Name: use of a battery of chemical and ecotoxicological methods for the assessment of the efficacy of wastewater treatment processes to remove estrogenic potency
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Description: Watch this Scientific Journal Video about Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency at

Projects presented in this paper were funded by Severn Trent Water and Brunel University London. The authors would like to thank Alan Henshaw and John Churchley for providing field and laboratory assistance. T.J.C. thanks the Heinz Endowments for support. M.R.M. thanks the Steinbrenner Institute for a Steinbrenner Doctoral Fellowship and Carnegie Mellon University for a Presidential Fellowship.

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The authors have nothing to disclose.

废水处理厂是用EDC地表水污染的主要途径。去除常规的，先进的或新出现的治疗过程的内分泌活性的效力的评估需要使用的各种化学和生物测定的。用非靶向和靶向分析化学分析提供了关于除去各组分的功效的定性或定量的数据，因此，允许对环境质量标准生产或预测分析的化合物或化合物的混合物无影响浓度的评估。 从下面的治疗和未知的生物活性成分的废水中存在的物质的不完全矿化所得转化产物的产生限制单独化学测试是有用的。 A 在体内和体外生物测定结合分析化学家组合RY筛选提供了一个有用的工具箱新兴废水处理过程，以确定的EDC去除的效力。这些测试时，除了传统的水质参数和其他毒理学和微生物学终点进行允许现有和新兴的废水处理技术的一个重要的评价。 要注意的是酵母基于雌激素的屏幕（ 例如 ，是）是很重要的并不是唯一的体外测定，以确定化学品和废水的雌激素效能。一些稳定转染基于哺乳动物细胞的测定法，已经开发过，对ER-CALUX 27和与人乳腺癌细胞或子宫颈肿瘤细胞hERα-的HeLa-9903 28分别为例子。的是已经相比类似哺乳动物基于细胞的测定法，并已发现具有可重复性的一个可比较的水平高，真阳性和真阴性雌激素的识别率29本书虽然啊它有时被认为是稍微较不敏感的27。基于酵母报告分析的一个优点是，在不与哺乳动物细胞培养显著经验的实验室的是，可以更容易地通过，因为它需要较少的严格的生物控制措施和无菌技术（可置于工作台顶部，如果必要，可以进行是） 。人细胞为基础的分析也需要的 CO 2孵化器和光度计相比，在是使用的标准培养箱和酶标仪。两个基于酵母的雌激素记者分析（YES， 酿酒酵母和A-YES，Arxula adeninivorans）的ISO 19040的认证目前正在实验室间的小径"水质-水和废水的雌激素潜在的测定"突出产业在这些技术的兴趣。 有一些所描述的方法的局限性，其中包括的潜在的污染的采样，样品存储和分析来自现场或实验室环境或人类污染（ 例如，增塑剂，表面活性剂，个人护理产品）始发雌激素作用物质中的样品。这种类型中的YES测定（或其他基于细胞的报告分析）污染将提升的背景和影响使用该测定的。存储在塑料瓶的水样或溶剂极易造成假阳性。假阴性也备受关注既是LCMS / MS和YES检测要求SPE到雌激素到集中检测水平。基质中，SPE吸附剂和洗脱溶剂的选择可影响萃取效率和类型洗脱化合物。使用C18 SPE柱，使用在此协议中描述的条件可以产生负偏压的提取，作为高极性和碱性化合物会通过吸附剂进行很差保留。此外，该协议要求从甲烷洗脱YES洗脱液重建醇通过蒸发乙醇至干产生挥发性化合物的损失出资者氮。其结果是该协议可以提供低估测试的样品的雌激素活性。考虑到是检测未知的或意外的化合物可能会被错过，因为他们没有被提取或他们因蒸发而丢失时，这些限制是特别重要的。此外，LCMS / MS技术利用了内标来校正恢复;这种方法不能与YES测定中使用。 体内流出物的测试显著局限性包括相比体外方法进行评估所需的高成本和时间。目前使用的鱼胚胎试验检测雌激素活性是有限的。然而，已经有了一些成功的生产雌激素反应转基因发光斑马鱼胚胎30，这可能会对未来的应用。黑头呆鱼（在此protoc使用醇）是一种常见的实验室物种和VTG诱导雄性鱼是雌激素暴露的证据充分的生物标志物和废水流出物雌激素22或其它的雌激素化合物或其混合物31的量化度量。对内分泌干扰物质的OECD测试规定已使用成人黑头呆鱼，青鳉日本和斑马鱼32,33验证，与VTG是在所有三种雌激素暴露的敏感生物标志物。然而，VTG感应不直接相关的生殖障碍，因此废水曝光的生态后果，如严重雌雄同体蟑螂3所示。在另一方面，蟑螂是不是生态毒理学研究的一个经典的"实验室物种，由于其大尺寸，长的生成时间（2 - 3年才能达到性成熟），生殖风格;组产卵（繁殖）发生一年一次，难识别从女性的男性（比在其他产卵季节）。然而，这通常雌雄异体物种已被很好的研究在英国，这是因为雌激素废水排放的下游，雄鱼展出扰动他们的内分泌（ 例如 ，在他们的血液女性特有的卵黄蛋白原的存在）和组织病理学（ovotestes发现-开发鸡蛋睾丸和/或女性生殖管道）5,6。因此，这些协议的应用前景，罗奇（或类似物质）可能是一个有益的野生物种定点显示，如果废水质量（并降低雌激素）真正的改善河流接受先进的污水处理被看见。它们也可以在配管系统的端部来监视从试验规模的工厂7技术上改进的废水。当考虑在体内废水评估要使用的物种有相对快速和受控的测试用实验室物种之间的折衷相比长场为主，但更多的环境有关​​，使用本地物种进行测试。然而，这种在体内的评价是成本高，只应视为最后一组使用以下化学分析评估，试验和体外试验。 所述的协议中的关键步骤包括制备和样品和玻璃器皿的处理（ 即，瓶和取样设备应用适当的表面活性清洁剂预先处理），以避免从环境污染物包括限制样品接触与塑料和样品的污染能产生误报的其他材料。设计和构建水族馆鱼曝光系统时，这是非常重要的。理想的情况是水族馆（住宅存量和期间暴露）应从材料建成具有低吸附32以最小的污染风险。不锈钢可以用于流出物或水储水槽。而一个玻璃结构的坦克是优选的鱼缸（因为这也提供了鱼便于观察）。使用低等级塑料管或管道的，应避免32，PVC 34和ABS可以当&#39;适当调味&#39;被使用， 即，留在运行稀释水中至少12小时，使用前以浸出任何污染物。医用级硅管已经在我们的蠕动泵输送化学品和废水/稀释水的坦克工厂成功应用。除了考虑建筑雌激素污染和养殖系统的运行，它考虑鱼的饮食也很重要;许多鱼类礼仪食品已被认为是雌激素对鱼类。因此它来测试任何食物为活动（ 例如 ，在酵母雌激素屏幕，查看贝雷斯福德等人 14）在这些类型的研究使用它们之前是重要的。 故障排除的描述，如果质量保证样本，包括多个行程，实验室和溶剂空白分析一起阳性对照和实际样品，以消除假阳性和假阴性结果是简化了化学分析或YES测定协议。正（ 例如 ，EE2）和负极（稀释仅水）控制应也总是在体内测定可用于证实预期生物标志物或端点（ 即 ，VTG或组织病理学）的灵敏度，并且允许任何意外污染待检测（ 例如，从实验装置，饮食，或稀释水域）。在协议的任何修改之前应进行任何研究来验证。 与进入通过污水处理厂的污水对环境的雌激素化合物的更严格的法规它是设想更有效的污水处理技术将需要开发。在这个手稿中描述的测试电池恭维生态毒理学和化​​学评估测试通常用于污水处理厂的污水排放。因此，这种类型的测试应使废水技术开发商与设备操作员，整体的电池的应用前景实行最生态安全设计考虑的最佳方法同时删除特定的调节雌激素类化学和整体的生物活性。

