Lac Barombi Et Mangrove De L‘Estuaire Du Wouri

AIX MARSEILLE UNIVERSITÉ École doctorale des sciences de l’environnement Laboratoire Chimie Environnement – UMR CNRS 7376 – Équipe TRAME Centre de Recherche et d’Enseignement de Géosciences de l’Environnement UMR CNRS UMR CNRS 7330, UMR IRD 161, Collège de France

THÈSE DE DOCTORAT Pour obtenir le grade de DOCTEUR DE L’UNIVERSITÉ D’AIX-MARSEILLE Discipline : Science de l’Environnement Spécialité : Chimie de l’environnement

Présentée par Kevin MBUSNUM GWETH Le 08 octobre 2020

Évaluation des Contaminants Organiques Hydrophobes dans deux Environnements Aquatiques en Afrique Centrale, Cameroun : Lac Barombi et Mangrove de l‘Estuaire du Wouri

Composition du Jury :

Mme Catherine GONZALEZ École des mines d’Ales Rapporteure M. Baghdad OUDDANE Professeur, Université de Lille Rapporteur Mme Guillemette MENOT Professeur, ENS de Lyon Examinatrice M. Jacques ETAME Professeur, IUT Université de Douala Examinateur Mme Aurore ZALOUK-VERGNOUX MCF, HDR Université de Nantes Examinatrice Mme Laure MALLERET MCF, Aix Marseille Université Co-directrice de thèse M. Pierre DESCHAMPS Chargé de Recherche, Aix Marseille Université Co-directeur de thèse M. Pierre DOUMENQ Professeur, Aix Marseille Université Directeur de thèse M. Jean-Jacques BRAUN Directeur de Recherche, IRD Membre invité M. Olivier DHONT Professeur des Universités, Université de Membre invité Paris1 Panthéon-Sorbonne

Acknowledgements

This thesis has been one of the most challenging and best life changing experiences in my life so far. Beyond the rich academic experience, I learned so much about human relationships and self-awareness. In this adventure I met many wonderful people that have contributed in making me a better version of myself and this is an experience I am ready and to relive in order to acquire all this knowledge. I will first thank the Almighty God for keeping in good health, safe and the journey mercies granted to me throughout this thesis. I express my gratitude to the members of jury; Pr. Catherine Gonzalez and Pr. Bagdhad Ouddane as rapporteur for the validation of this work and the consideration giving to it. Your comments and observations helped to improve its quality. I was honoured to have you; Pr. Guillemette Menot as jury chair. I lack words to express my gratitude for your unconditional and endless support from the beginning of this adventure. Thank you for your valuable comments, advices and constant motivation. You have been there for me in happy and tough times. I endlessly say thank you! I thank Dr. Aurore Zalouk-Vergnoux for taking part in the evaluation of this work and your valuable comments and suggestions. I thank Pr. Jacques Etame for the examination of this work, your interest and consideration for this work. I greatly appreciate your comments, observations and suggestions. I would like to express my sincere gratitude to my thesis supervisors. Working with three supervisors was challenging but very beneficial. To Pr. Pierre Doumenq, thank you Sir, for giving me this exceptional opportunity to learn by your side. Despite your heavy schedule, I greatly appreciate every effort to make time for us to carry this work forward. At the beginning, it wasn’t quite easy, but I gradually adapted to suit your way of working. Your great advices and words of encouragement kept me going. You are an inspiration to hard work and achievement; you have my deepest admiration and respect. To Dr. Pierre Deschamps without whom this thesis would not have been possible. I equally lack words to express my gratitude. Thank you for trusting me, it was a great boost of confidence and energy to overcome the encountered challenges. I appreciate your academic rigor and the relevance in your comments. You always did what was necessary to make things and your openness made me feel at ease. I have learned so much from your mentorship throughout these years. I endlessly say thank you! To Dr. Laure Malleret, I express my profound gratitude for your huge support and kindness. I greatly appreciate your listening ability, understanding and pedagogic approach in our discussions. Somehow difficult for me at the beginning, despite your busy schedule, you made time to clarify the questions, worries and doubts I had concerning certain aspects of this work. I had a sense of satisfaction at the end of every working session. Also, with time I discovered you have a great sense of humanity. I am very grateful to Dr. Jean-Jacques Braun for the multidimensional support from the beginning and future prospects related to this work. I am thankful to Pr. Olivier Dhont for the interest you have shown to this subject matter, the valuable advices and unforgettable moments during the Lake Barombi sampling campaign. Special thanks to Dr. Pascal Prudent for being part of my thesis committee and her active contribution to the realization of trace metal analyses which unfortunately does not appear in this thesis. This work is valorized in the article entitled “Assessment of organochlorinated pesticides, polyaromatic hydrocarbons and trace metals in water, soil and sediments of Lake Barombi watershed, Southwest Region of ii

Cameroon. Special thanks to Dr. Yannick Garcin who open my way to this adventure. Thank you for giving me this great opportunity since 2015 to join the amazing team of researchers for the Lake Barombi sampling campaign. Thanks for your valuable advices and support from the beginning to the end of this thesis. I am grateful to Dr. Frank Torre, for giving me the opportunity through the MedNet Program of excellence that enabled my immersion in this rewarding journey. Thanks to Laurent Vassalo, for his training and assistance on the measurement TOC in soil and sediments samples. Thanks to Dr. Raphael Onguene, for the warm welcome at the University of Douala and JEAI-RELIFORME laboratory. Thanks to Pr. Henri Wortham (LCE Director) for welcoming me into LCE as a master’s intern and PhD student. This thesis was supported by the Institute of Recherche for Development (IRD), Service de Coopération et d’Action Culturelle (SCAC) of the French Embassy in Cameroon and Ecosystèmes Continentaux et Risques Environnementaux (ECCOREV). I express my profound gratitude to all the staff of Aix-Marseille University (AMU) in Laboratoire de Chimie de l’Environment (LCE), the IRA and TRAME teams. I am very grateful to Pr. Pascal Wong-Wah-Chung, an exceptional person. Every morning we greeted each other, It was a boost of energy and motivation for the day. I learned from you that self-discipline and organization are essential for achieving set objectives. Thanks for your valuable advices, great sense of humour and comprehension of cultural diversity. To Dr. Laurence Asia (Loloe) and Stephanie Lebarillier (Steph) thanks for your warm welcome at LCE and training on various analytical technics and equipments (ASE, SPE, GC-MS, HPLC etc…). You were always ready and available to help when I needed assistance. Thanks for your daily encouragements and attention. Thanks to Dr. Anne Piram and Dr. Stephanie Rossignol for the interesting discussions we had on various topics, your encouragements and moment we shared. Thanks to Cecile Langlois for her promptness and precious help for administrative procedures. I wish you a quick recovery. Thanks to Max Bresson and Jean François Barbion for your assistance when needed, wish you recover as soon as possible. Thanks to Pr. Gilbert Mille for his encouragement and discussion on polyaromatic hydrocarbons, may your Soul Rest in Peace. Thanks to Frederique, Lionel and Sylvain for the interesting discussions and encouragement. I express my sincere gratitude to my PhD colleagues at LCE, I wish you’ll the very best for the completion of your thesis. To Nuning Vita Hidayati my sister from Indonesia, thanks for your encouragements and support in various forms. We shared joyful and painful moments together, you are exceptional. All the best for the end of your thesis, you will make it. Clementine Côte, you were always available to help and supported me from the beginning to the end. You have done a great job in your thesis, all the best for the end of your thesis and future career. Houssein Louati, your kindness and respect are overwhelming. You were always willing to help. Stay as you are my friend. Imen, for your outstanding work on pesticides for the Wouri Estuary Mangrove during you master’s internship which is part of this thesis. I am grateful for your concern and unforgettable moments we shared. El Mountassir El Mouchtari, my brother from Morocco. I am thankful for your support in many forms and joke cracking moments. You have a great heart, stay as you are! Mathilde Godéré, an exceptional person with a great heart. I greatly appreciate the time you made out of your busy schedule to help me. I also thank you for the trips to and from the laboratory when it was possible. Maria

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Masry, you were able to put a smile on my face during hard times. Thanks for your kindness, you were always there for me, you are an amazing person. Mathilde Chantreux, a wonderful person with a great sense of humanity and comprehension of cultural diversity, I enjoyed the interesting philosophical discussions we had and was touched by your kindness. Carole Abdel-Nour, a lovely and caring person with a great heart. Thanks for your concern, the delicious banana cakes, and the breathing exercises. Dr. Perrine Branchet, we have known each other for a while but I was touched by your kindness. Thanks for the huge support you brought to me and the time you dedicated to help me. Dr. Camille Grandclément and Dr. Fanny Desbiolles, thank you for the interesting discussions we had and the encouragements. I am grateful to the former and present staff and PHD students of Aix-Marseille University in CEREGE; Pr. Olivier Bellier (CEREGE Director), for having accepted me into the laboratory. I am thankful to Pr. Nicolas Thouveny (OSU Pytheas Director), Pr. Bruno Hamelin, Pr. Julio Gonçalves, Pr. Christine Vallet-Coulomb, Dr. Florence Sylvestre, Hélène Mariot, Wulfran Barthelemy, Abel Guihou, Nicolas Godeau, Aladin Andrisoa, Mahamat Nour Abdallah, Souleyman Abba, Yacoub Abdallah Nassour, Jennifer Weil- Accardo and Chloé Poulin. I express my profound gratitude to Dr. Thomas Stieglitz for your multifaceted support and unforgettable moments we shared. I am very grateful to Isabelle Hammad, an exceptional person with a great heart. Thanks for your prompt response to all my concerns regarding procedures with the Environmental Sciences doctoral school (ED 251). I thank Laboratoire De Mecanique Modelisation et Procedes Propres (M2P2) - UMR 7340 M2P2 for the welcome and the opportunity to receive training on the measurement of TOC on solid samples and performing measurements on my soil and sediment samples. I therefore thank Pr. Olivier Boutin and Jean-Paul Nisteron for making this possible in the best conditions. Special thanks to Ms Henriette Massusi, Head of Laboratoire d’Analyse Géochimique des Eaux (LAGE) in Cameroon and the entire staff of LAGE for their assistance throughout my stay at the laboratory. I am very grateful to the students of the University of Douala that assisted me throughout my sampling campaign in the Wouri Estuary Mangrove; Vanessa Ndikontar, Didier Bonga and Ulrich Bilounga. Finally, I take this opportunity to express my profound gratitude to my lovely family that has always been the solid foundation on which my strength, motivation and determination are built since my tender age. To my lovely father and mother, words are not enough to express my gratitude for your unconditional love, support and valuable sacrifice in making me who I am. One of my life goals has always been to make you extremely happy and proud of me. You know how much I love you. To my siblings; Collins, Leonard, Audrey and in-law Oriane, thanks for the unconditional love and endless support. You are what I have as most precious. I am very grateful to my lovely and caring aunt Elizabeth, for her unconditional love and support. I am equally thankful to Daniel and Veronique for the support, joyful and unforgettable moments we shared. I thank all my longtime friends who have always supported me; Doh Munoh, Sube Ebah, Kongolo Sora, Kelly Ameck, Mpude Diobe, Manly Ngong, Emmanuel Ngwe, Richard Tabot, Ariane Messomo, Marcel Mbaw, Lionel Tochap, Ulrich Nguemdjo, Midrel Tchouta, Paul Gerard GBETKOM, Brina Tchibinda, Linda, Nadia Dempowo, Tah Boris.

Table of Contents

Table of Contents ...... ii

List of figures ...... vii

List of tables ...... ix

List of abbreviations ...... xi

General Introduction ...... 1

CHAPTER 1: Bibliographic Review ...... 12 1.1. The Stockholm Convention on Persistent Organic Pollutants ...... 13 1.2 Institutional and legislative framework of POPs in Africa ...... 17 1.3. Import and usage of Persistent Organic Pollutants in Africa ...... 21 1.4. Presentation of the Targeted Persistent Organic Pollutants ...... 27 1.4.1 Organochlorinated pesticides ...... 27 1.4.2 Polychlorinated Biphenyls ...... 35 1.4.3 Polyaromatic hydrocarbons ...... 37 1.5. The Physical-Chemical properties of target POPs ...... 42 1.5.1. Vapour pressure and solubility in water ...... 43 1.5.2. Henry’s law constant ...... 44 1.5.3. Octanol-water, octanol-air and organic carbon-water partition coefficients ...... 44 1.5.4. Chemical reactivity and half-lives ...... 45 1.6 Ecotoxicological impact of sediment contaminants (OCPs, PCBs and PAHs) ...... 53 1.7. Effects of organochlorinated pesticides, polychlorobiphenyls and polyaromatic hydrocarbons on human health ...... 63 I.7.1. Cancer ...... 63 1.7.2. Disruptive Effects ...... 64 1.7.3. Neurodevelopment effects ...... 65 1.7.4. Reproductive Effects ...... 66 1.8 Review on environmental levels of target POPs in aquatic environments of Africa (Article review) ...... 67 Abstract ...... 67 1.8.1 Introduction ...... 68 1.8.1. Persistent Organic Pollutants in Africa ...... 71 1.8.2. Methodology ...... 75 1.8.3. Environmental levels and ecotoxicological implication of POPs in African aquatic environments .... 78 1.8.5 Underexamined areas, future research and recommendations ...... 104 1.8.6 Limitations of the review ...... 106

CHAPTER 2: Study Sites ...... 99 2.1. Lake Barombi Watershed ...... 109 2.1.1 Geographical context ...... 109 2.1.2 Anthropic activities ...... 112

2.2. Wouri Estuary Mangrove ...... 113 2.2.1. Geographical context ...... 113 2.2.2. Hydrographic network ...... 117 2.2.3. Fauna and Flora ...... 121 2.2.4. Anthropic activities ...... 121

CHAPTER 3: Material and Methods ...... 126 3.1. Methodological approach in the determination of PAHs, PCBs and OCPs in African aquatic environments ...... 127 3.2 On-site survey of pesticides commercialization and uses in Kumba and Barombi Mbo ...... 130 3.3. Sample Collection ...... 131 3.3.2. Wouri Estuary Mangrove ...... 132 3.3.3. Chemical reagents, materials and instruments ...... 133 3.4. Sample pre-treatment ...... 136 3.4.2. Soils and sediments ...... 138 3.5. Extraction and concentration ...... 139 3.5.1. Water ...... 139 3.5.2 Soils and sediments ...... 141 3.6. Activation of Copper for sediment desulphurization ...... 145 3.7. Clean up and fractionation ...... 145 3.8. Instrumental analysis ...... 147 3.8.1. Polyaromatic hydrocarbons ...... 147 3.8.2. Chlorinated pesticides and polychlorinated biphenyls ...... 148 3.8.3. Quality control and quality assurance ...... 148

CHAPTER 4: Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon ...... 159 Abstract ...... 160 4.1 Introduction ...... 161 4.2 Material and methods ...... 164 4.2.1 Study area ...... 164 4.2.2 Survey on pesticide commercialization and uses in the Kumba market and Barombi Mbo watershed ...... 166 4.2.3. Pre-treatment and extraction ...... 169 4.2.4. Instrumental analysis ...... 170 4.2.5. Determination of Total Organic Carbon ...... 171 4.2.6. Quality assurance and quality control ...... 171 4.3 Results and Discussion ...... 172 4.3.1 Pesticide commercialization and uses ...... 172 4.3.2. Total Organic Carbon content ...... 175 4.3.3. Levels and distribution of PAHs and OCPs ...... 175 4.4.4. Comparison of OCPs levels with Sediment Quality Guidelines ...... 190 4.4.5. Comparison with other lacustrine environments worldwide ...... 191 4.5. Conclusion ...... 195 vi

CHAPTER 5: Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance ...... 196 Abstract ...... 197 5.1. Introduction ...... 198 5.2. Materials and methods ...... 201 5.2.1 Study area ...... 201 5.2.2. Sample collection, pre-treatment and contents of moisture and organic carbon ...... 203 5.2.3. Reagents and standards ...... 204 5.2.4. PAHs, PCBs and CLPs extraction ...... 205 5.2.5. PAHs, PCBs and CLPs analyses ...... 206 5.2.6. Quality Assurance and Control (QA/QC) ...... 207 5.3. Results and discussion ...... 208 5.3.1. Spatial distribution and profiles of PAHs, PCBs and CLPs ...... 208 5.3.2 Comparison to Worldwide estuary and mangrove areas ...... 229 5.3.3 Ecotoxicological significance ...... 235 5.4. Conclusion ...... 238 5.5 Conclusive statement on Chapter 4 and Chapter 5 ...... 239

General conclusion, Perspectives and Recommendations ...... 241 General conclusion ...... 242 Perspectives ...... 247 Recommendations ...... 248

Résumé en Français ...... 261

Bibliography ...... 283

APPENDICES ...... 308

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List of figures

Figure 1. A typical persistent organic pollutant time trend ...... 22 Figure 2. Chemical structures of targeted organochlorinated Pesticides ...... 29 Figure 3. Chemical structures of targeted chlorinated organophosphorus pesticides ...... 32 Figure 4. Chemical structures of the targeted chloroacetamide herbicides ...... 35 Figure 5. General formula of polychlorinated biphenyls ...... 36 Figure 6. Chemical structures of the targeted polychlorinated biphenyls ...... 37 Figure 7. The 16 priority polyaromatic hydrocarbons designated by the US EPA ...... 41 Figure 8. Number of published articles examined for OCPs, PCBs and PAHs in aquatic environments of Africa from 1998 to 2019 ...... 76 Figure 9. Distribution of study sites for OCPs, PCBs and PAHs for aquatic environments in Africa ...... 77 Figure 10. Concentration of POPs (OCPs, PCBs and PAHs) in sediment (ng/g, dry weight) of African aquatic environments ...... 83 Figure 11. Concentration of POPs (OCPs, PCBs and PAHs) in fish (ng/g, lipid weight) of African aquatic environments ...... 95 Figure 12. Concentration of POPs (OCPs, PCBs and PAHs) in water (μg/L) of African aquatic environments. ... 101 Figure 13. Location of a) Cameroon, b) Southwest region and c) Lake Barombi Watershed ...... 110 Figure 14. Location of Lake Barombi and the city of Kumba ...... 111 Figure 15. Location of the Wouri Estuary mangrove in the Cameroun coast ...... 115 Figure 16. Cameroun Estuary mangrove soil profiles ...... 116 Figure 17. Major rivers in and around the city of Douala ...... 118 Figure 18. Hydrographic networks and watersheds in the city of Douala...... 120 Figure 19. Localisation of industries in the city of Douala ...... 123 Figure 20. Municipal waste dump in Dinde, Koweït City (Crique Docteur zone) ...... 125 Figure 21. Sampling of surface soil (left) and b) Lake sediments (right) in the Lake Barombi Watershed ...... 132 Figure 22. Sample collection from the Wouri Estuary Mangrove ...... 133 Figure 23. Summary of the major methodological steps employed in the determination of all studied compounds...... 137 Figure 24. Filtration device (Buchner funnel) for water samples ...... 138 Figure 25. Pre-treatment material for soil and sediment samples ...... 139 Figure 26. Extraction device for pesticides in water samples ...... 141 Figure 27. Accelerated Solvent Extraction Device (ASE 350 Dionnex) and components of the ASE cell ...... 142 Figure 28. Summary of the ASE procedure ...... 144 Figure 29. Evaporation of ASE extracts with a Liebisch Labortechnik evaporator ...... 145 Figure 30. Solid Phase Extraction, clean-up and fractionation of sample extracts ...... 146 Figure 31. Location of the study area (a) Africa (b) South West region of Cameroon and (c) Lake Barombi Mbo ...... 165 vii

Figure 32. Distribution pattern (%) of PAHs in the Lake Barombi Mbo Watershed ...... 179 Figure 33. Cross-plot of PAH ratios (PHE/ANT versus FL/PYR) for samples of the Lake Barombi Mbo Watershed ...... 181 Figure 34. Profile and detection frequencies of OCPs in soils of LBM watershed ...... 187 Figure 35. Pattern and detection frequencies of OCPs in stream sediments of LBM watershed ...... 188 Figure 36. Pattern and detection frequencies of OCPs in Lake sediments of LBM watershed ...... 188 Figure 37. Location of the Wouri Estuary mangrove (a) World map, (b) Cameroon map and (c) Sampling stations ...... 203 Figure 38. Percentage distribution of PAHs and PCBs in sediments of the Wouri Estuary Mangrove ...... 215 Figure 39. Cross-plot of PAH ratios for Wouri Estuary Mangrove sediments: PHE/Ant versus FL/PYR BaA/(BaA+CHRY) and IP/(IP+B(g,h,i)P) versus ANT/(ANT+PHE) ...... 217 Figure 40. Concentration ranges and frequency of detection of chlorinated pesticides in the Wouri Estuary Mangrove ...... 221 Figure 41. Spatial distribution of PAHs, PCBs and CLPs in sediments of the Wouri Estuary Mangrove ...... 224 Figure 42. Comparison of DDT and PCB concentrations in sediments of the WEM with Effect-based Sediment Quality Guidelines ...... 237

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List of tables

Table 1. Major conventions prohibiting hazardous chemicals and wastes with signatories and parties worldwide ...... 14 Table 2. List of POPs regulated under the Stockholm Convention and chemicals under revision ...... 15 Table 3. Regional regulations on the import, use, disposal and management of POPs in Africa ...... 17 Table 4. Regulatory measures implemented by some African countries on the import, use, disposal and management of POPs in Africa ...... 19 Table 5. Regulatory situation of POPs in Cameroon ...... 20 Table 6. Pesticide import (tonnes) in 2009, in Cameroon from Europe, Africa out of CEMAC zone and the rest of the world (through the Douala port) ...... 25 Table 7. Distribution of obsolete pesticides (tonnes) in the regions of Cameroon ...... 26 Table 8. Summary of physical-chemical properties for chlorinated pesticides (Mackay et al., 2006) ...... 48 Table 9. Summary of physical-chemical properties for polychlorinated biphenyls (Mackay et al., 2006) ...... 50 Table 10. Summary of physical-chemical properties for polyaromatic hydrocarbons (Mackay et al., 2006) ...... 51 Table 11. Half-life classes of PAHs, PCBs and Pesticides (Mackay et al., 2006) ...... 46 Table 12. Summary of sediment quality guidelines for PAHs in aquatic ecosystems that reflect TECs (concentrations below which adverse biological effects are unlikely to be observed) ...... 57 Table 13. Summary of sediment quality guidelines for OCPs and PCBs that reflect TECs (concentrations above which adverse biological effects are likely observed) ...... 58 Table 14. Consensus based empirical SQGs approach ...... 59 Table 15. Summary of the most used empirical and theoretical approaches to set sediment quality guidelines, with their advantages and limitations...... 61 Table 16. Summary of OCPs, PCBs and PAHs levels in sediments of African aquatic environments ...... 84 Table 17. Summary of OCPs, PCBs and PAHs levels in biota of African aquatic environments ...... 96 Table 18. Summary of OCPs, PCBs and PAHs levels in water of African aquatic environments ...... 102 Table 19. Summary of methodological approaches employed to determine OCPs, PCBs and PAHs in African aquatic environments ...... 311 Table 20. Characteristics of products, chemicals and materials used ...... 134 Table 21. Characteristics of products, chemicals and materials used (continued) ...... 135 Table 22. Characteristics of products, chemicals and materials used (continued) ...... 135 Table 23. Summary of ASE extraction parameters for studied compounds ...... 142 Table 24. Parameters for analyses of PAHs by HPLC-PFD ...... 147 Table 25. Identification parameters for the studied compounds in GC-MS SIM mode ...... 152 Table 26. Summary of method validation parameters for studied compounds with Certified Reference Materials (CRM860 and CNS391) for the Lake Barombi study ...... 154 Table 27. Localisation and site description of sampling stations from the Lake Barombi Watershed ...... 167 Table 28. Pesticides registered at the Kumba market and dumped packages in farms ...... 174 ix

Table 29. PAH concentrations (ng/g of dry weight) in soils and sediments of LBM watershed with Sediments Quality Guidelines (Long et al., 1995) ...... 177 Table 30. Concentration of OCPs (ng/g) of OCPs in soils. stream and lake sediments from LBM watershed and Sediment Quality Guidelines (Long et al., 1995) ...... 183 Table 31. Summary of OCPs and PAHs concentrations (ng/g) in sediments (dry weight) of lacustrine environment worldwide ...... 193 Table 32. PAH concentrations (ng/g of dry weight) in sediments of the WEM with Sediments Quality Guidelines (Long et al., 1995) ...... 211 Table 33. Concentration of CLPs and PCBs (ng/g of dry weight) in sediments of the Wouri Estuary Mangrove 225 Table 34. Summary of POP levels (ng/g) in sediments (dry weight) of coastal environments (estuaries and mangroves) worldwide ...... 232

List of abbreviations

ASE – Accelerated Solvent Extraction Ace – Acetone Acn – Acetonitrile ATSDR – Agency of Toxic Substances and Disease Register BDL – below the detection limit BFRs – Brominated flame retardants CVL – Cameroon Volcanic line CRM – Certified Reference Material- CLPs – Chlorinated Pesticides CAHs – Chloroacetamide Herbicides COPPs – Chlorinated organophosphorus pesticides and Dcm – Dichloromethane DDT - Dichlorodiphenyltrichloroethane CEMAC – Economic and Monetary Community of Central Africa ERL -– Effects range low ERM – Effect range median ECD – Electron Capture Detector FAO – Food and Agriculture Organization FLD – Fluorescence Detector GC-MS – Gas Chromatography-Mass Spectrometry GDP – Gross Domestic Product Hex – Hexane HCB – Hexachlorobenzene HPLC – High Performance Liquid Chromatography HOCs – Hydrophobic Organic Contaminants IARC – International Agency for Research on Cancer ICES – International Council for the Exploration of the Sea IUPAC – International Union of Pure and Applied Chemistry LBW – Lake Barombi Watershed LEL – Lowest effect level LOD – Limit of detection xi

LLE – Liquid-Liquid Extraction MET – Minimal effect threshold MINADER - Ministère de l'Agriculture et du Développement Rural MINEPDED – Ministère de l’Environnement, de la Protection de la Nature et du Développement Durable MINHDU – Ministère de l’Habitat et du Développement Urbain NIP – National Implementation Plans NOAA – National Oceanic and Atmospheric Administration

KOW – Octanol-water partition coefficient

KOC – Organic carbon-water partition coefficient OCPs – Organochlorinated Pesticides POPs – Persistent Organic Pollutants PAHs – Polyaromatic hydrocarbons PBDEs – Polybrominated diphenyl ethers PCBs – Polychlorinated biphenyls PCDDs – Polychlorinated dibenzo-p-dioxins PCDFs – Polychlorinated dibenzofurans PAD – Port Authority of Douala PEC – Probable effect concentration PEL– Probable effects level SQGs – Sediment Quality Guidelines SIM – Selected Ion Monitoring SEL – Severe effect level SPE –solid-phase extraction SAICM – Strategic Approach to International Chemicals Management TEC – Threshold effect concentration TEL – Threshold effect level TOC – Total Organic Carbon TET – Toxic effect threshold TEF – Toxic Equivalent Factor UNEP – United Nations Environmental Program US EPA - United States Environmental Protection Agency WEM – Wouri Estuary Mangrove

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General Introduction

CHAPTER I :Bibliographic Review

Over the past three decades, the striking growth in the production and use of chemicals has raised concerns among international organizations, governments and the general public on their potential threats to human and environmental health.

Anthropic activities such as the extensive use of pesticides, petroleum and organic solvents has led to the wide-area dissemination of contaminants in the environment known as Hydrophobic Organic Contaminants (HOCs). Environmental contamination from historical or recent use of HOCs is known, documented and now widespread globally (Cui et al., 2013). Owing to their toxicity, persistence and potential bioaccumulation properties, HOCs have been classified as Persistent Organic

Pollutants (POPs) under the Stockholm Convention (adopted in 2001). POPs include pesticides like DDT, lindane, endosulfan, polychlorinated biphenyls (PCBs) polyaromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), brominated flame retardants (BFRs) such as polybrominated diphenyl ethers (PBDEs). The last two decades have been marked by an increase in research interests and efforts both in the development of analytical methods and the determination of these compounds in various environmental compartments.

Since the 1970s, restrictions were imposed on the production and use of organochlorine compounds in most developed nations while there was continuous use in developing countries. Later on, in the late 1980s, prohibitions were established in most developing countries. Till date despite these restrictions, these compounds remain ubiquitous in the environment. In line with restrictive regulations established in developed nations, significant decrease in levels of some POPs were observed 3

CHAPTER I :Bibliographic Review

(except Asia), where they had been mainly produced and used. Besides that, increasing or more prevalent levels of POPs have been reported for regions of the southern hemisphere (where they have not been produced) (Bommanna and

Kurunthachalam, 1994; Gioia et al., 2008; Jones and de Voogt, 1999;

Shunthirasingham et al., 2010) Owing to their persistence, they possess strong abilities of long-range transport, POPs are able to move from source regions to remote areas of the globe where they have not been used or produced et al., 2001); in air and seawater of the Atlantic Ocean (Gioia et al., 2008; Jaward et al., 2004),

Lake Gerio in Nigeria (Mazlan et al., 2017).

Previous studies have revealed both occurrence and adverse effects of POPs on various terrestrial and aquatic ecosystems worldwide (Bansal, 2019; Merhaby et al., 2019; Abdel-Shafy and Mansour, 2016; Brits et al., 2016; Ribeiro et al., 2016; Net et al., 2015; Rabodonirina et al., 2015). The fragile ecology of aquatic environments

(oceans, rivers, lakes, mangroves, streams, estuaries) is particularly sensitive to pollution and subjected to constant and increasing pressure from human activities.

POP contamination in these ecosystems may emanate from accidental spills, spray- drift, reaching and/or runoff from discharge of municipal, households, pharmaceutical and industrial wastes, agricultural effluents and nonpoint pollution sources. Due to their hydrophobic nature, POPs preferentially accumulate on organic rich phases such as sediments and fatty tissues of living organisms (bioaccumulation) (Jacob and

Cherian, 2013), leading to biomagnification along the food chain. In addition, the presence of POPs in the environment resulting from activities like pesticide application is associated with effects on non-target organisms. (Jayaraj et al., 2016),

CHAPTER I :Bibliographic Review reported that only 0.3 % of applied pesticides reaches the target pest while 99.7 % are dispersed in the environment.

Among aquatic ecosystems, mangroves are one of the most productive and biologically diverse ecosystems on earth. They are the most dominant coastal ecosystems and provide important ecosystemic, economic and socio-cultural benefits such as nurseries, breeding sites of marine and arboreal life fertilisation, stabilisation, filtration, regulation of microclimate, food chain support, timber and no- timber forest products to support rural economies. Moreover, they act as physical shelter and a buffer from episodic severe storms, river flows, and large waves (Duke,

2011). Despite these benefits, globally 9,736 km2 and 1,389 km2 of mangroves have been lost and degraded respectively since • 1996. Current data suggest an average loss rate of 0.21 % annually from 1996 to 2016 (IUCN, 2020). It worth an estimated

33,000 – 57,000 US $ per hectare, mangroves are being degraded, lost, or poorly restored at an alarming rate (Duke et al., 2014). According to (Maciel-Souza et al.,

2006), mangroves are at risk from acute/point and chronic pollution when associated with ports and petrochemical industries where repeated spills are common. Recent studies worldwide have reported mangrove contamination by POPs (Chai et al.,

2019; Qiu et al., 2019; Assunção et al., 2017; Alegria et al., 2016; Fusi et al., 2016;

Bayen, 2012; Bodin et al., 2011).

Lake ecosystems are in a similar way are greatly affected by population growth, increasing agricultural and industrial development. Lakes are threatened by activities related to the massive exploitation of halieutic resources, reclamation of land from marshes of lakes, various wastewater discharges, construction of water conservancy and tourism. Lakes often contain high pollution levels relative to the 5

CHAPTER I :Bibliographic Review environment. This is because rivers and streams drain pollutants from the surrounding landscape and concentrate in lakes, acting as pollution sink. Thus, lakes reflect the processes and activities that operate around them. Recent studies worldwide have revealed POP contamination in lakes (Kampire et al., 2017;

Mawussi, 2016; Kafilzadeh, 2015; Polder et al., 2014; Quiroz et al., 2010) and the use of chemicals like pesticides for fishing in African lakes (Kuranchie-Mensah et al.,

2012; Wasswa et al., 2011; Ntow, 2005). Aquatic sediments therefore act as natural archives and can be used in the reconstruction pollution history. Sediments that accumulate in mangroves are potential repositories of anthropogenic pollution due to high total organic carbon, anaerobic properties, rapid turnover and burial

(bioturbation) (Vane et al., 2009; Tam and Yao, 2002), thus making them preferential sites for deposition and accumulation of pollutants (Raza et al., 2013). Likewise, aquatic organisms that are particularly sensitive to pollution could serve as bioindicators of pollution.

The current African context is characterized by rapid demographic growth (2.5 billion people by 2050 according to UN estimate), most economies relying on agriculture, increasing industrial and agricultural development, inadequate facilities for waste management coupled with poor implementation of regulations are drivers of threats to the environmental quality of aquatic ecosystems and human health.

Besides that, knowledge and data on POPs are scarce in Africa. Given that the existing data mainly focuses on OCPs in and around highly urbanized and agricultural areas, it is important that attention should be given to other groups of

POPs and remote areas. This raises concern on the environmental quality of rich and

CHAPTER I :Bibliographic Review fragile ecosystems listed as protected areas, as it is the case for most African aquatic environments. This triggers various questionings on:

i) What is the level of anthropization of environments undergoing strong

anthropogenic pressure such as coastal areas and conversely remote

areas related to POPs contamination in Africa?

ii) What are the environmental levels of POPs in African aquatic

environments and the toxic effects associated with these levels?

iii) What occurrence of POPs pertains to historical or recent uses and acute

or chronic contamination in Africa?

iv) What is the contamination status of African ecosystems when compared

to similar ecosystems worldwide?

It is within this framework that the aim of this thesis is to carry out an assessment of OCPs, PCBs and PAHs in two aquatic environments undergoing very dissimilar anthropogenic pressures in Cameroon. One subjected to little human pressure, the

Lake Barombi watershed (LBW) and the other site underdoing huge human pressure, the Wouri estuary mangrove (WEM). The sites were chosen based on some of the following criteria:

Lake Barombi watershed (LBW) is a near-pristine and remote area of about

415 hectares with a population of about 400 people, who depend entirely on the resources of the lake and the surrounding forest. Since 1940, this watershed was classified as the Barombi Mbo forest reserve and the Lake Barombi designated as the second Ramsar site1 of Cameroon in 2006. The later serves as the main source

1 A Ramsar site is a wetlands of international importance identified by contracting parties to the Ramsar Convention (signed in 1971) and an advisory board because they meet one or more of the Ramsar Criteria in terms of ecology, botany, zoology, limnology or hydrology Some of the criteria are if the wetland contains a 7

CHAPTER I :Bibliographic Review of drinking water supply to the nearby town of Kumba and its environs. Unfortunately, till today this forest reserve lacks a management plan, consequently the lake subjected to overfishing and to some extent fishing with chemicals (Balgah and

Kimengsi, 2011). Other major activities carried within this watershed include i) the spraying of pesticides on smallholder farms (cash crops and mostly food crops) eventually washed down to the lake, ii) slash and burn farming and iii) disposal of pesticide packages in farms after use. The nearby town of Kumba undergoes rapid urbanization leading to more acquisition of farmland in the LBW and greater reliance on its resources. Consequently, the interest of studying this site was i) to carry out a baseline study to assess the level of anthropogenic impact with respect to OCPs in water, soil and sediments and PAHs in soil and sediments of the LBW.

The Wouri Estuary Mangrove (WEM) at proximity to the city of Douala, the most populated city (about 3 million people) of Cameroon and first industrial centre of the Economic and Monetary Community of Central Africa (CEMAC). The Wouri estuary hosts the Port Authority of Douala, the largest port of Central Africa. The mangrove area suffers from the discharge of municipal wastes and industrial effluents, the use of pesticides in vector control of diseases (malaria) and peri-urban agriculture, vehicular emissions from dense traffic, increasing deforestation of the mangrove for settlement, fuel wood and smoking fish and sand extraction. (Essombè

Edimo, 2007) indicated that, since independence (1960), industrial development, demographic growth and land use increasingly occur in the outskirts or peri-urban areas of Douala. With a poor and inadequate waste management system, the

representative, rare, or unique example of a natural or near-natural wetland type found within the appropriate biogeographic region or the wetland supports vulnerable, endangered, or critically endangered species or threatened ecological communities. 8

CHAPTER I :Bibliographic Review aforementioned activities in Douala pose serious threats to the environmental quality of the estuary and mangrove ecosystem. Therefore, the aim at this site was to evaluate the level of OCPs, PCBs, and PAHs in sediments of the WEM and evaluate the likelihood of adverse biological effects associated with sediment contamination.

Besides, chlorinated organophosphorus pesticides (COPPs) and Chloroacetamide

Herbicides (CAHs), for reasons of high environmental levels, high detection frequencies and extensive use in tropical areas

This dissertation consists of five chapters as follows:

CHAPTER 1 introduces Persistent Organic Pollutants and gives summary of the legislative framework that governs the management of POPs in Africa with emphasis on Cameroon. This was followed by account estimated production, import/export, usage of POPs worldwide and in Africa. A description of the selected of target compounds and their physico-chemical properties. A description of the assessment of ecological impacts of sediment contamination and health effects in humans. Then a review of POPs in aquatic environments of Africa.

CHAPTER 2 makes a comprehensive description of the two study sites, presenting their peculiarities and characteristics in terms of biodiversity, physical and geographical setting as rationale for studying them.

CHAPTER 3 gives a summary of the methodological approaches employed in the determination of POPs in aquatic environments of Africa. Followed by a description of the materials used and methodology employed in the determination of target compounds in samples from both study sites.

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CHAPTER 4 presents assessment of organochlorinated pesticides and polyaromatic hydrocarbons in water, soil and sediments of Lake Barombi watershed,

Southwest Region of Cameroon

CHAPTER 5 presents the assessment of organochlorinated pesticides, polychlorinated biphenyls and polyaromatic hydrocarbons in sediments of the Wouri

Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance.

This thesis is concluded with a highlight of the major findings of this work and prospective that may be addressed for future studies in the same region of the world or other aquatic environment worldwide. Lastly, a summary of this dissertation is presented in French.

This thesis produced 3 articles as follows:

PAPER 1: Persistent Organic Pollutants in Sediments of the Wouri Estuary

Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance (accepted for publication in Marine Pollution Bulletin on the 15 June 2020).

PAPER 2: A critical review of Persistent Organic Pollutants in Aquatic environments of Africa: Organochlorinated Pesticides, Polychlorobiphenyls and Polyaromatic

Hydrocarbons (to be submitted).

PAPER 3: The health status of Cameroon volcanic lakes: Assessment of

Organochlorinated Pesticides and Polyaromatic Hydrocarbon contamination of

Water, Soil and Sediments – The case of Barombi Mbo (to be submitted).

The first phase of this research on the Lake Barombi Watershed was carried out in the framework of the “CAMEROON POLLUTION” project funded by ECCOREV and extended to a second phase on the Wouri Estuary Mangrove. This work is a fruit 10

CHAPTER I :Bibliographic Review of the collaboration between Laboratoire de Chimie de l’Environnement (LCE) and

Centre Européen de Recherches et d’Enseignements en Geosciences de l’Environnement (CEREGE).

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CHAPTER 1: Bibliographic Review

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1.1. The Stockholm Convention on Persistent Organic Pollutants

Rachel Carlson in 1962, gave the first alert in her book entitled the

“silent spring” to the public on the detrimental effects related to the indiscriminate use of synthetic organochlorine pesticides such as DDT

(Dichlorodiphenyltrichloroethane) on non-target organisms (Carson, 1962). In an effort to protect human health and the environment from hazardous chemicals and wastes, 3 major multilateral agreements were put in place by international organizations (table 1). Firstly, the Basel convention adopted in 1989, in response to a public outcry following the discovery, in Africa and other parts of the developing world of deposits of toxic wastes imported from abroad in the 1980s (www.basel.int/).

This was following major international scandals related with hazardous waste trade; such as the transport of 8000 barrels of hazardous wastes including

Polychlorobiphenyls (PCBs) and toxic solvents from Italy to Nigeria in 1988. The dumping of a portion of toxic incinerator ash from a barge (transporting 14000 tons of toxic ash) in Haiti from Philadelphia (Westervelt and Beckham, 2015). Secondly, the Rotterdam Convention adopted in 1998, to promote shared responsibility and cooperative efforts among Parties in the international trade and contribute to the environmentally sound management of hazardous chemicals (www.pic.int/). Thirdly and most recently, the Stockholm Convention on Persistent Organic Pollutants

(POPs) in response to the urgent need for global action to protect human health and the environment (chm.pops.int/). All these international agreements had a converging conclusion on the fact that countries, lacking adequate infrastructure to monitor the import and use of such substances, are particularly vulnerable, in reference to developing nations and African countries in particular. 13

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Table 1. Major conventions prohibiting hazardous chemicals and wastes with signatories

and parties worldwide

Adoption Signatory Number Treaty/Agreement Chemicals (Entry into Force) states of parties chemical waste, Basel Convention: 22-03-1989 radioactive waste, Control of transboundary municipal solid waste, (5-05-1992) 53 187 movements of hazardous asbestos incinerator ash, wastes and their disposal and old tires Rotterdam Convention: 36 pesticides, 5 severely Prior informed consent hazardous pesticide 10-09-1998 procedure for certain formulations and 15 (24-02-2004) 72 161 hazardous chemicals and industrial chemicals pesticides in international trade 8 pesticides 2 industrial chemicals Stockholm Convention: 22-05-2001 2 By-products 152 182 Persistent organic pollutants (17-05-2004) 16 new POPs (2017) 3 chemicals under review

The Stockholm convention defines Persistent Organic Pollutants (POPs) as

chemical substances that are persistent in the environment, bioaccumulate along the

food chain, undergo long-range transport and pose a risk of adverse effects to human

health and the environment (Lambert et al., 2011). This convention initially

designated 12 POPs (table 2) known as the ‘’Dirty Dozen’’ in two major categories 1)

Intentionally produced chemicals; often used in agriculture, disease control,

manufacturing or industrial processes and 2) Unintentionally produced chemicals;

that result from some industrial processes and combustion. Intentionally produced

chemicals were classified in two groups; Pesticides (namely, aldrin, chlordane, DDT,

dieldrin, endrin, heptachlor, Hexachlorobenzene, mirex, toxaphene) and Industrial

chemicals (Hexachlorobenzene (HCB), Polychlorinated Biphenyls (PCBs) while

unintentionally produced chemicals belong to the group of by-products 14

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(Polychlorinated dibenzo-p-dioxins (PCDDs) and Polychlorinated dibenzofurans

(PCDFs), Hexachlorobenzene (HCB), Polychlorinated Biphenyls (PCBs) (OITA US

EPA, 2014). Prior to the Stockholm Convention, the Aarhus Protocol on POPs

(adoption 1998) listed Polyaromatic hydrocarbons (PAHs) as POPs. The United

States Environmental Protection Agency (US EPA) added PAHs to the list of US EPA

POPs (Wirnkor et al., 2019). It placed 16 PAHs on the priority pollutant list over the other PAH compounds (OITA US EPA, 2014). They were later also classified as

POPs with an assigned Toxic Equivalent Factor (TEF) or Relative potency

(www.eugris.info). The focal point of the Stockholm Convention was to eliminate or reduce the release of POPs in the environment. Currently, there are 16 chemicals or groups of chemicals added in 2017 to the initial list of POPs following the amendments in Annexes A, B, C and 3 chemicals under review (table 2).

Table 2. List of POPs regulated under the Stockholm Convention and chemicals under

revision

Chemical Group Annex

Aldrin P A

Chlordane P A

Dieldrin P A

Endrin P A

Heptachlor P A

Hexachlorobenzene P and I A

Mirex P A

Toxaphene P A

12 Initial POPs Polychlorobyphenyles (PCBs) I A

Dichlorodiphenyltrichloroethane (DDT) P B

Polychlorinated dibenzo-p-dioxins (PCDDs) and P C dibenzofurans (PCDFs)

Hexaclorobenzene U C

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Polychlorobyphenyles (PCBs) U C

Chemical Group Annex

Alpha HexachlorocycloHexane P A

BetaHexachlorocycloHexane P A

Chlordecone P A

Lindane P A

Pentachlorophenol and its salts and esters P A

Technical endosulfan and its related isomers P A

Decabromodiphenyl ether (Commercial mixture, c- I A DecaBDE)

Hexabromobiphenyl I A

Hexabromodiphenyl ether and heptabromodiphenyl I A ether (commercial octabromodiphenyl ether) 16 New 16 POPs New Perfluorooctane sulfonic acid (PFOS), its salts and I B perfluorooctane sulfonyl fluoride (PFOSF)

Short-chain chlorinated paraffins (SCCPs) I A

Tetrabromodiphenyl ether and pentabromodiphenyl I A ether (commercial pentabromodiphenyl ether)

Hexabromocyclododecane I and U A

Hexachlorobutadiene I and U A and C

Polychlorinated naphthalenes I and U A and C

Pentachlorobenzene P, I and U A and C

PerfluoroHexane sulfonic acid (PFHxS), its salts and I NI

PFHxS-related compounds

Dechlorane Plus I NI

Methoxychlor P NI Under revision

Groups: P = Pesticides, I = Industrial chemicals, U = Unintentionally produced, NI = No indication

Annex A: Parties must take measures to eliminate the production and use of the chemicals. Specific exemptions for use or production are listed in the Annex and apply only to Parties that register for them.

Annex B: Parties must take measures to restrict the production and use of the chemicals in light of any applicable acceptable purposes and/or specific exemptions listed in the Annex.

Annex C: Parties must take measures to reduce the unintentional release of chemicals with the goal of continuous minimization and, where feasible, ultimate elimination.

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1.2 Institutional and legislative framework of POPs in Africa

After the introduction of regulation process in the late 1980s in developing

countries including Africa, there has been expansion in terms of the number of

molecules and countries involved over the years as shown in table 3. Regulations in

African countries are mostly available for pesticides (OCPs) but very limited for PCBs

and almost inexistent for other POPs like PAHs, PBDEs, PCDDs and PCDFs. This

may be because pesticides have been used extensively for agriculture and public

health in Africa relative to other POPs. This could also be supported by the fact

Africa’s economy is inherently dependent on agriculture contributing over 32 % of the

continent’s Gross Domestic Product (GDP).

Table 3. Regional regulations on the import, use, disposal and management of POPs in

Africa

Date of Aim Regulation creation State(s) Reference Comment (Country, city)

Establishment Assessment and control of Marine Pollution in the Mediterranean 1975 Algeria, Responsible for the follow up work Egypt, related to the implementation of the MED POL Wagner et al., Morocco Land-Based Sources Protocol, the programme Adoption 1980 2013 and Protocol for the Protection of the Tunisia Mediterranean Sea against Pollution from Land-Based Sources/Activities Entry into force and of the dumping and Hazardous 1983 Wastes Protocols

signed 1991 29 to help achieve the objectives of the signatories Basel Convention Bamako entry into force UNEP, 2017a Convention in 1998 and 25 prohibition of importing any hazardous parties to and toxic (including radioactive) wastes (Mali,Bamako) date. into Africa Permanent Interstate An agreement was signed on a 13 Committee for Signed 1992 regulation governing the homologation Diarra, 1999 countries Drought Control in of pesticides the Sahel Southern Work and Health 2004 It is a collaborative research initiative African in Southern Africa covering community capacity-building Developme WASHA, 2009 (WAHSA) program and surveillance on pesticide toxicity nt 17

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(Botswana, Community and risk communication on hazardous Gaberone) (SADC) chemicals.

Date of creation Aim Regulation State(s) Reference (Country, city) Comment

to remove all obsolete pesticides, All African including Persistent Organic Pollutants www.fao.org/ African Stockpile countries (POPs), from the Africa continent over 2005 ag/obstocks.ht Programme (ASP) a period of 12 to 15 years, while also (54 m countries) helping countries prevent future accumulation

Adoption https://www.cp Comité Inter-Etats Central Coordination and harmonization of the ac- des Pesticides de 2007 African management of agricultural inputs in cemac.org/a- l’Afrique Centrale Tchad, Countries Central Africa propos/historiq (CPAC) Ndjamena ue/ https://www.cp Laboratoire Inter- Central Contribute to the sanitation of ac- Etats d’Analyse African agricultural production and science Adoption cemac.org/wp- des Pesticides de Countries development in terms of technical content/upload l’Afrique Centrale 2008 analyses of agrochemical products in (8 s/2018/06/CIP (LIEAP) the Central African sub-region countries) Special2.pdf Basel and Stockholm It aims to strengthen the capacity of its Conventions 2012 members in implementing the Regional Centre conventions in chemicals as waste chm.pops.int/ for African clusters by mobilizing different English-speaking South Africa academic and research institutions countries (BCRC- located in the member states SCRC)

Historically, in Kenya, the Public Health Act of the British government

legislation to protect human beings and regulate the use of pesticides by farmers in

was enacted in 1921 (Wandiga, 2001). Nowadays, many African countries have

established various laws, regulations and are signatories of most international

conventions relative to the management of chemical substances. For instance, a

National Implementation Plan (NIP) for the Stockholm Convention and Rotterdam

Convention National Action Plan have been drafted by all signatory African countries.

At national level, various institutions, decrees and orders have been put in place by

CHAPTER I :Bibliographic Review

most African countries to regulate the import, use and disposal of hazardous

chemicals (table 4).

Table 4. Regulatory measures implemented by some African countries on the import, use,

disposal and management of POPs in Africa

Date of Country Institution, project Aim, role, responsibility or task References creation Department of Agriculture, conduct/facilitate risk assessment for every chemical Forestry and Fisheries - before registration and records incidents relating to (DAAF) and Department of chemicals. Health (DoH) South Africa UNEP- administrative mechanism which provides a FAO, 2009 National Committee for foundation for the coordination of the activities Chemicals Management - required to fulfil the country’s obligations under the (NCCM) Rotterdam Convention and the Multi-stakeholder Committee for Chemicals Management (MCCM) national guidelines and standards for Environmental Federal Environmental 1991 Pollution Control regarding industrial effluents, FEPA, 1991 Protection Agency (FEPA) gaseous emission and hazardous wastes etc. Nigeria Its mission is to protect the public health by ensuring National Agency for Food the safety of drugs, food, chemicals, pesticides, Akpotaire, and Drugs Administration 1993 cosmetics and related products in both Nigeria and 2013 and Control (NAFDAC) Cameroun national body in charge of developing strategies for Egyptian Environmental El Zarka, 1982 environmental welfare including the management of Affairs Agency (EEAA) 1999 hazardous chemicals development of an integrated system for a more Egypt The Egyptian-German efficient hazardous waste management and a legal twinning project on 2008 to Wagner et framework on waste and hazardous substance hazardous substances and 2011 al., 2013 management that is harmonised with relevant EU waste regulations is in charge of the management of chemical products National Commission for tackled through the life cycle (Import, export, the Management of Senegal 2002 transport, production, storage, commercialization and Seck, 2016 Chemical Products use). It equally ensures the application of (CNGPC) international codes and conventions

Cameroon (at the junction of western and central Africa) is part of many

international and regional agreements and treaties on the management of hazardous

chemicals. In Cameroon, the Basel Convention was ratified on the 09th February

2001 and entered into force on the 10th May 2001; the Rotterdam Convention was

signed on the 11th September 1998, ratified on the 20th May 2002 and entered into

CHAPTER I :Bibliographic Review force on the 24th February 2004. The Stockholm Convention on POPs was signed on the 05th September 2001, ratified on the 19th May 2005 and entered into force on the

17th August 2009. The country adopted the Strategic Approach to International

Chemicals Management (SAICM) in 2006, that aims to foster the sound management of chemicals. At the national level various laws and regulations have been put forth in the management of hazardous chemicals and wastes (Table 5) but currently there is still no specific regulation regarding unintentionally produced POPs such as PAHs,

PCDDs and PCDFs (UNEP-POPS-NIP-Cameroon, 2016) or new POPs such as

PBDEs and SCCPs.

Table 5. Regulatory situation of POPs in Cameroon

Products Regulation in force

Prohibited by order N°0020/A/MINAGRI/DIRAGRI/SDPV/SDPV Dieldrin, Heptachlor of 07/01/1989

Mirex, DDT, Enrin, Aldrin, Prohibited by law N°90/013 of 10th August 1990, decree Chlordane, Toxaphene N°92/23/PM of 25th May 1992 and order Hexachlorobenzene N°0020/A/MINAGRI/DPA/SDPV/SPP/BCIP of 07/05/1998

Order n°020/A/MINAGRI/DPA/SDPV/SPP/BCIP of 7th May 1998 Non homologated Pesticides on the prohibition of some pesticides for agricultural use

Order n°057/05/A/MINADER/SG/DPA/SDPV/LAD of 22nd Lindane August 2005 on the prohibition of pesticide formulations composed of Lindane

Order n°071/08/D/MINADER/SG/DRCQ/SDRP/SRP of 17th July Endosulfan 2008 on the prohibition of some pesticides on cocoa

Polychlorobiphenyls, Prohibited by decree N°2011/2581/PM of 23 August 2011 on Chlordecone, the regulation of toxic and/or hazardous chemical substances Pentachlorobenzene

Source: UNEP-POPs-NIP-CAMEROON, 2016

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1.3. Import and usage of Persistent Organic Pollutants in Africa

Although the production of most POPs has been deliberate, there is uncertainty and poor knowledge on global and/or regional usage and total quantity entering the environment (Jacob and Cherian, 2013; Jones and de Voogt, 1999).

This might be due to unintentional production and the existence of new POPs.

However, more data is available for North America, Europe and China compared to

Africa. This data is of fundamental importance for effective source control measures, national/regional/global environmental inventories, budgets and models. Figure 1 illustrates the emission profile of POPs used in Europe and North America but may be unrepresentative of the global emission profile, especially when the chemical was used extensively out of these regions, especially in the tropics or following a global shift in the place of production (Jones and de Voogt, 1999). The same author states that for some new POPs like BFRs and chlorinated benzyltoluenes (used as PCB substitutes) a similar pattern may have been observed but more compressed in time, probably illustrating a better reactivity in restrictive regulatory policies at present time.

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Figure 1. A typical persistent organic pollutant time trend (Jones and de Voogt, 1999).

For the last thirty years, the export of agrarian and agro-industrial raw materials has raised the economic growth of Africa. Since the mid-1950s, synthetic pesticides have been used in Africa and their application has increased over the years in public health, agriculture and livestock development but the precise date of introduction is not known. Information of the quantities of POPs products imported and used in Africa is very scarce except for pesticides where little is known. However, in Africa, PCBs have been used in transformers, electrical equipments, ship painting and other industrial applications (Barakat et al., 2002). PAHs being ubiquitous compounds and one of the most widespread contaminants owing to their diverse origin (natural and anthropogenic origin), makes data acquisition very complex. The illegal movement of e-waste and some banned or unregistered hazardous pesticides from developed to developing nations banned under the Basel Convention and

Rotterdam convention were one of the major contributions to the presence of POPs in Africa (Gioia et al., 2014). For instance, the Basel Action Network (BAN) estimated

CHAPTER I :Bibliographic Review that about 45 % of e-wastes imports in Africa were from the European Union, 45 % from the U.S. and the remaining 10 % from Japan, Korea, Finland, Germany,

Norway, Netherlands, Italy and Singapore (Dave et al., 2016).

According to the FAO statistical database, in 2016 Africa imported pesticides for approximately 1 590 160.326 USD (FAOSTAT, 2018). The top 10 African countries by import value of pesticides in decreasing order are: Nigeria, South Africa,

Ghana, Ivory Coast, Egypt, Kenya, Cameroon, Tanzania, Ethiopia and Guinea (Guy

Bertrand, 2019). The same author indicates that, in 2011, the volume of pesticides imported in Ghana was 20.747 tons (9.216 tons of insecticides, 8986 tons of herbicides and 2.545 tons of fungicides) while in 2016, Egypt used over 10.600 tons of pesticides. Nowadays, most pesticides formulations imported in Africa are; glyphosate, paraquat, lambda-cyhalothrin, mancozeb, atrazine and Imidacloprid. The current absence of organochlorinated pesticides in African markets is obviously due to established regulations worldwide.

Cameroon is located between latitudes 1° and 13° N and longitudes 8° and

16° E. It is at the extreme north-eastern end of the Gulf of Guinea, bordering

Equatorial Guinea, Gabon, and Congo to the south; Nigeria to the west; the Central

African Republic and Chad to the east and a narrow portion of Lake Chad to the north. With a population of about 25 million inhabitants, it covers a surface area of

475.650 km2. It is one of the countries in Africa whose geographical position provides many opportunities for diverse agricultural production. There is no chemical industry in Cameroon and chemical products are mainly used in three sectors; i) the agricultural sector as inputs ii) industrial sector as raw materials and/or finished

CHAPTER I :Bibliographic Review products and iii) public health as disinfectant and/or medicinal products (MINEPDED-

UNITAR, 2013). However, these uses are often poorly controlled and inadequate.

Agriculture contributes to about 35 % of Cameroon’s Gross Domestic product

(GDP) and accounts for almost 70 % of the national labour force (Kimengsi and

Muluh, 2013). The growing threat of pests and diseases has led to a reduction in agricultural productivity. Souop, (2000) reported important crop losses like cocoa due to the black pod disease ranging from 30 to 100 %, coffee crop losses due to scolytids are 30 %, sorghum crop losses due to striga 100 %, cassava and yam crop losses caused by viral disease 80 to 100 % (Souop, 2000). Consequently, pesticide use is revealed as indispensable to improve agricultural productivity. As a result of growing industrialization and urbanisation, industrial pollution is becoming a serious problem in Cameroon. Lack and/or inadequate facilities for waste management coupled with poor enforcement of legislation, leads to uncontrolled discharge of industrial wastes into waterways (MINEPDED-UNITAR, 2013).

Between 1963 and 1989, most pesticides used were subsidized at 100 % by the state. The latter was responsible for purchase, storage and distribution of the majority of pesticides used. With the advent of the economic crisis in the 1980s, the state withdrew from the acquisition and distribution of phytosanitary products

(including pesticides) to focus on regulation and control. This gave rise to a liberalization of pesticide markets to the private sector (World Bank, 2007). Parastatal

Institutions such as; SODECAO and SODECOTON also bought, stored and used chemicals that have been approved by the government. In Africa, reliable official statistics on pesticide are scarce due to disorganized marketing, distribution and poor

CHAPTER I :Bibliographic Review legislative control. As a result, it is difficult to determine the types of market, their sizes and the fraction of pesticides in use.

In 2009, Cameroon imported 2898.4 tonnes (57 %) of pesticides from Europe,

196.5 tonnes (3.9 %) from Africa out of the CEMAC zone and 1986.5 tonnes from the rest of the world (MINADER/DESA/AGRI-STAT, 2012). The most abundant pesticides imported were Herbicides < Fungicides < Insecticides. The volume of pesticides imported in Cameroon and the use of phytosanitary products in the regions of Cameroun are shown in table 6.

