Synergistic toxicity of pyroxasulfone and pendimethalin on Eisenia fetida: an integrated biomarker response and molecular docking analysis.

Herbicide exposure poses potential risks to soil-dwelling organisms, particularly earthworms that play critical roles in soil health. This study evaluated the individual and combined toxic effects of pyroxasulfone and pendimethalin on Eisenia fetida through acute toxicity, oxidative stress biomarkers, histopathology, body weight, Integrated Biomarker Response (IBRv2) and molecular docking analyses. Although both herbicides exhibited relatively low acute lethality individually, pendimethalin (LC50 430.3 mg kg-1) was more toxic than pyroxasulfone (LC50 611.4 mg kg⁻1), while their combination showed enhanced toxicity (LC50 302.3 mg kg-1). Sub-chronic exposure significantly (p < 0.05) altered antioxidant (superoxide dismutase, catalase, peroxidase) and detoxification (glutathione S-transferase) enzyme activities and elevated malondialdehyde content. Across enzyme activities, inhibition was greater under co-exposure (11.34-49.52%) than under individual treatments (5.95-34.10%), with maximal effects at 21 days followed by partial recovery. Body weight generally increased under individual treatments, whereas co-exposure produced greater alterations, with initial weight gain in some treatments followed by progressive weight loss. Histopathological damage was also more pronounced under combined exposure, including muscular disruption, intestinal alteration and cellular pyknosis. IBRv2 indices indicated a greater impact of pendimethalin (5.38-18.40) than pyroxasulfone (3.34-14.40), while combined exposure produced the highest values (8.77-22.90). GST was the most responsive biomarker based on measured activity and dominant IBRv2 contribution, whereas molecular docking provided structural information on predicted herbicide-enzyme interactions. These findings demonstrate that combined exposure elicits greater sub-lethal effects that are not predicted by acute toxicity alone, highlighting the importance of mixture toxicity and integrative biomarkers in soil ecotoxicological risk assessment.