Combined exposure to microplastics and PFASs on earthworms: size and concentration effects

Microplastics (MPs) can act as carriers for per- and polyfluoroalkyl substances (PFASs), exerting both direct and indirect effects on soil organisms. However, few studies have systematically evaluated the combined effects of co‑exposure to MPs and PFASs. In this study, earthworms (Eisenia fetida) were exposed to polystyrene (PS) MPs of different sizes (30 nm and 10 μm) and PFASs for 21 days to investigate the toxicological mechanisms of single and combined exposure. The results showed that earthworm survival rates remained above 80% in all treatment groups throughout the 21‑day exposure period (p > 0.05). However, the earthworm growth rate (K_gr) was significantly inhibited (p < 0.05), with negative growth observed on days 14 and 21. At the end of the experiment, the K_gr in the combined exposure groups decreased by 65.53% compared to the control (CK) and by 59.86% compared to the single‑exposure groups. Histological observations revealed more severe damage to the earthworm epidermis and intestine under combined exposure, characterized by large vacuoles beneath the epidermis, varying degrees of atrophy in the circular muscle layer and intestinal epithelium, loosely arranged cells, and damaged villus structures. Regarding oxidative stress indicators, combined exposure significantly aggravated oxidative stress in earthworms. Specifically, compared to single exposure to MPs or PFASs, the combined treatment groups exhibited significantly increased activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as elevated contents of glutathione (GSH) and malondialdehyde (MDA). Notably, the size and concentration of MPs influenced the combined effects of PFASs and MPs on earthworms, with the strongest effects observed in the treatment with 30 nm MPs at 100 mg kg^-1. Furthermore, 16S rRNA sequencing results showed that co‑exposure to MPs and PFASs induced gut microbiota dysbiosis in earthworms. At the phylum level, the relative abundance of Proteobacteria increased significantly, while that of Actinobacteriota and Firmicutes decreased. Such alterations in microbial community structure may further affect the physiological functions and health status of earthworms. This study provides data support for the environmental and toxicological risk assessment of MPs and PFASs in soil ecosystems by investigating their effects on earthworms.