The ecological risks of biochar-derived dissolved organic matter (DOM) to soil invertebrates at different organismal levels remains limited. This study comprehensively explored the ecological risks of biochar-derived DOM on earthworm gut through assessments of enzyme activity response, histopathology, gut microbiomes, and metabolomics. Results demonstrated that DOM disturbed the digestive enzymes in earthworm, especially for 10% DOM300 groups. The integrated biomarker response v2 (IBRv2) indicated that the perturbation of earthworm digestive enzymes induced by DOM was both time-dependent and dose-dependent. Pathological observations revealed that 10% DOM300 damaged intestinal epithelium and digestive lumen of earthworms. The significant damage and injury to earthworms caused by DOM300 due to its higher concentrations of heavy metal ions and organic substrates (e.g., toluene, hexane, butanamide, and hexanamide) compared to DOM500 and DOM700. Analysis of 16S rRNA from the gut microbiota showed a significant decrease in genera (Verminephrobacter, Bacillus, and Microbacteriaceae) associated with inflammation, disease, and detoxification processes. Furthermore, 10% DOM300 caused the abnormality of metabolites, such as glutamate, fumaric acid, pyruvate, and citric acid, which were involved in energy metabolism, These findings contributed to improve our understanding of the toxic mechanism of biochar DOM from multiple perspectives.
