Integrated biomarker responses and multivariate assessment of polyethylene microplastics toxicity in the endemic freshwater fish Tor putitora

Microplastics (MPs) in freshwater environments are a significant ecological threat; however, their toxicological impact on endemic fish species is not well documented. This study aimed to assess the hazardous effects of polyethylene microplastics (PE-MPs) on endemic freshwater fish Tor putitora through a comprehensive assessment of integrated biomarkers. Fingerlings of T. putitora were exposed to PE-MPs at concentrations of 0, 0.1, 1, and 10 mg/L for 15 and 30 days. The findings revealed significant changes in hemato-biochemical, antioxidant, immunological, and neurotoxic biomarkers in fish exposed to PE-MPs, which varied with time and dosage. There was a notable (p < 0.05) reduction in red blood cells, hemoglobin, hematocrit, and platelet counts, whereas total leukocyte counts significantly (p < 0.05) increased in fish subjected to PE-MPs. Moreover, the levels of total protein, triglycerides, Fe, Na, and Cl⁻ were reduced, whereas glucose, urea, creatinine, Ca, K, and liver enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase) were increased in the treated groups. The increase in cortisol and thyroid stimulating hormone levels, coupled with a decline in tri-iodothyronine, thyroxine and insulin levels, suggests that PE-MPs caused stress in fish. Oxidative stress induced by PE-MPs was demonstrated by decreased catalase, superoxide dismutase, elevated malondialdehyde and glutathione activities in both gill and liver tissues. The immunological response in fish exposed to PE-MPs is enhanced through the induction of nitric oxide, respiratory burst, and lysozyme activity. Additionally, exposure to PE-MPs reduced acetylcholinesterase activity, underscoring the neurotoxic effects associated with PE-MPs. Correlation matrix and principal component analyses were employed to explore inter-biomarker relationships and to visualize treatment-specific clustering of responses. The findings indicate that particulate plastic pollution exerts harmful effects on fish, which may subsequently impact human health through the food chain. Future work should aim to enhance plastic waste control and strengthen pollution regulations in freshwater systems, particularly those supporting endemic fish species. Research should examine long-term effects of MPs and interactions with other pollutants like heavy metals and pharmaceuticals using omics tools and field studies.