Urea Co-exposure Increases Glyphosate Toxicity in Earthworms: Evidence from Species with Distinct Sensitivities

Glyphosate is among the most widely used herbicides worldwide and is considered to have adverse effects on soil ecosystems. Simultaneous application of glyphosate and urea frequently occurs in agricultural management, yet the ecological risk of their co-exposure to earthworms remains unexplored. In this study, epigeic <i>Eisenia fetida</i> (<i>E. fetida</i>) and anecic <i>Metaphire guillelmi</i> (<i>M. guillelmi</i>) were exposed for 28 days to soils treated with glyphosate (1 or 10 mg/kg), urea (240 kg N/ha), or their combination. Compared to glyphosate alone, co-exposure with glyphosate and urea increased glyphosate residues in earthworm gut contents by 9.89-40.23% and reduced the body weight of the earthworm by 17.86-21.05%. Furthermore, co-exposure aggravated intestinal tissue damage, sharply decreased digestive enzyme activity, upregulated <i>lysozyme</i> expression by more than 150.28%, and raised LPS content by over 10.08%. In addition, co-exposure intensified gut microbiota dysbiosis, with the inflammatory marker phylum <i>Proteobacteria</i> increasing by 6.98-17.52%. Besides these same impacts, <i>E. fetida</i> exhibited more severe oxidative damage and energy metabolic disorders under co-exposure, while <i>M. guillelmi</i> showed greater sensitivity in intestinal tissue and microbial responses. Partial least-squares path modeling confirmed that co-exposure primarily intensified growth toxicity in <i>E. fetida</i> via oxidative damage, and in <i>M. guillelmi</i> through increased intestinal permeability. These findings highlight the toxicological risks of combined fertilizer and glyphosate exposure in earthworms and underscore species-specific responses, providing new insights for ecological risk assessment.