Funding was provided by State Water Resources Control Board (Agreements No. 10-096-250 and 14-090-270). We thank S. Abbasi, M. Connor, S. Engelage, K. North, J. Armstrong, S. Asato, M. Dojiri, D. Schlenk, S. Snyder, S. Westerheide, B. Escher, F. Leusch, G. Pelanek, K. Bi, and J. Printen. The authors declare no conflict of interest, and reference to trade names does not imply endorsement.

1.收集和处理水样（从埃舍尔等修改。9） <ol><li>填充含有1g叠氮化钠和50mg抗坏血酸与感兴趣水样品的顶部干净的1L的琥珀色玻璃瓶中。 72小时内，在4℃储存样品和过程。 注:叠氮化钠是剧毒，必须谨慎处理。使用护具（眼睛/面部，手套，服装）和正常工作的通风橱权衡。不要使用金属铲进行称量。 </li><li>通过一1.6微米玻璃纤维过滤器，然后通过在5-10毫升/分钟的流速预处理固相萃取（SPE）柱通过样品。调整真空泵的压力来控制流速。 </li><li>真空干燥该盒15分钟。 </li><li>用10毫升甲醇，随后用10毫升丙酮中洗脱盒:己烷（1:1，体积/体积）。 </li><li>下的高纯度氮气的轻柔气流集中洗脱液至〜1毫升溶剂交换通过加入500微升二甲基亚砜（DMSO），并蒸发该萃取液下降到500微升。 </li><li>转移提取物到琥珀色玻璃自动进样瓶中。储存于-20℃。 </li></ol> 2.准备分析具体参考化学和水提取物的稀释液<ol><li> 制备9稀释的校准曲线。 <ol><li>使在100％DMSO测定具体的参考，化学品的储液。最终浓度必须为2微米17β雌二醇对雌激素受体分析，和100μM的地塞米松为GR测定。存储储备溶液在-20℃。 </li><li>添加15微升适当的参考化学库存到285微升测定培养基的在无菌管（管＃1）。通过上下吹打混匀。 </li><li>加入200μl5％DMSO在测定培养基中的溶液在8个附加管（管＃2-9）。 </li><li>通过浴缸传输距离地铁1号100微升等分至管＃2È＃9以执行3倍稀释系列。每个稀释样品必须彻底用吸管取等分试样，并将其加入到下一个管之前进行混合。 </li></ol></li><li>由190微升检测培养基混合10微升DMSO的准备溶剂对照样本。 </li><li>制备四种稀释液每个水提取物。 <ol><li>在第一管中，加入5μL水提取物的95微升分析培养基调匀。加入50μl5％DMSO在检测培养基中的其他三个管。 </li><li>通过从一个管转移50微升溶液到下执行2倍稀释。 </li></ol></li></ol> 3.准备细胞悬液进行FRET生物测定<ol><li>根据制造商的说明准备在测定特定媒体。测定培养基必须储存在4℃下，并在使用前水浴加热到37℃。 </li><li>以ER或GR分裂被捕细胞的小瓶出低温无ZER并通过将小瓶在37℃水浴中2分钟，轻轻摇动快速解冻细胞。净化用70％乙醇小瓶，并将其放置在II级生物安全柜。 </li><li>打开使用无菌技术的小瓶中，并将细胞转移到10ml测定培养基中。 </li><li>离心5分钟200×g的。 </li><li>使用无菌玻璃吸管吸出上清液和重悬细胞沉淀于6ml测定培养基中。 </li><li>混合5微升细胞悬浮液与5μl的一个至关重要的染色液并加入等分试样到计数室。 </li><li>算使用光学显微镜或自动细胞计数器的活细胞的数目，并估计活细胞的密度在细胞悬浮液中。稀释如果必要，得到每ml测定培养基550000活细胞的最终密度。 </li></ol> 4.在96孔黑色壁透明底板板细胞及加稀水提取物<ol><li>创建板布局包括了9点检测特定的校准曲线，QA / QC样品和稀释倍数为每个水提取物。一个96孔板布局的一个例子示于图1。 </li><li>添加90微升测定培养基的到复制无细胞对照孔。 </li><li>倒入细胞悬浮在无菌移液贮存器，并添加90微升的细胞悬浮液，使用多道移液器其它井。 </li><li>加入10微升第2步到适当的孔中制备的稀释的样品。每孔DMSO的最终浓度必须为最大0.5％。 </li><li>覆盖有盖板，并放置在一个5％CO 2培养箱在37℃下16小时。 </li></ol><img alt="图1" src="/files/ftp_upload/54725/54725fig1.jpg" /> 图1:96孔板布局的实施例的多穴板被设计为包括一个测定特定的校准曲线，3种类型QA / QC控制（仅介质，细胞在清洁中，细胞在DMSO飙升介质），每个水提取物稀释4。每个控制和水提取物的稀释一式三份井进行了分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.准备装载解决方案，并添加到每口井<ol><li>温育之后，使板平衡至室温。对于此FRET生物测定，步骤5.2通过5.6应在不存在直接的光的情况下进行。 </li><li>根据制造商的说明制备6×加载溶液。 </li><li>加入20μl的6倍加载溶液的各孔中。 </li><li>添加10微升的细胞活力试剂至各孔以评价稀释的水提取物的细胞毒性。 </li><li>密封采用铝胶膜板。 </li><li>孵育在室温在黑暗中板2小时。 </li></ol> 6.测量细胞毒性和内分泌活动响应<ol><li>设置了以下制造商的说明底部阅读功能酶标仪。 </li><li>对于FRET转录生物测定，测量蓝色（（前）/发射器（EM）波长四百六十零分之四百○九纳米激发）和绿色（409例/ 530 EM NM）渠道的荧光。 </li><li>对于细胞毒性试验，测量在560例/ 590 EM纳米荧光。 </li></ol> 7.评估QA / QC检查以确定数据质量<ol><li>比较无细胞（媒体只）和细胞仅（在清洁测定培养基的细胞）对照的平均生荧光。唯一的媒体背景荧光应该比仅细胞控制的响应至少低25％。 </li><li>处理原始数据FRET。对于这两种"蓝"和"绿"的数据集，从含有以及所有细胞中减去无细胞对照孔的平均荧光。计算蓝/绿比每个实验井。 </li><li>唯一比较细胞和细胞DMSO对照的蓝/绿比值。这些控件的荧光值应为15％的相对标准偏差（RSD）内。 </li><li>积测定特定的校准曲线作为对表示为日志分子量（日志M）的样品浓度蓝/绿比值。计算斜率，R 2和登录EC 50（50％有效浓度）。校准参数应于表1中所列的预期范围之内。 </li><li>计算RSD为所有一式三份井。重复中的变异性应低于20％。 </li><li>对于细胞毒性数据（560实施例/ 590 EM纳米），从所有含细胞的孔中减去无细胞控制平均（即媒体背景）。 </li><li>计算各样品的平均扣除背景荧光。积所得的荧光数据如DMSO对照的百分比。样品稀释不应该表现出相比于细胞仅与DMSO对照的20％以上的细胞死亡。 </li></ol> 8.数据分析<ol><li>计算检测限（LOD）的测定极限的最低校准响应加上响应的平均值的两个标准偏差。 </li><li>为了呈现出剂量反应的样品，获得了欧共体50 &#160;使用10和50中的参考化学的效应％浓度之间的校准曲线的线性回归的斜率的水萃取物。 </li><li>用计算公式生物分析当量浓度（BEQ）:参考化学BEQ = EC 50 / EC水样50。 </li></ol>

