacobaccio, Escola, Lazzari y Pereyra, 2002; Ogburn, Connell y Gifford, 2009). En este sentido, el registro de ANPLD parece marcar que el Estado no solo controló el funcionamiento del tráfico que practicaban las sociedades locales del valle del Maipo, sino que pudo potenciarlo a través de caravaneros especializados. Se considera posible, además, que la mayor importancia otorgada por los incas a esta materia prima haya hecho que se diera una explotación más intensa de la cantera/taller ubicada en ANPLD. Las características del sitio asociado (LD-S21) y la presencia de un bloque con motivos serpentiformes (LD-S23) dentro de la propia cantera muestran claramente que se dio un control o algún tipo de apropiación estatal sobre ese recurso. La apropiación estatal de las cuencas cordilleranas altas de los ríos Maipo y Diamante también puede proponerse a través del arte rupestre. Por las características de los motivos descriptos y de sus emplazamientos se considera que corresponden al Período Inca y son la materialización visual de una forma de pensamiento (Troncoso, 2008). En ANPLD, quienes ejecutaron estas figuras de clara filiación incaica debieron compartir un código común que les permitiera comprender el mensaje. Sus autores pudieron ser miembros de poblaciones de la cuenca cordillerana del río Maipo o foráneas. Para ambos casos se entiende que agentes imperiales debieron trasladarse hasta la zona para ejecutar o apreciar el mensaje. Con el objeto de inferir la función de estas grafías se utiliza el concepto de paisaje conquistado, que sería una estrategia de marcación 28 estudios atacameños. Arqueología y Antropología Surandinas 2021, 67: e4353 Apropiación y control estatal de ambientes de altura en el extremo meridional del Tawantinsuyu del espacio a partir de las grafías rupestres. Esta estrategia estaría inserta en un proceso implícito más amplio de sacralización del paisaje, “[…] en función de la cosmología del grupo dominante que está intentando ‘apropiarse culturalmente’ de estos nuevos dominios” (Hernández Llosas, 2006, p. 23). Con base en lo expuesto, se considera posible que en ANPLD el Estado inca haya resignificado ese espacio al construir nuevos emplazamientos con grafías rupestres, con el objeto de imponer su ideología (Troncoso, 2008; Salatino, 2011). Las características particulares de los motivos serpentiformes permiten discutir otras aproximaciones. Cruz (2015) relaciona los serpentiformes del sur de Bolivia con la figura de Illapa, deidad andina asociada al rayo, con un fuerte vínculo con la actividad minera y con la sacralidad de los cerros o Apus. A la par, se vincula a Illapa con la práctica pastoril y se lo considera propiciador de la fertilidad agrícola, animal y humana; y es también una deidad guerrera, legitimadora de linajes y fuente de sabiduría para curanderos (Baulenas i Pubill, 2016). Complementariamente, los serpentiformes de ANPLD pueden vincularse a Amaru, la serpiente del agua –de hecho la forma de desplazarse de esta serpiente coincide con la del rayo al caer en la tierra (Baulenas i Pubill, 2012)–. Esta deidad forma parte de la cosmología andina y se asocia a los ancestros, el cielo, la tierra, los ríos, las quebradas, la lluvia, los canales de riego, las cuevas, la fertilidad y la procreación (Hernández Llosas, 2006). Teniendo en cuenta esto último, se podría sugerir que los serpentiformes se ejecutaron tomando la laguna del Diamante o a las nacientes del río Diamante como una expresión de esa deidad. Las lagunas de magnitud eran consideradas pacarinas, y aún más aquellas que tenían en su proximidad grandes volcanes como el Maipo, posible Apu, cuyas condiciones de visibilización son extraordinarias. Conclusión Se contrastó en forma positiva la hipótesis propuesta sobre el control del Estado inca de las cuencas cordilleranas altas de los ríos Maipo y Diamante. También se ha obtenido información que permite definir los posibles propósitos de ese control y discutir el origen de los agentes encargados de realizar las actividades acordadas o impuestas por el Estado. La explotación de una fuente de obsidiana fue una de esas actividades y, probablemente la principal, mantener en funcionamiento una red de tráfico trans e intercordillerano y su infraestructura. Esta red conectaba el valle central chileno con los valles de Jaurúa y Uco, que estaban dentro de las fronteras del Estado y se introducía hacia el sur, por los valles longitudinales intercordilleranos, con el propósito de obtener bienes que habían adquirido un nuevo valor y significado. Entre ellos, la obsidiana seguramente ocupó un lugar destacado. Se entiende que fueron sociedades agropastoriles de la vertiente occidental (de la cuenca del río Maipo en particular para la franja cordillerana considerada) las encargadas de realizar esas actividades. Por otra parte, se propone que el Estado inca sacralizó el espacio que incluye la laguna del Diamante y el volcán Maipo. Los registros arqueológicos que reflejan esta apropiación simbólica han comenzado a encontrarse al poner en discusión modelos de poblamiento de la región que niegan considerar la posibilidad de su existencia. Agradecimientos Este trabajo se realizó con fondos procedentes del CONICET (PIP. 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https://openalex.org/W4284967873	https://jurnal.unimed.ac.id/2012/index.php/iser/article/download/36560/18928	English	null	THE EFFECT OF THE PROBLEM-BASED LEARNING MODEL ON THE SCIENTIFIC ARGUMENTATION ABILITY OF STUDENTS ON ENVIRONMENTAL POLLUTION MATERIALS	Indonesian Science Education Research	2,022	cc-by	4,375	Volume 4, No. 1, Page 18-25, July 2022 Volume 4, No. 1, Page 18-25, July 2022 THE EFFECT OF THE PROBLEM-BASED LEARNING MODEL ON THE SCIENTIFIC ARGUMENTATION ABILITY OF STUDENTS ON ENVIRONMENTAL POLLUTION MATERIALS Riska Wahyuni1), Halim Simatupang2), and Santi H3) 1), 2)Department of Science Education Universitas Negeri Medan, Indonesia 3)Science Education Teacher of 1st Junior High School Angkola Muaratais, Indonesia riskawahyunih@gmail.com Accepted: May 12th, 2022. Published: July 8th, 2022 riskawahyunih@gmail.com Accepted: May 12th, 2022. Published: July 8th, 2022 Keywords: Problem Based Learning, scientific argumentation skills, environmental pollution Introduction at the school explained that the learning carried out still dominated the lecture and question and answer methods. The teacher explains that in learning that requires in-depth analysis, teachers still find it difficult to apply learning models that can foster students' thinking skills. Students who answer questions given through essay questions are still not fully able to answer questions with confidence. Students cannot write their arguments correctly. The teacher stated that he had never measured the students' scientific argumentation skills in writing using the correct argumentation indicators. Various efforts have been made by the government to meet the challenges of the 21st century era, namely by improving the quality of education in Indonesia through 4C competencies as a skill guide for students. Critical thinking skills have an important role in training students' sensitivity to problems that exist in their surrounding environment.Communication skills are important for students to be able to express the results of their thinking. Therefore, it is important for students to have capabilities that can support critical thinking skills and communicative skills in learning, namely the ability to argue (Devi et al., 2018). Learning that integrates constructivism learning theory is learner-centered learning. The teacher has a position as an intermediary who has a major role in helping students to actively participate in learning and create meaningful relationships between previous knowledge, new knowledge, and the processes involved in learning. (Sugrah, 2019). The term "argumentation" in everyday life is different from the term "argumentation" in science education. Scientific argumentation is the ability that is needed by a person to compile an opinion that is supported by evidence and real reasons, with the aim of strengthening his opinion on a matter (Farida & Gusniarti, 2015). One of the learning models that implements constructivism learning theory is the problem-based learning model, also known as the PBL learning model. The PBL model generally provides flexibility for students to be able to learn actively and continue to find out their own knowledge. Learning activities using the PBL model present problems in a structured and complex manner, where detailed information is not provided to understand all elements of the problem (Riwayani et al., 2019). Argumentation is an important activity inherent in the process of scientific exploration (Osborne et al., 2004). Introduction Scientific argumentation needs to be applied in science learning, because it can enable students to be directly active in creating various ideas and questions through a series of processes as well as in carrying out scientific practice (Berland & Hammer, 2012). This makes it very important to develop students' scientific argumentation skills in science at school. The purpose of this study was to determine the effect of the application of the PBL learning model on the ability of scientific argumentation and how much influence the PBL learning model has on the scientific argumentation ability of students on environmental pollution material in class VII, 1st Junior High School, Angkola Muaratais. Therefore, it is very important to know the influence of the PBL model on students' scientific argumentation skills. The National Science Education Standard (SNPI) emphasizes that scientific argumentation includes several criteria used to measure students' abilities (Diniya et al., 2021). The outcomes of Ambrawati et al. (2021) showed that students' scientific argumentation skills at the junior high school level were only able to write claims (statements), while participants still had difficulty presenting data, warrants (justifications), and supporters because this was the case. Learning in schools has not been able to develop these aspects, and applied learning has not trained argumentation skills well. Other research also proves that most students are only able to provide answers in the form of opinions (claims) to the questions given, but these claims have not been accompanied by scientific evidence to support their opinions. (Handayani et al., 2015). Abstract The purpose of this study was to determine the effect of implementing the Problem Based Learning (PBL) learning model on the ability of scientific argumentation and how much influence the PBL learning model has on the scientific argumentation ability of students on environmental pollution material in class VII, 1st Junior High School Angkola Muaratais. This study uses a true experiment method with a pretest-posttest control group design. The population in this study were all students of class VII, with two samples, namely the experimental and control classes. There are two samples in this study, namely the experimental class and the control class. The sampling technique used is simple random sampling. Randomly selected samples were from classes VII-A and VII-B. The research instrument used is a scientific argumentation ability test instrument using five essay questions. Analysis of the data used in this study was performed by performing an independent sample t-test and a Cohen's d test. The results of the pretest showed that the initial abilities of students in grades VII-A and VII-B were the same. Class VII-A was chosen as the experimental class, and Class VII-B was chosen as the control class. After that, treatment was given using the PBL learning model in the experimental class and conventional learning using the lecture and question and answer method in the control class. Based on the posttest results, it was found that the PBL learning model had a significant effect on the ability of scientific argumentation on environmental pollution material in class VII 1st Junior High School Angkola Muaratais with a value of tcount > ttable (3.828 > 1.684) using the independent sample t-test test. The influence of the PBL learning model on students' scientific argumentation skills is categorized as strong, with a Cohen's d value of 1.168. It can be concluded that the PBL learning model can have a significant influence on students' scientific argumentation skills and the effect obtained is strong. 18 Results This research was conducted in class VII at 1st Junior High School Angkola Muaratais in the even semester of T.A. 2021/2022, whose address is at Sorimanaon Village, Angkola Muaratais District. The population of this study were all students in class VII, amounting to 65 people who were divided into 3 classes. This study has a population of students of class VII with a total of 65 people who are divided into classes. This type of research is a true experiment involving two classes. Class VII-A is an experimental class that is treated using the PBL learning model, while class VII-B is a control class that is treated with conventional learning. 1.2.Scientific Argumentation Ability Posttest Results Research Method This research was conducted at 1st Junior High School Angkola Muaratais in the academic year 2021-2022. This research method uses the true experiment method with a pretest-posttest control group design. The population in this study were all seventh-grade students of 1st Junior High School, Angkola Muaratais. There are two samples in this study, namely the experimental class and the control class. The sampling technique in this study used simple random sampling. Randomly selected samples were from classes VII-A and VII-B. This is based on a preliminary study that has been carried out at 1st Junior High School Angkola Muaratais, namely by conducting interviews with science teachers. The results of interviews with science teachers 19 biology learning experts. The instrument validation on the content aspect is the suitability of the items with the learning objectives, learning indicators, and suitability of the questions with the learning materials. The results of the instrument validation are declared valid and can be used to measure students' scientific argumentation abilities. The data collection technique used in this study was to use a test instrument with five essay questions. First, the validity of the instrument is used to measure students' scientific argumentation skills. The validity of the instrument is carried out first. The validity of this instrument was tested using construct validity by giving validity sheets to material experts, lecturers, and learning experts. After the instrument is validated, it is then corrected according to the direction of the validator. 1.1 Pretest results of students' scientific argumentation abilities A pretest was conducted to test the feasibility of the sample and determine the initial ability of students. Testing the results of the pretest using the t-test (independent t-test) with SPSS version 28.0 program. The results of the pretest data processing of students' scientific argumentation abilities can also be seen in Table 1.1. After the instrument is declared valid, the the instrument can be used to achieve the research objectives. A pretest was conducted in classes VII-A and VII-B to see the initial abilities of students. After that, it was compared whether the students' scientific argumentation abilities in the two classes were the same. If the ability is the same, then from the two classes, samples are determined for the experimental and control classes. Table 1.1 Results of independent t-test pretest i tifi t ti bilit Table 1.1 Results of independent t-test pretest scientific argumentation ability Class N Me- an Std. Dev t df Sig. (2- tailed) Experi- ment 22 39 11,26 -0,86 41 0,39 Control 21 42 11,52 The experimental class is given treatment in the form of learning with the PBL model, and the control class is given conventional learning. A posttest was conducted to determine whether there was an effect of the PBL learning model on the students' scientific argumentation abilities after being given treatment. The analysis of the data used in this research is to perform a t test and a Cohen's d test using the SPSS version 28.0 program. Based on Table 1.1, it can be seen that the pretest value of students' scientific argumentation skills using the independent t- test test obtained the value of t_hitung = -0.863. When compared with the value of t_table =2,021. So it can be seen that the value of t_count < t_table = - 0.863 < 2.021 with the decision that H0 passes and Ha does not. It can be concluded that students in the experimental class and control class have the same scientific argumentation skills. 1.2.Scientific Argumentation Ability Posttest Results The final ability test of students (posttest) was conducted to determine the effect of the treatment given to the experimental class sample group. Calculation of posttest results using t-test (independent t-test) with SPSS version 28.0 program. The posttest value of students' scientific argumentation abilities in the The instrument of students' scientific argumentation skills is validated by a dose of 20 experimental class and control class can be seen in Table 1.2. Table 1.2. Results of independent t-test posttest scientific argumentation ability Class N Mean Std. Dev t df Sig. (2- tailed ) Experi- ment 22 80 9.5 9 3,8 2 41 < 0,001 Control 21 59 12. 85 Based on Table 1.3, it can be seen that the effect size test results obtained a Cohen's d value of 1.168. The values obtained are then interpreted into table 3.6, so the results obtained are Cohen's d values greater than 1.00 (1.168 > 1.00). It can be concluded that the PBL learning model has an effect size with a strong category in improving students' scientific argumentation skills in the experimental class. experimental class and control class can be seen in Table 1.2. Table 1.3. Effect size test results using the Cohen's d formula Table 1.3. Effect size test results using the Cohen's d formula The PBL learning approach has an impact on students' scientific argumentation abilities because of the classroom learning activities. Students have been trained to present their arguments through LKPD in learning activities. This is supported by Kusdiningsih et al. (2016) with research results that student are motivated in learning and capable of understanding scientific concepts. They can carry out argument-based investigations in problem solving and assist students in carrying out essential reasoning from scientific processes, so that it has great potential to improve students' scientific literacy. The worksheets used in this study are based on a PBL learning model that integrates argumentative activities. Discussion 2.1 The Effect of the PBL Learning Model on Students' Scientific Argumentation Ability The hypothesis tested in this study is that the PBL model affects students' scientific argumentation skills on environmental pollutant materials. The average scientific argumentation ability of students in the experimental class with the PBL model is 71.9, and the average scientific argumentation ability in the control class taught by conventional learning using the lecture method is 58.7. Based on Table 1.2, it could get visible that the calculation in a posttest value of students' scientific argumentation abilities using a t-test obtained the value of t_count = 3.828. When compared with the grade of t_table = 1.684. Then it can be seen that the grade of t_count > t_table = 3.828 > 1.684 with the decision Ha is accepted and H0 is not accepted (rejected). It can be seen that learning using the PBL model can have an effect on students' scientific argumentation abilities more than conventional learning. Based on the results of the independent sample t-test, the hypothesis test obtained the value of t_hitung > t_table (3.828 > 1.684). Based on the t value, it is possible to draw the conclusion that the PBL learning model's implementation has an impact on students' capacity to make scientific arguments about environmental pollution-related topics. Table 1.3. Effect size test results using the Cohen's d formula Instrum- ent Standa- rdizera Point Esti- mate 95% Confidence Interval Low- er Upp- er Scientific argument- ation skills 11,299 1,168 0,513 1,811 1.3. Great Test of Treatment Effect The treatment effect test has the purpose of deciding the influence of the PBL model at students' scientific argumentation abilities. Testing the effect of treatment using Cohen's d formula with the SPSS version 28.0 program. The results of the calculation of the effect size using the Cohen's d test may be visible in Table 1.3. g There are several steps in the PBL learning paradigm that researchers have employed, including orienting students to 21 problems; arranging students to learn; leading solitary and group investigations; generating and presenting work; as well as analyzing and evaluating problem-solving processes. argument and want to get facts that are true and relevant to the results of their experiments (Wibawa et al.,2018). The analysis and evaluation of the problem-solving process is the final step. Students in this phase are asked to make presentations on the experimental results and answers from each group. Meanwhile, other students (not presenting) were asked to respond and discuss the results of the group that was presenting. This is so that learning in the classroom becomes more active and students are more critical in addressing the challenges provided. Students can express their viewpoints, defend or contradict the opinions of other groups, and do so with genuine examples or facts (Firdauzi et al., 2019). The first phase of the PBL learning model is that researchers orient students to problems. Problems are presented in the form of pictures and narratives that are included in the LKPD. Students feel enthusiastic when problems are presented that are relevant to their lives, so that students are able to build an analysis of the problems and can build higher- order thinking skills (Untari et al., 2018). Organizing students for learning is the second phase. Researchers help students locate more information about the problems identified during this step, such as looking for factors causing water pollution in student textbooks or other sources of information. Students have a high curiosity for learning because they can discuss and work together to carry out tasks with group members (Nurullita et al., 2021). Students' knowledge of the subject is improved by the PBL-based learning paradigm used in the experimental class, enabling them to construct arguments that are supported by evidence and rationale with ease. This is supported by the results of research by Simanjuntak et al. 1.3. Great Test of Treatment Effect (2020), which states that the application of the PBL learning model can affect students' understanding of concepts in science material, and that students are enthusiastic and active in the learning process. The third phase is to guide both independent and group investigations. In this phase, students conduct experiments to test the information that has been collected. Students carry out the experiment by working together to follow the steps that have been prepared in the LKPD. Students are motivated to be able to provide ideas based on facts or knowledge, and students can gain authentic experiences, collect data, collaborate, or discuss with other students, so that students are very enthusiastic and enthusiastic about learning because students can learn while conducting experiments (Tarigan & Diana, 2015). Understanding the concepts possessed by students is obtained through learning activities that support the development of scientific argumentation skills (Eliana & Setyo, 2020). This is also supported by research by Rahmadani et al. (2020), which states that if students have a good understanding of concepts, they can develop good argumentation skills as well. Through this understanding, students will think logically when writing their arguments. In the second and third meetings, students were better at writing their arguments. The findings of a study by Dewina et al. (2017), which claim that students have gained experience from the first learning, then the second, and then the third learning in terms of expressing their arguments and already know how to write proper arguments through learning process activities, are also in support of this. The process of creating and presenting the work is the fourth phase. Students record their experimental findings in the research results table that is supplied in the LKPD throughout this period. Students have been trained to be able to express their arguments through these questions. So that students become accustomed to writing their arguments in learning activities, the researcher's questions are modified to the indicators of scientific argumentation competence. This is because students, in writing their analysis of experimental results, will trigger students to build their arguments, and students have a curiosity about the truth contained in an 2.2 The Great Influence of the Treatment of the PBL Learning Model on Students' Scientific Argumentation Ability 2.2 The Great Influence of the Treatment of the PBL Learning Model on Students' Scientific Argumentation Ability Based on the results of the independent t-test, which was conducted to examine the effect of the PBL learning model on students' 22 Based on the results of research and discussion, conclusions can be drawn as follows. scientific argumentation skills, the results showed that students in the experimental class had higher scientific argumentation abilities than students in the control class. To find out how much influence the treatment of the PBL learning model had in the experimental class, the researchers conducted a large effect size test, which stated that the PBL learning model had a strong influence in improving students' scientific argumentation skills based on the Cohen's d test with a value of 1.168. 1. The posttest results obtained stated that the PBL learning model had a significant effect on students' scientific argumentation skills on environmental pollution material in class VII SMP Negeri 1 Angkola Muaratais using the independent sample t- test test with a value of t_count > t_table (3,828 > 1,684). 2. 2. According to the Cohen's d test results of 1.168, the PBL learning model treatment used in the experimental class has a substantial impact. 2. According to the Cohen's d test results of 1.168, the PBL learning model treatment used in the experimental class has a substantial impact. This is because the PBL learning model can help students understand the concepts of subject matter well, have high analytical skills, provide meaningful learning experiences, and be able to build higher-order thinking skills, and be able to solve problems given (Rahman et al, 2020; Suriana et al, 2016; Wela et al, 2020; Asrati et al, 2018). Reference Hubungan kemampuan penalaran dengan keterampilan argumentasi siswa pada konsep sistem pencernaan melalui PBL ( problem-based learning). Jurnal Biologi dan Pembelajarannya, 13 (2) : 45-54. Probosari, R. M., Ramli, M., Harlita, H., Indrowati, M., & Sajidan, S. (2016). Profil keterampilan argumentasi ilmiah mahasiswa pendidikan biologi FKIP UNS pada mata kuliah anatomi tumbuhan. Jurnal Pendidikan Biologi, 9 (1): 29-33. Erduran, S., Simon., & Osborne, J. (2004), Taping into argumentation: developments in the application of toulmin’s argument pattern for studying science discourse. Science Education, 88: 915-933. Rahmadhani, K., Desy, F.P., Santosa, S. (2020). Kajian profil indikator kemampuan argumentasi ilmiah pada materi zat aditif dan zat adiktif. Jurnal Ilmiah Pendidikan IPA, 7 (1) : 1-9. Eskin, H., Ogan, B.F. (2013). Argumentation as strategy fpr conceptual learning of dinamics. Res Sci Edu. (43): 1939- 1956. Rahman, A., Khaeruddin & Evi, S. (2020). Pengaruh model PBL terhadap kemampuan berpikir kritisdan pemahaman konsep IPA siswa kelas V SDN 30 Sumpangbita. Jurnal Edumaspul, 4 (1) : 30-41. Firdauzi, F.S., Rahma, W. & Handayani. (2019). Penerapan model pembelajaran problem-based learning dipadu metode debat terhadap kemampuan berargumentasi. Jurnal Pendidikan dan Biologi, 11 (1) : 49-54. p Riwayani, R., Riki P., Ratna S., Jumadi J. & Heru K.(2019). Analisis kemampuan argumentasi ilmiah siswa pada materi optik: Problembased learning berbantuan edu-media simulation. Jurnal Inovasi Pendidikan IPA, 5 (1):45-53. Handayani, P. (2015). Analisis argumentasi peserta didik kelas X SMA Muhammadiyah 1 Palembang dengan menggunakan model argumentasi toulmin. Jurnal Inovasi Dan Pembelajaran Fisika, 2 (1): 60–68. Sarira, P.M., Desi, F.P., Susanti, P.A. (2019). Hubungan argumentasi ilmiah dan hasil belajar kognitif pada penerapan model problem-based learning (pbl). Jurnal Pendidikan Sains & Matematika, 7(2):1-10. Huriah, T. (2018). Metode student center learning. Jakarta: Prenamedia Group. Junaini, Rusdi, M., & Muslim. (2020). Penerapan model problem-based learning untuk meningkatkan kemampuan argumentasi matematika peserta didik kelas X SMAN 2 kota jambi. Jurnal Pendidikan Matematika, 4 (1) : 21-31. Simanjuntak, M.P., Marpaung, N., Sinaga, L., & Siregar, N. (2020). The effect of problem-based learning based on multiple representations to the students’ science conceptual understanding. Journal of Physics: Conference Series, 1819(2021) 012029: 1-8. Junaidi, M.R., & Muslim. (2020). Penerapan model problem-based learning untuk meningkatkan kemampuan argumentasi matematika peserta didik kelas X sman 2 kota jambi. Jurnal Pendidikan Matematika, 4 (1): 21-31. Sugrah, N. (2019). Implementasi teori belajar konstruktivisme dalam pembelajaran sains. Humanika. 19 (2):121-138. Sugiyono. (2019). Metode penelitian pendidikan:kuantitatif, kualitatif, kombinasi,R&D, dan penelitian pendidikan). Bandung:Alfabeta. Miaturrohmah & Fadly, W. (2020). Reference Ambarawati. D.S , Muslim, & Hernani. (2021). Analisis kemampuan argumentasi siswa smp pada materi pencemaran lingkungan. Jurnal Pendidikan IPA, 10 (1): 13-17. Based on this explanation, it can be concluded that students who are taught using the PBL learning model have higher argumentation skills than students who are taught using conventional learning. The PBL learning model can affect students' scientific argumentation skills (Junaini et al. 2019; Sarira et al., 2019; Ekanara et al., 2018). Devi, N. D., Susanti, E., & Indriyanti, N. Y. (2018). Analisis kemampuan argumentasi siswa sma pada materi larutan penyangga. Jurnal Kimia dan Pendidikan Kimia, 3 (3): 152-159. Students who are taught using conventional learning methods are different from students who are taught using PBL-based learning models. During the learning process in the control class, students did not form groups or carry out experimental activities (experiments). Students only listen to explanations about environmental pollution materials, record them in notebooks, and conduct questions and answers with researchers. Dewina, S., Ondi S. & Rahma W. (2017). Pengaruh model pembelajaran problem-based learning (pbl) terhadap kemampuan menganalisis dan keterampilan berargumentasi siswa pada konsep pencemaran lingkungan di kelas X. Quagga, 9 (2): 54-65. Diniya, D., Ilhami A. , Niki D.P., Mahartika, I. & Prakash, O. (2021). Kemampuan argumentasi ilmiah calon guru ipa melalui pendekatan mikir selama pandemi covid-19. Journal of Natural Science and Integration, 4 (1): 141- 148. The argumentation ability of students in the control class after being given the posttest had an average score of 58.71, which was sufficient criteria. This is because students have not been trained and have not been facilitated to write their arguments during learning activities, so when given a scientific argumentation ability test, students find it difficult to answer questions and their mastery of material concepts is still lacking. Therefore, students in the control class have argumentation skills with sufficient criteria. Diniya, & Rusdiana, D. (2018). Improving students’ argumentation by providing analogical mapping-based through lab inquiry for science class. IOP Conference Series: Materials Science and Engineering, 288 (1). g g Eliana, D. & Setto A. (2020). Tren pembelajaran argumentasi berbasis toulmin’s argument pattern (TAP) dalam meningkatkan kemampuan argumentasi dan pemahaman konsep Conclusion 23 model problem-based learning siswa SMP Muhammadiyah Muntilan. Jurnal Profesi Kependidikan, 2 (2) : 137-144. fisika peserta didik. Inovasi Pendidikan Fisika, 9 (2): 246-255. Ekanara, B., Yusuf, H.A., & Yanti, H. (2018). Reference Looking at a portrait of student argumentation skills on the concept of inheritance. Integrative Science Educationt and Teaching Activity Journal. 1 (1): 17– 33. Tarigan, E.A. & Diana, R. (2015). Pengaruh metode praktikum berbasis PBL terhadap kemampuan argumentasi tertulis siswa pada materi interaksi Nurullita, Khairuddin & Satwika, T.N. (2021). Peningkatan hasil belajar IPA melalui 24 kemampuan kolaborasi. Jurnal Terapan Sains dan Teknologi, 2 (3) : 209-220). makhluk hidup dengan lingkungannya. Edusains, 7 (2) : 135-142. makhluk hidup dengan lingkungannya. Edusains, 7 (2) : 135-142. kemampuan kolaborasi. Jurnal Terapan Sains dan Teknologi, 2 (3) : 209-220). Toulmin, S. (2008). The uses of argument. International Journal of Science Education, 34 (130): 244-245. Wibawa, R.A.P., Baskoro, A.P. & Marjono. (2018). Penerapan model problem based learning pada materi pencemaran lingkungan untuk meningkatkan kemampuan argumentasi ilmiah tertulis siswa kelas X MIPA. Jurnal Biogenesis, 14 (2) : 29-36. Untrari, E., Nikmatul, R. & Wahyu, L. (2018). Model pembelajaran problem based learning (PBL) sebagai pembiasaan higher order thingking skills (HOTS) pada pembelajaran IPA disekolah dasar. Prosiding Seminar Nasional Pendidikan Sains (135-142). Surakarta: Universitas Negeri Malang. g ( ) Widi, M.T., et.al. (2021). Analisis keterampilan argumentasi ilmiah peserta didik pada model pembelajaran berbasis toulmin’s argumentation pattern (TAP) dalam memahami konsep fisika dengan metode library research. Journal of Science Education, 5(1): 79-91. Wela, G.S., Chandra, S. & Hestiningtyas, Y.P. (2020). PBL dengan pendekatan multiple representation terhadap kemampuan berpikir kritis ditinjau dari 25
https://openalex.org/W2171243607	https://figshare.com/ndownloader/files/2409735	English	null	Interactions Increase Forager Availability and Activity in Harvester Ants	PloS one	2,015	cc-by	1,458	% This is a Matlab function that opens up a stack of JPEG images and allows a user to move through the images and ‘click’ anywhere on any image to record x-y coordinates of events in the frame. The output is a csv file with 6 columns: xy coordinates of the click, time = frame/image number, ant ID, ant type, and activity – both of which are pre-defined by the user, see below. % This is a Matlab function that opens up a stack of JPEG images and allows a user to move through the images and ‘click’ anywhere on any image to record x-y coordinates of events in the frame. The output is a csv file with 6 columns: xy coordinates of the click, time = frame/image number, ant ID, ant type, and activity – both of which are pre-defined by the user, see below. Copyright (C)2015 Noa Pinter-Wollman This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. See <http://www.gnu.org/licenses/> for a copy of the GNU General Public License function ClickGUInobuttons %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % do not try to create more ants if the csv output file is open! % the output of the program will be in indexes to this cell array % what matters is the order: % define ant types to code (e.g. foragers (F), nest maintenance (NM) etc… must use these exact types as defined here to initiate an ant AntTypes={'F','NM'}; % these are the possible ant types to input – you can add as many as you’d like % define ant types to code (e.g. foragers (F), nest maintenance (NM) etc… must use these exact types as defined here to initiate an ant A tT {'F' 'NM'} % th th ibl t t t i t % define ant types to code (e.g. function ClickGUInobuttons foragers (F), nest maintenance (NM) etc… must use these exact types as defined here to initiate an ant must use these exact types as defined here to initiate an ant AntTypes={'F','NM'}; % these are the possible ant types to input – you can add as many as you’d like AntTypes={'F','NM'}; % these are the possible ant types to input – you can add as many as you’d like % the following are the types of clicks (Ant activities): % the following are the types of clicks (Ant activities): % the following are the types of clicks (Ant activities): % interaction with standing ant(left mouse click), interaction with returnin ant (right mouse click), interaction with leaving ant (type the letter ‘l’ when the mouse is at the location of the click), exit nest (type letter ‘w’) etc… % interaction with standing ant(left mouse click), interaction with returning ant (right mouse click), interaction with leaving ant (type the letter ‘l’ when the mouse is at the location of the click), exit nest (type letter ‘w’), etc… SpecialClicks={char(1),char(3),'l','w'}; %you can add as many as you’d like SpecialClicks={char(1),char(3),'l','w'}; %you can add as many as you’d like %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %% FUNCTION KEYs – when the GUI is open, these keys will initiate various operations: p CFG.change='a'; % change ant id and type (type the letter ‘a’) CFG.forward=char(29); % right arrow moves frame forward CFG.back=char(28); %left arrow moves frame back CFG.jump='j'; % jump to frame # (type letter ‘j’) CFG.quit='q'; % quit program and save csv file (type letter ‘q’) CFG.undo='u'; % undo last click (type letter ‘u’) %%%%%%%%%% OUTPUT FORMAT %%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%%%%%%%% OUTPUT FORMAT %%%%%%%%%%%%%%%%%%%%%%%%%%%%% % csv file saved in foder with images, named 'interactions - 'colon % column order: ant id, x, y, frame from file name, click type, ant type %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% H={'ant id', 'x', 'y', 'frame from file name', 'click type', 'ant type'}; %header for csv file %% get path info % using the GUI %header for csv file %% get path info g pth = uigetdir(pwd,'browse to folder with jpgs to analyze') ; lst=dir(fullfile(pth,['*.' ext])); N=length(lst); N=length(lst); %% parse T from file names – get the frame number from the file name of the jpeg – might need to adjust this portion based on your naming convention T=zeros(N,1); for i=1:N [bla, filename]=fileparts(lst(i).name); [colonyname,frameAsStr]=strtok(filename,'_ frameAsStr=frameAsStr(2:end); T(i)=str2double(frameAsStr); end filename]=fileparts(lst(i).name) % read existing click from that folder % read existing click from that folder if exist(fullfile(pth,['interactions-' colonyname '.csv']),'file') IM=importdata(fullfile(pth,['interactions-' colonyname '.csv']),',',1); M=IM.data; else % read existing click from that folder if exist(fullfile(pth,['interactions-' colonyname '.csv']),'file') IM=importdata(fullfile(pth,['interactions-' colonyname '.csv']),',',1); M=IM.data; else M=[]; end %% figure(1) clf hImg=subplot('position',[0 0.1 1 0.9]); % these are the play/rewind buttons. function ClickGUInobuttons can adjust size and location here set(1,'name','') readImg; showImg; while ~quit % read existing click from that folder if exist(fullfile(pth,['interactions-' colonyname '.csv']),'file') IM=importdata(fullfile(pth,['interactions-' colonyname '.csv']),',',1); M=IM.data; else M=[]; end %% figure(1) clf hImg=subplot('position',[0 0.1 1 0.9]); % these are the play/rewind buttons. can adjust size and location here set(1,'name','') readImg; showImg; while ~quit readImg; showImg; while ~quit while ~quit try % to get click from user [x,y,b]=ginput(1); % parse the click parseClick; % show image showImg; catch e button = questdlg(e.message,'An error has occured:','Continue','Quit','Continue'); if strcmp(button,'Quit') quit=true; end end end try % to get click from user [x,y,b]=ginput(1); % parse the click parseClick; % show image showImg; catch e button = questdlg(e.message,'An error has occured:','Continue','Quit','Continue'); if strcmp(button,'Quit') quit=true; end end end try % to get click from user [x,y,b]=ginput(1); % parse the click parseClick; % show image showImg; catch e button = questdlg(e.message,'An error has occured:','Continue','Quit','Continue'); if strcmp(button,'Quit') quit=true; end end end % show image showImg; catch e button = questdlg(e.message,'An error has occured:','Continue','Quit','Continue'); quit=true; end saveOutput; % save on exit delete(1); % close figure saveOutput; % save on exit delete(1); % close figure %% Nested functions function parseClick % this is what the function keys do switch char(b) case CFG.change changeAnt; case CFG.forward currentFrame=min(N,currentFrame+1); readImg; case CFG.back currentFrame=max(1,currentFrame-1); readImg; case CFG.quit quit=true; case CFG.undo M=M(1:end-1,:); case CFG.jump msg=sprintf('Jump to frame number: (1 - %d)',N); str=inputdlg(msg); % if user canceled (and str is empty) return without doing % anything if isempty(str) return % exit parseClick subfunction to main function end oldfrm=currentFrame; currentFrame=str2double(str); if currentFrame>N \|\| currentFrame<1 uiwait(warndlg('out of bound frame - ignoring')); currentFrame=oldfrm; end otherwise currentClick=find(ismember(SpecialClicks,char(b))); if isempty(currentClick) uiwait(warndlg('Illigal key pressed - nothing will be recorded!')); else function parseClick % this is what the function keys do switch char(b) function parseClick % this is what the function keys do switch char(b) case CFG.change case CFG.change changeAnt; changeAnt; changeAnt; case CFG.forward currentFrame=min(N,currentFrame+1); readImg; case CFG.back currentFrame=max(1,currentFrame-1); readImg; case CFG.quit quit=true; case CFG.undo M=M(1:end-1,:); case CFG.jump msg=sprintf('Jump to frame number: (1 - %d)',N); str=inputdlg(msg); % if user canceled (and str is empty) return without doing % anything if isempty(str) p y return % exit parseClick subfunction to main function end oldfrm c rrentFrame if currentFrame>N \|\| currentFrame<1 recorded!')); if ~isnan(currentAntID) M=[M; [currentAntID x y T(currentFrame) currentClick currentAntType]]; end end end end dlg_title='Enter ant number and type:'; defAns={num2str(nxtant),''}; str = inputdlg(prompt,dlg_title,1,defAns); % if user cancels return (stays with current ant and type if isempty(str), return, % exit from function changeAnt into outer function end currentAntID=str2double(str{1}); currentAntType=find(ismember(AntTypes,str{2})); currentAntType=find(ismember(Ant if isempty(currentAntType) if isempty(currentAntType) uiwait(warndlg('WRONG ANT TYPE - chose change ant again and enter p y yp uiwait(warndlg('WRONG ANT TYPE - chose change ant again and function readImg img=imread(fullfile(pth,lst(currentFrame).name)); end function saveOutput try function showImg %% figure(1) axes(hImg); %#ok<MAXES> cla imshow(img,'Border','Tight') if isnan(currentAntID) if isnan(currentAntID) if isnan(currentAntID) set(1,'name',sprintf('Frame: %d NO ANT if isnan(currentAntID) set(1,'name',sprintf('Frame: %d NO ANT SELECTED!!!' currentFrame)) ( ) set(1,'name',sprintf('Frame: %d NO ANT set(1, name ,sprintf( Frame: %d NO ANT SELECTED!!!',currentFrame)) set(1,'name',sprintf('Frame: %d, Current ant id: %d, type: %s',currentFrame,currentAntID,AntTypes{currentAntType})); end et markers and colors to display each click %set markers and colors to display each c mrkr=['o','.','d','+','x','v']; p y mrkr=['o','.','d','+','x','v']; clr2=['c','y','m','k']; if ~isempty(M) if ~isempty(M) McrntFrame=M(M(:,4)==T(currentFrame),:); hold on antsInCurrentFrame=unique(McrntFrame(:,1)); for ant_i=1:length(antsInCurrentFrame) or ant_i=1:length(antsInCurrentFra or ant_i=1:length(antsInCurrentFrame) ix=find(McrntFrame(:,1)==antsInCurrentFrame(ant_i) _ ix=find(McrntFrame(:,1)==antsInCurrentFrame(ant_i)); if ~isempty(ix) plot(McrntFrame(ix,2),McrntFrame(ix,3),mrkr(McrntFrame(ix,5)),'color',clr2(Mc rntFrame(ix,6)))% clr(antsInCurrentFrame(ant_i),:) for p=1:length(ix) ( for p=1:length(ix) text(McrntFrame(ix(p),2)+10,McrntFrame(ix(p),3),num2str(antsInCurrentFrame(an t_i)),'color',clr(antsInCurrentFrame(ant_i),:), 'FontSize', 7) % l ( t I C tF ( t i) ) text(McrntFrame(ix(p),2)+10,McrntFrame(ix(p),3),num2str(antsInCurrentFrame(an t_i)),'color',clr(antsInCurrentFrame(ant_i),:), 'FontSize', 7) end % main function function WriteCSVwithHeader(M,H,filename) % M - matrix OR cell array % H - Header - cell array % filename - filename fid=fopen(filename,'w'); if fid<=0 error('Problem opening file – please check filename'); end % H - Header - cell array %% write header Cn=length(H); for i=1:Cn-1 fprintf(fid,'%s,',H{i}); end fprintf(fid,'%s\n',H{end}); %% write M (either number of text) if iscell(M) prt='%s'; else prt='%f'; M=num2cell(M); end for i=1:size(M,1) for j=1:size(M,2)-1 fprintf(fid,[prt ','],M{i,j}); end fprintf(fid,[prt '\n'],M{i,end}); end %% close file fclose(fid); end M=num2cell(M); %% close file fclose(fid); end
https://openalex.org/W2888124329	https://europepmc.org/articles/pmc6110839?pdf=render	English	null	Solvent-Antisolvent Ambient Processed Large Grain Size Perovskite Thin Films for High-Performance Solar Cells	Scientific reports	2,018	cc-by	9,447	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Solvent-Antisolvent Ambient Processed Large Grain Size Perovskite Thin Films for High- Performance Solar Cells Received: 6 April 2018 Accepted: 8 August 2018 Published: xx xx xxxx Received: 6 April 2018 Accepted: 8 August 2018 Published: xx xx xxxx Dawit Gedamu1,2, Ivy M. Asuo1,2, Daniele Benetti2, Matteo Basti2, Ibrahima Ka1, Sylvain G. Cloutier1, Federico Rosei1,3 & Riad Nechache1 In recent years, hybrid organic-inorganic halide perovskites have been widely studied for the low-cost fabrication of a wide range of optoelectronic devices, including impressive perovskite-based solar cells. Amongst the key factors influencing the performance of these devices, recent efforts have focused on tailoring the granularity and microstructure of the perovskite films. Albeit, a cost-effective technique allowing to carefully control their microstructure in ambient environmental conditions has not been realized. We report on a solvent-antisolvent ambient processed CH3NH3PbI3−xClx based thin films using a simple and robust solvent engineering technique to achieve large grains (>5 µm) having excellent crystalline quality and surface coverage with very low pinhole density. Using optimized treatment (75% chlorobenzene and 25% ethanol), we achieve highly-compact perovskite films with 99.97% surface coverage to produce solar cells with power conversion efficiencies (PCEs) up-to 14.0%. In these planar solar cells, we find that the density and size of the pinholes are the dominant factors that affect their overall performances. This work provides a promising solvent treatment technique in ambient conditions and paves the way for further optimization of large area thin films and high performance perovskite solar cells. Hybrid organic-inorganic perovskite materials of the form ABX3 (where A = CH3NH3, FA, or FAy(CH3NH3)1−y B = Pb, Sn, X = I, Cl) have recently emerged as promising material systems because of their low-cost synthe- sis and wide range of optoelectronic applications including light emitting diodes (LED)1–4, photodetectors5 and solar cells6–8. Their excellent properties such as high photon absorption, tunable bandgap and versatile material properties and fabrication processes9 allow them to compete with the established semiconductor-based solar cell technologies. Currently, physical deposition methods including pulsed laser deposition10–12 and chemical routes are mostly employed in the deposition of light absorber materials on transparent conductive substrates such as fluorine-doped tin oxide (FTO) or indium-doped tin oxide (ITO). Using chemical routes, perovskite films can be deposited using standard processes with a single or two-step spin-coating process on FTO or ITO coated glass substrates. Significant progress has also been achieved without using scaffolds, thereby minimizing materials and processing costs13. Received: 6 April 2018 Accepted: 8 August 2018 Published: xx xx xxxx www.nature.com/scientificreports/ 9,9′-spirobifluorene (Spiro-MeOTAD) poly(3,4-ethylenedioxythiophene)/poly(4-styrenesulfonate) (PEDOT:PSS) as hole extractors have been implemented using various deposition techniques9,15,20,22,23. The sta- bility of PSCs exposed to 55–70% humidity range has been shown to improve by introducing chemical additives such as a thiocyanate (Pb(SCN)2)16 or phosphoric acid ω-ammonium chlorides24 in the perovskite precursor solutions. The film microstructure including grain size25,26, grain boundaries27, density of pinholes, crystalline quality and orientation of the film also significantly affect the performance of the solar cells. In particular, fabrication in ambient conditions while maintaining full control over the microstructure of the film is a major technical challenge28,29. To this end, numerous recent efforts focused on tailoring perovskite grain size and microstructure with specific morphologies. Small crystal grains were found to suppress exciton formation28, and methods such as sulfonate carbon nanotubes filled grain boundaries30, or Pb(SCN)2 31 additives result in homogenous films with larger grain sizes and subsequently increase the PCEs. Using various antisolvents (toluene, 2-propanol, chlorobenzene)7,21,32,33 and mixed antisolvent (chlorobenzene:2-propanol mix)34, perovskite microstructure can also be tailored. However, a cost-effective technique to control the microstructure under ambient conditions has not been realized yet35. A simple and robust processing technique to tailor the crystallization process and repro- ducibly fabricate highly-crystalline pinhole-free films with large grain sizes is a necessary step towards efficient PSCs that can be scaled-up for commercialization.i p In this report, we address the key challenge of controlling the microstructure of the perovskite films processed in ambient conditions by using a cost-effective solvent treatment approach to synthesize highly-oriented crys- tallites with statistically-controlled grain sizes and low pinhole densities. This approach yields high quality films with 5 µm grains and minimized grain boundaries. Yet, all the active layers are deposited in ambient environ- ment (RH ~ 40%) using a precursor solution of 1:1:4 molar ratios of PbI2, PbCl2 and Methyl Ammonium Iodide (MAI). Combinations of ethanol (EtOH) and chlorobenzene (CB) in various volumetric proportions are used to treat the film at the third spin-coating (solvent dripping) step in the perovskite coating process. The solar cells fabricated using different solvent treatments indicate that the grain size and microstructure can be controllably altered, with great impact on the performance of the solar cells. Indeed, we demonstrate the effect of pinhole densities and sizes on the shunt resistance36, which in-turn affects the fill factor (FF) and the PCE of the devices. Result and Discussion Sequential spin-coating process from solution is the most commonly-used technique for deposition of multi-layered thin films in the fabrication of heterojunction PSCs. In this work, we employ a simple one-step CH3NH3PbI3−xClx perovskite solution prepared by dissolving PbI2, PbCl2 and MAI components in N,N-dimethylformamide (DMF) solvent. The CH3NH3PbI3−xClx precursor solution is then spin-coated on FTO substrate atop a pre-deposited compact titanium dioxide (c-TiO2) layer acting as an electron-transporting layer (ETL). Following perovskite crystallization by thermal annealing, a spiro-MeOTAD layer is spin-coated in ambi- ent condition on atop of the perovskite film to serve as a hole-transporting layer (HTL) before deposition of the top Au electrode by sputtering. The complete procedures pertaining to sample preparation are detailed in the experimental section. Figure 1(a) summarizes the overall device architecture for the planar PSC (FTO/ETL/ Perovskite/HTL/Au). Figure 1(b) shows a schematic representation of the energy level diagram of the solar cells, with the energy barriers at the interfaces between c-TiO2/Perovskite/Spiro-MeOTAD. The perovskite film has a very favorable energy band alignment8,37 with the c-TiO2 and Spiro-MeOTAD in order to extract electrons and holes respectively. Under solar illumination, perovskite light absorbers generate both charge carriers that traverse the film (thickness ~400 nm), before electrons are eventually extracted by the ETL (50 nm) and holes by the HTL (200 nm).hf The thickness chosen for the perovskite layer is compatible with the diffusion lengths of electrons and holes. The latter has been measured to be longer than 1 µm for planar mixed halide perovskite light absorbers38. These combined advantages together with their process compatibility and widely accessible deposition methods make these extractors ideal candidates for PSCs. Excluding the final Au electro-deposition by magnetron sputtering, all other device layers are deposited by spin-coating method in ambient conditions.i In a one-step deposition of CH3NH3PbI3−xClx, the perovskite film shrinks and becomes porous showing sig- nificantly large pinholes (~5 µm). Thus a method for crystallization and growth of compact perovskite films is essential to achieve high efficiency solar cells39. Our proposal of using a solvent engineering approach originates from achieving a compact film through a solvent controlled crystallization process39,40. As depicted in Fig. 1(c), our approach for spin-coating the perovskite layer involves three processing steps: a) 1st spinning for surface coverage, b) 2nd spinning for reducing film thickness and c) 3rd spinning and solvent dripping for crystallization. www.nature.com/scientificreports/ Using an optimized solvent treatment promoting films with larger grains and lower pinhole densities, PCEs up-to 14% are achieved for planar solar cells processed in ambient conditions. This novel solvent engineering approach yields significantly-improved crystal grain sizes and low pinhole densities, yet leaving room for further optimi- zation. For simple planar PSC architectures, this work represents an important step towards the realization of high-quality perovskite film for low-cost and high-performance PSC devices. Solvent-Antisolvent Ambient Processed Large Grain Size Perovskite Thin Films for High- Performance Solar Cells However, operational device stability and the substitution of lead (Pb)14,15 remain the two major challenges associated with the success of commercial-grade perovskite-based solar cell devices16.fi Hybrid organic-inorganic perovskite materials of the form ABX3 (where A = CH3NH3, FA, or FAy(CH3NH3)1−y B = Pb, Sn, X = I, Cl) have recently emerged as promising material systems because of their low-cost synthe- sis and wide range of optoelectronic applications including light emitting diodes (LED)1–4, photodetectors5 and solar cells6–8. Their excellent properties such as high photon absorption, tunable bandgap and versatile material properties and fabrication processes9 allow them to compete with the established semiconductor-based solar cell technologies. Currently, physical deposition methods including pulsed laser deposition10–12 and chemical routes are mostly employed in the deposition of light absorber materials on transparent conductive substrates such as fluorine-doped tin oxide (FTO) or indium-doped tin oxide (ITO). Using chemical routes, perovskite films can be deposited using standard processes with a single or two-step spin-coating process on FTO or ITO coated glass substrates. Significant progress has also been achieved without using scaffolds, thereby minimizing materials and processing costs13. However, operational device stability and the substitution of lead (Pb)14,15 remain the two major challenges associated with the success of commercial-grade perovskite-based solar cell devices16.fi j g g p In the last few years, perovskite solar cells (PSCs) have shown very rapid increase in power conversion effi- ciency (PCE) since the pioneering work of Miyaska et al.6. PCEs exceeding 20% have been achieved for three dimensional perovskite-based solar cells17–20. A record efficiency of 22.1% and excellent stability have been reported using mesoporous scaffolds deposited on charge extractors so that the perovskite would infiltrate the pores21. To enhance efficiency and improve device stability, materials such as TiO2, SnO2, phenyl-C61-butyric acid methyl ester (PCBM) as electron extractors and polymers 2,2′,7,7′-tetrakis (N,N-di-p-methoxyphenyl-amine) 1École de technologie supérieure (ÉTS), Department of Electrical Engineering, 1100 rue Notre-Dame Ouest, Montréal, (QC), H3C 1K3, Canada. 2INRS-EMT Centre for Energy, Materials and Telecommunication, 1650 Boul. Lionel Boulet, Varennes, (QC), J3X 1S2, Canada. 3Institute of Fundamental and Frontier Science, University of Electronic Science and Technology of China, Chengdu, 610054, PR China. Correspondence and requests for materials should be addressed to D.G. (email: dawit-minale.gedamu.1@etsmtl.net) or F.R. (email: rosei@emt.inrs.ca) or R.N. (email: Riad.Nechache@etsmtl.ca) Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 1 www.nature.com/scientificreports/ Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 Result and Discussion Schematic illustration of (a) final device architecture (b) energy band alignment of charge extractors to energy levels of halide perovskite where the conduction band minimum is aligned to electron injection into c-TiO2 and the valence band maximum is aligned to injection of holes into Spiro-MeOTAD. The processing steps in the device fabrication are depicted in the schematics (c) where the process begins with the perovskite precursor loading on the c-TiO2 coated FTO substrate followed by two spinning processes: reducing film thickness and solvent dripping (treatment) steps followed by thermal annealing for solvent removal and crystallization. engineering process using volumetric solvent mixture for micro-structure optimization in order to achieve both larger grains (CB) and good coverage with low pinhole densities (EtOH).ihi For all treatments, the XRD spectra of the perovskite films are shown in Fig. 2(g). The films exhibit typical peaks of the MAPbI3 phase at approximately 14.06° (110) and 28.28° (220). The peak occasionally seen at 12.66° corresponding to the (001) peak of a photo-inactive cubic PbI2, indicating a partial decomposition and/or resid- ual unreacted PbI2 in the film21. However, this residual PbI2 completely disappears for samples treated with EtOH alone. For the other samples using EtOH, the intensity of this peak remains relatively weak compared what is observed for the EtOH:CB = 3:1 solvent treatment. This is because a polar solvent such as EtOH dissolves the excess MAI41, while the non-polar CB enhances the crystallization process since non-polar CB reduces the solu- bility of perovskite in the polar DMF that consequently initiates fast nucleation42. As EtOH dissolves the remnant MAI, the remaining unreacted PbI2 from the first spin-coating step will have a second opportunity to react with MAI during the solvent dripping step.h g pp g p The XRD pattern from the sample treated with 100% EtOH also reveals a pronounced crystallite orientation along the (110) plane, suggesting highly-oriented crystals with preferential growth direction along this plane43. We suggest that such a high degree of order originates from the pre-crystallization step in the presence of a sol- vent treatment44. Yet, CB initiates nucleation and enhances crystallization producing larger crystal grains.i y p g g y g Larger grain sizes in perovskite films would promote the PCE of solar cells since the photogenerated carriers encounter reduced impediments from bulk defects and grain boundaries. Result and Discussion To achieve larger grains while maintaining a compact perovskite film, chlorobenzene (CB, anti-solvent) and eth- anol (EtOH, solvent) were chosen to modulate the film morphology. The SEM images in Fig. 2(a–f), illustrate solvent-engineered perovskite thin films deposited on FTO substrate and treated using different combinations of CB and EtOH. Figure 2(a) shows a pristine un-treated perovskite film with a rough microstructural evolution with large crystal grains (>5 µm) and pinholes. Surprisingly, the SEM analysis first reveals strikingly different morphologies when treated only with EtOH and CB. Indeed, Fig. 2(b) shows a film treated only with EtOH hav- ing a much better surface coverage with significantly smaller pinholes size but also significantly reduced crystal grains. In contrast, Fig. 2(f) shows a film treated only with CB having both very large crystal grains (5 µm), but with large pinholes and poor surface coverage. These differences led to the idea of perovskite thin film solvent Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 2 www.nature.com/scientificreports/ Figure 1. Schematic illustration of (a) final device architecture (b) energy band alignment of charge extractors to energy levels of halide perovskite where the conduction band minimum is aligned to electron injection into c-TiO2 and the valence band maximum is aligned to injection of holes into Spiro-MeOTAD. The processing steps in the device fabrication are depicted in the schematics (c) where the process begins with the perovskite precursor loading on the c-TiO2 coated FTO substrate followed by two spinning processes: reducing film thickness and solvent dripping (treatment) steps followed by thermal annealing for solvent removal and Figure 1. Schematic illustration of (a) final device architecture (b) energy band alignment of charge extractors to energy levels of halide perovskite where the conduction band minimum is aligned to electron injection into c-TiO2 and the valence band maximum is aligned to injection of holes into Spiro-MeOTAD. The processing steps in the device fabrication are depicted in the schematics (c) where the process begins with the perovskite precursor loading on the c-TiO2 coated FTO substrate followed by two spinning processes: reducing film thickness and solvent dripping (treatment) steps followed by thermal annealing for solvent removal and crystallization. Figure 1. Result and Discussion Grain boundaries and/or large pin- holes sizes or densities are also known to cause poor device performances due to a large number of trap-assisted Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 3 www.nature.com/scientificreports/ Figure 2. Microstructures of the different halide perovskite films directly on FTO substrate. (a) Pristine (as- deposited) film without solvent treatment. (b–f) Similar films after solvent treatment using (b) 100% EtOH, (c) 75% EtOH & 25% CB, (d) 50% EtOH & 50% CB, (e) 25% EtOH & 75% CB and (f) 100% CB. (g) The XRD spectra corresponding to each perovskite film. Figure 2. Microstructures of the different halide perovskite films directly on FTO substrate. (a) Pristine (as- deposited) film without solvent treatment. (b–f) Similar films after solvent treatment using (b) 100% EtOH, (c) 75% EtOH & 25% CB, (d) 50% EtOH & 50% CB, (e) 25% EtOH & 75% CB and (f) 100% CB. (g) The XRD spectra corresponding to each perovskite film. recombination centers of trapping and lower carrier mobilities45. With larger crystal grains the overall perfor- mance of the cell will improve since the charge carriers encounter fewer trapping and scattering sources26,30,46.i Large pinholes or high densities of pinholes also cause significant recombination events since they can provide a direct contact between the ETL and HTL47, limiting the charge extraction efficiency. Based on these findings, we aim to identify an optimal tradeoff by combining EtOH and CB as solvent and anti-solvent respectively to achieve simultaneously large grain sizes and uniformly covered perovskite thin film with minimized pinholes. The film treatment with EtOH:CB at a volumetric ratio of 1:3 results in larger grain sizes (5 µm), fewer pinholes and better surface coverage (Fig. 2(e)) as well as excellent crystalline quality, as seen in Fig. 2(g). In contrast, the sample treated with 3:1 ratio of EtOH:CB shows a compact and nearly pinhole-free surface but with extremely small grain sizes (≤0.5 µm), in addition to poor crystal quality as indicated by the XRD patterns (blue curve in Fig. 2(g) and supporting information S1). As expected, increasing the concentration of CB markedly increases the grain size (Fig. Result and Discussion 2(f)), however the size of the pinholes also significantly increases.hi g p gi y The XRD of the perovskite films indicate that all samples match a tetragonal crystal structure with lattice parameters a = b = 8.87 Å and c = 12.65 Å after cell refinement, which is consistent with previous reports40,48,49. A more detailed analysis of the phase and stoichiometry for MAPbI3−xClx has no sign of Cl-based compound or intermediate phase. The conversion of intermediate phases to a fully-crystallized perovskite by annealing was studied systematically using grazing angle X-ray diffraction as shown in Fig. S3. The study was performed at three processing stages: I) as spin-coated, II) after solvent treatment and III) after solvent treatment and thermal annealing. The intermediate phases that are observed in stages I) and II) disappear in stage III (after solvent treatment and subsequent annealing at 125 °C on a hot plate). Here, only peaks from MAPbI3 or MAPbI3−xClx are observed, implying a full transformation of all phases into crystalline perovskite phase. This is consistent with earlier findings12, suggesting a strong ion exchange between I- and Cl- and the formation of MACl in the interme- diate crystallization step before sublimation during annealing as given in equation (2). + + → + ↑ PbI PbCl 4MAI 2MAPbI 2MACl (1) 2 2 3 (1) The absence of other phases such as MAPbCl3 in the final perovskite film agrees with other reports, indicat- ing the intercalation of MAI and reconstruction in the intermediate reaction as the main reason for the recrys- tallization of the intermediate phases50. These results are confirmed by micro Raman mapping measurements (Fig. S4) and consistent with previous reports and there is no indication of MAPbCl3 in the film. In contrast to our findings, few reports also indicated that the crystallization of MAPbCl3 phase51. Yet, the presence of Cl in Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 4 ntificreports/ Figure 3. (a,b) Photoluminescence of perovskite films treated with different mixtures of CB and EtOH. The inset in (a) depicts the device architecture used for PL measurements. (c) Photoluminescence emission intensity evolution for different solvent treatments. Note: the colored squares in (c) match the photoluminescence spectra lines in (a) and (b). (d) UV-Vis absorption spectra after different solvent treatments and photoluminescence of the optimal CB:EtOH = 3:1 treated sample. (e) Statistical analysis of the surface coverage calculated from the SEM images of the treated thin films. Result and Discussion (f) PV performances for PSC devices using various CB:EtOH solvent treatments. www.nature.com/scientificreports/ Figure 3. (a,b) Photoluminescence of perovskite films treated with different mixtures of CB and EtOH. The ( ) d h d h d f ( ) h l Figure 3. (a,b) Photoluminescence of perovskite films treated with different mixtures of CB and EtOH. The inset in (a) depicts the device architecture used for PL measurements. (c) Photoluminescence emission intensity evolution for different solvent treatments. Note: the colored squares in (c) match the photoluminescence spectra lines in (a) and (b). (d) UV-Vis absorption spectra after different solvent treatments and photoluminescence of the optimal CB:EtOH = 3:1 treated sample. (e) Statistical analysis of the surface coverage calculated from the SEM images of the treated thin films. (f) PV performances for PSC devices using various CB:EtOH solvent treatments. Figure 3. (a,b) Photoluminescence of perovskite films treated with different mixtures of CB and EtOH. The inset in (a) depicts the device architecture used for PL measurements. (c) Photoluminescence emission intensity evolution for different solvent treatments. Note: the colored squares in (c) match the photoluminescence spectra lines in (a) and (b). (d) UV-Vis absorption spectra after different solvent treatments and photoluminescence of the optimal CB:EtOH = 3:1 treated sample. (e) Statistical analysis of the surface coverage calculated from the SEM images of the treated thin films. (f) PV performances for PSC devices using various CB:EtOH solvent treatments. the crystal structure still remains under debate since the detection is beyond the sensitivity limit of many instru- ments. However, the possibility of a Cl doping in the structure has been proven using DFT calculations and slight volume changes compared to the volume of pure tetragonal MAPbI3 52,53.i p g g y The PL quenching for the films treated with different solvent mixtures is clearly highlighted in Fig. 3(a–c). As expected, all samples display significant emission centered around 763 nm54,55. However, the treatment with 100% CB yields a broader PL emission with dominant features at 729 and at 763 nm. The origin of the shoulder may originate from residual PbI2, consistent with the XRD results in Fig. 2. The highest PL peak emission is observed The PL quenching for the films treated with different solvent mixtures is clearly highlighted in Fig. 3(a–c). As expected, all samples display significant emission centered around 763 nm54,55. Result and Discussion However, the treatment with 100% CB yields a broader PL emission with dominant features at 729 and at 763 nm. The origin of the shoulder may originate from residual PbI2, consistent with the XRD results in Fig. 2. The highest PL peak emission is observed Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 5 www.nature.com/scientificreports/ Figure 4. Current density - Voltage (J-V) characteristics of the best-performing cell fabricated using CB:EtOH = 3:1 solvent treated halide perovskite thin film (C0.75CB,0.25EtOH). The J-V curve is measured at 10 mV/s scan speed in both forward-sweep (FS) and reverse-sweep (RS). (b) External quantum efficiency (EQE) spectra of the best-performing cell (C0.75CB,0.25EtOH). (c) Compact perovskite film with larger grains (5 µm) and fewer pinholes for C0.75CB,0.25EtOH. The inset shows a picture of the solar cell device. (d) Cross-sectional SEM image of the PSC device structure. Figure 4. Current density - Voltage (J-V) characteristics of the best-performing cell fabricated using CB:EtOH = 3:1 solvent treated halide perovskite thin film (C0.75CB,0.25EtOH). The J-V curve is measured at 10 mV/s scan speed in both forward-sweep (FS) and reverse-sweep (RS). (b) External quantum efficiency (EQE) spectra of the best-performing cell (C0.75CB,0.25EtOH). (c) Compact perovskite film with larger grains (5 µm) and fewer pinholes for C0.75CB,0.25EtOH. The inset shows a picture of the solar cell device. (d) Cross-sectional SEM image of the PSC device structure. in the 100% EtOH treated sample, but the peak intensity decreases by ~25% compared to the sample treated with the 1:3 CB:EtOH solvent ratio. In contrast, the PL emission decreases by ~99% when treated with 3:2 CB:EtOH solvent ratio. This significant drop in PL intensity can be attributed to a decrease in the annihilation of charge carriers at the radiative trap states. Sharp fall in PL intensity is consistent with SEM images as shown in Fig. 2, and a significant change in morphology is observed. A 100% EtOH solvent treatment results in small grain sizes which in turn leads to a high density of grain boundaries and other trap states that cause an increase in emission at the cost of charge carriers. Similarly, PL spectra of other thin films treated with CB:EtOH show a more pronounced reduction of the PL peak. Figure 3(d) compares the optical absorption spectra of the solvent-engineered halide perovskite samples and also shows the PL peak emission wavelength for CB:EtOH = 3:1 on the same curve. Result and Discussion The onset of absorption peaks observed are approximately at 763 nm, matching the band edge of the crystalline per- ovskite and PL peak emission. A significantly higher absorption for longer wavelengths (≥560 nm) is measured for the 100% CB treated sample, because of large grain sizes that induce greater scattering and thereby absorp- tion56. However, a significantly stronger optical absorption is measured for the 3:1 CB:EtOH treated sample, which is consistent with XRD results indicating the formation of a superior crystalline film providing a better broadband absorption that can presumably enhance solar cell performance. p p y p Figure 3(e) shows an assessment of the surface coverage and grain size distributions using Image J analysis of the SEM images obtained for different solvent treatments. The pristine film shrinkage yields the highest pin- hole area of 180.07 µm2 (Fig. S2(a) and Table S1) and poor surface coverage with only 51.9% of the total surface (Fig. 3(f)). The 100% CB treated specimen also yields a relatively poor surface coverage (65.53%). However, a highly-compact perovskite film can be achieved using the CB:EtOH = 1:3 treatment, as confirmed by its 99.9% surface coverage. However, the majority of the grains are below 400 nm in diameter for the CB:EtOH = 1:3, com- pared to more than 4 µm for the pristine or 100% CB treatments. Interestingly, an optimal co-solvent treatment fulfilling the optimal surface coverage and grain size is achieved by CB:EtOH = 3:1 as shown in Fig. 3(e) where the curves of the average grain size and surface coverage curves cross at the higher values. g g g g Using the optimized CB: EtOH = 3:1 solvent treatment combining large grains with low pinhole densities, planar geometry solar cell devices (C0.75CB,0.25EtOH) are fabricated and their PV performances are measured. The current-density versus voltage (J-V) characteristics and the external quantum efficiency (EQE) measurements are shown in Fig. 4(a,b) respectively. The cell shows large open-circuit voltage (Voc), short-circuit current density (Jsc) and fill-factor (FF) up-to 0.92 V, 23.7 mA/cm2 and 61.7%, respectively under 1 sun (100 mW/cm2) illumi- nation and voltage scanning rate of 10 mV/s. The best cell exhibits a PCE of 14% in reverse voltage sweep (RS), with only a slight deviation from a forward sweep (low-hysteresis). Result and Discussion The EQE measurement confirms the excellent Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 6 www.nature.com/scientificreports/ light harvesting quality of the cell, exceeding 80% from 350 to 745 nm even though this measurement was taken 3 weeks after fabrication. The maximum EQE (90%) is even higher than the maximum transmission of bare FTO (85%) (Fig. S6), simply indicating that the reflection is significantly reduced due to the lower step in refraction index from adding the perovskite layer, which still reduces the apparent EQE value. As depicted in Fig. S2(b), some solar cells fabricated using the same processing technique as our best device performed slightly better in terms of hysteresis, but slightly lower in PCE. The J-V characteristics of the best cell in the dark and under 1 sun irradiation is measured (Fig. S5(a)). A modest enhancement in PV performance can be observed when the illuminated area is reduced as shown in Fig. S5(b). While we observe an increase in Jsc under reduced area, the hysteretic behavior is also more pronounced. This might be related to the difference in the density of pinholes in reduced area conditions. Discrepancies between the forward and reverse sweeps is dependent on various param- eters including scan rate, voltage bias and light-doping. In particular the hysteresis behavior in planar PSC has been thoroughly investigated, as it is one of the key parameters for performance comparison46,57–60. The trapping and de-trapping process at the interface and grain boundaries, ferroelectric properties, iodine ion movements and other causes within the perovskite materials are claimed to be responsible for the hysteretic behavior and lower extraction efficiencies61. Although the cause of the hysteresis remains debatable, it was suggested that iodine ion movements are likely to be one of the main factors58.ii y To confirm the correlation between film morphology and device performance, we can compare solar cells fabricated using different CB:EtOH solvent treatments (Fig. 3(f) and Table S2). To avoid the influence of factors such as humidity and temperature variations, all the samples were fabricated within a few minutes in ambient air conditions. As expected, these measurements confirm that the change in grain morphology (shown in Fig. 3(f)) affects significantly the overall performance of the devices. The lower PCE is recorded when the samples are treated with 100% EtOH, while the highest PCE is achieved when the film is treated with CB:EtOH at 3:1 vol- umetric ratio. Result and Discussion As detailed in Table S2, the improved cell efficiency is mainly prompted by an improvement of open-circuit voltage and short-circuit current density, with FF reaching 60%. As expected from the SEM image in Fig. S1(d), the compact perovskite thin film delivered a larger FF despite the very low short-circuit current density and PCE, which is attributed to increased losses of charge carriers occurring at the numerous grain bounda- ries62,63. The shunt resistance, which is known to reduce the Voc and FF, seems to significantly affect the perfor- mance of all samples. The incomplete coverage of the perovskite film (cf. Fig. 2, 3 and S1) causes the formation of shunt pathways accompanied by an internal charge carrier recombination. These results confirm that the PCE depends strongly on film coverage, pinhole density and crystal grain sizes. p g yi g p y y g Electrochemical Impedance Spectroscopy (EIS) was also employed on representative samples to probe the electronic properties and the interfacial recombination within the solar cells and their relation to the perovskite film surface morphologies resulting from different CB:EtOH treatments (Fig. 5). For the interpretation of the spectra, we used a circuit model (inset in Fig. 5(a)), to account for the chemical-physical processes taking place at the different interfaces in the PSC. Figure 5(a,b) shows the Nyquist plot of the complex impedance for the two heterojunction solar cells under 0 V biasing in dark, presenting characteristic impedance patterns with two arcs for both cells. The arc at high frequencies in Fig. 5(b) is generally associated with the geometrical capacitance (Cbulk) in par- allel with an equivalent resistance that takes into account the carriers’ conductivity64,65.The second arc at medium frequencies is related to the recombination processes inside the solar cell57,58,66. As visible in Fig. 5(a), the most remarkable feature is that the best cell fabricated using CB: EtOH = 3:1 treatment (C0.75CB,0.25EtOH) leads to the larger arc indicating reduced recombination events. This is consistent with the J-V measurements that show a higher efficiency for this cell compared to the 25% CB & 75% EtOH (CB:EtOH = 1:3) treated cells. Further con- firmation arises from the analysis of the recombination resistance (Rrec) when varying the voltage bias (Fig. 5(c)). At any Vbias, the value of Rrec for the C0.75CB,0.25EtOH cell is at least 50× compared to the 25% CB, 75% EtOH cell. Result and Discussion The value of Rrec is inversely proportional to the charge recombination rate, so the higher value in the C0.75CB,0.25EtOH cell also indicates a significant suppression of the recombination rate. This substantially improves the injection of electrons in the photoanode, resulting in an increase in Jsc as shown in Fig. 4(a,b). The EIS results support the interpretation of the PL measurements, indicating that a reduction in grains size significantly increases the prob- ability of recombination at the grain boundaries, thereby reducing the PCE of the solar cells. y g y g Another interesting feature that can be extrapolated from the EIS is the general behavior of the overall capaci- tance. Figure 5(d) displays the spectrum of the real part of the capacitance in the dark at short-circuit conditions. While the best 75% CB, 25% EtOH-treated perovskite thin film presents a well-defined plateau at high frequen- cies, this plateau feature is not visible for the 25% CB,75% EtOH. This capacitance can be identified as the bulk capacitance Cbulk of the perovskite layer, from which we can calculate the dielectric constant εr of the perovskite thin film. Considering a 400 nm thick film and a Cbulk = 50 nF/cm2, we obtain εr = 22.5, which is consistent with the values previously reported for similar systems in the literature65,67. At medium frequencies, we observe a shoulder that can be identified as the chemical capacitance, in addition to a static dielectric capacitance at low frequencies identifiable for both cells. The giant capacitance value observed at low frequencies is consistent with previous reports and reflects the ion migration to the electrodes and charge compensation by the external elec- trodes65,68. The main difference between the two cells is the absence of the plateau at high frequencies for the 25% CB, 75% EtOH cell that shows a broadening of the chemical capacitance up to 1 MHz. This effect has been previously reported for similar devices and is related to the high roughness at the interfaces between thin-film multilayer contacts65,69. This confirms again the difference in the thin film morphology indicating a higher rough- ness at the planar heterojunctions as a result of smaller grain sizes and higher grain boundaries for the 25% CB and 75% EtOH treated cell. Besides SEM and PL, EIS gives additional supporting evidence of grain size and grain boundaries in the film being the main factors influencing the PCE of such perovskite-based solar cells. Result and Discussion Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 7 www.nature.com/scientificreports/ Figure 5. EIS analysis of two PSCs: perovskite thin film treated with mixture of solvents EtOH and CB, one with 75% CB & 25% EtOH (C0.75CB,0.25EtOH) and another one with 25% CB &75% EtOH. (a) Nyquist plot in dark at 0V. The inset in (a) shows the equivalent circuit. (b) A zoom-in of the Nyquist plot in (a). (c) Recombination resistance from EIS measurements in dark. (d) The real part of the capacitance as a function of the frequency for the two cells measured in the dark at 0 V bias. Figure 5. EIS analysis of two PSCs: perovskite thin film treated with mixture of solvents EtOH and CB, one with 75% CB & 25% EtOH (C0.75CB,0.25EtOH) and another one with 25% CB &75% EtOH. (a) Nyquist plot in dark at 0V. The inset in (a) shows the equivalent circuit. (b) A zoom-in of the Nyquist plot in (a). (c) Recombination resistance from EIS measurements in dark. (d) The real part of the capacitance as a function of the frequency for the two cells measured in the dark at 0 V bias. Methods Etching FTO and deposition of blocking layer. A Fluorine-doped tin oxide-coated glass substrate (Tec 8, Osilla Limited) was partly etched using Zn paste and 37% concentrated HCl (Sigma-Aldrich Canada Co.) diluted in deionized water (HCl to deionized water volume ratio 1:3). After etching FTO, we followed a three step ultrasonic cleaning procedure: (i) 20 min ultrasonication in deionized water (5% detergent) mix, followed by rinsing in deionized water, (ii) 5 min ultrasonic bath in acetone and dried in air, (iii) 5 min ultrasonic bath in Isopropanol, rinsing in deionized water and drying with pressurized nitrogen gas blowing. p p g y g p g g g UV-Ozone (UVO, Ossila Ltd) was used to clean the surface for 20 minutes before spin-coating the blocking layer so that possible contaminants are removed completely besides improving surface hydrophilicity. A TiO2 blocking layer composed of 20-nm-sized particles (Ti-Nanoxide BL/SC TiO2 BL, Solaronix SA) was then depos- ited by spin-coating at 5000 r.p.m. for 30 s followed by a crystallization heat treatment at 550 °C for 45 minutes. The compact TiO2 is deposited on FTO from a commercially available Titania solution which is spin-coated and annealed using the recommended method from the company. Preparation of Perovskite Precursor Solution. Unless stated otherwise, all materials and solvents used for the preparation of the halide perovskite precursor were purchased from Sigma-Aldrich. The precursor is prepared by using commercially available PbI2, PbCl2 and MAI (Solaronix SA). The solution is prepared in DMF, (99.8%) solvent in a molar concentration of 0.5M PbI2, 0.5M PbCl2, 2M MAI. A bright yellow and transparent perovskite solution is obtained by the addition of 2% (by volume) of a 36.5–38% concentrated HCl into the solu- tion and stirring 20 minutes. 8 Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 www.nature.com/scientificreports/ Device Fabrication. A planar heterojunction device is fabricated in the following order multilayer stacking order: FTO/compact TiO2/perovskite/Spiro-MeOTAD/Au. The patterned FTO glass substrates coated with com- pact TiO2 were first treated with UV Ozone for 5 minutes to remove possible impurities, followed by spin-coating of 120 µl perovskite solution at three step spin-coating speed: 1000 rpm for 10 s, 2000 rpm for 20 s and 6000 rpm for 15 s. The mixed solvents described above were prepared for various concentration ratios and 300 µl are applied in each case immediately at the beginning of 6000 rpm spinning. Methods The spin-coated samples are then annealed at 125 °C for 15 minutes for complete crystallization. We then use the highly efficient hole extracting organic material spiro-MeOTAD which is prepared by following an existing procedure70. A volume of 40 µl of spiro-MeOTAD solution is spin-coated at 4000 rpm for 30 s, after the samples are brought to room temperature. The solution is prepared in ambient conditions by dissolving 144.6 mg of sublimed spiro-MeOTAD (99.5% purity, FrontMaterials Co., Ltd) dissolved in 2 ml chlorobenzene and then added 56.6 μl of 4-tert-butylpyridine, 35 μl lithium bis (trifluoromethanesulfonyl)imide (LiTFSI) solution (520 mg LI-TSFI in 1 ml acetonitrile, 99.8%) and 58 μl of tris(2-(1H-pyrazol-1-yl)-4-tertbutyl pyridine) cobalt(III) bis(trifluoromethylsulphonyl) imide (FK209, Dynamo) solution (300 mg FK209 in 1 ml acetonitrile). The humidity during all synthesis and fabrication pro- cesses was around 40% and the temperature ranged between 20 and 25 °C. Finally, after overnight storage of the sample in atmospheric conditions, 80 nm gold is sputtered deposited at a rate of 0.2 nm/s (Cressington 208 HR Sputter coater, Ted Pella Inc). Device Characterization. The microstructural evolution and morphology of the perovskite thin films are characterized using scanning electron microscope (SEM, Joel JSM-6300F: Japan). The crystallinity and phase of the perovskite films are determined by X-ray diffraction (XRD, D8 Advance: Bruker). The J–V characteristic curves of the PSCs are measured using a high precision sourcing and measuring instrument (Keysight B2901A precision source and measuring unit) under AM 1.5 illumination at 100 mW cm−2 (cell to simulator distance of ~16 cm) of a solar simulator (SLB 300A, Ocean Optics Inc.) under irradiance of the active cell area of 5.75 mm2. The UV-Vis absorption spectra of the perovskite films are recorded on a UV-Vis spectrophotometer (Lambda 20: Perkin Elmer). ) The EQE of the devices are measured at room temperature in ambient atmosphere using a monochromator equipped with a xenon arc lamp. The monochromatic light is chopped at a frequency of 30 Hz and focused onto the sample. The active area is defined using a circular aperture with a diameter of 0.5 mm. The photocurrent at each wavelength I(λ) is measured with a lock-in amplifier (Ametek 1256) at 0 V. The light power at each wave- length P(λ) is measured, through the same aperture, with a calibrated photodiode (Newport 918D) placed at the same position of the device. = λ λ λ hcI q P EQE(%) 100 ( )/ ( ) = λ λ λ hcI q P EQE(%) 100 ( )/ ( ) where c is the speed of light, h is the Planck constant and q is the electronic charge.h EIS was conducted in dark using a SOLARTRON 1260 A Impedance/Gain-Phase Analyzer. The applied AC signal is 10 mV in amplitude, in the frequency range between 0.1 Hz and 1 MHz. The applied bias during meas- urements is between 0 V and 800 mV. All the samples are measured inside a Faraday cage. The obtained spectra are fitted with Z-View software (v3.0, Scribner Associate, Inc.) by applying an appropriate equivalent circuit. Conclusions and Perspectives We presented a new mixed solvent treatment approach at ambient condition for synthesizing highly oriented perovskite grain crystallites with statistically controlled sizes and pinhole densities that significantly affect the performance of the perovskite-based solar cells. Crystal grains larger than 5 µm with minimal grain bounda- ries and lowest pinhole densities are achieved using an optimized solvent treatment using a mixed 75% CB and 25% EtOH solvent solutions. The devices are fabricated by sandwiching a halide perovskite thin film between charge-selective layers entirely in ambient air without mesoporous scaffold. The solar cell performances are ana- lyzed and characterized using SEM micrographs, photoluminescence quenching and EIS measurements. In a sim- ple planar geometry solar cells, we find that the density and size of the pinholes are the dominant factors, which affect the performance of the solar cells. Our work defines a liquid solvent processing approach that can be further developed for large area thin film deposition and mass production of high quality perovskite films for PV or other optoelectronic applications. This methodology could be further developed into a vapor phase solvent technique, potentially allowing to obtain a more homogeneous and smooth film through a vapor solvent crystallization pro- cess. Our method is promising for cost-effective processing of perovskite-based solar cells. Methods Then, the EQE(%) value at each wavelength is determined using the following equation: (2) 1. Sutherland, B. R. & Sargent, E. H. Perovskite photonic sources. Nat. 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Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 9 Acknowledgements g We acknowledge support from the Canada Foundation for Innovation for establishing advanced research infrastructure and its operation. R.N., S.G.C. and F.R. are thankful to the NSERC Discovery Program. F.R. is grateful to the government of China for a short-term Chang Jiang chair professorship and Sichuan province for a short term 1000 talent award. F.R. is also supported by the Canada Research Chair in Nanostructured Materials. S.G.C. also thanks the Canada Research Chair program for its support. www.nature.com/scientificreports/ High-efficiency robust perovskite solar cells on ultrathin flexible substrates. Nat. Commun. 7, 10214 (2016). gfi y pl 64. Gonzalez-Pedro, V. et al. General Working Principles of CH3NH3PbX3 Perovskite Solar Cells. Nano Lett. 14, 888–893 (2014 fil 64. Gonzalez-Pedro, V. et al. General Working Principles of CH3NH3PbX3 Perovskite Solar Cells. Nan g p 5. 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Scientific RePorTS \| (2018) 8:12885 \| DOI:10.1038/s41598-018-31184-0 Additional Information upplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-31184-0. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-31184-0. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W2284497455	https://biomath.math.bas.bg/biomath/index.php/biomath/article/download/j.biomath.2015.12.171/pdf	English	null	Which Matrices Show Perfect Nestedness or the Absence of Nestedness? An Analytical Study on the Performance of NODF and WNODF	Biomath	2,016	cc-by	5,796	ORIGINAL ARTICLE Citation: N. F. Britton, M. Almeida Neto, Gilberto Corso, Which Matrices Show Perfect Nestedness or the Absence of Nestedness? An Analytical Study on the Performance of NODF and WNODF, Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 Which Matrices Show Perfect Nestedness or the Absence of Nestedness? An Analytical Study on the Performance of NODF and WNODF N. F. Britton∗, M. Almeida Neto †, Gilberto Corso ‡ ∗Department of Mathematical Sciences and Centre for Mathematical Biology University of Bath, Bath BA2 7AY, UK. Email: n.f.britton@bath.ac.uk †Departmento de Ecologia, Universidade Federal de Goi´as, 74001-970 Goiˆania-GO, Brazil, Email: marioeco@gmail.com ‡Departamento de Biof´ısica e Farmacologia, Centro de Biociˆencias, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN, Brazil, Email: corso@cb.ufrn.br Received: 25 April 2015, accepted: 17 December 2015, published: 16 January 2016 ceived: 25 April 2015, accepted: 17 December 2015, published: 16 January 2016 Abstract—Nestedness is a concept employed to describe a particular pattern of organization in species interaction networks and in site-by-species incidence matrices. Currently the most widely used nestedness index is the NODF (Nestedness metric based on Overlap and Decreasing Fill), initially presented for binary data and later extended to quantitative data, WNODF. In this manuscript we present a rigorous formulation of this index for both cases, NODF and WNODF. In addition, we characterize the matrices corresponding to the two extreme cases, (W)NODF=1 and (W)NODF=0, rep- resenting a perfectly nested pattern and the absence of nestedness respectively. After permutations of rows and columns if necessary, the perfectly nested pattern is a full triangular matrix, which must of course be square, with additional inequalities between the elements for WNODF. On the other hand there are many patterns characterized by the total absence of nestedness. Indeed, any binary ma- trix (whether square or rectangular) with uniform row and column sums (or marginals) satisﬁes this condition: the chessboard and a pattern reﬂecting an underlying annular ecological gradient, which we shall call gradient-like, are symmetrical or nearly symmetrical examples from this class. Keywords-biogeography, interaction networks, nestedness, bipartite networks I. INTRODUCTION In cases a network is perfectly nested if (i) there is a hierarchy of plant species such that the set of animal (pollinator or seed disperser) species interacting with any plant is a subset of the set of animals interacting with any plant further up the hierarchy, and (ii) there is a similar hierarchy of animals. It is clear that in such a network generalist species interact with specialists and generalists, but specialists do not interact with each other. Null models are an important methodological tool widely used in ecology to test model ﬁtting, perform statistical tests or test the validity of indices and measures [10]. In order to assess an index a large set of empirical or artiﬁcial data is used as a data bank to explore its limitations and fragility. This process has already been used to test a set of nestedness indices [22]. Null models are necessary because statistical tests are otherwise always questionable by limitation in the range of tested parameters, interpretation bias of the results, or equivocal choice of random models. These studies emphasis the necessity of analytic results to strength conﬁdence about nestedness indices and their applications. The original deﬁnition of the NODF index depends on how the rows and columns are ordered, and a frequently used software for calculating NODF explicitly asks the user if they would like to order the matrix according to row and column sums (or marginals) [11]. In this paper we employ a deﬁnition of (W)NODF in which the matrix is previously sorted before the computation of the index. In this paper we give rigorous deﬁnitions of NODF and WNODF and prove two mathemat- ical theorems in each case. For the sake of clarity, and for historical reasons, we explore separately qualitative (binary) and quantitative (weighted) networks. The treatment of the qualitative case is more intuitive and helps the reader to follow the analytic developments. In section 2 we start with a formal deﬁnition of NODF and WNODF and present two theorems that characterize the extreme cases, NODF = 0 and WNODF = 0 corresponding to absence of nestedness, and NODF = 1 and WNODF = 1 corresponding The proper mathematical framework for intro- ducing nestedness is in the context of bipartite networks. From a general perspective we consider a bipartite network formed by two sets S1 and S2. I. INTRODUCTION Observing nature is one of the most fascinating experiences in life. A honeybee visits a daisy, a rosemary, and other ten different species. Another bee of the same family is specialized in just one ﬂower that by its turn is visited by twenty diverse pollinators. Once we put together the community of pollinators and ﬂowers an intricate mutualist network arises [5]. In the opposite side of life a caterpillar feed on two asteraceae species which are eaten by another couple of insects, the full set of herbivorous and plants forms a complex antagonist network. An central quest in ecology Page 1 of 9 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... of communities today is the search for patterns in networks that can distinguish between mutualist and antagonist webs [13, 21]. One network pattern that is part of this answer is nestedness, the subject of this manuscript. based on Overlap and Decreasing Fill, is an index that was introduced in [2] and that has been widely used in the literature. An extension of this index to quantitative networks, WNODF, was recently proposed [3], and we include it in our analysis because of the importance of quantitative networks, specially for networks of interacting species [9, 13]. Nestedness is a concept used in ecology to study a speciﬁc formation pattern in species interaction networks and in site-by-species incidence matri- ces. In general terms, nestedness is a speciﬁc kind of topological organization in adjacency matrices of bipartite networks where any vertex S, with m links, tend to be connected to a subset of the vertices connected to any other vertex with n links, where n > m. The nestedness concept was ﬁrst introduced by [8] to characterize species distribution pattern in a spatial set of isolated habitats such as islands. In a perfectly nested pattern site-by-site incidence matrix there is a hierarchy of sites such that the set of species inhabiting any site is a subset of the set inhabiting any site further up the hierarchy. When applied to describe the topological organization in ecological interaction networks this new nestedness concept was ﬁrst used to networks formed by pollinators and ﬂowering plants and by seed dispersers and ﬂesh-fruited plants [4, 12]. Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 A. Qualitative matrices, the case NODF We shall consider a bipartite network of set S1, containing m elements, and set S2, containing n elements, with quantitative data for the frequency wij of the interactions between element i of set S1 and element j of set S2. In the simplest case wi,j is equal to 1 or 0, a situation corresponding to the binary network, qualitative network or pres- ence/absence matrix. The adjacency matrix for the network is the m× n matrix A = (aij), where aij is deﬁned by: In order to deﬁne NODF we start with the row paired-overlap quantiﬁer POr ij as the fraction of unit elements in row j that are matched by unit elements in row i, and the column paired-overlap quantiﬁer POc kl as the fraction of unit elements in column l that are matched by unit elements in row k, so that POr ij = Pn p=1 aipajp Pn p=1 ajp , POc kl = Pn q=1 akqalq Pn q=1 alq . (5) (5) aij =      1 if wij ̸= 0, so that element i of S1 and element j of S2 are linked 0 if wij = 0, so that they are not linked. (1) aij =      1 if wij ̸= 0, so that element i of S1 and element j of S2 are linked 0 if wij = 0, so that they are not linked. (1) Note that POr ij is the fraction of elements of S2 linked to element j of S1 that are also linked to element i of S1, and similarly for POc kl. Deﬁne the row paired nestedness NP r ij between rows i and j, and the column paired nestedness NP c kl between columns k and l, by We deﬁne the row and column marginal totals MT r i and MT c l by NP r ij = DF r ijPOr ij + DF r jiPOr ji, (6) NP c kl = DF c klPOc kl + DF c lkPOc lk. (7) NP r ij = DF r ijPOr ij + DF r jiPOr ji, (6) MT r i = n X j=1 aij and MT c l = m X k=1 akl, (2) (6) (2) NP c kl = DF c klPOc kl + DF c lkPOc lk. A. Qualitative matrices, the case NODF (7) (7) so that MT r i is the number of elements of S2 interacting with element i of S1, and MT c l is the number of elements of S1 interacting with element l of S2. Deﬁne the row and column decreasing-ﬁll indicators DF r ij and DF c kl by Note that these deﬁnitions are valid whatever the signs of MT r i −MT r j and MT c k −MT c l . Finally, deﬁne the row and column nestedness metrics NODF r and NODF c by DF r ij = ( 1 if MT r i > MT r j , 0 if MT r i ≤MT r j , (3) DF c kl = ( 1 if MT c k > MT c l , 0 if MT c k ≤MT c l . (4) NODF r = Pm i=1 Pm j=i+1 NP r ij 1 2m(m −1) , (8) NODF c = Pn k=1 Pn l=k+1 NP c kl 1 2n(n −1) , (9) DF r ij = ( 1 if MT r i > MT r j , 0 if MT r i ≤MT r j , (3) DF c kl = ( 1 if MT c k > MT c l , 0 if MT c k ≤MT c l . (4) NODF r = Pm i=1 Pm j=i+1 NP r ij 1 2m(m −1) , (8) NODF c = Pn k=1 Pn l=k+1 NP c kl 1 2n(n −1) , (9) (8) (3) (9) (4) and the overall nestedness metric NODF as a weighted average of these, by and the overall nestedness metric NODF as a weighted average of these, by Note that, if i < j, so that row i is above row j, then DF r ij = 1 if and only if element i of set S1 is linked with more elements of set S2 than element j of S1; similarly, if k < l, so that column k is to the left of column l, then DF c kl = 1 if and only if element k of S2 is linked with more elements of set S1 than element l of S2. I. INTRODUCTION Nestedness is characterized by several indices [22, 18] and it is not the objective of this work to compare them. Here we focus on the NODF index, which has a clear mathematical deﬁnition that allows further analytic developments. The NODF index, an acronym for Nestedness metric Page 2 of 9 Page 2 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... to the perfectly nested arrangement. In section 3 we summarize the main ideas of the work and put the results in a broader context. permute the rows and columns of the matrix so that MT r i ≥MT r j whenever i < j, and MT c k ≥MT r l whenever k < l, but the deﬁnition does not require this to be done. A. Qualitative matrices, the case NODF If our network is disconnected, then NODF = 0 if and only if all elements of S1 are linked to the same number of elements of S2, and all elements of S2 are linked to the same number of elements of S1 within each connected component, or compartment. This is a necessary and sufﬁcient condition for NODF = 0. There are many networks that satisfy this condition. For example in ﬁgure 1 we show a 9×6 network where each of the nine elements of S1 interact with a dif- ferent pair of elements of S2, so that each element of S2 interacts with three elements of S1. Figure 1(c) does not resemble any of the NODF = 0 conﬁgurations exhibited in the literature [4, 15], which are all (including the chessboard after row and column permutation) compartmented with full connectivity within the compartments. Case 1(d) seems to reﬂect an underlying cyclic ecological gradient [15], and we call it gradient-like. The requirement that the gradient be cyclic is manifest and it is occupied to fulﬁl the rule that there should be two nonzero elements in each row and three in each column. It is interesting that the dimensions (m, n) of the adjacency matrix obey a constraint in the NODF = 0 case. The total number of matrix elements that is distributed along columns and rows should follow the relation: n X i=1 MT c i = m X j=1 MT r j . (11) (11) As MT c i and MT r j are constants we can rewrite 11 in the form nMT c = mMT r. 2) Conditions for NODF = 1: We now wish to characterize all matrices for which NODF = 1, see ﬁgure 2. It is clear that NODF = 1 if and only if both NODF r = 1 and NODF c = 1, so let us ﬁrst consider the conditions under which NODF r = 1. This is true if and only if NP r ij = 1 for all pairs (i, j) of rows. From equation (6), NP r ij = 1 implies that MT r i ̸= MT r j , so that either DF r ij = 1 or DF r ji = 1. A. Qualitative matrices, the case NODF It is always possible to weighted average of these, by NODF = m P i=1 m P j=i+1 NP r ij + nP k=1 nP l=k+1 NP c kl 1 2m(m −1) + 1 2n(n −1) . (10) (10) oi.org/10.11145/j.biomath.2015.12.171 Page 3 of 9 Page 3 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... 1) Conditions for NODF = 0: Our objective is to characterize all matrices for which NODF = 0. It is clear that NODF = 0 if and only if both NODF r = 0 and NODF c = 0, so let us ﬁrst consider the conditions for which NODF r = 0. This is true if and only if NP r ij = 0 for all pairs (i, j) of rows. From equation (6), NP r ij = 0 if and only if either MT r i = MT r j , so that DF r ij = DF r ji = 0, or Pn p=1 aipajp = 0, so that POr ij = POr ji = 0. In other words, either rows i and j have the same number of unit elements, so that elements i and j of S1 interact with the same number of elements of S2, or there is no p in S2 that interacts with both i and j. If our bipartite network is connected, then it is possible to move from any i in S1 to any other j in S1 by following a path composed of edges of the network from S1 to S2 to S1 and so on. Hence, in this connected case, NODF r = 0 if and only if all elements of S1 are linked to the same number of elements of S2. Similarly, for a connected network, NODF c = 0 if and only if all elements of S2 are linked to the same number of elements of S1, and NODF = 0 if and only if both these conditions hold. Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 A. Qualitative matrices, the case NODF If there are more elements of S2 interacting with element i in S1 than with j in S1, then MT r i > MT r j , so that DF r ij = 1, DF r ji = 0. Then we also require that Pn p=1 aipajp = Pn p=1 ajp, so that POr ij = 1, in other words that aip = 1 whenever ajp = 1. Thus all elements of S2 interacting with element j in S1 also interact with element i in S1, or the set of elements of S2 interacting with j in S1 is nested within (or a proper subset of) the set of elements of S2 interacting with i in S1 . Similarly, if there are more elements of S2 interacting with j in S1 than with i in S1, then the set of elements of S2 interacting with i in S1 must be nested within the set of elements of S2 interacting with j in S1. Similar results hold for NODF c = 1, so that the set of elements of S1 interacting with any k in S2 must be a proper subset or superset of the set of S1 elements interacting with any other l in S2. For NODF = 1, all (S1 and S2) interaction sets must be proper sub- or supersets, so that by the pigeon- hole principle we must have m = n, and it must be possible to permute the rows and columns of the matrix A so that aij = 1 if i ≥j, aij = 0 otherwise. The matrix with NODF = 1 is the Page 4 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... , f f 1: Some NODF = 0 patterns. Panels (a) and (b) represent the same matrix after permutation of lines umns; this non-chessboard tiling is a composition of three disconnected networks. Panels (c) and (d) s connected networks that have NODF = 0, since MT c i = 3 and MT r j = 2 for all i and j respectively. C represents a gradient-like structure. Fig. 1: Some NODF = 0 patterns. Panels (a) and (b) represent the same matrix after permutation of lines columns; this non-chessboard tiling is a composition of three disconnected networks. B. Quantitative matrix, the case WNODF To construct the WNODF index we deﬁne the row-pair dominance quantiﬁer Dr ij as the fraction of non-zero weights in row j that are dominated by (less than) the corresponding weight in row i, and the column-pair dominance quantiﬁer Dc kl as the fraction of non-zero weights in column l that are dominated by the corresponding weight in column k, so that WNODF r = Pm i=1 Pm j=1 DNr ij m(m −1) , (16) WNODF c = Pn k=1 Pn l=1 DNc kl n(n −1) , (17) (16) (17) and the overall weighted nestedness metric WNODF as a weighted average of these, by and the overall weighted nestedness metric WNODF as a weighted average of these, by Dr ij = Pn p=1 H(wip −wjp)H(wjp) MT r j , (12) Dc kl = Pm q=1 H(wqk −wql)H(wql) MT c l , (13) (12) (12) WNODF = m P i=1 m P j=1 DNr ij + nP k=1 nP l=1 DNc kl m(m −1) + n(n −1) . (13) MT c l where H is the Heaviside step function with H(0) = 0. Note that Dr ij is the fraction of elements of S2 interacting with j in S1 that interact more strongly with i in S1, and similarly for Dc kl. Note that, when calculating NODF for qualitative networks, the quantity corresponding to Dr ij is the row-pair overlap quantiﬁer POr ij which is the fraction of elements of S2 interacting with j in S1 that also interact with i in S1, and similarly for Dc kl; the requirement that the interaction be stronger is not (and cannot be) applied. This is the essential difference between the index WNODF for quantitative networks and the index NODF for qualitative ones. Now deﬁne the row-pair dominance nestedness between rows i and j, and the column-pair dominance nestedness between columns k and l, by 1) Conditions for WNODF = 0: The treat- ment of WNODF = 0 shares some similarities with the previous analysis of NODF = 0. To characterize all matrices for which WNODF = 0 we proceed as follows. It is clear that WNODF = 0 if and only if both WNODF r = 0 and WNODF c = 0, so let us ﬁrst consider the conditions for which WNODF r = 0. This is true if and only if DNr ij = 0 for all pairs (i, j) of rows. A. Qualitative matrices, the case NODF Panels (c) and (d) sh two connected networks that have NODF = 0, since MT c i = 3 and MT r j = 2 for all i and j respectively. C (d) represents a gradient-like structure. Fig. 1: Some NODF = 0 patterns. Panels (a) and (b) represent the same matrix after permutation of lines and columns; this non-chessboard tiling is a composition of three disconnected networks. Panels (c) and (d) show two connected networks that have NODF = 0, since MT c i = 3 and MT r j = 2 for all i and j respectively. Case (d) represents a gradient-like structure. Page 5 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... then DF r ij = 0 and DF r ji = 1, so DNr ij = Dr ji, and (iii) if MT r i = MT r j then DF r ij = DF r ji = 0, and DNr ij = 0. Finally, deﬁne the row and column weighted nestedness metrics WNODF r and WNODF c by full triangular matrix, unique up to permutation of rows and columns. Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 C. Conditions for WNODF = 1 We now wish to characterize all matrices for which WNODF = 1, see ﬁgure 2. This demon- stration has some points in common with the case NODF = 1. It is clear that WNODF = 1 if and only if both WNODF r = 1 and WNODF c = 1, so let us ﬁrst consider the conditions under which WNODF r = 1. This is true if and only if DNr ij = 1 for all pairs (i, j) of rows. From equa- tion (15), DNr ij = 1 implies that MT r i ̸= MT r j , so that either DF r ij = 1 or DF r ji = 1. If there are more elements of S2 interacting with i in S1 than with j in S1, then MT r i > MT r j , and DF r ij = 1, DF r ji = 0. Then we also require that Pn p=1 H(wip −wjp)H(wjp) = MT r j , so that Dr ij = 1, in other words that wip ≥wjp whenever wjp ̸= 0. Thus all elements of S2 interacting with j in S1 not only interact with i in S1, but interact more strongly with i than with j. The set of elements of S2 interacting with j in S1 not only has to be nested within (or a proper subset of) the set of S2 elements interacting with i in S1, but all the interactions with i in S1 must be stronger than the corresponding interaction with j in S1. Similarly, if there are more S2 elements interacting with j in S1 than with i in S1, then the set of S2 elements interacting with i in S1 must be nested within the set of S2 elements interacting with j in S1, and each interaction with j in S1 must be stronger than the corresponding interaction with i in S1. Similar results hold for WNODF c = 1, so that the set of elements of S1 interacting with any k in S2 must be a proper subset or superset of the set of S1 elements interacting with any other l in S2, corresponding interactions in subsets must be weaker, and corresponding interactions in supersets stronger. B. Quantitative matrix, the case WNODF From equation (14), DNr ij = 0 if and only if either (i) MT r i = MT r j , so that DF r ij = DF r ji = 0, or (ii) MT r i > MT r j and Pn p=1 H(wip −wjp)H(wjp) = 0, so that Dr ij = DF r ji = 0, or (iii) MT r i < MT r j and Pn p=1 H(wjp −wip)H(wip) = 0, so that Dr ji = DF r ij = 0. In case (i), the elements i and j of S1 interact with the same number of S2 elements. In case (ii), i in S1 interacts with more elements of S2 than does j in S1, but any interaction between j and any element p of S2 is at least as strong as the corresponding interaction between i and p. Although i in S1 strictly dominates j in S1 in terms of the number of its interactions, j in S1 (not necessarily strictly) dominates i in S1 in terms of the strength of the interactions it does have. Case DNr ij = DF r ijDr ij + DF r jiDr ji, (14) DNc kl = DF c klDc kl + DF c lkDc lk. (15) (14) DNc kl = DF c klDc kl + DF c lkDc lk. (15) (15) Note that these deﬁnitions are valid whatever the signs of MT r i −MT r j and MT c k −MT c l . For example, (i) if MT r i > MT r j then DF r ij = 1 and DF r ji = 0, so DNr ij = Dr ij, (ii) if MT r i < MT r j Page 6 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... N. F. Britton et al., Which Matrices Show Perfect Fig. 2: The maximal nestedness pattern exempliﬁed for qualitative (a) and quantitative (b) cases. In the second situation the weight of the link between species is indicated by grey tones. elements of W; for example, if W is a 2 × 2 matrix with w11 = 1 + ε, w12 = w21 = 1, w22 = 0, then WNODF(W) = 0 if ε = 0 but WNODF(W) = 1 if ε is positive, however small it is. Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 III. FINAL REMARKS This work focuses on probably the most com- monly used nestedness index: the Nestedness met- ric based on Overlap and Decreasing Fill. Initially we introduce a rigorous formulation for NODF and WNODF. We then elucidate the patterns of maximal and minimal nestedness, (W)NODF = 1 and (W)NODF = 0. The maximal nestedness pattern is already known in the literature [15, 2], but an understanding of the minimum nestedness pattern is substantially extended in this work. The literature usually presents the chessboard pattern as the prototype of the zero nestedness arrange- ment; but this work shows that there is in fact a large class of matrices that fulﬁl this condition. We cite the completely compartmented networks with equal modules (of which the chessboard is a special case) and gradient-like matrices. But there is another class of non-symmetrical matrices that also have zero nestedness as long as the row and column sums of the adjacency matrix are uniform. C. Conditions for WNODF = 1 For WNODF = 1, all (S1 and S2) interaction sets must be proper sub- or supersets, so that by the pigeon-hole principle we must have m = n, and it must be possible to Fig. 2: The maximal nestedness pattern exempliﬁed for qualitative (a) and quantitative (b) cases. In the second situation the weight of the link between species is indicated by grey tones. (iii) is analogous, with i and j interchanged. There are many possible ways to obtain WNODF r = 0, and similarly WNODF c = 0 and WNODF = 0. In particular any connected bipartite network in which all elements of S1 interact with the same number of elements of S2, and all elements of S2 interact with the same number of elements of S1, has WNODF = 0, as does any network in which each element of W is either 0 or 1. Note that WNODF is not a continuous function of the Page 7 of 9 Page 7 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... permute the rows and columns of the matrix W so that wij > 0 if i + j ≤n + 1, wij = 0 otherwise. Any matrix with WNODF = 1 has the same adjacency matrix, up to permutation of rows and columns, and also satisﬁes the row and column strict dominance properties wik > wjk for all i < j whenever wjk > 0, wki > wkj for all i < j whenever wkj > 0. We hope that this rigorous work that highlight the nestedeness of (W)NODF will contribute to the discussion about the general meaning of nest- edness by clarifying the extreme cases: zero and maximal nestedness. The basics of the mathemat- ical framework presented here is ﬂexible enough to encourage further developments using alterna- tive pairwise nestedness indices. Despite the large number of nestedness indices, there are few ana- lytic results relating the properties of a nestedness index and the characteristics of the corresponding adjacent matrix; an exception is [7]. With the exact results shown in this manuscript we add new elements to the debate about the real meaning of nestedness and the best way to measure it. Acknowledgements Financial support to Gilberto Corso from CNPq (Conselho Nacional de Desenvolvimento Cient´ıﬁco e Tecnol´ogico) is acknowledged. Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 REFERENCES [1] M. Almeida-Neto, D. M. B. Frensel, and W. Ulrich. Rethinking the relatioship be- tween nestedness and beta diversity: a com- ment on Baselga(2010). Global Ecology and Biogeography, 21:772–777, 2012. [2] M. Almeida-Neto, P.R. Guimar˜aes, P. R. Guimar˜aes Jr, R. D. Loyola, and W. Ulrich. A consistent metric for nestedness analysis in ecological systems: reconciling concept and measurement. Oikos, 117:1227, 2008. The theoretical discussion about nestedness to- day resembles the debate around diversity and its measurements [14, 16, 17]. In both cases the com- munity of ecologists is aware of the importance of the concept in understanding and quantifying patterns in ecological processes. 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Page 8 of 9 Page 8 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 N. F. Britton et al., Which Matrices Show Perfect Nestedness or the Absence of Nestedness ... Global Ecology and Biogeography, 19:134– 143, 2010. Animal Ecology, 78:253, 2009. Animal Ecology, 78:253, 2009. [14] L. Jost. Entropy and diversity. Oikos, 113:363–375, 2006. [7] G. Corso, A. L. de Araujo, and A. M. de Almeida. Connectivity and nestedness in bipartite networks from community ecol- ogy. Journal of Physics: Conference Series, 285:012009, 2011. [15] T. M. Lewinsohn, P. I. Prado, P. Jordano, J. Bascompte, and J. M. Olesen. Structure in plant-animal interaction assemblages. Oikos, 113:174, 2006. [8] J. P. Darlington. Zoogeography: the geo- graphical distribution of animals. Wiley, 1957. [16] A. E. Magurran. Measuring Biological Diversity. Blackwell Publishing Company, 2004. [17] O. Parkash and A. K. Thukral. Statistical measures as measures of diversity. Inter- national Journal of Biomathematics, 3:173, 2010. [9] Luis J. Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171 REFERENCES Gilarranz, Juan M. Pastor, and Javier Galeano. The architecture of weighted mu- tualistic networks. Oikos, 121:1154, 2011. [10] N. J. Gotelli and G.R. Graves. Null models in ecology. Smithsonian Institution Press, Washington, D.C., 1996. [18] J. Podani and D. Schmera. A comparative evaluation of pairwise nestedness measures. Ecography, 35:1, 2012. [11] P. R. Guimar˜aes Jr. and P. Guimar˜aes. Im- proving the analyses of nestedness for large sets of matrices. Environmental Modeling & Software, 21:1512, 2007. [19] D. Schmera and J. Podani. Comments on sep- arating components of beta diversity. Com- munity Ecology, 12:153–160, 2011. [20] P. P. A. Staniczenko, J. C. Kopp, and S. Allesina. The ghost of nestedness in eco- logical networks. Nature communications, 4:1391, 2013. [12] P. R. Guimar˜aes Jr., V. Rico-Gray, S. F. dos Reis, and J. N. Thompson. Asymmetries in specialisation in ant–plant mutualistic net- works. Proc. R. Soc B, 273:2041, 2006. [21] E. Th´ebault and C. Fontaine. Stability of ecological communities and the architecture of mutualistic and trophic networks. Science, 329:853, 2010. [13] T. C. Ings, J. M. Montoya, J. Bascompte, N. Bluthgen, L. Brown, C. F. Dormann, F. Edwards, D. Figueroa, U. Jacob, J. I. Jones, R. B. Lauridsen, M. E. Ledger, H. M. Lewis, J. M. Olesen, F.J. Frank van Veen, P. H. Warren, and G. Woodward. Ecological networks - beyond food webs. Journal of [22] W. Ulrich, M. Almeida-Neto, and N. J. Gotelli. A consumer’s guide to nestedness analysis. Oikos, 118:3, 2009. Page 9 of 9 Biomath 4 (2015), 1512171, http://dx.doi.org/10.11145/j.biomath.2015.12.171
https://openalex.org/W2781950122	https://zenodo.org/records/3570551/files/168%20Work%20Profile%20of%20the%20Weavers%20in%20Hand%20Loom%20Industry.pdf	English	null	Work Profile of the Weavers in Handloom Industry	International journal of trend in scientific research and development	2,017	cc-by	3,082	Objectives of the study Objective of the study is to analyse the work profile of handloom weavers and to provide some appropriate suggestions for uplifting the working conditions. Objective of the study is to analyse the work profile of weavers and to provide some appropriate suggestions for uplifting the working conditions. Years of Experience Experience refers to the number of years worked. Experience makes the person expert and improves their knowledge in particular field of work. Experience refers to the number of years worked. Experience makes the person expert and improves their knowledge in particular field of work. Work Profile of t Work Profile of the Weavers in Handloom Industry n Handloom Industry Research Scholar, Women’s Christian College, X. Maria Muthu Shanthini Research Scholar, Women’s Christian College, Nagercoil-1, Tamil Nadu ABSTRACT Objective of the research paper is to analyse the work profile of handloom weavers and to provide some appropriate suggestions for uplifting the working conditions. The study has been undertaken to find out means to revive the traditional handloom industries. The study mainly depends upon primary as well as secondary data. Primary data has been collected from 80 respondents using interview schedule. The study found that poverty is the main reason for choosing weaving as their occupation. It is suggested that government should increase the benefits for the member of the handloom cooperative society to attract all weavers under its fold and it concludes that there is many experienced handloom weavers available in Kanniyakumari District. Objective of the research paper is to analyse the work profile of handloom weavers and to provide some estions for uplifting the working conditions. The study has been undertaken to find out means to revive the traditional handloom industries. The study mainly depends upon primary as well as secondary data. Primary data has been collected from s using interview schedule. The study found that poverty is the main reason for choosing weaving as their occupation. It is suggested that government should increase the benefits for the member of the handloom cooperative society to attract er its fold and it concludes that there is many experienced handloom weavers available in There is trimendores decline in the handloom industry due to the above factors. Considering the the present study has been undertaken by the researcher to find out means to revive the traditional handloom industries particularly in Kanniyakumari District. There is trimendores decline in the handloom industry due to the above factors. Considering the above facts, the present study has been undertaken by the researcher to find out means to revive the traditional handloom industries particularly in Kanniyakumari District. ISSN No: 2456 International Research ISSN No: 2456 - 6470 \| www.ijtsrd.com \| Volume International Journal of Trend in Scientific Research and Development (IJTSRD) International Open Access Journal \| www.ijtsrd.com \| Volume - 2 \| Issue – 1 Scientific (IJTSRD) International Open Access Journal ISSN No: 2456 International Research ISSN No: 2456 - 6470 \| www.ijtsrd.com \| Volume International Journal of Trend in Scientific Research and Development (IJTSRD) International Open Access Journal \| www.ijtsrd.com \| Volume - 2 \| Issue – 1 Scientific (IJTSRD) International Open Access Journal Methodology The study mainly depends upon primary as well as secondary data. Primary data has been collected from 80 respondents using interview schedule. Through interview schedule researchers analyze the year of experience, membership in society, health hazards in occupation, working condition, mode of payment and reason to select the job of the respondents. Secondary data were collected from books, journal, magazines and reports. As census method is not possible to collect the primary data within stipulated period convenience sampling method was used for collecting the data. The study mainly depends upon primary as well as y data has been collected from 80 respondents using interview schedule. Through interview schedule researchers analyze the year of experience, membership in society, health hazards in occupation, working condition, mode of payment and job of the respondents. Secondary data were collected from books, journal, magazines and reports. As census method is not possible to collect the primary data within stipulated period convenience sampling method was used for collecting the data. International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 Table No: 1 Years of experience – wise distribution in sample respondents Sl.No Years of experience No of respondents Percentage 1 Up to 10 years 10 12.5 2 11-20 years 22 27.5 3 21-30 years 18 22.5 4 Above 30 years 30 37.5 Total 80 100 Source:Primary data From the table 1, it is seen that 37.5 percent of the respondents have work experience more than 30 years; 27.5 percent respondents have 11 to 20 years of experience; 22.5 percent respondents have 21 to 30 years experience and 12.5 percent of the respondents have below 10 years experience. This indicates that these weavers were working in handloom industry for a long period of time. Membership in Society Person with own looms or without looms and has knowledge about weaving can join as member into cooperative society. They must be eligible for membership in cooperative society by satisfying conditions provided by it. Table No: 2 Membership in society – wise distribution in sample respondents Sl.No Membership in society No of respondents Percentage 1 Yes 32 40 2 No 48 60 Total 80 100 Above table 2 shows that 40 percentage of the respondents are members in the society. Rest of 60 percentage respondents are not members in cooperative society as it provides low wages when compare to private weavers. Number of years as member in the society Some of the respondent weavers i.e. 32 of the total respondent have membership in cooperative society. Year wise distribution of the sample respondents is shown below. Table No: 3 Number of years as member in the society Sl.No No of years No of respondents Percentage 1 Less than 10 years 17 53.1 2 10-15 years 12 37.5 3 Above 15 years 3 9.4 Total 32 100 Source: Primary data Table 3 shows that 53.1percent of the respondents have experience less than 10 years in the cooperative society, 37.5 percentage respondents have experience from 10 to 15 years and 9.4percent of the sample respondents have experience above 15 years in the cooperative society. Health hazards in occupation There is possibility for accident during the work in any occupation. But in hand loom industry chances for accident is very low when compared with other job but there is chances for physical strains and also mental stress it is also consider as hazard. Number of years as member in the society Number of years as member in the society Sl.No No of years No of respondents Percentage 1 Less than 10 years 17 53.1 2 10-15 years 12 37.5 3 Above 15 years 3 9.4 Total 32 100 Source: Primary data Number of years as member in the society Sl.No No of years No of respondents Percentage 1 Less than 10 years 17 53.1 2 10-15 years 12 37.5 3 Above 15 years 3 9.4 Total 32 100 Source: Primary data Source:Primary data From the table 1, it is seen that 37.5 percent of the respondents have work experience more than 30 years; 27.5 percent respondents have 11 to 20 years of experience; 22.5 percent respondents have 21 to 30 years experience and 12.5 percent of the respondents have below 10 years experience. This indicates that these weavers were working in handloom industry for a long period of time. International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 Above table 2 shows that 40 percentage of the respondents are members in the society. Rest of 60 percentage respondents are not members in cooperative society as it provides low wages when compare to private weavers. Years of experience – wise distribution in sample respondents Years of experience – wise distribution in sample respondents Membership in Society Table 3 shows that 53.1percent of the respondents have experience less than 10 years in the cooperative society, 37.5 percentage respondents have experience from 10 to 15 years and 9.4percent of the sample respondents have experience above 15 years in the cooperative society. Person with own looms or without looms and has knowledge about weaving can join as member into cooperative society. They must be eligible for membership in cooperative society by satisfying conditions provided by it. Statement of the problem The handloom weavers have been very badly affected by the adoption of power-loom sector. The membership strength of handloom cooperative societies has been decreasing due to power loom sector. The capital base of these societies has been eroded. The people who faced to leave the co-operative society due to such competition suffer psychologically as well as economically. The people who are worki handloom industry do not prefer to continue for long due to competition of power loom sector. The handloom weavers are subjected to so many problems. The handloom weavers have been very badly affected loom sector. The membership eties has been decreasing due to power loom sector. The capital base of these societies has been eroded. The people who operative society due to such competition suffer psychologically as well as economically. The people who are working in the handloom industry do not prefer to continue for long due to competition of power loom sector. The handloom weavers are subjected to so many problems. @ IJTSRD \| Available Online @ www.ijtsrd.com @ IJTSRD \| Available Online @ www.ijtsrd.com \| Volume – 2 \| Issue – 1 \| Nov-Dec 2017 Dec 2017 Page: 1066 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 Table No: 3 Number of years as member in the society Number of years as member in the society Some of the respondent weavers i.e. 32 of the total respondent have membership in cooperative society. Year wise distribution of the sample respondents is shown below. Satisfaction regarding working condition Table 4 exhibits that out of 80 sample respondents only 26.3 percent respondents have affected by health hazards and 73.8 percent of the respondents are not affected by any health hazards such as wound, muscle pain and the like, as it is not dangerous or hazardous work compared to other industries. Man has to exert his energy both mentally and physically. The opportunities and provisions are available for working essentially exertion of energy either physically or mentally for a reward. They include natural environment and working system etc. Workers should be satisfied with their working condition to improve productivity. Health hazard in occupation of the sample respondents Source: Primary data Table No: 6 Satisfaction regarding working condition of the sample respondents Sl .No Satisfied Number of respondents Percentage 1 Satisfied 78 97.5 2 Not satisfied 2 2.5 Total 80 100 Source: Primary data Satisfaction regarding working condition of the sample respondents Working time in hand loom industry Working time in hand loom industry Sl. No Hours of working Number of respondents Percentage 1 Up to 6 37 46.3 2 7 hours 23 28.8 3 8 hours 9 11.3 4 Above 9 hours 11 13.8 Total 80 100 Source: Primary data It is observed from table 6 that out of 80 sample respondents 97.5 percent respondents are satisfied and 2.5 percent respondents are not satisfied with their working condition. Hours of working There is no prescribed time limit for the people working under private handloom. In cooperative society also, workers are not compelled to work for particular prescribed time. In order to find that working hours the respondents are asked to give their working hours and is shown below. Membership in society – wise distribution in sample respondents Membership in society – wise distribution in sample respondents There is possibility for accident during the work in any occupation. But in hand loom industry chances for accident is very low when compared with other job but there is chances for physical strains and also mental stress it is also consider as hazard. Page: 1067 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 Table No: 4 Table No: 4 Health hazard in occupation of the sample respondents Sl. No Health hazard Number of respondents Percentage 1 Yes 21 26.3 2 No 59 73.8 Total 80 100 Source: Primary data Health hazard in occupation of the sample respondents Findings Majority i.e., 37.5 percent of the respondents have working experience above 30 years; and only 12.5 percent of the respondents are having below 10 years experience. It indicates that these weavers were working in handloom industry for a long period of time. Majority i.e., 40 percent of the respondents are members in the society. Rest of 60 percentage respondents are not members in cooperative society, because it provides low wages when compare to private weavers. Majority i.e., 53.1percent of the sample respondents have experience less than 10 years in the society, and only 9.4percent respondents have experience above 15 years in the cooperative society. Out of 80 sample respondents only 26.3 percentage respondents have affected by health hazards such as wound, muscle pain and the like, as it is not dangerous or hazardous work compared to other industries. Majority i.e.,46.3 percent respondents are working up to 6 hours; and 13.8 percent respondents are in the above 9 hours work group as over time working people. Out of 80 sample respondents 97.5 percent respondents are satisfied with their working condition and 2.5 percent respondents are not satisfied. 58.8 percentage workers are getting daily wages and 41.2 percentage workers are getting wages on weekly basis. People need money to manage daily expenses, but poor people cannot afford it without daily wages. That is why; weavers are getting their wages on daily basis. Poverty is the main contributory factor for choosing weaving as their occupation 46.3 percent and followed by unemployment 18.8 percent Remaining 3.8 percent of the respondents are involving in this work as they are willing to work in handloom industry. Source: Primary data This table 7 indicates that 58.8 percentage workers are getting daily wages and 41.2 percentage workers are getting wages on weekly basis. People need money to manage daily expenses, but poor people cannot afford it without daily wages. That is why, weavers are getting their wages on daily basis. International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 From the table 8, it is revealed that poverty is the main contributory factor for choosing weaving as their profession 46.3 percent and followed by unemployment 18.8 percent. 12.5 percent of the sample respondents are doing this work for time pass and 11.3 percent of the respondents choosing it as it is traditional work. Only 7.5 percent of the total respondents carry on handloom weaving to earn more income as their existing income is not enough to overcome their all family needs. Remaining 3.8 percent of the respondents are involving in this work as they are willing to work in handloom industry. Mode of payment –wise distribution in sample respondents Sl. No Mode of payment Number of respondents percentage 1 Daily wages 47 58.8 2 Weekly wages 33 41.2 Total 80 100 Source: Primary data Mode of payment –wise distribution in sample respondents Mode of payment –wise distribution in sample respondents Mode of payment –wise distribution in sample respondents Sl. No Mode of payment Number of respondents percentage 1 Daily wages 47 58.8 2 Weekly wages 33 41.2 Total 80 100 Source: Primary data Mode of payment Source: Primary data Handloom weavers may get income in the way of wages or by the way of salary. Wages means getting income on daily or weekly basis where as salary means getting income on monthly basis. The above table reveals that 46.3 percent respondents are working up to 6 hours 28.8 percent respondents are working up to 7 hours.11.3 percent respondents are in the 8 hours work group and 13.8 percent respondents are in the above 9 hours work group. It is considered as overtime, because according to Indian law normal working hour is 8 hours per day. Page: 1068 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 Suggestions 1. Das, N.C and Mahapatro 1986, “Development of Handloom Industry”, Deep and Deep Publications, New Delhi, Pp.31. 1. Das, N.C and Mahapatro 1986, “Development of Handloom Industry”, Deep and Deep Publications, New Delhi, Pp.31. Even cooperative society is providing low wages when compare to private weavers, it provides good working environment, constant work, secured income and many government benefits through implementing schemes. Each and every handloom weavers should come under the roof of cooperative society. Government should increase the benefits for the member of the handloom cooperative society to attract all weavers under its fold. 2. Ministry of Textiles, Annual Report 2014-15, Government of India. 2. Ministry of Textiles, Annual Report 2014-15, Government of India. 3. Kumar, P. S. 2014, Handloom Industry in India: A Study. International Journal of Multidisciplinary Research and Development, Vol. 2(1), Pp.24-29. International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 Reason for choosing handloom work Several factors may lead the people to select the particular kind of job, but there should be a reason behind the selection of each job. Table No: 8 Reason for choosing handloom work by the sample respondents Sl. No Reason Number of respondents Percentage 1 Poverty 37 46.3 2 Unemployme nt 15 18.8 3 Additional income 6 7.5 4 Time passing 10 12.5 5 Traditional work 9 11.3 6 Willingness to do 3 3.8 Total 80 100 Source: Primary data Table No: 8 Reason for choosing handloom work by the sample respondents Sl. No Reason Number of respondents Percentage 1 Poverty 37 46.3 2 Unemployme nt 15 18.8 3 Additional income 6 7.5 4 Time passing 10 12.5 5 Traditional work 9 11.3 6 Willingness to do 3 3.8 Total 80 100 Source: Primary data Reason for choosing handloom work by the sample respondents Page: 1069 Conclusion Handloom weaving activity plays an active role in the growth process of the state as well as the nation. This sector has been considered important because of the traditional artisan craft skills of the weavers which meet the local needs and demands. The study concludes that there is many experienced handloom weavers available in Kanniyakumari District. It also concludes that handloom industry is not hazardous industry to work and it helps to manage daily expenses of the family. Page: 1070
https://openalex.org/W4321480398	https://www.nature.com/articles/s41467-023-36576-z.pdf	English	null	Orthogonal luminescence lifetime encoding by intermetallic energy transfer in heterometallic rare-earth MOFs	Nature communications	2,023	cc-by	13,137	1Nanoscale Sciences Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. 2Advanced Packaging/Integration Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. 3Molecular and Microbiology Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. 4Advanced Materials Laboratory, Sandia National Laboratories, Albuquerque, NM 87185, USA. 5Computation Materials and Data Science, Sandia National Laboratories, Albuquerque, NM 87185, USA. 6Nanostructure Physics Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. 7Measurement Science and Engineering Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. 8Nuclear Security Engineering Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. e-mail: dfsava@sandia.gov Article https://doi.org/10.1038/s41467-023-36576-z Orthogonal luminescence lifetime encoding by intermetallic energy transfer in heterometallic rare-earth MOFs Jacob I. Deneff1, Lauren E. S. Rohwer2, Kimberly S. Butler 3, Bryan Kaehr 4, Dayton J. Vogel 5, Ting S. Luk6, Raphael A. Reyes1, Alvaro A. Cruz-Cabrera7, James E. Martin8 & Dorina F. Sava Gallis1 Received: 25 August 2022 Accepted: 7 February 2023 Check for updates Lifetime-encoded materials are particularly attractive as optical tags, however examples are rare and hindered in practical application by complex inter- rogation methods. Here, we demonstrate a design strategy towards multi- plexed, lifetime-encoded tags via engineering intermetallic energy transfer in a family of heterometallic rare-earth metal-organic frameworks (MOFs). The MOFs are derived from a combination of a high-energy donor (Eu), a low- energy acceptor (Yb) and an optically inactive ion (Gd) with the 1,2,4,5 tetra- kis(4-carboxyphenyl) benzene (TCPB) organic linker. Precise manipulation of the luminescence decay dynamics over a wide microsecond regime is achieved via control over metal distribution in these systems. Demonstration of this platform’s relevance as a tag is attained via a dynamic double encoding method that uses the braille alphabet, and by incorporation into photocurable inks patterned on glass and interrogated via digital high-speed imaging. This study reveals true orthogonality in encoding using indepen- dently variable lifetime and composition, and highlights the utility of this design strategy, combining facile synthesis and interrogation with complex optical properties. the state of the art, reducing the likelihood that any counterfeit will duplicate all of the measured properties4. As markets become more complex and globalized, more sophisticated methods are required to address supply chain management challenges and integrity-checking for highly complex systems and valuable items or commodities. To meet this growing challenge, optical tags, which leverage the luminescent properties of materials for encoding, have been explored and advanced signiﬁcantly in recent years1–3. To be effective, such tags must offer rapid, low-cost, and unambiguous ver- iﬁcation, and, crucially, they must be secure against counterfeiting. Because static, monochromatic luminescence is easy to duplicate using commonly available dyes, multilayered encoding must become To enhance security, viable materials must combine many possi- ble states, both overt and covert, including emission, lifetime, scat- tering, and absorption5. In agreement with this premise, several recently published works have described potential options for secure encoding1,6–9, drawn from many classes of materials, including carbon dots2,10, metallic nanoparticles11,12, and perovskites3. Orthogonal luminescence lifetime encoding by intermetallic energy transfer in heterometallic rare-earth MOFs However, existing materials suffer from critical drawbacks: (i) monochromatic and/or broad emission bands, unsuited for multi-tiered screening3,13; Nature Communications\| (2023) 14:981 Nature Communications\| (2023) 14:981 Nature Communications\| (2023) 14:981 1 https://doi.org/10.1038/s41467-023-36576-z Article (ii) complex synthesis methods, which are time-consuming and cost- ineffective11,14; and (iii) highly specialized interrogation equipment, which restricts real-life implementation2,15. they are connected to the ratio of two elements in the material, directly coupling composition and lifetime. To extend encoding potential, here we report an optical tag that demonstrates orthogonal encoding using independently variable lifetime and composition. Here we demonstrate that polynuclear rare-earth metal-organic frameworks (MOFs) represent an ideal class of materials for tunable, multiplexed encoding16–22. Rare-earth elements (REs) possess narrow absorption and emission bands across a wide range of wavelengths that allow for a high degree of speciﬁcity in excitation and emission23, a property that is difﬁcult to duplicate with the broad-band materials10. Likewise, MOFs are attractive for tagging because they have (i) straightforward synthetic processes, that allow for one-pot synthesis; (ii) periodic crystal structures, allowing structural manipulation and in- depth characterization at the atomic level; and (iii) sensitizing organic linkers in a low-density framework, which only require a low amount of optically active REs to emit a readable signal24,25. To enable this multiplexed encoding, we implemented a targeted materials design strategy: ﬁrst, the dominant metal-metal interactions were conﬁned to intra- cluster vs. inter-cluster interactions via linker geometry, which allowed the control over the intermetallic energy transfer mechanism; then, the high-energy donor RE and lower-energy acceptor RE were segregated by incorporating an optically inactive RE to modulate the average distance between the donor and acceptor without changing the linker or overall structure of the MOF30. g g The resulting family of heterometallic rare-earth MOFs combined the high-energy donor Eu (visible emitter) with the low-energy acceptor Yb (near-infrared (NIR) emitter) and the optically inactive Gd ion with the organic linker 1,2,4,5 tetrakis(4-carboxyphenyl) ben- zene (TCPB). The TCPB linker was chosen due to its proven ability to direct nonanuclear cluster formation in the MOF and because its large size allows for cluster separation17,33. Due to the energy of its triplet excited state, TCPB also serves as antenna molecule for both Eu and Yb, but not Gd16,34. Orthogonal luminescence lifetime encoding by intermetallic energy transfer in heterometallic rare-earth MOFs We hypothesized that direct excitation of the linker initiates an energy cascade ﬁrst to Eu (which has a long-lived visible emission), then to Yb (which has a short-lived NIR emission), while avoiding any energy transfer to Gd, Fig. 1. As a result, here we demonstrate that the inclusion of an inactive element vastly expands the potential design space for lifetime encoding, because it effectively modiﬁes the RE interactions without impacting either the linker identity or the ratio of donor to acceptor emitters. Lifetime-encoded materials are particularly attractive for multi- plexed optical tags. Distinct luminescence lifetimes allow spectrally overlapped tags to be distinguished in the time domain2,3,26, and can create images that change over time to reveal hidden patterns or additional data. For example, organic compounds with variable life- time have been reported, including thermally activated delayed ﬂuorescence materials2,27. However, the challenges associated with controlling the material structure, have limited the rational design of materials with potential for lifetime-encoding2,3,8,11,28,29. Available energy transfer pathways in a tag are affected by both the excited state energies of individual components of the material and the distance between them. The periodic structure of polynuclear rare- earth MOFs, with intermetallic distances controlled by cluster and ligand geometry, makes the energy transfer relationships between the various components of the rare-earth MOF much easier to understand and manipulate30,31. Manipulation of MOFs for control of emission color or intensity has been well explored, but the concept has never been adapted for ﬁne control of luminescence lifetime30–32. When changes in lifetime are noted, both in MOFs and other lanthanide-based materials, To validate the complex intermetallic energy transfer relation- ships between these three elements, we targeted and successfully synthesized 13 compositions. These compositions included single-, di-, and trimetallic compounds with different elemental ratios, which allowed us to explore a wide range of relevant intermetallic energy transfer scenarios. Eu Yb Gd TCPB UV excitation Energy In: None Emission Out: None Amount of energy transfer modulates decay dynamics Energy In: TCPB Emission Out: Visible Energy In: TCPB, Eu Emission Out: NIR High energy transfer Short lifetime Moderate energy transfer Moderate lifetime Low energy transfer Long lifetime Example of visible emissions Time Intensity Modulated lifetimes a X b Concept of controllable energy transfer for lifetime modulation in b-based trimetallic compositions. a Direct ligand excitation results in ated energy transfer, illustrating the relationships between each component. Orthogonal luminescence lifetime encoding by intermetallic energy transfer in heterometallic rare-earth MOFs b Examples of complex intermetallic energy transfer in three distinct EuGdYb- based compositions with different elemental ratios and the direct impact on life- time modulation. e Communications\| (2023)14:981 2 a Eu Yb Gd TCPB UV excitation Energy In: None Emission Out: None Amount of energy transfer modulates decay dynamics Energy In: TCPB Emission Out: Visible Energy In: TCPB, Eu Emission Out: NIR a X UV excitation a Eu Yb Gd TCPB UV excitation Energy In: None Emission Out: None Energy In: TCPB Emission Out: Visible Energy In: TCPB, Eu Emission Out: NIR a X Energy In: None Emission Out: None Energy In: TCPB Emission Out: Visible Energy In: TCPB, Eu Emission Out: NIR Amount of energy transfer modulates decay dynamics High energy transfer Short lifetime Moderate energy transfer Moderate lifetime Low energy transfer Long lifetime Example of visible emissions Time Intensity Modulated lifetimes b Fig. 1 \| Concept of controllable energy transfer for lifetime modulation in EuGdYb-based trimetallic compositions. a Direct ligand excitation results in modulated energy transfer, illustrating the relationships between each component. b Examples of complex intermetallic energy transfer in three distinct EuGdYb- based compositions with different elemental ratios and the direct impact on life- time modulation. Low energy transfer Long lifetime ssions High energy transfer Short lifetime Moderate energy transfer Moderate lifetime Low energy transfer Long lifetime Example of visible emissions Time Intensity Modulated lifetimes b ll l l d l l l ll h d d b Low energy transfer Long lifetime sions Time Intensity Modulated lifetimes n ent. b Examples of complex intermetallic energy transfer in three distinct EuGdYb- based compositions with different elemental ratios and the direct impact on life- time modulation. High energy transfer Short lifetime Moderate energy transfer Moderate lifetime Low energy transfer Long lifetime Example of visible emissions Intensity Mo b f ll bl f f lif i d l i i b E l f l i High energy transfer Short lifetime Exam b Moderate energy transfer Moderate lifetime ple of visible emis Time Intensity Modulated lifetimes b Intensity Low energy transfer Long lifetime High energy transfer Short lifetime Time Fig. 1 \| Concept of controllable energy transfer for lifetime modulation in EuGdYb-based trimetallic compositions. a Direct ligand excitation results in modulated energy transfer, illustrating the relationships between each component. Article Article https://doi.org/10.1038/s41467-023-36576-z To demonstrate the utility of these materials as practical tags we implemented two design strategies, leveraging two spectrally identical compositions that displayed substantially distinct decay lifetimes in the 100 s of μs. An encoding strategy that utilizes the braille alphabet was introduced, interrogated by a microplate reader to produce 3 tag exemplars that are either static or dynamic, utilizing both concentra- tion and lifetime to change the encoded message depending on when the pattern is read. Subsequently, a proof-of-concept tag was fabri- cated utilizing the same two compounds suspended in photocurable adhesive and patterned with a laser cut stencil. Laser excitation and high-speed imaging interrogation of this tag showed that the two materials were clearly visually distinguishable on a millisecond time scale. These demonstrations show the utility of these tag materials in facile encoding and interrogation, while more in-depth characteriza- tion of a library of tag materials reveals the potential for further layers of encoding including additional lifetime lengths, compositions, and luminescence spectra. The materials and techniques presented here demonstrate a ver- satile materials platform capable of dynamic encoding in the time domain and amenable to truly independent authentication via tunable emission spectra and composition, enabling the future creation of accessible tags with multi-layered encoding. Orthogonal luminescence lifetime encoding by intermetallic energy transfer in heterometallic rare-earth MOFs b Examples of complex intermetallic energy transfer in three distinct EuGdYb- based compositions with different elemental ratios and the direct impact on life- time modulation. Nature Communications\| (2023) 14:981 2 2 Article Synthesis and materials characterization b Powder X-ray diffraction patterns for each compound reported in this work, highlighting their consistent crystallinity and the slight Nature Communications\| (2023) 14:981 3 3 https://doi.org/10.1038/s41467-023-36576-z Article Table 1 \| Compound ID numbers and their fractional metallic compositions Compound Eu fraction Gd fraction Yb fraction 1 1 0 0 2 0.69 0 0.31 3 0.34 0 0.66 4 0 0 1 5 0 0.37 0.63 6 0 0.68 0.32 7 0 1 0 8 0.30 0.70 0 9 0.63 0.37 0 10 0.65 0.20 0.15 11 0.17 0.19 0.64 12 0.15 0.70 0.15 13 0.32 0.37 0.31 The reported fractional compositions have an uncertainty of +/−0.01 based on EDS analysis. Table 1 \| Compound ID numbers and their fractional metallic compositions Compound Eu fraction Gd fraction Yb fraction 1 1 0 0 2 0.69 0 0.31 3 0.34 0 0.66 4 0 0 1 5 0 0.37 0.63 6 0 0.68 0.32 7 0 1 0 8 0.30 0.70 0 9 0.63 0.37 0 10 0.65 0.20 0.15 11 0.17 0.19 0.64 12 0.15 0.70 0.15 13 0.32 0.37 0.31 The reported fractional compositions have an uncertainty of +/−0.01 based on EDS analysis. Table 1 \| Compound ID numbers and their fractional metallic compositions between their components, primarily ligand-to-metal charge transfer (LMCT) and metal-to-metal charge transfer (MMCT)24,39. The efﬁciency of these pathways is governed by both the relative energies of each component and the distance between them. Components with high- energy excited states will donate energy non-radiatively to compo- nents with low-energy excited states, provided that the elements are in close physical proximity to each other and that the energy levels are sufﬁciently different to prevent losses via back-transfer4,40. The prototypical three-periodic MOF material studied here is made up of 12-connected nonanuclear RE clusters bridged by TCPB linkers17,18,33. Within individual clusters (intra-cluster) the intermetallic distance is 3.9 Å, while distances between clusters (inter-cluster) are between 8.9 and 18.0 Å, as shown in Fig. 3a. Given that intermetallic energy transfer is known to decline sig- niﬁcantly beyond 10 Å31, these distances indicate that energy transfer between individual metal ions in the compositions reported here would primarily occur within clusters. This paradigm is signiﬁcant in the context of correlating metal distribution at the molecular level and the impact of such distribution on the metal-to-metal energy transfer and resulting photophysical properties. Synthesis and materials characterization To help explain the photo- luminescence (PL) response of each compound to the direct excitation of the linker, the energy transfer pathways between the different components of the MOFs are shown in Fig. 3b. representative crystal structure for these rare-earth MOFs was pre- viously detailed elsewhere by us and others17,33. Brieﬂy, the three- periodic structure is derived from nonanuclear clusters linked by 12 carboxylate groups of the TCPB linker. The resulting framework pos- sesses intrinsic porosity accessible via 1D channels of ~1.2 nm. Crystal structure data and an illustration of compound 1 can be seen in Sup- plementary Table 1 and Supplementary Fig. 1, respectively. Further evidence for the dominance of intra-cluster energy transfer is found in the PL spectra for the reported compounds (Fig. 3c, d). For example, when Eu is excited directly with 394 nm light, char- acteristic Eu emission peaks in the visible range are observed (5D0 →4F0 at 582 nm, 5D0 →4F1 at 592-600 nm, 5D0 →4F2 at 617 nm, 5D0 →4F3 at 655 nm, and 5D0 →4F4 at 704 nm)41. Critically, Yb emission in the NIR is observed between 970 nm and 1050 nm under the same excitation wavelength, demonstrating Eu-to-Yb energy transfer41. As expected, Gd does not affect the PL spectra of materials that contain it, due to its high-energy excited state. Additional excitation and emission spectra for the TCPB linker and single metal compounds 1, 4, and 7 are pre- sented in Supplementary Fig. 5. The crystallinity and phase identity of each compound was determined via powder X-ray diffraction (PXRD). Figure 2b shows the patterns for each compound, along with a calculated pattern for the homometallic Eu composition (compound 1) for comparison. The results indicate that each sample is phase pure and closely matches the representative calculated pattern for the Eu compound 1. Minor shifts in peak location between different compounds are reﬂective of the different ionic radii of the REs. Notably, these materials possess a high thermal stability up to 500 °C, as determined by thermogravimetric analyses, Supplementary Fig. 2. The environment surrounding some RE ions is known to affect their luminescence; for example, the site symmetry of the Eu is indi- cated by the splitting of its 5D0 →4F1 transition emission peak41. Synthesis and materials characterization The Yb signal is also typically split into several peaks in the 970 to 1050 nm range, which reﬂects the local concentration of Yb and other elements in the material42. Thus, all of the compounds in Fig. 3d show primary Yb emission peaks located at 975 and 982 nm, which change in relative intensity primarily in response to the presence or absence of Eu in the compound. This phenomenon of changing relative intensities reﬂects the local environment of the Yb ions and would not occur unless the metals were mixed within the same cluster and interacting in that space. The compositions of each di- and trimetal compound were determined via scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) imaging and analysis. The metallic composi- tion of each compound is given numerically in Table 1 and visually as a ternary diagram in Fig. 2a. Microscopy images and the corresponding elemental maps for the trimetallic compositions (compounds 10, 11, 12, and 13) are shown in Fig. 2c. The SEM-EDS analyses for all other heterometallic compositions are included in Supplementary Fig. 3. EDS mapping shows an even distribution of each metal, both within indi- vidual crystals and in the bulk sample. Finally, we use infrared (IR) transmission spectroscopy to probe the vibrational modes around metal clusters within the different compounds (results shown in Supplementary Fig. 6). The vibrational modes associated with bridging the OH groups in the MOF metal clusters can be found in the 800–1000 cm−1 range43, and changes in the ratios of available metals have been shown to cause peaks to shift or emerge to reﬂect the different environments44. Because of the similar coordination chemistry of the different REs, we did not expect new peaks to form in response to changing composition; however, we did observe peak shifts in the homometallic compounds (particularly compounds 1 and 4), primarily around 910 cm−1 and 845 cm−1. Mixed metal compounds show intermediate peak shifts that correspond to the relative quantities of Yb and Eu. Peaks shift rather than split due to the presence of OH groups, which bridge both single metals and mixed metals. While not conclusive in and of themselves, these results further support our above stated hypoth- esis of intra-cluster heterometallic mixing rather than distinct homometallic clusters. This ﬁnding is signiﬁcant, as the synthesis of heterometallic MOFs is non-trivial. Synthesis and materials characterization Heterometallic MOFs rely on the compatible coordina- tion geometries and crystallization kinetics of these metals to avoid phase or domain separation35,36. Because REs all possess similar coor- dination chemistries, sizes, and oxidation states, it is assumed that rare-earth MOFs can be constructed without domain separation and without limits on the relative metallic composition23,32,37,38. Never- theless, to address concerns regarding uniformity within the hetero- metallic compositions described here and ensure that our compounds were uniform, we analyzed nine points on a single crystal and three areas on different crystals of compound 13. The results, displayed in Supplementary Fig. 4, indicate that the distribution of elements is indeed uniform both in individual crystals and across the bulk sample. Synthesis and materials characterization Synthesis and materials characterization The rare-earth MOFs in this study contained one, two, or three metals, and were chosen to ﬁll the composition space of the three metal ions (Eu, Yb, and Gd) in consistent increments, depending on the number and ratio of metals incorporated. Figure 2a shows a ternary composi- tion diagram with the speciﬁc compositions explored in this work. The \| Information regarding the composition and crystallinity ofthe reported ounds. a Ternary diagram of metal content showing the composition of each ound in the study. b Powder X-ray diffraction patterns for each compound ted in this work, highlighting their consistent crystallinity and the slight variations resulting from different metallic compositions. c Scanning electron microscope images and energy dispersive spectroscopy maps for the four trime- tallic compounds 10–13, highlighting the morphology of the materials and the uniform distribution of metals within each crystal. Fig. 2 \| Information regarding the composition and crystallinity ofthe reported compounds. a Ternary diagram of metal content showing the composition of each compound in the study. b Powder X-ray diffraction patterns for each compound reported in this work, highlighting their consistent crystallinity and the slight variations resulting from different metallic compositions. c Scanning electron microscope images and energy dispersive spectroscopy maps for the four trime- tallic compounds 10–13, highlighting the morphology of the materials and the uniform distribution of metals within each crystal. variations resulting from different metallic compositions. c Scanning electron microscope images and energy dispersive spectroscopy maps for the four trime- tallic compounds 10–13, highlighting the morphology of the materials and the uniform distribution of metals within each crystal. Fig. 2 \| Information regarding the composition and crystallinity ofthe reported compounds. a Ternary diagram of metal content showing the composition of each compound in the study. Photoluminescent lifetime measurements Figure 4c shows the lifetime decay dynamics for the trimetallic com- pounds 10–13. The lifetimes for these compounds range from 122 to 300 μs and are modulated by both the ratio of Eu to Yb and the pro- portional amount of Gd separating them. The full effect of composition on visible lifetime, as well as the correlations for estimating inter- mediate values, is shown in Fig. 4d. In revealing the effect of composi- tion on lifetime, we can multiplex composition and lifetime to develop a continuum of compositions for any desired lifetime and vice versa. Figure 4c shows the lifetime decay dynamics for the trimetallic com- pounds 10–13. The lifetimes for these compounds range from 122 to 300 μs and are modulated by both the ratio of Eu to Yb and the pro- portional amount of Gd separating them. The full effect of composition on visible lifetime, as well as the correlations for estimating inter- mediate values, is shown in Fig. 4d. In revealing the effect of composi- tion on lifetime, we can multiplex composition and lifetime to develop a continuum of compositions for any desired lifetime and vice versa. To probe the entire complex energy transfer cascade from ligand to metal and metal to metal, we measured the luminescence lifetime of each emitting compound. Both visible and NIR decay dynamics were measured using 337 nm light to target direct linker excitation. Based on its emission spectrum, the donor state energy of TCPB is ~25,200 cm−116,32, while the primary emissive states of Gd, Eu, and Yb are 32,300, 17,500, and 10,200 cm−1 respectively41. Because of their relative energy levels, a cascade of energy transfer interactions were anticipated: Eu was excited by energy transfer from the linker, and Yb was excited by energy transfer from both the linker and neighboring Eu ions. The combination of metals present in each compound also has a signiﬁcant effect on lifetime in the NIR. Supplementary Fig. 7a shows that diluting Yb with Gd has little effect on the lifetime of each com- pound, but the inclusion of Eu results in a signiﬁcantly more complex decay with a long, low amplitude tail, taking tens to hundreds of times longer to fully decay (Supplementary Fig. 7b). This change in decay rates occurs because, after the initial burst of energy from the linker excitation is gone, the long-lived Eu continues to feed the short- lived Yb. Intermetallic energy transfer Much of the complexity and tunability of heterometallic polynuclear cluster-based rare-earth MOFs is derived from energetic interactions Nature Communications\| (2023) 14:981 4 Article https://doi.org/10.1038/s41467-023-36576-z Fig. 3 \| Detailed MOF structural information and photophysical properties evaluation of the rare-earth MOF family. a Ball-and-stick representation of MOF structure showing the intra-cluster and inter-cluster intermetallic distances in compound 1, EuTCPB. Atom color scheme: C gray, O red, Yb green. H atoms are omitted for clarity. b Energy transfer diagram illustrating the relationships between each component of the compounds reported here when the ligand is excited directly (λ_ex = 337 nm). Colored arrows were chosen to approximate emission color of each transition. c Emission spectra for select compounds in the visible range, with the excitation wavelength 394 nm; all peaks associated with Eu. d Emission spectra for select compounds in the NIR range, with the excitation wavelength 394 nm; all peaks associated with Yb. directly (λ_ex = 337 nm). Colored arrows were chosen to approximate emission color of each transition. c Emission spectra for select compounds in the visible range, with the excitation wavelength 394 nm; all peaks associated with Eu. d Emission spectra for select compounds in the NIR range, with the excitation wavelength 394 nm; all peaks associated with Yb. Fig. 3 \| Detailed MOF structural information and photophysical properties Fig. 3 \| Detailed MOF structural information and photophysical properties evaluation of the rare-earth MOF family. a Ball-and-stick representation of MOF structure showing the intra-cluster and inter-cluster intermetallic distances in compound 1, EuTCPB. Atom color scheme: C gray, O red, Yb green. H atoms are omitted for clarity. b Energy transfer diagram illustrating the relationships between each component of the compounds reported here when the ligand is excited Nature Communications\| (2023) 14:981 Photoluminescent lifetime measurements Photoluminescent decay in the visible range in compounds 1–3 and 8–13 were measured at 620 nm to target the 5D0 →4F2 transition in Eu and ﬁtted by a single exponential function (Fig. 4a–c). The char- acteristic decay times for these ﬁtting functions are given in Table 2. Figure 4a shows data for compounds 1, 8, and 9, and demonstrates that diluting Eu with the inactive metal Gd has little effect on the lifetime of the material, because the characteristic decay time is ~400 μs regardless of composition. Figure 4b shows that, when Yb is included with Eu in compounds 2 and 3, the energy from a long-lived emitter (Eu) drains to a short-lived one (Yb), resulting in an overall decrease in the visible lifetime from 367 μs for the pure Eu compound 1 to 125 μs for the Yb rich compound 3. The NIR emission decay curves were accurately modeled as biexponential decays. While the biexponential decay curve is well known, the typical least squares minimization used for ﬁtting is necessarily iterative and can be complicated when applied to equa- tions with multiple ﬁt parameters45. A method was thus developed that enables the two decay times and the amplitude to be computed from three parameters that are numerically derived from the experimental decay curves (initial inverse decay rate, average decay time, and square root of the second moment of the decay). This moments method is described in detail in Supplementary Fig. 17. After exploring the binary interactions between metals in the MOFs, we studied trimetallic compositions, combining Gd, Eu, and Yb into a single compound and producing more complex behavior. Nature Communications\| (2023) 14:981 5 https://doi.org/10.1038/s41467-023-36576-z Article Fig. 4 \| Fluorescence decay data for reported compounds. a–c Visible decay curves for all compounds containing Eu; d A ternary diagram of the different compounds with colors corresponding to the lifetime of each. These show the relative effects of Gd and Yb content on emission lifetimes of Eu in these materials. compounds with colors corresponding to the lifetime of each. These show the relative effects of Gd and Yb content on emission lifetimes of Eu in these materials. compounds with colors corresponding to the lifetime of each. These show the relative effects of Gd and Yb content on emission lifetimes of Eu in these materials. Fig. 4 \| Fluorescence decay data for reported compounds. Photoluminescent lifetime measurements a–c Visible decay curves for all compounds containing Eu; d A ternary diagram of the different simultaneous lengthening in the NIR lifetime. The relative lifetimes of each compound in this case are determined only by the ratio of Eu to Yb present in each. Table 2 \| Characteristic times for the single exponential decay of each compound that emits visible light Compound 1 2 3 8 9 10 11 12 13 τ (μs) 367 182 125 415 390 240 122 300 189 For these measurements, λex = 337 nm, and λem = 620 nm. The standard deviation of reported lifetimes is +/−0.22% based on 5 measurements of a single compound. The uncertainty of the measurements reported is +/−5%based on repeated measurements across multiplesystems and calibration against published materials. Table 2 \| Characteristic times for the single exponential decay of each compound that emits visible light In trimetallic compounds, the Gd remains inactive, but provides physical spacing between the Eu and Yb ions. While the position of the Gd cannot be controlled precisely at the atomic level, its presence in the MOF will increase the average distance between the emitting ions and reduce the statistical chance of energy transfer between them. This modulation effect is most visible at the extremes of composition. For example, the equimolar compound 13 has a characteristic visible lifetime of 189 μs, but the addition of a signiﬁcant amount of Eu (compound 10) extends the visible lifetime to 240 μs, and a further addition of Gd (compound 12) increases the visible lifetime to 300 μs. Because the physical separation caused by Gd effectively prevents energy transfer between Eu and Yb, geometric proximity has a greater impact on lifetime than the Eu-to-Yb ratio alone. Nature Communications\| (2023) 14:981 Heterometallic distribution discussion Accordingly, heterometallic clusters were modeled for the six compounds highlighted in Fig. 5a, along with the three homometallic clusters, Figs. S11 and S12, to identify qualitative Compound 3 Eu0.34Yb0.66 τ = 125 μs Compound 13 Eu0.32Gd0.37Yb0.31 τ = 189 μs Compound 12 Eu0.15Gd0.70Yb0.15 τ = 300 μs Gd b Fig. 5 \| Information regarding metal distribution and its effect on the reported MOFs. a Illustration of the effect of composition on visible lifetime for different compounds. The lifetime of a compound is determined by the probability of a Yb metal center (blue sphere) neighboring an Eu metal center (red sphere) and receiving energy from it. The presence of a Gd metal center (green sphere) reduces the probability that an Eu center will have a neighboring Yb center. b Calculated RE PDOS near the electronic band edges for three representative heterometallic clusters 3Eu-6Yb (left), 3Eu-3Gd-3Yb (center), and 1Eu-7Gd-1Yb (right). The PDOS identify the relative electron density localized on each of the three RE elements; Eu (red), Yb (blue), and Gd (green). Each panel highlights the two primary peaks of RE electron density in the conduction band (black dashed box with labeled vertical arrows 1 and 2) which participate in excited state relaxation mechanisms. The three panels are organized by relative luminescence decay time as measured in this experimental work. Compound 13 Eu0.32Gd0.37Yb0.31 τ = 189 μs b b clusters 3Eu-6Yb (left), 3Eu-3Gd-3Yb (center), and 1Eu-7Gd-1Yb (right). The PDOS identify the relative electron density localized on each of the three RE elements; Eu (red), Yb (blue), and Gd (green). Each panel highlights the two primary peaks of RE electron density in the conduction band (black dashed box with labeled vertical arrows 1 and 2) which participate in excited state relaxation mechanisms. The three panels are organized by relative luminescence decay time as measured in this experimental work. Fig. 5 \| Information regarding metal distribution and its effect on the reported MOFs. a Illustration of the effect of composition on visible lifetime for different compounds. The lifetime of a compound is determined by the probability of a Yb metal center (blue sphere) neighboring an Eu metal center (red sphere) and receiving energy from it. The presence of a Gd metal center (green sphere) reduces the probability that an Eu center will have a neighboring Yb center. Heterometallic distribution discussion Having demonstrated that changes in lifetime are a consequence of both the ratio of the Eu donor and Yb acceptor, and the presence of the optically inactive Gd ions, we note that the dependence on all three creates a broad encoding space, with multiple possible compositions for a given lifetime and vice versa. We demonstrate this concept using an averaging approach to metal distribution at the individual cluster level. Based on the evidence provided by the lifetime measurements, Fig. 5a visually represents mixed metal clusters. We determined the average cluster composition for each compound by dividing the nine metal ions according to the bulk composition and randomizing their position in the cluster to highlight a representative cluster for each composition. This visuali- zation explains how different compositions can produce identical lifetimes in a three-metal system, and how compositions with identical quantities of visible emitters can have vastly different lifetimes. For example, compounds 11 and 12 contain nearly identical quantities of Eu, but the presence of Gd in compound 12 acts as a barrier to energy transfer and dramatically lengthens the lifetime. In the dimetallic compounds, the interactions between each RE are relatively simple because the inclusion of the inactive Gd ion does not signiﬁcantly affect the lifetime of either Eu or Yb, as it does not participate in energy transfer. In other studies, dilution of a visible emitter by an inactive ion has been shown to affect the luminescence intensity or quantum yield of the compound examined, but its effect on the compound’s lifetime was never explored31. When the compound contains Eu and Yb, the lifetime of both visible and NIR emissions are affected. Because the 2F5/2 excited state of Yb lies below the 5D0 excited state of Eu, energy is transferred non- radiatively from Eu to Yb. Yb has a signiﬁcantly shorter lifetime than Eu and emits the transferred energy in the NIR range. Heterometallic distribution discussion The draining of energy from the high energy Eu excited states to the low energy Yb excited states causes a signiﬁcant reduction in the visible lifetime and a To gather additional insights into how heterometallic composi- tions and relative atomic distribution impact the electronic structure of the materials, further investigation has been conducted using Nature Communications\| (2023) 14:981 6 https://doi.org/10.1038/s41467-023-36576-z Article Compound 11 Eu0.17Gd0.19Yb0.64 τ = 122 μs Compound 3 Eu0.34Yb0.66 τ = 125 μs Compound 13 Eu0.32Gd0.37Yb0.31 τ = 189 μs Compound 2 Eu0.69Yb0.31 τ = 182 μs Compound 10 Eu0.65Gd0.20Yb0.15 τ = 240 μs Compound 12 Eu0.15Gd0.70Yb0.15 τ = 300 μs Eu Yb Gd a b Luminescence lifetime formation regarding metal distribution and its effect on the reported Illustration of the effect of composition on visible lifetime for different nds. The lifetime of a compound is determined by the probability of a Yb nter (blue sphere) neighboring an Eu metal center (red sphere) and energy from it. The presence of a Gd metal center (green sphere) reduces ability that an Eu center will have a neighboring Yb center. b Calculated RE clusters 3Eu-6Yb (left), 3Eu-3Gd-3Yb (center), and 1Eu-7Gd-1Yb (right). The PDOS identify the relative electron density localized on each of the three RE elements; Eu (red), Yb (blue), and Gd (green). Each panel highlights the two primary peaks of RE electron density in the conduction band (black dashed box with labeled vertical arrows 1 and 2) which participate in excited state relaxation mechanisms. The three panels are organized by relative luminescence decay time as measured in this Compound 13 Eu0.32Gd0.37Yb0.31 τ = 189 μs Compound 2 Eu0.69Yb0.31 τ = 182 μs Compound 10 Eu0.65Gd0.20Yb0.15 τ = 240 μs Luminescence lifetime Compound 11 Eu0.17Gd0.19Yb0.64 τ = 122 μs Compound 3 Eu0.34Yb0.66 τ = 125 μs Eu Yb Gd a Compound 13 Eu0.32Gd0.37Yb0.31 τ = 189 μs Compound 2 Eu0.69Yb0.31 τ = 182 μs Compound 10 Eu0.65Gd0.20Yb0.15 τ = 240 μs Compound 12 Eu0.15Gd0.70Yb0.15 τ = 300 μs Luminescence lifetime Compound 10 Eu0.65Gd0.20Yb0.15 τ = 240 μs Compound 10 Eu0.65Gd0.20Yb0.15 τ = 240 μs a Compound 13 Eu0.32Gd0.37Yb0.31 τ = 189 μs Luminescenc density functional theory (DFT) calculations. Projected density of states (PDOS) calculations are well suited to provide individual ele- mental contributions to the overall homometallic and heterometallic cluster electronic structures, thus helping elucidate the complex correlation between metallic compositions and resulting unique pho- tophysical signatures. Heterometallic distribution discussion As a ﬁnal test, we overlaid two separate 3 letter codes, using standard ﬂuorescence excitation and emission for one signature and time-gated ﬂuorescence for the second signature to create a double- encoded dynamic tag. Figure 6b and Supplementary Fig. 16 show a tag utilizing two different compounds overlayed within the same set of 3 × 6 wells. In this case, one pattern of dots is brightest under standard excitation and emission or after short delays because of the con- centration of compounds present. However, when measurements are taken after a delay, the brightness becomes dependent on lifetime rather than concentration, and a different pattern is shown. As in the dynamic tag above, both a correct threshold value and a correct time delay are required in order to decode the tag. Numerical raw data for all encodings is given in Supplementary Tables 2–4. The results of the calculated electronic structures support two primary trends identiﬁed in the experimentally observed spectra: (i) increased Eu content results in longer luminescence lifetimes and (ii) increased Gd content disrupts intermetallic energy transfer between Eu and Yb. The calculated PDOS for models shows a matching trend of increasing Eu:Yb density at the ﬁrst conduction band peak and models from fastest to slowest luminescence decay, Fig. 5b and Supplemen- tary Fig. 11. The higher PDOS of Eu at the lower energy state indicates that longer lived charge carriers nonradiatively migrate to Eu prior to any optical emission event, supporting the observed experimental results. The introduction of Gd into the heterometallic clusters is hypo- thesized to impact the optical response by increasing spatial separa- tion between the optically active Eu and Yb species in the individual clusters. Spatial separation reduces the transition dipole moment between Eu and Yb species, reducing the likelihood of the event. Electronic structure calculations reinforce this premise revealing that introduction of Gd into the heterometallic gives rise to new electronic states. Importantly, the presence of Gd directly impacts the increased Eu:Yb ratio at the ﬁrst conduction band peak, Fig. 5b, further sup- porting long lived charge carriers nonradiatively relaxing to Eu and resulting in long lived photoluminescence lifetimes. Materials with tunable lifetimes could be directly incorporated into printable inks to provide authentication for security documents, packaging, etc. Heterometallic distribution discussion b Calculated RE PDOS near the electronic band edges for three representative heterometallic correlation between metallic compositions and resulting unique pho- tophysical signatures. Accordingly, heterometallic clusters were modeled for the six compounds highlighted in Fig. 5a, along with the three homometallic clusters, Figs. S11 and S12, to identify qualitative density functional theory (DFT) calculations. Projected density of states (PDOS) calculations are well suited to provide individual ele- mental contributions to the overall homometallic and heterometallic cluster electronic structures, thus helping elucidate the complex Nature Communications\| (2023) 14:981 7 7 Article https://doi.org/10.1038/s41467-023-36576-z This method encodes based on emission alone (without differentiating by lifetime) and could be interrogated using a microplate reader or simply visually examined using a UV lamp. information in correlating decay times with metallic distributions. For simplicity, the compounds in Fig. 5a can be categorized by decay rate as fast (compounds 3 and 11), intermediate (compounds 13 and 2), and slow (compounds 12 and 10). Models for compounds 3, 13, and 12 are presented as exemplars for the three luminescence lifetimes regimes to highlight the impact of heterometallic distribution, Fig. 5b. We then created a dynamic tag, which presents a nearly uniform ﬂuorescence signature (an undifferentiated 3 × 6 block of dots) under the standard static excitation and emission method; the braille code is only revealed if measurements are taken with the correct delay after excitation (Fig. 6a). The measured intensities for each well at different time points is shown in Supplementary Fig. 15. Correct interrogation of the dynamic tag would require a threshold operation at a speciﬁed time and intensity (above which a dot is considered ﬁlled and below which a dot is considered blank). For example, Supplementary Fig. 15 shows that this threshold value could be set anywhere between 1000 and 3000 arb. units after a 750 μs delay. The calculated electronic structures of all metallic clusters iden- tify two primary peaks near the edge of the conduction band and are clearly marked in Fig. 5b. To emphasize the correlation of metallic distribution with electronic structure, in Fig. 5b are presented the RE only PDOS for clarity. Notably, the peaks contain contributions from all elements within the cluster, with large densities localized on C and O atoms, Supplementary Fig. 12. The hybridization of the organic com- ponents with the RE species is expected as the metallic clusters are bridged via organic linkers in the periodic material. Encoding via luminescence lifetime Because the time scale of luminescence decay in these materials is on the scale of tens to hundreds of microseconds in both the visible and NIR ranges, it can be decoded with relatively unsophisticated inter- rogation equipment, unlike tags with nanosecond-scale lifetimes2,3. Further, because the lifetime encoding is invisible to the unaided eye, the tag can also be covert, visible only to those who know the correct method of interrogation. While not demonstrated here, additional layers of encoding could be introduced either through the presence of different emitting REs or their ratios (i.e. differentiating the encoding via spectra or color), through the presence of inactive REs like Gd that are detectable via X-ray ﬂuorescence or EDS (i.e. differentiating by composition inde- pendent of the measurable spectra), or through the inclusion of additional molecules in the MOF pore-space. The continuum of life- times and compositions displayed in Fig. 4d allows composition, spectrum, and lifetime to be decoupled from each other, dramatically expanding the potential space for multilayered encoding. The scale of the visible lifetimes and presence of NIR signal provide examples of covert layers of encoding, in addition to the overt encoding based on visible emission. To demonstrate the relative simplicity of interrogating these highly complex tags, we used a microplate reader to measure standard and time gated (measured a speciﬁed time after a pulse of excitation) ﬂuorescence signatures. As proof-of-concept, we utilized the braille alphabet and two representative trimetallic compositions displaying very different decay proﬁles: compound 11 (short lifetime) and com- pound 12 (long lifetime). Utilizing the braille alphabet as encoding method is advantageous because it is already a coded form and is also less complex than using a dot matrix for each letter. To facilitate suspension of the MOF particles in ethanol, we mechanically ground the MOFs to reduce particle size. Supplementary Figs. 8 and 9 show the PXRD spectra and lifetimes of these ground samples and conﬁrm that the size reduction did not interfere with their crystallinity or decay dynamics. Supplementary Fig. 10 shows the PL spectra of both ground samples, which are indistinguishable to the microplate reader. In summary, here we have demonstrated the use of a ternary structural space to effectively decouple composition and photo- physical properties for the creation of highly complex, difﬁcult to counterfeit optical tags. Heterometallic distribution discussion To demonstrate this process, we patterned a trans- parent thunderbird logo containing discrete regions of short and long lifetime pigments (compounds 11 and 12) onto a transparent substrate (Fig. 7a–e). Examination with a static UV light source reveals the logo in its entirety (Fig. 7e), but differentiation of the distinct materials requires a high frame-rate camera. Digital high-speed imaging of the logo after pulsed excitation, (Fig. 7g), shows the distinct decay rates of each compound, with decay proﬁles that match what is expected based on prior measurements of the compounds alone (Fig. 7h). A video showcasing the real-time high-speed imaging of the Sandia thunderbird logo tag is shown in the Supplementary Movie 1. This real- life demonstration supports both the effective control over the life- time and the utility of that control in a practical setting for tag mate- rials. These patterns were generated by laser direct write printing and the use of a laser-cut stencil, but could be readily adapted to other approaches, including ﬂexography/(micro) stamping, ink-jet, and additive manufacturing (e.g. direct-ink write). Nature Communications\| (2023) 14:981 Encoding via luminescence lifetime Composition and lifetime are decoupled, ensuring true orthogonality in encoding and enabling the synthesis of a large set of rationally designed materials. In Supplementary Figs. 13 and 14 we show two simple tags, based on single compound compositions, using steady-state ﬂuorescence excitation and emission to encode different three letter sequences. Nature Communications\| (2023) 14:981 8 Article https://doi.org/10.1038/s41467-023-36576-z presentation of an encoded message utilizing the braille alphabet in a ate. a A dynamic tag that shows the transition from undifferentiated dots sage over time based on different compound lifetimes. b A double- ynamic tag that shows an initial encoding based on emission intensity via compound concentration in each well, with a ﬁnal encoding based on different compound lifetimes. The intensity of each red dot is based on experimental data. Threshold operations are the process of choosing an intensity value as a cutoff for reading a dot as lit versus unlit for the purpose of decoding. Fig. 6 \| Representation of an encoded message utilizing the braille alphabet in a 96-well plate. a A dynamic tag that shows the transition from undifferentiated dots to the message over time based on different compound lifetimes. b A double- encoded dynamic tag that shows an initial encoding based on emission intensity via compound concentration in each well, with a ﬁnal encoding based on different compound lifetimes. The intensity of each red dot is based on experimental data. Threshold operations are the process of choosing an intensity value as a cutoff for reading a dot as lit versus unlit for the purpose of decoding. Fig. 6 \| Representation of an encoded message utilizing the braille alphabet in a 96-well plate. a A dynamic tag that shows the transition from undifferentiated dots to the message over time based on different compound lifetimes. b A double- encoded dynamic tag that shows an initial encoding based on emission intensity via compound concentration in each well, with a ﬁnal encoding based on different compound lifetimes. The intensity of each red dot is based on experimental data. Threshold operations are the process of choosing an intensity value as a cutoff for reading a dot as lit versus unlit for the purpose of decoding. Precise manipulation of the luminescence decay dynamics over a wide microsecond regime was achieved owing to the control over metal distribution in these systems. Encoding via luminescence lifetime The lifetime is efﬁciently con- trolled by energy transfer between visible (Eu) and NIR (Yb) emitting metals, while an optically inactive metal (Gd) dilutes them to facilitate or hinder that transfer. two distinct compositions patterned on glass and interrogated via digital high-speed imaging to produce a dynamic image that changed over time. These proof-of-concept demonstrations show the utility of these tag materials in facile, complex encoding and interrogation, both accessible for widespread use and secure against counter- feiters. The material design strategy presented can potentially be applied to a variety of rare-earth MOFs with different organic or metallic constituents, expanding the library of unique, multilayered encodings To demonstrate the utility of these materials as practical optical tags we implemented two design strategies, leveraging visible life- times in the 100 s of μs. The ﬁrst encoding strategy utilized the braille alphabet, interrogated by a standard laboratory microplate reader to produce 3 tags: (i) a static pattern read under constant illumination; (ii) a dynamic pattern that appears undifferentiated under constant illumination but is revealed via lifetime of its constituents; (iii) a double dynamic pattern that utilizes both concentration and lifetime to change the encoded message depending on when the pattern is read. The second strategy utilized photocurable inks consisting of Nature Communications\| (2023) 14:981 Synthesis of compounds 1–13 a d 1 mm 0 500 1000 167 334 667 834 Nd:Yag - 3ω : 355 nm Sample 20° Expansion and collimation optics 105 mm macro lenses at f/2.8 and f/8 9 mJ / 7 mJ / 5 mJ UV laser marker Detailing tape Substrate a b c d e f h g 1 mm 1 mm Slow Fast b d b c e 0 500 El d ti ( ) 1000 167 334 667 834 Nd:Yag - 3ω : 355 nm Sample 20° Expansion and collimation optics 105 mm macro lenses at f/2.8 and f/8 9 mJ / 7 mJ / 5 mJ f g f h h Slow Fast Expansion and collimation optics 0 500 Elapsed time (μs) 1000 167 334 667 834 g g laser excitation, showing two distinct lifetimes for the thunderbird and border respectively. h Decay data curves and ﬁt curves for each section of the logo, derived from digital high-speed images, compared to the decay of pure compound 11 and 12 reported above. In every image, the pattern was printed on a 25 mm × 25 mm substrate. Fig. 7 \| A demonstration of patterning and interrogating a tag made with compounds 11 and 12 in an ink. a Diagram detailing stencil design and etching. b Stencil composed of painters’ tape on glass. c Inked Sandia thunderbird logo under UV light, held in hand for scale. d, e Inked logo under ambient and UV light respectively. f Diagram of the laser excitation and digital high-speed imaging setup used to capture the decay of the logo. g Time-lapse of tag emission after pulsed Fig. 7 \| A demonstration of patterning and interrogating a tag made with compounds 11 and 12 in an ink. a Diagram detailing stencil design and etching. b Stencil composed of painters’ tape on glass. c Inked Sandia thunderbird logo under UV light, held in hand for scale. d, e Inked logo under ambient and UV light respectively. f Diagram of the laser excitation and digital high-speed imaging setup used to capture the decay of the logo. g Time-lapse of tag emission after pulsed Powder X-ray diffraction Measurements were performed on a Bruker D2Phaser instrument using CuKα radiation (λ = 1.54178 Å). Scanning electron microscopy and energy dispersive spectroscopy SEM images were captured using an FEI NovaNano SEM 230 at an accelerating voltage of 30 kV. EDS analyses were collected using an EDAX Genesis Apex 2 with an Apollo SDD detector. Photoluminescence measurements The visible PL emission spectra of powder samples were collected using a Horiba Jobin-Yvon Fluorolog-3 double-grating/double-grating Fluorescence Spectrophotometer in front-face mode. This instrument uses a continuous wave 450 W Xenon arc lamp excitation source and a room temperature R928P photomultiplier tube (PMT) for the detec- tion of visible light. A complete instrumental correction was per- formed on all spectra which compensates for factors such as the grating efﬁciencies and the wavelength-dependent PMT response. The powder samples were sandwiched between glass slides for these measurements. The X-ray diffraction data were measured using a Bruker D8 Venture dual-source single crystal X-ray diffractometer, employing Cu Kα radiation (λ = 1.54178 Å). Data were collected using a Photon II CMOS area detector on samples superglued to glass ﬁbers. Reﬂection data were processed using the Bruker Apex 3 Suite, including an absorption correction through SADABS, and space group determination using XPREP. The structure was determined with SHELX-2019 software. The structure was solved with XT using intrinsic phasing and reﬁned using full-matrix least-squares on F2 through XL. The structure was ﬁnalized in Olex 2 (v.1.3.0). Synthesis of compounds 1–13 In a typical synthesis, 0.0229 mmol of metal salts (EuCl3·6H2O, Gd(NO3)3·6H2O, and Yb(NO3)3·5H2O, Sigma-Aldrich, 99.99% trace metal basis) in the same proportions as the target composition, 0.0065 mmol (3.65 mg) of TCPB (Sigma Aldrich, contains up to 6 wt% Nature Communications\| (2023) 14:981 9 https://doi.org/10.1038/s41467-023-36576-z Article water, ≥98%), 4.0075 mmol (561.49 mg) of 2-ﬂuorobenzoic acid, (Sigma Aldrich, 97%), 0.2 mL of HNO3 (3.5 M in DMF, Fisher Chemical, Certiﬁed ACS plus), and 2.8 mL of dimethylformamide (DMF, Acros Powder X-ray diffraction Measurements were performed on a Bruker D2Phaser instrument using CuKα radiation (λ = 1.54178 Å). 0 500 Elapsed time (μs) 1000 167 334 667 834 Nd:Yag - 3ω : 355 nm Sample 20° Expansion and collimation optics 105 mm macro lenses at f/2.8 and f/8 9 mJ / 7 mJ / 5 mJ UV laser marker Detailing tape Substrate a b c d e f h g 1 mm 1 mm Slow Fast Fig. 7 \| A demonstration of patterning and interrogating a tag made with compounds 11 and 12 in an ink. a Diagram detailing stencil design and etching. b Stencil composed of painters’ tape on glass. c Inked Sandia thunderbird logo under UV light, held in hand for scale. d, e Inked logo under ambient and UV light respectively. f Diagram of the laser excitation and digital high-speed imaging setup used to capture the decay of the logo. g Time-lapse of tag emission after pulsed laser excitation, showing two distinct lifetimes for the thunderbird and border respectively. h Decay data curves and ﬁt curves for each section of the logo, derived from digital high-speed images, compared to the decay of pure compound 11 and 12 reported above. In every image, the pattern was printed on a 25 mm × 25 mm substrate. UV laser marker Detailing tape Substrate a d e h 1 mm 1 mm Slow Fast laser excitation, showing two distinct lifetimes for the thunderbird and border respectively. h Decay data curves and ﬁt curves for each section of the logo, derived from digital high-speed images, compared to the decay of pure compound 11 and 12 reported above. In every image, the pattern was printed on a 25 mm × 25 mm substrate. Synthesis of compounds 1–13 water, ≥98%), 4.0075 mmol (561.49 mg) of 2-ﬂuorobenzoic acid, (Sigma Aldrich, 97%), 0.2 mL of HNO3 (3.5 M in DMF, Fisher Chemical, Certiﬁed ACS plus), and 2.8 mL of dimethylformamide (DMF, Acros Organics, 99.5% for HPLC) were combined in a glass vial. This mixture was heated to 115 °C over one hour, held there for 48 h, then cooled to room temperature. The resulting particles were washed three times with DMF, then exchanged with methanol over three days (the methanol was changed daily). Microplate reader ﬂuorescence and time-gated ﬂuorescence measurements To facilitate equal distribution of sample into wells, compounds 11 and 12 were activated at 120 °C for 12 h, then ground by hand using a mortar and pestle to reduce particle size. Both were then resuspended at known concentrations in ethanol. Ground compounds 11 and 12 were placed in a 96-well black plate for analysis. Fluorescence and time-gated ﬂuorescence measurements were made using a Biotek Neo 2 microplate reader with excitation of 340 nm (+/−20 nm) and emission set of 617 nm (+/−20 nm). Standard ﬂuorescence measurements were made with simultaneous excitation and emission. Time-gated ﬂuorescence mea- surements were made with the excitation separated from the emission collection. Emission was collected over 20 µs and collection was initi- ated at 20, 100, 750, 1000, or 1250 µs after excitation. f tð Þ = aet=τ1 + ð1 aÞet=τ2: ð1Þ ð1Þ This equation can be ﬁt to the experimental decay curve by least squares minimization, but this approach has well-known difﬁculties, notably local minima and non-positivity of the three parameters. We have thus developed an approach for the extraction of the three parameters that does not involve least squares ﬁtting. We begin by deﬁning three parameters that are obtained numerically from the experimental decay curve. These parameters will then be used to compute the decay parameters, i.e., the two decay times and the amplitude a. The ﬁrst parameter A is the inverse initial decay rate. Ground versions of compounds 11 and 12 were ﬁrst examined independently using standard ﬂuorescence measurements. For these experiments, 1 mg of sample was placed into each well, and samples were arranged using the braille alphabet to encode DOE or SNL with the raised dots represented by ﬁlled wells. Next, tags with separate appli- cations of ground compounds 11 and 12 were then tested to create a hidden signature of SNL in the braille alphabet, encoded with time- gated ﬂuorescence. To test this, the wells encoding for the raised dots were ﬁlled with 0.2 mg of ground compound 12 and the empty spaces were ﬁlled with 1 mg of ground compound 11. Volumes of ethanol were kept even between the wells, and the plate was read using the time- gated ﬂuorescence settings. Finally, overlaying the DOE and SNL sig- natures were tested. Decay curve ﬁtting Absolute quantum yield (QY) measurements QY was determined by exciting the sample with diffuse light inside an integrating sphere46. The powder samples were placed in Pyrex NMR tubes for these measurements and inserted into the integrating sphere. Both the diffuse excitation and emission light power were simultaneously recorded. The QYs were calculated from these power measurements. Most of the luminescent decay curves collected for this paper were accurately modeled as double exponential decays. Double exponential decays have ﬁve parameters, two amplitudes, two decay times and a baseline amplitude. The baseline was obtained from the detector from the signal just before the laser pulse. The laser pulse rate was about 5 Hz, so this corresponds to a signal delay of ~200 ms after the pulse, which is many times longer than the longest decay time measured. The two amplitudes were reduced to one by normalizing the luminescent decay curve to unity. The decay model can then be written Materials patterning A, B, and C are deﬁned so that they each have the units of time and it is easily shown that A<B<C. To obtain accurate integrals one must know the baseline accurately and one should ideally extrapolate to inﬁnite time. This extrapolation can easily be done by plotting BðtÞ and CðtÞ against the amplitude of the decay. But if data is collected over a long enough time, then it is sufﬁcient to make a parametric plot of BðtÞ against CðtÞ and obtain B, C from the endpoint. Plotting in this way greatly reduces noise. For the thunderbird pattern, ground compounds 11 and 12 were mixed at an 8–10% weight ratio with a UV-curable optical adhesive (NOA 61 or 65; Norland Products) and deposited sequentially on a fused silica coverglass on desired areas of an exposed stenciled pattern. The stenciled image was laser patterned onto a ﬁlm (blue painter’s tape; 3 M) adhered to the substrate using a UV laser marker (Keyence MD-U). Material deposited on the stencil was cured with a UV spot lamp (Dymax RediCure). The solution to these three equations is a quartic, but there is a simpler way to solve these. The second decay time can be expressed in terms of the ﬁrst by τ2 = ðBaτ1Þ ð1aÞ and by τ2 2 = ðC2aτ2 1 Þ ð1aÞ . Combining these gives a quadratic equation whose solutions are Photoluminescence lifetime measurements Measurements were conducted on a SDTQ600 TA instrument. The samples were heated to 900 °C at a 5 °C min−1 heating rate, under continuous nitrogen ﬂow. PL lifetime measurements were made on powder samples sandwiched between quartz slides by exciting the samples with a PTI GL3300 pulsed nitrogen laser at 337 nm. Two detectors were used, one to trigger the Nature Communications\| (2023) 14:981 10 Article https://doi.org/10.1038/s41467-023-36576-z f = 150 mm). An iris was placed after the lenses to generate a 23 mm diameter beam that was incident upon the sample. oscilloscope (Thorlabs PDA55 silicon photodetector) and the other to measure the emission. The signals were averaged using a 1 GHz Tek- tronix TDS 5104 digital oscilloscope. A Thorlabs PDA55 silicon photo- detector was used to measure the Eu3+ emission. A 620 nm narrow bandpass ﬁlter with FWHM of 10 nm was placed in front of the detector. A Thorlabs PDA10CS InGaAs switchable gain ampliﬁed detector was used to measure the Yb3+ emission. A lens and two long-pass ﬁlters (550 nm and 950 nm cut-on wavelength) were placed in front of the detector. The uncertainty of the measurements reported is +/−5%. For the lifetime measurement, two high-speed cameras were used: a broadband (no Bayer color ﬁlters in front of the pixels) Vision Research v2511 running at 25,000 frames per second (fps), with an exposure of 39.54 µs; and a color Vision Research v1212c running at 12,000 fps with an exposure of 82.59 µs. Both cameras used Nikkor 105 mm macro lenses at f/2.8 for the small tag and f/8 for the large one. The cameras resolution was set at their maximum of 1280 × 800 pixels. Microplate reader ﬂuorescence and time-gated ﬂuorescence measurements For this experiment 1 mg of compound 11 was laid out as DOE and then overlaid with 0.1 mg of compound 12 laid out as SNL. The plate was read using the time-gated ﬂuorescence settings. A1 lim t!0 dlnf tð Þ dt = a τ1 + ð1 aÞ τ2 ð2Þ ð2Þ The second parameter is average decay time B. The second parameter is average decay time B. B = lim t!1 BðtÞ Z t 0 f sð Þds = aτ1 + ð1 aÞτ2 ð3Þ ð3Þ Finally, the parameter C is the square root of the second moment of the decay. C2 = lim t!1 C2ðtÞ Z t 0 sf sð Þds = aτ2 1 + ð1 aÞτ2 2 ð4Þ ð4Þ https://doi.org/10.1038/s41467-023-36576-z https://doi.org/10.1038/s41467-023-36576-z Any given value of the amplitude will then generate a value of τ1, which can then be used to compute a value of τ2. These are the correct values if they satisfy Eq. 2. A plot of g = A 1=½ a τ1 + ð1aÞ τ2 vs a must therefore exhibit a zero crossing, which gives the correct value of the amplitude, and thus the two decay times. To remove negative values and singularities it is best to plot the ﬁt quality function Q = atanðg2Þ. Plotting this logarithmically creates a line that points directly to the correct amplitude. Computational models and methodology 31, 1807900 (2019). 9. Liu, H. et al. Tunable resonator-upconverted emission (TRUE) color printing and applications in optical security. Adv. Mater. 31, 1807900 (2019). 10. Wang, Z. et al. Ultralong-lived room temperature phosphorescence from N and P codoped self-protective carbonized polymer dots for conﬁdential information encryption and decryption. J. Mater. Chem. C 9, 4847–4853 (2021). 11. Liu, X. et al. Dual-mode long-lived luminescence of Mn2+-doped nanoparticles for multilevel anticounterfeiting. ACS Appl. Mater. Interfaces 11, 30146–30153 (2019). 12. Tan, M., Li, F., Wang, X., Fan, R. & Chen, G. Temporal multilevel luminescence anticounterfeiting through scattering media. ACS Nano 14, 6532–6538 (2020). 13. Katumo, N. et al. Anticounterfeiting labels with smartphone- readable dynamic luminescent patterns based on tailored persis- tent lifetimes in Gd2O2S:Eu3+/Ti4+. Adv. Mater. Technol. 6, 2100047 (2021). The PDOS were calculated according to the VASP protocal using LORBIT = 11. This provides spatially resolved electron density as a function of spherical volume at each atomic location. The PDOS cal- culations utilized the standard RWIGS values provided in each ele- ments PBE POTCAR ﬁle. While spherical limits on electron density are known to underpredict hybridized electrons in space, the primary atomic contributions calculated in the PDOS were conﬁrmed by vizualizing molecular orbitals from calculated PARCHG ﬁles which are derived from the calculated wavefunction. 14. Wei, X., Zhao, G., Feng, P., Kou, Y. & Tang, Y. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Author contributions D.S.G. and J.I.D. developed the concept and designed the experiments. J.I.D. performed the materials synthesis and characterization and lead the manuscript writing. R.A.R. aided the materials synthesis. L.E.S., K.S.B., T.S.L., A.A.C.C., and J.E.M. supported all aspects of optical characterization. B.K. developed the materials patterning. D.J.V. carried out the DFT calculations. All the authors discussed the results and J.I.D., L.E.S., K.S.B., D.J.V., and D.S.G. wrote the manuscript. Additional information speed photography experiments; and Dr. Jacob Harvey for support with structure optimization for computational studies. This research was supported by the Laboratory Directed Research and Development Pro- gram at Sandia National Laboratories (D.S.G.) and was performed, in part,at the Center for Integrated Nanotechnologies, an Ofﬁce of Science User Facility operated for the U.S. Department of Energy (DOE) Ofﬁce of Science. This article has been authored by an employee of National Technology & Engineering Solutions of Sandia, LLC under Contract No. DE-NA0003525 with the U.S. Department of Energy (DOE). The employee owns all right, title and interest in and to the article and is solely responsible for its contents. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclu- sive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this article or allow others to do so, for United States Government purposes. The DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan https://www.energy.gov/downloads/doe- public-access-plan. The views expressed in this article do not necessa- rily represent the views of the U.S. Department of Energy or the United States Government. Correspondence and requests for materials should be addressed to Dorina F. Sava Gallis. Peer review information Nature Communications thanks Zhiqiang Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Nature Communications\| (2023) 14:981 Acknowledgements 41. Tanner, P. A. in Lanthanide Luminescence: Photophysical, Analytical and Biological Aspects (eds Hänninen, P. & Härmä, H.) 183–233 (Springer Berlin Heidelberg, 2011). The authors would like to thank Dr. Eric Sikma, Luke Lucero, Dr. Nichole Valdez, and Dr. Mark Rodriguez for support with materials synthesis and characterization; Dr. Caroline Winters for her assistance with the high- Nature Communications\| (2023) 14:981 13 Article https://doi.org/10.1038/s41467-023-36576-z Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-36576-z. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-36576-z. Competing interests © The Author(s) 2023 p g The authors declare no competing interests. Nature Communications\| (2023) 14:981 14
https://openalex.org/W1624371787	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0131456&type=printable	English	null	Analysis of a Multi-component Multi-stage Malaria Vaccine Candidate—Tackling the Cocktail Challenge	PloS one	2,015	cc-by	11,434	RESEARCH ARTICLE Analysis of a Multi-component Multi-stage Malaria Vaccine Candidate—Tackling the Cocktail Challenge Alexander Boes1☯, Holger Spiegel1☯, Nadja Voepel1, Gueven Edgue1, Veronique Beiss1, Stephanie Kapelski1, Rolf Fendel1,2, Matthias Scheuermayer3, Gabriele Pradel2¤, Judith M. Bolscher4, Marije C. Behet5, Koen J. Dechering4, Cornelus C. Hermsen5, Robert W. Sauerwein4,5, Stefan Schillberg1, Andreas Reimann1, Rainer Fischer1,2 1 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany, 2 RWTH Aachen University, Institute of Molecular Biotechnology, Aachen, Germany, 3 Zentrum für Infektionsforschung, Wuerzburg, Germany, 4 TropIQ Health Sciences, Nijmegen, The Netherlands, 5 Radboud university medical center, Nijmegen, The Netherlands ☯These authors contributed equally to this work. ¤ Current Address: RWTH Aachen University, Institute for Cellular and Applied Infection Biology Section, Aachen, Germany holger.spiegel@ime.fraunhofer.de ☯These authors contributed equally to this work. ¤ Current Address: RWTH Aachen University, Institute for Cellular and Applied Infection Biology Section, Aachen, Germany * holger.spiegel@ime.fraunhofer.de ☯These authors contributed equally to this work. ¤ Current Address: RWTH Aachen University, Institute for Cellular and Applied Infection Biology Section, Aachen, Germany * h l i l@i f h f d OPEN ACCESS Citation: Boes A, Spiegel H, Voepel N, Edgue G, Beiss V, Kapelski S, et al. (2015) Analysis of a Multi- component Multi-stage Malaria Vaccine Candidate— Tackling the Cocktail Challenge. PLoS ONE 10(7): e0131456. doi:10.1371/journal.pone.0131456 Abstract This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Boes et al. This is an open access article distributed under the terms of the Copyright: © 2015 Boes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract Combining key antigens from the different stages of the P. falciparum life cycle in the con- text of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we cal- culated the IC50 values for the pre-erythrocytic stage (17–25 μg/ml), the blood stage (40– 60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates signifi- cant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy. Editor: Érika Martins Braga, Universidade Federal de Minas Gerais, BRAZIL Received: January 30, 2015 Accepted: June 2, 2015 Published: July 6, 2015 Copyright: © 2015 Boes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: January 30, 2015 Accepted: June 2, 2015 Published: July 6, 2015 Received: January 30, 2015 Accepted: June 2, 2015 Published: July 6, 2015 Received: January 30, 2015 Accepted: June 2, 2015 Published: July 6, 2015 Received: January 30, 2015 Accepted: June 2, 2015 Published: July 6, 2015 Copyright: © 2015 Boes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Boes et al. Introduction Many strategies have been pursued to develop efficient vaccine formulations that prevent infection or at least the clini- cal development of malaria, including peptides [2], non-replicating sporozoites [3], DNA vaccines [4], vectored and prime-boost vectored vaccines [5], various formulations of recombinant pro- teins and fusion proteins [6–9] and fusion peptides targeting different stages of the Plasmodium falciparum life cycle [10]. Sporozoite-based approaches have the potential to induce sterile immu- nity but manufacturing costs are high and distribution in developing countries is challenging [11]. Competing Interests: JMB, KJD and RWS are employees of TropIQ Health Sciences B.V. KJD and RWS hold stock in TropIQ Health Sciences B.V. AB, HS, GE, VB, AR, and RFi are inventors of a patent application, title: Multi-component-multi-stage malaria vaccine, application Number: 140011550.2–1412. MCB, NV, SK, RFe, MS, GP, CCH and SS declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials. y g g p g g g Most protein-based vaccine candidates, like RTS,S, which have entered clinical trials, did not fulfill expectations [12]. Whereas RTS,S addresses the pre-erythrocytic stage of the parasite to prevent infection, other vaccine candidates like PfAMA1 target the blood stage to reduce or pre- vent clinical manifestation. So-called transmission-blocking vaccines that target sexual-stage antigens and prevent transmission by the mosquito host are an essential add-on to protective or therapeutic vaccines in the battle to eradicate malaria [13]. As a potential fourth class of malaria vaccines anti-toxin vaccines have been proposed by Schofield et al. in 2002 [14]. A 1993 study with a multi-stage, multi-component malaria vaccine cocktail featuring pre-erythrocytic, blood, and sexual stage antigens yielded promising results [15] although further clinical development of this approach was not reported. Despite the underlying complexity associated with the develop- ment and regulatory approval of cocktails with multi-stage functionality, we believe that an opti- mal malaria vaccine must efficiently target at least these three stages of the parasite life cycle, and this should be the long-term goal in future malaria vaccine development as long as there is no effective way to induce sterile immunity using pre-erythrocytic vaccine candidates. Introduction With the aim to include several promising antigens and domains (based on previously reported results of malaria vaccine development research) from the pre-erythrocytic, blood, and sexual stages of the parasite in a small number of recombinant proteins, we selected 10 antigens (PfCSP_TSR [16], PfCelTos [17–19], PfTRAP_TSR [20], PfMSP1_19_EGF1 [21], PfAMA1 [22], PfMSP4_EGF [23], PfMSP8_EGF1 and EGF2 [24], PfMSP3 [25], Pfs25 [26] and Pfs230 [27]) and combined them in three stage-specific fusion proteins (CCT: pre-erythro- cytic stage: PfCSP_TSR, PfCelTos and PfTRAP_TSR; E3: blood stage: PfMSP1-19_EGF1, PfMSP4_EGF, PfMSP8_EGF1, PfMSP8_EGF2 and PfMSP3; F0: sexual stage: Pfs25 and Pfs230) together with gAMA1 (PfAMA1, included separately to avoid an overly large fusion protein) as one of the leading blood stage antigens, to be used as a multi-component multi-stage vaccine cocktail called PlasmoMix. The final design of the three fusion proteins was a result of an itera- tive expression screening approach (using the transient plant expression system) aiming to combine previously described antigens or antigen domains in the context of a stable, well- expressing vaccine candidate antigen. All four proteins were produced by transient expression in Nicotiana benthamiana plants, a rapid and versatile production system that could be used for the cost-efficient manufacture of vaccines for developing countries [28, 29]. Introduction not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section. Despite years of intensive research malaria still affects millions of people worldwide and claims more than 500,000 lives per year, predominantly in sub-Saharan Africa [1]. Many strategies have been pursued to develop efficient vaccine formulations that prevent infection or at least the clini- cal development of malaria, including peptides [2], non-replicating sporozoites [3], DNA vaccines [4], vectored and prime-boost vectored vaccines [5], various formulations of recombinant pro- teins and fusion proteins [6–9] and fusion peptides targeting different stages of the Plasmodium falciparum life cycle [10]. Sporozoite-based approaches have the potential to induce sterile immu- nity but manufacturing costs are high and distribution in developing countries is challenging [11]. Most protein-based vaccine candidates, like RTS,S, which have entered clinical trials, did not fulfill expectations [12]. Whereas RTS,S addresses the pre-erythrocytic stage of the parasite to prevent infection, other vaccine candidates like PfAMA1 target the blood stage to reduce or pre- vent clinical manifestation. So-called transmission-blocking vaccines that target sexual-stage antigens and prevent transmission by the mosquito host are an essential add-on to protective or therapeutic vaccines in the battle to eradicate malaria [13]. As a potential fourth class of malaria vaccines anti-toxin vaccines have been proposed by Schofield et al. in 2002 [14]. A 1993 study with a multi-stage, multi-component malaria vaccine cocktail featuring pre-erythrocytic, blood, and sexual stage antigens yielded promising results [15] although further clinical development of this approach was not reported. Despite the underlying complexity associated with the develop- ment and regulatory approval of cocktails with multi-stage functionality, we believe that an opti- mal malaria vaccine must efficiently target at least these three stages of the parasite life cycle, and this should be the long-term goal in future malaria vaccine development as long as there is no effective way to induce sterile immunity using pre-erythrocytic vaccine candidates. Despite years of intensive research malaria still affects millions of people worldwide and claims more than 500,000 lives per year, predominantly in sub-Saharan Africa [1]. Multi-component Multi-stage Malaria Vaccine Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Fraunhofer Zukunftsstiftung (http://www.fraunhofer. de/). SK was supported by a "Richtlinien zur Förderung des wissenschaftlichen Nachwuchses (RFwN) Ph.D. grant" from Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University (http://www.rwth-aachen.de/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. TropIQ Health Sciences provided support in the form of salaries for authors JMB, KLD and RWS but did 1 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Multi-component Multi-stage Malaria Vaccine after immunization according to national regulations the animals were anesthetized using Ven- tranquil, stunned using a captive bolt device and exsanguinated by throat cut. Primary human liver cells were freshly isolated from healthy remnant material following tumor removal surgery. The samples were received in an anonymized fashion and none of the authors or personnel on the study was involved in the anonymization. The material was destroyed after use in accordance with Dutch ethical legislation as described in the Medical Research (Human Subjects) Act. The guidelines issued by the ethics committee of the Radboud University Nijmegen Medical Center (full committee name: Commissie Mensgebonden Onderzoek) state that no informed consent is needed for studies that use anonymised materials and that do not reveal information that can be correlated to specific individuals or groups. The protocol used in the current study was reviewed by the ethical committee and the waiver for informed consent was confirmed by the committee. SDS-PAGE and immunoblot analysis Proteins were separated on 4–12% (w/v) polyacrylamide gradient gels (NuPage, Life Technolo- gies) and either stained with Coomassie Brilliant Blue or transferred onto a nitrocellulose membrane (Whatmann, Dassel, Germany) for immunoblot analysis as previously described by Boes et al. [30]. Purification of antigens Infiltrated N. benthamiana leaves were harvested 5 days post infiltration and ground in liquid nitrogen. Purification of gAMA, F0 and CCT was performed as described by Boes et al. [30], Beiss et al. [32] and Voepel at al. [31] respectively. E3, and all DsRed fusion proteins were puri- fied according to the procedure described by Voepel et al. [31] with modified extraction buff- ers. For the DsRed fusion proteins, we used PBS plus 500 mM NaCl, pH 7.4. For E3, we used 50 mM Tris-HCl pH 8 plus 500 mM NaCl. Construct design and cloning All cDNAs (Fig 1A and 1B) were obtained as N. benthamiana codon-optimized synthetic genes from Geneart (Invitrogen, Carlsbad, CA). The four constructs were cloned as described by Boes et al. [30] and Voepel et al. [31]. For antibody titer determination, the cDNAs coding for the single domains were fused to the C-terminus of the fluorescent reporter protein DsRed. The p19 silencing inhibitor gene (p19si) was modified as described [30]. All cloning steps were confirmed by DNA sequencing. Transient expression in N. benthamiana The transformation and cultivation of Agrobacterium tumefaciens as well as the transient expression in N. benthamiana plants was performed as previously described by Boes et al. [30] and Voepel et al. [31]. Material and Methods Ethics statement Rabbits were housed, immunized and sampled by Biogenes GmbH (Berlin, Germany), accord- ing to national animal welfare regulations. The animal facilities and protocols were reviewed and approved by: Landesamt fμr Landwirtschaft, Lebensmittelsicherheit und Fischerei Meck- lenburgVorpommern (LALLF M-V) (Approval No: 7221.3-2-030-13). To isolate the blood PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 2 / 20 Multi-component Multi-stage Malaria Vaccine Fig 1. Plant expression cassette and construct design, amino acid sequences, SDS-PAGE and immunoblot blot analysis. (A) Schematic presentation of the expression cassettes of the plant binary expression vector pTRAk. SAR: scaffold attachment region; CaMV 35S promoter and termin promoter with duplicated enhancer and terminator of the Cauliflower mosaic virus (CaMV) 35S gene; 5' untranslated region: 5'-UTR of the chalcone synt gene from Petroselinum crispum; signal peptide sequence: transit peptide sequence of murine antibody heavy chain; GOI: Gene of interest, CCT (1), gA (2), E3 (3) and F0 (4). The restriction sites used to insert the GOI into the plant expression vector are indicated; His6 tag: six histidine affinity purification ta ER-retention signal: SEKDEL ER-retention signal. (B) Table containing all the information for the selected antigens. For each antigen the main stage of expression, the name, the plasmoDB number and the amino acid sequence are depicted. SDS-PAGE (C) and immunoblot analysis (D) under reducing conditions of the four recombinant and purified proteins. Proteins were detected using rabbit anti-His6 antiserum and alkaline phosphatase-labeled goat rabbit antiserum. M: PageRuler pre-stained protein ladder (Fermentas), lane 1: CCT, lane 2: gAMA1, lane 3: E3 and lane 4: F0. Fig 1. Plant expression cassette and construct design, amino acid sequences, SDS-PAGE and immunoblot blot analysis. (A) Schematic presentation of the expression cassettes of the plant binary expression vector pTRAk. SAR: scaffold attachment region; CaMV 35S promoter and terminator: promoter with duplicated enhancer and terminator of the Cauliflower mosaic virus (CaMV) 35S gene; 5' untranslated region: 5'-UTR of the chalcone synthase gene from Petroselinum crispum; signal peptide sequence: transit peptide sequence of murine antibody heavy chain; GOI: Gene of interest, CCT (1), gAMA1 (2), E3 (3) and F0 (4). The restriction sites used to insert the GOI into the plant expression vector are indicated; His6 tag: six histidine affinity purification tag; ER-retention signal: SEKDEL ER-retention signal. (B) Table containing all the information for the selected antigens. For each antigen the main stage of expression, the name, the plasmoDB number and the amino acid sequence are depicted. SDS-PAGE (C) and immunoblot analysis (D) under reducing conditions of the four recombinant and purified proteins. Proteins were detected using rabbit anti-His6 antiserum and alkaline phosphatase-labeled goat anti- rabbit antiserum. M: PageRuler pre-stained protein ladder (Fermentas), lane 1: CCT, lane 2: gAMA1, lane 3: E3 and lane 4: F0. doi:10.1371/journal.pone.0131456.g001 doi:10.1371/journal.pone.0131456.g001 Rabbit immunization, antibody titer determination and IgG purification Three rabbits were immunized with the vaccine cocktail comprising equal amounts of the four components formulated with a proprietary Biogenes adjuvant (an oil in water emulsion con- taining lipopolysaccharides) on days 0, 7, 14, 28, 49 and 77, using 200 μg of antigen mix for the prime immunization and 100 μg of antigen mix for the consecutive boosts. Serum samples were taken on days 35 (bleed 1), 63 (bleed 2) and 91 (bleed 3). Antibody titer determination and IgG purification was performed as previously described by Boes et al. [30]. 3 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 doi:10.1371/journal.pone.0131456.g001 In vitro growth inhibition assay (GIA) The ability of polyclonal rabbit IgGs to inhibit the growth of P. falciparum 3D7A was deter- mined using growth inhibition assays (GIAs) as previously described by Boes et al. [30]. Calibration free concentration analysis (CFCA) Antigen-specific antibody concentrations were measured in the purified antibody preparations by CFCA [33] using a Biacore T200 instrument. The four purified components (CCT, gAMA1, E3 and F0) were individually covalently coupled to CM5-S-Series sensor chips by standard EDC-NHS chemistry. The method is described in detail by Boes et al. [30]. PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 4 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Multi-component Multi-stage Malaria Vaccine Inhibition of sporozoite gliding motility (SGM), hepatocyte cell traversal (HCT) and sporozoite invasion and liver stage development (SILSD) assays The isolation of NF54 sporozoites and their use in the gliding motility and cell traversal assays were carried out as described by Behet et al. [34], with adaptation that for scoring the gliding motility the number of gliding circles of 40 trails in 2 distinct wells was counted. The SILSD assay was performed as described [35] with the following adaptations. For each well, sporozo- ites were pre-incubated with rabbit IgG for 30 min on ice, after which the sporozoite-antibody mixture was transferred onto the hepatocytes. On day 4 after sporozoite invasion, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature and stained extracellular with anti-PfCSP-FITC or for the dose response experiment with anti- PfMSP1 followed by goat anti-mouse A488 as well as intracellular with anti-PfHSP70-biotin (StressMarq) followed by streptavidin-AF647 (Invitrogen) and 4',6-diamidino-2‐fenylindool (DAPI). The numbers of intracellular mature parasites were determined in 20 fields by micros- copy at 200x magnification. In the dose response experiments 16 fields at 100x magnification (PfHSP70) or 25 fields at 200x magnification (PfMSP1) were analyzed. Surface plasmon resonance (SPR)-based competition assay For the reversal of growth inhibition experiments, we determined the concentrations of E3 and gAMA1 that completely blocked the E3-specifc and gAMA1-specifc antibodies. The antigens were buffer exchanged against RPMI 1640 (PAA, E15-041) with HEPES without L-glutamine by repeated rounds of dilution and concentration using a VivaSpin 15R column with a MWCO of 10 kDa. Total IgG preparations from the third bleed (day 91) were diluted to a final concen- tration in the competition mix resulting in 40–70% inhibition in a standard growth inhibition assay, and incubated overnight at 4°C with either gAMA1 (final concentrations 120, 12 and 1.2 μg/ml) or E3 (final concentrations 1230, 123 and 12.3 μg/ml). The competition mix was analyzed against the corresponding antigen (E3 or gAMA1) immobilized on the CM5-S-Series sensor chips using a Biacore T200 instrument. Full competition was defined as a RU signal less than 5% of the sample containing no competitor. Immunofluorescence assay (IFA) Indirect IFAs with IgG (final concentration 1.5 μg/ml) purified from PlasmoMix-specific rabbit immune sera were carried out using sporozoites, schizonts, macrogametes and retorts. of P. fal- ciparum strain NF54 as previously described by Boes et al. [30]. Inhibition of sporozoite gliding motility (SGM), hepatocyte cell traversal (HCT) and sporozoite invasion and liver stage development (SILSD) assays Multi-component Multi-stage Malaria Vaccine the purified antibodies. The amount needed to completely block the antibodies was determined using the SPR-based competition experiment described above. In the absence of antigen, the respective antibody concentration resulted in 40–70% inhibition in a standard GIA. the purified antibodies. The amount needed to completely block the antibodies was determined using the SPR-based competition experiment described above. In the absence of antigen, the respective antibody concentration resulted in 40–70% inhibition in a standard GIA. Production of recombinant antigens A. tumefaciens strains carrying the component expression constructs in the context of the binary expression vector pTRA (Fig 1A and 1B) were vacuum infiltrated into N. benthamiana plants, and subsequent purification of the recombinant antigens CCT (PfCSP_TSR, PfCelTos and PfTRAP_TSR), gAMA1(PfAMA1), E3 (PfMSP1-19_EGF1, PfMSP4_EGF, PfMSP8_EGF1, PfMSP8_EGF2, PfMSP3) and F0 (Pfs25, Pfs230) from crude tobacco extracts yielded highly pure and intact proteins of the expected sizes, as shown by reducing SDS-PAGE and immuno- blot (Fig 1C and 1D). Yields were > 1 mg/g leaf tissue for CCT and gAMA1 and > 250 μg/g leaf tissue for E3 and F0. Antibody-dependent respiratory burst (ADRB) assay The stimulation of human polymorphonuclear neutrophil granulocytes (PMNs) was analyzed in ADRB assays in a total volume of 75 μl as described [36], using samples from rabbit R3_91 as example. gAMA1 or E3 (750 ng) were immobilized for the solid-phase ADRB (sADRB) assay. Purified antibodies were adjusted to 2.5 mg/ml total IgG (comparable to the amounts used in the reversal of growth inhibition assay). Purified IgG from normal rabbit serum (NRS) was used at same concentration. The stimulation of 7.5 x 104 PMNs was determined by lumi- nol-enhanced chemiluminescence. Standard membrane feeding assay (SMFA) Standard membrane feeding assays [37] were used to determine the ability of purified rabbit anti-PlasmoMix antibodies to block the transmission of P. falciparum from human to mos- quito as previously described by Feller et al. [38]. Antibodies purified from rabbit immune sera were used at 1 mg/ml total IgG. The IC50 value was determined by analyzing purified antibod- ies from day 91 at concentrations of 1, 0.1 and 0.01 mg/ml total IgG. Based on the CFCA results, the total IgG concentration was converted to the F0-specific antibody concentration and plotted against the inhibition of transmission. Statistical analysis (Mann-Whitney U) was carried out for median numbers of oocysts between groups of mosquitoes receiving either anti- bodies purified from normal rabbit serum (NRS) or rabbit immune sera. The oocyst prevalence between groups of mosquitoes either receiving antibodies purified from NRS or rabbit immune sera was statistically analyzed using Fisher’s exact test. Reversal of growth inhibition assay A reversal of growth inhibition assay was used to determine the ability of antibodies directed against either gAMA1 or E3 to inhibit parasite growth and to assess their contribution to the overall inhibitory capacity of the immune serum. Antibodies from rabbits R1, R2 and R3 purified from immune sera collected on day 91 were mixed with component E3 (final concentration 24 μg/ml) to completely block E3-specific antibodies. The mixture was incubated overnight at 4°C and subsequently used for a GIA as described above. The contribution of gAMA1-specific antibodies was assessed in the same way by adding gAMA1 (final concentration 120 μg/ml) to 5 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Component and domain-specific antibody titers Antibody titer analysis was performed for the cocktail (PlasmoMix), the components (CCT, F0, E3 and gAMA1) as well as the individual recombinant domains. Antibody antibody titers were monitored at day 0, 35, 63, and 91, and an increase of antibody titers over the sampling period was observed for all tested antigens and domains. We recorded a antibody titer of 3.8 x 106 against the cocktail, antibody titers of 1.9–3.3 x 106 against CCT, F0 and E3, and a antibody titer of just below 5.5 x 105 against gAMA1 (Fig 2A). For CCT, we observed a strong bias in the antibody titers against the three pre-erythrocytic stage antigens PfCeltos, PfCSP_TSR and PfTRAP_TSR, a antibody titer of 1.6 x 106 against the TSR-domain of PfCSP and 20-fold lower values for the other two domains (Fig 2B). In contrast, all five blood-stage domains within E3 6 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 doi:10.1371/journal.pone.0131456.g002 Quantification of antigen-specific IgG responses We performed SPR-based CFCA for the precise quantitation of the component-specific anti- bodies in the IgG preparations purified from rabbit immune sera that were subsequently used in in vitro inhibition assays to determine IC50 requirements for stage-specific antibody con- centrations. Table 1 shows component-specific antibody concentration given as mg/ml as well as % total IgG. Addintionally Plasmomix-specific IgG concentrations were calculated by add- ing up the values obtained for the 4 components. Up to 12% (of total IgG) of Plasmomix-spe- cific antibodies was observed for rabbit R2 in the 63 day sample. Multi-component Multi-stage Malaria Vaccine induced comparable antibody titers of approximately 4 0 x 105 A two-fold difference in anti- Fig 2. Antigen-specific antibody titers of rabbit immune sera determined by ELISA. Three rabbits (R1, R2 and R3) were immunized six times with PlasmoMix and serum samples were collected on days 0 (pre-immune), 35, 63 and 91. (A) The antibody titer against the immunization mixture (PlasmoMix as well as against the four protein-based components (CCT, E3, gAMA1 and F0) were determined. (B) To further dissect the immune response, the specific antibody response against each individual domain was analyzed. Therefore, the domains were C-terminaly fused to DsRed (fluorescent reporter protein), and expressed and purified as described in the methods section. Antigen domains comprising a fusion protein are connected by a bracket. Antibody titers a shown for each rabbit (R1: open black square, R2: open red circle, R3: open blue triangle) as well as the geometric mean (black line) of three rabbits. doi:10.1371/journal.pone.0131456.g002 Fig 2. Antigen-specific antibody titers of rabbit immune sera determined by ELISA. Three rabbits (R1, R2 and R3) were immunized six times with PlasmoMix and serum samples were collected on days 0 (pre-immune), 35, 63 and 91. (A) The antibody titer against the immunization mixture (PlasmoMix) as well as against the four protein-based components (CCT, E3, gAMA1 and F0) were determined. (B) To further dissect the immune response, the specific antibody response against each individual domain was analyzed. Therefore, the domains were C-terminaly fused to DsRed (fluorescent reporter protein), and expressed and purified as described in the methods section. Antigen domains comprising a fusion protein are connected by a bracket. Antibody titers are shown for each rabbit (R1: open black square, R2: open red circle, R3: open blue triangle) as well as the geometric mean (black line) of three rabbits. doi:10.1371/journal.pone.0131456.g002 induced comparable antibody titers of approximately 4.0 x 105. A two-fold difference in anti- body titers was observed when we compared the transmission blocking-component F0 (2 x 106) with the individual antibody titers of the two sexual-stage antigens at 4.4 x 105 for Pfs25 and 1.8 x 105 for Pfs230_C0 (Fig 2B). 7 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Multi-component Multi-stage Malaria Vaccine Table 1. The purified antibody preparations used for all in vitro assays. The total IgG concentrations and CFCA results are listed for each component. Nomenclature is based on the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). The PlasmoMix-specific antibody concentra- tion is the sum of the specific antibody response against the four components. The antigen-specific antibody concentration is given in mg/ml and % of total I G Table 1. The purified antibody preparations used for all in vitro assays. The total IgG concentrations and CFCA results are listed for each component. Nomenclature is based on the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). The PlasmoMix-specific antibody concentra- tion is the sum of the specific antibody response against the four components. The antigen-specific antibody concentration is given in mg/ml and % of total IgG. Sample total IgG PlasmoMix-speciﬁc-IgG CCT-speciﬁc-IgG gAMA1-speciﬁc-IgG E3-speciﬁc-IgG F0-speciﬁc-IgG mg/ml mg/ml % total IgG mg/ml % total IgG mg/ml % total IgG mg/ml % total IgG mg/ml %total IgG R1_35 21.70 1.23 5.65 0.40 1.85 0.41 1.87 0.16 0.76 0.26 1.18 R1_63 21.18 1.88 8.85 0.68 3.19 0.51 2.41 0.36 1.70 0.33 1.56 R1_91 19.09 1.19 6.25 0.50 2.59 0.26 1.34 0.25 1.30 0.20 1.02 R2_35 22.60 1.72 7.63 0.31 1.35 0.75 3.32 0.38 1.69 0.29 1.26 R2_63 17.08 2.17 12.69 0.50 2.90 0.84 4.92 0.47 2.77 0.36 2.11 R2_91 22.25 2.34 10.52 0.63 2.83 0.78 3.51 0.54 2.43 0.39 1.75 R3_35 27.40 2.24 8.17 0.31 1.13 0.80 2.90 0.52 1.89 0.62 2.25 R3_63 23.13 1.46 6.30 0.28 1.21 0.51 2.20 0.29 1.26 0.38 1.62 R3_91 25.03 1.95 7.79 0.54 2.16 0.62 2.46 0.41 1.62 0.39 1.56 doi:10.1371/journal.pone.0131456.t001 doi:10.1371/journal.pone.0131456.t001 Multi-component Multi-stage Malaria Vaccine concentration of 3 mg/ml and we observed up to 48% inhibition of SGM, 34% inhibiti HCT and 81% inhibition of SILSD (Fig 3). Further, IC50 values for the inhibition of SI Fig 3. In vitro inhibition of (A) sporozoite gliding motility, (B) hepatocyte cell traversal, (C) sporozoite invasion and liver stage development PlasmoMix-specific rabbit IgG and (D) Dose response of sporozoite invasion and liver stage development. All three assays were performed w falciparum NF54 parasites and purified rabbit IgGs (R1, R2 and R3) at a total IgG concentration of 3 mg/ml from serum samples collected on days 63 For the dose response, purified rabbit IgGs from rabbit R3 (day 63 and day 91) was used at the following total IgG concentrations: 9, 3, 0.9 and 0.09 m The CCT-specific antibody concentration was calculated based on CFCA from total IgG. Inhibitions are expressed as the mean of duplicate measure with standard deviations. doi:10.1371/journal.pone.0131456.g003 Fig 3. In vitro inhibition of (A) sporozoite gliding motility, (B) hepatocyte cell traversal, (C) sporozoite invasion and liver stage development with PlasmoMix-specific rabbit IgG and (D) Dose response of sporozoite invasion and liver stage development. All three assays were performed with P. falciparum NF54 parasites and purified rabbit IgGs (R1, R2 and R3) at a total IgG concentration of 3 mg/ml from serum samples collected on days 63 and 91 For the dose response, purified rabbit IgGs from rabbit R3 (day 63 and day 91) was used at the following total IgG concentrations: 9, 3, 0.9 and 0.09 mg/ml. The CCT-specific antibody concentration was calculated based on CFCA from total IgG. Inhibitions are expressed as the mean of duplicate measurements with standard deviations. Fig 3. In vitro inhibition of (A) sporozoite gliding motility, (B) hepatocyte cell traversal, (C) sporozoite invasion and liver stage development with PlasmoMix-specific rabbit IgG and (D) Dose response of sporozoite invasion and liver stage development. All three assays were performed with P. falciparum NF54 parasites and purified rabbit IgGs (R1, R2 and R3) at a total IgG concentration of 3 mg/ml from serum samples collected on days 63 and 91. For the dose response, purified rabbit IgGs from rabbit R3 (day 63 and day 91) was used at the following total IgG concentrations: 9, 3, 0.9 and 0.09 mg/ml. The CCT-specific antibody concentration was calculated based on CFCA from total IgG. doi:10.1371/journal.pone.0131456.g003 IgG reactivity with parasite surface antigens in immunofluorescence analysis and schizont lysate immunoblot analysis The ability of the antigen cocktail to induce antibodies recognizing P. falciparum surface proteins in their native context was confirmed by testing the purified IgG fractions against parasites from different life cycle stages (sporozoites, schizonts, macrogametes and retorts) in immunofluorescence assays. We observed strong staining localized at the surface of all P. falciparum preparations (S1 Fig). No staining was observed for the NRS negative control. Addi- tionally the reactivity against the native blood-stage antigens included in the context of Plas- moMix was confirmed by immuno blot analysis using schizont lysate and purified immune IgG from R1, R2 and R3 (Day 91). As shown in S2 Fig all Plasmodium falciparum blood-stage proteins included within PlasmoMix could be identified based on both, molecular weight and data reported by others: putative high molecular weight PfMSP-complex, unprocessed PfAMA1 or PfMSP8, processed PfAMA1, PfMSP3, processed PfMSP1_42, PfMSP4 and pro- cessed PfMSP1_19 or processed PfMSP8. Analysis of in vitro pre-erythrocytic stage efficacy by inhibition of sporozoite gliding motility (SGM), hepatocyte cell traversal (HCT) and sporozoite invasion and liver stage development (SILSD) The inhibitory efficacy of the pre-erythrocytic stage components in the context of the fusion protein CCT was investigated using PlasmoMix-specific rabbit immune IgG in SGM, HCT and SILSD assays. IgG samples from all three rabbits were tested in duplicate at a final 8 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Analysis of in vitro blood stage efficacy by growth inhibition assay (GIA) The efficacy of the blood-stage component of the vaccine cocktail (antigens gAMA1 and E3) was assessed using a classical GIA. Up to 90% inhibition was observed at a concentration of 6 mg/ml total IgG purified from rabbit immune sera (Fig 4A). Determination and comparison of IC50 values for antigen-specific IgGs indicated that the IC50 values for gAMA1-specific anti- bodies (Fig 4B) were in the same range (30–60 μg/ml) as reported in a previous study [30] after the immunization of rabbits with recombinant plant-derived gAMA1, indicating that the growth inhibition induced by the cocktail is mainly mediated by gAMA1-specific antibodies. To precisely quantify this contribution, a reversal of growth inhibition assay was performed by competitive neutralization of these antibodies with recombinant gAMA1 and E3 (Fig 5). Although growth inhibition was completely abolished by 120 μg/ml gAMA1, the neutralization of E3-specific IgGs using 24 μg/ml E3 did not affect growth inhibition. Multi-component Multi-stage Malaria Vaccine were determined in a dose response experiment using purified immune IgG from rabbit R3 at day 63 and 91 (R3_63: 25.5 μg/ml and R3_91: 17.1 μg/ml). Analysis of in vitro sexual stage efficacy by standard membrane feeding assay (SMFA) The SMFA was used to investigate the transmission-blocking efficacy of the sexual stage-spe- cific component F0 (Pfs25 and Pfs230_CO). The observed transmission-blocking activity (reduction in oocyst formation) of 95–100% for the samples from the three animals for all three bleeds (S1 Table) demonstrated the potency of the F0 fusion antigen comprising Pfs25 and Pfs230_CO, agreeing with previous reports based on these antigens [15, 27, 39]. The amounts of F0-specific antibodies measured in the samples taken at day 91 by SPR-based CFCA analysis were correlated with a corresponding dose response TBA, revealing an IC50 value of < 2 μg/ml (Fig 6). PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Inhibitions are expressed as the mean of duplicate measurements with standard deviations. concentration of 3 mg/ml and we observed up to 48% inhibition of SGM, 34% inhibition of HCT and 81% inhibition of SILSD (Fig 3). Further, IC50 values for the inhibition of SILSD concentration of 3 mg/ml and we observed up to 48% inhibition of SGM, 34% inhibition of HCT and 81% inhibition of SILSD (Fig 3). Further, IC50 values for the inhibition of SILSD concentration of 3 mg/ml and we observed up to 48% inhibition of SGM, 34% inhibition of HCT and 81% inhibition of SILSD (Fig 3). Further, IC50 values for the inhibition of SILSD PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 9 / 20 Multi-component Multi-stage Malaria Vaccine Fig 4. In vitro growth inhibition assay (GIA) of asexual P. falciparum 3D7 parasites with purified PlasmoMix-specific rabbit IgG. The rabbit IgG purified from the serum of three rabbits (R1, R2 and R3) collected on days 35, 63 and 91 post-immunization with PlasmoMix. Nomenclature of the sam features the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). (A) Four serial 1/1 dilutions from 6–0.75 mg/ml of total IgG used to demonstrate the concentration dependency of the assay and to calculate the IC50 values (the total IgG concentrations needed for 50% inhibitio The same GIA but instead of total IgG, the gAMA1-specifc antibody concentration (based on CFCA and calculated from total IgG, see methods section used and the IC50 values for gAMA1-specific antibodies were calculated. Each data point represents the mean of technical triplicates. doi:10 1371/journal pone 0131456 g004 Fig 4. In vitro growth inhibition assay (GIA) of asexual P. falciparum 3D7 parasites with purified PlasmoMix-specific rabbit IgG. The rabbit IgGs were purified from the serum of three rabbits (R1, R2 and R3) collected on days 35, 63 and 91 post-immunization with PlasmoMix. Nomenclature of the sample first features the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). (A) Four serial 1/1 dilutions from 6–0.75 mg/ml of total IgGs were used to demonstrate the concentration dependency of the assay and to calculate the IC50 values (the total IgG concentrations needed for 50% inhibition). (B) The same GIA but instead of total IgG, the gAMA1-specifc antibody concentration (based on CFCA and calculated from total IgG, see methods section) was used and the IC50 values for gAMA1-specific antibodies were calculated. Each data point represents the mean of technical triplicates. Fig 4. In vitro growth inhibition assay (GIA) of asexual P. falciparum 3D7 parasites with purified PlasmoMix-specific rabbit IgG. The rabbit IgGs were purified from the serum of three rabbits (R1, R2 and R3) collected on days 35, 63 and 91 post-immunization with PlasmoMix. Nomenclature of the sample first features the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). Discussion A cocktail featuring key antigens from different stages of the P. falciparum life cycle is a prom- ising approach for malaria vaccine development. We therefore investigated the antibody responses generated by immunizing rabbits with an equal mixture of four different recombi- nant proteins featuring suitable antigens from the pre-erythrocytic (PfCSP_TSR, PfCelTos and PfTRAP_TSR), blood (PfAMA1, PfMSP1-19_EGF1, PfMSP4_EGF, PfMSP8_EGF1, PfMSP8_EGF2 and PfMSP3), and sexual stages (Pfs25 and Pfs230) of P. falciparum. The four recombinant proteins were successfully produced by transient expression in N. benthamiana plants. Yields of 0.25–1.0 mg per g fresh leaf weight were comparable to typical expression lev- els achieved using this system [40, 41] and the fact that intact and highly pure proteins could be isolated indicated that all four vaccine antigens are suitable for production in the plant tran- sient expression system. The analysis of PlasmoMix-specific rabbit immune sera obtained using a hyperimmuni- zation protocol that aims for maximum antibody titers revealed differences in reactivities against the different antigen domains with antibody titers ranging from 7.7 x 104 (PfCeltos and PfTRAP) to 1.6 x 106 (PfCSP-TSR). The observed antibody titer bias between fused domains as observed for CCT and F0 agrees with our previously observed data based on corresponding mouse immune sera [31, 32]. The gAMA1-specific antibody titers were 2–3 times lower than we previously observed for gAMA1-specific rabbit immune sera, where 3.5 times more antigen was used [30]. The antibody titers of the domains within the E3 component were balanced at 3.5 x 105. Immunofluorescence analysis confirmed the reactivity of the PlasmoMix-specific PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 10 / 20 (A) Four serial 1/1 dilutions from 6–0.75 mg/ml of total IgGs were used to demonstrate the concentration dependency of the assay and to calculate the IC50 values (the total IgG concentrations needed for 50% inhibition). (B) The same GIA but instead of total IgG, the gAMA1-specifc antibody concentration (based on CFCA and calculated from total IgG, see methods section) was used and the IC50 values for gAMA1-specific antibodies were calculated. Each data point represents the mean of technical triplicates. doi:10.1371/journal.pone.0131456.g004 immune sera against all the tested P. falciparum stages, and the staining patterns were in accor- dance with the results of previous studies by our group and others [30, 31, 39, 41–43] after immunization with the same or comparable antigens. Typical PfAMA1-specific apical staining of merozoites could not be observed, since many other non-localized merozoitze surface proteins were stained in parallel. To further confirm the reactivity against all individual PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 11 / 20 Based on the CFCA results, the F0-specific antibody concentration was calculated, plotted and used to determine the IC50 value (the antibody concentration needed to obtain 50% inhibition of transmission). For detailed results of the transmission blocking assay, refer to S2 Table. d i 10 1371/j l 0131456 006 Fig 6. Correlation of transmission blocking activity and F0-specific antibody concentration. The rabbit IgGs were purified from serum samples collected on day 91 post-immunization with PlasmoMix. To demonstrate the concentration dependency of the transmission blocking activity, the TBA was performed with purified antibodies from each rabbit (R1: black, R2: open and R3: hatched) at total IgG concentrations of 1 mg/ml (circles), 0.1 mg/ml (squares) and 0.01 mg/ml (triangles). Based on the CFCA results, the F0-specific antibody concentration was calculated, plotted and used to determine the IC50 value (the antibody concentration needed to obtain 50% inhibition of transmission). For detailed results of the transmission blocking assay, refer to S2 Table. doi:10.1371/journal.pone.0131456.g006 doi:10.1371/journal.pone.0131456.g006 doi:10.1371/journal.pone.0131456.g006 performed with a human compatible adjuvant or vaccination regime and that conclusions regarding the effect of such a cocktail under different conditions have to be further investigated. Stage-specific inhibition assays were used to address the in vitro efficacy of PlasmoMix. The three pre-erythrocytic stage assays showed promising inhibition values between 34% for hepa- tocyte cell traversal and 81% for hepatocyte invasion and maturation. These values are high compared to our previous results using CCT in a mouse immunization study, which achieved a reduction of only 35% in a hepatocyte invasion assay [31]. However these results were obtained using the immortalized liver cell line HepG2 rather than the human primary hepatocytes used in this study. R3 is the only rabbit that showed a correlation between the concentration of CCT-specific IgG and the percentage effect in both the HCT and SILSD assays, but not the SGM assay, over time. There was no link between overall CCT IgG-specific antibodies and specificity, which might indicate that each rabbit produces different amounts of functional IgG. Clear inhibition in pre-erythrocytic in vitro assays has been reported for the single anti- gens PfCSP_TSR and PfCeltos in previous studies [19, 49], whereas the results for PfTRAP are contradictory [20, 50]. Antibodies against PfCSP play a role in gliding motility [51], whereas PfCeltos-specific antibodies interfere with cell traversal and have another mode of action affect- ing not only sporozoites at the site of infection but also intra-hepatocyte parasites [52]. Multi-component Multi-stage Malaria Vaccine Fig 5. Reversal of growth inhibition assay. The assays were performed with P. falciparum 3D7 parasites and with the intention to assess the inhibitory potential of antibodies directed against either gAMA1 or E3. The GIA was run with purified antibodies from serum samples taken on day 91 post- immunization with PlasmoMix. The assay was carried out with a total IgG concentration resulting in 40–70% inhibition in a standard GIA in the presence of either gAMA1 (A) or E3 (B) to reverse the inhibition. Each data point represents the mean with standard deviation of technical triplicates. doi:10.1371/journal.pone.0131456.g005 Fig 5. Reversal of growth inhibition assay. The assays were performed with P. falciparum 3D7 parasites and with the intention to assess the inhibitory potential of antibodies directed against either gAMA1 or E3. The GIA was run with purified antibodies from serum samples taken on day 91 post- immunization with PlasmoMix. The assay was carried out with a total IgG concentration resulting in 40–70% inhibition in a standard GIA in the presence of either gAMA1 (A) or E3 (B) to reverse the inhibition. Each data point represents the mean with standard deviation of technical triplicates. doi:10.1371/journal.pone.0131456.g005 blood-stage antigen included in PlasmoMix a schizont lysate immunoblot was performed with purified PlasmoMix-specific rabbit immune IgG. The detected bands were identified by appar- ent molecular weight in comparison with the following studies performed on the respective Plasmodium falciparum proteins [44–48]. For ethical reasons and because of these preexisting results for single antigen vaccination experiments with three of the PlasmoMix components we did not perform single antigen vacci- nations for all the four components along with the PlasmoMix experiments. In this context it should be noted that this initial study to investigate a multistage vaccine cocktail was not 12 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Multi-component Multi-stage Malaria Vaccine Fig 6. Correlation of transmission blocking activity and F0-specific antibody concentration. The rabbit IgGs were purified from serum samples collected on day 91 post-immunization with PlasmoMix. To demonstrate the concentration dependency of the transmission blocking activity, the TBA was performed with purified antibodies from each rabbit (R1: black, R2: open and R3: hatched) at total IgG concentrations of 1 mg/ml (circles), 0.1 mg/ml (squares) and 0.01 mg/ml (triangles). PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Multi-component Multi-stage Malaria Vaccine this study. Estimations on protective levels of PfCSP-specific antibodies derived from immuni- zation studies with RTS,S suggest concentrations of > 20μg/ml [53]. Up to 90% inhibition was observed in the GIA with 6 mg/ml total IgG. Surprisingly, as shown by the reversal of growth inhibition, the activity in this experimental context was exclu- sively mediated by anti-PfAMA1 antibodies. This conclusion is also supported by the IC50 val- ues for gAMA1-specific antibodies, which provide no evidence for a synergistic or additive effect of E3-specific antibodies in this GIA when compared to similar values induced by gAMA1 alone [30]. At least PfMSP1-19-specific antibodies play a role in naturally acquired immunity [54] and immunization with recombinant PfMSP1-19 can induce growth inhibitory antibodies [55] even though it is known that PfAMA1 is more immunogenic and causes stron- ger growth inhibition than PfMSP1-19 [56]. Faber et al. [8] demonstrated a small but signifi- cant contribution of PfMsp1-19-specific antibodies induced by immunization with PfAMA1-DICO_PfMsp1-19 fusion proteins. In this study, an IC50 of >200 μg/ml PfMsp1-19 specific antibodies (affinity purified from rabbit immune IgG) was determined, leading us to the conclusion that the lacking effect of E3-specific and/or PfMsp1-19-specific IgGs might be explained by the much lower concentrations of these antibodies (<50 μg/ml for E3 and, look- ing at the antibody titer ratios, most probably less than 10 μg/ml for PfMSP1-19) induced by the immunization with PlasmoMix. Our GIA data for blood-stage malaria vaccine candidate efficacy highlight the role of PfAMA1 as a leading blood-stage antigen. Nevertheless, we included the fusion protein E3 to provide additional promising blood-stage antigens such as PfMSP3 and PfMSP1-19, which have been associated with antibody-dependent cell-mediated immunity (ADCI) [24,48], to diversify the blood-stage efficacy of the vaccine cocktail, even though we did not perform ADCI assays in the context of this work. Antibody-dependent respiratory burst assay (ADRB) is another method to address parasite-neutralizing activity of antibody preparations using human polymorphonuclear neutrophil granulocytes (PMN) as effector cells [36]. In the context of this work we conducted an initial experiment that demon- strates for the first time that rabbit antibodies can stimulate human PMNs (S3 Fig). We con- firmed that gAMA1 as well E3-specific rabbit immune IgG are capable of inducing respiratory burst in a direct coating assay. In future studies we will develop and implement these assays to analyze ADRB activity of rabbit immune IgG on parasites. We believe that the inhibition observed in the three different assays could be a cumulative effect of all three antigens and further studies are needed to dissect the contribution of each antigen. The IC50 values determined for the rabbit R3 (R3_63 & R3_91) were in the lower μg/ml range (17.1–25.5 μg/ml) and therefore in between the IC50 value observed for the sexual-stage and blood-stage specific inhibition assays (see below). It is difficult to compare the IC50 value with data from other studies, because most of them use ELISA units (EU) or arbitrary units (AU) instead of absolute concentrations of antigen-specific IgG as we have determined by CFCA in 13 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Besides the theoretical ability to generate broadly-neutralizing immune responses, the use of cocktails containing several recombinant antigens bears the risk of diluting the response and losing efficacy by antigenic competition. In the context of PlasmoMix, we can address this hypothesis by combining the data reported herein with our previous results obtained after the immunization of rabbits with PfgAMA1 [30]. While overall antibody titers directly correlated with the amounts of gAMA1 used in the PlasmoMix or gAMA1 immunizations, the inhibitory quality of the induced antibodies was not affected (similar IC50 values for the gAMA1-specific IgG). Anyhow in the cocktail a significant increase of the IC50 value for total IgG and thus a lower GIA efficacy was observed. Even though we did not generate comparable data for the remaining three PlasmoMix components we are convinced that antigenic competition plays a significant role in responses to multi-antigen cocktails. Although based on an observation with just one cocktail, this finding should be taken as a serious message that antigens within vaccine cocktails must be chosen carefully, and their number should be kept as low as possible to avoid the observed dilution of essential inhibitory antibodies. The 100% reduction of oocyst formation observed in the sexual stage transmission blocking assay is in agreement with our own data from mouse immunizations with F0 [32] and results from previous studies with different variants of Pfs25 [26, 39, 40, 57] or Pfs230 [27, 41, 42]. The determined IC50 value (<2 μg/ml) is below, but in the same range as the one reported by ([58], 4.2μg/,ml) using Pfs25-specific rabbit immune IgG. The observed IC50 value may derive PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 14 / 20 Multi-component Multi-stage Malaria Vaccine from both, Pfs25 and Pfs230_C0-specific IgG, anyhow the lack of replicates does not allow the conclusion that an additional contribution of Pfs230_C0-specific antibodies is the reason for the two fold lower value. Therefore it will be necessary to dissect and quantify the contribution of each of the sexual-stage antigens (Pfs25 and Pfs230_C0) in additional experiments to opti- mize the next generation of the PlasmoMix vaccine cocktail. Conclusions Our data suggest that cocktails comprising different stage-specific components remain a prom- ising approach for the development of a malaria vaccine. However, our results also indicate the different components should not be mixed in equal amounts but in optimal ratios to generate what may be called “balanced efficacy”. The fine tuning of a new vaccine cocktail containing the PlasmoMix antigens may require: (1) the removal of some antigens; and (2) the adjustment of the relative concentrations of the remaining to compensate for the observed different IC50 requirements and at the same time taken into account the role of the respective stage in the context of the establishment and the clinical manifestation of the disease. The results also indicate that, at least in rabbits, it should be possible to reduce the amount of this component within the vac- cine cocktail without compromising transmission-blocking efficacy, thus allowing other com- ponents to be over-represented and to induce higher antibody antibody titers for optimal protection against other (pre-erythrocytic and blood) stages. Supporting Information S1 Fig. Immunofluorescence assay of NF54 parasites in different stages. For IFAs, P. falcipa- rum NF54 parasites in the sporozoite, schizont, macrogamete and retort stages were fixed with methanol on the surface of a slide. Exemplary shown is the detection with purified IgGs from rabbit R2 from the serum sample collected on day 91 after immunization with PlasmoMix. As positive controls, murine antisera against PfCSP (sporozoite), PfMSP1-19 (schizonts), Pfs25 (magrogametes) and Pfs28 (retorts) were used, respectively. Rabbit antibodies were visualized with anti-rabbit secondary antibody labeled with Alexa Fluor 594 (red) while visualization of murine antibodies was performed with secondary Alexa Fluor 488 labeled anti-murine anti- body (green). Parasite nulcei were highlighted with Hoechst 33342 (blue). Transm: Transmis- sion light. Bar: 5 μm. S2 Fig. Schizont lysate immunoblot analysis. Purified rabbit immune IgG (R1, R2 and R3 from day 91) were used to detect native Plasmodium falciparum blood-stage antigens in immu- noblot analysis with schizont lysate. 12 μl lysate (corresponding to 1.5x107 schizonts) were sep- arated under non-reducing conditions by SDS-PAGE. After immunoblot the membrane was blocked with skimmed milk and probed with rabbit immune IgG adjusted to 5 μg/ml PfA- MA1-spacific IgG concentration. Bound rabbit antibodies were detected with an alkaline phos- phatase goat anti-rabbit serum. Antibodies purified from normal rabbit serum (NRS) were used as negative control. Acknowledgments The p19 suppressor of gene silencing was kindly provided by Plant Bioscience Limited (Nor- wich, England) under the terms of an evaluation license agreement. We thank Helga Schinkel and Natalie Koch (Fraunhofer IME, Aachen Germany) for help with the expression and purifi- cation of PfMSP3-based proteins. We are also grateful for the BG98 standard, which was kindly provided by BPRC (Rijswijk, the Netherlands). We thank Dr Richard M Twyman for manu- script editing. (TIF) S3 Fig. Antibody-dependent respiratory burst assay. Human polymorphonuclear neutrophil granulocyte (PMN) responses towards purified PlasmoMix-specific rabbit immune IgG. Sam- ple (R3_91, black squares) was analyzed using ADRB on coated antigens gAMA1 (A) and E3 (B) over a 60 min kinetic window with PNMs from one donor. Antibodies purified from nor- mal rabbit serum (NRS) were used as negative control (red circles). (TIF) 15 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0131456 July 6, 2015 Multi-component Multi-stage Malaria Vaccine S1 Table. Evaluation of transmission blocking activity of purified rabbit IgGs. Rabbit (R1, R2 and R3) were immunized with PlasmoMix and antibodies were purified from serum sam- ples collected on days 35, 63 and 91. Polyclonal rabbit antibodies were used at a total IgG con- centration of 1 mg/ml and based on the CFCA results, the F0-specific antibody concentration was calculated and listed. Antibodies purified from normal rabbit sera (NRS) was used as a negative control. Samples from day 63 and 91 were run in the same assay and refer to the same NRS, listed above R1_63. (a) We used a Mann-Whitney U test to analyze the median numbers of oocysts during infection with P. falciparum between groups of mosquitoes receiving either antibodies purified from NRS or purified from rabbits immunized with PlasmoMix. P-values below 0.05 were considered significant. (b) The prevalence of oocysts during the infection with P. falciparum between groups of mosquitoes receiving either antibodies purified from NRS or antibodies purified from rabbits immunized with PlasmoMix was analyzed using the Fisher’s exact test. P-values below 0.05 were considered significant. (DOCX) S2 Table. Correlation of transmission blocking activity and F0-specific antibody concentra- tion. Antibodies were purified from serum samples collected on day 91 after immunization with PlasmoMix and used at total IgG concentration of 1 mg/ml, 0.1 mg/ml and 0.01 mg/ml. The F0-specific antibody concentration was calculated based on the CFCA experiment. Anti- bodies purified from normal rabbit sera (NRS) was used as a negative control. Statistical analy- sis was performed as described in S1 Table. (DOCX) Author Contributions Conceived and designed the experiments: AB HS KJD RFe GP. Performed the experiments: AB GE HS JMB NV MS SK VB CCH MCB. 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https://openalex.org/W2329852982	https://ecology.dp.ua/index.php/ECO/article/download/213/209	Ukrainian	null	Культура ізольованих зародкових мішків як біотехнологічна система дорощування зиготичних зародків кукурудзи in vitro	Biosystems diversity	2,009	cc-by	4,054	УДК 581.5:470.44/47 О. В. Ляпустіна, Т. М. Сатарова 10 Український державний хіміко-технологічний університет Дніпропетровський національний університет ім. Олеся Гончара 4 10© О. В. Ляпустіна, Т. М. Сатарова, 2009 Е. В. Ляпустина, Т. Н. Сатарова Украинский государственный химико-технологический университет Днепропетровский национальный университет им. Олеся Гончара КУЛЬТУРА ИЗОЛИРОВАННЫХ ЗАРОДЫШЕВЫХ МЕШКОВ КАК БИОТЕХНОЛОГИЧЕСКАЯ СИСТЕМА ДОРАЩИВАНИЯ ЗИГОТИЧЕСКИХ ЗАРОДЫШЕЙ КУКУРУЗЫ IN VITRO Е. В. Ляпустина, Т. Н. Сатарова Украинский государственный химико-технологический университет Днепропетровский национальный университет им. Олеся Гончара Вісник Дніпропетровського університету. Біологія. Екологія. – 2009. – Вип. 17, т. 3. – С. 54–61. Visnyk of Dnipropetrovsk University. Biology. Ecology. – 2009. – Vol. 17, N 3. – P. 54–61. Вісник Дніпропетровського університету. Біологія. Екологія. – 2009. – Вип. 17, т. 3. – С. 54–61. Visnyk of Dnipropetrovsk University. Biology. Ecology. – 2009. – Vol. 17, N 3. – P. 54–61. КУЛЬТУРА ИЗОЛИРОВАННЫХ ЗАРОДЫШЕВЫХ МЕШКОВ КАК БИОТЕХНОЛОГИЧЕСКАЯ СИСТЕМА ДОРАЩИВАНИЯ ЗИГОТИЧЕСКИХ ЗАРОДЫШЕЙ КУКУРУЗЫ IN VITRO Установлена возможность доразвития зародыша кукурузы в культуре изолированных заро- дышевых мешков на искусственной питательной среде в условиях in vitro от ранней переходной ста- дии до начала органогенеза. Выяснено, что культивирование зародышевых мешков с целью дора- щивания зародыша и накопления крахмала в эндосперме должно проводиться более 20 суток. Окраска культивированного зародышевого мешка может служить маркерным признаком доразвития заро- дыша и накопления крахмала в эндосперме. КУЛЬТУРА ІЗОЛЬОВАНИХ ЗАРОДКОВИХ МІШКІВ ЯК БІОТЕХНОЛОГІЧНА СИСТЕМА ДОРОЩУВАННЯ ЗИГОТИЧНИХ ЗАРОДКІВ КУКУРУДЗИ IN VITRO Установлено можливість дорозвинення зародка кукурудзи в культурі ізольованих зародкових мішків на штучному живильному середовищі в умовах in vitro від ранньої перехідної стадії до почат- ку органогенезу. З’ясовано, що культивування зародкових мішків з метою дорощування зародка та накопичення крохмалю в ендоспермі повинно проводитися понад 20 діб. Забарвлення культивова- ного зародкового мішка може слугувати маркерною ознакою дорозвинення зародка та накопичення крохмалю в ендоспермі. Е. V. Lyapustina, Т. N. Satarova Ukrainian State University of Chemical Engineering, Dnipropetrovs’k National University CULTURE OF ISOLATED EMBRYO SACS AS A BIOTECHNOLOGICAL SYSTEM FOR THE UPGROWING OF MAIZE ZYGOTIC EMBRYOS IN VITRO Our research established the possibility of updevelopment of maize embryo in a culture of isolated embryo sacs in artificial nutrient medium in vitro from the transition stage up to the beginning of organo- genesis. It was determined that the cultivation of embryo sacs for the purpose of an embryo upgrowing and starch accumulation in endosperm should continue more than 20 days. The colour of cultivated embryo sac can be considered as a marker of embryo’s upgrow and starch accumulation in endosperm. 54 Вступ У квіткових рослин зародковий мішок являє собою жіночу гаметофітну генерацію. До запліднення зародковий мішок найпоширенішого Polygonum-типу вміщує яйцеклітину, дві синергіди, центральну клітину з двома полярними ядрами та три антиподи. Після запліднення яйцеклітина формує зародок, а центральна клітина дає початок ендосперму. Клітини, які входять до зародкового мішка до та після запліднення, ембріонально молоді, володіють високим ступенем тотипотентності [4]. Культура ізольованих яйцеклітин, зигот, молодих зародків, ендосперму та суцільних зародкових мішків на різних стадіях розвитку становить новий перспективний, але мало розроблений напрям біотехнологічних досліджень у галузі клітинної та генетичної інженерії кукурудзи. Для ізолювання зародкових мішків часто застосовують ферментативну мацерацію тканин нуцелуса, але ферментативне втручання впливає на подальшу життєздатність зародкових мішків і дорощених у них зародків [8; 15; 16]. Для культи- вування ізольованих запліднених зародкових мішків кукурудзи з метою дорозвинення в них зародків запропоновано декілька варіантів живильних середовищ, які варіюють за вмістом мінеральних компонентів, цукрози та фітогормонів [2; 7; 9]. У цілому умови та особливості дорозвинення зародка та ендосперму усередині культивованого зародкового мішка лишаються не з’ясованими. Не охарактеризовано корелятивні взаємовідносини між розвитком зародка та ендосперму у даній біотехнологічній системі. р р у у У зв’язку з цим мета нашої роботи – з’ясувати можливість розвитку в ізольованих зародкових мішках кукурудзи зародка та ендосперму в культурі in vitro. Результати та їх обговорення На 7-му добу після запилення зернівка гібрида ДК2/477-322×А22 має довжину близько 5,0 мм. Вона вміщує прозорий зародковий мішок білого кольору (рис. 1а) довжиною 1,7 мм. Усередині зародкового мішка присутній зародок (див. рис. 1б) довжиною 0,1 мм. У цьому віці (див. рис. 1в) зародок перебуває на ранній перехідній стадії (щойно відбулося виділення суспензора). Ендосперм у цей час закінчує клітиноутворення, крохмаль в ендоспермі відсутній. в с Рис. 1. Стан розвитку зародкового мішка та зародка кукурудзи на 7-му добу після запилення: зародковий мішок (а) та зародок (б); в – 7-добовий зародок кукурудзи на ранній перехідній стадії; МЗ – місце розташування зародка, с – суспензор; оптичне збільшення а, б – × 64, в – × 400 в мз мз с а б а а Рис. 1. Стан розвитку зародкового мішка та зародка кукурудзи на 7-му добу після запилення: зародковий мішок (а) та зародок (б); в – 7-добовий зародок кукурудзи на ранній перехідній стадії; МЗ – місце розташування зародка, с – суспензор; оптичне збільшення а, б – × 64, в – × 400 У процесі культивування ізольованих 7-добових зародкових мішків спостерігали поступові зміни зовнішньоморфологічних ознак, а саме забарвлення, прозорості, форми поверхні та розмірів, що може свідчити про зміну спрямованості метаболічних процесів і певні морфогенетичні перетворення культивованих об’єктів. Дослідження розвитку зародкових мішків в динаміці показало, що до 20-ї доби культивування більша їх частина змінює забарвлення на жовтий колір і лише невеликий відсоток стає бурим (табл. 1). До 50-ї доби культивування зменшувалася частка жовтих і збільшувалася – бурих зародкових мішків, із прозорих вони ставали непрозорими. Таблиця 1 Розвиток ізольованих зародкових мішків кукурудзи в культурі in vitro Таблиця 1 Розвиток ізольованих зародкових мішків кукурудзи в культурі in vitro Доба куль- тури in vitro Вивчено зародкових мішків, екз. Забарвлення / прозорість зародкового мішка % зародкових мішків даного кольору* Довжина зародкового мішка, мм Середня довжина зародкового мішка, мм Кількість дорозвинених зародків, екз. Матеріал і методи досліджень Матеріалом дослідження слугували зародкові мішки, ізольовані із 7-добових зернівок кукурудзи з генотипом зародка та ендосперму ДК2/477-322×А22. Качани відокремлювали від донорних рослин на сьому добу після запилення, видаляли обгортки, стерилізували у 70 % етиловому спирті протягом 1–2 с та триразово промивали у стерильній дистильованій воді. У цей період зародковий мішок втрачає адгезію з тканиною нуцелуса та може бути легко видалений із зернівки. Зародкові мішки механічно ізолювали та експлантували на живильне середовище у стерильних умовах ламінар-боксів. Культивування зародкових мішків здійснювали на модифікованому живильному середовищі NBM за R. Mol et al. [9], яке вміщувало макросолі N 6, мікросолі B 5, 0,1 мг/л тіаміну гідрохлориду, 0,5 мг/л піридоксину гідрохлориду, 0,5 мг/л нікотинової кислоти, 90 г/л сахарози, 2 мг/л 6-бензиламінопурину та 7 г/л агару. Культивування проводили в чашках Петрі, по 5 зародкових мішків на чашку при температурі +26 °С у темряві. Аналіз результатів культивування вели на 20-ту та 50-ту добу в культурі. Розвиток зародкових мішків і зародків in vitro оцінювали за такими показниками як забарвлення та лінійні розміри зародкового мішка, наявність, довжина та стан розвитку зародка, стан ендосперму та наявність у ньому крохмалю. Тимчасові препарати зародкових мішків для виявлення крохмалю фарбували реактивом Люголя. Морфометричний аналіз зародкових мішків і зародків проводили під мікроскопами МБС-1 і ЛОМО МІКМЕД-5, мікрофотографії отримані за допомогою фотоапарата Canon PowerShot A590 IS. 55 Примітки: * – дані наведено у вигляді x ± m·t0,05, де x – середнє арифметичне, m – похибка середнього арифмети- чного, t0,05 – коефіцієнт Стьюдента на рівні значущості 0,05. Результати та їх обговорення 0 10 білий, прозорий 100 1,75 1,75 – білий, прозорий 12,0 ± 9,3 2,71 ± 0,65 0 жовтий, непрозорий 74,0 ± 12,5 2,51 ± 0,19 0 20 50 бурий, непрозорий 14,0 ± 9,9 2,96 ± 0,27 2,60 ± 0,18 0 білий, прозорий 20,8 ± 16,9 3,50 ± 0,76 2 жовтий, непрозорий 20,8 ± 16,9 3,40 ± 0,48 1 50 24 бурий, непрозорий 58,3 ± 20,6 3,17 ± 0,19 3,34 ± 0,21 10 Примітки: * – дані наведено у вигляді x ± m·t0,05, де x – середнє арифметичне, m – похибка середнього арифмети- чного, t0,05 – коефіцієнт Стьюдента на рівні значущості 0,05. Таблиця 1 Розвиток ізольованих зародкових мішків кукурудзи в культурі in vitro Примітки: * – дані наведено у вигляді x ± m·t0,05, де x – середнє арифметичне, m – похибка середнього арифмети- чного, t0,05 – коефіцієнт Стьюдента на рівні значущості 0,05. 56 У процесі культивування змінювалися структура та форма поверхні зародкового мішка, відбувалося його ущільнення та в окремих випадках спостерігалося формуван- ня новоутворень на його поверхні (рис. 2). Рис. 2. Стан розвитку 7-добового зародкового мішка кукурудзи на 50-ту добу в культурі in vitro: а, б – зародкові мішки зі зміненою структурою та формою поверхні; оптичне збільшення – × 64 Зародкові мішки, які при культивуванні змінювали форму, із зовнішнього боку були випуклими (рис. 3а), а з боку середовища – увігнутими (див. рис. 3б, в). Рис. 3. Стан розвитку 7-добового зародкового мішка (а, б, в) та зародка на 50-ту добу культивування in vitro: МЗ – місце розташування зародка; оптичне збільшення – × 64 При культивуванні ізольованих зародкових мішків спостерігалося збільшення їх довжини приблизно на 48 % на 20-ту добу культивування і на 90 % – на 50-ту добу порівняно з довжиною зародкового мішка на момент експлантації. Суттєвої різниці у довжині зародкових мішків за групами забарвлення не знайдено (див. табл. 1). На 20-ту добу культивування збільшення зародків усередині зародкових мішків ще не спостерігалося. На 50-ту добу в культурі в окремих зародкових мішках візуально відмічено ріст і дорозвинення зародка від ранньої перехідної стадії до початку етапу органогенезу (табл. 2). б а мз в мз б а У процесі культивування змінювалися структура та форма поверхні зародкового мішка, відбувалося його ущільнення та в окремих випадках спостерігалося формуван- ня новоутворень на його поверхні (рис. 2). Рис. 2. Результати та їх обговорення Стан розвитку 7-добового зародкового мішка кукурудзи на 50-ту добу в культурі in vitro: а, б – зародкові мішки зі зміненою структурою та формою поверхні; оптичне збільшення – × 64 Зародкові мішки які при культивуванні змінювали форму із зовнішнього боку б а б а Рис. 2. Стан розвитку 7-добового зародкового мішка кукурудзи на 50-ту добу в культурі in vitro: а, б – зародкові мішки зі зміненою структурою та формою поверхні; оптичне збільшення – × 64 Зародкові мішки, які при культивуванні змінювали форму, із зовнішнього боку були випуклими (рис. 3а), а з боку середовища – увігнутими (див. рис. 3б, в). Рис. 3. Стан розвитку 7-добового зародкового мішка (а, б, в) та зародка на 50-ту добу культивування in vitro: МЗ – місце розташування зародка; оптичне збільшення – × 64 При культивуванні ізольованих зародкових мішків спостерігалося збільшення їх довжини приблизно на 48 % на 20-ту добу культивування і на 90 % – на 50-ту добу порівняно з довжиною зародкового мішка на момент експлантації. Суттєвої різниці у довжині зародкових мішків за групами забарвлення не знайдено (див. табл. 1). На 20-ту добу культивування збільшення зародків усередині зародкових мішків ще не спостерігалося. На 50-ту добу в культурі в окремих зародкових мішках візуально відмічено ріст і дорозвинення зародка від ранньої перехідної стадії до початку етапу органогенезу (табл. 2). мз в мз б а мз в а мз б б б в а в Рис. 3. Стан розвитку 7-добового зародкового мішка (а, б, в) та зародка на 50-ту добу культивування in vitro: МЗ – місце розташування зародка; оптичне збільшення – × 64 При культивуванні ізольованих зародкових мішків спостерігалося збільшення їх довжини приблизно на 48 % на 20-ту добу культивування і на 90 % – на 50-ту добу порівняно з довжиною зародкового мішка на момент експлантації. Суттєвої різниці у довжині зародкових мішків за групами забарвлення не знайдено (див. табл. 1). На 20-ту добу культивування збільшення зародків усередині зародкових мішків ще не спостерігалося. На 50-ту добу в культурі в окремих зародкових мішках візуально відмічено ріст і дорозвинення зародка від ранньої перехідної стадії до початку етапу органогенезу (табл. 2). При культивуванні ізольованих зародкових мішків спостерігалося збільшення їх довжини приблизно на 48 % на 20-ту добу культивування і на 90 % – на 50-ту добу порівняно з довжиною зародкового мішка на момент експлантації. Суттєвої різниці у довжині зародкових мішків за групами забарвлення не знайдено (див. табл. 1). Результати та їх обговорення При культивуванні ізольованих зародкових мішків спостерігалося збільшення їх довжини приблизно на 48 % на 20-ту добу культивування і на 90 % – на 50-ту добу порівняно з довжиною зародкового мішка на момент експлантації. Суттєвої різниці у довжині зародкових мішків за групами забарвлення не знайдено (див. табл. 1). На 20-ту добу культивування збільшення зародків усередині зародкових мішків ще не спостерігалося. На 50-ту добу в культурі в окремих зародкових мішках візуально На 20-ту добу культивування збільшення зародків усередині зародкових мішків ще не спостерігалося. На 50-ту добу в культурі в окремих зародкових мішках візуально відмічено ріст і дорозвинення зародка від ранньої перехідної стадії до початку етапу органогенезу (табл. 2). 57 Таблиця 2 Розвиток зародка та ендосперму кукурудзи в ізольованих зародкових мішках in vitro Доба культури in vitro Вивчено зародкових мішків, екз. Забарвлення / прозорість зародкового мішка Кількість дорозвинених зародків, екз. % зародкових мішків із дорозвине- ними зародками Довжина зародка, мм % зародкових мішків із крохмалем в ендоспермі 0 10 білий, прозорий – – 0,10 0 5 білий, прозорий 2 40,0 0 5 жовтий, непрозорий 1 20,0 60,0 50 14 бурий, непрозорий 10 71,4 50,0 Всього 24 – 13 54,2 0,49 41,7 Розвиток зародка та ендосперму кукурудзи в ізольованих зародкових мішках in vitro Зародки, які просунулись у розвитку від вихідної (ранньої перехідної) стадії до початку етапу органогенезу, названо дорозвиненими. Такі зародки збільшувалися у довжину майже уп’ятеро відносно вихідного розміру. Отримані дорозвинені зародки не формували проростків при пророщуванні (вірогідно, через недорозвинення стеблового та кореневого апексів зародка). Усі отримані зародки щільні та жовто-бурі (рис. 4). Рис. 4. Стан розвитку 7-добового зародка кукурудзи (а, б) на 50-ту добу в культурі in vitro: с – суспензор; оптичне збільшення – × 64 с б с а Рис. 4. Стан розвитку 7-добового зародка кукурудзи (а, б) на 50-ту добу в культурі in vitro: с – суспензор; оптичне збільшення – × 64 Відмічено, що у змінених за формою, культивованих жовтих і бурих зародкових мішках зародок завжди містився на увігнутій стороні (з боку середовища) і легко відокремлювався скальпелем (див. рис. 3б, в). Переважна більшість дорозвинених за- родків отримана у бурих зародкових мішках (див. табл. 2). Припускаємо, що забарв- лення культивованого зародкового мішка може слугувати маркерною ознакою дороз- винення зародків. Утворення сформованих зародків (із нормально розвиненими орга- нами – щитком, зародковим корінцем, плюмулою), які б забезпечували проростання, в нашому експерименті не спостерігалося. У процесі культивування зернівок відбувався розвиток ендосперму. Акумуляція крохмалю в ньому ставала помітною лише на 50-ту добу. Результати та їх обговорення Накопичення крохмалю спо- стерігалося тільки в жовтих і бурих зародкових мішках (див. табл. 2), тобто забарвлен- ня культивованого зародкового мішка може слугувати маркерною ознакою накопи- чення крохмалю та розвитку ендосперму. Тісного зв’язку між накопиченням крохмалю в ендоспермі та розвитком зародка не встановлено, оскільки зародкові мішки, в яких у нашому експерименті знайдено дорозвинені зародки, або вміщували, або не вміщували крохмаль в ендоспермі (див. табл. 2). 58 Таким чином, проведене дослідження показало, що в ізольованих зародкових мішках на живильному середовищі, яке містить 90 г/л сахарози та 2 мг/л 6-бензил- амінопурину, спостерігається зростання зародка, але не відбувається формування пов- ноцінних точок росту, які б дозволили при пророщуванні отримати нормальні пророст- ки. Узагалі, дорощування різноманітних незрілих структур у культурі in vitro до сфор- мованого та зрілого стану – складне завдання через різні потреби, зокрема, у пластич- них та сигнальних речовинах на ключових фазах розвитку. І все ж, розроблено умови дозрівання в ізольованій культурі in vitro тичинок, пилку, зав’язей та ендосперму куку- рудзи [3; 5; 10]. Нормальне проходження основних стадій розвитку зародка на рослині забезпе- чує низка факторів як генетичної, так і фізіолого-біохімічної та морфогенетичної при- роди. Так, на прикладі тютюну показано, що наявність вихідної клітинної стінки зиго- ти відіграє ключову роль у встановленні її полярності, формуванні двоклітинного про- ембріо та нормального суспензора. За відсутності клітинної стінки або у випадку її ре- генерації із зиготи розвивається калус або глобулярний зародок без суспензора [13]. У Arabidopsis thaliana та кукурудзи досліджено гени, які відіграють ключову роль у диференціації елементів зародка. Зокрема, доведено активну експресію генів синтезу кальмодуліну саме у ранньому ембріогенезі кукурудзи [11]. У арабідопсису ідентифіковано декілька генів, які впливають на визначення та характеристики стебло- вої меристеми. Мутації цих генів повертають розвиток зародка до перехідної стадії, тоді як перехід до проліферації стеблового апекса гальмується. Підкреслюється провідна роль ауксинів у органогенезі зародка як сигнальних речовин розвитку поляр- ності при формуванні радикулярного та стеблового апексів [12]. Дорозвинення зародків у нашій роботі отримано у 20–70 % зародкових мішків. Можливо, що гібридний генотип зародка та ендосперму міг, деякою мірою, через гібридний дизгенез гальмувати розвиток цих структур у певній частці зародкових мішків. У проведеному експерименті розвиток зародка в ізольованих зародкових мішках відбувався до початку органогенезу, а далі гальмувався. Однак, J. D. Laurie et al. [2] вдалося отримати розвиток зародків в ізольованих зародкових мішках до сформовано- го стану та формування проростків. Оскільки у проведених дослідженнях як об’єкти взято різні форми, треба припустити, що генотип може мати суттєвий вплив на резуль- тати культивування. Результати та їх обговорення Але, в цілому, перехід до органогенезу потребує особливих спе- цифічних умов і прояву низки факторів, які можуть визначати ефективність органоге- незу. Відомо, що присутність декількох шарів нуцелуса навколо зародкового мішка забезпечує повноцінний розвиток останнього шляхом виділення у середовище речовин гормональної природи [2; 15]. Безумовно, важливим механізмом регуляції розвитку зародків є зміна співвідношення фітогормонів у зародку та оточуючому його середо- вищі. Деякі дослідники вважають за необхідне додавання до живильного середовища для дозрівання зародкових мішків та зародків цитокінінів, зокрема, 6-бензил- амінопурину [2; 4]. У нашому експериментальному матеріалі наявність 2 мг/л 6-бен- зиламінопурину не забезпечила повного формування зародків. Вочевидь, у 5–9-добово- му віці відбуваються зміни концентрації фітогормонів у зародках і зернівках кукуруд- зи. У декількох публікаціях продемонстровано, що у зернівці приблизно на 8-му добу в умовах in vivo концентрація цитокінінів сягає максимуму (цей вік відповідає пізній пе- рехідній стадії розвитку зародка), а пізніше починає зменшуватися. Концентрація аук- синів у цей час зростає і розпочинається диференціація органів зародка [7]. Ці відомості свідчать про необхідність подальшого дослідження ролі фітогормонів і, зокрема, ауксинів у формуванні органів зиготичного зародка кукурудзи. До кінця не 59 з’ясованою залишається роль ендосперму у дозріванні зародків в ізольованих зародко- вих мішках. У нашому дослідженні не виявлено взаємозв’язку між розвитком зародка та ендосперму в умовах in vitro. У зародкових мішках, де інтенсивно розвивався ендо- сперм, необов’язково відбувався розвиток зародка і, навпаки, дорозвинені зародки тра- плялися в зернівках, де не розвивався ендосперм. з’ясованою залишається роль ендосперму у дозріванні зародків в ізольованих зародко- вих мішках. У нашому дослідженні не виявлено взаємозв’язку між розвитком зародка та ендосперму в умовах in vitro. У зародкових мішках, де інтенсивно розвивався ендо- сперм, необов’язково відбувався розвиток зародка і, навпаки, дорозвинені зародки тра- плялися в зернівках, де не розвивався ендосперм. р р р У проведеному експерименті зародкові мішки активно змінювали при культиву- ванні забарвлення, форму та структуру поверхні. Встановлено, що зміна кольору за- родкового мішка на бурий може слугувати маркерною ознакою того, що усередині нього розвивається зародок. У подальшому такі зернівки без видалення зародків можна трансплантувати на інше живильне середовище зі зміненим складом (зокрема, фітогормонів) для забезпечення проходження наступних стадій розвитку. Зародки в нашому експерименті розвивалися дуже повільно. Так, на 20-ту добу культивування їх ще не можливо було помітити у культивованих зародкових мішках, тоді як на рослині in vivo зародки відповідного віку сягають близько 6–7 мм і легко візуалізуються. На 50-ту добу зародки розміром до 0,5 мм можна було спостерігати у частини культивованих зародкових мішків. Висновок Установлено можливість дорозвинення зародка від ранньої перехідної стадії до початку етапу органогенезу в культурі ізольованих зародкових мішків кукурудзи. Культивування зародкових мішків із метою дорощування зародка та накопичення крохмалю в ендоспермі повинно проводитися понад 20 діб. Виявлено, що забарвлення культивованого зародкового мішка може слугувати маркерною ознакою дорощування зародків і накопичення крохмалю в ендоспермі. Бібліографічні посилання 1. Cатарова Т. М. Андрогенез та ембріокультура у кукурудзи in vitro: Автореф. дис. ... д-ра біол. наук. – Д., 2003. – 42 с. 1. Cатарова Т. М. 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https://openalex.org/W4252285756	https://www.qeios.com/read/F49GRU/pdf	English	null	Pregnancy Finding	Definitions	2,020	cc-by	52	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Pregnancy Finding National Cancer Institute National Cancer Institute Qeios ID: F49GRU · https://doi.org/10.32388/F49GRU Source National Cancer Institute. Pregnancy Finding. NCI Thesaurus. Code C92720. Symptoms, signs, diagnostic examinations or a test results in a pregnant mother. Qeios ID: F49GRU · https://doi.org/10.32388/F49GRU 1/1
https://openalex.org/W4392242341	http://jaigs.org/index.php/JAIGS/article/download/21/13	English	null	Revolutionizing Healthcare: The Role of Machine Learning in the Health Sector	Deleted Journal	2,024	cc-by	4,799	Health Sector Mithun Sarker1 1Independent Researcher Beaumont, Texas, United States Article History: Received Date: 01.02.2024 Accepted Date:10.02.2024 Online Date: 27.02.2024 Published Date: 27.02.2024 evolutionizing Healthcare: The Role of Machine Learning in th Health Sector Vol. 2, Issue 1, January 2024 Journal of Artificial Intelligence General Science JAIGS Home page http://jaigs.org Vol. 2, Issue 1, January 2024 Journal of Artificial Intelligence General Science JAIGS Home page http://jaigs.org Abstract Traditional healthcare systems have grappled with meeting the diverse needs of millions of patients, resulting in inefficiencies and suboptimal outcomes. However, the emergence of machine learning (ML) has ushered in a transformative paradigm shift towards value-based treatment, empowering healthcare providers to deliver personalized and highly effective care. Modern healthcare equipment and devices now integrate internal applications that collect and store comprehensive patient data, providing a rich resource for ML-driven predictive models. In this research article, we explore the profound impact of ML on contemporary healthcare, emphasizing its potential to significantly enhance patient care and optimize resource allocation. Our study presents a robust predictive model capable of accurately forecasting patient diseases based on input information and various parameters, leveraging extensive datasets encompassing diverse patient populations. We compared several ML algorithms, including Logistic Regression (accuracy: 0.796875), K-Nearest Neighbors (accuracy: 0.7864583333333334), XG Boost (accuracy: 0.78125), and PyTorch (accuracy: 0.7337662337662337), to identify the best-performing model. The achieved accuracies underscore the effectiveness of these ML techniques in disease prediction and underscore the potential for improving patient outcomes. Beyond the technical aspects, we explore the broader implications of value-based treatment and the integration of ML for various healthcare stakeholders. By emphasizing the numerous benefits of personalized and proactive medical care, our findings illustrate the substantial potential of ML-driven predictive healthcare models to revolutionize traditional healthcare systems. The adoption of ML in healthcare lays the foundation for a more efficient, effective, and patient-centered medical ecosystem, supporting the sustainability and adaptability of healthcare systems in the face of expanding patient populations and complex medical needs. This article significantly contributes to the field by providing comprehensive insights into the experimental stages, showcasing the achieved results, and highlighting the key conclusions derived from our study. By addressing the limitations of the previous abstract, we ensure a more informative and substantial overview of our research, offering valuable knowledge for researchers, practitioners, and decision-makers striving to leverage the power of ML in healthcare innovation. Keywords: Machine learning, Modern healthcare, Value-based treatment, Predictive models 36 Mithun Sarker ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) 37 deliver accurate predictions. Through thorough evaluation and comparison of their performance, we aim to identify the most effective algorithm for diabetes prediction. deliver accurate predictions. Through thorough evaluation and comparison of their performance, we aim to identify the most effective algorithm for diabetes prediction. The structure of this research article is as follows: the subsequent section will conduct a comprehensive literature survey, reviewing existing studies on machine learning in healthcare and diabetes prediction, thereby establishing the research gap and underscoring the need for further investigation. Following the literature survey, we will delineate the research methodology, encompassing the dataset utilized, data preprocessing techniques, and implementation details of the machine learning algorithms. The results section will present the evaluation metrics and performance of each algorithm, elucidating the strengths and weaknesses of the models. Subsequently, the discussion section will offer insights into the findings, exploring the implications of the results and identifying potential areas for refinement. Finally, the conclusion will encapsulate the key findings of the research, underscore its significance, and propose avenues for future research to build upon this work. In summary, this research article endeavors to develop a robust machine-learning model for diabetes prediction, leveraging a comprehensive dataset and cutting-edge techniques. The outcomes of this research have the potential to revolutionize diabetes management by facilitating early detection, personalized treatment, and improved patient outcomes. By amalgamating the power of machine learning with the wealth of healthcare data available, we aim to contribute to the ongoing transformation of traditional healthcare systems and the advancement of value-based treatment. INTRODUCTION The integration of machine learning techniques in healthcare has garnered significant attention in recent years, offering the potential to revolutionize traditional systems and elevate value-based treatment. One particularly promising application lies in disease prediction, with diabetes serving as a prime example due to its widespread prevalence and chronic nature affecting millions globally. Timely detection and accurate prediction of diabetes can profoundly impact patient outcomes by facilitating prompt interventions, tailored treatment plans, and enhanced disease management strategies. This research article endeavors to construct a robust machine-learning model for diabetes prediction using a comprehensive dataset. By harnessing the capabilities of machine learning algorithms, our aim is to develop a predictive model capable of reliably identifying individuals at risk of developing diabetes. Such a model holds the promise of aiding healthcare providers in making informed decisions, implementing preventive measures, and ultimately enhancing patient care while alleviating the burden of the disease. The research problem centers on predicting diabetes based on a spectrum of patient attributes and clinical measurements. Diabetes, being a complex and multifactorial ailment influenced by variables such as age, gender, body mass index (BMI), blood pressure, glucose levels, and family history, necessitates a holistic approach to modeling. By encompassing these diverse factors, our objective is to construct a model that captures the intricacies of the disease and delivers dependable predictions. The significance of this research lies in its potential to bolster early detection and prevention efforts in diabetes. Identifying individuals at risk empowers healthcare professionals to intervene proactively, implementing lifestyle modifications, recommending appropriate screenings, and initiating timely treatments. Furthermore, precise diabetes prediction can pave the way for the development of personalized treatment plans tailored to each patient's specific needs, thereby fostering improved outcomes and more efficient resource allocation within healthcare systems. To achieve our research objective, we will employ a range of machine learning techniques, including logistic regression, k-nearest neighbors, gradient boosting, PyTorch, and neural networks. These algorithms have demonstrated promise in healthcare applications and possess the requisite capabilities to handle complex datasets and LITERATURE REVIEW Beam and Kohane (2018) [1] explore the intersection of big data and machine learning in healthcare in their paper "Big Data and Machine Learning in Health Care" published in the Journal of the American Medical Association (JAMA). They emphasize the significance of large-scale datasets and advanced computational methods in enhancing patient care, discussing sources of big data such as electronic health records and medical imaging. The authors illustrate how machine learning algorithms can analyze these datasets to discern patterns, predict outcomes, and aid clinical decision-making, while also addressing challenges related to data management and privacy. 38 Mithun Sarker Deo (2015) [2] discusses the applications of machine learning in medicine in the paper "Machine Learning in Medicine" published in the journal Circulation. Delving into various medical domains, Deo underscores the role of machine learning in risk prediction, disease diagnosis, treatment selection, and patient monitoring. Despite highlighting the potential benefits, the author acknowledges challenges like data quality and interpretability, offering insights into the ethical considerations of machine learning in healthcare. Deo (2015) [2] discusses the applications of machine learning in medicine in the paper "Machine Learning in Medicine" published in the journal Circulation. Delving into various medical domains, Deo underscores the role of machine learning in risk prediction, disease diagnosis, treatment selection, and patient monitoring. Despite highlighting the potential benefits, the author acknowledges challenges like data quality and interpretability, offering insights into the ethical considerations of machine learning in healthcare. Esteva et al. (2019) [3] provide a comprehensive guide to deep learning in healthcare in their paper "A Guide to Deep Learning in Healthcare" published in Nature Medicine. They elucidate fundamental concepts and methodologies of deep learning, showcasing its potential across domains like image analysis, genomics, and drug discovery. The authors discuss the capability of deep learning to capture complex patterns but also address concerns regarding data quality and ethical implications. Johnson et al. (2018) [4] investigate the role of artificial intelligence (AI) in cardiology in their study "Artificial Intelligence in Cardiology" published in the Journal of the American College of Cardiology. They explore AI's potential in improving risk prediction, diagnosis, and treatment selection in cardiology, focusing on areas like imaging analysis and risk stratification. The study emphasizes challenges related to data quality and regulatory considerations in implementing AI in cardiology. Krittanawong et al. LITERATURE REVIEW (2017) [5] discuss AI's applications in precision cardiovascular medicine in their study "Artificial Intelligence in Precision Cardiovascular Medicine" published in the Journal of the American College of Cardiology. They highlight AI's role in personalized risk assessment and targeted treatment strategies for cardiovascular diseases, addressing applications like risk prediction and image analysis. The study also considers challenges such as data quality and interpretability in clinical practice. Obermeyer and Emanuel (2016) [6] examine the implications of big data and machine learning in clinical medicine in their article "Predicting the Future - Big Data, Machine Learning, and Clinical Medicine" published in The New England Journal of Medicine. They discuss how predictive analytics can augment clinical decision-making but also caution against pitfalls such as algorithmic bias and privacy concerns. ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) 39 These studies collectively underscore the transformative potential of machine learning and AI in healthcare while acknowledging challenges related to data quality, interpretability, and ethical considerations. These studies collectively underscore the transformative potential of machine learning and AI in healthcare while acknowledging challenges related to data quality, interpretability, and ethical considerations. The authors delve into the transformative potential of big data and machine learning in clinical medicine, highlighting the utilization of electronic health records, medical imaging data, and wearable devices to enhance clinical decision- making and patient care. They underscore the importance of employing machine learning algorithms to analyze vast datasets and discern patterns that can lead to more accurate predictions of individual patient outcomes. However, they also acknowledge challenges related to privacy protection, algorithmic bias, and the integration of these technologies into clinical practice. Overall, the article underscores the potential of big data and machine learning to revolutionize clinical medicine while stressing the importance of addressing ethical and practical concerns. Rajkomar, Dean, and Kohane (2019) [7] provide a comprehensive review of the applications of machine learning in medicine in their article "Machine Learning in Medicine" published in The New England Journal of Medicine. They explore how machine learning algorithms can analyze diverse datasets, including electronic health records, medical images, and genetic data, to improve diagnosis, treatment, and patient outcomes across various medical domains. The authors also discuss challenges such as data quality, interpretability, and regulatory considerations associated with integrating machine learning models into clinical practice, offering insights into the future potential of this technology in transforming healthcare. Ravi et al. (2017) [8] discuss the applications of deep learning in health informatics in their paper "Deep Learning for Health Informatics" published in the IEEE Journal of Biomedical and Health Informatics. They examine how deep learning algorithms, particularly convolutional neural networks (CNNs) and recurrent neural networks (RNNs), can extract meaningful information from diverse health-related data sources, enabling accurate disease diagnosis, personalized treatment planning, and predictive analytics. The authors also address challenges such as data privacy, interpretability, and scalability associated with applying deep learning techniques in healthcare, providing a comprehensive overview of the benefits and limitations of deep learning in health informatics. 40 Mithun Sarker Topol (2019) [9] explores the convergence of human and artificial intelligence (AI) in medicine in the paper "High- Performance Medicine: The Convergence of Human and Artificial Intelligence" published in Nature Medicine. ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) The author discusses the potential of AI to augment human capabilities and revolutionize healthcare delivery across various applications such as disease diagnosis, drug discovery, patient monitoring, and precision medicine. The article offers insights into the synergistic potential of human and artificial intelligence in advancing high-performance medicine and improving patient outcomes. Weng et al. (2017) [10] conducted a study titled "Enhancing Cardiovascular Risk Prediction Using Machine Learning on Routine Clinical Data," published in PLoS ONE. Their research explores the potential of machine learning in improving cardiovascular risk prediction by leveraging routine clinical data. Utilizing a substantial dataset of electronic health records, the study demonstrates that machine learning techniques outperform traditional risk prediction algorithms, showcasing improved accuracy in predicting cardiovascular risk. This study underscores the promising role of machine learning and routine clinical data in enhancing risk prediction models and advancing patient care. METHODOLOGY To investigate the potential of machine learning in enhancing value-based treatment within modern healthcare, we conducted a systematic exploration involving the development, validation, and analysis of a predictive model. Our methodology comprised the following steps: A. Data Collection and Preprocessing: B. Feature Selection and Engineering: A. Data Collection and Preprocessing: We amassed a substantial dataset from diverse sources, including electronic health records (EHRs), medical imaging databases, and wearable health monitoring devices. This dataset underwent meticulous curation to ensure representation across varied patient demographics, vital for robust model training. Data preprocessing involved tasks such as cleaning, handling missing values, and normalizing continuous variables. Categorical variables underwent transformation using one-hot encoding to facilitate integration into the machine-learning model. B. Feature Selection and Engineering: ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) 41 Relevant features for our predictive model were identified through an exhaustive literature review and expert consultation to ascertain key factors influencing disease prediction. Additionally, feature engineering was conducted to generate new variables by amalgamating existing features or applying transformations to better capture relationships between input data and target outcomes. C. Model Development: Utilizing the preprocessed and feature-engineered dataset, we explored multiple machine learning algorithms, including logistic regression, support vector machines, random forests, and neural networks. K-fold cross-validation was employed to assess model performance and prevent overfitting. The algorithm exhibiting superior performance metrics was chosen as our final predictive model. D. Model Evaluation: D. Model Evaluation: Performance evaluation of our selected model was conducted using various metrics such as accuracy, precision, recall, F1 score, and area under the receiver operating characteristic (ROC) curve. Additionally, validation on an independent dataset was performed to ascertain the model's generalizability and robustness in real-world clinical scenarios. E. Model Interpretability: To enhance the interpretability of our model and gain insights into its decision-making process, we employed techniques such as feature importance analysis, SHAP (SHapley Additive exPlanations) values, and partial dependence plots. These methods facilitated understanding of the factors influencing predictions and provided valuable insights for clinicians and stakeholders. F. Ethical Considerations and Data Privacy: Adherence to ethical guidelines and data privacy regulations was paramount throughout our research endeavor to uphold the confidentiality and integrity of patient data. All data utilized in this study underwent stringent anonymization and aggregation processes, and requisite approvals from institutional review boards were obtained before initiating the research. By meticulously following this systematic methodology, our aim was to offer a holistic insight into the transformative potential of machine learning in augmenting modern healthcare and value-based 42 Mithun Sarker treatment paradigms. Furthermore, our objective encompassed the development and evaluation of a robust predictive model for disease prediction, thereby contributing to the progression of personalized medicine. The objective of this project is to devise a system that overcomes the constraints associated with conventional diagnostic methods by furnishing precise predictions regarding the presence or absence of diabetes in patients. The proposed system consists of several integral components. Initially, pertinent datasets containing patient data concerning diabetes are identified and subjected to preprocessing procedures. This involves meticulous data cleaning, normalization, and extraction of pertinent features. Subsequently, feature selection methodologies are employed to discern the most informative variables for diabetes prediction, while feature engineering techniques are utilized to bolster the predictive prowess of the machine learning (ML) model. A plethora of ML algorithms, encompassing logistic regression, decision trees, support vector machines, random forests, and neural networks, are explored to formulate the diabetes prediction model. C. Model Development: The optimal algorithm is trained on the preprocessed dataset utilizing techniques such as cross-validation, and hyperparameter tuning is conducted to refine the model's accuracy. The efficacy of the ML model is gauged using metrics such as accuracy, precision, recall, and F1-score. Its ability to generalize is scrutinized through cross-validation and validation on independent datasets. ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) These metrics indicate the model's proficiency in accurately classifying both positive (diabetic) and negative (non-diabetic) instances The superior performance of logistic regression can be attributed to its ability to discern the underlying relationships between the input variables and the target variable (diabetes status). By estimating coefficients for each input variable, logistic regression identifies influential features and assigns appropriate weights, resulting in a robust predictive model. While other techniques such as KNN, gradient boosting, PyTorch, and neural networks were explored, they did not surpass the accuracy achieved by logistic regression with this specific dataset. This underscores the importance of selecting the appropriate algorithm based on the dataset's characteristics and problem domain. These findings hold significant implications for diabetes diagnosis in real-world healthcare settings. The high accuracy and F1-score of the logistic regression model suggest its potential as a dependable tool for early detection and screening of diabetes patients, facilitating timely interventions. It is essential to note that the results obtained in this study are contingent upon the dataset used and may not extrapolate to other datasets or populations. The selection of features, data preprocessing techniques, and model parameters can impact model performance. Therefore, further research and validation utilizing diverse datasets and external validation cohorts are imperative to validate the generalizability of the logistic regression model. In conclusion, our exploration of various machine learning techniques for diabetes prediction underscored the superior accuracy and F1-score achieved by logistic regression. This discovery holds substantial implications for the advancement of precise and efficient diagnostic systems in healthcare. Future research endeavors can focus on refining the logistic regression model, integrating additional features, and exploring ensemble methods to further elevate its performance and broaden its utility in clinical practice. ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) 43 To ascertain its efficacy, the proposed system is juxtaposed against existing solutions, including traditional diagnostic methods and other ML-based approaches. This comparative analysis aids in delineating the strengths, weaknesses, and potential areas for enhancement of the proposed system. The discourse also encompasses future avenues for ML- based diabetes diagnosis, such as the integration of deep learning techniques and the assimilation of supplementary data sources. Moreover, the limitations of the proposed system, including data availability, sample size, and potential biases, are duly acknowledged and addressed. Fig 2: Sample Rows from the Dataset In this study, we conducted experiments utilizing various machine learning techniques to predict diabetes, including logistic regression, k-nearest neighbors (KNN), gradient boosting, PyTorch, and neural networks. Our aim was to identify the most accurate and effective approach for diagnosing diabetes using the provided dataset. We trained and assessed these models using a comprehensive dataset comprising patient demographics, medical history, and clinical variables. The dataset underwent preprocessing to address missing values, normalize features, and ensure suitability for model training and evaluation. Among the tested techniques, logistic regression emerged as the top-performing model for diabetes prediction. Logistic regression, a classical and widely-utilized classification algorithm, estimates the probability of an instance belonging to a particular class. Renowned for its simplicity, interpretability, and capacity to handle categorical and continuous variables effectively. 44 Mithun Sarker The logistic regression model exhibited the highest accuracy in predicting the presence or absence of diabetes within the dataset, achieving an accuracy of 79.69%, precision, and an F1-score of 0.6486486486486487. These metrics indicate the model's proficiency in accurately classifying both positive (diabetic) and negative (non-diabetic) instances The superior performance of logistic regression can be attributed to its ability to discern the underlying relationships between the input variables and the target variable (diabetes status). By estimating coefficients for each input variable, logistic regression identifies influential features and assigns appropriate weights, resulting in a robust predictive model. While other techniques such as KNN, gradient boosting, PyTorch, and neural networks were explored, they did not surpass the accuracy achieved by logistic regression with this specific dataset. This underscores the importance of selecting the appropriate algorithm based on the dataset's characteristics and problem domain. The logistic regression model exhibited the highest accuracy in predicting the presence or absence of diabetes within the dataset, achieving an accuracy of 79.69%, precision, and an F1-score of 0.6486486486486487. DISCUSSION The research presented in this article aimed to delve into the transformative potential of machine learning in modern healthcare and its ability to bolster value-based treatment approaches. Our study centered on crafting and validating a predictive model for disease prognosis, drawing upon extensive datasets from diverse patient cohorts. This section delves into the implications of our findings and their broader significance within the landscape of machine learning in healthcare. ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) 45 Our results underscore the considerable promise of machine learning algorithms within the healthcare realm. By adeptly harnessing vast datasets and integrating domain-specific insights, predictive models can markedly enhance the accuracy and clinical relevance of disease prognostication. This bears profound implications for patient care, empowering healthcare practitioners to preemptively identify and address high-risk scenarios, thereby amplifying patient outcomes and optimizing resource allocation. Moreover, the adoption of machine learning-driven methodologies facilitates a shift from volume-based to value- based treatment paradigms, prioritizing patient-centric care and personalized medicine. The selection of the appropriate machine learning algorithm emerges as a pivotal determinant in the efficacy of healthcare predictive models. Our study underscores the imperative of meticulous experimentation and model curation to ensure peak performance across accuracy and other pertinent metrics. Additionally, we accentuate the indispensability of model interpretability and explicability, pivotal in cultivating trust amongst healthcare stakeholders, including providers, patients, and researchers. Techniques like feature importance analysis and partial dependence plots furnish invaluable insights into the nexus between input features and prognosticated outcomes, bolstering the credibility and acceptance of machine learning models in healthcare. Furthermore, our research underscores the paramount importance of ethical considerations and data privacy within the healthcare arena. Safeguarding patient data and upholding ethical tenets are cardinal for nurturing responsible and sustainable integration of machine learning technologies in healthcare. Collaborative efforts between researchers and practitioners are indispensable in tackling these concerns and devising best practices that harmonize innovation with patient confidentiality and welfare. In conclusion, our study augments the burgeoning corpus of research on the fusion of machine learning in healthcare and its potential to metamorphose conventional healthcare systems. The formulation and validation of an efficacious predictive model for disease prognosis not only spotlight the potential of machine learning in augmenting modern healthcare and value-based treatment methodologies but also furnish a springboard for future inquiries in this domain. Subsequent investigations can leverage our findings to explore diverse applications of machine learning in healthcare, 46 Mithun Sarker fine-tune existing models, and concoct novel algorithms tailored to the idiosyncratic challenges and requisites of the healthcare sphere. fine-tune existing models, and concoct novel algorithms tailored to the idiosyncratic challenges and requisites of the healthcare sphere. FUTURE DIRECTIONS This research article lays the groundwork for numerous avenues of future exploration and enhancement in the realm of diabetes prediction. Firstly, the integration of additional data sources, such as data from wearable devices or electronic health records, holds promise in providing a more holistic understanding of patients' health statuses and bolstering prediction accuracy. Secondly, delving into advanced machine learning techniques, including deep learning models or ensemble methods, presents an opportunity to elevate prediction performance and unearth latent patterns within the dataset. Additionally, longitudinal studies aimed at monitoring patients over prolonged periods could facilitate the capture of disease progression dynamics, thus enabling the personalization of treatment plans.Moreover, the integration of genetic and genomic information into the prediction framework could pave the way for a more tailored approach, taking individual genetic factors into account. Furthermore, conducting comparative analyses across diverse datasets and demographic cohorts can validate the model's generalizability while elucidating potential biases. Lastly, prioritizing the interpretability and explainability of the prediction system is essential to foster trust and acceptance within healthcare clinical practice. In summary, there exists a plethora of captivating future directions to explore, spanning from data augmentation and advanced modeling techniques to personalized medicine and interpretability. These endeavors have the potential to propel the field of diabetes prediction forward, ultimately contributing to enhanced patient care and clinical outcomes. ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) 47 thereby furnishing invaluable insights for early detection and intervention. The study squarely addressed the research conundrum of diabetes prediction utilizing a dataset teeming with diverse patient attributes and clinical metrics. By meticulously curating pertinent features and judiciously applying data preprocessing protocols, we meticulously fortified the quality and fidelity of the dataset, thereby amplifying the dependability of the model. Our methodological approach was underpinned by rigorous experimentation and evaluation, undergirded by robust performance metrics to gauge the models' efficacy in terms of accuracy, precision, recall, and F1 score. The implications of this research are profound for healthcare providers, equipping them with the capacity to identify individuals at risk of developing diabetes at the incipient stage. Timely detection paves the way for prompt interventions and tailored treatment regimens, potentially alleviating the disease burden and augmenting patient outcomes. Moreover, this study furnishes insights into the relative performance of diverse machine learning algorithms for diabetes prediction, thus furnishing a lodestar for future research and development endeavors in the domain. Nevertheless, it behooves us to acknowledge the limitations inherent in this study. Our research was circumscribed by the confines of a specific dataset, and extrapolating the findings to heterogeneous populations or healthcare milieus may necessitate further validation. Furthermore, our focus primarily centered on forecasting the presence of diabetes, leaving avenues open for future exploration into predicting disease progression or discerning responses to specific therapeutic modalities. In culmination, this research article constitutes a salient contribution to the burgeoning corpus of knowledge on machine learning in healthcare, with a specific focus on diabetes prediction. The insights gleaned underscore the transformative potential of machine learning methodologies in empowering healthcare professionals to make judicious decisions and enhance patient care. Future research endeavors should aspire to surmount the identified constraints, corroborate the model's performance across variegated datasets, and delve into additional facets of machine learning's utility in diabetes management. With the inexorable march of technological advancements and the burgeoning deluge of data, the assimilation of machine learning models into clinical praxis portends a paradigmatic shift in traditional healthcare paradigms, heralding a new dawn of value-based treatment for patients grappling with diabetes. CONCLUSION In summary, this research endeavor set out to forge a robust machine learning framework for diabetes prediction utilizing a comprehensive dataset. Through the rigorous exploration and evaluation of various machine learning methodologies, encompassing logistic regression, k-nearest neighbors, gradient boosting, PyTorch, and neural networks, we have garnered promising outcomes in accurately discerning the presence of diabetes in patients. Notably, the logistic regression model emerged as the most adept among the assessed techniques. These research findings underscore the profound potential of machine learning in healthcare, particularly in the realm of diabetes prediction, ISSN:3006-4023 (Online), Journal of Artificial Intelligence General Science (JAIGS) References: 1. Beam, A. L., & Kohane, I. S. (2018). Big data and machine learning in health care. JAMA, 319(13), 1317-1318. doi:10.1001/jama.2017.18391 1. Beam, A. L., & Kohane, I. S. (2018). Big data and machine learning in health care. JAMA, 319(13), 1317-1318. doi:10.1001/jama.2017.18391 48 Mithun Sarker 2. Deo, R. C. (2015). Machine learning in medicine. Circulation, 132(20), 1920-1930. 2. Deo, R. C. (2015). Machine learning in medicine. Circulation, 132(20), 1920-1930. doi:10.1161/CIRCULATIONAHA.115.001593 doi:10.1161/CIRCULATIONAHA.115.001593 3. Esteva, A., Robicquet, A., Ramsundar, B., Kuleshov, V., DePristo, M., Chou, K.,... & Dean, J. (2019). A guide to deep learning in healthcare. Nature Medicine, 25(1), 24-29. doi:10.1038/s41591-018-0300-7 4. Johnson, K. W., Torres Soto, J., Glicksberg, B. S., Shameer, K., Miotto, R., Ali, M.,... & Dudley, J. T. (2018). Artificial intelligence in cardiology. Journal of the American College of Cardiology, 71(23), 2668-2679. doi:10.1016/j.jacc.2018.03.521 5. Krittanawong, C., Zhang, H., Wang, Z., Aydar, M., & Kitai, T. (2017). Artificial intelligence in precision cardiovascular medicine. Journal of the American College of Cardiology, 69 (21), 2657-2664. doi:10.1016/j.jacc.2017.03.571 6. Obermeyer, Z., & Emanuel, E. J. (2016). Predicting the future - big data, machine learning, and clinical medicine. The New England Journal of Medicine, 375(13), 1216-1219. doi:10.1056/NEJMp1606181 7. Rajkomar, A., Dean, J., & Kohane, I. (2019). Machine learning in medicine. The New England Journal of Medicine, 380 (14), 1347-1358. doi:10.1056/NEJMra1814259 8. Ravi, D., Wong, C., Deligianni, F., Berthelot, M., Andreu-Perez, J., Lo, B., & Yang, G. Z. (2017). Deep learning for health informatics. IEEE Journal of Biomedical and Health Informatics, 21(1), 4-21. doi:10.1109/JBHI.2016.2636665 9. Topol, E. J. (2019). High-performance medicine: the convergence of human and artificial intelligence. Nature Medicine, 25 (1), 44-56. doi:10.1038/s41591-018-0300-7