关于内分泌干扰物对野生动物的生殖健康产生不利影响的担忧已导致欧盟在水框架指令（WFD）"观察名单"上放置两个雌激素物质（雌二醇和炔雌醇）。 EDC包括各种化学类，包括天然和合成的类固醇的雌激素，药物，杀虫剂和工业化学品和消费产品上的野生动物已知的不利影响的成分的。这些化合物的一些可能潜在地影响人类健康1。 有研究表明，污水处理厂的污水是雌激素对鱼类2并因此很多受纳水体也雌激素对鱼类3。这是首先通过在英国的全国调查表明，野生雄鱼的血液和前期高位表明卵黄蛋白原增加浓度（女卵黄蛋白前体4）阴阳的价（发展雄鱼睾丸鸡蛋和/或女性生殖管道）的正常雌雄异体鱼种5,6。 传统的污水处理通常是由经过初步筛选后一级和二级处理从而消除溶解和悬浮有机物的一个三阶段的过程。除去个别的EDC的功效依赖于物质的物理化学性质和所采用的治疗方法的有效性。对于通过吸附和生物降解许多EDC去除可以显著，但不完整的。三级处理，如砂滤，可在采用先进的氧化（例如臭氧）增加，而先进的治疗EDC拆除7有效或活性炭能有效地接近完全去除7实现。 任何新技术的污水处理NEE评估DS确定EDC除去所提出的方法的功效。测试，包括有针对性的化学分析毒理学一起测试， 使用体内和体外生物测定的电池，为此提供了全面的数据。虽然监管是非常有用的，有针对性的化学分析只能提供有关监测化合物（和具体的代谢物）数据。生物测定另外允许的副产品代谢物和治疗产生的废水变换的不利影响，否则将被未被发现-8,9-"检测"。本文介绍了使用化学和生态毒性实验室检测的电池的评估除去原油的雌激素效力和处理后的污水和受纳水体的一些先进和新兴的污水处理工艺的功效。

