Transcriptomic and Metabolomic Analyses Reveal Intestinal Lipid Metabolic Disturbance in Loaches Co-Exposed to Polystyrene Nanoplastics and Imidacloprid.

Polystyrene nanoplastics (PS-NPs; nominal diameter, 100 nm; unmodified and negatively charged) and imidacloprid (IMI) are common freshwater contaminants, but little is known about their combined toxicity in benthic fish. In this study, loaches (Misgurnus anguillicaudatus) were exposed for 21 days to clean water, 100 μg/L PS-NPs, 78 μg/L IMI, or 78 μg/L IMI combined with 25, 50, or 100 μg/L PS-NPs. Each treatment contained 90 fish distributed among three replicate tanks. Survival, hepatic oxidative-stress indices, inflammatory gene expression, histopathology, and intestinal transcriptomic and metabolomic profiles were evaluated. Survival decreased across the co-exposure groups, with a significant overall difference among treatments (log-rank test, p = 0.0027). Hepatic CAT, SOD, and GSH levels decreased, whereas MDA increased, with the strongest changes observed in the higher PS-NP co-exposure groups. Pro-inflammatory genes, including IL-1β, IL-6, IL-8, and TNF-α, were upregulated, whereas IL-4 and IL-10 were downregulated. Histopathological injury of the gill, intestine, and liver was most pronounced in the NIH group. Transcriptomic analysis identified changes in pathways related to digestion, cell-cycle regulation, cholesterol metabolism, and lipid metabolism, while metabolomic analysis revealed alterations in glycerophospholipid, sphingolipid, fatty-acid, and carbohydrate metabolism. HMGCS1, CEL, CYP8B1, APOB, and APOC1 showed treatment-associated expression changes, with APOB displaying a non-linear response across the co-exposure groups. Overall, PS-NP co-exposure was associated with stronger IMI-related adverse responses under the tested conditions and substantial disruption of intestinal lipid homeostasis.