【作者】 梁宏武；

【导师】 邱立红； 周志强；

【作者基本信息】 中国农业大学 ， 农药学， 2014， 博士

【摘要】 手性农药在农业生产过程中的广泛使用,使其不可避免地进入到生物体、土壤和水体等环境系统中。手性农药的特点在于其对映异构体的物理、化学性质基本相同,但在具有手性特征的生物体内或环境中往往会表现出不同的生物活性、生态毒理及环境行为。然而,大多数手性农药至今仍是以外消旋体形式生产、销售和施用。为此,手性农药的使用与环境安全成为近几年新的热点问题。目前,有关手性农药对映异构体的环境行为和生态毒性研究还十分有限。本论文对几种典型手性农药对映体的环境行为和对非靶标水生生物的毒性进行研究。主要研究内容和结果如下：首先,利用高效液相色谱串联质谱结合反相手性固定相法,建立了三唑类手性杀菌剂氟环唑在葡萄与土壤中对映体水平的残留分析方法,进一步通过田间茎叶喷雾法开展了氟环唑在大田条件下葡萄与土壤中的立体选择性行为研究。结果表明：氟环唑对映体在葡萄果实内的降解符合一级动力学方程(R2>0.92),并且在喷药后2h就出现明显的立体选择性。在葡萄果实内,(-)-氟环唑平均半衰期为9.3天,而其对映体(+)-氟环唑的平均半衰期为13.2天,说明优先降解(-)-氟环唑,随着施药后时间的推移,造成葡萄果实中(+)-氟环唑的富集。但是,氟环唑对映体在土壤中的降解却不符合一级动力学方程,降解大致可以分为3个阶段：上升期(0d-3d)、快速降解期(3d-7d)和相对稳定期(7d-60d)；通过对映体EF值的计算,发现其土壤降解过程中也存在明显立体选择性,而且同样是(-)-氟环唑降解快于(+)-氟环唑,易造成土壤中(+)-氟环唑的富集。其次,比较了纤维素-三(3,5-二甲基苯基氨基甲酸酯)和直链淀粉-三[(s)-α-甲基苯基氨基甲酸酯]两种手性固定相对手性三唑类杀菌剂三唑酮(TF)及其手性代谢物三唑醇(TN)共6个对映体的分离效果,结果表明纤维素-三(3,5-二甲基苯基氨基甲酸酯)能实现三唑酮和三唑醇共6个对映体的较好分离。通过进一步对改性剂种类和含量的优化,最终获得了三唑酮和三唑醇对映体同时手性分离的最佳色谱条件,并利用液相色谱串联质谱结合反相手性固定相法,建立了三唑酮及其代谢产物三唑醇在麦粒、麦秆和土壤中同时提取、净化及分析的方法。在实际大田生产施药方式下,进行了三唑酮在麦粒、麦秆和土壤中的立体选择性降解代谢研究。结果表明：(1)在麦粒中三唑酮和三唑醇的残留量低于检测方法的最低定量限。(2)三唑酮在麦秆中降解迅速,两个对映体的降解趋势符合一级反应动力学规律,通过考察三唑酮的降解半衰期和EF值发现,在三个试验地中,S-(+)-三唑酮和R-(-)-三唑酮降解速率均没有显著性差别,即三唑酮在麦秆中的降解不具有立体选择性。(3)三唑酮两个对映体在土壤中的降解趋势同样也符合一级反应动力学规律,通过比较其半衰期和EF值来衡量对映体在土壤中的立体选择性强度,发现三唑酮对映体在北京和郑州采集的土壤中存在立体选择性降解,均为R-(-)-三唑酮被优先降解,造成S-(+)-三唑酮在土壤中富集。(4)进一步考查了三唑酮在麦秆和土壤中转化成三唑醇4个对映体的规律,发现三地实验中,麦秆中三唑醇4个对映体生成浓度的大小顺序依次为SR-(-)-TN> RR-(+)TN>RS-(+)-TN>SS-(-)-TN.但是,在土壤中4个对映体的生成浓度大小顺序则是RR-(+)TN>SS-(-)-tn>sr-(-)-tn>RS-(+)-TN第三,开展了己唑醇外消旋体及两个光学纯对映体对大型溞、斑马鱼(胚胎、仔鱼、成鱼)两种非靶标水生生物的急性毒性差异研究。结果表明：己唑醇外消旋体及两个光学纯对映体对两种水生生物的急性毒性以及对斑马鱼胚胎的发育毒性都存在立体选择性,其急性毒性及致畸效应大小顺序是：(-)-己唑醇>Rac-己唑醇>(+)-己唑醇。同时,斑马鱼胚胎、仔鱼、成鱼对己唑醇及其对映体的敏感性不同,且有较明显的差异,总体上96h的LCso大小顺序为胚胎>仔鱼(3d)>成鱼,即成鱼对己唑醇及其对映体更敏感,胚胎的敏感性最低。最后,开展了新型噁二嗪类杀虫剂茚虫威在斑马鱼体内的选择性富集以及不同对映体对斑马鱼胚胎-仔鱼的发育毒性研究。结果表明：(1)斑马鱼对茚虫威的富集存在明显立体选择性,其优先富集的是(-)-R-茚虫威；对映体在斑马鱼体内的代谢较快,在清水中恢复饲养阶段,(-)-R-茚虫威和(+)-S-茚虫威的平均半衰期分别为4.2和2.4d。(2)茚虫威外消旋体及两个光学纯对映体对斑马鱼胚胎-仔鱼发育的影响存在显著性差异。以斑马鱼的胚胎卵内自主运动、心跳次数、孵化率和致畸率为检测终点,发现茚虫威存在显著的对映体选择性毒性,其毒性大小顺序为：(-)-R-茚虫威>(+)-S-茚虫威>外消旋体。更多还原