<table><tbody><tr><td>Wellwash Versa plate washer</td><td>Thermo Scientific</td><td>5165010</td><td></td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>SpectraMax 340PC</td><td></td></tr><tr><td>Incubator</td><td>Memmert</td><td>INB 400</td><td>37 &amp;#176;C incubation required for carp assay</td></tr><tr><td>Fisherbrand whirlimixer</td><td>Fisher Scientific</td><td>13214789</td><td></td></tr><tr><td>Icemaker</td><td>Scotsman</td><td>AF80</td><td></td></tr><tr><td>12-Channel F1 digital multichannel pipette</td><td>Thermo Scientific Finnpipette</td><td>4661070</td><td></td></tr><tr><td>ELISA kits</td><td>Biosense Laboratories</td><td>V01018401-096 (Fathead minnow)&lt;br/&gt; V01003402-096 (Carp)</td><td></td></tr><tr><td>Microfuge tubes, 0.5 ml</td><td>Alpha labs</td><td>LW2372</td><td></td></tr><tr><td>Microfuge tubes, 1.5 ml</td><td>Alpha labs</td><td>LW2375</td><td></td></tr><tr><td>Sulphuric acid, 95-98%</td><td>Sigma-Aldrich</td><td>258105</td><td></td></tr><tr><td>&lt;strong&gt;Histology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tissue processor</td><td>Leica Biosystems</td><td>TP1020</td><td></td></tr><tr><td>Wax dispenser</td><td>Thermo Scientific Raymond Lamb</td><td>E66HC</td><td></td></tr><tr><td>Metal embedding mold</td><td>Leica Biosystems</td><td>Various</td><td></td></tr><tr><td>Hot plate</td><td>Thermo Scientific Shandon</td><td>3120063</td><td></td></tr><tr><td>Cold plate (EG1150 C)</td><td>Leica Biosystems</td><td>14038838037</td><td></td></tr><tr><td>Heated forceps (EG F)</td><td>Leica Biosystems</td><td>14038835824</td><td></td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>RM2235</td><td></td></tr><tr><td>Paraffin section floatation&amp;nbsp; bath</td><td>Electrothermal</td><td>MH8517</td><td></td></tr><tr><td>Slide drying bench</td><td>Electrothermal</td><td>MH6616</td><td></td></tr><tr><td>Stainmate automated stainer</td><td>Thermo Scientific Shandon</td><td>E103/S10L</td><td></td></tr><tr><td>Cassettes, Histosette II, biopsy</td><td>Simport</td><td>M493</td><td></td></tr><tr><td>Paraffin wax</td><td>Thermo Scientific Raymond Lamb</td><td>W1</td><td></td></tr><tr><td>Histo-Clear II</td><td>National Diagnostics</td><td>HS-202</td><td></td></tr><tr><td>IMS (ethanol mix), IDA99</td><td>Tennants</td><td>ID440</td><td></td></tr><tr><td>Polysine adhesion slides</td><td>Thermo Scientific Gerhard Menzel</td><td>J2800AMNZ</td><td></td></tr><tr><td>Cover slips, 22x50 mm</td><td>VWR</td><td>631-0137</td><td></td></tr><tr><td>Histomount</td><td>National Diagnostics</td><td>HS-103</td><td></td></tr><tr><td>Haematoxylin Harris GURR</td><td>VWR</td><td>351945S</td><td></td></tr><tr><td>Eosin, 1%, aqueous</td><td>Pyramid Inovation</td><td>S20007-E</td><td></td></tr><tr><td>Fisherbrand slide boxes</td><td>Fisher Scientific</td><td>11701486</td><td></td></tr><tr><td>Microtome blades, MB35</td><td>Thermo Scientific Shandon</td><td>3050835</td><td></td></tr><tr><td>Bouin&amp;rsquo;s solution</td><td>Sigma Aldrich</td><td>HT10132-1L</td><td></td></tr><tr><td>&lt;strong&gt;Yeast screen&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Flow cabinet</td><td>Labcaire Systems Ltd</td><td>SC12R</td><td></td></tr><tr><td>Cooled incubator</td><td>LMS Cooled Incubator</td><td>303</td><td></td></tr><tr><td>Incubator</td><td>Memmert</td><td>INB 400</td><td></td></tr><tr><td>Shaker</td><td>Grant</td><td>PSU-10i</td><td></td></tr><tr><td>Fisherbrand whirlimixer</td><td>Fisher Scientific</td><td>13214789</td><td></td></tr><tr><td>Plate