Polyethylene microplastics (PE-MPs) are commonly found alongside fungicides in farmland soils. However, the toxic effects of varying PE-MP sizes and concentrations on soil fauna in fungicide-contaminated soils are unclear. This study aimed to investigate the impact of different PE-MP sizes (13, 48, and 150 mu m) and concentrations (0.05% and 0.25%) on tebuconazole accumulation, oxidative stress, and gut bacteria in earthworms. The results indicated that earthworms exposed to MP13-H accumulated the highest tebuconazole on day 7, 19.77 % higher than tebuconazole alone, 7.27 % higher than MP48-H, and 10.30 % higher than MP150-H. MP13-H led to the most severe oxidative stress, significantly increasing the oxidative biomarkers catalase and peroxidase in earthworms. After 28 days, the expression of glutathione sulfotransferase genes was the lowest in the MP13-H group, while the antibacterial defense gene heat shock protein 70 and translationally controlled tumor protein were the highest, indicating severe DNA damage and increased toxicity to earthworms. Further, 150-mu m PE-MPs caused the most severe damage to the intestinal epithelium. Moreover, PE-MPs induced an increase in the abundance of specific gut bacterial community associated with oxidative stress. The study suggested that PE-MPs changed the migration of fungicides to earthworms, induced oxidative stress, altered gene expression, and modified the gut microbiota, thereby increasing the risk to earthworms.

Freeze-thaw (FT) aging can change the physicochemical characteristics of microplastics (MPs). The toxic impacts of FT-aged-MPs to soil invertebrates are poorly understood. Here the toxic mechanisms of FT-aged-MPs were investigated in earthworms after 28 d exposure. Results showed that FT 50 mu m PE-MPs significantly increased reactive oxygen species (ROS) by 5.78-9.04 % compared to pristine 50 mu m PE-MPs (41.80-45.05 ng/mgprot), whereas FT 500 mu m PE-MPs reduced ROS by 7.52-7.87 % compared to pristine 500 mu m PE-MPs (51.44-54.46 ng/ mgprot). FT-PP-MPs significantly increased ROS and malondialdehyde (MDA) content in earthworms by 14.82-44.06 % and 46.75-110.21 %, respectively, compared to pristine PP-MPs (40.56-44.66 ng/mgprot, 0.41-2.53 nmol/mgprot). FT-aged PE- and PP-MPs caused more severe tissue damage to earthworms. FT-aged PE-MPs increased the alpha diversity of the gut flora of earthworms compared to pristine MPs. Earthworm guts exposed to FT-aged-MPs were enriched with differential microbial genera of contaminant degradation capacity. FT-PE-MPs affected membrane translocation by up-regulating lipids and lipid-like molecules, whereas FTPP-MPs changed xenobiotic biodegradation and metabolism by down-regulating organoheterocyclic compounds compared to the pristine PE- and PP-MPs. This study concludes that FT-aged MPs cause greater toxicity to earthworms compared to pristine MPs.

Polyethylene microplastics (PE MPs) are the main MPs in agricultural soils and undergo oxidation upon environmental exposure. However, the influence of MP oxidation on phytotoxicity (especially for crop fruit) is still limited. This study aimed to explore the effect of PE MP oxidation on crop toxicity. Herein, a combination of plant phenotyping, metabolomic, and transcriptomic approaches was used to evaluate the effects of lowoxidation PE (LOPE) and high-oxidation PE (HOPE) on wheat growth, grain quality, and related molecular mechanisms using pot experiments. The results showed that HOPE induced a stronger inhibition of wheat growth and reduction in protein content and mineral elements than LOPE. This was accompanied by root ultrastructural damage and downregulation of carbohydrate metabolism, translation, nutrient reservoir activity, and metal ion binding gene expression. Compared with HOPE, LOPE activated a stronger plant defense response by reducing the starch content by 22.87 %, increasing soluble sugar content by 44.93 %, and upregulating antioxidant enzyme genes and crucial metabolic pathways (e.g., starch and sucrose, linoleic acid, and phenylalanine metabolism). The presence of PE MPs in the environment exacerbates crop growth inhibition and fruit quality deterioration, highlighting the need to consider the environmental and food safety implications of MPs in agricultural soils.