Table 6. Pesticide import (tonnes) in 2009, in Cameroon from Europe, Africa out of CEMAC

zone and the rest of the world (through the Douala port)

Product Europe Africa out of CEMAC zone Rest of the World Insecticide 503.5 173.5 475.3 Herbicide 1063.6 23 1041.1 Fungicides 97.7 - 263.1 Nematicide - Insecticide 34.6 12 Domestique - Insecticide 42.7 157.2 Fungicide - Insecticide 132.5 21.8

Nematicide 24.3 - - Growth regulators 108.6 - 16 Molluscicide 15 - - TOTAL 2898.4 196.5 1986.5 Source: (MINADER/DESA/AGRI-STAT, 2012)

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In the framework of the African Stockpile Programme (2005), obsolete pesticide stocks of banned chemicals under the Stockholm Convention were identified in 7 out of the 10 regions of Cameroon (MINEPDED-UNITAR, 2013) as shown in table 7. It illustrates that the highest obsolete pesticide stock was for Lindane < Endosulfan < Dieldrin < DDT. The highest agricultural regions namely, the Far North, North West and West region recorded the highest quantity of obsolete pesticides representing 23.1, 20.8 and 17.7 % respectively. However, this data was not exhaustive considering the biased nature and time lag of some surveys.

Table 7. Distribution of obsolete pesticides (tonnes) in the regions of Cameroon

Regions

Far Nord Pesticides Centre East Littoral South West Total North West DDT NOP NOP NOP NOP 0.15 NOP NOP 0.15

Lindane NOP 0.4 NOP NOP 0.4 0.35 0.31 1.4

Dieldrin NOP 0.2 NOP NOP NOP NOP NOP 0.2

Endosulfan 0,3 NOP 0.2 0.15 0.2 NOP 0.15 1.0

Total 0.3 0.6 0.2 0.15 0.7 0.35 0.5 2.7

Percentage 12.4 23.1 6.8 5.8 20.8 13.4 17.7

Source: MINEPDED-UNITAR, 2013. NOP = No obsolete pesticide

In the framework of the National Implementation Plan (NIP) of the Stockholm

Convention on POPs launched in 2009, a preliminary PCB survey by colourimetry

(Analyser L 2000) carried out in 10 regions, showed that 16 companies in Cameroon owned equipment or materials (transformers, condensers, dielectric oils and liquids) contaminated with PCBs. These equipments were present in all regions, but located mainly in the Littoral (37 %), Centre (22 %) and Southwest (16 %) regions (UNEP-

POPS-NIP-Cameroon, 2016). According to the same report, in 2011 six alarming categories of sources of unintentional emission of POPs were identified: forest and 26

CHAPTER I :Bibliographic Review savanna fires (54 %), incineration of hospital wastes (18.3 %), dump fill and landfill fires (15.2 %), uncontrolled combustion of household waste and agricultural residues

(11 %). Despite the absence of a specific legislation dealing with e‑waste in

Cameroon, various existing laws can be read to impact on e‑waste (GISWatch,

2010). Nevertheless, sites of public disposal of industrial wastes are strongly suspected of being contaminated by polychlorinated biphenyls (PCBs) and other

POPs. Some of these sites include the Ngousso “open pit” (Yaounde), sites at

Makepe (Douala) and Nkolfoulou (Soa, Yaounde) (GISWatch, 2010).

1.4. Presentation of the Targeted Persistent Organic Pollutants

The main compounds examined in this work are the Organochlorinated pesticides (OCPs), the Polychlorinated Biphenyls (PCBs) and the Polyaromatic

Hydrocarbons (PAHs). They fall into each category of POPs regulated under the

Stockholm Convention. For reasons of high environmental levels, high detection frequencies and extensive use in tropical areas, chlorinated organophosphorus pesticides (COPPs) and Chloroacetamide Herbicides (CAHs) were equally studied.

1.4.1 Organochlorinated pesticides

Organochlorinated Pesticides (OCPs) are synthetic pesticides that belong to the group of chlorinated hydrocarbons derivatives (figure 2). They have been extensively used worldwide in agriculture and public health from 1940s to 1970s

(Jayaraj et al., 2016). According to the same author, 40 % of all pesticides used belong to the class of organochlorines. The most representative compounds include

CHAPTER I :Bibliographic Review

DDT, lindane, chlordane, dieldrin, methoxychlor, benzene Hexachloride, toxaphene, mirex and kepone. Dichlorodiphenyltrichloroethane (DDT) was the first synthetic organic pesticide and most popular chemical favoured for its low cost and broad- spectrum activity against insect pests of agriculture and human health (Zacharia and

Tano, 2011). DDT was extensively used as an agricultural insecticide on over 334 different agricultural crops and against 240 pests (Juc, 2006).

In 1955, the World Health Organisation (WHO) started malaria eradication programs in countries with low to moderate transmission rates worldwide, highly relying on DDT for mosquito control. The program eliminated the disease in parts of northern Africa, Taiwan, Caribbean, Balkans and northern region of Australia (Mendis et al., 2009) and later on used in other parts of the globe. Most OCPs are banned worldwide but are still used legally or illegally in some developing countries. For instance, in 2007 the WHO recommends the use of DDT only for indoor residual spraying in malaria endemic regions such as most countries in sub-Saharan Africa

(WHO/HTM/GMP, 2011).

OCPs have long half-lives ranging from about 60 days to 15 years. DDT

(insecticide) is highly hazardous and highly persistent with a half-life between 2 to 15 years while endosulfan (insecticide) is highly hazardous and moderately persistent with a half-life of about 50 days (Jayaraj et al., 2016). Endodulfan is one of the most abundant OCP in the global atmosphere (Weber et al., 2010). Previous studies in

Africa have revealed endosulfan to be one of the most abundant and frequently detected OCPs (Mohammed et al., 2017; Ibigbami et al., 2015) alongside with DDT and Hexachlorohexanes (HCHs) including lindane (ϒ-HCH). Organochlorine compounds were the first pesticides to be studied because of their widespread use, 28

CHAPTER I :Bibliographic Review persistence and effects on human health, but in the last twenty years there has been a significant increase in the use of less persistent compounds with greater acute toxicity such as organophosphorus insecticides (Aprea et al., 2000).

Cl Cl Cl

Cl Cl Cl Cl Cl Cl

Cl Cl Cl a-HCH b-HCH g-HCH Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl Cl d -HCH p-p' DDT Cl Cl Cl Cl

Cl Cl Cl Cl

p-p' DDE p-p' DDD

Figure 2. Chemical structures of targeted organochlorinated Pesticides

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Cl Cl Cl Cl Cl Cl

O Cl

Cl Cl endrin Cl aldrin Cl Cl

Cl Cl Cl H

H Cl H H Cl H Cl Cl H H Cl O O Cl Cl H H Cl endrin aldehyde H dieldrin

Cl Cl Cl H Cl Cl Cl Cl Cl

H Cl H Cl Cl H O Cl heptachlor heptachlor epoxide

Figure 2. Chemical structures of targeted organochlorinated pesticides

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Cl Cl Cl Cl Cl Cl Cl Cl

Cl Cl Cl O Cl O O a-endosulfan O -endosulfan S S b O O

Cl H Cl Cl Cl Cl Cl H Cl Cl Cl Cl Cl Cl O O S O Cl endosulfan sulfate H Cl O a-chlordane

Cl Cl Cl Cl

Cl Cl

g-chlordane Cl

Figure 2. Chemical structures of targeted organochlorinated pesticides

Organophosphorus Pesticides (OPPs) are synthetic esters, amides, or thiol derivatives of the phosphoric, phosphonic, phosphorothioic, or phosphonothioic acids (figure 3) (Stoytcheva and Zlatev, 2011). They are less persistent in the environment, not subject to bioaccumulation and biomagnification and release no 31

CHAPTER I :Bibliographic Review toxic by-products in the environment. In the 1930s, organophosphates were used as insecticides, but they were developed by the German military as neurotoxins in World

War II (Ghorab, 2015). OPPs are popular because of their broad spectrum of applications and potent toxicity to insects, their relative inexpensive costs, and their decreased likelihood for pest resistance (Barr et al., 2004). Some of the widely used pesticides include glyphosate, Malathion, chlorpyrifos, chlorfenvinphos.

Cl Cl S O CH3 P O O Cl N

Chlorpyriphos-ethyl CH3

Cl O O CH3 P O O

Cl Cl CH3 Chlorfenvinphos

Figure 3. Chemical structures of targeted chlorinated organophosphorus pesticides

Chlorpyrifos is a broad-spectrum, chlorinated organophosphate insecticide, acaricide and nematicide. Chlorpyrifos is the common name for the chemical 0,0- diethyl 0-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate. It was first registered as insecticide in 1965 in the US in agriculture and non-agricultural areas (Christensen et al., 2009). Since its registration, the US EPA has reviewed chlorpyrifos several

CHAPTER I :Bibliographic Review times for tolerance reassessment and reregistration (OCSPP US EPA, 2014).

Chlorfenvinphos can be described as an enol ester derived from dichloroacetophenone and diethylphosphonic acid. It has been widely used as insecticides and acaricide for controlling fleas and ticks on domestic pets and other animals. Chlorfenvinphos has been included in many products since its first use in

1963. More uses were registered between 1963 and 1970 as fly spray, larvicide and surface spray. In the early 1980s, chlorfenvinphos was registered for additional uses in a dust formulation for use in dog kennels and in dog collars for the control of fleas and ticks (EPA, 1995). It was commonly used until 1991 when all products containing chlorfenvinphos were cancelled in the US.

Chloroacetamide is a chlorinated aliphatic amide (figure 4), one of the most widely used groups of herbicides, characterized by an excellent efficiency against many annual grass weeds and certain, mainly small-seeded, dicotyledonous weeds in a variety of major crops, such as corn, cotton, rice, and soybean (Lamberth, 2016).

They are mainly used in preemergent and early post emergent treatment.

Chloroacetamide have been equally used as preservative in cosmetic products.

Chloroacetamide herbicides (CAHs) were discovered in the early 1950s at Monsanto, where their severe inhibition to germinating seeds were found at relatively higher rates but weakly active at lower use rates (Hamm, 1974). A short time later, the selectivity of these herbicides was discovered, when it was observed that N- (2,5- dichlorophenyl) chloroacetamide strongly prevented the grass seed germination with little or no effect against broadleaf seeds, marking the birth of Chloroacetamide

Herbicides (Lamberth, 2016). This period matched with an acute need for annual grass control over 60 years ago.

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In 1962, alachlor was commercialized and it was the first herbicide of this group which was not irritating and efficient in all kinds of soils for the reliable preemergent broad-spectrum control of both grasses and broad-leaved weeds in corn and soybeans (Hamm, 1974). The two most important chloroacetamide herbicides, metolachlor and acetochlor were launched in the market 1976 and 1994 respectively. In 1996, the chiral switch of metolachlor by its active principle (S)- metolachlor, significantly reduced the chemical load to the environment (Lamberth,

2016).

In 2012, France through the French National Agency for the Safety of

Medicines and Health products (ANSM) banned the production, import, export and placing in the market of cosmetics containing chloroacetamide. According to this agency, chloroacetamide was found to have harmful effects on human fertility

(reprotoxic). In 2016, following a consultation of the Scientific Committee on

Consumer Safety (SCCS), decided to prohibit all substances (cosmeticsdesign- europe.com, 2016). The use of chloroacetamide was prohibited as pesticide, cosmetics and personal care products since 2009 in Canada.

OCH3

O N Cl

N O Cl

alachlor metolachlor

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Figure 4. Chemical structures of the targeted chloroacetamide herbicides

1.4.2 Polychlorinated Biphenyls

Polychlorinated Biphenyls (PCBs) are a group of synthetic chemical compounds in which 2 to 10 chlorine atoms are attached to the biphenyl molecule

(figure 5). Over 209 different polychlorinated biphenyls are possible, differing from each other by substitution position and level of chlorination. PCBs are included on the OSPAR List of Chemicals for Priority Action due to their toxicity, persistence, potential to bioaccumulate. The analysis of the International Council for the

Exploration of the Sea (ICES) 7 PCBs (Indicator PCBS) in sediments and Biota is a mandatory requirement of the OSPAR Coordinated Environmental Monitoring

Program (CEMP) since 1998 (Webster et al., 2013). There is very limited information on specific congener toxicity because toxicity testing has been carried out on specific commercial mixtures known as Aroclors, identified by a four-digit numbering such that the first two digits indicate the product series or type of mixture designated by

Monsanto while the last two digits indicate the percentage of chlorine by mass in the mixture.

PCBs were mainly produced from 1930 to 1977, under the trademark name

Aroclor by Monsanto Corporation in the U.S. Different Aroclors were used at different periods for different applications. Before the 1950’s, Aroclor 1260 and 1254 were the main mixtures used in the manufacturing of electrical equipments. Between 1950 and

1960’s the main mixture used was Aroclor 1242 until phased out in 1971 replaced by

Aroclor 1016. Between 1957 and 1970, 12 different types of Aroclors with chlorine atoms ranging from 21 to 60 % were produced in the U.S. The production of PCBs

CHAPTER I :Bibliographic Review in the U.S. was stopped in August 1977, but before that 99 % of PCBs used in the

U.S. industries were produced by Monsanto Company.

The global production of PCBs from their first synthesis in the 1920 has been estimated in the order of 106 tonnes but information on usage is very limited. The production and use of PCBs mainly occurred in the US, Europe, and Russia, and approximately 97 % of this was used in the Northern Hemisphere (Gioia et al., 2014).

About 10 000 tons of PCBs were produced In China, from 1965 to 1975, known as

PCB3 and PCB5. About 10 % of this cumulative tonnage was employed as additives to paint, with the overwhelming majority being utilized in electrical industry (as dielectric fluid in capacitors and transformers) and in carbon-free copy papers (Yang et al., 1997; Jing et al., 1992). Their use was banned in 1980s. However, even today, a large proportion of PCB-containing old transformers and capacitors still remain in use in China, which resulted in continued environmental input (from both deliberate and accidental sources) and as a pool of PCBs remaining in many environment compartments.

3 2 2' 3'

4 4'

n(Cl) (Cl) 5 6 6' 5' n

Figure 5. General formula of polychlorinated biphenyls

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Cl Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl Cl PCB 52 Cl PCB 101 PCB 118 PCB 28

Cl Cl Cl

Cl Cl Cl Cl Cl Cl Cl PCB 138 PCB 153 PCB 180

Figure 6. Chemical structures of the targeted polychlorinated biphenyls

1.4.3 Polyaromatic hydrocarbons

Polyaromatic hydrocarbons are a class of organic compounds comprised of two or more benzene rings bonded in linear, cluster, or angular arrangements (figure

7a and b) with 2 or more fused carbon rings that have substituted groups attached

(Adeniji et al., 2018). They are rapidly adsorbed onto particles and show a high environmental persistence and bioaccumulation potential (Rocher et al., 2006).

About 660 parent PAH compounds (solely made up of fused aromatic rings without 37

CHAPTER I :Bibliographic Review alkyl groups) have been listed by (Sander and Wise, 1997). Far in the 18th century, a higher rate of skin cancer was observed among roofers, who were exposed to sooth.

In 1947, the relationship between lung cancer and working conditions of gas industry workers and those working with coal tar was established (IARC, 2010). It was found then that induction of cancer was caused by PAHs present in coal tar and soot (IARC,

2010). In 1983, the International Agency for Research on Cancer acknowledged 30

PAHs as carcinogenic to humans. In 1997, the U.S. EPA defined 16 PAHs to be highly toxic and recommended the analysis of their concentration. They have been classified among the most hazardous and persistent organic pollutants by the US

EPA, Agency of Toxic Substances and Disease Register (ATSDR), International

Agency for Research on Cancer (IARC) and the European Commission (EC). Many studies have examined the 16 priority U.S. EPA PAHs due to their recognized toxicity by the scientific community in the late 1960s and early 70s (Vera Samburova et al.,

2017). These include; Naphthalene (NA), Acenaphthylene (ACY), Acenaphthene

(ACE), Fluorene (F), Phenanthrene (PHE), Anthracene (ANT), Fluoranthene (Fl),

Pyrene (PYR), Benzo(a)Anthracene (BaA), Chrysene (CHRY),

Benzo(b)fluoranthene (B(b)Fl), Benzo(k)fluoranthene (B(k)Fl), Benzo(a)pyrene

(BaP), Dibenzo(a,h)anthracene (DBA), Benzo(g,h,i)perylene (B(g,h,i)P),

Indeno(1,2,3-cd)pyrene (IP).

PAHs originate from anthropogenic or natural sources. Those originating from natural sources can either be from the chemical or biological transformation of natural organic matter, biological processes or natural sources such as oils seeps from crude oil deposits, forest fires, erosion of ancient sediments and volcanoes (Stogiannidis and Laane, 2015). PAHs of anthropic origin are formed either by incomplete

CHAPTER I :Bibliographic Review combustion or thermal alteration of organic matter (Budzinski et al., 1997). However, anthropogenic emissions predominate over natural emissions through the use of petroleum products, incomplete combustion of fossil fuels, biofuels or other forms of organic matter (Abdel-Shafy and Mansour, 2016; Stogiannidis and Laane, 2015).

PAHs can be classified according to their temperature of formation or their origin. Pyrogenic PAHs originating from different pyrolysis substrates such as fossil fuels; petrogenic PAHs from petroleum-related sources and natural PAHs of biogenic or diagenetic origin (Boehm and Page, 2007). Pyrogenic PAHs form when fuels and other organic matter are incompletely or inefficiently combusted or pyrolyzed at moderate to high temperatures (> 400°C) over very short time intervals, while

Petrogenic PAHs are formed by the geochemical alteration of organic matter at moderate temperature (50 - 150°C) and pressure over very long (geologic) timescales.

According to their molecular weights, the 16 PAHs have been classified into 2 groups; Low Molecular Weight PAHs (LPAHs) from 2 to 3 rings (Naphthalene (NA),

Acenaphthylene, Acenaphthene (ACE), Fluorene, Phenanthrene and High

Molecular Weight PAHs (HPAHs) with 4 to 6 rings (Anthracene, Fluoranthene,

Pyrene, Benzo(a)Anthracene, Chrysene, Benzo(b)fluoranthene,

Benzo(k)fluoranthene, Benzo(a)pyrene, Dibenzo(a,h)anthracene,

Benzo(g,h,i)perylene, Indeno(1,2,3-cd)pyrene). Major anthropogenic sources of

PAHs include residential heating, coal gasification and liquefying plants, carbon black, coal-tar pitch and asphalt production, coke and aluminium production, catalytic cracking towers and related activities in petroleum refineries as well as and motor vehicle exhaust (Abdel-Shafy and Mansour, 2016). 39

CHAPTER I :Bibliographic Review

Acenaphtene

Naphtalene Acenaphtylene

Phenanthrene Fluorene

Pyrene Anthracene

Fluroanthene Benzo(a)pyrene

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Figure 7. The 16 priority polyaromatic hydrocarbons designated by the US EPA

Chrysene Benzo(a)anthracene

Benzo(b)fluoranthene Benzo(k)fluoranthene

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Dibenzo(a,h)anthracene Benzo(g,h,i)perylene

Indeno(1,2,3-cd)pyrene

Figure 7. The 16 priority polyaromatic hydrocarbons designated by the US EPA (continued)

1.5. The Physical-Chemical properties of target POPs

The physical and chemical properties of organic chemicals are accountable for major differences between their behaviour in the environment. A large amount of environmental physical and chemical data on organic chemicals have been compiled and reported by literature (Mackay et al., 2006) and the US EPA. There are various properties that control their persistence in the environment, the phases in which they will preferentially concentrate (dissolved, gaseous and solid) and bioaccumulate along the food chain. This work focuses on the key physical and chemical properties that control the fate of POPs as they are transported in different environmental

CHAPTER I :Bibliographic Review compartments (biota, water, air, soils and sediments). Despite the significant variation of environmental temperatures, values of the physical and chemical properties in literature are commonly reported at standard temperature of 20 or 25

°C (Mackay et al., 2006).

1.5.1. Vapour pressure and solubility in water

Vapour pressure (VP) and solubility in water (SW) are known as “saturation” properties. They are measurements of the maximum capacity that a fluid has for dissolved chemical. Vapor pressure P (Pa) can be viewed as a “solubility in air,” the corresponding concentration C (mol/m3) being P/RT where R is the ideal gas constant (8.314 J/mol.K) and T is absolute temperature (K). Although most chemicals are present in the environment at concentrations well below saturation, these concentrations are useful for estimating air-water partition coefficients as ratios of saturation values. It is usually assumed that the same partition coefficient applies at lower sub-saturation concentrations. Therefore, vapor pressure and solubility provide estimates of the air-water partition coefficient KAW, the dimensionless ratio of concentration in air (mass/volume) to that in water.

SW for PAHs are higher than those of OCPs and PCBs and decrease with increasing number of aromatic rings or molecular weight. This indicates that low molecular weight (LMW) compounds will be predominant in the dissolved phase while high molecular weight (HMW) will preferentially accumulate in suspended solid matter (SSM) and sediments. Due to the high lipophilic nature of PCBs, OCPs and

CHAPTER I :Bibliographic Review

HMW PAHs, their dissolved phases generally represent a smaller fraction of the total

PCB, OCP and PAH fractions respectively (Net-David, 2016).

1.5.2. Henry’s law constant

Henry’s law constant H (Pa.m3/mol) is the ratio of partial pressure in air (Pa) to the concentration in water (mol/m3). Both express the relative air-water partitioning tendency. When solubility and vapor pressure are both low in magnitude, it is preferable to measure Henry’s law constant directly.

1.5.3. Octanol-water, octanol-air and organic carbon-water partition coefficients

The octanol-water partition coefficient (KOW) provides a direct estimate of hydrophobicity/lipophilicity or of partitioning tendency from water to organic media such as lipids, waxes and natural organic matter such as humin or humic acid. It is critical as a method of estimating organic carbon-water partition coefficient (KOC), the usual correlation invoked being that of (Karickhoff, 1981):

KOC = 0.41 KOW ………………………… Equation 1

(Seth et al., 1999) suggested that a better correlation is

KOC = 0.35 KOW ………………………… Equation 2

The error limits on KOC, resulting from differences in the nature of organic matter, are a factor of 2.5 in both directions, i.e. the coefficient 0.35 may vary from

0.14 to 0.88. KOC is an important parameter, which describes the potential for movement or mobility of pesticides in soil, sediment and groundwater. Owing to the 44

CHAPTER I :Bibliographic Review structural complexity of the agrochemical molecules studied, the above simple relationship which considers only the chemical’s hydrophobicity may fail for polar and ionic compounds. The effects of pH, soil properties, mineral surfaces and other factors influencing sorption become important. Other quantities, KD (sorption partition coefficient to the whole soil on a dry weight basis) and KOM (organic matter-water partition coefficient) are also commonly used to describe the extent of sorption. KOM is often estimated as 0.56 KOC, implying that organic matter is 56 % carbon. KOW is also used to estimate equilibrium fish-water bioconcentration factor KB, or BCF using a correlation similar to that of (Mackay, 1982)

KB = 0.05 KOW ………………………… Equation 3 where the term 0.05 corresponds to a lipid content of the fish of 5%.

For OCPs, PCBs and PAHs the Log KOW and Log KOC increase with increasing number of cycles or molecular weights, suggesting that POPs with high molecular weights are preferentially adsorbed on solid matrices such as suspended matter, sediments, soil and aquatic organisms, making them stable in the environment (Net-

David, 2016). Thus, making these compounds ubiquitous in rivers and coastal environments. (Rabodonirina et al., 2015) reported strong correlations between distributions of PAHs, PCBs and Log KOW or Log KOC.

1.5.4. Chemical reactivity and half-lives

The characterization of chemical reactivity generally presents a challenging problem in environmental science. While radioisotopes have fixed half-lives, the half- life of a chemical in the environment depends not only on the intrinsic properties of

CHAPTER I :Bibliographic Review the chemical, but also on the nature of the environmental compartments. Factors such as sunlight intensity, hydroxyl radical concentration and the nature of the microbial community, as well as temperature, affect the chemical’s half-life so it is impossible (and misleading) to document a single reliable half-life. (Mackay et al.,

2006) proposed that the best approach is to suggest a semi-quantitative classification of half-lives into groups or ranges, assuming average environmental conditions to apply (table 8). Obviously, a different class will generally apply between compartments such as in air and bottom sediment. The half-lives apply to the reaction rates of parent compounds, metabolites have different properties and require a different assessment. The classes in this compilation were used for chemical reactivity in a single medium such as water. These times were divided logarithmically with a factor of approximately 3 between adjacent classes and with the state of knowledge it was probably misleading to divide the classes into minor groupings; in fact, a single chemical is likely to experience half-lives ranging over three classes, depending on season (Mackay et al., 2006). These half-lives apply to the reaction of the parent substance (Table 8).

Table 8. Half-life classes of PAHs, PCBs and Pesticides (Mackay et al., 2006)

Class Mean half-life (hours) Range (hours)

1 5 < 10 2 17 (~ 1 day) 10–30 3 55 (~ 2 days) 30–100 4 170 (~ 1 week) 100–300 5 550 (~ 3 weeks) 300–1,000 6 1700 (~ 2 months) 1,000–3,000 7 5500 (~ 8 months) 3,000–10,000

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8 17000 (~ 2 years) 10,000–30,00 9 55000 (~ 6 years) > 30,000

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Table 9. Summary of physical-chemical properties for chlorinated pesticides (Mackay et al., 2006)

Molecular Sol. Log Henry’s Molecular MP VP Log Compound Weight (g/m3) KOC Law Constant Half-life Class at 25 °C formula S KOW (°C) (P /Pa) (g/mol) (H/Pa.m3/mol) Air Water Soil Sed.

–3 –2 Aldrin C12H8Cl6 364.9 104 5 x 10 2 x 10 3.01 2.61 91.2 4 8 8 9

–4 –2 p,p’-DDD C14H10Cl4 320.0 109.5 1.3 x 10 5 x 10 5.5 5.0 0.6 - - - -

–4 –2 p,p’-DDE C14H8Cl4 318.0 89 8.6 x 10 4 x 10 5.7 5.0 7.9 4 9 9 9

–5 –3 p,p’-DDT C14H9Cl5 354.4 108.5 2.0 x 10 5.5 x 10 6.19 5.4 2.3 4 7 8 9

–4 –1 Dieldrin C12H8Cl6O 380.9 175.5 5 x 10 2 x 10 5.20 4.08 1.1 4 8 8 9

–5 –1 Endrin C12H8Cl6O 380.9 - 2 x 10 2 x 10 5.2 4 0.03 - - - -

–3 α-HCH C6H6Cl6 290.8 158 3 x 10 1 3.81 3.81 0.87 - - - -

–5 β-HCH C6H6Cl6 290.8 309 4 x 10 0.1 3.8 3.36 0.116 - - - -

–3 γ-HCH C6H6Cl6 290.8 112.5 3.7 x 10 7.3 3.7 3.0 0.149 5 8 8 9

–3 δ HCH C6H6Cl6 290.8 141.5 2 x 10 8 4.14 - 0.0727

–3 –2 Heptachlor C10H5Cl7 373.3 95.5 5.3 x 10 5 x 10 5.27 4.38 353.4 3 5 6 7

–1 Heptachlor Epoxide C10H5Cl7O 389.3 160 - 3.5 x 10 5.0 4.0 -

–4 –2 α-Chlordane C10H6Cl8 409.7 131 4 x 10 5 x 10 6.0 5.5 0.342 4 8 8 9

–4 –2 γ-Chlordane C10H6Cl8 409.7 106 5.2 x 10 5.6 x 10 6.0 5.5 0.262 4 8 8 9

–3 –1 Endosulfan C9H6Cl6O3S 406.9 106 1.3 x 10 5 x 10 3.6 4.09 - - - - -

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–3 –1 α-Endosulfan C9H6Cl6O3S 406.9 106 1.3 x 10 5 x 10 3.62 3.4 - - - - -

–3 –1 β-Endosulfan C9H6Cl6O3S 406.9 208 6.1 x 10 4.5 x 10 3.83 3.5 - - - - -

Molecular Sol. Log Henry’s MP VP Molecular Weight 3 Log KOC Compound (g/m ) Law Constant Half-life Class at 25 °C formula (°C) (PS/Pa) KOW (g/mol) 3 (H/Pa.m /mol) Air Water Soil Sed.

Endosulfan sulfate C9H6Cl6O4S 422.9 181 ------

–3 Alachlor C14H20ClNO2 269.7 40 2.0 x 10 240 2.8 2.23 0.0022 1 8 6 6

–3 –3 Metolachlor C15H22ClNO2 283.7 liquid 4.2 x 10 430 3.13 2.26 2.33 x 10 4 6 6 7

–3 Chlorpyriphos C9H11Cl3NO3PS 350.5 42 2.2 x 10 0.73 4.92 3.78 1.09 2 4 4 6

–4 –4 Chlorfenvinphos C12H14Cl3O4P 359.5 - 1.0 x 10 124 3.82 2.47 2.90 x 10 - - - -

p,p’-DDD = 1,1-Dichloro-2,2-bis (4-chlorophenyl)ethane, p,p’-DDE = 1,1-dichloro-2,2-bis-(p-chlorophenyl)-ethylene , p,p’-DDT = 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)-ethane, α-HCH = alpha-HexachlorocycloHexane, β-HCH = Beta-HexachlorocycloHexane, γ-HCH = Gamma-HexachlorocycloHexane, δ HCH = Delta-Hexachlorocyclohexane, M.P =

Melting point, BP = Boiling point, VP = Vapour pressure, Sol. = Solubility, KOW = octanol-water partition coefficient, Log KOC = organic carbon-water partition coefficient

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Table 10. Summary of physical-chemical properties for polychlorinated biphenyls (Mackay et al., 2006)

Henry’s Half-life class at 25 °C IUPAC M.W. BP MP VP Sol. Congener Log KOW Law Constant No. S 3 (g/mol) (°C) (°C) (P /Pa) (g/m ) Air Water Soil Sed. (H/Pa.m3/mol) 28 2,4,4’ 257.5 337 57 - 1.6 x 10–1 5.8 - 5 8 9 9 52 2,2’,5,5’ 291.9 360 87 4.9 x 10–4 3 x 10–2 6.1 47.69 6 9 9 9

101 2,2’,4,5,5’ 326.4 381 78 1.1 x 10–4 1 x 10–2 6.4 35.58 6 9 9 9

118 2,2’,4,4,5’ 326.4 381 107 3.x 10–4 – 9.3 x 10–3 4 x 10–3 – 2 x 10–2 6.2-6.50 24.8-151.4 6 9 9 9

138 2,2’,3,4,4’,5’ 360.8 400 80 2 x 10–5 – 1.6 x 10–3 4 x 10–4 – 7.4 x 10–4 6.7-7.30 11.9-818 7 9 9 9

153 2,2’,4,4’,5,5’ 360.8 400 103 1.19 x 10–4 1 x 10–3 6.9 42.94 7 9 9 9

180 2,2’,3,4,4’,5,5’ 395.323 417 110 2.7 x 10–5 4.5 x 10–5 – 2 x 10– 6.7-7.0 5.40 7 9 9 9 4

IUPAC = International Union of Pure and Applied Chemistry, M.P = Melting point, BP = Boiling point, VP = Vapour pressure, Sol. = Solubility, KOW = octanol-water partition coefficient

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Table 11. Summary of physical-chemical properties for polyaromatic hydrocarbons (Mackay et al., 2006)

Henry’s Half-life class at 25 °C N° MW BP MP VP Sol. Log Molecular Law Constant Compound rings (g/mol) (°C) (°C) (PS/Pa) (g/m3) KOW formula (H/Pa.m3/mol) Air Water Soil Sed.

NA 2 C10H8 128.1 217.9 80.26 10.4 31 3.37 43.00 2 4 6 7

ACY 3 C12H8 150.1 280 91.8 0.9 16.1 4.00 8.396 - 5 7 8

ACE 3 C12H10 154.2 279 93.4 0.3 3.80 3.92 12.17 3 5 7 8

F 3 C13H10 166.2 295 114.77 0.09 1.90 4.18 7.873 3 5 7 8 LMW

PHE 3 C14H10 178.2 340 99.24 0.02 1.10 4.57 3.240 3 5 7 8

ANT 3 C14H10 178.2 339.9 215.76 0.001 0.045 4.54 3.961 3 5 7 8

FL 4 C16H10 202.2 384 110.19 0.00123 0.26 5.22 0.957 4 6 8 9

PYR 4 C16H10 202.2 404 150.62 0.0006 0.132 5.18 0.919 4 6 8 9

–5 BaA 4 C18H12 228.2 438 160.5 2.80 x 10 0.011 5.91 0.581 4 6 8 9

–7 CHRY 4 C18H12 228.2 448 255.5 5.70 x 10 0.002 5.60 0.065 4 6 8 9

BaFL 5 C20H12 252.3 481 168 - 0.0015 5.80 - 4 6 8 9

–8 BkFL 5 C20H12 252.3 480 217 5. x 10 0.0008 6.00 0.016 4 6 8 9 HMW

–7 BaP 5 C20H12 252.3 495 181.1 7.00 x 10 0.0038 6.04 0.046 4 6 8 9

–10 –4 DA 5 C22H14 278.3 524 269.5 3.70 x 10 0.0006 6.75 1.72 x 10 4 6 8 9

BgP 6 C22H12 276.330 - 272.5 - 0.00026 6.50 - - - - _

IP 6 C22H12 276.330 - 162 ------LMW=Low Molecular Weight, HMW=High Molecular weight, Abb.= Abbreviation, ACE= Acenaphthene, F= Fluorene, PHE= Phenanthrene, ANT= Anthracene, Fl= Fluoranthene, PYR= Pyrene, BaA= Benzoa)Anthracene, CHRY= Chrysene, B(b)Fl= Benzo(b)fluoranthene, B(k)Fl= Benzo(k)fluoranthene, BaP= Benzo(a)pyrene, DBA=

CHAPTER I :Bibliographic Review

Dibenzo(a,h)anthracene, B(g,h,i)P = Benzo(g,h,i)pyrelene, IP= Indeno(1,2,3-cd)pyrene, MP = Melting point, BP q= Boiling point, VP = Vapour pressure, Sol. = Solubility KOW = octanol-water partition coefficient

CHAPTER I :Bibliographic Review

1.6 Ecotoxicological impact of sediment contaminants (OCPs, PCBs and PAHs)

Since the early 1970s, the protection of aquatic resources has been a major concern that has raised public awareness. There has been a need to protect fresh water and marine ecosystems from pesticide runoff, pharmaceutical wastes, oil spills and various types of plastic wastes. The restriction of chemicals from occurring in water at concentrations above the known “safe” chronic concentrations is the fundamental premise that has been used to protect water quality (Adams et al.,

1992). This approach has equally been considered for aquatic sediments.

Historically, several assumptions have been made or implied in the development and application of environmental safety assessments and water quality criteria. These assessments were solely based on the factor that introduced chemicals were generally water soluble, limiting impact analysis to the water column (pelagic zone)

(Dickson et al., 1987). According to the same author, following the development of the Priority Pollutant List in 1976, including a wide range of organic and inorganic chemicals, investigation revealed that they were water insoluble. This introduced the second factor; the “irreversibility” of chemicals sorbed to sediments, including terms like bioavailability and portioning as keywords of environmental assessment.

Sediment quality assessment is quite more complex than water quality assessment because it considers site-specific parameters that do not apply to water such as sediment characteristics; deposition, erosion and compaction; bioavailability; sorption kinetics and bioturbation (Adams et al., 1992).

The need to develop sediment assessment approaches arose from the acknowledgement that freshwater and marine sediments in the United States were

CHAPTER I :Bibliographic Review contaminated with different levels of organic and inorganic chemicals. The US EPA is one of the first governmental agencies to produce reports on Water and Sediment

Quality Criteria since the early 1970s. The most commonly reported chemicals included polyaromatic hydrocarbons, some pesticides and chlorinated organics

(Adams et al., 1992) because they have very long decay half-lives leading to high levels of contamination over long periods of time.

In this work, emphasis would be laid on aquatic sediment quality. This is because hydrophobic organics have an affinity for particulate matter when they enter aquatic ecosystems, acting as repositories for physical debris and “sinks” for various chemicals (Ntow, 2005). Aquatic sediments provide a pathway for the consumption of these chemicals by higher aquatic life, wildlife and humans (Adams et al., 1992).

According to the same author, sediments are considered a major route of exposure for numerous aquatic species, providing a substrate for many economically important species such as fish, shrimp, crabs, mussels, lobsters and crayfish. Aquatic sediments (top 10 to 20 cm) represent a relatively small volume of the entire aquatic ecosystem, and thus high concentrations of sediment-bound chemicals tend to be found on a relatively localized basis (Dickson et al., 1987). One of the primary efforts that addressed the emerging technical and regulatory issues on sediments was a

“Pellston” workshop in 1984 on the “Fate and Effects of sediment-Bound Chemicals in Aquatic systems”.

Over the past 40 years several methods have been developed for evaluating the environmental safety of aquatic sediments. Before the 1980s, the level of sediment contamination was evaluated by comparing the bulk chemical concentration in a sediment sample to background or reference values. However, 54

CHAPTER I :Bibliographic Review this approach provided limited insight into the ecosystem impact of sediment contaminants. Since then, Sediment Quality Guidelines (SQGs) have tried to integrate biological response/effects in their derivation approach (Burton, 2002).

Various empirical and theoretical methods have been employed to derive SQGs.

i) Empirical approach

1. Empirical methods permit to establish the relationship between sediment and toxic effects in order to develop effects-based Sediment quality Guidelines. The recent generation of empirically SQGs was developed based on the analyses of matching field sediment chemistry coupled with field or biological effects data (Long et al., 2006; Burton, 2002). They are mostly based on frequency distribution and suitable for prediction of biological effects in most marine and freshwater ecosystems. The empirically based approaches have made use of large data sets, that are continuously expanded. The method of determination of threshold effects for each approach differs, but most of them are similar.

These approaches include the Effects range (Ingersoll et al., 1996; Long and

Morgan, 1991), Effect level (Ingersoll et al., 1996; Smith et al., 1996) and Apparent

Effects threshold (Cubbage et al., 1997) and Screening Level concentration (Persaud et al., 1993). Generally, two threshold levels were set for each approach as shown in table 12 and table 13, one below which effects toxic effects will occur (the lowest effect level (LEL), threshold effect level (TEL), effects range low (ERL), minimal effect threshold (MET), and threshold effect concentration (TEC)), and one above which toxic effects are likely to occur (the severe effect level (SEL), probable effects level

CHAPTER I :Bibliographic Review

(PEL), effect range median (ERM), toxic effect threshold (TET), and probable effect concentration (PEC).

The apparent threshold Effect Approach is based on finding the sediment concentration above which significant biological effects will always occur. The main advantage of this approach is that it assumes a direct cause-effect relationship between sediment concentrations of a contaminant and the occurrence of significant biological effects without no assumption of contaminant availability (Persaud et al.,

1993).

The ERLs and ERMs were set at the lower 10 percentiles and 50 percentiles

(Median) in the data distribution respectively. This was derived from the matching of synoptically collected sediment chemistry and bioassay data from marine and estuarine sediments through the United States (about 200 samples) by the National

Oceanic and Atmospheric Administration (NOAA) in the National Status and Trends

(NS&T) Program (Long and Morgan, 1991).

The TEL and PEL were determined by calculating the geometric mean of the

15th percentile and 20th percentile of the no effect data set respectively from chemical and biological data from freshwater sediment throughout North America. These two thresholds delineate three ranges of chemical concentrations, a minimal effect range

(concentrations equal to and below the TEL), a possible effect range (concentrations above TEL but below PEL), and a probable effect range (concentrations above PEL); rarely, occasionally and frequently associated with adverse biological effects (Smith et al., 1996). TEL and PEL are quite similar to ERM and ERL values.

CHAPTER I :Bibliographic Review

Table 8. Summary of sediment quality guidelines for PAHs in aquatic ecosystems that

reflect TECs (concentrations below which adverse biological effects are unlikely to be

observed)

Threshold Effect Concentrations Probable Effect Concentrations Compound ERL TEL LEL MET CB TEC ERM PEL CB PEC SEL Acenaphthene 16 NG NG NG NG 500 NG NG NG Acenaphthylene 44 NG NG NG NG 640 NG NG NG Anthracene 85.3 NG 220 NG 57 1100 NG NG NG Fluorene 19 NG 190 NG 77.4 540 NG NG NG Naphthalene 160 NG NG 400 176 2100 NG NG NG Phenanthrene 240 41.9 560 400 204 1500 515 1170 9500 LPAHs 552 NG 320 NG NG 3160 NG NG NG B(a)Anthracene 261 31.7 370 400 108 1600 385 1050 14800 Benzo(b)fluoranthene NG NG NG NG NG NG NG NG NG Benzo(k)fluoranthene NG NG NG NG NG NG NG NG NG Benzo(a)pyrene 430 31.9 370 500 150 1600 782 1450 14400 Dibenzo(a,h)anthracene 63.4 NG 60 NG 33.0 260 NG NG NG Chrysene 384 57.1 340 600 166 2800 862 1290 4600 Fluoranthene 600 111 750 600 432 5100 2355 12230 10200 Pyrene 665 53 490 700 195 2600 875 2230 8500 HPAHs 1700 NG NG NG NG 9600 NG NG NG Total PAHs 4022 NG 4000 NG 1610 447892 NG 22800 100000 Reference a b c d e a b e c* a= Long et al., 1995 (Marine and estuarine sediments), b = smith et al., 1996 (Freshwater sediments), c= Persaud et al., 1993, c* = Persaud et al., 1993 (assuming 1% toxicity), d = EC and MENVIQ 1992, e= MacDonald et al., 2000 (Freshwater sediments), TEL= Threshold effect level; ERL= Effects range low; LEL= Lowest effect level; MET=Minimal effect threshold; CB=Consensus Based; TEC=threshold effect concentration; LPAHs=Low molecular-weight PAHs; HPAHs, High molecular-weight PAHs; NG, no guideline

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Table 9. Summary of sediment quality guidelines for OCPs and PCBs that reflect TECs

(concentrations above which adverse biological effects are likely observed)

Threshold Effect Concentrations Probable Effect Concentrations ERL TEL LEL MET CB TEC ERM PEL CB PEC SEL Chlordane 0.5 4.5 7 7 3.24 6 8.9 17.6 60 Dieldrin 0.02 2.85 2 2 1.90 8 6.67 61.8 910 Total DDD 2 3.54 8 10 4.88 20 8.51 28.0 60 Total DDE 2 1.42 5 7 3.16 15 6.75 31.3 190 Total DDT 1 NG 8 9 4.16 7 NG 62.9 710 Total DDTs 3 7 7 NG 5.28 350 4450 572 120 Endrin 0.02 2.67 3 8 2.22 45 62.4 207 1300 Heptachlor Epoxide NG 0.6 5 5 2.47 NG 2.74 16 50 Lindane (γ-HCH) NG 0.94 3 3 2.37 NG 1.38 4.99 10 Total PCBs 50 24.1 70 200 59.8 400 277 676 5300 Reference a b c d e a b e c* a= Long et al 1995 (Marine and estuarine sediments), b = smith et al 1996 (Freshwater sediments), c= Persaud et al 1993, c* = Persaud et al 1993 (assuming 1% toxicity), d = EC and MENVIQ 1992, e= MacDonald et al., 2000 (Freshwater sediments), TEL= Threshold effect level; ERL= Effects range low; LEL= Lowest effect level; MET=Minimal effect threshold; CB=Consensus Based; TEC=threshold effect concentration; DDD, dichlorodiphenyldichloroethane; DDE, dichlorodiphenyldichloroethylene; DDT, dichlorodiphenyltrichloroethane; PCBs, polychlorinated biphenyls, NG, no guideline

Later, a large degree of congruence when comparing SQGs enabled the development of “Consensus-Based Sediment Quality Guidelines” for freshwater sediments (MacDonald et al., 2000), marine and estuarine sediments exclusively for

PAHs (Swartz, 1999). Consensus based empirical SQGs approach is a method, which introduced the Threshold Effect Concentration (TEC). TEC is defined as the contaminant concentration below which a toxic effect is not expected and is formulated using a TEL-type approach (Threshold Effect Level). The Probable Effect

Concentration (PEC) is defined as the concentration above which a toxic response is expected and is formulated using a PEL-type approach. TEC and PEC are the

CHAPTER I :Bibliographic Review

geometric means of the guidelines such as an EqP-based sediment quality advisory

level (SQAL), ERLs, LELs, TELs, METs, SELs, ERMs, and PELs. The Midpoint Effect

Concentration (MEC) is developed by taking the average of the TEC and PEC as

shown in table 14. A site-specific TEC and PEC could also be formulated on the basis

of laboratory test data for the contaminants and benthos present at a site.

Table 10. Consensus based empirical SQGs approach

Predictive TEC Level of MEC Level of PEC Predictive Concern Concern toxicity toxicity Level 1 CBSQG Level 2 MEC = TEC + PEC/2 Level 3 CBSQG Level 4 (lowest) Value Value (Highest)

≤ TEC >TEC ≤ >MEC ≤ >PEC MEC PEC TEC = Threshold Effect Concentration, MEC = Midpoint Effect Concentration, PEC = Probable Effect Concentration, CBSQG = Consensus-Based Sediment Quality Guideline

ii) Theoretical approach

Theoretically based approaches, that account for differences in bioavailability

via equilibrium partitioning (EqP), that is using organic carbon or acid volatile

sulphides. It attempts to describe bioavailability by suggesting that interstitial water

concentrations represent the primary route of exposure for aquatic organisms, and

the EqP calculation can be used to determine chemical concentrations (Di Toro et

al., 1991). Fundamentally, the sediment criterion is determined as follows:

Sediment criterion = WQCUS EPA x KOW x OCs ………………………… Equation 4

Where:

WQCUS EPA = US EPA Water Quality Criteria

KOW is the octanol-water partition coefficient

CHAPTER I :Bibliographic Review

OCs = Sediment Organic Carbon

From this, the toxicity of chemicals could be directly linked to the US EPA water quality data base, and the concentration of chemicals in sediments above which interstitial water would produce toxic effects (Burton, 2002). The normalization of sediments based on their organic carbon content allows to largely account for differences in bioavailability (toxicity) of chemicals. Though the EqP approach has been promoted by the US EPA, it is rarely used or is only used in support of other assessment methods.

Both empirical and theoretical approaches have their advantages and limitations as shown in table 15. Relatively, numerous empirical approaches have been reported based on the total concentration of chemicals in sediments and the occurrence of effects on benthic macroinvertebrates. These approaches rely on actual field and laboratory data, examining potential adverse effects to benthic organisms, when exposed to site contaminated sediment.

These assessment methods could be a major contribution in decision making regarding the protection of aquatic ecosystem and human health, the disposal of dredging and open water sediments, the control of pollution source and the remediation of contaminated sites.

CHAPTER I :Bibliographic Review

Table 11. Summary of the most used empirical and theoretical approaches to set sediment quality guidelines, with their advantages and

limitations.

SQG Approach Advantages Limitations Threshold below Threshold above Reference which toxic effects which toxic effects will rarely occur are likely to occur Effects Effects Range- Effects Range- Long and Morgan Range Low (ERL) Median (ERM) 1991 Minimal effect Toxic effect EC, MENVIQ, - Based on total sediment --Bioavailability not well addressed threshold (MET) threshold (TET) 1992 concentration Threshold-Effects Probable-Effects - Do not establish cause and effect Smith et al., 1996 - large data base for field Level (TEL) Level (PEL) - Varying rates of false positive and negative and laboratory tests Persaud et al., Effects Level results Lowest Severe - prediction of toxic effects 1993 - Most data for metals effect level (LEL) effect level (SEL) Empirical -simple to use - Difficult to differentiate effects from a mixture of chemicals Screening-Level Concentration for - Swartz, 1999 - grey area between thresholds PAHs Consensus- Based Poor documentation of formulations

Evaluation Threshold effect Probable effect MacDonald et al., concentration concentration 2000 (TEC) (PEC) - Predicts bioavailability -More important to understand assumptions like sorption and pore water -Suitable for organic Equilibrium chemicals - More difficult to use Di Toro et al., Theoretical - - Partitioning 1991 -Theoretical formulation - May be underestimated, since it does not consider sediment ingestion as a route of -Attempt to account for exposure bioavailability changes with

CHAPTER I :Bibliographic Review respect to changes in - Does not consider many factors that influence sediment organic matter bioavailability

CHAPTER I :Bibliographic Review

1.7. Effects of organochlorinated pesticides, polychlorobiphenyls and polyaromatic hydrocarbons on human health

There is universal scientific consensus that persistent organic pollutants (POPs) occur globally, are hazardous, and that implementation of the Stockholm Convention should be a high priority for all countries (Damstra, 2002). Humans are exposed to these chemicals through; consumption of contaminated food and the environment

(water, air, soil and sediments). The health effects linked to these exposures depend on; the specific compound, level of exposure (dosage), time and duration of exposure and of the individual. One of the major limits of human studies associated with OCPs,

PCBs and PAHs is the unavailability or limited availability of historical exposure data.

In cases where data was scarce or unavailable the adverse effects observed in laboratory animals were reported. A range of health effects have been reported by various studies as follows.

I.7.1. Cancer

Exposure to DDT has been linked to pancreatic cancer and non-Hodgkin’s lymphoma and exposures early in life is associated with an increased breast cancer risk later in life (Cohn et al., 2007; Garabrant et al., 1992). The majority of organochlorine pesticides, such as mirex, chlordane and toxaphene, are known to be carcinogenic as well. For instance, a study carried in India on women from agricultural areas revealed that, women with breast cancer had much higher total organochlorine pesticide concentrations in their blood (Dich et al., 1997). A study by (Multigner et al.,

2010) found a positive relationship between exposure to chlordecone and the risk of prostate cancer.

CHAPTER I :Bibliographic Review

Retrospective cohort mortality studies in workers exposed during capacitor manufacturing and repairing revealed PCB-related cancers at various parts, specifically, the skin (melanoma), the liver, intestines and biliary tract (ATSDR, 2002).

Liver cancer in Yusho victims appear to support occupational hepatocarcinogen city data (Miyata et al., 1985). The same study found no statistically significant (p<0.05) increased mortality from cancer of the stomach or oesophagus. However, no clear relation has been found between environmental exposures to PCBs and cancer in tissues such as, the brain, hematopoietic and lymphatic systems. Case-control studies of the general population are inconclusive with respect to associations between environmental exposures to PCBs and risk of breast cancer or non-Hodgkin’s lymphoma, although there are preliminary indications that particular subgroups of women may be at increased risk for breast cancer (ATSDR, 2002).

Benzo(a)pyrene is known to be the most carcinogenic PAH. The most prominent health effect to be expected as a result of inhalation exposure to PAHs is a high risk of lung cancer (Kim et al., 2013). The WHO guidance of 2003 set the unit risk for lung cancer of B(a)P at 87 x 10-6 ng/m3 for lifetime exposure. The guideline values for many WHO members states for B(a)P vary between 0.1 and 1.3 ng/m3 (Abdel-Shafy and Mansour, 2016).

1.7.2. Disruptive Effects

I) Thyroid disruption

Thyroid hormones are critical for normal growth and development in foetuses, infants, and small children. OCPs have been found to alter levels of maternal thyroid

CHAPTER I :Bibliographic Review hormones during pregnancy. Women with HCB concentrations that ranged from 7.5 -

841.0 ppb (ng/mL) had altered thyroid hormone levels (Chevrier et al., 2008). In female rats an increase of hepatocellular carcinomas and a concomitant decrease in mammary gland tumors, tumors of the thyroid, and of the pituitary were noted following long-term exposure to technical mixtures of PCB, such as Aroclor 1260 and Chlophen

A-60 (Kimbrough, 1995).

ii) Endocrine disruption

Due to their strong potential to bind to estrogen or andogen receptors, OCPs act principally by interfering with natural hormones. Most effects linked to endocrine disrupting effects are linked to OCPs and affect reproductive function (Oskam et al.,

2003). For instance, exposure to OCPs (in particular DDT) were associated with a statistically significant higher rate of prostate cancer among farmers in a multi-site case-control study carried out in five rural areas between 1990–1992 in Italy (Settimi et al., 2003). PCBs may affect steroid hormone metabolism through PXR, indicating both direct endocrine disrupting effects through PXR agonistic activity and possible secondary effects on a wide variety of steroid hormones as a result of altered metabolism (Masuyama et al., 2000).

1.7.3. Neurodevelopment effects

There exist several indicators that exposure to organochlorinated compounds

(OCPs and PCBs) disrupts normal development. Cassidy et al., (1994), showed that prenatal exposure to the organochlorine pesticide chlordane has been linked to

CHAPTER I :Bibliographic Review reduction of testosterone in adult female rats and behavioural changes in both sexes.

Exposure to OCPs is associated with neurodevelopmental health effects in humans and has been linked to decreased psychomotor function and mental function, including memory, attention, and verbal skills in children. (Korrick and Sagiv, 2008). There is also some evidence that organochlorine pesticide exposure is associated with the development of autism, although this is based on limited research (Bryson et al., 2007).

Various studies have reported that prenatal PCB exposure was associated with neonatal hypotonia, hyporeflexia lower Psychomotor Developmental Index (PDI) scores on the Bayley Scales of Infant Development (BSID) from 3 to 24 months

(Gladen et al., 1988; Koopman-Esseboom et al., 1996; Rogan et al., 1986; Rogan and

Gladen, 1991). The exposure to PAH pollution during pregnancy is associated with low birth weight, premature delivery and heart malformations (Abdel-Shafy and Mansour,

2016).

1.7.4. Reproductive Effects

In humans, maternal concentrations of DDE (a metabolite of DDT) above 10 ppb (µg/l) are associated with preterm birth and babies’ size. The higher the concentration of DDE in the mother’s blood, the more likely she was to have a preterm birth and the baby was more likely to be small for its gestation age (Longnecker et al.,

2001). (Pocar et al., 2003) showed that human oocytes collected from follicles with high PAH levels had fewer cell divisions after in vitro fertilization. Adverse reproductive system effects associated with in utero DDT or DDE exposure in male animals include reduced penis size, reduced testosterone levels, reduced male rat anogenital distance and low sperm density (Bhatia et al., 2005) A study of women being occupationally 66

CHAPTER I :Bibliographic Review exposed to high levels of PCBs suggested a relationship between PCBs exposure and reduced birth weight and shortened gestational age of their babies (Ostrowski et al.,

1997).

1.8 Review on environmental levels of target POPs in aquatic environments of Africa (Article review)

Abstract

This review examines the current environmental levels of organochlorinated pesticides (OCPs), Polychlorinated Biphenyls (PCBs) and Polyaromatic Hydrocarbons

(PAHs) in aquatic environments of Africa based on 62 published articles from 1998 to

2019. Though currently a huge number of articles have been published and numerous studies carried out in various parts of the world, there is still scarcity of knowledge related to Persistent Organic Pollutants (POPs) contamination in Africa. This study reveals that levels of POPs were relatively higher in sediments and fish compared to water, which is justified by the hydrophobic or lipophilic nature of these compounds.

The highest concentrations of POPs in all studied samples (sediments, biota and water) were recorded in areas associated with high anthropogenic pressure (high urbanization, industrial and agricultural activities). For PAHs, high levels were also linked to areas of petroleum exploitation, harbour and shipping activities. Generally, the most abundant OCPs were DDTs, HCHs, endosulfan and aldrin showing higher concentrations than their respective metabolites. DDTs and HCHs revealed both recent and historical uses mainly attributed to agriculture and disease vector control.

High molecular weight compounds were predominant over low molecular compounds

CHAPTER I :Bibliographic Review due to higher persistence in the environment. In most cas, PCB profiles indicated the historical use of highly chlorinated PCB mixtures, Aroclor 1254 and Aroclor 1260 (most used PCBs) mostly used as dielectric fluid in transformers. PAH ratios commonly revealed mixed sources of pyrolytic and petrogenic sources dominated by pyrolytic sources. Among the studied fish species, Tilapia zillii and the catfish were the most studied and contaminated edible fish species which represent an important source of protein (among most consumed by the local population) and income to local populations. POP concentrations in African aquatic environments are generally lower but somehow comparable to other aquatic ecosystems worldwide. This review identifies knowledge gaps and suggests new research insights to address the problem of data scarcity, improve the monitoring and assessment of POPs in Africa.

Keywords: POPs, water, sediments, biota, aquatic, Africa

1.8.1 Introduction

The adoption and ratification of the Stockholm Convention on Persistent

Organic Pollutants (POPs) by most African states, the development of global monitoring programs, the toxicity and ubiquitous nature of these compounds coupled with the significant scarcity of data in Africa, raises a pressing and increasing need to provide information on the state of POP contamination. In the past 20 years, over

100.000 research and review articles have been published worldwide on POPs against about 10.000 in Africa (ScienceDirect data base, 2020). This is all the more important due to the rapid demographic growth of Africa, with the population of sub-Saharan

Africa expected to double by 2050 (99 % increase) (UN DESA, 2019). This clearly

CHAPTER I :Bibliographic Review indicates increasing anthropogenic pressure on various ecosystems. Consequently, a necessity to provide the current state of knowledge on POP contamination in Africa.

Persistent Organic Pollutants (POPs) are compounds with toxic properties, persistent

(long half-lives), hydrophobic (water-loving) and lipophilic (fat-loving), which bioaccumulate/biomagnify in food chains/food webs and undergo long range transport

(Lambert et al., 2011). The Stockholm Convention in 2004, initially banned 12 intentionally and non-intentionally produced compounds called Persistent Organic pollutants (POPs). This convention was ratified by 182 parties including many African countries. The aforementioned compounds were also referred to as the ‘dirty dozen’ and classified in three major groups, pesticides (aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, Hexachlorobenzene, mirex, toxaphene) and industrial chemicals

(Hexachlorobenzene (HCB), Polychlorinated Biphenyls (PCBs) and by-products

(Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans

(PCDFs), Hexachlorobenzene (HCB), Polychlorinated Biphenyls (PCBs). Nowadays, other compounds with similar properties such as Polyaromatic Hydrocarbons (PAHs) have been classified as POPs with Toxic Equivalent Factor (TEF) or Relative Potency

(REP) (Eljarrat and Barceló, 2003).

Prior to the Stockholm convention, these chemicals were banned since the

1970s and 1980s in most northern hemisphere countries, where there was continuous production and export to developing countries leading to an extensive use between the

1970s and 1980s (Wiktelius and Edwards, 1997). In addition, the illegal transboundary movements of hazardous wastes banned under the Basel Convention (adopted in

1998) have also remarkably contributed to the presence of POPs in Africa (Gioia et al.,

2014). Prohibitions were operated in developing countries in the early 1990s to 2000s.

CHAPTER I :Bibliographic Review

Many years after the ban, these compounds are still detected in various compartments of the environment suggesting continuous human exposure. According to (Kaiser et al., 2016), modern human development has increased output of POPs into the natural environment from urbanization, agricultural and industrial activities. In Africa, rapid population growth and high urbanization rates have resulted in a recent expansion of cities in the absence of proper planning and without adequate waste disposal facilities

(Yabe et al., 2010). The global use of insecticides like endosulfan, DDT, lindane and specifically in Africa countries have been significant. More so, the economy of most countries in the African continent rely on agriculture, facing various threats such as pests and diseases. The latter makes the use of phytosanitary products such as pesticides indispensable to improve agricultural yields and preserve human health through disease vector control. In Africa, the sources of POPs are mostly related to industrial processes, open burning of wastes, leakages from electricity generation plants, transformers and obsolete stockpiles of pesticides (Ssebugere et al., 2019).

Nowadays, these activities or practices have been prohibited or regulated in most developed nations but unfortunately some are still taking place in Africa.

