Environmentally Persistent Free Radicals on Aged Polyamide Microplastics Drive Neurotoxicity in Eisenia fetida via a Glutamate-Centered Cascade

Nitrogen-containing microplastics (N-MPs) are widespread in soil and often exhibit distinct environmental behaviors and effects owing to their unique elemental composition and molecular structure. However, their toxicity to soil organisms remains poorly understood. To address this knowledge gap, the toxicity of pristine and aged polyamides (PA) was evaluated in earthworms (Eisenia fetida), a representative soil invertebrate. At 0.01% (w/w), aged PA increased the magnitude of body-weight loss, MDA levels, and AChE activity by 24.4%, 11.6%, and 24.1%, respectively, relative to pristine PA-MPs. Greater accumulation of aged PA-MPs was accompanied by enhanced oxidative damage and neurotoxicity. Integrated multiomics, quantitative real-time PCR, and biomarker analyses revealed that oxidative stress-mediated membrane damage promoted glutamate accumulation, receptor overactivation, Ca2+ overload, synaptic injury, and ultimately neurotoxicity. Pharmacological interventions and transmission electron microscopy of the ventral nerve cord provided functional and morphological support for this cascade. Radical quenching and comparative leachate bioassays further supported the important contribution of environmentally persistent free radicals in aged PA-MPs to neuronal damage. These findings define a mechanistic pathway for aged microplastic-induced neurotoxicity and provide a basis for assessing the ecological risks of aged N-MPs in soils.