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University Microfilms International 300 North Zeeb Road Ann Arbor, Michigan 48106 USA St. John’s Road, Tyler's Green High Wycombe, Bucks, England HP10 8HR 7619507 ESTENIK, JOHN FRANCIS TOXICITY STUDIES OF INSECTICIDES WITH LABORATORY COLONIES OF MIDGE LARVAE, CHIRONOMUS RIPARIUS (= CHIRONOMUS THUMMI), AND MOSOUITO LARVAE, AEDES AEGYPTI, AND THE IN VITRO CHARACTERIZATION OF ALDRIN EPOXIDATION IN THE MIDGE. THE OHIO STATE UNIVERSITY, P H #D,, 1978 TOXICITY STUDIES OF INSECTICIDES WITH LABORATORY COLONIES OF MIDGE LARVAE, CHIRONOMUS RIPARIUS (= CHIRONOMUS THUMMI), AND MOSQUITO LARVAE, AEDES AEGYPTI, AND THE IN VITRO CHARACTERIZATION OF ALDRIN EPOXIDATION IN THE MIDGE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By John F. Estenik, B. S., M. S The Ohio State University 1978 Reading Committee: L. S. Putnam N. Wilson Britt '/ Co-Advisors” William J. Collins Department of Zoology ACKNOWLEDGEMENTS I would like to thank my co-advisors, Drs. N. W. Britt and William J. Collins. Dr. Britt, whose help allowed me the freedom to define my graduate program, was appreciated. Dr. Collin's suggestions, interest, and help throughout all aspects of this research project and manuscript preparation is deeply appreciated. I would like to thank Dr. L. S. Putnam for helping me return to graduate school and making this all pos sible. I would like to thank Drs. W. B. Parrish and S. I. Lustick, Department of Zoology, Dr. IV. Foster, Department of Entomology, and Drs. G. S. Serif, D. H. Ives, and Means, Department of Biochemistry, for the use of laboratory space and instrumentation during various aspects of this research project. I would also like to thank members of the Zoology and Entomology Departments who helped and encouraged me throughout my studies. Finally, I would like to thank my parents and my grandmother, Mrs. F. Rose, for their help, encouragement, and financial aid throughout piy graduate studies. VITA November 11, 1940 Born - Canton, Ohio 1962 B. S., John Carroll University, Cleveland, Ohio 1964 M. S., John Carroll University, Cleveland, Ohio 1964-1967 Research Assistant, Case Western Reserve University, Department of Radiation Biology, Cleveland, Ohio 1967-1970 Instructor, Cuyahoga Community College, Department of Biology, Cleveland, Ohio 1971-1975 Teaching Associate, Ohio State University, Department of Zoology 1975-1976 Teaching Associate, Ohio State University, School of Nursing PUBLICATIONS Estenik, J._F. and P. F. Federle, "The Susceptibility of an Adult Damselfly Ischnura verticalis to Selected Insecticides". The Ohio Jour, of Science, March, 19 75. FIELDS OF STUDY Major Field: Aquatic Biology - Toxicology TABLE OF CONTENTS Page ACKNOWLEDGEMENTS......................................... ii VITA .................................................. iii LIST OF TABLES...............................................vii LIST OF F I G U R E S ......................................... ix I. INTRODUCTION AND LITERATURE REVIEW .............. I A. The Fate of Pesticides in an Aquatic Organism ....................................... I B. The Biological Oxidation of Insecticides - 5 C. Chironomus riparius Meigen as a Test O r g a n i s m ................................... 10 D. Objectives .................................. 14 II. METHODS AND MATERIALS.............................. 16 A. Insect Culture ...................... 16 B. Chemicals.................................... 17 C. Immersion Toxicity: Assay Procedure, Criteria of Response and Data Analysis . 18 D. Midge and Mosquito Preparation for All Experiments Except Immersion Toxicity A s s a y s ...................................... 21 E. Homogenizing Procedure ...................... 21 F. Extraction Procedure ......................... 