Aquatic environments tend to be sinks or reservoirs of various pollutants and

POPs in particular, due to their hydrophobic nature making them preferably bind to suspended particulate matter and accumulate in the bottom sediments of water ecosystems or easily accumulate in fatty tissues of aquatic biota (Ssebugere et al.,

2019; Widenfalk, 2002). Rivers, lakes, estuaries and oceans are highly vulnerable to

POP contamination owing to their proximity to densely populated areas, agricultural or industrial zones. This exerts a huge anthropogenic pressure on these ecosystems because the majority of local populations especially in developing countries rely on the

CHAPTER I :Bibliographic Review numerous services they provide. These services include, drinking water sources, important source of protein (fish, shrimps), irrigation water for agriculture and water for animal husbandry and water for cooling engines. For instance, Lake Chad Ramsar located at the southern fringe of the Sahara Desert bordered by 4 countries Cameroon,

Nigeria, Chad and Niger. The Congo River and its tributaries representing 25% of the renewable water supply of Africa (Verhaert et al., 2013). Unfortunately, they are often used as waste dumps for various types of wastes (household, municipal, agricultural, industrial and pharmaceutical). Organochlorinated pesticides, polychlorobiphenyls and/or polyaromatic hydrocarbons have been studied in important freshwater ecosystems of Africa such as the Congo River, Lake Bosomtwi (Ramsar site) the largest natural lake in West Africa, Lake Burullus. The Congo river basin is the second- largest watershed in the world representing 25 % of Africa’s renewable water supply.

Wetlands of international importance (Ramsar sites) such as Lake Chad located at the southern fringe of the Sahara Desert bordered by 4 countries Cameroon, Nigeria, Chad and Niger. Lake Volta is the most important inland water resource in Ghana and supplies electrical energy. It is an extension of the Volta River which originates from

Burkina Faso, Ivory Coast and Togo flowing through many farming regions. Lake

Manzala largest of Egypt’s Mediterranean coastal lakes and the most productive for fisheries. It is an internationally registered Important Bird Area. Lake Qarun Egypt is the third-largest lake in Egypt and one of the oldest lakes in the world.

1.8.1. Persistent Organic Pollutants in Africa

In Africa, data on import, use, exposure and health effects of POPs is poorly documented but more available for pesticides than other groups of POPs (Industrial 71

CHAPTER I :Bibliographic Review chemicals and by-products). There is greater accessibility for such data on conventional insecticides such as DDT, HCHs (lindane) and endosulfan which have been extensively used for agriculture and public health. Practically no such information is available for ubiquitous pollutants such as PCBs and PAHs which could be produced intentionally and non-intentionally through incomplete combustion of biomass. One of the most common use of pesticides in Africa is the fight against vectors of diseases like malaria which causes about 1 million deaths each year (mostly children and pregnant woman), nine in ten of these deaths are in Africa (Yamey and Attaran, 2003).

Eckhardt et al., (2007) pointed out biomass burning as one of the sources of PCBs in the environment. Africa represents the largest source of biomass burning through forest and savanna fires, firewood combustion, open burning of household and municipal waste containing and producing hazardous chemicals electronic waste, lubricants, textiles, solvents and pharmaceutical wastes. In addition, the presence of many petroleum exploitation industries, oil refineries, intense harbour and shipping activities in Africa are a great deal of interest for information on the historic and trend of these pollutants in Africa. Previous studies in Africa, reported the use persistent and highly toxic organochlorinated insecticides such as lindane (γ-HCH) known as

Gammalin, DDT and endosulfan for fishing in Nigeria (Akoto et al., 2013), Cameroon

(Balgah and Kimengsi, 2011), Uganda (Wasswa et al., 2011) and Ghana (Ntow, 2005).

Despite the ban of DDT and HCH in Nigeria (one of the highest pesticide users in sub-

Saharan Africa), they are still used by many farmers for agricultural production and few countries other African countries are still involved in the large-scale production, use and export of lindane (Mazlan et al., 2017).

CHAPTER I :Bibliographic Review

Polychlorinated biphenyls (PCBs) were extensively used between the 1950s and 1970s as coolants and lubricants in transformers, generators, and capacitors contained in electrical and electronic products, hydraulic and heat exchange fluids due to their insulating capacity, heat stability, and low-burning capacity (Gioia et al., 2014).

They have also been used as plasticizers and sealing agents in rubber, especially in polyvinyl chloride plastics used to coat electrical wiring, adhesives, paints, and inks.

Increasing industrialization in Africa has favoured the import of new and/or used products such as electronic equipments. In the context of lack or insufficient waste management facilities (recycling and treatment), a huge amount of e-wastes is generated. The latter is mostly incinerated or openly burned to recover certain parts or disassembled leading to leakages (Gioia et al., 2014) or openly disposed or discharged in the environment. Besides the fact that PCBs have not been produced nor highly used in Africa, surprisingly high levels of 7 indicator PCBs (Σ7PCBs) were reported from samples collected 400 km off the West African coast at concentrations of

200 pg/m3 and 190 pg/m3 in 2005 and 2008 respectively (Gioia et al., 2011; 2008). The monitoring of PCB levels using passive air samples recorded monthly average concentration of 100 pg/m3 and more in urban/industrial sites of South Africa, Senegal,

Kenya, Egypt, Democratic Republic of Congo, Ghana, Mali, and Sudan, comparable to those in US and European cities. Likewise (Gioia et al., 2008) reported air

3 concentrations in excess of Σ7PCBs at 100 pg/m in Ivory Coast and Gambia. (Mazlan et al., 2017) reported that insecticides have never been used in Lake Gerio (Adamawa state, Nigeria) but DDT levels were found between 6.1 – 31.3 μg/L.

Nowadays levels of POPs in developed nations have decreased due to strict adherence to international restrictions on their usage and emissions. Conversely, rising

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POP levels are observed in Africa due to low awareness on their harmful effects, poor enforcement of legislation, illegal entry of some banned products, lack of adequate waste management systems, inadequate storage and disposal facilities, absence or inadequate protective equipment during handling or use, misuse of these chemicals, open burning of waste, inappropriate disposal of hazardous wastes, the reuse of empty containers of pesticides and lubricating oil (motor oil), ship dismantling, the presence of old stock piles and obsolete pesticides. The aforementioned factors contribute to increasing the risk of POP and particularly pesticide exposure to most people in the continent. This is supported by the fact that many African countries do not recognize diseases related to POP exposures like pesticides as professional diseases. It has been reported that about 2.2 million people in Nigerian states are at risk as a result of exposure from various agricultural inputs such as pesticides and other related chemicals (Mazlan et al., 2017). In addition, there are indications of continuing shift in primary emission sources of POPs such as PCBs from regions where they have been manufactured to regions of the southern hemisphere such as Africa (Gioia et al., 2014)

The paucity of knowledge and data in Africa could be justified by limited funding, insufficient infrastructure (well-equipped laboratories) and qualified personnel to perform the analyses. This explains why the analyses of almost all studies of POPs in

Africa are mainly carried out laboratories found in North America, Europe and Asia.

However, between 2016 and 2017 there 20 African laboratories from South Africa,

Nigeria, Ghana, Egypt, Uganda, Mali, Ethiopia, Tanzania, Senegal, Morocco,

Madagascar participated in the ‘Bi-ennial Global Interlaboratory Assessment on

Persistent Organic Pollutants, 3rd Round’ (UNEP, 2017b). Review papers on POPs in

Africa are very few and mostly recently published such as; Brits et al., (2016) : analysis

CHAPTER I :Bibliographic Review of brominated flame retardants and their environmental levels; Katima et al., (2017) : brominated flame retardants in the environment with emphasis on atmospheric levels;

Merhaby et al., (2019) : sediment pollution by PAHs and PCBs in the Mediterranean basin; Mazlan et al., (2017) : the status of persistent organic pesticide residues in water and food and their effects on environment and farmers (Nigeria); Gioia et al., (2014) : the environmental levels Polychlorinated biphenyls in Africa; Mochungong et al.,

(2015) : DDTs, PCBs and PBDEs contamination in Africa, Latin America and South- southeast Asia; and Wiktelius and Edwards, (1997): on organochlorine insecticide residues in African fauna (1971 - 1995).

The aim of this review is to provide an overview of current data on the environmental levels of POPs in aquatic environments of Africa for organochlorinated pesticides (OCPs), polychlorobiphenyls (PCBs) and polyaromatic hydrocarbons

(PAHs). It covers a total of 62 research articles published in the past 21 years (1998 to 2019). This review highlights underexamined areas and increase awareness on the impact of POPs in aquatic ecosystems of Africa countries characterized by insufficient or inadequate facilities for the disposal and elimination of these pollutants. To the best of our knowledge, this review is the first examines various groups of POPs (OCPs,

PCBs and PAHs) in a wide range of matrices (sediments, biota and water) in aquatic environments of Africa.

1.8.2. Methodology i) Selection criteria

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The compounds of interest (OCPs, PCBs and PAHs) examined were selected relying on their toxicity to humans and aquatic life, the quantity imported, used, abundance and frequency of detection reported by previous studies in carried out

Africa. Based on the paramount importance of aquatic ecosystems and few data available in Africa, this work focused on rivers, streams, lakes, estuaries and coastal areas. Over 63 articles published articles from 1998 to 2019 related to OCPs, PCBs and PAHs in aquatic environments of Africa were examined (figure 8). The latter was obtained by screening through various scientific research databases such as

ScienceDirect, Web of Science, PubMed, web search engines (google scholar) and scientific research networking sites (ResearchGate).

Figure 8. Number of published articles examined for OCPs, PCBs and PAHs in aquatic

environments of Africa from 1998 to 2019

Though this literature is not exhaustive, it is representative and gives a good picture of the distribution of studies carried out in African aquatic ecosystems related 76

CHAPTER I :Bibliographic Review to OCPs, PCBs and PAHs as shown in figure 9. A total of 18 out of 54 African countries were examined for published research articles representing a ratio of 1:3 countries for the study of POPs in African aquatic ecosystems. The majority of studies were carried out in Nigeria (12), Egypt (9), South Africa (9), Ghana (6) and Tunisia (5), representing

67 % of the number of published articles.

Figure 9. Distribution of study sites for OCPs, PCBs and PAHs for aquatic environments in

Africa ii) Data Treatment

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In cases where the minimum concentrations were below the detection limit

(BDL), the limit of detection (LOD) was considered for convenience of data presentation. Specifications on the number of sampling stations, samples, species, target analytes used in computation for each group of POPs in different matrices was indicated (table 16, 17 and 18). Data on concentration was expressed to one significant digit, to overcome constraints of analytical variability and ease data presentation. The mean and median values of concentrations were represented by (x̅) and (x̄) respectively. Concentration of studied compounds were reported as dry weight (dw) for sediments; lipid weight (lw), wet weight (ww) or dry weight (if indicated) for biota.

Lipid-normalized concentrations were presented for biota because of the suitability in comparing exposure concentrations across tissue specimens or across studied populations (Morgan and Roan, 1970). The summary of OCP, PCB and PAH concentrations were graphically represented as box and whisker plots or boxplots where:

1.8.3. Environmental levels of POPs in African aquatic environments

i) Sediments

The mean ΣOCPs levels in sediments of African aquatic environments were generally < 200 ng/g dw; lakes (x ̅ = 196.5 ng/g, x̄ = 82.9 ng/g), coastline (x ̅ = 189.9 ng/g, x ̄ = 16.4 ng/g) except rivers (x̅ = 1319.2 ng/g, x̄ = 105.6 ng/g) (Appendix 1).

Mohammed et al., 2017 reported seasonal (rainy and dry season) ΣOCPs concentrations in sediments within the Komadugu River basin in Yobo state, Nigeria

CHAPTER I :Bibliographic Review between 1030 – 12000 ng/g dw. Dieldrin was the most abundant compound at

8870 ng/g dw. The highest pesticide load was recorded in the rainy season (39 %), the harmattan 2 period (32 %) and dry season (29 %) due to the high use of pesticide for agriculture during the rainy season. Ibigbami et al., (2015) found ΣOCPs levels in sediments of the Ogbese River in Ekitti state, Nigeria ranging from 211 - 1957 ng/g dw.

Aldrin and endosulfan were higher than dieldrin and endosulfan sulfate their respective metabolites. DDT was assigned to historical use and long-range transport. However,

Ben Salem et al., (2017) recently reported higher levels of OCPs in the Ichkeul Lake-

Bizerte lagoon complex in Tunisia between 34 - 2021 ng/g dw (1576 ng/g). Lindane, dieldrin and endrin were the most represented in the sediments of Ichkeul Lake. The results showed that Lake Ichkeul relatively had higher levels of OCPs than the Bizerte lagoon. Appendix 1 shows that the mean ΣDDTs African aquatic environments were <

50 ng/g dw; lakes (x̅ = 61.3 ng/g, x̄ = 4.4 ng/g), rivers (x ̅ = 40.8 ng/g, x ̄ = 22.6 ng/g), coastline (x ̅ = 14.0 ng/g, x ̄ = 9.2 ng/g) and dams/reservoirs (x̅ = 2.8 ng/g, x̄ = 2.8 ng/g).

The same illustration shows that the mean of ΣHCHs were equally < 50 ng/g dw; lakes

(x̅ = 13.5 ng/g, x ̄ = 4.0 ng/g), rivers (x ̅ = 48.9 ng/g, x ̄ = 6.2 ng/g), coastline (x̅ = 16.9 ng/g, x ̄ = 2.8 ng/g) and dams/reservoirs (x̅ = 4.3 ng/g, x̄ = 4.3 ng/g). Said et al., (2008) reported DDT levels in sediments of Lake Burullus in Egypt ranging from 46.3 -

656.5 ng/g. OCPs were more abundant than PCBs and their major source was attributed to municipal and/or agricultural waste mainly from Kafr El Sheikh

Governorate.

2 Harmattan is a cool dry wind that blows from the northeast or east in the western Sahara and is strongest in late fall and winter (late November to mid-March). It usually carries large amounts of dust, which it transports hundreds of kilometres out over the Atlantic Ocean (https://www.britannica.com/science/harmattan). 79

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The previously reported ΣOCP concentrations in African aquatic environments were higher than those reported for sediments from Cortiou; an outfall receiving sewage from the city of Marseille with about 1.5 million inhabitants, 2.01 - 254.8 ng/g dw (Wafo et al., 2006) and 1.2 – 190.6 ng/g dw (Syakti et al., 2012). However, the latter were comparable to OCPs in sediments of urban rivers in the Congo Basin

(Democratic Republic of Congo) ranging between 1.2 - 270.6 ng/g (Kilunga et al.,

2017). High levels of HCHs were recorded in sediments of the Ogbese River in Ekitti state, Nigeria between 83.7 - 618 ng/g dw (Ibigbami et al., 2015). This was comparable to HCHs levels found in sediments of the Baiyangdian Lake, North China (4.7 -

679 ng/g dw) (Hu et al., 2010). Said et al., (2008) reported HCHs levels in sediments of Lake Burullus, Egypt from BDL - 114.7 ng/g. Low DDTs levels values were found in the were found in sediments of the Tekeze River dam in Ethiopia between 4.2 -

6.2 ng/g (Ga, 2016) and Aiber reservoir in Nigeria at 0.5 - 6.2 ng/g (Olutona et al.,

2014) The lowest HCHs levels were found in sediments of the Petite Côte and the

Sine-Saloum Estuary in Senegal ranging from 0.1 - 1.9 ng/g (Bodin et al., 2011a). This is lower than the HCHs levels recorded in sediments of the Minjiang River Estuary,

China (2.99 - 16.21 ng/g) (Zhang et al., 2003).

The concentration of ΣPCBs in sediments of African aquatic environments as shown in Appendix 1., illustrates that the mean of ΣPCBs were < 80 ng/g dw; lakes (x ̅

= 38.6 ng/g, x ̄ = 11.7 ng/g), rivers (x ̅ = 75.6 ng/g, x̄ = 10.5 ng/g) and coastline (x̅ = 58.9 ng/g, x ̄ = 9.7 ng/g). Barakat et al., 2013, 2002 reported total PCBs levels in sediments along the Mediterranean coast of Egypt between 0.9 - 1210 ng/g dw. These concentrations were comparable to PCB levels in sediments of Cortiou, Marseille 12.7

- 1559.3 ng/g (Wafo et al., 2006). Ragab et al., 2016 reported ΣPCB levels in

CHAPTER I :Bibliographic Review sediments of the Red Sea coast of Egypt between 29 - 366 ng/g. The total PCB levels in the Aqaba Gulf, the Suez Gulf and the Red Sea proper were 412 ng/g dw, 456 ng/g dw and 1028 ng/g dw respectively. The most abundant PCB congeners were CB 138>

180> 52> 153. PCB 138 and PCB 153 are the main constituents of Aroclor 1254 and

1260 respectively (ATSDR, 2002). Higher PCB levels in most contaminated sites were associated with direct industrial and domestic discharge. Samia et al., 2018 reported

PCBs levels in sediments of the El bey river system, Tunisia between 39.6 - 643.9 ng/g dw (x ̅ = 643.9 ng/g). The PCB patterns were different at each site but high molecular weight PCBs (HPCBs) were dominant after wastewater effluent sources and

Grombalia City indicating industrial input. Higher PCB contaminations were recorded at stations close to industrial areas of Bouargroub and Soliman, as well as wastewater discharge locations in Soliman. This PCB contamination was much lower than that found in sediments of Lambro River, northern Italy (10 - 3054 ng/g dw) (Bettinetti et al., 2003).

In urban rivers of the Congo river basin, DR Congo, total PCB concentration ranged between 11.1 - 169.3 ng/g dw (x ̅ = 54.9 ng/g) (Kilunga et al., 2017). Penta and hepta chloropropyls (PCB 153> 138> 180> 149> 101) were the most abundant congeners. This pattern indicates a dominant and historical use of Aroclor 1260, contrary to that found by Verhaert et al., 2013) in the Congo river basin indicating the dominant use of Aroclor 1254. Bodin et al., (2011) reported seasonal (wet and dry season) Σ7PCB levels in sediments of the Fadiouth and Falia Estuary, Senegal from

0.3 - 19.1 ng/g. Levels in the wet season were 2 to 16 times higher than the dry season due to the strong washout of PCB residues from inland to the marine ecosystem. The dominant congeners were CB 153, 138 and 180, representing 55 to 65% of Σ7PCBs.

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This contamination was not likely to cause adverse biological effects to molluscs or potential risks to humans. Higher PCB levels were found at stations located close to industrialized and densely populated areas. These levels were lower than the concentrations recorded in sediments of the Minjiang river Estuary, China (Zhang et al., 2003).

The range of mean ΣPAHs concentrations in sediments of African aquatic environments is higher than those of ΣOCPs and ΣPCBs shown in (Appendix 1). The mean ΣPAHs levels is generally < 10 000 ng/g dw for lakes (x ̅ = 2601.7 ng/g, x̄ =

2601.7 ng/g), rivers (x ̅ = 694.2 ng/g, x ̄ = 258.9 ng/g) and coastline (x ̅ = 9279.7 ng/g, x̄

= 10583.8 ng/g). High PAHs levels were reported for epipelic and benthic sediments of the Iko River Estuary mangrove in Nigeria between 6100 - 35270 ng/g (Essien et al., 2011). HPAHs (4 – 6 rings) were dominant representing more than 60 % of total

PAHs. The major PAH source was pyrolytic over petrogenic sources. These levels were higher than the Daliao river estuary, China (272 - 1,607 ng/g) (Men et al., 2009) and the Gironde Estuary, France (19 - 4888 ng/g) (Budzinski et al., 1997). Barakat et al., (2011) reported PAH levels in sediments of the Mediterranean coast of Egypt varying from 13.5 - 22600 ng/g. Most samples showed a mixture of petrogenic and pyrolytic sources. HPAHs (4 -6 rings) were dominant over LPAHs (2 - 3 rings). Higher levels were recorded at industrialized and urbanized regions. These levels were comparable to the PAH levels found in sediments of the West Mediterranean Sea

(French Riviera, Corsica, Sardinia) between 1.5 - 20440 ng/g dw (Baumard et al.,

1998a, 1998b). (Mohammed et al., 2017 ; Ibigbami et al., 2015). Adeniji et al., (2019) reported seasonal levels of PAHs in sediments of the Buffalo river estuary in South

Africa between 1107 - 22310 ng/g. Total PAH concentrations were higher in summer

CHAPTER I :Bibliographic Review than autumn. High molecular weight PAHs were dominant (LPAHs/HPAHs < 1) indicating a dominance of pyrolytic sources. These levels were higher than those found in the Pearl river delta (estuary) (408 - 10811 ng/g). In the Ichkeul Lake-Bizerte lagoon complex, Tunisia, PAH level ranged from 122 - 19600 ng/g dw (Ben Salem et al.,

2017). Barakat et al., (2002) reported levels of PAHs in sediments of Lake Manzala in

Egypt from 256 - 9910 ng/g (x ̅ = 1480 ng/g). LPAHs (2 - 3 rings) from petrogenic origin were predominant over HPAHs (4 - 6 rings) from pyrogenic origin. The highest PAH contamination was associated to industrialized, urbanized and shipping activities.

Generally, Rivers show the highest mean concentration of ΣPOPs in sediments followed by coastlines, lakes, dams and reservoirs of African aquatic environments as shown in figure 10.

Figure 10. Concentration of POPs (OCPs, PCBs and PAHs) in sediment (ng/g, dry weight) of

African aquatic environments (Mean concentrations in Appendix 1).

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Table 12. Summary of OCPs, PCBs and PAHs levels in sediments of African aquatic environments

Number of Sediments Aquatic Country Main activities Stations Concentration range (ng/g) of dry weight (dw) Reference environment (samples) DDTs HCHs OCPs 7 PCBs 16 PAHs South North End Lake Industrial 13 ND ND ND 1.6 – 3.1 ND Kampire et al., 2017 Africa Agriculture, fishing, Lake Togo Togo 8 (48) <0.001 - 1.49 I 0.001 - 1.37 E NC NA NA Mawussi et al 2016 disease vector control Lake Noukoué and Benin Disease vector control, -

Lake Victoria Uganda Industrial 4 NA ND NA 0.4 – 0.8 a NA Ssebugere et al., 2014

R Agricultural, industrial, K W 1.5 - 137.2 Lake Qarun Egypt 34 0.1 – 5.88 0.1 – 62.6 1.0 - 164.8 NA Barakat et al. 2013a urbanization J

Y W A

Lakes Lake Bosomtwi Ghana Agriculture, fishing 10 1.8 – 7.0 0.75 – 3.4 NC 4.1 – 19.2 NA Afful 2013 Fishing, industrial, Lake Maryut Egypt 11 ND ND ND ND 246 - 9910 Barakat et al. 2012 urbanization Lake Victoria Uganda Agriculture, fishing 3 (117) * 4.2 ± 3.8 K 2.8 ± 2 D NC ND ND Wasswa J. et al, 2011

Lake Bosomtwi Ghana Agriculture, fishing (50) 4.41 H 6.8 E NC ND ND Darko et al. 2008 140.2 – 4.6 to Lake Burullus Egypt Agriculture, fishing 12 46.3 - 656.5N 0.3 - 114.7D ND Saïd et al 2008 811.0 213.9

Lake Volta Ghana Agriculture, fishing 6 (36) 61.3±42.8I 2.3±1.4 NC ND ND Ntow 2005 Awash River Basin Ethiopia Agriculture, industrial 46 1.99–139.68Y 2.41–15.9 W 6.6 – 206.1 0.9 – 26.6 ND Dirbaba et al 2018

South 0.0004 – 0.006 Ga-Selati River Agriculture, mining 3 0.2 – 80 N NC 0.2 – 0.25 Z NA Govaerts et al., 2018 Africa W Nairobi River Kenya Industrial, agricultural 3(54) NC NC 0.01 - 41.9 NA NA Ndunda et al., 2018 industrial, urbanization, 39.6 - 240 - El bey River system Tunisia 9 (9) NA NA NA Samia et al 2018 agricultural 643.9 5362.2

Rivers and Streams and Rivers urban Rivers, DR Industrial, urbanization, 11.1 - 22.6 - 3 1.2 – 270.6 ND 21.6 – 146.8 Kilunga et al., 2017 Kinshasa Congo agriculture 169.3 495.3

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Komadugu River 1030 – Nigeria Agriculture, fishing 5 NA NA NA NA Mohammed et al., 2017 basin 12,000

Number of Sediments Aquatic Country Main activities Stations Concentration range (ng/g) of dry weight (dw) Reference environment (samples) DDTs HCHs OCPs 7 PCBs 16 PAHs

Ovia River Nigeria Agriculture, fishing 3(54) F NA NA NA NA 5.3 - 573.3 Tongo et al. 2017 Industrial, petroleum Imo river Nigeria 4 NA NA NA NA 28 – 117 B Oyo-Ita et al., 2016 refinery, fishing DR 1.38 – 34.48 – Congo River basin Industrial, urbanization 5 0.6 – 3.3 K NA NC Mwanamoki et al. 2014 Congo 13.31 63.89 Ogbese River Nigeria Agricultural, fishing 5 5.1 – 77.7 83.7 - 618 211 – 1957 NA NA Ibigbami et al., 2015

Ondo State Rivers Nigeria Agricultural 11 (11) (DS) 0.1 - 71.3 H 0.03 – 22.9 1.3 – 208.3 NA NA Okoya et al., 2013 DR Agricultural, industrial, Congo River basin 5 (15) 0.01 – 0.4 N 0.01 – 0.4 D NC 0.01 – 1.4 T NA Verhaert V. et al., 2013 Congo urbanization Kuranchie-Mensah et al. Densu River basin Ghana Agriculture, fishing 3 (15) 0.1 – 0.2 F 0.5 – 0.6 NC NA NA 2012

Yala/Nzoia River Kenya Agriculture, vector control 9 (DS) 9.5F 16.0E BDL-24.54 NA NA Musa et al. 2011 Ashanti Region Ghana Agricultural 42 0.46 ± 0.24G 3.2 ± 0.6E NC NA NA Ntow 2001 Streams Fishing, Mussel farming Lake Ichkeul- (LI) 122 – Tunisia 28 1 - 14 K 12 – 209 W 34 - 2021 0.04 - 10.7 Ben Salem et al 2017 Bizerte lagoon Harbour, urbanization, 19,600 complex industrial, agricultural, oil refinery (BL)

Bahiret el Bibane Agricultural, fishing, Tunisia lagoon industrial, urbanization N E et al areas (10) 0.1 – 20.3 0.3 – 4.1 4.4 – 20.3 29.5 – 88.2 ND Barhoumi 2016 Harbour, urbanization, Bizerte Lagoon Tunisia industrial, agricultural, oil 18 0.3 - 11.5 ND NC 0.8–14.6 L ND Barhoumi et al 2014 refinery

Estuaries, gulfs and other coastal coastal other and gulfs Estuaries, South 1107 – Buffalo River Estuary Industrial, harbour (19) NA NA NA NA Adeniji et al 2019 Africa 22310

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Number of Aquatic Reference Country Main activities Stations Concentration range (ng/g) of dry weight (dw) environment (samples) DDTs HCHs OCPs 7 PCBs 16 PAHs Fadiouth and Falia Senegal Agricultural 2 0.3-15.9 0.1–1.9E 1.2-18.2O 0.3 – 19.1 NA Bodin et al., 2011 EstuaryR Iko River Estuary 6100 - Nigeria Oil spillage, agricultural NA NA NA NA Essien et al 2011 Mangrove 35270 Tanzania coastal Tanzania Industrial, agricultural 7 0.1 - 8.6 0.2 – 5.3D ND 2.3-3.7C NA Kruitwagen et al., 2008 mangrove 15.7 - Coast of Djibouti City Djibouti Oil spills, Industrial 11 NA NA NA NA Madhi Ahmed et al., 2017 3760.1 Agricultural, industrial, Red Sea coast Egypt 16 1 – 47 H 1 - 7 E 16 - 106 29 - 366 NA Ragab et al 2016 urbanization

Agricultural, industrial, Atlantic coast Morocco 5 (40) 5.7 – 17.3 2.9 – 6.8 13.2 - 27.5 NA NA Benbakhta et al 2014 urbanization Agricultural, industrial, Mediterranean coast Egypt 26 0.07 - 81.5 K 0.25 – 1.88 W 0.3 – 16.8 0.29 - 377 NA Barakat 2013a urbanization Agricultural, industrial, 13.5 - Mediterranean coast Egypt 26 NA NA NA NA Barakat 2011 urbanization 22600 Indian Ocean Coast Kenya Agricultural, industrial 4 (36) 3.2 - 7.0 2.3 – 16.0 NC NA NA Barasa et al., 2007

Northwestern Red Industrial, fishing, Egypt 16 F NA NA NA NA 0.7 – 456.9 Aly Salem et al., 2014 Sea aquaculture, shipping Napoleon gulf Uganda Industrial, urbanization 4 (24) NA ND NC 0.4 – 0.8 NA Ssebugere et al., 2014 Industrial, urbanization, Alexandra Harbour Egypt 23 0.25 - 885 0.4 - 2.1 NC 0.9 - 1210 NA Barakat 2002 agricultural

Agriculture, vector control, Tekeze River Dam Ethiopia 2 (24) 4.2 - 6.2 H 3.1 - 14.2E NC NA NA GA 2016 fishing s ands Agriculture, fishing, Aiba Reservoir Nigeria 4 0.5 ± 1.2 H 0.02 ± 0.05E NC NA NA Olutona et al., 2014 reservoirs Dam urbanization ND= Not detected, NA = Not analysed, NC =Not computable *=sediment cores, BDL= below detection limit, DS = Dry season, R = Ramsar site, NC = not computable, I = Invertebrates, S = seal, F = fish, m = molluscs, A = 6 PCBs, B = 13 PAHs, C= 17 PAHs, D= 3HCHs, E = γ-HCH, G = p,p’ -DDE, H = 1 DDTs, I = 2 DDTs, J = 29 PCBs, k=5

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DDTs, L = 10 PCBs, M= 18 OCPs, N=6 DDTs, O = 14 OCPs, P = 2 HCHs, Q = 10 PCBs, T = 21 PCBs, U = 12 PCBs, V = 21 OCPs, W = 4 HCHs, Y = 3 DDTs, Z = 31 PCBs, AA = 20 OCPs, AB = 5 PAHs

CHAPTER I :Bibliographic Review ii) Biota

According to Fürst et al., (1990), dietary intake has been recognized as the main route of uptake of POPs by humans, contributing to more than 90 % of the total daily exposure, 90 % of which is of animal origin, which brings out the importance of this compounds to be studied in biota. More so, higher concentrations of POPs could be found in biota due to bioaccumulation and biomagnification of these pollutants resulting from high affinity with fatty tissues. The majority of studies on OCPs, PCBs and PAHs in biota of African aquatic environments have mostly been carried out on fish and to a lesser extent on invertebrates (snails, shrimps, mussels). The mean ΣOCPs levels in fish of African aquatic environments were < 800 ng/g lw; lakes (x̅ = 18.8 ng/g, x̄ = 11.6 ng/g), rivers (x̅ = 517.3 ng/g, x ̄ = 517.3 ng/g) and coastline (x̅ = 787.6 ng/g, x̄ = 787.6 ng/g) (Appendix 1).

(Adeyemi et al., 2008) recorded high levels of OCPs in 3 fish species from the

Lagos lagoon between 182.4 - 1906.8 ng/g lw (x̅ = 1155.2 ng/g). These concentrations were higher than those reported for OCPs in 24 fish species in the Taihu lake (third largest shallow lake in China) ranging from 121 – 904 ng/g lw (Wang et al., 2012). The

Lagos lagoon is one of the most threatened water resources in Nigeria by anthropogenic activities comprising of agriculture, industrialization and inadequate disposal of waste in the city of Lagos (largest city in Nigeria with over 13 million people).

Total OCPs levels were higher in Tillapia zillii (red belly tilapia) ranging from 1671.5 -

1906.9 ng/g lw (x̅ = 1789.2 ng/g lw) compared to Ethmalosa fimbriata (bonga shad or bonga) 182.4 - 1797.5 ng/g lw (x̅ = 990 ng/g) and Chrysichthys nigrodigitatus (cat fish) from 299.8 - 1073.5 ng/g lw (x̄ = 686.65 ng/g). In this study, HCHs concentrations ranged between 88 - 1254.3 ng/g lw (x̄ = 712 ng/g). HCHs were the only compounds 88

CHAPTER I :Bibliographic Review above the residue limit of 5 ppm set by the Codex Alimentarius commission of FAO-

WHO, 1997. Total DDT levels in all fish species ranged from 21.9 - 250 ng/g lw (x̄ =

712.1 ng/g). Urban and industrial waste discharge into the lagoon was identified as one of the major sources of pollution. (Mohammed et al., 2017) found seasonal OCPs concentrations between 250 - 420 ng/g ww in four fish species (Claria sanguillaris,

Tilapia zillii, Synodontisbudgetti, and Heterotis niloticus) from Komadugu River basin of Yobe State in Nigeria. The highest mean OCP level was found in Tillapia zillii (x̄ =

2760 ng/g ww) and the lowest in Heterotis niloticus (x̄ = 990 ng/g). DDTs and HCHs were not analysed in this study. The concentrations of aldrin, dieldrin and endosulfan in most of the fish samples were much higher than the WHO and FAO (Codex, 2009) prescribed maximum residue limits (MRL) of 0.2 mg/kg (aldrin an dieldrin), 0.1 mg/kg

(endosulfan) and the acceptable daily intake values (ADIs) of 0.0001 mg/kg (aldrin an dieldrin) and 0.006 mg/kg (endosulfan).

Ibigbami et al., (2015) found mean total OCPs concentrations in fish varying between 139 - 490 ng/g ww from five fish species (Heterotis niloticus, Oreochromis niloticus, Tilapia zillii, Chrysichthys nigrodigitatus and Clarias gariepinus) in the

Ogbese river, Nigeria. The mean total OCP levels were higher in Chrysichthys nigrodigitatus and lowest in Heterotis niloticus (Ibigbami et al., 2015). The OCP concentrations found in the Ogbese river were lower than those recorded in smallmouth bass (40 - 320 ng/g ww) in from the lower Willamette River, Oregon, USA

(Sethajintanin et al., 2004) but comparable to OCP levels found in the than those recorded in Saury, mollet, white fish, perch, carp, gurnet, bull trout (2.6 -133.5 ng/g ww) of the Qiantang River, China (Zhou et al., 2007). Appendix 1. shows that mean

ΣDDTs levels in fish of African aquatic environments were < 1000 ng/g lw; lakes (x̅ =

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329 ng/g, x̄ = 24 ng/g), rivers (x̅ = 972 ng/g, x̄ = 522.3 ng/g) and coastline (x̅ = 409.9 ng/g, x̄ = 470 ng/g). For HCHs, mean levels were < 500 ng/g lw; lakes (x̅ = 29.4 ng/g, x̄ = 0.7 ng/g), rivers (x̅ = 104.2 ng/g, x̄ = 68.4 ng/g) and coastline (x ̅ = 407.7 ng/g, x̄ =

30.5 ng/g). In the Ogbese River, HCHs and DDTs levels vary between 21.7 -

115.2 ng/g ww (x̄ = 58 ng/g) and 6.8 - 32.3 ng/g ww (x̄ = 14.5 ng/g) respectively. These concentration of DDTs were comparable to those found in muscles of fish (Abramos brama, Rutilus rutilusheckeli, Perca fluviatilis, Chondrostomanasus, Carasius auratus gibelio, Cyprinus carpio) in the Dniester River in Moldova ranging from 3.1–34.8 ng/g ww (Sapozhnikova et al., 2005). DDT levels in the Ogbese River was lower than those reported for the Qiantang River in China for 4 fish species (Saury, mollet, white fish, perch, carp, gurnet, bulltrout) ranging from 2.6 – 133.5 ng/g ww (Zhou et al., 2007).

Endosulfan I was the most abundant at concentrations varying between 8.54 -

66.0 ng/g ww (x̄ = 30.0 ng/g). It was the only OCP that exceeded the Maximum

Residue Limits (MRLs) of 50 ng/g ww prescribed by the European Union and

FAO/WHO (Codex, 2009). Kruitwagen et al., (2008) reported DDT and HCH levels in barred mudskipper (Periophthalmus argentilineatus Valenciennes) from the Tanzania coastal mangrove between 37 - 1080 ng/g lw and 18 - 2570 ng/g lw respectively.

These HCH concentrations are exclusively for lindane (ϒ-HCH). (Mustafa et al., 2019) reported ΣDDTs in fish from flood plain of Sonargaon upazila in Bangladesh for Corica soborna (7.5 ± 0.8 ng/g ww), Puntius sophore (x̄ = 7.55 ± 0.5 ng/g ww), Heteropnuestes fossilis (x̄ = 12.8 ± 1.2 ng/g ww), Glossoobius giuris (x̄ = 14.01 ± 1.6 ng/g ww), Mystus vittatus (20.5 ± 0.8 ng/g ww), Nandus nandus (x̄ = 32.5 ± 3.7 ng/g ww), Chewa

Pseudapocryptus elongatus (x̄ = 41.9 ± 3.6 ng/g ww). Adu-Kumi et al., 2010 reported high DDTs levels in 2 fish species (red belly tilapia and catfish) from Lake Volta, Lake

CHAPTER I :Bibliographic Review

Bosomtwi, Lake Weija at concentration ranging from 28.7 - 2205.5 lw (x̄ = 440.9 ng/g).

In Tilapia, all HCHs concentrations were below detection limits (BDL) in all lakes except lindane (ϒ-HCH) in Catfish between 0.6 - 0.8 ng/g lw (x̄ = 0.7 ng/g). Apart from chlordane compounds, catfish from Lake Volta and the Weija Lake had more of other

OCPs than tilapia. This could suggest effective ban of lindane in Ghana and therefore no use for agriculture or fishing in the three lakes. Based on concentrations OCPs measured in the tilapia, this latter study revealed that Lake Bosumtwi was a relatively more polluted freshwater body than Weija Lake and Lake Volta (Veljanoska-

Sarafiloska et al., 2011) found ΣDDTs in the muscle tissue of the Barbus peloponnesius, Valenciennes (1842) in Lake Ohrid (Macedonia/Albania) ranging from

7.6 – 9.7 ng/g ww. Rahmawati et al., (2013) reported total OCP concentration in catfish from ponds in the Citarium watershed from 40 – 149 ng/g ww and mean concentrations of DDTs (x̄ = 3.7 ng/g ww) and lindane (x̄ = 8 ng/g ww).

Verhaert et al., (2013) reported DDTs and HCHs levels in rivers of the Congo

River basin, from six fish species consumed by the local population, Marcusenius sp.,

Shoulderspot catfish (Schilbe marmoratus), Blackline glass catfish (Schilbe grenfelli),

Bigeye squeaker (Synodontis alberti), Spot-tail robber (Brycinus imberi) and Sharktail distichodus (Distichodus fasciolatus) ranged from 0.01 - 504 ng/g and 0.01 - 66 ng/g lw respectively. The DDT and metabolites profile indicate a historical DDT use. In invertebrates, the shrimps Caridina africana and Macrobrachium sp., two species of apple snail (Lanistes cf. ovum and Pila sp.) lower concentrations were reported for

ΣDDTs and ΣHCHs and ranging from 0.01 - 27, 0.01 - 46 and 0.01 - 507 ng/g lw respectively. Lindane was the dominant HCH isomer in both fish (62 % of ΣHCHs) and invertebrates (52 % of ΣHCHs).

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The mean of ΣPCB levels in fish from African aquatic environments were < 1000 ng/g lw for lakes (x ̅ = 29.9 ng/g, x̄ = 24.1 ng/g) and coastlines (x ̅ = 971 ng/g, x̄ = 350 ng/g (Appendix 1). (Kruitwagen et al., 2008) reported ΣPCBs levels in fish tissues

(barred mudskipper) between 84 - 5040 ng/g lw from the Tanzanian coast. The dominant congeners were CB 153, CB 138, and CB 180, which belong to the group of

HexaCB (5 chlorine atoms), HexaCB (5 chlorine atoms) and HeptaCB (7 chlorine atoms) respectively. These are highly toxic and lipophilic PCB congeners that could easily bioaccumulate or biomagnify through the food web. Their presence was attributed to plastic manufacturers located near Dar es Salaam (Tanzania) or the use of outboard engines in the semi closed bay because of the absence of industries in this area. Verhaert et al., 2013 reported Σ10PCBs in six fish species and invertebrates

(shrimps and snails) from 0.01 - 3664 ng/g and 0.01 - 507 ng/g lw respectively. The dominant congeners were penta and Hexa-CBs, CB 153, 149, 101 and 138, for sediment, fish and invertebrates, indicating dominance of technical PCB mixtures close to Aroclor 1254. The use of PCB contaminated oil in old engines and power transformers on boats and in industrial and agricultural activities were suggested as the potential source of PCB contamination in this area (Cooper, 2005).

In Hartbeespoort Dam, South Africa, Rimayi and Chimuka, (2019) reported

Σ16PAHs in different parts (muscle, liver, spleen and kidney) of the carp (Cyprinus carpio) between 184000 – 885000 ng/g wet weight (ww) and the catfish (Clarias gariepinus) between 397 – 2149 ng/g ww. The PAH profile in both fish species was dominated by LPAHs over HPAHs, indicating a strong petrogenic contamination source. Similarly, the dominance of LPAHs was observed in edible fishes, bighead carp (Aristichthys nobilis) and silver carp (Hypophthal- michthys molitrix), from the

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Poyang Lake (largest freshwater lake) in China. PAH levels in the Poyang Lake ranged from 105 to 513 ng/g ww and from 53.9 to 401 ng/g ww in different tissues of bighead carp and silver carp, respectively.

Barhoumi et al., (2016) found concentrations of Σ15PAHs in fish, eels (Anguilla anguilla) and mussels (Mytilus galloprovincialis) of the Bizerte lagoon (Tunisia) ranging from 114.5 - 133.7 ng/g (x̅ = 124.6 ng/g) and 107.4 - 430.7 ng/g (x̅ = 264.8 ng/g) respectively. The PAH composition was dominated by LPAHs (2 - 3 rings) accounting for 59 of ΣPAHs while HPAHs (4 - 6 rings) accounted for 41% of ΣPAHs. PAH ratios indicated mixed pyrolytic and petrogenic sources. Surprisingly the most volatile PAH,

Naphthalene (57.9 ± 26.5 - 68.6 ± 27.0 ng/g) was the most abundant congener in fish and mussels’ tissues. This was attributed to the oil refinery found 10 Km north of the lagoon. For fish, these levels were much lower than those recorded in Anguilla anguilla

(muscle) Camargue Biosphere Reserve in France, 2370 ± 1250 - 90,500 ± 45,900 ng/g

(Roche et al., 2002) and comparable to that found in Anguilla anguilla (muscle). From the Vaccarès lagoon, France with mean concentrations of 313.65 ng/g (Buet et al.,

2006). For mussels, these concentrations were higher than those found in Mytilus galloprovincialis from the Gironde estuary, France between 49.8–101.5 ng/g (Bodin et al., 2011) but comparable to Mytilus edulis from Seine estuary, France 147 - 447

(Rocher et al., 2006) and lower than Mytilus galloprovincialis from the San Diego Bay,

USA (1831 - 23,985 ng/g) (Anderson et al., 1999).

In the Ovia river, Σ17PAHs PAHs levels in fish (Clarias gariepinus) varied between 10 - 914.4 ng/g ww (x̅ = 378.56 ± 274.05 ng/g) (Tongo et al., 2017). The pattern of PAHs was dominated by LPAHs (2 - 3 rings) representing 80 % ΣPAHs

While HPAHs (4 - 5) represented 20 % ΣPAHs. PAH ratios indicated mixed pyrolytic 93

CHAPTER I :Bibliographic Review and petrogenic origin dominated by petrogenic sources. This was attributed to oil exploitation and production activities carried out in this area, such that oil spills often occur due to vandalism on pipelines.

The variation of POPs concentrations in fish species could be due to differences in their feeding habit and metabolic characteristics. Tillapia sp is the most studied fish species in aquatic environments in Africa. Tilapias are middle to top feeders and primarily herbivorous cichlidae, its diet is dominated by phytoplankton (Chlorophyceae,

Cyanophyceae and Euglenophyceae) or benthic algae. The catfish species are bottom to middle feeders. The African sharptooth catfish (Clarias gariepinus) represents the most common species in the aquatic ecosystem, it feeds on insects, namely chiromidae and detritus while bonga shad or bonga (Ethmalosa fimbrata) feeds on detritus, phytoplankton and sand grains depending on the size. Ibigbami et al., (2015) showed evidence of POPs enrichment and bioaccumulation in fish and sediments compared to water. The aforementioned fish species represent an important source of food and revenue for fishing communities in many African countries. For instance, the

Bonga is highly consumed in West and Central Africa, where it is often smoke-dried for preservation and taste. Generally, Fish and invertebrates in Africa show moderate pollution levels for PAHs according to the contamination levels assigned by (Baumard et al., 1998).

Generally, coastlines show the highest mean concentration of ΣPOPs in fish followed by coastlines, lakes, dams and reservoirs of African aquatic environments as shown in figure 11.

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Figure 11. Concentration of POPs (OCPs, PCBs and PAHs) in fish (ng/g, lipid weight) of

African aquatic environments (Mean concentrations in Appendix 1).

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Table 13. Summary of OCPs, PCBs and PAHs levels in biota of African aquatic environments

Aquatic Country Main activities Number of Fish and invertebrates Stations environment Concentration range (ng/g) of lipid weight Reference (species) DDTs HCHs OCPs 7 PCBs 16 PAHs Lake Chad Nigeria Agricultural, industrial (4) F NC NC 0.5 – 8.3 NA NA Akan and Chellube 2014

Lake Noukoué, Benin Disease vector control (6) F 183 – 580 K 4 – 28 E 275 - 786 NA NA Yehouenou et al 2014 Cotonou Lagoon

Lake Victoria Uganda Industrial 4 F ND 0.02 – 0.05 ND 0.04 – 0.7 ND Ssebugere et al., 2014

Lake Awassa Ethiopia Agricultural, industrial, (3) F 1.8 – 21.3N NA 1.8 – 21.3 NA NA Yohannes et al., 2013 urbanization

Lake Volta, Lake Ghana Agriculture, fishing, urbanization 3 (6) F 28.7 - 2205.5 0.60 - 0.83 W - NA NA Adu-Kumi 2010 Bosomtwi, Lake Weija N

Lake Bosomtwi Ghana Agriculture, fishing (50) F 3.6 0.1 E NC ND ND Darko et al. 2008

Lake Burullus Egypt Agriculture, fishing (2) F 2.8 – 45.1 N ww 0.4 – 16.7 E. ww 4.1 – 76.6 ww 3.3 – 44.8 ww ND Saïd et al 2008

Lake Victoria Tanzania Agricultural, fishing 13 (2) 3800 AC NA NC NA NA Henry and Kishimba, 2006

Lake Tanganyika Burundi Fishing, industrial (7) F 68.3 – 909.1 21.2 – 288.2 D NC 24.3 – 106.4 NA Manirakiza et al 2002 N

Ga-Selati River South Africa Agriculture, mining 3 (5) F 4.4 – 5643 N

3 (1) I 2 – 133 N < BDL W NC < BDL W NA

Niger River Nigeria Agricultural 4 (10) F 24.4 – 1561 Y 29.3 – 393 W 1006 - 4302 AA NA NA Unyimadu et al., 2018 (WW)

Ovia River Nigeria Agriculture, fishing 3(54) F NA NA NA NA 10 - 914.2 WW Tongo et al. 2017

Komadugu River basin Nigeria Agriculture, fishing (4) F NA NA 20 - 1420 WW NA NA Mohammed et al., 2017

Ogbese River Nigeria Agricultural, fishing (5) F 6.8 – 32.3 WW 21.6 -115.2D 139 - 490 WW NA NA Ibigbami et al., 2015 WW

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Aquatic Country Main activities Number of Fish and invertebrates Stations environment Concentration range (ng/g) of lipid weight Reference (species) DDTs HCHs OCPs 7 PCBs 16 PAHs

Congo River basin DR Congo Agricultural, industrial, (6) F 0.1 – 504N 0.1 – 66D NC 0.1 - 3664 Q Verhaert V. et al., urbanization 2013

(2) I 0.1 – 27 N 0.1 – 46 D NC 0.1 – 507 Q NA

Saldanha Bay South Africa Mining, industrial, crude oil (2) m 0.7 – 0.9 H - - 0.7 – 48 Q 1 – 13.4 AB Firth 2019 imports

Bizerte Lagoon Tunisia Harbour, urbanization, industrial, 5 (50) F NA NA NA NA 114.5 - 133.7 X Barhoumi et al., 2016 agricultural, oil refinery 5 (30) m NA NA NA NA 107.4 - 430.7 X Lagos Lagoon Nigeria Industrial, fishing, urbanization (3) F 21.9 - 250.9 88 - 1285.5 182.4 - 1906.8 NA NA Adeyemi et al 2008

Fadiouth and Falia Senegal Agricultural 2 (5) m 1.6 – 15.6 0.1 - 2.1 1.9 – 17.8 O 5.7 – 38.0 NA Bodin et al., 2011 EstuaryR

Tanzania coastal Tanzania Industrial, agricultural 6 (1) F 37 - 1080 18 – 2570 E NC 84 - 5040 Q NA Kruitwagen et al., mangrove 2008

Napoleon Gulf Uganda Industrial, urbanization 4 (1) F NA 15.0 to 46.0 D NC 0.04 – 0.7A NA Ssebugere et al., 2014

Cape cross Namibia Agricultural Seal (1) F 11 - 1115 3 – 19 E NC 4 - 697 NA Vetter et al., 1999

Hartbeespoort Dam South Africa Fishing (2) F NA NA NA 1.8 – 592.4 18400 - 2149 Rimayi 2019 Z

Tekeze Dam Ethiopia Agriculture, vector control, fishing 2 (7) F 3.5 - 5.6 H 0.65-1.47E NC NA NA GA 2016 ND= Not detected, NA = Not analysed, NC =Not computable, BDL= below detection limit, DS = Dry season, R = Ramsar site, NC = not computable, I = Invertebrates, S = seal, WW = Wet weight, F = fish, m = molluscs, A = 6 PCBs, B = 13 PAHs, C= 17 PAHs, D= 3HCHs, E = γ-HCH, G = p,p’ -DDE, H = 1 DDTs, I = 2 DDTs, J = 29 PCBs, k=5 DDTs, L = 10 PCBs, M= 18 OCPs, N=6 DDTs, O = 14 OCPs, P = 2 HCHs, Q = 10 PCBs, T = 21 PCBs, U = 12 PCBs, V = 21 OCPs, W = 4 HCHs, X = 15 PAHs, Y = 3 DDTs, Z = 31 PCBs, AA = 20 OCPs, AB = 5 PAHs

CHAPTER I :Bibliographic Review iii) Water

Generally, limited knowledge is available for POPs in African water resources.

In most studies, the concentrations were below detection limit by reason of their hydrophobicity making them preferentially adsorbed to particulate matter and deposited in sediments. However, based on the studies examined, the mean concentrations of ΣOCPs in water of African aquatic environments were < 1400 μg/L; lakes (x̅ = 196.5 μg/L, x ̄ = 82.9 μg/L), rivers (x ̅ = 1319.2 μg/L, x ̄ = 105.6 μg/L) and coastline (x ̅ = 189.9 μg/L, x ̄ = 16.3 μg/L) Appendix 1. The mean concentration of

ΣDDTs in water of African aquatic environments was < 150 μg/L for lakes (x̅ = 148.2

μg/L, x ̄ = 3.2 μg/L), rivers (x ̅ = 55.1 μg/L, x ̄ = 1.2 μg/L) and Dams (x ̅ = 0.1 μg/L, x ̄ = 0.1

μg/L) Appendix 1. The mean concentration of ΣHCHs in water of African aquatic environments was < 30 μg/L for lakes (x ̅ = 0.2 ng/g, x̄ = 0.17 ng/g) and rivers (x̅ = 27.6 ng/g, x ̄ = 8.9 ng/g) Appendix 1. (Unyimadu et al., 2018) reported ΣOCPs, ΣDDTs and

ΣHCHs levels in water from the Niger River between 0.02 - 1.9 μg/L, 0.006 – 0.6 and

0.002 – 0.06 respectively. The concentrations of ΣOCPs in the Niger river were comparable to those Minjiang River Estuary in China (0.2 - 1.8 μg/L) (Zhang et al.,

2003), Taihu Lake in China (0.031–1.2 μg/L) (Wu et al., 2014) but higher than ΣOCPs the Chenab river in Pakistan (0.008 – 0.76 μg/L). (Ibigbami et al., 2015) reported concentrations of ΣOCPs, ΣDDTs and ΣHCHS between 7 - 42.3 μg/L, 0.6 - 3.9 μg/L

(x̅ = 2.6 μg/L) and 2 - 15.8 μg/L (x ̅ = 8.8 μg/L) respectively in the Ogbese River, Nigeria.

The mean concentrations of the most abundant OCPs were, β-HCH (x ̅ = 7.45 μg/L), sulfate (x̅ = 6.28 μg/L), endosulfan II (x ̅ = 4.45 μg/L), heptachlor epoxide (x ̅ = 2.73 μg/L) and lindane (x ̄ = 2.08 μg/L). The concentration of parent compounds such as aldrin and p,p’ DDT were higher than their metabolites dieldrin and p,p’ DDD, which might

CHAPTER I :Bibliographic Review suggest recent use despite their ban in the 1990s in most of African countries. The mean concentrations of 10 out of the 15 pesticide residues detected were above the maximum acceptable concentration of 0.1 μg/L set by the European Union for the protection of the aquatic environment and drinking water but very low compared to the allowable level of 10 μg/L set by the Federal Environmental Protection Agency (FEPA)

1991, in Nigeria. The occurrence of OCPs in this area was assigned to previous and current use of the pesticides, run-off from various agricultural areas, washing of materials or containers after pesticide application. (Mohammed et al., 2017) found seasonal levels of ΣOCPs in water from 0.67 - 2.55 μg/L in the Komadugu River basin in Yobo state, Nigeria. Regardless of the fact that DDTs and HCH were not determined, dieldrin was the most abundant OCP followed by aldrin. The highest levels of OCPs were recorded during the rainy season compared to the harmattan and dry season.

Generally, these levels were higher than DDT and HCH concentrations in Haihe River ranging from BDL - 1.2 μg/L (x ̅ = 0.002 μg/L) and 0.04 - 2.1 μg/L (x ̅ = = 0.2 μg/L) respectively (Wang et al., 2010). Levels of DDT and HCHs in African waters were generally lower than those found in the Taihu Lake (China) for DDT (BDL – 9.3 μg/L) and HCH (BDL – 36.0 μg/L) (Feng et al., 2003) and the Pearl River delta for DDT (2.9

- 5.5 μg/L) (x ̅ = 3.9 μg/L) except HCHs (0.002 - 0.005 μg/L) (x ̅ = 0.004 μg/L) (Guan et al., 2009).

The mean concentrations of ΣPCBs in water of African aquatic environments were < 150 μg/L for lakes (x ̅ = 2.25 ng/g, x̄ = 2.25 ng/g) and rivers (x̅ = 141.25 ng/g, x̄

= 141.25 ng/g) Appendix 1. Samia et al., 2018 reported ΣPCB levels in surface water from the El Bey River system varying from 90 to 470 μg/L with the detection of the congeners CB 28, 52, 101, 153, 138, 180, and 209. The main sources of pollution were

CHAPTER I :Bibliographic Review suggested to be domestic and industrial wastewater evacuations. Along the course of the Buffalo River, Yahaya et al., (2017) reported seasonal (autumn and summer) ΣPCB levels from 0.005 - 0.3 μg/L. The congener PCB 101 was the most abundant at almost every site. Out of 19 PCB congeners, 0 to 13 congeners were detected in autumn and

2 to 9 PCB congeners in summer. The ΣPCB concentrations at all stations during summer were below the World Health Organization (WHO) permissible level of

500 ng/L recommended for humans but were exceeded at all the sampling sites in autumn except one site.

(Samia et al., 2018) reported PAH levels in surface water from the El bey river system in Tunisia between 370 - 9910 μg/L. Naphthalene, fluorene, fluoranthene, and anthracene were detected with a predominance of LPAHs between 75 and 100 % of

ΣPAHs. In surface water of the Buffalo River Estuary, South Africa, (Adeniji et al., 2019) recorded Σ16PAHs ranging between 14.9 - 206 μg/L (76.1 ± 11.0 μg/L).

Benzo(a)anthracene (90 %) was the most frequently detected and acenaphthylene (7

%) the least detected. Naphthalene, chrysene, and benzo(a)pyrene exceeded their maximum allowable concentrations (MAC) in fresh and marine waters (CCME, 2008).

Benzo(a)pyrene equally exceeded Permissible limit of 0.2 μg/L prescribed by the

Agency for Toxic Substances and Disease Registry (ATSDR, 2009). A dominance of

HPAHs (65 %) was observed over LPAHs (35 %) and Carcinogenic PAHs (CPAHs) contributing over 47 % of ΣPAHs. PAH ratios revealed a predominant pyrolytic origin over the petrogenic origin. The petrogenic origin was attributed to possible leakage of fuel from two-stroke engines of the fishing boats and yacht in the area. In summer

(64.21 μg/L) higher mean concentration of ΣPAHs recorded compared to autumn

(57.33 μg/L).

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Generally, rivers show the highest mean concentration of ΣPOPs in water than lakes of African aquatic environments as shown in figure 12.

Figure 12. Concentration of POPs (OCPs, PCBs and PAHs) in water (μg/L) of African

aquatic environments(Mean concentrations in Appendix 1).