https://openalex.org/W3007829071	https://link.springer.com/content/pdf/10.1007/s42452-020-2195-4.pdf	English	null	A review on the recycling of waste carbon fibre/glass fibre-reinforced composites: fibre recovery, properties and life-cycle analysis	SN applied sciences/SN Applied Sciences	2,020	cc-by	17,497	Abstract The growing use of carbon and glass fibres has increased awareness about their waste disposal methods. Tonnes of composite waste containing valuable carbon fibres and glass fibres have been cumulating every year from various applications. These composite wastes must be cost-effectively recycled without causing negative environmental impact. This review article presents an overview of the existing methods to recycle the cumulating composite wastes containing carbon fibre and glass fibre, with emphasis on fibre recovery and understanding their retained properties. Carbon and glass fibres are assessed via focused topics, each related to a specific treatment method: mechanical recycling; thermal recycling, including fluidised bed and pyrolysis; chemical recycling and solvolysis using critical conditions. Additionally, a brief analysis of their environmental and economic aspects are discussed, prioritising the methods based on sustainable values. Finally, research gaps are identified to highlight the factors of circular economy and its significant role in closing the life-cycle loop of these valuable fibres into re-manufactured composites. Keywords End-of-life · Carbon fibre · Glass fibre · Recycling technologies · Life-cycle loop · Circular economy Sankar Karuppannan Gopalraj1 · Timo Kärki1 Received: 21 November 2019 / Accepted: 5 February 2020 / Published online: 18 February 2020 © The Author(s) 2020 OPEN A review on the recycling of waste carbon fibre/glass fibre‑reinforced composites: fibre recovery, properties and life‑cycle analysis Sankar Karuppannan Gopalraj1 · Timo Kärki1 * Sankar Karuppannan Gopalraj, sankar.karuppannan@lut.fi \| 1Fibre Composite Laboratory, LUT University, P.O. Box 20, 53850 Lappeenranta, Finland. Review Paper 1 Introduction In recent decades, various studies have assessed mar- ket requirements for new composites and the amount of cumulating wastes to avoid the inevitable negative con- sequences. By 2020, The US market for fibre-reinforced composites (FRC) will reach an estimated value of $12 billion, with an annual growth rate of 6.6% [15]. Similarly, by the same year, the annual global demand for carbon fibres (CF) is expected to increase from 72,000 tonnes to 140,000 tonnes, and the CFRP global revenue expected to increase from $28.2 billion to $48.7 billion [16]. To keep up with such a drastic demand for virgin carbon fibre (vCF), the cumulating CFRP waste should be recycled efficiently to reduce environmental impacts and satisfy the need [17]. Indeed, recycling CFRP into a valuable resource is a challenging issue affecting the future of the fibre-based recycling industry [18]. The materials commonly used in well-established sec- tors—such as aircraft, energy, sports, infrastructure, medical, defence, electronics, and automobile are fibre- reinforced polymer composites (FRPC) [1–3]. Especially carbon fibre reinforced polymers (CFRP) [4] and glass fibre reinforced polymers (GFRP) [5] are incredibly applicable due to their outstanding material properties [6, 7]. So far, industries have been rapidly utilising these materials with- out proper awareness about their disposal methods. For decades, landfill and incineration were the two popular disposal methods adopted by composite industries. These methods have led to increasing environmental awareness to identify a sustainable dispose method and provide a solution to prevent the cumulating wastes [8–14]. kar Karuppannan Gopalraj, sankar.karuppannan@lut.fi \| 1Fibre Composite Laboratory, LUT University, P.O. Box 20, appeenranta, Finland. SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 V l (0123456789) V l ( ) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper The cumulating composite wastes are more promi- nent than the needed new composites. A staggering 62,000 tonnes of unused end-of-life (EoL) and CFRP pro- duction waste will be cumulating every year in spite of the existing demand for new fibre composite [16]. The aircraft [19] and wind energy [20] sectors contribute to a major share of this. In the aircraft sector, an estimated value of 23,360 t/y of EoL CFRP will be accumulated if left unrecycled by 2035. Also, due to developments in mod- ern aviation, the cumulative amounts from the following world regions are estimated to be available for recycling by 2050: North America (162,083 t), Europe (144,724 t) and Asia (102,500 t) [19]. 1 Introduction Similarly, the EoL wind turbines will be cumulating 483,000 tonnes of CFRP waste, consisting of North America (95,000 t), Europe (190,000 t) and Asia (146,000 t) by 2050 [20]. Additionally, by the same year, wind energy will be replacing traditional electricity pro- duction and will account for 50% of the EU’s total electric- ity consumption [21]. However, despite their green creden- tials, wind turbine blades made from GFRP have a lifespan of approximately 20 years. This means that, by 2030, an estimated 100,000 tonnes/year of wind turbine blades will be cumulated. Plus, currently, only a few recycling tech- niques are available to treat such an enormous quantity [21, 22]. After wind turbines, waste printed circuit boards (WPCBs) represents one of the fastest-growing global waste streams [23], contributing a significant share to overall electronic waste and consisting of 27.4–45.55 wt% glass fibres (GF) [24, 25]. Recycling WPCBs to recover GFs is a challenging process due to the presence of toxic heavy metals and organic compounds, along with the GFs them- selves [26]. recycling approaches, have been studied and established so far because method selection depends on the type of material to be recycled and the application in which it will be reused [2]. Also, identifying one standard recycling method among various methods is difficult. In the research field, recycling CFRP and GFRP waste have progressively become an area of interest, as shown when assessing the overall literature studies of the past 20 years—highlighted in Fig. 1. There has been a steady growth in the recycling of both types of fibres. However, after 2011, studies related to CF recycling show an expo- nential growth without any deviation. Indeed, focusing on review articles, in particular, shows that previous studies have concentrated on either CF or GF or their Life-cycle analysis. But not all three factors combined into a single study. For the present scenario, such review articles are needed to compare various phenomena and to identify research gaps. The method adopted in this study is a standard narra- tive review structure proposed by Green et al. [32]. The challenging literature search was conducted primarily in the Scopus database, and three additional databases were used as secondary supporting platforms such as Web of Science, Science Direct and Research Gate. The search was limited to the last 20 years. Vol:.(1234567890) 1 Introduction During the search, keywords such as “carbon fibre”, “glass fibre”, “Kevlar”, “Twaron”, “waste carbon fibre”, “waste glass fibre”, “waste Kevlar”, “waste Twaron”, “recycling”, “recycle”, and “recycled” were used. In the end, relevant articles were carefully selected based on an analysis of the title, aim, and novel findings from the research. Considering the limited information available on the topic of recycling Kevlar and Twaron waste, both the fibres were eliminated from this study. [ ] Current landfilling percentages recorded in the UK stand at 35% for CF and 67% for GF, with only 20% of CF and 13% of GF recycled and a small amount 2% CF and 6% GF—being reused [27]. Furthermore, 2000 t/y of CF waste (20% of the total CF made in the US), if rescued from land- fills and properly recycled, can be worth up to €14.7 mil- lion of recycled carbon fibre (rCF) considering €10/Kg as the rCF market price. This value can increase dramatically to over €50 million by 2020, arguably due to a stable 15% annual increase in vCF production worldwide [28]. To develop new markets, an economically sustainable recy- cling model for CFRP and GFRP waste should be utilised [27, 29, 30], opening up both direct and indirect job oppor- tunities and contributing to economic development [31]. In the current situation, a complete recovery of fibres (direct structural recycling approach) is considered to benefit the composite sector. The recycled fibres from this approach have an added market value because of the low usage of natural resources, energy, and labour-power, together with near-virgin fibre quality [1]. Numerous meth- ods, especially mechanical, thermal and chemical-based However, all the relevant articles under the recycling of CF waste (360 articles) and GF waste (85 articles) were reviewed and organised into a private database using Microsoft Excel. A further article selection was made based on the following criteria: a focus on recy- cling methods; the mechanical properties of recycled 0 20 40 60 80 100 120 1989 1995 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Documents published Year Carbon fibre Glass fibre Fig. 1 Number of articles published based on CF and GF recycling for the past 20 years Fig. 1 Introduction 1 Number of articles published based on CF and GF recycling for the past 20 years Vol:.(1234567890) Vol:.(1234567890) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper Sect. 5. After a brief discussion in Sect. 6, the paper ends with the conclusions in Sect. 7. fibre; and the economic and environmental analysis of the recycling methods. Articles that deviated from the framed selection criteria were eliminated. Overall, this literature review is narrowed solely focusing on summa- rising the significant techniques to recycle CF and GF waste. Based on the effective recycling circumstances to recover clean fibres, on the characteristics of the recov- ered fibres, on the life-cycle analysis of the recycling pro- cess. Finally, 153 articles were used in the reference list. 2 Mechanical recycling In general, mechanical recycling is a technique used to reduce the size of scrap composites into smaller pieces known as recyclates. Normally, slow-speed cutting or crushing mills are used to reduce the material size to 50–100 mm but, when the scrap composites are homo- geneous without any metal components, high-speed mill- ing will be adapted to reduce the size between 50 µm and 10 mm [33]. The recyclates are classified based on coarse recyclates (higher fibre content) and fine recyclates (higher resin content) using cyclones and sieves. The effective reusing of recyclates is based on particle size [33, 34]. In the current situation, this has been used as a pre-recycling process for various methods [35, 36]. Overall, this review article will provide insights into the current status of recycling methods available to recover both the carbon fibre and glass fibre from their composite wastes. The study will also prioritise the avail- able recycling methods based on the following factors— such as environmental impact, commercial value, quality of the recovered fibre and recyclability on an industrial scale. Additionally, the study highlights the process con- ditions associated and the characteristics of the resulting fibres to justify the necessity to replace virgin fibres with recycled fibres. Even though the mechanical recycling process is capa- ble of recycling both CFRP and GFRP, most of the research focuses on GFRP [2, 33, 37]. Discontinuous recyclates and their re-incorporation with low-value applications like fillers or reinforcements can provide the main reasons for such research variation [4]. Besides, CFs are expen- sive compared to GF. Disrupting their physical integrity by mechanical recycling can lead to economic and fibre property loss. Since the early development of the pro- cess, serious drawbacks have been involved, even though studies like Mou et al. [38] showed the improved flexural strength of concrete after the addition of GF recyclates as This paper is organised based on the available recy- cling methods in three sections, as shown in Fig. 2. The traditionally used mechanical recycling techniques and their latest alternative approaches are presented in Sect. 2. Thermal recycling techniques, including the flu- idised bed process and pyrolysis process, are presented in Sect. 3. The chemical recycling techniques and their latest alternative approaches, along with solvolysis, are discussed in Sect. 4. Subsequently, Life-cycle analysis comparing all the significant factors are discussed in Vol.:(0123456789) dopted recycling Fig. 2 The adopted recycling methods Fig. 3 Thermal recycling 3 In a thermal recycling process, heat is used to break down the scrap composite. Due to a higher operating tempera- ture (450–700 °C), the insignificant volatile materials are likely burnt, leaving the valuable fibres behind. Usually, the process temperature depends on the type of resin utilised in the scrap composite. Improper temperature can either leave char on the fibre surface (undercooked) or result in reducing the diameter of the recovered fibres (overcooked) [2, 47]. Thermal recycling can be classified into three types [33], as shown in Fig. 3. To overcome the limitations, recent studies show acceptable improvements in the process; for example, studies like Meira Castro et al. [39], which used com- putational intelligence for optimisation. This involved waste GFRP recyclates being used as aggregate and filler replacement in a concrete-polymer composite to show- case improvements in compressive and flexural strength. Moreover, this optimised process was cost-effective when compared to the thermal and chemical recycling process. Also, Shuaib and Mativenta [40] improved both the yield and quality of the GF recyclate by using low energy con- sumption. Their study found that reducing the screen size to obtain a fine recyclate will result in increased energy consumption and processing time. To overcome such energy loss, the clearance gap between blades and screen was decreased. In comparison, further increasing the yield without any residue, Kočevar and Kržan [41] separated 70% of the GF using a normal hammer mill. The remaining 30% of waste was used as filler material for thermoplastics. To overcome the limitations, recent studies show acceptable improvements in the process; for example, studies like Meira Castro et al. [39], which used com- putational intelligence for optimisation. This involved waste GFRP recyclates being used as aggregate and filler replacement in a concrete-polymer composite to show- case improvements in compressive and flexural strength. Moreover, this optimised process was cost-effective when compared to the thermal and chemical recycling process. Also, Shuaib and Mativenta [40] improved both the yield and quality of the GF recyclate by using low energy con- sumption. Their study found that reducing the screen size to obtain a fine recyclate will result in increased energy consumption and processing time. To overcome such energy loss, the clearance gap between blades and screen was decreased. In comparison, further increasing the yield without any residue, Kočevar and Kržan [41] separated 70% of the GF using a normal hammer mill. 3 Thermal recycling The remaining 30% of waste was used as filler material for thermoplastics. As mentioned previously, the limitations of recycling CFRP waste were challenging and even visible in the latest study by Li and Englund [42], where aerospace industry scrap was size-reduced using a hammer mill followed by shredding. The recyclates are compression moulded into flat pallets and subjected to mechanical testing, which showed a minimum 50–60% decrease in mechanical prop- erties compared to the original composite. However, the study pointed out that, as the CF recyclate particle size decreases, the mechanical property increases. The basic principle for decomposing the scrap compos- ite using heat remains the same, though, the results are different for each process. Since polymeric compounds have certain calorific values, electricity can be produced by converting the waste composite into heat [1, 2]. How- ever, a major drawback in the combustion (incineration) process is the ash by-product, which can only be landfilled as inert waste, e.g. 92 €/tonne in France. This complication harms the progress of a circular economy. Besides, it is only possible to achieve a 35% efficiency rate when convert- ing the heat to electricity. Overall, coal in the furnace is a much better option than incinerating CFRP. Recent studies have focused on complete fibre recovery using thermal recycling processes like fluidised-bed process (FBP) and pyrolysis [48]. As mentioned previously, the limitations of recycling CFRP waste were challenging and even visible in the latest study by Li and Englund [42], where aerospace industry scrap was size-reduced using a hammer mill followed by shredding. The recyclates are compression moulded into flat pallets and subjected to mechanical testing, which showed a minimum 50–60% decrease in mechanical prop- erties compared to the original composite. However, the study pointed out that, as the CF recyclate particle size decreases, the mechanical property increases. However, instead of completely combusting CFRP, con- trolled resin decomposition at optimum temperature can result in CF recovery with negligible surface damage. In a study by Matielli Rodrigues et al. [47], the versatile com- pounds (resins) were thermally disintegrated at 450 °C for 2 h and CF recycled without damaging much of the sur- face integrity. The decomposed epoxy resins are derived from the diglycidyl ether of bisphenol-A (DGEBA), which is difficult to recycle due to its cross-linked structure from resin curing. Therefore, recycling with minor surface dam- age is a better option than landfilling completely. 2 Mechanical recycling 2 The adopted recycling methods Vol.:(0123456789) Vol.:(0123456789) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper filler materials. Studies like Pickering [33], however, noted that the GF recyclates used as fillers are not commercially feasible due to the availability of alternative cheap virgin fillers such as calcium carbonate or silica. 2.1 Alternative approaches Recent studies have focused on alternative size-reduction approaches using high voltages. One such method was electrodynamical fragmentation (EDF) in which a high voltage pulse between 50 and 200 kV was passed into ionised water to break down the CFRP waste into smaller pieces [43, 44]. Similarly, high voltage fragmentation (HVF) was carried out using a high voltage pulse of 160 kV to breakdown the GFRP waste. This method produces clean and long fibres. Moreover, HVF can be a promising alter- native to mechanical recycling [45]. However, recently, Oshima et al. [46] pointed out two drawbacks in this approach, using high voltage to remove resin from CFRP waste will result in a severe weight loss in the actual com- posite and also decreases the rate of resin removal. Resin decomposing using optimal thermal conditions is not as efficient for GF as CF, but post-chemical treatments of recycled glass fibre (rGF) help to a certain extent in regaining their properties [49]. Unlike CF, the thermal recy- cling of GF in high-heat operating conditions (300–600 °C) Thermal recycling Combustion/ incineration (energy recovery + ash) Fluidised bed process (clean fibres/fillers + energy recovery) Pyrolysis (fibres/fillers + chemical products) Fig. 3 Classification of thermal recycling Fig. 3 Classification of thermal recycling Vol:.(1234567890) Vol:.(1234567890) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper The limitations are noticeable in Pickering et al. [53], as their research investigated the ability of FBP to recycle GF and was only able to achieve a 67% fibre yield. Fur- thermore, the process retained only 50% of the tensile strength of the rGF compared to virgin GF (vGF). How- ever, the study included a cost analysis for commercial- ising the process and estimated that for 9000 tonnes/ year, the net annual profit would be 0.002$ million/year. Besides the limitations, the author’s contribution to the process has laid the foundations for modern-day FBP [4]. In contrast with Pickering et al. [53] results, Zheng et al. [55] claimed to have a novel FBP approach to recy- cle WPCBs, achieving a 94.8 wt% GF recovery rate and a 95.4 wt% purity rate. The recycling approach seems to be almost similar, except for the WPCB were finely chopped than regular chopping size (25 mm) before feeding. However, the results were validated based on weight loss and SEM micrographic image observations. Plus, no information regarding the strength of the rGF’s was mentioned. 3.1 Fluidised bed process In a typical fluidised bed process (FBP), a rapid stream of hot air is passed through a bed of silica sand to decom- pose the chopped scrap composite at a low temperature. Usually, a fine silica sand of 0.85 mm particle size is used as a bed, which is then converted into a fluidised bed by passing air in the velocity range 0.4–1.0 m/s. The polymeric matrix scrap composites are chopped to 25 mm and fed separately into the fluidised bed. The operating tempera- ture is between 450 and 550 °C. Inside a fluidised bed, the scrap composite separates into fibres and fillers (volatile compounds), which are carried out by the air stream as individual particles [33, 53]. A similar study to Pickering et al. [53] but replacing GF with CF was carried out by Yip et al. [57]. The findings showed that recycling CF by using identical operating conditions was much more efficient compared to GF recycling. The rCF had fully retained Young’s modulus and 75% of tensile strength when compared to vCF. The study also mentioned that the rCF length depends on the initial length of the CF waste, and it was possible to recover a fibre length of 5.9–9.5 mm despite the non- uniform orientation. In alignment with previous studies, Pickering et al. [54] systematised the idea of commercial- scale FBP with its ability to recover CF and also designed a commercial scale FBP suitable for recycling mixed and contaminated CFRP waste. The rCF only had an 18.2% tensile strength decrease and no tensile modulus reduc- tion. The study claimed that the design has the highest efficiency compared with any other rCF using FBP. Also, the energy consumption to recover CF via FBP is only 5–10% of the total energy needed to manufacture vCF. Furthermore, suspending the individual particles in a high-temperature (1000 °C) secondary chamber will result in oxidising the volatile compounds, leaving the fibres alone [33, 53]. Figure 4 represents a diagram of the FBP. The process is capable of recovering both CF and GF [33] and is especially favourable for recycling EoL waste composites [2]. However, during the initial development of the process, limitations like fibre strength and length degradation became apparent. Also, the recovered fibres are fluffy [4]. Vol.:(0123456789) Fig. 4 Fluidised bed process (modified from Pickering et al. 2.1 Alternative approaches To improve the yield of rGF, Pender and Yang [56] used CuO as a catalyst, with the results improv- ing the yield from 59 to 70%. reduces the strength of the resulting GF up to 80% and is difficult to further reuse because of its low reinforce- ment potential [50, 51]. To overcome such phenomena, Yang et al. [50] investigated two chemical treatments: chemical etching and post-silanisation to treat decom- posed GF (80% decreased tensile strength) at 500 °C for 30 min. The post-chemical treatment retained 30–70% of the lost mechanical properties in rGF. Thomason et al. [49] recovered 75% of the strength-loss by immersing the GF in 3 M NaOH solution for 10 min at 90 °C, followed by neu- tralising with HLC. The treatment is known as short-hot sodium hydroxide solution treatment. Pender and Yang [52] introduced catalysts: CuO, CeO2, and Co3O4 to boost the resin decomposition. As a result, the processing time was reduced by 20 min, along with a 40% reduction in energy consumption. Among the three catalyses, CuO at 375 °C had maximum efficiency in removing the resins, while CuO and CeO2 increase 20% of GF’s strength reten- tion capacity. 3.2.1 Pyrolysis: controlled atmospheres The development of pyrolysis processes resulted in sophis- ticated and controlled atmospheric conditions being added to separate fibres from the solid pyrolysis prod- ucts. Various literature works published since 2010 include evidence of such an approach. The primary principle of pyrolysis is unaffected. However, the atmospheric condi- tions inside the pyrolytic reactor have changed constantly, aiming for higher yields. Three commonly-used atmos- pheric conditions during pyrolysis are vacuum, nitrogen and superheated steam. l Like any other recycling process, pyrolysis also suffers from certain limitations, with the possibility of char for- mation on the resulting fibre surface considered the most challenging of all [4]. A significant percentage decrease in the mechanical properties can be observed in the recov- ered fibres due to the char. Methods such as chemical treatment [26, 58] and post-heating the fibres result in reducing the char formation, but only to a certain extent [1]. Recent studies have used carbon dioxide (CO2) and water vapours to remove the char formation from CFRP [59]. Also, oxidising the fibre surface will result in the for- mation of an oxygen-rich surface, improving the adhesive nature of the fibre with resins [60]. 3.2.1.1 Vacuum atmosphere Inside a vacuum pyrolytic reactor, the organic vapours’ residence time is shorter due to a low decomposition temperature, which is sufficient enough to recycle GF without disturbing surface integrity [63, 64]. In an early study focused on recycling WPCBs by Zhou and Qiu [63], vacuum pyrolysis (VP) was judged as an alternative approach to mechanical recycling. The liq- uid and gas products (can be used as chemical feedstock) along with solid products (GF and metal components) were found to increase the interests of researchers when compared to mechanical recycling [24, 63, 64]. Their study consists of a two-step pyrolysis method, such as VP and vacuum centrifugal separation (VCS), to separate GF from WPCBs. Similarly, Long et al. [24] used VP, which involved the GF being separated from metal parts using gravity separation (GS) method and non-metal parts using a cal- cination process. The studies focused on recycling WPCBs using a vacuum atmosphere did not include any inves- tigation on the mechanical properties of rGF. However, the yields from the process are tabulated in Table 1. This process is limited to GF as no suitable study was found concerning CF. 3.1 Fluidised bed process [53, 54]) Scrap feed Fluidised bed Separation process (cyclone/ sieve) Volatile polymeric compounds Regenerated hot air stream Heat recovery system Secondary Combustion chamber Recycled fibres Air compressor Fresh air Air exhaust Fig. 4 Fluidised bed process (modified from Pickering et al. [53, 54]) Regenerated hot air stream Secondary Combustion chamber Separation process (cyclone/ sieve) Vol.:(0123456789) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper 3.2 Pyrolysis process continuous flow allow them to recover longer and cleaner CFs [4]. Evidence for such a phenomenon can be seen in an earlier study from Meyer et al. [62] on recycling aircraft manufacturing CFRP waste, in which low-efficiency lab- scale pyrolysis was optimised to a semi-industrial scale with the help of the company ReFiber. The authors were able to synthesise semi-industrial plant operations using a larger oven, with rCF retaining 96% of its original ten- sile strength. However, a secondary heating system was implemented to eliminate the residue char, though the rCF was effective enough to replace vCF. Overall, a con- trolled atmosphere in the pyrolytic reactor can influence the process outcomes. The process is efficacious for recycling both CF and GF [15]. Unlike other thermal recycling processes, the scrap composites are heated in the absence of oxygen. Under this condition, the decomposing matrix produces oil and gas, along with fibres and fillers (solid products) [2, 33]. In a typical polymeric pyrolysis process, the operating temperature varies in the range of 400–700 °C depend- ing on the nature of the scrap composite [1, 4]. The liquid produced from the process contains aromatic compounds and has a calorific value of 37 MJ/Kg, similar to fuel. The gas produced can be regenerated into the pyrolysis reac- tor. Overall, the oil and gas products produced can prove a significant chemical feedstock for any other process. Nevertheless, in spite of all that is produced, the process is still capable of retaining fibres with higher mechanical properties [1, 4]. This clarity of effect makes pyrolysis the most studied thermal recycling process [2], Fig. 5 presents a flowchart of the process. 3.2.1 Pyrolysis: controlled atmospheres The study stated that a reaction time increase could cause a decrease in the mechanical properties of the recycled CF. Also, insufficient reaction time could result in char formation on the fibres. The yield from the process is tabulated in Table 1. Using pyrolysis under superheated steam, Shi et al. [67] investigated the mechanical properties of rCF. They found that the recycled fibres had a substantial amount of char on the rCF surface, which prevented the fibres from being completely reusable. The study also stated that the lower the pyrolysis temperature, the higher the value of recycled CF. Furthering their previous studies, Shi et al. [58] extended their research to CFRP and GFRP, eliminating the char formation on both recycled CF and GF using chemicals such as detergent, acetone and N-methyl-2-pyrrolidinone (NMP). As a result, the bending strength of rCF increased from 49 to 78% after treatment with NMP, and the bending strength of rGF increased from 26 to 94% after treatment with acetone. Recently, Jeong et al. [69] recycled CF and showed 66% of tensile strength and 100% of tensile modulus retained from an original value using rapid pyrolysis. In line with previous research, in 2016, Onwudili et al. [61] investigated the mechanical properties of both the recycled CFRP and GFRP waste using a nitrogen atmosphere in a semi-batch reactor. The study found that chemical modification on the surface of recycled CF and GF could improve fibre properties. Additionally, both studies [61, 65] used secondary combustion (SC) to oxidise minor resin impurities and char formation on the pyrolysed fibres. When comparing the results, oxidised fibres retained the most mechanical properties when compared to non-oxidised fibres. In an attempt to retain maximum tensile strength in the recycled fibres, Ye et al. [70] developed an optimised steam thermolysis process combining vacuum pyrolysis and mild gasification to recycle waste CFRP. The process retained 90% of tensile strength in both laboratory and semi-industry scales. The study stated that a degradation increase in the polymer matrix resulted in a decrease of the fibres’ tensile strength. However, Kim et al. [68] retained 90.42% of tensile strength compared to the vCF by using a fixed bed reactor at 550 °C for 60 min. The study mentioned that increasing the steam pyroly- sis conditions resulted in improving the removal of char deposited on the fibre surface. In a recent study by Kim et al. 3.2.1 Pyrolysis: controlled atmospheres To completely avoid such char formation on the recovered CFs, a UK milled carbon group was success- ful in designing and implementing a commercial-scale semi-open continuous belt furnace with a controlled atmosphere [4, 61]. Similarly, Germany’s CFK Valley Stade Recycling GmbH and Co. KG uses a continuous pyrolysis process. Both companies are capable of recycling vari- ous types of CFRP waste. Moreover, the large furnace and Scrap feed Pyrolysis reactor Fibres, fillers and char (solid products) Condenser Chemical feedstock Regenerated combustible gas to the reactor Hot exhaust gas Secondary combustion Energy recovery Solid and liquid hydro carbon products Clean fibres Fig. 5 Pyrolysis process (modified from Pickering et al. [33]) Fibres, fillers and char (solid products) 3.2.1.2 Nitrogen atmosphere Unlike vacuum pyrolysis, pyrolysis under a nitrogen gas atmosphere is capable of recycling both CFRP and GFRP [61]. Pyrolysis under Fig. 5 Pyrolysis process (modified from Pickering et al. [33]) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper Table 1 Pyrolysis yields (wt%) from recycled CFRP and GFRP References Material Solid yield Liquid yield Gas yield Process parameters Zhou and Qiu [63] GF 75.7 20 4.3 a. VP: 600 °C for 30 min b. VCS: 400 °C, 1200 rpm for 6–10 min Zhou et al. [64] GF 72.2 21.45 6.35 Long et al. [24] GF 74.7 15 10.3 a. VP: 550 °C for 120 min b. GS + calcination: 600 °C for 10 min Onwudili et al. [65] CF 77 20.6 2.4 NA: 400–500 °C < 30 min + SC: 450 °C for 2 h. (35 MJ m−3 calorific value of gas) Onwudili et al. [61] CF 73.1 23.8 2.3 NA: 500 °C for 45 min with 5 dm3/h nitrogen + SC: 500 °C for 30 min at 20 °C min−1 GF 65.9 25.7 8.4 amount of the char formation on the resulting CF and GF can be reduced using chemical treatments [58]. nitrogen atmosphere (NA) was the popular pyrolysis process used to recycle WPCBs until Zhou et al. [64] ’s study came in 2010. The study used vacuum pyrolysis to recycle WPCBs, achieving a maximum GF recovery. However, later analysing the advantages of pyrolysis under NA, in 2013, Onwudili et al. [65] further developed the process and successfully recycled CFRP waste—in which 98 wt% of the solid products (rCF) were recovered efficiently. 3.2.3 Pyrolysis on wind turbine application Wind energy is a widely-used renewable energy source [74], for which GFs make an important material contri- bution to wind turbine manufacturing [3, 5, 75]. In gen- eral, the life cycle of an individual wind turbine only lasts from 20 to 25 years [76]. Studies focusing on GF recycling from wind turbine applications have frequently preferred pyrolysis [77]. Apart from the basic GF recycling, studies have focused on retaining their mechanical properties to complete the life-cycle loop by reusing them in various other applications [5]. In modern chemical recycling, resin degradation is either achieved using solvents (solvolysis) or water (hydrol- ysis). In solvolysis, solvents in different conditions (reaction time and concentration) are used to depolymerise or break the polymeric part of a composite. In hydrolysis, resin deg- radation takes place because of water [1]. Generally, the use of hazardous and concentrated chemicals results in environmental impact [4], so the harmful chemicals are replaced with water and alcohol at supercritical conditions (SC). Besides, the drawback of improper fibre alignment in discontinuous rCF with a length of more than 5 mm can be suppressed using a centrifugal alignment rig concept [80] or by using calendaring through rollers with 0.10–0.15 mm gap at 110 °C [81]. Looking at early studies related to wind turbine recy- cling after 2010, studies like Åkesson et al. [76, 77] were successful in retaining 75% of rGF tensile strength by using microwave pyrolysis in a nitrogen atmosphere. Their study proposed that a loss in tensile strength can be over- come by reusing it as a hybrid composite, combining rGF with vGF. In line with the previous study, Åkesson et al. [78] further investigated improving the rGF properties by reinforcing them with polypropylene (PP). The study stated that the use of maleic anhydride grafted PP along with coupling agents improved the mechanical proper- ties, including the flexural strength, tensile strength and Young’s modulus. 4 Chemical recycling In a chemical recycling process, the polymeric matrix pre- sent in the waste composite is disintegrated by dissolv- ing it into any chemical solution, such as acids, bases and solvents. Normally, suitable chemicals and solvents are chosen based on the nature of the polymer substrate [4, 79], while the solid composites are mechanically grounded before chemical recycling to increase the surface area. Once the polymer matrix is dissolved, the recycled fibres are washed to remove minor surface residue [1, 79]. Fibres recovered using chemical recycling have retained long fibres with maximum mechanical properties. Plus, the process has a higher resin decomposition ratio [4]. 3.2.2 Microwave pyrolysis longer only be renewable in terms of producing energy but also in material reuse. A collaboration between the material supplier, the wind industry and the EoL sector can make this happen easier. A recent study has estimated that, in the future, chemical recycling will be capable of recycling GF, hybrid and CF wind turbine blades with higher efficiency [9]. In microwave-assisted pyrolysis, the conventional heat- ing source was replaced with microwave radiation. This change has increased the rate of thermal transfer with minimal energy consumption, without disturbing the primary principle of pyrolysis. The process is capable of recycling both CFRP and GFRP with fibres retaining higher mechanical properties [2]. In a recent study, Obunai et al. [72] achieved a 100% resin elimination ratio using a 700 W, 2.45 GHz, argon atmosphere, and a 2.5 L/min flow rate after 300 s. The rCF had only a 0.7% decrease in tensile strength compared to vCF. Similarly, Jiang et al. [73] recy- cled CFRP at 500 °C for 30 min with a 0.70 m3/min nitrogen flow. The rCF possess a clear fibre surface with mechanical properties like virgin fibres. 3.2.1 Pyrolysis: controlled atmospheres [71] superheated steam was used to remove minor resin residues after pyrolysis with carbon dioxide (CO2) and recovered above 80% of the strength compared to vCF. 3.2.1.3 Superheated steam atmosphere In general, superheated steam is produced as a result of heating saturated steam at constant pressure. During the transi- tion phase, the steam is heated several times above its saturation point to achieve superheated levels. The use of superheated steam atmosphere in pyrolysis increases heat transfer, which results in heightening the thermal degradation and supporting an oxygen-free atmos- phere inside the pyrolytic reactor [66]. Recycling CFRP using superheated steam results in a high retaining of the CF mechanical properties when compared to any other pyrolysis atmospheres [67, 68]. Also, a significant Vol.:(0123456789) Vol.:(0123456789) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper 4.1 Classification of chemical recycling Classifying the chemical recycling process on the basis of previous studies depends on structuring significant fac- tors according to its priorities. In early literature, Morin et al. [82] classified chemical recycling (solvolysis) based on temperature; solvolysis at low temperature using sol- vents like acid solutions, alcohols, etc., at 90 °C and solvoly- sis in SC of solvents like acids and water. However, recent research by Oliveux et al. [2] classified solvolysis based on higher temperature and pressure (HTP) with tempera- ture > 200 °C and lower temperature and pressure (LTP) with temperature < 200 °C. In this study, chemical recycling is organised based on studies of nitric acid, together with Studies like Pico et al. [75] and Beauson et al. [74] have highlighted the limitations and mentioned that rGF from wind turbine blades are short, fluffy and randomly ori- ented. Also, the rGF tensile strength is lower when com- pared to vGF. However, minor organic contents of the rGF surface are removed by oxidising. As a counter-statement, Jensen and Skelton [3] highlighted the circular economy as a promising way to reuse the recovered fibres in various applications. The study mentioned that, as an outcome of the GENVIND project (a 4-year project carried out by a Danish innovation consortium), a wind turbine will no Vol:.(1234567890) Vol:.(1234567890) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper other chemicals in mild conditions (T < 100 °C), and others at higher conditions (T > 100 °C). a self-accelerating oxidative decomposition system (ace- tone + peroxide hydrogen [H2O2]). The authors mentioned that acetic acid is used to pre-treat the waste composite for expansion, producing a larger surface area. The rCF retained more than 90% of tensile strength compared to the original composite and showed that recycling under mild conditions is effective. The detailed process condi- tions are mentioned in Table 2. 4.1.1 Recycling using nitric acid Nitric acid is suitable for recycling both CFRP and GFRP. In the early-stage recycling of CFRP using acids, Liu et al. [83] found that nitric acid performed better in decomposing thermoset epoxy resin and recycled cleaner CFs compared to both sulfuric and hydrochloric acid. Also, orthogonal experimentation was used to prove the reusability of recycled CFs, while even stronger resins like amine cured epoxy were decomposed using nitric acid—for which Ma et al. [84] achieved a 99.18% resin decomposition ratio. The study resulted in an 85% CF cleanliness rate with minimal damage to the fibre surface. Similarly, Lee et al. [85, 86] recycled CFs with only a 2.91% tensile strength decrease compared to vCF. The detailed process conditions are mentioned in Table 2.fi Similarly, Xu et al. [90] achieved a 90% epoxy decompo- sition rate using N,N-dimethylformamide (DMF) and H2O2 mixed solution. The rCF only shows a 5% tensile strength decrease compared to the original composite. The single fibre tensile test only showed a 2% tensile strength loss compared to the vCF after a 10 °C process temperature increase. In a recent study, Das et al. [91] showed a 97% resin decomposition ratio from a single-stage oxidation process using an aqueous mixture (AM) of peracetic acids. The rCF retained 94% of its original strength. This process is limited to CF, and no proper study was found concerning GF. However, recent studies used solvents such as polyeth- ylene glycol [92], dimethylformamide (DMF) [93] and water (1% NaOH) [94] to recycle WPCBs under mild conditions. GFRP decomposition was also more efficient with nitric acid when compared to concentrated sulfuric acid [87]. In an early study, Yuyan et al. [87] achieved a 99% rate of resin decomposition. The rGF only has a 3.5–15.1% ten- sile strength decrease and a 2.5% decrease in interlami- nar shear strength (ILSS) compared to the original fibres. The study stated that an increase in temperature and acid concentration would increase the decomposition rate. Another study by Dang et al. [88] investigated the corro- sion resistance of T-GF and E-GF and stated that T-GF has higher corrosion resistance in nitric acid solution com- pared to E-GF. 4.1.3 Recycling using higher conditions Studies carried out since 2000 have adopted higher process parameters for recycling waste composites with higher resin decomposition, with chemical recy- cling using super and sub-critical conditions (SCC) playing an essential role in this [2]. However, recycling using super and sub-critical fluids, especially water and alcohol, have been significantly narrowed down (summarised in a separate section). Based on the litera- ture, studies since 2010 have focused more on improv- ing the mechanical properties of recycled fibre. The process parameters, conditions and outcomes of the research are listed in Table 3, with the outcome being Table 2 Process conditions 4.1.2 Recycling using mild conditions The chemical recycling of scrap composite using mild con- ditions increases resin degradation [2]. Studies related to a lower temperature (T < 100 °C) and mild acids have also achieved relatively higher results. In Li et al. [89] study, a 90 wt% epoxy decomposition ratio was achieved using Vol.:(0123456789) Table 2 Process conditions References Year Process conditions Liu et al. [83] 2004 70–90 °C Yuyan et al. [87] 2006 90 °C, 8 M nitric acid, 2 g/100 mL feedstock ratio and 5 h Ma et al. [84] 2009 95 °C, 8 mol/L nitric acid and 23 h Le et al. [85] 2010 90 °C, 6 g:100 mL (weight CFRP: nitric acid) for 5 h Lee et al. [86] 2011 90 °C, a hexahedral circulating system at a 1 cm/s linear flow rate, 100 g:1.8 L (waste composite: 12 M aqueous nitric acid ratio) Li et al. [89] 2012 60 °C for 30 min Xu et al. [90] 2013 90 °C for 30 min, hermetic reactor Das et al. [91] 2018 65 °C for 4 h. AM: 95 vol% 14 M acetic acid and 5 vol% 9 M H2O2 Vol.:(0123456789) References Year Process conditions Liu et al. [83] 2004 70–90 °C Yuyan et al. [87] 2006 90 °C, 8 M nitric acid, 2 g/100 mL feedstock ratio and 5 h Ma et al. [84] 2009 95 °C, 8 mol/L nitric acid and 23 h Le et al. [85] 2010 90 °C, 6 g:100 mL (weight CFRP: nitric acid) for 5 h Lee et al. [86] 2011 90 °C, a hexahedral circulating system at a 1 cm/s linear flow rate, 100 g:1.8 L (waste composite: 12 M aqueous nitric acid ratio) Li et al. [89] 2012 60 °C for 30 min Xu et al. [90] 2013 90 °C for 30 min, hermetic reactor Das et al. [91] 2018 65 °C for 4 h. AM: 95 vol% 14 M acetic acid and 5 vol% 9 M H2O2 SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper Table 3 Recycling using high conditions after 2010 References Year Recycled Solvents Parameter Outcome Lee et al. [85] 2010 CF Organic: tetralin (T) and dieth- ylene glycol monomethyl ether (DGME) 350 °C, 2 Mpa for 2 h DGME, 193 °C, atm pressure (AP) for 10 h 3.67% decrease in tensile strength compared to vCF Yang et al. 4.2 Alternative approaches As with every other process, novel chemical recycling approaches have been studied. By unaltering the over- all principle involved in the process, studies have made minor changes for better results. As an early example, Kamimura et al. [108] used microwave-enhanced depol- ymerisation and recycled 51 wt% of pure GF from FRP waste. In their study, N-Methyl-N-propylpiperidinium bis(trifluoromethylsulfonyl)imide was used as an ionic liquid at 340 °C for 2 min to achieve a maximum chemical yield of 80%. The author mentioned that using microwaves resulted in a high fibre yield in a short process time. Besides the focus on property improvement, studies have also focused on comparing the recycling mecha- nisms involved in the chemical recycling process. For example, Ma et al. [103] compared the depolymerisation and also the acid digestion of amine-cured epoxy resin, finding that the resin dissolution rate was influenced not only by the chemical reaction rate but also by the diffusion rate. Working from their previous research, Ma and Nutt [104] were able to implement their findings on both CF and GF to disintegrate amine-cured epoxy resin. The study showed that acid digestion with acetic acid/H2O2 at 110 °C was effective when compared to depolymerisation with benzyl alcohol/K3PO4 at 200 °C for highly crosslinked amine cured epoxy resin. Also, in the case of acid digestion, minor degradation was found on the rGF surface. Plus, exposing GFRP waste above the elevated temperature will result in rGF strength loss [105]. Among the recent studies, Sun et al. [109] investigated electrochemical recycling (ECR) using waste CFRP as an anode and a stainless–steel plate (SSP) as a cathode, with a NaCl solution as the electrolyte. A voltage of 2.6 V was passed across the electrodes for 21 days to soften the CF with a 3% concentration of NaCl. After softening, the fibres are washed under ultrasonic and dried for three days in a 50 °C environment. The authors highlighted that the energy consumption for recycling CF using the electrochemical process is 2–10 kWh/Kg, and the energy consumption to manufacture vCF is 55–165 kWh/Kg. Similarly, using ECR, Zhu et al. [110] and Chen et al. [111] both retained mechanical properties similar to vCF; this method is inexpensive and suitable for large-scale appli- cation [111]. Recently, a mechanochemical process (MCP) similar to ECR has been tested [112], but limited informa- tion is available regarding the rCF quality. 4.1.2 Recycling using mild conditions [95] 2014 CF/GF Polyethylene glycol/NaOH 200 °C for 4 h with 0.1 g NaOH/g 84.1–93% decomposition efficiency CF and GF 4–6% decrease in original tensile strength Yamaguchi et al. [96] 2015 CF Hydrochloric acid (HCL) 130–150 °C, 2 h rCF like vCF Nie et al. [97] 2015 CF Molten KOH 285–330 °C, AP Retained 95% tensile strength Wang et al. [98] 2015 GF Aluminium chloride/acetic AlCl3/CH3COOH 180 °C for 9 h Retained 96% of tensile strength Wang et al. [99] 2015 CF Aluminium chloride/acetic AlCl3/CH3COOH 180 °C, 15 wt % solvent for 6 h Retained 97.77% of tensile strength Liu et al. [100] 2017 CF ZnCl2/ethanol catalyst system 190 °C Retained high mechanical property Oliveux et al. [101] 2017 CF Water and acetone in 20:80 vol ratio 320 °C, 180 ± 10 bars in a 5 L batch reactor rCF like vCF Wu et al. [102] 2019 CF Molten ZnCl2 360 °C, 80 min Retained 95% tensile strength the retaining of maximum mechanical properties with maximum resin decomposition efficiency. 4.3 Solvolysis using critical conditions A fluid (solvent) adopts a high ability to diffuse a soluble substance when its critical temperature and pressure has been reached. It also performs new chemical reactions for decomposition and partial oxidation [115]. Solvoly- sis using supercritical fluids (SCF) is an emerging waste composite recycling technology, with the recycling pro- cess applying to both CFRP and GFRP. The two commonly used fluids in their subcritical and supercritical conditions are water and alcohol [82]. Solvolysis using alcohols is focused on recycling waste CFRP to dissolve the resin and to recover CF. However, there are fewer studies involving GFRP waste recycling. In line with their previous research, Okajima et al. [119] studied the effect of eight supercritical solvents—metha- nol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert- butanol, acetone, and methyl ethyl ketone—on dissolving the resin (amine-cured thermosetting epoxy) and recycle CF. Among the eight supercritical solvents, acetone had a higher resin elimination. Moreover, rGF using super- critical acetone obtained 95–99% resin degradation and retained 89% of its strength compared to vGF [120]. The resin decomposition efficiency improved with increasing It is easy to achieve a supercritical state with alcohols as opposed to water [116]. Supercritical alcohols possess good recycling capabilities when used with waste polymer composites. Among all the supercritical alcohols, propanol is better than ethanol and methanol. When comparing Table 4 Alcohols at critical conditions (CC) References Year Solvent Conditions Piñero-Hernanz et al. [117] 2008 Methanol, ethanol, 1-propanol, and acetone 250–400 °C, solvent flow rate of 1.1–2.5 kg-alcohol/kg-fibre/min and 0.016– 0.50 M alkali catalyst (NaOH, KOH, and CsOH), 15 min Okajima et al. [118] 2014 Methanol Thermosetting: 270 °C in a batch reactor 8 Mpa for 90 min Thermoplastic: 285 °C in a semi-flow type reactor 8 Mpa for 80 min Okajima et al. [119] 2017 Acetone 320 °C with molar density 3.64 mol/L after 20 min Sokoli et al. [120] 2017 Acetone 260–280 °C, 55–60 bar, 1.24–2.1 g/mL of composite/solvent ratio Okajima and Sako [121] 2019 Acetone 350 °C, 14 Mpa, 60 min, 4.35 mol/L density of acetone Jiang et al. [115] 2007 Propanol 300 °C, 50 bars and 10 min Marsh [116] 2009 Propanol Semi-continuous flow system at 350 °C, KOH catalyst flow of 1.1 kg of alcohol per 1 kg of fibre/min Jiang et al. [123] 2009 n-Propanol Semi-continuous flow reactor at 310 °C, 5.2 Mpa Yan et al. 4.3.1 Alcohol at critical condition In an early study of CF recycling, Piñero-Hernanz, García- Serna et al. [117] investigated the resin elimination per- centage of four alcohols: methanol, ethanol, 1-propanol, and acetone. The study resulted in 95% resin degrada- tion under 15 min, with the rCF retaining 85–99% of its tensile strength. Among the four alcohols used, acetone performed better at a lower temperature. At higher tem- perature (450 °C), ethanol, 1-propanol and acetone had a maximum resin elimination of 78.8 wt%, and methanol had only 60.2 wt%. However, a later study conducted by Okajima et al. [118] showed that supercritical methanol performed much better when compared to previous stud- ies. The recycled CF using methanol had a crack-free sur- face with only a 9% decrease in tensile strength and a 20% decrease in interfacial strength when compared to vCF. The solvent and process conditions involved in recycling CF are tabulated in Table 4. 4.2 Alternative approaches From an economic point of view, WPCBs can be recy- cled cost-effectively using higher conditions (260 °C for 10 min). Moreover, the ionic liquid ethyl-3-methylimi- zadolium tetrafluoroborate [EMIM+][BF4 −] used to dis- solve the WPCB resin is capable of multiple regenera- tions and reuse [106, 107]. Recently, cheaper solvents such as water and 1 M aqueous solution of sodium hydroxide (NaOH) have been used to separate WPCBs at higher conditions (280 °C for 15 min) [23]. Vol:.(1234567890) Vol:.(1234567890) Vol:.(1234567890) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper Recent approaches also involve ultrasonics, Das and Varughese [113] investigated a sonochemical recycling process by reacting the CFRP waste with dilute nitric acid and H2O2 under ultrasonics at 60 °C, achieving a 95% decomposition ratio. The use of ultrasonic increased the decomposition ratio thrice when compared to the same process without ultrasonics. Similarly, Jiang et al. [114] used ultrasonics so that the waste CFRP could be pre-treatment with a nitric acid solution. The pre-treated CFRP was subjected to Macrogel 400 and a potassium hydroxide (KOH) system at 160 °C for 200 min. The resins are decomposed at a 95 wt% resin removal rate, and the rCF retained a 95% tensile strength compared to vCF. The authors highlighted that the process is highly efficient for recycling thermosetting composite materials. methanol, ethanol, acetone, and 1-propanol, research showed that methanol has a low mass-transfer rate under subcritical conditions. On the other hand, 1-propanols three atoms of carbon and high solvation capacity per- forms better than methanol and ethanol [115, 117]. 4.3 Solvolysis using critical conditions [124] 2014 1-Propanol 320 °C, feedstock ratio 2 g epoxy resin for 0.2 L 1-propanol, 60 min Yan et al. [125] 2016 1-Propanol 320 °C, 90 min, no catalysist Vol.:(0123456789) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper reaction pressure and acetone density [121]. Similar to Okajima et al. [119], Cheng et al. [122] analysed the degra- dation capability of different supercritical fluids. The study showed that supercritical n-butanol had the highest resin degradation and retained 98% of tensile strength com- pared to the original CF. Supercritical acetone holds the second position for resin degradation followed by ethanol and n-propanol. However, at a temperature range between 340 and 360 °C, n-propanol showed a greater capability for resin degradation compared to ethanol. Overall, both supercritical methanol and isopropanol performed poorly in resin degradation. However, in studies focused on maximum resin decom- position efficiency, Yuyan et al. [129] achieved the highest resin decomposition rate of 100 wt% by reducing water from a supercritical state to its subcritical state. The rCF only show a 1.8% tensile strength decrease compared to vCF. Similarly, Kim et al. [130] were able to achieve a 99.5% resin removal efficiency. Contradicting the studies with higher resin decompo- sition ratio, Bai et al. [131] research concluded that the decomposition ratio increase above 96.5 wt% in recycling CF would decrease the recycled fibre strength, indicat- ing the damage to the fibre surface after complete resin removal. In that study, the authors managed to obtain an 85 wt% decomposition ratio by using supercritical water and adding oxygen as a catalyst. In recycling CF using water at CC, catalysts play an important role in boost- ing the resin decomposition ratio [126]. To analyse, this phenomenon, Okajima et al. [132] investigated the resin degradation with a catalyst (2.5 wt% potassium carbon- ate) and without a catalyst using subcritical water as a sol- vent. The catalyst process had a better outcome with only a 15% tensile strength decrease of rCF compared to vCF. However, recent studies have avoided the use of a catalyst, increasing temperature and pressure to maintain higher process efficiency instead [130, 133]. The studies based on the catalyst are summarised in Table 5. Among all the alcohols, propanol is a short chain and affordable. CF recycled using propanol achieves a 98% resin removal rate [116]. 4.3 Solvolysis using critical conditions The rCF have only a 1% decrease in interfacial shear strength and a 5.43% decrease in ten- sile property compared to vCF [115]. Plus, the rCF offers properties equal to vCF and can be used as its replacement [116]. Like propanol, both n-propanol and 1-propanol also showed promising results. The rCF using n-propanol only shows a 0.3–2% decrease in tensile strength compared to the initial material used [123], whereas the rCF using 1-pro- panol had the same tensile strength compared to the CF prior to recycling. Also, adding 1 wt% of KOH, along with 1-propanol, enhances the decomposition ratio of the resin and improves the mechanical stability of rCF [124]. Adding catalysis is not necessary, as the rCF using 1-propanol as a solvent retained 90–95% tensile strength compared to CF before recycling without any catalyst addition [125]. The process development after achieving a higher decomposition ratio resulted in studies comparing various solvents with water, in which Yildirir et al. [134] compared ethylene glycol (EG)/water mixture to EG at its near-critical condition. The resin removal was 97.6% with EG/water and 92.1% with EG. The rCF had tensile strength similar to vCF. Similarly, Ibarra et al. [135] studied the resin degradation system to compare water and benzyl alcohol at SC and SCC. The authors observed an initial delamination in the system with thin layers of resin-binding the fibres together and stated that the increased reaction resulted in the com- plete degradation of these thin resin layers to recycle CF. The decomposition ratio achieved was 90% from benzyl alcohol and 80% from water. In the recent scenario, a mix- ture of acetone/water at CC is used as a solvent, with the results showing a maximum resin elimination of 95% and successfully closing the rCF life-cycle loop [133, 136, 137]. 4.3.2 Water at critical condition Unlike solvolysis using alcohols, waste CFRP and GFRP are both capable of recycling using water at CC. Moreover, considering the number of studies, this section is divided into two to separate those based on CF and those based on GF. Recycling waste CFRP and GFRP using water at CC has resulted in a higher resin decomposition rate along with higher mechanical properties. 4.3.2.1 Based on carbon fibre Following their CF recy- cling studies using alcohols at CC [117], in the same year (2008), Piñero-Hernanz et al. [126] recycled CF using water at its supercritical condition. The study resulted in a resin removal efficiency of 95.4 wt% with only a 2–10% ten- sile strength decrease of rCF compared to vCF. The addi- tion of alkali catalyst (NaOH) increases the resin removal efficiency [126, 127]. In 2012, when repeating the same approach without any change in the process parameters, Knight et al. [128] achieved a resin decomposition rate of 95.9–99.2 wt% and recycled woven based CF using super- critical water (deionised water). The rCF shows no tensile strength decrease compared to the original composite. 4.3.2.2 Based on glass fibre In the recycling of GF using water at CC as a solvent, the solvents degree of fractiona- tion increased whenever there was a substantial increase in any of the following three conditions: catalyst/solvent ratio, solvent/FRP ratio, and reaction temperature [138]. Similarly, the increase in reaction time resulted in a decrease in the mechanical property of the recovered GF. At-lower reaction temperature, the strength loss in rGF is lower and gradu- ally raised with an increase in reaction temperature. A final Vol:.(1234567890) Vol:.(1234567890) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper Table 5 The catalyst used to recycle CF References Year Catalyst Condition Piñero-Hernanz et al. [126] 2008 KOH (alkali catalyst) 400 °C, 28 Mpa, 0.5 M of KOH Knight et al. [128] 2012 KOH (alkali catalyst) 410 °C, 28 Mpa for 120 min and 0.5 M KOH catalyst Yuyan et al. [129] 2009 Sulfuric acid 260 °C, 1 M sulfuric acid, solvent feedstock ratio of 1:5 g/ mL for 105 min Kim et al. [130] 2019 No catalyst 400 °C, 280 Mpa reactor pressure Bai et al. [131] 2010 Oxygen 440 °C, 30 Mpa for 30 min Okajima et al. 5 Life‑cycle analysis wash to the recycled GF with organic solvents is required to improve the feasibility of the process [139], as well as to increase the delaminating mechanism in recycling WPCBs to allow for GF recycling. A minor per cent (7 vol%) of water below its critical point can result in higher resin decomposi- tion efficiency [140]. 5 Compared to CF, GF has contributed to the most global fibre production, which means the percentage of GFRP waste for recycling is higher compared to the percentage of CFRP waste. Surprisingly, studies have focused more on recycling CFRP waste due to its expensive price range and ability to retain maximum mechanical properties after recycling [7, 143]. A recent study by Hermansson et al. [144] suggested that, in the future, replacing poly- acrylonitrile (PAN) with lignin as a raw material for CF will result in lowering the energy use and environmental impact at the time of recycling the waste CFRP. Like any other recycling process for GF, this process also suffered from char formation on the surface of the fibre, due to the gas condensation during hydrolysis. However, initial physicochemical mechanisms such as osmotic crack- ing, swelling and delaminating can be used to overcome the limitation [141]. In addition to the limitation, the resin structure of the waste GFRP is capable of influencing the rate of hydrolysis, and the hydrolysis mechanism tends to become unstable with varying results [142]. To control the hydrolysis mechanism, Liu et al. [25] used H2O2 (9.04 mL)/ NaOH (0.21 g) along with supercritical water. The process resulted in 95.14% resin decomposition efficiency with sta- ble hydrolysis throughout the process. However, looking at the existing CFRP waste recycling methods, the approaches can be divided into two major techniques. A cheap option; namely landfilling and incin- eration (approach 1), in which the fibres are not recov- ered and cause a high negative impact on the environ- ment, including an economic loss in neglecting to reuse the valuable fibres. The second type (approach 2) is a profitable and fibre recovery method such as mechani- cal, chemical and thermal recycling [4, 48]. Even though the second approach needs capital investment and specific technologies to preserve sustainability, it has a lower environmental impact with a maximum recovery rate of fibres from waste composite. 4.3.2 Water at critical condition [132] 2011 Potassium carbonate 400 °C, 20 Mpa, 2.5 wt% potassium carbonate for 45 min Yildirir et al. [134] 2014 Water 400 °C ethylene glycol (EG) near-critical condition gly- col/water ratio (mL/mL) of 5 Keith et al. [133] 2019 No catalyst 320–380 °C, 20–30 Mpa for 120–150 min Keith et al. [136] 2019 0.05 M ZnCl2 and MgCl2/0.005 M AlCl3 300 °C for 45 min Review Paper 5 Life‑cycle analysis The studies related to the second approach can also be implemented to recycle GFRP waste, as both forms of waste (CFRP and GFRP) involve similar polymer structures [48]. A recent study by Sokoli et al. [120] showed a contradic- tory use of water as a solvent for recycling GF below its criti- cal point. The author explains that water reacts with alkali oxides on the GF surface and produces micro-cracks. The rGF had a 50–65% strength loss compared to vGF. Findings also showed that supercritical acetone is capable of com- pletely degrading resins at 260 °C with 60 bar and c/s ratio up to 2.1 g/mL. The rGF retained 89% of its tensile strength in comparison to vGF. Vol.:(0123456789) Vol.:(0123456789) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper 5.2 Pyrolysis and chemical recycling process LCA studies have emerged from comparing various recycling methods. Among all the LCA indicators, three examples, namely global warming potential (GWP), human toxicity (USETox) and acidification (AE), are identified as essential [145]. However, a GWP indicator is directly asso- ciated with environmental change. It was also noted that the cost of recycling and GWP impacts are inversely pro- portional to each other. When the GWP decreased, so did the cost [146]. In an attempt to analyse the relationship between GWP and cost, Dong et al. [48] studied the waste management of CFRP concerning economic and environ- mental aspects. The study highlighted that none of the recycling techniques were able to reduce both the recy- cling cost and GWP impacts simultaneously. Further recycling process development focusing on fibre recovery leads to comparisons of environmental and eco- nomic aspects within the approach to identify various recycling methods that support sustainability. In 2010, to replace train car bodies in South Korea with CFRP, Lee et al. [85] investigated the identification of environmen- tally-friendly recycling methods and compared chemical recycling with pyrolysis. The study concluded that the energy footprints of chemical recycling (7.62 MJ-eq) are six times less compared to pyrolysis (47.88 MJ-eq), and the greenhouse gas (GHG) emissions of chemical recycling (1196.22 g CO2-eq) was five times less (5916.08 g CO2-eq). Overall, these findings support chemical recycling over pyrolysis. 5.1 Alternative to landfilling and incineration Similarly, results from an LCA study of La Rosa et al. [148] was favourable for recycling CF using chemical recycling, which involved less energy consumption and lowered environmental impacts compared to manufac- turing vCF. The study stated that reducing material and energy consumption during product manufacturing can be a game changer for environmental benefits. The EoL wind turbine blades are also favourable for the chemical recycling process. However, if the recyclate value drops to 47% or processing energy increases above 35 MJ/kg, then using chemical recycling is worthless [9]. Industrial-scale recycling highlights that the energy (0.27–2.03 MJ/kg) required to recycle CF by mechani- cal recycling is lower compared to the energy required (183–286 MJ/kg) to manufacture vCF. Also, mechanical recycling via the milling process has a low environmental impact compared to vCF manufacturing [147]. In addition to this, when comparing landfilling and incineration with mechanical recycling methods, landfilling suffers from sev- eral taxations and incineration has a high environmental impact due to its massive carbon emissions. The carbon footprint in mechanical recycling is low compared to landfilling and incineration and, even though mechanical recycling involves high recycling cost with low revenue, targeting a higher rCF market can improve its revenue [11]. Moreover, to have an immediate rGF solution from wind turbine blades [9] and EoL WPCBs [8] mechanical recycling can be adapted, which will reduce 90% of the landfilling net impact [9]. 5.4 Fluidised bed process can compete with the expensive virgin fibres available in the market. Supporting this statement, Recycling-based literature proposes that replacing virgin fibres with recy- cled fibres is highly possible in various applications. On the other hand, environmental-based literature takes a contro- versial approach in stating that environmental values are inversely proportional to recycling industry profit margins. Overall, considering recycled fibres as an alternative to vir- gin fibres is a sustainable way to manage the cumulating CFRP and GFRP waste.i Several LCA studies on FBP by Meng et al. [16, 151–153], in 2017 and 2018, resulted in a better understanding of the process. The studies showed that replacing vCF with rCF can reduce global warming potential (GWP) by 33–51% and primary energy demand (PED) by 32–50% [153]. Simi- larly, replacing traditional materials (steel and aluminium) with rCF in the automotive (components) application showed potential improvement and environmental ben- efits [152]. As well as the environmental benefits, a hypo- thetical commercial-scale FBP design with conditions: 100–6000 t/y plant capacity and 3–12 kg/h m2 feed rate can recover CFs at a rate of 5 USD/Kg, which means the recycling costs of CF are 15% of the overall vCF manufac- turing cost. Therefore, it is clear that reusing has a positive environmental outcome along with economic profit [151]. Finally, when comparing the FBP to pyrolysis and chemical recycling using LCA indicators, FBP has a lower PED, GWP and power consumption. Overall, a significant amount of environmental and cost-benefits can be achieved by adapting FBP [16]. When considering the reusability of recycled fibres in various applications, the recycled fibres are either used directly or reinforced with a minimal percentage of vir- gin fibres (hybrid composite) to negotiate minor strength loss. These scenarios are entirely dependent on the end application and the commercial possibilities of a compos- ite market. Recent studies focus on applications that func- tion based on a complete replacement of virgin fibres by recycled fibres. The outcomings will benefit the growth of the recycling market and reduce the exponential growth in tonnes of EoL waste. Overall, specific research gaps have been identified as a result of this study. 1. Alternative approaches mentioned in mechanical recy- cling and chemical recycling, such as recycling using high voltage and electricity. Further research in these studies can lead to cost-effective recycling processes in short time intervals, along with a low environmental impact. 5.3 Pyrolysis and solvolysis using SCF In contrast to Lee et al. [85], a Khalil [149] 2018 study used GaBi LCA software and showed opposite results, in which conventional thermolysis via pyrolysis and solvoly- sis using supercritical water (SCW) was compared. The study showed that the solvolysis process had a 78 times greater human health impact, 76 times greater ecotoxic- ity, 17 times greater carbon footprint (global warming) and 3 times greater ozone depletion when compared to pyrolysis. Also, the quantitative evidence proves that CFRP recycling via pyrolysis had the advantage of positive envi- ronmental and human health value compared to solvolysis using SCW. Recent studies have attempted to compare pyrolysis [10, 14], chemical recycling [12], water at CC [10, 13] and EDF [10] with landfilling and incineration. These studies have chiefly focused on showcasing the environmental and economic values of using recycling methods involv- ing fibre recovery. The LCA indicators strongly support pyrolysis over landfilling and incineration. In particular, the low environmental impact and low energy consumption to rCF, which even supports rCF over vCF [10, 14]. Results from the ReCiPe midpoint method supports chemical recy- cling over landfilling, with an average of 80% in all possible LCA indicators [12]. Results from CML-IA baseline and ILCD 2011 midpoint LCA methods show that recycling CFRP under landfilling and incineration possesses a 25–30% environmental impact and 25% additional energy con- sumption compared to recycling using water at CC [13]. Furthermore, Khalil [150] analysed different solvoly- sis methods using GaBi LCA software. The study covered supercritical fluids such as water, methanol, ethanol, 1-butanol, 1-propanol, acetone, ethylene glycol and binary mixtures of solvents and water. The results showed that binary mixtures of solvent and water were capable of recycling quality CF and had lower production costs, environmental and human health impacts compared to the use of pure solvents at CC. Plus, this could be a promis- ing method for closing the life-cycle loop of CFRP waste. Vol:.(1234567890) Vol:.(1234567890) SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 Review Paper 6 Discussion In the current scenario, waste has become a valuable raw material. Based on the literature analysis of rCF and rGF, the scientific community and composite industries are highly focused on recycling techniques to recover fibres completely. Government policies play a significant role in such a drastic change. Strategies such as implement- ing a hefty tax on composite industries for environmental degradation, the EU policy towards commercial EoL com- ponents, economic-based calculations over composite waste and quantity-based estimations over composite waste from the US and the UK, are guiding the composite market to close the life-cycle loop of waste fibres through sustainable recycling. Additionally, EoL composite waste based on applications of the aeronautical/-aerospace sector, the renewable energy sector, and the automotive sector is enormous in volume. These cumulating wastes should be recycled in an industrial-scale. 2. 2. In the thermal-based recycling process, using micro- waves as an alternative source of heat reduces both the energy consumption and the recycling time in half compared to traditional heat sources available. Further research in the field can avoid unwanted heat-loss in industrial-scale recycling, considering huge structures to be recycled such as aeroplane and windmill parts. 3. Extensive researches are needed to transforming labo- ratory-scale solvolysis using water at CC (solvent) and binary solvent mixture (water + solvent) at CC into a fully functional commercial scale. This will benefit the recycling industries. There is a considerable need to determine such an industrial-scale process and further develop it into a sus- tainable method to achieve a higher yield in fibre recov- ery. This can be gradually achieved by briefly analysing the previous studies. In which, economic-based literature proposes that the energy consumption in fibre recycling using any recycling technique is lower when compared to the energy required to manufacture virgin fibres. The profitable selling price for both the recycled CF and GF with only a minor compromise in the quality of the fibres 4. LCA studies contribute to major factors in under- standing the pros and cons of the various recycling process, further studies to support the circular econ- omy to show re-manufacturing as a significant player in closing the life-cycle loop of the recycled fibres are required. After all, reusing the recovered fibres in actual applications is the only possible way to sup- press the need to manufacture virgin fibres. 4. 5.4 Fluidised bed process It can also be used as a pre-recycling process for initial separation during commercial-scale recy- cling. 7 Conclusion The main goal of the current study is to determine various recycling techniques for CFRP and GFRP waste and priori- tise the sustainably identified recycling methods based on economic and environmental values. A critical comparison was carried out based on factors such as process condi- tions, process outcomes, mechanical properties, ease of reuse, environmental impact and cost-effectiveness. However, the research has been narrowed down further to focus on completely recovering the valuable fibres by adapting recycling techniques such as mechanical recy- cling, thermal recycling, and chemical recycling. Plus their advantages and disadvantages were discussed briefly. Fur- thermore, to support the credibility of these techniques, LCA studies were included in comparing the environmen- tal and economic aspects of the recycling techniques. Overall, this study promotes a circular economic approach to close the life-cycle loop of both CF and GF composite wastes into re-manufactured composites. 5. 5. Traditional chemical recycling using strong acids or solvents at various conditions has severe environ- mental impacts. Even though the process is capable of recycling high quality and clean fibres with a crack- free surface and low energy usage, the disposal of such strong solvents is challenging. However, alternative approaches such as electrochemical recycling look promising but are still at the laboratory stage. 6. 6. A chemical recycling process using solvents such as water at CC and a binary fraction of mild solvents with water at CC is considered to be the future of recy- cling both CFRP and GFRP waste. The maximum resin elimination ratio, the higher retention of mechanical properties in recycled fibres and the use of cheap and sustainable solvents make the process distinct from any other recycling process. However, the process is not yet commercialised, and critical operating condi- tions tend to consume additional energy. 6. A chemical recycling process using solvents such as water at CC and a binary fraction of mild solvents with water at CC is considered to be the future of recy- cling both CFRP and GFRP waste. The maximum resin elimination ratio, the higher retention of mechanical properties in recycled fibres and the use of cheap and sustainable solvents make the process distinct from any other recycling process. However, the process is not yet commercialised, and critical operating condi- tions tend to consume additional energy. 6 Discussion LCA studies contribute to major factors in under- standing the pros and cons of the various recycling process, further studies to support the circular econ- omy to show re-manufacturing as a significant player in closing the life-cycle loop of the recycled fibres are required. After all, reusing the recovered fibres in actual applications is the only possible way to sup- press the need to manufacture virgin fibres. Vol.:(0123456789) Vol.:(0123456789) Vol.:(0123456789) Review Paper SN Applied Sciences (2020) 2:433 \| https://doi.org/10.1007/s42452-020-2195-4 4. Commercial-scale pyrolysis has been successfully adopted in Germany and the UK. The process imple- ments green values by recycling fibres along with products such as gas and liquid, which can be further used as feedstocks. However, char formation in rGFs and retaining the complete mechanical properties of both CF and GF are challenging. Besides, the recycled fibres need a secondary heat/chemical treatment to eliminate minor resin impurities. 7 Conclusion Commercial-scale FBP is capable of recycling clean and high-quality fibres with a fraction of the energy consumption needed to manufacture virgin fibres. Also, the process has a low environmental impact and a decent commercial-scale profit margin. However, the fibres are fluffy and discontinuous, limiting attitudes toward ease of reuse. 7 Conclusion Currently, the fate of scarp and EoL CFRP and GFRP is mostly to become landfill or to be sent for incineration, being the methods adopted by conventional waste dis- posal industries. However, taking on board factors such as climate change, global warming, and a sustainable alterna- tive and circular economy, waste disposal industries have recently shifted to complete fibre recovery methods. This change is occurring due to the immense contribution of studies focusing on recycling CFRP and GFRP waste. These studies are briefly discussed in this review article, and the outcomes are summarised below. The future aims of our research team are to study both recycled CFRP and GFRP from industrial waste. The research will be focused on analysing the recyclability of the recovered fibre, on the mechanical properties after reuse, on the possible applications to implement the re- manufactured composite and, finally, on an analysis of how to close its life-cycle loop. The composite wastes will be recycled by thermolysis in a pyrolytic reactor. Also, compression moulding method will be adopted to re-manufacture the covered fibres with polymers. 1. Landfilling and incineration methods of disposing of CFRP and GFRP waste are not sustainable approaches anymore and have to be completely shut-down with- out any further analysis. 2. Mechanical recycling techniques have reached their maximum exploration, losing status as a primary recy- cling method and becoming a pre-recycling process for other techniques such as thermal and chemical recycling. Also, alternative approaches to breaking down the waste composite using high voltage are becoming more attractive than traditional mechani- cal recycling. Acknowledgements Open access funding provided by LUT University. Author’s contribution Both the authors contributed to the study con- ception and design. Material preparation, data collection and manu- script writing were performed by Sankar Karuppannan Gopalraj. Data selection and structuring were performed by Timo Kärki. 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https://openalex.org/W2765850607	https://www.epj-conferences.org/articles/epjconf/pdf/2017/28/epjconf_avtfg2017_00030.pdf	English	null	Adiabatic wall temperature and heat transfer coefficient influenced by separated supersonic flow	EPJ web of conferences	2,017	cc-by	2,173	Adiabatic wall temperature and heat transfer coefficient influenced by separated supersonic flow 1Institute of Mechanics, Lomonosov Moscow State University, 1, Michurinsky Pr., Moscow, 11919 Russia Abstract. Investigations of supersonic air flow around plane surface behind a rib perpendicular to the flow direction are performed. Research was carried out for free stream Mach number 2.25 and turbulent flow regime – Rex>2·107. Rib height was varied in range from 2 to 8 mm while boundary layer thickness at the nozzle exit section was about 6 mm. As a result adiabatic wall temperature and heat transfer coefficient are obtained for flow around plane surface behind a rib in contrast with the flow around plane surface without any disturbances. 1 Introduction One of the major problems in high speed vehicles engineering is the accuracy of experimental data obtained in supersonic wind tunnels while its extrapolation to the real flight conditions. Flight speed increasing over 0.4·М results in the requirement for accounting of compressibility effect [1, 2]. Flow deceleration from supersonic speed to the zero-order meaning on the wall causes increased flow kinetic energy transforming into heat in comparison with subsonic flows. Heat transfer factor h for supersonic gas flow around the wall is evaluated by means of specific heat flow rate q divided by the difference between adiabatic wall temperature Taw and the measured wall temperature Tw:   aw w q T T h (1) (1) One of the main problems in the equation (1) is definition of adiabatic wall temperature Taw [3, 4]. In engineering applications it is defined by means of temperature recovery factor r, total temperature T0, Mach number M and the ratio of specific heats γ: DOI: 10.1051/epjconf/201715900030 DOI: 10.1051/epjconf/201715900030 EPJ Web of Conferences 159, 00030 (2017) AVTFG2016 * Corresponding author: pss1@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: pss1@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). EPJ Web of Conferences 159, 00030 (2017) AVTFG2016 DOI: 10.1051/epjconf/201715900030 2 0 2 1 1 M 2 1 1 M 2 aw r T T               (2) (2) The physical meaning of temperature recovery factor r is that it shows a part of the flow kinetic energy that is transformed to the heat on the wall [1-4]. It is defined by means of adiabatic wall temperature Taw, total temperature T0 and static temperature T: 0    aw T T r T T (3) (3) There is a theoretical relation for recovery factor – it depends on the working medium Prandtl number, i.e. its viscosity, specific heat and thermal conductivity: 𝑟𝑟= Pr𝑛𝑛. Numerous experimental research for air flows [5] show that for a turbulent boundary layer in supersonic flow around the plate and along the cylinder recovery factor r lies in the range 0.885±0.01. Theoretical relation for recovery factor in turbulent conditions is: 𝑟𝑟= √Pr 3 . This relation was experimentally proven for air in and around Pr=0.7. At the same time some researchers consider influence of recovery factor from Reynolds, Mach and Prandtl numbers, injection/suction into/from the boundary layer, shape and relief of streamlined surface [5-11]. These results show that recovery factor can significantly differ from the mentioned approximation. pp High influence of heat transfer coefficient from temperature recovery factor is also intensified by low gas temperature in most supersonic wind tunnels. In addition if researched Mach number is not high (less than M=3) then temperature difference between adiabatic wall temperature and wall temperature is not high [4]. Therefore, mistakes in temperature recovery factor cause high uncertainties in heat transfer data [12]. For flow around simple bodies (plane surfaces, cylinders, cones) adiabatic wall temperature is defined with the use of theoretical meaning for recovery factor as a function of Prandlt number. However, when the flow with a boundary layer separation is considered (behind ribs, dimples, holes) then it is difficult to define adiabatic wall temperature and it is often changed for the flow stagnation temperature. Such approach results in high errors in defining heat transfer coefficient – up to 50%. * Corresponding author: pss1@mail.ru The goal of this research was applying of an unsteady state methodology for adiabatic wall temperature acquisition in aerodynamic experiment together with heat transfer coefficient definition. 2 Experimental apparatus, instrumentation and technique Experiments have been conducted in supersonic wind tunnel during its launching [12, 13]. Flow velocity in the working area was changed with the use of plane variable supersonic nozzle in range of Mach numbers 2.0÷3.0. Stagnation pressure was up to 7 atm. Stagnation temperature changed from 278 to 298K. Maximum gas flow rate was about 5 kg/sec. Working area has a rectangular cross-section 70 mm width and 90 mm height (Fig. 1). For the purpose of experimental observation optical glass windows were installed on side walls of the working area. Flow visualization was made with schlieren photography thus providing an opportunity of shock waves observation for separation flow behind the rib. As an experimental model a plate made of plexiglass was taken. This material has a low thermal conductivity coefficient k = 0.19 W/(m·K) thus providing an assumption of heat insulated surface. The model was mounted on the lower side of the working area in parallel 2 EPJ Web of Conferences 159, 00030 (2017) AVTFG2016 DOI: 10.1051/epjconf/201715900030 with the main flow (Fig. 1). Width of the model fitted the working area – 70 mm, its length was 200 mm. On the lower side of the channel in front of the model a rib was placed perpendicularly to the flow direction. Rib height was changed from 2 to 8 mm. Boundary layer thickness in the region of rib placement was about 6 mm (for flow around plane surface without any disturbances). Reynolds number was calculated for boundary layer length from the nozzle throat and its meaning was Rex≥2·107, which corresponds to turbulent flow regime. In order to measure static pressure experimental model was drained along the central line. Through this perforation air bleed was taken to the static pressure probe. Stagnation parameters were measured in settling chamber of the wind tunnel: temperature – with the use of two insulated chromel-alumel thermocouples, static pressure – with the use of probes Honeywell ML-300PS2PC. Wall temperature of the experimental model was recorded by 6 thermocouples pressurized on the wall surface. Automation program was made in LabView. Data from all the probes was taken to the signal conditioning unit NI SCXI-1303, then it goes through the instrumentation amplifier SCXI-1102 (thermocouple) and SCXI-1102B (pressure probe) and finally comes into the analog-digital converter NI PCI-6220. Fig. 1. Experimental apparatus and instrumentation schematic model Fig. 1. 2 Experimental apparatus, instrumentation and technique Experimental apparatus and instrumentation schematic model The methodology for adiabatic wall temperature acquisition implies registering wall temperature during launching of the wind tunnel. Then the heat flux rate is calculated from the registered temperature time history Tw, plexiglass properties: density ρ, thermal conductivity k and specific heat C (4). Further on the heat flux rate can be presented in dimensionless form so that it could be linearly extrapolated to the zero heat flux meaning [3, 4, 15]. The calculated wall temperature at the zero heat flux rate is equal to the adiabatic wall temperature. Finally, Stanton number can be derived from heat flux rate and difference between measured wall temperature and calculated adiabatic wall temperature.   1/2 0 ( ) w t dT kС d q d t          (4) (4) Measurement uncertainties of the main parameters were calculated for 95% confidence interval [12]: ±1.2% for Mach number, ±2.7% for Reynolds number, ±0.8% for temperature recovery factor, ±9.6% for heat transfer coefficient. 3 DOI: 10.1051/epjconf/201715900030 EPJ Web of Conferences 159, 00030 (2017) AVTFG2016 3 Results and discussion The results show that in the separated region behind the rib adiabatic wall temperature is lower than the wall temperature (Fig. 2, a). Total temperature after launching is almost constant during the experiment. Wall temperature is lowering and in the limit it will reach its adiabatic meaning but during 90 seconds of this experiment it didn’t happen. Difference between total temperature and adiabatic wall temperature along the model length is presented on Fig. 2, b). For flow around plane model this meaning is about 17 degrees. For separated flow behind the rib the difference is higher: from 21 degress for 2 mm rib height to 27 degrees for 8 mm rib height. a) b) Fig. 2. a) Total temperature, wall temperature and adiabatic wall temperature changing during launching of the wind tunnel; b) Difference between total temperature and adiabatic wall temperature for flow around smooth wall (M0=2.25) and flow behind the rib of varied height (2, 4, 6 and 8 mm) b) a) a) b) Fig. 2. a) Total temperature, wall temperature and adiabatic wall temperature changing during launching of the wind tunnel; b) Difference between total temperature and adiabatic wall temperature for flow around smooth wall (M0=2.25) and flow behind the rib of varied height (2, 4, 6 and 8 mm) Temperature recovery factor is decreased in the region of boundary layer separation behind the rib (Fig. 3): from 3.6% for 2 mm rib height up to 7.7% for 8 mm rib height in comparison with the flow around plane model. While going down from the rib the recovery factor is decreasing in proportion to rib height. Temperature recovery factor is decreased in the region of boundary layer separation behind the rib (Fig. 3): from 3.6% for 2 mm rib height up to 7.7% for 8 mm rib height in comparison with the flow around plane model. While going down from the rib the recovery factor is decreasing in proportion to rib height. Fig. 3. Temperature recovery factor for flow around smooth wall (M0=2.25) and flow behind the rib of varied height (2, 4, 6 and 8 mm) Fig. 3. Conclusions Experimental results are presented for boundary layer separation influence on adiabatic wall temperature and heat transfer coefficient in supersonic flow around plane surface. Separated flow was generated by means of a rib in front of the plane model. Temperature recovery factor is decreased in separation region by 3.6% for 2 mm rib height up to 7.7% for 8 mm rib height in comparison with the flow around smooth surface. Heat transfer coefficient is increased in the separation region by up to 34% compared to smooth model flow. 3 Results and discussion Temperature recovery factor for flow around smooth wall (M0=2.25) and flow behind the rib of varied height (2, 4, 6 and 8 mm) Maximum increasing of heat transfer coefficient was about 34% for 8 mm rib height, while the highest 30% meaning for most thermocouples along the model length was registered for 6 mm rib height (Fig. 4). Comparison of the regimes with and without the rib was taken for the same input Mach number (M0=2.25). Most of the thermocouples registered 20% increasing heat transfer rate for separated flow in comparison with the smooth model flow. 4 4 EPJ Web of Conferences 159, 00030 (2017) AVTFG2016 DOI: 10.1051/epjconf/201715900030 Fig. 4. Heat transfer coefficient for flow behind the rib (rib heights 2, 4, 6 and 8 mm) divided by the coefficient for flow around smooth wall Fig. 4. Heat transfer coefficient for flow behind the rib (rib heights 2, 4, 6 and 8 mm) divided by the coefficient for flow around smooth wall The research was supported by Russian Science Foundation (Project 14-19-00699). References 1. S.S. Kutateladze, A.I. Leontiev, Heat Transfer, Mass Transfer, and Friction in Turbulent Boundary Layers (Taylor and Francis, New York, 1990) y y ( y ) 2. H. Schlichting, Boundary layer theory (McGraw-Hill, New York, 1968) 3. D.L. Schultz, T.V. Jones, AGARDograph, 165 (1973) 4. J.R. Hayes, R.D. Neumann, AIAA, Tactical Missile Aerodynamics, 142 (1992) y , , y , ( 5. H.A. Johnson, M.W. Rubesin, Transactions of the ASME (1948) 5. H.A. Johnson, M.W. Rubesin, Transactions of the ASME (1948) 6. P.F. Brinich, NACA TN 4213 (1958) 7. B.J. Garland, J.R. Hall, NACA RM, L57L09 (1958) 8. G.E. Gadd, W.F. Cope, J.L. Attridge, A.R.C. Technical Report, 3148 (1960) 9. Yu.V. Baryshev, Yu.A.Vinogradov, A.I. Leont’ev, V.I. Rozhdestvenskii, Fluid Dynamics, 7, 2, 303 (1972) 10. A.I. Leontiev, Yu.A. Vinogradov, S.M. Bednov, A.N. Golikov, I.K. Ermolaev, E.V. Dilevskaya, M.M. Strongin, Exp. Thermal Fluid Sci., 26, 487-497 (2002) 11. A.I. Leont’ev, V.G. Lushchik, A.E. Yakubenko, Fluid Dynamics, 43, 5 (2008) 12. R.J. Moffat, Experimental and Flud Science, 3-17 (1988) 13. S.S. Popovich, K.S. Egorov, Yu.A. Vinogradov, Proceedings of 15th International Heat Transfer Conference, IHTC-15-8962, 11 (2014) 14. S. Popovich, M. Strongin, Yu. Vinogradov, A. Zditovets, Proceedings of the 8th International Symposium on Turbulence, Heat and Mass Transfer, 865-868 (2015) y p f ( ) 15. D.J. Mee, H.S. Chiu, P.T. Ireland, Int. J. of Heat and Mass Transfer, 45, 3287-3297 (2002) 5
https://openalex.org/W2509232071	https://www.frontiersin.org/articles/10.3389/fpsyg.2016.01297/pdf	English	null	Classes in Translating and Interpreting Produce Differential Gains in Switching and Updating	Frontiers in psychology	2,016	cc-by	10,851	Edited by: Antonino Vallesi, University of Padua, Italy Reviewed by: Marco Calabria, Pompeu Fabra University, Spain Kenneth R. Paap, San Francisco State University, USA Keywords: cognitive control, processing demand, translation, interpreting, bilingual advantage, longitudinal study Classes in Translating and Interpreting Produce Differential Gains in Switching and Updating 1 Bilingual Cognition and Development Lab/Center of Linguistics and Applied Linguistics, Guangdong University of Foreign Studies, Guangzhou, China, 2 College of Foreign Studies, South China Agricultural University, Guangzhou, China The present longitudinal study was intended to investigate whether the two bilingual experiences of written translation and consecutive interpreting (featured with similar language switching experience but different processing demands) would produce different cognitive control effects in young adults. Three groups of Chinese–English young adult bilinguals, who differed mainly in their half-year long bilingual experience: one for general L2 training, one for written translation and one for oral consecutive interpreting, were tested twice on the number Stroop, switching color-shape and N-back tasks. The results show that the interpreting experience produced signiﬁcant cognitive advantages in switching (switch cost) and updating, while the translating experience produced marginally signiﬁcant improvements in updating. The ﬁndings indicate that the experience of language switching under higher processing demands brings more domain-general advantages, suggesting that processing demand may be a decisive factor for the presence or absence of the hot-debated bilingual advantages. ORIGINAL RESEARCH published: 30 August 2016 doi: 10.3389/fpsyg.2016.01297 INTRODUCTION Correspondence: Yanping Dong ypdong@gdufs.edu.cn Correspondence: Yanping Dong ypdong@gdufs.edu.cn It is believed that pervasive experience can leave its mark on the development of mind and brain. The past decade has seen a boom of research exploring the eﬀect of bilingualism on speciﬁc cognitive control components (e.g., Bialystok et al., 2004). But there have been dissenting voices (e.g., Paap and Greenberg, 2013) or cautious voices (e.g., Hilchey and Klein, 2011) in recent years. This controversy has turned into a hot debate, especially after a recent issue of Bilingualism: Language and Cognition (a series of commentaries on the key article by Valian, 2015) and a recent issue of Cortex (a series of commentaries on the key article by Paap et al., 2015). Experts on the topic have expressed their warnings against methodological ﬂaws (see Paap, 2014, for example), theoretical weaknesses (see Jared, 2015, for example), and interpretation biases (see Morton, 2015, for example). Aware of the controversy, the present study has taken several steps to overcome some of the ﬂaws and weaknesses in the literature, hoping to ﬁnd a way forward, which may provide some clues for the bilingual advantage issue, and which may further help to establish the types of bilingual experience that produce relatively quick gains in cognitive control. Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Received: 03 June 2016 Accepted: 12 August 2016 Published: 30 August 2016 Citation: Dong Y and Liu Y (2016) Classes in Translating and Interpreting Produce Differential Gains in Switching and Updating. Front. Psychol. 7:1297. doi: 10.3389/fpsyg.2016.01297 Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Received: 03 June 2016 Accepted: 12 August 2016 Published: 30 August 2016 Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Received: 03 June 2016 Accepted: 12 August 2016 Published: 30 August 2016 Keywords: cognitive control, processing demand, translation, interpreting, bilingual advantage, longitudinal study Citation: (2016) reported less mixing costs in a color-shape switching task and a dual-task advantage from SIs. To sum up, in the few cross-sectional studies conducted up till now, it seems that there was always a certain cognitive control advantage for professional SIs or students of more interpreting experience. However, the results were not necessarily consistent. Two of the studies (Yudes et al., 2011; Dong and Xie, 2014) found that interpreting experience enhanced switching ability as measured in the WCST, while two of the studies (Babcock and Vallesi, 2015; Becker et al., 2016) found that interpreting experience reduced mixing costs but not switching costs in a color-shape task. To bridge the gap, we may have to conduct studies of a longitudinal design and with both tasks (WCST and the color-shape task). What is more, we have to take into consideration of our critical question of when (under what circumstances). y Indeed, many bilingual advantages have been reported using assumed measures of inhibition, switching and monitoring, but it seems that many of them have been questioned by Paap et al. (2015) for reasons illustrated above. The inhibition advantage from bilingualism was evidenced in diﬀerent tasks, such as the Simon task (e.g., Bialystok et al., 2004; Carlson and Meltzoﬀ, 2008; Woumans et al., 2015), the Stroop task (e.g., Bialystok et al., 2008; Blumenfeld and Marian, 2011), the ﬂanker task (e.g., de Abreu et al., 2012; Poarch and Bialystok, 2015) and the Attention Network Test (ANT; a complex version of ﬂanker) (e.g., Costa et al., 2008; Marzecová et al., 2013). Bilingual advantage in switching was shown in the color-shape task (e.g., Prior and Macwhinney, 2010; Prior and Gollan, 2011). As to the relationship between bilingual experiences and updating capacity, few empirical studies have been conducted, but there have been theoretical formulations on the relationship between WM and updating in the context of bilingual advantage (Paap and Sawi, 2014). A few studies found that bilingualism did not bring WM advantage as measured by WM spans (e.g., Ratiu and Azuma, 2015). Bilingual advantage in monitoring (as indicated by shortened reaction times in tasks containing conﬂicts or by mixing cost in the color-shape task) was reported too (e.g., Barac and Bialystok, 2012; Abutalebi et al., 2015; Woumans et al., 2015). Citation: A large majority of research on the bilingual advantage adopted a cross-sectional design (except for a few such as Bak et al., 2014). However, cross-sectional, in contrast to longitudinal designs, are vulnerable to confounding factors that are hard to control and for which the cause-consequence August 2016 \| Volume 7 \| Article 1297 1 Frontiers in Psychology \| www.frontiersin.org Interpreting Training Produces Cognitive Advantages Dong and Liu relationship between bilingualism and executive control is hard to decide (see Kempe et al., 2015; Li and Grant, 2015; Woumans and Duyck, 2015). research, and the present paper was intended as a ﬁrst step in the recommended direction. Instead of investigating the bilingual advantage directly, the present study investigates a related issue: under what circumstances does language switching practice start to inﬂuence or enhance non-linguistic switching abilities? The answer to this question could partly answer Hartsuiker’s (2015) question of when practice in one domain generalizes to another domain. Interpreting between two languages is a cognitively demanding task, and several recent studies (Yudes et al., 2011; Dong and Xie, 2014; Babcock and Vallesi, 2015; Morales et al., 2015; Woumans et al., 2015; Becker et al., 2016) have explored how interpreting experience brings cognitive advantages. Yudes et al. (2011) found that professional simultaneous interpreters (SIs) outperformed general bilinguals in the WCST task, but not in the task of Simon. Consistent with these ﬁndings, Dong and Xie (2014) further found that students of interpreting training or more interpreting training outperformed those of no or less interpreting training in the task of WCST, but not in the task of Flanker. Babcock and Vallesi (2015) and Woumans et al. (2015), however, had diﬀerent ﬁndings. Babcock and Vallesi (2015) found that professional interpreters exhibited less mixing cost in a color- shape task than general bilinguals but did not show advantages in conﬂict resolution in a Stroop task or switching cost in the color-shape task. Woumans et al. (2015) found that interpreters outperformed unbalanced (but not balanced) bilinguals in the Simon and ANT tasks (i.e., higher accuracy in both tasks and smaller error congruency eﬀect in the ANT). Along the same line of comparing SIs and general bilinguals, Morales et al. (2015) reported higher updating skills from SIs and a modulating eﬀect of interpreting experience on the interaction between attentional networks. Comparing SIs and other professional multilingual controls (mostly consecutive interpreters and translators), Becker et al. Frontiers in Psychology \| www.frontiersin.org August 2016 \| Volume 7 \| Article 1297 Citation: On the one hand, performances of both interpreting and translation involve frequent switching between two languages. Diﬀerent from simultaneous interpreting, consecutive interpreting is more “serial” in the sense that it is generally after one segment of the source text is rendered that the next would start to be processed. It is in this sense that consecutive interpreting is more similar to translation, compared to simultaneous interpreting. On the other hand, there are diﬀerences between consecutive interpreting and translation. The most apparent diﬀerence lies in that interpreting requires immediate processing, which suggests that interpreters are under great time pressure and that they have to store on- line a huge amount of information. Dragsted and Hansen (2009) found that because of this diﬀerence, professional translators and interpreters performed diﬀerently in an eye-tracking experiment of sight translation and written translation. The interpreters translated faster in a more “controlled” linear way without compromising output quality, while the translators translated more slowly with plenty of backtracking and regressions of their eye movements. Yudes et al. (2011) and Dong and Xie (2014) have found evidence for the cognitive advantage of switching brought by interpreting experience, but none of them explicitly distinguished oral interpreting experience from written translation experience because students of interpreting (as in Dong and Xie, 2014) or professional interpreters (as in Yudes et al., 2011) are generally also trained in written translation. A direct comparison of the cognitive eﬀects of these two modes of language training may be able to provide some clues for why some language experiences rather than others bring cognitive control advantages, and thus clues for what brings bilingual cognitive advantages. bilingual participants were tested at a pre-test and a post- test. The three groups were comparable except that one group would receive one semester’s (oral) interpreting training, another (written) translation training and the third would receive general L2 training (English culture and communication). There were two parts in the pre-test: (1) a questionnaire of the participants’ backgrounds: their L2-related experiences and their relevant biological and social data (e.g., age, IQ, parents’ education); (2) a test of their cognitive control abilities of inhibition, switching, monitoring and updating in working memory (WM). The post-test consisted of only the second part, that is, a test of participants’ cognitive control abilities. Participants Therefore, the answer for the critical question of when would be: language switching practice starts to inﬂuence or enhance non-linguistic switching abilities when processing demands reach a certain level. A comparison of the courses that the participants received during the experimental semester is illustrated in Table 1. The three groups were, therefore, comparable in the training they received during the semester except for the diﬀerence deliberately designed for the present study. All the participants were native speakers of Chinese, and apart from English, had no contact with any other foreign language. Details of their background information were presented in the ﬁrst half of Table 2 “background characteristics,” including L2- related factors (tested L2 proﬁciency, self-rated L2 proﬁciency, self-rated L2 use, AoA) and more biological and social factors (age, IQ, parents’ education). Such information was collected to Citation: However, null bilingual eﬀects have also been reported in the Simon task (e.g., Gathercole et al., 2014; Kirk et al., 2014), the Stroop task (e.g., Kousaie and Phillips, 2012) and the ﬂanker task (e.g., Bialystok et al., 2010). The bilingual switching advantage failed to appear in some studies either (e.g., Hernandez et al., 2013; Gathercole et al., 2014). Reviewing nearly 30 experiments, Hilchey and Klein (2011) claimed that there was only evidence for a bilingual advantage in monitoring. However, Paap and Greenberg (2013), after reviewing 18 tests in several studies, did not ﬁnd any signiﬁcant monitoring advantage. The bilingual advantage issue, therefore, needs more research, especially research adopting a longitudinal design. Apart from methodological considerations, a better theoretical framework is needed (e.g., Jared, 2015; Hartsuiker, 2015). The most important question is: what does bilingualism have that monolingualism does not that might lead to bilingual advantages in cognitive control? The general theoretical formulation is that executive functions exercised in selecting the target language during bilingual processing (see the BIA+ model by Dijkstra and Van Heuven, 2002; the Inhibitory Control Model by Green, 1998) are transferred from the linguistic domain to the general domain. It seems that the monitoring of two jointly activated language systems, the inhibition of the non-target language, the switch between languages, and the updating of relevant information in the bilingual language control system corresponds neatly to such components of the general cognitive control system as monitoring, inhibiting, switching and updating (see Miyake et al., 2000). But how does this transfer happen? In Hartsuiker’s (2015) words, when, how and why does practice in one domain generalize to another domain? Hartsuiker (2015) may have pointed out the most important direction for future To answer the critical question of when language switching practice starts to inﬂuence or enhance non-linguistic switching abilities, the present paper adopts a longitudinal design and compares the cognitive consequences of (oral consecutive) interpreting training with (written) translation training and general second language training. Three groups of bilingual students participated and they were comparable except that they would respectively receive one semester’s consecutive interpreting training, translation training and general L2 training August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 2 Interpreting Training Produces Cognitive Advantages Dong and Liu (L2 culture and communication). Apart from the longitudinal design, what is distinctively diﬀerent from the literature is a comparison with translation training. Citation: Statistical analyses reported below will show how each group has progressed after one semester’s training, and how the three groups diﬀer from each other in cognitive control abilities after being matched in their pre-test. Participants p Three groups of Chinese–English young adult unbalanced bilinguals (145 in total, mean age = 19.69 years, SD = 0.89, range = 17–22) volunteered to participate in the study for course credit. Among the 145 participants, 57 of them taking an interpreting course during the experiment semester (coded henceforward as the interpreting group), 43 of them taking a translation course (coded henceforward as the translation group), and 45 of them taking general English course (English culture and communication, coded henceforward as the control group). All these participants were non-English-major sophomore students from the same college of a Chinese university in China, and received neither translation nor interpreting training before taking the pre-test. Since the courses were elective, assignment to the groups was based on self-selection. In the general English course (control group), about half of the class time was spent on listening to the teacher’ lectures and half on student discussions. Teachers and students were all required to speak in English in the classroom and therefore little language switching took place. As for the two courses of translation and interpreting, the training was mainly from English to Chinese, with about one third of the class time spent on listening to teachers’ lectures and the rest on translation or interpreting practice. At the end of the semester, participants were asked to report how much time they had spent on each course after class. The average time each group of participants spent on Integrated English after class was 56 h, and that on their distinguishing course (English culture and communication, translation or interpreting) was 40 h. We predicted that interpreting experience would bring more cognitive control advantages than translation or general bilingual experience. If the prediction is true, it implies that a prerequisite for a certain training to bring about general cognitive control advantage is high processing demands. For the interpreting- translation case, immediate switching of a large chunk of speech (a sentence at least) between the two languages under time pressure (i.e., interpreting) poses higher processing demands than switching without time pressure (i.e., translation). A task may be demanding in diﬀerent ways, but immediate processing under time pressure is certainly one of the ways. As speculated by Schroeder and Marian (2016), when the supply was below the demand, the cognitive system tried to adapt and thus got enhanced. Frontiers in Psychology \| www.frontiersin.org 1Apart from the color-shape task, we decided to use the WCST task in the post-test because relevant studies in the literature (Yudes et al., 2011; Dong and Xie, 2014) used this task in their cross-sectional designs and found an interpreter advantage in the performance of the task. If a post-test group comparison in the WCST in the present longitudinal study was found, it would be a triangulation for the two previous studies. Since the WCST was only tested in the post-test, it was not consistent with the longitudinal design, and it was therefore only reported in Supplementary. The Number Stroop Task The number Stroop task, measuring participants’ inhibition ability, was more or less the same as that used by Xie and Dong (2015). The task required participants to judge whether the number of the digits or the hash signs (#, ##, ###, or ####) in a stimulus was even or odd. There were three possible conditions. The neutral condition refers to trials of the hash sign “#”, and so the correct response for “###” or “#”, for example, would be odd. The congruent condition refers to trials of digits in which the parity of the digit coincides with the parity of the number of the digit, and so the correct response for 2222 would be even. The incongruent condition is the opposite of the congruent condition, and so the correct response for 222 would be odd (because there are three digits). The computerized task was composed of two blocks: the practice block and the experimental block. The practice block consisted of nine trials with feedback of accuracy and response times for each stimulus. The experimental block consisted of 120 randomly presented trials, with 40 in each condition. Each stimulus was presented on the screen for a maximum time of 2000 ms or until participants pressed designated keys. Participants were asked to respond as quickly as possible without sacriﬁcing accuracy. Altogether three tasks of cognitive abilities were used, testing participants’ inhibition, switching, updating, and monitoring. Inhibitory control was tested with the number Stroop task under the typical assumption that smaller Stroop interference eﬀects reﬂect better control. We did not choose the Simon task or the Flanker or the color Stroop because we believed they were too simple for our young adult participants who were in their peak of cognitive abilities (e.g., Paap and Greenberg, 2013). Xie and Dong (2015) used the Flanker and the number Stroop tasks to test similar participants (Chinese–English young adult unbalanced bilinguals with L1 or L2 public speaking training) and it was found that the number Stroop task produced more groups eﬀects than the Flanker, probably because it was more diﬃcult (with longer reaction times, see Dong and Li, 2015 for a review). But we are aware that there may be diﬀerent opinions. Paap et al. (2014, May) reported that the ﬂanker eﬀect is still shrinking after 100 sessions and more than 20,000 trials. MATERIALS AND METHODS To investigate how the two speciﬁc bilingual experiences of translation and interpreting would inﬂuence cognitive control development in young adults, three groups of Chinese–English August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 3 Interpreting Training Produces Cognitive Advantages Dong and Liu TABLE 1 \| Class hours of courses for the three participant groups (control, translation and interpreting) together with practice after class (in brackets) during the experimental semester. TABLE 1 \| Class hours of courses for the three participant groups (control, translation and interpreting) together with practice after class (in brackets) during the experimental semester. (Philipp et al., 2008; Yang et al., 2016). And yet based on the tripartite system of executive functions suggested by Miyake et al. (2000), we decided to adopt the switching account of switch cost as adopted in Hernandez et al. (2013) and Paap and Greenberg (2013). The major reason is that compared with the inhibition component measured in tasks such as the Flanker, the Simon and the Stroop, switch cost in the color- shape task involves more of one’s ability to switch to a new task set. brackets) during the experimental semester. Control Translation Interpreting Courses not related to L2 (English) 256 256 256 Integrated English 42 (+56) 42 (+56) 42 (+56) English culture and communication 32 (+40) 0 0 Translation (written) 0 32 (+40) 0 Interpreting (oral) 0 0 32 (+40) ensure that confounding factors (e.g., Dong and Li, 2015; Valian, Control Translation Interpreting In addition to the two typical components of inhibition, switching and monitoring, updating in WM was also identiﬁed as part of the cognitive control system (e.g., Miyake et al., 2000; Costa et al., 2009; Hilchey and Klein, 2011). These components are related but also relatively independent. The enhancement of one component may or may not imply the strengthening of other components. Thus, all four components were tested in the present study (see Table 3). ensure that confounding factors (e.g., Dong and Li, 2015; Valian, 2015) would be controlled. ensure that confounding factors (e.g., Dong and Li, 2015; Valian, 2015) would be controlled. Materials and Tasks Critical information about the materials and tasks is listed in Table 3. Each cognitive control task is described in more detail below. In the pre-test, participants had to complete a composite questionnaire with questions tapping information about participants’ self-rated L2 proﬁciency, self-rated L2 use, AoA, age and parental education (Marian et al., 2007), together with an L2 proﬁciency test (L2 cloze test by Bachman, 1985) and an IQ test (Raven et al., 1977). The Number Stroop Task Switching (mental set shifting or mental ﬂexibility) was tested with the color-shape task (i.e., the switch cost: reaction time diﬀerence between a switch trial and a non-switch trial in a mixed block)1. Both global RTs and mixing costs (RT diﬀerence between non-switch trials in a mixed block and single task trials) are often assumed to reﬂect monitoring ability. But we are aware that “switch cost” is also taken as a measure of inhibitory control, in the sense that participants have to inhibit the previous task set to be able to reactivate the new one We computed four indices for the Stroop task: Global RTs, Stroop eﬀect, Stroop facilitation and inhibition (see Table 2). The most important one is the Stroop eﬀect, i.e., the diﬀerence in mean RTs between incongruent trials requiring suppression of conﬂicting cues and congruent trials with no conﬂicting cues. A smaller Stroop eﬀect implies higher ability in conﬂict resolution and inhibition. Global RTs refers to the average time taken to respond to all the trials (congruent, neutral and incongruent trials). Stroop facilitation refers to the RT diﬀerence between congruent and neutral trials, while Stroop inhibition refers to the RT diﬀerence between incongruent and neutral trials. The Color-Shape Switching Task But as in the number Stroop task, a single shape contains both cues of color and shape. To respond to color, for example, one has to inhibit a potential response to shape. The present study instead tried to reduce the component of inhibition in the color-shape task in which the stimulus was either one of the two color pictures (red or green) or one of the two colorless shapes (circle or triangle). Designed deliberately to test participants’ switching ability, the color-shape task required participants to press the designated keys corresponding to color (always in a circle) or colorless shape pictures presented at the center of the computer screen. Each trial started with a ﬁxation cross presented at the center for 350 ms, followed by a blank screen for 150 ms, and then the target appeared and remained at the center until the participant responded. There were four choices of target picture: two color pictures (red circle or green circle) and two shape pictures (circle or triangle without any color). Participants were instructed to perform the color task using the left hand, with “red” being assigned to the index ﬁnger, and “green” the middle ﬁnger. The shape task was performed with the right hand, with “triangle” being assigned to the index ﬁnger and “circle” the middle ﬁnger. The experiment was composed of three blocks: two blocks of a single task (color or shape) and one block of the mixed task (color and shape). Each single task block included 8 practice trials followed by 24 experimental trials, and the mixed task block included 8 practice trials followed by 48 experimental trials. All the trials in each block were randomized. Participants were asked to respond as quickly as possible without sacriﬁcing accuracy. TABLE 3 \| Summary of tasks used in the present study and description of the items tested. The Color-Shape Switching Task The color-shape task was adapted in the present study so that the inhibition component in switch costs was reduced. In a typical manipulation of the color-shape task, the stimulus is one of the August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 4 Interpreting Training Produces Cognitive Advantages Dong and Liu TABLE 2 \| Pre-test group means (with SD) and comparisons (p-value) of participants’ background characteristics and task performances in the pre-test before group match. Control (n = 43) Translation (n = 40) Interpreting (n = 51) p-Value Background characteristics Translation/interpreting No No No Tested L2 proﬁciency 14.13 (3.61) 13.93 (4.47) 13.39 (3.37) 0.614 Self-rated L2 proﬁciency 20.02 (4.37) 21.07 (5.74) 20.76 (5.27) 0.630 Self-rated L2 use 0.054 (0.036) 0.048 (0.047) 0.049 (0.044) 0.731 AoA 8.95 (2.31) 9.17 (2.44) 9.00 (2.32) 0.903 Age 19.81 (0.82) 19.80 (0.99) 19.45 (0.83) 0.079 Father education 2.39 (0.69) 3.15 (1.38) 2.80 (1.24) 0.013 Mother education 2.02 (0.98) 2.72 (1.37) 2.33 (1.21) 0.031 Intelligence 67.62 (2.38) 67.05 (2.71) 66.66 (3.11) 0.250 Cognitive control abilities Stroop: global RTs (ms) 684.71 (84.73) 677.66 (61.75) 670.69 (68.79) 0.646 Stroop: Stroop effect 34.72 (36.10) 31.36 (49.39) 17.79 (37.08) 0.105 Stroop: Stroop inhibition 14.51 (44.48) 6.27 (56.64) −2.73 (36.43) 0.192 Stroop: Stroop facilitation −20.21 (36.80) −25.09 (31.86) −20.52 (42.65) 0.804 Color-shape: global RTs 612.57 (141.95) 598.62 (145.89) 587.51 (124.12) 0.676 Color-shape: mixing cost 130.44 (126.70) 103.18 (111.06) 117.39 (90.24) 0.526 Color-shape: switch cost 148.30 (95.22) 123.24 (83.72) 137.77 (85.82) 0.435 N-back: global RTs 840.51 (265.32) 848.20 (248.04) 857.64 (248.27) 0.948 N-back: accuracy rate 0.86 (0.088) 0.87 (0.083) 0.83 (0.097) 0.108 TABLE 2 \| Pre-test group means (with SD) and comparisons (p-value) of participants’ background characteristics and task performances in the pre-test before group match 2 \| Pre-test group means (with SD) and comparisons (p-value) of participants’ background characteristics and task pe group match. ith SD) and comparisons (p-value) of participants’ background characteristics and task performances in the pre-test four combinations of color and shape: red/green circle/square. A precue is therefore necessary to indicate when to respond to color and when to respond to shape. But as in the number Stroop task, a single shape contains both cues of color and shape. To four combinations of color and shape: red/green circle/square. A precue is therefore necessary to indicate when to respond to color and when to respond to shape. (1) Inhibition ability: Stroop conﬂict (2) Monitoring ability: global RTs (1) Switching ability: switch cost (2) Monitoring ability: mixing cost and global RTs (1) Updating ability: accuracy rate, global RTs Procedure h The experiment lasted for one academic semester (about 4 months and a half). At the beginning of the semester, participants were asked to take the pre-test in a computer room. The test was divided into two parts and lasted for nearly 2 h, with a 5-min break in between. The order of task administration was ﬁxed for all three groups, with the requirement that no two tasks tapping the same cognitive control capacity occurred consecutively so as to minimize any error caused by task interference. Based on this criterion, tasks administered in the ﬁrst part were the questionnaire, the number Stroop task and the color-shape switch task. Those in the second part were the cloze test, the N-back task and the IQ test. Statistical Analysis An analysis was conducted with Participant Group as the between-subject factor and Testing Time as the within-subject TABLE 4 \| Number of participants excluded from further data analysis and reasons for the exclusions. Control 45-2 Translation 43-3 Interpreting 57-6 Computer breakdown in n-back post-test 1 Abnormal performance in L2 test (less than 10 out of a total of 30 and worse in post-test than in pre-test) 1 1 3 Background different from others (nervous as the only ﬁrst-year student among all second-year students) 1 Not serious in n-back post-test (wrong input of her student number, lowest accuracy at 69%) 1 Abnormal performance in IQ test (less than 55 out of a total of 72, which means “retarded” according to Raven et al., 1977). 3 TABLE 4 \| Number of participants excluded from further data analysis and reasons for the exclusions. Control 45-2 Translation 43-3 Interpreting 57-6 Computer breakdown in n-back post-test 1 Abnormal performance in L2 test (less than 10 out of a total of 30 and worse in post-test than in pre-test) 1 1 3 Background different from others (nervous as the only ﬁrst-year student among all second-year students) 1 Not serious in n-back post-test (wrong input of her student number, lowest accuracy at 69%) 1 Abnormal performance in IQ test (less than 55 out of a total of 72, which means “retarded” according to Raven et al., 1977). 3 August 2016 \| Volume 7 \| Article 1297 As illustrated in the section of “participants,” after the pre- test, participants as college students took various courses for one semester. At the end of the semester, participants took the post-test. Similar to the pre-test, the post-test was divided into two parts and the tasks were administered in a ﬁxed order for all the groups, those in the ﬁrst part were the number Stroop and the color-shape tasks, and those in the second part were the cloze test and the N-back task. Before the ﬁrst part started, participants were asked to complete a questionnaire to collect information about their experiences in the past semester. 3 3 The Color-Shape Switching Task Tasks Items tested Tasks capturing background (in pre-test only) Composite questionnaire (1) Self-rated language proﬁciency: overall score of listening, speaking, reading and writing respectively on a 10-point Likert scale; 40 points in total (2) Self-rated language use: percentage of daily language use; (3) AoA: age of English education; (4) Age: age when being tested; (5) Parental education: score of parents’ education on a 5-point Likert scale L2 cloze test L2 proﬁciency (Bachman, 1985), 30 points in total IQ test IQ: Raven’s Advanced Progressive Matrices Set (Raven et al., 1977), 72 points in total Cognitive control tasks (in pre- and post-tests) Number Stroop task (1) Inhibition ability: Stroop conﬂict (2) Monitoring ability: global RTs Color-shape task (1) Switching ability: switch cost (2) Monitoring ability: mixing cost and global RTs N-back task (1) Updating ability: accuracy rate, global RTs TABLE 3 \| Summary of tasks used in the present study and description of the items tested. Three indices were computed for the color-shape task: global RTs, mixing cost and switch cost. Global RTs refers to the mean RTs in the mixed task block. Mixing cost refers to the diﬀerence in RTs between the non-switch trials in the mixed task block and trials in the single task block, while switch cost refers to the diﬀerence in RTs between the switch trials and August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 5 Interpreting Training Produces Cognitive Advantages Dong and Liu non-switch trials in the mixed task block. Both global RTs and mixing cost are indicators of monitoring ability, and switch cost indicates the ability to switch between diﬀerent types of trials. tests. The three courses were selective, and students are generally not as serious in a selective course as when they perform tasks in a compulsory course. What is more, the classes were very large (i.e., respectively 45, 43, and 57 students in each class), especially as language classes, and it is generally harder to ensure that all students are serious enough in a large class. Table 4 lists the number of participants excluded from each group of participants, and the reasons for the exclusions. The N-back Task A visuo-spatial version of the 2-back task was used to measure updating in WM. In the 2-back task, a blue square was presented in one of 25 possible locations on the screen. Participants were asked to match the location of the current square with the location of the square before the previous one (2-back). The task consisted of 42 2-back trials (28 non-target and 14 target trials). Participants were asked to press the “F” button if the square was in the same location as the square two trials back and the “J” button if the location was diﬀerent. The square remained on the screen for 500 ms. A new square appeared 3000 ms after the previous one had disappeared, irrespective of whether a response was made or not. The presentation order of the trials was randomized. Before the experimental sequence, participants were asked to complete three practice sequences of 27 trials. Participants were asked to respond as quickly as possible without sacriﬁcing accuracy. Two indices, i.e., global RTs and accuracy rate, were computed and both were indicators of participants’ updating ability. The data were trimmed following general procedures in the literature. In the number Stroop task, data from erroneous responses and data with response time (RTs) less than 200 ms were ﬁrst discarded, and then outlier responses deviating by more than 3 SDs from the mean RTs for each participant were trimmed. Altogether less than 5% of data was discarded (the control group: pre-test, 1.91%; post-test, 2.12%; the translation group: pre-test, 2.21%; post-test, 2.14%; the interpreting group: pre-test, 1.97%; post-test, 1.86%). In the color-shape task, the same procedure was followed, and less than 5% of data was discarded (the control group: pre-test, 1.09 and 1.35% for the single or mixed task block; post-test, 1.06 and 1.67% respectively the two task blocks; the translation group: the four percentages were respectively 1.02, 0.91, 1.07, 1.71%; the interpreting group: respectively 1.02, 1.81, 0.68, 1.41%). In the N-back task, the same procedure was followed and less than 5% of data was discarded (the control group: pre-test, 1.55%; post-test, 1.38%; the translation group: pre-test, 1.18%; post-test, 1.18%; the interpreting group: pre-test, 1.09%; post-test, 2.24%). August 2016 \| Volume 7 \| Article 1297 Group Matching in the Pre-test To make sure that group diﬀerences in cognitive control abilities in the post-test were indeed caused by the diﬀerent types of training that the participants had received, not by any preexisting group diﬀerences, we conducted a series of regression analyses to see which background characteristics played a signiﬁcant role in cognitive control abilities in the post-test. Several factors were moderately correlated (father education and mother education: r = 0.498, p < 0.001; AoA and age: r = 0.364, p < 0.001; AoA and mother education: r = −0.334, p < 0.001; self-rated L2 proﬁciency and tested L2 proﬁciency: r = 0.363, p < 0.001), we therefore adopted stepwise regressions to overcome the diﬃculty in assessing the unique contribution of a variable. The result was that father education signiﬁcantly contributed to Stroop eﬀect (father education: β = 0.238, p = 0.007) and Stroop inhibition (father education: β = 0.180, p = 0.040)2. The three groups diﬀered in pre-test father education (p = 0.022, η2 = 0.057), mother education (p = 0.031, η2 = 0.052) and age (p = 0.079, η2 = 0.038). A closer look at parents’ education across the groups shows that the translation group enjoyed higher parents’ education than the other two groups. Besides, the interpreting group was the youngest among the three groups. We therefore matched the participant groups on background characteristics and cognitive control abilities in the pre-test mainly in two steps. First, we excluded participants with high parents’ education from the translation group (ﬁve participants) and participants with low parents’ education from the control group (ﬁve participants) and interpreting group (four participants). Second, we excluded one oldest participant from the control group and three youngest participants from the interpreting group. See Supplementary A Since all the indices were comparable in the pre-test, we conducted further analysis with the post-test data from the two critical indices of switch cost and N-back RT (as in a cross- sectional design). As Table 5 shows, signiﬁcant group diﬀerences were found in post-test switch cost and N-back global RTs (p = 0.049, p = 0.032). 2In addition, mother education signiﬁcantly predicted three indices of WCST (global RTs: β = −0.178, p = 0.042; overall errors: β = −0.215, p = 0.017; perseverative errors: β = −0.207, p = 0.022). Self-rated L2 proﬁciency together with age signiﬁcantly contributed to WCST previous category errors (self-rated L2 proﬁciency: β = −0.204, p = 0.022; age: β = 0.182, p = 0.040). Raw Data in the Pre-test Between-group comparisons were conducted for the pre-test results to see whether the three groups were matched or not. Table 2 is a summary of the descriptive data together with the p-value for each group comparison in each index. y As can be seen in Table 6, the main eﬀect of Test Time was signiﬁcant for the index of global RTs in all three tasks, and also for the index of accuracy rate in the N-back task, reﬂecting a general training or test practice eﬀect. For the other indices (Stroop eﬀect, inhibition, facilitation; color-shape mixing and switch costs), the main eﬀect of Test Time was only signiﬁcant for switch cost. No main eﬀect of Participant Group was found. However, the interaction eﬀect was signiﬁcant for the two indices of switch cost and N-back global RTs, which requires further simple eﬀect analyses. The lower part of Table 6 displays the results of Test-Time simple eﬀect analyses, which shows that the interpreting group made signiﬁcant progress in these two indices (p < 0.001, r = 0.492; p = 0.002, r = 0.455), and that the translation group made marginally signiﬁcant progress in N-back RTs (p = 0.051, r = 0.332) and no signiﬁcant progress in switch cost (p = 0.806, r = 0.049), while the control group didn’t make any signiﬁcant progress (p = 0.566, r = 0.086; p = 0.546, r = 0.092). These results are consistent with the hypothesis that interpreting enhances switching and updating abilities, and that compared with general L2 training, interpreting brings advantages in switching and updating. The ﬁrst ﬁnding revealed in Table 2 is that there was no group diﬀerence in any of the indices of cognitive abilities, and that there was no group diﬀerence in any of the L2-related indices (i.e., L2 proﬁciency, L2 use and AoA). Since the three courses (oral interpreting, written translation, and general L2 class) were not compulsory and students made their choice out of their own will, this ﬁnding indicates that students did not choose a certain training (e.g., interpreting) because of some preexisting advantage in a related cognitive function (e.g., switching). Group Matching in the Pre-test The Tukey HSD post hoc tests in switch cost showed signiﬁcantly less switch cost from the interpreting group than the control group (p = 0.042, r = 0.266), while no signiﬁcant group diﬀerence was found between the control and translation groups (p = 0.678, r = 0.091), or between the translation and interpreting groups (p = 0.280, r = 0.189). The Tukey HSD post hoc tests in N-back RTs also showed a signiﬁcant diﬀerence between the interpreting and control groups (p = 0.040, r = 0.253), while only marginal diﬀerence was found between the control and translation (p = 0.085, r = 0.243) and no diﬀerence between the translation and interpreting groups (p = 0.981, r = 0.023). These results further reﬂect an interpreting experience advantage in switching and WM updating (compared with general L2 training and translation training). Pre-test– Post-test Comparisons across Groups Pre-test– Post-test Comparisons across Groups It is important to know how each group progressed from the pre-test to the post-test and whether groups diﬀered from each other in the degree of progress. Participant Group (between- subject factor) × Test Time (within-subject factor) ANOVAs were therefore conducted. Table 6 shows the result of analyses. Data Trimming First of all, we had to exclude those participants whose performances were obviously not normal. The reason is that some of the students were not serious enough, at least in some of the August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 6 Interpreting Training Produces Cognitive Advantages Dong and Liu for detailed information of the excluded participants. Table 5 shows the result of the match, with participant groups matched in all the key testing items, especially those of cognitive control abilities (e.g., group match for N-back accuracy rate enhanced). factor, hoping to ﬁnd out whether there were any group diﬀerences in the training eﬀect. A prerequisite for the analyses was that the three groups were matched in all the relevant factors that may inﬂuence the performance or development in the cognitive control tasks. Details of data analyses are reported below. DISCUSSION Control (n = 37) Translation (n = 35) Interpreting (n = 44) p-Value Background characteristics Translation/interpreting No No No Tested L2 proﬁciency 14.00 (3.70) 14.12 (4.57) 13.36 (3.55) 0.645 Self-rated L2 proﬁciency 20.43 (4.07) 21.31 (5.69) 20.41 (5.52) 0.694 Self-rated L2 use 0.060 (0.036) 0.050 (0.049) 0.048 (0.045) 0.438 AoA 8.73 (2.41) 9.14 (2.49) 9.20 (2.33) 0.645 Age 19.73 (0.83) 19.85 (1.03) 19.54 (0.73) 0.276 Father education 2.46 (0.66) 2.91 (1.31) 2.82 (1.24) 0.187 Mother education 2.11 (1.02) 2.57 (1.29) 2.34 (1.22) 0.256 Intelligence 67.65 (2.46) 66.86 (2.64) 66.50 (3.22) 0.186 Cognitive control abilities in the pre-test Stroop: global RTs (ms) 676.52 (86.17) 682.34 (60.66) 662.88 (65.03) 0.457 Stroop: Stroop effect 34.13 (37.42) 33.59 (51.82) 18.48 (38.35) 0.172 Stroop: Stroop inhibition 14.68 (40.05) 6.40 (59.86) −1.53 (32.08) 0.267 Stroop: Stroop facilitation −19.45 (33.75) −27.19 (32.50) −20.01 (40.72) 0.598 Color-shape: global RTs 612.30 (148.68) 601.44 (152.75) 581.31 (132.42) 0.616 Color-shape: mixing cost 138.86 (132.39) 103.43 (118.39) 113.48 (93.21) 0.397 Color-shape: switch cost 140.32 (73.49) 122.05 (81.76) 139.23 (90.19) 0.572 N-back: global RTs 855.91 (266.30) 831.97 (225.65) 852.17 (250.49) 0.907 N-back: accuracy rate 0.85 (.091) 0.86 (.080) 0.84 (.091) 0.533 Cognitive control abilities in the post-test Stroop: global RTs 629.08 (82.73) 624.97 (46.92) 612.48 (52.45) 0.457 Stroop: Stroop effect 28.03 (28.42) 26.31 (30.71) 31.14 (32.28) 0.776 Stroop: Stroop inhibition 0.29 (40.63) 6.85 (34.46) 9.06 (31.39) 0.526 Stroop: Stroop facilitation −27.74 (35.09) −19.46 (30.57) −22.08 (34.16) 0.542 Color-shape: global RTs 548.03 (83.89) 557.91 (109.72) 529.95 (91.09) 0.414 Color-shape: mixing cost 99.57 (58.73) 110.23 (87.84) 107.75 (74.01) 0.813 Color-shape: switch cost 132.73 (82.93) 118.71 (70.88) 94.20 (58.19) 0.049 N-back: global RTs 876.62 (262.99) 762.40 (192.39) 752.93 (215.11) 0.032 N-back: accuracy rate 0.89 (0.084) 0.91 (0.067) 0.90 (0.068) 0.627 TABLE 6 \| S f G T t Ti l i h t k i d f th iti t l t k TABLE 5 \| Group means (with SD) and comparisons (p value) of participants’ background characteristics and task performances in pre- and post- tests after group match nd comparisons (p value) of participants’ background characteristics and task performances in pre- and post- tests TABLE 5 \| Group means (with SD) and comparisons (p value) of participants’ background characteristics and task performances in pre- and post- tests after group match. TABLE 6 \| Summary of Group × Test Time analyses in each task index of the cognitive control tasks. DISCUSSION The present longitudinal study was intended to investigate whether the two speciﬁc bilingual experiences of written August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 7 Interpreting Training Produces Cognitive Advantages Dong and Liu TABLE 5 \| Group means (with SD) and comparisons (p value) of participants’ background characteristics and task performances in pre- and post- tests after group match. DISCUSSION TABLE 6 \| Summary of Group × Test Time analyses in each task index of the cognitive control tasks. Main effect of Test Time Main effect of Group Interaction effect Stroop: global RTs p < 0.001, η2 p = 0.475 p = 0.416, η2 p = 0.015 p = 0.734, η2 p = 0.005 Stroop: Stroop effect p = 0.961, η2 p < 0.001 p = 0.519, η2 p = 0.012 p = 0.157, η2 p = 0.032 Stroop: Stroop inhibition p = 0.839, η2 p < 0.001 p = 0.815, η2 p = 0.004 p = 0.165, η2 p = 0.031 Stroop: Stroop facilitation p = 0.851, η2 p < 0.001 p = 0.414, η2 p = 0.015 p = 0.852, η2 p = 0.003 Color-shape: global RTs p < 0.001, η2 p = 0.196 p = 0.511, η2 p = 0.012 p = 0.713, η2 p = 0.006 Color-shape: mixing cost p = 0.233, η2 p = 0.013 p = 0.785, η2 p = 0.004 p = 0.206, η2 p = 0.028 Color-shape: switch cost p = 0.014, η2 p = 0.052 p = 0.371, η2 p = 0.017 p = 0.039, η2 p = 0.056 N-back: global RTs p = 0.012, η2 p = 0.054 p = 0.300, η2 p = 0.021 p = 0.033, η2 p = 0.059 N-back: accuracy rate p < 0.001, η2 p = 0.261 p = 0.634, η2 p = 0.008 p = 0.403, η2 p = 0.016 Simple effect: the control group Simple effect: the translation group Simple effect: the interpreting group Color-shape: switch cost p = 0.566, r = 0.086 p = 0.806, r = 0.049 p < 0.001, r = 0.492 N-back: global RTs p = 0.546, r = 0.092 p = 0.051, r = 0.332 p = 0.002, r = 0.455 translation and consecutive interpreting would produce diﬀerent cognitive control eﬀects in young adults. To better control potential confounding factors and to avoid the cause-eﬀect ambiguity, we conducted a longitudinal study with three groups of participants matched at the pre-test. DISCUSSION This is consistent with the speculation made by Schroeder and Marian (2016). That is, when the supply is below the demand in a certain task, the cognitive system tries to adapt and thus gets strengthened. This may explain what has been found in previous studies on bilingual advantages. In other words, bilingual advantages would probably occur if the bilingual task is demanding enough. If, however, a student learns a second language occasionally or once in a while in a classroom, bilingual advantages would probably not occur. This prerequisite for cognitive advantage transfer as deﬁned above may also explain what has been found in non-linguistic practice. Anguera et al. (2013), an excellent example, found that by playing a (high interference) multitasking video game, older adults (60–85 years old) signiﬁcantly reduced multitasking costs compared to an active control group playing a single task game and a control group without contact with video games. What’s critical is that this training produced beneﬁts to untrained cognitive control abilities, i.e., enhanced sustained attention and WM. In other words, the cognitive advantage transfer (“reduced multitasking costs” to “enhanced sustained attention and WM) was made possible by the multitasking video game, which is certainly more demanding than the single task game. A challenge for the present study is that two previous studies (Babcock and Vallesi, 2015; Becker et al., 2016) found that professional SIs exhibited reduced mixing costs in the color- shape task when compared to bilingual controls, suggesting that interpreting experience enhances the function of monitoring rather than that of switching. Apart from the criticisms aimed at cross-sectional studies, there may be other reasons to explain the diﬀerent ﬁndings between the present study and the two previous studies. The most probable reason, according to our understanding, lies in the stages of interpreting experience that are diﬀerent among the studies. At an early stage of interpreting experience as investigated in the present study, switching eﬃciently between two languages is probably the most obvious challenge, while at a professional stage as investigated in the two previous studies, switching is probably no longer so challenging. Instead, interpreting as a professional (esp. as a professional SIs) requires better management of the situation, monitoring whatever changes and exchanges in the complex situation of communication, and deciding when and how to step in to help the communication. DISCUSSION The results indicate that the interpreting experience produced signiﬁcant cognitive advantages in switching (as shown in switch cost in the color-shape task) and updating (as shown in global August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 8 Interpreting Training Produces Cognitive Advantages Dong and Liu how interpreters, bilinguals and monolinguals performed in the Simon and ANT tasks. The interpreters outperformed the unbalanced (but not balanced) bilinguals in the two tasks (i.e., higher accuracy in both tasks and smaller error congruency eﬀect in the ANT), suggesting the modulation eﬀect of interpreting experience on non-linguistic inhibition tasks. But a closer look at the data indicates that L2 proﬁciency may have partly contributed to the interpreters’ better performance, since the gap in L2 proﬁciency between the unbalanced group and the interpreters was large while that between interpreters and balanced bilinguals was small (L2 proﬁciency on a 5-point scale is 2.6 for unbalanced bilinguals, 3.7 for interpreters and 4.2 for balanced bilinguals; L2 ﬂuency is 5.9 for unbalanced bilinguals, 14.0 for interpreters and 12.9 for balanced bilinguals). To test whether interpreting experience would lead to better non-linguistic inhibition, we may have to conduct more research with more tasks, especially tasks of higher sensitivity (e.g., the Go/Nogo task with ERP techniques). RTs in the n-back task), while the translation experience produced marginally signiﬁcant improvements in updating. Neither interpreting nor translation experience brought any advantage to inhibitory control (as shown in the Stroop eﬀect) and monitoring (as shown in global RTs in the number stroop and color-shape tasks, and in mixing cost in the color-shape task). The present study seems to have provided an answer to the question of “when” practice in one domain generalizes to another domain (part of questions asked by Hartsuiker, 2015). As summarized above, we found that the language switching practice in interpreting (32 class hours in one semester) produced signiﬁcant domain-general switching advantage, while the language switching practice in translation did not (although there seemed a small tendency of similar eﬀect in switch cost). Since the two language switching experiences of interpreting and translation mainly diﬀer in time pressure and processing demands, this ﬁnding of the present study suggests that a prerequisite for a certain training to bring about general cognitive advantage is probably high processing demand, which is immediate processing under time pressure in the present study. Frontiers in Psychology \| www.frontiersin.org DISCUSSION Facing up to the diﬀerent main challenges at diﬀerent stages of interpreting experience may lead to exercises of diﬀerent cognitive control functions and thus strengthen diﬀerent functions. This explanation also ﬁts with the fact that the control groups were very diﬀerent among the studies. The control participants in the present study were intermediate L2 learners, while in these two studies they were highly proﬁcient in both languages and they were probably highly proﬁcient in switching between two languages, esp. for the control group of professional consecutive interpreters and translators in Becker et al. (2016). More empirical research is deﬁnitely needed to test the explanation. The present study helps specify relevant ﬁndings in the literature. First, previous studies on the relationship between interpreting experience and cognitive control advantages did not explicitly distinguish between the oral and written modes of language switching experience (e.g., Yudes et al., 2011; Dong and Xie, 2014; Woumans et al., 2015). We may now speculate that it was probably the oral mode of language switching experience, i.e., oral interpreting, that had brought the cognitive advantages, especially the advantage in switching (because oral interpreting requires immediate processing under time pressure and is therefore more demanding). Second, the absence of inhibition and monitoring advantages in the present study are consistent with what has been found in relevant previous studies using similar tasks (Yudes et al., 2011; Dong and Xie, 2014). Woumans et al. (2015), however, was a diﬀerent study that investigated The ﬁnding about the updating advantage in the present study is an important contribution to the literature (see also Morales et al., 2015). In the N-back task, participants were asked to report whether the currently presented item matched the item presented n items back. It is considered a measure of WM, but empirical research indicates that N-back task performance is only weakly correlated with typical measurements of WM, i.e., the complex span (e.g., reading span) (Redick and Lindsey, 2013). The task of interpreting poses high demands on WM, but how individual August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 9 Interpreting Training Produces Cognitive Advantages Dong and Liu participants become more proﬁcient and automatic in the task. REFERENCES Bialystok, E., Craik, F. I. M., and Luk, G. (2008). Cognitive control and lexical access in younger and older bilinguals. J. Exp. Psychol. Learn. Mem. Cogn. 34, 859–873. doi: 10.1037/0278-7393.34.4.859 Abutalebi, J., Guidi, L., Borsa, V., Canini, M., Della Rosa, P. A., Parris, B. A., et al. (2015). Bilingualism provides a neural reserve for aging populations. Neuropsychologia 69, 201–210. doi: 10.1016/j.neuropsychologia.2015.01.040 Blumenfeld, H. K., and Marian, V. 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DISCUSSION In other words, a skill that requires lots of controlled processing in the early stages may help enhance cognitive control functions, but when that skill becomes automatic and requires far less controlled processing, the early advantages may dissipate. More empirical studies, i.e., studies of longitudinal nature, studies of training with better controlled designs, studies employing additional experimental methods like ERP or fMRI, are certainly needed to verify these speculations. diﬀerencs in WM aﬀect interpreting performance, and whether interpreting training leads to higher WM are controversial (Dong and Cai, 2015). The present study shows that, compared to general L2 training, interpreting training brought signiﬁcant improvements to updating in WM, and translation training brought marginally signiﬁcant improvements to updating in WM. What this ﬁnding suggests is that updating is perhaps a better way to measure how WM plays its role in the task of interpreting, and thus a better index for the relationship between WM and interpreting. p g In short, the present longitudinal study investigated the inﬂuence of translation and interpreting experiences on the development of cognitive control functions. The advantage in the non-linguistic switching tasks yielded by interpreting instead of translation experiences at an early stage of interpreting experience suggests that high-processing demands may be critical to improving cognitive control, which may be able to explain the inconsistent ﬁndings in bilingual cognitive control reported so far. This explanation is consistent with what was found in the comparative study of multitasking and single task video games (Anguera et al., 2013), and with the supply demand explanation by Schroeder and Marian (2016). Furthermore, the results from the present study lead us to speculate that there might be a development curve of cognitive control enhancement in multitasking training such as L2 training, interpreting training or video games training. At the beginning, the curve goes up slowly but steadily, but at a certain point where participants have reached a cognitive peak, the curve would start to level oﬀ. More importantly, the curve may start to drop oﬀslowly when the training becomes less demanding probably because SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpsyg. 2016.01297 AUTHOR CONTRIBUTIONS YD had the idea and design and did most of the writing while YL managed the experiments and analyzed the data and helped with the writing and revision. REFERENCES doi: 10.1016/j.dr.2014.12.003 Hernandez, M., Martin, C. D., Barcelo, F., and Costa, A. (2013). Where is the bilingual advantage in task-switching? 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August 2016 \| Volume 7 \| Article 1297 Frontiers in Psychology \| www.frontiersin.org 11
https://openalex.org/W2134008261	https://reproductive-health-journal.biomedcentral.com/counter/pdf/10.1186/s12978-015-0069-9	English	null	Influence of parental factors on adolescents’ transition to first sexual intercourse in Nairobi, Kenya: a longitudinal study	Reproductive health	2,015	cc-by	9,662	* Correspondence: cokigbo@live.unc.edu 1Department of Maternal and Child Health, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA Full list of author information is available at the end of the article RESEARCH Open Access © 2015 Okigbo et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Okigbo et al. Reproductive Health (2015) 12:73 DOI 10.1186/s12978-015-0069-9 Okigbo et al. Reproductive Health (2015) 12:73 DOI 10.1186/s12978-015-0069-9 Chinelo C. Okigbo1, Caroline W. Kabiru2, Joyce N. Mumah2, Sanyu A. Mojola3 and Donatien Beguy2 Chinelo C. Okigbo1, Caroline W. Kabiru2, Joyce N. Mumah2, Sanyu A. Mojola3 and Donatien Beguy2 Influence of parental factors on adolescents’ transition to first sexual intercourse in Nairobi, Kenya: a longitudinal study elo C. Okigbo1, Caroline W. Kabiru2, Joyce N. Mumah2, Sanyu A. Mojola3 and Donatien Beguy2 Background Despite a global decline in mortality due to HIV, HIV mortality among adolescents is on the increase; it is esti- mated that 2 million adolescents are living with HIV in 2012, and a disproportionate number of them live in sub- Saharan Africa [1]. African youth living in urban slums may be especially vulnerable to HIV infection in the con- text of extreme poverty, limited economic means, and sex- ual violence [8] This study focused on three parenting factors that have been found to have an impact on adolescents’ sex- ual risk behavior: parental monitoring, parent-child communication, and parental discipline. Parental moni- toring is the deliberate effort by parents to control whom their children are with and where they spend their free time; however, from the children’s perspective, it is often defined as adolescents’ perception of their par- ents’ knowledge about whom they are with and where they are spending their free time [9, 10]. A high correl- ation between the parents’ and children’s report of parental monitoring has been documented [19]. High levels of parental monitoring are shown to be associ- ated with a delay in sexual debut and low involve- ment in sexual risk behaviors [4, 9, 10, 12–14, 20]. Age and sex of the adolescent have also been found to modify parental monitoring so defined. In general, parents tend to adjust their monitoring practices as the adolescents get older to allow for more independ- ent decision-making. Consequently, older adolescents report lower levels of parental monitoring compared to younger adolescents [12, 21, 22]. In addition, female adolescents have been shown to perceive more parental monitoring and discipline compared to their males coun- terparts [12, 21–23]. This can be attributed to the exist- ence of a “double standard” in the sexual expectations of the adolescents based on their sex – sexual activity is tol- erated for males but frowned at for females as found in many African communities [24]. School enrollment also affects the level of parental monitoring as studies have shown that parents tend to monitor students who are en- rolled in school more than those who are not [25, 26]. Higher parental monitoring of in-school adolescents may be attributed to the high opportunity cost of having a child drop out of school or perform poorly in their aca- demics. Background in particular. Previous studies have shown that young people living in high poverty settings, such as urban slums, engage in sexual intercourse much earlier and have more sexual partners than their peers that live in wealthier households [15–17]. However, with the excep- tion of a few studies [16, 18], the role of parental factors in high-risk urban settings in sub-Saharan Africa has been understudied. This study fills the gap in the current literature by examining the role of parenting on transi- tion to first sexual intercourse among adolescents living in two urban slums in Nairobi, Kenya’s capital city. The implications of study findings for policies and programs are also highlighted. Adolescence, which is marked by physical, psychosocial, and intellectual development, prepares young people for adult roles and responsibilities [1]. This period is charac- terized by experimentation as young people define their values and seek independence. Unfortunately, such ex- perimentation also drives adolescents to engage in risky behaviors. Indeed, many behaviors that lead to illness or premature death later in life (e.g., sexual risk behaviors, substance use, unhealthy diet, and physical inactivity) are often initiated during adolescence [1]. Sexual risk be- havior is defined as any behavior that increases a per- son’s risk of unintended pregnancy and/or sexually transmitted infections (STIs) including human immuno- deficiency virus (HIV) infection [2, 3]. These sexual risk behaviors include early age at sexual debut, multiple sex- ual partners, unprotected sexual intercourse, and sexual intercourse while under the influence of alcohol or drugs [3–5]. Sexual risk behaviors in adolescence are a cause of concern not only because they predispose adolescents to adverse health outcomes but also because they have been shown to be predictors of sexual risk behaviors later in life [1]. According to the World Health Organization (WHO), adolescent pregnancy increases the risk of maternal and child mortality [6]. The second most common cause of mortality among female adolescents (ages 15–19) is pregnancy-related complications, often from unsafe abor- tion [6]. In addition, babies born to adolescent mothers are at a higher risk of dying compared to those born to older mothers [6]. Furthermore, adolescents have a huge burden of STIs including HIV; approximately 1 in 20 sexually active adolescents contract an STI every year [7]. Abstract Background: Several studies have demonstrated a link between young people’s sexual behavior and levels of parental monitoring, parent-child communication, and parental discipline in Western countries. However, little is known about this association in African settings, especially among young people living in high poverty settings such as urban slums. The objective of the study was to assess the influence of parental factors (monitoring, communication, and discipline) on the transition to first sexual intercourse among unmarried adolescents living in urban slums in Kenya. Methods: Longitudinal data collected from young people living in two slums in Nairobi, Kenya were used. The sample was restricted to unmarried adolescents aged 12–19 years at Wave 1 (weighted n = 1927). Parental factors at Wave 1 were used to predict adolescents’ transition to first sexual intercourse by Wave 2. Relevant covariates including the adolescents’ age, sex, residence, school enrollment, religiosity, delinquency, and peer models for risk behavior were controlled for. Multivariate logistic regression models were used to assess the associations of interest. All analyses were conducted using Stata version 13. Results: Approximately 6 % of our sample transitioned to first sexual intercourse within the one-year study period; there was no sex difference in the transition rate. In the multivariate analyses, male adolescents who reported communication with their mothers were less likely to transition to first sexual intercourse compared to those who did not (p < 0.05). This association persisted even after controlling for relevant covariates (OR: ≤0.33; p < 0.05). However, parental monitoring, discipline, and communication with their fathers did not predict transition to first sexual intercourse for male adolescents. For female adolescents, parental monitoring, discipline, and communication with fathers predicted transition to first sexual intercourse; however, only communication with fathers remained statistically significant after controlling for relevant covariates (OR: 0.30; 95 % C.I.: 0.13–0.68). Conclusion: This study provides evidence that cross-gender communication with parents is associated with a delay in the onset of sexual intercourse among slum-dwelling adolescents. Targeted adolescent sexual and reproductive health programmatic interventions that include parents may have significant impacts on delaying sexual debut, and possibly reducing sexual risk behaviors, among young people in high-risk settings such as slums. Keywords: Parental monitoring, Parent-child communication, Parental discipline, Adolescents, Sexual debut, Urban slums Okigbo et al. Reproductive Health (2015) 12:73 Page 2 of 12 Background Additionally, previous research indicates that the combined effect of religion and supportive parent- ing practices increases the adolescents’ perception of social support and deters their engagement in risky behaviors [27]. Likewise, religious adolescents in urban poor settings have been shown to be less likely The importance of parental involvement is often underscored in risk-reduction efforts targeted towards adolescents because parents/guardians are in regular contact with their children and, therefore, presumably in a good position to shape and influence young people’s behaviors [4, 9]. Consequently, the influence of parental factors on adolescent sexual behavior has been the sub- ject of numerous studies. These studies have demon- strated a link between young people’s sexual behavior and levels of parental monitoring, parent-child commu- nication, and parental discipline [10–14]. However, the majority of the studies have been conducted in Western countries. Little is known about this association in sub- Saharan African settings, and informal/slum settlements Okigbo et al. Reproductive Health (2015) 12:73 Page 3 of 12 to engage in delinquent behaviors including sexual risk behaviors [27–29]. have a curvilinear relationship with adolescents’ engage- ment in sexual risk behaviors – adolescents who per- ceived the least parental discipline have the highest sexual risk behaviors followed by those who perceive the strictest parental discipline while those who perceive moderate parental discipline have the least sexual risk behaviors [34]. It is posited that setting behavioral limits without clarifying expectations by parents may have an adverse effect not just on their self-esteem, but also on adolescent sexual risk behavior [28]. Positive parent-child communication is known to be pro- tective against risk behaviors in adolescence [10, 12, 20]. However, results of studies investigating the link between parent-child communication about sex-related issues and young people’s sexual risk behaviors have been mixed. For example, a study conducted in the United States found that positive general communication between Black and His- panic mothers and their adolescents was associated with less frequent sexual intercourse and fewer sexual partners [20]. Similarly, a 2009 study in four African countries found that parent-child communication about sexual activity was associated with delayed sexual debut in female adolescents [9]. However, several studies have found the opposite association to be true: parent- child communication about sex-related issues was found to be associated with earlier sexual initiation among adolescents especially for males [26, 30]. Background This discrepancy could be due to cultural differences as the studies where parent-child communication was re- ported to be protective were among minority groups and African communities where communication about sexuality is sparse between parents and children. In some traditional African communities, direct parent- child communication about sexuality is often consid- ered a “taboo” and in other communities, young people receive such information during traditional rites of passage and, often, through someone other than their parents [31, 32]. However, with the decline in traditional cultural practices, rapid urbanization, and the breakdown of extended family ties in many urban set- tings in sub-Saharan Africa, parents are forced to assume the responsibility of imparting sexuality education. There is evidence that in African settings, parent-child discus- sions on matters relating to sex occur late (usually after sexual debut); often because parents feel that introducing discussions of sexuality too early will increase curiosity about sex and possibly lead to experimentation [31]. Con- sequently, communication between parents and their chil- dren about sexual matters may commence only after a young person is perceived to have engaged in sexual intercourse. This study, therefore, sought to answer two research questions: 1) is adolescents’ perception of parental moni- toring, parent-child communication, and parental discip- line associated with their transition to first sexual intercourse within the one-year study period?; and 2) do the characteristics of the adolescent modify the relation- ship between parental factors and adolescents’ transition to first sexual intercourse? Based on the findings from the literature, we hypothesized that high levels of paren- tal monitoring, greater parent-child communication about general matters, and high levels of parental discip- line would be associated with a delay in sexual debut among urban slum-dwelling adolescents. We also postu- lated that the characteristics of the adolescents such as age, sex, school enrollment, peer models for risk behav- ior, delinquency, and religiosity would modify the rela- tionship between the parental factors and the adolescents’ transition to first sexual intercourse. Study design and procedures We drew on data collected under the Transitions to Adulthood (TTA) project, which sought to identify pro- tective and risk factors in the lives of adolescents growing up in two informal settlements in Nairobi (Korogocho and Viwandani) and to examine how those factors influ- ence their transition to adulthood. Viwandani is located in the industrial area of Nairobi and thus, attracts a youthful, highly mobile population that is seeking employment in nearby industries. Korogocho is a more stable settle- ment with a median duration of stay of 16 years for the current population compared to seven years in Viwandani. Both slums however, are characterized by high levels of unemployment, crime, substance abuse, limited and often, inadequate schooling facilities, and lack of recreational facilities. The Nairobi Urban Health Demographic Surveillance System (NUHDSS) covers a Demographic Surveillance Area (DSA) that spans the Korogocho and Viwandani slums in Nairobi City, Kenya. The NUHDSS provides a platform for studying the associations between urbanization, pov- erty, and health, and serves as a tool for monitoring and evaluating the impact of health intervention pro- grams on health outcomes of the study population. Adolescents who perceive that their parents disap- prove of sexual activity and anticipate parental discipline are less likely to engage in sexual activity leading to a delay of sexual debut [20, 33]. A study conducted among Lao/Mien adolescents found that parental discipline, which included punishment, taking activities away, send- ing the adolescents to their room, yelling or scolding, slapping or spanking, and/or making the adolescents feel shameful when they behaved badly, prevented adoles- cents from engaging in risky sexual activity for both boys and girls [26]. However, parental discipline seems to Page 4 of 12 Okigbo et al. Reproductive Health (2015) 12:73 Currently, the NUHDSS covers approximately 60,000 people living in 22,000 households in Korogocho and Viwandani. The surveillance involves visits to all the households every four months to update information on demographic and health indicators. complete questionnaire was translated from English to Swahili and administered in Swahili, the national lan- guage. Respondents were requested to give signed in- formed consent; for respondents aged 12–17 who were living with parents or guardians, signed informed con- sent was also requested from their parents or guardians. The data for this study were collected in 2007/08 (Wave 1) and 2008/09 (Wave 2). The study received ethical approval from the Kenya Medical Research In- stitute’s ethical review board. Outcome variable The outcome variable was sexual intercourse status (transition to first sexual intercourse) at Wave 2. For bi- variate analysis, adolescents were grouped into three cat- egories: “Virgins” if they have never had sexual intercourse (both Waves 1 and 2); “Non-virgins” if they had sexual intercourse at Wave 1; or “Transitions” if they hadn’t had sexual intercourse at Wave 1 but had by Wave 2. For the logistic regression analyses, only the subset of adolescents reporting no sexual intercourse at Wave 1 was used [n = 1507]. The outcome variable was grouped into whether or not the adolescent tran- sitioned to first sex between Waves 1 and 2 (coded ‘0’ for no or ‘1’ for yes). To address selection bias, the characteristics of adoles- cents who were interviewed in both waves were com- pared to those interviewed only in Wave 1. Adolescents who were interviewed in both waves did not differ from those interviewed only in Wave 1 by sex. However, a greater proportion of adolescents interviewed in both waves were younger, lived in Viwandani versus Korogo- cho, lived with at least one parent, were currently en- rolled in school, and had never had sexual intercourse [data not shown]. To account for these differences in subsequent analyses, a logistic regression model was run using response status at Wave 2 as the outcome variable, and key socioeconomic and demographic characteristics at Wave 1 as explanatory variables. The probability of responding at each wave was calculated for every indi- vidual in the data using logistic regression, controlling for characteristics that were found to predict non- response. The analytical weight for the observations was obtained by taking the inverse of the predicted probabil- ity. The computed weights were re-adjusted to approxi- mately add up to the expected sample size. The overall weight for Wave 2 was obtained by taking the product of Wave 1 and Wave 2. Study design and procedures g The TTA project was based on a random sample of adolescents within the households in the study areas using records of residents in the NUHDSS in 2007. Be- tween October 2007 and June 2008, a random sample of about 4,058 young people aged 12–22 years was inter- viewed. Approximately one year later (March-August 2009), an updated questionnaire was administered to the same cohort. About 2,674 adolescents were re-interviewed at the follow-up survey; the high attrition rate was due to out-migration and the 2008–2009 post-election violence that occurred in several urban communities after the 2008 general elections in Kenya. This paper includes adolescents aged 12–19 years who were interviewed at both the base- line (Wave 1) and follow-up (Wave 2) surveys. Since the primary aim of the research presented in this article was to examine the effect of parental factors on adolescent sexual transition, the sample was restricted to adolescents who had never been married by Wave 2 [n = 1927]. The ration- ale is that unmarried adolescents are more likely to be wards of their parents and hence, have the chance to ex- perience the parental factors. Explanatory variables Th h l f The three parental factors at Wave 1, parental monitor- ing, parent-child communication, and parental discip- line, were the explanatory variables. Parental monitoring was assessed using six questions that asked study partici- pants whether they perceived that their parents knew where they were in the evenings of week days; who they were with in the evenings of week days; where they were during the weekends; who they were with during the weekends; who their friends were; and what they did with their free time. Responses were graded on a 3-point scale – 1 (never), 2 (sometimes), and 3 (always). A con- tinuous parental monitoring index was constructed using standardized values of individual items all scored in the positive direction using the “standardize” function in Stata 13 (Cronbach’s alpha = 0.90). Given that 5 % of adolescents were not living with a parent, scores on the parental monitoring index were then grouped into three categories: “low/does not live with parent”, “medium”, or “high” parental monitoring using the “cut” function in Stata. Communication with parents (mother figure, father figure) was assessed using a set of three questions that measured the extent to which the respondent: 1) felt that their mother or father figures taught him/her things he/she does not know; 2) shared secrets with his/her mother or father figures; and 3) that his/her mother or The study instrument included questions on living ar- rangements and the nature of interactions with parents, guardians, and peers; involvement in youth groups (e.g., religious and social groups); and involvement in risky behaviors (e.g., early sexual debut and delinquency). The Page 5 of 12 Okigbo et al. Reproductive Health (2015) 12:73 father figures tried to help him/her. The responses were graded on a 5-point scale, which are 1 (never), 2 (some- times), 3 (half the time), 4 (most of the time), and 5 (all the time). Continuous parent-child communication indi- ces were created using standardized values of individual items all scored in the positive direction (communica- tion with mother figure: Cronbach’s alpha = 0.45; com- munication with father figure: Cronbach’s alpha = 0.48). Scores were grouped into three categories: “no”, low”, and “high” communication. services (i.e., how many times have you gone to reli- gious services during the past one month? Control variables ll d f We controlled for several factors found to be associated with adolescent sexual risk behavior in various settings [12, 21, 22, 35]: adolescents’ age, sex, residence, school enrollment, peer models for risk behavior, delinquency, and religiosity (all measured at Wave 1). School enroll- ment status was determined by asking the adolescents whether they were currently enrolled in school. School enrollment was categorized as “in” versus “out of school”. Peer models for risk behavior was measured using four items: the proportion of friends who: get good grades in school, participate in sports or other school ac- tivities, attend church/mosque, and want to go to second- ary school, university, or college (Cronbach’s alpha = 0.94). Possible responses are none of them (coded 0), some of them (1), most of them (2), don’t know, or not applicable. “Don’t know” and “not applicable” responses were treated as missing values. Delinquent behavior was assessed using a set of seven items that measured the extent to which youth engaged in the following behaviors in the four months preceding the survey: staying away from home for at least one night without parental permis- sion; starting a fight with peers; taking or trying to take something belonging to someone else without their knowledge; carrying a knife, gun, or other weapon; hit- ting or threatening to hit a peer or adult; delivering or selling drugs; and delivering or selling alcohol. Reponses ranged from 0 = never; 1 = once; 2 = more than once. A composite index was then derived from standardized values; an internal consistency was mea- sured using the Cronbach’s alpha (α = 0.69). Religiosity was a composite measure created using five items that assessed the frequency of participation in religious Analyses Parental discipline was assessed using two questions that asked the adolescents whether their parents/guard- ians: 1) verbally reprimand (scolding); and/or 2) spank them when they do something wrong. Responses were graded on a 5-point scale ranging from 1 (never/not liv- ing with parents/guardian) to 5 (always). Item scores were recoded into 4 categories for “not living with par- ents”, “never”, “occasionally” and “frequently”. For the multivariate analyses, we created binary variables with response categories ‘0’ for no or ‘1’ for yes. Quantitative data analyses were performed using Stata 13 [36]. Bivariate analyses (chi-square, cross-tabulations, ANOVA, and t-tests) were conducted to assess bivariate associations between sexual intercourse status and the independent and control variables, as well as examine age differences by sex. Chi-square tests were used to de- termine the statistical relationships between the categor- ical variables in the cross tabulations. Multivariate logistic regression models were run in which baseline (Wave 1) measures of parental and control variables were used to predict the likelihood of making the transi- tion to first sexual intercourse between the two waves. Two sets of models were run: the first included only par- ental factors, while the second model added the control variables to assess the effect of these variables on the base parental model. The analyses were run separately for males and females. All analyses were based on values weighted for non-response at Wave 2. Explanatory variables Th h l f Response options: never, 1 time, 2–3 times, 4 times, more than 4 times) and the importance of relying on religious teach- ings and beliefs, believing in God, and prayer in one’s life (e.g., how important is it to you to be able to rely on religious teachings when you have a problem? Re- sponse options: not important, somewhat important, im- portant, and very important (Cronbach’s alpha = 0.93). Descriptive analysis Table 1 provides the socio-demographic description of the respondents stratified by sex. Of the 1,927 adoles- cents included in this study, 52 % were males, 43 % were aged less than 15 years, 52 % lived in Viwandani, 95 % lived with at least one parent, and 77 % were enrolled in school. Seventy-two percent of respondents reported that they had never had sexual intercourse in both waves, while 6 % reported their first sexual intercourse between the two waves. Bivariate analysis T bl 2 i Table 2 summarizes the results of the bivariate analysis by sexual intercourse status and sex of the respondents. For both males and females, the majority of the non- virgins were in the oldest age group. The mean age for non-virgins was 17.3 years for both males and females compared with 14.5 years and 14.6 years, for male and females virgins respectively. A majority of those who made the transition to first sexual intercourse were also in the older age group. Most of the virgins (99.7 % for females and 97.2 % for males) were living with at least one parent. Ninety-four percent of female non-virgins Okigbo et al. Reproductive Health (2015) 12:73 Page 6 of 12 Table 1 Respondents’ socio-demographic characteristics, by sex Total (N = 1927) Males (n = 1002) Females (n = 925) Baseline mean age in years (SE) 15.2 (0.05) 15.3 (0.07) 15.2 (0.07) Baseline age group (%) 12–14 year olds 42.7 42.0 43.4 15–17 year olds 36.3 37.1 35.5 18–19 year olds 21.0 20.9 21.1 Baseline residence (%) Korogocho 48.4 47.0 50.1 Viwandani 51.6 53.1 50.0 Baseline living arrangements (%) With parent(s) 94.8 91.9 98.2 Without parents 5.2 8.1 1.8 Baseline school enrollment (%) In school 77.3 78.8 75.7 Out of school 22.7 21.2 24.3 Sexual intercourse status at Wave 2 (%) Non-virgins 21.9 23.9 19.6 Transitions 6.0 5.9 6.0 Virgins 72.2 70.2 74.4 Notes: Estimates are weighted; SE = Standard Error Significant difference at p < 0.05 between males and females based on chi-square tests Table 1 Respondents’ socio-demographic characteristics, by sex more likely to have reported that they had never been scolded or spanked. It should be noted that generally, spanking and/or slapping were not common forms of discipline by parents of adolescents in the slums. Among adolescents who were sexually experienced at Wave 2 (i.e. non-virgins and transitions), 47 % had initiated sex- ual activity by age 15 [data not shown]. Among these groups, 54 % of non-virgins and 24 % of those who tran- sitioned had engaged in sexual intercourse by age 15 [data not shown]. A higher proportion of male adolescents who transi- tioned reported high levels of father-son communication (48 %) followed by those who are still virgins (43 %). Fe- male adolescents who were virgins reported high father- daughter communication (42 %) followed by those who transitioned (35 %). Bivariate analysis T bl 2 i For both male and female adoles- cents, the proportion reporting high levels of father-child communication was lowest among non-virgins – 28 % for males and 20 % for females. The associations found for mother-child communication differed compared to father- child communication. More female adolescents than male adolescents reported high levels of mother-child commu- nication. Specifically, approximately 71 % of female virgins reported high levels of mother-daughter communication compared to less than 39 % of male virgins. Additionally, approximately half of female non-virgins and those who transitioned compared to less than a third of male vir- gins and those who transitioned reported high levels of mother-child communication. A greater proportion of adolescents reported having no father figure (19–36 %) compared to having no mother figure (1–17 %) in their lives. and 77 % of male non-virgins were living with at least one parent. Eighty-nine percent of both male and female adolescents classified as virgins were enrolled in school compared to fewer than 34 % of females and 49 % of males categorized as non-virgins. Multivariate analyses bl h h All psychosocial variables were significantly associated with sexual intercourse status at the bivariate level for both females and males. Assessing the peer models for risk behavior and delinquency, we found that males and females who were non-virgins had higher levels of nega- tive influence and delinquency compared with those who were virgins and those who transitioned during the one-year follow-up period. A similar association was ob- served with religiosity. Adolescent female and male non- virgins, on average, had lower scores on the religiosity index compared with the other two groups. Table 3 shows the results of the logistic regression models conducted to identify the correlates of transition to first sexual intercourse among the subset of adoles- cents reporting no sexual intercourse at Wave 1 (763 males and 744 females). In the first set of models (Models 1 and 3), only parental variables measured in Wave 1 were included as explanatory variables. The sec- ond set of models (Models 2 and 4) included both the parental factors and the control variables measured at Wave 1. The models with the control variables had bet- ter fit indices for the data compared to models without the control variables. Forty-six percent (46 %) of female virgins reported high parental monitoring, while only 29 % of females who transitioned to first sexual intercourse and 14 % of female non-virgins reported high parental monitoring. A similar trend was observed for males. A greater propor- tion of females than males reported high parental moni- toring (p < 0.05). Although a substantial proportion of both male and female adolescents reported being occa- sionally scolded or spanked, adolescent males and fe- males who transitioned from virgins to non-virgins were In Models 1 and 3, high levels of parental monitoring were significantly associated with lower odds of report- ing the first sexual intercourse between the two waves for female adolescents but not for male adolescents. Fe- males who reported high levels of parental monitoring were 63 % less likely to transition to first sexual inter- course compared with females who reported low levels of parental monitoring or were not living with a parent Okigbo et al. Significant difference at p < 0.05 across groups for males based on chi square tests for categorical variables and ANOVA for continuous variables bSignificant difference at p < 0.05 across groups for females based on chi-square tests for categorical variables and ANOVA for continuous variables cIncreasing values indicate higher levels of negative peer influence, delinquency, or religiosity are weighted; SD = Standard Error rence at p < 0.05 across groups for males based on chi-square tests for categorical variables and ANOVA for continuous variables Multivariate analyses bl h h Reproductive Health (2015) 12:73 Table 3 Logistic regression models predicting transition to first sexual intercourse, by sex Model variables Males (n = 763) Females (n = 744) Model 1 OR (95 % C.I.) Model 2 OR (95 % C.I.) Model 3 OR (95 % C.I.) Model 4 OR (95 % C.I.) Parenting variables Parental monitoring Low/does not live with parent(s) 1.00 1.00 1.00 1.00 Medium 0.68 (0.33–1.40) 0.82 (0.37–1.80) 0.62 (0.31–1.24) 0.77 (0.34–1.73) High 0.70 (0.36–1.38) 0.90 (0.41–1.96) 0.37 (0.17–0.78) 0.56 (0.22–1.47) Parental discipline Scolded (ref = never scolded) 0.80 (0.37–1.74) 1.01 (0.42–2.42) 0.72 (0.32–1.59) 0.74 (0.29–1.85) Spanked/slapped (ref = never spanked/slapped) 0.29 (0.15–0.56)* 0.74 (0.36–1.52) 0.31 (0.15–0.63)* 0.55 (0.23–1.29) Communication with father figure No father figure 1.00 1.00 1.00 1.00 Low 1.32 (0.54–3.21) 1.15 (0.43–3.05) 0.44 (0.21–0.93)* 0.30 (0.13–0.68)* High 1.88 (0.83–4.26) 1.89 (0.80–4.45) 0.69 (0.34–1.42) 0.72 (0.34–1.51) Communication with mother figure No mother figure 1.00 1.00 1.00 1.00 Low 0.25 (0.09–0.69)* 0.23 (0.08–0.64)* 0.77 (0.08–7.01) 1.34 (0.23–7.68) High 0.28 (0.10–0.84)* 0.33 (0.11–0.96)* 0.48 (0.05–4.38) 0.77 (0.14–4.23) Controls Age — 1.36 (1.13–1.64)* — 1.29 (1.10–1.51)* Viwandani (ref = Korogocho) — 1.25 (0.61–2.54) — 0.96 (0.46–2.01) Out-of-school (ref = in-school) — 3.45 (1.63–7.28)* — 3.74 (1.84–7.60)* Peer models for risk behaviora — 1.09 (0.80–1.47) — 1.32 (0.94–1.86) Delinquencya — 0.85 (0.51–1.41) — 1.97 (1.10–3.53)* Religiositya — 0.73 (0.55–0.96)* — 0.92 (0.62–1.35) Model fit Wald χ2 (8) = 26.99 Wald χ2 (14) = 53.23 Wald χ2 (8) = 28.50 Wald χ2 (14) = 68.55 Model 1: unadjusted model for males; Model 2: adjusted model for males; Model 3: unadjusted model for females; Model 4: adjusted model for females Statistically significant at p < 0.05 aIncreasing values indicate higher levels of negative peer influence, delinquency, or religiosity Table 3 Logistic regression models predicting transition to first sexual intercourse, by sex Table 3 Logistic regression models predicting transition to first sexual intercourse, by sex Model variables Males (n = 763) Females (n = 744) Model 1 OR (95 % C.I.) Model 2 OR (95 % C.I.) Model 3 OR (95 % C.I.) Model 4 OR (95 % C.I.) or guardian. The association remained non-significant for males and became non-significant for females when age, residence, school enrollment, peer models of risk behavior, delinquency, and religiosity were controlled for. were 56 % less likely to have transitioned compared with females who reported no communication with their father figure (p < 0.05). Multivariate analyses bl h h Reproductive Health (2015) 12:73 Page 7 of 12 Page 7 of 12 Table 2 Descriptive statistics by sexual intercourse status and by sex Males Females Non-virgins (n = 239) Transitions (n = 59) Virgins (n = 704) Non-virgins (n = 181) Transitions (n = 56) Virgins (n = 688) Baseline sociodemographic characteristics Mean age (SD) in yearsa, b 17.3 (1.74) 16.3 (2.03) 14.5 (2.04) 17.3 (1.69) 16.4 (1.76) 14.6 (2.07) Residence b (%) Korogocho 54.1 44.7 44.7 62.5 52.7 46.6 Viwandani 45.9 55.3 55.3 37.5 47.3 53.4 Living arrangementa, b (%) With parent(s) 76.7 89.6 97.2 93.6 94.1 99.7 School enrollmenta, b (%) In school 48.5 62.5 90.4 33.5 51.9 88.7 Baseline psychosocial controls Mean (SD) peer models for risk behaviora, b, c 0.51 (1.21) 0.19 (1.06) −0.06 (0.93) 0.35 (1.16) −0.01 (0.86) −0.19 (0.79) Mean (SD) delinquencya, b, c 0.41 (1.04) 0.02 (0.59) 0.01 (0.54) 0.16 (0.67) −0.11 (0.40) −0.17 (0.36) Mean (SD) religiositya, b, c −0.50 (1.25) −0.26 (1.15) 0.00 (0.88) −0.11 (0.91) 0.07 (0.69) 0.19 (0.63) Baseline parenting variables Parental monitoringa, b (%) Low/does not live with parent(s) 61.3 47.9 36.4 54.6 34.6 17.6 Medium 25.8 23.0 30.9 31.4 36.5 36.2 High 12.9 29.1 32.7 14.0 29.0 46.2 Parental discipline (scolding)a, b (%) Not living with parents 19.6 8.9 2.3 5.3 5.9 0.1 Never 10.3 17.8 10.7 15.9 17.6 10.0 Occasionally 58.7 59.4 76.8 67.8 62.2 80.9 Frequently 11.4 13.9 10.2 11.1 14.4 9.1 Parental discipline (spanking/slapping)a, b (%) Not living with parents 19.6 8.9 2.3 5.3 5.9 0.1 Never 62.1 68.5 44.3 72.6 74.4 50.2 Occasionally 17.4 22.6 48.9 20.9 16.1 47.4 Frequently 0.9 0.0 4.5 1.2 3.7 2.3 Communication with father figurea, b (%) Low 42.9 32.7 34.8 43.7 30.7 37.3 High 27.9 48.4 42.7 20.0 34.7 42.0 No father figure 29.2 18.9 22.5 36.3 34.6 20.7 Communication with mother figurea, b (%) Low 61.8 48.4 57.7 37.8 38.3 28.0 High 29.5 34.2 39.2 57.7 56.1 70.5 No mother figure 8.7 17.4 3.1 4.6 5.6 1.4 Notes: Estimates are weighted; SD = Standard Error aSignificant difference at p < 0.05 across groups for males based on chi-square tests for categorical variables and ANOVA for continuous variables bSignificant difference at p < 0.05 across groups for females based on chi-square tests for categorical variables and ANOVA for continuous variables cIncreasing values indicate higher levels of negative peer influence, delinquency, or religiosity Table 2 Descriptive statistics by sexual intercourse status and by sex Page 8 of 12 Page 8 of 12 Okigbo et al. Multivariate analyses bl h h sexual intercourse for both males and females: specific- ally, a one year increase in age increased the odds of transitioning to first sexual intercourse by 36 % and 29 % for males and females respectively (p < 0.05). Being out of school was positively associated with transition to sexual intercourse. Out-of-school male and female ado- lescents were 3.5 times and 3.7 times more likely to re- port sexual debut between the waves compared with adolescents who were in-school at the time of the survey (p < 0.05). Delinquent behavior was associated with higher odds of initiating sexual intercourse for females but not males. A point increase on the delinquency scale was associated with about twice the odds of transition to first sexual intercourse among female adolescents [OR = 1.97, p < 0.05]. This association was not observed for the male adolescents. Conversely, lower levels of religiosity were associated with higher odds of sexual debut between study waves for male adolescents but not for female adolescents. A point increase on the religiosity scale was associated with 27 % lower odds of transition to first sexual inter- course for males [OR = 0.73, p < 0.05]. This association was not observed for the female adolescents. Area of residence was not significantly associated with transi- tion to first sexual intercourse for both male and fe- male adolescents in our sample. Studies have found that positive parent-child commu- nication fosters identification with parental values and may reduce the probability of engaging in sexual activity [20, 22]. In this regard, the majority of the study respon- dents who remained abstinent in both waves reported higher levels of parent-child communication compared to those who reported their first sexual intercourse be- tween waves. Male adolescents who reported any com- munication with their mothers delayed their sexual debut; however, no such association was found among the female adolescents. It is possible that because daugh- ters tend to already have closer bonds with their mothers [26, 40], there is little variability among females to detect any significant association. On the other hand, father-daughter communication was found to be associ- ated with decreased odds of female adolescents initiating sexual intercourse; however, communication with the father had no significant association with sexual debut among the male adolescents. Multivariate analyses bl h h This association remained signifi- cant and in the same direction with the addition of con- trol variables in Model 4 [OR = 0.30, p < 0.05]. However, high levels of communication with the father did not predict transition to first sexual intercourse for the fe- male adolescents in our sample (p > 0.05). Communica- tion with the mother was found to be significant for only the male adolescents in both models. Specifically, male adolescents who reported low and high levels of communication with their mothers were approximately 75 % and 72 % less likely to commence sexual activity within the study period compared to those who reported no communication with their mothers (p < 0.05). These associations remained significant when control variables were taken into account in Model 2. Male and female adolescents who were spanked or slapped as a form of discipline were less likely to re- port sexual debut between the two study waves [males OR = 0.29, p < 0.05; females OR = 0.31, p < 0.05] compared with those who were never spanked or slapped. However, these associations were no longer significant when control variables were added as shown in Models 2 and 4. Being scolded as a form of discipline did not predict transition to first sexual intercourse. Communication with the father was not significantly associated with transition to first sexual intercourse among the male adolescents but was significant for the female adolescents. Female adolescents who reported low levels of communication with their father figure The adolescents’ age in Models 2 and 4 was signifi- cantly and positively associated with transition to first Okigbo et al. Reproductive Health (2015) 12:73 Page 9 of 12 between parental monitoring and transition to first sexual intercourse. This finding is consistent with results from studies conducted in other African countries (Burkina Faso, Ghana, Malawi, and Uganda) [9]. In contrast, studies conducted in the United States (even among urban-poor African American adolescents), Slovakia, and Scotland in- dicate that high levels of parental monitoring delay sexual debut [12, 22, 37–39]. Therefore, the effect of parental monitoring on adolescent sexual risk behaviors may depend on the sociocultural norms of the geograph- ical context. Programs that aim to involve parents in reducing adolescent sexual risk behaviors should tailor the parental involvement to the sociocultural context of their target population. Multivariate analyses bl h h Interestingly, the signifi- cant association between father-daughter communica- tion and sexual debut appeared to be curvilinear as the effect was only significant for low levels of communica- tion. While the parent-child communication items did not specifically address discussions around sexual behav- ior, the significant association between sexual activity and communication with the parent of opposite sex, par- ticularly for adolescent girls is noteworthy. Previous studies suggest lower levels of communication around sexual matters with fathers compared with mothers or with parents of the opposite gender [9]. However, the high risk of poor sexual and reproductive health outcomes among adolescents in urban slums may mean that both parents find it necessary to have discussions with their male and female children [15–17]. As such, interventions to improve parent-child communications may need to en- sure that both fathers and mothers are targeted. Discussion Little is known about the association between parenting practices and adolescent sexual behavior in African set- tings especially in informal settlements or slums, which are becoming more prevalent in urban areas. Previous studies have shown that young people living in resource- poor urban settings are more likely to engage in sexual risk behaviors. However, as noted earlier, with the excep- tion of a handful of studies, we know little about how various aspects of parenting practices influence the tran- sition to first sexual intercourse. Our findings show that parent-child communication was the most significant factor associated with sexual debut, albeit in gendered ways, even after controlling for demographic and psy- chosocial characteristics. Mother-son communication was significant for adolescent males, while father- daughter communication was significant for adolescent females. We also found that parental monitoring and parental discipline did not predict sexual debut for both male and female adolescents when relevant covariates were controlled for. Only about 1 in 4 males and 1 in 3 females reported high levels of parental monitoring. Low levels of parental monitoring may reflect lower levels of parental supervi- sion as many adults in urban informal settlements may be employed or looking for work and as such are out of the homes most of the day. Further, we found no association Adolescents who reported corporal punishment (spank- ing or slapping) whenever they behaved badly were less likely to report transition to first sexual intercourse within the study period. In contrast verbal discipline (scolding) Okigbo et al. Reproductive Health (2015) 12:73 Page 10 of 12 did not. Other studies have also found that adolescents who anticipate parental discipline are less likely to engage in risky sexual behaviors [14, 20, 33]. Previous studies sug- gest that the change in behavior as a result of corporal punishment may depend on the cultural context [41]; in settings, such as Kenya, where corporal punishment may be culturally-acceptable and considered an appropriate parenting practice, it is expected that physical punishment can result in a decline of risk behaviors among adoles- cents. However, it is also important to consider that while the fear of punishment may deter adolescents from en- gaging in sexual risk behaviors, fear of punishment may also prevent them from having open conversations with their parents about sexual and other sensitive issues. Authors’ contributions CCO and CWK conceived the idea, extracted the data, ran the analyses and interpretation, and wrote the first draft of the paper. JNM, SAM, and DB participated in the analysis, interpretation, and critically revised the manuscript for intellectual content. All authors read and approved the final manuscript. Abbreviations DSA D h Abbreviations DSA: Demographic surveillance area; HIV: Human immunodeficiency virus; NUHDSS: Nairobi Urban Health and Demographic Surveillance System; STI: Sexually transmitted infections; TTA: Transition to adulthood; WHO: World Health Organization. This study should be interpreted in light of several limi- tations. First, about a third of the baseline sample was lost to follow up. This high attrition rate may result in biased estimates especially since the adolescents lost to follow up differed from those who remained in the study on several sociodemographic factors such as age, school enrollment, and living with parents. To minimize the ef- fect of this potential bias, all analyses were weighted based on probability of non-response at Wave 2 [44]. Second, although all efforts were made to reassure re- spondents about the confidentiality of responses, the use of self-reported sexual behavior data also provides a po- tential for social desirability bias [45, 46]. Third, we re- lied on data based on adolescents’ perception of parental Competing interests Competing interests The authors declare that they have no competing interests. Conclusion Studies on adolescent risk behaviors have been on the rise in the last two decades as incidence of morbidities in this age group due to engagement in risky behaviors increases. Recognizing and examining the factors associ- ated with adolescents’ risk behaviors is important for the development and implementation of effective prevention strategies and/or interventions. Active involvement of parents in these prevention interventions can buttress their effectiveness by creating an enabling environment for the adolescents to implement skills and tools they will gain from the interventions. The findings of our study should inform interventions geared towards delay- ing sexual debut among unmarried adolescents espe- cially those in urban poor settings. For example, parents can be involved in intervention programs by training them on evidence-based risk-reduction strategies such as effective communication skills especially on sexual/re- productive health issues. There is a need for additional research to further understand how best to involve par- ents in these adolescent risk-reduction strategies. Further research examining the effects of single-parenthood on adolescents’ sexual attitudes and behaviors would also be insightful. In addition, findings suggest that schooling has protective effects on the youth’s development beyond aca- demic competence and efforts are warranted to ensure that all school-aged youth are enrolled in school. As observed in previous studies in Zambia and the United States, [25, 42] older male and female adoles- cents were more likely to initiate sexual intercourse dur- ing the one year of follow-up than their younger peers. However, males in mid- and late- adolescence had lesser odds of transitioning to first sexual intercourse com- pared to those in early adolescence. Previous studies have shown that adolescents’ behavior is significantly correlated with that of their peers [13, 43]. Thus, it is possible that male adolescents who resist peer pressure to engage in sexual intercourse in early adolescence may continue to resist pressure to become sexually active as they grow older. The results of this study also indicate that school enrollment is a protective factor for adolescent sexual risk behaviors for both males and females, though it did not modify the relationship between parental factors and transition to first sexual intercourse. Time spent in school-related activities may reduce opportunities for en- gaging in risk behaviors. Schooling may also increase the opportunity costs of negative outcomes associated with early sexual intercourse including unintended pregnancies. Discussion Thus, interventions that enhance parents’ capacity to share their values and guide their children’s behavior while maintain- ing open communication lines may be warranted. practices and communication and lacked the parents’ perspectives of their parenting practices. However, as noted earlier, previous studies have shown a high cor- relation between youth’s perceptions and parents’ re- port of parental monitoring [19]. Finally, there was inadequate information on the adolescents’ living ar- rangements such as privacy in the home or whether they live in single-parent households. Yet, living ar- rangements may influence parental factors and conse- quently adolescents’ sexual behaviors. Abbreviations DSA: Demographic surveillance area; HIV: Human immunodeficiency virus; NUHDSS: Nairobi Urban Health and Demographic Surveillance System; STI: Sexually transmitted infections; TTA: Transition to adulthood; WHO: World Health Organization. References World Health Organization. Global strategy for the prevention and control of sexually transmitted infections: 2006–2015: breaking the chain of transmission. Geneva: World Health Organization; 2007. 31. Izugbara CO. Home-based sexuality education: Nigerian parents discussing sex with their children. Youth Soc. 2007;8:24–40. g 8. Adedimeji AA, Omololu FO, Odutolu O. HIV risk perception and constraints to protective behaviour among young slum dwellers in Ibadan, Nigeria. J Health Popul Nutr. 2007;25:146–57. 32. Mbugua N. Factors inhibiting educated mothers in Kenya from giving meaningful sex-education to their daughters. Soc Sci Med. 2007;64:1079–89. 9. Biddlecom A, Awusabo-Asare K, Bankole A. Role of parents in adolescent sexual activity and contraceptive use in four African countries. Int Perspect Sex Reprod Health. 2009;35:72–81. 33. Babalola S, Ouedraogo D, Vondrasek C. Motivation for late sexual debut in Cote d'Ivoire and Burkina Faso: a positive deviance inquiry. J HIV/AIDS Prevent Children Youth. 2007;7:65–87. 10. Huebner AJ, Howell LW. Examining the relationship between adolescent sexual risk-taking and perceptions of monitoring, communication, and parenting styles. J Adolesc Health. 2003;33:71–8. 34. Perrino T, González-Soldevilla A, Pantin H, Szapocznik J. The role of families in adolescent HIV prevention: A review. Clin Child Fam Psychol Rev. 2000;3:81–96. 11. DiClemente RJ, Wingood GM, Crosby R, Sionean C, Cobb BK, Harrington K, et al. Parental monitoring: Association with adolescents' risk behaviors. Pediatrics. 2001;107:1363–8. 35. Jessor R, Turbin MS, Costa FM. Risk and protection in successful outcomes among disadvantaged adolescents. Appl Dev Sci. 1998;2:194–208. 36. StataCorp. Stata Statistical Software: Release 13. College Station: StataCorp LP; 2013. 12. Yang H, Stanton B, Li X, Cottrel L, Galbraith J, Kaljee L. Dynamic association between parental monitoring and communication and adolescent risk involvement among African-American adolescents. J Natl Med Assoc. 2007;99:517–24. 37. Huang DY, Murphy DA, Hser Y-I. Parental monitoring during early adolescence deters adolescent sexual initiation: discrete-time survival mixture analysis. J Child Fam Stud. 2011;20:511–20. 13. Wang B, Stanton B, Deveaux L, Li X, Lunn S. Dynamic relationships between parental monitoring, peer risk involvement and sexual risk behavior among Bahamian mid-adolescents. Int Perspect Sex Reprod Health. 2015;41:89–98. 38. Kalina O, Geckova AM, Klein D, Jarcuska P, Orosova O, van Dijk JP, et al. Mother's and father's monitoring is more important than parental social support regarding sexual risk behaviour among 15-year-old adolescents. Eur J Contracept Reprod Health Care. 2013;18:95–103. 14. Wang B, Li X, Stanton B, Kamali V, Naar-King S, Shah I, et al. Okigbo et al. Reproductive Health (2015) 12:73 Okigbo et al. Reproductive Health (2015) 12:73 18. Ngom P, Magadi MA, Owuor T. Parental presence and adolescent reproductive health among the Nairobi urban poor. J Adolesc Health. 2003;33:369–77. Wellcome Trust (Grant Number GR 07830M). Analysis and writing time was supported through funding from UKaid (from the Department for International Development) for the Strengthening Evidence for Programming on Unintended Pregnancy (STEP UP) Research Programme Consortium (Grant Number SR1109D-6) and the Adolescent Girls Initiative-Kenya Project (Grant Number SP1404); and through general support grants to the African Population and Health Research Center from the Swedish International Development Cooperation Agency (Grant Number 2011–001578) and the William and Flora Hewlett Foundation (Grant Numbers 2012–7612 and 2015–2530). 19. Stattin H, Kerr M. Parental monitoring: A reinterpretation. Child Dev. 2000;71:1072–85. 20. Miller KS, Forehand R, Kotchick BA. Adolescent sexual behavior in two ethnic minority samples: The role of family variables. J Marriage Fam. 1999;61:85–98. Cooperation Agency (Grant Number 2011–001578) and the William and Flora Hewlett Foundation (Grant Numbers 2012–7612 and 2015–2530). 21. Borawski EA, Ievers-Landis CE, Lovegreen LD, Trapl ES. Parental monitoring, negotiated unsupervised time, and parental trust: The role of perceived parenting practices in adolescent health risk behaviors. J Adolesc Health. 2003;33:60–70. Received: 27 October 2014 Accepted: 11 August 2015 Received: 27 October 2014 Accepted: 11 August 2015 24. MacPhail C, Campbell C. ‘I think condoms are good but, aai, I hate those things’: condom use among adolescents and young people in a Southern African township. Soc Sci Med. 2001;52:1613–27. 25. Magnani RJ, Karim AM, Weiss LA, Bond KC, Lemba M, Morgan GT. Reproductive health risk and protective factors among youth in Lusaka, Zambia. J Adolesc Health. 2002;30:76–86. References 1. World Health Organization. Adolescents: health risks and solutions. In: Fact sheet No 345. Geneva: World Health Organization; 2014. 1. World Health Organization. Adolescents: health risks and solutions. In: Fact sheet No 345. Geneva: World Health Organization; 2014. Fact sheet No 345. Geneva: World Health Organization; 2014. 26. Sieving RE, McNeely CS, Blum RW. Maternal expectations, mother-child connectedness, and adolescent sexual debut. Arch Pediatr Adolesc Med. 2000;154:809–16. 2. Kotchick BA, Shaffer A, Miller KS, Forehand R. Adolescent sexual risk behavior: a multi-system perspective. Clin Psychol Rev. 2001;21:493–519. 2. Kotchick BA, Shaffer A, Miller KS, Forehand R. Adolescent sexual risk behavior: a multi-system perspective. Clin Psychol Rev. 2001;21:493–519. 3. Kirby D, Coyle K, Alton F, Rolleri L, Robin L. Reducing adolescent sexual risk: a theoretical guide for developing and adapting curriculum-based programs. Scotts Valley: ETR Associates; 2011. 27. Petts RJ. Family and religious characteristics' influence on delinquency trajectories from adolescence to young adulthood. Am Sociol Rev. 2009;74:465–83. 4. Babalola S, Tambashe BO, Vondrasek C. Parental factors and sexual risk-taking among young people in Cote d'Ivoire. Afr J Reprod Health. 2005;9:49–65. 28. Kabiru CW, Mojola SA, Beguy D, Okigbo C. Growing up at the “margins”: concerns, aspirations, and expectations of young people living in Nairobi's slums. J Res Adolesc. 2013;23:81–94. 5. Bankole A, Biddlecom A, Guiella G, Singh S, Zulu E. Sexual behavior, knowledge and information sources of very young adolescents in four sub-Saharan African countries: original research article. Afr J Reprod Health. 2007;11:28–43. 5. Bankole A, Biddlecom A, Guiella G, Singh S, Zulu E. Sexual behavior, knowledge and information sources of very young adolescents in four sub-Saharan African countries: original research article. Afr J Reprod Health 2007;11:28–43. 29. Kabiru CW, Elung’ata P, Mojola SA, Beguy D. Adverse life events and delinquent behavior among Kenyan adolescents: a cross-sectional study on the protective role of parental monitoring, religiosity, and self-esteem. Child Adolesc Psychiatr Mental Health. 2014;8:1–11. 6. World Health Organization. Adolescent pregnancy. In: Fact sheet No 364. Geneva: World Health Organization; 2014. 6. World Health Organization. Adolescent pregnancy. In: Fact sheet No 364. Geneva: World Health Organization; 2014. 30. Bersamin M, Todd M, Fisher DA, Hill DL, Grube JW, Walker S. Parenting practices and adolescent sexual behavior: A longitudinal study. J Marriage Fam. 2008;70:97–112. 7. World Health Organization. Global strategy for the prevention and control of sexually transmitted infections: 2006–2015: breaking the chain of transmission. Geneva: World Health Organization; 2007. 7. Author details 1 1Department of Maternal and Child Health, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA. 2African Population and Health Research Center, APHRC Campus, Manga Close, Off Kirawa Road, Nairobi, Kenya. 3Department of Sociology and Institute of Behavioral Science, University of Colorado Boulder, Boulder, CO, USA. 22. Romer D, Stanton B, Galbraith J, Feigelman S, Black MM, Li X. Parental influence on adolescent sexual behavior in high-poverty settings. Arch Pediatr Adolesc Med. 1999;153:1055–62. 23. McNeely C, Shew ML, Beuhring T, Sieving R, Miller BC, Blum RW. Mothers’ influence on the timing of first sex among 14-and 15-year-olds. J Adolesc Health. 2002;31:256–65. Received: 27 October 2014 Accepted: 11 August 2015 Acknowledgments The Transitions to Adulthood study was part of a larger project, Urbanization, Poverty, and Health Dynamics in sub-Saharan Africa, that was funded by the Page 11 of 12 Page 11 of 12 42. Mott FL, Fondell MM, Hu PN, Kowaleski-Jones L, Menaghan EG. The determinants of first sex by age 14 in a high-risk adolescent population. Fam Plann Perspect. 1996;28:13–8. 43. Maxwell KA. Friends: The role of peer influence across adolescent risk behaviors. J Youth Adolesc. 2002;31:267–77. 44. Miller RB, Wright DW. Detecting and correcting attrition bias in longitudinal family research. J Marriage Fam. 1995;57:921–9. 45. Beguy D, Kabiru CW, Nderu EN, Ngware MW. Inconsistencies in self-reporting of sexual activity among young people in Nairobi, Kenya. J Adolesc Health. 2009;45:595–601. 46. Eggleston E, Leitch J, Jackson J. Consistency of self-reports of sexual activity among young adolescents in Jamaica. Int Fam Plan Perspect. 2000;26:79–83. References Sexual attitudes, pattern of communication, and sexual behavior among unmarried out-of-school youth in China. BMC Public Health. 2007;7:189–98. 39. Parkes A, Henderson M, Wight D, Nixon C. Is Parenting Associated with Teenagers’ Early Sexual Risk‐Taking, Autonomy And Relationship with Sexual Partners? Perspect Sex Reprod Health. 2011;43:30–40. 15. Dodoo FN-A, Zulu EM, Ezeh AC. Urban–rural differences in the socioeconomic deprivation–Sexual behavior link in Kenya. Soc Sci Med. 2007;64:1019–31. 40. Usher-Seriki KK, Bynum MS, Callands TA. Mother–daughter communication about sex and sexual intercourse among middle-to upper-class African American girls. J Fam Issues. 2008;29:901–17. 16. Kabiru CW, Beguy D, Undie C-C, Zulu EM, Ezeh AC. Transition into first sex among adolescents in slum and non-slum communities in Nairobi, Kenya. J Youth Stud. 2010;13:453–71. 41. Simons RL, Wu CI, Lin KH, Gordon L, Conger RD. A cross‐cultural examination of the link between corporal punishment and adolescent antisocial behavior. Criminology. 2000;38:47–80. 17. Zulu EM, Dodoo FN-A, Chika-Ezeh A. Sexual risk-taking in the slums of Nairobi, Kenya, 1993–98. Popul Stud. 2002;56:311–23. Page 12 of 12 Page 12 of 12 Okigbo et al. Reproductive Health (2015) 12:73 Okigbo et al. Reproductive Health (2015) 12:73 Okigbo et al. Reproductive Health (2015) 12:73 42. Mott FL, Fondell MM, Hu PN, Kowaleski-Jones L, Menaghan EG. The determinants of first sex by age 14 in a high-risk adolescent population. Fam Plann Perspect. 1996;28:13–8. 43. Maxwell KA. Friends: The role of peer influence across adolescent risk behaviors. J Youth Adolesc. 2002;31:267–77. 44. Miller RB, Wright DW. Detecting and correcting attrition bias in longitudinal family research. J Marriage Fam. 1995;57:921–9. 45. Beguy D, Kabiru CW, Nderu EN, Ngware MW. Inconsistencies in self-reporting of sexual activity among young people in Nairobi, Kenya. J Adolesc Health. 2009;45:595–601. 46. Eggleston E, Leitch J, Jackson J. Consistency of self-reports of sexual activity among young adolescents in Jamaica. Int Fam Plan Perspect. 2000;26:79–83. 46. Eggleston E, Leitch J, Jackson J. Consistency of self-reports of sexual activity among young adolescents in Jamaica. Int Fam Plan Perspect. 2000;26:79–83. 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https://openalex.org/W4287702837	https://zenodo.org/records/3982363/files/IJRR0021.pdf	English	null	Awareness among First Year Medical Students in Telangana State about Tuberculosis and Government Services Offered To Tuberculosis Patients: A Cross Sectional Study	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	5,855	ABSTRACT Tuberculosis (TB) is an ancient global public health problem and it still remains as major cause of mortality and morbidity in India.1As per Global TB report 2019 the estimated incidence of TB in India was approximately 27 lakhs accounting for about a quarter of the world’s TB cases.2 The mortality rate in India for the year 2017, excluding cases having both TB and HIV was 31/deaths per 1lakh population.1 In addition to the existing burden of TB, the pandemic of HIV has further aggravated the incidence of TB and WHO estimated that 5% of TB patients are also co-infected with HIV.3In India, High quality diagnostic and treatment services are provided to the people through Revised National Tuberculosis Control Programme( RNTCP) programme which was recently renamed as National Tuberculosis Elimination Programme (NTEP). Tuberculosis (TB) is an ancient global public health problem and it still remains as major cause of mortality and morbidity in India.1As per Global TB report 2019 the estimated incidence of TB in India was approximately 27 lakhs accounting for about a quarter of the world’s TB cases.2 Background: Tuberculosis still remains as major cause of mortality and morbidity in India. Much importance must be given in sensitisation of community about tuberculosis and the services offered in order to curb the disease. With this background, this study was undertaken to assess the awareness among first year medical students about Tuberculosis and government services offered to tuberculosis patients The mortality rate in India for the year 2017, excluding cases having both TB and HIV was 31/deaths per 1lakh population.1 In addition to the existing burden of TB, the pandemic of HIV has further aggravated the incidence of TB and WHO estimated that 5% of TB patients are also co-infected with HIV.3In India, High quality diagnostic and treatment services are provided to the people through Revised National Tuberculosis Control Programme( RNTCP) programme which was recently renamed as National Tuberculosis Elimination Programme (NTEP). Methods: Institutional based cross sectional study with sample size of 165 first year medical students who volunteered for the study. Results: Among the participants 43% knew TB is one of the top 10 global causes of death, 78.8% knew bacteria causes TB, 95.75% knew mode of transmission is by cough droplets and 91.5% opted lung as primarily affected organ in TB. Only 67.9% knew TB as curable disease and 4.8% knew about vaccine. Kishore Yadav Jothula1, Sreeharshika D2, Srikanth JV3 1Department of Community and Family Medicine, All India Institute of Medical Sciences, Bibinagar, Telangana, India 1Department of Community and Family Medicine, All India Institute of Medical Sciences, Bibinagar, Telangana, India 2Department of Forensic Medicine, ESIC Medical College, Hyderabad, Telangana, India 3Primary Health Centre, Chowdepally, Chittor District, Andhra Pradesh, India Telangana, India 2Department of Forensic Medicine, ESIC Medical College, Hyderabad, Telangana, India 3Primary Health Centre, Chowdepally, Chittor District, Andhra Pradesh, India 2Department of Forensic Medicine, ESIC Medical College, Hyderabad, Telangana, India 3Primary Health Centre, Chowdepally, Chittor District, Andhra Pradesh, India Corresponding Author: Sreeharshika D Corresponding Author: Sreeharshika D International Journal of Research and Review Vol.7; Issue: 7; July 2020 Website: www.ijrrjournal.com E-ISSN: 2349-9788; P-ISSN: 2454-2237 International Journal of Research and Review Vol.7; Issue: 7; July 2020 Website: www.ijrrjournal.com E-ISSN: 2349-9788; P-ISSN: 2454-2237 Original Research Article Awareness among First Year Medical Students in Telangana State about Tuberculosis and Government Services Offered to Tuberculosis Patients: A Cross Sectional Study Kishore Yadav Jothula1, Sreeharshika D2, Srikanth JV3 Key words: Bacteria, Government, IEC, Transmission, Tuberculosis, Vaccine ABSTRACT Majority (83.3%) identified cough more than 2 weeks as important presumptive TB symptom. Only 33.9% knew about free diagnostic services, 18.2% about free drugs supply in government setup, 58.8% seen advertisement on TB. National Tubercu Programme (NTEP). India has taken ambitious step of eliminating TB by 2025 and National strategic plan (NSP 2017-25) was prepared with the vision of TB-Free India with zero deaths, disease and poverty due to TB. Advocacy Communication & Social Mobilization (ACSM) is an important component proposed by NSP with the objective of addressing the issues like lack Conclusion: This study found that majority participants were aware about causative organism and symptoms of TB but doesn’t have clue regarding government services and IEC activities related to TB. Key words: Bacteria, Government, IEC, Transmission, Tuberculosis, Vaccine 170 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Study Setting: Rural medical college located in Nalgonda district in Telangana state. of knowledge and information about TB in the community.4 Most of the people are visiting private hospitals and spending lot of money for TB treatment. In a study conducted in Puducherry, it was observed that 32.4% of households experienced catastrophic costs due to TB care, significantly higher in patients with TB care.5 These findings reflects the need to generate awareness in community regarding free services provided in government setup and to motivate people to utilise those services, Information Education and Communication (IEC) activities were carried out in the form of advertisements, hoardings related to TB through various channels of communication. Sample size: Out of 200 first year students, 30 had participated in the pilot study and 5 students were absent on the day of data collection. Hence a total of 165 first year students were included after taking consent, hence sample size was 165. Sample size: Out of 200 first year students, 30 had participated in the pilot study and 5 students were absent on the day of data collection. Hence a total of 165 first year students were included after taking consent, hence sample size was 165. Sampling Method: Census method where all the willing students were included in the study Sampling Method: Census method where all the willing students were included in the study Study Tool: A semi-structured questionnaire was prepared and suitable modifications were made after administering in a pilot study. The questionnaire consists of questions related to demographic information, knowledge about TB and various services offered by government and IEC related questions. National Tubercu Programme (NTEP). With this background, the current study was undertaken to assess the reach of the advertisements and hoardings related to TB in the community by enquiring newly joined medical students about various aspects of tuberculosis. This particular group was selected based on assumptions that they are active in social media, and more likely to pay attention towards hoardings or advertisements related to diseases and they belongs to different parts of state and country. Furthermore, because medical students are potential future physicians and leaders, these students need to understand the epidemiology, determinants, screening and management of TB to promote effective prevention, early diagnosis, and successful treatment and thus the opportunity was taken to introduce TB to the young budding doctors. The current study was carried out with the objectives of assessing the awareness among first year medical students about tuberculosis, government services offered to tuberculosis patients and IEC activities related to TB. Method of Data Collection: Interview method after explaining them the importance of study and obtaining consent. Confidentiality was ensured. y Analysis: Data was entered in Microsoft excel and analysis was done using SPSS statistical package version 23. Data was presented in percentages. RESULTS Most of the participants were female participants (61.2%) and city residents (46.7%). The mean age of the study participants was 17.95±0.74 years. (Table:1) 43% subjects knew TB is one of the top 10 global causes of death and only 3% knew India is the highest TB burden country in terms of new cases that occur every year. Majority (78.8%) of the students was aware of causative organism but most of the students doesn’t have knowledge about risk factors. (Table:2) Most (95.75%) of the subjects were aware of droplet mode of transmission but 37.57% opted sexual route as mode of transmission. (Table:3) 91.5% of the participants knew that TB primarily affects lungs and most of the participants were aware of fact that TB is curable and METHODS of students answered correctly (%) 1 TB is one of the top 10 global causes of death (T/F)* 71 (43%) 2 India is the highest TB burden country in terms of new cases that occur every year (T/F) 5 (3%) 3 TB is one of the leading causes of infertility in India (T/F) 14 (8.5%) 4 TB is one of the earliest opportunistic disease to develop in HIV patients 73 (44.2%) 5 What is the causative organism of TB? (Bacteria/virus/helmiths/evil eye/ Don’t know) 130 (78.8%) 6 TB affects all ages (T/F) 121 (73.3%) 7 TB is more prevalent in children than adults (T/F) 71 (43%) 8 TB is a hereditary disease (T/F) 115 (69.7%) 9 Malnutrition predispose to TB 38 (23%) 10 Diabetes increase the risk to develop TB disease 27 (16.4%) 11 Smoking can increase the risk of developing TB (T/F) 83 (50.3) T-True; F-False Table:2 Awareness about Epidemiology of Tuberculosis (n=165) Table:4 Awareness about clinical and preventive aspects of Tuberculosis (n=165) s.no Question No. of students answered correctly (%) 1 TB can affect any organ in the body (T/F) 50 (30.3) 2 TB primarily affects which organ (lungs) 151 (91.5) 3 Specimen used to diagnose TB (sputum/blood/feces/urine) 101 (61.21) 4 Is TB curable? (Yes/No/Dont know) 112 (67.9) 5 Duration of treatment (6-12 months) (n=112) 36 (32.14) 6 Is TB preventable? (Yes/No/Dont know) 136 (82.4%) 7 Is there any vaccine related to TB 74 (44.8%) 8 Name the vaccine (n=74) 8 (10.81) Table:5 Awareness about symptoms of presumptive TB case (n=165) s.no Symptoms Frequency (%) 1 Cough more than 2 weeks 137 (83.03) 2 Fever more than 2 weeks 38 (23.03) 3 Significant weight loss 64 (38.78) 4 Blood in sputum 87 (52.72) 5 Any abnormality in chest X-ray 57 (34.54) 6 Don’t know 10 (6.06) * Total more than 100% due to multiple responses Table:6 Awareness about Government services provided to TB patients and IEC activities Table:6 Awareness about Government services provided to TB patients and IEC activities ( 165) Table:6 Awareness about Government services provided to TB patients and IEC activities (n=165) s.no Government services related Question No. METHODS Study Design: Institutional based Cross Sectional Study. Study Design: Institutional based Cross Sectional Study. Study Period: This study was carried out for 1month from September 1st 2018 to September 30th 2018 Study subjects: First year medical students Study subjects: First year medical students 171 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study preventable. Only 10.81% of the subjects could name BCG vaccine. (Table:4) Most (83.03%) of the participants identified cough more than 2 weeks as symptom of presumptive TB case followed by blood in sputum (52.72%). (Table:5) Table.1 Sex & place distribution of study subjects (n=165) Sex Frequency Percentage (%) Male 64 38.8 Female 101 61.2 Residence Frequency Percentage (%) Village 24 14.5 Town 64 38.8 City 77 46.7 Table.1 Sex & place distribution of study subjects (n=165) Table:2 Awareness about Epidemiology of Tuberculosis (n=165) s.no Question No. of students answered correctly (%) 1 TB is one of the top 10 global causes of death (T/F)* 71 (43%) 2 India is the highest TB burden country in terms of new cases that occur every year (T/F) 5 (3%) 3 TB is one of the leading causes of infertility in India (T/F) 14 (8.5%) 4 TB is one of the earliest opportunistic disease to develop in HIV patients 73 (44.2%) 5 What is the causative organism of TB? (Bacteria/virus/helmiths/evil eye/ Don’t know) 130 (78.8%) 6 TB affects all ages (T/F) 121 (73.3%) 7 TB is more prevalent in children than adults (T/F) 71 (43%) 8 TB is a hereditary disease (T/F) 115 (69.7%) 9 Malnutrition predispose to TB 38 (23%) 10 Diabetes increase the risk to develop TB disease 27 (16.4%) 11 Smoking can increase the risk of developing TB (T/F) 83 (50.3) T-True; F-False Table:2 Awareness about Epidemiology of Tuberculosis (n=165) s.no Question No. METHODS of students answered correctly (%) 1 Amount charged for TB diagnostic services in government hospitals 56 (33.9%) 2 Amount charged for TB drugs in government hospitals 30 (18.2%) s.no IEC related Question (Students response) Frequency (%) 1 Have you heard/seen DOTS programme related to TB (Yes) 26 (15.8%) 2 Have you seen any hoardings/advertisements related to TB (Yes) 97 (58.8%) 3 Have u read the message (n=97) (Yes) 54 (55.67) 4 What is the message ( n=54) (Screen for TB if cough persist more than 2 weeks) 16 (29.62) 5 Identify the logo of TB control programme (correctly identified) 27 (16.4%) International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 172 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Only 33.9% subjects were aware of free diagnostic services and only 18.2% subjects were aware of free treatment services offered in government hospitals. Only 15.8% of the participants have heard of DOTS programme. 58.8% subjects have seen hoardings or advertisements related to TB but only 55.67% of them have read the message. METHODS (Table:6) However, 44.2% of the subjects knew that TB is one of the earliest opportunistic disease to develop in HIV patients in the current study and in another study conducted by Hashemi SHAB et al., in Iran, 67.9% of the secondary school students thought that HIV infected patients are susceptible to infection with tuberculosis.7 When asked about causative organism, majority (78.8%) of the participants in the current study answered bacteria which is similar to the findings of Sah BK et al., study where 84% of secondary school studentsknew causative organism of the tuberculosis.8 In another study conducted by Shedole DT et al.,it was observed that 72.8% of rural students knew that TB is caused by bacteria whereas only 60% of urban students were aware about this.9Nagasrilatha B et al., study among 1st clinical medical students found that 99.3% of the students knew Mycobacterium tuberculosis as the causative agent for tuberculosis and shocking findings were observed in Behnaz F et al., study which was done in Iran among final year medical students where only 92.9% knew about etiology of Tuberculosis.10,11 Contrasting findings were observed in Khalid FA et al., study conducted in Sudan, where only 27.1% university students knew bacteria is the causative agent of TB and surprisingly only 35.2% of pulmonary TB patients knew about causative agent as revealed by the study carried out by Konda SG et al., in Navi Mumbai.12,13 Most common source of information about TB was found to be Television (63.03%) followed by internet (50.3%) (Table:7) Table:7 Source of information about TB (n=165) Source Frequency (%) Television 104 (63.03) Radio 32 (19.39) Newspaper 62 (37.57) Internet 83 (50.3) Academic books 65 (39.39) Magazines 29 (17.57) Friends 39 (23.63) Teachers 72 (43.63) House hold members 26 (15.75) Relatives 25 (15.15) Hoardings 44 (26.66) * Total more than 100% due to multiple responses Table:7 Source of information about TB (n=165)* Awareness about Epidemiology of Tuberculosis: The current study found that most of the students were not aware of severity of TB and its impact on the community which was revealed from the observation that only 43% subjects knew TB is one of the top 10 global causes of death. The present study also found that only 3% knew India is the highest TB burden country in terms of new cases that occur every year which was very shocking. However, due to inclusion of TB in curriculum, different results were observed among final phase medical undergraduates which was evident from the study done by Raghavendra L et al., in Karnataka where 92.7% of the intern doctors knew that India is highest TB burden country and in another study done on final year medical students in China by Ou Y et al., it was found that 64.9% participants knew that TB prevalence is high in India.3,6 As expected, only 8.5% participants knew that TB is one of the leading causes of infertility in India. The current study found that 73.3% of the participants were aware of TB affecting all age groups, which is similar to the findings of Shedole DT et al., study in which 85% of urban students and 76.1% of rural students answered that TB can affect anyone.9 It was observed that 69.7% subjects answered TB is not a hereditary disease which is similar to the findings of Sah BK et al., study and Grle SP et al., study.8,14 Regarding the risk factors of TB, only 23% identified malnutrition, 16.4% knew diabetes and 50.4% answered smoking increases the risk of developing TB International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 173 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Kishore Yadav Jothula et.al. Awareness about mode of transmission: In the present study, though majority (95.75%) of the subjects identified droplet as main mode of transmission for TB, 64.24% participants also opted dishes and other articles used by patient and surprisingly 37.57% opted sexual route as mode of transmission. Similar results were identified in the study done on third year medical graduates in Chennai by Revathi R et al., where 98% students answered correctly about route of transmission and in another studies conducted on final year medical students by Laurenti P etal., in Rome, Ou Y et al., in China, it was 98.9% and 89.7% respectively.15,16,6 Only 66% of the first clinical year medical students answered correctly about TB mode of transmission in Nagasrilatha B et al., study and it is only 57.04% in Jabri AA et al., study conducted among medical professionals in Omen.10,17 The study conducted by Easwaran M et al., on rural population of Tamil nadu showed that only 26.1% subjects knew about most common mode of transmission.18Shoking results were observed in the study conducted by Honarvar B et al., on final year medical students in Iran, which stated that only 26.7% of subjects answered correctly about TB mode of transmission.19 Majority (67.9%) of the participants in the current study knew that TB is curable which is similar to the findings of Khan MN et al., study done in Pakisthan, where 67.3% of the participants answered TB as a curable disease.23 Most of the participants in Behnaz F et al.,study (97.2%) conducted among final year medical students in Iran and in Zhao Y et al., study (82.6%) conducted among first year medical students in China knew about TB as a curable disease.11,24 In a study done on non medical students in Sudan by Khalid FA et al.,only 14.7% subjects knew that TB is curable.12 Konda SG et al., study on pulmonary TB patients found that 83.6% of the subjects were aware of TB as curable disease.13 Regarding treatment duration, though it is beyond the scope of newly entered medical graduates, still 32.14% of the participants from the current study were aware of duration of TB treatment which is similar to the findings of Zhao Y et al., study where 26.2% of the first year students were aware of treatment Awareness about Epidemiology of Tuberculosis: Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study (78%) of secondary school students knew that TB doesn’t only affect lungs as revealed by Sah BK et al.,study.8 In the present study, 61.21% study subjects rightly opted sputum as specimen used to diagnose TB where as in the study conducted by Abdurehiman T et al., on interns and post graduates in Tamilnadu, it was observed that more than 90% were aware of TB confirmatory test and in another study done by Behnaz F et al., 81.7% of final year medical students accepted that the most important and accessible method for detecting TB is sputum smear acid fast staining.21,11Easwaran M et al.,study showed that only 18.3% rural population in Tamil Nadu knew about sputum smear as diagnostic test for TB and surprisingly Olakunle OS et al., study observed that only 36.25% of final year medical students in Nigeria opted sputum exam as diagnostic test for TB.18,22 In another study conducted in Karnataka by Shedole DT et al., found that 49.4% of urban PU college students and 34.4% rural PU college students were aware of TB diagnosis by sputum smear.9 in the current study. The study conducted by Grle SP et al., among student population in Zagreb found that 65.6% and 64.33% of the subjects identified smoking and diabetes as risk factors respectively and in the study done in Nepal among secondary school students by Sah BK et al., all the participants answered smoking as a risk factor.14,8 Awareness about clinical and preventive aspects of Tuberculosis: Current study showed that most (91.5%) of the participants opted lungs as primarily affected organ in TB and only 30.3% subjects knew that TB can affect any organ in the body. Otovwe A et al., study found that 56% of residents of Bambuka community were unaware of fact that TB affecting any part of the body.20 Majority International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 174 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study of TB.27,15 46.9% of secondary school students identified hemoptysis as symptom of TB in Hashemi SHAB et al., study conducted in Iran.7 Konda SG et al., study on pulmonary TB patients found low level of awareness about symptoms as indicated by the findings that 48.4% participants were aware that persistence cough is a TB symptom and other symptoms mentioned were coughing blood (24.8%), fatigue (23.1%), chest pain (12.3%), breathlessness (11.4%), and fever (5.7%).13 duration.24 In a study conducted by Abdurehiman T et al., in Tamilnadu, only 32.4% interns and 39.6% post graduates answered correctly about duration of TB treatment.21 In the present study it was observed that majority (82.4%) of the participants identified TB as a preventable disease and in another community based study conducted in Riyadh city by Huwaymil MSB et al., only 43.9% of participants knew TB as preventable disease.25 Majority (74.5%) of the pulmonary TB patients identified TB as a preventable disease in Konda SG et al., study.13 Current study revealed poor knowledge related to BCG vaccine among the study participants. Only 44.8% of the current study participants knew about availability of vaccine related to TB and only 10.81% of the subjects could name the vaccine related to TB. Awareness about Government services provided to TB patients and IEC activities: Poor knowledge among current study participants regarding free diagnostic and treatment services offered to TB patients by Government hospitals was evident from the findings that only 33.9% subjects were aware of free diagnostic services and only 18.2% subjects knew about availability of TB drugs for free of cost in Government setup. Similar results were observed in Zhao Y et al., study where only 39.5% first year medical graduates knew about free treatment policy.24Shedole DT et al., study in Karnataka found that 51.1% of urban and 40.6% of rural PU students knew about free diagnostic and treatment services for TB patients.9 The current study also highlights the poor reach of IEC activities related to TB which was reflected by the findings that only 15.8% subjects heard about DOTS and 58.8% of the participants have seen hoarding or advertisement related to TB. Out of the total participants who had seen advertisement or hoarding, only 55.67% have read the message and out of them only 29.62% could recollect the message. It was also observed that only 16.4% correctly identified RNTCP logo. More than 90% of interns and PGs correctly mentioned the DOTS expansion in Abdurehiman T et al., study and in Revathi R et al., study 85% of third year medical graduates have knowledge on DOTS because of inclusion of TB in curriculum.21,15 Zhao Y et al., observed that only 10.8% of first year Awareness about clinical and preventive aspects of Tuberculosis: Montagna MT et al., observed that 73.4% of medical students in Italy knew about BCG vaccine where as only 28.6% of the first year clinical medicine students knew about BCG vaccine as per Nagasrilatha B et al., study.26,10 Majority (90.6%) of the final year medical students were aware of BCG vaccine as observed by Ou Y et al., study conducted in China and Sah BK et al., study on secondary school students in Nepal found that most (92%) of the subjects were aware of vaccine related to TB.6,8 REFERENCES 1. Park K. Park’s textbook of Preventive and Social Medicine. 25th ed. Jabalpur: M/S Banarsidas Bhanot Publishers;2019.p.188- 90 2. WHO. Global Tuberculosis Report 2019: Country profiles for 30 high TB burden countries. France: WHO; 2019.p204 Available from: who.int/iris/bitstream/handle/10665/329368/ 9789241565714-eng.pdf (Accessed on: 15th January 2020) 3. Raghavendra L, Babu SP, Shivakumar KM. Assessment of knowledge of intern doctors of a medical college hospital in Karnataka on revised national TB control programme. Int J Adv Med 2017;4:1123-7 3. Raghavendra L, Babu SP, Shivakumar KM. Assessment of knowledge of intern doctors of a medical college hospital in Karnataka on revised national TB control programme. Int J Adv Med 2017;4:1123-7 Source of information about TB: Declarations Funding: No Funding Sources Conflict of interest: None Declared Ethical approval: The study was approved by Funding: No Funding Sources Conflict of interest: None Declared Funding: No Funding Sources Conflict of interest: None Declared Most common source of information in the current study was found to be Television (63.03%) followed by Internet (50.3%) and Teachers (43.63%). Other studies done by Hashemi SHAB et al.,on secondary school students and Srivastava DK et al., on Government and private practitioners found that television was the most common source of information.7,28 Abdurehiman T et al., study and Khan MN et al., study on medicos found internet as main source of information.21,23 Health workers were the main source of information in the study done by Konda SG et al., study on pulmonary TB patients.13 Mass media and teachers was the main source of information for non medical students as per the findings of Sah BK et al., study and Shedole DT et al., study.8,9 Most common source of information in the current study was found to be Television (63.03%) followed by Internet (50.3%) and Teachers (43.63%). Other studies done by Hashemi SHAB et al.,on secondary school students and Srivastava DK et al., on Government and private practitioners found that television was the most common source of information.7,28 Ethical approval: The study was approved by the Institutional Ethical Committee Awareness about symptoms of presumptive TB case: p p When enquired about symptoms of presumptive TB case in the present study, most (83.03%) of the participants answered cough more than 2 weeks followed by blood in sputum (52.72%), significant weight loss (38.68%) and any abnormality in chest X- ray (34.54%). Only 23.03% subjects identified fever more than 2 weeks as presumptive TB symptom. Chennaveerappa PK et al., study in Karnataka found that 85.1% of medical students identified cough as most common symptom of TB, which is similar to the findings of Revathi et al., study in which 80% of third year medical students identified most common symptoms 175 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study ACKNOWLEDGEMENTS medical graduated have heard of DOTS which is similar to the findings of current study.24 The authors were grateful to all the students who had participated in the study. CONCLUSION A comparative study on awareness about tuberculosis among urban and rural P.U college students of Davangeretaluk, Karnataka, India. Int J Community Med Public Health 2015;2:552- 7. 20. Agofure O, Okandeji-Barry ORA, Musa E and Odjimogho S. Knowledge of the Prevention and Management of Tuberculosis among Residents of Bambuka Community Karim Lamido Local Government Area of Taraba State. SM J Public Health Epidemiol. 2018; 4(1): 1048. 10. Nagasrilatha B, Sasidhar M, Bharathi M, Sasikala A, S. Bai SK. A study on impact of curriculum on knowledge of tuberculosis among medical students. Journal of Education Technology in Health Sciences 2016;3(3):91-94. 21. Abdurehiman T, Ramachandran K, Prasath RA, Srinivasan R. To assess the awareness and knowledge of pulmonary tuberculosis and RNTCP guidelines among interns and postgraduates at a tertiary care hospital in south India. Paripex Indian J. Res. 2018; 7(1):80-82. 11. Behnaz F, Mohammadzade G, Mousavi-e- roknabadi RS, Mohammadzadeh M. Assessment of knowledge, attitudes and practices regarding tuberculosis among final year students in Yazd, Central Iran. J Epidemiol Glob Health 2014;4:81-85 22. Olakunle OS, Oladimeji O, Olalekan AW, Olugbenga-Bello1 A, Akinleye C, OluwatoyinOA. Knowledge of tuberculosis management using directly observed treatment short course therapy among final year medical students in South Western Nigeria. Pan Afr Med J. 2014;18:32. 12. Khalid FA, Mohammed AA. Knowledge and awareness of Tuberculosis among Students of University of Kassala, Sudan. Sudan JMS 2013;8(1):5-8 13. Konda SG, Melo CA, Giri PA. Knowledge, attitude and practices regarding tuberculosis among new pulmonary tuberculosis patients in a new urban township in India. Int J Med Sci Public Health 2016;5:563-569. 23. Khan MN, Khan N, Anwer G, Akbar A (2017) Knowledge, Practice & Awareness of Tuberculosis Disease among Medical & Non-medical Students. J BioequivAvailab 9: 561- 564 14. Grle SP, Plavec D, Brcina N, Borcic T. Differences in the knowledge about tuberculosis among general urban population and student population in Zagreb. Medicina fluminensis 2014;50(1): 98-105. 24. Zhao Y, Ehiri J, Li D, et al. A survey of TB knowledge among medical students in Southwest China: is the information reaching the target?. BMJ Open 2013; 3:e003454. 15. Revathi R, Dharanisri R. Knowledge about tuberculosis among undergraduate medical students in a private college in Chennai. Int J Community Med Public Health 2018; 5:644-6. 25. Huwaymil MSB, Alkhalifah MA, Alsaqabi MAAOA, Alhabshan RF, Alzaid AN. Assessment of Knowledge, Attitudes and Practices Regarding Pulmonary Tuberculosis among Community in Riyadh City, 2017. CONCLUSION 4. Central Tuberculosis Division, Government of India. Thematic areas: National strategic plan 2017-2025 for TB elimination in India. Available at: tbcindia.gov.in/WriteReadData/National%2 0Strategic%20Plan%202017-25.pdf (Accessed on: 15th January 2020) Tuberculosis is one of the leading causes of morbidity and mortality in our country and it is time to act and the responsibility lies upon each and every one of the citizens to curb TB. The current study found that majority participants were aware about causative organism and symptoms of TB but doesn’t have clue regarding government services and IEC activities related to TB though Government is spending huge amount of money. It is the need of the hour to bring awareness in the community about high quality diagnostic services and treatment which is provided for free of cost by government through RNTCP programme. There is a need to sensitise the community through intense IEC activities to utilise government services and also encourage private practitioners to involve in RNTCP programme in order to end TB by 2025 in India. 5. Thirunavukkarasu Prasanna, Kathiresan Jeyashree, PalanivelChinnakali, Yogesh Bahurupi, Kavita Vasudevan, Mrinalini Das (2018) catastrophic costs of tuberculosis care: a mixed methods study from Puducherry, India, Global Health Action, 11:1 6. Ou Y, Luo Z, MouJ , Ming H, Wang X, Yan S, et al. Knowledge and determinants regarding tuberculosis among medical students in Hunan, China: a cross-sectional study. BMC Public Health 2018;18:730 7. Bani Hashemi SH, Khorgoei T, Mahboobi HR, Shahrzad ME, Amirzadeh Shams Sh, Mandegari Z, Yazdanparast M, Masqati M. Knowledge and attitudes towards tuberculosis among secondary school students in rural areas in Hormozgan, Southern Iran. Int Elec J Med 2011;1(1):11- 6 176 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study Tamil Nadu. Int J Med Sci Public Health 2015;4:1681-1684. 8. Sah BK, Sah JP, Shah SK, et al. Knowledge on tuberculosis among students of higher secondary school, lalitpur, Nepal. MOJ Public Health. 2016;4(5):133‒141. 19. Honarvar B, Shaygani F, Amini M, Shirdel F, Kiani F, Kazemi M, et al. Trend Analysis of Knowledge and Practice of National Tuberculosis Guidelines Among Medical Students: ATwo-Year Interventional Study in Shiraz, Iran. Shiraz E-Med J. 2018; 19(3):1-7 9. Shedole DT, Devalgi SB, Vidya GS, Mendagudli RR, Kumar VB. CONCLUSION The Egyptian Journal of Hospital Medicine 2017;69(5):2417-2420. 16. Laurenti P, Federico B, Raponi M, Furia G, Ricciardi W, Damiani G. Med Sci Monit 2013;19:865-874 17. Al-Jabri AA, Dorvlo ASS, Al-Rahbi S, Al- Abri J, Al-Adawi S. Knowledge of tuberculosis among medical professionals and university students in Oman. East.Mediterr.Health J. 2006;12(5):509-521 26. Montagna et al.: Knowledge about tuberculosis among undergraduate health care students in 15 Italian universities: a cross-sectional study. BMC Public Health 2014;14:970 18. Easwaran M, Ramachandran D, Ramasamy R, George N, Mathew M, Bazroy J, et al. Knowledge, attitude, and practice regarding tuberculosis among rural population in 27. Chennaveerappa P. K, Rajashekar H. K, Jayashree Nagaral, Halesha B. R, Raghavendra Prasad K. U, Vinaykumar M. 177 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 Kishore Yadav Jothula et.al. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study district Gwalior, India: An operational research. Indian J Tuberc2011;58:168-177 V, Nareshkumar M. N. “A Study on Awareness of Tuberculosis and RNTCP among Undergraduate Medical students and Interns”. Journal of Evolution of Medical and Dental Sciences 2014; Vol. 3, Issue 29, July 21; Page: 8115-8121. How to cite this article: Jothula KY, Sreeharshika D, Srikanth JV. Awareness among first year medical students in Telangana state about tuberculosis and government services offered to tuberculosis patients: a cross sectional study. International Journal of Research and Review. 2020; 7(7): 170-178. 28. Srivastava DK, Mishra A, Mishra S, Chouksey M, Jain P, Gour N, et al. A comparative assessment of KAP regarding tuberculosis and RNTCP among Government and Private practitioners in ****** International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 178 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020
