【作者】 刘甜甜；

【导师】 鲁润华； 周志强； 刁金玲；

【作者基本信息】 中国农业大学 ， 农药学， 2014， 博士

【摘要】 农药的广泛使用对农作物的增产起到了重要作用,但同时大量农药的使用也对环境生态系统造成了污染。农药在生产或使用过程中,可通排污、淋溶及直接施用等方式进入水生态系统,污染水体,对水生生物产生毒害作用。常用的农药中有一百多种具有手性,且大多数以外消旋体的形式生产使用,而手性农药对映体表现出来的生物活性往往是不同的,甚至是截然相反的。因此,在对映体水平上进行污染物的环境安全评估更准确可靠,是环境科学领域一个新的发展方向。颤蚓是一种典型的底栖动物,其对污染的环境有很强的耐受性,对水质和底质两方面的污染反应都敏感,常被作为考察水体污染程度的指示生物。本研究选择颤蚓作为研究对象,评价了4种常用手性农药在颤蚓体内的选择性富集、代谢,以及颤蚓的存在对农药对映体在水环境中的分布、代谢和降解行为产生的影响。同时测定在手性农药胁迫下颤蚓生理指标发生的变化,进一步对选择性产生的机理进行初步探讨。这是关于手性农药对映体在颤蚓体内选择性行为的首次报道。选定4种常用手性农药,氟虫腈、水胺硫磷、三唑酮和禾草灵,利用高效液相色谱手性固定相(CDMPC)法建立了氟虫腈、水胺硫磷和禾草灵及其代谢产物禾草酸对映体的分离方法,利用气相色谱手性固定相法对三唑酮及其代谢产物三唑醇的六个立体异构体进行了同时拆分。经溶剂萃取、SPE净化,建立了农药对映体在颤蚓、土壤和水样本中的残留分析检测方法。利用水体染毒和底层基质染毒两种培养方式对选定手性农药的富集、代谢行为进行了研究。结果显示,在两种方式培养颤蚓的富集实验过程中,氟虫腈、水胺硫磷和三唑酮均能在颤蚓样本中检出,表明颤蚓在不同的生活环境下对该3种农药均有一定程度的富集。而在禾草灵富集试验中,颤蚓样本中未检测到禾草灵,但检测到了其水解产物禾草酸。进一步实验得出,由于颤蚓的存在禾草灵在水中快速水解为禾草酸,所以本文仅研究禾草酸对映体在颤蚓中的富集行为。氟虫腈、水胺硫磷、三唑酮、三唑醇和禾草酸在染毒水培养富集过程中具有明显的对映体选择性,颤蚓体内的浓度R-(-)-氟虫腈>S-(+)-氟虫腈、(-)-水胺硫磷>(+)-水胺硫磷、S-三唑酮>R-三唑酮、(1R,2S)-三唑醇>(1S,2R)-三唑醇>(1R,2R)-三唑醇>(1S,2S)-三唑醇、R-(+)-禾草酸>S-(-)-禾草酸。而在底层基质染毒培养试验中,只有氟虫腈和三唑醇富集过程中具有明显的对映体选择性,推测颤蚓在不同暴露方式下通过不同的途径富集环境中的农药,不同富集途径中参与的手性选择因子不同导致选择性水平出现差异。对颤蚓体内三唑酮和三唑醇的代谢行为进行了研究,发现三唑酮的代谢速率较三唑醇快,经水培养的代谢过程也具有对映体选择性。在选定农药在培养基质中的分布和代谢行为研究中发现,氟虫腈、水胺硫磷和三唑酮在底层基质中均表现出明显的代谢。在添加颤蚓的底层基质中农药的代谢速率显著高于对照组,说明除去土壤微生物的代谢作用外,颤蚓表现出对底层基质污染的净化作用。而对上层水中农药的检测发现,颤蚓的存在有利于底层基质中的农药向上层水扩散,使底层固体基质中的污染物得以释放,可能对水系统中上层水生生物造成胁迫。实验中发现,禾草灵在水体中可发生自然水解,降解速率低且不存在对映体选择性,颤蚓的存在不仅可以显著加速禾草灵的水解,而且使水解过程发生显著的对映体选择性,生成R-(+)-禾草酸浓度显著高于S-(-)-禾草酸。为了探讨选择性产生的原因,进行了光学纯禾草灵及禾草酸在水中单体培养试验。结果发现,禾草灵在水解过程中会出现构型翻转,而禾草酸对映体在实验过程中保持光学稳定,表明选择性的产生是由于禾草灵两个对映体之间转化速率的差异所致。利用颤蚓匀浆液制备了颤蚓的粗酶液,并建立了粗酶液蛋白含量、MDA含量、CAT, GST及GR酶活性测定方法。利用水培养染毒,研究了氟虫腈及其对映体对颤蚓CAT和GR酶活性的影响,结果发现氟虫腈及其对映异构体对颤蚓抗氧化酶的影响较小,在实验选定浓度下酶活性均未检测到显著变化；在水胺硫磷及其对映体对颤蚓CAT和GR酶活性及MDA含量的影响研究中发现,暴露浓度与酶活性之间不存在明显的剂量-效应线性关系,但在同一浓度下,对映体之间对抗氧化酶活性影响存在显著差异,对酶活影响程度的大小为(+)-ICP> Rac-ICP≥(-)-ICP, MDA含量变化不显著；在三唑酮及三唑醇对颤蚓CAT、GST、GR酶活性和MDA含量的影响研究中发现：对于三唑酮而言,CAT和GR酶活性均与浓度存在显著的剂量-效应关系,但与暴露时间之间存在的规律不如浓度显著,另外经三唑酮处理后,颤蚓体内MDA的含量显著增多,但三唑酮对颤蚓体内总蛋白含量和GST酶活性的影响不显著。对于三唑醇来说,CAT、GST和GR酶的活性均与浓度之间存在剂量-效应关系,同时CAT和GST酶活性与暴露时间存在一定的关系,GR酶活性随时间变化不显著,三唑醇对MDA含量的影响不如三唑酮显著。更多还原

