Toxicity Of Commonly Used Agricultural Fungicides On Embryos Of Frogs (Xenopus Tropicalis) And Their Joint Effects

In recent decades, amphibian population decline has attracted wide attentions. Extensive using of agrochemicals is considered an important factor of amphibian decline. As a kind of main types of pesticides, fungicides use is amazingly increasing in recent years, especially new types of agricultural fungicides, such as strobilurin and SDHI fungicides. However, the relative eco-toxicology data of agricultural fungicides is very poor:especially little is known about developmental toxicity of these new fungicides on amphibians. In this study, developmental toxicity of six commonly used fungicides on embryos of Xenopus tropicalis were fully studied, in order to assess the ecological risk of fungicides on amphibians. These fungicides are trifloxystrobin and azoxystrobin, isopyrazam and bixafen, tebuconazole and myclobutanil, which stand for strobilurins, SDHI and triazole fungicides, respectively.Firstly, FETAX experiments were respectively performed for ten fungicides; and the actual exposure concentrations of these fungicides were assayed with high performance liquid chromatography (HPLC) analysis. Results showed that six fungicides presented lethal effects and the growth inhibition on embryos of X. tropicalis to different degrees. Medium lethal concentrations (LC50) of strobilurin fungicides, trifloxystrobin and azoxystrobin were 30.37μg/L and 196.59μg/L, respectively; LC50 of SDHI fungicides, isopyrazam and bixafen were 2.87mg/L and 1.84mg/L; LC50 of triazole fungicides, tebuconazole and myclobutanil were more than 9 mg/L. The results indicate that strobilurins are highly toxic to amphibian embryos at mg/L level, which is close to environmentally relevant concentrations. SDHI fungicides can induce lethal toxicity on embryos at high concentrations. The two triazole fungicides have no effect on the embryos survival at environmental concentrations.Secondly, teratogenic toxic effects and abnormal phenotype induced by six fungicides were fully analyzed. Medium teratogenic concentrations (TC50) of trifloxy strobin and azoxystrobin were 2.28μg/L and 84.13μg/L; TC50 of Isopyrazam and Bixafen were 0.31 mg/L 和 0.14 mg/L: TC50 of Tebuconazole and Myclobutanil. were 2.76 mg/L 和 8.98 mg/L. Strobilurin and SDHI fungicides showed strong teratogenic toxicity on embryos of X. tropicalis. Moreover, they presented the similar concentration-effect relationships and teratogenic phenotypes. The main teratogenic phenotypes include microcephaly, narrow fin, displacedcement gland, turbid lens of eyes, hypopigmentation and enlarged proctodaeum. The teratogenic index of strobilurin and SDHI fungicides is in the range of 2.3 and 13.3, which indicates strong teratogenic toxicity. Otherwise, tebuconazole and myclobutanil induced slight malformation of embryos at concentrations much higher than environmental concentrations.Thirdly, joint toxicity effects of strobilurins and SDHI fungicides on embryos were investigated using exposure design of equal toxicity. After several groups joint exposure to three strobilurin fungicides (pyriclostrobin. trifloxystrobin. azoxystrobin), the additive index (AI) values were in the range -0.12 to 10.11, by analysis of both lethal and teratogenic concentrations, which indicates synergistic or additive effect of strobilurins on embryos of X. tropicalis. After joint exposure to the SDHI fungicides, AI values were 0.79 and 99, which shows obvious synergistic effects of developmental toxicity. While AI values were in the range-1.34 to 0.03, indicating antagonism or additive effects of the joint exposure between strobilurins and SDHI fungicides. The synergistic or additive effects of these fungicides can enhance its harm to amphibians, and induce lethal and teratogenic toxicity to amphibian at low concentration levels.In the present study, comparative toxicology was performed between three commonly used types of fungicides. We found that strobilurin fungicides can induce lethality or teratogenicity on amphibian embryos at environmental concentration levels. This finding reveals the ecological risk of strobilurins. The synergistic or additive effect of these fungicides was also uncovered. It suggests that these fungicides can induce animal death and teratogenic toxicity at lower concentration levels. Strobilurin and SDHI fungicides showed the similar malformation phenotype. which provides crucial clues and supports for further exploring teratogenic mechanisms of mitochondrial targeting pesticides.