When Herbicides Interact: Synergistic, Additive, and Antagonistic Effects of 2,4-D- and Glyphosate-Based Herbicides in Physalaemus cuvieri Tadpoles

Abstract Considering the ecological relevance of amphibians and the widespread agricultural use of herbicide mixtures, investigations addressing their joint toxicological effects are increasingly necessary. Here, we investigated the effects of a mixture of two commercially formulated herbicides widely used in combination in agriculture—2,4-D-based herbicide (DBH) and a glyphosate-based herbicide (GBH)—on tadpoles of Physalaemus cuvieri , a bioindicator amphibian species. Chronic tests were performed using environmentally relevant concentrations and the maximum concentrations permitted in water bodies by current legislation. The mixture of these two pesticides impaired tadpole survival and induced abnormal behavior, characterized by distinct responses patterns. Nearly half of the analyzed tadpoles exhibited malformation by the end of assay. The integrated analysis of survival, behavior, and malformation data revealed antagonistic, additive and synergistic interactions between DBH and GBH in Physalaemus cuvieri tadpoles. These findings demonstrate that the herbicide mixture is nonetheless lethal to the tested species, inducing changes that can compromise its survival and overall living conditions, which may lead to significant biological impacts.