Prioritization of Contaminants of Emerging Concern in wastewater treatment plant discharges using transcriptional effects in caged fish. Pimephales promelas

We applied an effects-based approach to determine if the impact of specific chemicals in mixtures discharged by waste water treatment plants could be linked to well-defined gene expression changes in exposed fish. Fathead minnows were deployed in cages for 2, 4, or 8 days at three locations near two WWTP discharge sites in the Saint Louis Bay, Duluth, MN and one upstream control site. The impact of 51 chemicals detected in the surface water on gene expression in ovaries of caged fish was determined using exposure activity ratios, existing knowledge on chemical:gene relationships, and analysis of gene expression co-variance with surface water chemistry. Thirty-two chemicals could be linked to gene expression and 12 were confirmed by other sources such as the Comparative Toxicogenomics Database (CTD). Chemical:gene associations determined by co-variation were supported by exposure activity ratios sufficient to cause biological effects, known chemical:gene associations from CTD, and upstream regulator analysis of the Ingenuity knowledge base. The analyses provided multiple lines of evidence that bisphenol A and estrone are likely sources of estrogenic effects on gene expression found at the study sites. Overall design: Fish were deployed at four sites in the St Louis River near Duluth, MN, USA. One site upstream of the WWTPs (Fond du Lac, FDL; N 46° 39.517, W 92° 16.951) was chosen as ‘reference’ location. Two sites were located near the Western Lake Superior Sanitary District (WLSSD) discharge. One (WLSSD-1; N 46° 45.460, W 92° 7.227) was located 10 m from the discharge, while the second (WLSSD-2; N 46° 45.347, W 92° 7.213) was located further in the main channel, 100 m from the discharge. A fourth site was located near the Superior Municipal Treatment Plant discharge (SMTP; N 46° 43.758, W 92° 4.125). A detailed description of the caging system can be found in Kahl et al.32. Briefly, three buoys anchored to the bottom sediment at each site. Three cages of fish, each containing 6 male and 6 female adult fathead minnows (5-6 month old) were attached to a buoy and its anchor, suspending cages at a depth of 1-2 m. After 2, 4, or 8 d of exposure, fish from two cages, each from a separate buoy, were transferred into separate buckets containing surface water collected at the appropriate site, and transported to the laboratory (transit time <60 min) for sampling. Animals were not fed during transit nor in the field. Fish were individually anesthetized and euthanized with MS-222 (Argent, Redmond, WA, USA), weighed, and evaluated for any external lesions. Blood and tissues (liver, gonad) were collected and stored at -80°C until extracted and analyzed. The normalized data were not available at the time of submission and therefore not included in these records.

本研究采用基于效应的研究方法，旨在探究污水处理厂（waste water treatment plant, WWTP）排放混合液中特定化学品的影响，是否与暴露鱼类的明确基因表达变化存在关联。研究将黑头软口鲦（fathead minnow）置于笼中，于美国明尼苏达州杜鲁斯市圣路易斯湾的两处WWTP排放点附近的三个点位，以及一处上游对照点位，分别暴露2、4或8天。

本研究通过暴露活性比、已报道的化学品-基因关联知识，以及基因表达与地表水化学特征的协方差分析，明确了地表水中检出的51种化学品对笼养鱼类卵巢基因表达的影响。其中32种化学品与基因表达变化存在关联，另有12种经《比较毒理基因组学数据库》（Comparative Toxicogenomics Database, CTD）等其他来源验证。通过协方差分析得到的化学品-基因关联，得到了足以引发生物学效应的暴露活性比、CTD中已收录的化学品-基因关联，以及Ingenuity知识库的上游调控因子分析结果的支持。本分析提供了多维度证据，表明双酚A与雌酮极有可能是研究点位中观察到的雌激素类基因表达效应的来源。

试验设计概况：试验鱼被部署于美国明尼苏达州杜鲁斯市附近圣路易斯河的4个点位。其中WWTP上游的点位（Fond du Lac, FDL；北纬46°39.517，西经92°16.951）被选为“参照点位”。两处点位位于苏必利尔湖西区卫生区（Western Lake Superior Sanitary District, WLSSD）排放口附近：第一处（WLSSD-1；北纬46°45.460，西经92°7.227）距排放口仅10米，第二处（WLSSD-2；北纬46°45.347，西经92°7.213）则位于主河道内，距排放口100米。第四处点位位于苏必利尔市市政污水处理厂（Superior Municipal Treatment Plant, SMTP）排放口附近。

笼养系统的详细说明可参见Kahl等（文献32）的研究。简要而言，每个点位布设3个锚定在底泥上的浮标。每个浮标及其锚具上挂载3组试验笼，每组笼内饲养6尾成年雄性与6尾成年雌性黑头软口鲦（5-6月龄），将试验笼悬挂于水下1-2米处。在暴露2、4或8天后，从两个不同浮标的试验笼中分别取出试验鱼，转移至盛有对应点位采集的地表水的独立桶中，转运至实验室（转运时间<60分钟）进行样本采集。试验鱼在野外暴露及转运过程中均未投喂饲料。对每条试验鱼单独使用MS-222（美国华盛顿州雷德蒙德市Argent公司产品）进行麻醉并处死，称量体重并检查是否存在外部损伤。采集血液与组织（肝脏、性腺）样本，置于-80℃冰箱保存，直至后续提取与分析。提交本数据集时，归一化处理后的基因表达数据尚未获取，因此未纳入本次记录。