MOLECULAR BIOMARKER STUDIES ON ECOTOXICOLOGICAL IMPACT OF POLLUTANTS ON THE MARINE GASTROPODS ALONG THE GOA COAST JACKY BHAGAT A Thesis submitted to Goa University for the Award of the Degree of DOCTOR OF PHILOSOPHY in MARINE SCIENCES Research Guide: Dr. B. S. Ingole, Professor & Chief Scientist, CSIR-National Institute of Oceanography Dona Paula, Goa - 403004 Co-guide: Dr. A. Sarkar Former Senior Principal Scientist CSIR-National Institute of Oceanography Dona Paula, Goa - 403004 Place of Work: CSIR-National Institute of Oceanography Dona Paula, Goa - 403004 Details of Ph.D. Registration: No. 201109255, 15 November, 2011 I | Page Statement As required under the University ordinance OB-9.9 (v-vi), I state that this thesis entitled "Molecular Biomarker Studies On Ecotoxicological Impact Of Pollutants On The Marine Gastropods Along The Goa Coast" is my original contribution and it has not been submitted on any previous occasion. The literature related to the problem investigated has been cited. Due acknowledgements have been made wherever facilities and suggestions have been availed of. JACKY BHAGAT CSIR-National Institute of Oceanography, Goa. 3rd April, 2017 II | Page Certificate This is to certify that the thesis entitled "Molecular Biomarker Studies On Ecotoxicological Impact Of Pollutants On The Marine Gastropods Along The Goa Coast" submitted by Shri Jacky Bhagat for the award of the degree of Doctor of Philosophy in Marine Science is based on original studies carried out by him under my supervision. The thesis or any part thereof has not been previously submitted for any other degree or diploma in any institution. Dr. B. S. Ingole Research Supervisor CSIR-National Institute of Oceanography, Dona Paula, Goa 3rd April, 2017 III | Page List of Papers Published Papers from thesis: 1. Bhagat J*, Sarkar A, Vashistha D, Vipin Singh, Laxmi Raiker, Ingole BS 2017. Integrated Biomarker Responses In Marine Gastropod (Nerita Chamaeleon) From Goa Coast, India. Invertebrate Survival Journal 14, 18-31. IF=0.754 (http://www.isj.unimo.it/articoli/ISJ452.pdf) 2. Bhagat J*, Ingole BS, Singh N. 2016. Glutathione S-transferase, catalase, superoxide dismutase, glutathione peroxidase, and lipid peroxidation as a biomarker of oxidative stress in snails: A review. Invertebrate Survival Journal 13, 333-349. IF=0.754 (http://www.isj.unimo.it/articoli/ISJ444.pdf) 3. Bhagat J*, Sarkar A, Ingole BS 2016. DNA damage and oxidative stress in marine gastropod, Morula granulata exposed to phenanthrene. Water air soil pollution (2016), 227:114 IF=1.551 (https://link.springer.com/article/10.1007/s11270-016-2815-1) 4. Bhagat J*, Ingole BS 2015. Genotoxic potency of mercuric chloride in gill cells of marine gastropod Planaxis sulcatus using comet assay. Environmental science and pollution research 22 (14):10758-10768. IF=2.828 (http://www.sciencedirect.com/science/article/pii/S0147651314001638) 5. Sarkar A, Bhagat J*, Ingole BS, Rao DP, Markad VL 2015. Genotoxicity of cadmium chloride in marine gastropod, Nerita chamaeleon using comet assay and alkaline unwinding assay. Environmental Toxicology, 30 (2): 177-187. IF=3.197 (http://onlinelibrary.wiley.com/doi/10.1002/tox.21883/full) 6. Sarkar A*, Bhagat J, Sarker S 2014. Evaluation of impairment of DNA in marine gastropod, Morula granulata as a biomarker of marine pollution, Ecotoxicology and Environmental Safety 106, 253–261. IF=2.762. (http://www.sciencedirect.com/science/article/pii/S0147651314001638) 7. Bhagat J*, Ingole B, Sarkar A, Gunjikar M 2012. Measurement of DNA damage in Planaxis sulcatus as a biomarker of genotoxicity. The Ecoscan, 1, 01-04, 2012, 219-223. (http://theecoscan.in/journalpdf/spl2012_v1- 38%20jacky%20bhagat.pdf) IV | Page 8. Bhagat J*, Singh K, Rai P, Raizada G, Sarkar A 2012. Alkaline unwinding assay for the assessment of DNA damage in Planaxis sulcatus from Goa coast. Nebio, 3, 5, 34-36. (http://www.nebio.info/2012/12/nebio-special-issue-ebi- 2012.html) *corresponding author Submitted for Publications from thesis: • Bhagat J*, Ingole BS. Effects of benzo(k)fluoranthene, a polycyclic aromatic hydrocarbon, on DNA damage and oxidative stress in marine gastropod Morula granulata. Toxicological & Environmental Chemistry (under review, GTEC-2016-0617) • Bhagat J*, A Sarkar, Ingole BS. Seasonal and spatial variability of oxidative stress biomarkers in marine gastropod Nerita chamaeleon in relation to polycyclic aromatic hydrocarbon. Ecotoxicology and Environmental Safety (under review, EES S16 01650) Papers presented in International Conference/Symposium Bhagat J*, Ingole BS. Effects of benzo(k)fluoranthene, a polycyclic aromatic hydrocarbon, on DNA damage and oxidative stress in marine gastropod Morula granulata, IC3-2015. Bhagat J*, Sarkar A, Singh V, Raiker L, Ingole BS. Integrated biomarker responses in marine gastropod (Nerita chamaeleon) environmentally exposed to polycyclic aromatic hydrocarbons, IIOE-2015. Bhagat J, Ingole BS. Assessment of genotoxic potency of mercuric chloride in gill cells of marine gastropod Planaxis sulcatus using