The widespread use of plastic agricultural films necessitates a thorough evaluation of environmental risks posed by soil microplastics (MPs). While the intestinal tract is a critical site for MP interactions in soil organisms, current research predominantly focuses on overall physiological responses, overlooking organ-specific toxic mechanisms. To address this gap, we exposed earthworms (Eisenia fetida) to polyethylene (PE) and biodegradable polylactic acid (PLA) MPs sourced from agricultural films at an environmentally realistic concentration of 1.0 g/kg. Incorporating natural earthworm mobility, we designed two exposure scenarios: migration from clean to contaminated soil (scenario A) and vice versa (scenario B). Machine learning-driven image analysis and phenotypic profiling revealed that PE induced more severe intestinal lesions than PLA, adversely affecting intestinal immune functions. Furthermore, PE resulted in greater oxidative damage and significantly activated immune proteins such as melanin and antimicrobial peptides through reprograming immune-related gene and protein pathways. Conversely, PLA predominantly disrupted intestinal digestive and absorptive functions, though the gut microbial community partially mitigated damage through structural and compositional adaptation. Compared with scenario A, earthworms in scenario B exhibited reduced tissue damage, enhanced digestive enzyme activity, and upregulated energy-related metabolites and cell proliferation genes, indicating partial recovery from MP-induced intestinal dysfunction. These findings elucidate the distinct toxicity mechanisms of conventional and biodegradable agricultural MPs on soil organisms, while the scenario-based approach advances risk assessment by aligning experimental design with real-world ecological behaviors.

Pesticides and microplastics are common pollutants in soil environments, adversely affecting soil organisms. However, the combined toxicological effects of aged microplastics and pesticides on soil organisms are still unclear. In this study, we systematically studied the toxicological effects of azoxystrobin and four different aged polyethylene (PE) microplastics on earthworms ( Eisenia fetida ). The purpose was to evaluate the effects of aging microplastics on the toxicity of microplastics-pesticides combinations on earthworms. The results showed that different -aged PE microplastics promoted azoxystrobin accumulation in earthworms. Meanwhile, combined exposure to azoxystrobin and aged PE microplastics decreased the body weight of earthworms. Besides, both single and combined exposure to azoxystrobin and aged PE microplastics could lead to oxidative damage in earthworms. Further studies revealed that azoxystrobin and aged PE microplastics damage the intestinal structure and function of earthworms. Additionally, the combination of different aged PE microplastics and azoxystrobin was more toxic on earthworms than single exposures. The PE microplastics subjected to mechanical wear, ultraviolet radiation, and acid aging exhibited the strongest toxicity enhancement effects on earthworms. This high toxicity may be related to the modification of PE microplastics caused by aging. In summary, these results demonstrated the enhancing effects of aged PE microplastics on the toxicity of pesticides to earthworms. More importantly, aged PE microplastics exhibited stronger toxicity -enhancing effects in the early exposure stages. This study provides important data supporting the impact of different aged PE microplastics on the environmental risks of pesticides.