Combined nephrotoxic effects of fluoride, water hardness, and microcystin-LR associated with CKDu in Sri Lanka: a zebrafish (Danio rerio) model approach.

Chronic Kidney Disease of unknown etiology (CKDu) is a major public health concern in several agricultural regions of Sri Lanka. In areas with a high prevalence of CKDu, elevated concentrations of fluoride and water hardness have been consistently reported in domestic wells, along with emerging evidence of cyanotoxin contamination. This study investigated the potential renal toxicity associated with environmentally relevant concentrations of fluoride, water hardness, and microcystin-LR (MC-LR) using a zebrafish (Danio rerio) model. Twenty groundwater samples were collected from the CKDu-endemic Padaviya region in the North Central Province. Mean fluoride and total hardness concentrations were 1.12 ± 0.64 mg L-1 and 424.2 ± 126.1 mg L-1, respectively, while MC-LR was detected in 45% of the samples (mean: 0.96 µg L-1). Zebrafish embryos (2 h post fertilisation) were exposed to the concentrations reflecting maximum and minimum field observation values. Single exposures to fluoride, hardness, and MC-LR resulted in moderate mortality and developmental abnormalities, whereas combined exposure produced significantly higher mortality, 50.0 ± 1.15% (p < 0.05), and increased incidence of oedema, spinal curvature, and growth retardation in larvae. Histopathological examinations of larvae exposed to the combined treatment revealed noticeable pronephric alterations, including tubular vacuolation, cellular swelling, and inflammation indicative of renal damage. Chronic exposure to zebrafish resulted in significant upregulation in Kidney Injury Molecule (KIM-1) gene expression, representing renal tubular injury. These findings suggest that the combined chronic exposure to multiple well water contaminants may induce nephrotoxic effects at concentrations relevant to CKDu-endemic surroundings. Although the results do not establish direct causation, they highlight the importance of considering mixture toxicity in environmental risk assessment and drinking-water management in CKDu-affected regions to address the health issue.