Divergent metal bioaccumulation and toxicity responses under metal-antibiotic co-exposure are associated with gut microbiota shifts in Eisenia fetida

Combined metal and antibiotic pollution (CMAP) is increasingly prevalent in agricultural soils, yet its joint toxicity to soil invertebrates remains mechanistically unclear. Using Eisenia fetida as a model organism, we examined associations among gut microbial-community shifts, metal body burden, and toxicological responses under Cd-ciprofloxacin (Cd-CIP) and Pb-norfloxacin (Pb-NOR) co-exposure. Bioaccumulation, biochemical and cellular responses, gut microbiome profiling, density functional theory (DFT) calculations, and quantitative adverse outcome pathway (qAOP) modeling were integrated to evaluate the thermodynamic plausibility of direct metal-antibiotic complexation and characterize associations among microbial-community changes, metal body burden, and toxicity responses. Cd-CIP co-exposure was associated with increased Cd body burden and enhanced toxicity responses, whereas Pb-NOR co-exposure was associated with reduced Pb body burden and attenuated adverse effects. DFT calculations indicated weak thermodynamic stability of the modeled Cd-CIP and Pb-NOR complexes under gut-relevant conditions, with monodentate keto coordination susceptible to competition from water and organic ligands. Microbiome analyses further revealed that CIP exposure was associated with suppression of metal-detoxifying taxa and higher Cd body burden, whereas NOR exposure was associated with enrichment of Bacillus spp. and lower Pb body burden. These differences in metal body burden were accompanied by changes in ROS-related oxidative stress, energy metabolism, apoptosis, and growth responses, which were quantitatively organized within the qAOP frameworks. Collectively, the observed associations among gut microbial composition, metal body burden, and toxicity responses highlight the potential relevance of host–microbiome interactions in the ecological risk assessment of CMAP.