Advancing the zebrafish embryo test for endocrine disruptor screening using micro‐injection: ethinyl estradiol as a case study


Fish (embryo) toxicity test guidelines are mostly based on aquatic exposures. However, in some cases, other exposure routes can be more practical and relevant. Micro‐injection into the yolk of fish embryos could offer a particular advantage for administering hydrophobic compounds, such as many endocrine disruptors. Single dose micro‐injection was compared to continuous aquatic exposure in terms of compound accumulation and biological responses. 17α‐ethinyl estradiol (EE2) was used as a model compound. First, the optimal solvent and droplet size were optimized, and needle variation was assessed. Next, biological endpoints were evaluated. The accumulated internal dose of EE2 decreased over time in both exposure scenarios. Estrogen receptor (ER) activation was concentration/injected dose dependent, increased daily and was related to esr2b transcription. Vtg1 and cyp19a1b transcription was induced in both scenarios, but the cyp19a1b transcription pattern differed between routes. Injection caused an increase of cyp19a1b transcripts from 48 hours post fertilization (hpf) onwards, while after aquatic exposure the main increase occurred between 96 and 120 hpf. Some malformations only occurred after injection, while others were present for both scenarios. We conclude that responses can differ between exposure routes and therefore micro‐injection is not a direct substitute for, but can be complementary to aquatic exposure. Nevertheless, vtg1 and cyp19a1b transcription and ER activation are suitable biomarkers for endocrine disruptor screening in both scenarios.

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