shaker</td><td>Heidolph Titramax 100</td><td>544-11200-00</td><td></td></tr><tr><td>12-Channel F1 digital multichannel pipette</td><td>Thermo Scientific Finnpipette</td><td>4661070</td><td></td></tr><tr><td>12-channel pipette, electronic</td><td>Sartorius</td><td>735441</td><td></td></tr><tr><td>96-well flat-bottom microplates</td><td>MP Biomedicals&lt;br/&gt; Thermo Scientific Nunc&lt;br/&gt; Sarstedt</td><td>76-232-05&lt;br/&gt; 260860&lt;br/&gt; 82.1581.001</td><td>We have found that these multiwell plates all produce low backgrounds</td></tr><tr><td>HPLC grade water</td><td>Rathburn</td><td>RH1020</td><td></td></tr><tr><td>Absolute ethanol</td><td>Hayman Kimia</td><td>F200238</td><td></td></tr><tr><td>Potassium phosphate monobasic anhydrous</td><td>Sigma-Aldrich</td><td>P-5655</td><td></td></tr><tr><td>Ammonium sulphate</td><td>Sigma-Aldrich</td><td>A-2939</td><td></td></tr><tr><td>Potassium hydroxide, pellets</td><td>Sigma-Aldrich</td><td>P-1767</td><td></td></tr><tr><td>Magnesium sulfate, anhydrous</td><td>Sigma-Aldrich</td><td>M-2643</td><td></td></tr><tr><td>Iron(III) sulfate</td><td>Sigma-Aldrich</td><td>307718</td><td></td></tr><tr><td>L-Leucine</td><td>Sigma-Aldrich</td><td>L-8912</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Aldrich</td><td>H-6034</td><td></td></tr><tr><td>Adenine</td><td>Sigma-Aldrich</td><td>A-2786</td><td></td></tr><tr><td>L-Argenine, hydrochloride</td><td>Sigma-Aldrich</td><td>A-6969</td><td></td></tr><tr><td>L-Methionine</td><td>Sigma-Aldrich</td><td>M-5308</td><td></td></tr><tr><td>L-Tyrosine</td><td>Sigma-Aldrich</td><td>T-8566</td><td></td></tr><tr><td>L-Isoleucine</td><td>Sigma-Aldrich</td><td>I-7403</td><td></td></tr><tr><td>L-Lysine, hydrochloride</td><td>Sigma-Aldrich</td><td>L-8662</td><td></td></tr><tr><td>L-Phenylalanine</td><td>Sigma-Aldrich</td><td>P-5482</td><td></td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G-8415</td><td></td></tr><tr><td>L-Valine</td><td>Sigma-Aldrich</td><td>V-0513</td><td></td></tr><tr><td>L-Serine</td><td>Sigma-Aldrich</td><td>S-4311</td><td></td></tr><tr><td>Thiamine, hydrochloride</td><td>Sigma-Aldrich</td><td>T-1270</td><td></td></tr><tr><td>Pyridoxine</td><td>Sigma-Aldrich</td><td>P-5669</td><td></td></tr><tr><td>D-Pantothenic acid, hemicalcium salt</td><td>Sigma-Aldrich</td><td>P-5155</td><td></td></tr><tr><td>Inositol</td><td>Sigma-Aldrich</td><td>I-5125</td><td></td></tr><tr><td>D-Biotin</td><td>Sigma-Aldrich</td><td>B-4639</td><td></td></tr><tr><td>D-(+)-Glucose anhydrous; mixed anomers</td><td>Sigma-Aldrich</td><td>G-7021</td><td></td></tr><tr><td>L-Aspartic acid</td><td>Sigma-Aldrich</td><td>A-4534</td><td></td></tr><tr><td>L-Threonine</td><td>Sigma-Aldrich</td><td>T-8441</td><td></td></tr><tr><td>Copper(II) sulfate, anhydrous</td><td>Sigma-Aldrich</td><td>C-1297</td><td></td></tr><tr><td>Chlorophenolred-&amp;#946;-D galactopyranoside (CPRG)</td><td>Sigma-Aldrich</td><td>10884308001</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G-2025</td><td></td></tr><tr><td>17 &amp;beta;-Estradiol</td><td>Sigma-Aldrich</td><td>E-8875</td><td></td></tr><tr><td>&lt;strong&gt;Steroids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Ammonia solution</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Ethylacetate</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Copper(II) nitrate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Acetone</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Dichloromethane</td><td>Rathburn</td><td></td><td></td></tr><tr><td>2,4,16,16-d&lt;sub&gt;4&lt;/sub&gt;-17&amp;#946;-estradiol</td><td>CDN