comet assay, presented at International Conference ICFA-2014. Bhagat J*, Ingole BS. Assessment of genotoxic damage and lipid peroxidation in marine gastropod Morula granulata exposed to phenanthrene, presented at 4th International Conference ICEES-2014. Bhagat J*, Ingole B, Sarkar A, Gunjikar M 2012. Measurement of DNA damage in Planaxis sulcatus as a biomarker of genotoxicity, ICAIECs-2012. V | Page Sarkar A, Bhagat J, Tegur PM* and Manu B, Application of Molecular Biomarker Technique for Assessment of the Impact of Genotoxic Contaminants in the Marine Environment, ICCTEM-2012. Bhagat J, Sarker G*, Rajgopal TV, Raizada G, Vashistha D and Sarkar A, Strategies for Prevention and Control of Water Pollution - An overview, Presented at International Conference on Ecotoxicological and Environmental Science (ICEES), Miramar Residency, Goa, India, 28-30 Nov, 2011 Sarkar A, Bhagat J* and Deepti Vashistha, Measurement of DNA Integrity In Morula granulata as a Biomarker of Genotoxicants along the Goa Coast, Presented at ICEES-2011. Papers presented in International Conference/Symposium Bhagat J, Singh K*, Rai P, Raizada G, Sarkar A, Alkaline unwinding assay for the assessment of DNA damage in Planaxis sulcatus from Goa coast, Presented at EBI-2012. Bhagat J*, Ingole BS, Sarkar A and Rao DP, Comet assay for the measurement of DNA damage in marine gastropods exposed to genobiotic pollutants, Presented at the EBI-2012. Bhagat J*, Sarkar A, Vashistha D, Mesquita A, Inhibition of AChE Activity in Marine Gastropod as a Biomarker of Coastal Pollution by Neurotoxic Contaminants, NCCIECM-2012. *presenting author VI | Page Acknowledgements I am deeply indebted to my thesis supervisor, Dr. B. S. Ingole for his encouragement and unwavering support in this work. Without his guidance and support, this thesis would not have been possible. I also thank him for believing in me, and for going the extra mile when it was necessary, in order to make this happen. I have greatly benefited from his knowledge and experience. I wish to express my profound thanks to Dr. Anupam Sarkar, for bringing me to this wonderful institute and acting as my research guide and then as a co-guide during my stay at CSIR-NIO. His positive criticism, suggestions and valuable comments has left everlasting impression on me. His initiative, constant support and help has lead to the successful completion of my present investigation. I would like to thank Dr. S. W. A. Naqwi, Director, CSIR-National Institute of Oceanography, Goa for providing me with all the necessary facilities to carry out my research work. The financial support by Department of Biotechnology, New Delhi in the form of Junior and Senior Research Fellowship is highly acknowledged. I also would like to thank CSIR for providing the infrastructure and the necessary facilities to conduct my research work. My heartfelt thanks to Dr. S. R. Shetye, Vice Chancellor, Goa University for giving me the opportunity to undergo the Ph.D. degree. I express my special thanks to Dr. R. Roy, Department of Zoology, Goa University and Dr. C. Mohandas, CSIR-NIO for their valuable suggestions given during my SRF assessment. My appreciation goes to Dr. S. S. Sawant, Scientist, CSIR-NIO for helping me in PAH analysis and Mithila Bhat for mercury analysis. I extend my sincere thanks to Dr. Ramaiah, Head of Department, BOD, Mrs. Analia Mesquita, Head of VII | Page Department, COD, Dr. C. U. Rivonkar, Head of Department of Marine Science, Goa University and Mr. D.P. Rao, Scientist, CSIR-NIO for their valuable suggestions and continuous help in furtherance of my research work. Many thanks to Mr. Prabhu, Mr. Madan, Mr. S.R. Sahu and Mr. Mithun at CSIR-NIO, Library for providing necessary support during my research work. I feel immense pleasure in expressing my special thanks to all the staffs from COD, BOD and ITG section, who directly or indirectly helped me during the completion of this work. I would also like to thank Nisha for helping me in thesis formatting and Haris, Afreen for all the suggestions in my thesis preparation, printing and binding. I would like to express my sincere appreciation to Vijay Markad for teaching me some of the important biomarker techniques. I would also like to thank my colleagues and friends, Deepti, Laxmi, Sutapa, Priyamvada, Moon, Geetika, Priyanka, Vipin, Manasi and everyone from benthos lab for assisting me in my studies and field work. I am also thankful to my roommate Vijay Rawat, Azraj Dahihande and Gobardhan Sahoo for their valuable friendship. No acknowledgement will be complete without expressing my gratitude towards my loving parents for their love and support in every walk of life. JACKY BHAGAT CSIR-National Institute of Oceanography, Goa. 3rd April, 2017 VIII | Page Table Of Contents STATEMENT II CERTIFICATE III LIST OF PAPERS IV ACKNOWLEDGEMENTS VI LIST OF TABLES XI LIST OF FIGURES XII ABBREVIATIONS XVII CHAPTER 1 - INTRODUCTION 1 CHAPTER 2 - REVIEW OF LITERATURE 5 2.1. Environment Pollutants 5 2.1.1. Polycyclic aromatic hydrocarbons (PAHs) 5 2.1.2.