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The authors have nothing to disclose.

环境雌激素，如17β雌二醇（E2），这些化学品在纳克/升浓度23,24权证筛查的有案可稽效力。在这项研究中，废水排放对ER响应（BEQ范围:2.3至17毫微克的E2 / L）的比报道的从澳大利亚污水处理厂20二级出水稍高，而BEQs地表水（&lt;0.5至4纳克E2 / L的）为报道的表面和雨水别处（&lt;1至11毫微克的E2 / L），16的范围内。尽管地表水样本中测得ER活动水平低，此法代表了水质评价是有关的筛选端点。雌激素，如双酚A和烷基酚表面活性剂往往在水生环境中检测到的，但它们难以用常规的分析化学量化。 ER-细胞测定还可以证明对当前和新注册的农药筛选是有用的，如由Kojima等25。 在GR活性通过幅度在这项研究中（ 表2）所分析的所有4流出物样品中的顺序超过了ER活性。这种趋势与在废水流出物14以前的研究结果一致，并为GR-BEQs范围这里报告略高但使用相同的体外生物测定12媲美报道其他二级污水。使用不同的细胞生物测定法，用于从在日本污水处理厂的污水为GR-BEQs范围较低（&lt;3至78纳克塞米松/ L）的比所报道的，在本研究22二级出水。与注册的30纳克塞米松/ L（ 表2，试样E）一种最大GR响应的单个表面的水样的例外，其余表面的水样中的GR活性相一致的低活性报告为荷兰表面水域21 。然而，环境即时为GR活性化学物质条约较少很好的特点比ER活性化学物质26，使得潜在的评估高阶效应是一个挑战。 与传统的化学分析，坚持书面协议，并采用基于绩效的QA测量验证/ QC方法最大限度地提高数据质量和稳定性。虽然适用于水质评价细胞生物测定验证将继续发展和完善，在Mehinto 等 14首概述并应用到这项研究的QA / QC参数的测量表明，我们的结果预先设定的标准值范围内为好（ 表1 ）。这些生物测定的校准曲线参数反映那些用于校准其它分析方案（ 例如，GC-MS），通过加入该评估的细胞（ 例如，细胞毒性）的可行性准则表示的主要区别。另一种操作以高通量形式使用细胞成为可能不同的是，示出了当生物活性测定（ 图2），所期望的浓度依赖性响应多个样品稀释的包容。在审查样品测量，下ER和GR回应:相对于三个二级污水样本三级废水（样本D）（AC）为我们的生物测定筛查结果的相对准确性补充证据表2。控制的法团或表示感兴趣的矩阵的参考样本，在这种情况下的水，将进一步加强，以比较内，特别是在整个测量实体生物测定结果的能力。 在这项研究中所用的雌激素受体和GR反式激活的细胞系的响应分别表示相对于所述强激动剂E2和地塞米松的活性，。这允许生物活性的定量估算表示的SA生物测定当量浓度（BEQ）。这进一步使得化学品的研究，有助于生物测定的反应， 即 BEQs与通过传统的化学分析确定各个激动剂浓度直接比较。这个概念强调剂生物筛选的在指导病原体，也称为效应定向分析的诊断调查附加实用工具。 由于SPE常规地用于隔离的宽范围内的水的药物和其它的"新兴污染物"的，我们使用的先前公布的协议9，20，其特色设计在水中捕获亲水和疏水的化学物质的吸附剂。尽管我们认为，所有的生物活性有机化合物定量采集使用这种技术，更多的工作需要充分规范和验证这种提取方法的性能。细胞测定的在这项研究中所用的类型可以呈现另一个限制。这些化验测定污染物和核受体之间的相互作用，不考虑其他可能的相互作用，如化工，膜受体。因此，有可能的是这里测定的筛选应答低估。样提取，没有标准化的方法来分析细胞测定数据和不同的方法可能会产生不同的结果。在这项研究中使用的校正曲线的下半部分中的响应进行建模线性回归可能并不适用于所有的细胞测定法，特别是如果仿照响应延伸超出在本研究中所指定的相对窄的范围内。其他调查上述问题，才能更好地理解到这些工具可以作为一个强大的监测工具被应用的程度。 而大部分的基础工作正在奠定了ER和GR作为内分泌活性化学物质的屏幕，研究人员正在扩大范围生物分析工具，包括行动等相关模式， 例如 ，androgenicity，遗传毒性，神经毒性和免疫毒性9,27。更完整的工具箱将加强筛查生物活性的化学物质，其中包括可能出现28，现在和未来，在各种水生环境19已知的和/或未知的化学物质转化产品的能力。