Isotopes</td><td></td><td></td></tr><tr><td>2,4,16,16-d&lt;sub&gt;4&lt;/sub&gt;-estrone</td><td>CDN Isotopes</td><td></td><td></td></tr><tr><td>2,4,16,16-d&lt;sub&gt;4&lt;/sub&gt;-17&amp;#945;-ethynyl oestradiol</td><td>CDN Isotopes</td><td></td><td></td></tr><tr><td>17&amp;#946;-estradiol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Estrone</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>17&amp;#945;-ethynyl oestradiol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Hexane</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Styrene divinyl benzene cartridge (Isolute ENV+) solid phase extraction cartridge (200 mg/6 ml)</td><td>Biotage</td><td></td><td></td></tr><tr><td>Isolute aminopropyl solid phase extraction cartridge (500 mg/6 ml)</td><td>Biotage</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fish study&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>orange-white silicon manifold tubing 0.63 bore pk 6</td><td>Watson Marlow</td><td>982.0063.000</td><td></td></tr><tr><td>straight connectors for 0.5/0.8 bore pk 20</td><td>Watson Marlow</td><td>999.2008.000</td><td></td></tr><tr><td>pumsil silicon tubing 0.8 bore 15 m</td><td>Watson Marlow</td><td>913.A008.016</td><td></td></tr><tr><td>200 series multi-channel persitaltic pump</td><td>Watson Marlow</td><td>205CA</td><td></td></tr><tr><td>Silicone tubing x 15 m (dosing tanks)</td><td>VWR</td><td>SFM1-3250</td><td></td></tr><tr><td>silicone tubing x 15 m (large for inflow/outflow)</td><td>VWR</td><td>SFM1-5450</td><td></td></tr><tr><td>2.5 L glass winchester pk 4</td><td>Fisher Scienctific</td><td>BTF-505-050B</td><td></td></tr><tr><td>magnetic stir bar 51 x 8 mm pk 10</td><td>Fisher Scienctific</td><td>FB55595&amp;nbsp;</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (MS222)</td><td>Sigma Aldrich</td><td>E10521-10G</td><td></td></tr><tr><td>17&amp;alpha;-Ethynylestradiol</td><td>Sigma Aldrich</td><td>E4876-100MG</td><td></td></tr><tr><td>Absolute ethanol</td><td>Hayman Kimia</td><td>F200238</td><td></td></tr><tr><td>&lt;strong&gt;SPE&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1/8 inch PTFE tubes &#039;straws&#039; colour coded pk 4</td><td>Sigma Aldrich</td><td>57276</td><td></td></tr><tr><td>disposable liners for manifold</td><td>Sigma Aldrich</td><td>57059</td><td></td></tr><tr><td>filtration tubes without frits 6 ml pk 30</td><td>Sigma Aldrich</td><td>57242</td><td></td></tr><tr><td>reservior adaptors pk 12</td><td>Sigma Aldrich</td><td>57020-U</td><td></td></tr><tr><td>stainless steel weight for manifold pk 4</td><td>Sigma Aldrich</td><td>57278</td><td></td></tr><tr><td>male Luer plug for manifold pk 12</td><td>Sigma Aldrich</td><td>504351</td><td></td></tr><tr><td>SPE Vacuum Manifold</td><td>Sigma Aldrich</td><td>57265</td><td></td></tr><tr><td>stop cocks for extraction mainfold (supelco) pk 12</td><td>Waters</td><td>WAT054806</td><td></td></tr><tr><td>Sep-Pak Plus C18 cartridge box 50&amp;nbsp;</td><td>Waters</td><td>WAT020515</td><td></td></tr><tr><td>Methanol HPLC grade 2.5 L</td><td>Fisher Scientific</td><td>M/4056/17</td><td></td></tr><tr><td>7 ml glass vials with lids (58 x 17 mm) pk 399</td><td>Fisher Scientific</td><td>TUL-520-031K</td><td></td></tr><tr><td>Absolute ethanol</td><td>Hayman Kimia</td><td>F200238</td><td></td></tr><tr><td>vacuum pump, &lt;em&gt;e.g.&lt;/em&gt;, VP Series Vacuum Pump</td><td>Camlab</td><td>1136915</td><td></td></tr></tbody></table>

