Bioaccumulation, biotransformation and hormesis effects of 6PPD-Q in Eisenia fetida: Impacts on metabolism and gut microbiota.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is an oxidative byproduct of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, a widely used tire antioxidant, and has emerged as an environmental pollutant of increasing concern. However, its environmental behavior and effects in soil ecosystems remain poorly characterized. This study focused on earthworm Eisenia fetida to reveal the bioaccumulation, biotransformation, and hormesis effects of 6PPD-Q. Findings indicated that 6PPD-Q mainly accumulated in intestinal tissues of E. fetida with concentration of 3465.2 ± 470.8 ng/g. A total of 32 transformation products were identified by UPLC-Orbitrap-MS/MS, including dehydrogenation, methylation, bond and quinone cleavage products that have not been reported in soil animals before. Earthworms showed a more complex detoxification network than aquatic organisms. Notably, the methylation product TP312 and the dehydrogenation product TP296 were predicted to have enhanced toxicity. Non-targeted metabolomics and 16S rRNA sequencing analysis indicated that 6PPD-Q induced dose-dependent hormesis effects. At low concentrations, 6PPD-Q stimulated energy metabolism, membrane function and antioxidant defense in E. fetida. In contrast, high concentrations of 6PPD-Q resulted in functional inhibition. Gut microbiota played a key role in mediating both 6PPD-Q biotransformation and the observed host hormesis. This study contributes vital insights regarding environmental fate of 6PPD-Q in soil ecosystems, and provides valuable reference for assessing its biological health risks.