Prothioconazole induced stereoselective developmental toxicity and liver injury in zebrafish embryos via ferroptosis.

The widespread use of prothioconazole (PTCZ), a globally applied triazole fungicide, raises concerns regarding ecological risks from environmental residues and highlights the critical gap in pesticide safety assessment concerning enantiomeric differences in toxicity. This study investigated the stereoselective toxicity and molecular mechanisms of PTCZ enantiomers in an aquatic model using a zebrafish embryo exposure system. The toxic effects were systematically analyzed through multidimensional endpoint assessments, which examined developmental malformations, liver histopathology, lipid metabolism indicators, and lipid peroxidation. The underlying molecular mechanisms were explored through GPX4 immunofluorescence, as well as qPCR and Western blot analyses of ferroptosis-related genes. A ferroptosis inhibitor rescue experiment utilizing Ferrostatin-1 was conducted to investigate the role of ferroptosis in the observed toxicity. Our findings demonstrate that the S-(+)-PTCZ enantiomer induced significantly more severe developmental toxicity and liver injury compared to its counterpart. Mechanistically, S-(+)-PTCZ triggered hepatic damage by activating the lipid peroxidation-ferroptosis axis, as evidenced by inhibition of GPX4 protein expression and an upregulation of the pro-ferroptotic gene acsl4. Crucially, Ferrostatin-1 significantly reversed these effects, reducing lipid peroxidation. Our results confirm that traditional risk assessments based on the racemate (Rac-PTCZ) would substantially underestimate the actual environmental risk posed by the highly non-target bioactive S-(+)-enantiomer. This work provides a critical theoretical basis for the precise regulation and low-toxicity design of chiral pesticides.