Bioaccumulation, Ecotoxicity, and Microbial Responses in Hoplobatrachus rugulosus Tadpoles Following Co-Exposure to Imidacloprid and Microplastics

Agricultural organic pollutants have been identified as a key factor contributing to amphibian population decline, particularly during early developmental stages when tadpoles are frequently exposed to neonicotinoids (NEOs) and microplastics (MPs). In this study, <i>Hoplobatrachus rugulosus</i> tadpoles were exposed to imidacloprid (IMI: 0.045, 0.45, and 4.5 mg L^-1) and polyethylene-derived MPs (10 mg L^-1) from agricultural mulch films, both individually and in combination. We systematically evaluated acute toxicity, bioaccumulation, developmental and oxidative stress responses, and changes in the skin and gut microbiota. The results showed that the 96 h median lethal concentration (LC₅₀) of IMI was 44.8 mg L^-1 in the IMI-only group and was 40.5 mg L^-1 in the IMI + MPs group, indicating the negligible impact of MPs on acute toxicity. However, in the highest co-exposure group (IMI4.5 + MPs), tadpole body length and weight decreased by 14.7% and 22.6%, respectively, alongside marked changes in oxidative stress, whereby catalase (CAT) and superoxide dismutase (SOD) activities were suppressed, while malondialdehyde (MDA) levels increased by 35%, indicating elevated lipid peroxidation. Furthermore, the micronucleus frequency in erythrocytes was significantly elevated, suggesting genotoxic effects. Microbial community analysis revealed significant shifts in the relative abundance of gut and skin microbiota under IMI + MPs exposure, with a notable enrichment of <i>Proteobacteria</i>, <i>Fusarium</i>, <i>Actinomycetota</i>, and <i>Bacteroidota</i>, indicating the disruption of host-microbiome interactions. This study proposes a comprehensive multi-tiered assessment framework encompassing environmental exposure, bioaccumulation, toxicological endpoints, oxidative stress biomarkers, and microbiome shifts. Our findings provide new mechanistic insights and quantitative evidence on the compound threats posed by IMI and MPs to amphibians in aquatic environments.