Toxic effects of flufenacet on zebrafish at various developmental stages

Flufenacet is an aryloxy acetamide herbicide. The potential risks of flufenacet to the aquatic ecosystem remain unclear. In this study, the acute toxicity and developmental toxic effects of flufenacet on zebrafish (Danio rerio) were assessed at three different life stages: embryo, larvae, and adult. Larvae at 3 days posthatch and adult zebrafish exhibited higher sensitivity to flufenacet than embryos. The 96-hr median lethal concentration values ranked as embryos (9.79 ± 1.22 mg/L) > adults (4.36 ± 0.56 mg/L) and larvae (3.89 ± 0.98 mg/L), highlighting larvae as the most sensitive life stage. Flufenacet exhibited moderate acute toxicity to adult zebrafish. Flufenacet exposure induced various developmental abnormalities in zebrafish, including increased mortality, delayed hatching, reduced voluntary movement, inhibited hatching rate, shortened body length, bent spine, and edema in the pericardial and yolk sac regions. Additionally, the expression levels of ache, mbp, gap43, and syn2a were dose-dependently downregulated following exposure to various concentrations of flufenacet, indicating neurotoxic effects in zebrafish embryos. Specifically, the expression of gata4 and nkx2.5 was significantly downregulated only in the 5 mg/L of flufenacet treatment group, whereas tbx5 and myh6 expression showed a dose-dependent significant downregulation. The myl7 expression was significantly upregulated in a dose-dependent manner, suggesting that flufenacet may induce zebrafish cardiac dysplasia through modulation of cardiac-related genes (nkx2.5, tbx5, gata4, myl7, and myh6). Expression of hypothalamus-pituitary-thyroid-axis-related genes (crh, tshβ, tra, trb, and dio2) was significantly downregulated in a dose-dependent manner, indicating potential endocrine disruption of the thyroid gland in zebrafish embryos. These results contribute additional evidence regarding the aquatic toxicity of flufenacet, which is crucial for environmental risk assessment.