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Table 14. Summary of OCPs, PCBs and PAHs levels in water of African aquatic environments

Number of Water Stations Aquatic Mean concentration range (mean concentration) (μg/L) Country Main activities (Number References environment of DDTs HCHs OCPs 7 PCBs 16PAHs samples) Lake Togo Togo Agriculture, fishing, disease 8 (48) ND <0.001 – NC NA NA Mawussi et al 2016 vector control 0.93 E

Lake Bosomtwi Ghana Agriculture, fishing 10 <0.01 – 0.01 – 0.45 W NC 1.09 – NA Afful 2013 6.35 Y 5.87 A

Lake Bosomtwi Ghana Agriculture, fishing (50) 0.01 H 0.1 E NC ND ND Darko et al. 2008

Lakes Lake Burullus Egypt Agriculture, fishing 4 (12) 0.00007 – BDL – 0.5 0.0002 – 0.001 – ND Saïd et al 2008 0.9 1.6 1.9 Lake Volta Ghana Agriculture, fishing 6 (180) ND 0.008 ± NC ND ND Ntow 2005 0.005

El bey River system Tunisia industrial, urbanization, 13 (13) NA NA NA 90 - 470 370 - 9910 Samia et al 2018 agricultural

Niger River Nigeria Agricultural 3 0.006 – 1.6 – 61.3 0.02 – 1.9 NA NA Unyimadu et al., 2018 0.56 et al Ovia River Nigeria Agriculture, fishing 3(54) NA NA NA NA 2.3 - 25.8 Tongo . 2017 Nairobi River Kenya Industrial, agricultural 3(54) NC NC BDL - 0.09 NA NA Ndunda et al., 2018

Buffalo River South Africa Agricultural, industrial, 6 NA NA NA 0.1– 4.9 NA Yahaya et al. 2017 urbanization Komadugu River Nigeria Agricultural, fishing 5 NA NA 0.67 - 2.55 NA NA Mohammed et al., basin 2017 Rivers and Streams and Rivers Ogbese River Nigeria Agricultural, fishing 5 0.6 – 3.9 2 – 15.8 7 – 42.3 NA NA Ibigbami et al., 2015

Ondo State Rivers Nigeria Agricultural 11 (11) 0.02 ± ND NC NA NA Okoya et al., 2013 (DS) 0.01H

Yala/Nzoia River Kenya Agricultural, vector control 9 (DS) BDL BDL BDL NA NA Musa et al. 2011

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Densu River basin Ghana Agriculture, fishing 3 (15) 0.01 – 0.03 0.05 – 0.1 NC NA NA Kuranchie-Mensah et al. 2012 Number of Stations Aquatic Water Country Main activities (Number environment Mean concentration range (mean concentration) (μg/L) References of samples) DDTs HCHs OCPs 7 PCBs 16PAHs

Ashanti region Ghana Agricultural (50) < 0.1G 0.095 E NC NA NA Ntow 2001 streams Buffalo River Estuary South Africa Industrial, harbour 5 (60) NA NA NA NA 14.91 – Adeniji et al 2019 206

Swartkops and South Africa Industrial, agricultural 10 0.001 – 0.0007 – 0.2 0.2 – 0.25 NA NA Olisah et al., 2019 Sundays River 0.08 Y W M Estuaries Estuaries Tekeze Dam Ethiopia Agriculture, vector control, 2 (24) 0.1- 0.2H 0.4 - 1.5E NC NA NA GA 2016

fishing Aiba reservoir Nigeria Agriculture, fishing, 4 0.05 ± ND NC NA NA Olutona et al., 2014 urbanization 0.08H Dams and and Dams Reservoirs ND= Not detected, NA = Not analysed, NC =Not computable, BDL= below detection limit, DS = Dry season, R = Ramsar site, NC = not computable, A = 6 PCBs, B = 13 PAHs, C= 17 PAHs, D= 3HCHs, E = γ-HCH, G = p,p’ -DDE, H = 1 DDTs, I = 2 DDTs, J = 29 PCBs, k=5 DDTs, L = 10 PCBs, M= 18 OCPs, N=6 DDTs, O = 14 OCPs, P = 2 HCHs, Q = 10 PCBs, T = 21 PCBs, U = 12 PCBs, V = 21 OCPs, W = 4 HCHs, X = 15 PAHs, Y = 3 DDTs, Z = 31 PCBs, AA = 20 OCPs, AB = 5 PAHs

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1.8.5 Underexamined areas, future research and recommendations

The term underexamined areas here is polysemous as it refers to limited number of studies in terms of geographical coverage (study sites) and examined environmental issues (target POPs and studied matrices). There is a widespread lack of data on POPs in most of African countries such that about one in three African countries have published scientific studies on POPs in aquatic environment. Many studies present limited temporal and spatial coverage for sample collection. Most studies are conducted as single sampling events in localized areas which provide only a snapshot of the pollution in the area of interest. There is lack of knowledge on

POPs in remote areas because most studies are focused on highly urbanized, industrialized and agricultural areas. Worldwide, POPs have been found in areas where they have not been used or produced as a result of their ability of long-range transport and persistence. Therefore, future studies should focus on the evaluation of POPs in remote areas. There is a need for more studies in order to tackle the aspect of spatial and temporal coverage. It is necessary that continuous monitoring studies be carried in areas which have been identified as hotspots of pollution and for chemicals like DDT which are still recommended to be used for the fight against the malaria vector disease. Long-term monitoring stands as a more effective means of assessing pollution in aquatic ecosystems.

Very few data are available for emerging POPs such as polybrominated diphenyl ethers (PBDEs), also known as Brominated Flame Retardants (BFRs).

BFRs are compounds added to products such as carpets, upholstered furniture, electronic devices and curtains. Likewise, attention should be paid on unintentionally produced POPs such as polychlorinated dibenzo-p-dioxins (PCDDs) and 104

CHAPTER I :Bibliographic Review dibenzofurans (PCDFs) and Short-chain chlorinated paraffins (SCCPs) used as lubricants in metalworking applications and in polyvinyl chloride (PVC) processing.

They are used as plasticizers and flame retardants in a variety of applications, including in paints, adhesives and sealants, leather fat liquors, plastics, rubber, textiles and polymeric materials. Over the past decades, the aforementioned products have been increasingly imported in Africa and these compounds used in various applications, thus a potential risk to human and environmental health. More interest has been focused on organochlorinated pesticides when compared to other groups of pesticides such as organophosphates, pyrethroids, carbamates and neonicotinoids of which few studies have revealed high environmental levels in Africa and harmful effects to human and living organisms.

A very limited number of studies exist on the levels of POPs in human body fluids (milk, serum, blood and urine) and almost inexistent knowledge on the human clinical effects related to POP exposure or poisoning in Africa. Considered as semi- volatile organic compounds, POPs undergo considerable exchanges between various compartments of the biosphere (lithosphere, hydrosphere and atmosphere).

Thus, future studies should focus on the occurrence and levels of POPs in the atmosphere. Despite the fact that the economies of most African countries are dependent on agriculture, few studies have been carried out to determine the levels of POPs in soils and food crops (Akoto et al., 2013) particularly pesticides on which have been detected in food crops from local markets above prescribed Maximum

Residue Limit (MRL) (Mazlan et al., 2017). More of such data is needed, added to the fact that one of the major routes of exposure to POPs by humans is through food

105

CHAPTER I :Bibliographic Review intake. In general, data on the import, production and/or use of pesticides and other

POPs is poorly documented and not readily accessible.

1.8.6 Limitations of the review

This review encountered some drawbacks such as limited knowledge available on the quality control and quality assurance parameters employed to validate the reported results. The comparison of OCPs, PCBs and PAHs levels particularly for biota was complex due to differences in species, trophic and developmental level, body parts and unit of concentrations (wet weight, lipid weight and dry weight).

Furthermore, the absence of risk assessment criteria or quality guidelines specific to

African aquatic ecosystems for data comparison and interpretation. Information on analyses in African laboratories is not known because almost all published articles did not indicate the laboratories in which laboratory analyses were carried out.

Conclusion

The present review is a good representative of current OCPs, PCBs and PAHs contamination in aquatic environments of Africa. The presence of OCPs, PCBs and

PAHs in African aquatic environments indicates continuous exposure of aquatic organisms and humans to these contaminants. This is of great concern in a context of rapid demographic growth, urbanization, industrial and agricultural development.

Africa is characterized by an overall scarcity of data on POPs mostly available for countries such as Nigeria, Egypt, South Africa, Ghana and Tunisia. Though contamination levels are generally low for sediments, biota and water, sediment contamination for instance is associated with concentration ranges within which

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CHAPTER I :Bibliographic Review adverse biological effects will occur occasionally or may occur frequently on aquatic organisms. Generally, the highest levels of POP contamination were recorded at stations close to shipping, industrial activities and urban areas. The most common sources of POP contamination included, the discharge of agricultural or industrial effluents, the discharge of household and municipal wastes and to a certain extent fishing with chemicals. Besides, contamination due to petroleum or oil refinery and harbour activities were common in coastal zones.

African aquatic environments are subjected to the same anthropogenic pressure rapid urbanization, agriculture and increasing industrial development. This review reaffirms the findings of previous studies on the fact that higher concentrations of POPs are associated with high human settlement, urbanization centres, estuaries and marine sediments near major metropolitan areas. Generally, PCB and PAH profiles in all studied samples were dominated by high molecular weight compounds indicating their persistence of contamination in African aquatic environment. The highest contamination levels were recorded in or at areas close to industrial, agricultural and urbanized areas. This study brings to light perspectives on the need for future studies related to POP contamination in other African countries with inadequate waste management systems, similar or different demographic growth and urbanization patterns, agricultural and industrial activities.

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CHAPTER 2 : Study Sites

2.1. Lake Barombi Watershed

2.1.1 Geographical context

The Lake Barombi Mbo watershed (LBW) is located northwest of the city of

Kumba, southwest region of Cameroon (figures 13 and 14) at 04⁰39'362'' N,

009⁰24'360'' E and 311 m above sea level. It hosts a 1 million-year-old volcanic and explosive crater lake located along the Cameroon Volcanic line (CVL) known as

Barombi Mbo, “Mbo” meaning lake in local language, which is the largest (2.5 km wide and 111 m deep) volcanic lake in west and central Africa (Asaah et al., 2020;

Tabot et al., 2016) The Lake, which was designated second Ramsar site of

Cameroon in 2006 by UNESCO, is famous for the occurrence of 12 endemic fish species out of 15 in the lake such as Sarotherodon linnellii (Unga sp.), Pungu maclareni and Cichlids. It equally hosts an endemic specie of freshwater sponges

(Corvospongilla thysi) and species of shrimp (Caridina sp), rendering it one of the places with the highest densities of endemic species per area in the world (Schliewen and Tanjong, 2006). Lake Barombi Mbo is found within the forest reserve created in

1940 (Order No.17 of 16/02/1940, British Southern Cameroon) in accordance with the forestry Ordinance of 1938 and more recently has assumed the status of a Forest

Reserve (Law No. 94/01 of January 1994 and Decree No. 95/55 of August 1995)

(Fonge et al., 2019).

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Figure 13. Location of a) Cameroon, b) Southwest region and c) Lake Barombi Watershed

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Figure 14. Location of Lake Barombi and the city of Kumba

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The climate in Kumba region is an equatorial type generally very hot and humid with two seasons of which a long dry season (November to February), a long rainy season (March to June), a short dry season (July to August) and a short but intense rainy season (September to November). The average annual rainfall varies between 3000 and 4000 mm, with a relative humidity between 70 and 80 % and mean temperature range of 24 to 35 °C (Fonge et al., 2019).

With regards to pedology and geology, Kumba is characterized by soils resulting from alteration of crystalline rocks between Kumba and Nguti, hydromorphic soils and clay-silt between Nguti and Bachuo Akagbe (towards the northern part of

Kumba at about 99 km and 159 km respectively). However, based on the composite fragments contained in its pyroclastic deposits, it is likely that the maar cuts through a geological succession composed by a granite-gneissic formation, sandstone, and basaltic lava flows; the same formations that make up the Kumba volcanic field

(Tchamabé et al., 2014).

2.1.2 Anthropic activities

The adjacent village to the lake is known as Barombi Mbo village. It is situated some 100 m from the lake with a total population of over 350 persons almost entirely dependent on the lake and the forest reserve for their livelihoods (Balgah and

Kimengsi, 2011). The lake represents an important sacred site to the Barombi tribe: the social and cultural life of the Barombi Mbo people is intimately linked to the use of the resources of the lake through fishing, farming and a means of transport

(Appendix 2). The main anthropic activities carried out in the LBMW are agriculture: slash and burn farming (Appendix 3), spraying of pesticides, fishing using gill nets

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(Appendix 4) and sometimes with chemicals (Balgah and Kimengsi, 2011). Appendix

5 and 20 shows the dumping of pesticides on farms after use. Mixed cropping is carried on the same plot for crops such as bananas and cocoa (Appendix 6) or palm oil (Appendix 7). (Fonge et al., 2019) reported an active forest conversion into farmland which in turn affects the lake and its inlets.

Lake Barombi Mbo is the main source of drinking water supply to the nearby town of Kumba, which is experiencing rapid demographic growth and urbanization.

The population in Kumba was 144 000 according to the census results of 2005

(Balgah and Kimengsi, 2011). In this regard, its population is expected to reach 450

000 inhabitants by 2027 (Kumba Urban Council, 2012). This growing population presents an increase in demand for farmland, timber (TFPs) and Non-Timber Forest

Products (NTFPs), fuel wood, and fishery.

2.2. Wouri Estuary Mangrove

2.2.1. Geographical, Geomorphological and hydrodynamic characteristics

The Wouri Estuary Mangrove is located in the Gulf of Guinea close to

Cameroon’s largest and most populated city known as Douala (figure 15). The coastal city of Douala occupies a surface area of 932 km2 with a population of about

3 million inhabitants. Between 1987 and 2012, its annual growth rate was 6.3 % and tripled between 2005 and 2017 (Shores et al., 2019). The population of Douala is expected to reach 4.7 million inhabitants in 2020 (UN DESA, 2019). This city acts as the economic capital and contributes over to over 33% of the Cameroon’s Gross

Domestic Product (GDP) representing about 50% of Central Africa’s GDP. Over 90

% of industries in Cameroon are found in and around Douala and are mainly located 113

CHAPTER 2 : Study Sites in the Bonaberi and Bassa industrial zones. This city deals with 95 % of the seaborne trade and the port of Douala is the largest port in Central Africa. Douala stands as the first industrial centre in Cameroon and the Economic and Monetary Community of Central Africa (CEMAC).

The littoral region presents a hot and humid equatorial climate. It is subjected to the Gulf of Guinea monsoon flux and exhibit two climates; a wet season from April to October, with July and August as the rainiest months, and a dry season from

November to March. It supports high annual rainfall with mean precipitation of 4000 mm, a mean annual temperature of 26.7 °C and mean relative humidity of about 80

% (Fonge, 2011). According to Din et al., 2017, winds almost give a constant direction to coastal currents that carry and deposit large amounts of materials (mud, clay, sand) in the “Cameroun mouths” where due to silting up permanent dredging of the

Douala Port channel is carried out.

The texture of mangrove sediments is rarely homogeneous, often characterized by a succession of clay beds on surface and sandy beds in depth or by alternating of these two layers (Din et al., 2017) as shown in figure 16. Because of the permanent flooding by tides, chemical ripening (transformation of sediments into soil) is more advanced than the physical. The pH is very fluctuating between two successive stages of engorgement (high tide) and drying (low tide). The mangrove soils are generally characterized by a high C/N ratio due to the slowing of the biological activity, induced from anoxia.

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Figure 15. Location of the Wouri Estuary mangrove in the Cameroun coast

(source: Din et al., 2017)

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Figure 16. Cameroun Estuary mangrove soil profiles (source: Din et al., 2017)

The landform of Douala is that of coastal lowlands with low altitudes generally ranging from 1 m on the coast to 40 m in the city (MINHDU, 2014). The basin of the littoral region is composed of sedimentary deposits from which yellow ferrallitic and marine alluvial soils developed. Ferralitic soils that constitute the plateau originate from sandy sedimentary rocks or sandy clays, but marine alluvial soils result from recent deposits and are colonized by the mangrove. These latter are less or non- consolidated soils, that display variable textures according to the condition of formation of the deposits.

The Wouri estuary is a large area (2300 km2) consisting of extensive mangrove swamps, mudflats and creeks. The estuary forms the entrance to the

Wouri river and the Douala port. The Douala estuary is known to host a large number 116

CHAPTER 2 : Study Sites of migrating water birds. To the north of the Estuary, the coast becomes rocky as it skirts Mount Cameroon, the highest mountain in West Africa. The Wouri estuary is, however, one of the main wetlands on the coast of Cameroon subjected to anthropic activities mainly; intense industrial and port activities, fishing, sand extraction, dense traffic, felling of mangrove forest for settlement and fish smoking. In fact, felling of mangrove forest leads to the destruction of habitats and makes the coast vulnerable to erosion and floods.

2.2.2. Hydrology

Mangroves of the Cameroon Estuary are often watered by brackish water resulting from the dilution of seawater by fresh water from rivers and heavy rainfall.

The estuary waters present a stratification with continental waters dominance, warm and rich in nutrients and sediment loads that float on marine water, saltier, colder and clearer. In Cameroon, tide regime is semi-diurnal in with a maximum amplitude that is approximately 3 m high in the Douala port (Wouri river). The local stability of salinity in the Wouri estuary mangrove by the existence of stabilizing factors is explained by

Baltzer et al. (1995).

The Wouri River runs through Douala, 24 km (15 miles) away from the sea. It is one of the most important coastal rivers of Cameroon with an annual flow of 311 m3/s emptying itself into the Atlantic Ocean. The Wouri watershed in Douala has a surface area of about 11700 km2, with an important marine forebay invaded by tides.

Its continental part is drained by two main tributaries; the Nkam and Makombe

(Appendix 8). Currently, the Wouri River faces serious navigation problems on its

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CHAPTER 2 : Study Sites channel due to high alluviation downstream of Yabassi (Appendix 8) and the invasion of its bank by macrophytes. The Wouri river separates the city of Douala into two parts such that the Wouri Brigde creates a link between the residential Douala and

Bassa Industrial zone at the east with the Bonaberi industrial zone at the west as shown in figure 17.

Figure 17. Major rivers in and around the city of Douala

The Mgoua watershed hosts the major part of the Bassa I.Z., drained by the

Mgoua river at the heart of this watershed (figure 18). The Mgoua river has various ramifications in the town of Douala; owing to areas crossed, materials and wastes that it carries towards the Crique Docteur area (southeast limit of Douala) to join the

Wouri river in the estuary area. It is known as the “black river” owing to its blackish

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CHAPTER 2 : Study Sites color (Appendix 9). The other part of the Bassa I.Z. is found in the Tongo Bassa watershed, drained by the Tongo Bassa river which flows into the mangrove area at the level of Djebale Island (Northern limit of Douala) (figure 18). The Dibamba catchment has a surface area of 2400 km2 found south of the Wouri watershed. It is drained by the Dibamba river which crosses a region composed of hills west of the

Sanaga River (largest river in Cameroon).

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Figure 18. Hydrographic networks and watersheds in the city of Douala. (Source; Douala Urban Council 2018)

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2.2.3. Fauna and Flora

The report of the water bird census on the Cameroonian Coast in 2006, preconized that the Wouri Estuary should be listed as a Ramsar site in order that management procedures should be put in place and enforced (Ajonina et al. 2007).

This is because the Wouri estuary exceeds the 1 % Ramsar criterion for bird species listed on the Ramsar convention and a very important breeding colony of a near- threatened bird species. For instance, the number of African Skimmers (Rynchops flavirostris) met the Ramsar criterion of more than 1 % of the regional or global population of a species at a site (Delany and Scott, 2006). The same report indicates that the Wouri Estuary exceeds the criterion for congregations of waterbirds (more than 20, 000 waterbirds at a site). This was previously demonstrated in studies 1998 and 1999 in the Wouri Estuary (West et al. 2002). The most abundant bird species include the Little Egret, Royal Tern, African Skimmers (Rynchops flavirostris), White

Pelicans, Squacco (Ardeola ralloides) and Green-back Herons. Notwithstanding, nowadays the Wouri Estuary has not been added to the list of Ramsar sites in

Cameroon.

The Wouri Estuary hosts various fish and shrimp species , these include the bonga (ethmalosa fimbriata), catfish (Arius heudelotii Valenciennes 1840 &

Siluriforms), bobo croaker or nyendi (Pseudotolithus elongates), Threadfin

(Galoides), shad or munyanya (Ilisha africana), Tilapia (Oreochromis niloticus), a wide range of invertebrates such as the tiger crayfish (Penacus kerathusus), the estuarine white shrimp or njanga (Palaemon), crabs (families Sesarmidae (95 %)

Grapsidae (5 %), Gecarcinidae and Ocypodidae (0.15 %), Portunidae (0.1 %))and

Ocypodidae) and molluscs (Potamididae (45.4 %), Pachymelaniidae (28 %),

121 CHAPTER 2 : Study Sites

Melanopsidae (18.5 %), the Neritidae (5.2 %), Onchidiidae, (0.06 %)) (Gemuh 2017;

Ngo-Massou et al 2012). The most consumed and valuable species by the population of Douala and other regions of Cameroon are Penacus kerathusus, Ethmalosa fimbriata, Arius heudelotii Valenciennes 1840 and Palaemon.

The main mangrove stands in Cameroon are located in the deltas of the Cross river and Wouri. The herbaceous stratum represents less than 1 % of the vegetation.

The pioneer and dominant species is the Rhizophora racemosa forming tall gallery forest of about 40 m (Van Campo and Bengo, 2004). Other species such as

Rhizophora mangle and Rhizophora harrisonii are found at the boundary between

Rhizophora and Avicennia stands reaching about 3 to 6 m.

2.2.4. Anthropic activities

The city of Douala is characterized by a dense industrial network, transport harbor and commercial activities. Industrial activities are carried out in two major areas, the Bassa and Bonaberi industrial zones (I.Z.) (figure 19), located on the East and West banks of the Wouri River respectively. Both cover a surface area of 342 hectares and are managed by the Industrial Zones Development and Management

Authority (MAGZI). The main industries located in the Bassa industrial zone include textile (CICAM), metallurgy (ALUBASSA), agrofood (CHOCOCAM, CAMLAIT),

Cosmetics (EUROCOMESTIC S.A), PVC industries (CMC plastique, PLASTICAM), glass industry (SOCAVER). In 2010, AES-SONEL (electricity company) constructed

2 PCB storage transformer warehouses in Douala in accordance with international standards (UNEP-POPS-NIP-Cameroon, 2016). Before then, illegal sales of transformers were done without prior testing or transformers were abandoned in

122 CHAPTER 2 : Study Sites inadequate sites. The same report revealed that AES-SONEL which is presently known as ENEO was found to be the highest holder of PCB contaminated equipment and transformers in Cameroon. Besides the Wouri Bridge is found a cement plant

(Dangote Cement Company).

Figure 19. Localisation of industries in the city of Douala

Near the Dibamba river there are various PVC industries (Deeplast, Prima

Deeplast, Parlite Foods Sarl, BATIMETAL AFRIQUE, CMC Plastique, HEVECAM)

Chemical plants (Cinpharm, SCIMPOS SA) and paper mill (PACK INDUSTRY S.A).

At proximity to the Douala port and Airport there are PVC industries (SGMC, Les

Grands Complexes Chimiques d’Afrique (LGCCA SA)), chemical plants (Cameroon

Breweries Limited Factory).

The Bonaberi I.Z. is located in the aquatic area of the lagoon which encroaches the on the lagoon (Bonaberi swamps). The main industries include Salt

123 CHAPTER 2 : Study Sites industry (SOCARSEL), cement factory (CIMENCAM), PVC industries (OK Plast

Cam, PETCAM, Super plastic), chemical plants (Fimex International), metallurgy (LP

Industrie Mambanda). Raw effluents from the different industries are directly discharged in the Wouri river through canalization. The dust and gaseous wastes are emitted in the atmosphere through chimneys (Appendix 10).

There exist other areas like Bois de singes, Youpwe and Koweit city with high urbanization and human activities close to the Crique Docteur. They represent major potential sources of pollution to the Wouri Estuary Mangrove due to the presence of municipal waste dumps, sewage and sludge disposal directly connected to the mangrove area at the level of the Crique Docteur which subsequently empties in the estuary. The sewage collected from septic tanks of homes in Douala by trucks are discharged in open basins without prior treatment (figure 20). The capacity of these basins and the volume of waste received daily does not allow the bacterial oxidation.

Consequently, this waste flows towards cultivation areas where they are recovered as fertilizer. From a topographic point of view, the land is progressively lower towards the mangrove, becoming increasingly flooded. This favour the drainage of river water and effluents towards the Crique Docteur and turned along the creek and houses.

124 CHAPTER 2 : Study Sites

Figure 20. Municipal waste dump in Dinde, Koweït City(Crique Docteur zone) (MINHDU,

2014)

The Port Authority of Douala (PAD) and the Douala International Airport represent a major concern interest regarding organic pollution in Douala and particularly in the nearby mangrove. The principal activities include transport, storage, maintenance of transport and handling equipment which could be a potential source of air, water and soil pollution by petroleum products (hydrocarbons) and other organic pollutants. Besides the discharge of effluent could Douala is made up of sandy soils which do not favour urban or peri-urban agriculture compared to other cities like Yaounde, Bafoussam and Bamenda with good pedological conditions.

However, studies have shown that urban and peri-urban agriculture in Cameroon cities like Yaounde and Douala have promoted increasing mosquitoes’ resistance to insecticides (Papito, 2017; Antonio-Nkondjio et al., 2015), water and soil pollution due to improper use of fertilizers and pesticides (Gockowski et al., 2003; Nguegang,

2008; Prain et al., 2010). Urban agriculture is carried out in upscale neighbourhoods like Bonanjo and Bonapriso (Douala) in the form of gardening while fresh vegetables are cultivated in areas like Matanda around Bonassama (northern half of the Wouri estuary). According to (Yemmafouo, 2014), almost half of the neighbourhoods in the northern part of Douala have been subjected to intense gardening

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126 CHAPTER 3 : Material and Methods

Despite the repetition of certain parts of this chapter in the rest of the document

(Chapter 4 and 5), we thought it necessary to have a separate chapter on Material and methods in order to allow clear, further and more detailed explanation of data and discussions that were abbreviated in conformity with publication in scientific journals. Following the review of persistent organic pollutants and polyaromatic hydrocarbons levels in aquatic environments of Africa (Chapter 1, section 1.5), the first part of this chapter presents a summary of the methodological approaches employed for the determination of PAHs, PCBs and OCPs in Africa. This is due to a scarcity of information and absence of reviews on methods and analytical techniques employed to determine POPs in Africa.

3.1. Methodological approach in the determination of PAHs, PCBs and OCPs in African aquatic environments

From the bibliographic study presented in section 1.8, Appendix 11 summarizes the sampling, treatment, extraction and analytical methods employed in the determination of PAHs, PCBs and OCPs in African aquatic environments. It shows that the most studied matrices are sediments, followed by biota particularly fish and lastly water. This could be justified by the fact that sediments act as archives for various pollutants and could be a potential source of release to the environment.

POPs have high binding interactions (lipophilic and hydrophobic properties) nature with solid matrices (sediments), and fatty tissues of living organisms or escape from the aqueous phase to the atmosphere, therefore represent major routes of exposure

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CHAPTER 3 : Material and Methods to humans and along the food chain. Also, it is important to underline that sediments, biota and water are suitable for routine monitoring of environmental pollutants. In

Africa, few studies have been carried out in other important matrices such as air, human fluids (breast milk, blood, urine), food crops and meats.

Sediments are commonly sampled by grab sampling and coring within a depth of 0 to 10 cm. Biota is often sampled with nets, manually or bought from local markets while water is collected by grab sampling in amber glass bottles or glass bottles (1 to

2 L) wrapped with aluminum foil. All samples are stored in ice chests at temperatures between 4 ° and -20 °C in the dark to avoid photodegradation. The Sediments are usually air-dried, oven-dried, freeze-dried or lyophilized (- 40 °C) to a lesser extent, ground and sieved at < 1 mm. Fish is mostly filleted to obtain the muscle tissues and invertebrates separated from their shells, freeze-dried or lyophilized and mixed with anhydrous sodium sulphate to remove residual moisture. Filtration of water (0.45 μm fiber glass filters) to remove particulate matter and acidified with concentrated nitric acid or sulphuric acid prevent its degradation by microorganisms.

Soxhlet extraction is the most common extraction method is for sediments and biota though it is a cost-effective compared to more recent methods such as accelerated solvent extraction (ASE), automated soxhlet, sonication and supercritical fluid extraction (SFE). Liquid-liquid extraction or solid-phase extraction are employed for water samples. Generally, OCPs and PCBs were extracted with a mixture of

Hexane/Acetone while PAHs were extracted with a mixture of

Hexane/Dichloromethane.

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Sample clean-up or fractionation (in the case of simultaneous analyses of

OCPs and PCBs) is usually carried out on packed chromatographic columns with activated silica or alumina, activated silica gel or activated florisil, with anhydrous sodium sulphate and copper powder. Copper powder was used to eliminate sulphur compounds in sediments and in some cases, mercury was used for the same purpose.

The most common separation and quantification technique used for OCPs and

PCBs analysis is Gas Chromatography-Electron Capture Detector (GC-ECD) and

Gas Chromatography-Mass Spectrometry (GC-MS) while Gas Chromatography-

Mass Spectrometry (GC-MS) and High Performance Liquid Chromatography with

Fluorescence Detector (HPLC-FLD) have been mostly employed for PAHs analyses.

Fused-silica capillary columns where mostly used for OCPs, PCBs and PAH analyses.

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3.2 On-site survey of pesticides commercialization and uses in Kumba and

Barombi Mbo

In some African countries like Senegal, Nigeria and Cameroon the existence of POPs has been documented and potential sources identified such as the existence of obsolete stockpiles and illegal international traffic. This is supported by the fact that for instance in Senegal out of 300 pesticides sold, only 189 were authorized in

June 2002 (Diop, 2014). The fact that products like pesticides are banned does not mean end of exposure or end of use or absence. This inquiry was carried out at two chosen sites: at the market of Kumba city and the Barombi Mbo watershed. It was designed in the form of semi-structured interviews, questionnaires, and field observations. The targeted informants were members of the association

“Agrochemical Union” and farmers in the locality.

The “Agrochemical Union” association was composed of all vendors of crop protection products in the town of Kumba. The President and a member of the association were interviewed and issued questionnaires on issues concerning the acquisition, commercialization and distribution of pesticides in this area. Three farmers in Barombi Mbo were interviewed on the methods of application and periodicity in pesticide use. An observatory stroll was as well done in order to identify and register the types of pesticides sold and used in Kumba. A sample of the questionnaires issued to pesticide retailers or sellers in the Kumba market and farmers in Lake Barombi watershed are presented in Appendix 12.

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3.3. Sample Collection

3.3.1. Lake Barombi Mbo Watershed

Sampling was carried out during the dry season between the 5th to the 6th of

March 2016 in the Lake Barombi Mbo watershed. Prior to sample collection, GPS coordinates and altitude of each sampling point was recorded using a GPS Handset

(Garmin eTrex 30). Besides, physicochemical parameters of water samples

(Temperature, pH, Electrical Conductivity) were measured “in situ” using a pre- calibrated WTW multiparameter probe (Multi 350i). Water samples were collected at

4 stations within the first 10 cm of the lake surface using clean 2.5 L amber glass bottles rinsed three times with the water at the site before sampling. Similar bottles were used to sample 4 stations of flowing streams serving as inlets to the lake. Soil samples were collected, after litter removal, within the superior 5 cm of the soil surface using a stainless-steel spoon and labelled aluminium containers with an aluminium lid (figure 21 a). A total of 11 stations were sampled from cocoa mainly, bananas and oil palm farms and 1 sample collected as control (blank), where no crop was cultivated. Each sample was carefully examined to remove stones and plant material. Stream sediments were sampled at 4 stations from the bed of flowing streams (inlets). Furthermore, lake sediments were sampled from 2 stations using an interface corer Uwitech system (figure 21 b). Slices of lake sediments were collected from different depths using a meter tape and stainless-steel spatula. All collected samples were transported to Yaounde for preservation.

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Figure 21. Sampling of surface soil (left) and b) Lake sediments (right) in the Lake Barombi

Watershed

3.3.2. Wouri Estuary Mangrove

From the 23rd of November to the 28th of December 2017, superficial sediments (0-5 cm) were collected throughout the Wouri Mangrove. Sampling was carried out by manual coring using a PVC tube (10 cm diameter, 50 cm length) with the help of a motorboat. At each preselected site, duplicate samples were collected closer to the banks (shallow waters) owing to low sediment depth. The tube was rinsed several times with water at each sample point and immersed gently through the water column and the water-sediment interface. The upper end of the tube was corked to provide suction in order to maintain the sample in the tube. The tube was raised gradually and gently from the sediment-water interface and the lower end corked simultaneously. The upper cork was removed, and the sample steadily extruded with the help of a piston placed on the lower cork. The first 5 cm was sampled using a stainless spatula in pre-labelled aluminium trays boxes of 0.5 L

(figure 22). Coarse debris, plant material and shells of microbenthic organisms were

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CHAPTER 3 : Material and Methods removed. The samples were put in a cooler with icepacks. The samples were later put in a freezer at -18 °C at the Laboratory of the “Jeune Équipe Associée à l’IRD"

(JEAI) at Doula University. The frozen samples were transported from Douala to Aix- en-Provence at Laboratoire Chimie de l’Environnement (LCE) for further treatment.

Figure 22. Sample collection from the Wouri Estuary Mangrove

3.3.3. Chemical reagents, materials and instruments

Table 19 below describes the characteristics and quality of all products, chemicals and materials used in this work. Besides, the suppliers or manufacturers are also indicated.

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Table 19. Characteristics of products, chemicals and materials used

Supplier/ Products/Chemicals Characteristics/quality Manufacturer

Methanol Technical grade VWR Chemicals dichloromethane n-Hexane Suprasolv grade Merck, Pessac, France Acetone Methanol Pesticide 8081 standard mix 1x1 ml, 200 μg/mL in Hexane: toluene Sigma-Aldrich (50:50) Dr. Ehrenstorfer PCB mix 3,1 mL at 10 ng/g in Laboratories (Augsburg, isooctane Germany) PAH calibration mix at 10 μg/mL -1 Supelco in acetonitrile individual pesticide (4,4’ DDT, 4,4’ Dr. Ehrenstorfer DDD, 4,4’ DDE, α-Endosulfan, Laboratories (Augsburg, Endrin and Endrin Ketone Germany individual pesticides Analytical standard (Chlorfenvinphos, chlorpyriphos- LGC Standards GmbH D- ethyl, alachlor and metolachlor) at 46485 Wesel 100 μg/mL in Hexane Dr. Ehrenstorfer 4,4’ DDT D8 Laboratories (Augsburg, Germany) 4,4’ DDE D8 CDN Isotopes, Canada PCB 156 D3, 1 ml at 100 μg/mL in isooctane Cluzeau info labo (C.I.L) PCB 116 D5, 10 mL at 10 μg/mL -1 in isooctane

Hydrochloric acid (35 %) Fisher Scientific, Marseille, pure grade Nitric acid (69 %) France Phosphoric acid (85 %) pure grade VWR (France) Activated neutral alumina 63-200 Aluminium oxide, Ecochrom™ VWR International µm

Fine copper powder 1 Kg Particle size < 63 μm, Quality level = 300, bulk density = 1290 Merck (Pessac, France) (>230 mesh ASTM) Kg/m3 CRM860, Pesticides- Loamy sand 50 g, pH 7.48, suitable for GC and Sigma-Aldrich soil HPLC

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CRM CNS391, 50 g; PAHs, PCBs and Pesticides on freshwater 50 g, suitable for GC and HPLC (RTC, USA). sediment

Table 20. Characteristics of products, chemicals and materials used (continued)

Material Quality Supplier/Manufacturer Mega Bond Elut Florisil 1 g, 6 ml, 40 μm Agilent Technologies (USA) SPE cartridges Discovery® DSC-18 1g, 6 ml, C18 (Octadecyl), 70 Å pore Supelco SPE Tube size, reversed phase Fontainebleau sand 5 Kg VWR International

Vials Amber glass, 2.0 mL Agilent Technologies (Les Conical, 250 μL pulled point glass, Ulis, France) Vial Insert Insert size: 5.6 x 31 mm Thermo Fisher Scientific (Illkirch, Pasteur pipette Glass unplugged, 230 mm France)

Norm-ject 3.0 mL, Luer Lock Henke Sass Wolf by VWR Syringe (Fontenay-sous-Bois, France) Syringe filter Ø 25 mm, PTFE membrane 0.2 μm VWR (Fontenay-sous-Bois, France) Braun by VWR (Fontenay-sous- Needles Fine Ject 20 G x 1”,0.9 x 25 mm TW/LB Bois, France) Table 21. Characteristics of products, chemicals and materials used (continued)

Supplier/ Material Characteristics Manufacturer adjustable up to 1,300 °C, adjustable up to Analytik Jena (Jena, TOC solid module HT1300 1,300 °C, patented VITA technology Germany) Cell sizes:1, 5, 11, 22, and 33 mL, Accelerated Solvent Extractor- Collection vial: 40 – 60 mL, Operating Dionex 200 (Dionex) pressure (35–200 Bar); Operating temperature: 40-125 °C Cell sizes: 10, 60, 34 and 100 mL, Accelerated Solvent Extractor- Collection vessel: 60 - 250 ml, Operating Dionex 350 (Dionex) temperature: 40-125 °C, Extraction pressure:1500 Psi 11 and 33 mL (stainless steel) ASE cells Dionex 60 and 100 mL (zirconium) Martin Christ CHRIST ALPHA 1-4 lyophilizer Single chamber, Shelf temperature -25 °C Gefriertrocknungsanla to +60 °C, Shelf diameter 200 mm gen GmbH, Germany VisiprepTM SPE Vacuum 12-port model, disposable liner Supelco, Sigma Aldrich Manifold

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Superior evaporation and fixed heating LiebischTM Labortechnik block, 12 positions, operating temperature, Fischer scientific evaporator RT-200 °C 30 m L X 0.25 μm i.d. X 0.25 mm film Elite 5MS GC column PerkinElmer thickness Supplier/ Material Characteristics Manufacturer Turbomass software, oven programmable GC Clarus 600 and MS Clarus temperature, autosampler, programmable 600 C split/splitless capillary injector Chromera software, Autosampler, Binary PerkinElmer FX6 LC or LC pump, Flexar Solvent Manager with 3- PerkinElmer PerkinElmer Flexar HPLC channel Vacuum Degassing, Dual Binary solvent selection 4 channel data mode; 248 - 280 nm PerkinElmer Altus UPLC A-30 (excitation wavelength, λex) and 375 - 462 FL and UV detector nm (emission wavelength, λem) Rxi – XLB capillary column 30 m x 0.25 mm i.d. x 0.25 μm Restek Pursuit 5 PAH column 250 mm x 4.6 mm i.d. x and 5 μm Agilent

3.4. Sample pre-treatment

All samples (water, sediment and soils) were treated, extracted and analysed according to the schematic diagram presented in figure 23. These methodological steps where employed in the determination of OCPs, PCBs and PAHs.

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Figure 23. Summary of the major methodological steps employed in the determination of all

studied compounds.

3.4.1. Water

Prior to the sampling campaign, the Solid Phase Extraction (SPE, C18, 6 mL,

500 mg) cartridges were conditioned at LCE (France) with 6 mL of Hexane (HEX),

6 mL of methanol (MeOH) and 6 mL of milliQ water. After conditioning, they were filled with milliQ water, carefully sealed with parafilm, aluminium foil and transported in a non-refrigerated box “Laboratoire d’Analyse Geochimique des Eaux” (LAGE) in

Cameroon. Water samples were collected in prelabelled 2.5 L amber glass bottles.

Because Onsite extraction was not possible in Barombi Mbo, water samples were filtered and extracted 3 days after sampling at LAGE. This was performed according to Sandstrom, 1996 mentioning that if onsite extraction is not possible, pesticide

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CHAPTER 3 : Material and Methods samples (water) should be processed through an SPE column within 4 days of collection.

Filtration of water samples (1L) has been done systematically to prevent clogging and blockage of SPE columns by particulate material. Filtration was carried out using a Buchner funnel through glass microfiber filters (GFF, 0.7 µm, diameter

42.5 mm) previously rinsed with distilled water (figure 24), oven-dried (1 hour at 105

°C) and weighted. After filtration, the filters were oven dried (1 hour at 105 °C) and weighted to determine the mass of suspended solids.

Figure 24. Filtration device (Buchner funnel) for water samples

3.4.2. Soils and sediments

For Barombi Mbo, soil and sediment samples were oven dried at 45 °C, ground using a ceramic mortar and pestle, sieved at < 2 mm with a stainless-steel sieve and homogenised (Figure 25). Lake sediments (sediment cores) were dried by lyophilization (-41 °C, 0,310 mbar) for 48 hours using a CHRIST ALPHA 1-4 138

CHAPTER 3 : Material and Methods lyophilizer. Mangrove sediments from the Wouri Estuary were thawed under a laboratory hood and oven dried at 40 °C for 44 hours. Dry sediments were ground and sieved as previously mentioned

Figure 25. Pre-treatment material for soil and sediment samples (mortar grinding and 2mm

sieving)

3.5. Extraction and concentration

3.5.1. Water

Extraction of pesticides in water samples from LBM was processed by “Onsite”

Solid-Phase Extraction (SPE) through a commercial column containing pesticide- specific sorbents (C18 cartridges). This was done in preference to laboratory SPE because, onsite solid-phase extraction (SPE) is useful, especially at remote sites, pesticides isolated on the sorbent are less susceptible to degradation than when in water and SPE cartridges are less expensive and more convenient to transport (ship) than water samples in bulky containers (Sandstrom et al., 2001). According to

(Andrade-Eiroa et al., 2016) the advantage of SPE over Liquid-Liquid Extraction

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(LLE) is that, SPE can extract a wide range of organic analytes (from non-polar to very polar analytes) from a large variety of samples and their products of degradation.

A volume of 500 mL of filtered water was spiked with 50 μl of 4,4’ DDE D8 at

2 mg/L in Ace as surrogates. The C18 cartridges were mounted on the Buchner funnel connected to a vacuum pump (Figure 26). The samples were passed through the cartridges at a flow rate of about 10 mL/min. Distilled water from LAGE was treated similarly to serve as blank samples. After sample percolation, the adsorbent

(stationary phase) of the cartridges were vacuum dried for 30 minutes, well properly sealed with parafilm, aluminium foil and air transported in a refrigerated box to LCE

(France) for subsequent treatment. A similar protocol was applied by for water samples collected from Lebanon to France (El-Osmani et al., 2014). Barion et al.,

2018 reported that both methods of wrapping aluminium foil were suitable for the preservation of OCPs for SPE disks storage at 4 °C and -18 °C up to 30 days.

Elution was carried out with 20 mL of Hexane at a flow rate of 10 mL/min. The eluate was concentrated to 1 mL for analysis.

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CHAPTER 3 : Material and Methods

Figure 26. Extraction device for pesticides in water samples

3.5.2 Soils and sediments

The extraction of soils and sediments was carried out by Accelerated Solvent

Extraction (ASE) using a Dionex ASE 200 (figure 27). ASE is an automated extraction technique that uses elevated temperature and pressure to achieve extraction from solid and semi-solid matrices in very short periods. Its extraction cell (stainless steel) design allows S/L extractions at elevated pressures (1500 psi) to maintain the solvents as liquids at temperatures above their "normal" boiling points. The advantage of this method compared to others like Microwave Assisted Extraction

(MAE) is that temperature and pressure are controlled independently for each cell regardless of the solvent used, the moisture or mineral content of the sample, or any characteristic of the matrix that might affect the actual extraction temperature.

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Figure 27. Accelerated Solvent Extraction Device (ASE 350 Dionnex) and components of

the ASE cell

The ASE extraction conditions with "in-cell" clean-up was carried out according to (Kanzari et al., 2015) for all studied compounds . In-cell clean-up is a technique that combine extraction and clean up in a single step, eliminating post- extraction clean-up steps there by reducing the volume of solvent use and wastes, consequently saving time for analysis (table 22). This technique permits the selective removal of interferences during extraction. This in-cell clean-up was performed using two adsorbents; activated neutral alumina and acid activated (35 % HCl) copper (as described in 2.6.2). Alumina allows the removal of lipids, chlorophyll, petroleum and waste while copper enables the removal of interfering sulfur compounds, for instance elemental sulfur (common component of sediments).

Table 22. Summary of ASE extraction parameters for studied compounds 142

CHAPTER 3 : Material and Methods

CLPs and PCBs HAPs

Mass of sample 10 g 10 g

Cell Volume 33 mL 33 mL

Solvent (volume/volume) Hex/Ace (1:1) Hex/Dcm (1:1)

Temperature (°C) 100 150

Pre-heating (minutes) 1 1

Heating (minutes) 5 7

Pressure (Psi) 1500 1500

Static (minutes) 7 5

Flush (%) 100 75

Purge (seconds) 60 2

Cycle 2 1

The ASE cells were prepared such that soil or sediment samples were mixed with baked neutral alumina and acid activated copper to perform in-cell clean-up. The sample proportion: activated alumina: activated cooper was 2:1:1 and the addition of a little amount of precleaned and pyrolyzed Fontainebleau sand. The different components were mixed together, the cell was half filled, in order to add the surrogates; 4,4’ DDE-D8 at 2000 pg/μL for OCPs in LBM samples, 4,4-DDE D8 and

PCB 156 D3 at 100 pg/μL for CLPs and PCBs in WEM samples. The cell was filled up with the rest of the mixture, packed down gently and the remaining space filled with Fontainebleau sand. Extraction cells of different sizes were used according to the quantity of the sample available. Generally, cells of 33 mL were used for extraction of soils (10 g) and surface sediments (10 g). Due to less quantity of samples available for superficial sediment cores (LBM), cells of 22 mL and 1 mL were

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CHAPTER 3 : Material and Methods used when 5 g and 2 g of samples were obtainable respectively. Figure 28 show the major steps in ASE.

Figure 28. Summary of the ASE procedure

All extracts were then collected in 60 mL glass vials, concentrated under a gentle air flux of nitrogen to a drop (figure 29). Concentrated extracts of Pesticides and PCBs were exchanged in 1 mL of DCM, the tubes well rinsed and directly transferred in 2 mL amber glass vials. Those of PAHs were exchanged in 1 mL of

DCM, evaporated to a drop and exchanged in 1 mL of ACN. The tubes were well rinsed and filtered through a Ø 25 mm, PTFE membrane 0.2 μm and directly transferred in a 2 mL amber injection vial. The CLPs and PCB (WEM) extracts in 1 mL of DCM were subjected to the clean-up procedure described below.

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Figure 29. Evaporation of ASE extracts with a Liebisch Labortechnik evaporator

3.6. Activation of Copper for sediment desulphurization

Copper was activated in order to eliminate sulphur compounds in sediment samples. About 30 g of fine copper powder was measured in a beaker and a mixture of 100 mL of 35 % hydrochloric acid (HCl) and MilliQ water (1:3). A magnet was placed in the beaker and the mixture agitated with a magnetic agitator for 30 minutes.

The mixture was allowed to decant, and the supernatant discarded. The remaining copper was successively rinsed by agitation four times with 100 mL of distilled water, methanol and dichloromethane. The mixture was allowed to decant after each rinse.

After the last rinse, the remaining copper was dried under the hood for 24 hrs.

3.7. Clean up and fractionation

The adsorbents used for the clean-up were activated alumina, activated copper, C18 and Florisil (synthetic magnesium silicate). As previously described C18 cartridges were used for extraction and clean-up of water samples, thus saving 145

CHAPTER 3 : Material and Methods money, time, solvents and avoid additional clean-up steps. Alumina and activated copper permitted to carry out an in-cell clean-up as follows: the preconcentrated

CLPs and PCB extracts (WEM) were cleaned up and fractionated using Solid Phase

Extraction (SPE) Florisil cartridges (1 g, 6 mL) as shown in figure 30. This method was carried out according to the US-EPA 3620 C method in order to separate CLPs and PCBs in two distinct fractions. The first elution, fraction 1 (F1), was done with

2.5 mL of Hex (100 %) and the second elution fraction 2 (F2), with 5 mL of Hex /Ace

(80/20 v:v). This fractionation permitted to obtain all PCBs in F1 and most of the pesticides in F2 with few highly polar/non-polar pesticides between F1 and F2. The

F1 and F2 eluates were concentrated under a gentle flux of nitrogen to a drop and exchanged in 1 mL Dcm and transferred into a 2 mL amber glass vial.

Figure 30. Solid Phase Extraction, clean-up and fractionation of sample extracts

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CHAPTER 3 : Material and Methods

Prior to analyses, a solution of 4.4’ DDT D8 at 10 pg/μL (LBM), PCB 116-D5 and 4.4’ DDT D8 (WEM) at 40 pg/μL as internal standard was added to the 1 mL solution.

3.8. Instrumental analysis

3.8.1. Polyaromatic hydrocarbons

PAHs were analysed by High Performance liquid Chromatography with

Fluorescence programmable Detector (HPLC-PFD) using a PerkinElmer Flexar LC.

Separation was performed on an Agilent Technologies Pursuit 5 PAH column

(250 mm x 4.6 mm i.d. x and 5 μm). The elution conditions are shown in table 23.

Quantification was performed by a PerkinElmer Altus UPLC A-30 FL detector in a 4-

channel data mode; 248 - 280 nm (excitation wavelength, λex) and 375 - 462 nm

(emission wavelength, λem). The PerkinElmer Altus UPLC A-30 UV detector was set at 229 nm solely for the quantification of Acenaphthene.

Table 23. Parameters for analyses of PAHs by HPLC-PFD

Injector Column Solvent Solvent FLD Time Flow temp. temp. rate A (%) B (%) (min) mL/min Channel λex λem (° C) (° C) Acn Water 10 30 0.1 1.2 50 50 A 248 375 NV NV 12.0 1.2 100 100 B 270 324 NV NV 12.0 1.2 100 100 C 270 385 NV NV 4.0 1.2 50 50 D 280 462 NV NV 3.0 1.2 50 50 NV NV NV temp. = temperature, min = minutes, Acn = Acetonitrile, FLD = Fluorescence detector, λex = excitation wavelength, λem = emission wavelength (nm), NV = No value

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3.8.2. Chlorinated pesticides and polychlorinated biphenyls

Analyses of CLPs and PCBs was carried out by Gas Chromatography-Mass

Spectrometry (GC-MS) in Selected Ion Monitoring mode (SIM) using a PerkinElmer

Clarus GC Clarus 600 and MS Clarus 600 C equipped with a Restek Rxi – XLB;

(30 m x 0.25 mm i.d. x 0.25 μm) capillary column. Identification and quantification of

CLPs and PCBs were based on that previously described by (Kanzari et al., 2012).

Prior to the injection, 4.4’ DDT D8 and PCB 116 D5 were added at 40 pg/μL for both

CLPs and PCBs. A volume of 1 µL was injected in spitless mode with helium as carrier gas with a constant flow rate of 1 mL/min. The temperature of the injector was driven from 50 °C (iso 0.1 min) to 250 °C (200 °C min - 1 - Iso 10 min). The GC oven was programmed from 70 °C (iso 2 min) to 175 °C (10 °C/min - iso 4 min) to 320 °C (5

°C/min - iso 1 min). Iso here means the same temperature for the indicated time.

3.8.3. Quality control and quality assurance

High purity chemicals, extraction solvents, standard solutions and acids were used of suprasolv grade, analytical standard and pure grade respectively (table 20).

As indicated by (Muir and Sverko, 2006), a standard Quality assurance (QA) step in the analysis of PCB/OCPs (POPs) is to include surrogate recovery standards in each sample. Recovery studies were carried out by fortifying real samples and studied matrices (certified reference materials) with stable isotope labeled surrogate and internal standards; 4,4-DDE D8 and 4,4’ DDT-D8 for pesticides, PCB 156 D3 and

PCB 116 D5 at for PCBs respectively. Stable labeled isotope surrogate standards were used for better precision and accuracy of analysis (to correct recoveries, matrix

148

CHAPTER 3 : Material and Methods effect) and suitability with the separation and detection system employed (Reddy,

2017).

Analytical methods were validated prior to sample analyses using certified reference materials such as CRM CNS391 (50 g, PAHs, PCBs and Pesticides on freshwater sediment) and CRM860 (Pesticides-loamy sand soil) that were supplied by RTC (USA) and Sigma-Aldrich (France) respectively. For reasons of quantification, commercial mixtures were mostly used, and individual standards added at known quantities to complete the lacking analytes. Procedural blanks were prepared similarly to samples and simultaneously extracted and analyzed to check for contamination. Method performance characteristics such as limits of detection

(LOD) and limits of quantification (LOQ) were determined from the lowest point of the calibration curve (Appendix 13 and14). LOD referred to a signal-to-noise ratio (S/N) of 3 and LOQ to a S/N of 10. Quality control and quality assurance measures for each study area are discussed in chapter 3 and chapter 4.

For Lake Barombi Mbo analysis of each batch of samples comprised of a procedural blank (Fontainebleau sand), solvent blanks, random injection of standards, a matrix-spike sample and Certified Reference Materials (CRM) for soil and sediments: CRM860 and CRM CNS391 respectively. The latter were processed similarly as the field samples and analyzed simultaneously. Procedural blanks were evaluated using the criteria that blank levels of target analytes should be at least three times below the detection limit (signal-to-noise ratio 3:1). The limit of detection ranged from 0.04 to 0.71 ng/g corresponding to three times the signal to noise ratio for all studied compounds. The mean recoveries of surrogate standard 4,4’-DDE D8, in soil and sediment CRMs (n=3) was 103 ± 2 % and 85 ± 7 % respectively. If 149

CHAPTER 3 : Material and Methods surrogate recoveries differ from more than ± 15 % for a specific analyte and the analyte as present in the sample extract, sample extraction was reinjected to ensure an accurate concentration (EPA/SW-846 Methods, 2006). Identification and quantification were performed by external calibration method and good linearity was obtained with a determination coefficient greater than 0.98 for each calibration curve.

The following conditions had to be met for an unequivocal identification and quantification of the analytes: (1) retention time matching that of the standard compound within ± 0.15 min; (2) signal-to-noise ratio greater than 3:1; (3) detection of the qualifier ions when compounds were analyzed by GC/MS (table 24). All concentrations were reported on dry mass. The accuracy of the method was tested by the analyses of studied compounds in the CRM yielding recoveries of ranging from

62 to 118 % and 88 to 141 % for OCPs in soil and sediment CRMs while recoveries for PAHs ranged from 69 to 95 % in sediment CRM (table 25). All reported concentrations were adjusted for analyte recoveries.

For the WEM procedural blanks composed of glass beads and Fontainebleau sand, and certified reference material (CRM CNS 391 for PAHs, PCBs and pesticides on sediment) were processed for each batch of 7 extractions in the same procedures used for field samples in this study. Procedural blanks were evaluated using the criteria that blank levels of target analytes should be at least three times below the detection limit (signal-to-noise ratio 3:1). The accuracy of the method was tested by analyzing native pollutants in certified sediment. Obtained recoveries were 62-94% for pesticides, 76-94% for PCBs and 69-95% for PAHs (table 26). Inter-day replicates

(n=7) lead to relative standard deviations of 3-14% for pesticides, 3-9% for PCBs and

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CHAPTER 3 : Material and Methods

3-12% for PAHs. The reported limit of detection was set as the concentration level yielding to a signal-to-noise ratio of 3:1.

Prior to each extraction, 4,4-DDE D8 and PCB 156 D3 were added as surrogate standards at 100 pg/μL to check the analytical procedure for CLPs and

PCBs, respectively. Mean ± SD recoveries were 85±7 % and 91±5 % for 4,4-DDE

D8 and PCB 156 D3, respectively. If surrogates’ recoveries differ from more than ±15

%, sample extraction was redone. Instrumental QC was performed by regular analyses of solvent blanks and random injection of standards. Measured values were not deviating more than 15 % from the theoretical values. The following conditions had to be met for an unequivocal identification and quantification of the analytes: (1) retention time matching that of the standard compound within ± 0.15 min, (2) signal- to-noise ratio greater than 3:1, (3) detection of the qualifier ions when compounds were analyzed by GC/MS. All concentrations are reported on dry mass.

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Table 24. Identification parameters for the studied compounds in GC-MS SIM mode

Lake Barombi Wouri Estuary Mangrove Peak Peak identification RT identification RT Ions m/z (amu): Fraction Ions m/z (amu): Compound Quantifier ; (min) (min) Quantifier ; qualifier1 ; qualifier1 ; qualifier2 qualifier2 F1 F2 α HCH 181 ; 183 ; 219 11.6 183 ; 109 ; 181 17.10 x x β HCH 181 ; 183 ; 219 12.57 183 ; 181 ; 217 18.60 x x γ HCH 181 ; 183 ; 219 12.72 183 ; 181 ; 217 20.08 x δ HCH 181 ; 183 ; 219 13.79 181 ; 183 ; 217 20.94 x Heptachlor 272 ; 274 ; 374 15.39 272 ; 274 ; 270 20.90 x Aldrin 263 ; 261 ; 265 16.78 263 ; 265 ; 261 22.05 x x Heptachlor epoxide 353 ; 355 ; 388 18.38 353 ; 351 ; 357 23.63 x isomer B α + β Chlordane 373 ; 375 ; 410 19.36 - - - - α-Endosulfan 373 ; 375 ; 195 19.91 241 ; 195 ; 197 24.86 x 254 ; 326 ; 4,4’ DDE d8SS 254 ; 256 ; 326 20.87 25.50 x 256 4,4' DDE 246 ; 248 ; 318 20.93 246 ; 248 ; 318 25.57 x Dieldrin 79 ; 81 ; 279 21.05 263 ; 237 ; 235 25.68 x Endrin 79 ; 81 ; 279 21.91 263 ; 265 ; 261 26.34 x Β-Endosulfan 195 ; 239 ; 241 22.39 - 27.25 x 4,4’ DDT d8IS 243 ; 245 ; 328 22,66 - 27.98 x 4,4' DDT+DDD 235 ; 165 ; 237 22.74 - - - Endrin Aldehyde 345 ; 248 ; 250 23.03 281 ; 279 ; 280 27.60 x Endosulfan sulfate 272 ; 274 ; 387 24.07 272 ; 274 ; 212 28.53 Methoxychlor 227 ; 228 ; 310 24.98 - - x β-Chlordane - - 375 ; 373 ; 371 24.71 x α-Chlordane - - 373 ; 375 ; 371 24.83 x 4,4' DDD - - 235 ; 237 ; 165 27.15 x x 4,4' DDT - - 235 ; 237 ;165 28.06 x x Endrin Ketone - - 317 ; 319 ; 315 29.74 x Alachlor - - 160 ; 188 ; 269 20.82 x x Metolachlor - - 162; 146 ; 163 22.19 x Chlorpyrifos-ethyl - - 197; 199 ;314 22.27 x Chlorfenvinphos - - 267 ; 269 ; 323 23.73 x

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PCB 28 - - 256 ; 186 ; 258 20.84 x Lake Barombi Wouri Estuary Mangrove

Peak Peak identification identification Fractio RT RT Ions m/z Ions m/z Compound (amu): (min) (amu): (min) Quantifier ; Quantifier ; qualifier1 ; qualifier1 ; F1 F2 qualifier2 qualifier2 PCB 53 - - 292 ; 220 ; 222 21.83 x PCB 101 - - 326 ; 256 ; 254 24,68 x PCB 116 D5IS - - 331 ; 333 ; 329 25.39 x PCB 118 - - 326 ; 328 ; 324 26.78 x PCB 138 - - 360 ; 362 ; 358 27.22 x PCB 153 - - 360 ; 290 ; 362 28.08 x PCB 156 D3SS - - 363 ; 365 ; 293 29.90 x PCB 180 - - 394 ; 324 ; 396 30.14 x Naphthalene - - 270-324 nm 9.07 - - Acenaphthylene - - 229 nm (UV) 9.88 - - Acenaphthene - - 270-324 nm 11.02 - - Fluorene - - 270-324 nm 11.27 - - Phenanthrene - - 248-375 nm 12.10 - - Anthracene - - 248-375 nm 12.91 - - Fluoranthene - - 280-462 nm 13.74 - - Pyrene - - 270-485 nm 14.40 - - Benzo(a)Anthracene - - 270-485 nm 16.20 - - Chrysene - - 270-485 nm 16.75 - - Benzo(b)Fluoranthene - - 290-430 nm 18.52 - - Benzo(k)Fluoranthene - - 290-430 nm 19.62 - - Benzo(a)Pyrene - - 290-430 nm 20.95 - - Dibenzo(a,h)Anthracene - - 290-430 nm 22.90 - - Benzo(g,h,i)Perylene - - 290-430 nm 24.88 - - Indeno(1.2.3-cd)Pyrene - - 274-507 nm 26.23 - - RT= Retention time, SIM = Single Ion Monitoring, SS = surrogate standard, IS = Internal standard, min = minutes

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Table 25. Summary of method validation parameters for studied compounds with Certified Reference Materials (CRM860 and CNS391) for the Lake

Barombi study

Lake Barombi study Measured Compounds Reference Reference value Soil LODs (ng/g) S/N=3 Value Soil Value CRM- Measured value Recove CRM860 Recovery Soil and sediment Acronym CRM860 CNS931 CRM-CNS931 Mean ry (%) (n=47) (ng/g) (%) (n=3) -1 (Cal. curve 5-500 Mean±SD (n=30) ±SD (n=3) (µg.kg ) (n=3) (µg/kg) pg/µL) (n=3) (µg/kg) Aldrin Aldrin NP ND ND 16.2 (±1.34) 16.35 (±3.65) 101 0.77 α-Endodulfan α-Endo 91.5 (±6.77) 64.9 (± 3.6) 81.8 14.20 (1.32) ND ND 0.58 β-Endosulfan β-Endo 111 (±7.75) ND ND NP ND ND 0.05 Dichlorodiphenyltrichloroet hane + 4.4’-DDT + 165 (±10.36) 186.1(±4.4) 112.0 21.40A 24.10 (±6.86) 141 0.04 Dichlorodiphenyldichloroet 4.4’-DDD hane Dichlorodiphenyltrichloroet N 4.4’-DDT NP ND 10.20 (±1.27) ND ND ND hane D Dichlorodiphenyldichloroet N 4.4’-DDD NP ND 13.90 (±0.98) ND ND ND hane D

Dichlorodiphényldichloroet 4.4’-DDE 70.8 (±4.37) 44.0 (±15.2) 90.5 18.80 (±1.23) 24.34 (±1.83) 129 0.16 hylene Dieldrin Dield 79.7 (±4.40) 94.0 (±15.4) 118.0 25.70 (±2.00) 33.78 (±0.73) 131 0.26 Pesticides Endrin Endrin 75.3 (±6.09) ND ND 10.40 (±2.14) ND ND 0.71 Endrin ketone Endr K. 119 (±9.95) ND ND NP ND ND ND

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Alpha- α-HCH 115 (±8.87) 106.2 (±21.1) 92.4 37.10 (±3.31) 34.86 (±9.77) 94 0.19 Hexachlorocyclohexane Lake Barombi study Measured Reference Reference Compounds value Soil LODs (ng/g) S/N=3 Value Soil Value CRM- Measured value Recove CRM860 Recovery Soil and sediment Acronym CRM860 CNS931 CRM-CNS931 Mean ry (%) (%) (n=3) (n=47) (ng/g) Mean±SD (n=30) ±SD (n=3) (µg.kg -1) (n=3) (Cal. curve (µg/kg) 5-500 pg/µL) (n=3) (µg/kg) Beta- β-HCH 109 (±10.8) 105.0 (±16.3) 96.3 21.10 (±2.05) ND ND 1.07 Hexachlorocyclohexane Gamma- γ-HCH 83.3 (±6.09) 72.12 (±13.5) 86.6 9.50 (±0.72) 11.23 (±3.56) 118 0.42 Hexachlorocyclohexane Delta- δ HCH 65.7 (±4.57) 52.6 (±19.4) 80.0 NP ND ND 0.68 Hexachlorocyclohexane Heptachlor Epoxide Hepta E. 106 (±6.43) 136.4 (±15.2) 77.9 33.10 (±2.40) 44.57 (±9.37) 134 0.30 Heptaclor Hepta NP ND ND 6.54 (±1.67) ND ND 6.05 Methoxychlor Metol 96.6 (±8.60) 87.2 (±8.9) 90.2 NP ND ND 0.72 α + γ-Chlordane α + γ-Chlord 175 (±11.37) 99.8 (±11.6) 94.2 NP ND NM 0.26 α-Chlordane α-Chlord NP ND ND NP ND ND ND γ-Chlordane γ-Chlord NP ND ND NP ND ND Endrin Aldehyde* End A. 50.2 (±6.09) ND ND NP ND ND 0.3 Endosulfan sulphate Endo S. 58.6 (±4.16) ND ND NP ND ND 2.13 A = extractions carried out the same day (intra-day repeatability), B = extractions carried out in 4 days (inter-day repeatability), C = (n=2). *Absent in the Certified Reference Material, NM-Not mentioned, S/N= Signal to Noise, CVR = Coefficient of variation (inter-day-repeatability).

