Toxic Effects of Triazole Fungicide Difenoconazole on the Early Development of African Clawed Frog, Xenopus laevis

Abstract

We investigated the toxic effects of difenoconazole on the development in the African clawed frog, Xenopus laevis. To test the toxic effects, frog embryo teratogenesis assays using Xenopus were performed. Embryos were exposed to various concentrations of difenoconazole (0-30 μM). LC100 for difenoconazole was 30 μM, and the LC50 determined by probit analysis was 27.19 μM. Exposure to difenoconazole concentrations ≥5 μM resulted in 10 different types of severe external malformation. Histological examinations revealed dysplasia of the eye, heart, liver, somatic muscle, and swelling of the pronephric ducts. The tissue-specific toxic effects were investigated with an animal cap assay. Blood cells were normally induced at a high frequency by mSCF and activin A. However, the induction of blood cells was strongly inhibited by the addition of difenoconazole. Electron micrographs of tested embryos showed the degeneration of somatic muscle and the shrinkage of microvilli on pronephric duct. The gene expression of cultivated animal cap explants was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). It revealed that the expression of the blood-specific marker(β -globin Ⅱ) and muscle-specific marker (XMA) were more strongly inhibited than the neural-specific marker(XEn2) by the addition of difenoconazole.