Herbicide 2,4-D induces oxidative stress-independent genotoxicity and hepatic alterations in Dendropsophus minutus tadpoles

Abstract

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is widely used in Brazilian and global agriculture and is classified as a potential neurotoxic, carcinogenic, and endocrine-disrupting agent. This study evaluated the acute effects of 2,4-D on Dendropsophus minutus tadpoles by integrating genotoxic, mutagenic, oxidative status, histopathological, and ultrastructural biomarkers. Tadpoles at Gosner stage 25 were exposed for 96 h to three concentrations of the herbicide (2, 4, and 8 µg/L), including the limit allowed by CONAMA Resolution No. 357/2005. Evaluations included the Comet assay, presence of micronuclei and nuclear abnormalities, antioxidant enzyme activity and oxidative stress markers, hepatic histological alterations, and mitochondria–endoplasmic reticulum (ER) association by transmission electron microscopy. Results revealed genotoxic and mutagenic damage even at the lowest tested concentrations, as well as histological liver alterations such as sinusoidal dilation and hepatocyte vacuolization. Mitochondrial deformation and increased association with the ER were also observed, indicating possible cellular dysfunction. Conversely, no significant changes were found in antioxidant enzymes and oxidative stress markers. The integration of multiple lines of evidence demonstrates that 2,4-D, even at legally permitted concentrations, can cause relevant sublethal effects in tadpoles. These findings highlight the urgent need to review legal herbicide concentration limits in water bodies by incorporating more sensitive ecotoxicological approaches that better represent aquatic biodiversity.