Waterborne Tebuconazole Exposure Induces Male-Biased Sex Differentiation in Zebrafish (Danio rerio) Larvae via Aromatase Inhibition

Tebuconazole is a widely used fungicide for various crops that targets sterol 14-alpha-demethylase (CYP51) in fungi. However, attention has shifted to aromatase (CYP19) due to limited research indicating its reproductive impact on aquatic organisms. Herein, zebrafish were exposed to 0.5 mg/L tebuconazole at different developmental stages. The proportion of males increased significantly after long-term exposure during the sex differentiation phase (0-60, 5-60, and 19-60 days postfertilization (dpf)). Testosterone levels increased and 17 beta-estradiol and cyp19a1a expression levels decreased during the 5-60 dpf exposure, while the sex ratio was equally distributed on coexposure with 50 ng/L 17 beta-estradiol. Chemically activated luciferase gene expression bioassays determined that the male-biased sex differentiation was not caused by tebuconazole directly binding to sex hormone receptors. Protein expression and phosphorylation levels were specifically altered in the vascular endothelial growth factor signaling pathway despite excluding the possibility of tebuconazole directly interacting with kinases. Aromatase was selected for potential target analysis. Molecular docking and aromatase activity assays demonstrated the interactions between tebuconazole and aromatase, highlighting that tebuconazole poses a threat to fish populations by inducing a gender imbalance.