use of a battery of chemical and ecotoxicological methods for the assessment of the efficacy of wastewater treatment processes to remove estrogenic potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

试图了解的改进废水处理过程的影响或确定最合适的技术在现有污水处理厂相对于除去排出废水的内分泌干扰活性的功效加装设备作为三级处理，不仅需要键化学的测定即进入工作，但需要分解产物也可以具有内分泌干扰活性的分析组件。在生活污水流出物，目前最雌激素物质是类固醇激素，雌酮（E1），17β雌二醇（E2）和17α炔雌醇（EE2）5,8。类固醇的雌激素主要从体内排出作为非活性结合物16,17的混合物。这些结合雌激素基本上在污水处理系统由细菌活动deconjugated和污水处理厂发生进一步退化。该deconjugated AR类固醇e。通过吸附从废水流中去除污泥或导致形成二次处理过程中降解，首先改造的副产品，并最终完全矿化可能发生的初始​​活性组分。的流出流中的所有单个化合物的化学分析将是困难的，耗时的和昂贵的，并且不会涵盖存在于样品中未知的活性成分。此外，各成分的雌激素贡献的总和将只提供所分析的化合物的样品的累积雌激素效力的指示。这是转化过程产生未知的雌激素物质或者其中流入是工业来源的风险。 在体内 和体外生态毒理学生物测定组合化学分析提供了在混合物如处理过的污水中的溶液到未知的雌激素组分的存在， 在体外测定，如酵母雌血肌酐EEN（YES），已被广泛用于确定污水流出物的雌激素活性，并帮助识别处理的样品8,18,19的活性成分。然而， 在体内的体外试验比较可以显著11，新工艺相对于治疗内分泌干扰效能的综合评估，需要化学和生态毒理学测试电池。 可使用随后的样品的提取，浓缩和分析前提取物的清理化学分析来实现的个别处理厂或过程是否从废水流中除去活性化合物A的确定，使用LCMS（/ MS）或GCMS最多进行（/ MS）的方法。从化学分析获得的数据可以被用来确定是否符合个别预测无影响浓度（PNEC）20或环境质量标准S（EQS）特定个人化合物21，因此这种方法对于法规遵从数据至关重要。此外，定位或非靶向化学分析方法允许用生物学方法，它提供了一个总的响应相比单个化合物或异构体的鉴定和定量。因此化学分析方法允许进行离散化合物的评估，以满足和解决个别处理厂的基础上，这些废水处理的挑战。有研究表明，虽然除去合成激素的EE2趋于较少有效的常规废水处理（ 例如，活性污泥处理厂）可能是在去除天然类固醇激素的高度有效的。使用利用的技术，如臭氧先进的治疗领域的研究中，颗粒活性炭（GAC）和膜已经证实，尽管成本高，它们可以作为尾管的溶液以除去EE2至低于预计值icted效果水平和低于检测限。 图3显示了EE2的使用GAC在中试规模的城市污水处理厂的拆除。在中试规模在用管GAC治疗结束市政污水处理厂进行的研究还显示以下的GAC在雌激素效力减少使用如图4中可见的酵母雌激素屏幕（是）进行测定。 <img alt="图3" src="/files/ftp_upload/54243/54243fig3.jpg" /> 图3.实例字段数据显示以下高级三级处理去除炔雌醇（A）样品是从污水处理厂以下为以下保存样品描述的步骤常规（活性污泥）处理收集。 （B）样品固相萃取，净化后用正相SPE去除干扰物质提取，凝胶渗透色谱法。 （C）的清洁浓缩提取物浓缩至小体积并在MRM模式使用负离子电LCMS / MS进行分析。结果是使用采用同位素标记的内标内部标准化计算。在所示的例子中，EE2是在0.1纳克/升的预测没有影响水平（PNEC）以上的浓度存在于最终的ASP流出物并用活性炭和臭氧去除（O 3），以对环境安全的浓度。 <a href="https://www.jove.com/files/ftp_upload/54243/54243fig3large.jpg" target="_blank">请按此查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54243/54243fig4.jpg" /> 图4.照片一个酵母雌激素屏幕（是）试验板（A）中示出从黄色到红色的颜色变化，与该样品的雌激素活性。从YES分析板显示创建的图形校正吸光度（540雌二醇标准（B）的纳米），活性污泥处理的流出物（ASP）和粒状活性炭（GAC）处理过的废水流出物的样品（C）将每个样品一式两份进行测试。 