Growth, Survivability, and Reproductive Effects of Pulse-Dosed Endosulfan on Jordanella floridae (Florida flagfish) Over One Complete Life-cycle. by Lindsay Alexandra Beyger A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Masters in Applied Bioscience in The Faculty of Science University of Ontario Institute of Technology August, 2009 © Lindsay Beyger, 2009 Abstract Endosulfan is a commonly used organochlorine in Durham Region, Ontario Canada which has known toxic effects on non-target organisms including fish. This research investigated the effects of endosulfan on Florida flagfish (Jordanella floridae), using both continuous and pulse-exposure. The 96 hour continuous exposure LC50 in larval flagfish was 4.35 µg/L; sub-lethal observations included hyperactivity, convulsions, and some axis malformation. The effects of a 4 hour endosulfan pulse-exposure on 7-8 day-old larval growth, reproduction, and survivability were investigated over one full life-cycle. The 4 hour pulse-exposure LC50 value for larval flagfish was 49.7 µg/L; there were no growth or reproductive effects of endosulfan pulse-exposure up to the highest exposure concentration of 10 µg/L. Thus, the life-cycle 4-h pulse-exposure no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were 3.2 and 10 µg/L endosulfan, respectively, due to significantly higher mortality. Keywords: Endosulfan, Life-cycle, Reproduction, Flagfish ii Acknowledgements The contributions of many people’s advice, support, and friendship have been an invaluable tool in the completion of this thesis: Dr. Douglas Holdway, my supervisor, for his endless supply of knowledge, his continual support and guidance, and his constant motivation from beginning to end of this project. Dr. Rodrigo Orrego for his insurmountable knowledge and advice, his priceless hands on training, and his great friendship. John Guchardi for his unparalleled technical and logistical assistance, and his constant support and friendship. Mathumai Ganeshakumar and Jeff Armour for their support, friendship, and endless supply of good times. Rachelle Krause for her perpetual wisdom in maintaining and breeding flagfish. My family and Adam for their unconditional support, encouragement, and love. iii Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii Index to Figures ................................................................................................................ vii Index to Tables ................................................................................................................. viii List of Abbreviations .......................................................................................................... x 1.0 Introduction ................................................................................................................. 1 1.1 Aims .......................................................................................................................... 2 2.0 Literature Review ....................................................................................................... 3 2.1 Toxicity Testing ........................................................................................................ 3 2.2 The Use of Pulse-exposure and Full life-cycle Studies ............................................ 4 2.3 Fish Physiology ......................................................................................................... 5 2.3.1 Basic Uptake and Elimination Physiology......................................................... 5 2.3.2 Basic Reproductive Physiology ......................................................................... 6 2.4 Jordanella floridae (Florida Flagfish) ...................................................................... 7 2.4.1 Classification, Habitat, and Characteristics ....................................................... 7 2.4.2 Success / Validity as Test Species ..................................................................... 8 2.4.3 Behavioural Characterization & Breeding Patterns ........................................... 9 2.4.4 Examples of Use & Sensitivity ........................................................................ 11 2.5 Endosulfan .............................................................................................................. 11 2.5.1 Organochlorine Insecticides ............................................................................. 11 2.5.2 General Use - Use in Ontario ........................................................................... 13 2.5.3 Endosulfan Fate in Environment ..................................................................... 14 2.5.3.1 Water ......................................................................................................... 14 2.5.3.2 Soil ............................................................................................................ 15 2.5.3.3 Air ............................................................................................................. 15 2.5.4 Metabolites / Degradation Pathway ................................................................. 15 2.5.5 Endosulfan Mode of Action ............................................................................. 16 2.5.6 Endocrine Disruption ....................................................................................... 17 2.6 Toxicity of Endosulfan to Fish ............................................................................... 20 2.6.1 Acute Toxicity ................................................................................................. 20 iv 2.6.2 Chronic Toxicity .............................................................................................. 21 2.7 Rationale ................................................................................................................. 22 3.0 Materials and Methods ............................................................................................. 23 3.1 Laboratory Fish ....................................................................................................... 23 3.2 Chemicals ................................................................................................................ 23 3.3 Feed ......................................................................................................................... 23 3.4 Water Parameters .................................................................................................... 24 3.4.1 Nitrate, Nitrite, Chlorine, Alkalinity, Hardness ............................................... 24 3.4.2 Dissolved Oxygen ............................................................................................ 24 3.4.3 Temperature, pH, and Conductivity ................................................................. 25 3.5 Breeding Tank Set-up ............................................................................................. 26 3.6 Egg Collection ........................................................................................................ 26 3.7 Larval Rearing ........................................................................................................ 26 3.8 Glassware Cleaning ................................................................................................ 27 3.9 Water Analysis ........................................................................................................ 27 3.10 Statistical Analysis ................................................................................................ 27 3.11 Experiment #1: 96-h Continuous Exposure LC50 Study ....................................... 28 3.12 Experiment #2: 2, 4, 8-h Pulse-exposure Study .................................................... 28 3.13 Experiment #3: 4-h Pulse-exposure LC50 Study ................................................... 29 3.14 Experiment #4: 4-h Pulse-exposure Sub-lethal Study .......................................... 29 3.15 Experiment #5: Full Life-Cycle Study .................................................................. 30 3.15.1 Egg Collection ............................................................................................... 30 3.15.2 Pulse-exposure and First Growth Measurement ............................................ 30 3.15.3 Transfer to 10 L Aquaria and Second Growth Measurement ........................ 31 3.15.4 Transfer to 70 L Aquaria ................................................................................ 31 3.15.5 Thinning, Egg Collection, and Larval Rearing .............................................. 32 3.15.6 Dissections ..................................................................................................... 33 4.0 Results ........................................................................................................................ 34 4.1 96-h Continuous exposure LC50 Study ................................................................... 34 4.1.1 Abiotic Factors ................................................................................................. 34 4.1.2 Toxicity Values ................................................................................................ 34 v 4.2 2, 4, 8-h Pulse-exposure