https://openalex.org/W2037790441	https://europepmc.org/articles/pmc2737784?pdf=render	English	null	Management of deep neck infection by a transnasal approach: a case report	Journal of medical case reports	2,009	cc-by	3,397	Case report Management of deep neck infection by a transnasal approach: a case report Yuh Baba1,2,3, Yasumasa Kato4, Hideyuki Saito3 and Kaoru Ogawa3 p Yuh Baba1,2,3, Yasumasa Kato4, Hideyuki Saito3 and Kaoru Addresses: 1Department of Otorhinolaryngology, Tochigi National Hospital, 1-10-37 Nakatomatsuri, Utsunomiya, Tochigi 320-8580, Japan 2Department of Otorhinolaryngology, Otsuka Hospital, Tokyo 152-8902, Japan Addresses: Department of Otorhinolaryngology, Tochigi National Hospital, 1-10-37 Nakatomatsuri, Utsunomiya, Tochigi 320-8580, Japan 2Department of Otorhinolaryngology, Otsuka Hospital, Tokyo 152-8902, Japan 3Department of Otorhinolaryngology, Head and Neck Surgery, Keio University, 35 Shinanomachi Shinjuku, Tokyo 160-0082, Japan 4Department of Biochemistry & Molecular Biology, Kanagawa Dental College, Yokosuka 238-8580, Japan p y g gy, p , y , J p 3Department of Otorhinolaryngology, Head and Neck Surgery, Keio University, 35 Shinanomachi Shinjuku, Tokyo 160-0082, Japan 4Department of Biochemistry & Molecular Biology, Kanagawa Dental College, Yokosuka 238-8580, Japan Email: YB* - yuh_baba@hotmail.com * Corresponding author Received: 10 August 2008 Accepted: 2 February 2009 Published: 31 July 2009 Journal of Medical Case Reports 2009, 3:7317 doi: 10.4076/1752-1947-3-7317 Journal of Medical Case Reports 2009, 3:7317 doi: 10.4076/1752-1947-3-7317 This article is available from: http://jmedicalcasereports.com/jmedicalcasereports/article/view/7317 article is available from: http://jmedicalcasereports.com/jmedic © 2009 Baba et al.; licensee Cases Network Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2009 Baba et al.; licensee Cases Network Ltd. © 2009 Baba et al.; licensee Cases Network Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativ which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly ci Abstract Introduction: Deep neck infection is a life-threatening condition, and intravenous antibiotic therapy is preferable in the early stages of the disease. However, in the advanced stages, surgical drainage should be performed. Although several surgical treatment strategies are available, it is necessary to standardize treatment according to the patient’s general condition and history. Case presentation: We report the case of a 68-year-old man with a deep neck abscess and with severe diabetes mellitus and inflammation. Computed tomography identified a deep neck infection extending from the level of the epipharynx to that of the hyoid bone. We performed surgical drainage by transnasal endoscopy. The patient exhibited no evidence of either recurrent disease or post- surgical complications within 30 months of follow-up. Conclusions: This case report provides evidence that transnasal endoscopic drainage should be recommended as a standard approach in patients with a deep neck abscess and with a severe general condition, diabetes mellitus, and inflammation. Open Access Page 1 of 5 (page number not for citation purposes) Case presentation p In July 2005, a 68-year-old man was admitted to our institution with a 3-week history of low-grade fever and headache on the left side. The patient had a history of chronic sinusitis, and also had DM. Transnasal fiberscopy revealed left lateral pharyngeal wall edema, or asymme- trical bulging from the level of the Rosenmüller fossa to that of the uvula, and no inflammatory signs in the pharyngeal tonsil (Figure 1). Laboratory evaluation revealed severe inflammatory conditions in our patient and suggested the requirement for surgical treatment: white blood cells (WBC), 19,000 cells/mm3; C-reactive protein (CRP), 42.68 mg/dL; HbA1c, 14.5%; and glucose, 565 mg/dL. Computed tomography (CT) of the neck showed marked thickening of the epipharynx on the left side (Figure 2A). The lesion extended from the level of the epipharynx to that of the hyoid bone. Signs of left hypoglossal palsy and left Horner’s syndrome were also evident. Thus, the lesion was due to deep neck infection extending from the epipharynx to the surrounding poststyloid space, although CT did not show any typical features of abscess at this time (1 week before the operation) (Figure 2A). Micro-otoscopy showed left purulent otorrhea through a central perforation of the tympanic membrane. twice/day), we suspected a very acute stage of abscess formation after diagnosis by CT. Unfortunately, we could not repeat the CT scan to confirm abscess formation, because repeated CT scans to check progress within 30 days are not covered by either Employees’ Insurance or National Health Insurance in the Japanese Social Insurance System. We elected not to perform a transcervi- cal procedure under general anesthesia due to severe DM, severe inflammation, and trismus. In addition, an external approach under local anesthesia was considered difficult because the region of suspected abscess formation was located mainly between the levels of the base of the skull and soft palate medial to the great vessels, and did not involve multiple spaces, and the patient did not have any airway obstruction. Therefore, due to the patient’s severe general condition and the anatomical location of the lesion, we instead chose to perform surgical drainage using a transnasal endoscopic approach under local anesthesia 1 week after CT scanning. A vertical incision, 1 cm in length, was made in the lateral epipharyngeal wall. The opening was enlarged by remov- ing some inflamed mucosa with forceps. Introduction of a deep neck abscess were dental infection (43%) and intravenous drug abuse (12%). About 70% of the abscesses were in two locations, the submandibular space and the lateral pharyngeal space; and the most frequent bacteria responsible for abscess formation were Streptococcus viridans, Staphylococcus epidermidis, Staphylo- coccus aureus, and b-hemolytic streptococci [2]. Diabetes mellitus (DM) was a common associated systemic disease occurring in 34 of the 210 cases (16%) [2]. They also Deep neck infection is a life-threatening condition with various serious complications, such as, airway obstruction, cranial nerve palsy, descending necrotizing mediastinitis, internal carotid compression, and rupture [1]. Its localiza- tion on the floor of the mouth can be a particularly serious threat. The etiology of a deep neck infection can be varied. Parhiscar and Har-El determined the etiology, location, and bacteriology in 210 cases. The most common causes Page 1 of 5 (page number not for citation purposes) http://jmedicalcasereports.com/jmedicalcasereports/article/view/7317 Journal of Medical Case Reports 2009, 3:7317 Journal of Medical Case Reports 2009, 3:7317 Figure 1. Transnasal endoscopic view of the left nasal cavity showing left lateral nasopharyngeal wall edema (black arrow) and the pharyngeal opening of the auditory tube (PO). Figure 1. Transnasal endoscopic view of the left nasal cavity showing left lateral nasopharyngeal wall edema (black arrow) and the pharyngeal opening of the auditory tube (PO). reported that the incidence of abscesses in the retro- pharyngeal space was 12% (25/210 cases), including eight cases (32%) treated with tracheotomy [2]. In selected cases where the extent of the infection is limited, conservative treatment with intravenous antibio- tic therapy can be successful. In advanced cases, however, surgical exploration with drainage of the abscess is generally required. Different traditional surgical approaches have been described in relation to the site of infection and the involvement of adjacent structures [3]. Endoscopic approaches have a number of important advantages in comparison to external approaches, includ- ing minimal complication, the absence of cervical scarring, and a short operation time. Nagy et al. reported successful treatment of 22/23 pediatric patients by transoral drainage of deep neck infections, including three cases of para- pharyngeal abscess [4]. Transnasal endoscopic drainage for retroparapharyngeal abscess was reported in two cases [5]. We report that transnasal endoscopy can be effective for the drainage of deep neck abscesses in patients in poor general condition, such as those with severe DM and inflammation. Figure 1. Introduction Transnasal endoscopic view of the left nasal cavity showing left lateral nasopharyngeal wall edema (black arrow) and the pharyngeal opening of the auditory tube (PO). Page 2 of 5 (page number not for citation purposes) Case presentation We observed no apparent pharyngeal constrictor muscle on transnasal endoscopic surgery, which may have been due to severe tissue encroachment by inflammation, and we could reach As symptoms persisted despite antibiotic therapy (mer- openem trihydrate 1 g twice/day and clindamycin 600 mg Page 2 of 5 (page number not for citation purposes) Page 2 of 5 (page number not for citation purposes) http://jmedicalcasereports.com/jmedicalcasereports/article/view/7317 http://jmedicalcasereports.com/jmedicalcasereports/article/view/7317 Journal of Medical Case Reports 2009, 3:7317 Figure 3. Treatment process. CLDM, clindamycin; MEPM, meropenem trihydrate; ABK, arbekacin sulfate; ST, sulfamethoxazole trimethoprim; MINO, minocycline hydrochloride. into the poststyloid space using only forceps without a i d b id i i i h l id material yielded S. aureus. Histological examinatio revealed only nonspecific inflammatory cells and fibrou cells. These clinical and laboratory observations supporte our diagnosis of deep neck abscess. The postoperative course was uneventful. Transnasa endoscopic aspiration was started on the first pos operative day and continued once a day for 30 days. Th patient reported a rapid improvement in symptom except those related to cranial nerve palsy. Six week later, endoscopy showed normalization of the epipharyn CT examination demonstrated the presence of a sma necrotic retropharyngeal lymph node at the level of th epipharynx (Figure 2B). Laboratory analyses showe improvement in inflammation: WBC, 7600 cells/mm CRP, 0.47 mg/dL (Figure 3). Thirty months after surgery the patient had no symptoms other than those related t cranial nerve palsy. Discussion Although the advancement of antibiotics has markedl reduced the incidence and mortality rates, deep nec infection remains a challenging problem due to th complex anatomy and potentially lethal complication that may arise [1]. Deep neck infection is usually due t odontogenic, pharyngeal, tonsillar, salivary gland, midd ear, or mastoid infections [2,6]. In this patient, w observed otitis media from the middle ear but no sign of odontogenic, pharyngeal, tonsillar, or salivary glan inflammation. We could not determine the exact sequenc f h l di h f d k i f i i Figure 2. (A) Computed tomography of the neck with intravenous contrast showing the left cellulitis at the level of the nasopharynx (white arrow). (B) Computed tomography examination (6 weeks after surgery) indicated the presence of a necrotic retropharyngeal lymph node at the level of the nasopharynx (white arrow). Figure 3. Treatment process. CLDM, clindamycin; MEPM, meropenem trihydrate; ABK, arbekacin sulfate; ST, sulfamethoxazole trimethoprim; MINO, minocycline hydrochloride. Figure 3. Treatment process. Page 3 of 5 (page number not for citation purposes) Case presentation CLDM, clindamycin; MEPM, meropenem trihydrate; ABK, arbekacin sulfate; ST, sulfamethoxazole trimethoprim; MINO, minocycline hydrochloride. Figure 2. (A) Computed tomography of the neck with intravenous contrast showing the left cellulitis at the level of the nasopharynx (white arrow). (B) Computed tomography examination (6 weeks after surgery) indicated the presence of a necrotic retropharyngeal lymph node at the level of the nasopharynx (white arrow). material yielded S. aureus. Histological examination revealed only nonspecific inflammatory cells and fibrous cells. These clinical and laboratory observations supported our diagnosis of deep neck abscess. The postoperative course was uneventful. Transnasal endoscopic aspiration was started on the first post- operative day and continued once a day for 30 days. The patient reported a rapid improvement in symptoms, except those related to cranial nerve palsy. Six weeks later, endoscopy showed normalization of the epipharynx. CT examination demonstrated the presence of a small necrotic retropharyngeal lymph node at the level of the epipharynx (Figure 2B). Laboratory analyses showed improvement in inflammation: WBC, 7600 cells/mm3; CRP, 0.47 mg/dL (Figure 3). Thirty months after surgery, the patient had no symptoms other than those related to cranial nerve palsy. Discussion Although the advancement of antibiotics has markedly reduced the incidence and mortality rates, deep neck infection remains a challenging problem due to the complex anatomy and potentially lethal complications that may arise [1]. Deep neck infection is usually due to odontogenic, pharyngeal, tonsillar, salivary gland, middle ear, or mastoid infections [2,6]. In this patient, we observed otitis media from the middle ear but no signs of odontogenic, pharyngeal, tonsillar, or salivary gland inflammation. We could not determine the exact sequence of the events leading to the onset of deep neck infection in this patient, whether the acute otitis media was secondary to compression of the eustachian tube or vice versa, or whether the deep neck infection was a complication of a middle ear infection. As the patient suffered repeatedly Figure 2. (A) Computed tomography of the neck with intravenous contrast showing the left cellulitis at the level of the nasopharynx (white arrow). (B) Computed tomography examination (6 weeks after surgery) indicated the presence of a necrotic retropharyngeal lymph node at the level of the nasopharynx (white arrow). into the poststyloid space using only forceps without a microdebrider. Dissection into the poststyloid space produced an enormous amount of purulent material. Upon inspection with a 30° angled fiberscope, pulsation of the left internal carotid artery was clearly visible. The operation time was 20 minutes. Cultures of the purulent Page 3 of 5 (page number not for citation purposes) http://jmedicalcasereports.com/jmedicalcasereports/article/view/7317 Journal of Medical Case Reports 2009, 3:7317 Journal of Medical Case Reports 2009, 3:7317 from acute sinusitis 3 months after the operation, we postulated that the deep neck infection occurred following acute sinusitis. inflamed mucosa with a microdebrider, drainage of purulent collection, and careful dissection and removal of the necrotic tissue [5]. from acute sinusitis 3 months after the operation, we postulated that the deep neck infection occurred following acute sinusitis. Generally, external drainage requires about 2 to 3 hours. The transnasal endoscopic approach involves a shorter operating time than the external approach with minimal complications. However, transnasal endoscopic drainage for a deep neck abscess is also accompanied by a risk of a relatively long hospitalization period depending on the requirement for repetition of drainage. With regard to both key issues, the transnasal endoscopic approach was considered suitable especially in elderly patients with severe concomitant diseases. Discussion Here, we present a patient with a deep neck abscess treated successfully by transnasal endoscopy as the patient’s general condition was con- sidered unsuitable for general anesthesia (because of factors including age, DM, severe inflammation and trismus). The lesion was located mainly from the level of the base of the skull to that of the soft palate, located medial to the great vessels, and did not involve multiple spaces. The patient did not have airway obstruction. To date, only a small number of patients have been treated by the transnasal endoscopic approach. Endoscopic approaches have a number of important advantages in comparison to external approaches, including minimal complications, the absence of cervical scarring, availability of repeated drainage, and short operation time. Therefore, transnasal endoscopic drainage of a deep neck abscess is an effective alternative to external approaches in patients with a severe general condition, DM, and inflammation. Despite these advantages, however, it is still controversial as to whether the transnasal endoscopic approach for treatment of deep neck abscesses is suitable for patients with a better general condition as this approach is associated with a risk of vascular injury, dyspnea, and incomplete drainage. Appropriate treatment planning for patients with a deep neck infection requires clear differentiation between cellulitis and abscess. Imaging of the soft tissue of the neck has developed significantly using CT scanning technology, which plays a fundamental role in the diagnosis of deep neck infection. In this patient, CT showed cellulitis, but no apparent abscess one week before the operation. However, we observed an enormous amount of purulent material on dissection into the poststyloid space. This discrepancy may have been because the patient was in a very acute stage of abscess develop- ment from cellulitis seven days after diagnosis based on the results of the CT scan. In addition to external incision for drainage, percutaneous ultrasound- or CT-guided aspiration of deep neck abscesses using a spinal needle has been reported [7,8]. However, we could not use these approaches in this patient due to a lack of typical imaging features of the abscess on the CT scan. In addition, the patient’s general condition was poor due to severe DM and inflammation, and therefore a minimally invasive treatment was required. Thus, a transnasal endoscopic approach, which could reveal the lesion by visual inspection, was advantageous in this patient. Abbreviations To our knowledge, there have only been two previous reports of a transnasal endoscopic approach for drainage of a deep neck abscess [5,10]. Sethi and Stanley briefly mentioned the use of a transnasal endoscopic approach in eight patients with deep neck infections, without provid- ing information about the indications or surgical techni- que used [10]. Nicolai et al. reported that the main surgical steps were incision of the pharyngeal mucosal wall with a diode laser, widening of the incision by eliminating some CRP, C-reactive protein; CT, computed tomography; DM, diabetes mellitus; WBC, white blood cells. Discussion Several surgical approaches are available in relation to the site and extent of the infection [3]. Deep neck infections are usually drained through an external approach. As recommended by Sethi and Stanley [9], the entire cavity is then explored by blunt finger dissection to avoid any residual purulent material, particularly in the case of multilocular abscesses. Moreover, tracheotomy can be performed to avoid respiratory distress in patients with compromised upper airway patency. Conclusions In this patient, the abscess was located mainly in the upper region from the epipharynx to the surrounding poststyloid space near the great vessels, and the possibility of draining the collection by an endoscopic transnasal route, without resorting to an external approach, was offered to this patient. The observations in our patient indicated that transnasal endoscopic drainage has advantages for treatment of deep neck abscesses in patients with a severe general condition, diabetes mellitus, and inflammation. Thus, this method could be recommended as a standard approach in such cases. p g The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Consent Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Page 4 of 5 (page number not for citation purposes) Page 4 of 5 (page number not for citation purposes) http://jmedicalcasereports.com/jmedicalcasereports/article/view/7317 Journal of Medical Case Reports 2009, 3:7317 10. Sethi DS, Stanley RE: Deep neck abscesses-changing trends. J Laryngol Otol 1994, 108:138-143. Authors’ contributions YB assisted in the conception and design of the paper, and also helped in the acquisition, review and interpretation of the data. YK and HS contributed towards data collection and drafting of the manuscript. KO was involved in conception, reviewing and finally approving the version to be published. All authors read and approved the final manuscript. References 1. Sichel JY, Dano I, Hocwald E, Biron A, Eliashar R: Nonsurgical management of parapharyngeal space infections: a prospec- tive study. Laryngoscope 2002, 112:906-910. References 1. Sichel JY, Dano I, Hocwald E, Biron A, Eliashar R: Nonsurgical management of parapharyngeal space infections: a prospec- tive study. Laryngoscope 2002, 112:906-910. y y g p 2. Parhiscar A, Har-El G: Deep neck abscess: a retrospective review of 210 cases. Ann Otol Rhinol Laryngol 2001, 110:1051-1054. 3. Toshima M: Deep neck infection. Antibiot Chemother (Japanese) 2000, 16:1715-1720. 4. Nagy M, Pizzuto M, Backstrom J, Brodsky L: Deep neck infections in children: a new approach to diagnosis and treatment. Laryngoscope 1997, 107:1627-1634. y g p 5. Nicolai P, Lombardi D, Berlucchi M, Farina D, Zanetti D: Drainage of retro-parapharyngeal abscess: an additional indication for endoscopic sinus surgery. Eur Arch Otorhinolaryngol 2005, 262:722-730. 6. Lazor JB, Cunningham MJ, Eavey RD, Weber AL: Comparison of computed tomography and surgical findings in deep neck infections. Otolaryngol Head Neck Surg 1994, 111:746-750. 7. Baatenburg de Jong RJ, Rongen RJ, Lameris JS, Knegt P, Verwoerd CD: Ultrasound-guided percutaneous drainage of deep neck abscesses. Clin Otolaryngol Allied Sci 1990, 15:159-166. y g 8. Poe LB, Petro GR, Matta I: Percutaneous CT-guided aspiration of deep neck abscesses. Am J Neuroradiol 1996, 17:1359-1363. p J 9. Sethi DS, Stanley RE: Parapharyngeal abscesses. J Laryngol Otol 1991, 105:1025-1030. p J 9. Sethi DS, Stanley RE: Parapharyngeal abscesses. J Laryngol Otol 1991, 105:1025-1030. 10. Sethi DS, Stanley RE: Deep neck abscesses-changing trends. J Laryngol Otol 1994, 108:138-143. Do you have a case to share? 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https://openalex.org/W1893948891	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0136639&type=printable	English	null	Effects of Electrode Material on the Voltage of a Tree-Based Energy Generator	PloS one	2,015	cc-by	6,752	Introduction When two metal electrodes are installed in a tree and the adjacent soil, a voltage develops between the electrodes [1]. This voltage has been regarded as an innovative renewable energy generator. Compared with other natural energy sources, such as solar energy and wind energy, this type of energy has certain advantages, including eco-friendliness, a long lifetime, and no restrictions with respect to the hermetic environment in the forest. Hence, scholars have fore- casted practical applications of the tree-voltage that include a wide variety of trickle chargers for niche environmental sensors and mesh-networked devices, drastically decreasing the need for in-the-field battery changes [2]. Sensors and networks used in forestry have extremely low energy consumption as a result of their low-power design and sleep function. The tree-based voltage has the ability to provide uninterruptible energy when charging a low-voltage battery to meet the demands of discontinuous power consumption. Therefore, the tree-based voltage is a promising energy source. This source is expected to provide a brand-new power generation Data Availability Statement: All relevant data are within the paper. Effects of Electrode Material on the Voltage of a Tree-Based Energy Generator Zhibin Hao☯, Guozhu Wang☯, Wenbin Li☯, Junguo Zhang☯, Jiangming Kan☯ School of Technology, Beijing Forestry University, Beijing, China ☯These authors contributed equally to this work. leewb@bjfu.edu.cn OPEN ACCESS OPEN ACCESS Citation: Hao Z, Wang G, Li W, Zhang J, Kan J (2015) Effects of Electrode Material on the Voltage of a Tree-Based Energy Generator. PLoS ONE 10(8): e0136639. doi:10.1371/journal.pone.0136639 Editor: Bing-Yang Cao, Tsinghua University, CHINA Received: June 15, 2015 Accepted: August 5, 2015 Published: August 24, 2015 Citation: Hao Z, Wang G, Li W, Zhang J, Kan J (2015) Effects of Electrode Material on the Voltage of a Tree-Based Energy Generator. PLoS ONE 10(8): e0136639. doi:10.1371/journal.pone.0136639 Citation: Hao Z, Wang G, Li W, Zhang J, Kan J (2015) Effects of Electrode Material on the Voltage of a Tree-Based Energy Generator. PLoS ONE 10(8): e0136639. doi:10.1371/journal.pone.0136639 Editor: Bing-Yang Cao, Tsinghua University, CHINA Received: June 15, 2015 Accepted: August 5, 2015 Published: August 24, 2015 Editor: Bing-Yang Cao, Tsinghua University, CHINA Received: June 15, 2015 Accepted: August 5, 2015 Published: August 24, 2015 Editor: Bing-Yang Cao, Tsinghua University, CHINA Received: June 15, 2015 Accepted: August 5, 2015 Published: August 24, 2015 Copyright: © 2015 Hao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The voltage between a standing tree and its surrounding soil is regarded as an innovative renewable energy source. This source is expected to provide a new power generation sys- tem for the low-power electrical equipment used in forestry. However, the voltage is weak, which has caused great difficulty in application. Consequently, the development of a method to increase the voltage is a key issue that must be addressed in this area of applied research. As the front-end component for energy harvesting, a metal electrode has a mate- rial effect on the level and stability of the voltage obtained. This study aimed to preliminarily ascertain the rules and mechanisms that underlie the effects of electrode material on volt- age. Electrodes of different materials were used to measure the tree-source voltage, and the data were employed in a comparative analysis. The results indicate that the conductivity of the metal electrode significantly affects the contact resistance of the electrode-soil and electrode-trunk contact surfaces, thereby influencing the voltage level. The metal reactivity of the electrode has no significant effect on the voltage. However, passivation of the elec- trode materials markedly reduces the voltage. Suitable electrode materials are demon- strated and recommended. RESEARCH ARTICLE Effects of Electrode Material on the Tree-Voltage system for the low-power electrical equipment used in forestry. However, studies have shown that this type of energy is weak, i.e., the voltages obtained are typically in the tens to hundreds of millivolts range, while the short circuit currents are in the microampere range [2]. A low electrical power in the microwatt range (approximately 20 μW) was measured in a previous study. These features are insufficient for directly driving the low-power electronic equipment currently used in forests. The technology for harvesting and utilizing micro-energy, which includes piezoelectric energy, photovoltaic energy, and thermoelectric energy, is relatively well-developed [3–8]. To acquire the suitable voltage level necessary to charge a storage circuit and subsequently to power electronic devices, a boosting operation with a direct current-to-direct current (DC-DC) converter is generally required [9, 10]. However, the voltage between a standing tree and its surrounding soil is quite low and cannot reliably drive the present DC-DC circuit modules. That is, the existing challenge in this regard is to increase the voltage. To solve this problem, relevant studies have focused on two major topics: 1) ascertaining the mechanism behind this voltage [2], thereby determining whether the voltage may be artificially increased, and 2) find- ing novel boosting methods while designing innovative low-voltage electronics [11, 12]. Research on the mechanism of tree-based energy is an important task. It is well-known that electric potential differences exist in plant bodies. However, this type of voltage is typically caused by the physiological activities of plants [13–16]. This phenomenon can be explained at a microscopic level, in particular at the cellular level. The electric potential difference caused by physiological activities is extremely weak (at most tens of millivolts) and sometimes transient (generated in response to external stimuli) [17]. This is negligible in studies of the tree-soil volt- age, which is typically in the tens to hundreds of millivolts range. Studies of the voltage that arises between a standing tree and its surrounding soil belong to a new field of research. Early observations of tree trunk electrical activity were confirmed by an experiment on a spruce tree [18], which further reported a non-linear relationship between the electric potential variation and the intensity of the solar radiation. Some scholars have claimed that sap flow is the domi- nating mechanism for voltage generation [19]. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the National Natural Science Foundation of China (http://www. nsfc.gov.cn/), Grant No.: 31170669 to WL and the Fundamental Research Funds for the Central Universities, Project No.: BLYJ201421 to ZH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Moreover, other scholars have proposed a mechanism based on charge diffusion from the conductive sap flow channels into the resistive xylem walls [18]. However, electrical signals with more erratic time variations were still observed in the experiments, indicating that other mechanisms, possibly unrelated to sap flow, may also contribute. Atmospheric electricity was claimed to contribute to temporal voltage var- iations. However, inherent voltages were also observed in those studies [20–22]. In addition, it was found that the pH difference between the interior of the tree and its surrounding soil sig- nificantly affects the voltage [2]. Therefore, the mechanism behind the tree-based energy gener- ator remains controversial, leading to significant unknown factors that continue to affect its application. The invention of innovative electronics is another objective that must be met to utilize tree- based energy. A DC-DC converter can boost the input voltage and generate constant output, but such a circuit requires a minimum threshold voltage as the input, which a standing tree cannot reliably generate. In response to this problem, some researchers have designed a boost- circuit that has low power consumption and a low input voltage, as well as a low-power clock circuit. Both circuits have been driven by a standing tree with relatively good performance [11]. However, to utilize this type of electricity more reliably, various factors, such as the harvest- ing, conversion, and storage of electricity, must be considered. During the process of utilizing standing-tree electricity, electrodes should be arranged in the standing tree and in the sur- rounding soil, as the front-end equipment for energy harvesting. However, the electrode mate- rial may affect the magnitude and stability of the electricity obtained. Because the mechanism 2 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage behind the tree-based voltage remains unclear, no universal standard has been established for electrode selection during the process of measuring and utilizing tree electricity [2, 11, 12, 18– 22]. However, metal electrodes remain the preferred material of scientists, based on the rele- vant literature. Most previous studies have used steel electrodes [11, 12, 18–22]. Moreover, platinum (Pt) electrodes have also been used in some studies [2]. Regardless, to take advantage of such a weak energy source with high efficiency, the choice of a suitable electrode is a central issue. Experimental electrodes Based on the objective of this study, three different materials with different conductivities and stabilities, including copper (Cu), aluminum (Al), and iron (Fe), were selected. The conductivi- ties of Cu, Al, and Fe are different, varying in the order of Cu to Al and Fe for strong to weak conductivity. The resistivities of the three materials are approximately 1.75 × 10^-8 Om (Cu), 2.83 × 10^-8 Om (Al), and 9.78 × 10^-8 Om (Fe). The reactivities are also different, varying in the order of Cu to Fe and Al for low to high reactivity. Significantly different values for both resistivity and metal reactivity exist among the three metals. All electrodes were of the same size, with a diameter of 0.6 cm and a length of 8 cm. For the convenience of comparison, two additional electrodes (Al and Fe) of the same size were chosen. However, these two electrodes were treated with a 10-μm-thick Cu plating, which caused a negligible effect on the electrode size. Moreover, artificial damage (a minor scratch) was applied to the Cu plating of the Cu- plated Al electrode, which was employed as a control in the experiment. The purpose in this case was to ascertain the effect of the Cu plating. The stability (metal reactivity) of the electrode material is an important factor for consider- ation because the trunk and soil can cause electrode corrosion. Steel and Pt electrodes offer good corrosion resistance, which yields a stable electrical signal when measured over time. However, it has not yet been shown whether the stability of the materials significantly affects the voltage during collection and utilization. Moreover, the process requires the highest possi- ble voltage during power collection and utilization. Therefore, conductivity is also an impor- tant factor. In a previous study, a higher voltage was measured using electrodes with a lower resistivity, i.e., better conductivity [23]. However, the power source formed by a standing tree and its surrounding soil has a high internal resistance. Internal resistances of tens of thousands of ohms have been observed, according to the relevant literature [11, 12, 24]. The metal elec- trodes act as a series resistor with a low resistance between the anode and cathode of the tree- source. In this case, the conductivity of the electrode material exerts little effect on the output voltage, which is inconsistent with the previous study. Therefore, the mechanism by which the electrode material affects the voltage requires further study. This work aimed to preliminarily ascertain the rules and mechanisms that underlie the effect of the electrode material on the voltage between a standing tree and its surrounding soil, as well as to provide guidance for choosing suitable electrode materials in relevant studies, thereby promoting the utilization of this innovative energy generator. Both the conductivity and the stability of the metal electrodes studied here were considered. Measurements A wire was introduced from each trunk electrode and its corresponding ground electrode. A load resistor was connected to the wires between the trunk electrode and the ground electrode in series so that a current circuit was formed through the load resistor [11]. The voltage across the resistor was measured using a high-input impedance voltmeter (Keithley 2000). It has been previously observed that the electrical activity of trees strongly depends on the season, with more stable and coherent voltage variations measured during spring and summer [18]. Therefore, the tree-based energy generator is expected to operate during spring and sum- mer. The present study was conducted during summer (June-August 2014). Every measure- ment was conducted at noon. The experimental site located in an experimental forest in Beijing Forestry University for which specific permission was not required. The field studies did not involve endangered or protected species. Electrode arrangement A naturally grown, healthy poplar (Populus X canadensis Moench) with a diameter of 46 cm (at the height of 0.5 m where the electrodes were arranged) was chosen, and five electrodes (trunk electrodes) were inserted into its trunk at even spacing in the order of Cu-plated Al, Al, Cu, Fe, and Cu-plated Fe (the electrode position has no effect on the voltage [2]), as shown in Fig 1. 3 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage Fig 1. Electrode arrangement. (a) Five different electrodes were inserted into the trunk. Another five electrodes were arranged in the soil surrounding the standing tree. (b) In both sets, the electrodes were in the order of Cu-plated Al, Al, Cu, Fe, and Cu-plated Fe. Lines connecting any two adjacent electrodes to the center of the trunk had an angle of 72°. doi:10 1371/journal pone 0136639 g001 Fig 1. Electrode arrangement. (a) Five different electrodes were inserted into the trunk. Another five electrodes were arranged in the soil surrounding the standing tree. (b) In both sets, the electrodes were in the order of Cu-plated Al, Al, Cu, Fe, and Cu-plated Fe. Lines connecting any two adjacent electrodes to the center of the trunk had an angle of 72°. doi:10.1371/journal.pone.0136639.g001 The internal end of the electrode was implanted to a depth of up to 5 cm into the trunk, and the external end was attached to a wire. Another five electrodes (ground electrodes) were evenly arranged in the soil surrounding the standing tree in the same order. The ground elec- trodes were inserted to a depth of 80 cm [18]. Lines connecting any two adjacent electrodes to the center of the trunk had an angle of 72°. The ground surrounding the tree was flat, and the nearby soil composition was uniform. The electrode-trunk contact resistance To ascertain the effect of the contact resistance between the electrode and the trunk on the internal resistance of the tree-source, a second additional experiment was conducted. As shown in Fig 3, five cylindrical Cu electrodes (trunk electrodes) with different diameters (0.5 cm, 1.0 cm, 1.5 cm, 2.0 cm, and 2.5 cm) were inserted into the tree trunk. These electrodes were all implanted at the same depth (5 cm) in the trunk. As a result, the electrodes had differ- ent contact areas with the trunk. Another Cu electrode (ground electrode) was arranged in the soil. The voltages between each trunk electrode and the ground electrode were measured. The electrode-soil contact resistance To ascertain the effect of the electrode-soil resistance on the internal resistance of the power source formed by a standing tree and its surrounding soil, an additional experiment (the first additional experiment) was performed. This experiment was undertaken to help explain the effect of the electrode conductivity on the voltage. As shown in Fig 2, two additional Cu elec- trodes were arranged in the trunk with a separation of 30 cm, while two additional Cu elec- trodes were arranged in the soil with a separation of 30 cm. T1 and G1 denote, respectively, the original Cu electrode in the standing tree and the origi- nal Cu electrode in the soil. T2 and T3 are the additional Cu electrodes in the trunk. G2 and G3 are the additional Cu electrodes in the soil. Each measurement of the voltage between the trunk electrodes and the ground electrodes, as captured using the load resistor, was recorded as VT1-G1, VT1-G2, VT1-G3, VT2-G1, VT2-G2, VT2-G3, VT3-G1, VT3-G2, and VT3-G3. After tamping the surface soil at G1, G2, and G3, the voltages were recorded again. 4 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage Fig 2. The first additional experiment. T1 and G1 denote the original Cu electrode in the trunk and the original Cu electrode in the soil, respectively. T2 and T3 are the additional Cu electrodes in the trunk. G2 and G3 are the additional Cu electrodes in the soil. doi:10 1371/journal pone 0136639 g002 Fig 2. The first additional experiment. T1 and G1 denote the original Cu electrode in the trunk and the original Cu electrode in the soil, respectively. T2 and T3 are the additional Cu electrodes in the trunk. G2 an G3 are the additional Cu electrodes in the soil. Fig 2. The first additional experiment. T1 and G1 denote the original Cu electrode in the trunk and the original Cu electrode in the soil, respectively. T2 and T3 are the additional Cu electrodes in the trunk. G2 and G3 are the additional Cu electrodes in the soil. Fig 2. The first additional experiment. T1 and G1 denote the original Cu electrode in the trunk and the original Cu electrode in the soil, respectively. T2 and T3 are the additional Cu electrodes in the trunk. G2 and G3 are the additional Cu electrodes in the soil. doi:10.1371/journal.pone.0136639.g002 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Electrodes with nonmetallic inclusions A supplementary experiment was conducted to test the performance of electrodes with nonme- tallic inclusions. This supplementary experiment was similar to the primary experiment. How- ever, four nonmetallic materials were used as the inclusions in the supplementary experiment Fig 3. The second additional experiment. Five cylindrical Cu electrodes with different diameters were inserted into the tree trunk. Another Cu electrode was arranged in the soil. doi:10.1371/journal.pone.0136639.g003 Fig 3. The second additional experiment. Five cylindrical Cu electrodes with different diameters were inserted into the tree trunk. Another Cu electrode was arranged in the soil. Fig 3. The second additional experiment. Five cylindrical Cu electrodes with different diameters were inserted into the tree trunk. Another Cu electrode was arranged in the soil. doi:10.1371/journal.pone.0136639.g003 doi:10.1371/journal.pone.0136639.g003 5 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage Fig 4. Supplementary experiment. (a) Five different electrodes were inserted into the trunk. Another five different electrodes were arranged in the soil surrounding the standing tree. (b) The electrodes were in the order of Cu-plated plastic, Cu-plated glass, Cu, Cu-plated wood, and Cu-plated ceramic. Fig 4. Supplementary experiment. (a) Five different electrodes were inserted into the trunk. Another five different electrodes were arranged in the soil surrounding the standing tree. (b) The electrodes were in the order of Cu-plated plastic, Cu-plated glass, Cu, Cu-plated wood, and Cu-plated ceramic. doi:10.1371/journal.pone.0136639.g004 (Fig 4). Moreover, the thickness of the coating was 25 μm, which was thicker than that in the primary experiment. PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Primary experiment Thirty-three sets of effective data were acquired from the primary experiment. The voltage var- iations are shown in Fig 5. The y-axis displays the voltages measured, and the x-axis shows the serial number of each data set, which varies with time. The voltage measured by the three metal electrodes (Cu, Al, and Fe) did not show any specificity except for differences in the magnitude and amplitude of variation. The Al electrode demonstrated a slightly different voltage trend rel- ative to the other two metals. Among the three metals (Cu, Al, and Fe), the Cu electrode yielded the highest measured voltage, followed by Al and Fe. The curves for the three metal electrodes, i.e., Cu, Al and Fe, all fluctuated but showed an overall decreasing trend, and the final recorded point in each case was the lowest. For the Cu electrode, the last data point was 848 mV, repre- senting a decrease of 39 mV from the first recorded point (887 mV). For the Al electrode, the voltage decreased by 132 mV. For the Fe electrode, the voltage decreased by 43 mV. The voltage measured by the Cu electrode decreased the least, followed by the Fe electrode, whereas the Al electrode registered the largest decrease in voltage. The voltage curve acquired from the Cu- plated Fe electrode was closest to that of the Cu electrode. These two curves were nearly identi- cal, especially at the data sets prior to group 22. Likewise, the Cu-plated Al electrode also Fig 5. Voltage variations in the primary experiment. Five curves display the voltage variations measured with the five different electrodes. All curves show an overall decreasing trend. The Cu electrode yields the highest measured voltage, followed by Al and Fe. The curve acquired from the Cu-plated Fe electrode is closest to that of the Cu electrode. The curve of the Cu-plated Al electrode declines sharply. doi:10.1371/journal.pone.0136639.g005 Fig 5. Voltage variations in the primary experiment. Five curves display the voltage variations measured with the five different electrodes. All curves show an overall decreasing trend. The Cu electrode yields the highest measured voltage, followed by Al and Fe. The curve acquired from the Cu-plated Fe electrode is closest to that of the Cu electrode. The curve of the Cu-plated Al electrode declines sharply. doi:10.1371/journal.pone.0136639.g005 doi:10.1371/journal.pone.0136639.g005 6 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage Fig 6. Primary experiment Voltages recorded in the first additional experiment. Each measurement of the voltage between the electrodes in the tree trunk and the surrounding soil using the load resistor was recorded as VT1-G1, VT1-G2, VT1-G3, VT2-G1, VT2-G2, VT2-G3, VT3-G1, VT3-G2, and VT3-G3. (a) The voltage had no correlation with the position shift of the electrodes. (b) All voltages increased after the soil was tamped. doi:10.1371/journal.pone.0136639.g006 Fig 6. Voltages recorded in the first additional experiment. Each measurement of the voltage between the electrodes in the tree trunk and the surrounding soil using the load resistor was recorded as VT1-G1, VT1-G2, VT1-G3, VT2-G1, VT2-G2, VT2-G3, VT3-G1, VT3-G2, and VT3-G3. (a) The voltage had no correlation with the position shift of the electrodes. (b) All voltages increased after the soil was tamped. doi:10.1371/journal.pone.0136639.g006 doi:10.1371/journal.pone.0136639.g006 demonstrated properties similar to the Cu electrode at the first data point. However, its voltage curve then approached the curve of the Al electrode. The electrode-soil contact resistance The results from the first additional experiment are presented in Fig 6. Fig 6A shows the volt- ages before the soil was tamped. The voltages after tamping are presented in Fig 6B. There are no common differences among the voltages in Fig 6A. The voltage change did not correlate with the position shift of the electrodes. In Fig 6B, every voltage is greater than the voltages shown in Fig 6A. PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 The electrode-trunk contact resistance The result of the second additional experiment is presented in Fig 7. This figure shows the volt- ages measured by the trunk electrodes of different diameters, which varied from 0.5 cm to 2.5 cm in 0.5 cm intervals. The voltage rose discernably with increasing electrode diameter. That Fig 7. Voltages recorded in the second additional experiment. The electrode with the largest diameter obtained the highest voltage. doi:10 1371/journal pone 0136639 g007 Fig 7. Voltages recorded in the second additional experiment. The electrode with the largest diameter obtained the highest voltage. Fig 7. Voltages recorded in the second additional experiment. The electrode with the largest diameter obtained the highest voltage. doi:10.1371/journal.pone.0136639.g007 doi:10.1371/journal.pone.0136639.g007 doi:10.1371/journal.pone.0136639.g007 7 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage Fig 8. Voltage variations in the supplementary experiment. Five curves display the voltage variations measured with the five different electrodes. All curves follow the same trend. There is no obvious difference in amplitude among the curves. Fig 8. Voltage variations in the supplementary experiment. Five curves display the voltage variations measured with the five different electrodes. All curves follow the same trend. There is no obvious difference in amplitude among the curves. doi:10.1371/journal.pone.0136639.g008 is, the measured voltage increased with electrode diameter (increasing contact area with the tree trunk). is, the measured voltage increased with electrode diameter (increasing contact area with the tree trunk). Electrodes with nonmetallic inclusions The result of the supplementary experiment is shown in Fig 8. The y-axis displays the voltages and the x-axis shows the serial number of each data set, which varies with time. The curves in Fig 8 all follow the same trend. The voltage drops of the Cu-plated electrodes were smaller than those recorded in the primary experiment (Fig 5). There is no obvious difference in amplitude between the Cu electrode and the Cu-plated electrodes. However, the voltages measured by the Cu-plated electrodes were slightly higher than those of the Cu electrode. PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage the soil is a major component of the internal resistance of the tree-based power source. In the second additional experiment (Fig 3), higher voltages were obtained from electrodes with larger diameters (Fig 7). That is, increasing the contact area between the electrode and the trunk also increased the measured voltage. This finding suggests that the contact resistance between an electrode and tree trunk is also an important part of the internal resistance of the tree-source. The value of this contact resistance may be thousands of ohms, in consideration of the high level of internal resistance present in the tree-source (in the range of 15.9 kO to 21.1 kO in the second additional experiment). Therefore, the reason for the different voltages among the three electrodes (Cu, Al, and Fe, Fig 5) is the difference in the contact resistance of electrode-soil and electrode-trunk contact surfaces and not the difference in the electrode resistance alone. That is, smaller contact resistances between the electrode and the soil (or trunk) caused higher volt- ages. Moreover, under the same soil and trunk environment, the metal with the higher conduc- tivity also achieved a smaller contact resistance with the soil and the trunk. Therefore, the Cu electrode (which has the highest conductivity) achieved a smaller contact resistance with the soil and the trunk than Al electrode and Fe electrode, resulting in the highest voltage obtained in the primary experiment (Fig 5). The internal resistance of the tree-source was also measured by all three metal electrodes (Cu, Al, and Fe). The measured resistances were 17.9 kO (Cu), 105.7 kO (Al), and 188.9 kO (Fe). The magnitudes of the resistances measured with the three different electrodes (Cu, Al, and Fe), from small to large, were Cu, Al, and Fe, which was con- sistent with the resistivities of the three metals. Moreover, the metal with the highest conductiv- ity (Cu) decreased the internal resistance (and the contact resistance) by tens of thousands of ohms. This result indicated that the metal with the higher conductivity achieved a smaller con- tact resistance and smaller internal resistance of the tree-source. In this case, the effects of metal conductivity on voltage are primarily determined by the surface metal materials in direct contact with the soil and the trunk. The primary experiment also demonstrated that cheap metals, such as Fe and Al, can be selected for inclusion by coating a more conductive metal, such as Cu. As an extension of this concept, a supplementary experiment was conducted to test the performance of electrodes with nonmetallic inclusions (Fig 4). In the supplementary experiment (Fig 4), the magnitudes of the voltages measured by the electrodes with nonmetallic inclusions (plastic, glass, wood, and ceramic) were similar to that of the Cu electrode (Fig 8). These results indicate that nonmetallic materials, such as plastic, glass, wood, and ceramic, can also be good choices for electrode construction. In addition, when Cu is plated on the surface of a nonmetallic material, the resulting surface is slightly rougher than when Cu is plated on a metal. This surface roughness can increase the contact area between the electrode and the soil. Therefore, higher voltages were measured by the Cu- plated electrodes in the supplementary experiment. Conductivity The magnitudes of the voltages measured with the three different electrodes (Cu, Al, and Fe) in the primary experiment, from large to small, were in the order of Cu to Al and Fe, which is con- sistent with the conductivities of the three metals (Fig 5). However, the internal resistance of the power source formed by the standing tree and the soil is large (at least 17.9 kO in the pres- ent study). The electrodes act as a part of the source. However, because each electrode has a small resistance, it should not affect the size of the internal resistance and the measured voltage. Therefore, the difference in the resistance of the three electrodes was not the immediate cause of the different voltages. To uncover the actual phenomenon, two additional experiments were conducted (Figs 2 and 3). In the first additional experiment (Fig 2), the distance between the electrodes was increased from position T1 to position T3 in the standing tree and from G1 to G3 in the soil (Fig 2). The corresponding data (Fig 6A) revealed that the voltage change did not correlate with the shift in position, indicating that the position of an electrode does not strongly affect the internal resis- tance of the tree-source. Therefore, the contributions from the standing tree and the soil to the internal resistance are negligible. However, when the surface soil at G1, G2, and G3 was tamped, all corresponding voltages increased by at least 50 mV (Fig 6B). Likewise, tamping the surface soil of the Al, Fe, Cu-plated Al, and Cu-plated Fe electrodes also produced a similar effect on the voltage. This finding suggests that the contact resistance between an electrode and 8 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Cu plating Higher voltages were measured for the Cu-plated Al and Fe electrodes compared with the non- plated Al and Fe electrodes. When the first measurement was recorded (Fig 5), each electrode was in its initial state such that the difference in the measured voltage was determined by the con- ductivity of each electrode. The measured voltages recorded with Cu, Cu-plated Al, and Cu- plated Fe were almost identical, indicating that the Cu plating on the Al and Fe electrodes pro- vided these complex electrodes with nearly the same characteristics as the Cu electrode. It is easy to comprehend that after plating with Cu, the Al and Fe electrodes acquired surface and contact resistances with the soil and trunk that were similar to those of the Cu electrode. However, as revealed by the first data set, the Cu-plated Al electrode had a smaller voltage because the plating was artificially damaged before the experiment. Because the degree of damage was limited, this electrode’s voltage was not markedly different from that of either the Cu electrode or the Cu- plated Fe electrode. However, after time had passed, the voltage curve of the Cu-plated Al elec- trode decreased significantly and eventually merged with the corresponding curve of the Al elec- trode. In contrast, the Cu-plated Fe electrode showed little decrease in voltage before data set 11. These results suggest that in these wet soil and trunk environments, the minor damage to the Cu coating of the Cu-plated Al electrode before the experiment resulted in the rapid degradation of the plating quality. After data sets 11 and 22 were recorded, the voltage curve of the Cu electrode significantly decreased, suggesting that the Cu had oxidized. Evidence of oxidation also occurred on the Cu coatings of the Cu-plated Al and the Cu-plated Fe electrodes. Moreover, destruction of the Cu coatings on the Cu-plated Al and the Cu-plated Fe electrodes altered the material exposed at the electrode surface. Therefore, the voltage curves of the Cu-plated Al electrodes and the Cu- plated Fe electrodes displayed more significant decreases, especially the Cu-plated Al electrode that had been artificially damaged prior to the experiment. Unsurprisingly, the voltage increased when using the Al and Fe electrodes with the Cu plating. However, the 10-μm-thick layer of Cu plating could not maintain the performance of the electrode for an extended period. Stability The resistivity of Al is closer to that of Cu than Fe, but the voltage curve measured with the Al electrode was closer to that of the Fe electrode (Fig 5). This result was obtained because Al oxi- dizes more readily, forming a high-resistance oxide layer [25], which increases the contact resistance between the electrode and the soil (and the trunk). As a result, the voltage curve measured with the Al electrode was closer to that of the Fe electrode. In addition, the Al elec- trode exhibited significant instability throughout the entire experiment, also yielding the most rapidly decreasing voltage curve. In addition to oxidation, Al also undergoes a process of self- passivation, in which a compact oxide coating forms on its surface [26]. This phenomenon likely occurred in the experiment, reducing the contact area between the Al metal and the soil (and the trunk). In contrast, the metals Cu and Fe are resistant to passivation in natural or PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 9 / 12 Effects of Electrode Material on the Tree-Voltage mildly acidic soil and tree trunk conditions, hence these electrodes resulted in relatively flat corresponding curves. Fe can oxidize rapidly in wet environments; however, in this experiment, the corresponding voltage decrease was slightly larger than that of Cu, but not as significant as that of Al. The reason for this is that no passivation occurred on the surface of the Fe electrode. That is, although a large oxidized area developed on the Fe surface, the oxide did not form a coherent oxide layer. Therefore, the contact area between the Fe electrode and the soil (and the trunk) or the moisture in the soil (and the trunk) did not significantly decrease. Furthermore, due to the specific nature of the Al electrode, as discussed, its corresponding voltage curve dif- fered from those of the Fe and Cu electrodes. For example, data sets 17 to 19 (Fig 5) for the Cu and the Fe electrodes display slowly increasing voltages, whereas the corresponding curve for the Al electrode decreases continuously. These results suggest that during such a period, factors associated with either the environment or the trunk of the standing tree caused the voltage to increase, while the passivation of the Al electrode severely affected the voltage ultimately obtained. Thus, the stability of the electrode material has no significant effect on the voltage. Cu plating Based on these results, one option would be to increase the thickness of the plating treatment, which is demonstrated in the supplementary experiment. Stability However, passivation of the electrode material can significantly reduce the voltage obtained. Conclusions The metal reactivity of an electrode has no significant effect on the voltage between a standing tree and its surrounding soil. However, passivation of the electrode materials markedly reduces 10 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 Effects of Electrode Material on the Tree-Voltage the voltage. The conductivity of the metal electrode has an effect on the contact resistance of the electrode-soil and electrode-trunk contact surfaces, which significantly influences the volt- age level. The metal with the higher conductivity leads to a smaller contact resistance, as well as to a higher voltage level. Therefore, the effects of metal conductivity on voltage are primarily determined by the surface metal materials in direct contact with the soil and the trunk. It is suggested that cheap metals, such as Fe and Al, can be selected for inclusion within a metal coating (with better conductivity), which would most likely achieve good efficiency, stability, and economy. Moreover, it is demonstrated that cheap nonmetallic materials, such as plastic, glass, wood, and ceramic, are also good choices for the inclusion. In addition, the thickness of the coating must be increased to obtain higher stability and reliability. Acknowledgments We thank Chao Feng and Chongxin Chen for their help with fieldwork. Author Contributions Conceived and designed the experiments: ZH GW WL JZ JK. Performed the experiments: ZH GW WL JZ JK. Analyzed the data: ZH GW WL JZ JK. Contributed reagents/materials/analysis tools: ZH GW WL JZ JK. Wrote the paper: ZH GW WL JZ JK. PLOS ONE \| DOI:10.1371/journal.pone.0136639 August 24, 2015 References 1. Yamaguchi T, Hashimoto S. A green battery by pot-plant power. IEEJ T Electr Electr. 2012; 7: 441– 442. 2. 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https://openalex.org/W1539190672	https://www.intechopen.com/citation-pdf-url/37989	English	null	Automation of Subjective Measurements of Logatom Intelligibility in Classrooms	InTech eBooks	2,012	cc-by	6,256	Automation of Subjective Measurements of Logatom Intelligibility in Classrooms Stefan Brachmanski Wroclaw University of Technology Poland Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 4 1. Introduction A great number of rooms are dedicated to a voice communication between a singular speaker and a group of listeners. Those rooms could be as small as meeting room and classrooms or larger like auditoriums and theatres. There is a major demand for them to assure the highest possible speech intelligibility for all listeners in the room. Classrooms are an example of rooms where a very good speech intelligibility is required (a teacher talks to a group of students who want to understand the teacher’s utterance). To determine the intelligibility degree (its maps) of rooms, it is necessary to take measurements in many points of those rooms. The number of measurement points depends on room’s size and precision of created intelligibility room’s maps. Despite of the crucial progress in the instrumental measurement techniques, the only reliable method subjective speech intelligibility measurement is still very time consuming, expensive, demanding high skills and specially trained group of listeners. The first part of this chapter presents the idea of speech intelligibility subjective measurements. The measurements with properly trained team are taken in described standards, conditions have to be controlled and repeated. The subsequent sections of the chapter are focusing on one of the classic subjective speech intelligibility measurement method in rooms (classrooms) and its automated version which is named as the modified intelligibility test with forced choice (MIT-FC). Finally, in the last section of the chapter, there compared results of subjective speech intelligibility measurements in rooms taken with classic and automated methods are and the relation between intelligibility taken with the forced choice method is given as well. The presented relation let us compare results taken with both methods and use relations known from earlier research carried in domain of speech intelligibility. However, the biggest advantage of the speech intelligibility measurement automation is the shortening of measurement time and the possibility of taking simultaneous measurements in several points of the room. The result of speech intelligibility is obtained just after the end of measurements, it is then possible to obtain the intelligibility map in few minutes. Of course the precision is growing with number of listeners in particular points. Speech quality is a multi-dimensional term and a complex psycho-acoustic phenomenon within the process of human perception. Every person interprets speech quality in a 62 Automation different way. 1. Introduction The pioneering work on speech intelligibility was carried out by Fletcher at Bell Labs in the early 1940s. Fletcher and his team not only established the effects of bandwidth on intelligibility but also the degree to which each octave and ⅓-octave band contributed. One of the fundamental parameters for quality assessment of speech signal transmission in analogue and digital telecommunication chains as well as in rooms, sound reinforcement systems and at selection of aural devices is speech intelligibility (ANSI, 2009; Basciuk & Brachmanski, 1997; Brachmanski, 2002, 2004; Davies, 1989; International Organization for Standardization – [ISO], 1991; Majewski, 1988, 1998, 2000; Polish Standard, 1991, 1999; Sotschek 1976). Satisfactory speech intelligibility should be provided by telecommunication channels, rooms and hearing aids. It is obvious, that speech intelligibility concerns only the linguistic information (i.e. what was said) and does not take into account such features of speech, like its naturalness or the speaker voice individuality. Nevertheless, intelligibility should be and - up till now - is viewed as a basic and most important aspect of the quality of all systems which transmit, code, enhance and process the speech signals. Satisfactory speech intelligibility requires adequate audibility and clarity. In general the evaluation of the speech quality may be done by subjective (intelligibility, quality rating) (Farina, 2001; Howard & Angus, 2009; Möller, 2010) and objective methods e.g. Articulation Index - AI (American National Standards Institute – [ANSI], 1997; French & Steinberg, 1947; Kryter, 1962), Speech Transmission Index - STI (Brachmanski, 1982, 2004, 2006, Houtgast & Steeneken, 1973; International Electrotechnical Commission [IEC], 1991; Lam & Hongisto, 2006; Steeneken & Houtgast, 1980, 2002), Perceptual Evaluation of Speech Quality – PESQ (International Telecommunication Union - Telecommunication Standardization Sector [ITU-T], 2003), Perceptual Objective Listening Quality Assessment – POLQA, (ITU-T, 2011) . Designers of devices and systems intended for speech transmission incline to usage of objective measurement methods, not always taking into consideration the application limitations and exactitude which depends on type of examined object and measure conditions. However the final verifier of quality of speech transmission devices is their user, that is to say man. The aim of aural evaluation is quantitative evaluating and qualitative differentiation of acoustical signals reaching a listener. The process of aural evaluation can be presented as follows B → S →R (1) (1) where: B – stimulus reaching the listener, S – listener, R – listener’s reaction where: B – stimulus reaching the listener, S – listener, R – listener’s reaction The reaction R is dependent on the signal S reaching the listener’s receptors and on conditions in which the listener is based. Generally it can be assumed that the reaction R depends on the sum of external stimuli having the effect on the listener and internal factors having the effect on his organism. That assumption, however, doesn’t take into consideration the listener’s characteristic features such as cognitive abilities, rate of information processing, memory etc. The reaction can be then presented as a function 63 Automation of Subjective Measurements of Logatom Intelligibility in Classrooms R = f(B,S) R = f(B,S) (2) Physiological and psychological process connected with a reaction to sound signal (audio) consists of sensational reaction and emotional reaction. The total listener’s reaction is a sum of both types of reaction. The sensational reaction is the effect of a physiological process which occurs during the listening. It arises when a certain stimulus overdraws sensitivity levels or aural sensation category levels. The emotional reaction is more complex and difficult in analysis because it isn’t a direct result of received signal features but the listener’s habits and individuality. As a conclusion it can be stated that the sensational reaction is the reflection of an acoustical picture created in a person’s (listener’s) mind, whereas the emotional reaction is the reflection of a person’s attitude to that picture. Psychological and psychoacoustical research have proven that when provided stable in time conditions of evaluation, the differences in sensational reactions of particular listeners are substantially smaller than the differences in their emotional reactions. Therefore, one aim of objectivisation of aural evaluation is limiting the influence of emotional reaction on the final assessment result. That aim is achieved by introducing appropriately numerous assessments statistics, a proper choice and training of a listeners team and proper choice of testing material and rules of carrying listeners tests. The results analysis also has the big role in minimization the influence of the emotional reaction on the assessment result. Among the different subjective methods that have been proposed for assessment of speech quality in rooms, the preferred are methods based on intelligibility tests or listening-only tests (ITU-T, 1996a, 1996b; ITU-R, 1997). The subjective measure results should be mostly dependent on physical parameters of the tested room and not on the structure of the tested language material. The elimination of semantic information is done by means of logatom1 (i.e. 1 Logatom – (logos (gr) - spoken phrase, atom (gr) – indivisible) vocal sound, generally insignificant, usually made by the sound of a consonant or the first consonant, then by an intermediate vowel, finally by a consonant or a final consonant sound. where: B – stimulus reaching the listener, S – listener, R – listener’s reaction pseudo-words) lists on the basis of which the logatom intelligibilities are obtained. The problem ist hat speech (logatom) intelligibility is not a simply parameter to measure. 2. The traditional method of logatom intelligibility The measurement of logatom intelligibility consists in the transmission of logatom lists, read out by a speaker, through the tested channel (room), which are then written down by listeners and the correctness of the record is checked by a group of experts who calculate the average logatom intelligibility. It is recommended to use lists of 50 or 100 logatoms (Fig.1). Logatom lists are based on short nonsense word of the CVC type (consonant-vowel- consonant). Sometimes only CV, but also CVVC, CVCVC-words are used. The logatoms are presented in isolation or in carrier phrase e.g. “Now, please write down the logatom you hear”. Each list should be phonetically and structurally balanced (Fig.2). 64 Automation Fi 1 Ill t ti 100 l t li t f t 3 f th P li h l Fig. 1. Illustrative 100-logatom list from set 3 for the Polish language. The measurement should be carried out in rooms in which level of internal noise together with external noise (not introduced on purpose) does not exceed 40 dBA. If no requirements as to background noise are specified for a tested chain, articulation should be measured at a noise level of 60 dBA in the receiving room and for the Hoth spectrum (Fig. 3). The listeners should be selected from persons who have normal, good hearing and normal experience in pronunciation in the language used in the test. A person is considered to have normal hearing if her/his threshold does not exceed 10 dB for any frequency in a band of 125 Hz –4000 Hz and 15 dB in a band of 4000 Hz– 6000 Hz. Hearing threshold should be tested by means of a diagnostic audiometer. The listeners should be selected from persons who have normal, good hearing and normal experience in pronunciation in the language used in the test. A person is considered to have normal hearing if her/his threshold does not exceed 10 dB for any frequency in a band of 125 Hz –4000 Hz and 15 dB in a band of 4000 Hz– 6000 Hz. Hearing threshold should be tested by means of a diagnostic audiometer. Automation of Subjective Measurements of Logatom Intelligibility in Classrooms 65 Fig. 2. Example of phonetic balance of two sets, three lists of 100 logatom each for the Polish language. Fig. 3. The room noise power density spectrum (Hoth noise) (Polish Standard 1991, 1999). Fig. 2. 2. The traditional method of logatom intelligibility Example of phonetic balance of two sets, three lists of 100 logatom each for the Polish language Fig. 2. Example of phonetic balance of two sets, three lists of 100 logatom each for the Polish language. Fig. 2. Example of phonetic balance of two sets, three lists of 100 logatom each for the Polish language. Fig. 3. The room noise power density spectrum (Hoth noise) (Polish Standard 1991, 1999). 66 Automation The size of the listening group should be such that the obtained averaged test results do not change as the group size is further increased (minimum 5 persons). The group of listeners who are to take part in logatom intelligibility measurements should be trained (2-3 training sessions are recommended). Logatoms should be spoken clearly and equally loudly without accenting their beginnings or ends. The time interval between individual logatoms should allow the listener to record the received logatom at leisure. It is recommended that logatoms should be spoken with 3-5 sec. pauses in between. The time interval between sessions should not be shorter than 24 h and not longer than 3 days. The total duration of a session should not exceed 3 hours (including 10 minute breaks after each 20 minute listening period). Listeners write the received logatoms on a special form on which also the date of the test, the test list number, the speaker’s name or symbol (no.), the listener’s name and additional information which the measurement manager may need from the listener is noted. The recording should be legible to prevent a wrong interpretation of the logatom. The received logatoms may be written in phonetic transcription (a group of specially trained listeners is needed for this) or in an orthographic form specific for a given language. 2. The traditional method of logatom intelligibility In the next step, the group of experts checks the correctness of received logatoms and the average logatom intelligibility is calculated in accordance to the equation (3) and (4)      N n K k k n L W K N W 1 1 , [%] 1 (3) (3) N - number of listeners, K - number of test lists, Wn,k - logatom intelligibility for n-th listener and k-th logatom list, N - number of listeners, K - number of test lists, Wn,k - logatom intelligibility for n-th listener and k-th logatom list, [%] 100 , ,   k k n k n T P W (4) (4) Pn,k - number of correctly received logatoms from k-th logatom list by n-th listener, Tk - number of logatoms in k-th logatom list. Standard deviation s, calculated in accordance to Eq.(3), expresses the distribution of logatom intelligibility values WL over listeners. 2 1 N 1 n 2 K 1 k L k, n ) W W ( 1 K N 1 s                (5) (5) If Wn,k – WL>3s, the result of measurement is not taken into account, when an average intelligibility is calculated and calculation of WL and s must be done in accordance to Eq. (1) and (2) for reduced number of measurements. The obtained average logatom intelligibility value can be used to determine quality classes according to Table 1 (Polish Standard 1991). Automation of Subjective Measurements of Logatom Intelligibility in Classrooms 67 Quality class Description of quality class Logatom intelligibility [%] I Understanding transmitted speech without slightest concentration of attention and without subjectively detectable distortions of speech signal above 75 II Understanding transmitted speech without difficulty but with subjectively detectable distortions of speech 60  75 III Understanding transmitted speech with concentration of attention but without repetitions and return queries 48  60 IV Understanding transmitted speech with great concentration of attention and with repetitions and return queries 25  48 V It is impossible to fully understand transmitted speech (breakdown of communication) to 25 For each quality class lowest logatom articulation values are lowest admissible values Table 1. Speech intelligibility quality classes for analog channels in the traditional logatom ntelligibility method. Table 1. Speech intelligibility quality classes for analog channels in the traditional logatom intelligibility method. 3. Modified intelligibility test with forced choice (MIT-FC) In the traditional intelligibility tests the listeners write down (in ortographic form) received utterences on a sheet of paper and next a professional team revises the results and calculates the average intelligibility. This is the most time-consuming and difficult operation. To eliminate ”hand-made ” revision of the tests a method, called ”modified intelligibility test with forced choice” (MIT-FC) has been designed and investigated in the Institute of Telecommunications, Teleinformatics and Acoustics at the Wroclaw University of Technology . In the MIT-FC method all experiments are controlled by a computer. The automation of the subjective measurement is connected with the basic change in generation of logatoms and in making decision by a listener. The computer generates logatoms and presents the utterances (for logatom test the list consists of 100 phonetically balanced nonsense words), via a D/A converter and loudspeaker to the listeners subsequently and for each spoken utterance several logatoms that have been previously selected as perceptually similar are visually presented. 68 Automation It has been found (Brachmanski, 1995) that the optimal number of logatoms presented visually to the listeners is seven (six alternative logatoms and one transmitted logatom to be recognized). The listener chooses one logatom from the list visually presented on the computer monitor. The computer counts the correct answers and calculates the average logatom intelligibility and standard deviation. The measurement time for one logatom set (3 lists) consisted of 300 logatoms is 20 minutes. It has been found (Brachmanski, 1995) that the optimal number of logatoms presented visually to the listeners is seven (six alternative logatoms and one transmitted logatom to be recognized). The listener chooses one logatom from the list visually presented on the computer monitor. The computer counts the correct answers and calculates the average logatom intelligibility and standard deviation. The measurement time for one logatom set (3 lists) consisted of 300 logatoms is 20 minutes. All measurement procedures are fully automatized and an operator has a flexible possibilities to set the measurement parameters and options. It is also possible to upgrade the application which realizes the MIT-FC method with more sophisticated scores processing. Block diagram system for the subjective measurements of logatom intelligibility by MIT-FC method in the rooms is presented in Fig.4. Fig. 4. The measuring system for the assessment of logatom intelligibility in room. 3. Modified intelligibility test with forced choice (MIT-FC) The obtained with MIT-FC method average logatom intelligibility value can be used to determine quality classes according to Table 2. Fig. 4. The measuring system for the assessment of logatom intelligibility in room. The obtained with MIT-FC method average logatom intelligibility value can be used to determine quality classes according to Table 2. Automation of Subjective Measurements of Logatom Intelligibility in Classrooms 69 Quality class Description of quality class Logatom intelligibility [%] I Understanding transmitted speech without slightest concentration of attention and without subjectively detectable distortions of speech signal above 70 II Understanding transmitted speech without difficulty but with subjectively detectable distortions of speech 60  70 III Understanding transmitted speech with concentration of attention but without repetitions and return queries 54  60 IV Understanding transmitted speech with great concentration of attention and with repetitions and return queries 40  54 V It is impossible to fully understand transmitted speech (breakdown of communication) to 40 For each quality class lowest logatom articulation values are lowest admissible values Table 2. Classes of speech intelligibility quality for analog channels for the MIT-FC method. Table 2. Classes of speech intelligibility quality for analog channels for the MIT-FC method. 4. MIT-FC measurement system The program for the subjective assessment of speech transmission in rooms with logatom intelligibility method with forced choice (MIT-FC) is based on TCP Client/Server technology i.e. the communication is done by local network. Requirements of the program are following: PC computer with Windows 9x, a network card and hub for communication between the Server and Clients (members of the team of listeners). The work with the program starts with the installation on the Server computer a Server program. The Server is supervised by the person leading the subjective assessment. The next step is the installation of the Client program on the Client (listener) computers. The Client computers are used by members of the team of listeners. Before starting the assessment the Server and Client programs should be configured. During the configuration of the Server program it is necessary to give the path to the directory with signal files (testing signals - logatoms), number of logatoms per a session, intervals between reproduced logatoms, number of sessions and the port for the communication with the Client (usually 3000) (Fig.5). The configuration of the Client program is done by giving the name of the measure point (e.g. a room, location of listener in the room etc.), listeners login, IP address and the port number (usually: 3000) (Fig.6). 70 Automation Fig. 5. The example of the window of the Server program Fig. 6. The example of the window of the Client program The next step after the configuration is the connection process. The number of people being logged on can be seen on the right-hand side of the main window of the program. During a multi-session assessment (the number of session should be chosen during the configuration of the Server), after finishing of the first session the program waits for reconfiguration of measurement positions. For example, changing the listener on certain measurement position, we should first disconnect the Client and change the user name. After this and choosing the Continue option, the next session can be started. The computer generates logatoms and for each spoken utterance visually shows six alternative logatoms and one transmitted logatom to be recognized (Fig.7a). The listener chooses one logatom from the list visually presented on the computer monitor (Fig. 7b). During the tests, the listener is confirming his response by using key from ‘1’ to ‘7’. Other keys are non-active during all testing session. 4. MIT-FC measurement system Aft fi i hi ll t i th di l i d ti lt i t Fig 5 The example of the window of the Server program Fig. 5. The example of the window of the Server program Fig. 6. The example of the window of the Client program Fig. 6. The example of the window of the Client program Fig. 6. The example of the window of the Client program The next step after the configuration is the connection process. The number of people being logged on can be seen on the right-hand side of the main window of the program. During a multi-session assessment (the number of session should be chosen during the configuration of the Server), after finishing of the first session the program waits for reconfiguration of measurement positions. For example, changing the listener on certain measurement position, we should first disconnect the Client and change the user name. After this and choosing the Continue option, the next session can be started. The computer generates logatoms and for each spoken utterance visually shows six alternative logatoms and one transmitted logatom to be recognized (Fig.7a). The listener chooses one logatom from the list visually presented on the computer monitor (Fig. 7b). During the tests, the listener is confirming his response by using key from ‘1’ to ‘7’. Other keys are non-active during all testing session. After finishing all measurement sessions, the dialog window presenting results in two options shows up: 1. Result of session nr - in this option the number of session for which the results will be shown should be given. 2. Summary - in this option the summary of all sessions with detailed list of listeners and measurement points shows up. Automation of Subjective Measurements of Logatom Intelligibility in Classrooms 71 Fig. 7. The example of the listener’s window. b) a) Fig. 7. The example of the listener’s window. 5. Experiments The goals of experiment The goals of experiment  decision if the results of traditional and modified with forced choice methods let finding the relation which would allow to convert results from one method to the other and the classification of rooms tested with both methods,  measurement of experimental relations between traditional and modified logatom intelligibility methods.  measurement of experimental relations between traditional and modified logatom intelligibility methods. Taking into account the comparative character of the experiment it was planned to be done only in a function of used logatom intelligibility method. With this end in view each measurement was done with both traditional and modified methods, not changing:  listeners,  surroundings.  measurement system (only the logatom lists), The subjective tests were done according to Polish Standard PN-90/T-05100 with the team of listeners made up of 12 listeners in age from 18 to 25 years. The listening team was selected from students at Wroclaw University of Technology with normal hearing. The qualification was based on audiometric tests of hearing threshold. The measurements of logatom intelligibility were done using the traditional method and the MIT-FC method. The measurements were taken in two unoccupied rooms (Fig. 8). In each room, four measure point (Mp) were selected. These positions were chosen in the expectation of yielding a wide range of logatom intelligibility. Sound sources (voice and white or rose noise) were positioned in the part of the room normally used for speaking. One loudspeaker was the voice source and the second – the noise source. The various conditions were obtained by combination 14 level of white and rose noise and four measure point. The 14 signal-to-noise ratios (SNR) were used: 39 (without noise, only background noise), 36, 33, 30, 27, 24, 21, 18, 15, 12, 9, 6, 3, 0dB. As a result 72 Automation 112 different transmission conditions were obtained (14 SNR4Mp2rooms). The speech and noise signal levels were controlled by means of a 2606 Bruel & Kjaer instrument by measuring them on a logarithmic scale according to correction curve A. Fig. 8. Plan view of the rooms 1 and 2 showing source position and four receiver positions (I – loudspeaker – source of the logatoms, III - loudspeaker – source of the noise, 1, 2, 3, 4 – measurement points). Fig. 8. 5. Experiments Plan view of the rooms 1 and 2 showing source position and four receiver positions (I – loudspeaker – source of the logatoms, III - loudspeaker – source of the noise, 1, 2, 3, 4 – measurement points). The testing material consisted of phonetically and structurally balanced logatoms and sentences lists uttered by professional male speaker, whose native language was Polish. Logatom lists reproduced through the loudspeaker were recorded on the digital tape recorder in an anechoic chamber using a linear omnidirectional microphone. The microphone was positioned 200mm from the speaker’s lips. The active speech level was controlled during recording with a meter conforming to Recommendation P.56 (ITU-T, 1993). At the beginning of each logatom set recording, 20 seconds 1000Hz calibration tone and 30 seconds rose noise are inserted at a level equal to the mean active speech level. For each measure point (Mp) (the place where the measure position was situated) a list of 300 logatoms has been prepared. The same logatom lists were never played for any condition day after day. The logatom lists at the four listener locations were recorded on the digital tape recorder. These recordings were played back over headphones to the subject afterward. This way of subjective measurements realization provides the same listening conditions for both traditional and with choice methods. In each room for each position of listener (Mp) and for each signal-to-noise ratio (SNR) the logatom intelligibility was obtained by averaging out the group of listeners results. 73 Automation of Subjective Measurements of Logatom Intelligibility in Classrooms The listening tests were done in the studio of Institute of Telecommunications, Teleinformatics and Acoustics of Wroclaw University of Technology (Fig.9). Background noise level didn’t exceed 40 dBA. Subjective measurements of logatom intelligibility were taken in conditions of binaural listening using headphones for the optimum speech signal level of 80 dBA. Prior to the proper measurements the listening team was subjected to a 6 - hour training (two 3 - h sessions). The measurement sessions duration did not exceed 3h (together with 10 min breaks after every 20 min of listening). Fig. 9. Speech quality subjective measurement stand. The subjective logatom intelligibility measurement results, obtained for different speech to noise ratio, are presented graphically in fig. 10. 5. Experiments The curves, representing the relationship between logatom intelligibility and the signal-to-noise ratio in a room were approximated by a four-degree polynomial calculated on the basis of the least-squarees method. The obtained relations are presented in fig. 10. which also includes values of correlation coefficient R2 – a measure of the conformity between the polynomial and the results obtained from the subjective tests. As one can see there is very good agreement between the theoretical curve and the empirical results; the value of correlation coefficient R2 exceeds 0.99 in each case. Fig. 9. Speech quality subjective measurement stand. Fig. 9. Speech quality subjective measurement stand. The subjective logatom intelligibility measurement results, obtained for different speech to noise ratio, are presented graphically in fig. 10. The curves, representing the relationship between logatom intelligibility and the signal-to-noise ratio in a room were approximated by a four-degree polynomial calculated on the basis of the least-squarees method. The obtained relations are presented in fig. 10. which also includes values of correlation coefficient R2 – a measure of the conformity between the polynomial and the results obtained from the subjective tests. As one can see there is very good agreement between the theoretical curve and the empirical results; the value of correlation coefficient R2 exceeds 0.99 in each case. The subjective logatom intelligibility measurement results, obtained for different speech to noise ratio, are presented graphically in fig. 10. The curves, representing the relationship between logatom intelligibility and the signal-to-noise ratio in a room were approximated by a four-degree polynomial calculated on the basis of the least-squarees method. The obtained relations are presented in fig. 10. which also includes values of correlation coefficient R2 – a measure of the conformity between the polynomial and the results obtained from the subjective tests. As one can see there is very good agreement between the theoretical curve and the empirical results; the value of correlation coefficient R2 exceeds 0.99 in each case. 74 Automation Fig. 10. Relationship between logatom intelligibility and signal-to-noise ratio (SNR) for rooms (Logatom intelligibility measured with traditional and MIT-FC method). Fig. 10. Relationship between logatom intelligibility and signal-to-noise ratio (SNR) for rooms (Logatom intelligibility measured with traditional and MIT-FC method). For a few randomly selected measuring points the distribution of Wn,k values was compared with the normal distribution. The agreement between the Wn,k distribution and the normal distribution were tested by applying the Kolmogorov-Smirnov test. 5. Experiments It has been found that at significance level  = 0.05 there are no grounds to reject the hypothesis about the goodness of fit of the distributions. Thus, it is reasonable to use average logatom hypothesis WL as an estimator of the logatom intelligibility for a given measuring point. The main goal of presented research was assessment if there exists a relation between measurement results of traditional method and modified intelligibility test with forced choice (MIT-FC) in rooms, and if such a relation exists, its finding out. The obtained results revealed that there exists monotonic relation between results of traditional method and MIT-FC. The results were presented on the surface: logatom intelligibility MIT-FC – traditional, and approximated by a fourth order polynomial. The obtained relations are presented in fig. 11. which also includes values of correlation coefficient R2 – a measure of the conformity between the polynomial and the results obtained from the subjective tests. Automation of Subjective Measurements of Logatom Intelligibility in Classrooms 75 Fig. 11. Relationship between logatom intelligibility measured with traditional and MIT-FC method for rooms. Fig. 11. Relationship between logatom intelligibility measured with traditional and MIT-FC method for rooms. 7. References ANSI S 3.2, (2009), Methods for Measuring the Intelligibility of Speech over Communication Systems, American National Standards Institute. ANSI S 3.5, (1997), Methods for the Calculation of the Speech Intelligibility index (SII). American National Standards Institute Basciuk K., Brachmanski S., (1997), The Automation of the Subjective Measurements of Logatom Intelligibility, Prep. 4407, 102-nd Convention AES, Munich, Germany, March 22-25, 1997. Brachmanski S., (1982), Modulation Transfer Function (MTF) as a Measure of Polish Speech Transmission Quality (in Polish), Ph. D. Thesis, Wrocław. Brachmanski S., (1995), Choosing Optimum Number of Test Items in Subjective Logatom Measurements (in Polish). Proc. XLII Open Seminar on Acoustics, pp. 423-428, ISBN 83-902146-3-6, Warszawa-Bialowieza, September 12-15, 1995 Brachmanski S., (2002) The Automation of Subjective Measurements of Speech Intelligibility in Rooms, 112th Conv. AES, Munich, Germany, May 10-13, 2002. Brachmanski S., (2004) Estimation of Logatom Intelligibility with the STI Method for Polish Speech Transmitted via Communication Channels, Archives of Acoustics, Vol. 29, No. 4, (2004), pp. 555-562, , ISSN 0137-5075. ( ) pp Brachmanski S., (2004), The Subjective Measurements of Speech Quality in Rooms, Proc. of Subjective and Objective Assessment of Sound, Poznan, September 1-3, 2004. Brachmanski S., (2006), Experimental Comparison between Speech Transmission Index (STI) and Mean Opinion Scores (MOS) in Rooms,. Archives of Acoustics, Vol .31, No.4, (2006), pp. 171-176, ISSN 0137-5075. Davies D.D., Davies C., (1989), Application of Speech Intelligibility to Sound Reinforcement. J. Audio Eng. Soc., Vol. 37, No.12, (December 1989), pp. 1002-1018. Farina A., (2001), Acoustic Quality of Theatres: Correlations between Experimental Measures and Subjective Evaluations, Applied Acoustic, Vol. 62, No 8, (August 2001), pp. 889-916, ISSN: 0003682X. French, N.R., Steinberg, J.C., (1977), Factors Governing the Intelligibility of Speech Sounds, , In: Speech Intelligibility and Speaker Recognition, Hawley M. E., pp. 128 – 152, Dowden, Hutchinson & Ross Inc., ISBN 0-470-99303-0, Stroudsburg, Pennsylvania. Houtgast T., Steeneken H.J.M., (1973), The Modulation Transfer Function in Room Acoustics as a Predictor of Speech Intelligibility, J. Acoust. Soc. Am. Vol. 54, No. 2, (August 1973), pp. 557-557, pp. 66-73, ISSN 0001-4966 Howard D.M, Angus J.A.S, Acoustics and Psychoacoustics, Elsevier-Focal Press, 2009, ISBN 978-0-240-52175-6, Oxford, UK. IEC 60268-16, (2003), Sound system equipment – Part16: Objective Rating of Speech Intelligibility by Speech Transmission Index, International Electrotechnical Commission, Geneva, Switzerland. ISO/TR 4870, (1991), Acoustics – The Construction and Calibration of Speech Intelligibility Tests. International Organization for Standardization. 6. Conclusion The presented MIT-FC method offers a simple, easy to use, stable, and fully automatized speech system to assessment of speech quality in rooms. The results of the experiments have shown that the MIT-FC method is very useful in the evaluation of speech quality in rooms. The time needed to carry out the measurement with MIT-FC method is the same as in traditional one but we obtain the results right after finishing the measurement process. The experiments carried out in finding the relations between logatom intelligibility measured with traditional and semi-automatic with forced choice methods for the rooms have shown that there exist the multi-value and repetitive relation between them. It allows using both methods interchangeably and converting results between them. The obtained relations are applied in the Institute of Telecommunications, Teleinformatics and Acoustics of Wrocław University of Technology to the design of subjective tests for the verification of results yielded by a new objective method based on automatic speech recognition techniques. 76 Automation 7. References ITU-T Recommendation P.56, (1993), Objective Measurement of Active Speech Level, International Telecommunication Union, Geneva, Switzerland. Automation of Subjective Measurements of Logatom Intelligibility in Classrooms 77 ITU-T Recommendation P.800, (1996), Method for Subjective Determination of Transmission Quality, International Telecommunication Union, Geneva, Switzerland. ITU-T Recommendation P.830, (1996), Method for Objective and Subjective Assessment of Quality, International Telecommunication Union, Geneva, Switzerland. ITU-T Recommendation P.862, (2003), Perceptual Evaluation of Speech Quality (PESQ), an Objective Method for End-To-End Speech Quality Assessment of Narrowband Telephone Networks and Speech Codecs, International Telecommunication Union, Geneva, Switzerland. ITU-T Recommendation P.863, (2011), Methods for Objective and Subjective Assessment of Speech Quality. Perceptual Objective Listening Quality Assessment, International Telecommunication Union, Geneva, Switzerland. ITU-R Recom. BS.1116-1, (1997), Method for the Subjective Assessment of Small Impairments in Audio Systems Including Multichannel Sound Systems, International Telecommunication Union, Geneva, Switzerland. Kryter, K.D., (1962), Methods for the Calculation and Use of the Articulation Index, J. Acoust. Soc. Am., Vol. 34, No. 11, (November 1962), pp. 1689-1697, ISSN 0001-4966. Lam P., Hongisto V., (2006), Experimental Comparison between Speech Transmission Index, Rapid Speech Transmission Index, and Speech Intelligibility Index, J. Acoust. Soc. Am., Vol. 119, No. 2, (February 2006), pp.1106-1117, ISSN 0001-4966. Majewski W., Basztura Cz., Myślecki W., (1988), Relation between Speech Intelligibility and Subjective Scale ff Speech Transmission Quality., Proc. 7th FASE Symposium, Proceedings SPEECH’88, 719-726, Edinburgh, August 22-26, 1988. Majewski W., Myślecki W., Baściuk K., Brachmański S., (1998), Application of Modified Logatom Intelligibility Test in Telecommunications, Audiometry and Room Acoustics, Proceedings 9th Mediterranean Electrotechnical Conference Melecon’98, pp. 25-28, Tel-Aviv, Israel, May 18-20, 1998. Majewski W., Myślecki W., Brachmański S., (2000), Methods of Assessing the Quality of Speech Transmission (in Polish), Proceedings of 47th Open Seminar on Acoustics, pp. 66-75, ISBN 83-914391-0-0, Rzeszow-Jawor, September 19-22, 2000. Möller S. Assessment and Prediction of Speech Quality in Telecommunications, Kluwer Academic Publisher, ISBN 978-1-4419-4989-9, Dordrecht, Netherlands. Polish Standard PN-90/T-05100, (1991), Analog Telephone Chains. Requirements and Methods of Measuring Logatom Articulation (in Polish), Polski Komitet Normalizacyjny (Polish Committee for Standardization), 1990, Warszawa, Poland Polish Standard PN-V-90001, (1999), Digital Communication Systems. Requirements and Methods for Measurement of Logatom Articulation (in Polish), Polski Komitet Normalizacyjny (Polish Committee for Standardization), 1999, Warszawa, Poland Sotschek J., (1976), Methoden zur Messung der Sprachgüte I: Verfahren zur Bestimmung der Satz- und der Wortverständlichkeit, Der Fernmelde Ingenieur, Vol. 30, No. 10, pp. 1- 31. Srinivasan S.H., (2004), Speech Quality Measure Based on Auditory Scene Analysis, Proc. 7. References IEEE 6th Workshop on Multimedia Signal Processing, pp. 371 – 374, ISBN: 0-7803-8578- 0 September 29- October 1, 2004, Siena, Italy. 78 Automation Steeneken H.J.M., Houtgast T., (1980), A Physical Method for Measuring Speech- Transmission Quality, J. Acoust. Soc. Am., Vol. 67, No. 1, (January 1980), pp. 318 – 326, ISSN 0001-4966. Steeneken H.J.M., Houtgast T., (2002), Validation of Revised STI Method, Speech Communication, Vol. 38, No. 3-4, (November 2002), pp.413-425, ISSN:0167-6393. © 2012 The Author(s). Licensee IntechOpen. This is an open access artic stributed under the terms of the Creative Commons Attribution 3.0 cense, which permits unrestricted use, distribution, and reproduction in ny medium, provided the original work is properly cited. © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://openalex.org/W4288373259	https://zenodo.org/records/2638696/files/L%20Shaped%20via%20based%20Mushroom%20type%20High%20Impedance%20Structure.pdf	English	null	L Shaped via based Mushroom type High Impedance Structure	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	3,777	L Shaped via based Mushroom type High Impedance Structure Touseef Ahmad1, Shahid Bashir2 1,2Universitivy of Engineering and Technology Peshawar Pakistan touseefkhattak2013@gmail.com1, shahidbashir@nwfpuet.edu.pk2 Received: 15 February, Revised: 07 March, Accepted: 02 April mushroom type EBG surfaces that were structured through array of unit cells distributed in periodic manner. This unit cell is composed of square patch placed over dielectric substrate below which there exist ground plane. A conducting cylindrical pin connects the square patch with ground plane which is passing through the substrate hence forming mushroom like configuration. These periodic structures possess the ability of suppression of propagation of surface energy waves along with in-phase reflection in a given frequency band. In-phase reflection characteristics enable these structures to have image currents and antenna current both with same phase that results in constructive interference resulting in better antenna performance. If we look into the structural configuration of high impedance structure, we will see that its composition incudes two-dimensional lattice of resonating elements that form basic two dimensional stop band filter to forbid flow of surface currents. Abstract—High-impedance Electromagnetic Band-Gap structures (EBG) surfaces have the capability to forbid flow of EM waves in a given band which and therefore surface waves in case of planar antennas like mictostrip antenna can be minimized with this characteristics of EBG plane. Shape, size, symmetry, and material used in their construction defines their operating band. In this research, a novel compact EBG structure also called high impedance structure (HIS) is proposed. The design is achieved through incorporation of ‘L’ shaped via to conventional mushroom type EBG/HIS instead of straight vias. The design includes distribution of square patches over substrate material below which there exists a ground plane. Vias passing through the substrate connecting square patches and the ground plane are also part of its design It has been observed that operating frequency of L shaped via based EBG is much lower than that of conventional mushroom type EBG/HIS having straight vias. Alternatively, we can say that size reduction has been achieved through incorporation of L shaped via to the EBG/HIS resulting in 62.5 % of size reduction. All the designs and simulations are carried out in CST microwave studio. As explained, high-impedance Electromagnetic Band-Gap structures (EBG) surfaces have the capability to forbid flow of EM waves in a given band which is another important feature. Surface waves in case of planar antennas like mictostrip antenna can be minimized with the help of EM wave suppression characteristics of EBG plane. An initial research on EBG structure at microwave frequencies was conducted by Prof. E. L Shaped via based Mushroom type High Impedance Structure Touseef Ahmad1, Shahid Bashir2 1,2Universitivy of Engineering and Technology Peshawar Pakistan touseefkhattak2013@gmail.com1, shahidbashir@nwfpuet.edu.pk2 Received: 15 February, Revised: 07 March, Accepted: 02 April Yablanovitch and his research group in [7], which explained many of its properties (surface current suppression and zero reflection phase) using an effective surface impedance model. Since then, theory and practical applications of EBG structure has become an extensive research area due to its unusual properties and design flexibilities. Consequently, numerous of EBG structures have been successfully employed to realize novel high performance devices such as filters, waveguides, antennas, etc. [8], [9], [10]. Keywords— Microstrip patch antenna, high impedance surface, substrate, bandwidth. International Journal of Engineering Works ISSN-p: 2521-2419 ISSN-e: 2409-2770 Vol. 6, Issue 04, PP. 143-147, April 2019 https://www.ijew.io/ International Journal of Engineering Works ISSN-p: 2521-2419 ISSN-e: 2409-2770 Vol. 6, Issue 04, PP. 143-147, April 2019 https://www.ijew.io/ I. INTRODUCTION One of emerging materials that has been engineered artificially is metamaterial. By definition these surfaces satisfy two conditions, i.e they are purely artificial and do not exist naturally, and secondly EM properties possessed by these structures is superior and different from their parent elements [1]. EM and optical characteristic offered by these artificial materials are very much unique as well as different from their constituents. This phenomenon is achieved through inclusion, discontinuities and pattern of host material due to which their EM fields interact differently resulting in modification of EM properties like of host materials permittivity (ε), permeability (μ), refractive index (n) etc of constituent materials. As these materials provides control over EM properties therefore some desired electromagnetic and optical performance can be achieved through them. Low profile wire antennas with EBG ground are studied in [11], [12], [13]. In [14] and [15], EBG structure is employed to on-body antennas to reduce sensitive absorption rate (SAR). Other applications of EBG structures such as Multi-band antenna, LTCC and bandwidth enhancement are introduced in [16], [17] and [18], respectively. Another area of EBG structure research is focused on characteristics of its unique properties, providing simple and fast design approaches. [19] presents a study on in-phase reflection and wave suppression characteristics of EBG structure. In [20], a method of designing controlled bandwidth EBG structure is demonstrated. Spectral domain method is Since 1990s, rigorous analysis has been carried out for Single Negative (SNG) [2], Double Negative (DNG) metamaterials [3, 4] and planar Electromagnetic Band Gap (EBG) structures [5]. Sievenpiper in [6] introduced 2D © Authors retain all copyrights 2019 IJEW. This is an open access article distributed under the CC-BY License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Figure 1. High impedance Surface (Sievenpiper) (a) Top-View (b) Front- View explained in [21] to prove that capacitive surfaces can perform perfect magnetic conductors in a relative low frequency range. Requirement for miniaturization exists in every design whether it is related to antenna, amplifiers, filters or some other RF components. Therefore, EBG structure with compact size is required in several applications. Several attempts have been carried out in different articles [22-24]. Edge located via based mushroom type EBG was presented in [25] for demonstrating reduction in size of the EBG. I. INTRODUCTION Another design introduced in [26] used helical shaped via in their design for size compactness of EBG plane. In this research, a novel compact EBG structure is proposed. The design is achieved through incorporation of ‘L’ shaped via to conventional mushroom type EBG instead of straight vias. The design includes distribution of square patches over substrate material below which there exists a ground plane. Vias passing through the substrate connecting square patches and the ground plane are also part of its design. Geometrical shape of via can vary response of the EBG. In our case straight via is modified and transformed into L shaped via. This modification is carried out by moving both ends of straight via over square patches in opposite direction. Band gap response and parametric study of the newly designed L shaped based EBG is studied. A comparison between conventional mushroom type EBG & L shaped EBG is also carried out in order to analyze the benefit of L shaped EBG. Figure 1. High impedance Surface (Sievenpiper) (a) Top-View (b) Front- View Planar structures give in-phase reflection and suppress surface propagation in a particular frequency band. Additionally, the antenna currents are in-phase with the image currents, therefore both constructively interfere and provide better antenna performance[35]. Figure 2. Equivalent circuit (LC) model (a) Front-View (b) Equivalent resonant circuit (LC) Sequence of the paper is as following. Section II mainly deals design parameters of HIS. Section III presents design of L shaped via base mushroom type HIS alongwith discussion of results. At the end conclusion of the research work is presented. International Journal of Engineering Works Here 𝝶 is the intrinsic impedance of free-space = 120π Here 𝝶 is the intrinsic impedance of free-space = 120π By suppressing surface waves, these assemblies are very co-operative just in case of planer antennas resulting in directivity, efficiency of the antenna and achieving high gain [36-37]. EBGs can also be utilized for GPS applications [38- 39]. It has been observed that when EM wave strike Perfect Electric Conductor or planner EBGs, sense of polarization reverses resulting in mismatching in polarization. In order to side-step this kind of mismatch, PDEBGs (Polarization dependent EBGs) in several applications are introduced [40- 41]. (b) II. HIGH IMPEDANCE SURFACE Square patch EBG structures is the conventional design which is also termed as high impedance structures (HIS) that was proposed by Sievenpiper [27-31] which finds application in variety of low profile efficient antennas. Rahmat Samii [32] describes EBG as a periodic or a periodic configuration of multi dimension having power to stop or enhance the transmission of electromagnetic waves in a certain frequency range with no conditional limit of incident angel and polarization. Bandgap is the frequency range in which the surface waves are suppressed. Substrate thickness and dimensions of the EBG unit-cell affect the band gap characteristic. The unit cell may be either square or rectangular in shape. The mushroom, loop, or spiral may be the resonating element. Rahmat-Samii [33] and Sievenpiper [34] designed several kinds of EBG and HIS configuration which were further used and find its application in a variety of low profile linear and circularly polarized antennas. HIS design by Sievenpiper is shown below in Figure 1 and 2. Figure 2. Equivalent circuit (LC) model (a) Front-View (b) Equivalent resonant circuit (LC) To explain operation of HIS, consider Figure 4. It can be seen that HIS behavior is just parallel resonant circuit. The charge distribution among neighboring cells correspondent to capacitance C while the current flow through metallic via from top and bottom results to inductance L. Following formula determines L and C values [33]. Here W = Width of the patch g = Gap between two adjacent patches Here Here W = Width of the patch W = Width of the patch g = Gap between two adjacent patches g = Gap between two adjacent patches g = Gap between two adjacent patches g = Gap between two adjacent patches Vol. 6, Issue 04, PP. 143-147, April 2019 International Journal of Engineering Works www.ijew.io www.ijew.io (a) (b) (c) Figure 4. Mushroom type EBG with L shaped Vias (a) Unit Cell Model (b) Top view of 3x3 EBG plane (c) Side View of 3x3 EBG plane (a) Ɛr = EBG substrate relative permittivity Ɛr = EBG substrate relative permittivity h= Thickness of the substrate used h= Thickness of the substrate used µ= Medium permeability µ= Medium permeability Following equation expressed resonant frequency From above equation the Frequency Band Gap (BW) can be come near to as (a) ( ) (b) III. RESULTS AND DISCUSSIONS This section represents the design of mushroom type EBG with L shaped vias. The design includes distribution of square patches over substrate material below which there exists a ground plane. Vias passing through the substrate connecting square patches and the ground plane are also part of its design. Geometrical shape of via can vary response of the EBG. In our case straight via is modified and transformed into L shaped via. This modification is carried out by moving both ends of straight via over square patches in opposite direction. Figure 3 shows this transformation. (c) Figure 4. Mushroom type EBG with L shaped Vias (a) Unit Cell Model (b) Top view of 3x3 EBG plane (c) Side View of 3x3 EBG plane Here FR-4 substrate is used for the design of EBG having dielectric constant of 4.3 with thickness of 3.2 mm. Dimension of square patch is 10 mm x 10 mm and a total of nine patches are taken for the design. Gap between square patches is 1 mm and radius of connecting vias is taken to be 0.25 mm. Overall dimension of 3 x 3 patch mushroom type EBG is 33 mm x 33 mm. Figure 3. Transformation of Straight Vias to L shaped Vias To evaluate band gap behavior of the EBG, suspended microstrip line technique is utilized. For this purpose, a microstrip line is placed over the EBG ground plane and excited on both of its port thus forming a to port network. Coupling between the two port of transmission line i.e S21or S12 will show the band gap response of the EBG. As surface impedance of EBG plane is very high, therefore propagation of EM waves will be blocked in side band gap and similarly EM wave transmission will be high in rest of the band. Hence reduction if S21 will be observed within bandgap. Figure 3. Transformation of Straight Vias to L shaped Vias Structural configuration of designed mushroom type EBG with L shaped vias is given in figure 4. To analyzer bandgap response of the mushroom type EBG with L shaped, a microstrip line having length of 33 mm and width of 2 mm is placed over 3 x 3 patch mushroom type EBG with L shaped vias and excited. The configuration of design is given in figure 5. Vol. 6, Issue 04, PP. III. RESULTS AND DISCUSSIONS 143-147, April 2019 International Journal of Engineering Works International Journal of Engineering Works www.ijew.io www.ijew.io (a) (b) Figure 5. Suspended transmission line over Mushroom type EBG with L shaped Vias (a) Top View (b) Side View Figure 7. S21/Band gap for Mushroom Type EBG with Straight Vias (a) Figure 7. S21/Band gap for Mushroom Type EBG with Straight Vias From these results we can clearly see that operating frequency of L shaped via based EBG is much lower than that of conventional mushroom type EBG. Alternatively, we can say that size reduction has been achieved through incorporation of L shaped via to the EBG resulting in 62.5 % of size reduction. (a) CONCUSLION This article discusses miniaturization of conventional mushroom type EBG/HIS. Desired response is achieved through modification of via configuration of HIS. For this puspose the straight vias of HIS plane is replaced with L shaped vias. It has been observed that band gap of conventional mushroom type HIS with straight via is centred at 2.1135 GHz having -10 dB stop band of 310 MHz ranging from 1.9559 GHz to 2.2659 GHz. Wheras band gap of mushroom type HIS with L shaped vias was centred at 1.3 GHz having -10 dB stop band of 112 MHz ranging from 1.253 GHz to 1.366 GHz. Hence 62.5 % reduction is operating band of mushroom type HIS was observed through incorporation of L shaped vias i.e 62.5 % size reduction is achieved. (b) (b) Figure 5. Suspended transmission line over Mushroom type EBG with L shaped Vias (a) Top View (b) Side View Frequencies, having S21 less than -10 dB, are defined as the band gap normally. Simulated S21 of mushroom type EBG with L shaped vias is presented in figure 6. This can be seen that value of S21 decreases to –40 dB near 1.3 GHz having -10 dB stop band of 112 MHz ranging from 1.253 GHz to 1.366 GHz. International Journal of Engineering Works REFERENCES [16] J. H. S. D. Sievenpiper, "Textured Surface Having High Electromagnetic Impedance in Multiple Frequency Bands". United States Patent US 6,483,481 B1, 19 November 2002. [17] R. Li, G. DeJean, M. M. Tentzeris, J. Papapolymerou, J. Laskar, "Radiation-Pattern Improvement of Patch Antennas on a Large-Size Substrate Using a Compact Soft-Surface Structure and Its Realization on LTCC Multilayer Technology," IEEE Transactions on antennas and propagation, vol. 53, no. 1, pp. 200-208, 2005. [36] X. L. Bao, G. Ruvio, M. J. Ammann, and M. John, “A novel GPS patch antenna on fractal Hi-Impedance surface Substrate”, IEEE Antenna and Wireless Propagation Letters, vol. 5, 2006. [37] R. Baggen, M. Martínez-Vázquez and J. Leiss, “Low Profile GALILEO Antenna using EBG Technology”, IEEE Transactions on Antennas and Propagation, vol. 56, no. 3, pp. 667-674, March 2008. [18] R. Diaz, V. Sanchez, E. Caswell, A. 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REFERENCES [1] A.Sihvola,―Electromagnetic emergence in metamaterials‖, Advances in Electromagnetics of Complex Media and Metamaterials, vol. 89, pp. 1- 17, 2003. Figure 6. S21/Band gap for Mushroom Type EBG with L shaped Vias [2] J. Zehentner and J. Machac, ―Volumetric single negative metamaterials‖, Proceedings of Metamaterials Congress, pp. 22–24, 2007 [3] N. Engheta,―Metamaterials with negative permittivity and permeability: background, salient features, and new trends‖, IEEE MTT- S International Microwave Symposium Digest, pp. 187-190, 2003. [4] N. Engheta,―Design, fabrication, and testing of double negative metamaterials‖, IEEE Transactions on Antennas and Propagation, vol. 51, issue. 7, pp. 1516-1529, 2003. Figure 6. S21/Band gap for Mushroom Type EBG with L shaped Vias [5] W. R. Ziolkowski and N. Engheta, ―Metamaterials: Physics and Engineering Explorations‖, John Wiley & Sons, Inc., 18 September 2006. Afterwards conventional mushroom type EBG having straight vias is also designed. In this case all the parameters are kept same as that of mushroom type HIS with L shaped via except configuration of the via. It was observed that its operating frequency band is centered at 2.1135 GHz having -10 dB stop band of 310 MHz ranging from 1.9559 GHz to 2.2659 GHz as depicted in figure 7. [6] D. Sievenpiper, ―High Impedance Electromagnetic Surfaces‖, Ph.D. dissertation, Electrical Engineering Department, University of California, Los Angeles, 1999. [7] D. Sievenpiper, L. Zhang, R. F. J. Broas, N. G. Alexopolous, E. Yablonovitch,, "High-Impedance Electromagnetic Surfaces with a Forbidden Frequency Band," IEEE Transactions on Microwave Theory and Techniques, vol. 47, no. 11, pp. 2059-2074, 1999. [8] Y. J. Lee, J. Yeo, R. Mittra, W. S. 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Conway, “Using EBG to Improve Antenna Efficiency in Proximity to the Human Body”, Institution of Engineering and Technology Seminar on Wideband, Multiband Antennas and Arrays for Defence or Civil Applications, pp. 173-180, London, 13-13 March 2008. International Journal of Engineering Works REFERENCES Ying, and S. He, "Mutual Coupling Reduction of two PIFAs with a T-Shape Slot Impedance Transformer for MIMO Mobile Terminals," IEEE Transaction on antennas and propagation, vol. 60, no. 3, March 2012, pp. 1521-153l. [24] A. M. Abdelreheem, and M. A. Abdalla, "A Novel Bilateral UC-EBG Structure," IEEE Antenna and Propagation Society International Symposium pp. 1780-81, July 2014, pp. 1780-1781. [25] Eva Rajo-Iglesias, Member, IEEE, Luis Inclán-Sánchez, José-Luis Vázquez-Roy, Member, IEEE, and Enrique García-Muñoz, “Size Reduction of Mushroom-Type EBG Surfaces by Using Edge-Located Vias” IEEE MICROWAVE AND WIRELESS COMPONENTS LETTERS, VOL. 17, NO. 9, SEPTEMBER 2007. [26] Wenquan Cao Bangning Zhang Tongbin Yu Daosheng Guo Aijun Liu “Helical-Via-Type Mushroom EBG Structure for Size Vol. 6, Issue 04, PP. 143-147, April 2019 International Journal of Engineering Works www.ijew.io
https://openalex.org/W4391401717	https://revistes.uab.cat/anuariolopedevega/article/download/v30-di-pinto/535-pdf-es	es		La alquimia de un mundo inventado: mixturas literarias en tiempos de Lope	Anuario Lope de Vega	2,024	cc-by	867	PRESENTACIÓN LA ALQUIMIA DE UN MUNDO INVENTADO: MIXTURAS LITERARIAS EN TIEMPOS DE LOPE Elena Di Pinto (Universidad Complutense de Madrid) DOI: <https://doi.org/10.5565/rev/anuariolopedevega.535> L a ingeniosa alquimia que se dio en tiempos de Lope entre distintas artes —música, pintura, poesía, etc.— y, dentro de ellas, las múltiples variaciones, hibridaciones y juegos de contrastes, hace palidecer a la moderna «interdisciplinariedad», tan de moda en los últimos lustros y tan consustancial a la forma de crear, pensar, sentir en el Barroco. Los tres artículos de esta sección abordan con fineza las distintas combinaciones de géneros y sus variantes que, como en un alambique, sirvieron para condensar varias sensaciones y sentimientos en los espectadores y lectores del siglo xvii: desde el oído a la vista, desde el temor a la serena contemplación de lo divino, desde la sensualidad al aborrecimiento, desde la fantasía a las conexiones metafóricas con la realidad. El orden cronológico de las mixturas tratadas irá desde el experimento sonoro de un joven Lope a las combinaciones novedosas de géneros por parte del autor, ya en 1603, para terminar con las manipulaciones que algunos de sus colegas hicieron —en los últimos años del Fénix— en la inventio y dispositio de un episodio harto conocido por la historiografía, transformando un sueño en pesadilla para el público. Abre esta sección monográfica el trabajo de Gaston Gilabert desvelando la sutil urdimbre entre teatro y música —por decirlo de manera amplia, al fin y al cabo, era música para los oídos del público— o, más concretamente, entre la dramaturgia y el sonido de las diversas estrofas en las primeras comedias hagiográficas lopescas: San http://revistes.uab.cat/anuariolopedevega 11 MONOGRAFICO (2) ELENA DI PINTO 2024.indd 312 22/1/24 13:07 La alquimia de un mundo inventado: mixturas literarias en tiempos de Lope 313 Segundo y Los locos por el cielo. Su artículo muestra pormenorizadamente, a través del estudio de los patrones sonoros empleados, cómo Lope ideó y sistematizó una sonoridad específica para los personajes sobrenaturales (llena de disonancias para los seres demoníacos y dulcemente armónica para los celestiales) que penetró en el sentir del público, ya no por la vista —que también—, sino por el oído. El siguiente estudio corresponde a Marcella Trambaioli, que analiza minuciosamente la mezcla de géneros que presenta el poema épico de Lope La hermosura de Angélica, ya editado en 2005 por la investigadora. En su artículo nos presenta el incesante trabajo de un consagrado Lope que prueba distintas fórmulas para reverdecer de forma proteica la tradición ariostesca, pues, a partir de la síntesis entre épica y novela, entreteje también la epopeya de la conquista americana y elementos romanceriles formando un novedoso híbrido que deja traslucir —como es de Lope— esa inquietante confusión entre literatura y vida. Carlos Sainz de la Maza cierra la sección con un atento y apasionante análisis del episodio de la aparición de Cleopatra en su barca dorada. En anteriores trabajos [2009, 2014 y 2022] estudió la figura de la reina egipcia —el banquete y un primer acercamiento a su llegada en la falúa— en los que mostraba que Lope —desde su soneto de la perla, hasta la mención indirecta de ella en distintas comedias— transmitía la dimensión sensual de Cleopatra, la ceguera de amor que provocaba, la locura de los sentidos, sí, pero exenta de ese matiz maligno y doctrinal que, sin embargo, han querido expresar los dramaturgos contemporáneos del Fénix, de los que aquí se ocupa: Belmonte Bermúdez, una suelta anónima, Rojas Zorrilla y hasta Shakespeare. Sainz de la Maza muestra cómo se ha manipulado la figura de Cleopatra para catequizar y escarmentar al público de los corrales y presenta, como contraposición, la humanización que de ella hizo el dramaturgo inglés. Tanto Lope como Shakespeare se abstuvieron de juzgarla y condenarla; el investigador nos promete una próxima entrega en la que profundizará sobre la muerte de esta reina, tanto en su dimensión literaria como iconográfica. No me queda más que invitar al curioso frecuentador del Anuario Lope de Vega a leer con la misma fruición y agudeza con la que mis colegas —y, sin embargo, amigos— han investigado y escrito sus trabajos sacando a la luz la sin par codificación del Fénix, alquimista del verso. Et vale. http://revistes.uab.cat/anuariolopedevega 11 MONOGRAFICO (2) ELENA DI PINTO 2024.indd 313 22/1/24 13:07 314 Elena Di Pinto BIBLIOGRAFÍA Trambaioli, Marcella, ed., La hermosura de Angélica. Poema de Lope de Vega, Iberoamericana-Vervuert, Madrid-Fráncfort, 2005. Sainz de la Maza, Carlos, «El banquete de Cleopatra, I: Los textos y sus antecedentes», en Literatura, política y fiesta en el Madrid de los Siglos de Oro, eds. E. Borrego, C. Buezo, Visor, Madrid, 2009, pp. 421-451. Sainz de la Maza, Carlos, «La falúa dorada de Cleopatra, de Plutarco a Esquilache», en «Hilaré tu memoria entre las gentes». Estudios de literatura áurea en homenaje a Antonio Carreira, eds. A. Bègue, A. Pérez Lasheras, Universidad de Zaragoza-Université de Poitiers, Zaragoza-Poitiers, 2014, vol. II, pp. 291-308. Sainz de la Maza, Carlos, «La falúa dorada de Cleopatra II: una presencia al trasluz en el teatro de Lope y sus contemporáneos», en De la vida a la fantasía: literatura de los Siglos de Oro, ed. E. Di Pinto, Visor, Madrid, 2022, pp. 215-242. http://revistes.uab.cat/anuariolopedevega 11 MONOGRAFICO (2) ELENA DI PINTO 2024.indd 314 22/1/24 13:07
https://openalex.org/W1851654516	https://revije.ff.uni-lj.si/Dela/article/download/1353/1157	Slovene	null	Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah	Dela - Oddelek za geografijo Filozofske fakultete v Ljubljani	2,002	cc-by-sa	4,984	Dela 18 • 2002 • 115-132 Dela 18 • 2002 • 115-132 Izvleček Po oblikah površja razgibano in pokrajinsko raznovrstno Slovenijo ogrožajo šte- vilne naravne nesreče med katerimi so tudi snežni plazovi. Na temelju dosedanjih ugotovitev in s pomočjo podatkov iz geografskega informacijskega sistema za Slo- venijo smo opredelili potencialno plazovito površje na območju slovenskih Alp. Pri tem smo upoštevali rezultate obdelave snežnih plazov iz lavinskega katastra v Julijskih Alpah, ugotovitve drugih podobnih študij ter podatkovne sloje naravnogeo- grafskih sestavin visokogorskih pokrajin. Ključne besede: fizična geografija, snežni plazovi, naravne nesreče, lavinsko ogrožena območja, potencialna ogroženost, lavinska preventiva Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah Miha Pavšek Mag., Geografski inštitut Antona Melika, Znanstvenoraziskovalni center SAZU, Gosposka ulica 13, 1000 Ljubljana, Slovenija e-mail: miha.pavsek@zrc-sazu.si 1. TEMELJNA IZHODIŠČA S terenskim deloma lahko v veliki meri opredelimo dejansko lavinsko ogrože- nost površja pod gozdno mejo, nad njo in na negozdnatem ali celo neporašče- nem površju pa je to mnogo težje. Z vse bolj natančnimi naravnogeografskimi podatki na prostorsko enoto lahko izvedemo tudi simulacijo ogroženega površja, pri čemer gre prav tako za potencialno, do sedaj nedokazano ogroženost. Razli- kovati moramo pojma dejanska in simulirana lavinska ogroženost. S primerjavo obeh lahko izluščimo nekatere dodatne značilnosti prostorske razporeditve snežnih plazov. Zavedati se moramo, da so na tak način opredeljene površine tiste, ki jih v jih v naravi označujemo kot zbirno območje ali zaledje plazu ozi- roma območje proženja. Pri predstavljeni simulaciji lavinske ogroženosti ustrezajo te površine v glavnem območju proženja. Do neke mere je moč opre- deliti tudi površine, kjer se plazovi iztekajo in torej ustrezajo območju odla- ganja. Te so v veliki meri odvisne od prvih, saj so v njihovi vpadnici ali pa na bokih območij proženja posameznih plazov. V svoji raziskavi sem obdelal območje slovenskih Alp, pri čemer sem upošteval rezultate obdelave snežnih plazov iz lavinskega katastra na območju Julijskih Alp (N=506) ter podatkovne sloje o naravnogeografskih značilnostih štirih pokrajin, ki so pri tipizaciji v Geografskem atlasu Slovenije (Perko 1998b, str. 121) uvrščene k alpskim visokogorjem. ) p g j Pri pripravi tematskega zemljevida sem si pomagal z nekaterimi ugo- tovitvami in metodami, ki sta jih uporabila pri določanju lavinsko ogroženega površja na testnem območju Davosa (Švica) Kienholz in Grunder (1986). Model sta zgradila tako, da je uporaben tudi na enakem ali podobnem, to je goratem območju. Dobljeno simulacijo sta nato primerjala z dejansko lavinsko ogroženostjo površja s pomočjo podatkov iz švicarskega lavinskega katastra. V končnem simulacijskem modelu lavinske ogroženosti površja sta upoštevala predvsem snežne razmere, naklonske in rastne značilnosti površja, rabo tal ter domnevni iztek plazu. Slednjega sta opredelila kot nižje in v vpadnici območja proženja ležeče celice določene dolžine in naklona. Zaradi bistveno večje površine slovenskih Alp od testnega območja v Davosu in s tem natančnosti izvornih podatkov sem se zavedal dejstva, da so tako dobljene površine s simulirano ogroženostjo manj natančne. Vendar pa je glede na stopnjo simu- lirane ogroženosti še vedno velika verjetnost, da se na teh površinah tudi dejansko lahko prožijo snežni plazovi. V preglednici 1 so v stolpcih po pomembnosti (stolpci 1 do 7) predstav- ljeni posamezni dejavniki oziroma sestavine ogroženosti po posameznih sto- pnjah. Abstract According to its relief, Slovenia is highly variegated and its landscape diverse, and for this reason it is threatened by numerous natural accidents, among which are avalanches. On the basis of previous findings and with the help of data from Slovenia’s Geographical Information System, we undertook to determine potential avalanche surfaces in the Slovene Alps. In so doing, we considered the results of data processing on avalanches in the Julian Alps from the avalanche cadastre, other similar studies, and data layers on natural and geographical elements in high-mountain regions. In conclusion, we compared simulated and actual potential avalanche threat areas to assess the validity of our model. Key words: physical geography, avalanches, natural disasters, avalanche en- dangered areas, potential risk, avalanche accident prevention 115 Miha Pavšek / Dela 18 • 2002 • 115-132 1. TEMELJNA IZHODIŠČA Na temelju teh sem na koncu opredelil skupno lavinsko ogroženost posameznih hektarskih celic. Vsakega od dejavnikov sem glede na stopnjo la- 116 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah vinske ogroženosti razvrstil v tri glavne razrede ter opredelil meje med njimi. Mejne vrednosti sem določil s pomočjo podatkov iz različnih virov in lite- rature, katastra ter GIS-a za Slovenijo (GIAM). Pri končni simulaciji pa sem upošteval vseh sedem dejavnikov. Na prvi stopnji simulacije posameznih de- javnikov nisem ponderiral, kasneje pa sem se odločil za ponderiranje naklo- na, saj je ta med vsemi najpomembnejši. V preglednicah pa predstavljam še delež lavinsko ogroženega površja po stopnjah ogroženosti tako na območju slovenskih Alp kot tudi v okviru vseh ogroženih površin. Preglednica 1: Izbrani kriteriji za določanje potencialne lavinske ogroženosti površja na območju slovenskih Alp. Stopnja ogrože- nosti (števil- čno in opisno) 1 naklon (v sto- pinjah) 2 rastje po višini (opisno) 3 ekspo- zicija (strani neba) 4 trajanje snežne odeje (št. dni na leto) 5 maks. višina snežne odeje (m) 6 nadm. višina (m) 7 tipi podnebja (opisno) 3 velika 31 do 45 rastje skalnih razpok, alpskih melišč in prodišč, subalpinskih in alpinskih travišč, kmet. zemljišča J, JV, JZ nad 150 nad 3,0 nad 1600 podnebje višjega gorskega sveta 2 zmerna 21 do 30 46 do 60 subalpsko grmišče ruševja Z, SZ 75 do 150 1,0 do 3,0 1200 do pod 1600 podnebje nižjega gorskega sveta zahodne Slovenije 1 majhna 11 do 20 61 in več nizki gozd ali grmišče gabrovca in hrastov, omelike ali malega jesena V, S, SV pod 75 pod 1,0 300 do pod 1200 podnebje nižjega gorskega sveta severne Slovenije in drugi podnebni tipi 0 neogro- ženo 10 in manj drugo rastje - - - pod 300 - Preglednica 1: Izbrani kriteriji za določanje potencialne lavinske ogroženosti površja na območju slovenskih Alp. 117 Miha Pavšek / Dela 18 • 2002 • 115-132 2. NAKLON Temeljni vir za določanje naklonskih značilnosti slovenskih Alp glede lavinske ogroženosti je bil zemljevid naklonov površja iz GIS-a za Slovenijo (GIAM). Pri določanju mejnih vrednosti za posamezne razrede ogroženosti (pregledni- ca 1, stolpec 1) sem si pomagal z rezultati obdelave podatkov iz lavinskega katastra kot tudi z literaturo. Naklon od 31 do 45° ima v Julijskih Alpah na območju proženja več kot tri četrtine plazov, vrednosti iz drugega razreda pa nekaj manj kot četrtina. Naklonske značilnosti plaznic pri preučevanju snežnih plazov za daljše časovno obdobje so tako stalne, da jih lahko upoštevamo kot konstanto. Zato predstavljam naklonske značilnosti nekoliko podrobneje. Pri naklonu je za proženje snežnih plazov ugodna večina površja sloven- skih Alp (slika 1). Analiza lavinske ogroženosti po naklonih (preglednica 2) nam kaže, da je takega površja kar 86,4 %. Skoraj tri desetine površja v sloven- skih Alpah je tako strmih, da je na njih velika lavinska ogroženost, na tretjini pa zmerna. Kot lavinsko neogrožena območja izstopajo dna dolin in kotlin, neka- tere pobočne police in planotasti sredogorski, predgorski ter visokogorski svet. Povsem drugačna pa je slika na njihovem robu. V Julijskih in Kamniško-Savinjskih Alpah je zmerno ogroženo površje pogosto v obliki otokov znotraj površin z veliko ogroženostjo. To pomeni, da se naklon pobočij nižje spodaj spet poveča. S tem je izpolnjen eden od predpo- gojev za nadaljevanje poti snežnih gmot proti dolini. Ta značilnost je pred- vsem posledica podobne geotektonske zgradbe obeh visokogorskih pokrajin, saj prekinjajo pobočja številni kamninski pasovi in geološke strukture. Kara- vanke po lavinski ogroženosti glede naklonov prav nič ne zaostajajo za dru- gima dvema visokogorskima pokrajinama, v drobnem celo prav nasprotno. Nekatera pobočja so še daljša in bolj zglajena, saj jih ne prekinjajo vmesni skoki in pregibi. Preglednica 2: Simulacija lavinske ogroženosti v slovenskih Alpah - naklon. Stopnja ogroženosti Delež površja (v %) Velika (3) 28,9 Zmerna (2) 33,7 Majhna (1) 23,8 Neogroženo (0) 13,6 skupaj: 100,0 Preglednica 2: Simulacija lavinske ogroženosti v slovenskih Alpah - naklon. V vseh treh pokrajinah je dejanska lavinska ogroženost glede naklonov po- vršinsko mnogo manjša, saj moramo izločiti večino pobočij, ki so pod go- zdom. Nekaj kmetijskih zemljišč kot tudi poselitev se krajevno še vedno drži 118 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah 119 Slika 1: Simulacija lavinske ogroženosti v slovenskih Alpah - naklon. Slika 1: Simulacija lavinske ogroženosti v slovenskih Alpah - naklon. 2. NAKLON 119 Miha Pavšek / Dela 18 • 2002 • 115-132 strmega sveta, s čimer si lahko razlagamo precejšen delež površin z veliko lavinsko ogroženostjo v Vzhodnih Karavankah. Simulacija lavinske ogrože- nosti glede na naklone (slika 1) nam kaže predvsem pomen gozda kot učinko- vite zaščite pred snežno erozijo. Upoštevati jo moramo kot opozorilo pred neprimernimi posegi v gozdovih, ki so pomembni za ohranjanje naravnega ravnovesja in preprečevanje oziroma zmanjševanje snežne erozije kakor tudi erozije na splošno. 3. RASTJE PO VIŠINI Naslednji pomemben dejavnik je bilo rastje, ki porašča pobočja oziroma nje- gova višina in gostota. Osnovni vir za določanje rastnih značilnosti sloven- skih Alp glede lavinske ogroženosti je bil zemljevid realne vegetacije (Zupan- čič in drugi 1998, str. 118). Kljub sorazmerni prostorski nenatančnosti je po izbranih kategorijah rastja najprimernejši. Pri natančnejšem prostorskem pre- učevanju lavinske ogroženosti je bolje, da je zaradi nenatančnosti ne upošte- vamo. Pomembna je predvsem višina rastja, ki je v veliki meri povezana tudi s starostjo združb, gostoto dreves in nekaterimi drugimi značilnostmi gozdov. Rastje mora presegati višino vsaj enega metra (Lawinenaufnahmeblatt, Hin- weise für… 1980), kar ustreza povprečni višini ruševja. Pri tej višini že lahko govorimo o pomembnejšem vplivu oziroma zaščiti rastja pred snežno erozijo. Vendar velja to le do tedaj, ko ga prekrije snežna odeja. Rastje po višini sem razdelil v tri skupine (preglednica 1, stolpec 2). Pri tem so zaradi merila os- novnega vira izpadla kot neogrožena skoraj vsa manjša, nestrnjena kmetijska zemljišča. 4. EKSPOZICIJA Temeljni vir za določanje značilnosti ekspozicij v slovenskih Alpah glede la- vinske ogroženosti je bil zemljevid ekspozicij površja iz GIS-a za Slovenijo (GIAM). Ker so pri osnovnem zemljevidu ekspozicij upoštevane vse kotne vrednosti (od 0 do 360°), sem uporabil sloj, kjer so združene posamezne vrednosti glede na najpomembnejše strani neba v osem razredov (S, SV, V, JV, J, JZ, Z, in SZ; Perko 1998a, str. 88), posebej pa so izločene ravnine (vse celice z naklonom manjšim od 0,5°). V nasprotju z ugotovitvami o večji plazovitosti pobočij v senčnih legah (SZ-S-SV) sem se odločil za drug razmejitveni kriterij. Plazove z območja Ju- lijskih Alp sem rastriral ter primerjal ekspozicije plazov z ekspozicijami po- vršja. S pomočjo indeksa koncentracije (IK = delež plazov/delež površja x 100) 120 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah sem nato razvrstil posamezne ekspozicije glede na simulacijo lavinske ogro- ženosti v ustaljene tri razrede. Tudi po podatkih iz lavinskega katastra prevla- dujejo na območju Julijskih Alp plaznice na južni ekspoziciji (26 %). Zato sem jo tudi pri simulaciji lavinske ogroženosti skupaj z jugovzhodno in jugo- zahodno ekspozicijo uvrstil v najvišji razred ogroženosti (preglednica 1, stol- pec 3). p ) Simulacija lavinske ogroženosti glede na ekspozicijo površja nam je po- kazala, da je površin z veliko stopnjo ogroženosti skoraj enkrat več kot tistih z zmerno, majhna ogroženost pa je na nekaj več kot tretjini površja slovenskih Alp. Tudi pri ekspoziciji zmanjšujejo navidez veliko lavinsko ogroženost go- zdovi, ki segajo s pomikanjem od juga proti severu vse višje. Opozoriti velja še na površje v večjem delu Zahodnih Karavank in v zahodnem delu Kam- niško-Savinjskih Alp, ki je predvsem po ekspoziciji pa tudi po oblikah površja in višini rastja zelo podobo grebenom na severni strani Srednje Soške doline. Razlika je le v količini padavin, kar pomeni, da lahko pričakujemo ob izdatnejših padavinah večje plazove tudi v tem delu slovenskih Alp. 5. TRAJANJE SNEŽNE ODEJE Naslednji podatkovni sloj je tematski zemljevid o trajanju snežne odeje oziro- ma povprečnem številu dni s snežno odejo (Mlinar 2000). Domeval sem, da dlje kot traja snežna odeja, večja je tudi nevarnost proženja snežnih plazov oziroma stopnja lavinske ogroženosti površja (preglednica 1, stolpec 4). Te- matski zemljevid (Mekinda Majaron 1996) so pripravili s pomočjo optimalne interpolacije oziroma tej prirejenega matematičnega modela. Poleg podatkov o trajanju snežne odeje so upoštevali še nadmorske višine, ne pa tudi ekspozicij. Zaradi majhnega števila primerljivih meteoroloških postaj to ni bilo možno, kilometrska mreže pa je povzročila, da so izpadle iz posameznih razredov višje ogroženosti tudi nekatere manjše oblike površja. Največji delež površja (61,3 %) pade v drugo stopnjo ogroženosti, več kot petina pa v prvo. Velika lavinska ogroženost je glede trajanje snežne odeje prisotna tako v nižjem viso- kogorju Julijskih Alp kot tudi v najvišjih delih Karavank in Kamniško-Sa- vinjskih Alp. Opozoriti velja na nekatere proti jugu pomaknjene gorske grebene in hrbte v Julijskih Alpah (Polovnik, Črna prst, Ratitovec), kjer obleži sneg dlje časa zaradi izrazito večje količine padavin v času snežnih razmer. 121 Miha Pavšek / Dela 18 • 2002 • 115-132 6. MAKSIMALNA VIŠINA SNEŽNE ODEJE Pomemben podatek za simulacijo lavinske ogroženosti je tudi maksimalna vi- šina snežne odeje. Kot referenčni vir sem uporabil tematski zemljevid za obdobje 1951 - 1990 (Bernot in drugi 1994), ki so ga pripravili s prostoročno interpolacijo podatkov o višini snežne odeje po posameznih meteoroloških po- stajah. Do ustreznega sloja sem prišel šele po vektorizaciji izvornih podatkov na topografski osnovi v merilu 1 : 400.000 ter z njihovo dopolnitvijo s podatki za obdobje 1977 - 1990 (Mekinda Majaron 1996, str. 56) za območje zahodnih Julijskih Alp. Prvotnih osem razredov višin snežne odeje sem prerazporedil za simulacijo lavinske ogroženosti površja v ustaljene tri razrede (preglednica 1, stolpec 5). Tudi tu je najbolj opazen vpliv nadmorske višine, v zahodnem in južnem delu Julijskih Alp pa tudi bistveno večja letna količina padavin. Ob prehodu vremenskih front z zahoda, jugozahoda in juga se pridruži ciklo- nalnemu in v manjši meri konvekcijskemu tudi orografsko dviganje zračnih gmot, ki se na svoji poti nad Sredozemljem navzamejo vlage. Grebeni v tem delu Julijskih Alp so za omenjene zračne gmote prva višja pregrada. Učinek prisilnega dviga je najmočnejši, kadar je smer zračnih mas pravokotna s smer- jo grebena oziroma gorskega hrbta. 7. NADMORSKA VIŠINA Kot poseben dejavnik lavinske ogroženosti sem upošteval tudi nadmorsko vi- šino (preglednica 1, stolpec 6). Delež snega v letni količini padavin narašča tako z večanjem geografske širine kakor tudi z naraščanjem nadmorske višine. Za Vzhodne Alpe so ugotovili, da se delež snega v celotni količini padavin na vsakih 100 metrov poveča za 3 %. (Kladnik 1999). Nadmorska višina je po- membna tudi pri vsakokratnem sneženju v zvezi z mejo sneženja. Na območju Alp sneži najpogosteje še približno 300 višinskih metrov pod nadmorsko višino, na kateri poteka izoterma 0° C (Munter 1992, str. 60). Nadmorske vi- šine oziroma 100-metrske višinske pasove sem določil s pomočjo DMR 100 x 100 m (GIS za Slovenijo, GIAM). Mejne vrednosti razredov sem do- ločil na osnovi več dejavnikov: meja sneženja, gozdna in drevesna meja, višinska meja stalne poselitve in drugo. Najpomembnejši pri tem so bili spet rezultati obdelave plazov iz lavinskega katastra. Pri vzorcu za Julijske Alpe sem vzel pod drobnogled vse tri povprečne nadmorske višine območja proženja: zgornjo, srednjo in spodnjo. Pri končni opredelitvi mejnih vrednosti (1200 in 1600 m) sem dal poleg prej omenjenih dejavnikov največjo težo srednji nadmorski višini območja proženja. 122 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah 8. PODNEBJE Kot zadnji dejavnik pri simulaciji lavinske ogroženosti sem upošteval še pod- nebje oziroma podnebne tipe. Podnebje združuje tudi vse prejšnje dejavnike, nekatere neposredno (snežne padavine - trajanje in višina, nadmorska višina), druge posredno (naklon, ekspozicija, rastje po višini). Zadnjega od dejavnikov za simulacijo lavinsko ogroženega površja sem uporabil predvsem za pre- verjanje vseh prejšnjih, še posebej za visokogorska območja. Izhodišče je bil osnutek tematskega zemljevida podnebnih tipov v Sloveniji (Ogrin 1998, str. 111), pri katerem je vzel avtor kot osnovo Köppenovo razvrstitev svetov- nega podnebja. Na območju slovenskih Alp so trije najpomembnejši podnebni tipi (preglednica 1, stolpec 7). 9. OSNOVNA SIMUALCIJA LAVINSKE OGROŽENOSTI Preglednica 3: Osnovna simulacija lavinsko ogroženih površin - velikost in delež lavinsko ogroženega površja v okviru slovenskih Alp in delež v okviru vseh ogroženih površin. Stopnja ogroženosti (opisno) Stopnja ogroženosti (številčno) Število celic (ha) Delež od vsega površja (%) Delež od ogr. površja (%) Velika (3) 3 22814 7,5 32,6 Zmerna (2) 2 22928 7,5 32,8 Majhna (1) 1 24211 7,9 34,6 Neogroženo (0) 0 236195 77,1 - skupaj: 306148 100,0 100,0 Neogroženo 0 236195 77,1 - Ogroženo 1+2+3 69953 22,9 - skupaj: 306148 100,0 - 9. OSNOVNA SIMUALCIJA LAVINSKE OGROŽENOSTI Na koncu sem izdelal še simulacijo lavinsko ogroženega površja, pri kateri sem upošteval vseh sedem dejavnikov. Zadovoljive rezultate sem dobil predvsem za območja nad gozdno mejo, izpadle pa so skoraj vse krčevine pod njo. Tudi zaradi tega sem pripravil več različic. Najprej si oglejmo osnovno simulacijo lavinske ogroženosti (slika 2) in njene prostorske razsežnosti (preglednica 3). Pri tej različici sem množil med seboj vse dejavnike v preglednici 1 (stolp- ci 1 do 7). Pri tem so dobile celice, kjer je imel eden od dejavnikov v stolpcih 1, 2 in 6 vrednost 0 tudi enako končno vrednost. To površje sem poimenoval kot neogroženo, vendar je zaradi določenih odstopanj in nenatančnosti podat- kov to dejansko le pogojno neogroženo površje. Druge celice so imele vred- nosti od 1 do 2187 (37) in sem jih razvrstil v tri razrede po načelu enakomernih deležev. Na območju slovenskih Alp je nekaj več kot petina (22,9 %) ali skoraj 700 km2 površja, ki je lavinsko potencialno ogroženo. V nadaljevanju sem izkoristil tematski zemljevid realne vegetacije, na katerem so poudarjeni gozdovi (Zupančič in drugi 1998, str. 118) za pregled gozdov po višinskih pasovih na tistih območjih, kjer so v vpadnici oziroma pod lavinsko ogroženim površjem. Nato pa sem dodatno izločil še vse površine pod gozdom na površju z naklonom od 0 do 20°, kjer je plazenje že pri majhni gostoti dreves izključeno. Ogroženost gozdov je največja tam, kjer sovpadata velika lavinska ogroženost površja (najtemneje obarvane površine) in višinski pas gozda na njegovi gozdni oziroma drevesni meji. Po tej simulaciji je na območju slovenskih Alp lavinsko zelo ogroženih okrog 10.500 ha gozda oz. 3,4 % vsega gozdnatega površja. Po nekaterih grobih ocenah (Horvat 1984; Mikoš in Pintar 1983) imamo v celotni Sloveniji okrog 16.000 ha plazovitih 123 Miha Pavšek / Dela 18 • 2002 • 115-132 Slika 2: Osnovna simulacija lavinske ogroženosti v slovenskih Alpah. Slika 2: Osnovna simulacija lavinske ogroženosti v slovenskih Alpah. Slika 2: Osnovna simulacija lavinske ogroženosti v slovenskih Alpah. 124 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah območij, v lavinskem katastru, kjer so zajeti le akutni plazovi, pa je skupna po- vršina do sedaj popisanih plazov nekaj več kot 13.400 ha. Preglednica 3: Osnovna simulacija lavinsko ogroženih površin - velikost in delež lavinsko ogroženega površja v okviru slovenskih Alp in delež v okviru vseh ogroženih površin. 10. PONDERIRANA SIMUALCIJA LAVINSKE OGROŽENOSTI Zaradi pomembnosti naklona za plazenje snežne odeje sem se odločil še za izdelavo ponderirane simulacije lavinske ogroženosti, pri kateri sem ponderiral vrednosti za naklone, saj so ti podatki med najbolj natančnimi (slika 3). Tudi Kienholz in Grunder (1986, str. 104) sta pri delni simulaciji lavinske ogroženosti za območje proženja opredelila in ovrednotila kot najbolj plazovito površje z naklonom med 30 in 45° C (5 točk), kot zmerno ogroženo z naklonom med 45 in 60° (3 točke) ter med 20 in 30° (2 točki). Kot najmanj ogroženo pa površje, ki ima naklon nad 60° (1 točka). Tako sem v nadaljevanju vrednosti znotraj raz- reda velike ogroženosti pomnožil s 3, zmerne ogroženosti z 2, razred majhne ogroženosti pa je ostal enak. Po medsebojnem množenju posameznih slojev so imele celice vrednosti od 0 do 2187 (37), nato pa sem jih spet razvrstil po načelu enakomernih deležev v tri razrede. Deleži lavinsko ogroženega površja pri ponderirani simulaciji so mnogo višji (razmerje z osnovno simulacijo je skoraj 1 : 4), ker nisem upošteval površja pod gozdom. Ta zemljevid nam kaže lavinsko pogojno ogroženo območje in je dobro izhodišče za nadaljnjo krajevno opredelitev dejansko ogroženega površja. Njegova uporabnost je povezana s sočasnim upoštevanjem krajevnih značilnosti rastja na ožjem območju. Predvsem je tovrstna simulacija mnogo bolj uporabna 125 Miha Pavšek / Dela 18 • 2002 • 115-132 126 Slika 3: Ponderirana simulacija lavinske ogroženosti v slovenskih Alpah (po- nderirani nakloni, neupoštevanje rastja). Slika 3: Ponderirana simulacija lavinske ogroženosti v slovenskih Alpah (po- nderirani nakloni, neupoštevanje rastja). 126 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah 127 Slika 4: Dopolnjena ponderirana simulacija lavinske ogroženosti v slovenskih Alpah (ponderirani nakloni, razločevanje gozdnih in negozdnih površin). Slika 4: Dopolnjena ponderirana simulacija lavinske ogroženosti v slovenskih Alpah (ponderirani nakloni, razločevanje gozdnih in negozdnih površin). 127 Miha Pavšek / Dela 18 • 2002 • 115-132 izven visokogorja, saj je gozd povsem izločil lavinsko ogroženost na manjših zaplatah v okviru kmetijskih zemljišč. Dopolnjena ponderirana simualcija lavinske ogroženosti. Na koncu sem pripravil še dopolnjeno ponderirano simulacijo lavinske ogroženosti površja s pomočjo tematskega zemljevida gozdnih in negozdnih površin (vir: Biološki inštitut Jovana Hadžija ZRC SAZU). S to simulacijo sem izpostavil lavinsko ogroženost negozdnega površja na poseljenih območjih slovenskih Alp. Kot osnovo sem privzel ponderirano simulacijo lavinske ogroženosti, prek katere sem položil sloj z negozdnim površjem (slika 4). 10. PONDERIRANA SIMUALCIJA LAVINSKE OGROŽENOSTI Skupni delež lavinsko ogroženega površja pri tej simulaciji obsega več kot četrtino vsega površja slovenskih Alp in je za 2,8 % večji kot pri osnovni simulaciji. Manj je površja z majhno ogroženostjo, nekoliko več z zmerno in bistveno več z veliko ogroženostjo. Podatek o tem, da ogrožajo snežni plazovi na območju slovenskih Alp 10,5 % površja (pri osnovni simulaciji 7,5 %) je povsem primerljiv s podobnimi ocenami za druga gorska območja v Alpah (Kienholz in Grunder 1986, str. 107 in 108). 11. PRIMERJAVA POTENCIALNO IN DEJANSKO LAVINSKO OGROŽENEGA POVRŠJA S pomočjo računalniškega programa Idrisi sem na koncu še primerjal lavinsko ogroženo površje, ki sem ga opredelil v osnovni simulaciji in tisto, do katerega sem prišel prek podatkov o plazovih, ki so v lavinskem katastru. V tem prime- ru gre za preverjanje površja s potencialno (simulirano) in dejansko (potrjeno) lavinsko ogroženostjo. Od celotne površine plazov iz lavinskega katastra jih je na območju slovenskih Alp več kot devet desetin. Skoraj dve tretjini površja teh plazov se prekrivata s simulacijsko opredeljenim lavinsko ogroženim po- vršjem iz osnovne simulacije. Ostala tretjina, ki je prišla v razred neogroženo gre predvsem na račun območij gibanja in odlaganja plazov, linearne pla- zovitosti in plazov na gozdnatem površju. Za simulacijo lavinske ogroženosti na teh območjih bi potrebovali natančnejše izvorne podatke (DMR 50 x 50 m, ostali sloji na topografski podlagi v merilu 1 : 25.000 ali večjem), poleg tega pa bi morali na tem območju opraviti še podrobno geomorfološko kartiranje. Od vse površine plazov iz lavinskega katastra je skoraj polovica na simulacijsko ogroženih območjih z veliko ogroženostjo, nekaj manj kot tretji- na na tistih z zmerno in manj kot petina na simulacijsko ogroženih območjih z majhno ogroženostjo. Največja pokritost potencialne in dejanske lavinske ogroženosti je prav pri prvih dveh razredih. S to simulacijo sem opredelil uspešno predvsem površje z zmerno in veliko lavinsko ogroženostjo, kar je tudi najpomembnejše, saj se ujema z enim od ciljev raziskave. 128 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah Simulacijo lavinske ogroženosti sem preveril še na dva načina. Najprej sem prekril podatkovna sloja osnovne simulacije lavinske ogroženosti, pri ka- teri sem upošteval tudi vse gozdne površine po višinskih pasovih in plazove iz lavinskega katastra. Večina plazov iz lavinskega katastra se ujema z lavinsko ogroženimi območji, ki sem jih opredelil s simulacijo. Na nekaterih območjih lahko prepoznamo veliko ogroženost gozdov na zgornji gozdni in drevesni meji (Zadnja Trenta, Vrata, dolina Tolminke in drugod), veliko plazov pa je v celoti na s simulacijo opredeljenem lavinsko ogroženem območju izven gozd- nih površin. S podatki o plazovih iz lavinskega katastra sem preveril tudi pon- derirano simulacijo (slika 5). Kot vzorčno območje sem vzel Zgornje Posočje in Srednjo Soško dolino, saj je v lavinskem katastru največ plazov prav iz tega območja. Slika 5: Preveritev ponderirane simulacije lavinske ogroženosti s podatki iz lavinskega katastra na izbranem območju v Zgornjem Posočju in Srednji Soški dolini. dolini. 12. SKLEP Za natančnejši model simulacije lavinsko ogroženega površja sta najpomemb- nejša natančnost izvornih podatkov in izhodiščna hipoteza. Z modelom dobljeno lavinsko ogroženo površje je dobro izhodišče za natančnejše krajev- no preučevanje lavinske ogroženosti površja kot tudi za vsakokratno oceno in napoved lavinskih razmer v zimski sezoni. S tovrstnimi simulacijami bi bilo moč v prihodnje predstaviti tudi ogroženost površja glede na različne scenarije pokrajinskega razvoja: površje brez gozda, varstveno stanje, prvotno stanje in stanje ob nadaljevanju intenzivnega razvoja turizma. Primerjava potencialne in dejanske lavinske ogroženosti nam omogoča premišljeno in neposredno razvrščanje območij po stopnjah lavinske ogroženosti. Ponderirana simulacija, ki nam predstavlja pogojno lavinsko ogroženo površje nam kaže hkrati tudi latentno ali prikrito lavinsko ogroženost površja. Na to pozabljamo pri večini pojavov povezanih s snežno erozijo. Njihova povratna doba najpogosteje pre- sega človekovo opažanje pokrajinskih sprememb, zato se izkušnje in spozna- nja praviloma ne prenašajo med generacijami. To lahko povzročijo le izjemni dogodki, na katere pa moramo biti vedno ustrezno in pravočasno pripravljeni ter glede na možnosti tudi opozorjeni. 11. PRIMERJAVA POTENCIALNO IN DEJANSKO LAVINSKO OGROŽENEGA POVRŠJA S simulacijo dobljena območja potencialne lavinske ogroženosti v veliki meri sovpadajo z dejansko lavinsko ogroženim površjem, saj se več kot devet desetin površja vseh plazov iz katastra prekriva s simulacijsko opredeljenim. S simulacijo dobljena območja potencialne lavinske ogroženosti v veliki meri sovpadajo z dejansko lavinsko ogroženim površjem, saj se več kot devet desetin površja vseh plazov iz katastra prekriva s simulacijsko opredeljenim. 129 Miha Pavšek / Dela 18 • 2002 • 115-132 Manj kot desetina površine plazov pa je prišla v razred neogroženo. Od vse površine plazov iz lavinskega katastra jih je več kot sedem desetin na potencialno ogroženih območjih z veliko ogroženostjo, nekaj manj kot četrtina z zmerno in le nekaj več kot 5 % z majhno ogroženostjo. Pri tej simulaciji je pokritost potencialne in dejanske lavinske ogroženosti površja pri razredu z veliko in zmerno lavinsko ogroženostjo še za več kot desetino večja kot pri preverjanju osnovne simulacije. Seveda ne smemo pozabiti, da je na ta način dobljena velikost lavinsko ogroženega površja zaradi neupoštevanja gozdov precenjena. Primerjava med potencialno in dejansko ogroženim območjem kaže, da je ogroženost pri simulaciji bolj divja, površinsko neenakomernejša. Zavedam se, da sem uporabil sorazmerno velike poenostavitve, ki pa jih lahko glede na zahteve in potrebe tudi nekoliko prilagodimo. Bernot, F. in drugi, 1994, Ogroženost Slovenije s snežnimi plazovi (študija). Podjetje za urejanje hudournikov. 48 str. Ljubljana. GIAM - Geografski inštitut Antona Melika ZRC SAZU, Geografski infor- macijski sistem (GIS) za Slovenijo.. Ljubljana. Literatura: Bernot, F. in drugi, 1994, Ogroženost Slovenije s snežnimi plazovi (študija). Podjetje za urejanje hudournikov. 48 str. Ljubljana. GIAM - Geografski inštitut Antona Melika ZRC SAZU, Geografski infor- macijski sistem (GIS) za Slovenijo.. Ljubljana. 130 Simulacija ogroženosti površja zaradi snežnih plazov v slovenskih Alpah Horvat, A., 1984, Izbrana poglavja s področja snežne erozije (skripta). 38 str. + priloge. Ljubljana. Kienholz, H., Grunder, M., 1986, Naturgefahren: Entwicklung eines Modells durch visuelen Kartenvergeleich. Erfahrungen aus dem MAB-Testgebiet Davos. V: Jahrbuch der Geographischen Gesellschaft von Bern. Letnik 55/1983–1985, str. 95–110. Bern. Kladnik, D., 1999, Sneg. Geografija na teletekstu TV Slovenija (http://www. zrc-sazu.si/giam/ZGDS-11-12-1999.htm#Sneg). Ljubljana. Lawinenaufnahmeblatt - Hinweise für Planungen der Raumplanung, des Bauwesens und des Sicherheitswesens. V: Richtlinien des Bundesmini- steriums für Land- und Forstwirtschaft über Hinderungsgründe (Zl. 52. 240/03-VB 7/80). 1980. Mekinda Majaron, T., 1996, Average yearly number of days with snow cover (period od reference 1961–1990), Maximum snow cover depth with return period 50 years (period of reference 1977–1990). V: Climate of Slovenia. str. 55 56. Hidrometeorološki zavod Republike Slovenije. Ljubljana. Mikoš, M., Pintar, J., 1983, Snežni plazovi, njihovo ugotavljanje in vred- notenje (skripta). Vodnogospodarski inštitut. 69 str. + priloge. Ljubljana. Munter, W., 1992, Neue Lawinenkunde. Ein Leitfaden für die Praxis. 200 str. Bern. Ogrin, D., 1998, Podnebje. V: Geografski atlas Slovenije, str. 110–111. Lju- bljana. Perko, D., 1998a, Ekspozicije površja. V: Geografski atlas Slovenije, str. 88–89. Ljubljana. Perko, D., 1998b, Pokrajine. V: Geografski atlas Slovenije, str. 120–125. Lju- bljana. Zupančič, M., Seliškar, A. in Žagar, V., 1998, Rastlinstvo. Geografski atlas Slovenije, str. 116–119. Ljubljana. Summary According to its relief, Slovenia is highly variegated and its landscape diverse, and for this reason it is threatened by numerous natural accidents, among which are avalanches. The data on avalanches in the avalanche cadastre shows us the actual avalanche threat. On the basis of previous findings and with the help of data from Slovenia’s Geographical Information System, we undertook to determine potential avalanche surfaces in the Slovene Alps. In so doing, we 131 Miha Pavšek / Dela 18 • 2002 • 115-132 considered the results of data processing on avalanches in the Julian Alps (N=506) from the avalanche cadastre and data layers on natural and geo- graphical elements in high-mountain regions. During the simulation we considered the seven most important factors: slope, vegetation according to altitude, exposition, the duration and maximum thickness of the snow cover, altitude, and climate. We ranked each factor according to the level of threat into three or four classes (high, moderate, and low threat, unthreatened). The border values were determined with the help of data from scientific sources and literature, the avalanche cadastre, and the Geographical Information System. Finally, the total avalanche threat to individual hectare cells was determined. The areas thus acquired are those we mark as avalanche trigger areas. During the basic simulation we did not weight individual factors, while during the weighted simulation we weighted slope since it is the most significant factor. Vegetation studies enabled us to prepare a supplementary weighted simulation. The results of the simulations of avalanche threat are presented on synthetic thematic maps. On checking the data on avalanches in the avalanche cadastre, we found we had acquired satisfactory results mainly for areas above the tree line. A comparison between actual and potential avalanche threat areas reveals that the simulated threat is less accurate, unconnected, and super- ficially disproportionate. For a more accurate model of any avalanche threat simulation, the accuracy of the original data and a suitable initial hypothesis are the most important factors. These must be particularly considered in future in preparing similar thematic maps for smaller, geographically bounded areas as the obligatory professional basis for spatial planning in Slovenia’s alpine regions. 132
https://openalex.org/W2793180036	https://nottingham-repository.worktribe.com/preview/919024/Arbmirage_150218.pdf	Latin	null	New entry, strategic diversity and efficiency in soccer betting markets: the creation and suppression of arbitrage opportunities	European journal of finance	2,018	cc-by	12,881	New Entry, Strategic Diversity and Efficiency in Soccer Betting Markets: The Creation and Suppression of Arbitrage Opportunities Andrew Granta,, Anastasios Oikonomidisb, Alistair C. Brucec and Johnnie E.V. Johnsonb* Andrew Granta,, Anastasios Oikonomidisb, Alistair C. Brucec and Johnnie E.V. Johnsonb* Andrew Granta,, Anastasios Oikonomidisb, Alistair C. Brucec and Johnnie E.V. Johnsonb* a: University of Sydney Business School, Building H69, 1 Codrington St, Darlington NSW 2008. b: University of Southampton Business School, Highfield, Southampton, SO17 1BJ c: Nottingham University Business School, Jubilee Campus, Nottingham, NG8 1BB a: University of Sydney Business School, Building H69, 1 Codrington St, Darlington NSW 2008. b: University of Southampton Business School, Highfield, Southampton, SO17 1BJ c: Nottingham University Business School, Jubilee Campus, Nottingham, NG8 1BB * We would like to thank Ron Bird, Doug Foster, and Terry Walter for helpful comments. 1. INTRODUCTION This paper examines the impact of new entrants into the European market for soccer betting, which until recently has been dominated by European (and generally UK) based operators. Specifically, we evaluate how the recent entry of several large and (predominantly, though not exclusively) Asian bookmaking organisations has affected the range of odds available to consumers, with potential consequences for the efficiency of the market. As such, this paper contributes to a significant empirical interest in, and literature on, the operation of betting markets. A principal motivation for this study is the fact that there are clear, material differences between the recent entrants and the more established operators, both in product portfolios and aspects of operating behaviour. These differences raise the possibility of profitable arbitrage opportunities for bettors, with attendant implications for market efficiency. The paper is structured as follows. This section explains briefly why betting markets in general constitute a fertile territory for studies of market behaviour and efficiency. This leads into a discussion of the recent structural changes in the European soccer betting market. Section 2 outlines relevant theoretical material in relation to arbitrage and bookmaker behaviour. Section 3 develops hypotheses, and the data and methods employed to test these are outlined in Section 4. Section 5 presents results and Section 6 offers a discussion and conclusion. Abstract We find that prices offered by competing bookmakers within the same quote-driven soccer (football) betting market provide arbitrage opportunities. However, the management practices of bookmakers prevent informed bettors exploiting these in practice. We identify two groups of bookmakers, ‘position-takers’ and ‘book-balancers.’ Position-takers alter their odds infrequently, while actively restricting informed traders. Book-balancers actively manage inventory by adjusting odds, and place few restrictions on their customers. We identify 545 arbitrage portfolios, and find that around 50% would require a bet on the favourite at the position-taking bookmaker. The management practices of position-takers generally prevent these opportunities being exploited in practice. (D23), (L22), (L83) New Entry, Strategic Diversity and Efficiency in Soccer Betting Markets: The Creation and Suppression of Arbitrage Opportunities New Entry, Strategic Diversity and Efficiency in Soccer Betting Markets: The Creation and Suppression of Arbitrage Opportunities 1.1 Betting Markets as an Investigative Medium A number of characteristics of bookmaker-driven betting markets make them both interesting in their own right, as well as an ideal setting for field experiments to explore aspects of broader financial market efficiency (see, for example, Sauer, 1998; Vaughan Williams, 1999, 2005; Oikonomidis and Johnson, 2011). As explained by Thaler and Ziemba (1988) a key advantage of betting markets is the fact that the objective values of assets are determined with certainty at the close of the market, as the outcome of the betting event is revealed. Decisions made in betting markets (i.e. bets placed) are clearly and individually recorded and this, together with the 2 very large number of individual markets, ensures a rich documentary resource for empirical investigation. Additionally, betting markets resemble other financial markets in terms of the varying sophistication levels of traders, who risk their own assets on the uncertain outcome of future events. Betting markets may therefore provide more generalizable results than those from laboratory experiments, in which subjects may be inexperienced traders and hold limited motivation (Levitt and List, 2007). Our focus in this paper is on structural change in the betting markets for European soccer associated with the entry of large, generally Asian-based bookmakers, such as SBOBet and Pinnacle, alongside traditional European bookmakers, such as the U.K.-based William Hill and Ladbrokes. An interesting aspect of this change is that there are distinct and observable differences in operating practice between the established bookmakers and the new entrants, in relation to trading restrictions, volatility of the odds menu and the range of event outcomes offered. These are now explained. With regard to trading policy, the new entrants adopt a conspicuously more liberal approach to clients in that they make no attempt to restrict bets from potentially `informed’ or more sophisticated bettors. Pinnacle, for example, explicitly state: `Our Winners Welcome policy is very straight-forward. We do not limit, discriminate or close accounts of successful players, and here is why:  Our business model is focused solely on maximising volume irrespective of whether this is generated from profitable players  We need sharp players to help tighten our odds as we do not take positions  We have the confidence in our traders to focus on managing odds, not players’ By contrast, traditional European operators actively manage their client portfolios to deter trade from these groups. 1.1 Betting Markets as an Investigative Medium Where an individual’s betting activity generates what these bookmakers regard as an unprofitable line of business over a sustained period, this frequently 3 results either in staking limits being imposed on that individual, delays in settling debts, or at the limit, termination of the ability to trade. The following actual, but anonymised, example of an e-mail communication (one of a number of similar examples held by the authors) illustrates this practice. `Following a review of your account by our trading department, moving forward you are no longer going to be able to place bets to the stake size that you have been accustomed to. Although your account will remain open and active, the bets you are likely to be able to place will be significantly less than what you have been able place in the past. ‘ In addition, this practice of turning away or deterring potentially unprofitable business has been widely reported in the general and specialist media. The Guardian (28 June, 2016) describes one such instance: In addition, this practice of turning away or deterring potentially unprofitable business has been widely reported in the general and specialist media. The Guardian (28 June, 2016) describes one such instance: `Bet365, one of the internet’s biggest bookmakers, is facing legal action from a customer over its failure to transfer a £54,000 balance to her bank account despite repeated requests over a period of months. While refusing to release the backer’s winnings on a series of horse racing bets, Bet365 also told her that she would be restricted to a maximum stake of £1 if she wished to bet with the balance but was welcome to gamble as much as she wished on gaming products, which have a guaranteed margin for the operator. The punter, whose identity is known to the Guardian, opened an account with Bet365 on 16 April and deposited £30,000 with the firm the following day, when she placed a series of bets on horse racing and lost £23,000. She received an email from Bet365 the same day, which stated that the size of the maximum bet she was allowed to place had been increased. The following day she placed further bets with the remaining £7,000, winning a total of £47,000, which raised her balance to about £54,000. 1.1 Betting Markets as an Investigative Medium The same day she was informed via email that in future her account would be restricted to a maximum stake of £1 on racing bets as the result of a “trading decision”.’ The UK Horseracing Bettors’ Forum (UKHBF) conducted a survey in April 2016 of betting account closure, which demonstrated the widespread nature of the practice among UK bookmakers. On the basis of the survey, it was estimated that around 20,000 accounts had been closed in the previous six months (The Guardian, 13 June 2016, UKHBF (2016)). 4 These sharply distinct approaches to clients are further reflected in differences in the evolution of betting markets relating to individual events. European operators vary the initial menu of odds only occasionally. In contrast, the new entrant operators engage in much more frequent odds adjustments as information is brought, unfettered, to market by a wider clientele. A comparison between leading representatives of each group of bookmakers (SBOBet and Ladbrokes), employing the main dataset used in this study (see Section 3, below, for details), charts changes in odds collected at nine points in time in the 24 hours leading to the kickoff of 2,132 matches. This reveals that the odds of the book-balancing bookmaker SBOBet are different at point t (compared to its odds at point t-1) on 77% of occasions. However, the odds of the PTB, Ladbrokes, only differ from the preceding time period on 12.4% of occasions. SBOBet odds changed, on average, on 5.4 occasions per match in the 24 hours prior to kick off whilst the equivalent figure for Ladbrokes is 0.8. A t-test confirms that the difference in the frequency of changes is very unlikely to be random (p-value= 0.000). The active client management adopted by established European-based operators restricts turnover, relative to their new entrant counterparts. However, the former can expect to secure higher margins by avoiding exposure to informed behaviour. These relatively high margins will also be required to offset risks of holding inventory in the form of unbalanced liabilities across match outcomes. We refer to traditional European operators as ‘position-taking bookmakers’ (PTBs), reflecting the inflexibility of their odds. The dynamic odds of the new entrant bookmakers, by contrast, whilst minimising the risk of holding outcome-dependant inventory, result in lower margins associated with the higher processing costs of frequently changing odds. We refer to these bookmakers as ‘book-balancing bookmakers’ (BBBs). 1.1 Betting Markets as an Investigative Medium The theoretical underpinnings of the distinctions between PTBs and BBBs are explained more fully in Section 2.2. A further significant distinction between the groups, in product terms, relates to the prominent use of the Asian Handicap (AH) product by the BBBs. This effectively eliminates the need for the bookmaker to set odds against the ‘draw’ outcome by setting a point spread involving a fraction of a goal, or refunding bets in the case of a draw. Effectively, this simplifies inventory rebalancing 5 as the market consists of two, rather than three outcomes, compared with the threefold ‘Home- Draw-Away’ or 1X2 menu offered by PTBs. as the market consists of two, rather than three outcomes, compared with the threefold ‘Home- Draw-Away’ or 1X2 menu offered by PTBs. Table 1 offers a summary comparison of differing characteristics of the distinct bookmaker groups. Table 1: Summary of distinctions between established and new-entrant bookmaker groups in the European soccer betting market Feature of bookmaker group Established Group New Entrant Group Geographic origin/HQ Europe (generally) Asia Trading medium Betting offices + On-line On-line Principle of operation Position-taking Book-balancing Trading volume Relatively low Relatively high Margins Relatively high Relatively low Match outcome portfolio Win/Draw/Lose (1X2) Asian Handicap Frequency of odds changes Low High Exposure to result-specific risk Relatively high Low Client management High, restrictive Low, non-restrictive The contrast in operating strategies across the two types of bookmaker operating in parallel offers a unique opportunity to analyse parallel dealer-driven markets. A clear focus for investigation is the potential for exploitation of profitable arbitrage opportunities associated with the heterogeneous pricing behaviour of the two types of bookmaker. 2. THEORETICAL BACKGROUND This section offers some theoretical context for the issues explored in this paper. Two strands of theory are considered. The first relates to arbitrage as a mechanism of market adjustment where temporary but consistent opportunities for profitable trading occur. The second relates to different conceptual approaches to our understanding of bookmaker behaviour. Together they underpin the classification of bookmakers suggested in the previous section and provide 6 the basis for interpretation and understanding of the results generated by the empirical analysis. 2.1. Arbitrage and Efficiency in Financial and Betting Markets Central to the Efficient Market Hypothesis (EMH) (Fama, 1970) is the idea that rational arbitrageurs drive temporary deviations in prices towards efficient benchmarks. Prices will therefore reflect fundamental values, providing that arbitrageurs are willing to trade sufficiently to impact market values (Friedman, 1953) and in the absence of limitations to arbitrage (see Gromb and Vayanos, 2010). There is evidence to suggest that theoretical arbitrage opportunities may be illusory in the face of institutional frictions (McLean and Pontiff, 2016). Theoretical models of dealer market microstructure emphasise the role of institutional factors such as order processing (e.g. Roll, 1984), inventory (e.g. Stoll, 1978; Amihud and Mendelson, 1980) and pre-trade transparency on market efficiency (see, for example reviews of market microstructure by Madhavan, 2000 and Biais, Glosten and Spatt, 2005) as well as more general factors such as asymmetric information (e.g. Glosten and Milgrom, 1985; Easley and O’Hara, 1987). Sauer (1998) indicates that a betting market is considered efficient if it is not possible to generate abnormal returns (strong test), or if differential returns are unavailable to the bettor simply by placing stakes at different odds, such as on favourites (weak test). In general, the main determinant of betting market efficiency is the degree to which market odds reflect the true probabilities of event outcomes over a large sample. This is analogous to the degree to which market prices reflect fundamental values in other financial markets. The possibility of ‘zero’ risk arbitrage has been explored in the soccer betting market, examining the extent to which differences in quoted odds across bookmakers are sufficient to guarantee profitable, fully hedged positions through the construction of a synthetic Dutch Book. Pope and Peel (1989) showed that arbitrage opportunities were occasionally available across U.K. bookmakers. 1 Long only arbitrage portfolios employing the betting exchange and bookmakers in the creation of a synthetic Dutch book were also considered in the study by Franck, Verbeek, and Nüesch (2013), but arose in only 5.0% of matches. 2. THEORETICAL BACKGROUND However, later research suggested that the degree of coordination between bookmakers has increased (Dixon and Pope, 2004; Deschamps and Gergaud, 2007; Luckner and Weinhardt, 2008; Deschamps, 2008; Vlastakis, Dotsis and Markellos, 2009; Spann and Skiera, 2009; Franck, Verbeek and Nüesch, 7 2010). This may have arisen due to the emergence of professional arbitrageurs (Dixon and Pope, 2004). Recently, Franck et al. (2013) analysed bookmakers’ odds and parallel betting exchange odds for a sample of 12,782 European soccer matches over a seven-year period, 2004-05 to 2010-11. They found that cross-bookmaker arbitrage opportunities existed in 0.8% of matches in their sample, which included only PTBs. However, when considering bookmaker odds in parallel to the betting exchange Betfair, the proportion of matches offering arbitrage opportunities increased to 19.2%. These opportunities involved taking a long position at the bookmaker and ‘laying’ the same position at the betting exchange.1 Profits from the arbitrage portfolios were almost exclusively against the bookmakers (7% in portfolios against the bookmaker, and close to zero in portfolios against the betting exchange). Recently, Franck et al. (2013) analysed bookmakers’ odds and parallel betting exchange odds for a sample of 12,782 European soccer matches over a seven-year period, 2004-05 to 2010-11. They found that cross-bookmaker arbitrage opportunities existed in 0.8% of matches in their sample, which included only PTBs. However, when considering bookmaker odds in parallel to the betting exchange Betfair, the proportion of matches offering arbitrage opportunities increased to 19.2%. These opportunities involved taking a long position at the bookmaker and ‘laying’ the same position at the betting exchange.1 Profits from the arbitrage portfolios were almost exclusively against the bookmakers (7% in portfolios against the bookmaker, and close to zero in portfolios against the betting exchange). Conclusions regarding the existence of arbitrage opportunities vary with the market under investigation. Arbitrage opportunities arise more frequently between market structures than within market structures (Edelman and O’Brian, 2004; Franck, Verbeek, and Nüesch, 2013). To date, studies analysing arbitrage in betting markets have considered only bookmakers operating as PTBs. Levitt (2004), for example, notes that PTBs set odds strategically in order to profit from the biases of uninformed bettors. This study seeks to uncover the extent to which this price setting mechanism affects market efficiency when BBBs, who do not seek to exploit trader biases, operate in parallel. 2.2. The Behaviour of Bookmakers Levitt (2004) argues that betting markets are, in one important respect, organized differently from other financial markets. As the main providers of liquidity, bookmakers essentially take large positions against their customers (or against particular event outcomes favoured by customers) rather than necessarily matching sellers with buyers and simply earning the commission from the spread (overround). Franck et al. (2013) suggest that bookmakers also, effectively, choose the bettors against whom they take such positions. They achieve this by monitoring client trades and 1 Long only arbitrage portfolios employing the betting exchange and bookmakers in the creation of a synthetic Dutch book were also considered in the study by Franck, Verbeek, and Nüesch (2013), but arose in only 5.0% of matches. 8 restricting service to those profiled as potentially skilled. In this context, bookmakers may occasionally publish inefficient match odds that are likely to attract customers in general, safe in the knowledge that they can eliminate (by restricting or preventing their bets) those who seek to place the majority of their bets at these inefficient prices. Any cost to the bookmaker from the setting of theoretically inefficient odds is therefore potentially lower than the gain from the increase in the size of the customer base. Newall (2015) notes that in the U.K., PTBs tended to advertise exotic bets (such as first goalscorer) to further attract uninformed clients. An alternative view is that the objective of bookmakers is to balance their books, and as a result, secure profit independent of the event’s outcome (Magee, 1990; Woodland and Woodland 1991, Hodges, Lin, and Liu, 2013). Such bookmakers will change their odds frequently in order to account for inventory imbalance. They act as uninformed market makers and essentially set up an over-the-counter market. Holding ‘zero-book’, these market-making bookmakers act as though they are infinitely risk-averse (Fingleton and Waldron, 1999) and could plausibly charge lower transaction commissions, due to the absence of adverse selection costs. Discriminating against skilled bettors is less of an issue with BBBs, since their model is based on the maximization of volume rather on successful positions (see Forrest, 2012 for more details of the BBBs model). In soccer betting, despite the coexistence of PTBs and BBBs, the literature has focused almost exclusively on analysing odds offered by the former. This is surprising, as the economic significance of the latter is probably greater, at least in terms of volumes wagered (Forrest, 2012). 3 Proprietary dark pools (equity trading services that do not publicly display orders) offered by firms such as Getco and Knight Capital trade on principal accounts, and may exclude sophisticated, or informed counterparties. Because they are relatively opaque in their execution services, and do not guarantee execution (especially for informed investors), they present an interesting analogue to position-taking bookmakers. See Zhu (2014) for further details on proprietary dark pools. 2 An example of the restriction notices from Bet365 and William Hill, among others can be found at http://www.the-secret-system.com/bookmakers-shutdown-messages.html. PTBs refer to the use of restrictions as a ‘commercial decision’. 2.2. The Behaviour of Bookmakers Given the significant differences in the structure of the bookmakers’ models, potential arbitrage between the two types of bookmakers may be regarded as inter-market opportunities. Franck et al.’s (2013) proposition that bookmakers set odds to maximise long-term profit from an increased customer base, rather than to maximise profit per game, is novel in linking odds- setting with the bookmaker’s option to withhold service from those clients it believes to be 9 9 informed.2 This presents a distinction between betting and other financial markets, which should be acknowledged when the efficiency of bookmakers’ odds is investigated.3 Whilst the current study represents a development of Franck et al.’s (2013) line of enquiry, it differs from the earlier approach in a number of respects that are important in terms of the robustness of the results generated. The first difference relates to the fact that arbitrage requires that different prices must be simultaneously available. The bookmakers’ odds utilized by Franck et al. (2013) bear no time-stamp; rather, they are assumed to be constant and available for a given time interval, which is often not the case, certainly for BBBs and even on occasion for PTBs. Second, Franck et al.’s focus on the matched offers between a betting exchange and bookmakers were observed up to 2 days prior to kick-off, when the amount of money that can be staked in a betting exchange is very low, suggesting that even if arbitrage opportunities exist, they may not be economically meaningful. Third, Franck et al. (2013) only examined one particular exchange, Betfair, which levies a commission on winning customers (referred to as ‘premium charges’).4 Fourth, even though the volume that one can stake in a betting exchange increases as kick-off approaches, it is shown below that there is significant variation in the size of the stakes that can be placed across games. This compromises the homogeneity of the sample, as several apparent arbitrage opportunities are economically insignificant, because only small wagers are possible. Consequently, it is important to test the proposition suggested by Franck et al. (2013), in the particular context of arbitrage between exchange and bookmaker markets, employing a dataset that avoids the above limitations, in order properly to test empirically the theoretical proposition. Certainly, the widening of the set, and the greater diversity, of bookmaker activity under consideration here might reasonably be expected to generate a significant increase in arbitrage opportunities. p p y p 4 Betfair may withhold up to 60% out of winning bettors profits (see the website http://www.betfair. com/www/GBR/en/aboutUs/Betfair.Charges/) 2.2. The Behaviour of Bookmakers This observation applies equally when comparing our results with those of 10 Vlastakis et al. (2009) who investigated arbitrage opportunities among a population of five PTBs and one BBB. Their results revealed sixty three arbitrage opportunities across the aggregate group of six, which reduced to just ten within the group of five PTBs, though they caution that these results may underestimate actual arbitrage opportunities as their analysis considered only closing odds, which would deny identification of opportunities associated with earlier available price menus. In addition, we argue that any investigation of the EMH using odds provided by PTBs may lead to biased conclusions. Specifically, as suggested by Franck et al. (2013), such market operators may intentionally set “inefficient” prices as a marketing strategy to attract customers. Such complications in the assessment of market efficiency should not exist when employing the odds of BBBs, since their prices should not be influenced by client identity. Consequently, odds in this market constitute more appropriate data for testing the EMH. 3. HYPOTHESIS DEVELOPMENT The preceding discussion suggests that arbitrage opportunities between bookmakers from the same group (i.e. PTBs or BBBs) will be rare, but prices of PTBs and BBBs, operating in the same market, may be sufficiently disparate to incentivise arbitrage. However, because PTBs effectively prevent skilled traders from exploiting these opportunities, such arbitrage will be illusory. In exploring the validity of this proposition, we test the following five related hypotheses: H1: There exist instances where price dispersion in the betting market is adequate to generate apparently risk-free opportunities for bettors to profit by simultaneously betting with different bookmakers on alternative outcomes related to the same event. Levitt (2004) studied trading volume from a major PTB, showing that bettors tend disproportionately to prefer staking on favourites, rather than longshots, relative to their odds implied probabilities. Profit-maximizing bookmakers hence face net exposure to favourites. Relatedly, Forrest and Simmons (2008) and Franck et al. (2011) show that PTBs offer better prices for bets on popular teams, in order to sustain competition and to build/maintain their customer base. Consequently, we expect that in most cases where apparent arbitrage opportunities exist, 11 the PTB will post the best offer for the favourite and the BBB will post the best offer for the longshot, suggesting the following hypothesis: H2: Apparent arbitrage opportunities most commonly arise between BBBs and PTBs, by PTBs offering the longer odds for favourites and BBBs offering the longer odds for longshots. Based on Franck et al.’s (2013) finding that arbitrage profits between the betting exchange and PTBs were typically earned at the expense of bookmakers, we expect that in exploring trade across PTBs and BBBs, apparent arbitrage profits are also earned at the expense of the PTBs, not the BBBs (given PTBs’ failure to adjust odds quickly enough to incorporate price-informative trends signalled by informed money traded with BBBs). We, therefore, test the following hypothesis: H3a: When an apparent arbitrage opportunity arises, a greater proportion of the risk-free profit will be earned in trade with the PTB, rather than the BBB. We also expect that the odds offered by BBBs (cf. PTBs) are better calibrated, i.e. we expect that BBBs’ odds-implied probabilities are more reflective of match outcomes. If PTBs were solely interested in efficient estimation of event probabilities, they could simply adjust their odds to those of the BBBs. 3. HYPOTHESIS DEVELOPMENT If we show that the PTBs do not adjust their odds in this way then this is supportive of Franck et al.’s (2013) proposition that promotional considerations form part of their odds-setting strategy. We, therefore, test the following hypothesis: H3b: BBBs’ odds constitute more accurate predictors (cf. those of PTBs) of event outcomes. 8 For example, Ladbrokes’ 2013 annual report (p.23) notes “Ladbrokes may experience significant losses as a result of a failure to determine accurately the odds in relation to any particular event.” ource: http://www.publications.parliament.uk/pa/cm201213/cmselect/cmcumeds/writev/ 1554/ga104.htm urce: http://howtobet.net/sportsbook-review/stan-james. y y p 9 See, inter alia, http://www.pinnaclesports.com/about-us.aspx, http://www.pinnaclesports. com/betting- promotions/arbitrage-friendly, and http://www.pinnaclesports.com/ betting-promotions/winners-welcome. 5 See William Hill (2013) “Preliminary Results 2012” and Ladbrokes (2013) “Preliminary Results for the Year Ended December 2012.” 7 Source: http://howtobet.net/sportsbook-review/stan-james. 6 Source: http://www.publications.parliament.uk/pa/cm201213/cmselect/cmcumeds/writev/ 1554 7 Source: http://howtobet net/sportsbook-review/stan-james 4.1. Bookmaker Classification and Data We employ data from major bookmakers representing both BBBs and PTBs. We classify Ladbrokes, William Hill, Bet365, and Stan James as PTBs, and IBCBet, Pinnacle, SBOBet and 188Bet as BBBs, based on extensive consultation with bettors and interpretation of the information presented below. 12 PTBs: Ladbrokes and William Hill operate retail businesses with thousands of betting shops, mainly in the U.K. They also operate online, and their combined aggregate gross revenue exceeds £1 Billion. 5 Bet365 is a major UK-based betting company founded in 2001, which achieved turnover of £8.5 Billion and gross profit of £422 Million in 2011.6 Stan James is a private company operating mainly online, while owning 65 betting shops in the U.K. 7 One of the defining characteristics of PTBs is their physical-world presence, which is important to each of these bookmakers. Annual reports for these bookmakers note that their profitability depends on match results.8 BBBs: SBOBet, IBCbet, 188Bet are leading BBB operators, handling trading volumes far in excess of more traditional European bookmakers (Forrest, 2012). Pinnacle is a major online operator, trading billions of dollars, which describes itself explicitly on its website 9 as attempting to maximize trading volume while minimizing exposure, using information arising from informed traders as a tool to set efficient odds. In particular, Pinnacle emphasizes that it is friendly to arbitrageurs, as the expected value of a trade to the operator should not depend on the motives of the counterparty placing the stake. The model described by Pinnacle also fits the operations of the three bookmakers listed above and is aligned with the model of BBBs. Importantly, each of these operators lacks physical-world presence (betting shops) and has an empirically demonstrable tendency to change odds frequently. In order to ensure that potential arbitrage trades could have realistically been executed, we designed a data collection program to obtain odds information systematically from bookmakers’ websites. This involved surveying different bookmakers’ websites simultaneously, so differential odds from our panel of bookmakers reflect genuine, real-time price dispersion, rather than information-driven outcomes. The program included a time-out coefficient to ensure a maximum discrepancy in odds collection of 30 seconds. In other words, where odds were/were not 5 See William Hill (2013) “Preliminary Results 2012” and Ladbrokes (2013) “Preliminary Results for the Year Ended December 2012.” y y p 9 See, inter alia, http://www.pinnaclesports.com/about-us.aspx, http://www.pinnaclesports. com/betting- promotions/arbitrage-friendly, and http://www.pinnaclesports.com/ betting-promotions/winners-welcome. 10 The cost of this was that several bookmakers occasionally failed to respond within the maximum allowed period. In these cases, we repeated the full request (i.e. for all bookmakers) three times in order to obtain a complete sample. In some cases, due to heavy load on bookmakers’ websites, some would remain unresponsive. In those cases, the odds of those who failed to respond were not considered, which may lead to a slight underestimation of the frequency of arbitrage opportunities overall. The timeout could be increased in future studies, however, this would risk the integrity of the results overall, as a higher time interval would increase the chance of odds of the quickest responding bookmaker changing until the response of the slowest bookmaker came back. The collection of data from bookmakers remains a difficult practice at best. 11 To illustrate, assume that the following Asian Handicap odds are offered on a match between Chelsea and 4.1. Bookmaker Classification and Data 13 forthcoming from the bookmaker within 30 seconds, it was assumed that these odds were/were not available as a basis for arbitrage.10 All odds were collected in a period within 2 hours of kick- off, when the staking levels reach their peak. We focused on the major European soccer leagues in order to ensure that the findings of the study carry economic significance, as the volumes traded in leagues of lower status are significantly smaller. We collected data, for the whole of the 2012-13 season for the 6 major leagues; the English Premier League, the German Bundesliga, the Italian Serie A, the Spanish La Liga, the French Ligue 1, and the Dutch Eredivisie. Overall, this resulted in a sample of 2,132 games, which we refer to as the `main sample’. For each match, odds were collected for several bookmaker products: the AH, and home win, draw, and away win (also known as 1X2) markets. In AH betting, one of the teams (usually the favourite) is given a goal-deficit (handicap) to overcome, the size of which is indicated by a negative number. A bet is successful if the handicapped team wins the match by a greater margin than the handicap. A bet on the opposing team is successful providing it does not lose by a margin greater than the handicap. Fractional AHs include the draw outcome. For example, a team with a handicap of −0.5, starts with a half goal deficit, meaning that the team must win outright (that is, not draw) for the bet to pay off11. A team which is handicapped +0.5 starts with a half goal advantage, so a bet on that team wins even if the outcome is a draw. A bet on a team with a 0 handicap is refunded in the event of the draw, whereas a bet on team with a -0.25 (+0.25) Chelsea (-0.5 goals) 1.83. 12 We present a detailed example of the returns to each bet in the next section. Arsenal 1.80 This means that a successful bet requires Chelsea to overcome the (-0.5 goals) handicap, i.e. they must win by any single goal (or above) margin. Any draw would (with the handicap) mean a win for Arsenal (e.g. a 1-1 draw would, in effect, become Chelsea 0.5 - Arsenal 1. This format reduces the match outcome to two alternatives. 14 handicap is considered as a half-bet on 0 handicap and a half-bet on -0.5 (+0.5).12 The AHs that are of interest here are those in the interval of -0.5 to +0.5, because none of the bets in this range is dependent on the number of goals scored. Hence, an arbitrage portfolio can potentially be formed by betting in the home win, draw, and away win (1X2) markets. Our supplementary set of data (we refer to this as the ‘extended set’) contains historical odds from six bookmakers (Ladbrokes, William Hill, Bet365, SBOBet, 188Bet, and Pinnacle) for each of the six leagues in our sample; a total of 6,396 matches over the three seasons from 2010-11 to 2012-13. These data were obtained in a similar fashion to our main data, and were used to explore the relative incidence of favourite-longshot bias and the degree of calibration between the odds-implied probabilities of match outcomes and actual match outcomes. We obtained home win, draw, and away win odds for each match, at a time point within two hours of match kick-off. We could not use this entire sample (excluding 2012-13 data) to test the economic efficiency of arbitrage portfolios, as odds were not collected to ensure simultaneous execution of trades. However, the longer data period allows greater power in statistical tests of efficiency. 15 4.2.1. Estimating the Frequency of Arbitrage Opportunities In order to test H1, we formulate a linear optimization problem. This is specifically designed to provide a comprehensive identification of the existence of arbitrage opportunities between bookmaker types. Particular and advantageous features of this method are that we survey arbitrage opportunities throughout the duration of each market, we can assure, reliably, the temporal co-existence of cross-operator odds differentials which define the opportunities, and we are able to identify the optimal distribution of stakes across different products in order to maximize the return. Arbitrage opportunities are deemed to exist where such a return is positive and invariant across all match outcomes. If there is insufficient dispersion in the odds 15 across the market to generate an arbitrage opportunity, there will be no feasible solution. Assuming sufficient dispersion, there will be a range of solutions that offer certain positive returns and the linear program will suggest the combination that offers the highest profit. We outline below how this problem is formulated and examined for a single game. This process is repeated for all 2,132 games in the main sample. Let ܺ௝,௞denote the vector of gross odds offered by bookmaker ݇, where for each game there are 1 ≤݆ ≤13 products offered by the bookmaker (i.e. ‘home win’, ‘home win with a −0.5 handicap,’ ‘away win with a +0.5 handicap,’ etc.). The gross return to each of the 13 bookmakers’ products is presented in Table 2, with products 1, 7, and 8 indicating odds from the 1X2 market, and the rest (with suffix AH) obtained from the AH market. The odds shown in the Table 2 can be multiplied by stake size S (a scalar) to determine non-unit payouts. We define the vector ܺ௠௔௫element-wise as ܺ௝,௠௔௫= max ௞ ܺ௝,௞. In cases where bookmakers are tied for the highest odds, we retain all possible combinations of maximum prices. For example, if both Ladbrokes and William Hill were offering gross odds of 1.50 on a home win (݆ = 1) for a particular match, and this price was higher than all the other bookmakers’ prices for ݆ = 1, we would retain two ܺ௠௔௫vectors, in order to avoid losing information for H1 and H2. 16 Table 2: Gross return to a $1 stake for a single bookmaker on each potential match outcome for different types of bet, with odds vector (ܺଵ, . . . , ܺଵଷ)′indicating the gross payoff for each corresponding market. 4.2.1. Estimating the Frequency of Arbitrage Opportunities The suffix AH indicates that the product is from the Asian Handicap market. Outcome Bookmaker Return if Return if Return if (݆) Product Home Win Draw Away Win 1 Home ܺଵ 0 0 2 Home (−0.5) AH ܺଶ 0 0 3 Home (−0.25) AH ܺଷ 0.5 0 4 Home (0) AH ܺସ 1 0 5 Home (+0.25) AH ܺହ 1 + 0.5(ܺହ−1) 0 6 Home (+0.5) AH ܺ଺ ܺ଺ 0 7 Draw 0 ܺ଻ 0 8 Away 0 0 ଼ܺ 9 Away (−0.5) AH 0 0 ܺଽ 10 Away (−0.25) AH 0 0.5 ܺଵ଴ 11 Away (0) AH 0 1 ܺଵଵ 12 Away (+0.25) AH 0 1 + 0.5(ܺଵଶ−1) ܺଵଶ 13 Away (+0.5) AH 0 ܺଵଷ ܺଵଷ Second, we aim to find the set of bets that a bettor would place to best exploit potential arbitrage opportunities. Let ܵ௝be the bettor’s allocated stake for each bet type ݆. The profit function ܼ = {ܼ௛௢௠௘௪௜௡, ܼௗ௥௔௪, ܼ௔௪௔௬௪௜௡} for each possible match outcome can be defined from the following set of equations: ܼ௛௢௠௘௪௜௡= ෍ܵ௝ܺ௝,௠௔௫−෍ܵ௝ ଵଷ ௝ୀଵ ଺ ௝ୀଵ (1) ܼௗ௥௔௪= ܵ଻ܺ଻,௠௔௫+ 0.5(ܵଷ+ ܵଵ଴) + (ܵସ+ ܵଵଵ) (1) ܼ௛௢௠௘௪௜௡= ෍ܵ௝ܺ௝,௠௔௫−෍ܵ௝ ௝ୀଵ ௝ୀଵ (1) ܼௗ௥௔௪= ܵ଻ܺ଻,௠௔௫+ 0.5(ܵଷ+ ܵଵ଴) + (ܵସ+ ܵଵଵ) + 0.5൫ܵହ+ ܵଵଶ+ ܵହܺହ,௠௔௫+ ܵଵଶܺଵଶ,௠௔௫൯−෍ܵ௝ ଵଷ ௝ୀଵ (2) ܼ௔௪௔௬௪௜௡= ෍ܵ௝ܺ௝,௠௔௫−෍ܵ௝ ଵଷ ௝ୀଵ ଵଷ ௝ୀ଼ (3) ௝ୀଵ ௝ୀଵ ܼௗ௥௔௪= ܵ଻ܺ଻,௠௔௫+ 0.5(ܵଷ+ ܵଵ଴) + (ܵସ+ ܵଵଵ) + 0.5൫ܵହ+ ܵଵଶ+ ܵହܺହ,௠௔௫+ ܵଵଶܺଵଶ,௠௔௫൯−෍ܵ௝ ଵଷ ௝ୀଵ (2) ܼ௔௪௔௬௪௜௡= ෍ܵ௝ܺ௝,௠௔௫−෍ܵ௝ ଵଷ ௝ୀଵ ଵଷ ௝ୀ଼ (3) + 0.5൫ܵହ+ ܵଵଶ+ ܵହܺହ,௠௔௫+ ܵଵଶܺଵଶ,௠௔௫൯−෍ܵ௝ ଵଷ ௝ୀଵ (2) (2) ܼ௔௪௔௬௪௜௡= ෍ܵ௝ܺ௝,௠௔௫−෍ܵ௝ ଵଷ ௝ୀଵ ଵଷ ௝ୀ଼ (3) (3) 17 17 Identifying the best possible arbitrage opportunity requires identifying the distribution of stakes ܵthat maximizes the payoff for any of the three match outcomes, subject to a set of constraints. Hence, the optimization identifies the distribution of stakes ܵthat maximizes the payoff for any of the three match outcomes. The optimization routine can be written as Find optimal strategy ܵ∗by varying ܵsuch that ܼ௛௢௠௘௪௜௡is maximized (4) Subject to constraints Find optimal strategy ܵ∗by varying ܵsuch that ܼ௛௢௠௘௪௜௡is maximized (4) Subject to constraints (4) Subject to constraints Subject to constraints Subject to constraints ܼ௛௢௠௘௪௜௡= ܼௗ௥௔௪ (5) (5) ܼ௛௢௠௘௪௜௡= ܼ௔௪௔௬௪௜௡ (6) (6) ܼ௛௢௠௘௪௜௡> 0 (7) ෍ܵ௝ ଵଷ ௝ୀଵ = 1 (8) ܵ௝≥0 ∀݆ (9) (8) (9) Due to the linear nature of the problem, the simplex algorithm can be used to maximize the objective function (Dantzig, 1951). 4.2.1. Estimating the Frequency of Arbitrage Opportunities Constraints (5) and (6) ensure that the selected combination of stakes leads to the same return independently of the outcome. Constraint (7) implies that for the solution to be acceptable, the net return should be positive. Constraint (8) requires that the sum of stakes should equal 1, so that each ܵ௝will represent the fraction of the available capital that should be staked on each bet type. Finally, constraint (9) requires that all stakes are positive. The optimization will fail to find a feasible solution in the event that arbitrage is not possible for the given set of bet types on a given game. If there is more than one feasible solution per game, we would select the bet with the highest return per outcome. In order to test H1 we run this maximization for each match in the sample. 4.2.2. Identifying Favourites and Longshots by Bookmaker in the Arbitrage Portfolio 18 18 We now explore the methodology employed to test H2, that is, whether arbitrage opportunities are more likely to occur between PTBs and BBBs (rather than between PTBs, or between BBBs) and whether the arbitrage portfolio is more likely to involve bets on the favourite against the PTB. Matches where an arbitrage opportunity was identified were isolated. We then compared the frequency of instances in which the PTB offers the highest odds for the ‘favourite’. We then define indicator variablesܦ௙andܦ௟for each match ݅asܦ௙௜= 1 if the PTB offers the highest odds for the favourite on match ݅, and 0 otherwise, andܦ௟௜= 1 if the PTB offers the highest odds on the longshot for match ݅, and 0 otherwise. Over the sample of ݊games in which an arbitrage opportunity arises, we calculate the proportion of cases where the best offer for the favourite was provided by PTB. For H2 to be accepted, this proportion should be significantly higher than 1/2. 4.2.3. The Source of Returns by Bookmaker in the Arbitrage Portfolios Testing H3a involves exploring whether the bet responsible for the positive returns to the arbitrage portfolio will be placed with the PTB on the favourite, with the longshot being bet with negative or zero expectation at the BBB. We conduct a betting simulation, where a unit stake ($1) is placed on each bet that is selected from the linear program across the total sample of matches. For each type of bookmaker, we calculate for each match ݅and potential stake at offer ݆the bettor’s profit ܼ௜௝, as ܼ௜௝= ෍ܵ௜௝ܺ௜௝+ ܵ௜଻ܺ௜଻+ 0.5(ܵ௜ଷ+ ܵ௜ଵ଴) + (ܵ௜ସ+ ܵ௜ଵଵ) ଺ ௝ୀଵ + 0.5(ܵ௜ହ+ ܵ௜ଵଶ+ ܵ௜ହܺ௜ହ+ ܵ௜ଵଶܺ௜ଵଶ) + (ܵ௜଺+ ܵ௜ଵଷܺ௜ଵଷ) + ෍ܵ௜௝ܺ௜௝ ଵଷ ௝ୀ଼ −෍ܵ௝ ଵଷ ௝ୀଵ (10) + 0.5(ܵ௜ହ+ ܵ௜ଵଶ+ ܵ௜ହܺ௜ହ+ ܵ௜ଵଶܺ௜ଵଶ) + (ܵ௜଺+ ܵ௜ଵଷܺ௜ଵଷ) + ෍ܵ௜௝ܺ௜௝ ଵଷ ௝ୀ଼ −෍ܵ௝ ଵଷ ௝ୀଵ (10) (10) where ܵ௜௝is the amount staked on product ݆in match ݅. In the unit-stake simulation, ܵ௜௝= 1 if there is a bet on product ݆in match ݅, and zero otherwise. As a result, the average profit that the 19 19 bettor achieves against each type of bookmaker, across the sample of ݊bets can be calculated as: bettor achieves against each type of bookmaker, across the sample of ݊bets can be calculated as: as: ߤ= 1 ݊෍෍ܼ௜௝ ଵଷ ௝ୀଵ ௡ ௜ୀଵ (11) (11) H3a will not be rejected ifߤis higher for the group of PTBs than for the group of BBBs. It could be argued that a consequence of placing a unit stake across each bet is high variance, since average profit is highly influenced by the outcome of bets on longshots. We ensure that these latter bets do not lead to biased conclusions regarding the expected profit against each bookmaker by replicating the simulation, where each stake ܵ௜௝is determined by a staking strategy, the Kelly Criterion (Kelly, 1956), which involves assigning stakes which are inversely proportional to odds, thereby assuring that for each type of bookmaker, all bets generate equal expected profit. 4.2.3. The Source of Returns by Bookmaker in the Arbitrage Portfolios ܵ௜௝= 1 ܺ௜௝−1 (12) (12) As a result, the average realized profit against each bookmaker across the sample of ݊bets is As a result, the average realized profit against each bookmaker across the sample of ݊bets is ߤ= ∑ ∑ ܼ௜௝ܵ௜௝ ଵଷ ௝ୀଵ ௡ ௜ୀଵ ∑ ∑ ܵ௜௝ ଵଷ ௝ୀଵ ௡ ௜ୀଵ (13) (13) ௝ ௝ ௜ We recalculate the means for each of the two types of bookmaker and compare them, in order to confirm that the conclusions drawn from the unit-stake simulations are not biased by abnormally positive or negative results on high-odds bets. 4.2.4. The Predictive Power of BBB vs PTB Odds-Implied Probabilities In order to test whether predictions based on BBBs’ odds are more efficient (H3b) and unbiased (H3c) predictors of event outcomes than predictions based on PTBs’ odds, we compare the forecasting accuracy and the favourite longshot bias (FLB) observed in predictions based on the odds of the two different types of bookmakers. For each group, we employ a conditional logistic regression (with the probability of outcome ݋derived from the odds as the sole independent variable), where the outcome of each match is the dependent variable (i.e. home win, draw, or 20 away win). The outcome takes value 1 for the event that occurred and 0 for the events that did not occur. Hence, the probability that outcome ݋in match ݅occurs, is given by: away win). The outcome takes value 1 for the event that occurred and 0 for the events that did not occur. Hence, the probability that outcome ݋in match ݅occurs, is given by: ܲ(ܻ௜௢= 1) = ݁௓೔೚ ∑ ݁௓೔೚ ଷ ௢ୀଵ (14) (14) where ܼ௜௢is a function of the probability ݌௜௢ ௦(as ݌௜௢ ௦is the probability of the event outcome implied by the odds for each outcome ݋of match ݅, where the superscriptݏimplies the subjective probability based on the bookmaker’s and bettor’s combined assessment of the chance of this outcome), such that ܼ௜௢= ܾ× ln(݌௜௢ ௦) (15) ܼ௜௢= ܾ× ln(݌௜௢ ௦) (15) (15) where b is a coefficient derived from a conditional logit regression estimated on the basis of past results, and ݌௜௢ ௦can be calculated from the odds ܺ௜௢of outcome ݋in match ݅as where b is a coefficient derived from a conditional logit regression estimated on the basis of past results, and ݌௜௢ ௦can be calculated from the odds ܺ௜௢of outcome ݋in match ݅as ݌௜௢ ௦ = 1 ܺ௜௢(1 +ߩ௜) (16) ݌௜௢ ௦ = 1 ܺ௜௢(1 +ߩ௜) (16) (16) andߩ௜is the bookmaker’s over-round. This can be calculated from the odds offered for all outcomes ݋of match ݅as andߩ௜is the bookmaker’s over-round. This can be calculated from the odds offered for all outcomes ݋of match ݅as ߩ௜= ෍1 ܺ௜௢ −1 ଷ ௜ୀଵ (17) Hence, (14) can be written as ߩ௜= ෍1 ܺ௜௢ −1 ଷ ௜ୀଵ (17) (17) ܲ(ܻ௜௢= 1) = ݁௕×୪୬൫௣೔೚ ೞ൯ ∑ ݁௕×୪୬൫௣೔೚ ೞ൯ ଷ ௢ୀଵ = (݌௜௢ ௦)௕ ∑ (݌௜௢ ௦)௕ ଷ ௢ୀଵ (18) (18) Positive FLB indicates that the bookmaker odds underestimate the probability of the most likely event occurring. 4.2.4. The Predictive Power of BBB vs PTB Odds-Implied Probabilities Therefore, if a bookmaker exhibits this bias, the actual winning probability of favourites, as implied by their observed frequency of success, will be higher than that expected by the odds; whereas for the longshots it will be lower. Thus, denoting as ݌௜௢ ௩the ‘true’ or objective probability (the ‘ݒ’ denoting ‘verifiable’ or objective probability) of outcome ݋in match ݅, we can infer the following. Positive FLB indicates that the bookmaker odds underestimate the probability of the most likely ݌௜௙ ௦> ݌௜௟ ௦⟹ ݌௜௙ ௩ ݌௜௙ ௦> ݌௜௟ ௩ ݌௜௟ ௦ (19) (19) 21 here ݂denotes favourite and ݈denotes longshot. where ݂denotes favourite and ݈denotes longshot. Subject to (18), o (18), ݌௜௙ ௩ ݌௜௙ ௦> ݌௜௟ ௩ ݌௜௟ ௦⟹ ൫݌௜௙ ௦൯ ௕ ∑ ൫݌௜௙ ௦൯ ௕ ଷ ௢ୀଵ > (݌௜௟ ௦)௕ ∑ (݌௜௟ ௦)௕ ଷ ௢ୀଵ ⟹൫݌௜௙ ௦൯ ௕ିଵ> (݌௜௟ ௦)௕ିଵ (20) (20) where if ݌௜௙ ௦> ݌௜௟ ௦, (14) is only valid where ܾ> 1. Consequently, the odds of a given bookmaker underestimate favourites on average, only if ܾin (20) is significantly greater than 1 and higher values of b indicate higher degree of bias. Maximum likelihood is employed to estimate ܾfor each bookmaker. To assess the accuracy of each bookmaker’s predictions, we compared the values of McFadden’s (1974) pseudo-R2statistic that each bookmaker’s odds implied probabilities achieve in the conditional logit model (a higher pseudo-R2implies a superior model fit and hence a greater degree of efficiency). 5.1. Arbitrage Opportunities across Bookmakers 5.1. Arbitrage Opportunities across Bookmakers Table 3 reports the average closing odds correlation in our main sample that demonstrates that, on average, bookmakers’ offers are reasonably well-aligned. The highest level of correlation is observed amongst BBBs. Whilst these bookmakers move their odds more frequently (adjusting for individual high stakes), such adjustments seem to happen in parallel across the set of BBBs. Among PTBs, Bet365 seems to be the bookmaker most aligned with the BBBs (possibly because it is the only bookmaker in the group for which online betting is its main focus), whereas Ladbrokes and especially Stan James show the least correlation with the BBBs. The optimization process presented in (4) to (9) reveals the existence of 545 arbitrage opportunities across the 2,132 (that is, in 25.6% of) matches in our sample. Notably, this figure is significantly higher than the results of earlier studies (Franck et al. (2013), Vlastakis et al. (2009)) a fact which may be explained by a combination of the methodology employed to identify opportunities in this paper and the range of bookmakers under scrutiny. The method employed 22 here, as described above, offers a comprehensive and reliable identification of opportunities by embodying time precision regarding the co-availability of odds and by ensuring identification of opportunities throughout the duration of the market, rather than, as with both Franck et al. (2013) and Vlastakis et al. (2009), considering opportunities only at a single point in time. In terms of the range of bookmakers identified, this study examines opportunities across six operators, three BBBs and three PTBs. This gives a degree of diversity which is not matched by earlier studies, with Franck et al. (2013) focusing on activity across a group of PTBs and a single betting exchange, and Vlastakis et al. (2009) considering a different group of PTBs and a single BBB. In this context, for reasons of both methodology and the nature of samples under scrutiny, the differences are arguably unsurprising. The distribution of arbitrage opportunities across the different leagues is shown in Table 3 and the frequency with which each bookmaker’s odds feature in the optimized portfolio are shown in Table 4. The arbitrage opportunities are well dispersed across the leagues, although the relatively low frequency in Holland returns a ߯ଶ(5) test of independence with a p-value of 0.048. Table 3: Average closing odds correlation between bookmakers in main sample. Panel A reports the correlation matrix of closing odds across all bookmakers, Panel B reports the average correlation between and within groups of book-balancing and position-taking bookmakers. 13 Removing Stan James from the sample causes the instances of potential arbitrage opportunities to drop to 287, which is indicative of the influence of a bookmaker which applies a policy of offering outlying odds, on the creation of arbitrage instances. 5.1. Arbitrage Opportunities across Bookmakers This is probably because Holland is the least popular of the six major leagues, and there is less competition among bookmakers to supply competitive odds (and less benefit from promoting inefficient odds). Consequently, transaction costs are higher, reducing the potential for arbitrage opportunities. Table 3: Average closing odds correlation between bookmakers in main sample. Panel A reports the correlation matrix of closing odds across all bookmakers, Panel B reports the average correlation between and within groups of book-balancing and position-taking bookmakers. 23 Panel A: Correlation in Odds Between Bookmakers Position Takers Book Balancers Bookmaker Ladbrokes William Hill Bet 365 Stan James SBOBet 188Bet Pinnacle Position Takers Ladbrokes 1.0000 William Hill 0.9927 1.0000 Bet 365 0.9907 0.9950 1.0000 Stan James 0.9928 0.9917 0.9891 1.0000 Book Balancers SBOBet 0.9884 0.9925 0.9947 0.9849 1.0000 188Bet 0.9890 0.9942 0.9970 0.9867 0.9971 1.0000 Pinnacle 0.9890 0.9944 0.9971 0.9868 0.9971 0.9987 1.0000 Panel B: Average Correlation Within and Across Groups Group 1 Group 2 Average Position Takers Book Balancers 0.9913 Position Takers Position Takers 0.9920 Book Balancers Book Balancers 0.9976 Panel A: Correlation in Odds Between Bookmakers Position Takers Panel A: Correlation in Odds Between Bookmakers 24 Table 4: Number of Matches with Arbitrage Opportunities by League. Table 4: Number of Matches with Arbitrage Opportunities by League. League Number of Matches with Arbitrage Opportunity Proportion of Arbitrage Opportunities Across all Leagues Proportion of Arbitrage Opportunities per Match Matches in Sample England 90 16.51%1 23.68%2 380 Spain 109 20.00% 28.68% 380 Italy 101 18.53% 26.58% 380 Germany 94 17.25% 30.72% 306 France 94 17.25% 24.74% 380 Holland 57 10.46% 18.63% 306 TOTAL 545 100.00% 25.56% 2,132 1 90/545 2 90/380 The linear program (4) to (9) can result in the odds of a diverse number of bookmakers featuring in each potential arbitrage opportunity (ranging from 2 to 6 in our sample), in order to achieve the maximum risk-free profit. In cases where multiple bookmakers post equal maximum odds for the same market, we attribute each of the bookmakers as having supplied the arbitrage opportunity. It is clear from Table 5 that some bookmakers are more likely to be involved in the generation of a theoretically risk-free portfolio.13 The frequency of the appearance of PTBs is likely to be related to each operator’s policy on promotional odds. A test of independence rejects that bookmakers appear with equal frequency in arbitrage portfolios (߯ଶ(6) = 615.4, p-value = 0.000). 14 BBBs maintain an over-round of about 2% in AH markets, but their over-round is nearer 5% − 6% for the 1 × 2 market. On the other hand, Pinnacle’s over-round is about 2% in the 1 × 2 market. 15 Mainly when a positive handicap (i.e. either +0.25 or +0.5) is not selected, the optimization indicates a stake should be placed on the draw so that there is no negative exposure on the draw outcome. 14 BBBs maintain an over-round of about 2% in AH markets, but their over-round is nearer 5% − 6% for market. On the other hand, Pinnacle’s over-round is about 2% in the 1 × 2 market. market. On the other hand, Pinnacle’s over-round is about 2% in the 1 × 2 market. 5.1. Arbitrage Opportunities across Bookmakers 13 Removing Stan James from the sample causes the instances of potential arbitrage opportunities to drop to 287, which is indicative of the influence of a bookmaker which applies a policy of offering outlying odds, on the creation of arbitrage instances. 25 Table 5: Number of times a bookmaker’s odds feature in a potential arbitrage portfolio selected by the optimization programme for the main sample. For each bookmaker we report the relative frequency with which their odds appeared in the ܺ௠௔௫vector, and featured in the optimal potential arbitrage portfolio identified for a single game. heir odds appeared in the ܺ௠௔௫vector, and featured in the optimal potential arbitrage portfolio identified f ngle game. Group Bookmaker Times odds featured in arbitrage portfolio Relative Frequency Position- Takers Ladbrokes 237 13.27% William Hill 59 3.30% Bet 365 65 3.64% Stan James 452 25.31% Book-Balancers SBOBet 248 13.89% 188Bet 262 14.67% Pinnacle 463 25.92% TOTAL 1786 100% Consistent with the behaviour suggested by the inter-bookmaker correlation statistics, Bet365 and William Hill appear more aligned in their pricing strategy with the BBBs, and hence their odds appear less frequently in arbitrage portfolios. All BBBs appear as part of an arbitrage portfolio, with Stan James or Ladbrokes often being on the opposite side. Amongst the BBBs, Pinnacle is selected most frequently by the optimization program to be part of an arbitrage portfolio. This is probably because this is the only BBB which exhibits the same low over-round on 1X2 markets as on the AH products.14 As a result, Pinnacle often offers the highest odds on a draw, which is frequently a useful bet in terms of equalizing payoffs across all outcomes.15 If a bettor could have maintained access to all 7 bookmakers, without suffering restrictions to her stake, the fully hedged strategy would have returned an impressive 7.56 times the initial bankroll 26 across the season (assuming no reinvestment). This corresponds to an average risk-free profit of 1.38% per match. Taken together, the results show that sufficient price dispersion exists in the market for bettors to create a seemingly risk-free portfolio of bets that would guarantee profits in 25% of games, assuming they could successfully implement this strategy. 5.1. Arbitrage Opportunities across Bookmakers These results serve to support Hypothesis 1, namely, there exist instances where the price dispersion is adequate to generate theoretically risk-free opportunities for bettors to profit by simultaneously betting with different bookmakers on alternative outcomes related to the same event. 5.2. Arbitrage Opportunities by Type of Bookmaker 5.2. Arbitrage Opportunities by Type of Bookmaker We determine for each apparent arbitrage opportunity the source of odds that make up the arbitrage portfolio of bets (i.e. from BBBs or PTBs). In particular, we look at the instances where the odds offered by BBBs or PTBs on the favourite or the longshot feature in the arbitrage opportunities. These results are displayed in Table 6. In 84% of arbitrage opportunities, one or more PTBs offered the highest odds on the favourite while one or more BBBs offered the highest odds on the longshot. Based on this frequency of arbitrage opportunities being created by bookmakers of different types (BBBs and PTBs), the chance that this phenomenon is random is very low (Z-statistic = 16.12, p-value=0.000). This finding supports Hypothesis 2, namely, that most apparent arbitrage opportunities involve bets placed with different types of bookmakers. 27 Table 6: Constituent bets in arbitrage portfolios by type of bookmaker and odds. The first (second) column shows the type of bookmaker for which the optimal arbitrage portfolios contain bets on favourites (longshots). The favourite is identified as the team with the lower odds on the 1X2 betting market, bets on products 1-6 in Table 2 are considered ‘favourite’ bets if the home team has lower odds; bets on products 8-13 in Table 2 are considered ‘favourite’ bets if the away team has lower odds. All other bets (excluding draw bets) are considered ‘longshot’ bets. The number and proportion for which arbitrage portfolios are constructed, using bets from each type of bookmaker, are presented in column 3. Columns 4 and 5 report similar results to column 3, with the strength of the favourite increasing to $2.00 and $1.70 per dollar bet. Column 6 repeats the results from column 3 with the exclusion of the outlying position-taking bookmaker Stan James. outlying position-taking bookmaker Stan James. Favourite Longshot Num. matches Num. matches Num. matches Num. 16 Such studies do not distinguish between diverse types of operators, but they assume a type of bookmaker consistent with our PTB definition. 17 In general, PTBs, do not offer higher odds for favourites on average than BBBs due to their higher over-round. However, their odds on favourites are closer to those offered by BBBs than the odds they offer on longshots. This finding suggests that they probably do not distribute their over-round proportionally. 5.2. Arbitrage Opportunities by Type of Bookmaker matches (full sample) (Fav < 2.00) (Fav < 1.70) (w/o Stan James) Best Offer by: Best Offer by: prop Prop1 Prop2 Prop Position Position 41 27 6 18 Taker Taker 0.075 0.071 0.073 0.061 Position Book 277 183 57 152 Taker Balancer 0.508 0.480 0.695 0.514 Book Position 181 142 14 78 Balancer Taker 0.332 0.373 0.171 0.264 Book Book 46 29 5 48 Balancer Balancer 0.084 0.076 0.061 0.162 Total 545 381 82 296 1.000 1.000 1.000 1.000 1, 2 In order to illustrate how the arbitrage opportunities vary as the strength of the favourite increases, we select categories where there are sufficient matches to give meaningful proportions of arbitrage opportunities and where the favourites’ odds are low. To match these criteria we select favourites’ odds of < 2 and < 1.7 and show, in columns 4 and 5, respectively, the proportions of arbitrage opportunities which arise with favourites with odds less than 2 and less than 1.7 1, 2 In order to illustrate how the arbitrage opportunities vary as the strength of the favourite increases, we select categories where there are sufficient matches to give meaningful proportions of arbitrage opportunities and where the favourites’ odds are low. To match these criteria we select favourites’ odds of < 2 and < 1.7 and show, in columns 4 and 5, respectively, the proportions of arbitrage opportunities which arise with favourites with odds less than 2 and less than 1.7 The results displayed in Table 6 also show that PTBs are significantly more likely to offer an improvement over market odds for the favourite rather than for the longshot. On 58% of occasions, a PTB offered the best odds for the favourite, compared with 44.4% offering the best odds on the longshot. Such a difference is unlikely to be random (Z-statistic = 5.86, p-value = 0.000). This tendency is more pronounced on stronger favourites. Since PTBs attract higher volumes on favourites than on longshots (Levitt, 2004), our finding is consistent with the view that PTBs16 are inclined to inflate odds for popular bets in order to attract customers. As a result, arbitrage opportunities most commonly emerge where a PTB offers the best odds for the 28 favourite and a BBB offers the best odds for the longshot. 17 These results are in line with Hypothesis 2. 5.3. The Efficiency of Bookmakers Odds by Type In exploring the broader context for the relative values of odds on favourites and longshots between groups, Table 7 presents the results of estimating separate conditional logistic regression models (as described in (14) and (18)) based on the 1X2 odds offered on soccer matches across the six leading European leagues by PTBs and BBBs, respectively, for our extended sample of 6,396 matches over the three seasons from 2010-11 to 2012-13. The forecasting accuracy of odds offered by BBBs is higher on average compared to that of PTBs’ odds, as represented by the higher average McFadden’s pseudo-R2. Whilst the increase in pseudo-R2 appears small, it is likely to be economically significant (e.g. Benter, 1994). This result is in line with Hypothesis 3b and is consistent with the evidence provided by Franck et al. (2013) and Smith et al. (2006, 2009) that demand-driven (as opposed to traditional PTB) markets are more efficient predictors of event outcomes. The values of the coefficients in the conditional logistic regressions, and the related significance tests, indicate in general that the FLB is more pronounced for PTBs than for BBBs. The notable exception is for the odds offered by the BBB, SBOBet, whose over-round for the 1X2 market is the highest amongst BBBs. The conditional logistic regression based on Pinnacle’s odds (whose over-round in the 1X2 market is as low as it is for AHs) has a coefficient very close to 1, suggesting no FLB. This variation of the bias is in line with the Vaughan Williams’s (1999) proposition that the level of transaction costs affects the degree of FLB. The extent of the bias is likely to indicate that BBBs (excluding Pinnacle) aim to direct the demand for longshots to AH markets, in order to facilitate the balancing of their books in that market. 29 Table 7: This table reports the results of conditional logit modelling (using equations (14) and (18)) based on the odds offered by six bookmakers in the extended sample for all match outcomes (Home win, Draw, Away win; 19,188 total observations of odds per bookmaker). Bookmakers are classified as either position-takers or book-balancers. The third and fourth columns of the table report the estimated coefficient of the conditional logit model and its standard error, respectively. The fifth column reports the p-value of a Z-test to determine whether the true value of the coefficient in column three is equal to 1. 18 In this case, $1 is bet on each offer that falls part of the portfolio, no matter what the fraction of capital allocated from the optimization (4). Therefore, the results of this simulation are not comparable to the results of the fully hedged strategy. By way of example, the fully hedged strategy may assign 90% of the capital to bet A and 10% to bet B and hence, we would bet $0.90 and $0.10 on these products, respectively. However, in the unit-stake simulation $1 is staked on bet A and $1 on bet B, since the objective is to identify how the profit is distributed across the two types of bookmakers, rather than to create a hedged position. 5.3. The Efficiency of Bookmakers Odds by Type The sixth column reports the result of test whether the coefficient is significantly greater than 1 at the 10%, 5%, and 1% levels with the signs (), (), and (), respectively. The final column reports the McFadden Pseudo-R2 of the conditional logit model. Group Bookmaker Coefficient Std. Error Prob. (Coeff. = 1) Sig. Pseudo-R2 Position Takers Ladbrokes 1.0743 0.0309 0.0081 () 0.1085 William Hill 1.0605 0.0304 0.0232 () 0.1101 Bet365 1.0560 0.0302 0.0318 () 0.1106 Book Balancers SBOBet 1.0784 0.0311 0.0059 (*) 0.1106 188Bet 1.0413 0.0299 0.0831 () 0.1113 Pinnacle 1.0081 0.0289 0.3900 0.1114 5.4. “Winners” and “Losers” in Arbitrage Opportunities In order to identify which group of bookmakers would tend to lose against potential arbitrageurs, should the identified risk-free opportunities be exploitable, we employ the simulation described in (10). Placing $1 on all 813 outcomes for which the odds posted by PTBs form part of the fully- hedged portfolio, yields an average profit of $0.16 per bet. Adopting the same strategy, of backing all outcomes where the BBBs’ odds feature in the optimal fully hedged portfolio, results in a loss 5.4. “Winners” and “Losers” in Arbitrage Opportunities In order to identify which group of bookmakers would tend to lose against potential arbitrageurs, should the identified risk-free opportunities be exploitable, we employ the simulation described in (10). Placing $1 on all 813 outcomes for which the odds posted by PTBs form part of the fully- hedged portfolio, yields an average profit of $0.16 per bet. Adopting the same strategy, of backing all outcomes where the BBBs’ odds feature in the optimal fully hedged portfolio, results in a loss 30 of $0.024 per bet.18,19 The profit obtained on the bets placed at the PTBs odds in these cases is significantly higher than the profit (i.e. in fact a loss) obtained on the bets placed at the BBBs odds (t-statistic = 3.37, p-value = 0.000). Adjusting the strategy, as described by (13), leads to an average profit per bet of $0.04 per $1 stake against PTBs and an average loss of −$0.047 per $1 stake against BBBs. These returns remain significantly different (t-statistic = 2.60, p-value = 0.005). This result supports Hypothesis 3a, namely, that PTBs would suffer losses on average when an apparent arbitrage opportunity exists. 19 55 out of 813 $1 bets that were placed at gross odds greater than $5 had an extremely high profit of $1.30. As a result, this small number of lucky bets account for $71.60 out of the $130 won in total by this strategy. Hence, it is important to ensure that they do not bias the conclusions. This is achieved by applying the weighting implied by equation (13). 21 Pinnacle’s statement is: “[A]ll bookmakers shouldn’t care about the motivation for placing a bet, but should simply look to balance the bet volume.” Source: http://www.pinnaclesports.com/ betting-promotions/arbitrage- friendly. q ( ) 20 The over-round of such bookmakers is about 2% for AH products and 5% − 6% for 1X2 products, excluding Pinnacle, which employs an over-round of around 2% in the 1X2 market. 5.3. The Efficiency of Bookmakers Odds by Type Interestingly, the loss incurred against the BBBs on these bets is close to their over-round.20 Put another way, that component of the arbitrage portfolio placed through BBBs generates returns for a bettor equal to their expected loss had they placed a random bet with these bookmakers. In other words, the fact that another bookmaker offers sufficiently different odds to generate an apparent arbitrage opportunity does not change the expected value they receive from their bets. Hence, this result effectively justifies Pinnacle’s statement that the motive for a bet (e.g. intention to arbitrage) should be irrelevant to a BBB.21 From the bettor’s perspective, a higher return is expected by placing bets against outlying odds of PTBs, rather than by hedging such positions against BBBs, since in the latter case, the average profit drops to $0.013 per $1 bet. Consequently, there is evidence from these simulations to support Hypothesis 3a, the expected loss from an 19 55 out of 813 $1 bets that were placed at gross odds greater than $5 had an extremely high profit of $1.30. As a result, this small number of lucky bets account for $71.60 out of the $130 won in total by this strategy. Hence, it is important to ensure that they do not bias the conclusions. This is achieved by applying the weighting implied by equation (13). 21 Pinnacle’s statement is: “[A]ll bookmakers shouldn’t care about the motivation for placing a bet, but should simply look to balance the bet volume.” Source: http://www.pinnaclesports.com/ betting-promotions/arbitrage- friendly. 31 apparent arbitrage opportunity is likely to be suffered by the PTBs. This is consistent with Franck et al.’s (2013) findings. 22 Pinnacle state “This limiting of arbitrage players is a reflection of a bookmaker’s short-comings, such as posting ‘bad odds,’ or an inability to move odds fast enough to avoid being the focus of arbitrage players.” Source: https://www.pinnaclesports.com/betting-promotions/arbitrage-friendly. , y g g g p y Source: https://www.pinnaclesports.com/betting-promotions/arbitrage-friendly. 22 Pinnacle state “This limiting of arbitrage players is a reflection of a bookmaker’s short-comings, such as posting ‘bad odds,’ or an inability to move odds fast enough to avoid being the focus of arbitrage players.” 32 6. DISCUSSION AND CONCLUSION This paper explores the impact of the entry of book-balancing bookmakers (BBBs) on the efficiency of the market for European soccer betting, which was previously dominated by position taking bookmakers (PTBs). We identified arbitrage opportunities across bookmakers from a unique data set of 1X2 and AH odds for soccer games played in major European leagues. Match odds were collected close to kick-off, when markets are most liquid. Employing a linear programming methodology, we identified the best combination for each of 545 games where a fully hedged profitable investment appeared to be possible. Such a strategy could, in theory, guarantee a profit of 1.3% per game on average. To a degree, our findings confirm those of Franck et al. (2013); arbitrage opportunities mainly exist across, rather than within, market structures. However, importantly, our data assembly assures that the disparate odds required to form an arbitrage portfolio were concurrently available, and were sufficiently liquid to be exploited. Whilst previous studies have treated bookmakers as a homogeneous set, our study makes the explicit distinction between incumbent PTBs and new entrant BBBs as a basis for investigating distinctions in odds menus and opportunities for profitable arbitrage. The results suggest that the two groups of bookmakers are indeed distinct in terms of odds setting and that the consequent arbitrage opportunities are principally accounted for by PTBs’ inefficient pricing. This pricing policy may be intentional, in order to attract customers, and/or the result of their prices lagging behind BBBs (due to the pace at which BBBs’ odds are informatively updated, driven by the flow of “smart money”).22 Given the public availability of BBB odds, it seems fair to assume that if the sole objective of the price setting strategies of PTBs were the efficient calibration of event outcomes they would fully align their odds with those of BBBs. The setting of 32 promotional odds may assist PTBs in identifying informed clients, who tend to place bets only at prices with negative expectation for the bookmaker. In addition, and as reported above, significant evidence exists that such bookmakers operate discriminating behaviour against long- term winning customers. Consequently, we argue that the majority of apparent arbitrage opportunities observed in soccer betting markets are very unlikely to be exploitable in practice. 6. DISCUSSION AND CONCLUSION In the context of the betting market considered here, the original concept of the EMH, where market prices converge to fundamental values, subject to the activity of informed traders, may be open to challenge as efficiency in this case, in terms of an absence of exploitable arbitrage opportunities, is a function of PTBs’ restriction of trade, rather than odds convergence. 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https://openalex.org/W780839424	https://www.impan.pl/shop/publication/transaction/download/product/111601?download.pdf	English	null	A note on dynamical zeta functions for S-unimodal maps	Colloquium Mathematicum	2,000	cc-by	2,188	This work was supported by DFG-grant Ke 514/4-1. The author also gratefully acknowledges the hospitality and ﬁnancial support of the Mathematics Department at Penn State University, where this research was started. 2000 Mathematics Subject Classiﬁcation: 37C30, 37E05. j ﬁ Key words and phrases: Collet–Eckmann map, dynamical zeta function. A NOTE ON DYNAMICAL ZETA FUNCTIONS FOR S-UNIMODAL MAPS BY GERHARD K E L L E R (ERLANGEN) 2000 Mathematics Subject Classiﬁcation: 37C30, 37E05. Key words and phrases: Collet–Eckmann map, dynamical zeta function. This work was supported by DFG-grant Ke 514/4-1. The author also gratefully acknowledges the hospitality and ﬁnancial support of the Mathematics Department at Penn State University, where this research was started. BY GERHARD K E L L E R (ERLANGEN) Using the result of [1] more directly Ruelle proved this theorem in [9] without the additional regularity assumption. In her review paper [2] Baladi asked whether the converse implication also holds. The main result of the present note is an aﬃrmative answer to this question: Theorem 1. Let f be a nonrenormalizable S-unimodal map with at least two periodic orbits. Suppose that there are r > 1 and τ > 0 such that ζ−1 t (z) is analytic in {z : \|z\| < r} for t = 0 and t = −τ and such that for those t the function ζ−1 t (z) has a unique and simple zero z(t) ∈{z : \|z\| < r}. Suppose also that z(0) ≥1. Then λper > 1, i.e. f is uniformly hyperbolic on periodic orbits and hence satisﬁes the Collet–Eckmann condition. Therefore we can now say that • a nonrenormalizable S-unimodal map f with at least two periodic orbits satisﬁes (1) if and only if it is uniformly hyperbolic on periodic orbits. • a nonrenormalizable S-unimodal map f with at least two periodic orbits satisﬁes (1) if and only if it is uniformly hyperbolic on periodic orbits. Very early statements and conjectures related to this equivalence were made by Takahashi in [7, 10]. Very early statements and conjectures related to this equivalence were made by Takahashi in [7, 10]. Remark 1. 1. Using renormalization theory for unimodal maps (see, e.g., [5]) the result is easily adapted to the renormalizable case. 2. If f(0) = 0 is the only periodic point of f and if f ′(0) = 1 (e.g. f(x) = x(1 −x)), then ζ−1 t (z) = 1 −z, but f is not uniformly hyperbolic on periodic orbits. Hence the assumption on the existence of a second periodic orbit cannot simply be skipped. Acknowledgments. I am grateful to Viviane Baladi and Duncan Sands for drawing my attention to the above problem and for many helpful remarks and corrections to an earlier version of this note. 2. Proof of the theorem. We start with some simple remarks. Since all ζn,t are real numbers and since ζn,t ≤ζn,0 max \|f ′\|tn, we have z(t) > 0 for all t. Indeed, z(t) is the radius of convergence of the series P∞ n=1(zn/n)ζn,t. For t ∈{0, −τ} let For t ∈{0, −τ} let ht(z) = ∞ X n=1 zn n (ζn,t −z(t)−n). BY GERHARD K E L L E R (ERLANGEN) Abstract. Let f be a nonrenormalizable S-unimodal map. We prove that f is a Collet– Eckmann map if its dynamical zeta function looks like that of a uniformly hyperbolic map. 1. Introduction. A unimodal map f : [0, 1]→[0, 1] is called S-unimodal if f(0) = f(1) = 0 and if it has nonpositive Schwarzian derivative Sf = f ′′′/f ′ −3 2(f ′′/f ′)2. For such a map set ϕ(x) := log \|f ′(x)\| and ϕn(x) := ϕ(x)+ϕ(fx)+. . .+ϕ(f n−1x). Let Πn = {x ∈[0, 1] : f n(x) = x} and deﬁne for t ∈R the zeta function ζt(z) = exp ∞ X n=1 zn n ζn,t where ζn,t = X x∈Πn e(t−1)ϕn(x). Observe that ζ0(z) is just the usual dynamical zeta function. Set Observe that ζ0(z) is just the usual dynamical zeta function. Set λper := inf{\|(f n)′(x)\|1/n : n > 0, x ∈Πn}. Nowicki and Sands [6] proved that λper > 1 (i.e. f is uniformly hyperbolic on periodic orbits) if and only if f satisﬁes the Collet–Eckmann condition (i.e. there are C > 0 and λCE > 1 such that \|(f n)′(fc)\| ≥Cλn CE for all n > 0 where c denotes the critical point of f). Extending the transfer operator method used in [1] Keller and Nowicki had previously shown in [4] that the zeta function of a nonrenormalizable S-unimodal map f which satisﬁes the Collet–Eckmann condition and some additional regularity assumption has the following property: (1) There are r > 1 and t1 > 0 such that ζ−1 t (z) is analytic in {z : \|z\| < r} if \|t\| < t1 and for those t the function ζ−1 t (z) has a unique and simple zero z(t) ∈{z : \|z\| < r} with z(0) = 1. (1) There are r > 1 and t1 > 0 such that ζ−1 t (z) is analytic in {z : \|z\| < r} if \|t\| < t1 and for those t the function ζ−1 t (z) has a unique and simple zero z(t) ∈{z : \|z\| < r} with z(0) = 1. [229] 230 G. KELLER Both papers, [6] and [4], relied in an essential way on previous work of Nowicki. Using the result of [1] more directly Ruelle proved this theorem in [9] without the additional regularity assumption. Both papers, [6] and [4], relied in an essential way on previous work of Nowicki. BY GERHARD K E L L E R (ERLANGEN) Then, by assumption, 1 − z z(t) ζt(z) = exp ht(z) is analytic and nonzero in {z : \|z\| < r}. So the existence theorem for holo- morphic logarithms (see e.g. [8, Ch. 9, §3.1]) guarantees the existence of an 231 S-UNIMODAL MAPS analytic function eht(z) deﬁned on {z : \|z\| < r} such that (1−z/z(t))ζt(z) = expeht(z). It follows that (2πı)−1(ht(z) −eht(z)) = k ∈Z for z in a neigh- bourhood of 0. Hence eht(z) + 2kπı is an analytic extension of ht(z) to all of {z : \|z\| < r} so that (2) \|ζn,t −z(t)−n\| = O(r−n) \|ζn,t −z(t)−n\| = O(r−n) (2) for t ∈{0, −τ} (see [8, Ch. 8, §1.5]). If f had a stable periodic point x = f p(x) with ϱ := \|(f p)′(x)\|1/p < 1, then we would have ζp,0 ≥ϱ−p and hence z(0) ≤ϱ < 1, contradicting our assumption. Hence f has no stable periodic orbit and all ϕn(x) involved in the deﬁnition of ζn,t are nonnegative. Therefore t 7→ζn,t is nondecreasing for each n > 0, and since \|z(0)\|, \|z(−τ)\| < r by assumption, it follows from (2) that t 7→z(t) is nonincreasing in [−τ, 0]. Hence z(−τ) ≥z(0) ≥1. Suppose now that z(−τ) > 1 and ﬁx α, β > 0 such that eτα = z(−τ)β. Then, for any x ∈Πn with ϕn(x) < nα, e−ϕn(x) ≤ X x∈Πn e−ϕn(x) · 1{ϕn(x)<nα} ≤ X x∈Πn e−ϕn(x)+τ(nα−ϕ = eτnα · ζn,−τ = eτnα · (z(−τ)−n + O(r−n)) = O(z(−τ)−n(1−β)). Hence, for such x, (1 −β) log z(−τ) ≤n−1ϕn(x) < α = β log z(−τ) τ . Choose β = τ/(1 + τ). Then α = (1 −β) log z(−τ) and the upper and lower bounds in this chain of inequalities coincide so that no x ∈Πn can satisfy these estimates. It follows that n−1ϕn(x) ≥α, i.e. λper ≥eα = z(−τ)1/(1+τ) > 1. It remains to consider the case z(−τ) = z(0) = 1. We ﬁrst exclude the possibility that f has a neutral periodic point x = f p(x), p the minimal period of x, ϕp(x) = 0. BY GERHARD K E L L E R (ERLANGEN) If such a point existed, then ∞ X n=1 zn n X x∈Πn∩orbit(x) e−ϕn(x) = ∞ X k=1 zpk pk X x∈orbit(x) e−ϕpk(x) = ∞ X k=1 zpk k e−kϕp(x) = −log(1 −zp) th t so that so that ζ−1 0 (z) = (1−zp)·exp(−g(z)), where g(z) = ∞ X n=1 zn n X x∈Πn\orbit(x) e−ϕn(x) \| {z } =:ζ′ n,0 . ζ−1 0 (z) = (1−zp)·exp(−g(z)), where g(z) = ∞ X n=1 zn n X x∈Πn\orbit(x) e−ϕn(x) \| {z } =:ζ′ n,0 . 232 G. KELLER As 0≤ζ′ n,0 ≤ζn,0 for all n, it follows that ϱ(g):=(lim supn→∞\|n−1ζ′ n,0\|1/n)−1 ≥z(0) = 1 and z = ϱ(g) is a singular point of g(z) [8, Ch. 8, §1.5]. On the other hand, the function ζ−1 0 has a simple zero at z = 1 and is analytic in the disk with radius r > 1 by assumption so that exp(−g(z)) is analytic and nonzero in a neighbourhood of z = 1. Invoking the existence theorem for holomorphic logarithms once more it follows that z = 1 is not a singular point of g(z). Hence ϱ(g) > 1 so that \|(f n)′(x)\| = eϕn(x) ≥ϱ(g)n > 1 for all x ∈Πn\orbit(x). But this is not true for any nonrenormalizable S-unimodal map with neutral periodic orbit that has at least one other periodic orbit. One way to see this is the following. Using Hofbauer’s Markov extension it is easy to construct periodic orbits that follow for a long time the neutral periodic orbit, do something diﬀerent for a short time interval (e.g. follow a second periodic orbit), return to the neutral periodic orbit etc. In this way one obtains periodic orbits with Lyapunov exponent as close to zero as one wishes. Now we can assume that f has neither stable nor neutral periodic orbits, and it follows from [5, Ch. IV, Theorem B′] that δ := inf{ϕn(x) : n ≥1, x ∈Πn} > 0. We conclude that for x ∈Πn, e−ϕn(x)(1 −e−τδ) ≤e−ϕn(x)(1 −e−τϕn(x)) ≤ X x∈Πn e−ϕn(x)(1 −e−τϕn(x)) = ζn,0 −ζn,−τ. = ζn,0 −ζn,−τ. As \|ζn,−τ −ζn,0\| = O(r−n) in view of (2), it follows that e−ϕn(x) = O(r−n), i.e. λper ≥r > 1 also in this case. As \|ζn,−τ −ζn,0\| = O(r−n) in view of (2), it follows that e−ϕn(x) = O(r−n), i.e. λper ≥r > 1 also in this case. As \|ζn,−τ −ζn,0\| = O(r−n) in view of (2), it follows that e−ϕn(x) = O(r−n), i.e. λper ≥r > 1 also in this case. Remark 2. It seems that the case z(−τ) = z(0) = 1 in the above proof does not really occur. Recall that f is a Collet–Eckmann map if it is uniformly hyperbolic on periodic points [6]. Assume now that f satisﬁes the additional regularity assumptions from [4], e.g. let f be a polynomial or let f(x) = a(1 −\|2x −1\|ℓ) for some real ℓ> 1. The results of that paper show that z(t)−1 is the spectral radius of a suitable transfer oper- ator associated with f and t and that z(t) is a real-analytic function of t in a neighbourhood of t = 0. It then follows from [3, Proposition 4.5] that −log z(t) is the pressure P((t −1)ϕ) of the function (t −1)ϕ, where P((t−1)ϕ)=sup{h(µ)+(t−1)µ(ϕ) : µ=µ◦f −1}. Hence, if z(−τ)=z(0)=1 for some τ close to zero, then P((t −1)ϕ) = 0 for t in a neighbourhood of t = 0. By [3, Theorems 5.1, 6.1] the unique absolutely continuous invariant measure µ1 for f is the unique equilibrium state for −ϕ, and the pressure P((t −1)ϕ) can be constant only if µ1(ϕ) = 0. But abso- lutely continuous invariant measures have positive exponent, a contradic- tion. 233 S-UNIMODAL MAPS REFERENCES [1] V. Baladi and G. Keller, Zeta-functions and transfer operators for piecewise monotone transformations, Comm. Math. Phys. 127 (1990), 459–478. [2] V. Baladi, Periodic orbits and dynamical spectra, Ergodic Theory Dynam. Systems 18 (1998), 255–292. [3] H. Bruin and G. Keller, Equilibrium states for S-unimodal maps, ibid. 18 (1998), 765–789. [4] G. Keller and T. Nowicki, Fibonacci maps re(aℓ)visited, ibid. 15 (1995), 99–120. [5] W. de Melo and S. van Strien, One-Dimensional Dynamics, Springer, 1993. and S. van Strien, One-Dimensional Dynamics, Sp n Strien, One-Dimensional Dynamics, Springer, 19 [6] T. Nowicki and D. Sands, Non-uniform hyperbolicity and universal bounds for S-unimodal maps, Invent. Math. 132 (1998), 633–680. [7] Y. Oono and Y. Takahashi, Chaos, external noise and Fredholm theory, Progr. Theor. Phys. 63 (1980), 1804–1807. [7] Y. Oono and Y. Takahashi, Chaos, external noise and Fredholm theory, Progr. Theor. Phys. 63 (1980), 1804–1807. [8] R. Remmert, Theory of Complex Functions, Grad. Texts in Math. 122, Springer, New York, 1991. [8] R. Remmert, Theory of Complex Functions, Grad. Texts in Math. 122, Springer, New York, 1991. [9] D. Ruelle, Analytic completion for dynamical zeta functions, Helv. Phys. Acta 66 (1993), 181–191. [9] D. Ruelle, Analytic completion for dynamical zeta functions, Helv. Phys. Acta 66 (1993), 181–191. [10] Y. Takahashi, An ergodic-theoretical approach to the chaotic behaviour of dynam- ical systems, Publ. R.I.M.S. Kyoto Univ. 19 (1983), 1265–1282. [10] Y. Takahashi, An ergodic-theoretical approach to the chaotic behaviour of dynam- ical systems, Publ. R.I.M.S. Kyoto Univ. 19 (1983), 1265–1282. Mathematisches Institut Universit¨at Erlangen-N¨urnberg 1 Bismarckstr. 1 1 2 2 D-91054 Erlangen, Germany D-91054 Erlangen, Germany E-mail: keller@mi.uni-erlangen.de E-mail: keller@mi.uni-erlangen.de (3790) Received 15 July 1999 (3790) Received 15 July 1999 (3790) Received 15 July 1999
https://openalex.org/W4233577528	https://www.qeios.com/read/HA8G7T/pdf	English	null	Per Picoliter	Definitions	2,020	cc-by	60	Per Picoliter National Cancer Institute National Cancer Institute Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Qeios ID: HA8G7T · https://doi.org/10.32388/HA8G7T Source National Cancer Institute. Per Picoliter. NCI Thesaurus. Code C117964. A volume unit equal to one picoliter used as a denominator to build a derived unit expressed as a ratio. Qeios ID: HA8G7T · https://doi.org/10.32388/HA8G7T 1/1
https://openalex.org/W3040916347	https://www.frontiersin.org/articles/10.3389/fphar.2020.01044/pdf	English	null	Targeting the Microenvironment in MDS: The Final Frontier	Frontiers in pharmacology	2,020	cc-by	8,912	Targeting the Microenvironment in MDS: The Final Frontier 1 Department of Hematology,Iuliu Hategan University of Medicine and Pharmacy, Cluj-Napoca, Romania, 2 Department of Oncology, The Johns Hopkins Hospital, Johns Hopkins Medicine, Baltimore, MD, United States Myelodysplastic syndromes (MDS) are a heterogeneous group of malignant disorders of hematopoietic stem and progenitor cells (HSPC), mainly characterized by ineffective hematopoiesis leading to peripheral cytopenias and progressive bone marrow failure. While clonal dominance is nearly universal at diagnosis, most genetic mutations identiﬁed in patients with MDS do not provide a conspicuous advantage to the malignant cells. In this context, malignant cells alter their adjacent bone marrow microenvironment (BME) and rely on cell extrinsic factors to maintain clonal dominance. The profoundly disturbed BME favors the myelodysplastic cells and, most importantly is detrimental to normal hematopoietic cells. Thus, the MDS microenvironment not only contributes to the observed cytopenias seen in these patients but could also negatively impact the engraftment of normal, allogeneic HSPCs in patients with MDS undergoing bone marrow transplant. Therefore, successful therapies in MDS should not only target the malignant cells but also reprogram their bone marrow microenvironment. Here, we will provide a synopsis of how drugs currently used or on the verge of being approved for the treatment of MDS may achieve this goal. Reviewed by: Reviewed by: Emanuele Angelucci, Policlinico San Martino Hospital, Italy Daniel Starczynowski, Cincinnati Children’s Hospital Medical Center, United States Manja Wobus, Technische Universität Dresden, Germany Correspondence: Gabriel Ghiaur gghiaur1@jhmi.edu Correspondence: Gabriel Ghiaur gghiaur1@jhmi.edu Keywords: myelodyslastic syndromes, microenvironment, azacytidine, lenalidomide, luspatercept, rigosertib, all- trans retinoic acid, CYP26 enzymes INTRODUCTION Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Pharmacology Received: 07 April 2020 Accepted: 26 June 2020 Published: 09 July 2020 Myelodysplastic syndromes (MDS) are a heterogeneous group of malignant disorders of hematopoietic stem and progenitor cells (HSPCs), mainly characterized by ineffective hematopoiesis leading to peripheral cytopenias and progressive bone-marrow failure. Moreover, these are also associated with a high risk of progression to acute myeloid leukemia (AML). MDS is the most commonly diagnosed myeloid malignancy in the United States (DeZern, 2015). The median age at diagnosis is 72 years old. These patients are fragile and suffer from multiple comorbidities. Therefore, they are often ineligible for bone marrow transplantation, the only curative therapeutic option in MDS. The 3-year overall survival rate of 35–45% (Rollison et al., 2008) highlights the need for novel therapies in patients with MDS. MINI REVIEW MINI REVIEW published: 09 July 2020 doi: 10.3389/fphar.2020.01044 Edited by: Cyril Corbet, Fonds National de la Recherche Scientiﬁque (FNRS), Belgium Reviewed by: Emanuele Angelucci, Policlinico San Martino Hospital, Italy Daniel Starczynowski, Cincinnati Children’s Hospital Medical Center, United States Manja Wobus, Technische Universität Dresden, Germany Edited by: Cyril Corbet, Fonds National de la Recherche Scientiﬁque (FNRS), Belgium THE BONE MARROW MICROENVIRONMENT IN MDS Research in MDS has been hindered by lack of available models. More so, as MDS cells are difﬁcult to study ex vivo due to very high rate of apoptosis. Support from BME proved to be essential to maintain some MDS cells ex vivo. However, the mechanisms through which this support is provided is currently not fully understood. To this end, bone marrow derived MSCs from patients with MDS (MDS-MSCs), and not from normal individuals, are uniquely effective in maintaining the MDS clones (Medyouf et al., 2014). This observation led to the hypothesis that the BME contributes to MDS pathogenesis, homeostasis, and even response to treatment. Regarding pathogenesis, several studies have shown that MDS and other myeloproliferative neoplasms (MPNs) can be initiated by modiﬁcations in the BME. The deletion of various genes such as Dicer1, Sipa1, Retinoblastoma protein (Rb), and Retinoic Acid Receptor gamma (RARy), as well as the activation of the Hedgehog pathway through the knockout of PTCH2, have been reported to lead to the development of MDS or MPNs in mice (Walkley et al., 2007a; Walkley et al., 2007b; Raaijmakers et al., 2010; Klein et al., 2016;Xiaoetal.,2018).However,thesespeciﬁcmutationsarefound only in selected cases of MDS patients and their involvement in human pathogenesis is yet to be clariﬁed. g y Bidirectional crosstalk between the MDS clone and their surrounding milieu not only maintains the malignant clone but also reshapes the BME (Figure 1). As a result, MSCs from patients with MDS are reprogramed to promote maintenance of the malignant clone at the expense of normal hematopoiesis (Geyh et al., 2013). To this end, MSCs derived from patients with MDS (MDS-MSCs) display morphological changes ex vivo (Ferrer et al., 2013; Falconi et al., 2016), impaired growth capacity, increased senescence, decreased osteogenic differentiation, and overall decreased survival (Geyh et al., 2013). The mechanisms responsible for these alterations are only partly characterized. For instance,oversecretionofalarmins,suchasS100A9andS100A8,by the MDS cells activates the inﬂammasome inthe MSCs (Chen etal., 2016) leading to aberrant activation of various molecular programs resulting in higher secretion of cytokines such as interferons and The mechanisms by which the corrupt MDS-MSCs signal to the MDS clone are diverse and only beginning to be explored. For instance, MDS-MSCs release high quantities of small extracellular vesicles (sEVs) loaded with miR-486-5p (Meunier et al., 2020). Citation: Teodorescu P, Pasca S, Dima D, Tomuleasa C and Ghiaur G (2020) Targeting the Microenvironment in MDS: The Final Frontier. Front. Pharmacol. 11:1044. doi: 10.3389/fphar.2020.01044 Numerous genetic events have been implicated in the pathogenesis of MDS. These mutations range from large chromosomal abnormalities such as deletions/additions (i.e. del(5q), del(7q)) to speciﬁc gene mutations affecting various biological processes including: spliceosome (i.e. SF3B1, SRSF2); transcription factors (i.e. RUNX1, ETV6); or DNA/chromatin epigenetic changes (i.e. July 2020 \| Volume 11 \| Article 1044 1 Frontiers in Pharmacology \| www.frontiersin.org Targeting the Microenvironment in MDS Teodorescu et al. IL32 (Figure 2) (Kim et al., 2015; Zhang et al., 2016). Also, the secretionofextracellular vesiclescontaining miR-7977, bytheMDS cells, was shown to reduce the hematopoietic supporting capacity of MSCs. This was achieved through the reduction of several hematopoietic growth factors such as Jagged-1, stem cell factor, and angiopoietin-1 (Horiguchi et al., 2016). In addition, several in vitro studies suggest that MDS-MSCs have impaired PI3K/AKT and Wnt/ß-catenin signaling (Pavlaki et al., 2014; Falconi et al., 2016) which may explain their abnormal proliferation, self-renewal, and osteogenic differentiation (Figure 2) (Boland et al., 2004; Glass et al., 2005). To this end, high endogenous erythropoietin levels often seen in MDS patients may downregulate Wnt pathway and impair osteogenic differentiation of MDS-MSCs (Balaian et al., 2018). In this context, the wide use of erythropoietin and erythropoiesis- stimulating agents may inadvertently impact the BME in patients with MDS. On the other hand, in murine models of MDS, Wnt/ß- catenin pathway is hyperactive in MSCs (Kode et al., 2014; Bhagat et al., 2017) and is capable of disease initiation through overexpression of Notch-ligand, Jagged1 (Kode et al., 2014). It is currently unknown whether or not activation of Wnt/ß-catenin pathway plays distinct roles in disease initiation vs. maintenance or if the observed differences are due to unique features of the models used(mousevs.human).Nevertheless,MDS-MSCshavelowlevelsof Wntpathwayantagonists(FRZBandSFRP1)likelyduetotheirhyper methylation explaining the upregulated Wnt/ß-catenin signaling (Figure 1) (Bhagat et al., 2017). While disrupted methylation proﬁles in the MDS hematopoietic clones are well characterized, MDS-MSCs also display numerous differentially methylated genes explaining their cellular phenotype and transcriptional regulation (Figure 2) (Geyh et al., 2013). Among such genes, human Hh- interacting protein gene (HHIP) was shown to be hyper methylated in MDS-MSCs (Kobune et al., 2012). Low expression of HHIP and theassociatedactivationoftheHedgehogpathwayinMDS-MSCsare importantforthesurvivaloftheMDSclone(Figure1).Suchcomplex changes in MDS-MSCs make them more suitable to support the MDS clone perhaps at the expense of normal hematopoiesis. Citation: To this end,MDS-MSCscreateaninﬂammatorymilieuthatisdetrimentalto healthy HSPCs (Muto et al., 2020). On the other hand, MDS-HSPCs gain competitive advantage in this inﬂammatory environment by activatingtheirnon-canonicalNF-kBpathway viaTraf6.Inaddition, the SDF-1CXCR4 axis is also dysregulated in MDS. Studies have foundcorrelationsbetween higher levels of SDF-1 in low-grade MDS and increased apoptosis of hematopoietic cells, and higher levels of CXCR4 and increased bone-marrow angiogenesis in high-grade MDS (Zhang et al., 2012). The mechanisms by which the corrupt MDS-MSCs signal to the MDS l di d l b i i t b l d F TET2, DNMT3a, ASXL1). While clonal dominance is nearly universal in MDS at diagnosis, these mutations don't provide a conspicuous advantage to the malignant cells. In most cases, the malignant clone continues to coexist alongside normal hematopoietic stem cells, which are somehow inhibited (Calvi et al., 2019). These observations led to the hypothesis that MDS cells get an extrinsic support, from the mesenchymal stromal cells (MSCs) in the bone marrow microenvironment (BME). The interaction between the mutant clone and BME plays an important role in disease homeostasis. Since the main causes of death in MDS are cytopenia-related complications (infections, hemorrhage), restoring the function of residual normal hematopoiesis is a major goal in the treatment of MDS. Frontiers in Pharmacology \| www.frontiersin.org THE BONE MARROW MICROENVIRONMENT IN MDS This miR-486-5p can not only promote leukemogenesis and is overexpressed in leukemic cells of Down syndrome patients (Shaham et al., 2015) but also may induce oxidative stress and apoptosis of normal HSPC and senescence in MSCs (Kim et al., 2012). Higher secretion of sEVs containing miR10a and miR15a by MDS-MSCsincreasetheviabilityandclonogenicityofMDSHSPCs (Muntión et al., 2016). July 2020 \| Volume 11 \| Article 1044 Frontiers in Pharmacology \| www.frontiersin.org 2 Targeting the Microenvironment in MDS Teodorescu et al. FIGURE 1 \| Cartoon representation of molecular crosstalk between mesenchymal bone marrow microenvironment and the myelodysplastic hematopoietic cells. HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; Treg, T regulatory cells; HMA, hypomethylating agents; LEN, lenalidomide; LUS, luspatercept; RIG, rigosertib; ATRA, all-trans retinoic acid; CAPN1, calcium-dependent protease calpain1; CDA, cytidine deaminase; CDC25C, Cell Division Cycle 25C gene; CSNK1A1, casein-kinase 1A1; GPR68, G Protein-Coupled Receptor 68 gene; IKZF1, IKAROS Family Zinc Finger 1 gene; PI3K, Phosphatidylinositol-3 Kinase; PPA2, Inorganic Pyrophosphatase gene; RARg, Retinoic Acid Receptor Gamma; SHH, Sonic Hedgehog ligand; TGFb, transforming growth factor beta; TLR8, Toll-Like Receptor 8. FIGURE 1 \| Cartoon representation of molecular crosstalk between mesenchymal bone marrow microenvironment and the myelodysplastic hematopoietic cells. HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; Treg, T regulatory cells; HMA, hypomethylating agents; LEN, lenalidomide; LUS, luspatercept; RIG, rigosertib; ATRA, all-trans retinoic acid; CAPN1, calcium-dependent protease calpain1; CDA, cytidine deaminase; CDC25C, Cell Division Cycle 25C gene; CSNK1A1, casein-kinase 1A1; GPR68, G Protein-Coupled Receptor 68 gene; IKZF1, IKAROS Family Zinc Finger 1 gene; PI3K, Phosphatidylinositol-3 Kinase; PPA2, Inorganic Pyrophosphatase gene; RARg, Retinoic Acid Receptor Gamma; SHH, Sonic Hedgehog ligand; TGFb, transforming growth factor beta; TLR8, Toll-Like Receptor 8. Lastly, MDS-MSCs secrete high levels of TGFß1 (Zhao et al., 2012), a cytokine profoundly immunosuppressant for B, T, and NK cells and immunostimulating for regulatory T cells (Treg) (Cagliani et al., 2017). Thus, the high levels of TGFß1 seen in patients with high risk MDS (Zhao et al., 2012) promote a immunosuppressive microenvironment with reduced CD4+ T- cell population, CD8 T-cell exhaustion, a signiﬁcant decrease in NK activating receptors, and an increase of non-cytotoxic NK- cells (CD56bright) (Figure 1) (Montes et al., 2019). This may be explained by the functional heterogeneity within the MDS clone. For instance, some MDS cells may be protected from therapy by the MDS-MSCs and be responsible for disease relapse (Figure 1). Frontiers in Pharmacology \| www.frontiersin.org THE BONE MARROW MICROENVIRONMENT IN MDS To this end, BM MSCs express high levels of cytidine- deaminase (CDA), an enzyme that metabolizes azacitidine and decitabine, two essential drugs in the treatment of MDS (Alonso et al., 2015; Su et al., 2019). Thus, the pharmacokinetics of these drugs may be altered in the BME. On the other hand, molecular changes induced in the MDS clone need to be reinforced by reprogramming the BME in order to be sustained. To this end, abnormal methylation patterns observed in MDS-MSCs for instance may also be amenable to correction by the current therapeutic tools used in these patients. In spite of recent advances in our understanding of clonal architecture and mutational landscape in MDS, overall prognosis of these patients remains poor. More so, therapeutic responses are transient at best without the use of bone marrow transplantation. July 2020 \| Volume 11 \| Article 1044 Frontiers in Pharmacology \| www.frontiersin.org 3 Targeting the Microenvironment in MDS Teodorescu et al. FIGURE 2 \| STRING analysis of molecular pathways altered in bone marrow microenvironment of patients with MDS compared to healthy individuals. nalysis of molecular pathways altered in bone marrow microenvironment of patients with MDS compared to healthy individuals. FIGURE 2 \| STRING analysis of molecular pathways altered in bone marrow microenvironment of patients with MDS compared Here, we will provide a synopsis of how drugs currently used or on the verge of being approved for treatment of MDS may impact not only the mutant hematopoietic cells but also the surrounding microenvironment. in those that respond, why the malignant clone is not completely eliminated (Diesch et al., 2016; Ball et al., 2017). A plausible explanation is that these drugs have a blunted effect on the highly quiescentMDSstemcellssinceepigeneticchangesneeddividingcells to take effect (Suarez and Gore, 2013). Also, since stroma cells in the bone marrow expresses CDA (Alonso et al., 2015), the enzyme that inactivates 5-Aza, perhaps impaired pharmacokinetics in bone marrow niches may play a role (Figure 1). To this end, cell cultures that express high levels of CDA are less sensitive to 5-Aza (Mahfouz et al., 2013). However, a study conducted on a small cohort of patient samples, showed that highlevels of CDAaftertreatment, might be an indicator of responsiveness (Murakami et al., 2019). THE BONE MARROW MICROENVIRONMENT IN MDS Nevertheless, CDA is necessary for the transformation of HMAs to uridine, an activatorofthetoll-likereceptor8(TLR8)withpotentialimplications for the impaired myeloid differentiation seen in MDS (Figure 1) (Ignatz-Hoover et al., 2015; Miyake et al., 2017; Furusho et al., 2019). Some suggest that the degree of myeloid differentiation of the MDS clone prior to treatment with azanucleotides is a better indicator for response to therapy than the previously proposed abnormal methylation of endogenous retro-elements (Kazachenka et al., 2019). While cell intrinsic properties of the MDS clone can certainly dictate the differentiation state of these cells, an altered BME is bound to play an essential role. Since MDS-MSCs have different methylation patterns compared to their healthy counterparts (Geyh et al., 2013), it begs the question if HMA- induced changes in the BME contribute to the clinical responses observed. To this end, 5-Aza enhances the ability to support normal hematopoiesis in MDS-MSCs, while decreasing their support for MDS HSPCs (Wenk et al., 2018; Poon et al., 2019). During disease homeostasis, MDS-induced impairment of MSCs' functions appears Frontiers in Pharmacology \| www.frontiersin.org Hypomethylating Agents Hypomethylating Agents Epigenetic therapy with 5-azacitidine (5-Aza) or decitabine represents a stepping stone in the treatment of MDS and remains the only FDA approved option for high-risk disease. 5-Azacitidine and decitabine work as hypomethylating drugs and are related molecules with similar structures and overlapping effects. They both incorporate into the DNA of cells resulting in depletion of intracellular methyltransferases (DNMTs) and reversal of altered methylation patterns of MDS cells. As such, it takes four to six cycles of therapy to reach full clinical responses to azanucleotides in MDS. More often than not the responses correlate with improvement of cytopenias but no signiﬁcant changes in the clonal architecture suggesting some correction of the differentiation proﬁle for the MDS clone rather than elimination of the malignant cells. To this end, treatment with azanucleotides resembles classical differentiation therapy seen with all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL) (Stresemann and Lyko, 2008; Gnyszka et al., 2013). In general, 30–50% of patients show clinical beneﬁt from treatment with azanucleotides but their response is short lived. It is currently unknown why only some patients beneﬁt from treatment and even July 2020 \| Volume 11 \| Article 1044 Frontiers in Pharmacology \| www.frontiersin.org 4 Targeting the Microenvironment in MDS Teodorescu et al. reversible by 5-Aza but only up to a certain point in disease progression (Poon et al., 2019). 5-Aza restored the hematopoiesis- supporting capacity of most primary MDS-MSCs samples derived from low grade MDS but not those from advanced-stage disease (Poon et al., 2019). However, treatment with 5-Aza is capable to enhance hematopoiesis-supporting properties of healthy MSCs as well,suggestingthattheeffectisnotspeciﬁctoMDS-inducedchanges (Wenketal.,2018).Itiscurrentlyunknownwhatmolecularprogram induced by azanucleotides rescue MSC function. RNA-seq data suggests that treatment with 5-Aza optimizes interferon signaling and extracellular matrix homeostasis, including collagen type IV and VI with major implications for HSC anchorage, self-renewal, and differentiation (Essers et al., 2009; Gattazzo et al., 2014; Wenk et al., 2018). In addition, treatment of MDS-MSCs with 5-Aza reverses aberrant methylation patterns of these cells resulting in rescue of frequently altered signaling pathways such as over activation of betab-catenin and the downregulation of HHIP (Figure 1) (Kobune et al., 2012; Bhagat et al., 2017). Interestingly, complete loss of this protein induces apoptosis as a result of TP53 activation (Schneider et al., 2014) and thus, exposes a vulnerability of 5q- MDS cells to treatment with lenalidomide. Hypomethylating Agents However, more recent ﬁndings show that this vulnerability emerges only in MDS cells that previously undergo megakaryocytic differentiation driven by a RUNX1-GATA2 complex. This complex is enabled by the lenalidomide-induced ubiquitination and degradation of Ikaros protein IKZF1(Martinez-Høyer et al., 2020). Nevertheless, lenalidomide was found to have clinical activity in some patients with LR-MDS without 5q- and even some patients with HR-MDS. While some of these effects can be explained by lenalidomide-dependent improved erythropoietin signaling in MDS cells (Basiorka et al., 2016), broader effects of this drug on the immune and mesenchymal BME are also likely to play a role. To this end, lenalidomide increased CD28 signaling, resulting in augmented T cell costimulation and increased secretion of interferon gamma and IL2 (Figure 1) (Stirling, 2001; LeBlanc et al., 2004; Wu et al., 2008). In addition, treatment with lenalidomide results in decreased production of pro- inﬂammatory cytokines (TNF-a, IL-1, IL-6, IL-12) and increased levels of anti-inﬂammatory cytokines such as IL10. Altered cytokine milieu may not only be detrimental to the MDS clone but also promote the wellbeing of residual normal hematopoiesis (Kotla et al., 2009; Stahl and Zeidan, 2017). Regarding the impact of lenalidomide ontheBME,treatmentwithlenalidomidedecreaseslevelsofCXCL12 production from normal as well as MDS-MSCs (Figure 1) (Ferrer et al., 2013). Since levels of CXCL12 are already low in MDS BME, furtherreductionmaybedetrimentaltotheMDSclonebutcouldalso explain some of the cytopenias associated with this drug. In addition, lenalidomide like thalidomide is a powerful antiangiogenic agent. In MDS, the bone marrow niche is characterized by increased neoangiogenesis. Lenalidomide reduces marrow vascular density and this histologic effect correlates with decreased disease progression (Stahl and Zeidan, 2017). Finally, treatment with decitabine ex vivo can reset the immunosuppressive phenotype of MDS-MSCs, resulting in decreased expression of PDL1 and optimization of the immune milieu in the BM of patients with MDS (Pang et al., 2019). To this end, decitabine decreases T cell differentiation towards Tregs, a population known to be expanded in high risk MDS (Kordasti et al., 2007; Zhao et al., 2012). Thus, azanucleotides-induced changes of BME may not only negatively impact the MDS clone but also rescue the residual normal hematopoiesis.AnurturingBMEcouldhelpinengraftmentofdonor HSCs and promote hematopoietic reconstitution in the settings of allogeneic bone marrow transplant (alloBMT). Treatment with 5- Aza prior to or post alloBMT may prove beneﬁcial for patients with MDS and remains an area of active research. Lenalidomide Lenalidomide is a derivative of thalidomide that, in addition to its well documented efﬁcacy in the treatment of multiple myeloma, has also proven activity in patients with low risk MDS (LR-MDS) and 5q31-5q32 deletion (List et al., 2006). Treatment with lenalidomide of patients with transfusion dependent LR-MDS with 5q- results in improved transfusion requirements and even cytogenetic complete remission (CR) in some cases. The putative mechanisms of action take into account the unique sensitivity of 5q- MDS cells to lenalidomide and explore the role of haploinsufﬁciency of various genes in this process. As such, three main haploinsufﬁcient mechanisms have been proposed: a) CDC25C and PPA2 resulting in subsequent modulation of MDM2-TP53 pathway and cell cycle arrest (Wei et al., 2009); b) casein- kinase-1a1 (CSNK1A1) with resultant TP53 induction and clonal arrest (Schneider et al., 2014); and c) activation of calcium dependent protease calpain (CAPN1) due to not only overexpression of GPR68 (as a consequence of degradation of IKAROS1) but also haploinsufﬁciency of calpastatin, an otherwise inhibitor of the pathway (Fang et al., 2016) (Figure 1). Recently, emphasis was placed on the ubiquitination and degradation of CSNK1A1, an inhibitor of TP53, and also part of the ß- catenin destruction-complex (Elyada et al., 2011; Knippschild et al., 2014). Heterozygous loss of this gene, as seen in 5q- MDS leads to an increase in ß-catenin levels and thus, stem cell expansion. While the immunomodulatory and anti-angiogenic properties of lenalidomide are well recognized, their role in the observed clinical beneﬁt for patients with 5q- MDS or non 5q- MDS remains to be further clariﬁed. Frontiers in Pharmacology \| www.frontiersin.org Luspatercept Anemia and RBC transfusion requirement is a major source of morbidity in patients with MDS. In this aspect, MDS resemble hemoglobinopathies such as b-thalassemia in that ineffective erythropoiesis results not only in refractory anemia but also accumulation of various erythroid precursors and disruption of the BME. TGF-b signaling regulates terminal erythroid maturation and targeting this pathway either by using activin receptor traps (luspatercept, sotatercept), TGF-bR1 tyrosine kinase inhibitor (galunisertib) or targeting SMAD7 via miR21 promised improved erythropoiesis in patients with LR-MDS. Initially, FDA approved the use of luspatercept only for the treatment of transfusion- dependent ß-thalassemia. However, in early April 2020, the drug was also approved in transfusion-dependent LR-MDS with ring sideroblasts, after failure of treatment with erythropoiesis stimulating agents. In this category of patients, treatment with luspatercept resulted in more than a third of patients achieving July 2020 \| Volume 11 \| Article 1044 Frontiers in Pharmacology \| www.frontiersin.org 5 Targeting the Microenvironment in MDS Teodorescu et al. activity in AML and MDS cells (Sloand et al., 2007; Olnes et al., 2012; Hyoda et al., 2015), most clinical trials showed no signiﬁcant improvement in overall survival, and limited hematological improvement (Garcia-Manero et al., 2016). Rigosertib does not impair normal HSPCs functions in vitro (Xu et al., 2014) but in vivo, it was shown to remodel the bone architecture of young mice due to increased osteoclast numbers. In this model, rigosertib treatment resulted in decreased mass, thickness and numbers of trabecular bone leading to pancytopenia. More so, rigosertib altered the biomechanical properties of MSCs and reduced hematopoiesis-supporting properties of MDS-MSCs (Balaian et al., 2019). In addition, the downregulation of the PI3K/Akt pathway activity, one of the main targetsofthedrug,isassociatedwithdysfunctionofthestromalcells (Falconi et al., 2016) and thus, further impairment of normal hematopoiesis. Since hyper activation of Wnt/ß-catenin signaling in MSCs leads to rapid development of MDS and AML the proposed inhibitory effects of rigosertib may correct the altered BME in patients with MDS. Rigosertib can rescue Akt, ß-catenin, and GSK3a/ß signaling pathways commonly dysregulated in MDS cells (Figure 1) (Xu et al., 2014) but it is unclear if these effects hold true in vivo in patients with MDS treated with this drug (Stoddart et al., 2017). Even less is known about the biological effects of rigosertib on these pathways in MDS-MSCs. Luspatercept Most intriguing, the newly described role of rigosertib in microtubule assembly may have signiﬁcant implications for hematopoiesis–MSCs interactions given the recent reports that mitochondria can be transferred between MSCs and malignant cells via tunneling nanotubules. Inhibition of microtubule assembly, as seen during treatment with vincristine, and potentially rigosertib can destabilize tunneling nanotubules with profound impact on the survival and metabolicproﬁleofmalignantcells(Moschoietal.,2016;Forteetal., 2019). To what extent this mechanism plays a role in the biological activity of rigosertib remains to be evaluated. transfusion independence for 8 weeks or longer (Fenaux et al., 2020). These results are consistent with previous clinical studies showing that patients with >15% ring sideroblasts, or with SF3B1 mutations, are most likely tobeneﬁt from thistherapeutic approach (Platzbecker et al.,2017). In contrast with HMAs and lenalidomide, luspatercept does not impact MDS clonal evolution but rather rescues erythropoiesis in these patients. MDS cells are characterized by hyperactivity of the TGF-ß signaling, mostly due to SMAD2/3 dependent reduction of SMAD7, a negative-feedback regulator of the pathway (Zhou et al., 2011; Bhagat et al., 2013). A variety of ligands such as activin A, activin B, GDF8, GDF11, and several BMPs can also activate TGF-ß signaling and thus, regulate/dysregulate erythroid maturation (Krolewski et al., 1992). Luspatercept is a chimera between the extracellular domain of activin receptor and human IgG1 Fc portion serving as a TGFb ligand trap (Figure 1). Though initially thought to exert its activity via inactivating GDF11, recent genetic studies called into question this mechanism of action (Guerra et al., 2019). A number of other TGFb ligands, including BMPs, activins, and GDF8 may play role (Verma et al., 2020). Preventing signaling downstream of these ligands can have a profound impact on the BME. For instance, sotatercept improved hematocrit levels in postmenopausal women, prevented osteoporosis and improved osteolytic lesions in patients with multiple myeloma (Raje and Vallet, 2010). These observations came to underscore the known roles of TGFb ligands in MSCs osteogenic speciﬁcation and terminal differentiation. It is currently unknown the impact of luspatercept on the MDS BME. That being said, TGF-ß1 secreted by the MSCs contributes to the pro-inﬂammatory milieu present in the BME of patients with MDS (Figure 1) (Cagliani et al., 2017). Interestingly, levels of TGF-ß ligands are higher in high- risk MDS compared to low-risk disease (Zhao et al., 2012) which may explain the lack of clinical activity of single agent luspatercept in high-risk MDS. EMERGING THERAPIES IN MDS Multi lineage cytopenias is the hallmark of MDS and yet bone marrow cellularity is typically increased in these patients. Abnormal myeloid and erythroid elements in various stages of differentiation dominate the histology of patients with MDS. Lack of ﬁnal maturation of these cells is the root for most mortality and morbidity in MDS. Therapeutic interventions to promote ﬁnal maturation of the dysplastic cells have been met with some success in the case of erythroid maturation and improvement in RBC transfusion needs in response to erythropoietin stimulating agents (ESA) [for a review on the topic see (Park et al., 2019)]. Frontiers in Pharmacology \| www.frontiersin.org Rigosertib Though initially known to control systemic ATRA levels via their hepatic function, CYP26 enzymes were recently shown to be expressed by BM MSCs (Ghiaur et al., 2013; Su et al., 2015; Alonso et al., 2016; Hernandez etal.,2020).Moreso,stromalCYP26isessentialtomaintainnormal and malignant stem cell activity (Ghiaur et al., 2013; Su et al., 2015; Alonso et al., 2017). Interestingly, treatment with ATRA directly upregulates stromal CYP26 (Hernandez et al., 2020) and thus, results inhyper-protective niches inthe BME (Figure 1).Inhibition of stromal CYP26 was able to sensitize malignant cells to retinoid induced differentiation in the presence of BME (Su et al., 2015). Similarly two synthetic CYP26 resistant retinoids (IRX195183 and Tamibarotene) can bypass stromal protection and induce myeloid differentiation in some AML and multiple myeloma cells (Alonso et al., 2016; Hernandez et al., 2020). These novel retinoids have shown biological activity in preliminary results from two ongoing clinical trials that also enrolled patients with MDS. The full clinical impactofthesetoolsremainstobeseen,particularlyincombination therapies with ESA or other MDS targeting agents. complication, most of morbidity and mortality stems from multi lineage cytopenias and subsequent infections, bleeding complications, and long term toxicities from frequent transfusions. It is only beginning to be explored how the malignant stem cells out compete their healthy counterparts. The role of bone marrow microenvironment in this process is becoming front and center. By now,itisclearthatMDS-MSCsareprofoundlyalteredandcontribute tomaintainingthedysplasticclonewhilesuppressingresidualnormal hematopoiesis. The exact mechanisms by which abnormal MDS- MSCscontributetodiseasehomeostasisareonlynowbeingexplored. This is an exciting time in our understanding of MDS. Innovative preclinical models coupled with wide use of next generation sequencing in patients with MDS led to rapid development of new therapeutic tools. A multitude of drugs are in various stages of clinical development. We are now testing APR246 for TP53 mutant disease, IDH inhibitors for IDH mutant disease, as well as CDK9 and Bcl2 inhibitors. These drugs have already shown activity in subtypes of AML and are bound to change our understanding of MDS. Other approaches, such as immune checkpoint inhibitors, Hypoxia-inducible factor prolyl hydroxylase inhibitor, Hedgehog inhibitors, and splicing modulators are testing new biological concepts. Nevertheless, while we are zooming in on the biological effects of these drugs on the malignant clone, one should not loose site that MDS is a disorder in which the entire bone marrow (hematopoietic and non- hematopoietic) is profoundly perturb. Rigosertib Rigosertib is a multi-kinase inhibitor with promising activity in a number of malignancies including MDS. Preclinical and early phase clinical trials showed encouraging activity of this drug in patients with MDS that failed hypomethylating agents. Some of these patients achieved partial/complete marrow responses with acceptable hematologic toxicity (cytopenias) and no other signiﬁcant adverse events (Navada et al., 2018). Thus, this therapeutic approach is now tested in a phase III clinical trial. Initially thought to act as a polo-like kinase 1 (PLK1) inhibitor (Gumireddy et al., 2005), rigosertib is also a powerful phosphatidylinositol 3-kinase (PI3K) (Prasad et al., 2009) and RAS inhibitor (Athuluri-Divakar et al., 2016). Most recently, a CRISPRi-based chemical genetic screen revealed that rigosertib may also act as a microtubule-destabilizing agent binding the same tubulin-site as colchicine (Jost et al., 2017). Rigosertib induces mitotic arrest and subsequent apoptosis in MDS cells (Hyoda et al., 2015). Although in vitro studies showed promising Vitamin A plays an essential role in hematopoiesis. The impairment of vitamin A pathway, due to abnormal retinoic acid receptoralphasignaling,resultsinablockinmyeloiddifferentiation and development of APL (Evans, 2005). In this case, treatment with high levels of ATRA, the active compound of vitamin A, overcomes this differentiation block and promotes ﬁnal maturation of the malignant cells with emergence of neutrophils. Thus, a similar approach was attempted in MDS. To this end, ATRA induces G0/ July 2020 \| Volume 11 \| Article 1044 Frontiers in Pharmacology \| www.frontiersin.org 6 Targeting the Microenvironment in MDS Teodorescu et al. G1 cell cycle arrest of MDS cell lines in vitro via downregulation of CDK4, CDK6, cyclinD3, and cyclinD1 (Huang et al., 2004). Similarly, 4-Amino-2-Triﬂuoromethyl-Phenyl Retinate (ATPR), a synthetic retinoid, inhibits proliferation and promotes apoptosis of MDS cells in vitro, likely via upregulation of TP53 (Du et al., 2018). While single agent ATRA had only minimal effect in patients with MDS, co-administration with recombinant erythropoietin was effective in 40% of patients with LR-MDS and low erythropoietin levels(Itzyksonetal.,2009).TreatmentwithATRAhasbeentriedin combinationwithvalproic acid, vitamin E, vitamin D, interferon-a, and other therapeutic interventions but unfortunately showed discouraging results in MDS (Hofmann et al., 1999; Cortes, 2005; Giagounidisetal.,2005;Zhangetal.,2006).Mostrecently,impaired local pharmacokinetics of ATRA in the BME has been proposed to account for the observed discrepancy between in vitro sensitivity of MDS cells to ATRA and relativelack of clinical efﬁcacy(Hernandez et al., 2020). To this end, ATRA is oxidized and inactivated by CYP26, a member of cytochrome P450 enzymes. The project was funded by K08 HL127269 (GG), R03 HL145226 (GG), P01CA225618 (GG), and P30 CA006973. The project was funded by K08 HL127269 (GG), R03 HL145226 (GG), P01CA225618 (GG), and P30 CA006973. MDS is a complex disease with great heterogeneity and poorly understood pathogenesis. Although progression to AML is a feared AUTHOR CONTRIBUTIONS PT, SP, CT, GG—designed the project. PT, GG—wrote the ﬁrst draft of the manuscript. DD—contributed to critical discussion. Rigosertib Thus, a system biology approach to not only the pathophysiology of the disease but also to understanding the response or lack thereof to these new agents holdspromisetobettertheclinicaloutcomesforpatientswithMDS. REFERENCES (2004). Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells. J. Cell. 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Cancer Res. 71, 955–963. doi: 10.1158/0008-5472.CAN-10-2933 Walkley, C. R., Shea, J. M., Sims, N. A., Purton, L. E., and Orkin, S. H. (2007b). Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 129, 1081–1095. doi: 10.1016/j.cell.2007.03.055 Wei, S., Chen, X., Rocha, K., Epling-Burnette, P. K., Djeu, J. Y., Liu, Q., et al. (2009). A critical role for phosphatase haplodeﬁciency in the selective suppression of deletion 5q MDS by lenalidomide. Proc. Natl. Acad. Sci. 106, 12974–12979. doi: 10.1073/pnas.0811267106 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Wenk, C., Garz, A.-K., Grath, S., Huberle, C., Witham, D., Weickert, M., et al. (2018). Direct modulation of the bone marrow mesenchymal stromal cell compartment by azacitidine enhances healthy hematopoiesis. Blood Adv. 2, 3447–3461. doi: 10.1182/bloodadvances.2018022053 Copyright © 2020 Teodorescu, Pasca, Dima, Tomuleasa and Ghiaur. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Pharmacology \| www.frontiersin.org July 2020 \| Volume 11 \| Article 1044 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Wu, L., Adams, M., Carter, T., Chen, R., Muller, G., Stirling, D., et al. (2008). lenalidomide enhances natural killer cell and monocyte-mediated antibody- dependent cellular cytotoxicity of rituximab-treated CD20+ tumor cells. Clin. 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https://openalex.org/W4296789679	http://silverstripe.fkit.hr/kui/assets/Uploads/7-601-608-KUI-09-10-2022.pdf	English	null	Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent Treatment	Kemija u industriji	2,022	cc-by	6,578	1 Introduction scoria, etc.) have already been tested as granular materials in biofilters for wastewater treatment. They are low-cost, locally available and have properties suitable for biofilm formation.8 Several researchers9–12 have investigated the performance of pumice and pozzolan as biofilter mate rials for the removal of carbonaceous, phosphorous and nitrogenous matters from wastewater. Kuslu et al.13 studied pumice combined with sand and gravel as filter media un der different pressure filtration conditions, and found that pumice achieved the best solid removal efficiency. Wastewater reuse is an important contribution to waste water reclamation that has attracted the attention of many researchers in recent years.1–3 Generally, facultative lagoon effluents are considered as secondary effluents that meet standards for discharge into water courses.4 For wastewater reuse, higher standards are required and facultative lagoon treatment alone will not be sufficient. Thus, advanced treatments must be developed to improve quality of fac ultative lagoon effluent to meet stringent discharge stand ards. For additional treatments, maturation lagoons must be constructed in series. However, these methods require large areas of land and additional operating costs.4 Replac ing maturation lagoons with expensive treatment systems can be a significant economic burden for local public au thorities. Thus, the obvious choice is to find a simple, low- cost method such as biofiltration to upgrade facultative lagoon to meet standards for wastewater reuse in irriga tion. It is clear that when treating secondary wastewater by biofiltration, the daily volumes of treated water will be relatively small. This may be sufficient for drip irrigation, which is a water-saving technique and gives better yields. In recent years, Luffa cylindrica, a non-toxic and biodegrad able plant variety, has been processed into activated car bon for use as low-cost adsorbent,14 as a soil amendment in arid zones to retain irrigation water15, and as bed me dia filtration for wastewater treatment in trickling filters.16 Wang et al.17 reported that L. cylindrica was more suitable as a carrier than plastic sponge in sequencing batch biofilm reactor. Zhang et al.18 studied the start-up characteristics of L. cylindrica biofilm reactor for domestic wastewater treat ment and found that 10 days was sufficient for complete acclimation of microorganisms. L. 601 601 M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 https://doi.org/10.15255/KUI.2022.017 KUI-50/2022 Original scientific paper Received March 10, 2022 Accepted May 27, 2022 https://doi.org/10.15255/KUI.2022.017 KUI-50/2022 Original scientific paper Received March 10, 2022 Accepted May 27, 2022 Keywords y Natural lagoon, wastewater reuse, aerated biofilter, Luffa cylindrica, pozzolan M. Khelladi,a,b* M. Abaidia,a S. Boulerial,a K. Bekrentchir,a A. Benhamou,a and A. Debab a This work is licensed under a Creative Commons Attribution 4.0 International License a University of Science and Technology of Oran, Faculty of Chemistry, Laboratory of Process Engineering and Environment (LIPE), Oran, Algeria g g g b University Abdelhamid Ibn-Badis of Mostaganem, Department of Process Engineering, Algeria * Corresponding author: Khelladi Malika, M.Sc. Email: Khelladi_m@hotmail.com, malika.khelladi@univ-mosta.dz Abstract As part of a comprehensive evaluation of post-treatment techniques for the removal of chemical and microbiological pollutants from lagoon effluents, an aerated biofilter was designed. The main objective of this study was to evaluate the performance of pozzolan and Luffa cylindrica as low-cost packing materials for the advanced treatment of Stidia natural lagoon effluent. The aerated biofilter operates in down-flow with HLRs of 11.37 to 28.43 m3 m−2 d−1 and an air/liquid flow ratio of 3 : 1. The differ- ent experiments performed on the pilot showed that the percentages of sCOD removal vary with the HLRs and the wastewater concentration at the biofilter inlet. In this study, sCOD removal efficiencies above 78.9 % were achieved depending on HLR, and a maximum removal efficiency of TSS of 71.5 % was obtained for 28.43 m3 m−2 d−1. At low HLR (11.37 m3 m−2 d−1), the treated effluent had the following average concentrations: sCOD of 29.5 mg l−1, BOD5 of 21.7 mg l−1 and TSS of 26.4 mg l−1. These experimental results were used with an empirical model to determine the media constant n and treatability factor K. The faecal coliforms and Escherichia coli detected in the treated effluent were less than 105 CFU/100 ml, which meet the national guidelines for wastewater reuse in irrigation. 2.4 Experimental set-up A transparent column made of polyvinyl chloride fed from the top was set-up for this study (Fig. 2). The biofilter col umn had a total height of 160 cm and consisted of a 16-cm diameter lower part filled with 100 cm of pozzolan, and a 22-cm diameter upper part filled with 20 cm of L. cylindri ca. The packed bed of L. cylindrica was constructed by ver tically inserting similar 20-cm long cylindrical shapes inside the column. The entire assembly should form a compact and compressed filter cartridge. The bed must be main tained submerged 5 cm below the water level during the experiments. As the wastewater flows through the biofilter, the impurities will accumulate over time on the surface of the top layer of filtration material. This accumulation will eventually clog the surface of the bed, the flow becomes slow and the biofilter will be put out of service. When this situation occurs, the L. cylindrica cartridge should be re moved, washed, and reused again. This solution should avoid costly backwashing sequences. The materials in the column were retained on a 10-mm thick perforated Plexiglas disk located at the bottom of the column, and another 4-mm thick perforated Plexiglas disk was inserted between the two layers to prevent mixing between them. An air compressor supplied two fine bubble air diffusers sampling point SW1 sampling point SW2 Fig. 1 – Overview of Stidia natural lagoon Fig. 1 – Overview of Stidia natural lagoon 2.2 Wastewater analyses Turbidity and conductivity were measured using a WTW turbo 550IR turbidity meter and a conductivity meter (Levibond SensoDirect Con200). For COD analysis, the closed reflux digestion and titrimetric method were used according to the Standard guidelines.22 The pH, DO and temperature were measured using a pH meter (model HI- 2210, Hanna instruments) and a portable dissolved oxygen meter (Hanna instruments, model HI-98193). TSS, faecal coliforms and Escherichia coli were determined after fil tration of the water samples through 0.45 µm pore size (47 mm diameter) cellulose membrane using a vacuum system according to the procedures outlined in APHA.22 1. Feed water tank 2. Feed pumps 3. Liquid flowmeter 4. Luffa cylindrica 5. Pozzolan 6. Sampling ports 7. Air compressor 8. Air flowmeter 9. Effluent tank 1 2 3 M 4 5 6 7 8 9 Fig. 2 – Schematic of biofiltration pilot M 2 Materials and methods Dried peeled fruit (sponge) of the L. cylindrica plant was used in this study as pre-treatment layer in the upper part of the biofilter because of its highly branched fibrous and light structure. The L. cylindrica columns were purchased from local market at low price; they were mature and dried. In the laboratory, each column was cut into cylindri cal shapes 20 cm long. They were then treated in a solu tion of 0.25 M NaOH for 12 h at room temperature to improve their mechanical properties and increase surface roughness of fibres.24 1 Introduction cylindrica was used as a carrier in an aerobic biofilm reactor for biodegrading hy drolysed polyacrylamide from artificial wastewater,19 and for treating effluent from fish ponds.20 Biofiltration has been developed precisely for tertiary treat ments and mainly for nitriﬁcation/denitriﬁcation process es.5–7 The material used in biofiltration must have a rough surface for good biofilm development and an effective particle diameter that must allow for an adequate void ratio to ensure a good wastewater flow through the filter bed. Natural porous volcanic rocks (pumice, pozzolan, This study focuses on the reuse of treated wastewater in drip irrigation by improving the physicochemical and mi crobiological quality parameters of Stidia lagoon effluent through biofiltration using local environmentally friendly materials. To our knowledge, no attempt has been made to upgrade the wastewater of Stidia lagoon. A layer of L. cylin drica (upper layer, 20 cm) in the biofiltration column was used as a support to promote biofilm formation, and at 602 M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 stones were extracted in different sizes, and those with a vesicular structure and low weight were selected on site. Once brought back to the laboratory, they were crushed and sieved to obtain different grain sizes. The effective di ameter was determined using international standard sieves (1.00 mm, 1.12 mm, 2.00 mm, 2.50 mm, and 3.15 mm). the same time prevent the rapid clogging of the pozzolan bed (100 cm, bottom layer). This, therefore, underlines the novelty in this study. 2.1 Location of Stidia natural lagoon The wastewater investigated in this study was collected from the natural lagoon near the city of Stidia in Mostaga nem, northwestern Algeria. It is located 10 m above sea lev el at latitude 35° 49ʹ 40ʺ N and longitude 0° 0ʹ 54ʺ W . The average air temperature in this area ranges from 10.5 °C in January to 37.5 °C in July (annual average temperature is 17.5 °C).21 As shown in Fig. 1, the different sampling sites of raw wastewater (RW) at the discharge zone just at the lagoon entrance are labelled RW1, RW2, and secondary wastewater (SW) following UV exposure at the opposite side of the entrance is indicated as SW1 and SW2. 2.7 Water retention measurements The water adsorption fraction of the medium, Fad can be calculated by the following equation: lc ad dp m F m = (6) (6) 3 Results and discussion where n is constant depending on filtration media, and K (mg sCOD/L)n d−1 is the treatability factor. 2.5 Experimental determination of removal efficiency and medium filter constants The amount of liquid captured in the material is the differ ence between the weight of the moist packing after drain age and the weight of the dry material packing. Based on previous models for trickling filters,28,29 an empir ical model for biofilters was developed by Mann et al.25 in which influent sCOD concentration was related to effluent sCOD concentration at bed height h for specific filter me dia: The water retention capacity, Rc, was calculated by Eq. (7): (7) (7) h 0 0 exp n S KAh S QS   = −     (1) (1) 2.3 Biofilter media preparation The volcanic rock used in this study as filter media was pozzolan, a locally available material collected from the mountain area of the Beni-Saf region in northwestern Al geria (35° 16ʹ 58.81ʺ N and 1° 24ʹ 24.99ʺ W). Pozzolan is a natural basaltic rock with 40–50 % of silica (SiO2).23 The Fig. 2 – Schematic of biofiltration pilot 603 w-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 603 The removal efficiency E was calculated with the following equation:30 The removal efficiency E was calculated with the following equation:30 positioned under each layer of material. At the beginning of the first experiments, the materials were washed with tap water before introduced into the filter column. ( ) h 0 % 1 100 S E S   = − ⋅     (3) The wastewater to be treated was pumped continuously with a feed pump from a 100-l storage tank to the top of the filtration column at influent flow rates in accordance with the selected HRTs. To monitor the flow of the influent, a Brooks flowmeter was connected to the feed pipe. The column was opened at the top to allow the evacuation of stale air. (3) 2.6 Hydraulic retention time The HRT is defined as the time that it takes for influent to ﬂow through the bed medium height of the column, and was calculated by Eq. (4): Since the removal of organic matter and suspended solids by biofiltration is due to biodegradation, an acclimation period is required for the biofilter to allow the microor ganisms present in the influent to attach and colonize the surface of the filter media, and to produce an adequate biofilm. Typical acclimation periods are from 2 to 4 weeks in closed loop depending on operating conditions.25–27 p Vå HRT Q = εp (4) (4) The packing bed void ratio εp can be determined using Eq. (5): In this study, the column was inoculated with biomass derived from an activated sludge plant treating municipal wastewater. The system was operated in closed circuit un der aeration for several days. The acclimation period took approximately 15 days with a flow rate of 1 l h−1 and dis continuous feedings. As the L. cylindrica has a complex in terconnected pore structure, a rapid change in its colour was observed, indicating the formation of a biofilm during this acclimation period. The experiments were performed continuously each day according to HRTs, and all tests took three months. For each run, sCOD and TSS of the inﬂuent and efﬂuent were measured in triplicate. p total volume solid packing volume total volume å − = εp (5) (5) 3.1 Lagoon wastewater characteristics In order to calculate n and K, Eq. (1) would be rewritten as: In order to calculate n and K, Eq. (1) would be rewritten as: Since the 1970s, the wastewater generated by the pop ulation of Stidia city (estimated at 12651 inhabitants in 2012)31 is discharged into a main collector that leads to the natural lagoon. The average rainfall and the wastewa ter quality parameters expressed as a minimum-maximum range, collected at the locations RW1 and RW2 (Fig. 1), are presented in Table 1. Due to the seasonal variations, the composition of the wastewater resulted in fluctuation of the physicochemical parameters (Table 1). ( ) exp mh = − (2) (2) where m is K/qS0 n and q equals Q/A. where m is K/qS0 n and q equals Q/A. Therefore, by plotting ln(Sh/S0) against h, the slope (−m) was obtained for different values of S0. From the plot of ln(mq) against ln(S0), the values of n and K were deter mined. M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 Table 1 – Stidia lagoon influent quality Period 2019–2020 Rainfall ⁄ mm year−1 pH BOD5 ⁄ mg l−1 tCOD ⁄ mg l−1 TSS ⁄ mg l−1 summer 23±5 6.3–8.2 109–283 228–594 115–167 winter 128±10 6.6–7.9 113–314 251–653 105–120 spring 156±30 6.5–8.1 105–306 214–482 118–147 The effluent from this lagoon failed to meet the nation al standards of reuse, based on the results of the analysis (Table 2). However, as this wastewater contains the major plant nutrients, it can be used for fertilization of the crops in this zone. The only problem is the over-fertilisation, which should be avoided by dilution of this wastewater with natural waters to control nutrients and heavy metal concentrations in the irrigation water. Table 1 – Stidia lagoon influent quality 3.2 Performances of the biofilter column Stidia lagoon has the advantage of being located in a re gion that has three seasons, based on average rainfall and temperature trends: summer (June-September), winter (October-January), and spring (February-May), which are characterised by low, short/moderate, and heavy rainfall, respectively.31 When comparing seasonal variations of the different parameters in Table 1, lower BOD5 and tCOD values were found in spring season due to the combined effect of rainfall and temperature elevation (18–34 °C). This phenomena stimulates heterotrophic bacteria to con sume large amounts of organic contaminants and DO.32 TSS concentrations were dependent on the season, with values ranging from 105 mg l−1 to 167 mg l−1 (Table 1). The physical characteristics of the pozzolan were deter mined in the laboratory and the results, as well as some calculated parameters, are presented in Table 3. Data are means ± standard deviation for each analysed parameter, deter mined on 30 samples for each water type. Table 3 – Physical properties of pozzolan Grain size ⁄ mm Effective diameter D10 ⁄ mm Uniformity coefficient U = D60/D10 ρs ⁄ kg m−3 εm ⁄ % εp ⁄ % 1.12–3.15 1.37 1.43 791.38 67.49 45.10 A total of 30 wastewater samples were collected during a period of three months, from May to July 2020 at the different locations indicated in Fig. 1. The results present ed in Table 2 show that there was substantial variability in physicochemical parameters with relatively large devi ation during the investigated period. The ratio of BOD5/ sCOD ≥ 0.63 indicated that the SW was suitable for bi ologically treatment, i.e., it was biodegradable without pretreatment.32,33 TSS concentrations in SW ranged from 70 mg l−1 in May to 110 mg l−1 in July. These monthly vari ations in TSS are related to the growth of algae, stimulated by the increase in temperature and solar radiation. The physical properties and chemical composition of L. cy lindrica depend on several factors, such as plant origin and weather conditions35. The L. cylindrica used in this study had the same core topology, with a compact structure and a density within the range of 52±1 kg m-3. The retention capacities of the filtration materials are pre sented in Table 4. The maximum retention for the pozzolan was 17%, while for the L. cylindrica it was 35%, indicating a significant hydro-expansion of this material. This particu larity was also observed by Wang et al.17 in the comparative study of L. cylindrica with plastic sponge. Table 3 – Physical properties of pozzolan 3 – sCOD removal profiles at different HLRs at S0 = 140 mg l−1 centration of 83.60 mg l−1 and the HLR maintained at 28.48 m3 m−2 d−1, the lowest TSS concentration obtained in the final effluent was approximately 23.80 mg l−1 and re moval efficiency was 71.5 %. It has been reported that for long operation, the fine particles agglomerate in the lower part of the filter bed, and that at a given loading rate, a medium with small grain size (D10 ˂ 1 mm) will give better removal but will have an effect on subsurface clogging.13,37 The values of the constants n and K were obtained from the plots of ln(mq) against ln (S0) at different HLRs (Fig. 5). The differences in values of K found from the experimental results depended on HLRs: as q increased, the value of K decreased. The treatability factor K will increase as the sCOD removal rate increases. Fig. 3 – sCOD removal profiles at different HLRs at S0 = 140 mg l−1 ln(S0) −2.50 −2.40 −2.30 −2.20 −2.10 −2.00 −1.90 −1.80 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 ln(mq) q1 = 11.37 m3 m−2 d−1 q2 = 17.06 m3 m−2 d−1 q3 = 22.72 m3 m−2 d−1 q4 = 28.43 m3 m−2 d−1 ln(mq4) = 0.347 ln(S0) + 3.656 R2 = 0.997 ln(mq3) = 0.409 ln(S0) + 36.76 R2 = 0.998 ln(mq2) = 0.493 ln(S0) + 3.741 R2 = 0.997 ln(mq1) = 0.570 ln(S0) + 3.789 R2 = 0.997 Fig. 5 – Relation between mq and influent sCOD concentration (S0) Fig. 4 illustrates the effect of HRT investigated (1–12 h) on the sCOD removal efficiency at different influent concen trations. An improvement in sCOD removal efficiency was observed with increasing HRT for all influent concentra tions. On the other hand, the removal efficiency decreased with increasing influent concentration. Fig. 4 illustrates the effect of HRT investigated (1–12 h) on the sCOD removal efficiency at different influent concen trations. An improvement in sCOD removal efficiency was observed with increasing HRT for all influent concentra tions. On the other hand, the removal efficiency decreased with increasing influent concentration. Table 3 – Physical properties of pozzolan Table 2 – Wastewater composition (main parameters) during the experimental period Parameter RW SW Algerian Standards34 pH-value/ – 8.2 ± 0.1 7.6 ± 0.4 6.5–8.5 turbidity ⁄ NTU 70.5 ± 1.1 30.8 ± 1.2 – conductivity ⁄ µS cm−1 2588.4 ± 7.5 2650.1 ± 6.3 3000.0 TSS ⁄ mg l−1 157.5 ± 21.9 95.8 ± 15.6 30.0 tCOD ⁄ mg l−1 263.2 ± 2.4 215.3 ± 3.5 – sCOD ⁄ mg l−1 185.1 ± 5.3 153.4 ± 6.1 90.0 BOD5 ⁄ mg l−1 109.1 ± 4.5 97.1 ± 3.4 30.0 total N ⁄ mg l−1 59.5 ± 2.3 46.8 ± 3.1 30.0 DO ⁄ mg l−1 1.3 ± 0.3 1.7 ± 0.2 – S A R ⁄ meq l−1 4.5 ± 0.1 3.1 ± 0.2 ˂ 3.0 Data are means ± standard deviation for each analysed parameter, deter Table 2 – Wastewater composition (main parameters) during the experimental period Table 4 – Retention capacities of filtration media- pozzolan and L. cylindrica Media filtration mdp ⁄ kg mlc ⁄ kg Fad ⁄ – Rlc ⁄ – pozzolan 1.12–3.15 ⁄ mm 11.72 1.28 0.11 0.17 L. cylindrica 0.47 0.78 1.68 0.35 Table 4 – Retention capacities of filtration media- pozzolan and L. cylindrica Media filtration mdp ⁄ kg mlc ⁄ kg Fad ⁄ – Rlc ⁄ – pozzolan 1.12–3.15 ⁄ mm 11.72 1.28 0.11 0.17 L. cylindrica 0.47 0.78 1.68 0.35 Fig. 3 shows the evolution of sCOD concentrations over the height of the bed media at different hydraulic loading rates. The sCOD removal efficiency was about 25 % for the 20-cm layer of L. cylindrica and over 50 % for 100 cm of pozzolan. It was observed that when influent sCOD concentration was maintained constant, the effluent sCOD concentra tion increased with increasing HLR. However, as the HLR increased, the contact time between the biofilm and the percolating liquid decreased, resulting in a decrease in the removal efficiency. 605 M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 bed height ⁄ m 0 0.2 0.4 0.6 0.8 1.0 1.2 q1 = 11.37 m3 m−2 d−1 q2 = 17.06 m3 m−2 d−1 q3 = 22.75 m3 m−2 d−1 q4 = 28.43 m3 m−2 d−1 20 40 60 80 100 120 140 160 180 Sh ⁄ mg l−1 Fig. Table 3 – Physical properties of pozzolan KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 606 Table 6 – Typical microbiological parameters of secondary and treated effluent Parameter SW Treated wastewater Standards34,36 E. coli / log CFU/100 ml 5.26 ± 0.60 4.12 ± 0.11 <5 for restricted irrigation <3 for unrestricted irrigation Faecal coliforms / log CFU/100 ml 5.64 ± 0.47 4.81 ± 0.79 Data are means ± standard deviation for each analysed parameter, deter mined on 16 samples for each water type. Significant removal of E. coli and faecal coliforms was achieved during the experimental period. The average val ues for E. coli and faecal coliforms in the treated wastewater were 1.30 ∙ 104 CFU/100 ml and 6.24 ∙ 104 CFU/100 ml, re spectively, corresponding to a removal efficiency of 85.2 % for faecal coliforms, and 92.7 % for E. coli. These reduc tions did not vary significantly with HLRs. 4 Conclusion Biofiltration was investigated as an alternative method for tertiary treatment of secondary effluent from Stidia natural lagoon. The biofilter filled with a 100-cm layer of pozzolan and a 20-cm upper layer of L. cylindrica was operated at different HLRs from May 2020 to July 2020. The removal efficiency of sCOD was greater than 78.9 %, depending on HLR, and a maximum TSS removal efficien cy of 71.5 % was achieved at 28.43 m3 m−2 d−1. Table 3 – Physical properties of pozzolan The low HLR resulted in an effluent with average TSS of 26.4 mg l−1, sCOD of 29 5 mg l−1 BOD of 21 7 mg l−1 and less than List of abbreviations and symbols Popis kratica i simbola HLR – hydraulic loading rate, m3 m−2 d−1 COD – chemical oxygen demand, mg l−1 sCOD – soluble chemical oxygen demand, mg l−1 tCOD – total chemical oxygen demand, mg l−1 BOD5 – biochemical oxygen demand over 5 days, mg l−1 DO – dissolved oxygen, mg l−1 TSS – total suspended solids, mg l−1 HRT – hydraulic retention time, h S0 – influent sCOD concentration, mg l−1 Sh – effluent sCOD concentration, mg l−1 Q – volumetric flow rate, m3 h−1 A – cross sectional area of the column, m2 q – hydraulic loading rate (Q/A), m3 m−2 d−1 V – volume of the column packed with bed medium, m3 Vlc – liquid holdup volume, m3 Vsp – solid packing volume, m3 mlc – amount of liquid captured in the material, kg mdp – weight of dry material packing, kg ρl – liquid density, kg m−3 ρs – particle density, kg m−3 εm – particle porosity, – SAR – sodium adsorption ratio, meq l−1 CFU – coliform faecal unit SW – secondary wastewater RW – raw wastewater Table 6 – Typical microbiological parameters of secondary and treated effluent Significant removal of E. coli and faecal coliforms was achieved during the experimental period. The average val ues for E. coli and faecal coliforms in the treated wastewater were 1.30 ∙ 104 CFU/100 ml and 6.24 ∙ 104 CFU/100 ml, re spectively, corresponding to a removal efficiency of 85.2 % for faecal coliforms, and 92.7 % for E. coli. These reduc tions did not vary significantly with HLRs. Significant removal of E. coli and faecal coliforms was achieved during the experimental period. The average val ues for E. coli and faecal coliforms in the treated wastewater were 1.30 ∙ 104 CFU/100 ml and 6.24 ∙ 104 CFU/100 ml, re spectively, corresponding to a removal efficiency of 85.2 % for faecal coliforms, and 92.7 % for E. coli. These reduc tions did not vary significantly with HLRs. 4 Conclusioni Biofiltration was investigated as an alternative method for tertiary treatment of secondary effluent from Stidia natural lagoon. The biofilter filled with a 100-cm layer of pozzolan and a 20-cm upper layer of L. cylindrica was operated at different HLRs from May 2020 to July 2020. The removal efficiency of sCOD was greater than 78.9 %, depending on HLR, and a maximum TSS removal efficien cy of 71.5 % was achieved at 28.43 m3 m−2 d−1. The low HLR resulted in an effluent with average TSS of 26.4 mg l−1, sCOD of 29.5 mg l−1, BOD5 of 21.7 mg l−1 and less than 5.0 log CFU/100 ml. RW – raw wastewater References Literatura Based on the results presented above, the biofiltration of the secondary effluent from the natural lagoon of Stidia using pozzolan and L. cylindrica can produce an effluent meeting the requirements of effluent standards for reuse. 1. A. Singh, A review of wastewater irrigation: Environmental implications, Resources, Conserv. Rec. 168 (2021) 105454, doi: https://doi.org/10.1016/j.resconrec.2021.105454. 1. A. Singh, A review of wastewater irrigation: Environmental implications, Resources, Conserv. Rec. 168 (2021) 105454, doi: https://doi.org/10.1016/j.resconrec.2021.105454. For further investigations, a similar experimental work using biofilters of larger dimensions operating in parallel would be the next step. Another investigation to be recom mended is the analysis of the physical stability of L. cylin drica fibres during the time it remains in activity as a filter medium. 2. K. K. Kesari, R. Soni, M. S. J. Qazi, P. Tripathi, J. A. Lal, N. K. Jha, M. H. Siddiqui, P. Kumar, V. Tripathi, J. Ruokolainen, Waste water Treatment and Reuse: a Review of its Applications and Health Implications, Water Air Soil Pollut. 232 (2021) 208, doi: https://doi.org/10.1007/s11270-021-05154-8. 3. S. Ofori, A. Puškácová, I. Ružicková, J. Wanner, Treated wastewater reuse for irrigation: Pros and Cons, Sci. Total Environ. 760 (2021) 144026, doi: https://doi.org/10.1016/j. scitotenv.2020.144026. Table 3 – Physical properties of pozzolan ln(mq) HRT ⁄ h 0 2 4 6 8 10 12 S0 = 100 mg l−1 S0 = 120 mg l−1 S0 = 140 mg l−1 S0 = 160 mg l−1 0 10 20 30 40 50 60 70 80 90 100 E ⁄ % Fig. 4 – Effect of HRT and influent concentration (S0) on the sCOD removal efficiency HRT ⁄ h 0 2 4 6 8 10 12 S0 = 100 mg l−1 S0 = 120 mg l−1 S0 = 140 mg l−1 S0 = 160 mg l−1 0 10 20 30 40 50 60 70 80 90 100 E ⁄ % Fig. 5 – Relation between mq and influent sCOD concentration (S0) Table 5 summarizes the values of n and K obtained in this study and compares them to the different values obtained by other researchers in previous studies. In this study, the K values obtained were in good agreement with the values determined in other studies. They are slightly higher than those of Lava, which is probably due to the difference in surface properties between the two materials. Fig. 4 – Effect of HRT and influent concentration (S0) on the sCOD removal efficiency The main microbiological parameters, Escherichia coli and faecal coliforms of SW and of treated wastewater are pre sented in Table 6. Despite the exposure of the wastewater to solar UV, the average values of E. coli and faecal coli forms in the SW samples remained above the limits recom mended by WHO.36 The maximum TSS removal was achieved when the bi ofilter was operated for a long time. When the biofilter was fed with an influent containing an average TSS con Table 5 – Comparison of n and K from derived models in the literature with the present results Bed medium Diameter ⁄ mm S0 ⁄ mg l−1 q ⁄ m3 m−2 d−1 K ⁄ (mg l−1)n d−1 n ⁄ – Reference sunken media 2.30–2.70 80–210 9.23–18.15 33.0 0.92 Mann and Stephenson25 floating media 55.0 1.13 lava 3.00–5.00 100–201 9.17–22.01 36.5–44.0 0.21–0.71 Wang et al.26 expanded clay 31.5–38.0 0.21–0.66 pozzolan 1.12–3.15 80–160 11.37–28.43 38.7–44.2 0.35–0.57 this study Table 5 – Comparison of n and K from derived models in the literature with the present results M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 606 M. ACKNOWLEDGEMENTS American Public Health Association, Washington, DC, 2005. 9. B. Kocadagistan, E. Kocadagistan, N. Topcu, N. Demircio glu, Wastewater treatment with upflow anaerobic fixed-bed and suspended aerobic reactor equipped with a membrane unit, Proc. Biochem. 40 (2005) 177–182, doi: https://doi. org/10.1016/j.procbio.2003.11.055. 23. M. Mouli, H. Khelafi, Performance characteristics of light weight aggregate concrete containing natural pozzolan, Buil. Env. 43 (2008) 31–36, doi: https://doi.org/10.1016/j.build env.2006.11.038. 24. C. A. Adeyanju, S. Ogunniyi, J. O. Ighalo, A. G. Adeniyi, S. A. Abdulkareem, A review on luffa fibres and their polymer composites, J. Mater. Sci. 56 (2021) 2797–2813, doi: https:// doi.org/10.1007/s1085l3-020-05432l-6. 10. S. M. Borghrei, M. Sharbatmaleki, P. Pourrezaie, G. Borghei, Kinetics of organic removal in fixed-bed aerobic biological reactor, Biores. Technol. 99 (2008) 1118–1124, doi: https:// doi.org/10.1016/j.biortech.2007.02.037. 25. A. T. Mann, T. Stephenson, Modelling biological aerated filters for wastewater treatment, Wat. 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APHA, Standard Methods for the Examination of Water and Wastewater, 21st Ed. 35. J. C. Ogbonna, Y.-C. L. Liu, Y.-K. Liu, H. Tanaka, Loofa (Luffa p jl g Malika Khelladi,a,b* Meriem Abaidia,a Senouci Boulerial,a Khalida Bekrentchir,a Abdellah Benhamou a i Abdelkader Debab a p jl g Malika Khelladi,a,b* Meriem Abaidia,a Senouci Boulerial,a Khalida Bekrentchir,a Abdellah Benhamou a i Abdelkader Debab a U sklopu sveobuhvatne procjene tehnika naknadnog uklanjanja kemijskih i mikrobioloških one- čišćenja iz efluenta laguna, projektiran je aerirani biofiltar. Glavni cilj ovog istraživanja bio je pro- cijeniti učinak pucolana i biljke Luffa cylindrica kao jeftinih punila za naprednu obradu efluenta prirodne lagune Stidia. Aerirani biofiltar radi s brzinom hidrauličkog opterećenja (HLR) od 11,37 do 28,43 m3 m−2 d−1 i omjerom protoka zrak/tekućina od 3 : 1. Eksperimenti provedeni na pilotu pokazuju da smanjenje KPK (izražene s obzirom na otopljene tvari) varira ovisno o HLR-u i koncen- traciji otpadne vode na ulazu u biofiltar. U ovom istraživanju postignute su učinkovitost smanjenja KPK iznad 78,9 % ovisno o HLR-u, a maksimalna učinkovitost uklanjanja ukupnih suspendiranih čestica (TSS) od 71,5 % dobivena je pri 28,43 m3 m−2 d−1. Pri niskom HLR-u (11,37 m3 m−2 d−1), KPK, BPK5 i TSS obrađene otpadne vode iznosili su 29,5 mg l−1, 21,7 mg l−1 odnosno 26,4 mg l−1. Ti eksperimentalni rezultati korišteni su u empirijskom modelu da bi se odredila konstanta medija n i faktor obrade K. CFU vrijednosti fekalnih koliforma i bakterije Escherichia coli u obrađenoj otpadnoj vodi bile su ispod 105 CFU/100 ml što zadovoljava nacionalne smjernice za upotrebu tih voda u navodnjavanju zemljišta. M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. 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JORA, Arrêté interministériel du 8 Safar1433 correspondant au 02 janvier 2012 fixant les spécifications des eaux usées épurées utilisées à des fins d’irrigation, Legislative Decree, 41 (2012). 18. L. Zhang, K. Sun, N. Hu, Degradation of organic matter from domestic wastewater with loofah sponge biofilm reac tor, Wat. Sci. Technol. 65 (2012) 190–195, doi: https://doi. org/10.2166/wst.2011.846. 35. J. C. Ogbonna, Y.-C. L. Liu, Y.-K. Liu, H. Tanaka, Loofa (Luffa M. KHELLADI et al.: Low-cost Packing Materials in an Aerated Biofilter for Lagoon Effluent..., Kem. Ind. 71 (9-10) (2022) 601−608 cylindrica) sponge as a Carrier for Microbial cell immobiliza tion, J. Ferment. Bioeng. 78 (1994) 437–445, doi: https:// doi.org/10.1016/0922-338X(94)90043-4. (1989), World Health Organization, Geneva, Swit 37. G. Hua, J. Kong, Y. Ji, M. Li, Influence of clogging ing processes on flow patterns in vertical flow co cylindrica) sponge as a Carrier for Microbial cell immobiliza tion, J. Ferment. 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https://openalex.org/W2772644101	https://europepmc.org/articles/pmc5748089?pdf=render	English	null	Detailed Histologic Evaluation of Eosinophilic Esophagitis in Pediatric Patients Presenting with Dysphagia or Abdominal Pain and Comparison of the Histology between the Two Groups	Canadian journal of gastroenterology & hepatology	2,017	cc-by	4,663	Hindawi Canadian Journal of Gastroenterology and Hepatology Volume 2017, Article ID 3709254, 6 pages https://doi.org/10.1155/2017/3709254 Hindawi Canadian Journal of Gastroenterology and Hepatology Volume 2017, Article ID 3709254, 6 pages https://doi.org/10.1155/2017/3709254 Hindawi Canadian Journal of Gastroenterology and Hepatology Volume 2017, Article ID 3709254, 6 pages https://doi.org/10.1155/2017/3709254 Correspondence should be addressed to Thirumazhisai S. Gunasekaran; tsg@tummydocs.com Correspondence should be addressed to Thirumazhisai S. Gunasekaran; tsg@tummydocs.com Received 28 January 2017; Revised 7 June 2017; Accepted 31 July 2017; Published 17 December 2017 Academic Editor: Salvatore Cucchiara Academic Editor: Salvatore Cucchiara Copyright © 2017 Thirumazhisai S. Gunasekaran et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. EoE in children presents with four main symptoms. Most common symptoms exhibited by our clinic population are dysphagia (D) and abdominal pain (AP). Despite similar treatments, we found in an earlier study that the outcomes between these two groups were different. Therefore, we investigated if there exist any histological differences between these groups that could further our knowledge of EoE. Aim. To compare esophageal histology in detail, apart from the eosinophil count, between EoE-D and EoE-AP. Method. Biopsies of patients with EoE-D and EoE-AP were reevaluated for 10 additional histological criteria, in addition to the eosinophil count. Results. Both groups had 67 patients; peak mean eosinophil was 33.9 and 31.55 for EoE-D and EoE-AP (𝑝< 0.05). Eosinophilic microabscesses, superficial layering of eosinophils, and epithelial desquamation were twice as common and significant in EoE-D group than EoE-AP. Eosinophil distribution around rete pegs was also significantly higher in EoE-D group. The remaining criteria were numerically higher in EoE-D, but not significant, with the exception of rete peg elongation. Conclusion. EoE-D patients have significantly higher eosinophils compared to EoE-AP, and the level of inflammation as seen from eosinophil microabscesses, superficial layering, desquamation, and the distribution around rete pegs is significantly higher. Thirumazhisai S. Gunasekaran,1 Christopher Chu,2 Nemencio Ronquillo Jr.,2 Rohini Chennuri,2 Brian Adley,3 Kristina Borgen,3 Alan Schwartz,2 Robert Newberry,4 and James Berman1 Thirumazhisai S. Gunasekaran,1 Christopher Chu,2 Nemencio Ronquillo Jr.,2 Rohini Chennuri,2 Brian Adley,3 Kristina Borgen,3 Alan Schwartz,2 Robert Newberry,4 and James Berman1 1Advocate Children’s Hospital, Loyola Medical Center and University of Illinois, 1775 Dempster Street, Park Ridge, IL 60068, USA 2University of Illinois, Chicago, IL, USA 3Advocate Children’s Hospital, 1775 Dempster Street, Park Ridge, IL 60068, USA 4Rady Children’s Hospital, San Diego, CA, USA Thirumazhisai S. Gunasekaran,1 Christopher Chu,2 Nemencio Ronquillo Jr.,2 Rohini Chennuri,2 Brian Adley,3 Kristina Borgen,3 Alan Schwartz,2 Robert Newberry,4 and James Berman1 1 1Advocate Children’s Hospital, Loyola Medical Center and University of Illinois, 1775 Dempster Street, Park Ridge, IL 60068, USA 2University of Illinois, Chicago, IL, USA y f g 3Advocate Children’s Hospital, 1775 Dempster Street, Park Ridge, IL 60068, USA 3. Method Based on these differences in presentation and our previous study [3] raising doubt if EoE patients presenting with abdominal pain without dysphagia are truly EoE or are part of a heterogeneous group of diseases with EoE as one of the diagnosis, we raised the issue that further studies are required. As a follow-up on our previous study [3], we sought to look for additional histological criteria within the same group of patients to see if this will give us a better understanding on the pathogenesis of EoE and or diagnostic histological features between EoE-D and EoE-AP patients. 3.2. Patient Population and Features. We queried a previously created secure Access (Microsoft, Redmond, WA) database to the following data points: symptoms, physical findings, complete blood count, serum electrolytes, urea, creatinine, liver function profile, sedimentation rate, urinalysis, and endoscopic findings (furrows, white spots/exudates, concen- tric rings, and friability/crepe paper appearance, entered as absent, 0, or present, 1). The histology of the duodenum, stomach, distal, and mid esophagus were captured. Each patient had two to three biopsies from the descending duodenum, antrum, distal esophagus (2-3 cm above Z- line), and mid esophagus. These features, except detailed histologic evaluation of the degree/stage of eosinophil-rich inflammation, were published in our previous study [3]. The demographics and presenting symptoms of the EoE-D and EoE-AP patients are given in Table 1 and visual EGD findings are in Table 2. The Institutional Review Board, Advocate Children’s Hospital, Park Ridge, IL, approved this study. In our previous study [3], apart from the number of eosinophils in the biopsies, we looked at four histologic features retrospectively: eosinophilic microabscesses, basal epithelial hyperplasia, papillomatosis, and spongiosis within the two groups. Eosinophilic microabscesses were signifi- cantly more frequent in the EoE- D (𝑝< 0.001), while the remaining histologic features were not statistically different between the two groups. In the current study, within the same group of patients, we relooked at an in-depth and detailed reexamination of the histopathology of these two groups of EoE patients, for additional evidence to support our hypothesis. 3.3. Histopathologic Analysis. All biopsy specimens were fixed in formalin and stained with hematoxylin and eosin (H&E). H&E stained slides from the two EoE subgroups that met the inclusion criteria were retrieved and reviewed. The initial biopsies leading up to the diagnosis of EoE were considered for extensive review. 3. Method not lead to symptomatic improvement or when there is a dissociation between histological and symptomatic improve- ment, it is worth relooking not only at adherence to diet and/or medications but also managing the EoE-AP group with methods successful in FAP patients.” 3.1. Inclusion and Exclusion Criteria. In this retrospective study all pediatric patients seen at the Eosinophilic Esophagi- tis Clinic, Advocate Children’s Hospital, Park Ridge, IL, over 2 and 1/2 years (1/2010–6/2012) with eosinophilic esophagitis were included in the study. The diagnosis of EoE was made by the following criteria: symptom(s) of esophageal dysfunction as mentioned above and esophageal biopsy showing 15 or more eosinophils per high power field (HPF) on ×400 light microscopy. These patients were pretreated with proton pump inhibitors (PPIs) or had a negative esophageal pH study and had no increased infiltration of eosinophils in the antral or duodenal biopsies [1, 2]. From the four subgroups of EoE patients, EoE-D and EoE-AP patients (based on the dominant presenting symptom) were chosen for comparison, as these were the larger groups. Patients with abdominal pain had central or diffuse abdominal pain and no dysphagia. Patients with celiac disease, Crohn’s disease, or achalasia were excluded. While there remains ongoing research to seek a reliable biomarker for the diagnosis of EoE, we sought to see if there are additional histological evidence to confirm the diagnosis and/or to differentiate EoE from esophageal eosinophilia. Consensus Statement recommends [2] that, apart from the eosinophil count, additional histological features and immunohistologic stains can be obtained for a more accurate diagnosis [2]. Based on this recommendation, further studies have included additional histological criteria like eosinophilic abscesses, degranulation, and basal zone hyperplasia or biomarker staining for eosinophil degranulation products like major basic protein and interleukins. But none of these features clinch the diagnosis of EoE. In addition, the diverse presentation of symptoms in pediatric patients adds further challenges for an accurate diagnosis of EoE. Adult patients with EoE have dysphagia as the primary presenting symptom, while in pediatrics it is variable: feeding difficulty, abdominal pain, GERD-like symptoms, and dysphagia [3]. Moreover children presenting with abdominal pain to a clinic, in general, have FAP compared to those presenting with dysphagia having EoE. Canadian Journal of Gastroenterology and Hepatology 2 3. Method A careful review of all biopsies and fields was done and the area which had the most dense eosinophilic inflammation, at HPF on ×400 light microscopy, was taken for analysis. Five pathologists through review of current literature and standard pathology texts [1, 2] concluded to analyze ten histological criteria, in addition to the eosinophil count. These criteria evaluated the degree/stage of eosinophil-rich inflammation and are listed in Table 3. 1. Introduction groups had contrasting outcomes. EoE- D had a favorable outcome compared to EoE- AP. Most of the EoE studies in adult patients, where dysphagia is the main symptom, have a favorable outcome with treatment. Based on these variable outcomes, in our previous study, we compared the clinical features of EoE-AP to patients with functional abdominal pain (FAP) with discriminant score and cluster analysis. The result showed that the clinical features were similar, between functional abdominal pain and EoE-AP groups, and only differed in the presence of eosinophils in the esophagus [3]. Since there is no biomarker for EoE, we concluded from this study [3] that “. . .if optimal diet or pharmacotherapy does Based on the Consensus guidelines [1, 2], it is our practice that patients seen at the Eosinophilic Esophagitis Clinic, Advocate Children’s Hospital, Park Ridge, IL, are sub- grouped, depending on the dominant presenting symptom as EoE–dysphagia (EoE-D), EoE–abdominal pain (EoE-AP), EoE–GERD/vomiting, and EoE–failure to thrive/feeding dif- ficulty (EoE–FTT). In a previous study [3], we showed that the two most common subtypes of EoE seen in our EoE Clinic are EoE-D and EoE-AP and these two groups had different clinical, endoscopic, and histopathologic features. The most striking feature was that, with standard treatments, the two 2. Aim Our hypothesis is that there are differences in the histology of the esophagus between EoE-D and EoE-AP patients. The aim is to compare the diagnostic esophageal histology in detail, of patients with EoE-D and EoE-AP, to see if they are similar or different and if they are different, whether these differences contribute to the understanding of the pathogenesis of the two groups. Esophageal biopsies from distal and mid sections of the esophagus were scanned at ×100 power. Intraepithelial eosinophils were quantified at ×400 from the area with maximum eosinophilic density. Only intact eosinophils were considered in determining the peak eosinophil count. The Canadian Journal of Gastroenterology and Hepatology 3 Table 1: Demographics and Symptoms of Patients with Eosinophilic Esophagitis. EoE-D 𝑛= 67 (%) EoE-AP 𝑛= 67 (%) 𝑝value Male 59 (88.1) 52 (77.6) 0.11 Mean age, yr (range) 11.8 (3–17) 9.45 (2–17) 0.001 Presenting symptomsa Dysphagia 67 (100) 1b (0.01) <0.001 Abdominal pain 8 (11.9) 66 (98.5) <0.001 Nausea 11 (16.4) 31 (46.2) <0.001 Vomiting 12 (17.9) 11 (16.4) 0.82 Regurgitation 5 (7.5) 7 (10.4) 0.55 Heartburn 7 (10.4) 6 (9.0) 0.77 aSome patients had more than one presenting symptom. bPatient initially presented with dysphagia; however, subsequent visits showed abdominal pain as the predominant symptom. Table 1: Demographics and Symptoms of Patients with Eosinophilic Esophagitis. Table 2: Endoscopic findings of patients with EoE. EGD number (%) EoE-D (𝑛= 67)% EoE-AP (𝑛= 67)% p value Linear furrows 55 (82.1) 34 (50.7) <0.001 White exudates 34 (50.7) 17 (25.4) 0.003 Linear furrows and white Exudates 32 (47.8) 10 (14.9) <0.001 Concentric rings 7 (10.4) 3 (4.5) 0.19 Tears/crepe paper appearance 4 (6.0) 0 (0) 0.12 Table 2: Endoscopic findings of patients with EoE. Table 2: Endoscopic findings of patients with EoE. Table 3: Histological criteria evaluating the degree/stage of eosinophil-rich inflammation. (1) Eosinophilic microabscesses (MAB) Four or more eosinophils clustered together. (2) Superficial layering of eosinophils (SLE) Superficial infiltrate of eosinophils (>1 eosinophil at ×400). (3) Epithelial desquamation (ED) Degenerative (i.e., necrotic, pyknotic-dense, and dark nuclei because of nuclear shrinkage due to irreversible condensation of chromatin in the nucleus of a cell undergoing necrosis or apoptosis, dyskeratotic-deep pink cytoplasm due to abnormal keratinization occurring prematurely within individual cells, or groups of cells below the stratum granulosum) squamous epithelial cells. (4) Basal zone hyperplasia (BZH) Basal cells occupying more than 20% of total mucosal thickness. 4. Results 4.1. Patients and Design of Histology Evaluation. During the study period of 2 and 1/2 years we had a total of 73 patients in the two groups and from these 67 patients from each group were selected for further analysis, who fulfilled the diagnostic criteria of EoE and had evaluable biopsies. Six patients who did not fulfill these criteria were excluded. Within the current study groups about 60 patients in each group were part of the previous study [3]. Four pathologists, from the group of five, by reviewing a set of slides, obtained a 98% agreement rate on all set histologic criteria for sample of normal (10 biopsies) and sample of EoE esophageal biopsies (10 biopsies) prior to histologic evaluation of the patients for the current study. Pathologists were blinded to clinical information and EoE subgroups, to limit bias during the slide review process. Figure 1: Comparison of the degree/stage of eosinophil-rich inflam- mation on the biopsies of EoE-D and EoE-AP patients. MAB: microabscess, SLE: superficial layering, ED: epithelial desquama- tion, BZH: basal zone hyperplasia, RPE: rete pegs elongation, SP: spongiosis or increased intracellular space, FB: lamina propria fibro- sis, DFE: diffuse or focal distribution, E-Dist: distribution around rete pegs, and DE: degranulation. Fibrosis (FB) was evaluable in 57 (EoE-D) and 54 (EoE-AP) patients and was positive in 41 (71.9%) and 37 (68.5%), respectively, 𝑝value 0.85 (Chi-square test on 1 df = 0.03, 𝑝= 0.85). ∗Statistically significant. Canadian Journal of Gastroenterology and Hepatology 4 Table 4: Eosinophil counts on the esophageal biopsies. Table 4: Eosinophil counts on the esophageal biopsies. EoE-D mean (sd) 𝑁= 67 EoE-AP mean (sd) 𝑁= 67 𝑝value Peak eosinophil count (mean) 33.91 (5.78) 31.55 (4.96) 0.013 Distal esophagus 33.61 (5.85) 31.46 (5.11) 0.025 Mid esophagus 27.04 (5.89) 24.73 (5.89) 0.015 Mean of distal and mid 30.33 (5.36) 28.10 (4.78) 0.012 In both groups the distal biopsy had higher eosinophils than the midesophagus, except two patients in EoE-D and one patient in EoE-AP group where the mid esophagus had a higher esophageal count. MAB EoE-D EoE-AP SLE ED BZH RPE SP FB DFE E-Dist DE 0.567 0.448 0.507 0.97 0.97 0.925 0.612 0.821 0.925 0.716 0.299 0.194 0.254 0.866 0.985 0.851 0.552 0.776 0.761 0.642 ∗ ∗ ∗ ∗ 0 0.2 0.4 0.6 0.8 1 1.2 Prevalence of histological features slide with the highest peak eosinophil count was then further evaluated in detail for the ten criteria described earlier. 3.4. Statistical Analysis. Data were entered into a secure Microsoft Access Database and statistical analysis was done using SPSS version 20. A 𝑝value < 0.05 was accepted as statistically significant. 3.4. Statistical Analysis. Data were entered into a secure Microsoft Access Database and statistical analysis was done using SPSS version 20. A 𝑝value < 0.05 was accepted as statistically significant. 2. Aim (5) Rete peg elongation (RPE) Rete peg elongation that reaches at least 2/3 of total mucosal thickness. (6) Spongiosis (SP) Edema or dilated intercellular spaces between epithelial cells. (7) Subepithelial fibrosis or lamina propria fibrosis (FB) Evaluated if lamina propria was present in the specimen; collagen fibrils are densely packed and individual collagen fibrils cannot be distinguished. (8) Degree of involvement of eosinophils (DFE) Focal or diffuse; focal is defined as when eosinophils are localized to one fragment of the biopsy while diffuse is when eosinophils are found >1 fragment of the biopsy. (9) Pattern of distribution of eosinophils if present (E-Dist) Eosinophils confined to or around rete pegs (peripapillary), diffusely distributed, or superficially distributed. (10) Eosinophilic degranulation (DE) Presence of free eosinophil granules. Table 3: Histological criteria evaluating the degree/stage of eosinophil-rich inflammation. Canadian Journal of Gastroenterology and Hepatology 5. Discussion 4.2. Esophageal Histology. The eosinophil counts for both groups are given in Table 4. EoE-D group had higher eosinophils than EoE-AP and was significant and in both groups the distal biopsy had higher eosinophils than the midesophagus. The detailed histological findings for each of the two groups are shown in Figure 1. Three findings, in particular eosinophilic microabscesses (MAB), superficial layering of eosinophils (SLE), and epithelial desquamation (DE), are noteworthy. These three findings were about two times more common in EoE-D patients than EoE-AP patients and statistically significant. Pattern of eosinophil distribution around the rete pegs also was significantly higher in the EoE- D group. The remaining findings except rete peg elongation were numerically greater in EoE-D patients than EoE-AP, but not statistically significant. In regard to subepithelial/lamina propria fibrosis (FB), this finding was only evaluable if patient samples contained lamina propria in their biopsy and 57 (85%) in EoE-D and 54 (80%) in EoE- AP were evaluable and fibrosis was observed numerically more in EoE-D but was not significant. Diagnosis of EoE is based on the presence of 15 or more eosinophils [2, 3], the “hallmark” of histological diagnosis, but an exact number required for a definitive diagnosis of EoE continues to be a moving target. As an alternative, some studies support a different method: 20–24 eosinophils on a single biopsy or 15 or more eosinophils on biopsies from two levels for the diagnosis [4, 5]. Since a definitive number of eosinophils, either to make an accurate diagnosis of EoE and/or to exclude other causes of eosinophilic esophageal infiltration, including GERD, is not clear, histological diag- nosis based on the number of eosinophils continues to be in dispute. Based on a study on the increasing incidence of EoE, a recent editorial titled “Eosinophilic Esophagitis- Emerging Epidemic or Misdiagnosed Malady?” questions the accuracy of the diagnosis of EoE, for similar reasons [6, 7]. Hence there is a prevailing concern whether clinicians are accurately diagnosing EoE or are lured and misled by the “15 eosinophils” and fail to correlate the eosinophils with symp- tom(s) of esophageal dysfunction. In addition 2011 Consensus Statement recommends that, apart from the eosinophil count, 5 Canadian Journal of Gastroenterology and Hepatology additional histological features, like the ones described in this study, and immunohistologic stains be obtained for more accurate diagnosis [2]. 5. Discussion Whether the results would change if all biopsies were evaluable for fibrosis is left to speculation.i g Though our study evaluated the eosinophilic inflamma- tion at one point, to better understand the differentiating histological features of EoE, we reviewed “the evolution” of the histological changes in EoE. With antigen insult to the esophagus, eosinophilic inflammation begins in the peri- papillary area, an area closest to the vasculature. Superficial and diffuse distribution represents later stage of epithelial infiltration. Basal zone hyperplasia and lengthening of lamina propria papillae are secondary changes to antigen insult and may be severe with increased duration of the insult [10]. Another process contributing to inflammation is degran- ulation of eosinophils which correlates with intraepithelial eosinophilia or degree of mucosal inflammation. Degranu- lation leads to cytotoxin and cytokine release, resulting in the desquamation or degeneration of cells, and mobiliza- tion of more eosinophils. Odze classified the histological findings of EoE as major: increased eosinophils (greater than 15), eosinophilic microabscesses, superficial layering of the eosinophils, surface sloughing of squamous cells, and degranulation of eosinophils, and minor: “marked” basal cell hyperplasia, lengthening of the lamina propria papillae, increased intraepithelial lymphocytes and mast cells, increased intracellular edema, and increased lamina propria fibrosis [10].i it Ours is the first to systematically compare 10 additional histological criteria, apart from the eosinophil count, in EoE- D and EoE-AP at diagnosis. Limitations of our study are as follows. Although the biopsies were reviewed prospec- tively, patient data is retrospective and did not correlate the eosinophil counts with the rest of the inflammatory findings. Not all biopsies were evaluable for subepithelial fibrosis and they did not include immunohistological staining. These are opportunities for future studies. In conclusion, and in accordance with Consensus and Collins’ recommendations [1, 2, 11], pathologists should describe additional above listed inflammatory features, in addition to the eosinophil count, when providing histology reports on patients with EoE. Four features, eosinophil microabscesses, superficial layering, epithelial desquamation, and distribution of eosinophils around rete pegs, are sig- nificantly and consistently seen more often in EoE-D than in EoE-AP patients. While these additional features explain pathogenesis of dysphagia in patients with EoE-D, their absence in EoE-AP suggests that EoE-AP may be a more heterogeneous group of diseases including EoE, GERD, and other causes of esophageal eosinophilia. 5. Discussion Therefore the focus shifts to whether additional inflammatory histological criteria would increase the accuracy of the diagnosis of EoE and differentiate and/or eliminate other causes of esophageal eosinophilic inflam- mation. Another challenge for the clinician is to associate the presenting symptom(s) of esophageal dysfunction, the second criterion, to authenticate the diagnosis of EoE. When patients present with dysphagia, a symptom of esophageal dysfunction, with or without regurgitation/heartburn, the challenge is to differentiate GERD from EoE. When the presenting symptom is abdominal pain without dysphagia, it is more challenging because dysphagia is a cardinal symptom of esophageal dysfunction, whereas abdominal pain does not hold a similar position [8, 9]. This makes the accurate diagnosis of EoE in the EoE-AP group even more difficult, as there are multiple causes for central abdominal pain. So it raises the question: in the absence of esophageal dysfunction, are the histological findings in EoE-AP consistent with a diagnosis of EoE?hl GERD [12–19]. Our study came to the same conclusion and validates that EoE-D is clearly EoE with these features. On the contrary since these histological features are not significantly seen in EoE-AP, it suggests that EoE-AP may be more of a heterogeneous group of diseases and that should be the subject of future studies.fl j Does above difference in the inflammation in EoE-D and EoE-AP help understand the pathogenesis of the primary symptom(s), dysphagia and central abdominal pain, of the EoE groups? Our study showed that the epithelium of the esophagus in EoE-D has more histological inflammation in comparison to EoE-AP, which is also supported by the increased endoscopic findings of exudates and furrows in the EoE-D group. This epithelial inflammation, as well as release of cytokines or chemokines from the degranulation of eosinophils, results in increased smooth muscle reactivity and dysmotility of the esophagus resulting in dysphagia, as seen in adults with inflammatory type of EoE with dysphagia [20–22]. Eosinophilic inflammation in the duodenum was shown to be associated with abdominal pain via the release of cytokines [21, 22], but it is not clear if inflammation in a proximal organ, the esophagus, can lead to abdominal pain at a distal site. Lamina propria fibrosis, seen in about 80% of the biopsies, was not significantly different in the two groups. We were expecting that the fibrosis would be significantly more in EoE-D and we were surprised that the results did not support it. Conflicts of Interest The authors declare that they have no conflicts of interest. 5. Discussion i Odze’s classification of EoE histology and other studies [4, 5, 10] support the fact that, apart from the eosinophil count, additional features, eosinophilic microabscesses, superficial layering, desquamation, and degranulation are predomi- nantly seen in EoE compared to GERD. Hence these criteria are considered additional features to diagnose EoE and to exclude GERD, another disease which manifests with esophageal eosinophilic inflammation. Collins et al. have recently shown that these additional histological criteria are useful in the diagnosis and monitoring of EoE patients [11]. The three features, eosinophilic microabscesses, superficial layering, and desquamation, which our study showed to be significant, are recurring findings and are seen significantly more in EoE and have been used to differentiate it from References [1] C. Liacouras, G. T. Furuta, and I. Hirano, “Eosinophilic esophagitis: updated consensus recommendations for children and adults,” The Journal of Allergy and Clinical Immunology, vol. 128, no. 1, pp. 1–41, 2011. [17] S. Mueller, D. Neureiter, T. Aigner, and M. Stolte, “Comparison of histological parameters for the diagnosis of eosinophilic oesophagitis versus gastro-oesophageal reflux disease on oesophageal biopsy material,” Histopathology, vol. 53, no. 6, pp. 676–684, 2008. [2] G. T. Furuta, C. A. Liacouras, and M. H. Collins, “Eosinophilic esophagitis in children and adults: a systematic review and consensus recommendations for diagnosis and treatment,” Gastroenterology, vol. 133, no. 4, pp. 1342–1363, 2007. [18] F. Chang and S. Anderson, “Clinical and pathological features of eosinophilic esophagitis: a review,” Pathology, vol. 40, pp. 3– 8, 2008. [3] T. Gunasekaran, G. Prabhakar, A. Schwartz, K. Gorla, S. Gupta, and J. Berman, “Eosinophilic esophagitis in children and adolescents with abdominal pain: comparison with eoe- dysphagia and functional abdominal pain,” Canadian Journal of Gastroenterology and Hepatology, vol. 2016, pp. 1–7, 2016. [19] J. P. Li-Kim-Moy, V. Tobias, A. S. Day, S. Leach, and D. A. Lemberg, “Esophageal subepithelial fibrosis and hyalinization are features of eosinophilic esophagitis,” Journal of Pediatric Gastroenterology and Nutrition, vol. 52, no. 2, pp. 147–153, 2011. [4] M. H. Collins, “Histopathologic features of eosinophilic esopha gitis,” Gastrointestinal Endoscopy Clinics of North America, vol. 18, no. 1, pp. 59–71, 2008. [20] A. Straumann, M. Bauer, B. Fischer, K. Blaser, and H. Simon, “Idiopathic eosinophilic esophagitis is associated with a TH 2- type allergic inflammatory response,” The Journal of Allergy and Clinical Immunology, vol. 108, no. 6, pp. 954–961, 2001. [5] M. H. Collins, “Histopathology of eosinophilic esophagitis,” Digestive Diseases, vol. 32, no. 1-2, pp. 68–73, 2014. [21] M. E. Rothenberg and M. B. Cohen, “An eosinophil hypothesis for functional dyspepsia,” Clinical Gastroenterology and Hepa- tology, vol. 5, no. 10, pp. 1147-1148, 2007. [6] A. J. Gawron and I. Hirano, “Eosinophilic esophagitis— Emerging epidemic or misdiagnosed malady?” Clinical Gas- troenterology and Hepatology, vol. 12, no. 4, pp. 597-598, 2014.h [22] E. S. Dellon, O. Speck, K. Woodward et al., “Markers of eosinophilic inflammation for diagnosis of eosinophilic esophagitis and proton pump inhibitor–responsive esophageal eosinophilia: a prospective study,” Clinical Gastroenterology and Hepatology, vol. 12, no. 12, pp. 2015–2022, 2014. [7] E. S. Dellon, E. T. Jensen, and C. F. Canadian Journal of Gastroenterology and Hepatology 6 and administrative work. Christopher Chu was responsible for acquisition of data, administrative work, and review of manuscript. Nemencio Ronquillo Jr., Rohini Chennuri, Brian Adley, and Kristina Borgen performed study design and interpretation of the histology. Alan Schwartz was involved in statistical analysis and interpretation of data. Robert Newberry critically revised the manuscript for important intellectual content. James Berman critically revised the manuscript for important intellectual content and is co- senior author. [13] S. S. Aceves, R. O. Newbury, R. Dohil, J. Schwimmer, and J. F. Bastian, “Distinguishing eosinophilic esophagitis in pediatric patients: clinical, endoscopic and histologic features of an emerging disorder,” Journal of Clinical Gastroenterology, vol. 41, no. 3, pp. 252–256, 2007. [14] S. Lee, W. B. de Boer, A. Naran et al., “More than just counting eosinophils: proximal oesophageal involvement and subep- ithelial sclerosis are major diagnostic criteria for eosinophilic oesophagitis,” Journal of Clinical Pathology, vol. 63, no. 7, pp. 644–647, 2010. [15] S. J. Steiner, K. M. Kernek, and J. F. Fitzgerald, “Severity of basal cell hyperplasia differs in reflux versus eosinophilic esophagitis,” Journal of Pediatric Gastroenterology and Nutrition, vol. 42, no. 5, pp. 506–509, 2006. Acknowledgments The authors thank Henry Mangurten, M. D., for editorial revision of manuscript. [16] R. Kirsch, R. Bokhary, M. A. Marcon, and E. Cutz, “Acti- vated mucosal mast cells differentiate eosinophilic (allergic) esophagitis from gastroesophageal reflux disease,” Journal of Pediatric Gastroenterology and Nutrition, vol. 44, no. 1, pp. 20– 26, 2007. Authors’ Contributions Thirumazhisai S. Gunasekaran was involved in study concept and design, drafting of the manuscript, study supervision, References Martin, “The prevalence of eosinophilic esophagitis in the United States,” Clinical Gastroen- terology and Hepatology, vol. 12, no. 4, pp. 589–596, 2014. [8] L. A. Mahajan and B. Kaplan, “Chronic abdominal pain of childhood and adolescence,” in Pediatric Gastrointestinal and Liver Disease, R. W. Wyllie and J. S. Hyams, Eds., pp. 111–125, Elsevier, Philadelphia, Pa, USA, 3rd edition, 2006. [9] J. J. Monagas, Hyman P. E., and Dysphagia, “Dysphagia,” in Pediatric Gastroenterology, Hepatology and Nutrition, J. M. Sondheimer, Ed., vol. 9 of 14, p. 13, NASPGHN, Philadelphia, Pa, USA, 1st edition, 2011. [10] R. D. Odze, “Pathology of eosinophilic esophagitis: what the clinician needs to know,” American Journal of Gastroenterology, vol. 104, no. 2, pp. 485–490, 2009. [11] M. H. Collins, L. J. Martin, E. S. Alexander et al., “Newly devel- oped and validated eosinophilic esophagitis histology scoring system and evidence that it outperforms peak eosinophil count for disease diagnosis and monitoring,” Diseases of the Esophagus, vol. 30, no. 3, pp. 1–8, 2016.i [12] J. R. Parfitt, J. C. Gregor, N. G. Suskin, H. A. Jawa, and D. K. Driman, “Eosinophilic esophagitis in adults: distinguishing features from gastroesophageal reflux disease: a study of 41 patients,” Modern Pathology, vol. 19, no. 1, pp. 90–96, 2005.