22 i v TABLE OF CONTENTS (CONTINUED) Page G. Chromatographic Analysis................. 23 H. Absorptive Uptake and Insecticide Loss. 24 I. Assay of Whole Body Homogenate for Aldrin Epoxidation ........................ 26 J. Microsome Preparation ...................... 29 K. Assay of Subcellular Fractions for Aldrin Epoxidation ........................... 30 L. Protein Analysis........................... 31 III. R E S U L T S ............................................. 32 A. Toxicity Assays ............................. 32 B. Absorptive Uptake and Insecticide Loss. 41 C. Assay of Whole Body Homogenate for Aldrin Epoxidation........................... 71 D. Assay of Subcellular Fractions for Aldrin Epoxidation........................ 90 IV. DISCUSSION AND CONCLUSIONS...................... 93 A. Toxicity Assays . ........................ 95 B. Absorptive Uptake and Insecticide Loss. 107 C. Assay of lVhole Body Homogenates for Aldrin Epoxidation............................. 115 D. Assay of Subcellular Fractions for Aldrin Epoxidation............................. 12 7 E. Conclusions.................................... 134 v TABLE OF CONTENTS (CONTINUED) Page LITERATURE CITED............................................ 138 vi LIST OF TABLES Table Page 1 Toxicity of Selected Insecticides With and Without Synergist to Fourth Instar Larval Chironomus riparius.................................. 33 2 Toxicity of Selected Insecticides With and Without Synergist to Late Third and Fourth Instar Larval Aedes aegypti.........................3? 3 The Absorption of Selected Insecticides by C. riparius Larvae and P u p a e ...................... 43 4 The Absorption of Selected Insecticides by A. aegypti Larvae and Pupae........................ 45 5 Depuration of Dieldrin by C. riparius Larvae . 72 6 The Uptake and Metabolism of Aldrin by Ligatured riparius Larvae 7 3 7 Aldrin Epoxidase Requirements of C. riparius Whole Body Homogenates............................. 74 8 The Effect of Substrate Concentration (Aldrin) and BSA Upon in vitro Aldrin Epoxidase of C. riparius Whole Body Homogenates ......................................... g7 9 Aldrin Epoxidase Requirements of C. riparius Whole Body Homogenates Under Optimum Conditions........................................... 89 10 Effect of FMN, FAD, and PBO Upon in^ vitro C. riparius Aldrin Epoxidase Whole Body Homogenates........................................... 91 11 Subcellular Localization of Aldrin Epoxidase Activity in C. riparius Larvae ................... 92 via Li ST OF TABLES (CONTINUED) Table Page 12 Reported 24 hr LCs q 's (ppb) of Susceptible Laboratory Strains of A. aegypti to Representative Insecticides........................ 98 13 Comparative Rates of Aldrin Epoxidation by Insect Microsomes (pmoles of Dieldrin/min/ mg protein).......................................... 131 LIST OF FIGURES Figure Page 1 The Fate of a Xenobiotic in an Aquatic Organism.................................. 3 2 The Metabolism of a Xenobiotic by an Aquatic Organism.................................. .. 4 3 * The Effect of Exposure Time on in vivo Insecticide Absorption and Metabolism by C_. riparius Larvae Immersed in 10 ppb Aldrin . 48 4 The Effect of Exposure Time on ill vivo Insecticide Absorption and Metabolism by A. aegypti Larvae Immersed in 10 ppb Aldrin . 51 5 The Effect of Exposure Time on the in vivo Hourly Rate of Insecticide Absorption and Metabolism/£. riparius Larva Immersed in 10 ppb Aldrin .......................................... 53 6 The Effect of Exposure Time on the in vivo Hourly Rate of Insecticide Absorption and Metabolism/A. aegypti Larva Immersed in 10 ppb Aldrin .......................................... 55 7 Dieldrin Production as a Function of Total Aldrin Absorbed for Larvae Immersed in 10 ppb A l d r i n ...........................................