Nanoplastics in the soil environment: toxicity of polystyrene nanoparticles in the soil centipede Lithobius forficatus (Myriapoda, Chilopoda)

Polystyrene nanoparticles (PS-NPs) are among the most common types of nanoparticles in the environment (e.g., soil), and passing through cell membranes, they can accumulate within an animal's body. Based on our experience with cultivation and cytotoxicity studies on the soil centipede Lithobius forficatus, we decided to analyze cytotoxicological parameters induced (or not) by PS-NPs present in the soil. Thus, the aim was to determine whether PS-NPs, after entering the cytoplasm, could affect organelles in midgut epithelial cells, induce changes in storage material, and activate autophagy. Individuals of L. forficatus were cultured in soil contaminated with PS-NPs at concentrations of 10 and 100 mg·kg⁻¹ for different periods, thus the following experimental groups were provided: 10L1,10L3, and 10L6 – the animals cultured in soil with 100 mg/kg dry mass of PS-NPs for 1, 3, and 6 months respectively; 100L1, 100L3, and 100L6 - animals cultured in soil with 100 mg/kg dry mass of PS-NPs for 1, 3, and 6 months respectively. An isolated midgut was prepared for ultrastructural analysis using transmission electron microscopy, which revealed changes in midgut cells depending on PS-NPs concentration and the duration of animal exposure. Confocal microscopy and histochemical reactions were used to analyze changes in storage materials and autophagy activation. The quantitative analysis was conducted using flow cytometry. The changes observed in midgut cells in animals cultured in soil contaminated with PS-NPs, depend on both nanoparticle concentration and exposure time. We observed an increase in the number of ROS-positive cells accompanied by increased vacuolization in midgut cells and changes in storage material levels. Thus, a clear link between oxidative stress, vacuolization, autophagy, and the accumulation of storage materials in maintaining midgut cell homeostasis in the presence of NPs in the soil was suggested. However, further studies on cell death, cell proliferation, and antioxidant systems are needed.