An Early–Life Stage Alternative Testing Strategy for Assessing the Impacts of Environmental Chemicals in Birds

Abstract

Early-life stage (ELS) toxicity tests are recognized as an advancement over current testing methodologies in terms of cost, animal use, and biological relevance. However, standardized ELS tests are not presently available for some vertebrate taxa, including birds. The present study describes a Japanese quail (Coturnix japonica) ELS test that is a promising candidate for standardization and applies it to test 8 environmental chemicals (ethinylestradiol, benzo[a]pyrene, chlorpyrifos, fluoxetine, lead(II)nitrate, trenbolone, seleno-L-methionine, hexabromocyclododecane). Individual chemicals were injected into the air cell of unincubated Japanese quail eggs at 3 concentrations, all predicted to cause ≤20% mortality. Survival to embryonic day 16 was consistently high (>90%) among the vehicle-injected controls. All chemicals, except ethinylestradiol, were detected in liver tissue, most at concentrations suggestive of embryonic clearance. Adverse effects were observed for 5 of the 8 chemicals; chlorpyrifos (41.1 µg/g) significantly increased developmental abnormalities and decreased embryo and gallbladder mass. Ethinylestradiol (54.2 µg/g) and hexabromocyclododecane (0.02 µg/g) decreased embryo mass and tarsus length, respectively. Benzo[a]pyrene (0.83 µg/g) and fluoxetine hydrochloride (32.7 µg/g) exceeded the 20% mortality cutoff. No effects were observed following lead(II)nitrate, seleno-L-methionine, or trenbolone exposure up to 10.7, 0.07, and 4.4 µg/g, respectively. Overall, our ELS approach was time-and cost-effective, caused minimal mortality in controls, effectively delivered diverse chemicals to the embryo, and permitted identification of apical outcomes, all of which provide support toward standardization.

Effects on Apical Outcomes of Regulatory Relevance of Early‐Life Stage Exposure of Double‐Crested Cormorant Embryos to 4 Environmental Chemicals

“…; reported purity ≥95%) were included in the double‐crested cormorant egg injection studies: BaP, chlorpyrifos, FLX, and EE2. Dimethyl sulfoxide (DMSO; Sigma‐Aldrich) was the solvent selected to prepare stock and working solutions; a detailed justification for selecting DMSO for early‐life stage avian egg injection studies can be found in Farhat et al (2020). Based on the Japanese quail early‐life stage toxicity study by Farhat et al (2020), we selected a high dose that we assumed would not exceed 20% lethality of the exposed organisms.…”

“…Dimethyl sulfoxide (DMSO; Sigma‐Aldrich) was the solvent selected to prepare stock and working solutions; a detailed justification for selecting DMSO for early‐life stage avian egg injection studies can be found in Farhat et al (2020). Based on the Japanese quail early‐life stage toxicity study by Farhat et al (2020), we selected a high dose that we assumed would not exceed 20% lethality of the exposed organisms. Wherever possible, nominal concentrations were chosen to match our previous study, although actual concentrations may differ.…”

“…Morphological measurements including embryo mass, liver mass, gallbladder mass, head + bill length, and tarsus length were obtained according to Farhat et al (2020). Blood and plasma samples were also collected based on Farhat et al (2020) for genetic sex determination and biochemical analyses, respectively. Finally, a pool of 3 whole livers was collected for chemical analysis to permit comparisons of tissue burdens at 2 different developmental stages.…”

“…Test chemical concentrations in dosing solutions and pooled liver tissues of ED14 and ED26 double‐crested cormorant embryos were determined by SGS‐AXYS Analytical Services using gas chromatography–mass spectrometry (for BaP and chlorpyrifos) or liquid chromatography–tandem MS (for FLX and EE2) as detailed in Farhat et al (2020). Standardized methods with the inclusion of quality control samples and certified reference materials were used for all analyses.…”

“…While a substantial amount of data for laboratory model avian species is critical to establish a baseline response, our group has been challenged by government regulators with regard to the applicability of our findings to the assessment of risk to wild avian species that are naturally exposed to chemical stressors. In the present study, we evaluated whether the Japanese quail early‐life stage test (Farhat et al 2020) could be successfully adapted to a wild species, double‐crested cormorant, to determine the effects of 4 environmental chemicals: benzo[ a ]pyrene (BaP), chlorpyrifos, fluoxetine hydrochloride (FLX), and ethinyl estradiol (EE2). In addition, we compared our results with those previously reported for the same 4 chemicals in early‐life stage Japanese quail.…”