DETERMINATION OF BIOCONCENTRATION POTENTIAL OF SELECTED PHARMACEUTICALS IN FATHEAD MINNOW, Pimephales promelas, AND CHANNEL CATFISH, Ictalurus punctatus Gopinath C. Nallani, B.Sc., M.Sc. Dissertation Prepared for the Degree of DOCTOR OF PHILOSOPHY UNIVERSITY OF NORTH TEXAS December 2010 APPROVED: Barney J. Venables, Major Professor Duane B. Huggett, Committee Member Edward M. Dzialowski, Committee Member Aaron P. Roberts, Committee Member Steven J. Wolverton, Committee Member Arthur J. Goven, Chair of the Department of Biological Sciences James D. Meernik, Acting Dean of the Robert B. Toulouse School of Graduate Studies Nallani, Gopinath C., Determination of bioconcentration potential of selected pharmaceuticals in fathead minnow, Pimephales promelas, and channel catfish, Ictalurus punctatus. Doctor of Philosophy (Biology), December, 2010, 233 pp, 32 tables, 55 illustrations, references, 195 titles. The primary objective of this work was to determine the tissue-specific bioconcentration factors (BCFs) of the selected pharmaceuticals: norethindrone (NET), ibuprofen (IBU), verapamil (VER), clozapine (CLZ) and fenofibrate (FFB) in two freshwater fishes: fathead minnow and channel catfish. BCF tests on fathead followed the standard OECD 42-day test while a 14-day abridged test design was used in catfish exposures. Additional objectives included a) comparing the measured BCFs to the US EPA’s BCFWIN model predicted values, b) comparing the BCF results from the standard and reduced tests, and c) prediction of chronic risk of the pharmaceuticals in fish using the human therapeutic plasma concentrations. The results indicated that all the pharmaceuticals, except IBU, have the potential for accumulation in fish. Estimated BCFs for NET, VER and FFB may not be significant in view of the current regulatory trigger level (BCF ≥ 2000); however, CLZ’s BCF in the liver had approached the criterion level. Significant differences were noticed in the tissue-specific uptake levels of the pharmaceuticals with the following general trend: (liver/kidney) > (gill/brain) > (heart/muscle) > plasma. IBU uptake was highest in the plasma. When compared to the measured BCFs, predicted values for NET, IBU, VER and FFB were slightly overestimated but did not differ largely. However, the measured BCF of CLZ in the liver was approximately two-orders of magnitude higher than the predicted level. The tissue- BCFs for the two species were not widely different indicating the potential usefulness of the reduced BCF test. Comparison of fish and human plasma levels indicated that NET, CLZ and VER have the potential to cause chronic effects in fish. Copyright 2010 by Gopinath C. Nallani ii ACKNOWLEDGMENTS The dissertation work outlined here was sponsored by Pfizer Global Research and Development and the financial assistance for this project is appreciated. I would like to express my sincerest gratitude to my major advisor Dr. Venables for his encouragement, guidance, support and extreme patience throughout my graduate career at UNT. He is undoubtedly the most influential person in my academic career and I have been fortunate to work under his advisement. I want to thank Dr. Huggett for his endless enthusiasm, supervision and professional help. I am particularly thankful for his insights and discussions during the course of the project. Many thanks to my other committee members Drs. Dzialowski, Roberts and Wolverton for instilling interdisciplinary thinking that helped broaden my scientific perspective. I thank all the fellow graduate students especially Peter and Regina for their valuable contribution to the successful completion this work. I appreciate the timely services provided by the Dept. of Biological Sciences and the IAS administrative staff. I thank the Graduate School for the financial assistance in the form of TA as well as the travel awards that helped presenting my research in the national and regional conferences. I extend my gratitude to UNT for the rewarding and fulfilling experience. I am particularly indebted to my family for the unflinching faith on my abilities and their constant support and encouragement. Special thanks to my Mom, Vijayalaxmi for her love and affection. Thanks to my brothers, sister-in-law and aunt. Finally, I thank my wife Srilaxmi for her love, caring, understanding and patience throughout the course of the doctoral work. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................................................................................................. iii LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ...................................................................................................................... xii LIST OF ABBREVIATIONS ...................................................................................................... xix CHAPTER 1 INTRODUCTION .....................................................................................................1 1.1 Aims and Objectives ................................................................................................4 1.2 Dissertation Structure...............................................................................................5 CHAPTER 2 PHARMACEUTICALS: EMERGING ENVIRONMENTAL CONTAMINANTS ..............................................................................................................................................7 2.1 Usage, Sources and Occurrence ...............................................................................7 2.1.1 Usage of Pharmaceuticals ............................................................................7 2.1.2 Sources .........................................................................................................9 2.1.3 Occurrence .................................................................................................11 2.2 Fate and Transport .................................................................................................14 2.2.1 Biotic Transformations ..............................................................................15 2.2.2 Abiotic Transformations ............................................................................17 2.3 Environmental Significance and Ecotoxicity .........................................................19 2.3.1 Significance................................................................................................19 2.3.2 Ecotoxicity .................................................................................................21 2.4 Environmental Risk Assessment (ERA) ................................................................29 2.4.1 EMEA Approach .......................................................................................30 2.4.2 US EPA/FDA Approach ............................................................................33 2.4.3 Environment Canada (EC) Approach ........................................................34 2.4.4 Concerns over the Current ERA Approaches ............................................35 CHAPTER 3 BIOCONCENTRATION ........................................................................................36 3.1 Terminology and Regulatory Importance ..............................................................36 3.1.1 Bioconcentration ........................................................................................36 iv 3.1.2 Bioaccumulation ........................................................................................37 3.1.3 Biomagnification........................................................................................37 3.1.4 Regulatory Importance of BCF/BAF Assessments ...................................38 3.2 BCF Models ...........................................................................................................40 3.3 Prioritization of Pharmaceuticals ...........................................................................43 3.3.1 Prioritization using Mammalian Data ........................................................44 3.3.2 Fish Plasma Model (FPM) .........................................................................45 3.4 Bioconcentration of Pharmaceuticals ....................................................................49 CHAPTER 4 MATERIALS AND METHODS ............................................................................51 4.1 Selection of Pharmaceuticals and Test Species .....................................................51 4.1.1 Pharmaceuticals .........................................................................................51 4.1.2 Selection of Test Species ...........................................................................52 4.2 Chemicals and Reagents ........................................................................................53 4.3 Fish Exposures .......................................................................................................54 4.3.1 Animals and Housing .................................................................................54 4.3.2 Flow-Through Exposure System ...............................................................54 4.4 BCF Experiments ...................................................................................................56 4.4.1 Fathead Tests .............................................................................................57 4.4.2 Catfish Tests...............................................................................................57