【Abstract】 Widespread application of chiral pesticides results in their occurrence in the organism, plant, soil, water and other environmental systems. In general, the enantiomers of chiral compounds are different in biological activities, enantioselective behavior and eoctoxicity because of their different interactions with enzymes and other naturally occurring chiral molecules. However, most of the chiral pesticides are still marketed and applied in racemates. Consequently, the application and envinormental safety of chiral pesticides have been the new concern of scientific researches. So far, the environmental behaviors and eoctoxicity of chiral pesticides have received limited research. In this study, the enantiomeric separation, environmental behaviors, bioaccumulation and biotoxicity of several typical chiral pesticides were investigated.(1) A sensitive and convenient chiral liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for measuring epoxiconazole stereoisomers in grape and soil. Then the proposed method was successfully applied to investigate the possible stereoselective degradation of Rac-epoxiconazole in grape and soil under field conditions. The results of field experiment showed that the dissipations of epoxiconazole stereoisomers in grape followed first-order kinetics (R2>0.92) and stereoselectivity occurred in2h after spraying. The (-)-stereoisomer of expoxiconazole with half-life of9.3d degraded faster than (+)-stereoisomer with that of13.2d, which would resulted in relative enrichment of (+)-stereoisomer in grape. However, the stereoisomeric dissipations of expoxiconazole in soil were triphasic ("increase-decrease-steady") with lower dissipation rates, although it also occurred with preferential degradation of (-)-stereoisomer under field condition.(2) The separation of triadimefon (TF) and its major metabolite triadimenol (TN) including their simultaneous stereoseparation was studied; using two different polysaccharide-type chiral stationary phases inculding Cellulose-tris(3,5-dimethyphenylcarbamate)(CDMPC) and Amylose-tris(3,5-dimethylphenylcarbamate)(ADMPC) on reverse-phase high-performance liquid chromatography. Compared with two chiral stationary phases, CDMPC exhibited higher resolving ability for TF and TN. And after the optimization of other chromatographic conditions such as the mobile phase composition, a good enantioseparation of two enantiomers of TF and four stereoisomers of TN was successfully achieved. Then we developed a sensitive and rapid analytical method for simultaneous determination of TF and TN stereoisomers in wheat, straw, and soil by LC-MS/MS.Racemic triadimefon (TF) was applied to wheat and soil at three sites (Beijing, Huaibei, and Zhengzhou in China) under open field conditions. Its enantioselective degradation and stereoselective transformation to the major metabolite, triadimenol (TN), in wheat straw, grain and soil were investigated. At all three sites, the degradation of TF enantiomers in straw and soil followed first-order kinetics, In soil from Beijing and Zhengzhou,,R-(—)-TF was preferentially degraded; however, preferential enantioselective degradations were not observed in soil from Huaibei or in the straw from all sites. There were noticeable differences in the stereoselective formation of TN stereoisomers in all straw and soil samples. Different TN concentrations were found in the order of SR-(-)-TN> RR-(+)-TN>RS-(+)-TN>SS-H-TN in straw, and RR-(+)-TN>SS-(-)-TN> SR-(-)-TN> RS-(+)-TN in soil. Neither TF nor TN was found in wheat grain at harvest.(3)The toxicity of each isomer and racemate of hexaconazole (HZ) to two aquatic orgnisms including Daphnia magna (D. magna), adult zebrafish (Danio rerio), zebrafish embryos and larvae (3d old) were tested. For the aquatic organisms metioned above, significant enantioselectivity were observed in the acute toxicity and development toxicity among the enantiomers of HZ. The order of the acute toxicity to the aquatic organisms of the enantiomers of HZ was (-)-HZ> Rac-HZ>(+)-HZ. At specified stages (24,48,72,96and120hour postfertilization), spontaneous movement, survival, heart beats and hatching as well as non-lethal malformation like crooked body or edema were recorded in detail in zebrafish embryo tests. The results indicated that the (-)-enantiomer of HZ enantioselectively inhibited of hatching and heart beats of embryos and induced significantly more crooked body, yolk sac edema, pericardial edema than (+)-enantiomer. Moreover,(-)-enantiomer also enantioselectively caused morphological impairments, and inhibited the free swimming of the larvae of zebrafish.(4) The enantioselective bioaccumulation of indoxacarb in adult zabrafish and the effects of two indoxacarb enantiomers on zebrafish embryos and larvae were studied. The results demonstrated that the accumulation of (-)-R-indoxacarb was approximately13-fold more than that of (+)-S-indoxacarb in adult zabrafish, indicating a clear enantioselectivity of indoxacarb in bioaccumulation. The depuration of the two enantiomers followed first-order kinetics and the average half-lives of (-)-R-indoxacarb and (+)-S-indoxacarb were4.2and2.4d, respectively.The results also indicated that (-)-R-indoxacarb was more toxic than (+)-S-indoxacarb to embryo-larval zebrafish in causing mortality, inhibition of hatching and heart beats, and in inducing yolk sac edema, pericardial edema and crooked body. The enantioselective toxicity of indoxacrb was consistent with its enantioselectivity in bioaccumulation. The Rac-indoxacarb had the least potency of development toxicity as compared to its two enantiomers.更多还原