Endocrine Disrupting Effects of Halogenated Chemicals on Fish

Halogenated chemicals are commonly used in a variety of everyday products such as cookware, electronics, furniture and baby products. Some of these chemicals have been found to negatively affect development and endocrine disruption. Therefore, there is a need for the development of next generation replacements that are less toxic; however, more studies need to be conducted to ensure their safety. The main objective of this dissertation was to assess the endocrine disrupting potential of halogenated and next generation chemicals, singly and in mixtures, in fish. We tested three well studied halogens, perfluorooctanoic acid (PFOA), tris (1,3-dichloro-2-propyl) phosphate (TDCPP) and tetrabromobisphenal A (TBBPA), and two lessstudied next generation chemicals, 9,10-Dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (DOPO) and perfluorobutyric acid (PFBA). We used zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) as models to evaluate the toxicity of these chemicals in mixtures as well as their individual endocrine disrupting potential, looking specifically at thyroid and estrogen disruption. Using zebrafish embryos, we first identified the lethal concentration (LC50) under 96 hour exposures. Chemical LC50 values ranged from 1.3-13,795 ppm. Next, we tested the toxicity of tertiary mixtures containing the estimated LC50 values for each chemical. We found that chemicals within these mixtures displayed concentration addition suggesting a similar mode of toxic action. Importantly, next generation chemicals were between 5 to 760 times less acutely toxic singly and in mixtures than their first generation counterpart. Next, we evaluated the potential thyroid disruption of old use and next generation halogenated chemicals by using thyroid induced swim bladder development and thyroid related gene expression as endpoints in zebrafish larvae and juveniles. Zebrafish embryos were exposed to three old use compounds (PFOA, TBBPA and TDCPP) and two next generation chemicals (DOPO and PFBA). Subchronic (0 to 6 days post fertilization (dpf)) and chronic (0 to 28 dpf) exposures were conducted at 1% of the concentration known to kill 50% (LC50) of the population. At 6 dpf, zebrafish exposed to all halogenated chemicals, both old use and next generation, had smaller posterior swim bladder and increased expression in thyroid peroxidase, tpo and two swim bladder surfactant proteins, sp-a and sp-c. These results mirrored the effects of thyroid hormone exposed positive controls. Fish exposed to a TPO inhibitor (methimazole, MMI) had a decrease in tpo expression levels at 28 dpf. Effects on the anterior swim bladder at 28 dpf, after exposure to MMI as well as both old and new halogenated chemicals, were the same, i.e., absence of SB in ~50% of fish, which were also of smaller body size. Finally, we assessed sex differences of halogenated compounds for thyroid and estrogenic disruption. Japanese medaka (strain SK2MC) are an excellent model for these studies since sex can be determined non-destructively a few hours post fertilization. Medaka embryos were exposed to TDCPP, PFOA and its next generation alternative PFBA. A negative water control was tested along with the reference chemicals MMI and T3. Fish were exposed to sublethal concentrations, TDCPP (0.019 ppm), PFOA (4.7 ppm) and PFBA (137 ppm), throughout embryo development until 48 hours post hatch. Changes in swim bladder surface area and in expression levels of thyroid and estrogenic related genes were quantified. There were no effects on swim bladder surface area in males; however, an upregulation of estrogenic related genes was observed after exposure to TDCPP, PFOA and MMI. Females displayed significantly larger swim bladders after exposure to all chemicals with exception of T3 which caused the opposite effect. Females exposed to TDCPP and PFOA had increased expression of vitellogenin and exposure to PFOA upregulated expression of multiple thyroid related genes. Overall our results suggest that males have increased expression of estrogenic genes when exposed to these halogenated chemicals and females are more susceptible to thyroid induced swim bladder dysfunction. These results are important as they stress the importance of sex when assessing the endocrine disrupting potential of chemicals. All in all, our results support earlier findings that the halogenated chemicals studied act as endocrine disrupting chemicals, specifically causing disruption to the thyroid and estrogen systems. However, with the exception of TBBPA and TDCPP, the concentrations tested (~5-137 ppm) are not likely to be found in the environment. We further show the importance of examining sex-specific endocrine effects and that Japanese medaka are a better model when assessing endocrine disrupting chemicals. This work sheds light for the need of next generation chemicals that are hopefully safer for the environment; however, there is also a need to fully assess the chemicals prior to production to ensure safety. We found that even the next generation chemicals in this study can cause developmental delays and endocrine disruption at early stages in fish. Future studies should assess the mechanism linking estrogen disruption with swim bladder dysfunction as well as confirm the compensatory effect of the surfactants on swim bladder dysfunction in both fish models tested