Neurotoxic effects of binary and ternary fungicide mixtures in developing zebrafish: A multi-endpoint assessment

Folpet (F), penconazole (P), and metrafenone (M) are commonly used fungicides. Their toxicological effects, particularly in mixtures, are not sufficiently understood. Zebrafish embryos were treated for 96 hpf with single compounds, binary and ternary mixtures at three environmentally relevant concentrations (0.25, 0.5, and 1 mg/L), evaluating developmental and craniofacial alterations, apoptosis, behaviour and expression of genes related to cell cycle, brain/craniofacial development and thyroid hormone system. Single fungicides displayed different activities; for all mixtures, we recorded dose-addition for the craniofacial M-PQ angle and synergism for head deformation. Increased hyperactivity was observed at the highest dose of M and F and the mixtures P+M and P+M+F. These same mixtures induced apoptosis at pericardial level. The use of EGFP-Neurod transgenic zebrafish and the ToxPI tool confirmed the greater neurotoxicity of the mixtures compared to single compounds, highlighting the importance of assessing the combined effects of pesticides to improve risk assessment.