Chronic Exposure to Pesticides in Amphibians: Assessing the Effects of Flupyradifurone and 2,4-D on the Development of Discoglossus galganoi and Pelophylax perezi

Amphibians are among the most threatened vertebrate taxa globally, yet remain underrepresented in pesticide risk assessments. We evaluated the chronic effects of the herbicide 2,4-D and the insecticide flupyradifurone—alone and in combination—on larval development in two amphibian species with contrasting ecologies: Discoglossus galganoi and Pelophylax perezi . Larvae were exposed throughout development to a range of concentrations (0.01–100 mg/L), and endpoints included survival, metamorphic timing, body condition, and malformations. Pelophylax perezi showed limited sensitivity, with effects only at the highest flupyradifurone concentration (100 mg/L). In contrast, D. galganoi displayed marked susceptibility to both pesticides and their mixtures, including elevated mortality, growth inhibition, delayed metamorphosis, and increased malformations—particularly under 2,4-D exposure. These species-specific responses underscore the limitations of current vertebrate surrogate models and the need for amphibian-inclusive risk assessments, particularly under realistic exposure scenarios involving pesticide mixtures. • Tadpoles were chronically exposed from larval stages through metamorphosis. Species from ephemeral habitats showed higher sensitivity to pesticides. • 2,4-D significantly increased larval malformations and reduced survival. • No major mixture effects were observed.