【作者】 张青；

【导师】 王鸣华；

【作者基本信息】 南京农业大学 ， 农药学， 2017， 博士

【摘要】 手性农药对映体具有相同的物理属性,主要以外消旋体的形式存在,因此手性农药在开发过程中相对较难实现不对称合成和手性分离。当手性农药进入动植物体和环境中,大部分手性农药对映体表现出显著的立体选择性降解、生物活性、毒性和生态毒理的差异,手性农药的潜在安全风险引起广泛的关注。因此,传统的农药生态风险评估方式面临着巨大挑战。在对映体水平开展手性农药的环境行为,以及研究对映体之间立体选择性环境行为、活性、毒性和生态风险差异,可为手性农药提供系统准确的环境风险评估参考数据。本论文建立了乙虫腈对映体的拆分和手性残留分析方法,开展了乙虫腈在蔬菜及土壤中的环境行为研究,探讨了乙虫腈对映体立体选择性活性、急性毒性和生态毒理,同时采用电生理实验技术,研究了乙虫腈对映体与氯离子通道的靶标蛋白结合差异。主要研究内容和结论如下:(1)在反相HPLC条件下,建立了乙虫腈对映体的手性拆分的仪器分析方法,同时考察了不同流动相组成和柱温对乙虫腈手性拆分的影响。最终选择甲醇:水=65:35(v/v)作为流动相,流速0.7mLmin-1,柱温为35℃,检测波长225nm,对映体达到较好的分离效果,分离度达到2.522;通过比较实际测定的圆二色光谱吸收谱图与计算的谱图,确定了乙虫腈对映体的绝对立体构型。(2)建立了乙虫腈对映体在蔬菜、水果和土壤样品中的手性分析方法。通过优化的QuEchERS快速提取方法,结合弗罗里硅土固相萃取柱净化样品,系统的评估了该方法的精确度和准确度。添加回收结果显示,乙虫腈对映体在六种不同基质中的平均回收率在83.5%-104.8%,相对标准偏差为1.7%-8.0%。该方法对乙虫腈两个对映体在土壤和蔬菜中的检测限在0.008 mg kg-1,在水果中的检测限0.012 mg kg-1,定量限在0.019-0.049 mg kg-1之间。该分析方法能充分的满足食品和环境样品中手性农药乙虫腈对映体水平上检测要求。(3)研究了乙虫腈对映体在大鼠肝微粒体的作用下具有显著的立体选择性降解现象。随着孵化时间的增加,EF值不断增加,在30 min时EF值能达到0.62。同时发现对映体之间存在对映体转化现象,R-乙虫腈可以转化为S-乙虫腈,然而S-乙虫腈不能转化为R-乙虫腈。(4)开展了乙虫腈对映体在农作物中立体选择性环境行为研究。乙虫腈对映体在四种蔬菜中的降解均符合一级动力学规律,R-乙虫腈在黄瓜、番茄、菠菜和田间土壤中均优先降解,导致S-乙虫腈的富集,EF值在0.65到0.90范围。然而在青菜中的立体选择性行为相反,S-乙虫腈在青菜中优先降解,iR-体容易富集,EF值达到0.15。可能由于植物体存在不同的代谢酶系,导致对映体在不同的植物中表现出不同的立体选择性代谢行为。(5)首次开展了乙虫腈对映体在五种不同土壤样品中立体选择性环境行为研究。乙虫腈对映体在五地土壤中均能够发生显著的立体选择性降解行为,在120天时,南京和广东的土壤中仅有S-体存在,检测不到R-体。在180 d时,江西、海南和吉林土壤的EF值分别能达到0.89、0.90、0.74。通过进一步的实验研究,采用光学纯乙虫腈对映体在吉林土壤中的孵化实验,结果发现R-乙虫腈能逐渐转化为S-乙虫腈,乙虫腈的立体选择性行为是由对映体的转化导致。(6)开展了乙虫腈外消旋体和对映体对褐飞虱(Nilaparvata lugens)和豌豆蚜虫(Acyrthosiphon pisum)的杀虫活性和机理研究。通过室内生物测定,发现iR-乙虫腈活性分别是S-乙虫腈的4.3和2.2倍,外消旋体杀虫活性介于两个对映体之间。然而通过乙虫腈不同对映体和外消旋体与氯离子通道GABAC1受体的结合,发现对映体和外消旋体之间不存在显著的差异。乙虫腈对映体之间的显著活性差异,可能是手性农药乙虫腈具有选择性进入昆虫体内,或者两者在生物体内的代谢速度不一致,导致手性农药表现出立体选择性活性,进一步推动了手性农药立体选择活性差异机理的研究。(7)开展了乙虫腈外消旋体和对映体对羊角月牙藻(Selenastrum capriconrnutu)、赤子爱胜蚓(Eiseniafoetida)和稻螟赤眼蜂(Trichogramma japonicum Ashmead)急性毒性研究。关于羊角月牙藻的毒性测定,外消旋体的毒性比两个对映体的毒性都高,其中if对映体毒性较低,其毒性大小顺序均为:Rac>S>R-乙虫腈。外消旋体是S对映体毒性的1.55倍,是R对映体毒性的2.95倍。对于赤子爱胜蚓的毒性,对映体之间的毒性顺序与羊角月牙藻的完全不同,其中外消旋体对蚯蚓的毒性最小,然而S和iR体毒性相对较大,S和R体毒性差异不太明显。对于天敌非靶标生物稻螟赤眼蜂,S-乙虫腈毒性较高,其次为乙虫腈外消旋体,最低的为R-乙虫腈,其中S-乙虫腈毒性是iR-体的近6.0倍。对于不同的非靶标生物体,由于手性农药与生物体内的降解行为和结合方式有差异,导致乙虫腈对映体表现出完全不一样的毒理现象。(8)通过高通量测序,经过分析不同乙虫腈对映体处理后的土壤样品,土壤细菌群落组成在门水平上具有显著的差异。通过PCoA分析,进一步开展了不同乙虫腈对映体在土壤中对细菌群落组成结构的分析,在不同时间点以及不同构型的乙虫腈处理后,土壤细菌群落区系变异度具有显著的差异,说明细菌的群落组成变异与药剂的处理存在密切的关系。通过细菌群落网络结构分析,S-体处理后土壤网络的微生物连接和交互比体处理后土壤网络结构更紧密,R-体对细菌群落结构的影响较大。(9)采用高通量测序,比较快速的筛选出一些可能导致手性农药乙虫腈立体选择性环境行为的相关菌株的门和属。然后通过OTU的分析,进一步缩小了产生立体选择性行为的菌株范围,进一步确定到菌株的种。研究发现Phenylobacterium属以及OTU-10、23、32、38、135和228与乙虫腈的不同构型的降解和立体选择性行为关系非常密切。为筛选出导致立体选择性降解和转化的相关菌株提供了重要的捷径,也为寻找不对称合成手性农药酶系的寻找提供了重要参考。更多还原

【Abstract】 The enantiomers of chiral pesticides have similar physical and chemical properties in achiral environments.Chiral pesticides are primarily used as mixtures of enantiomers,or racemates.It is hard to asymmetric synthesis and chiral separation of chiral pesticide in the development process.However,most of stereoisomers always show differ biological activity,toxicity and ecotoxicology in chiral environment.The risk assessment of chiral pesticide caused the extensive concern.Therefore,the achiral analysis of chiral pesticides are caused great challenge