ASP和GAC废水提取和使用第1节中列出的SPE方法集中<a href="https://www.jove.com/files/ftp_upload/54243/54243fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 臭氧也是从常规治疗的污水处理厂去除类固醇雌激素和雌激素活性高效。臭氧是能够氧化多种有机污染物和溶解的有机物质的废水样品中，并提供消毒性能。臭氧化的有效性取决于水的特性如pH，有机物量和臭氧的施用剂量。由常规处理除去不良的雌激素可以是从废水0.8和2毫克O 3 /毫克之间DOC去除剂量。臭氧是一种选择性的氧化剂，其与富电子网站（不饱和碳 - 碳键，芳族化合物，包括芳族醇），这使得适用于一个号码的EDC的击穿臭氧反应。然而，单独的化合物的消除并不必然导致对原始化合物的完全矿化。有机物质以下臭氧可能的副产物，其中包括一些低分子量的转化产生的中间体或转化氧化，极性类型的化合物如醛，酮，羧酸，酮基羧酸，和溴化的化合物。实例包括溴酸盐，甲醛，乙醛和羧酸。 采用体内 和体外生物测定已经表明，尽管臭氧仅部分氧化一些化学物质，将得到的主要转变产物具有较低estrogENIC效力并因此臭氧在一个较高的去除雌激素活性的适当剂量的结果的应用程序。 一个废水处理额外的主要优点是在受纳水雄鱼女性化的减少;产生不利影响，可导致减少的生育3。使用鱼体内研究（ 例如 ，蟑螂或黑头呆鱼）暴露于废水显示雌性生殖细胞或卵母细胞的雄性鱼的精巢（ 例如，如在图5中看到）。阴阳或男性VTG缺席或鱼类以下先进的治疗，如海关总署7或臭氧22显著减少。这些研究表明，臭氧化过程中产生的变换的产品没有雌激素，然而，这并没有解决所产生的流出物的毒性。此问题已在其他研究中得到解决，例如通过马格德堡等的研究。 <s达&gt; 23这表明副产品臭氧氧化是有毒的虹鳟鱼，但这种毒性可以通过下游砂滤以下臭氧被删除。 <img alt="图5" src="/files/ftp_upload/54243/54243fig5.jpg" /> 图5.正常男性（A）和阴阳的显微照片（B，C）从成年罗奇（ 拟鲤 ）性腺在基于实地评估。显微照片-A 暴露于废水 ，描绘了正常男性睾丸的组织切片。显微照片-B和-C，描绘了阴阳公鱼的组织切片，被暴露在活性污泥法处理废水排放六个月。箭头指示存在于睾丸组织卵母细胞。比例尺为100μm在每个显微照片。 <a href="https://www.jove.com/files/ftp_upload/54243/54243fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 12,24在有机微双氧水氧化- 26。 TAML与H 2 O 2活化剂，有效地降解EE2和其他类固醇雌激素在纯实验室用水，以及在城市污水处理厂的污水和尖刺尿样12英寸实验室的研究，示出TAML / H 2 O 2的处理提供了高类固醇的雌激素去除包括EE2去除和实质上降低使用是生物测定和质体外测定雌激素活性LLY减少鱼女性化在使用VTG生物测定（ 图1和图6）测量体内 。 <img alt="图6" src="/files/ftp_upload/54243/54243fig6.jpg" /> 图6.平均EE2浓度和雌激素活性处理和未处理的罐水域（A）和基线血浆卵黄蛋白原并暴露雄鱼（B）的A）EE2浓度（ng / L，深蓝色条）通过LCMS / MS测量，雌激素活性（EE2当量纳克/升，深绿色条）通过体外酵母雌激素屏幕测量（YES）。B）血浆卵黄蛋白原（毫微克/毫升，淡蓝色的条）浓度男性黑头呆鱼通过定量酶测定 - 连接的免疫吸附测定（ELISA）。报告为EE2化学分析结果&lt;0.03纳克/升EE2（ 即，高于检测限（LOD）低级）被视为具有一半的LOD（即 </青霉&gt; 0.015纳克/升EE2），用于在平均值，标准误差和统计分析的计算中使用。 EE2和雌激素活性是测得的平均浓度在取样21天的暴露。等离子VTG是之前曝光（基线），并在21天后曝光测量。治疗制度包括的;阴性对照组（仅稀释水），EE2 + H 2 O 2 + TAML，EE2 + H 2 O 2和EE2只。在图-A误差棒代表平均值的标准误差，在图-B误差棒代表标准偏差。以上图-B条字母代表统计相似。这个数字已经从米尔斯等人修改。12 <a href="https://www.jove.com/files/ftp_upload/54243/54243fig6large.jpg" target="_blank">，请点击这里查看本图的放大版本。</a>

