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Combined exposure to polyethylene microplastics, copper, and Di(2-Ethylhexyl) phthalate reconfigures component-specific internal exposure profiles and triggers cross-level biological responses in earthworms.
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Earthworms were exposed to microplastics, copper, and a plastic chemical called DEHP, alone and combined. Combined exposure changed how much of each substance built up in the body, and caused more harm overall: higher death rates, weight loss, reproductive problems, and changes in gut bacteria and body chemistry compared to single exposures.
Microplastics, metals, and plastic additives commonly coexist in soils, but how their interactions reconfigure internal exposure and relate to biological responses across multiple levels We exposed Eisenia fetida to polyethylene microplastics (PE), copper (Cu), and di(2-ethylhexyl) phthalate (DEHP) singly and in binary and ternary combinations, integrating internal exposure, organismal phenotypes, redox biomarkers, histopathology, gut microbiota, and untargeted metabolomics. Combined exposure reconfigured rather than uniformly increased internal exposure: under ternary exposure, whole-body Cu decreased from 17.96 to 10.34 mg kg⁻¹ , whereas DEHP increased from 2735.76 to 8523.19 μg kg⁻¹ , while gut-retained PE varied among mixtures. Ternary exposure produced the strongest organismal effects, with mortality reaching 34%, body-weight loss 18.16%, reproductive and locomotor impairment, and ROS increasing 4.9-fold over the control. Gut bacterial responses were contaminant-specific: PE increased Chao1 richness from 1808 to 2579, while community composition differed among treatments (PERMANOVA, R² = 0.482, P = 0.001) and assembly shifted markedly under PE + DEHP and DEHP + Cu. Predicted microbial functions differed among treatments (R² = 0.525), with internal PE and Cu burdens significantly associated with KO profiles. Metabolomics identified 100, 66, and 124 candidate metabolites under PE, Cu, and DEHP, respectively, while Cu showed the clearest FDR-supported pathway enrichment. These findings reveal component-specific internal exposure linked to host, microbial, and metabolic responses.
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