【作者】 张昌朋；

【导师】 郑永权；

【作者基本信息】 中国农业科学院 ， 农药学， 2010， 博士

【摘要】 咪唑乙烟酸是咪唑啉酮类除草剂,主要用于大豆田防除一年生和多年生禾本科杂草及阔叶杂草。该除草剂是长残留除草剂,其长期大量重复使用不仅对后茬作物产生药害,形成所谓的“癌症田”,而且破坏了土壤生态系统和资源可持续利用。本文结合田间调查及室内培养试验研究了咪唑乙烟酸影响土壤微生物多样性的机制,探讨了除草剂咪唑乙烟酸在土壤中的消解动态、吸附解吸附等环境行为。主要研究结果如下:1.建立了土壤中咪唑乙烟酸残留量的超高液相色谱-串联四级杆质谱测定方法。该方法采用固相萃取（SPE）为样品前处理方法,超高效液相色谱-串联四极杆质谱联用（UPLC-MS/MS）检测土壤中咪唑乙烟酸的残留量。土壤样品经0.1 mol L-1的氯化铵与氨水缓冲液（pH=10）超声提取、C18 SPE柱净化后,应用超高效液相色谱串联四级杆质谱仪多离子反应监测（MRM）定量检测,分别以碎片离子m/z 290>176和m/z 290>245进行定性,m/z 290>245外标法定量。在0.01～0.5 mg kg^-1添加水平范围内咪唑乙烟酸的平均添加回收率在83.47%～101.70%之间;相对标准偏差在4.15%～5.28%之间。该方法对咪唑乙烟酸的定量检出限（LOQ）为0.075μg kg^-1。该方法灵敏度高,操作简单,定量准确,可用于土壤中咪唑乙烟酸的残留分析及大量样品的快速检测。2.采用田间调查试验和室内培养试验相结合,研究了咪唑乙烟酸对土壤微生物多样性影响的机制,为了解和预测土壤质量、土壤生态系统安全及农药生态风险评估提供了重要的科学依据。采集同一地点不同施药历史的大豆田土壤,分别测定其残留量、微生物生物量碳和PLFA含量。结果表明,施药历史1年和2年的土壤中咪唑乙烟酸的残留量分别为1.62μg kg^-1和1.79μg kg^-1。施药历史2年的土壤中微生物生物量碳和总的PLFAs含量显著高于空白对照。施药历史2年的土壤中用PLFAs表征的细菌、真菌、革兰氏阴性菌、革兰氏阳性菌显著高于未施药土壤。施药历史1年和2年的土壤中表征微生物群落的GN/GP值和压力指数显著高于未施药土壤。主成分分析结果表明3种不同施药历史的土壤微生物群落结构之间存在着显著差异。田间调查结果表明除草剂咪唑乙烟酸在田间推荐施用量的条件下,显著改变了土壤微生物生物量和微生物群落结构。2种土壤（soil HS,粘壤土;soil QL,壤土）在培养箱中以一定的温湿度预培养后,添加一定浓度（0.1、1和10 mg kg -1）的咪唑乙烟酸。添加后一定的时间间隔采集土样,分别测定其残留量、PLFA含量和微生物生物量碳。结果表明,除草剂咪唑乙烟酸在培养最初一段时间显著改变了土壤微生物的群落结构。咪唑乙烟酸在2种土壤中的消解动态符合一级动力学方程,在2种土壤中的半衰期为63.0～111.8d;咪唑乙烟酸在2种土壤中降解的较慢,尤其是在土壤HS（具有更低的pH值）更是如此。不同浓度的咪唑乙烟酸显著降低了土壤微生物的生物量（微生物生物量碳和总的PLFA含量）。在2种土壤中,GN/GP和fungi/bacteria值随着除草剂浓度的增加而降低。高浓度的咪唑乙烟酸处理(1 mg kg^-1、10 mg kg -1)显著提高了2种土壤的压力指数（cyc17:0 + cyc19:0）/（16:1ω7c + 18:1ω7c）。主成分分析的结果表明除草剂咪唑乙烟酸改变了土壤微生物的群落结构。在0.1～10 mg kg -1范围内对2种不同类型土壤微生物群落的影响是一致的,且恢复周期相同（基本上都在培养60d时恢复）。3.采用批量平衡法研究了咪唑乙烟酸在2种土壤中的吸附、解吸附动力学以及吸附、解吸附等温曲线。吸附、解吸附动力学试验结果表明,咪唑乙烟酸在2种土壤中的分配系数（Kd）分别为3.42和2.01,吸附率分别为40.60%和29.19%,吸附能力顺序为土壤HS >土壤QL;解吸附分配常数（Kdes）分别为19.85和12.83,解吸附能力相反,土壤HS <土壤QL。吸附、解吸附等温曲线试验结果表明,咪唑乙烟酸在2种土壤中的吸附等温曲线均符合Freundlich等温方程,吸附系数（KFads）分别为8.33和1.33,吸附能力顺序为土壤HS >土壤QL;咪唑乙烟酸在2种土壤中的回归系数1/n均小于1,属于L?型吸附等温线,即随着农药浓度的增加,其吸附率反而降低。以上结果都说明了土壤HS对咪唑乙烟酸的吸附能力较土壤QL强。更多还原

