An Integrated Approach Combining Regulatory Tests in tg ( cyp19a1b:GFP ) Zebrafish Embryos to Assess Toxicity, Developmental Effects, and Estrogenic Activity of Chemicals: A Case Study with Bisphenol A Substitutes

The use of efficient testing strategies for chemical hazard assessment is a current challenge in supporting regulatory requirements. Herein, we combined two zebrafish eleuthero-embryo assays, a refined Fish Embryo Acute Toxicity assay (FET, OECD TG 236) and the EASZY assay (OECD TG 250), both using transgenic tg(cyp19a1b:GFP) embryos, to jointly assess acute toxicity, developmental effects, and estrogenic activity of bisphenol A (BPA) and ten substitutes. Several bisphenols were more toxic than BPA, inducing developmental effects on zebrafish embryos, with some showing potential teratogenicity. Increased GFP intensity was also detected for ten bisphenols, suggesting estrogenic activity. FET results guided the selection of sublethal concentrations for the EASZY assay, which confirmed estrogenic activity for all bisphenols except TCBPA, most being more estrogenic than BPA in vivo and requiring functional zfERs to induce brain aromatase. All bisphenols also activated zfERβ2 in a zebrafish-specific in vitro reporter gene assay, except BPS-MAE and BPS-MPE, which only induced brain aromatase in vivo. Overall, the combined FET and EASZY assays efficiently generated relevant data for hazard assessment of chemicals and provided further evidence that bisphenols modulate ER-dependent cyp19a1b expression during early zebrafish brain development, raising concerns about their potential short- and long-term adverse effects.