The combined pollution of cadmium (Cd) and enrofloxacin (ENR) in aquatic environments represents a critical issue in environmental toxicology. Using zebrafish as model organisms, we systematically investigated the combined toxicity of Cd and ENR through both acute (96-h) and chronic (20-d) exposure experiments. Our results demonstrated significant synergistic effects: co-exposure reduced the 96-h LC50 values from 89.12 mg/L (Cd alone) and 190.11 mg/L (ENR alone) to 46.35 mg/L and 99.39 mg/L, respectively (combined effect index = 0.96). Chronic exposure revealed that ENR enhanced Cd accumulation in the liver, intestine, and muscle tissues by 1.11–2.33-fold compared to single Cd exposure. Oxidative stress markers showed dynamic temporal changes, with superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities initially increasing by 1.34–7.06-fold, 0.98–3.28-fold, and 1.53–3.65-fold at 8 d, respectively, followed by 9.9–48.98% reductions after 20 d of exposure. Malondialdehyde (MDA) levels progressively accumulated, reaching up to 4.06-fold higher than controls. Notably, co-exposure elevated oxidative stress by 11.24–34.48% relative to single exposures. The 16S rDNA sequencing analysis indicated that Cd exposure significantly reduced the α-diversity of zebrafish gut microbiota (57–63% decrease in Shannon index), characterized by a 16–20% reduction in beneficial Cetobacterium and a 44–114% increase in pathogenic Aeromonas abundance. The combined exposure further exacerbated these gut microbiota dysbiosis patterns. These findings provide crucial evidence for ecological risk assessment, suggesting that current environmental standards based on single-pollutant evaluations may substantially underestimate the actual risks of heavy metal-antibiotic co-contamination in aquatic ecosystems.

As a highly effective acaricide, etoxazole is widely used in agricultural production, but its toxicological effects on soil organisms remain unclear. Based on nuclear magnetic resonance (NMR) metabolomics technology, in this study, we systematically investigated the sublethal responses of earthworms (Eisenia fetida) to etoxazole. The results showed that etoxazole exposure significantly altered the endogenous metabolic profiles in earthworms, with 19 and 20 metabolites significantly changed after 2 and 14 d of exposure, respectively. Trimethylamine N-oxide exhibited specific changes, indicating that it may be a potential biomarker for exposure to etoxazole. KEGG pathway analysis revealed that five metabolic pathways were notably affected after 2 and 14 days of etoxazole exposure. These pathways were primarily associated with energy conversion, protein and amino acid synthesis and metabolism, carbohydrate metabolism, and nucleic acid and DNA synthesis. Overall, etoxazole exposure notably altered the endogenous metabolic profiles of earthworms. This study was of great significance for comprehensively understanding the potential hazards that etoxazole poses to soil ecosystems and provides important information for environmental monitoring and ecological risk assessment.