Effects of Polyethylene Microplastic Particle Size and Concentration on Earthworm Fitness and Gut Microbial Functional Potential

Polyethylene microplastics (PE MPs) occur widely in soils, but how particle size and concentration relate to earthworm physiological responses and gut bacterial communities remains unclear. We exposed Eisenia fetida to four nominal PE particle sizes (15, 45, 100, and 200 μm) at three concentrations (0.5%, 2%, and 5%, w/w) for 30 days. Mortality, container-level mean body-weight change, cocoon production, gut bacterial communities, and PICRUSt2-predicted functional potential were assessed. Mean body-weight change was lower in all 12 PE MP treatments than in the control (Dunnett-adjusted p < 0.001), with the most negative mean value (−19.50 ± 3.80%) observed at 15 μm and 5%. Particle size and concentration had significant main effects on body-weight change, but no significant interaction. Mortality and cocoon production did not differ significantly from the control after adjustment. Neither Chao1 richness nor Shannon diversity differed significantly among groups. Global bacterial community differences were detected (PERMANOVA, R2 = 0.393, p = 0.0001), although unequal dispersion (PERMDISP, p = 0.0069) and nonsignificant BH-adjusted pairwise comparisons limited their interpretation. None of the ten dominant phyla or genera tested differed significantly among groups after false-discovery-rate correction, while LEfSe yielded exploratory discriminatory taxa. Only three KEGG Level 2 and six Level 3 treatment–control comparisons met the BH-adjusted significance threshold within their respective comparison families. Overall, container-level body-weight change showed the most consistent treatment–control differences, whereas evidence for treatment-specific gut bacterial and predicted functional changes was more limited under these high-exposure laboratory conditions.