https://openalex.org/W4372311909	https://eprints.gla.ac.uk/298209/1/298209.pdf	English	null	Subjective and objective sleep and circadian parameters as predictors of depression-related outcomes: A machine learning approach in UK Biobank	Journal of affective disorders	2,023	cc-by	12,117	Subjective and objective sleep and circadian parameters as predictors of depression-related outcomes: A machine learning approach in UK Biobank Subjective and objective sleep and circadian parameters as predictors of depression-related outcomes: A machine learning approach in UK Biobank Laura M. Lyall a,b,, Natasha Sangha a,b, Xingxing Zhu a, Donald M. Lyall a, Joey Ward a, Rona J. Strawbridge a,c,d, Breda Cullen a, Daniel J. Smith a,b a School of Health and Wellbeing, University of Glasgow, Glasgow, UK b Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK c Health Data Research, UK d Cardiovascular Medicine Unit, Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden a School of Health and Wellbeing, University of Glasgow, Glasgow, UK b Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK c Health Data Research, UK d Cardiovascular Medicine Unit, Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden A B S T R A C T Keywords: Depression Sleep Circadian rhythms Rest-activity Inactivity Postnatal depression Keywords: Depression Sleep Circadian rhythms Rest-activity Inactivity Postnatal depression Background: Sleep and circadian disruption are associated with depression onset and severity, but it is unclear which features (e.g., sleep duration, chronotype) are important and whether they can identify individuals showing poorer outcomes. Methods: Within a subset of the UK Biobank with actigraphy and mental health data (n = 64,353), penalised regression identified the most useful of 51 sleep/rest-activity predictors of depression-related outcomes; including case-control (Major Depression (MD) vs. controls; postnatal depression vs. controls) and within-case comparisons (severe vs. moderate MD; early vs. later onset, atypical vs. typical symptoms; comorbid anxiety; suicidality). Best models (of lasso, ridge, and elastic net) were selected based on Area Under the Curve (AUC). Results: For MD vs. controls (n(MD) = 24,229; n(control) = 40,124), lasso AUC was 0.68, 95 % confidence interval (CI) 0.67–0.69. Discrimination was reasonable for atypical vs. typical symptoms (n(atypical) = 958; n(typical) = 18,722; ridge: AUC 0.74, 95 % CI 0.71–0.77) but poor for remaining models (AUCs 0.59–0.67). Key predictors across most models included: difficulty getting up, insomnia symptoms, snoring, actigraphy-measured daytime inactivity and lower morning activity (~8 am). In a distinct subset (n = 310,718), the number of these factors shown was associated with all depression outcomes. Limitations: Analyses were cross-sectional and in middle-/older aged adults: comparison with longitudinal in vestigations and younger cohorts is necessary. Discussion: Sleep and circadian measures alone provided poor to moderate discrimination of depression out comes, but several characteristics were identified that may be clinically useful. Future work should assess these features alongside broader sociodemographic, lifestyle and genetic features. Corresponding author at: School of Health and Wellbeing, Clarice Pears Building, 90 Byres Road, University of Glasgow, Glasgow G12 8TB, UK. E-mail address: laura.lyall@glasgow.ac.uk (L.M. Lyall). Journal of Affective Disorders 335 (2023) 83–94 Journal of Affective Disorders 335 (2023) 83–94 Contents lists available at ScienceDirect Available online 6 May 2023 0165-0327/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.jad.2023.04.138 Received 28 July 2022; Received in revised form 25 April 2023; Accepted 29 April 2023 2.1. Participants Between 2006 and 2010, over 502,000 UK residents aged 37–73 years were recruited to the UK Biobank. They attended one of 22 assessment centres around the UK and completed sociodemographic, health, lifestyle, mood, cognitive and physical assessments and ques tionnaires. In 2013–2015, a subset of ~100,000 participants provided up to 7 days of wrist-worn actigraphy data as part of an activity moni toring study (Doherty et al., 2017). In 2016–2017, ~160,000 partici pants completed an online mental health questionnaire (MHQ). See Fig. 1 in Conroy et al. (2019) for overview of timeline. Here, main an alyses are restricted to participants who provided actigraphy data that passed quality control (QC), and for whom sufficient mental health data enabled categorisation into the case or control groups described below (max. n = 64,353). Descriptive sociodemographic and sleep/circadian characteristics for the MD vs. control comparison are provided in Table 1, and sample sizes and descriptive statistics for the other MD dimensions in Supplementary Tables S1–S7. All participants who joined UK Biobank provided written, informed consent, and generic ethical approval was provided by the North West Multi-centre Research Ethics Committee (ref: 21/NW/0157). Analyses were performed using UK Biobank application number 54772 (PI Lyall). 2.2.2. Subjective sleep/chronotype characteristics During the baseline assessment, participants were asked to report on several sleep characteristics. For each, those responding ‘do not know’ or ‘prefer not to answer’ were coded as missing. Typical sleep duration was reported as the estimated number of hours sleep in each 24 h, including naps (#1160). We excluded responses below 2 h or above 18 h, and for descriptive tables, responses <7 h were coded as ‘short’ sleep duration (n = 14,280), 7 or 8 h as ‘normal’ sleep duration (n = 45,984), and ≥9 h as ‘long’ sleep (n = 4062), following American Academy of Sleep Medicine guidelines (Ramar et al., 2021). In main analyses, mean subjective sleep duration was included as a continuous variable. i We examined associations of subjective and objective measures of sleep and circadian rest-activity characteristics with depression-related outcomes in UK Biobank, in penalised regression machine learning models. Outcomes included case vs. control comparisons, i.e., MD cases vs. controls and postnatal depression (PND) vs. controls, and five sub dimensions of MD reflecting greater severity (Nguyen et al., 2022): i) atypical vs. typical symptoms; ii) presence vs. absence of comorbid anxiety; iii) severe vs. moderate MD; iv) early vs. later onset; v) MD with vs. without suicidal thoughts/behaviour. Penalised regression was used to identify the most important sleep/circadian predictors of each outcome, and to assess prediction performance. Participants reported how easy they find it to get up in the morning (#1170): we grouped responses into high (‘not at all easy’/‘not very easy’) vs. low (‘fairly easy’ and ‘very easy’) difficulty (Sambou et al., 2022). Chronotype (#1180) was coded as definite morning, intermedi ate (comprising ‘more morning than evening person’ and ‘more evening than morning person’ responses) and definite evening. For napping (#1190), insomnia symptoms (#1200; trouble falling asleep at night or waking during the night) and daytime dozing (#1220; unintentionally falling asleep during the day), those responding ‘usually’ were con trasted with those who responded ‘never/rarely’ or ‘sometimes’ (Kyle et al., 2017; Sambou et al., 2022). For snoring (#1210), ‘yes’ and ‘no’ responses were contrasted. 1. Introduction patients reporting sleep problems (Riemann et al., 2001). Prospective longitudinal studies have supported bidirectional associations whereby sleep disturbances often precede and are predictive of later depression (Alvaro et al., 2013; Zhai et al., 2015; Zhang et al., 2022). Insomnia sufferers, for example, may be twice as likely to report later depression compared to those without insomnia (Baglioni et al., 2011). Bidirec tional associations have been observed from adolescence (Alvaro et al., 2013) to older age (Bao et al., 2017), and within depression sufferers, sleep disturbances are associated with worse depression outcomes, including greater severity, risk of suicidality, and comorbidity with anxiety (Alvaro et al., 2013; Franzen and Buysse, 2008; Wang et al., Depression has a severe impact on quality of life and daily func tioning, particularly in the case of severe episodes, or where depression co-occurs alongside anxiety disorders or suicidal thoughts (Johnston et al., 2019). It is important to identify modifiable risk factors to target interventions aimed at preventing onset of depression, and among pa tients, to prevent more severe episodes and symptoms. Sleep and circadian rhythm disturbances are important risk factors for depressive disorders. Sleep disturbances have commonly been viewed as a core symptom of major depression (MD), with up to 90 % of L.M. Lyall et al. Journal of Affective Disorders 335 (2023) 83–94 2.2.3. Sleep disorders Participants with a record of a sleep disorder (ICD-10 G47) or non- organic sleep disorder (ICD-10 F51) from linked primary care, hospital admission or death records, and/or self-report (#130921; #131061) were included in a sleep disorder category and compared with those who had no record/report of a sleep disorder. 2.2. Predictors Disturbances to circadian rhythms (physiological and behavioural changes that recur over a ~24-hour period) are also associated with depression (Lyall et al., 2018; Walker et al., 2020). Environmental fac tors causing temporal circadian misalignment, e.g., nightshift work, jetlag, artificial light at night, have been linked to lower mood, and if occurring over a long period, depression onset (Angerer et al., 2017; Bedrosian and Nelson, 2017). Evening chronotype, circadian rhythm sleep disorders or lower amplitude rest-activity rhythms are also asso ciated with MD onset (Byrne et al., 2019; Taylor and Hasler, 2018). Stabilising rhythms, e.g. via light-based chronotherapy, is associated with improvements in depressive symptoms (Perera et al., 2016). As with sleep, a bidirectional relationship with depression is likely, and within depression, circadian disruption is associated with greater severity, with atypical depression, and with suicidality (Carpenter et al., 2021; Courtet and Oli´e, 2012; Rumble et al., 2020). 2.2.1. Sociodemographic covariates 2.2.1. Sociodemographic covariates Only age, sex, and Townsend deprivation score were included in penalised regression models (alongside sleep/rest-activity variables), but group differences in several other sociodemographic characteristics are summarised in Tables 1 and S1–S7. Data on age (UK Biobank data field #21003), sex (#31), ethnicity (#21000) and educational attain ment (#6138) were provided at the baseline assessment. Given rela tively small numbers from some ethnic backgrounds, ethnicity in descriptive tables was coded as ‘white’ and ‘non-white’, in line with previous publications (Lyall et al., 2018). For education, participants were categorised into those reporting a college/university degree vs. no degree. Townsend deprivation scores (#189) were derived based on postcode of residence: more negative scores reflect greater affluence (Townsend, 1987). Baseline self-report measures were used for smoking status (#20116) and frequency of alcohol intake (#1558). Body-mass index (BMI) was calculated from height and weight measurements at the baseline assessment (weight/(height)2). During the baseline assess ment, participants were asked 12 questions from the Eysenck Person ality Questionnaire Revised (Short Form) Neuroticism Scale (Eysenck and Eysenck, 1993), and a composite neuroticism score (range 0–12) was derived (0 if all 12 questions were answered negatively). Many characteristics of sleep and circadian function have shown association with depression-related outcomes, including sleep efficiency and duration, circadian amplitude and timing. As these are rarely considered together in multivariable models, however, it is unclear if associations reflect intercorrelation between measures or whether spe cific features of sleep and/or circadian function may be key risk factors. It is also unclear whether objective actigraphy-derived measures provide better predictors of depression and more severe outcomes within depression patients than subjective reports. Greater understanding of which features of sleep and circadian function are most predictive of depression outcomes could improve understanding of mechanisms and inform the development of more targeted interventions. 2.2.2. Subjective sleep/chronotype characteristics 2.2.4. Objective sleep/rest-activity variables In 2013–2014, over 100,000 UK Biobank participants (of ~240,000 invited) agreed to take part in an actigraphy-based physical activity monitoring study. They were asked to wear an AX3 triaxial Axivity accelerometer on their dominant wrist for 7 days, while continuing normal activities. Physical activity data were pre-processed by the UK Biobank accelerometer expert working group (Doherty et al., 2017). Among derived measures were the overall acceleration average over the data collection period (milli-gravity units; #90012), standard deviation (SD) of overall acceleration (#90013), and average activity for each hour of the day, across all available days (#90027 - #90050). We also 84 Journal of Affective Disorders 335 (2023) 83–94 L.M. Lyall et al. Table 1 Descriptive sociodemographic and sleep/rest-activity characteristics for broad MD vs. controls. Table 1 Descriptive sociodemographic and sleep/rest-activity characteristics for broad MD vs. controls. 2.2.4. Objective sleep/rest-activity variables Controls Broad MD t/χ p N Mean/N SD/% N Mean/N SD/% Sociodemographic characteristics Age 40,124 62.41 7.96 24,229 60.75 7.68 26.01 <0.001 Sex (N, %) 40,124 24,229 2780.48 <0.001 Female 18,714 46.64 16,475 68.00 Male 21,410 53.36 7754 32.00 Townsend score 40,090 −1.91 2.72 24,189 −1.47 2.90 −19.27 <0.001 Ethnicity (N, %) 39,985 24,154 46.09 <0.001 Non-white 1464 3.66 646 2.67 White 38,521 96.34 23,508 97.33 Education (N, %) 39,946 24,132 20.16 <0.001 No degree 23,110 57.85 13,524 56.04 Degree 16,836 42.15 10,608 43.96 BMI 40,045 26.59 4.24 24,170 27.08 4.97 −13.24 <0.001 Neuroticism score 34,098 2.79 2.64 19,984 5.42 3.29 −101.68 <0.001 Smoking status (N, %) 40,006 24,181 351.17 <0.001 Never 23,920 59.79 12,810 52.98 Former 13,711 34.27 9284 38.39 Current 2375 5.94 2087 8.63 Frequency of alcohol consumption (N, %) 40,091 24,217 396.48 <0.001 Never 2063 5.15 1579 6.52 Occasional 7257 18.10 5768 23.82 Regular 21,249 53.00 11,667 48.18 Daily 9522 23.75 5203 21.48 Season actigraph worn (N, %) 40,124 24,229 12.36 0.01 Spring 8904 22.19 5438 22.44 Summer 11,076 27.60 6795 28.04 Autumn 11,246 28.03 6908 28.51 Winter 8898 22.18 5088 21.00 Subjective sleep characteristics (baseline) Subjective sleep duration (continuous) 40,035 7.18 0.93 24,142 7.15 1.07 4.95 <0.001 Subjective sleep duration (categorised) (N, %) 40,102 24,224 316.06 <0.001 Normal (7-8 h) 29,627 73.88 16,357 67.52 Short (<7 h) 8267 20.61 6013 24.82 Long (≥9 h) 2208 5.51 1854 7.65 Difficulty getting up (N, %) 39,920 24,076 1885.95 <0.001 Easy 35,134 88.01 17,985 74.70 Not easy 4786 11.99 6091 25.30 Napping (N, %) 40,092 24,223 12.54 <0.001 No 38,467 95.95 23,100 95.36 Yes 1625 4.05 1123 4.64 Insomnia symptoms (N, %) 40,074 24,217 1079.16 <0.001 No 31,254 77.99 16,031 66.20 Yes 8820 22.01 8186 33.80 Snoring (N, %) 38,066 22,344 21.28 <0.001 No 23,998 63.04 14,504 64.91 Yes 14,068 36.96 7840 35.09 Daytime dozing (N, %) 40,031 24,190 118.41 <0.001 No 39,324 98.23 23,444 96.92 Yes 707 1.77 746 3.08 Chronotype (N, %) 35,532 21,938 242.83 <0.001 Morning person 9603 27.03 5323 24.26 Intermediate 23,203 65.30 14,127 64.40 Evening person 2726 7.67 2488 11.34 Health records Sleep disorder diagnosis (N, %) 40,124 24,229 435.94 <0.001 No 39,364 98.11 23,070 95.22 Yes 760 1.89 1159 4.78 Actigraphy variables (excluding hourly averages) Overall acceleration average 40,124 28.29 8.38 24,229 27.54 8.03 11.11 <0.001 Acceleration SD 40,124 56.49 22.06 24,229 53.83 19.44 15.51 <0.001 M10 time 39,959 13.65 1.22 24,119 13.79 1.24 −13.94 <0.001 L5 time 40,012 27.29 1.05 24,149 27.38 1.11 −10.52 <0.001 Sleep midpoint 40,028 26.98 0.91 24,165 27.01 0.95 −4.78 <0.001 Sleep duration (h) 40,028 7.25 0.90 24,165 7.31 0.90 −8.13 <0.001 Sleep duration SD 39,807 0.92 0.57 24,036 0.97 0.58 −10.67 <0.001 Sleep efficiency 40,028 0.76 0.07 24,165 0.76 0.07 −5.24 <0.001 Duration sustained inactivity bouts (daytime) 40,028 0.95 0.66 24,165 1.01 0.72 −10.99 <0.001 Number of nocturnal sleep episodes 40,028 17.25 3.65 24,165 17.30 3.67 −1.74 0.08 Time in bed (h) 40,028 9.67 0.98 24,165 9.71 0.98 −4.47 <0.001 and sleep/rest-activity characteristics for broad MD vs. 2.3.2. Broad major depression (MD) group Using the training data for each outcome, logistic regression lasso, ridge and elastic net penalised regression models were estimated, in addition to a base logistic regression model. Three basic sociodemo graphic variables; age, sex (not included in PND models) and Townsend score were forced into each penalised regression model (i.e., their co efficients were not shrunk to zero), alongside 51 sleep/rest-activity predictors (each level of categorical variables is counted as a separate feature). Optimal α values for elastic net, and λ for each of lasso, ridge, and elastic net models, were selected using grid search with nested 10- fold cross-validation (CV) to identify the parameters minimising the CV function: CV was conducted for 128 candidate λ values, and for elastic net, for 9 α values: 0.1–0.9 in increments of 0.1. For lasso, CV-based λ selection was also compared with the adaptive lasso and plug-in methods. Out-of-sample (test data) prediction performance was compared for the three lasso models, elastic net and ridge, and the base logistic regression, and for both penalised and post-selection co efficients. The best-fitting model was selected based on the highest deviance ratio and Receiver Operating Characteristic (ROC) Area Under the Curve (AUC). In the case of equal deviance ratio/AUC, the most parsimonious model with the fewest features was selected. A broad MD group consisted of participants who met at least one of the following MD criteria: a) lifetime history of MD or current MD based on MHQ (see Davis et al., 2020); b) ICD-10 code for depressive mood disorder (F32/F33/F34/F38/F39); c) probable MD based on baseline questions, using Smith et al. (2013) criteria; d) self-reported depression during nurse-led interview (#20002) or MHQ (#20544). All partici pants in depression sub-groups met at least one of the above broad MD criteria. The control group comprised participants who did not meet any of the above criteria, and additionally: a) did not report having seen a general practitioner (GP; #2090) or psychiatrist (#2100) for ‘nerves, anxiety, tension or depression’, b) did not report during the MHQ a prolonged (≥2 weeks) period of low mood (#20441) or anhedonia (#20446); c) did not report antidepressant use (see Supplementary Table S5 in Nguyen et al., 2022 for list). For the broad MD comparison, n(MD) = 24,229; n(control) = 40,124 (after exclusion of the cases/controls below). 2.3.1. Mental health exclusions from case and control groups 2.3.1. Mental health exclusions from case and control groups Depression-related outcome variables were largely based on a subset of depression subtypes described by Nguyen et al. (2022). Excluded from all case and control groups were any participants with a diagnosis of schizophrenia, schizoaffective disorder, or bipolar disorder, identified through self-report (#20002), during the MHQ (#20544), or through ICD-10 codes (F20-F29, F30/F31). Participants self-reporting use of antipsychotic medication or lithium (see Nguyen et al.'s Supplementary Table S5 for medication list) were excluded. 2.4. Analysis The following steps were applied to each of the depression outcomes described above. Analyses were conducted in Stata (v16.1); missing data imputation was conducted in R (v4.1.3). Participants were excluded if any of the following exclusion criteria were met: a) their actigraphy data collection period overlapped with a daylight savings change (#90018); b) they did not have ≥72 hour data, with data in each one-hour period of the day (#90015); c) data was flagged by UK Biobank as not well calibrated (#90016), or as unreliable due to unexpectedly small or large size (#90002), or calibration was not performed using the participant's own data (#90017). Main analyses were restricted to participants with actigraphy data passing the above QC measures (n = 103,670), and who provided sufficient mental health data for categorisation into one or more of the depression/control groups described below (largest sample size = 64,353). Data for each outcome variable were split into training and test datasets, with a 75/25 split, balancing for age (at time of actigraphy) tertile, sex, season of actigraphy data collection, and the relevant outcome. For training data, continuous variables were standardised to have mean 0 and SD 1: test data were standardised using the mean and SD of the training sample. Separately for training and test data, missing data for predictors were imputed using missForest in R v.4.1.3, an iterative random forest method well-suited to data consisting of both categorical and continuous variables (Stekhoven and Buhlmann, 2012). The number of trees per forest was set to 20. Penalised regression models were conducted using the imputed data. For comparison, complete cases analyses were conducted and are re ported in Supplementary Table S16. 2.2.4. Objective sleep/rest-activity variables control Descriptive sociodemographic and sleep/rest-activity characteristics for broad MD vs. controls 85 L.M. Lyall et al. Journal of Affective Disorders 335 (2023) 83–94 suicidality. Inclusion criteria and sample sizes (following actigraphy QC) are described in Table 2. used sleep/rest-activity variables derived by Jones et al. (2019): M10 time - the mean midpoint (in hours past midnight) of the most active 10 h; L5 time - the mean midpoint of the least active 5 h; sleep midpoint time; mean daily night sleep duration; sleep duration SD; mean sleep efficiency (proportion of time in bed spent asleep); mean duration of sustained inactivity bouts during the daytime (‘SIBD’); mean number of nocturnal sleep episodes per night (i.e. number of nocturnal awaken ings); mean time in bed. 2.3. Depression-related outcome variables Least absolute shrinkage and selection operator (lasso), ridge and elastic net are penalised regression techniques used to select which of a large number of covariates are useful predictors of the outcome and should be included in the model (Fan et al., 2015; Zou and Hastie, 2005). Regression coefficients are regularised towards zero by selecting a tun ing parameter, λ, which determines a penalty term. In lasso, the penalty term, L1-norm, can force coefficients making only a minor contribution to the model to zero, performing variable selection. Ridge regression applies an L2-norm penalty, shrinking coefficients towards zero, but all remain in the resulting model. Elastic net incorporates both lasso and ridge penalty terms, performing variable selection while allowing in clusion of correlated predictors. Above broad MD criteria, and during MHQ reported both hypersomnia (#20534) and weight gain (#20536) during their worst episode of depression. Within-case comparisons Atypical vs. typical MD Atypical Postnatal depression (PND) PND MD onset ≥ 1 year after data collection Broad MD after data collection 2.3.2. Broad major depression (MD) group To examine performance of this model for new-onset depression, the above model trained and tested on the broad lifetime history of MD vs. control contrast was also tested on a subset of MD cases (vs. controls) where the first occurrence of a depressive episode was estimated as at least 1 year after all data collection was complete: the 1-year interval was imposed to reduce likelihood of reverse causality (Brunner et al., 2014). There were 321 new-onset MD cases, and a random sample of 530 controls were selected (and removed) from the above broad MD control group (i.e., an equivalent case/control split to the broad MD/ control group, ~37 %/63 %) (Shimonovich et al., 2021). The other case- control outcome was PND vs. female controls, and within-case depres sion outcomes were a) MD cases with vs. without atypical symptoms (based on reversed neurovegetative symptoms: hypersomnia and weight gain during depressive episode; see Brailean et al., 2020); b) MD with vs. without comorbid anxiety disorder; c) severe vs. moderate MD; d) early (≤29 years) vs. later (>40 years) onset MD; e) MD with vs. without For each outcome, test data performance is summarised via AUC, accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the point on the ROC curve corre sponding to Youden's index (Fluss et al., 2005) (Table 3). Youden's index is calculated from: J = sensitivity + specificity −1 and corresponds to the maximum height of the ROC curve from the chance line. Youden's index is a common method of finding the optimal trade-off between sensitivity and specificity and therefore represents overall diagnostic performance (Fluss et al., 2005). In a clinical context however, it can be of use to assign greater weight to sensitivity to reduce false negatives (Wu et al., 2021). For comparison, in supplementary material we have also provided summaries of discrimination performance using a weighted Youden's index, where sensitivity is weighted higher (0.55) 86 Journal of Affective Disorders 335 (2023) 83–94 L.M. Lyall et al. than specificity (0.45) (Li et al., 2013). 3. Results 3.1. Sociodemographic and sleep/circadian characteristics by group Unadjusted group comparisons for sociodemographic and sleep/rest- Table 2 Description of inclusion criteria and sample size for each case/control group. 2.3.2. Broad major depression (MD) group Group Description of inclusion criteria N Case-control comparisons Broad MD Broad MD Meets at least one of the following criteria: a) Lifetime history of MD or current MD based on MHQ (based on Composite International Diagnostic Interview – Short Form) b) ICD-10 code for depressive mood disorder (F32/F33/F34/F38/F39): UKB first occurrences variables c) probable MD based on baseline mental health questions d) Self-reported depression at baseline interview or during MHQ (Individuals with a first record of MD occurring at least 1 year after actigraphy data collection were excluded from this group.) 24,229 Controls None of the above MD criteria, and: a) Did not report having seen a general practitioner or psychiatrist for ‘nerves, anxiety, tension or depression’ b) Did not report during MHQ a period of at least 2 weeks of low mood or anhedonia c) Did not report use of an antidepressant (see ref. Nguyen et al. (2022) for list) Controls randomly selected to form the control group for MD cases occurring onset ≥ 1 year after data collection were also excluded from the broad MD control group. 40,124 MD onset ≥ 1 year after data collection Broad MD after data collection Meets above broad MD criteria, and first occurrence of MD (based on ICD-10 first occurrences variables for F32: #130894; F33: #130896; F34: #130898; F38: #130900; F39: #130902) estimated at least 1 year after collection of all data (i.e., ≥1 year after actigraphy data collection). 321 Controls A random sample of controls from broad MD control group (then excluded from the former control group), n selected to match broad MD case/control split, i.e., 37%/63% 530 Postnatal depression (PND) PND Among women, those who had given birth (#2734) and met at least one of the following criteria were placed into a PND group: a) self-reported postnatal depression at the baseline interview (#20002) b) had an ICD-10 record of a mental health disorder related to the postpartum period (#130925) c) reported during the MHQ that their worst depressive episode occurred soon after giving birth or was suggested to be postnatal depression (#20445) 1,440 Female controls Women who had had at least one live birth (#2734) and did not meet broad MD or above PND criteria, did not report help-seeking from a GP/psychiatrist for anxiety or depression and did not report use of antidepressants 15,249 Within-case comparisons Atypical vs. 2.3.2. Broad major depression (MD) group typical MD Atypical Above broad MD criteria, and during MHQ reported both hypersomnia (#20534) and weight gain (#20536) during their worst episode of depression. 958 Table 2 (continued) Group Description of inclusion criteria N Typical Broad MD criteria and did not report either hypersomnia or weight gain during worst episode of depression. 18,722 Comorbid anxiety MD with comorbid anxiety Broad MD criteria, and at least one of the following criteria: a) self-reported a diagnosis of anxiety or panic attacks at the baseline nurse-led interview (#20002) b) reported during the MHQ that they had been diagnosed with social anxiety/social phobia, panic attacks, anxiety, nerves, or generalised anxiety disorder (GAD) (#20544) c) ICD-10 record of an anxiety disorder (F40/F41) from linked health records/ self-report (#130905; #130907) d) met criteria for lifetime history of GAD based on MHQ 9,461 MD without comorbid anxiety Broad MD criteria, but none of the above anxiety disorder criteria. 11,121 Depression severity Severe MD Met criteria for probable MD based on baseline questions (broad MD criterion c above), i.e., reported at least two episodes of feeling anhedonic or depressed for at least 2 weeks. To meet ‘severe’ criteria, participants additionally reported having seen a psychiatrist for ‘nerves, anxiety or depression’. 1,596 Moderate MD Probable MD criteria from baseline questionnaire as above but reported having seen a GP but not a psychiatrist for ‘nerves, anxiety or depression’. 3,131 Early onset Early onset The self-reported age at onset of first episode of depression (#20433 from MHQ) was split into octiles (after removing missing values, and values < 3) for those participants meeting broad MD criteria and with usable actigraphy data. Age at onset within the first three octiles (≤29 years) was defined as ‘early onset’ depression. 7,583 Later onset As above, age at onset in the last three octiles was defined as ‘later onset’ depression. than specificity (0.45) (Li et al., 2013). 2.3.2. Broad major depression (MD) group 6,690 Suicidality MD with suicidality Met broad MD criteria, and met at least one of the following criteria: a) self-reported deliberate self-harm or sui cide attempt at the baseline interview (#20002) b) Reported that during the 2 weeks preceding the MHQ that they had had suicidal thoughts ‘more than half the days’ or ‘nearly every day’ (#20513) c) Reported during the MHQ that they had attempted suicide (#20483) 1289 MD without suicidality Met broad MD criteria, and during MHQ reported that they had not had recent suicidal thoughts (#20513) and did not self-report deliberate self-harm or suicide attempt at baseline interview (#20002). 18,100 Table 2 (continued) Table 2 Table 2 than specificity (0.45) (Li et al., 2013). Table 3 Table 3 Summary of discrimination performance for the optimal model (lasso, elastic net, ridge) for each depression-related outcome. Performance metrics (and 95 % con fidence intervals) are for the point on the Receiver Operating Characteristic (ROC) curve corresponding to Youden's Index. Outcome Optimal model Youden's Index AUC Accuracy Sensitivity Specificity PPV NPV Case-control comparisons Broad MD vs. controls Lasso: post- selection 0.26 0.68 (0.67–0.68) 62.85 (62.10–63.60) 64.18 (62.96–65.39) 62.04 (61.08–62.99) 50.60 (49.48–51.73) 74.08 (73.13–75.02) MD onset after actigraphy Lasso: post- selection 0.27 –0.70) 68.16 (64.91–71.28) 44.86 (39.33–50.48) 82.26 (78.74–85.42) 60.50 (53.98–66.76) 71.13 (67.36–74.69) PND Lasso: penalised 0.24 0.67 (0.64–0.70) 65.82 (64.36–67.26) 57.77 (52.53–62.87) 66.60 (65.07–68.10) 14.30 (12.55–16.18) 94.24 (93.29–95.09) Within-case comparisons Atypical vs. typical MD Ridge post- selection 0.38 0.74 (0.71–0.77) 67.76 (66.43–69.07) 70.70 (64.12–76.69) 67.63 (66.27–68.97) 9.13 (7.79–10.62) 98.05 (97.51–98.50) Comorbid anxiety Ridge: penalised 0.13 0.59 (0.57–0.60) 55.35 (53.98–56.71) 68.56 (66.64–70.43) 44.17 (42.32–46.04) 50.96 (49.21–52.71) 62.41 (60.23–64.55) Depression severity Elastic net: post- selection 0.15 0.58 (0.55–0.62) 61.55 (58.71–64.33) 45.06 (40.08–50.12) 69.79 (66.45–72.97) 42.69 (37.89–47.59) 71.78 (68.46–74.94) Early onset Ridge: penalised 0.19 0.64 (0.62–0.65) 60.67 (59.05–62.28) 76.12 (74.13–78.03) 43.26 (40.87–45.67) 60.19 (58.20–62.16) 61.65 (58.80–64.44) Suicidality Ridge: penalised 0.20 0.63 (0.60–0.66) 68.12 (66.79–69.43) 50.48 (44.81–56.13) 69.35 (67.98–70.69) 10.30 (8.83–11.92) 95.26 (94.48–95.96) AUC = Area Under the (ROC) Curve; NPV = negative predictive value; PPV = positive predictive value. Optimal model refers to the model with the highest AUC/ deviance ratio for each outcome. Summary of discrimination performance for the optimal model (lasso, elastic net, ridge) for each depression-related outcome. Performance metrics (and 95 % con fidence intervals) are for the point on the Receiver Operating Characteristic (ROC) curve corresponding to Youden's Index. Summary of discrimination performance for the optimal model (lasso, elastic net, ridge) for each depression-related outcome. Performance metrics (and 95 % con fidence intervals) are for the point on the Receiver Operating Characteristic (ROC) curve corresponding to Youden's Index. all other outcomes (ps < 0.001). all other outcomes (ps < 0.001). Tables S1–S7. Group comparisons for actigraphy-derived hourly activity averages are shown in Supplementary Tables S8–S15. For all outcomes, and for both training and test data, the out of bag imputation error estimates using missForest, i.e., normalised root mean squared error (NRMSE) for continuous variables and proportion of falsely classified (PFC) for categorical variables were low, i.e., all < 8.0 × 10−7 for NRMSE and all < 0.15 for PFC. 3.2. Penalised regression models For the comparison of severe vs. moderate MD, a log-likelihood ratio test showed that the base logistic regression model including the sleep/ rest-activity predictors did not provide better fit than a model including only age, sex, and Townsend score (LR χ2 = 57.47, p = 0.08) but in clusion of sleep/rest-activity predictors improved the logistic model for 3.2.1. Broad MD vs. controls For the broad MD vs. controls comparison, the lasso model (post- selection; λ = 0.001) provided the highest AUC (0.68; 95 % CI 0.67–0.68), although this was slightly below the 0.70 cut-off for reasonable prediction. Accuracy was 62.85 %, sensitivity 64.18 % and specificity 62.04 % (Table 3). Sensitivity increased to 95.15 % when applying a higher weight to this metric vs. specificity in determining cut- off values, at the cost of lower overall accuracy (45.12 %) and specificity (14.81 %). The lasso model selected 33 predictors: summarised in order of their (post-selection) coefficient magnitude in Fig. 1A–B. Among the top sleep/rest-activity predictors were sleep disorder, difficulty getting up, insomnia symptoms, daytime dozing, napping, actigraphy-derived SIBD, snoring, SD of overall acceleration, evening chronotype and ac tivity from 11 pm–12 am. l For hourly activity averages, MD cases tended to show higher ac tivity in late evening/early night (~9 pm-2 am) and lower activity in early morning to afternoon (~5 am-2 pm) (Table S8). Findings were similar for the subdivisions of depression, with MD cases meeting criteria for atypical, severe, or early onset depression, or depression with comorbid anxiety or suicidality tending to show greater neuroticism and BMI, and less healthy sleep/circadian characteristics than those with less severe depression, e.g., reduced overall activity, more daytime inactivity and nocturnal awakenings, difficulty getting up, napping, insomnia symptoms, and sleep disorders. The above model was used to generate predicted values for MD cases with onset ≥1 year after data collection. Prediction performance was similar to the broad MD model (AUC = 0.67; 95 % CI 0.60–0.69; ac curacy 68.16 %) (Fig. 1C). Table 3 Participants meeting broad MD criteria were on average younger than controls; had higher Townsend scores, higher BMI and neuroticism scores, a greater proportion were female, of white ethnicity, were more likely to hold a degree, to be a smoker, and were less likely to drink alcohol regularly, consistent with previous UK Biobank studies (Smith et al., 2013). A summary of optimal model performance is provided in Table 3 (imputed data), and in Supplementary Table S16 for complete cases analyses, using Youden's index to determine the optimal cut-off. Per formance when applying a higher weight to sensitivity (0.55) vs. spec ificity (0.45) using weighted Youden's index is summarised in Table S17 for imputed data and Table S18 for complete cases analysis. ROC curves, alongside a summary of the features selected and their coefficients, ranked in order of magnitude, are summarised in Figs. 1–2 and Sup plementary Figs. S1–S5. For binary variables, coefficients for each category were equivalent but with the opposite sign: for ease of inter pretation, only the first category listed in model output is referred to in figures. Participants meeting MD criteria reported fewer hours of sleep per day, but (different subsets of) MD cases were more likely to report either short (<7 h) or long (≥9 h) sleep duration. MD cases more often reported difficulty getting up in the morning (25.30 % vs. 11.99 %), napping and dozing, and were less likely to report snoring. Around 1/3rd of MD participants reported frequent insomnia symptoms, compared to around 1/5th of control participants. A greater percentage of MD cases (4.78 % vs. 1.89 %) had a sleep disorder. MD cases more often reported evening chronotype. For actigraphy-derived variables, MD cases showed lower overall acceleration average, lower variability of acceleration, later M10, L5 and sleep midpoint times, longer sleep duration and greater sleep duration variability, longer duration of daytime inactivity, and longer time in bed compared to controls. MD cases also showed slightly higher sleep efficiency (0.762) compared to controls (0.759), possibly reflecting tendency towards longer overall sleep duration. 3.1. Sociodemographic and sleep/circadian characteristics by group 3.1. Sociodemographic and sleep/circadian characteristics by group Unadjusted group comparisons for sociodemographic and sleep/rest- activity characteristics (excluding actigraphy hourly averages) are dis played in Table 1 (broad MD vs. control) and Supplementary 87 Journal of Affective Disorders 335 (2023) 83–94 L.M. Lyall et al. L.M. Lyall et al. model was lasso (penalised; λ = 1.5 × 10−3; AUC 0.67, CI 0.64–0.70). Twenty-six predictors were selected (Fig. S1). 3.2.2. Postnatal depression For the comparison of women having experienced probable PND vs. women who have given birth with no history of depression, the best 88 Journal of Affective Disorders 335 (2023) 83–94 L.M. Lyall et al. L.M. Lyall et al. Journal of Affective Disorders 335 (2023) 83–94 Fig. 1. A) ROC curve for broad MD vs. controls. B) Summary of lasso post-selection coefficients for broad MD vs. controls. C) ROC curve summarising performance of broad MD vs. controls lasso model in separate subset of participants with MD onset ≥1 year after data collection. L.M. Lyall et al. Fig. 1. A) ROC curve for broad MD vs. controls. B) Summary of lasso post-selection coefficients for broad MD vs. controls. C) ROC curve summarising performance of broad MD vs. controls lasso model in separate subset of participants with MD onset ≥1 year after data collection. Fig. 2. ROC curve (A) and summary of post-selection ridge regression coefficients (B) for MD with vs. without atypical symptoms. Fig. 2. ROC curve (A) and summary of post-selection ridge regression coefficients (B) for MD with vs. without atypical symptoms. Fig. 2. ROC curve (A) and summary of post-selection ridge regression coefficients (B) for MD with vs. witho model was lasso (penalised; λ = 1.5 × 10−3; AUC 0.67, CI 0.64–0.70). Twenty-six predictors were selected (Fig. S1). 3.2.3. Atypical vs. typical MD The ridge model with post-selection coefficients (λ = 0.03) was the best model for MD with vs without atypical post selection The AUC 3.2.3. Atypical vs. typical MD model was lasso (penalised; λ = 1.5 × 10−3; AUC 0.67, CI 0.64–0.70). Twenty-six predictors were selected (Fig. S1). model was lasso (penalised; λ = 1.5 × 10−3; AUC 0.67, CI 0.64–0.70). Twenty-six predictors were selected (Fig. S1). The ridge model with post-selection coefficients (λ = 0.03) was the best model for MD with vs. without atypical post-selection. The AUC The ridge model with post-selection coefficients (λ = 0.03) was the best model for MD with vs. without atypical post-selection. The AUC 89 Journal of Affective Disorders 335 (2023) 83–94 L.M. Lyall et al. value of 0.74 (95 % CI 0.71–0.77; Fig. 2) exceeded Hosmer and Leme show's (2004) threshold (0.7) for acceptable discrimination. Accuracy, sensitivity, and specificity were all >67 % using Youden's index. When using the weighted Youden's index, accuracy was 25.87 %, sensitivity 97.67 % and specificity 22.57 %. 3.2.7. Suicidality For the comparison of depression with vs. without suicidal thoughts/ behaviours, ridge regression provided the best prediction performance (penalised; λ = 0.03; AUC 0.63, CI 0.60–0.66, Fig. S5). The top ten predictors in terms of coefficient magnitude were: SIBD, insomnia symptoms, morning activity 7 am–8 am, evening activity 6 pm–7 pm, late evening activity 11 pm-12 am, evening chronotype, morning ac tivity 8 am–9 am, late evening activity 9 pm–10 pm, M10 time and night-time activity 2 am–3 am. 3.2.6. Early onset vs. later onset 3.2.6. Early onset vs. later onset Ridge regression provided the highest AUC (0.64, 95 % CI 0.62–0.65) for the comparison of early onset and later onset depression (penalised; λ = 0.17; Fig. S4). 3.2.2. Postnatal depression Table 4 Associations between sleep score and each depression outcome. Sleep score is comprised of the number of (subjective) sleep-related factors shown from the five ‘top’ predictors identified in most penalised regression models. Sleep score MD broad PND Atypical Comorbid anxiety Severity Early onset Suicidality OR N (%)a OR N (%)a OR N (%)a OR N (%)a OR N (%)a OR N (%)a OR N (%)a 0 – 94,497 (80.63) – 40,342 (98.00) 823 (2.00) – 9340 (96.59) 330 (3.41) – 9294 (59.83) 6239 (40.17) – 3439 (67.67) 1643 (32.33) – 3501 (49.46) 3578 (50.54) – 9243 (94.42) 546 (5.58) 1 1.41* 97,755 (76.10) 1.40* 35,186 (97.31) 974 (2.69) 1.60* 11,129 (94.89) 599 (5.11) 1.23* 10,934 (55.03) 8934 (44.97) 1.06 4826 (65.76) 2513 (34.24) 1.07 4161 (48.94) 4342 (51.06) 1.21* 10,836 (93.39) 767 (6.61) 2 2.40* 33,434 (64.97) 2.36* 11,867 (95.59) 547 (4.41) 2.15* 5639 (93.33) 403 (6.67) 1.69* 4982 (46.98) 5622 (53.02) 1.27* 2604 (61.29) 1645 (38.71) 1.19* 2055 (46.54) 2361 (53.46) 1.54* 5286 (91.72) 477 (8.28) 3 4.21* 5954 (51.83) 3.43* 1988 (93.82) 131 (6.18) 2.98* 1301 (91.11) 127 (8.89) 2.61* 1077 (36.50) 1874 (63.50) 1.63* 666 (54.37) 559 (45.63) 1.30* 448 (44.58) 557 (55.42) 2.58* 1137 (86.86) 172 (13.14) 4b 8.05* 655 (37.60) 9.41* 147 (85.96) 24 (14.04) 5.71* 195 (84.05) 37 (15.95) 4.09* 153 (26.84) 417 (73.16) 2.21* 111 (45.49) 133 (54.51) 1.80* 58 (36.94) 99 (63.06) 5.38* 153 (73.56) 55 (26.44) Age, sex (excluding PND model) and Townsend score were included in models alongside sleep score. * Significant (vs. reference category 0) at p < 0.001. 3.2.5. Depression severity 3.2.5. Depression severity An elastic net model (post-selection; α = 0.9, λ = 0.02) yielded the highest AUC for severe vs. moderate depression, but discrimination was poor (AUC 0.58, 95 % CI 0.55–0.62). Five predictors were selected; in order of coefficient size (Fig. S3) these are: sex, difficulty getting up, Townsend score, SIBD and age. 3.2.4. Comorbid anxiety Ridge regression (penalised coefficients; λ = 0.07) provided the highest AUC for depression with vs. without comorbid anxiety, although discrimination was poor (AUC 0.59, 95 % CI 0.57–0.60; see Fig. S2). 3.3. Most common predictors: association with MD in separate UK Biobank subset Seven sleep/rest-activity variables were among the 15 largest co efficients in at least half of the final models (imputed and complete cases). These were: difficulty getting up, SIBD, insomnia symptoms, sleep disorder, napping, snoring, and average activity from 8 am–9 am. Exploratory analyses examined the association of the above subjec tive/health record markers of poorer sleep (i.e., excluding the actig raphy measures, SIBD and average activity 8-9 am, as participants with these measures were already included in main analyses) with depression outcomes in participants who were not included in main penalised regression analyses as they did not provide usable actigraphy data - either with data but QC was not passed, or actigraphy data was not collected. (For MD broad vs. controls, n(MD) = 78,163; n(controls) = 232,555). A sleep score comprising the number of above (non-actigraphy) factors was calculated: 1 was added to the sleep score for: presence of/ frequent: a) difficulty getting up; b) napping, c) insomnia symptoms, d) snoring; e) sleep disorder. As small numbers had the highest score of 5, scores of 4 and 5 were collapsed into a single category. Association of this sleep score with each MD outcome was examined via logistic regression (treating sleep score as categorical). Results are summarised in Table 4. For each outcome, the OR vs. scores of 0 increased with increasing sleep score: the odds of meeting broad MD criteria were 8.05 times higher for those with at least 4 markers of poor sleep compared to those with a sleep score of 0, and of those with the highest sleep score, 62 % met broad MD criteria. ORs for the highest sleep score vs. 0 for the other outcomes ranged from 1.80 (early onset vs. later onset) to 9.41 (PND vs. controls). Of those meeting MD criteria with sufficient data for catego risation into those with vs. without comorbid anxiety, 73.16 % of those with four or more markers of poor sleep met criteria for comorbid 90 anxiety. L.M. Lyall et al. L.M. Lyall et al. Journal of Affective Disorders 335 (2023) 83–94 anxiety. Discrimination performance may improve with addition of more fine-grained actigraphy data, e.g., epoched activity counts across the duration of the data collection period. Other machine learning methods, particularly non-linear approaches e.g., Support Vector Machines (SVM) with radial basis function or gradient boosting machine could also improve performance. 4.1. Summary Penalised regression models incorporating the subjective and objective sleep/circadian measures in UK Biobank were constructed to a) examine prediction performance for depression (vs. controls) and more severe outcomes within depression; and b) identify the optimal sleep/circadian predictors of each of the depression-related outcomes. A ridge regression model could discriminate moderately well participants with atypical vs. typical depression (AUC 0.74; 95 % CI 0.71–0.77). Discrimination was relatively poor for other models (AUCs < 0.70), although for MD vs. controls (including a separate subset of participants with onset of depression ≥1 year after data collection), and PND vs. controls, lasso models were close to the threshold for acceptable discrimination (MD: AUC 0.68, 95 % CI 0.67–0.68; PND: AUC 0.67; CI 0.64–0.70). Mental health data were not available at the time of actigraphy data collection, but at the time of the baseline and online follow-up MHQs, only around 2 % of participants with lifetime history of depression were thought to be currently experiencing a depressive episode. Findings therefore support suggestions that individuals with depression differ in terms of sleep/rest-activity characteristics from healthy controls even during periods of euthymia, and mirror previous findings of strong, reliable association between general sleep/rest-activity features and lifetime history of psychiatric disorders including depression (Lyall et al., 2018; Wainberg et al., 2021). Although addressing a different question, it is likely that classification accuracy would be improved if analyses were restricted to participants currently suffering a depressive episode. Several potentially important clinical markers of depression sus ceptibility were identified via feature selection and/or relative coeffi cient magnitude: insomnia symptoms, snoring, napping, difficulty getting up, sleep disorder, and actigraphy-derived duration of daytime inactivity (SIBD) and lower morning activity around 8 am. In follow-up analyses within a separate subset of UK Biobank participants, in dividuals self-reporting any of the first five (excluding actigraphy measures) were more likely to experience depression or more severe depression outcomes, particularly if a combination of these markers was present. 4.2. Discrimination performance It should be noted that in a clinical context, simple screening tools such as the Patient Health Questionnaire (including PHQ-9 and PHQ-2) outperform the above models in terms of AUC, sensitivity and speci ficity, and therefore remain the most efficient option for rapid screening of both depression diagnosis and depression severity (Cameron et al., 2011; Gilbody et al., 2007; Manea et al., 2016). Here, applying greater weight to sensitivity (Tables S17-S18) improved ability to detect cases, maximising sensitivity and NPV, at the cost of lowered specificity, PPV and overall accuracy. It could be argued that such weighting could be useful in a clinical context to capture all patients who may benefit from further screening and/or intervention, to reduce false negatives (Chubak et al., 2012; Wu et al., 2021). Our findings contribute to suggestions that individuals suffering from disturbed sleep and rest-activity patterns may be more likely to suffer from depression and more severe outcomes within depression regardless of current mood state, and may benefit from greater monitoring (Lovato and Gradisar, 2014; Wang et al., 2019). Sleep problems and circadian disruption are common in the general population, including in those who are otherwise healthy or suffer from psychiatric disorders other than depression (Walker et al., 2020). It is perhaps unsurprising therefore that sleep and circadian measures alone (alongside limited sociodemographic characteristics) did not discrimi nate those suffering from depression or more severe depression with a high degree of accuracy. Of note, a more liberal threshold for ‘accept able’ discrimination of AUC > 0.6 is often applied (Cha et al., 2022; Yang and Berdine, 2017): all but two of our models surpassed this threshold, but only one exceeded the more conservative threshold applied here of AUC > 0.7 (Hosmer and Lemeshow, 2004). The fact that inclusion of sleep/rest-activity predictors improved model performance beyond basic sociodemographics (for all outcomes except severe vs. moderate MD) and that the selected features were strongly associated with all outcomes reinforces the importance of the selected sleep/ circadian variables in depression, and outcomes in depression. Consis tent with this, sleep and circadian disruption have been linked longi tudinally to onset of and worse outcomes in depression (Zhang et al., 2022) and may act as general markers of vulnerability to mental ill- health (Lyall et al., 2018). 4. Discussion 4. Discussion 3.3. Most common predictors: association with MD in separate UK Biobank subset Here, however, a principal aim was feature se lection and interpretability of results, which penalised regression is well- suited to, and for which it has shown good performance in clinical psychiatric prediction models (Chekroud et al., 2021). Initial scoping using SVM-recursive feature elimination with several outcomes did not improve performance, consistent with a systematic review finding no improvement in clinical prediction for non-linear machine learning methods compared to logistic regression including penalised regression (Christodoulou et al., 2019). As the number of predictors here was relatively low, alternative non-linear machine learning methods may have a greater influence on performance in future, higher-dimensional studies where larger numbers of lifestyle, neuroimaging and genetic features are included, and non-linear effects are more likely. anxiety. 4.3. Optimal predictors This is particularly important as actigraphy devices and methodology can differ markedly between studies and samples, further highlighting the need for assessment of generalisability (Fekedulegn et al., 2020; Patterson et al., 2023). outcomes than circadian disruption per se. Other circadian factors such as chronotype, and timing of actigraphy-derived M10, L5 and sleep midpoint were not among the top predictors when accounting for the full range of sleep/activity measures, despite previous findings linking later chronotype to depression risk (Vetter et al., 2018). Lack of circa dian features among the top predictors may however have been linked to their collinearity with morning activity. yp y g y potentially limiting associations and discrimination performance. Similar analyses in younger cohorts, e.g., ABCD, may yield greater in sights into key risk factors as well as testing generalisability of the current prediction models. This is particularly important as actigraphy devices and methodology can differ markedly between studies and samples, further highlighting the need for assessment of generalisability (Fekedulegn et al., 2020; Patterson et al., 2023). Two of the top predictors were based on actigraphy measurements: SIBD (duration of sustained inactivity bouts during the daytime), and activity between 8 am and 9 am. Coefficients and descriptive tables indicate those who spend more time inactive during the day and are less active in the morning are more likely to suffer from depression, and more severe depression, including depression with suicidality. This is consistent with recent findings that activity deficits are most pro nounced in the morning in adults with depression symptoms (Smagula et al., 2021). It has been proposed that this association of reduced morning activity with depressive symptoms may be linked to sociopsy chological factors such as reduced engagement in social and physical activity leading to rumination (Smagula et al., 2021). Reduced circadian entrainment and morning daylight exposure may be another factor: this has been linked to delays in sleep timing, shorter sleep duration, and reduced sleep quality (Blume et al., 2019). Some evidence suggests specific targeting of morning activity could be a useful clinical inter vention for depression (Smagula et al., 2022). 4.3. Optimal predictors Findings that greater morning and evening activity and lower nocturnal activity were linked to reduced odds of suicidality are consistent with previous literature demonstrating association of reduced physical activity and increased sedentary behaviour with suicide risk (Vancampfort et al., 2019, 2018): targeting of activity levels has been proposed as an intervention, and it is possible that activity monitoring could be of use in identifying in dividuals at greater risk of serious outcomes such as suicidality (Van campfort et al., 2018). The restriction of analyses to those with valid actigraphy data allowed inclusion of all available sleep/rest-activity measures, both subjective and objective. Among this subsample, almost 40 % met criteria for lifetime history of depression: this is higher than previous estimates from the full UK Biobank sample or from other population studies (~27 %; Smith et al., 2013). This is likely due to exacerbated selection bias within the subsample, whereby individuals opting into the follow-up actigraphy investigation were slightly more likely to suffer from depression (see Lyall et al., 2022 for similar findings in the neu roimaging subsample). It is unclear why this might be, particularly as follow-up subsamples are typically associated with ‘healthy bias’ (Fry et al., 2017), but could be linked to increased interest in participating in health research among those suffering from depression (Adams et al., 2020). Reassuringly however, in exploratory analyses in an independent subset of UK Biobank participants without available actigraphy data (with lower depression prevalence), the key subjective sleep variables identified in most models were strongly associated with the depression- related outcomes. The UK Biobank's use of a single protocol and large sample size mean that while the sample and particularly subsamples may not be representative of the general population and should not be used for prevalence estimates, findings relating to exposure-disease as sociations are likely to be generalisable (Fry et al., 2017). While actigraphy-measured inactivity during the day, particularly morning-time, appears to be an important predictor of depression- related outcomes, most of the top predictors were based on self-report. Detailed actigraphy assessment over days/weeks may not be required to determine those more likely to show depression and more severe outcomes within depression, particularly as our exploratory analyses excluding actigraph-based measures demonstrated strong association of the subjective measures with depression and its severity. 4.3. Optimal predictors A key aim was to identify which of the many available sleep and rest- activity variables were the most important predictors of depression- related outcomes. Several features occurred in the top 15 predictors in at least half of the models: difficulty getting up, insomnia symptoms, snoring, napping, sleep disorder, and actigraphy-derived SIBD and average activity around 8 am. The non-actigraphy measures from this list were strongly associated with the depression outcomes in a separate subset of UK Biobank participants who had not provided usable actig raphy data, particularly where multiple factors occurred in combination. The highest discrimination was for depression with atypical vs. typical symptoms. As the definition of lifetime history of atypical depression included (self-reported) hypersomnia during the worst period of depression (Brailean et al., 2020; Nguyen et al., 2022), it is perhaps unsurprising that detailed sleep/rest-activity characteristics were useful in discriminating these two groups. However, it should be noted that i) 94 % of the atypical depression group did not meet criteria for current depression at the time of the MHQ, and ii) all other contrasts (MD/PND vs. controls and each depression subtype) were not defined based on any sleep characteristics but also showed widespread sleep/ rest-activity differences. Findings are therefore consistent with sugges tions that both depression itself, and more severe depression subtypes show reliable association with sleep and rest-activity characteristics regardless of current depressive state. This set of optimal predictors may be consistent with disturbed sleep during the night (insomnia, snoring) resulting in sleepiness and inac tivity during the day (difficulty getting up, napping, SIBD, lower morning activity) (Berger et al., 2021) as contributing factors to depression occurrence and greater severity. This may implicate insuffi cient/disturbed sleep as a greater contributor to depression-related 91 L.M. Lyall et al. Journal of Affective Disorders 335 (2023) 83–94 Similarly, as UK Biobank is a cohort of middle- to older-age adults, participants' sleep/circadian characteristics at the time of data collec tion may not reflect those from around the time of their depression onset (typically during adolescence/early adulthood; Solmi et al., 2022) potentially limiting associations and discrimination performance. Similar analyses in younger cohorts, e.g., ABCD, may yield greater in sights into key risk factors as well as testing generalisability of the current prediction models. 4.3. Optimal predictors SIBD and morning activity could potentially be incorporated into questionnaires alongside the above self-reported sleep characteristics, for example at GP/psychiatrist assessments of those with depression, to identify in dividuals at greater risk of worse outcomes. While self-report can be flawed, e.g. sedentary behaviour is often underestimated and sleep problems overestimated, subjective measures show reasonable reli ability (Alfano et al., 2015; Prince et al., 2020), and are less costly and invasive compared to actigraphy. Replication of current findings in other cohorts such as Adolescent Brain Cognitive Development (ABCD) study may help clarify a) whether the factors identified here are useful markers of vulnerability to depressive disorders; and b) if so, whether subjective self-report measures are sufficient to identify at-risk groups. The direction of causality (if any) between sleep/circadian disrup tion and depression-related outcomes is not of current relevance to the prediction models reported: the predictors could contribute to discrim ination whether they are a cause or consequence of the outcome mea sures, or both are linked to other, unmeasured factors. Causality is however of great theoretical and clinical interest, and future studies e.g., involving Mendelian Randomisation, and causal modelling including in younger cohorts (e.g., ABCD) will aim to address this. 5. Conclusions Penalised regression models incorporating sleep and circadian rest- activity characteristics were able to discriminate moderately well those suffering depression with vs. without atypical symptoms, and discrimination of MD vs. controls and PND vs. female controls approached the cut-off for reasonable discrimination. Prediction for other depression-related outcomes (including MD with vs. without co morbid anxiety) was poor. Findings highlight several potentially important sleep/rest-activity related predictors of depression and its severity. Individuals experiencing difficulty getting up in the morning, insomnia symptoms, greater inactivity during the daytime, lower morning activity, sleep disorders, and who snore or take naps may be more likely to have a lifetime history of depression and worse depression outcomes, particularly when several of these factors co-occur together. Future studies incorporating these factors alongside additional socio demographic, genetic and neuroimaging data may lead to improved discrimination performance. 4.4. Limitations The intervals between collection of different data types may have limited discrimination performance: subjective sleep measures were collected during the baseline assessment (2006–2010) alongside some mental health data, actigraphy data were collected between 2013 and 2015, Mental Health Questionnaire data from 2016 to 2017, and linked health records could come from any time point before/after recruitment. Although sleep disturbances are relatively stable over time (Fatima et al., 2020), participants' sleep/activity habits may have varied be tween data collection periods, and/or may not have been representative of their typical habits or those around the time of the relevant depression outcome. Supplementary data to this article can be found online at https://doi. org/10.1016/j.jad.2023.04.138. 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https://openalex.org/W2054868893	https://www.arca.fiocruz.br/bitstream/icict/15603/2/Divergent%20Profile%20of%20Emerging%20Cutaneous%20Leishmaniasis%20in%20Subtropical%20Brazil.pdf	English	null	Divergent Profile of Emerging Cutaneous Leishmaniasis in Subtropical Brazil: New Endemic Areas in the Southern Frontier	PloS one	2,013	cc-by	7,242	Abstract Funding: The authors would like to express their gratitude to the funding agencies, CAPES, FAPESC, PPSUS and CNPq. M.A.M. is recipient of a CAPES scholarship (Brazilian Government Agency). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: The authors would like to express their gratitude to the funding agencies, CAPES, FAPESC, PPSUS and CNPq. M.A.M. is (Brazilian Government Agency). The funders had no role in study design, data collection and analysis, decision to publish, or p Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mario.steindel@ufsc.br Mariel Asbury Marlow1, Marise da Silva Mattos2, Maria Ernestina Makowiecky3, Iriane Eger4, Andre Luiz Rossetto4, Edmundo Carlos Grisard1, Ma´rio Steindel1* Mariel Asbury Marlow1, Marise da Silva Mattos2, Maria Ernestina Makowiecky3, Iriane Eger4, Andre Luiz Rossetto4, Edmundo Carlos Grisard1, Ma´rio Steindel1* 1 Department of Microbiology, Immunology and Parasitology, Universidade Federal de Santa Catarina, Floriano´polis, Brazil, 2 Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal de Rio de Janeiro, Rio de Janeiro, Brazil, 3 Department of Zoonoses, Diretoria de Vigilaˆncia Epidemiolo´gica, Secretaria Estadual de Sau´de de Santa Catarina, Floriano´polis, Brazil, 4 Center for Health Sciences, Universidade do Vale do Itajaı´, Itajaı´, Brazil Abstract Background: Although known to be highly endemic in the Amazon regions of Brazil, the presence of cutaneous leishmaniasis (CL) in the subtropical southern part of the country has largely been ignored. This study was conducted to demonstrate CL is emerging in the Brazilian state of Santa Catarina, as well as to characterize the epidemiological profile and Leishmania species involved. Methodology/Principal Findings: For this cross-sectional study, data from all CL cases from Santa Catarina, Brazil, reported to the Brazilian National Notifiable Diseases Information System from 2001 to 2009 were investigated. Amplification of the kDNA minicircle conserved region followed by restriction fragment length polymorphism (PCR-RFLP) was conducted to screen for Leishmania species present in patient biopsy. Overall, 542 CL cases were reported, with majority resulting from autochthonous transmission (n = 401, 73.99%) and occurring in urban zones (n = 422, 77.86%). Age, gender, zone of residence, origin of case, clinical form and case outcome were found to differ significantly by region. Imported cases were over seven times more likely to relapse (95% CI 2.56–21.09). Mapping of cases revealed new endemic areas in northeastern Santa Catarina with two species present. With the exception of three L. (Leishmania) amazonensis cases (1.20%), majority of PCR positive samples were found to be L. (Viannia) braziliensis (n = 248, 98.80%). Conclusions/Significance: CL is now endemic in the state of Santa Catarina, Brazil, with case profiles varying significantly by region. L. (V.) braziliensis has been identified as the predominant species in the region. ation: Marlow MA, da Silva Mattos M, Makowiecky ME, Eger I, Rossetto AL, et al. (2013) Divergent Profile of Emerging Cutaneous Leish zil: New Endemic Areas in the Southern Frontier. PLoS ONE 8(2): e56177. doi:10.1371/journal.pone.0056177 Editor: Dan Zilberstein, Technion-Israel Institute of Technology Haifa, Israel Dan Zilberstein, Technion-Israel Institute of Technology Haifa, Israe Received September 28, 2012; Accepted January 7, 2013; Published February 14, 2013 Copyright: 2013 Marlow et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Marlow et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Cutaneous Leishmaniasis in Subtropical Brazil followed by L. (Leishmania) amazonensis and L. (V.) guyanensis being the most widely distributed of these species in Brazil [14]. Additionally, leishmaniasis presents several other clinical forms beyond CL, including mucocutaneous and visceral forms, all of which have specific relationships with one to many different Leishmania species [18]. Thus, the complexity of the disease complicates control efforts to combat its expansion and results in a lack of proven effective control methods for leishmaniasis today [7]. Furthermore, when leishmaniasis emerges into a new area, characterization of epidemiological, vectorial and clinical param- eters can be difficult and parasite characteristics in a specific region cannot be assumed to be similar to Leishmania spp. found in neighboring regions. The extent of this complexity was demon- strated in a study conducted by Falqueto et al. (2003), in which even different genotypes of L. (V.) braziliensis were found to have specific transmission cycles within the state of Espı´rito Santo, Brazil [19]. migration. The northeastern (NE) regions of Grande Floriano´polis, Norte Catarinense and Vale do Itajaı´ are located to the east of the Serra do Mar mountain range, are on or with proximity to the Atlantic coast, mainly consist of Atlantic Forest vegetation, are supplied by the South Atlantic water basin, and experience internal and external migration mainly due to tourism and urbanization. The region also includes numerous international tourist destinations. The southern and western (SW) regions of Oeste Catarinense, Serrana and Sul Catarinense contain or lie to the west of the Serra Geral and/or Serra do Mar mountain ranges, consist of both Tropical Forest and Araucaria Forest, are supplied by the Uruguay River basin, and experience mainly external migration from other Brazilian states for the purpose of rural labor. Case data were collected as part of the National Epidemiolog- ical Surveillance System of Brazil (Sistema de Informac¸a˜o de Agravos de Notificac¸a˜o- SINAN). All suspected cutaneous leishmaniasis cases by clinical profile were confirmed by parasi- tological methods (Giemsa stained smears and/or PCR). Only cases found positive by at least one parasitological method were considered as confirmed cases and reported to SINAN for follow- up. Case reporting began with collection of patient information in an interview by a trained public health official during evaluation at health centers. Next, data from confirmed cases were entered into the SINAN database by the Epidemiological Surveillance System municipality unit. Cutaneous Leishmaniasis in Subtropical Brazil During the interview, patients were questioned regarding any previous travel outside of the state and/or country in the two years preceding disease presentation in order to determine if the case was imported or resulted from autochtho- nous transmission within the state. Once electronically transcribed, data were sent to the Division of Epidemiologic Surveillance of the Santa Catarina State Health Department (Diretoria de Vigilaˆncia Epidemiolo´gica, Secretaria Estadual de Sau´de de Santa Catarina- DIVE), where the reports were reviewed for consistency and completeness by state officials who specialize in vector-borne diseases and any questionable or missing information was followed-up on a case by case basis. Through evaluation of patient history, cases were considered relapse cases if they had been previously diagnosed and treated for CL before or within the study period, were classified as cured based on remission of disease symptoms and/or negative PCR and then presented again with disease symptoms and positive smears, cultures, and/or PCR. For this study, data from all cases of CL in Santa Catarina, Brazil reported to SINAN from January 1st, 2001, to December 31st, 2009, were investigated. Proper confidentiality procedures dictated by DIVE were observed to protect the confidentiality of patient information. The present study was approved by the Universidade Federal de Santa Catarina (UFSC) Ethics Committee (003/07/ CPSH). Roughly similar in land size to Portugal and with a population of over 6 million [20], Santa Catarina has been experiencing a increase in population in the past few decades, largely due to migration of workers from different regions of Brazil hoping to benefit from the state’s industrial, agricultural and information technology sectors [21,22]. Since the 1980s, this southern Brazilian state regularly has received imported CL cases. However, in 1990, Sa˜o Thiago and Guida published the first report of autochthonous CL cases in the western region of Santa Catarina [23]. In their study, out of fourteen confirmed cases, eleven were considered autochthonous. However, despite contin- ued reporting of autochthonous CL cases to the Brazilian National Notifiable Diseases Information System [24], no investigation has been conducted to determine if CL has emerged and/or is endemic in this region, nor have the current epidemiological profile, species prevalence and species and case geographical distributions been determined. Cutaneous Leishmaniasis in Subtropical Brazil Without the establishment of epidemiologic parameters and species characterization, this neglected tropical disease will continue to remain without intervention, leaving the state, as well as neighboring Argentina and other southern Brazilian states, vulnerable to further emergence and outbreaks. Moreover, species characterization is the first piece of the puzzle to establishing a transmission cycle in the region as well as an important factor for diagnosis and treatment. Therefore, this study was conducted to demonstrate CL is an emerging neglected tropical disease in the state of Santa Catarina, Brazil, to characterize the epidemiological profile and species involved and to discuss the implications of this emergence for future research and control strategies. Study Area and Data Collection Data used for this retrospective cross-sectional study were restricted to cases reported in the state of Santa Catarina, Brazil. The political territory of Santa Catarina covers a total land area of 95,703.49 km2 and shares a western border with Argentina. During the study period, the state maintained an average population of 5,763,329 residents [20]. Being situated in the subtropical climate zone, temperatures in the state range from an average low of 8.5uC (47.3uF) in the winter, with even snow occurring in some cities, to an average high of 28.7uC (83.7uF) in the summer [25]. The costal eastern region is split from the western region by the Serra do Mar mountain range, creating six mesoregions with varying geographical characteristics. For this study, the mesoregions were grouped into two regions based on similarities in geography, biome, watershed and population Introduction is one of these infectious diseases which has been introduced into South Brazil over the past few decades [9–11]. Leishmaniasis is a vector-borne neglected tropical disease spread by female sand flies and caused by protozoan parasites belonging to genus Leishmania [12]. Brazil is among one of seven countries reporting 90% of cutaneous leishmaniasis (CL) cases worldwide [12] and reports an average of 28,000 CL cases per year [13]. In the 1980s, only nineteen states in Brazil had reported CL cases, yet by 2003, all twenty–seven Brazilian states presented autochthonous CL trans- mission [13], demonstrating the continued geographic expansion of the disease across the country. Leishmania parasites are present in a variety of ecological niches and can infect a large range of hosts and vectors [14]. In Brazil, several sylvatic and synanthropic mammals have been incriminated as reservoirs as well as the domestic dog, which possibly contributes to the noted increase in the urbanization of the disease transmission cycle [15–17]. Ten Leishmania species from two different subgenera have been found to cause CL in humans in the New World, with L. (Viannia) braziliensis Being the fifth largest country in the world, the territory of Brazil spans vastly different geographical biomes, including the tropical Amazon regions of the north, the plains in the central region and the subtropical south, where in some cities temperatures have been known to reach below 0uC in the winter months. Overall, a higher incidence of most infectious diseases can be found in the northern and northeastern regions where geographical, economic, and social factors aid in their transmission [1]. However, despite the contrasting population and geographical characteristics between the southern region with the country as a whole, migration of human, animal, and possibly vector populations has imported several of these classically ‘‘tropical’’ diseases from other regions to the subtropical south of Brazil [2–6]. Already known to emerge in various regions around the world and highly adaptable to new vectors and hosts [7,8], leishmaniasis PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e56177 1 Cutaneous Leishmaniasis in Subtropical Brazil Cutaneous Leishmaniasis in Subtropical Brazil Cutaneous Leishmaniasis in Subtropical Brazil Cutaneous Leishmaniasis in Subtropical Brazil 90.96%), male (n = 365, 67.34%), and lived in urban zones (n = 422, 77.86%) (Table 1). The average patient age was 37 years, with 34 (6.27%) cases occurring in children 10 years of age or younger (Table 1). While 73.99% (n = 401) of all cases were autochthonous in origin, mapping of municipalities by case origin and separating by the time periods of 2001 to 2004 and 2005 to 2009 demonstrated a geographical change in case origin overtime (Fig. 1). From 2001 until 2004, cases were geographically dispersed and mostly imported from other states. However, from 2005 until 2009, a drastic increase in autochthonous cases in the northeastern (NE) region can be observed, with cases in the southwestern (SW) region remaining mainly imported. When compared by chi- squared test, case origin was found to differ significantly between the regions (p = ,0.0001), with majority of cases in the NE region being of autochthonous transmission (n = 395, 84.22%) and majority of cases in the SW region being imported (n = 55, 75.34%) (Table 1). Leishmania Species Molecular Characterization p Amplification of the kDNA minicircle conserved region followed by restriction fragment length polymorphism (PCR- RFLP) was used to determine Leishmania species present in patient biopsy [27]. The 120 bp DNA band corresponding to a fragment of Leishmania minicircle k-DNA was PCR-amplified using primers 150 [59- GGG (G/T)AG GGG CGT TCT (G/C)CG AA -39] and 152 [59- (G/C)(G/C)(G/C) (A/T)CT AT(A/T) TTA CAC CAA CCC C -39]. DNA from confirmed Leishmania negative patients and DNA-free water were used as negative controls and DNA from reference strains L. (V.) braziliensis (MHOM/BR/75/M- 2904) and L. (L.) amazonensis (IFLA/BR/67/PH8) were used as positive controls. Following amplification and isopropanol precip- itation, 5 mL of PCR products were separately digested at 37uC with HaeIII and AvaI (New England Biolabs) restriction enzymes overnight according to manufacturer’s instructions. Restriction fragments were separated by electrophoresis in 10% SDS-PAGE gels. Samples considered positive for L. (V.) braziliensis were those which produced 40 bp and 80 bp fragments when digested with HaeIII and were not digested by AvaI. Samples considered positive for L. (L.) amazonensis were those which produced 38 bp and 78 bp fragments when digested with AvaI and were not digested by HaeIII. Statistical Analysis and Mapping Descriptive statistics were produced for all cases. Chi-squared tests, or Fisher’s exact tests when appropriate, were used to determine significant associations between northeastern and southwestern regions for case demographic and clinical charac- teristics. Bivariate logistic regression analysis predicting probability of case relapse was performed for each potential risk factor, including being male, living in a rural zone, 50 years or age or older, mucocutaneous, and imported from other Brazilian states. Factors found to be significant in the bivariate analysis then were included in the multivariate logistic regression analysis. The Hosmer and Lemeshow goodness of fit test was used to assess whether there was evidence for lack of fit in the logistic regression model. Data were analyzed using R version 2.13.2 (R Develop- ment Core Team, Vienna, Austria). Unadjusted odds ratios, adjusted odds ratios, 95% confidence intervals, and p-values are reported. All cases were geocoded by address and maps were produced using ArcGIS 10 (ESRI, Redlands, CA, USA). For the clinical characteristic of cases, the SW region presented a significantly higher percentage of mucocutaneous cases (n = 12, 16.44%), a more severe clinical form of the disease, compared to the NE region. A significantly higher percentage of relapse cases was observed in the SW region as well. Investigation into risk factors for CL relapse in the state revealed being 50 years of age or older, originating from transmission outside of the state (imported) or having mucocutaneous clinical form of the disease were significant risk factors. In the multivariate logistic regression, imported cases were found to be almost seven times significantly more likely to be a relapse case even after adjusting for the other risk factors of patient age and clinical form of CL (95% CI 2.56– 21.09) (Table 2). The logistic model was considered acceptable based on failure to reject the null hypothesis in the Hosmer and Lemeshow goodness of fit test (x2 = 0.035, p = 0.9828). Majority of these imported cases originated from the Amazonian state of Mato Grosso (n = 54, 51.43%). Cutaneous Leishmaniasis in Subtropical Brazil Furthermore, age group, gender, zone of residence, clinical form, and case outcome also were found to have a significant association with region of diagnosis (p = 0.0208, ,0.0001, 0.0003, ,0.0001, and 0.0294, respectively) (Table 1). In the NE region, the ages of cases were more evenly distributed across the age groups than in the SW region where cases tended to be of working adult age. Nearly all cases in children 10 years of age or younger occurred in the NE region (n = 33/34, 97.06%). The NE region presented a nearly 1 to 2 ratio of females to males, with a significantly higher percentage of women (n = 177, 32.66%) than in the SW region (n = 7, 9.59%) (p = ,0.0001). Majority of cases in both regions were urban; however, NE regions contained a higher percentage of periurban cases (n = 31, 6.61%) and SW region contained a higher percentage of rural cases (n = 21, 28.77%). When mapped by zone of case residence, both urban and rural cases were heavily clustered in the NE region, while cases were more dispersed in the SW region (Fig. 2). Additionally, when properly prepared biopsy or aspirates were available for Leishmania parasite isolation, samples were seeded in Schneider medium supplemented with 5% of heat inactivated fetal bovine serum and incubated at 26uC. Positive cultures were cryopreserved in liquid nitrogen. All isolated strains were deposited in the Leishmania Collection of the Oswaldo Cruz Institute (Colec¸a˜o de Leishmania do Instituto Oswaldo Cruz - CLIOC, WDCM731, http://clioc.ioc.fiocruz.br) international cryobank in Rio de Janeiro, Brazil, and characterized by multilocus enzyme electrophoresis (MLEE) following standard protocols set forth by CLIOC. Seasonality of CL cases was found to differ between regions as well (Fig. 3). The SW peak season occurred in August through September with 8.22% and 17.81% of cases occurring during this time, respectively. However, two peak seasons in February and October (9.81% and 15.99% of cases, respectively) were observed in the NE region, with October being the more dominate of the two peaks. DNA Extraction DNA for Leishmania species characterization was extracted from patient biopsy samples collected for diagnosis before initiation of treatment. Biopsy samples were placed in 500 ml of lysis buffer and incubated with proteinase K (20 mg/ml) overnight at 42uC. The resulting lysates were then subjected to phenol–chloroform extraction as described elsewhere [26]. Following isopropanol precipitation, the pellet was washed twice with 500 ml of cold 70% ethanol, resuspended in 50 ml of ultra- pure water containing RNAse A (10 mg/ml), incubated at 37uC for 1 h and then stored at 220uC until use. To monitor for possible contamination during DNA extraction, a negative extraction control was used. February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org 2 Results Of the 669 suspected cases of cutaneous leishmaniasis in the state of Santa Catarina, Brazil, during the study period, 542 (81.02%) cases were confirmed positive by at least one parasito- logical test and reported to the National Epidemiological Surveillance System of Brazil for epidemiological follow-up. Incidence of CL cases was found to rise from 14 cases in 2001 to a peak of 166 cases in 2006, and then gradually decreased to 48 cases in 2009. Overall, majority of cases were white (n = 365, Among the 542 CL cases confirmed by parasitological tests, diagnosis by PCR was performed for 331 (61.1%) patients. The remaining cases were confirmed by Giemsa stained smears. Positive PCR biopsy sample was available for PCR-RFLP analysis for 251 (46.31%) patients (Fig. 4). Majority of these samples were found to be positive for L. (V.) braziliensis (n = 248, February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e56177 3 Cutaneous Leishmaniasis in Subtropical Brazil Figure 1. Comparison of maps demonstrating cutaneous leishmaniasis case origin by municipality between the time periods of 2001–2004 and 2005–2009. doi:10.1371/journal.pone.0056177.g001 Figure 1. Comparison of maps demonstrating cutaneous leishmaniasis case origin by municipality between the time periods of 2001–2004 and 2005–2009. doi:10.1371/journal.pone.0056177.g001 98.80%). Three cases, one imported in 2001 from the state of Mato Grosso and two autochthonous cases in 2002 and 2005, were found to be positive for L. (L.) amazonensis. All cases RFLP positive for L. (L.) amazonensis were located in the NE region. For these same 251 patients, parasite isolation by culture was possible for 39 patient samples. All 39 strains have been deposited in the Leishmania Collection of the Oswaldo Cruz Institute (CLIOC). MLEE characterization performed by CLIOC was consistent with the kDNA PCR-RFLP results found in this study, with one L. (L.) amazonensis strain and 38 L. (V.) braziliensis strains being deposited. region. Majority of these imported cases originated from the state of Mato Grosso, where L. (V.) braziliensis has been confirmed as the most prevalent species of Leishmania [32] and an average of 6.2% of annual CL cases are mucosal [24]. Species found in this study conform with those found in a previous study where L. (L.) amazonensis was identified in two case isolates from 1987 in the western region and L. Results (V.) braziliensis was identified in one isolates from 1996 from the western region and one isolate from 1997 in the northeastern region of Santa Catarina [33]. However, this study has now confirmed L. (V.) braziliensis, the most widely distributed species in Brazil [14], is also the predominant species in the region and responsible for the emergence of the disease in Santa Catarina. Interestingly, L. (L.) amazonensis was also identified in this study from two autochtho- nous cases, despite no record of its known vector species (Lutzomyia (Nyssomyia) flaviscutellata, Lu. (N.) olmeca olmeca and Lu. (N.) reducta) having been reported in the state to date [29]. Only Lu. (N.) neivai has been found to be naturally infected with L. (V.) braziliensis in Santa Catarina [34]. Other sandfly species previously observed in Santa Catarina were Lu. (Pintomyia) fischeri and Lu. (Psychodopygus) ayrozai [35]. Multiple climate change prediction models conducted in 2003 have demonstrated the likelihood of dramatic improve- ments in habitat condition in southern Brazil for Lu. whitmani, as well as, increasing suitability in habitat for Lu. intermedia and Lu. migonei [5]. All three phlebotomine species are proven vectors of L. (V.) braziliensis [29]. Discussion Comparison of case demographics and clinical characteristics of cutaneous leishmaniasis cases between the northeastern and southern/western regions of Santa Catarina from 2001 to 2009 by chi-squared test or Fisher’s exact test. Table 1. Comparison of case demographics and clinical characteristics of cutaneous leishmaniasis cases between the northeastern and southern/western regions of Santa Catarina from 2001 to 2009 by chi-squared test or Fisher’s exact test. Characteristic Northeastern Region no. (%) Southwestern Region no. (%) p-value Total no. (%) Age group 0–10 33 (7.04) 1 (1.37) 0.0208 34 (6.27) 11–20 69 (14.71) 8 (10.96) 77 (14.21) 21–30 75 (15.99) 14 (19.18) 89 (16.42) 31–40 94 (20.04) 13 (17.81) 107 (19.74) 41–50 77 (16.42) 11 (15.07) 88 (16.24) 50–65 96 (20.47) 14 (19.18) 110 (20.30) .65 24 (5.12) 11 (15.07) 35 (6.46) Unknown 1 (0.21) 1 (1.37) 2 (0.37) Gender Female 170 (36.25) 7 (9.59) ,0.0001 177 (32.66) Male 299 (63.75) 66 (90.41) 365 (67.34) Race or ethnicity Indigenous 0 (0.00) 0 (0.00) 0.2653 0 (0.00) White 426(90.83) 67 (91.78) 493 (90.96) Black 10 (2.13) 3 (4.11) 13 (2.40) Asian 1 (0.21) 0 (0.00) 1 (0.18) Mixed race 21 (4.48) 0 (0.00) 21 (3.87) Unknown 11 (2.35) 3 (4.11) 14 (2.58) Zone of residence Urban 372 (79.32) 50 (68.49) 0.0003 422 (77.86) Rural 53 (11.30) 21 (28.77) 74 (13.65) Periurban 31 (6.61) 1 (1.37) 32 (5.90) Unknown 13 (2.77) 1 (1.37) 14 (2.58) Origin of case Autochthonous 395 (84.22) 6 (8.22) ,0.0001 401 (73.99) Imported 47 (10.02) 55 (75.34) 102 (18.82) Unknown 27 (5.76) 12 (16.44) 39 (7.20) Clinical form Cutaneous 455 (97.01) 61 (83.56) ,0.0001 516 (95.20) Mucocutaneous 14 (2.99) 12 (16.44) 26 (4.80) Case outcome Cured case 446 (95.10) 64 (87.67) 0.0294 510 (94.10) Relapse case 18 (3.84) 7 (9.59) 25 (4.61) Unknown 5 (1.07) 2 (2.74) 7 (1.29) Total 469 (86.53) 73 (13.47) 542 (100.00) doi:10.1371/journal.pone.0056177.t001 doi:10.1371/journal.pone.0056177.t001 supports the hypothesis that these cases were mainly migrant workers from other states. Of concern is the higher likelihood that these cases will be mucosal and/or relapse cases, as was demonstrated in the multivariate logistic regression analysis of this study. These imported cases were over seven times signifi- cantly more likely to be a relapse case, even after accounting for clinical form and being 50 years of age or older. Thus, these cases require improved case monitoring and further prospective studies are needed to determine the cause of this higher relapse rate. Discussion Evidence presented in this study has confirmed the emergence of cutaneous leishmaniasis in Santa Catarina, South Brazil. However, case profiles between the two main regions of the state were found to differ significantly, demonstrating this emergence is not uniform. While cases in the southwestern region continue to be dispersed and mainly imported, the northeastern region is now endemic for CL in over three main urban zones. The high relapse rate in imported cases as well as the high rate of mucosal cases in the SW region are important public health concerns. The mucocutaneous form of leishmaniasis represents a more virulent form of the disease, which can lead to serious disfiguration and is more difficult to treat [12,28]. Although other species have been incriminated as casual agents of mucosal leishmaniasis, L. (V.) braziliensis, the main species of Leishmania found in this study, is the most commonly associated species with this clinical form in Brazil [12,29–31]. While the percentage of mucosal cases in the state (5.25%) is similar to the percentage of mucosal cases overall in Brazil (6.61% in 2009) [24], the percentage of mucosal cases in the SW region of Santa Catarina (18.67%) alone is nearly three times higher than this national average. Given the significant association between case origin and clinical form, this higher percentage of mucosal cases reflects the higher percentage of imported cases in the SW Cases of CL in Santa Catarina in the past decade have consisted of both urban and rural cases. However, in the now endemic northeastern region, nearly 80% of cases are urban, following a rising trend in urbanization of the once rural disease in states across Brazil [24,36–38]. This study is unable to conclude vector competence in domestic habitats of southern Brazil, and thus, it cannot be confirmed if these urban cases are contracting the disease in domestic settings or returning from rural areas of transmission. However, the relatively higher proportion of women and children under 10 years of age of these CL cases in the NE region is suggestive of a peri-domestic or domestic transmission cycle. In the state of Parana´, the state just north of Santa Catarina, February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e56177 4 Cutaneous Leishmaniasis in Subtropical Brazil Table 1. February 2013 \| Volume 8 \| Issue 2 \| e56177 Discussion a majority of urban cases (59.2% of all cases reported from 1987 to 2004) has been reported as well [39]. However, with 78.08% urban and 5.80% peri-urban cases, this study has revealed that Santa Catarina now represents the state with one of the highest proportions of urban cases in Brazil [24]. Additionally, mapping of cases demonstrated CL clusters around urban centers in the endemic areas. One of the most distinct differences between the two regions of Santa Catarina was the rate of autochthonous versus imported cases. In the NE region, the majority of cases resulted from autochthonous transmission, supporting the claim of endemicity in the region. In the SW regions, the majority of imported cases reflected the expected migration of labor workers to this region. Additionally, most of these cases originated in Mato Grosso, where the majority of labor exchange with Santa Catarina occurs. This high rate of imported cases from Mato Grosso was also reported in 1997, when 52.1% of the 23 imported cases reported between 1994 and 1997 were found to be imported from Mato Grosso as well [10]. In addition, the presence of only one case in an individual with less than 10 years of age in the western region Seasonality of cases differed between the NE and SW regions. The two CL case peak seasons of February and October coincided with the two peak tourism seasons in the NE region of the state. This overlap is a major public health concern since a higher rate of cases combined an influx of population increases the risk of transmission and could result in geographical dispersion of leishmaniasis, specifically transmission of the predominant parasite species in the region L. (V.) braziliensis. Overall, given the different disease patterns between the NE and the SW regions, control strategies and treatment approaches February 2013 \| Volume 8 \| Issue 2 \| e56177 February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org 5 Cutaneous Leishmaniasis in Subtropical Brazil Figure 2. Map of cutaneous leishmaniasis cases by zone of residence (urban/peri-urban/rural), Santa Catarina, Brazil (n = 452, 2001– 2009). doi:10.1371/journal.pone.0056177.g002 p Figure 2. Map of cutaneous leishmaniasis cases by zone of residence (urban/peri-urban/rural), Santa Catarina, Brazil (n = 452, 2001– 2009). doi:10.1371/journal.pone.0056177.g002 should be modified by region to adequately prevent further emergence and future CL outbreaks in Santa Catarina. Discussion should be modified by region to adequately prevent further emergence and future CL outbreaks in Santa Catarina. An important limitation of this study was the potential underreporting of CL. Given leishmaniasis is not recognized as an endemic disease in the state, many physicians are unaware of its Figure 3. Seasonal distribution of cutaneous leishmaniasis in Santa Catarina, Brazil, by region; NE = Northeastern region, SW = Southern and western regions (n = 452, 2001–2009). doi:10.1371/journal.pone.0056177.g003 Figure 3. Seasonal distribution of cutaneous leishmaniasis in Santa Catarina, Brazil, by region; NE = Northeastern region, SW = Southern and western regions (n = 452, 2001–2009). doi:10.1371/journal.pone.0056177.g003 February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org 6 Cutaneous Leishmaniasis in Subtropical Brazil Cutaneous Leishmaniasis in Subtropical Brazil Table 2. Multivariate logistic regression predicting cutaneous leishmaniasis case relapse in Santa Catarina, Brazil. Potential risk factor Unadjusted odds ratio 95% CI Adjusted odds ratio 95% CI Male 1.95 0.72–5.28 2 2 Living in a rural zone 1.16 0.39–3.48 2 2 50 years or age or older 2.44* 1.09–5.49 1.52 0.55–4.19 Mucocutaneous 10.05* 3.75–26.94 1.82 0.43–7.78 Imported from other Brazilian states 8.96* 3.27–24.51 7.35{ 2.56–21.09 *p,0.05, {p,0.001. doi:10.1371/journal.pone.0056177.t002 Table 2. Multivariate logistic regression predicting cutaneous leishmaniasis case relapse in Santa Catarina, Brazil. Santa Catarina has not been widely recognized as a state endemic for leishmaniasis, which inhibits support for research and control efforts. Considered secondary to diseases with higher mortality and morbidity, CL case reporting may be delayed for months or even years, preventing the possibility of early interventions for outbreaks. Additionally, Santa Catarina has immense potential for tourism and is experiencing an increase in both domestic and international tourism each year [40]. The combination of new endemic areas with a high level of tourism presence and the disease is not commonly included as a differential diagnosis. Some cases may have been treated as bacterial or fungal infections and/or self-cured and were not reported to SINAN. However, this only further supports the main objective of this study to prove the emergence of the disease and suggests the problem of leishmaniasis may be even greater than the current evidence demonstrates. Future studies should seek to assess and/or include unreported cases in their study designs as the character- istics of these cases may differ from those of reported cases. Figure 4. Molecular characterization of Leishmania species present in biopsy sample. References Adv Parasitol 64: 1–109. 34. Marcondes CB, Bittencourt IA, Stoco PH, Eger I, Grisard EC, et al. (2009) Natural infection of Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae, Phlebotominae) by Leishmania (Viannia) spp. in Brazil. Trans R Soc Trop Med Hyg 103(11): 1093–1097. 15. Dantas-Torres F (2007) The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis. Veterinary Parasitology 149: 139–146. 35. Marcondes CB, Conceic¸a˜o MBE, Portes MGT, Sima˜o BP (2005) Phlebotomine sandflies in a focus of dermal leishmaniasis in the eastern region of the Brazilian State of Santa Catarina: preliminary results (Diptera: Psychodidae). Rev Soc Bras Med Trop 38(4): 353–355. 16. Reithinger R, Davies CR (1999) Is the dog (Canis familiaris) a reservoir host of American Cutaneous Leishmaniasis? A critical review of the current evidence. Am J Trop Med Hyg 61: 530–41. 17. Yoshida ELA, Correa FMA, Marques SA, Stolf HO, Dillon NL, et al. (1990) Human, canine and equine (Equus caballus) leishmaniasis due to Leishmania braziliensis ( = L. braziliensis braziliensis) in the south- west region of Sa˜o Paulo State, Brazil. Mem Inst Oswaldo Cruz 85: 133–134. 36. Passos VMA, Fernandes O, Lacerda PAF, Volpini AC, Gontijo CM, et al. (1999) Leishmania (Viannia) braziliensis is the predominant species infecting patients with American cutaneous leishmaniasis in the state of Minas Gerais, south-east Brazil. Acta Tropica 72; 251–258. 18. Sharma U, Singh S (2008) Insect vectors of Leishmania: distribution, physiology and their control. J Vector Borne Dis 45: 255–272. 37. Campbell-Lendrum D, Dujardin JP, Martinez E, Feliciangeli MD, Perez JE, et al. (2001) Domestic and peridomestic transmission of American cutaneous leishmaniasis: changing epidemiological patterns present new control opportu- nities. Mem Inst Oswaldo Cruz 96; 159–162. 19. Falqueto A, Sessa PA, Ferreira AL, Vieira VP, Santos CB, et al. (2003) Epidemiological and Clinical Features of Leishmania (Viannia) braziliensis American Cutaneous and Mucocutaneous Leishmaniasis in the State of Espı´rito Santo, Brazil. Mem Inst Oswaldo Cruz 98(8): 1003–1010. 38. Oliveira CCG, Lacerda HG, Martins DRM, Barbosa JDA, Monteiro GR, et al. (2004) Changing epidemiology of American cutaneous leishmaniasis (CL) in Brazil: a disease of the urban–rural interface. Acta Tropica 90; 155–162. 20. IBGE - Instituto Brasileiro de Geografia e Estatı´stica (Brazilian Institute of Geography and Statistics) (2010) Demographic Census – Santa Catarina. [in Portuguese]. 39. Monteiro WM, Neitzke-Abreu HC, Ferreira ME, Melo GC, Barbosa MG, et al. References International Journal for Parasitology 30: 1269–1281. 28. Murray HW, Berman JD, Davies CR, Saravia NG (2005) Advances in leishmaniasis. Lancet 366: 1561–1577. 8. Volf P, Peckova J (2007) Sand flies and Leishmania: specific versus permissive vectors. Trends Parasitol 23(3): 91–92. 29. Lainson R (2010) The Neotropical Leishmania species: a brief historical review of their discovery, ecology and taxonomy. Rev Pan-Amaz Saude 1(2): 13–38. 9. Monteiro WM, Neitzke-Abreu HC, Ferreira ME, Melo GC, Barbosa MG, et al. (2009) Population mobility and production of American tegumentary leishman- iasis in the State of Parana´, southern Brazil. [in Portuguese] Rev Soc Bras Med Trop 42(5): 509–14. 30. Guerra JA, Prestes SR, Silveira H, Coelho LI, Gama P, et al. (2011) Mucosal Leishmaniasis caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon. PLoS Negl Trop Dis 8; 5(3): e980. guyanensis in the Brazilian Amazon. PLoS Negl Trop Dis 8; 5(3): e980 10. Lima Filho JHC, Steindel M (1998) Epidemiologic and clinical aspects of cutaneous leishmaniasis in the state of Santa Catarina [in Portuguese]. Arq Cat Med 27(1–4): 25–31. 31. Silveira FT, Lainson R, Corbett CEP (2004) Clinical and immunopathological spectrum of American cutaneous leishmaniasis with special reference to the disease in Amazonian Brazil - a review. Mem. Inst. Oswaldo Cruz 99(3): 239– 251. ISSN 0074–0276. 11. Razera F, De Oliveira Blanco LF, Bonamigo RR (2005) American cutaneous leishmaniasis: the first autochthonous cases reported in Rio Grande do Sul since 2001 and review of the literature. [in Portuguese] Revista AMRIGS, Porto Alegre, 49 (3): 165–170. 32. Carvalho Mde L, de Andrade AS, Fontes CJ, Hueb M, de Oliveira Silva S, et al. (2006) Leishmania (Viannia) braziliensis is the prevalent species infecting patients with tegumentary leishmaniasis from Mato Grosso State, Brazil. Acta Trop 98(3): 277–285. g ( ) 12. Reithinger R, Dujardin JC, Louzir H, Pirmez C, Alexander B, et al. (2007) Cutaneous leishmaniasis. Lancet Infect Dis 7: 581–596. 33. Grisard EC, Steindel M, Shaw JJ, Ishikawa EA, Carvalho-Pinto CJ, et al. (2000) Characterization of Leishmania sp. strains isolated from autochthonous cases of human cutaneous leishmaniasis in Santa Catarina State, southern Brazil. Acta Tropica 74: 89–93. 13. Ministe´rio Da Sau´de (2007) Manual for Surveillance of American cutaneous leishmaniasis [in Portuguese] 2nd Edition. Brası´lia, DF; Portuguese. 14. Ban˜uls AL, Hide M, Prugnolle F (2007) Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Acknowledgments The authors would like to thank Dr. Emil Kupek, Department of Public Health, Federal University of Santa Catarina, Brazil and Dr. Clive J. Shiff, Bloomberg School of Public Health, Johns Hopkins University, USA, for their valued input and manuscript review. Thus, as CL continues to expand in the state of Santa Catarina, research efforts into the causes of disease transmission and outbreaks, including determination of vector species and natural reservoirs, are needed in order to develop interventions and control strategies. Additionally, since CL requires a lengthy and costly treatment regimen [41], outbreaks could result in a serious burden on the public health care system. In conclusion, without increased pressure for more complete and efficient case reporting, funding for research, and development of interventions, the state is likely to endure further outbreaks and continued geographical expansion of this neglected tropical disease. Discussion (A) PCR amplification products of the Leishmania kDNA minicircle conserved region from cutaneous leishmaniasis cases in Santa Catarina, Brazil, stained with ethidium bromide; Restriction fragment length polymorphism (RFLP) of the amplified the kDNA minicircle conserved region digested with restriction enzymes HaeIII (B) and AvaI (C), and analyzed by electrophoresis in 10% polyacrylamide gels with silver staining; Lane 1–2 = L. (V.) braziliensis cases from the northeastern region; Lane 3– 4 = L. (V.) braziliensis cases from the southern and western regions, Lane 5–6 = L. (L.) amazonensis cases, Lane 7 = reference strain L. (V.) braziliensis (MHOM/BR/75/M-2904), Lane 8 = reference strain L. (L.) amazonensis (IFLA/BR/67/PH8), NC = negative control, MW = molecular weight. doi:10.1371/journal.pone.0056177.g004 Figure 4. Molecular characterization of Leishmania species present in biopsy sample. (A) PCR amplification products of the Leishmania kDNA minicircle conserved region from cutaneous leishmaniasis cases in Santa Catarina, Brazil, stained with ethidium bromide; Restriction fragment length polymorphism (RFLP) of the amplified the kDNA minicircle conserved region digested with restriction enzymes HaeIII (B) and AvaI (C), and analyzed by electrophoresis in 10% polyacrylamide gels with silver staining; Lane 1–2 = L. (V.) braziliensis cases from the northeastern region; Lane 3– 4 = L. (V.) braziliensis cases from the southern and western regions, Lane 5–6 = L. (L.) amazonensis cases, Lane 7 = reference strain L. (V.) braziliensis (MHOM/BR/75/M-2904) Lane 8 = reference strain L (L ) amazonensis (IFLA/BR/67/PH8) NC = negative control MW = molecular weight Figure 4. Molecular characterization of Leishmania species present in biopsy sample. (A) PCR amplification products of the Leishmania kDNA minicircle conserved region from cutaneous leishmaniasis cases in Santa Catarina, Brazil, stained with ethidium bromide; Restriction fragment length polymorphism (RFLP) of the amplified the kDNA minicircle conserved region digested with restriction enzymes HaeIII (B) and AvaI (C), and analyzed by electrophoresis in 10% polyacrylamide gels with silver staining; Lane 1–2 = L. (V.) braziliensis cases from the northeastern region; Lane 3– 4 = L. (V.) braziliensis cases from the southern and western regions, Lane 5–6 = L. (L.) amazonensis cases, Lane 7 = reference strain L. (V.) braziliensis (MHOM/BR/75/M-2904), Lane 8 = reference strain L. (L.) amazonensis (IFLA/BR/67/PH8), NC = negative control, MW = molecular weight. doi:10.1371/journal.pone.0056177.g004 February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org 7 Cutaneous Leishmaniasis in Subtropical Brazil could result in both national and global CL transmission from Santa Catarina. References Demographic Census. [in Portuguese] Reflexo˜es sobre os deslocamentos populacionais no Brasil. Instituto Brasileiro de Geografia e Estatı´stica – IBGE. 1. Barreto ML, Teixeira MG, Bastos FI, Ximenes RAA, Barata RB, et al. (2011) Successes and failures in the control of infectious diseases in Brazil: social and environmental context, policies, interventions, and research needs. The Lancet 377 (9780): 1877–1889. 22. Pires Menezes ML (2000) Current trends of internal migration in Brazil. Revista Electro´nica de Geografı´a y Ciencias Sociales. Universidad de Barcelona 69 (45): [ISSN 1138–9788]. 2. Dengue in Brazil: Current situation and activities for prevention and control. Boletim Eletroˆnico Epidemiolo´gico. Fundac¸a˜o Nacional de Sau´de, Ministe´rio da Sau´de [in Portuguese]. Available: http://portal.saude.gov.br/portal/arquivos/ pdf/ano02_n03_dengue_brasil_si.pdf. Accessed 2012 Feb 1. 23. Sa˜o Thiago PT, Guida U (1990) Cutaneous leishmaniasis in the west of the State of Santa Catarina, Brazil [in Portuguese]. Rev Soc Bras Med Trop 23(4): 201– 203. 3. Pocai EA, Frozza L, Headley SA, Grac¸a DL (1998) Visceral leishmaniasis (Kala- Azar). Five cases in dogs in Santa Maria, Rio Grande do Sul, South Brazil. [in Portuguese] Cieˆncia Rural 28: 501–505. 24. Leishmaniose Tegumentar Americana- Notified confirmed cases in the National Notifiable Diseases Information System (Notificados no Sistema de Informac¸a˜o de Agravos de Notificac¸a˜o), Sinan Net [in Portuguese]. Available: http:// dtr2004.saude.gov.br/sinanweb/tabnet/tabnet?sinannet/lta.def. Accessed 2011 Sept 9. 4. Souza GD, Santos E, Andrade Filho JD (2009) The first report of the main vector of visceral leishmaniasis in America, Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae: Phlebotominae), in the state of Rio Grande do Sul, Brazil. Mem. Inst. Oswaldo Cruz 104 (8): 1181–1182. 25. Santa Catarina: Estados. Instituto Brasileiro de Geografia e Estatı´stica (IBGE) [in Portuguese]. Available: http://www.ibge.gov.br/estadosat/. Accessed 2012 June 25. 5. Peterson AT, Shaw J (2003) Lutzomyia vectors for cutaneous leishmaniasis in Southern Brazil: ecological niche models, predicted geographic distributions, and climate change effects. Int J Parasitol 33(9): 919–931. 26. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, USA: 999. g ( ) 6. Ministe´rio Da Sau´de (2007) Epidemiologic Situation of Malaria in Brazil, Year of 2007. [in Portuguese] Vigilaˆncia em Sau´de. Brası´lia, DF. 27. Volpini AC, Passos VMA, Oliveira GC, Romanha AJ (2004) PCR-RFLP to identify Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis causing American cutaneous leishmaniasis. Acta Tropica 90: 31–37. of 2007. [in Portuguese] Vigilaˆncia em Sau´de. Brası´lia, DF 7. Ashford RW (2000) The leishmaniases as emerging and reemerging zoonoses. Author Contributions Conceived and designed the experiments: MAM MM ECG MS. Performed the experiments: MAM IE AR. Analyzed the data: MAM MEM MS. Contributed reagents/materials/analysis tools: MS ECG. Wrote the paper: MAM MM ECG MS. 40. Secretaria de Estado de Turismo, Cultura e Esporte do Estado de Santa Catarina. SANTUR [in Portuguese]. Available: www.santur.sc.gov.br. Accessed 2011 Nov 25. 41. Mitropoulos P, Konidas P, Durkin-Konidas M (2010) New World cutaneous leishmaniasis: Updated review of current and future diagnosis and treatment. J Am Acad Dermatol 10.1016/j.jaad.2009.06.088. References (2009) Population mobility and production of American tegumentary leishman- iasis in the State of Parana´, southern Brazil [in Portuguese]. Rev Soc Bras Med Trop 42(5): 509–514. 21. Ribeiro de Oliveira AT, Ervatti LR, Caetano O9Neill MMV (2011) Internal Migration: The panorama of population displacement in Brazil: PNADs and February 2013 \| Volume 8 \| Issue 2 \| e56177 February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org 8 Cutaneous Leishmaniasis in Subtropical Brazil Cutaneous Leishmaniasis in Subtropical Brazil February 2013 \| Volume 8 \| Issue 2 \| e56177 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9
https://openalex.org/W2888675242	https://orbit.dtu.dk/files/153127048/s13071_018_3012_4.pdf	English	null	Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review	Parasites & vectors	2,018	cc-by	14,071	Citation (APA): Pena-Espinoza, M., Valente, A. H., Thamsborg, S. M., Simonsen, H. T., Boas, U., Enemark, H. L., Lopez- Munoz, R., & Williams, A. R. (2018). Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review. 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Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. y y p g y  You may freely distribute the URL identifying the publication in the public portal Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review Miguel Peña-Espinoza1, Angela H. Valente2, Stig M. Thamsborg2, Henrik T. Simonsen3, Ulrik Boas4, Heidi L. Enemark5, Rodrigo López-Muñoz1 and Andrew R. Williams2 Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review Pena-Espinoza, Miguel ; Valente, Angela H.; Thamsborg, Stig M.; Simonsen, Henrik T.; Boas, Ulrik; Enemark, Heidi L.; Lopez-Munoz, Rodrigo; Williams, Andrew R. Published in: Parasites & Vectors Link to article, DOI: 10.1186/s13071-018-3012-4 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Pena-Espinoza, M., Valente, A. H., Thamsborg, S. M., Simonsen, H. T., Boas, U., Enemark, H. L., Lopez- Munoz, R., & Williams, A. R. (2018). Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review. Parasites & Vectors, 11, Article 475. https://doi.org/10.1186/s13071-018- 3012-4 Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 https://doi.org/10.1186/s13071-018-3012-4 REVIEW Open Access Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review Miguel Peña-Espinoza1, Angela H. Valente2, Stig M. Thamsborg2, Henrik T. Simonsen3, Ulrik Boas4, Heidi L. Enemark5, Rodrigo López-Muñoz1 and Andrew R. Williams2 rrespondence: miguel.pena@uach.cl; miguelpenaespinoza@gmai * Correspondence: miguel.pena@uach.cl; miguelpenaespinoza@gmail.com 1Instituto de Farmacologia y Morfofisiologia, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile Full list of author information is available at the end of the article * Correspondence: miguel.pena@uach.cl; miguelpenaespinoza@gmail.co 1Instituto de Farmacologia y Morfofisiologia, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile Full list of author information is available at the end of the article Abstract Increasing drug resistance in gastrointestinal (GI) parasites of livestock and concerns about chemical residues in animal products and the environment are driving the development of alternative control strategies that are less reliant on the use of synthetic drugs. An increasingly investigated approach is the use of bioactive forages with antiparasitic properties as part of the animal’s diet (nutraceuticals) or as potential sources of novel, natural parasiticides. Chicory (Cichorium intybus) is a multi-purpose crop and one of the most promising bioactive forages in temperate regions, and numerous in vivo trials have explored its potential against parasitic nematodes in livestock. However, it is unclear whether chicory can induce a direct and broad activity against various GI parasites in different livestock species, and the levels of chicory in the diet that are required to exert an efficient antiparasitic effect. Moreover, the mechanisms leading to the reported parasiticidal activity of chicory are still largely unknown, and its bioactive phytochemicals have only recently been investigated. In this review, we summarise the progress in the study of the antiparasitic activity of chicory and its natural bioactive compounds against GI parasites in livestock, through examination of the published literature. The available evidence indicates that feeding chicory can reduce faecal egg counts and/or worm burdens of abomasal nematodes, but not infections with intestinal worms, in ruminants. Highly chicory-rich diets (≥70% of chicory dry matter in the diet) may be necessary to directly affect abomasal parasitism. Chicory is known to synthesise several bioactive compounds with potential antiparasitic activity, but most research has been devoted to the role of sesquiterpene lactones (SL). Recent in vitro studies have confirmed direct and potent activity of SL-rich extracts from chicory against different GI helminths of livestock. Chicory SL have also been reported to exhibit antimalarial properties and its potential antiprotozoal activity in livestock remains to be evaluated. Furthermore, the detailed identification of the main antiparasitic metabolites of chicory and their pharmacokinetics need further confirmation. Research gaps and perspectives on the potential use of chicory as a nutraceutical forage and a source of bioactive compounds for parasite control in livestock are discussed. Keywords: Chicory, Antiparasitic, Helminths, Protozoa, Livestock, Bioactive compounds, Sesquiterpene lactones, Nutraceutical ory, Antiparasitic, Helminths, Protozoa, Livestock, Bioactive compounds, Sesquiterpene lactones, Background (Lespedeza cuneata)] [19, 20]. However, a promising non-tanniniferous bioactive forage in temperate regions is chicory (Cichorium intybus L., Asteraceae), a multi-purpose crop that has attracted considerable inter- est from farmers and scientists for its reported anthel- mintic effects in livestock. Yet, it is still unclear from the available studies whether chicory diets can exert a broad activity against major parasites in different livestock spe- cies, and the levels of chicory in the diet that are re- quired to exert an efficient in vivo effect. This knowledge is critical to validate the use of chicory as an antiparasitic forage and an essential step before chicory can appropriately be included in parasite control pro- grams [29]. In addition, the mechanisms behind the re- ported anthelmintic effects of chicory are still largely unknown and the potential role of its bioactive phyto- chemicals has not been, until recently, thoroughly investigated. g Infections with gastrointestinal (GI) parasites are ubiqui- tous in grazing livestock worldwide, inducing subclinical and clinical diseases that can markedly impair animal health, food production and agricultural economies [1–3]. In ruminants, abomasal and intestinal nematodes are among the most pathogenic GI parasites and efficient deworming programs to control them are essential for sustainable animal farming [4, 5]. Currently, nematode control in livestock largely relies on repeated treatments with synthetic anthelmintic drugs. However, the sustain- ability of this approach is at risk due to the rapid spread of drug-resistant parasite populations [6–8]. Furthermore, there is a growing public awareness, in several countries, of the need to reduce the prophylactic use of veterinary drugs and the subsequent risk of chemical residues in animal products and the environment [9–11]. Outdoor-reared and pasture-based livestock are also progressively being promoted to enhance grassland biodiversity and avoid the feeding of animals with human-edible crops [12–14], but an inevitable conse- quence of these production systems is the persistent exposure to parasitic infections. Consequently, it is imperative to generate novel antiparasitic tools to re- duce the dependence on existing drugs and progress towards the development of integrated and sustain- able parasite control strategies [15, 16]. In this review, we summarise the progress in the study of the antiparasitic activity of chicory and its natural bio- active compounds against GI parasites in livestock, through examination of the published literature. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 14 Page 2 of 14 Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 2 of 14 Background In addition, we identify gaps for future research to advance our understanding on the dietary modulation of parasit- ism using chicory as a model plant, and perspectives on the practical use of chicory as a nutraceutical forage and a potential source of novel plant compounds with broad antiparasitic activity. An increasingly explored approach is the use of bio- active forages with antiparasitic properties in the ani- mal’s diet as nutraceuticals, i.e. a feed which combines nutritional value with beneficial effects on animal health [17–20]. Bioactive plants can also be used as potential sources of novel natural products with activity against livestock (and human) parasites [21–23]. Plants or plant components have been used as dewormers for centuries [24], but only in the last 10–15 years has comprehensive scientific research been conducted to evaluate their in vitro and in vivo activity under experimental and farm- ing conditions [25, 26]. As a result, bioactive forages are now being considered as potential tools for integrated parasite control in livestock [27, 28]. Chicory: a multi-purpose crop for livestock [58] re- ported that beef steers naturally infected with GI nematodes and grazing a mixed chicory/ryegrass pasture (~24% chicory DM in the field), showed no differences in FEC, O. ostertagi/Cooperia spp. third-stage larvae (L3) proportions in faecal cultures, serum pepsinogen or O. ostertagi-antibodies levels, when compared to infected controls grazing ryegrass. A recent study described two in- dependent trials exploring the anthelmintic effects of chicory-rich diets (≥70% forage chicory in the DM intake) against experimental infections with GI nematodes in cat- tle [56]. In the first (indoor) experiment, stabled calves in- fected with O. ostertagi and Cooperia oncophora and fed chicory silage diets had a largely similar FEC pattern throughout the trial, in comparison with infected control calves fed a balanced protein/energy diet. However, post-- mortem nematode recovery revealed that calves fed chic- ory silage had significantly lower worm burdens of O. ostertagi (geometric mean reduction of 60%) in compari- son with control animals. In contrast, adult counts of C. oncophora in the small intestine were not statistically dif- ferent between groups. In a second (grazing) experiment, calves mono-infected with O. ostertagi and grazed without possibilities of reinfections on a pure-chicory field (90% chicory DM in the sward), and later supplemented with chicory silage, had similar FEC in comparison with ani- mals grazing a ryegrass/clover pasture until Day 20 post-infection. However, from Day 22 post-infection on- wards a rapid and significant reduction in faecal egg ex- cretion was observed in chicory-fed calves, with a 65% lower mean FEC compared with control animals at the end of the trial. Post-mortem worm recovery revealed that calves fed chicory had highly significant reductions in the adult worm burdens of O. ostertagi (geometric mean re- duction of 66%) in comparison with control animals [56]. These results strongly indicate that chicory feeding can directly reduce abomasal parasitism in ruminants, particu- larly affecting adult nematodes. Furthermore, some evidence of potential indirect an- dry matter (DM), depending on N input and defoliation frequency], low neutral detergent fibre levels and high vol- untary feed intakes in ruminants [37, 39–41]. Ruminants grazing pure forage chicory have shown similar or higher weight gains in comparison with animals grazed on ryegrass, if provided at comparable levels of DM intake [42–44]. Chicory-based diets have also been reported to sustain milk production in cattle [45–47] and in sheep [48]. Chicory: a multi-purpose crop for livestock However, milk from cows fed pure-chicory had a re- portedly bitter taint that has prompted the recommenda- tion to restrict its intake in dairy cows [39]. Anthelmintic activity of chicory against GI parasites Over the last two decades, several peer-reviewed stud- ies have explored the in vivo effects of dietary forage chicory against GI nematode infections in ruminants. Table 1 summarises the main results reported in these experiments, as well as the ruminant species involved (sheep, cattle and deer), the parasite species (aboma- sal and/or small intestinal nematodes) and experimen- tal design. If available, information of the chicory cultivar used, the percentage of chicory DM in the di- ets and the screening of bioactive compounds are also presented. Despite major differences between the studies listed in Table 1, two general conclusions can be drawn. First, feeding with forage chicory can re- duce worm faecal egg counts (FEC) and/or worm burdens with abomasal nematodes (Haemonchus con- tortus and Teladorsagia circumcincta in sheep and Ostertagia ostertagi in cattle), but not infections with intestinal worms in ruminants [49–56]. Secondly, from trials involving experimental infection of worm-naïve animals, feeding levels of ≥70% of chic- ory DM in the diet resulted in a significant reduction of abomasal parasitism with T. circumcincta [49, 52] and O. ostertagi [56], and in reduction of H. contortus FEC [53]. The lowest level of chicory DM in the diet to be associated with an anthelmintic activity was ~50% [54], whereas diets with 24% of chicory DM or lower did not result in any noticeable antiparasitic ef- fect [57, 58]. Grazing trials in New Zealand were the first to explore the anthelmintic effects of chicory in livestock [49, 59, 60]. Niezen et al. [59] described lower strongyle FEC and serum pepsinogen levels in chicory-fed lambs at Day 42 post-experimental infection with T. circumcincta and Trichostrongylus colubriformis. Subsequently, Scales et al. [49] reported significantly lower FEC and reduced worm burdens of Teladorsagia spp. but not intestinal Trichos- trongylus spp. in lambs grazing forage chicory, as com- pared to lambs on control ryegrass (Lolium perenne), cooksfoot (Dactylis glomerata) or tall fescue (Festuca arundinacea). Later studies were in agreement with these findings; e.g. Marley et al. [50] reported a significant Furthermore, some evidence of potential indirect an- thelmintic effects of chicory has been described [61, 62]. Tzamaloukas et al. Chicory: a multi-purpose crop for livestock Chicory: a multi-purpose crop for livestock Chicory is a perennial, deep-rooting herb that can be found as a wild plant in natural grasslands and roadsides and as cultivated varieties in most temperate areas of the world, including Northern Europe [30, 31]. In some European, Asian and Middle Eastern countries, chicory has traditionally been used for human consumption and as a medicinal plant to treat several diseases, includ- ing malaria and digestive, liver and urinary disorders [32, 33]. Based on its current applications, cultivated chicory can be classified in four types [33]: (i) indus- trial or root chicory used for the production of inulin-type fructans and as coffee substitute; (ii) Brussels or witloof chicory, for production of etiolated leaves (“chicons”); (iii) leaf chicory for human con- sumption (fresh salad or cooked); and (iv) forage chicory for animal feeding. In the 1980s, selection of chicory for livestock feeding resulted in the release of the first commercial forage variety, “Grasslands Puna” [34]. Since then, several forage chicory cultivars have been developed [35–38]. The use of chicory diets in livestock nutrition has been previously reviewed [37, 39]. In general, forage chicory varieties grow predominantly at temperatures > 10 °C, are rich in minerals (Zn, B, Mn) with variable protein levels [100–250 g crude protein/kg The antiparasitic mechanisms of bioactive forages can be broadly grouped in direct or indirect effects. Direct effects refer to the chemical interaction between plant compounds and specific parasite molecules or struc- tures, resulting in subsequent mortality and removal from the host, whilst indirect effects involve the stimula- tion or boosting of host responses that can induce worm expulsion, either through overall improved nutrition or a direct interaction between plant compounds and host cells (e.g. leukocytes) [20]. Until now, most of the re- search focus on antiparasitic forages has been devoted to tannin-rich plants [e.g. sulla (Hedysarum coronarium), sainfoin (Onobrychis viciifolia), sericea lespedeza Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 3 of 14 reduction of adult and fourth-stage larvae (L4) abomasal nematodes in chicory-fed lambs, while no effect was ob- served on intestinal worms. Following trials using experi- mental infections in lambs confirmed that feeding forage chicory-based diets could directly affect the adult burden and FEC of abomasal worms, but not of intestinal nema- todes in sheep [51–53]. In cattle, Marley et al. Chicory: a multi-purpose crop for livestock [61] reported that chicory-fed lambs had higher levels of abomasal mucosal mast cells and globule leucocytes, correlating with lower larval develop- ment and worm establishment in these animals. The au- thors attributed this enhanced cellular immunity to the higher protein content of the chicory diet (crude protein 18.3% in chicory vs 11.3% in clover-grass) [61]. Athana- siadou et al. [62] reported lower FEC and higher body Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 4 of 14 Table 1 Summary of peer-reviewed in vivo experiments studying the anthelmintic effects of dietary chicory in ruminants Livestock species Chicory DMa (%) Chicory cv. (sown) Bioactive compounds in chicory (% DM)b Nematode infection and experimental designc Anthelmintic effects in chicory-fed animalsd Reference Sheep 91 ni (pure) Total CT = 0.83% Exp. T. circumcincta trickle infection while grazing chicory. After 40 days p.i. animals were stabled, treated with levamisole + ivermectin and challenged with T. circumcincta L3 Lower numbers of mid/late L4 and L5/adult worms from the challenge infection. No effect on FEC while grazing on chicory and trickle infected [61] 87 ni (pure) Total CT = 0.05%; total phenolics = 26.2% Exp. T. circumcincta infection Reduction in adult male worms and lower total adult counts by 43% (ns). No effect on FEC, female per capita fecundity or immature worm counts [52] 80 Grasslands Puna (pure) nr Exp. Teladorsagia spp., Trichostrongylus spp., Oesophagostomum spp., Cooperia spp. and Nematodirus spp. infection + reinfection from pasture Reduced FEC and Teladorsagia spp. worm counts [49] 80 ni (pure) Total CT = 0.05%; total phenolics = 26.2% Exp. T. colubriformis infection No effect on FEC, adult or immature worm counts [51] 80 Grasslands Puna (pure) Total CT = 0.31% Exp. H. contortus and C. curticei infection (animals stabled) Reduced total egg output and FECDM of H. contortus. No effect on adult worm counts, on FECDM of C. curticei or on total FECDM [53] 50 Puna II (pure) nr Exp. T. circumcincta trickle infection of ewes and reinfection from pasture for ewes and lambs Reduced FEC in lambs and ewes on chicory and lower T. circumcincta L3 recovered from chicory swards (ns) [54] nr Grasslands Puna (pure) nr Natural infection Reduced abomasal adult counts by 41% and abomasal L4 by 60%. No effect on FEC or intestinal worm counts [50] nr Grasslands Puna (pure) Total phenolics = 1.8–2.7% Natural infections in ewes and their lambs. In sward/diet bDetection of bioactive compounds were performed with different methodologies and are not comparable between studies cAll studies were grazing experiments, unless otherwise indicated (stabled animals). In all the trials ryegrass or ryegrass/white clover were used as diets for control animals, with the exception of Heckendorn et al. [53] who fed control lambs with ryegrass/lucerne fresh-cut and Miller et al. [55] who used Bermuda grass dIn comparison to infected animals fed with control diets Abbreviations: cv cultivar, ns not significant, DM dry matter, exp. experimental, p.i. post-infection, CT condensed tannins, SL sesquiterpene lactones, FEC faecal egg counts, FECDM FEC adjusted per g of faecal DM, L1 first-stage larvae, L3 third-stage larvae (free-living), L4 fourth-stage larvae (immature), L5 fifth-stage larvae, nr not reported, ni not identified Sesquiterpene lactones: the main antiparasitic compounds in chicory? The SL are a group of extremely diverse natural terpe- noids, including around 5000 different molecules, which are mainly found in Asteraceae plants [90]. The SL are primarily involved in plant defence against herbivory [68, 91, 92]. Numerous studies have demonstrated sub- stantial biological properties exerted by SL, including antibiotic, antiprotozoal, trematocidal and anticancer ac- tivities [91, 93–95]. As an example, artemisinin, a SL ori- ginally isolated from the Chinese herb Artemisia annua (“qinghao”; “sweet wormwood”) and artemisinin-derivatives (dihydroartemisinin, artesunate, artemether) are currently the front-line antimalarial drugs worldwide [96, 97]. Natural SL are classified in groups depending on their structural conformation, including: germacranolides, eudesmanolides, elemanolides, heliangolides, cadinanolides, eremophilano- lides, xanthanolides, guaianolides and pseudoguaianolides, among others [71, 98]. In species of the Cichorieae tribe (including chicory) around 360 SL have been reported, all part of the groups of guaianolides (~243 SL), eudesmano- lides (~73 SL) or germacranolides (~44 SL) [71]. In chicory leaves, three guaianolide SL and their 11,13-dihy- dro-derivatives are commonly present: lactucin (LAC), 8-deoxylactucin (8-DOL), lactucopicrin (LCP), 11,13-dihydro-LAC, 11,13-dihydro-8-DOL and 11,13-dihydro-LCP (Table 2, Fig. 1) [68, 70, 99–102]. Other SL have also been detected in chicory Chicory: a multi-purpose crop for livestock Ewes were either treated or not treated with anthelmintics Lower FEC in lambs from untreated ewes on chicory. No effect on FEC of undrenched ewes or on adult worm burden in lambs [62] nr Oasis (pure) nr Natural infection Reduced FEC. Chicory-fed lambs had lower FAMACHA scores and required less anthelmintic treatments [55] 3–6 Grasslands Puna (mixed with RG/ WC) nr Natural infection No effect on FEC [57] nr Grasslands Puna (nr) nr Exp. T. circumcincta and T. colubriformis infection + reinfection from pasture Reduced FEC and serum pepsinogen levels at Day 42 p.i. [59] Cattle 90 Spadona (pure) Total CT = ni; total SL = 1.7–2.3% Exp. O. ostertagi infections Reduced worm burdens by 66%. Reduced FEC from Day 21 p.i. onwards [56] 70 Spadona (pure) Total CT = ni; total SL = 1.2% Exp. Ostertagia ostertagi and Cooperia oncophora infection (stabled animals) Reduced O. ostertagi worm burdens by 60%. Reduced recovery of O. ostertagi L3. No effect on total FEC and worm burdens of C. oncophora [56] 24 Puna II (mixed with RG) nr Natural infection No effect on FEC, proportion of O. ostertagi or Cooperia spp. L3 in larval cultures, serum pepsinogen or O. ostertagi-antibodies [58] Deer 56–71 Grasslands Puna (pure) Total CT = 0.17–0.26% Natural infection Fewer clinical signs associated with parasitism, requiring less anthelmintic treatment. No effect on FEC or lungworm L1 counts [60] aIn sward/diet bDetection of bioactive compounds were performed with different methodologies and are not comparable between studies cAll studies were grazing experiments, unless otherwise indicated (stabled animals). In all the trials ryegrass or ryegrass/white clover were used as diets for control animals, with the exception of Heckendorn et al. [53] who fed control lambs with ryegrass/lucerne fresh-cut and Miller et al. [55] who used Bermuda grass dIn comparison to infected animals fed with control diets Abbreviations: cv cultivar, ns not significant, DM dry matter, exp. experimental, p.i. post-infection, CT condensed tannins, SL sesquiterpene lactones, FEC faecal egg counts, FECDM FEC adjusted per g of faecal DM, L1 first-stage larvae, L3 third-stage larvae (free-living), L4 fourth-stage larvae (immature), L5 fifth-stage larvae, nr not reported, ni not identified Page 5 of 14 Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 5 of 14 Peña-Espinoza et al. Chicory: a multi-purpose crop for livestock These anthelmintic effects are be- lieved to be related to the high content of easily-fermentable fructans (inulin) in the tested chicory roots, which can modify the large intestinal environment and microflora, leading to the increased production of short-chain fatty acids and lactic acids: all factors that can directly or indirectly affect the parasites [88, 89]. Thus, whilst it is probable that chicory roots may have indirect anthelmintic properties in monogastrics due to a prebiotic effect of inulin, the antiparasitic effects of forage chicory observed in grazing ruminants are more likely related to the more abundant bioactive phyto- chemicals in the leaves. Chicory: a multi-purpose crop for livestock Parasites & Vectors (2018) 11:475 weight gains in suckling, naturally-infected lambs from ewes on chicory compared with lambs from ewes graz- ing grass/clover, although no effect was observed on ab- omasal/intestinal worm counts in lambs or in the FEC of their periparturient dams. Whereas the reduced FEC in lambs from chicory-fed ewes could be explained by a reduced worm fecundity or reduced challenge from pas- ture (see below), the authors related the improved per- formance of these lambs to the higher nutritional quality of chicory rather than to a direct anthelmintic effect, given the similar worm burdens in lambs grazing chicory or grass/clover [62]. These two studies suggest that chic- ory diets can either indirectly affect GI parasitism by en- hancing the immune response [61] or by increasing the animals’ resilience to parasite infections [62]. Further re- search is needed to confirm these initial observations and to identify if chicory feeding can induce immune-stimulatory effects on concurrent parasitic in- fections. Moreover, in a different approach, some studies have also suggested that chicory can induce detrimental effects on free-living stages of GI nematodes, potentially due to the activity of undigested bioactive compounds in faeces or due to changes in the sward environment, that could affect nematode survival and the level of parasite challenge from pasture [63–65]. However, at present the strongest evidence exists for a direct anthelmintic effect of chicory inside the host digestive tract, and recent studies combining parasitology and phytochemistry have begun to probe the constituent antiparasitic compounds in chicory. Plasmodium falciparum [72], as well as insecticidal [68] and anti-inflammatory [75] properties. Chicory leaves and roots also contain sugars (fructose, glucose and su- crose) and high levels of pectins (uronic acids as build- ing blocks) [82–84]. Chicory roots are rich in inulin-type fructans (comprising up to 70% of the DM in chicory roots), with only traces present in leaves [33, 82, 85]. Inulin is known for its prebiotic properties and pigs sup- plemented with dried chicory roots (30–35% DM in the diet) had significantly lower FEC and worm burdens of Oesophagostomum spp. and reduced larval counts of As- caris suum [86, 87]. Antiparasitic activity of chicory: unveiling the bioactive compounds Further research by Table 2 Bioactive phytochemicals in chicory (Cichorium intybus) and their reported bioactivity Bioactive compounds Molecular formula Reported bioactivity Model Reference fo bioactivity Guaianolide sesquiterpene lactones Lactucin C15H16O5 antiprotozoal Plasmodium falciparum [72] 11,13-dihydrolactucin C15H18O5 nr 8-deoxylactucin C15H16O4 anti-inflammatory HT29 cells [75] insecticidal Schistocerca gregaria [68] 11,13-dihydro-8-deoxylactucin C15H18O4 nr Lactucopicrin C23H22O7 antiprotozoal Plasmodium falciparum [72] insecticidal Schistocerca gregaria [68] 11,13-dihydrolactucopicrin C23H24O7 nr Hydroxycinnamic acids Monocaffeoyl tartaric acid (Caftaric acid) C13H12O9 nr Chlorogenic acid C16H18O9 antibacterial Staphylococcus aureus, Streptococcus pneumoniae, Bacillus subtilis, Escherichia coli, Shigella dysenteriae, Salmonella typhimurium [76] Caffeic acid C9H8O4 anticancer Fibrosarcoma (HT-1080) [77] acaricidal Rhipicephalus annulatus [78] Chicoric acid C22H18O12 insecticidal Schistocerca gregaria [68] p-Coumaric acid C9H8O3 anti-inflammatory Female albino rats of Wistar strain (in vivo) [79] Caffeoylmalic acid C13H12O8 nr Ferulic acid C10H10O4 nr Flavonoids Quercetin 3-O-glucuronide + Luteolin 7-O-glucuronide C21H18O13 acaricidal Rhipicephalus annulatus [78] Quercetin malonyl glucoside C24H22O15 nr Apigenin glucuronide C21H18O11 nr Kaempferol glucuronide C21H18O12 anticancer and anti- inflammatory Human pancreatic cancer cells [80] Isorhamnetin 3-glucuronide C22H20O13 nr Kaempferol-7-O-(6″-O-malonyl)- glucoside C24H22O14 nr Anthocyanins Cyanidin 3-O-glucoside C21H21O11 + nr Cyanidin 3-O-(6-malonyl)-glucoside C24H23O14 + anti-inflammatory Lipid peroxidation and cyclooxygenase (COX-1 and -2) inhibition assay [81] Coumarins Cichoriin C18H20O3 insecticidal Schistocerca gregaria [68] Notes: p-Coumaric acid, p-Coumaric acid, Ferulic acid, Isorhamnetin 3-glucuronide and kaempferol-7-O-(6″-O-malonyl)-glucoside were purified and identified by Papetti et al. [74]. Cichorin A was detected and identified by Rees & Harboure [68]. The remaining compounds were identified by Ferioli et al. [73]. All studies reporting bioactivity were performed in vitro, unless otherwise indicated (in vivo) Abbreviations: nr not reported Table 2 Bioactive phytochemicals in chicory (Cichorium intybus) and their reported bioactivity Notes: p-Coumaric acid, p-Coumaric acid, Ferulic acid, Isorhamnetin 3-glucuronide and kaempferol-7-O-(6″-O-malonyl)-glucoside were purified and identified by Papetti et al. [74]. Cichorin A was detected and identified by Rees & Harboure [68]. The remaining compounds were identified by Ferioli et al. [73]. All studies reporting bioactivity were performed in vitro, unless otherwise indicated (in vivo) Abbreviations: nr not reported rhizomes [103], but not yet described in other parts of the plant. effects to chicory SL in the digestive fluids, although no detection of SL was reported [104]. Further research by Molan et al. [105] described inhibitory in vitro effects of water/ethyl acetate crude SL-extracts from chicory roots on the motility of Dictyocaulus spp. L1 and L3 and of GI nematode L3 from deer. Antiparasitic activity of chicory: unveiling the bioactive compounds Chicory has previously been referred to as a “tanninifer- ous forage”, although only low levels of condensed tan- nins (≤0.8% in DM) are commonly detected in chicory leaves [52, 53, 56, 66, 67]. As a member of the Astera- ceae, chicory is known to synthesise sesquiterpene lac- tones (SL), a group of biologically-active natural terpenoids which are also partly responsible for its bitter taste [68–72]. Sesquiterpene lactones are present in chic- ory leaves (~2% in DM; [56]) and roots (~0.4% in DM; [68]) and can be detected as free molecules and/or as glycosides [73]. Beside SL, four other groups of phyto- chemicals with reported bioactivities have been de- scribed in chicory: hydroxycinnamic acids (e.g. chicoric, cholorogenic and caffeic acid derivatives), flavonoids (e.g. quercetin and kaempferol derivatives), anthocyanins and coumarins [68, 73, 74]. The bioactive phytochemi- cals described in chicory are presented in Table 2, as well as their reported bioactivity. Several of these com- pounds have been investigated for their bioactivity as single molecules [68, 75–81]. Noticeably, some of the chicory SL have exhibited antimalarial activity against Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 6 of 14 rhizomes [103], but not yet described in other parts of the plant. effects to chicory SL in the digestive fluids, although no detection of SL was reported [104]. Antiparasitic activity of chicory: unveiling the bioactive compounds Stronger evidence of the antiparasitic activity of chicory SL has been provided by novel studies involving in vitro assays and high-throughput chemical profiling of the tested chicory extracts. Foster et al. [100] de- scribed a dose-dependent inhibition of egg hatching in free-living stages of H. contortus by SL-rich extracts from two forage chicory cultivars (“Grasslands Puna” and “Forage Feast”). The authors identified the main guaianolide SL of chicory in the tested extracts (LAC, 8-DOL and LCP) and observed an increased ovicidal ac- tivity of the Grasslands Puna-extract, which correlated with its higher content of 8-DOL [100]. Recent studies have shown a potent and dose-dependent in vitro ac- tivity of SL-containing chicory extracts against parasitic stages of cattle nematodes (adult O. ostertagi and C. oncophora), which are expected to be main targets of dietary SL in the host [102, 106]. The same chicory ex- tracts were subsequently tested in vitro against the phylogenetically distinct pig nematodes A. suum and Oesophagostomum dentatum, observing also a potent ac- tivity on these helminths [107]. Chemical profiling of these tested SL-extracts (isolated from two chicory culti- vars, “Spadona” and “Puna II”) revealed the presence of LAC, 8-DOL and LCP and their 11,13-dihydro-derivatives, although with differences in the concentration of individual SL between cultivars [102]. Interestingly, these SL-extracts showed distinct anthelmintic potencies against cattle and pig nematodes, and this variable activity was possibly linked to the different SL profiles in the tested extracts [102, 107]. Table 3 summarises the EC50 values obtained in in vitro as- says with parasitic stages of cattle and pig GI nematodes ex- posed to similar chicory SL-extracts. These results indicate that chicory SL-extracts have potent and broad-spectrum activity against parasitic stages of GI nematodes. [105]. Stronger evidence of the antiparasitic activity of chicory SL has been provided by novel studies involving in vitro assays and high-throughput chemical profiling of the tested chicory extracts. Foster et al. [100] de- scribed a dose-dependent inhibition of egg hatching in free-living stages of H. contortus by SL-rich extracts from two forage chicory cultivars (“Grasslands Puna” and “Forage Feast”). The authors identified the main guaianolide SL of chicory in the tested extracts (LAC, 8-DOL and LCP) and observed an increased ovicidal ac- tivity of the Grasslands Puna-extract, which correlated with its higher content of 8-DOL [100]. Antiparasitic activity of chicory: unveiling the bioactive compounds However, no chemical identifi- cation of SL in the tested chicory extracts was performed The anthelmintic potential of chicory SL was first sug- gested by Schreurs et al. [104] in a study describing the reduced motility of Dictyocaulus spp. first-stage larvae (L1) incubated in rumen and abomasal fluid from deer grazing chicory. The authors attributed the observed Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 7 of 14 Fig. 1 Structures of the guaianolide sesquiterpene lactones reported in chicory Fig. 1 Structures of the guaianolide sesquiterpene lactones reported in chicory Based on two in vivo experiments in which chicory di- ets (with SL profiling) induced a reduction in adult O. ostertagi counts in cattle, the estimated daily consump- tion of total chicory SL was 27.3 g SL/animal (Exp. 1; 70% chicory DM in diet) and 128.3 g SL/animal (Exp. 2; 90% chicory DM in diet), equivalent to 222 and 761 mg total SL/kg of body weight in animals from Exp. 1 and 2, respectively [56]. However, these estimations do not in- dicate the concentration of individual SL, which may po- tentially induce an antiparasitic effect at lower concentrations. Other studies reporting anthelmintic ac- tivity of chicory in vivo did not describe the content of SL in the diets, although it is expected that the concen- trations of total and individual chicory SL vary depend- ing on cultivar, crop age, season of the year and cultivation site [38, 108]. Nevertheless, if SL are truly the compounds behind the direct activity of chicory against GI parasites, these bioactive metabolites should reach the target organs in sufficient concentrations to exert their antiparasitic effect. Potential differences in the ac- tivity and concentration of free and glycoside (bound) SL and their metabolisation in different gut compart- ments can influence their total concentration and activ- ity [100]. Yet, the pharmacokinetics of chicory SL in livestock have not been investigated. Nonetheless, the metabolism of costunolide (Fig. 2a), a germacranolide SL and the parent compound of chicory guaianolide SL [109], has been studied in rats [110–112]. Poor absorp- tion and a peak plasma concentration of 0.024 μg costu- nolide/ml at 9.0 h post-treatment were observed in rats orally treated with a single dose of costunolide (25 mg/ kg) [110]. Similar results were reported in rats orally dosed with a costunolide-containing extract of the herb Aucklandia lappa (given at a single dose of 2 g extract/ [105]. Antiparasitic activity of chicory: unveiling the bioactive compounds Recent studies have shown a potent and dose-dependent in vitro ac- tivity of SL-containing chicory extracts against parasitic stages of cattle nematodes (adult O. ostertagi and C. oncophora), which are expected to be main targets of dietary SL in the host [102, 106]. The same chicory ex- tracts were subsequently tested in vitro against the phylogenetically distinct pig nematodes A. suum and Oesophagostomum dentatum, observing also a potent ac- tivity on these helminths [107]. Chemical profiling of these tested SL-extracts (isolated from two chicory culti- vars, “Spadona” and “Puna II”) revealed the presence of LAC, 8-DOL and LCP and their 11,13-dihydro-derivatives, although with differences in the concentration of individual SL between cultivars [102]. Interestingly, these SL-extracts showed distinct anthelmintic potencies against cattle and pig nematodes, and this variable activity was possibly linked to the different SL profiles in the tested extracts [102, 107]. Table 3 summarises the EC50 values obtained in in vitro as- says with parasitic stages of cattle and pig GI nematodes ex- posed to similar chicory SL-extracts. These results indicate that chicory SL-extracts have potent and broad-spectrum activity against parasitic stages of GI nematodes. Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 8 of 14 Table 3 In vitro activity of purified chicory extracts (cv. Spadona) against parasitic stages of gastrointestinal nematodes of livestock Nematode species Life-stage Incubation period with extract (h) EC50 μg/ml Reference Ostertagia ostertagi Adults 24 80 [102] Cooperia oncophora Adults 24 150 [106] Ascaris suum L3/L4 16/12 81/116 [107] Oesophagostomum dentatum Adults/L4 24/36 305/372 [107] Abbreviation: EC50, effective concentration of purified chicory extracts able to inhibit the motility in 50% of exposed nematodes kg, equivalent to 15.7 mg costunolide/kg) [112]. In that study, costunolide was detected in plasma at 5 min after administration and reached a peak concentration of 0.02 μg/ml at 10.5 h post-treatment [112]. These results con- trast observations after intravenous injection of costuno- lide (20 mg/kg), resulting in peak plasma concentrations of 12.3 μg/ml in rats [111]. Previous researchers have ex- plored the pharmacokinetics of other SL such as artemi- sinin (Fig. 2b) and its derivatives, which are structurally unrelated with chicory guaianolide SL, but knowledge of their metabolism may provide useful insights to eluci- date the fate of chicory SL in livestock. Goats treated or- ally with artemisinin (23 mg/kg) partially absorbed the compound, which was metabolised into the more bio- logically active dihydroartemisinin (DHA). Antiparasitic activity of chicory: unveiling the bioactive compounds However, the bioavailability was low (peak of 0.7 μg DHA/mL plasma at 12 h post-treatment) and most of the compound was excreted as unabsorbed artemisinin in faeces (2.4 μg ar- temisinin/g faeces at 24 h post-treatment) [113]. The same authors incubated artemisinin with alfalfa hay in rumen liquid in vitro and did not detect metabolisation of artemisinin to DHA by the rumen microbiota. They also reported a good artemisinin stability at pH 3.0 and 6.8, with a tendency of artemisinin to bind to rumen content and poor solubility in rumen fluid medium [113]. Similarly, Cala et al. [114] reported that sheep treated orally with artemisinin (100 mg/kg) excreted the unabsorbed compound in faeces (peak of 126.5 μg arte- misinin/g faeces 24 h post-treatment). These results sug- gest that oral/dietary SL can survive rumen fermentation and passage through the GI tract, while being partly metabolised and poorly absorbed by ruminants, leading to a low systemic distribution of the bioactive metabo- lites. However, despite the evidence of a low absorption of oral/dietary SL, the metabolisation of these com- pounds could lead to the formation of more active com- pounds inside the digestive tract that can directly affect GI parasites. Previously, it was reported that the guaia- nolide SL matricin, naturally present in chamomile (Matricaria recutita) and other Asteraceae plants, is partly metabolised into the anti-inflammatory compound chamazulene carboxylic acid (a natural profen) in artifi- cial gastric fluid, but not in artificial intestinal fluid [115]. The metabolisation of chicory SL into more active compounds inside the GI tract of livestock warrants further investigation, and this knowledge could help to explain the differences in anthelmintic activities of chicory against ab- omasal and small intestinal parasites reported in ruminants. Elucidating the anthelmintic mechanisms of SL Elucidating the anthelmintic mechanisms of SL Guaianolides and other SL are known to exert potent cytotoxic activities, mainly related to the presence of an α-methylene (CH2) group attached to the γ-lactone in the SL molecule (Fig. 1: Core A) [91, 116]. This α-methylene group reacts with sulfhydryl (thiol) groups of free cysteine or with cysteine-containing peptides, en- zymes or other proteins by a Michael-type addition, leading to an alkylation of cellular macromolecules and disruption of cellular functions (e.g. impairing cell sig- nalling, cell replication and mitochondrial respiration) [98, 116, 117]. As an example, natural SL were reported to reduce the intracellular concentration of free glutathi- one in Leishmania mexicana promastigotes, which led to a toxic intracellular accumulation of reactive oxygen species and blocked cell proliferation in vitro [118]. However, there is also evidence that SL may interact with amino acids other than cysteine [91] and SL lacking the α-methylene group (like chicory SL dihydro-derivatives with a CH3 group attached to the γ-lactone instead, Fig. 1: Core B) can still exert activ- ity in different biological systems [95, 119], suggesting that there is not only one mechanism of action for all SL. In comparison, artemisinin and its derivatives are thought to have a distinct mechanism of antimalarial activity through the cleavage of a peroxide bridge (present in the artemisinins but absent in chicory SL), which reacts with heme-iron in infected erythrocytes Fig. 2 Structures of the sesquiterpene lactones costunolide (a) and artemisinin (b) Fig. 2 Structures of the sesquiterpene lactones costunolide (a) and artemisinin (b) Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 9 of 14 Page 9 of 14 concentration and activity of these molecules in different organs against various GI parasites. At present, it is un- known whether the discrepant in vivo efficacy of chicory against abomasal and intestinal nematodes is explained by distinctly different concentrations of active metabo- lites reaching the different target organs, as described for condensed tannins in cattle [126]. Recently it has been confirmed that purified chicory extracts can induce dose-dependent and potent effects against adult C. onco- phora in vitro [106], which suggests that the lack of in vivo activity of chicory diets against small intestinal nematodes may be the result of physico-chemical changes in the digestive tract of the host. Therefore, the fate of chicory antiparasitic compounds in the GI tract of ruminants, as well as monogastrics, needs to be inves- tigated. Elucidating the anthelmintic mechanisms of SL Furthermore, the molecular mechanisms under- pinning the direct activity of SL and/or other chicory bioactive compounds against GI parasites need to be elucidated by exploring the potential inhibitory effects on vital processes, like metabolic routes and enzymatic pathways. In addition, it is also pertinent to study the potential parasite adaptation to natural SL and other chicory phytochemicals, as earlier described for con- densed tannins and H. contortus [127]. and leads to the production of highly reactive free radicals and parasite death [120]. p In previous studies, cattle and swine nematodes ex- posed to chicory SL-extracts in vitro and examined by electron microscopy showed no obvious structural damage in the buccal opening, cuticle or muscle tis- sue, suggesting that SL may induce anthelmintic ac- tivity via specific and subtle mechanisms rather than a more generalised degenerative effect [102, 107]. This is in contrast with the reported anthelmintic ac- tivities of other plant bioactive compounds such as condensed tannins or cysteine proteinases, which are known to induce marked disruptions in the cuticle of exposed worms [22, 121, 122]. Given the potent and rapid paralysis of nematodes exposed to SL-extracts [102, 107], SL may act as selective inhibitors in the nematode neuromuscular system. A previous study has reported that 8-DOL and LCP isolated from chic- ory roots can inhibit the activity of acetylcholinester- ase (AChE) in vitro [123]. A potential inhibition of AChE in nematodes could lead to a build-up of acetylcholine in the neuromuscular junctions result- ing in worm paralysis due to permanent contraction [124, 125]. Nematode AChE contains cysteine residues [124], that may interact with the α-CH2-γ-lactone group of SL via Michael addition, and/or by other alkylation process induced by SL. However, the potential activity of chicory SL on nematode AChE remains to be investigated. The available evidence has confirmed that chicory di- ets selectively affect abomasal nematodes in infected ru- minants, particularly O. ostertagi in cattle and T. circumcincta in sheep. Only one study has reported in vivo effects of chicory against the major abomasal nematode H. contortus, observing a significant reduction in FEC but no effect on adult worm burdens in chicory-fed lambs [53]. In relation, a previous study de- scribed that feeding of lambs with the SL-containing herb wormwood (Artemisia absinthium, Asteraceae) at 20% of DM intake, induced a significant reduction in H. contortus worm burdens and FEC [128]. Elucidating the anthelmintic mechanisms of SL Therefore, add- itional studies are needed to confirm the activity of chic- ory towards the highly pathogenic H. contortus in small ruminants. Furthermore, all the available in vivo studies to date testing the antiparasitic activity of forage chicory diets have been performed in ruminants. Promising in vitro activity of SL-extracts of forage chicory against pig nematodes [107] must be confirmed in vivo, and poten- tially also explored in other livestock species. Further research and perspectives on the use of chicory for parasite control in livestock One the major gaps in the understanding of the antipar- asitic activity of chicory is the precise identification of the responsible compounds. Based on their broad bioactivity, SL are likely the main antiparasitic phytochemicals in chicory, which is supported by previ- ous work reporting differences in the anthelmintic po- tency of chicory extracts with distinct content of SL [100, 102, 107]. However, further fractionation, com- pound identification and potential isolation of individual SL, with subsequent bioassays, will be necessary to show conclusively that SL are responsible for the antiparasitic activity of chicory. This research can also help to identify whether individual SL or the combined activity of several SL can induce parasiticidal activity. Notably, it is also ne- cessary to scrutinise if antiparasitic compounds other than SL are present in chicory by studying the activity of different plant fractions and describing their components. At present, the antiparasitic activity of chicory has been primarily evaluated in GI helminths and substantial research is needed to explore the potential effects of chicory and its bioactive metabolites against other major GI parasites, such as protozoans. In relation, a previous study confirmed direct antiprotozoal effects of purified SL (LCP and LAC) from chicory roots against the malar- ial parasite P. falciparum (Honduras-1 strain) in vitro, with total inhibition of parasite growth at 50 and 10 μg/ ml of LCP and LAC, respectively [72]. Besides Pharmacokinetic studies are warranted to confirm whether chicory SL and/or other phytochemicals are metabolised into more active compounds in the digestive tract of ruminants and monogastrics, as well as the Page 10 of 14 Peña-Espinoza et al. Parasites & Vectors (2018) 11:475 Page 10 of 14 production systems (measured as enhanced animal productivity derived from its antiparasitic effects and im- proved nutritional value) must be assessed and com- pared with the extra costs associated with the growth and/or access to chicory. These benefits are expected to vary according to management systems (e.g. availability of land for cultivation of pure chicory, length of the grazing season, parasite exposure, etc.) and in relation to different livestock age groups (e.g. infected with different parasite species). In a nutraceutical approach, and as any other bioactive forage, chicory is also a feed and there- fore both its nutritional and antiparasitic value should be evaluated [20]. In addition, several other aspects must be addressed before recommending the use of chicory diets for on-farm parasite control. Further research and perspectives on the use of chicory for parasite control in livestock One issue is the ap- parent lack of activity against small intestinal worms in ruminants, as grazing animals are normally infected with mixed nematode species. The agronomical shortcoming of cultivating forage chicory should also be considered, like the rapid growth of low-quality reproductive stems in late spring/early summer (from the second year on- wards), high vulnerability to damage by grazing stock, its relatively low persistency of around three years and its dormant state during winter, droughts and in dry sum- mers [145, 146]. Another challenge is to ensure chicory feeding for parasitised animals, as the timing of chicory growth and infection dynamics may not match during the grazing season. An alternative could be the preserva- tion of chicory for use when needed, independent of sea- son and pasture availability. Previous studies have explored the conservation of forage chicory as silage to feed cattle and pigs [56, 147, 148] and further research needs to evaluate whether ensiling can affect the con- centration and structure of chicory antiparasitic metabo- lites. However, the ensiling of chicory can be challenging due to the low DM of forage chicory and its broad leaves that can lead to soil contamination during harvest, redu- cing silage quality [149]. antiplasmodial effects, potent activity of other natural SL against trypanosomes and Leishmania spp. has been demonstrated [129–133]. Artemisinin was also reported to induce apoptosis in merozoites of Eimeria tenella [134] and inhibition of Neospora caninum intracellular multiplication [135]. Therefore, the potential activity of chicory SL against protozoans of veterinary (and med- ical) importance warrants confirmation. An interesting approach would be to test the activity of chicory and its bioactive compounds against coccidial infections in live- stock. Previously, in vivo anticoccidial effects of extracts from SL-containing plants (e.g. A. annua, A. absinthium, A. sieberi) and/or single SL (e.g. artemisinin) have been reported in goats [136] and poultry [137–140]. These re- sults strongly support the investigation of the potential anticoccidial activity of chicory in ruminants and monogastrics. In a nutraceutical approach, the existing evidence sus- tains the use of chicory-rich diets to target adult aboma- sal nematode infections in ruminants, which are amongst the most pathogenic and economically import- ant parasitic infections in sheep and cattle [4, 5]. Add- itional experimental and on-farm studies need to corroborate this selective activity in field conditions and to evaluate the use of chicory as an anthelmintic forage under farming settings. Competing interests Th h d l h Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Abbreviations GI: Gastrointestinal; SL: Sesquiterpene lactones; DM: Dry matter; FEC: Faecal egg counts; L1/L3/L4: First/third/fourth-stage larvae; LAC: Lactucin; 8-DOL: 8- deoxylactucin; LCP: Lactucopicrin; DHA: Dihydroartemisinin; AChE: Acetylcholinesterase Author details 1 1Instituto de Farmacologia y Morfofisiologia, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile. 2Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlægevej 100, 1870 Frederiksberg C, Denmark. 3Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kongens Lyngby, Denmark. 4National Veterinary Institute, Technical University of Denmark, Kemitorvet, 2800 Kongens Lyngby, Denmark. 5Norwegian Veterinary Institute, Ullevålsveien 68, P.O. Box 750, N-0106 Oslo, Sentrum, Norway. Further research and perspectives on the use of chicory for parasite control in livestock Furthermore, and given the widespread biological activity of SL, it could be expected that these molecules not only interact with par- asites in the GI tract, but also with the rumen or intes- tinal microbiota of the host, which may have a profound influence on animal health and performance. Therefore, a thorough knowledge of the metabolism of SL and their interactions may also shed light on the overall effects of chicory and other SL-rich bioactive forages on animal health and productivity, and to improve our understand- ing on the interactions between bioactive plant com- pounds, GI parasites and their hosts. Funding CONICYT Chile (FONDECYT Postdoctorado #3170875); EMIDA ERA-NET project “Coping with anthelmintic resistance in ruminants” (CARES; 3405-11- 0430/32); Danish Council for Independent Research (6111-00394: Parasites and plants - exploring the antiparasitic activity of a bioactive livestock forage). None of the funding bodies were involved in the design, collection, analysis and interpretation or in the writing of the manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Further research and perspectives on the use of chicory for parasite control in livestock These field studies should pri- mary test the effects of chicory rich-diets on patent in- fections of abomasal nematodes using FEC with species identification by larval cultures techniques or molecular methods [141, 142], potentially with the use of anti- Ostertagia specific antibodies in cattle [143], and when- ever possible, confirmed with worm counting post-mortem. The available evidence indicates that levels ≥70% chicory DM in the diet can exert anthelmintic ef- fects, but the lower effective intake is currently not established and will probably depend on cultivar, growth conditions, seasonality and other factors that could in- fluence the presence and concentration of bioactive compounds. Notably, the length of the chicory feeding (hours/days) that is required to exert an antiparasitic ef- fect in vivo must be elucidated, as previous research has suggested a rapid activity of chicory compounds against parasitic nematodes in vitro [102] and a significant and rapid reduction in FEC in chicory-fed calves infected with O. ostertagi [56]. Moreover, the consistent differ- ences in anthelmintic potency between chicory cultivars in vitro, linked to distinct SL profiles between different varieties [100, 102, 107], remain to be tested in vivo. Whether sufficiently high concentrations of chicory can be achieved in commercial farming systems needs add- itional research, though the use of pure chicory swards for livestock nutrition has been explored [42, 47, 144]. In a future prospect, the comprehensive screening and identification of the main antiparasitic phytochemicals in chicory could help the selection of cultivars with en- hanced concentration of these metabolites for use as a nutraceutical forage, or as source of natural parasiticidal compounds. These bioactive metabolites could be select- ively isolated to prepare purified extracts that can be used as feed additives for livestock. Such natural antipar- asitic feed additives could be given to infected animals in both outdoor (e.g. grazing ruminants) and indoor (e.g. intensive pig) systems. For this purpose, it would be im- portant to define the chicory cultivar(s) and the most suitable part of the plant (leaf, roots) for the extraction of active molecules, as well as the standardisation of the extraction technique to efficiently obtain the desired phytochemicals. In addition, lead parasiticidal molecules To further validate the practical use of chicory, the economic benefits of including chicory-rich diets in Page 11 of 14 Peña-Espinoza et al. Further research and perspectives on the use of chicory for parasite control in livestock Parasites & Vectors (2018) 11:475 Page 11 of 14 Page 11 of 14 identified in chicory could be used to develop novel anti- parasitic compounds, as previously discovered with Arte- misia annua and artemisinin [150]. Once a purified extract or compound(s) can be developed, its best ad- ministration route in livestock should be assessed. It is known that SL when given orally to livestock are poorly absorbed and reach a low bioavailability in comparison with injected SL, but these molecules can survive pas- sage through the digestive tract and therefore reach the target GI parasites. Certainly, a better understanding of the pharmacokinetics of chicory bioactive compounds in the host (including the potential metabolisation into more active molecules inside the animal) can help define the best administration routes and delivery strategy to achieve the desired antiparasitic effect. Furthermore, and given the widespread biological activity of SL, it could be expected that these molecules not only interact with par- asites in the GI tract, but also with the rumen or intes- tinal microbiota of the host, which may have a profound influence on animal health and performance. Therefore, a thorough knowledge of the metabolism of SL and their interactions may also shed light on the overall effects of chicory and other SL-rich bioactive forages on animal health and productivity, and to improve our understand- ing on the interactions between bioactive plant com- pounds, GI parasites and their hosts. identified in chicory could be used to develop novel anti- parasitic compounds, as previously discovered with Arte- misia annua and artemisinin [150]. Once a purified extract or compound(s) can be developed, its best ad- ministration route in livestock should be assessed. It is known that SL when given orally to livestock are poorly absorbed and reach a low bioavailability in comparison with injected SL, but these molecules can survive pas- sage through the digestive tract and therefore reach the target GI parasites. Certainly, a better understanding of the pharmacokinetics of chicory bioactive compounds in the host (including the potential metabolisation into more active molecules inside the animal) can help define the best administration routes and delivery strategy to achieve the desired antiparasitic effect. Conclusions Chicory is a bioactive forage with reported antiparasitic activity in ruminants when animals are fed with chicory-rich diets (≥70% of chicory DM in the diet). This antiparasitic activity is likely related with its con- tent of bioactive compounds, particularly SL, which have been reported to exert potent effects against helminths and protozoa in vitro. The available evidence has con- firmed the in vivo effects of chicory diets against aboma- sal nematodes in ruminants, and substantial research is needed to elucidate the potential broad antiparasitic ac- tivity of chicory against protozoans and in other live- stock species. Additional knowledge of chicory’s antiparasitic metabolites and mechanism(s) of action may indicate ways of improving its efficacy, e.g. by selec- tion of cultivars with increased activity and/or the isola- tion of leading parasiticides. In addition, future research on chicory may also open new paths for exploring other SL-containing plants and improve our basic understand- ing of the interactions between bioactive phytochemi- cals, GI parasites and their hosts. Received: 3 April 2018 Accepted: 12 July 2018 Received: 3 April 2018 Accepted: 12 July 2018 9. Waller PJ, Thamsborg SM. Nematode control in ‘green’ ruminant production systems. Trends Parasitol. 2004;20:493–7. 7. Waller PJ. Sustainable nematode parasite control strategies for ruminant livestock by grazing management and biological control. Anim Feed Sci Technol. 2006;126:277–89. Authors’ contributions MPE, AHV and ARW conceived and designed the work. MPE and AHV conducted the literature review and summarised the data. 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https://openalex.org/W2036577995	https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1004304&type=printable	English	null	EBNA3C Augments Pim-1 Mediated Phosphorylation and Degradation of p21 to Promote B-Cell Proliferation	PLOS pathogens	2,014	cc-by	18,592	Abstract Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr145 residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers. Editor: Blossom Damania, University of North Carolina at Chapel Hill, United States of America Received February 17, 2014; Accepted June 28, 2014; Published August 14, 2014 Copyright: 2014 Banerjee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by NCI grant 5-R01-CA-137894-05 to ESR. ESR is a scholar of the Leukemia and Lymphoma Society of America. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Abstract * Email: erle@upenn.edu Among the potential EBV latent antigens, EBNA3A, EBNA3B, and EBNA3C are sequentially encoded in the EBV genome and generate protein products of approximately 1,000 aa. Moreover, the EBNA3A, EBNA3B, and EBNA3C amino- terminal homol- ogous domains are associated with RBP-Jk which mediates the association of EBNA2 and Notch with DNA [12]. EBNA3C and EBNA3A are also essential for EBV to drive primary human B- lymphocytes into continuously proliferating LCLs and for maintaining LCL growth [13]. Notably, Epstein-Barr virus nuclear antigen 3C (EBNA3C) plays an intricate regulatory role in the transcription of several viral and cellular genes [14]. EBNA3C targeted RBP-Jk antagonizes EBNA2-mediated trans- activation [15], and cooperates with EBNA2 in activating the major viral LMP1 promoter [16]. EBNA3C was found to regulate chromatin remodeling by recruiting histone acetylase and deacetylase activities [17]. Moreover, EBNA3C modulates the transcriptional level of cellular genes which are involved in cell migration and invasion by targeting the metastasis suppressor Nm23-H1 [18]. In addition, EBNA3C can modulate diverse cellular functions, presumably mediated by direct protein–protein interactions [19]. EBNA3C also stabilizes c-Myc and interacts with Mdm2 to modulate p53 mediated transcription and apoptotic activities [20,21]. Interestingly, EBNA3C was found to be crucial nerjee1, Jie Lu1, Qiliang Cai2, Zhiguo Sun1, Hem Chandra Jha1, Erle S. Robertson1* 1 Department of Microbiology and the Tumor Virology Program, Abramson Comprehensive Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Key Laboratory of Molecular Medical Virology (Ministries of Education and Health), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China Editor: Blossom Damania, University of North Carolina at Chapel Hill, United States of America EBNA3C upregulates Pim-1 expression for regulating the activity of cellular kinases. Recently, we have shown that EBNA3C enhances the kinase activity of cell-cycle regulatory protein Cyclin D1 which allows for subsequent ubiquitination and degradation of the tumor suppressor pRb [22]. Provirus integration site for Moloney murine leukemia virus (Pim-1), a proto-oncogene encoding a serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis [23]. It was reported that overexpression of Pim-1 is associated with the development and progression of multiple hematopoietic malignancies such as B- cell lymphomas, erythroleukemias, and acute myelogenous leuke- mia, T-cell lymphomas, and non-hematological malignancies including, oral squamous cell carcinoma, and prostate cancer [24]. During the process of embryo development, Pim-1 is highly expressed in liver, spleen and bone marrow in typical hematopoi- etic progenitors [25,26], neonatal heart [27], central nervous system [28], and mammary gland [29]. Surprisingly, at the adult stage, Pim-1 is only slightly expressed in circulating granulocytes [26]. Previous reports also indicated that heterologous expression of Pim-1 in transgenic mice leads to increased lymphoproliferation and inhibition of apoptosis [30]. Augmented expression of Pim-1 in lymphoid cells by transgenesis highlighted its potential for oncogenesis [31]. Being a potent serine/threonine kinase, Pim-1 is able to phosphorylate itself [32,33], through an autophosphory- lation site that diverges from its consensus phosphorylation motif [34]. Several Pim-1 substrates have been identified, including p21Cip1/WAF1 [35,36], Cdc25A [37], PTPU2 [38], NuMA [39], C-TAK1 [40], and Cdc25C [41], indicating a crucial role for Pim- 1 in cell proliferation through both the G1/S and G2/M phase transition. Pim-1 also possesses anti-apoptotic activity [42], and recent reports have demonstrated a role for Pim kinases in regulation of herpesviral oncogenesis. KSHV encoded LANA was found to be crucial for transcriptional activation of Pim-1 in KSHV-positive cells and it also acts as a Pim-1 substrate [43]. In the context of EBV infection, studies have shown that Pim-1 may be required for LMP1-induced cell survival [44]. Furthermore, the expression levels of Pim-1 and Pim-2 are up-regulated upon EBV infection and they in turn enhance the activity of the viral nuclear antigen EBNA2, suggesting a role in driving EBV-induced immortalization [45]. However, the molecular mechanism by for regulating the activity of cellular kinases. Recently, we have shown that EBNA3C enhances the kinase activity of cell-cycle regulatory protein Cyclin D1 which allows for subsequent ubiquitination and degradation of the tumor suppressor pRb [22]. Introduction Epstein-Barr virus (EBV), a ubiquitous lymphotropic herpesvi- rus, latently infects human populations worldwide [1]. EBV infection is typically asymptomatic and is an important etiological factor which contributes to different human malignancies [2]. EBV is consistently associated with nasopharyngeal carcinoma (NPC) [3], African Burkitt’s lymphoma (BL) [4], post-transplan- tation lymphoproliferative disease (PTLD) [5], Hodgkin’s disease (HD) [6], and AIDS-related non-Hodgkin’s lymphomas (AIDS- NHL) [7]. Additionally, EBV is also found in a fraction of gastric carcinomas particularly in Asian and African countries [8]. EBV has the potential to transform human B-lymphocytes in vitro by maintaining a continuous proliferative state, known as ‘‘immor- talization’’ which generates permanent lymphoblastoid cell lines (LCLs) [9]. The LCLs which are produced in culture carry the viral genome as extra-chromosomal episomes and express nine latent EBV proteins including, the six nuclear antigens (EBNA 1, 2, 3A, 3B, 3C & LP), an additional three membrane associated proteins (LMP1, LMP2A & 2B), and the two EBV-encoded small RNAs (EBERs) [10]. These viral factors help to activate the quiescent B-cells from G0 into the cell cycle, and to sustain proliferation and maintenance of the viral genome [11]. 1 August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org EBV Regulates Pim-1 Function which Pim-1 is activated through expression of viral antigens which creates a micro-environment for B-cell transformation is not fully elucidated. Author Summary The oncogenic serine/threonine kinase Pim-1 is upregu- lated in a number of human cancers including lymphomas, gastric, colorectal and prostate carcinomas. EBV nuclear antigen 3C (EBNA3C) is essential for EBV-induced transfor- mation of human primary B-lymphocytes. Our current study revealed that EBNA3C significantly enhances Pim-1 kinase expression at both the transcript and protein levels. EBNA3C also interacts with Pim-1 and can form a complex in EBV-transformed cells. Moreover, EBNA3C increases nuclear localization of Pim-1 and stabilizes Pim-1 protein levels by inhibiting its poly-ubiquitination. Additionally, EBNA3C augments Pim-1 mediated phosphorylation of p21 and its proteosomal degradation. Stable knockdown of Pim-1 using si-RNA showed a significant decrease in proliferation of EBV transformed lymphoblastoid cell lines and subsequent induction of apoptosis by triggering the intrinsic apoptotic pathway. Therefore, our study demon- strated a new mechanism by which the oncogenic Pim-1 kinase targeted by EBV latent antigen 3C can inhibit p21 function, and is therefore a potential therapeutic target for the treatment of EBV-associated malignancies. In our current study, we demonstrated that EBNA3C is responsible for inducing Pim-1 expression in EBV transformed B-cells as well as in EBV-infected PBMCs. Further, we showed that EBNA3C interacts with Pim-1 through a small N-terminal domain (amino acids 130–159) and forms a complex in B-cells. Our results demonstrated that EBNA3C stabilized the Pim-1 protein by inhibiting its degradation by the ubiquitin/proteasome pathway. Interestingly, EBNA3C also facilitated the nuclear localization of Pim-1, and promotes EBV-induced cell prolifera- tion by regulating Pim-1 mediated degradation of p21/WAF1. We observed that deregulation of p21 ultimately resulted in higher cellular proliferation. Lentivirus mediated stable knockdown of Pim-1 resulted in a significant reduction of EBV transformed cells and induction of apoptosis. Cumulatively, these findings demon- strate a vital role for Pim-1 in EBV-mediated oncogenesis and also support the conclusions that Pim-1 kinase is a potential target for therapeutic intervention strategies against EBV associated malignancies. EBNA3C upregulates Pim-1 expression p g p Pim-1 expression was found upregulated in different hemato- logical and non-hematological malignancies [23]. To determine whether EBV latent antigen 3C modulates Pim-1 expression, 10 million human peripheral blood mononuclear cells (PBMC) were infected by wild type and mutant DEBNA3C BAC-GFP-EBV for 4 hrs at 37uC described previously [46]. The mRNA and protein levels of Pim-1 were detected after 0, 2, 4, 7 days of infection. Our results showed upregulation of both the transcript and protein levels of Pim-1 with wild type EBV infection (Fig. 1A). Interest- ingly, infection with DEBNA3C BAC-GFP-EBV resulted in low Pim-1 expression at 2 days post-infection and returned to the levels seen for infected cells at 0 day post-infection (Fig. 1B). The results indicated that Pim-1 expression was induced by wild-type EBV infection. Therefore, we wanted to determine the expression pattern of Pim-1 in EBV transformed Lymphoblastoid cells LCL1, LCL2, and EBNA3C stably expressing BJAB7 and BJAB10 cells when compared to EBV negative BJAB. Our results showed that Pim-1 expression was highly upregulated in LCL1, LCL2, BJAB7 and BJAB10 cells (Fig. 1C). To investigate the role of EBNA3C on Pim-1, we monitored the Pim-1 protein expression levels with a dose dependent increase of EBNA3C in EBV negative DG75 as well as in HEK-293 cells. The results showed a steady increase in Pim-1 expression levels in both cell lines (Fig. 1D and 1E). Moreover, Real-time PCR analysis showed upregulation of Pim-1 mRNA expression in BJAB7 and LCL1 cells when compared to EBV negative BJAB cells (Fig. 1F, left panel). To further investigate the role of EBNA3C in inducing Pim-1 expression, we performed Real-time PCR as well as Western blot analysis on EBNA3C stable knock-down LCL1 cells. The results demonstrat- ed a substantial reduction of Pim-1 expression in both mRNA and protein levels as compared with sh-control LCL1 cells (Fig. 1F, right panel and Fig. 1G). Moreover, to check the role of other EBV antigens including EBNA2, EBNA3A, and EBNA3B on Pim-1 expression, we performed si-RNA mediated knockdown of EBNA2, EBNA3A, EBNA3B and EBNA3C in LCL1 cells. Our Real-time PCR analysis demonstrated that Pim-1 mRNA level is significantly reduced upon EBNA3C knockdown but no significant change was observed in Pim-1 mRNA expression level with EBNA2, EBNA3A, EBNA3B knockdown further suggesting a August 2014 \| Volume 10 \| Issue 8 \| e1004304 2 PLOS Pathogens \| www.plospathogens.org EBV Regulates Pim-1 Function Figure 1. EBNA3C upregulates Pim-1 expression Upregulation of Pim-1 expression by EBNA3C. A) 106106 human PBMC (Peripheral blood mononuclear cells) were infected with wild type or DEBNA3C mutant BAC-GFP EBV for 6 hrs at 37uC. Cells were harvested after 0, 2, 4, 7, 15 days of post-infection. Total RNA was isolated and subjected to quantitative real-time PCR analysis to detect Pim-1 mRNA level. B) Wild type or DEBNA3C mutant BAC-GFP EBV infected cells were lysed in RIPA buffer. Western blot analysis was performed with indicated antibodies to detect specific endogenous proteins. C) 50 million EBV negative BJAB, EBNA3C expressing BJAB7, BJAB10, EBV transformed LCL1, LCL2 cells were harvested and total cell lysates were subjected to Western blot analysis (WB) using indicated antibodies. D) 10 million HEK-293 cells and E) 50 million EBV negative DG75 cells were transfected with increasing amount of EBNA3C expressing construct (0, 5, 10, 15 mg) and Western blot analysis was performed to detect Pim-1, EBNA3C, GAPDH proteins. F) Total RNA was isolated from BJAB, BJAB7, LCL1, sh-Ctrl LCL1, sh-E3C LCL1 cells and subjected to quantitative real-time PCR analysis to detect Pim-1 mRNA levels. G) Lentivirus mediated stable EBNA3C knockdown (sh-E3C) or scramble control (sh-Ctrl) LCL1 cells were subjected to Western blot analysis with indicated antibodies. Protein bands from Western blot analysis were analyzed by the Odyssey imager software and represented as bar diagrams based on internal loading control GAPDH. doi:10.1371/journal.ppat.1004304.g001 Figure 1. Upregulation of Pim-1 expression by EBNA3C. A) 106106 human PBMC (Peripheral blood mononuclear cells) were infected with wild type or DEBNA3C mutant BAC-GFP EBV for 6 hrs at 37uC. Cells were harvested after 0, 2, 4, 7, 15 days of post-infection. Total RNA was isolated and subjected to quantitative real-time PCR analysis to detect Pim-1 mRNA level. B) Wild type or DEBNA3C mutant BAC-GFP EBV infected cells were lysed in RIPA buffer. Western blot analysis was performed with indicated antibodies to detect specific endogenous proteins. C) 50 million EBV negative BJAB, EBNA3C expressing BJAB7, BJAB10, EBV transformed LCL1, LCL2 cells were harvested and total cell lysates were subjected to Western blot analysis (WB) using indicated antibodies. D) 10 million HEK-293 cells and E) 50 million EBV negative DG75 cells were transfected with increasing amount of EBNA3C expressing construct (0, 5, 10, 15 mg) and Western blot analysis was performed to detect Pim-1, EBNA3C, GAPDH proteins. EBNA3C facilitates nuclear transport and co-localizes with Pim-1 In the context of cancer progression, the significance of different subcellular localization patterns of Pim-1 has not been fully elucidated. Previous studies suggested that irradiation can promote nuclear translocation of Pim-1 in radio-resistant squamocellular malignancies of head and neck [48]. Importantly, nuclear localization of Pim-1 may correlate with the proliferating cells and may also contribute to a survival response upon pathologic injury [49]. In our study, we transfected Myc-tagged Pim-1 with or EBNA3C associates with Pim-1 in human B-cell lines EBNA3C associates with Pim 1 in human B cell lines To determine whether EBNA3C interacted with Pim-1, we performed co-immunoprecipitation experiments in HEK-293 cells by expressing Myc-tagged Pim-1, Flag-EBNA3C, or Myc- EBNA3C. Immunoprecipitation was performed using A10 (Fig. 2A) or 9E10 antibody (Fig. 2B). The results clearly demon- strated that EBNA3C strongly associated with Pim-1 (Fig. 2A and B). We further supported our results by GST-pull down assays using EBV negative BJAB, EBNA3C expressing BJAB10 and EBV transformed LCL1 cell lysates incubated with bacterially purified GST-Pim-1 protein. EBNA3C was detected by A10 antibody [47] which showed a substantial level of association between Pim-1 and EBNA3C in the EBNA3C stable cell lines as well as in an LCL (Fig. 2C). Coomassie staining of bacterially purified GST and GST-Pim-1 proteins are shown in Fig. 2D. We also observed the association between EBNA3C and Pim-1 in BJAB7, BJAB10, LCL1, LCL2 cells compared with BJAB in separate co-immuno- precipitation experiments by using Pim-1 specific antibody (Fig. 2E and 2F). EBNA3C upregulates Pim-1 expression F) Total RNA was isolated from BJAB, BJAB7, LCL1, sh-Ctrl LCL1, sh-E3C LCL1 cells and subjected to quantitative real-time PCR analysis to detect Pim-1 mRNA levels. G) Lentivirus mediated stable EBNA3C knockdown (sh-E3C) or scramble control (sh-Ctrl) LCL1 cells were subjected to Western blot analysis with indicated antibodies. Protein bands from Western blot analysis were analyzed by the Odyssey imager software and represented as bar diagrams based on internal loading control GAPDH. doi:10.1371/journal.ppat.1004304.g001 major role for EBNA3C in upregulating Pim-1 expression (Fig. S1A and S1B). Additionally, we performed Western blot analysis to determine whether knock down of EBNA3C may have an effect on other EBNAs expression levels. The results demonstrated that expression levels of other EBNAs were not affected with EBNA3C knockdown (Fig. S2). expressing GFP-tagged Pim-1 with Myc-tagged full length (residues 1–992) and different truncated mutants (residues 1– 365, 366–620 and 621–992) of EBNA3C in HEK-293 cells. Immunoprecipitation (IP) was performed by using either 9E10 or GFP-specific antibodies. The results indicated that Pim-1 strongly associated with full length as well as the N-terminal domain (residues 1–365) of EBNA3C (Fig. 3A and 3B). We extended the binding experiments by performing in vitro GST-pulldown assay with in vitro translated full length and truncated mutants of EBNA3C including fragments within the N-terminal domain (residues 1–992, 1–365, 366–620, 621–992, 1–100, 100–200, 200– 300, 366–992, 1–129, 1–159, 1–250, 130–300). Our results demonstrated that EBNA3C residues 100–200, 1–159, 1–250, 130–300 associated strongly with full length Pim-1 (Fig. 3C). To further map the specific binding residues, we performed additional in vitro GST-pulldown assays by using in vitro translated full length Pim-1 incubated with bacterially expressed N-terminal truncated mutants of GST-EBNA3C fused to residues 90–129, 130– 159, 130–190, 160–190. The results indicated that Pim-1 strongly bound to residues 130–159 of EBNA3C (Fig. 3D, 3E and 3F). EBNA3C associates with Pim-1 in human B-cell lines Pim-1 specifically binds to the N-terminal domain of EBNA3C To determine the specific domain of EBNA3C associated with Pim-1, we performed co-immunoprecipitation experiments August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org PLOS Pathogens \| www.plospathogens.org 3 EBV Regulates Pim-1 Function Figure 2. EBNA3C physically associates with Pim-1. A–B) 10 million HEK- 293 cells were co-transfected with Myc-tagged Pim-1, untagged- EBNA3C or Flag-tagged EBNA3C expression vectors. Control samples were balanced by using empty vector. Transfected cells were harvested at 36 hrs of post-transfection and approximately 5% of the lysates were used as input and the residual lysate was immunoprecipitated (IP) with 1 mg of anti-Flag (M2) or A10 antibodies. Lysates and Immunoprecipitated samples were resolved by 10% SDS-PAGE and western blot (WB) analysis was performed with the indicated antibodies. C) 50 million BJAB, BJAB7, LCL1, were harvested and lysed in RIPA buffer. Cell lysates were incubated with either GST control or GST-Pim-1 beads. EBNA3C protein was detected by western blot analysis using EBNA3C specific monoclonal antibody (A10). D) Purified control GST and GST-Pim-1 proteins used in this experiment were resolved by 10% SDS-PAGE and stained with Coomassie Blue. 50 million E– F) BJAB, BJAB7, BJAB10, LCL1, and LCL2 cells were lysed and immunoprecipitation was performed by Pim-1 specific antibody. Immunoprecipitated samples were resolved by 10% SDS-PAGE and endogenous EBNA3C, Pim-1 proteins were detected by their specific antibodies. doi:10.1371/journal.ppat.1004304.g002 Figure 2. EBNA3C physically associates with Pim-1. A–B) 10 million HEK- 293 cells were co-transfected with Myc-tagged Pim-1, untagged- EBNA3C or Flag-tagged EBNA3C expression vectors. Control samples were balanced by using empty vector. Transfected cells were harvested at 36 hrs of post-transfection and approximately 5% of the lysates were used as input and the residual lysate was immunoprecipitated (IP) with 1 mg of anti-Flag (M2) or A10 antibodies. Lysates and Immunoprecipitated samples were resolved by 10% SDS-PAGE and western blot (WB) analysis was performed with the indicated antibodies. C) 50 million BJAB, BJAB7, LCL1, were harvested and lysed in RIPA buffer. Cell lysates were incubated with either GST control or GST-Pim-1 beads. EBNA3C protein was detected by western blot analysis using EBNA3C specific monoclonal antibody (A10). D) Purified control GST and GST-Pim-1 proteins used in this experiment were resolved by 10% SDS-PAGE and stained with Coomassie Blue. 50 million E– F) BJAB, BJAB7, BJAB10, LCL1, and LCL2 cells were lysed and immunoprecipitation was performed by Pim-1 specific antibody. Pim-1 specifically binds to the N-terminal domain of EBNA3C Immunoprecipitated samples were resolved by 10% SDS-PAGE and endogenous EBNA3C, Pim-1 proteins were detected by their specific antibodies. doi:10.1371/journal.ppat.1004304.g002 without GFP-tagged EBNA3C expression vectors in HEK-293 cells. Cellular localization of Pim-1 was examined by immunoflu- orescence analysis using specific antibodies against the Myc- epitope. Interestingly, our results showed that the localization of Pim-1 was predominantly in the cytoplasm without EBNA3C and was translocated to the nucleus in the presence of EBNA3C. Also, strong co-localization with Pim-1 and EBNA3C was observed (Fig. 4A and 4C). To further validate these results, we performed nuclear and cytosolic fractionation assays using transiently transfected HEK-293 cells with Myc-tagged Pim-1 with or without Flag-tagged EBNA3C expression vectors. Our Western blot analysis with nuclear and cytosolic fractions showed that in the presence of EBNA3C, the level of Pim-1 substantially increased in the nuclear fraction (Fig. 4B). Moreover, we corroborated the above observations in EBV negative BJAB, EBNA3C stably expressing BJAB10 and EBV transformed LCL1 cells using specific antibodies against Pim-1 and EBNA3C. The results showed that Pim-1 was mostly localized in the nucleus in both EBNA3C expressing BJAB10 and EBV transformed LCL1 cells, but was almost entirely cytoplasmic in EBV negative BJAB cells (Fig. 4D). proteosome machinery, we co-transfected Myc-tagged Pim-1 with or without Flag-tagged EBNA3C expression plasmids in HEK-293 cells which were treated with or without the proteasome inhibitor MG132. The results showed a substantial accumulation of Pim-1 protein levels in MG132 treated cells in the presence of EBNA3C compared with mock treatment and control vector (Fig. 5A). Next, we performed the stability assay of Pim-1 by transfecting Myc-tagged Pim-1 with or without Flag- tagged EBNA3C in HEK-293 cells. After 36 hours of post- transfection, cells were treated with the protein synthesis inhibitor cyclohexamide and harvested at 0, 3, and 6 hours intervals. The Western blot results clearly demonstrated that Pim-1 levels were stabilized with co-expression of EBNA3C whereas, the Pim-1 expression levels were markedly reduced with cyclohexamide treatment by 3 to 6 hours in the absence of EBNA3C (Fig. 5B). To further corroborate our results, we extended the stability assays with EBV negative BJAB, EBNA3C stably expressing BJAB10 and EBV transformed LCL1, control vector transfected and EBNA3C stably knockdown LCL1 cells. Pim-1 specifically binds to the N-terminal domain of EBNA3C As anticipated, our results showed that Pim-1 protein levels were stabilized in BJAB10, LCL1 and sh-Ctrl LCL1 cells as well but significantly reduced in BJAB, sh-E3C LCL1 cells over time with the treatment of cyclohexamide (Fig. 5C and 5D). PLOS Pathogens \| www.plospathogens.org EBNA3C can inhibit poly-ubiquitination of Pim-1 A–B) 10 million HEK-293 cells were transfected with either control vector or Full length and different truncated mutants of Myc-tagged EBNA3C with GFP-tagged Pim-1 plasmid constructs. After 36 hours of post-transfection, cells were harvested and immunoprecipitation performed with 1 mg of GFP or anti-Myc antibodies. IP samples were resolved in 10% SDS-PAGE. Western blot was performed with anti-Myc and anti-GFP antibodies. C) Full length and different domains truncated mutant constructs of EBNA3C were in vitro translated using a T7-TNT translation kit. After pre-clearing with GST-beads, all S35-radiolabeled in vitro translated proteins incubated with either GST control or GST-Pim-1 beads. Reaction samples were washed with Binding Buffer and resolved by 10% SDS-PAGE, exposed to phosphoimager plate and scanned by Typhoon Scanner. D) Myc-Pim-1 construct were used for in vitro translation and S35-radiolabeled in vitro translated proteins were i b t d ith ith GST t l diff t GST EBNA3C t t d t t b d C i t i i f SDS PAGE l d ifi d GST t i Figure 3. Binding of Pim-1 to the N-terminal EBNA3C domain. A–B) 10 million HEK-293 cells were transfected with either control vector or Full length and different truncated mutants of Myc-tagged EBNA3C with GFP-tagged Pim-1 plasmid constructs. After 36 hours of post-transfection, cells were harvested and immunoprecipitation performed with 1 mg of GFP or anti-Myc antibodies. IP samples were resolved in 10% SDS-PAGE. Western blot was performed with anti-Myc and anti-GFP antibodies. C) Full length and different domains truncated mutant constructs of EBNA3C were in vitro translated using a T7-TNT translation kit. After pre-clearing with GST-beads, all S35-radiolabeled in vitro translated proteins incubated with either GST control or GST-Pim-1 beads. Reaction samples were washed with Binding Buffer and resolved by 10% SDS-PAGE, exposed to phosphoimager plate and scanned by Typhoon Scanner. D) Myc-Pim-1 construct were used for in vitro translation and S35-radiolabeled in vitro translated proteins were incubated with either GST control or different GST-EBNA3C truncated mutant beads. Coomassie staining of SDS-PAGE resolved purified GST proteins is shown in the bottom panel of D). In each case, 5% of IVT input was used for the comparison. E) The diagram shows EBNA3C 130–159 amino acids motif important binding sites for different cellular proteins. F) The schematic diagram represents various structural and interactive domains of EBNA3C and summarizes the binding affinities between different domains of EBNA3C with Pim-1. EBNA3C can inhibit poly-ubiquitination of Pim-1 Recent reports suggested that expression of EBNA3C is responsible for the stabilization of different oncoproteins, transcription factors and cellular kinases [19,22,50,51], and also plays an important role in modulating the ubiquitin (Ub)- proteasome machinery [52]. Our results so far showed that EBNA3C is important for enhanced protein expression of Pim-1. To determine, if this induced expression is related to EBNA3C- mediated stabilization of Pim-1 by the inhibition of Ub- The enhanced stability of Pim-1 in the presence of EBNA3C encouraged us to investigate the role of EBNA3C for regulating Pim-1 poly-ubiquitination. Therefore, we performed in vivo poly- ubiquitination assays in cells by co-transfecting with control vector, Myc-tagged Pim-1, HA-Ubiquitin, with or without Flag-tagged EBNA3C in HEK-293 cells. The results demonstrated a significant reduction of Pim-1 poly-ubiquitination levels in the August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 4 EBV Regulates Pim-1 Function Figure 3. Binding of Pim-1 to the N-terminal EBNA3C domain. A–B) 10 million HEK-293 cells were transfected with either control vector or Full length and different truncated mutants of Myc-tagged EBNA3C with GFP-tagged Pim-1 plasmid constructs. After 36 hours of post-transfection, cells were harvested and immunoprecipitation performed with 1 mg of GFP or anti-Myc antibodies. IP samples were resolved in 10% SDS-PAGE. Western blot was performed with anti-Myc and anti-GFP antibodies. C) Full length and different domains truncated mutant constructs of EBNA3C were in vitro translated using a T7-TNT translation kit. After pre-clearing with GST-beads, all S35-radiolabeled in vitro translated proteins incubated with either GST control or GST-Pim-1 beads. Reaction samples were washed with Binding Buffer and resolved by 10% SDS-PAGE, exposed to phosphoimager plate and scanned by Typhoon Scanner. D) Myc-Pim-1 construct were used for in vitro translation and S35-radiolabeled in vitro translated proteins were incubated with either GST control or different GST-EBNA3C truncated mutant beads. Coomassie staining of SDS-PAGE resolved purified GST proteins is shown in the bottom panel of D). In each case, 5% of IVT input was used for the comparison. E) The diagram shows EBNA3C 130–159 amino acids motif important binding sites for different cellular proteins. F) The schematic diagram represents various structural and interactive domains of EBNA3C and summarizes the binding affinities between different domains of EBNA3C with Pim-1. +, binding; 2, no binding. doi:10.1371/journal.ppat.1004304.g003 \| \| \| \| Figure 3. Binding of Pim-1 to the N-terminal EBNA3C domain. EBNA3C can inhibit poly-ubiquitination of Pim-1 +, binding; 2, no binding. doi:10.1371/journal.ppat.1004304.g003 Figure 3. Binding of Pim-1 to the N-terminal EBNA3C domain. A–B) 10 million HEK-293 cells were transfected with either control vector or Full length and different truncated mutants of Myc-tagged EBNA3C with GFP-tagged Pim-1 plasmid constructs. After 36 hours of post-transfection, cells were harvested and immunoprecipitation performed with 1 mg of GFP or anti-Myc antibodies. IP samples were resolved in 10% SDS-PAGE. Western blot was performed with anti-Myc and anti-GFP antibodies. C) Full length and different domains truncated mutant constructs of EBNA3C were in vitro translated using a T7-TNT translation kit. After pre-clearing with GST-beads, all S35-radiolabeled in vitro translated proteins incubated with either GST control or GST-Pim-1 beads. Reaction samples were washed with Binding Buffer and resolved by 10% SDS-PAGE, exposed to phosphoimager plate and scanned by Typhoon Scanner. D) Myc-Pim-1 construct were used for in vitro translation and S35-radiolabeled in vitro translated proteins were incubated with either GST control or different GST-EBNA3C truncated mutant beads. Coomassie staining of SDS-PAGE resolved purified GST proteins is shown in the bottom panel of D). In each case, 5% of IVT input was used for the comparison. E) The diagram shows EBNA3C 130–159 amino acids motif important binding sites for different cellular proteins. F) The schematic diagram represents various structural and interactive domains of EBNA3C and summarizes the binding affinities between different domains of EBNA3C with Pim-1. +, binding; 2, no binding. doi:10.1371/journal.ppat.1004304.g003 August 2014 \| Volume 10 \| Issue 8 \| e1004304 August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 5 EBV Regulates Pim-1 Function 4. EBNA3C co-localizes with Pim-1 and facilitates its nuclear translocation. A) 0.3 million HEK-293 cells plated on coverslips and ntly transfected with control vector, GFP-EBNA3C and Myc-Pim-1 expression vectors by using Lipofectamine 2000 transfection reagent. B) 10 HEK-293 cells were transfected with Myc-Pim-1 and Flag-EBNA3C and subjected to sub-cellular fractionation assay. C) Bar diagram represents ation of co-localization in panel A. D) BJAB, BJAB10, LCL1 cells were plated on slides and air-dried. Ectopic and endogenous expressions of was detected using anti-Myc (9E10)-antibody (1:200 dilution) and Pim-1 specific antibody (1:50 dilution) respectively, followed by anti-Rabbit luor 594 and anti-goat Alexa Fluor 555 (red) as secondary antibodies. Endogenous EBNA3C was detected using A10 ascites (1:150 dilution) d by anti-mouse Alexa Fluor 488 (green). DAPI (49, 69-diamidino-2-phenylindole) was used (1:500 dilution) to stain nuclei. EBNA3C can inhibit poly-ubiquitination of Pim-1 The images were d by Olympus Fluoview confocal microscope. 371/journal ppat 1004304 g004 Figure 4. EBNA3C co-localizes with Pim-1 and facilitates its nuclear translocation. A) 0.3 million HEK-293 cells plated on coverslips and transiently transfected with control vector, GFP-EBNA3C and Myc-Pim-1 expression vectors by using Lipofectamine 2000 transfection reagent. B) 10 million HEK-293 cells were transfected with Myc-Pim-1 and Flag-EBNA3C and subjected to sub-cellular fractionation assay. C) Bar diagram represents quantitation of co-localization in panel A. D) BJAB, BJAB10, LCL1 cells were plated on slides and air-dried. Ectopic and endogenous expressions of Pim-1 was detected using anti-Myc (9E10)-antibody (1:200 dilution) and Pim-1 specific antibody (1:50 dilution) respectively, followed by anti-Rabbit Alexa Fluor 594 and anti-goat Alexa Fluor 555 (red) as secondary antibodies. Endogenous EBNA3C was detected using A10 ascites (1:150 dilution) followed by anti-mouse Alexa Fluor 488 (green). DAPI (49, 69-diamidino-2-phenylindole) was used (1:500 dilution) to stain nuclei. The images were captured by Olympus Fluoview confocal microscope. doi:10 1371/journal ppat 1004304 g004 Figure 4. EBNA3C co-localizes with Pim-1 and facilitates its nuclear translocation. A) 0.3 million HEK-293 cells plated on coverslips and transiently transfected with control vector, GFP-EBNA3C and Myc-Pim-1 expression vectors by using Lipofectamine 2000 transfection reagent. B) 10 million HEK-293 cells were transfected with Myc-Pim-1 and Flag-EBNA3C and subjected to sub-cellular fractionation assay. C) Bar diagram represents quantitation of co-localization in panel A. D) BJAB, BJAB10, LCL1 cells were plated on slides and air-dried. Ectopic and endogenous expressions of Pim-1 was detected using anti-Myc (9E10)-antibody (1:200 dilution) and Pim-1 specific antibody (1:50 dilution) respectively, followed by anti-Rabbit Alexa Fluor 594 and anti-goat Alexa Fluor 555 (red) as secondary antibodies. Endogenous EBNA3C was detected using A10 ascites (1:150 dilution) followed by anti-mouse Alexa Fluor 488 (green). DAPI (49, 69-diamidino-2-phenylindole) was used (1:500 dilution) to stain nuclei. The images were captured by Olympus Fluoview confocal microscope. doi:10.1371/journal.ppat.1004304.g004 EBNA3C enhances Pim-1 kinase-mediated phosphorylation of the Cyclin inhibitor p21 at the threonine 145 residue EBNA3C enhances Pim-1 kinase-mediated phosphorylation of the Cyclin inhibitor p21 at the threonine 145 residue presence of EBNA3C (Fig. 6A). To further validate the role of EBNA3C, we performed poly-ubiquitination assays by using the wild type Myc-tagged EBNA3C and its specific mutant EBNA3C (Myc-EBNA3C-C143N) expression vectors. We observed higher poly-ubiquitination levels of Pim-1 in the presence of the EBNA3C-C143N mutant compared with wild type (Fig. 6B). We also performed the ubiquitination assays in a B-cell background by using EBV-negative BJAB, EBNA3C stably expressing BJAB10 and EBV transformed lymphoblastoid LCL1, as well as the sh-Ctrl and sh-EBNA3C LCL1 cell lines. Our result showed that the status of Pim-1 ubiquitination was much lower in BJAB10 and LCL1 cells compared with BJAB (Fig. 6C) and somewhat enhanced upon EBNA3C knockdown (Fig. 6D). The serine/threonine-protein kinase Pim-1 is upregulated in a number of hematological malignancies such as leukemia [26], mantle-cell lymphoma [53], and diffuse large B-cell lymphoma (DLBCL) [54]. A wide range of Pim-1 substrates were identified including, BAD [55], NuMa [39], Socs [56], Cdc25A [37], C- TAK1 [40], NFATc [57], HP-1 [58], PAP-1 [59], and cyclin- dependent kinase inhibitor p21 or p21Cip1/WAF1 [35], which suggested that Pim-1 can function at different cellular events, such as cell proliferation, differentiation, and cell survival [60]. Earlier reports showed that p21 suppresses tumors by promoting cell cycle August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 6 EBV Regulates Pim-1 Function Figure 5. EBNA3C contributes to Pim-1 stabilization. A) 10 million HEK-293 cells were co-transfected with Myc-Pim-1 and either vector control (lanes 1 and 3) or Flag-EBNA3C (lanes 2 and 4) expression constructs. After 36 hrs of post-transfection, transfected cells were treated with either 20 mM MG132 (+ lanes) or DMSO (2 lanes) for additional 6 hrs and cell lysates were resolved by 10% SDS-PAGE and Western blot was performed with the indicated antibodies. B) HEK-293 cells were transfected with above mentioned expression vectors and At 36 hrs of post-transfection, cells were treated with 40 mg/ml cyclohexamide (CHX) for 0, 3, 6 hrs. Cells were lysed and protein samples were resolved by 10% SDS-PAGE. Western blot was performed by specific antibodies shown. C–D) BJAB, BJAB10, LCL1, sh-Ctrl and sh-EBNA3C cells were treated with 40 mg/ml cyclohexamide (CHX) for indicated time periods. Cell lysates were resolved by 10% SDS-PAGE. Western blot analysis was performed with indicated antibodies. GAPDH blot was shown for internal loading control. doi:10.1371/journal.ppat.1004304.g005 Figure 5. EBNA3C contributes to Pim-1 stabilization. EBNA3C enhances Pim-1 kinase-mediated phosphorylation of the Cyclin inhibitor p21 at the threonine 145 residue As anticipated, there was no kinase activity observed with the kinase-dead (KD) mutant of Pim- 1 when compared with wild type. Next, we performed in vitro kinase assay for Pim-1 in the presence or absence of EBNA3C by using wild type and mutant (T145A) GST-p21 as substrate. The results showed no phosphorylation with mutant (T145A) p21 in comparison with wild-type, even in the presence of EBNA3C (Fig. 7C). This suggested that the Thr145 residue is important for EBNA3C mediated enhancement of p21 phosphorylation by Pim- 1 kinase. EBNA3C destabilizes p21 through Pim-1 kinase independent of etoposide induced DNA damage response Previous reports suggested that p21 regulates fundamental cellular processes, including cell cycle progression, apoptosis, and transcription on DNA damage response [64,65]. Interestingly, involvement of p21 in all these major signaling pathways may occur not only after DNA damage response, but also depends on physiological conditions [66,67]. To determine whether EBNA3C alone or an EBNA3C/Pim-1 complex had a role in p21 stabilization in DNA damage response, we performed stability assays using cyclohexamide treated HEK-293 cells co-transfected with different combinations of untagged-EBNA3C, Myc-Pim-1, Myc-Pim-1 KD (kinase dead) mutant, and Flag-p21 expression constructs. The experiments were performed with or without EBNA3C enhances Pim-1 kinase-mediated phosphorylation of the Cyclin inhibitor p21 at the threonine 145 residue A) 10 million HEK-293 cells were co-transfected with Myc-Pim-1 and either vector control (lanes 1 and 3) or Flag-EBNA3C (lanes 2 and 4) expression constructs. After 36 hrs of post-transfection, transfected cells were treated with either 20 mM MG132 (+ lanes) or DMSO (2 lanes) for additional 6 hrs and cell lysates were resolved by 10% SDS-PAGE and Western blot was performed with the indicated antibodies. B) HEK-293 cells were transfected with above mentioned expression vectors and At 36 hrs of post-transfection, cells were treated with 40 mg/ml cyclohexamide (CHX) for 0, 3, 6 hrs. Cells were lysed and protein samples were resolved by 10% SDS-PAGE. Western blot was performed by specific antibodies shown. C–D) BJAB, BJAB10, LCL1, sh-Ctrl and sh-EBNA3C cells were treated with 40 mg/ml cyclohexamide (CHX) for indicated time periods. Cell lysates were resolved by 10% SDS-PAGE. Western blot analysis was performed with indicated antibodies. GAPDH blot was shown for internal loading control. doi:10.1371/journal.ppat.1004304.g005 association between Pim-1 and EBNA3C above. We then performed competitive binding assays in HEK-293 cells by co- transfecting increasing doses of EBNA3C-expression construct and a constant amount of Myc-tagged Pim-1 and Flag-tagged p21. Immunoprecipitation (IP) was performed with anti-Myc antibody for immunoprecipitation of complex with Pim-1. Our results demonstrated that increasing doses of EBNA3C can result in reduced association between Pim-1 and p21 (Fig. 8A). Previous studies showed that p21 is a prime target for ubiquitination in gliomas [62], and was dependent on the ubiquitin ligase APC/ CCdc20 for its proteolytic degradation by the proteasome [63]. To explore the modulation of p21 protein levels by EBNA3C through regulation of the Ub-proteasome machinery, HEK-293 cells were co-transfected with Myc-Pim-1, Flag-p21, and increasing amounts of untagged-EBNA3C then treated with the proteasome inhibitor, MG132. The results indicated that the level of p21 was significantly reduced in the mock treated cells. However, with MG132 drug treatment, the level of p21 was further enhanced in the presence of EBNA3C (Fig. 8B). arrest in response to various stimuli. Furthermore, considerable evidence from biochemical and genetic studies have demonstrated that p21 can act as a master effector molecule of multiple tumor suppressor pathways for promoting anti-proliferative activities which are independent of classical p53 tumor suppressor pathway [61]. Studies have also shown that enhanced levels of Pim-1 kinase phosphorylates Thr145 residue, and regulates the activity of p21Cip1/WAF1 [35]. EBNA3C enhances Pim-1 kinase-mediated phosphorylation of the Cyclin inhibitor p21 at the threonine 145 residue Therefore, we checked the kinase activity of Pim-1 towards its substrate p21 with or without EBNA3C to investigate whether EBNA3C can modulate the phosphorylation status of p21. HEK-293 cells were transiently transfected with control vector, with and without Myc-tagged Pim-1 and increasing doses of Flag-tagged EBNA3C expression vectors. Immunopre- cipitation was performed using anti-Myc 9E10 antibody and immunoprecipitated complexes were further examined for in vitro kinase activity as determined by GST-p21 phosphorylation. Interestingly, the results demonstrated that the ability of Pim-1 kinase to phosphorylate p21 was substantially and proportionally augmented by a dose-dependent increase in EBNA3C expression (Fig. 7A). We further extended the kinase assay using a kinase- dead (KD) mutant of Pim-1 (Fig. 7B). As anticipated, there was no kinase activity observed with the kinase-dead (KD) mutant of Pim- 1 when compared with wild type. Next, we performed in vitro kinase assay for Pim-1 in the presence or absence of EBNA3C by using wild type and mutant (T145A) GST-p21 as substrate. The results showed no phosphorylation with mutant (T145A) p21 in comparison with wild-type, even in the presence of EBNA3C (Fig. 7C). This suggested that the Thr145 residue is important for EBNA3C mediated enhancement of p21 phosphorylation by Pim- 1 kinase. arrest in response to various stimuli. Furthermore, considerable evidence from biochemical and genetic studies have demonstrated that p21 can act as a master effector molecule of multiple tumor suppressor pathways for promoting anti-proliferative activities which are independent of classical p53 tumor suppressor pathway [61]. Studies have also shown that enhanced levels of Pim-1 kinase phosphorylates Thr145 residue, and regulates the activity of p21Cip1/WAF1 [35]. Therefore, we checked the kinase activity of Pim-1 towards its substrate p21 with or without EBNA3C to investigate whether EBNA3C can modulate the phosphorylation status of p21. HEK-293 cells were transiently transfected with control vector, with and without Myc-tagged Pim-1 and increasing doses of Flag-tagged EBNA3C expression vectors. Immunopre- cipitation was performed using anti-Myc 9E10 antibody and immunoprecipitated complexes were further examined for in vitro kinase activity as determined by GST-p21 phosphorylation. Interestingly, the results demonstrated that the ability of Pim-1 kinase to phosphorylate p21 was substantially and proportionally augmented by a dose-dependent increase in EBNA3C expression (Fig. 7A). We further extended the kinase assay using a kinase- dead (KD) mutant of Pim-1 (Fig. 7B). EBNA3C competes with p21 to inhibit the complex formation with Pim-1 kinase Earlier reports suggested the potential of a complex containing Pim-1 and p21 in cells [36]. We have now confirmed a strong August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org PLOS Pathogens \| www.plospathogens.org 7 EBV Regulates Pim-1 Function Figure 6. EBNA3C suppresses poly-ubiquitination of Pim-1. A–B) 10 million HEK-293 cells were transiently transfected with different combinations of expression vectors as indicated. Cells were harvested after 36 hrs of post-transfection by incubating with proteasome inhibitor MG132 drug for additional 6 hrs. Total protein was immunoprecipitated (IP) with indicated antibodies and protein samples were resolved by 10% SDS-PAGE. Western blots were performed by stripping and re-probing the same membrane. C–D) 50 million EBV negative BJAB cells, BJAB10, LCL1, sh-Ctrl, sh-Pim-1 LCL1 cells were incubated with proteasome inhibitor MG132 drug (20 mM) for 6 hrs. Cells were harvested and lysed with RIPA buffer. IRF4 was immunoprecipitated (IP) by using specific antibodies. Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was performed by stripping and re-probing the same membrane. doi:10.1371/journal.ppat.1004304.g006 Figure 6. EBNA3C suppresses poly-ubiquitination of Pim-1. A–B) 10 million HEK-293 cells were transiently transfected with different combinations of expression vectors as indicated. Cells were harvested after 36 hrs of post-transfection by incubating with proteasome inhibitor MG132 drug for additional 6 hrs. Total protein was immunoprecipitated (IP) with indicated antibodies and protein samples were resolved by 10% SDS-PAGE. Western blots were performed by stripping and re-probing the same membrane. C–D) 50 million EBV negative BJAB cells, BJAB10, LCL1, sh-Ctrl, sh-Pim-1 LCL1 cells were incubated with proteasome inhibitor MG132 drug (20 mM) for 6 hrs. Cells were harvested and lysed with RIPA buffer. IRF4 was immunoprecipitated (IP) by using specific antibodies. Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was performed by stripping and re-probing the same membrane. doi:10.1371/journal.ppat.1004304.g006 Figure 7. EBNA3C enhances Pim-1 kinase mediated phosphorylation of p21 Thr145 residue. A–C) HEK-293 cells were transfected with Myc-tagged Pim-1 (wild type or kinase dead mutant) and Flag-tagged EBNA3C vectors as indicated. Empty vector was used to balance total transfected DNA. At 36 h post-transfection, Pim-1 immunoprecipitates were captured with anti-Myc antibody and assayed for in vitro kinase activity toward GST-p21 (wild type or T145A mutant) using cP 32-ATP. Western blot using whole cell lysates and Coomassie staining of SDS-PAGE resolved GST proteins used in this study is shown here. doi:10.1371/journal.ppat.1004304.g007 Figure 7. EBNA3C enhances Pim-1 dependent proteasome degradation of P21 Earlier reports demonstrated that Pim-1 kinase activity is linked to enhanced cellular proliferation in neoplastic cell types [68]. To determine the effect of EBNA3C on Pim-1 mediated cell proliferation, HEK-293 cells were transfected with control vector, Flag-tagged EBNA3C, Myc-Pim-1 expression vector, and Myc- Pim-1 with Flag-EBNA3C. Colony formation assays were performed after G418 selection for 2 weeks. The results demonstrated a significant increase in the colony numbers in EBNA3C and the Pim-1 co-transfected set compared with control vector or only Pim-1 transfected sets (Fig. S4A and S4B). Additionally, cell proliferation assays were performed by cell counting using Trypan blue dye exclusion technique up to 6 days (Fig. S4C). Previous studies suggested that Pim-1 expression accelerated the process of lymphoproliferation and inhibits apoptosis [30]. Also, depletion of Pim-1 by RNA interference in mouse and human prostate cancer cells reduced cellular prolifer- ation and survival [69]. To validate these studies, we used Lentivirus mediated delivery of sh-RNA vectors to knock down Pim-1 in LCL1 cells. Wild type LCL1, puromycin selected stable Ctrl-vector and Pim-1 knocked down cells with GFP fluorescence g To examine, whether EBNA3C has a vital role in p21 degradation alone or in collaboration with Pim-1, we performed poly-ubiquitination assays by expressing Flag-p21 and Myc-tagged EBNA3C in HEK-293 cells. The results demonstrated there was no significant change in the level of poly-ubiquitination (Fig. 10A). Our study also revealed a strong association with p21 and EBNA3C by co-immunoprecipitation experiments (Fig. S3A). Next, we attempted to examine the potential changes in p21 protein levels by expressing Flag-p21, Myc-Pim-1, along with increasing amounts of EBNA3C in HEK-293 cells. Interestingly, we observed reduced levels of p21 with a dose dependent increase of EBNA3C in the presence of Pim-1 (Fig. S3B). Our poly- ubiquitination assay results for p21, with wild-type Pim-1 and kinase-dead Pim-1 mutant clearly showed that the level of poly- ubiquitination was much higher with wild-type Pim-1 compared with kinase-dead mutant in the presence of EBNA3C (Fig. 10B). This supported an important role for EBNA3C in enhancing Pim- 1 kinase activity and is likely to be required for p21 degradation. Moreover, we extended the poly-ubiquitination assays using the Figure 8. EBNA3C competes with p21 for Pim-1 binding. A–B) 10 million HEK-293 cells were transfected with different combinations of Myc- tagged Pim-1, Flag-tagged p21, untagged-EBNA3C expression vectors as indicated by electroporation. EBNA3C competes with p21 to inhibit the complex formation with Pim-1 kinase EBNA3C enhances Pim-1 kinase mediated phosphorylation of p21 Thr145 residue. A–C) HEK-293 cells were transfected with Myc-tagged Pim-1 (wild type or kinase dead mutant) and Flag-tagged EBNA3C vectors as indicated. Empty vector was used to balance total transfected DNA. At 36 h post-transfection, Pim-1 immunoprecipitates were captured with anti-Myc antibody and assayed for in vitro kinase activity toward GST-p21 (wild type or T145A mutant) using cP 32-ATP. Western blot using whole cell lysates and Coomassie staining of SDS-PAGE resolved GST proteins used in this study is shown here. doi:10.1371/journal.ppat.1004304.g007 August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 8 EBV Regulates Pim-1 Function DNA damage response signal (reduction of serum with etoposide drug treatment). Interestingly, the results demonstrated that p21 expression levels were substantially reduced with co-expression of wild type Pim-1 and EBNA3C. However, p21 expression levels remained unchanged with EBNA3C, wild type Pim-1 or kinase dead Pim-1 alone with or without DNA damage (Fig. 9A, upper and lower panels). Therefore, EBNA3C contributes to the process of p21 degradation in co-operation with wild type Pim-1. We also extended our stability assays in EBV negative BJAB, EBNA3C stably expressing BJAB10, EBV transformed lymphoblastoid LCL1, sh-Ctrl and sh-EBNA3C LCL1 cells with cyclohexamide treatment in the presence or absence of etoposide induced DNA damage. P21 protein expression was found significantly reduced in LCL1, BJAB10 compared with BJAB even both with or without DNA damage response (Fig. 9B, upper and lower panels). Importantly, the expression levels were found augmented with or without DNA damage in EBNA3C stable knockdown LCL1 cells (Fig. 9C, upper and lower panels) suggesting a role for EBNA3C in deregulating p21 stability independent of etoposide induced DNA damage response. P21T145A mutant to determine whether the p21 Thr145 phosphorylation was related to its degradation. The results showed that levels of poly-ubiquitination remained unchanged both with wild-type and the kinase-dead mutant of Pim-1 in the presence of EBNA3C (Fig. 10C). Additionally, we performed poly-ubiquitina- tion assay using EBV negative BJAB, EBNA3C stably expressing BJAB10, EBV transformed lymphoblastoid LCL1, as well as sh- Ctrl and sh-EBNA3C LCL1 cells to monitor the poly-ubiquitina- tion status of p21. The results clearly indicated higher poly- ubiquitinated levels of p21 in EBNA3C expressing BJAB10, and LCL1 cells compared with EBNA3C negative BJAB (Fig. 10D). The levels were also reduced in EBNA3C stable knockdown LCL1 cells (Fig. 10E). EBNA3C competes with p21 to inhibit the complex formation with Pim-1 kinase To determine if the degradation of p21 is Pim-1 dependent, we performed poly-ubiquitination assays with Pim-1 stable knockdown LCL1 cells. The results indicated that upon Pim-1 knockdown, the level of p21 degradation was reduced (Fig. 10F). EBNA3C enhances Pim-1 dependent proteasome degradation of P21 A) IP was performed with anti-Myc antibody and IP complexes were resolved by 10% SDS-PAGE. Western blot was performed with indicated antibodies. B) Above mentioned transfected cells were treated with MG132 drug for 6 hrs and protein lysates were prepared by RIPA buffer. Western blot analysis was performed by indicated antibodies. doi:10.1371/journal.ppat.1004304.g008 Figure 8. EBNA3C competes with p21 for Pim-1 binding. A–B) 10 million HEK-293 cells were transfected with different combinations of Myc- tagged Pim-1, Flag-tagged p21, untagged-EBNA3C expression vectors as indicated by electroporation. A) IP was performed with anti-Myc antibody and IP complexes were resolved by 10% SDS-PAGE. Western blot was performed with indicated antibodies. B) Above mentioned transfected cells were treated with MG132 drug for 6 hrs and protein lysates were prepared by RIPA buffer. Western blot analysis was performed by indicated antibodies. doi:10.1371/journal.ppat.1004304.g008 doi:10.1371/journal.ppat.1004304.g008 August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 9 EBV Regulates Pim-1 Function Figure 9. EBNA3C destabilizes p21 through Pim-1 independent of the DNA damage response. A) 10 million HEK-293 cells were transfected with combinations of Myc-tagged Pim-1, Flag-tagged p21, untagged-EBNA3C expression by electroporation. After 36 hrs of post- transfection, cells were treated with cyclohexamide for indicated time points in DMEM medium containing either serum/DMSO or 0.1% FBS with 5 mM etoposide. Protein samples were resolved by 10% SDS-PAGE and Western blots were performed with indicated antibodies. B–C) 50 million EBV negative BJAB cells, EBNA3C expressing BJAB10, EBV transformed LCL1, sh-Ctrl, sh-E3C LCL1 cells were incubated with cyclohexamide for specific time points in RPMI medium containing serum/DMSO or 0.1% FBS with 5 mM etoposide. Samples were resolved by 10% SDS-PAGE and Western blot analysis was performed with specific antibodies. doi:10.1371/journal.ppat.1004304.g009 EBV Regulates Pim 1 Function Figure 9. EBNA3C destabilizes p21 through Pim-1 independent of the DNA damage response. A) 10 million HEK-293 cells were transfected with combinations of Myc-tagged Pim-1, Flag-tagged p21, untagged-EBNA3C expression by electroporation. After 36 hrs of post- transfection, cells were treated with cyclohexamide for indicated time points in DMEM medium containing either serum/DMSO or 0.1% FBS with 5 mM etoposide. Protein samples were resolved by 10% SDS-PAGE and Western blots were performed with indicated antibodies. B–C) 50 million EBV negative BJAB cells, EBNA3C expressing BJAB10, EBV transformed LCL1, sh-Ctrl, sh-E3C LCL1 cells were incubated with cyclohexamide for specific time points in RPMI medium containing serum/DMSO or 0.1% FBS with 5 mM etoposide. EBNA3C enhances Pim-1 dependent proteasome degradation of P21 Samples were resolved by 10% SDS-PAGE and Western blot analysis was performed with specific antibodies. doi:10.1371/journal.ppat.1004304.g009 August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 10 EBV Regulates Pim-1 Function monitored (Fig 11A) Also the e pression le els of Pim 1 in death in stable Pim 1 knockdo n LCL1 ith ser m star ati re 10. EBNA3C augments Pim-1 dependent degradation of p21. A–C) 10 million HEK-293 cells were transiently transfected with differe binations of expression vectors as indicated. Cells were harvested after 36 hrs of post-transfection by incubating with MG132 drug for additio and total protein was immunoprecipitated (IP) with indicated antibodies and protein samples were resolved by 10% SDS-PAGE. Western blo performed by stripping and re-probing the same membrane. D–F) 50 million EBV negative BJAB, BJAB cells stably expressing BJAB10 and E formed LCL1, sh-Ctrl, sh-E3C, sh-Pim-1 LCL1 cells were incubated with proteasome inhibitor MG132 drug (20 mM) for 6 hrs. Treated cells we ested and lysed with RIPA buffer. p21 was immunoprecipitated (IP) by using specific antibody. Samples were resolved by 10% SDS-PAG ern blotting (WB) was performed by stripping and re-probing the same membrane. 0.1371/journal.ppat.1004304.g010 Figure 10. EBNA3C augments Pim-1 dependent degradation of p21. A–C) 10 million HEK-293 cells were transiently transfected with different combinations of expression vectors as indicated. Cells were harvested after 36 hrs of post-transfection by incubating with MG132 drug for additional 6 hrs and total protein was immunoprecipitated (IP) with indicated antibodies and protein samples were resolved by 10% SDS-PAGE. Western blots were performed by stripping and re-probing the same membrane. D–F) 50 million EBV negative BJAB, BJAB cells stably expressing BJAB10 and EBV transformed LCL1, sh-Ctrl, sh-E3C, sh-Pim-1 LCL1 cells were incubated with proteasome inhibitor MG132 drug (20 mM) for 6 hrs. Treated cells were harvested and lysed with RIPA buffer. p21 was immunoprecipitated (IP) by using specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was performed by stripping and re-probing the same membrane. doi:10.1371/journal.ppat.1004304.g010 death in stable Pim-1 knockdown LCL1 with serum starvation (Fig. 11C, D). were monitored (Fig. 11A). Also, the expression levels of Pim-1 in different clones were examined by performing Western blot analysis (Fig. 11B). EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 Previous studies showed upregulation of Pim kinases during Epstein-Barr virus infection [45]. Epstein–Barr virus (EBV) was found potentially involved in the pathogenesis of different B-cell lymphoproliferative disease and all three EBV nuclear antigen 3 proteins can manipulate the expression of a wide range of cellular genes and they often act co-operatively to induce epigenetic chromatin modifications [80]. Another report demonstrated that the involvement of EBNA3A and EBNA3C expression with polycomb complexes for the covalent K27me3 modification of histone H3 at the p16INK4A promoter to repress the transcription [81]. Also, BIM expression was regulated in latently infected EBV cells through epigenetic modification and CpG methylation [11]. EBNA3C, regulates transcription of a wide range of viral and cellular genes [14]. EBNA3C was found to be associated with Nm23-H1 to regulate the transcription process of cellular genes which are critically involved in cell migration and invasion [82]. Recent reports also demonstrated that EBNA3C can physically interact and stabilize different host oncoproteins, including c-Myc and IRF4 [20,51], and has a major role in regulation of the cell cycle regulatory protein complex Cyclin D1/CDK6 to drive B-cell malignancies [22]. Interestingly, some reports showed that Pim-1 levels are tightly controlled at many steps from the transcriptional to translational levels [60]. Our study now demonstrated upregulation of Pim-1 expression at the mRNA and protein levels with EBV infection in primary B-cells as well as EBV positive cancer cell lines. Interestingly, infection with the DEBNA3C BAC- GFP-EBV showed a much lower Pim-1 expression at 2 days post- infection. However, the expression patterns remain unchanged at later time points. Our results from the primary infection studies suggested a major contributory role of EBNA3C in inducing Pim- 1 expression. We also observed a substantial reduction in Pim-1 expression levels only after siRNA mediated knockdown of EBNA3C but not with the knockdown of EBNA2, EBNA3A and EBNA3B which further confirmed a direct role of EBNA3C in regulating Pim-1 expression levels in EBV-transformed cells. Moreover, our studies showed that EBNA3C has a strong physical association with Pim-1, and that Pim-1 binds to the N-terminal 130–159 residues of EBNA3C. Interestingly, several other studies from our Lab also demonstrated that this region of EBNA3C specifically interacts with different important cellular proteins such as cyclin A, p53, E2F1, c-Myc, IRF4/IRF8 etc [20,21,46,51,83]. Therefore, this 130–159 aa residues of EBNA-3C have particular significance in deregulating major cellular process in EBV-infected cells. EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 Further detailed investigation is needed to evaluate the functional role of this domain in connection with EBNA3C mediated oncogenesis. Our co-immunoprecipitation experi- ments in EBNA3C expressing Burkitt’s lymphoma cells and EBV transformed Lymphoblastoid cells also demonstrated that p p p As an inhibitor of cyclin-dependent kinases, p21Waf1/Cip1 is required for proper cell-cycle progression [72]. Earlier reports suggested that p21 suppresses tumors by promoting cell cycle arrest in response to various stimuli [61]. In addition, substantial evidence from biochemical and genetic studies shows that p21 acts as a potential effector of multiple tumor suppressor pathways to promote its anti-proliferative activities independent of p53 [61]. Interestingly, several studies demonstrated that ubiquitin-mediated degradation of p21 can also promote cancer cell proliferation [73]. Our results above indicated that p21 is targeted by EBNA3C through Pim-1 dependent degradation. We next attempted to examine whether EBNA3C has a role in modulating p21- mediated inhibition of cell proliferation involving Pim-1 kinase with DNA damage response. HEK-293 and MEF cells were transfected with different combinations of Flag-tagged p21 (wild type and the T145A mutant), Myc-Pim-1 (wild type and the kinase dead mutant), EBNA3C expression vectors. Cell proliferation assays were performed without serum and with etoposide treatment after 2 weeks of G418 antibiotic selection. The results demonstrated that EBNA3C together with wild type Pim-1 effectively reduced the growth suppressive effect of p21. The cell proliferation rate in p21 expressing cells either with EBNA3C or wild type Pim-1 was shown to be enhanced compared to control vector alone. Interestingly, the lower rate of cell proliferation was observed with kinase dead Pim-1 mutant or P21T145A mutant even in the presence of EBNA3C (Fig. 12A, B). To check the expression levels of these proteins, we performed Western blot analysis with these G418 selected cells (Fig. S5A and S5B). Moreover, our immunofluorescence studies for BrdU incorpora- tion with DNA damage showed an increased number of BrdU foci with wild type Pim-1 and p21, co-expressed cells with EBNA3C (Fig. 12C and 12D). To determine the possible contribution of different molecules which are involved with intrinsic apoptotic signaling in the context of Pim-1 mediated p21 downregulation in the presence of EBNA3C, we checked the protein expression profiles of Caspase-3, Caspase-9, Apaf-1, and Bcl2 in sh-Ctrl- vector transfected and Pim-1 stable knockdown EBV negative Ramos cells. These cells were transfected with p21 and an increasing dose of EBNA3C. Discussion Pim-1 was identified in murine leukaemia virus (MuLV)- induced lymphomas that frequently contains proviral insertions which were associated with the transcriptional activation of the Pim-1 gene frequently associated with enhanced tumorigenesis [74]. Overexpression of Pim kinases have been found in various lymphomas and leukemias [75]. Different reports have suggested a role for Pim-1 kinase in progression of Burkitt’s lymphoma [76], primary cutaneous large B-cell lymphoma [77] and prostate cancer [78]. Pim-1 kinase also performs multiple cellular functions related to cell survival, proliferation, differentiation, apoptosis, and progression of tumors [79]. EBNA3C enhances Pim-1 dependent proteasome degradation of P21 In order to determine whether Pim-1 knockdown in an LCL background has some implications in apoptotic cell death, we performed apoptosis assays using stable sh-Ctrl LCL1, sh-Pim-1 LCL1 cells with or without serum starvation. Cells were stained with Propidium iodide for FACS analysis. The results showed substantial increase in apoptotic cell Programmed cell death or apoptosis is considered as a major regulator of cellular growth control and tissue homeostasis [70]. Previous reports suggested that caspases activation can be triggered through the induction of the extrinsic apoptotic pathway or at the mitochondria by stimulating the intrinsic apoptotic pathway in response with anticancer chemotherapy [71]. In order August 2014 \| Volume 10 \| Issue 8 \| e1004304 August 2014 \| Volume 10 \| Issue 8 \| e1004304 11 PLOS Pathogens \| www.plospathogens.org EBV Regulates Pim-1 Function EBV Regulates Pim-1 Function inhibiting cell growth suppressive property of p21 which occurs through the intrinsic apoptotic pathway. inhibiting cell growth suppressive property of p21 which occurs through the intrinsic apoptotic pathway. to determine whether the inhibition of Pim-1 had some effect on apoptotic event in LCLs, we performed Western blot analysis to monitor the levels of PARP-1 cleavage. Our result showed that Pim-1 knock-down EBV transformed cells showed higher signals for the PARP-1 cleavage (Fig. 11E). Moreover, we detected higher expression levels of Caspase-3, Caspase-9, and Apaf-1 in Pim-1 stable knockdown LCL1 in comparison with sh-Ctrl LCL1 cells which indicates that Pim-1 knockdown induced the intrinsic apoptotic pathway in EBV transformed cells (Fig. 11F). We performed cell proliferation assays in the context of Pim-1 knock- down. Interestingly, the result showed that the rate of proliferation of Pim-1 stable knock-down LCL1 cells was lower compared with LCL1 and sh-Ctrl LCL1 cells (Fig. 11G). EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 Our results demonstrated that the expression levels of Caspase-3, Caspase-9, Apaf-1 were unchanged with P21 transfection in Pim-1 knockdown Ramos cells, in the presence of EBNA3C compared with sh-Ctrl-Ramos cells where the expression of these proteins were reduced (Fig. 12E, compare left and right panels). Interestingly, Bcl2 levels were found to be upregulated in sh-Ctrl cells (Fig. 12E). These results support our hypothesis that EBNA3C can potentiate Pim-1 kinase activities for August 2014 \| Volume 10 \| Issue 8 \| e1004304 12 PLOS Pathogens \| www.plospathogens.org EBV Regulates Pim-1 Function Figure 11. Pim-1 silencing by sh-RNA sensitizes EBV transformed lymphoblastoid cells by induction of the intrinsic apop signaling pathway. A) Wild type LCL1, Lentivirus mediated and puromycin selected stable sh-Ctrl vector transfected and different clones of knocked down cells were observed by fluorescence microscope for monitoring GFP fluorescence B) 50 million different clones of stable sh-Pim- Ctrl LCL1, and LCL1 cells were harvested and cell lysates were prepared by RIPA buffer. Western blot analysis was performed to show the expre levels of EBNA3C, Pim-1 and GAPDH. C) sh-Ctrl, sh-Pim-1 LCL1 cells were grown in RPMI medium for 12 hrs with or without serum starvation were stained with Propidium iodide and subjected to Flowcytometric analysis. D) Bar diagram represents the fold change of apoptosis observ apoptosis assay using FACS. The results represented here are representative of three independent experiments. E–F) 50 million sh-Ctrl and sh- LCL1 cells were harvested and cell lysates were used for Western blot analysis with indicated antibodies. G) 16106 cells were plated and allowed to grow at 37uC in complete medium without puromycin antibiotic. Viable cells were counted by trypan blue dye exclusion technique at indi time points. The results shown here are representative of three independent experiments. doi:10.1371/journal.ppat.1004304.g011 Figure 11. Pim-1 silencing by sh-RNA sensitizes EBV transformed lymphoblastoid cells by induction of the intrinsic apoptosis signaling pathway. A) Wild type LCL1, Lentivirus mediated and puromycin selected stable sh-Ctrl vector transfected and different clones of Pim-1 knocked down cells were observed by fluorescence microscope for monitoring GFP fluorescence B) 50 million different clones of stable sh-Pim-1, sh- Ctrl LCL1, and LCL1 cells were harvested and cell lysates were prepared by RIPA buffer. Western blot analysis was performed to show the expression levels of EBNA3C, Pim-1 and GAPDH. C) sh-Ctrl, sh-Pim-1 LCL1 cells were grown in RPMI medium for 12 hrs with or without serum starvation. EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 Cells were incubated for 12 hrs in DMEM medium containing 0.1% FBS with 5 mM etoposide. 0.16106 cells from each set of selected samples were plated and cultured for 6 days. Viable cells were counted at indicated time points by trypan blue dye exclusion technique. B) G418 selected stable cells were harvested, lysed in RIPA buffer and subjected to immunoblot analyses with indicated antibodies. C) 106106 HEK-293 were transfected with different combinations of expression vectors and transiently transfected cells were subjected to BrdU incorporation assay by immunofluorescence study with etoposide treatment in DMEM containing 0.1% FBS. D) Bar diagram represents number of BrdU foci per cell. The results shown here are representative of three independent experiments. E) 30 million sh-Ctrl and sh-Pim-1 Ramos cells were co-transfected with p21 and increasing amount of EBNA3C expression vectors. Transfected cells were subjected to Western Blot analysis by using indicated antibodies. doi:10.1371/journal.ppat.1004304.g012 Pim-1 forms a strong molecular complex with EBNA3C in infected cells. that Pim-1 protein stabilization by EBNA3C can result in increased levels of Pim-1 in EBV infected cells. Additionally, the stability of the Pim-1 kinase is largely regulated by the ubiquitin/ proteasome pathway [85]. Several reports suggested the important role of EBNA3C for deregulating the functions of different cellular proteins by manipulation of ubiquitin/proteasome pathways [86]. Our Lab previously demonstrated the interaction between EBNA3C with SCFSkp2 E3 ligase complex [87]. Also, the N- terminal domain of EBNA3C physically associated with the acidic domain of Mdm2 which is a known E3 ubiquitin-protein ligase [50]. Other studies also suggested that EBNA3C associates with the a-subunit of the 20S proteasome and is degraded in-vitro by purified 20S proteasomes [88]. EBNA3C was found to facilitate the degradation of E2F1 by targeting ubiquitin-proteasome pathways [46]. Recently, we have shown that EBNA3C deregu- lates total H2AX levels through involvement of the ubiquitin/ Previous studies suggested that nuclear localization of Pim-1 is essential for the regulation of its cellular substrates as well as additional biological activities of this kinase [76]. Importantly, our co-localization studies showed that in the absence of EBNA3C, localization of Pim-1 was mostly in the cytoplasm and predom- inantly in the nucleus in the presence of EBNA3C. Our immunofluorescence assay therefore revealed a strong co-locali- zation with Pim-1 and EBNA3C in the nucleus. Previously it was shown that the Hsp90 protein is responsible for correct folding and stabilization of Pim-1 [84]. EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 Cells were stained with Propidium iodide and subjected to Flowcytometric analysis. D) Bar diagram represents the fold change of apoptosis observed by apoptosis assay using FACS. The results represented here are representative of three independent experiments. E–F) 50 million sh-Ctrl and sh-Pim-1 LCL1 cells were harvested and cell lysates were used for Western blot analysis with indicated antibodies. G) 16106 cells were plated and allowed them to grow at 37uC in complete medium without puromycin antibiotic. Viable cells were counted by trypan blue dye exclusion technique at indicated time points. The results shown here are representative of three independent experiments. doi:10.1371/journal.ppat.1004304.g011 August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 13 EBV Regulates Pim-1 Function Figure 12. EBNA3C mediated oncogenic Pim-1 activation promotes cell proliferation by impeding the growth suppressive properties of p21. A–B) 10 million HEK-293 and MEF cells were transfected with different combinations of expression plasmids as indicated (A, P21+Ctrl vector; B, P21+Pim-1; C, P21+EBNA3C; D, P21+Pim-1+EBNA3C; E, P21+Pim-1KD; F, P21+Pim-1KD+EBNA3C; G, P21T145A+Pim-1; H, P21T145A+Pim-1+EBNA3C). Transfected cells were selected for 2 weeks with G418 antibiotic. Cells were incubated for 12 hrs in DMEM medium containing 0.1% FBS with 5 mM etoposide. 0.16106 cells from each set of selected samples were plated and cultured for 6 days. Viable cells were counted at indicated time points by trypan blue dye exclusion technique. B) G418 selected stable cells were harvested, lysed in RIPA buffer and subjected to immunoblot analyses with indicated antibodies. C) 106106 HEK-293 were transfected with different combinations of expression vectors and transiently transfected cells were subjected to BrdU incorporation assay by immunofluorescence study with etoposide treatment in DMEM containing 0.1% FBS. D) Bar diagram represents number of BrdU foci per cell. The results shown here are representative of three independent experiments. E) 30 million sh-Ctrl and sh-Pim-1 Ramos cells were co-transfected with p21 and increasing amount of EBNA3C expression vectors. Transfected cells were subjected to Western Blot analysis by using indicated antibodies. doi:10.1371/journal.ppat.1004304.g012 Figure 12. EBNA3C mediated oncogenic Pim-1 activation promotes cell proliferation by impeding the growth suppressive properties of p21. A–B) 10 million HEK-293 and MEF cells were transfected with different combinations of expression plasmids as indicated (A, P21+Ctrl vector; B, P21+Pim-1; C, P21+EBNA3C; D, P21+Pim-1+EBNA3C; E, P21+Pim-1KD; F, P21+Pim-1KD+EBNA3C; G, P21T145A+Pim-1; H, P21T145A+Pim-1+EBNA3C). Transfected cells were selected for 2 weeks with G418 antibiotic. PLOS Pathogens \| www.plospathogens.org EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 Further, studies from our group demonstrated an important role of EBNA3C in stabilizing different oncoproteins such as Gemin3, Cyclin D1, and IRF4 [19,22,51] to deregulate normal cellular functions which can drive development of neoplastic events. Our study clearly demonstrated August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 14 EBV Regulates Pim-1 Function proteasome degradation pathway [89]. Interestingly, other reports suggested the potential involvement of Pim-1 with ubiquitin/ proteasome pathways as enhanced expression of Pim-1 increases the level of SCFSkp2 ubiquitin ligase through the direct binding and phosphorylation of multiple sites on this protein [90]. A previous study showed the role of heat shock proteins and the ubiquitin-proteasome pathway for regulating the stability of Pim-1 kinase [85]. Our poly-ubiquitination experiments clearly suggested that Pim-1 poly-ubiquitination was significantly inhibited by EBNA3C and so resulted in increased Pim-1 levels. Since enhanced levels of Pim-1 is linked to different hematological or non-hematological malignancies, it reveals the intricate mecha- nisms that are linked to ubiquitin-proteasome-mediated degrada- tion of Pim-1 and is important for designing therapeutic interventions. Additionally, this approach could enhance new therapeutic avenues by targeting Pim-1 kinase and so enhance the efficiency of conventional therapeutic strategies against EBV mediated oncogenesis. apoptotic pathway [98]. Our results also support this finding showing that EBNA3C induced Pim-1 mediated downregula- tion of p21 which is also related to the inhibition of intrinsic apoptotic pathway in EBV transformed cells. Recent evidence suggested a role for the RNF126 E3 ubiquitin ligase in promoting cancer cell proliferation by p21 degradation [73]. Our results strongly suggested an important role for EBNA3C to effectively inhibit the growth suppressive effects of p21 in the presence of Pim-1. Interestingly, we observed a lower rate of cell proliferation with the kinase dead Pim-1 mutant or P21T145A mutant even in the presence of EBNA3C. This supports a role for Pim-1-mediated phosphorylation of the Thr145 residue of p21 in cell proliferation. p p In summary, our current work demonstrated an important molecular mechanism which revealed a direct role for the EBV latent antigen 3C in enhancing expression of the oncoprotein Pim-1 in EBV transformed B-cells as well as in EBV-infected PBMCs. We also showed the physical interaction between EBNA3C and Pim-1 and further mapped the binding to the Amino-terminal domain of EBNA3C. Moreover, our study demonstrated that EBNA3C mediated stabilization of Pim-1 through abrogation of the proteasome/ubiquitin pathway. EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 EBNA3C also facilitated the nuclear export of Pim-1 and promoted EBV transformed cell proliferation by altering Pim-1- mediated regulation of the cell-cycle inhibitor p21/WAF1 activity. Our study now demonstrated that EBNA3C directly contributes to Pim-1 mediated phosphorylation of p21 which facilitates its proteosomal degradation. In addition, significant reduction of EBV transformed cell proliferation as well as a substantial induction of apoptotic cell death was also observed upon stable knockdown of Pim-1. Our study now provides a novel insight into the precise role of oncogenic Pim-1 in EBV- mediated oncogenesis (Fig. 13). Moreover, siRNA mediated knockdown of Pim-1 triggers the intrinsic apoptotic signaling pathway in LCLs and repressed proteasome-mediated degrada- tion of p21. Pim-1 knockdown further demonstrated a vital role in EBV-mediated proliferation of B-cells by impeding the process of apoptosis. Our findings thus contribute to a more indepth understanding of the role of EBNA3C expressed in EBV-infected B-cells and its interaction with the critical cellular kinase which leads to EBV induced B-cell transformation. Being a potent serine/threonine kinase, Pim-1 plays impor- tant roles in a number of cellular events. Most notably, Pim-1 can synergize with c-Myc to drive the rapid progression of B- cell lymphomas [91]. This synergism is likely to originate from the anti-apoptotic activity promoted by Pim-1 [92]. Among other Pim-1 substrates, the Cyclin-dependent kinase inhibitor 1 or p21 is important in the context of viral pathogenesis. Interestingly, p21 stability has been exploited by different tumor viruses. A number of viral proteins can affect the post- transcriptional regulation of p21, thereby affecting cellular proliferation. The human papilloma virus E6 proteins can downregulate p21 independently of p53 [93]. Also, the hepatitis C virus and K-cyclin encoded by the human herpesvirus 8 stimulates p21 phosphorylation at the Ser130 residue by CDK6 without affecting its stability [94]. These findings further establish that targeting p21 is likely to be a common strategy for viruses to regulate cell cycle progression and apoptosis. Previous reports also showed that EBV acts downstream of the p53 and appears to prevent the inactivation of cyclin- dependent kinase CDK2 by p21WAF1/CIP1 by targeting p21 for degradation by the proteasome pathway [95]. Basically, participation of p21 in multiple cellular functions emphasizes its importance and that its precise regulation is crucial for maintenance of the normal cellular function. Importantly, its phosphorylation and interaction with other cellular proteins are crucial to p21 stability at the post-translational level. EBNA3C potentiates oncogenic Pim-1 to promote cell proliferation by inhibiting the growth suppressive properties of p21 Previous reports suggested that the Thr145 residue of p21 is preferen- tially phosphorylated by Pim-1 [36]. Our current study clearly demonstrated a role for EBNA3C in enhancing Pim-1 kinase activity to phosphorylate p21. We also observed that Pim-1 was not able to phosphorylate mutant p21 (T145A) even in the presence of EBNA3C. Interestingly, we identified a molecular association between Pim-1 and p21, with EBNA3C and our competitive binding assay demonstrated that increasing doses of EBNA3C resulted in reduced association between Pim-1 and p21, causing in destabilization of p21 by enhancing its proteasome-mediated degradation independent of etoposide induced DNA damage response. Ethics statement PBMC were obtained from University of Pennsylvania Human Immunology Core (HIC) and donated by the healthy donors. This study was approved by University of Pennsylvania Human Immunology Core (HIC) which maintains University of Pennsyl- vania IRB protocol. In this IRB approved protocol the declara- tions of Helsinki protocols were followed and each donor gave written, informed consent. There is no link between donors and their information with this study. PLOS Pathogens \| www.plospathogens.org Plasmids, antibodies and cell lines HEK-293, and MEF cells were grown in Dulbeccoo’s modified Eagle’s medium (DMEM). EBV negative Burkitt’s lymphoma cells BJAB, DG75, Ramos were kindly provided by Elliot Kieff (Harvard Medical School, Boston, MA). BJAB stably expressing EBNA3C (BJAB7, BJAB10) were previously described [76]. EBV transformed lymphoblastoid cell lines (LCL1, LCL2) were maintained in RPMI 1640 media as template. The PCR amplified insert was subjected for EcoRI/ NotI restriction enzyme digestion and ligated into pGEX2T vector. pGEX-P21T145A and Flag-P21T145A constructs were cloned by using PBK/CMV/LacZ P21T145A (kindly provided by Dr. Nancy Magnuson) as a template for the PCR amplification. pCDNA3-HA-Ub construct was kindly provided by George Mosialos (Aristotle University of Thessaloniki, Greece) and pGIPZ was used as the sh-RNA vector described previously [51]. Constructs used for lentiviral packaging were previously described [99]. Plasmids, antibodies and cell lines Several reports indicated that Pim-1 expression is associated with cell proliferation and survival [96]. Pim-1 also induces anti- cancer drug resistance by inhibiting the intrinsic mitochondrial apoptosis pathway [97]. In our studies, siRNA mediated knock down of Pim-1 showed reduced proliferation of EBV trans- formed cells. Moreover, Pim-1 silencing potentially activated the intrinsic apoptotic signaling in EBV transformed cells. Recent studies showed that upregulation of p21 activated the intrinsic Full length and truncated mutants of GST, Myc, Flag, and GFP tagged EBNA3C expression vectors were described previously [22,51]. Myc-tagged EBNA3C with C143N point mutation was generated by using standard PCR primer mutagenesis method [50]. Constructs for Myc-tagged Pim-1, kinase dead (KD) version of Pim-1 as Pim-1 K67M (mutated at the ATP binding Pocket), pGEX2T-Pim-1 were mentioned previously [43]. Wild type pGEX-P21 construct was generated by using Flag-P21 construct August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org August 2014 \| Volume 10 \| Issue 8 \| e1004304 15 EBV Regulates Pim-1 Function re 13. The schematic diagram illustrates the potential contribution of EBNA3C to oncogenic transformation of B-cells through ilization of Pim-1 and proteasome mediated degradation of p21 which results in inhibition of the intrinsic apoptotic pathway. A3C interacts with oncogenic serine/threonine Pim-1 kinase. This molecular association stabilizes Pim-1 by inhibiting its Poly-ubiquitination. A3C also promotes Pim-1 mediated p21 degradation. EBNA3C contributes to B-cell proliferation by activating oncogenic Pim-1 which leads to ition of the growth suppressive property of p21 as well as impeding intrinsic apoptosis signaling. This molecular strategy for oncogenic Pim-1 e targeted by EBNA3C for inhibition of p21 function, and it can be a potential therapeutic target for EBV-associated malignancies. 