Chronic toxicity of pandemic-driven chloroquine diphosphate use in aquatic ecosystems: zebrafish (Danio rerio) as a model species.

Chloroquine diphosphate (CQD) is used for treating systemic and endemic diseases, with increased consumption during the COVID-19 pandemic. Its widespread use has led to increased detection in aquatic environments, where previous studies have reported oxidative stress and physiological disturbances in aquatic organisms. This study evaluated the chronic effects of CQD on zebrafish (Danio rerio) exposed for 21 days to environmentally relevant concentrations (0.6, 1.8, 5.4, 16.2, and 48.6 µg/L). Biomarkers related to metabolism (proteins, glucose, triglycerides), mucosal immunity (immunoglobulins), oxidative stress (protein carbonylation and lipid peroxidation), detoxification (glutathione S-transferase), and enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase), as well as histopathological alterations in gills, liver, and kidneys, were evaluated (p < 0.05). Alterations in metabolic parameters were observed, including decreased in the body from 1.8 µg/L and increase in the brain from 5.4 µg/L. Immunoglobulin levels increased at 5.4 and enzymatic activities increased from 1.8 µg/L. Oxidative stress increased from 0.6 µg/L in the body and 1.8 µg/L in the brain. Histopathological lesions were observed at all concentrations, with increasing severity at higher concentrations. Principal component analysis (PCA) showed concentration-dependent effects, with biochemical and oxidative stress biomarkers driving variance and histopathological alterations in gills, liver, and kidneys associated with higher CQD levels, while correlation analysis linked metabolic and biochemical markers to liver and kidney damage, with inverse relationships between energy reserves and oxidative stress and tissue alterations. These findings indicate that CQD may induce early biochemical and structural alterations in fish at environmentally relevant levels.