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Table 26. Summary of method validation parameters for studied compounds with the Certified Reference Material (CRM CNS391) for the Wouri

Estuary Mangrove study

Wouri Estuary Mangrove LODs (ng/g) Compounds Measured value Reference Value Measured value CRM Recovery RSD S/N=3 CRM Mean ±SD CRM (n=30) Mean ±SD (n=7) B (%) (%) Acronym (n=3) A Cal. curve

(µg/kg) (µg/kg) (n=7) N=7 (µg/kg) (0.1 – 100 pg/µL) Aldrin Aldrin 16.2 (±1.34) 15.58 (±0.13) 15.27 (±0.55) 94 3.60 0.12 α-Endodulfan α-Endo 14.20 (1.32) 13.90 (±0.09) 13.43 (±0.35) 95 2.61 0.58 Dichlorodiphenyldichloroethane 4.4’-DDD 13.90 (±0.98) 13.36 (±0.10) 13.21 (±0.44) 95 3.33 0.05 Dichlorodiphényldichloroethylene 4.4’-DDE 18.80 (±1.23) 18.07 (±0.05) 17.98 (±0.32) 96 1.78 0.04 Dichlorodiphenyltrichloroethane 4.4’-DDT 10.20 (±1.27) 9.91 (±0.04) C 9.61(±0.27) 94 2.81 0.16 Dieldrin Dield 25.70 (±2.00) 24.27 (±0.14) 24.08 (±0.53) 94 2.20 0.26

Endrin Endrin 10.40 (±2.14) 9.87 (±0.09) 9.62 (±0.38) 93 3.95 0.71 Alpha-Hexachlorocyclohexane α-HCH 37.10 (±3.31) 36.25 (±0.22) 35.98 (±0.59) 97 1.64 0.23

Pesticides Beta-Hexachlorocyclohexane β-HCH 21.10 (±2.05) 20.72 (±0.08) 20.22 (±0.36) 96 1.78 0.16 Gamma-Hexachlorocyclohexane γ-HCH 9.50 (±0.72) 9.05 (±0.04) 8.96 (±0.29) 94 3.24 0.20 Heptaclor Hepta 6.54 (±1.67) 6.04 (±0.05) 5.98 (±0.25) 91 4.18 0.20 Heptachlor Epoxide Hepta E. 33.10 (±2.40) 31.89 (±0.11) 31.55 (±0.61) 95 1.93 0.15 Delta-Hexachlorocyclohexane δ HCH * NM NM NM NM NM 0.21 Alachlor* Alach NM NM NM NM NM 0.12

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Metolachlor* Metol NM NM NM NM NM 0.4 Chlorpyriphos-ethyl* Chlorp NM NM NM NM NM 0.16 Wouri Estuary Mangrove LODs (ng/g) Measured value Compounds Reference Value Measured value CRM Recovery RSD S/N=3 CRM Mean ±SD CRM (n=30) Mean ±SD (n=7) B (%) (%) Acronym (n=3) A Cal. curve (µg/kg) (µg/kg) (n=7) N=7 (µg/kg) (0.1 – 100 pg/µL) Chlorfenvinphos* Chlorf NM NM NM NM NM 0.32 γ-Chlordane* γ-Chlord NM NM NM NM NM 0.13 α-Chlordane α-Chlord NM NM NM NM NM 0.27 Endrin Aldehyde* End A. NM NM NM NM NM 0.42 Endosulfan sulphate Endo S. NM NM NM NM NM 0.35 Endrin ketone* End K. NM NM NM 95 NM 0.39 2,4,4-Trichlorobiphenyl PCB28 44.9 (±3.31) 43.02 (±0.09) 42.77 (±0.44) 97 1.03 0.02 2,2,5,5-Tetrachlorobiphenyl PCB52 64.4 (±4.23) 62.90 (±0.06) 62.42 (±0.43) 95 0.70 0.07 2,2,4,5,5-Pentachlorobiphenyl PCB101 45.7 (±3.13) 43.81 (±0.04) 43.54 (±0.44) 95 1.02 0.06

2,2,4,4,5-Pentachlorobiphenyl PCB118 24.04 (±1.31) 23.10 (±0.08) 22.72 (±0.37) 98 1.62 0.06

PCBs 2,2,4,4,5,5-Hexachlorobiphenyl PCB153 50.1 (±2.59) 50.23 (±0.06) 48.85 (±0.51) 96 1.05 0.13 2,2,3,4,4,5-Hexachlorobiphenyl PCB138 34.6 (±2.68) 33,65 (±0,08) 33,17 (±0,34) 98 1,01 0,02 2,2,3,4,4,5,5-Pentachlorobiphenyl PCB180 54,7 (±0,13) 53,92 (±0,10) 53,49 (±0,49) 90 0,92 0,07 Naphthalene NA 464 (±39.9) 413.98 (±2.93) 417.09 (±3.72) 87 4.07 0.039

Acenaphtylene ACY 53.4 (±10.8) 48.70 (±1.31) 48.98 (±1.73) 92 3.53 0.590

PAHs Acenaphthene ACE 29.9 (±6.43) 27.00 (±0.61) 27.38 (±1.01) 95 3.69 0.021

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Fluorene F 409 (±42.3) 385.08 (±1.56) 387.66 (±2.01) 98 0.52 0.007 Phenanthrene PHE 660 (±34.5) 640.40 (±1.66) 645.40 (±2.04) 92 0.32 0.006 Wouri Estuary Mangrove LODs (ng/g) Compounds Measured value Reference Value Measured value CRM Recovery RSD S/N=3 CRM Mean ±SD CRM (n=30) Mean ±SD (n=7) B (%) (%) Acronym (n=3) A Cal. curve (µg/kg) (µg/kg) (n=7) N=7 (µg/kg) (0.1 – 100 pg/µL) Anthracene ANT 15 (±3.35) 14.41 (±0.50) 13.74 (±0.79) 98 2.72 0.002 Fluoranthene FL 557 (±29.5) 515.17 (±1.36) 544.70 (±3.16) 98 0.58 0.005 Pyrene PYR 331 (±31.6) 323.59 (±1.63) 324.04 (±2.75) 96 0.85 0.003 Benzo(a)Anthracene BaA 338 (±26.6) 317.60 (±1.95) 323.11 (±2.51) 93 0.78 0.002 Chrysene CHRY 376 (±13.1) 349.13 (±1.38) 351.00 (±2.30) 96 0.66 0.002 Benzo(b)Fluoranthene BbFL 210 (±8.09) 202.65 (±0.64) 202.29 (±0.93) 93 0.46 0.011 Benzo(k)Fluoranthene BkFL 300 (±11.6) 279.91 (±3.19) 278.53 (±2.28) 93 0.82 0.002 Benzo(a)Pyrene BaP 38.2 (±4.77) 34.48 (±0.83) 35.42 (±0.45) 97 1.27 0.004 Dibenzo(a,h)Anthracene DBA 294 (±11.8) 286.53 (±2.25) 283.91 (±2.78) 92 0.98 0.008 Benzo(g,h,i)Perylene BgP 139 (±10.1) 128.87 (±1.33) 127.26 (±2.41) 96 1.90 0.017 Indeno(1,2,3-cd)Pyrene IP 235(±12.0) 225.29 (±1.77) 226.32 (±2.90) 95 1.28 0.028 A = extractions carried out the same day (intra-day repeatability), B = extractions carried out in 4 days (inter-day repeatability), C = (n=2). *Absent in the Certified Reference Material, NM- Not mentioned, S/N= Signal to Noise, CVR = Coefficient of variation (inter-day-repeatability).

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CHAPTER 4: Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon

CHAPTER 4 : Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon

Abstract

This study evaluates the level of contamination and spatial distribution of 18 organochlorinated pesticides (OCPs) and 16 US EPA priority polyaromatic hydrocarbons

(PAHs) in the Lake Barombi watershed (LBW), the largest and deepest volcanic lake of

Cameroon. Water, sediment cores, superficial soil and sediments from the lake, farms and streams (inlets) were sampled in March 2016. Accelerated Solvent Extraction (ASE) and Solid

Phase Extraction (SPE) were employed for soils, sediments and water respectively. In water, all studied OCPs were below the limit of detection while 8 OCPs were measured in soils and sediments. The abundance of OCPs in all samples in increasing order was, endosulfan (β- endosulfan + endosulfan sulfate), HCHs (Σ α-HCH + β-HCH +δ -HCH + γ-HCH), dieldrin and aldrin. HCHs had the highest frequency of detection with concentrations ranging from

(HPAHs) (4 – 6 rings) were more abundant than Low Molecular Weight PAHs (LPAHs) (2 – 3 rings) representing 74 % and 26 % respectively. Selected PAH ratios showed predominant pyrolytic sources of PAHs specifically grass, wood or coal combustion. The level contamination of LBM was lower than most lakes worldwide. Sediment Quality Guidelines for individual chemicals indicated low ecological risks to aquatic life. The presence of OCPs and PAHs in this watershed was mainly related to agricultural activities (spraying of pesticides, slash and burn farming) and fishing with chemicals.

Keywords: organochlorinated pesticides, polyaromatic hydrocarbons, sediments, soil, agriculture, Cameroon, Lake

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4.1 Introduction

Anthropic activities such as agriculture, industrialization and urbanization have directly or indirectly led to adverse effects on human health and environmental degradation. Pesticides have been extensively used in agriculture to improve agricultural yield and disease vector control in public health. Among other groups of pesticides, organochlorine pesticides (OCPs) have been used as insecticides have been used worldwide and particularly in Africa in the cultivation of cash crops such as coffee, oil palm, cotton and cocoa because of their broad spectrum activity and cost effectiveness (Isogai et al., 2018). Despite their benefits, various studies have reported their toxicity to humans and other living organisms regarding cancer, neurodevelopmental effects, reproductive effect, thyroid and endocrine disruption

(Bornman et al., 2017; Chevrier et al., 2008; Korrick and Sagiv, 2008; Garabrant et al.,

1992) and pose serious threats to biodiversity (Smeti et al., 2019; Mrema et al., 2013).

Likewise, a group of ubiquitous organic compounds mainly originating from anthropogenic sources such as fossil fuels and combustion (Essien et al., 2011;

Yunker et al., 2002) known as Polyaromatic hydrocarbons of which some are carcinogenic and mutagenic (Adeniji et al., 2018; Abdel-Shafy and Mansour, 2016;

Quiroz et al., 2010; Budzinski et al., 1997).

For many decades OCPs have been banned worldwide and classified as Persistent

Organic Pollutants (POPs) by the Stockholm Convention on POPs while PAHs have recently been added to the list of US EPA POPs (Wirnkor et al., 2019). POPs (OCPs and some PAHs) are highly toxic, resistant to degradation, undergo long range transport and readily accumulate in fatty tissues of living organisms by bioaccumulation

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CHAPTER 4 : Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon along the food chain (Breivik et al., 2011; Jones and de Voogt, 1999). Despite their prohibition, they are still currently detected in humans and various environmental compartments worldwide and particularly in Africa (Adeniji et al., 2019; Adekunle et al.,

2018; Basweti et al., 2018; Ndunda et al., 2018; Kilunga et al., 2017; Yahaya et al.,

2017). By contrast, the Stockholm Convention under the World Health Organization

(WHO) allowed, in 2006, the reuse of the insecticide DDT exclusively for public health reasons (indoor spraying for disease vector control) in regions affected by malaria.

Over the past years, notwithstanding the growing interest on persistent organic pollutant contamination there is still paucity of data and knowledge gaps in Africa

(Ssebugere et al., 2019; Isogai et al., 2018; Mochungong et al., 2015).

In Cameroon, a central African country whose economy essentially relies on agriculture, important crop losses have been reported for cocoa, coffee, sorghum, cassava and yam due to the black pod disease, scolytids, striga and viral diseases respectively (Souop, 2000). These crops represent the main cash and food crops in

Cameroon, such that pesticide use has been largely promoted in agri-food industry as well as in small family-owned farms. In a bordering country Nigeria, Okoya et al. (2013) reported OCP contamination of water and sediments from cocoa growing areas of

Ondo State (Southwestern Nigeria). In addition to pesticide use, slash and burn agriculture is one of the most common farming practice in tropical humid forest zones, the primary causes of land degradation and deforestation (Gay‐des‐Combes et al.,

2017; Hauser and Norgrove, 2013; Styger et al., 2007; Tinker et al., 1996). This could represent a significant source of PAHs in these regions through the burning of wood and grass.

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Aquatic environments (oceans, seas, rivers, streams, lakes, estuaries and wetlands) act as major repositories for various types of contaminants including OCPs and PAHs. Among these ecosystems, lakes happen to be more prone to pollution than rivers as they lack a flushing effect and insufficient dilution effect of large water bodies like seas. Few studies in Africa have reported contamination of lakes by OCPs and/or

PAHs; Lake Ichkeul, Tunisia (Ben Salem et al., 2017), Lake Qarun, Egypt, (Barakat et al., 2013), Lake Maryut, Egypt (Barakat et al., 2012), Lake Victoria, Uganda side

(Wasswa et al., 2011), Lake Bosomtwi, Ghana (Darko et al., 2008), Lake Barullus

Egypt (Said et al., 2008), Lake Volta, Ghana (Ntow, 2005) and Lake Tangayinka,

Burundi (Manirakiza et al., 2002)

The present work investigates the Barombi Lake and its watershed located South-

West Cameroon, with the aim to establish the baseline level of OCPs and PAHs in an ecosystem that undergoes increasing anthropogenic pressure. A previous study has shown that this volcanic lake and its watershed have already recorded anthropogenic environmental disturbances occurring long before the industrial revolution, 2600 years ago (Garcin et al., 2018). Nowadays, the main anthropogenic activities include, cash crop and food crop farming around the lake (mainly slash and burn farming with the use of pesticides on cocoa, banana, oil palm) and harvesting of various timber and non-timber forest products. Fishing is also highly carried out using fishing nets and chemicals such as Gamaline (about 2 tons per year) (Balgah and Kimengsi, 2011),

These activities are very common on the many lacustrine water body systems that characterized the Cameroon volcanic Lake (Kling et al., 1987). Thus, the Barombi watershed can be considered as a good analogue of the such kind of ecosystem for

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Hence, this study aims at (i) evaluate the level of anthropogenic impact on the LBW regarding OCPs in water, soil, stream, lake sediments and PAHs in soil and sediments

(ii) to determine the concentrations and distribution of these contaminants throughout the watershed in all studied matrices (iii) to acquire information on pesticide commercialization and use in the town of Kumba and LBW. To the best of our knowledge no previous study has been carried out to determine the level of pesticide contamination in this area, and more generally in such kind of lacustrine ecosystems in Cameroon. Hence, the outcome of this work would serve as baseline data on the level, distribution and toxicological in of OCPs and PAHs in water, soil and sediments of Lake Barombi and its watershed

4.2 Material and methods

4.2.1 Study area

Lake Barombi is famous for hosting 12 endemic fish species, it was designated second Ramsar site of Cameroon in 2006 by the UNESCO (Ramsar site no. 1643)

(figure 31). These include, freshwater sponges, Corvospongilla thysi and Cichlides;

Sarotherodon linnellii (Unga sp.), Pungu maclareni (critically endangered), rendering it one of the places with the highest densities of endemic species per area in the world

(Schliewen and Tanjong, 2006). The area around the lake was designated as a forest reserve since 1940 as the lake is the main source of drinking water for the town of

Kumba. The Barombi Mbo village (adjacent to the lake) is at 100 m from the lake with

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Figure 31. Location of the study area (a) Africa (b) South West region of Cameroon and (c)

Lake Barombi Mbo

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4.2.2 Survey on pesticide commercialization and uses in the Kumba market and Barombi Mbo watershed

An inquiry was carried out in the Kumba market and Barombi village in the form of semi-structured interviews and questionnaires along with field observations. The targeted informants were members of the “Agrochemical Union” association which brings together all retailers of phytosanitary products in the town of Kumba.

Questionnaires and semi-structured interviews were issued to three members of this association in the Kumba namely; the president of the association (retailer) and two members (retailers) of the association on the acquisition, commercialization and distribution of pesticides in Kumba. At the Barombi Mbo catchment, farmers were interrogated on the methods of application and periodicity of pesticide use.

i) Sampling

The location, description of each sampling station and number of samples for each studied matrix is presented in table 27. A total of 8 water samples from the lake and small streams (inlets) were collected within the first 10 cm in prelabelled 2.5 L amber glass bottles. Due to the impossibility of onsite extraction, filtration and extraction was done 3 days after sampling in accordance with (Sandstrom et al., 2001). The water samples (1 L) were filtered was using a Buchner funnel through glass microfiber filters

(GFF, 0.7 µm, diameter 42.5 mm) previously rinsed with distilled water and oven-dried

(1 hour at 105 °C).

11 soil samples were collected within the first 5 cm of the soil surface using a stainless-steel spoon in prelabelled aluminium boxes, after the removal of litter and 167

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coarse materials. Likewise, stream sediments were collected from the bed of flowing

streams (inlets). Finally, sediment cores of 0 -10 cm corresponding to station 4 and 25

were collected using an interface Uwitec coring system and for each core, intervals of

2 cm were sampled in plastic bags. All collected sampled were stored at ambient

outdoor temperature (about 25 °C) in a cooler.

Table 27. Localisation and site description of sampling stations from the Lake Barombi

Watershed

N° Sample code Longitude Latitude Alt.(m) Site description

1 Lake 1 9° 24' 25.92" 4° 39' 16.56" 309 Towards lake outlet

2 Lake 2 9° 24' 16.56" 4° 39' 39.6" - Close to the centre of the lake 3 Lake 3 9° 23' 52.08" 4° 39' 29.16" - Towards farming zone

4 Lake 4 9° 23' 42" 4° 40' 8.04" - Towards village

5 Stream 9° 23' 18.24" 4° 40' 28.92 325 Upstream of the village Water catchment 6 Stream school 9° 23' 21.84" 4° 40' 20.64" 317 Close to the village 7 Inlet 1A 9° 23' 59.28" 4° 40' 18.12" 305 Besides a cocoa farm

8 Inlet 2A 9° 24' 32.76" 4°40' 11.28" 310 Besides a Banana farm 9 Soil FZA 9° 23' 29.4" 4° 39' 55.8" 312 Forest zone, dry soil, abundant litter, cultivated 10 Soil Palm FZ-H 9° 23' 26.16" 4° 39' 43.92" 324 Forest zone, Humid soil, herbicide use

11 Soil Bank FZ 9° 23' 28.32" 4° 39' 42.12" 349 Forest zone, Steep slope, humid soil

12 Soil FZ CP 9° 23' 16.08" 4°39' 24.12" 323 Forest zone, dry soil, cultivated and productive 13 Soil FZ NC-P 9° 23' 19.32" 4°39' 32.76" 317 Forest zone, dry soil, dry vegetation,

non-cultivated

14 Soil Soil FZ NC 9° 23' 19.68" 4° 39' 32.4" 317 Forest zone, non-cultivated

15 Burned Palm FZ 9° 23' 34.44" 4°40' 8.76" 314 Forest zone, close to the Barombi village 16 Soil UV-NV 9° 23' 18.24" 4° 40' 39.36" 347 Upstream the village, no vegetation

17 Cocoa UV-V 9° 23' 22.2" 4° 40' 20.28" 349 Upstream the village, vegetation

18 Soil inlet 1 9° 23' 59.28" 4° 40' 18.12" 305 Cacao farm, humid soil

19 Soil inlet 2 9° 24' 32.76" 4° 40' 11.28" 310 Banana farm, humid soil

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N° Alt.

Sample code Longitude Latitude Site description (m)

20 Catchment 9° 23' 18.24" 4° 40' 28.92" 325 Upstream the village

21 School 9° 23' 21.84" 4° 40' 20.64" 317 Close to Barombi village

22 Inlet 1 9° 23' 59.28" 4° 40' 18.12" 305 Close to Cocoa Farm

23 Inlet 2 A 9° 24' 32.76" 4° 40' 11.28" 310 Close to Banana farms Stream sediments 24 Lake sed. 1 9° 23' 34.08" 4° 39' 46.44" 312 Towards lake bank and farming zone

25 Lake sed. 2 9° 23' 47.04" 4° 40' 5.16" 319 Towards lake bank and Barombi Lake village Alt. = Altitude, FZ = Farming zone, C = Cultivated, NC = Not cultivated, CP = Cultivated and Productive, UZ = Upper zone, NV = No vegetation

ii) Reagents and standards

All solvents; n-Hexane (Hex), acetone (Ace), dichloromethane (Dcm), methanol

(MeOH) and acetonitrile (Acn) were of suprasolv grade purchased from Merck

(Pessac, France). Nitric acid (69 %), hydrochloric acid (35 %) and phosphoric acid

(85 %) of pure grade were provided by Fisher Scientific (Marseille, France) and VWR

(France). PAH calibration mix at 10 μg/mL in acetonitrile was supplied by Supelco,

Pesticide 8081 standard mix 1 mL at 200 μg/mL in Hexane: toluene (50:50) form

Sigma-Aldrich (France). Pesticide 8081 standard mix, 1x1 ml, 200 μgml-1 in Hexane :

toluene (50:50) form Sigma-Aldrich (France), individual pesticide (4,4’ DDT, 4,4’ DDD,

4,4’ DDT d8, α-Endosulfan, Endrin and Endrin Ketone), individual surrogate; 4,4’ DDE

d8 (CDN Isotopes, Canada) and internal standard ; 4,4’ DDT d8 were obtained from

Dr. Ehrenstorfer Laboratories (Augsburg, Germany). Alumina and copper powder

(>230 mesh) were purchased from VWR International and Merck (Pessac, France)

respectively. Two Certified Reference Materials, CRM CNS391 (PAHs, PCBs and

Pesticides on freshwater sediment) and CRM860 (Pesticides-loamy sand soil) were 169

Elut Florisil cartridges (1 g, 6 ml) were supplied by Agilent Technologies (USA). The surrogate standards 4,4’ DDT D8 and 4,4’ DDE D8 were from Dr. Ehrenstorfer

Laboratories (Augsburg, Germany) and CDN Isotopes, Canada respectively.

4.2.3. Pre-treatment and extraction

Soil and sediment samples were oven dried at 45 °C, sieved at < 2 mm and homogenised. The sampled sediment cores were dried by lyophilization (- 41 °C,

0.310 mbar) for 48 H using a CHRIST ALPHA 1-4 lyophilizer.

Pesticides residues were extracted from water by Solid Phase Extraction based on method described by (Kouzayha et al., 2012). Prior to extraction, 500 mL of filtered water was spiked with 50 μL of 4,4’-DDE D8 at 2 mg/L as surrogate standard. The latter was passed through preconditioned C-18 cartridges (6 ml, 500 mg) with 6 mL of

Hexane (Hex), 6 mL of methanol (MeOH) and 6 mL of milli Q water. After percolation, the adsorbent was vacuum dried for 30 minutes. The SPE cartridges were properly sealed with parafilm and aluminium foil. Elution was carried out with 20 mL of Hexane within 7 days of extraction as prescribed by (Sandstrom et al., 2001).

In soil and sediments OCPs were extracted by accelerated solvent extraction according to the method described by (Kanzari et al., 2012). A 10 g dry weight sample was thoroughly mixed with baked neutral alumina and acid activated copper (sulfur removal) to perform an in-cell clean-up in the proportion 2:1:1 respectively. The latter was spiked with 100 µL of 4,4’-DDE D8 at 2 mg/L. The extraction solvent was a mixture

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2 cycles. Extracts were concentrated under a gentle stream of nitrogen and exchanged in 1 mL of dichloromethane.

4.2.4. Instrumental analysis

Organochlorine pesticides were analysed by Gas Chromatography-Mass

Spectrometry (GC-MS) using a PerkinElmer Clarus GC Clarus 600 and MS Clarus 600

C equipped with a Restek Rxi – XLB; (30 m x 0.25 mm i.d. x 0.25 μm) capillary column.

Prior to the injection, a standard solution of 4,4’-DDT D8 at 40 pg/μL was added as internal standard. A volume of 1 µL was injected in spitless mode with helium as carrier gas with a constant flow rate of 1 mL/min. The temperature of the injector was driven from 50 °C (iso 0.1 min) to 250 °C (200 °C min - 1 - Iso 10 min). The GC oven was programmed from 70 °C (iso 2 min) to 175 °C (10 °C/min - iso 4 min) to 320 °C

(5 °C/min - iso 1 min). Identification and quantification of CLPs and PCBs were based on that previously described by (Kanzari et al., 2012).

PAHs were analysed by High Pressure Liquid Chromatography with

Fluorescence Programmable Detector (HPLC-PFD) using a PerkinElmer FX6 LC or

PerkinElmer Flexar LC. Separations were performed on an Agilent Pursuit 5 PAH column (250 mm x 4.6 mm i.d. x and 5 μm). The elution solvent gradient was as follows; a mobile phase with ACN/Water (50:50), flow rate 1.2 mL/min, injector and column temperature of 10 °C and 30 °C respectively was applied. ACN concentration was gradually increased to 100 % after 12 min and maintained for the next 12 min, 171

CHAPTER 4 : Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon decreased to 50 % after 4 min and finally maintained for the last 3 min. Quantification was performed by a PerkinElmer Altus UPLC A-30 FL detector in a 4 channel data

mode; 248 - 280 nm (excitation wavelength, λex) and 375 - 462 nm (emission

wavelength, λem). The PerkinElmer Altus UPLC A-30 UV detector was set at 229 nm solely for the quantification of Acenaphthene.

4.2.5. Determination of Total Organic Carbon

Total Carbon (TC) and Total Inorganic Carbon (TIC) were determined separately using a TOC solid module HT1300, such that the difference results in Total

Organic Carbon (TOC). For TC measurement, 50 mg of sediments were weighed in a ceramic boat, introduced in a furnace and combusted at 950 °C under a flux of oxygen.

A pre-calibrated (CaCO3 12 % Carbon) non-dispersive infrared detector enabled the quantification of emitted CO2. For TIC measurement, 100 mg of sediments were weighed in a conical flask and reacted with 2 mL of 40 % phosphoric acid, heated at

83 °C and agitated at 350 rpm. The amount of emitted CO2 was quantified as above.

4.2.6. Quality assurance and quality control

Quality assurance and quality control was cautiously carried out as described in

Mbusnum et al., 2020 (See section 3.8.3). Analysis of each batch of samples comprised of a procedural blank (Fontainebleau sand), solvent blanks, random injection of standards, a matrix-spike sample and Certified Reference Materials (CRM) for soil and sediments: CRM860 and CRM CNS391 respectively. The latter were processed similarly as the field samples and analysed simultaneously. Procedural blanks were evaluated using the criteria that blank levels of target analytes should be 172

CHAPTER 4 : Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon at least three times below the detection limit (signal-to-noise ratio 3:1). The accuracy of the method was tested by the analyses of studied compounds in the CRM yielding recoveries of ranging from 62 to 118 % and 88 to 141 % for OCPs in soil and sediment

CRMs while recoveries for PAHs ranged from 69 to 95 % in sediment CRM. All reported concentrations were adjusted for analyte recoveries. The limit of detection ranged from 0.04 to 0.71 ng/g corresponding to three times the signal to noise ratio for all studied compounds. The mean recoveries of surrogate standard 4,4’-DDE D8, in soil and sediment CRMs (n=3) was 103 ± 2 % and 85 ± 7 % respectively. If surrogate recoveries differ from more than ±15% for a specific analyte and the analyte as present in the sample extract, sample extraction was reinjected to ensure an accurate concentration (EPA/SW-846 Methods, 2006). Identification and quantification were performed by external calibration method and good linearity was obtained with a determination coefficient greater than 0.98 for each calibration curve. The following conditions had to be met for an unequivocal identification and quantification of the analytes: (1) retention time matching that of the standard compound within ± 0.15 min;

(2) signal-to-noise ratio greater than 3:1; (3) detection of the qualifier ions when compounds were analysed by GC/MS. All concentrations were reported on dry mass.

4.3 Results and Discussion

4.3.1 Pesticide commercialization and uses

The list of pesticides registered at the Kumba market and dumped packages in farms is given in Table 28 and Appendix 15 and 16 respectively. The results of the survey reveal that the most common pesticides/active ingredients sold in the Kumba

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CHAPTER 4 : Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon market are fungicides/metalaxyl+copper oxides or hydroxides, insecticides/chlorpyriphos, herbicides/glyphosate. The findings of the survey and field observations were in line with (Gockowski and Mva Mva, 2001), who reported that the proportion of fungicide: insecticides: herbicide spraying in the southwest region of

Cameroon was 5.5: 4.3: 2.5 respectively. Fungicides are mostly applied during the rainy season on cocoa, insecticides on fruits and vegetables whereas herbicides are mostly applied on various crops (oil palm, vegetables). The rainy season (September to November) corresponds to the period that supports the growth of phytophthora sp,

(pathogen of cocoa pods), cocoa harvest and peak demand of fungicides.

The survey revealed that vendors in Kumba get pesticides from the same registered suppliers mostly located in the economic capital, most populated and industrialized city of Cameroon known as Douala. The average storage period in shops was two years and after the expiry date the products were returned to suppliers in exchange for new products. Pesticides are often sold with Individual protective equipment’s (IPE) (masks, gloves, goggles) but farmers have limited income and knowledge on the effects of these pesticides, as such do not buy them. Pesticide packages such as sachets were directly dumped in farms after use while pesticide jerrycans were washed and reused. Most farm sizes around the watershed range between 10,000 to 40,000 m2 and the majority are owned by the urban population in

Kumba who progressively acquire more land, together with the population growth and increasing urbanization. Despite the absence of banned pesticides in the Kumba market and farms in Barombi Mbo village, other pesticides were sold in the Kumba market such as chlorpyrifos (insecticide), imidacloprid (insecticide) and glyphosate

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(herbicide). Chlorpyrifos and imidacloprid both act on the nervous system of insects

and belong to the class of chemicals called organophosphate pesticides and

neonicotinoids respectively. According to the WHO classification, they both belong to

the toxicity class II, suggesting their moderate hazardous nature (WHO, 2009).

Glyphosate (non-selective herbicide) is the most used pesticide worldwide by volume

and belongs to the toxicity class III (slightly hazardous) based on the WHO

classification (WHO, 2009).

Table 28. Pesticides registered at the Kumba market and dumped packages in farms

Active Season Trade Name Prescription Crops Mode of action Ingredient used Contact and Callomil Plus 72 WP Fungicide Metalaxyl+ Copper Oxide Cocoa Rainy Systemic Contact and Apromil 72 WP Fungicide Metalaxyl+ Copper Oxide Cocao Rainy Systemic Metacide Super 66 Contact and Fungicide Metalaxyl+ Copper Oxide Cocoa Rainy WP Systemic Contact and Metalm 72 WP Fungicide Metalaxyl+ Copper Oxide Cocao Rainy Systemic Ridomil Gold Plus 66 Fungicide Metalaxyl+ Copper Oxide Cocao Systemic Rainy WP Contact and Agro Comet Fungicide Metalaxyl+ Copper Oxide Cocoa Rainy Systemic Fungi off 720 WP Fungicide Metalaxyl+ Copper Oxide Cocoa Systemic Rainy Ametoctradine+ Overgo Fungicide Cocoa Systemic Rainy Dimethomorphe Hydrox Super WG Fungicide Copper hydroxide Cocoa Contact Rainy Nordox 75 WG Fungicide Cuprous oxide Various crops Contact Rainy Fungicide and Kocide 2000 WG Copper hydroxide Various crops - Rainy bactericide Cacaocide 2010 75 Cocoa and Fungicides Copper hydroxide - Rainy WP Banana Contact and Actara 25 WG Insecticide Thiamethoxam Rainy systemic Capsidor 50 SC Insecticide Fipronil Cocoa - Rainy

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Pychlorex 480 EC Insecticide Chlorpyriphos Vegetables - Rainy

Pyriforce Insecticide Chlorpyriphos Coffee - Rainy

Fruits and Cypercal Insecticide Cypermithrin Contact Rainy vegetables Imidaclopride + Lambda Crops and Contact and Gamalin 80 EC Insecticide Rainy Cyhalothrin vegetables systemic Chlorcot 480 EC Insecticide Chlorpyriphos-ethyl Tomato - Rainy Round up Herbicide Glyphosate Vegetables Systemic Dry Herbextra 720 SL Herbicide 2,4-D Amine salt Oil palm Systemic Dry Casse-tout Herbicide Glyphosate Various crops - Dry Finish 68 SG Herbicide Glyphosate Oil palm - Dry Glycot Herbicide Glyphosate Cotton - Dry

4.3.2. Total Organic Carbon content

TOC ranged from 1.4 to 7.0 ng/g, 0.9 to 1.9 ng/g and 9.0 to 9.7 ng/g in soils,

sediments and sediment cores. Higher concentrations of TOC in sediments may be

due to the wash down of organic matter from farmlands and surrounding vegetation

into streams (inlets) and/or the lake. This is favoured by the nature of the lake

characterized by steep slopes surrounding the lake.

4.3.3. Levels and distribution of PAHs and OCPs

i) Polyaromatic Hydrocarbons

Levels of PAHs ranged from 149.7 to 154.7 ng/g, 115.5 to 116.6 ng/g and 81.3

to 108.0 ng/g, in lake sediments, soils and sediments of inlets respectively (Table 29).

In terms of total PAH concentrations, no significant difference was observed between

the different sample categories. In sediment cores, the highest levels were highest is

observed in core 4 (149 -169 ng/g) maybe because this core shows the highest Total

Organic Carbon (TOC) content (table 30) and it is the nearest from the largest part of

the farming zone where slash and burn farming is carried out (Appendix 3). The same

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Table 29. PAH concentrations (ng/g of dry weight) in soils and sediments of LBM watershed with Sediments Quality Guidelines

(Long et al., 1995)

NA ACY ACE F PHE ANT FL PYR BaA CHRY B(b)FL B(k)FL BaP DBA B(ghi)P IP Σ16PAHs LPAHs/ ΣCPAHs TEQ ΣComPAHs/ (2) (3) (3) (3) (3) (3) (4) (4) (4) (4) (5) (5) (5) (5) (6) (6) HPAHs (%) CPAHs ΣPAHs Soil inlet 1 0.5 3.0 2.0 4.6 9.1 8.0 14.1 12.0 13.2 11.9 2.7 5.6 5.2 6.7 8.0 9.1 115.5 0.3 54.4 14.5 0.72 Soil inlet 2 0.7 3.8 2.9 2.8 8.6 7.1 13.8 11.8 11.5 10.0 7.5 4.6 7.1 4.4 10.1 10.0 116.6 0.3 55.1 14.4 0.69 TPAHs 4 5.9 8.7 13 17.9 15.2 31.8 29.8 22.7 26.3 14.8 14.7 14.4 16.1 15.2 22.9 ND ND ND ND ND Sed inlet 1 1.5 1.0 2.8 2.9 5.7 4.3 9.0 8.5 7.9 6.8 3.3 4.6 6.1 5.8 4.3 6.7 81.3 0.3 41.2 13.8 0.71 Sed inlet 2 2.0 3.7 1.0 1.5 5.1 3.9 10.8 10.3 5.6 5.7 5.4 6.5 5.1 6.6 3.9 7.1 84.1 0.3 42.0 13.6 0.74 Sed catch 0.5 1.2 4.9 8.6 7.1 7.0 12.0 11.0 9.2 13.8 6.1 3.6 3.2 3.7 7.0 9.1 108.0 0.4 48.7 9.4 0.70 TPAHs 1.2 6.8 4.9 7.4 17.7 15.1 27.9 23.8 24.7 21.9 10.2 10.2 12.3 11.1 18.1 19.1 ND ND ND ND ND Lake sed. 1 2.9 8.6 8.2 3.0 10.0 7.1 16.4 14.6 20.2 16.2 9.9 3.0 8.8 8.7 7.1 10.0 154.7 0.3 76.7 21.5 0.69 Lake sed. 2 4.2 7.6 4.8 4.7 8.0 6.9 14.0 13.5 16.7 14.2 5.9 6.7 10.1 5.2 12.7 13.9 149.2 0.3 72.7 19.0 0.72 TPAHs 7.1 16.2 13 7.7 18 14 30.4 28.1 36.9 30.4 15.8 9.7 18.9 13.9 19.8 23.9 ND ND ND ND ND TEL NG NG NG NG 41.9 NG 111 53 31.7 57.1 NG NG 31.9 NG NG NG ND ND ND ND ND ERL 340 NG NG 35 225 85 600 350 230 400 NG NG 400 60 NG NG 4000 ND ND ND ND LEL NG NG NG 190 560 220 750 490 320 340 NG NG 370 60 NG NG 4000 ND ND ND ND PEL NG NG NG NG 515 NG 2355 875 385 862 NG NG 782 NG NG NG ND ND ND ND ND ERM 2100 NG NG 640 1380 960 3600 2200 1600 2800 NG NG 2500 260 NG NG 35000 ND ND ND ND ( )= number of rings, * = Upper limit, NA= Naphthalene, ACY= Acenaphthylene, ACE= Acenaphthene, F= Fluorene, PHE= Phenanthrene, ANT= Anthracene, Fl= Fluoranthene, PYR= Pyrene, BaA= Benzo(a)Anthracene, CHRY= Chrysene, B(b)Fl= Benzo(b)fluoranthene, B(k)Fl= Benzo(k)fluoranthene, BaP= Benzo(a)pyrene, DBA= Dibenzo(a,h)anthracene, B(g,h,i)P = Benzo(g,h,i)pyrelene, IP= Indeno(1,2,3-cd)pyrene, HPAHs= High Molecular Weight PAHs, LPAHs= Low Molecular Weight LPAHs, ∑PAHs = Sum of 16 PAHs, TPAHs = Sum of individual PAHs, CPAHs= Carcinogenic PAHs, LPAHs= Low Molecular Weight PAHs, HPAHs= High Molecular Weight PAHs, TEQ CPAHs= Toxic Equivalents of CPAHs, ΣComPAHs= Sum of combustion PAHs, ERL= Effect range Low, LEL = Lowest effect level, (Persaud et al. 1993), ERM = Effect Range Median, TEL = Threshold effect level; dry weight (Smith et al. 1996), ERL = Effect range low; dry weight (Long and Morgan 1991), LEL = Lowest effect level, dry weight (Persaud et al. 1993), NG= No guideline, ND = Not determined 178

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TOC in soils and sediments of the LBMW show poor correlation with PAH levels.

This could be explained by the correlation between PAHs and sediment OC, only significant for highly contaminated sites, where the total PAH concentrations were >

2000 ng/g (Simpson et al., 1996). This indicates that the distribution of PAHs in sediments of the LBM is not controlled by TOC content.

The most abundant PAHs in increasing order were Fluoranthene (Fl),

Benzo(a)Anthracene (B(a)A), Pyrene (PYR), Chrysene (CHR) , Indeno (1,2,3-cd)

Pyrene (IP), Phenanthrene (PHE), Benzo(g,h,i)perylene (B(g,h,i)P) and Anthracene

(ANT). These PAHs are referred as High Molecular Weight PAHs (HPAHs) except PHE and ANT referred as Low Molecular Weight PAHs. In all samples, we observed a similar profile for HPAHs (4-6 rings) relative to LPAHs (2-3 rings) at all stations contributing on average to 74 % and 26 % of total PAHs respectively (figure 32).

HPAHs are strongly carcinogenic, mutagenic (Stogiannidis and Laane, 2015) and more sorbed in solid matrices as a result of lower water solubility (SW), higher hydrophobicity (KOW and KOC) (Samia et al., 2018). A similar profile dominated by

HPAHs was found in sediments of Lake Burullus, Egypt (Barakat et al., 2011) but contrary to Lake Manzala where PAH profiles were generally dominated by LPAHs

(Barakat et al., 2013).

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Figure 32. Distribution pattern (%) of PAHs in the Lake Barombi Mbo Watershed

The predominance of FL, PYR and PHE in soils and sediments of the LBM watershed, indicates PAH contamination from pyrolytic sources (Page and Boehm,

1999; Budzinski et al., 1997). According to Stogiannidis and Laane (2015), open-flame combustion increases the relative abundance of PHE-ANT, FL, PYR. Therefore, this suggests that PAHs in LBW could be associated to slash and burn agricultural activities carried out in this watershed. Previous studies worldwide have used characteristic PAH ratios to appraise PAH sources like FL/PYR and PHE/ANT to discriminate between pyrolytic and petrogenic origin of PAHs (Budzinski et al., 1997; Yunker et al., 2002).

The pyrolytic origin of PAH contamination in the LBM watershed could be comforted

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IP/Σ(IP+B(g,h,i)P) > 0.5 reveal that PAHs in the LBM watershed are solely from pyrolytic sources and more specifically combustion of biomass (grass, wood and coal)

(Yunker et al., 2002) as shown in figure 34. The combustion origin of PAHs is supported by the ratio between the sum of combustion PAHs and total PAHs

(ΣComPAHs/ΣPAHs) of 0.7 for all studied matrices (table 29). These results are consistent with the fact the main source of combustion in the LBW is burning of grass and wood in slash and burn agricultural practices which is predominant in this area.

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Figure 33. Cross-plot of PAH ratios (PHE/ANT versus FL/PYR) for samples of the Lake

Barombi Mbo Watershed

Figure 34. Cross-plot of PAH ratios (BaA/(BaA+CHRY) and IP/(IP+B(g,h,i)P) versus

ANT/(ANT+PHE) for samples of the Lake Barombi Mbo Watershed 182

OCPs residues in all water samples were below the limit of detection (< LOD).

Similar results were reported for OCPs in water samples (rainy and dry seasons) from the Yala/Nzoia River in Lake Victoria basin, Kenya (Musa et al., 2011). Furthermore, in cocoa producing areas of Ondo state in Nigeria, OCPs residues were measured in one out of eleven surface water samples at trace concentrations (<0.01 !g/L, detection limit) for the wet and dry season (Okoya et al., 2013). This approves the hydrophobic nature of these compounds and their tendency to associate with soils and sediments upon release or atmospheric deposition.

Concentrations of OCPs in LBM watershed ranged from 3 to 175 ng/g, 4 to 31 ng/g and 42 to 58 ng/g in soils, stream sediments and lake sediments (Table 30). The abundance of OCPs in the LBM watershed was in decreasing order, endosulfan (sum of β-endosulfan and endosulfan sulfate) > HCHs (sum of α, β, γ and δ-HCH) > Dieldrin

> Aldrin. The most frequently detected compounds were HCHs with concentrations ranging from

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Table 30. Concentration of OCPs (ng/g) of OCPs in soils. stream and lake sediments from LBM watershed and Sediment Quality Guidelines (Long et al., 1995)

Concentration of OCPs (ng/g) β- Sample α-HCH β-HCH γ-HCH δ-HCH ∑HCHs α/γ-HCH Ald Diel Endo. s. ∑OCPs TOC (%) endo. Soil FZ 0.4 0.3 0.4 0.6 1.7 1.1

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Concentration of OCPs (ng/g) β- Sample α-HCH β-HCH γ-HCH δ-HCH ∑HCHs α/γ-HCH Ald Diel Endo. s. ∑OCPs TOC (%) endo. Sediment Inlet 1 0.5 0.2

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The most abundant pesticide in all studied matrices of the LBM watershed was endosulfan (sum of β-endosulfan and endosulfan sulfate) with concentrations over 10 to 100 times that of other compounds (

(>> 200 days) compared to α-endosulfan (> 200 days) (Guerin, 2001). In Cameroon, endosulfan was commercialized with the trade names, Sultan 500 EC,

Thiofanex 500 EC, thioplant 50 WP, Caiman rouge and Calthio E, and totally banned in July 2008. This could suggest the recent use of endosulfan in the Lake Barombi

Watershed.

Lindane (γ-HCH) is the HCH isomer with the highest pesticidal activity. It has been used as agricultural pesticide and medicinally in the treatment of lice and scabies. The highest concentration of lindane were measured in core 2 (2.3 ng/g) and core 1 (2.1 ng/g) sediment samples. High lindane concentrations were also found in station 16

(1.5 ng/g) which corresponds to soil from a cacoa farm with no vegetation (Soil UV

NV), station 18 (1.4 ng/g) and station 23 (1.0 ng/g) (table 30). The absence of vegetation at station 16 could indicate the use of herbicides and other pesticides such

186

Fc 320 and totally banned in August 2005. Commercial HCHs were manufactured in two formulations: technical HCHs (α-HCH (60–70 %), β-HCH (5–12 %), γ-HCH (10–

12 %) and δ-HCH (6–10 %)) and lindane (>99 % γ-HCH) (Willett et al., 1998).

Considering differences in the removal rates of HCH isomers, the ratio of α/γ-HCH has been used as an indication of the application age (Tao et al., 2005; Zhang et al., 2011).

The α/γ-HCH ratio in LBM watershed ranges between 0.3 to 1.5, indicating a recent use of lindane rather than technical HCH.

The presence of HCHs is generally related to agricultural activities (Kruitwagen et al., 2008). The occurrence of OCPs in LBW watershed is mainly associated to agricultural activities supported by the fact that it is located in a remote, closed basin.

The presence lindane in Lake Barombi could also be associated with fishing since the use of Gammalin (lindane) for fishing in lake Barombi was previously by (Balgah and

Kimengsi, 2011). The use of gammalin (lindane) for fishing was equally reported in other African lakes: the Volta lake (Ntow, 2005), Lake Victoria (Wasswa et al., 2011) and Densu river basin (Kuranchie-Mensah et al., 2012).

The recent use of endosulfan and lindane in soil and sediments of the LBW could be justified by the fact that a national inventory revealed that lindane and endosulfan represented 55 % and 37 % of total pesticide stocks in Cameroon respectively (UNEP-

POPS-NIP-Cameroon, 2016) in the report of the National Implementation Plan of the

Stockholm Convention on Persistent Organic Pollutants of 2012.

In lake sediment, Aldrin was detected in core 2 at concentration below 1.9 ng/g. In soils and sediments of inlets its concentration was below 0.6 ng/g. Dieldrin was 187

CHAPTER 4 : Assessment of Organochlorinated Pesticides and Polyaromatic Hydrocarbons in Water, Soil and Sediments of Lake Barombi Mbo Watershed, Southwest Region of Cameroon detected in 6 soil samples (Station 9, 10, 14, 17, 18 and 19) and in core 2 at concentrations not exceeding 1.6 ng/g and 1.7 ng/g respectively (table 30). Low detection frequencies and concentrations (fig. 35, 36 and 37) of aldrin (commercialized as Aldrex, Aldrex 30, Aldrite, Aldrosol, Drinox, Octalene, Seedrin) and dieldrin commercialized as; Alvit, Dieldrex, Dieldrite, Octalox, Panoram D 31) in all samples could be related to the total ban these two pesticides in Cameroon since January 1989.

Figure 35. Profile and detection frequencies of OCPs in soils of LBM watershed

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Figure 36. Pattern and detection frequencies of OCPs in stream sediments of LBM

watershed

Figure 37. Pattern and detection frequencies of OCPs in Lake sediments of LBM watershed

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The OCPs detected in the LBW are all insecticides and their presence could mainly be attributed to agricultural activities through the spraying of farms to control crop pests and diseases. Besides, the high detection frequency (100 %) of lindane (γ-HCH) in lake sediments suggests an input from fishing. None of the pesticides detected in the

LBW was sold in the Kumba market and no packages of these pesticides were found in farms.

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4.4.4. Comparison of OCPs levels with Sediment Quality Guidelines

The ecological risk assessment of sediments from the LBMW was performed according to Sediment Quality Guidelines for pesticides and polyaromatic hydrocarbons in freshwater ecosystems (Smith et al., 1996; Persaud et al., 1993; Long and Morgan, 1991). At Lowest Effect Level (LEL), sediments are considered to be clean to marginally polluted, that is no effects on the majority of sediment-dwelling organisms are expected below this concentration (Persaud et al., 1993). Threshold

Effect Level (TEL) stands for the concentration below which adverse effects are expected to occur only rarely, Probable Effect Level (PEL) represents the concentration above which adverse effects are expected to occur frequently (Smith et al., 1996). ERL represents a concentration below which adverse effects rarely observed and ERM represents the concentration above which adverse effects would frequently occur on sediment dwelling organisms (Long and Morgan, 1991).

Generally, levels of lindane and dieldrin in LBW are below the selected sediment quality guidelines (table 30). For lindane, sediments of Inlet 2 were above TEL, suggesting concentrations occasionally associated to adverse biological effects. Core

1 and 2 were above PEL, suggesting concentrations frequently associated to adverse biological effects. For Dieldrin, the sediments of Core 2 were above TEL, suggesting concentrations occasionally associated to adverse biological effects.

As soils are concerned, ecological risk was assessed with the use of Soil Quality

Standards regulations (FAO, 2007). All soils from LBW were below the contaminant limit (maximum limit, dry weight) in habitat and agricultural soils for the studied OCPs

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4.4.5. Comparison with other lacustrine environments worldwide

The comparison between levels of OCPs and PAHs in lakes worldwide is presented in table 31. The concentration of endosulfan (sum of β-endosulfan and endosulfan sulfate) in LBM (

(6.00 ± 4.36 ng/g). The concentration of lindane in sediments of LBM watershed

(0.30 – 0.57 ng/g) were higher than those observed in sediments from the Weija Lake,

Densu river basin, Ghana (0.36 – 0.80 ng/g) (Kuranchie-Mensah et al., 2012) and Lake

Ohrid, Macedonia/Albania (MDL – 0.21 ng/g) (Veljanoska-Sarafiloska et al., 2011), but lower than that measured in sediments of: Lake Tashk, Iran (5.64 – 8.47 ng/g)

(Kafilzadeh, 2015); Lake Qarun, Egypt (0.09 – 100.1 ng/g); (Barakat et al., 2013); Lake

Parishan, Iran (3.06 – 14.21 ng/g) (Kafilzadeh et al., 2012); Lake Bosomtwi, Ghana

(2.03 – 13.94 ng/g) (Darko et al., 2008); Lake Burullus along the Egyptian

Mediterranean Sea coasts (0.30 – 22.84 ng/g) (Said et al., 2008) and the Volta Lake, largest Lake in Ghana (2.30 ± 1.40 ng/g) (Ntow, 2005).

The mean concentrations measured in sediments of LBM were similar to those of

Lake Bosomtwi for aldrin (0.65 ng/g) but higher than dieldrin (0.072 ng/g) (Darko et al.,

2008). Correspondingly, in sediments levels of aldrin were lower than those of dieldrin

(its metabolite). Aldrin and dieldrin were also not detected in water samples of Lake

Bosomtwi. (Kuranchie-Mensah et al., 2012). According to the same author, aldrin was extensively used in cocoa, therefore justifying the presence of aldrin in inlet and lake 192

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Table 31. Summary of OCPs and PAHs concentrations (ng/g) in sediments (dry weight) of lacustrine environment worldwide

Sampling Depth Concentration range (ng/g) of dry weight (dw) Country Study area Main activities References Stations (cm) ΣDDTs ΣHCHs Endo Aldrin Dieldrin ΣOCPs PAHs Lake Barombi < DL – Cameroon Agriculture, Fishing 6 0-10

17.0 - Uganda Lake Victoria Industrial, fishing 4 (53) <13 NA NA NA NA NA NA Kerebba et al., 2017 80.2 Industrial, Congo Lake Ma Vallée 4 6 < 0.05LQ NA < 0.05LQ < 0.05LQ < 0.05LQ < 0.05LQ NA Mwanamoki et al. 2014 commercial ND – Egypt Lake Quran Industrial, agricultural 24 0-5 n.d. – 5.9 0.1 – 62.6M 0.1 – 18.7 ND – 5.4 1 – 164.8 NA Barakat et al., 2013 12.2

Yala/Nzoia E A D A C A A A Agriculture, disease 29.1A 9.2 8.9 12.7 59 164.8 Kenya River (Lake 9 - NA Musa et al., 2011 vector control E B D B C B B B Victoria basin) 26.9 B 23.2 18.5 12.7 24.5 135.8 2.8 ± 2 6 ± 4.4 3.2 ± Uganda Lake Victoria Agriculture, fishing 3 (117) 0-20* 4.2 ± 3.8K 3.8 ± 3.5 NA NA Wasswa et al, 2011 (mn) (mn) 1.85 Ghana Lake Bosomtwi Agriculture, fishing 50 3.5 – 26.4 2 -13.9 F NA NA NA NA NA Darko et al. 2008 46.3 –

2.3±1.4 Ghana Volta lake Agriculture, fishing 6 (36) - 61.3±42.8G 0.7C NA NA 64.34H NA Ntow, 2005 (mn) Shallow Lakes Agriculture, indstrial, 4 – Spain Mediterranean 48 - 3-65 ND ND NA ND 1-73 Hijosa-Valsero et al., 2016 urbanization 4286 region Veljanoska-Sarafiloska et Macedonia Lake Ohrid Agriculture, industrial 6 - 2.4 – 4.3E 0.7 – 1.3D 0.3 – 0.6 NA NA - al., 2011 Baiyangdian 12.1–15.8 101.3– China Industrial 19 0-5 2.2–3.1I 9.8–12.8D NA NA NA Hu et al., 2010 Lake O 322.8

Iran Lake Tashk Agriculture 4 (48) Sr 4.1 – 10.9 5.6 – 8.5F 9.1 – 15.3 NA NA - Kafilzadeh, 2015

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( ) = number of samples, *=sediment core, A=Wet season, B=Dry season, C= (α+βEndosulfan+Endosulfan sulfate), D= 3HCHs, , E =3 DDTs, , F= γ-HCH , G=2 DDTs, H= 6 OCPs, I= 5DDTs, , M= 4HCHs , N=6 DDTs, O= 8 OCPs, LOD = Limit of Detection, LQ = Limit of Quantification, mn = mean, ND = Not detected, NA = Not analyzed

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4.5. Conclusion

This study has shown that activities within lake catchments could define or influence their chemical profiles. The presence of OCPs and PAHs at quite low levels in different compartments of the Lake Barombi watershed (water, soil and sediments) was mainly attributed to agricultural activities (spraying of crops for pest and disease control, slash and burn farming) and fishing with the use of chemicals like lindane. The fact that OCP levels were below the limit of detection in all water samples is an indication of low toxicity risks to biodiversity in the lake and the local population. The most abundant and frequently detected pesticides, endosulfan and lindane indicate recent uses while they have been banned in Cameroon in 2005 and 2008 respectively. This could ascertain their persistence in the environment particularly in soil and sediment matrices or chronic uses before their prohibition. Higher OCPs and PAHs in sediments (lake and streams) could represent a potential source of dispersion of these contaminants via sediments to the water column which might constituting toxicological risks to aquatic organisms like endemic fish species in the lake and the population of Kumba.

Thus, the importance of long-term monitoring of this ecosystem, added to increasing anthropic pressure from the rapid urbanization and demographic growth of the town of

Kumba. Levels of OCP and PAH contamination in the LBMW watershed was lower than most lakes worldwide. The state of anthropization related to OCPs and PAHs of

Lake Barombi Mbo presents a picture of the state of contamination of volcanic lakes in

Cameroon. It is therefore necessary to carry out similar studies with temporal and spatial considerations in order to establish a monitoring network of these lakes with various ecosystemic benefits and reliance of local populations.

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CHAPTER 5: Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance

This chapter has been published in the journal “Marine Pollution Bulletin”

(Volume 160, July 2020 111542) https://doi.org/10.1016/j.marpolbul.2020.111542

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Abstract

The anthropogenic impact in the Wouri Estuary Mangrove located in the rapidly developing urban area of Doula, Cameroun, Africa, was studied. A set of 45 Persistent

Organic Pollutant were analysed in surficial mangrove sediments at 21 stations.