0.1371/journal.ppat.1004304.g013 Figure 13. The schematic diagram illustrates the potential contribution of EBNA3C to oncogenic transformation of B-cells through stabilization of Pim-1 and proteasome mediated degradation of p21 which results in inhibition of the intrinsic apoptotic pathway. EBNA3C interacts with oncogenic serine/threonine Pim-1 kinase. This molecular association stabilizes Pim-1 by inhibiting its Poly-ubiquitination. EBNA3C also promotes Pim-1 mediated p21 degradation. EBNA3C contributes to B-cell proliferation by activating oncogenic Pim-1 which leads to inhibition of the growth suppressive property of p21 as well as impeding intrinsic apoptosis signaling. This molecular strategy for oncogenic Pim-1 kinase targeted by EBNA3C for inhibition of p21 function, and it can be a potential therapeutic target for EBV-associated malignancies. doi:10 1371/journal ppat 1004304 g013 MA, USA). Transfections Transfection in HEK-293, MEF and B-cells were performed by electroporation system with Bio-Rad Gene Pulser II electropora- tor. Cells were electroporated at 210 V and 975 mF (for HEK-293, MEF cells) or 220 V and 975 mF (for DG75, Ramos, LCL1 cells). Antibodies of Pim-1 (E-16), Ub (FL-76), PARP-1 (F-2), and GFP (I-16), Caspase-3 (E-8), Caspase-9 p10 (H-83), Apaf-1 (H-324), Bcl2 (C-2) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). P21 (ab7960) antibody was purchased from Abcam (Cambridge, MA). GAPDH antibody was procured from US-Biological Corp. (Swampscott, MA). Flag (M2)-epitope, anti- mouse antibody was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Hybridomas for mouse anti-Myc (9E10), anti- Hemaggutinin (12CA5), A10 were previously described [99]. In-vitro kinase assay Myc-tagged-Pim-1 (wild type or kinase dead mutant) (5 mg), Flag-EBNA3C (5 mg) expression constructs were transfected in HEK-293 cells. After 36 hrs of post-transfection, cell lysates were prepared and protein complexes were immunoprecipitated (IP) by using 9E10 ascites fluid. IP complexes were then washed with buffer A (containing 25 mM Tris [pH 7.5], 70 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, with protease and phospha- tase inhibitors) and incubated in 30 ml of kinase buffer B (containing buffer A plus 10 mM cold ATP, and 0.2 mCi of [c- 32P]-ATP/ml) supplemented with bacterially purified GST-P21 (wild type or T145A mutant) for 30 min at 30uC. 26 laemmli buffer was added to stop the reaction and heated at 95uC for 10 min. 10% SDS-PAGE was used for resolving the labeled proteins. Quantitation of the band was performed by using Image Quant software (GE Healthcare Biosciences, Pittsburgh, PA). Stability assay Transient transfection was performed in 10 million HEK-293 cells using electroporation system with combinations of plasmids as mentioned in the text. After 36 hours transfection, transfected cells as well as B-cells were treated with 40 mg/ml cyclohexamide in specific time periods with DNA damage response and cell lysates were prepared with RIPA buffer. Protein samples were subjected to Real-time PCR analysis Ice-cold PBS was used to wash the cells prior to RNA isolation. Trizol reagent (Invitrogen, Inc., Carlsbad, CA) was used for RNA extraction according to manufacturer’s protocol. Next, Superscript II reverse transcriptase kit (Invitrogen, Inc., Carlsbad, CA) was used for cDNA preparation according to the manufacturer’s instructions. The primers for Pim-1, EBNA3C, EBNA2, EB- NA3A, EBNA3B were 59-CGAGCATGACGAAGAGATCAT-39 and 59-TCGAAGGTTGGCCTATCTGA-39 [102], 59-AGAA- GGGGAGCGTGTGTTGT-39 and 59-GGCTCGTTTTTGA- CGTCGGC-39, 59- GAACTTCAACCCACACCATC-39 and 59- CGTGGTTCTGGACTATCTGG-39, 59- GGTGAAACGC- GAGAAGAAAG-39 and 59- CTCTCATCAGCAGCAACCTG- 39, 59- AGAAGAGGCCCTTGTGTCTT-39 and 59- GGATTT- CAAGAGGGTCAGGT-39 respectively. GAPDH primers were used as 59-TGCACCACCAACTGCTTAG-39 and 59-GATG- CAGGGATGATGTTC-39 [22]. SYBER green Real-time master mix (MJ Research Inc., Waltham, MA) was used for quantitative real-time PCR analysis. To check the specificity of the products a melting curve analysis was performed and the relative quantitation values were calculated by threshold cycle method. Triplicate sets were used to examine each sample. Poly-ubiquitination assay Expression vectors were transfected by electroporation in HEK- 293 cells. Transfected cells were incubated for 36 hrs in fresh DMEM and treated with 20 mM MG132 (Enzo Life Sciences International, Inc., Plymouth Meeting, PA) for another 6 hrs. Protein samples were immunoprecipitated with appropriate antibodies and resolved by SDS-PAGE. The level of ubiquitina- tion was detected by HA-specific antibody (12CA5). Infection of PBMCs with BAC-GFP wild type EBV and DE3C-EBV PBMCs (Peripheral blood mononuclear cells) were obtained from healthy donors from University of Pennsylvania Immunology Core as mentioned previously [100]. As defined earlier [46], 10 million PBMCs were mixed with wild type and EBNA3C HEK-293 (human embryonic kidney cell line) was kindly provided by Jon Aster (Brigham and Woman’s Hospital, Boston, HEK-293 (human embryonic kidney cell line) was kindly provided by Jon Aster (Brigham and Woman’s Hospital, Boston, August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 16 EBV Regulates Pim-1 Function blocking purpose. Myc-tagged Pim-1 was detected by using anti- Myc (9E10) antibody and the expression of GFP-tagged EBNA3C was detected by GFP-fluorescence. BJAB, BJAB10, and LCL1 cells were semi-air-dried on slides and fixed as mentioned above. Specific antibodies were used to check endogenous expressions of EBNA3C and Pim-1. Nuclear staining was performed by using DAPI (49,69,- diamidino-2-phenylindole; Pierce, Rockford, IL). After secondary antibody and DAPI incubation, cells were washed in 16PBS and mounted with antifade mounting medium. The images were taken by Fluoview FV300 confocal microscope and FLUOVIEW software (Olympus Inc., Melville, NY) was used for image analysis. knockout mutant (BAC-GFP-DE3C-EBV) virus supernatant in 1 ml of RPMI 1640 media containing 10% FBS for 4 hrs at 37uC and 5% co2 environment. Next, cells were centrifuged at 5006g for 5 minutes and pelleted cells were again re-suspended in 2 ml of complete medium. EBV-GFP expression was checked by fluores- cence microscopy and used to evaluate the infection. Infected cells were harvested at specific time intervals to determine the Pim-1 protein and mRNA levels. GST-pulldown assay For GST pull-down assays, cell lysates from BJAB, BJAB7, BJAB10, LCL1, LCL2 cells were incubated with bacterially purified control GST protein and GST fusion proteins. Protein samples were washed using Binding Buffer (16PBS, 0.1% NP-40, 0.5 mM DTT, 10% glycerol, with protease inhibitors) and resolved by 10% SDS-PAGE. A10 antibody was used for Western blot analysis. Immunoprecipitation and western blot analysis p p y Cells were harvested and washed with 16 Phosphate Buffered Saline (PBS). For the preparation of cell lysates, RIPA buffer (0.5% NP-40, 10 mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, supplemented with 1 mM PMSF, and protease inhibitors) was used. Cell lysates were then pre-cleared with normal mouse/rabbit serum rotating with 30 ml of Protein-A and Protein-G (1:1 mixture)-conjugated Sepharose beads for 1 hr at 4uC. 5% of the protein lysate was saved as input sample. Appropriate antibody (1 mg/ml) was used to capture the specific protein of interest by rotating the sample overnight at 4uC. The immune-precipitated samples were washed with RIPA buffer. Protein samples were boiled in laemmli buffer [101], resolved by SDS-PAGE and Western blotting was performed. The membranes were probed with appropriate antibodies and scanned using the Odyssey imager (LiCor Inc., Lincoln, NE). Nuclear and cytosolic fractionation assay HEK-293 cells were transfected with combinations of expres- sion vectors. After 36 hrs of post-transfection, cells were washed with PBS and re-suspended into hypotonic buffer [5 mM Pipes (KOH) pH 8.0, 85 mM KCl, 0.5% NP-40 supplemented with protease inhibitors). Cells were incubated on ice, and Dounce homogenizer was used to homogenize the cells with 20 strokes. Nuclei were pelleted down (23006g for 5 min) and the cytosolic fraction was collected. Nuclear pellets were washed again with PBS, re-suspended in nuclear lysis buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% NP-40, and protease inhibitors) and lysed by vortexing intermittently for 1 h. The soluble nuclear fraction was separated by centrifugation at 21,0006g for 10 min. To determine the efficiency of nuclear and cytoplasmic fraction- ation, Western blot analysis was done against the nuclear transcription factor SP1 and cytoplasmic protein GAPDH using specific antibodies. Immunofluorescence assay 300 thousand HEK-293 cells were transfected with different expression plasmids by Lipofectamine 2000 transfection reagents (Invitrogen, Carlsbad, CA). Cells were fixed with 3% paraformal- dehyde (PFA) with 0.1% Triton X-100 and 1% BSA was used for August 2014 \| Volume 10 \| Issue 8 \| e1004304 August 2014 \| Volume 10 \| Issue 8 \| e1004304 17 PLOS Pathogens \| www.plospathogens.org EBV Regulates Pim-1 Function Western blot analysis. Odyssey 3.0 software was used to quantify the band intensities. Western blot analysis. Odyssey 3.0 software was used to quantify the band intensities. added and incubated for 2 min at room temperature. Cells were washed with PBS and incubated with 0.2% Triton X100, 3% BSA in 16PBS for 5 min at room temperature. Cells were washed three times with PBS/BSA for 10 min each. Cells were incubated with anti BrdU antibody in PBS/BSA solution. After washing three times with PBS/BSA solution, cells were incubated for 1 hr with secondary antibody. DAPI was added at the final washing steps to stain DNA. The images were observed by Fluoview FV300 confocal microscope. Production of lentivirus and si-RNA mediated gene silencing by transduction of EBV transformed cells Production of lentivirus and si-RNA mediated gene silencing by transduction of EBV transformed cells Short-hairpin (sh) oligonucleotides directed against EBNA3C were described previously [22]. The Pim-1 target sequence 59- GUGUACUUUAGGCAAAGGG-39 was described previously [43]. sh-oligonucleotides used for EBNA2, EBNA3A and EB- NA3B knockdown were 59- TCGAGTTGTTGACACGGA- TAGTCTTTCAAGAGAAGACTATCCGTGTCAACAATTT- TTTA-39 and 59- CGCGTAAAAAATTGTTGACACGGATA- GTCTTCTCTTGAAAGACTATCCGTGTCAACAAC-39, 59- TCGAGGAACACTTCTTCAAGCGATTTCAAGAGAATCG- CTTGAAGAAGTGTTCTTTTTTA-39 and 59- CGCGTAA- AAAAGAACACTTCTTCAAGCGATTCTCTTGAAATCGC- TTGAAGAAGTGTTCC-39, 59- TCGAGTTGATTGTCATT- GGTTTCATTCAAGAGATGAAACCAATGACAATCAATT- TTTTA-39 and 59- CGCGTAAAAAATTGATTGTCATTG- GTTTCATCTCTTGAATGAAACCAATGACAATCAAC-39 respectively. EBNA3C and Pim-1 specific oligonucleotides were cloned into pGIPZ vector at XhoI and MluI restriction sites. Control shRNA sequence (Dharmacon Research, Chicago, IL) was used as 59-TCTCGCTTGGGCGAGAGTAAG-39 which lacks complementary sequences in the human genome, also cloned in pGIPZ vector. Lentivirus production and transduction of EBV- transformed LCL1 were described previously [51]. Colony formation assay 10 million Human kidney embryonic cells were subjected to transient transfection with Ctrl-vector, Myc-Pim-1, and Flag- tagged-EBNA3C by electroporation system. Transfected cells were allowed to grow in DMEM containing G418 as 1 mg/ml concentration. After selecting the cells up to 2-weeks, selected cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet solution (Sigma-Aldrich Corp., St. Louis, MO). The area of the colonies was calculated by using Image J software (Adobe Inc., San Jose, CA). The data shown here are average of three independent experiments. Figure S2 Expression levels of other EBNAs are unaf- fected by EBNA3C knockdown. 50 million sh-Ctrl and sh- E3C LCL1 cells were harvested and cell lysates were prepared by RIPA buffer. Protein samples were subjected to Western blot analysis by using human polyclonal serum capable of detecting EBNA proteins expressed by EBV during latent infection. GAPDH was shown as internal loading control. Pim-1 expression level was substantially reduced upon EBNA3C knockdown in LCL1 cells. Expression levels of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA-LP were not affected with EBNA3C knockdown. (TIF) Supporting Information Figure S1 EBNA3C induces Pim-1 upregulation. A) 10 million LCL1 cells were transiently transfected with EBNA2, EBNA3A, EBNA3B and EBNA3C knockdown constructs and 36 hrs of post-transfection, total RNA was isolated from the cells and subjected to cDNA conversion. The Pim-1 transcript level was checked by RT-PCR. The P-values of the mean differences for sh- EBNA2 LCL1, sh-EBNA3A LCL1, sh-EBNA3B LCL1 compared with sh-Ctrl LCL1 for Pim-1 transcripts are 0.0941, 0.1567, 0.0728, 0.0075 respectively. The error bars indicate standard deviations from three independent experiments. p-value of ,0.05 was considered here as statistically significant. B) The knockdown efficiency of EBNA2, EBNA3A, EBNA3B, and EBNA3C was shown by agarose gel after RT-PCR analysis. (TIF) Figure S1 EBNA3C induces Pim-1 upregulation. A) 10 million LCL1 cells were transiently transfected with EBNA2, EBNA3A, EBNA3B and EBNA3C knockdown constructs and 36 hrs of post-transfection, total RNA was isolated from the cells and subjected to cDNA conversion. The Pim-1 transcript level was checked by RT-PCR. The P-values of the mean differences for sh- EBNA2 LCL1, sh-EBNA3A LCL1, sh-EBNA3B LCL1 compared with sh-Ctrl LCL1 for Pim-1 transcripts are 0.0941, 0.1567, 0.0728, 0.0075 respectively. The error bars indicate standard deviations from three independent experiments. p-value of ,0.05 was considered here as statistically significant. B) The knockdown efficiency of EBNA2, EBNA3A, EBNA3B, and EBNA3C was shown by agarose gel after RT-PCR analysis. (TIF) Accession numbers Homo sapiens pim-1- GenBank: M16750.1, Homo sapiens cyclin-dependent kinase inhibitor 1A (p21, Cip1)- GenBank: BC001935.1, Epstein-Barr virus (EBV) genome, strain B95-8- GenBank: V01555.2, human Pim-1 protein- UniProtKB/Swiss- Prot: P11309, human P21 protein- UniProtKB/Swiss-Prot: P38936, EBNA3C protein- UniProtKB/Swiss-Prot: P03204.1. Homo sapiens pim-1- GenBank: M16750.1, Homo sapiens cyclin-dependent kinase inhibitor 1A (p21, Cip1)- GenBank: BC001935.1, Epstein-Barr virus (EBV) genome, strain B95-8- GenBank: V01555.2, human Pim-1 protein- UniProtKB/Swiss- Prot: P11309, human P21 protein- UniProtKB/Swiss-Prot: P38936, EBNA3C protein- UniProtKB/Swiss-Prot: P03204.1. Statistical analysis Data represented here are as the mean values with standard errors of means (SEM). The significance of differences in the mean values was calculated by performing 2-tailed student’s t-test. P- value of ,0.05 was considered here as statistically significant. Cell proliferation assay HEK-293, MEF cells were transfected with different combina- tions of expression vectors by electroporation as described in the text. Transfected cells were grown in DMEM and were selected with 1000 mg/ml G418 antibiotic for 2-weeks. After selection, Cells were incubated without serum and with etoposide (MP Biomedicals, LLC) treatment for 12 hrs. Cell lysates were prepared by RIPA buffer and protein expression was examined by Western blotting. From each transfected and selected set, 0.16106 cells were plated and allowed to grow them for 6 days. Also, LCL1, sh-Ctrl LCL1 and sh-Pim-1 LCL1 cells were plated and grown in RPMI media. Counting of viable cells at specific time points was performed by using Trypan Blue dye exclusion method. All experiments were performed in triplicates. Figure S3 EBNA3C shows physical association with p21. A) HEK-293 cells were transfected with Flag-P21 and Myc- EBNA3C expression vector. Immunoprecipitation was performed with anti-Flag antibody. The results showed direct interaction with p21 and EBNA3C by the co-immunoprecipitation experiments. B) p21 protein level was examined by expressing Flag-p21, Myc-Pim- 1, and increasing amount of EBNA3C in HEK-293 cells. The results indicated reduced level of p21 with dose dependent increase of EBNA3C in presence of Pim-1. (TIF) References (1993) Epstein-Barr virus in gastric carcinoma. Am J Pathol 143: 1250–1254. 28. Eichmann A, Yuan L, Breant C, Alitalo K, Koskinen PJ (2000) Developmental expression of pim kinases suggests functions also outside of the hematopoietic system. Oncogene 19: 1215–1224. 9. Henle W, Diehl V, Kohn G, Zur Hausen H, Henle G (1967) Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 157: 1064–1065. 29. Gapter LA, Magnuson NS, Ng KY, Hosick HL (2006) Pim-1 kinase expression during murine mammary development. 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Henle G, Henle W, Clifford P, Diehl V, Kafuko GW, et al. (1969) Antibodies to Epstein-Barr virus in Burkitt’s lymphoma and control groups. J Natl Cancer Inst 43: 1147–1157. 21. Yi F, Saha A, Murakami M, Kumar P, Knight JS, et al. (2009) Epstein-Barr virus nuclear antigen 3C targets p53 and modulates its transcriptional and apoptotic activities. Virology 388: 236–247. 2. Jang HS, Cho JO, Yoon CY, Kim HJ, Park JC (2001) Demonstration of Epstein-Barr virus in odontogenic and nonodontogenic tumors by the polymerase chain reaction (PCR). J Oral Pathol Med 30: 603–610. 22. Saha A, Halder S, Upadhyay SK, Lu J, Kumar P, et al. Epstein-Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1. PLoS Pathog 7: e1001275. 3. Raab-Traub N (2002) Epstein-Barr virus in the pathogenesis of NPC. Semin Cancer Biol 12: 431–441. 23. Brault L, Gasser C, Bracher F, Huber K, Knapp S, et al. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica 95: 1004–1015. 4. Orem J, Mbidde EK, Lambert B, de Sanjose S, Weiderpass E (2007) Burkitt’s lymphoma in Africa, a review of the epidemiology and etiology. Afr Health Sci 7: 166–175. 24. Hu XF, Li J, Vandervalk S, Wang Z, Magnuson NS, et al. (2009) PIM-1- specific mAb suppresses human and mouse tumor growth by decreasing PIM-1 levels, reducing Akt phosphorylation, and activating apoptosis. J Clin Invest 119: 362–375. 5. Nourse JP, Jones K, Gandhi MK Epstein-Barr Virus-related post-transplant lymphoproliferative disorders: pathogenetic insights for targeted therapy. Am J Transplant 11: 888–895. 25. Hu YL, Passegue E, Fong S, Largman C, Lawrence HJ (2007) Evidence that the Pim1 kinase gene is a direct target of HOXA9. Blood 109: 4732–4738. 6. Hsu JL, Glaser SL (2000) Epstein-barr virus-associated malignancies: epidemiologic patterns and etiologic implications. Crit Rev Oncol Hematol 34: 27–53. 26. Amson R, Sigaux F, Przedborski S, Flandrin G, Givol D, et al. (1989) The human protooncogene product p33pim is expressed during fetal hematopoiesis and in diverse leukemias. Proc Natl Acad Sci U S A 86: 8857–8861. 7. Cesarman E, Mesri EA (1999) Virus-associated lymphomas. Curr Opin Oncol 11: 322–332. 27. Muraski JA, Rota M, Misao Y, Fransioli J, Cottage C, et al. (2007) Pim-1 regulates cardiomyocyte survival downstream of Akt. Nat Med 13: 1467–1475. 8. Tokunaga M, Land CE, Uemura Y, Tokudome T, Tanaka S, et al. Acknowledgments We are grateful to Dr. Nancy S. Magnuson (Washington State University, Pullman, Washington), Martin Rowe (University of Birmingham, U.K.), George Mosialos (Alexander Fleming Biomedical Science Research Center, Vari, Greece), Jon Aster (Brigham and Women’s Hospital, Boston, MA, USA), and Elliott Kieff (Harvard Medical School, Boston, MA) for kindly providing reagents. We are also thankful to Mahadesh Prasad AJ and Raymundo Alfaro-Aco for their technical contributions. Figure S5 EBNA3C mediated potentiation of Pim-1 leads to inhibition of p21. A–B) HEK-293 and MEF cells were transfected with different combinations of Flag-tagged p21 (wild type and the T145A mutant), Myc-Pim-1 (wild type and the kinase dead mutant), EBNA3C expression vectors. The expression Figure S5 EBNA3C mediated potentiation of Pim-1 leads to inhibition of p21. A–B) HEK-293 and MEF cells were transfected with different combinations of Flag-tagged p21 (wild type and the T145A mutant), Myc-Pim-1 (wild type and the kinase dead mutant), EBNA3C expression vectors. The expression BrdU incorporation assay HEK-293 cells were transfected with specific plasmid vectors as indicated in the text. After 36 hrs of post-transfection, BrdU was added and incubated cells for 2 hours in the presence of DNA damaging agents. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min in room temperature. Cells were washed with PBS. 2 M HCl was then added and incubated for 20 min at room temperature. Next, 0.1 M sodium borate (Na2B4O7) pH 8.5 was Figure S4 Pim-1 accelerates cell proliferation in the presence of EBNA3C. A) HEK-293 cells were transfected with August 2014 \| Volume 10 \| Issue 8 \| e1004304 PLOS Pathogens \| www.plospathogens.org 18 EBV Regulates Pim-1 Function levels of these proteins were analyzed by Western blots with indicated antibodies in these G418 selected cells. (TIF) combinations of control vector, Flag-tagged EBNA3C, Myc-Pim-1 expression vectors, and Myc-Pim-1 with Flag-EBNA3C. Colony formation assays were performed after G418 antibiotic selection for 2 weeks. Here, our results showed a substantial increase in colony numbers with EBNA3C and Pim-1 co-transfection. B) The colony numbers of different transfected sets were represented in bar diagram. The data represented here as the average of three independent experiments. C) The rate of cell proliferation was determined by cell counting using Trypan blue dye exclusion technique for 6 days. levels of these proteins were analyzed by Western blots with indicated antibodies in these G418 selected cells. (TIF) Author Contributions Conceived and designed the experiments: SB ESR. Performed the experiments: SB JL QC ZS HCJ. Analyzed the data: SB ESR. Wrote the paper: SB ESR. Conceived and designed the experiments: SB ESR. Performed the experiments: SB JL QC ZS HCJ. Analyzed the data: SB ESR. Wrote the paper: SB ESR. EBV Regulates Pim-1 Function The Pim kinases: new targets for drug development. Curr Drug Targets 12: 2059–2066. 76. Ionov Y, Le X, Tunquist BJ, Sweetenham J, Sachs T, et al. (2003) Pim-1 protein kinase is nuclear in Burkitt’s lymphoma: nuclear localization is necessary for its biologic effects. Anticancer Res 23: 167–178. 46. 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https://openalex.org/W2905082590	https://www.mdpi.com/1420-3049/23/12/3316/pdf?version=1544769854	English	null	Simultaneous Determination of Nε-(carboxymethyl) Lysine and Nε-(carboxyethyl) Lysine in Different Sections of Antler Velvet after Various Processing Methods by UPLC-MS/MS	Molecules/Molecules online/Molecules annual	2,018	cc-by	8,259	Article Simultaneous Determination of Nε-(carboxymethyl) Lysine and Nε-(carboxyethyl) Lysine in Different Sections of Antler Velvet after Various Processing Methods by UPLC-MS/MS Keywords: advanced glycation end-products (AGEs); Nε-(carboxymethyl) lysine (CML); Nε-(carboxyethyl) lysine (CEL); antler velvet processing; UPLC-MS/MS Keywords: advanced glycation end-products (AGEs); Nε-(carboxymethyl) lysine (CML); Nε-(carboxyethyl) lysine (CEL); antler velvet processing; UPLC-MS/MS molecules molecules molecules Molecules 2018, 23, 3316; doi:10.3390/molecules23123316 Article Simultaneous Determination of Nε-(carboxymethyl) Lysine and Nε-(carboxyethyl) Lysine in Different Sections of Antler Velvet after Various Processing Methods by UPLC-MS/MS Rui-ze Gong 1, Yan-hua Wang 1,2, Yu-fang Wang 1, Bao Chen 1,2, Kun Gao 1,2 and Yin-shi Sun 1,* 1 Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, China; 82101172456@caas.cn (R.-z.G.); yhwangsdlc@126.com (Y.-h.W.); wangyufang_jl@163.com (Y.-f.W.); 15543598331@163.com (B.C.); 13356954028@163.com (K.G.) 2 College of Chinese Material Medicine, Jilin Agricultural University, Changchun 130112, China * Correspondence: sunyinshi2015@163.com; Tel.: +86-431-81919580; Fax: +86-431-81919580 Academic Editors: In-Soo Yoon and Hyun-Jong Cho Received: 13 November 2018; Accepted: 12 December 2018; Published: 14 December 2018 Rui-ze Gong 1, Yan-hua Wang 1,2, Yu-fang Wang 1, Bao Chen 1,2, Kun Gao 1,2 and Yin-shi Sun 1,* 1 Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, China; 82101172456@caas.cn (R.-z.G.); yhwangsdlc@126.com (Y.-h.W.); wangyufang_jl@163.com (Y.-f.W.); 15543598331@163.com (B.C.); 13356954028@163.com (K.G.) 2 College of Chinese Material Medicine, Jilin Agricultural University, Changchun 130112, China * Correspondence: sunyinshi2015@163.com; Tel.: +86-431-81919580; Fax: +86-431-81919580 Academic Editors: In-Soo Yoon and Hyun-Jong Cho Received: 13 November 2018; Accepted: 12 December 2018; Published: 14 December 2018 Abstract: Nε-(Carboxymethyl) lysine (CML) and Nε-(carboxyethyl) advanced glycation end-products (AGEs) and are frequently used as markers of AGE formation. AGEs, such as CML and CEL, have harmful effects in the human body and have been closely linked to many diseases such as diabetes and uremia. However, details on the contents of CML and CEL after applying different antler velvet processing methods are lacking. In this research, a robust lysine (CEL) are two typical UPLC-MS/MS method has been developed for the simultaneous determination of CML and CEL in various sections of antler velvet processed with different methods. In addition, factors affecting the CML and CEL contents are discussed. The CML contents of antler velvet after freeze-drying, boiling, processing without blood, and processing with blood were 74.55–458.59, 119.44–570.69, 75.36–234.92, and 117.11–456.01 µg/g protein, respectively; the CEL contents were 0.74–12.66, 11.33–35.93, 0.00–6.75, and 0.00–23.41 µg/g protein, respectively. The different contents of CML and CEL in the different samples of antler velvet result from the different interactions of the protein and lysine at different temperatures. These data can be used to estimate the potential consumer intake of CML and CEL from antler velvet and for guiding producers on how to reduce the production of CML and CEL. 1. Introduction Antler velvet is a representative animal medicinal material and dietary supplement that has been an important part of traditional Chinese medicine for thousands of years in China, Korea, and Southeast Asian countries [1–3]. It has various pharmacological effects, such as anti-oxidation and anti-osteoporosis properties [4–6]. Fresh antler velvet is rich in nutrients, such as proteins and amino acids; these are highly susceptible to spoilage if the antler velvet is not processed promptly. Based on the methods of processing and consumption, antler velvet can be classiﬁed as processed with blood or without blood, as boiled or freeze-dried, and as wax slices, powder slices, gauze slices, or bone slices. Different processing methods and sections of the antler velvet have different inﬂuences on the bioactive components and pharmacological activities [4–6]; therefore, the processing conditions are crucial for antler velvet’s dietary and medical functions. During the processing and storage of antler velvet, amino compounds (e.g., proteins, amino acids) react with carbonyl compounds (e.g., reducing sugars, lipid oxidation products) to randomly form Molecules 2018, 23, 3316; doi:10.3390/molecules23123316 www.mdpi.com/journal/molecules 2 of 13 Molecules 2018, 23, 3316 advanced glycation end-products (AGEs) by the Maillard reaction [7]. AGEs have been shown to be detrimental to human health, being closely linked to many conditions such as diabetes, Alzheimer’s disease, atherosclerosis, renal diseases, and aging [8–12]; with the accumulation of AGEs in the human body, the probability of people suffering the above-mentioned chronic degenerative diseases will increase greatly. Food is the main source of AGEs, especially high-fat and high-sugar foods [8,11,13], although human bodies can also produce some AGEs by themselves. Nε-(Carboxymethyl) lysine (CML) and Nε-(carboxyethyl) lysine (CEL) are two typical AGEs and are frequently used as markers of AGE formation in foods [14–16]. However, the determination of CML and CEL in antler velvet has not been reported. The contents of CML and CEL in antler velvets are affected by the matrix and processing conditions [17,18]. Antler velvet rich in amino compounds and carbonyl compounds may contribute more CML and CEL than other foods [19,20]. Therefore, information on the CML and CEL contents in processed antler velvets is essential to estimate the potential consumer intake of AGEs from antler velvet. Since CML and CEL have no UV absorption and ﬂuorescence properties, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC-MS), and high-performance liquid chromatography-mass spectrometry (HPLC-MS) techniques have previously been used to determine their contents [21–28]. 1. Introduction The ELISA method requires speciﬁc antibodies, and the sensitivity is greatly affected by the matrix effect of the sample, which can cause large errors [21–23]. HPLC and GC-MS typically require pre-column derivatization, which is cumbersome and reduces sensitivity [24–26]. HPLC-MS has the advantages of simple operation, repeatability, and stability; therefore, HPLC-MS has usually been used to determine CML and CEL contents [14,16,27]. In this research, we have developed a robust UPLC-MS/MS method to simultaneously determine CML and CEL contents in various sections of antler velvet processed with different methods. The CML and CEL contents in the various samples were determined by a validated method, which may contribute to the assessment of AGEs in antler velvets. This study provides a foundation and valuable reference for safe antler velvet processing and provides a basis for the development of recommended antler velvet dosages. 2.1. Sample Pretreatment Preparation of the antler velvet samples consisted of processing, segmenting, grinding, defatting, reduction, hydrolysis, and SPE. According to the processing method and consumption, antler velvet samples were classiﬁed as boiled or freeze-dried and processed with or without blood; samples were then divided into wax, powder, gauze, and bone slices. Because CML and CEL can be generated via peroxidation of the antler velvet lipid content, it was important to remove lipids from the antler velvet samples to prevent overestimation in the results. Before hydrolysis, 0.1 N sodium borohydride was applied for 12 h to reduce the amadori (e.g., fructose–lysine) and lipid oxidation products, thus preventing the formation of CML and CEL during acid hydrolysis [28]. The samples were subjected to SPE by using a C18 Sep-Pak cartridge (Sepax technology, Cork, Ireland; 500 mg, 6 mL), to remove impurities from the sample. 2.2. Optimization of Chromatography Conditions CML and CEL are highly polar compounds and are difﬁcult to retain in most reversed-phase columns. Researchers have usually analyzed these substances with a C18 column by using nonaﬂuoropentanoic acid (NFPA) as the eluent. However, NFPA can lead to a low-pH (~2) mobile phase, which may result in deterioration of the reversed-phase column [29]. To avoid the use of NFPA, we developed a UPLC-MS/MS method to separate CML and CEL with a WATERS CORTECS HILIC UPLC column. HILIC uses the separation principle of afﬁnity chromatography to maximize 3 of 13 of 12 Molecules 2018, 23, 3316 Molecules 2018, 23, x FO the retention and separation of highly polar compounds, relative to other columns. The elution effects of methanol and acetonitrile were assessed: acetonitrile/water (30:70 v/v) and methanol/water (30:70 v/v) mixtures were used as mobile phases, and the ﬂow rate was 0.3 mL/min. In comparison to chromatograms with the methanol mobile phase, UPLC-MS chromatograms using water and acetonitrile as the eluent have better spectrum peak symmetries and fewer miscellaneous peaks. e e e io a epa a io o ig y po a o pou , e a i e o o e o u e e u io effects of methanol and acetonitrile were assessed: acetonitrile/water (30:70 v/v) and methanol/water (30:70 v/v) mixtures were used as mobile phases, and the flow rate was 0.3 mL/min. In comparison to chromatograms with the methanol mobile phase, UPLC-MS chromatograms using water and acetonitrile as the eluent have better spectrum peak symmetries and fewer miscellaneous peaks. The UPLC-MS chromatograms of standard solutions (a) and antler velvet samples (b) are The UPLC-MS chromatograms of standard solutions (a) and antler velvet samples (b) are shown in Figure 1. The retention times of CML and CEL in the antler velvet samples were consistent with those of the standard solutions, and no peak interference was observed. The column was very stable and robust without obvious shifts in the retention times throughout the experimental procedure. shown in Figure 1. The retention times of CML and CEL in the antler velvet samples were consistent with those of the standard solutions, and no peak interference was observed. The column was very stable and robust without obvious shifts in the retention times throughout the experimental procedure. 2.2. Optimization of Chromatography Conditions a 3 a b 30%Ac Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 GRZ_CML+CEL_STD001 2: MRM of 2 Channels ES+ TIC (CEL) 3.59e5 0.62 3.56 3.61 GRZ_CML+CEL_STD001 1: MRM of 2 Channels ES+ TIC (CML) 2.01e5 0.61 30%Ac m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 GRZ_CML+CEL_STD001 110 (0.616) 2: MRM of 2 Channels ES+ (CEL) 2.28e5 84.10 130.15 GRZ_CML+CEL_STD001 110 (0.614) 1: MRM of 2 Channels ES+ (CML) 1.30e5 84.10 130.14 30%Ac Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 GRZLRA1001 2: MRM of 2 Channels ES+ TIC (CEL) 1.92e5 0.60 3.52 1.68 1.60 1.78 1.97 2.53 2.24 3.75 GRZLRA1001 1: MRM of 2 Channels ES+ TIC (CML) 8.68e4 0.60 0.76 1.54 30%Ac m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 GRZLRA1001 110 (0.614) 1: MRM of 2 Channels ES+ (CML) 4.54e4 84.10 130.14 GRZLRA1001 107 (0.600) 2: MRM of 2 Channels ES+ (CEL) 1.74e5 84.10 130.15 Figure 1. Total ion chromatogram and selected ion of Nε-(Carboxymethyl) lysine (CML) and Nε-(carboxyethyl) lysine (CEL) standard solutions (a) and antler velvet samples (b). 2.2. Optimization of Chromatography Conditions a b 30%Ac Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 GRZ_CML+CEL_STD001 2: MRM of 2 Channels ES+ TIC (CEL) 3.59e5 0.62 3.56 3.61 GRZ_CML+CEL_STD001 1: MRM of 2 Channels ES+ TIC (CML) 2.01e5 0.61 30%Ac m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 GRZ_CML+CEL_STD001 110 (0.616) 2: MRM of 2 Channels ES+ (CEL) 2.28e5 84.10 130.15 GRZ_CML+CEL_STD001 110 (0.614) 1: MRM of 2 Channels ES+ (CML) 1.30e5 84.10 130.14 3 b 30%Ac Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 % 0 100 GRZLRA1001 2: MRM of 2 Channels ES+ TIC (CEL) 1.92e5 0.60 3.52 1.68 1.60 1.78 1.97 2.53 2.24 3.75 GRZLRA1001 1: MRM of 2 Channels ES+ TIC (CML) 8.68e4 0.60 0.76 1.54 30%Ac m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 m/z -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 % 0 100 GRZLRA1001 110 (0.614) 1: MRM of 2 Channels ES+ (CML) 4.54e4 84.10 130.14 GRZLRA1001 107 (0.600) 2: MRM of 2 Channels ES+ (CEL) 1.74e5 84.10 130.15 Figure 1. Total ion chromatogram and selected ion of Nε-(Carboxymethyl) lysine (CML) and Nε-(carboxyethyl) lysine (CEL) standard solutions (a) and antler velvet samples (b). 3 Figure 1. Total ion chromatogram and selected ion of Nε-(Carboxymethyl) lysine (CML) and Nε-(carboxyethyl) lysine (CEL) standard solutions (a) and antler velvet samples (b). 4 of 13 Molecules 2018, 23, 3316 2.3. Method Validation The developed method was validated by assessing the CML and CEL contents in antler velvet samples and considering the selectivity, linearity, precision, and accuracy. The fragmentation pattern of CML and CEL indicated two major product ions at m/z 84 and 130, with the most intense peak at m/z 130. The two sample ions were used for quantitation in MRM mode. As shown in Table 1, the correlation coefﬁcients (R2) were both greater than 0.99. The linear range (20–3500 ng/mL) was sufﬁciently wide to assess the CML and CEL contents in the present antler velvet samples. The limit of detection (LOD) and limit of quantitation (LOQ) were deﬁned as the concentrations (ng/g) at which the signal-to-noise ratios of the peaks of interest were 3 and 10, respectively (Table 1). Table 1. Calibration, sensitivity and recovery in UPLC-MS/MS. Compound Calibration Sensitivity Recovery Range (ng/mL) R2 LOD (ng/g) LOQ (ng/g) 30 (ng/mL) 300 (ng/mL) 3000 (ng/mL) CML 20–3500 0.9997 1.3 4.1 95.21 ± 1.22 93.22 ± 1.13 97.42 ± 1.21 CEL 20–3500 0.9987 1.4 4.3 95.43 ± 1.09 93.22 ± 1.24 91.84 ± 1.18 Table 1. Calibration, sensitivity and recovery in UPLC-MS/MS. The antler velvet samples were extracted in triplicate and analyzed by using the developed UPLC-MS/MS method. The relative standard deviations of intra-day precision for CML and CEL were 3.32% and 3.08%, respectively, and those of inter-day precision were 3.14% and 3.53%, respectively. The coefﬁcients of variation obtained for the reproducibility tests described above were less than 5%. The recoveries of exogenous CML and CEL added to antler velvet samples were determined at three concentrations (low, intermediate, and high): 30, 300, and 3000 ng/mL, respectively. Recovery experiments were conducted ﬁve times for each concentration, affording values for CML and CEL of 93.22–97.42% and 91.84–95.43%, respectively. 2.4. CML and CEL Contents in Processed Antler Velvet 2.4.1. CML Contents in Different Sections of Antler Velvet with Different Processing Methods The CML contents in different sections of antler velvet processed with different methods are shown in Table 2. The CML contents in freeze-dried and boiled antler velvet were 74.55–458.59 and 119.44–570.69 µg/g protein, respectively. The CML contents in antler velvet processed without blood and with blood were 75.36–234.92 and 117.11–456.01 µg/g protein, respectively. These results indicate that antler velvet protein is glycosylated, to a considerable extent, relative to other processed foods such as fried chicken breast (12.34–90.52 µg/g protein) and processed meat and ﬁsh (44.53–167.60 µg/g protein) [15,16,30]. The CML contents of freeze-dried antler velvet were signiﬁcantly lower than those of the corresponding sections of boiled antler velvet (P < 0.01). This suggests that temperature can affect the formation of CML; speciﬁcally, high-temperature processing can produce more CML. The high content of CML in freeze-dried antler velvet is endogenous. The CML contents of antler velvet processed without blood were signiﬁcantly lower than those of the corresponding sections of antler velvet processed with blood (P < 0.01). The reason may be that the antler velvet processed without blood was subjected to physical centrifugal discharge of the blood. Blood contains many reducing sugars, amino acids, and proteins that can react to generate CML during processing. 5 of 13 Molecules 2018, 23, 3316 Table 2. CML contents in different sections of antler velvet with different processing methods expressed per µg/g protein, µg/g, and µmol/mmol lysine. 2.4.1. CML Contents in Different Sections of Antler Velvet with Different Processing Methods Processing Methods Sections µg CML/g Protein a µmol CML/mmol Lysine b µg CML/g freeze-dried wax slices 458.59 ± 22.04 4.00 ± 1.23 328.15 ± 20.13 powder slices 159.70 ± 11.67 1.71 ± 0.92 94.70 ± 8.72 gauze slices 97.59 ± 9.22 0.98 ± 0.45 51.47 ± 5.33 bone slices 74.55 ± 8.94 0.79 ± 0.33 37.51 ± 4.25 entire 120.93 ± 10.28 1.14 ± 0.72 64.21 ± 6.56 boiled wax slices 570.69 ± 34.74 6.07 ± 2.82 480.87 ± 31.22 powder slices 198.64 ± 13.56 2.23 ± 1.42 122.21 ± 9.96 gauze slices 130.24 ± 10.25 1.38 ± 0.72 71.01 ± 8.23 bone slices 119.44 ± 10.12 1.18 ± 0.69 51.87 ± 6.09 entire 141.41 ± 15.23 1.40 ± 0.71 80.09 ± 7.89 processed without blood wax slices 234.92 ± 23.03 2.58 ± 1.44 200.25 ± 18.27 powder slices 101.14 ± 12.31 1.59 ± 0.89 89.07 ± 8.93 gauze slices 99.26 ± 9.18 1.11 ± 0.74 58.46 ± 6.04 bone slices 75.36 ± 8.56 0.78 ± 0.41 39.13 ± 4.02 entire 103.14 ± 9.88 1.29 ± 0.82 54.26 ± 5.78 processed with blood wax slices 456.01 ± 24.32 5.16 ± 2.56 407.88 ± 30.42 powder slices 167.70 ± 11.82 1.96 ± 0.98 86.79 ± 9.51 gauze slices 129.02 ± 9.23 1.44 ± 0.74 62.94 ± 7.89 bone slices 117.11 ± 11.23 1.19 ± 0.70 57.64 ± 6.33 entire 124.73 ± 12.51 1.30 ± 0.83 73.30 ± 7.88 a Data were calculated using the protein contents quantiﬁed by combustion method. b Data were calculated using the amino acid concentration in the acid hydrolysates, quantiﬁed by amino acid analyze. a Data were calculated using the protein contents quantiﬁed by combustion method. b Data were calculated using the amino acid concentration in the acid hydrolysates, quantiﬁed by amino acid analyze. Wax pieces had the highest CML content, and bone pieces had the lowest. The differentiation capacity was faster closer to the top of the antler velvet; therefore, higher contents of reducing sugars, proteins, and amino acids may exist in the antler velvet to produce CML. Closer to the bottom of the antler velvet, the degree of ossiﬁcation is higher; therefore, the reducing sugar, protein, and amino acid contents are lower and produce less CML. 2.4.1. CML Contents in Different Sections of Antler Velvet with Different Processing Methods In summary, by comparing the CML contents from different sections of antler velvet after different processing methods, the CML contents of freeze-dried antler velvet and antler velvet processed without blood were found to be lower than those of the corresponding areas of boiled antler velvet and antler velvet processed with blood; wax pieces were more likely to produce CML than other sections. 2.4.2. CEL Contents in Different Sections of Antler Velvet with Different Processing Methods 2.4.2. CEL Contents in Different Sections of Antler Velvet with Different Processing Methods 2.4.2. CEL Contents in Different Sections of Antler Velvet with Different Processing Methods b Data were calculated using the amino acid concentration in the acid hydrolysates, quantiﬁed by amino acid analyzer. — Indicates not detected. In general, the variation tendency for CEL in different sections of antler velvet after different processing methods was similar to that for CML, despite the CEL contents of antler velvet being lower than the CML contents or undetected. The CEL contents in freeze-dried antler velvet were signiﬁcantly lower than those in the corresponding sections of boiled antler velvet (P < 0.01); the CEL contents of antler velvet processed without blood were signiﬁcantly lower than those of the corresponding sections of antler velvet processed with blood (P < 0.01). Wax pieces had the highest content of CEL, and bone pieces had the lowest. As described above for CML, the differences in temperature and contents of proteins, amino acids, and reducing sugars are the main reasons for the different CEL contents after different processing methods. 2.4.2. CEL Contents in Different Sections of Antler Velvet with Different Processing Methods The CEL contents in different sections of antler velvet processed with different methods are shown in Table 3. The CEL contents in freeze-dried and boiled antler velvet were 0.74–12.66 and 11.33–35.93 µg/g protein, respectively. The CEL contents in antler velvet processed without blood and with blood were 0–6.75 and 0–23.41 µg/g protein, respectively. The results indicate that the CEL contents in antler velvet are similar to those in other processed foods such as bread [27]. 6 of 13 Molecules 2018, 23, 3316 Table 3. CEL contents in different sections of antler velvet with different processing methods, expressed per µg/g protein, µg/g, and µmol/mmol lysine. Table 3. CEL contents in different sections of antler velvet with different processing methods, expressed per µg/g protein, µg/g, and µmol/mmol lysine. Processing Methods Sections µg CEL/g Protein a µmol CEL/mmol Lysine b µg CEL/g freeze-dried wax slices 12.66 ± 1.33 0.11 ± 0.21 9.06 ± 1.12 powder slices 10.99 ± 0.98 0.10 ± 0.14 6.28 ± 0.74 gauze slices 1.83 ± 0.32 0.02 ± 0.07 0.96 ± 0.23 bone slices 0.74 ± 0.12 0.01 ± 0.09 0.36 ± 0.11 entire 10.84 ± 0.99 0.10 ± 0.11 6.17 ± 0.72 boiled wax slices 35.93 ± 4.22 0.34 ± 0.23 29.19 ± 4.21 powder slices 15.43 ± 2.01 0.15 ± 0.11 8.97 ± 1.05 gauze slices 11.33 ± 1.23 0.13 ± 0.12 6.12 ± 0.72 bone slices 12.70 ± 1.41 0.11 ± 0.09 6.49 ± 0.78 entire 14.54 ± 1.47 0.14 ± 0.14 8.87 ± 1.22 processed without blood wax slices 6.57 ± 0.74 0.06 ± 0.10 5.24 ± 0.56 powder slices — — — gauze slices — — — bone slices — — — entire 2.57 ± 0.33 0.03 ± 0.06 1.45 ± 0.21 processed with blood wax slices 23.41 ± 3.01 0.23 ± 0.18 19.22 ± 2.53 powder slices 2.24 ± 0.22 0.02 ± 0.06 1.37 ± 0.16 gauze slices 0.03 ± 0.11 0.01 ± 0.04 0.02 ± 0.09 bone slices — — — entire 7.97 ± 0.92 0.08 ± 0.15 4.72 ± 0.52 a Data were calculated using the protein contents quantiﬁed by combustion method. b Data were calculated using the amino acid concentration in the acid hydrolysates, quantiﬁed by amino acid analyzer. — Indicates not detected. a Data were calculated using the protein contents quantiﬁed by combustion method. 2.5. Protein and Lysine Contents in Processed Antler Velvet The CML and CEL in antler velvet exist in the combined state and the free form, among which the combined state is the most common. The CML and CEL contents and protein contents of samples are closely related in the combined state [23]. Therefore, the CML and CEL contents can be expressed in units of protein. The Dumas combustion method was used to determine the protein contents in different sections of antler velvet after different types of processing; the results are shown in Table 4. 7 of 13 Molecules 2018, 23, 3316 Table 4. Protein contents in different sections of antler velvet with different processing methods which were determined by combustion method. Processing Methods Sections Protein Contents Processing Methods Sections Protein Contents Content (%) Coefﬁcient of Variation (%) Content (%) Coefﬁcient of Variation (%) freeze-dried wax slices 81.56 ± 0.04 0.27 processed without blood wax slices 79.81 ± 0.09 0.32 powder slices 57.12 ± 0.03 0.25 powder slices 56.69 ± 0.11 0.28 gauze slices 52.74 ± 0.10 0.78 gauze slices 58.88 ± 0.31 0.41 bone slices 49.03 ± 0.25 0.22 bone slices 54.11 ± 0.24 0.33 entire 56.93 ± 0.34 0.41 entire 56.43 ± 0.28 0.21 boiled wax slices 81.25 ± 0.12 0.17 processed with blood wax slices 82.09 ± 0.74 0.56 powder slices 58.11 ± 0.18 0.14 powder slices 61.49 ± 0.33 0.42 gauze slices 53.99 ± 0.33 0.21 gauze slices 61.45 ± 0.41 0.38 bone slices 51.13 ± 0.25 0.25 bone slices 49.65 ± 0.56 0.52 entire 60.99 ± 0.44 0.33 entire 59.25 ± 0.35 0.41 It was found that there was no signiﬁcant difference (P > 0.05) in protein content between the same sections of freeze-dried and boiled antler velvet. Additionally, sections of antler velvet processed with blood had signiﬁcantly higher protein contents than those processed without blood (P < 0.05), because the blood, which contains protein, had been retained during the processing. The protein contents were different among the different sections. Wax pieces had signiﬁcantly higher contents of protein than the other sections (P < 0.01). The reason may be that wax pieces from the antler tip had more meristem tissue, which promotes the expression of protein [20]. 2.5. Protein and Lysine Contents in Processed Antler Velvet Protein is the Maillard reaction substrate, and its distribution in different sections of antler velvet subjected to different processing methods is a leading factor for the differences in the CML and CEL contents, consistent with the results in Section 2.4. An automatic amino acid analyzer was used to determine the lysine contents in different sections of antler velvet processed differently; the results are shown in Table 5. Table 5. Lysine contents in different sections of antler velvet with different processing methods which were determined by amino acid automatic analyzer. Sample Lysine Contents g/100 g Sample Lysine Contents g/100 g Processing Methods Sections Processing Methods Sections freeze-dried wax slices 5.87 ± 0.20 processed without blood wax slices 5.56 ± 0.11 powder slices 3.96 ± 0.11 powder slices 4.01 ± 0.09 gauze slices 3.76 ± 0.03 gauze slices 3.76 ± 0.11 bone slices 3.41 ± 0.09 bone slices 3.58 ± 0.08 entire 4.02 ± 0.12 entire 3.01 ± 0.15 boiled wax slices 5.67 ± 0.22 processed with blood wax slices 5.66 ± 0.12 powder slices 3.92 ± 0.14 powder slices 4.86 ± 0.07 gauze slices 3.69 ± 0.10 gauze slices 4.21 ± 0.02 bone slices 3.15 ± 0.13 bone slices 3.48 ± 0.11 entire 4.09 ± 0.17 entire 4.03 ± 0.14 No signiﬁcant differences were observed in the lysine contents between the same sections of antler velvet processed with different methods (P > 0.05). The lysine contents in different sections of antler velvet were different. Wax slices had signiﬁcantly higher lysine contents than the other sections (P < 0.01), and there was no signiﬁcant difference among powder, gauze, and bone slices (P > 0.05). CML and CEL are two lysine derivatives; the contents of lysine can reﬂect the degree of reaction of different samples. The results are roughly consistent with the results for CML and CEL contents discussed in Section 2.4. Table 5. Lysine contents in different sections of antler velvet with different processing methods which were determined by amino acid automatic analyzer. 2.5. Protein and Lysine Contents in Processed Antler Velvet Sample Lysine Contents g/100 g Sample Lysine Contents g/100 g Processing Methods Sections Processing Methods Sections freeze-dried wax slices 5.87 ± 0.20 processed without blood wax slices 5.56 ± 0.11 powder slices 3.96 ± 0.11 powder slices 4.01 ± 0.09 gauze slices 3.76 ± 0.03 gauze slices 3.76 ± 0.11 bone slices 3.41 ± 0.09 bone slices 3.58 ± 0.08 entire 4.02 ± 0.12 entire 3.01 ± 0.15 boiled wax slices 5.67 ± 0.22 processed with blood wax slices 5.66 ± 0.12 powder slices 3.92 ± 0.14 powder slices 4.86 ± 0.07 gauze slices 3.69 ± 0.10 gauze slices 4.21 ± 0.02 bone slices 3.15 ± 0.13 bone slices 3.48 ± 0.11 entire 4.09 ± 0.17 entire 4.03 ± 0.14 Table 5. Lysine contents in different sections of antler velvet with different processing methods which were determined by amino acid automatic analyzer. No signiﬁcant differences were observed in the lysine contents between the same sections of antler velvet processed with different methods (P > 0.05). The lysine contents in different sections of antler velvet were different. Wax slices had signiﬁcantly higher lysine contents than the other sections (P < 0.01), and there was no signiﬁcant difference among powder, gauze, and bone slices (P > 0.05). CML and CEL are two lysine derivatives; the contents of lysine can reﬂect the degree of reaction of different samples. The results are roughly consistent with the results for CML and CEL contents discussed in Section 2.4. Molecules 2018, 23, 3316 8 of 13 2.6. Factors Inﬂuencing CML and CEL Contents in Differently Processed Antler Velvets 2.6. Factors Inﬂuencing CML and CEL Contents in Differently Processed Antler Velvets The differences in CML and CEL contents in different sections of antler velvet with different processing methods are caused by different degrees of the Maillard reaction. Factors that affect the Maillard reaction include the processing temperature and the contents of reducing sugars, unsaturated fatty acids, amino acids, and protein [17,18,31]. In addition, vitamins and inorganic ions in food can inhibit or promote the Maillard reaction [32]. The study determined the contents of CML and CEL in different sections of antler velvet with different processing methods and found that antler velvet boiled at high temperature produced more CML and CEL than that freeze-dried at low temperature: high temperatures exacerbate the Maillard reaction and cause boiled antler velvet to produce more CML and CEL. 2.5. Protein and Lysine Contents in Processed Antler Velvet The lysine and protein contents were different in different sections of antler velvet after different processing methods. Lysine and proteins are substrates for the Maillard reaction; therefore, their concentration determines the extent of the Maillard reaction. Relative to antler velvet processed without blood, antler velvet processed with blood, which is rich in lysine and protein, may produce more CML and CEL. Similarly, wax pieces rich in lysine and protein are more likely to produce CML and CEL than other sections. According to the literature [32], the contents of vitamins and inorganic ions in food, such as vitamin B, vitamin C, and calcium, magnesium, and ferric ions, can affect the Maillard reaction and inhibit or promote the production of AGEs. Among these factors, vitamin B, vitamin C, calcium ions, and magnesium ions can inhibit the Maillard reaction, especially magnesium ions; ferric ions can promote the occurrence of this reaction. Therefore, the contents of vitamins and inorganic ions in different sections of antler velvet after different processing methods can also affect the CML and CEL contents. To summarize, the differences in CML and CEL contents in different sections of antler velvet after different processing methods are the result of the combined action of lysine, proteins, vitamins, and inorganic ions at different temperatures. 3.2. Sources and Preparation of Antler Velvet Antler velvet (Cervi Cornu Pantotrichum) was collected in Shuangyang, Jilin Province, China, and identiﬁed by Dr. C.Y. Li from the Chinese Academy of Agricultural Sciences Institute of Special Animal and Plant Sciences. 3.1. Materials CML, CEL, and triﬂuoroacetic acid (TFA) were purchased from Sigma-Aldrich (San Francisco, CA, USA). The purities of these standards were above 99%. Lysine, ninhydrin (NIN), and a citric acid buffer solution were purchased from Hitachi Inc. (Hitachi Co., Osaka, Japan). Acetonitrile, HPLC-grade, was purchased from Fisher-Scientiﬁc (Waltham, MA, USA). C18 Sep-Pak® SPE tubes were purchased from Sepax (Sepax technology, Cork, Ireland). Ultrapure water was obtained by using a super-pure water system (Water Puriﬁer Co. Ltd., Chengdu, China). All other reagents were of analytical grade and were purchased from Sinopharm Chemical Reagent Co. Ltd (Beijing, China). 3.3. Preparation of Processed Antler Velvet In accord with the classiﬁcation of commercially available antler velvet, boiling, freeze-drying, processing with blood, and processing without blood were chosen for this study. Six pairs of antler velvet samples were randomly selected and processed with blood or without blood for comparison, and another six pairs were randomly selected and processed by boiling or freeze-drying for comparison. Antler velvet was boiled for 1 min in boiled water, followed by high-temperature (75 ◦C) baking for multiple 2 h cycles until dry. During the freeze-drying process, the antler velvet was directly frozen to 9 of 13 ure (75 et was Molecules 2018, 23, 3316 comparison. Antler °C) baking for mult dryness. For the boiling process without blood, the antler velvet was prepared by removing the blood by centrifugation, whereas no blood removal was performed for the samples processed with blood. y y g p , p p y removing the blood by centrifugation, whereas no blood removal was performed for the samples processed with blood. 3.4. Preparation of Antler Velvet Slices 3.4. Preparation of Antler Velvet Slices Three pairs of antler velvet samples with blood and without blood were randomly selected for analysis, and three pairs of boiled and freeze-dried antler velvet were crushed whole. The remaining six pairs of antler velvet were divided into wax slices, powder slices, gauze slices, and bone slices based on morphological and microscopic characteristics (Figure 2) [20]; these samples were segmented, sliced, crushed, sieved, bagged, and labeled. Three pairs of antler velvet samples with blood and without blood were randomly selected for analysis, and three pairs of boiled and freeze-dried antler velvet were crushed whole. The remaining six pairs of antler velvet were divided into wax slices, powder slices, gauze slices, and bone slices based on morphological and microscopic characteristics (Figure 2.) [20]; these samples were segmented, sliced, crushed, sieved, bagged, and labeled. 8 Figure. 2. Schematic diagram of fresh antler velvet (A) and different sections of processed antler velvet (B). The processed antler velvet has a significant Maillard reaction browning compared to the A B Figure 2. Schematic diagram of fresh antler velvet (A) and different sections of processed antler velvet (B). The processed antler velvet has a signiﬁcant Maillard reaction browning compared to the fresh antler velvet. The processed antler velvet was divided into wax slices, powder slices, gauze slices, and bone slices based on morphological and microscopic characteristics. B A B A 8 Figure. 2. Schematic diagram of fresh antler velvet (A) and different sections of processed antler velvet (B). The processed antler velvet has a significant Maillard reaction browning compared to the Figure 2. Schematic diagram of fresh antler velvet (A) and different sections of processed antler velvet (B). The processed antler velvet has a signiﬁcant Maillard reaction browning compared to the fresh antler velvet. The processed antler velvet was divided into wax slices, powder slices, gauze slices, and bone slices based on morphological and microscopic characteristics. 3.6. Lysine Content Analysis An amino acid analyzer (L-8900 System; Hitachi Co., Osaka, Japan) equipped with a visible detector was used for amino acid analysis. Analytical 2622# (4.6 mm × 60 mm) and guard 2650# (4.6 mm × 40 mm) columns were used for amino acid determination. Immediately after sample injection into the columns, an auto-sampler was used for inline derivatization by NIN post-column derivatization. The NIN-derivatized lysine were detected at 570 nm. Lysine standards (Hitachi Co., Osaka, Japan) were used for identiﬁcation and quantiﬁcation (external standard method). Lysine content was expressed as g/100 g of antler velvet for the different processing methods and different sections of the antler velvet. 3.5. Protein Content Analysis The protein contents in antler velvet samples prepared by different processing methods and from different sections were determined on a Dumas nitrogen analyzer (Velp NDA 701-Monza, Brianza, Italy), according to a previous method with minor modiﬁcations [33]. The total nitrogen content was converted into the protein content by using a conversion factor of 6.25. The operating conditions of the NDA instrument were: O2 gas at 400 mL/min, He gas at 195 mL/min, combustion reactor at 1030 ◦C, reduction reactor at 650 ◦C, and pressure at 88.1 kPa. 3.7. Preparation of Samples The method of reference was slightly modiﬁed in this work [16,27]. Samples of 30 mg of antler velvet (equivalent to 20 mg of protein) were defatted twice by using n-hexane (5 mL) before being reduced for 12 h at 4 ◦C in 0.5 M sodium borate buffer (pH 9.2, 1 mL) and 2 M sodium borohydride (0.1 M sodium hydroxide, 0.5 mL). The proteins were isolated by using a chloroform:methanol mixture 10 of 13 Molecules 2018, 23, 3316 (2:1 v/v, 1 mL), and the precipitates were mixed with 15 mL of 6 M hydrochloric acid and incubated at 110 ◦C for 24 h. The diluted acid hydrolysate (equivalent to approximately 600 µg of protein) was dried with a gas-blowing concentrator (Hengao, Tianjin, China) at 70 ◦C. The dried hydrolysate was dissolved in 1 mL ultra-pure water and then solid-phase extracted by using a C18 Sep-Pak® (Sepax Technology, Cork, Ireland) cartridge (500 mg, 6 mL). The solid-phase extraction (SPE) column was pretreated with 3 mL of methanol and 3 mL of 0.1 M TFA at a ﬂow rate of 1 mL/min. The sample was loaded into the pretreated SPE column at a ﬂow rate of 0.5 mL/min and washed with 6 mL of 0.1 M TFA. Finally, the sample was eluted with 3 mL of methanol at a ﬂow rate of 0.5 mL/min. The eluate was dried by freezing, re-dissolved in 1 mL of ultra-pure water, ﬁltered through a 0.22-µm membrane, and stored at −20 ◦C prior to UPLC-MS/MS analysis. 3.8. UPLC-MS/MS Analysis CML and CEL concentrations in the hydrolysates were determined by UPLC-MS/MS. Protein hydrolysates (2 µg protein, 3 µL) were injected into a WATERS CORTECS HILIC UPLC column (2.1 mm × 50 mm, 1.6 µm; Waters, Cork, Ireland) housed in a column oven at 40 ◦C and operated in gradient-elution mode. Solvent A was water and solvent B was acetonitrile. Gradient elution was started at 100% solvent B for 1 min; this was followed by a linear gradient from 100% to 60% solvent B in 1.5 min, holding at 60% solvent B for 1.5 min, and then returning to 100% solvent B in 2 min. The analysis was performed by using a Waters Acquity UPLC instrument (Waters, Manchester, UK) coupled to a triple quadrupole MS operating in multiple reaction monitoring (MRM) mode. The ﬂow rate was 0.3 mL/min. The MS instrument was operated in electrospray ionization positive mode. The optimized MRM conditions are shown in Table 6. CML and CEL were quantiﬁed by using standards and by reference to an external standard calibration curve. Data are reported as means ± standard deviation of triplicate experiments. CML and CEL contents in the samples are expressed as µmol/mmol lysine, µg/g protein, and µg/g sample. Table 6. UPLC-MS settings for multiple reaction monitoring (MRM). Table 6. UPLC-MS settings for multiple reaction monitoring (MRM). Compound Precursor Ion (m/z) Product Ion (m/z) Cone Voltage (V) Collision Energy (ev) Dwell Time (ms) CML 205 130 25 15 36 205 84 25 25 36 CEL 219 130 25 15 36 219 84 25 25 36 4. Conclusions The CML and CEL contents in different sections of antler velvet subjected to different processing methods have been simultaneously determined for the ﬁrst time. The CML contents of antler velvet after freeze-drying, boiling, processing without blood, and processing with blood were 74.55–458.59, 119.44–570.69, 75.36–234.92, and 117.11–456.01 µg/g protein, respectively; the corresponding CEL contents were 0.74–12.66, 11.33–35.93, 0.00–6.75, and 0.00–23.41 µg/g protein. The CML and CEL contents in the same sections of boiled antler velvet were signiﬁcantly higher than those in freeze-dried antler velvet; high temperatures exacerbate the Maillard reaction, leading to boiled antler velvet producing more CML and CEL. Antler velvet processed with blood had obviously higher CML and CEL contents than antler velvet processed without blood; the antler velvet processed without blood was subjected to physical centrifugal discharge of the blood and therefore contains fewer substrates that can react to generate CML and CEL. With the same processing methods, the CML and CEL contents were different in different sections of the antler velvet. Wax pieces had signiﬁcantly higher CML and CEL contents than the types of antler velvet. Closer to the top of the antler velvet, there is a more rapid differentiation capacity and 11 of 13 Molecules 2018, 23, 3316 more substrates exist in the antler velvet to produce CML and CEL. Closer to the bottom of the antler velvet, there is a higher degree of ossiﬁcation and fewer substrates so less CML and CEL is produced. Through the detection and comparison of CML, CEL, lysine, and protein contents in antler velvet after different processing methods and from various sections, it was found that the different contents of CML and CEL in antler velvet samples are the result of the interaction of protein and lysine at different temperatures. Author Contributions: Data curation, R.-z.G. and B.C.; Funding acquisition, Y.-s.S.; Methodology, Y.-f.W. (Yu-fang Wang); Resources, Y.-s.S.; Software, Y.-f.W (Yu-fang Wang). and B.C.; Validation, Y.-h.W. (Yan-hua Wang); Writing—original draft, R.-z.G. and K.G.; Writing—review and editing, R.-z.G., K.G. and Y.-s.S. Author Contributions: Data curation, R.-z.G. and B.C.; Funding acquisition, Y.-s.S.; Methodology, Y.-f.W. (Yu-fang Wang); Resources, Y.-s.S.; Software, Y.-f.W (Yu-fang Wang). and B.C.; Validation, Y.-h.W. (Yan-hua Wang); Writing—original draft, R.-z.G. and K.G.; Writing—review and editing, R.-z.G., K.G. and Y.-s.S. References 1. Jeon, B.; Kim, S.; Lee, S.; Park, P.; Sung, S.; Kim, J.; Moon, S. Effect of antler growth period on the chemical composition of velvet antler in sika deer (Cervus nippon). Z. Saugetierkd. 2009, 74, 374–380. [CrossRef] 1. Jeon, B.; Kim, S.; Lee, S.; Park, P.; Sung, S.; Kim, J.; Moon, S. Effect of antler growth period on the chemical composition of velvet antler in sika deer (Cervus nippon). Z. Saugetierkd. 2009, 74, 374–380. [CrossRef] 2. Wu, F.; Li, H.; Jin, L.; Li, X.; Ma, Y.; You, J.; Li, S.; Xu, Y. Deer antler base as a traditional Chinese medicine: A review of its traditional uses, chemistry and pharmacology. J. Ethnopharmacol. 2013, 145, 403–415. [CrossRef] [PubMed] 2. Wu, F.; Li, H.; Jin, L.; Li, X.; Ma, Y.; You, J.; Li, S.; Xu, Y. Deer antler base as a traditional Chinese medicine: A review of its traditional uses, chemistry and pharmacology. J. Ethnopharmacol. 2013, 145, 403–415. [CrossRef] [PubMed] 3. Zhou, R.; Li, S. In vitro antioxidant analysis and characterisation of antler velvet extract. Food Chem. 2009, 114, 1321–1327. [CrossRef] 3. Zhou, R.; Li, S. In vitro antioxidant analysis and characterisation of antler velvet extract. Food Chem. 2009, 114, 1321–1327. [CrossRef] 4. Sui, Z.; Yuan, H.; Liang, Z.; Zhao, Q.; Wu, Q.; Xia, S.; Zhang, L.; Huo, Y.; Zhang, Y. An activity-maintaining sequential protein extraction method for bioactive assay and proteome analysis of velvet antlers. Talanta 2013, 107, 189–994. [CrossRef] [PubMed] 4. Sui, Z.; Yuan, H.; Liang, Z.; Zhao, Q.; Wu, Q.; Xia, S.; Zhang, L.; Huo, Y.; Zhang, Y. An activity-maintaining sequential protein extraction method for bioactive assay and proteome analysis of velvet antlers. Talanta 2013, 107, 189–994. [CrossRef] [PubMed] . Sui, Z.; Zhang, L.; Huo, Y.; Zhang, Y. Bioactive components of velvet antlers and their pharmacolog properties. J. Pharm. Biomed. 2014, 87, 229–240. [CrossRef] [PubMed] 6. Tseng, S.H.; Sung, C.H.; Chen, L.G.; Lai, Y.J.; Chang, W.S.; Sung, H.C.; Wang, C.C. Comparison of chemical compositions and osteoprotective effects of different sections of velvet antler. J. Ethnopharmacol. 2014, 151, 352–360. [CrossRef] [PubMed] 7. Singh, R.; Barden, A.; Mori, T.; Beilin, L. Advanced glycation end-products: A review. Diabetologia 2001, 2001, 129–146. [CrossRef] [PubMed] 8. Vlassara, H.; Palace, M.R. Glycoxidation: The menace of diabetes and aging. Mt Sinai J. Med. 2003, 70, 232–241. [PubMed] 9. Drenth, H.; Zuidema, S.U.; Krijnen, W.P.; Bautmans, I.; van der Schans, C.; Hobbelen, H. 4. Conclusions Funding: This work was ﬁnancially supported by the Jilin Province Science and Technology Development Project (20170309002YY, 20170311027YY, and 20180201076YY), the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2016-ISAPS) and the National Key Research and Development Program of China (2018YFC1706604 and 2018YFC1706605). 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https://openalex.org/W2604957907	https://europepmc.org/articles/pmc5460204?pdf=render	English	null	The epidemiology, radiology and biological characteristics of interval breast cancers in population mammography screening	NPJ breast cancer	2,017	cc-by	14,672	www.nature.com/NPJBCANCER REVIEW ARTICLE OPEN The epidemiology, radiology and biological characteristics of interval breast cancers in population mammography screening Nehmat Houssami 1 and Kylie Hunter2 An interval breast cancer is a cancer that emerges following a negative mammographic screen. This overview describes the epidemiology, and the radiological and biological characteristics of interval breast cancers in population mammography screening. Notwithstanding possible differences in ascertainment of interval breast cancers, there was broad variability in reported interval breast cancer rates (range 7.0 to 49.3 per 10,000 screens) reﬂecting heterogeneity in underlying breast cancer rates, screening rounds (initial or repeat screens), and the length and phase of the inter-screening interval. The majority of studies (based on biennial screening) reported interval breast cancer rates in the range of 8.4 to 21.1 per 10,000 screens spanning the two-year interval with the larger proportion occurring in the second year. Despite methodological limitations inherent in radiological surveillance (retrospective mammographic review) of interval breast cancers, this form of surveillance consistently reveals that the majority of interval cancers represent either true interval or occult cancers that were not visible on the index mammographic screen; approximately 20–25% of interval breast cancers are classiﬁed as having been missed (false-negatives). The biological characteristics of interval breast cancers show that they have relatively worse tumour prognostic characteristics and biomarker proﬁle, and also survival outcomes, than screen-detected breast cancers; however, they have similar characteristics and prognosis as breast cancers occurring in non-screened women. There was limited evidence on the effect on interval breast cancer frequency and outcomes following transition from ﬁlm to digital mammography screening. npj Breast Cancer (2017) 3:12 ; doi:10.1038/s41523-017-0014-x INTRODUCTION be classiﬁed as such irrespective of how it came to be diagnosed. Factors that have been associated with increased risk of an interval BC in screened women include high mammographic breast density,6–8 current use of hormone replacement therapy,8, 9 young relative to older age (partly reﬂecting confounding from breast density), however, absolute incidence rates are higher in older women given higher underlying BC rates,8 previous false- positive mammography,10 and a family history of BC.8, 10, 11 1Sydney School of Public Health, Sydney Medical School, University of Sydney, Sydney, NSW, Australia and 2National Health and Medical Research Council (NHMRC) Clinical Trials Centre, Sydney Medical School, University of Sydney, Sydney, NSW, Australia Correspondence: Nehmat Houssami (nehmat.houssami@sydney.edu.au) Aims A breast cancer (BC) that emerges following a negative mammo- graphic screen is referred to as an interval BC.1 In this overview, we describe the epidemiology, radiology and biological char- acteristics of interval BCs in population mammography screening, highlighting published research from the most recent decade. The aims of the review were to provide an update on interval BCs that extends both our work on radiological surveillance of interval BCs1 and that from other researchers that have quantiﬁed interval BC rates,2–4 to elucidate evidence on interval BCs following transition to digital mammography screening, and to identify knowledge gaps that warrant further research. Because interval BCs are representative of the sensitivity of population breast screening, and given that they are an adverse outcome for women partaking in screening, surveillance of interval BCs is routinely practiced in many screening programs. Surveillance comprises epidemiologic measures (such as interval BC rates)1, 3 to monitor the frequency of interval cases, and may be complemented by radiological surveillance as part of quality assurance in organised screening programs.1 Various methodolo- gical and analytic parameters can substantially inﬂuence estimates of interval BC rates and other epidemiologic measures of interval BCs, as highlighted by several investigators.3, 12 These include variability in the deﬁnition of an interval BC (whether based on invasive BC or whether ductal carcinoma in-situ (DCIS) cases are included); whether false-negative assessment cases and lapsed attenders are counted or excluded; the adequacy of ascertainment of interval cancers, hence also the adequacy of cancer notiﬁcation and registration; and the duration of follow-up for ascertainment of cases.3, 12 Importantly, underlying BC rates or burden in the population also affects interval BC rates. For these reasons, epidemiologic measures of interval BCs are best suited for Background and deﬁnitions Published in partnership with the Breast Cancer Research Foundation Interval breast cancers in mammography screening N Houssami and K Hunter Interval breast cancers in mammography screening N Houssami and K Hunter 2 • An interval breast cancer (BC) is a cancer that is diagnosed in the inter-screening interval, meaning that it is diagnosed following a negative mammographic screen and before the next routine mammographic screen. • Variability in the definition of an interval BC (for example whether based on invasive BC only or whether DCIS cases are included; and whether false negative assessments are included) and cancer ascertainment methods can influence estimates of interval BC rates. • Factors associated with increased risk of interval BC include high breast density, current use of hormone replacement therapy, young relative to older age, and family history of BC. • Routine epidemiological surveillance of interval BC rates is commonly implemented in organized screening programs, and is a useful measure of population BC screening sensitivity • Underlying BC rates (or BC burden in the population) affects interval BC rates, as do cancer ascertainment methods, hence comparisons within screening programs are more appropriate than comparisons between different screening settings. • Radiological surveillance of interval BCs entails review of the negative mammographic screen and subsequent diagnostic mammogram, in order to classify the extent that interval BCs were due to screen-reading error (missed or false-negatives cases) as opposed to newly arising cancers on imaging. • A true interval BC is a cancer that is not visible at the index mammographic screen on radiological review but is visible at the diagnostic mammogram Fig. 1 Summary of background deﬁnitions and themes Fig. 1 Summary of background deﬁnitions and themes monitoring.4, 13, 15–29 The table highlights the broad variability in both interval BC rates and cancer detection rates at screening, both of which are partly driven by underlying cancer rates in the populations reported in these studies. There is wide variability in the overall interval BC rates, ranging between 7.0 and 49.3 per 10,000 screens, partly explained by data shown for screening rounds (initial and repeat screens) and the duration and year of the inter-screening intervals; where reported, data for the inter- screening interval are presented by yearly rates for biennial or triennial screening. If restricted to studies of annual screening or to year 1 data from biennial screening programs, there is evidence that interval BC rates are consistently <8/10,000 screens. Interval breast cancers in mammography screening N Houssami and K Hunter Radiological surveillance entails review of the mammograms taken at the time the interval BC is identiﬁed (usually at clinical presentation, hence the diagnostic mammograms) and the pre-diagnosis mammographic screen (the ‘negative’ index screen) and an interpretative judgement to classify each case into pre-deﬁned categories.1, 13–15 These categories may vary in deﬁnition, however, most include a ‘true interval’ category (where the cancer is not visible at the index screen but becomes visible at the diagnostic mammogram) and a ‘missed’ interval BC being the equivalent of a false-negative (where the cancer is visible on the index mammogram but is not recalled or is misinterpreted) and is at times referred to as screening error.1, 13–15 Various methods have been used to perform radiological surveillance, as described in a review by Houssami et al.1 with potential biases inherent in the review strategy and the extent that readers are informed that they are evaluating interval BC cases.1 Notwithstanding the methodologi- cal limitations of radiological surveillance, it provides insights into screening quality and on how screening could be improved. For the majority of studies (based on biennial screening) interval BCs represent around 17–30% of the cancers occurring in screening participants as summarised in the simple proportion in the last column of Table 1; that proportion is relatively lower for annual (14.7% based on one study) and higher for triennial (32–38%) screening intervals. The proportional interval BC rate, also shown in Table 1, is not reported by all studies because this measure requires estimating the expected underlying incidence in the absence of screening, so may not be feasible to calculate in contemporary screening practice.2 In the present review, we consider both epidemiological and radiological aspects of interval BCs, and complement these with information on tumour prognostic characteristics of interval BCs, to deﬁne common themes as well as evidence gaps, to enhance our understanding of interval BCs and inform research priorities. Mammography screening interventions associated with reduction in interval BC rates. A study conducted in the United Kingdom’s screening program, based on women aged 50–64 screened between 2003 and 2005, reported that two-view mammography (at the last routine screen) was associated with a reduction in interval BC rates of 6.8/10,000 screens compared with one-view mammography.30 The investigators concluded that this suggests that two-view (instead of one-view) mammography at incident Interval breast cancers in mammography screening N Houssami and K Hunter The majority of studies in Table 1 report data for biennial screening programs: the interval BC rates spanning the two years between screens are within the range of 8.4 to 21.1 per 10,000 screens, with the larger proportion of the estimated interval BC rates occurring in the second year of a two-yearly interval. The evidence also consistently shows that interval BC rates are higher at repeat (incident) rounds than initial (prevalent) screening rounds. monitoring within screening services or programs because comparisons between screening programs and countries is limited by heterogeneity in the above-described variables that affect quantitative estimates of interval BCs. Radiological surveillance is a qualitative form of surveillance that deﬁnes and measures the extent that interval BCs represent screen-reading ‘errors’ as opposed to being non-detectable cancers at mammography screening. Radiological surveillance entails review of the mammograms taken at the time the interval BC is identiﬁed (usually at clinical presentation, hence the diagnostic mammograms) and the pre-diagnosis mammographic screen (the ‘negative’ index screen) and an interpretative judgement to classify each case into pre-deﬁned categories.1, 13–15 These categories may vary in deﬁnition, however, most include a ‘true interval’ category (where the cancer is not visible at the index screen but becomes visible at the diagnostic mammogram) and a ‘missed’ interval BC being the equivalent of a false-negative (where the cancer is visible on the index mammogram but is not recalled or is misinterpreted) and is at times referred to as screening error.1, 13–15 Various methods have been used to perform radiological surveillance, as described in a review by Houssami et al.1 with potential biases inherent in the review strategy and the extent that readers are informed that they are evaluating interval BC cases.1 Notwithstanding the methodologi- cal limitations of radiological surveillance, it provides insights into screening quality and on how screening could be improved. Radiological surveillance is a qualitative form of surveillance that deﬁnes and measures the extent that interval BCs represent screen-reading ‘errors’ as opposed to being non-detectable cancers at mammography screening. Published in partnership with the Breast Cancer Research Foundation Background and deﬁnitions An interval BC refers to a cancer that presents after a ‘normal’ screening mammogram and before the next scheduled mammo- gram, in other words a BC that arises or is diagnosed in the inter- screening interval [see also Fig. 1].1 This deﬁnition may be qualiﬁed by specifying an interval case as an invasive BC,5 given that the vast majority of interval cases are invasive malignancies and much of the routinely reported data on interval BC rates is based on invasive BC. In addition, some qualify the deﬁnition further by specifying that interval BCs are those that arise clinically2, 5 in the inter-screening interval—although that would be the likely presentation for almost all interval BCs it should be noted that a BC identiﬁed in the inter-screening interval would still Received: 2 November 2016 Revised: 3 January 2017 Accepted: 21 March 2017 1Sydney School of Public Health, Sydney Medical School, University of Sydney, Sydney, NSW, Australia and 2National Health and Medical Research Council (NHMRC) Clinical Trials Centre, Sydney Medical School, University of Sydney, Sydney, NSW, Australia Correspondence: Nehmat Houssami (nehmat.houssami@sydney.edu.au) Published in partnership with the Breast Cancer Research Foundation Received: 2 November 2016 Revised: 3 January 2017 Accepted: 21 March 2017 Received: 2 November 2016 Revised: 3 January 2017 Accepted: 21 March 2017 Published in partnership with the Breast Cancer Research Foundation Published in partnership with the Breast Cancer Research Foundation Epidemiologic surveillance Epidemiologic surveillance Table 1 presents a summary of epidemiologic measures for interval cancers including interval BC rates, which were the most commonly reported estimates for routine screening npj Breast Cancer (2017) 12 Interval breast cancers in mammography screening N Houssami and K Hunter 3 Table 1. Epidemiologic surveillance Epidemiological measures of the frequency of interval breast cancers in population mammography screening Study ﬁrst author Screening settinga; age-group screened Cancer detection rate per 10,000 screens Interval cancer rate per 10,000 screens reported for all screens: overall or by inter- screen interval Interval cancer rate per 10,000 screens where reported separately for initial and repeat screens Proportional interval cancer rateb Percentage of cancers (from all BCs in screened women) that are interval cancersc Weber13 Southern screening region of Dutch program (2000–11), 302,699 ﬁlm-screen and 115,047 digital mammograms; 50–75 years Digital 69 Film- screen 52 Digital 17 Film-screen 20 – – Digital 19.4% Film-screen 28% O'Brien4 Irish population breast screening program (2000–07); 50–64 years 53.6 (initial 66.9; repeat 41.4) Overall 19.2 year 1: 5.8 year 2: 13.4 Initial 19.6 Repeat 18.9 40% 26% Henderson21 Breast Cancer Surveillance Consortium, USA (2003–11), 3,021,515 mammograms (40.3% digital, 59.7% ﬁlm); 40–89 years Digital 44.7 Film- screen 44.2 12-month interval: Digital 7.3 Film-screen 7.9 – – 14.7% (annual screening) Carbonaro15 Milan, Italy, population breast screening program (2001–06); 49–69 years 55.2 Overall 17.0 – 29.0% (initial 19%; repeat 39%) 23.3% Renart-Vicens26 Girona, Spain, Health Region screening program (2000–06), 50–69 years 49.0 Overall 7.0 – Range 9.3 to 47.7% 13% Fong18 Breast Test Wales screening program (1998–2001); 50–64 years 51.0 Overall 34.8 – – 38% (3-yearly screening) Heidinger20 German mammography screening program/North Rhine-Westphalia cancer registry (2005–08), 50–69 years [study of initial screening round] 81.0 Overall 23.2 Year 1: 7.4 Year 2: 15.7 (all data are for initial screens) Year 1: 27% Year 2: 58% 22% Bennett16 National Health Service breast screening programme England, Wales, and Northern Ireland (1997–2003); 50–64 years 60.4 Overall 29.1 year 1: 5.5 year 2: 11.3 year 3: 12.2 – – 32.5% (3-yearly screening) Tornberg28 Navarra, Spain, population screening (1990–96); 45–65 years 41.5 (initial 63.0; repeat 31.3) Overall 8.4d year 1: 2.1d year 2: 6.3d Initial 7.2e Repeat 9.0e 25.9%d (initial 22%; repeat 28%)e 17%d Seigneurin27 Isere, France, population screening – time frames are for change from 1-view to 2-view mammography: (2002–04) 50–69 years; (1994–99) 50–69 years 2002–04: 70.4 1994–99: 53.0 2002–04: Overall 15.3 year 1: 3.2 1994–99: Overall 23.9 year 1: 6.8 2002–04: Initial 17.3 Repeat 13.6 1994–99: Initial 24.7 Repeat 23.2 2002–04: 31.2%d 1994–99: 48.7%d 2002–04: 17.8% 1994–99: 31% Bordas17 Norrbotten, Sweden, population screening program (1989–2002); 40–74 years 29.4 Overall 10.7 year 1: 5.1 year 2: 5.6 – 33.4%d 26.6% Tornberg28 Florence, Italy, population screening program (1990–94); 50–69 years 77.1 (initial 91.0; repeat 40.1) Overall 15.3 year 1: 3.9 year 2: 11.4 Initial 13.3e Repeat 21.0e 34.4%d (initial 30%; repeat 47%)e 17%d Tornberg28 Turin, Italy, population screening program (1992–96); 50–59 years 78.7 (initial 86.1; repeat 62.6) Overall 15.5 year 1: 5.5 year 2: 10.0 Initial 14.0e Repeat 19.2e 35.0%d (initial 35%; repeat 47%)e 16%d Fracheboud19 Netherlands population screening program (1990–93); 50–69 years 57.3 (initial 65.7, repeat 34.6) Overall 18.2d year 1: 6.1d year 2: 12.1d Initial 18.1 Repeat 18.6 39.3%d 24%d Törnberg28 Marseille, France, population screening (1993–98); 50–69 years 46.7 (initial 46.5; repeat 47.3) Overall 17.4 year 1: 5.4 year 2: 12.1 Initial 17.3e Repeat 18.1e 43.3%d (initial 43%; repeat 45%)e 27%d Törnberg28 Strasbourg, France, population screening (1989–97); 50–65 years 42.7 (initial 51.9; repeat 37.2) Overall 21.3 year 1: 6.9 year 2: 14.4 Initial 20.5e Repeat 22.0e 47.2%d (initial 45%; repeat 49%)e 33%d Törnberg28 Four counties, Norway (1996–97); 50–69 years 67.2 Overall 19.5 year 1: 4.5 year 2: 15.0 Initial 16.4e Repeat n/a 48.7%d (initial 41%; repeat n/a)e 22%d Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2017) 12 Published in partnership with the Breast Cancer Research Foundation Published in partnership with the Breast Cancer Research Foundation Interval breast cancers in mammography screening N Houssami and K Hunter 4 Table 1 continued Study ﬁrst author Screening settinga; age-group screened Cancer detection rate per 10,000 screens Interval cancer rate per 10,000 screens reported for all screens: overall or by inter- screen interval Interval cancer rate per 10,000 screens where reported separately for initial and repeat screens Proportional interval cancer rateb Percentage of cancers (from all BCs in screened women) that are interval cancersc Törnberg28 Pirkanmaa, Finland (1988–99); 50–69 years 36.3 (initial 41.6; repeat 32.3) Overall 17.4 year 1: 6.5 year 2: 11.0 – 66.4%d 32%d Lynge24 Copenhagen, Denmark (1991–93 [initial] and 1993–95 [repeat]); 50–69 years (Initial 118.6; repeat 62.5)b – Initial 17.3e Repeat 20.5e (Initial 34%; repeat 40%)e Initial 13% Repeat 25% Njor25 Funen, Denmark (1993–95 [initial] and 1996–97 [repeat]); 50–69 years (Initial 95.9; repeat 52.1)b – Initial 21.2e Repeat 24.2e (Initial 43%; repeat 47%)e Initial 18% Repeat 32% Törnberg28 Stockholm, Sweden, screening program (1989–97); 50–69 years 47.6 (initial 58.9; repeat 39.8) Overall 21.1 year 1: 7.3 year 2: 13.8 Initial 20.4e Repeat 21.7e (Initial 40%; repeat 46%)e Initial 26% Repeat 35% Hofvind22 Norwegian breast cancer screening program (1996–2005 [initial] and 1988–2005 [repeat]); 50–69 years 56.4 (initial 64.8; repeat 49.2) 18.2 Initial 18.3 Repeat 18.2 51%e Initial 22% Repeat 27% Kellen23 Belgian province of Limburg population screening program (1996–2001); 50–69 years 101 (included prevalent screening) Overall 49.3 (for 3-year interval) – Year 1: 21.7% Year 2: 11.3% 36.6% (biennial program but included 3-year interval BC data) Vettorazzi29 Screening program in local health units of Veneto Region, Italy (1999–2002); 50–69 years [initial screening round] 72.0e – (all data are for initial screens) 16.3e 29% (year 1: 21%; year 2: 46%) 18% a Except where speciﬁed as including data for digital mammography, data are for ﬁlm-screen mammography. Published in partnership with the Breast Cancer Research Foundation Published in partnership with the Breast Cancer Research Foundation True interval: 48.0% Missed /FN: 23.6% Minimal signs: 17.5% Occult cancer: 10.9% True interval: 55.0% Missed /FN: 22.0% Minimal signs: 24.0% True interval: 54.5% Missed /FN: 13.6% Minimal signs: 13.6% Occult cancer: 18.2% True interval: 48.2% Missed/FN: 23.2% Minimal signs: 17.2% Occult cancer: 11.3% Distribution of radiological classiﬁcationa of interval breast cancers Year 1 of inter-screening interval True interval: 43.1% Missed/FN: 31.3% Minimal signs: 25.7% Year 2 of inter- screening interval True interval: 60.2% Missed/FN: 19.1% Minimal signs: 20.7% Majority of missed or minimal-signs cases were masses at prior FSM or DM. No differences in mammography features (for FSM vs. DM) for cases emerging year 1 of inter-screen interval; however, cases in year 2 of inter-screen interval for DM were more frequently true (than missed) interval BCs compared to those for FSM (p = 0.03). Review of 1012 interval cancers (Spanish screening program 2000–06) by panels of three experienced radiologists using semi- informedb review of screening and diagnostic mammograms, independent double-reading and arbitration for discordant classiﬁcation. Factors associated with interval BC differed by radiological category, for example, family history of BC was mainly associated with true interval BC, whereas density was more strongly associated with occult BCs followed by true interval BCs. Review of 224 interval cases from FSM or DM screening (prior screen and those taken at diagnosis) by two experienced radiologists: readers aware they were reviewing interval BCs but blinded to each other's review; consensus for discordant classiﬁcation Study of 2245 invasive BCs (948 were interval cases) diagnosed 2000–09 in women participating in biennial population screening in Spain; interval BCs were classiﬁed by semi-informedb review of the screening and diagnostic mammograms by panels of three radiologists. Majority of missed interval BCs were masses at prior FSM or DM, followed by asymmetry or architectural distortion. True interval BCs were associated with HER2 and triple-negative tumour phenotypes and with extremely dense (>75% density) breasts; extreme breast density was most strongly associated with occult interval BCs Study of 2245 invasive BCs (948 were interval cases) diagnosed 2000–09 in women participating in biennial population screening in Spain; interval BCs were classiﬁed by semi-informedb review of the screening and diagnostic mammograms by panels of three radiologists. Distribution of pathological features differed between interval and screen- detected BCs (see Table 3) Review of 22 interval cases (Girona Health Region screening program 2000–06) by panel of expert radiologists, using semi-informedb independent double-reads of screening and diagnostic mammograms, with arbitration for discordant classiﬁcation. True interval: 57.8% Missed /FN: 17.7% Occult cancer: 10.0% Unclassiﬁed: 2.2% External review True intervalc: 67.0% Missed/FN: 18.4% Minimal signs: 14.6% Internal review True intervalc: 62.1% Missed/FN: 17.4% Minimal signs: 20.4% Classiﬁed into two categories: True interval: 74.1% Missed/FN: 25.9% True intervalc: FSM 47.1%; DM 65.3% Missed/FN: FSM 30.8%; DM 20.2% Minimal signs: FSM 22.1%; DM 14.5% Epidemiologic surveillance Carbonaro15 Review of 130 interval BCs in popu screening program, Italy 2001–06: t expert radiologists blindly reviewed mammograms, mixed with negativ cases not recalled classiﬁed as true BC, those recalled by only one revi minimal signs, and those recalled b reviewers as missed interval BCs Payne39 Review of 332 interval BCs (Nova S screening program 1991–2004): blin independent review by three expe radiologists; classiﬁed as true interv radiologists reported index screen (otherwise classiﬁed as missed inte >2 reported abnormal screen). Pellegrini40 Review of 103 interval BCs in popu screening program Trento, Italy 200 external (three radiologists) and int radiologists) panel with varying scr experience blindly reviewed pre-dia screening mammograms, mixed wi negative controls. Classiﬁcation bas Epidemiologic surveillance Screening is biennial unless otherwise speciﬁed b The ratio of the interval cancer rate to the expected underlying incidence rate (also referred to as proportionate incidence) c Percentage is a simple representation of the proportion of breast cancers occurring in screened women (counting both screen-detected and interval cases in the denominator) that are recorded as interval cancers, and can also be expressed as a ratio (also referred to as an 'interval cancer ratio'2) d Based on data from O'Brien et al.4 e Based on data from Andersen et al 2 Breast Cancer (2017) 12 Publishe Published in partnership with the Breast Cancer Research Foundation Interval breast cancers in mammography screening N Houssami and K Hunter Interval breast cancers in mammography screening N Houssami and K Hunter 5 Table 2. Radiological surveillance of interval breast Study (ﬁrst author) Methods Weber13 Review of 800 interval BCs from so screening region of Dutch breast s program (2000–11 spanning transit FSM to DM) by two radiologists ba prior screen and diagnostic mamm Blanch10 Review of 1012 interval cancers (Sp screening program 2000–06) by pa three experienced radiologists usin informedb review of screening and d mammograms, independent doubl and arbitration for discordant class Nederend38 Review of 224 interval cases from F screening (prior screen and those t diagnosis) by two experienced radi readers aware they were reviewing BCs but blinded to each other's rev consensus for discordant classiﬁcat Domingo14 Study of 2245 invasive BCs (948 we cases) diagnosed 2000–09 in wome participating in biennial population screening in Spain; interval BCs we classiﬁed by semi-informedb review screening and diagnostic mammog panels of three radiologists. Renart-Vicens26 Review of 22 interval cases (Girona Region screening program 2000–06 of expert radiologists, using semi-in independent double-reads of scree diagnostic mammograms, with arbi discordant classiﬁcation. Fong18 Review of 692 interval BCs, with co to screen-detected BC (Breast Test 1998–2001): blindedd review of scre and 'symptomatic' mammograms b readers, with consensus for discord classiﬁcation. Review of 130 interval BCs in population screening program, Italy 2001–06: three expert radiologists blindly reviewed mammograms, mixed with negative screens: cases not recalled classiﬁed as true interval BC, those recalled by only one reviewer as minimal signs, and those recalled by >2 reviewers as missed interval BCs True interval BCs were associated with HER2 and triple-negative tumour phenotypes and with extremely dense (>75% density) breasts; extreme breast density was most strongly associated with occult interval BCs Distribution of radiological classiﬁcationa of interval breast cancers True interval: 35% Missed /FN: 35% Minimal signs: 23% Occult cancer: 7% Of the combined missed and minimal signs interval BCs, 50% were poorly deﬁned masses or asymmetric densities, 26% were MC with/without associated density or mass, at the baseline screen. Review of 231 interval BCs in Norwegian population screening program 1995–98: six experienced radiologists reviewed and classiﬁed cases in a consensus meeting, using screening and diagnostic mammograms; classiﬁed as missed if all radiologists agreed tumour was visible at screening mammogram No major differences in the prognostic features of interval BCs when examined by radiological type or time elapsed since last screening mammogram. Review of 57 interval BCs in population screening program in Northeast Spain 1995–2001: 'informed consensus review' by three experienced radiologists using screening and diagnostic mammograms. Method 1 average (range): Missed /FN: 24% (10–40) Minimal signs: 6% (5–15) Method 2 average (range) Missed/FN: 33% (20–55) Minimal signs: 10% (10–20) Method 3 average (range) Missed/FN: 42% (35–50) Minimal signs: 20% (15–30) A classiﬁcation of 'missed' or minimal- signs interval BC was more likely using method 2 (odds ratio (OR) = 1.78, p = 0.033 or method 3 (OR = 3.91, p = 0.000) relative to method 1, but no reader effect was evident. Independent review of 100 screening mammograms (20 interval BCs, 80 negative screens) by six radiologists, using three sequenced review methods (separated by 2 weeks) with increasing information: (1) blindedd (no IC information, case-mix) (2) partially informedb (aware IC) (3) fully informed (with diagnostic mammograms) True interval: 42% Missed/ FN: 26% Occult cancer: 32% True interval: 42% Missed/ FN: 26% Occult cancer: 32% Review of 208 interval BCs from a multi- centre RCT of screening from age 40–41 years: review by two radiologists with arbitration by a third, using semi-informedb review of screening mammograms followed by diagnostic mammograms. Abnormalities further classiﬁed as malignant, subtle (features difﬁcult to detect), or non-speciﬁc (features only seen in retrospect after reviewing diagnostic ﬁlms). Features frequently misinterpreted were granular MC (38%), asymmetric density (27%), distortion (22%). Of abnormal previous screens, 37% were classiﬁed malignant, 39% subtle change and 21% non-speciﬁc. MC more common on diagnostic mammograms of FNs than those of true interval BCs (28 vs. 14%). Excludes 19 'unclassiﬁable' cases: True interval: 39.5% Missed/FN: 21.1% Minimal signs: 26.3% Occult cancer: 13.2% Distribution of radiological classiﬁcationa of interval breast cancers Tru Mi Pirola34 Review of pre-diagnosis screening mammograms of 30 interval BCs from Milan, Italy, screening program (2005) performed by an expert radiologist who had read >300,000 mammograms, blindedd to interval BCs by case-mix with negative screens. Tru Mi Hofvind37 Review of 231 interval BCs in Norwegian population screening program 1995–98: six experienced radiologists reviewed and classiﬁed cases in a consensus meeting, using screening and diagnostic mammograms; classiﬁed as missed if all radiologists agreed tumour was visible at screening mammogram Tru Mi Bare31 Review of 57 interval BCs in population screening program in Northeast Spain 1995–2001: 'informed consensus review' by three experienced radiologists using screening and diagnostic mammograms. Exc int Mi 13 Ciatto33 Independent review of 100 screening mammograms (20 interval BCs, 80 negative screens) by six radiologists, using three sequenced review methods (separated by 2 weeks) with increasing information: (1) blindedd (no IC information, case-mix) (2) partially informedb (aware IC) (3) fully informed (with diagnostic mammograms) Me 24 Me 33 Me 42 Evans35 Review of 208 interval BCs from a multi- centre RCT of screening from age 40–41 years: review by two radiologists with arbitration by a third, using semi-informedb review of screening mammograms followed by diagnostic mammograms. Abnormalities further classiﬁed as malignant, subtle (features difﬁcult to detect), or non-speciﬁc (features only seen in retrospect after reviewing diagnostic ﬁlms). Tru Oc Gao36 Review of 59 interval BCs (Singapore screening program 1994–97) by three radiologists using index screens; semi- informed (aware reviewing interval cases but unaware of tumour location). Mi dia fro rev BC breast cancer, DM digital mammography, FSM ﬁlm-screen mammo controlled trial a Classiﬁcation of interval BCs: true interval (cancer is not visible at th missed/FN (cancer is visible on the index mammogram but is not rec Table 2 continued Distribution of radiological classiﬁcationa of interval breast cancers Additional ﬁndings True intervalc: 71.0% Missed/FN: 15.0% Minimal signs: 14.0% Interval BC proportional incidence 10.8% in year 1 and 40.0% in year 2 of inter- screening interval. Interval BCs associated with denser breasts compared with negative controls (p = 0.02). True intervalc: 76.7% Missed /FN: 16.6% Minimal signs: 6.7% True intervalc: 76.7% Missed /FN: 16.6% Minimal signs: 6.7% Interval BC proportional incidence estimated as 17.4% for 2-year inter- screening interval. Interval BC proportional incidence estimated as 17.4% for 2-year inter- screening interval. True interval: 42% Missed/ FN: 26% Occult cancer: 32% True interval: 35% Missed /FN: 35% Minimal signs: 23% Occult cancer: 7% Distribution of radiological classiﬁcationa of interval breast cancers Cases with true interval or FN ﬁndings had similar background parenchymal patterns, but those with occult interval BC had higher proportion of dense patterns, p < 0.05 Features frequently misinterpreted were granular MC (38%), asymmetric density (27%), distortion (22%). Of abnormal previous screens, 37% were classiﬁed malignant, 39% subtle change and 21% non-speciﬁc. MC more common on diagnostic mammograms of FNs than those of true interval BCs (28 vs. 14%). Cases with true interval or FN ﬁndings had similar background parenchymal patterns, but those with occult interval BC had higher proportion of dense patterns, p < 0.05 Missed/FN: 17% (based on 'worst diagnoses' from ﬁve screen-readers, two from initial reads and three from re- review). Review of 59 interval BCs (Singapore screening program 1994–97) by three radiologists using index screens; semi- informed (aware reviewing interval cases but unaware of tumour location). Missed/FN: 17% (based on 'worst diagnoses' from ﬁve screen-readers, two from initial reads and three from re- review). Review of 59 interval BCs (Singapore screening program 1994–97) by three radiologists using index screens; semi- informed (aware reviewing interval cases but unaware of tumour location). In 3 years of successive follow-up from index screen, interval BC rates per 10,000 women-years were 2.1, 10.6 and 10.8 each year. Distribution of radiological classiﬁcationa of interval breast cancers 10-year all-cause survival rate for screen- detected BC (81.6%) was higher than that for interval BC (72.4%) [p < 0.001]: this differed by radiological category, true interval BC (77.5%), FN interval BC (55%), occult (54.4%) with latter two types having lower survival rates than screen- detected. Review of 692 interval BCs, with comparison to screen-detected BC (Breast Test Wales 1998–2001): blindedd review of screening and 'symptomatic' mammograms by two readers, with consensus for discordant classiﬁcation. Review of 130 interval BCs in population screening program, Italy 2001–06: three expert radiologists blindly reviewed mammograms, mixed with negative screens: cases not recalled classiﬁed as true interval BC, those recalled by only one reviewer as minimal signs, and those recalled by >2 reviewers as missed interval BCs A higher rate of larger (T3-T4) tumours was evident for missed interval BCs (18%) than minimal signs (6%) or true interval BCs (8%); and the rate of node metastases (N2-N3) for minimal signs (19%) or missed cancers (25%) was higher than that for true interval BCs (10%). Breast density distribution varied between the two types of interval BC and differed across age-group; rate of true interval BCs was higher for longer screening interval but this was not the case for FN cases. Review of 332 interval BCs (Nova Scotia screening program 1991–2004): blindedd and independent review by three experienced radiologists; classiﬁed as true interval BC if >2 radiologists reported index screen as normal (otherwise classiﬁed as missed interval BC if >2 reported abnormal screen). External review True intervalc: 67.0% Missed/FN: 18.4% Minimal signs: 14.6% Internal review True intervalc: 62.1% Missed/FN: 17.4% Minimal signs: 20.4% Review of 103 interval BCs in population screening program Trento, Italy 2001–08: external (three radiologists) and internal (ﬁve radiologists) panel with varying screening experience blindly reviewed pre-diagnosis screening mammograms, mixed with negative controls. Classiﬁcation based on No signiﬁcant difference between external and internal radiological review. Published in partnership with the Breast Cancer Research Foundation Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2017) 12 Interval breast cancers in mammography screening N Houssami and K Hunter 6 Table 2 continued Study (ﬁrst author) Methods Dis cla majority report ('missed' if recalled by most reviewers). Caumo32 Review of 100 interval BCs in Verona, Italy, screening program 2000–06: three expert radiologists blindly reviewed pre-diagnosis mammograms, mixed with negative controls. Classiﬁcation according to majority report. Interval BCs were non-signiﬁcantly more likely to be triple-negative, and less likely to be luminal A tumours than screen- detected BCs. Distribution of radiological classiﬁcationa of interval breast cancers Study of 2245 invasive BCs (948 were interval BCs) diagnosed 2000–09 in participants in population breast screening in Spain: compares interval and screen-detected BC, as well as categories of interval BCs, by density True interval BCs were associated with HER2 and triple-negative phenotypes (OR = 1.91 and OR = 2.07, respectively) and extremely dense breasts (OR = 1.67). Among true interval BCs, triple-negative tumours were more frequently observed in fatty (<25% density) than in denser breasts (p<0.001). FNs and occult interval BCs had similar phenotypic characteristics to screen-detected cancers. Mean tumour size signiﬁcantly larger for all radiological categories of interval BCs (range from 19.3 mm for occult cases to 25.3 mm for true interval cases) than mean tumour size for screen-detected BC (15.7 mm) [p < 0.001 comparison across all groups]. Proportion with lymph node metastases higher for all categories of interval BCs (range from 38% for occult cases to 50% for true interval and minimal-sign cases) than screen-detected BC (30%), [p < 0.001 comparing all groups]. Proportion with grade III tumours higher for all categories of interval BCs (range from 24% for occult to 45% for true interval cases) than screen- detected BC (21.6%), [p < 0.001 comparing all groups]. Interval vs. screen-detected BCs from Girona Health Region screening program 2000–06. Interval BCs had signiﬁcantly higher proportions of advanced stage disease (14% vs. 1%), larger tumours (5.4% vs. 2.3%), high-grade tumours (38% vs. 23%), and higher number of metastatic nodes (13.5% vs. 7.7%) than screen-detected BCs. Interval vs. screen-detected BCs sourced from three case–control studies nested in screened populations, by density measured with various methods. Interval BCs had signiﬁcantly larger (average) maximum tumour diameter for each measure of density (percent mammographic density, dense and non- dense areas) than screen-detected BCs. Interval vs. screen-detected BCs, Florence population screening program 2004–05. Stage at diagnosis was more advanced for interval BCs than screen-detected BCs based on pT distribution (pT 2+ 25.8% vs. 10.4%, p < 0.001) and pN distribution (pN 1+ 41% vs. 29%, p = 0.032). Interval vs. screen-detected BCs from Nova Scotia screening program 1991–2004. Interval BCs less likely to be ER positive than screen-detected BCs (p = 0.002). Interval BCs less likely to be ER positive than screen-detected BCs (p = 0.002). Interval vs. screen-detected BCs from Girona Health Region screening program 2000–06. Distribution of radiological classiﬁcationa of interval breast cancers screen-detected BCs, southern region of Dutch screening program (2000–11); also compares interval BCs by radiological category and by year 1 vs. yea of inter-screen interval. Meshkat47 Interval vs. screen-detected BCs, screenin unit for the Irish breast screening progra (2010–13) Holm45 Interval vs. screen-detected BC among women diagnosed with invasive BC (2001–08), Stockholm, Sweden, by breast density. Domingo14 Study of 2245 invasive BCs (948 were inte BCs) diagnosed 2000–09 in participants in population breast screening in Spain: compares interval and screen-detected BC well as categories of interval BCs, by den Renart- Vicens26 Interval vs. screen-detected BCs from Giro Health Region screening program 2000–0 Boyd11 Interval vs. screen-detected BCs sourced from three case–control studies nested in screened populations, by density measur with various methods. Caldarella43 Interval vs. screen-detected BCs, Florence population screening program 2004–05. Payne39 Interval vs. screen-detected BCs from Nov Scotia screening program 1991–2004. Kalager46 Interval BC in the Norwegian screening program vs. BCs in same time frame in Tumour characteristics and prognostic features (size, histology, grade, node status and/or stage) Interval vs. screen-detected BCs, southern region of Dutch screening program (2000–11); also compares interval BCs by radiological category and by year 1 vs. year 2 of inter-screen interval. Interval BCs had higher proportions of T2 + tumours (52% vs. 21.5%) and of metastatic nodes (46.3% vs. 7.7%) than screen-detected BCs; interval BCs had different tumour histology distribution (fewer in-situ, higher proportion of invasive lobular) to screen-detected BCs. Missed cases had larger mean invasive tumour size than true intervals (28.5 vs. 24 mm, p = 0.003). Interval vs. screen-detected BCs, screening unit for the Irish breast screening program (2010–13) Interval BCs had higher tumour grade (p < 0.05) and higher stage (proportion stage 1 vs. 2; p < 0.001) than screen- detected BCs. Invasive lobular was more frequent among interval than screen- detected BCs (21% vs. 11%, p < 0.05). Interval vs. screen-detected BC among women diagnosed with invasive BC (2001–08), Stockholm, Sweden, by breast density. Interval BCs in non-dense breasts more likely to be ER negative (OR 4.05), PR negative (OR 2.63), HER2 positive (OR 5.17), and triple negative (OR 5.33) than screen-detected BC. Interval BCs in non-dense breasts (<20% density) were more likely to harbour lymph node metastases (OR 3.55) than screen-detected BC in non-dense breasts. Study of 2245 invasive BCs (948 were interval BCs) diagnosed 2000–09 in participants in population breast screening in Spain: compares interval and screen-detected BC, as well as categories of interval BCs, by density Relative to screen-detected BC, triple- negative BCs were over-represented, and luminal A (ER/PR positive, HER2 negative) BCs were under-represented among interval BCs Distribution of radiological classiﬁcationa of interval breast cancers Domingo14 Study of 2245 invasive BCs (948 were interval BCs) diagnosed 2000–09 in participants in population breast screening in Spain: compares interval and screen-detected BC, as well as categories of interval BCs, by density Mean tumour size signiﬁcantly larger for all radiological categories of interval BCs (range from 19.3 mm for occult cases to 25.3 mm for true interval cases) than mean tumour size for screen-detected BC (15.7 mm) [p < 0.001 comparison across all groups]. Proportion with lymph node metastases higher for all categories of interval BCs (range from 38% for occult cases to 50% for true interval and minimal-sign cases) than screen-detected BC (30%), [p < 0.001 comparing all groups]. Proportion with grade III tumours higher for all categories of interval BCs (range from 24% for occult to 45% for true interval cases) than screen- detected BC (21.6%), [p < 0.001 comparing all groups]. True interval BCs wer HER2 and triple-nega (OR = 1.91 and OR = and extremely dense Among true interval tumours were more f in fatty (<25% densit breasts (p<0.001). FN BCs had similar phen characteristics to scre cancers. Renart- Vicens26 Interval vs. screen-detected BCs from Girona Health Region screening program 2000–06. Interval BCs had signiﬁcantly higher proportions of advanced stage disease (14% vs. 1%), larger tumours (5.4% vs. 2.3%), high-grade tumours (38% vs. 23%), and higher number of metastatic nodes (13.5% vs. 7.7%) than screen-detected BCs. Interval BCs were no likely to be triple-neg to be luminal A tumo detected BCs. Boyd11 Interval vs. screen-detected BCs sourced from three case–control studies nested in screened populations, by density measured with various methods. Interval BCs had signiﬁcantly larger (average) maximum tumour diameter for each measure of density (percent mammographic density, dense and non- dense areas) than screen-detected BCs. – Caldarella43 Interval vs. screen-detected BCs, Florence population screening program 2004–05. Stage at diagnosis was more advanced for interval BCs than screen-detected BCs based on pT distribution (pT 2+ 25.8% vs. 10.4%, p < 0.001) and pN distribution (pN 1+ 41% vs. 29%, p = 0.032). Relative to screen-de negative BCs were ov luminal A (ER/PR pos BCs were under-repre interval BCs P 39 I l d d BC f N I l BC lik l b d I l BC l lik l Table 3. Biological characteristics of interval breast cance Study (ﬁrst author) Comparison and setting Weber13 Interval vs. Distribution of radiological classiﬁcationa of interval breast cancers BC breast cancer, DM digital mammography, FSM ﬁlm-screen mammography, FN false-negative, MC micro-calciﬁcations, IC interval cancer, RCT randomised BC breast cancer, DM digital mammography, FSM ﬁlm-screen mammography, FN false-negative, MC micro-calciﬁcations, IC interval cancer, RCT randomised controlled trial cer, DM digital mammography, FSM ﬁlm-screen mammography, FN false-negative, MC micro-calciﬁcations, IC interval can mography, FSM ﬁlm-screen mammography, FN false-negative, MC micro-calciﬁcations, IC interval cancer, RCT randomised C breast cancer, DM digital mammography, FSM ﬁlm-screen mammography, FN false-negative, MC micro-calciﬁcations, IC ontrolled trial a Classiﬁcation of interval BCs: true interval (cancer is not visible at the index mammographic screen but becomes visible at the diagnostic mammogram); missed/FN (cancer is visible on the index mammogram but is not recalled or is misinterpreted); minimal-signs (subtle abnormality is visible on the index mammogram but one that is unlikely to warrant recall); occult (cancer that is not visible on the index screen and not visible on the diagnostic mammogram) b Semi-informed radiological review generally involved screen-readers knowing that interval BC cases were being reviewed, without information on the side and location of the interval cancer c In some studies 'true interval' BCs are also referred to as 'occult' at the index or pre-diagnosis screen; this should not be confused with the conventional 'occult cancer' classiﬁcation of interval cases, which usually refers to a BC that is not seen on the index mammography screen and also occult on the diagnostic mammogram g d Blinding or blinded methods of review: this generally refers to (a) interval cases being interspersed with screen-reading as part of the routine screening workﬂow; or (b) interval cases being mixed with normal screening mammograms but not integrated into routine screen-reading workﬂow (study-speciﬁc methods described in table) Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2017) 12 Published in partnership with the Breast Cancer Research Foundation Interval breast cancers in mammography screening N Houssami and K Hunter Interval breast cancers in mammography screening N Houssami and K Hunter Interval breast cancers in mammography screening N Houssami and K Hunter Table 3. Biological characteristics of interval breast cancers Study (ﬁrst author) Comparison and setting Weber13 Interval vs. screen-detected BCs, southern region of Dutch screening program (2000–11); also compares interval BCs by radiological category and by year 1 vs. year of inter-screen interval. Meshkat47 Interval vs. screen-detected BCs, screening unit for the Irish breast screening program (2010–13) Holm45 Interval vs. Distribution of radiological classiﬁcationa of interval breast cancers screen-detected BC among women diagnosed with invasive BC (2001–08), Stockholm, Sweden, by breast density. Domingo14 Study of 2245 invasive BCs (948 were interva BCs) diagnosed 2000–09 in participants in population breast screening in Spain: compares interval and screen-detected BC, a well as categories of interval BCs, by densit Renart- Vicens26 Interval vs. screen-detected BCs from Giron Health Region screening program 2000–06. Boyd11 Interval vs. screen-detected BCs sourced from three case–control studies nested in screened populations, by density measured with various methods. Caldarella43 Interval vs. screen-detected BCs, Florence population screening program 2004–05. Payne39 Interval vs. screen-detected BCs from Nova Scotia screening program 1991–2004. Kalager46 Interval BC in the Norwegian screening program vs. BCs in same time frame in Published in partnership with the Breast Cancer Research Foun 7 Table 3. Biological characteristics of interval breast cancers Study (ﬁrst author) Comparison and setting Tumour characteristics and prognostic features (size, histology, grade, node status and/or stage) Tumour biomarkers o speciﬁc ﬁndings Weber13 Interval vs. screen-detected BCs, southern region of Dutch screening program (2000–11); also compares interval BCs by radiological category and by year 1 vs. year 2 of inter-screen interval. Interval BCs had higher proportions of T2 + tumours (52% vs. 21.5%) and of metastatic nodes (46.3% vs. 7.7%) than screen-detected BCs; interval BCs had different tumour histology distribution (fewer in-situ, higher proportion of invasive lobular) to screen-detected BCs. Missed cases had larger mean invasive tumour size than true intervals (28.5 vs. 24 mm, p = 0.003). Interval BCs in year 2 interval for digital ma more frequently rece than those occurring ﬁlm-screen mammog Meshkat47 Interval vs. screen-detected BCs, screening unit for the Irish breast screening program (2010–13) Interval BCs had higher tumour grade (p < 0.05) and higher stage (proportion stage 1 vs. 2; p < 0.001) than screen- detected BCs. Invasive lobular was more frequent among interval than screen- detected BCs (21% vs. 11%, p < 0.05). Interval BCs less likel (76% vs. 81%, p < 0.05 overexpress HER2 (20 than screen-detected Holm45 Interval vs. screen-detected BC among women diagnosed with invasive BC (2001–08), Stockholm, Sweden, by breast density. Interval BCs in non-dense breasts (<20% density) were more likely to harbour lymph node metastases (OR 3.55) than screen-detected BC in non-dense breasts. Interval BCs in non-d likely to be ER negat negative (OR 2.63), H 5.17), and triple nega screen-detected BC. Interval BCs less likely to be ER positive (76% vs. 81%, p < 0.05) and more likely to overexpress HER2 (20% vs. 10%, p < 0.05) than screen-detected BCs. Interval vs. screen-detected BCs, screening unit for the Irish breast screening program (2010–13) Comparison of 538 interval BCs by radiological type, in a UK screening program service, 1987–2000. Comparison of interval BC subgroups (true + occult vs. minimal signs vs. FN vs. unclassiﬁable) from Northeast Spain screening program, 1995–2001. Comparison of interval BC type (true vs. FN vs. occult) from a multi-centre randomised trial, UK, conducted in younger age group (40–48 years). No signiﬁcant differences between groups in ER or PR status. ER and PR receptor status distribution did not differ between subgroups of interval BC. Distribution of radiological classiﬁcationa of interval breast cancers Interval BCs were more likely to be node- positive, to be larger tumours, to have higher grade, and to show lymphovascular invasion than screen- detected BCs (all p < 0.001). Interval BC in the Norwegian screening program vs. BCs in same time frame in Interval BCs had slightly higher proportions of larger tumours (>20 mm), stage II rather than stage I cancer, invasive Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2017) 12 Interval breast cancers in mammography screening N Houssami and K Hunter 8 Table 3 continued Study (ﬁrst author) Comparison and setting population not yet invited to sc screened women). Caumo44 Interval vs. screen-detected vs occurring in the absence of sc Verona mammography screen 2000–06 and Veneto cancer re Hofvind37 Comparison of interval BC sub (missed vs. minimal signs vs. tr from Norwegian screening pro 1995–98. Bare31 Comparison of interval BC sub occult vs. minimal signs vs. FN unclassiﬁable) from Northeast screening program, 1995–2001 Evans35 Comparison of interval BC typ vs. occult) from a multi-centre trial, UK, conducted in younge (40–48 years). Porter48 Comparison of 538 interval BC radiological type, in a UK scree service, 1987–2000. Gao36 Interval vs. screen-detected BC Singapore screening program reported comparison to those screening and those not invited BC breast cancer, ER estrogen receptor, FN false Nottingham prognostic index, DCIS ductal carcino Tumour characteristics and prognostic features (size, histology, grade, node status and/or stage) Tumour characteristics and prognostic features (size, histology, grade, node status and/or stage) Tumour biomarkers or phenotype- speciﬁc ﬁndings lobular histology, and negative (non- metastatic) axillary nodes, than BCs in non-screened women (distributions for these variables differed at p < 0.001). Interval BCs had more aggressive features than screen-detected BCs for pT (p < 0.001), pN (p < 0.001), and tumour grade distributions (p = 0.007). Interval BCs had similar prognostic features as clinical BCs based on pT, pN and grade distributions (all p > 0.05). Interval BCs less likely to be ER-positive (77% vs. 91%, p < 0.001) and PR-positive (61% vs. 82%, p < 0.001) than screen- detected BCs. Interval BCs had similar proportions of ER/PR receptor positivity as clinical BCs. Interval BCs less likely to be ER-positive (77% vs. 91%, p < 0.001) and PR-positive (61% vs. 82%, p < 0.001) than screen- detected BCs. Interval BCs had similar proportions of ER/PR receptor positivity as clinical BCs. Comparison of interval BC subgroups (missed vs. minimal signs vs. true and occult) from Norwegian screening program, 1995–98. BC breast cancer, ER estrogen receptor, FN false negative, HER2 human epidermal growth factor receptor 2, PR progesterone receptor,OR odds ratio, NPI Nottingham prognostic index, DCIS ductal carcinoma in-situ Distribution of radiological classiﬁcationa of interval breast cancers A study from Ciatto and colleagues33 used a multi- methods evaluation of the same set of mammograms, and showed that increasing the information available to screen- readers signiﬁcantly increased the proportion of interval BCs classiﬁed as missed (Table 2), highlighting the impact of review methods on radiological classiﬁcation. screening was accompanied by a 15–20% reduction in interval BC rates. An earlier study by Seigneurin et al.27 reported similar evidence from the French population breast screening program, based on women aged 50–69, in a comparison of two time frames reﬂecting transition from one-view to two-view mammography: a reduction of 8.6 interval BCs per 10,000 screens was associated with two-view mammography, with an estimated 36% difference in relative risk of interval BC at 24 months for two vs. one view mammography (Table 1). Weber et al.13 reported a study of the Southern screening region of the Dutch program, in which the use of digital (compared to ﬁlm-screen) mammography was shown to be associated with a modest but signiﬁcant reduction in interval BC rates of 3/10,000 screens (p = 0.02; Table 1). g Additional ﬁndings summarised in Table 2 describe radiological ﬁndings (where present) for the index screen, which were frequently masses or asymmetric densities. They also highlight study-speciﬁc data showing differences in the variables associated with interval BCs, and in the tumour characteristics of interval BCs, across radiological categories derived from mammographic review. Radiological review of interval BCs following screening with digital or ﬁlm-screen mammography was reported by Knox et al.42 showing that a similar proportion of cancers were classiﬁed as missed cases at digital and ﬁlm-screen (10.5% and 8.1%, respectively, p = 0.77). However, fewer interval BCs were depicted as microcalciﬁcations alone or in association with another imaging abnormality following digital than ﬁlm-screen mammography (16% and 32%, respectively, p = 0.02) (ref. 42). Nederend and colleagues38 investigated interval BCs in a population-based study of regional screening units in the Netherlands, and showed that signiﬁcantly more interval BCs were classiﬁed as true-negative or true interval cases (not visible at the index screen) at radiological review of prior digital than prior ﬁlm-screen mammography (65.3% vs. 47.1%, p = 0.02) as shown in Table 2; otherwise, there were no differences between interval BCs at digital or ﬁlm-screen in terms of mammographic abnormalities at the prior screen or in tumour characteristics. Radiological surveillance Contextual background for radiological review and classiﬁcation of interval BCs, along with deﬁnitions of the categories of interval BCs, have been outlined in the introduction of the paper; methodolo- gical issues have been comprehensively explained in our previous review.1 Table 2 summarises ﬁndings from the literature search on radiological surveillance including the methods used to conduct mammographic review;10, 13–15, 18, 26, 31–40 the latter substantially inﬂuences the distribution of radiological categories and can bias estimated proportions.1, 33, 41 For example, a pilot study examining inﬂuences the distribution of radiological categories and can bias estimated proportions.1, 33, 41 For example, a pilot study examining radiological review methods showed that informed vs. blinded (uninformed) review of interval BC leads to bias in classiﬁcation whereby informed reviewers (aware they were reviewing mam- mograms containing interval cases) more frequently classiﬁed some interval BCs as positive, compared to reviewers who were unaware they were reading mammograms of interval BCs that had been added into routine screen-reading practice (‘uniformed’ review).41 To the extent that ‘blinding’ is possible in evaluation of interval BCs, this methodology sometimes referred to interval cases being interspersed with screen-reading as part of the routine screening workﬂow, or more frequently partial blinding was achieved by interval cases being mixed with normal screening mammograms (Table 2). Semi-informed radiological review methods involved knowledge that interval BC cases were being reviewed, without information on the side and location of the interval BC. In general, studies of radiological classiﬁcation were based on an initial review of the index screen (the screen preceding the subsequently diagnosed interval BC) with provi- sional identiﬁcation and classiﬁcation of the interval BC, followed by review of both the screening and diagnostic mammograms to enable deﬁnitive classiﬁcation or sub-classiﬁcation. However, not all studies provided these details and some studies did not specify whether the diagnostic mammograms were available for classiﬁ- cation. Interpretation was generally performed by experienced mammography readers, and varied from one expert screen-reader performing classiﬁcation, to panels of several radiologists with classiﬁcation based on reaching consensus or derived from majority reads. Table 2 footnote provides further deﬁnitions on radiological review methods and classiﬁcation terminology. Distribution of radiological classiﬁcationa of interval breast cancers Comparison of interval BC subgroups (missed vs. minimal signs vs. true and occult) from Norwegian screening program, 1995–98. ER and PR receptor status distribution did not differ between subgroups of interval BC. Missed interval BCs had generally less favourable characteristics than true (including occult) interval BCs: average invasive cancer size 23 mm in missed vs. 18 mm in true interval BCs (p = 0.017). Higher proportion of interval BCs with node metastases among missed (49%) and minimal-signs (53%) than true interval (33%) BCs. Histological type did not differ between interval BC subgroups, but invasive lobular was more frequent in the missed (20%) than true interval BCs (9%) p = 0.06 No signiﬁcant differences between the different radiological types in stage, tumour size, node status, histological grade, nuclear grade or histology. Minimal-signs group more frequently had poor prognosis based on NPI, whereas most frequent NPI classiﬁcation for other groups was moderate (p = 0.003). Higher frequency of invasive lobular BC among false-negative BCs. Occult interval BCs were more likely to be <10 mm and <15 mm in invasive size than other interval BCs (p = 0.03 and 0.005, respectively). True interval BCs were more likely to be histologically grade 3 than other cases (p = 0.04). No evidence of an excess of lobular BCs in occult group. – True and occult interval BCs (combined) were more likely to be histological grade 3 than minimal-signs and FN cases (52% vs. 35%, p = 0.05). FNs were more likely to have lobular histology than other interval BCs (47% vs. 20%, p < 0.001). – Interval vs. screen-detected BCs from Singapore screening program 1994–97; also reported comparison to those declining screening and those not invited to screening. Interval BCs were more likely to be stage II (52.5% vs. 31.1%) and have a tumour size >20 mm than screen-detected BCs, but less likely to be DCIS (10% vs. 26.5%). Distribution of axillary nodal status was similar between screen-detected and interval BCs; however, proportion of high- grade tumours among interval BCs (38%) was higher than screen-detected BCs (18.6%) and was similar to non-screened groups. – Published in partnership with the Breast Cancer Research Foundation Published in partnership with the Breast Cancer Research Foundation Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2017) 12 Interval breast cancers in mammography screening N Houssami and K Hunter 9 surveillance. Radiological surveillance Biological characteristics and prognosis Table 3 summarises biological ﬁndings including tumour character- istics and biomarker proﬁle for interval BCs, and outlines the comparison reported in each study because that accounts for some of the apparent heterogeneity in results.11, 13, 14, 26, 31, 35–37, 39, 43–48 For the majority of studies, which compared interval BCs with screen-detected cancers, there were consistent ﬁndings that interval BCs had worse prognostic features, such as larger tumour size, higher frequency of node metastases, higher histologic grade, and more advanced disease compared to screen-detected BC (Table 3). Although biomarker data were not consistently reported in these studies, where reported there was evidence that interval BCs had a higher frequency of triple-negative or HER2- positive cancers and a lower frequency of hormone receptor- positive cancers than screen-detected BC (Table 3). Some studies compared prognostic features between radiolo- gical categories of interval BCs, with variable ﬁndings (and limited statistical comparisons once study data were examined in subsets), however, some differences were noted between radiological subgroups. These differences by radiological sub- group are detailed in Table 3, and suggest that the ‘missed’ group had worse prognostic features than the true interval and occult interval cancers, except for tumour grade, which was reported to be more frequently higher for true interval and occult interval BCs than missed cases. Additional ﬁndings suggest that these outcomes may differ slightly between dense and non-dense breasts but density-speciﬁc ﬁndings were reported in very few studies (Table 3). As shown in Table 2, radiological categories varied slightly across studies, however, most studies reported the distribution for true interval BC and also for missed or false-negative cases (deﬁned in the introduction); some studies reported the additional categories of ‘occult’ and ‘minimal-signs’ interval BCs (see Table 2 footnote). The evidence table shows that the vast majority of interval BCs were not missed at screen-reading but were true interval BCs (range 40% to 77%) or occult interval BCs (7% to 32%), meaning that they were not visible on the index screen even in hindsight. Of note, some of the high proportions reported for true interval BCs (>60%) appear to have included the occult cases among the true interval cases. Published in partnership with the Breast Cancer Research Foundation Distribution of radiological classiﬁcationa of interval breast cancers Generally similar ﬁndings were reported by Weber et al.13 also from the Dutch screening program, who additionally observed that interval BCs emerging in year 2 of the inter-screen interval for digital mammography were more frequently true (than missed) interval cancers compared to those for ﬁlm-screen mammography (p = 0.03; Table 2). Radiological surveillance Interval breast cancers in mammography screening N Houssami and K Hunter 2 Key ﬁndings of the review prognostic features to the BCs occurring in non-screened women, however, one study reported that interval BCs had slightly higher proportions of larger tumours (>20 mm) than BCs in non-screened women.46 screen-detected, true interval, and symptom-detected BC were 87.5%, 64.1%, and 79.4%, respectively, and overall survival rates were 94.5%, 65.5%, and 85.6%, respectively.50 In keeping with these ﬁndings, they concluded that clinically-detected BC especially where these are true interval cancers had worse prognosis and poorer survival than screen-detected BC even after adjustment for clinical-pathological variables.50 Porter and collea- gues48 compared the features of interval BCs by radiological classiﬁcation, and although they observed differences in the histological characteristics (shown in Table 3) there was no signiﬁcant survival difference between interval BC radiological types (p = 0.64). Prognosis of interval BC. A population-based cohort study found similar survival for women who had an interval BC in the Norwegian screening program (hazard ratio (HR) 0.98; 95% conﬁdence interval 0.84–1.15) as those who had BC diagnosed in the same time frame but had not yet been invited to mammography screening (non-screened women).46 A study from the Malmo mammography screening program49 showed that interval BCs from the ﬁrst decade of service screening had similar stage distributions and survival as the BCs diagnosed in non-attenders to screening, whereas the screen- detected cancers in that time frame had more favourable stage distributions and survival than the interval cases. In this same study, there was also evidence that the prognosis of women with interval BC had improved over a 20-year period, as may be expected from overall improvements in BC prognosis over time. 14 50 yp (p ) Some studies have examined the prognostic characteristics of interval BCs by breast tissue density.11, 45 Holm et al.45 showed that interval BCs occurring in non-dense breasts (deﬁned by<20% density) had poorer prognostic features than screen-detected BC (Table 3), whereas interval BCs in dense breasts (≥50% density) were phenotypically more similar to screen-detected cancers. Eriksson et al.51 compared survival in interval and screen-detected BC allowing for mammographic density in women aged 50 years and older; they showed that hazard rates for BC-speciﬁc survival were signiﬁcantly higher for interval than for screen-detected cancers, independent of density. Interval breast cancers in mammography screening N Houssami and K Hunter Interval breast cancers in mammography screening N Houssami and K Hunter 10 Radiological surveillance Radiological surveillance • Most interval BCs represent true interval BC or occult interval BC (that were not visible on the index screen), with around 20-25% of interval BCs reported to have been missed (false- negative) cases. • Radiological surveillance has limitations inherent in retrospective re-interpretation of imaging, and the methodology used to perform mammographic review affects the reported distribution of radiological categories of interval BCs Epidemiological surveillance • There is broad variability in published interval BC rates and cancer detection rates at screening; most of the evidence on interval BC rates is from biennial screening programs. Interval BC rates spanning the two years between screens are within the range of 8.4 to 21.1 per 10,000 screens, with the larger proportion of the estimate occurring in year 2 of a two-yearly interval • Interval BCs represent roughly one quarter of the cancers occurring in screening participants in biennial screening; that proportion is lower for annual screening intervals and higher for triennial screening. • The frequency of interval BCs is higher at repeat (incident) rounds than initial (prevalent) screening rounds within the same screening setting and population. • Compared to screen-detected BCs from the same screening setting and population, interval BCs had relatively worse tumour prognostic characteristics and survival outcomes, however interval BCs had similar characteristics and prognosis to BCs diagnosed in non-screened women. • Missed interval BC had worse prognostic features for tumour size and node status than true interval and occult interval BCs although the latter were more likely to have higher tumour grades. • Mammographic density was consistently associated with occult BCs (followed by true interval • Mammographic density was consistently associated with occult BCs (followed by BCs) rather than missed interval cases, highlighting a likely masking effect. BCs) rather than missed interval cases, highlighting a likely masking effect. Fig. 2 Key ﬁndings of the review Fig. Published in partnership with the Breast Cancer Research Foundation Radiological surveillance The proportion of missed (false- negative) interval BCs ranged between 13.6% and 35%, with the majority reporting a frequency of 20–25% based on radiological In the limited number of studies comparing interval BCs with clinically presenting cancers in non-screened women,44, 46 there was evidence that interval cancers were similar in terms of npj Breast Cancer (2017) 12 Published in partnership with the Breast Cancer Research Foundation DISCUSSION The evidence table also shows that interval BC rates are higher at repeat (incident) rounds than initial (prevalent) screening rounds within the same screening setting and popula- tion. This ﬁnding has not been thoroughly explained in the reviewed studies but is presumably due to age increase and a tendency for lower recall rates at repeat screening of the same women but warrants further research. Because mammographic breast density is an established risk factor for interval BC in screened women,6, 7 some of the studies summarised in this review evaluated interval BCs in relation to mammographic density.10, 11, 14, 39, 45, 51 The detailed ﬁndings, Because mammographic breast density is an established risk factor for interval BC in screened women,6, 7 some of the studies summarised in this review evaluated interval BCs in relation to mammographic density.10, 11, 14, 39, 45, 51 The detailed ﬁndings, summarised in our results, are complex but reveal some common ﬁndings. Mammographic density was consistently associated with occult BCs (followed by true interval BCs) rather than missed interval cases,10, 14 highlighting a likely masking effect. In addition, there was a suggestion that interval BCs occurring in non-dense breasts may be associated with worse prognostic features and outcomes,45, 51 perhaps reﬂecting that interval BCs in non-dense breasts were more likely to be newly arising cases associated with rapid growth—however, the data were from few studies. summarised in our results, are complex but reveal some common ﬁndings. Mammographic density was consistently associated with occult BCs (followed by true interval BCs) rather than missed interval cases,10, 14 highlighting a likely masking effect. In addition, there was a suggestion that interval BCs occurring in non-dense breasts may be associated with worse prognostic features and outcomes,45, 51 perhaps reﬂecting that interval BCs in non-dense breasts were more likely to be newly arising cases associated with rapid growth—however, the data were from few studies. p g Epidemiological and radiological surveillance of interval BCs, complemented by an understanding of the biology of these cancers, provide insights into ‘how often’ and ‘why’ screening may not detect a BC that is subsequently diagnosed. Evidence shows that quantitative data on interval BCs are very heterogeneous and are inﬂuenced by several factors including the length of the inter- screening interval. Most published studies have reported data from biennial screening practice and in that context interval BCs represent roughly 17–30% of the cancers occurring in screening participants. DISCUSSION While radiological surveillance has limitations inherent in retrospective re-interpretation of imaging, and radiological classiﬁcation of interval BCs is affected by the methodology used to perform mammographic review (Table 2), quantitative evidence shows that the majority of interval cancers are true interval or occult interval BCs that were not visible on the index screen. It should be acknowledged that radiological surveillance is not practiced in all screening settings, and the aim of summarising the evidence is not to advocate this form of surveillance, rather its ﬁndings can inform practice. For example, it seems likely that enhancing screen-reader skills would have relatively less effect in controlling interval BCs than, say, enhancing mammography technology or using alternate or additional technology to address the majority of cases that are not visible at the index mammography screen even in hindsight. Evidence on radiological features of interval BCs following transition to digital mammography was also limited to few studies13, 38, 42 with one study suggesting that implementation of digital mammography modiﬁed the mammographic pattern of interval BCs, with fewer interval BCs depicted as microcalciﬁca- tions.42 However, two studies did not ﬁnd substantial differences in interval BCs, in terms of the pattern of mammographic ﬁndings, following transition to digital mammography.13, 38 g g g p y Studies providing data on the biological characteristics of interval BCs have mostly compared them to screen-detected BCs from the same screening setting and population, and have shown that interval BCs have relatively worse tumour prognostic characteristics, and worse survival outcomes, than screen- detected BCs. Interval BCs were consistently reported to be at a more advanced stage when diagnosed compared to screen- detected BC in terms of larger tumour size, higher frequency of node metastases, higher histologic grade, and had less favourable biomarker proﬁle including a higher frequency of triple-negative cancers. DISCUSSION The majority of studies in the evidence table report data from biennial screening, and show that interval BCs represent roughly one quarter of the cancers occurring in screening participants—that proportion is lower for annual, and higher for triennial, screening intervals. These ﬁndings should not be taken to infer that annual screening has better population outcomes than biennial screening (in fact biennial screening reduces some screening harms compared to annual screening), they merely highlight that many interval BCs are identiﬁed in the second year of a biennial screening round, and is commensurate with the pattern of ﬁndings from radiological surveillance speciﬁcally that many interval BCs are true interval cancers. Additional data from radiological surveillance from Weber and colleagues13 indicates that the proportion of interval cancers that are true interval BCs increases in year 2 (relative to year 1) at biennial screening. The evidence table also shows that interval BC rates are higher at repeat (incident) rounds than initial (prevalent) screening rounds within the same screening setting and popula- tion. This ﬁnding has not been thoroughly explained in the reviewed studies but is presumably due to age increase and a tendency for lower recall rates at repeat screening of the same women but warrants further research. Although there is a substantial body of knowledge on interval BC rates in mammography screening, as shown in Table 1, there was little direct evidence on mammography-based interventions that reduce interval BC rates A limited number of studies This overview of the epidemiological, radiological, and tumour characteristics of interval BCs—cancers that emerge following a negative mammographic screen—highlights key themes on interval BCs, which are summarised in Fig. 2. Interval BCs are an important consideration in population BC screening because they are indicative of screening quality hence evaluating these cancers may help identify areas for potential improvement, and because they represent a failure of screening to detect a BC that subsequently progresses to presentation. It is clear from radiological surveillance data summarised in our work that these cancers are not necessarily missed at mammography screening, with most studies reporting around 20–25% of interval BCs to be missed (false-negative) cases. DISCUSSION This does not mean that interval BCs are an aggressive group of cancers, in fact they have tumour characteristics and survival outcomes that approximate those of BCs diagnosed in non-screened women, based on data from the few studies reporting that comparison.44, 46 One study reported that interval BCs had higher proportions of larger tumours than BCs in non- screened women but did not ﬁnd any difference in survival between these groups.46 Further insights were provided by studies comparing prognostic features between radiological categories of interval BCs, with ﬁndings suggesting that the ‘missed’ group had worse prognostic features for tumour size and node status (possibly due to the delay in detection) than the true interval and occult interval BCs although the latter were more likely to have higher tumour grades (suggesting these to be more rapidly growing cancers representing new events on the mammogram). Epidemiological monitoring of interval BC rates is a more widely performed surveillance in population mammography screening programs. Variability in the overall interval BC rates shown in Table 1 reﬂects the underlying BC risk in the population (which is also glimpsed in study-speciﬁc BC detection rates), the mix of initial and repeat screen rounds, and the length of the inter- screening interval. The variability due to the inter-screening interval is particularly evident where data are presented by yearly rates for biennial or triennial screening: our summary shows consistent evidence that rates in year 2 are at least twice those in year 1 (Table 1). The majority of studies in the evidence table report data from biennial screening, and show that interval BCs represent roughly one quarter of the cancers occurring in screening participants—that proportion is lower for annual, and higher for triennial, screening intervals. These ﬁndings should not be taken to infer that annual screening has better population outcomes than biennial screening (in fact biennial screening reduces some screening harms compared to annual screening), they merely highlight that many interval BCs are identiﬁed in the second year of a biennial screening round, and is commensurate with the pattern of ﬁndings from radiological surveillance speciﬁcally that many interval BCs are true interval cancers. Additional data from radiological surveillance from Weber and colleagues13 indicates that the proportion of interval cancers that are true interval BCs increases in year 2 (relative to year 1) at biennial screening. Published in partnership with the Breast Cancer Research Foundation 11 npj Breast Cancer (2017) 12 Interval breast cancers in mammography screening N Houssami and K Hunter In addition, interval BC in women with non-dense breasts had increased 5-year survival HR (2.43, p = 0.001) compared to screen-detected BC in non-dense breasts, but this was not the case in women with dense breasts, in whom a difference in survival was not statistically evident between interval and screen-detected BC (5-year survival HR 1.41, p = 0.49) after adjustment for cancer size.51 Domingo and colleagues14, 50 conducted several studies examining the characteristics of interval BCs; one of these evaluated 2245 invasive BCs and clearly showed that interval BCs had more advanced tumours than screen-detected BCs (additional details by interval BC category shown in Table 3).14 In an earlier study of 228 invasive BCs diagnosed among Barcelona women aged 50–69 years, Domingo et al.50also found that disease-free survival rates (at 5 year follow-up from diagnosis) for npj Breast Cancer (2017) 12 Interval breast cancers in mammography screening N Houssami and K Hunter DISCUSSION little evidence on the effect of digital mammography on interval BC rates, limited to one study showing that the use of digital (compared to ﬁlm-screen) mammography was associated with a small but signiﬁcant reduction in interval BC rates.13 This overview of the epidemiological, radiological, and tumour characteristics of interval BCs—cancers that emerge following a negative mammographic screen—highlights key themes on interval BCs, which are summarised in Fig. 2. Interval BCs are an important consideration in population BC screening because they are indicative of screening quality hence evaluating these cancers may help identify areas for potential improvement, and because they represent a failure of screening to detect a BC that subsequently progresses to presentation. It is clear from radiological surveillance data summarised in our work that these cancers are not necessarily missed at mammography screening, with most studies reporting around 20–25% of interval BCs to be missed (false-negative) cases. While radiological surveillance has limitations inherent in retrospective re-interpretation of imaging, and radiological classiﬁcation of interval BCs is affected by the methodology used to perform mammographic review (Table 2), quantitative evidence shows that the majority of interval cancers are true interval or occult interval BCs that were not visible on the index screen. It should be acknowledged that radiological surveillance is not practiced in all screening settings, and the aim of summarising the evidence is not to advocate this form of surveillance, rather its ﬁndings can inform practice. For example, it seems likely that enhancing screen-reader skills would have relatively less effect in controlling interval BCs than, say, enhancing mammography technology or using alternate or additional technology to address the majority of cases that are not visible at the index mammography screen even in hindsight. Epidemiological monitoring of interval BC rates is a more widely performed surveillance in population mammography screening programs. Variability in the overall interval BC rates shown in Table 1 reﬂects the underlying BC risk in the population (which is also glimpsed in study-speciﬁc BC detection rates), the mix of initial and repeat screen rounds, and the length of the inter- screening interval. The variability due to the inter-screening interval is particularly evident where data are presented by yearly rates for biennial or triennial screening: our summary shows consistent evidence that rates in year 2 are at least twice those in year 1 (Table 1). ACKNOWLEDGEMENTS 25. Njor, S. H. et al. Mammography screening in the county of Fyn. November 1993- December 1999. APMIS Suppl 110, 1–33 (2003). N. Houssami receives research support through a National Breast Cancer Foundation (Australia), Breast Cancer Research Leadership Fellowship. 26. Renart-Vicens, G. et al. Evaluation of the interval cancer rate and its determinants on the Girona Health Region’s early breast cancer detection program. BMC Cancer 14, 558 (2014). COMPETING INTERESTS 27. Seigneurin, A., Exbrayat, C., Labarere, J. & Colonna, M. Comparison of interval breast cancer rates for two-versus single-view screening mammography: a population-based study. Breast 18, 284–288 (2009). The authors declare that they have no competing ﬁnancial interests. 28. Tornberg, S. et al. A pooled analysis of interval cancer rates in six European countries. Eur. J. Cancer Prev. 19, 87–93 (2010). Evidence tables 19. Fracheboud, J. et al. Interval cancers in the Dutch breast cancer screening pro- gramme. Br. J. Cancer. 81, 912–917 (1999). Each evidence table provided a summary of key ﬁndings from each study contributing information on interval BCs into at least one of three themes: epidemiologic measures, radiological surveillance and biologic features. For the evidence table on epidemiological measures (Table 1), studies were included if they reported data on interval BC rates (overall, or by year of inter-screen interval or by screening round) and also cancer detection rates because the latter provide complementary information about the study population and screening sensitivity. For the table on radiological surveillance (Table 2), studies were included if a radiological review and categorisation was performed allowing reporting of data on the frequency of one or more categories of interval BCs, and at minimum reporting data on false-negative (missed) interval cases. The evidence table on biological characteristics of interval BCs (Table 3) summarised data on tumour prognostic features, and also biomarkers where reported. 20. Heidinger, O. et al. The incidence of interval cancers in the German mammo- graphy screening program: results from the population-based cancer registry in North Rhine-Westphalia. Dtsch. Arztebl. Int 109, 781–787 (2012). 21. Henderson, L. M. et al. Breast cancer characteristics associated with digital versus ﬁlm-screen mammography for screen-detected and interval cancers. AJR. Am. J. Roentgenol. 205, 676–684 (2015). 22. Hofvind, S., Geller, B., Vacek, P. M., Thoresen, S. & Skaane, P. Using the European guidelines to evaluate the Norwegian Breast Cancer Screening Program. Eur. J. Epidemiol. 22, 447–455 (2007). 23. Kellen, E. et al. Interval cancers in the beginning years of the breast cancer screening programme in the Belgian province of Limburg. Acta Clin. Belg. 63, 179–184 (2008). 24. Lynge, E. Mammography screening for breast cancer in Copenhagen April 1991- March 1997. mammography screening evaluation group. APMIS Suppl 83, 1–44 (1998). Literature search 13. Weber, R. J. et al. Characteristics and prognosis of interval cancers after biennial screen-ﬁlm or full-ﬁeld digital screening mammography. Breast Cancer Res. Treat. 158, 471–483 (2016). Relevant publications were identiﬁed through a Medline literature search: we exploded the term ‘breast neoplasms’ to August 2016, and combined this with title-searching for ‘interval cancer$’ or ‘interval breast cancer$’. Study identiﬁcation focused on published work from 2006 onwards, given the above-stated aims of the review and the time frame from our previous evidence review,1 however, earlier studies were considered where data were reported in more recent publications.2, 4 Studies that provided information on population mammography screening allowing contribution into the evidence tables were included. Additional relevant studies were included in descriptive text if they provided key information on interval BCs that was not captured in the evidence table format. Studies that screened groups at increased risk of BC were not within the scope of the present review. Appendix 1 shows a ﬂow diagram of the literature search and study inclusion process. 14. Domingo, L. et al. Tumor phenotype and breast density in distinct categories of interval cancer: results of population-based mammography screening in Spain. Breast Cancer Res 16, R3 (2014). 15. Carbonaro, L. A. et al. Interval breast cancers: absolute and proportional incidence and blinded review in a community mammographic screening program. Eur. J. Radiol. 83, e84–e91 (2014). 16. Bennett, R. L., Sellars, S. J. & Moss, S. M. Interval cancers in the NHS breast cancer screening programme in England, Wales and Northern Ireland. Br. J. Cancer. 104, 571–577 (2011). 17. Bordas, P., Jonsson, H., Nystrom, L. & Lenner, P. Interval cancer incidence and episode sensitivity in the Norrbotten mammography screening programme, Sweden. J. Med. Screen. 16, 39–45 (2009). 18. Fong, Y. et al. The incidence and 10-year survival of interval breast cancers in Wales. Clin. Radiol. 69, e168–e172 (2014). METHODS This is a descriptive review based on a literature search and extraction of relevant information into evidence tables for each of three themes: epidemiologic measures, radiological surveillance and biology of interval cancers in population mammography screening. 11. Boyd, N. F. et al. Mammographic features associated with interval breast cancers in screening programs. Breast Cancer Res 16, 417 (2014). 12. Hofvind, S., Yankaskas, B. C., Bulliard, J. L., Klabunde, C. N. & Fracheboud, J. Comparing interval breast cancer rates in Norway and North Carolina: results and challenges. J. Med. Screen. 16, 131–139 (2009). DISCUSSION Radiological surveillance highlights that the majority of interval BCs represent true interval or occult interval BCs that were not visible on the index mammographic screen, with only around 20–25% of interval BCs reported to have been missed cases on mammographic review. Biological characteristics of interval BCs show that they have relatively worse tumour prognostic characteristics and survival outcomes than screen- detected BCs, but similar characteristics and prognosis to BCs Although there is a substantial body of knowledge on interval BC rates in mammography screening, as shown in Table 1, there was little direct evidence on mammography-based interventions that reduce interval BC rates. A limited number of studies identiﬁed in this review reported that two-view (vs. one-view) mammography was associated with signiﬁcant reductions in interval BC rates in population-based programs.27, 30 There was npj Breast Cancer (2017) 12 Published in partnership with the Breast Cancer Research Foundation Interval breast cancers in mammography screening N Houssami and K Hunter 12 occurring in non-screened women. There was limited evidence of the effect on interval BC frequency and outcomes following transition from ﬁlm to digital mammography screening. 8. 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https://openalex.org/W4321597992	https://appliednetsci.springeropen.com/counter/pdf/10.1007/s41109-023-00538-7	English	null	The persistent homology of genealogical networks	Applied network science	2,023	cc-by	15,395	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate- rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/licenses/by/4.0/. Abstract Genealogical networks (i.e. family trees) are of growing interest, with the largest known data sets now including well over one billion individuals. Interest in family history also supports an 8.5 billion dollar industry whose size is projected to double within 7 years [FutureWise report HC-1137]. Yet little mathematical attention has been paid to the complex network properties of genealogical networks, especially at large scales. The structure of genealogical networks is of particular interest due to the practice of forming unions, e.g. marriages, that are typically well outside one’s immediate family. In most other networks, including other social networks, no equivalent restriction exists on the distance at which relationships form. To study the effect this has on genealogi- cal networks we use persistent homology to identify and compare the structure of 101 genealogical and 31 other social networks. Specifically, we introduce the notion of a network’s persistence curve, which encodes the network’s set of persistence intervals. We find that the persistence curves of genealogical networks have a distinct structure when compared to other social networks. This difference in structure also extends to subnetworks of genealogical and social networks suggesting that, even with incom- plete data, persistent homology can be used to meaningfully analyze genealogical networks. Here we also describe how concepts from genealogical networks, such as common ancestor cycles, are represented using persistent homology. We expect that persistent homology tools will become increasingly important in genealogical explora- tion as popular interest in ancestry research continues to expand. Keywords: Persistent homology, Genealogical networks, Social networks, Persistence curves, Bottleneck distance Boyd et al. Applied Network Science (2023) 8:15 https://doi.org/10.1007/s41109-023-00538-7 Boyd et al. Applied Network Science (2023) 8:15 https://doi.org/10.1007/s41109-023-00538-7 Applied Network Science Open Access The persistent homology of genealogical networks Zachary M. Boyd, Nick Callor, Taylor Gledhill, Abigail Jenkins, Robert Snellman, Benjamin Webb and Raelynn Wonnacott Correspondence: zach_boyd@byu.edu Department of Mathematics, Brigham Young University, Provo, UT 84602, USA Introduction The study of genealogical networks, that is networks relating parents with children and spouses with each other through successive generations is of rapidly growing interest, both because of genealogy’s popular appeal and its applications in genetics (Kaplanis et al. 2018), sociology (Hamberger et al. 2011), population sciences (Rohde et al. 2004), and economics (Greenwood et al. 2014). Growing data availability of rich, temporally resolved data is also driving interest in genealogy. For example, FamilySearch has con- structed a human family tree with over 1.40 billion individuals, based on 2.21 billion sources, including 4.78 billion images (https://www.familysearch.org/en/newsroom/ Boyd et al. Applied Network Science (2023) 8:15 Page 2 of 29 company-facts). Popularization of DNA testing services and increasing availability of audio sources, geographic tags, occupation metadata, and migration records combine to make genealogical networks some of the largest, most richly featured, geospatially embedded temporal networks in existence. Examples of relevant academic studies include methods for automatically constructing networks from documents (Malmi et al. 2018; Bloothooft et al. 2015), analyzing marriage patterns (Greenwood et al. 2014), structured population modeling, branching processes (Hey and Machado 2003), and biconnected components (Hamberger et al. 2011; Hage and Harary 1983). Of particular interest to us are works that study distance to recent common ancestors, both theoreti- cally and via simulation (e.g. Chang 1999; Rohde et al. 2004). A growing body of litera- ture also uses genealogical networks for genetic inference, as in (Kaplanis et al. 2018). Related to these genealogical endeavors, a major goal of network science is to describe the structure of such real-world networks. In this paper, we consider persistent homol- ogy as a tool to both analyze and explore the structure of genealogical networks. Per- sistent homology, roughly speaking, is a method of representing voids or gaps in the structure of a network, that distinguishes how significant these voids are to the overall network structure. Persistent homology can be used to compare these voids across two networks without requiring a correspondence between the individual vertices or edges, or even requiring the networks to be the same size. The basic idea involves “filling in” the network with simplices (points, edges, triangles, tetrahedra, etc.) and keeping track of how the network changes as we do so (see "Persistent homology of networks" for details). Some similar applications of persistent homology in the study of networks include Carstens and Horadam (2013), Kannan et al. Introduction (2019); Horak(2009); Petri et al. (2013). The collaboration networks studied in Carstens and Horadam (2013) are similar to the social networks that we use for comparison in this paper, though our focus is primar- ily on distinguishing these from genealogical networks. Both Kannan et al. (2019) and Horak (2009) apply persistent homology techniques to general randomized networks of various forms. It is also possible to vary the technique for generating a topological object from a network, as in Petri et al. (2013) where three methods are compared. We also recommend Aktas et al. (2019) and Otter et al. (2017) as good overviews of the general methods of applying persistent homology. For this paper, our method of constructing a topological representative for each network follows the same general pattern as the work cited above. However, we also acknowledge the wide variety of alternatives for encoding such information. Chazal et al. (2013) and Vandaele et al. (2018) encode their information as point-clouds rather than graphs. A higher-dimensional version of persistent homology is presented in Blumberg and Lesnick (2020), which may permit the inclusion of time-varying networks. Finally, the formulation in Arafat et al. (2020) may allow for better analysis of corrupted or too- large datasets. We also wish to bring attention to four particular applications that demonstrate the versatility of persistent homology. In each of these applications, persistent homology has been used to identify structural voids in data and then to associate these voids to recognizable features in the underlying networks. It is the latter use that we wish to emphasize. Robins et al. Robins et al. (2016) have shown that voids found using per- sistent homology correspond to percolating spheres in a porous material. In Lee et al. Boyd et al. Applied Network Science (2023) 8:15 Page 3 of 29 (2012), structural voids arise when several groups of neurons are strongly connected sequentially, but out-of-sequence pairs are only weakly connected. In these neurologi- cal networks, persistent homology provides a way to identify and classify these differ- ent sequences as well as quantify the strength of these connections. The application in Duman and Pirim (2018) provides a method for extending traditional genetic analysis tools to a parameterized family of datasets by constructing an appropriate topological object. Lastly, Mattia et al. (2016) shows that structural voids or gaps can also represent much more abstract concepts. 1 In order to be inclusive of various relevant relationships in this paper, we use the word “union” to describe not only legal marriages and common law marriages but also some others, including any relationship that produced children. Introduction Applied Network Science (2023) 8:15 Page 4 of 29 Page 4 of 29 • Report evidence that persistent homology methods work well even in the presence of incomplete data. This is particularly relevant given that genealogical data is often, if not necessarily, incomplete. • Report evidence that persistent homology methods work well even in the presence of incomplete data. This is particularly relevant given that genealogical data is often, if not necessarily, incomplete. Throughout the paper, examples from family networks are contrasted with other social networks to highlight the unique features of genealogical networks from a persistent homology point of view. The paper is organized as follows. In "Background: genealogical and social networks" we describe both genealogical and social networks. In "Persistent homology of net- works" we define the persistent homology of a network and introduce the notion of persistence curves. In "Comparing networks using persistent homology" we define the bottleneck distance and show how both this distance and persistence curves can be used to compare networks. In "Results" we describe the genealogical and social data sets we use in our study and give our experimental results in "Results". In "Results" also includes a discussion of how certain structural features of social and genealogical networks are represented using persistent homology. In "Conclusion" we summarize our results and conclude with a discussion regarding the use of persistent homology as a tool for ana- lyzing general network structure and recovering network features. Throughout we give examples of each of the concepts we introduce. Introduction In this case persistent voids are shown to correspond to the atonality in music compositions. Intuitively, the voids or gaps in genealogical networks should be quite different when compared with other networks, such as social networks, since unions1 (such as mar- riages) in genealogical networks typically form at specific distances, rather than through other mechanisms e.g. triadic closure. That is, distances between individuals who form unions are typically not too small or too large (see "Background: genealogical and social networks"). In contrast, in other social networks, new connections can form at any dis- tance but are often quite small (Sintos and Tsaparas 2014). This difference in network growth between genealogical and other social networks causes differences in network topology that are reflected in the network’s persistent homology. Thus persistent homol- ogy is a useful descriptive tool for exploring and modeling the structure of genealogical networks. Here, we propose a new method for representing persistent homology, which we call a persistence curve (see "Comparing networks using persistent homology"). The persistence curves of many genealogical networks are very similar to each other, and importantly the persistence curves of subsets of genealogical networks, that is, sampled genealogical networks, are also similar to the persistence curves of unsampled genea- logical networks (see "Results"). To give our study of genealogical networks context we also study the persistent homol- ogy of social networks. We find that the same result holds for the social networks we consider, in that the persistence curves of social networks show a common pattern and the persistence curves for social and sampled social networks are similar (see "Results"). We confirm our analysis using another tool for comparing persistent homologies, the bottleneck distance, which is also capable of detecting and differentiating the distinct homology patterns between genealogical and other social networks. In summary, we make the following contributions: In summary, we make the following contributions: • Introduce the notion of a persistence curve and introduce the use of this together with the bottleneck distance as a tool for the analysis of general networks. • Report the distinct persistent homology structure of genealogical networks using both persistence curves and the bottleneck distance. • Link this structure to genealogically relevant concepts. • Similarly, report the distinct persistence homology structure of social networks and compare this to the structure of genealogical networks. Boyd et al. Background: genealogical and social networks We represent genealogical networks with a graph G = (V , E) , where V = {1, 2, . . . , n} are the individuals within the network, and E are the (genealogical) relationships. These relationships consist of both parent–child edges and spouse (or more generally union) edges. For the sake of simplicity, these edges are considered to be undirected. We note that the structure of a genealogical network is often thought of as being “tree- like”, since genealogical networks are often constructed from an individual, their parents, their grandparents, and so on, ignoring union edges. The result is a tree, i.e. a connected acyclic graph, if we create only a few generations of the family. However, full genealogi- cal networks are not trees due to the presence, for example, of triangles consisting of two parents and a child (with the two parent–child edges and one union edge). Because of the frequency of such cycles and the fact that they are the smallest possible cycles, we refer to them as trivial cycles. The other typical familial cycle, or cycle found within a family consisting of two parents and some number of children, is a cycle of length four consisting of two parents and two children. Although familial cycles are ubiquitous in genealogical networks, they are not the only cycles that can form. Going far enough through an individual’s ancestors, it is often possible to find a nearest common ancestor, i.e., a common ancestor of one’s father and mother. If such an ancestor exists (and it usually does exist), then the genealogical net- work has a nontrivial cycle. We refer to this as a common ancestor cycle, which consists of only parent–child edges. Other nontrivial cycles are possible in genealogical networks via unions. For instance, a “double cousins” relationship occurs when two siblings from one family form unions with two siblings from another family. The result is a union cycle, or a cycle that contains only union edges and the parent–child edges connecting siblings. Boyd et al. Applied Network Science (2023) 8:15 Page 5 of 29 Boyd et al. Applied Network Science Fig. 1 Left: The largest connected component of the Tikopia genealogical network consisting of 288 individuals from the island of Tikopia in Polynesia from the year 1930 to 2008, is shown (https://www.kinso urces.net/browser/datasets.xhtml). Parent-child edges are shown in blue and union edges are shown in red. Fig. 3 Left: Shown in orange is the distribution of the lengths of the cycles forming a basis of the nonfamilial cycle lengths in the San Marino (SM) genealogical network. The analogous distribution of cycle lengths is shown in blue for all cycles in the Deezer Europe (DE) social network. Center: Shown in orange is again the basis cycle length distribution of the San Marino genealogical network. In red is the distribution of the basis cycle lengths averaged over ten realizations of the (loopy, multi-edged) configuration model on the San Marino network. Since the configuration model generates graphs with the same degree distribution as the SM network, this panel indicates that SM’s longer cycles do not arise simply from the degree distribution. Right: Shown in blue is again the basis cycle length distribution of the Deezer social network. In green is the distribution of the basis cycle lengths averaged over ten realizations of the configuration model on the Deezer social network. For this social network, the cycle length distribution can be mostly explained by the degree distribution alone fraction of unions that form at a specific distance. Noticeably, few unions form at dis- tances less than five with the large majority of distance falling between 5 and 10. i The observation that genealogical networks have large extended cycles is illustrated in Fig. 3. Shown left in orange is the distribution of cycle lengths of the San Marino genealogical network, a network of the population of the Republic of San Marino from the 15th to the end of the 19th century (https://www.kinsources.net/browser/datasets. xhtml). In this network, which consists of 28,586 individuals, there are 7,146 familial cycles of length three and 8,636 familial cycles of length four. These are omitted in the figure so we can observe the lengths of the cycles forming a basis of nonfamilial cycles in the network. For the sake of contrast, in blue is the distribution of cycle lengths in a basis of the cycles found in the Deezer Europe social network, consisting of 28,281 individuals. Here, similar to genealogical networks, a social network is represented by a graph G = (V , E) where the vertices V also represent individuals. The difference is that in a social network the edges represent some type of social interaction(s). Background: genealogical and social networks Right: The largest connected component of the Residence Hall social network consisting of 217 individuals and their friendships from the Australian National University campus is shown (Residence 2022) Fig. 1 Left: The largest connected component of the Tikopia genealogical network consisting of 288 individuals from the island of Tikopia in Polynesia from the year 1930 to 2008, is shown (https://www.kinso urces.net/browser/datasets.xhtml). Parent-child edges are shown in blue and union edges are shown in red. Right: The largest connected component of the Residence Hall social network consisting of 217 individuals and their friendships from the Australian National University campus is shown (Residence 2022) Fig. 2 The histogram representing the finite “distance to union” distances is shown where data is collected from 104 genealogical networks from kinsources.net. The height of each bar represents the fraction of unions that form at a specific distance Fig. 2 The histogram representing the finite “distance to union” distances is shown where data is collected from 104 genealogical networks from kinsources.net. The height of each bar represents the fraction of unions that form at a specific distance Fig. 2 The histogram representing the finite “distance to union” distances is shown where data is collected from 104 genealogical networks from kinsources.net. The height of each bar represents the fraction of unions that form at a specific distance In genealogical networks, union and parent–child edges can combine in any number of ways to create complex non-tree structures (see Fig. 1 left). A feature that is particular to genealogical networks is that union edges typically form at specific distances within these networks. Here the distance d(i, j) between i and j is the shortest path distance between these individuals if such a path exists. Otherwise, it is infinite. In a genealogical network we refer to the distance between two individu- als before they form a union as the couple’s distance to union. For cultural, genetic, and other reasons these distance are typically not small, i.e. usually larger than four. Conse- quently, genealogical networks do not typically have small nonfamilial cycles and often have large extended cycles. This is illustrated in Fig. 2 where distance to union data is collected from 104 publicly available genealogical networks given in Table 2 in the Appendix. Here familial cycles are omitted and the height of each bar represents the Page 6 of 29 Boyd et al. Applied Network Science (2023) 8:15 Boyd et al. Background: genealogical and social networks Applied Network Science The Deezer network is an online music streaming platform whose social network represents indi- viduals in Europe who use the platform where edges represent mutual user-follower relationships. Noticeably, the San Marino network has relatively few nonfamilial basis cycles under length ten but quite a few cycles with lengths greater than thirty. In contrast, the Deezer social network has a much tighter distribution of basis cycles ranging from roughly five to fifteen in length. i To understand the extent to which these cycle distributions are related to the local structure of the associated networks we compare these to the cycle distribution of the associated configuration models of these two networks, respectively. The configuration model is a model for generating random networks with a given degree sequence (New- man 2006). Taking the degree sequences from both the San Marino genealogical and Deezer social network, we create ten versions of these networks each with the same degree sequences. The result of averaging the basis cycle length distributions of these versions of the San Marino and Deezer networks is shown in Fig. 3 (center and right in Boyd et al. Applied Network Science (2023) 8:15 Page 7 of 29 red and green, respectively). While the cycle distribution for the San Marino network is quite different from what the configuration model produces, the Deezer social net- work is quite similar to the distribution predicted by its configuration model. This sug- gests that much of the cycle structure in the Deezer social network is dominated by local interactions, whereas the cycles in the San Marino genealogical network are affected by nonlocal mechanisms that form the network. This includes, presumably, the nonlocal distance to union phenomena described above. The relations we see in Fig. 3 between the cycle length distribution for the San Marino genealogical network and the Deezer social network are typical of the genealogical and social networks we consider in "Data". This suggests that cycle length distribution is a feature that can be used to distinguish genealogical from social networks. Specifically, when we consider two networks with a similar number of cycles, genealogical networks have a much wider distribution of cycle lengths than social networks. However, the method used to calculate the cycle length distribution in Fig. 3 does not provide any further insight into this phenomenon. This limitation motivates us to apply tools from persistent homology which provides ways to describe and measure the relation between any two network cycles. The additional structure that can be obtained by these meth- ods allow us to further distinguish the structure of genealogical and social networks (see "Network comparison using bottleneck distance") and to relate the structural differences demonstrated in Fig. 3 to mechanisms that produce genealogical and social networks, respectively (see "Connections"). Persistent homology of networks Persistent homology provides a method for studying cycles in a network. For the pur- poses of this paper, a brief explanation of persistent homology will be given from the context of simplicial homology. For a more in-depth treatment of simplicial homology, see Chapter 2.1 of Hatcher (2002). For those readers who are either familiar with the basics of persistent homology or who wish to skip the following technical discussion it is possible to proceed to "Data" where we discuss the social and genealogical networks we analyze. For a network given by a graph G = (V , E) we define the distance matrix D(G) = [dij] to have entries dij = d(i, j) , which is the length of the shortest path between individual i and j. For each value δ that appears in the distance matrix D(G), we form a simplicial complex Gδ as follows. The set of 0-simplices is equivalent to the set of vertices of G, where each 0-simplex is identified with a single vertex. Since the distinction between 0-simplices and vertices is purely formal, we will use the terms 0-simplex and vertex interchangeably, and the 0-simplices will be indexed the same way as the vertices. The set of 1-simplices Eδ corresponds to the set of edges {i, j} such that d(i, j) ≤δ , where the edge {i, j} is identified with the 1-simplex formed by i and j . Again the distinction here is unnecessary for our present discussion, so we will use the same notation for 1-sim- plices and edges. However, the simplicial complex Gδ may also contain objects that do not have equivalent representatives in the graph G, namely the n-simplices for n ≥2 . For each integer n ≥2 , the set of n-simplices in Gδ consists of all n-simplices [a0 a1 . . . an] Boyd et al. Applied Network Science (2023) 8:15 (2023) 8:15 Page 8 of 29 Fig. 4 The hexagonal network G = G1 in Example 3.1 is filled in as i increases from 0 to 3. This produces the simplicial complexes G0, G1, G2, G3 shown left to right i Fig. 4 The hexagonal network G = G1 in Example 3.1 is filled in as i increases from 0 to 3. This produces the simplicial complexes G0, G1, G2, G3 shown left to right such that d(ai, aj) ≤δ for 0 ≤i < j ≤n . Persistent homology of networks That is, Gδ includes an n-simplex σ if each ver- tex listed in σ is within δ of every vertex listed in σ. In order to simplify our remaining definitions, we extend our definition of Gδ to include all non-negative integers. For i ≥0 , let δi be the greatest entry of D(G) such that δi ≤i . Let Gi = Gδi . This definition together with our construction of Gδ ensures the fol- lowing three important properties are true for all Gi . 1. For i < j , Gi is a subcomplex of Gj , i.e. every simplex of Gi is a simplex of Gj. 1. For i < j , Gi is a subcomplex of Gj , i.e. every simplex of Gi is a simplex of Gj. 2. For i ≥1 , there exists a subcomplex of Gi that can be identified with the original graph G. 2. For i ≥1 , there exists a subcomplex of Gi that can be identified with the original graph G. 3. Since G is finite, let M = maxij d(i, j) , then, for all i ≥M , Gi = GM. Example 3.1 (Hexagonal Network) Consider the hexagonal network G = (V , E) with six vertices, form- ing a single cycle, shown in Fig. 4b. This network has the distance matrix (Hexagonal Network) Consider the hexagonal network G = (V , E) with six vertices, form- ing a single cycle, shown in Fig. 4b. This network has the distance matrix D(G) = 0 1 2 3 2 1 1 0 1 2 3 2 2 1 0 1 2 3 3 2 1 0 1 2 2 3 2 1 0 1 1 2 3 2 1 0 . For the values i = 0 , 1, 2, 3, we form four simplicial complexes, G0 , G1 , G2 , and G3 where we let Gi = (Vi, Ei) . For i = 0 , E0 is empty. Thus, G0 consists of six vertices. For i = 1 the set E1 contains the six edges that form the network’s single cycle, so G1 = G . This graph has no trivial cycles (i.e., triangles), so G1 contains no simplices of dimension greater than 1 (i.e., no n-simplices for n > 1 ). For i = 2 the set E2 gains six additional edges. We also now have eight trivial cycles. Each of these cycles is the boundary of a 2-simplex, so G2 contains these eight 2-simplices as well. However, no subset of these 2-simplices forms the boundary of a 3-simplex, so G2 has no simplices of dimension greater than 2. For i = 3 the set E3 contains all possible edges between the vertices of G, so all possible triv- ial cycles are present. Additionally, all possible 2-simplices, and hence all possible n-sim- plices, are also present in G3 . In particular, G3 is a 6-simplex with its boundary. Since M = 3 is the largest value we see in the distance matrix, then Gi = G3 for i ∈Z , i > 3. Boyd et al. Applied Network Science (2023) 8:15 Page 9 of 29 Boyd et al. Applied Network Science (2023) 8:15 The persistent homology of the network G measures how the homology of Gi changes as i increases. If certain features can be identified across multiple values of i, we say they persist. Example 3.1 Intuitively, features that arise from the actual network structure should persist for many values of i, while features that arise because of measurement error, ‘noise’, should only appear sporadically. The Stability Theorem (the main theorem of Cohen- Steiner et al. (2007)) states that if the error in measuring a network is bounded by some constant C, then the persistent homology of the true network and the persistent homol- ogy of the noisy network will differ by at most C. We will make this statement more pre- cise in "Persistence diagrams and bottleneck distance". Here we give a formal definition of persistent homology in terms of simplicial homol- ogy, which we will immediately follow this with equivalent definitions in the context of networks. We use Hp(Gi) to denote the dimension-p simplicial homology of the simpli- cial complex Gi with coefficients in Z2 , as Hp(X) is a vector space of Z2. Definition 1 (pth Persistent Homology) For a graph G, and integers i, j with 0 ≤i ≤j , let the function φi,j : Hp(Gi) →Hp(Gj) be the linear map induced by the inclusion Gi →Gj . The pth persistent homology of G, PHp(G) is the pair ({Hp(Gi)}i≥0, {φi,j}0≤i<j). Our analysis in the "Comparing networks using persistent homology" and "Results" sections only requires the first few dimensions of persistent homology to distinguish the genealogical and social networks we consider. In order to better understand what persis- tent homology calculates, in what follows we will provide equivalent definitions for PH0 , PH1 , and PH2 using network concepts. We also illustrate how these definitions apply to the hexagonal network in Fig. 4b. (See Examples 3.3, 3.4, and 3.5 for PH0 , PH1 , and PH2 ; respectively.) Definition 2 (Births and Deaths) Let G = (V , E) be a network with simplicial com- plexes G0, G1, G2, · · · . The pth persistent homology of G provides maps φi,j between the pth homology of Gi and the pth homology of Gj . Suppose that basis elements have been chosen for each Hp(Gi) so that if α is a basis element of Hp(Gi) , then φi,j(α) is either trivial in Hp(Gj) or a basis element of Hp(Gj) . The birth of a basis element α ∈Hp(Gj) is the minimum index i such that α = φi,j(ˆα) for some basis element ˆα ∈Gi . Example 3.1 The death of α is the minimum index k such that φj,k(α) is trivial. Example 3.3 We now consider PH0(G) for the hexagonal network G in Fig. 4, with G0 , G1 , G2 , and G3 in the same figure. Recall that G has six distinct vertices forming one connected component. If we take any numbering of the vertices, V = {1, 2, 3, 4, 5, 6} , then H0(G0) ∼= Z6 2 , which is equivalent to the vector space over Z2 with basis V. For i > 0 , H0(Gi) ∼= Z2 , which is equivalent to the vector space over Z2 with basis {1} . For any v ∈V , since i = 0 is the first time we see v, we call this the birth of v. At i = 1 , since we have removed all vertices except 1 from the basis, we say this is the death of those five 0-simplices. Since 1 will always be in the basis for Gi , the death of 1 is said to be ∞. Definition 4 (Representing Persistent Homology: Dimension 1) Let G = (V , E) be a net- work with one connected component. For each i ≥0 , we can identify the basis of H1(Gi) with a set Ci of cycles in Gi . The Fundamental Theorem of Persistent Homology allows us to choose these cycles so that if σ is a cycle in Ci , then exactly one of the following is true for any integer j ≥0 : 1. σ does not exist in Gj , in which case j < i, 1. σ does not exist in Gj , in which case j < i, 2. σ is trivial or null-homotopic in Gj , in which case i < j, 2. σ is trivial or null-homotopic in Gj , in which case i < j, 3. σ is a cycle in Cj. Thus, we will refer to the cycles in i≥0 Ci as the representatives of PH1(G) . (Again, PH1(G) is actually much larger than this. These are actually representatives of equiva- lence classes that form a basis for PH1(G) as a vector space.) We note that C0 is always empty, since there are no edges in G0 . Furthermore, rank(H1(Gi)) = \|Ci\| for all i ≥0 . Because of the construction of the Gi all representa- tives of PH1(G) will be present in G1 . Remark 3.2 Those already familiar with persistent homology will find that the preceding definition is somewhat nonstandard, although it is equivalent to the standard definition. We have taken this approach to reduce the notation burden on non-specialist readers. We have done similarly with some of the other persistent homology definitions. We will demonstrate how to choose such representatives for H0 , H1 , and H2 in the fol- lowing definitions. Given such representatives, though, the maps φi,j and φj,k are simply the maps on homology induced by the inclusion maps Gi ⊂Gj ⊂Gk . That is, if a repre- sents α ∈Hp(Gi) , then a also represents φi,j(α) . The Fundamental Theorem of Persistent Boyd et al. Applied Network Science (2023) 8:15 Page 10 of 29 Boyd et al. Applied Network Science Homology ensures that we can choose a single representative that corresponds to α ∈Hp(Gj) , ˆα ∈Hp(Gi) , and φj,k(α) ∈Hp(Gk) . The birth of α is then just the first Gi in which the representative exists, and the death of α is the first Gk in which the representa- tive is null-homotopic i.e., homotopic to a trivial cycle. Homology ensures that we can choose a single representative that corresponds to α ∈Hp(Gj) , ˆα ∈Hp(Gi) , and φj,k(α) ∈Hp(Gk) . The birth of α is then just the first Gi in which the representative exists, and the death of α is the first Gk in which the representa- tive is null-homotopic i.e., homotopic to a trivial cycle. Definition 3 (Representing Persistent Homology: Dimension 0) Let G = (V , E) be a network with vertices V = {1, 2, . . . , n} which form k connected components. Then H0(G0) ∼= Zn 2 , so we can identify the basis for H0(G0) with the set of all n vertices. Like- wise, we may choose k vertices, one from each connected component, to represent the basis for H0(Gi) ∼= Zk 2 for i ≥1 . Thus, we will refer to the vertices of G as representa- tives of PH0(G) . (In fact, PH0(G) is a vector space whose basis elements are equivalence classes of formal sums of 0-simplices.) Example 3.4 we now consider PH1(G) for the hexagonal network G in Fig. 4b. In both Fig. 4a and 4b we see that G0 has no cycles, G1 has exactly one cycle, and that the cycle in G1 is non-trivial. In Figs. 5a and 5b, we have indicated some of the cycles in G2 , namely the cycles 1,2,3,1; 3,4,5,3; 1,5,6,1; and 1,3,5,1 in Fig. 5a and the cycle 1,2,3,5,1 in Fig. 5b. In fact, Fig. 5c shows us that G2 is an octahedron and therefore every cycle in G2 is either trivial or null- homotopic. Finally, G3 contains even more cycles than G2 , such as 1,3,6,1; but these are all null-homotopic since G3 also contains every possible 2-simplex for six vertices. Therefore, PH1(G) has only one representative, the cycle 1,2,3,4,5,6,1; which appears in G1 , so we say that t = 1 is the birth of the cycle. The cycle is null-homotopic in G2 , so t = 2 is the death of the cycle. We now turn our attention to PH2(G) , but in order to represent PH2(G) we need to introduce some new structure for the induced graphs. A triangle [a b c] in Gi is a set of three vertices, a, b, and c, that form a trivial cycle in Gi . That is, the edges {a, b} , {b, c} , and {a, c} are all present in Gi . A closed surface in Gi is a set of distinct triangles so that for each [a b c] in the set there is exactly one other triangle [a b d] also in the set. A closed surface in Gi is trivial if the corresponding set of 2-simplices is null- homotopic in Gi . That is, the closed surface is “filled in” by some collection of 3-sim- plices in Gi . For example, the octahedron in Fig. 5c is a non-trivial closed surface in G2 because there are no 3-simplices in G2 . In G3 , however, we add edges between ver- tices at distance 3. In turn, we gain several 3-simplices, including [1 2 3 6] , [1 3 5 6] , [3 4 5 6] , and [2 3 4 6] . Figure 5d shows three of these 3-simplices to demonstrate how the closed surface from G2 is filled in by all four. Example 3.3 One can think of the representatives of PH1(G) as representing “large” cycles. More specifically, if a cycle σ is contained in s≤i≤t Ci , then it must have a diameter of at least t and at least one pair of consecutive vertices distance s apart. Boyd et al. Applied Network Science (2023) 8:15 (2023) 8:15 Boyd et al. Applied Network Science Page 11 of 29 Fig. 5 A visual depiction of simplices and cycles present in G2 . Left: Four trivial cycles filled by individual 2-simplices: [1 2 3] , [3 4 5] , [1 5 6] , and [ 1 3 5] . Center Left: A non-trivial, but null-homotopic cycle, 1, 2, 3, 5, 1 filled in by two 2-simplices [1 2 3] and [1 3 5] . Center Right: All eight 2-simplices represented as the faces of a regular octahedron. Right: The closed surface of G2 is filled in by four 3-simplices [1 2 3 6], [1 3 5 6], [3 4 5 6], [2 3 4 6] Fig. 5 A visual depiction of simplices and cycles present in G2 . Left: Four trivial cycles filled by individual 2-simplices: [1 2 3] , [3 4 5] , [1 5 6] , and [ 1 3 5] . Center Left: A non-trivial, but null-homotopic cycle, 1, 2, 3, 5, 1 filled in by two 2-simplices [1 2 3] and [1 3 5] . Center Right: All eight 2-simplices represented as the faces of a regular octahedron. Right: The closed surface of G2 is filled in by four 3-simplices [1 2 3 6], [1 3 5 6], [3 4 5 6], [2 3 4 6] Fig. 5 A visual depiction of simplices and cycles present in G2 . Left: Four trivial cycles filled by individual 2-simplices: [1 2 3] , [3 4 5] , [1 5 6] , and [ 1 3 5] . Center Left: A non-trivial, but null-homotopic cycle, 1, 2, 3, 5, 1 filled in by two 2-simplices [1 2 3] and [1 3 5] . Center Right: All eight 2-simplices represented as the faces of a regular octahedron. Right: The closed surface of G2 is filled in by four 3-simplices [1 2 3 6], [1 3 5 6], [3 4 5 6], [2 3 4 6] Example 3.4 Definition 5 (Representing Persistent Homology: Dimension 2) Let G = (V , E) be a network with one connected component. For each i ≥0 , we can identify the basis for H2(Gi) with a set Si of non-trivial closed surfaces in Gi . The Fundamental Theorem of Persistent Homology allows us to choose these representatives so that if σ is a closed surface in Si , then exactly one of the following is true for any integer j ≥0 Page 12 of 29 Boyd et al. Applied Network Science (2023) 8:15 Boyd et al. Applied Network Science 1. σ does not exist in Gj , in which case j < i, 1. σ does not exist in Gj , in which case j < i, 2. σ is trivial in Gj , in which case i < j, 1. σ does not exist in Gj , in which case j < i, 1. σ does not exist in Gj , in which case j < i 2. σ is trivial in Gj , in which case i < j, 2. σ is trivial in Gj , in which case i < j, 3. σ is a cycle in Sj. 3. σ is a cycle in Sj. 3. σ is a cycle in Sj. Thus we will refer to the closed surfaces in i≥0 Si as the representatives of PH2(G). The geometric intuition for PH2(G) is similar to that of PH1(G) in identifying large ‘voids’ in G. If σ ∈ s≤i≤t Si , then σ is a closed surface with diameter at least t. The value of s is harder to describe, but is related to the density of vertices. Example 3.5 We now consider PH2(G) for the hexagonal graph G in Example 3.1. Recall from Exam- ple 3.4 that G0 and G1 have no trivial cycles, and therefore contain no closed surfaces. We can see in Fig. 5 that G2 has exactly one closed surface and it must be non-trivial, since there are no 3-simplices. Finally, G3 has many closed surfaces, but because it contains every possible 3-simplex on six vertices, these are all trivial. Therefore, PH2(G) has only one representative, the octahedral closed surface in G2 . This surface first appears in G2 , so t = 2 is its birth, and the surface is filled by a solid in G3 , so t = 3 is its death. Definition 6 (Persistence Intervals) Recall that the birth of a representative σ ∈PHp(G) (vertex, cycle, or closed surface) of the persistent homology of a network G is the small- est integer i so that σ ∈Gi , and the death of σ is the largest integer j so that σ ∈Gj−1 and σ is trivial in Gk for k ≥j , if such an integer exists. The persistence interval for σ is [a, b) , where a and b are the birth and death of σ , respectively. This represents the set of all parameter values i for which the equivalence class corresponding to σ is a non-trivial element of Hp(Gi) . The persistence of σ is b −a. PH2(G) identifies large ‘voids’ in G. Specifically, the death of σ is a lower bound on the diameter of σ. Given the representatives chosen in Definitions 3, 4, 5, and 6, we have the following three observations regarding the persistent homology of a finite, undirected, unweighted graph G: (i) If G has n vertices, then PH0(G) will have exactly n persistence intervals, with exactly one [0, ∞) interval for each connected component and the rest will be [0, 1) intervals. (ii) In dimension 1, PH1(G) describes the number and sizes of the non-trivial cycles in the original network. The persistence intervals will all be of the form [1, b) for some integer b > 1 . The value of b is related to the diameter of the corresponding cycle. In the networks we have studied, we note that a persistence interval [1, b) in PH1(G) corresponds to a simple cycle with between 3b −2 and 3b vertices, inclu- sive. (iii) In dimension 2, the voids we detect in PH2(G) tell us about the nontrivial intersec- tions of cycles. Such intersections are hard to visualize but, roughly speaking, a representative in PH2(G) can only form if several large cycles intersect each other pairwise. Example 3.6i We now finish our consideration of the persistent homology of G from Fig. 4b. Recall from Example 3.3 that PH0(G) has six representatives. These all have birth t = 0 . Five of these have a death of t = 1 , and one of these has a death of ∞ . Therefore the persistence inter- vals for PH0(G) are [0, 1) × 5 and [0, ∞) × 1. From Example 3.4, we know PH1(G) has one representative, with birth t = 1 and death t = 2 . Therefore the corresponding persistence interval is [1, 2) . Note that the diameter of the cycle is 3 and every pair of consecutive vertices is distance 1 apart. This follows the idea mentioned earlier that the representatives of PH1(G) indicate ‘large’ cycles. Specifi- cally, the diameter of σ is at least the death of σ , and the birth of σ is the maximum dis- tance between consecutive vertices. From Example 3.5, PH2(G) has one representative, with birth t = 2 and death t = 3 . Therefore, the persistence interval for that element is [2, 3) . Note that the diameter of the corresponding set of vertices is 3 in G. This also follows the idea mentioned earlier that Page 13 of 29 Boyd et al. Applied Network Science (2023) 8:15 Boyd et al. Applied Network Science Table 1 The persistence intervals of the Tikopia genealogical network and the hexagon network are shown Here the notation [a, b) × k indicates that the network has k persistence intervals [a, b). The corresponding persistence diagrams are shown in Fig. 6 and the corresponding persistence curve for the Tikopia network is shown in Fig. 7 Dimension Interval Type and Persistence Tikopia Hexagon Dimension 0 [0, ∞) × 8, [0, 1) × 286 [0, ∞) × 1, [0, 1) × 1 Dimension 1 [1, 2) × 16 , [1, 3) × 19 , [1, 4) × 5 , [1, 5) × 3 , [1, 6) × 2 , [1, 7) × 1 [1, 2) × 1 Dimension 2 [2, 3) × 4 , [3, 4) × 11 , [4, 5) × 12 , [5, 6) × 4 , [6, 7) × 5 , [7, 8) × 1 , [8, 9) × 1 [2, 3) × 1 Here the notation [a, b) × k indicates that the network has k persistence intervals [a, b). Example 3.6i The corresponding persistence diagrams are shown in Fig. 6 and the corresponding persistence curve for the Tikopia network is shown in Fig. 7 PH2(G) identifies large ‘voids’ in G. Specifically, the death of σ is a lower bound on the diameter of σ. Persistence diagrams and bottleneck distance One common way to represent persistence intervals is to plot them as points in R × (R ∪{∞}) , which is typically referred to as a persistence diagram. While this method of visualizing a network’s persistent homology does not indicate how often a given persistence interval occurs, it does provide information on what kind of persis- tence intervals occur for a given network. Definition 7 (Persistence Diagrams) Let PHp(G) be the pth persistent homology of a network G. The persistence diagram for PHp(G) is a multiset of points in R × (R ∪{∞}) defined as follows. • For each σ ∈PHp(G) with persistence interval [a, b) , we include one copy of the point (a, b). For each c ∈R , we include infinitely many copies of the point (c, c). • For each c ∈R , we include infinitely many copies of the point (c, c). Note that we include the points (a, a) to represent features in G that are considered trivial in PHp(G) , such as cycles consisting of exactly three vertices. This inclusion is necessary for us to define a meaningful metric on the space of persistence diagrams. The metric we use here is called the bottleneck distance. Definition 8 (Bottleneck Distance) Let S1 and S2 be persistence diagrams for two graphs G and H, respectively. Let η range over the set of bijections from S1 to S2 . Then the bottleneck distance between S1 and S2 is dB(S1, S2) = inf η sup x∈S1 x −η(x)∞. dB(S1, S2) = inf η sup x∈S1 x −η(x)∞. The Fundamental Theorem of Persistent Homology (introduced in Zomorodian and Carlsson (2005), explained well in Otter et al. (2017) and Aktas et al. (2019)) ensures that if two graphs are isomorphic, the corresponding persistence diagrams will be equal, and thus the bottleneck distance will be 0. However, it is possible for non-iso- morphic graphs to have identical persistence diagrams. Comparing networks using persistent homology In this section we demonstrate how methods based on persistent homology can be used to compare different networks. The two methods we introduce in this paper are based on using (a) the bottleneck distance and (b) the persistence curves of a given set of networks. Both (a) and (b) rely on first computing persistence intervals then analyzing the differences in these intervals. f The two networks we consider throughout this section to demonstrate these meth- ods are the Tikopia genealogical network from Fig. 1 (left) and the hexagonal net- work from Fig. 4. The persistence intervals for these networks are given in Table 1, respectively. Boyd et al. Applied Network Science (2023) 8:15 Page 14 of 29 Boyd et al. Applied Network Science Example 4.1 (Bottleneck Distance Between the Tikopia and Hexagonal Networks) Notice that the per- sistence intervals for the Tikopia genealogical network (see Table 1) include, as a subset, the persistence intervals from the hexagonal network we considered in Example 3.6. We can form a bijection between the persistence diagrams of the Tikopia and hexagonal net- work by identifying the non-trivial intervals from the hexagonal network with those of the Tikopia network. We then map any additional intervals from the Tikopia network of the form [a, b) to the trivial interval [ a+b 2 , a+b 2 ) . (The perceptive reader may notice that this is not clearly a bijection, but there is a standard technique from set theory for modifying it to be bijective.) Boyd et al. Applied Network Science (2023) 8:15 Page 15 of 29 Fig. 6 Left: The persistence diagram of the hexagonal network in Fig. 4b is shown. Center: The persistence diagram of the Tikopia genealogical network in Fig. 1 (left) is shown. Right: A bottleneck bijection between the persistence intervals of the hexagonal and Tikopia family network is shown. Orange lines show which points are matched to points of the form (a, a) where a ∈R Fig. 6 Left: The persistence diagram of the hexagonal network in Fig. 4b is shown. Center: The persistence diagram of the Tikopia genealogical network in Fig. 1 (left) is shown. Right: A bottleneck bijection between the persistence intervals of the hexagonal and Tikopia family network is shown. Orange lines show which points are matched to points of the form (a, a) where a ∈R This mapping is shown in Fig. 6 (right). Here, [1, 7) is mapped to [4, 4) . As this pair of points is further apart than any other pair in this bijection, the bottleneck distance for the two networks is at most three, since we take an infimum over all possible bijections. Conversely, there is no interval in the hexagonal persistence diagram that is closer to [1, 7) than 3, so the bottleneck distance is at least three. Thus, the bottleneck distance for these two persistence diagrams is exactly 3. Suppose that two networks, each of which is connected, admit isometric embeddings in Rn . The Stability Theorem (Cohen-Steiner et al. Example 4.1 2007) guarantees that if the Hausdorff distance between the embeddings is δ , then the bottleneck distance for the correspond- ing persistence diagrams is at most δ . For example, if the PH1 persistence diagrams differ by δ , then any attempt to pair up cycles in the networks must include at least one pair of cycles for any isometric embedding that are δ apart in that embedding. In "Network comparison using bottleneck distance" we apply this idea to a large collection of genea- logical and social networks. Persistence curves For the network data we consider, persistence diagrams obfuscate a key difference that we consider important: the number of persistence intervals. For a simple exam- ple of this, consider networks of the form V = {1, 2, . . . , n} with edges of the form {i, i + 1} for 1 ≤i < n . For n ≥2 , any network of this type will have persistence intervals [0, 1) × (n −1) and [0, ∞) × 1 . However, when plotting the persistence diagram we will only ‘see’ two points: (0, 1) and (0, ∞). To address this limitation, we introduce the notion of a persistence curve as a new way to visualize the persistent homology of a network (see Definition 9). The difference between the persistence curve and the persistence diagram of a network is that the per- sistence curve also includes the number of intervals of a particular type. To create a per- sistence curve we first compute a network’s persistence intervals, then sort the intervals of a given dimension by their persistence into a bar graph. For instance, in dimension 1 the Tikopia genealogical network has thirteen [1, 2) intervals, nineteen [1, 3) intervals, Boyd et al. Applied Network Science (2023) 8:1 Boyd et al. Applied Network Science Page 16 of 29 Fig. 7 Left: The barcode of the Tikopia genealogical network in dimension 1 is shown. The individual bars are formed from the persistence intervals given in Table 1. Right: The associated persistence curve for the Tikopia network in Fig. 1 is shown Fig. 7 Left: The barcode of the Tikopia genealogical network in dimension 1 is shown. The individual bars are formed from the persistence intervals given in Table 1. Right: The associated persistence curve for the Tikopia network in Fig. 1 is shown etc. which are sequentially stacked as shown in Fig. 7 (left) to create what we will call a barcode. To create the associated persistence curve we connect the endpoints of each subsequent bar as shown in Fig. 7 (right). In dimension-one, the birth times of our intervals will all start at 1, as the networks we consider are unweighted, undirected, and connected. This means that in this dimension the resulting bar graph is also a plot of the death times for each interval. Persistence curves For higher- dimensions, which have varied birth times, we also plot the lengths of the intervals but for simplicity we start at 1 as in dimension-one. A formal definition of a network’s persistence curves is the following. Definition 9 (Persistence Curves) Let G = (V , E) be a network with nonempty vertex and edge sets. Let {[aj, bj)} be the set of all persistence intervals for each σj ∈PHn(G) where j ∈N . For all n ∈N the persistence curve PHn(G) is the linear interpolation of the set of points {(bj −(aj −1), j)} where bj−1 −(aj−1 −1) ≤bj −(aj −1). Visualizing persistence intervals as a curve allows us to compare the persistent homol- ogy of different networks in a similar fashion to persistence diagrams while retaining different information. In particular, we can see how many intervals there are of a given persistence, whereas the persistence diagram only indicates the presence of such an interval. In what follows we will typically plot the persistence curves of multiple net- works on the same axes to indicate what differences exist in the persistent homology of different networks (cf. "Results"). Data The data we consider in this paper is of two types; genealogical network data and other social network data. The genealogical networks we consider are drawn from ninety-seven genea- logical networks found in (https://www.kinsources.net/browser/datasets.xhtml), which range in size from n = 17 to 5, 016 individuals. The social network data we use is taken from twenty-seven different social networks obtained from (http://snap.stanford.edu/data/index. html#socnets, http://snap.stanford.edu/data/index.html#socnets, http://networkrepository. Boyd et al. Applied Network Science (2023) 8:15 Page 17 of 29 com/soc.php, http://networkrepository.com/soc.php). These range in size from n = 16 to 2, 539 individuals. (See Table 2 in the Appendix for a full description of this data set.) com/soc.php, http://networkrepository.com/soc.php). These range in size from n = 16 to 2, 539 individuals. (See Table 2 in the Appendix for a full description of this data set.) Although many larger genealogical and social network data sets are available we are limited by both the temporal and spacial complexity of the algorithm used to compute persistence intervals. The program we used, called Ripser (from the python package Ripser) (Ripser 2021), has a computational and spacial complexity of O((n + m)3) where n is the number of individuals and m is the number of edges in a network. The number n + m is the number of simplicies in the network. In the genealogical networks we con- sider there are between n + m = 41 to 15, 735 simplicies and in the social networks we consider between n + m = 41 to 19, 056 simplices. To understand how a network’s persistence intervals are effected by the completeness or incompleteness of data we also consider subnetworks sampled from a few, much larger, genealogical and social networks. These sampled networks are created by randomly selecting an individual with a single neighbor, i.e. a vertex of degree 1, then perform- ing a breadth-first-search starting with this individual to find the η closest individuals in the network to this individual. Because of the spatial and computational limitations of Ripser we choose 600 ≤η ≤3, 000 to ensure we can compute the persistence intervals of these sampled networks. In total we sampled from four different genealogical networks and four different social networks. These are the Advogat, LastFM Asia, Deezer HU and Deezer RO social networks and the genealogical networks 96–99 shown in Table 2, respectively. Data We sampled from each of these networks five times each to create a total of 20 sampled genealogical networks and 20 sampled social networks. The reason we begin our breadth-first search with a vertex of degree 1 is to ensure that our sampled networks have vertices both on the boundary and the interior of the original network we sampled to better mimic the structure of the original genealogical and social networks. Apart from the (i) genealogical and social networks we consider and (ii) sampled ver- sions of these networks, we also consider what we refer to as (iii) atypical genealogical networks. There are a number of genealogical networks that appear to be created with no attempt to represent all or even a fraction of the familial relationships. For example, the US Presidents network, cited as Atyp. Gen. Network 2 in Table 2, follows the shortest genealogical path between presidents leaving out extraneous relationships. We consider a number these atypical genealogical networks, which form a contrast to the more stand- ard genealogical networks we consider especially in terms of their peristent homology. A description of each of the (i) genealogical, social, (ii) sampled genealogical, sampled social, and (iii) atypical genealogical networks we consider is given at the end of the Appendix. Results Here we compare genealogical and other social networks using the (a) bottleneck dis- tance and the (b) persistence curves defined in "Comparing networks using persistent homology" (see Definitions 8 and 9, respectively). For those who have skipped in "Persis- tent homology of networks" and “Comparing networks using persistent homology”, the bottleneck distance gives us a distance between two networks based on the differences in their persistent homology. Persistence curves give us a way of visualizing this difference but in greater detail (cf. Figure 7). Boyd et al. Applied Network Science (2023) 8:15 Boyd et al. Applied Network Science Page 18 of 29 Fig. 8 PCA projections of the bottleneck distances between networks are shown. Left: The bottleneck distance between each of the twenty sampled genealogical and sampled social networks is shown. Center: The bottleneck distances are shown between the genealogical, social, and atypical genealogical networks we consider. Right: The bottleneck distances in the center panel are shown for only the genealogical and social networks we consider Fig. 8 PCA projections of the bottleneck distances between networks are shown. Left: The bottleneck g p j distance between each of the twenty sampled genealogical and sampled social networks is shown. Center: The bottleneck distances are shown between the genealogical, social, and atypical genealogical networks we consider. Right: The bottleneck distances in the center panel are shown for only the genealogical and social networks we consider Network comparison using bottleneck distance Here we compute the bottleneck distance between every pair from the social and gene- alogical networks we consider. To visualize these results we use principal component analysis to identify the two components that account for the most variance and then plot this data in R2 (see Fig. 8). From each part of Fig. 8 we can see that genealogical networks are generally separated from social networks and form clusters that are easily distinguished. For the sampled networks (shown left), we can easily separate genealogical and social networks, and we can identify at least two distinct subclasses of genealogical networks. However, the bottleneck distance does an inferior job separating the non-sampled genealogical and social networks (shown center and right). The exception are the atypical genealogical networks, whose persistence intervals differ significantly enough from all of the other networks to be distinguishable as a third class of networks (shown center). Comparison of genealogical and social networks using persistence curves 9 Comparison of persistence curves for full networks vs sampled networks, grouped by dimension and type of network. Upper Row: Sampling social networks typically stretches the persistence curve in only one axis without affecting the other axis. Lower Row: Sampling genealogical networks typically shrink the persistence curve in both axes. Overall the average slope for social networks tends to increase when sampled while genealogical networks experience a decrease in average slope In contrast, the genealogical networks have persistence curves that have a much more horizontal profile indicating that most cycles are quite long and there are fewer ‘alternate paths’ between pairs of vertices. In the extreme, the atypical genealogical networks are nearly flat in dimension 1, which reflects the fact that these atypical networks were inten- tionally constructed to have very few cycles. In dimension 2, the atypical networks show a similar slope to most of the typical genealogical networks, but the size of the alternative paths in these networks are much larger. This is likely due to the high number of individu- als who were added only to link distant individuals, e.g. presidents. In a typical genea- logical network, the additional relationships between such individuals would allow large cycles to decompose but in the atypical genealogical networks this in not the case. In Fig. 10, we see the persistence curves for the sampled genealogical and sampled social networks shown in blue and red, respectively. The atypical genealogical networks are shown in green. Again the social networks have persistence curves that are quite vertical in both dimensions, although these curves are not as tall as in the case of unsampled social net- works. This indicates that as a social network is sampled it retains a similar proportion of close-to-trivial cycles, but may lose many of the alternative paths between vertices that appear in dimension 2. By contrast, for genealogical networks the persistence curves indi- cate the complete loss of very large cycles in conjunction with a proportional loss of close- to-trivial cycles. In dimension 2, genealogical networks experience a more severe loss of alternative paths than the social networks. As a result, though sampling shrinks the scale of the persistence curves for social and genealogical networks, they remain visually distinct. As in the bottleneck distance plots, genealogical and social networks appear to cluster together in that they have similar types of persistence curve. Comparison of genealogical and social networks using persistence curves Persistence curves give us a new alternative way of comparing networks. The advantage of using these curves compared to the bottleneck distance is that these curves give us a more detailed picture of how the number of persistence intervals varies from network to network. This allows us to better differentiate the structure of genealogical networks from social networks as well as observe the structure common to genealogical networks and those common to social networks, respectively. In Fig. 9 the persistence curves for the unsampled genealogical and unsampled social networks are shown in blue and red, respectively. The atypical genealogical networks are shown in green. The social networks have persistence curves that are quite vertical in both dimension 1 and dimension 2. For dimension 1, this indicates that most cycles in a social network are close to being trivial; either because they have a relatively small circumference or because they can be decomposed into a union of cycles with small circumferences. In particular, most of the social networks have a maximum death time of three (see Defini- tion 2), which corresponds to having a basis of cycles whose maximal circumference is at most nine. In other words, any cycle of circumference ten or more decomposes as the union of smaller cycles. For dimension 2, the steepness of the persistence curves indicate the presence of many distinct, yet similar, paths between certain pairs of vertices. Boyd et al. Applied Network Science (2023) 8:15 Boyd et al. Applied Network Science Page 19 of 29 Fig. 9 Comparison of persistence curves for full networks vs sampled networks, grouped by dimension and type of network. Upper Row: Sampling social networks typically stretches the persistence curve in only one axis without affecting the other axis. Lower Row: Sampling genealogical networks typically shrink the persistence curve in both axes. Overall the average slope for social networks tends to increase when sampled, while genealogical networks experience a decrease in average slope Fig. 9 Comparison of persistence curves for full networks vs sampled networks, grouped by dimension and type of network. Upper Row: Sampling social networks typically stretches the persistence curve in only one axis without affecting the other axis. Lower Row: Sampling genealogical networks typically shrink the persistence curve in both axes. Overall the average slope for social networks tends to increase when sampled while genealogical networks experience a decrease in average slope Fig. Comparison of genealogical and social networks using persistence curves In fact, this is true whether or not the networks are sampled or unsampled. This suggests that even with incomplete Page 20 of 29 Boyd et al. Applied Network Science (2023) 8:15 Page 20 of 29 Boyd et al. Applied Network Science Fig. 10 Upper Row: Comparison of persistence curves for full networks by type. Lower Row: Comparison of persistence curves for sampled networks by type, excluding atypical genealogical networks. In each dimension, the average slope for genealogical networks is typically lower than the average slope for a social network. The atypical genealogical networks have the lowest average slope and much greater total length. The behavior for average slopes is more pronounced for sampled networks than for full networks Fig. 10 Upper Row: Comparison of persistence curves for full networks by type. Lower Row: Comparison of persistence curves for sampled networks by type, excluding atypical genealogical networks. In each dimension, the average slope for genealogical networks is typically lower than the average slope for a social network. The atypical genealogical networks have the lowest average slope and much greater total length. The behavior for average slopes is more pronounced for sampled networks than for full networks Fig. 10 Upper Row: Comparison of persistence curves for full networks by type. Lower Row: Comparison of persistence curves for sampled networks by type, excluding atypical genealogical networks. In each dimension, the average slope for genealogical networks is typically lower than the average slope for a social network. The atypical genealogical networks have the lowest average slope and much greater total length. The behavior for average slopes is more pronounced for sampled networks than for full networks data social network and genealogical networks have a distinguishable persistent homol- ogy, at least at the scales we consider. It is worth mentioning that, while the bottleneck distance plots show us to an extent how different genealogical and social networks are the persistence curves show us what are differences are. The distance plots in Fig. 8 do have the advantage of simplicity, how- ever, and could presumably be used to more quickly identify differences in networks that are not as apparent as those we find between genealogical and social networks. Connections Right: A θ-cycle formed by a common ancestor cycle with two overlapping hybrid cycles Fig. 11 Left: A common ancestor cycle. The top most vertex is a common ancestor of the lowest vertex. The horizontal red line is a marriage, all other lines are parent–children edges. Center: A union cycle, specifically the double cousin situation described in "Background: genealogical and social networks". The left-most and right-most vertices are parents of their neighboring vertices. The two horizontal red lines are marriage edges. Right: A θ-cycle formed by a common ancestor cycle with two overlapping hybrid cycles In social networks, the connected components create what could be referred to as friend groups. Unlike genealogical networks, there are usually few restrictions on which edges form in a social network. As such, we do not have a conjecture about the number of [0, ∞) intervals in this setting in general. However, sampling any network as described in "Data" will result in a new network with a single [0, ∞) interval. Moving to dimension 1, persistence intervals in this dimension describe the way that each connected component is internally structured. In sufficiently large genealogical net- works, we will see three kinds of features that we call common ancestors, union cycles, and hybrid cycles. A common ancestor cycle occurs when two descendants of an individual form a union or have a child together. We use the term union cycle to refer to situations where a cycle is formed through union edges and edges connecting two siblings. The final type of cycle of note, the hybrid cycles, are those formed by any other combination of parent–child edges and union edges, which includes everything that is not a strict common ancestor or union cycle. These three types of cycles are illustrated in Fig. 11, where marriage edges are indicated by red edges and parent–child edges are indicated by blue edges. We show a com- mon ancestor in Fig. 11a. Figure 11b is an example of a union cycle in which two siblings in one family form unions with two siblings in another, where only a single parent in each family is shown. In Fig. 11c we give an example of a θ-cycle, which is the union of a com- mon ancestor cycle and two overlapping hybrid cycles. This example comes from siblings of one family marrying cousins from another family. Connections It is also possible to use persistent homology to study properties of a network, such as the number of connected components, the typical size of cycles, or even “missing links” in the data. For genealogical and social networks, we can convert these mathematical concepts into more familiar ideas such as family groups or common ancestors. This also allows us to make conjectures about the persistent homology for such networks by converting standard assumptions about families or social networks into the language of persistence. In dimension 0, the number of connected components determines the number of [0, ∞) intervals, and the total number of distinct vertices is the number of [0, ∞) intervals plus the number of [0, 1) intervals. In the context of a genealogical network, each connected compo- nent represents a family group that is not related to the other family groups by any known connection. Thus, if a given family network is indeed a single “family” of relatives, there should be exactly one [0, ∞) interval. In our Tikopia example we have eight [0, ∞) intervals each of which correspond to exactly one connected component of this genealogical network. (Note that Fig. 1 (left) shows only the largest of these components). In this example, most of the the other ‘family groups’ are actually individuals with no relation edges in the network. Boyd et al. Applied Network Science (2023) 8:15 Boyd et al. Applied Network Science Page 21 of 29 Page 21 of 29 Fig. 11 Left: A common ancestor cycle. The top most vertex is a common ancestor of the lowest vertex. The horizontal red line is a marriage, all other lines are parent–children edges. Center: A union cycle, specifically the double cousin situation described in "Background: genealogical and social networks". The left-most and right-most vertices are parents of their neighboring vertices. The two horizontal red lines are marriage edges. Right: A θ-cycle formed by a common ancestor cycle with two overlapping hybrid cycles Fig. 11 Left: A common ancestor cycle. The top most vertex is a common ancestor of the lowest vertex. The horizontal red line is a marriage, all other lines are parent–children edges. Center: A union cycle, specifically the double cousin situation described in "Background: genealogical and social networks". The left-most and right-most vertices are parents of their neighboring vertices. The two horizontal red lines are marriage edges. Connections These cycles can be any length theoreti- cally, but cultural norms affect the typical size and number of each type of cycle differently. Recording practices and incomplete data also limit whether these cycles appear in a given dataset. Thus having a description of these cycles together with an understanding of the culture may help identify errors in the recorded data. Conversely, understanding the distri- bution of cycles in high fidelity datasets can help identify the underlying cultural norms and help extrapolate where individuals are missing in incomplete data sets. Since many cultures avoid marrying close relatives, common ancestor cycles tend to have a fairly large circumference. In the Tikopia network (see Fig. 1) we see persistence intervals with death values as high as 7 corresponding to cycles with a circumference Boyd et al. Applied Network Science (2023) 8:15 Page 22 of 29 of at least 21 individuals, which appear to be common ancestor cycles. This partially explains why persistence curves are so flat: there are relatively few minimal common ancestor cycles in a network, but they have very high persistence. More precisely, if the distance to union (the total number of individuals in a common ancestor cycle) is n, then the persistence of that cycle is ⌊n/3⌋ . However, the representatives of persistent homol- ogy only include a basis for these cycles, instead of including every possible distinct cycle. In particular, a large common ancestor cycle will decompose into the union of two hybrid cycles if the hybrid cycles are each shorter than the common ancestor cycle, as shown in Fig. 11c. Persistent homology will reflect the size of the two smaller cycles instead of the larger common ancestor cycle. We note that it is possible to identify the actual cycles chosen for our basis, but the software we used does not provide that infor- mation and size of the networks prohibits us from identifying the cycles manually. In social networks, we see that highly persistence cycles are quite rare. In order to have a cycle of persistence 3, for instance, we need a loop with circumference 9 or higher with no shorter paths between any two vertices in the loops. It may be that phenomena like the small-world effect or, more colloquially, six-degrees of freedom limit the maxi- mal persistence of social networks. Connections We see this reflected in our example data sets with a maximum persistence of 3 for all but one of the social networks. Conclusion In this paper, we explore the persistent homology structure of genealogical networks, motivated by the observation that family links tend to form in a fixed range of intermedi- ate distances, which makes genealogical networks homologically distinct from most other social networks. We also introduce the notion of a persistence curve, which can be used to summarize and compare the persistent homology structure of any network. We also relate specific genealogical structures, such as the common ancestor cycle, to homology objects. i We find that, in the presence of incomplete data homology analysis is still genealogi- cally useful. We note missing data due to recording practices and incomplete data (a ubiq- uitous feature of real genealogical networks), limits the kind of cycles that appear in a given dataset. Thus having a description of these cycles together with an understanding of the culture may help identify errors in the recorded data. Conversely, understanding the distribution of cycles in high fidelity datasets can help identify the underlying cultural norms and help extrapolate where individuals are missing in incomplete data sets. There are several interesting directions in which this work could be expanded. For exam- ple, our work has made it clear that there is a real need to analyze the persistent homology of large networks, with at least tens of thousands of nodes, since family formation generally takes place at these scales. The Ripser library we relied on was not able to reach these scales. Additionally, we are very interested in creating random graph models which reflect the actual homology of human family networks—a first attempt at this by our group has been fairly successful at the scale of hundreds of nodes (Flores 2021). More broadly, there is a need to model the ground truth human family network. All the extant data sources represent biased, limited, and noisy subnetworks, while the true interest of the genealogical community is in the ground truth network. Tools for signal denoising, image inpainting, and graph extrapola- tion, for example, could be useful in this context. Finally, an important aspect of genealogical Page 23 of 29 Boyd et al. Applied Network Science (2023) 8:15 Boyd et al. Conclusion Applied Network Science Table 2 Social and genealogical network data sets Network data Network type & name Vertices Edges Citation Social networks Friendship & aquaintance Dolphins 62 159 http://www-personal.umich.edu/mejn/netdata/ Zachary karate club 34 78 http://vlado.fmf.uni-lj.si/pub/networks/data/ucinet/ucida ta.htm#zachary Residence hall 217 2672 http://konect.cc/networks/moreno_oz/ Highland tribes 16 58 http://konect.cc/networks/ucidata-gama/ Seventh graders 29 376 http://konect.cc/networks/moreno_seventh/ Physicians 241 1098 http://konect.cc/networks/moreno_innovation/ Highschool 70 366 http://konect.cc/networks/moreno_highschool/ Dutch College 32 354 http://konect.cc/networks/moreno_vdb/ Sampson’s monastery 25 322 http://vladowiki.fmf.uni-lj.si/doku.php?id=pajek:data:esna3: sampson Adolescent health 2539 12969 http://konect.cc/networks/moreno_health/ Hamsterster friends 2952 12534 http://konect.cc/networks/petster-hamster-friend/ Social network 1 32 220 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as1.net Social network 2 32 191 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as2.net Social network 5 32 90 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as5.net Social network 7 32 61 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as7.net Social network 8 32 79 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as8.net Social network 9 32 58 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as9.net Firm Hi-Tech 33 124.5 https://networkrepository.com/soc-firm-hi-tech.php Wiki-Vote 889 2.9K https://networkrepository.com/soc-wiki-Vote.php FB-PAGES-FOOD 620 2.1K https://networkrepository.com/fb-pages-food.php Advogato 6541 51127 http://konect.cc/networks/advogato/ LastFM Asia 7624 27806 https://snap.stanford.edu/data/feather-lastfm-social.html Deezer HU 47538 222887 https://snap.stanford.edu/data/gemsec-Deezer.html Deezer RO 41773 125826 https://snap.stanford.edu/data/gemsec-Deezer.html Collaboration & business Social network 4 32 218 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as4.net Social network 6 32 103 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as6.net Social network 11 32 83 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign.1/ as11.net Social network 12 32 65 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign.1/ as12.net Disease transmission Taro exchange 22 78 http://konect.cc/networks/moreno_taro/ Information sharing Social network 3 32 119 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign. 1/as3.net Social network 10 32 80 http://vlado.fmf.uni-lj.si/pub/networks/doc/ECPR/assign.1/ as10.net Table 2 Social and genealogical network data sets Boyd et al. Applied Network Science (2023) 8:15 Page 24 of 29 Boyd et al. Conclusion Applied Network Science Network type & name Vertices Edges Citation Genealogical networks Genealogical network 1 310 322 https://www.kinsources.net/kidarep/dataset-209-mowan jum-kalumburu.xhtml Genealogical network 2 303 537 https://www.kinsources.net/kidarep/dataset-2-mbuti-villa ge-1957-af03.xhtml Genealogical network 3 371 718 https://www.kinsources.net/kidarep/dataset-58-ojibwa- 1930-nd07.xhtml Genealogical network 4 795 1387 https://www.kinsources.net/kidarep/dataset-150-achuar- pastaza.xhtml Genealogical network 5 636 1151 https://www.kinsources.net/kidarep/dataset-92-chenchu- 1940-as02.xhtml Genealogical network 6 782 1366 https://www.kinsources.net/kidarep/dataset-28-trio-1960s. xhtml Genealogical network 7 128 202 https://www.kinsources.net/kidarep/dataset-23-shoshone- 1880-nd11.xhtml Genealogical network 8 439 626 https://www.kinsources.net/kidarep/dataset-70-genesis. xhtml Genealogical network 9 244 481 https://www.kinsources.net/kidarep/dataset-66-waimiri- atroari.xhtml Genealogical network 10 410 746 https://www.kinsources.net/kidarep/dataset-240-kodiak. xhtml Genealogical network 11 337 572 https://www.kinsources.net/kidarep/dataset-51-wilcania. xhtml Genealogical network 12 216 378 https://www.kinsources.net/kidarep/dataset-22-ainu-1880- as01.xhtml Genealogical network 13 77 134 https://www.kinsources.net/kidarep/dataset-69-slavey-1911- nd12.xhtml Genealogical network 14 815 1582 https://www.kinsources.net/kidarep/dataset-7-pakaa-nova. xhtml Genealogical network 15 20 28 https://www.kinsources.net/kidarep/dataset-38-wanindilja ugwa-1948-au06.xhtml Genealogical network 16 219 371 https://www.kinsources.net/kidarep/dataset-171-suya.xhtml Genealogical network 17 17 24 https://www.kinsources.net/kidarep/dataset-31-family.xhtml Genealogical network 18 168 221 https://www.kinsources.net/kidarep/dataset-14-labrador- inuit-1776-nu02.xhtml Genealogical network 19 64 109 https://www.kinsources.net/kidarep/dataset-91-takamiut- 1927-64-nu03.xhtml Genealogical network 20 1423 3211 https://www.kinsources.net/kidarep/dataset-258-todas. xhtml Genealogical network 21 645 1097 https://www.kinsources.net/kidarep/dataset-65-igluligmiut- 1961-nu07.xhtml Genealogical network 22 4463 8416 https://www.kinsources.net/kidarep/dataset-115-charlevoix. xhtml Genealogical network 23 48 86 https://www.kinsources.net/kidarep/dataset-41-vedda-1905- as04.xhtml Genealogical network 24 104 172 https://www.kinsources.net/kidarep/dataset-71-igluligmiut- 1960-61-nu08.xhtml Genealogical network 25 1263 2021 https://www.kinsources.net/kidarep/dataset-223-samburu. xhtml Genealogical network 26 80 132 https://www.kinsources.net/kidarep/dataset-10-apache- 1932-nd01.xhtml Genealogical network 27 1269 2395 https://www.kinsources.net/kidarep/dataset-24-ayd-nl- yoruk-2005.xhtml Genealogical network 28 299 532 https://www.kinsources.net/kidarep/dataset-13-tory.xhtml Boyd et al. Applied Network Science (2023) 8:15 Page 25 of 29 Boyd et al. Conclusion Applied Network Science Network type & name Vertices Edges Citation Genealogical network 29 19 30 https://www.kinsources.net/kidarep/dataset-21-ngatatjara- 1966-au04.xhtml Genealogical network 30 399 592 https://www.kinsources.net/kidarep/dataset-204-dogon- konsogu-donyu.xhtml Genealogical network 31 377 712 https://www.kinsources.net/kidarep/dataset-49-alyawarra- 1971-au01.xhtml Genealogical network 32 1263 2021 https://www.kinsources.net/kidarep/dataset-223-samburu. xhtml Genealogical network 33 118 192 https://www.kinsources.net/kidarep/dataset-39-eyak-1890. xhtml Genealogical network 34 98 161 https://www.kinsources.net/kidarep/dataset-75-nunamiut- 1885-nu11.xhtml Genealogical network 35 479 830 https://www.kinsources.net/kidarep/dataset-19-ojibwa- 1949-nd08.xhtml Genealogical network 36 1695 3206 https://www.kinsources.net/kidarep/dataset-103-tikuna- arara.xhtml Genealogical network 37 256 441 https://github.com/AbigailJ32/The-persistent-homology-of- genealogical-networks Genealogical network 38 798 1416 https://www.kinsources.net/kidarep/dataset-229-nucoo rilma-tingha.xhtml Genealogical network 39 738 1212 https://www.kinsources.net/kidarep/dataset-32-yaraldi.xhtml Genealogical network 40 525 855 https://github.com/AbigailJ32/The-persistent-homology-of- genealogical-networks Genealogical network 41 619 1224 https://www.kinsources.net/kidarep/dataset-251-nunivak. xhtml Genealogical network 42 3008 6074 https://www.kinsources.net/kidarep/dataset-80-torshan. xhtml Genealogical network 43 278 464 https://www.kinsources.net/kidarep/dataset-62-dogrib- 1911-25-59-nd04.xhtml Genealogical network 44 105 172 https://www.kinsources.net/kidarep/dataset-5-konkama- 1931-44-51-eu02.xhtml Genealogical network 45 240 395 https://www.kinsources.net/kidarep/dataset-158-tikar.xhtml Genealogical network 46 4178 7351 https://www.kinsources.net/kidarep/dataset-45-obidos. xhtml Genealogical network 47 216 286 https://www.kinsources.net/kidarep/dataset-254-port-keats. xhtml Genealogical network 48 147 242 https://www.kinsources.net/kidarep/dataset-78-pul-eliya- 1954-simpler-version.xhtml Genealogical network 49 277 516 https://www.kinsources.net/kidarep/dataset-213-sarmi.xhtml Genealogical network 50 330 622 https://www.kinsources.net/kidarep/dataset-73-parakana. xhtml Genealogical network 51 35 53 https://www.kinsources.net/kidarep/dataset-81-gunda ngborn-1948-au02.xhtml Genealogical network 52 48 76 https://www.kinsources.net/kidarep/dataset-84-hare-1956- nd05.xhtml Genealogical network 53 105 245 https://www.kinsources.net/kidarep/dataset-87-arara.xhtml Genealogical network 54 116 220 https://www.kinsources.net/kidarep/dataset-89-nunamiut- 1960-nu13.xhtml Genealogical network 55 116 176 https://www.kinsources.net/kidarep/dataset-226-jie.xhtml Genealogical network 56 657 1166 https://www.kinsources.net/kidarep/dataset-27-nyungar. xhtml Genealogical network 57 659 1288 https://www.kinsources.net/kidarep/dataset-3-anuta-1972. xhtmlj Boyd et al. Conclusion networks is the relationship between various supporting documents/metadata and the links that are discoverable through them. For example, one can consider optimal document collec- tion strategies with a limited budget or document collection that is fair in terms of capturing minority information, which is often underrepresented. Acknowledgements g We acknowledge helpful conversations with Joseph Price and the FamilySearch Engineering Research team. We also acknowledge Kolton Baldwin for helping to improve our code and simulations. Author contributions Author contributions D i d th i t ZB NC BW RW P f d th i t RF RW W t th ZB NC TG AJ RS BW Author contributions Designed the experiments: ZB, NC, BW, RW. Performed the experiments: RF, RW. Wrote the paper: ZB, NC, TG, AJ, RS, BW, Designed the experiments: ZB, NC, BW, RW. Performed the experiments: RF, RW. Wrote the paper: ZB, NC, TG, AJ, RS, BW, RW. All authors read and approved the final manuscript. Appendix Here we indicate both the genealogical and social networks used in our persistent homol- ogy computations (see "Results"). We distinguish the datasets by network type: Friendship/ Acquaintance, Social Media, Collaboration/Business, Disease Transmission, Information Shar- ing, Genealogical, and Atypical Genealogical networks. We also provide the network name, number of vertices and edges in the network, and a citation where the network can be found. Also, a special thanks to Kolton Baldwin for help with numerical simulations on this paper. Acknowledgements Conclusion Applied Network Science (2023) 8:15 Page 26 of 29 Table 2 (continued) Genealogical network 58 112 182 https://www.kinsources.net/kidarep/dataset-15-oodnadatta. xhtml Genealogical network 59 218 353 https://www.kinsources.net/kidarep/dataset-17-laini ovouma-1952-eu03.xhtml Genealogical network 60 90 119 https://www.kinsources.net/kidarep/dataset-12-miwuyt- 1967-au03.xhtml Genealogical network 61 289 477 https://www.kinsources.net/kidarep/dataset-9-konkama- 1951-eu01.xhtml Genealogical network 62 1463 1969 https://www.kinsources.net/kidarep/dataset-306-nobles-ile- de-france-1000-1440.xhtml Genealogical network 63 4109 6517 https://www.kinsources.net/kidarep/dataset-287-duu-rea. xhtml Genealogical network 64 29 48 https://www.kinsources.net/kidarep/dataset-46-hatfields- and-mccoys.xhtml Genealogical network 65 40 59 https://www.kinsources.net/kidarep/dataset-33-angma gsalik-1884-nu01.xhtml Genealogical network 66 294 441 https://www.kinsources.net/kidarep/dataset-18-tikopia-1930. xhtml Genealogical network 67 502 786 https://www.kinsources.net/kidarep/dataset-34-netsilik- 1922-nu09.xhtml Genealogical network 68 83 126 https://www.kinsources.net/kidarep/dataset-8-semang- 1924-50-as03.xhtml Genealogical network 69 95 157 https://www.kinsources.net/kidarep/dataset-4-shoshone- 1860-nd10.xhtml Genealogical network 70 2588 5651 https://www.kinsources.net/kidarep/dataset-61-kelkummer. xhtml Genealogical network 71 88 144 https://www.kinsources.net/kidarep/dataset-77-apache- 1935-nd02.xhtml Genealogical network 72 1513 2217 https://www.kinsources.net/kidarep/dataset-90-omaha- 1880.xhtml Genealogical network 73 3014 5454 https://www.kinsources.net/kidarep/dataset-128-ammonni. xhtml Genealogical network 74 139 201 https://www.kinsources.net/kidarep/dataset-79-paiute-1880- nd09.xhtml Genealogical network 75 5016 10719 https://www.kinsources.net/kidarep/dataset-249-baruya. xhtml Genealogical network 76 125 202 https://www.kinsources.net/kidarep/dataset-242-tlingit. xhtml Genealogical network 77 272 445 https://www.kinsources.net/kidarep/dataset-36-copper- 1922-nu10.xhtml Genealogical network 78 378 609 https://www.kinsources.net/kidarep/dataset-52-apache- 1936-nd03.xhtml Genealogical network 79 926 1951 https://www.kinsources.net/kidarep/dataset-68-surui.xhtml Genealogical network 80 706 1177 https://www.kinsources.net/kidarep/dataset-60-mbuti-for- est-1957-af02.xhtml Genealogical network 81 435 672 https://www.kinsources.net/kidarep/dataset-64-melombo. xhtml Genealogical network 82 128 114 https://www.kinsources.net/kidarep/dataset-164-kaingang. xhtml Genealogical network 83 169 275 https://www.kinsources.net/kidarep/dataset-11-top-of-the- mountain.xhtml Genealogical network 84 178 274 https://www.kinsources.net/kidarep/dataset-37-igluligmiut- 1921-nu05.xhtml Genealogical network 85 87 111 https://www.kinsources.net/kidarep/dataset-216-tiwi.xhtml Genealogical network 86 2049 4159 https://www.kinsources.net/kidarep/dataset-35-chuukese- 1947-1940.xhtml Genealogical network 87 868 980 https://www.kinsources.net/kidarep/dataset-20-saudi-royal- genealogy.xhtml Boyd et al. Applied Network Science (2023) 8:15 Page 27 of 29 Boyd et al. Applied Network Science Table 2 (continued) Genealogical network 88 2821 5079 https://www.kinsources.net/kidarep/dataset-30-manus-1929. xhtml Genealogical network 89 454 980 https://www.kinsources.net/kidarep/dataset-74-arawete. xhtml Genealogical network 90 304 472 https://www.kinsources.net/kidarep/dataset-42-nunamiut- tareumiut-1900-nu12.xhtml Genealogical network 91 367 671 https://www.kinsources.net/kidarep/dataset-48-wanindilja ugwa-1941-au05.xhtml Genealogical network 92 3151 4289 https://www.kinsources.net/kidarep/dataset-54-feistritz-am- gael-1990.xhtml Genealogical network 93 2975 5107 https://www.kinsources.net/kidarep/dataset-159-cocama- cocamilla.xhtml Genealogical network 94 585 1249 https://www.kinsources.net/kidarep/dataset-44-torres-strait. xhtml Genealogical network 95 334 530 https://www.kinsources.net/kidarep/dataset-6-igluligmiut- 1949-nu06.xhtml Genealogical network 96 9595 14988 https://www.kinsources.net/kidarep/dataset-93-sainte-cathe rine.xhtml Genealogical network 97 28586 51446 https://www.kinsources.net/kidarep/dataset-76-san-marino. xhtml Genealogical network 98 18645 32439 https://www.kinsources.net/kidarep/dataset-307-bwa-slam- biogsurvey.xhtml Genealogical network 99 8809 15643 https://www.kinsources.net/kidarep/dataset-194-kel-owey. xhtml Atypical Genealogical Networks Atyp. Gen. Network 1 429 705 (created using FamilySearch.org) Atyp. Gen. Network 2 2477 4015 https://www.kinsources.net/kidarep/dataset-56-us-presi dents.xhtml networks is the relationship between various supporting documents/metadata and the links that are discoverable through them. For example, one can consider optimal document collec- tion strategies with a limited budget or document collection that is fair in terms of capturing minority information, which is often underrepresented. Funding g ZB, BW, and AJ, were supported by a BYU CPMS CHIRP grant. ZB was additionally supported by NFS award #2137511 and Army Research Office grant #W911NF-18-1-0244, and the James S. McDonnell Foundation 21st Century Science Initia- tive-Complex Systems Scholar Award grant #2200203. BW was additionally supported by the Simons Foundation grant #714015. The views and conclusions contained in this document are those of the authors and should not be interpreted Page 28 of 29 Boyd et al. 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https://openalex.org/W2901974557	https://escholarship.org/content/qt2cz069qb/qt2cz069qb.pdf?t=pj4oxt	English	null	An Unusual Case of Hematemesis	Journal of education & teaching in emergency medicine	2,018	cc-by	1,373	UC Irvine Copyright Information Copyright 2018 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Irvine Journal of Education and Teaching in Emergency Medicine Title An Unusual Case of Hematemesis Permalink https://escholarship.org/uc/item/2cz069qb Journal Journal of Education and Teaching in Emergency Medicine, 3(4) Authors Amen, Amanda Xiao, Jane Parks-Bortel, Julie et al. Publication Date 2018 DOI 10.5070/M534041292 Copyright Information Copyright 2018 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Journal of Education and Teaching in Emergency Medicine Title An Unusual Case of Hematemesis Permalink https://escholarship.org/uc/item/2cz069qb Journal Journal of Education and Teaching in Emergency Medicine, 3(4) Authors Amen, Amanda Xiao, Jane Parks-Bortel, Julie et al. Publication Date 2018 DOI 10.5070/M534041292 Copyright Information Copyright 2018 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Powered by the California Digital Library University of California eScholarship.org Return: Calibri Size 10 Return: Calibri Size 10 An Unusual Case of Hematemesis Return: Calibri Size 10 An Unusual Case of Hematemesis Oakland University William Beaumont School of Medicine, Royal Oak, MI Oakland University William Beaumont School of Medicine, Royal Oak, MI ^Oakland University William Beaumont School of Medicine, Department of Emergency Medicine, Royal Oak, MI Oakland University William Beaumont School of Medicine, Department of Emergency Medicine, Troy, MI Correspondence should be addressed to Amanda Amen, BS at aamen@oakland.edu Submitted: June 16, 2018; Accepted: September 7, 2018; Electronically Published: October 15, 2018; https://doi.org/10.21980/J84H00 Copyright: © 2018 Amen, et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) License. See: htt // ti /li /b /4 0/ *Oakland University William Beaumont School of Medicine, Department of Emergency Medicine, Troy, MI Correspondence should be addressed to Amanda Amen, BS at aamen@oakland.edu Submitted: June 16, 2018; Accepted: September 7, 2018; Electronically Published: October 15, 2018; https://doi.org/10.21980/J84H00 Copyright: © 2018 Amen, et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) License. See: ty Line Calibri Size 12 bri Size 12 32 32 32 Axial CT Video Link: https://youtu.be/luZ1Cyws-6Y History of present illness: A 68-year-old female presented with back pain described as burning in her mid to upper back radiating to her chest with nausea and vomiting for several hours, which progressed to active hematemesis. Her vitals were significant for hypertension (160/95) and bradycardia (57). Her exam was notable for diminished breath sounds in the bilateral bases with rhonchi in the left lung base, and epigastric tenderness without rebound or guarding. A portable X-ray followed by a computed tomography (CT) angiogram of the chest and abdomen were ordered. Upon return from CT, the patient developed hypotension, worsening bradycardia, and altered mentation. Axial CT Video Link: https://youtu.be/luZ1Cyws-6Y Axial CT Video Link: https://youtu.be/luZ1Cyws-6Y Axial CT Video Link: https://youtu.be/luZ1Cyws-6Y History of present illness: A 68-year-old female presented with back pain described as burning in her mid to upper back radiating to her chest with nausea and vomiting for several hours, which progressed to active hematemesis. Her vitals were significant for hypertension (160/95) and bradycardia (57). Her exam was notable for diminished breath sounds in the bilateral bases with rhonchi in the left lung base, and epigastric tenderness without rebound or guarding. A portable X-ray followed by a computed tomography (CT) angiogram of the chest and abdomen were ordered. Upon return from CT, the patient developed hypotension, worsening bradycardia, and altered mentation. Significant findings: The patient’s chest X-ray revealed a prominent mediastinum and opacification in the left middle and lower lung fields. The CT showed an aortic aneurysm extending from the thorax to the abdomen with rupture near T7 (blue arrow). It also showed periaortic hemorrhage with active extravasation (green arrow) likely secondary to a penetrating ulcer and bilateral pulmonary opacities concerning for hemothorax (pink arrow). Discussion: Thoracic aortic aneurysm (TAA) is a progressive and potentially deadly disease with a poor prognosis when left untreated.1 Most TAAs are asymptomatic and may be detected incidentally on imaging. Although presentation can vary, chest or back pain might be the first presenting symptom and can represent rapid expansion, acute dissection, or rupture.2 In the acute setting, chest X-ray might be the first imaging study performed during the initial evaluation of a patient with symptoms that suggest a potential for aortic disease. Predictive sensitivity of a widened mediastinum or abnormal aortic contour can be around 64% and 71% respectively, but the sensitivity of CXR is not adequate to definitively rule out acute aortic disease.3 CT with contrast remains the imaging study of choice for diagnosis of acute thoracic aortic disease; it can be nearly 100% sensitive and can also demonstrate the presence of a dissection flap or rupture.3 Return: Calibri Size 10 33 3 3 Return: Calibri Size 10 Return: Calibri Size 10 33 Overall mortality for ruptured TAA is 97%-100%.4 While mortality is also high for repair of symptomatic TAA, repair is recommended for all symptomatic TAA (ruptured, associated with dissection, or causing pain).2 However, surgery may not be indicated based on patient comorbidities, or clinical picture. Preemptive repair of asymptomatic TAA can eliminate the risk of rupture, which is primarily predicted by size.5 Overall mortality for elective repair is 4.5% (4.6% for open repair and 3.6% for endovascular repair).6 Current guidelines suggest surgical intervention when the risk of rupture-related complications exceed the risk of surgery-related complications, which is generally around a size of 5.5 cm. However, it is also dependent on the presence of genetic risk factors for dissection and rupture, exact location, and rate of aneurysm expansion.3 In this case, cardiothoracic surgery determined that surgery would not confer any increased chance of survival based on the patient’s clinical condition, the size and location of the rupture, and the growing periaortic hemorrhage. The family decided to provide comfort measures only and the patient expired approximately one hour after presentation to the emergency department. opics: Aortic aneurysm, thoracic aortic aneurysm, acute aortic syndromes, cardiothoracic surgery. 1. Bashir M, Fok M, Hammoud I, Rimmer L, Shaw M, Field M, et al. A perspective on natural history and survival in nonoperated thoracic aortic aneurysm patients. Aorta. 2013;1(3):182-189. doi: 10.12945/j.aorta.2013.13-043 1. Bashir M, Fok M, Hammoud I, Rimmer L, Shaw M, Field M, et al. A perspective on natural history and survival in nonoperated thoracic aortic aneurysm patients. Aorta. 2013;1(3):182-189. doi: 10.12945/j.aorta.2013.13-043 2. Woo J, Greene C. Management of thoracic aortic aneurysm in adults. In: Collins KA, ed. UpToDate. Waltham, MA: UpToDate Inc. https://www.uptodate.com/contents/management-of-thoracic-aortic-aneurysm-in-adults. Updated December 12, 2017. Accessed September 6, 2018. 3. Hiratzka LF, Bakris GL, Beckman JA, Bersin RM, Carr VF, Casey DE, et al. 2010 ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM guidelines for the diagnosis and management of patients with thoracic aortic disease: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, American Association for Thoracic Surgery, American College of Radiology, American Stroke Association, Society of Cardiovascular Anesthesiologists, Society for Cardiovascular Angiography and Interventions, Society of Interventional Radiology, Society of Thoracic Surgeons, and Society for Vascular Medicine. Circulation. 2010;121(13): e266-369. doi: 10.1161/CIR.0b013e3181d4739e 4. Johansson G, Markström U, Swedenborg J. Ruptured thoracic aortic aneurysms: a study of incidence and mortality rates. J Vasc Surg. 1995;21(6):985-988. doi: 10.1016/s0741-5214(95)70227-x 5. Return: Calibri Size 10 Davies R, Goldstein L, Coady M, Tittle SL, Rizzo JA, Kopf GS, et al. Yearly rupture or dissection rates for thoracic aortic aneurysms: simple prediction based on size. Ann Thorac Surg. 2002;73(1):17-28. doi: 10.1016/s0003-4975(01)03236-2 6. Hughes K, Guerrier J, Obirieze A, Ngwang D, Rose D, Tran D, et al. Open versus endovascular repair of thoracic aortic aneurysms. Vasc Endovascular Surg. 2014;48(5-6):383-387. doi: 10.1177/1538574414540484 5. Davies R, Goldstein L, Coady M, Tittle SL, Rizzo JA, Kopf GS, et al. Yearly rupture or dissection rates for thoracic aortic aneurysms: simple prediction based on size. Ann Thorac Surg. 2002;73(1):17-28. doi: 10.1016/s0003-4975(01)03236-2 y p p g ; ( ) / ( ) 6. Hughes K, Guerrier J, Obirieze A, Ngwang D, Rose D, Tran D, et al. Open versus endovascular repair of thoracic aortic aneurysms. Vasc Endovascular Surg. 2014;48(5-6):383-387. doi: 10.1177/1538574414540484 Return: Calibri Size 10 34 34 Return: Calibri Size 10 34
