基于生物富集转化过程及其仿生技术的水中抗雄激素活性物质鉴别评价研究

The wide spread of antiandrogenic (AA) contaminants is one of the important factors leading to the occurrence of feminized male fish, and generates potential threat to the aquatic organism and even for regional drinking water security. Since the structures and species of AA contaminants are not clear, the identification of key active contaminant is desiderated to carry out. In order to explore the key factor that revealing the aquatic process of AA contaminants and the further identification and evaluation, this project tends to :(1) study the accumulation and transformation of AA contaminants in typical aquatic organism, and revealing the effects of the dynamic process to the activity of AA contaminants; (2) develop the bionic techniques according to the accumulation and transformation process of the contaminants based on passive sampling and in vitro degradation; (3) combine mass spectrum, chromatography and activity simulation for the stepped identification and risk assessment of non-target AA contaminants, on the basis of quantitative analysis of the relationship between physiochemical properties and chromatographic characteristic as well as the AA activities; (4) finally establish the identification and evaluation techniques of AA contaminants in aquatic environment including bionic accumulation and transformation, stepped identification of non-target compounds and risk assessment. Moreover, this technique will be put into use in surface water in the lower reaches of Yangtze River to find out the main AA contaminants, their spatial distribution and potential risk, which will support the management of regional hormone contamination.

抗雄激素活性物质在水环境中广泛分布，是导致雄鱼雌性化的重要因素，对水生生物甚至区域饮用水安全构成潜在威胁。但其结构、种类尚不明确，亟需开展关键活性物质的鉴别研究。围绕如何真实反映抗雄激素活性物质的水环境过程并加以鉴别评估这一关键问题，本项目拟研究抗雄激素活性物质在典型水生生物中的富集转化过程，揭示该动力学过程对抗雄激素活性的影响；在此基础上，研发基于被动采样与体外降解的活性物质富集转化仿生技术；并联合质谱、色谱、活性模拟，定量解析活性物质物化参数与色谱特征及活性效应的关系，从而实现非目标抗雄激素活性物质的逐级鉴别与风险评估；最终构建起集富集转化仿生、非目标物逐级鉴别、风险评估为一体的水中抗雄激素活性物质鉴别评价技术，并应用于长江下游地表水，明确其关键抗雄激素活性物质种类、空间分布及潜在风险，为区域环境激素污染控制提供科学依据。

本项目针对水体中关键毒物的鉴别过程鲜少考虑水生生物富集转化作用的突出问题，建立了基于鱼体富集转化特征的水体中雄激素活性效应评价与毒物鉴别技术。首先，研究了水体中典型雄激素活性物质双酚类和酞酸酯类物质在鱼体内的富集转化过程，发现生物体内的雄激素物质分布特征与水体有显著差异，极性(logKow<2)和中等极性（logKow:4-8）的物质具有较高的鱼体富集能力。鱼肝中识别出转化产物389种，涉及16种转化反应类型，包括N-乙酰化、氧化和酮形成等；在此基础上，基于多种商用材料（聚甲醛，POM；低密度聚乙烯，LDPE；硅胶，Silicone；聚二甲氧硅烷，PDMS），发现POM 和LDPE最符合雄激素物质在鱼体的富集特征，尤其是对中等极性雄激素物质，与鱼体富集相似性分别达到71±8%和69±6%，在此基础上引入的鱼肝S9体外降解，形成了集被动富集和体外代谢为一体的仿生富集转化方法，可获得鱼肝中64%的特征差异峰；继而，建立了非目标性雄激素物质的逐级识别方法，利用基于OPLS的特征峰提取，可从数万种检出峰中快速识别差异物质，在雄激素活性物质作用机制研究的基础上，发现受体-配体-共因子的相互作用定性、定量地决定了雄激素活性大小，并据此建立了雄激素活性物质结构与效应关系，实现了活性物质的高效虚拟筛选，进而有效去除非目标性雄激素物质鉴别过程中超过90%的假阳性结果，时间节省50%以上，提高了40%以上致毒物的鉴定等级，从而实现非目标抗性雄激素物质的逐级鉴别；最终，将构建的方法应用于在太湖典型区域地表水中，发现LDPE与POM相比，在野外情况下与实际生物富集更为接近，基于LDPE作为仿生富集材料，应用上述毒性评价与毒物鉴别方法，识别出太湖漫山地区雄激素活性物质污染最为严重，高达11.6 mol*kg-1氟他胺当量，其次为大浦、湖州与七都等地区，基于逐级识别方法，从5万余个峰中识别出有效应点位的68个非目标毒物，经过分级验证，其中14个达到一级，毒性贡献最高的是灭多虫、灭虫菊和戊唑醇。本研究为激素污染区域识别与优控物质清单建立提供了有力的技术支持。

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