TOXICANT INDUCED ANTIOXIDANT ACTIVITY IN OREOCHROMIS MOSSAMBICUS (Peters) Thesis submitted to the Cochin University of Science and Technology In partial fulfilment of the requirements for the degree of Doctor of Philosophy in Marine Biology Under the Faculty of Marine Science By KESAVAN K. DEPARTMENT OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY SCHOOL OF MARINE SCIENCES COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY KOCHI – 682 016 MARCH 2010 This is to certify that the thesis entitled “TOXICANT INDUCED ANTIOXIDANT ACTIVITY IN OREOCHROMIS MOSSAMBICUS (Peters)” is an authentic record of the research work carried out by Sri. Kesavan. K, under my scientific supervision and guidance in the Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Marine Biology of Cochin University of Science and Technology and that no part of the thesis has been presented before for the award of any other degree, diploma or associateship in any University. Prof. Dr. N. RAVINDRANATHA MENON Hon. Director and Emeritus Professor Centre for Integrated Management of Coastal Zones School of Marine Sciences Cochin University of Science and Technology Kochi - 682016 March 2010. Kochi – 682 016. Declaration I hereby declare that the thesis entitled “TOXICANT INDUCED ANTIOXIDANT ACTIVITY IN OREOCHROMIS MOSSAMBICUS (Peters)” is a genuine record of the research work done by me under the scientific guidance and supervision of Prof. Dr. N. Ravindranatha Menon, Hon. Director and Emeritus Professor, Centre for Integrated Management of Coastal Zones, School of Marine Sciences, Cochin University of Science and Technology, Kochi and that this has not previously formed the basis for the award of any degree, diploma or associateship in any University. KESAVAN K. Kochi – 16 March, 2010. Acknowledgements I wish to convey my ardent and humble sense of indebtedness to my Supervising Guide Prof. Dr. N.R. Menon, Emeritus Professor and Hon. Director, C- IMCOZ, School of Marine Sciences, Cochin University of Science and Technology for his untiring guidance, invaluable advices, intellectual contributions, critical suggestions and constant encouragement all through the tenure of my research work and preparation of the manuscript. Besides, the consideration and affection he has showered upon me during the course of my work is gratefully acknowledged; deep from my inner heart. I record my unfeigned obligations to Dr. N. Chandramohanakumar, Professor and Head, Department of Chemical Oceanography, School of Marine Sciences, CUSAT, for his whole hearted support during the concluding phase of my research work. I am thankful to Prof. Dr. K. Mohankumar, former Dean of Faculty of Marine Sciences and Prof. Dr. H.S. Ram Mohan, Director and Dean, School of Marine Sciences for their immense support during the tenure of my studies. I am grateful to Prof. Dr. Aneykutty Joseph, Head, Department of Marine Biology, Microbiology and Biochemistry, CUSAT, since she has contemplated a lot in the successful completion of my research work. I sincerely thank Dr. A.V. Saramma, former Head, Department of Marine Biology, Microbiology and Biochemistry, CUSAT, for her kind considerations especially when I was suffering from serious infirmity happened halfway between the progresses of my laboratory works. I would like to express my whole hearted gratitude to Prof. Dr. Babu Philip, Department of Marine Biology, Microbiology and Biochemistry, CUSAT, for his timely suggestions which helped me a lot for making modifications of laboratory experiments wherever necessary. Thanks are due to Dr. Rosamma Philip, Department of Marine Biology, Microbiology and Biochemistry, CUSAT for providing advanced instrumentation facilities. Sincere thanks are also extended to Dr. C. K. Radhakrishnan, retired Professor of the Department. I realize the value of unlimited scientific suggestions proposed by Dr. K.C. George, Senior Scientist, CMFRI, Kochi, in finalizing the histological studies. His valuable suggestions are thankfully acknowledged. The sisterly affection and backing imparted by Dr. N. Nandini Menon during the preliminary strides of this assignment is specially remembered with great fervor. I wish to sincerely thank Dr. K. Suresh for his invaluable recommendations when I was in the formative phase of this work. I am extremely glad to thankfully scribe and remember the precious help and back up extended by Dr. A. Biju, Principal, MES Asmabi College, P. Vemballur, during the finishing phases of the documentation. Sincere help and cooperation provided by the administrative staff of Dept. Marine Biology, Microbiology and Biochemistry is also acknowledged. The unfailing help and cooperation offered by my friends; Mr. Harisankar, H.S and Mr. K.B. Padmakumar are specially acknowledged. Besides, I wish to consign on record my deepest thanks to my friends; Ms. Anupama Nair and Ms. Bindya Bhargavan who have extended all possible support during the period of laboratory works. Sincere supports afforded by my dear friend Mr. K.U. Abdul Jaleel and my colleague Mr. Shibu A Nair are remembered with adoration. I would like to thank Mr. Anilkumar, P.R, Ms. Smitha Banu, Ms. Remya Varadarajan and Ms. Soja Louis who have helped me in some way or other during the tenure of this work. I wish to express my deep sense of gratitude to University Grants Commission for providing Junior Research Fellowship during the initial stages of the work and Teacher Fellowship when it approached its completion. I extend my sincere thanks to the Principal and Management of MES Asmabi College, P. Vemballur for sanctioning me to continue the Ph.D programme under the Faculty Improvement Programme of University Grants Commission. My deepest thanks are due to my Mother and Father for their love and blessings throughout the schedule of my research work. The fruitful conclusion of my studies would not have been a reality