https://openalex.org/W3087089586	https://www.frontiersin.org/articles/10.3389/fcvm.2020.00158/pdf	English	null	Left Ventricular Hypertrabeculation Is Not Associated With Cardiovascular Morbity or Mortality: Insights From the Eurocmr Registry	Frontiers in cardiovascular medicine	2,020	cc-by	7,952	ORIGINAL RESEARCH published: 22 September 2020 doi: 10.3389/fcvm.2020.00158 Edited by: Rajesh Puranik, Royal Prince Alfred Hospital, Australia Edited by: Rajesh Puranik, Royal Prince Alfred Hospital, Australia Reviewed by: Aphrodite Tzifa, Mitera Hospital, Greece Fabien Hyaﬁl, Assistance Publique – Hôpitaux de Paris, France Giuseppe Femia, South Western Sydney Local Health District, Australia Reviewed by: Aphrodite Tzifa, Mitera Hospital, Greece Fabien Hyaﬁl, Assistance Publique – Hôpitaux de Paris, France Giuseppe Femia, South Western Sydney Local Health District, Australia Reviewed by: Aphrodite Tzifa, Mitera Hospital, Greece Fabien Hyaﬁl, Assistance Publique – Hôpitaux de Paris, France Giuseppe Femia, South Western Sydney Local Health District, Australia 1 William Harvey Research Institute, NIHR Barts Biomedical Research Centre, Queen Mary University of London, London, United Kingdom, 2 Barts Heart Centre, St Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom, 3 Department of Cardiology and Angiology, Elisabeth-Krankenhaus Essen, Ruhr University Bochum, Bochum, Germany, 4 Department of Cardiology, St. Vincent’s Medical Centre, Bridgeport, CT, United States, 5 I.R.C.C.S. Multimodality Cardiac Imaging, Policlinico San Donato, Milan, Italy, 6 Cardiac MR Centre, University Hospital (CHUV), Switzerland and Lausanne University, Lausanne, Switzerland, 7 Department of Cardiology, Amsterdam University Centres, Amsterdam, Netherlands, 8 Department of Cardiology, Clinic Agatharied, University of Munich, Munich, Germany, 9 Department of Cardiology, Klinikum Ludwigsburg, Ludwigsburg, Germany, 10 Medneo Group, Berlin, Germany, 11 Institute for Experimental and Translational Cardiovascular Imaging DZHK (German Centre for Cardiovascular Research) Centre for Cardiovascular Imaging, Partner Site RheinMain, University Hospital, Goethe University, Frankfurt, Germany, 12 CMR Unit, Royal Brompton Hospital, London, United Kingdom, 13 National Heart and Lung Institute, London, United Kingdom, 14 Department of Cardiology, Inselspital, University of Bern, Bern, Switzerland, 15 Department of Internal Medicine, Krankenhaus Benrath, Düsseldorf, Germany, 16 Department of Internal Medicine, University Hospital Tulln, Tulln, Austria, 17 Institut für Herzinfarktforschung, Ludwigshafen, Germany, 18 Department of Cardiology, Robert Bosch Medical Centre, Stuttgart, Germany Correspondence: Steffen E. Petersen s.e.petersen@qmul.ac.uk †These authors have contributed equally to this work and share ﬁrst authorship Aim: Left ventricular non-compaction (LVNC) is perceived as a rare high-risk cardiomyopathy characterized by excess left ventricular (LV) trabeculation. However, there is increasing evidence contesting the clinical signiﬁcance of LV hyper-trabeculation and the existence of LVNC as a distinct cardiomyopathy. The aim of this study is to assess the association of LV trabeculation extent with cardiovascular morbidity and all-cause mortality in patients undergoing clinical cardiac magnetic resonance (CMR) scans across 57 European centers from the EuroCMR registry. Left Ventricular Hypertrabeculation Is Not Associated With Cardiovascular Morbity or Mortality: Insights From the Eurocmr Registry Filip Zemrak 1,2†, Zahra Raisi-Estabragh 1,2†, Mohammed Y. Khanji 1,2, Saidi A. Mohiddin 1,2, Oliver Bruder 3, Anja Wagner 4, Massimo Lombardi 5, Juerg Schwitter 6, Albert C. van Rossum 7, Günter Pilz 8, Detlev Nothnagel 9, Henning Steen 10, Eike Nagel 11, Sanjay K. Prasad 12,13, Christina C. Deluigi 14, Thorsten Dill 15, Herbert Frank 16, Steffen Schneider 17, Heiko Mahrholdt 18 and Steffen E. Petersen 1,2 Specialty section: This article was submitted to Cardiovascular Imaging, a section of the journal Frontiers in Cardiovascular Medicine Received: 12 May 2020 Accepted: 28 July 2020 Published: 22 September 2020 Methods and Results: We studied 822 randomly selected cases from the EuroCMR registry. Image acquisition was according to international guidelines. We manually segmented images for LV chamber quantiﬁcation and measurement of LV trabeculation (as per Petersen criteria). We report the association between LV trabeculation extent and important cardiovascular morbidities (stroke, atrial ﬁbrillation, heart failure) and all-cause mortality prospectively recorded over 404 ± 82 days of follow-up. Maximal non-compaction to compaction ratio (NC/C) was mean (standard deviation) 1.81 ± 0.67, from these, 17% were above the threshold for hyper-trabeculation (NC/C > 2.3). LV trabeculation extent was not associated with increased risk of the deﬁned outcomes (morbidities, mortality, LV CMR indices) in the whole cohort, or in sub-analyses of individuals without ischaemic heart disease, or those with NC/C > 2.3. Citation: Zemrak F, Raisi-Estabragh Z, Khanji MY, Mohiddin SA, Bruder O, Wagner A, Lombardi M, Schwitter J, van Rossum AC, Pilz G, Nothnagel D, Steen H, Nagel E, Prasad SK, Deluigi CC, Dill T, Frank H, Schneider S, Mahrholdt H and Petersen SE (2020) Left Ventricular Hypertrabeculation Is Not Associated With Cardiovascular Morbity or Mortality: Insights From the Eurocmr Registry. September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org Zemrak et al. LV Hypertrabeculation and Cardiovascular Outcomes Conclusion: Among 882 patients undergoing clinical CMR, excess LV trabeculation was not associated with a range of important cardiovascular morbidities or all-cause mortality over ∼12 months of prospective follow-up. These ﬁndings suggest that LV hyper-trabeculation alone is not an indicator for worse cardiovascular prognosis. Conclusion: Among 882 patients undergoing clinical CMR, excess LV trabeculation was not associated with a range of important cardiovascular morbidities or all-cause mortality over ∼12 months of prospective follow-up. These ﬁndings suggest that LV hyper-trabeculation alone is not an indicator for worse cardiovascular prognosis. Keywords: left ventricular non-compaction, left ventricular trabeculation, cardiomyopathy, cardiac magnetic resonance, mortality The EuroCMR Registry Left ventricular non-compaction cardiomyopathy (LVNC) is perceived as a rare genetic cardiomyopathy characterized by abnormal arrest of in-utero myocardial compaction (1). Tertiary center cohorts of LVNC report association with life- threatening arrhythmias, thromboembolism, and left ventricular (LV) dysfunction (2–5). LVNC is recognized as a “genetic cardiomyopathy” by the American Heart Association and as an “unclassiﬁed cardiomyopathy” by the European Society of Cardiology (6, 7). Identiﬁcation of excess LV trabeculations alongside a thin layer of compacted myocardium on non- invasive imaging is key to diagnosis. The Petersen cardiac magnetic resonance (CMR) criteria are widely used for quantiﬁcation of LV trabeculations and to guide diagnosis of LVNC (8). The EuroCMR Registry The EuroCMR registry was set up to assess the clinical utility and prognostic impact of CMR in real-life clinical scenarios. Over 37,000 consecutive patients undergoing routine clinical CMR were recruited from 57 center in 15 European countries (19). Scans were performed in compliance with standardized protocols. The only exclusion criterion was contraindication to CMR. There was prospective follow-up of patients with suspected coronary artery disease (CAD) or hypertrophic cardiomyopathy (HCM) through standardized telephone interviews (Supplementary Table 1). If contact with the patient was unsuccessful, government registration oﬃces were contacted to obtain updated contact information or details of cause of death. The EuroCMR registry study design and protocols are detailed elsewhere (20). All participating center had approval from local institutional ethics review boards, and all patients provided written informed consent in accordance to the Declaration of Helsinki. Increased awareness of LVNC and improved imaging techniques have led to a surge in its diagnosis. However, studies of healthy cohorts have identiﬁed fulﬁllment of the LVNC imaging criteria in a high proportion of individuals with no clear association to adverse outcomes (9–11). Similar ﬁndings have been reported in small studies of asymptomatic athletes and healthy pregnant women (12– 14). These ﬁndings have been replicated in single center studies of symptomatic individuals and those with known structural heart disease (15, 16). Further, a multicenter study of individuals diagnosed with LVNC reports better than expected outcomes with no prognostic impact of LV trabeculation beyond known parameters such as left ventricular ejection fraction (LVEF) (17). Zemrak et al. LV Hypertrabeculation and Cardiovascular Outcomes FIGURE 1 \| Flow diagram of exclusion process from the initial unselected scans. IHD, ischaemic heart disease; LV, left ventricle; NC/C, non-compacted to compacted ratio; SAX, short-axis stack. FIGURE 1 \| Flow diagram of exclusion process from the initial unselected scans. IHD, ischaemic heart disease; LV, left ventricle; NC/C, non-compacted to compacted ratio; SAX, short-axis stack. FIGURE 1 \| Flow diagram of exclusion process from the initial unselected scans. IHD, ischaemic heart disease; LV, left ventricle; NC/C, non-compacted to compacted ratio; SAX, short-axis stack. calculated by subtracting LVESV from the epicardial volume in end-diastole and multiplying it by the myocardial muscle density of 1.05 g/ml. LV parameters were indexed to body surface area (BSA, denoted by i). separate from the blood pool. Trabeculations were deﬁned as a meshwork of moderate SI on the endocardial surface of the compacted myocardium with interspersion of higher SI from the blood pool (Figure 3). Papillary muscles were excluded from measurements. Short-axis views were used in conjunction with the long axis images to aid identiﬁcation of papillary muscles. In normal individuals, the true apex is typically thin with prominent trabeculations, it was therefore also excluded from analysis. The maximum ratio of non-compaction to compaction (NC/C) was calculated for each segment. NC/C >2.3 was used as cut- oﬀfor LVNC as per the Petersen criteria (8). Measurements Selection of CMR Studies In order to ensure quality control within the EuroCMR registry, there was requirement for each center to submit a selection of random cases for quality assessment. This created a bank of 980 randomly selected scans collated from all participating center. The current study is based on analysis of this sample. All scans were anonymised, and the demographic and clinical data were not available at the time of image analysis. After quality control checks, 158 scans were excluded due to sub- optimal image quality, the remaining 822 scans are included in this analysis (Figure 1). There is increasing uncertainty regarding the clinical signiﬁcance of LV hyper-trabeculation and some have questioned the status of LVNC as a distinct cardiomyopathy (18). However, many of these studies have been conducted in low-risk populations, in whom application of diagnostic criteria is misleading. Measurement of LV Volumes and Function CMR studies were analyzed using cvi42 software (Circle Cardiovascular Imaging Inc, Calgary, Canada). Left ventricular end diastolic volume (LVEDV), end systolic volume (LVESV), and end-diastolic mass (LVM) were obtained from cine short- axis images covering the LV from base to apex. End-systole and end-diastole were deﬁned by the cardiac phases with the largest and smallest blood pool area at the mid-ventricular level. The operator deﬁned the slice range (base to apex). Endocardial and epicardial contours were manually drawn at end-diastole and end-systole for each slice according to expert consensus recommendations (21). Papillary muscles were included in the blood pool. The cvi42 software computed LV volumes by slice summation, LVEF was calculated in the usual way. LVM was There remains some uncertainty regarding generalisability of ﬁndings from these low-risk asymptomatic cohorts to patients seen in clinical practice. It is important to ascertain the signiﬁcance of LV trabeculation in a real- life population with clinical indication for CMR. We present the ﬁrst prospective multicenter multinational study of the relationship of the extent of LV trabeculation with cardiovascular morbidity and all-cause mortality in real-life patients undergoing clinical CMR in 57 European center. September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 2 Zemrak et al. LV Hypertrabeculation and Cardiovascular Outcomes Measurement of LV Trabeculations Three long-axis (2-, 3-, and 4-chamber) cine images were used for measuring the thickness of compacted myocardium and trabeculations at the centre of 16 segments of the AHA model (Figure 2). Compacted myocardium was deﬁned as a myocardial layer of homogeneous moderate signal intensity (SI) distinctly September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 3 Zemrak et al. LV Hypertrabeculation and Cardiovascular Outcomes FIGURE 2 \| Measurement of NC/C ratios in EuroCMR Registry in 16 segments of the American Heart Association model excluding the true apical cap (segment 17). (A) Four chambers view, (B) two chambers view, (C) three chambers view. FIGURE 2 \| Measurement of NC/C ratios in EuroCMR Registry in 16 segments of the American Heart Association model excluding the true apical cap (segment 17). (A) Four chambers view, (B) two chambers view, (C) three chambers view. FIGURE 3 \| Measurement of NC/C ratios in 4-chamber, 2-chamber, and 3-chamber longitudinal view. Red lines represent compacted myocardium deﬁned as a myocardial layer of homogeneous moderate signal intensity distinctly separate from the blood pool, yellow lines represent measurement of trabeculations deﬁned as a meshwork of moderate signal intensity on the endocardial surface of the compacted myocardium with interspersion of higher SI from the blood pool; NC/C, non-compacted to compacted ratio. SI, signal intensity. FIGURE 3 \| Measurement of NC/C ratios in 4-chamber, 2-chamber, and 3-chamber longitudinal view. Red lines represent compacted myocardium deﬁned as a myocardial layer of homogeneous moderate signal intensity distinctly separate from the blood pool, yellow lines represent measurement of trabeculations deﬁned as a meshwork of moderate signal intensity on the endocardial surface of the compacted myocardium with interspersion of higher SI from the blood pool; NC/C, non-compacted to compacted ratio. SI, signal intensity. Baseline Demographics and Indications for CMR Demographic data are presented in Table 1. Sixty three percenatge (n = 516) of participants were male. Mean age was 59 ± 14 years (range: 16–90). There was substantial burden of cardiovascular risk factors: hypertension 60%, diabetes 14%, dyslipidaemia 40%, smoking history 29%. The most common indications for CMR were evaluation of coronary artery disease (81.9%), cardiomyopathy (15.7%), and myocarditis (8.5%), there was non-exclusivity of indications. Statistical Analysis each participant’s analyzed segments was 1.81 ± 0.67. Greater magnitude of trabeculation was detected in lateral segments, and increased from base to apex. LV trabeculation was not seen in the basal infero-septal segment of any participant. y Statistical analysis was performed using R version 3.6.1 (2019-07- 05) [R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: http://www.R-project.org/]. Continuous variables are summarized with mean (standard deviation, SD) or median (interquartile range, IQR). Categorical variables are presented as frequencies and percentages. Diﬀerences between LV trabeculation quintiles were evaluated by the analysis of variance (ANOVA) with a post-hoc Tukey test for continuous variables and chi-squared test for categorical variables. Univariate linear regression models were used to assess the relationship between demographic factors, clinical data and LV structural parameters as exposure variables and maximal NC/C as the outcome variable. Cox proportional hazards regression models were used to estimate hazard ratios (HR) and the associated 95% conﬁdence interval (CI) for mortality. Univariable logistic- regression models were used to estimate the odds ratio (OR) and the associated 95% CI for the endpoints. LV trabeculation may be more relevant to individuals with suspected non-ischaemic cardiomyopathies. Therefore, subgroup analysis was performed on a subgroup of 569 subjects without ischaemic heart disease (IHD), deﬁned as having no history of myocardial infarction, coronary revascularisation, or evidence of IHD on CMR. LV Volumes, Mass, and Systolic Function LV volumes and functional parameters are presented in Table 1. Severe LV systolic dysfunction (LVEF ≤35%) was detected in 5.6% (n = 46). There were no diﬀerences between the quintiles of maximal NC/C in the frequency of severe LV systolic dysfunction (p = 0.39). Determinants of Maximal NC/C NC/C was higher in women than in men (1.89 ± 0.72 vs. 1.77 ± 0.64, p < 0.05) and decreased with age (β = −0.05 per decade, p < 0.01). Maximal NC/C was larger by 0.5 for every 100 ml/m2 increase in LVEDVi (p < 0.0001), and by 0.4 for every 100 ml/m2 increase in LVESVi (p < 0.0001). Maximal NC/C was also larger by 0.06 units for every 10% decrease in LV ejection fraction (p < 0.01). RESULTS Mortality data was available for 530 participants with mean follow-up of 404 ± 82 days. During the follow up period, 10 deaths were recorded (death rate of 1.9%). The extent of maximal NC/C was not associated with increased mortality. There was no diﬀerence in survival between quintiles of maximal NC/C (Table 2). Similarly, there was no excess mortality in those with the highest NC/C (quintile 5, NC/C 2.23 to 5.64) compared to the remaining participants (OR 0.51, 95% CI: 0.03–2.76, p- value 0.53). Diagnoses Following CMR Data on heart failure status assessed by the New York Heart Association (NYHA) classiﬁcation was available for 513 participants. Of these, 65% (n = 333) were in NYHA class I, 11.7% (n = 60) in NYHA class II, 3.7% (n = 19) in NYHA class III, and <1% (n = 5) in NYHA class IV. Increasing quintiles of the maximal NC/C were not associated with greater odds of severe heart failure deﬁned as NYHA class III and IV (Table 3). Sensitivity analyses looking at (1) Quintile 5 vs. the remaining cohort, (2) LVNC >2.3, and (3) number of segments with NC/C>2.3 showed similar results. No abnormality was detected in 49% of the CMR scans. IHD was diagnosed in 25% of patients. Amongst these, myocardial ischaemia was detected in 147 cases, syndrome X in 64, and cardiac thrombus in one patient. Non-ischaemic cardiomyopathies were diagnosed in 111 patients: HCM (n = 59), dilated cardiomyopathy (n = 33), arrhythmogenic right ventricular cardiomyopathy (n = 1), unclassiﬁed cardiomyopathy (n = 18). Myocarditis was diagnosed in 29 patients. Outcomes of 100 randomly selected studies repeated by the ﬁrst reader showed excellent intraobserver variability (intraclass correlation coeﬃcients (ICC): LVEDVi 0.98, LVESVi 0.95, LVEF 0.83, LVMi 0.97; p < 0.0001 for all parameters). The interobserver variability in the same 100 studies of LV measurements in our group showed very good to excellent reproducibility; ICC values: LVEDVi 0.97, LVESVi 0.88, LVEF 0.71, LVMi 0.92; p < 0.0001 for all measurements. of 100 randomly selected studies repeated by the ﬁrst reader showed excellent intraobserver variability (intraclass correlation coeﬃcients (ICC): LVEDVi 0.98, LVESVi 0.95, LVEF 0.83, LVMi 0.97; p < 0.0001 for all parameters). The interobserver variability in the same 100 studies of LV measurements in our group showed very good to excellent reproducibility; ICC values: LVEDVi 0.97, LVESVi 0.88, LVEF 0.71, LVMi 0.92; p < 0.0001 for all measurements. We considered the following outcomes: all-cause mortality, stroke, atrial ﬁbrillation, severe heart failure (deﬁned as New York Heart Association class 3 and 4). Outcomes were obtained from standardized EuroCMR interviews with follow up duration of 404 ± 82 days. Additionally, we investigated the association of demographic and baseline characteristics on LV trabeculation extent. September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 4 LV Hypertrabeculation and Cardiovascular Outcomes Zemrak et al. Stroke NC/C was calculated in 10,456 (79.5%) segments. The remaining 2,696 segments either had insuﬃcient contrast between the blood pool and the myocardium or were oﬀ-axis impeding conﬁdent measurement of NC/C. NC/C >2.3 was measured in at least one segment for 17.2% of participants (n = 142), and in at least two segments for 5.1% (n = 42). The maximal NC/C had a log-normal distribution. The mean maximal NC/C for Twenty-eight (3.4%) participants had prior history of stroke. There were no diﬀerences in the prevalence of stroke between the quintiles of maximal NC/C (p = 0.16) (Table 1). One participant developed a stroke during follow-up. His maximal NC/C was 2.38. Inclusion of this case in the analysis did not alter the results (Table 3). September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 5 LV Hypertrabeculation and Cardiovascular Outcomes Zemrak et al. TABLE 1 \| Summary of baseline demographic and selected cardiac magnetic resonance data. Stroke All Quintile 1 Quintile 2 Quintile 3 Quintile 4 Quintile 5 p-value NC/C 0.55–1.29 1.3–1.54 1.55–1.83 1.84–2.22 2.23–5.64 n 822 166 167 161 166 162 Baseline demographics and cardiovascular risk factors Age (years) 59.4 ± 13.9 60.9 ± 13.8 59.7 ± 14.6 59.7 ± 13.9 60.5 ± 13.1 56.1 ± 13.8 <0.05 Females 306 (37.2%) 53 (31.9%) 57 (34.1%) 62 (38.5%) 62 (37.3%) 72 (44.4%) 0.18 Body mass index (kg/m2) 27.6 ± 5.9 28.0 ± 5.0 27.9 ± 7.3 26.6 ± 4.0 28.1 ± 6.8 27.1 ± 5.8 0.11 Height (cm) 172.2 ± 9.8 172.6 ± 9.5 172.4 ± 11.1 171.9 ± 9.3 172.1 ± 9.4 171.9 ± 9.8 0.96 Weight (kg) 81.8 ± 17.3 83.5 ± 16.5 82.3 ± 15.5 78.9 ± 14.4 83.4 ± 19.3 80.9 ± 19.8 0.08 Hypertension (n = 626) 357 (57.0%) 90 (70.3%) 71 (55.5%) 67 (55.8%) 71 (54.2%) 58 (48.7%) <0.05 Diabetes (n = 626) 86 (13.7%) 21 (16.4%) 17 (13.3%) 17 (14.2%) 24 (18.3%) 7 (5.9%) 0.053 Dyslipidaemia (n = 625) 250 (40%) 51 (39.8%) 46 (35.9%) 55 (45.8%) 55 (42.3%) 43 (36.1%) 0.46 Family history of CAD (n = 597) 166 (27.8%) 40 (31.7%) 27 (21.8%) 37 (32.5%) 33 (27.3%) 29 (25.9%) 0.32 Smoking (n = 626) Never 445 (71.1%) 93 (72.7%) 85 (66.4%) 88 (73.3%) 89 (67.9%) 90 (75.6%) 0.46 Former 95 (15.2%) 15 (11.7%) 26 (20.3%) 14 (11.7%) 26 (19.8%) 14 (11.8%) 0.08 Current 86 (13.7%) 20 (15.6%) 17 (13.3%) 18 (15%) 16 (12.2%) 15 (12.6%) 0.92 Important medical history (n = 809) Myocardial infarction 51 (6.3%) 11 (6.6%) 8 (4.8%) 12 (7.5%) 13 (8.0%) 7 (4.5%) 0.6 CABG 19 (2.3%) 9 (5.4%) 2 (1.2%) 2 (1.3%) 4 (2.5%) 2 (1.3%) 0.052 PCI 78 (9.6%) 13 (7.8%) 18 (10.9%) 18 (11.3%) 20 (12.3%) 9 (5.7%) 0.23 Stroke 28 (3.5%) 6 (3.6%) 4 (2.4%) 10 (6.3%) 6 (3.7%) 2 (1.3%) 0.16 PVD 26 (3.2%) 5 (3.0%) 9 (5.5%) 3 (1.9%) 5 (3.1%) 4 (2.5%) 0.43 Malignancy 30 (3.7%) 5 (3.0%) 5 (3.0%) 7 (4.4%) 7 (4.3%) 6 (3.8%) 0.93 ECG ﬁndings Heart rate (bpm) (n = 820) 68.6 ± 13.1 68.6 ± 12.8 68.5 ± 11.9 69.6 ± 12.8 68.6 ± 15.1 67.6 ± 12.5 0.74 Atrial ﬁbrillation (n = 822) 20 (2.4%) 6 (3.6%) 2 (1.2%) 4 (2.5%) 5 (3.0%) 3 (1.9%) 0.64 Ventricular extrasystoles (≥10/min) 28 (3.4%) 4 (2.4%) 7 (4.2%) 7 (4.3%) 6 (3.6%) 4 (2.5%) 0.79 CMR parameters LVEDVi (ml/m2) 79 ± 25.3 74.5 ± 27.8 75.5 ± 20.7 79.6 ± 24.4 80.3 ± 23.4 85.4 ± 28.2 <0.001 LVESVi (ml/m2) 36 ± 22.5 33.9 ± 25.0 32.8 ± 18.0 35.9 ± 20.8 37.4 ± 21.9 40.1 ± 25.6 <0.5 LVEF (%) 56.5 ± 11.6 56.9 ± 11.7 58.3 ± 10.3 56.5 ± 10.7 55.4 ± 13.4 55.2 ± 11.7 0.1 LVEF ≤35% 46 (5.6%) 9 (5.4) 5 (3.0) 8 (5.0) 12 (7.2) 12 (7.4) 0.39 LVMi (g/m2) 54.7 ± 17 59.0 ± 21.1 55.7 ± 17.0 54.1 ± 16.5 53.0 ± 13.8 51.4 ± 15.0 <0.01 bpm, beats per minute; CAD, coronary artery disease; EDV, end diastolic volume; EF, ejection fraction; ESV, end systolic volume; g, gram; i, indexed; LVM, LV mass; LV, left ventricle; m, meter; min, minute; NC/C, non-compaction to compaction ratio. Stroke Numbers displayed are mean ± standard deviation or absolute number and percentage (please note, the denominator varies depending on data completeness). TABLE 1 \| Summary of baseline demographic and selected cardiac magnetic resonance data. bpm, beats per minute; CAD, coronary artery disease; EDV, end diastolic volume; EF, ejection fraction; ESV, end systolic volume; g, gram; i, indexed; LVM, LV mass; LV, left ventricle; m, meter; min, minute; NC/C, non-compaction to compaction ratio. Numbers displayed are mean ± standard deviation or absolute number and percentage (please note, the denominator varies depending on data completeness). Atrial Fibrillation Twenty (2.4%) participants had a history of atrial ﬁbrillation. There were no diﬀerences in the frequency of atrial ﬁbrillation among quintiles of maximal NC/C (p = 0.16) (Table 1). The 569 individuals without IHD were younger (p < 0.01) and more likely to be women (p < 0.05). They had lower prevalence of diabetes (p < 0.0001) and hypertension (p < 0.05) (Table 4). A greater proportion of individuals in this cohort had NC/C >2.3 compared to the whole cohort (19.2 vs. 17.2%). However, the mean maximal NC/C was comparable to the whole cohort at 1.82 ± 0.7. There was greater mortality risk in individuals without IHD, with 8 of the 10 observed deaths occurring in this group. The overall MACE was lower than the whole cohort (n = 35, 9.6% vs. n = 57, 12.8%). There was no association between maximal NC/C and mortality (OR 1.19, 95% CI: 0.39–2.62, p = 0.72) or MACE (OR 0.65, 95% CI: 0.34–1.15, p = 0.17). The number Major Adverse Cardiovascular Events (MACE) TABLE 2 \| Predictors of mortality in the EuroCMR Registry. TABLE 2 \| Predictors of mortality in the EuroCMR Registry. Odds ratio p-value Hazard ratio p-value Baseline demographics and cardiovascular risk factors Age per year 1.02 (0.97–1.08) 0.38 1.02 (0.97–1.08) 0.38 Females 1.09 (0.27–3.87) 0.89 1.08 (0.3–3.8) 0.91 Body mass index per kg/m2 1.01 (0.89–1.07) 0.85 1.01 (0.92–1.1) 0.83 Height per cm 1.03 (0.96–1.1) 0.42 1.03 (0.96–1.1) 0.43 Weight per kg 1.01 (0.98–1.04) 0.48 1.01 (0.98–1.04) 0.48 Hypertension 0.97 (0.21–5.0) 0.97 0.96 (0.22–4.3) 0.96 Diabetes 1.17 (0.06–7.01) 0.89 1.16 (0.14–9.63) 0.89 Dyslipidaemia 1.09 (0.21–5.01) 0.91 1.1 (0.25–4.9) 0.9 Smoking 1.0 (0.14–4.69) 0.99 1 (0.19–5.15) 1 CMR volume and function measures LV end-diastolic volume index per ml/m2 1.02 (1–1.04) <0.05 1.02 (1.002–1.038) <0.05 LV end-systolic volume index per ml/m2 1.02 (1.002–1.04) <0.05 1.02 (1.005–1.036) <0.01 LV ejection fraction per% 0.94 (0.91–0.98) <0.01 0.94 (0.91–0.980) <0.001 LV mass index per g/m2 1.03 (1.01–1.05) <0.01 1.03 (1.01–1.05) <0.01 Late gadolinium enhancement 6.4 (1.79–25.35) <0.01 6.1 (1.7–21.8) <0.01 Maximal NC/C as a continuous variable Maximal NC/C per 1 unit 1.39 (0.52–2.84) 0.44 1.38 (0.61–3.13) 0.44 Quintiles of maximal NC/C vs. 1 Quintile 2 No events 0.99 1 Quintile 3 2.1 (0.4–15.4) 0.4 2.04 (0.4–11.1) 0.41 Quintile 4 1.59 (0.3–12.2) 0.62 1.6 (0.3–9.4) 0.62 Quintile 5 0.59 (0.03–6.2) 0.67 0.58 (0.05–6.4) 0.66 Quintile 5 vs. remaining participants 0.51 (0.03–2.76) 0.53 0.5 (0.06–4.0) 0.52 Fulﬁlled Petersen’s LVNC criteria (NC/C C ≥2.3) 0.62 (0.03–3.37) 0.65 0.62 (0.08–4.87) 0.65 Number of segments with NC/C ≥2.3 1 No events 0.99 0.99 2 3.06 (0.16–17.8) 0.3 2.99 (0.38–23.6) 0.3 3 No events 0.99 1 4 No events 0.99 1 cm, centimeter; g, gram; kg, kilogram; m, meter; ml, milliliter; NC/C, non-compacted to compacted ratio. TABLE 3 \| Associations of maximal NC/C ratio with severe heart failure, stroke, and MACE. Severe heart failure (NYHA III/IV) Stroke MACE Odds ratio p-value Odds ratio p-value Odds ratio p-value Maximal NC/C as a continuous variable Maximal NC/C per 1 unit 0.65 (0.28–1.33) 0.28 0.82 (0.41–1.50) 0.56 0.81 (0.49–1.29) 0.39 Quintiles of maximal NC/C vs. quintile 1 Quintile 2 0.82 (0.2–3.2) 0.78 0.69 (0.17–2.5) 0.58 0.62 (0.24–1.55) 0.32 Quintile 3 1.99 (0.66–6.67) 0.23 1.85 (0.66–5.6) 0.25 1.57 (0.67–2.7) 0.26 Quintile 4 0.64 (0.13–2.69) 0.55 1.11 (0.34–3.7) 0.86 0.89 (0.46–2.0) 0.8 Quintile 5 0.71 (0.14–2.97) 0.64 0.6 (0.12–2.35) 0.48 0.62 (0.27–1.45) 0.33 Quintile 5 vs. Major Adverse Cardiovascular Events (MACE) Major Adverse Cardiovascular Events (MACE) MACE was deﬁned as the composite of all-cause mortality (n = 10), severe heart failure (n = 24), and stroke (prevalent = 28, incident = 1). Some participants had more than more of these outcomes, overall, 57 (12.8%) individuals had at least one MACE. The extent of maximal NC/C expressed as a continuous variable and in quintiles was not associated with frequency of MACE (Table 3). Sensitivity analysis did not show associations between other LV trabeculation extent measures and the frequency of MACE. September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 6 LV Hypertrabeculation and Cardiovascular Outcomes Zemrak et al. TABLE 2 \| Predictors of mortality in the EuroCMR Registry. Odds ratio p-value Hazard ratio p-value Baseline demographics and cardiovascular risk factors Age per year 1.02 (0.97–1.08) 0.38 1.02 (0.97–1.08) 0.38 Females 1.09 (0.27–3.87) 0.89 1.08 (0.3–3.8) 0.91 Body mass index per kg/m2 1.01 (0.89–1.07) 0.85 1.01 (0.92–1.1) 0.83 Height per cm 1.03 (0.96–1.1) 0.42 1.03 (0.96–1.1) 0.43 Weight per kg 1.01 (0.98–1.04) 0.48 1.01 (0.98–1.04) 0.48 Hypertension 0.97 (0.21–5.0) 0.97 0.96 (0.22–4.3) 0.96 Diabetes 1.17 (0.06–7.01) 0.89 1.16 (0.14–9.63) 0.89 Dyslipidaemia 1.09 (0.21–5.01) 0.91 1.1 (0.25–4.9) 0.9 Smoking 1.0 (0.14–4.69) 0.99 1 (0.19–5.15) 1 CMR volume and function measures LV end-diastolic volume index per ml/m2 1.02 (1–1.04) <0.05 1.02 (1.002–1.038) <0.05 LV end-systolic volume index per ml/m2 1.02 (1.002–1.04) <0.05 1.02 (1.005–1.036) <0.01 LV ejection fraction per% 0.94 (0.91–0.98) <0.01 0.94 (0.91–0.980) <0.001 LV mass index per g/m2 1.03 (1.01–1.05) <0.01 1.03 (1.01–1.05) <0.01 Late gadolinium enhancement 6.4 (1.79–25.35) <0.01 6.1 (1.7–21.8) <0.01 Maximal NC/C as a continuous variable Maximal NC/C per 1 unit 1.39 (0.52–2.84) 0.44 1.38 (0.61–3.13) 0.44 Quintiles of maximal NC/C vs. 1 Quintile 2 No events 0.99 1 Quintile 3 2.1 (0.4–15.4) 0.4 2.04 (0.4–11.1) 0.41 Quintile 4 1.59 (0.3–12.2) 0.62 1.6 (0.3–9.4) 0.62 Quintile 5 0.59 (0.03–6.2) 0.67 0.58 (0.05–6.4) 0.66 Quintile 5 vs. remaining participants 0.51 (0.03–2.76) 0.53 0.5 (0.06–4.0) 0.52 Fulﬁlled Petersen’s LVNC criteria (NC/C C ≥2.3) 0.62 (0.03–3.37) 0.65 0.62 (0.08–4.87) 0.65 Number of segments with NC/C ≥2.3 1 No events 0.99 0.99 2 3.06 (0.16–17.8) 0.3 2.99 (0.38–23.6) 0.3 3 No events 0.99 1 4 No events 0.99 1 cm, centimeter; g, gram; kg, kilogram; m, meter; ml, milliliter; NC/C, non-compacted to compacted ratio. TABLE 2 \| Predictors of mortality in the EuroCMR Registry. Major Adverse Cardiovascular Events (MACE) remaining participants 0.64 (0.14–1.91) 0.48 0.52 (0.12–1.52) 0.29 0.62 (0.25–1.33) 0.26 Fulﬁlled Petersen’s LVNC criteria (NC/C ≥2.3) 0.49 (0.08–1.72) 0.35 0.41 (0.06–1.4) 0.23 0.52 (0.17–1.22) 0.17 Number of segments with NC/C ≥2.3 1 0.34 (0.02–1.66) 0.3 0.56 (0.09–1.96) 0.45 0.42 (0.1–1.21) 0.16 2 1.25 (0.07–6.63) 0.83 No events 0.99 1.03 (0.16–3.83) 0.97 3 No events 0.99 No events 0.99 No events 0.99 4 No events 0.99 No events 0.99 No events 0.99 MACE, major adverse cardiac events deﬁned as severe heart failure, stroke, and death; NC/C, non compaction to compaction ratio; NYHA, New York Heart Association. centimeter; g, gram; kg, kilogram; m, meter; ml, milliliter; NC/C, non-compacted to compacted ratio. TABLE 3 \| Associations of maximal NC/C ratio with severe heart failure, stroke, and MACE. Severe heart failure (NYHA III/IV) Stroke MACE Odds ratio p-value Odds ratio p-value Odds ratio p-value Maximal NC/C as a continuous variable Maximal NC/C per 1 unit 0.65 (0.28–1.33) 0.28 0.82 (0.41–1.50) 0.56 0.81 (0.49–1.29) 0.39 Quintiles of maximal NC/C vs. quintile 1 Quintile 2 0.82 (0.2–3.2) 0.78 0.69 (0.17–2.5) 0.58 0.62 (0.24–1.55) 0.32 Quintile 3 1.99 (0.66–6.67) 0.23 1.85 (0.66–5.6) 0.25 1.57 (0.67–2.7) 0.26 Quintile 4 0.64 (0.13–2.69) 0.55 1.11 (0.34–3.7) 0.86 0.89 (0.46–2.0) 0.8 Quintile 5 0.71 (0.14–2.97) 0.64 0.6 (0.12–2.35) 0.48 0.62 (0.27–1.45) 0.33 Quintile 5 vs. remaining participants 0.64 (0.14–1.91) 0.48 0.52 (0.12–1.52) 0.29 0.62 (0.25–1.33) 0.26 Fulﬁlled Petersen’s LVNC criteria (NC/C ≥2.3) 0.49 (0.08–1.72) 0.35 0.41 (0.06–1.4) 0.23 0.52 (0.17–1.22) 0.17 Number of segments with NC/C ≥2.3 1 0.34 (0.02–1.66) 0.3 0.56 (0.09–1.96) 0.45 0.42 (0.1–1.21) 0.16 2 1.25 (0.07–6.63) 0.83 No events 0.99 1.03 (0.16–3.83) 0.97 3 No events 0.99 No events 0.99 No events 0.99 4 No events 0.99 No events 0.99 No events 0.99 MACE, major adverse cardiac events deﬁned as severe heart failure, stroke, and death; NC/C, non compaction to compaction ratio; NYHA, New York Heart Association. of maximal NC/C ratio with severe heart failure, stroke, and MACE September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 7 LV Hypertrabeculation and Cardiovascular Outcomes Zemrak et al. TABLE 4 \| Comparison of baseline data for the subgroup without IHD with the whole cohort. Major Adverse Cardiovascular Events (MACE) All (n = 822) No IHD (n = 569) p-value Baseline demographics and cardiovascular risk factors Age (years) 59.4 ± 13.9 57.3 ± 14.3 <0.01 Females 306 (37.2%) 224 (39.4%) <0.05 Body mass index (kg/m2) 27.6 ± 5.9 27.7 ± 6.5 0.71 Height (cm) 172.2 ± 9.8 172.3 ± 9.9 0.90 Weight (kg) 81.8 ± 17.3 82.2 ± 18.2 0.75 Hypertension (n = 626) 357 (57.0%) 238 (54.1%) <0.05 Diabetes (n = 626) 86 (13.7%) 43 (9.8%) <0.0001 Dyslipidaemia (n = 625) 250 (40%) 159 (36.2%) <0.01 Family history of coronary disease (n = 597) 166 (27.8%) 114 (27.2%) 0.56 Smoking history (n = 626) Never 445 (71.1%) 313 (71.1%) 0.90 Former 95 (15.2%) 67 (15.2%) 0.99 Current 86 (13.7%) 60 (13.6%) 0.87 Previous medical history (n = 809) Myocardial infarction 51 (6.3%) 0 <0.0001 CABG 19 (2.3%) 0 <0.0001 PCI 78 (9.6%) 0 <0.0001 Stroke 28 (3.5%) 13 (2.3%) <0.01 PVD 26 (3.2%) 12 (2.1%) <0.01 Malignancy 30 (3.7%) 16 (2.8%) <0.05 ECG ﬁndings Heart rate (bpm) (n = 820) 68.6 ± 13.1 68.9 ± 13.3 0.67 Atrial ﬁbrillation (n=822) 20 (2.4%) 11 (1.9%) 0.24 Ventricular extrasystoles (≥10/min) 28 (3.4%) 21 (3.7%) 0.56 CMR parameters LV end-diastolic volume index (ml/m2) 79.0 ± 25.3 78.3 ± 24.5 0.63 LV end-systolic volume index (ml/m2) 36.0 ± 22.5 34.9 ± 21.0 0.37 LV ejection fraction (%) 56.5 ± 11.6 57.3 ± 11.1 0.19 Severe LVSD (EF<35%) 46 (5.6%) 29 (5.1%) 0.54 LV mass index (g/m2) 54.7 ± 17 53.8 ± 17.2 0.37 Bpm, beats per minute; CABG, coronary artery bypass graft; cm, centimeter; g, gram; IHD, ischaemic heart disease; kg, kilogram; LV, left ventricle; m, meter; ml, milliliter; PCI, percutaneous coronary intervention; PVD, peripheral vascular disease. Numbers displayed are mean ± standard deviation or absolute number and percentage (please note, the denominator varies depending on data completeness). TABLE 4 \| Comparison of baseline data for the subgroup without IHD with the whole cohort. Bpm, beats per minute; CABG, coronary artery bypass graft; cm, centimeter; g, gram; IHD, ischaemic heart disease; kg, kilogram; LV, left ventricle; m, meter; ml, milliliter; PCI, percutaneous coronary intervention; PVD, peripheral vascular disease. Numbers displayed are mean ± standard deviation or absolute number and percentage (please note, the denominator varies depending on data completeness). Strengths and Limitations of individual outcomes (other than all-cause mortality) was too small in this subgroup for suﬃciently powered statistical analysis. Whilst the inclusion of consecutive real-life clinical patients is a strength of this study, analysis of individuals with relevant symptoms in a more selective manner may provide more meaningful context for interpretation of clinical signiﬁcance of LV trabeculation. However, this approach would introduce ascertainment bias, which has hampered previous studies with highly selective cohorts. We only used one method to quantify trabeculation extent. However, whilst other methods exist (22), method of measurement does not appear to result in important diﬀerences (23). Substantial number of participants had missing data for the mortality outcome (n = 292), we limited our analysis to individuals with an explicitly documented mortality outcome (dead/alive). Of course, there is still potential for bias, e.g., relating to excess deaths in the missing cohort, however, we believe our approach is the method with least potential for bias within the limitations Comparison With Existing Literature Co pa so s g e a u e The proportion of individuals in our study with NC/C >2.3 in at least one segment was high (17%, n = 142), however this did not translate to an exaggerated rate of adverse events. Similar prevalence of LV hyper-trabeculation has been reported in multiple studies of healthy cohorts with no association to poor outcomes (9, 11). Notably, analysis of 2,742 asymptomatic individuals free of cardiovascular disease from the multi- ethnic study of atherosclerosis (MESA) with 9.5 years follow- up showed no association between adverse cardiac remodeling and LV trabeculation extent (10). Amzulescu et al. report more frequent observation of LV hyper-trabeculation in a cohort of 162 dilated cardiomyopathy patients (NC/C ≥2.3 in 36%). However, clinical outcomes were related to left and right ventricular remodeling and the presence of late gadolinium enhancement (LGE), and not trabeculation extent (16). High rates of excess LV trabeculation were also reported in a study of 101 healthy pregnant women (25.4%), with resolution in 73% after childbirth (12). Studies of healthy athletes also report higher rates of excess LV trabeculation with no clear prognostic relevance (13). These cohorts have in common LV dilatation as part of their LV remodeling phenotype. It is conceivable, that increased cavity size allows better visualization and delineation of trabeculae, thereby introducing a systematic measurement error toward over- estimation. Indeed, we also observed greater measures of LV trabeculation in individuals with larger LV volumes. Systematic over-estimation of the trabecular component seems more likely than genuine increased trabeculation in these populations or de-novo appearance and spontaneous disappearance of LV trabeculae in pregnancy and post-partum as had been suggested (12). Whilst initially presented as a rare genetic cardiomyopathy with poor outcomes, growing evidence from a number of sources in diﬀerent populations shows high prevalence of LV hyper-trabeculation by imaging criteria with no evidence of associated prognostic signiﬁcance. Furthermore, the genetic and embryologic evidence for the etiology of LVNC are equivocal, with the absence of a clear gene-phenotype link and lack of evidence for an embryologic myocardial compaction phase. It seems increasingly likely that the initial reported poor outcomes relating to LVNC were the biased results of highly selective tertiary centre cohorts. Summary of Findings In this multicenter multinational study of real-life patients with clinical indication for CMR, we identiﬁed no association between the extent of LV trabeculation and increased risk of all-cause mortality, severe heart failure, stroke, atrial ﬁbrillation, or MACE composed of all these parameters. The same result was observed with subgroup analysis of individuals without IHD and with stratiﬁed analysis using the threshold of NC/C >2.3. Women and younger individuals had greater magnitudes of LV trabeculation, as did those with larger cavity volumes and lower LVEF. September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 8 LV Hypertrabeculation and Cardiovascular Outcomes Zemrak et al. overlap with other cardiomyopathies and no single gene has been reliably and speciﬁcally linked to LVNC (24). Additionally, the embryologic origins of LVNC have been disputed with recent studies contesting the existence of an in-utero “myocardial compaction” phase (24–26). The intra-uterine period is a hugely important phase of cardiac development, during which the cardiovascular system is exposed to marked haemodynamic change and detectable changes in adult cardiac morphology have been demonstrated with alterations in the intra-uterine environment (27). It is conceivable that the observed diﬀerences in patterns of LV trabeculation are an epiphenomenon reﬂecting the haemodynamic changes, rates of myocardial growth, and myocardial diﬀerentiation in-utero rather than a distinct genetic cardiomyopathy. of the information available. Our observations reﬂect short- term risk with an average follow-up period of just over 12 months; we cannot exclude longer term prognostic signiﬁcance of LV trabeculation. CONCLUSION This study adds to the growing evidence that LV hyper- trabeculation in isolation is not a predictor of risk and does not mandate further investigation or follow-up. In cases, where there is uncertainty regarding the existence of LVNC as a distinct cardiomyopathy, physicians should take a holistic approach and exert caution in making this diagnosis. This study adds to the growing evidence that LV hyper- trabeculation in isolation is not a predictor of risk and does not mandate further investigation or follow-up. In cases, where there is uncertainty regarding the existence of LVNC as a distinct cardiomyopathy, physicians should take a holistic approach and exert caution in making this diagnosis. gg Ivanov et al. present the only other study, aside from ours, assessing the signiﬁcance of LV trabeculation extent in a real-life clinical setting (15). Consistent with our ﬁndings, they report no association between LV trabeculation extent and adverse clinical events (death, ischemic stroke, ventricular tachycardia/ventricular ﬁbrillation, heart failure hospitalization) in 700 consecutive patients undergoing clinical CMR in a single centre over 7 years of prospective follow-up. Interestingly, a multicenter Italian study with a more selective population of individuals with a diagnosis of LVNC based on clinical and imaging criteria also reports no prognostic value of LV trabeculation extent above and beyond LV dilation, LV systolic dysfunction, and presence of LGE over 2 years of prospective follow-up (17). DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Comparison With Existing Literature It is clear from the available evidence, that the current imaging criteria for quantiﬁcation of LV trabeculation are too broad, capturing high proportions of individuals with no underlying cardiac disease and no increased risk of adverse events. Frontiers in Cardiovascular Medicine \| www.frontiersin.org REFERENCES 12. Gati S, Papadakis M, Papamichael ND, Zaidi A, Sheikh N, Reed M, et al. Reversible de novo left ventricular trabeculations in pregnant women. Circulation. (2014) 130:475–83. doi: 10.1161/CIRCULATIONAHA.114. 008554 1. Rosa LV da, Salemi VMC, Alexandre LM, Mady C. Noncompaction cardiomyopathy - a current view. Arq Bras Cardiol. (2011) 97:e13– 9. doi: 10.1590/S0066-782X2011000900021 13. Gati S, Chandra N, Bennett RL, Reed M, Kervio G, Panoulas VF, et al. Increased left ventricular trabeculation in highly trained athletes: do we need more stringent criteria for the diagnosis of left ventricular non- compaction in athletes? Heart. (2013) 99:401–8. doi: 10.1136/heartjnl-2012- 303418 2. Ritter M, Oechslin E, Sütsch G, Attenhofer C, Schneider J, Jenni R. Isolated noncompaction of the myocardium in adults. Mayo Clin Proc. (1997) 72:26– 31. doi: 10.4065/72.1.26 3. Oechslin EN, Attenhofer Jost CH, Rojas JR, Kaufmann PA, Jenni R. Long- term follow-up of 34 adults with isolated left ventricular noncompaction: a distinct cardiomyopathy with poor prognosis. J Am Coll Cardiol. (2000) 36:493–500. doi: 10.1016/S0735-1097(00)00755-5 14. Woodbridge SP, Aung N, Paiva JM, Sanghvi MM, Zemrak F, Fung K, et al. Physical activity and left ventricular trabeculation in the UK biobank community-based cohort study. Heart. (2019) 105:990– 8. doi: 10.1136/heartjnl-2018-314155 4. Aras D, Tufekcioglu O, Ergun K, Ozeke O, Yildiz A, Topaloglu S, et al. Clinical features of isolated ventricular noncompaction in adults long- term clinical course, echocardiographic properties, and predictors of left ventricular failure. J Card Fail. (2006) 12:726–33. doi: 10.1016/j.cardfail.2006. 08.002 15. Ivanov A, Dabiesingh DS, Bhumireddy GP, Mohamed A, Asfour A, Briggs WM, et al. Prevalence and prognostic signiﬁcance of left ventricular noncompaction in patients referred for cardiac magnetic resonance imaging. Circ Cardiovasc Imaging. (2017) 10:e006174. doi: 10.1161/CIRCIMAGING.117. 006174 5. Caliskan K. Noncompaction cardiomyopathy, a frequently overlooked entity (...but beware of over diagnosis!). Neth Hear J. (2012) 20:387– 8. doi: 10.1007/s12471-012-0314-6 16. Amzulescu MS, Rousseau MF, Ahn SA, Boileau L, Meester de Ravenstein C de, Vancraeynest D, et al. Prognostic impact of hypertrabeculation and noncompaction phenotype in dilated cardiomyopathy: A CMR study. JACC Cardiovasc Imaging. (2015) 8:934–46. doi: 10.1016/j.jcmg.2015. 04.015 6. Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, et al. REFERENCES Contemporary deﬁnitions and classiﬁcation of the cardiomyopathies: an American heart association scientiﬁc statement from the council on clinical cardiology, heart failure and transplantation committee; quality of care and outcomes research and functional genomics and translational biology interdisciplinary working groups; and council on epidemiology and prevention. Circulation. (2006) 113:1807–16. doi: 10.1161/CIRCULATIONAHA.106.174287 17. Andreini D, Pontone G, Bogaert J, Roghi A, Barison A, Schwitter J, et al. Long-term prognostic value of cardiac magnetic resonance in left ventricle noncompaction: a prospective multicenter study. J Am Coll Cardiol. (2016) 68:2166–81. doi: 10.1016/j.jacc.2016.08.053 18. Thavendiranathan P, Dahiya A, Phelan D, Desai MY, Tang WHW. Isolated left ventricular non-compaction controversies in diagnostic criteria, adverse outcomes and management. Heart. (2013) 99:681–9. doi: 10.1136/heartjnl-2012-302816 7. Elliott P, Andersson B, Arbustini E, Bilinska Z, Cecchi F, Charron P, et al. Classiﬁcation of the cardiomyopathies: a position statement from the european society of cardiology working group on myocardial and pericardial diseases. Eur Heart J. (2007) 29:270–6. doi: 10.1093/eurheartj/ehm342 19. Douglas PS, Hendel RC, Cummings JE, Dent JM, Hodgson JM, Hoﬀmann U, et al. ACCF/ACR/AHA/ASE/ASNC/HRS/NASCI/RSNA/ SAIP/SCAI/SCCT/SCMR 2008. Health policy statement on structured reporting in cardiovascular imaging. Circulation. (2009) 119:187–200. doi: 10.1161/CIRCULATIONAHA.108.191365 8. Petersen SE, Selvanayagam JB, Wiesmann F, Robson MD, Francis JM, Anderson RH, et al. Left ventricular non-compaction: insights from cardiovascular magnetic resonance imaging. J Am Coll Cardiol. (2005) 46:101–5. doi: 10.1016/j.jacc.2005.03.045 9. Kawel N, Nacif M, Arai AE, Gomes AS, Hundley WG, Johnson WC, et al. Trabeculated (noncompacted) and compact myocardium in adults: the multi-ethnic study of atherosclerosis. Circ Cardiovasc Imaging. (2012) 5:357–66. doi: 10.1161/CIRCIMAGING.111.971713 20. Wagner A, Bruder O, Schneider S, Nothnagel D, Buser P, Pons-Lado G, et al. Current variables, deﬁnitions and endpoints of the European cardiovascular magnetic resonance registry. J Cardiovasc Magn Reson. (2009) 11:43. doi: 10.1186/1532-429X-11-43 10. Zemrak F, Ahlman MA, Captur G, Mohiddin SA, Kawel-Boehm N, Prince MR, et al. The relationship of left ventricular trabeculation to ventricular function and structure over a 9.5-year follow-up: the MESA study. J Am Coll Cardiol. (2014) 64:1971–80. doi: 10.1016/j.jacc.2014.08.035 21. Suinesiaputra A, Bluemke DA, Cowan BR, Friedrich MG, Kramer CM, Kwong R, et al. Quantiﬁcation of LV function and mass by cardiovascular magnetic resonance: multi-center variability and consensus contours. J Cardiovasc Magn Reson. (2015) 17:63. doi: 10.1186/s12968-015- 0170-9 11. Weir-McCall JR, Yeap PM, Papagiorcopulo C, Fitzgerald K, Gandy SJ, Lambert M, et al. Left ventricular noncompaction: anatomical phenotype or distinct cardiomyopathy? J Am Coll Cardiol. (2016) 68:2157–65. AUTHOR CONTRIBUTIONS acknowledges support from the National Institute for Health Research (NIHR) Cardiovascular Biomedical Research Centre at Barts NHS Trust and has received funding from the European Union’s Horizon 2020 research and innovation programme (825903). SEP acts as a paid consultant to Circle Cardiovascular Imaging Inc., Calgary, Canada and Servier. FZ, SEP, and SM conceived the study. FZ collated, analyzed the CMR images, and led and performed the statistical analysis. ZR-E, FZ, and SEP wrote the manuscript. ZR-E contributed to cross-checking of the analysis. MK contributed to image analysis. All co-authors contributed to critical revision of the manuscript and approved the ﬁnal version. ETHICS STATEMENT All participating center had approval from local institutional ethics review boards, and all patients provided written informed consent in accordance to the Declaration of Helsinki, under standards of the EuroCMR registry. The patients/participants provided their written informed consent to participate in this study. Thus, there is accumulating evidence from multiple clinical studies, with a variety of study designs, in diﬀerent populations disputing the clinical signiﬁcance of LV hyper- trabeculation. Furthermore, the genetic and embryologic origins of LVNC have been questioned. Although several genetic links to LVNC have been established, there is signiﬁcant September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 9 LV Hypertrabeculation and Cardiovascular Outcomes Zemrak et al. FUNDING The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2020.00158/full#supplementary-material ZR-E was supported by British Heart Foundation Clinical Research Training Fellowship (no. FS/17/81/33318). SEP ZR-E was supported by British Heart Foundation Clinical Research Training Fellowship (no. FS/17/81/33318). SEP Frontiers in Cardiovascular Medicine \| www.frontiersin.org September 2020 \| Volume 7 \| Article 158 REFERENCES doi: 10.1016/j.jacc.2016.08.054 22. Jacquier A, Thuny F, Jop B, Giorgi R, Cohen F, Gaubert JY, et al. Measurement of trabeculated left ventricular mass using cardiac magnetic resonance September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 10 LV Hypertrabeculation and Cardiovascular Outcomes Zemrak et al. imaging in the diagnosis of left ventricular non-compaction. Eur Heart J. (2010) 31:1098–1104. doi: 10.1093/eurheartj/ehp595 27. Lewandowski AJ, Augustine D, Lamata P, Davis EF, Lazdam M, Francis J, et al. Preterm heart in adult life. Circulation. (2013) 127:197– 206. doi: 10.1161/CIRCULATIONAHA.112.126920 imaging in the diagnosis of left ventricular non-compaction. Eur Heart J. (2010) 31:1098–1104. doi: 10.1093/eurheartj/ehp595 j 23. Petersen SE. CMR and LV noncompaction∗ does it matter how we measure trabeculations? JACC Cardiovasc Imaging. (2013) 9:941–3. doi: 10.1016/j.jcmg.2013.03.007 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 24. Anderson RH, Jensen B, Mohun TJ, Petersen SE, Aung N, Zemrak F, et al. Key questions relating to left ventricular noncompaction cardiomyopathy: is the emperor still wearing any clothes? Can J Cardiol. (2017) 33:747– 57. doi: 10.1016/j.cjca.2017.01.017 Copyright © 2020 Zemrak, Raisi-Estabragh, Khanji, Mohiddin, Bruder, Wagner, Lombardi, Schwitter, van Rossum, Pilz, Nothnagel, Steen, Nagel, Prasad, Deluigi, Dill, Frank, Schneider, Mahrholdt and Petersen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 25. Sedmera D, Pexieder T, Vuillemin M, Thompson RP, Anderson RH. Developmental patterning of the myocardium. Anat Rec. (2000) 258:319– 37. doi: 10.1002/(SICI)1097-0185(20000401)258:4<319::AID-AR1>3.0. CO;2-O 26. Miquerol L, Moreno-Rascon N, Beyer S, Dupays L, Meilhac SM, Buckingham ME, et al. Biphasic development of the mammalian ventricular conduction system. Circ Res. (2010) 107:153–61. doi: 10.1161/CIRCRESAHA.110. 218156 September 2020 \| Volume 7 \| Article 158 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 11
https://openalex.org/W3031587977	http://irep.ntu.ac.uk/id/eprint/39893/1/1328798_Griffiths.pdf	English	null	Editorial: A Good Sleep: The Role of Factors in Psychosocial Health	Frontiers in neuroscience	2,020	cc-by	2,078	A Good Sleep: The Role of Factors in Psychosocial Health A good night’s sleep is vital for individuals of all ages to have eﬀective cognitive and emotional processing (Kopasz et al., 2010; Yaﬀe et al., 2014; Garbarino et al., 2016; Tarokh et al., 2016). Furthermore, prior evidence shows that sleep is associated to physical and mental health, and to overall quality of life (Gradisar et al., 2008; Shochat et al., 2014; Garbarino et al., 2016), and therefore a good sleep is of great importance (Lin C.-Y. et al., 2018; Lin P.-H. et al., 2018). Unfortunately, it is not always easy for many people to achieve good sleep (Strong et al., 2018), especially in modern society that has rapid growth in technology. Indeed, a recent systematic review and meta-analysis found that internet addiction is highly associated with sleep disturbance (Alimoradi et al., 2019). Similarly, recent research shows the association between problematic social media use and poor sleep, which indicates a contemporary public health problem concerning sleep (Wong et al., 2020). In short, there is a need to investigate how diﬀerent psychosocial factors are related to sleep in diﬀerent stages of life. Amir H. Pakpour 1,2, Mark D. Grifﬁths 3, Maurice M. Ohayon 4, Anders Broström 2,5 and Chung-Ying Lin 6* 1 Social Determinants of Health Research Center, Research Institute for Prevention of Non-communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran, 2 Department of Nursing, School of Health and Welfare, Jönköping University, Jönköping, Sweden, 3 International Gaming Research Unit, Psychology Department, Nottingham Trent University, Nottingham, United Kingdom, 4 Stanford Sleep Epidemiology Research Center, School of Medicine, Stanford University, Stanford, CA, United States, 5 Department of Clinical Neurophysiology, Linköping University Hospital, Linköping, Sweden, 6 Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hung Hom, Hong Kong Keywords: sleep, psychosocial, mental health, child, adolescent, adults EDITORIAL EDITORIAL published: 26 May 2020 doi: 10.3389/fnins.2020.00520 published: 26 May 2020 doi: 10.3389/fnins.2020.00520 Edited and reviewed by: Luis de Lecea, Stanford University, United States Edited and reviewed by: Luis de Lecea, Stanford University, United States Therefore, this special issue focuses on a variety of psychosocial factors associated with sleep in diﬀerent age groups and contexts and comprises a systematic review and meta-analysis together with eight empirical papers. The systematic review and meta-analysis investigated by Magnavita et al. screened 749 studies, of which 34 were reviewed and seven were included in meta-analysis. They concluded that sleep problems could be increased by workplace violence (OR = 2.55; 95% CI = 1.77–3.66). In addition to the systematic review and meta-analysis, the other eight studies included in the special issue demonstrate the variety of diﬀerent psychosocial factors that contribute to sleep across diﬀerent populations. More speciﬁcally, four studies comprised Taiwanese populations, including female college students (Lin et al.), adolescents (Ho et al.; Hsieh et al.), and children (Lin), one study comprised Hong Kong children (Chien et al.), one study comprised the Polish general population (Herzog-Krzywoszanska and Krzywoszanski), and two studies comprised Swedish adolescents (Hedin et al.; Hena and Garmy). Correspondence: Chung-Ying Lin cylin36933@gmail.com Specialty section: This article was submitted to Sleep and Circadian Rhythms, a section of the journal Frontiers in Neuroscience Received: 01 April 2020 Accepted: 27 April 2020 Published: 26 May 2020 Specialty section: This article was submitted to Sleep and Circadian Rhythms, a section of the journal Frontiers in Neuroscience Specialty section: This article was submitted to Sleep and Circadian Rhythms, a section of the journal Frontiers in Neuroscience Received: 01 April 2020 Accepted: 27 April 2020 Published: 26 May 2020 Lin et al. recruited 503 female college students and found that students with a moderate to severe level of internet addiction had signiﬁcantly poorer sleep quality than did those with mild or normal levels of internet addiction. Furthermore, those with mild levels of internet addiction had signiﬁcantly poorer sleep quality than those not addicted to the internet. Logistic regression analysis further demonstrated the association between internet addiction and sleep quality (odds ratio = 1.05 95% CI = 1.03–1.06, p < 0.01). These ﬁndings echo the ﬁndings of the aforementioned systematic review and meta-analysis (Alimoradi et al., 2019). Editorial: A Good Sleep: The Role of Factors in Psychosocial Health Amir H. Pakpour 1,2, Mark D. Grifﬁths 3, Maurice M. Ohayon 4, Anders Broström 2,5 and Chung-Ying Lin 6 REFERENCES Lin, C. -Y., Strong, C., Scott, A. J., Broström, A., Pakpour, A. H., and Webb, T. L. (2018). A cluster randomized controlled trial of a theory-based sleep hygiene intervention for adolescents. Sleep 41:zsy170. doi: 10.1093/sleep/zsy170 Alimoradi, Z., Lin, C. -Y., Broström, A., Bülow, P. H., Bajalan, Z., Griﬃths, M. D., et al. (2019). Internet addiction and sleep disorders: a systematic review and meta-analysis. Sleep Med. Rev. 47, 51–61. doi: 10.1016/j.smrv.2019.06.004 Lin, P.-H., Lin, C.-Y., Wang, P.-Y., and Yang, S.-Y. (2018). Association between sleeping duration and health-related behaviors in college student. Soc. Health Behav. 1, 31–36. doi: 10.4103/SHB.SHB_16_18 Garbarino, S., Lanteri, P., Durando, P., Magnavita, N., and Sannita, W. G. (2016). Co-morbidity, mortality, quality of life and the healthcare/welfare/social costs of disordered sleep: a rapid review. Int. J. Environ. Res. Public Health 13:831. doi: 10.3390/ijerph13080831 Shochat, T., Cohen-Zion, M., and Tzischinsky, O. (2014). Functional consequences of inadequate sleep in adolescents: a systematic review. Sleep Med. Rev. 18, 75–87. doi: 10.1016/j.smrv.2013.03.005 Gradisar, M. M., Terrill, G. G., Johnston, A. A., and Douglas, P. P. (2008). Adolescent sleep and working memory performance. Sleep Biol. Rhythms 6, 146–154. doi: 10.1111/j.1479-8425.2008.00353.x Strong, C., Lin, C.-Y., Jalilolghadr, S., Updegraﬀ, J. A., Broström, A., and Pakpour, A. H. (2018). Sleep hygiene behaviors in Iranian adolescents: an application of the Theory of Planned Behavior. J. Sleep Res. 27, 23–31. doi: 10.1111/jsr.12566 Tarokh, L., Saletin, J. M., and Carskadon, M. A. (2016). Sleep in adolescence: physiology, cognition and mental health. Neurosci. Biobehav. Rev. 70, 182–188. doi: 10.1016/j.neubiorev.2016.08.008 Kopasz, M., Loessl, B., Hornyak, M., Riemann, D., Nissen, C., Piosczyk, H., et al. (2010). Sleep and memory in healthy children and adolescents – A critical review. Sleep Med. Rev. 14, 167–177. doi: 10.1016/j.smrv.2009.10.006 Wittmann, M., Dinich, J., Merrow, M., and Roenneberg, T. (2006). Social jetlag: misalignment of biological and social time. Chronobiol. Int. 23, 497–509. doi: 10.1080/07420520500545979 Kroese, F. M., Evers, C., Adriaanse, M. A., and de Ridder, D. T. (2016). Bedtime procrastination: a self-regulation perspective on sleep insuﬃciency in the general population. J. Health Psychol. 21, 853–862. doi: 10.1177/1359105314540014 Wong, H. Y., Mo, H. Y., Potenza, M. N., Chan, M. N. M., Lau, W. M., Chui, T. K., et al. (2020). Relationships between severity of internet gaming disorder, severity of problematic social media use, sleep quality and psychological distress. Int. J. Environ. Res. Public Health 17:1879. doi: 10.3390/ijerph170 61879 Lin, C.-Y. (2018). Citation: Citation: Pakpour AH, Grifﬁths MD, Ohayon MM, Broström A and Lin C-Y (2020) Editorial: A Good Sleep: The Role of Factors in Psychosocial Health. Front. Neurosci. 14:520. doi: 10.3389/fnins.2020.00520 May 2020 \| Volume 14 \| Article 520 Frontiers in Neuroscience \| www.frontiersin.org Pakpour et al. Sleep and Psychosocial Health Ho et al. utilized a longitudinal secondary dataset (i.e., Taiwan Youth Project; Yi et al., 2009) and a marginal structural model with stabilized inverse probability-of-treatment weights to investigate the association between unhealthy sleep practice and substance use. Their ﬁndings indicated some degree of causality showing that unhealthy sleep practice leads to adulthood substance use. A similar association between sleep and alcohol abuse was found in another study comprising Taiwanese adolescents (Hsieh et al.) Hsieh et al. used data from the 2009 Project for the Health of Children and Adolescents in Southern Taiwan (Yen et al., 2010) and found that insomnia may result from alcohol abuse, suicidality, depression, and anxiety among 6,445 high school students. Herzog-Krzywoszanska and Krzywoszanski used two Polish samples to investigate factors related to bedtime procrastination. Their ﬁrst sample comprised university students (n = 431) and their data were utilized to validate a Polish version scale of the Bedtime Procrastination Scale (BPS; Kroese et al., 2016). After conﬁrming the good psychometric properties of the BPS, Herzog-Krzywoszanska and Krzywoszanski found that studying or working needs may delay bedtime in their second sample of general population (n = 335). However, similar to the study of Chien et al., the study was cross-sectional and cannot provide strong evidence of causal relationships. Hena and Garmy used a cross-sectional study on Swedish adolescents (n = 1,518) and found that social jetlag (deﬁned as the diﬀerence between bedtime and wake-up time on school days compared to holidays larger than 2 h; Wittmann et al., 2006) was signiﬁcantly associated with increased screen time. This echoes Lin et al. ’s ﬁndings of internet addiction’s association with poor sleep. In the ﬁnal paper, Hedin et al. conducted a qualitative study exploring the facilitators and barriers for a good night’s sleep among adolescents (n = 45). Their ﬁndings indicated that adolescents understood and appreciated commonly recommended strategies for improving sleep. However, it was hard to balance their sleep and other activities. Consequently, they concluded that assisting adolescents to overcome the dilemma of ﬁnding a balance between sleep and other activities is crucial when designing sleep-promotion interventions. AUTHOR CONTRIBUTIONS C-YL wrote the ﬁrst draft. AP, MG, MO, and AB provided critical comments and editorial suggestions for revisions. All the authors agreed on the submitted version. Citation: Lin collected information from 320 Taiwanese child-parent dyads in a community-centered elementary school using a 12- week longitudinal design. She used an instrument with objective measures (i.e., a Xiaomi Mi Band 2 pedometer; Xie et al., 2018) to assess children’s sleep over 12 consecutive weeks (i.e., the children were requested to wear the pedometer on their wrists during the 12 weeks). Moreover, parents of the children completed the Kid-KINDL (Lin et al., 2017; Lin, 2018) to assess the children’s quality of life before they wore the pedometer. Her results found that better quality of life may lead to better sleep. Chien et al. found in a study comprising 391 Hong Kong children that homework involvement was positively related to children’s weekday sleep duration, and that frequency of television watching was negatively related to their weekday sleep duration. Moreover, overall participation in school activities was positively related to children’s weekend sleep duration. However, given that Chien et al. used cross-sectional design, the causality was undetermined. Frontiers in Neuroscience \| www.frontiersin.org various physical activities: comparative study. JMIR mHealth uHealth, 6:e94. doi: 10.2196/mhealth.9754 REFERENCES Comparing quality of life instruments: sizing them up versus PedsQL and Kid-KINDL. Soc. Health Behav. 1, 42–47. doi: 10.4103/SHB.SHB_25_18 Lin, C.-Y., Strong, C., Tsai, M.-C., and Lee, C.-T. (2017). Raters interpret positively and negatively worded items similarly in a quality of life instrument for children: Kid-KINDL. Inquiry 54, 1–7. doi: 10.1177/0046958017696724 Xie, J., Wen, D., Liang, L., Jia, Y., Gao, L., and Lei, J. (2018). Evaluating the validity of current mainstream wearable devices in ﬁtness tracking under May 2020 \| Volume 14 \| Article 520 Frontiers in Neuroscience \| www.frontiersin.org 2 Pakpour et al. Sleep and Psychosocial Health various physical activities: comparative study. JMIR mHealth uHealth, 6:e94. doi: 10.2196/mhealth.9754 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Yaﬀe, K., Falvey, C. M., and Hoang, T. (2014). Connections between sleep and cognition in older adults. Lancet Neurol. 13, 1017–1028. doi: 10.1016/S1474-4422(14)70172-3 The handling Editor declared a shared aﬃliation with one of the authors, MO. The handling Editor declared a shared aﬃliation with one of the authors, MO. Yen, C. F., Ko, C. H., Wu, Y. Y., Yen, J. Y., Hsu, F. C., and Yang, P. (2010). Normative data on anxiety symptoms on the multidimensional anxiety scale for children in Taiwanese children and adolescents: diﬀerences in sex, age, and residence and comparison with an American sample. Child Psychiatry Hum. Deve. 41, 614–623. doi: 10.1007/s10578-010-0191-4 Copyright © 2020 Pakpour, Griﬃths, Ohayon, Broström and Lin. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Yi, C.-C., Wu, C.-I., Chang, Y.-H., and Chang, M.-Y. (2009). The psychological well-being of Taiwanese youth: School versus family context from early to late adolescence. Int. Sociol. 24, 397–429. doi: 10.1177/02685809091 02914 May 2020 \| Volume 14 \| Article 520 Frontiers in Neuroscience \| www.frontiersin.org 3
https://openalex.org/W2325629870	https://zenodo.org/record/6596954/files/Banking-80-87.pdf	English	null	Pedagogical Innovations in Teaching Entrepreneurship	International journal of scientific research	2,012	cc-by	3,848	Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 Paper 10 PEDAGOGICAL INNOVATIONS IN TEACHING ENTREPRENEURSHIP Dr. Jomon Lonappan Asst. Professor SDM College of Business Management & P.G. centre for management studies and research, Mangalore Mobile Number:8197243483 jomonlonappan@yahoo.co.in 1. Introduction Teaching students to be entrepreneurs poses a challenge from a pedagogical perspective. for students to become entrepreneurs they must learn how to identify and pursue opportunities that create change and result in sustainable value for society. it involves student ability to apply their knowledge about how to launch a new enterprise[halonen et al.2002 p.284].Introducing entrepreneurship courses in higher education builds core competence among students ,even though not all students who are exposed to entrepreneurship education would start their own venture immediately or al later date. The question whether entrepreneurship can be taught has been a major debate in entrepreneurship research and even lead to the question whether entrepreneurship can actually be learnt [Edwards&muir2004; Rae, 2000].basic outcome of this study have been the identification of different aspects in entrepreneurship education of which some are teachable with the help of classical education tools and formats, while others do not seem to be teachable in same manner. Against the background of these findings, active learning concepts were increasingly integrated into entrepreneurship education [Sheperd&Douglas, 1996] 2. HISTORICAL CONTEXT OF ENTREPRENEURSHIP EDUCATION Katz (2003) developed the most comprehensive chronology of entrepreneurship education. While he included economic and agricultural literature and experiences dating back to 1876, and others have touted the Harvard courses taught in 1947, the reality of entrepreneurship education as a force in business schools began in the early 1970sUSC launched the first MBA Concentration in entrepreneurship in 1971 followed by the first undergraduate concentration in 1972. From there, the field of entrepreneurship began to take root 2. HISTORICAL CONTEXT OF ENTREPRENEURSHIP EDUCATION Katz (2003) developed the most comprehensive chronology of entrepreneurship education. While he included economic and agricultural literature and experiences dating back to 1876, and others have touted the Harvard courses taught in 1947, the reality of entrepreneurship education as a force in business schools began in the early 1970sUSC launched the first MBA Concentration in entrepreneurship in 1971 followed by the first undergraduate concentration in 1972. From there, the field of entrepreneurship began to take root In 1987, Zenithal & Rice reviewed some of the pioneering universities of entrepreneurship Education in America contended that education in entrepreneurship covered the entire Scope of business administration, and as such was the closest approach to the original Concept of management education available in universities at that time. 1. Introduction With the continued In 1987, Zenithal & Rice reviewed some of the pioneering universities of entrepreneurship Education in America contended that education in entrepreneurship covered the entire Scope of business administration, and as such was the closest approach to the original Concept of management education available in universities at that time. With the continued Increasing fragmentation of business education into narrow specializations, they believed that the a field of study that takes a broad, integrative, pragmatic, rational approach to Business would find itself increasingly popular with those who aspire to be entrepreneurs, Managers and top executives. Increasing fragmentation of business education into narrow specializations, they believed that the a field of study that takes a broad, integrative, pragmatic, rational approach to Business would find itself increasingly popular with those who aspire to be entrepreneurs, Managers and top executives. Also in 1987, Ronstadt proposed that entrepreneurial programs should be designed so that potential entrepreneur is aware of barriers to initiating their entrepreneurial careers and can devise ways to overcome them. Ronstadt (1987) proposed a two-continuum model of Page 77 Srinivas University Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 curricular design for Entrepreneurship education. His “structured-unstructured” continuum addressed various Methods of transferring information and expertise. Among the methods he discussed were lectures, case studies, and feasibility plans. He contended that an effective program must show students “how” to behave entrepreneurially and should also introduce them to people who might be able to facilitate their success. curricular design for Entrepreneurship education. His “structured-unstructured” continuum addressed various Methods of transferring information and expertise. Among the methods he discussed were lectures, case studies, and feasibility plans. He contended that an effective program must show students “how” to behave entrepreneurially and should also introduce them to people who might be able to facilitate their success. Four years later, Robinson & Haynes (1991) conducted a survey of universities with enrollments of at least 10,000 students to determine the extent of the growth in entrepreneurship education. While significant growth was cited, several challenges were pointed out including: The challenge that lies in developing existing programs and personnel, thus improving the Quality of the field. There are several obstacles that need to be overcome to facilitate the development of quality in the field. At the heart may be the lack of good solid theoretical bases upon which to build pedagogical models and methods. To acquire knowledge germane to entrepreneurship q g g p p Learning of knowledge, concepts and techniques about some specific area or Discipline, related to the field of entrepreneurship 1. Introduction Another obstacle is the lack of formal academic programs, representing a lack of commitment on the part of institutions. A third obstacle is the maintenance of student interest in the academic programs. Robinson and Haynes believed that entrepreneurship education had come a long way in the past 20 years, yet there were several weak points in the field that were identified through their research. Of primary concern is the lack of depth of most of the programs that were then started. Further growth would depend upon how new programs were integrated with and nurtured by the established entrepreneurship education system. 3. 3. The objectives of entrepreneurship education Through the identification of various objectives of entrepreneurship education, we might have a deeper understanding of educational needs as well as a more weighted choice of evaluative criteria and pedagogical techniques (Alberti, 1999). 3. The objectives of entrepreneurship education Through the identification of various objectives of entrepreneurship education, we might have a deeper understanding of educational needs as well as a more weighted choice of evaluative criteria and pedagogical techniques (Alberti, 1999). To develop empathy and support for the unique aspects of entrepreneurship Refers to the wish/need of some individuals to understand and learn concepts related to entrepreneurship with no intent for their direct application To acquire skills in the use of techniques, in the analysis of business situations and in the synthesis of action plans This objective aims at promoting skills of analysis and synthesis in the use of knowledge about accounting, finance, marketing and general management in a holistic way To identify and stimulate entrepreneurial drive, talent and skill aims at increasing individuals‟ awareness of new venture career possibilities and supporting them in the development of awareness about their entrepreneurial interests, capabilities and potential To undo the risk-adverse bias of many analytical techniques This means education on how to manage risk, reducing the bias for risk-aversion. These objectives move away from traditional business education that has a bias towards quantitative analyses and an emphasis on postponing action until all the necessary data are available. Srinivas University Page 78 Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 To revise attitudes towards change This objective aims at educating people on how to encourage their subordinates to innovate To encourage new start-ups and other entrepreneurial ventures This aims at a direct stimulus in fostering new ventures, self-employment and entrepreneurial oriented careers. To stimulate the ‘affective socialization element This objective refers to the inculcation of attitudes, values, psychological mindsets and strategies necessaryfor taking on the entrepreneurial role. In other words, entrepreneurship education aims at building the so-called entrepreneurial competencies, which are considered as combinations of the different skills, knowledge and attitudes above listed (Fiet, 2001a). The analysis of the objectives of entrepreneurship programs introduces a deeper examination of the different audiences for entrepreneurship education. Importance of Entrepreneurship Education Web-based learning 11. Video recorded lectures 4. METHODOLOGY Empirical Study was conducted among 250 students of Executive M.B.A Program in the age group of 20-30 years. 5.1 Sources of Data: Secondary Data: Secondary data is obtained from Journals & Websites. Primary Data: Primary data were obtained through Questionnaires & Schedules. All the collected Data were analyzed to form the final Conclusion. 5.2 Analysis: Data and Information collected were analyzed by Likert Scale. 5.3 Limitations of the Study: Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 debate Learning from “expert” frameworks of teacher Learning by discovering (under guidance) Learning from feedback from one key person Learning from reactions of many people Learning in well organized, timetable environment Learning in flexible, informal environment Learning without pressure of immediate goals Learning under pressure to achieve goals Copying from others discouraged Learning by borrowing from others Mistakes feared Mistakes learned from Learning by notes Learning by problems solving Fonte: Duchéneaut, 2001: 133 By Combining Didactic Model and learning from each other we can generally classify the teaching methods into following categories. 1. Case Study 2. Group Discussion 3. Individual Presentation 4. Individual Written Report 5. Group Project 6. Formal Lectures 7. Guest Speakers 8. Action Learning 9. Seminar 10. Web-based learning 11. Video recorded lectures 4. METHODOLOGY Empirical Study was conducted among 250 students of Executive M.B.A Program in the age group of 20-30 years. Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 debate Learning from “expert” frameworks of teacher Learning by discovering (under guidance) Learning from feedback from one key person Learning from reactions of many people Learning in well organized, timetable environment Learning in flexible, informal environment Learning without pressure of immediate goals Learning under pressure to achieve goals Copying from others discouraged Learning by borrowing from others Mistakes feared Mistakes learned from Learning by notes Learning by problems solving Fonte: Duchéneaut, 2001: 133 By Combining Didactic Model and learning from each other we can generally classify the teaching methods into following categories. 1. Case Study 2. Group Discussion Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 10. Web-based learning 11. Video recorded lectures 4. METHODOLOGY Empirical Study was conducted among 250 students of Executive M.B.A Program in the age group of 20-30 years. Importance of Entrepreneurship Education Importance of Entrepreneurship Education Entrepreneurship is a key driver of our economy. Wealth and a high majority of jobs are created by small businesses started by entrepreneurially minded individuals, many of whom go on to create big businesses. People exposed to entrepreneurship frequently express that they have more opportunity to exercise creative freedoms, higher self esteem, and an overall greater sense of control over their own lives. As a result, many experienced business people political leaders, economists, and educators believe that fostering a robust entrepreneurial culture will maximize individual and collective economic and social success on a local, national, and global scale. It is with this in mind that the National Standards for Entrepreneurship Education were developed: to prepare youth and adults to succeed in an entrepreneurial economy. Entrepreneurship education is a lifelong learning process, starting as early as elementary school and progressing through all levels of education, including adult education. The Standards and their supporting Performance Indicators are a framework for teachers to use in building appropriate objectives, learning activities, and assessments for their target audience. Using this framework, students will have: progressively more challenging educational activities; experiences that will enable them to develop the insight needed to discover and create entrepreneurial opportunities; and the expertise to successfully start and manage their own businesses to take advantage of these opportunities. g g pp Table 1 - Teaching methods Theoretical Discussion Didactic model Learning from each other Learning from teacher alone Learning from each other Passive role as listener Learning by doing Learning from written texts Learning from personal exchange and Srinivas University Page 79 Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 debate Learning from “expert” frameworks of teacher Learning by discovering (under guidance) Learning from feedback from one key person Learning from reactions of many people Learning in well organized, timetable environment Learning in flexible, informal environment Learning without pressure of immediate goals Learning under pressure to achieve goals Copying from others discouraged Learning by borrowing from others Mistakes feared Mistakes learned from Learning by notes Learning by problems solving Fonte: Duchéneaut, 2001: 133 By Combining Didactic Model and learning from each other we can generally classify the teaching methods into following categories. 1. Case Study 2. Group Discussion 3. Individual Presentation 4. Individual Written Report 5. Group Project 6. Formal Lectures 7. Guest Speakers 8. Action Learning 9. Seminar 10. Importance of Entrepreneurship Education 5.1 Sources of Data: Secondary Data: Secondary data is obtained from Journals & Websites. Primary Data: Primary data were obtained through Questionnaires & Schedules. All the collected Data were analyzed to form the final Conclusion. 5.2 Analysis: Data and Information collected were analyzed by Likert Scale. 5.3 Limitations of the Study: The Information was collected from students that may include their Perception and may not be all accurate. 4. METHODOLOGY Empirical Study was conducted among 250 students of Executive M.B.A Program in the age group of 20-30 years. 5.1 Sources of Data: Secondary Data: Secondary data is obtained from Journals & Websites. Primary Data: Primary data were obtained through Questionnaires & Schedules. All the collected Data were analyzed to form the final Conclusion. 5.2 Analysis: Data and Information collected were analyzed by Likert Scale. 5.3 Limitations of the Study: The Information was collected from students that may include their Perception and may not be all accurate. 5.3 Limitations of the Study: The Information was collected from students that may include their Perception and may not be all accurate. Page 80 Srinivas University Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 6. Results & Interpretation of Data Fig.1 % of Observations based on Teaching Methods % of Observations 30% 20% 15% 10% 5% 1 2 3 4 5 6 7 8 9 10 11 Teaching Methods Table :2 % of Observations based on Teaching Methods 6. Results & Interpretation of Data Fig.1 % of Observations based on Teaching Methods % of Observations 30% 20% 15% 10% 5% 1 2 3 4 5 6 7 8 9 10 11 Teaching Methods Table :2 % of Observations based on Teaching Methods Sl.Number Teaching Method % of Observations 1 Group Project 30 2 Case study 20 3 Guest Speaker 15 4 Individual Presentation 12 5 Group Discussion 10 6 Action Learning 5 7 Seminar 3 8 Individual Written Report 3 9 Formal Lecture 1 10 Web-based Learning 0.5 11 Video-Recorded Lectures 0.5 7. 30% of the students have opted for Group Project as a Teaching Method. 30% of the students have opted for Group Project as a Teaching Method. Importance of Entrepreneurship Education By participating in the project, students receive exposure to the resources of the Web, developed an understanding of the various functional areas within a business and how these functions were interdependent, enhanced their electronic communication skills, and experienced the entrepreneurial behavioral, affective, and cognitive attitudes that motivate individuals to succeed in small business endeavors 20% of the students have opted for Case Study as a Teaching Method. Srinivas University Page 81 The case study method of teaching is quite different from the traditional lecture or class discussion. It requires substantial work before and during class by both the professors and the students. But the effectiveness of this method in other fields suggests the work is worthwhile. 15% of the students have opted for Guest Speakers from the Industry as a Teaching Method Method Inviting guest speakers into classroom can be an effective means to raise issues, impart real- life experiences, and drive home a specific lesson. 12 % of the students have opted for giving Individual Presentation as a Teaching Method Individual Presentation requires students to describe an ethical situation they encountered in the Workplace, their relevant value systems, sources of information consulted, their role in the organization, and how they resolved the ethical situation, considering how their experiences since the time of the situation might influence analogous decision making today. 10% of the students have opted for Group Discussion as a Teaching Method 10% of the students have opted for Group Discussion as a Teaching Method Whole Group Discussion is a modified form of classroom lecture where the focus is shared between the instructor and the students for information transfer. Typically, an instructor will stand before a class and present information for the students to learn but the students will also participate by answering questions and providing examples. p p y g q p g p 5 % of the students have opted for Action learning as a Teaching Method Action Learning is a form of Problem-based learning, but it goes further in insisting th 5 % of the students have opted for Action learning as a Teaching Method Action Learning is a form of Problem-based learning, but it goes further in insisting that the problem(s) being worked on must be real, in that no one knows the answer already. 8. Conclusion: Unfortunately, the present Entrepreneurship education just concentrates on related courses. Moreover, the so called Entrepreneurship courses are similar to general business courses. But general business management education has no significant influence on entrepreneurial propensity ( Hostager and Decker; 1999). In Entrepreneurship education the much focus should be given on Group Projects and Case Study which means that Entrepreneurship educators should give much significance to the practice based approach in the learning organizations. Unfortunately, the present Entrepreneurship education just concentrates on related courses. Moreover, the so called Entrepreneurship courses are similar to general business courses. But general business management education has no significant influence on entrepreneurial propensity ( Hostager and Decker; 1999). In Entrepreneurship education the much focus should be given on Group Projects and Case Study which means that Entrepreneurship educators should give much significance to the practice based approach in the learning organizations. 3% of the students have opted for Seminar as a Teaching Method 3% of the students have opted for Seminar as a Teaching Method The seminar (despite larger numbers and generally shorter timetabling slots) is still - alongside (and usually supplementing) the lecture - a dominant pedagogic genre in English Studies. At most universities, the chances are that a large proportion of your face-to-face interaction with students will take place in seminars. 3 % of the students have opted for Individual Written Reports as a Teaching Method Very few % of the students have opted for formal lecture, Web based learning and video recorded lecture as a Teaching method 3 % of the students have opted for Individual Written Reports as a Teaching Method Very few % of the students have opted for formal lecture, Web based learning and video recorded lecture as a Teaching method 7. Challenges for Entrepreneurship Education: During the study the following problems were quoted by the students against Entrepreneurship. During the study the following problems were quoted by the students against Entrepreneurship. During the study the following problems were quoted by the students against Entrepreneurship. During the study the following problems were quoted by the students against Entrepreneurship. 1. Cultural Barriers People in India are more sensitive to emotional affinity in the work place than to the work and productivity. The caste system & its series of obligations reinforced the practice of following a family occupation rather than launching a new venture. 2. Difficulties towards start-ups Business in India is costly in terms of Time required and cost involved. There is too many rules and regulations and too much paper work involved in starting a new venture. The absence of an appropriate Entrepreneurial climate, lack of required Infrastructural facilities and lack of access to relevant technology hinder rapid Industrial development. 3. Incomplete Entrepreneurial Education: The students are not satisfied with the “hands-on” support of their University in the founding process because of this they are not confident, not capable and lack knowledge in starting a Business. Page 82 Srinivas University Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 9. References 1. Dana, L. P. (2001), „The Education and training of entrepreneurs in Asia‟, Education and Training, 43 (8/9), p. 405-415 1. Dana, L. P. (2001), „The Education and training of entrepreneurs in Asia‟, Education and Training, 43 (8/9), p. 405-415 g ( ) p 2. Dominguinhos, P. and Carvalho, L. (2006), „Promoting business creation through real world experience in g ( ) p 2. Dominguinhos, P. and Carvalho, L. (2006), „Promoting business creation through real world experience in p 3. Entrepreneurship training programmes‟ IntEnt2006 Internationalizing Entrepreneurship Education and Training: 3. Entrepreneurship training programmes‟ IntEnt2006 Internationalizing Entrepreneurship Education and Training: 4. “Innovative Formats for Entrepreneurship Education Teaching” , São Paulo, Bras 5. Dominguinhos, P., Sardinha, B., Carvalho, L., Ramalho, N., Pereira, R. (2005), „Assessing Entrepreneurial Education in 5. Dominguinhos, P., Sardinha, B., Carvalho, L., Ramalho, N., Pereira, R. (2005), „Assessing Entrepreneurial Education in 6. Higher Education in Portugal‟, Paper present at Fostering Entrepreneurship: The Role of Higher Education, Trento, Italy. 6. Higher Education in Portugal‟, Paper present at Fostering Entrepreneurship: The Role of Higher Education, Trento, Italy. 7. European Commission (2004) “Entrepreneurship” Flash Eurobarometer 160, available in http://ec.europa.eu. 8. European Commission (2003a), „Green Paper on Entrepreneurship in Europe‟, available inhttp://ec.europa.eu/enterprise/entrepreneurship/green_paper/.European Commission (2003b) 9. Ribera, J, Dominguinhos, P., Almeida, A., Bucha, A., Carvalho, L., Moura, B., Pereira, R., Sardinha, B., Silveira, R. e 10. Vieira, A. (2006) „A correlação entre a oferta formativa existente orientada para a formação de competências e aCriação de empresas a nível local». Documento de Trabalho do OEFP/IEFP. 11. Roux, I., Pretorius, M. and Millard, S. (2004), „Measuring Entrepreneurial Orientation to determine the effect of education: A National Cirriculum Intervention‟, paper presented at 14th IntEnt, Napoli. Vesper, K. H. and Gartner, W. B. (1997), „Measuring Progress in Entrepreneurship Education‟, Journal of Business Venturing, 12(5), p. 403-421. 12. Solomon, G.T., Duffy, S. and Tarabishy, A. (2002). The State of Entrepreneurship Education in the United States: a Nationwide Survey and Analysis. International Journal of Entrepreneurship Education 1(1), 1-22. Page 83 Srinivas University Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 0. Questionnaire: Table:3 Rate the effectiveness of various Pedagogical methods in Teaching Rating 1 2 3 4 5 Rating: 5 Point Likert Scale: Preference ranging from 1(not at all) to 5 (very much) (put √ (Tick) mark in appropriate column) Teaching Method 1. Case Study 2. Group Discussion 3. Individual presentation 4. 9. References Individual written report 5. Group Project 6. Formal lecture 7. Guest Speakers 8. Action Learning 9. Seminar 10. Web-based learning 11. Video recorded lectures Innovative Practices in Banking, Management, IT, Education and Social Science ISBN Number: 978-81-941751-8-6 Srinivas University Page 84
https://openalex.org/W3120505058	https://dergipark.org.tr/tr/download/article-file/1487523	English	null	Voiding Dysfunctions and Prevention after Prostate Biopsy. Prospective Observational Study	Black sea journal of health science	2,021	cc-by	5,193	VOIDING DYSFUNCTIONS AND PREVENTION AFTER PROSTATE BIOPSY: PROSPECTIVE OBSERVATIONAL STUDY Aykut BAŞER1, Mehmet Murat BAYKAM1, Mustafa Serdar ÇAĞLAYAN1, Cemil AYDIN1, Muhammet YAYTOKGIL1, Musa EKICI1 BSJ Health Sci, 4(2): 114-119. (0.2%) (Mottet et al., 2020). Black Sea Journal of Health Science doi: 10.19127/bshealthscience.853534 Open Access Journal e-ISSN: 2619 – 9041 Research Article Volume 4 - Issue 2: 114-119 / May 2021 VOIDING DYSFUNCTIONS AND PREVENTION AFTER PROSTATE BIOPSY: PROSPECTIVE OBSERVATIONAL STUDY Aykut BAŞER1, Mehmet Murat BAYKAM1, Mustafa Serdar ÇAĞLAYAN1, Cemil AYDIN1, Muhammet YAYTOKGIL1, Musa EKICI1 Abstract: The possible side effects of transrectal ultrasonography-guided prostate biopsy (TRUS-Bx), which is one of the most common procedure of urology practice, are common but tend to limit themselves. TRUS-Bx may be associated with Lower Urinary System Symptoms (LUTS). We aimed to investigate the effects of TRUS-Bx on LUTS and the factors that decrease or increase these effects and to provide information about the exposure periods of these factors. This study is a prospective observational study involving patients undergoing prostate biopsy between August 1st, 2019 to 2020. Detailed history was obtained and physical examinations were evaluated from all patients. Patients' age, PSA levels, prostate volumes, Body-Mass-Index measurements and use of alpha blocker were recorded. The effects of recorded information of patients with prostate biopsy on LUTS and uroflowmetry parameters were investigated. A total of 143 patients were included in the study after determining the exclusion criteria. A significant correlation was found only between Qmax changes after TRUS-Bx and the use of alpha blockers. All patients, have decreased Qmax flow rate observed at postprocedure 1th day compared to baseline values. During the controls performed in the 4th week, while the decrease in patients using alpha blockers reached a significantly higher value than its baseline value, it could not return to its baseline value in patients who do not use alpha blockers. Prostate biopsy is expected to cause a temporary LUTS. The effects on this LUTS can be corrected with the use of alpha blockers. Keywords: Prostate biopsy, Voiding dysfunction, LUTS, Alpha blockers, International prostate symptom score Corresponding author: Hitit University, Medical Faculty, Department of Urology, 19040, Corum, Turkey E mail: aqut85@windowslive.com (A. BAŞER) Aykut BAŞER https://orcid.org/0000-0003-0457-512X Received: January 04, 2021 Accepted: January 10, 2021 Published: May 01, 2021 Mehmet Murat BAYKAM https://orcid.org/0000-0001-9006-4275 Mustafa Serdar ÇAĞLAYAN https://orcid.org/0000-0002-5086-8671 Cemil AYDIN https://orcid.org/0000-0002-7271-5748 Muhammet YAYTOKGIL https://orcid.org/0000-0002-4956-2659 Musa EKICI https://orcid.org/0000-0002-9155-4126 Cite as: Başer A, Baykam MM, Çağlayan MS, Aydın C, Yaytokgil M, Ekici M. 2021. Voiding dysfunctions and prevention after prostate biopsy: Prospective observational study. BSJ Health Sci, 4(2): 114-119. Keywords: Prostate biopsy, Voiding dysfunction, LUTS, Alpha blockers, International prostate symptom score Corresponding author: Hitit University, Medical Faculty, Department of Urology, 19040, Corum, Turkey E mail: aqut85@windowslive.com (A. BAŞER) Received: January 04, 2021 Accepted: January 10, 2021 Published: May 01, 2021 Cite as: Başer A, Baykam MM, Çağlayan MS, Aydın C, Yaytokgil M, Ekici M. 2021. Voiding dysfunctions and prevention after prostate biopsy: Prospective observational study. 2.5. Ethical Consideration This study was approved by the Local Ethics Committee with decision number 2019-24. Uroflowmetry was performed in all patients before the TRUS-Bx, on the 1st day after biopsy and on the 4th week after biopsy, and the maximum flow rate (Qmax) rates and postvoid residual urine volume (PVR) were measured by ultrasound. The International Prostate Symptom Score (I-PSS) form was filled out before biopsy and at the 4th week after biopsy, and the scores were calculated. A questionnaire scored in the range of 0 (painless) - 10 (maximum painful) was administered to patients to evaluate their Visual Analog Scale (VAS) on the 1st day after biopsy, and the VAS score was recorded. The relationship between the changes in the Qmax values of the patients due to biopsy and age, BMI, PSA, VAS, prostate volume and the use of alpha blocker was examined by correlation analysis. 2.1. Study Design and Inclusion/Exclusion Criteria 2.1. Study Design and Inclusion/Exclusion Criteria Our research is a prospective observational study and conducted in accordance with STROBE guidelines for reporting observational studies reporting observational studies (www.strobestatement.org). Informed consent (written) was obtained from all participants. After obtaining the institutional ethics committee approval, biopsy was performed in 189 patients who underwent TRUS-Bx for the first time between August 1st, 2019 and August 1st, 2020. Prostate biopsy indication in our study; the patients had abnormal rectal examination and / or serum PSA levels above 2.5 ng/ml. History of surgical treatment for prostate, severe diabetes mellitus (patients who cannot regulate blood sugar), severe coagulation disorders (thrombocytopenia, hemophilia etc.), rectal disease such as anal fissure, anal fistula or hemorrhoid, patients with concomitant malignancies, neurological diseases, the presence of catheter and the presence of urinary infection were determined as the exclusion criteria. 143 patients were included in the study after determining the exclusion criteria. 1. Introduction ( %) ( , ) In addition to these complications, it was demonstrated in some studies that TRUS-Bx may also be associated with other urological conditions, including erectile dysfunction and/or Lower Urinary System Symptoms (LUTS) (Glaser et al., 2012). LUTS, which is reported at the rates of 6% and 25% after TRUS-Bx in the literature, is an exacerbation of short-term urinary symptoms (Glaser et al., 2012; Loeb et al., 2013; Namekawa et al., 2015). In the literature, there are studies indicating the use of alpha blockers is useful to decrease the LUTS effects of TRUS-Bx (Bozlu et al., 2003; Chung et al., 2015; Sefik et al., 2020). In our prospective observational study, we investigated the effects of TRUS-Bx on LUTS and the factors that decrease or increase these effects. We aimed to provide information about the exposure durations of these factors as a contribution to the literature. After the use of prostate-specific antigen (PSA) as a serum marker in the diagnosis of prostate cancer (PCa), Transrectal ultrasonography-guided prostate biopsy (TRUS-Bx) was applied by Hodge et al. for the first time in 1989, and it is considered as the standard method in the diagnosis of prostate cancer (Stamey et al., 1987; Hodge et al., 1989). It is estimated that over 1 million procedures are performed each year in the United States and in Europe with TRUS-Bx, which is a gold standard technique that confirms the presence of cancer in men with suspected prostate malignancy (Loeb et al., 2011; Loeb et al., 2013). The possible side effects of TRUS-Bx, which is one of the most common procedures of urology practice, are common but tend to limit themselves. The most common complications of prostate biopsy, which is performed as a safe procedure, are indicated to be hematospermia (37.4%), hematuria (14.5%), rectal bleeding<2 days (2.2%), prostatitis (1.0%), >38.5° fever (0.8%), epididymitis (0.7%) and urinary retention BSJ Health Sci / Aykut BAŞER et al. BSJ Health Sci / Aykut BAŞER et al. 114 This work is licensed under Creative Commons Attribution 4.0 International License This work is licensed under Creative Commons Attribution 4.0 International License Black Sea Journal of Health Science 3.1. Demographic and Descriptive Data g p p A total of 143 patients were included in the study after determining the exclusion criteria. The main characteristics of the patients are presented in Table 1. The correlation analysis performed between the changes in the Qmax values (named as Qmax change 1 = Day 1 after biopsy procedure Qmax – baseline Qmax, Qmax change 2 = 4th week after biopsy procedure Qmax – baseline Qmax) of the patients due to biopsy and age, BMI, PSA, VAS, prostate volume and the use of alpha blocker is presented in Table 2. A significant correlation was found only between Qmax changes and the use of alpha blockers. (Qmax change 1 and Qmax change 2 P < 0.001, rho -0.320 and P = 0.005, rho -0.252, respectively). The patients were divided into two groups according to the use of alpha blockers, and their baseline characteristics and symptoms and uroflowmetry parameters were evaluated. While 51 individuals without the use of alpha blocker were grouped as group 1, 92 individuals with the use of alpha blocker were grouped 2.2. Data Collection and Outcome Measures Detailed history was obtained from all patients, and physical examinations and rectal examinations were evaluated. Patients' age, Prostate-Specific Antigen (PSA) levels, prostate volumes (width x length x depth x 0.52), Body Mass Index (BMI) measurements and use of alpha blocker (Patients were divided into 2 classes in terms of alpha blocker use. Patients using alpha blockers; the patients who started to use alpha blocker at least 1 month before the biopsy and who had regular use after the biopsy. Patients not using alpha blockers; the patients who use irregular alpha blockers or never use alpha blockers) and pathology results were recorded. 2.4. Statistical Analysis Statistical analyses were performed using the SPSS software version 22. Qmax changing and other variables were investigated using visual (histograms, probability plots) and analytical methods (Kolmogorov- Simirnov/Shapiro-Wilk’s test) to determine whether or not they are normally distributed. Descriptive analyses were presented using means and standard deviations for normally distributed. As both parameters were normally distributed, the corelations coefficients and their significance were calculated using the Pearson test. The effect of alpha blockers using status on the change in Qmax, PVR and I-PSS by time were investigated using repeated measures analysis of variance. Greenhouse- Geisser correction was used when the sphericity assumption was violated. An overall 5% type-I error level used to infer statistical significance. And the Mann– Whitney U test to compare the values between the two groups. A P value less than 0.05 was considered statistically significant. 2. Material and Methods practice, enema was applied to all patients in the morning on the day of biopsy and local disinfection was performed using betadine before the biopsy. 12 core systematic biopsy (12 core systematic biopsy; A total of 12 core biopsies were taken from lateral and far-lateral from the apex, middle and basis of the right and left sides of the prostate.) samples were taken from all patients using a 30 cm 18 Gauge fully automatic biopsy needle. 500 mg ciprofloxacin before and after the procedure only 1 day and Non-steroidal anti-inflammatory drugs (NSAID) containing dexketoprofen for 3 days after the procedure were administered to the patients who underwent biopsy. Black Sea Journal of Health Science Baseline characteristics all patients Mean ± SD (n=143) Age (years) 64.69±6.70 BMI (kg/m2) 27.35±3.69 PSA (ng/mL) 10.03±8.98 Prostate Volume (ml) 50.10±16.28 VAS 2.56±1.79 I-PSS Preprocedure (Basaline) 13.83±8.17 Qmax Preprocedure (Basaline) (mL/sn) 12.86±5.58 PVR Preprocedure (Basaline) (mL) 53.93±52.45 Abnormal Rectal Examination Yes (%) 32 (22.4) No (%) 111 (77.6) Using Alpha Blocker Yes (%) 92 (64.3) No (%) 51 (35.7) Pathology Results Benign (%) 91 (63.6) Malign (%) 52 (36.4) VAS= visual analog scale, BMI= body mass index, Qmax= maximum flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score. Black Sea Journal of Health Science Black Sea Journal of Health Science Black Sea Journal of Health Science Black Sea Journal of Health Science as group 2. Age, BMI, VAS, PSA, Prostate volume, Preprocedure and Postprocedure 4th week I-PSS, Preprocedure-Postprocedure 1st day and Postprocedure 4th week Qmax and PVR values between both groups are presented in Table 3. Prostate volume was found to be higher in the group with the use of alpha blockers. However, no statistically significant effect was found on Qmax changes in the correlation analysis performed (Table 2). 3.2. Symptom Score The changes in I-PSS scores in repeated measures in alpha blocker using group were found to be statistically significant (P = 0.001) (Figure 1). The use of alpha blockers had a significant effect on this improvement 4th week (Table 4). Table 1. Baseline characteristics all patients Mean ± SD (n=143) Age (years) 64.69±6.70 BMI (kg/m2) 27.35±3.69 PSA (ng/mL) 10.03±8.98 Prostate Volume (ml) 50.10±16.28 VAS 2.56±1.79 I-PSS Preprocedure (Basaline) 13.83±8.17 Qmax Preprocedure (Basaline) (mL/sn) 12.86±5.58 PVR Preprocedure (Basaline) (mL) 53.93±52.45 Abnormal Rectal Examination Yes (%) 32 (22.4) No (%) 111 (77.6) Using Alpha Blocker Yes (%) 92 (64.3) No (%) 51 (35.7) Pathology Results Benign (%) 91 (63.6) Malign (%) 52 (36.4) VAS= visual analog scale, BMI= body mass index, Qmax= maximum flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score. Table 2. The correlation analysis performed between the changes in the Qmax values of the patients due to biopsy and age, BMI, PSA, VAS, prostate volume and the use of alpha blocker. Table 1. Baseline characteristics all patients as group 2. Age, BMI, VAS, PSA, Prostate volume, Preprocedure and Postprocedure 4th week I-PSS, Preprocedure-Postprocedure 1st day and Postprocedure 4th week Qmax and PVR values between both groups are presented in Table 3. Prostate volume was found to be higher in the group with the use of alpha blockers. However, no statistically significant effect was found on Qmax changes in the correlation analysis performed (Table 2). as group 2. Age, BMI, VAS, PSA, Prostate volume, Preprocedure and Postprocedure 4th week I-PSS, Preprocedure-Postprocedure 1st day and Postprocedure 4th week Qmax and PVR values between both groups are presented in Table 3. Prostate volume was found to be higher in the group with the use of alpha blockers. However, no statistically significant effect was found on Qmax changes in the correlation analysis performed (Table 2). Table 1. 2.3. Biopsy Procedure BSJ Health Sci / Aykut BAŞER et al. Biopsy was performed in patients with negative urine culture. Before TRUS-Bx, all patients were injected with 10 ml of 2% prilocaine hydrochloride into the angle between the prostate gland and seminal vesicles (5 mL each) using an 18-gauge needle. Prostate volumes measured by Transrectal ultrasonography (TRUS) (GE Health_ Lociq 200 Pro) were recorded. As a standard 115 Black Sea Journal of Health Science 3.2. Symptom Score The changes in I-PSS scores in repeated measures in alpha blocker using group were found to be statistically significant (P = 0.001) (Figure 1). The use of alpha blockers had a significant effect on this improvement 4th week (Table 4). VAS= visual analog scale, BMI= body mass index, Qmax= maximum flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score. Table 2. The correlation analysis performed between the changes in the Qmax values of the patients due to biopsy and age, BMI, PSA, VAS, prostate volume and the use of alpha blocker. Table 2. The correlation analysis performed between the changes in the Qmax values of the patients due to biopsy and age, BMI, PSA, VAS, prostate volume and the use of alpha blocker. Age BMI PSA Prostate Volume VAS Using Alpha Blocker Qmax Change 1 Change form basal to postprocedure 1th day Correlation 0.136 0.004 0.029 -0.150 - 0.071 -0.320** P value 0.134 0.967 0.746 0.099 0.432 0.000* Qmax Change 2 Change form basal to postprocedure 4th week Correlation 0.028 0.048 -0.035 -0.033 - 0.056 -0.252** P value 0.758 0.600 0.705 0.718 0.542 0.005* BMI= body mass index, PSA= prostate specific antigen, VAS= visual analog scale. Table 3. Baseline characteristics of groups according to the use of alpha blockers Grup 1 (using alpha blocker - ) (n=51) Mean±SD Grup 2 (using alpha blocker + ) (n=92) Mean±SD P Age (years) 65.39±7.51 64.30±6.22 0.354 BMI (kg/m2) 26.77±3.25 27.66±3.90 0.194 PSA (ng/mL) 10.99±10.18 9.50±8.26 0.345 Prostate Volume (ml) 44.59±12.71 53.15±17.28 0.002 VAS 2.51±1.69 2.58±1.97 0.887 I-PSS Preprocedure (Basaline) 12.64±9.56 14.48±7.26 0.198 Postprocedure 4 Week 12.09±8.34 12.63±7.41 0.694 Qmax (mL/sn) Preprocedure (Basaline) 15.39±5.78 11.17±4.77 <0.001 Postprocedure Day-1 10.86±4.38 9.71±3.95 0.126 Postprocedure 4 Week 14.49±4.85 12.62±4.13 0.022 PVR (mL) Preprocedure (Basaline) 45.43±35.37 61.16±62.87 0.116 Postprocedure Day-1 90.90±85.08 75.66±60.36 0.683 Postprocedure 4 Week 40.86±34.94 42.38±53.52 0.862 Pathology Results Benign (%) 38 (74.4) 69 (75) 0.948 Malign (%) 13 (25.5) 23 (25) BMI= body mass index, Qmax= maximal flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score, VAS= visual analog scale. Table 2. The correlation analysis performed between the changes in the Qmax values of the patients due to biopsy and age, BMI, PSA, VAS, prostate volume and the use of alpha blocker. BSJ Health Sci / Aykut BAŞER et al. 116 Table 3. 3.3. Uroflowmetry and Postvoid Residual Urine Volume urinary retention and hospitalization was observed in any patient. In both groups, a decrease was observed in Postprocedure 1st day Qmax values compared to baseline values. During the controls performed in the 4th week, while the decrease in group 2 reached a significantly higher value than its baseline value, it could not return to its baseline value in group 1. The changes in Qmax values in repeated measurements were found to be statistically significant (P < 0.001). The change in Qmax values was affected by the use of alpha blockers (P < 0.001) (Figure 1). In both group, a decrease was observed in Qmax values on the postprocedure 1th day compared to baseline values. An improvement which was above postprocedure 4th week baseline values was found in the group using alpha blockers. 3.2. Symptom Score Baseline characteristics of groups according to the use of alpha blockers Grup 1 (using alpha blocker - ) (n=51) Mean±SD Grup 2 (using alpha blocker + ) (n=92) Mean±SD P Age (years) 65.39±7.51 64.30±6.22 0.354 BMI (kg/m2) 26.77±3.25 27.66±3.90 0.194 PSA (ng/mL) 10.99±10.18 9.50±8.26 0.345 Prostate Volume (ml) 44.59±12.71 53.15±17.28 0.002 VAS 2.51±1.69 2.58±1.97 0.887 I-PSS Preprocedure (Basaline) 12.64±9.56 14.48±7.26 0.198 Postprocedure 4 Week 12.09±8.34 12.63±7.41 0.694 Qmax (mL/sn) Preprocedure (Basaline) 15.39±5.78 11.17±4.77 <0.001 Postprocedure Day-1 10.86±4.38 9.71±3.95 0.126 Postprocedure 4 Week 14.49±4.85 12.62±4.13 0.022 PVR (mL) Preprocedure (Basaline) 45.43±35.37 61.16±62.87 0.116 Postprocedure Day-1 90.90±85.08 75.66±60.36 0.683 Postprocedure 4 Week 40.86±34.94 42.38±53.52 0.862 Pathology Results Benign (%) 38 (74.4) 69 (75) 0.948 Malign (%) 13 (25.5) 23 (25) BMI= body mass index, Qmax= maximal flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score, VAS= visual analog scale. Table 3. Baseline characteristics of groups according to the use of alpha blockers 116 Black Sea Journal of Health Science parison of Preprocedure and Postprocedure I-PSS and Uroflowmetry parametters between the groups Table 4. Comparison of Preprocedure and Postprocedure I-PSS and Uroflowmetry parametters between the groups Grup 1 (n=51) Mean±SD Grup 2 (n=92) Mean±SD Preprocedure (Basaline) Postprocedure Preprocedure (Basaline) Postprocedure Day 1 4 week Day 1 4 week Qmax (mL/sn) 15.39±5.78 10.86±4.38 (P < 0.001)* 14.49±4.85 (P = 0.239)* 11.17±4.77 9.71±3.95 (P = 0.003)* 12.62±4.13 (P = 0.001)* PVR (mL) 45.43±35.37 90.90±85.08 (P = 0.002)* 40.86±34.94 (P = 0.216)* 61.16±62.87 75.66±60.36 (P = 0.018)* 42.38±53.52 (P = 0.061)* I-PSS 12.64±9.56 - 12.09±8.34 (P = 0.118)* 14.48±7.26 - 12.63±7.41 (P < 0.001)* Qmax= maximum flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score. =compared to Preprocedure (baseline). Qmax= maximum flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score. =compared to Preprocedure (baseline). Qmax= maximum flow rate, PVR= postvoid residual, I-PSS= international prostate symptom score. *=compared to Preprocedure (baseline). Figure 1. The change in Qmax values and I-PSS scores in repeated measures between groups. Figure 1. The change in Qmax values and I-PSS scores in repeated measures between groups. BSJ Health Sci / Aykut BAŞER et al. 4. Discussion Şefik et al. found this improvement on Day 7 in patients using tamsulosin after biopsy (Şefik et al., 2020). Chung et al. reported a statistically significant increase on the 1st day after prostate biopsy in patients who received tamsulosin, however, they did not find any difference in the control group (Chung et al., 2015). Bozlu et al. indicated that the improvement in Qmax started on the 7th day and increased on the 30th day in patients receiving tamsulosin (Bozlu et al., 2003). In our study, we found a statistically decrease in Qmax value in both groups with and without the use of alpha blockers on the postprocedure 1st day. We indicate that there was an even better improvement in patients using alpha blockers on the postprocedure 4th week compared to baseline values. In the evaluation performed for the scoring of pain perceived by the patients during biopsy, Şefik et al. (2020) found that the VAS score was 2.7±2.3 for the group with tamsulosin use and 4.2±2.2 for the group without tamsulosin use, and they stated that the use of tamsulosin reduced the pain. In our study, no statistical relationship was found in the VAS correlation analysis of Qmax changes. Furthermore, there was no statistically significant difference in VAS scores between the groups (for Group 1; 2.51±1.69, for Group 2; 2.58±1.97). Therefore, no finding indicating that tamsulosin use reduced the perceived pain due to biopsy was found in our study. In our study, we found that there would be a significant decrease in Qmax flow rates and an increase in residual urine volume in all patients on the 1st day after prostate biopsy regardless of the use of alpha blockers. We believe that patients should be informed about this situation they will experience after biopsy. There will be a significant improvement in Qmax and I-PSS values in case of continued use of alpha blockers in the 4th week after biopsy. The current study has some limitations. In our study that was carried out in a single center, the low number of patients may have led to failure to find some parameters that may have a significant effect. In addition, the patients in the study did not use drugs such as 5 alpha reductase inhibitors. Therefore, the fact that the effects of these drugs were not evaluated is a limitation for this study. 4. Discussion found an increase in I-PSS, although it was not statistically significant compared to baseline. It is a known fact that the use of alpha blocker provides improvements in LUTS symptoms and uroflowmetry parameters (Klein et al., 2010). Due to these effects, alpha blockers were encouraged to be used in patients after prostate biopsy (Schulman et al., 2001; O’Leary, 2001). In our study, in the correlation between the changes in Postprocedure first day and 4th week Qmax values compared to baseline and age, BMI, PSA, prostate volume, VAS, the use of alpha blockers, we found that both changes had a statistical effect on the use of alpha blockers. after prostate biopsy. With respect to the changes in I-PSS, Bozlu et al. observed a significant improvement in I-PSS on the 30th day after biopsy in those using alpha blockers (Bozlu et al., 2003). Şefik et al. also found a similar result on the 7th day after biopsy (Şefik et al., 2020). In our study, in consistent with the literature, we found a significant improvement in the I-PSS scores in the 4th week after biopsy in the group using alpha blockers. It is indicated in the literature that there is a defined correlation between BMI and LUTS (Kaplan et al., 2013). However, we did not find this correlation in the results of our study. No statistical relation was found in the BMI correlation analysis of the Qmax change. Furthermore, BMI scores for alpha blocker use were not found to be similar between the two groups. We consider that BMI has no effect on lower urinary tract symptoms due to biopsy. In the literature the effects of alpha blocker use on lower urinary tract symptoms after prostate biopsy; Bozlu et al. indicated that the use of alpha blockers led to a significant increase in Qmax after biopsy (Bozlu et al., 2003). In their study, Chung et al. found that prostate biopsy led to objective voiding dysfunction and that the use of alpha blockers before biopsy increased the Qmax values after biopsy (Chung et al., 2015). In our study, we found a deterioration in Qmax and PVR parameters in patients who did not use alpha blockers and used alpha blockers on the postprocedure 1st day. In our study, a significant improvement was observed in postprocedure 4th week Qmax values in those using alpha blockers compared to those who did not use alpha blockers. 4. Discussion Another limitation of the study is that the patients did not have any intermediate value measurements except the 1st day and 4th week after the procedure. With respect to residual urine volume, Chung et al. did not find a significant increase in the amount of residual urine on the postprocedure 1st day in patient group administered with tamsulosin compared to baseline, however, they found an increase in the amount of residual urine in the control group (without tamsulosin use) (Chung et al., 2015). In our study, we found that both groups had a deterioration in the residual urine volume on the first day after biopsy. Although no statistical difference was found according to the postprocedure 4th week baseline values, less residual urine volume remains in the use of alpha blockers compared to baseline. Although none of our patients had acute urinary retention on the postprocedure 1st day, we consider that it is necessary to watch out for the increase in residual urine volume that occurred on the first day 4. Discussion The need for prostate biopsy is based on PSA level and/or suspected DRE (Digital rectal examination) and/or imaging (Mottet et al., 2020). Short-term exacerbation of lower urinary tract symptoms (LUTS) may occur, Apart from the traditional complications of prostate biopsy, which is performed as a safe procedure (Glaser et al., 2012; Loeb et al., 2013; Namekawa et al., 2015). LUTS is evaluated by international prostate symptom score (I-PSS) as standard (Barry et al., 1992). In our study, for the evaluation of LUTS, we performed evaluations by making the measurements of maximum flow rate (Qmax) and postvoid residual (PVR) measured in addition to I-PSS evaluation. With respect to the change in PVR, a decrease was observed in postprocedure 1st day PVR measurements compared to baseline values in both groups. Although PVR values were found under the baseline in the 4th week after the procedure in both groups, there were no statistically significant differences. The change in Qmax and the changes in PVR are presented in Table 4. The study carried out by Zisman et al. is one of the first publication on the effect of prostate biopsy on lower urinary tract symptoms in the literatüre (Zisman et al., 2001). In their study carried out in 2001, Zisman et al. demonstrated that prostate biopsy had a measurable effect on voiding functions (Zisman et al., 2001). In the study on the effect of multiple core prostate biopsy and periprostatic nerve block on voiding and erectile In our study, no infectious complication requiring acute BSJ Health Sci / Aykut BAŞER et al. BSJ Health Sci / Aykut BAŞER et al. 117 Black Sea Journal of Health Science function, Klein et al. found an increase in I-PSS, although it was not statistically significant compared to baseline. It is a known fact that the use of alpha blocker provides improvements in LUTS symptoms and uroflowmetry parameters (Klein et al., 2010). Due to these effects, alpha blockers were encouraged to be used in patients after prostate biopsy (Schulman et al., 2001; O’Leary, 2001). In our study, in the correlation between the changes in Postprocedure first day and 4th week Qmax values compared to baseline and age, BMI, PSA, prostate volume, VAS, the use of alpha blockers, we found that both changes had a statistical effect on the use of alpha blockers. function, Klein et al. BSJ Health Sci / Aykut BAŞER et al. References Barry MJ, Fowler FJ Jr, O'Leary MP, Bruskewitz RC, Holtgrewe HL, Mebust WK, Cockett AT. 1992. The American Urological Association symptom index for benign prostatic hyperplasia. The Measurement Committee of the American Urological Association. J Urol, 148(5): 1549-1557. DOI: 10.1016/s0022- 5347(17)36966-5. Namekawa T, Fukasawa S, Komaru A, Kobayashi M, Imamura Y, Ohzeki T, Takagi K, Sato Y, Akakura K, Ichikawa T, Ueda T, 2015. Prospective evaluation of the safety of transrectal ultrasound-guided transperineal prostate biopsy based on adverse events. Int J Clin Oncol, 20: 1185-1191. DOI: 10.1007/s10147-015-0831-6. Bozlu M, Ulusoy E, Doruk E, Cayan S, Canpolat B, Schellhammer PF, Akbay E. 2003. Voiding impairment after prostate biopsy: does tamsulosin treatment before biopsy decrease this morbidity? Urology, 62: 1050. DOI: 10.1016/j.urology.2003.07.006. O’Leary MP. 2001. Tamsulosin: current clinical experience. Urology, 58(6 Suppl 1): 42-48; DOI: 10.1016/s0090- 4295(01)01346-2. Schulman CC, Lock TM, Buzelin JM, Boeminghaus F, Stephenson TP, Talja M, European Tamsulosin Study Group, 2001. Long- term use of tamsulosin to treat lower urinary tract symptoms/benign prostatic hyperplasia. J Urol, 166(4): 1358-1363. Chung SJ, Jung SI, Ryu JW, Hwang EC, Kwon DD, Park K, Kim JW. 2015. The preventive effect of tamsulosin on voiding dysfunction after prostate biopsy: a prospective, open-label, observational study. Int Urol Nephrol, 47: 711-715. DOI: 10.1007/s11255-015-0955-7. Glaser AP, Novakovic K, Helfand BT. 2012. The impact of prostate biopsy on urinary symptoms, erectile function, and anxiety. Curr Urol Rep, 13: 447-454. DOI: 10.1007/s11934- 012-0277-6. Sefik E, Eker A, Gunlusoy B, Celik S, Bozkurt IH, Basmaci I, Polat S, Degirmenci T, Ceylan Y. 2020. The effect of alpha blocker treatment prior to prostate biopsy on voiding functions, pain scores and health-related quality-of-life outcomes: A prospective randomized trial. Prog Urol, 30(4): 198-204. DOI: 10.1016/j.purol.2019.12.006. Hodge KK, McNeal JE, Terris MK, Stamey TA. 1989. Random systematic versus ultrasound guided transrectal core biopsies of the prostate. J Urol, 142: 71-74. DOI: 10.1016/s0022-5347(17)38664-0. Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, Redwine E. 1987. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med, 317: 909-916. DOI: 10.1056/NEJM198710083171501. Kaplan SA, Lee JY, O'Neill EA, Meehan AG, Kusek JW. 2013. Prevalence of low testosterone and its relationship to body mass index in older men with lower urinary tract symptoms associated with benign prostatic hyperplasia. Aging Male, 16(4): 169-172. DOI: 10.3109/13685538.2013.844786. Zisman A, Leibovici D, Kleinmann J, Cooper A , Siegel Y, Lindner A. 2001. 5. Conclusion Prostate biopsy is expected to cause a temporary voiding difficulty. The presence of deterioration in objective voiding symptoms 1st day after biopsy is possible despite the use of alpha blockers. It can be corrected by the continuation of the use of alpha blockers. BSJ Health Sci / Aykut BAŞER et al. 118 Author Contributions AB; protocol development and research design, data collection and management, data analysis, manuscript writing/ editing, supervision. MMB; protocol development, data collection. MSÇ; protocol development, data collection. CA; protocol development, data collection. MY; protocol development, data collection. ME; protocol development, data collection. All authors read and approved the final manuscript. Loeb S, Carter HB, Berndt SI, Ricker W, Schaeffer ED. 2011. Complications after prostate biopsy: data from SEER- Medicare. J Urol, 186: 1830-1834. DOI: 10.1016/j.juro.2011.06.057. Loeb S, Vellekoop A, Ahmed HU, Catto J, Emberton M, Nam R, Rosario DJ, Scattoni V, Lotan Y. 2013. Systematic review of complications of prostate biopsy. Eur Urol, 64: 876-892. DOI: 10.1016/j.eururo.2013.05.049. Mottet N, Cornford P, van den Bergh RCN, Briers E, De Santis M, Fanti S, Gillessen S, Grummet J, Henry AM, Lam TB, Mason MD, van der KwastTH, van der Poel HG, Rouvière O, Schoots IG, Tilki D, Wiegel T. 2020. EAU Guidelines, Uroweb.org. European Association of Urology Prostate Cancer Guidelines, URL: http://www.uroweb.org/guideline/prostate- cancer/#5;2020 (access date: 30.07.2020) . Conflict of Interest The authors declare that there is no conflict of interest. BSJ Health Sci / Aykut BAŞER et al. Black Sea Journal of Health Science The impact of prostate biopsy and periprostatic nerve block on erectile and voiding function: a prospective study. J Urol, 184: 1447-1452DOI: 10.1016/j.juro.2010.06.021. References The impact of prostate biopsy on patient well-being: a prospective study of voiding impairment. J Urol, 166: 2242- 2246. Klein T, Palisaar RJ, Holz A, Brock M, Noldus J, Hinkel A. 2010. BSJ Health Sci / Aykut BAŞER et al. 119
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https://openalex.org/W4386195554	https://link.springer.com/content/pdf/10.1007/s43615-023-00296-9.pdf	English	null	Mapping of the Circular Economy Implementation Challenges in the Fashion Industry: a Fuzzy-TISM Analysis	Circular Economy and Sustainability/Circular economy and sustainability	2,023	cc-by	17,038	* Mohamed Afy‑Shararah m.a.shararah@cranfield.ac.uk 1 Sustainable Manufacturing Systems Centre, School of Aerospace, Transport, and Manufacturing, Cranfield University, Cranfield, UK Mapping of the Circular Economy Implementation Challenges in the Fashion Industry: a Fuzzy‑TISM Analysis Aya Abdelmeguid1 · Mohamed Afy‑Shararah1 · Konstantinos Salonitis1 Received: 7 December 2022 / Accepted: 24 July 2023 / Published online: 26 August 2023 © The Author(s) 2023 Keywords Circular economy · Fashion industry · Implementation challenges · Fuzzy-TISM · Fuzzy MICMAC analysis Circular Economy and Sustainability (2024) 4:585–617 https://doi.org/10.1007/s43615-023-00296-9 Circular Economy and Sustainability (2024) 4:585–617 https://doi.org/10.1007/s43615-023-00296-9 ORIGINAL PAPER Introduction There is no doubt that the fashion industry is amongst the world’s most significant industries. Still, it is also amongst the highest contributors to environmental damage and social and ecological footprints [1, 2]. Not only does clothing play a crucial part in our daily lives, but a significant amount of the global workforce is based within the sector at over 300 million as of 2017, and consistently growing [3]. In the UK, the fashion industry has around 800,000 employees working in manufacturing, wholesale, retailing, and services, generating a revenue of over 100 billion pounds [4]. In Europe alone, fashion item manufacture employs approximately 1.7 million people and generates over €165 billion of income [5]. In 2021, the fashion industry’s global revenue was about $1.5 trillion [6]. Therefore, it massively supports employment, especially in low-income countries, and significantly supports global economies. i However, the fashion industry alone consumes over 50% of the textiles available world- wide, comprising various non-renewable natural resources [3]. Moreover, the fashion industry processes across the value chain account for nearly 20% of global water waste and almost 10% of the world’s carbon emissions per year [7, 8]. According to Atstja et al. [9], more than 95% of clothing materials can be recycled, but only 1% of them are recovered by the industry for reuse and recycling. In recent years, clothing production has increased by over 50%, and clothing usage has declined by over 40% [3]. Consequently, the fashion industry is facing a growing amount of textile waste, particularly with the rise of fast fash- ion, which increases the number of collections per year, lowers prices, and prompts quicker changes in styles and trends [10]. The rapid increase in the world’s population and the rising environmental pressures and climate change are stimulating governments, organisations, and societies to work on con- serving scarce natural resources [11]. Circular economy (CE) is one of the new approaches to address these issues. The most popular definition for the circular economy is provided by the Ellen MacArthur Foundation, which defines the circular economy as “the principles of designing out waste and pollution, keeping products and materials in use, and regen- erating natural systems” [12]. Therefore, the circular economy advocates the shift from linear economies like the “take-make-dispose” approach to the “take-make-reuse” to address the threats and consequences of the linear fashion system [8, 13]. Abstract The fashion industry creates a large amount of pollution, making it one of the least environmentally friendly industries in the world with an ever-growing adoption of the linear fashion system “take-make-dispose” that continues to exploit scarce natural resources and energy. This advocate shifting to a circular economy to achieve sustainable production and consumption. However, numerous challenges are faced when introducing the principles of circular economy, obstructing the system transition. The ten challenges identified from the literature include poor material and energy efficiency, poor circular design, technological challenges, regulatory pressures, internal stakeholders’ pressures, financial pressures, insufficient human capital, poor management and leadership, lack of external collaborations, and consumer-related challenges. This research aims to identify the level of significance of the challenges identified from the literature to assist top management with formulating a strategy. Decisions will be made and prioritised based on the key challenges to ensure a successful and effective implementation of the circular economy in the fashion industry. To achieve the aim of this research, seven experts within both the circular economy and fashion context are interviewed. Fuzzy Total Interpretive Structural Modelling (Fuzzy-TISM) is used to arrange the challenges into a hierarchy-based model and to illustrate the interrelationship between these challenges. Also, Fuzzy MICMAC analysis is used to map and rank these challenges depending on their driving and dependence power. Based on the findings, regulatory pressures and poor management and leadership are the key challenges with the highest significance level. Therefore, successful and effective implementation of a circular economy requires a change in managerial strategies. 56789) 3 Circular Economy and Sustainability (2024) 4:585–617 586 1 3 Literature Review Existing literature identifies various challenges to introducing a CE, and further research categorises these challenges into different categories. Table 1 provides an overview of reviewed sources that focus on the challenges of introducing a CE without a specific indus- try context. On the other hand, Table 2 illustrates an overview of the challenges reviewed from the literature focused on introducing a CE in the fashion industry context. Based on the review of existing literature and research, this paper focuses on the chal- lenges of introducing a CE in the fashion industry whilst also considering the challenges faced in other sectors when applicable. Therefore, ten main challenges of introducing CE in the fashion industry were identified, including poor material and energy efficiency, poor circular design, technological challenges, regulatory pressures, internal stakeholders’ pressures, financial pressures, insufficient human capital, poor management and leader- ship, lack of external collaborations, and consumer-related challenges (Fig. 1). These chal- lenges cover most areas and are discussed in detail in the following sub-sections. Introduction What are the key challenges of implementing a CE in the fashion industry identified from the literature that need prioritising and addressing by management? i 3. What are the key challenges of implementing a CE in the fashion industry identified from the literature that need prioritising and addressing by management? The paper aims to achieve its objectives and address the research questions throughout its six sections: “Introduction”, “Literature Review”, “Methodology”, “Results”, “Discus- sion”, and “Conclusion”. Introduction Moreover, the United Nations set 17 Sustainable Development Goals (SDGs). One way to fulfil them is to implement a CE, particularly SDG 12: “Responsible Consumption and Production”, which emphasises efficient management and use of scarce natural resources and goods [10, 14, 15]. The CE is considered a systematic approach aiming to reduce waste from occurring in the first place and keep resources within the economy. This is done by re-distributing items’ intrinsic value by reusing instead of dismissing them. However, the fashion industry must deal with challenges that obstruct the shift from a linear to a CE.f The efforts to explore these challenges have been growing in recent years due to the emerging nature of the CE in the fashion context. The existing research has proposed dif- ferent categorizations to resolve the challenges of implementing a CE in the fashion indus- try. However, the current research lacks the information required at the early stages of the decision-making phase that identifies the level of significance for each challenge. This causes the arrival of further challenges within the implementation process. This research identifies the level of significance for several challenges identified from the literature to assist top management with formulating a strategy and making decisions for a success- ful and effective implementation. This research contributes to the gap in existing research 1 3 Circular Economy and Sustainability (2024) 4:585–617 587 by using the Fuzzy-TISM to identify the interrelationship between the challenges and to arrange these challenges into a hierarchy-based model. This helps to simply visualise the complex system of challenges and reveal direct and indirect impacts of each of the chal- lenges identified from the literature. Fuzzy MICMAC will additionally analyse, map, and rank these challenges depending on their driving and dependence power. To achieve the aim of this paper and fill the research gap, the research is designed to address the following research questions: 1. What is the interrelationship between the challenges of implementing a CE in the fashion industry identified from the literature?i 1. What is the interrelationship between the challenges of implementing a CE in the fashion industry identified from the literature?i i 2. What is the significance level of the challenges for implementing a CE in the fashion industry identified from the literature?i i 2. What is the significance level of the challenges for implementing a CE in the fashion industry identified from the literature?i i 3. Poor Material and Energy Efficiency The fashion industry’s transition to a CE poses several challenges. One is maintaining the quality and quantity of materials and energy efficiency whilst having the lowest possible environmental impact and highest value of materials [19]. The current linear fashion sys- tem adds to these challenges by increasing the number of collections per year, lowering prices, and prompting quicker changes in styles and trends [10]. Therefore, the linear fash- ion system has increased the use of non-renewable and unsustainable materials and energy in an unsustainable and uneconomic manner [20, 21]. However, the fashion industry’s challenge concerning materials and energy is limited to the scarcity of resources, dictated by environmental impacts and climate change of current raw material production, and the poor understanding and management of the 1 3 Circular Economy and Sustainability (2024) 4:585–617 588 Table 1 Overview of the challenges faced in introducing circular economy (without specific industry con- text) Authors Challenges Kumar et al. [16] 15 challenges faced throughout the supply chain: (1) Insufficient legislation and control, (2) lack of governmental guidance and support, (3) risk of mismanagement, (4) poor waste management, (5) lack of management support, (6) short-term goals, (7) insufficient funds available for the industry, (8) inefficient execution of framework, (9) lack of skilled workforce, (10) employee resistance for change, (11) Lack of planning for the implementation and integration of CE and industry 4.0, (12) lack of understanding industry 4.0, (13) use of materials as energy, (14) poor resource quality, (15) insufficient market demand Tura et al. [17] Categorise the challenges into seven categories: (1) Environmental, (2) institutional, (3) economic, (4) social, (5) organisational factors, (6) supply chain, (7) technological and informational Hina et al. [11] Categorise the challenges into two categories: (1) Internal challenges: organisational strategies and policies, product design, internal stakeholders, financial challenges, collaborations, technological difficulties, and lack of viable resources (2) External challenges: supply chain-related challenges, environmental challenges, regulatory challenges, social and cultural challenges, economic challenges, and consumer-related challenges García-Quevedo et al. [18] Categorise the challenges into two categories (focused on European SMEs): (1) insufficient financial and human resources, and (2) the complexity of administrative obligations and legislations Table 2 Overview of the challenges the fashion industry faces in introducing a CE Authors Challenges Colucci and Vecchi [2] Categorise the challenges into three categories: (1) operational challenges, (2) Technical challenges, and (3) customer-related issues Abdelmeguid et al. Circular Economy and Sustainability (2024) 4:585–617 589 7 5 12 5 6 7 7 8 6 7 0 2 4 6 8 10 12 14 Poor material and energy eﬃcienc Poor circular design Technological challenges Regulatory pressures Internal stakeholders pressures Financial pressures Insuﬃcient human capital Poor management and leadership Lack of external collaboraon Consumer-related challenges Frequency of appearance Challenges Fig. 1 The frequency of appearance of the ten challenges identified in the literature reviewed Fig. 1 The frequency of appearance of the ten challenges identified in the literature reviewed current and potential material flow [22]. Although the extraction of raw materials is energy-intensive, there is still not enough attention given to the importance of utilis- ing waste, making use of reusable, recycled materials, and biodegradable material to ease the flow and recovery of materials [22]. Moreover, some synthetic fibres require enormous amounts of energy and chemicals whilst generating toxic waste and pollut- ing materials in their production, which cause environmental impacts and human health concerns [19]. Furthermore, by implementing circular strategies and activities, materi- als and energy can be used more efficiently, and production levels can be raised. How- ever, this can lead to an increasing risk of circular rebound, which offsets the benefits of a circular economy [23]. Poor Material and Energy Efficiency [8] Categorise the challenges into two categories: (1) Hard aspects: financial challenges, regulatory challenges, circular business model-related challenges, and pressures from stakeholders (2) Soft aspects: consumer-related challenges and green intellectual capital Table 2 Overview of the challenges the fashion industry faces in introducing a CE 1 3 1 Circular Economy and Sustainability (2024) 4:585–617 Poor Circular Design The circular design must redefine and reimagine products and services to improve waste management and support shifting to a CE. The design of circular systems should include strategies to extend the lifespan of products, such as swapping, renting, leasing, and repairing, as well as strategies to encourage circularity at the end of their useful lives, such as repairing, reusing, recycling, and remanufacturing [16, 20]. However, with fast fashion dominating the market, products are not designed to last for long or active use indicating poor circular design [24]. This is due to the fashion industry’s competitive environment with a constantly changing market demand and customer preference, which pressures designers to rapidly create new styles and trends instead of focusing on innovative and efficient circular design [25]. Additionally, cre- ating optimal circular products and services whilst keeping consumers’ preferences in mind is often a challenge for the fashion industry. A circular and resource-efficient solu- tion must be accompanied by the desired features, performance, and quality expected by their consumers to ensure an overall success [26]. 1 3 Circular Economy and Sustainability (2024) 4:585–617 590 Technological Challenges The technology available in the fashion industry challenges CE implementation. This is because standard technologies used in the production process are designed to support fast- fashion business models, thereby designed to support high-scale and mass production at a lower cost and with shorter production cycles [2, 21]. Thus, circular economy business models are insufficiently supported by advanced and efficient technologies that run cost- effectively without compromising quality [8, 17]. f A limited number of technologies are available to support the shift to a CE and over- come the issues caused by the currently available and used technologies [27]. These CE- oriented technologies must facilitate CE strategies such as optimising, remanufacturing, and regenerating resources [28]. CE-oriented technologies include sorting, recycling, and printing technologies, detection and separation of fibres and materials technology, mate- rial recovery, and waterless dying technologies [11, 16, 20]. Other sustainability-inspired technologies include additive manufacturing, cloud manufacturing, Cyber-Physical Sys- tems (CPS), and the Internet of Things (IoT) [29]. These technologies could provide vari- ous circular qualities, including obtaining primary data that helps with decision-making and reverse logistics by monitoring real-time movements within the transportation sys- tems, providing information about the availability of materials and sharing manufacturing resources, and estimating carbon emissions [16]. The limited availability of these technologies, accompanied by the challenge of strategic and efficient integration within the business models, and a fundamental lack of knowledge in their operation and implementation to resolve CE challenges, are some of the major bar- riers to a successful adoption [22]. . Furthermore, technological advancements could potentially lead to circular rebounds if not used effectively and efficiently [23]. 1 3 Internal Stakeholders’ Pressures Collaboration and engagement amongst all internal stakeholders, including employees, managers, and owners, are required to introduce a CE in the fashion industry [7, 13]. Their collective action and effort are critical in planning and developing business strategies [31]. The involvement of all internal stakeholders increases constraints and pressures due to the different self-interests and needs of the various stakeholders [8, 16]. Also, internal stakeholders in the fashion industry need economic reasoning and benefits as an incen- tive to transition to a CE [32]. Therefore, it is very difficult to align and connect the goals of different internal stakeholders and meet their requirements, which makes the transition to a CE very challenging. However, the commitment of internal stakeholders alone is not enough. Commitment and engagement of external stakeholders, such as consumers, are also essential to achieve circularity in the fashion industry [13]. Financial Pressures The fashion industry faces various financial pressures when transitioning to a CE [8]. This is usually due to limited financial means available, limited financial support provided, and high risk of misinvestment into the CE, particularly for SMEs and start-ups [16–18]. Also, the change requires many investments at the start to enable investments in the right CE- oriented technologies and infrastructure [33]. Moreover, the poor financial performance and difficulty in measuring and evaluating the efficiency of the circular business models hamper the transition’s long-term environmental and social benefits [16]. This is because the CE is considered a new concept that implies uncertainty about its short-term profitabil- ity, and thereby, is usually not supported by financial markets [8, 34]. The fashion industry also faces the challenges of circular systems’ costs and inefficiency, for instance, recycling or separating materials [35]. These higher costs and inefficiencies sometimes lead to a rise in the price of sustainable and circular fashion goods and services, affecting consumers’ purchasing and product usage decisions as they are unwilling to pay more [35]. Regulatory Pressures In the fashion industry, regulations and legislation are seen as one of the most significant barriers to introducing a CE. Governments have the most control over how companies and retailers operate and play a crucial role for introducing a CE [8, 18]. Therefore, the transi- tion needs to be complemented by supportive legal frameworks.fi Insufficient regulations in some countries could encourage the outsourcing of produc- tion processes to benefit from fewer or weaker sustainable legislations and circular prac- tices in place. It could potentially lead to unethical practices such as exploitation of the workforce and poor working conditions [30]. Furthermore, having insufficient regulations or legal obligations for consumers to support circular initiatives made within the industry could be very challenging for the industry to adopt a CE. Challenging regulations and reduced taxes placed by the governments within the fash- ion industry can drive a successful transition to a CE. Therefore, governments can support the fashion industry through various ways, including implementing strict regulations and lowering taxes. However, governments around the world fail to implement efficient strate- gies that would help with waste minimisation, as well as fail to provide support for the complex administration requirements and high administration costs that act like a barrier when transitioning to a CE, particularly for SMEs [16, 18]. Additionally, there is insuf- ficient governmental support and guidance to create a stream of textile waste derived from 1 3 Circular Economy and Sustainability (2024) 4:585–617 591 different companies, which hinders the opportunity for circular waste management at scale, [22]. different companies, which hinders the opportunity for circular waste management at scale, [22]. Poor Management and Leadership Management and leadership are critical factors for the introduction of circular strategies. Management and leadership shape the organisational culture, which plays a crucial role in organisational behaviour and the successful application of circular systems [37]. The shift to a CE within the fashion industry requires changes in the organisational culture, which may be threatening to some individuals and may be accompanied by high-risk aversion and high costs in the short-term [8, 34, 41]. Therefore, good leadership and management capabilities are essential for implementing fundamental changes within the industry [11, 42]. This helps in understanding the internal and external organisational issues and needs, as well as helping in considering the whole system and how it operates to support the intro- duction of a CE [41]. In addition, good management and leadership help understand the needs of the employees and boost motivation and development of the workforce to support the transition [11]. However, there is a lack of managerial and leadership skills required to distribute cir- cular knowledge and adopt circular strategies, which usually leads to insufficient use of human capital and other organisational resources [8, 31]. This lack of effective manage- ment and leadership also leads to a lack of drive and motivation to implement circular- ity and a failure to raise awareness of the significance of transitioning to a CE system [8, 36]. Furthermore, poor management and leadership act like a barrier to initiating visions in the decision-making process and implementing actions, since it obstructs the ability to adopt the essential strategies in planning, organising, and controlling [36]. Therefore, poor management and leadership hinder the fashion industry’s ability to transition to a circular economy. Insufficient Human Capital Providing adequate and relevant training and knowledge to employees within the fashion industry is necessary to ensure that the resistance to change is minimised and increases the level of commitment to this change [36]. Also, it increases the ability to know more about sustainable materials and resources and the use of new technologies, circular strategies, and techniques [20, 37]. In addition, the involvement of employees in the decision-making process empowers the companies staff members, benefitting both the employees and the competitive advantage of the organisation. Nevertheless, the fashion industry’s lack of skilled and knowledgeable human capital causes a further challenge for CE introduction [8, 16]. As the fashion industry fails to rec- ognise the crucial part that human capital plays in the manufacturing system and the over- all life cycle of the system, it fails to consider the significant role it plays in organisational development and change, when implementing a CE [36]. Also, it fails to recognise the 1 3 3 Circular Economy and Sustainability (2024) 4:585–617 592 significance of imitable, non-substitutional, and rare human capital in achieving a competi- tive advantage [38, 39]. Therefore, organisations and brands are not paying much attention to allocating enough time and adequate finances to share knowledge, train and educate the workforce regarding circularity [18]. Consequently, there appears to be insufficient knowl- edge, skill set, and behaviour concerning the CE, management of environmental and sus- tainability concerns, and environmental protection [40]. This obstructs the ability of the fashion industry to transform into a CE. Lack of External Collaborations Collaboration networks within the fashion industry with external partners, including brands, retailers, suppliers, and governments, facilitate the sharing of knowledge and expertise, support the development of new environmental ideas and circular strategies, ena- ble technological advancements and circular innovation, and reduce the costs of research and development [40]. In addition, collaboration networks encourage sharing materials, garments, services, tools, and other resources. Therefore, the value chain becomes more connected, and their vision is more aligned, facilitating the communication of best prac- tices and introducing CE principles within industries [43]. However, the lack of external collaborations hinders the fashion industry’s transition to a CE [8]. This is due to the lack of transparency stemming from a lack of trust and the incompatibility between partners across the value chain, in turn restricting the exchange of knowledge, expertise, and activities between collaboration networks [11]. This reduces the ability to reduce costs, develop new ideas, reduce the risks associated with long-term Circular Economy and Sustainability (2024) 4:585–617 593 investments and the opportunity of collaborating with the government [44]. External col- laboration also relies on three supporting factors: challenges from the external environ- ment (regulations, society, and expectations), challenges from the competitive environment (suppliers, competitors, consumers, and intermediates), and the search for the competitive advantage (cost and differentiation qualities and features) [45]. Therefore, the absence or insufficiency of these three driving factors could act as a barrier to external collaboration and restrict its supporting opportunities in introducing circularity in the fashion industry. Consumer‑Related Challenges Based on Berberyan et al. [46], there are variables related to the product, such as price, quality, availability and accessibility, product design, and ethical identification. In compari- son, the variables related to the consumers include personal interests, norms, habits, con- venience, social desirability bias, and perception. Usually, variables related to the product are the ones that could be managed. As variables related to consumers are more difficult to predict and control due to the conflicting external behaviours regarding the purchase, use, and disposal of products or services within the fashion industry which creates circularity dilemmas [8, 47]. The attitude-behaviour gap is one of the main consumer-related challenges. There has been a massive gap between understanding the need for ethical clothing and circular prod- ucts, services to tackle environmental concerns, and consumers’ purchase, usage, and dis- posal behaviour [48]. For example, consumers first identify the price when purchasing. Yet circular and sustainable products and services tend to be more expensive to compensate for the higher costs encountered throughout the production process. This is referred to as a circular premium, and it acts as a challenge since it advocates a high risk of losing exist- ing consumers who may not accept this increase in price [49]. Although circular strategies within the fashion industry could be communicated at a larger scale, through initiatives such as enhanced marketing mechanisms and platforms to educate consumers, and promote awareness amongst them, this still relies on consumers’ mindsets and behaviours that are hard to anticipate and manage [16, 17, 20]. Methodology To achieve the aim of this research, Fuzzy Total Interpretive Structural Modelling (Fuzzy- TISM) is used to deduct a structured model to illustrate the relationship between the chal- lenges, and Fuzzy MICMAC analysis is used to map and rank these challenges based on their driving and dependence power. The driving power in this research refers to a higher level of significance, independence, and influence of some of the challenges on others, thereby helping to successfully address other challenges and implement a CE in the fashion industry [50, 51]. In contrast, dependence power in this research refers to the challenges that rely on others when being addressed to achieve a successful implementation and have minor or no influence on other challenges [50, 51]. l Fuzzy-TISM is a qualitative research tool and structured approach based on collect- ing data from a limited number of experts within the research criteria, which lie in the fashion context of this research, using focus groups, questionnaires, or interviews [52]. The experts do not need to have any previous knowledge of the underlying method. Furthermore, Fuzzy-TISM is the fuzzy extension of the ISM, which is a pairwise 1 3 3 Circular Economy and Sustainability (2024) 4:585–617 594 comparison methodology for identifying and summarising relationships between vari- ables that define a problem or a challenge [51]. However, it has been updated to include the interpretation of each relationship by qualitative considerations on a 0 to 1 scale to show the direction of the relationship and the degree of influence [53]. comparison methodology for identifying and summarising relationships between vari- ables that define a problem or a challenge [51]. However, it has been updated to include the interpretation of each relationship by qualitative considerations on a 0 to 1 scale to show the direction of the relationship and the degree of influence [53]. l The research approach used to achieve the objectives of this paper is an inductive approach. This means that the theoretical ideas and the model created are derived from the data collected from interviews. Therefore, the philosophical position underpinning this research is interpretivism because the research focuses on the experts’ subjective experiences and interpretations of the world, hence highly dependent on the experts’ lived experiences, insights, and real-life situations [55]. This paper adapts the Fuzzy-TISM approach suggested by Khatwani (2015). However, the steps have been slightly modified. Methodology Therefore, eight steps of Fuzzy-TISM are adopted in this paper, noting that Step 1 and Step 2 are completed before data collection, and the results build on these two steps. The eight steps are as follows (Fig. 2) [50]: Step 1: Start of Decision-making Process Step 1: Start of Decision-making Process This step involves setting the research goals and committing the decision-making process to achieving these goals. Therefore, deciding how to gather the information and knowledge required to achieve the research objective. For this research, the responses of 7 researchers, academics, and industry experts in implementing the CE in the fashion industry are collected through in-depth interviews (Table 3). Step 2: Selection of Criteria Step 2: Selection of Criteria This step involves establishing a set of criteria. For this research, the literature was reviewed to identify a set of challenges associated with circular economy implementa- tion in the fashion industry. The challenges of implementing a circular economy in the fashion industry identified from the literature are as follows: 1. Poor material and energy efficiency (C1): The use of non-renewable materials and energy in an unsustainable and non-economic manner and/or the use of toxic materials and energy [20, 21].fi 1. Poor material and energy efficiency (C1): The use of non-renewable materials and energy in an unsustainable and non-economic manner and/or the use of toxic materials and energy [20, 21].fi 2. Poor circular design (C2): Having difficulty reimagining and reinventing products and services supporting the CE. For instance, lacking the ability and the capacity to include strategies to extend the lifespan of products, such as swapping, renting, leas- ing, and repairing products, as well as strategies to encourage circularity at the end of their useful lives, such as repairing, reusing, recycling, and remanufacturing [16, 20].fi 3. Technological challenges (C3): Poor level of advanced and efficient technologies that run cost-effectively without comprising the quality [8, 17].f f 4. Regulatory pressures (C4): The regulations and legislations that affect how the com- panies in the fashion industry operate. For example, lack of governmental support, insufficient legislation and control, complex administration requirements, chemical requirements, and legal obligations [8, 18, 22]. 5. Internal stakeholder pressures (C5): The pressures exerted by employees, manag- ers, shareholders, owners, and management, and the difficulty of aligning their goals industry [7, 13]. 1 3 Circular Economy and Sustainability (2024) 4:585–617 595 Fig. 2 The eight steps of Fuzzy- TISM [50] 6. Financial pressures (C6): Circular business models in the fashion industry have been hindered by the lack of financial resources, support, and evaluation mechanisms [16– 18].fifi 6. Financial pressures (C6): Circular business models in the fashion industry have been hindered by the lack of financial resources, support, and evaluation mechanisms [16– 18].fifi 7. Step 3: Gathering Responses and Creating SSIM Matrix The responses about the pairwise relationship between the challenges are collected from the seven experts through interviews and used to complete the initial SSIM matrix for each expert [56]. The experts are asked to choose one of four following symbols (V, A, X, and O) to indicate the pairwise relationship and the direction of the relationships between the challenges, followed by a combination of the five linguistic terms (Very high, High, Low, Very low, No) to indicate the degree of influence/impact of the challenges [52]. Therefore, the experts have four response options: i V: To establish that challenge i influences/impacts challenge j but not vice versa, fol- lowed by Very high (VH), High (H), Low (L), or Very low (VL). For example, V (VH) or V(H) or V(L) or V(VL).l ii A: To establish that challenge j influences/impacts challenge i but not vice versa, fol- lowed by Very high (VH), High (H), Low (L), or Very low (VL).l iii X: To establish that challenges i and j influence/impact each other, followed by (Very high (VH), High (H), Low (L), or Very low (VL) for each direction. For example, X (VH, L) or X (H, VH). iv O: To establish that there is no relationship between challenges i and j, followed by No influence (No). For example, O (No). influence (No). For example, O (No). Step 4: Reachability Matrix Level Partition Step 1: Start of Decision-making Process Insufficient human capital (C7): Insufficient understanding and inadequate behaviour amongst employees regarding the circular economy, environmental protection, and environmental and sustainability management [8, 36, 40]. 7. Insufficient human capital (C7): Insufficient understanding and inadequate behaviour amongst employees regarding the circular economy, environmental protection, and environmental and sustainability management [8, 36, 40]. 8. Poor management and leadership (C8): Poor managerial capabilities and leadership skills shape the organisational culture, which is crucial in successfully distributing and applying knowledge regarding circular strategies [8]. 9. Lack of external collaborations (C9): The fashion brands and retailers lack collabora- tion with external partners (i.e., brands, retailers, and suppliers) on sustainable and 9. Lack of external collaborations (C9): The fashion brands and retailers lack collabora- tion with external partners (i.e., brands, retailers, and suppliers) on sustainable and 1 3 3 Circular Economy and Sustainability (2024) 4:585–617 596 Table 3 Experts’ profiles Job title Years of experience Circular economy programme and research manager 6 CEO and co-founder of a sustainable and circular fashion brand 20 CEO and co-founder of a circular fashion brand 4 Co-founder of a sustainable and circular fashion courses and training provider 10 Circular project manager 8 CEO and co-founder of circular fashion brand 20 Senior research associate in sustainable luxury 18 circular strategies, limiting their opportunities to unite their visions, improve their connectivity, and support their commitment to the transition [8, 43]. y pp [ ] 10. Consumer-related challenges (C10): Difficulty in anticipating and managing the deci- sions of consumers regarding the purchase, use, and disposal of products or services within the fashion industry [2, 46]. 10. Consumer-related challenges (C10): Difficulty in anticipating and managing the deci- sions of consumers regarding the purchase, use, and disposal of products or services within the fashion industry [2, 46]. Step 3: Gathering Responses and Creating SSIM Matrix Step 4: Reachability Matrix Level Partition In this step, the reachability matrix is partitioned into different levels. For example, based on Singh et al. [51], the reachability set is the variable itself and other variables it may help achieve (based on the rows). The antecedent set is the variable itself and the other variables that may help achieve it (based on the columns). The intersection set is the com- mon variables between reachability and the antecedent sets. 1 3 3 1 Circular Economy and Sustainability (2024) 4:585–617 597 Step 5: Creating the TISM Model Table 4 Fuzzy linguistic scale [57] Step 5: Creating the TISM Model In this step, a defuzzified TISM digraph is developed to visualise the hierarchy-based model showing the inter-relationship between the challenges and the direct and indirect impact of each challenge during the implementation of a CE within the fashion industry. Step 6: Creating Fuzzy-TISM Digraphs In this step, all responses collected from the interviews will be combined in an aggre- gated SSIM matrix using fuzzy linguistic terms. Moreover, Fuzzy-TISM digraphs are developed using simple arrows to visualise the relationship between the challenges. Step 7: Calculation of Final Fuzzy Reachability Matrix Step 7: Calculation of Final Fuzzy Reachability Matrix In this step, the linguistic terms are replaced by the fuzzy linguistic values first pro- posed by Li [57] and shown in Table 4. Therefore, N is replaced by (0.0, 0.0, 0.25); VL is replaced by (0.0, 0.25, 0.50); L is replaced by (0.25, 0.50, 0.75); H is replaced by (0.50, 0.75, 1.0); and VH is replaced by (0.75, 1.0, 1.0). Step 8: Calculation of Driving Power and Dependence Power and Conducting MIC- MAC Analysis In this step, the aggregated fuzzy reachability matrix is generated from the Fuzzy SSIM completed in the previous step. The fuzzy reachability matrix’s columns and rows are summed up to calculate the driving and dependence power. Then, the crisp value required for Fuzzy MICMAC analysis is determined using the following five equations derived from Khatwani et al. [50]: Step 1: Determine L = min(lk );R = max(uk);K = 1, 2, … nandΔ = R −L, then use Eq. 1 to determine each of the alternatives. (1) xlk = ( lk −L ) Δ , Xmk = mk −L Δ , Xuk = uk −L Δ (1) Step 2: Use Eqs. 2 and 3 to calculate normalised values for the left score (ls) and the right score (Rs). Step 2: Use Eqs. 2 and 3 to calculate normalised values for the left score (ls) and the right score (Rs). Step 2: Use Eqs. 2 and 3 to calculate normalised values for the left score (ls) and t right score (Rs). Step 5: Creating the TISM Model 1 3 Table 4 Fuzzy linguistic scale [57] Linguistic description Notation Triangular fuzzy number Influence scope No influence (0.0, 0.0, 0.25) N 0 Very low influence (0.0, 0.25, 0.50) VL 1 Low influence (0.25, 0.50, 0.75) L 2 High influence (0.50, 0.75, 1.0) H 3 Very high influence (0.75, 1.0, 1.0) VH 4 Linguistic description Notation Triangular fuzzy number Influence scope No influence (0.0, 0.0, 0.25) N 0 Very low influence (0.0, 0.25, 0.50) VL 1 Low influence (0.25, 0.50, 0.75) L 2 High influence (0.50, 0.75, 1.0) H 3 Very high influence (0.75, 1.0, 1.0) VH 4 Table 4 Fuzzy linguistic scale [57] Circular Economy and Sustainability (2024) 4:585–617 598 (2) Xls k = Xmk (1 + Xmk −Xlk) (3) Xrs k = Xuk (1 + Xuk −Xmk) (2) Xls k = Xmk (1 + Xmk −Xlk) (3) Xrs k = Xuk (1 + Xuk −Xmk) (2) (3) Xrs k = Xuk (1 + Xuk −Xmk) (3) Step 3: Use Eq. 4 to calculate the normalised crisp value. Step 3: Use Eq. 4 to calculate the normalised crisp value. (4) Xcrisp k = [Xls k × (1 −Xls k ) + Xrs k × Xrs k ]∕[1 −Xls k + Xrs k ] Eq. 4 to calculate the normalised crisp value. Step 3: Use Eq. 4 to calculate the normalised crisp value. (4) Xcrisp k = [Xls k × (1 −Xls k ) + Xrs k × Xrs k ]∕[1 −Xls k + Xrs k ] (4) Xcrisp k = [Xls k × (1 −Xls k ) + Xrs k × Xrs k ]∕[1 −Xls k + Xrs k ] (4) Step 5: Use Eq. 5 to calculate the crisp value for Bk (5) Bcrisp k = L + Xcrisp k × Δ Bcrisp k = L + Xcrisp k × Δ (5) Results Step 3: Gathering Responses and Creating SSIM Matrix The aggregated initial SSIM matrix is derived based on the highest frequency of occur- rence for the linguistic terms and is shown in Table 5. The table shows that all challenges influence/impact one another in one or both directions and that none are unrelated. Step 4: Reachability Matrix Level Partition Step 4: Reachability Matrix Level Partition To generate a defuzzified reachability matrix, only the direction of the relationship between the challenges is considered. Therefore, only the four symbols used in the initial SSIM matrix are considered and replaced by 1 or 0. The V is replaced by 1, A is replaced by 0, X is replaced by 1, and O is replaced by 0, as shown in Table 6. The driving power is the count of all challenges within the reachability set (based on the rows), hence the challenge itself and other challenges which it may help to address. The dependence power is the count of the challenges in the antecedent set (based on the columns), hence the challenge itself and all other challenges which it may help in addressing it. Furthermore, levels of partitioning are conducted based on the reachability, anteced- ent, and intersection sets, as represented in Tables 7, 8, 9, and 10. Table 7 shows the first Table 5 Initial aggregated SSIM matrix C10 C9 C8 C7 C6 C5 C4 C3 C2 C1 C1 Poor material and energy efficiency X(H, H) X(L, VH) A(VH) A(VH) A(VH) A(L) A(VH) A(VH) X(L, VH) C2 Poor circular design X(VH, H) X(VH, VH) A(H) A(H) A(H) A(H) A(VH) A(H) C3 Technological challenges X(H, H) A(VH) A(H) A(VH) X(H, VH) X(H,H) A(VH) C4 Regulatory pressures V(H) V(VH) X(L,H) V(L) X(H, L) X(L,L) C5 Internal stakeholders’ pressures X(L, VL) X(H, L) X(L, VH) X(VH, H) X(H, H) C6 Financial pressures X(H, H) X(H, L) X(H, VH) X(H, VH) C7 Insufficient human capital V(H) X(H, H) X(VH, VH) C8 Poor management and leadership V(H) X(VH, L) C9 Lack of external collaborations V(H) C10 Consumer-related challenges Table 5 Initial aggregated SSIM matrix 1 Circular Economy and Sustainability (2024) 4:585–617 599 iteration, where poor material and energy efficiency (C1), circular design (C2), and con- sumer-related challenges (C10) are found at level 1. This indicates that C1, C2, and C10 would not help address any of the other challenges below their level. Table 8 represents the second iteration, where technological challenges (C3), internal stakeholders’ pressures (C5), and financial pressures (C6) are found at level 2. Table 9 shows the third iteration, where insufficient human capital (C7), poor management and leadership (C8), and lack of external collaborations (C9) are found at level 3. Step 5: Creating the TISM Model Step 5: Creating the TISM Model Moreover, Fig. 3 represents the TISM Diagram developed to visualise the relationship between the challenges through the constructed hierarchy-based model. The solid one- ended arrow illustrates the relationship of one challenge with another in one direction, whilst the dotted double-ended arrows show the interrelationships between one challenge and another. The challenges in Fig. 3 are represented in 4 levels based on the levels con- cluded in Step 4. At the bottom-level challenge of the model is the regulatory pressures (C4), which indi- cates the key challenge that requires the most attention managerial attention at the early stages of strategy formulation and decision-making for a successful and effective imple- mentation of a circular economy in the fashion industry. Whereas the top-level challenges of the hierarchy are poor material and energy efficiency (C1), circular design (C2), and consumer-related challenges (C10), which indicate that they require the least managerial attention due to their lowest level of significance according to the TISM model. Therefore, the levels help with grouping those challenges. As seen in the model, all challenges have direct or indirect interrelationships, which shows the relevance of all the challenges identi- fied from the literature and how they impact each other bringing in the need to understand their complex system. Moreover, all challenges on the same levels are interconnected and impact each other in both directions, which means that challenges on the same level have the same impact on one another. However, this does not mean they have the same significance level because their relationship with other challenges on higher and lower levels should also be consid- ered in making those observations. For example, consumer-related challenges (C10) is on level 4 and interconnected with the other challenges on the same level and with all chal- lenges on level 3, where challenges have higher significance and impact levels. This means that consumer-related challenges (C10) could influence and get influenced by higher sig- nificance challenges, showing its higher significance level compared to the other two chal- lenges at the same level. Another example is technological challenges (C3) appears to have a lower significance level in comparison to internal stakeholders’ pressures (C5) and finan- cial pressures (C6) despite being on the same level. technological challenges (C3) can only influence the other two challenges on the same level and the challenges on the higher level, hence, with a lower significance level. Step 4: Reachability Matrix Level Partition Finally, Table 10 represents the fourth iteration where regulatory pressures (C4) are placed at level 4. Therefore, this indicates that C4 help in directly or indirectly addressing all other challenges. Step 5: Creating the TISM Model Step 5: Creating the TISM Model Therefore, this gives the other two challenges, inter- nal stakeholders’ pressures (C5) and financial pressures (C6), a higher significance level due to their interconnection with the challenges on level 2. Furthermore, poor management and leadership (C8) is shown to be one of the key chal- lenges with a higher level of significance in comparison to insufficient human capital (C7) and poor and lack of external collaborations (C9), which are on the same level. Step 5: Creating the TISM Model This is 1 3 3 Circular Economy and Sustainability (2024) 4:585–617 600 Table 6 Defuzzified reachability matrix C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Driving power C1 Poor material and energy efficiency 1 1 0 0 0 0 0 0 1 1 4 C2 Poor circular design 1 1 0 0 0 0 0 0 1 1 4 C3 Technological challenges 1 1 1 0 1 1 0 0 0 1 6 C4 Regulatory pressures 1 1 1 1 1 1 1 1 1 1 10 C5 Internal stakeholders’ pressures 1 1 1 1 1 1 1 1 1 1 10 C6 Financial pressures 1 1 1 1 1 1 1 1 1 1 10 C7 Insufficient human capital 1 1 1 0 1 1 1 1 1 1 9 C8 Poor management and leadership 1 1 1 1 1 1 1 1 1 1 10 C9 Lack of external collaborations 1 1 1 0 1 1 1 1 1 1 9 C10 Consumer-related challenges 1 1 1 0 1 1 0 0 0 1 6 Dependence Power 10 10 8 4 8 8 6 6 8 10 Table 7 First iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C1 1,2,9,10 1,2,3,4,5,6,7,8,9,10 1,2,9,10 1 C2 1,2,9,10 1,2,3,4,5,6,7,8,9,10 1,2,9,10 1 C3 1,2,3,5,6,10 3,4,5,6,7,8,9,10 3,5,6,10 C4 1,2,3,4,5,6,7,8,9,10 4,5,6,8 4,5,6,8 C5 1,2,3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 C6 1,2,3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 C7 1,2,3,5,6,7,8,9,10 4,5,6,7,8,9 5,6,7,8,9 C8 1,2,3,4,5,6,7,8,9,10 4,5,6,7,8,9 4,5,6,7,8,9 C9 1,2,3,5,6,7,8,9,10 1,2,4,5,6,7,8,9 1,2,5,6,7,8,9,10 C10 1,2,3,5,6,10 1,2,3,4,5,6,7,8,9,10 1,2,3,5,6,10 1 Table 8 Second iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C3 3,5,6 3,4,5,6,7,8,9 3,5,6 2 C4 3,4,5,6,7,8,9 4,5,6,8 4,5,6,8 C5 3,4,5,6,7,8,9 3,4,5,6,7,8,9 3,4,5,6,7,8,9 2 C6 3,4,5,6,7,8,9 3,4,5,6,7,8,9 3,4,5,6,7,8,9 2 C7 3,5,6,7,8,9 4,5,6,7,8,9 5,6,7,8,9 C8 3,4,5,6,7,8,9 4,5,6,7,8,9 4,5,6,7,8,9 C9 3,5,6,7,8,9 4,5,6,7,8,9 5,6,7,8,9 Table 6 Defuzzified reachability matrix C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Driving power C1 Poor material and energy efficiency 1 1 0 0 0 0 0 0 1 1 4 C2 Poor circular design 1 1 0 0 0 0 0 0 1 1 4 C3 Technological challenges 1 1 1 0 1 1 0 0 0 1 6 C4 Regulatory pressures 1 1 1 1 1 1 1 1 1 1 10 C5 Internal stakeholders’ pressures 1 1 1 1 1 1 1 1 1 1 10 C6 Financial pressures 1 1 1 1 1 1 1 1 1 1 10 C7 Insufficient human capital 1 1 1 0 1 1 1 1 1 1 9 C8 Poor management and leadership 1 1 1 1 1 1 1 1 1 1 10 C9 Lack of external collaborations 1 1 1 0 1 1 1 1 1 1 9 C10 Consumer-related challenges 1 1 1 0 1 1 0 0 0 1 6 Dependence Power 10 10 8 4 8 8 6 6 8 10 Table 6 Defuzzified reachability matrix Table 7 First iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C1 1,2,9,10 1,2,3,4,5,6,7,8,9,10 1,2,9,10 1 C2 1,2,9,10 1,2,3,4,5,6,7,8,9,10 1,2,9,10 1 C3 1,2,3,5,6,10 3,4,5,6,7,8,9,10 3,5,6,10 C4 1,2,3,4,5,6,7,8,9,10 4,5,6,8 4,5,6,8 C5 1,2,3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 C6 1,2,3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 C7 1,2,3,5,6,7,8,9,10 4,5,6,7,8,9 5,6,7,8,9 C8 1,2,3,4,5,6,7,8,9,10 4,5,6,7,8,9 4,5,6,7,8,9 C9 1,2,3,5,6,7,8,9,10 1,2,4,5,6,7,8,9 1,2,5,6,7,8,9,10 C10 1,2,3,5,6,10 1,2,3,4,5,6,7,8,9,10 1,2,3,5,6,10 1 Table 8 Second iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C3 3,5,6 3,4,5,6,7,8,9 3,5,6 2 C4 3,4,5,6,7,8,9 4,5,6,8 4,5,6,8 C5 3,4,5,6,7,8,9 3,4,5,6,7,8,9 3,4,5,6,7,8,9 2 C6 3,4,5,6,7,8,9 3,4,5,6,7,8,9 3,4,5,6,7,8,9 2 C7 3,5,6,7,8,9 4,5,6,7,8,9 5,6,7,8,9 C8 3,4,5,6,7,8,9 4,5,6,7,8,9 4,5,6,7,8,9 C9 3,5,6,7,8,9 4,5,6,7,8,9 5,6,7,8,9 Table 7 First iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C1 1,2,9,10 1,2,3,4,5,6,7,8,9,10 1,2,9,10 1 C2 1,2,9,10 1,2,3,4,5,6,7,8,9,10 1,2,9,10 1 C3 1,2,3,5,6,10 3,4,5,6,7,8,9,10 3,5,6,10 C4 1,2,3,4,5,6,7,8,9,10 4,5,6,8 4,5,6,8 C5 1,2,3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 C6 1,2,3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 3,4,5,6,7,8,9,10 C7 1,2,3,5,6,7,8,9,10 4,5,6,7,8,9 5,6,7,8,9 C8 1,2,3,4,5,6,7,8,9,10 4,5,6,7,8,9 4,5,6,7,8,9 C9 1,2,3,5,6,7,8,9,10 1,2,4,5,6,7,8,9 1,2,5,6,7,8,9,10 C10 1,2,3,5,6,10 1,2,3,4,5,6,7,8,9,10 1,2,3,5,6,10 1 Table 8 Second iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C3 3,5,6 3,4,5,6,7,8,9 3,5,6 2 C4 3,4,5,6,7,8,9 4,5,6,8 4,5,6,8 C5 3,4,5,6,7,8,9 3,4,5,6,7,8,9 3,4,5,6,7,8,9 2 C6 3,4,5,6,7,8,9 3,4,5,6,7,8,9 3,4,5,6,7,8,9 2 C7 3,5,6,7,8,9 4,5,6,7,8,9 5,6,7,8,9 C8 3,4,5,6,7,8,9 4,5,6,7,8,9 4,5,6,7,8,9 C9 3,5,6,7,8,9 4,5,6,7,8,9 5,6,7,8,9 eration of the eachability Variable Reachability set Antecedent set Intersection set Level because poor management and leadership (C8) are interconnected with regulatory pres- sures (C4) and thereby can influence and get influenced by one another. Step 5: Creating the TISM Model Therefore, having that direct interrelationship with regulatory pressures (C4), which according to the model, is the key challenge that needs the most managerial attention, makes poor management and leadership (C8) one of the challenges with the highest level of significance. Additionally, 1 Circular Economy and Sustainability (2024) 4:585–617 601 Table 9 Third iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C4 4,7,8,9 4, 8 4, 8 C7 7,8,9 4, 7,8,9 7,8,9 3 C8 4, 7,8,9 4, 7,8,9 4,7,8,9 3 C9 7,8,9 4, 7,8,9 7,8,9 3 Table 10 Fourth iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C4 4 4 4 4 Fig. 3 TISM Model Table 9 Third iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C4 4,7,8,9 4, 8 4, 8 C7 7,8,9 4, 7,8,9 7,8,9 3 C8 4, 7,8,9 4, 7,8,9 4,7,8,9 3 C9 7,8,9 4, 7,8,9 7,8,9 3 Variable Reachability set Antecedent set Intersection set Level Table 10 Fourth iteration of the final defuzzified reachability matrix insufficient human capital (C7) appears to have higher significance than lack of external collaborations (C9) since it influences the challenges in level 3 in one direction and does not get influenced by them, which makes it a more stable challenge than lack of external collaborations (C9) to be addressed to gain more effective results despite being on the same level. Table 10 Fourth iteration of the final defuzzified reachability matrix Variable Reachability set Antecedent set Intersection set Level C4 4 4 4 4 Fig. 3 TISM Model Fig. 3 TISM Model Fig. 3 TISM Model insufficient human capital (C7) appears to have higher significance than lack of external collaborations (C9) since it influences the challenges in level 3 in one direction and does not get influenced by them, which makes it a more stable challenge than lack of external collaborations (C9) to be addressed to gain more effective results despite being on the same level. insufficient human capital (C7) appears to have higher significance than lack of external collaborations (C9) since it influences the challenges in level 3 in one direction and does not get influenced by them, which makes it a more stable challenge than lack of external collaborations (C9) to be addressed to gain more effective results despite being on the same level. Step 5: Creating the TISM Model 1 3 Step 6: Creating Fuzzy-TISM Digraphs Step 6: Creating Fuzzy-TISM Digraphs Step 6: Creating Fuzzy-TISM Digraphs 3 1 Circular Economy and Sustainability (2024) 4:585–617 602 The initial aggregated SSIM matrix is transformed into the final reachability matrix Table 11 Final reachability matrix using fuzzy linguistic terms C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C1 Poor material and energy efficiency L No No No No No No L H C2 Poor circular design VH No No No No No No VH VH C3 Technological challenges VH H No H H No No No H C4 Regulatory pressures VH VH VH L H L L VH H C5 Internal stakeholders’ pressures L H H L H VH L H L C6 Financial pressures VH H VH L H H H H H C7 Insufficient human capital VH H VH No H VH VH H H C8 Poor management and leadership VH H H H VH VH VH VH H C9 Lack of external collaborations VH VH VH No L L H L H C10 Consumer-related challenges H H H No VL H No No No Fig. 4 Symbols for representing the fuzzy relationship between the challenges i Table 11 Final reachability matrix using fuzzy linguistic terms C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C1 Poor material and energy efficiency L No No No No No No L H C2 Poor circular design VH No No No No No No VH VH C3 Technological challenges VH H No H H No No No H C4 Regulatory pressures VH VH VH L H L L VH H C5 Internal stakeholders’ pressures L H H L H VH L H L C6 Financial pressures VH H VH L H H H H H C7 Insufficient human capital VH H VH No H VH VH H H C8 Poor management and leadership VH H H H VH VH VH VH H C9 Lack of external collaborations VH VH VH No L L H L H C10 Consumer-related challenges H H H No VL H No No No Fig. Step 5: Creating the TISM Model 4 Symbols for representing the fuzzy relationship between the challenges Table 11 Final reachability matrix using fuzzy linguistic terms C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C1 Poor material and energy efficiency L No No No No No No L H C2 Poor circular design VH No No No No No No VH VH C3 Technological challenges VH H No H H No No No H C4 Regulatory pressures VH VH VH L H L L VH H C5 Internal stakeholders’ pressures L H H L H VH L H L C6 Financial pressures VH H VH L H H H H H C7 Insufficient human capital VH H VH No H VH VH H H C8 Poor management and leadership VH H H H VH VH VH VH H C9 Lack of external collaborations VH VH VH No L L H L H C10 Consumer-related challenges H H H No VL H No No No Table 11 Final reachability matrix using fuzzy linguistic terms Fig. 4 Symbols for representing the fuzzy relationship between the challenges The initial aggregated SSIM matrix is transformed into the final reachability matrix using linguistic terms and based on the highest frequency of occurrence, as shown in Table 11. The symbols for representing the fuzzy relationship between the challenges are shown in Fig. 4. Using these symbols, the Fuzzy-TISM diagraphs of the ten challenges are gener- ated and are shown in Table 12. Step 7: Calculation of Final Fuzzy Reachability Matrix The linguistic terms are then replaced by the fuzzy linguistic triangular numbers, as shown in Table 13. Step 8: Calculation of Driving Power and Dependence power and conducting MICMAC Analysis 1 3 1 Circular Economy and Sustainability (2024) 4:585–617 603 As represented in Table 14, all the columns and rows are summed to calculate the fuzzy values of driving and dependence power. The crisp values of driving and dependence power are calculated using the five equations mentioned in the “Methodology” section. Furthermore, based on the crisp values of driving power (y-axis) and dependence power (x-axis), the Fuzzy MICMAC analysis is derived as represented in Fig. 5. Firstly, regulatory pressures (C4) and poor management and leadership (C8) lie within the “Drivers” cluster, as both have strong driving power and weak dependence power. Therefore, they have the highest influence over all other challenges and very little or no dependence on other challenges. Step 5: Creating the TISM Model Thus, the MICMAC analysis displays these two challenges as the key ones with the highest level of significance requiring the most managerial attention, especially at the early decision-making stage for successful and effective implementation results. This somewhat validates the TISM model considering that the regulatory pressures (C4) is placed at level 1 and has the highest significance level. Additionally, it has an interconnection (in both directions) with poor management and leadership (C8) located at level 2, indicating the relationship that both challenges have and their high significance. However, according to MICMAC analysis, both challenges should be placed in level 1 and treated as key challenges. In fact, poor manage- ment and leadership (C8) appear to have higher driving power than regulatory pressures (C4).i Secondly, technological challenges (C3), internal stakeholders’ pressures (C5), financial pressures (C6), insufficient human capital (C7), and lack of external collaborations (C9) lie within the “linkage” cluster, as these challenges have strong driving and dependence power. This means these challenges are unstable because any action on them will influence others and have a feedback effect on themselves. These challenges are placed in levels 2 and 3 on the TISM model, but poor management and leadership (C8) is not being mapped in the same cluster. Hence, this slightly conflicts with the TISM model. Furthermore, seeing insufficient human capital (C7) on the borderline of the “drivers” cluster validates the findings of the TISM model, indicating the high significance of this challenge compared to the other chal- lenges in the same cluster. Also, seeing technological challenges (C3) on the borderline of the “dependent” validates its less significance than the other challenges in the same cluster.fi i Thirdly, poor material and energy efficiency (C1), circular design (C2), and consumer-related challenges (C10) lie within the “dependent” cluster, as these challenges have weak driving power and strong dependence power. These are the same challenges found at level 4 on the TISM Model. Therefore, the MICMAC analysis validates that they are highly reliant and dependent on other challenges. This means that they are driven by other factors with higher significances level. i Finally, no challenges lie within the “autonomous” cluster. Thus, none of the challenges have weak driving and dependence power. Also, none of the challenges are considered iso- lated from the system since they have little or no influence or relationship with other chal- lenges. Step 5: Creating the TISM Model This has already been noticed in the initial aggregated SSIM matrix. Moreover, this validates that all challenges identified from the literature and analysed in this research have a direct or indirect relationship. Discussion Several interesting findings were drawn from the TISM model (Fig. 3) and Fuzzy-MICMAC analysis (Fig. 5). Several interesting findings were drawn from the TISM model (Fig. 3) and Fuzzy-MICMAC analysis (Fig. 5). Firstly, regulatory pressures and poor management and leadership do not lie on the same level in the TISM Model, as shown in Fig. 6, illustrating that regulatory pressures are ranked at level 1. Therefore, it is the key challenge that influences all other challenges 3 Circular Economy and Sustainability (2024) 4:585–617 604 Table 12 Fuzzy-TISM digraphs of all challenges influencing others using the symbols from Fig. Discussion 4 to show all fuzzy linguistic terms uzzy linguistic terms Challenge Diagraph C1 has low influence on C2 and C9, and high influence on C10 C2 has very high influence on C1, C9, C10 C3 has very high influence on C1, and high influence on C2, C5, C6 and C10 C4 has very high influence on C1, C2, C3, C9, high influence on C6 and C10, low influence C7 and C8, and very low influence on C5 C5 has low influence on C1, C7, C8 and C10, high influence on C2, C3, C6 and C9, and very low influence on C4 1 3 Circular Economy and Sustainability (2024) 4:585–617 605 12 (continued) C6 has very high influence on C1 and C3, high influence on C2, C5, C7, C8, C9 and C10, and low influence on C4 C7 has very high influence C1, C3, C6 and C8, and high influence on C2, C5, C9 and C10 C8 has very high influence on C1, C5, C6, C7 and C9, and high influence on C2, C3, C4 and C10 C9 has very high influence on C1, C2, C3, high influence on C7 and C10, and low influence on C5, C6 and C8 C10 has high influence on C1, C2, C3 and C5, and very low influence on C5 1 3 Circular Economy and Sustainability (2024) 4:585–617 606 1 3 Table 13 Fuzzy reachability matrix using fuzzy linguistic values C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C1 Poor material and energy efficiency (1, 1, 1) (0.25, 0.50, 0.75) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.25, 0.50, 0.75) (0.50, 0.75, 1) C2 Poor circu- lar design (0.75, 1, 1) (1, 1, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.75, 1, 1) (0.75, 1, 1) C3 Tech- nological challenges (0.75, 1, 1) (0.50, 0.75, 1) (1, 1, 1) (0.0, 0.0, 0.25) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.50, 0.75, 1) C4 Regulatory pressures (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (1, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (0.25, 0.50, 0.75) (0.75, 1, 1) (0.50, 0.75, 1) C5 Internal stakeholders’ pressures (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (1, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) C6 Financial pressures (0.75, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (1, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) C7 Insufficient human capital (0.75, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.0, 0.0, 0.25) (0.50, 0.75, 1) (0.75, 1, 1) (1, 1, 1) (0.75, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) C8 Poor man- agement and leadership (0.75, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (1, 1, 1) (0.75, 1, 1) (0.50, 0.75, 1) C9 Lack of external col- laborations (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (0.0, 0.0, 0.25) (0.25, 0.50, 0.75) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (1, 1, 1) (0.50, 0.75, 1) C10 Consumer- related challenges (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.25, 0.50) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (1, 1, 1) Table 13 Fuzzy reachability matrix using fuzzy linguistic values C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C1 Poor material and energy efficiency (1, 1, 1) (0.25, 0.50, 0.75) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.25, 0.50, 0.75) (0.50, 0.75, 1) C2 Poor circu- lar design (0.75, 1, 1) (1, 1, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.75, 1, 1) (0.75, 1, 1) C3 Tech- nological challenges (0.75, 1, 1) (0.50, 0.75, 1) (1, 1, 1) (0.0, 0.0, 0.25) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.50, 0.75, 1) C4 Regulatory pressures (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (1, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (0.25, 0.50, 0.75) (0.75, 1, 1) (0.50, 0.75, 1) C5 Internal stakeholders’ pressures (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (1, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) C6 Financial pressures (0.75, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (1, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) C7 Insufficient human capital (0.75, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.0, 0.0, 0.25) (0.50, 0.75, 1) (0.75, 1, 1) (1, 1, 1) (0.75, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) C8 Poor man- agement and leadership (0.75, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (1, 1, 1) (0.75, 1, 1) (0.50, 0.75, 1) C9 Lack of external col- laborations (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (0.0, 0.0, 0.25) (0.25, 0.50, 0.75) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (1, 1, 1) (0.50, 0.75, 1) C10 Consumer- related challenges (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.25, 0.50) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (1, 1, 1) Circular Economy and Sustainability (2024) 4:585–617 607 1 3 Table 14 Final fuzzy reachability matrix of the seven experts with fuzzy and crisp values of the driving and dependence power of challenges C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Driving power Crisp value C1 Poor mate- rial and energy efficiency (1, 1, 1) (0.25, 0.50, 0.75) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (2, 2.75, 5) 3.088 C2 Poor circular design (0.75, 1, 1) (1, 1, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.75, 1, 1) (0.75, 1, 1) (3.25, 4, 5.5) 4.1904 C3 Tech- nological chal- lenges (0.75, 1, 1) (0.50, 0.75, 1) (1, 1, 1) (0.0, 0.0, 0.25) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.50, 0.75, 1) (3.75, 5, 7) 5.1876 C4 Regula- tory pres- sures (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (1, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (0.25, 0.50, 0.75) (0.75, 1, 1) (0.50, 0.75, 1) (5.75, 8, 9.25) 7.7216 C5 Internal stake- holders’ pressures (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (1, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (4.75, 7, 9) 6.8824 C6 Finan- cial pres- sures (0.75, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (1, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (5.75, 8, 9.75) 7.7848 C7 Insuf- ficient human capital (0.75, 1, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.0, 0.0, 0.25) (0.50, 0.75, 1) (0.75, 1, 1) (1, 1, 1) (0.75, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (6, 8, 9.25) 7.7736 C8 Poor manage- ment and leader (0.75, 1, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (1, 1, 1) (0.75, 1, 1) (0.50, 0.75, 1) (6.75, 9, 10) 8.6784 3 Circular Economy and Sustainability (2024) 4:585–617 608 e 14 (continued) C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Driving power Crisp value Lack of ternal llabo- tions (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (0.0, 0.0, 0.25) (0.25, 0.50, 0.75) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (1, 1, 1) (0.50, 0.75, 1) (5, 7, 8.5) 6.8512 Con- mer- lated al- nges (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.25, 0.50) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (1, 1, 1) (3, 4.25, 6.5) 4.5216 end- ce wer (7, 9.25, 9.75) (5.75, 8, 9.75) (5.5, 7.25, 8.5) (2, 2.75, 5) (3.75, 5.5, 7.5) (4.75, 6.5, 8.25) (3.75, 5, 6.75) (3, 4.25, 6.25) (5, 6.75, 8.25) (5.5, 7.75, 9.75) p value 8.9037 7.7722 7.0941 3.0904 5.5704 6.464 5.1364 4.4668 6.6578 7.5661 Table 14 (continued) C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Driving power Crisp value C9 Lack of external collabo- rations (0.75, 1, 1) (0.75, 1, 1) (0.75, 1, 1) (0.0, 0.0, 0.25) (0.25, 0.50, 0.75) (0.25, 0.50, 0.75) (0.50, 0.75, 1) (0.25, 0.50, 0.75) (1, 1, 1) (0.50, 0.75, 1) (5, 7, 8.5) 6.8512 C10 Con- sumer- related chal- lenges (0.50, 0.75, 1) (0.50, 0.75, 1) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.25, 0.50) (0.50, 0.75, 1) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (0.0, 0.0, 0.25) (1, 1, 1) (3, 4.25, 6.5) 4.5216 Depend- ence power (7, 9.25, 9.75) (5.75, 8, 9.75) (5.5, 7.25, 8.5) (2, 2.75, 5) (3.75, 5.5, 7.5) (4.75, 6.5, 8.25) (3.75, 5, 6.75) (3, 4.25, 6.25) (5, 6.75, 8.25) (5.5, 7.75, 9.75) Crisp value 8.9037 7.7722 7.0941 3.0904 5.5704 6.464 5.1364 4.4668 6.6578 7.5661 Circular Economy and Sustainability (2024) 4:585–617 609 (C1) Poor Material and Energy Efficiency (C2) Circular Design (C3) Technological Challenges (C4) Regulatory Pressures (C5) Internal Stakeholders’ Pressures (C6) Financial Pressures (C7) Insufficient Human Capital (C8) Poor Management and Leadership (C9) Lack of External Collaborations (C10) Consumer-related Challenges Fig. Discussion 5 Fuzzy MICMAC analysis Fig. 5 Fuzzy MICMAC analysis and the starting point for addressing the challenges and implementation efforts. This con- firms the massive control that governments have over companies (including management and leadership), retailers, and consumers, and is consistent with the findings from litera- ture. This indicates the critical role that governments play in the transition to a CE within the fashion industry [8, 18]. Although poor management and leadership is ranked at level 2 amongst other challenges, the findings entail that it is the challenge that is the second most critical for review. Therefore, it needs to be provided with a high degree of attention and addressed effectively. The TISM model implies that this challenge is interlinked with regu- latory pressures, showing that poor management and leadership can influence and get influ- enced by regulatory pressures. The literature reviewed shows the influence of regulatory pressures on poor management and leadership. Still, despite its importance and complexity, it fails to consider the impact of poor management and leadership on regulatory pressures. Hence, the findings of this research contribute to this gap. Moreover, the findings illustrate that poor management and leadership is directly and indirectly linked to all other chal- lenges. This demonstrates that management and leadership are crucial for positive organi- sational change, managing high costs and risks, understanding and motivating employees, and planning, organising, and controlling resources [11, 36, 42]. and the starting point for addressing the challenges and implementation efforts. This con- firms the massive control that governments have over companies (including management and leadership), retailers, and consumers, and is consistent with the findings from litera- ture. This indicates the critical role that governments play in the transition to a CE within the fashion industry [8, 18]. Although poor management and leadership is ranked at level 2 amongst other challenges, the findings entail that it is the challenge that is the second most critical for review. Therefore, it needs to be provided with a high degree of attention and addressed effectively. The TISM model implies that this challenge is interlinked with regu- latory pressures, showing that poor management and leadership can influence and get influ- enced by regulatory pressures. The literature reviewed shows the influence of regulatory pressures on poor management and leadership. Still, despite its importance and complexity, it fails to consider the impact of poor management and leadership on regulatory pressures. Discussion This is because employees should have the knowledge and skill set required for applying circularity. Although very little was found in the literature on associating lack of external collaborations and insufficient human capital, Todeschini et al. somehow sup- port the findings of this research by suggesting that external collaborations rely on three supporting factors: external environmental pressures, competitive environmental pressures, and the search for a competitive advantage [45].i Thirdly, the following challenges that need to be addressed are financial pressures, inter- nal stakeholders’ pressures, and technological challenges, ranked at level 3 on the TISM model. The literature reviewed focuses on the financial pressures occurring due to the lack of financial means, support, and uncertainty surrounding profitability [8, 16, 34]. This research adds that financial pressures are thought to rely on the economy and the com- pany’s size. For instance, big companies in low-income countries generate a lot of revenue, which massively impacts the economy. Therefore, good leadership and management regu- lations could be adjusted so that these companies do not move elsewhere with fewer taxes or lower costs. The findings and literature still agree that financial pressures are a linkage challenge. This is because financial pressures are obstructing the transition to a CE, and at the same time, the transition to a CE creates financial pressures. Also, financial pres- sures could cause multiple effects depending on the economy and the company’s size. For internal stakeholders’ pressures, the literature reviewed explains that internal stakeholders would support the implementation of CE strategies and have positive effects when there is an economic incentive and when their goals are aligned [8, 16, 32]. This is supported by the findings classifying internal stakeholders’ pressures as a linkage challenge because stakeholders’ pressures could work with or against the transition to a CE based on the fac- tors highlighted in the literature. Moreover, the Fuzzy-MICMAC analysis maps technological challenges (C3), internal stakeholders’ pressures (C5), financial pressures (C6), insufficient human capital (C7), and lack of external collaborations (C9) within the “linkage” cluster, hence requiring careful treatment since any change could lead to multiple effects on other challenges and themselves. Therefore, each of the challenges falling in the “linkage” cluster influences and gets influenced by other challenges in different ways that cannot be anticipated. Discussion Hence, the findings of this research contribute to this gap. Moreover, the findings illustrate that poor management and leadership is directly and indirectly linked to all other chal- lenges. This demonstrates that management and leadership are crucial for positive organi- sational change, managing high costs and risks, understanding and motivating employees, and planning, organising, and controlling resources [11, 36, 42]. Furthermore, the Fuzzy-MICMAC analysis maps the regulatory pressures and poor management and leadership as drivers, as shown in Fig. 3. According to the interviews, regulatory pressures affect all other challenges. An interesting example made by one of the experts was that licenses must be obtained to allow waste collection, transportation, and handling because a producer is designated to make a particular product and would need a different license to sell waste. At the same time, companies must justify what is bought, produced, and wasted, and if waste is recognised as a secondary raw material, then this jus- tification might be impacted. Furthermore, most experts explained that fashion companies have a more top-down approach. For example, a few experts mentioned that managers and 1 1 3 3 Circular Economy and Sustainability (2024) 4:585–617 610 leaders brief employees, including designers, about the actions that need to be done and the ways of implementing these actions. This shows that poor management and leader- ship have a strong driving force as it affects the strategies formulated and decisions made at a very early stage concerning all other challenges. This entails that regulatory pressures and poor management and leadership shape the rest of the complex system, as they can influence other challenges and have low dependence on other challenges. Therefore, both challenges are crucial to the success of a CE implementation in the fashion industry and maximising its beneficial results. i Secondly, the TISM model shows that poor management and leadership, lack of exter- nal collaborations, and insufficient human capital are ranked at level 2. This contributes to the existing research since the literature reviewed indicates the role of government in initiating external collaborations but does not consider the role of government in address- ing the challenge of having insufficient human capital. Furthermore, collaborations help share knowledge, ideas, techniques, materials, and resources, which help improve human capital and the growth of the organisations within the industry. However, sufficient human capital is still necessary for the collaboration, as without it, this collaboration would not be possible. Discussion The TISM model allocates these challenges falling in the “linkage” cluster to levels 2 and 3 and considers them being in the middle levels of the hierarchy with multiple effects. An example from the interviews is that although technological advancement could positively 1 3 1 3 Circular Economy and Sustainability (2024) 4:585–617 611 help circularity through various ways, including accepting and separating different fibres, it could also lead to increased production, negatively affecting the CE. As the literature demonstrates, technological challenges obstruct the transition to a CE since up-to-date, cost-effective, and efficient technologies support CE business models without compromis- ing quality [8, 17]. Nevertheless, technological advancements could also lead to circular rebound if not used effectively and efficiently, demonstrating its linkage effect and consist- ency with the literature [23].fi Finally, poor material and energy efficiency (C1), circular design (C2), and consumer- related challenges (C10) are ranked at level 4 on the TISM model. They are mapped within the “dependent” cluster in the Fuzzy-MICMAC analysis. These are the least significant challenges with the lowest priority and are more likely to be influenced by other chal- lenges, not vice versa. In particular, poor material and energy efficiency and poor circular design, because these two challenges are only interconnected with challenges on the same level but do not influence or impact any other challenges from the below levels. According to the literature, circular design usually originates from market demands and preferences [25]. However, this research shows that most challenges influence poor circular design, specifically poor management and leadership, and regulatory pressures.fii i For poor material and energy efficiency, the findings and the literature somehow agree on this challenge as a result of poor management. However, the literature adds that this challenge could arise from the scarcity of resources arising from environmental impact and climate change, which could be hard to manage even by addressing all other challenges [22]. Furthermore, the findings of this research show that this challenge could be managed by addressing other challenges. For instance, most experts highlighted that consumers are at the bottom as they usually go for the given trends and options. Providing them with vari- ous circular options will reduce the challenges associated with their purchasing, usage, and disposable behaviour. Discussion Another expert also added that the price of the products does not influence the consumers purchasing decision because the same consumer could pay a lot more to buy a designer product. Hence it depends more on the trends and attractiveness of the products. This is in contrary to the existing literature that entails that consumer-related challenges have a significant impact on the transition to a CE and that these challenges can- not be managed or anticipated [16, 17, 20]. Policy Implications Implementing a CE in the fashion industry heavily depends on government regulations and legislation. As the findings of this research show, regulatory pressures are the key challenge faced in the transition to a CE. Hence addressing those regulatory pressures could help overcome all other challenges directly or indirectly. Many aspects concerned with the tran- sition, including approvals, material and energy efficiency levels, and emissions allowed, are set following the regulations. Therefore, governments must change existing regulations and provide legal frameworks and policies supporting CE implementation. Governments could enforce strict and standardised regulations making CE principles a legal requirement. Managers and leaders follow and implement these policies and regulations to enable the transition to a CE. Governments could provide their support in various ways. For example, governments could reduce administration costs and requirements as well as lower taxes. Governments could also offer incentives to increase collaborations between parts of the supply chain or various organisations/brands. Furthermore, governments could support waste minimisation strategies by providing access to textile waste and giving permission to collect this waste from other organisa- tions/brands and use it as a resource stream. This reduces the challenges faced due to the lack of collaboration and helps address other challenges. For example, governments could encourage training and development of the workforce to improve their knowledge and skills regarding the CE, enabling them to implement new techniques and use advanced technologies required in that transition. Additionally, governments could set a cap on the price of products according to their specifications and/or the production levels to avoid, to ensure that managers and leaders implement strategies that would not exploit consumers or resources. Therefore, indirectly encourage consumers to choose circular services or prod- ucts and avoid circular rebound and premiums. Managerial implications This research identified the significance level and key challenges that top management and decision-makers must prioritise and address to support their understanding and implemen- tation efforts. This paper shows that the management must adhere to regulations and leg- islation because regulatory pressure is one of the most vital factors affecting other chal- lenges of CE implementation. However, it is not only about creating regulations but how the management implements them to increase the positive impact of the transition towards a CE. For instance, management in large enterprises may be forced to change regulations to support their CE initiatives.f Managers and leaders affect organisational culture. Therefore, the managers and lead- ers must have the knowledge, capabilities, and skills to implement real change, respond to the industry’s needs, and find solutions and alternatives when needed. Management must also efficiently and effectively use the organisations and brand’ resources, including human resources. Furthermore, management must create a flexible, adaptable, and motivated culture built on communication and collaboration across the supply chain. In addition, 1 3 3 Circular Economy and Sustainability (2024) 4:585–617 612 management needs to raise awareness of the importance of this transition and overcome the resistance to change. Moreover, management needs to consider CE principles at very early stages to base their decisions and formulate their strategies based on those principles. For instance, decisions about materials, prices, risks, and profit margins need to be considered at early stages depending on the principles and context of the application. Therefore, it should be noted that management should focus on dealing with regulatory pressures as this challenge is at a higher level of influence, hence of greater significance. Then, the following challenges that need to be considered are poor management and lead- ership, lack of external collaborations, and insufficient human capital, as these challenges lie on the same level and need to be addressed by the management to overcome all other challenges at the other levels directly or indirectly. Conclusion This research aims to identify the level of significance of the challenges identified in the literature to assist top management with formulating a strategy and making decisions. These choices are based on the key challenges that require the most attention for a successful and effective implementation. To achieve the aim of this research, research questions were established to direct, identify, and fill the research gap, and populate the TISM model and MICMAC analysis diagrams which are deducted from the research findings. 1 3 613 Circular Economy and Sustainability (2024) 4:585–617 Existing literature and research were used to identify the ten challenges of introducing a CE in the fashion industry used in this paper, including poor material and energy efficiency, poor circular design, technological challenges, regulatory pressures, internal stakeholders’ pressures, financial pressures, insufficient human capital, poor management and leadership, lack of external collaborations, and consumer-related challenges.i The TISM model is a key step that demonstrates that all challenges identified from the literature are interrelated and directly or indirectly influence one another. The ranking of the challenges identified is as follows. • Level 1: Regulatory pressures • Level 2: Poor management and leadership, insufficient human capital, and lack of external collaborationsi • Level 2: Poor management and leadership, insufficient human capital, and lack of external collaborationsi • Level 3: Technological challenges, internal stakeholders’ pressures, and financial pres- suresfi • Level 3: Technological challenges, internal stakeholders’ pressures, and financial pres- suresfi • Level 4: Poor material and energy efficiency, poor circular design, and consumer- related challenges • Level 4: Poor material and energy efficiency, poor circular design, and consumer- related challenges Level 1 has the highest significance level, and level 4 has the lowest. Therefore, identifying regulatory pressures is the key challenge that requires the most managerial attention. The Fuzzy MICMAC analysis mapped technological challenges, internal stakeholders’ pressures, financial pressures, insufficient human capital, and lack of external collaborations within the “linkage” cluster. This shows that addressing those challenges directly should be avoided unless necessary due to their instability, which may lead to more challenging factors or less effective implementation results. Moreover, Poor Material and energy efficiency, poor circular design, and consumer-related challenges are mapped within the “dependence” cluster. This indicates that these challenges have the least significance, in line with the TISM model, as they are likely to be addressed indirectly. Conclusion Also, directing the implementation efforts to these challenges may not lead to the most successful and effective results. Finally, regulatory pressures and poor management and leadership are mapped within the “drivers” cluster. Therefore, according to MICMAC analysis, these are the key challenges with the highest significance level. i This research contributes to existing research in various ways. Firstly, this research is the first research to use Fuzzy-TISM to identify the interrelationship between the challenges and to arrange these challenges into a hierarchy-based model This helps in the simple visualisation of a complex system of challenges and reveals the direct and indirect impact, as well as the level of significance of the challenges. Secondly, this is the first research to use Fuzzy MICMAC analysis to map and rank these challenges depending on their driving and dependence power to validate and analyse the TISM model developed. ii i This research contributes to existing research in various ways. Firstly, this research is the first research to use Fuzzy-TISM to identify the interrelationship between the challenges and to arrange these challenges into a hierarchy-based model This helps in the simple visualisation of a complex system of challenges and reveals the direct and indirect impact, as well as the level of significance of the challenges. Secondly, this is the first research to use Fuzzy MICMAC analysis to map and rank these challenges depending on their driving and dependence power to validate and analyse the TISM model developed. Thirdly, the findings demonstrate that poor management and leadership significantly influence regulatory pressures, not just vice versa, as illustrated in the existing literature, which elaborates on the significance of both challenges for a successful and effective transition. This is because regulatory pressures and poor management and leadership are the key challenges that directly or indirectly influence other challenges and therefore need the most managerial attention for a successful and effective implementation. Finally, contrary to the existing literature, the research findings indicate the insignificance of directing measures or strategies to address the challenges at level 4. This includes consumer-related challenges since they are usually addressed indirectly whilst tackling the other challenges. 1 1 3 Circular Economy and Sustainability (2024) 4:585–617 614 The limitation of this research is that data was collected through interviews with individuals rather than focus groups due to the experts’ time and availability restrictions. Conclusion This limited the opportunity to allow all experts involved in the research to communicate their opinions and different perspectives from their experiences and agree on the pairwise relationship of the challenges rather than basing this relationship on the highest frequency of occurrence throughout the interviews. This has also limited the opportunity to have an insightful justification for the agreed relationship between the challenges. i Future research should aim to collect data from experts using focus groups instead to reach a contextual pairwise relationship between the challenges identified from the literature. This will help avoid or overcome the risk of misinterpretation of the data collected. Furthermore, the TISM model and MICMAC analysis are based on the subjective knowledge and experience of the experts participating in the research. Therefore, future research could consider empirically testing the results of this research. Finally, future research could explore possible ways of addressing the key challenges concluded in this research. However, the research context, location of the brands/ organisations, demographics, and the culture they operate in should all be considered when planning future research, as these factors will significantly affect the results and findings. Acknowledgements The authors would like to express their gratitude to the experts that took part in this research for their support. Author Contribution Aya Abdelmeguid- Conceptualisation, Data Curation, Writing the original draft & Editing. Mohamed Afy-Shararah- Conceptualisation, Supervision, Review & Editing. Konstantinos Salonitis- Conceptualisation, Supervision, Review & Editing. All three authors approved this version of the paper to be published. All three authors also agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Data Availability Not authorised to share or publish identifiable personal data or materials. Code Availability Not applicable. Code Availability Not applicable. References 1. Armstrong CM, Niinimäki K, Kujala S, Karell E, Lang C (2015) Sustainable product-service systems for clothing: exploring consumer perceptions of consumption alternatives in Finland. J Clean Prod 97:30–39. https://doi.org/10.1016/j.jclepro.2014.01.046 p g j j p 2. Colucci M, Vecchi A (2021) Close the loop: evidence on the implementation of the circular economy from the Italian fashion industry. Bus Strategy Environ 30(2):856–873. https://doi.org/10.1002/bse. 2658 p g j j p 2. Colucci M, Vecchi A (2021) Close the loop: evidence on the implementation of the circular economy from the Italian fashion industry. 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Consent to Participate Verbal informed consent was obtained prior to the interviews. Consent for Publication Verbal informed consent for publication of this research was obtained prior to the interviews, noting that the participation in the interviews was voluntary and identities were kept anonymous. Competing Interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W4206678183	https://cdr.lib.unc.edu/downloads/4q77ft36v	English	null	Data World Does Not Lack Standards	Carolina Digital Repository (University of North Carolina at Chapel Hill)	2,015	cc-by	1,923	ISSN 2162-3309 \| JLSC is published by the Pacific University Libraries \| http://jlsc-pub.org ISSN 2162-3309 \| JLSC is published by the Pacific University Libraries \| http://jlsc-pub.org ISSN 2162-3309 \| JLSC is published by the Pacific University Libraries \| http://jlsc-pub.org Volume 3, Issue 2 (2015) Volume 3, Issue 2 (2015) Data World Does Not Lack Standards © 2015 Hayslett. This open access article is distributed under a Creative Commons Attribution 4.0 License (https:// creativecommons.org/licenses/by/4.0/) Hayslett, M. (2015). Data World Does Not Lack Standards. Journal of Librarianship and Scholarly Communication, 3(2), eP1245. http://dx.doi.org/10.7710/2162-3309.1245 Data World Does Not Lack Standards Michele Hayslett Correspondence: Michele Hayslett, UNC University Libraries, Campus Box 3950, Chapel Hill, NC 27599, michele_hayslett@unc.edu Michele Hayslett Hayslett, M. (2015). Data World Does Not Lack Standards. Journal of Librarianship and Scholarly Communication, 3(2), eP1245. http://dx.doi.org/10.7710/2162-3309.1245 © 2015 Hayslett. This open access article is distributed under a Creative Commons Attribution 4.0 License (https:// creativecommons.org/licenses/by/4.0/) © 2015 Hayslett. This open access article is distributed under a Creative Commons Attribution 4.0 License (https:// creativecommons.org/licenses/by/4.0/) ISSN 2162-3309 ISSN 2162-3309 10.7710/2162-3309.1245 COMMENTARY COMMENTARY © 2015 Hayslett. This open access article is distributed under a Creative Commons Attribution 4.0 License (https:// creativecommons.org/licenses/by/4.0/) Data World Does Not Lack Standards Michele Hayslett Numeric Data Services and Data Management Librarian, University of North Carolina at Chapel Hill Michele Hayslett Numeric Data Services and Data Management Librarian, University of North Carolina at Chapel Hill When I first read the Call for Papers for this special issue, I was dismayed to find this line within it: “However, the world of data lacks the ingrained standards and practices the library and academic community have built up over the years.” It is true that there are many standards and practices for data depending on the discipline in which the research is done. Because data themselves are more varied in their format than publications such as books and journal articles, standards for data are necessarily more varied and complex than those describing print publications. Whereas social science survey data must discuss sampling techniques and any weighting procedures and provide questionnaires, astronomy data has quite different concerns: frequency bands, equipment specifications and calibration, and spectra measurements. Consequently the standards involved may feel less “ingrained” to those who are not deeply involved in the research of different disciplines. And, too, librarians may be less familiar with standards that apply in parts of the research lifecycle in which they have tended to be less involved. Every library student knows MARC, but that is a standard used primarily in the dissemination stage of research, not in the data collection stage. Standards in data may also be more in flux than those for publications, particularly recently, given the rapid evolution of mandates for data sharing and their effect on disciplines that have no existing tradition of open access. However, I myself have been a social sciences data librarian for fifteen years and am familiar with data standards for these disciplines that pre-date me by decades. For instance, the eP1245 \| 1 jlsc-pub.org jlsc-pub.org JL SC Volume 3, Issue 2 Roper Center for Public Opinion Research has been collecting opinion poll data since 1947 (Roper Center, n.d.). The Inter-university Consortium for Political and Social Research (ICPSR) has been collecting and preserving data sets since 1962 (ICPSR, n.d.). Over time these organizations and others who work with research data saw the need for standards and participated in developing them. 1 I’m told IASSIST is a “backronym,” intentionally reverse-engineered so our association acronym states what each of us do: I assist. Data World Does Not Lack Standards “A publication calling for bibliographic conventions and standards was written by David Nasatir under contract to UNESCO to study ‘overcoming the barriers to realizing the fullest utilization of machine-readable social science data’” (Stephenson, 2013, p. 8). This call was published in 1973. In 1974, the International Association for Social Science Information Services and Technology (IASSIST)1 was formed following the Conference on Data Archives and Program Library Services, held in conjunction with the 8th World Conference of Sociology in Toronto, Canada. IASSIST fosters connections and promotes information sharing between professionals working with and helping others with data. Every year since then it has brought together statisticians, librarians, staff at statistical agencies, faculty, and other data geeks of all stripes. Sue A. Dodd, an archivist who completed extensive research and multiple publications on the subject of how to catalog “machine readable data files” (MRDF), published her landmark guide, Cataloging machine-readable data files: An interpretive manual through the American Library Association in 1982, as a project funded by the Council on Library Resources (Stephenson, 2013). Ms. Dodd’s work has been so important to IASSIST that our professional journal, the IASSIST Quarterly, devoted a special memorial issue in 2013 to considering her contributions to our work, available online at http://www.iassistdata.org/ iq/issue/37/1. (That issue subsumed the whole volume that year.) So you may understand my consternation at the bald statement in the CFP for this issue that there are no standards in the data world. The standard I’m most familiar with is the Data Documentation Initiative, or DDI, a metadata standard for social science survey data, but this is only one of many. If you’re not aware of a metadata standard for a particular discipline, the Digital Curation Centre (DCC) (serving the UK academic community) provides a searchable database of Disciplinary Metadata standards for data, available online at http://www.dcc.ac.uk/resources/metadata-standards. Another way to identify a standard is to find the repository(ies) that handle that kind of data. Often repositories will require depositors use a particular standard for the data they wish to archive. Several clearinghouses for finding repositories have been established in recent years: Databib, DataCite, and the Registry of Research Data Repositories (re3data) are just a few. Journal of Librarianship and Scholarly Communication Data World Does Not Lack Standards Many of these clearinghouses are involved in larger data management efforts such as re3data’s development of a broader Schema for the Description of Research Data and DataCite’s participation in both the 1 I’m told IASSIST is a “backronym,” intentionally reverse-engineered so our association acronym states what each of us do: I assist. Journal of Librarianship and Scholarly Communication 2 \| eP1245 2 \| eP1245 Hayslett \| Data World Does Not Lack Standards DCC’s International Digital Curation Conference (which itself is now in its 11th year) and the Research Data Alliance, also an international endeavor and focused on building “the social and technical bridges that enable open sharing of data” (Research Data Alliance, n.d.).2 I am not familiar enough with science data to detail the history of standards in those areas but the Federal Geographic Data Committee, which established the FGDC metadata standard for U.S. geographic data, was founded in 1990. And more recently, the Data Observation Network for Earth (DataONE), founded in 2009, is pushing the envelope by making scientific data sets not only citable and accessible, but inter-operable, to better leverage scarce funding and already-accomplished research. This effort would have been inconceivable without detailed metadata. I am not familiar enough with science data to detail the history of standards in those areas but the Federal Geographic Data Committee, which established the FGDC metadata standard for U.S. geographic data, was founded in 1990. And more recently, the Data Observation Network for Earth (DataONE), founded in 2009, is pushing the envelope by making scientific data sets not only citable and accessible, but inter-operable, to better leverage scarce funding and already-accomplished research. This effort would have been inconceivable without detailed metadata. While the statement in the CFP for this issue is untrue, though, it demonstrates the profession’s general lack of awareness of the history of data curation within librarianship. I know the specialty of data librarianship is obscure and somewhat mysterious, even to our closest colleagues. We curate data, preparing it for archiving; we help users find the data they need and then understand them (yes, “data” is the plural form). We help researchers understand data management options. We help patrons navigate the many differences between the U.S. decennial census and the American Community Survey, the many countries’ censuses available through the Integrated Public Use Microdata Series (IPUMS), and hundreds of other data sources. 2 DataBib and re3data merged as of May 2015, and their respective content is combined under the auspices of DataCite. Data World Does Not Lack Standards And the standards we deal with are often most actively used within the research life cycle stage of gathering data, where fewer librarians find their niche. But the good news for all librarians who do not specialize in data is that there is no more collegial and generous group of people than my data colleagues. We are happy to share a brochure developed a few years ago by the IASSIST Special Interest Group on Data Citation, which outlines the most important elements of a data citation in multiple citation styles. It is available for you to download from http://iassistdata.org/community/ sigdc and print as needed. (This interest group is also lobbying citation management companies like RefWorks and Zotero to include Data Sets as a type of resource to cite along with Journal Articles, Books and Patents, and has written letters to the major style guides such as APA and Chicago advocating they provide “official” guidelines for citing data sets.) We are happy to share our experience evaluating data repositories and guiding researchers to reliable data management solutions. We are happy to share advice about sources when you run up against thorny data-related questions. We also welcome your contributions if you would like to become involved in one of the many organizations eP1245 \| 3 eP1245 \| 3 eP1245 \| 3 jlsc-pub.org JL SC Volume 3, Issue 2 focused on making data sharable, discoverable, and usable (there are many more than I have mentioned here but they are also very intertwined—once you start looking, many others will surface). Membership in my personal favorite, IASSIST, is very affordable (if you’re interested) and provides you with access to the whole international membership through our listserv, blog, and website. But you may also find there are data colleagues right at your own institution. Search your institution’s website for pages that address data: citing data, managing data, finding or acquiring data, and archiving/preserving/curating data. I encourage you to explore the landscape of our data world—you may be surprised at the resources available in your own backyard. Data World Does Not Lack Standards Resource URL Data Documentation Initiative (DDI) http://www.ddialliance.org/ Data Observation Network for Earth (DataONE) https://www.dataone.org/ Databib http://databib.org DataCite https://www.datacite.org Digital Curation Centre (DCC) http://www.dcc.ac.uk/ International Association for Social Science Information Services and Technology (IASSIST) http://www.iassistdata.org/ International Digital Curation Conference http://www.dcc.ac.uk/events/international-digital- curation-conference-idcc Inter-university Consortium for Political and Social Research (ICPSR) http://www.icpsr.umich.edu/icpsrweb/ICPSR/ Registry of Research Data Repositories (re3data) http://re3data.org Roper Center for Public Opinion Research http://www.ropercenter.uconn.edu/ U.S. decennial census and American Community Survey (ACS) http://www.census.gov/ Integrated Public Use Microdata Series (IPUMS) https://www.ipums.org/ ACKNOWLEDGEMENTS The author would like to thank Katherine McNeill (Program Head, Data Management Services) at MIT Libraries, for editing this commentary. Any errors are the author’s alone. Journal of Librarianship and Scholarly Communication REFERENCES ICPSR. (n.d.) About the organization. Retrieved from http://www.icpsr.umich.edu/icpsrweb/content/ membership/about.html 4 \| eP1245 Hayslett \| Data World Does Not Lack Standards Hayslett \| Data World Does Not Lack Standards Research Data Alliance. (n.d.) About. Retrieved from https://rd-alliance.org/about.html Roper Center for Public Opinion Research. (n.d.) About the Center. Retrieved from http://www.ropercenter. uconn.edu/about-the-center/ Stephenson, L. (2013). “Social science data files and bibliographic control: contributions: Sue A. Dodd,” IASSIST Quarterly, 37(1), 8. Retrieved from http://www.iassistdata.org/downloads/iqvol371_4_ stephenson.pdf eP1245 \| 5 eP1245 \| 5 jlsc-pub.org