for the risk assessment of pesticides.In recent years,stereoselectivity of bioactivity,toxicity and risk assessment have attracted increasing attention.The systemic assessments of the stereoisomers of chiral pesticide are very important to make a more accurate benefit-risk evaluation.In the present study,the enantioselective degradation of chiral insecticides ethiprole in greenhouse vegetables and five soils were investigated.And the optical pure enantiomer of ethiprole were also selected to investigate their enantioselective bioactivity,toxicology and ecotoxicology to some insects,nontarget organisms and microbial diversity.Furthermore,the enantiomer of ethiprole are against Musca Rdlac GABACls were also investigated.The main results were listed as followed:1.The separation of ethiprole enantiomers was performed using reversed-phase HPLC.The influences of different mobile phase composition and temperature of chiral column were discussed for chiral separation of ethiprole.Satisfactory separation was obtained by using a mobile phase of methanol water(65:35,v/v)at ultraviolet(UV)detection 225 nm,the flow rate was 0.7 mL min-1,and the column temperaturewas set as 35 ℃.And the Rs was arrived 2.522.The absolute configuration of ethiprole enantiomers was determined through the comparison of experimental and predicted ECD spectra.2.A simple and reliable method for the simultaneous determination of ethiprole enantiomers in fruits,vegetables,and soil samples was successfully established.An Florisil solid phase extraction(SPE)column was used by modified QuEchERS method for the cleanup of the samples.The method was evaluated by the specificity,matrix effect,linearity,precision and accuracy.The mean recoveries of two enantiomers ranged from 83.5-104.8%.The limit of detections(LODs)for two enantiomers in the six matrices were 0.008-0.012 mg kg-1,whereas the limit of quantifications(LOQs)were 0.019-0.049 mg kg-1.The RSD was ranged from 1.7%to 8.0%.The analytical method meet the requirement for simultaneous determination of ethiprole enantiomers in food and environmental samples.3.Stereoselective kinetic dissipation of ethiprole enantiomers in rat liver microsomes were investigated.The stereoselective degradation was occurred in rat liver microsomes.The EF values was decrease and was finally reached to 0.62.There is enantioconvergence was occurred in the incubation experiment.Only the(R)-ethiprole could translate into(S)-ethiprole.4.Stereoselective degradation of ethiprole enantiomers for vegetables was studied under field conditions.The degradation of ethiprole on four vegetables were accorded to first order kinetics.The(R)-ethiprole was preferentially degraded in cucumber,tomato,spinach and soil,and the EF values was ranged from 0.65 to 0.90.However,the degradation(S)-ethiprole was faster in pak choi,the EF values was finally reach to 0.15.There are different enzyme in different kinds of plants,which caused different stereoselective degradation of ethiprole.5.It was first time to report the stereoselective degradation of ethiprole in five different kinds of soil.The significant