Critical review: Considerations in applying avian egg injection studies to characterize effects of hydrophobic organic compounds on wild birds

Egg injection has been used for decades to determine embryonic mortality and developmental effects of chemical exposures in birds. Specific egg injection methods affect how well these studies replicate the process of chemical delivery to the embryo via maternal deposition, yet few data are available to compare exposure-response relationships between egg injection and maternal transfer studies. This information gap creates uncertainty when considering egg injection studies for assessment of potential adverse effects in wild birds. Focusing on hydrophobic organic compounds, we evaluated avian egg injection methods and potential methodological artifacts, through critical literature review and analyses of egg injection and maternal transfer data. Parameters that can affect embryotoxicity outcomes in egg injection studies include injection site, incubation orientation, type of vehicle, injection timing, injection volume, and different combinations of these parameters. Data to quantitatively compare embryotoxicity between egg injection and maternal transfer studies in the same species were identified only for a few polychlorinated biphenyl (PCB) mixtures and only for two egg injection methods, while comparisons among additional egg injection methods (without comparable maternal transfer toxicity data) were possible for certain PCB and dioxin congeners. Additionally, chemical uptake patterns in eggs and embryos were reviewed for different study designs. Overall, one egg injection study demonstrably replicated the exposure-response relationship from a comparable maternal transfer study, while median lethal doses (LD₅₀s) varied up to 8-fold for the same congener across egg injection methods. Risk assessors should carefully consider the potential for over- or underestimation of toxicity when applying egg injection toxicity data during toxicity reference value development, and researchers should prioritize realistic representation of wild bird exposures when designing and standardizing egg injection protocols.