Impact of Well Water Hardness and Fluoride on Chronic Kidney Disease of Unknown Aetiology in Sri Lanka– Zebrafish as an Animal Model

Chronic kidney disease of unknown etiology (CKDu) is a significant health problem in Sri Lanka. Several hypotheses, such as soil geology, pesticide exposure, cyanotoxins, and prolonged dehydration, have been suggested as possible contributors to CKDu; however, the precise etiology remains unclear. This study aimed to investigate the potential impact of groundwater hardness and fluoride content on CKDu using zebrafish (Danio rerio) as an animal model. Sixty well water samples were randomly collected during the dry season from CKDu endemic regions in Sri Lanka, including Galnewa, Rajanganaya, and Medirigiriya in the North Central Province, Dehiattakandiya in the Eastern Province, and Agunukolapelassa (non-endemic area) in the Southern Province as a control. Water pH, conductivity, and dissolved oxygen were measured onsite and within the Sri Lankan Drinking Water Quality Standards (SLS 614: 2013). The highest mean water hardness (285.2 ppm) was recorded in Medirigiriya, and the highest mean fluoride (1.24 ppm) was recorded in Dehiattakandiya, both of which are identified as CKDu high-prevalence areas. The acute toxic effects of these parameters were assessed using zebrafish (Danio rerio) embryos. Ninety embryos were exposed up to 96 h post-fertilization (hpf) to water samples representing the highest and lowest water hardness and fluoride concentrations, as single and combined solutions. Embryonic mortality rate in combined exposure (highest hardness and fluoride) was 49.0±0.58%, while hardness alone was 16.7±0.58% and fluoride alone was 18.8±1.15%. Statistical analysis revealed a significant synergistic effect of hardness and fluoride combination (p< 0.05) on mortality compared to single exposure. Subsequently, combined exposure caused developmental delays (> 65%) and morphological abnormalities, such as bent body axis and yolk sac edema. Fluorescence images indicated that the damaged premature pronephros in the combined exposure group emitted bright green fluorescence compared to that in the single exposure group. Thus, the findings suggest that the synergistic nephrotoxic effect of high water hardness and fluoride can be a prominent cause of CKDu etiology in Sri Lanka.