Chlorinated Pesticides (CLPs), Polychlorinated Biphenyls (PCBs) and Polyaromatic

Hydrocarbons (PAHs) have concentrations ranging from 2.2 – 27.4, 1.7 – 31.6 and 83

– 544 ng/g, respectively. The most abundant CLPs were endosulfan, alachlor, heptachlor, lindane (γ-HCH) and DDT, which metabolites pattern revealed recent use.

Selected PAHs diagnostic ratios show pyrolytic input predominantly. The sum of 7 carcinogenic PAHs (ΣC-PAHs) represented 30 to 50% of Total PAHs (TPAHs).

According to effect-based sediment quality guidelines, the studied POPs levels imply low to moderate predictive biological toxicity. This study contributes to depict how fare water resources are shifting within what is now termed the Anthropocene due to increasing local pressures in African developing countries.

Keywords: Pesticides, Polyaromatic Hydrocarbons, Polychlorobiphenyls, Sediments,

Mangrove, Africa.

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5.1. Introduction

Alongside the greenhouse gases leading to climate warming, the pollution of the earth's surface does contribute as one of the main components of the global change.

The entering into the newly called Anthropocene, characterized by man's predominant influence on the earth system, including the global dispersion of different types of pollutants in all the environmental compartments, represents a major concern for environmental and human health. In particular, the management of coastal areas is becoming a growing issue.

Mangrove forests are one of the most threatened world’s coastal ecosystems acting as a fragile connection between marine and fresh water ecosystems (Maiti and

Chowdhury, 2013; Duke, 2011). They provide numerous ecosystem services ranging from habitats, food, nursery and breeding areas to marine and arboreal life, firewood, timber and non-timber forest products to local populations. Besides, they play an important role in nutrient cycling, pollution sink, stabilization and protection of coastlines from tsunamis and hurricanes (Ajonina, 2018; Pernot et al., 2015).

Anthropogenic pressure on mangroves, such as pollution, deforestation, dam, has risen over the years (Maiti and Chowdhury, 2013) and modify the functioning of mangroves. Beyond the preservation aspect, this may act as an opportunity to use them as biological indicators of anthropogenic pressures, in particular for water quality.

In this trend, two compartments of the mangrove are generally examined: the mangrove stand and the sediment. Sediments that accumulate in mangroves are potential sinks of anthropogenic pollution due to high total organic content, anaerobia, rapid turnover and burial (bioturbation) (Raza et al., 2013; Vane et al., 2009; Tam and

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Yao, 2002). Contaminated sediments can further act as chronic sources of pollution.

Actually, contaminants can be remobilized by microorganisms, consumed and retained the benthos and consequently biomagnified through aquatic food chains to higher trophic levels (Minh et al., 2007; Ross and Birnbaum, 2003). Hence, the assessment of contaminant levels in mangrove sediments could provide essential information on the chemical status and ecological risks linked.

Since the 2000's, several researchers pointed out the occurrence of diverse organic and inorganic anthropogenic contaminants including pharmaceutical products, endocrine disruptors, industrial chemicals and pesticides in mangrove compartments worldwide (Net et al., 2015; Kouzayha et al., 2013; Maiti and Chowdhury, 2013; Bayen,

2012; Lewis et al., 2011; Bayen et al., 2005; Zheng et al., 2000). But, the lack of data on contaminant levels in African aquatic ecosystems is often noted (Gioia et al., 2014,

Ssebugere et al. 2019, Fernandez et al. 2007, Brits et al., 2016). African mangrove covers, however, about 3.2 million hectares (20 %), making it the second largest worldwide mangrove ecosystem, following Asia (42 %) (Giri et al., 2011). Among other anthropogenic pressure and threats, they suffer in the last decades intense pollution due to large urban and harbour centres rapidly grown in their vicinity (Lagos and Port

Harcourt, Nigeria; Douala, Cameroun; Monbassa, Kenya; Abidjan, Ivory Coast; Dar Es

Salaam and Tanga, Tanzania; Dakar, Senegal; Durban, South Africa; Djibouti, Djibouti;

Suez Canal and Alexandria , Egypt; Tangier, Morocco; Port Luanda, Angola; Takoradi and Tema Harbour, Ghana). Rapid population growth and recent expansion of cities with inadequate planning and environmental management facilities fostered increasing input of various contaminants in the environment, including the Persistent Organic

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Pollutants (POPs) through urbanisation, agriculture and industrialization (Kaiser et al.,

2016). POPs are defined as compounds with toxic properties, a proven resistance to degradation, long-range transport and bioaccumulation potential in fatty tissues of living organisms (Lu et al., 2013; Jones and de Voogt, 1999). Although prohibited from the 1970s and 1980s in Europe and North America, their bans in Africa occurred only recently in signatory countries of the Stockholm Convention.

The present study focuses on the Wouri Estuary Mangrove (WEM) surrounding the Douala city, Cameroon. It follows a previous study carried out by Fusi et al., 2016 at two stations in the Wouri estuary (Bois des singes and Wouri Bridge) on macrobenthos (molluscs and crabs), assessing the ecological status and level of chemical pollution by anthropogenic contaminants, namely DDT and its metabolites

(DDE, DDD), bis(2- ethylHexyl)phthalate (DEHP), 10 polyaromatic hydrocarbons

(PAHs), 6 polychlorobiphenyls (PCBs), 16 heavy metals and 4 sterols. Based on statistical analysis, this study revealed significant sensitivity of macrobenthos to the target compounds and raised the concern that these compounds could potentially be spread throughout the mangrove zone. The need for further information on the levels, spatial distribution and toxicity risk of anthropogenic contaminants throughout the

Wouri Estuary Mangrove motivated the present study. It aims to be a pilot survey of anthropization level of the WEM by Persistent Organic Pollutants, carried out on 21 stations to ensure an extensive geographical coverage of the study area. To investigate the level of anthropization, a broad range of compounds (45) were analysed in the sediment compartment namely: a list of 22 chlorinated pesticides (CLPs) including 18 Organochlorinated Pesticides (OCPs), the 7 Polychlorinated Biphenyles

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Hydrocarbons (PAHs), which are non-intentionally produced POP. Though not classified as POPs, among the 22 CLPs targeted, 2 Organophosphorus Pesticides

(OPPs: chlorpyrifos, chlorfenvinphos) and 2 Chloroacetamide Herbicides (CAHs: alachlor, metolachlor) were investigated because of their toxicity and extensive use in

Cameroon for subsistence farming (Ntiendjui et al., 2009). In addition, alachlor and metolachlor were one of the most abundant and highly detected pesticides in surface water from urban and peri-urban areas of Yaounde (capital city, Cameroon) (Branchet et al., 2018).The sources of the studied compounds were discussed, and the likelihood of toxicity to aquatic organisms and human health was assessed using Sediments

Quality Guidelines (SQGs). Thereafter, our findings are then compared with those of other studies related to worldwide coastal environments (estuaries, mangroves, bays and lagoons).

5.2. Materials and methods

5.2.1 Study area

The present study was carried out in the Wouri estuary mangrove (WEM) located between 4° 03’ N and 9° 42’ E and covering 650 km 2 along the Cameroonian coastline in the Gulf of Guinea (figure 38). In 2007, the Foundation Working Group International

Wader and Waterfowl Research (WIWO) carried out a water bird census concluding that WEM qualifies as wetlands of international importance (Ramsar site) according to criteria of the Ramsar convention (Van der Waarde, 2007). This region has a warm and humid equatorial climate (mean annual temperature of 27 °C and average annual 202

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance precipitation of 4000 mm). The dominant tree species in this mangrove forest are

Rhizophora racemosa and Avicennia africana. This study mainly focuses on the area located between the Wouri and Dibamba rivers, acting as administrative borders of the city of Douala (figure 37). This town is the economic capital and the most populated city of Cameroon. Its present population is about 3 million inhabitants and is expected to reach 4 million inhabitants by 2030. This rapid urbanisation exerts a strong pressure on the mangrove ecosystem. The WEM forest is thus highly exploited for its halieutic resources, firewood, sand extraction and house construction (Longonje N. Simon,

2012). New quarters of the town are now expanding on the mangrove that equally serves as waste dumps for household and municipal sludge and sewage. Peri-urban agriculture has led to the conversion of mangroves into agricultural ponds and favours the cultivation of various crops such as maize, banana, oil palm, green vegetables and sugar cane. The Douala seaport is central Africa’s largest port and serves most landlocked countries of the African Sub-Saharan region. Douala is characterised by two major industrial zones known as Bassa and Bonaberi on both sides of the Wouri

River and a dense traffic in the city centres. These activities in conjunction with laxity in the implementation of laws protecting the environment and mangroves in particular, lead to continuous huge pressure exerted on the WEM ecosystem.

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Figure 38. Location of the Wouri Estuary mangrove (a) World map, (b) Cameroon map and

5.2.2. Sample collection, pre-treatment and contents of moisture and organic carbon

Superficial sediments (0 - 5 cm n=21) were collected by manual coring between

November and December 2017 (dry season). The study area was divided into four mangrove zones (figure 37): i) upstream of Douala (UD) (stations 1 to 3), ii) Bonaberi and Bassa Industrial Zones (BB) (stations 4 to 12), iii) Crique Docteur (CD)

(stations 13 to 19) and iv) Dibamba River (DR) (stations 20 to 21). During the sampling campaign, field observations revealed the presence of car tyres, used oil containers buried into sediments, dock and boating activities at the Douala harbor, commercial

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance fishery, waste dumps and effluent discharges. After collection, samples were transferred in aluminium trays of 0.5 L and stored at – 18 °C until pre-treatment. After thawing, samples were oven dried at 40 °C, ground and sieved at < 2 mm.

Moisture content was determined according to the standard method ISO 11465

(ISO 11465, 1993). Total Carbon (TC) and Total Inorganic Carbon (TIC) were determined separately using a TOC solid module HT1300, such that the difference results in Total Organic Carbon (TOC). For TC measurement, 50 mg of sediments were weighed in a ceramic boat, introduced in a furnace and combusted at 950 °C under a flux of oxygen. A pre-calibrated (CaCO3 12 % Carbon) non-dispersive infrared detector enabled the quantification of emitted CO2. For TIC measurement, 100 mg of sediments were weighed in a conical flask and reacted with 2 mL of 40 % phosphoric acid, heated at 83 °C and agitated at 350 rpm. The amount of emitted CO2 was quantified as above.

5.2.3. Reagents and standards

Solvents of suprasolv grade; n-Hexane (Hex), acetone (ACE), dichloromethane

(DCM), methanol (MeOH) and acetonitrile (ACN) were purchased from Merck (Pessac,

France). Hydrochloric acid (35 %), Nitric acid (69 %) and Phosphoric acid (85 %) of pure grade were from Fisher Scientific (Marseille, France) and VWR (France) respectively. PAH calibration mix at 10 μg/mL -1 in acetonitrile was supplied by

Supelco, Pesticide 8081 standard mix at 200 μg/mL -1 in Hexane: toluene (50:50) were obtained from Sigma-Aldrich and PCB mix at 10 ng/g in isooctane was provided by Dr.

Ehrenstorfer Laboratories (Augsburg, Germany). The individual pesticides

(Chlorfenvinphos, chlorpyriphos-ethyl, alachlor and metolachlor) at 100 μg/mL in

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Hexane were supplied by LGC Standards GmbH D-46485 Wesel. Alumina and copper powder (>230 mesh) were purchased from VWR International and Merck (Pessac,

France), respectively. Certified reference material RTC, CNS391-050, 50 g of PAHs,

PCBs and Pesticides on sediment was obtained from Merck. Agilent Mega Bond Elut

Florisil cartridges, 1 g, 6 ml were supplied by Agilent Technologies.

5.2.4. PAHs, PCBs and CLPs extraction

The extraction of 10 g of sediment was performed by Accelerated Solvent Extraction

(ASE Dionex 350). CLPs and PCBs extraction, adapted from (Villaverde et al., 2008), included a temperature of 100 °C and a static time of 5 min (2 cycles) at a pressure of

1500 psi. The solvent used was a mixture of Hex/Ace (1/1) with a flush volume of 60% and a purge time of 90 s. PAHs were extracted in the same way, but with a mixture of

Hex/Dcm (1/1) at temperature and pressure of 150°C and 1500 psi, respectively. The final extract was always in the range of 45-55 mL when using a thimble size of 33 mL.

An in-cell clean-up was performed by mixing the sample with 4 g of activated alumina for PAHs extraction, to eliminate polar compounds and with 3 g of activated copper for

CLPs and PCBs extraction, to remove sulphur compounds (Kanzari et al., 2012). The

PAH extracts were concentrated to a drop under a gentle flux of nitrogen and exchanged in 0.5 mL Acn for further analysis. The CLPs and PCB extracts were concentrated to a drop under a gentle flux of nitrogen, exchanged in 1 mL Hex and subjected to a clean-up and fractionation procedure using Solid Phase Extraction

(SPE) with Florisil cartridges (1g, 6 mL), according to the US-EPA 3620 C method. The elution procedure was optimized as follows: the cartridge was washed by 10 mL of Hex

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance before loading the 1 mL CLPs and PCBs extract. PCBs were eluted in the first fraction

(F1) with 2.5 mL of Hex (100 %). The targeted pesticides were eluted in the second fraction (F2) with 5mL of Hex/Ace (80/20 v:v), apart from the most hydrophobics ones

(i.e. α et β HCH, DDD, DDT), which were recovered in both fractions. Fractions F1 and

F2 were concentrated to 0.25-0.5 mL before subsequent instrumental analysis.

5.2.5. PAHs, PCBs and CLPs analyses

PAHs were analysed by high pressure liquid chromatography with fluorescence programmable detector (HPLC-PFD) using a PerkinElmer Flexar FX6 LC and a

PerkinElmer Altus A-30 FL detector. Separation was performed on an Agilent

Technologies Pursuit 5 PAH column (250 mm x 4.6 mm i.d. x and 5 μm). The elution conditions were as follows; a mobile phase with ACN/Water (50:50), flow rate

1.2 mL/min, injector and column temperature of 10 °C and 30 °C respectively was applied. For the separation, ACN rate was gradually increased to 100 % after 12 min and maintained for the next 12 min and decreased to 50 % for post-run after 4 min and finally maintained for 3 min for conditioning. The fluorescence detector was

programmed in a 4 channels data mode: 248 - 280 nm (excitation wavelength, λex) and

324 - 462 nm (emission wavelength, λem) for selective detection of each PAH (see

Table 22). The PerkinElmer Altus A-30 UV detector was set at 229 nm to enable the quantification of acenaphthene. Subsequently, data were processed by Empower 3 software. Quantification was based on a twelve-point calibration curve plotted between

1-1000 ng/mL.

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Analyses of CLPs and PCBs was carried out by Gas Chromatography-Mass

Spectrometry (GC-MS) using a Perkin Elmer Clarus 600 GC and Clarus 600 C MS, equipped with a Restek Rxi – XLB; (30 m x 0.25 mm i.d. x 0.25 μm) capillary column.

Prior to the injection, internal standard of PCB 116 D5 were added at 40 pg/μL. A volume of 1 µL was injected in spitless mode with helium as carrier gas at a constant flow rate of 1 mL/min. The temperature of the injector was driven from 50 °C (iso

0.1 min) to 250 °C (200 °C min - 1 - Iso 10 min). The GC oven was programmed from

70 °C (iso 2 min) to 175 °C (10 °C/min - iso 4 min) to 320 °C (5 °C/min - iso 10 min).

Source ionization was performed by electronic impact at an ionization energy of 70 eV and a temperature of 250°C. The temperature of the transfer line was set at 280°C.

The mass spectrometer was operated in SIM mode (Single Ion Monitoring) targeted three specific ions (one quantifier and two qualifiers) for each studied compound (see

Table 23). Subsequently, data were processed with TurboMass Version 5.4.1 software using the NIST MS Search Version 2.0 library. Quantification was based on the internal standard (PCB 116 D5) calibration curves obtained from the analysis of standard solutions at seven concentration levels (0.1, 0.5, 1. 5, 10, 20 and 100 pg/μl).

5.2.6. Quality Assurance and Control (QA/QC)

All analytical data were subjected to strict quality assurance and control. Procedural blanks composed of glass beads and Fontainebleau sand, and certified reference material (CRM CNS 391 for PAHs, PCBs and pesticides on sediment) were processed for each batch of 7 extractions in the same procedures used for field samples in this study. Procedural blanks were evaluated using the criteria that blank levels of target

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance analytes should be at least three times below the detection limit (signal-to-noise ratio

3:1). The accuracy of the method was tested by analysing native pollutants in certified sediment. Obtained recoveries were 62-94 % for pesticides, 76-94 % for PCBs and

69-95 % for PAHs (see section 3.8.3). Inter-day replicates (n=7) lead to relative standard deviations of 3-14 % for pesticides, 3-9 % for PCBs and 3-12 % for PAHs.

The reported limit of detection (See section 3.8.3) was set as the concentration level yielding to a signal-to-noise ratio of 3:1.

Prior to each extraction, 4,4-DDE D8 and PCB 156 D3 were added as surrogate standards at 100 pg/μL to check the analytical procedure for CLPs and PCBs, respectively. Mean ± SD recoveries were 85±7 % and 91±5 % for 4,4-DDE D8 and

PCB 156 D3, respectively. If surrogates’ recoveries differ from more than ±15 %, sample extraction was redone.

Instrumental QC was performed by regular analyses of solvent blanks and random injection of standards. Measured values were not deviating more than 15% from the theoretical values. The following conditions had to be met for an unequivocal identification and quantification of the analytes: (1) retention time matching that of the standard compound within ±0.15 min, (2) signal-to-noise ratio greater than 3:1, (3) detection of the qualifier ions when compounds were analysed by GC/MS. All concentrations are reported on dry mass.

5.3. Results and discussion

5.3.1. Spatial distribution and profiles of PAHs, PCBs and CLPs i) Polyaromatic Hydrocarbons (PAHs)

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The concentration of PAHs ranged from 83.35 (station 8) to 544.31 ng/g (station

17) with mean and median of 218.79 and 185.0 ng/g, respectively (Table 32). Lower concentrations of PAHs close to 100 ng/g were measured upstream of Douala (stations

1 – 3). This may be attributed to the fact that these stations are located upstream the anthropogenic pressure and the probable tidal flushing of pollutants adsorbed on sediment particles. In the Bonaberi and Bassa Industrial zones, station 9 and 11 had the highest PAHs concentrations of respectively 299.44 and 251.51 ng/g. PAHs input is probably due to massive burning (station 9) and effluent discharge (station 11).

Station 9 and 11 are located at the vicinity of a cement plant in the Bonaberi industrial zone where gaseous wastes from chimneys and industrial effluents are directly released in the mangrove area. At proximity to both stations is also an oil and gas company and massive burning of mollusc shells, locally used for liming to neutralize acidic soils in order to improve plant growth.

At the Crique Doctor, were determined at stations 13, 18 and 19 contained PAHs concentrations of 279.08 to 376.97 ng/g, which can be explained by the vicinity of

Douala port and by wastes discharges. The most contaminated stations 15 and 17 with

PAHs levels of 405.83 and 544.31 ng/g can be linked to exhaust emission from boating activities during commercial fishery and from waste dumps. The Crique Doctor area receives the discharge of sludge, sewage, municipal wastes. The sales of petroleum products and automobile repair garage are also located here. In addition, the Mgoua river known as the “black river” draining part of the Bassa industrial zone discharges in this area. Solange et al., (2014) reported high PAH levels (480000 – 333000 ng/g)

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(Nettoycam) located in the Bassa industrial zone.

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Table 32. PAH concentrations (ng/g of dry weight) in sediments of the WEM with Sediments Quality Guidelines (Long et al., 1995)

NA ACY ACE F PHE ANT FL PYR BaA CHRY B(b)FL B(k)FL BaP DBA B(ghi)P IP ∑PAHs LPAHs/ ΣCPAHs TEQ TOC (SD) CPAHs/ (%) (2) (3) (3) (3) (3) (3) (4) (4) (4) (4) (5) (5) (5) (5) (6) (6) HPAHs (%) TPAHs (%)

1 12.0 15.4 1.4 4.5 15.1 2.3 11.4 10.8 6.5 0.6 4.0 0.9 4.0 0.9 0.8 1.1 91.7 1.2 19.6 6.7 3.3 (0.02)

2 14.7 25.0 2.6 2.4 9.1 3.5 6.0 6.3 4.0 2.6 1.8 0.6 2.4 1.7 13.2 18.8 114.7 1.0 27.8 5.8 7.5 (0.13)

3 12.5 17.7 2.0 2.2 10.5 1.4 12.0 3.5 16.8 9.5 14.2 0.7 0.6 1.8 2.8 3.6 111.9 0.7 42.2 5.3 4.4 (0.02)

4 8.8 28.9 1.7 4.1 9.8 3.6 12.5 18.1 6.9 3.5 1.4 0.6 0.8 1.4 13.4 35.9 151.4 0.6 33.4 4.4 6.3 (0.15)

5 12.4 24.7 1.7 3.2 12.1 2.6 90.6 20.4 7.2 1.9 1.2 1.6 0.6 1.5 1.2 2.8 185.5 0.4 9.1 1.8 3.9 (0.04)

6 21.9 14.7 2.5 2.6 2.4 0.9 14.5 2.6 4.2 6.1 10.8 0.6 2.8 4.6 1.8 1.2 94.1 0.9 32.1 9.6 7.1 (0.08)

7 20.2 27.1 1.5 5.3 18.0 1.2 6.4 2.6 13.6 1.3 25.9 0.7 0.9 1.3 0.7 12.5 139.2 1.0 40.4 5.4 6.5 (0.11)

8 6.5 9.1 7.2 2.3 7.4 2.3 3.9 9.8 5.3 3.3 6.5 2.9 2.2 0.9 10.5 3.5 83.4 0.7 29.4 5.6 6.0 (0.08)

9 28.5 28.7 1.9 6.8 25.1 6.2 33.5 40.4 29.9 21.0 26.1 11.7 13.8 3.6 17.0 5.2 299.4 0.5 37.2 7.9 6.4 (0.18)

10 11.6 15.8 1.2 5.2 15.7 3.7 23.4 22.6 14.5 10.1 13.9 7.2 10.2 1.5 3.0 3.6 163.2 0.5 37.3 9.1 6.0 (0.05)

11 25.3 9.9 2.4 6.6 16.3 5.3 25.0 63.2 16.6 18.4 11.5 7.8 14.6 2.6 17.0 9.0 251.6 0.4 32.0 8.3 6.5 (0.15)

12 13.2 20.8 2.4 5.7 29.5 3.8 23.0 29.1 9.1 8.0 16.0 5.0 7.9 1.8 10.7 8.6 194.5 0.6 28.9 6.7 6.9 (0.16)

13 20.2 27.1 1.9 4.8 18.1 5.1 25.3 29.1 18.3 15.5 68.0 6.6 10.2 3.3 11.0 14.8 279.1 0.4 48.9 8.5 5.6 (0.03)

14 10.3 24.3 1.6 4.8 10.5 4.6 4.4 28.6 10.8 5.8 1.8 0.7 1.2 0.9 10.2 23.5 144.0 0.6 31.1 4.0 5.5 (0.12)

15 9.9 10.0 2.4 7.1 29.4 7.9 31.2 68.5 45.9 34.1 87.5 11.4 16.6 3.9 34.4 5.8 405.8 0.2 50.5 8.5 6.7 (0.14)

16 13.5 32.1 2.0 5.8 21.0 6.2 31.7 42.6 28.1 4.9 2.3 4.3 1.1 2.0 17.9 21.7 237.2 0.5 27.2 3.5 6.9 (0.10)

17 64.5 23.1 2.6 11.9 32.5 8.7 47.7 60.7 58.9 47.2 102.5 16.1 28.8 6.3 31.7 1.4 544.3 0.4 48.0 9.5 6.6 (0.46)

18 23.1 21.0 1.6 10.3 26.8 16.8 35.4 73.5 9.6 5.3 6.6 8.8 2.1 2.2 20.8 26.5 290.3 0.5 21.0 3.0 10 (0.02)

19 14.8 30.5 2.2 8.8 26.5 8.2 39.5 52.0 39.1 24.9 52.5 15.4 2.4 3.7 26.2 30.5 377.0 0.3 44.7 4.9 8.3 (0.08)

20 10.5 12.3 2.5 4.1 17.8 5.4 45.5 39.2 23.2 17.4 25.0 11.7 18.2 3.6 17.7 8.2 262.2 0.3 40.9 10.5 6.4 (0.14)

21 7.0 23.0 1.7 3.9 10.1 4.3 12.9 59.2 11.7 1.5 1.6 4.0 7.1 1.8 8.8 15.4 173.9 0.4 24.7 6.8 8.1 (0.06)

Min 6.5 9.1 1.2 2.2 2.4 0.9 3.9 2.6 4.0 0.6 1.2 0.6 0.6 0.9 0.8 1.1 83.4 NA 9.1 1.8 NA

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Max 64.5 32.1 2.6 11.9 32.5 16.8 47.7 68.5 58.9 47.2 102.5 16.1 28.8 6.3 34.4 35.9 544.3 NA 50.5 9.5 NA

NA ACY ACE F PHE ANT FL PYR BaA CHRY B(b)F B(k)F BaP DBA B(ghi)P IP ∑PAHs LPAHs/ ΣCPAH TEQ TOC (SD) L L s CPAHs/ (%) (2) (3) (3) (3) (3) (3) (4) (4) (4) (4) (5) (5) (6) (6) HPAHs (5) (5) (%) TPAHs (%)

Mean 17.2 21.0 2.2 5.4 17.0 5.0 25.5 32.5 18.1 11.6 22.9 5.7 7.1 2.4 13.1 12.1 218.7 NA 33.6 6.5 NA

Median 13.2 23.0 2.0 4.8 15.7 4.3 23.4 29.1 13.6 6.1 11.5 4.3 2.8 1.8 11.0 8.6 185.5 NA 32.1 6.7 NA

TPAHs 361.3 440.9 46.8 112.5 363.4 104.0 535.8 682.9 380. 242.5 480.9 119.1 148. 51.1 270.7 253. NA NA NA NA NA 3 3 4

ERL 160 44 16 19 240 85.3 600 665 261 384 NA NA 430 63.4 NA NA 4022 NA NA NA NA

ERM 2100 640 500 540 1500 1100 5100 2600 160 2800 NA NA 160 260 NA NA 44792 NA NA NA NA 0 0

() = number of rings, NA = Naphthalene, ACY = Acenaphthylene, ACE = Acenaphthene, F = Fluorene, PHE = Phenanthrene, ANT = Anthracene, Fl = Fluoranthene, ` PYR = Pyrene, BaA = Benzo(a)Anthracene, CHRY = Chrysene, B(b)Fl = Benzo(b)fluoranthene, B(k)Fl = Benzo(k)fluoranthene, BaP = Benzo(a)pyrene, DBA = Dibenzo(a,h)anthracene, B(g,h,i)P = Benzo(g,h,i)pyrelene, IP = Indeno(1,2,3-cd)pyrene, HPAHs = High Molecular Weight PAHs, LPAHs = Low Molecular Weight PAHs, ΣPAHs = Sum of 16 PAHs, TPAHs = Sum of individual PAHs, CPAHs = Carcinogenic PAHs, LPAHs = Low Molecular Weight PAHs, HPAHs = High Molecular Weight PAHs, TEQ CPAHs = Toxic Equivalents of CPAHs, ERL = Effect range Low, ERM = Effect Range Median, SD = Standard deviation. NA = not applicable

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According to a classification scale proposed by (Baumard et al., 1998b) 8 stations out of the 21 studied are moderately contaminated (100–1000 ng/g dry weight), while only three stations (1, 6, 8) are slightly contaminated, with a total PAH concentration of 0–100 ng/g dry weight, and none were corresponding to high contamination levels (1000 – 5000 ng/g dry weight).

TOC content ranged from 3.27 (station 1) to 10.13 % (station 18) with mean and median very close of 6.43 % and 6.48 % respectively, indicating a symmetrical distribution of the values (table 32). The rather high organic contents are typical of mangrove sediments and can originate through three main inputs: mangrove plants

(annual litter fall and underground roots), sediment trapping of suspended matter from coastal waters and benthic animals (Kristensen et al., 2008; Yong et al., 2011). TOC is observed to increase from upstream to downstream of the Wouri river. This is due to the deposit of coarser material upstream where sand extraction is mostly carried out whereas fine particles with higher TOC content are deposited downstream where the energy of the rivers is low.

In contrast to studies that reported a positive correlation between sediment organic carbon and concentration of POPs in mangroves (Tam and Yao, 2002; Vane et al., 2009), sediments of the WEM show poor correlations (R2 = 0.35) for TOC against

PAHs. This therefore suggests that the distribution and concentrations of PAHs in sediments of the WEM are not controlled or driven by the sediment’s OC content.

Similarly, a poor correlation was found between TOC and PAH concentrations in surface coastal sediments of the Gulf of Mexico (Wang et al., 2014). According to

(Simpson et al., 1996), correlation between PAHs and sediments’ OC was only

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> 2000 ng/g.

Concerning the distribution of the different compounds studied, tri-aromatic (15-

35 %) and tetra-aromatic (20-60 %) compounds are mostly accumulated in surface sediments (figure 39). The most abundant PAHs in increasing order were Pyrene

(PYR), Fluoranthene (FL), Benzo(b)Fluoranthene (B(b)FL), Acenaphthylene (ACY) and Benzo(a)Anthracene (B(a)A) referred to as High Molecular Weight PAHs (HPAHs) with 4 to 6 rings, except ACY referred as Low Molecular Weight PAHs (LPAHs) with 2 rings (Table 32).

PAH source characterisation was based on source specific diagnostic ratios such as PHE/ANT and FL/PYR distinguishing between pyrolytic (<10 and >1, respectively) and petrogenic (>10 and <1, respectively) origins in sediments (Budzinski et al., 1997). The same authors stated that, these two ratios must be studied simultaneously so as to provide a good estimate of PAH sources.

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Figure 39. Percentage distribution of PAHs and PCBs in sediments of the Wouri Estuary

Mangrove

The predominance of FL, PYR and to a lesser extent PHE (Table 32) observed in WEM sediments indicates the pyrolytic source of the contamination (Page and

Boehm, 1999). PHE/ANT values lower than 10 were measured in all stations and 33

% of the stations satisfy FLY/PYR < 1 (figure 39) expressing a predominance of pyrolytic sources. This is reinforced by the PAH distribution dominated by HPAH

(ΣLPAHs/ΣHPAHs < 1 in 85 % of the stations (Table 32). HPAHs from pyrolytic sources enter aquatic environments through contaminated soil or direct deposition

(Stogiannidis and Laane, 2015; Morillo et al., 2007; Budzinski et al., 1997), pointing out that this could be the major sources of PAHs in the WEM. Similar results were obtained by Aly Salem et al., (2014) for sediments of the Egyptian Red Sea coast (Suez

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Gulf, Aqaba Gulf and Red sea). Other ratios like BaA/Σ(BaA+CHRY) and

ANT/Σ(ANT+PHE) enable to distinguish between combustion and petroleum sources while IP/Σ(IP+B(g,h,i)P) enable the discrimination between various pyrolytic sources

(de Almeida et al., 2018; Mahdi Ahmed et al., 2017; Barakat et al., 2013, 2011; Asia et al., 2009; Vane et al., 2009; Mille et al., 2006; Yunker et al., 2002) (figure 40). These ratios BaA/Σ(BaA+CHRY) > 0.35, ANT/Σ(ANT+PHE) > 0.10 and IP/Σ(IP+B(g,h,i)P) >

0.5, reveal that the dominant pyrolytic source of PAHs in sediments of the WEM corresponds to the combustion of biomass (grass, wood and coal) and petroleum. This could be supported by burning activities such as dense traffic, harbour activities fish smoking, sustained by mangrove wood taking place in the study area.

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Figure 40. Cross-plot of PAH ratios for Wouri Estuary Mangrove sediments: PHE/Ant versus

FL/PYR (top) BaA/(BaA+CHRY) and IP/(IP+B(g,h,i)P) versus ANT/(ANT+PHE) (bottom) ii) Polychlorobiphenyls

The concentrations of ΣPCBs range from 1.7 (station 2) to 31.6 ng/g (station 10) with mean and median of 16.0 and 17.1 ng/g (Table 33). The highest PCB concentrations were measured at the Bonaberi and Bassa industrial zones (stations 9,

10) and to a lesser extend at Crique Docteur (stations 16, 17, 18). A recent study by 218

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(Ouabo et al., 2018) showed high PCB levels in soils from three informal recycling sites in Douala, located in the Bonaberi and Douala I.Z. As aforementioned, Crique Doctor is also a hub for the disposal of untreated wastes (municipal, household, sewage and slugde). During the sampling campaign, old car tyres, various plastic wastes, electronic wastes (e-wastes) were noticed and could spread out PCBs during their decomposition. Station 20 found along the Dibamba river shows also one of the highest

PCB levels (25.8 ng/g). The outfall in this river of industrial effluents from the Bassa industrial zone housing various plastic industries, pen producing, and textile industry can explain these findings.

The PCB pattern is dominated by hexachlorobiphenyls (PCB 138 and PCB 153) and pentachlorobiphenyls (PCB 101 and PCB 118) representing a total of 47 % and

30 % of the ΣPCBs (figure 39 above). This could be due to recent and/or chronic PCB contamination and to the higher persistence of highly chlorinated PCBs in sediments

(Bodin et al., 2011). This can be supported by the fact that despite the ban of PCB import in Cameroon since 2011, it’s use is authorized till 2025 but wastes should be eliminated before 2028 on the national territory (UNEP-POPS-NIP-Cameroon, 2016).

Our results furthermore revealed the use of highly chlorinated PCB mixtures such as

Aroclor 1254 (electrical capacitors and transformers, vacuum pumps, hydraulic fluids, plasticiser resins) and 1260 (electrical transformers and hydraulic fluid) (54 and 60 % chlorine by weight respectively), Phenoclor DP6 or Clophen A60. Similar PCB patterns were identified in sediments of the Hong Kong coastal mangrove (Tam and Yao, 2002),

Senegal Estuary mangroves (Bodin et al., 2011) and Congo River Basin (Kilunga et al., 2017; Verhaert et al., 2013).

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In the Wouri mangrove zone, with the exception of endrin, all the pesticides targeted in our study were detected at least once above method detection limit (MDL)

(see figure 41). The presence of normally prohibited substances, such as aldrin, heptachlor, 4,4'-DDT, 4,4'-DDD, 4,4'-DDE, chlordane and dieldrin must be highlighted.

The latter have also been found in several other African countries (Benin, Nigeria,

Tanzania, Mali). This can be explained by the sale and illegal disposal of stocks of pesticides that are now banned but not destroyed and also by the persistent nature of some of these compounds. DDT and metabolites, two metabolites of lindane, as well as alachlor are the most frequently detected pesticides (frequency above 50 %). (Fusi et al., 2016) also mention the presence of DDT and metabolites in sediments in the same area. It is only since 2002 that DDT has been banned in Cameroon. High detection frequency of DDT and metabolites (4,4’ DDT, 4,4’ DDD and 4,4’ DDE) was similarly found in sediments from the Atlantic coast of Morocco (Benbakhta et al., 2014) and it was attributed to its long half-life (T1/2= 2 - 25 years) (Chattopadhyay and

Chattopadhyay, 2015) and its recent and/or chronic use. Contrarily, the relative low detection frequency of parent lindane (γ-HCH, detected on 29 % of cases) while its metabolites (α and δ-HCH) were present on more than 50 % of stations. Its prohibition in Cameroon’s territory dates back to 2007 (UNEP-POPS-NIP-Cameroon, 2016).

Therefore large detection of metabolites indicates HCH extensive degradation, relatively short half-life (T1/2 » 6 years) (Mackay and Mackay, 2006) and significant historical use. Early ban in 1989 (UNEP-POPS-NIP-Cameroon, 2016) and rapid degradation could also explain dieldrin’s low concentration and detection frequency.

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90 % of this molecule was found to degrade within one month in tropical regions (Bodin et al., 2011).

To assess the level of contamination in the area, figure 40 shows for each pesticide, the distribution over the 21 stations in three classes of pesticide concentration (< 0.1, [0.1-1], > 1 ng/g). For the majority of the stations, it can be observed that these compounds are detected at very low concentrations, even below the MDL (<0.1 ng/g), especially trans-chlordane, which is detected at about 40% of the stations but always at very low levels. On the other hand, in nearly 50% of cases, alachlor and endosulfan were quantified at non-negligible concentrations (> 1 ng/g).

Similarly, the results presented by the Stockholm Convention national implementation plan report indicated that endosulfan persists in Cameroon and is also present in several African countries, such as Mali, Senegal and Togo. In addition, in about 30 % of the samples, HCH, aldrin, metolachlor, heptachlor, cis-chlordane, DDT and metabolites were detected at intermediate levels between 0.1 and 1 ng/g.

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Figure 41. Concentration ranges and frequency of detection of chlorinated pesticides in the

Wouri Estuary Mangrove

CLP concentrations ranged from 2.2 (station 12) to 29.1 ng/g (station 19 and 21)

(mean and median values 10.6 and 10.0 ng/g, respectively) (Table 33). The contamination was mainly in endosulfan (station 2, 6, 10, 13-17, 19, 21), alachlor

(station 1-4, 9-10, 15, 18-20), heptachlor (station 1, 3, 5, 16, 21), DDT and metabolites

(spread over all stations). Highest levels of the sum of pesticides were encountered in stations 19 (Bois des singes) and 21 (Dibamba river). This is explained by the activities that characterize the latter zone, such as (1) treatment against vectors of diseases such as malaria in the Ndogpassi, Nylon and Village districts, which are close to the

Dibamba River which flows into the Wouri estuary, (2) peri-urban agricultural activities practiced in the Yassa area which is in connectivity with Dibamba hydrosystem. At Bois des singes (station 19), the presence of municipal wastewater disposal in basins without treatment could explained the high level measured. The use of pesticides in

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance peri-urban agricultural activities and disease vector control such as malaria was reported by (Antonio-Nkondjio et al., 2011). Moreover, Nfotabong-Atheull et al., (2013) reported that mangrove stands around Douala have been converted into agricultural areas for the cultivation of maize, bean, green vegetables, sugar cane and banana.

Furthermore, the development of agriculture upstream of the estuarine zone (UD area) could likely be due to low level of salinity due to high rate of sea water dilution. This can contribute to concentrations of pesticides accounting to around 10 ng/g measured at stations 1, 2 and 3.

For OCPs, the concentration of ΣDDTs (4.4’ DDT, 4.4’ DDD, 4.4’ DDE) ranged from 0.1 to 5.0 ng/g (mean and median of 1.0 to 0.5 ng/g respectively) whereas concentrations of ΣHCHs (α, δ, β and γ isomers) ranged from 0.4 to 3.1 ng/g (mean and median of 1.2 and 1.1 ng/g, respectively). Endosulfan (α-Endosulfan + Endosulfan sulphate) was the most abundant pesticide found in WEM sediments (concentrations from 0.95 to 18.5 ng/g and mean and median values of 4.7 and 2.6 ng/g respectively).

High levels could be explained by its broad spectrum as insecticide and its large atmospheric pattern transport (Weber et al., 2010). Our results suggest its recent use, which is congruent with its relatively recent prohibition since 2008 in Cameroon. -

Endosulfan concentrations were higher than those of its toxic metabolite, endosulfan sulfate (ratio between 3 to 75), indicating a slow rate of endosulfan degradation

(Kuranchie-Mensah et al., 2012) and relatively recent use. Mean concentration of endosulfan sulfate was higher in WEM (0.97 ng/g) than values previously reported in

Ghana (Kuranchie-Mensah et al., 2012). Similarly α-Endosulfan exhibited higher concentrations in WEM sediments (~ 3.2 ng/g) than in the Atlantic coast of Morocco

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Ghana (Kuranchie-Mensah et al., 2012), but lower than those in the Ogbese river sediments in Nigeria (Ibigbami et al., 2015).

OPP concentrations were rather low, ranging from 0.4 – 0.9 for chlorpyriphos- ethyl and

It is globally used as an insecticide against soil insects, termites and sometimes for the control of anopheles mosquitoes acting as vectors for malaria. In Cameroon, chlorpyrifos-ethyl is an insecticide frequently used by farmers in the cultivation of maize

(Antonio-Nkondjio et al., 2011; Nwane et al., 2009), its low concentration and detection frequency in WEM sediments are consistent with a relatively high water solubility and very short half-life (24 days) in seawater-sediment system as described previously

(Howard, 1991).

Levels of CAHs ranged from

Residue Limit in 22 out of 72 food samples from the western highlands of Cameroon were previously detected (Galani et al., 2018).

Figure 42 below presents the spatial distribution and order of magnitude of

PAHs to PCBs and CLPs; 20: 4: 5 respectively. The Crique Docteur and the Bonaberi and Bassa industrial area show the highest levels of contamination for the studied compounds due to point and non-point pollution sources; the increase in settlement of various industries in these areas and Urbanization. In addition, the complex

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Mgoua and Dibamba river) that discharge in the Cameroon estuary. And lastly, this is supported by winds giving a constant direction to coastal currents that carry and deposit large amounts of materials (mud, clay, sand), nutrients and pollutants in the

“Cameroun mouths” (Cameroun Estuary) towards sites like the where silting up occurs leading to permanent dredging of the Douala Port channel (Din et al., 2017). The hydrodynamic conditions of the Cameroon estuary could explain the dispersion of

POPs throughout the WEM as proven by the present study.

Figure 42. Spatial distribution of PAHs, PCBs and CLPs in sediments of the Wouri Estuary

Mangrove

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Table 33. Concentration of CLPs and PCBs (ng/g of dry weight) in sediments of the Wouri Estuary Mangrove

CAHs OPPs OCPs PCBs ΣPenta- Station Alach Metol ∑CAHs Chlorp Chlorf ∑OPPs ∑Endos ∑Chlord ∑Endrin ∑Hepta Aldrin Dield ∑HCHs ∑DDTs ∑OCPs ∑PCBs Hexa CBs (%) 1 3.0 ND 3.0 ND ND ND 1.0 0.2 ND 3.1 0.3 ND 0.4 ND 8.1 4.1 79 2 4.2 1.0 5.2 ND ND ND 4.0 ND ND 0.3 ND ND 1.0 0.3 10.7 1.7 78 3 2.0 ND 2.0 0.8 ND 0.8 ND ND ND 6.0 1.0 ND ND 1.8 11.5 13.1 87 4 1.0 ND 1.0 ND ND ND ND 0.2 ND 0.3 0.6 ND 3.1 0.7 5.9 13.3 81 5 ND 0.9 0.9 ND ND ND ND 0.3 ND 3.5 0.5 ND ND 5.0 10.0 17.1 89 6 ND 0.2 0.2 ND ND ND 10.2 0.2 0.6 0.3 ND ND 1.8 0.5 13.7 4.4 53 7 ND ND 0.0 ND ND ND 1.6 0.2 ND 0.8 1.0 ND 1.2 1.1 5.9 23.7 82 8 0.5 0.1 0.7 ND ND ND ND ND ND 1.0 ND ND 0.5 0.6 2.8 10.8 75 9 1.6 ND 1.6 ND ND ND ND ND ND 0.3 0.5 ND 1.6 0.1 4.0 30.4 76 10 2.0 ND 2.0 ND ND ND 4.3 ND 2.2 0.3 0.5 1.1 1.0 0.7 11.9 31.6 71 11 ND 1.7 1.7 ND ND ND ND 0.2 ND ND 0.7 ND 1.8 1.2 5.5 17.1 84 12 ND ND ND ND ND ND ND ND ND 0.3 0.4 ND 1.1 0.4 2.2 6.6 82 13 ND 0.4 0.4 ND 0.3 0.3 11.3 0.2 ND ND ND ND 3.1 0.5 15.8 19.9 77 14 0.6 0.2 0.8 ND ND ND 11.1 ND ND ND ND 0.1 0.5 0.4 13.0 5.3 51 15 3.5 ND 3.5 ND ND ND 4.6 ND 0.2 ND ND ND 1.7 0.4 10.4 12.8 82 16 1.1 ND 1.1 0.9 ND 0.9 4.4 0.2 ND 4.3 1.9 ND 0.6 1.2 14.6 30.1 76 17 0.6 0.2 0.8 ND ND ND 11.1 ND ND ND ND 0.1 0.5 0.4 13.0 20.0 76 18 4.9 ND 4.9 ND ND ND 2.6 ND ND ND ND ND 2.1 0.4 10.0 22.7 80

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19 4.0 0.2 4.2 ND ND ND 18.5 ND 4.5 ND ND ND 1.6 0.4 29.1 18.3 74

CAHs OPPs OCPs PCBs ΣPenta- Station Alach Metol ∑CAHs Chlorp Chlorf ∑OPPs ∑Endos ∑Chlord ∑Endrin ∑Hepta Aldrin Dield ∑HCHs ∑DDTs ∑OCPs ∑PCBs Hexa CBs (%) 20 2.3 ND 2.3 ND ND ND ND ND 0.6 1.2 ND ND ND 1.7 5.8 25.8 87 21 ND ND ND 0.4 ND 0.4 15.1 0.2 1.7 5.5 ND ND 2.3 3.9 29.1 7.8 83

Min ND ND - ND ND - ND ND ND ND ND ND ND ND - 1.7 79 Max 4.9 1.7 5.2 0.9 0.3 0.9 18.5 0.2 4.5 4.3 1.9 1.1 3.1 5.0 29.1 31.6 78 Mean 2.2 0.5 1.7 0.7 0.3 0.1 7.7 0.2 1.6 1.9 0.7 0.4 1.4 1.1 11.1 0.2 87 Median 2.0 0.2 1.1 0.8 0.3 0.0 4.6 0.2 1.2 0.9 0.5 0.1 1.4 0.6 10.4 0.1 81

ND = Not determined, CAHs = Chloroacetinamide Herbicides, OPPs = Organophosphate Pesticides, OCPs = Organochlorine Pesticides, Alach = Alachlor, Metol = Metolachlor, Chlorp = Chlorpyrifos-ethyl, Chlorf = Chlrorfenvimphos, ∑Endo = Endosulfan I + Endosulfan sulphate, ∑Chlord = Trans + Cis Chlordane, ∑Hepta = Heptachlor + Heptachlor epoxide, Dield = Dieldrin, ∑HCHs = α-HCH + δ-HCH + β-HCH + γ-HCH, ∑DDTs = 4,4’ DDT + 4,4’ DDE + 4,4’ DDD, ∑PCBs= sum of 7 indicator PCBs (CB28, CB52, CB101, CB118, CB138, CB153 and CB180), ∑Penta-Hexa CBs = Sum of Penta CBs (CB101, CB118) and CBs (CB138, CB153)

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Although DDE is generally the most widely detected and most resistant to biotransformation compound in the environment (Chattopadhyay and Chattopadhyay,

2015), the abundance pattern of DDTs in the WEM showed that 4,4’ DDT > 4,4’ DDE >

4,4’ DDD, indicating a recent use of DDT according to Nozar, (2013) and prevailing anoxic conditions of sediments that favour the formation of DDD. In addition, this pattern could be observed due to higher solubility of DDT metabolites (DDE and DDD)

(Chattopadhyay and Chattopadhyay, 2015). To assess the recent DDT use, diagnostic ratios (4.4’ DDE + 4.4’ DDD) / 4.4’ DDT) were computed for stations where all the three compounds (DDT, DDE and DDD) were present (i.e. stations 5, 8, 11, 12, 14, 20 and 21having calculated ratios of 0.08, 0.35, 2.00, 1.18, 0.40, 0.09 and 0.06, respectively). Except for stations 11 and 12 located on the western bank of the Wouri river, the ratios lower than one sign a recent use of DDT (Zhang et al., 2011). Similar results were found in the mangrove of South Iran Nozar (2013), in line with the recent recommendation by the World Health Organization (WHO) for the reintroduction of

DDT for vector control in some malaria endemic settings, such as Africa (Coleman et al., 2008).

The diagnostic ratio (α/γ-HCH) between 3 and 7 indicates the use of technical

HCH (Zhang et al., 2011) while values lower than 3 indicates lindane use (Tao et al.,

2005). Furthermore, low or high α/γ-HCH values were found to reflect current lindane or historical HCHs uses, respectively (Tao et al., 2005). Due to absence of α and γ isomers in most samples of the WEM, computed α/γ-HCH ratios were <3 for three stations (4, 8 and 21). The pattern α > δ > β > γ of lindane isomers suggested an extensive degradation of lindane into its metabolites, reflected by lindane low half-life

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(90 days, (Sang and Petrovic, 1999)) and its degradation speeded up by anaerobic conditions (Fiedler et al., 1990). However, despite an absence of homologation and a ban, a stock of lindane still remained in 2005 (UNEP-POPS-NIP-Cameroon, 2016).

Similar to the Mumbai mangroves, this study shows that despite the restriction on the use of DDT and lindane in Cameroon, they are found to be persistent in mangrove sediments, usage after prohibition to sell off stock or even nowadays

(especially DDT allowed by WHO for malaria vector control).

5.3.2 Comparison to Worldwide estuary and mangrove areas

Compared to the previous study carried out in this area (Fusi et al., 2016, samples collected in 2009) the concentrations of 10 PAHs and 6 PCBs congeners in 2 stations of sediments (BS and WB) were below 300 ng/g and 20 ng/g of sediment dry weight, respectively. Main conclusions indicated no statistical differences among sites

(Wouri Bridge and Bois des singes), belts (Avicenia sp, Pandanus sp, Rhizophora sp) and sampling depths (0 - 10 and 10 - 20 cm). Out of the 21 stations examined in the present study, we showed also that the distribution of the contamination is relatively homogenous in the area. Some spots of pollution were nevertheless noticed in the industrial zones of Bonaberi and Bassa and Crique Docteur.

Levels of PAHs contamination have not really changed between 2009 (< 300 ng/g) and 2017 (83-544 ng/g). Conversely, higher PCBs levels were measured increasing from about 5 ng/g in 2009 to 20-30 ng/g in 2017. These compounds were banned very recently (August 2011) in Cameroun. Moreover, PCBs can enter the environment through wastes disposal and can be linked to the rapid growth of

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance urbanization and industrialization in Douala during the past decade. The opposite trend is noticed for DDE, which has dropped down from 10 - 40 ng/g in 2009 to level below

1.72 ng/g in 2017. In Fusi et al. 2016, DDT was not detected while 4,4’ DDE was the only metabolite detected at concentrations higher than 3 ng/g. The same author found

4,4’ DDE at mean concentration of 30 ng/g (n = 7, depth of 0 - 10 cm) while this study presents very low mean concentrations of 0.28 ng/g (n = 21, depth of 0 - 5 cm) for

DDE. This large discrepancy could be associated to sand extraction activities highly carried in this area, which remobilize these contaminants in the water column.

Furthermore, a return to DDT is on the horizon in some African countries to combat malaria and the WHO has declared itself in favour of a resumption of its use by spraying in homes. The recent usage of DDT for malaria vector control corroborates our data showing the prevalence of the parent compound (DDT) over its metabolites (DDD and

DDE).

On a global scale, the level of POPs in sediments of mangrove and estuaries areas across the world are presented in table 34. On a worldwide scale, PAH contamination in the WEM was higher than mangrove sediments of the Malaysian

Peninsular (Raza et al., 2013), Imo river estuary and mangrove (Nigeria) (Oyo-Ita et al., 2016), the Minjiang river estuary (China) and the Rembau–Linggi estuary

(Malaysia) and the Douro River estuary (Portugal). But concentrations of PAHs in sediments of the WEM were lower than those of other mangroves located in Djibouti,

Nigeria and Martinique. The highest PAH contamination was found in sediments of the

Iko River estuary mangrove (6100 - 35270 ng/g), mangroves of Bay of Fort-de -France

(18000 - 112000 ng/g), Gironde Estuary (1000 - 2000 ng/g).

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Levels of HCHs in the WEM are within the range of concentrations found in sediments of other studies worldwide are higher than those in sediments of the South

China mangrove reserves (Qiu et al., 2019), Falia and Fadiouth estuary mangroves in

Senegal (Bodin et al., 2011). For DDTs, concentrations are generally lower than those found in sediments of studies worldwide. Levels of PCBs in sediments of the WEM are higher than most mangrove areas such as, mangrove and coastal sediments in the

Persian Gulf (Nozar, 2013; de Mora et al., 2010), mangrove sediments of the

Tanzanian coast (Kruitwagen et al., 2008), Hong Kong mangrove sediments (Tam and

Yao, 2002), S. Buloh and S. Khatib Bongsu Mangroves in Singapore (Bayen et al.,

2005), Sunderban mangrove in India (Binelli et al., 2009). The results of this study can serve as an early warning of current PCBs contamination in the WEM. Many entry points for these compounds have been mentioned in our work, such as improper e- wastes disposal, wastewaters outfall, and growing industrial activities. The management of this area should be adjusted in the face of these particular pressures.

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Table 34. Summary of POP levels (ng/g) in sediments (dry weight) of coastal environments (estuaries and mangroves) worldwide

Concentration Concentration range (ng/g) of dry weight (dw) Study area and Sampling Depth range (ng/g) Country Main activities References Environment Stations (cm) of dry weight ΣDDTs ΣHCHs ΣOCPs Σ7PCBs (dw)

2.21- Cameroon Wouri Estuary Mangrove Industrial, harbour 21 0 - 5 0.1-0.5 0.4 – 3.1 1.7- 31.6 83 - 544 This study 27.4E

15.7 - Madhi Ahmed Djibouti Djibouti-city Mangrove Oil spills, Industrial 11 0-10 NA NA NA NA 3760.1 2017

Cameroon Wouri Estuary Mangrove Industrial, harbour 2 0 - 20 10 - 40A NA - 2.6 – 6.3G 85 – 250B Fusi M. et al. 2016

Nigeria Imo River Estuary Industrial, Petroleum 5 0 - 3 NA NA NA NA 28 - 64 Oyo-Ita et al 2016

Fadiouth and Falia Senegal Agricultural 2 0 - 5 0.3-15.9 0.1–1.9N 1.2-18.2O 0.3 – 19.1 NA Bodin et al., 2011 MangroveR

Iko River Estuary Oil spillage, 6100 - Nigeria 0-10 NA NA NA NA Essien et al 2011 Mangrove Agricultural 35270

Tanzanian coast Industrial, 0.2 – Kruitwagen et al., Tanzania 7 0 - 5 0.1-8.6 ND 2.3-3.7C NA mangrove agricultural 5.3D 2008

Mangroves os Southern Industrial, Assunção et al. Brazil 9 0-10 NA NA NA NA 6.8 - 437.3 Brazil Mariculture 2017

Oil refinery, solid Mangroves 0.42 - Puerto Rico Mangroves of Jobos Bay waste landfill, 18 0 - 1 NA NA NA NA Alegria et al 2016 1232 pharmaceutical

Agricultural, Mangroves of Bay of 18000 - Martinique industrial, nautical, 16 0 - 10 NA NA NA NA Mille et al., 2006 Fort-de-France 112000 transport

Guanabara bay Industrial, 0.7 – 28.4 – 17.8 – Brazil 5 0 - 3 10.6 - 37.4K NA Souza et al., 2008 Mangroves Petrochemical plants 2.0N 216.6O 184.2

Mangroves of Nanliu Industrial,

Industrial, South China Mangroves Agricultural, 0.01 - 0.08 - China 12 0 -1 1.14 - 6.82 - NA Qiu et al 2019 reserves Aquaculture, 0.16 0.23 Harbour

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Industrial, India Sunderban mangrove agricultural, 7* 0 - 30 NA NA NA 0.5 – 26.8 NA Binelli et al 2009 aquaculture, harbour

S. Buloh and S. Khatib Singapore Aquaculture 2 2 - 7 <0.1 - 0.9W 1.2 – 6.0 - 0.6 – 1.9T NA Bayen et al. 2005 Bongsu Mangroves

Imo river coastal Industrial, Petroleum Nigeria 5 0 - 5 NA NA NA NA 51 – 90X Oyo-Ita et al 2016 mangrove refineries

Indian Coast of Kenya Agricultural, 16.1- Kenya 4 0 - 5 2.3 – 42.0 - NA NA Barasa et al., 2007 Estuary industrial 1445N

Wastewater, oil Portugal Douro River estuary 4 50 NA NA NA NA 59 - 156.45 Rocha et al., 2011 refinery

UK Mersey Estuary Industrial 14* 0 - 100 NA NA NA 36 - 1406 626 - 3766 Vane et al., 2007

Agriculture, Malaysia Rembau–Linggi estuary 21 NA NA NA NA 20 - 112 Raza et al. 2013 ubanization

1000 - Budzinski et al.

France Gironde estuary Industrial 24 0 - 2 NA NA NA NA 2000U 1997

Industrial, China Beibu Gulf 1* 0 - 45 ND-0.5V NA - ND 38 - 74 Kaiser et al 2016 agricultural Estuaries Northern Bohai and Agricultural, 52.3- China 35 0 - 10 NA NA NA NA Jiao et al., 2012 Yellow seas Industrial 1870.6

Industrial, oil, steel China Dalliao River Estuary 35 0 - 5 NA NA NA NA 272 - 1607 Men et al 2009 factory

Industrial, 28.8– 15.14– China Minjiang river estuary 9 1 1.6–13.1 3.0–16.2 NA Zhang et al. 2003 agricultural 52.1O 57.9F

Industrial, 0.003 – Bhattacharya et India Hugli Estuary, Sunderban 1 Surface 0.003 - 0.1S NA NA NA agricultural 0.3 al., 2003 A=DDE, B=10 PAHs, C=10 PCBs, D= 2HCHs, E=18 OCPs, F=21 PCBs, G=6 PCBs, *=sediment core, H=40 PCBs, i=11 PCBs, J=13PCBs, K= 6DDTs, L= 3HCHs, M= , N= γ- HCH, O= 14 OCPs, P= 28 PCBs, Q= 11OCPs, , R=Ramsar site, S= 5DDTs , T = 40PCBs, , U= 17 PAHs V= 4DDTs, W= 2DDTs, X = 13 PAHs, ΣDDTs = (DDT+DDE+DDD), ΣHCHs = (α-HCH+ δ-HCH+β-HCH+γ-HCH), NA= Not analyzed, ND = Not detected, Y = 15 PAHs, DL = Detection Limit

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5.3.3 Ecotoxicological significance i) Implication for aquatic organisms

The appraisal of sediment quality and ecological risk assessment from the WEM was carried out following sediment quality guidelines (SQGs) developed for marine and estuarine sediments (Long et al., 1998, 1995).. These SQGs express the incidence of adverse biological effects to be observed and or predicted in aquatic organisms closely associated with sediments. Two approaches were applied: the

Effect Range Low (ERL) and Effect Range Median (ERM) values as well as the

Threshold Effect Level (TEL) and Probable Effect Level (PEL). These values delimit three concentration ranges: (1) concentrations below ERL and/or TEL representing minimal effect ranges below which adverse biological effects would be rarely observed,

(2) concentrations equivalent to and above ERL and TEL representing possible-effect ranges within which adverse effects will occur and (3) concentrations equivalent to and above ERM and PEL representing probable-effect ranges within which biological effects are frequently observed. (Long et al., 1998, 1995).

