Comparative Toxicity Of Difenaconazole And Its Chiral Isomers On Two Aquatic Organisms

Difenoconazole, a triazole fungicide, can destroy the cellular structure and function by inhibiting the biosynthesis of ergosterol in bacterial cell. There are two chirality carbon atoms in difenoconazole which make four of different isomers of difenoconazole. Japanese medaka and daphnia magna are model organisms. They have been widely studied in science experiment because of their large amout of reproduction, short life circle and easy raising charactors. In this experiment, different toxicology effects of difenoconazole and its chiral isomers on Japanese medaka and daphnia magna were studied.Based on reported literature, the absolute configuration of difenoconazole chiral isomers were identified by their NMR (NOESY,’H NMR and13CNMR) and optical activity.At1.00mg/L, hatchibility of Japanese medaka embryo were inhibited by all the difenoconazole isomers except (2R,4S)-difenoconazole. Daily hatchability of Japanese medaka embryo were inhibited by rac-difenoconazole,(2S,4R)-difenoconazole,(2R,4R)-difenoconazole and (2S,4S)-difenoconazole at0.03,0.17and0.50mg/L. Japanese medaka embryo treated with rac-difenoconazole and its chiral isomers reached100%hachibility in an order of their concentration value except (2R,4R)-difenoconazole. Different from rac-difenoconazole and other difenoconazole chiral isomers,(2R,4S)-difenoconazole can promote the hatchability of Japaneses medaka embryo at0.03and0.17mg/L.As with the blank control, the peak of hatching period were observed on day10-13when treated with different concentrations of (2R,4S)-difenoconazole and (2R,4R)-difenoconazole. At0.03and0.17mg/L, Japanese medaka embryo treated with rac-difenoconazole had the same peak of hatching period as the blank control; while at0.50mg/L, its peak hatching rate were observed on day18-21, however, no significant difference were found. The peak of hatching period of Japanese medaka embryo were not found when treated with0.03and0.50mg/L (2S,4R)-difenoconazole.(2S,4S)-difenoconazole delayed the peak of hatching period of Japanese medaka embryo at0.50mg/L.Potetial effects of rac-difenoconazole and its chiral isomers on hatching enzyme gene expression of Japanese medaka were also studied. Results showed that at0.17mg/L, when treated with rac-difenoconazole, expression of HCE, LCE were both significantly inhibited. When treated with (2R,4S)-difenoconazole, gene expression of HCE were inhibited at0.50mg/L. Expression of HCE were inhibited at all of the three concentrations designed in our test when Japanese medaka embryo were treated with (2S,4S)-difenoconazole. Endocrine effects of rac-difenoconazole and its chiral isomers on male Japanese medaka were also evaluated. Amount of the Vtg and Vtg1 gene expression in liver of male Japanese medaka were studied. Results showed that, difenoconazole and its chiral isomers significantly increased the amount of Vtg in male Japanese medaka. Vtg1gene was also up-regulated when treated with rac-difenoconazole and its chiral isomers.Acute toxicity and antioxidase activities of daphnia magna were studied when treated with difenoconazole and its chiral isomers. According to our experiment, rac-difenoconazole and its chiral isomers were all high toxicity to daphnia magna. The toxicity order was:(2R,4S)-difenoconazole<[(2R,4R)-difenoconazole;(2S,4R)-difenoconazole; rac-difenoconazole]<(2S,4S)-difenoconazole. CAT activity were significantly inhibited when treated with (2S,4S)-difenoconazole.