without the understanding, endurance, love and sacrifices from my beloved wife and my little daughter; Varsha K. Namboothiri. Most of all, the heavenly blessings from the Supreme Power has guarded and guided me all through the hardships when I was in pursuit of this assignment. Kesavan. K PREFACE The presence of a xenobiotic compound in an aquatic ecosystem does not, by itself, indicate injurious effects. Connections must be established between external levels of exposure, internal levels of tissue contamination and early adverse effects. Many of the hydrophobic organic compounds and their metabolites, which contaminate aquatic ecosystem, have yet to be identified and their impact on aquatic life has yet to be determined. Therefore, the exposure, fate and effects of chemical contaminants or pollutants in the aquatic ecosystem have been extensively studied by environmental toxicologists. Deleterious effects on populations are often difficult to detect in feral organisms since many of these effects tend to manifest only after prolonged cycles of ontogeny. Often the damages become conceivable very late in the ecosystem, thus rendering remedial actions pointless. In an environmental context, biomarkers offer promise as sensitive indicators demonstrating that toxicants have entered organisms, have been distributed in tissues, and are eliciting a toxic effect at critical targets. In this respect, it is also interesting to study the development and application of sensitive laboratory bioassays, based upon the responses of biomarkers. Bioassays offer many advantages for comparing the relative toxicity of specific chemicals or specific effluents. Histopathological techniques are a rapid, sensitive, reliable and comparatively inexpensive tool for the assessment of stress responses to xenobiotics. Cytological and histopathological alterations provide a direct record of trace effect. Evaluation of histopathological manifestations provides insight into the degree of stress, susceptibility and adaptive capability of the stressed organism. The route that the toxicant takes during its metabolisation often dictates the choice of organs for examining the effect of xenobiotics. In this context, gill, liver and the kidney of teleost are the best suited organ system to analyse both the biochemical and histopathological aberrations due to xenobiotic stress. Fish are of special concern because of the properties of aquatic environment and its relationship with native organisms. An aquatic environment is characterized by marked spatial and temporal heterogeneity of its physicochemical parameters and processes. Oreochromis mossambicus (Peters), the cichlid species known by the common name tilapia, forms the experimental animal in the present investigation because of its year round availability, adaptability to varied situations and sensitivity to toxicant exposures. Antioxidant defense studies on oxidative stress in fish open a number of research lines aimed at providing greater knowledge of fish physiology and toxicology. In addition, such studies would provide more precise information concerning the response of antioxidant defenses in different species under various circumstances as well as on the regulatory mechanisms of this response. Such future studies will, no doubt, benefit aspects related to fish farming and aquaculture practices. …....(cid:89)(cid:90)….... CONTENTS Page No List of Tables List of Figures Abbreviations & Symbols Chapter -1 INTRODUCTION............................................................................01 - 06 Chapter -2 REVIEW OF LITERATURE.............................................................07 - 31 Chapter -3 LETHAL TOXICITY STUDIES ......................................................32 - 41 3.1 Introduction 32 3.2 Material and methods 33 3.2.1 Test animals 33 3.2.2 Laboratory conditioning of test animals 33 3.2.3 Toxicants 34 3.2.3.1 Copper 35 3.2.3.2 Metacid 50 35 3.2.3.3 Malachite green 35 3.2.4 Studies on Lethal Toxicity 36 3.2.4.1 Lethal Toxic Responses to Copper 36 3.2.4.2 Lethal Toxic responses to Metacid 50 37 3.2.4.3 Response to malachite green 37 3.3 Results 38 3.4 Discussion 40 Chapter -4 SUBLETHAL TOXICITY................................................................42 - 162 4.1 Introduction 42 4.1.1 Oxidative Stress and Antioxidant Defense Mechanisms 43 4.1.2 Target Organs 49 4.2 Material and Methods 50 4.2.1 General protocol adopted for Sublethal Toxicity Studies. 50 4.2.2 Assays of Lipid peroxidation, Antioxidant Enzymes and Estimation of Protein. 52 4.2.2.1 Assay of Lipid peroxidation. 52 4.2.2.2 Assay of Antioxidant Enzymes 52 4.2.2.3 Estimation of Endogenous Antioxidants 55 4.2.3 Scheme Adopted for Histopathology 56 4.2.4 Statistical Analysis 58 4.3 Results 59 4.3.1 Effects of Exposure to Copper 59 4.3.2 Effects of Exposure to Malachite Green 88 4.3.3 Effects of Exposure to Metacid 50 114 4.4 Discussion 138 Chapter -5 EFFECT OF VITAMIN C ON TOXICITY.........................................162 - 269 5.1 Introduction 163 5.2 Material and Methods 164 5.3 Results 165 5.3.1 Effect of Vitamin C on Copper Toxicity. 165 5.3.2 Effect of Vitamin C on Malachite green toxicity. 193 5.3.3 Effect of Vitamin C on Toxicity of Metacid 50 226 5.4 Discussion 258 Chapter -6 SUMMARY AND CONCLUSION ..................................................270 - 274 BIBLIOGRAPHY ..........................................................i - xxvi APPENDIX …....(cid:89)(cid:90)….... Abbreviations & Symbols used in the thesis % percentage & and / per µg microgram µl microlitre µM micro molar CAT catalase CDNB 1-chloro-2,4-dinitrobenzene cm centimeter Cu copper e.g example EDTA ethylene diamine tetra acetic acid Fig. Figure. g gram GSH glutathione (reduced) HCl hydrochloric acid hrs. hours i.e that is I.U International Units kg kilogram l litre L-1 per litre M molar mal. green malachite green