For ΣPAHs, all stations fall to very low levels below the ERL concentrations

(4000 ng/g) indicating minimal probability of effects. At these concentrations, adverse biological effects would be rarely observed. In 71 % of stations, ΣPCB concentrations were lower than the ERL and TEL threshold values (figure 42), expressing concentrations corresponding to minimal probability for toxic effects to occur. However,

29 % of the sites located in the Bonaberi and Bassa Industrial zones (station 7,9 and

10) and Crique Docteur (stations 16, 18 and 20) exhibited concentrations associated with an occasional probability of toxic effects to occur. For ΣDDTs, up to 80 % of

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance stations have concentrations below ERL and TEL (figure 42) thus associated with a minimal probability for toxic effects to occur, whereas 20 % of stations fall between

ERL and ERM and 10 % between TEL and PEL indicating concentrations that were occasionally associated to adverse biological effects. Following the aforementioned results, sediments throughout the study area predominantly represent concentrations within which adverse biological effects would be rarely observed. To a lesser extent, sediments mostly found in the Crique Docteur, Bonaberi and Bassa Industrial zones represent possible-effect ranges within which adverse biological effects will occur.

None of the analysed stations have concentrations equivalent to ERM or PEL for all studied compounds (figure 43). This is consistent with the findings of (Fusi et al.,

2016), who observed that the relationship between macrobenthos assemblages and

POPs (PAHs and 4,4’ DDE) was not significant accounting for a total variation below

10 %.

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Figure 43. Comparison of DDT and PCB concentrations in sediments of the WEM with

Effect-based Sediment Quality Guidelines ii) Implication for Human Health

The pattern of PAHs in the WEM sediments was dominated by (HPAHs) (see

Table 32), indicating the presence of strongly carcinogenic and mutagenic PAHs

(Karlsson and Viklander, 2008; Laane et al., 2005), apportioning for approximately 25

– 50 % of the total PAH.

Carcinogenicity of WEM sediments was assessed using Toxic Equivalent

Concentrations (TEQs) of carcinogenic PAHs (C-PAHs) (Raeisi et al., 2016; Aly Salem et al., 2014; Nasher et al., 2013; Chen and Chen, 2011; Qiao et al., 2006). Among the

7 compounds, B(a)P is the only PAH for which toxicological data are sufficient to derive a carcinogenic potency factor (Aly Salem et al., 2014; Nasher et al., 2013; Hu et al.,

2011; Peters et al., 1999). Hence, the carcinogenicity of other PAHs was expressed relative to that of B(a)P. For the 7 C-PAHs, the Toxic Equivalent Factors (TEF) according to the US EPA is given in bracket: Chr (0.001), B(a)A (0.1), B(b)F (0.1),

B(k)F (0.1), B(a)P (1), IP (0.1) and DB(a,h)A (1). The total TEQs for C-PAHs was defined by:

ΣTEQ C − PAHs = ∑.(Ci × TEFi) ………………………… Equation 5 where, Ci is the concentration and TEFi the toxic equivalent factor of PAHi relative to

B(a)P. The ΣTEQ C-PAHs ranged from 3.2 – 51.5 ng/g with mean and median of 14.9 and 9.0 ng/g respectively. Higher TEQs were found in station 17, followed by the stations 15, 20 and 13 located in the Crique Docteur (CD) area. This could be attributed to industrial, harbour and airport activities coupled with the dense traffic in the city of 238

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Douala. The highest contributors of PAHs carcinogenicity include Benzo(a)Pyrene

(47 %), Dibenz(a,h)Anthracene (16 %), Benzo(b)Fluoranthene (15 %),

Benzo(a)Anthracene (12 %). According to Dickhut et al., (2000), motor vehicles are major sources of these C-PAHs especially in highly populated areas.

5.4. Conclusion

The present study provides comprehensive information on the level of Wouri

Estuary Mangrove anthropization relative to POPs and associated toxicity risks.

Despite the ban of some of these compounds over the last 20 years in Cameroon, their presence in mangrove sediments evidenced their recent and/or chronic uses and also pressures from local as well as global scales. However, WEM sediments generally show low pollution levels and toxicity risks to aquatic organisms. The dispersion of

POPs throughout the WEM and identified pollution hotspots; Crique Docteur and the

Bonaberi and Bassa Industrial area is related to point and non-point pollution sources coupled with hydrodynamic processes in the Cameroun estuary. The concentrations of PAHs and CLPs measured are in the same order of magnitude of those determined in mangroves of tropical developing countries and corroborate data published on soil or aquatic organisms in the vicinity of the studied stations. Levels of PCBs are higher than those reported in similar environment and could be associated with an occasional probability of toxic effects.

The outcome of this work could help reinforce the action plan set up in the framework of the National implementation plan of the Stockholm Convention in

Cameroon launched since 2012. It contributes in raising awareness for the

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5.5 Conclusive statement on Chapter 4 and Chapter 5

Almost all targeted compounds were present in both study sites while OCPs in water were below detection limit in Lake Barombi. The aforementioned results affirm that the WEM is subjected to higher anthropogenic pressure showing higher POP contamination (OCPs and PAHs) than the LBW. The levels of ΣOCPs in sediments were comparable between the WEM (2.2 – 29.1 ng/g) and LBW (4.3 – 57.7 ng/g) but slightly higher in LBW may be due to the predominance of agricultural activities, despite similar TOC content for the WEM (3.3 – 10.1 %) and LBW (0.9 – 9.7 %). It is important to note that only 9 OCPs out of 18 OCPs were detected in sediments and soils of the

LBW while all OCPs where found in sediments of the WEM. TPAHs concentrations in the WEM were about 5 times higher than those in the LBW may be due to vehicular emissions due to dense traffic, port activities, petroleum exploitation, effluent discharges and emissions from the Bonaberi and Bassa industrial zones, open burning of household wastes and municipal waste discharge in the mangrove.

The two study sites provide various ecosystemic benefits which need to be preserved. Overtime both ecosystems are altered by city expansion, rapid demographic growth and urbanization, the city of Kumba in the case of the LBW and

Douala for the WEM. These represent serious threats to the state and wellbeing of these very important ecosystems. Lake Barombi watershed is a protected area; forest reserve and a Ramsar site but the absence of a management plan favours the degradation of this fragile ecosystem. Recent studies have reported increasing

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance anthropogenic pressure on the Lake Barombi watershed. According to (Fonge et al.,

2019), the predominant dense forest has been converted to new land uses, such that

90.4 % of dense forest is lost in favour of open forests, cocoa farms and mosaic forests.

Therefore, there is a pressing need to implement policies, regulations and management plans for the conservation of the status and functions of these environments.

Endosulfan was the most abundant pesticide in both study sites followed by lindane (γ-HCH). Common pesticides found on both sites were endosulfan (α or β endosulfan and endosulfan sulphate), HCHs (α-HCH, δ-HCH, β-HCH, γ-HCH), aldrin and dieldrin. DDT was only found in sediments of the WEM. In the framework of the

National Implementation Plan (NIP) of the Stockholm Convention in Cameroon, this is coherent with the survey carried out in 2012 reporting that a stockpile of 3 tons remaining were endosulfan, lindane, dieldrin and DDT, with 151 kg of DDT as POPs and obsolete pesticides. The same report indicates that is endosulfan was found to be persistent in Cameroon and other African countries such as Mali, Senegal and Togo.

This supports the idea that that some POPs could have been extensively used or still used after their ban in Cameroon and similarly in other African countries.

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General conclusion, Perspectives and Recommendations

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General conclusion

This work demonstrates that the concern about the protection of human health and the environment against POP contamination is a current and highly topical issue globally, especially in developing countries which appear to be more vulnerable due to lack, insufficient or inadequate infrastructures to manage these chemicals. This thesis contributes in filling the gaps of data scarcity in Africa and raising awareness for the strengthening of regulations regarding anthropogenic pollutants and POPs in particular. More so, the findings of this work will help reinforce the action plan set up in the framework of the National Implementation Plan of the Stockholm Convention on

POPs in Cameroon launched since 2012. The present African context of rapid demographic growth, urbanization, industrial and agricultural development favours the presence and exposure of humans and biodiversity to POP contamination in highly urbanized or remote areas. Programs such as the Africa Stockpiles Programme (ASP) launched since 2005 and implemented for over 10 - 15 years helped to clean up stockpiles of POPs (over 50,000 tonnes of obsolete pesticides and associated wastes) and provided local capacity building to prevent future build-ups. Nevertheless, the occurrence and levels of these compounds in the environment requires the implementation of strong measures, continuous awareness raising and the provision of adequate facilities to prevent their recurrence. In addition, despite the legal arsenal available in various African countries, its effective implementation remains a serious issue.

The bibliographic overview indicates that the state of knowledge on POPs in

Africa is still very scarce and needs more focus in terms of mobilization of resources

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance for the establishment of adequate infrastructures and equipments that will promote scientific research and favour the publication of more research studies. The review equally reveals a ratio of about one in three countries where scientific research studies have been published. This implies that the contamination status of POPs of many protected areas, coastal environments, industrial and agricultural areas, highly urbanized and remote areas in Africa is not known.

In spite of the fact that these chemicals have been banned worldwide over 20 years ago, they remain ubiquitous in the environment particularly in aquatic ecosystems acting as potential sinks. Aquatic ecosystems in Africa are subjected to the similar anthropogenic threats and pollution hotspots are commonly related to areas around high human settlements, agricultural and industrial areas. Activities mainly associated with high POP (OCPs, PCBs and PAHs) levels in Africa include, open discharge and burning of household and municipal wastes, washouts of agricultural effluents, fishing with chemicals and vector control of diseases such as malaria and typhus, petroleum exploitation and harbour activities. Although POP levels in African aquatic environments are relatively low compared to similar ecosystems worldwide, literature reveals that aquatic sediments in Africa are associated with concentration ranges within which toxic effects will occasionally and/or may frequently occur on aquatic organisms. In addition, the characteristic ratios of compounds such as DDTs

(DDT, DDE and DDD) and HCHs (α-HCH, β-HCH, γ-HCH (lindane) and δ HCH) indicate recent uses and or chronic uses. Endosulfan is one of the most abundant and frequently detected OCP in Africa, followed by lindane and DDT. This suggests that

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance some of these chemicals could still be illegally sold and used in some African countries or their presence is due high persistence and/or chronic contamination.

Highly consumed fish species such as the Tilapia (Tillapia zillii, Oreochromis niloticus), catfish (Chrysichthys nigrodigitatus, Claria sanguillaris, Clarias gariepinus,

Schilbe grenfelli, Schilbe marmoratus), carp (Cyprinus carpio), bonga shad or bonga

(Ethmalosa fimbriata), Mudskippers (Periophthalmus argentilineatus) Valenciennes,

1837), African arowana or Nile arowana (Heterotis niloticus) are the most studied and most contaminated species in Africa. Likewise, studies on edible invertebrates such as shrimps (Caridina Africana, Macrobrachium sp.), mussels (Mytilus galloprovincialis) have shown POP contamination. Thus, representing a major route of exposure to the local population that highly depend on source resources as a source of food and income. Due to the highly hydrophobic nature of some POPs, their levels in water were commonly below detection limit but in some cases above acceptable levels for individual compounds set by the World Health Organization (WHO) and European

Union for the protection of the aquatic environment and drinking water.

The pesticide survey in the Kumba market and LBW proves the absence of banned pesticides under the Stockholm Convention. This shows some level effectiveness in the implementation of regulations to protect human health and the environment. The most common pesticides sold and used in the LBW are mostly for cocoa and banana. These include, fungicides (metalaxyl+copper oxides or hydroxides), insecticides (chlorpyriphos, imidacloprid) and herbicides (glyphosate).

Even though they have not been classified as POPs, previous studies have shown their detrimental effects on humans and biodiversity. Due to lack of knowledge,

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance pesticide packages are poorly managed after use such that empty packages are dumped directly in farms and pesticide preparation is done close to the lake.

The presence of POPs (OCPs and PAHs) in an area of low human impact like the LBW shows the importance of carrying out such studies in remote areas. The non- detection of OCPs in water indicates low risk for the population which directly consumes this water. Nevertheless, sediment contamination represents a low toxicological risk to fish from this lake which is highly consumed by the local population.

It is necessary that to set a management plan for the Barombi Mbo Forest so as to limit the anthropogenic pressure in this area. With its very rich and fragile biodiversity of 15 fish species, 12 of which are endemic species of sponges and shrimp, the government should reinforce legislation to prevent the continuous use or illegal entry of banned chemicals. This study confirms that Lakes in Africa, some of which have the status

Ramsar site, are commonly contaminated with POPs such as, Congo (Lake Ma

Vallée), Egypt (Lake Quran, Lake Maryut, Lake Barullus), Ghana (Lake Bosomtwi,

Volta Lake), Togo (Lake Togo), Benin (Lake Noukoué), Ethiopia (Lake Awassa),

Burundi (Lake Tanganyika), Nigeria (Lake Chad), Uganda, Kenya and Tanzania (Lake

Victoria)

Three groups of widely distributed POPs (OCPs, PCBs and PAHs) and two groups of highly detected pesticides in Africa (chlorinated organophosphorus insecticides and chloroacetamide herbicides) were detected in sediments of the WEM hosting the largest port in Central Africa and located close to the industrial center of

Cameroon. With a wider geographical coverage, the findings of this study approved the hypothesis put forward by Fusi et al., (2016) on the spread of POPs (OCPs, PCBs

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance and PAHs) contamination throughout the WEM. This distribution could be similar for other groups of POPs such as dioxins and new POPs that have similar physical and chemical properties. The population of Douala (about 3 million) is not among first ten most populated cities in Africa, this suggests an analogous or higher contamination of

POPs in densely populated and urbanized cities, industrial centres and large harbours in Africa such as Lagos (21 million), Cairo (20.4 million), Kinshasa (13.3million),

Luanda (13.3 million), Nairobi (6.5 million), Mogadishu (6.0 million), Abidjan

(4.7 million), Alexandria, Addis Ababa, Johannesburg. The presence of pharmaceutical wastes (used drip bags) and various e-wastes suggest in the WEM suggest the presence of other groups of POPs such as PBDE’s and SCCPs. No such study has been carried out in Cameroon and very few in Africa.

In most African countries and Cameroon in particular electronic wastes (e)- wastes) are poorly managed either due to lack, insufficient or inadequate facilities for the treatment and disposal. E-waste is one of the rapidly growing global problem because it is reported that only 15 to 20 % of the global e-wastes (50 million tonnes) are properly recycled. This is comforted by the fact that in recent years, there has been a massive import of old or used and new electronic equipments in Africa. More so,

Guiyu (china) is the largest e-waste collector worldwide, followed by Accra (Ghana) and Lagos (Nigeria) in Africa which certainly lack the facilities to manage such wastes.

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Perspectives

For the continuity of this work, it would be necessary to focus on other POPs e.g. dioxines and new or emerging POPs the Wouri Estuary Mangrove. Also, both

POPs that have been detected in sediments and new POPs should be analysed in aquatic food such as; fish, mussels, crabs, in order to complement the findings of this study and those of Fusi et al., 2016. Sediment cores could be sampled at lower depths in order to analyse these POPs and determine a historic of POP contamination in the

WEM especially at hot spots.

With coastal population growth and urbanization, comparative studies could be carried out throughout Cameroon mangroves (274,918 ha) located close to densely populated cities, like the Tiko estuary (38,715 ha) occupying the central part of the

Cameroon Estuary ii) Rio Del Rey zone close to the Wouri Estuary Mangrove (WEM) close to Nigeria (169,459 ha, 127,000 inhabitants) and iii) The Kribi-Campo zone (592 ha).

The current African context needs a monitoring of protected areas such as wetlands (swamps and marshes, lakes and lagoons, mangroves and coral reefs, bogs and peatlands) which are one of the most valuable ecosystems on earth regarding

POP contamination. Also, air is one of the major pathways of POPs in the environment and route of contamination to humans, thus monitoring studies should equally focus on the determination of these pollutants in air.

Although most studies of POPs in Africa have focused on pesticides, other aforementioned POPs with similar properties and harmful effects need the same attention. Pesticides such as DDT whose use has been reintroduced by the World

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Health Organization for indoor spraying in certain regions affected by malaria should be continuously monitored in humans and various environmental matrices.

It is necessary to set environmental quality criteria, guidelines or standards for

African aquatic environments in order to have a better appraisal of pollution status and toxicity risk assessment. This is because the set guidelines will be more representative of the physico-chemical and biological environments of Africa.

Recommendations

As recommendations, sound environmental management plans should be established for both the Lake Barombi Mbo forest reserve and WEM to help protect these fragile ecosystems sustainably from growing anthropogenic pressure. This will enable the protection the unique biodiversity and preserve the touristic value of Lake

Barombi. This will favour the protection of near-threatened waterbird species and important waterbird diversity in the Wouri Estuary providing opportunities for birding tourism.

Continuous sensitization should be done to importers, sellers and users of hazardous chemicals on their appropriate use, disposal and dangers to human health and the environment.

Faced with poverty and rooted with traditional farming practices such as slash and burn farming which cause serious environmental degradation (greenhouse gases, land degradation, production of PAHs), farmers could diversify their sources of income or develop alternative farming practices like more permanent agricultural crops which will avoid constant burning of vegetation.

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African states should put in place (common) regulations, reinforce existing ones and adequate facilities and infrastructures for the management of waste especially hazardous waste.

Resources should be mobilized for more research to be carried out in Africa in order to provide more data on the occurrence of environmental pollutants and POPs in particular.

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Résumé en Français

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance

Introduction générale

La croissance remarquable dans la production et l’utilisation des produits chimiques au cours des trois dernières décennies a suscité des préoccupations au sein des organisations internationales, les gouvernements et le grand public sur leurs

éventuelles menaces sur la santé humaine et l’environnement. Des activités anthropiques telles que l’utilisation des pesticides, du pétrole et des solvants organiques ont conduit à la dissémination de ces contaminants dans l’environnement connu sous le nom de Contaminants Organiques Hydrophobes (HOCs) (Gavrilescu,

2005). La contamination environnementale due à l’utilisation ancienne ou récente des

HOCs est connue, documentée et est maintenant généralisée à l’échelle mondiale (Cui et al., 2013). En raison de leur toxicité, persistance et propriétés potentielles de bioaccumulation, certains de ces composés ont été nommés « Polluants Organique

Persistants » (POPs) par la Convention de Stockholm (adoptée en 2001). Les POPs comprennent des pesticides organochlorés (OCPs) tels que le DDT, le lindane et l’endosulfan, les polychlorobiphényles (PCBs), les hydrocarbures polyaromatiques

(PAHs), les dibenzo-p-dioxines (PCDDs), les dibenzofuranes (PCDFs) ou encore les retardateurs de flammes bromés tels que les polybromodiphényléthers (PBDEs). Les deux dernières décennies ont été marquées par une augmentation de l’intérêt pour ces molécules et des travaux en lien, aussi bien sur le plan du développement de méthodes que sur celui de la détermination de ces composés dans les différents compartiments de l’environnement.

Depuis les années 1970s, des interdictions ont été imposées sur les composés organochlorés dans les pays développés alors qu’ils faisaient encore l’objet

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Jusqu’à ce jour, malgré ces interdictions, ces composés demeurent répandus dans l’environnement. Liées aux mesures restrictives appliquées dans les pays de l’hémisphère nord, des baisses sensibles du niveau de certains POPs a été observée

(hormis pour l’Asie), là où ils ont été principalement produits et utilisés. A côté de cela une augmentation et/ou des niveaux plus importants ont été rapportés dans les pays du sud (où ils n’ont pas été produits) (Shunthirasingham et al., 2010 ; Gioia et al., 2008

; Jones and de Voogt, 1999 ; Bommanna and Kurunthachalam, 1994). En raison de leur persistance notamment, ils ont de fortes capacités de transport à grande distance.

De fait, les POPs sont capables de se déplacer des régions sources vers les zones les plus éloignées du monde où ils n’ont ni été utilisés ni produit, tels que l’océan atlantique (Gioia et al., 2008; Jaward et al., 2004), le Lac Gerio, Nigeria (Mazlan et al.,

2017).

Des études antérieures montrent à la fois la présence et les effets néfastes des POPs sur les écosystèmes terrestre et aquatiques (Bansal, 2019 ; Merhaby et al., 2019 ;

Abdel-Shafy and Mansour, 2016 ; Brits et al., 2016 ; Ribeiro et al., 2016 ; Net et al.,

2015 ; Rabodonirina et al., 2015). Les fragiles écosystèmes des environnements aquatiques (océans, rivières, lacs, mangroves, ruisseaux, estuaires) sont particulièrement sensibles aux pollutions et sont soumis depuis des décennies à une pression constante et croissante des activités humaines. La contamination de ces

écosystèmes par les POPs provient des déversements accidentels, pulvérisation, ruissellement et/ou lessivage des sols urbains et agricoles, des décharges de déchets

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance municipaux et industriels, des effluents agricoles et autres sources diffuses de pollution. La nature hydrophobe de ces contaminants leur permet de s’accumuler sur les phases riches en matière organique telles que les sédiments et les tissus graisseux des organismes vivants (bioaccumulation) (Jacob and Cherian, 2013) qui conduit à une bioamplification le long de la chaine alimentaire.

Les mangroves sont des écosystèmes aquatiques qui comptent parmi les plus productifs et présentant les plus hauts niveaux de biodiversité. Elles fournissent des services écosystémiques, économiques et socioculturels tels que les sites de reproduction des organismes aquatiques, la stabilisation, filtration, régulation du microclimat, source alimentaire, le bois et produits forestiers non ligneux. Malgré ces bienfaits, les données récentes estiment un taux de perte moyen annuelle de 0.3 % de 1996 à 2016.

(https://www.iucn.org/sites/dev/files/content/documents/mangroveloss-brief-4pp-

19.10.low_.pdf). La dégradation, perte et mauvaise gestion des mangroves est estimée à 33 000 – 57 000 $ US par hectare (Duke et al., 2014). Selon Maciel-Souza et al., (2006), les mangroves sont vulnérables aux pollutions aiguës et chroniques lorsqu’elles sont associées aux ports et industries pétrochimiques. D’une de manière analogue, les lacs sont grandement affectés par les activités anthropiques liées à la croissance démographique, au développement agricole et industriel grandissant.

Ceci est associé à l’exploitation massive des ressources halieutiques, l’assèchement de terres sur des zones marécageuses, la conservation de l’eau et le tourisme. Des

études récentes dans le monde ont ont révélées la contamination des lacs par les

POPs (Kampire et al., 2017 ; Mawussi, 2016 ; Kafilzadeh, 2015 ; Polder et al., 2014 ;

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Quiroz et al., 2010) et l’utilisation des produits chimiques notamment les pesticides organochlorés pour la pêche dans les lacs en Afrique (Kuranchie-Mensah et al.,

2012a; Wasswa et al., 2011a; Ntow, 2005). Les sédiments aquatiques agissent comme des archives naturelles et peuvent être utilisés pour reconstruire l’historique de la pollution. De même, les organismes aquatiques, particulièrement sensibles à la pollution, peuvent servir de bioindicateurs.

Le contexte Africain actuel de forte croissance démographique, les économies dépendantes de l’agriculture, le fort développement industriel et agricole, des

équipements inadéquats pour la gestion de déchets associés à l’application peu rigoureuse des règlementations sont des moteurs de menaces sur les écosystèmes aquatiques et la santé humaine. En outre, les connaissances et données sur les POPs en Afrique sont rares. Étant donné que les données existantes sont principalement axées sur les OCPs dans et autour des zones fortement urbanisées et des zones agricoles, il est important que l’attention soit accordée aux autres groupes de POPs et aux zones plus reculées. Cela suscite des préoccupations sur la qualité environnementale des écosystèmes riches et fragiles inscrits comme zones protégées, comme c’est le cas pour beaucoup, d’écosystèmes aquatiques d’Afrique.

Ceci amène plusieurs questions fondamentales : i) Quelle est le niveau d’anthropisation des milieux subissant de fortes pressions anthropiques et réciproquement les zones reculées liée aux contaminations en

Afrique. ii) Quelles sont les teneurs environnementales des POPs dans les environnements aquatiques d’Afrique et les effets toxiques qui pourrait être associé à ces teneurs.

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CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance iii) Quelle présence de POPs en Afrique correspond aux utilisations historiques ou récentes et contaminations aiguës ou chroniques. iv) Quelle est l’état de contamination des écosystèmes en Afrique par rapport aux

écosystèmes semblable dans le monde.

C’est dans ce cadre que s’inscrit cette recherche doctorale, au travers de l’évaluation des niveaux de OCPs, PCBs et PAHs dans les sédiments de deux environnements aquatiques du Cameroun soumis à des pressions anthropiques très différentes. L’un des sites, le bassin versant du Lac Barombi (LBW) est faiblement anthropisé et alors que l’autre site est la mangrove de l’estuaire du Wouri (MEW) — Douala qui subit une pression humain considérable.

Les justifications pour le choix de ces sites sont les suivantes : i) Le bassin versant du Lac Barombi (LBW) est une zone reculée et quasi-vierge de

415 hectares avec une population d’environ 400 personnes, lesquelles dépendent entièrement des ressources du Lac et de la forêt environnante. Depuis 1940, ce bassin versant a été désigné comme réserve forestière et, en2006, le lac Barombi a été classé deuxième site Ramsar3 du Cameroun. Ce dernier sert enfin de principale source d’approvisionnement en eaux potable de la ville voisine de Kumba et ses environs.

Malheureusement jusqu’à ce jour, cette réserve forestière n’a pas de plan de gestion

(Balgah and Kimengsi, 2011). L’intérêt d’étudier de ce site préservé était également

3 Un site Ramsar est la désignation d’une « zone humide d’importance internationale » inscrite sur la liste établie par la Convention de Ramsar par un État partie. Ce traité international a été adopté le 2 février 1971 pour la conservation et l’utilisation durable des zones humides, qui vise à enrayer leur dégradation ou disparition, aujourd’hui et demain, en reconnaissant leurs fonctions écologiques ainsi que leur valeur économique, culturelle, scientifique et récréative sous la désignation de site Ramsar 266

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance de pouvoir effectuer c une étude de référence pour évaluer le niveau d’anthropisation liée aux OCPs dans les eaux, sols, sédiments et PAHs dans les sols et sédiments. ii) La mangrove de l’estuaire du Wouri (WEM) est à proximité de la ville de Douala, la plus peuplée du Cameroun (environ 3 millions d’habitants) et premier centre industriel de la Communauté Économique et Monétaire de l’Afrique Centrale (CEMAC).

L’estuaire du Wouri héberge le plus grand port d’Afrique centrale, le Port Autonome de Douala (PAD). L’objectif de ce site était d’évaluer le niveau de OCPs, PCBs et

PAHs dans les sédiments de WEM sur une vaste étendue. En outre, les pesticides organophosphorés chlorés (Chlorpyrifos et Chlorfenvinphos) et herbicide de la familles chloroacétamide (alachlore et metolahlore) ont été étudiés a raison des teneurs environnementales élevés, forte détection et utilisation intensive dans les régions tropicales (Akan et al., 2014; Ntiendjui et al., 2009; Laabs et al., 2002).

CHAPITRE 1 : SYNTHÈSE BIBLIOGRAPHIQUE

I.1 Convention de Stockholm sur les Polluants Organiques Persistants

La convention de Stockholm définit un polluant organique persistant comme une substance chimique qui persiste dans l’environnement, se bioaccumule le long de la chaine alimentaire, se transporte à grande distance et pose des risques d’effets néfastes sur la santé humaine et l’environnement (Lambert et al., 2011). Initialement,

12 composés ont été désignés POPs dans trois catégories ; les pesticides, les

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POPs ont été ajoutés en 2017 et 3 nouvelles molécules sont en cours d’examen.

1.2 Cadre Institutionnel et législatif des POPs en Afrique

Au fil des ans, les règlements sur les produits chimiques dangereux se sont élargis en termes de nombre de molécules et pays concernés. Les règlementations en Afrique sont pour la plupart des pays existants pour les pesticides mais très limitées pour les

PCBs et quasi absentes pour les PAHs. Dans tous les pays africains signataires de la

Convention de Rotterdam et de la Convention de Stockholm, un plan national de mise en œuvre et un plan d’action national ont été respectivement élaborés. Aux niveaux nationaux, des institutions, décrets et arrêtés ont été mis en place pour réguler les importations, l’utilisation et l’élimination des produits chimiques dangereux.

Le Cameroun a ratifié de nombreux accords et traités internationaux et régionaux sur la gestion des produits chimiques dangereux. Il s’agit notamment de la Convention de

Rotterdam (1998) et de la Convention de Stockholm (2001) respectivement entrées en vigueur en 2004 et 2009. Le Cameroun a adopté en 2016, l’approche stratégique de la gestion internationale des produits chimiques (ASGIPC). Comme dans la plupart des pays africains, au Cameroun il n’existe toujours pas de règlementation sur les

POPs dont la production est involontaire tels que les PAHs, les dioxines

(polychlorodibenzodioxines ou PCDDs) et furanes (polychlorodibenzofuranes ou

PCDFs). En raison de la crise économique des années 90, l’état du Cameroun s’est

4 The dirty dozen : aldrine, chlordane, DDT, dieldrine, dioxines, endrine, furanes, heptachlor, Hexachlorobenzène, mirex, PCBs, et toxaphene 268

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance désengagé de l’acquisition et de la distribution des intrants agricoles (dont des pesticides) pour se consacrer à ses missions régaliennes à savoir ; la formation, l’encadrement des producteurs et le contrôle de la qualité des intrants importés et distribués par le secteur privé.

1.3 Importation et Utilisation des Polluants Organiques Persistants en Afrique

Ces trente dernières années, l’exportation des matières premières agro-industrielles a permis d’entretenir la croissance économique de bon nombre de pays africains.

Depuis le milieu des années 50, date à laquelle a débuté leur commercialisation à grande échelle, l’utilisation des pesticides synthétiques a augmenté au fil des années.

En Afrique, les POPS les mieux documentés sont les pesticides. Il est beaucoup plus difficile de trouver des données disponibles sur les autres POPs.

Les mouvements transfrontaliers illégaux de déchets électroniques, produits chimiques interdits ou pesticides dangereux non homologués des pays du nord vers les pays du sud ont été l’une des contributions majeures à l’introduction des POPs en

Afrique (Gioia et al., 2014). L’importation massive de pesticides pour les activités agricoles et des questions de santé publique contribuent aussi à la présence de ces produits dangereux dans l’environnement. Selon la base statistique du Programme

Alimentaire Mondial (PAM), l’Afrique a importé en 2016 une valeur en pesticides d’environ 1 590 160 USD (FAOSTAT, 2018). Les dix premiers pays africains en termes de valeurs d’importation de pesticides (ordre décroissant) sont ; le Nigeria, l’Afrique du

Sud, le Ghana, la Côte d’ivoire, l’Égypt., le Kenya, le Cameroun, la Tanzanie, l’Éthiopie et la Guinée (Guy Bertrand, 2019). 269

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Au Cameroun l’agriculture contribue à environs 35 % du produit intérieur brut (PIB) et représente jusqu’à 70 % de la main-d’œuvre nationale (Kimengsi and Muluh, 2013).

La menace grandissante des ravageurs et maladies conduit à une diminution de la productivité agricole, obligeant à l’utilisation des produits phytosanitaires tels que les pesticides. En 2009, le Cameroun a importé 2898,4 tonnes (57 %) de pesticides venant d’Europe, 196,5 tonnes (3,9 %) de l’Afrique hors de la zone CEMAC et 1986,5 tonnes en provenance du reste du monde (MINADER/DESA/AGRI-STAT, 2012). Dans le cadre du Programme africain relatif à l’élimination des stocks de POPs et autres pesticides obsolètes (2005), des stocks de pesticides interdits par la Convention de

Stockholm ont été retrouvés dans 7 des 10 régions du Cameroun (MINEPDED-

UNITAR, 2013). Le plus grand stock était du Lindane < Endosulfan < Dieldrin et le

DDT. Les stocks les important ont été retrouvés dans les plus grandes régions agricoles notamment la région du Grand-Nord, le Nord-ouest et l’Ouest représentant respectivement 23,1, 20,8 et 17,7 %. De même, en 2009, une enquête préliminaire dans le cadre du Plan National de Mise en Œuvre (PNM) de la Convention de

Stockholm a révélé que 16 entreprises détenaient des équipements (transformateurs, condensateurs, fûts de liquide, séchoir électrique, disjoncteurs, coupe-circuits, EPI et matériels de laboratoire) étaient souillés de PCBs. Ces équipements étaient présents dans toutes les régions principalement dans la région du Littoral (37 %), Centre (22 %) et Sud-ouest (16 %) (UNEP-POPs-NIP-CAMEROON, 2016). Du fait de l’absence de toute règlementations particulières sur les déchets électroniques et industriels, les sites de dépôts se sont multipliés et sont fortement soupçonnés d’être contaminés par les PCBs et autres POPs. On peut citer notamment la fosse ouverte de « Ngousso »

(Yaoundé), les sites de Nkolfoulou (Yaoundé) et Makepe (Douala) (GISWatch, 2010). 270

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1.4 Impact écotoxicologique des contaminants (OCPs, PCBs et PAHs) des sédiments

Au cours des 4 dernières décennies, de nombreuses approches ont été mises en place pour évaluer le risque environnemental lié à la contamination des sédiments aquatiques. Avant les années 80, on évaluait le risque en comparant la concentration absolue du polluant dans un échantillon de sédiment à des valeurs dites de « fond » ou de « référence ». Au-delà du fait qu’il est parfois compliqué de trouver une valeur de fond ou de référence, il est évident que cette approche ne fournissait qu’un aperçu très limité de l’impact des contaminants sédimentaires sur les écosystèmes. Les recommandations pour la qualité des sédiments (SQGs) ont ensuite essayé d’intégrer la réponse/effet biologique aux simples données de concentration (Burton, 2002). De nombreuses méthodes empiriques et théoriques ont été déployées pour déterminer ces SQGs. Les approches empiriques permettent d’établir le lien entre la chimie des sédiments et les données relatives aux effets biologiques (prédiction des effets se basant sur des approches statistiques) en déterminants des seuils de concentration critiques. Les méthodes théoriques reposent plutôt sur de la modélisation, en prenant en compte les différences de biodisponibilité via le partage à l’équilibre (EqP) en suggérant que l’eau interstitielle du sédiment représente la voie principale d’exposition des organismes aquatiques Di Toro n1991.

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1.5 Effets des pesticides organochlorés, polychlorobiphényles et hydrocarbures polyaromatiques sur la santé humaine

Comme pour les autres organismes vivants, la principale voie d’exposition des êtres humains est l’ingestion. Les conséquences sanitaires liées à ces expositions dépendent du composé, du niveau d’exposition (dose), de la durée de l’exposition et de l’individu. L’une des principales limites des études humaines liées aux POPs, c’est la disponibilité vraiment limitée des données sur les antécédents d’expositions. Les différents effets décrits dans la littérature sont : i) cancers (Abdel-Shafy and Mansour,

2016) (Kim et al., 2013), (Multigner, 2010) (ATSDR, 2002) ii) perturbation thyroïdienne

(Chevrier et al., 2008, Kimbrough, 1995) et perturbation endocrinienne Oskam et al.,.,

2003) (Settimi, 2003), iii) effets neurotoxiques (Abdel-Shafy and Mansour, 2016),

Korrick et al., 2008), Roberts et al., (2007) troubles reproductifs (Bhatia et al., 2005),

(Longnecker et al., 2001). Pocar et al., 2003)

1.6 Review sur les teneurs environnementales des POPs cibles sur les environnements aquatiques d’Afrique. (Article de revue)

L’étude de 62 articles relatifs à l’Afrique de 1998 à 2019 sur les OCPs, PCBs, et PAHs démontre la rareté des données sur Afrique. Les teneurs en POPs sont de façon générale plus élevées dans les sédiments et biote (poissons) que dans les eaux. Ceci est explicable par la nature hydrophobe de ces composés qui entraine la phase solide comme matrice de prédominance. Sans surprise, c’est dans les zones présentant les plus fortes pressions anthropiques (forte urbanisation, activités industrielles et agricoles) que l’on a mesuré les teneurs les plus importantes. Pour les DDTs et HCHs 272

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance on constate à la fois des profils d’utilisation récente et des contaminations historiques.

Les profils de PCBs révèlent dans la plupart des cas une utilisation historique de mélanges techniques fortement chlorés du type Aroclor 1254 et Aroclor 1260 qui ont d’ailleurs été les plus utilisés. Les indices basés sur des rapports d’abondance de

PAHs révèlent des sources mixtes de PAHs pyrolytiques et pétrogéniques. Parmi les espèces de poissons étudiées, le Tilapia zilli et les poissons-chats (parmi les plus consommés par les populations locales) faisaient partie des espèces les plus contaminées. Au final les concentrations mesurées sont généralement plutôt faibles mais comparables aux écosystèmes équivalents dans le monde.

CHAPITRE 2 : SITES D’ÉTUDE

2.2.1 Le Bassin versant du Barombi Mbo

Le Bassin versant du Lac Barombi situé au Sud-ouest du Cameroun héberge un Lac de cratère situé sur la Ligne Volcanique du Cameroun de l’appelé Barombi Mbo. Il est le plus grand Lac volcanique d’Afrique de l’Ouest et d’Afrique Centrale mesurant

2,5 km de diamètre et 111 m de profondeur (Asaah et al., 2020; Tabot et al., 2016). Il a été désigné comme site Ramsar par l’UNESCO en 2006, à cause de la présence de

12 espèces endémique de poisson sur 15 espèces recensées dans le Lac tels que

Sarotherodon linnellii (Unga sp.), Pungu maclareni et divers Cichlidés. De même, il héberge une espèce endémique de spongilles (Corvospongilla thysi) et de crevette

(Caridina sp). Le Lac Barombi se trouve dans la réserve forestière du Barombi créée en 1940. Le village Barombi (350 habitants) est situé à environ 100 m du Lac et dépend entièrement de ses ressources. Les activités anthropiques majeures sont l’agriculture 273

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(agriculture sur brûlis, pulvérisation des pesticides sur le cacao, banane et palmier à huile), la pêche à l’aide des filets et même parfois en utilisant des produits chimiques

(Balgah and Kimengsi, 2011).

La ville voisine de Kumba a une croissance démographique très rapide, ce qui entraine une pression grandissante pour les ressources du lac, les terres agricoles, le bois et autres produits forestiers.

2.2.2 Mangrove Estuaire du Wouri

Le fleuve du Wouri est l’une des principales rivières côtières du Cameroun avec un débit annuel de 311 m3/s. Il sépare la ville de Douala en deux parties et se jette dans l’Océan atlantique. La mangrove de l’Estuaire du Wouri (MEW) située dans le Golfe de Guinée à proximité immédiate de la ville de Douala, la plus peuplée du Cameroun, héberge plus de 90 % des industries camerounaises. Les deux zones industrielles principales sont la zone de Bassa et la zone de Bonaberi. Le Port Autonome de Douala

(PAD) assure près de 95 % du trafic portuaire national camerounais. La MEW occupe une superficie d’environ 2300 km2 de l’estuaire du fleuve Wouri et est directement au contact du PAD. L’estuaire de Douala est par ailleurs un site d’une grande biodiversité hébergeant un grand nombre d’oiseaux aquatiques sédentaires et migrateurs. Il existe

également de très nombreuses espèces de poissons, crustacés (crevettes, crabes) et mollusques qui sont fortement consommés par la population de Douala et des autres villes du Cameroun. Un rapport sur le recensement des oiseaux aquatiques montre que la zone de l’estuaire du Wouri remplit pleinement les critères RAMSAR et préconise que ce site soit inscrit comme site RAMSAR (Delany and Scott, 2006). Les 274

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance activités anthropiques majeures sont des activités industrielles et portuaires intenses, la pêche, l’extraction du sable, l’abattage des mangroves (bois de chauffe et fumage du poisson, a visée résidentielle) et trafic routier particulièrement dense. Les industries principales sont agroalimentaires, métallurgiques, cimenterie, plasturgie et chimiques

L’agriculture périurbaine est aussi pratiquée, ce qui favorise la résistance des moustiques aux insecticides (Papito 2017 ; Antonio-Nkondjio 2011, 2015). Il existe des décharges de déchets municipaux et de boues de vidanges sur la limite Sud de Douala dans la zone appelée « Crique Docteur » principalement dans les quartiers « Bois de singes », « Youpwe » et « Koweit city » impactés par une urbanisation grandissante. Le fleuve du Wouri est l’une des principales rivières côtières du Cameroun avec un débit annuel de 311 m3/s. Il sépare la ville de Douala en deux parties et se jette dans l’Océan

Atlantique.

CHAPITRE 3 :

3.1 Enquête sur la commercialisation et l’utilisation des pesticides à Kumba et le bassin versant du Barombi Mbo

Cette enquête a été effectuée sur deux sites, le marché de Kumba et le bassin versant du Barombi Mbo. Elle a été conçue sous forme d’entretiens semi-directifs, questionnaires et observations de terrains. Les agriculteurs ont été questionnés sur la fréquence et les méthodes d’applications des pesticides. Le président de l’association

« Agrochemical Union » et un membre de l’association ont été questionnés sur l’acquisition, la commercialisation et distribution des pesticides. Cette association regroupe tous les vendeurs de produits phytosanitaires de la ville de Kumba. De plus, 275

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance une enquête terrain a été effectuée dans le marché de la ville afin d’identifier et enregistrer les pesticides vendus et utilisés tout autour de la ville de Kumba.

3.1 Échantillonnage

3.1.1 Bassin versant du Lac Barombi

L’échantillonnage a été effectué en mars 2016 (fin de la saison sèche). Des

échantillons d’eau du Lac ont été prélevés dans des bouteilles ambrées de 2,5 L (4 stations) et au niveau de ruisseaux (affluents du Lac) (3 stations). Les sols ont été prélevés dans les champs (9 stations) et à proximité des ruisseaux (2 stations) à l’aide d’une spatule en inox. Les sédiments ont été prélevés dans de façon identique pour les ruisseaux (4 stations) et le Lac (2 stations) et également par carottage (interface corer Uwitech system). Des tranches de carottes (0 -10 cm) ont ensuite été prélevées et transportées au LAGE à Yaoundé pour les congeler.

3.1.2 Mangrove estuaire du Wouri

L’échantillonnage dans la mangrove a été effectué en décembre 2017 (saison sèche).

Les sédiments ont été prélevés par carottage manuel dans des barquettes en aluminium de 0,5 L et conservé à - 18 °C au laboratoire de la « Jeune Équipe Associée

à l’IRD » (JEAI), Université de Douala. Les échantillons congelés ont été transportés de Douala à Aix-en-Provence au Laboratoire Chimie de l’Environnement (LCE) pour y

être traités.

3.2 Traitement et extraction des échantillons

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3.2.1 Eaux

Les échantillons d’eaux du Barombi Mbo ont été filtrés à travers des filtres en microfibres de verre (GFF, 0,7 µm, diamètre 42.5 mm) et extrait par extraction en

phase solide (SPE) sur des cartouches C18 préconditionnées. Après percolation, l’adsorbant (phase stationnaire) a été séché sous vide en 30 minutes, correctement scellé avec du parafilm, papier aluminium et transporté au LCE dans des glacières.

L’élution a été effectuée avec 20 mL d’Hexane et concentré à 1 mL pour analyse.

3.2.1 Sols et sédiments

L’extraction accélérée par solvant (ASE) couplée à une purification directe « en cellule » (in-cell cleanup) a été employée pour les échantillons solides (sols et sédiments) des deux sites. Cette extraction/purification a été réalisée avec de l’alumine activée (5 g) et du cuivre activé à l’acide (5 g) mélangés avec l’échantillon (10 g) dans la cellule. Les échantillons du LBMW ont été dopés avec des étalons marqués de

4,4’ DDE-D8 at 2000 pg/μL pour les OCPs et ceux du WEM avec du 4,4 — DDE D8 et du PCB 156 D3 à 100 pg/μL pour les pesticides chlorés (PCLs) et PCBs. Les conditions d’extraction des composés ciblés sont adaptées de travaux antérieurs du laboratoire (Kanzari et al., 2015). Pour l’analyse des PCLs et PCBs, l’extrait a été concentré et repris dans 1 mL de dichlorométhane. Pour l’analyse des PAHs l’extrait a été concentré, repris dans 1 mL d’acétonitrile et passé sur un filtre à membrane de

PTFE. Les cartouches C18 ont permis le fractionnement des PCLs et PCBs en deux fractions (F1 et F2) après élution avec 2,5 mL d’Hexane et 5 mL d’Hexane/acétone respectivement selon la méthode US-EPA 3620 C. 277

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3.3 Analyses instrumentales

L’analyse des PAHs a été réalisée par chromatographie liquide à haute performance avec détection par fluorescence programmée (HPLC-PFD) à l’aide d’un PerkinElmer

Flexar LC. La séparation a été réalisée avec une colonne Agilent Technologies

Pursuit 5 PAH (250 mm x 4,6 mm i. d. x 5 μm). L’analyse des PCLs et PCBs a été effectuée par chromatographie en phase gazeuse/spectrométrie de masse en mode

SIM (Single Ion Monitoring) à l’aide d’un PerkinElmer Clarus GC Clarus 600 et MS

Clarus 600 C équipés d’une colonne capillaire Restek Rxi — XLB ; (30 m x 0,25 mm i. d. x 0,25 μm). Avant l’injection 40 pg/μL de 4,4’ DDT D8 et PCB 116 D5 ont été ajoutés respectivement pour l’analyse des PCLs et PCBs comme étalon interne.

L’identification et quantification ont été effectuées selon (Kanzari et al., 2012).

3.4 Contrôle et assurance de la qualité

Les méthodes analytiques ont été validées à l’aide de matériaux de références certifiés

(CRM) notamment le CRM CNS391 (50 g, PAHs, PCBs and Pesticides on freshwater sediment) et CRM860 (Pesticides-loamy sand soil). Pour l’étude du LBW, la limite de détection (LDD) varie entre 0.04 et 0,71 ng/g (signal/bruit = 3) et les limites de quantification (LOQ) déterminées pour des rapports signal/bruit = 10. Le taux de récupération moyen d’étalon marqué 4,4’— DDE D8 dans les échantillons de sols et sédiments était respectivement de 103 ± 2 % et 85 ± 7 %. Pour l’étude de WEM, la

LOD varie entre 0,04 – 0,71 ng/g (CLPs), 0,02 – 0,13 ng/g (PCBs) et 0,002 – 0,04 ng/g.

(PAHs). Le taux de récupération moyen d’étalon marqué 4,4’— DDE D8 et 278

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PCB 156 D3 dans les échantillons sédiments était respectivement de 85 ± 7 % et 91

± 5 %.

CHAPITRE 4 État sanitaire des Lacs volcanique du Cameroun : Évaluation des

Pesticides et Hydrocarbures Polyaromatiques dans les eaux, sols et sédiments.

Le cas du Barombi Mbo

Cette étude révèle que les pesticides interdits ; classifiés comme POPs et non homologués ne sont pas vendu dans le marché de Kumba. Les pesticides les plus courants sont les fongicides (metalaxyle et oxydes ou hydroxydes de cuivre), insecticides (chlorpyrifos, imidaclopride) et herbicides (glyphosate). Les fongicides sont appliqués pendant la saison sèche sur le cacao, les insecticides sur les fruits et légumes, les herbicides sur plusieurs cultures notamment le palmier à huile et les légumes. La durée moyenne de stockage des produits phytosanitaires dans les boutiques est de deux ans. Après cette période, ils sont retournés aux fournisseurs pour de nouveaux produits. Les pesticides sont obtenus des mêmes fournisseurs agrées et vendus avec des Equipements de Protection Individuelle (EPI) que beaucoup d’agriculteurs n’achètent ni n’utilisent du fait de leurs connaissances limitées sur les effets néfastes de ces produits et/ou de leur manque de moyens.

L’abondance des OCPs (ordre décroissant) est ; endosulfan (β-endosulfan + endosulfan sulfate)> HCHs (Σ α-HCH + β-HCH +δ -HCH + γ-HCH) > dieldrine> aldrine. Les plus fréquemment détectés sont les HCHs avec des concentrations variant de < LDD — 5,1 ng/g, 0,9 - 2,2 ng/g et 1,4 - 2,0 ng/g respectivement pour les sols, 279

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance sédiments de ruisseaux (affluents) et carottes. Le ratio α/γ-HCH (0,3 - 1,3) indique une utilisation récente du lindane (γ-HCH). Les teneurs en PAHs varient de 106 - 112 ng/g,

72 - 96 ng/g, et 144 - 151 ng/g dans les sols, sédiments de ruisseaux (affluents) et carottes. Les PAHs à poids moléculaire plus élevés (HPAHs) (4 – 6 cycles) étaient plus abondants que les PAHs à faibles poids moléculaires (LPAHs) (2 – 3 cycles) représentant respectivement 74 % et 26 %. Des rapports et indices sélectionnés de

PAHs ont montré une prédominance des PAHs de sources pyrolytiques plus précisément la combustion d’herbes, du bois ou le charbon. La présence des OCPs et

PAHs dans ce bassin versant est principalement liée aux activités agricoles : pulvérisation des cultures et pêche avec des produits chimiques (notamment le lindane) pour les OCPs et agriculture sur brûlis pour les PAHs. Les SQGs pour les sédiments démontrent un faible risque de toxicité pour les organismes aquatiques. Au final, le LBW présente des faibles teneurs en OCPs et PAHs par rapport aux autres

Lacs dans le monde.

CHAP 5 : Polluants organiques persistants dans les sédiments de la Mangrove

Estuaire du Wouri, Cameroun : teneurs, profils et implication écotoxicologique.

Les teneurs en pesticides chlorés (PCLs), PCBs et PAHs varient respectivement de .2

– 27.4, 1.7 – 31.6 et 83 – 544 ng/g. Les PCLs les plus abondants étaient l’endosulfan, l’alachlor, l’heptachlor, le lindane (γ-HCH) et le DDT avec des profils de métabolites qui révèlent une utilisation récente. Les profils des PCBs étaient dominés par des 280

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance

PCBs à fort poids moléculaire, Hexachlorobiphényles (PCB 138 and PCB 153) et pentachlorobiphényles (PCB 101 and PCB 118) représentant respectivement 47 % et

30 % des PCBs totaux. Ceci indique une contamination récente et/ou chronique et une persistance des PCBs fortement chlorés dans les sédiments. Des ratios d’PAHs sélectionnés montrent une contribution prédominante des PAHs de sources pyrolytiques correspondant plus spécifiquement à la combustion de biomasse (herbes, bois ou charbon) et du pétrole. La somme des PAHs cancérigènes (ΣC-PAHs) représentait 30 à 50 % des PAHs totaux (TPAHs). Les SQGs montrent que les teneurs en POPs relevées sont associées à des toxicités prédites faibles à modérées.

Conclusion générale, perspectives et recommandations

Cette étude démontre que la protection de l’homme et l’environnement face à la contamination des POPs est un sujet d’actualité et d’une importance capitale, particulièrement pour les pays en voie de développement qui semblent plus vulnérables en raison du manque d’infrastructures adéquates et adaptées pour gérer ces produits chimiques. Malgré, l’arsenal règlementaire mis en place par plusieurs pays africains, la mise en application effective de ces textes demeure un sérieux problème. Au-delà de la nécessité absolue de mobiliser des ressources pour la mise en place des infrastructures et équipements adéquats à une gestion raisonnée et durable, l’étude bibliographique que nous avons éffectuée démontre une rareté de données en Afrique liée aux POPs, et enfin le besoin de promouvoir la recherche scientifique et de favoriser la publication des articles de recherches. Les teneurs en

POPs dans les environnements aquatiques en Afrique sont pour l’instant

281

CHAPTER 5 : Persistent Organic Pollutants in Sediments of the Wouri Estuary Mangrove, Cameroon: Levels, Patterns and Ecotoxicological Significance généralement faibles par rapport aux autres environnements aquatiques dans le monde. Ces gammes de concentrations sont généralement associées aux effets toxiques qui se produisent occasionnellement et/ou fréquemment. Les espèces aquatiques les plus consommées et de grande importance économique sont les plus contaminées en POPs notamment le tilapia (Tillapia zillii, Oreochromis niloticus), poisson-chat (Chrysichthys nigrodigitatus, Claria sanguillaris, Clarias gariepinus,

Schilbe grenfelli, Schilbe marmoratus), carpe (Cyprinus carpio) et bonga (Ethmalosa fimbriata), crevette (Caridina Africana, Macrobrachium sp.) et moules (Mytilus galloprovincialis).

L’absence constatée des pesticides interdits dans le marché de Kumba démontre l’efficacité des efforts de l’état du Cameroun à appliquer les règlementations.

Néanmoins, la présence des POPs aussi bien dans le WEM qu’au niveau du LBW pourrait indiquer une utilisation chronique et/ou récente liée à la présence des anciens stocks ou entrée frauduleuse de ces produits.

Pour la continuité de ce travail, il est nécessaire de s’intéresser à d’autres POPs tels que dioxines et les POPs émergents pour lesquels il n’existe pas encore de règlementations spécifiques au Cameroun. En raison de la rareté de données, des

études similaires pourraient être menées dans d’autres écosystèmes aquatiques en

Afrique. Le contexte africain actuel et à venir exige une surveillance accrue de ces

écosystèmes fragiles et précieux face à une pression anthropique qui ne peut que croitre

282

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Appendices

APPENDICES

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Appendices

Appendix 1. Summary data of POPs in aquatic environments of Africa

Rivers (number of studies) Mean Median (mean concentrations of POPs) Sediments (ng/g) dw DDTs (10) 70.9 40.1 135.9 2.0 41.4 35.7 0.2 0.2 9.5 0.5 33,6 22,6

HCHs (8) 9.2 350.9 11.5 0.2 0.6 16.0 3.2 ND ND ND 48,9 6,2

OCPs (6) 106.4 84.2 21.0 6515.0 1084.0 104.8 ND ND ND ND 1319,2 105,6

7 PCBs (6) 13.8 0.2 341.8 90.2 7.2 0.7 ND ND ND ND 75,6 10,5

16 PAHs (5) 2801.1 259.0 289.3 72.5 49.2 ND ND ND ND ND 694,2 259,0

Fish (ng/g) Lw DDTs (4) 2823.7 792.7 19.6 252.1 ND ND ND ND ND ND 972,0 522,4

HCHs (3) 211.2 68.4 33.1 ND ND ND ND ND ND ND 104,2 68,4

OCPs (2) 720.0 314.5 ND ND ND ND ND ND ND ND 517,3 517,3

7 PCBs (1) 1832.1 ND ND ND ND ND ND ND ND ND ND ND

Water (μg/L) DDTs (6) 42.3 286.2 2.3 0.02 0.02 0.1 ND ND ND ND 55,1 1,2

HCHs (5) 97.4 31.5 8.9 0.1 0.01 ND ND ND ND ND 27,6 8,9

OCPs (4) 133.0 968.0 1.6 24.7 ND ND ND ND ND ND 281,8 78,8

7 PCBs (2) 280.0 2.5 ND ND ND ND ND ND ND ND 141,3 141,3

16 PAHs (2) 5140.0 14.1 ND ND ND ND ND ND ND ND 2577,0 2577,0

311

Coastlines (number of studies) Mean Median (mean concentrations of POPs) Sediments (ng/g) dw DDTs (10) 7.5 10.2 5.9 8.1 4.4 24.0 11.5 40.8 5.1 442.6 56,0 9,2

HCHs (9) 110.5 2.2 1.0 2.8 4.0 4.9 1.1 9.2 1.3 ND 15,2 2,8

OCPs (6) 1027.5 12.4 9.7 61.0 20.4 8.6 ND ND ND ND 189,9 16,4

7 PCBs (9) 5.4 58.9 7.7 9.7 3.0 197.5 188.6 0.6 605.5 ND 119,6 9,7

16 PAHs (6) 9861.0 11708.5 20685.0 1887.9 11306.8 228.8 ND ND ND ND 9279,7 10583,9

Fish (ng/g) Lw DDTs (4) 381.5 136.4 558.5 563.0 ND ND ND ND ND ND 409,9 470,0

HCHs (5) 16.0 686.8 1294.0 30.5 11.0 ND ND ND ND ND 407,7 30,5

OCPs (2) 530.5 1044.6 ND ND ND ND ND ND ND ND 787,6 787,6

7 PCBs (3) 2562.0 0.4 350.5 ND ND ND ND ND ND ND 971,0 350,5

16 PAHs (1) 124.1 ND ND ND ND ND ND ND ND ND ND ND

Lakes (number of studies) Mean Median (mean concentrations of POPs) Sediments (ng/g) dw DDTs (7) 4.4 0.7 3.0 4.2 4.4 351.4 61.3 ND ND ND 61,3 4,4

HCHs (8) 5.2 2.1 0.7 31.4 2.8 6.8 57.2 2.3 ND ND 13,5 4,0

OCPs (3) 31.0 82.9 475.6 ND ND ND ND ND ND ND 196,5 82,9

7 PCBs (5) 2.4 11.7 0.6 69.4 109.3 ND ND ND ND ND 38,6 11,7

16 PAHs (2) 125.3 5078.0 ND ND ND ND ND ND ND ND 2601,7 2601,7

312

Fish (ng/g) Lw DDTs (5) 11.6 1117.1 3.6 24.0 488.7 ND ND ND ND ND 329,0 24,0

HCHs (5) 0.0 0.7 0.1 1.0 145.2 ND ND ND ND ND 29,4 0,7

Lakes (number of studies) Mean Median (mean concentrations of POPs) OCPs (3) 4.4 11.6 40.4 ND ND ND ND ND ND ND 18,8 11,6

7 PCBs (3) 0.4 24.1 65.4 ND ND ND ND ND ND ND 29,9 24,1

μ Water ( g/L) DDTs (3) 3.2 0.01 441.4 ND ND ND ND ND ND ND 148,2 3,2 HCHs (4) 0.5 0.1 0.01 0.2 ND ND ND ND ND ND 0,2 0,2

OCPs (1) 0.8 ND ND ND ND ND ND ND ND ND 0,8 ND

7 PCBs (2) 1.0 3.5 ND ND ND ND ND ND ND ND 2,2 2,2

16 PAHs (1) 110.5 ND ND ND ND ND ND ND ND ND ND ND

Dams. Reservoirs (number of studies) Mean Median

(mean concentrations of POPs) Sediments (ng/g) dw DDTs (2) 5.2 0.5 ND ND ND ND ND ND ND ND 2,9 2,9 HCHs (2) 8.7 0.02 ND ND ND ND ND ND ND ND 4,3 4,3 Fish (ng/g) Lw DDTs (1) 4.6 ND ND ND ND ND ND ND ND ND ND ND HCHs (1) 1.1 ND ND ND ND ND ND ND ND ND ND ND 7 PCBs (1) 297.1 ND ND ND ND ND ND ND ND ND ND ND

16 PAHs (1) 1166.5 ND ND ND ND ND ND ND ND ND ND ND

Water (μg/L) DDTs (2) 0.2 0.1 ND ND ND ND ND ND ND ND 0,1 0,1 HCHs (1) 1.0 ND ND ND ND ND ND ND ND ND ND ND

313

Dw =dry weight, Lw = lipid weight, ND = No data

314

Appendices

Appendix 2. Transport of food crops from farms through Lake Barombi Mbo

Appendix 3. Slash and burn farming in the Lake Barombi Mbo Watershed

311

312

Appendices

Appendix 4. Fishing with gill nets in Lake Barombi Mbo

Appendix 5. Dumping of pesticide packages on farmlands after use

311

Appendix 6. Mixed cropping of banana and cocoa in the Lake Barombi Mbo Watershed.

Appendix 7. Palm oil plantations in the Lake Barombi Mbo watershed.