stereoselective degradation were observed,there is no(S)-ethiprole in Nanjing and Guangdong at 120 d.The EF values of soil in Jiangxi,Hainan and Jilin was reached to 0.11,0.10 and 0.26 at 180 d.For the incubation of pure enantiomers in Jilin soil,it was show that the(R)-ethiprole could translate into(S)-ethiprole.Therefore,the stereoselective behavior of ethiprole are caused by the enantioconvergence of enantiomers.6.The stereoselective bioactivity of ethiprole enantiomers were studied on Nilaparvata lugens and Acyrthosiphon pisum.The bioactivity of(R)-ethiprole was 2.2-4.3 times higher than that of(S)-ethiprole.And the rac-ethiprole was between them.The enantiomer of ethiprole are almost the same to against Musca Rdlac GABAC1s.The stereoselective bioactivity may be caused be the stereoselective absorption and degradation by the insects.This results have promoted further the mechanism of stereoselective bioactivity for chiral pesticide.7.The stereoselective toxicology of ethiprole enantiomers were studied onPseudokirchneriella subcapitata,Eisenia foetida and Trichogramma japonicum Ashmead.The rac-ethiprole have the high toxicity for the Pseudokirchneriella subcapitata,and the toxicity was follow the order:Rac->S->R-ethiprole.The toxicity of Rac-ethiprole was 1.55 and 2.95 times higher than the R and S-ethiprole.The toxicity is pretty different for Eisenia foetida.The Rac-ethiprole have the low toxicity and there have not different between the enantiomers.And the toxicity of S-ethiprole was around 6 times higher than S-ethiprole for Trichogramma japonicum Ashmead.The enantiomers are combined different for different protein crystal,which caused the different toxicity for different nontarget organism.8.The abundant phyla of soil microbial community are significantly different with the different treatment of ethiprole enantiomers in soil by high-throughput sequencing.Principal coordinates analysis of bacterial communities based on Bray-Curtis distance among different treatments.The coefficient of variance are significantly different with different time and enantiomers.It was show that the ethiprole have a close relationship with soil microbial community.’Network analysis showed that S-ethiprole application induced the more complex and closely linked microbial networks,which mainly dominated by bacteria,and the interactions within module and among mudules were better than R-ethiprole.And the R-ethiprole was behavior more damaging for soil microbial community.9.The high-throughput sequencing method is easy to find the microorganism phyla and genus which could enantioselective degradation and enantioconvergence of chiral ethiprole.The further OTU analytical method was tried to find out the microorganism species.It was show that the Phenylobacterium、OTU-10、23、32、38、135 and 228 have a close relationship with enantioselective degradation of chiral ethiprole.It is not only important to find out the enantioselective degradation and enantioconvergence of chiral ethiprole microorganism species,but also find out a good enzyme to achieve the asymmetric synthesis of chiral pesticide.更多还原