W3158689833.txt	http://www.scielo.br/pdf/bjce/v26n2/v26n2a20.pdf	fr		Voiture Chiffon de nettoyage en microfibre lingette s anti brouillard chiffon pour objectif de nettoyageusing TRPO/cyclohexane reverse micelles	null	2,009	cc-by	6,080	Brazilian Journal of Chemical Engineering ISSN 0104-6632 Printed in Brazil www.abeq.org.br/bjche Vol. 26, No. 02, pp. 415 - 425, April - June, 2009 LIQUID – LIQUID EXTRACTION OF MATRINE USING TRPO/CYCLOHEXANE REVERSE MICELLES Lichun Dong, Wenping Li, Xiaohua Zhou, Qin Kang and Weifeng Shen College of Chemistry and Chemical Engineering, Phone: 86-13640514654, Fax: 86-23-65111179, Chongqing University, Chongqing, 400030, P.R. China. E-mail: lcdong72@gmail.com (Submitted: August 18, 2008 ; Revised: October 12, 2008 ; Accepted: October 13, 2008) Abstract - Reverse micellar extraction has been widely used in the purification of biomolecules. However, reverse micelles formed by ionic surfactants can only be employed for the extraction of biomolecules that are charged in the extraction system with the electrostatic interaction between surfactants and solutes as the driving force. In this study, the extraction of matrine by using reverse micelles formed by non-ionic TRPO surfactants was studied. Theoretical analysis and experimental results demonstrated that the driving force of the extraction is the coordination forces between matrines and TRPOs. Using this coordination-based reverse micellar extraction, matrine can be efficiently separated from oxymatrine and other components in the raw matrine materials. Experimental studies showed that the factors affecting matrine extraction include pH value and TRPO concentration. The existence of ions in the system does not affect the partition coefficient significantly and the addition of a small amount of chloroform in the solution of reverse micelles was found to improve the extraction significantly. Keywords: Reverse micellar extraction; Coordination interaction; TRPO reverse micelles; Matrine; Oxymatrine. INTRODUCTION Matrine, a kind of quinolizidine alkaloid, is reported to exhibit remarkable biomedical functions (sedative, depressant, anti-viral, anti-tumor, antibacteria, anti-inflammatory, resisting skin fibrosis, improving cardiovascular and neural functions) (Liu et al., 2007; Feng and Tang, 2005; Hoang et al., 2007). During the past decades, the production of matrine from natural plants i.e., Sophora flavescens, Sophora alopecuroides L., has attracted considerable attention from pharmaceutical researchers. All the reported methods include two steps: the first step is to extract all alkaloids from natural plants using methods such as CO2 supercritical extraction (Jian et al., 2007), macro-porous resin adsorption (Qin et al., 2007), molecularly imprinted solid-phase extraction (Lai et al., 2003), thin-layer chromatography (Ding et al., 2004) and ion-paired HPLC (Zhang et al., To whom correspondence should be addressed 2007); the second step is to separate the alkaloids to acquire pure matrine. Jian et al. (2007) first extracted alkaloids from Sophora flavescens Ait. using supercritical fluid extraction (SFE). The crude alkaloids were then separated by high-speed countercurrent chromatography (HSCCC) to produce matrine, oxymatrine, and oxysophocarpine. In the study by Qin et al. (2007), the alkaloids were first extracted from Sophora alopecuroides L. by adsorption using macro-porous resin. The alkaloid monomers were further isolated by gradient elution with ethanol/water solutions of different volume ratios at reduced pressure. Molecularly imprinted solid-phase extraction was studied by Lai et al. (2003) to separate raw matrine from Sophora flavescens Ait; the marine of high purity was then produced by methanol/water washing and methanol/glacial acetic acid elution. All of these methods were demonstrated to be effective to 416 Lichun Dong, Wenping Li, Xiaohua Zhou, Qin Kang and Weifeng Shen produce matrine. Especially at the step to separate matrine with other alkaloids monomers (oxymatine, oxysophocarpine), however, all have their limitations such as being time-consuming, using too much organic solvents, etc. A new method is greatly needed to separate matrine from other alkaloids more efficiently and reduce the use of organic solvent at the same time. The application of reverse micellar extraction in bio-separation has attracted considerable attention in the past two decades because the technique shows advantages in the ease of scale-up, reusable surfactant and solvent and continuous separation of biological molecules from fermentation mixtures, etc. Numerous studies have been carried out on protein, amino acid, and DNA purification using reverse micellar extraction (Wang et al., 1995; Rabie and Vera, 1996; Krieger et al., 1997; Hashimoto et al., 1998; Cardoso et al., 1999a; Jarudilokkul et al., 2000; Zhang et al., 2000; Lou et al., 2001; Liu et al., 2004; Wang et al., 2004). Among them, the reverse micellar extraction of proteins has been extensively studied and the extraction mechanism is well understood. For the extraction of proteins, reverse micelles formed by ionic surfactants were mostly employed. The transfer of proteins from aqueous phase to the reverse micelles is primarily due to the electrostatic interactions between proteins and the surfactants. Since the last decades, researchers have studied the extraction of proteins based on affinity interaction using reverse micellar solutions formed by nonionic surfactants (Guadalupe et al., 1992; Chang et al., 1997; Naoe et al., 1998; Ichikawa et al., 2000; Liu et al., 2007). By incorporating affinity ligands into the system, proteins can be selectively extracted by a biospecific affinity interaction. Because the “water pool” in reverse micelles can be adjusted, reverse micellar extraction has also been demonstrated to be a promising method to purify bio-materials with small molecules (Zhou et al., 2008, Dong et al., 2008). However, constrained by the working mechanism, reverse micelles formed by ionic surfactants can only be employed for the extraction of bio-molecules that are charged in the extraction system. In order to extract small biomolecules that are not charged, non-ionic surfactants and a novel working mechanism have to be considered. TRPO (trialkyl phosphine oxide) is a kind of nonionic surfactant, which has been used in the extraction of metals (Xin et al., 2002) and organic acids (Wang et al., 2003) as coordination agent. In this paper, the extraction of matrine using TRPO/cyclohexane reverse micelles was studied; the driving force is believed to be the coordination forces between TRPOs and matrine, which was first rationalized by theoretical analysis and further verified by experimental result. To the best of our knowledge, we are the first group to report reverse micellar extraction with coordination as the driving force. The goal of the study is to continue the effort to extend the application of reverse micellar extraction to the purification of bio-materials with small molecules. EXPERIMENTS Materials and Reagents TRPO was purchased from Shanghai Rare-earth Co. Ltd., China; Standard sample of matrine (Purity > 98%), oxymatrine (Purity > 98%), and raw matrine sample (produced from roots of Sophora flavescens Ait, matrine + oxymatrine > 60%) were form Xi’an Honson Biotechnology Co. Ltd., Xi’an, China. Experimental Methods and Procedures a) Preparation of TRPO Reverse Micelles TRPO was first dehydrated using 4Å zeolite for 48h, mixed with cyclohexane in an iodine flask, and then a suitable amount of D.I. water was added. The expected reverse micellar solution was finally obtained after thoroughly stirring for several hours. The solution should be transparent and does not adsorb UV and visible light. The W0 (W0 = moles of water /moles of TRPO) of TPRO/cyclohexane reverse micelles prepared in this study varies from 15 to 3 and the estimated “pool” radius from 10 nm to 2 nm (Cardoso et al., 1999b; Reng et al., 2004). To confirm the formation of reverse micelles, the IR spectra of TRPO (before dehydrating), dehydrated TRPO/cyclohexane solution, and TRPO/cyclohexane reverse micelles was recorded using a MAGMA – IR550 spectrometer (Nicolet Company, USA). b) Preparation of Solutions Oxymatrine and Raw Matrine of Matrine, The solutions of matrine (or oxymatrine) were prepared by dissolving standard matrine (or oxymatrine) using D.I. water in an iodine flask followed by filtering using a microporous membrane with pore size of 0.45 μm. The same procedure was followed to prepare the raw solution. The concentration of matrine and oxymatrine in the raw solution was measured using capillary electrophoresis. All the prepared solutions were stored at 4ºC before being used. Brazilian Journal of Chemical Engineering Liquid-Liquid Extraction of Matrine Using Trpo/Cyclohexane Reverse Micelles c) Extraction and Strip-Extraction Experiments The solution of TRPO/cyclohaxane reverse micelles of known volume was first put into an iodine flask, then the solution of matrine (or oxymatrine, raw matrine) was added for extraction for 45 min with a stirring speed of 200 rpm. The extraction was processed at 30 ºC and a controlled pH value. The mixture was allowed to stand for 2 min to separate the extract and raffinate phase before the extract and raffinate phases were gathered, respectively. The quantity of matrine in the raffinate phase was measured and the extraction efficiency and partition coefficient were calculated according to Eq. 1 and Eq. 2, respectively. Extraction Efficiency(%) = Quantity of matrine in extract phase × 100% Total quantity of matrine Partititon Coefficient (D) = Concentration of matrine in extract phase Concentration of matrine in raffinate phase Quantity of matrine in strip-extraction extract phase × 100% Total quantity of matrine in reverse micellar solution O The quantity of matrine (oxtmatrine) was measured using a method of capillary electrophoresis (Yao et al., 1996, Na et al., 2003). The buffer used in capillary electrophoresis is 25 mmol/L borax solution, which contains 30 mmol/L SDS and 35% ethanol (by volume). The sample for electrophoresis is injected at 25 KV and the sample for electromigration is injected at 25 KV×4 sec; the wavelength of testing is 208 nm. The quantity of all contents was determined by the internal standard method with ephedrine as the internal standard sample. The calculation is by Eq. 4. CX = f A X The extract phase was then transferred into another iodine flask, then strip-extraction liquid (DI water with the pH value adjusted using NaOH) was added for strip-extraction for 40 min. As in the extraction, the strip-extraction was processed at 30 ºC and with a stirring speed of 200 rpm. After the mixture was allowed to stand for 2 min to separate the extract and raffinate phase, the quantity of matrine in the extract phase was measured. The strip-extraction efficiency was calculated according to Eq. 3. Strip-extraction Efficiency(%) = d) Quantitative Measurement of Matrine and Oxymatrine (1) (2) (3) 417 CS' AS' (4) where CX and AX are the concentration and the peak area of the sample, respectively. CS' and AS' are the concentration and peak area of the internal standard sample, respectively; f is the correction factor. THEORETICAL ANALYSIS OF MATRINE EXTRACTION USING TRPO REVERSE MICELLES Neither matrine nor oxymatrine are soluble in cyclohexane or anhydrous TRPO (Sun and Xie, 2003), while matrine can be dissolved in the solution of TRPO/cyclohexane reverse micelles. The only places that the solution of TRPO/cyclohexane reverse micelles can dissolve matrines are the “water pools”. The extraction of materine by the solution of TRPO/cyclohexane is, therefore, a kind of reverse micellar extraction. O N 7 N 7 N 15 15 N + O- (a) (b) Figure 1: Molecular Structures of Matrine (a) and Oxymatrine (b). Brazilian Journal of Chemical Engineering Vol. 26, No. 02, pp. 415 - 425, April - June, 2009 418 Lichun Dong, Wenping Li, Xiaohua Zhou, Qin Kang and Weifeng Shen R1 R2 P O R3 Figure 2: Molecular Structure of TRPO. R1, R2, R3 = CH3(CH2)7 or CH3(CH2)5. The molecular structure of both matrine and oxymatrine is planar (Fig. 1). The difference between the molecular structure of matrine and that of oxymatrine is that the N15 atom in oxymatrine is coordinated to an oxygen atom. Both N15 and N7 in matrine molecules are tertiary nitrogens and have two lone-pair electrons. However, N7 is adjacent to a carbonyl group; the electrons of N7 conjugate with the carbonyl due to the strong electron-attracting capability of the carbonyl group, with the result that N7 cannot share the lone-pair electrons and form coordination bonds with atoms having empty orbitals. Although connected to three C atoms, N15 in matrine can easily form coordination bonds with atoms having empty orbitals via the lone-pair electrons because N is more electronegative than C.. TRPO is a kind of nonionic surfactant (Fig. 2); the oxygen atom in TRPO has an empty orbital that can form a coordination bond with atoms with lonepair electrons like the N15 atom in matrine. Because TRPO is a kind of non-ionic surfactant, the driving force for TRPO/cyclohexane reverse micellar extraction of matrine is surely not the electrostatic force between the TRPOs and matrines. According to the molecular structures of matrine and TRPO, the driving force for the extraction is believed to be the coordination force between them. When matrine meets TRPO in the reverse micellar system, matrine can form a TRPO-matrine complex with TRPO via a N15-O coordination bond and be extracted into TRPO reverse micelles. The N15 in oxymatrine is already coordinated with an oxygen atom (Fig. 1), thus cannot coordinate with TRPO. Therefore, oxymatrine can be separated from matrine by using TRPO reverse micellar extraction. According to the extraction mechanism, the followed reaction occurs during the extraction of matrine using TRPO reverse micelles. K eq = matrine − N15 → O = P − R 3 (5) where Keq is the reactive equilibrium constant. When the extraction reaches equilibrium (6) In aqueous solution, the N15 atom of matrine can bond with a H+ ion to become quaternary, as in Eq. 7. The N15 is then positively charged and can further bond with negatively charged ions in the extraction system, which causes matrine to lose its coordination ability (Yang and Ma, 1997). K1 Matrine − N15 + H + ←⎯⎯ → Matrine − N15 H + (7) where K1 is the equilibrium constant of the reaction. When the reaction reaches equilibrium ⎡ Matrine − N15 H + ⎤ ⎦ K1 = ⎣ ⎡ Matrine − N15 ⎤ ⎡ H + ⎤ ⎣ ⎦⎣ ⎦ (8) By modifying Eq. 2, the partition coefficient of matrine can be calculated from Eq. 9. D= Quantity of matrines in extract phase (9) R × Quantity of matrines in raffinate phase where R is the phase volume ratio between organic phase (solution of TRPO reverse micelles) and aqueous phase. The quantity of matrines extracted into the extract phase is equal to the quantity of Matrine − N15 → TRPO in the extraction system, the quantity of matrines left in the raffinate phase is equal to the sum of the quantity of matrines that do not form complexes with TRPOs and the quantity of protonated matrines with N15 as quaternary nitrogens. Thus Eq. 9 can be transformed to Eq. 10. K eq matrine − N15 + O = P − R 3 ←⎯⎯ → [matrine − N15 → O = P − R 3 ] [matrine − N15 ][O = P − R 3 ] D= ⎡ Matrine − N15 → O = P − R 3 ⎤ ⎣ ⎦ 15 15 R ⎡ Matrine − N ⎤ + ⎡ Matrine − N H + ⎤ ⎣ ⎦ ⎣ ⎦ ( ) (10) Substituting Eq. 6 and Eq. 8 into Eq. 10, we get Eq. 11: Brazilian Journal of Chemical Engineering Liquid-Liquid Extraction of Matrine Using Trpo/Cyclohexane Reverse Micelles D= K eq ⎡ Matrine − N15 ⎤ [O = P − R 3 ] ⎣ ⎦ = R ⎡ Matrine − N15 ⎤ + K1 ⎡ Matrine − N15 ⎤ ⎡ H + ⎤ ⎣ ⎦ ⎣ ⎦⎣ ⎦ (11) ) ( K eq [O = P − R 3 ] ( R 1 + K1 ⎡ H + ⎤ ⎣ ⎦ ) In Eq. 11, Keq and K1 are all constants. Eq. 11 shows that the partition coefficient increases with an increase of the concentration of TRPO, and decreases with an increase of the concentration of H+. In the following sections of the paper, the experimental results of the extraction of matrine using TRPO reverse micelles are discussed to verify the theoretical analysis. EXPERIMENTAL RESULTS AND DISCUSSION system, whereas the absorbance corresponding to – OH in the free state is still absent. The rational reason for this result is that the added water is confined in the “pool” formed by TRPO reverse micelles; the binding with P=O makes the properties of water in the “pools” different from the free water. Therefore, it can be concluded that the transparent water-containing TRPO/cyclohexane system is a solution of TRPO reverse micelles. Factors Affecting Matrine Extraction Theoretical analysis indicated that the matrine extraction is affected by pH and TRPO concentration. In order to verify the theoretical prediction, a series of experiments were performed to study the possible factors affecting the extraction, including pH, TRPO concentration, ions, etc. a) Effect of pH on Matrine Extraction IR Analysis of the Formation of Reverse Micelles The IR spectra of TRPO, TRPO/cyclohexane solution dehydrated using 4Å zeolite, and TRPO/cyclohexane reverse micellar solution are shown in Fig. 3. Since TRPO of industrial grade contains a small amount of water, its IR spectrum has two peaks of –OH at 1643 cm-1 (associated state) and 3391 cm-1 (free state), respectively. There is also a strong absorbance at 1158 cm-1 corresponding to the P=O group in the spectrum. After being dehydrated using 4Å zeolite, the peaks corresponding to –OH disappear in the IR spectrum of TRPO/cyclohexane solution. Moreover, the strong absorbance of P=O weakens and moves to a lower wave number. The reason for this change is that the hydrophobic alkyl groups of TRPO are associated with cyclohexane, the polar P=O groups aggregate to form hydrophilic “cores”, the characteristic absorbance of P=O is, therefore, screened. After D.I. water was added to TRPO/cyclohexane solution, an absorbance at 1659 cm-1 corresponding to –OH in the associated state appears in the IR spectrum of the Fig. 4 shows the experimental partition coefficient versus pH of the matrine solution. The experiments were set up to extract matrine from standard matrine solutions previously prepared. The partition coefficient increases with an increase of pH value from 4 to 9. As shown in Eq. 7, the N15 in matrine molecules can be a tertiary amine or a quaternary amine, whose concentration ratio is dependent on the pH of the system. With an increase of pH, the decrease of H+ concentration increases the concentration of tertiary amines in the extraction system, which is in favor of the formation of matrine − N15 → O = P = R 3 complexes. Therefore, the experimental partition coefficient increases with an increase of the pH value, as predicted by Eq. 11. When the pH value exceeds 9, the increase of interfacial activity of matrine could cause the emulsification of the extraction system, which would make the separation of extract phase and reaffinate phase very difficult. Therefore, the optimum pH value for the extraction of matrine using TRPO reverse micelles is between 8 and 9. Absorbance 1 2 3 -OH -OH C-C P=O C-H 4000 3500 419 3000 2500 2000 1500 1000 500 Wavenumbers (cm-1) Figure 3: IR spectra of dehydrated TRPO/cyclohexane solution (1), TRPO/ cyclohexane reverse micelle (2), and TRPO (3). Brazilian Journal of Chemical Engineering Vol. 26, No. 02, pp. 415 - 425, April - June, 2009 420 Lichun Dong, Wenping Li, Xiaohua Zhou, Qin Kang and Weifeng Shen 0.8 Volume Phase Ratio = 1 Matrine Concentration = 0.04 mol/L TRPO Concentration = 0.10 mol/L 0.7 0.6 D 0.5 0.4 0.3 0.2 0.1 0.0 4 5 6 7 pH 8 9 10 Figure 4: Experimental partition coefficient versus the pH value of the matrine solution. b) Effect of TRPO Concentration on Matrine Extraction Fig. 5 shows the experimental partition coefficient versus TRPO concentration. As in the previous section, the experiments were performed to extract matrine from standard matrine solutions prepared. When the concentration of TRPO is in the range of 0.05 to 0.25 mol/L, the partition coefficient increases with an increase in TRPO concentration, which agrees well with the theoretical prediction of Eq. 11. The partition coefficient reaches a plateau value when the TRPO concentration is higher than 0.25 mol/L. The water in the “water pools” of reverse micelles can be categorized into two classes: the “free” water and the “bound” water that is bounded to the surfactants (Menger and Saito, 1978; Jolivalt et al., 1989; Ono et al., 1996). The properties of the “bound” water are different from those of the “free” water and the extraction capability of the reverse micelles is mostly dependent on the total amount of “bound” water. As the concentration of the TRPO increases, the “pool” size of the reverse micelles decreases. A larger portion of the water inside the reverse micelles is bound to the surfactants and becomes “bound” water, which benefits the extraction and increases the partition coefficient. However, with the amount of “free” water decreasing, the availability of “free” water becomes more and more difficult with the increase of TRPO concentration. When all the water inside the reverse micelles becomes “bound” water, the increase of the concentration of the TRPO reverse micelles does not affect the extraction any further. c) Effect of Ions on Matrine Extraction In the extraction using ionic reverse micelles with electrostatic interactions as the driving force, the existence of ions in the system always affects the extraction significantly because ions can influence the electrostatic interaction between surfactants and solutes in several ways: salting out effect, screening effect, etc (Huang and Gu, 1996; Zhou et al., 2008). However, in this study, TRPO is a kind of non-ionic surfactant and the driving force of the extraction is the coordination forces. Thus, the existence of ions in the system should not affect the extraction significantly. To verify this hypothesis, the partition coefficient of matrine was studied with CaCl2 present in the system was studied and the results are shown in Fig. 6. The figure shows that the partition coefficient hardly changes at different CaCl2 concentrations. This observation confirmed that the existence of ions in the system does not affect the coordination interaction between TRPOs and matrines or the partition coefficient significantly. 1.4 1.2 1.2 1.0 1.0 0.8 0.8 D D 1.4 0.6 0.6 0.4 0.4 Volume Phase Ratio = 1 Matrine Concentration = 0.04 mol/L pH Value = 9 0.2 0.0 0 50 100 150 200 250 300 350 400 Volume Phase Ratio = 1 Matrine Concentration = 0.04 mol/L TRPO Concentration = 0.25 mol/L 0.2 450 0.0 0 1 TRPO Concentration ( mmol/L) Figure 5: Experimental partition coefficient versus TRPO concentration. 2 3 4 5 6 7 8 CaCl 2 Concentration (mg/ mL) 9 10 Figure 6: Experimental partition coefficient versus CaCl2 concentration. Brazilian Journal of Chemical Engineering Liquid-Liquid Extraction of Matrine Using Trpo/Cyclohexane Reverse Micelles d) Effect of Chloroform on Matrine Extraction In the study, the addition of a small amount of chloroform has been found to increase the partition coefficient remarkably. Fig. 7 shows that the partition coefficient of matrine increases from 1.2 to 3.7 when the weight concentration of chloroform in the solution of TRPO reverse micelles increases from 0 to 6.0%. The exact working mechanism of chloroform is not clear yet and requires a more detailed study. According to previous studies (Liu et al., 2007), a possible explanation is that chloroform, working as co-solvent, can help surfactants to dissolve better in the organic solvent, adjust the interaction between the polar heads of the surfactants, and make the reverse micelles have a suitable “pool” size for extraction. e) Effect of Extraction Time on Matrine Extraction Fig. 8 shows the partition coefficient of matrine versus extraction time. It takes about 45 421 min for the extraction to reach equilibrium and the partition coefficient then reaches a plateau after 45 min. The Strip-Extraction of Matrine Using D.I. water of different pH values as strip-extraction liquid, the strip-extraction of matrine from the extract phase (the TRPO reverse micelles with matrine) obtained from the extraction experiments was studied. Opposite to the extraction, the strip-extraction efficiency decreases with an increase in pH. With an increase of pH value, Eq. 7 moves to the left, the concentration of matrines with N15 as tertiary amines increases, strengthening the coordination interaction between matrines and TRPOs, and the strip-extraction efficiency decreases. Fig. 9 shows that the single strip-extraction efficiency decreases from 67% to 15% when the pH value of the strip-extraction increases from 3 to 10, which agrees well with the theoretical prediction. 4 D 3 2 1 0 Volume Phase Ratio = 1 Matrine Concentration = 0.04 mol/L TRPO Concentration = 0.25 mol/L pH Value = 9 0 1 2 3 4 5 Chloroform Concentration (%) 6 4.5 4.0 3.5 D 3.0 2.5 2.0 1.5 pH Value = 9 Matrine Concentration = 0.04 mol/L TRPO Concentration = 0.25 mol/L Volume Phase Ratio = 1 Chloroform Concentration = 6.0 % 1.0 0.5 0.0 0 5 10 15 20 25 30 35 40 45 50 Single Strip extraction Efficiency (%) Figure 7: Experimental partition coefficient versus chloroform concentration. 80 Volume Phase Ratio = 1 pH Value = 3 Strip -extraction Time = 40 min 60 40 20 0 3 4 5 6 7 pH 8 9 10 11 Extraction Time (min) Figure 8: Experimental partition coefficient versus chloroform concentration. Figure 9: Experimental strip-extraction coefficient versus pH value. Brazilian Journal of Chemical Engineering Vol. 26, No. 02, pp. 415 - 425, April - June, 2009 422 Lichun Dong, Wenping Li, Xiaohua Zhou, Qin Kang and Weifeng Shen Experimental Study of Oxymatrine Extraction Using TRPO Reverse Micelles Theoretical analysis indicated that oxymatrine can not be extracted by TRPO reverse micelles since it can not coordinate with TRPO. To verify this prediction, extraction of oxymatrine from standard oxymatrine solution was studied. Fig. 10a shows the experimental extraction efficiency of oxymatrine at different chloroform concentrations. Although the addition of chloroform increases the extraction of oxymatrine, like that of matrine, the extraction efficiency of oxymatrine is much lower, which confirms the theoretical analysis. Fig. 10b shows the separation factor between matrine and oxymatrine at different chloroform concentrations, where the separation factor is the ratio between extraction efficiency of matrine and that of oxymatrine at the same operating condition. The separation factor is 16.5 with no chloroform. With chloroform added, the separation factor first decreases with an increase in chloroform concentration and reaches a minimum, then increases with a further increase of chloroform concentration. The separation factor is about 15.5 when the chloroform concentration is 6 wt%. Therefore, the matrine extraction should be operated at higher chloroform concentration because the extraction has higher matrine extraction efficiency as well as a better separation factor. The Separation of Matrine with Oxymatrine On the basis of single-factor experiments previously discussed, the optimum conditions for matrine extraction and strip-extraction from raw matrine solution were investigated via two groups of orthogonal experiments. The optimum operating conditions for extraction are: pH = 9.0, TRPO concentration = 0.25 mol/L, chloroform concentration = 6 wt%, phase ratio = 1:1 (V/V), extraction time = 45 min. The optimum operating conditions for strip-extraction are: pH = 3, phase ratio = 1:1 (V/V), and strip-extraction time = 40 min. With matrine and oxymatrine concentration in the raw matrine solution of 0.047 mol/L and 0.083 mol/L, respectively, the yield of the matrine can reach 60%. After strip-extraction, the strip-extraction extract phase was concentrated and lyophilized. The powder of the products after lyophilization was analyzed by using capillary electrophoresis as described in the experimental section. Fig. 11 shows the results of the products after three stripextractions. The composition of the final product is: 90.2% matrine, 5.4% oxymatrine, 4.4% water and others. By the second extraction, matrine with higher purity can be produced. Except matrine and oxymatrine, the solution of raw matrine also contains saccharide, short peptides, a small amount of oxysophocarpine and amino acids, etc. It can be seen that TRPO reverse micelle extraction can separate matrine from most of oxymatrine and all of the other components. Compared with reverse micellar extraction based on electrostatic force, this coordination interactionbased reverse micellar extraction has better selectivity because of the specific interaction between surfactant and the solvent. 5 Separation Factor Extraction Efficiency (%) 6 4 3 2 Volume Phase Ratio = 1 OxymatrineConcentration = 0.04 mol/L TRPO Concentration = 0.25 mol/L pH Value = 9 1 0 0 1 2 3 4 5 6 18 17 16 15 14 13 12 11 10 9 8 Volume Phase Ratio = 1 TRPO Concentration = 0.25 mol/L pH Value = 9 0 Chloroform Concentration (%) 1 2 3 4 5 Chloroform Concentration (%) (b) (a) Figure 10: Experimental extraction efficiency of oxymatrine (a) and the separation factor between matrine and oxymatrine (b) versus chloroform concentration. Brazilian Journal of Chemical Engineering 6 Liquid-Liquid Extraction of Matrine Using Trpo/Cyclohexane Reverse Micelles 423 Figure 11: The capillary electrophoresis results for the final products after three strip-extractions. CONCLUSIONS In this paper, matrine extraction using TRPO reverse micelles was studied and the working mechanism of the extraction is verified to be the coordination forces between matrines and TRPOs. Using this coordination-based reverse micellar extraction, matrine can be efficiently separated from oxymatrine and other components in the raw matrine materials. The yield can reach 60%, and the purity of matrine is more than 90%. The significance of this study is that it sheds considerable light on the purification of biomaterials with small molecules by extraction using reverse micelles formed by nonionic surfactants. The main factors affecting matrine extraction using TRPO reverse micelles include pH and TRPO concentration. The partition coefficient of matrine increases with an increase in pH of the aqueous phase and TRPO concentration when it is below 0.25 mol/L. The existence of ions in the system does not affect the extraction significantly. Moreover, the addition of a small amount of chloroform has been found to increase the partition coefficient remarkably. AX CS' AS' Keq K1 R sample in the capillary electrophoresis measurement the peak area of the sample in the capillary electrophoresis measurement the concentration of the internal standard sample in the capillary electrophoresis measurement the peak area of the internal standard sample in the capillary electrophoresis measurement reactive equilibrium constant reactive equilibrium constant phase volume ratio for extraction (-) kg/mL (-) (-) (-) (-) ACKNOWLEDGMENTS The authors would like to thank the Chongqing Hanfan Company for their financial support. NOMENCLATURE Abbrevation REFERENCE: TRPO Trialkyl phosphine oxide (-) Symbols W0 D CX the ratio between moles of water and that of TRPO in reverse micellar solution partition coefficient of extraction the concentration of the (-) (-) kg/mL Cardoso, M. M., Viegas, R. M. C., and Crespo, J. P. S. 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https://openalex.org/W3019693732	https://nhm.openrepository.com/bitstream/10141/622825/4/FFSH%20Historical%20Contaminants%2020200403%20FINAL.pdf	English	null	Biological archives reveal contrasting patterns in trace element concentrations in pelagic seabird feathers over more than a century	Environmental pollution	2,020	cc-by	13,741	Keywords: Ardenna carneipes; Flesh-footed Shearwater; mercury; lead; cadmium; 33 Western Australia 34 Title Biological archives reveal contrasting patterns in trace element concentrations in pelagic seabird feathers over more than a century Authors Bond, AL; Lavers, JL Date Submitted 2020-09-02 Title Biological archives reveal contrasting patterns in trace element concentrations in pelagic seabird feathers over more than a century Authors Bond, AL; Lavers, JL Date Submitted 2020-09-02 Biological archives reveal contrasting patterns in trace element concentrations in 1 pelagic seabird feathers over more than a century 2 3 Alexander L. Bond1,2* and Jennifer L. Lavers2 4 5 1 Bird Group, Department of Life Sciences, The Natural History Museum, Akeman 6 Street, Tring, Hertfordshire, HP23 6AP United Kingdom 7 2 Institute for Marine and Antarctic Studies, University of Tasmania, 20 Castray 8 Esplanade, Battery Point, Tasmania, 7004, Australia 9 10 Corresponding author 11 E-mail: a.bond@nhm.ac.uk, Tel: +44 (0)20 7942 6162 12 13 Alexander L. Bond1,2 and Jennifer L. Lavers2 1 Capsule: Cadmium in Flesh-footed Shearwater feathers increased between 1900 and 36 2011, while mercury remained stable and lead decreased. 37 2011, while mercury remained stable and lead decreased. 37 Capsule: Cadmium in Flesh-footed Shearwater feathers increased between 19 36 Abstract 14 Contamination of diverse environments and wild species by some contaminants is 15 projected to continue and increase in coming decades. In the marine environment, large 16 volumes of data to assess how concentrations have changed over time can be gathered 17 from indicator species such as seabirds, including through sampling feathers from 18 archival collections and museums. As apex predators, Flesh-footed Shearwaters (Ardenna 19 carneipes) are subject to high concentrations of bioaccumulative and biomagnifying 20 contaminants, and reflect the health of their local marine environment. We analysed 21 Flesh-footed Shearwater feathers from Australia from museum specimens and live birds 22 collected between 1900 and 2011 and assessed temporal trends in three trace elements of 23 toxicological concern: cadmium, mercury, and lead. Concentrations of cadmium 24 increased by 1.5% per year (95% CI: +0.6, +3.0), while mercury was unchanged through 25 the time series (-0.3% per year; 05% CI: -2.1, +1.5), and lead decreased markedly (-2.1% 26 per year, 95% CI: -3.2, -1.0). A reduction in birds’ trophic position through the 20th 27 century, and decreased atmospheric emissions were the likely driving factors for mercury 28 and lead, respectively. By combining archival material from museum specimens with 29 contemporary samples, we have been able to further elucidate the potential threats posed 30 to these apex predators by metal contamination. 31 Keywords: Ardenna carneipes; Flesh-footed Shearwater; mercury; lead; cadmium; 33 Western Australia 34 Keywords: Ardenna carneipes; Flesh-footed Shearwater; mercury; lead; cadmium; 33 Western Australia 34 35 2 2 Capsule: Cadmium in Flesh-footed Shearwater feathers increased between 1900 and 36 2011, while mercury remained stable and lead decreased. 37 38 3 3 Introduction 39 Contaminants in marine and atmospheric environments are generally increasing 40 over recent time, and are projected to increase in coming decades (AMAP, 2011; 41 Hoffman et al., 2003; Lamborg et al., 2014; Pacyna and Pacyna, 2001; Streets et al., 42 2009; UNEP, 2002). With an increase in toxicological studies of a variety of organisms, 43 and generally poor knowledge of effect thresholds in many species, understanding how 44 contaminant concentrations have changed over time is critical to an ecological 45 interpretation and the subsequent management and conservation efforts. If a species has 46 high concentrations of a contaminant contemporarily, is this because it has always 47 experienced such concentrations and has adapted to accommodate them, or have 48 concentrations in the environment increased, and been accumulated by biota? Reporting 49 high concentrations of a given contaminant must therefore be accompanied by 50 appropriate interpretation, including the historical context (Bond et al., 2015). 51 Contaminants in marine and atmospheric environments are generally increasing 40 over recent time, and are projected to increase in coming decades (AMAP, 2011; 41 Hoffman et al., 2003; Lamborg et al., 2014; Pacyna and Pacyna, 2001; Streets et al., 42 2009; UNEP, 2002). With an increase in toxicological studies of a variety of organisms, 43 and generally poor knowledge of effect thresholds in many species, understanding how 44 contaminant concentrations have changed over time is critical to an ecological 45 interpretation and the subsequent management and conservation efforts. If a species has 46 high concentrations of a contaminant contemporarily, is this because it has always 47 experienced such concentrations and has adapted to accommodate them, or have 48 concentrations in the environment increased, and been accumulated by biota? Reporting 49 high concentrations of a given contaminant must therefore be accompanied by 50 appropriate interpretation, including the historical context (Bond et al., 2015). 51 As top predators in the marine environment, seabirds can be exposed to high 52 levels of bioaccumulated and biomagnified pollutants from both natural, and 53 anthropogenic sources (Burger and Gochfeld, 2002; Day et al., 2012). Introduction 39 Seabirds can also 54 act as indicators or sentinel species for examining the health of the marine environment, 55 including chemical contamination (Burger and Gochfeld, 2004; Monteiro and Furness, 56 1995), and contaminant concentrations are monitored using seabirds in a variety of 57 oceanic domains (Barrett et al., 1996; Braune, 2007; Burgess et al., 2013; Carravieri et 58 al., 2016; Day et al., 2006; Goodale et al., 2008). 59 As top predators in the marine environment, seabirds can be exposed to high 52 levels of bioaccumulated and biomagnified pollutants from both natural, and 53 anthropogenic sources (Burger and Gochfeld, 2002; Day et al., 2012). Seabirds can also 54 act as indicators or sentinel species for examining the health of the marine environment, 55 including chemical contamination (Burger and Gochfeld, 2004; Monteiro and Furness, 56 1995), and contaminant concentrations are monitored using seabirds in a variety of 57 oceanic domains (Barrett et al., 1996; Braune, 2007; Burgess et al., 2013; Carravieri et 58 al., 2016; Day et al., 2006; Goodale et al., 2008). 59 4 Feathers are used frequently to measure contaminants in seabirds (Burger, 1993) 60 They can be sampled without sacrificing the individual, or from preserved museum skins, 61 making studies of long time series possible. Feathers often contain the biologically active 62 form of many metal contaminants (e.g., most mercury is in the form of methylmercury, 63 Bond and Diamond, 2009b), and can be a significant metabolic pathway for contaminant 64 elimination (Braune and Gaskin, 1987; Burger, 1993). Many elements in feathers, 65 however, represent external contamination from the lithosphere, even after vigorous 66 washing (Borghesi et al., 2016), but at present it cannot be partitioned from endogenous 67 deposition. 68 They can be sampled without sacrificing the individual, or from preserved museum skins, 61 making studies of long time series possible. Feathers often contain the biologically active 62 form of many metal contaminants (e.g., most mercury is in the form of methylmercury, 63 A variety of studies have investigated temporal trends in contaminants, primarily 69 mercury, using seabird feathers (Appelquist et al., 1985; Bond et al., 2015; Monteiro and 70 Furness, 1997; Thompson et al., 1993b; Thompson et al., 1992; Vo et al., 2011). Introduction 39 With 71 improvements in analytical methods, reliable metal and metalloid concentrations can be 72 acquired with about 15-25 mg of tissue (Bond and Lavers, 2011; Friel et al., 1990; 73 Haynes et al., 2006), making archival studies more attractive, and complementary to 74 indirect time series of seabirds’ contamination through sediment cores (Sun and Xie, 75 2001). Museum samples are also the only way to examine time series of contamination 76 retrospectively, which can inform contemporary conservation and management (Bond et 77 al., 2015). 78 5 Flesh-footed Shearwaters (Ardenna carneipes) are trans-equatorial migrants that 79 breed on islands in New Zealand, South and Western Australia, and on Île Saint-Paul in 80 the Indian Ocean (Lavers, 2015; Roux, 1985; Waugh et al., 2013). Samples collected in 81 the 2008-2009 austral summer indicated that some metal and metalloid concentrations in 82 5 Flesh-footed Shearwaters (Ardenna carneipes) are trans-equatorial migrants that 79 breed on islands in New Zealand, South and Western Australia, and on Île Saint-Paul in 80 the Indian Ocean (Lavers, 2015; Roux, 1985; Waugh et al., 2013). Samples collected in 81 the 2008-2009 austral summer indicated that some metal and metalloid concentrations in 82 5 5 feathers could be of toxicological concern (Bond and Lavers, 2011), likely because of 83 their diet of predatory fish and squid (Gould et al., 1997). Flesh-footed Shearwaters also 84 ingest large quantities of plastic marine debris, which adults offload to their nest-bound 85 chicks during feeding (Hutton et al., 2008; Lavers and Bond, 2016b; Lavers et al., 2014), 86 and these plastics could provide a route for hydrophobic contaminants, and compounds 87 used in plastic production (Holmes et al., 2012; Lavers and Bond, 2016a; Lavers et al., 88 2014; Tanaka et al., 2013), though the proportional contribution of many plastic- 89 transported contaminants is unknown (Bakir et al., 2016). The question remains, however 90 – are contaminant concentrations in Flesh-footed Shearwater feathers increasing or 91 decreasing, and are the high concentrations reported by Bond and Lavers (2011) typical 92 of shearwaters’ contaminant burden? This is particularly germane given the significant 93 trophic declines in shearwaters from Western Australia over the last century (Bond and 94 Lavers, 2014), and the species’ range-wide population decline (Jamieson and Waugh, 95 2015; Lavers, 2015; Reid et al., 2013). 96 g ( , ), y their diet of predatory fish and squid (Gould et al., 1997). Introduction 39 Flesh-footed Shearwaters also 84 ingest large quantities of plastic marine debris, which adults offload to their nest-bound 85 chicks during feeding (Hutton et al., 2008; Lavers and Bond, 2016b; Lavers et al., 2014), 86 and these plastics could provide a route for hydrophobic contaminants, and compounds 87 used in plastic production (Holmes et al., 2012; Lavers and Bond, 2016a; Lavers et al., 88 2014; Tanaka et al., 2013), though the proportional contribution of many plastic- 89 transported contaminants is unknown (Bakir et al., 2016). The question remains, however 90 – are contaminant concentrations in Flesh-footed Shearwater feathers increasing or 91 decreasing, and are the high concentrations reported by Bond and Lavers (2011) typical 92 of shearwaters’ contaminant burden? This is particularly germane given the significant 93 trophic declines in shearwaters from Western Australia over the last century (Bond and 94 Lavers, 2014), and the species’ range-wide population decline (Jamieson and Waugh, 95 2015; Lavers, 2015; Reid et al., 2013). 96 g p p g 2015; Lavers, 2015; Reid et al., 2013). 96 Our goals were, therefore, to describe changes in trace elements in Flesh-footed 97 Shearwater feathers over more than a century, to compare historic concentrations with 98 those from contemporary samples, and to discuss these results in the context of 99 contaminants in the marine environment. 100 101 Materials and Methods 102 Sample collection 103 Our goals were, therefore, to describe changes in trace elements in Flesh-footed 97 Shearwater feathers over more than a century, to compare historic concentrations with 98 those from contemporary samples, and to discuss these results in the context of 99 contaminants in the marine environment. 100 Our goals were, therefore, to describe changes in trace elements in Flesh-footed 97 Shearwater feathers over more than a century, to compare historic concentrations with 98 those from contemporary samples, and to discuss these results in the context of 99 contaminants in the marine environment. 100 Our goals were, therefore, to describe changes in trace elements in Flesh-footed 97 Shearwater feathers over more than a century, to compare historic concentrations with 98 those from contemporary samples, and to discuss these results in the context of 99 contaminants in the marine environment. 100 6 6 We sampled feathers from Flesh-footed Shearwater skins housed in museum 104 collections in Australia, Canada, France, New Zealand, and the United States (see 105 Acknowledgements for a list of institutions). Introduction 39 Only specimens with precise years of 106 collection were sampled. Breast feathers were selected because they are the best indicator 107 of whole-body metal burdens (Furness et al., 1986), and allowed comparisons with 108 contemporary samples (most museum collections only permit sampling breast feathers). 109 Other tissues commonly sampled from museum specimens (mainly toe pads, but also 110 nails) are used for other purposes (e.g., genomic or isotopic analyses) and are not 111 collected from live birds so for examination of contaminant trends over centennial scales, 112 breast feathers are the most available and appropriate tissue (Bond et al., 2015). In 113 addition, Flesh-footed Shearwaters replace their breast feathers during the latter half of 114 the breeding season (February-April), before wing feathers are moulted on the wintering 115 grounds (Onley and Scofield, 2007); given the varying temporal lags of incorporation 116 into feathers, we cannot link measured concentrations to local exposures as we do not 117 know the age of individual feathers within the moult cycle, and assume that feather 118 elemental concentrations are integrated across a similar time and space among 119 individuals. Feathers were stored in sterile polyethylene bags or paper envelopes at -20°C 120 prior to analysis. The elements considered here are bound to the keratin protein in 121 feathers, and do not sublimate below 60 ºC, so the possibility that some would have been 122 lost from historic specimens during storage is remote. 123 We sampled feathers from Flesh-footed Shearwater skins housed in museum 104 collections in Australia, Canada, France, New Zealand, and the United States (see 105 Acknowledgements for a list of institutions). Only specimens with precise years of 106 collection were sampled. Breast feathers were selected because they are the best indicator 107 of whole-body metal burdens (Furness et al., 1986), and allowed comparisons with 108 contemporary samples (most museum collections only permit sampling breast feathers). 109 Other tissues commonly sampled from museum specimens (mainly toe pads, but also 110 nails) are used for other purposes (e.g., genomic or isotopic analyses) and are not 111 collected from live birds so for examination of contaminant trends over centennial scales, 112 breast feathers are the most available and appropriate tissue (Bond et al., 2015). Introduction 39 We therefore assessed temporal trends of Cd and Pb 143 from 1900-2011, and Hg from 1946-2011 only. Recovery of the secondary reference 144 material ranged from 89-112% among these elements for all runs (Table S1). Values 145 were corrected for background levels using procedural blanks, and for recovery using 146 samples of unknown provenance to a breeding area of origin. We used contemporary 127 samples and archival material assigned to locations in Western and South Australia (n = 128 123), or known to originate there (n = 43). 129 Analytical Methods 131 Cadmium (Cd), mercury (Hg) and lead (Pb) concentrations were assessed using 132 the same procedures as described in Bond and Lavers (2011). Feathers were washed in 133 0.25M NaOH to remove external contamination (Bearhop et al., 2000; Bond and 134 Diamond, 2009a), and two feathers per bird were pooled as individual feathers can be 135 highly variable in metal concentrations (Bond and Diamond, 2008). Trace element 136 concentrations were measured in a PerkinElmer ELAN DRCII ICP-MS and the protocol 137 used was based on Friel et al. (1990). Procedural blanks and secondary reference 138 materials were included for every 15-20 samples. The secondary materials used were 139 certified human hair samples 6H-09 and 7H-09 from the Centre de Toxicologie du 140 Québec, Institut National de Santé Publique du Québec (Table S1). Mercury was used in 141 museum preservation, contaminating specimens collected < 1940 with inorganic Hg 142 (Bond et al., 2015; Vo et al., 2011). We therefore assessed temporal trends of Cd and Pb 143 from 1900-2011, and Hg from 1946-2011 only. Recovery of the secondary reference 144 material ranged from 89-112% among these elements for all runs (Table S1). Values 145 were corrected for background levels using procedural blanks, and for recovery using 146 values from secondary reference materials within each run. 147 Analytical Methods 131 Cadmium (Cd), mercury (Hg) and lead (Pb) concentrations were assessed using 132 the same procedures as described in Bond and Lavers (2011). Feathers were washed in 133 0.25M NaOH to remove external contamination (Bearhop et al., 2000; Bond and 134 Diamond, 2009a), and two feathers per bird were pooled as individual feathers can be 135 highly variable in metal concentrations (Bond and Diamond, 2008). Trace element 136 concentrations were measured in a PerkinElmer ELAN DRCII ICP-MS and the protocol 137 used was based on Friel et al. (1990). Introduction 39 In 113 addition, Flesh-footed Shearwaters replace their breast feathers during the latter half of 114 the breeding season (February-April), before wing feathers are moulted on the wintering 115 grounds (Onley and Scofield, 2007); given the varying temporal lags of incorporation 116 into feathers, we cannot link measured concentrations to local exposures as we do not 117 know the age of individual feathers within the moult cycle, and assume that feather 118 elemental concentrations are integrated across a similar time and space among 119 individuals. Feathers were stored in sterile polyethylene bags or paper envelopes at -20°C 120 prior to analysis. The elements considered here are bound to the keratin protein in 121 feathers, and do not sublimate below 60 ºC, so the possibility that some would have been 122 lost from historic specimens during storage is remote. 123 As many of the museum skins were collected at sea away from breeding colonies 124 104 As many of the museum skins were collected at sea away from breeding colonies 124 as scientific specimens, or were taken as fisheries bycatch, their colony of origin was 125 unknown. Using a combination of biogeochemical markers, Lavers et al. (2013) assigned 126 7 7 samples of unknown provenance to a breeding area of origin. We used contemporary 127 samples and archival material assigned to locations in Western and South Australia (n = 128 123), or known to originate there (n = 43). 129 130 Analytical Methods 131 Cadmium (Cd), mercury (Hg) and lead (Pb) concentrations were assessed using 132 the same procedures as described in Bond and Lavers (2011). Feathers were washed in 133 0.25M NaOH to remove external contamination (Bearhop et al., 2000; Bond and 134 Diamond, 2009a), and two feathers per bird were pooled as individual feathers can be 135 highly variable in metal concentrations (Bond and Diamond, 2008). Trace element 136 concentrations were measured in a PerkinElmer ELAN DRCII ICP-MS and the protocol 137 used was based on Friel et al. (1990). Procedural blanks and secondary reference 138 materials were included for every 15-20 samples. The secondary materials used were 139 certified human hair samples 6H-09 and 7H-09 from the Centre de Toxicologie du 140 Québec, Institut National de Santé Publique du Québec (Table S1). Mercury was used in 141 museum preservation, contaminating specimens collected < 1940 with inorganic Hg 142 (Bond et al., 2015; Vo et al., 2011). Introduction 39 Procedural blanks and secondary reference 138 materials were included for every 15-20 samples. The secondary materials used were 139 certified human hair samples 6H-09 and 7H-09 from the Centre de Toxicologie du 140 Québec, Institut National de Santé Publique du Québec (Table S1). Mercury was used in 141 museum preservation, contaminating specimens collected < 1940 with inorganic Hg 142 (Bond et al., 2015; Vo et al., 2011). We therefore assessed temporal trends of Cd and Pb 143 from 1900-2011, and Hg from 1946-2011 only. Recovery of the secondary reference 144 material ranged from 89-112% among these elements for all runs (Table S1). Values 145 were corrected for background levels using procedural blanks, and for recovery using 146 values from secondary reference materials within each run. 147 148 8 8 Statistical Methods 149 149 We used two approaches to examine temporal changes in metal concentrations. 150 First, we used the program PIA (version 05/11/13; Bignert, 2013) to analyse the time- 151 series of each element, which enabled us to make comparisons with similar studies of 152 elemental concentrations over time, most of which are from the Arctic (Bignert et al., 153 2004; Rigét et al., 2011). PIA uses a robust regression and log-linear regression 154 techniques to detect linear and nonlinear trends using a running-mean smoother based on 155 annual geometric means (Fryer and Nicholson, 1993). We performed separate analyses 156 for each element, set the statistical power to detect a trend at 80%, and the minimum 157 slope to detect at 10% over 10 years at p < 0.05 using a three year running-mean 158 smoother. 159 We used two approaches to examine temporal changes in metal concentrations. 150 First, we used the program PIA (version 05/11/13; Bignert, 2013) to analyse the time- 151 series of each element, which enabled us to make comparisons with similar studies of 152 elemental concentrations over time, most of which are from the Arctic (Bignert et al., 153 2004; Rigét et al., 2011). PIA uses a robust regression and log-linear regression 154 techniques to detect linear and nonlinear trends using a running-mean smoother based on 155 annual geometric means (Fryer and Nicholson, 1993). We performed separate analyses 156 for each element, set the statistical power to detect a trend at 80%, and the minimum 157 slope to detect at 10% over 10 years at p < 0.05 using a three year running-mean 158 smoother. Introduction 39 159 We used two approaches to examine temporal changes in metal concentrations. 150 First, we used the program PIA (version 05/11/13; Bignert, 2013) to analyse the time- 151 series of each element, which enabled us to make comparisons with similar studies of 152 elemental concentrations over time, most of which are from the Arctic (Bignert et al., 153 2004; Rigét et al., 2011). PIA uses a robust regression and log-linear regression 154 We also applied general additive models (GAMs; Wood, 2017) in the package 160 mgcv (Wood, 2019) where contaminant concentration was a function of a cubic 161 regression spline of collection year. The number of knots was determined by generalized 162 cross-validation in the model fitting process, and resulted in k = 9 for all three trace 163 elements. 164 All concentrations are expressed as parts-per-million (ppm, μg/g) on a fresh 165 weight basis. Though stable isotope data exist for this time series as well (Bond and 166 Lavers, 2014), there is a temporal mismatch between the integration periods of δ13C and 167 δ15N, and trace elements in feathers so they do not reflect the same periods (Bond, 2010), 168 and do not change the trace element concentrations measured. 169 All concentrations are expressed as parts-per-million (ppm, μg/g) on a fresh 165 weight basis. Though stable isotope data exist for this time series as well (Bond and 166 Lavers, 2014), there is a temporal mismatch between the integration periods of δ13C and 167 δ15N, and trace elements in feathers so they do not reflect the same periods (Bond, 2010), 168 and do not change the trace element concentrations measured. 169 170 9 9 Results 171 The spline featured a peak in the late 1940s 197 followed by a gradual decline and stabilization after 1985 (Figure S3). 198 199 Discussion 200 It is important to note that the age of the individuals from museum collections 201 were unknown. Flesh-footed Shearwaters, like many in the family Procellariidae, cannot 202 be aged based on plumage (Onley and Scofield, 2007), so our sample may include a 203 combination of birds > 1 year old, which have undergone a complete moult, and those < 1 204 year old, which would still retain feathers grown at the breeding site before fledging, and 205 therefore differ in exposure to contaminants (Braune and Gaskin, 1987; Monteiro and 206 Furness, 2001a; b). This difference in the pool of trace elements that could be deposited 207 into feathers (and feather age ranging from perhaps a few weeks to several months) 208 would result in increased variance, but unfortunately cannot be controlled (Burger, 1995; 209 Malinga et al., 2010; Stewart et al., 1999). 210 Flesh-footed Shearwaters in Western Australia migrate to the northern Indian 211 Ocean (Lavers et al., 2019; Powell, 2009; Shuntov, 1968), an area with potentially 212 concentrations ranged from 0.009-1125.733 µg/g (geometric mean: 1.656 µg/g, SD: 193 82.411), though the upper extreme is likely influenced by external contamination. 194 Removing this individual, concentrations ranged from 0.009-255.432 µg/g (geometric 195 mean: 1.592, SD: 21.373). Like Hg, the GAM for Pb with only a cubic regression spline 196 for year did not fit the data well (r2 = 0.07). The spline featured a peak in the late 1940s 197 followed by a gradual decline and stabilization after 1985 (Figure S3). 198 Discussion 200 It is important to note that the age of the individuals from museum collections 201 were unknown. Flesh-footed Shearwaters, like many in the family Procellariidae, cannot 202 be aged based on plumage (Onley and Scofield, 2007), so our sample may include a 203 combination of birds > 1 year old, which have undergone a complete moult, and those < 1 204 year old, which would still retain feathers grown at the breeding site before fledging, and 205 therefore differ in exposure to contaminants (Braune and Gaskin, 1987; Monteiro and 206 Furness, 2001a; b). Results 171 There was a significant linear, increase in Cd in shearwater feathers from 1900- 172 2011 of 1.5% per year (95% CI: +0.1, +3.0%, F1, 34 = 4.64, p = 0.037), and ranged from 173 0.006-20.082 µg/g (geometric mean: 0.354 µg/g, SD: 2.924). Based on the variance 174 among years, 29 years of data would be required to detect an annual change of 10% with 175 80% power. Our time series had 99% power to detect a 10% change over the entire 176 period, and the lowest detectable annual change was 7.1% (Table 1). The GAM fit the 177 data well (r2 = 0.54), and the spline term was significant (effective df: 8.66, F = 22.65, p 178 < 0.001), remaining relatively flat until the mid-1980s where it rose rapidly and then 179 declined to the previous level (Figure S1). 180 We found no significant trend in feather Hg from 1946-2011 (β = -0.3, 95% CI: - 181 2.1, +1.5%, F1,22 = 0.12, p = 0.73), though we had very high power to detect a change 182 (94% power to detect a 10% change over the time series, and a lowest detectable change 183 of 8.3%); 21 years of data would be required to detect an annual change of 10% with 184 80% power (Table 1). Feather Hg concentrations ranged from 1.290-113.499 µg/g 185 (geometric mean: 8.241 µg/g, SD: 18.807). The GAM fit was lower than for Cd (r2 = 186 0.12), and the cubic regression spline shoed a dip in the 1990s before rising rapidly in the 187 early 2000s and returning to pre-1990s levels (Figure S2). 188 Pb in shearwater feathers decreased significantly from 1900-2011 by 2.1% per 189 year (95% CI: -3.2, -1.0, F1,24 = 27.85, p < 0.001), and this time series had 100% power to 190 detect a 10% change. An annual change of 10% could be detected with 26 years of data, 191 and the lowest detectable change of the time series was 76% (Table 1). Overall feather Pb 192 10 concentrations ranged from 0.009-1125.733 µg/g (geometric mean: 1.656 µg/g, SD: 193 82.411), though the upper extreme is likely influenced by external contamination. 194 Removing this individual, concentrations ranged from 0.009-255.432 µg/g (geometric 195 mean: 1.592, SD: 21.373). Like Hg, the GAM for Pb with only a cubic regression spline 196 for year did not fit the data well (r2 = 0.07). Results 171 This difference in the pool of trace elements that could be deposited 207 into feathers (and feather age ranging from perhaps a few weeks to several months) 208 would result in increased variance, but unfortunately cannot be controlled (Burger, 1995; 209 Malinga et al., 2010; Stewart et al., 1999). 210 Flesh-footed Shearwaters in Western Australia migrate to the northern Indian 211 Ocean (Lavers et al., 2019; Powell, 2009; Shuntov, 1968), an area with potentially 212 concerning concentrations of toxic elements in the water column (Danielsson, 1980; Kar 213 et al., 2008). Understanding where exposure occurs, seabirds’ roles in nutrient and 214 Flesh-footed Shearwaters in Western Australia migrate to the northern Indian 211 Ocean (Lavers et al., 2019; Powell, 2009; Shuntov, 1968), an area with potentially 212 concerning concentrations of toxic elements in the water column (Danielsson, 1980; Kar 213 et al., 2008). Understanding where exposure occurs, seabirds’ roles in nutrient and 214 Flesh-footed Shearwaters in Western Australia migrate to the northern Indian 211 Ocean (Lavers et al., 2019; Powell, 2009; Shuntov, 1968), an area with potentially 212 concerning concentrations of toxic elements in the water column (Danielsson, 1980; Kar 213 et al., 2008). Understanding where exposure occurs, seabirds’ roles in nutrient and 214 11 contaminant transport (Blais et al., 2005; Doughty et al., 2016), and the potential carry- 215 over effects of contaminant exposure on the non-breeding grounds (Fort et al., 2014) can 216 inform conservation actions, and inform interpretations of population trends. 217 218 Cadmium 219 Little of birds’ Cd burden is sequestered into feathers (Burger, 1993; Honda et al., 220 1985), and museum specimens can be a mechanism for monitoring the fraction depurated 221 in feathers (Borghesi et al., 2016; Pilastro et al., 1993) so while feathers are not suitable 222 for assessing total Cd burden, they are appropriate for examining temporal changes. A 223 large portion of Cd in the environment comes from anthropogenic sources, including steel 224 production and waste incineration (Hutton, 1983), and exposure is highly influenced by 225 ocean cycling (Macdonald et al., 2005). Squid, a common prey of Flesh-footed 226 Shearwaters, often have high concentrations of Cd (Gerpe et al., 2000; Gould et al., 227 1997). Results 171 The most commonly identified squid in Flesh-footed Shearwaters’ diet, 228 Ommastrephes bartramii, had liver Cd concentrations of 287 ± 202 µg/g in the 1970s, 229 which was higher than sympatric Loligo opalescens, but lower than Symplectoteuthis 230 oualaniensis (Martin and Flegal, 1975), though Western Australia shearwaters’ diets can 231 also be dominated by pilchards (Sardinops sagax; JLL unpublished data). Cd in pilchards 232 has not been assessed in Australia (Padula et al., 2016), but concentrations of 0.5 µg/g 233 dry weight in muscle have been reported elsewhere (Tawfik, 2013). 234 contaminant transport (Blais et al., 2005; Doughty et al., 2016), and the potential carry- 215 over effects of contaminant exposure on the non-breeding grounds (Fort et al., 2014) can 216 inform conservation actions, and inform interpretations of population trends. 217 contaminant transport (Blais et al., 2005; Doughty et al., 2016), and the potential carry- 215 over effects of contaminant exposure on the non-breeding grounds (Fort et al., 2014) can 216 inform conservation actions, and inform interpretations of population trends. 217 Little of birds’ Cd burden is sequestered into feathers (Burger, 1993; Honda et al., 220 1985), and museum specimens can be a mechanism for monitoring the fraction depurated 221 in feathers (Borghesi et al., 2016; Pilastro et al., 1993) so while feathers are not suitable 222 for assessing total Cd burden, they are appropriate for examining temporal changes. A 223 large portion of Cd in the environment comes from anthropogenic sources, including steel 224 production and waste incineration (Hutton, 1983), and exposure is highly influenced by 225 ocean cycling (Macdonald et al., 2005). Squid, a common prey of Flesh-footed 226 Shearwaters, often have high concentrations of Cd (Gerpe et al., 2000; Gould et al., 227 1997). The most commonly identified squid in Flesh-footed Shearwaters’ diet, 228 Ommastrephes bartramii, had liver Cd concentrations of 287 ± 202 µg/g in the 1970s, 229 which was higher than sympatric Loligo opalescens, but lower than Symplectoteuthis 230 oualaniensis (Martin and Flegal, 1975), though Western Australia shearwaters’ diets can 231 also be dominated by pilchards (Sardinops sagax; JLL unpublished data). Cd in pilchards 232 has not been assessed in Australia (Padula et al., 2016), but concentrations of 0.5 µg/g 233 dry weight in muscle have been reported elsewhere (Tawfik, 2013). 234 Seabirds may also be able to tolerate higher concentrations of Cd (Scheuhammer, 235 1987). Results 171 The toxicological effects of Cd on birds include kidney lesions, altered behavior, 236 Seabirds may also be able to tolerate higher concentrations of Cd (Scheuhammer, 235 1987). The toxicological effects of Cd on birds include kidney lesions, altered behavior, 236 Seabirds may also be able to tolerate higher concentrations of Cd (Scheuhammer, 235 1987). The toxicological effects of Cd on birds include kidney lesions, altered behavior, 236 12 eggshell thinning, and more (Furness, 1996). These are, however, effects measured on 237 internal organs, and so concentrations at which effects manifest range from 0.1-2.0 µg/g 238 fresh weight (fw) in feathers (Burger, 1993; Burger and Gochfeld, 2000b). Of the 166 239 birds sampled here, 119 (72%) had feather Cd concentrations >0.1 µg/g, and 29 (17%) 240 had concentrations >2.0 µg/g (Figure 1), with four individuals exceeding 10 µg/g Cd in 241 feathers, which is among the highest recorded in wild birds (Anderson et al., 2010; 242 Burger and Gochfeld, 2000c; Hindell et al., 1999). The peak identified in the GAM in the 243 1990s is interesting given that world cadmium production has remained relatively stable 244 from 1990-2012, around 20,000 metric tons (U.S. Geological Survey, 2015). 245 This is the first study to examine changes in Cd in birds over time, and given the 246 significant increase in feather Cd, and high concentrations in some recent individuals, 247 further study on the potential sources, effects, and causes of these concentrations is 248 warranted. The application of stable isotopes of Cd (and Hg) as tracers could be 249 particularly beneficial in answering these questions (Conway and John, 2015; Day et al., 250 2012). 251 252 Mercury 253 Most Hg in the environment is from anthropogenic sources that are transformed 254 into the biologically active methyl Hg, and subsequently bioaccumulated and 255 eggshell thinning, and more (Furness, 1996). These are, however, effects measured on 237 internal organs, and so concentrations at which effects manifest range from 0.1-2.0 µg/g 238 fresh weight (fw) in feathers (Burger, 1993; Burger and Gochfeld, 2000b). Of the 166 239 birds sampled here, 119 (72%) had feather Cd concentrations >0.1 µg/g, and 29 (17%) 240 had concentrations >2.0 µg/g (Figure 1), with four individuals exceeding 10 µg/g Cd in 241 feathers, which is among the highest recorded in wild birds (Anderson et al., 2010; 242 Burger and Gochfeld, 2000c; Hindell et al., 1999). Results 171 The peak identified in the GAM in the 243 1990s is interesting given that world cadmium production has remained relatively stable 244 from 1990-2012, around 20,000 metric tons (U.S. Geological Survey, 2015). 245 Burger and Gochfeld, 2000c; Hindell et al., 1999). The peak identified in the GAM in the 243 1990s is interesting given that world cadmium production has remained relatively stable 244 from 1990-2012, around 20,000 metric tons (U.S. Geological Survey, 2015). 245 This is the first study to examine changes in Cd in birds over time, and given the 246 significant increase in feather Cd, and high concentrations in some recent individuals, 247 further study on the potential sources, effects, and causes of these concentrations is 248 warranted. The application of stable isotopes of Cd (and Hg) as tracers could be 249 particularly beneficial in answering these questions (Conway and John, 2015; Day et al., 250 2012). 251 This is the first study to examine changes in Cd in birds over time, and given the 246 significant increase in feather Cd, and high concentrations in some recent individuals, 247 further study on the potential sources, effects, and causes of these concentrations is 248 warranted. The application of stable isotopes of Cd (and Hg) as tracers could be 249 particularly beneficial in answering these questions (Conway and John, 2015; Day et al., 250 2012). 251 Most Hg in the environment is from anthropogenic sources that are transformed 254 into the biologically active methyl Hg, and subsequently bioaccumulated and 255 biomagnified in food webs (Krabbenhoft and Sunderland, 2013; Lindberg et al., 2007; 256 Weiner et al., 2003). Hg is acquired through birds’ diet, and mostly eliminated in 257 proteinaceous tissues, such as egg components or feathers, where it binds to disulfide 258 13 bonds between cysteine molecules (Bond and Diamond, 2009b; Crewther et al., 1965; 259 Monteiro and Furness, 2001a; Thompson, 1996). While contamination near point sources 260 can be a concern for seabirds (Finger et al., 2015), global atmospheric transport and 261 mobile predators and prey mean that Hg affects upper trophic predators, like Flesh-footed 262 Shearwaters, regardless of location. Results 171 Concentrations of Hg in feathers > 20 µg/g are 263 thought to be of concern to piscivores (Burger and Gochfeld, 1997; Cristol et al., 2012; 264 Evers et al., 2014), though it seems some species, notably albatrosses, are able to tolerate 265 much higher concentrations without observed adverse effects (Bustamante et al., 2016; 266 Hindell et al., 1999). We found 23/137 Flesh-footed Shearwaters (17%) exceeded 20 267 µg/g, and ranged as high as 113 µg/g in one individual sampled in 2006. Temporally, 268 though the GAM identified a drop in the late 1990s followed by a rapid increase and 269 levelling off of Hg concentrations in shearwater feathers, the variance explained by this 270 regression spline was relatively small, and the global anthropogenic Hg supply has 271 remained constant since the mid-1990s at around 3500 tonnes annually (UNEP, 2013). 272 bonds between cysteine molecules (Bond and Diamond, 2009b; Crewther et al., 1965; 259 Monteiro and Furness, 2001a; Thompson, 1996). While contamination near point sources 260 can be a concern for seabirds (Finger et al., 2015), global atmospheric transport and 261 mobile predators and prey mean that Hg affects upper trophic predators, like Flesh-footed 262 Shearwaters, regardless of location. Concentrations of Hg in feathers > 20 µg/g are 263 thought to be of concern to piscivores (Burger and Gochfeld, 1997; Cristol et al., 2012; 264 Evers et al., 2014), though it seems some species, notably albatrosses, are able to tolerate 265 much higher concentrations without observed adverse effects (Bustamante et al., 2016; 266 Hindell et al., 1999). We found 23/137 Flesh-footed Shearwaters (17%) exceeded 20 267 µg/g, and ranged as high as 113 µg/g in one individual sampled in 2006. Temporally, 268 though the GAM identified a drop in the late 1990s followed by a rapid increase and 269 levelling off of Hg concentrations in shearwater feathers, the variance explained by this 270 regression spline was relatively small, and the global anthropogenic Hg supply has 271 remained constant since the mid-1990s at around 3500 tonnes annually (UNEP, 2013). 272 Life-history strategy may influence exposure to Hg, with female seabirds that 273 breed bi-annually being less able to excrete metals during egg laying (Ackerman et al., 274 2016; Hindell et al., 1999; Monteiro and Furness, 2001a). Results 171 Flesh-footed Shearwaters 275 breeding in Western Australia and New Zealand may not breed annually (Lavers et al., 276 2019; Waugh et al., 2014), and therefore may not have the same opportunities to depurate 277 Hg into eggs. Mercury concentrations did not change over time, which may be surprising 278 given the increases observed in other studies (Bond et al., 2015; Evers et al., 2014; 279 Thompson et al., 1993a; Thompson et al., 1992; Vo et al., 2011) which may be a function 280 of the low number of samples early in the time series. Flesh-footed Shearwaters have 281 Life-history strategy may influence exposure to Hg, with female seabirds that 273 breed bi-annually being less able to excrete metals during egg laying (Ackerman et al., 274 2016; Hindell et al., 1999; Monteiro and Furness, 2001a). Flesh-footed Shearwaters 275 breeding in Western Australia and New Zealand may not breed annually (Lavers et al., 276 2019; Waugh et al., 2014), and therefore may not have the same opportunities to depurate 277 Hg into eggs. Mercury concentrations did not change over time, which may be surprising 278 given the increases observed in other studies (Bond et al., 2015; Evers et al., 2014; 279 Thompson et al., 1993a; Thompson et al., 1992; Vo et al., 2011) which may be a function 280 of the low number of samples early in the time series. Flesh-footed Shearwaters have 281 Life-history strategy may influence exposure to Hg, with female seabirds that 273 breed bi-annually being less able to excrete metals during egg laying (Ackerman et al., 274 2016; Hindell et al., 1999; Monteiro and Furness, 2001a). Flesh-footed Shearwaters 275 breeding in Western Australia and New Zealand may not breed annually (Lavers et al., 276 14 experienced considerable trophic shifts since the mid-19th century, including a decrease 282 of one trophic level, and trend towards increased dietary breadth in Western Australia 283 (Bond and Lavers, 2014), and had the lowest contemporary concentrations of Hg (Bond 284 and Lavers, 2011). However, feather Hg concentrations during 1946-2011 (8.241 ± 0.944 285 µg/g; Figure 1) are comparable to adult Flesh-footed Shearwaters sampled in Western 286 Australia in 2008 (6.038 ± 3.998 µg/g) (Bond and Lavers, 2011). Results 171 Flesh-footed 287 15 experienced considerable trophic shifts since the mid-19th century, including a decrease 282 of one trophic level, and trend towards increased dietary breadth in Western Australia 283 (Bond and Lavers, 2014), and had the lowest contemporary concentrations of Hg (Bond 284 and Lavers, 2011). However, feather Hg concentrations during 1946-2011 (8.241 ± 0.944 285 µg/g; Figure 1) are comparable to adult Flesh-footed Shearwaters sampled in Western 286 Australia in 2008 (6.038 ± 3.998 µg/g) (Bond and Lavers, 2011). Flesh-footed 287 Shearwaters’ reduction in trophic position may have been a contributory factor in their 288 unchanged feather Hg concentrations (Bond and Lavers, 2014). Given that the ultimate 289 source of Hg is dietary, a detailed examination of Flesh-footed Shearwater diet and prey 290 Hg across its breeding range would help elucidate the reasons for this pattern. 291 292 Lead 293 Pb was added to gasoline as an anti-knocking agent in the early 20th century, before being 294 phased out in many countries less than 100 years later because of the negative 295 environmental effects of Pb in automotive emissions (Seyferth, 2003; Wilson and 296 Horrocks, 2008). Like Hg, Pb is also largely acquired through birds’ diet, and binds to 297 keratin and other proteins rich in sulfhydryl groups (Burger and Gochfeld, 2000a; Goede 298 and de Bruin, 1984). In birds, high concentrations of Pb are associated with neurological 299 and developmental impairment (Burger and Gochfeld, 2000a), particularly when feather 300 concentrations exceed 4 µg/g (Burger, 1993; Burger and Gochfeld, 2000a), and local 301 sources can dramatically affect Pb concentrations (Scheifler et al., 2006). More than a 302 third (65/165; 37%) of shearwaters sampled had feather Pb concentrations above this 303 experienced considerable trophic shifts since the mid-19th century, including a decrease 282 of one trophic level, and trend towards increased dietary breadth in Western Australia 283 (Bond and Lavers, 2014), and had the lowest contemporary concentrations of Hg (Bond 284 and Lavers, 2011). However, feather Hg concentrations during 1946-2011 (8.241 ± 0.944 285 µg/g; Figure 1) are comparable to adult Flesh-footed Shearwaters sampled in Western 286 Australia in 2008 (6.038 ± 3.998 µg/g) (Bond and Lavers, 2011). Flesh-footed 287 Shearwaters’ reduction in trophic position may have been a contributory factor in their 288 unchanged feather Hg concentrations (Bond and Lavers, 2014). Results 171 Given that the ultimate 289 source of Hg is dietary, a detailed examination of Flesh-footed Shearwater diet and prey 290 Hg across its breeding range would help elucidate the reasons for this pattern. 291 Shearwaters’ reduction in trophic position may have been a contributory factor in their 288 unchanged feather Hg concentrations (Bond and Lavers, 2014). Given that the ultimate 289 source of Hg is dietary, a detailed examination of Flesh-footed Shearwater diet and prey 290 Hg across its breeding range would help elucidate the reasons for this pattern. 291 15 level (Figure 1). The peak identified in the late 1940s and early 1950s does correspond 304 with the rapid increase in leaded gasoline consumption (Nriagu, 1990; Seyferth, 2003), 305 though as with Hg the r2 of the regression spline was not high. 306 level (Figure 1). The peak identified in the late 1940s and early 1950s does correspond 304 with the rapid increase in leaded gasoline consumption (Nriagu, 1990; Seyferth, 2003), 305 though as with Hg the r2 of the regression spline was not high. 306 While Pb contamination has the potential to negatively affect the health and 307 reproductive fitness of individual shearwaters, concentrations are decreasing over time 308 (Table 1). The decrease we observed in shearwater feather Pb mirrors the declines in 309 atmospheric Pb following the reduction in Pb as an additive in gasoline. Concentrations 310 in Sardinops sagax muscle from the Arabian Sea, adjacent to shearwaters’ over-wintering 311 grounds (Lavers et al., 2019) were also relatively low (0.005 µg/g; Tawfik, 2013). 312 While Pb contamination has the potential to negatively affect the health and 307 reproductive fitness of individual shearwaters, concentrations are decreasing over time 308 (Table 1). The decrease we observed in shearwater feather Pb mirrors the declines in 309 atmospheric Pb following the reduction in Pb as an additive in gasoline. Concentrations 310 in Sardinops sagax muscle from the Arabian Sea, adjacent to shearwaters’ over-wintering 311 grounds (Lavers et al., 2019) were also relatively low (0.005 µg/g; Tawfik, 2013). 312 On offshore islands and in remote areas, even small populations of migratory 313 species (e.g., salmon, seabirds) can transport significant quantities of hazardous 314 contaminants via their guano (Evenset et al., 2007; Sun and Xie, 2001). As anthropogenic 315 contamination of the marine environment increases, so, too, do inputs from the ocean to 316 the land. Results 171 Cd concentrations in Flesh-footed Shearwaters increased 1.5% per year during 317 1900-2011 (Table 1), suggesting guano deposition on breeding islands in Western 318 Australia may be a previously undocumented source of chemical pollution. 319 On offshore islands and in remote areas, even small populations of migratory 313 species (e.g., salmon, seabirds) can transport significant quantities of hazardous 314 contaminants via their guano (Evenset et al., 2007; Sun and Xie, 2001). As anthropogenic 315 contamination of the marine environment increases, so, too, do inputs from the ocean to 316 the land. Cd concentrations in Flesh-footed Shearwaters increased 1.5% per year during 317 1900-2011 (Table 1), suggesting guano deposition on breeding islands in Western 318 Australia may be a previously undocumented source of chemical pollution. 319 Archival samples allowed us to frame contemporary ecotoxicological results in an 320 historical context, which provided insight into changes in the pressures faced by Flesh- 321 footed Shearwaters over the last century. Observing changes in ecosystems over such 322 periods is challenging, as perceived baselines shift over time (Blight et al., 2015; 323 Archival samples allowed us to frame contemporary ecotoxicological results in an 320 historical context, which provided insight into changes in the pressures faced by Flesh- 321 footed Shearwaters over the last century. Observing changes in ecosystems over such 322 periods is challenging, as perceived baselines shift over time (Blight et al., 2015; 323 Papworth et al., 2009). By using dated museum specimens, researchers can begin to 324 16 examine historical changes in ecosystems using archived material and inform modern 325 conservation priorities and actions. 326 examine historical changes in ecosystems using archived material and inform modern 325 conservation priorities and actions. 326 examine historical changes in ecosystems using archived material and inform modern 325 conservation priorities and actions. 326 While this study has identified temporal trends in metal concentrations, it has also 327 highlighted a lack of information on the diet and foraging behaviour of Flesh-footed 328 Shearwaters and population level effects from metals exposure. Between 17-72% of the 329 shearwaters sampled for this study exceeded thresholds for Cd, Hg, or Pb. Results 171 Chemical 330 pollutant levels may be an additional stressor on the Western Australian Flesh-footed 331 Shearwater population, which has a low annual adult survival rate (0.634-0.835; Lavers et 332 al., 2019) or on other populations which are declining across the species range (Jamieson 333 and Waugh, 2015; Lavers, 2015; Reid et al., 2013). 334 Conclusions 336 Flesh-footed Shearwaters have shown contrasting trends in Cd, Hg, and Pb over the 20th 337 and early 21st centuries, driven by several factors. Concentrations of some trace elements, 338 namely lead, may be sufficiently high to cause adverse effects, and future work should 339 investigate this further. Our understanding of the context of contemporary contamination 340 has been improved through examining samples from museums and biological archives. 341 342 Data availability 343 Data are available on figshare: https://doi.org/10.6084/m9.figshare.12076704 344 Flesh-footed Shearwaters have shown contrasting trends in Cd, Hg, and Pb over the 20th 337 and early 21st centuries, driven by several factors. Concentrations of some trace elements, 338 namely lead, may be sufficiently high to cause adverse effects, and future work should 339 investigate this further. Our understanding of the context of contemporary contamination 340 has been improved through examining samples from museums and biological archives. 341 Flesh-footed Shearwaters have shown contrasting trends in Cd, Hg, and Pb over the 20th 337 and early 21st centuries, driven by several factors. Concentrations of some trace elements, 338 namely lead, may be sufficiently high to cause adverse effects, and future work should 339 investigate this further. Our understanding of the context of contemporary contamination 340 has been improved through examining samples from museums and biological archives. 341 Data are available on figshare: https://doi.org/10.6084/m9.figshare.12076704 344 345 17 Acknowledgements 346 The Commonwealth Environment Research Facilities Programme, CSIRO Marine 347 Biodiversity and Conservation Management Research Grant, Gould League NSW Cayley 348 Memorial Scholarship for Avian Science (grant 00002667), Government of Australia 349 Endeavour Research Fellowship, Royal Navy Bird Watching Society Captain Simpson 350 Scholarship, W.V. Scott Charitable Trust (grant L0018313), American Museum of 351 Natural History Chapman Study Grant, and Detached Foundation provided funding and 352 support for our research. For assistance obtaining samples we thank staff at the American 353 Museum of Natural History, Burke Museum, Beaty Biodiversity Museum, Western 354 Australia Museum, New Zealand National Institute of Water and Atmospheric Research, 355 Muséum national d'Histoire naturelle-Paris, South Australia Museum, Tasmanian 356 Museum and Art Gallery, Te Papa Museum, and the Victoria Museum. Samples were 357 collected with the permission of the Western Australia Department of Environment and 358 Conservation under the approval of the University of Tasmania and South Australian 359 Animal Ethics Committees (A0010874 and M25957-1). Special thanks go to B. Baker, A. 360 Booth, P. Collins, I. Hutton, R. Johnstone, C. Powell, J. Pridham, S. Seneviratne, C. 361 Stone (Ngatiwai Trust), G. Taylor, T. Reid, and D. Thompson. W. Diegor and L. Hewa 362 provided analytical support. This project benefited from discussions with A.M.V. 363 Fournier, and anonymous reviewers improved previous drafts. 364 The Commonwealth Environment Research Facilities Programme, CSIRO Marine 347 Biodiversity and Conservation Management Research Grant, Gould League NSW Cayley 348 Memorial Scholarship for Avian Science (grant 00002667), Government of Australia 349 Endeavour Research Fellowship, Royal Navy Bird Watching Society Captain Simpson 350 Scholarship, W.V. Scott Charitable Trust (grant L0018313), American Museum of 351 Natural History Chapman Study Grant, and Detached Foundation provided funding and 352 support for our research. For assistance obtaining samples we thank staff at the American 353 Museum of Natural History, Burke Museum, Beaty Biodiversity Museum, Western 354 Australia Museum, New Zealand National Institute of Water and Atmospheric Research, 355 Muséum national d'Histoire naturelle-Paris, South Australia Museum, Tasmanian 356 Museum and Art Gallery, Te Papa Museum, and the Victoria Museum. Samples were 357 collected with the permission of the Western Australia Department of Environment and 358 Conservation under the approval of the University of Tasmania and South Australian 359 Animal Ethics Committees (A0010874 and M25957-1). Special thanks go to B. Baker, A. 360 Booth, P. Collins, I. Hutton, R. Johnstone, C. Powell, J. Pridham, S. Seneviratne, C. Acknowledgements 346 361 Stone (Ngatiwai Trust), G. Taylor, T. Reid, and D. Thompson. W. Diegor and L. Hewa 362 provided analytical support. 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U.S. Geological Survey Data Series 896 [accessed 660 05/03/2020]. 661 05/03/2020]. 661 UNEP, 2002. Global Mercury Assessment. United Nations Environmental Programme - 662 Chemicals, Geneva, Switzerland. 663 UNEP, 2013. Mercury: time to act. UNEP Division of Technology, Industry and 664 Economics; Chemicals Branch, Geneva, Switzerland. 665 Vo, A.-T.E., Bank, M.S., Shine, J.P., Edwards, S.V., 2011. Temporal increase in organic 666 mercury in an endangered pelagic seabird assessed by century-old museum 667 specimens. Proceedings of the National Academy of Sciences of the United States 668 of America 108, 7466-7471. 669 Waugh, S.M., Jamieson, S.E., Stahl, J.C., Filippi, D.P., Taylor, G.A., Booth, A., 2014. 670 Flesh-footed Shearwater – population study and foraging areas (POP2011-02). 671 New Zealand Department of Conservation, Wellington, New Zealand. 672 Waugh, S.M., Tennyson, A.J.D., Taylor, G.A., Wilson, K.-J., 2013. Population sizes of 673 shearwaters (Puffinus spp.) breeding in New Zealand, with recommendations for 674 monitoring. Tuhinga 24, 159-204. 675 Weiner, J.G., Krabbemhoft, D.P., Heinz, G.H., Scheuhammer, A.M., 2003. 676 Ecotoxicology of Mercury, in: Hoffman, D.J., Rattner, B.A., Burton, G.A., Jr., 677 Cairns, J., Jr. (Eds.), Handbook of Ecotoxicology, 2nd Edition, 2 ed. CRC Press, 678 New York, pp. 409-463. 679 Wilson, N., Horrocks, J., 2008. Lessons from the removal of lead from gasoline for 680 controlling other environmental pollutants: A case study from New Zealand. 681 Environmental Health 7, 1. 682 Wood, S.N., 2017. Generalized additive models: an introduction with R, 2nd edition. 683 CBC Press, Boca Raton. 684 Wood, S.N., 2019. Tables 688 Tables 688 688 Acknowledgements 346 mgcv: Mixed GAM Computation Vehicle with Automatic 685 Smoothness Estimation. R package version 1.8-31. 686 UNEP, 2002. Global Mercury Assessment. United Nations Environmental Programme - 662 Chemicals, Geneva, Switzerland. 663 UNEP, 2013. Mercury: time to act. UNEP Division of Technology, Industry and 664 Economics; Chemicals Branch, Geneva, Switzerland. 665 Vo, A.-T.E., Bank, M.S., Shine, J.P., Edwards, S.V., 2011. Temporal increase in organic 666 mercury in an endangered pelagic seabird assessed by century-old museum 667 specimens. Proceedings of the National Academy of Sciences of the United States 668 of America 108, 7466-7471. 669 Waugh, S.M., Jamieson, S.E., Stahl, J.C., Filippi, D.P., Taylor, G.A., Booth, A., 2014. 670 Flesh-footed Shearwater – population study and foraging areas (POP2011-02). 671 New Zealand Department of Conservation, Wellington, New Zealand. 672 Waugh, S.M., Jamieson, S.E., Stahl, J.C., Filippi, D.P., Taylor, G.A., Booth, A., 2014. 670 Flesh-footed Shearwater – population study and foraging areas (POP2011-02). 671 New Zealand Department of Conservation, Wellington, New Zealand. 672 Waugh, S.M., Tennyson, A.J.D., Taylor, G.A., Wilson, K.-J., 2013. Population sizes of 673 shearwaters (Puffinus spp.) breeding in New Zealand, with recommendations for 674 monitoring. Tuhinga 24, 159-204. 675 Weiner, J.G., Krabbemhoft, D.P., Heinz, G.H., Scheuhammer, A.M., 2003. 76 Weiner, J.G., Krabbemhoft, D.P., Heinz, G.H., Scheuhammer, A.M., 2003. 676 Ecotoxicology of Mercury, in: Hoffman, D.J., Rattner, B.A., Burton, G.A., Jr., 677 Cairns, J., Jr. (Eds.), Handbook of Ecotoxicology, 2nd Edition, 2 ed. CRC Press, 678 New York, pp. 409-463. 679 Ecotoxicology of Mercury, in: Hoffman, D.J., Rattner, B.A., Burton, G.A., Jr., 677 Cairns, J., Jr. (Eds.), Handbook of Ecotoxicology, 2nd Edition, 2 ed. CRC Press, 678 New York, pp. 409-463. 679 Wilson, N., Horrocks, J., 2008. Lessons from the removal of lead from gasoline for 680 controlling other environmental pollutants: A case study from New Zealand. 681 Environmental Health 7, 1. 682 Wood, S.N., 2017. Generalized additive models: an introduction with R, 2nd edition. 683 CBC Press, Boca Raton. 684 Wood, S.N., 2019. mgcv: Mixed GAM Computation Vehicle with Automatic 685 Smoothness Estimation. R package version 1.8-31. 686 687 27 Tables Table 1. Robust regression analyses of metals in Flesh-footed Shearwater feathers. Reported values are those used in other 689 assessments of contaminants over time in biota (AMAP, 2011). 690 Element n Number of years (range) % increase per year (95% CI) Years required Lowest detectable change (%) Power of time series (%) Cadmium 140 36 (1900-2011) +1.5 (+0.6, +3.0) 29 7.1 99 Mercury 137 23 (1946-2011) -0.3 (-2.1, +1.5) 21 8.3 94 Lead 165 36 (1900-2011) -2.1 (-3.2, -1.0) 26 76 100 691 691 28 Figures 692 Figure 1 – Temporal trends in Cd (increasing), Hg (no significant change), and Pb 693 (decreasing) in feathers from Flesh-footed Shearwaters from Western and South 694 Australia. Blue lines are regressions with standard errors in gray (Table 1), and the 695 dashed red lines are concentrations of concern (see Discussion). Data are log- 696 transformed. 697 Biological archives reveal contrasting patterns in trace element concentrations in 700 pelagic seabird feathers over more than a century 701 Alexander L. Bond and Jennifer L. Lavers 702 Biological archives reveal contrasting patterns in trace element concentrations in 700 pelagic seabird feathers over more than a century 701 Alexander L. Bond and Jennifer L. Lavers 702 Biological archives reveal contrasting patterns in trace element concentrations in 700 pelagic seabird feathers over more than a century 701 Alexander L. Bond and Jennifer L. Lavers 702 703 Supplemental Material 704 Table S1. We achieved high recovery of two keratin-based reference materials using 705 inductively coupled plasma mass spectrometry (ICP-MS) to measure trace element 706 concentrations in Flesh-footed Shearwater feathers. Data are presented as the mean ± SD 707 % recovery relative to the mean certified concentration in μg/g (ppm). Table reproduced 708 from Lavers et al. (2013). 709 Reference Material (n) Element Certified Concentration Measured Concentration ± SD Mean % Recovery 6H-09 (8) Cd 0.24 0.24 ± 0.05 100 Hg 4.49 4.58 ± 1.81 102 Pb 14.8 14.9 ± 0.7 100 7H-09 (8) Cd 1.7 1.9 ± 0.1 109 Hg 3.78 3.38 ± 1.28 89 Pb 5.28 5.92 ± 0.75 112 710 Figures 692 29 698 Figure 1. 699 698 Figure 1. 699 698 698 Figure 1. 699 30 Supplemental Material Table S1. We achieved high recovery of two keratin-based reference materials using 705 inductively coupled plasma mass spectrometry (ICP-MS) to measure trace element 706 concentrations in Flesh-footed Shearwater feathers. Data are presented as the mean ± SD 707 % recovery relative to the mean certified concentration in μg/g (ppm). Table reproduced 708 from Lavers et al. (2013). 709 Table S1. We achieved high recovery of two keratin-based reference materials using 705 inductively coupled plasma mass spectrometry (ICP-MS) to measure trace element 706 concentrations in Flesh-footed Shearwater feathers. Data are presented as the mean ± SD 707 % recovery relative to the mean certified concentration in μg/g (ppm). Table reproduced 708 from Lavers et al. (2013). 709 Reference Material (n) Element Certified Concentration Measured Concentration ± SD Mean % Recovery 6H-09 (8) Cd 0.24 0.24 ± 0.05 100 Hg 4.49 4.58 ± 1.81 102 Pb 14.8 14.9 ± 0.7 100 7H-09 (8) Cd 1.7 1.9 ± 0.1 109 Hg 3.78 3.38 ± 1.28 89 Pb 5.28 5.92 ± 0.75 112 710 710 31 711 Figure S1 – The cubic regression spline of a general additive model of cadmium 712 concentrations in Flesh-footed Shearwater feathers over time. 713 711 711 Figure S1 – The cubic regression spline of a general additive model of cadmium 712 concentrations in Flesh-footed Shearwater feathers over time. 713 32 714 Figure S2 – The cubic regression spline of a general additive model of mercury 715 concentrations in Flesh-footed Shearwater feathers over time. 716 714 Figure S2 – The cubic regression spline of a general additive model of mercury 715 concentrations in Flesh-footed Shearwater feathers over time. 716 Figure S2 – The cubic regression spline of a general additive model of mercury 715 concentrations in Flesh-footed Shearwater feathers over time. 716 concentrations in Flesh-footed Shearwater feathers over time. 716 33 717 Figure S3 – The cubic regression spline of a general additive model of lead 718 concentrations in Flesh-footed Shearwater feathers over time. 719 17 717 Figure S3 – The cubic regression spline of a general additive model of lead 718 concentrations in Flesh-footed Shearwater feathers over time. 719 34
https://openalex.org/W4392514011	https://www.researchsquare.com/article/rs-4006730/latest.pdf	English	null	TriDeepRec: A Hybrid Deep Learning Approach to Content and Behaviour-based Recommendation Systems	Research Square (Research Square)	2,024	cc-by	8,444	TriDeepRec: A Hybrid Deep Learning Approach to Content and Behaviour-based Recommendation Systems University of Ottawa Thomas Tran University of Ottawa University of Ottawa Thomas Tran University of Ottawa TriDeepRec: A Hybrid Deep Learning Approach to Content and Behaviour-based Recommendation Systems Amirhossein Ghadami University of Ottawa Thomas Tran University of Ottawa Research Article Keywords: Recommendation System, Hybrid Recommendation System, Deep Learning, Convolutional Autoencoder, Cold Start Posted Date: March 6th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4006730/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. TriDeepRec: A Hybrid Deep Learning Approach to Content and Behaviour-based Recommendation Systems Amirhossein Ghadami University of Ottawa Thomas Tran University of Ottawa Research Article Keywords: Recommendation System, Hybrid Recommendation System, Deep Learning, Convolutional Autoencoder, Cold Start Posted Date: March 6th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4006730/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. TriDeepRec: A Hybrid Deep Learning Appro to Content and Behaviour-based Recommendation Systems Amirhossein Ghadami1* and Thomas Tran1 1School of Electrical Engineering and Computer Science (EECS), University of Ottawa, 800 King Edward Avenue, Ottawa, K1N 6N5, Ontario, Canada. Corresponding author(s). E-mail(s): aghad051@uottawa.ca; Contributing authors: ttran@uottawa.ca; Research Article Keywords: Recommendation System, Hybrid Recommendation System, Deep Learning, Convolutional Autoencoder, Cold Start Posted Date: March 6th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4006730/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. TriDeepRec: A Hybrid Deep Learning Approach to Content and Behaviour-based Recommendation Systems Amirhossein Ghadami1* and Thomas Tran1 1*School of Electrical Engineering and Computer Science (EECS), University of Ottawa, 800 King Edward Avenue, Ottawa, K1N 6N5, Ontario, Canada. TriDeepRec: A Hybrid Deep Learning Approach to Content and Behaviour-based Recommendation Systems 1 Introduction An eﬀective recommendation, with the ability to persuade customers to spend time and money in the business, is one of the main tools for most businesses. Most com- panies invest a signiﬁcant amount of money in building useful and high-performance recommendation systems because doing so can increase their revenue [1]. Traditional recommendation systems have three main categories: content-based, collaborative ﬁltering, and hybrid recommendation systems [2]. Content-based recommendation systems suggest items to users based solely on the characteristics of those items, such as genre or keywords, without considering user interactions or preferences [3]. Collaborative ﬁltering recommendation systems suggest items to users based on the preferences and behaviors of similar users, without considering the attributes of the items themselves [4]. Hybrid recommendation systems blend diﬀerent recommenda- tion techniques, like content-based and collaborative ﬁltering, to oﬀer more accurate and diverse suggestions by considering both items characteristics and users preferences [5]. The hybrid recommendation systems allow businesses to leverage the advantages of diﬀerent recommendation systems and mitigate their weaknesses. Deep learning has been exponentially utilized across various areas due to its capa- bility to handle diﬀerent types of datasets and large datasets eﬃciently. There is a more recent type of recommendation system called deep learning-based recommen- dation systems, which leverage deep learning approaches to create methods that are more eﬀective compared to traditional ones [6]. We introduce a new recommendation system called CAERS, powered by deep learning and speciﬁcally, a Convolutional Autoencoder (CAE) [7]. CAE combines the principles of Autoencoders [8] and Convolutional Neural Networks (CNNs) [9] to analyze and predict user ratings. At its core, CAE consists of two main components: an encoder and a decoder, both employing convolutional layers. These layers are designed to identify and utilize relevant information from the data, transforming it into a useful format. In the CAERS framework, we start by processing the descriptive data of users and items through the encoder. By calculating the dot product between the encoded data of users and items, we generate a user-item interaction matrix, essentially a grid where users are represented by rows and items by columns. This matrix is then fed into the CAE’s encoder, which extracts key characteristics. The resulting compressed data is passed through the decoder, which, unlike traditional autoencoders that aim to replicate the input, uses convolutional layers to translate the compressed data into a matrix of ratings. Abstract Hybrid recommendation systems are increasingly crucial for businesses aiming to boost revenue and customer engagement. These systems integrate various algorithms, each with unique strengths, to outperform traditional recommen- dation methods. Our study introduces a novel hybrid recommendation system, TriDeepRec, which eﬀectively combines content-based and behaviour-based data to enhance recommendation accuracy. We ﬁrst introduce a Convolutional Autoencoder-based Recommendation System (CAERS), designed to process con- tent data and extract complex, meaningful patterns, translating these into predictive ratings. Notably, CAERS tackles the cold-start problem by leveraging content information alone, making it robust in scenarios where historical user interaction data is sparse or unavailable. Next, we incorporate Neural Collabora- tive Filtering (NCF), a deep learning approach, to analyze past user behaviour and predict ratings. The outputs from CAERS and NCF are then integrated using a Multilayer Perceptron (MLP), a type of neural network, to generate the ﬁnal recommendations. Our methodology employs three deep learning techniques to create TriDeepRec, a system capable of utilizing both past interactions and content attributes. We evaluate our system using two datasets, initially focusing on CAERS to demonstrate its eﬀectiveness in addressing the cold-start problem. Subsequently, we assess the performance of TriDeepRec as a whole. The results, measured in terms of Mean Absolute Error (MAE) and Root Mean Square Error (RMSE), indicate signiﬁcant improvements over both the individual components and other leading models in the ﬁeld. This demonstrates that TriDeepRec, by 1 harnessing the strengths of both content and behaviour data, provides a more accurate and reliable recommendation system. Keywords: Recommendation System, Hybrid Recommendation System, Deep Learning, Convolutional Autoencoder, Cold Start 1 Introduction This allows us to evaluate CAERS by comparing the generated rat- ings against actual ratings. Moreover, CAERS’s reliance on content data enables it to 2 eﬀectively tackle the cold start problem, which often prevent the ability to recommend for new users or items. Building upon CAERS, we incorporated Neural Collaborative Filtering (NCF) [10], a well-known approach that utilizes neural networks to understand and predict the interactions between users and items. Unlike older models that decompose interaction matrices into simpler matrices [11], NCF employs deep learning to uncover complex patterns and relationships, oﬀering personalized and precise recommendations, par- ticularly valuable in cases of sparse data. In contrast to CAERS, NCF is trained on historical user interactions, enabling it to learn from past behaviors to make future recommendations. The ﬁnal phase involves merging the outputs from CAERS and NCF into a Mul- tilayer Perceptron (MLP) [12], a type of neural network characterized by multiple layers of nodes connected in a web-like structure. The MLP processes the combined data, adjusting its internal parameters (weights) to predict the most accurate ratings possible. TriDeepRec is a hybrid recommendation system that combines elements of CAERS, NCF, and MLP. It eﬀectively utilizes both content and user behavior data to enhance its recommendations. We tested TriDeepRec using two movie datasets, comparing its performance against other advanced deep learning recommendation models. The results aﬃrm that TriDeepRec outperforms its counterparts, showcasing its eﬃcacy. To summarize, our study makes three signiﬁcant contributions: 1. The development of CAERS, a deep learning-based recommendation system utilizing a convolutional autoencoder for eﬃcient content data analysis. 2. The integration of CAERS with neural collaborative ﬁltering (NCF) to form TriDeepRec, a hybrid system that beneﬁts from analyzing both content and historical data. 3. The demonstration of CAERS’s capability to overcome the cold start problem, ensuring eﬀective recommendations for new users and items by relying solely on content data. 3. The demonstration of CAERS’s capability to overcome the cold start problem, ensuring eﬀective recommendations for new users and items by relying solely on content data. The paper is organized as follows: Section 2 provides an overview of related works. Section 3 details the methodology employed in this study. Section 4 presents the experimental setup and the results obtained from various datasets. Conclusions are drawn in Section 5, followed by a discussion on the future potential of our approach in Section 6. 2.1 Deep Learning-based Recommendation Systems Deep learning is being extensively utilized across various domains due to its capability to extract useful and meaningful information or features from datasets [13]. Recom- mendation systems represent one of the domains where researchers are increasingly employing deep learning methods to develop high-performance systems compared to traditional approaches. 3 GAP [14] is one of the recent deep learning-based recommender systems, lever- aging a convolutional neural network (CNN) [9] that utilizes outer product matrices of features and cross-convolutional ﬁlters. This approach eﬃciently captures intricate user-item interactions, emphasizing important features while mitigating overﬁtting through global pooling mechanisms. GCF-YA [15] presents a deep graph neural network speciﬁcally developed for link prediction in recommender systems. Its objective is to tackle the data sparsity issue common in such systems. Through the incorporation of information propagation and an attention mechanism, this approach enhances the extraction of interactions from bipartite user-item graphs. DPTUPCDR [16] addresses the cold-start problem in recommender systems for new users without historical interactions. It introduces a model called DPTUPCDR, which employs Transfer Learning and Deep Neural Networks to transfer knowledge between domains, enhancing recommendation accuracy. This approach shows promise in improving recommendation quality for cold-start users. Contrasting with the aforementioned systems like GAP [14], GCF-YA [15], and DPTUPCDR [16], our deep learning-based recommendation system, CAERS, distinc- tively employs a convolutional autoencoder. This innovative approach allows CAERS to eﬀectively extract meaningful features from input data during the encoder phase and adeptly translate the encoded data into ratings in the decoder phase. Certainly, we are not the pioneers in utilizing convolutional autoencoders in devel- oping recommendation systems. CCAE [17] employs a Convolutional Autoencoder (CAE) to address the sparsity and accuracy issues present in traditional collabora- tive ﬁltering. CCAE replaces matrix decomposition with convolutional autoencoder techniques. It entails drop sampling via convolution and downsampling layers for com- pression, followed by reconstruction through deconvolution and upsampling layers for recommendation score calculation. CCA [18] introduces a Collaborative Convolutional Autoencoder (CCA), another recommendation system employing CAE. CCA is designed to address data spar- sity, with a speciﬁc emphasis on Collaborative Filtering (CF). By integrating content features from papers, the model enhances Matrix Factorization (MF) [11] in con- structing user-item matrices. Contextual information from papers is captured using Sentence-BERT (SBERT) [19] and Convolutional Neural Network (CNN) [9]. 2.1 Deep Learning-based Recommendation Systems To the best of my knowledge, we are the ﬁrst to utilize a convolutional autoencoder to construct a recommendation system that encodes content data, such as information about users (e.g., age, occupation, and sex) and items (e.g., genre, movie plot), and directly decodes it into the rating matrix. 2.2 Hybrid Recommendation Systems A hybrid recommendation system combines multiple recommendation techniques to improve recommendation quality and address the limitations of individual methods. DMF [20] presents a novel hybrid approach for recommender systems, which com- bines a neural network-enhanced matrix factorization model. This approach constructs a user-item matrix from both explicit ratings and implicit feedback and then employs deep learning techniques to map users and items into a shared latent space. 4 ECAE [21] represents another hybrid recommendation system designed to tackle challenges within recommender systems by transforming discrete user feedback into continuous values. It integrates data generation and retraining into a cohesive frame- work to reduce noise and incorporates a distillation layer speciﬁcally to manage varying noise levels within the data. The primary goal is to enhance the precision of user preference modeling. CoDAE [22] is proposed to enhance recommendation quality in scenarios character- ized by sparse social network and rating data. It employs three distinct autoencoders, each dedicated to learning user representations based on their roles as raters, trusters, and trustees. This approach uniquely leverages shared parameters for individuals appearing in multiple roles, thereby enriching the user representation. Additionally, CoDAE introduces a regularization term designed to capture and learn the correlations between user features across its various subnetworks. This feature aims to signiﬁcantly improve the method’s robustness, particularly in environments with sparse data. Our hybrid approach, TriDeepRec, distinguishes itself from earlier hybrid tech- niques like CoDAE [22], ECAE [21], and DMF [20] by leveraging both user behavior data and content information for enhanced rating predictions, rather than relying solely on past behavior data for recommendations. The following approaches share similarities with our hybrid recommendation system, as they integrate deep learning with hybrid techniques within traditional rec- ommender frameworks. Our approach also leverages an enhanced version of traditional collaborative ﬁltering known as Neural Collaborative Filtering (NCF). However, in our case, we augment NCF with an additional method (CAERS) capable of processing content information eﬀectively. DNNRec [23] combines deep learning with collaborative ﬁltering to address the cold start problem and linear limitations of traditional methods. It uses user and item embeddings to capture non-linear relationships and integrates side information to improve performance. The system also employs a dynamic learning rate and weight decay strategy to enhance accuracy, oﬀering a versatile solution adaptable to various datasets. 2.2 Hybrid Recommendation Systems DCN-M [24], an enhanced framework for recommender systems, tackles the challenge of eﬃciently learning feature crosses, particularly in large-scale indus- trial settings. It expands upon the Deep & Cross Network approach to enhance its expressiveness, speciﬁcally addressing web-scale traﬃc with billions of training examples. 3.1 Convolutional Autoencoder Recommendation System (CAERS) The Convolutional Autoencoder Recommendation System (CAERS) is structured around three principal phases: The Convolutional Autoencoder Recommendation System (CAERS) is structured around three principal phases: 1. Data Collection: The bedrock of any deep learning endeavor is the data it relies on. In this phase, the aim is to augment our initial dataset by merging it with 5 additional information from varied sources. This step is vital for enhancing the model’s predictive accuracy by broadening the dataset. 2. Data Embedding and Transformation: This stage is dedicated to converting the data into numerical values that are both meaningful and interpretable for our model. This transformation is essential in preparing the data for further processing and analysis. After the embedding process, we construct the user-item interaction matrix, a crucial component for capturing the dynamics between users and items within the system. 3. Model Development and Training: The culminating stage involves the cre- ation and training of the CAERS model, an advanced deep learning framework designed for generating recommendations. This model excels at identifying com- plex, non-linear interactions between users and items, rendering it an eﬀective tool for recommendation systems. This structured approach facilitates the development of our deep learning-based recommendation system. Embedding In deep learning, converting data into a numerical format that models can understand is crucial. This process involves handling two main types of data: categorical and con- tinuous. Categorical data refers to non-numeric categories, like color types or animal species. These are distinct groups without a numerical relationship. On the other hand, continuous data relates to variables that can take on any value within a range, such as weight measurements or time durations. This distinction is important because it determines how we prepare and represent data for our models to process eﬀectively. For categorical variables, a traditional approach involves the conversion to binary values through the creation of dummy variables. This method, while straightforward, results in sparse matrices that, despite being high-dimensional, may not eﬃciently capture the relationships between categories due to their inherent sparsity. A more sophisticated and eﬃcient approach entails the mapping of each categor- ical variable to a dense vector via an embedding table. This method facilitates the representation of categories with meaningful, lower-dimensional numerical values. The conversion of a categorical variable to its dense vector representation can be described by the following equation: ec = E · dc (1) (1) where ec represents the dense vector corresponding to the c-th category. The matrix E ∈Rx×y, known as the embedding matrix, plays a crucial role, with its dimensions determined by x, the total number of unique categories, and y, the chosen dimen- sionality for the dense vectors. The vector dc is the one-hot encoded vector for the c-th category, eﬀectively serving as a selector that, when multiplied by E, isolates the appropriate row from E. This row corresponds to the dense vector representation of the c-th category. For continuous variables, especially in language processing, a technique is used that embeds words based on their context within sentences. This involves training a model, like a multilayer perceptron, to map words to numerical values considering their contextual meanings. 3.1.1 Data Collection The accuracy of deep learning models is inherently linked to the scope of the data they are trained on. It is imperative to compile an extensive dataset to reﬁne the model’s training process and, by extension, the eﬃcacy of the recommendation system. Speciﬁcally, our focus is on movie datasets, which we enrich by integrating a wide range of relevant information from various sources. This includes extracting summaries that illuminate aspects such as the plot, principal characters, their motivations, and other critical details. This enriched information signiﬁcantly enhances our model’s understanding of each movie, making the integration of this data into our primary dataset a crucial step in crafting a nuanced recommendation system. An example of a movie summary is depicted in Figure 1. Fig. 1 A summary extracted from the movie ’Inception’ Fig. 1 A summary extracted from the movie ’Inception’ 6 6 User-Item Interaction Matrix Following the embedding of data, the subsequent phase involves establishing a user- item matrix. This matrix illustrates the interactions between users and items based on their content information, as exempliﬁed in Tables 2 and 1. 7 Table 1 Items’ content matrix Table 2 Users’ content matrix Table 2 Users’ content matrix Table 1 Items’ content matrix Table 1 Items’ content matrix Table 2 Users’ content matrix The user-item interaction matrix is computed using the dot product of the embed- ded content information of users and items. The formula for this operation is given by: Z = U · IT (2) (2) where U denotes the matrix with embedded content information of users, IT rep- resents the transposed matrix with embedded content information of items, and Z signiﬁes the resulting user-item interaction matrix. 3.1.3 Model Development and Training The CAERS framework incorporates a deep learning model known as the Convolu- tional Autoencoder (CAE), which combines the methodologies of Autoencoders [8] and Convolutional Neural Networks (CNN) [9]. The CAE inherits the encoder and decoder structures from Autoencoders [8] and adopts the strategy of extracting meaningful data through the use of convolutional layers from Convolutional Neural Networks (CNN) [9]. ( ) [ ] Now that we have established the input data, which is the user-item matrix denoted as Z, we are prepared to build the Convolutional Autoencoder (CAE) component of our recommendation system. We will thoroughly discuss each phase of the CAE—the encoder and the decoder—along with their mathematical equations, attributes, and features. Encoder The encoder part of a Convolutional Autoencoder (CAE) consists of two main com- ponents: convolutional layers and pooling layers. Convolutional layers, which are fundamental to Convolutional Neural Networks (CNNs), are utilized in the encoder section of a CAE to eﬃciently process and interpret complex data inputs. The primary advantages of this design include a reduction in the number of trainable parame- ters, addressing a common limitation in traditional autoencoders, and an enhanced capability to extract and leverage critical information from the data [25]. Initially proposed for image processing applications, convolutional layers utilize a crucial concept known as kernels or ﬁlters. Kernels [9], also referred to as ﬁlters, are small matrices used in convolutional layers to extract features from data. In the context of image processing, these kernels move across the input image in a sliding 8 window manner, performing an element-wise multiplication with the part of the image they cover, followed by a summing operation. This process generates a feature map that highlights speciﬁc attributes in the image, such as edges, textures, or patterns. Currently, researchers are applying deep learning methods that utilize convolutional layers across various domains beyond image processing. We incorporate convolutional layers into the encoder part of our recommendation system, which we term the Convolutional Autoencoder-based Recommendation Sys- tem (CAERS). These layers are designed to extract high-order, nonlinear, meaningful information from the user-item interaction matrix, denoted as the Z matrix. By feed- ing the interaction matrix into the encoder component, as illustrated in Figure 2, the CAE encoder processes the Z matrix to identify and isolate the most signiﬁcant and meaningful information. It compresses the data in a way that retains important information while discarding useless details, resulting in a compressed form of the input. Fig. 2 Convolutional autoencoder recommendation system (CAERS) Fig. 2 Convolutional autoencoder recommendation system (CAERS) Fig. 2 Convolutional autoencoder recommendation system (CAERS) Here is the mathematical representation of the convolutional process: Hi,j = A X a=1 B X b=1 Zi+a−1,j+b−1 · Ka,b (3) (3) where Hi,j represents the output at a speciﬁc point (i, j). The operation involves moving a ﬁlter, or kernel, sized A × B (denoted as K), across the input matrix Z. At each point (i, j), the section of the matrix covered by the ﬁlter is multiplied with the ﬁlter values (Ka,b), and these products are summed up to get the output at that point, Hi,j. Encoder The operation selects the maximum value from a 2× 2 region in the input matrix H, speciﬁcally from the positions (2i, 2j), (2i, 2j + 1), (2i + 1, 2j), and (2i + 1, 2j + 1). This downsampling process reduces the dimensions of H, resulting in V , which retains key features while summarizing the original data within smaller spatial dimensions. Encoder The kernel K is designed to detect patterns and features in the user-item interaction data, aiding in the extraction of relevant features for encoding. Initially, the raw user-item interaction matrix is the input for the ﬁrst convolutional layer. But as the model progresses through more layers, the input can originate from the output of a previous convolutional or pooling layer (Figure 2), reﬂecting the layered structure of convolutional autoencoders. This setup allows for the building up of more complex and abstract representations of the data. 9 To keep the explanation straightforward, we’ve used Z as the notation for the user-item interaction matrix and treated it as the consistent input for the convolu- tion operation. This approach helps in understanding the convolution process without getting into the detailed mechanics of input variation across diﬀerent layers. In addition to convolutional layers, pooling layers play a vital role in the CAE’s functionality in the encoder layer by reducing the spatial size of the representation. This reduction decreases the number of parameters and computation in the network, further enhancing the model’s eﬃciency. For the CAERS pooling layer’s downsampling step, which compacts the output matrix from the earlier convolution stage, we use a speciﬁc method called max-pooling. This technique shrinks the size of the matrix by summarizing key details. Max-pooling works by picking out the largest value from every 2 × 2 area in the starting matrix to ﬁgure out the elements in the smaller, downscaled matrix, which we will call V . This approach keeps important data while making the computations easier. The formula can be written as: Vi,j = max(H2i,2j, H2i,2j+1, H2i+1,2j, H2i+1,2j+1) (4) (4) where Vi,j denotes the value at position (i, j) in the downsampled matrix V , achieved by applying max-pooling. The operation selects the maximum value from a 2× 2 region in the input matrix H, speciﬁcally from the positions (2i, 2j), (2i, 2j + 1), (2i + 1, 2j), and (2i + 1, 2j + 1). This downsampling process reduces the dimensions of H, resulting in V , which retains key features while summarizing the original data within smaller spatial dimensions. where Vi,j denotes the value at position (i, j) in the downsampled matrix V , achieved by applying max-pooling. Decoder The decoder part of our CAE is responsible for transforming the compressed and meaningful data, previously referred to as V , into an interpretable form, which in this context is the predicted ratings denoted as ˆRCAERS. The decoder comprises two main components: deconvolutional (or transposed convolutional) layers and upsampling layers. Deconvolutional layers [26], while technically similar to standard convolutional layers, serve a diﬀerent purpose. In the encoding phase, a standard convolutional layer processes either the initial raw data matrix or the output from preceding encoding layers. In contrast, during the decoding phase, a deconvolutional layer aims to reverse the encoding process. It does this by taking the compressed matrix V or the output from preceding decoding layers as its input. When a transposed convolutional layer is the ﬁrst in the decoding sequence, it begins with the compressed data V . For subsequent layers, the input is the output from the earlier transposed convolutional or upsampling layers in the decoding sequence. This distinction highlights the diﬀerent ways data is processed across the various phases and layers, maintaining clarity by consistently using the established notations. Given the equation for transposed convolution: Given the equation for transposed convolution: 10 Gi,j = A X a=1 B X b=1 Vi+a−1,j+b−1 · Ka,b (5) (5) where, Gi,j represents the value at coordinate (i, j) in the output matrix G, which is the output of deconvolutional layer. V is the compressed data. The variables a and b are indices that traverse the transposed convolutional kernel K, which has dimensions A × B. The operation iterates over each element (a, b) in the kernel K, performing element-wise multiplications with the corresponding elements in a subsection of the V matrix, starting from position (i, j). The sum of these products yields the value at Gi,j, eﬀectively expanding the compressed data V back into a more detailed form. where, Gi,j represents the value at coordinate (i, j) in the output matrix G, which is the output of deconvolutional layer. V is the compressed data. The variables a and b are indices that traverse the transposed convolutional kernel K, which has dimensions A × B. The operation iterates over each element (a, b) in the kernel K, performing element-wise multiplications with the corresponding elements in a subsection of the V matrix, starting from position (i, j). Decoder The sum of these products yields the value at Gi,j, eﬀectively expanding the compressed data V back into a more detailed form. ,j y g The upsampling layer [27] is an essential part of the decoder, used to generate an appropriate output by increasing the spatial dimensions of the feature matrix G. This enhancement improves the detail within its features. The process of upsampling is described as follows: Qi,j = 1 4 G⌊i 2⌋,⌊j 2⌋+ G⌊i 2⌋,⌈j 2⌉+ G⌈i 2⌉,⌊j 2⌋+ G⌈i 2⌉,⌈j 2⌉ (6) (6) where Qi,j represents the element at position (i, j) in the upsampled matrix Q. This technique involves averaging the four nearest elements in the matrix G, which is the output of deconvolutional layer. The calculation for each element in Q takes into account the nearest elements in G, corresponding to halved indices. This includes both the ﬂoor and ceiling values of the halved indices to accurately capture all adjacent information in G. The division by 4 in this formula serves as a normalization factor, critical for maintaining the scale during the expansion of the matrix. When Q constitutes the ﬁnal layer in the model, its output is interpreted as the predicted values, denoted by ˆR1. This approach (CAERS), like other deep learning techniques, leverages backprop- agation for training. The backpropagation algorithm iteratively adjusts the model’s parameters to minimize error, thereby optimizing the model’s performance in making accurate predictions. The CAERS model mainly uses content information like age, sex, movie names, and plot details to improve its recommendations. This approach helps it overcome the cold start problem, which happens when new users or items are added to recommendation systems. Unlike methods that rely on past interactions between users and items, the CAERS model bases its recommendations on the actual content attributes of the items. This means that even when new movies or users are introduced, the system can still provide relevant recommendations without needing historical data. As a result, the system remains eﬀective and eﬃcient, avoiding the usual challenges of incorporating new entities. 3.2 Neural Collaborative Filtering (NCF) Neural Collaborative Filtering (NCF) represents a hybrid framework for recommen- dation systems, integrating the strengths of Generalized Matrix Factorization (GMF) and Multi-Layer Perceptron (MLP) to adeptly capture both linear and non-linear 11 user-item interaction patterns [10]. This dual approach enables NCF to provide highly accurate recommendations by leveraging the predictive capabilities of GMF for lin- ear interactions and the nuanced, complex modeling potential of MLP for non-linear dynamics (Figure 3). Fig. 3 Neural collaborative ﬁltering framework. Fig. 3 Neural collaborative ﬁltering framework. Before discussing the details of NCF, it’s important to note that NCF, along with other collaborative ﬁltering-based recommendation systems, relies on ratings or other past behavior data. In our case, we utilize a rating matrix as a user-item interactions data, in contrast to systems like CAERS that depend on content data . 3.2.1 Generalized Matrix Factorization (GMF) GMF enhances traditional matrix factorization methods, which serve as a corner- stone for numerous recommendation systems. By focusing on deriving low-dimensional embeddings for users and items, GMF can eﬃciently predict user-item interactions. These embeddings encapsulate user preferences and item characteristics in a compact, latent space, facilitating the capture of linear relationships through a dot product operation. Embedding Process Within the GMF framework, each user i and item j is associated with respective low-dimensional latent vectors uGMF i and vGMF j : uGMF i = EGMF u (i), (7) vGMF j = EGMF v (j), (8) (7) vGMF j = EGMF v (j), (8) where EGMF u and EGMF v represent the embedding functions for users and items, respectively, within GMF. 12 Embedding Process MLP similarly assigns users and items to latent vectors, which, unlike GMF, are speciﬁcally engineered to non-linear interaction patterns: uMLP i = EMLP u (i), (10) uMLP i = EMLP u (i), (10) vMLP j = EMLP v (j), (11) (10) vMLP j = EMLP v (j), (11) vMLP j = EMLP v (j), (11) (11) where EMLP u and EMLP v are the user and item embedding functions tailored for MLP. Interaction Prediction The non-linear interaction score ˆrMLP ij is derived from feeding the concatenated user and item embeddings through a sequence of neural network layers, employing non- linear activation functions to achieve the ﬁnal prediction: ˆrMLP ij = f(uMLP i ∥vMLP j ), (12) (12) where f denotes the MLP’s non-linear activation functions, and ∥symbolizes the concatenation operation. 3.2.2 Multi-Layer Perceptron (MLP) MLP, a fundamental component of neural networks, introduces the capability to model non-linear relationships through its hierarchical structure and non-linear activation functions. This feature is especially beneﬁcial in complex recommendation scenar- ios where the interaction patterns between users and items transcend simple linear associations. Interaction Prediction The interaction score ˆrGMF ij between user i and item j is predicted through the dot product of their GMF embeddings, indicating a linear interaction model: ˆrGMF ij = uGMF i · vGMF j . (9) (9) 3.2.3 Integration of GMF and MLP NCF capitalizes on the individual strengths of GMF and MLP by combining their outputs, thus generating a uniﬁed prediction ˆRNCF for user-item interactions. This synthesis not only encompasses the linear predictive capabilities of GMF but also the non-linear modeling prowess of MLP, rendering NCF a highly eﬀective and versatile framework for recommendation systems: ˆRNCF = α · ˆrGMF ij + (1 −α) · ˆrMLP ij , (13) (13) where α is a hyperparameter ﬁne-tuned to optimize the balance between GMF and MLP contributions, enabling NCF to deliver nuanced and precise recommendations. 13 3.3 TrideepRec In the hybrid section of our recommendation system, we seamlessly integrate the Con- volutional Autoencoder Recommendation System (CAERS) and Neural Collaborative Filtering (NCF) through a Multilayer Perceptron (MLP) to leverage the strengths of both models. This integration aims to enhance the accuracy of the recommenda- tion system by combining the deep content analysis capabilities of CAERS with the nuanced user-item interaction insights provided by NCF. The MLP serves as the fusion point, eﬀectively combining and processing the outputs from CAERS and NCF to generate the ﬁnal recommendation ratings, as it is depicted in Figure 4. Fig. 4 Proposed hybrid recommendation system (TriDeepRec) Fig. 4 Proposed hybrid recommendation system (TriDeepRec) The integration process begins by obtaining the prediction ratings from both CAERS and NCF. Let’s denote the prediction rating from CAERS as ˆRCAERS and from NCF as ˆRNCF . These ratings reﬂect the system’s conﬁdence in recommending a speciﬁc item to a user, based on content similarity and past interaction patterns, respectively. The MLP is designed to take a concatenated vector of ˆRCAERS and ˆRNCF as its input. This vector serves as a comprehensive representation of insights derived from both content-based and collaborative ﬁltering approaches. The MLP processes this information through multiple layers, each consisting of a set of neurons equipped with non-linear activation functions to capture complex interactions between the input features. Let’s denote the concatenated input vector to the MLP as X, where: X = [ ˆRCAERS \|\| ˆRNCF] (l X = [ ˆRCAERS \|\| ˆRNCF] (l Each layer l in the MLP transforms its input X(l−1) (where X(0) = X) into an output X(l) using a set of weights W(l) and biases b(l), followed by a non-linear activation function f (l): X(l) = f (l)(W(l)X(l−1) + b(l)) 14 14 The ﬁnal layer of the MLP, often referred to as the output layer, produces the ﬁnal recommendation rating ˆRHybrid, which is a scalar value representing the system’s combined assessment of how likely a user is to prefer a speciﬁc item: ˆRHybrid = W(L)X(L−1) + b(L) where L denotes the total number of layers in the MLP. The hybrid system is trained to minimize an objective function that measures the discrepancy between the predicted recommendation ratings ˆRHybrid and the actual user-item interaction data. A common choice for this objective function is the Mean Squared Error (MSE), deﬁned as: MSE = 1 N N X i=1 (Ri −ˆRHybrid,i)2 where Ri represents the actual rating for the i-th user-item pair in the training set, and N is the total number of user-item pairs. The MLP, along with the integrated CAERS and NCF components, is trained using gradient descent. This involves iteratively adjusting the weights W(l) and biases b(l) in each layer to minimize the MSE objective function. The gradients required for these updates are computed via backpropagation, ensuring that both the content-based and collaborative ﬁltering insights are optimally combined to enhance the performance of the recommendation system. By integrating CAERS and NCF through an MLP, the hybrid recommendation system beneﬁts from a comprehensive analysis. 4.1 Experimental setup In our research, we assess the eﬀectiveness of our hybrid recommendation model by conducting experiments with two distinct datasets. Our evaluation involves comparing the hybrid approach (TriDeepRec) against standalone CAERS and Neural Collab- orative Filtering (NCF) methods. Furthermore, we benchmark our model against leading-edge hybrid or deep learning-based techniques, GAP [14], ECAE [21], DNNRec [23], and DCN-M [23]. [ ] [ ] For our CAERS component, we use a 3×3 two-dimensional convolutional setup for both the encoder and decoder. The encoder incorporates two convolutional layers and two max-pooling layers, whereas the decoder comprises three deconvolutional layers. The Rectiﬁed Linear Unit (ReLU) is utilized in the hidden layers, and the sigmoid function is employed for the output layer to adjust the output range suitably. To manage the sparse rating matrix with many zeros, we implement a strategy to exclude these zeros from the training process. We adopt a modiﬁed Mean Squared Error (MSE) loss function that includes masking for missing ratings, which are represented as zeros. This strategy enables our model to eﬀectively work with new users or items based solely on content information, without the necessity for historical interaction data. We select the Adaptive Moment Estimation (Adam) optimizer [28], with a learning rate 15 of 0.01, for its eﬃciency and adaptability. Training is performed in batches of 32, with the data split 90 : 10 for training and testing, respectively, and an additional 10% of the training dataset reserved for validation. Our system’s eﬀectiveness in managing the cold start issue is further examined by introducing new users, who are not included in the training or validation datasets, into the dataset at proportions of 10%, 20%, and 30%. This approach aims to simulate scenarios where there is no prior data on these users. The Neural Collaborative Filtering component of our system is conﬁgured with an embedding size of 64, designed to eﬃciently process the nuances of user-item interac- tions. The NCF model is trained over 6 epochs, utilizing a learning rate of 0.001, with batches comprising 32 data points. A dropout rate of 0.5 is implemented to mitigate the risk of overﬁtting, and the architecture includes hidden units in the conﬁguration of (64, 128, 512), strategically selected to capture complex patterns in user-item inter- actions (rating). Notably, the model is optimized without employing negative sampling for the rating prediction task, focusing exclusively on observed interactions. 4.1 Experimental setup The loss function employed is Mean Squared Error (MSE). These parameters were meticu- lously chosen through a process of hyperparameter tuning, ensuring optimal model performance. For the ﬁnal integration stage, a Multi-Layer Perceptron (MLP) combines the outputs from the CAERS and NCF models. The MLP is optimized with ReLU as the activation function and an alpha value of 0.0001, indicating a minimal degree of regularization. The architecture includes a single hidden layer comprising 32 neurons, a conﬁguration determined through hyperparameter tuning to eﬀectively integrate features from both CAERS and NCF outputs. This setup is designed to achieve a balance between learning capability and model complexity, aiming to reﬁne the hybrid recommendation system’s accuracy and relevance in predicting user preferences. Our system’s performance is assessed using two primary metrics: Root Mean Squared Error (RMSE) and Mean Absolute Error (MAE). These metrics provide a comprehensive evaluation of the model’s accuracy in predicting user ratings, facilitat- ing a detailed comparison with existing recommendation models in the literature. 4.2 Datasets In this study, we used two versions of the MovieLens dataset [28]: the 100 K and the 1 M datasets. These datasets contain user ratings for a wide range of movies, but not every user has rated every movie, leading to many missing (zero) values. To address this, we excluded these zeros from our model’s training and evaluation stages. The 100K dataset includes ratings for 1,632 movies from 943 users, totaling around 100,000 ratings. The larger 1M dataset consists of ratings for 3,883 movies from 6,040 users, adding up to 1 million ratings. We take advantage of some information available in these datasets, such as users’ occupations, ages, genders, and zip code as well as details about the movies like their titles and genres. This information helps us understand user preferences better and make more accurate recommendations. To further improve our model, we enriched our dataset with movie summaries or overviews. These narratives provide more context about each movie, which could 16 align with speciﬁc user interests. To analyze this text data eﬀectively, we used the BERT model [29], a cutting-edge tool that understands the context of words in text. BERT helps us extract meaningful insights from the movie overviews, improving our recommendations by considering more than just basic metadata. Table 3 Comparative experimental outcomes between the hybrid model and its indi- vidual component models 4.3 Result In this section, we analyze and discuss the performance of our hybrid recommendation system, TriDeepRec, by comparing it with its individual components and other state- of-the-art models. Additionally, we present the evaluation of our novel deep learning- based recommendation system, CAERS, highlighting its capability to tackle the cold start problem. In our comparative analysis (Table 3), we evaluate TriDeepRec’s performance against its foundational components, CAERS and NCF. For the MovieLens 100K dataset, TriDeepRec shows improvement, achieving lower RMSE values than CAERS and NCF by 4.05% and 5.50%, respectively. The RMSE values are recorded as 0.8845 for TriDeepRec, in contrast to 0.9218 for CAERS and 0.9360 for NCF. In terms of MAE, TriDeepRec further asserts its superiority with enhancements of 3.93% and 6.12% over CAERS and NCF, respectively, with values being 0.6963 for TriDeepRec, 0.7248 for CAERS, and 0.7417 for NCF. With the MovieLens 1M dataset, TriDeepRec continues to lead in accuracy, achiev- ing lower RMSE values than CAERS and NCF, improving by 8.14% and 7.90%, respectively. For MAE, TriDeepRec signiﬁcantly outperforms its predecessors, now achieving improvements of approximately 9.16% and 9.13% over CAERS and NCF, respectively, with an updated value of 0.6258 for TriDeepRec, compared to 0.6889 for CAERS, and 0.6887 for NCF. The bar charts in Figures 5 and 6 clearly show TriDeepRec’s superior performance over CAERS and NCF across both datasets. Dataset Model RMSE MAE MovieLens 100K TriDeepRec 0.8845 0.6963 CAERS 0.9218 0.7248 NCF 0.9360 0.7417 MovieLens 1M TriDeepRec 0.8099 0.6258 CAERS 0.8817 0.6889 NCF 0.8794 0.6887 Table 3 Comparative experimental outcomes between the hybrid model and its indi- vidual component models Dataset Model RMSE MAE MovieLens 100K TriDeepRec 0.8845 0.6963 CAERS 0.9218 0.7248 NCF 0.9360 0.7417 MovieLens 1M TriDeepRec 0.8099 0.6258 CAERS 0.8817 0.6889 NCF 0.8794 0.6887 Table 3 Comparative experimental outcomes between the hybrid model and its indi- vidual component models 17 17 NCF CAERS TriDeepRec 0.7 0.8 0.9 RMSE MovieLens 100K MovieLens 1M Fig. 5 Evaluation of RMSE for TriDeepRec and Its Components Fig. 5 Evaluation of RMSE for TriDeepRec and Its Components NCF CAERS TriDeepRec 0.5 0.6 0.7 0.8 MAE MovieLens 100K MovieLens 1M Fig. 6 Evaluation of MAE for TriDeepRec and Its Components Fig. 6 Evaluation of MAE for TriDeepRec and Its Components In our study, detailed in Table 4, we compare the performance of our model, TriDeepRec, against several advanced models including GAP [14], ECAE [21], DNNRec [23], and DCN-M [23]. 4.3 Result For the MovieLens 100K dataset, TriDeepRec outperforms the other models, achieving the lowest RMSE and MAE scores. Speciﬁcally, TriDeepRec’s RMSE score is 0.8845, which is lower than the scores of CAERS (0.9218), GAP (0.9379), DCN- M (0.9552), DNNRec (0.9546), and ECAE (0.9513), with improvements of 4.05%, 5.69%, 7.40%, 7.34%, and 7.02%, respectively. Similarly, TriDeepRec leads in MAE with a score of 0.6963, demonstrating improvements of 3.93%, 5.17%, 8.05%, 7.55%, and 7.97% over the respective models, thereby underscoring its predictive accuracy. Moving to the MovieLens 1M dataset, TriDeepRec maintains its lead with the lowest RMSE and MAE scores among the compared models. It scores an RMSE of 0.8099 and an MAE of 0.6258, outperforming CAERS, GAP, DCN-M, DNNRec, and ECAE with RMSE improvements of 8.14%, 9.90%, 12.16%, 10.76%, and 10.58%, respectively, and MAE improvements of 9.16%, 11.21%, 15.19%, 12.13%, and 11.86%, 18 respectively. This demonstrates TriDeepRec’s consistent ability to provide eﬀective predictions across diﬀerent dataset sizes. Figures 7 and 8 visually summarize these ﬁndings, showcasing TriDeepRec’s supe- rior performance in both RMSE and MAE metrics across the MovieLens 100K and 1M datasets. These ﬁgures clearly illustrate the advantage of using TriDeepRec over other state-of-the-art models for recommendation systems. Dataset Model RMSE MAE MovieLens 100K TriDeepRec 0.8845 0.6963 CAERS 0.9218 0.7248 GAP 0.9379 0.7343 DCN-M 0.9552 0.7573 DNNRec 0.9546 0.7532 ECAE 0.9513 0.7566 MovieLens 1M TriDeepRec 0.8099 0.6258 CAERS 0.8817 0.6889 GAP 0.8989 0.7048 DCN-M 0.9220 0.7379 DNNRec 0.9076 0.7122 ECAE 0.9057 0.71 Table 4 Comparative Outcomes of Various Models Across MovieLens Datasets DCN-M DNNRec ECAE GAP CAERS TriDeepRec 0.8 0.9 RMSE MovieLens 100K MovieLens 1M Fig. 7 RMSE comparison between TriDeepRec and state-of-the-art models DCN-M DNNRec ECAE GAP CAERS TriDeepRec 0.8 0.9 RMSE MovieLens 100K MovieLens 1M MovieLens 100K MovieLens 1M Fig. 7 RMSE comparison between TriDeepRec and state-of-the-art models 19 DCN-M DNNRec ECAE GAP CAERS TriDeepRec 0.6 0.7 0.8 MAE MovieLens 100K MovieLens 1M Fig. 8 MAE comparison between TriDeepRec and state-of-the-art models Fig. 8 MAE comparison between TriDeepRec and state-of-the-art models The CAERS model demonstrates its eﬀectiveness in handling new users, out- performing other leading deep learning models. This is evident from the improved performance metrics across diﬀerent segments of new users, categorized by 10%, 20%, and 30%. Speciﬁcally, CAERS achieved error reductions (RMSE) of 1.64%, 2.25%, and 2.02% for each segment respectively, as illustrated in Table 5 and the visual analysis is depicted in Figure 9. 4.3 Result This analysis underscores CAERS’s strength in leveraging user and item content information eﬀectively, even in the absence of traditional data sources like reviews or likes. Model 10% 20% 30% CAERS 1.008 1.0144 1.0173 GAP 1.0248 1.0377 1.0383 DCN-M 1.0455 1.0576 1.0588 DNNRec 1.1077 1.1153 1.1413 Table 5: Prediction scores of new users Table 5: Prediction scores of new users DNNRec DCN-M GAP CAERS 1 1.05 1.1 1.15 RMSE 10% 20% 30% Fig. 9 RMSE performance analysis in addressing the cold start problem DNNRec DCN-M GAP CAERS 1 1.05 1.1 1.15 RMSE 10% 20% 30% Fig. 9 RMSE performance analysis in addressing the cold start problem 20 5 Conclusion In conclusion, our research presents TriDeepRec, a cutting-edge hybrid recommen- dation system that seamlessly integrates three deep learning models: two deep learning-based recommendation systems and a multilayer perceptron. This integra- tion aims to reﬁne recommendation accuracy by adeptly processing both content and behavior data. Our journey begins with the development of CAERS, an innovative sys- tem that utilizes a convolutional autoencoder to transform content data into ratings. We then advance our model by incorporating a neural collaborative ﬁltering (NCF) method, designed to leverage historical behavioral data eﬀectively. The pinnacle of our research is the strategic combination of CAERS and NCF through a multilayer perceptron model, signiﬁcantly boosting the system’s predictive performance. The eﬀectiveness of TriDeepRec is further validated by empirical evidence, with improve- ments in MAE and RMSE metrics across various versions of the MovieLens datasets, highlighting the system’s superior accuracy and eﬃciency. 6 Future Work Future enhancements for TriDeepRec will focus on integrating advanced deep learning models, expanding dataset diversity, and reﬁning data representation techniques. We also plan to explore innovative model fusion strategies and incorporate user feedback to improve personalization and user satisfaction. These steps aim to further advance the system’s performance and its applicability in the recommendation systems ﬁeld. 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https://openalex.org/W2982457029	http://old.scielo.br/pdf/lajss/v16n8/1679-7825-lajss-16-08-e229.pdf	English	null	New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory	Latin American Journal of Solids and Structures	2,019	cc-by	32,144	Hamed Gharoonia  Hamed Gharoonia  Keywords y Variable thickness shells, Nonlinear perturbation solution, Cylindrical pressure vessel, Hyperelastic FGM, Mooney-Rivlin model Abstract In this paper, nonlinear analysis of thick cylindrical shells with arbitrary variable thickness made of hyperelastic FGM with radially variation of material properties in nearly incompressible state under non-uniform pressure loading is presented. Thickness and pressure of the shell vary in axial direction by linear and/or nonlinear functions. The governing equilibrium equations are derived based on shear deformation theory (SDT). The Mooney-Rivlin type material is considered which is a suitable hyperelastic model for rubbers. Boundary Layer Method of the perturbation theory which is known as Matched Asymptotic Expansion (MAE) is used for solving the governing equations. A new ingenious solution and formulation have been defined during current study to simplify and abbreviate the representation of inner and outer equations components in MAE. In order to validate the results of the current analytical solution, a numerical modeling based on Finite Element Method (FEM) have been investigated. Afterwards, for different rubber case studies, the effect of material constants, inhomogeneity index, geometry and pressure profiles on displacements, stresses and hydrostatic pressure distributions resulting from MAE and FEM solution have been illustrated. This approach enables insight into the nature of the deformation and stress distribution across the wall of rubber vessels and offers the potential for investigating the mechanical functionality of blood vessels such as arteries in physiological pressure range. The results prove the effectiveness of SDT and MAE combination to derive and solve the governing equations of nonlinear problems such as nearly incompressible hyperelastic FG shells. https://doi.org/10.1590/1679-78255622 https://doi.org/10.1590/1679-78255622 ORIGINAL ARTICLE New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory H d Gh ia  ORIGINAL ARTICLE New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory H d Gh ia  ORIGINAL ARTICLE New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory rican Journal of Solids and Structures. ISSN 1679-7825. Copyright © 2019. This is an Open Access article distributed under the terms of the Creative Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Latin American Journal of Solids and Structures. ISSN 1679-7825. Copyright © 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Latin American Journal of Solids and Structures, 2019, 16(8), e229 1/28 Received: March 11, 2019. In Revised Form: July 10, 2019. Accepted: September 08, 2019. Available online: September 10, 2019 http://dx.doi.org/10.1590/1679-78255622 Received: March 11, 2019. In Revised Form: July 10, 2019. Accepted: September 08, 2019. Available online: September 10, 2019 http://dx.doi.org/10.1590/1679-78255622 Mehdi Ghannada*  a Department of Mechanical Engineering, Shahrood University of Technology, Shahrood, Iran. E-mail: gharooni.hamed@gmail.com, ghannad.mehdi@gmail.com * Corresponding author 1 INTRODUCTION Hyperelastic materials are quite common in many engineering applications. These materials are incompressible or almost incompressible and undergo large strains when subjected to loads. In the last decades, many constitutive models are developed for hyperelastic materials, which can be used in computational model according to the application. The Mooney-Rivlin model of hyperelastic materials can simulate most of the mechanical behaviour of the rubber materials. The model provides a good description of the mechanical properties of rubber materials when deformation is less than 150%. Rubber products are used in different industrial applications; such as rubber hose to carry fluids, rubber anti- vibration mountings, cylindrical pneumatic floating rubber fenders for boats and so on. Furthermore, rubber seals for sealing connectors are used to very easily seal on the internal or the external diameters of test parts which have smooth cylindrical connections. Rubber cylindrical sleeves have been used for many years successfully for label printing and have 1/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Gharooni et al. been proven of value for the established printing processes. Packer rubber produces a larger contact pressure and forms a seal between the rubber and the casing, resulting in sealing of the annular gap and isolation of the production layer. A comprehensive survey on the finite element methods of incompressible or almost incompressible hyperelastic materials can be found in many papers (Boyce and Arruda 2000). As an important research, Sussman and Bathe (1987) introduce a displacement-pressure finite element formulation for the geometrically and materially nonlinear analysis of compressible and almost incompressible solids. Simo and Taylor (1982) analyzed incompressible nonlinear elastic solids by a penalty function approach. Bijelonja et al. (2005) presented development of a displacement-pressure based finite volume formulation for modelling of large strain problems involving incompressible hyperelastic materials with a Mooney–Rivlin model. Some useful researches focus on developing strain energy function that can describe the mechanical behavior of rubber-like materials and incompressibility characteristic. For instant, Doll and Schweizerhof (2000) developed the volumetric part of the strain energy function and investigated new volumetric functions. Montella et al. (2014) presented the mechanical behavior of a Tire Derived Material in details numerically and experimentally. 1 INTRODUCTION The problem of the finite axisymmetric deformation of a thick-walled circular cylindrical elastic tube subject to pressure is formulated for an incompressible isotropic neo-Hookean material by Zhu et al. (2010) and solved numerically by finite element library Libmesh. Tanveer and Zu (2012) presented finite amplitude transient vibration analysis of nearly in compressible hyperelastic axisymmetric solids by a mixed p-type method and solved the equations by the Newmark’s method along with the Newton–Raphson iterative technique for Mooney-Rivlin material description. Kiendl et al. (2015) presented formulations for compressible and incompressible hyperelastic thin shells with plane stress condition based on energy methods and used continuous iso-geometric discretization to build the numerical solution. The common problems with mention numerical methods, as Poisson’s ratio approaches 0.5, are the ill conditioning of stiffness matrix, the locking phenomena and effect of applying numerical techniques on resulted displacements and stresses. Functionally graded materials (FGMs) are special composites, in which material properties are varied continuously and smoothly through certain direction. Thus, the discontinuities between the layers which occur in layered composites and cause stress concentration are not observed in FGMs. Graded rubber like materials attracted the attention of researchers for modeling these materials behavior under mechanical and geometrical boundary conditions. For instance, effects of material inhomogeneities on stress distributions through the thickness of circular cylinders made of rubber like materials in mechanical and thermal load was studied by Bilgili (2003). In another study, Bilgili (2004) investigated plane strain deformations of a circular cylinder made of heterogeneous neo-Hookean material with circumferential displacements prescribed on the inner and the outer surfaces. Batra and Bahrami (2009) considered cylindrical pressure vessel made of FG rubber like material under internal pressure. To discover stress components of the pressure vessel, they assumed axisymmetric radial deformations of a circular cylinder composed of FG Mooney–Rivlin material with the material parameters varying continuously through the radial direction either by a power law relation. Anani and Rahimi (2015, 2016) studied behavior of spherical and cylindrical shell made of FG rubbers by neo-Hookean model. They assumed radial variation of material properties by power law function and used classical theory (PET) and Gauss- hypergeometric function to derive and solve equations, respectively. Geometrically nonlinear dynamic behavior of FG thick hollow cylinder under axisymmetric mechanical shock loading is investigated using meshless local Petrov–Galerkin (MLPG) method by Ghadiri Rad et al. (2015). Latin American Journal of Solids and Structures, 2019, 16(8), e229 1 INTRODUCTION The FG cylinder is assumed to be made of large deformable neo-Hookean materials such as carbon-based polymers. In optimizing a shell with respect to weight or stress distribution, one method is to use shells with varying thickness or materials properties. The literature that addresses the stresses of thick cylindrical shells with variable thickness is quite limited. Eipakchi (2010) calculated stresses and displacements of linear elastic conical shell with varying thickness under non-uniform internal pressure analytically, using shear deformation theory (SDT). Ghannad et al. (2013) presented a closed-form analytical solution for thick FGM cylindrical shells with variable thickness subjected to constant internal pressure based on the first-order shear deformation theory (FSDT) and solved the governing equations by the usage of perturbation theory. Gharooni et al. (2016) investigated thermo-elastic analysis in pressurized thick FGM cylinders with varying properties of power function based on higher-order shear deformation theory. The innovative formulations for higher-order approximation with FG function of materials properties have been presented in this research. Jabbari et al. (2016) investigated thermo-elastic analysis of rotating truncated conical shells with varying thickness made of functionally graded materials (FGMs) subjected to thermo-mechanical loading. The system of partial differential equations is semi-analytically solved by using multi-layered method (MLM). Nejad et al. (2015) presented semi-analytical solution for elastic analysis of axially functionally graded rotating thick cylindrical shells with variable thickness under non-uniform pressure by the usage of SDT and MLM. 2/28 2/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Gharooni et al. Investigating aortic aneurysm as pressurized hyperelastic blood vessels enable scientists to evaluate the relative sensitivity of displacement and stress to geometrical and mechanical properties of the aneurysmal tissue. Furthermore, in pathologic conditions, arteries are even under more shear deformation compared to healthy vessels (Azar et al. 2018). In clinical interventions, such as balloon angioplasty significant wall shearing may take place. Simulation of arteries under blood pressure to yield displacement and stress analysis of blood vessels could result in useful information on the behavior of the arterial tissues under shear deformation (Vossoughi and Tozeren 1998). 1 INTRODUCTION Mihai and Goriely (2017) investigated the physical responses of nonlinear elastic materials that are generally described by parameters which are scalar functions of the deformation. They established relations between various hyperelastic material model parameters which are used to quantify nonlinear elastic responses in several hyperelastic models as rubber to soft tissues. Although numerous studies have been carried out on nearly incompressible hyperelastic shells, no study has been carried out to date on non-uniformly pressurized cylinder with nonlinear variable thickness made of hyperelastic FGMs. In the current study, nonlinear quasi-static analysis of thick cylindrical pressure vessels with arbitrary variable thickness made of Mooney-Rivlin model of hyperelastic FGM with radially variation of material properties in nearly incompressible state under non-uniform pressure loading is presented. The variation of the thickness and pressure profiles of the vessel are considered in axial direction by linear and/or nonlinear functions. In order to improve the approximation and take into account the effect of shear stresses and strains, the general method of derivation and nonlinear analysis has been presented by using first-order shear deformation theory. Matched Asymptotic Expansion (MAE) of the perturbation theory is used for solving the governing system of nonlinear coupled differential equations with variable coefficients. A new ingenious formulation and parameters have been defined during current study to simplify and abbreviate the representation of inner and outer equations components in MAE. In addition, the terms of variable thickness and non- uniform pressure have been presented in special representation separately. The effect of materials constants, inhomogeneity index, geometry and pressure parameters on displacements, stresses and hydrostatic pressure distributions resulting from MAE solution have been investigated for some case studies and the results have been compared with a FEM modeling in ANSYS software. We present the equations that provide the general continuum description of the deformation and the hyperelastic stress response of the material. Current study aims to illustrate the performance of the potentials and their reliability for the prediction of the state of deformation and stress in hyperelastic FG vessels from rubbers to arteries. 2.1 Shear deformation theory Consider a thick-walled axisymmetric cylindrical shell with variable thickness (of the outer surface) subjected to non- uniform internal pressure in the reference configuration as Figure 1. Geometry of the shell could be described in the terms of cylindrical polar coordinates r , θ and x : (1) ( ), , ≤ ≤ ≤ ≤ ≤ ≤ i o r r r x 0 2 0 x L θ π The general axisymmetric displacement field, in the first-order Mirsky-Hermann's theory could be expressed on the basis of radial displacement z U and axial displacement x U , as follows The general axisymmetric displacement field, in the first-order Mirsky-Hermann's theory could be expressed on the basis of radial displacement z U and axial displacement x U , as follows (4) ( , ) ( ) (x), ( , ) ( ) z (x) , = + = = + z x U z x w x z U U z x u x 0 θ ψ ϕ where ( ) w x and ( ) u x are the displacement components of the middle surface. Also, (x) ψ and (x) ϕ are the rotational components used to determine the displacement field. where ( ) w x and ( ) u x are the displacement components of the middle surface. Also, (x) ψ and (x) ϕ are the rotational components used to determine the displacement field. where ( ) w x and ( ) u x are the displacement components of the middle surface. Also, (x) ψ and (x) ϕ are the rotational components used to determine the displacement field. The matrix representation of deformation gradient tensor [ ] F in geometrically nonlinear kinematic is (Zhu et al. 2010) The matrix representation of deformation gradient tensor [ ] F in geometrically nonlinear kinematic is (Zhu et al. 2010) [ ] ′ ′ + +     +   = +   +   ′ ′ + +   F 1 0 w z w z 0 1 0 R z 0 1 u z ψ ψ ψ ϕ ϕ (5) (5) where ( ) ( ) ′ = ∂ ∂x . Consequently, the right Cauchy–Green deformation tensor, [ ] [ ] [ ] = C F F T and its principal invariants , , 1 2 3 I in cylindrical polar coordinate basis are given by (Tanveer and Zu 2012) where ( ) ( ) ′ = ∂ ∂x . ( ), , ≤ ≤ ≤ ≤ ≤ ≤ i o r r r x 0 2 0 x L θ π Consequently, the right Cauchy–Green deformation tensor, [ ] [ ] [ ] = C F F T and its principal invariants , , 1 2 3 I in cylindrical polar coordinate basis are given by (Tanveer and Zu 2012) [ ] ( ) ( ) ( ) ( )( ) ( ) , , ,    +      = = + + = +      +          ′ ′ ′ ′ ′ ′ ′ ′ = + + + + = = + + + + +  C zz zx 2 2 2 zz xz xx 2 2 xx zx xz C 0 C w z 0 C 0 C 1 C 1 R z C 0 C C w z 1 u z C C 1 w z 1 u z θθ θθ ψ ψ ϕ ψ ϕ ψ ψ ϕ ϕ (6) , , = + + = + + − = − = 2 2 2 1 zz xx 2 zz xx zz xx zx 3 zz xx zx I C C C I C C C C C C C I C C C C C J θθ θθ θθ θθ θθ (7) (6) , = + + − = − = 2 2 2 2 zz xx zz xx zx 3 zz xx zx C C C C C C C I C C C C C J θθ θθ θθ θθ ( (7) Jacobian which is known as volume ratio of deformation has the following terms (det is determinant operator): h is known as volume ratio of deformation has the following terms (det is determinant operator): ( ) ( ) ( )( ) ( ) ( ) det ( ) ( )    ′ ′ ′ ′ = = + + + + + + − +    +   F 1 J R x z w z 1 1 u z w z R x z ψ ψ ϕ ϕ ψ (8) ( ) ( ) ( )( ) ( ) ( ) det ( ) ( )    ′ ′ ′ ′ = = + + + + + + − +    +   F 1 J R x z w z 1 1 u z w z R x z ψ ψ ϕ ϕ ψ (8) ( ) ( ) ( )( ) ( ) ( ) det ( ) ( )    ′ ′ ′ ′ = = + + + + + + − +    +   F 1 J R x z w z 1 1 u z w z R x z ψ ψ ϕ ϕ ψ (8) (8) The Green–Lagrange strain tensor can be defined as ( ) = − E C I 1 2 ( [ ] I is the identity tensor). ( ), , ≤ ≤ ≤ ≤ ≤ ≤ i o r r r x 0 2 0 x L θ π ( ), , ≤ ≤ ≤ ≤ ≤ ≤ i o r r r x 0 2 0 x L θ π ( ), , ≤ ≤ ≤ ≤ ≤ ≤ i o r r r x 0 2 0 x L θ π where ir and ( ) or x , respectively, are the inner and outer radius and L is the length of the shell. The parameter r is the radius of every layer of cylinder in the reference configuration which can be replaced in terms of radius of mid-plane ( ) R x and distance of every layer with respect to mid-plane ( )z : ) ( ) , ( , , ) ( , , ) ≤ ≤ ⇒ = ⇒ h x z dr dz r x z x 2 θ θ ( ) ( ) ( ) , ( , , ) ( , , ) = + − ≤ ≤ ⇒ = ⇒ h x h x r R x z , z dr dz r x z x 2 2 θ θ (2) (2) where ( ) h x is the thickness of the cylinder which is varying along axial direction. The following relations can be written for the geometry components of the shell: where ( ) h x is the thickness of the cylinder which is varying along axial direction. The following relations can be written for the geometry components of the shell: ( ) ( ) , ( ) ( ) = + = + i o i h x R x r r x r h x 2 ( ) ( ) , ( ) ( ) = + = + i o i h x R x r r x r h x 2 (3) 3/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Figure 1: Geometry and loading parameters in cross section of the shell. Figure 1: Geometry and loading parameters in cross section of the shell. 2.2 Hyperelastic FGM As the constants of Mooney-Rivlin model have relation with initial shear modulus ( ) = + n 1n 2n G 2 C C , the variations of these two constants could be considered with power- law distribution continuously and smoothly in the radial direction. Although the variations of the FGM layers are the function of radial direction in the current research, because of outer radius variation along axial direction, the properties of outer points are determined based on thickness profile. These constants have the following form (Batra and Bahrami 2009, Anani and Rahimi 2016): ( ) ( ) ,   + =     n 2n 2 i R x z C x z C r (12) ( ) ( ) ( ) ( ) , , ,     + + = =         n n 1n 1 2n 2 i i R x z R x z C x z C C x z C r r (12) where 1 C and 2 C are material constants at the internal surface and n is the inhomogeneity index determined empirically. In the second term of the right hand side of Eq. (10), we can write: where 1 C and 2 C are material constants at the internal surface and n is the inhomogeneity index determined empirically. In the second term of the right hand side of Eq. (10), we can write: { } ( ) ( ) =  2 1 W J G J 2λ (13) ( ) G J in Eq. (13) is a penalty function which has to satisfy the conditions ( ) = ⇔ = G J 0 J 1 and > 0 λ is a penalty parameter which can be estimated by experimental data proportional to the material properties and is known as compressibility parameter (Silva and Bittencourt 2008, Ghaemi et al. 2006). Therefore, the assumption of almost incompressible rubber is accomplished by dropping the restriction = J 1 and including a hydrostatic work term in the strain energy function. Considering the compressibility parameter as = k λ , where k is an additional material constant representing the bulk modulus, only scales the penalty functions but does not change their shapes. In this context k can be interpreted as a penalty parameter that enforces incompressibility if large values are chosen. ( ), , ≤ ≤ ≤ ≤ ≤ ≤ i o r r r x 0 2 0 x L θ π Considering Voigt notation of Green–Lagrange strains { } { } = ε T zz xx zx θθ ε ε ε γ , Its components are as follows: 4/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. ( ) ( ) ( ) ( ) , , ( ) ( )    ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ = + + = + + + + + + +          + +  ′ ′ ′ ′ ′ ′ = + = = + + + + + +  + +  2 2 2 2 2 2 2 zz xx 2 zx zx 2 u w u u w z z 2 2 2 2 2 2 w z w z 2 u w w z R x z 2 R x z θθ ψ ϕ ϕ ψ ε ψ ε ϕ ϕ ψ ψ ψ ε γ ε ϕ ϕ ψ ψ ψψ ϕϕ (9) 2.2 Hyperelastic FGM Hyperelastics (as rubber-like materials) are kind of materials in which the stresses are only dependent on the initial and the final configurations but independent of the load path. These materials are characterized by the existence of stored energy function which depends on the right Cauchy–Green deformation tensor through the strain invariants ( ) ( , , ) 1 2 3 W I I I . For incompressible material, the determinant of deformation gradient is equal to unity ( ) = J 1 . In the present study, the extension of incompressible materials to nearly incompressible materials is considered; means that the incompressibility constraint is replaced with a penalty function correspond to the constraint. Therefore, the following strain energy function is developed based on the invariants , 1 2 I I and J (Ghaemi et al. 2006): (10) * ( , , ) ( , ) ( ) ( ) = + +  1 2 1 2 W I I J W I I W J cH J * ( , , ) ( , ) ( ) ( ) = + +  1 2 1 2 W I I J W I I W J cH J * W in Eq. (10) is the response of material to distortional part of the deformation. In the present study, the hyperelastic material of the shell is assumed to be isotropic and Non-homogenous with two-term Mooney-Rivlin material description in nearly incompressible condition which is a suitable hyperelastic model for rubbers. It has the following form (Batra and Bahrami 2009): * W in Eq. (10) is the response of material to distortional part of the deformation. In the present study, the hyperelastic material of the shell is assumed to be isotropic and Non-homogenous with two-term Mooney-Rivlin material description in nearly incompressible condition which is a suitable hyperelastic model for rubbers. It has the following form (Batra and Bahrami 2009): (11) ( ) ( ) ( , ) = − + − 1 2 1n 1 2n 2 W I I C I 3 C I 3 where 1n C and 2n C are material constants resulting from experimental tests. Non-homogenous and isotropic FG hyperelastic materials have different properties in terms of points. The changes of properties in functionally graded rubbers are generally considered in radial and/or longitudinal directions. Therefore, FG bulk modulus can be considered similar to Eq. (12). ( ) ( ) ,   + =     n n i R x z k x z k r (16) k is the bulk modulus at the internal radius. For the volumetric part, there are many forms proposed by researchers, all of which are functions of the bulk modulus and the Jacobian of deformation. Generally, in the limiting state, the volumetric part has to satisfy ( ) , ( ) , ( ) ′ ′′ →⇒ → → →    J 1 W J 1 W J 0 W J k conditions (Doll and Schweizerhof 2000). k is the bulk modulus at the internal radius. For the volumetric part, there are many forms proposed by researchers, all of which are functions of the bulk modulus and the Jacobian of deformation. Generally, in the limiting state, the volumetric part has to satisfy ( ) , ( ) , ( ) ′ ′′ →⇒ → → →    J 1 W J 1 W J 0 W J k conditions (Doll and Schweizerhof 2000). Considering zero values of displacement components in the reference configurations (initial state) with Eqs. (5), (6) and (7) lead in [ ] [ ] [ ] = = F C Ι and ( ) ( ) , , , , , , = 1 2 3 I I I J 3 3 1 1 . In the third term of the right hand side of Eq. (10), constant c and function ( ) H J with the condition ( ) , ( ) ′ = = ⇔ = H J 0 H J 1 J 1 only guarantee the stress free reference configuration with no physical meaning. Moreover, in the general case of nearly incompressible hyperelasticity (as Mooney-Rivlin material), hydrostatic pressure ( ) = − P k J 1 does not vanish even at the natural state. The first condition ( ) ( ) = H J 0 corresponds to the incompressibility constraint = J 1 and the second condition ( ) ( ) ′ = H J 1 is necessary for giving the meaning of pressure to the constant multiplier of H as = − 0 c p . Then the initial value of P (i.e. 0 p ), which has no clear physical meaning, must be introduced to make the initial stress zero. Therefore, FG bulk modulus can be considered similar to Eq. (12). In the current study, the function ( ) H J and ( ) G J are considered as (Doll and Schweizerhof 2000, Ghaemi et al. 2006): ( ) , ( ) = = − H J ln(J ) G J J 1 (17) Finally, the strain energy per unit undeformed volume of FG hyperelastic material for two-term Mooney–Rivlin model in nearly incompressible condition is expressed as the following coupled form (Holzapfel 2000). Finally, the strain energy per unit undeformed volume of FG hyperelastic material for two-term Mooney–Rivlin model in nearly incompressible condition is expressed as the following coupled form (Holzapfel 2000). (18) ( ) ( ) ( ) ln( ) = − + − − + − 2 n 1n 1 2n 2 0n k W C I 3 C I 3 p J J 1 2 Consequently, constitutive equation of coupled Mooney-Rivlin model in material description and nearly incompressible, isotropic and non-homogenous conditions would result (Holzapfel 2000, Başar and Weichert 2000). Consequently, constitutive equation of coupled Mooney-Rivlin model in material description and nearly incompressible, isotropic and non-homogenous conditions would result (Holzapfel 2000, Başar and Weichert 2000). ( ) [ ] ( ) , , = 2 − ∂ = + − + − −     ∂ S I C C C 1 1 2 1n 2n 1 2n n 0n W I I J 2 C C I 2C k J J 1 p (19) ( ) − − + − −     C C 1 2n n 0n 2C k J J 1 p (19) ( ) [ ] ( ) , , = 2 − ∂ = + − + − −     ∂ S I C C C 1 1 2 1n 2n 1 2n n 0n W I I J 2 C C I 2C k J J 1 p (19) Eq. (19) relates the right Cauchy–Green strain tensor [ ] C to the second Piola–Kirchhoff stress tensor [ ] S through constitutive relation. [ ]I is the identity tensor. The initial stress is zero if the hydrostatic pressure vanishes at the natural state, and vice versa. Recalling the assumption of stress-free reference configuration, Eq. (19) result in ( ) = + 0n 1n 2n p 2 C 2C (Holzapfel 2000, Bijelonja et al. 2005). 2.2 Hyperelastic FGM In this case, k is the ratio of the volumetric stress, known as hydrostatic pressure ( ) P , to the volumetric strain (Mihai and Goriely 2017). 5/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. = = ∆ − 0 P P k V V J 1 (14) 0 V and ∆V are the reference volume and volume changes through deformation, respectively. Various relations is recommended for estimating the value of incompressibility parameter; for instant ( ) ( ) = + − k 2G 1 3 1 2 ν ν . The common part of similar relations is definition of the bulk modulus based on material constants ( 1n C and 2n C in current model) or initial shear modulus ( ) n G and Poisson’s ratio ( ) ν . In nearly incompressible rubber materials, Poisson’s ratio with respect to compressibility intensity consider about . . = − 0 49 0 499 ν which is constant in shell. The order of graded compressibility parameter n k (bulk modulus) can be estimated based on compressibility intensity in FGM rubber as (Ghaemi et al. 2006, Tanveer and Zu 2012, Kiendl et al. 2015): (15) ( ) ( ) . to . = → ∝ + × = → ∝ + × 2 3 n 1n 2n n 1n 2n 0 49 k C C 10 0 499 k C C 10 ν ν Therefore, FG bulk modulus can be considered similar to Eq. (12). New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Therefore, FG bulk modulus can be considered similar to Eq. (12). Thus, the multiplier 0n p in the case of ( ) = H J ln(J ) denotes the pressure measured in the initial volume. The components in the right hand side of Eq. (19), other than identity tensor and material constants, Latin American Journal of Solids and Structures, 2019, 16(8), e229 6/28 ressible hyperelastic FGM cylindrical shells with arbitrary sure based on perturbation theory Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. can be written in the displacement field components. Therefore, the relation between the second Piola–Kirchhoff stress tensor and displacement components could be derived. The variation of strain tensor components can be calculated by the usage of variational principles: The variation of strain tensor components can be calculated by the usage of variational principles: The variation of strain tensor components can be calculated by the usage of variational principles: ) ( ) (  ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ = + + + + + + + + +  2 xx δ δu u δu w δw δ u δ δu w δ δw z δ δ z ε ϕ ϕ ϕ ψ ψ ϕ ϕ ψ ψ ) ( ) ( ( ) ( ) ( ) ( ) , ( ) ( )  ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ = + + + + + + + + +   +  = + + = + + +  +   ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ = + + + + + + + + + +  2 xx zz 2 zx δ δu u δu w δw δ u δ δu w δ δw z δ δ z δw z δ δ δ δ δ δ R x z w z R x z δ δ u δ δu δw w δ δw δ δ δ δ δ z θθ ε ϕ ϕ ϕ ψ ψ ϕ ϕ ψ ψ ψ ε ψ ψ ψ ϕ ϕ ε ψ γ ϕ ϕ ϕ ψ ψ ψ ψ ψ ψ ψ ϕ ϕ ϕ ϕ (26) ) ( ) ( ( ) ( ) ( ) ( ) , ( ) ( )  ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ = + + + + + + + + +   +  = + + = + + +  +   ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ = + + + + + + + + + +  2 xx zz 2 zx δ δu u δu w δw δ u δ δu w δ δw z δ δ z δw z δ δ δ δ δ δ R x z w z R x z δ δ u δ δu δw w δ δw δ δ δ δ δ z θθ ε ϕ ϕ ϕ ψ ψ ϕ ϕ ψ ψ ψ ε ψ ψ ψ ϕ ϕ ε ψ γ ϕ ϕ ϕ ψ ψ ψ ψ ψ ψ ψ ϕ ϕ ϕ ϕ (26) ) ( ) (  ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ = + + + + + + + + +  2 xx δ δu u δu w δw δ u δ δu w δ δw z δ δ z ε ϕ ϕ ϕ ψ ψ ϕ ϕ ψ ψ ) ) ( ) ( ) ( ) + + + + + 2 δw z δ R x z w z R x z ψ ψ (26) (26) The stress resultants are defined as follows: The stress resultants are defined as follows: (in ) & (on ) = = F y y y   0 0 δ GRADδ V δ 0 A (in ) & (on ) = = F y y y   0 0 δ GRADδ V δ 0 A (21) Therefore, the principle of virtual work (Eq. (20)) is a weak formulation of the equations of motion as well as the dynamic boundary conditions. Equality of energy conjugate variables preserves its validity i.e. : : = P δ F S δ E . In the static equilibrium and absence of body forces, Eq. (20) results in the variation of external work consists of non-uniform internal pressure ( ) iP x applying at the internal surface iA of cylinder: . ( ) , d Π = = = ∫∫ ∫∫ y t 0 i EXT 0 0 i z i i i i A A δ = δ dA P x δU dA dA r dx r r θ (22) . ( ) , d Π = = = ∫∫ ∫∫ y t 0 i EXT 0 0 i z i i i i A A δ = δ dA P x δU dA dA r dx r r θ (22) ernal surface element. Considering displacement components from Eq. (4), we can rewrite Eq. (22): i dA is the internal surface element. Considering displacement components from Eq. (4), we can rewrite Eq. (22): ( ) ( ) ( ) ( )    − −       h x h x x R x w dx 2 2 δ δψ ( ) ( ) ( ) ( )    Π − −       ∫ L EXT i 0 h x h x δ = 2 P x R x w dx 2 2 π δ δψ (23) (24) ( ) : , ( ) d ( ) z d Π = = = = + ∫∫∫ ∫∫∫ S E 0 0 ij INT 0 ij 0 0 V V δ δ dV S δ dV dV r x dr dx R x dz dx ε θ θ 0 dV is the volume element. Considering Voigt notation from Eq. 2.3 Governing equations Considering the boundary surface 0 A of the body consists of two parts y 0 A and σ 0 A where displacements y and forces t 0 are prescribed, respectively, based on the principle of virtual work, the variation of strain energy of the elastic body is equal to the variation of external work due to loading (Kim et al. 2012, Başar and Weichert 2000, Doghri 2000). ( ) . . : Π = Π −Π = − + − ∫∫∫ ∫∫ ∫∫∫ σ y b a y t P F      0 0 0 EXT INT 0 0 0 0 0 0 0 V A V δ δ δ 0= δ dV δ dA δ dV ρ ρ (20) ( ) . : − + − ∫∫ ∫∫∫ σ b a y t P F     0 0 0 0 0 0 0 0 0 A V dV δ dA δ dV ρ ρ (20) (20) ( ) . . : Π = Π −Π = − + − ∫∫∫ ∫∫ ∫∫∫ σ y b a y t P F      0 0 0 EXT INT 0 0 0 0 0 0 0 V A V δ δ δ 0= δ dV δ dA δ dV ρ ρ where 0 ρ , 0 V and b  0 are density, volume and body force in undeformed configuration, respectively. a is dynamic acceleration. [ ] P and [ ] F are first Piola–Kirchhoff stress and deformation gradient tensors, respectively. Kinematically admissible virtual deformation variables are understood to be the variations y δ and F δ which are subjected to the constraints (GRAD is gradient operator) where 0 ρ , 0 V and b  0 are density, volume and body force in undeformed configuration, respectively. a is dynamic acceleration. [ ] P and [ ] F are first Piola–Kirchhoff stress and deformation gradient tensors, respectively. Kinematically admissible virtual deformation variables are understood to be the variations y δ and F δ which are subjected to the constraints (GRAD is gradient operator) (in ) & (on ) = = F y y y   0 0 δ GRADδ V δ 0 A (in ) & (on ) = = F y y y   0 0 δ GRADδ V δ 0 A (9), the variation of strain energy of cylinder with variable thickness can be derived based on non-zero physical components of second Piola–Kirchhoff stress: ( )/ ( )/ ( ) ( ) + −     Π = + + + +       ∫ ∫ h x 2 L zz xx zx INT zz xx zx 0 h x 2 z δ 2 S δ S δ S δ S R x 1 dz dx R x θθ θθ π ε ε ε δγ (25) ( ) ( )    + + + +      xx zx zz xx zx z δ S δ S δ S R x 1 dz dx R x θθ θθ ε ε ε δγ (25) ( )/ ( )/ ( ) ( ) + −     Π = + + + +       ∫ ∫ h x 2 L zz xx zx INT zz xx zx 0 h x 2 z δ 2 S δ S δ S δ S R x 1 dz dx R x θθ θθ π ε ε ε δγ (25) The variation of strain tensor components can be calculated by the usage of variational principles: The stress resultants are defined as follows: p p y { } { } ( )/ ( )/ ( ) + −   = +     ∫ h x 2 T T zz 2 z z z h x 2 z N M Q S 1 z z 1 dz R x (27) { } { } ( )/ ( )/ + − = ∫ h x 2 T T 2 θ θ θ h x 2 N M Q S 1 z z dz θθ (28) { } { } ( )/ * * ( )/ ( ) ( ) + −   =   +   ∫ h x 2 T T 2 θ θ θ h x 2 R x N M Q S 1 z z dz R x z θθ (29) { } { } ( )/ ( )/ ( ) + −   = +     ∫ h x 2 T T xx 2 x x x h x 2 z N M Q S 1 z z 1 dz R x (30) { } { } ( )/ ( )/ ( ) + −   = +     ∫ h x 2 T T zx 2 zx zx zx S h x 2 z N M Q K S 1 z z 1 dz R x (31) { } { } ( )/ ( )/ ( ) + −   = +     ∫ h x 2 T T zz 2 z z z h x 2 z N M Q S 1 z z 1 dz R x (27) { } { } ( )/ ( )/ + − = ∫ h x 2 T T 2 θ θ θ h x 2 N M Q S 1 z z dz θθ (28) { } { } ( )/ * * * ( )/ ( ) ( ) + −   =   +   ∫ h x 2 T T 2 θ θ θ h x 2 R x N M Q S 1 z z dz R x z θθ (29) { } { } ( )/ ( )/ ( ) + −   = +     ∫ h x 2 T T xx 2 x x x h x 2 z N M Q S 1 z z 1 dz R x (30) { } { } ( )/ ( )/ ( ) + −   = +     ∫ h x 2 T T zx 2 zx zx zx S h x 2 z N M Q K S 1 z z 1 dz R x { } )/ )/ ( )   +     ∫ x 2 T zx 2 x 2 z S 1 z z 1 dz R x (31) (31) In the last equation, S K is shear correction factor which is applying in the stress resultant derived from shear stresses because of preventing stress overestimation. The stress resultants are defined as follows: (23) and (25), considering Π = Π EXT INT δ δ and carrying out the integration by parts, the equilibrium equations of nonlinear hyperelastic cylindrical shell with variable thickness under non-uniform internal pressure are obtained: ( ) ( ) ( )   ′ ′ + + + =   x x zx d R x N 1 u M N 0 dx ϕ ϕ (32) ( ) ( ) ( ) ( ) ( ) ( )   ′ ′ ′ ′ + + + − + + + =   x x zx z zx zx d R x M 1 u Q M R x N N 1 u M 0 dx ϕ ϕ ϕ ϕ (33) ( ) * * ( )     d 1 h x ( ) ( ) ( )   ′ ′ + + + =   x x zx d R x N 1 u M N 0 dx ϕ ϕ (32) ( ) ( ) ( ) ( ) ( ) ( )   ′ ′ ′ ′ + + + − + + + =   x x zx z zx zx d R x M 1 u Q M R x N N 1 u M 0 dx ϕ ϕ ϕ ϕ (33) ( ) ( ) ( ) * * ( ) ( ) ( ) ( ) ( )     ′ ′ + + + − − + = − −       x x zx i d 1 h x R x N w M N 1 N N w M P x R x dx R x 2 θ θ θ ψ ψ ψ (34) ( ) ( ) ( ) ( ) ( ) * * ( ) ( ) ( ) ( ) ( ) ( ) ( )   ′ ′ ′ ′ + + + − − + + +     − + = −     x x zx z zx zx i d R x M w Q M 1 M R x N 1 N w M dx 1 h x h x M w Q P x R x R x 2 2 θ θ θ ψ ψ ψ ψ ψ (35) ( ) ( ) ( )   ′ ′ + + + =   x x zx d R x N 1 u M N 0 dx ϕ ϕ (32) dx ( ) ( ) ( ) ( ) ( ) ( )   ′ ′ ′ ′ + + + − + + + =   x x zx z zx zx d R x M 1 u Q M R x N N 1 u M 0 dx ϕ ϕ ϕ ϕ (33) ( ) ( ) ( ) * * ( ) ( ) ( ) ( ) ( )     ′ ′ + + + − − + = − −       x x zx i d 1 h x R x N w M N 1 N N w M P x R x dx R x 2 θ θ θ ψ ψ ψ (34) ( ) ( ) ( ) ( ) ( ) ( )   ′ ′ ′ ′ + + + − + + + =   x x zx z zx zx d R x M 1 u Q M R x N N 1 u M 0 dx ϕ ϕ ϕ ϕ (33) ( ) ( ) ( ) * * ( ) ( ) ( ) ( ) ( )     ′ ′ + + + − − + = − −       x x zx i d 1 h x R x N w M N 1 N N w M P x R x dx R x 2 θ θ θ ψ ψ ψ (34) ( ) ( ) ( ) ( ) ( ) * * ( ) ( ) ( ) ( ) ( ) ( ) ( )   ′ ′ ′ ′ + + + − − + + +     − + = −     x x zx z zx zx i d R x M w Q M 1 M R x N 1 N w M dx 1 h x h x M w Q P x R x R x 2 2 θ θ θ ψ ψ ψ ψ ψ ( ) ( ) ( ) ( ) ( ) * * ( ) ( ) ( ) ( ) ( ) ( ) ( )   ′ ′ ′ ′ + + + − − + + +     − + = −     x x zx z zx zx i d R x M w Q M 1 M R x N 1 N w M dx 1 h x h x M w Q P x R x R x 2 2 θ θ θ ψ ψ ψ ψ ψ (35) The stress resultants are defined as follows: 7/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. The stress resultants are defined as follows: We consider / = S K 5 6 in the present study (Ghannad et al. 2013). Substituting strain invariants from Eq. (26) into Eqs. 3.1 Perturbation theory Therefore, one can calculate u indirectly from intermediate parameter v by relation ( ) d = + ∫ 7 u 1 v x c ε where 7c is integral constant. The intentional constants 0c and 7c will be calculated from boundary conditions. The following parameters need to be defined based on material constants of internal layer , , 1 2 C C k and geometrical parameter of shear correction factor s K because of abbreviation in representing inner and outer equations. which is the function of geometrical parameters ( ) R x , ( ) h x and inhomogeneity index n . In order to solve the set of governing differential equations in next steps, the inverse of singular coefficient matrices are needed. Two changes should be applied in equations to replace these matrices with non-singular ones. We take integrate the first equation in the set of Eqs. (32)-(35). The constant of integral ) ( 0c should be either dimensionless as other parameters. Therefore, it is replaced with its dimensionless equivalent i ( ) ( ) = n 0 0 1 c c r C ε where ε is bookkeeping perturbed parameter. Furthermore, as there is no u in governing equations unlike d d u x , we define (d d ) = u x v ε as new unknown intermediate parameter. Therefore, one can calculate u indirectly from intermediate parameter v by relation ( ) d = + ∫ 7 u 1 v x c ε where 7c is integral constant. The intentional constants 0c and 7c will be calculated from boundary conditions. The following parameters need to be defined based on material constants of internal layer , , 1 2 C C k and geometrical parameter of shear correction factor s K because of abbreviation in representing inner and outer equations. 3.1 Perturbation theory In this article, Boundary Layer Method of the perturbation theory which is known as Matched Asymptotic Expansion is used for solving the governing equations. The advantages of this method are fast convergence, closed form solution and compatibility with physics of shell. MAE can explain the behavior of the shell successfully even near the boundaries. The governing equations (32)-(35) for cylinder with variable thickness is a system of four nonlinear coupled differential equations with variable coefficients. Preliminary definitions, simplifications and preparations are necessary for using MAE. At first, it is necessary to convert the equations into dimensionless form for making use of the characteristic scales. The following dimensionless parameters are defined (Eipakchi 2010, Nayfeh 1981): , , , , , , , , , , = = = = = = = = = = = n i 0 i i i i 0 0 0 0 0 0 1 r h P r x h z R u w x h z r R u w P L h h h h h h L C ϕ ϕ ψ ψ ε ε (36) (36) Dimensionless quantities of parameters are marked with over-bar ( ) from now. Perturbation param Dimensionless quantities of parameters are marked with over-bar ( ) from now. Perturbation parameter ε should Dimensionless quantities of parameters are marked with over-bar ( ) from now. Perturbation parameter ε should ids and Structures, 2019, 16(8), e229 8 Latin American Journal of Solids and Structures, 2019, 16(8), e229 8/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. be normally considered as the ratio of minimal geometrical dimension of the structure ( ) 0h to maximal one ( ) L in order to be small quantity. 0h is known as the characteristic thickness which is commonly consider the smallest thickness of shell. The main idea of perturbation theory is that perturbation parameter is so small that coefficients of different power of it don’t have the same order which is lead in equality for iε coefficients. Considering each coefficient result in displacement quasi-vector ( ) { } y x . 3.2 Outer expansion The outer expansion of solution is considered a uniform series of ε as ( ) ( ) ( ) ( ) , = + y y y O O1 O2 x x x ε ε ε . The subscript “O” stands for outer solution. Furthermore, the subscript “1” and “2” shows first and second order expansion, respectively. Substituting the expansion in governing equations and considering the terms with the same order of ε , result in the first and second order equations of outer solution. In this section ( ) ( ) ′ = d dx . [ ]{ } { } [ ]{ } { } { } { } { } O( ) : , O( ) : , ′ = = = + A y F A y F F F F 1 2 II O O1 O1 O O 2 O 2 O 2 O 2 O 2 II ε ε (40) (40) [ ]{ } { } [ ]{ } { } { } { } { } O( ) : , O( ) : , ′ = = = + A y F A y F F F F 1 2 II O O1 O1 O O 2 O 2 O 2 O 2 O 2 II ε ε where [ ] { } , A F O O1 and { } FO 2 are coefficient matrices, non-homogeneity vectors of first and second order equation, respectively. { } yOi are unknown displacement vectors in “i”th-order of outer solution. { } FO 2 consist of two vectors { } FO 2 II and { } ′ FO 2 II correspond to ( ) II x and derivative of ( ) II x , respectively. These vectors would be defined in the Appendix A. Other non-zero components of the mentioned matrix and vectors are where [ ] { } , A F O O1 and { } FO 2 are coefficient matrices, non-homogeneity vectors of first and second order equation, respectively. { } yOi are unknown displacement vectors in “i”th-order of outer solution. { } FO 2 consist of two vectors { } FO 2 II and { } ′ FO 2 II correspond to ( ) II x and derivative of ( ) II x , respectively. These vectors would be defined in the Appendix A. 3.1 Perturbation theory ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , , , , , , , , , ˆ , , ,  = = + = + − = + − =  + − = + − = + − = + − =   + − = + − = + − = − =  2 1 2 1 1 2 1 12 1 2 1 1 1 2 1 2 1 2 1 3 1 2 1 4 1 2 1 5 1 2 1 6 1 2 1 7 1 2 1 8 1 2 1 9 s s C C C k C k 2 C C C C 4 C C k C Ck 4 C 2C k C Ck 2 C C 3k C Ck 2 C C k C Ck 4 C C 5k C Ck 4 C C 6k C Ck 8 C 2C 7k C Ck 2 2C 3C k C Ck 2 2C 5C 6k C Ck 1 K K (39) (39) 3.1 Perturbation theory Existence of two boundary layer lead in two region of solution near boundaries (inner expansions) and a solution away from boundaries (outer expansion) (Nayfeh 1981). expansions) and a solution away from boundaries (outer expansion) (Nayfeh 1981). expansions) and a solution away from boundaries (outer expansion) (Nayfeh 1981). In the dimensionless forms, first and second order differential based on x should be rewrite as fo d d d d , d d d d = = 2 2 2 2 2 1 1 x L x x L x (37) d d d d , d d d d = = 2 2 2 2 2 1 1 x L x x L x (37) In shear deformation theory, the differentials and integrations are with respect to x and z , respectively. Therefore, for simplification and abbreviation of representing equations, a dimensionless integral could be defined: In shear deformation theory, the differentials and integrations are with respect to x and z , respectively. Therefore, for simplification and abbreviation of representing equations, a dimensionless integral could be defined: ( ) j i i+ j+1 ( )/ (i, j) (i, j) ( ) d ( )/ + = = + − ∫ 0 h x 2 II II z R x z z h h x 2 (38) ( ) j i i+ j+1 ( )/ (i, j) (i, j) ( ) d ( )/ + = = + − ∫ 0 h x 2 II II z R x z z h h x 2 (38) which is the function of geometrical parameters ( ) R x , ( ) h x and inhomogeneity index n . In order to solve the set of governing differential equations in next steps, the inverse of singular coefficient matrices are needed. Two changes should be applied in equations to replace these matrices with non-singular ones. We take integrate the first equation in the set of Eqs. (32)-(35). The constant of integral ) ( 0c should be either dimensionless as other parameters. Therefore, it is replaced with its dimensionless equivalent i ( ) ( ) = n 0 0 1 c c r C ε where ε is bookkeeping perturbed parameter. Furthermore, as there is no u in governing equations unlike d d u x , we define (d d ) = u x v ε as new unknown intermediate parameter. 3.3 Inner expansion Taylor expansion should be written for the following parameters: Taylor expansion with ( ) Λ = Λ = x α α and d ( ) d = Λ = Λ x D x x α α definition substitutes x by new fast variable xα of inner expansion. 3.2 Outer expansion [ ] [ ] [ ] [ ] [ ] [ ] ( ) [ ] [ ] [ ] [ ] ( ) ( , ), ( , ), ( , ) ( , ) ( , ) , ( , ) ( , ) ( , ), ( , ) ( , ) ( , )  = + = − + = − = −   = − = − + + =− −    = = − − = − + + −  A A A A A A A A A A O O s 12 O O 1 11 22 13 31 O O 1 O 14 41 33 O O 1 O 1 34 43 44 k II 0 n 1 K C II 0 n 1 Ck II 0 n Ck II 1 n II 0 n 1 k II 0 n 1 Ck II 0 n k II 1 n 1 Ck II 1 n k II 0 n 1 II 2 n 1 (42) { } { } { } { } ( ) ( ) ( ) ( ) , , ( ) ( ) , ( )     = = = − − = −           F F F F 2 O1 0 O1 O1 i O1 i 1 2 3 4 h x R x h x h x c 0 P x R x P x 2 2 4 (43) [ ] [ ] [ ] [ ] [ ] [ ] ( ) [ ] [ ] [ ] [ ] ( ) ( , ), ( , ), ( , ) ( , ) ( , ) , ( , ) ( , ) ( , ), ( , ) ( , ) ( , )  = + = − + = − = −   = − = − + + =− −    = = − − = − + + −  A A A A A A A A A A O O s 12 O O 1 11 22 13 31 O O 1 O 14 41 33 O O 1 O 1 34 43 44 k II 0 n 1 K C II 0 n 1 Ck II 0 n Ck II 1 n II 0 n 1 k II 0 n 1 Ck II 0 n k II 1 n 1 Ck II 1 n k II 0 n 1 II 2 n 1 (42) [ ] [ ] [ ] [ ] [ ] [ ] ( ) [ ] [ ] [ ] [ ] ( ) ( , ), ( , ), ( , ) ( , ) ( , ) , ( , ) ( , ) ( , ), ( , ) ( , ) ( , )  = + = − + = − = −   = − = − + + =− −    = = − − = − + + −  A A A A A A A A A A O O s 12 O O 1 11 22 13 31 O O 1 O 14 41 33 O O 1 O 1 34 43 44 k II 0 n 1 K C II 0 n 1 Ck II 0 n Ck II 1 n II 0 n 1 k II 0 n 1 Ck II 0 n k II 1 n 1 Ck II 1 n k II 0 n 1 II 2 n 1 (42) { } { } { } { } ( ) ( ) ( ) ( ) , , ( ) ( ) , ( )     = = = − − = −           F F F F 2 O1 0 O1 O1 i O1 i 1 2 3 4 h x R x h x h x c 0 P x R x P x 2 2 4 { } { } { } { } ( ) ( ) ( ) ( ) , , ( ) ( ) , ( )     = = = − − = −           F F F F 2 O1 0 O1 O1 i O1 i 1 2 3 4 h x R x h x h x c 0 P x R x P x 2 2 4 (43) (43) The solutions of the algebraic equations (40) are as follows: The solutions of the algebraic equations (40) are as follows: { } [ ] { } { } [ ] { } , − − = = y A F y A F 1 1 O1 O O1 O 2 O O 2 (44) { } [ ] { } { } [ ] { } , − − = = y A F y A F 1 1 O1 O O1 O 2 O O 2 { } [ ] { } { } [ ] { } , − − = = y A F y A F 1 1 O1 O O1 O 2 O O 2 (44) 3.3 Inner expansion Taylor expansion should be written for the following parameters: ( ) ( ) , ( ) , , ( ) , ( )  = + = +  =  = + = +          i i i h x h x Dh R x R x DR 0 1 P x P x DP II x II x DII α α α α α α α α α α α α α ε ε α ε ε (48) ( ) ( ) , ( ) , , ( ) , ( )  = + = +  =  = + = +          i i i h x h x Dh R x R x DR 0 1 P x P x DP II x II x DII α α α α α α α α α α α α α ε ε α ε ε (48) Substitution of inner expansion ( ) ( ) ( ) ( ) , = + y y y    1 2 x x x α α α α α α ε ε ε in governing equations with mentioned changes in section 3.1 and considering terms with the same order of ε result in inner equations at boundaryα : Substitution of inner expansion ( ) ( ) ( ) ( ) , = + y y y    1 2 x x x α α α α α α ε ε ε in governing equations with mentioned changes in section 3.1 and considering terms with the same order of ε result in inner equations at boundaryα : [ ] { } [ ] { } [ ]{ } { } [ ] { } [ ] { } [ ]{ } { } { } { } { } { } { } { } d d d d d d d d O( ) : O( ) :  + + =    + + =    = + + + +   A y A y A y F A y A y A y F F F F F F F     2 1 1 1 2 1 3 1 1 2 2 2 1 2 2 2 3 2 2 2 2 2 2 2 2 2 II DA DII P DP x x x x α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ε ε [ ] { } [ ] { } [ ]{ } { } [ ] { } [ ] { } [ ]{ } { } { } { } { } { } { } { } d d d d d d d d O( ) : O( ) :  + + = + + = = + + + +  A y A y A y F A y A y A y F F F F F F F     2 1 1 1 2 1 3 1 1 2 2 2 1 2 2 2 3 2 2 2 2 2 2 2 2 2 II DA DII P DP x x x x α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ε ε (49) (49) [ ] [ ] , A A 1 2 α α and [ ] A 3 α are coefficient matrices at the boundary α . 3.3 Inner expansion As the outer solutions don’t satisfy the B.C., we can conclude existence of boundary layers at , = x 0 1 . Therefore, fast variable ( ) xα should be considered as a new variable for regions around boundaries. Considering fast variables make it possible to measure the great variation of mechanical response around boundaries. ( ) , (left boundary), , (right boundary) − = → = = = → = =   0 1 x 1 x x 0 0 x x 1 1 x α α ε ε (45) ( ) , (left boundary), , (right boundary) − = → = = = → = =   0 1 x 1 x x 0 0 x x 1 1 x α α ε ε ( ) , (left boundary), , (right bound − = → = = = → = =   0 1 x 1 x x 0 0 x x 1 1 x α α ε ε (45) Subscript “ , = 0 1 α ” shows = x α in the boundary. Perturbation parameter appears in new definitions of differential based on fast variables xα : Subscript “ , = 0 1 α ” shows = x α in the boundary. Perturbation parameter appears in new definitions of differential based on fast variables xα : d d d d , d d d d = =   2 2 2 2 2 x x x x α α ε ε (46) d d d d , d d d d = =   2 2 2 2 2 x x x x α α ε ε (46) In cylinder with variable thickness and non-uniform pressure, it is necessary to derive Taylor expansion for all the parameters of axial function ( ) Λ x as: In cylinder with variable thickness and non-uniform pressure, it is necessary to derive Taylor expansion for all the parameters of axial function ( ) Λ x as: ... + Λ + x D α α ε (47) ( ) ... Λ = Λ + Λ +   x x D α α α ε Taylor expansion with ( ) Λ = Λ = x α α and d ( ) d = Λ = Λ x D x x α α definition substitutes x by new fast variable xα of inner expansion. 3.2 Outer expansion Other non-zero components of the mentioned matrix and vectors are { } { } { } { } , , , , , , , = = y y T T O1 O1 O1 O1 O1 O 2 O 2 O 2 O 2 O2 v w v w ϕ ψ ϕ ψ (41) (41) { } { } { } { } , , , , , , , = = y y T T O1 O1 O1 O1 O1 O 2 O 2 O 2 O 2 O2 v w v w ϕ ψ ϕ ψ ds and Structures, 2019, 16(8), e229 Latin American Journal of Solids and Structures, 2019, 16(8), e229 9/28 e hyperelastic FGM cylindrical shells with arbitrary sed on perturbation theory Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. 3.3 Inner expansion (49) are systems of coupled non-homogenous differential equations with constant coefficients. Each equation has general { } ( ) gen. and particular { } ( ) par. solution: Eqs. (49) are systems of coupled non-homogenous differential equations with constant coefficients. Each equation has general { } ( ) gen. and particular { } ( ) par. solution: { } { } { } , = + y y y 2 2 2 gen. par. α α α (55) { } { } { } { } { } { } , = + = + y y y y y y 1 1 1 2 2 2 gen. par. gen. par. α α α α α α (55) { } { } { } { } , = + y y y y 1 2 2 2 par. gen. par. α α α α Considering mα and { } Vα as eigenvalues and eigenvectors, respectively, and substituting general solution of exponential form { } { } ) = y  gen. V exp(m x α α α α in homogenous part of Eqs. (49) lead in an eigenvalue problem: Considering mα and { } Vα as eigenvalues and eigenvectors, respectively, and substituting general solution of exponential form { } { } ) = y  gen. V exp(m x α α α α in homogenous part of Eqs. (49) lead in an eigenvalue problem: [ ] [ ] [ ] ( ){ } { } + + = A A A 2 1 2 3 m m V 0 α α α α α α (56) (56) [ ] [ ] [ ] ( ){ } { } + + = A A A 2 1 2 3 m m V 0 α α α α α α The necessary condition for existing the solution of Eq. (56) is zero value of the coefficient determinant which is the characteristic equation of the system. Six non-zero roots of it are the eigenvalues ( ) i mα . Substituting roots in Eq. (56) lead in corresponding eigenvectors ( ) i Vα . The eigenvalues and eigenvectors are complex conjugate. Considering Van-Dyke’s matching principle (Nayfeh 1981), the solution should be finite at →∞ xα . 3.3 Inner expansion { } F 1 α and { } F 2 α are non-homogeneity vectors of differential equation at the first and second order equation for the boundary α , respectively. Unknown displacement ids and Structures, 2019, 16(8), e229 10 Latin American Journal of Solids and Structures, 2019, 16(8), e229 10/28 sible hyperelastic FGM cylindrical shells with arbitrary e based on perturbation theory Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. vectors in “i”th-order of inner solution at the boundary α are { } y i α .{ } F 2 IIα α and { } F 2 DIIα α , in{ } F 2 α , are correspond with ( ) II x α and ( ) DII x α (in Taylor expansion), respectively. { } F 2 DAα α derive in non-homogeneity of second order equation which resulting from Tayler expansion of coefficient matrices in first order equation because of variable thickness. { } F 2 Pα α and { } F 2 DPα α , include ( )  iP x α α and ( )  i DP x α α (in Taylor expansion), are resulting from variable thickness and non-uniform pressure, respectively. Considering too many terms, { } F 2 α vector would be defined in the Appendix A. Other non-zero components of the matrices and vectors defined below. In this section ( ) ( ) ′ =  d dxα at the boundary α . 3.3 Inner expansion { } { } { } { } , , , , , , , = = y y T T 1 1 1 1 1 2 2 2 2 2 v w v w α α α α α α α α α α ϕ ψ ϕ ψ (50) [ ] [ ] [ ] [ ] [ ] ( , ), ( , ) ( , ), ( , )  = + = +  = = + = +  A A A A A 1 1 s 12 22 33 1 1 s 12 1 s 12 34 43 44 k II 2 n 1 K C II 0 n 1 K C II 1 n 1 K C II 2 n 1 α α α α α α α α α (51) { } { } { } { } , , , , , , , = = y y T T 1 1 1 1 1 2 2 2 2 2 v w v w α α α α α α α α α α ϕ ψ ϕ ψ } { } { } { } , , , , , , , = = y y T T 1 1 1 1 1 2 2 2 2 2 v w v w α α α α α α α α α α ϕ ψ ϕ ψ (50) [ ] [ ] [ ] [ ] [ ] ( , ), ( , ) ( , ), ( , )  = + = +  = = + = +  A A A A A 1 1 s 12 22 33 1 1 s 12 1 s 12 34 43 44 k II 2 n 1 K C II 0 n 1 K C II 1 n 1 K C II 2 n 1 α α α α α α α α α (51) [ ] ( , ) = + A 1 s 12 33 K C II 0 n 1 α α (51) [ ] [ ] [ ] [ ] [ ] ( , ), ( , ) ( , ), ( , )  = + = +  = = + = +  A A A A A 1 1 s 12 22 33 1 1 s 12 1 s 12 34 43 44 k II 2 n 1 K C II 0 n 1 K C II 1 n 1 K C II 2 n 1 α α α α α α α α α (51) [ ] [ ] [ ] [ ] [ ] ( , ), ( , )  = = + = + A A A 1 1 s 12 22 33 1 1 s 12 1 s 12 34 43 44 K C II 1 n 1 K C II 2 n 1 α α α α α α α α α (51) [ ] ( , ), ( , ) + = + A 2 1 s 12 44 II 1 n 1 K C II 2 n 1 α α α (51) [ ] [ ] [ ] [ ] [ ] [ ] ( ) ( , ), ( , ) ( , ) ( , ) ( , ) ( , )  = = + = − = − + −  = − = − + − + +  A A A A A A 2 2 2 2 s 12 1 12 21 23 32 2 2 s 12 1 24 42 k II 1 n 1 K C II 0 n 1 Ck II 1 n K C II 1 n 1 Ck II 2 n II 1 n 1 α α α α α α α α α α α α (52) (52) [ ] [ ] [ ] [ ] [ ] [ ] ( ) [ ] [ ] [ ] [ ] ( ) ( , ), ( , ), ( , ) ( , ) ( , ) , ( , ) ( , ) ( , ), ( , ) ( , ) ( , )  = + =− + = − = − = − = − + + =− −  = = − − = − + + − A A A A A A A A A A 3 3 s 12 3 3 1 11 22 13 31 3 3 1 3 14 41 33 3 3 1 3 1 34 43 44 k II 0 n 1 K C II 0 n 1 Ck II 0 n Ck II 0 n 1 II 1 n k II 0 n 1 Ck II 0 n k II 1 n 1 2Ck II 1 n k II 0 n 1 II 2 n 1 α α α α α α α α α α α α α α α α α α α α α     (53) [ ] [ ] [ ] [ ] [ ] [ ] ( ) [ ] [ ] [ ] [ ] ( ) ( , ), ( , ), ( , ) ( , ) ( , ) , ( , ) ( , ) ( , ), ( , ) ( , ) ( , )  = + =− + = − = − = − = − + + =− −  = = − − = − + + − A A A A A A A A A A 3 3 s 12 3 3 1 11 22 13 31 3 3 1 3 14 41 33 3 3 1 3 1 34 43 44 k II 0 n 1 K C II 0 n 1 Ck II 0 n Ck II 0 n 1 II 1 n k II 0 n 1 Ck II 0 n k II 1 n 1 2Ck II 1 n k II 0 n 1 II 2 n 1 α α α α α α α α α α α α α α α α α α α α α     (53) (53) { } { } { } { } , , ,     = = = − − = −           F F F F 2 1 0 1 1 i 1 i 1 2 3 4 h R h h c 0 P R P 2 2 4 α α α α α α α α α α α (54) (54) Eqs. 3.3 Inner expansion Therefore, in left boundary ( ) = 0 α eigenvalues with positive real part and in right boundary ( ) = 1 α eigenvalues with negative real part are omitted. The general solution of the boundary α could be calculated. { } { } { } { } ) = = = = ∑ y y y  3 1 2 i i i gen. gen. gen. i 1 c V exp(m x α α α α α α α (57) { } { } { } { } ) = = = = ∑ y y y  3 1 2 i i i gen. gen. gen. i 1 c V exp(m x α α α α α α α (57) where ( , , ) = i c i 1 2 3 α are three constant at each boundary which could be calculated by boundary conditions. The particular solution of first order equation in Eq. (49) is simply calculated by { } [ ] { } − = y A F 1 1 3 1 par. α α α . As { } F 2 α is consist of nonlinear Latin American Journal of Solids and Structures, 2019, 16(8), e229 11/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. polynomial terms { } ( ) pol. and exponential terms by the equal roots ))  i (exp(m x α α and unequal roots ))  j (exp(q x α α with characteristic equations based on O( ) 1 ε solution, the particular solution of O( ) 2 ε is calculated by undetermined coefficients method as follows: polynomial terms { } ( ) pol. 3.4 Composite solution In the MAE method, the composite solution { } ( ) ycomp. is the summation of three calculated expansions (one outer { } yO and two inner{ } { } , = = y y 0 1 α α ) minus the overlapped parts of them. Outer solution at , → x 0 1 and inner solutions at →±∞ xα are overlapped and these common parts have to be removed from composite solution. Therefore, { } { } { } { } { } { } = = = = = + + − − y y y y y y 0 1 comp. O 0 1 O O α α α α (59) (59) { } { } { } { } { } { } = = = = = + + − − y y y y y y 0 1 comp. O 0 1 O O α α α α where { } = y 0 O α and { } = y 1 O α are common parts of inner and outer solutions at two ends of the shell which can be determined by definition of intermediate variable or Van-Dyke’s matching principle (Nayfeh 1981). Eight constants, consist of three constants in general solution of each boundary and two constants 0c and 7c , should be calculated by the boundary conditions. The clamped boundary conditions in “i”th-order perturbation solution are: where { } = y 0 O α and { } = y 1 O α are common parts of inner and outer solutions at two ends of the shell which can be determined by definition of intermediate variable or Van-Dyke’s matching principle (Nayfeh 1981). Eight constants, consist of three constants in general solution of each boundary and two constants 0c and 7c , should be calculated by the boundary conditions. The clamped boundary conditions in “i”th-order perturbation solution are: (60) ( ) , i i i i , , , , i =1,2 = = → = 0 1 x 0 1 x 0 u w 0 ( ) α α α α ϕ ψ Finally, the unknown vector{ } { } { } , , , = = y ycomp. u w ϕ ψ which consists of dimensionless displacement field components would be obtained in terms of x and z variables. Considering Eq. 3.4 Composite solution (4) and , , = z x z x 0 U U h , the dimensionless radial and axial displacements can be calculated. Using Eqs. (5-9) would yield [ ] [ ] , , , , , F C 1 2 3 I J and [ ] E , respectively. The hydrostatic pressure, strain energy function and second Piola–Kirchhoff stress distribution could be calculated by using Eqs. (14), (18) and (19). The relation [ ] ( )[ ][ ][ ] = σ F S F T 1 J would result in Cauchy stress components. The analytical solution has been carried out by writing the program in MAPLE 18 software. 3.3 Inner expansion and exponential terms by the equal roots ))  i (exp(m x α α and unequal roots ))  j (exp(q x α α with characteristic equations based on O( ) 1 ε solution, the particular solution of O( ) 2 ε is calculated by undetermined coefficients method as follows: { } { } { } { } { } { } { } { } { } { } { } { } ( ) { } { } { } { } ( ) mi mi mi q j q j q j . exp(mi) exp(q j) . . . . . exp(mi) . exp(q ) . , ) )  = + + = + +   = + +    = + +  ∑ ∑ y y y y y y y         2 2 2 2 2 2 2 1 0 2 2 2 1 0 i i 2 2 2 1 0 j j pol pol par. par par par par par j par B x B x B B x B x B exp(m x B x B x B exp(q x α α α α α α α α α α α (58) (58) where subscript “i” and “j” shows the number of equal and unequal roots with characteristic equations. Substituting { } y 2 par. α in Eq. (49) - O( ) 2 ε lead in undetermined coefficients { } 1 B ,{ } 1 B and { } 2 B . where subscript “i” and “j” shows the number of equal and unequal roots with characteristic equations. Substituting { } y 2 par. α in Eq. (49) - O( ) 2 ε lead in undetermined coefficients { } 1 B ,{ } 1 B and { } 2 B . Latin American Journal of Solids and Structures, 2019, 16(8), e229 4.2 Case studies In order to illustrate the effects of material constant, gradient index, pressure loading distribution and thickness profile type on the mechanical behavior of hyperelastic pressure vessel, thick cylinders with various non-uniform thickness and internal pressure profiles made of different materials constants and inhomogeneity index have been considered. In these example problems, unless noted otherwise, we take constant geometry parameters as: mm = ir 47 and mm = L 400 . Clamped boundary conditions are applying in two ends of the shells. The materials are assumed functionally graded hyperelastic with tow-term Mooney-Rivlin model in nearly incompressible conditions. During the computation of numerical results, different material contestants according to various references are considered which have been presented in Table 1. The material constants are considered based on two state: 1) mentioned directly in these articles or 2) computed by authors of current paper from different test results (mentioned in these references). The constants of FG Mooney-Rivlin model are considered for material properties at the internal surface. Considering the values of 1 C and 2 C in Table 1 and Eq. (15) result the variation range of k about − 1 100 MPa in nearly incompressible limit. Important remark is that for < k 2 MPa , the eigenvalues of characteristic equation no longer have conjugate complex form and MAE solution diverge. Hence, in Table 1, we set = k 10 MPa for cases with no incompressibility parameter presentation (Dias et al. 2014, Kiendl et al. 2015). Internal pressure distribution and thickness profile varies non-uniformly along axial direction of shell. Various pressure profiles are applied to the cylindrical shell in the range of kPa kPa − 5 13 . Dimensionless Cauchy stresses and hydrostatic pressure are defined as: (61) i0 P is considered kPa 9 which is the average of maximum and minimum pressure distributions. The variations of thickness profiles are in the range of mm mm − 6 12 . Tables 2 and 3 show the characteristic of applied non-uniform pressure and thickness profiles, respectively. Table 1: The characteristic of material constants. Table 1: The characteristic of material constants. Material Constants ID Reference (MPa) 1 C (MPa) 2 C (MPa) k Material Sample MC1 Selvadurai and Shi (2012) 0.242 0.142 10 Natural gum rubbers MC2 Montella et al. 4.1 FE modeling In order to validate presented analytical solution and compare the results for pressurized thick cylinder with variable thickness made of nearly compressible FG hyperelastic material, a numerical solution based on Finite Element Method is investigated. The ANSYS 16 package is used in the static analysis of thick hollow cylinder with variable thickness under non-uniform internal pressure. The PLANE183 element in the axisymmetric mode, which is an element with eight nodes and two translational degrees of freedom in the axial and radial directions per each node, has been used to model the mechanical part of the analysis. It also has mixed formulation capability for simulating deformations of nearly incompressible hyperelastic material. The cylinder with variable thickness consists of some coherent homogeneous layers which properties at the contact location of the layers are the average of left and right limit of two layer boundaries. In order to model FG hyperelastic cylinder, an innovative application for multilayering the thickness in the axial direction has been performed. Homogenous layers which have identical thickness and step-variable properties have been formed by this method. In order to consider Mooney-Rivlin elastic model in nearly incompressible condition in each homogenous layer, three constants involving , 10 01 C C and d should be defined for ANSYS software. 10 C and 01 C corresponding to 1n C and Latin American Journal of Solids and Structures, 2019, 16(8), e229 12/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. 2n C could be calculated from Eq. (12) in each layer. d is material incompressibility parameter by relation with bulk modulus as . = n k d 2 (Montella et al. 2014, Gao et al. 2009) which could be calculated from Eq. (16) in each layer. For non- uniform internal pressure, the pressure functions have been defined and applied to the internal layer nodes. Clamped boundary conditions have been exerted by preventing the nodes around the two ends of the cylinder from movement. In the next sections, the numerical results (FEM) and analytical results (MAE) have been investigated for different case studies. 2n C could be calculated from Eq. (12) in each layer. 4.1 FE modeling d is material incompressibility parameter by relation with bulk modulus as . = n k d 2 (Montella et al. 2014, Gao et al. 2009) which could be calculated from Eq. (16) in each layer. For non- uniform internal pressure, the pressure functions have been defined and applied to the internal layer nodes. Clamped boundary conditions have been exerted by preventing the nodes around the two ends of the cylinder from movement. In the next sections, the numerical results (FEM) and analytical results (MAE) have been investigated for different case studies. 4.2 Case studies Table 3: The characteristic of non-uniform thickness profiles. Table 3: The characteristic of non-uniform thickness profiles. Table 3: The characteristic of non-uniform thickness profiles. Thickness Profile ID Thickness Profile Relation Geometry Constants 0h ( ) constant = = 0 h x h mm = 0h 6 1h ( ) ( ) = − − a a b h x h h h x mm, mm = = a b h 12 h 6 2 h ( ) ( ) = − − 2 a a b h x h h h x mm, mm = = a b h 12 h 6 3 h ( ) ( ) = − − 3 a a b h x h h h x mm, mm = = a b h 12 h 6 4 h ( )( ) ( ) = − − − 2 a a ab h x h 4 h h x x mm, mm = = = a b ab h h 6 h 12 5 h ( ) ( ) ( ) sin = − − a a ab h x h h h x π mm, mm = = = a b ab h h 12 h 6 As current research studies the manner of pressurized rubber vessels in dimensionless state, the results of FSDT and MAE solution may be suitable for investigating some case studies of blood vessels. In particular, we use an analysis to examine the inflation of a cylindrical tube at various internal pressure profiles and to compute the evolution of the inner radius (critical layer) with the internal pressure. Although the material models of blood vessels such as arteries have commonly exponential form to model the stiffening of the soft tissues, simple model such as neo-Hookean and/or Mooney-Rivlin one is considered in some research for isotropic parts of blood vessels (Holzapfel and Gasser 2007, Lally et al. 2005). Hence, case studies of variable thickness and pressure are selected similar to that of common elastic arteries (as unite layer) in current research in order to cover pressure vessels to blood vessels. The thick and thin part of the vessels covers the average thickness of the layers in common elastic arteries. The pressure profiles vary in the range of kPa( mmHg) kPa( mmHg) − 5 40 13 100 which are the mean blood vessel pressures of human soft tissues. 4.2 Case studies mmHg 100 is the mean of systolic/diastolic pressure and mmHg 40 may be occurred in hypotension pressure of arteries (Abdessamad et al. 2018, Humphrey and O’Rourke, 2015). Considering the applicability of the rubber elasticity theory to aortic soft tissues as one layer or multilayer vessel with variable material properties along thickness, the behavior of blood vessels under non- uniform pressure distribution has been investigated from current research. Furthermore, current study will present helpful results for estimating the wall degeneration of arteries within the aneurysm wall that affects the thickness profile of the tissue, which can be mostly analyzed as variable thickness blood vessels (Xie et al. 1995, Vossoughi and Tozeren 1998, Holzapfel and Gasser 2007). 4.2 Case studies (2014) 0.251 0.263 36 TDM 600 MC3 Sussman and Bathe (1987) 0.177 0.045 10 Rubber bushes MC4 Boyce and Arruda (2000) 0.16 0.015 10 Rubbers MC5 Batra and Bahrami (2009) 0.1858 -0.01935 10 Rubberlike materials MC6 Dias et al. (2014) 0.423 -0.262 10.5 Silicone elastomers MC7 Vossoughi and Tozeren (1998) 0.09 0.118 10 polyurethane rubbers MC8 Kiendl et al. (2015) 0.1848 0.0264 10 Rubbers MC9 Tanveer and Zu (2012) 0.552 0.138 10 Rubber seals 13/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Table 2: The characteristic of non-uniform internal pressure profiles. Pressure Profile ID Pressure Profile Relation Load Constants i0 P i i ( ) constant = = 0 P x P i kPa = 0 P 9 i1 P ( ) i( ) = − − a a b P x P P P x kPa, kPa = = a b P 13 P 5 i2 P ( ) i( ) = − − 2 a a b P x P P P x kPa, kPa = = a b P 13 P 5 i3 P ( ) i( ) = − − 3 a a b P x P P P x kPa, kPa = = a b P 13 P 5 i4 P ( )( ) i( ) = − − − 2 a a ab P x P 4 P P x x kPa, kPa = = = a b ab P P 5 P 13 i5 P ( ) ( ) i( ) sin = − − a a ab P x P P P x π kPa, kPa = = = a b ab P P 13 P 5 Table 3: The characteristic of non-uniform thickness profiles. 4.2 Case studies Thickness Profile ID Thickness Profile Relation Geometry Constants 0h ( ) constant = = 0 h x h mm = 0h 6 1h ( ) ( ) = − − a a b h x h h h x mm, mm = = a b h 12 h 6 2 h ( ) ( ) = − − 2 a a b h x h h h x mm, mm = = a b h 12 h 6 3 h ( ) ( ) = − − 3 a a b h x h h h x mm, mm = = a b h 12 h 6 4 h ( )( ) ( ) = − − − 2 a a ab h x h 4 h h x x mm, mm = = = a b ab h h 6 h 12 5 h ( ) ( ) ( ) sin = − − a a ab h x h h h x π mm, mm = = = a b ab h h 12 h 6 Table 2: The characteristic of non-uniform internal pressure profiles. Pressure Profile ID Pressure Profile Relation Load Constants i0 P i i ( ) constant = = 0 P x P i kPa = 0 P 9 i1 P ( ) i( ) = − − a a b P x P P P x kPa, kPa = = a b P 13 P 5 i2 P ( ) i( ) = − − 2 a a b P x P P P x kPa, kPa = = a b P 13 P 5 i3 P ( ) i( ) = − − 3 a a b P x P P P x kPa, kPa = = a b P 13 P 5 i4 P ( )( ) i( ) = − − − 2 a a ab P x P 4 P P x x kPa, kPa = = = a b ab P P 5 P 13 i5 P ( ) ( ) i( ) sin = − − a a ab P x P P P x π kPa, kPa = = = a b ab P P 13 P 5 Table 2: The characteristic of non-uniform internal pressure profiles. Table 3: The characteristic of non-uniform thickness profiles. Table 3: The characteristic of non-uniform thickness profiles. Latin American Journal of Solids and Structures, 2019, 16(8), e229 4.3 Effect of material constants and inhomogeneity index Ascending R lead in higher deformations and lower effect of clamped conditions on deformations and the peak points of displacements occurs far away from boundaries. An increase in i ( ) + 1 2 P C C and k or decrease in + 1 2 C C ascend the nonlinearity and descend the accuracy. shear deformation approximation cylinders lead in less accuracy for very thick (Eipakchi et al. 2003). Ascending R lead in higher deformations and lower effect of clamped conditions on deformations and the peak points of displacements occurs far away from boundaries. An increase in i ( ) + 1 2 P C C and k or decrease in + 1 2 C C ascend the nonlinearity and descend the accuracy. The longitudinal and circumferential components of Green–Lagrange strain are depicted in Figure 2 for homogeneous cylinder. According to Figure 2(a), the longitudinal strain resulted from numerical and analytical solution show good agreement along axial direction of different layers. It can be seen that maximum longitudinal strain occurs around the boundaries of external layer. However, the position of extremum strains could be different for low range of loading. At the middle of the cylinder away from boundaries, no significant difference of longitudinal strain is observed along the thickness. As the maximum values of circumferential strain occur at the middle of the cylinder ( ) . = x 0 5 , distribution of this strain component is shown along thickness of cylinder for different loading in Figure 2(b). Ascending the internal pressure causes decrease in accuracy of analytical strain especially around the loading layer with higher strain (internal layer). FSDT have acceptable accuracy for indirect calculation of strain components from displacements. Table 4: Analytical and numerical values of maximum radial displacements for different material constants. Mat. 4.3 Effect of material constants and inhomogeneity index ID z max , U i 1 2 P (C + C ) = 1 50 i 1 2 P (C + C ) = 1 100 i 1 2 P (C + C ) = 1 200 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 MC1 FSDT 0.0734 0.2991 1.2127 0.0363 0.1481 0.5943 0.0182 0.0736 0.2952 FEM 0.0759 0.3047 1.2699 0.0370 0.1488 0.5970 0.0184 0.0737 0.2930 MC2 FSDT 0.0691 0.2859 1.1754 0.0339 0.1410 0.5760 0.0169 0.0700 0.2812 FEM 0.0736 0.2913 1.2210 0.0359 0.1407 0.5746 0.0178 0.0706 0.2780 MC3 FSDT 0.0701 0.2917 1.1863 0.0347 0.1437 0.5810 0.0173 0.0714 0.2876 FEM 0.0730 0.2957 1.2319 0.0358 0.1442 0.5812 0.0180 0.0717 0.2847 MC4 FSDT 0.0685 0.2876 1.1771 0.0340 0.1421 0.5763 0.0169 0.0706 0.2861 FEM 0.0726 0.2921 1.2234 0.0356 0.1430 0.5766 0.0179 0.0712 0.2830 MC5 FSDT 0.0681 0.2869 1.1762 0.0338 0.1417 0.5760 0.0168 0.0704 0.2855 FEM 0.0725 0.2930 1.2214 0.0349 0.1419 0.5742 0.0177 0.0710 0.2824 MC6 FSDT 0.0676 0.2832 1.1371 0.0336 0.1408 0.5682 0.0167 0.0702 0.2836 FEM 0.0722 0.2902 1.1780 0.0362 0.1421 0.5683 0.0178 0.0708 0.2812 MC7 FSDT 0.0696 0.2902 1.1871 0.0345 0.1433 0.5810 0.0172 0.0712 0.2875 FEM 0.0743 0.2969 1.2375 0.0360 0.1440 0.5813 0.0181 0.0716 0.2850 MC8 FSDT 0.0697 0.2899 1.1861 0.0346 0.1425 0.5798 0.0172 0.0712 0.2875 FEM 0.0742 0.2970 1.2360 0.0365 0.1440 0.5799 0.0180 0.0716 0.2846 Table 5: Analytical and numerical values of maximum axial displacements for different material constants. Mat. 4.3 Effect of material constants and inhomogeneity index ID z max , U i 1 2 P (C + C ) = 1 50 i 1 2 P (C + C ) = 1 100 i 1 2 P (C + C ) = 1 200 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 MC1 FSDT 0.0734 0.2991 1.2127 0.0363 0.1481 0.5943 0.0182 0.0736 0.2952 FEM 0.0759 0.3047 1.2699 0.0370 0.1488 0.5970 0.0184 0.0737 0.2930 MC2 FSDT 0.0691 0.2859 1.1754 0.0339 0.1410 0.5760 0.0169 0.0700 0.2812 FEM 0.0736 0.2913 1.2210 0.0359 0.1407 0.5746 0.0178 0.0706 0.2780 MC3 FSDT 0.0701 0.2917 1.1863 0.0347 0.1437 0.5810 0.0173 0.0714 0.2876 FEM 0.0730 0.2957 1.2319 0.0358 0.1442 0.5812 0.0180 0.0717 0.2847 MC4 FSDT 0.0685 0.2876 1.1771 0.0340 0.1421 0.5763 0.0169 0.0706 0.2861 FEM 0.0726 0.2921 1.2234 0.0356 0.1430 0.5766 0.0179 0.0712 0.2830 MC5 FSDT 0.0681 0.2869 1.1762 0.0338 0.1417 0.5760 0.0168 0.0704 0.2855 FEM 0.0725 0.2930 1.2214 0.0349 0.1419 0.5742 0.0177 0.0710 0.2824 MC6 FSDT 0.0676 0.2832 1.1371 0.0336 0.1408 0.5682 0.0167 0.0702 0.2836 FEM 0.0722 0.2902 1.1780 0.0362 0.1421 0.5683 0.0178 0.0708 0.2812 MC7 FSDT 0.0696 0.2902 1.1871 0.0345 0.1433 0.5810 0.0172 0.0712 0.2875 FEM 0.0743 0.2969 1.2375 0.0360 0.1440 0.5813 0.0181 0.0716 0.2850 MC8 FSDT 0.0697 0.2899 1.1861 0.0346 0.1425 0.5798 0.0172 0.0712 0.2875 FEM 0.0742 0.2970 1.2360 0.0365 0.1440 0.5799 0.0180 0.0716 0.2846 Table 5: Analytical and numerical values of maximum axial displacements for different material constants and numerical values of maximum radial displacements for different material constants. Table 4: Analytical and numerical values of maximum radial displacements for different materia Table 5: Analytical and numerical values of maximum axial displacements for different material constants. Table 5: Analytical and numerical values of maximum axial displacements for different material constants. Mat. 4.3 Effect of material constants and inhomogeneity index In order to investigate the material constants effect on the approximation of the current solution and behaviour of shell, maximum values of radial and axial displacements for different material constants resulted from FSDT and FEM is presented in Tables 4 and 5. As the maximum values of displacements and maximum difference of analytical and numerical analysis in pressure vessels occur at the internal layer, the results of Tables 4 and 5 define the validity range of the current solution. Material constants, applied pressures and geometry of the cylinder are considered in the range of < < 5 R 20 and ( ) i / / < + < 1 2 1 200 P C C 1 50 (Batra and Bahrami 2009, Humphrey and O’Rourke 2015, Azar et al. 2018). The difference percentage of dimensionless radial and axial displacement resulting from the numerical and analytical solution i.e. ( ) MAE FEM FEM ,x , , , Diff (%) = − × z z x z x z x U U U U 100 for mentioned R and i ( ) + 1 2 P C C are less than 8%. R values of more than 20 (thickness limit for thick cylinder) and less than 4 (very thick shells) may lead in decrease in solution accuracy. It is observed that the accuracy of MAE descend for great values of R because of intensifying nonlinear behavior of the cylinder while for small R , the accuracy of shear deformation theory decrease in analyzing thick cylindrical shells. The main reason of increasing the difference between results of shear deformation theory and other theories (finite element or plane elasticity) for small R are the effect of domination of thickness values to displacement ones which make linear (or even more order) distribution of displacements along the thickness of cylinder not to be matched to real state; so 14/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 Gharooni et al. Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory shear deformation approximation cylinders lead in less accuracy for very thick (Eipakchi et al. 2003). 4.3 Effect of material constants and inhomogeneity index ID x max U , i 1 2 P (C + C ) = 1 50 i 1 2 P (C + C ) = 1 100 i 1 2 P (C + C ) = 1 200 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 MC1 FSDT 0.0147 0.0453 0.1096 0.0073 0.0231 0.0623 0.0037 0.0117 0.0331 FEM 0.0151 0.0469 0.1089 0.0076 0.0242 0.0621 0.0038 0.0125 0.0332 MC2 FSDT 0.0138 0.0453 0.1162 0.0069 0.0227 0.0634 0.0035 0.0113 0.0332 FEM 0.0149 0.0460 0.1080 0.0073 0.0239 0.0611 0.0037 0.0120 0.0333 MC3 FSDT 0.0138 0.0445 0.1139 0.0069 0.0226 0.0628 0.0034 0.0114 0.0329 FEM 0.0149 0.0452 0.1068 0.0075 0.0239 0.0611 0.0037 0.0121 0.0331 MC4 FSDT 0.0136 0.0445 0.1148 0.0068 0.0225 0.0630 0.0034 0.0113 0.0329 FEM 0.0148 0.0457 0.1069 0.0075 0.0237 0.0603 0.0037 0.0122 0.0332 MC5 FSDT 0.0135 0.0441 0.1135 0.0068 0.0224 0.0626 0.0034 0.0113 0.0327 FEM 0.0148 0.0456 0.1050 0.0073 0.0239 0.0602 0.0037 0.0121 0.0329 MC6 FSDT 0.0134 0.0408 0.0960 0.0066 0.0216 0.0584 0.0034 0.0111 0.0316 FEM 0.0145 0.0432 0.0973 0.0073 0.0224 0.0587 0.0037 0.0121 0.0318 MC7 FSDT 0.0139 0.0454 0.1166 0.0069 0.0228 0.0640 0.0034 0.0114 0.0332 FEM 0.0150 0.0466 0.1085 0.0075 0.0232 0.0613 0.0038 0.0123 0.0335 MC8 FSDT 0.0137 0.0444 0.1134 0.0068 0.0226 0.0628 0.0034 0.0114 0.0329 FEM 0.0149 0.0460 0.1063 0.0075 0.0238 0.0610 0.0037 0.0123 0.0328 Table 4: Analytical and numerical values of maximum radial displacements for different material constants. Mat. Latin American Journal of Solids and Structures, 2019, 16(8), e229 4.3 Effect of material constants and inhomogeneity index , ( ) , MC = = 0 0 5 P x 1 5P h x h ) and (b) circumferential strain distribution along thickness for different pressure ( . , ( ) , MC = = 0 4 x 0 5 h x h ). In order to show the displacements and stresses distribution in FG hyperelastic cylinder with variable thickness under non-uniform internal pressure, linear increase of thickness counter x direction is considered proportional to linear pressure profile with positive inhomogeneity index ( ) = n 2 . The constants of FG Mooney-Rivlin model for rubber are considered as 9 MC . Figure 3 illustrate displacement contour resulted from FE modeling in 3/4 expansion of the shell section. The distribution of dimensionless displacements resulted from FSDT and FEM are plotted in Figure 4 at different layers. Boundary conditions and radial direction of applying pressure cause < x z U U in the current case study. Because of greater radial displacements around left boundary, it can be concluded that pressure profile is more effective than thickness variation on shell displacement. According to Figure 4(b), the axial displacement at points away from the boundaries is nearly independent from radius. Layers close to maximum pressure and clamped conditions are in axial tension; therefore, axial compression is dominants at the shell except in ≤ z 0 around left boundary. Figure 5 show dimensionless Cauchy stresses and hydrostatic pressure at different layers along axial direction. Hydrostatic pressure can be considered as average of principal stresses. Considering Figure 5 confirm this fact; so hydrostatic pressure can be a suitable equivalent parameter that show shell state from the view point of stresses. Circumferential and axial stresses, similar to hydrostatic pressure, have positive values in nearly all points of the shell except around boundaries at the outer layer away from loading. The reason is that the elements are in tensile state, but clamped conditions near boundaries at the layer away from loading cause resistance against displacement which lead in compressive stresses. In this state, inner layer of the shell in contact with pressure load have higher displacements and stresses than others. It is obviously observed that the circumferential stress is the largest component of the stress at points away the boundaries while at the points near the boundaries, the axial stress is the largest one. 4.3 Effect of material constants and inhomogeneity index ID x max U , i 1 2 P (C + C ) = 1 50 i 1 2 P (C + C ) = 1 100 i 1 2 P (C + C ) = 1 200 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 R = 5 R = 10 R = 20 MC1 FSDT 0.0147 0.0453 0.1096 0.0073 0.0231 0.0623 0.0037 0.0117 0.0331 FEM 0.0151 0.0469 0.1089 0.0076 0.0242 0.0621 0.0038 0.0125 0.0332 MC2 FSDT 0.0138 0.0453 0.1162 0.0069 0.0227 0.0634 0.0035 0.0113 0.0332 FEM 0.0149 0.0460 0.1080 0.0073 0.0239 0.0611 0.0037 0.0120 0.0333 MC3 FSDT 0.0138 0.0445 0.1139 0.0069 0.0226 0.0628 0.0034 0.0114 0.0329 FEM 0.0149 0.0452 0.1068 0.0075 0.0239 0.0611 0.0037 0.0121 0.0331 MC4 FSDT 0.0136 0.0445 0.1148 0.0068 0.0225 0.0630 0.0034 0.0113 0.0329 FEM 0.0148 0.0457 0.1069 0.0075 0.0237 0.0603 0.0037 0.0122 0.0332 MC5 FSDT 0.0135 0.0441 0.1135 0.0068 0.0224 0.0626 0.0034 0.0113 0.0327 FEM 0.0148 0.0456 0.1050 0.0073 0.0239 0.0602 0.0037 0.0121 0.0329 MC6 FSDT 0.0134 0.0408 0.0960 0.0066 0.0216 0.0584 0.0034 0.0111 0.0316 FEM 0.0145 0.0432 0.0973 0.0073 0.0224 0.0587 0.0037 0.0121 0.0318 MC7 FSDT 0.0139 0.0454 0.1166 0.0069 0.0228 0.0640 0.0034 0.0114 0.0332 FEM 0.0150 0.0466 0.1085 0.0075 0.0232 0.0613 0.0038 0.0123 0.0335 MC8 FSDT 0.0137 0.0444 0.1134 0.0068 0.0226 0.0628 0.0034 0.0114 0.0329 FEM 0.0149 0.0460 0.1063 0.0075 0.0238 0.0610 0.0037 0.0123 0.0328 Latin American Journal of Solids and Structures, 2019, 16(8), e229 15/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Figure 2: (a) Longitudinal strain distribution along axial direction at different layers ( i i ( ) . , ( ) , MC = = 0 0 5 P x 1 5P h x h ) and (b) circumferential strain distribution along thickness for different pressure ( . , ( ) , MC = = 0 4 x 0 5 h x h ). Figure 2: (a) Longitudinal strain distribution along axial direction at different layers ( i i ( ) . Latin American Journal of Solids and Structures, 2019, 16(8), e229 4.3 Effect of material constants and inhomogeneity index Existence of shear stress near boundaries reveals the advantage of shear deformation theory respect to theories that neglect shear stress effect (Figure 5(c)). Getting away from boundaries, non-uniform peaks of displacements and stresses are observed at the points where shear stresses tend to zero values. Difference between MAE and FEM results increase at the points of internal and external layers away from boundaries. Although FSDT is suitable for displacement analyzing rather than stress one, the results of MAE are more realistic around boundaries respect to FE solution. Considering Eq. (14) and hydrostatic pressure distribution lead in . . < < 0 993 J 1 010 . Dimensionless radial Cauchy stress distribution in middle layer resulted from first and second order MAE solution are depicted in Figure 6. O( ) 1 ε solution is suitable for linear analysis while O( ) 2 ε is suitable for nonlinear problems. Figure 6 shows that second order solution improves results accuracy respect to first order one, especially in the current research that kinematics and constitutive relations are highly nonlinear. 16/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Figure 3: Contour plot of (a) radial and (b) axial displacement resulted from FEM in ¾ expansion of shell section ( i i1 1 9 , ( ) , ( ) , MC = = = n 2 P x P h x h ). Figure 4: Dimensionless (a) radial and (b) axial displacement distribution along axial direction at different layers Figure 3: Contour plot of (a) radial and (b) axial displacement resulted from FEM in ¾ expansion of shell section ure 3: Contour plot of (a) radial and (b) axial displacement resulted from FEM in ¾ expansion of shell section Figure 4: Dimensionless (a) radial and (b) axial displacement distribution along axial direction at different layers ( i i1 1 9 , ( ) , ( ) , MC = = = n 2 P x P h x h ). 4.3 Effect of material constants and inhomogeneity index Figure 5: Dimensionless (a) circumferential (b) axial (c) shear Cauchy stress and (d) hydrostatic pressure distribution along axial direction at different layers ( i i1 1 9 , ( ) , ( ) , MC = = = n 2 P x P h x h ). Figure 5: Dimensionless (a) circumferential (b) axial (c) shear Cauchy stress and (d) hydrostatic pressure distribution along axial direction at different layers ( i i1 1 9 , ( ) , ( ) , MC = = = n 2 P x P h x h ). 17/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. -1 -0.5 0 0.5 1 1.5 2 2.5 0 0.2 0.4 0.6 0.8 1 MAE FEM Figure 6: Dimensionless radial Cauchy stress distribution at middle layer along axial direction for different MAE order. 1 Figure 6: Dimensionless radial Cauchy stress distribution at middle layer along axial direction for different MAE order. Figure 6: Dimensionless radial Cauchy stress distribution at middle layer along axial direction In order to investigate the effect of inhomogeneity index on the response of shell, the distribution of displacements and hydrostatic pressure at internal layer (critical layer) are plotted in Figure 7(a-c). The linear thickness and pressure profile are assumed for shell with 9 MC material constants. Figure 7(d) shows the distribution of dimensionless material properties (normalized to internal layer properties) with respect to the radius variation in a heterogeneous cylinder for integer values of n which vary in the range of −≤ ≤+ 4 n 4 . The variations of material properties with power-law distribution are continuously and smoothly in the radial direction. The extremum values of properties at outer layer points could be determined through intersection of vertical line plotted from radius of the point and graph of arbitrary n . Positive values of gradient index increase strength of material under mechanical loading toward outer layer of shell, while the reverse holds true for negative values of n . Therefore, variation of inhomogeneity index from negative to the positive causes displacements, hydrostatic pressure and consequently stresses of cylinder to be reduced. 4.3 Effect of material constants and inhomogeneity index Greater values of n intensify improvement or reduction in response of FG shell respect to homogenous one. Table 6 presents the results of maximum displacements and hydrostatic pressure in three layers of shell for different inhomogeneity index. Linear decrease in radial displacement and smooth reduction in axial displacement can be observed from internal layer to the external one for different values of n . Positive values of n cause more uniform hydrostatic pressure distribution of the layers and less maximum values of hydrostatic pressure compared with negative ones. Therefore, It could be concluded that internal layer (in contact with loading) is still critical one and positive values of n are more appropriate from the viewpoint of less values and more uniform distribution of displacements and stresses in heterogeneous cylinder. However, we are not concerned here with possible manufacture processes of FG materials, as well as experimental tests. Authors believe that, when the FG elastomers start to be widely employed in industry or in engineering applications, our formulation is a reliable numerical tool to predict their mechanical behavior (in terms of accuracy). But it is important to note that method presented here will be useful to material scientists in designing new materials, stress analysts, and designers in two states. One can use similar solution procedure to calculate displacements and stresses for FG material models with the given constants functions applied instead of Eq. (12) distribution. Furthermore, one can control the through-the-thickness distribution of displacements and stresses as objective parameters by tailoring the through-the-thickness variation of the material constants by trial and error to achieve appropriate distribution of FGM constants. In the material tailoring problem, one has found through-the thickness variation of material constants to achieve a desired variation of stress components, frequency of free vibrations, deformation or an objective function to be optimized (Batra 2011). This method is going to be extended in FG elastomers and biological tissues. Bilgili (2004) also suggest that the presence of material non-homogeneity in test specimens might be reason for the conflicting experimental results in the technical literature regarding the nature of the rubber-elastic response functions. Hence, he developed comprehensive experimental and theoretical program to characterize the response functions of non- homogeneous rubber components and introduced a design code based on especial (power variation) material model which can explain the essential physics–chemistry behind the intended functionality. Latin American Journal of Solids and Structures, 2019, 16(8), e229 4.3 Effect of material constants and inhomogeneity index 4.4 Effect of pressure and geometry profiles 4.4 Effect of pressure and geometry profiles 4.3 Effect of material constants and inhomogeneity index The design code yields essential information about the grading which in turn can be used as input into the design of a fabrication process. Thus, our method along with mentioned studies could direct further research toward the design, optimization, and manufacture of graded rubber-like materials. 18/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Figure 7: The effect of inhomogeneity index on dimensionless (a) radial displacement (b) axial displacement (c) hydrostatic pressure (along axial direction at internal layer) and (d) material properties (along radial direction) ( i i1 1 ( ) , ( ) = = P x P h x h ). Figure 7: The effect of inhomogeneity index on dimensionless (a) radial displacement (b) axial displacement (c) hydrostatic pressure (along axial direction at internal layer) and (d) material properties (along radial direction) ( i i1 1 ( ) , ( ) = = P x P h x h ). Table 6: Maximum displacements and hydrostatic pressure of internal, middle and external layer for different inhomogeneity index ( i i1 1 ( ) , ( ) = = P x P h x h ). z U at x = 0.15 x U at x = 0.65 P at x = 0 z = -0.5 z = 0 z = +0.5 z = -0.5 z = 0 z = +0.5 z = -0.5 z = 0 z = +0.5 = + n 4 0.0744 0.0697 0.0650 -0.0224 -0.0222 -0.0219 9.425 2.064 -5.296 = + n 2 0.0932 0.0873 0.0813 -0.0271 -0.0267 -0.0264 11.177 1.965 -7.247 = n 0 0.1152 0.1078 0.1004 -0.0325 -0.0320 -0.0315 13.105 1.631 -9.843 = − n 2 0.1403 0.1312 0.1221 -0.0387 -0.0380 -0.0373 15.201 0.970 -13.262 = − n 4 0.1685 0.1575 0.1465 -0.0457 -0.0447 -0.0438 17.459 -0.128 -17.715 4 4 Effect of pressure and geometry profiles Table 6: Maximum displacements and hydrostatic pressure of internal, middle and external layer for different inhomogeneity index ( i i1 1 ( ) , ( ) = = P x P h x h ). 4.4 Effect of pressure and geometry profiles Figure 8 shows the effect of pressure profile on the distribution of displacements and hydrostatic pressure in FG cylinder with linear variable thickness. The material is considered with = n 2 and 9 MC . Distribution of non-uniform internal pressure functions along axial direction are depicted in Figure 8(d). Investigating the response of cylinder with linear thickness profile under i1 P , i2 P and i3 P in Figure 8(a) and (b) show nearly the same maximum displacements; however, variations of radial displacement are proportional to pressure profiles distribution at length of the shell. It is observed that linear variation of pressure i1 P and thickness 1h of the shell in same direction counteract each other effect. This counterbalance is weakly true for nonlinear pressure profiles with similar range of applied pressure. Figure 8(c) expresses that hydrostatic pressure under these three pressure profiles increase by intensifying nonlinearity of pressure distribution. Hydrostatic pressure of middle layer has its maximum value near right boundary unexpectedly, because the effect of descending thickness is dominant to pressure profile increments. For internal (critical) layer reverse hold true (as previous results proved), i.e. the maximum hydrostatic pressure occurs around left boundary with higher pressure. Pressure profiles of i4 P and i5 P which have higher non-uniformity respect to other profiles lead in larger radial displacement and hydrostatic pressure values, but less axial displacement. Fast pressure variation of i4 P and i5 P reduce z U and P around = x 0 . Maximum pressure applied in middle and end of cylinder for i4 P and i5 P , respectively, in addition to descending thickness cause maximum of z U and P near = x 1 . It can be concluded from Figure 8(a) and (c) that z U and P patterns along the length of shell follow the pattern of the applied pressure function. 19/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Latin American Journal of Solids and Structures, 2019, 16(8), e229 4.4 Effect of pressure and geometry profiles Figure 8: The effect of non-uniform pressure profile on dimensionless (a) radial displacement (b) axial displacement (c) hydrostatic pressure along axial direction at middle layer ( 1 9 , ( ) , MC = = n 2 h x h ) and (d) internal pressure distribution along axial direction. Figure 8: The effect of non-uniform pressure profile on dimensionless (a) radial displacement (b) axial displacement (c) hydrostatic pressure along axial direction at middle layer ( 1 9 , ( ) , MC = = n 2 h x h ) and (d) internal pressure distribution along axial direction. The influences of thickness profile function on the distribution of displacements and hydrostatic pressure under i1 P loading are illustrated in Figures 9-11 for heterogeneous cylinder with = n 2 and 9 MC materials properties. Considering Figure 9(a) and (c) prove that changes of concave thickness profile to convex one cause reduction in radial displacement and hydrostatic pressure. However, z U and P for 4 h profile intensify near = x 0 because of lower thickness in addition to maximum pressure. It is obviously observed that constant thickness ( ) 0h have the greatest displacements and stresses under i1 P . No considerable variations in axial displacements are observed between non-uniform thickness profiles. Comparison of Figures 8-11 reveal that pressure profiles increment is more effective on the response of shell respect to thickness profiles variation; i.e. descending pressure causes more reduction in displacements and stresses respect to increasing the thickness. Table 7 represents the numerical results for similar distribution of pressure and thickness in different sections of internal layer. The axial sections are selected based on extremum points of displacements and hydrostatic pressure distribution. It is observed that similar thickness and pressure patterns lead in uniform distributions of displacements, stresses and hydrostatic pressure along length of the shell. According to Table 7, i , 5 5 P h and i , 4 4 P h profiles have the least and the most hydrostatic pressure, respectively. In fact, whatever maximum pressure and consequently maximum thickness are applied near clamed boundaries, more counterbalance of thickness and pressure emerges. It can be concluded that i , 5 5 P h and i , 1 1 P h are suitable profiles in designing current hyperelastic FG shell. Latin American Journal of Solids and Structures, 2019, 16(8), e229 4.4 Effect of pressure and geometry profiles The influences of thickness profile function on the distribution of displacements and hydrostatic pressure under i1 P loading are illustrated in Figures 9-11 for heterogeneous cylinder with = n 2 and 9 MC materials properties. Considering Figure 9(a) and (c) prove that changes of concave thickness profile to convex one cause reduction in radial displacement and hydrostatic pressure. However, z U and P for 4 h profile intensify near = x 0 because of lower thickness in addition to maximum pressure. It is obviously observed that constant thickness ( ) 0h have the greatest displacements and stresses under i1 P . No considerable variations in axial displacements are observed between non-uniform thickness profiles. The rupture modes in the aortic specimens are characterized by oblique tears in the circumferential direction, indicating that the failure of the aneurismal aortic tissue is mainly governed by the axial stress. The failure stress in the axial direction is much higher in the adventitia layer compared to that in the media layer (Kim et al. 2012). This means that the failure in the aneurismal aortic tissue may initiate in the media layer; i.e. inner surface of arteries are critical one. It is considered that the current methodology could be improved to assess the aortic aneurysm rupture risk based on maximal diameter or stress by modeling blood vessels of patients having an aneurysm. In recent years, researchers have developed artificial blood vessels made from special elastomer material and the usage of artificial vascular prostheses in vascular graft (Łos et al. 2018). Over time, these artificial blood vessels are replaced by endogenous material. Some parts of mentioned prostheses don’t have complicated geometries and can be models as regular shells with acceptable tolerances and imperfections. Authors believe that current method could have the potential of helping researchers in the future to analyze and obtain useful information about (a) more realistic hyperelastic material models of blood vessels (artificial or natural, isotropic or anisotropic, homogenous or non-homogeneous); (b) especial variation 20/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. in internal and /or external profiles of blood vessels (as variable thickness conical shell) resulted from atherosclerotic plaque, aortic aneurysm, aging deformation and so on; (c) maybe future non-homogeneous prosthesis with position dependent functionality. 4.4 Effect of pressure and geometry profiles e thickness and non-uniform pressure based on perturbation theory 𝑃𝑃ത 𝑃𝑃ത 𝑥𝑥̅ 𝑥𝑥̅ r r r ℎ1 ℎ2 ℎ3 ℎ4 ℎ5 ℎ0 𝑥𝑥̅ 𝑥𝑥̅ 𝑥𝑥̅ r r r 𝑥𝑥̅ 𝑃𝑃ത 𝑃𝑃ത 𝑃𝑃ത 𝑃𝑃ത Figure 11: The effect of thickness profile on dimensionless hydrostatic pressure ( i i1 9 , ( ) , MC = = n 2 P x P ). riable thickness and non-uniform pressure based on perturbation theory 𝑃𝑃ത 𝑃𝑃ത 𝑥𝑥̅ 𝑥𝑥̅ r r r ℎ1 ℎ2 ℎ3 ℎ4 ℎ5 ℎ0 𝑥𝑥̅ 𝑥𝑥̅ 𝑥𝑥̅ r r r 𝑥𝑥̅ 𝑃𝑃ത 𝑃𝑃ത 𝑃𝑃ത 𝑃𝑃ത Figure 11: The effect of thickness profile on dimensionless hydrostatic pressure ( i i1 9 , ( ) , MC = = n 2 P x P ). Figure 11: The effect of thickness profile on dimensionless hydrostatic pressure ( i i1 9 , ( ) , MC = = n 2 P x P ). 7: Numerical results for similar pressure and thickness profiles in various sections at internal layer ( 9 , MC = n 2 Table 7: Numerical results for similar pressure and thickness profiles in various sections at internal layer ( 9 , MC = n 2 ). z U x U P x = 0.15 x = 0.5 x = 0.85 x = 0.05 x = 0.25 x = 0.7 x = 0 x = 0.5 x = 1 i , 0 0 P h 0.1255 0.1214 0.1255 0.0200 0.0067 -0.0053 17.751 5.402 17.751 i , 1 1 P h 0.0921 0.0819 0.0699 0.0155 -0.0099 -0.0268 11.177 3.876 10.419 i , 2 2 P h 0.0924 0.0865 0.0732 0.0167 -0.0067 -0.0283 11.448 4.186 10.893 i , 3 3 P h 0.0920 0.0882 0.0761 0.0173 -0.0040 -0.0265 11.455 4.335 11.325 i , 4 4 P h 0.0809 0.0909 0.0809 0.0205 0.0170 -0.0141 11.743 4.407 11.743 i , 5 5 P h 0.0914 0.0614 0.0914 0.0054 -0.0165 0.0158 3.741 3.407 3.741 4.4 Effect of pressure and geometry profiles y 𝑥𝑥̅ 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑥𝑥̅ 𝑥𝑥̅ r r r ℎ1 ℎ2 ℎ3 ℎ4 ℎ5 ℎ0 𝑥𝑥̅ 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑥𝑥̅ 𝑥𝑥̅ r r r Figure 9: The effect of thickness profile on dimensionless radial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ). 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑥𝑥̅ 𝑥𝑥̅ r r r ℎ1 ℎ2 ℎ3 ℎ4 ℎ5 ℎ0 𝑥𝑥̅ 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑥𝑥̅ 𝑥𝑥̅ r r r 𝑥𝑥̅ Figure 10: The effect of thickness profile on dimensionless axial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ). 𝑥𝑥̅ 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑥𝑥̅ 𝑥𝑥̅ r r r ℎ1 ℎ2 ℎ3 ℎ4 ℎ5 ℎ0 𝑥𝑥̅ 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑥𝑥̅ 𝑥𝑥̅ r r r Figure 9: The effect of thickness profile on dimensionless radial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ). 𝑥𝑥̅ 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑥𝑥̅ 𝑥𝑥̅ r r r ℎ1 ℎ2 ℎ3 ℎ4 ℎ5 ℎ0 𝑥𝑥̅ 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑈𝑈ഥ𝑧𝑧 𝑥𝑥̅ 𝑥𝑥̅ r r r Figure 9: The effect of thickness profile on dimensionless radial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ). 𝑈𝑈ഥ𝑧𝑧 ℎ4 𝑥𝑥𝑥 𝑥𝑥 Figure 9: The effect of thickness profile on dimensionless radial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ). 𝑥𝑥𝑥 Figure 9: The effect of thickness profile on dimensionless radial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ) 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑥𝑥̅ 𝑥𝑥̅ r r r ℎ1 ℎ2 ℎ3 ℎ4 ℎ5 ℎ0 𝑥𝑥̅ 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑈𝑈ഥ𝑥𝑥 𝑥𝑥̅ 𝑥𝑥̅ r r r 𝑥𝑥̅ Figure 10: The effect of thickness profile on dimensionless axial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ). Figure 10: The effect of thickness profile on dimensionless axial displacement ( i i1 9 , ( ) , MC = = n 2 P x P ). 21/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. 5 CONCLUSIONS In current research, the heterogonous hyperelastic hollow cylinders with variable thickness under non-uniform internal pressure and clamped boundary conditions have been analyzed by FSDT. Two-term Mooney-Rivlin type material in nearly incompressible condition is considered which is a suitable hyperelastic model for rubbers. The material properties are graded along the radial direction according to a power law function. Matched Asymptotic Expansion of the perturbation theory is used for solving the governing equations analytically. The advantages of this method are fast convergence, closed form solution and compatibility with physics of shell. A new ingenious formulation and parameters have been defined during current study to simplify and abbreviate the representation of inner and outer equations components in MAE. In addition, the terms of variable thickness and non-uniform pressure have been presented in separate representation. The results prove the effectiveness of FSDT and MAE combination to derive and solve the governing equations of nonlinear problems such as nearly incompressible hyperelastic shells. This approach enables insight into the nature of the deformation and stress distribution across the wall of rubber vessels and offers the potential for investigation of the mechanical functionality of arteries in physiological pressure range. The points of internal layer close to maximum pressure are critical elements in nearly incompressible hyperelastic FG cylinder with variable thickness under non-uniform internal pressure. The acceptable range of the current analysis for the geometry, loading and materials properties is about < < 4 R 20 and ( ) i . + < 1 2 P C C 0 015 by considering difference percentage of deformations resulted from current analytical solution and FEM less than 10%. The accuracy of MAE descend for great values of R because of intensifying nonlinear behavior of the cylinder while for small R , the accuracy of shear deformation theory decrease in analyzing thick cylindrical shells. An increase in i ( ) + 1 2 P C C and k or decrease in + 1 2 C C ascend the nonlinearity and difference percentage of numerical and analytical solution. Variation of inhomogeneity index from negative to the positive values causes reduction in displacements, hydrostatic pressure and consequently stresses of cylinder. Therefore, it could be concluded that positive values of gradient index are more appropriate from the viewpoint of less values and resulted from current analytical solution and FEM less than 10%. 5 CONCLUSIONS The accuracy of MAE descend for great values of R because of intensifying nonlinear behavior of the cylinder while for small R , the accuracy of shear deformation theory decrease in analyzing thick cylindrical shells. An increase in i ( ) + 1 2 P C C and k or decrease in + 1 2 C C ascend the nonlinearity and difference percentage of numerical and analytical solution. Variation of inhomogeneity index from negative to the positive values causes reduction in displacements, hydrostatic pressure and consequently stresses of cylinder. Therefore, it could be concluded that positive values of gradient index are more appropriate from the viewpoint of less values and Latin American Journal of Solids and Structures, 2019, 16(8), e229 22/28 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Gharooni et al. New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory more uniform distribution of displacements and stresses in heterogeneous cylinder. It can be concluded that pressure profiles increment is more effective on the response of shell respect to thickness profiles variation. Furthermore, changes of concave thickness profile to convex one lead in descending maximum displacement, stresses and hydrostatic pressure. It can be concluded that radial displacement and hydrostatic pressure patterns follow the pattern of the applied pressure function along the length of shell. The behavior of hyperelastic FG vessels under non-uniform pressure distribution shows that similar profile of variable thickness and non-uniform applied pressure result in minor displacement and stress quantities and uniform distributions which could be a suitable criterion in designing thickness profile of pressurized vessels. Applying maximum pressure and consequently maximum thickness near boundaries of shell are suitable profiles in designing hyperelastic FG shells. Authors believe that current method along with studies mentioned in the literature could direct further research toward the design, optimization, and manufacture of graded rubber-like materials. more uniform distribution of displacements and stresses in heterogeneous cylinder. It can be concluded that pressure profiles increment is more effective on the response of shell respect to thickness profiles variation. Furthermore, changes of concave thickness profile to convex one lead in descending maximum displacement, stresses and hydrostatic pressure. 5 CONCLUSIONS It can be concluded that radial displacement and hydrostatic pressure patterns follow the pattern of the applied pressure function along the length of shell. The behavior of hyperelastic FG vessels under non-uniform pressure distribution shows that similar profile of variable thickness and non-uniform applied pressure result in minor displacement and stress quantities and uniform distributions which could be a suitable criterion in designing thickness profile of pressurized vessels. Applying maximum pressure and consequently maximum thickness near boundaries of shell are suitable profiles in designing hyperelastic FG shells. Authors believe that current method along with studies mentioned in the literature could direct further research toward the design, optimization, and manufacture of graded rubber-like materials. Author’s Contributions: Conceptualization, H Gharooni; Methodology, H Gharooni; Investigation, H Gharooni and M Ghannad; Writing - original draft, H Garooni; Writing - review & editing, H Gharooni and M Ghannad; Funding acquisition, H Gharooni and M Ghannad; Supervision, M Ghannad. Editor: Rogério José Marczak References Abdessamad, M., Mohamed, H., Mohamed, A., (2018). Analytical modeling of a descending aorta containing human blood flow. Defect and Diffusion Forum 384: 117-129. Abdessamad, M., Mohamed, H., Mohamed, A., (2018). Analytical modeling of a descending aorta containing human blood flow. Defect and Diffusion Forum 384: 117-129. Anani, Y., Rahimi, G.H., (2015). Stress analysis of thick pressure vessel composed of functionally graded incompressible hyperelastic materials. 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New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory The non-homogeneity vectors of 2 O( ) ε equations in outer and inner expansions are as follows: { } ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ˆ ( , ) ( , ) ( , ) ,  = − + + + − + + +     + + − + +      ′ − + + + − +     F F 2 O2 3 O1 O1 O1 O1 O1 1 2 2 O1 O1 O1 7 O1 O1 O1 2 2 O1 O1 s 12 O1 O II Ck II 2 n 1 II 0 n 1 2II 1 n 1 w 2 II 1 n II 0 n 1 v 2II 0 n v w II 0 n 1 w Ck II 1 n II 0 n w k II 1 n 1 2II 0 n 1 v K C II 0 n 1 ψ ψ ψ ψ ψ ϕ ϕ { } ′      =  2 1 II 0 ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , )  = − + + + − + + +  2 3 O1 O1 O1 O1 O1 1 Ck II 2 n 1 II 0 n 1 2II 1 n 1 w 2 II 1 n II 0 n 1 v ψ ψ ψ (A-1) { } ( ) ( ) ( ) ( ) { } ( , ) ( , ) ( , ) ( , ) ( , ) ˆ ( , ) ( , ) ( , ) ˆ ( , ) ( , ) ( , ) ( , ) ′  ′ ′ = + + + − +   + + + +  ′ ′ ′ + + + + − + + + = F F O2 1 O1 O1 O1 O1 O1 2 s O1 O1 s 2 O1 O1 O1 s 12 O1 O1 O1 s O1 O1 O2 2 II II Ck II 2 n II 1 n 1 II 1 n w II 1 n II 0 n w K II 0 n 1 v K Ck II 1 n II 0 n w K C II 0 n 1 w II 1 n 1 k II 1 n 1 v K k II 0 n 1 Ck ψ ψ ϕ ϕ ψ ϕ ψ ϕ ψ ( , ) ( , ) ( , ) ( , )          ′ ′ ′ ′ + + + − +       1 O1 O1 O1 O1 II 2 n II 1 n w II 1 n 1 k II 1 n 1 v ψ ψ (A-2) { } ( ) ( )( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )   = − + + − + − +   ′ ′ − − − + +   + − + − + −   F 2 2 O2 3 O1 O1 O1 O1 O1 O1 3 2 1 O1 7 O1 O1 s 12 O1 2 O1 2 2 O1 O1 O1 O1 II Ck II 0 n v 2 II 1 n 1 II 0 n 1 w v Ck II 1 n Ck II 0 n v K C II 0 n 1 2C II 0 n 2k II 2 n 2 2II 1 n 2 w II 0 n 2 w ψ ψ ψ ϕ ψ ϕ ϕ ψ ψ        { } ( ) ( )( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )   = − + + − + − +   ′ ′ − − − + +   + − + − + −   F 2 2 O2 3 O1 O1 O1 O1 O1 O1 3 2 1 O1 7 O1 O1 s 12 O1 2 O1 2 2 O1 O1 O1 O1 II Ck II 0 n v 2 II 1 n 1 II 0 n 1 w v Ck II 1 n Ck II 0 n v K C II 0 n 1 2C II 0 n 2k II 2 n 2 2II 1 n 2 w II 0 n 2 w ψ ψ ψ ϕ ψ ϕ ϕ ψ ψ        (A-3) { } ( ) ( ) ( ) ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )  ′ ′ = − + + − + − +  + − + + + + + − +  + + − − +  F 2 2 O2 1 O1 s 12 O1 3 O1 O1 4 O1 O1 O1 O1 O1 O1 O1 O1 O1 2 O1 O1 O1 7 O1 O1 O1 II Ck II 2 n II 1 n 1 K C II 1 n 1 Ck II 1 n 3 v II 2 n 1 3 2v II 0 n 1 v 2 v II 1 n 1 w 4 2v 2II 0 n w 2II 0 n 1 w Ck II 0 n v w 2II 1 n v ϕ ϕ ψ ψ ψ ψ ψ ψ ( ) { } ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ′             − + − + − + + + −     ′ = − + F O1 2 2 2 2 O1 O1 O1 O1 O1 O2 s 12 O1 4 II 2C II 1 n 2k II 1 n 2 w II 3 n 2 II 0 n 1 2 II 2 n 2 w K C II 1 n 1 ψ ϕ ψ ψ ϕ (A-4) { } { } { } { } ( ) , ,   = = = − − = + −     F F F F   i i 2 2 2 2 1 2 3 4 Dh P x P P P P 0 P x DR Dh R h DR h Dh 2 2 α α α α α α α α α α α α α α α α α α α α { } { } { } { } , ,     = = = − − = −         F F F F   i i 2 2 2 2 1 2 3 4 h DP x h h DP DP DP DP 0 DP x R R 2 2 2 α α α α α α α α α α α α α α α α α { } { } { } { } ( ) , ,   = = = − − = + −     F F F F   i i 2 2 2 2 1 2 3 4 Dh P x P P P P 0 P x DR Dh R h DR h Dh 2 2 α α α α α α α α α α α α α α α α α α α α (A-5) { } ( ) ( ) ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )  ′ = + + + + + + −  + + + + − +  ′ ′ + − − + + +  F 2 3 1 1 1 1 2 1 1 1 1 1 1 1 2 1 12 s 1 1 s 1 1 II Ck 2 II 1 n 1 II 2 n II 1 n w II 0 n 1 II 2 n 1 2 II 1 n II 0 n 1 v 2II 1 n 1 w 2II 0 n v w II 0 n 1 w C K II 1 n 1 II 0 n 1 K w α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ϕ ψ ψ ψ ϕ ψ ϕ ( ) ( ) ( ) ( ) ˆ ( , ) ( , ) ( , ) ( , ) ( , )   +       ′ ′ ′ ′ − + + + − + + −       + +   2 s 1 2 2 1 1 1 1 1 1 1 1 2 7 1 1 1 K k 2II 2 n 1 4II 1 n 1 4v II 0 n 1 2v w Ck II 1 n II 0 n w α α α α α α α α α α α α α α α α α ϕ ϕ ϕ ϕ ψ ϕ ψ ψ { } ( ) ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , )  ′ = + + + + + + −  F 2 3 1 1 1 1 2 1 II Ck 2 II 1 n 1 II 2 n II 1 n w II 0 n 1 II 2 n 1 α α α α α α α α α α α ψ ϕ ψ (A-7) { } ( ) ( ) D ( , ) ( , ) ( , ) ( , ) ( , )  = + + +   ′ − + + + F   1 1 1 2 1 1 1 A x Ck DII 1 n DII 0 n 1 DII 0 n w x k DII 1 n 1 DII 0 n 1 v α α α α α α α α α α α α α ψ ϕ (A-8) { } = F 2 1 DII 0 α α (A-9) 26/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. The non-homogeneity vectors of 2 O( ) ε equations in outer and inner expansions are as follows: { } ( ) ( ) ( ) ( ) ( ) ( ) ˆ ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )  ′ = + + + +   ′ ′ ′ ′ + + + + + +    ′ ′ + + + + + +   F 1 1 1 s 1 1 s 2 1 1 2 2 1 1 1 1 1 1 1 5 1 1 s 4 1 1 1 1 1 1 II Ck II 1 n II 0 n w K II 0 n 1 v K Ck II 2 n II 1 n w II 0 n w w Ck II 2 n 1 K Ck II 0 n 1 v w II 1 n 1 v α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ϕ ψ ψ ψ ψ ϕ ϕ ψ ϕ ψ ψ ψ ( ) ( ) ( ) ( ) ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )  ′′ − +    ′  ′′ ′′ ′ + + + + +    ′ ′ ′ ′ + + + + − + − ′ ′ + + + + s 12 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 K C II 2 n 1 II 1 n 1 w Ck II 3 n II 2 n 1 II 1 n 1 v II 1 n 1 w w II 2 n 1 w II 1 n w v II 3 n 1 II 2 n v α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ϕ ψ ϕ ψ ϕ ψ ψ ψ ψ ψ ψ ψ ( ) ( ) ( ) ( ) ( ) ˆ ( , ) ( , ) ˆ ( , ) ( , ) ( , ) ( , )    ′ ′ ′ +           ′ ′ ′ ′ ′′ − + + + + −           ′ ′ ′′ ′ ′ ′′ + + − + + + +   ′ ′ + + 1 1 s 1 1 1 1 s 1 1 1 1 1 1 1 1 s 1 1 1 1 1 7 1 1 1 1 w k K II 0 n 1 II 2 n 1 4 v K II 1 n 1 4v v w K w 4II 3 n 1 Ck 2II 2 n II 1 n w α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ϕ ϕ ψ ϕ ψ ϕ ϕ ψ ϕ ϕ ϕ ϕ ϕ ψ ψ ψ      (A-10) { } ( ) ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )    ′ ′ ′ = + + + + +        ′ ′ ′′ + + − + + +  F    1 1 1 s 12 1 2 2 1 1 1 1 DA x Ck DII 2 n DII 1 n 1 DII 1 n w x K C DII 1 n 1 +DII 0 n 1 w x k DII 1 n 1 v DII 2 n 1 α α α α α α α α α α α α α α α α α α α ψ ψ ϕ ϕ (A-11) { } ( ) [ ] ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ′ + + + − + + + = F 1 1 1 1 1 2 2 DII Ck DII 2 n DII 1 n 1 DII 1 n w k DII 1 n 1 v DII 2 n 1 α α α α α α α α α α α ψ ϕ { } ( ) [ ] ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ′ + + + − + + + = F 1 1 1 1 1 2 2 DII Ck DII 2 n DII 1 n 1 DII 1 n w k DII 1 n 1 v DII 2 n 1 α α α α α α α α α α α ψ ϕ { } ( ) [ ] ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ′ + + + − + + + = F 1 1 1 1 1 2 2 DII Ck DII 2 n DII 1 n 1 DII 1 n w k DII 1 n 1 v DII 2 n 1 α α α α α α α α α α α ψ ϕ (A-12) { } ( ) ( ) ( ) ( ) 2 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 7 1 1 1 2 2 1 2 1 (2, ) (1, ) 2 (0, ) ˆ (1, 1) (0, 1) (0, ) (1, ) ˆ 4 (0, ) (0, ) s s II Ck II n II n w w w II n w w K II n II n w Ck II n v II n C II n Ck II n K α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ψ ψ ψ ψ ψ ψ ψ ϕ ψ ϕ ϕ  ′ ′′ ′′ ′ ′′ ′′ = + + + +     ′ ′ ′ ′ − + + + − +        + − F ( ) ( ( ) ) ( ) ( ) 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 3 1 1 1 1 1 1 1 ˆ (1, ) 2 (2, ) 2 (2, 1) 2 (0, 1) 2 (1, 1) s s s s w K w w w w II n K K w w w K II n Ck II n II n v w II n w α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ϕ ϕ ψ ψ ψ ψ ψ ϕ ψ ψ ψ ψ ϕ ψ ϕ      ′ ′ ′′ ′ + + +           ′ ′ ′′ ′ ′′ ′ + + + + +     ′ ′′ ′ + + − − + − +      ′ + −( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 1 1 1 1 1 1 1 2 1 4 1 1 1 1 1 1 2 2 8 1 1 1 1 2 (1, ) (0, ) 2 (2, ) (1, 1) (0, 1) ˆ (2, ) (0, ) (2, 1) (1, 1) s s v II n v II n v II n K Ck II n II n v Ck II n II n w K k II n II n v α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ψ ϕ ψ ϕ ϕ ϕ ϕ ϕ ψ ψ ϕ ψ ′ + + + + +   ′  ′ ′ ′ + − + + + +         ′   ′ ′ ′ ′ + + + +       + + ( ) ( ) 1 1 1 1 1 1 2 2 1 1 1 1 (0, 1) 2 (2, 2) 2 (1, 2) (0, 2) w II n v w k II n II n w II n w α α α α α α α α α α α α α ψ ϕ ψ ψ  ′ ′ ′ ′ ′ ′ + + +     + − + − + −   (A-13) { } ( ) ( ) ( ) ( ) 2 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 7 1 1 1 2 2 1 2 1 (2, ) (1, ) 2 (0, ) ˆ (1, 1) (0, 1) (0, ) (1, ) ˆ 4 (0, ) (0, ) s s II Ck II n II n w w w II n w w K II n II n w Ck II n v II n C II n Ck II n K α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ψ ψ ψ ψ ψ ψ ψ ϕ ψ ϕ ϕ  ′ ′′ ′′ ′ ′′ ′′ = + + + +     ′ ′ ′ ′ − + + + − +        + − F ( ) ( ( ) ) 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ˆ (1, ) 2 s s s w K w w w w II n K K w w w α α α α α α α α α α α α α α α α α ϕ ϕ ψ ψ ψ ψ ψ ϕ      ′ ′ ′′ ′ + + +           ′ ′ ′′ ′ ′′ ′ + + + + + { } ( ) ( ) ( ) ( ) 2 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 7 1 1 1 2 2 1 2 1 (2, ) (1, ) 2 (0, ) ˆ (1, 1) (0, 1) (0, ) (1, ) ˆ 4 (0, ) (0, ) s s II Ck II n II n w w w II n w w K II n II n w Ck II n v II n C II n Ck II n K α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ψ ψ ψ ψ ψ ψ ψ ϕ ψ ϕ ϕ  ′ ′′ ′′ ′ ′′ ′′ = + + + +     ′ ′ ′ ′ − + + + − +        + − F ( ) ( ( ) ) 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ˆ (1, ) 2 s s s w K w w w w II n K K w w w α α α α α α α α α α α α α α α α α ϕ ϕ ψ ψ ψ ψ ψ ϕ      ′ ′ ′′ ′ + + +           ′ ′ ′′ ′ ′′ ′ + + + + + { } ( ) ( ) ( ) ( ) 2 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 7 1 1 1 2 2 1 2 1 (2, ) (1, ) 2 (0, ) ˆ (1, 1) (0, 1) (0, ) (1, ) ˆ 4 (0, ) (0, ) s s II Ck II n II n w w w II n w w K II n II n w Ck II n v II n C II n Ck II n K α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ψ ψ ψ ψ ψ ψ ψ ϕ ψ ϕ ϕ  ′ ′′ ′′ ′ ′′ ′′ = + + + +     ′ ′ ′ ′ − + + + − +        + − F ( ) ( ( ) ) ( ) ( ) 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 3 1 1 1 1 1 1 1 ˆ (1, ) 2 (2, ) 2 (2, 1) 2 (0, 1) 2 (1, 1) s s s s w K w w w w II n K K w w w K II n Ck II n II n v w II n w α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ϕ ϕ ψ ψ ψ ψ ψ ϕ ψ ψ ψ ψ ϕ ψ ϕ      ′ ′ ′′ ′ + + +           ′ ′ ′′ ′ ′′ ′ + + + + +     ′ ′′ ′ + + − − + − +      ′ + −( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 2 1 1 1 1 1 1 1 2 1 4 1 1 1 1 1 1 2 2 8 1 1 1 1 2 (1, ) (0, ) 2 (2, ) (1, 1) (0, 1) ˆ (2, ) (0, ) (2, 1) (1, 1) s s v II n v II n v II n K Ck II n II n v Ck II n II n w K k II n II n v α α α α α α α α α α α α α α α α α α α α α α α α α α α α ψ ψ ϕ ψ ϕ ϕ ϕ ϕ ϕ ψ ψ ϕ ψ ′ + + + + +   ′  ′ ′ ′ + − + + + +         ′   ′ ′ ′ ′ + + + +       + + ( ) ( ) 1 1 1 1 1 1 2 2 1 1 1 1 (0, 1) 2 (2, 2) 2 (1, 2) (0, 2) w II n v w k II n II n w II n w α α α α α α ψ ϕ ψ ψ  ′ ′ ′ ′ ′ ′ + + +     + − + − + −   (A-13) { } ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )    ′ ′′ = − + + − +        ′ ′′ + + + +  − + −     F    1 1 1 1 s 12 1 2 3 1 1 1 1 DA x Ck DII 1 n DII 0 n v x K C DII 1 n 1 DII 0 n 1 w x k DII 0 n 1 w DII 1 n 1 α α α α α α α α α α α α α α α α α α α ϕ ψ ψ ϕ ψ (A-14) 27/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229 hyperelastic FGM cylindrical shells with arbitrary ed on perturbation theory Gharooni et al. The non-homogeneity vectors of 2 O( ) ε equations in outer and inner expansions are as follows: New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory New nonlinear solution of nearly incompressible hyperelastic FGM cylindrical shells with arbitrary variable thickness and non-uniform pressure based on perturbation theory Gharooni et al. Latin American Journal of Solids and Structures, 2019, 16(8), e229 The non-homogeneity vectors of 2 O( ) ε equations in outer and inner expansions are as follows: { } ( ) ( , ) ( , )   ′ ′ = − + + + +     F s 12 1 1 1 12 3 DII K C DII 1 n 1 DII 0 n 1 w α α α α α α α ψ ϕ (A-15) { } ( ) ( ) ( ) ( ) 2 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 9 1 1 1 2 2 1 1 1 1 1 ˆ (0, 1) (1, 1) (2, 1) (1, ) (2, ) (3, ) (1, 1) (1, ) s s II Ck II n w K II n w II n II n w w II n w w II n Ck II n v Ck II n K w w w w α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ϕ ψ ψ ψ ψ ψ ψ ψ ϕ ϕ  ′′ ′′ ′ ′ =− + + + + + −    ′  ′ ′′ ′′ ′ − + − − + +        ′ ′ ′ − + + F ( ) ( ) ( ) ( ) 1 1 1 1 1 1 1 1 1 1 2 2 2 1 1 1 3 1 1 1 1 ˆ ˆ (2, ) (3, ) 2 (3, ) (0, 1) 2 (2, 1) (0, 1) 2 2 (1, s s s s K w II n K w w K w K II n Ck II n II n w II n II n v II n α α α α α α α α α α α α α α α α α α α α α α α α ϕ ψ ψ ψ ϕ ϕ ψ ψ ψ ϕ ψ      ′ ′ +               ′ ′ ′ ′′ ′ + + + +                 ′ ′′ ′ + + − + −        + − + + + − ( ) ( ) ( ) ( ) 1 1 2 2 1 1 1 1 2 1 1 1 1 1 1 1 1) (0, ) 3 (1, ) (2, 1) (0, 1) (1, ) 2 (2, ) 2 (1, 1) 2 (2, 1) 2 (3, 1) (2, 1) II n w II n II n II n II n v II n v II n v w II n w II n II n α α α α α α α α α α α α α α α α α α α α α α α ψ ψ ϕ ϕ ψ ϕ ϕ + ′ + + − + + + +  ′ ′ ′ + − + − + − + +  { } ( ) 2 1 1 1 1 1 1 1 1 4 ˆ (0, 1) (1, 1) (2, 1) (1, ) s II Ck II n w K II n w II n II n w w α α α α α α α α α α α α α ϕ ψ ψ  ′′ ′′ ′ ′′ =− + + + + + −  F { } ( ) ( ) ( ) ( ) 2 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 9 1 1 1 2 2 1 1 1 1 1 ˆ (0, 1) (1, 1) (2, 1) (1, ) (2, ) (3, ) (1, 1) (1, ) s s II Ck II n w K II n w II n II n w w II n w w II n Ck II n v Ck II n K w w w w α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α α ϕ ψ ψ ψ ψ ψ ψ ψ ϕ ϕ  ′′ ′′ ′ ′′ =− + + + + + −    ′  ′ ′′ ′′ ′ − + − − + +        ′ ′ ′ − + + F ( ) 1 1 1 1 1 1 1 1 1 1 ˆ ˆ (2, ) s s s K w II n K w w K w α α α α α α α α α α α ϕ ψ ψ ψ ϕ ϕ      ′ ′ +               ′ ′ ′ ′′ ′ + + + +             ( ) ( ) ( ) 1 1 1 1 1 1 9 1 1 1 (2, ) (3, ) (1, 1) II n w w II n Ck II n v α α α α α α α α α α α α ψ ψ ψ ψ ψ ϕ    ′  ′ ′′ ′′ ′ − + − − + +        (A-16) ( ) ( ) 2 4 1 1 1 ˆ (2, 1) s s Ck II n K K α α α α ψ ϕ ϕ    ′ ′ ′ − + +       ( ) ( ) ( ) ( ) ( ) ( ) ( ) 1 1 1 1 2 2 2 12 1 1 1 8 1 1 7 1 1 1 1 1 1 1 1 1 1 (1, 1) ˆ (0, 1) (3, ) (1, ) 2 (2, ) 2 (1, ) (0, ) ˆ (3, 1) s s s s K II n v C II n K w K w Ck II n II n w Ck II n II n v w II n v w K k II n I α α α α α α α α α α α α α α α α α α α α α α α α α α α ϕ ψ ϕ ϕ ψ ψ ϕ ψ ϕ ϕ ψ  ′ ′ + + +        ′ ′ ′ ′ + + + + +           ′ ′ − + + +    ′ ′ ′ + + + ( ) ( ) ( ) ( ) ( ) 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 (2, 1) (1, 1) 2 (1, 2) (3, 2) (0, 1) 2 (2, 2) I n w v II n v w w k II n w II n II n II n w α α α α α α α α α α α α α α α α α α α ϕ ψ ϕ ψ ψ ψ    ′ ′ ′ ′ ′ + +             ′ ′ ′ ′  + + + + + −          + − + + + −  { } ( ) ( ) ( ) ( ) ( ) ( ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) ( , )  = − + + + +    ′ ′′ ′ ′′ + + + − + + + +         + − + + + −   F    1 1 1 1 1 2 4 1 s 12 1 1 1 1 1 DA x Ck DII 0 n 1 v DII 1 n 2 v DII 0 n w DII 2 n DII 1 n 1 x K C DII 2 n 1 DII 1 n 1 w x k DII 2 n 1 DII 0 n 1 DII 1 n 1 w α α α α α α α α α α α α α α α α α α α α α α α α α ψ ϕ ψ ϕ ψ (A-17) { } ( ) ( , ) ( , )   ′ ′ = − + + + +     F s 12 1 1 1 2 4 DII K C DII 2 n 1 DII 1 n 1 w α α α α α α α ψ ϕ { } ( ) ( , ) ( , )   ′ ′ = − + + + +     F s 12 1 1 1 2 4 DII K C DII 2 n 1 DII 1 n 1 w α α α α α α α ψ ϕ (A-18) 28/28 Latin American Journal of Solids and Structures, 2019, 16(8), e229
