Studies On Biological Effect And Degradation Of Chiral Fungicide Oxathiapiprolin Enantiomers

Oxathiapiprolin is the first piperidine thiazolidine isoxazoline fungicide.It is widely used in fruits and vegetables to prevent and treat many diseases caused by oomycete pathogens.The commercial oxathiapiprolin is racemate now.Chiral pesticides often exhibit stereoselective bioactivity,toxicity and environmental behavior.The data obtained were incomplete and inaccurate by conventional risk evaluations for chiral pesticides,these pesticides can bring potential risks to environmental safety and human health.In this thesis,the novel fungicide oxathiapiprolin was selected as the target chiral pesticide.The bioactivity,ecotoxicity and degradation of the oxathiapiprolin enantiomers were evaluated.The result of this study will provide scientific basis for reducing the use of oxathiapiprolin,accurate risk assessment and development of highly effective and low risk single-enantiomer products.The main contents and conclusions of this paper were as follows:1.The separation method of oxathiapiprolin enantiomers was established and optimized using chiral chromatography column combined with ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry.The effect of factors such as chiral stationary phases,mobile phase,flow rate and column temperature on the separation of oxathiapiprolin enantiomers were investigated.The baseline separation of oxathiapiprolin enantiomers was achieved on Chiralpak IG-3 column with acetonitrile and0.1%formic acid aqueous solution（90:10,v/v）as the mobile phase at the flow rate of 0.6m L/min,and column temperature of 35°C within 10 min,and resolution was 1.53.The specific rotation and absolute configuration of oxathiapiprolin enantiomers was identified by optical rotation detector and electronic circular dichroism spectra,and the elution order of R-（-）-oxathiapiprolin and S-（+）-oxathiapiprolin.Chiral residue analysis methods of oxathiapiprolin enantiomers in vegetables,fruits and soil were established.The solvent standard curves and matrix-matched calibration curves from 0.01 to 1 mg/kg showed excellent linearity of all enantiomers with R²≥0.9936.The mean recoveries of R-（-）-oxathiapiprolin in the five matrices were 75.1%～108.8%,and the mean recoveries of S-（+）-oxathiapiprolin were 84.0%～109.5%,intraday and interday relative standard deviation were all less than 6.3%.The minimum detection concentration was 0.01 mg/kg.The method reached the requirements of pesticide residue detection.2.The mycelial growth rate method was used to evaluate the bioactivity of oxathiapiprolin enantiomers against six kinds of pathogens（Phytophthora capsici,Phytophthora melonis,Peronophythora litchi,Phytophthora sojae,Phytophthora infestans,Phytophtora nicotianae）.The bioactivity of R-（-）-oxathiapiprolin was 2.5 to 13.3-fold higher than that of S-（+）-oxathiapiprolin against six kinds of oomycetes.Except for Phytophthora sojae and Phytophthora nicotianae,the bioactivity of R-（-）-oxathiapiprolin was 1.4 to 1.9-fold higher than that of racemate against four kinds of oomycetes.The interaction between the oxathiapiprolin enantiomers and the target oxysterol binding protein was studied by computational chemistry technology.The results showed that R-（-）-oxathiapiprolin had a more stable binding conformation with oxysterol binding protein,which may lead to the higher biological activity.3.The acute toxicity and morphological differences of the oxathiapiprolin enantiomers on zebrafish embryos,larvae and adult fish were studied.Besides,the growth inhibition and photosynthetic pigment in aquatic plant（Chlorella pyrenoidosa,Soirodela polyrhiza）were investigated at an enantiomer level.The results showed that the order of toxicity of three aquatic organisms was R-（-）-oxathiapiprolin>rac-oxathiapiprolin>S-（+）-oxathiapiprolin.The zebrafish embryos were more sensitive to oxathiapiprolin enantiomers.After exposure to R-（-）-oxathiapiprolin,zebrafish embryos showed obvious hatching delay,inhibition or deformity.The contents of photosynthetic pigment were significantly reduced by R-（-）-oxathiapiprolin.4.The stereoselective degradation of oxathiapiprolin in tomatoes and peppers and stereoselective metabolism of rat liver microsomes were investigated.The results showed that R-（-）-oxathiapiprolin was preferentially degraded in tomato and pepper.The enantiomeric fraction values were 0.40 and 0.43,respectively.In addition,R-（-）-oxathiapiprolin was preferentially metabolized in rat liver microsomes,and the enantiomeric fraction was 0.38.The results of enzyme kinetic revealed that the V_max,K_mand C_lint values of R-（-）-oxathiapiprolin were 1.4,1.1 and 1.3 times of the S-enantiomer,respectively.The results indicated that the preferentially metabolized of R-（-）-oxathiapiprolin was observed,and the R-enantiomer performed better binding with the metabolic enzyme.5.The stereoselective degradation of oxathiapiprolin in four types of soils（Jilin black soil,Jiangsu yellow brown soil,Tibet brown soil,Jiangxi red soil）under aerobic,anaerobic and sterilized conditions.The degradation order of oxathiapiprolin were was Jilin black soil>Jiangsu yellow brown soil>Tibet brown soil>Jiangxi red soil.Furthermore,the higher the content of soil organic,the faster degradation of oxathiapiprolin enantiomers.The results showed that the stereoselective degradation was observed with R-（-）-oxathiapiprolin preferentially degraded in all soils under the aerobic condition.The enantiomeric fraction values were ranged from 0.28 to 0.42.The oxathiapiprolin enantiomers were degraded more slowly in anaerobic and sterilized conditions with no significant stereoselectivity.The results indicated that the aerobic microorganisms play a major role in the degradation and stereoselectivity of oxathiapiprolin.Therefore,the development of R-（-）-oxathiapiprolin to replace its racemate can reduce the use of pesticides,redusdcvce pesticide costs,and reduce ecological risks.