【Abstract】 Pesticides play very important role in increasing crop production, however, tons of pesticides enter into the environment as contaminants and affect the ecosystem greatly. In the process of production or use, pesticides can entery into aquatic ecological system via direct spraying, rain wash and surface runoff, causing toxic effect on aquatic organisms. As we kown that almost25%current used pesticides were chiral, they have two or more enantiomers. Previous studies have shown that the enantiomers of a chiral pesticide have identical physical and chemical properties but they may perform different behavior in the processes of absorption, accumulation and degradation when confront with chrial environment. This makes it necessary to study the behavior of the enantiomers of chiral pesticides individually to understand the effects more comprehensively on the environmental safety and public health. As a typical aquatic benthic organism, Tubifex tubifex always be found in ecosystems contaminated by organic matter with a low level of oxygenation. These worms are very widely distributed and frequently dominant in freshwater benthic communities. They have an intimate contraction with the solid phase and the pore water of the sediment, burrowing the anterior part in the sediment and undulating the posterior part in the overlying water. Because of the endobenthic lifestyle and stress resistance, tubifex has been designated as a representative freshwater benthic infauna for aquatic system bioassays. In the present work, we study the differences in bioaccumulation and degradation behaviors of individual enantiomers of four chiral pesticides in tubifex. We also evaluated the effects of the tubifex on diffusion and degradation of pesticides. This is the first report about enantioselective behaviorof chiral pesticides in tubifex.Four chiral pesticides were studied in this work and the enantiomers of pesticides were separated on high-performance liquid chromatography (HPLC) or gas chromatography (GC) based on chiral stationary phases (CSP).The enantiomers concentrations of the selected chiral pesticides in tubifex, soil and water samples were analysed by solvent extraction and SPE clean-up. Tubifex worm were cultivated in two different contamination systems, including spiked water and spiked soil, to compare the influence of different uptake routes on bioaccumulations and excretions. In bioaccumulation experiments, the results indicated that tubifex can accumulate fipronil, isocarbophos (ICP), triadimefon (TF) and triadimenol (TN). However, diclofop-methyl (DM) was not detected in worm during the whole accumulation process. Furhter studies showed that DM can be rapidly hydrolysis to diclofop acid (DC), resulting only the metabolite DC was detected in tubifex. The bioaccumulations of fipronil, ICP, TF, TN and DC were enantioselective in tubifex tissue in spiked water treatments, with the concentration orders R-(-)-fipronil>S-(+)-fipronil,(-)-ICP>(+)-ICP,S-TF>R-TF,(1R,2S)-TN>(1S,2R)-TN>(1R,2R)-TN>(1S,2S)-TN, R-(-)-DC> S-(+)-DC. However, in the spiked soil treatments, enantioselective accumulations were not detected except for fipronil and TN. It was speculated that, there were different chiral selective factor in different uptake routes. The excretions of TF and TN in tubifex were also studied, the results showed that the excretion rate of TF was higher than that of TN, what’s more the excretion of the two fungicides were also enantioselective in spiked water treatment.Studies about the the effects of the tubifex on diffusion and degradation of pesticides showed that, with the presence of tubifex worms, higher concentrations of pesticides in overlying water and lower concentrations in soil were detected than that in the absence of tubifex treatment during the whole exposure period. This means that tubifex has positive functions in pesticides’ diffusion from bottom solid substrate to overlying water and in the degradation of the soil-associated pesticides. We also found that, the present of tubifex can accelerated the hydrolysis of DM and make the hydrolytic process have enantioselectivity. Further studies showed that DC enantiomers were configuration?lly stable in water, however the DM enantiomers experienced inversion of configuration in hydrolytic process.Crude enzyme was prepared using tubifex homogenates, and methods that determining the content of protein and MDA, the enzyme activities of CAT, GST and GR were developed. Changes in CAT and GR activities were tested by exposing tubifex to solutions rac-fipronil and its enantiomers, respectively. The results showed there’s no significant effect on the enzyme activities. Studies on the ICP and its enantiomers showed that, there’s no obvious dose-response relationship, but at the same concentration, there’s significant effect of ICP on the activities of CAT and GR and the toxic effects were enantioselective, following the order (+)-ICP> Rac-ICP≥(-)-ICP. ICP has no significant influence on the content of MDA. For TF experiment, the changes of CAT and GR activities showed a significant dose-response relationship with TF. Meanwhile, there’s a significant increase for the content of MDA, but TF performed no effect on the content of protein and activity of GST. For TN experiment, there’s a significant dose-response relationship between the fungicide and activities of CAT, GST and GR. There’s also a significant increase for the content of MDA, but the level was lower than that of TF. Changes on GR activity were not sifnifincant.更多还原