Toxicity assessment of the insect repellent nootkatone in developing zebrafish (Danio rerio)

Nootkatone is a naturally occurring sesquiterpenoid found in grapefruit that stands out for its repellent activity. However, its use as a commercial repellent requires a better understanding of its effects on non-target organisms, particularly aquatic species. In this context, this study aimed to evaluate the effects of nootkatone (0.1–10,000 µg/L) on developing zebrafish by assessing a series of endpoints, including survival, deformities, hatching rate, behavior, mitochondrial function, oxidative stress, apoptosis, gene expression, and untargeted lipidomics and metabolomics. Survival remained above 80% at concentrations up to 5,000 µg/L. Complete mortality was observed at 7,500 µg/L, and survival dropped to approximately 10% at 10,000 µg/L. The LC₅₀ for zebrafish larvae at 7 dpf was estimated at 4,900 µg/L. Hatching success and deformity incidence were not affected up to 2,500 µg/L. However, at higher concentrations, hatching decreased while deformities increased. Locomotor activity was significantly impaired at 5,000 µg/L, while no changes were detected at lower concentrations or in anxiety-related behavior. At 10,000 µg/L, nootkatone increased non-mitochondrial respiration and reduced ATP-linked respiration, with no detectable effects at lower concentrations. In addition, reactive oxygen species levels and apoptosis remained unchanged across treatments. Similarly, transcript levels of genes associated with oxidative stress and metabolism were not altered. Lipidomic and metabolomic profiling identified 192 lipid species and 16 polar metabolites. However, none showed significant differences among treatments. Our results indicate that nootkatone exhibits low acute toxicity in developing zebrafish. Further studies should assess its long-term effects across aquatic organisms and other species, contributing to the development of environmentally safer insect repellents.