312

Appendix 8. Nkam and Makombe rivers (main tributaries of the Wouri river)

313

Source: United Councils and Cities of Cameroon (UCCC, 2014)

Appendix 9. Mgoua river “Black River” at Crique Docteur.

314

Source: (MINHDU, 2014)

Appendix 10. Discharge of gaseous waste and dust from a cement factory in Douala (Bonaberi I.Z)

Source: (MINHDU, 2014)

315

Appendices

Appendix 11. Summary of methodological approaches employed to determine OCPs, PCBs and PAHs in African aquatic environments

Sample, depth LOD Analyte Country, site, Sample size, extraction Clean-up/Fractionation (cm) Sampling method, Pre- Recovery Analytical technique ng/g or g/L Compound(s) treatment method μ References (Sampling (dw or lw) (%) date)

Sediment Interface corer, sampled OCPs at 2 cm interval, PE core GC-MS 103 ± 2s bags, oven dried 45 °C, Cameroon 0 – 10 lyophilisation (- 41 °C, 88 – 141 c Restek Rxi – XLB column (30 0.310 mbar) Lake Barombi (2016) m x 0.25 mm x 0.25 μm)

Mbo In-cell clean up, 10 g sediment/soil, 5 g of Soil and activated alumina, 5 g activated copper mixed in Stainless steel spoon, 0.002 – 0.59 This study 10 g, ASE, 23 mL Hex the extraction cell, (filtration of ASE extract through aluminum boxes, PAHs Stream /Dcm (1/1) 4 mm PTFE syringe for PAHs analysis) removal of litter and 85 ± 7s 16 PAHs sediment HPLC-PFD coarse material, oven 62 – 118 c 0 - 5 dried 45 °C, sieved (<2 Pursuit 5 PAH column (250 mm) (2016) mm x 4.6 mm x and 5 μm)

Sediment 69 – 95 This study Cameroon Manual Coring, oven 10 g, ASE, 23 mL Hex In-cell clean up, 10 g sediment, 4 g of activated HPLC-FLD/UV 0.002 – 0.59 Wouri estuary 0 - 5 dried, ground, sieved < 2 /Dcm (1/1), filtered with 4 alumina, 3 g activated copper mixed in the mm mm PTFE syringe extraction cell Pursuit 5 PAH column mangrove (2018) (250 mm x 4.6 mm x 5 m) μ 16 PAHs

Van Veen Grab sampler, 10-mm I.D. x 30 cm chromatographic South Africa Sediment wide-mouth bottles, 10 g, Na2SO4, Soxhlet, stored in ice- chest < 4 200 mL Dcm for 24 h, GC-FID 2015 Column, packed with a slurry prepared with 10 g NI 72.20 %, Buffalo River °C, air-dried in the dark extract mixed with Estuary activated silica gel in Dcm, about HP-5 fused silica column 5 days, crushed, sieved anhydrous Na2SO4 Adeniji 2019 0.5 mm mesh 2 cm of anhydrous Na2SO4 was added (top) silica (30 m × 0.320 mm i.d. × 0.250 gel. pre-elution, 20 mL of n-pentane, and eluant μm) 1 L precleaned glass 16 PAHs 500 mL extracted x3, LLE, discarded, elution 20 mL of n-pent, aliphatic and Water bottles, 6 M HCL 0.01 - n- Hex, extracts dried over aromatic fraction, elution 40 mL Dcm/pent (40:60), 79.53, % adjusted to pH<2, stored 2015-2016 anhydrous Na2SO4 exchanged in Dcm 0.03 in ice- chest < 4 °C

311

Republic of GC-MS Djibouti Sediment Alumina: silica column (1/1, w:w) Manual sampling, stored 5 g, ASE, 100 % Dcm, 20 Madhi Elite 5MS 0.01 - 0.18 84% - Djibouti city 0 - 5 4 °C, air dried 30 °C, min filtered, copper Ahmed et 102s sieved (< 2 mm) powder column al., 2017 (2016) (30 m 0.25 mm 0.25 m) 16 PAHs × × μ

D.R. Congo Activated copper, Congo river Sediment 5g, Soxhlet 20 % of GC-MS/MS LOQ basin, acetone in 80 % of Hex chromatographic column, 3 g silica gel, 16 mL Kilunga et 0 - 4 Manual sampling ZB-5ms column 0.5 (LPAHs) NI (v/v) for 4 h Hex, 35 mL Hex, 50 mL of Hex:/Dcm (v/v, 1:1). F1 al., 2017 (urban rivers) = PCBs, F2 = PBDEs, F1, F2 and F3 = PAHs and (2016) (60 m x 0.25 mm x 0.25 μm) 1.0 (HPAHs) 16 PAHs CLPs

Sediment Van veen grab 15 g, Sonication, Hex/Dcm Activated copper granules and alumina (1:2), silica Tongo et al. 3:2 for 5 h, 30g anhydrous gel glass column, 5 ml Hex, 30 mL Hex/Dcm 70:30 0.001– 78 - 102 0 - 2 sampler 2017 Na2SO4 (v/v) 0.003

Homogenized, soxhlet, 20 GC-FID Nigeria Water, hydrobios sampler, Column chromatography, activated silica gel, 0.0001– mL Hex/Dcm, 3:1 with NI filtered eluted with 40 mL n-Hex solvent DB-5 column 0.003 Ovia river, 1 m periodic venting

(60 m x 0.25 mm x 0.25 μm) 16 PAHs hand fishing net,

washed, 25 g of frozen whole-body tissue, Soxhlet for 5 mins, 1:2 (v/v) alumina: silica gel glass column elution 0.001–0.003 Fish NI homogenized for 20 Hex/Dcm 50 mL Hex ww mins, 5g anhydrous Na2SO4

Caught and kept in 80 L 5 mL of H2SO4 added to extract, vigorously shaken Tunisia plastic tank with air 2 min., aqueous layer discarded, repeated twice, HPLC-FLD Fish bubbling, muscle tissue rinse extract with water and NaHCO3 (5 %), glass Bizerte lagoon et dissected, freeze dried, 5 g, Soxhlet for 16 hrs, n- column (1 cm i.d reverse-phase C18 column Barhoumi 0.01 - 0.49 85 – 114 s al homogenized Hex/Ace (4:1) (4.6 250 mm, 5 m particle 2016b X 15 cm length), pieces of glass wool on glass frit, × μ size) 1 g activated alumina, florisil, anhydrous Na2SO4, 15 PAHs Mussel Scuba diving, ice boxes, soft tissues from shells, elution 40 mL of ethyl acetate/Hexane (1:1)

312

freeze dried, homogenized

Cameroon, Eijkelkamp Multisampler Sediment GC-MS Fusi et al., piston corer, dried, Soxhlet 2 ng/g Wouri estuary 0 - 10 and 2016 ground Florisil column SLB-5ms column 84 - 130 c mangrove, Hex/Ace (80:20) 0 - 20 (30 m x 0.25 mm x 0.25 m) 10 PAHs (<0.25 mm), sieved μ

Nigeria Adsorption chromatography, glass column packed, GC-MS

Sediment Van-Veen grab sampler, 1g, sonication for 30 min 1 g of anhydrous Na2SO4 (top), 2 g of activated Oyo-Ita et Imo River, DB-1HT column 60 - 130 al 0 - 3 freeze-dried with 15 mL Hex/Ace (1:1) alumina (middle) and glass wool (bottom), Hex (6 NI ., 2016 13 PAHs mL) (30 m x 0.25 mm x 0.10 μm) Egypt, GC-MS & GC-FID 30g, Sonication twice in 96 - 110.3 Northwestern Sediment0 Van Veen grab sampler, 0.02 ug/mL - Aly Salem et 100 ml n-Hex for 30 min silica/aluminum oxide column; 70 ml 9:1 Hex/Dcm HP-5 column c Red sea, - 5 dried, sieved (2mm) 0.1 ug/mL al., 2014 and 100 mL DCM (30 m x 0.32 mm x 0.17 m) 15 PAHs μ

D.R. Congo, Manual sampling 5g, Soxhlet GC-MS/MS Mwanamoki Sediment0 Activated copper, chromatographic column, 3 g Congo river 0.05 (LOQ) -6 Hex/Ace 80:20 (v/v) for 4 h Silica gel, 16 mL Hex, 35 mL Hex, 50 mL Hex/Dcm VF–Xms column NI et al. 2014 basin, (2013) (v/v, 1:1) (30 m x 0.25 mm x 0.25 μm) 16 PAHs

Grab sampling, stored in 15 g, mixed with 15 – 20 g Activated silica gel, activated the pre-combusted Egypt anhydrous Na2SO4, ASE, Alumina, elution aromatic hydrocarbons GC-MS amber glass jars glass Dcm, extract concentrated Barakat Lake Manzala Sediment jars with Teflon-lined 0.2 - 0.4 75 – 120 s in water bath and from column with 200 mL of PENT/Dcm (1/1, v/v), DB-5 MS column (30 m × 0.25 2013 lids, stored in the dark 39 PAHs exchanged with 2 mL of collected solvent concentrated and exchanged to 1 mm x 0.25 μm) -20 °C Hex mL of Hex

Egypt Grab sampling, stored in the pre-combusted 15 g, mixed with 15 – 20 g GC-MS Mediterranean Activated silica gel, activated Barakat anhydrous Na2SO4, ASE, Sediment amber glass jars glass DB-5 MS column (30 m 0.25 0.2 - 0.4 75 – 120 s coast Dcm, extract concentrated Alumina, elution aromatic hydrocarbons × 2011 jars with Teflon-lined mm x 0.25 μm) 39 PAHs lids, stored in the dark in water bath and

313

-20 °C exchanged with 2 mL of from column with 200 mL of PENT/Dcm (1/1, v/v), Hex collected solvent concentrated and exchanged to 1 mL of Hex

Sample, depth LOD Analyte Country, site, Sample size, extraction Clean-up/Fractionation (cm) Sampling method, Pre- Recovery Analytical technique ng/g or μg/L Compound(s) treatment method References (Sampling (dw or lw) (%) date)

Nigeria, GC-MS Iko river Sediment0 Gravity corer, freeze 50g, Soxhlet, 30g of deactivated alumina with 4.5% of Water; 50 HP-5MS column Essien J.P. NI NI estuary, -10 dried, sieved (2mm) 65◦C for 24 h, Dcm mL of Hex/Dcm 95:5 (30m x 0.25 mm) 2011 16 PAHs

Sediment Interface corer, sampled at 2 cm interval, PE 85 ± 7s Cameroon core bags, oven dried 45 °C, OCPs

Lake Barombi lyophilisation (- 41 °C, 10 g, ASE, 23 mL 62 – 118 c 0 - 10 0.310 mbar) GC-MS Mbo Hex/Ace (1/1), 20 min In-cell clean up, 10 g sediment/soil, 5 g of activated alumina, 5 g activated copper mixed in Restek Rxi – XLB column (30 0.04 – 0.71 Soil and Stainless steel spoon, the extraction cell This study m x 0.25 mm x 0.25 μm) OCPs 103 ± 2s 18 OCPs aluminium boxes, Stream removal of litter and 88 – 141 sediment coarse material, oven dried 45 °C, sieved (<2 CRM c 0 - 5 mm)

Cameroon (0.04 – 0.71) 77 – 94 Wouri estuary ASE, 10g, 23 mL Hex/Ace GC-MS Sediment Manual Coring, oven SPE florisil cartridges (1g, 6 mL), F1: 2.5 mL Hex, OCPs (PCBs) This study mangrove, (1:1) RRxi – XLB column 0 - 5 dried, ground, sieved F2: 5mL Hex/Ace (80/20; v:v) (0.02 – 0.13) 72 – 94 18 OCPs (30 m x 0.25 mm x 0.25 μm) PCBs (OCPs) 7 PCBs

Ethiopia Metallic core sampler, Backed 1g Anhydrous Na2SO4, 1g Florisil, 1g GC-ECD 0.01 – 0.3 Awash River 1g sample, Soxhlet, 1mL Sediment Composite sample, acidified silica gel, 1g Copper powder and ground HP-5MS column (OCPs Basin DCM Dirbaba et al aluminium foil, ice box, sample mixture (bottom to top). Column elution, (30 m 0.25 mm i.d. 0.25 mm) ) 76 ± 13 % 0 – 10 2018 16 OCPs dried, removal of coarse Soxhlet apparatus, 1 mL DCM, Concentrated 0.04 – 0.1 material and plant extract, died, redissolved in 100 μL n-Hex (PCBs) 7 PCBs debris, sieved (2 mm),

314

ground and sieved (0.3 mm), stored (4 °C)

20 g mixed with 60 g Sediment 100 g, Stainless steel Na2SO4, Soxhlet, ground, GC-ECD Kenya shovel, aluminum foil, dried, Soxhlet,130 mL n- 70 - 79 (2009) BPX 5 column (30 m x 0.25 Nairobi river stored at – 20 °C Hex/Ace (3:1, v:v), 16 h, 2 mL isooctane mm x 0.25 μm) Chromatographic column, 15 g deactivated Ndunda et 1 L, LLE, separating aluminum oxide, elution 167 mL of n-Hex, 2 mL 1.1 - 3.1 pg al., 2018 17 OCPs funnel, pH adjusted to 7 isooctane, activated copper (for sediments) Confirmatory test Water Grab sampling, 2.5 L with phosphate buffer, 100 amber glass bottles, g NaCl, 6O mL, vigorous second column different 70 - 85 (2009) stored at 4 °C shaking, extraction polarity BPX 50 (60 m x 0.25 repeated twice, anhydrous mm x 0.25 μm) Na2SO4, 2 mL isooctane

150 mL Hex/Dcm GC-ECD (for PCBs) Tunisia Sediment Collected and kept in pre-calcinated d (2:1. v/v) using Soxhlet for Activated silica gel and florisil column, elution 40 Elite column (15 m 0.25 m) Grombalia × μ 0 - 10 aluminium containers. 16 h, activated copper ml of Hex /Dcm (9:1. v/v) and 70 ml Dcm Samia et al Aquifer powder - - 2018 2 L pre-cleaned amber HPLC-SPDM2OA Diode array PAHs Water glass bottles. LLE, n-Hex and DEE detector PCBs (2009) (75:25 v/v) three times C18 column (250 L × 4.6 mm)

3 mL extract into separating funnel, 6 mL of South Africa Water 100 Grab sampling, 1 L H2SO4/Water (1:1; v/v), Decantation, 5 mL of Buffalo River 500 mL, 3 x 30 mL Dcm in GC-ECD mm deep amber glass bottles, aqueous KMnO added to the organic fraction, Yahaya et separating funnel, ₄ 0.005 – 0.3 70 - 92 acidified with 5 mL of glass chromatographic column, (30 cm x 10 mm HP-5 (30 m × 0.25 mm × 0.25 al. 2018 (2015 - anhydrous Na2SO4 HCl (1:1) I.D.), 4 g deactivated silica gel, 2 g anhydrous μm). 19 PCBs 2016) Na2SO4, pre-elution 10 mL Hex, Elution 40 mL Hex

Centrifuged 3 min at 2500 rpm, 10 mL of the Tunisia Van Veen 5g, QuEChERS, 4 mL supernatant transferred into another polypropylene 81 - 137 Sediment ultrapure water, manually OCPs Ichkeul lake Grab, placed in glass tube (15 mL capacity) GC-MS Ben Salem shaken, 20 mL Dcm/Ace - 2011 and bottles and were kept 2017 Bizerte lagoon (50:50, v:v), shaken containing 900 mg of MgSO4 and 150 mg primary DB5-MS UI column 2012 frozen at 20 °C, freeze- − vigorously by hand 1 min, secondary amine (PSA), shaken vigorously by dried, sieved 60 - 103 22 OCPs addition of citrate buffer hand for 30 s and centrifuged 315

12 PCBs salt mixture, shaken for 3 min at 2500 rpm, dried residue re-dissolved PAHs vigorously manually 5 min. with 1 mL CAN, kept at −20 °C 16 PAHs 76 - 131 PCBs

South Africa, 78.4 ± 4.4 Port Elizabeth, 20g, Soxhlet, 20g of 0.01 and anhydrous Na2SO4, 5 g Extract (1 mL), dissolved in 2 mL of Hex, 2 mL to 98 ± 2.7 North End Steel Van GC-MS 0.20 (LOD) H2SO4, anhydrous Na2SO4, A glass column Lake Sediment activated copper, 250 mL Veen grab sampler, Hex/Dcm, packed 10 g of activated florisil, 2 g of anhydrous (n=3) Kampire E DB-1ms et al 0 - 20 homogenized, air-dried, Na2SO4 (top). ., 2017 0.04 and ground, sieved (<1 mm) (1:1) v/v for 24 h (30 m x 0.25 mm x 0.25 m) 7 PCBs Elution 80 mL of Hexane 0.66 (LOQ) 98.6 ± 5.4s

Kilunga et D.R. Congo al Sediment Manual sampling 5 g, Soxhlet, Hex/Ace (4:1; GC-MS/MS LOQ ., 2017 Kinshasa v/v) for 4 h, activated Chromatographic column, 3 g of Silica gel, F1 (16 0 – 4 copper ZB-5ms column 0.02 Urban rivers mL Hex), F2 (35 mL Hex), F3 (50 mL Hex/Dcm)

(2016) (60 m x 0.25 mm x 0.25 μm) (4,4’-DDE) NI (1:1, v/v)

ZB-XLB column 26 OCPs

(20 m x 0.18 mm x 0.18 μm) 12 PCBs

Extract dissolved in 4 mL n-Hex, 8 mm florisil mini- 10 g, soxhlet, 150 mL column, 3.5 g of 7% deactivated florisil, 1 cm layer Nigeria Sediment (Boyd and Tucker, 1992) HEX/ACET (4:1; v/v), 8 h, of anhydrous Na2SO4 (top and bottom), washed 10 dissolved in 10 mL n-Hex mL n-Hex, elution 150 mL 30 % DEE in n-Hex Komadugu (v/v). GC-MS river Basin Mohammed 10 g deactivated silica gel, 10 mm (NI) NI NI et al., 2017 20 g of sample, 20 g chromatographic column, 3 g anhydrous Na2SO4, anhydrous Na2SO4, 5 g 5 OCPs Bought from local rinsed 10 mL (1:1; v:v) ethyl acetate/Dcm, elution Fish NaHC03, 100 mL 1:1 (v/v) fishermen 80 mL (1:1; v:v) ethyl acetate/Dcm and 50 mL (1:1; ethyl acetate/Dcm, mixed, v:v) ethyl acetate/Dcm re-extraction De-fattening (5 mL of Hex/Acn), 25 mL Hex

316

(Boyd and Tucker, 1992) 1 L, LLE, separating Water 1.5 L amber glass funnel, 50 mL n-Hex, bottles, stored in ice vigorous shaking 5 min, NI 0 - 3 box, sored at 4 °C, re-extraction of aqueous filtered phase, 50 mL n-Hex

South Africa Water Grab sampling, 1 L LOD 20 to amber glass bottles, GC -ECD Buffalo River 100 mm Glass column (30 cm x 10 mm I.D.), 5 g florisil, 2 g μ 60 ng/L acidified with 5 mL of 500 mL, 30 mL Hex/Ace Yahaya et deep layer of anhydrous Na2SO4, pre-elution 10 mL HP-5 capillary column (30 m x 76 – 93s al HCl (1:1) (1:1), anhydrous Na2SO4 LOQ 110 to . 2017 HEX, Elution 40 mL Hex 0.25 mm x 0.25 m) (2015 - μ 530 ng/L 17 OCPs 2016)

Tunisia GC-ECD Grab sampling, Bahiret el 5 g, Soxhlet, 90 mL Sediment aluminum jars, stored – Column, 5g activated florisil, 1 cm anhydrous PTE-5 Bibane lagoon Hex/Ace (3:1 v/v), 12 h, 20 °C, freeze dried, Na2SO4, washed 2 X 15 mL n-Hex, transferred to 0 – 10 H2SO4 (98 %), NaHC03 (5 (30 m × 0.32 mm, 0.32 μm) OCPs sieved (2 mm) column 10 mm, elution 50 mL Hex/Dcm (9:1; v/v) Barhoumi et %), separating funnel, and > 90s homogenized, stored 4 al 2016a (2011) activated copper 0.10 - 0.4 26 OCPs °C. HP1 (30 m × 0.32 mm, 0.25 μm) PCBs 12 PCBs 0.02 - 0.1 Columns

Cameroon Eijkelkamp Multisampler Wouri estuary piston corer, dried, Sediment ground GC-MS mangrove Soxhlet 0 - 10 and 70 and Fusi et al. (<0.25 mm), sieved Florisil column SLB-5ms column (30 m 0.25 0.5 (PCB) Hex/Ace (4:1) × 115 2016 0 - 20 mm 0.25 μm). 6 PCBs 3 OCPs

stored in an ice-chest at 10g, Soxhlet, 50g Ethiopia Sediment GCμ-ECD 4°C, air-dried anhydrous Na2SO4, 150 ml Pre-conditioned octadecyl C-18 columns, washed (Mwevura H, Othman C, Tekeze Dam of Hex/Ace (4:1 v/v) at 50 with 1 ml, 30% methanol and 1 ml ultrapure water, Mhehe L (2002) NI NI GA 2016 stored ice-chest at 4°C, Fish °C for 4 h, dissolved in 10 elution 5 x 0.5 mL Hex Organochlorine Pesticide ground mL Hex Residues in Edible Biota from

317

6 OCPs LLE APHA, 100 mL the Coastal Area of Dar es Hex/Ace (1:1 v/v) shaken Salaam City. Western Indian 800 mL, surface water, vigorously 2 min, 20g Ocean J Mar Sci 1: 91-96) Water filtered, stored in ice- NI anhydrous Na2SO4, re- chest at 4 °C extraction, 50 mL Hex/Dcm (1:1 v/v)

Stainless steel hand trowel, composite Sediment sample, pre-cleaned glass bottles wrapped 10 g, Soxhlet 8 h, 180 mL 0 - 10 0.001 87 - 104 s with aluminium foil, Hex Glass chromatographic columns were packed with (2015) water decanted, 4 g of deactivated SiO2, 0.5 g of Na2SO4 and 6 g of GCμ-ECD Togo homogenized, air dried alumina, 1 g Na2SO4, 1 g of activated charcoal respectively for water and sediment clean up. SGE BPX-5 of 60 m x 0.25 at ambient temperature Mawussi et Lake Togo Rinsed 10 ml of Hex, 40 ml Hex added to the mm and 0.25 μm) and al 2016 Randomly collected, concentrate in a ratio 2:1:1, Extract transferred equipped with 1 m retention OCPs composite sample, pre- 500 mL, separatory funnel, onto the clean-up column, eluate allowed to run gap (0.53 mm, deactivated) cleaned glass bottles 350 mL Hex, allowed 30 into a receiving flask, concentrated to dryness. Water wrapped with aluminium min for phase separation, Concentrate picked in 2 ml ethyl acetate 0.001 86 - 103 s 2015 foil, filtered through organic layer dried with watman GF/F filter (0.7μm porosity), stored 20 g anhydrous Na2SO4, at 4 °C

GC–MS 30 g, Soxhlet 8 h, 200 mL Grab sampling, pre- Egypt Sediment n-Hex/Dcm, copper Fractionation, glass column packed with 20 g fused-silica capillary column cleaned glass bottles powder florisil, elution 70 mL of n-Hex PCB fraction (F1). (30 m x 0.32 mm x 0.52 m) Ragab et al 12 OCPs 0 - 5 with wide mouth, then μ 92 – 104 c Elution 50 mL of n-Hex/Dcm (70 : 30) pesticide coated with DB-1 (5% diphenyl 2016 frozen and stored in the - fraction (F2). and 95% dimethyl 7 PCBs 2011 laboratory at -20 °C

polysiloxane)

Nigeria Column conditioning: 15 mL Hex, column (15 cm

(L) x 1 cm (i.d.)), packed with 2 g of deactivated GC-ECD Ogbese river Sediment et Ekman grab sampler, air silica gel, 1g anhydrous Na2SO4 (top), elution 20 Ibigbami HP5 MS column 0.15 83 - 98.7s al -dried, sieved 2 mm mL of Hex/DEE (1:1) ., 2015 20 g, Sonication (twice), (30 m x 0.32 mm x 0.25 μm) 15 OCPs 20g anhydrous Na2SO4,

318

50 mL Hex/Ace (1:1 v/v), 10-15 mins for 60 °C

Fish Sonication (twice) (15 Drag net method, mins), 10g, 25 g of homogenized head and NI anhydrous Na2SO4, 40 mL muscle tissue Hex/Ace (1:1)

100 mL, LLE, 50 mL Dcm, Water Grab method, acidified shaken vigorously for 2 NI 0.15 by conc. HNO3 to pH 2 min, settle 30 mins, 20 g anhydrous Na2SO4

Gill nets, dissected to Nigeria 20 g, shaking, 20g 10 g deactivated silica gel, 3g anhydrous Na2SO4, remove flesh, liver, GC-FLD anhydrous Na2SO4, 5g 8 mL ethyl acetate/Dcm, elution 80 mL ethyl Akan and Lake Chad, stomach and gills, Fish NaHC03, 100 mL (1:1) acetate/Dcm and 50 mL ethyl acetate/Dcm, de- 35% diphenyl/65% NI NI Chellube Baga stored in 4% formalin ethyl acetate/Dcm, fattening (50 mL (1:1) HexAcn), shaking 3 min, 25 2014 dimethyl polysiloxane column 11 OCPs decantation mL Hex 4 OPPs

GC-ECD Tunisia PTE-5 Bizerte Grab sampling, Sediment 5 g, ASE, H2SO4 (98 %), Lagoon aluminum foil, freeze- Column, 5g activated florisil, 1 cm anhydrous (30 m 0.32 mm, 0.32 m) 0.5 - 1 for OCPs NaHC03 (5 %), separating × μ Barhoumi et 0 – 10 dried, sieved (2 mm), Na2SO4, washed 2 X 15 mL n-Hex, transferred to and OCPs and funnel, activated copper 55.5 - al 2014 homogenized, stored 4 column 10 mm, elution 50 mL Hex/Dcm (9:1; v/v) PCBs (2011) HP1 (30 m 0.32 mm, 0.25 93.0s 4 OCPs °C × μm) 12 PCBs columns

GC-ECD Morocco Sediment stainless steel grab, 20g, Soxhlet PTFE bags, stored at - (UNEP/FAO), 8 h, 200 mL florisil column 87 – 103s fused silica capillary column Benbakhta Atlantic coast 0 - 3 20 °C, freeze-dried, n-Hex, treated with 0.12 - 0.40 chromatography (UNEP/FAO), partially RSD 2% et al 2014 homogenized, sieve (63 mercury to remove CP-Sil 8 CB (30 m x 0,25 mm 12 OCPs (2010) deactivated florisil, 2 cm Na2SO4 at the top to 14% μm) Sulphur compounds x and 0.25 μm) D.R. Congo Sediment 0.02 5g, Soxhlet, Hex/ACe (4:1) chromatographic column, 3 g of silica gel, F1 (16 River-reservoir Manual sampling GC-MS/MS Mwanamoki 0 - 6 (v/v) mL Hex), F2 (35 mL Hex), F3 (50 mL Hex/Dcm (4,4’ DDE) system (v/v, 1:1). ZB-5ms column NI et al., 2014 (2013) for 4 h, activated 26 OCPs (60 m x 0.25 mm x 0.25 μm) copper 12 PCBs

319

Nigeria Sediment Grab sampling, Column (15 cm × 1 cm i.d.), 5 g activated silica gel GC-ECD 3 aluminium foil, stored 4 20 g, Soxhlet extraction, in slurry of n-Hex, 0.5 cm Na2SO4 (top), pre- Aiba Reservoir 0 – 10 0.04 - 2.11 °C, air-dried, sieved (< Dcm at 40 ° C, 10 h elution 15 mL n-Hex, elution 2 x 10 mL, eluate column Zebron ZB-1701 (30m 2mm) dried with Na2SO4 × 0.25 mm × 0.25μm) (2012) Olutona et 81 - 95 al., 2014 20 OCPs 500 mL, LLE, separatory Water 2.5 L Winchester bottles, funnel, 10 mL Dcm, 87 - 95 (2012) conc. HNO3 (pH2) vigorous shaking, 30 mins,

anhydrous Na2SO4

Sediment 5 g, ASE, 20 mL, n- 0 – 5 Grab sampling, dried Hex/Ace (9:1 v/v), 2 mL of Column, glass wool (bottom), covered with 3 g Benin Isooctane (2002) aluminium oxide 15 % deactivated with water and Lake Noukoué 2 g, 200 mL acetone 10 conditioned with 4 mL petroleum GC-ECD Yehouenou min, 20 mL saturated 0.1 80 - 110 c Cotonou Ether/DEE (95:5 v/v), 17 mL petroleum et al 2014 Na2SO3 and 100 mL Lagoon Nets, stored at 4 °C, Fish petroleum ether, shaking ether–DEE was used to elute pesticides dissected, freeze dried, 9 OCPs 10 min, filtered and (2004) lyophilized, ground from the column, 1 mL isooctane was added washed with 500 mL deionised water, 20 mL NaCL.

HCHs HCHs 5g, freeze-dried, Soxhlet, Uganda Column bottom to top; 8 g acidified silica gel, 2 g HCHs 4 h; 100 mL 3:1 n- - Sediment Na2SO4, Elution 15 mL Hex, 10 mL Dcm Napoleon gulf Sediment corer, Hex/Ace GC-ECD 4–20 pg/g PCBs of Lake < 30 cm homogenized, for PCBs DB-5 column (60 m x 0.25 mm Victoria transferred in acetone 60.5– (2011) x 0.25 m) HCHs Pre-washed 60 mL of n-Hexane, multilayer column μ 97.2% 10 g, mixed with bottom to top: 2 g silica gel, 5 g 33 % silica gel- hydromatrix™(Varian), 3 OCPs ASE, 100 mL 3:1 v/v NaOH, 2 g silica gel, 5 g 44% silica gel-H2SO4, 10 PCBs mixture of n-Hex/Ace g 22% silica gel- H2SO4 and 5 g anhydrous 7 PCBs Na2SO4. Elution 60 mL n-Hex, transferred to MultiResidue-2 column (30 m Ssebugere Fish carbon column, Elution 100 mL n-Hex 0.01–0.32 Gill nets, 10 g muscle tissue, mixed x 0.25 mm x 0.20 μm) HCHs et al., 2014 (2011) with hydromatrix™ pg/g PCBs

320

Homogenized, (Varian), ASE, 100 mL 3:1 60.3 to transferred in acetone v/v n-Hex/Ace 80.1

Ghana Grab sampling, stored in Sonication, 2 h at 40 °C Sediment an ice-chest, 4 °C, dried GC-ECD Hex/Ace, concentrated for Lake at room temperature, Elution 10 mL Hex, 5 mL of 2:1 Hex/Diether, 0 - 20 clean up VF – 5mS column (40m x 0.25 Bosomtwi ground, sieved 500 m) concentrated almost to dryness, dissolved in 1.5 NI 96 -101 s Afful 2013 μ mm x 16 OCPs mL ethyl acetate 500 mL high-density LLE, Hex, concentrated for Water 0.25μm) 7 PCBs polyethylene containers clean up

20 g, shaking, 20g Nigeria Gill nets, dissected to anhydrous Na2SO4, 5g 10 g deactivated silica gel, 3g anhydrous Na2SO4, Fish remove flesh, liver, GC–MS NaHC03, 100 mL (1:1) 8 mL ethyl acetate/Dcm, elution 80 mL ethyl Alau Dam Akan et al., stomach and gills, ethyl acetate/Dcm, 20g acetate/Dcm and 50 mL ethyl acetate/Dcm, de- 35 % diphenyl/ 65% NI NI (2010 – stored in 4% formalin 2013 11 OCPs anhydrous Na2SO4, 20g fattening (50 mL (1:1) Hex/Acn), shaking 3 min, 25 2011) Dimethyl polysiloxane column NaHC03, shaking 10 min, mL Hex

decantation

Fractionation alumina: silica open column chromatography, column packed (bottom to top); GC–MS 87.8 - 107 s Lake Qarun Sediment Grab sampling, pre- combusted amber glass 1g backed anhydrous Na2SO4, 10 g, 1% OCPs 0.01 - 0.04 10g, Soxhlet, Dcm, 48 h, DB-5 MS Barakat OCPs 0 - 5 jar with Teflon lined lids, deactivated alumina OCPs and exchanged for Hex 91.2 - 102 s 2013a stored in the dark at -20 20 g 5% deactivated silica gel and 1 g anhydrous column (30 m x 0.25 mm x PCBs PCBs 2011 °C, freeze dried Na2SO4, activated Cu pellets at the top of the 0.25 μm) PCBs column, elution 200 mL of 1:1 n-Hex/Dcm

Grab sampling (Ekman GC-ECD Mediterranean dredge), clean Fractionation by alumina: silica open column coast polyethylene bag, stored chromatography. 20 g of activated silica gel was DB-5 bonded phase 0.25 Sediment in pre-combusted amber slurry pecked in Dcm over 10 g activated alumina, Barakat 10 g, ASE, Dcm 30 m x 0.25 mm I.D. fused (OCPs and 92 OCPs 0 - 5 glass jar with Teflon activated Cu pellets (top), Dcm replaced by PENT 2013a lined lids, stored in the by elution, elution (pesticide/PCB fraction) with 200 silica column with a 0.25 mm PCBs) PCBs dark at -20 °C, freeze mL of PENT/Dcm (1:1)

dried

321

91.0 ± 6.78s Egypt 85 - 101 c Fractionation alumina: silica open column Grab sampling, pre- GC–MS Lake Maryut Sediment chromatography, column packed (bottom to top); combusted amber glass 0.01 - 0.04 OCPs 10g, Soxhlet, Dcm, 48 h, 1g backed anhydrous Na2SO4, 10 g, 1% DB-5 MS fused silica column Barakat et 0 - 5 cm jar with Teflon lined lids, exchanged for Hex deactivated alumina, 20 g 5% deactivated silica (30 m x 0.25 mm, 0.25 m) for OCPs al. 2012 stored in the dark at -20 μ gel and 1 g anhydrous Na2SO4, activated Cu and PCBs 25 OCPs (2005) °C, freeze dried 94.4 ± pellets (top), elution 200 mL of 1:1 n-Hex/Dcm 29 PCBs 6.56s 84 – 108 c PCBs

Sediment 3g, Soxhlet, 5g, copper GC–MS 71-116 Grab sampling powder, 75 mL of Hex/Ace Acidified silica, 2g of activated copper on top (SRM (2010) (3:1) DB-5 column 0.01 – 0.05 1588b) DR Congo (30 m x 0.25 mm x 0.25 μm) Gill nets, filleted, 79 – 142 Fish skinned, caudal fines, Soxhlet, 0.2 – 6.2 g, 100 Congo River (OCPs) (PCBs) (2010) homogenized with mL Hex/Ace (3:1) for 2 h Verhaert V. Basin anhydrous Na2SO4 et al., 2013 15 OCPs Activated silica, 8g, elution: 20 mL Hex and 15 mL GC–MS 69 – 140 Dcm (OCPs) 33 PCBs Invertebrat Homogenized whole 0.1 – 4.1 g, Soxhlet, 100 HT-8 column 0.1 0. 4 es mL Hex/Ace (3:1) for 2 h body with anhydrous (25 m x 0.22 mm x 0.25 μm) (2010) Na2SO4 (PCBs) SRM 1945

Sediment 3g, SFE, 500 !L Thawed, air-dried at MeOH/Ace (2:3), 0 - 3 0.02 !g/g et al Nigeria ambient temperature, pressurized cell to 300 98 - 134 Okoya ., bars at 60 °C with SC-CO2 GC-ECD 2013 Ondo State sieved (2 mm) Zebron ZB column (30 cm x Rivers (density = 0.872 g/mL) NI 0.25 mm x 0.25 μm) 15 OCPs 1 L of pre-extracted water,

Surface Grab sampling (2.5 L), 5 LLE 0.01 85 - 103 water mL conc. H2SO4 3 x 15 mL of Dcm, anhydrous Na2SO4

322

Ethiopia 70 – 110s Bought from local 10 g of sample mixed with GC-ECD fishermen, thawed, anhydrous Na2SO4, pre- Glass column, 6 g activated florisil, anhydrous Lake Awassa Fish Yohannes et dissection for liver and washed Hex/Ace Na2SO4 (top), elution 80 mL Hex containing 25% ENV-8MS column 0.05 - 0.1 al., 2013 (2011) muscle, stored at – 20 Soxtherm, 150 mL diethyl ether 75 – 115 °C Hex/Ace (3:1; v:v) (30 m x 0.25 mm x 0.25 μm) 6 OCPs CRM

Grab sampling, well 10 g, Soxhlet, 50 g Sediment Ghana mixed, stored at -20 °C anhydrous Na2SO4, 150 ml 0.01 0 - 2 in aluminum foils, air- Hex/Ace (80:20; v:v) at 50 GC-ECD Densu River dried sieved 250 μm °C for 8h Activated florisil column 1.5 g, 0.5 g layer of Basin fused silica gel 79 – 96s Kuranchie- 1 L, LLE, 50 mL of n-Hex, anhydrous Na2SO4, elution 10 mL of n-Hex Mensah et 2.5 L amber glass separating funnel, shaken VF- 5 ms al . 2012 bottles, pre-filtered vigorously 5 min, re- 14 OCPs Water (30 m x 0.25 mm x 0.25 m) 0.001 through 0.45 μm fiber extraction of organic μ glass filters phase 2 x 50 mL of n-Hex, anhydrous Na2SO4

Sediment 1–2 g, MAE, Dcm, filtration Senegal Dried and ground of organic extract 0 - 5 Column of acidic silica Fadiouth and GC-ECD

Falia Estuary, gel column with activated copper and eluted with a Bodin et al., Molluscs soaked HP5-MS column NI NI pentane-Dcm mixture 2011 7 PCBs overnight in clean Biota aerated seawater, soft (60 m x 0.25 mm x 0.25 m) (90:10 v/v) μ 14 OCPs tissues, excised, freeze dried, ground

Orbital shaker, 50 mL Kenya Ekman grab sampler, Hex/Ace 1:1 for 12h, Sediment thaw 4 H, mixing, 25g + centrifugation at 4000 rpm GC-ECD Yala/Nzoia Florisil column NI 0 - 20 3.5 ml of 0.2 M NH4Cl, for 30 min, decantation, River settle for 15 min re-extraction twice with 25 88.62 - Musa et al. Lake Victoria mL Hex/Ace (1:1; v:v) CP-SIL 8CB column 97.53 2011 basin Grab sampling, 60 mL distilled Dcm, Florisil column topped with anhydrous sodium (15 m x 0.25 mm x 0.25 μm) 15 OCPs Water neutralize sample, 100 g shaken 2 min, settle for 30 sulphate, elution 200 ml of 6%, 15% and 50% DEE NI NaCl mins in Hex

323

10g (two portions);

dispersion method (Akerblom, 1995), 40 g of GC-ECD Uganda Na2SO4, successive Coring, wrapped in shaking, 1:1 (v/v), Gel permeation chromatography (GPC), DB-1 non-polar column (50 m 0.0022 - Lake Victoria Sediment aluminum foil, airtight Ace/cyHEX chromatographic tube (50 cm x 1 cm i.d.), elution x 0.53 mm) later interchanged Wasswa J. 0.0082 bags, stored at - 20 °C 14–25 mL ethyl acetate/CyHex (1:1 v/v) with a CP-Sil 19 CB semi- et al, 2011 0 - 20 cm (50, 30, 20, 20 mL), (ng/g) polar (30 m x 0.53 mm) extraction of aqueous 16 OCPs layer ethyl acetate/cyHex (50 mL; 15:18 v/v),

20 g of Na2SO4

Fish Ghana (2008) Bought from the local market, aluminium foil, H2SO4 clean-up step repeated, Hexane layer

Lake placed in PE bags, washed and separated with water, multi-layer 40 g, homogenized with GC-MS Bosomtwi, stored – 20 °C column ( Na2SO4 (2 g), 10% (w/w) silver nitrate– Na2SO4, Soxhlet, Dcm, Adu-Kumi silica (2 g), silica gel (2 g), 22% (w/w) potassium 0.01 - 0.3 50 – 120s Lake Weija concentration and DB-5ms capillary column (60 2010 hydroxide–silica gel (0.5 g), elution 100 mL Hex. dissolution in Hexane m x 0.25 mm x 0.25 μm) Eluate concentrated and separated. Elution Volta Lake Hex/Dcm (9/1) F1 for OCPs

17 OCPs

Nigeria SPE, glass chromatographic column (400 mm x 20 Stored at – 20 °C, 10 g, cold extraction GC-ECD Lagos Lagoon Fish mm I.d.), activated silica gel, sample extract dissection to remove mode, Dcm Adeyemi et dissolved in 5 mL n-Hex, elution 60 mL n-HEX. HP-5 column (30 m x 0.32 mm NI 78 - 80 al (2007) muscle tissue, 10 g 2008 x 0.25 m) ground with Na2SO4 μ 9 OCPs

Ghana GC-ECD 0.18-0.55 76 - 95 10 g, 50 g anhydrous (S) Lake Na2SO4, Soxhlet, 150 ml Pre-conditioned octadecyl C18 columns at a rate SPB-608 column (30 m x 0.32 Darko et al. Sediment Grab sampling, air-dried Bosomtwi Hex/Ace (80:20; v:v) at 50 of 2 mL/min, elution 5 x 0.5 mL Hex mm x 0.25 μm) 2008 °C for 4 h 6 OCPs 324

1 L, Solid Phase Pre-filtration (0.45 μm Extraction (SPE), Water 0.10-0.50 fiber glass filters), conditioned C-18 78 - 97 ng/l (W) 0 - 3 cartridges, elution 3x 5 mL 5 mL conc. H2SO4 Hex

Washed with distilled Soxhlet 78 – 95 Fish 0.70-3.00 water, ground ng/L ng/g (F)

Tanzania 3g, Soxhlet, 100 mL Air-dried at room 0.2 (S) Sediment Hex/Ace (3:1) for 2 h 6 g acidified silica, 3 g copper powder, elution 15 Kruitwagen Coastal temperature, sieved < NI et al 0 - 5 mL Hex and 10 mL Dcm ., 2008 mangrove 63 μm GC-ECD

HT-8 column (25 m x 0.22 mm Scoop nets 4 g, Soxhlet, 100 mL 8 g acidified silica, elution 15 mL Hex and 10 mL x 0.25 μm) 0.1 0.3 (F) 6 OCPs Fish NI Hex/Ace (3:1) for 2 h Dcm 10 PCBs

Sediments Grab sampling, stored - 30 g, Soxhlet, 250 mL 0 – 3 20 °C, freeze dried, HEX 8 h, re-extraction 8 h 0.2 (PCBs) ground, sieved 250 μm 250 mL Dcm Egypt (2006) Shaking with mercury Sulphur for Sulphur removal GC-ECD Lake Burullus Fish (sediments) Soxhlet, 10g muscle A fused silica capillary column (2 tissue, 30 g anhydrous 0.3 (OCPs) 96 - 106 Saïd et al - 20 g florisil, 10 g alumina, 1 g anhydrous Na2SO4, (50 m x 0.32 mm x 0.52 μm) species) Na2SO4, 200 mL Hex/Dcm PCBs 2008 13 OCPs elution 70 mL of n-Hex for PCB, coated with DB-1 (5% diphenyl (1:1) 8 h (2006) and 95% dimethyl 7 PCBs 50 mL Hex/Dcm (70:30; v/v) for OCPs. polysiloxane) Water 1 L, Field extraction, 3 x 1 m depth - 200 mL Dcm (2006)

Kenya Glass-wool plugged miniature GC-ECD Barasa et al Sediment Scooping, aluminium Soxhlet, 3 h, 100 mL of 85 NI foil, stored in a deep 2007 Indian ocean % Hex, 10 % acetone and columns (10 cm x 2 cm i.d.), 4 g of florisil, 2 cm SE-54 column (30 m x 0.25 86.2 - 0 - 5 freezer, 30 g of sample 5 % deionized water coast layer of anhydrous Na2SO4 (top), mm x 0.25 m) 94.4s mixed with 20 g μ 325

7 OCPs anhydrous Na2SO4, elution 10 mL Hex, 10 mL of 1 % acetone in Hex, sieved 63 μm 10 mL of 2% acetone in Hex. Sample, depth LOD Analyte Country, site, Sample size, extraction Clean-up/Fractionation (cm) Sampling method, Pre- Recovery Analytical technique ng/g or g/L Compound(s) treatment method μ References (Sampling (dw or lw) (%) date)

Collected from local The powder was extracted The sample extract (3 ml in each case) was fishermen, wrapped in with 4 successive portions passed through an Extrelut-3 disposable column, GC-ECD aluminium foil, deep- of Dcm (50 ml and 3 x 20 eluted with Acn saturated in Hex (4 x 5 mL), Non-polar (SE-30) and frozen to below -18 °C, mL) and the solvent combined eluate fractions concentrated to 2 mL, Tanzania Fish semipolar Henry and evaporated in vacuo after from which 1 mL was further cleaned by GPC 10g (fish fillet), 0.3 – 1.3 52 - 120 Kishimba Lake Victoria (1999) which the extract was using ethyl acetate:CyHex (1:1 v/v) as eluent, (OV-1701) columns (30 m x defrosted, minced fish, 2006 dissolved in 3.5 mL solvent was 0.32 mm x ground with Na2SO4, CyHex. cleaned with free-flowing evaporated to dryness, extract redissolved in 0.25 μm) powder. CyHex/Ace (9:1 v/v)

Sediment Grab sampling, Soxhlet, 5g, SPE bond elute LOQ (1995- aluminium foil, air-dried columns GC-ECD, capillary column 0.01 – 0.20 996) Ghana coated with SPB-5 Solid Phase Extraction 78 - 104s Ntow 2005 Volta Lake (30 x 0.53 x 1.5 μm) Water 1 L amber glass (SPE) (reference LOQ 6 OCPs Analytichem NI 0.3 m Bottles, stored at 4 °C 0.001 – 0.1 International 1987)

South Africa Sediment Plastic containers, dried, 10g, Soxhlet, 120 mL GC-ECD 88 – 109s Eastern Cape 0 - 5 cm ground, sieved Dcm, 10 h Province Chromatographic column (20 cm x 8 mm I.D.), 5g methyl 5% phenyl silicone Fatoki and activated silica gel, 0.5 mL anhydrous Na2SO4, column (30 m x 0.53mm x 2 5.5 – 20.6 Awofolu Rivers & Dam 2.5 L, Grab sampling in 1L, LLE, 3x 15 mL Dcm, elution 2x (10) mL Dcm μm) 2003 Water Winchester bottles, 5 mL anhydrous Na2SO4 71 – 101s

of H2SO4 15 OCPs

Egypt GC-ECD Grab sampling (Ekman Fractionation by alumina: silica open column Alexandria chromatography. 20 g of activated silica gel was DB-5 bonded phase Sediment dredge), clean Harbour polyethylene bag, stored slurry pecked in DCM over 10 g activated alumina, Barakat 10 g, Soxhlet, Dcm 30 m x 0.25 mm I.D. fused 0.25 ng/g NI in pre-combusted amber activated Cu pellets (top), Dcm replaced by PENT 2002 OCPs silica column with a 0.25 mm glass jar with Teflon by elution, elution (pesticide/PCB fraction) with 200 mL of PENT/Dcm (1:1) PCBs lined lids, stored in the

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dark at -20 °C, freeze dried

PCBs & acid-stable pesticides: 25 mL cartridge, 3 g activated alumina, (45% w/w) impregnated silica, Burundi Lake 6 ml Hex. Tanganyika GC-μ ECD Bought from local Non-acid stable pesticides: Fish 2g, Soxhlet, 2 h, 6 mL HT-8 column (25 m x 0.22 mm Manirakiza market, stored at – 20 0.1 – 0.5 NI et al (1999) Hex/Ace (3:1) 25 mL cartridge, 2 g activated alumina, 2g silica, 2 x 0.25 2002 21 OCPs °C in PE vessels g florisil, 15% KOH methanolic solution (50%, v/w), μm)

12 PCBs 1 g Na2SO4 (top), 6 mL Hex. Elution 2x5 mL Hex/Dcm (3:1) for each.

1 L, SPE, 2 mL/min, 1 ml 30 % (v/v) methanol Grab sampling, 1L 5 g, Soxhlet, 200 mL Ghana water-bed sediments MeOH, 8h, extract in water, 1 mL distilled water, air-dried LOQ Sediment aluminium foil, evaporation to dryness, 1 80 - 110 Ashanti decanted, mixed, air- mL MeOH, diluted 1 mL 15 min. elution GC-ECD 0.01 – 2.5 dried water Region (FAO/IAEA 1997) DB-5 column Ntow 2001 Streams 1 L, SPE, 2 mL/min, 1 ml (30 m x 0.53 mm, 1.5 μm) 30 % (v/v) MeOH in water, LOQ 1 L glass bottle and Water 1 mL distilled water, air- - 85 - 94 7 OCPs stoppered dried 10 – 2500 ng/L 15 min, elution, 1.5 ml Hex

GC-ECD Automated GPC Namibia columns CP-Sil 2 and CP-Sil Bio beads S-X3 combined with an Autoprep 1002 8/20% C18 both (50 m x 0.25 Cape Cross 1 – 2 g blubber, MAE, 8 Seal system, ethyl acetate and CyHex (1:1, v:v), Silica mm x 0.25 μm) mL of ethyl acetate/ Vetter et al., blubber NI GPC eluate, 2 ml of isooctane, isooctane extract, NI > 75 cycloHexane (1:1, v:v), 5 1999 placed on 10 mm i.d. glass column, 3 g 9 OCPs (1997) min 30 sec deactivated silica gel, elution 60 mL n-Hex (OCPs) GC-MS 8 PCBs PCB/CTT group separation CP-Sil 2 Fused silica column (50 m x 0.25 mm x 0.25 μm)

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C-Certified Reference Material (CRM), s-Matrix spiked, S-Sediment, F-Fish, d.w. -dry weight, l.w.-lipid weight, OCPs = Organochlorinated Pesticides, PCBs = Polychlorobiphenyls, PAHs = Polyaromatic Hydrocarbons, Conc. = concentrated, h = Hours, MeOH = Methanol, DEE= diethyl Ether, Dcm = Dichloromethane, LLE = Liquid- Liquid Extraction, CyHex = CycloHexane, n-Pent = n-Pentane, n-Hex = n-Hex, Ace = Acetone, min = minute(s), SPE = Solid Phase Extraction, RSD = Relative standard deviation, MAE = Microwave assisted extraction, sec = seconds, GPC = gel-permeation chromatography, FID = Flame Ionization detector, FLD = Fluorescence detector, NI = Not indicated, PE = Polyethylene

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Appendices

Appendix 12. Questionnaire issued to pesticide retailers or sellers in the Kumba market and farmers in Lake Barombi watershed

329

Appendices

330

Appendices

Appendix 13. Calibration curves of some PAH compounds

1200 y = 2,3887E-02x + 1,9548E+01 y = 5,3965E-03x + 1,1776E+01 R² = 9,9843E-01 R² = 9,9871E-01

L) 1000 μ 800 (pg/ 600

400

200 Concentration Concentration

0 0 50000 100000 150000 200000 Peak surface area ace229 ace320

1200 y = 9,2138E-05x + 4,4893E+00 y = 4,2061E-05x + 5,1048E+00 R² = 9,9887E-01 R² = 9,9906E-01 1000 L) μ 800 (pg/ 600 y = 1,3103E-05x + 2,4988E+00 R² = 9,9939E-01 400

Concentration Concentration 200

0 0 20000000 40000000 60000000 80000000 100000000 Peak surface area Phenanthrene Anthracene Fluoranthene

1200 y = 3,7411E-05x + 4,8780E+00 y = 2,4759E-05x + 4,5573E+00 R² = 9,9897E-01 R² = 9,9903E-01 1000 L) μ 800 y = 2,0187E-05x + 3,8005E+00 R² = 9,9919E-01 600

400

Concentration (pg/ Concentration 200

0 0 10000000 20000000 30000000 40000000 50000000 60000000 Peak surface area Pyrene Benzo(a)Anthracene Chrysene

331

Appendices

Appendix 13. Calibration curves of some PAH compounds

1200 y = 3,0428E-05x + 6,2492E+00 R² = 9,9888E-01 1000

800 y = 9,2253E-06x + 5,1246E+00 R² = 9,9892E-01 600 y = 6,2019E-06x + 5,1793E+00 400

Concentration R² = 9,9895E-01

200

0 0 50000000 100000000 150000000 200000000 Peak surface area Benzo(b)Fluoranthene Benzo(k)Fluoranthene Benzo(a)Pyrene

1200 y = 7,8427E-05x + 8,5551E+00 y = 2,6619E-05x + 4,5719E+00 R² = 9,9873E-01 R² = 9,9916E-01 1000

800

600 y = 2,6940E-05x + 6,2042E+00 400 R² = 9,9889E-01 Concentration 200

0 0 10000000 20000000 30000000 40000000 Aire

diBenzo(ah)Anthracene Benzo(ghi)Perylene Indeno(1,2,3-cd)Pyrene

332

Appendices

Appendix 14. Calibration curves of some PCB compounds

5 y = 0,0494x - 0,0429 R² = 0,9992 4

1 Peak area PCB28/PCB116D5 0 0 20 40 60 80 100

PCB28 Linéaire (PCB28)

2,5 y = 0,0245x - 0,022 R² = 0,9974 2

1,5 PCB101/PCB116D5 1

0,5 Peak area 0 0 20 40 60 80 100

PCB101 Linéaire (PCB101)

333

Appendices

2 1,8 y = 0,0175x - 0,0195 1,6 R² = 0,9973 1,4 1,2 1 0,8 PCB153/PCB116D5 0,6 0,4 0,2

Peak area 0 0 20 40 60 80 100

PCB153 Linéaire (PCB153)

Appendix 14. Calibration curves of some PCB compounds

1,6 1,4 y = 0,015x - 0,013 R² = 0,9972 1,2 1 0,8

PCB138/PCB116D5 0,6 0,4 0,2 Peak area 0 0 20 40 60 80 100 120

PCB138 Linéaire (PCB138)

1,2 y = 0,0105x - 0,0028 1 R² = 0,9969

0,8

0,6 PCB180/PCB116D5 0,4

0,2 Peak area 0 0 20 40 60 80 100 120

PCB180 Linéaire (PCB180)

334

Appendices

Appendix 15 a) and b) Some pesticides sold in the Kumba Market

335

Appendices

Appendix 16 a), b), c). Packages of used pesticides dumped in farms within the Barombi Mbo Watershed and d) well close to pesticide dump

a b

336

Appendices

c d

Appendix 17. Extraction of mangrove peat for construction (top left and right), open burning of household waste in an illegal landfill (bottom left) and incinerator for hospital waste at laquintinie hospital in Douala (bottom right)

Source : (MINHDU, 2014)

337

Appendices

Source : (MINEPDED, 2012)

Appendix 18. Effluent discharge around the Douala airport zone

Source: (MINHDU, 2014)

Appendix 19. Identification of OCPs in SIM mode from a standard solution at 100 pg/uL

(continued)

338

Appendices

Appendix 19. Identification of OCPs in SIM mode from a standard solution at 100 pg/uL

339

Appendices

Appendix 20. dentification of PCBs in SIM mode from a standard solution at 100 pg/uL

Appendix 15. Identification of PAHs from a standard solution at 100 pg/uL

340

Appendices

Appendix 19a. List of banned active ingredients in Cameroon

341

Appendices

Appendix 19b. List of banned active ingredients in Cameroon

342

Appendices

Appendix 19c. List of authorized pesticides in Cameroon

343

Appendices c

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Abstract

Persistent Organic Pollutants (POPs) are compounds that are highly toxic to living organisms, persistent in the environment, undergo long distance transport and accumulate in organic-rich phases (sediments and fatty tissues). Given the vulnerability of aquatic ecosystems to pollution and scarcity of data on POPs in Africa and, this thesis examines the level of Organochlorinated Pesticides (OCPs), Polychlorobiphenyls (PCBs) and Polyaromatic hydrocarbons (PAHs) in the Lake Barombi Watershed (LBW) and Wouri Estuary Mangrove (WEM) in Cameroon. In LBW, OCPs detected were endosulfan, hexachlorocyclohexane (HCHs), dieldrin and aldrin. No OCPs were detected in water and the most frequently detected were HCHs with higher levels in soil than sediments (stream and lake). The α/γ-HCH ratio indicated recent use of lindane. The presence of OCPs is attributed to agriculture and fishing. PAH levels were higher in lake sediments than other samples. Pyrolytic sources of PAHs were predominant specifically combustion of grass, wood or coal. Sediment Quality Guidelines (SQGs) indicated low ecological risks to aquatic life. In the WEM, PAHs levels in sediments were more abundant than Chlorinated Pesticides (CLPs) and PCBs. The most abundant CLPs were endosulfan, alachlor, heptachlor, lindane (γ-HCH) and DDT for which metabolites pattern revealed recent use. PAHs ratios showed a predominant pyrolytic input. The presence of POPs in the WEM is mainly attributed to disease vector control (malaria), municipal waste dumps, industrial emissions and effluents, open burning of wastes, petroleum exploitation and harbour activities. SQGs implied low to moderate predictive biological toxicity.

Keywords: Persistent Organic Pollutants, mangrove, lake, sediments, Africa, Cameroon

Résumé Les Polluants Organiques Persistants (POPs) sont des composés hautement toxiques, persistants dans l'environnement, une capacité de transport à grande distance et accumulation sur les phases riches en matière organique (sédiments et tissues graisseux). Compte tenu de la la vulnérabilité des écosystèmes aquatiques aux pollutions et la rareté des données sur les POPs en Afrique, cette thèse examine les niveaux de Pesticides organochlorés (POCs), Polychlorobiphényles (PCBs) et Hydrocarbures Polyaromatiques (HAPs) dans le bassin versant du Lac Barombi (LBW) et la Mangrove estuaire du Wouri (MEW). Dans le LBW, les POCs détectés étaient, l’endosulfan, l‘hexachlorocyclohexanes (HCHs), le dieldrine and l’aldrine. Aucun POCs n'a été détecté dans les eaux et les plus fréquemment détectés étaient les HCHs avec des teneurs plus élevées dans les sols par rapport au sédiments (ruisseaux et Lac). Le ratio α/γ-HCH ont indiqué l’utilisation récente du lindane (γ-HCH). La présence des OCPs a été attribué à l'agriculture et la pêche. Les teneurs en PAHs était plus élevé dans les sédiments lacustres que les autres échantillons. Les sources pyrolytiques de HAPs étaient dominantes plus spécifiquement la combustion d’herbes, du bois ou le charbon. Les recommandations pour la qualité des sédiments (SQGs) un faible risque de toxicité sur les organismes aquatiques. Dans les MEW, les teneurs en HAPs dans les sédiments étaient 10 fois plus élevés que les PCBs et Pesticides Chlorés (PCLs). Les PCLs les plus abondant étaient l’endosulfan, l’alachlor, l’heptachlor, le lindane (γ-HCH) et le DDT pour lesquels les métabolites indiquent une utilization récentes. La contribution des HAPs pyrolytique était dominante. La présence des POPs dans la MEW a été attribué à la lutte anti-vectorielle (paludisme), les décharges municipales, effluents et émissions industrielles, brûlage à ciel ouvert des déchets, l'exploitation pétrolière et activités portuaires

Mots clés : Polluants Organiques Persistants, mangrove, lake, sediments, Afrique, Cameroun

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