【Abstract】 Imazethapyr, imidazolinone herbicide, is mainly used in soybean fields to control the annual grass weeds, perennial grass weeds and broad-leaved weeds. The long-term use and large amounts use of imazethapyr, long residual herbicide, caused not only succeeding crop injury, forming the so-called "cancer field," but also damaged the soil ecosystem and the sustainable use of resources. In this work, the mechanism of imazethapyr affecting soil microbial diversity were studied by the combination of field surveys and culture experiments in laboratory, and the degradation dynamics, adsorption behavior, and desorption behavior of imazethapyr were studied in soils. The results are as follows:1. A method for the determination of imazethapyr residues in soil was estimated based on solid phase extraction and ultra-performance liquid chromatography-mass spectrometry （UPLC-MS/MS）. The samples were extracted with 0.1 mol L-1 NH₄Cl/NH₃·H₂O （pH=10） buffer by ultrasonic wave, and cleaned up by C18 SPE cartridge. The imazethapyr residues were analyzed by UPLC-MS/MS under multiple-reaction monitoring mode. The qualitative results were obtained based on retention time, the precursor ion and two daughter ions, and the quantitative results were on the intention of the characteristic m/z 290>176 ion and m/z 290>245 ion. Average recoveries of imazethapyr in soil samples were found in the range of 83.47%～101.70% at three spiking levels from 0.01 mg kg^-1 to 0.5 mg kg^-1 with relative standard deviations of 4.15%～5.28%. Limit of quantification of imazethapyr was 0.075μg kg^-1. The method is simple and suitable for the routine and confirmation analysis.2. The effects of imazethapyr on soil microbial diversity were studied by the combination of field surveys and culture experiments in laboratory, which provided important scientific evidence for the understanding and prediction of soil quality, soil ecosystem safety and ecological risk assessment of pesticides.In the field surveys, the soils were collected at the same location in different application history soybean field for the determination of imazethapyr residue, soil microbial PLFA content and soil microbial biomass C. The residue in was 1.62μg kg^-1 in the soil （soil 1） with 1 year history of imazethapyr application, and 1.79μg kg^-1 for the soil （soil 2） with 2 years history of imazethapyr application. The microbial biomass C and total PLFAs of soil 2 were much higher than the soil （blank soil） with no imazethapyr application. PLFA profiles showed that fatty acids for Gram-negative （GN） and Gram-positive （GP） bacteria, as well as total bacteria and total fungi in soil 2 were much higher than blank soil. GN/GP and the stress level were much more than the blank soil. Principal component analysis of the PLFAs showed that the microbial community structure differed substantially among the three different soybean field soils. The application of the herbicide imazethapyr to soybean fields clearly changed the soil microbial biomass and shifted the microbial community structure.In the culture experiments, two agricultural soils, a silty loam （HS） and a loamy soil （QL）, were spiked with imazethapyr (CK, 0.1, 1 and 10μg g^-1) and incubated for 1, 15, 30, 60, 90 and 120 days. In addition, untreated controls received only water. The soil microbial community structures were characterized by investigating the phospholipid fatty acids （PLFA） and microbial biomass C. The results showed that the dissipation kinetics of imazethapyr in the two soils were described using a first order kinetics model, the half-time were 63.0～111.8d; there was a slow decline in imazethapyr that leveled out at longer incubation times,especially in soil HS with lower pH. The soil microbial biomass C and total concentration of PLFA were reduced by the addition of imazethapyr. Imazethapyr addition also decreased the ratios of GN/GP and fungi/bacteria. A larger stress level, measured as the ratio of PLFA （cyc17:0 + cyc19:0）/（16:1ω7c + 18:1ω7c）, was found in the high concentration (1 and 10μg g^-1) herbicide treatment groups. Principal component analysis （PCA） of the PLFA clearly separated the treatments and incubation times. Both soils showed different total PLFA concentrations and ratios of GN/GP and fungi/bacteria, but similar changes in the PLFA pattern upon soil treatment. The soil microbial community structure was shifted by the addition of imazethapyr, which recovered after 60 days. Our results demonstrated that the addition of imazethapyr shifted the microbial community structure, but that it recovered after a period of incubation.3. Adsorption, desorption kinetics and adsorption, desorption isothermal experiments were studied using batch equilibrium method. The results of adsorption, desorption kinetics experiments showed that distribution coefficient （Kd） of imazethapyr in the two soils were 3.42 and 2.01, the adsorption rates were 40.60% and 29.19%, adsorption capacity was soil HS > soil QL. The desorption distribution constant （Kdes） were 19.85 and 12.83, and desorption capacity was soil HS < soil QL. Adsorption, desorption isotherm results showed that adsorption isotherms were described by Freundlich isotherm equation, adsorption coefficient （KFads） were 8.33 and 1.33, adsorption capacity was soil HS > soil QL. Regression coefficient 1/n of imazethapyr in the two soils were less than 1, which indicating Langmuir adsorption isotherm and the adsorption rate reducing with the increase of the concentration of pesticides. Our results demonstrated that imazethapyr adsorption was higher in soil HS than that of soil QL.更多还原