Multi-level biomarker responses in Eisenia fetida co-exposed to PE-MPs and DiBP: An ecological risk assessment based on physiological, biochemical, and molecular indicators

The widespread use of agricultural films has resulted in the pervasive accumulation of microplastics (MPs) and phthalates (PAEs) in soil, posing significant ecological toxicity risks to soil organisms. In this study, we investigated the toxic responses of Eisenia fetida following single and combined exposure to polyethylene microplastics (PE-MPs) and dibutyl phthalate (DiBP), covering survival, growth inhibition, oxidative stress, stress-related gene transcription, and histopathology. At the early exposure phase, co-stimulation by PE-MPs and DiBP markedly inhibited core antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), while triggering sharp compensatory elevation of glutathione S-transferase (GST) activity. Such antioxidant imbalance triggered early lipid peroxidation and cell membrane injury, evidenced by elevated malondialdehyde (MDA) concentrations. Although partial recovery of antioxidant enzyme activity was observed at day 28, obvious oxidative DNA damage was detected. This DNA injury disturbed genomic homeostasis, as reflected by significantly upregulated transcription of calreticulin (CRT) and heat shock protein 70 (Hsp70), alongside suppressed expression of translationally controlled tumor protein (TCTP). Two-way ANOVA verified prominent interactive effects between PE-MPs and DiBP at the gene transcription level, with synergistic and antagonistic effects alternating under different concentration combinations. At the tissue level, the combined exposure eventually induced severe pathological lesions in the epidermis, muscle layers and intestinal wall, accompanied by obvious body weight loss in earthworms. Collectively, these results provide mechanistic insights into the interactive toxic effects of coexisting PE-MPs and DiBP, and offer fundamental data to support ecological risk evaluation of mixed microplastic–plasticizer pollution in farmland soils.