Watch this Scientific Journal Video about Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency at

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

内分泌干​​扰物（EDC）构成重大风险到水生环境。城市污水处理厂的主要贡献者，以地表水的雌激素效力。本文提供的方法允许疗效和污水处理工艺的适用性相对于EDC去除的评估。

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

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Environment

14.0K 次观看.  Brunel University London. 内分泌干​​扰物（EDC）构成重大风险到水生环境。城市污水处理厂的主要贡献者，以地表水的雌激素效力。本文提供的方法允许疗效和污水处理工艺的适用性相对于EDC去除的评估。 

视频: Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

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环境科学

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

准备和测试阻抗为基础与RTgill-W1细胞饮用水进行快速评估水样进行毒性射流生物芯片

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

在用水筛查内分泌活动市售的<em&gt;体外</em&gt;式激活生物测定

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

用于去除与农业和城市径流中地表水毒性有关的污染物的植被处理系统

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation

Monitoring pharmaceuticals throughout the water cycle is becoming increasingly important for the aquatic environment and eventually for human health. Targeted and non-targeted analysis are today's means of choice. Although targeted analysis usually conducted with the help of a triple quadrupole mass spectrometer may be more sensitive, only compounds previously selected can be identified. The most powerful non-targeted analysis is performed through time of flight mass spectrometers (TOF-MS) extended by a quadrupole mass analyzer (Q), as used in this study. Preceded by solid phase extraction and high-performance liquid chromatography (HPLC), the non-targeted approach allows to detect all ionizable substances with high sensitivity and selectivity. Taking full advantage of the Q-TOF-MS instrument, tandem mass spectrometry (MS/MS) experiments accelerate and facilitate the identification while a targeted MS method enhances the sensitivity but relies on reference standards for identification purposes. The identification of four pharmaceuticals from Rhine river water is demonstrated. The Rhine river originates in Tomasee, Graub&#252;nden, Switzerland and flows into the North Sea, near Southern Bight, The Netherlands. Its length amounts to 1232.7 km. Since it is of prime interest to effectively eliminate pharmaceuticals from the water cycle, the effect UV-C irradiation is demonstrated on a laboratory scale. This method allows fast degradation of pharmaceuticals, which is exemplarily shown for the macrolide antibiotic erythromycin. Using the above HPLC-Q-TOF-MS method, concentration-time diagrams are obtained for the parent drug and their photodegradation products. After establishing the equations for first-order sequential reactions, computational fitting allows the determination of kinetic parameters, which might help to predict irradiation times and conditions when potentially considered as fourth stage within wastewater treatment.

We present a protocol for non-targeted analysis using time of flight mass spectrometry as a perfect tool to identify pharmaceuticals in waters. We demonstrate the application of UV irradiation for their elimination. Analysis involving irradiation, compound isolation, identification and kinetic modelling of the degradation profiles is illustrated.

高效液相色谱-ESI-Q-MS 与红霉素通过光致降解法鉴定水生环境中的药物

Monitoring pharmaceuticals throughout the water cycle is becoming increasingly important for the aquatic environment and eventually for human health. ...

Necropsy-based Wild Fish Health Assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

基于尸检的野生鱼类健康评估

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, &#945;- and &#946;-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

使用Tg（Vtg1:mcherry）斑马鱼胚胎测试内分泌干扰化合物的雌激素效应

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

胚胎暴露于低剂量毒物的长期行为和生殖后果

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

发育生物学

In Silico Modeling Method for Computational Aquatic Toxicology of Endocrine Disruptors: A Software-Based Approach Using QSAR Toolbox

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological systems. In particular, quantitative structure-activity relationship (QSAR) models have been increasingly applied to assess the environmental effects of a plethora of toxic materials. In recent years, some more highlighted types of toxicants are endocrine disruptors (EDs, which are chemicals that can interfere with any hormone-related metabolism). Because EDs may significantly affect animal development and reproduction, rapidly predicting the adverse effects of EDs using in silico techniques is required. This study presents an in silico method to generate prediction data on the effects of representative EDs in aquatic vertebrates, particularly fish species. The protocol describes an example utilizing the automated workflow of the QSAR Toolbox software developed by the Organization for Economic Co-operation and Development (OECD) to enable acute ecotoxicity predictions of EDs. As a result, the following are determined: (1) calculation of the numerical correlations between the concentration for 50% of lethality (LC50) and octanol-water partition coefficient (Kow), (2) output performances in which the LC50 values determined in experiments are compared to those generated by computations, and (3) the dependence of estrogen receptor binding affinity on the relationship between Kow and LC50.

Quantitative structure-activity relationship (QSAR) modeling is a representative bioinformatics-assisted method in toxicological screening. This protocol demonstrates how to computationally assess the risks of endocrine disruptors (EDs) in aquatic environments. Utilizing the OECD QSAR Toolbox, the protocol implements an in silico assay for analyzing toxicity of EDs in fish.

在内分泌干扰物计算水生毒理学的Silico建模方法:使用QSAR工具箱的基于软件的方法

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological ...

生物工程

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...