CHRONIC TOXICITY OF PHTHALATES, BISPHENOL A AND A CANADIAN BOTTLED WATER STORED UNDER DIFFERENT LIGHT REGIMES USING THE CNIDARIAN HYDRA VIRIDISSIMA© By: Mathumai Ganeshakumar A Thesis Submitted in Partial Fulfillment of the Requirements of the Degree of Masters in Applied Bioscience In The Faculty of Science Applied Bioscience MSc. 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Abstract: There is general concern whether human exposure to selected Canadian bottled waters and the chemicals which may leach from them, could potentially cause deleterious effects. This research was designed to determine whether exposures to bottled water and plastic leachates caused toxicity to the freshwater Cnidarian Hydra viridissima (green hydra). Three chemicals used in the production of polycarbonate and polyethylene plastics, bisphenol A (BPA) and two phthalate esters: dibutyl phthalate (DBP) and bis (2- ethylhexyl) phthalate (DEHP) along with one type of commercial bottled water were investigated. One brand of bottled water was analyzed over four months (stored in light and dark conditions) along with lab water similarly stored in glass, polycarbonate and polyethylene bottles. Following 2, 4, 8 and 16 weeks in each of the two treatments, hydra bioassays were conducted. Chronic toxicity tests were also conducted on the two phthalates and BPA. The chronic toxicity tests showed that BPA caused effects on hydra morphology and population at low doses and DBP and DEHP both showed signs of hormesis. Key words: Bottled water, dibutyl phthalate, bisphenol A, bis(2-ethylhexyl) phthalate, hydra ii Acknowledgements: First I would like to thank my supervisor Dr. Douglas Holdway for all of his encouragement, enthusiasm, advice and support throughout these two years. Thank you to Dr. Rodrigo Orrego for your support, much needed help in the laboratory and guidance. Thank you to John Guchardi for your help and great technical advice. Thank you to Lindsay Beyger and Jeff Armour for their friendship and laughter and helping me get through the difficult times. And to my colleagues in the grad office for their support and jokes. Finally thank you to my parents and brothers for their endless support which allowed me to survive this year. iii Table of Contents: Abstract ........................................................................................................................................... ii Acknowledgements ....................................................................................................................... iii List of Tables ................................................................................................................................ vii List of Figures...............................................................................................................................vii List of Abbreviations.....................................................................................................................xi 1.0 - Introduction ...................................................................................................................... - 1 - 2.0 - Comprehensive Literature Review ................................................................................. - 3 - 2.1 - Issue of Bottled Water .................................................................................................... - 3 - 2.2 - Phthalates ........................................................................................................................ - 7 - 2.2.1 - Toxicity of Bis(2-ethylhexyl) phthalate (DEHP) ...................................................... - 9 - 2.2.2 – Toxicity of Dibutyl Phthalate (DBP) ..................................................................... - 12 - 2.3 - Toxicity of Bisphenol A (BPA) .................................................................................... - 16 - 2.4 - Toxicity of 4-chlorophenol ........................................................................................... - 20 - 2.5 - Regulation of DEHP, DBP & BPA in Canada ............................................................ - 24 - 2.6 – An Introduction to Hydra ............................................................................................. - 27 - 2.6.1 - Hydra Characteristics and Physiology .................................................................. - 27 - 2.6.2 - Hydra Classification .............................................................................................. - 34 - 2.6.4 - The Advantages of Using Hydra in Ecotoxicology ................................................ - 36 - 3.0 - Research Objectives ....................................................................................................... - 38 - 4.0 - Methods ........................................................................................................................... - 39 - 4.1 - Laboratory Culturing of Hydra ..................................................................................... - 39 - 4.2 – Field Experiment .......................................................................................................... - 39 - 4.2.1 - Field Experiment Bioassays .................................................................................. - 40 - 4.2.2 – Field Experiment Chemical Analysis – Solid Phase Extraction (SPE) (Part External Lab) ................................................................................................................................... - 42 - iv 4.23 - Chemical Analysis – Gas Chromatography-Mass Spectrometry (External Lab) ... - 45 - 4.3 – In-Lab Experiments ..................................................................................................... - 45 - 4.3.1 - Range Finders for Bis(2-ethylhexyl) phthalate (DEHP), Dibutyl phthalate (DBP) & Bisphenol A (BPA) ............................................................................................................ - 46 - 4.3.2 - Reference Toxicant Test using 4-chlorophenol ..................................................... - 47 - 4.3.3 - Chronic Toxicity Tests for Dibutyl phthalate (DBP), Bis (2-ethylhexyl) phthalate (DEHP) and Bisphenol A (BPA) ....................................................................................... - 49 - 4.4 – Statistical Analysis of Results ...................................................................................... - 50 - 5.0 Results ................................................................................................................................ - 52 - 5.1 – Hydra Culture Establishment ....................................................................................... - 52 - 5.3 – Field Experiment Results ............................................................................................. - 59 - 5.3.1 – Field Experiment Bioassays .................................................................................. - 59 - 5.3.2 –Chemical Analysis Results (External Lab): ........................................................... - 94 - 5.4 – In Lab Experiment Results: ......................................................................................... - 96 - 5.4.1 – Range Finders: ...................................................................................................... - 96 - 5.4.2 – Bis (2-ethylhexyl) phthalate (DEHP) Chronic Toxicity Test ................................ - 96 - 5.4.3 – Dibutyl phthalate (DBP) Chronic Toxicity Test: ...................................................... - 97 - (Figure 39). The DBP chronic