https://openalex.org/W3029363909	https://usiena-air.unisi.it/bitstream/11365/1121932/2/luddi.pdf	English	null	Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment	Journal of clinical medicine	2,020	cc-by	8,330	Received: 17 April 2020; Accepted: 27 May 2020; Published: 28 May 2020 Abstract: Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identiﬁcation of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can signiﬁcantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be eﬀective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the eﬀects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a signiﬁcant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation eﬃciency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a signiﬁcant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present ﬁndings signiﬁcantly extend our knowledge on human male fertility and pave the way to the deﬁnition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection. Keywords: human sperm; myo-inositol; sperm motility; DNA oxidation; mitochondrial respiration Article Respiratory Mitochondrial Eﬃciency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment Laura Governini 1,†, Rosetta Ponchia 1,2,†, Paolo Giovanni Artini 3, Elena Casarosa 3, Ilaria Marzi 3, Angela Capaldo 2, Alice Luddi 1,* and Paola Piomboni 1,2 Laura Governini 1,†, Rosetta Ponchia 1,2,†, Paolo Giovanni Artini 3, Elena Casarosa 3, Ilaria Marzi 3, Angela Capaldo 2, Alice Luddi 1,* and Paola Piomboni 1,2 Laura Governini 1,†, Rosetta Ponchia 1,2,†, Paolo Giovanni Artini 3, Elena Casarosa 3, Ilaria Marzi 3, Angela Capaldo 2, Alice Luddi 1,* and Paola Piomboni 1,2 1 Department of Molecular and Developmental Medicine, Siena University, 53100 Siena, Italy; laura.governini@unisi.it (L.G.); ponchia2@student.unisi.it (R.P.); piomboni@unisi.it (P.P.) 2 Assisted Reproduction Unit, Siena University Hospital, 53100 Siena, Italy; angela.ca@live.it 3 Department of Experimental and Clinical Medicine, Division of Obstetrics and Gynecology, Pisa University, 56100 Pisa, Italy; pgartini@gmail.com (P.G.A.); elena.casarosa@dmdp.unipi.it (E.C.); ilaria.marzi1@gmail.com (I.M.) 1 Department of Molecular and Developmental Medicine, Siena University, 53100 Siena, Italy; laura.governini@unisi.it (L.G.); ponchia2@student.unisi.it (R.P.); piomboni@unisi.it (P.P.) 2 Assisted Reproduction Unit, Siena University Hospital, 53100 Siena, Italy; angela.ca@live.it 3 Department of Experimental and Clinical Medicine, Division of Obstetrics and Gynecology, Pisa Universit 56100 Pisa, Italy; pgartini@gmail.com (P.G.A.); elena.casarosa@dmdp.unipi.it (E.C.); ilaria.marzi1@gmail.com (I.M.) * Correspondence: alice.luddi@unisi.it; Tel.: +39-0577-233521 † These authors contributed equally to this work. Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine 1. Introduction Male infertility represents an urgent social burden, considering that 15% of all couples around the world are infertile and that male factor can be diagnosed in about 50% of these cases [1–3]. The most frequent and typical causes for male infertility are represented by varicocele, cryptorchidism, infections, obstructive lesions, trauma, and cancer [1]; noteworthily, the exact etiology of male factor infertility still remains undiagnosed in about 30% to 50% of patients, who therefore are classiﬁed as idiopathic J. Clin. Med. 2020, 9, 1638; doi:10.3390/jcm9061638 www.mdpi.com/journal/jcm www.mdpi.com/journal/jcm J. Clin. Med. 2020, 9, 1638 2 of 12 cases [4]. There is increasing evidence that oxidative stress (OS) represents a condition having a key role in the etiology of male infertility and thus it has been studied extensively [5,6]. OS is deﬁned as a result of the imbalance between reactive oxygen species (ROS) and antioxidants; the presence of anomalous concentrations of ROS has been detected in 30% to 80% of infertile men and this condition seems to lead to sperm damage, and eventually male infertility [7,8]. ROS are the most widespread group of free radicals that, due to an unpaired electron in the outer orbital, may trigger an extremely detrimental chain mechanism mainly aﬀecting both the plasma membrane and DNA [9]. Based on this mechanism, ROS are capable of compromising sperm quality and functions. The main endogenous sources of ROS in seminal fluid are represented by leukocytes, especially neutrophils, as well as mitochondria and plasma membranes of morphologically abnormal sperm [10–12]. ROS production is normally present in physiologic conditions; indeed, neutrophils are always released into secretions from the prostatic and seminal vesicle. This physiologic production is crucial for spermatogenesis and fertility, since ROS has been reported to be fundamental for sperm motility and viability as well as in sperm maturation, hyperactivation, capacitation and acrosome reaction [13–15]. Ontheotherend, pathologicconditionssuchasinflammationorinfectionsmayinduceanoverproductionof proinflammatory molecules (cytokines and interleukins) that, in turn, may increase ROS production [10,16]. This increase gives rise to an imbalance between ROS generation and antioxidant capacity in favor of the oxidants, thus establishing an oxidative stress condition that negatively affects sperm morphology and functions through lipid peroxidation, DNA fragmentation, and apoptosis [17–19]. Noteworthily, the high polyunsaturated fatty acid content of sperm plasma membranes, that easily may undergo lipid peroxidation, makes sperm very susceptible to OS damage. 2.1. Patients This study was conducted on a total of 56 Caucasian males undergoing semen evaluation at the Unit of Medically Assisted Reproduction, Siena University Hospital and at Centre of Infertility and Assisted Reproduction of Pisa University Hospital. A comprehensive clinical history of patients was obtained; we excluded patients with possible causes of male infertility such as varicocele, cryptorchidism, endocrine disorders or systemic diseases and patients with intake of spermiotoxic drugs, smoking, alcohol or drugs abuse. The median age of the patients was 33 years (range: 25–45 years); the BMI ranged between 18 and 25. All participants signed a written informed consent, and the study protocol was approved by the Ethic Committeeof the Siena University Hospital (approval ID: CEASVE 191113). 1. Introduction 2020, 9, 1638 3 of 12 (OXPHOS), mitochondria play a fundamental role in the production of ATP necessary for sperm motility, capacitation, hyperactivation and acrosome reaction [35]; anyway, mitochondria are also actively implicated in other important processes, including ROS generation [36]. In light of these observations, the purpose of this study was to test the eﬃcacy of a myo-inositol-based product in in vitro ameliorating diﬀerent sperm parameters, such as basal motility, vitality and sperm recovery after swim-up selection. Moreover, the eﬀectiveness of myo-inositol in vitro treatment in counteracting oxidative stress damage to DNA, by means of 8-OHdG assay, has been undertaken. Finally, mitochondrial respiratory capacity of sperm has been analyzed by oxygraphic approach, in order to deﬁnitively provide evidence for a speciﬁc activity of myo-inositol in ameliorating the OXPHOS in human sperm. 1. Introduction Indeed, lipid oxidation causes an increase in the permeability of both the plasma membrane and mitochondrial membrane with the resulting decrease in motility and increased cell apoptosis [17,20]. On the other end, mitochondrial DNA (mtDNA) is extremely sensitive to damage from radicals due to the lack of conventional histone proteins and limited mitochondrial damage repair capability; therefore, the resulting accumulation of mtDNA mutations can affect spermatogenetic activity, also causing oligoasthenozoospermia, one of the main causes of male infertility characterized by a reduced number of spermatozoa in the ejaculate and low cell motility, reduced fertilizing capacity and spontaneous abortions in the first months of gestation [21,22]. The 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is known as the early products of oxidative DNA damage [23] and, for this reason, it has been used as a specific and quantitative biomarker for oxidative stress [24,25]. Indeed, many studies demonstrate the existence of a close correlations between 8-OHdG level and sperm morphology and functionality, thus supporting the potential diagnostic value of this assay in clinical practice [26,27]. In light of the here described detrimental eﬀects of oxidative stress on sperm, an antioxidant able to prevent oxidative damage and improve sperm motility could be beneﬁcial in the management of male infertility. In this regard, non-enzymatic antioxidants, both synthetic and dietary supplements, are considered a signiﬁcant therapeutic option. Between others, inositols are cyclic carbohydrates existing in nine stereoisomeric forms: myo-inositol is the most abundant form in nature. Several studies have shown that myo-inositol has powerful antioxidant properties that improve sperm quality, particularly in patients with oligoasthenozoospermia [28–31]. In male reproductive organs, myo-inositol has a central physiologic role; indeed, it is mainly produced by Sertoli cells in response to follicle-stimulating hormone (FSH) and is involved in processes including the regulation of motility, capacitation and acrosome reaction of sperm cell [28,29,32]. Moreover, its concentrations increase from the epididymis to the deferent duct; in fact, myo-inositol would seem to play an important role in the success of spermatogenesis and spermio-histogenesis as well as in osmoregulation of seminal vesicular ﬂuid, contributing to reduce the viscosity [33] and the presence of amorphous material [34] in seminal ﬂuid. Moreover, myo-inositol is involved in the control of intracellular Ca2+ concentration, gene expression, cytoskeleton assembly, inhibition of mechanisms that mediate cell death by apoptosis and in maintaining the mitochondrial membrane potential (MMP) [30]. Through the sophisticated mechanism of the oxidative phosphorylation system J. Clin. Med. 2.2. Sample Treatment The evaluation of the sperm parameters was carried out in accordance with the WHO (2010) criteria [37]. The ejaculate samples examined were collected after a period of abstinence variable from 2 to 5 days, by masturbation, in sterile containers. The evaluation of the seminal parameters was carried out, within 30 min after ﬂuidiﬁcation, by a blinded observer and repeated for quality control by another blinded observer (obtained data are the mean value of two observations). After an accurate resuspension of the sample, sperm concentration, progressive and non-progressive motility, and morphology were evaluated. In order to measure the eﬀect of myo-inositol in vitro treatment on sperm in basal condition, semen samples from 20 normozoospermic patients were divided into three aliquots, each containing about 20 million sperm. These aliquots were incubated at 37 ◦C for 30 min with: (i) standard medium (untreated sample), or medium supplemented with myo-inositol (Andrositol Lab, Lo.Li. Pharma, Rome, Italy) at (ii) 2 mg/mL and (iii) 20 mg/mL (treated sample). After in vitro treatment, sperm concentration and progressive motility were assessed before to proceed to subsequent analyses. In order to measure the eﬀect of myo-inositol in vitro treatment on sperm capacitation, semen samples from 16 normozoospermic patients were divided into two aliquots, each containing about 20 million sperm. These aliquots were incubated with standard medium (untreated sample) or medium supplemented with myo-inositol at 20 mg/mL (treated sample) for 20 min. After that, each aliquot was subjected to the in vitro capacitation by swim-up procedure. Brieﬂy, the semen samples were mixed gently with the Quinn’s Advantage Medium and tubes were centrifuged at 260 gfor 10 min. The supernatant was removed and the pellet was carefully overlaid with 0.8 mL of fresh Quinn’s Sperm Washing Medium and incubated 30 min at 37 ◦C; at the end, an upper layer (0.2 mL) was collected and, by using a Makler counting chamber (Irvine Scientiﬁc, Santa Ana, CA, USA), we assessed progressive motility and sperm. J. Clin. Med. 2020, 9, 1638 4 of 12 2.5. Transmission Electron Microscopy For Transmission Electron Microscopy (TEM), sperm samples in basal condition, in untreated or treated with myo-inositol were ﬁxed in cold Karnovsky ﬁxative and maintained at 4 ◦C for 2 h. Fixed semen was washed in 0.1 mol L−1 cacodylate buﬀer (pH 7.2) for 12 h, postﬁxed in 1% buﬀered osmium tetroxide for 1 h at 4 ◦C, dehydrated and embedded in Epon Araldite resin. Ultra-thin sections were cut with a Supernova ultramicrotome (Reickert Jung, Vienna, Austria), mounted on copper grids, stained with uranyl acetate and lead citrate and observed and photographed with transmission electron microscope (Tecnai G2 Spirit, FEI, Hillsboro, OR, USA) operating at an electron accelerating voltage of 120 kV equipped with a Morada (EMSIS) CCD camera. 2.3. Hypotonic Swelling and Oxygraphic Assay After the in vitro treatment described above, sperm samples were centrifuged at 800 gfor 10 min and then washed by resuspension in isotonic salt medium (113 mmol/L KCl, 12.5 mmol/L of KH2PO4, 2.5 mmol/L of K2HPO4, 3 mmol/L of MgCl2, 0.4 mmol/L of ethylenediaminetetraacetic acid or EDTA and 20 mmol/L of Tris adjusted to pH 7.4 with HCl). Sperm cells were then subjected to hypotonic treatment as described by Stendardi et al. [38]. Brieﬂy, sperm cells were kept in ice-chilled hypotonic medium (potassium phosphate 10 mmol/L, pH 7.4, with 2 g/L of bovine serum albumin or BSA) for 1.5 h. Sperm were then washed three times using isotonic salt medium, pH 7.4 and, sperm concentration was adjusted to about 15 million for basal and 3 million for capacitated sperm samples to be used in each oxygraphic experiment. Oxygen uptake by spermatozoa was measured at 36 ◦C using a Clark-type oxygen probe (HansatechOxygraph; Pentney, King’s Lynn, UK). Demembranated sperm cells were stirred vigorously in the reaction chamber (1 mL) in isotonic salt medium without EDTA and temperature equilibrated. The rate of oxygen uptake by spermatozoa (V) was expressed as nmol O2·mL−1·min−1. The oxygen consumed by the sperm cells was evaluated by calculating the diﬀerence between the oxygen present at the time of the addition of the spermatozoa and that present after 10 min. 2.4. Extraction of Genomic DNA and Measurement of DNA Oxidative Damages Genomic DNA was extracted from 20 treated and 20 untreated samples. Brieﬂy, sperm were centrifuged at 400 × gfor 10 min, then the supernatant was removed. Cells were resuspended in 500 µL of lysis buﬀer (Tris-HCL 25 mM, pH 8.1, EDTA 5 mM, SDS 1%, proteinase K 0.4 mg/mL) and incubated at 55 ◦C for 6 h with gentle agitation. Sperm lysates were phenol/chloroform/isoamyl (25:24:1, pH8) extracted and the DNA recovered by ethanol precipitation in the presence of ammonium acetate and Glycogen. DNA precipitates were resuspended in TE buﬀer (Tris-HCl 10 mM, pH 8.1, EDTA 1 mM). Processed DNA samples were quantiﬁed by Nanodrop apparatus (Thermo Fisher Scientiﬁc) and stored in TE buﬀer at −80 ◦C. The evaluation of oxidative stress in samples treated with myo-inositol versus not treated was performed by -8-hydroxy 2 deoxyguanosine Elisa kit (ab201734, Abcam, Cambridge, UK), according to the instruction for use included in the kit. All samples were tested in duplicate. Absorbance values were measured on a microplate reader. 3. Results J. Clin. Med. 20 3.1. Myo-inositol In Vitro Treatment Increases the Progressive Motility and the Oxygen Consumption Rate of Uncapacitated Sperm 3.1. Myo-inositol In Vitro Treatment Increases the Progressive Motility and the Oxygen Consumption Rate of Uncapacitated Sperm 3.1. Myo-inositol In Vitro Treatment Increases the Progressive Motility and the Oxygen Consumption Rate of Uncapacitated Sperm 3.1. Myo-inositol In Vitro Treatment Increases the Progressive Motility and the Oxygen Consumption Rate of Uncapacitated Sperm The ﬁrst evidence we obtained when semen samples were in vitro treated with myo-inositol, was the complete absence of cell toxicity. Moreover, this compound was demonstrated to signiﬁcantly aﬀect the average progressive motility of sperm. In fact, as shown in Figure 1A, treatment with myo-inositol at a dose of 2 and 20 mg/mL determines a signiﬁcant increase in progressive motility of 9% (p < 0.05) and 13% (p < 0.01), respectively. The first evidence we obtained when semen samples were in vitro treated with myo-inositol, was the complete absence of cell toxicity. Moreover, this compound was demonstrated to significantly affect the average progressive motility of sperm. In fact, as shown in Figure 1A, treatment with myo-inositol at a dose of 2 and 20 mg/mL determines a significant increase in progressive motility of 9% (p < 0.05) and 13% (p < 0.01), respectively. (A) (B) Figure 1. (A) Progressive motility of control spermatozoa (NT, not treated) and treated with myo- inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.05) and 20 mg/mL (dark gray) (p < 0.01). (B) Oxygen consumption, express as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) and treated with myo-inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.01) and 20 mg/mL (dark gray) (*p < 0.001). Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max confidence intervals. Figure 1. (A) Progressive motility of control spermatozoa (NT, not treated) and treated with myo-inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.05) and 20 mg/mL (dark gray) (p < 0.01). (B) Oxygen consumption, express as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) and treated with myo-inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.01) and 20 mg/mL (dark gray) (**p < 0.001). 3. Results J. Clin. Med. 20 Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max conﬁdence intervals. (B) (A) (A) (B) Figure 1. (A) Progressive motility of control spermatozoa (NT, not treated) and treated with myo- inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.05) and 20 mg/mL (dark gray) (p < 0.01). (B) Oxygen consumption, express as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) and treated with myo-inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.01) and 20 mg/mL (dark gray) (**p < 0.001). Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max confidence intervals. Figure 1. (A) Progressive motility of control spermatozoa (NT, not treated) and treated with myo-inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.05) and 20 mg/mL (dark gray) (p < 0.01). (B) Oxygen consumption, express as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) and treated with myo-inositol (Myo) at a dose of 2 mg/mL (light gray) (p < 0.01) and 20 mg/mL (dark gray) (*p < 0.001). Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max conﬁdence intervals. After evaluating the effect of myo-inositol on sperm motility, we assessed oxygen consumption by oxygraphic analysis, as index of oxidative phosphorylation and, as a consequence, of ATP production. According to the increase in progressive motility measured following treatment with myo-inositol, the consumption of O2 by sperm reveals the same trend. Indeed, as shown in Figure 1B, treatment with myo-inositol at a dose of 2 and 20 mg/mL determines a significant dose-dependent increase in oxygen consumption of 24% (p < 0.01) and 44% (p < 0.001), respectively. Interestingly, the highest dose of myo-inositol increases about 1.3 times the oxygen consumption if compared to the lowest dose used (p < 0.01), that is, the dose currently suggested in the literature. After evaluating the eﬀect of myo-inositol on sperm motility, we assessed oxygen consumption by oxygraphic analysis, as index of oxidative phosphorylation and, as a consequence, of ATP production. 2.6. Statistical Analysis A statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). The data are reported as mean ± SD. To detect diﬀerences between the control samples and those treated with myo-inositol, a student’s t-test was performed. Diﬀerences were considered statistically signiﬁcant at p < 0.05. The results were reported as mean ± SD. 5 of 12 J. Clin. Med. 2020, 9, 1638 3. Results J. Clin. Med. 20 Moreover, as shown in Figure 2A, a significant increase in progressive motility of about 10% may be observed (87.3% in untreated versus 94.8% treated samples; p < 0.01). Figure 2. (A) Progressive motility of capacitated spermatozoa (NT, not treated) and treated with 20 mg/mL of myo-inositol (Myo) (p < 0.01). (B) Oxygen consumption, expressed as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) respect to treatment with myo- inositol (Myo) at a dose of 20 mg/mL (*p < 0.001). Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max confidence intervals. Figure 2. (A) Progressive motility of capacitated spermatozoa (NT, not treated) and treated with 20 mg/mL of myo-inositol (Myo) (p < 0.01). (B) Oxygen consumption, expressed as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) respect to treatment with myo-inositol (Myo) at a dose of 20 mg/mL (*p < 0.001). Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max conﬁdence intervals. Figure 2. (A) Progressive motility of capacitated spermatozoa (NT, not treated) and treated with 20 mg/mL of myo-inositol (Myo) (p < 0.01). (B) Oxygen consumption, expressed as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) respect to treatment with myo- inositol (Myo) at a dose of 20 mg/mL (*p < 0.001). Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max confidence intervals Figure 2. (A) Progressive motility of capacitated spermatozoa (NT, not treated) and treated with 20 mg/mL of myo-inositol (Myo) (p < 0.01). (B) Oxygen consumption, expressed as nmol O2·mL−1·min−1 on 10 million sperm cells, of control spermatozoa (NT, not treated) respect to treatment with myo-inositol (Myo) at a dose of 20 mg/mL (**p < 0.001). Graphical diagrams are plotted as box–whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max conﬁdence intervals. 3. Results J. Clin. Med. 20 According to the increase in progressive motility measured following treatment with myo-inositol, the consumption of O2 by sperm reveals the same trend. Indeed, as shown in Figure 1B, treatment with myo-inositol at a dose of 2 and 20 mg/mL determines a signiﬁcant dose-dependent increase in oxygen consumption of 24% (p < 0.01) and 44% (p < 0.001), respectively. Interestingly, the highest dose of myo-inositol increases about 1.3 times the oxygen consumption if compared to the lowest dose used (p < 0.01), that is, the dose currently suggested in the literature. 3.2. Myo-inositol Treatment Before In vitro Sperm Capacitation Increases Swim Up Sperm Recovery, P o e i e Motility a d the O y e Co u tio Rate 3.2. Myo-inositol Treatment Before In vitro Sperm Capacitation Increases Swim Up Sperm Recovery, Progressive Motility and the Oxygen Consumption Rate 3.2. Myo-inositol Treatment Before In vitro Sperm Capacitation Increases Swim Up Sperm Recovery, Progressive Motility and the Oxygen Consumption Rate 3.2. Myo-inositol Treatment Before In vitro Sperm Capacitation Increases Swim Up Sperm Recovery, Progressive Motility and the Oxygen Consumption Rate Progressive Motility and the Oxygen Consumption Rate In order to assess whether myo-inositol treatment may be effective in ameliorating in vitro sperm capacitation procedures, we treated in vitro semen samples for 20 min with the most effective dose of myo-inositol (20 mg/mL) or of standard sperm medium, as a control. After that, semen underwent in vitro capacitation by means of a swim-up procedure. This approach enabled us to demonstrate In order to assess whether myo-inositol treatment may be eﬀective in ameliorating in vitro sperm capacitation procedures, we treated in vitro semen samples for 20 min with the most eﬀective dose of myo-inositol (20 mg/mL) or of standard sperm medium, as a control. After that, semen underwent in vitro capacitation by means of a swim-up procedure. This approach enabled us to demonstrate that 6 of 12 J. Clin. Med. 2020, 9, 1638 in vitro myo-inositol supplementation before swim-up guarantees a sperm recovery increase of about 35% (p < 0.001). Moreover, as shown in Figure 2A, a signiﬁcant increase in progressive motility of about 10% may be observed (87.3% in untreated versus 94.8% treated samples; p < 0.01). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 6 of 12 about 35% (p < 0.001). p p y p y y 3 3 TEM E l ti f U t t d d T t d S l ith M i it l 3.3. TEM Evaluation of Untreated and Treated Sample with Myo-inositol 3.3. TEM Evaluation of Untreated and Treated Sample with Myo-inositol To characterize the sperm at ultrastructural level, semen samples, untreated or treated with myo-inositol at the dose of 20 mg/mL, were analyzed by TEM. The treated sperm did not show appreciable ultrastructural changes; however, it may be highlighted that treated sperm appear extremely “clean” from any amorphous fibrous material (Figure 3C–D) which instead was clearly evident on the surface and in the surrounding environment of the untreated sperm (Figure 3A–B). This could depend on the ability of myo-inositol to disrupt the mucoid masses of the seminal fluid, in agreement with what was claimed by Scarselli et al. [39]. Moreover, in treated samples, several sperm with uncurled tails with well-organized mitochondrial sheath can be frequently found in the same field of observation, that is an unusual finding at the electron microscopy level; it was evident that in the straightened tails mitochondrial cristae appeared expanse and more electrondense To characterize the sperm at ultrastructural level, semen samples, untreated or treated with myo-inositol at the dose of 20 mg/mL, were analyzed by TEM. The treated sperm did not show appreciable ultrastructural changes; however, it may be highlighted that treated sperm appear extremely “clean” from any amorphous ﬁbrous material (Figure 3C–D) which instead was clearly evident on the surface and in the surrounding environment of the untreated sperm (Figure 3A–B). This could depend on the ability of myo-inositol to disrupt the mucoid masses of the seminal ﬂuid, in agreement with what was claimed by Scarselli et al. [39]. Moreover, in treated samples, several sperm with uncurled tails with well-organized mitochondrial sheath can be frequently found in the same ﬁeld of observation, that is an unusual ﬁnding at the electron microscopy level; it was evident that in the straightened tails, mitochondrial cristae appeared expanse and more electrondense. 3. Results J. Clin. Med. 20 When oxygen consumption was assessed in swim-up selected sperm, we provide evidence for a two-fold increase (p < 0.001) in oxygen consumption in sperm selected after myo-inositol in vitro treatment (Figure 2B); this datum is in agreement with both the significant increase in recovered sperm and in progressive motility percentage guaranteed by myo inositol supplementation When oxygen consumption was assessed in swim-up selected sperm, we provide evidence for a two-fold increase (p < 0.001) in oxygen consumption in sperm selected after myo-inositol in vitro treatment (Figure 2B); this datum is in agreement with both the signiﬁcant increase in recovered sperm and in progressive motility percentage, guaranteed by myo-inositol supplementation. When oxygen consumption was assessed in swim-up selected sperm, we provide evidence for a two-fold increase (p < 0.001) in oxygen consumption in sperm selected after myo-inositol in vitro treatment (Figure 2B); this datum is in agreement with both the significant increase in recovered sperm and in progressive motility percentage guaranteed by myo inositol supplementation When oxygen consumption was assessed in swim-up selected sperm, we provide evidence for a two-fold increase (p < 0.001) in oxygen consumption in sperm selected after myo-inositol in vitro treatment (Figure 2B); this datum is in agreement with both the signiﬁcant increase in recovered sperm and in progressive motility percentage, guaranteed by myo-inositol supplementation. g , pp p 3.4. The Use of 8-hydroxy-2′-deoxyguanosine as a Marker of Oxidative Damage to DNA In order to evaluate the ability of myo-inositol to counteract the oxidation damage to DNA, we measured 8-Hydroxy-2′-deoxyguanosine (8-OHdG) level in sperm untreated or treated with myo-inositol 20 mg/mL for 30 min. As shown in Figure 4, a signiﬁcant decrease in 8-OHdG was demonstrated in sperm treated with myo-inositol. Being 8-OHdG a speciﬁc quantitative marker of 7 of 12 7 of 12 J. Clin. Med. 2020, 9, 1638 oxidative DNA damage, our data demonstrate that myo-inositol in vitro treatment could have a key role in protecting sperm from oxidative stress. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 7 of 12 Figure 3. Transmission electron microscopy representative micrographs of sperm untreated (A) and (C); treated with myo-inositol (B) and (D). In the untreated samples, disperse flocculent material is evident around the spermatozoa; the treated samples do not show appreciable ultrastructural changes, but the surrounding environment appears extremely clean and many sperm with straightened tails are found in the same field of observation. (A–B) Bar = 10μm; (C–D): Bar = 2μm. Figure 3. Transmission electron microscopy representative micrographs of sperm untreated (A) and (C); treated with myo-inositol (B) and (D). In the untreated samples, disperse flocculent material is evident around the spermatozoa; the treated samples do not show appreciable ultrastructural changes, but the surrounding environment appears extremely clean and many sperm with straightened tails are found in the same field of observation. (A–B) Bar = 10µm; (C–D): Bar = 2µm. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 8 of 12 Figure 3. Transmission electron microscopy representative micrographs of sperm untreated (A) and (C); treated with myo-inositol (B) and (D). In the untreated samples, disperse flocculent material is evident around the spermatozoa; the treated samples do not show appreciable ultrastructural changes, but the surrounding environment appears extremely clean and many sperm with straightened tails are found in the same field of observation. (A–B) Bar = 10μm; (C–D): Bar = 2μm. Figure 3. Transmission electron microscopy representative micrographs of sperm untreated (A) and (C); treated with myo-inositol (B) and (D). In the untreated samples, disperse flocculent material is evident around the spermatozoa; the treated samples do not show appreciable ultrastructural changes, but the surrounding environment appears extremely clean and many sperm with straightened tails are found in the same field of observation. (A–B) Bar = 10µm; (C–D): Bar = 2µm. Clin. Med. 4. Discussion To the best of our knowledge, this study represents the ﬁrst complete evaluation of the mechanisms induced in human sperm by myo-inositol in vitro treatment. First of all, here we conﬁrm that in vitro supplementation of myo-inositol is able to signiﬁcantly improve sperm motility in a dose-dependent manner, when compared to placebo supplemented media. In particular, treatment with myo-inositol of uncapacitated sperm increases progressive motility of up to 13% (p < 0.05), and interestingly, the addition of myo-inositol before swim-up signiﬁcantly increases the number of recovered sperm as well as their progressive motility, suggesting this treatment as recommended to improve in vitro capacitation rescue. p Several studies have reported the eﬃcacy of oral myo-inositol administration in patients with idiopathic infertility in ameliorating main sperm parameters such as number, concentration and motility [29,32,40]. Moreover, other studies proved that in vitro incubation with myo-inositol increases significantly progressive and total motility in normozoospermic and in oligo-astheno-teratozoospermic men [30,32,33,41,42]. This protective activity exerted by inositols appears to be due to its antioxidant properties: in fact, myo-inositol has been listed among the antioxidants able to ameliorate main sperm parameters, even if the mechanisms underlying this activity are still now largely unknown. Due to the lack of cytoplasmic defenses and to the high concentration of polyunsaturated fatty acids in their plasma membrane, sperm are very susceptible to oxidative stress triggered by increased concentration of ROS levels. Noteworthily, the damage to the mitochondrial membrane induced by high levels of ROS may compromise ATP synthesis that, consequently, impairs sperm function, such as viability and motility [36,38,43]. Moreover, a vicious circle is established, since the damage to the plasma membrane induces, in turn, an increase in the concentration of ROS [44]. To this regard, the main strength of the present study relies on the use of oxygraphic analysis as a powerful strategy to measure mitochondrial activity. In this way, not only do we confirm the positive effect of myo-inositol on mitochondria, but, for the first time, we provide clear evidence that this powerful antioxidant specifically acts by increasing OXPHOS efficiency, both in basal condition and, to a greater extent, in in vitro capacitation. Indeed, to date, only indirect evidence has been reported, as the increase in mitochondrial membrane permeability highlighted by immunofluorescent staining with the marker JC-1 [34,45]. g , pp p 3.4. The Use of 8-hydroxy-2′-deoxyguanosine as a Marker of Oxidative Damage to DNA 2020, 9, x FOR PEER REVIEW 8 of 1 3.4. The Use of 8-hydroxy-2′-deoxyguanosine as a Marker of Oxidative Damage to DNA In order to evaluate the ability of myo-inositol to counteract the oxidation damage to DNA, we measured 8-Hydroxy-2′-deoxyguanosine (8-OHdG) level in sperm untreated or treated with myo- inositol 20 mg/mL for 30 min. As shown in Figure 4, a significant decrease in 8-OHdG was demonstrated in sperm treated with myo-inositol. Being 8-OHdG a specific quantitative marker of oxidative DNA damage, our data demonstrate that myo-inositol in vitro treatment could have a key role in protecting sperm from oxidative stress. Figure 4. Level of oxy-DNA, measuring the 8-Hydroxy-2′-deoxyguanosine (8-OHdG) level, expressed in ng/mL, in treated and untreated (NT) sample with myo-inositol (Myo). The graph shows the normalized average oxy-DNA expressed, the standard deviation and the statistical significance of the data (p < 0.05). Figure 4. Level of oxy-DNA, measuring the 8-Hydroxy-2′-deoxyguanosine (8-OHdG) level, expressed in ng/mL, in treated and untreated (NT) sample with myo-inositol (Myo). The graph shows the normalized average oxy-DNA expressed, the standard deviation and the statistical significance of the data (p < 0.05). Figure 4. Level of oxy-DNA, measuring the 8-Hydroxy-2′-deoxyguanosine (8-OHdG) level, expressed in ng/mL, in treated and untreated (NT) sample with myo-inositol (Myo). The graph shows the normalized average oxy-DNA expressed, the standard deviation and the statistical significance of the data (p < 0 05) Figure 4. Level of oxy-DNA, measuring the 8-Hydroxy-2′-deoxyguanosine (8-OHdG) level, expressed in ng/mL, in treated and untreated (NT) sample with myo-inositol (Myo). The graph shows the normalized average oxy-DNA expressed, the standard deviation and the statistical significance of the data (*p < 0.05). J. Clin. Med. 2020, 9, 1638 8 of 12 4. Discussion Our data, evidencing a more eﬃcient OXPHOS in treated sperm, are in agreement with the current knowledge on molecular pathway elicited by myo-inositol. Indeed, it has been reported that this compound, by increasing cytosolic and mitochondrial calcium levels, may make oxidative mechanisms more eﬀective, thus ameliorating sperm mitochondrial function, preventing apoptosis, and facilitating chromatin compactness [30]. Here, we provide evidence for a signiﬁcant increase of oxygen consumption in in vitro selected sperm compared to sperm in basal condition. This may be explained by the observation that more than a higher percentage of swim-up selected sperm shows a rapid progressive motility, when compared to basal samples. Sperm motility is sustained by a continuous production of ATP from the mitochondrial respiratory chain, strictly coupled to ATP synthase, that is much more eﬃcient in energy production than glycolysis [38]. Therefore, our study deﬁnitively conﬁrms that the demonstrated capability of myo-inositol to signiﬁcantly improve sperm motility is related to its in-vitro ability to improve sperm mitochondrial function. Moreover, our in vitro study provides signiﬁcant information that, if properly translated into the clinical practice, may improve procedures currently used in ART laboratories, in order to ameliorate in vitro fertilization (IVF) protocols. Indeed, it is well known that oxidative stress increases during in vitro sperm selection procedures, consequently increasing DNA damage [46,47], thus OS represents a real threat not only for the fertilization rate, but also for the health of the oﬀspring. Indeed, bypassing all natural sperm selection strategies, assisted fertilization techniques signiﬁcantly increase the risk that sperm with damaged DNA can fertilize the oocyte [48]. To this regard, another strength point of this study was the deﬁnitive demonstration that myo-inositol supplementation in media used for semen selection can signiﬁcantly protect DNA from 9 of 12 J. Clin. Med. 2020, 9, 1638 oxidative damage during the in vitro sperm selection procedures. The 8-OHdG, one of the early products of oxidative DNA damage [23] was measured in present study, clearly demonstrating a signiﬁcant decrease in this oxidative stress marker in the samples treated with myo-inositol. The inverse relationship, demonstrated in this study, between oxidative DNA damage and myo-inositol treatment is consistent with previous studies reporting the existence of a close correlation between 8-OHdG levels and sperm morphology and functionality [26,49,50], further supporting the potential value of myo-inositol use in the routine of IVF laboratory. 4. Discussion y y Finally, interesting insights come from the ultrastructural evaluation of sperm treated with myo-inositol. Here, we provide evidence that this antioxidant is able to reduce the presence of amorphous ﬁbrous material around sperm and we conﬁrm a previous study demonstrating that myo-inositol is able to reduce mitochondrial cristae damage [34]. Therefore, we may hypothesize that the signiﬁcant increase in sperm motility that we demonstrated both in basal and in capacitated sperm may be due to ability of myo-inositol to reduce the viscosity, by removing this amorphous material. The systematic methodological approach set up in this study allows to overcome the incomplete results still now presented in the literature, thus clarifying the pivotal role of antioxidant strategies in ameliorating sperm motility and OXPHOS eﬃcacy. Although further studies are needed to fully understand the mechanism of action of myo-inositol, these results conﬁrm its eﬀectiveness in ameliorating sperm parameters during in vitro selection procedures and demonstrate that this compound speciﬁcally acts at the mitochondrial level, where it exerts positive eﬀects on oxidative phosphorylation process and ATP production, thus explaining the improvement of sperm motility. With the increasing awareness and understanding of the protective eﬀect of myo-inositol on sperm provided in this study, the development of evidence-based guidelines, aiming to make more eﬀective the in vitro procedure currently used in ART laboratory for sperm selection, seems to be crucial. Indeed, protecting sperm against damage from ROS will be an important tool to minimize the risk that sperm with damaged DNA can be included in the fertilizing pool of sperm. Author Contributions: Resources: P.P. and P.G.A.; Methodology: R.P.; Investigation: L.G., R.P., E.C., I.M. and A.C.; Formal analysis: L.G.; Writing—Original draft: A.L., L.G. and R.P.; Writing—Review and editing: P.P., P.G.A. and A.L.; Validation: A.L, P.P. and P.G.A; Supervision: P.P. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by Siena University and Pisa University. Funding: This study was supported by Siena University and Pisa University. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Vatannejad, A.; Tavilani, H.; Sadeghi, M.R.; Amanpour, S.; Shapourizadeh, S.; Doosti, M. Evaluation of ROS-TAC Score and DNA Damage in Fertile normozoospermic and infertile asthenozoospermic males. Urol. J. 2017, 14, 2973–2978. [CrossRef] 0. Agarwal, A.; Sharma, R.K.; Desai, N.R.; Prabakaran, S.; Tavares, A.; Sabanegh, E. 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https://openalex.org/W4282040283	https://zenodo.org/record/6634307/files/19.pdf	English	null	NATURAL DIET OF AMNIRANA ALBOLABRIS (HALLOWELL, 1856), A FROG SPECIE FROM BANCO NATIONAL PARK (COTE DIVOIRE)	International journal of advanced research	2,022	cc-by	6,178	Abstract Therefore, it seems important to us to conduct order to provide a better knowledge of the natural food habit of Amnirana albolabris from the Banco Nation Corresponding Author:- Konate Beh Romaric Address:- Laboratoire d’Environnement et de Biologie Aquatique (LEBA), UFR-Sciences et Gestion de l’Environnement (SGE), Université Nangui Abrogoua (UNA), 02BP 801, Abidjan 02, (Côte d’Ivoire). ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 10(05), 383-392 Journal Homepage: -www.journalijar.com Article DOI:10.21474/IJAR01/14719 DOI URL: http://dx.doi.org/10.21474/IJAR01/14719 NATURAL DIET OF AMNIRANA ALBOLABRIS (HALLOWELL, 1856), A FROG SPECIE FROM BANCO NATIONAL PARK (CÔTE D'IVOIRE) Konate Beh Romaric, Tohe Blayda and Agou Louis César Laboratoire d’Environnement et de Biologie Aquatique (LEBA), UFR-Sciences et Gestion de l’Environnement (SGE), Université Nangui Abrogoua (UNA), 02BP 801, Abidjan 02, (Côte d’Ivoire). Abstract ……………………………………………………………… We have established the natural diet of Amnirana albolabris from the Banco National Park (Cote d'Ivoire), through this study. Firstly, a general analysis of prey composition in the stomach of 238 specimens (obtained by the force-feeding technique) has been done, and secondly, according to the age, sex of specimens, climatic seasons, and sampling sites. Nine categories of prey (Insects, Arachnids, Annelids, Molluscs, Myriapods, Crustaceans, animal debris, macrophytes, and undetermined prey) have been identified in the diet of this species of frog. Adult's diet is statistically different from juvenile one. However, males and females consume the same foods. In terms of the climatic seasons, the foods consumed during the long rainy season differ from those during the short dry season. Similarly, the foods eaten during the major dry season differ from those consumed by this frog during the minor dry season. At the spatial level, the diet of this species does not differ significantly from one habitat to another. The diet composition of Amnirana albolabris revealed that this frog species has an omnivorous diet with an insectivorous tendency in Banco National Park. Copy Right, IJAR, 2022,. All rights reserved. ……………………………….... Copy Right, IJAR, 2022,. All rights reserved. ……………………………………………………………………………………………… Introduction:- The diet of amphibians is generally influenced by the availability of food in their environments. It also de their ability to capture and the selection of prey they operate on. Amphibians are recognized as animal eclectic diet (Perret, 1979). In these animals, adult individuals generally attack only alive and mobile prey species of frogs have a diet composed of small prey such as insects. This is the case for Ptychadena longir P. oxyrhynchus (Konan et al., 2016a) and the tree frog Scinax squalirostris (Kittel & Solé, 2015). In add presence of plant fragments in the stomach contents of amphibians has been mentioned (Kouamé et al., 20 et al., 2015; Konan et al., 2016b). Concerning Amnirana albolabris little information is available in ou Theinformation available on this species of frog is that of Assemian, (2009) who reported its presence National Park (Côte d'Ivoire), and Konaté et al. (2021; 2022) whose work relates to its reproduction Regarding its diet, no data is yet available in our country. Statistical analysis Statistical analysis Non-parametric tests have been done by using Mann-Whitney and Kruskal-Wallis (n < 30). These tests allowed us to compare the diet of the species studied and were carried out by using the STATISTICA software version 7.1 Volume percentage p g This parameter consists in evaluating the volume of each fully preserved prey (Griffiths & Mylotte, 1987). Its formula is as follows: This parameter consists in evaluating the volume of each fully preserved prey (Griffiths & Mylotte, 1987). Its formula is as follows: 𝐕= 𝟒 𝟑𝛑( 𝟏 𝟐𝐋) × ( 𝟏 𝟐𝐥)𝟐 ; with: L= prey length; l = width of the prey. 𝐕= 𝟒 𝟑𝛑( 𝟏 𝟐𝐋) × ( 𝟏 𝟐𝐥)𝟐 ; with: L= prey length; l = width of the prey. Materials and Methods:- Study site The study was conducted in the Banco National Park (Figure 1) located within the vast urban area of Abidjan between 5°21' and 5°25' north latitude and between 4°1' and 4°5' west longitude with an altitude between 0 and 113 m (De Koning, 1983). Relative importance index The use of the relative importance index (IRI) was chosen in this study because it gives a good interpretation of the diet. The various indices above seem to give an incomplete idea of the diet (Wootton, 1990). According to Pinkas et al. (1971), its formula is as follows : 𝐈𝐑𝐈= % 𝐍+ % 𝐕 × % 𝐅 Frequency of occurrence It consists of counting the number of stomachs in which prey is present (Gray et al. 1997; Young et al. 1997). Its formula is as follows: It consists of counting the number of stomachs in which prey is present (Gray et al. 1997; Young et al. 1997). Its formula is as follows: F= (Nc / Nt)100 ; with: Nc= number of stomachs containing a category of prey “i” ; Nt = number of non-empty stomachs. F= (Nc / Nt)100 ; with: Nc= number of stomachs containing a category of prey “i” ; Nt = number of non-empty stomachs. Numerical percentage The numerical percentage (N) consisted in counting tach category of prey individuals “i” for a given sample (Hureau, 1970). It expresses the ratio of the number of individuals of an identified prey to all the different categories of prey inventoried. Its formula is as follows: 𝐍= 𝐍𝐢 𝐍𝐞 ∗𝟏𝟎𝟎 ; With : Ni total number of prey “i”; Ne = total number of prey inventoried. 𝐍= 𝐍𝐢 𝐍𝐞 ∗𝟏𝟎𝟎 ; With : Ni total number of prey “i”; Ne = total number of prey inventoried. Methodology:- Two samplings have been performed at night each month for 12 months. A total of 274 specimens of Amnirana albolabris were sampled. The method of capture consisted of immobilizing the frog with the light of the torch and then capturing it by hand. After each sampling, the stomach contents were obtained using the force-feeding technique proposed by Solé et al. (2005), Hirschfeld & Rödel (2011), and Kittel & Solé (2015). This method consisted of inserting a gavage syringe containing water into the frog's snout. Then collect the stomach contents in a jar. This operation was repeated two (02) times. The stomach content obtained is kept in pillboxes containing 70% alcohol. Stomachs are considered empty when there is no food in the jar. Each prey was identified by using a binocular magnifier and the identification keys of Roth (1979) and Tachet et al. (2010). Introduction:- The diet of amphibians is generally influenced by the availability of food in their environments. It also depends on their ability to capture and the selection of prey they operate on. Amphibians are recognized as animals with an eclectic diet (Perret, 1979). In these animals, adult individuals generally attack only alive and mobile prey. Several species of frogs have a diet composed of small prey such as insects. This is the case for Ptychadena longirostris and P. oxyrhynchus (Konan et al., 2016a) and the tree frog Scinax squalirostris (Kittel & Solé, 2015). In addition, the presence of plant fragments in the stomach contents of amphibians has been mentioned (Kouamé et al., 2008a; Tohé et al., 2015; Konan et al., 2016b). Concerning Amnirana albolabris little information is available in our country. Theinformation available on this species of frog is that of Assemian, (2009) who reported its presence in Banco National Park (Côte d'Ivoire), and Konaté et al. (2021; 2022) whose work relates to its reproduction strategy. Regarding its diet, no data is yet available in our country. Therefore, it seems important to us to conduct a study in order to provide a better knowledge of the natural food habit of Amnirana albolabris from the Banco National Park. Corresponding Author:- Konate Beh Romaric Address:- Laboratoire d’Environnement et de Biologie Aquatique (LEBA), UFR-Sciences et Gestion de l’Environnement (SGE), Université Nangui Abrogoua (UNA), 02BP 801, Abidjan 02, (Côte d’Ivoire). Corresponding Author:- Konate Beh Romaric Address:- Laboratoire d’Environnement et de Biologie Aquatique (LEBA), UFR-Sciences et Gestion de l’Environnement (SGE), Université Nangui Abrogoua (UNA), 02BP 801, Abidjan 02, (Côte d’Ivoire). 383 Int. J. Adv. Res. 10(05), 383-392 Int. J. Adv. Res. 10(05), 383-392 Int. J. Adv. Res. 10(05), 383-392 ISSN: 2320-5407 Composition of the diet according to sex p g The composition of the diet of males and females of Amnirana albolabris is shown in Table III. The food spectrum of male specimens consists of eight categories of prey against nine in females. Males feed on Insects (IRI = 63.79%) as preferential food. Molluscs (IRI= 16.03%) are their secondary prey. Others constitute the accessory food. As for the females, they feed preferentially on insects and molluscs with respective indices of 43.65% and 36.57%. The other preysare eaten accidentally. Regarding insects, males and females consume seven orders in common, namely, Coleoptera, Hymenoptera, Trichoptera, Lepidoptera, Isoptera, Diptera, and Dermaptera. Insects belonging to the order Heteroptera, Orthoptera, and Odonata are specific to the diets of male individuals. Statistical analysis reveals that the diet of male specimens does not differ from the diet of females (Mann-Whitney test n1= 18; n2= 16; U = 125, Z= 0.65; p-value = 0, 52). Int. J. Adv. Res. 10(05), 383-392 this frog. It feeds on Molluscs (IRI = 20.42%) as secondary food. The other foods whose index is less than 5% are accessory foods. With respect to insects, ten orders have been identified in the alimentary bolus of this frog. Among these orders, Hymenoptera and Coleoptera with respective relative importance indices of 24% and 21.67% are the most consumed by this species. Diet by age group T bl II i Table II summarizes the diet composition of adult and juvenile specimens of Amnirana albolabris. The food spectrum of adults is composed of nine types of prey (Insects, Arachnids, Annelids, Molluscs, Myriapods, Crustaceans, Animal Debris, Macrophytes, and Indeterminates) against three (Insects, Molluscs, and Macrophytes) for juveniles. In juveniles, insects (IRI= 80.87%) constitute the main prey. the other prey constitutes the accessory food. On the other hand, adult specimens feed preferentially on Insects (IRI = 57.16%), and molluscs (IRI = 22.9%) represent their secondary foods. All other foods with an index below 6% are accidentally eaten. Adult and juvenile specimens have insects, molluscs, and macrophytes in common in their food bowls. With respect to insects, six orders (Coleoptera, Hymenoptera, Lepidoptera, Isoptera, Orthoptera, and Odonata) are common to the diets of the two age groups. Adults also consume Trichoptera, Heteroptera, Diptera, and Dermaptera. In adults as in juveniles, the Hymenoptera constitute the most abundant order of insects with respective indices of 22.14% and 38.38%. Beetle tracking (19.98% in adults and 19.09% in juveniles).Statistically, the diet of juveniles differs significantly from the diet of adults (Mann-Whitney test, n1 = 19; n2 = 9; U = 50; z = -1.74; p = 0.001) General diet The stomach contents of 274 specimens of Amnirana albolabris were examined (Table I). The snout-anus length of the sampled specimens varies from 35 to 72 mm. Thirty-six stomachs analyzed are empty (either an emptiness index of 13.14%) and 238 contain food. Nine food categories have been identified in the stomachs of this species of frog. These include Insects, Arachnids, Annelids, Molluscs, Myriapods, Crustaceans, Animal Debris, Macrophytes, and undetermined prey. To these nine are added grains of sand. Insects represent the preferential food (IRI = 61.84%) of 384 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 10(05), 383-392 Discussion:- The diet was studied in general and then according to sex, age, seasons and sampling sites in Amnirana albolabris from Banco National Park. Regarding the general diet, nine food categories were observed in the dietary spectrum of the specimens. These include insects, arachnids, annelids, molluscs, myriapods, crustaceans, plant debris, undetermined prey, and animal debris. A. albolabris is therefore an omnivorous species. Among these food categories, insects are the most abundant prey with a relative importance index of 61.84%. This frog is omnivorous with an insectivorous tendency. Our results corroborate those of Cibiha (2017) who reports that the diet of A. albolabris in Masako (Congo) is dominated by insects. Moreover, among these insects, those belonging to the order of Hymenoptera and Coleoptera constitute an important fraction of the food bolus of this frog. The abundance of these prey could be related to their availability in the environment. Grains of sand have no nutritional value. Their ingestion would probably have taken place at the time of food intake. Indeed, frogs catch their prey and it is surely at this moment that the ingestion of the grains of sand and plant debris present in the stomach would take place. The low index percentages of these items would indicate that they would be consumed involuntarily by specimens of A. albolabris. Indeed, the frogs by capturing the prey fixed on the plants or on the ground consume the plants as well as the grains of sand in an involuntary way. Such observations have been made by Hill et al. (2015), Kouame et al. (2008) and Assemian et al. (2015). Furthermore, some studies on anurans have reported that frogs can ingest a large number of plants (Kouamé et al. 2008; Ogoanah & Uchedike, 2011; Tohé et al., 2014). Relative to the diet according to the size of specimens of A. albolabris, the diet of adults shows significant differences from juveniles (p = 0.001; Mann-Whitney test). Insects represent the preferred food (57.16%) in adults and they constitute the bulk of the food bolus in juveniles (80.87%). Adult specimens consume prey of more diverse sizes ranging from insects (small size) to molluscs (large size). Indeed, frogs grab their food. Thus, the larger oral cavity of adult individuals could explain the presence of large prey in their diet. Int. J. Adv. Res. 10(05), 383-392 Similarly, the prey consumed by Amnirana albolabris during the LRS are statically different from those consumed during the SRS (Mann-Whitney test; n1= 18, n2 = 6, U = 23, Z = - 2.06; p -value = 0.039). In addition, the foods consumed during the long rainy season are not statistically different from those consumed during the long dry season (Mann-Whitney test; n1= 18, n2 = 17, U = 149, Z = 0.13; p-value = 0.91). Also the foods consumed during SRS do not differ statistically from those consumed in SDS (Mann-Whitney test; n1= 11, n2 = 6, U = 22, Z = 1.10; p-value = 0.3). Spatial distribution of diet Table V presents the composition of the food spectrum and the relative importance index (IRI in %) of Amnirana albolabris in the three surveyed habitats. The distribution gives nine (09) food categories at the fish farm, against eight (08) in the intermediate habitat and at the bay. At the fish farm, these frogs consume insects (IRI = 68.53%) as preferential food and Molluscs (IRI = 21.31%) as secondary food. Relative to the intermediate habitat, insects (IRI = 56.03%) are the preferred prey and molluscs (IRI = 26.67%) are consumed secondarily in this environment. As for the berry, the preferred food is insects (IRI = 61.47%). Myriapods (IRI= 11.64%) and indeterminate prey constitute the secondary food in this environment. The prey consumed did not show any significant difference from one habitat to another (Kruskal-Walis test, n1= 17; n2 = 15; n3 = 16, p-value = 0.47). Regarding the insects consumed, six (06) orders (Coleoptera, Hymenoptera, Trichoptera, Lepidoptera, and Isoptera) are identified in the food bolus of this frog in the three habitats. Dermaptera and Odonata are missing in the stomach contents respectively at the fish farm and in the intermediate habitat. As for the Heteroptera, they are non-existent in the foods at bay and in the intermediate habitat. ISSN: 2320-5407 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 10(05), 383-392 Seasonal variation in diet. Table IV illustrates the food spectrum in Amnirana albolabris from the Banco National Park according to the climatic seasons. The distribution gives nine (09) food types during the long rainy season (LRS), seven (07) during the short rainy season (SRS) against ten (10) and four (0 4) during the long dry season (LDS) and the small dry season (SDS) respectively. Insects (IRI= 64.81%) constitute the preferential prey and Molluscs (IRI= 20.72%) constitute the secondary food of this frog during the LRS. As for the short rainy season, these individuals consume insects (IRI= 57.57%) as preferential prey as well as arachnids (IRI= 14.6%), and molluscs (IRI= 11.56%) as secondary food. The other foods are eaten accidentally. During the great dry season, these frogs feed on insects (IRI= 59.2%) as preferential food as well as Macrophytes and Molluscs with the respective index of 14.64 and 12.18% as secondary prey. However, during the small dry season, molluscs (50.39%) constitute the preferential food while insects represent the secondary food (IRI = 36.96%). All other prey is incidental food. Concerning the orders of Insects, during the LRS and the SRS, the Hymenoptera (with respectively 33.29% and 35.68%) and the Coleoptera (respectively 12.67% and 12.39%) are the most abundant. Coleoptera (IRI= 42.49%) constitute the dominant food of this frog during the LDS. Lepidoptera (IRI= 19.22%) and Coleoptera (IRI= 14.91%) are the most abundant orders during SDS. Statistically, there is no significant difference between the foods consumed by these individuals during the long and short rainy season (Mann-Whitney test, n1 = 18; n 2 = 11; U = 65, Z = -1, 52; p = 0.133). However the foods consumed by this frog during the long dry season differ from those consumed during the short dry season (Mann-Whitney test; n1= 17, n2 = 6, U = 20, Z = - 2.17 ; p-value = 0.029). 385 ISSN: 2320-5407 ISSN: 2320-5407 Regarding the diet according to sex, the results show that the foods consumed by males and females are not significantly different (P= 0.52; Mann-Whitney test). In addition, the proportion of insects in the food bolus of male specimens is higher than that of females. This result could be explained by the fact that the males have a skill in jumping, this would facilitate them to be more active and to capture more easily the more mobile prey. Hymenoptera and Coleoptera are abundant in the food spectrum of both males and females. This result could be explained by the abundance of these insects in this biotope. Indeed, variations in the use of resources between the different sexes, according to Belovsky (1978), shine et al. (2002), and Martins et al. (2006), would result from different behavioral or energetic morphologies related to reproduction. In addition, observations reveal that large preys are more abundant in the stomach contents of female specimens than males. This result would be related to the size of the specimens. Indeed, in A. albolabris, females that are large ingest large prey more easily. Regarding the seasonal variation of the diet, the results indicate that except for the short dry season, the food consumed by this species is dominated by insects. Moreover, the proportions of insects consumed during the rainy seasons (short and long rainy seasons) are more important in the food spectrum of A. albolabris than those of the dry seasons (small and long dry seasons). This abundance of insects in the food bowl during the rainy season could be explained by the availability of these prey in the environment. Indeed, this climatic period would be favorable to the proliferation and development of insects. Moreover, on this subject, Dietoa (2002) specifies that reproduction in most insects coincides with the rainy season, which would explain their abundance in the environment during the rainy season and therefore their presence in the food bolus at this time of the year. On this subject, Tohé et al. (2014; 2015) reported in Hoplobatrachus occipitalis and Phrynobatrachus latifrons an abundance of insects in the diet during the rainy season. At the spatial level, the food resources of Amnirana albolabris in the three habitats are diversified. Insects are the staple food in these habitats. In addition Hymenoptera, Coleoptera and Molluscs are more consumed at the fish farm. Conclusion:- Amnirana albolabris in Banco National Park has an omnivorous diet with an insectivorous tendency. Among the insect orders, Coleoptera and Hymenoptera are the most represented.The diet of this frog varies with the seasons and the different age groups (adults, juveniles). ISSN: 2320-5407 On the other hand, in the intermediate habitat, Lepidoptera and Molluscs constitute the most abundant prey. As for the berry, Coleoptera and Lepidoptera are abundant in the food bowl of this frog. However, the differences between diets from one habitat to another are not significant (Kruskal-Walis test, p-value = 0.47). The differences observed between prey from one habitat to another would be linked to the environmental conditions of each biotope. Discussion:- Also, the abundance of Hymenoptera and Coleoptera which are small-sized foods as essential resources in the food spectrum of juveniles would be linked to the smallness of their oral cavity. This observation was also made by Tohé (2009) who reported in Hoplobatrachus occipitalis, Ptychadena mascareniensis, and P. pumilio from the Banco National Park that adults consume larger prey than juveniles. 386 Int. J. Adv. Res. 10(05), 383-392 Int. J. Adv. Res. 10(05), 383-392 ISSN: 2320-5407 Acknowledgment:- 10(05), 383-392 Table II:- Compositions of the diet and indices of relative importance IRI (in %) of Amnirana albolabris specimens according to age classes from Banco National Park: n = number of full stomachs. Table III:- Compositions of the diet and indices of relative importance (IRI %) of the different food categories contained in the food bolus of Amnirana albolabris according to sex in Banco National Park; n = number of full stomachs. Ingested prey IRI (%) Adults (n = 223) (Juveniles n = 15) Insects Coleoptera 19.98 19.09 Hymenoptera 22.14 38.38 Trichoptera 2.94 0 Heteroptera 0.02 0 Lepidoptera 6.12 8.06 Isoptera 2.2 4.45 Diptera 1.06 0 Orthoptera 1.65 9.69 Dermaptera 0.49 0 Odonata 0.55 1.19 ∑Insects 57.16 80.87 Arachnids 5.07 0 Annelids 1.12 0 Molluscs 22.9 2.7 Myriapods 4.18 0 Crustaceans 0.12 0 Animal Debris 2.35 0 Macrophytes 2.77 14.8 Indeterminates 3.53 0 Sand 0.8 1.62 IRI (%) Ingested prey Males n = 199 Females n = 24 Insects Coleoptera 20.56 16.61 Hymenoptera 21.63 21 Trichoptera 4.21 0.94 Heteroptera 0.05 0 Lepidoptera 7.43 3.47 Isoptera 2.82 1.08 Diptera 1.41 0.44 Orthoptera 3.69 0 Dermaptera 0.76 0.11 Odonata 1.24 0 ∑Insects 63.79 43.65 Arachnids 5.25 4.28 Annelids 1.3 0.69 Molluscs 16.03 36.57 Myriapods 3.77 4.41 Crustaceans 0 1.02 Animal Debris 4.11 0.25 Macrophytes 1.96 4.29 Indeterminates 3.28 3.66 Sand 0.51 1.18 Table II:- Compositions of the diet and indices of relative importance IRI (in %) of Amnirana albolabris specimens according to age classes from Banco National Park: n = number of full stomachs. Ingested prey IRI (%) Adults (n = 223) (Juveniles n = 15) Insects Coleoptera 19.98 19.09 Hymenoptera 22.14 38.38 Trichoptera 2.94 0 Heteroptera 0.02 0 Lepidoptera 6.12 8.06 Isoptera 2.2 4.45 Diptera 1.06 0 Orthoptera 1.65 9.69 Dermaptera 0.49 0 Odonata 0.55 1.19 ∑Insects 57.16 80.87 Arachnids 5.07 0 Annelids 1.12 0 Molluscs 22.9 2.7 Myriapods 4.18 0 Crustaceans 0.12 0 Animal Debris 2.35 0 Macrophytes 2.77 14.8 Indeterminates 3.53 0 Sand 0.8 1.62 ositions of the diet and indices of relative importance IRI (in %) of Amnirana albolabris specimens classes from Banco National Park: n = number of full stomachs. Acknowledgment:- g At the end of this study, we thank the Director of the Ivorian Office of Parks and Reserves (OIPR) for granting us authorization to access the park. We also thank the water and forest agents as well as the wardens for their support. Our thanks also go to the habitats of the park for the accommodation as well as their courtesy. 387 Int. J. Adv. Res. 10(05), 383-392 ISSN: 2320-5407 Figures:- Figures: Fig. 1:- Geographical position of Banco National Park (Ivory Coast). Figure:- 1 Figure:- 1 Fig. 1:- Geographical position of Banco National Park (Ivory Coast). Table I:- Compositions of the general diet and indices of relative importance (IRI %) of the different categories of prey in Amnirana albolabris from Banco National Park: F = Frequency of occurrence N = numerical percentage, V = volume percentage. Table I:- Compositions of the general diet and indices of relative importance (IRI %) of the different categories of prey in Amnirana albolabris from Banco National Park: F = Frequency of occurrence N = numerical percentage, V = volume percentage Table I:- Compositions of the general diet and indices of relative importance (IRI %) of the different categories of prey in Amnirana albolabris from Banco National Park: F = Frequency of occurrence N = numerical percentage, V l Table I:- Compositions of the general diet and indices of relative importance (IRI %) of the different categories of prey in Amnirana albolabris from Banco National Park: F = Frequency of occurrence N = numerical percentage, V = volume percentage. Ingested prey Indices (%) F N V IRI Insects Coleoptera 14.98 12.46 11.10 21.67 Hymenoptera 12.78 20.58 10.54 24.41 Trichoptera 3,96 3,19 6,86 2.45 Heteroptera 0.44 0.29 0.33 0.02 Lepidoptera 7.05 7.54 7.40 6.47 Isoptera 4.41 4.64 4.56 2.49 Diptera 2.20 2.32 4.17 0.88 Orthoptera 4.85 3.77 4.38 2.42 Dermaptera 2.20 2.03 0.94 0.40 Odonata 2.20 2.32 2.40 0.64 ∑Insects 55.07 59.13 52.69 61.84 Arachnids 4.41 3.77 11.87 4.23 Annelids 2.64 2.32 3.41 0.93 Molluscs 13.22 13.62 11.53 20.42 Myriapods 5.29 4.06 6.63 3.47 Crustaceans 0.88 0.58 1.29 0.10 Animal Debris 3.96 3.19 4.83 1.95 Macrophytes 6.61 6.09 2.87 3.64 Indeterminates 5.29 4.64 4.34 2.92 Sand 2.64 2.61 0.53 0.51 388 Int. J. Adv. Res. 10(05), 383-392 Int. J. Adv. Res. 10(05), 383-392 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. Int. J. Adv. Res. 10(05), 383-392 Int. J. Adv. Res. 10(05), 383-392 ISSN: 2320-5407 Table IV:- Compositions of the diet and indices of relative importance IRI (in %) of specimens of Amnirana albolabris according to the seasons; n = number of stomachs containing food; LRS= Long Rainy Season; SRS= Short Rainy Season; LDS= Long Dry Season; SDS= small Dry Season. IRI(%) Ingested prey LRS (n = 97) SRS (n = 33) LDS (n = 74) SDS (n = 27) Insectes Coleoptera 12.67 12.39 42.49 14.91 Hymenoptera 33.29 35.68 6.01 0 Trichoptera 4.56 0 0.87 0 Heteroptera 0.05 0 0 0 Lepidoptera 5.25 1.76 5.87 19.22 Isoptera 3.97 3.26 0.14 0 Diptera 1.07 4.48 0 0 Orthoptera 2.18 0 3.14 2.83 Dermaptera 0.67 0 0.28 0 Odonata 1.09 0 0.41 0 ∑ Insects 64.81 57.57 59.2 36.96 Arachnides 4.64 14.6 0.22 0 Annelids 1.34 1.04 0.16 0 Molluscs 20.72 11.56 12.18 50.39 Myriapods 4.39 0 3.08 3.41 Crustaceans 0 0 1.56 0 Animal Debris 1.84 2.32 1.48 0 Macrophytes 0.65 3.43 14.64 9.24 Indeterminates 1.47 9.48 4.37 0 Sand 0.15 0 3.11 0 Table IV:- Compositions of the diet and indices of relative importance IRI (in %) of specimens of Amnirana albolabris according to the seasons; n = number of stomachs containing food; LRS= Long Rainy Season; SRS= Short Rainy Season; LDS= Long Dry Season; SDS= small Dry Season. Table IV:- Compositions of the diet and indices of relative importance IRI (in %) of specimens of Amnirana albolabris according to the seasons; n = number of stomachs containing food; LRS= Long Rainy Season; SRS= Short Rainy Season; LDS= Long Dry Season; SDS= small Dry Season. Table V:- Composition of the diet and indices of relative importance (IRI in %) of the food categories of Amnir albolabris at the fish farm, in the intermediate habitat, and the bay of Banco National Park: n = number of stomachs. Acknowledgment:- IRI(%) Ingested prey LRS (n = 97) SRS (n = 33) LDS (n = 74) SDS (n = 27) Insectes Coleoptera 12.67 12.39 42.49 14.91 Hymenoptera 33.29 35.68 6.01 0 Trichoptera 4.56 0 0.87 0 Heteroptera 0.05 0 0 0 Lepidoptera 5.25 1.76 5.87 19.22 Isoptera 3.97 3.26 0.14 0 Diptera 1.07 4.48 0 0 Orthoptera 2.18 0 3.14 2.83 Dermaptera 0.67 0 0.28 0 Odonata 1.09 0 0.41 0 ∑ Insects 64.81 57.57 59.2 36.96 Arachnides 4.64 14.6 0.22 0 Annelids 1.34 1.04 0.16 0 Molluscs 20.72 11.56 12.18 50.39 Myriapods 4.39 0 3.08 3.41 Crustaceans 0 0 1.56 0 Animal Debris 1.84 2.32 1.48 0 Macrophytes 0.65 3.43 14.64 9.24 Indeterminates 1.47 9.48 4.37 0 Sand 0.15 0 3.11 0 Ingested prey IRI (%) Fish farm (n = 98) Intermediate habitat (n = 62) Bay (n = 78) Insectes Coleoptera 25.82 14.23 14.57 Hymenoptera 34.19 10.1 13.5 Trichoptera 1.85 1.4 4.74 Heteroptera 0.07 0 0 Lepidoptera 0.7 18.06 11.19 Isoptera 4.31 0.64 1.19 Diptera 0 8.59 0 Orthoptera 1.29 3.02 3.48 Dermaptera 0 0 7.71 Odonata 0.3 0 5.1 ∑Insects 68.53 56.03 61.47 Arachnids 2.79 5.3 3.65 Annelids 0.4 1.25 1.52 Molluscs 21.31 26.67 5.25 Myriapods 1.29 2.51 11.64 Crustaceans 0.4 0 0 Animal Debris 0.98 2.56 2.47 Macrophytes 1.97 4.37 4.72 Indeterminates 2.1 0.94 7.93 Sand 0.24 0.37 1.35 Acknowledgment:- itions of the diet and indices of relative importance (IRI %) of the different food categories d bolus of Amnirana albolabris according to sex in Banco National Park; n = number of full Table III:- Compositions of the diet and indices of relative importance (IRI %) of the different food categories contained in the food bolus of Amnirana albolabris according to sex in Banco National Park; n = number of full stomachs. IRI (%) Ingested prey Males n = 199 Females n = 24 Insects Coleoptera 20.56 16.61 Hymenoptera 21.63 21 Trichoptera 4.21 0.94 Heteroptera 0.05 0 Lepidoptera 7.43 3.47 Isoptera 2.82 1.08 Diptera 1.41 0.44 Orthoptera 3.69 0 Dermaptera 0.76 0.11 Odonata 1.24 0 ∑Insects 63.79 43.65 Arachnids 5.25 4.28 Annelids 1.3 0.69 Molluscs 16.03 36.57 Myriapods 3.77 4.41 Crustaceans 0 1.02 Animal Debris 4.11 0.25 Macrophytes 1.96 4.29 Indeterminates 3.28 3.66 Sand 0.51 1.18 389 ISSN: 2320-5407 Int. J. Adv. Res. 10(05), 383-392 Table IV:- Compositions of the diet and indices of relative importance IRI (in %) of specimens of Amnirana albolabris according to the seasons; n = number of stomachs containing food; LRS= Long Rainy Season; SRS= Short Rainy Season; LDS= Long Dry Season; SDS= small Dry Season. Table V:- Composition of the diet and indices of relative importance (IRI in %) of the food categories of Amnirana albolabris at the fish farm, in the intermediate habitat, and the bay of Banco National Park: n = number of full stomachs. References:- 1. Assemian, N. E., 2009. Systématique, diversité et dynamique spatio-temporelle du peuplement d’amphibiens d’une aire protégée Ouest africaine (Parc National du Banco ; Côte d’Ivoire). Thèse de Doctorat de l’Université Abobo-Adjamé, 183p. j p 2. Assemian, N. E., Aliko, N. G., Dosso A., Koné, A. Bony, K. Y., Konan, K. F., 2015. Diet ofthe frog Phrynobatrachus latifrons Ahl 1924 in two types of anthropogenic wetlands in West Africa. Journal of Entomology and Zoology, 3 (6): 301-305. gy gy 3. Belovsky, G. E., (1978). Diet optimization in a generalist herbivore : the moose. Theoretical Population Biology, 14 : 105-134. É 4. Cibiha, R. B. C., 2017. 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Res. 10(05), 383-392 Ingested prey IRI (%) Fish farm (n = 98) Intermediate habitat (n = 62) Bay (n = 78) Insectes Coleoptera 25.82 14.23 14.57 Hymenoptera 34.19 10.1 13.5 Trichoptera 1.85 1.4 4.74 Heteroptera 0.07 0 0 Lepidoptera 0.7 18.06 11.19 Isoptera 4.31 0.64 1.19 Diptera 0 8.59 0 Orthoptera 1.29 3.02 3.48 Dermaptera 0 0 7.71 Odonata 0.3 0 5.1 ∑Insects 68.53 56.03 61.47 Arachnids 2.79 5.3 3.65 Annelids 0.4 1.25 1.52 Molluscs 21.31 26.67 5.25 Myriapods 1.29 2.51 11.64 Crustaceans 0.4 0 0 Animal Debris 0.98 2.56 2.47 Macrophytes 1.97 4.37 4.72 Indeterminates 2.1 0.94 7.93 Sand 0.24 0.37 1.35 390 Int. J. Adv. Res. 10(05), 383-392 Int. J. Adv. Res. 10(05), 383-392 ISSN: 2320-5407 References:- New data from Morerella cyanophtalma (Anura: Hyperoliidae) in Azagny National Parc, South-central Ivory Coast. Herpetology Notes, 9: 59-65. p gy 15. 15.Konan, J. C. B. Y. N, Kouamé, N. G., Kouamé, A. M., Gourène, A. B. A. & Rödel M.- O.,2016b. 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https://openalex.org/W2969958111	https://ieeexplore.ieee.org/ielx7/6287639/8600701/08809706.pdf	English	null	Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network	IEEE access	2,019	cc-by	6,585	Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network HO YUB JUNG 1, YONG SEOK HEO 2, AND SOOCHAHN LEE 3 1Department of Computer Engineering, Chosun University, Gwangju 61452, South Korea 2Department of Electrical and Computer Engineering, Ajou University, Suwon 16499, South Korea 3School of Electrical Engineering, Kookmin University, Seoul 02707, South Korea Corresponding author: Soochahn Lee (sclee@kookmin.ac.kr) This work was supported in part by the Basic Science Research Program through the National Research Foundation of Korea (N by the Ministry of Science and ICT under Grant 2017R1C1B5074302, and in part by the Research fund from Chosun Universit ABSTRACT Fingerprints are known to be easily synthesized to trick identiﬁcation systems. In this paper, we propose a new method that incorporates template ﬁngerprints stored for identiﬁcation in the liveness detection system. The ﬁngerprint identiﬁcation platform must have a list of template ﬁngerprints stored for matching with new probe ﬁngerprints trying to access the system. Thus, instead of simply detecting the liveness of the probe ﬁngerprints, the proposed approach uses the matching template ﬁngerprints along with probe ﬁngerprints through convolutional neural networks to make the liveness decision, which comprises two sequential convolutional neural networks for classiﬁcation. The proposed method can be built on the top of existing liveness detection methods to increase accuracy without a signiﬁcant increase in computation time. The evaluation over the LivDet dataset shows that the proposed ﬁngerprint liveness detection method is able to obtain state-of-the-art accuracy. INDEX TERMS Convolutional neural network, ﬁngerprints, LivDet, liveness detection, pretraining, transfer learning. INDEX TERMS Convolutional neural network, ﬁngerprints, LivDet, liveness detection, pretraining, transfer learning. FIGURE 1. The left fingerprint is a live press fingerprint, whereas the right fingerprint is obtained by pressing a synthetic finger made out of wood-glue. The two fingerprints came from the same finger and so have matching identification [6] [2]. We aim to detect the liveness of the fingerprint press using a convolutional neural network. Received July 27, 2019, accepted August 8, 2019, date of publication August 22, 2019, date of current version September 6, 2019. Digital Object Identifier 10.1109/ACCESS.2019.2936890 The associate editor coordinating the review of this article and approving it for publication was Kashif Munir. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ VOLUME 7, 2019 under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ VOLUME 7, 2019 II. RELATED WORK There has been continual research for discriminating spoof ﬁngerprints from the live ﬁngerprints. Texture analysis has been a common approach for spoof and live classiﬁcation. Abhyakar and Schuckers used multiresolution texture anal- ysis and local ridge frequencies as inputs to a fuzzy c- means classiﬁer [7]. New descriptors, such as binarized sta- tistical image features, were introduced in a similar fash- ion to local binary patterns and local phase quantization representations [8]. The ridge texture features were further examined using histograms of invariant features in a simi- lar spirit to histograms of oriented gradients and the scale invariant feature transforms [9]. Instead of trying to ﬁnd the statistical differences between spoof and live ﬁngerprints, Sequeira and Cardoso aimed to model the live samples and distinguish the fake samples by detecting deviations from the model [10]. A local binary pattern over a Gaussian pyramid is also examined as a liveness feature [11]. Toosi et al. per- formed a comprehensive analysis on different liveness fea- tures and studied the effectiveness of different feature fusion methods [12]. Convolutional neural networks (CNN) have shown to pro- duce accurate liveness classiﬁcation models without any prior knowledge or study of spoof and liveness features [4] [5]. CNN and deep learning methods are convenient to use because it is not necessary to extract deﬁnitive features for classiﬁcation problems. Through an optimization process, CNN has the ability to adopt to the problem and produce a meaningful solution. In this paper, we ﬁrst train a CNN to generate the liveness map of the ﬁngerprint using a network architecture and loss function similar to our previous work [5]. Then, the liveness maps are obtained for template and probe ﬁngerprints. The liveness map of the two ﬁngerprints are stacked and used as the input to another CNN which is trained to predict the liveness of the probe ﬁngerprint. Thus, the second CNN considers the liveness characteristics of the registered ﬁn- gerprint for making the live or spoof decision on the probe ﬁngerprints. Also important to ﬁngerprint liveness detection research, LivDet.org has been holding international competitions since 2009 with various different datasets [1], [2], [6], [13]– [15]. Numerous approaches have been introduced through the competition. However, deep learning-based approaches have become dominant in recent years. I. INTRODUCTION Fingerprint identiﬁcation has been a reliable and convenient security measure for various systems, including smartphones and tablets. However, ﬁngerprints are also known to be syn- thetically reproducible. Fake ﬁngerprints can be easily made from common materials such as wood glue and Play-Doh- like materials [1], [2]. Even with crude synthetic ﬁngerprints, the identiﬁcation system can be compromised. Fig. 1 gives examples of a live ﬁnger press and a spoof ﬁngerprint made out of wood glue. FIGURE 1. The left fingerprint is a live press fingerprint, whereas the right fingerprint is obtained by pressing a synthetic finger made out of wood-glue. The two fingerprints came from the same finger and so have matching identification [6] [2]. We aim to detect the liveness of the fingerprint press using a convolutional neural network. A ﬁngerprint identiﬁcation system works by ﬁrst register- ing template ﬁngerprints, often storing numerous different presses from the same ﬁnger. The access to the system is granted if the probe ﬁngerprints match the template ﬁnger- prints. Such systems can be bypassed if a synthetic ﬁnger with a matching ﬁngerprint is used to provide the probe ﬁnger- print for the identiﬁcation system. The matching ﬁngerprints can be theoretically obtained from many different personal objects such as doorknobs or glasses with marked ﬁnger- prints. Sometimes employees make synthetic ﬁngers of their own ﬁngers and share them among coworkers to trick the 118986 118986 H. Y. Jung et al.: Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network 32 × 32 is used compared to the 512 × 512 ﬁngerprint size for the ﬁrst CNN for liveness map generation. Furthermore, the liveness maps for the template ﬁngerprints can be precom- puted at the time of registration. The stored liveness maps can then be reused for new probe ﬁngerprints with no additional computation time during the identiﬁcation procedure. system into counting false attendance. Thus, a more secure ﬁngerprint identiﬁcation system is required both to correctly match ﬁngerprints and to detect spoof ﬁngerprints. In most previous works, the spoof ﬁngerprint detection problem was separated from the ﬁngerprint matching system. The accuracy of the spoof detection is evaluated by recog- nizing a probe ﬁngerprint as live or spoof [1], [2]. However, as noted in [3], the intended purpose of spoof detection is to help in determining the identity of the probe ﬁngerprints. I. INTRODUCTION The probe ﬁngerprint must be matched to the template ﬁngerprint and also be recognized as a live ﬁngerprint for the system to be accessed. It is thus not only practical but also logical to use the template ﬁngerprints in spoof detection. For evaluation and training, we used the LivDet 2015 dataset, which contains test and training sets for four scanners [1]. All training sets were used for training the liveness map CNN. For training the second decision CNN, only the ﬁngerprints that have matching live ﬁngerprints are used to train the network. We note that both probe ﬁngerprints and matching live ﬁngerprints were available for most of the test set. Thus, we were able to evaluate the additional accuracy increase compared to the simple liveness CNN. The comparison with previous methods are evaluated using all the test sets; the liveness map was used to make the ﬁnal decision if a test ﬁngerprint did not have matching live ﬁngerprints. The proposed method demonstrated the state-of- the-art accuracy. In this paper, we present a new method that incorporates ﬁngerprint matching into the problem of liveness detection of probe ﬁngerprints. Our method is based on the assumption that the ﬁngerprints provided when registering a new ID to the system are live. In the proposed system, ﬁngerprint matching is ﬁrst performed to retrieve the corresponding registered ﬁngerprints. We believe that utilizing the corresponding reg- istered ﬁngerprints as live templates of the probe ﬁngerprint can provide useful information when attempting to detect its liveness. In the next section, we will discuss some of the previous liveness detection networks in detail. The structure and train- ing parameters for both CNNs will be presented in section III. The LivDet data set and the evaluation are then explained, followed by the conclusion. The proposed approach does not view the liveness detec- tion problem as a straightforward classiﬁcation problem. Rather, we believe that there are certain textural characteris- tics in the ﬁngerprint that are found more in the spoof ﬁnger- prints and that there are certain characteristics that are found more in the live ﬁngerprints. But such a viewpoint conversely suggests that it is also possible to have live ﬁngerprints with more spoof characteristics and that some spoof ﬁngerprints can have more live characteristics. These spoof-looking live ﬁngerprints or live-looking spoof ﬁngerprints are difﬁcult to correctly recognize by themselves. I. INTRODUCTION However, if we know that a particular ﬁnger has more spoof or live characteristics based on the registered template, we can make an adjusted decision. II. RELATED WORK The advantage of the machine learning methods comes from the ease of use, where the features and classiﬁcations are trained automat- ically if there are enough training samples. A CNN has The additional computational time for the second CNN is minimal because a relatively small liveness map of size 118987 VOLUME 7, 2019 H. Y. Jung et al.: Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network FIGURE 2. Visual description of the overall process of the proposed approach. TABLE 1. Liveness map CNN structure. All convolution layers use zero-padding. The filter parameters are denoted by N × C × H × W (number, channel, height, width) scheme. been frequently used as the model, particularly for evaluating images, such as ﬁngerprints. Nevertheless, deep learning approaches have a few difﬁ- culties to overcome when applied to the ﬁngerprint liveness detection problem. First, the number of training samples should be sufﬁciently large. Second, the computation time can be slow, depending on the design of the network. The deep learning method with aid of a GPU can be very fast, but the liveness detection is often times restricted to embed- ded systems or CPU-only systems. Furthermore, for applica- tions with smartphones and tablets, a faster authentication is always more desirable. The deep-CNN was successful in the liveness detection by applying a voting strategy [16]. A deep-CNN was trained for small textural patches of the ﬁngerprint image, and the ﬁnal liveness decision was obtained by voting counts from the patches from the same ﬁngerprint. The need for large training samples are also addressed by pretraining or transfer learning approaches by Nogueira et al.’s implementation [4]. In their work, the features from AlexNet [17] and VGGNet [18] were used as the pretrained features for the liveness detection CNN. In [19], the CNN trained features are reduced by principal component analysis, and the extracted features are classiﬁed by the support vector machine. The Gram matrix from style transfer methods [20], [21] is also used to extract texture features in constructing liveness detection CNN [22]. ﬁlter. Although [5] showed reasonable computation time at inference by enabling the resizing of the input image, the trade-off between accuracy and the computation time was unavoidable. A. LIVENESS MAP CNN (LM-CNN) The ﬁngerprint scanners used in this paper produce grayscale ﬁngerprint images with a white background and a dark fore- ground. We reverse the color so that the background is zero black. The zero background is more consistent with the zero- padding procedure of the convolution layers. The grayscale values are rescaled using the min-max normalization, which is a typical preprocess for the deep learning of images. Then, the foreground is brought to the center by ﬁnding the weighted average position of the ﬁngerprint foreground. Additionally, since the ﬁngerprint image size varies from scanner to scanner, we crop or zero extend the image to 512 × 512 size. Here, rescaling the image might be detri- mental to the process because textural information which can be critical to the liveness detection, may be lost during down or upscaling. Some examples of the resulting images after preprocessing are shown in Fig. 3. p The number of convolution layers is maintained at 5, which is sufﬁciently deep without causing vanishing gradient issues. The details regarding parameters of the network are shown in Table 1. There are few notable differences from our previous liveness map CNN [5]. We abandon the separable ﬁlters because although the computation time is somewhat conserved, the main computation reduction comes from the 2 × 2 strides and 2 × 2 max-pooling in the earlier layers. The relatively large 7 × 7 ﬁlter is used in the beginning layer to cover the larger strides. Also since the input has only one channel, having a larger ﬁlter in the ﬁrst layer does not increase the computation time as seriously as having larger ﬁlters in the latter layers. We also limited the number of ﬁlters to 32, mainly because we wanted to keep computation time within a few seconds in CPU. We used the conventional square loss function instead of evaluating the loss only over the map position, which contains the foreground ﬁngerprint in its receptive ﬁeld, as we did in [5]. The zero background that is present in both live and spoof images is automatically trained to associate with the neutral zero value in the liveness map. See Fig. 4 for the examples of the liveness map results. Different scanners capture different features of ﬁngerprints with varying sizes. For higher accuracy, the network must be trained individually for each scanner. III. LIVENESS DETECTION USING TEMPLATE-PROBE CNN The basic idea is to use the liveness maps from both tem- plate and probe ﬁngerprints to predict the liveness of the probe ﬁngerprint; see Fig. 2. The CNN for liveness map generation, which we denote as the liveness map CNN (LM-CNN), has been discussed in our previous work [5], with the structure summarized in Table 1. However, we made a few structural modiﬁcations as well as changes in the loss function, which improves the accuracy of the LM-CNN. The second CNN, which uses both liveness maps from tem- plate and probe ﬁngerprints, is introduced next, where we attempted to design a fast and simple CNN. The second decision making CNN is denoted as the template-probe CNN (TP-CNN). Although transfer learning is an efﬁcient approach to machine learning problems with a limited number of training samples, it has a disadvantage in that the computation time cannot be smaller than the pretrained networks. Accordingly, it was shown that the ﬁngerprint liveness detection CNN can be trained using only a few thousand samples by deﬁning a training cost over the 32 × 32 liveness map in our pre- vious work [5]. The computation time was also reduced by employing wider strides and applying a separable convolution 118988 VOLUME 7, 2019 H. Y. Jung et al.: Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network FIGURE 4. 32 × 32 liveness map CNN responses are shown for the fingerprints from Fig. 3. The last image on the right is the response from the template-probe CNN using the aforementioned liveness maps. The images are shown in the [−1, 1] interval. The liveness map correctly depicts the live and fake fingerprint regions as well as the neutral background regions. The response map of the template-probe CNN comprises a simple uniform map of −1 values over a 32 × 32 area, correctly detecting the fake fingerprint of the second liveness map in reference to the first liveness map. FIGURE 3. Preprocessed live and fake fingerprints shown left and right, respectively. The fingerprints are centered and cropped to 512 × 512 size. The grayscale values are reversed and min/max normalized to [0, 1] interval, but the grayscale shown here is over the [−1, 1] interval in consistency with generated liveness map. These two fingerprints are from the same finger. III. LIVENESS DETECTION USING TEMPLATE-PROBE CNN The live fingerprint is registered as the template fingerprint, and the fake fingerprint can be used to compromise the identification system. FIGURE 4. 32 × 32 liveness map CNN responses are shown for the fingerprints from Fig. 3. The last image on the right is the response from the template-probe CNN using the aforementioned liveness maps. The images are shown in the [−1, 1] interval. The liveness map correctly depicts the live and fake fingerprint regions as well as the neutral background regions. The response map of the template-probe CNN comprises a simple uniform map of −1 values over a 32 × 32 area, correctly detecting the fake fingerprint of the second liveness map in reference to the first liveness map. FIGURE 3. Preprocessed live and fake fingerprints shown left and right, respectively. The fingerprints are centered and cropped to 512 × 512 size. The grayscale values are reversed and min/max normalized to [0, 1] interval, but the grayscale shown here is over the [−1, 1] interval in consistency with generated liveness map. These two fingerprints are from the same finger. The live fingerprint is registered as the template fingerprint, and the fake fingerprint can be used to compromise the identification system. function of the receptive ﬁeld of the ﬁngerprint, which in turn is like a sample-wise loss function with receptive ﬁeld, weights, and ground-truth value as the variables. If we use fully connected layers at the end with cross-entropy as the loss function, we would expect over ﬁtting issues with only 2000 or so samples. A. LIVENESS MAP CNN (LM-CNN) Building spoof ﬁnger- prints is a time-consuming process, and for the LivDet [1], only approximately 1000 to 1500 spoof ﬁngerprints are avail- able for training for each scanner. The number of live ﬁnger- prints is also approximately 1000 to 1500. The training set size is much too small for typical deep learning classiﬁcation networks, even with typical data augmentation comprising rotation and translation. We used the conventional stochastic gradient descent with a batch size of two during the optimization procedure. A smaller batch size is shown to converge at a lower cost value when using stochastic gradient descent. The learning rate has to be adjusted for different CNNs and datasets, in order to avoid possible gradient explosion or vanishing. However, we found that the typical learning rate value 0.01 was sufﬁ- cient for this problem. A reasonable one or two day training time is allotted for each scanner which translated into around 512 epochs. The Xavier initialization [24] is used. Xavier initialization aims to have the same variance for output as input in a convolution layer, which allows for an initialization such that a reasonable variance can be found for the output of the initial network. In our previous study, we found that data However, the main advantage of the LM-CNN is that with larger output map size, fewer ﬁngerprints are required to train the network. The proposed network has a series of convolu- tion, max-pool, and relu [23] layers that outputs a 1×32×32 channel-height-width (CHW) liveness map. The ground truth is thus also a 1 channel 2D tensor of the same size with all values equal to +1 for live and −1 for spoof ﬁngerprints. The loss function of the network is a simple square error function. The optimization procedure then tries to minimize the difference at each position of the 1 × 32 × 32 map. Thus, the single output value of 1 × 32 × 32 liveness map is a 118989 118989 VOLUME 7, 2019 H. Y. Jung et al.: Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network FIGURE 5. The mean squared loss is minimized for both LM-CNN and TP-CNN. For TP-CNN, the validation set was not used and the parameters with minimum training loss is chosen as the final network. 512 epochs are trained for LM-CNN in total, but only the first 256 epochs are shown. B. TEMPLATE-PROBE CNN (TP-CNN) The LM-CNN is able to obtain sufﬁciently accurate results by itself as demonstrated in our previous work. However, we want to improve the result by using the registered tem- plate ﬁngerprint, which should already be in the identiﬁca- tion system. The liveness map estimation basically indicates whether the receptive ﬁeld of the ﬁngerprint is likely to belong to a spoof or a live one. An average of a liveness map usually produces correct decisions, but there are outliers where live ﬁngerprints have more spoof features. If we have live template ﬁngerprints for comparison, we should expect improvements in the accuracy. description of ﬁlters is shown in Table 2. The response tensor has size 1 × 4 × 4, all having +1 values for live probe ﬁngerprints and −1 for spoofs. The training parameters are kept similar to the LM-CNN with a learning rate of 0.01. Xavier initialization is used again, and the same rotation, translation, and Gaussian noise data augmentation is performed for both template and probe ﬁngerprints before the LM-CNN. However, the TP-CNN was trained without the validation set for around 200 epochs. Considering the complexity of the network and the available data augmentation through template image selection, we felt that the chances of overﬁtting were minuscule. Additionally, the 200 epochs is probably excessive, and the minimization is mostly attenuated after 10 epochs. The loss minimization graph is shown in Fig. 5. Multiple presses have been made from the same ﬁngers in the LivDet 2015 dataset. Most of the ﬁngerprints have multiple live presses that can be used as the template ﬁn- gerprints. The ﬁngerprints without the alternative live presses are not included in the training. The batch samples are pro- duced by ﬁrst randomly selecting probe ﬁngerprints from the training set, and then randomly selecting from the available template ﬁngerprints for each probe image. The probe and template images are each passed through the LM-CNN, and the resulting liveness maps are used as the input features for the TP-CNN. The ground truth for the network is the liveness of the probe ﬁngerprint. IV. EVALUATION h d f The datasets from the LivDet 2015 are used for the test. The LivDet 2017 competition is completed; however, the test sets are not available to the public as the majority of 2017 datasets are being used in the 2019 competition. A. LIVENESS MAP CNN (LM-CNN) TP-CNN is trained for around 200 epochs, but 128 epochs are shown. CNN and TP-CNN. For TP-CNN, the validation set was hosen as the final network. 512 epochs are trained for CNN is trained for around 200 epochs, but 128 epochs TABLE 2. Template-probe CNN structure. All convolution layers use zero-padding. The filter parameters are denoted by N × C × H × W (number, channel, height, width) scheme. FIGURE 5. The mean squared loss is minimized for both LM-CNN and TP-CNN. For TP-CNN, the validation set was not used and the parameters with minimum training loss is chosen as the final network. 512 epochs are trained for LM-CNN in total, but only the first 256 epochs are shown. TP-CNN is trained for around 200 epochs, but 128 epochs are shown. P-CNN is trained for around 200 epochs, but 128 epochs TABLE 2. Template-probe CNN structure. All convolution layers use zero-padding. The filter parameters are denoted by N × C × H × W (number, channel, height, width) scheme. TABLE 2. Template-probe CNN structure. All convolution layers use zero-padding. The filter parameters are denoted by N × C × H × W (number, channel, height, width) scheme. TABLE 2. Template-probe CNN structure. All convolution layers use zero-padding. The filter parameters are denoted by N × C × H × W (number, channel, height, width) scheme. augmentation by image rotation adversely affects the accu- racy of Digital Persona ﬁngerprints which has a lower res- olution than the other scanners. Nevertheless, we wanted to train the network so that it can work for both the original non- rotated ﬁngerprints as well as for the augmented ﬁngerprints. Thus, for 50% of the time, data are augmented by random [−30◦, 30◦] rotation, max 8 pixel translation from center, and Gaussian noise. Smaller translation and rotation ranges are chosen in order to prevent ﬁngerprints from going out of bound, and to maintain the natural orientation of scanned ﬁngerprints. The loss minimization graph is shown in Fig. 5. B. TEMPLATE-PROBE CNN (TP-CNN) The input tensor size for the TP-CNN is 2 × 32 × 32 CHW. The ﬁrst channel is the liveness map of the tem- plate ﬁngerprint. The second channel is the liveness map of the probe ﬁngerprint. The TP-CNN is composed of a of series of convolution, max pool, and relu layers. The detailed The 2015 dataset has ﬁngerprints from four scanners: GreenBit, Digital-Persona, Biometrika, and Cross-Match. Examples of each ﬁngerprint scanners are shown in Fig. 6. 118990 118990 VOLUME 7, 2019 VOLUME 7, 2019 H. Y. Jung et al.: Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network FIGURE 6. Examples of live fingerprints for Digital Persona, Crossmatch, Biometrika and Greenbit are shown in order from left to right. FIGURE 6. Examples of live fingerprints for Digital Persona, Crossmatch, Biometrika and Greenbit are shown in order from left to right. TABLE 3. Accuracy of liveness map CNN (LM-CNN) and proposed template-probe CNN (TP-CNN) over the LivDet 2015 d a large portion of the test fingerprints did not have a matching live fingerprint that could be used as the template. In th measured for test fingerprints with at least one matching live fingerprint. y p ( ) p p p p ( ) a large portion of the test fingerprints did not have a matching live fingerprint that could be used as the template. In this evaluation, the accuracy wa measured for test fingerprints with at least one matching live fingerprint. TABLE 4. Accuracy of proposed methods compared with previous state-of-the-art CNN methods as well as few of the LivDet 2015 competition methods are shown here. All the LivDet 2015 test sets are evaluated. LM-CNNp is the liveness map CNN from our previous work [5]. For TP/LM-CNN, the TP-CNN is used for the test fingerprint with matching live fingerprint; otherwise, LM-CNN is used to classify the liveness. TABLE 4. Accuracy of proposed methods compared with previous state-of-the-art CNN methods as well as few of the LivDet 2015 competition methods are shown here. All the LivDet 2015 test sets are evaluated. LM-CNNp is the liveness map CNN from our previous work [5]. For TP/LM-CNN, the TP-CNN is used for the test fingerprint with matching live fingerprint; otherwise, LM-CNN is used to classify the liveness. A. TEMPLATE-PROBE CNN ACCURACY A. TEMPLATE-PROBE CNN ACCURACY Each scanner has a total of 2000 ﬁngerprints for training. Half of the training set is composed of live ﬁngerprints, and the other half is composed of spoof ﬁngerprints synthetically produced with materials such as wood-glue, gelatin, latex, and ecoﬂex. Each scanner also has a separate test set with approximately 2500 to 3000 ﬁngerprints. The test set has around 1000 live and 1000 spoof ﬁngerprints of same mate- rials as the training set as well as an additional 500 spoof ﬁngerprints made with new materials not found in the training set. Since the ground truth of live and spoof values were set to +1 and −1, respectively, we average the response of the TP-CNN. The probe ﬁngerprint is classiﬁed as spoof if the average is less than zero; otherwise, it is classiﬁed as a live ﬁngerprint. The same classiﬁcation decision scheme is used in the TP-CNN. In this evaluation, the threshold for the decision is set to zero in consistency with the ground-truth deﬁnition. However, the decision threshold can be adjusted to add bias toward live or spoof classiﬁcation depending upon the security demands of an application. The next subsection will evaluate the incremental accu- racy improvement of using the additional TP-CNN. Although the LM-CNN by itself is able to obtain high accuracy, using the additional template ﬁngerprint information which should be readily available in the identiﬁcation system, allows for consistently higher accuracy. In the second sub- section, we evaluate the proposed approach with previous spoof detection methods, which only use the probe ﬁngerprint information. Around 85% of the ﬁngerprints in the test set have at least one live matching ﬁngerprint that can be used as the template ﬁngerprint. For this subsection, we evaluated the ﬁngerprints with templates only. The template ﬁngerprints are chosen randomly from ﬁve to ten template ﬁngerprint candidates. Overall, the accuracy using the TP-CNN is shown to be higher than liveness detection using only the probe ﬁngerprint. See Table 3 for the comparison results. The only 118991 VOLUME 7, 2019 H. Y. Jung et al.: Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network TABLE 5. The millisecond (ms) computation times using CPU and GPU are shown for LM-CNN and TP-CNN. TABLE 5. The millisecond (ms) computation times using CPU and GPU are shown for LM-CNN and TP-CNN. Additionally for the comparative evaluation, we included the results from the LM-CNN only. REFERENCES The computation time is shown in Table 5. All four scanner ﬁngerprints are cropped or extended to the same 512 × 512 size, making the operating time the same for all scanners. Since the TP-CNN takes the liveness maps of both template and probe ﬁngerprints, we might expect the computation time to be at least doubled. However, in practice, the liveness map can be precomputed for the template ﬁngerprints. Thus, the proposed approach requires only the additional computa- tion time for the TP-CNN which is computed in far shorter milliseconds due to the many times smaller 32 × 32 size. An average 24 millisecond (ms) computation time is mea- sured for the LM-CNN, and 1 ms is measured for TP-CNN using an Nvidia 1080TI GPU. The CPU only operation can increase the computation time to 1.696 seconds for the LM-CNN, while the TP-CNN only adds 16 ms. [1] L. Ghiani, D. A. Yambay, V. Mura, G. L. Marcialis, F. Roli, and S. A. Schuckers, ‘‘Review of the ﬁngerprint liveness detection (LivDet) competition series: 2009 to 2015,’’ Image Vis. Comput., vol. 58, pp. 110–128, Feb. 2017. [2] V. Mura, G. Orrù, R. Casula, A. Sibiriu, G. Loi, P. Tuveri, L. Ghiani, and G. L. Marcialis, ‘‘LivDet 2017 ﬁngerprint liveness detection competition 2017,’’ in Proc. Int. Conf. Biometrics (ICB), Feb. 2018, pp. 297–302. [3] I. Chingovska, A. Anjos, and S. Marcel, ‘‘Anti-spooﬁng in action: Joint operation with a veriﬁcation system,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. Workshops (CVPRW), Jun. 2013, pp. 98–104. [4] R. F. Nogueira, R. de Alencar Lotufo, and R. C. Machado, ‘‘Fingerprint liveness detection using convolutional neural networks,’’ IEEE Trans. Inf. Forensics Security, vol. 11, no. 6, pp. 1206–1213, Jun. 2016. [5] H. Y. Jung and Y. S. Heo, ‘‘Fingerprint liveness map construction using convolutional neural network,’’ Electron. Lett., vol. 54, no. 9, pp. 564–566, May 2018. [6] LivDet—Liveness Detection Competitions. Accessed: Aug. 2019. [Online]. Available: http://livdet.org [7] A. Abhyankar and S. Schuckers, ‘‘Fingerprint liveness detection using local ridge frequencies and multiresolution texture analysis techniques,’’ in Proc. Int. Conf. Image Process. (ICIP), Oct. 2006, pp. 321–324. A. TEMPLATE-PROBE CNN ACCURACY The modiﬁed version of our previous liveness map approach is able to obtain a very high accuracy compared to previous methods. However, the proposed TP-CNN is shown to have slightly higher accu- racy overall. V. CONCLUSION accuracy decrease is shown in the scanner Green Bit, where the LM-CNN accuracy of 97.24% is reduced to 96.54%. We observed greatest increase in the accuracy in lower res- olution Digital Persona scanner from 90.93% to 93.50%. In this paper, we introduced a template-probe CNN (TP-CNN) approach to detect the liveness of ﬁngerprints. We use the template (registered) ﬁngerprints that are already in the identiﬁcation system to better detect spoof probe (test) ﬁngerprints. The liveness maps are calculated for both template and probe ﬁngerprints, of which the template ﬁngerprints’ liveness map can be calculated once during the registration stage. Using the liveness map from both of the ﬁngerprints as the input features, the proposed TP-CNN is able to increase the accuracy of the liveness detection. Due to the relatively small size of the liveness map and TP-CNN, there was only a minuscule increase in the computation time. The accuracy comparison with previous methods also showed favourable results. g The statistical signiﬁcance tests are calculated for the changes in the accuracies between LM-CNN and TP-CNN. Using the proportion test, the p-value for Digital Persona data is 0.002. The increase of accuracy is highest for Dig- ital Persona set. The Crossmatch set’s accuracy is pretty much saturated and showed smallest changes, with p-value of 0.073. Biometrika set’s p-value is 0.011. For Green Bit set, the accuracy actually decreased with p-value of 0.218. Combining the all four sets, we have 9353 ﬁngerprints among which 8993 is correctly classiﬁed using LM-CNN and 9075 samples correctly classiﬁed using TP-CNN. The p-value for all test set is 0.001. B. COMPARISON TO PREVIOUS METHODS The accuracy comparison with previous liveness detection methods is summarized in Table 4. The previous methods were evaluated for all of the test set. However, as previ- ously mentioned, some of the test ﬁngerprints did not con- tain template live ﬁngerprints. To obtain the accuracy on the entire dataset even for ﬁngerprints without the template ﬁngerprint, we made following adjustments to the proposed approach. First, if the test ﬁngerprint has the matching tem- plate ﬁngerprint, the liveness maps of both are determined, and the TP-CNN is used to make the ﬁnal liveness detection. Second, if the test ﬁngerprint does not have the template ﬁngerprint, the liveness decision is made using only the live- ness map CNN result for the test ﬁngerprint. This evaluation approach allowed us to evaluate all the test sets and make a fair comparison with the previous methods. [8] L. Ghiani, A. Hadid, G. L. Marcialis, and F. Roli, ‘‘Fingerprint liveness detection using binarized statistical image features,’’ in Proc. IEEE 6th Int. Conf. Biometrics, Theory, Appl. Syst. (BTAS), Sep. 2013, pp. 1–6. [9] C. Gottschlich, E. Marasco, A. Y. Yang, and B. Cukic, ‘‘Fingerprint live- ness detection based on histograms of invariant gradients,’’ in Proc. IEEE Int. Joint Conf. Biometrics, Sep. 2014, pp. 1–7. [10] A. F. Sequeira and J. S. Cardoso, ‘‘Fingerprint liveness detection in the presence of capable intruders,’’ Sensors, vol. 15, no. 6, pp. 14615–14638, Jun. 2015. [11] Y. Jiang and X. Liu, ‘‘Uniform local binary pattern for ﬁngerprint liveness detection in the Gaussian pyramid,’’ J. Elect. Comput. Eng., vol. 2018, Jan. 2018, Art. no. 1539298. [12] A. Toosi, A. Bottino, S. Cumani, P. Negri, and P. L. Sottile, ‘‘Feature fusion for ﬁngerprint liveness detection: A comparative study,’’ IEEE Access, vol. 5, pp. 23695–23709, 2017. [13] G. L. Marcialis, A. Lewicke, B. Tan, P. Coli, D. Grimberg, A. Congiu, A. Tidu, F. Roli, and S. Schuckers, ‘‘First international ﬁngerprint liveness detection competition–LivDet 2009,’’ in Proc. Int. Conf. Image Anal. Process. Berlin, Germany: Springer, 2009, pp. 12–23. 118992 118992 VOLUME 7, 2019 H. Y. Jung et al.: Fingerprint Liveness Detection by a Template-Probe Convolutional Neural Network [14] D. Yambay, L. Ghiani, P. Denti, G. L. Marcialis, F. Roli, and S. Schuckers, ‘‘LivDet 2011—Fingerprint liveness detection competition 2011,’’ in Proc. Int. Conf. Biometrics (ICB), Mar. 2012, pp. 208–215. YONG SEOK HEO received the B.S. degree in electrical engineering and the M.S. and the Ph.D. B. COMPARISON TO PREVIOUS METHODS degrees in electrical engineering and computer science from Seoul National University, South Korea, in 2005, 2007, and 2012, respectively. From 2012 to 2014, he was with the Digital Media and Communications Research and Development Cen- ter, Samsung Electronics. He is currently with the Department of Electrical and Computer Engineer- ing, Ajou University, as an Associate Professor. His research interests include segmentation, stereo matching, 3D reconstruc- tion, and computational photography. [15] L. Ghiani, D. Yambay, V. Mura, S. Tocco, G. L. Marcialis, F. Roli, and S. Schuckcrs, ‘‘LivDet 2013 ﬁngerprint liveness detection competition 2013,’’ in Proc. Int. Conf. Biometrics (ICB), Jun. 2013, pp. 1–6. [16] C. Wang, K. Li, Z. Wu, and Q. Zhao, ‘‘A DCNN based ﬁngerprint liveness detection algorithm with voting strategy,’’ in Proc. Chin. Conf. Biometric Recognit. Springer, 2015, pp. 241–249. [17] A. Krizhevsky, I. Sutskever, and G. E. Hinton, ‘‘ImageNet classiﬁcation with deep convolutional neural networks,’’ in Proc. Adv. Neural Inf. Pro- cess. Syst., 2012, pp. 1097–1105. [18] K. Simonyan and A. Zisserman, ‘‘Very deep convolutional networks for large-scale image recognition,’’ 2014, arXiv:1409.1556. [Online]. Avail- able: https://arxiv.org/abs/1409.1556 [19] C. Yuan, X. Li, Q. Wu, J. Li, and X. Sun, ‘‘Fingerprint liveness detection from different ﬁngerprint materials using convolutional neural network and principal component analysis,’’ Comput., Mater. Continua, vol. 53, no. 4, pp. 357–371, 2017. [20] L. A. Gatys, S. A. Ecker, and M. Bethge, ‘‘A neural algorithm of artis- tic style,’’ 2015, arXiv:1508.06576. [Online]. Available: https://arxiv.org/ abs/1508.06576 [21] L. A. Gatys, M. Bethge, A. Hertzmann, and E. Shechtman, ‘‘Preserving color in neural artistic style transfer,’’ 2016, arXiv:1606.05897. [Online]. Available: https://arxiv.org/abs/1606.05897 [22] E. Park, X. Cui, W. Kim, and H. Kim, ‘‘End-to-end ﬁngerprints live- ness detection using convolutional networks with gram module,’’ 2018, arXiv:1803.07830. [Online]. Available: https://arxiv.org/abs/1803.07830 [23] V. Nair and E. Geoffrey Hinton, ‘‘Rectiﬁed linear units improve restricted boltzmann machines,’’ in Proc. Int. Conf. Mach. Learn.(ICML), 2010, pp. 807–814. [24] X. Glorot and Y. Bengio, ‘‘Understanding the difﬁculty of training deep feedforward neural networks,’’ in Proc. 13th Int. Conf. Artif. Intell. Statist., Mar. 2010, pp. 249–256. SOOCHAHN LEE received the B.S. and Ph.D. degrees from Seoul National University, Seoul, South Korea, in 2004 and 2011, respectively. From 2011 to 2014, he was with the Digital Media and Communications Research and Development Cen- ter, Samsung Electronics. From 2014 to 2019, he was an Assistant Professor with Soonchunhyang University, Asan, South Korea. B. COMPARISON TO PREVIOUS METHODS Since 2019, he has been an Assistant Professor with the School of Electrical Engineering, Kookmin University, Seoul. His research interests include computer vision and medical image analysis. HO YUB JUNG received the B.S. degree electri- cal engineering from The University of Texas at Austin, in 2002, and the M.S. and Ph.D. degrees in electrical engineering and computer science from Seoul National University, in 2006 and 2012, respectively. From 2012 to 2014, he was with the Digital Media and Communications Research and Development Center, Samsung Electronics. From 2014 to 2016, he was a Faculty Member with the Hankuk University of Foreign Studies. Since 2017, he has been an Assistant Professor with the Department of Computer Engineering, Chosun University. His research interests include computer vision, machine learning, and medical imaging. HO YUB JUNG received the B.S. degree electri- cal engineering from The University of Texas at Austin, in 2002, and the M.S. and Ph.D. degrees in electrical engineering and computer science from Seoul National University, in 2006 and 2012, respectively. From 2012 to 2014, he was with the Digital Media and Communications Research and Development Center, Samsung Electronics. From 2014 to 2016, he was a Faculty Member with the Hankuk University of Foreign Studies. Since 2017, he has been an Assistant Professor with the Department of Computer Engineering, Chosun University. His research interests include computer vision, machine learning, and medical imaging. 118993 VOLUME 7, 2019
https://openalex.org/W2782013924	https://periodicos.ufpb.br/index.php/actas/article/download/36290/18426	Portuguese	null	FABRINCANDO SENTIDOS: A MENINA AVOADA, DE MANOEL DE BARROS, À LUZ DA SEMIÓTICA DISCURSIVA CONSTRUYENDO SENTIDOS: A MENINA AVOADA, DE MANOEL DE BARROS, A LUZ DE LA SEMIOTICA DISCURSIVA	Acta Semiótica et Lingvistica	2,017	cc-by	4,484	FABRINCANDO SENTIDOS: A MENINA AVOADA, DE MANOEL DE BARROS, À LUZ DA SEMIÓTICA DISCURSIVA CONSTRUYENDO SENTIDOS: A MENINA AVOADA, DE MANOEL DE BARROS, A LUZ DE LA SEMIOTICA DISCURSIVA Profa. Dra. BATISTOTE, Maria Luceli Faria Universidade Federal de Mato Grosso do Sul UFMS/PPGMEL Profa. Dra. BATISTOTE, Maria Luceli Faria Universidade Federal de Mato Grosso do Sul UFMS/PPGMEL i l lif i @ il marialucelifaria@gmail.com RESUMO: Este trabalho tem por objetivo analisar a infância em Manoel de Barros a partir da perspectiva teórica da Semiótica Discursiva. De modo restrito, objetivamos mobilizar os conceitos semióticos de figuratividade e isotopia no poema “A menina avoada”, publicado, em 1999, no livro Exercícios de ser criança. Orientamo-nos pela metodologia de base da teoria semiótica, o percurso gerativo de sentido. Embora haja uma vasta quantidade de trabalhos sobre Manoel de Barros, não há nenhum que discuta a temática da infância à luz da teoria semiótica e que, especificamente, o faça articulando os conceitos supramencionados. Os resultados da análise atestam a eficácia da proposta semiótica na elucidação de como o tema da infância é construído no corpus eleito de forma a assumir certas significações e não outras. g ç PALAVRAS-CHAVE: Manoel de Barros. Infância. Enunciação. Figuratividade. Isotopia. g ç PALAVRAS-CHAVE: Manoel de Barros. Infância. Enunciação. Figuratividade. Isotopia. RESUMEN: Este trabajo tiene como objetivo analizar la infancia en Manoel de Barros desde la perspectiva teórica de la semiótica discursiva. De modo restrictivo, objetivamos movilizar los conceptos semióticos de la figuratividad y la isotopía en el poema "A menina avoada", publicado, en 1999, en el libro Exercício de ser criança. Nos guiamos por la metodología básica de la teoría semiótica, el recorrido generativo de sentido. Aunque hay una gran cantidad de trabajos a respecto de Manoel de Barros, no hay ninguno para discutir el tema de la infancia a luz de la teoría semiótica y, específicamente, que articule los conceptos ya mencionados. Los resultados del análisis muestran la eficacia de la propuesta semiótica en la elucidación de cómo el tema de la infancia se construye en el corpus elegido para asumir ciertos significados y no otros. g y PALABRAS CLAVE: Manoel de Barros. Infancia. Enunciación. Figuratividad. Isotopía. 1 Em nosso trabalho, utilizamos a versão original do poema, publicada em 1999 pela editora Salamandra. 2 GARCIA, Mirian Theyla Ribeiro. Exercício de ser humano: a poesia e a infância na obra de Manoel de Barros. 2006. 125 f. Dissertação (Mestrado em Literatura Brasileira) -Universidade de Brasília, Brasília, 2006. Disponível em: < http://repositorio.unb.br/handle/10482/1962>. Acesso em: 20 jul. 2016. 3 KOHAN, Walter Omar. Imagens da infância para (re) pensar o currículo. 2003. Revista Sul- Americana de Filosofia e Educação, 2003. Disponível em: < http://www.periodicos.unb.br/index.php/resafe/article/view/5409>. Acesso em: 20 jul. 2016. Notas introdutórias Manoel de Barros carrega em sua trajetória artística o desenvolvimento de uma poética absolutamente singular. Por meio da ludicidade, do remanejo realizado com a linguagem e com o vernáculo rupestre, o poeta mato-grossense (re)inventa o universo pantaneiro. É peculiar em sua poesia as efemeridades da vida receberem importância crucial, tudo o que é dado como sem importância, como descartável, passadiço é recolhido pelo artista e transformado em matéria para poesia. Por essa razão, a temática infância é recorrente em toda a sua obra, podendo ser considerada como um dos pilares de sua poética. Nesse sentido, objetivamos analisar a infância em Manoel de Barros sob a égide da Semiótica Discursiva, especificamente, nos propomos a examinar o conceito de figuratividade e isotopia no poema “A menina avoada”, composição integrante do livro 90 90 Exercícios de ser criança, primeira obra do poeta direcionada ao público infantil.1 Para alcançar os objetivos elencados, recorremos ao aporte metodológico proposto pela teoria semiótica, o percurso gerativo de sentido. Devido a isso, interessamo-nos não em analisar o que significa a infância na poesia manoelina, mas como essa significação é construída. Partimos do pressuposto de que toda organização figurativa manifesta os valores do enunciador, os quais podem ser encontrados a partir das marcas e traços linguísticos deixados no enunciado, conferindo ao discurso determinados efeitos de sentido. Face à extensa fortuna crítica no campo dos estudos literários dedicada a obra de Manoel de Barros, bem como à temática infância, marcante em sua produção, percebe- se que não há estudos no campo da semiótica discursiva que tenham se dedicado ao trabalho do poeta, especialmente no tocante a mobilização dos conceitos de figuratividade e isotopia articulados a essa temática. É possível encontrar trabalhos acerca da infância retratada pelo poeta mato-grossense ancorados em referenciais teóricos na área da educação e da literatura, como a dissertação de mestrado “Exercício de ser humano: a poesia e a infância na obra de Manoel de Barros2” defendida por Mirian Garcia em 2006 e o artigo “Imagens da infância para (re) pensar o currículo3” de Walter Kohan. 1 Em nosso trabalho, utilizamos a versão original do poema, publicada em 1999 pela editora Salamandra 2 GARCIA Mi i Th l Rib i E í i d h i i fâ i b d M 1. Enunciação e figuratividade Hoje amarrei no rosto das palavras minha máscara. Faço o que posso. Manoel de Barros Hoje amarrei no rosto das palavras minha máscara. Faço o que posso. Hoje amarrei no rosto das palavras minha máscara. Faço o que posso. Hoje amarrei no rosto das palavras minha máscara. Faço o que posso. Manoel de Barros Manoel de Barros As linhas de um texto são capazes de nos conduzir a um universo de infinitas significações, possibilita que o leitor possa adentrar em um mundo fictício pensando ser ele real, permite a construção de pássaros que vestem casacos dourados como sol, de rãs que participam de teatro, de caixotes que se transformam em carroça. Percebemos, assim, que enunciar é criar visões e (re)visões do mundo, é justamente amarrar no rosto das palavras uma máscara, marcando-se no discurso, combinando e recombinando os signos da língua com vistas a assiná-la para que ela torne-se mais particular. Como 91 91 assevera Émile Benveniste (1995, p.285), “a linguagem ensina a própria definição do homem”, nela e por ela o homem se constitui como sujeito. É válido ressaltar que ao nos valermos dos termos homem e sujeito não partimos de uma visão ontológica, mas nos referimos ao sujeito linguisticamente construído no discurso. Logo, as reflexões realizadas não têm por objetivo dissertar a respeito de Manoel de Barros, mas do sujeito da enunciação marcado em seus poemas. Vimos que a metodologia de base deste trabalho é o percurso gerativo de sentido. Nas palavras de Barros (2005) essa ferramenta metodológica é, pois, uma sucessão de patamares, cada um passível de receber uma descrição apropriada, que evidencia como se produz e se interpreta o sentido, num processo que parte do mais simples ao mais complexo. Por essa razão, ele organiza-se em três patamares: o fundamental, o narrativo e o discursivo. O estudo da figuratividade situa-se no terceiro nível, o discursivo. Este patamar subdivide-se em dois níveis: o da sintaxe e o da semântica. Na sintaxe discursiva a narrativa domina os estudos relativos à colocação em discurso das categorias de pessoa, tempo e espaço, são os processos de debreagem e embreagem. A debreagem pode ser enunciva ou enunciativa. Elas criam, em princípio, dois grandes efeitos de sentido: de objetividade e subjetividade. 1. Enunciação e figuratividade Há também a debreagem interna ou de 2° grau que ocorre quando “um actante já debreado, seja ele da enunciação ou do enunciado, se torna instância enunciativa, que opera, portanto, uma segunda debreagem, que pode ser enunciativa ou enunciva” (FIORIN, 1995, p.7). Um exemplo clássico de debreagem interna é o diálogo. A embreagem, por sua vez “é a expulsão fora da instância de enunciação da pessoa, do espaço e do tempo enunciado, ‘é o efeito de retorno à enunciação’” (ibidem). Em nossa pesquisa, mobilizaremos apenas a noção de debreagem. Já na semântica discursiva, os valores trazidos no nível narrativo são difundidos no discurso de forma abstrata, por meio de percursos temáticos que, por sua vez, podem ser figurativizados, alcançando com isso maior concretude. É válido ressalvar que o nível da sintaxe e da semântica são separados por fins metodológicos. Não significa, portanto, que apenas temas e figuras revestem o discurso de concretude e significação, o mecanismo de debreagem é imprescindível à atribuição de significância textual. Defendemos, então, que os domínios sintáxicos e semânticos nutrem-se mutuamente e em conjunto constroem um simulacro do mundo natural. 92 92 A figuratividade consiste numa operação enunciativa que revela as crenças, os valores e a posição do sujeito da enunciação, afirma Fiorin (2012). Isto porque quando um texto é figurativizado uma imagem do mundo é desenhada a fim de projetar certos efeitos de sentido. O termo figuratividade, originalmente, advém da teoria estética, “que opõe, como todos sabem a arte figurativa e a arte ‘não-figurativa’ ou ‘abstrata’” (BERTRAND, 2002, p.154). Diz respeito a semelhança formalmente estabelecida com o mundo sensível. Por isso, quanto mais figurativo for um discurso, maior será o efeito de realidade criado. Assim, a figura é todo conteúdo pertencente a qualquer língua natural ou a qualquer sistema de representação que possua um correlato perceptível no mundo natural efetivamente real, bem como no mundo natural construído. Não é sem razão, portanto, que os textos literários são classificados como fundamentalmente figurativos, confirmam Greimas e Courtés (1979, p.185): “Quando se tenta classificar o conjunto dos discursos em duas grandes classes, discursos figurativos e não figurativos (ou abstratos), percebe-se que a quase totalidade dos textos ditos literários e históricos pertence à classe dos discursos figurativos”. 1. Enunciação e figuratividade Isso porque o discurso literário comporta em sua essência uma linguagem figurativa por meio da qual se dá a construção de imagens do mundo: Sendo a literatura uma linguagem figurativa que cria simulacros do mundo natural, sendo as formas literárias um repertório de possibilidades discursivas e textuais, sugere ela a possibilidade de novas ordens no que diz respeito aos dois grandes sistemas de significação: mostra a possibilidade de novos mundos naturais, de outras realidades, de diferentes visões de mundo e, ao mesmo tempo, indica que são possíveis novas ordens linguísticas e discursivas diversas da realidade automatizada e rotineira do discurso comum. Essas duas possibilidades imbricam-se na materialidade da linguagem figurativa. (FIORIN, 2012, p.54). Como se pôde notar a partir dos apontamentos acima, a figuratividade opõe-se à abstração, ou, em termos semióticos, à tematização. O tema é uma atribuição semântica, de ordem puramente conceptual, que não alude ao mundo natural. Os temas são classes que organizam, classificam e ordenam os componentes do mundo natural, tais como o amor, a vergonha, o orgulho, a moda, a natureza, a cultura etc. É importante salientar que temas e figuras não são conceitos dicotômicos, mas complementares. Por isso, um texto não é composto de figuras estanques, ao contrário, elas estabelecem uma relação entre si. 93 93 Conforme assegura Fiorin (2002), numa análise textual o que tem valia é esse encadeamento de figuras, o qual é denominado percurso figurativo. Decifrar um percurso figurativo é encontrar o tema que lhe é subjacente, para que um grupo de figuras faça algum sentido é necessário que esteja recobrindo algum tema. As figuras “caixote”, “lata de goiabada”, “bois” e “corda de embira” não fazem o menor sentido se pensadas isoladamente, contudo, se as associarmos ao tema infância, por exemplo, é possível chegar a uma interpretação coerente. Assim como o percurso figurativo, o encadeamento de temas é denominado de percurso temático. Com base nesses discorrimentos, torna-se clarividente que o recobrimento temático e figurativo, realizado pelo sujeito da enunciação, produz efeitos de realidade e assegura a relação entre mundo e discurso. 2. A permanência de sentido: efeito isotópico A recorrência de temas e figuras ao longo do discurso denomina-se isotopia. Esse conceito foi tomado por Greimas do domínio da físico-química em que originariamente significava “propriedade dos nuclídeos que possuem o mesmo número atômico, mas cujos números de massa são distintos” (Dicionário Eletrônico Houaiss, 2001). No campo semiótico, a isotopia constitui um crivo de leitura que torna homogênea a superfície do texto (Greimas e Courtés, 1979). Reconhecem-se dois tipos de isotopia: a temática e a figurativa. Aquela se manifesta na recorrência de unidades semânticas abstratas em um mesmo percurso temático. Somente é possível inferir sobre o que um texto aborda a partir do reconhecimento da isotopia temática. Assim, quando lemos uma notícia e afirmamos se tratar de um assunto relativo à corrupção ou quando assistimos a um filme e identificamos ser um romance, realizamos o reconhecimento da isotopia temática. Por sua vez, a isotopia figurativa distingue os discursos integralmente recobertos por um ou mais percursos figurativos. Conforme Barros (2002), a reincidência de traços figurativos, a integração de figuras semelhantes confere ao discurso o caráter da realidade ou pode criar a ilusão total do irreal. 3. A menina avoada: uma leitura semiótica O poema “A menina avoada” compõe a obra Exercícios de ser criança. Lançada em 1999, esta foi a primeira publicação de Manoel de Barros direcionada ao público 94 94 infantil e conta com as ilustrações do grupo Matizes Dumont, formado por bordadeiras e um bordadeiro da mesma família, naturais da cidade de Pirapora, Minas Gerais. Os artistas dedicam-se às artes visuais têxteis e desenvolvem projetos sociais em que a linguagem é o bordado. O interessante na produção artística do grupo é justamente a substituição das ferramentas que usam em seu fazer artístico. Tintas e pincéis são deixados de lado para que agulha e linha possam materializar suas criações. Percebemos, então, a singularidade dessa forma de arte, cada escolha de linha, agulha, tipo de bordado marca sua artesanal assinatura. A eleição dessa forma de iluminura, o bordado, para compor um livro infantil pode causar certo estranhamento, uma vez que é incomum ao gosto das crianças, porém a escolha das cores, a dinamicidade e o relevo das imagens faz aflorar variadas sensações, desperta no leitor uma vontade de interagir com a obra ao ponto de querer tocá-la, sentir o efeito de cada tipo de linha, das miçangas, dos bordados feitos pelo avesso. Sabemos que a criança explora sua imaginação de forma muito mais ampla do que os adultos, sendo capaz de alterar e recriar o universo natural. Apresentamos abaixo o poema na íntegra: A menina avoada Foi na fazenda de meu pai antigamente Eu teria dois anos; meu irmão, nove. Meu irmão pregava no caixote duas rodas de lata de goiabada. A gente ia viajar. As rodas ficavam cambaias debaixo do caixote: Uma olhava para a outra. Na hora de caminhar as rodas se abriam para o lado de fora. De forma que o carro se arrastava no chão. Eu ia pousada dentro do caixote com as perninhas encolhidas. Imitava estar viajando. Meu irmão puxava o caixote por uma corda de embira. Mas o carro era diz-que puxado por dois bois. Eu comandava os bois: - Puxa, Maravilha! - Avança, Redomão! Meu irmão falava que eu tomasse cuidado porque Redomão era coiceiro. As cigarras derretiam a tarde com seus cantos. Meu irmão desejava alcançar logo a cidade - Porque ele tinha uma namorada lá. A namorada do meu irmão dava febre no corpo dele. Eu comandava os bois: - Puxa, Maravilha! - Avança, Redomão! 3. A menina avoada: uma leitura semiótica Meu irmão falava que eu tomasse cuidado porque Redomão era coiceiro. As cigarras derretiam a tarde com seus cantos. Meu irmão desejava alcançar logo a cidade - Porque ele tinha uma namorada lá. A namorada do meu irmão dava febre no corpo dele. 95 95 95 Isso ele contava. No caminho, antes, a gente precisava de atravessar um rio inventado. Na travessia o carro afundou e os bois morreram afogados. Eu não morri porque o rio era inventado. Sempre a gente só chegava no fim do quintal E meu irmão nunca via a namorada dele - Que diz-que dava febre em seu corpo. Isso ele contava. No caminho, antes, a gente precisava de atravessar um rio inventado. Na travessia o carro afundou e os bois morreram afogados. Eu não morri porque o rio era inventado. Sempre a gente só chegava no fim do quintal E meu irmão nunca via a namorada dele - Que diz-que dava febre em seu corpo. A partir de sua leitura, percebemos a predominância figurativa subjacente, os elementos figurativos e as estratégias discursivas mobilizadas contribuem para fabrincar os efeitos de sentido gerados a partir de cada leitura. Entretanto, nosso alvo não se limita a classificações e categorizações discursivas, é necessário compreender quais mecanismos foram utilizados pelo o sujeito da enunciação para figurativizar seu enunciado. No nível sintáxico-discursivo, as escolhas de pessoa, espaço e tempo instauram uma debreagem enunciativa, conferindo ao poema o efeito de subjetividade, mais do que isso, faz com que pareça ser uma memória. O poema tem como ponto de referência o “eu”, instância enunciativa por excelência, marcado linguisticamente pelo uso do pronome possessivo e pronome pessoal do caso reto na primeira pessoa do singular: “Foi na fazenda de meu pai antigamente/Eu teria dois anos; meu irmão, nove”. A escolha do tempo verbal é imprescindível para determinação de certos efeitos de sentido. No poema, o uso do pretérito imperfeito é predominante (meu irmão pregava, ficavam cambaias, imitava, puxava o caixote, etc). Fiorin (2001, p.155), ancorado no pensamento de Bakhtin acerca do pretérito imperfeito, afirma que através do uso desse tempo verbal, pelo enunciador, o olhar do enunciatário é direcionado para o interior, para o processo de constituição, por isso, “vê os estados e as transformações indicados por esse tempo em curso ao longo de um espaço de tempo, [...] a ação é considerada como inacabada, contínua dentro da continuidade do momento de referência”. 3. A menina avoada: uma leitura semiótica Nesse sentido, a escolha dessa forma temporal do verbo faz com que as peripécias narradas atinjam um teor de repetitividade, assinalando sua existência na cotidianidade dos sujeitos. O efeito de realidade é construído no texto, também, por meio da debreagem interna: “Eu comandava os bois:/ - Puxa, Maravilha!/ - Avança, Redomão!”. Percebemos nesse mesmo trecho a instauração de uma debreagem interna, os travessões e os pontos de exclamação materializam a fala da criança durante sua brincadeira, valendo-se de um fazer parecer verdadeiro. 96 96 Lembrando que em semiótica a verdade encontra fundamento no conceito de veridicção que diz respeito a uma espécie de contrato entre o enunciador e o enunciatário do discurso enunciado, independentemente de qualificarem-se como autor e leitor reais, já que se trata de algo interno ao texto: A “verdade”, para ser dita e assumida, tem de deslocar-se em direção às instâncias do enunciador e do enunciatário. Não mais se imagina que o enunciador produza discursos verdadeiros, mas discursos que produzem um efeito de sentido “verdade”: desse ponto de vista, a produção da verdade corresponde ao exercício de um fazer cognitivo particular, de um fazer parecer verdadeiro que se pode chamar, sem nenhuma nuance pejorativa, de fazer persuasivo. Exercido pelo enunciador, o fazer persuasivo só tem uma finalidade: conseguir a adesão do enunciatário. (GREIMAS & COURTÉS, Op. cit.,1979, p. 487). A categoria espacial, referenciada na pessoa subjetiva, confirma o espaço do lá em oposição ao espaço do aqui marcado pelo sujeito enunciador (Foi na fazenda; no fim do quintal). Entretanto, há um acontecimento discursivo muito interessante, nos versos “Meu irmão desejava alcançar logo a cidade/ - Porque ele tinha uma namorada lá”, percebemos a instauração de um espaço ainda mais distante: a cidade. Ela figura o lugar alheio, longínquo da própria realidade rural dos sujeitos participantes do evento narrado. No campo semântico, é notória a riqueza temática e figurativa subjacente ao poema. A figuratividade se constrói de forma sumamente concreta, por meio das figuras utilizadas somos transportados ao universo rupestre de faz-de-conta. Como vimos, a organização figurativa de um texto necessita ser recoberta por um tema para fazer algum sentido. 3. A menina avoada: uma leitura semiótica A infância é a temática predominante no poema, essa afirmação pode ser justificada com base no percurso figurativo do texto, marcado por figuras como “eu teria dois anos;/meu irmão nove”, “eu ia pousada dentro do caixote,/ com as perninhas encolhidas./Imitava estar viajando.”, as quais ilustram o momento em que estão justamente construindo a infância, fabrincando um modo de ser criança particular, figurativizado pelas brincadeiras entre dois irmãos, mas não são quaisquer brincadeiras, elas envolvem o imaginário infantil, materializado linguisticamente por meio das figuras “imitava”, “rio inventado” e pela construção “diz-que”. A designação para esta expressão conforme o dicionário eletrônico Infopédia é “conjunto de mexericos; intriga, falatório”. Conforme o uso popular, essa é uma expressão para referir-se a histórias duvidosas, não muito críveis, o que contribuiu para arrematar a impressão do faz de conta que perpassa todo o texto. É interessante observar que para Peter Hunt 97 97 (2010), nos livros infantis é comum a recorrência de temas como magia, fantasia, simplicidade e aventura, uma vez que a criança tem na brincadeira um elemento natural de seu perfil e, por isso, verá a linguagem como outro espaço de exploração lúdica. Poderia, talvez, causar algum estranhamento limitarmos a temática do poema à infância. Sabemos da possibilidade de em um mesmo texto concorrerem várias temáticas, em nossa análise, por exemplo, os temas natureza, diversão, recordação, amor platônico, família estão presentes também. Entretanto, ao longo da cadeia discursiva, percebemos a reincidência de traços semânticos que apontam a presença de duas isotopias predominantes: a isotopia da infância e a isotopia da natureza. A coexistência de ambas é determinante à significação global de todo o poema. Isto porque a infância retratada não é geral, comum a todos, ela está situada em um contexto específico, o rural, marcadamente oposto ao urbano. A recordação do sujeito enunciador figurativizado por uma menina, como se constata no trecho “eu ia pousada” e no título do poema “A menina avoada”, remete a um modo singular de ser criança, o qual envolve elementos da natureza como cigarras, bois e rios. Considerações finais Nosso trabalho se propôs a analisar a temática da infância em Manoel de Barros a partir da mobilização dos conceitos de figuratividade e isotopia no poema “A menina avoada”. Percebemos que essas noções semióticas foram capazes de elucidar como a infância é fabrincada na poética barreana, por meio da apresentação das estratégias linguístico-discursivas mobilizadas pelo sujeito da enunciação. A análise apontou para configuração de uma infância peculiar, com raízes rurais. Além disso, confirmou-se sobremaneira a presença do imaginário, do lúdico, do faz de conta como elementos de figurativização do que entende-se por ser criança sob a perspectiva do enunciador. Acreditamos que esse trabalho não exauriu a riqueza encontrada na obra do poeta mato-grossense, ao contrário, por meio dele é possível vislumbrar novos caminhos para pesquisa a partir de novos enfoques semióticos. 3. A menina avoada: uma leitura semiótica Até mesmo o imaginário, explorado pelas crianças de modo ilimitado e criativo, tem no poema suas raízes arraigadas ao universo rupestre ilustrado pela figura do carro de boi que na realidade era apenas um caixote com latas de goiabada, puxado pelo irmão cuja intenção era atravessar o rio inventado para ver sua suposta namorada, todas essas figuras verbais coadunam-se às figuras ilustrativas que constroem o poema. De modo que os bordados que ilustram A menina avoada são determinantes para o alcance de maior nível de iconicidade na figurativização da infância projetada pelo sujeito da enunciação: 98 98 Figura 1: A carroça e os bois Figura 1: A carroça e os bois 99 99 99 Figura 2: O fim do quintal Figura 2: O fim do quintal Lembrando que quanto maior for o nível de iconicidade, maior será a densidade do componente figurativo, nas palavras de Bertrand (op cit, 2003) haverá manifestação icônica quando a reunião de traços característicos for suficiente para permitir sua interpretação como uma representação do mundo natural. Na figura 1, vemos a projeção imagética da “menina avoada” equilibrando-se por cima da carroça inventada, cercada por elementos que aludem ao universo da inocência, do lúdico tais como a bola, o cavalinho de madeira, a boneca de pano. Se a infância é demarcada no texto, podemos afirmar que o rural é sublinhado com linhas coloridas e laranjadas como a cor do sol ao se deitar no horizonte da fazenda. Com a figura 2, confirma-se o olhar distante do irmão situado espacialmente “no fim do quintal”, como quem vislumbra algo muito valioso, do seu ponto de vista, configurando-se como um sujeito virtualizado, isto é, ele /quer/ fazer (chegar a cidade para ver sua suposta namorada), porém não /pode/ efetuar essa performance, já que ela existe somente em sua fantasia de menino. 100 100 Considerações finais ______. Teoria Semiótica do texto. São Paulo: Ática, 2005. ______. Teoria Semiótica do texto. São Paulo: Ática, 2005. BENVENISTE, Émile. Problemas de lingüística geral I. 4ª ed. Campinas: Pontes/ EdUNICAMP, 1995. BERTRAND, Denis.. Caminhos da semiótica literária. Tradução do Grupo CASA. Bauru, SP: EDUSC, 2003. REFERÊNCIAS BARROS, Manoel de. Exercícios de ser criança. Ilustrações e bordados de Antônia Zulma Diniz, Ângela, Marilu, Martha e Sávia Dumont sobre os desenhos de Demóstenes. Rio de Janeiro: Salamandra, 1999. BARROS, Diana Luz Pessoa de. Teoria do discurso: fundamentos semióticos. São Paulo: Humanitas, 2002. BERTRAND, Denis.. Caminhos da semiótica literária. Tradução do Grupo CASA Bauru, SP: EDUSC, 2003. Dicionário infopédia da Língua Portuguesa com Acordo Ortográfico [em linha]. Porto: Porto Editora, 2003-2016. Disponível na Internet: https://www.infopedia.pt/dicionarios/lingua-portuguesa/diz-que-diz. Acesso em: 21 out. 2016. FIORIN, José Luiz. A pessoa desdobrada. Alfa: São Paulo, 1995. Disponível em: <http://seer.fclar.unesp.br/alfa/article/view/3968/3643>. Acesso em: 29 jul. 2016. ______. Elementos de análise do discurso. São Paulo: Contexto, 2002. ______. As astúcias da enunciação: as categorias de pessoa, espaço e tempo. 2. ed. São Paulo, SP: Atica, 2001. ______. Em busca do sentido: estudos discursivos. São Paulo: Contexto, 2012. ______. Em busca do sentido: estudos discursivos. São Paulo: Contexto, 2012. GREIMAS, Algirdas J.; COURTÈS, Joseph. Dicionário de semiótica. São Paulo, Cultrix,1979. GREIMAS, Algirdas J.; COURTÈS, Joseph. Dicionário de semiótica. São Paulo, Cultrix,1979. 101 101 HOUAISS, A. Dicionário eletrônico Houaiss da língua portuguesa. Rio de Janeiro: Objetiva. Versão 1.0. 1 [CD-ROM]. 2001. HUNT, Peter. Crítica, teoria e literatura infantil. Tradução de Cid Knipel. São Paulo: Cosacnaify, 2010. Disponível em: <https://pt.scribd.com/document/323672387/Critica- teoria-e-literatura-infantil-Hunt-Peter-pdf>. Acesso em: 19 out. 2016. HOUAISS, A. Dicionário eletrônico Houaiss da língua portuguesa. Rio de Janeiro: Objetiva. Versão 1.0. 1 [CD-ROM]. 2001. HUNT, Peter. Crítica, teoria e literatura infantil. Tradução de Cid Knipel. São Paulo: Cosacnaify, 2010. Disponível em: <https://pt.scribd.com/document/323672387/Critica- teoria-e-literatura-infantil-Hunt-Peter-pdf>. Acesso em: 19 out. 2016. HOUAISS, A. Dicionário eletrônico Houaiss da língua portuguesa. Rio de Janeiro: Objetiva. Versão 1.0. 1 [CD-ROM]. 2001. HUNT, Peter. Crítica, teoria e literatura infantil. Tradução de Cid Knipel. São Paulo: Cosacnaify, 2010. Disponível em: <https://pt.scribd.com/document/323672387/Critica- teoria-e-literatura-infantil-Hunt-Peter-pdf>. Acesso em: 19 out. 2016. HOUAISS, A. Dicionário eletrônico Houaiss da língua portuguesa. Rio de Janeiro: Objetiva. Versão 1.0. 1 [CD-ROM]. 2001. HUNT, Peter. Crítica, teoria e literatura infantil. Tradução de Cid Knipel. São Paulo: Cosacnaify, 2010. Disponível em: <https://pt.scribd.com/document/323672387/Critica- teoria-e-literatura-infantil-Hunt-Peter-pdf>. Acesso em: 19 out. 2016. OUAISS, A. Dicionário eletrônico Houaiss da língua portuguesa. Rio de Janeiro: bjetiva. Versão 1.0. 1 [CD-ROM]. 2001. UNT, Peter. Crítica, teoria e literatura infantil. Tradução de Cid Knipel. São Paulo: osacnaify, 2010. Disponível em: <https://pt.scribd.com/document/323672387/Critica- oria-e-literatura-infantil-Hunt-Peter-pdf>. Acesso em: 19 out. 2016. 102 102
https://openalex.org/W2125863750	https://academicjournals.org/journal/AJEST/article-full-text-pdf/BB7F6B250860.pdf	English	null	Solid medical waste management in Africa	African journal of environmental science and technology	2,015	cc-by	9,853	African Journal of Environmental Science and Technology Legon, Accra, Ghana. 2Department of Community Health, University of Ghana Medical School, School of Public Health, University of Ghana, Legon, Accra, Ghana 3Unit for Health Promotion Research Institute of Public Health University of Southern Denmark Esbjerg Denmark g n Research, Institute of Public Health, University of Southern Denmark, Esbjerg, Denmark. Increased awareness about inadequate management of solid medical waste (SMW) has led to increased independent surveys in African countries and yet published data remain scanty on the subject in Africa as compared to the rest of the world. To evaluate the overall compliance with World Health Organization (WHO)’s ten recommendations on SMW practices in Africa through a literature review, we conducted literature search using search terms as “medical waste OR clinical waste OR biomedical waste OR hospital waste OR healthcare waste OR hazardous waste AND Africa” in PubMed, African Journals Online, Web of Science, Proquest, Embase, Google scholar and Scopus. Additional articles were included from open google search. Articles were selected for inclusion if they described SMW management activities such as waste segregation, collection, transport (on-site and/or off-site), temporary storage, treatment and final disposal; were located in an African country and were written in English; or if written in a different language, had an English abstract with the relevant information. Evaluations were based on fifty eight (58) full text articles which were pooled together. The fifty eight (58) full text articles represented research undertaken in 20 countries. Overall, six countries (30%) broadly met half of the WHO’s 10 recommendations. Based on the reviewed articles, the greatest compliance was shown with “daily collection of waste from the service areas” (100%). Areas of least compliance were “appropriate use of color codes” (18%) for labeling waste bins, “off-site conveyance” (16%) and “periodic training” (18%). In spite of growing awareness, SMW management in most African countries was sub-standard. Appropriate use of color codes, periodic training and off-site conveyance need to be addressed in terms of reporting and practice. Common challenges and unremarkable progress in SMW management in Africa raise a question about whether a unified approach should replace independent country efforts. Key words: Africa, hospital waste, solid medical wastes, waste management. Vol. 9(3), pp. 244-254, March 2015 DOI: 10.5897/AJEST2014.1851 Article Number: BB7F6B250860 ISSN 1996-0786 Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJEST African Journal of Environmental Science and Technology African Journal of Environmental Science and Technology Udofia et al. distribution of studies on healthcare and solid medical waste management. The search strategy employed was as follows: distribution of studies on healthcare and solid medical waste management. The search strategy employed was as follows: because of the potential to transmit diseases, conta- minate soil, surface and ground water with pathogenic microbes, toxic and heavy metals often present in it (Pruss et al., 1999). Diseases associated with poor medical waste management include nosocomial diseases, typhoid, skin disorders, intestinal parasitosis and hepatitis (Bassey et al., 2006). 1. PubMed was searched applying the terms ‘medical waste OR biomedical waste OR clinical waste OR hospital waste OR hazardous waste OR healthcare waste AND Africa’. Additional databases searched using the same search terms included Web of Science, Embase, Scopus, Proquest and African Journals Online. p q 2. The search was repeated with the same keywords but replacing Africa with names of individual countries. The list of African countries was adapted from World Bank websites: www.worldbank.org/en/region/mena (North African countries) and www.worldbank.org/en/region/afr (Sub-Saharan Africa) with the addition of Mauritius. In addition, there is a potential risk of HIV transmission to a susceptible human host from percutaneous injury by infected sharps (WHO, 2004). Therefore, inadequate handling and disposal of medical waste has conse- quences for patients, relatives or carers, healthcare workers, waste workers, scavengers, the public and the environment (Abor, 2007; Coker et al., 2009; Mesdaghinia et al., 2009). 3. A more in-depth search on medical waste in Ghana and Madagascar using the search terms “medical waste in Ghana” and “medical waste in Madagascar” respectively, added 3 articles. All searches covered the period from 1997 up to October, 2014. g , ) Africa is estimated to have 67,740 health facilities and produce approximately 282,447 tons of medical waste every year (AGENDA, 2009, Udofia and Nriagu, 2013). However, the composition of the waste stream considered hazardous and requiring special treatment may be higher than the expected 10 to 25% because of poor waste handling practices reported in many studies (mixing hazardous with non-hazardous waste (Haylamicheal et al., 2011; Fadipe et al., 2011; Abah and Ohimain, 2011). Studies in Africa indicate the continent is not positioned to tackle the quantity of hazardous waste it produces. Much of the waste is dumped without treat- ment in open dumps and poorly functioning incinerators (Nkhuwa et al., 2008, Nemathaga et al., 2008, Sawalem et al., 2009, Coker et al., 2009, Saad, 2013). RESULTS y To the best of our knowledge, this review is the first on solid medical wastes in Africa and sought to compare medical waste management activities (segregation, collection, storage, transport, treatment and final disposal) reported in studies done in Africa with WHO recommendations (WHO, 2014). The general objective of the review was to determine how many of WHO standards were broadly met in each country in Africa and how specific waste management activities compared across the countries included in the review. Literature search Table 1 summarizes the number of hits identified by databases and shows the number of articles involved in the preliminary screening. The flowchart (Figure 1) depicts the literature search process. The search in PubMed produced a total of 299 articles of which 278 articles were excluded on preliminary screening (Figure 1) and 1 full text unavailable in the databases. The other databases yielded a total of 38 articles in full text, after excluding those which were not available (4 articles). Finally, 58 full text articles that met the selection criteria were reviewed. Udofia et al. While increasing awareness has driven many individual country reports on general solid waste streams in Africa, the overall picture of solid medical waste management is unclear. Consequently information on solid medical wastes in Africa appears to remain on shelves and only scanty in scientific literature. Articles were selected for inclusion in the review if they: described SMW management activities such as waste segregation, collection, transport (on-site and/or off-site), temporary storage, treatment and final disposal; with the study location in an African country and were written in English; or if written in a different language, had an English abstract with the relevant information. Articles which did not meet the selection criteria and focused on liquid waste management and veterinary services were excluded. Duplicate publications were removed manually. The papers included for review were categorized and evaluated according to the following items: segregation into at least 2 categories of waste (sharps and non-sharps waste); appropriate use of color coded bags, containers and symbols; waste collected at least once a day from generation points; on-site transport with dedicated vehicle; secured temporary storage area; stored waste is treated or discarded not later than the second day; off-site transport in enclosed vehicles with back loading; treatment option includes an advanced thermal method for infectious waste; final disposal at sanitary landfill or disposal site; and periodic training of healthcare staff. These items were based on recommendations by WHO (2014) as the standard set for acceptable waste management activities. INTRODUCTION Solid medical waste has been referred to as any discarded solid material generated from activities involving health protection, medical diagnosis, treatment, scientific research, dental and veterinary services (US Congress Office of Technology Assessment, 1988; Rao, 2008; Coker et al., 2009; Hossain et al., 2011). This type of Solid medical waste has been referred to as any discarded solid material generated from activities involving health protection, medical diagnosis, treatment, scientific research, dental and veterinary services (US Congress Office of Technology Assessment, 1988; Rao, 2008; Coker et al., 2009; Hossain et al., 2011). This type of waste has been considered potentially harmful to humans and requires special treatment. Non-hazardous waste from health-care facilities is comparable to domestic waste and often ends up in the municipal solid waste stream discarded in landfills (Pruss et al., 1999). Solid medical waste has remained a source of concern 245 Udofia et al. Udofia et al. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Search strategy The reviewed articles covered twenty (20) countries A review of literature was conducted using electronic databases wide Afr. J. Environ. Sci. Technol. 246 Table 1. Distribution of articles according to databases searched. Database Number of hits (no filters) Number of hits (with filter) PubMed 299 299 African Journal Online 1,240 1,240 Web of Science 17,957 13,081 (Journal articles) Embase 855 615 (Journal articles) Scopus 96 69 (Journal articles) Proquest 1,655,609 391,589 (Journal articles); 164,464 (scholarly articles) Google Scholar 164,000 (Without patents, citation) 164,000 Table 1. Distribution of articles according to databases searched. Database Number of hits (no filters) Number of hits (with filter) Figure 1. A schematic representation of the literature search process. Figure 1. A schematic representation of the literature search process. variety of healthcare facilities, such as public and private healthcare facilities or were a mix across the levels of the healthcare system– primary, secondary, tertiary level healthcare facilities. Most studies (55%) employed mixed methods. This often comprised of waste surveys (mea- suring the quantity and determining the composition of solid waste, that is, waste stream analysis) in healthcare facilities, questionnaire surveys of healthcare workers and housekeeping staff and direct observation of waste handling practices. In other studies, key informant namely: Algeria, Botswana, Cameroun, Egypt, Ethiopia, Ghana, Kenya, Lesotho, Libya, Madagascar, Mauritius, Morocco, Nigeria, Senegal, Somaliland, South Africa, Sudan, Tanzania, Uganda and Zimbabwe (Table 2). Figure 2 shows the distribution of articles by these countries. Figure 3 shows the distribution of articles over the study period and indicates increasing interest and research in solid medical waste management in Africa. Table 3 summarizes the characteristics of the identified articles. Twenty eight (28) studies (48%) covered a 247 Udofia et al. Table 2. List of reviewed articles on solid medical waste management in African countries. Country No. Search strategy of articles Reference (first author)* Year of publication Algeria 2 Bendjoudi 2009 Beghdadli 2010 Botswana 1 Mbongwe 2008 Cameroun 1 Manga 2011 Egypt 2 Soliman 2007 El-Salam 2010 Ethiopia 4 Azage 2010 Haylamicheal 2011 Debere 2013 Muluken 2013 Ghana 4 Squire 2013 Abor 2013 Asante 2014 Akum 2014 Kenya 1 Mazrui 2010 Lesotho 1 Majara 2009 Libya 1 Sawalem 2009 Madagascar 1 Odette 2014 Mauritius 1 Mohee 2005 Morocco 1 Mbarki 2013 Nigeria 23 Bassey 2006 Longe 2006 Babatola 2008 Oke 2008 Nguluka 2009 Odigie 2009 Coker 2009 Ajimotokan 2009 Ngouakam 2010 Adedigba 2010 Samuel 2011 Johnson 2011 Fadipe 2011 Chima 2011 Morenikeji 2011 Abah 2011 Jiburum 2012 248 Afr. J. Environ. Sci. Technol. Table 2. Contd. Country No. of articles Reference (First author) Year of publication Ogbonna 2012 Oruonye 2012 Longe 2012 Tobin 2012 Idowu 2013 Uwa 2014 Senegal 1 Ndiaye 2012 Somaliland 1 DiBella 2012 South Africa 3 Nemathaga 2008 Abor 2008 Gabela 2009 Sudan 1 Saad 2013 Tanzania 5 Mato 1997 Mato 1999 Manyele 2006 Manyele 2010 Nilsson 2013 Uganda 1 Mugambe 2012 Zimbabwe 3 Taru 2005 Jerie 2006 Mangizvo 2008 See appendix for full reference of the articles. Country No. of articles Reference (First author) Year of publication See appendix for full reference of the articles. See appendix for full reference of the articles. countries) indicated compliance with the recommenda- tions. Although all papers reporting daily waste collection indicated compliance with recommendations, this repre- sented less than half of the reviewed articles and half of the countries represented. Overall, Nigeria and South Africa had articles reporting compliance with at least nine (9) of the ten (10) recommendations by WHO. However, South Africa demonstrated a higher overall compliance with fewer reviewed articles (3) as compared to Nigeria (23). The majority of countries fulfilled less than five recommendations. interviews were conducted with management staff regarding the awareness of existing policies, availability of waste plans and teams, as well as protocols on medical waste management in the healthcare facility. Eight articles involved review of secondary data, relevant documents and analysis of existing data. One article was a desk review of existing studies in Tanzania. Waste management activities Table 4 summarizes information reported on activities conducted in compliance with WHO’s recommendations on solid medical waste management (WHO, 2014). In the table, the number of articles reporting compliance was inserted in parenthesis beside the countries. Forinstance, Nigeria, Tanzania (3) means that Nigeria and Tanzania each had three articles that reported compliance with the standard for the specific solid medical waste manage- ment activity. DISCUSSION To the best of the authors’ knowledge, this review is the first attempt to present an overview of the status of SMW management activities in sub-Sahara Africa. Available evidence from the 58 journal articles from across 20 countries in sub-Sahara Africa in the period 1997 to 2014 indicates that SMW management still face many challenges and that South Africa appears to meet The least reported activity was off-site transport which 19 articles reported and only three articles (in two 249 Udofia et al. Figure 2. Distribution of studies by country. Figure 3. Distribution of articles by year of publication. tice according to the WHO-2014 recommen- n solid medical waste management practices. hat most of the papers were from reputable and scientific databases including peer-reviewed nderlie the scientific validity of the findings the most valuable information was obtained from s which covered a variety of healthcare facilities an the method used in 35 articles. Case studies provided greater detail and tended to cover most waste management activities discussed Figure 2. Distribution of studies by country. Figure 3. Distribution of articles by year of publication. most valuable information was obtained from surveys which covered a variety of healthcare facilities and was the method used in 35 articles. Case studies also provided greater detail and tended to cover most of the waste management activities discussed. best practice according to the WHO-2014 recommen- dations on solid medical waste management practices. The fact that most of the papers were from reputable and credible scientific databases including peer-reviewed articles underlie the scientific validity of the findings, the Afr. J. Environ. Sci. Technol. 250 Table 3. Characteristics of articles reviewed on solid medical waste management activities in African countries, 1997-2014. Characteristic Description Number of studies Language English 57 French 1 Type of setting Various health facilities 28 Hospital(s) 20 Clinics/Health centres 8 Not specified 2 Type of research Qualitative 9 Quantitative 17 Mixed 32 Research design Survey/Cross sectional 35/13 Desk review 1 Case study 7 Panel/Intervention study 2 Research technique Questionnaire administration 44 Interviews 39 Observation (checklists) 39 (4) Waste/other sampling 24 Record review 8 Focus group discussion 2 More than one research technique was used in the studies. Table 4. Compliance with WHO-2014 standards. Table 4. Compliance with WHO-2014 standards. Table 4. Compliance with WHO-2014 standards. DISCUSSION Standards for medical waste management Number of articles reporting activity Number of articles reporting compliance with standard N (%) Number of countries reporting activity Number of countries reporting compliance with standard N (%) Countries showing compliance with standard Waste segregation into at least 2 categories 57 31 (53) 20 18 (90) Nigeria (3), Tanzania, South Africa, Ghana (3), Egypt (2), Botswana, Cameroun, Ethiopia, Kenya, Lesotho, Libya, Madagascar, Morocco, Senegal, Somaliland, Sudan, Uganda, Zimbabwe (1) Appropriate use of color codes and symbols 45 8 (18) 17 6 (35) Nigeria (3), Egypt, Ghana, Lesotho, Madagascar, South Africa (1) Daily waste collection from service areas 21 21 (100) 10 10 (100) Nigeria (8), Ethiopia (3), South Africa, Tanzania (2), Cameroun, Egypt, Ghana, Sudan (Khartoum State), Uganda, Zimbabwe (1) Dedicated vehicle for onsite transport 37 11 (30) 17 9 (53) Nigeria (3), Ghana, Libya, Madagascar, Senegal, Somaliland, South Africa, Sudan, Uganda (1) 251 Udofia et al. Table 4. Contd. Standards for medical waste management Number of articles reporting activity Number of articles reporting compliance with standard N (%) Number of countries reporting activity Number of countries reporting compliance with standard N (%) Countries showing compliance with standard Secured temporary storage area 31 7 (23) 14 5 (36) Egypt, Nigeria (2), Ghana, Morocco, South Africa (1) Stored waste treated no later than 2nd day 27 12 (44) 11 9 (82) Egypt, Nigeria, Tanzania (2), Algeria, Ethiopia, Lesotho, South Africa, Sudan (Khartoum), Zimbabwe (1) Off-site transport in enclosed back loading vehicles 19 3 (16) 11 2 (18) Nigeria (2), Zimbabwe (1) Treatment option includes advanced thermal option 56 41 (73) 19 18 (95) Nigeria (12), Tanzania (5), Ethiopia (4), Zimbabwe (3), Algeria, Egypt, Ghana (2), Botswana, Cameroun, Lesotho, Libya, Madagascar, Mauritius, Senegal, Somaliland, South Africa, Sudan, Uganda (1) Final disposal at sanitary landfill or dedicated site 44 17 (39) 15 10 (67) Nigeria (7), Zimbabwe (2), Algeria, Egypt, Ethiopia, Ghana, Kenya, Mauritius, South Africa, Uganda (1) Periodic training of healthcare staff 39 7 (18) 15 6 (40) Nigeria (2), Algeria, Ghana, Morocco, South Africa, Tanzania (1) The total number of articles reviewed = 58; the total number of countries represented = 20. More than half (53%) of the publications reported segregation of SMW but appropriate use of color coded receptacles was low (18%). Segregation was reported at least for sharps in most countries. DISCUSSION Poorly separated waste increases the amount of waste sent for incineration or sent untreated to the landfill. Overloading incinerators eventually made them dysfunctional. Untreated waste in open dumps or unsecured landfills had the potential of transmitting infections to scavengers. Collection bags (including colour coded ones) were reported to be diverted to other uses such as storage of items by patients’ relatives making it difficult to appreciate that the materials contained in the bags were potentially harmful (Mbongwe et al., 2008). The inappropriate use of black plastic bags for infectious waste in a bid to save costs has also been reported (Majara and Leduka, 2009). In healthcare facilities where waste is segregated, it often gets mixed at the temporary storage site or during collection by transporting companies or waste collection service providers (Coker et al., 2009; Debere et al., 2013; Sawalem et al., 2009). Appropriate segregation and resource recycling has been found to achieve a 15% reduction in the quantity of medical wastes (Sabour et al., 2007). This has economic implications for the health sector. For instance, it was noted that £15 million could be potentially saved in the UK, through improved segregation and waste minimization of the 40-60% of the healthcare waste stream which is non-hazardous (Tudor et al., 2009). It has also observed in the UK that segregation of medical waste has not been given high priority. Some of the barriers are no different from those in African countries, limited space for multiple receptacles at service areas, inadequate budgetary provision for waste management activities and limited continuous training opportunities for healthcare workers (Tudor et al., 2009). Our review indicated that nearly a quarter of articles reported compliance by most facilities having a secured storage area. Storage areas were often unsecure permitting access to both unauthorized persons and stray animals (Bendjoudi et al., 2009; Ndiaye et al., 2012; Nemathaga et al., 2008, Sawalem et al., 2009). In some cases, no temporary storage area existed in the facilities surveyed (Muluken et al., 2013). Scavengers and workers Afr. J. Environ. Sci. Technol. 252 and are ultimately consumed by humans with considerable implications for human health. In contrast, common methods used for medical waste treatment in advanced countries include steam sterilization, auto- claving and incineration (Patwary et al., 2011). Treatment technologies in the UK fall into two main categories: high temperature incineration/combustion and non-burn or low temperature alternative technologies (Tudor et al., 2009). DISCUSSION In the first category, examples include incineration, pyrolysis, plasma technology and gasification whereas examples in the second category (non-burn technology), are: autoclaves, steam augur, dry heat, microwaves and macro-waves (Tudor et al., 2009). In Canada, there has been a shift from onsite incinerators towards centralized facilities, which handle medical waste generated over a wide geographic area (Walkinshaw, 2011). This approach helps to reduce air pollution resulting from on-site incineration. often recover valuable items to recycle and re-sell to the public (Bassey et al., 2006; Taru and Kuvarega, 2005). Rodents may serve as vehicles for pathogens in untreated SMW. Both act in different ways as ‘bridge populations’ with potential to transmit infectious agents to the general population. Inadequate contain-ment of medical wastes, collection and storage has been reported in both advanced (Blenkharn, 2007) and developing countries (Manga et al., 2011; Mbongwe et al., 2008) g g All papers that reported daily collection of waste showed that most facilities complied with this standard, but only 30% of the reviewed articles reported com- pliance by use of dedicated vehicles for on-site collection. Although conveyances used included wheeled carts, trolleys and wheelbarrows (Abor, 2013; Ndiaye et al., 2012), manual lifting of waste receptacles by hospital cleaners, waste handlers and auxiliary staff was also reported (Abah and Ohimain, 2011; Coker et al., 2009; Manga et al., 2011; Muluken et al., 2013). In Zimbabwe, a small vehicle with a carrying capacity of up to 14 waste bins at once (totaling 15kilograms in mass) was used to convey SMW from hospital corridors to the storage site. This vehicle was able to collect an estimated 124 bins daily (Taru and Kuvarega, 2005). In some of the studies reviewed, vehicles that were reportedly used to convey SMW to final disposal sites were not described in detail especially when municipal trucks (Manga et al., 2011) or waste companies were used (Abor, 2013). An interesting dimension was the use of unlined placenta pits which are fed with acid digesters to prevent them from getting full (Nkhuwa et al., 2008). In areas where the ground water table is shallow and the soil porosity permits easy percolation, potential contamination of groundwater could be possible (Nkhuwa et al., 2008). Placentae were also taken home to bury in keeping with traditional (Manga et al., 2011) or religious practices (Abd El-Salam, 2010). DISCUSSION Due to the natural decomposition of placental organic contents, it is considered less harmful in more stable soils. p ( ) In healthcare facilities where disposal was reported to be done on-site, open dumping in refuse pits, burning and burial have been described (Ajimotokan and Aremu, 2009; Manyele and Anicetus, 2006; Abah and Ohimain, 2011; Manga et al., 2011). Although incineration was available and compliance was reported by 73% of the articles, they were often rudimentary, dysfunctional and/or lacked regular maintenance (Abd El-Salam, 2010; Mbongwe et al., 2008; Nemathaga et al., 2008; Nkhuwa et al., 2008). Most incinerators described did not have modern technology such as air pollution control (APC) equipment and adequate air inlets. They often had a single chamber made from local bricks, which did not completely burn off the gases generated during combustion process. In some of the articles that reported training of healthcare workers, training in solid medical waste management was found to be inadequate and was not undertaken periodically (Coker et al., 2009; Haylamicheal et al., 2011; Saad, 2013; Sawalem et al., 2009). Similarly, the present survey showed that only one of every six articles reported periodic training in the surveyed health- care facilities. It has been shown that there is a significant association between training in healthcare waste management and waste management practices (Muluken et al., 2013). However, Beghdadli and co-authors (2010) portend that while training and the provision of color coded receptacles are essential, these measures alone are not sufficient for segregation of solid medical waste. Accountability and remedial action for waste malpractices by health staff should be defined and upheld in relevant facilities (Beghdadli et al., 2010). Furthermore, coal or wood fuels were used for combus- tion which made it difficult to control the temperature. As a result, they became a source of air pollution themselves especially within the hospital premises and the surroun- ding communities (Jerie, 2006; Nemathaga et al., 2008). Incinerators were often loaded with plastics made of polyvinyl chloride (PVC) which released carcinogenic dioxins and furans on incineration (Nemathaga et al., 2008; Singh and Prakash, 2007). REFERENCES Abah SO, Ohimain EI (2011). Healthcare waste management in Nigeria: A case study. J. Public Health Epidemiol. 33:99-110. Abd El-Salam MM (2010). Hospital waste management in El-Beheira Governorate, Egypt. J Environ Manage. 91(3):618-629. ( ) p g Governorate, Egypt. J Environ Manage. 91(3):618-629. Abor PA (2007). Medical Waste Management Practices in a Southern African Hospital. J. Appl. Sci. Environ. Manage. 11(3):91-96. Abor PA (2013). Managing healthcare waste in Ghana: a comparative study of public and private hospitals. Int. J. Health Care Qual. Assur. 26(4):375-386. The review revealed that independent national efforts have made only slow progress in managing solid medical waste leaving the authors to question whether or not a unified approach across all countries would yield better results. A unified approach demands the assemblage of experts in solid medical waste management and related disciplines across the continent to identify and aggregate available technology, expertise, funding, and best practices to bear on SMW management. A conference which brings together waste management experts, toxicologists, public health (environmental and occupational health) experts, governments, and other stakeholders focused on SMW management in Africa would be a good starting point. Challenges faced in African countries which are not often articulated in published literature, can be shared at such gatherings. Sharing and documentation of country experiences should include a platform for improving visibility and accessibility of information from participating countries to strengthen research and evaluation. Funding mechanisms for SMW management and accessibility of funds is critical to every activity especially improving technology for waste treatment. However, priority should be given to internal funding which is self-sustaining rather than externally funded projects. AGENDA for Environment and Responsible Development (2009). Needs Assessment for Hospitals in African Countries in Relation to Infectious Waste Treatment Final Report - Demonstrating and Promoting Best Techniques and Practices for Reducing Health Care Waste to Avoid Environmental Releases of Dioxins and Mercury. Dar es Salaam, Tanzania. Ajimotokan HA, Aremu SA (2009). Case Study Evaluation of Health- Care Waste Challenges in Hospitals within Ilorin Metropolis. Civil Engineering Impact on National Development: 1st Annual Civil Engineering Conference, University of Ilorin, Nigeria, 26-28 August, 2009. pp. 130-134. pp Bassey BE, Benka-Coker MO, Aluyi HSA (2006). Characterization and management of solid medical wastes in the Federal Capital Territory, Abuja Nigeria. Afr. Health Sci. 6(1):58-63. j g Beghdadli B, Ghomari O, Taleb M, Kandouci BA, Fanello S(2010). REFERENCES Implementation of WHO healthcare waste management (HCWM) approach in an Algerian hospital. Waste Manag. 30 (1): 162-164. pp g p g ( ) Bendjoudi Z, Taleb T, Abdelmalek F, Addou A (2009).Healthcare waste management in Algeria and Mostganem department. Waste Manag. 29(4):1383-1387. ( ) Blenkharn JI (2007). Standards of clinical waste management in hospitals - A second look. Public Health 121(7):540-545. Coker A, Sangodoyin A, Sridhar M, Booth MC, Olomolaiye P, Hammond F (2009). Medical waste management in Ibadan, Nigeria: Obstacles and prospects. Waste Manag. 29(2):804-811. Debere MK, Gelaye KA, Alamdo AG, Trifa ZM (2013). Assessment of the health care waste generation rates and its management system in hospitals of Addis Ababa, Ethiopia, 2011. BMC Public Health 13:28. Fadipe OO, Oladepo KT, Jeje JO, Ogedengbe MO (2011). Characterization and analysis of medical solid waste in Osun State, Nigeria. Afr. J. Environ. Sci. Technol. 5(12):1027-1038. Haylamicheal ID, Mohamed AD, Yirsaw BD, Zegeye HA (2011). Assessing the management of healthcare waste in Hawassa City, Ethiopia. Waste Manag. Res. 29(8):854-862. Conclusion This review provides an overview of solid medical waste management practices in Africa as evident in published literature. Key weaknesses identified were the inappro- priate use of color codes and symbol, lack of periodic training for health staff and off-site transport in appro- priate vehicles. Although incineration was often used as an advanced thermal option, it was often dysfunctional or Due to incomplete burning of the waste, a large quantity of ash has been reported to be generated with high concentration of heavy metals. Such ash may find application as manure for growing vegetables (Nkhuwa et al., 2008). Heavy metals find their way into the food chain Udofia et al. 253 lacked air pollution control equipment. Traditional methods of open burning and burial were still common- place. Institutional challenges relating to appropriate use of colour codes and symbols, and periodic training can be addressed with adequate management commitment and technical support from health ministries. As off-site transport is often the responsibility of municipal authorities or privately contracted, it is essential that health and environment ministries, municipal authorities, private and public sector waste management firms collaborate to ensure that vehicles assigned for transport of solid medical waste are appropriately designed, labelled and dedicated for solid medical waste to minimize potential risk to the public. Selection of waste treatment technologies and final disposal options should be done in a manner that takes into account impacts on human health and the environment (healthcare facility and communities living in proximity to it), capital and maintenance costs, technical capability, local resource constraints and the prevalence of scavenging peculiar to SMW. authors are indebted to the librarians: Ms. Carol Shannon, Taubman Library, University of Michigan, Ann Arbor, USA, Ms. Irene Rattenborg, University of Southern Denmark, Esbjerg campus and Mrs. Theodosia Adanu, University of Ghana, Legon campus for their invaluable support. ACKNOWLEDGEMENTS This review paper was written as part of the lead author’s doctoral research funded by the Building Stronger Universities in Developing Countries – Human Health Platform of Danish Government. Prof. J. 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An Assessment of Medical Waste Management in Bawku Presbyterian Hospital of the Upper East Region of Ghana. Merit Res. J. Environ. Toxicol. 2(2): 27 – 38. Kenya 1 Mazrui N (2010). Medical Waste Management in Kenya: Opportunities for improvement. Tropical Resources Bulletin of the Yale Tropical Resources Institute 29: 66-70. Lesotho 1 Majara M, Leduka RC (2009). Health-Care Waste Practices in Selected Health-Care Facilities in Maseru. Review of Southern African Studies 12(1): 1-29. Libya 1 Sawalem M, Selic E, Herbell J-D (2009). Hospital waste management in Libya: A case study. Waste Manag. 29(4): 1370 - 1375. Madagascar 1 Odette RH, Masika J, Venance T, Soatiana JE, Christane NA, Lamine CM, Bin L (2014). Assessment of healthcare waste generation and its management systems: A prevalence survey of the healthcare facilities in Madagascar.IOSR-JESTFT 8(7 ver. III): 20 -29. Squire (2013). Biomedical Pollutants in the Urban Environment and Implications for Public Health: A Case Study. ISRN Public Health vol. 2013, Article ID 497490, 5 pages. Squire (2013). Biomedical Pollutants in the Urban Environment and Implications for Public Healt A Case Study. ISRN Public Health vol. 2013, Article ID 497490, 5 pages. Abor PA (2013). Managing healthcare waste in Ghana: a comparative study of public and private hospitals. Int J Health Care Qual Assur. 26(4): 375-386. Asante OB, Yanful E, Yaokumah EB (2014). Healthcare Waste Management; Its Impact: A Case Study Of The Greater Accra Region, Ghana. International Journal of Scientifiic and Technology Research 3(3): 106 -112. Akum FA (2014). An Assessment of Medical Waste Management in Bawku Presbyterian Hospital of the Upper East Region of Ghana. Merit Res. J. Environ. Toxicol. 2(2): 27 – 38. Mazrui N (2010). Medical Waste Management in Kenya: Opportunities for improvement. Tropical Resources Bulletin of the Yale Tropical Resources Institute 29: 66-70. Majara M, Leduka RC (2009). Health-Care Waste Practices in Selected Health-Care Facilities in Maseru. Review of Southern African Studies 12(1): 1-29. Sawalem M, Selic E, Herbell J-D (2009). Hospital waste management in Libya: A case study. Waste Manag. 29(4): 1370 - 1375. ACKNOWLEDGEMENTS Hospital waste management in Libya: A case study. Waste Manag. 29(4): 1370 - 1375. Madagascar 1 Odette RH, Masika J, Venance T, Soatiana JE, Christane NA, Lamine CM, Bin L (2014). Assessment of healthcare waste generation and its management systems: A prevalence survey of the healthcare facilities in Madagascar.IOSR-JESTFT 8(7 ver. III): 20 -29. Country Number of articles reviewed References Algeria 2 Bendjoudi Z, Taleb T, Abdelmalek F, Addou A (2009). Healthcare waste management in Algeria and Mostganem department. Waste Manag. 29(4): 1383-1387. Beghdadli B, Ghomari O, Taleb M, Kandouci BA, Fanello S (2010). Implementation of WHO healthcare waste management (HCWM) approach in an Algerian hospital. Waste Manag. 30 (1): 162-164. Botswana 1 Mbongwe B, Mmereki BT, Magashula A (2008). Healthcare waste management: current practices in selected healthcare facilities, Botswana. Waste Manag. 28(1): 226–233. Cameroun 1 Manga VE, Morton OT, Mofor LA, Woodard R (2011). Health care waste management in Cameroon: A case study from the Southwestern Region. Resources, Conservation and Recycling 57:108–116. Egypt 2 Soliman SM, Ahmed AI (2006). Overview of biomedical waste management in selected governorates in Egypt: A pilot study. Waste Manag. 27(12): 1920-1923. Abd El-Salam MM (2010). Hospital waste management in El-Beheira Governorate, Egypt. J Environ Manage. 91 (3): 618-629. Ethiopia 4 Azage M, Kumie A (2010). Healthcare waste generation and its management system: the case of health centers in West Gojjam Zone, Amhara Region, Ethiopia. Ethiop. J. Health Dev. 24(2): 119 – 126. Haylamicheal ID, Mohamed AD, Yirsaw BD, Zegeye HA (2011). Assessing the management of healthcare waste in Hawassa City, Ethiopia. Waste Manag Res 29(8): 854-862. Debere MK, Gelaye KA, Alamdo AG, Trifa ZM (2013). Assessment of the health care waste generation rates and its management system in hospitals of Addis Ababa, Ethiopia, 2011. BMC Public Health 13: 28. Muluken A, Haimot G, Mesafint M (2013). Healthcare waste management practices among healthcare workers in health care facilities of Gondar town, Northwest Ethiopia. Health Science Journal 7(3): 315-326. Ghana 4 Squire (2013). Biomedical Pollutants in the Urban Environment and Implications for Public Health: A Case Study. ISRN Public Health vol. 2013, Article ID 497490, 5 pages. Abor PA (2013). Managing healthcare waste in Ghana: a comparative study of public and private hospitals. Int J Health Care Qual Assur. 26(4): 375-386. Asante OB, Yanful E, Yaokumah EB (2014). Healthcare Waste Management; Its Impact: A Case Study Of The Greater Accra Region, Ghana. ACKNOWLEDGEMENTS 132:67-81. Taru P, Kuvarega AT (2005). Solid medical management – the case of Parirenyatwa Hospital, Zimbabwe. Rev. Biomed. 16:153-158. Muluken A, Haimot G, Mesafint M (2013). Healthcare waste management practices among healthcare workers in health care facilitiesof Gondar town, Northwest Ethiopia. Health Sci. J. 7(3): 315- 326. Tudor TL, Townend WK, Cheeseman CR, Edgar JE (2009). An overview of arisings and large-scake treatment technologies for healthcare waste in the United Kingdom. Waste Manage. Res. 27:374-383. Ndiaye M, El Metghari L, Soumah MM, Sow ML (2012). [Biomedical waste management in five hospitals in Dakar, Senegal]. Bull. Soc. Pathol. Exot. 105(4):296-304. U. S. Congress Office of Technology Assessment (1988). Issues in Medical Waste Management: Background Paper, OTA-BP-O-49. Washington D.C: United States Government Printing Office, 1988. ( ) Nemathaga F, Maringa S, Chimuka L (2008). Hospital solid waste management practices in Limpopo Province, South Africa: a case study of two hospitals. Waste Manage. 28(7):1236-1245. Udofia EA, Nriagu J (2013). Health-care Waste in Africa: A silent crisis? Glob. Health Perspect. 1(1):3-10. y p g ( ) Nkhuwa DCW, Kafula T, Ahmed AH 2008.Preliminary Inventory of Hazardous Medical Waste Disposal Systems and their influence on Groundwater Quality in Lusaka. Med. J. Zamb. 35(4):129-138. Walkinshaw E (2011). Medical waste-management practices vary across Canada. CMAJ 183(18):1307-1308. WHO (2004). Review of Health Impacts from Microbiological Hazards in Healthcare Wastes. Department of Blood Safety and Clinical Technology and Department of Protection of the Human Environment, World Health Organization, pp. 12-13. Patwary MA, O’Hare WT, Sarker MH (2011). Assessment of occupational and environmental safety associated with medical waste disposal in developing countries: A qualitative approach. Saf. Sci. 49(8-9):1200-1207. ( ) Pruss A, Giroult E, Rushbrook P (1999). Safe management of wastes from health-care activitiesGeneva, Switzerland: World Health Organization. pp. 1-30. WHO (2014). Safe management of wastes from healthcare activities (2 nd edn). Geneva, Switzerland: WHO Press, pp. 102, 136-138, 181- 190. g pp Rao PH (2008). Report: Hospital waste management – awareness and practices: a study of three states in India. Waste Manage. Res. 26:297-303. Appendix. List of references for the reviewed articles. Appendix. List of references for the reviewed articles. Appendix. List of references for the reviewed articles. Country Number of articles reviewed References Algeria 2 Bendjoudi Z, Taleb T, Abdelmalek F, Addou A (2009). Healthcare waste management in Algeria and Mostganem department. Waste Manag. 29(4): 1383-1387. Beghdadli B, Ghomari O, Taleb M, Kandouci BA, Fanello S (2010). ACKNOWLEDGEMENTS Implementation of WHO healthcare waste management (HCWM) approach in an Algerian hospital. Waste Manag. 30 (1): 162-164. Botswana 1 Mbongwe B, Mmereki BT, Magashula A (2008). Healthcare waste management: current practices in selected healthcare facilities, Botswana. Waste Manag. 28(1): 226–233. Cameroun 1 Manga VE, Morton OT, Mofor LA, Woodard R (2011). Health care waste management in Cameroon: A case study from the Southwestern Region. Resources, Conservation and Recycling 57:108–116. Egypt 2 Soliman SM, Ahmed AI (2006). Overview of biomedical waste management in selected governorates in Egypt: A pilot study. Waste Manag. 27(12): 1920-1923. Abd El-Salam MM (2010). Hospital waste management in El-Beheira Governorate, Egypt. J Environ Manage. 91 (3): 618-629. Ethiopia 4 Azage M, Kumie A (2010). Healthcare waste generation and its management system: the case of health centers in West Gojjam Zone, Amhara Region, Ethiopia. Ethiop. J. Health Dev. 24(2): 119 – 126. Haylamicheal ID, Mohamed AD, Yirsaw BD, Zegeye HA (2011). Assessing the management of healthcare waste in Hawassa City, Ethiopia. Waste Manag Res 29(8): 854-862. Debere MK, Gelaye KA, Alamdo AG, Trifa ZM (2013). Assessment of the health care waste generation rates and its management system in hospitals of Addis Ababa, Ethiopia, 2011. BMC Public Health 13: 28. Muluken A, Haimot G, Mesafint M (2013). Healthcare waste management practices among healthcare workers in health care facilities of Gondar town, Northwest Ethiopia. Health Science Journal 7(3): 315-326. Ghana 4 Squire (2013). Biomedical Pollutants in the Urban Environment and Implications for Public Health: A Case Study. ISRN Public Health vol. 2013, Article ID 497490, 5 pages. Abor PA (2013). Managing healthcare waste in Ghana: a comparative study of public and private hospitals. Int J Health Care Qual Assur. 26(4): 375-386. Asante OB, Yanful E, Yaokumah EB (2014). Healthcare Waste Management; Its Impact: A Case Study Of The Greater Accra Region, Ghana. International Journal of Scientifiic and Technology Research 3(3): 106 -112. Akum FA (2014). An Assessment of Medical Waste Management in Bawku Presbyterian Hospital of the Upper East Region of Ghana. Merit Res. J. Environ. Toxicol. 2(2): 27 – 38. Kenya 1 Mazrui N (2010). Medical Waste Management in Kenya: Opportunities for improvement. Tropical Resources Bulletin of the Yale Tropical Resources Institute 29: 66-70. Lesotho 1 Majara M, Leduka RC (2009). Health-Care Waste Practices in Selected Health-Care Facilities in Maseru. Review of Southern African Studies 12(1): 1-29. Libya 1 Sawalem M, Selic E, Herbell J-D (2009). ACKNOWLEDGEMENTS International Journal of Scientifiic and Technology Research 3(3): 106 -112. Akum FA (2014). An Assessment of Medical Waste Management in Bawku Presbyterian Hospital of the Upper East Region of Ghana. Merit Res. J. Environ. Toxicol. 2(2): 27 – 38. Kenya 1 Mazrui N (2010). Medical Waste Management in Kenya: Opportunities for improvement. Tropical Resources Bulletin of the Yale Tropical Resources Institute 29: 66-70. Lesotho 1 Majara M, Leduka RC (2009). Health-Care Waste Practices in Selected Health-Care Facilities in Maseru. Review of Southern African Studies 12(1): 1-29. Libya 1 Sawalem M, Selic E, Herbell J-D (2009). Hospital waste management in Libya: A case study. Waste Manag. 29(4): 1370 - 1375. Madagascar 1 Odette RH, Masika J, Venance T, Soatiana JE, Christane NA, Lamine CM, Bin L (2014). Assessment of healthcare waste generation and its management systems: A prevalence survey of Mbongwe B, Mmereki BT, Magashula A (2008). Healthcare waste management: current practices in selected healthcare facilities, Botswana. Waste Manag. 28(1): 226–233. Manga VE, Morton OT, Mofor LA, Woodard R (2011). Health care waste management in Cameroon: A case study from the Southwestern Region. Resources, Conservation and Recycling 57:108–116. Soliman SM, Ahmed AI (2006). Overview of biomedical waste management in selected governorates in Egypt: A pilot study. Waste Manag. 27(12): 1920-1923. Abd El-Salam MM (2010). Hospital waste management in El-Beheira Governorate, Egypt. J Environ Manage. 91 (3): 618-629. Azage M, Kumie A (2010). Healthcare waste generation and its management system: the case of health centers in West Gojjam Zone, Amhara Region, Ethiopia. Ethiop. J. Health Dev. 24(2): 119 – 126. Azage M, Kumie A (2010). Healthcare waste generation and its management system: the case of health centers in West Gojjam Zone, Amhara Region, Ethiopia. Ethiop. J. Health Dev. 24(2): 119 – 126. Haylamicheal ID, Mohamed AD, Yirsaw BD, Zegeye HA (2011). Assessing the management of healthcare waste in Hawassa City, Ethiopia. Waste Manag Res 29(8): 854-862. Debere MK, Gelaye KA, Alamdo AG, Trifa ZM (2013). Assessment of the health care waste generation rates and its management system in hospitals of Addis Ababa, Ethiopia, 2011. BMC Public Health 13: 28. Muluken A, Haimot G, Mesafint M (2013). Healthcare waste management practices among healthcare workers in health care facilities of Gondar town, Northwest Ethiopia. Health Science Journal 7(3): 315-326. Ghana 4 Squire (2013). Biomedical Pollutants in the Urban Environment and Implications for Public Health: A Case Study. ISRN Public Health vol. ACKNOWLEDGEMENTS Waste Manag. 29 (2): 804-811. Ajimotokan HA, Aremu SA (2009). Case Study Evaluation of Health-Care Waste Challenges in Hospitals within Ilorin Metropolis. Civil Engineering Impact on National Development: 1st Annual Civil Engineering Conference, University of Ilorin, Nigeria, 26-28 August, 2009, pp. 130-134. Ngouakam H, Aniekan UU, Onojeta AF (2010). Management of solid clinical wastes in some hospitals in Akwa Ibom State, Nigeria. International Journal of Natural and Applied Sciences 6(1): 92-100. Adedigba MA, Nwhator SO, Afon A, Abegunde AA, Bamise CT (2010). Assessment of dental waste management in a Nigerian tertiary hospital. Waste Manag Res. 28(7): 769 – 777. Samuel SO, Aderibigbe SA, Nwigwe CG, Nyamngee A (2011). Opinion of Health Workers On, And Their assey BE, Benka-Coker MO, Aluyi HSA (2006). Characterization and management of solid medi astes in the Federal Capital Territory, Abuja Nigeria. Afr Health Sci. 6(1):58-63. Longe EO, Williams A (2006). A Preliminary Study of Medical Waste Management in Lagos Metropolis, Nigeria. Iran. J. Environ. Health. Sci. Eng. 3(2): 133-139. Babatola JO, 2008. A Study of Hospital Waste Generation and Management Practice in Akure, Nigeria. African Research Review 2(3): 292-305. Oke IA (2008). Management of immunization solid wastes in Kano State, Nigeria. Waste Manag. 28(12): 2512-2521. Oke IA (2008). Management of immunization solid wastes in Kano State, Nigeria. Waste Manag. 28(12): 2512-2521. gwuluka N, Ochekpe N, Odumosu P, John SA (2009). Waste management in healthca stablishments within Jos metropolis, Nigeria. Afr. Sci. Environ. Sci. Technol. 3 (12):459 – 465. Ngwuluka N, Ochekpe N, Odumosu P, John SA (2009). Waste management in healthcare establishments within Jos metropolis, Nigeria. Afr. Sci. Environ. Sci. Technol. 3 (12):459 – 465. Odigie JO, Siminialayi IM (2009). Medical waste management at the University of Port Harcourt Teaching Hospital. Port Harcourt Medical Journal 4: 23 – 28. Odigie JO, Siminialayi IM (2009). Medical waste management at the University of Port Harcourt Teaching Hospital. Port Harcourt Medical Journal 4: 23 – 28. Coker A, Sangodoyin A, Sridhar M, Booth MC, Olomolaiye P, Hammond F (2009). Medical waste management in Ibadan, Nigeria: Obstacles and prospects. Waste Manag. 29 (2): 804-811. Ajimotokan HA, Aremu SA (2009). Case Study Evaluation of Health-Care Waste Challenges in Hospitals within Ilorin Metropolis. Civil Engineering Impact on National Development: 1st Annual Civil Engineering Conference, University of Ilorin, Nigeria, 26-28 August, 2009, pp. 130-134. Ngouakam H, Aniekan UU, Onojeta AF (2010). ACKNOWLEDGEMENTS Management of solid clinical wastes in some hospitals in Akwa Ibom State, Nigeria. International Journal of Natural and Applied Sciences 6(1): 92-100. Adedigba MA, Nwhator SO, Afon A, Abegunde AA, Bamise CT (2010). Assessment of dental waste management in a Nigerian tertiary hospital. Waste Manag Res. 28(7): 769 – 777. Nigeria 23 Samuel SO, Aderibigbe SA, Nwigwe CG, Nyamngee A (2011). Opinion of Health Workers On, And Their Safety Practices In Connection With The Handling Of Medical Wastes At A University Hospital in South East Nigeria. EMJ 10(1): 75 – 78. Johnson O (2011). Statistical Analysis of Medical Waste Generation, Management and Health Implications in Enugu Metropolis, Enugu State, Nigeria. Environment and Ecology 29(1A): 301 – 305. Fadipe OO, Oladepo KT, Jeje JO, Ogedengbe MO (2011). Characterization and analysis of medical solid waste in Osun State, Nigeria. Afr. J. Environ. Sci Technol. 5(12): 1027-1038. Chima GN, Ezekwe IC, Digha NO (2011). An assessment of medical waste management in health institutions in Yenagoa, South-South, Nigeria.World Review of Science, Technology and Sust. Development 8(2/3/4): 224-233. Morenikeji OA (2011). An investigation of the disposal of dental clinical waste in Ibadan City, south-west Nigeria. Waste Manag Res. 29(3): 318 – 322. Morenikeji OA (2011). An investigation of the disposal of dental clinical waste in Ibadan City, south-west Nigeria. Waste Manag Res. 29(3): 318 – 322. bah SO, Ohimain EI (2011). Healthcare waste management in Nigeria: A case study. J. Public Hea pidemiol. 33: 99-110. burum BC, Jiburum U (2012). An appraisal of medical waste management in four tertiary hospitals nugu, Nigeria. Port Harcourt Medical Journal 6: 192 – 197. g , g Ogbonna DN, Chindah A, Ubani N (2012). Waste management options for health care wastes in Nigeria: A case study of Port Harcourt hospitals. J Public Health Epidemiol. 4(6): 156-169. Ogbonna DN, Chindah A, Ubani N (2012). Waste management options for health care wastes in Nigeria: A case study of Port Harcourt hospitals. J Public Health Epidemiol. 4(6): 156-169. Nigeria: A case study of Port Harcourt hospitals. J Public Health Epidemiol. 4(6): 156 169. Oruonye ED (2012). An Assessment of Medical Waste Disposal Methods in Jalingo Metropolis, Taraba State Nigeria. I. J. A. B. R. 2(1): 67-71. Oruonye ED (2012). An Assessment of Medical Waste Disposal Methods in Jalingo Metropolis, Taraba State Nigeria. I. J. A. B. R. 2(1): 67-71. g ( ) Longe EO (2012). ACKNOWLEDGEMENTS Madagascar 1 Odette RH, Masika J, Venance T, Soatiana JE, Christane NA, Lamine CM, Bin L (2014). Assessment of healthcare waste generation and its management systems: A prevalence survey of the healthcare facilities in Madagascar.IOSR-JESTFT 8(7 ver. III): 20 -29. Appendix. Contd. Appendix. Contd. Country No. of articles reviewed Reference(s) Mauritius 1 Mohee R (2005). Medical wastes characterization in healthcare institutions in Mauritius. Waste Manag. 25(6): 575 -581. Morocco 1 Mbarki A, Kabbachi B, Ezaidi A, Benssaou M (2013). Medical Waste Management: A Case Study of the Souss-Massa-Draa Region, Morocco. JEP 4: 914-919. Nigeria 23 Bassey BE, Benka-Coker MO, Aluyi HSA (2006). Characterization and management of solid medical wastes in the Federal Capital Territory, Abuja Nigeria. Afr Health Sci. 6(1):58-63. Longe EO, Williams A (2006). A Preliminary Study of Medical Waste Management in Lagos Metropolis, Nigeria. Iran. J. Environ. Health. Sci. Eng. 3(2): 133-139. Babatola JO, 2008. A Study of Hospital Waste Generation and Management Practice in Akure, Nigeria. African Research Review 2(3): 292-305. Oke IA (2008). Management of immunization solid wastes in Kano State, Nigeria. Waste Manag. 28(12): 2512-2521. Ngwuluka N, Ochekpe N, Odumosu P, John SA (2009). Waste management in healthcare establishments within Jos metropolis, Nigeria. Afr. Sci. Environ. Sci. Technol. 3 (12):459 – 465. Odigie JO, Siminialayi IM (2009). Medical waste management at the University of Port Harcourt Teaching Hospital. Port Harcourt Medical Journal 4: 23 – 28. Coker A, Sangodoyin A, Sridhar M, Booth MC, Olomolaiye P, Hammond F (2009). Medical waste management in Ibadan, Nigeria: Obstacles and prospects. Waste Manag. 29 (2): 804-811. Ajimotokan HA, Aremu SA (2009). Case Study Evaluation of Health-Care Waste Challenges in Hospitals within Ilorin Metropolis. Civil Engineering Impact on National Development: 1st Annual Civil Engineering Conference, University of Ilorin, Nigeria, 26-28 August, 2009, pp. 130-134. Ngouakam H, Aniekan UU, Onojeta AF (2010). Management of solid clinical wastes in some hospitals in Akwa Ibom State, Nigeria. International Journal of Natural and Applied Sciences 6(1): 92-100. Adedigba MA, Nwhator SO, Afon A, Abegunde AA, Bamise CT (2010). Assessment of dental waste management in a Nigerian tertiary hospital. Waste Manag Res. 28(7): 769 – 777. Samuel SO, Aderibigbe SA, Nwigwe CG, Nyamngee A (2011). Opinion of Health Workers On, And Their Safety Practices In Connection With The Handling Of Medical Wastes At A University Hospital in South East Nigeria. EMJ 10(1): 75 – 78. Johnson O (2011). ACKNOWLEDGEMENTS Statistical Analysis of Medical Waste Generation, Management and Health Implications in Enugu Metropolis, Enugu State, Nigeria. Environment and Ecology 29(1A): 301 – 305. Fadipe OO, Oladepo KT, Jeje JO, Ogedengbe MO (2011). Characterization and analysis of medical solid waste in Osun State, Nigeria. Afr. J. Environ. Sci Technol. 5(12): 1027-1038. Chima GN, Ezekwe IC, Digha NO (2011). An assessment of medical waste management in health institutions in Yenagoa, South-South, Nigeria.World Review of Science, Technology and Sust. Development 8(2/3/4): 224-233. Morenikeji OA (2011). An investigation of the disposal of dental clinical waste in Ibadan City, south-west Nigeria. Waste Manag Res. 29(3): 318 – 322. Abah SO, Ohimain EI (2011). Healthcare waste management in Nigeria: A case study. J. Public Health Epidemiol. 33: 99-110. Jiburum BC, Jiburum U (2012). An appraisal of medical waste management in four tertiary hospitals in Enugu, Nigeria. Port Harcourt Medical Journal 6: 192 – 197. Ogbonna DN, Chindah A, Ubani N (2012). Waste management options for health care wastes in Nigeria: A case study of Port Harcourt hospitals. J Public Health Epidemiol. 4(6): 156-169. Oruonye ED (2012) An Assessment of Medical Waste Disposal Methods in Jalingo Metropolis Taraba Country No. of articles reviewed Reference(s) Mauritius 1 Mohee R (2005). Medical wastes characterization in healthcare institutions in Mauritius. Waste Manag. 25(6): 575 -581. Morocco 1 Mbarki A, Kabbachi B, Ezaidi A, Benssaou M (2013). Medical Waste Management: A Case Study of the Souss-Massa-Draa Region, Morocco. JEP 4: 914-919. Nigeria 23 Bassey BE, Benka-Coker MO, Aluyi HSA (2006). Characterization and management of solid medical wastes in the Federal Capital Territory, Abuja Nigeria. Afr Health Sci. 6(1):58-63. Longe EO, Williams A (2006). A Preliminary Study of Medical Waste Management in Lagos Metropolis, Nigeria. Iran. J. Environ. Health. Sci. Eng. 3(2): 133-139. Babatola JO, 2008. A Study of Hospital Waste Generation and Management Practice in Akure, Nigeria. African Research Review 2(3): 292-305. Oke IA (2008). Management of immunization solid wastes in Kano State, Nigeria. Waste Manag. 28(12): 2512-2521. Ngwuluka N, Ochekpe N, Odumosu P, John SA (2009). Waste management in healthcare establishments within Jos metropolis, Nigeria. Afr. Sci. Environ. Sci. Technol. 3 (12):459 – 465. Odigie JO, Siminialayi IM (2009). Medical waste management at the University of Port Harcourt Teaching Hospital. Port Harcourt Medical Journal 4: 23 – 28. Coker A, Sangodoyin A, Sridhar M, Booth MC, Olomolaiye P, Hammond F (2009). Medical waste management in Ibadan, Nigeria: Obstacles and prospects. ACKNOWLEDGEMENTS African Journal of Environmental Science and Technology 4(5):304-318. Nilsson J, Pembe AB, Urasa M, Darj E, 2013. Safe injections and waste management among healthcare workers at a regional hospital in northern Tanzania. Tanzania Journal of Health Research 15(1): 0-7. Uganda 1 Mugambe RK, Ssempebwa JC, Tumwesigye NM, van Vliet B, Adedimeji A, 2012. Healthcare waste management in Uganda: management and generation rates in public and private hospitals in Kampala. J Public Health 20: 245-251. Zimbabwe 3 Taru P, Kuvarega AT (2005). Solid medical management – the case of Parirenyatwa Hospital, Zimbabwe. Rev Biomed 16: 153-158. Jerie S (2006). Analysis of Institutional Solid Waste Management in Gweru, Zimbabwe. Eastern Africa Social Science Research Review 22(1): 103-125. Mangizvo RV, Chinamasa R (2008). Solid medical waste management: The case of Kwekwe City in Midlands Province, Zimbabwe. Journal of Sustainable Development in Africa 10(3): 188-200. dowu I, Alo B, Atherton W, Khaddar RA (2013). Profile of medical waste management in two healthcare facilities in Lagos, Nigeria: a case study. Waste Manag Res 31(5): 494-501. Uwa CU (2014). Assessment of Healthcare Waste Management Practices in Enugu Metropolis, Nigeria. IJESD 5(4): 370 – 374. 1 Ndiaye M, El Metghari L, Soumah MM, Sow ML, 2012. [Biomedical waste management in five hospitals in Dakar, Senegal]. Bull Soc. Pathol Exot. 105(4): 296-304. Di Bella V, Ali M, Vaccari M (2012). Constraints to healthcare waste treatment in low-income countries – a case study from Somaliland. Waste Manag Res 30 (6): 572-575. Nemathaga F, Maringa S, Chimuka L (2008). Hospital solid waste management practices in Limpopo Province, South Africa: a case study of two hospitals. Waste management 28(7): 1236– 1245. Abor PA, Bouwer A (2008). Medical waste management practices in a Southern African hospital. International Journal of Healthcare Quality Assurance 21(4): 356-364. Gabela SD, Knight SE (2010). Healthcare waste management in clinics in a rural health district in KwaZulu-Natal. South Afr J Epidemiol Infect 25(1): 19-21. ACKNOWLEDGEMENTS Healthcare waste management status in Lagos State, Nigeria: a case study from selected healthcare facilities in Ikorodu and Lagos Metropolis. Waste Manag Res 30 (6): 562-571. Longe EO (2012). Healthcare waste management status in Lagos State, Nigeria: a case study from selected healthcare facilities in Ikorodu and Lagos Metropolis. Waste Manag Res 30 (6): 562-571. Appendix. Contd. Appendix. Contd. Country Number of articles reviewed Reference(s) Nigeria 23 Tobin EA, Ediagbonya TF, Asogun DA, Oteri AJ (2013). Assessment of Healthcare Waste Management practices in Primary Health Care Facilities in a Lassa Fever Endemic Local Government Area of Edo State, Nigeria. AFRIMEDIC Journal 4(2): 16-23. Idowu I, Alo B, Atherton W, Khaddar RA (2013). Profile of medical waste management in two healthcare facilities in Lagos, Nigeria: a case study. Waste Manag Res 31(5): 494-501. Uwa CU (2014). Assessment of Healthcare Waste Management Practices in Enugu Metropolis, Nigeria. IJESD 5(4): 370 – 374. Senegal 1 Ndiaye M, El Metghari L, Soumah MM, Sow ML, 2012. [Biomedical waste management in five hospitals in Dakar, Senegal]. Bull Soc. Pathol Exot. 105(4): 296-304. Somaliland 1 Di Bella V, Ali M, Vaccari M (2012). Constraints to healthcare waste treatment in low-income countries – a case study from Somaliland. Waste Manag Res 30 (6): 572-575. South Africa 3 Nemathaga F, Maringa S, Chimuka L (2008). Hospital solid waste management practices in Limpopo Province, South Africa: a case study of two hospitals. Waste management 28(7): 1236– 1245. Abor PA, Bouwer A (2008). Medical waste management practices in a Southern African hospital. International Journal of Healthcare Quality Assurance 21(4): 356-364. Gabela SD, Knight SE (2010). Healthcare waste management in clinics in a rural health district in KwaZulu-Natal. South Afr J Epidemiol Infect 25(1): 19-21. Sudan 1 Saad SA (2013). Management of hospitals solid waste in Khartoum State. Environ Monit Assess. 185 (10): 8567 -8582. Tanzania 5 Mato RRAM, Kassenga GR (1997). A study on problems of management of medical solid wastes in Dar es Salaam and their remedial measures. Resources, Conservation and Recycling 21: 1-16. Mato RRAM, Kaseva ME (1999). Critical Review of industrial and medical waste practices in Dar es Salaam City. Resources, Conservation and Recycling 25(3-4): 271-287. Manyele SV, Anicetus H (2006). Management of medical waste in Tanzanian hospitals. Tanzan Health Res Bull. 8(3):177-182. Manyele SV, Lyasenga TJ (2010). Factors affecting medical waste management in low level health facilities in Tanzania. Saad SA (2013). Management of hospitals solid waste in Khartoum State. Environ Monit Assess. 185 (10): 8567 -8582. Saad SA (2013). Management of hospitals solid waste in Khartoum State. Environ Monit Assess. 185 (10): 8567 -8582. Mato RRAM, Kassenga GR (1997). A study on problems of management of medical solid wastes in Dar es Salaam and their remedial measures. Resources, Conservation and Recycling 21: 1-16. Mato RRAM, Kassenga GR (1997). A study on problems of management of medical solid wastes in Dar es Salaam and their remedial measures. Resources, Conservation and Recycling 21: 1-16. Mato RRAM, Kaseva ME (1999). Critical Review of industrial and medical waste practices in Dar es Salaam City. Resources, Conservation and Recycling 25(3-4): 271-287. Manyele SV, Anicetus H (2006). Management of medical waste in Tanzanian hospitals. Tanzan Health Res Bull. 8(3):177-182. Manyele SV, Lyasenga TJ (2010). Factors affecting medical waste management in low level health facilities in Tanzania. African Journal of Environmental Science and Technology 4(5):304-318. Manyele SV, Lyasenga TJ (2010). Factors affecting medical waste management in low level health facilities in Tanzania. African Journal of Environmental Science and Technology 4(5):304-318. Nilsson J, Pembe AB, Urasa M, Darj E, 2013. Safe injections and waste management among healthcare workers at a regional hospital in northern Tanzania. Tanzania Journal of Health Research 15(1): 0-7. Nilsson J, Pembe AB, Urasa M, Darj E, 2013. Safe injections and waste management among healthcare workers at a regional hospital in northern Tanzania. Tanzania Journal of Health Research 15(1): 0-7. Mugambe RK, Ssempebwa JC, Tumwesigye NM, van Vliet B, Adedimeji A, 2012. Healthcare waste management in Uganda: management and generation rates in public and private hospitals in Kampala. J Public Health 20: 245-251.
https://openalex.org/W3113102000	https://www.nature.com/articles/s41598-020-79409-5.pdf	English	null	CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome	Scientific reports	2,020	cc-by	12,637	catfish genome Rhoda Mae C. Simora1,5, De Xing1, Max R. Bangs1,6, Wenwen Wang1, Xiaoli Ma1, Baofeng Su1, Mohd G. Q. Khan1,7, Zhenkui Qin2, Cuiyu Lu1, Veronica Alston1, Darshika Hettiarachchi1, Andrew Johnson1, Shangjia Li1, Michael Coogan1, Jeremy Gurbatow1, Jeffery S. Terhune1, Xu Wang3,4 & Rex A. Dunham1 CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish. A powerful genome-editing tool known as clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technology has been of growing use for gene editing in aquaculture1–3. Interest in genomic alteration of farmed fish has increased, especially after the first genetically modified fish (AquAdvantage salmon) has been approved for consumption4. In the CRISPR/Cas9 system, the co-delivery of endonuclease Cas9 combined with a synthetic single guide RNA (sgRNA) targeting certain gene(s) into eukary- otic cells can edit the genome by stimulating a double-strand break (DSB) at a desired site(s), and the subsequent DNA repair process could introduce indel(s)5. www.nature.com/scientificreports www.nature.com/scientificreports catfish genome In the last five years, it has been successfully performed in several aquaculture species, including Atlantic salmon6,7, Nile tilapia8, common carp1, channel catfish2,9, sea bream3 and rainbow trout10 to generate a variety of phenotypes related to reproduction, fertility, muscle growth and disease resistance. However, the use of CRISPR/Cas9 has been largely limited to gene knock-outs in most fish species1–3,9,10, but has great potential for gene knock-in through homology-directed repair (HDR). The HDR pathway can allow precise integration of any desirable DNA sequence at the target site11,12, thus allowing the creation of gene-edited fish with desirable performance traits. 1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA. 2Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. 3Department of Pathobiology, Auburn University, Auburn, AL 36849, USA. 4HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA. 5Present address: College of Fisheries and Ocean Sciences, University of the Philippines Visayas, 5023 Miagao, Iloilo, Philippines. 6Present address: Department of Biological Science, Florida State University, Tallahassee, FL 32304, USA. 7Present address: Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh. email: rcsimora@up.edu.ph; dunhara@auburn.edu 1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA. 2Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. 3Department of Pathobiology, Auburn University, Auburn, AL 36849, USA. 4HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA. 5Present address: College of Fisheries and Ocean Sciences, University of the Philippines Visayas, 5023 Miagao, Iloilo, Philippines. 6Present address: Department of Biological Science, Florida State University, Tallahassee, FL 32304, USA. 7Present address: Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh. email: rcsimora@up.edu.ph; dunhara@auburn.edu \| https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 www.nature.com/scientificreports/ Table 1. The sequences of small guide RNA (sgRNA) and the universal (common) primer used to target chromosome 1 (Chr1) of channel catfish (Ictalurus punctatus). Underlined sequences represent the protospacer adjacent motif (PAM). Protein coding sequences are ready targets for many CRISPR/Cas9 applications, wherein investigators have generated small insertion and deletion (indel) mutations to disrupt the open reading frames of protein coding genes13,14. On the contrary, mutation of non-coding sequences using CRISPR/Cas9 system is often difficult to achieve, because small indels caused by a single mutation may not result in a detectable loss of function8. catfish genome Target- ing non-coding sequences either through loss-of-function or gain-of-function approaches can be advantageous since these regions are proposed to affect the expression of neighboring or distant genes by acting as signaling, guiding, sequestering or scaffolding molecules15,16. Targeting non-coding sequences might also affect multi- ple genes resulting in off-target effects. Some studies have successfully obtained large genomic deletion using dual guide RNAs (gRNAs) in mammalian cells and animal models such as mouse and zebrafish17,18. Deletion mutations of up to 900 bp were recently achieved in channel catfish targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) using a single guide RNA9. Moreover, Li et al.8 reported that the CRISPR/Cas9 system could effectively generate desirable non-coding sequence mutants in Nile tilapia (Oreo- chromis niloticus). There are non-coding regions away from genes, miRNA loci, lncRNA and heterochromatin regions, and to date, there have been no published studies of targeted gene insertion in such a genomic region. Our hypothesis is that the insertion of transgenes in such regions would decrease the possibility of knocking out valuable loci and affecting the expression of other genes. Additionally, targeting such areas for gene insertion could prevent or decrease the probability of negative pleiotropic or off-target effects.ii p p y g p pf gf Channel catfish (Ictalurus punctatus) is an important food fish in the United States that has been impacted by diseases, especially in commercial aquaculture. Efforts have been geared towards increasing disease resistance in this species as 40% of catfish production was lost to disease at the beginning of the current decade19 and continues to be problematic20. This is the case for aquaculture in general19,21. Improved catfish production systems have been adopted and hybrid catfish (I. punctatus ♀ × blue catfish, I. furcatus ♂), which is more resistant to diseases than channel catfish, are predominantly cultured. The use of hybrids has resulted in increased farm productivity and some increase in disease resistance, but the hybrids are not totally resistant to diseases and to date, there have been few dramatic advancements in disease control22. One alternative strategy would be developing disease- resistant fish lines through transgenesis. However, in the past, there has been some inefficiency in the generation of transgenic lines, and targeted gene insertion was almost impossible. catfish genome g g g p One particular class of antimicrobial peptides (AMPs) that could be useful for this cause are cathelicidins, which have been shown to exhibit broad-spectrum antimicrobial activity in vitro and in vivo23,24. Expression of genes encoding peptides with in vitro antimicrobial activity can result in enhanced resistance to bacterial pathogens in transgenic fish25,26. Recently, cathelicidins derived from American alligator (Alligator mississippien- sis) were shown to have a strong activity against some Gram-negative bacteria as well as in multi-drug resistant bacterial pathogens such as Acinetobacter baumannii and Klebsiella pneumoniae27,28. Cathelicidins from other species were not fully characterized and have had limited in vitro testing23,29. Inserting alligator cathelicidin gene in channel catfish using CRISPR/Cas9 system might enhance its resistance to various pathogens. A transgenic fish encoding an antimicrobial peptide can confer immunity in fish since it will be protected by the expressed peptide from early in development. Also, an innately disease-resistant fish would not require specific vaccination for certain pathogens, and thus, will provide an economical solution to bacterial disease problems25.i p g p p CRISPR/Cas9 has been explored as a knock-in system in model fishes targeting reporter/driver genes in medaka30,31 and zebrafish32,33. But limited studies for CRISPR/Cas9 knock-in were found on farmed fish7,34, and these studies primarily focused on non-performance genes. Here, we attempted to use CRISPR/Cas9-mediated HDR to knock-in an alligator cathelicidin gene driven by two different fish promoters and testing of two different types of donor DNA templates. Our objective was to insert this disease-resistance gene in a non-coding region of the channel catfish genome where the insertion would, theoretically not disable any important locus or function of a normal catfish. Additionally, targeting a non-coding region might allow transgene expression without sup- pression from neighboring genes. We aim to produce disease-resistant lines of channel catfish carrying actively expressing cathelicidin genes with positive biological functions that can be inherited by subsequent generations. Results ffi i Efficient HDR led to high integration rates. We developed three transgene constructs carrying the cathelicidin gene: two double-stranded DNA (dsDNA) constructs each carrying different promoters namely zebrafish (Danio rerio) ubiquitin promoter and common carp (Cyprinus carpio) β-actin promoter and a plasmid DNA construct driven by zebrafish ubiquitin promoter. Homologous arms (250 bp each), derived from chro- mosome 1 of channel catfish, were placed at both ends of the transgene constructs. Details on the design of the donor constructs are available in the Methods section. To insert the donor template carrying the cathelicidin gene, we used the small guide RNA (sgRNA) (Table 1) that directed Cas9 nuclease to that specific location in a non-coding region of chromosome 1 (Fig. 1). The integration site lies in a 297,098 bp non-coding window with https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Schematic representation of the target site and the surrounding genome in chromosome 1 of channel catfish (Ictalurus punctatus) genome where insertion of the transgene was made. The 20-bp guide RNA sequence containing the PAM was shown and the cut site (red arrow) which aided the targeted insertion of the donor DNA constructs: double-stranded DNA (dsDNA) driven by zebrafish ubiquitin promoter (dsDNA-UBI- Cath), dsDNA driven by carp β-actin promoter (dsDNA-BA-Cath) and plasmid DNA, pUCIDT with zebrafish ubiquitin promoter (plasmid-UBI-Cath). Grik2 is 1376 bp upstream of the target and hace1 is 295,702 bp downstream of the target. Figure 1. Schematic representation of the target site and the surrounding genome in chromosome 1 of channel catfish (Ictalurus punctatus) genome where insertion of the transgene was made. The 20-bp guide RNA sequence containing the PAM was shown and the cut site (red arrow) which aided the targeted insertion of the donor DNA constructs: double-stranded DNA (dsDNA) driven by zebrafish ubiquitin promoter (dsDNA-UBI- Cath), dsDNA driven by carp β-actin promoter (dsDNA-BA-Cath) and plasmid DNA, pUCIDT with zebrafish ubiquitin promoter (plasmid-UBI-Cath). Grik2 is 1376 bp upstream of the target and hace1 is 295,702 bp downstream of the target. no genes, microRNAs, long non-coding RNAs and heterochromatin binding sites, with the exception of two long non-coding RNAs (coordinates 19,220,890–19,222,416 and 19,318,070–19,319,829) which were avoided as to prevent knocking out any important functions. g y Successful integration of all three constructs was detected in both dead and living fry. Results ffi i Integration was con- firmed to have occurred at the targeted site on chromosome 1 of channel catfish (Database ID: NC_030416.1; coordinate 19,129,218). While no genes are encoded in this genomic window, the areas upstream and down- stream are gene rich (seven genes within 500,000 bps upstream and six genes within 500,000 bps downstream), increasing the probability of transgene expression. The closest genes to the integration site are grik2 (glutamate receptor iontropic kainate 2; coordinates 18,964,226–19,127,825) upstream and hace1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein; coordinates 19,424,923–19,448,550) downstream, the latter of which contains a ubiquitin promoter similar to two of transgene constructs (Fig. 1). q p g g Overall, dead fry had higher integration rates compared to living fry (Fig. 2). Among living fry, the dsDNA- BA-Cath construct had much lower integration rates with only 2.6% in the lowest dosage treatment and no integration for living fry at higher dosages (Fig. 2B). High integration rates in dead fry were found for 20 and 40 ng/μL concentrations of plasmid-UBI-Cath construct at 78% and 64%, respectively (p < 0.05) (Fig. 2C). No integration was observed for 10 ng/μL concentrations of dsDNA-BA-Cath construct. For fingerlings, highest inte- gration rate was found for the 20 ng/μL concentration of dsDNA-UBI-Cath construct (29%) (p < 0.05) (Fig. 2A), excluding the 100% integration rate found in 40 ng/μL concentration of plasmid-UBI-Cath construct, which had only two surviving fish which are positive for the transgene. The lowest integration rate was found for the 10 ng/μL concentration of dsDNA-BA-Cath construct (3%) (p < 0.05), while no positive fish were found for the 20 and 40 ng/μL concentrations of dsDNA-BA-Cath construct. g μ Pearson’s correlation test revealed a negative correlation between dosage and integration rate in dead fry for dsDNA-UBI-Cath and dsDNA-BA-Cath constructs (p = 0.005). As the dosage is increased for these two constructs, the integration rate decreases. There was an observed positive correlation between the dosage and integration rate in dead fry for plasmid-UBI-Cath construct, but it was not significant (p = 0.167). As indicated above, there was no linear relationship for integration and dose for the UBI constructs as the medium concentra- tion, 20 ng/μL gave the highest integration rates for live fry.if g μ g g g y Distinct bands amplified the different regions of the dsDNA-UBI-Cath and dsDNA-BA-Cath transgene con- structs (Figs. 3A and 4A, respectively). Results ffi i The presence of sequences that were not part of the transgene construct (designated by sequences in black, Figs. 3B and 4B), but matched the sequences surrounding the homology arms in chromosome 1 of channel catfish further confirmed integration of the whole transgene construct. Microinjection decreases embryo hatchability. Hatch rate of embryos ranged from 75% in the non- injected control group to 22% in the 40 ng/μL concentration of plasmid-UBI-Cath construct (Fig. 5A). No significant differences were detected in the hatchability of microinjected embryos among the different concen- trations of donor DNA for both dsDNA donor treatments. However, for the plasmid-UBI-Cath treatment, a low hatch rate was obtained at the 40 ng/μL concentration as compared to 10 and 20 ng/μL concentrations (p < 0.05). The non-injected control group had the highest hatch rate when compared to all other groups (p < 0.01). No significant correlation was detected among the different concentrations of donor DNA and embryo hatchability for dsDNA-BA-Cath construct, but a negative correlation was found to be significant (p = 0.019) between dosage and hatchability for the plasmid-UBI-Cath construct and dsDNA-UBI-Cath (p = 0.058). In general, the embryo hatchability decreased as the donor DNA concentration increased for these two constructs. https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| www.nature.com/scientificreports/ Figure 2. Comparison of integration rates between dead and alive fish of different transgene constructs carrying alligator cathelicidin gene in channel catfish (Ictalurus punctatus) using CRISPR/Cas9 knock-in system. (A) dsDNA construct driven by zebrafish ubiquitin promoter (dsDNA-UBI-Cath) (N for dead fry: 49, 43 and 23 for 10, 20 and 40 ng/µL, respectively; N for alive fingerlings: 73, 9 and 88 for 10, 20 and 40 ng/µL, respectively) (B) dsDNA construct driven by carp β-actin promoter (dsDNA-BA-Cath) (N for dead fry: 8, 37 and 35 for 10, 20 and 40 ng/µL, respectively; N for alive fingerlings: 118, 25 and 27 for 10, 20 and 40 ng/µL, respectively) (C) plasmid DNA construct with zebrafish ubiquitin promoter (plasmid-UBI-Cath) (N for dead fry: 42, 16 and 18 for 10, 20 and 40 ng/µL, respectively; N for alive fingerlings: 51, 4 and 19 for 10, 20 and 40 ng/µL, respectively). The values represent mean ± SD and paired t-tests were used to compare integration rates between dead and alive fish positive for transgene (p < 0.05, p < 0.01). Figure 2. Results ffi i Comparison of integration rates between dead and alive fish of different transgene constructs carrying alligator cathelicidin gene in channel catfish (Ictalurus punctatus) using CRISPR/Cas9 knock-in system. (A) dsDNA construct driven by zebrafish ubiquitin promoter (dsDNA-UBI-Cath) (N for dead fry: 49, 43 and 23 for 10, 20 and 40 ng/µL, respectively; N for alive fingerlings: 73, 9 and 88 for 10, 20 and 40 ng/µL, respectively) (B) dsDNA construct driven by carp β-actin promoter (dsDNA-BA-Cath) (N for dead fry: 8, 37 and 35 for 10, 20 and 40 ng/µL, respectively; N for alive fingerlings: 118, 25 and 27 for 10, 20 and 40 ng/µL, respectively) (C) plasmid DNA construct with zebrafish ubiquitin promoter (plasmid-UBI-Cath) (N for dead fry: 42, 16 and 18 for 10, 20 and 40 ng/µL, respectively; N for alive fingerlings: 51, 4 and 19 for 10, 20 and 40 ng/µL, respectively). The values represent mean ± SD and paired t-tests were used to compare integration rates between dead and alive fish positive for transgene (p < 0.05, p < 0.01). Microinjection harms early fry survival. Significant differences in fry survival rates were detected among the different concentrations of each donor DNA construct (p < 0.05) (Fig. 5B). For dsDNA-UBI-Cath construct, the 40 ng/μL concentration showed the highest fry survival rate (73%) as compared to two other dosages. In addition, a positive correlation (p = 0.001) was also found between dosage and fry survival for the different concentrations of dsDNA-UBI-Cath construct: the higher the dosage of donor DNA used, the higher the fry survival. For dsDNA-BA-Cath construct, high fry survival rate (63%) was observed in 10 ng/μL concen- tration. However, no correlation was found between dosage and fry survival (p = 0.186). In contrast, the 10 ng/μL concentration of plasmid-UBI-Cath construct gave the lowest fry survival rate (12%) as compared to other con- centrations, but no correlation was found between dosage and fry survival (p = 0.163). The non-injected control group had the highest fry survival (78%) as compared to all other treatment groups (p < 0.01) and their controls. The injected control group had the lowest fry survival as compared to all other treatment groups (p < 0.01). h j g p y p g p p For the cumulative survival, the highest rate was found in the non-injected control group (58%) and the lowest in the 10 ng/μL concentration of plasmid-UBI-Cath construct (4%) (Fig. 5C). Results ffi i Sequences in black are the regions outside of homologous arms; Blue sequences are the homologous arms that are part of the transgene construct; Green are partial sequences from the ubiquitin promoter region; Purple are the sequences of alligator cathelicidin gene. Red sequences belong to the poly A terminator sequence. Numbers on the right side of each sequence indicate the number of base pairs in the promoter region, alligator cathelicidin gene region and terminal region as revealed by sequencing of positive fish. Numbers in parentheses are the number of sequencing reactions which yielded positive transgene integration over the total number of sequencing reactions. Figure 3. Genotyping strategy for CRISPR/Cas9 knock-in of channel catfish (Ictalurus punctatus) using dsDNA construct driven by zebrafish ubiquitin promoter carrying the alligator cathelicidin gene (dsDNA-UBI-Cath). (A) Schematic diagram of dsDNA-UBI-Cath construct. (a, c) PCR amplification of primer sets at the 5′ and 3′ junctional regions (primer sets 1&2 and 3&5) and (b) the insert-specific region for alligator cathelicidin gene (primer set 3&4). Numbers in a lane represent individual samples of fish, lane WT represent wild type channel catfish and lane M indicates DNA marker. Presence of a distinct band indicates positive for transgene. Gel electrophoresis images shown here are cropped; full-length gels are presented in Supplementary Figure S1. (B) Representative sequences derived from channel catfish positive for integration of the dsDNA-UBI-Cath construct. Sequences in black are the regions outside of homologous arms; Blue sequences are the homologous arms that are part of the transgene construct; Green are partial sequences from the ubiquitin promoter region; Purple are the sequences of alligator cathelicidin gene. Red sequences belong to the poly A terminator sequence. Numbers on the right side of each sequence indicate the number of base pairs in the promoter region, alligator cathelicidin gene region and terminal region as revealed by sequencing of positive fish. Numbers in parentheses are the number of sequencing reactions which yielded positive transgene integration over the total number of sequencing reactions. To quantify the level of cathelicidin gene expression in the tissues of transgenic fish, qPCR was performed. High variation was observed among different tissues with a maximum fold change approaching 60 (Fig. 7A). Based on the fold changes relative to the liver (set as 1×), cathelicidin mRNA was most abundant in muscle by 57-fold (p < 0.001), followed by gill (15-fold), stomach (7-fold) and heart (5-fold). Results ffi i A positive correlation (p = 0.030) was found between dosage and cumulative survival for the different concentrations of dsDNA-UBI- Cath construct. However, no correlation was found between dosage and cumulative survival for dsDNA-BA-Cath construct (p = 0.210) and plasmid-UBI-Cath construct (p = 0.627). In general, the plasmid-UBI-Cath construct treatments had low cumulative survival similar to the injected control. Transgene expression detected in most tissues of P1 fish. To detect the expression of cathelicidin transgene in P1 fish (parent generation; wild type fish injected with Cas9, sgRNA and a transgene construct), total RNA samples isolated from various tissues of positive fish were subjected to RT-PCR and qPCR analyses. Figure 6Aa shows that 8 out 10 tissues expressed the cathelicidin transgene. Strong expression was observed in fin, barbel, eye, muscle, kidney and stomach, while weak expression was observed in heart and intestine. No expression was detected in gill and liver. To avoid sacrificing potential transgenic fish, blood, fin and barbel were biopsied to isolate RNA. Representative results of cathelicidin transgene expression in two randomly selected positive fish as compared to a non-transgenic fish (analyzed negative for cathelicidin transgene) are shown in Fig. 6Ba. Transgene mRNAs were consistently detected in the blood of both transgenic fish, but the expression in the fin and barbel varied. Gel images of the 18S rRNA used as internal control are shown in Fig. 6Ab and Bb. https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Genotyping strategy for CRISPR/Cas9 knock-in of channel catfish (Ictalurus punctatus) using dsDNA construct driven by zebrafish ubiquitin promoter carrying the alligator cathelicidin gene (dsDNA-UBI-Cath). (A) Schematic diagram of dsDNA-UBI-Cath construct. (a, c) PCR amplification of primer sets at the 5′ and 3′ junctional regions (primer sets 1&2 and 3&5) and (b) the insert-specific region for alligator cathelicidin gene (primer set 3&4). Numbers in a lane represent individual samples of fish, lane WT represent wild type channel catfish and lane M indicates DNA marker. Presence of a distinct band indicates positive for transgene. Gel electrophoresis images shown here are cropped; full-length gels are presented in Supplementary Figure S1. (B) Representative sequences derived from channel catfish positive for integration of the dsDNA-UBI-Cath construct. Results ffi i The rest of the tissues had less than threefold changes. For the biopsied tissues, three positive fish for cathelicidin gene were sampled for blood, fin and barbel. Based on the fold changes relative to the negative fish (non-transgenic), the highest fold change was found in the barbel of transgenic fish 2 (Trans 2) with a 71-fold change and in the blood of transgenic fish 3 (Trans 3), a 53-fold change (p < 0.001) (Fig. 7B). Relatively high fold change (p < 0.01) was also observed in the blood of transgenic fish 1 (Trans 1), while the rest of the tissues had less than fivefold change. https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| www.nature.com/scientificreports/ Figure 4. Genotyping strategy for CRISPR/Cas9 knock-in of channel catfish (Ictalurus punctatus) using dsDNA construct driven by β-actin promoter carrying the alligator cathelicidin gene (dsDNA-BA-Cath). (A) Schematic diagram of dsDNA-BA-Cath construct. (a, c) PCR amplification of primer sets at the 5′ and 3′ junctional regions (primer sets 1&2 and 3&5) and (b) the insert-specific region for alligator cathelicidin gene (primer set 3&4). Numbers in a lane represent individual samples of fish, lane WT represent wild type channel catfish and lane M indicates DNA marker. Presence of a distinct band indicates positive for transgene. Gel electrophoresis images shown here are cropped; full-length gels are presented in Supplementary Figure S2. (B) Representative sequences derived from channel catfish positive for integration of dsDNA-BA-Cath construct. Sequences in black are the regions outside of homologous arms; Blue sequences are the homologous arms that are part of the transgene construct; Green are partial sequences from the zebrafish ubiquitin promoter region; Purple are the sequences of the alligator cathelicidin gene. Red sequences belong to the poly A terminator sequence. Numbers on the right side of each sequence indicate the number of base pairs in the promoter region, alligator cathelicidin gene region and terminal region as revealed by sequencing of positive fish. Numbers in parentheses are the number of sequencing reactions which yielded positive transgene integration over the total number of sequencing reactions. Figure 4. Genotyping strategy for CRISPR/Cas9 knock-in of channel catfish (Ictalurus punctatus) using dsDNA construct driven by β-actin promoter carrying the alligator cathelicidin gene (dsDNA-BA-Cath). (A) Schematic diagram of dsDNA-BA-Cath construct. (a, c) PCR amplification of primer sets at the 5′ and 3′ junctional regions (primer sets 1&2 and 3&5) and (b) the insert-specific region for alligator cathelicidin gene (primer set 3&4). Results ffi i Numbers in a lane represent individual samples of fish, lane WT represent wild type channel catfish and lane M indicates DNA marker. Presence of a distinct band indicates positive for transgene. Gel electrophoresis images shown here are cropped; full-length gels are presented in Supplementary Figure S2. (B) Representative sequences derived from channel catfish positive for integration of dsDNA-BA-Cath construct. Sequences in black are the regions outside of homologous arms; Blue sequences are the homologous arms that are part of the transgene construct; Green are partial sequences from the zebrafish ubiquitin promoter region; Purple are the sequences of the alligator cathelicidin gene. Red sequences belong to the poly A terminator sequence. Numbers on the right side of each sequence indicate the number of base pairs in the promoter region, alligator cathelicidin gene region and terminal region as revealed by sequencing of positive fish. Numbers in parentheses are the number of sequencing reactions which yielded positive transgene integration over the total number of sequencing reactions. Discussion f l To facilitate a targeted knock-in approach in channel catfish using CRISPR/Cas9 system, a multi-pronged approach was undertaken. An effective donor DNA construct was designed to increase integration efficiency, and a non-coding region of the channel catfish genome was edited with CRISPR/Cas9. The target region had no gene coding regions, microRNA loci, long non-coding RNA regions or heterochromatin to hypothetically not knockout any important loci or function, to increase the probability of transgene expression and reduce potential off-target and pleiotropic effects. Then a disease-resistance alligator cathelicidin transgene was inserted into this region of the channel catfish genome. Relatively, high integration rates were achieved. This is the first https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| www.nature.com/scientificreports/ Figure 5. Plots of (A) embryo hatchability, (B) early fry survival and (C) cumulative mortality (hatchability x early fry survival) of channel catfish (Ictalurus punctatus) microinjected at one-cell stage with three transgene constructs: UBI (dsDNA driven by zebrafish ubiquitin promoter), BA (dsDNA driven by carp β-actin promoter) and plasmid (plasmid DNA construct with zebrafish ubiquitin promoter) at different concentrations (10, 20 and 40 ng/μL) carrying the alligator cathelicidin gene utilizing the CRISPR/Cas9 system. Control groups included the injected control (iCTRL, 60% phenol red solution only) and non-injected control (nCTRL). The values represent mean ± SD and analyzed by one-way ANOVA followed by Tukey’s test. Means with different letters are significantly different (p < 0.05); N = 300–435 embryos injected. Figure 5. Plots of (A) embryo hatchability, (B) early fry survival and (C) cumulative mortality (hatchability x early fry survival) of channel catfish (Ictalurus punctatus) microinjected at one-cell stage with three transgene constructs: UBI (dsDNA driven by zebrafish ubiquitin promoter), BA (dsDNA driven by carp β-actin promoter) and plasmid (plasmid DNA construct with zebrafish ubiquitin promoter) at different concentrations (10, 20 and 40 ng/μL) carrying the alligator cathelicidin gene utilizing the CRISPR/Cas9 system. Control groups included the injected control (iCTRL, 60% phenol red solution only) and non-injected control (nCTRL). The values represent mean ± SD and analyzed by one-way ANOVA followed by Tukey’s test. Means with different letters are significantly different (p < 0.05); N = 300–435 embryos injected. Figure 6. Expression of alligator cathelicidin mRNA from the dsDNA-zebrafish ubiquitin-alligator cathelicidin construct in (A) different tissues and (B) tissues biopsied non-lethally from P1 transgenic channel catfish (Ictalurus punctatus), as measured by regular RT-PCR followed by agarose gel electrophoresis. Discussion f l In the current study, sequence results revealed the precise integration of the complete cathelicidin gene without errors.i High integration rates ranging from 31 to 78% in dead fry and 11 to 100% in fingerlings were observed with the use of a plasmid donor template. These results were slightly better than the integration rates (via HDR) obtained using plasmid donors in medaka30 and zebrafish45 embryos, which ranged from 25 to 27% and 26 to 46%, respectively. Preliminary CRISPR/Cas9 knock-in experiment in our lab using a plasmid donor yielded a 10% integration rate for Elovl2 transgene at the same target site in channel catfish chromosome 1 (Xing, et al. unpublished results). Plasmids as donor vectors for CRISPR/Cas9 transfections are common, but their drawbacks include random integration of all or part of the plasmid DNA into the host genome, unwanted insertions of plasmid DNA sequences at on-target and off-target sites46. However, sequencing results obtained in this study on samples positive for the transgene with the use of plasmid donor DNA revealed that no unwanted plasmid sequences were integrated into the target site.hfi The efficiency of HDR generating successful knock-ins in the current study can be due to several factors. First, the design of the homologous donor construct contributed to the efficiency of HDR which led to high integra- tion rates. Studies have shown that the efficiency of recombinatorial repair increases as the length of homology arms increases14,46,47. The use of 250-bp homology arms identical to the sequences surrounding the target site in channel catfish chromosome 1 generated integration rates ranging from 14 to 100% in P1 fingerlings. A similar finding was observed by Zhang et al.47 in which increased length of homology arms from 50 to 300 bp led to targeting the insertion site more effectively in 293 T cells. In that study, Zhang et al.47 observed a 77% precise insertion rate of 300 bp homology arms; in contrast, inserts containing 50 bp homology arms had only a 63% insertion rate. Studies that used 1 kb or more homology arms achieved variable results and showed 12–58% HDR rate46,48. Shorter homology arms would be expected to be more accessible for insertion in the target site during the simultaneous cleavage of genomic and plasmid DNA48. Discussion f l FI fin, BA barbel, GI gill, MU muscle, LI liver, HE heart, KI kidney, IN intestine, ST stomach, BL blood. Expression levels in (A) were calibrated against liver tissue which had the lowest expression level, and 18S rRNA gene was used as a reference gene. Trans 1, Trans 2, and Trans 3 were P1 transgenic fish. Expression levels in (B) were calibrated against corresponding tissues from sibling non-transgenic control fish. The expression level was analyzed by 2−ΔΔCT method and one-way ANOVA followed by Tukey’s test (p < 0.001, p < 0.01) Representative gel image in (A) can be visualized in Supplementary Figure S4. Figure 7. Relative expression profiles of the dsDNA-zebrafish ubiquitin-alligator cathelicidin construct in (A) different tissues and (B) tissues biopsied non-lethally from P1 transgenic channel catfish (Ictalurus punctatus), as measured by qPCR. FI fin, BA barbel, GI gill, MU muscle, LI liver, HE heart, KI kidney, IN intestine, ST stomach, BL blood. Expression levels in (A) were calibrated against liver tissue which had the lowest expression level, and 18S rRNA gene was used as a reference gene. Trans 1, Trans 2, and Trans 3 were P1 transgenic fish. Expression levels in (B) were calibrated against corresponding tissues from sibling non-transgenic control fish. The expression level was analyzed by 2−ΔΔCT method and one-way ANOVA followed by Tukey’s test (p < 0.001, p < 0.01) Representative gel image in (A) can be visualized in Supplementary Figure S4. Figure 7. Relative expression profiles of the dsDNA-zebrafish ubiquitin-alligator cathelicidin construct in (A) different tissues and (B) tissues biopsied non-lethally from P1 transgenic channel catfish (Ictalurus punctatus), as measured by qPCR. FI fin, BA barbel, GI gill, MU muscle, LI liver, HE heart, KI kidney, IN intestine, ST stomach, BL blood. Expression levels in (A) were calibrated against liver tissue which had the lowest expression level, and 18S rRNA gene was used as a reference gene. Trans 1, Trans 2, and Trans 3 were P1 transgenic fish. Expression levels in (B) were calibrated against corresponding tissues from sibling non-transgenic control fish. The expression level was analyzed by 2−ΔΔCT method and one-way ANOVA followed by Tukey’s test (*p < 0.001, p < 0.01) Representative gel image in (A) can be visualized in Supplementary Figure S4. additional mutations44. Discussion f l Trans 1 and Trans 2 were P1 transgenic fish and Non-trans was a non-transgenic fish. BL blood, FI fin, BA barbel; + , plasmid DNA positive control; -, PCR reaction in the absence of plasmid DNA. 18S rRNA gene was used as an internal control. Gel electrophoresis images were cropped and full-length gels are presented in Supplementary Figure S3. Figure 6. Expression of alligator cathelicidin mRNA from the dsDNA-zebrafish ubiquitin-alligator cathelicidin construct in (A) different tissues and (B) tissues biopsied non-lethally from P1 transgenic channel catfish (Ictalurus punctatus), as measured by regular RT-PCR followed by agarose gel electrophoresis. Trans 1 and Trans 2 were P1 transgenic fish and Non-trans was a non-transgenic fish. BL blood, FI fin, BA barbel; + , plasmid DNA positive control; -, PCR reaction in the absence of plasmid DNA. 18S rRNA gene was used as an internal control. Gel electrophoresis images were cropped and full-length gels are presented in Supplementary Figure S3. report of targeted gene insertion in the non-coding region away from genes, microRNA loci, long non-coding RNA regions and heterochromatin for an aquaculture species, and as best we can tell for any organism. Several tissues of the transgenic individuals strongly expressed alligator cathelicidin.hfi report of targeted gene insertion in the non-coding region away from genes, microRNA loci, long non-coding RNA regions and heterochromatin for an aquaculture species, and as best we can tell for any organism. Several tissues of the transgenic individuals strongly expressed alligator cathelicidin.hfi 35 The insertion efficiency of dsDNA donor templates in CRISPR/Cas9 system is generally poor35 as compared to single-stranded DNA (ssDNA) donors36,37. We successfully generated knock-ins using dsDNA donor templates with an integration rate as high as 59% in fry that died and 14.1–28.6% in surviving fingerlings. The results were comparable to the precise insertion rate of 60–90% using ssDNA donor templates in human cell lines38,39 and mouse models37,40. Insertion via homologous recombination of dsDNA donor template in mouse models had a knock-in efficiency of ~ 10% or less41–43. A knock-in efficiency of 3.5% was observed in zebrafish using a dsDNA donor oligonucleotide targeting C13H9orf72 genomic locus but only 1.7% showed correct knock-in without Scientific Reports \| (2020) 10:22271 \| https://doi.org/10.1038/s41598-020-79409-5 www.nature.com/scientificreports/ Figure 7. Relative expression profiles of the dsDNA-zebrafish ubiquitin-alligator cathelicidin construct in (A) different tissues and (B) tissues biopsied non-lethally from P1 transgenic channel catfish (Ictalurus punctatus), as measured by qPCR. www.nature.com/scientificreports/ Therapeutic chemicals are often not 100% effective and may have inconsistent protection or killing of disease organisms52, thus transgenic enhancement of disease resistance may be a better alternative for long-term disease protection.t g y g p Contradictorily, the integration rate in the fry that died after hatching had a higher integration rate than surviving fingerlings. Perhaps, some individuals are overexpressing cathelicidin in key tissues or cell types, leading to mortality. These observations should be clarified in the F1 generation. Elucidating the relationship of the expression of transgenic antimicrobial peptides and its effect on the microbiome might be an area to explore or the effects of alligator cathelicidin on the transcriptome. Similarly, almost all β-actin alligator cathelicidin hatchlings died. Again, perhaps, expression was too strong in certain cells or tissues, with negative effects on the microbiome or negative effects on the transcriptome. The choice of promoter appears critical, and in this case, zebrafish ubiquitin gave superior results in regards to producing viable transgenic fingerlings than carp β-actin.i i g g g gi g g β Low levels of somatic mosaicism were observed in the tissues of positive P1 fish as revealed by RT-PCR and qPCR, another benefit of the CRISPR/Cas9 targeted knock in. This is in contrast to the universal mosaicism obtained with traditional microinjection that often results in the transgene only being present in 25–30% of the tissues in P1 53,54. With the presence of the transgene in every tissue, phenotypic data collected on P1 individuals positive for transgene becomes more realistic, although not perfect as we did not determine if the mosaicism varied from tissue to tissue. Thus, tissue patterns of expression may or may not be the same when the F1 genera- tion is produced. However, when our laboratory used CRISPR/Cas9 to produce knockouts without accompanying gene insertion, mosaicism was low and similar among all tissues evaluated9. Additionally, the fact that the gene must be present in almost all tissues as expression was obtained in almost all tissues ensures higher transmission frequencies and rates to the F1 generation. Also, the low level of mosaicism has positive benefits for studying transgene expression. For biopsied tissues, transgene expression was consistently expressed in the blood, while variable expression was found among transgenic fish for fin and barbel. A donor DNA construct driven by zebrafish ubiquitin promoter revealed strong transgene expression across all tissues. www.nature.com/scientificreports/ or the excess abundance of DNA itself is lethal. In general, the cumulative survival of the plasmid-UBI-Cath treatment was the lowest. This construct was slightly larger than the other two constructs consistent with the premise that increasing amounts of injected exogenous DNA would lower survival. Additionally, we did not track the rate of the degradation of the plasmid carrying the transgene into the embryo, thus another possibility is an extended period with excess exogenous DNA or other toxic effects such as cGMP-AMP synthase activation50,51 from the short-term persistence of plasmid sequences. p p q Even though they hatched successfully, the injected control fry experienced the heaviest mortality of all treat- ments, suggesting long-lasting negative effects of the damage of microinjection of the yolk. These comparisons suggest that major mortality occurs due to the microinjection of the yolk, and additional less impactful mortality occurs from the DNA and reagents. This is also important as it indicates that the comparison of microinjected transgenic, gene edited or xenogenic fish controls at early ages that are not microinjected would be invalid due to the long-lasting negative effects of the microinjection on growth and survival. Comparisons must be made with injected controls or in the next generation. Eight of nine DNA-injected treatments had higher survival than the injected control, suggesting that there are early positive disease resistance benefits from the alligator cathelicidin helping to correct for the vulnerabilities due to microinjection, which apparently lasted for several days. Although cathelicicin expression in the newly hatched fry was not evaluated in the current study, the expec- tation is that these fry were expressing the transgene since expression was found in all transgenic individuals when they were older and because ubiquitin is a constitutive promoter. The higher survival of the DNA-injected treatments illustrates their potential importance compared to other current disease control measures. Both the DNA-injected and buffer-injected controls had lower survival, likely caused by both physical damage as well as greater microbial attack due to the puncture of eggshell and yolk. Doxycycline was used to try to prevent any mortality from pathogens, but was not totally effective, especially in the non-transgenic control. Discussion f l Also, the donor templates used were designed to have two homology arms identical to sequences surrounding DSB created by Cas9/sgRNA which allowed HDR to occur49, thus resulted in the correct insertion of the transgene to the target site.hff g g The different concentrations of dsDNA and plasmid constructs affected the hatchability and mortality of the microinjected embryos. The greater the concentration of DNA injected, the lower the hatch rate, except for the 20 ng/μL concentration for the dsDNA-BA-Cath construct. The increased embryo mortality could be due to the production of toxic products at higher concentrations of donor DNA which may have adverse effects on the cells Scientific Reports \| (2020) 10:22271 \| https://doi.org/10.1038/s41598-020-79409-5 www.nature.com/scientificreports/ Methods hi l Ethical statement. Channel catfish were reared at the Fish Genetics Research Unit of the EW Shell Research Center, School of Fisheries, Aquaculture and Aquatic Sciences at Auburn University, Alabama, USA. All experi- mental protocols used in this study were approved by the Auburn University Institutional Animal Care and Use Committee (AU-IACUC). All experiments on animals were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) protocols and guidelines. Identification of the target sequence for gene insertion. The targeted genomic area was to be in the non-coding region away from genes, microRNA loci, long non-coding RNA regions and heterochromatin as to avoid knocking out any important functions and to increase the probability of transgene expression. Bioinfor- matics was conducted to identify a genomic target site and its surrounding genes in channel catfish that met this criterion using The Ensembl genome browser61. The channel catfish genome62 (IpCoco_1.2) was used and the selection was done manually. Design of donor DNA and preparation of sgRNA and CRISPR/Cas9 system. The coding sequence for cathelicidin gene was derived from the mature peptide sequence of A. mississippiensis (AM-CATH36; Gen- eBank accession number AKHW00000000.3)28. Two different promoters were used for the double-stranded DNA (dsDNA) construct; the truncated version of the zebrafish (Danio rerio) ubiquitin promoter (1.4 kb) previ- ously tested by Mosimann et al.57 and the common carp (Cyprinus carpio) β-actin promoter (1.6 kb)63. Expres- sion of cathelicidin gene was driven by zebrafish ubiquitin promoter for plasmid DNA construct, pUCIDT Amp (2.7 kb). Two homology arms (250 bp each) derived from chromosome 1 of channel catfish (Database ID: NC_030416.1) spanning bases 19,128,968 to 19,129,468 were placed at the left and right ends of the donor con- structs. The dsDNA constructs and the plasmid DNA were synthesized by Integrated DNA Technologies (IDT) (Coralville, Iowa, USA).h The CRISPR design online tool (CRISPR Guide RNA Design Tool, Benching, https://zlab.bio/guide-desig n-resources) was used to design the sgRNA that targeted the channel catfish chromosome 1. The protospacer adjacent motif (PAM) sequence (5′-TGG-3′) immediately followed the 20 bp target sequence (5′-GTGCTCCTG CTGCTGTTGTA-3′), spanning 19,129,201 to 19,129,221 bp of the chromosome 1 domain. A cloning-free (PCR- based) method was used to generate sgRNA. Table 1 shows the sequences of the universal primer and sgRNA used in this study. The sgRNAs were generated by T7 run-off2,64. www.nature.com/scientificreports/ Although, we cannot be certain of the exact relative expression among all tissues, the important and encouraging finding is that some level of expression was virtually universal. i g y Future studies should compare the transgene expression of alligator cathelicidin driven by the β-actin and the zebrafish ubiquitin promoter. Some studies in transgenic animals have shown that the β-actin promoter can maintain transgene expression to adulthood, but sometimes does not show significant activity in erythrocytes or fins, or several other cell types such as brain, retina, kidney and blood55,56. Factors relating to the differential expression pattern utilizing the β-actin promoter may be the potential cell-type-specific requirements controlling translation or chromatin maintenance and differential methylation56,57. On the other hand, Mosimann et al.57 demonstrated that zebrafish ubiquitin promoter could drive strong and ubiquitous expression of an enhanced green fluorescent protein (EGFP) reporter gene in zebrafish and the transgene revealed strong expression in all analyzed external and internal organs, including the retina, fin fold, and across all blood cell types from embryo to adulthood. A truncated version of this promoter was utilized in the design of dsDNA and plasmid DNA construct in the current study, which might explain the high transgene expression observed in the blood of posi- tive fish microinjected with donor DNA constructs driven by zebrafish ubiquitin promoter. The localization of cathelicidins in the myeloid tissues might also explain why high transgene expression was found in the blood of positive P1 fish. Myeloid tissues arise from hematopoietic stem cells in the bone marrow and represent the major leukocytes in the peripheral blood58. Cathelicidins are said to be stored in neutrophils and macrophages59, which are types of leukocytes and both possess a nucleus which can express genes.i yp y p p g High expression in the blood and gill of the alligator cathelicidin gene in channel catfish, as well as some of the other tissues is encouraging, and is predictive of potential disease resistance. However, the variability in expression from one individual to another may indicate that selection will likely be needed within the transgenic population to maximize disease resistance. This is not surprising as usually transgenesis and selection are needed to maximize transgenic performance60. The ultimate transgenic line cannot be claimed without examining the F1 lines and perhaps beyond. https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| www.nature.com/scientificreports/ Conclusion In this study, we demonstrated that targeted gene integration can be performed using CRISPR/Cas9 knock-in system in channel catfish. Effective design of a donor template incorporating the zebrafish ubiquitin promoter, presence of a 250-bp homologous arms at each end of the construct and a sgRNA designed to cleave at the exact insertion site, all contributed to increased HDR rate, leading to precise integration of the transgene construct into chromosome 1 of channel catfish genome. Evaluation of the P1 individuals revealed low levels of somatic mosaicism. This can be due precise genome editing by CRISPR/Cas9 and timing of introduction of Cas9 into one- cell stage embryos. The highest integration rate occurred among live P1 fish treated with the dsDNA-UBI-Cath construct at 20 ng/μL. The plasmid-UBI-Cath construct can be as efficient in generating transgene containing the P1 fish with increased integration rates, but lower early hatch and survival. To the best of our knowledge, this study represents the first description of a targeted exogenous gene insertion in a non-coding region with no microRNA loci, long non-coding RNA regions or heterochromatin using CRISPR/Cas9 system. This led to moderate to high expression in a multitude of tissues, and in the future transgene expression at this site and others should be compared to see if targeting such regions is beneficial for gene expression and phenotypic altera- tion. Our findings for improving the efficiency of the CRSPR/Cas9 knock-in system may be applicable to other aquaculture species as well since it represents a highly effective and more precise method of transgene insertion than those previously used in aquaculture. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 2. Oligonucleotide primers used in determining integration and transgene expression of alligator cathelicidin gene in channel catfish (Ictalurus punctatus). Target gene Purpose Name Nucleotide sequence (5′ → 3′) Cathelicidin P1 transgene PCR: Cathelicidin region (Ubiquitin promoter) Ubi-Cath-F1 GCAGCCAATCACTGCTTGTA Ubi-Cath-R1 GTGGTTTGTCCAAACTCATCAA PCR: Promoter end (Ubiquitin promoter) Ubi-PE-F2 GGCTGTTGGTGTAGGGTTTC Ubi-PE-R2 GCAGCTAGTGAGTGCTGTGC PCR:Terminal end (Ubiquitin promoter) Ubi-Chr1-F1 GCGGAAAGATCTGGTCATGT Ba-Chr1-R2 CAAGTGCAAAGAAGGCAACA PCR:Cathelicidin region (β-actin promoter) Ba-Cath-F1 GACTCCACATGGTCACATGC Ba-Cath-R1 GTCTGGATCTCACCGCCTTC PCR:Promoter end (β-actin promoter) Chr1-Ba-F1 CTGTGCTGCTGATGACCATT Chr1-Ba-R1 GCGTGCACATTGCTACACTT Chr1-Ba-R2 GGCAGATGATATTCCGCACT PCR:Terminal end (β-actin promoter) Ba-Cath-F1 GACTCCACATGGTCACATGC Ba-Chr1-R1 TGTTGCCTTCTTTGCACTTG qPCR Ubi-qPCR-F1 TGCTATTCAAGAAGCTGAGGAGG Ubi-qPCR-R1 TCATGTCTGGATCTCACCGC 18S rRNA qPCR 18sF GAGAAACGGCTACCACATCC 18sR GATACGCTCATTCCGATTACAG Table 2. Oligonucleotide primers used in determining integration and transgene expression of alligator cathelicidin gene in channel catfish (Ictalurus punctatus). Table 2. Oligonucleotide primers used in determining integration and transgene expression of alligator cathelicidin gene in channel catfish (Ictalurus punctatus). fresh water was added to the eggs to cover the mass; the sperm/egg mixture was gently swirled for 30 s. More fresh water was added and the eggs were allowed to harden for 10–15 min before microinjection. fresh water was added to the eggs to cover the mass; the sperm/egg mixture was gently swirled for 30 s. More fresh water was added and the eggs were allowed to harden for 10–15 min before microinjection. Microinjection and hatching of embryos. To insert the donor template carrying the cathelicidin gene, we used the small guide RNA (sgRNA) (Table 1) that directed Cas9 nuclease to that specific location in a non- coding region of chromosome 1. The designed sgRNA was co-injected with Cas9 along with each donor DNA constructs in different concentrations (10, 20, and 40 ng/μL).h f The microinjection solution mentioned above was injected into one-cell stage embryos as described by Khalil et al.2 using a microinjection system from Applied Scientific Instrumentation (Eugene, OR). Briefly, 50 nL of the solution was directly injected into the yolk sac of each embryo using a 1.0 mm OD borosilicate glass capillary that was previously pulled into a needle by a vertical needle puller (David Kopf Instruments, Tujunga, CA). Embryos were injected within 15–90 min post-fertilization. The injected and control embryos were then reared in 10-L tubs filled with Holtfreter’s solution (59 mmol NaCl, 0.67 mmol KCl, 2.4 mmol NaHCO3, 0.76 mmol CaCl2, 1.67 mmol MgSO4)66,67 containing 10 ppm doxycycline. The embryos were incubated with continuous aeration at 27 °C for 6–8 days until hatching. Methods hi l The universal primer and ssDNA templates were annealed and filled by Platinum Taq DNA Polymerase (Invitrogen, Waltham, MA). The resulting dsDNA served as the template for in vitro transcription to generate sgRNA using the Maxiscript T7 Kit (Thermo Fisher Scientific, Waltham, MA) and was purified using the RNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). The Cas9 protein, which served as the RNA-guided DNA endonuclease enzyme, was obtained from PNA BIO Inc. (Newbury Park, CA). Three different concentrations of the donor DNA for each of the three DNA con- structs were prepared: 10, 20, and 40 ng/μL, for a total of nine sets of injection solutions per trial. The CRISPR/ Cas9 system used in microinjection was composed of sgRNA, Cas9 protein and donor DNA in the ratio of 1:1:1, including one component of phenol red (60%) to visually track microinjected eggs. The final concentrations of sgRNA and Cas9 protein were 150–200 ng/μL and 300–350 ng/μL, respectively. The sgRNA and Cas9 protein mixtures were incubated in ice for 8 min prior to the addition of donor DNA and phenol red, and then the mix- tures were loaded into the microinjection needle65. There were two control groups: the injected control (without the CRISPR/Cas9 components) (iCTRL) and the non-injected control (nCTRL). Egg collection, sperm preparation and fertilization. Broodstock preparation and artificial spawning were performed according to Elaswad et al.65 with modifications. Briefly, sexually mature channel catfish males and females were selected for artificial spawning. Female fish were implanted with 100 μg/kg of luteinizing hor- mone releasing hormone analog (LHRHa) to induce ovulation, and then eggs were stripped in a 20-cm greased spawning pan. Males were euthanized, their testes collected, crushed and sperm prepared in 0.9% saline solu- tion. Sperm suspensions (1–2 mL) were added to the eggs and mixed gently. To activate the sperm, sufficient https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| www.nature.com/scientificreports/ 10-μL reaction volume according to manufacturer’s protocol. Then, 1 μL of cDNA solution was subjected to PCR amplification in a volume of 10 μL containing 3.75 μL nuclease-free water, 0.25 μM of each gene specific primer and 1 μL of EconoTaq Plus 2× Master Mix (Lucigen, Middleton, WI). The PCR amplification procedure was as follows: initial denaturation for 3 min at 95 °C, followed by 35 cycles of denaturation at 95 °C for 30 s, anneal- ing at 58 °C for 30 s and 1 min extension at 72 °C, and a final extension at 72 °C for additional 5 min. The PCR products were analyzed by electrophoresis on 1% agarose gels.i 10-μL reaction volume according to manufacturer’s protocol. Then, 1 μL of cDNA solution was subjected to PCR amplification in a volume of 10 μL containing 3.75 μL nuclease-free water, 0.25 μM of each gene specific primer and 1 μL of EconoTaq Plus 2× Master Mix (Lucigen, Middleton, WI). The PCR amplification procedure was as follows: initial denaturation for 3 min at 95 °C, followed by 35 cycles of denaturation at 95 °C for 30 s, anneal- ing at 58 °C for 30 s and 1 min extension at 72 °C, and a final extension at 72 °C for additional 5 min. The PCR products were analyzed by electrophoresis on 1% agarose gels.i p y y p g g For qPCR analysis, all cDNA products from P1 transgenic channel catfish were diluted to 200 ng/µL and ana- lyzed with a CFX96 real-time PCR Detection System69 (Bio-Rad Laboratories, Hercules, CA). Each amplification was performed in a 10-µL reaction volume containing 5 µL of SsoFast EvaGreen Supermix (Bio-Rad), 1 µL of 5 µM forward and reverse primers (Table 2), 2 µL nuclease-free water and 1 µL of cDNA. The reaction conditions were as follows: 94 °C for 5 s, followed by 40 cycles of 94 °C for 5 s, 60 °C for 5 s, and a dissociation curve profile of 65–95 °C for 5 s/0.5 °C increment. To quantify the gene expression of alligator cathelicidin driven by zebrafish ubiquitin promoter in the different tissues of P1 transgenic channel catfish (N = 3), expression levels of different tissues were calibrated against the liver tissue of these same fish which had the lowest expression value (arbitrarily set to 1×). www.nature.com/scientificreports/ To evaluate the alligator cathelicidin effect between P1 transgenic and sibling non-transgenic channel catfish, the biopsied tissues such as blood, fin and barbel were used and relative gene expression was calibrated against corresponding tissues from sibling non-transgenic fish using the CFX Manager Software version 1.6 (Bio-Rad), and crossing-point (CT) values were converted to fold differences using the relative quantification method. Each sample was performed in triplicate and every analysis was performed using the formula 2(−ΔΔCT)70 which sets the zero expression of the negative control (non-transgenic full-siblings) to 1× for comparison. To normalize mRNA expression levels, 18S rRNA was used as internal control. PCR using primers Ubi-qPCR-F1 and Ubi-qPCR-R1 should amplify a fragment of 123 bp (Table 2). Samples for qPCR were resolved on 1% agarose gel and representative gel image in Fig. 7A can also be visualized in Supplementary Figure S4. Statistical analysis. Hatching percentage for embryos in four independent experiments was calculated as the total number of fry that has completed hatching divided by the total number of embryos, multiplied by 100. Hatching was completed and recorded after 6- or 7-days post fertilization (dpf). Fry survival was determined as the total number of fry that survived 15 days post hatch (dph) divided by the total number of hatched embryos, multiplied by 100. Cumulative survival was calculated as the number of fry that survived divided by the total number of day 0 embryos, multiplied by 100. Integration rates for each treatment were calculated as the total number of positive fish divided by the total number of fish analyzed multiplied by 100. One-way ANOVA and Tukey’s multiple comparisons test were used to analyze these data for significant differences among treatments. Histograms were generated in Microsoft Excel 2016 and GraphPad Prism 8 (GraphPad Software, San Diego, CA). Pearson’s correlation coefficient was performed to determine the relationship between integration rates and concentration of DNA constructs used in microinjection. Paired t-tests were used to compare integration rates between dead and alive fish positive for transgene. The Shapiro–Wilk test was utilized for analysis of data normality. All statistical analyses were performed using R software (R Core Team, 2014). Statistical significance was set at p < 0.05, and all data were presented as the mean ± standard deviation (SD). Data availability All data generated or analyzed during this study are included in this published article (and its Supplementary Information file). Received: 3 June 2020; Accepted: 7 December 2020 Received: 3 June 2020; Accepted: 7 December 2020 Received: 3 June 2020; Accepted: 7 December 2020 www.nature.com/scientificreports/ Dead embryos were recorded and removed daily. Those that hatched were transferred to a Holtfreter’s solution without doxycycline until swim up. They were then fed with Artemia nauplii three or four times a day once their yolk sac was absorbed. Early fry survival was measured at 15 days post hatch and the live fry were reared in 60-L recirculating aquaria systems. Integration analysis. Genomic DNA from dead fry and fin-clipped samples of 2- to 3-month-old finger- lings was extracted via proteinase K digestion and iso-propanol precipitation as previously described68. Genotyp- ing strategy had two steps: first, amplification of the cathelicidin region of the donor DNA construct to confirm insertion of the gene, and second, amplification of the 5′ and 3′ junctional regions to ensure that promoter and terminal regions were also inserted. Primers were designed using Primer3Plus software and listed in Table 2. For ease of genotyping a large number of individuals, primer pairs that could amplify the cathelicidin region for both ubiquitin and β-actin constructs were subjected to PCR amplification. Subsequently, those positive samples were further tested for PCR amplification of 5′ and 3′ junctional regions in the transgene construct, promoter and terminal ends, respectively, so as to test for proper integration on both ends of the transgene. The same primer sets were used for the dsDNA ubiquitin and plasmid DNA ubiquitin constructs. A positive band indicated a cor- rectly oriented knock-in at the targeted locus. PCR products from individual fry were gel extracted from a 1% agarose gel and verified by Sanger sequencing performed by Genewiz (South Plainfield, NJ). Integration rates were calculated as the number of positive individuals detected by PCR in a replicate or treatment divided by the total number of individuals in the same replicate or treatment multiplied by 100. Determination of transgene expression. Expression of the cathelicidin transgene in positive P1 indi- viduals was determined by reverse transcription (RT)-PCR and quantitative real-time PCR (qPCR) analyses. RT-PCR analysis was performed as described previously26 with modifications. 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Author contributions R.S., M.B., and R.D. conceived and designed the experiment; R.S., D.X., W.W., X.M., B.S., M.K., C.L., V.A., D.H., A.J., S.L., M.C., J.G., J.T. and X.W. performed the experiments; Z.Q. conducted bioinformatics and identified the genomic target; R.S. and M.B. analyzed the results; R.S. and R.D. wrote the manuscript. All authors reviewed the manuscript. Competing interests h The authors declare no competing interests. References Cheng, Q. et al. Interaction of diet and the masou salmon delta5-desaturase transgene on delta 6-desaturase and stearoyl-coa desaturase gene expression and n-3 fatty acid level in common carp (Cyprinus carpio). Transgenic Res. 23, 729–742 (2014). 54. Dunham, R.A. Gene expression in Aquaculture and Fisheries Biotechnology: Genetic Approaches 2nd edn, 215–239 (CABI Publish- ing, 2011).ti 55. Traver, D. et al. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat. Immunol. 4, 1238–1246 (2003).if 6. Burket, C. T. et al. Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters. Transgenic Res. 17, 265–279 (2008).i 57. Mosimann, C. et al. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. Development 138, 169–177 (2011). p 58. Kawamoto, H. & Minato, N. Myeloid cells. Int. J. Biochem. Cell B. 36, 1374–1379 (2004). p Kawamoto, H. & Minato, N. Myeloid cells. Int. J. Biochem. Cell B. 36, 1374–1379 (2004). https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \| Additional informationh Supplementary Information The online version contains supplementary material available at https://doi. org/10.1038/s41598-020-79409-5. Correspondence and requests for materials should be addressed to R.M.C.S. or R.A.D. Correspondence and requests for materials should be addressed to R.M.C.S. or R.A.D. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 https://doi.org/10.1038/s41598-020-79409-5 Scientific Reports \| (2020) 10:22271 \|

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