手性苯基吡唑类和新烟碱类杀虫剂对映体对蚯蚓的生态毒理差异及其机制研究

The enantiomers of chiral pesticides often have different toxicities to non-target organisms, however, this point is always ignored in conventional analysis with treating the enantiomers just as one compound. Consequently, the environmental risk evaluations of chiral pesticides based on traditional achiral methods are incomplete and nonspecific. After application, most of pesticides will eventually enter the soil. Then the effective way to solve the problem may well be conducting the study of environmental fate and ecotoxicology differences of chiral pesticide to soil biota in enantiomer level. In China, phenylpyrazole and neonicotinoid are two widely-used pesticides, which have posed a serious threat to ecological safety. Thus, three typical chiral insecticides, namely, ethiprole, flufiprole and dinotefuran were selected for the investigation. The enantioselective environmental fate of the three chiral pesticides in soil and earthworm were studied to illuminate the main metabolites and degradation products, and to find out the metabolic differences and influencing factors. Meanwhile, the ecotoxicology differences of enantiomers to earthworm and the mechanisms of enantioselectivity were also studied from different levels, including individual level, the cellular and subcellular level, the enzyme level, molecular and gene level. Carrying out this study is helpful not only to establish a more scientific and effective risk assessment system of chiral pesticides, but to provide a data support and theoretical basis for early warning detection, rational use and single-enantiomer pesticide development as well.

手性农药不同对映体对非靶标生物的毒性往往存在很大差异，目前人们在考察手性农药环境行为时常常忽略此点，极易造成环境风险评估结果不准确。农药施用后，绝大部分终将进入土壤。从对映体水平研究手性农药在土栖生物中的环境行为及生态毒理学差异是解决上述问题的有效途径。本课题针对农业生产中使用量大、生态安全问题较为突出的苯基吡唑类和新烟碱类手性杀虫剂，以乙虫腈、丁虫腈和呋虫胺为研究对象，首先考察其在土壤及蚯蚓体内的立体选择性降解、代谢行为及影响因素，通过鉴定主要代谢产物，阐明不同对映体在蚯蚓及土壤中的环境归趋，之后从不同角度（个体水平、细胞水平、亚细胞水平、组织水平、分子水平）研究手性农药对映体对蚯蚓的生态毒理学差异及其机制。本课题的开展有助于建立更为科学有效的手性农药风险评估体系,也为手性农药的环境风险预警、合理使用及单体研发等提供数据支持和理论依据。

手性农药对映体对非靶标生物的毒性可能存在很大差异，忽视此点进行的手性农药环境风险评估结果往往不够准确。农药施用后大部分进入土壤，对土壤中的蚯蚓产生毒害。从对映体水平研究手性农药在蚯蚓体内的环境行为及生态毒理学差异意义重大。本课题以农业生产中普遍使用的、生态安全问题较为突出的呋虫胺、乙虫腈和丁虫腈作为研究对象，筛选出Chiralpak®IF、Chiralcel®OD、Chiralpak®IC三种手性柱，优化分离条件，三种手性农药均达到了对映体分离。在此基础上，开发确证了蚯蚓、土壤基质中呋虫胺、丁虫腈的反相HPLC-MS/MS残留检测方法与乙虫腈的SFC-MS/MS残留检测方法，进一步探究了三种手性农药在土壤及蚯蚓体内的立体选择性累积、降解行为，结果发现三种手性农药在蚯蚓体内存在一定程度的对映体选择性累积行为，但不存在明显的富集现象，合理使用三种农药不会对蚯蚓产生毒害。急性毒性实验结果表明，呋虫胺对蚯蚓毒性较高，且存在对映体选择性毒性，毒性次序为S-(+)-呋虫胺＞rac-(±)- 呋虫胺＞R-(-)-呋虫胺；丁虫腈对蚯蚓低毒，毒性次序为R-(+)-丁虫腈＞ rac-(±)-丁虫腈＞S-(-)-丁虫腈；乙虫腈对蚯蚓急性毒性过低（14d-LC50高达5000.2mg/kg），没有从对映体水平做深入研究。课题从多个层面上探讨了呋虫胺、丁虫腈对映体对蚯蚓的选择性毒性差异机制。采用切片观察法比较了染毒蚯蚓组织细胞受损程度；通过酶活测定比较了染毒蚯蚓体内SOD、POD、CAT、MDA等活性变化趋势；通过彗星实验比较染毒蚯蚓体腔细胞DNA损伤及修复状况；利用荧光定量PCR技术检测了农药染毒对蚯蚓体内SOD、CAT、HSP70、TCTP等基因表达量的影响。实验结果显示，呋虫胺或丁虫腈对映体选择性急性毒性次序与染毒蚯蚓受药物抑制程度相符，蚯蚓所表现的对映体选择性毒性是相关酶的活性变化、DNA修复、基因表达等综合作用的结果。乙虫腈对蚯蚓的生态毒性研究结果表明，CAT酶、OTM值以及CAT基因表达量可作为土壤环境中乙虫腈污染指示物。本研究为手性农药环境风险预警、安全使用等提供数据支持和理论依据。