Toxicity testing is moving from animal-based studies to faster, more ethical in vitro approaches that focus on mechanistic toxicology. The use of bisphenol A (BPA) replacement compounds is increasing and there is limited toxicity data available for these compounds in avian species. The overall goals of this thesis were to: a) determine if avian cell lines are suitable alternatives to primary hepatocytes for chemical screening; b) generate toxicity data for five BPA replacement compounds: bis(4-hydroxyphenyl)methane (BPF), bis(3-allyl-4-hydroxyphenyl)sulfone (TGSH), 7-bis(4-hydroxyphenylthio)-3,5-dioxaheptane (DD-70), 2,2-bis(4-hydroxyphenyl)hexafluoropropane (BPAF) and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP) in three in vitro models: primary chicken embryonic hepatocytes (CEH), double-crested cormorant (DCCO) embryonic hepatocytes (DCEH) and chicken LMH cell line; and 3) prioritize two replacements for early-life stage testing (ELS). LMH cells cultured as 3D spheroids, as opposed to 2D monolayer, had enhanced mRNA expression and CYP1A activity and were therefore used for screening. Additionally, an immortalized DCCO hepatic cell line, DCH22, was established, which may be useful for future avian toxicity testing. DD-70 and BPAF were the most cytotoxic across the three in vitro models. TGSH and DD-70 altered expression of genes associated with multiple toxicity pathways, but not estrogen response, and are potential non-estrogenic replacements. BPAF, BPF and BPSIP are potential estrogenic replacements. In general, the replacements were more cytotoxic and/or transcriptionally active than BPA. There was species-specific variability in toxicity; the replacements were more transcriptionally active in CEH compared to DCEH. LMH spheroids were more sensitive to estrogenic endpoints than CEH. DD-70 and BPAF were prioritized for ELS studies based on in vitro results. All of the replacements modulated the expression of genes related to bile acid regulation in vitro and an increase in gallbladder mass was observed in chicken embryos after exposure to DD-70 or BPAF. Overall, this thesis evaluated the utility of LMH cells cultured as spheroids as an animal free alternative for chemical screening, established a DCCO cell line, and generated novel cytotoxicity and gene expression data for five BPA replacement compounds in three in vitro avian models and determined ELS toxicity of two replacement compounds.

Since their emergence in the early 1990s, neonicotinoid use has increased exponentially to make them the world's most prevalent insecticides. Although there is considerable research concerning the lethality of neonicotinoids, their sub-lethal and developmental effects are still being explored, especially with regards to non-mammalian species. The goal of this research was to investigate the effects of the neonicotinoid imidacloprid on the morphological and physiological development of northern bobwhite quail (Colinus virginianus). Bobwhite eggs (n = 650) were injected with imidacloprid concentrations of 0 (sham), 10, 50, 100 and 150 grams per kilogram of egg mass, which was administered at day 0 (pre-incubation), 3, 6, 9, or 12 of growth. Embryos were dissected on day 19 when they were weighed, staged, and examined for any overt structural deformities. Embryonic heart, liver, lungs and kidneys were also weighed and preserved for future use. Treated embryos exhibited increased frequency of severely deformed beaks and legs, as well as larger hearts and smaller lungs at the higher dosing concentrations. Some impacts are more pronounced in specific dosing periods, implying that there may be critical windows of development when embryos are highly susceptible to neonicotinoid exposure. This investigation suggests that imidacloprid could play a significant role in chick survival and declining quail populations in treated regions of the country.