Potential toxicological assessment of 4-Methylimidazole (4-MEI) in Ctenopharyngodon idella; LC50 determination, acute and sublethal effects on physiological, immunological, and histopathological indicators.

4-Methylimidazole (4-MEI) is a widely used industrial compound and a common byproduct of caramelization in various foods and beverages. It enters aquatic ecosystems through multiple ways and is a well-recognized toxic and potentially carcinogenic substance in humans. In this study, the median lethal concentration (LC₅₀) of 4-MEI for Ctenopharyngodon idella over 96 h was determined for the first time in fish and found to be 26.79 mg/L. Subsequently, to assess its toxicological impact, C. idella fingerlings were exposed to an acute concentration (LC₅₀ for 96 h) and two sublethal concentrations, 1/5th (5.35 mg/L) and 1/10th (2.69 mg/L) of the LC₅₀, over 35 days. Acute exposure resulted in significant adverse effects, including substantial increases in white blood cell count, total immunoglobulins, lysozyme activity, phagocytic activity, respiratory burst, and the enzymatic activities of AST, ALT, ALP, and LDH. There was also upregulation of key immune-related genes: TNF-α, Interleukin-1β, Lysozyme-C, and Lysozyme-G. In contrast, red blood cell count, hemoglobin levels, hematocrit, total protein, albumin, globulin, and the albumin-to-globulin ratio were significantly reduced. Sublethal exposures produced concentration-dependent toxic effects. Histological analysis revealed that fish exposed to acute concentration exhibited marked structural alterations (> 50%) in liver and gill tissues. However, the sublethal concentration exposure (1/5th and 1/10th of LC₅₀) induced moderate (< 50%) to mild (< 10%) histopathological changes, respectively. These findings demonstrate the toxic effects of 4-MEI on fish and highlight the urgent need for increased awareness regarding its environmental risks and impact on aquatic life.