Identification of chlorpyrifos and carbofuran residues in Kumari Beel, Bangladesh, and their coexposure effect on the cartilage development during zebrafish embryogenesis

Chlorpyrifos (organophosphorus) and carbofuran (carbamate) are widely used pesticides in Bangladesh, raising concerns about their environmental and ecotoxicological impacts, particularly on aquatic life. In this study, pesticide concentrations in water, soil sediment, and fish samples collected from Kumari Beel, Rajshahi, Bangladesh, were measured using HPLC analysis. Concentrations were highest in fish, ranging from 0.2097 to 0.2671 ppm. Concentrations in soil sediment and water ranged from 0.0789 to 0.1037 ppm and 0.0009 to 0.0011 ppm, respectively. Although the toxic effects of these pesticides are well-known, their specific impact on cartilage development remains poorly understood. To determine the effect of these pesticides on craniofacial cartilage, zebrafish embryos were exposed to individual and combined treatments of chlorpyrifos and carbofuran at concentrations up to 0.5 ppm. Single pesticide treatments did not affect body length, but coexposure significantly shortened body length and caused trunk curvature, indicating vertebral malformation. Alcian blue cartilage staining revealed that carbofuran and its coexposure with chlorpyrifos caused malformation in craniofacial cartilage, as manifested by significant alteration in the Meckel’s–palatoquadrate angle. Using EGFP-zTERF1 transgenic zebrafish, we found that coexposure to carbofuran and chlorpyrifos significantly affected the telomere diameter in the eye lens. RT-PCR analysis revealed that the cartilage-related genes chrdl2 and wisp2 were significantly affected by simultaneous pesticide exposure, where chrdl2 was upregulated, and wisp2 was downregulated. Overall, carbofuran and chlorpyrifos coexposure induce craniofacial cartilage malformation and alter associated gene regulations.