Integrated transcriptomics, metabolomics, network toxicology and phenotype-based validation unveil the ferroptosis-induced toxicity mechanism of methyl eugenol in zebrafish (Danio rerio)

• The environmental risk of methyl eugenol was assessed using the "1N+2T+3M" strategy. • Methyl eugenol induced ferroptosis in zebrafish resulting in liver and gill necrosis. • Multi-omics identified linoleic acid-driven lipid peroxidation as a critical pathway. • Network toxicology and molecular docking revealed ferroptosis-regulating targets. • The ferroptosis inhibitor reduced methyl eugenol-induced mortality in zebrafish. The aquatic toxicity mechanism of methyl eugenol (ME), an emerging insect sex pheromone-based pesticide, remains unclear. To address this issue, we established a strategy termed "1N (network toxicology) + 2T (toxicity assessment, transcriptomics) + 3M (metabolomics, molecular docking, mechanism verification)" to systematically investigate its toxicity, focusing on ferroptosis as a potential core mechanism. Toxicity assessment determined the 96-hour LC₅₀ of ME in zebrafish to be 2.536 mg/L and it caused marked histopathological damage in liver and gill tissues. Furthermore, integrated transcriptomic and metabolomic analysis revealed the co-enrichment of dysregulated genes and altered metabolites in ferroptosis-related pathways. Subsequently, network toxicology identified CDK1, TP53, CHEK2, and CTNNB1 as key ferroptosis-related targets of ME, and molecular docking confirmed their stable binding via hydrogen bonds with lysine residues. For mechanism verification, the ferroptosis inhibitor ferrostatin-1 (Fer-1) significantly reduced ME-induced mortality, alleviated tissue damage, and mitigated oxidative stress and lipid peroxidation. These findings demonstrated that ferroptosis was the primary toxicity mechanism of ME in zebrafish. Our study provides scientific evidence for the ecological risk assessment of ME and presents the "1N+2T+3M" strategy as an applicable framework for evaluating other pesticides.