Difenoconazole-based fungicide formulations interfere with the embryogenesis of the two aquatic vertebrate organisms Xenopus laevis and Danio rerio

Amphibians are among the most threatened vertebrate groups worldwide, with more than 40% of amphibian species currently at risk. Besides habitat loss, pesticide exposure is considered a major driver of this decline. While herbicides such as glyphosate have been widely studied, the effects of fungicides - particularly triazoles - remain underexplored. Difenoconazole (DFZ) is one of the most commonly used triazole fungicides, characterized by environmental persistence. However, its impact on amphibian and fish embryonic development has not been systematically investigated. This study examined lethal and sublethal effects of two DFZ-containing formulations - Difcor® (Difcor; professional use) and COMPO® Duaxo Universal Pilz-frei (Compo; non-professional use) - on Xenopus laevis embryos. Embryos were exposed to environmentally relevant (0.01-1 mg a.i./L) and higher concentrations (10-100 mg a.i./L) for 14 days. Endpoints included mortality, morphology, cardiac and neural development, oxidative stress, gene expression, cell proliferation, and apoptosis. Both formulations caused concentration-dependent mortality with LC50 values of 12.34 mg a.i./L (Difcor) and 16.28 mg a.i./L (Compo). Sublethal effects occurred from 0.01 mg a.i./L, including reduced body length, smaller head and eye areas, pericardial edema, and altered heart rate and mobility. Difcor additionally impaired neural and cardiac development, reduced mhcα expression, and increased proliferation and apoptosis. Complementary analyses with the formulation Difcor in Danio rerio embryos confirmed concentration-dependent mortality and revealed sublethal morphological changes such as peracardial edema. Overall, DFZ-containing formulations exert pronounced lethal and sublethal effects in amphibian and fish embryos, highlighting the need to include amphibians and fish more systematically in the environmental risk assessment of triazole fungicides.