Non-target metabolomic approach of the toxic effects of glyphosate in zebrafish (D. rerio)

Glyphosate (GLY) is the most widely used herbicide globally and is frequently detected in aquatic environments at low concentrations, raising concerns about its potential long-term effects on non-target organisms. However, the systemic metabolic disruptions of chronic GLY exposure in aquatic vertebrates remain poorly understood, especially at environmentally relevant concentrations. This study investigates the metabolic disruptions of GLY exposure in zebrafish (D. rerio) using a non-targeted metabolomic approach. Brain, gut, and fecal samples were analyzed after two weeks of exposure to environmentally relevant concentrations (0.3 and 3 μg L⁻¹). Metabolic profiling was performed using LC-HRMS data processed via the Regions of Interest Multivariate Curve Resolution (ROIMCR) method, resolving up to 70 components per tissue and capturing >99 % of the experimental variance. Direct MSident annotation revealed diverse biomolecules, including amino acids, sugars, nucleotides, and organic acids. Tentative identification yielded 92, 182, and 117 metabolites in the brain, gut, and feces, respectively, further confirmed by the KEGG database. Statistical analysis revealed significant metabolic differences between control and GLY-treated groups, particularly at higher concentrations. Significantly altered metabolites were observed in the brain (37), gut (75), and feces (31), respectively. Fifteen overlapping metabolites between the studied brain and gut tissues suggest a GLY-induced gut-brain axis disruption. Functional enrichment analysis showed down-regulation of pathways related to amino acid, lipid, and energy metabolism, especially in gut and brain tissues, while fecal metabolites were generally up-regulated. These results demonstrate that GLY exposure induces tissue-specific and systemic metabolic disturbances in zebrafish, potentially linked to oxidative stress and neurotoxicity, with implications for aquatic health and environmental risk assessment.