Extra-legged flies and several other transgenerational developmental defects induced by environmentally relevant concentrations of N-nitrosodimethylamine in Drosophila melanogaster

N-nitrosodimethylamine (NDMA) is a ubiquitous disinfection byproduct (DBP) and a probable human carcinogen produced by reactions between organic nitrogen precursors and dichloramine. NDMA is the most frequently found nitrosamine in drinking water. It has been detected in food, pharmaceuticals, and effluent discharges from industries, posing a serious threat to ecological and human health. Although NDMA-induced toxicity has been well-studied in animal species, information on its long-term effects is scarce. This study aimed to comprehensively investigate the effects of NDMA exposure at environmentally relevant concentrations on Drosophila melanogaster development over four generations. Third-instar larvae were fed NDMA (88.3 ng/L-723.5 μg/L) for 48 h and later emerged as adult flies (parental generation). Subsequent offspring (F₁-F₃) were grown under NDMA-free conditions. Deformed flies, predominantly females, were found in both the exposed and untreated generations. However, malformation rates were significant only in the F₂ and F₃ generations. The entire body of the fly was affected below 2826 ng/L NDMA, and most abnormalities occurred in the thoracic area, mainly in the legs. Surprisingly, eight-legged and seven-legged flies were detected in the F₂ and F₃ generations. The notable transgenerational developmental defects underscore the long-term toxicity of NDMA to D. melanogaster, suggesting that harmful effects can be transmitted to offspring when NDMA is removed. This study provides evidence that extra-legged organisms can stem from exposure to environmental pollutants.