当前水质监测的前提是要准确和精确地测量化学污染物的产生，作为暴露于野生动物和人类的代理的能力。然而，这种化学物质通过化学监测和评估模式不能跟上，我们所面临的不断变化的化学世界。当我们了解的命运和合成和天然化学物质的影响，我们继续寻找测量工具，解决预期生物的影响，而在同一时间的免疫化学品生产，使用和环保投入的变化。这样的工具是特别了解相关未知或新的化学品，并转化产物是否值得我们注意。此外，存在于水中的化学物质的复杂混合物由个体化学监测不善处理。因此，我们面临的现有监控工具箱地表水THA这些问题，现代化，更好地解决的挑战收不到处理后的废水污水和城市/雨水径流排放。 近年来，生物分析技术已经表明希望作为筛选工具水质评估。如果对通过称为起作用的化学反应尤其in&#39;vitro生物测定，动作1,2的具体模式是极大的兴趣，环境监测社区3。大量调查在体外生物测定用来量化饮用水，地表废水4-6的内分泌活动。此外，一些生物测定目标分子发起事件（ 例如，受体活化），其可以潜在地通过不利后果通路连接于有害影响分析7,8。 生物筛选水质评价的发展一直相对较快，与数百种不同的体外生物分析端点已被评估他们的实用9,10。目前，仅生物测定的少数已显示以达到良好的测量精度（实验室内）同时展现水质5,6-之间区分的能力。特别是对于处理后的废水污水，雌激素和糖皮质类固醇的发生已经成功占据了利用体外转录实验11,12。然而，大多数研究迄今为止已采用的生物测定，其细胞系是专有的（并且因此没有被广泛可得），需要连续护理和操纵，或两者兼而有之。这样一来，能够标准化的协议，进行实验室间的校准练习，并最终该筛选技术转移到水资源的社会仍然受阻。 通过美国ToxCast程序审核体外生物检测中的至少一个供应商是容易商购的13用"冻融4;格式。这些除法被捕细胞"试剂盒"已被证明是一种用于测定从水中提取的化学品代表不同程度的处理14的活性健壮。虽然商协议可用来筛选个别化学品或混合物的生物活性，其中一些需要修改，才可以被应用到的水样。处理后的废水出水15，雨水径流16，受纳水体17,18和最近的循环水19,20是感兴趣的水质社区水介质的最好的例子。 这项研究提供了一个单一的，标准化的协议使用市售测量水样中的内分泌活动，除被捕体外转录生物测定。我们通过后台，剂量响应和可重复性响应为TW的全面评估证明该协议的健壮性特别感兴趣雌激素与糖皮质激素受体转录（ER和GR，分别）邻端点。该协议在加州应用于处理后的废水污水和地表水幕样本淡水系统。

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">GeneBLAzer ER alpha DA assay kit</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">K1393</td>
			<td style="width:313px;">Kit includes ER division arrested (DA) cells and LiveBLAzer FRET loading kit.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">GeneBLAzer GR DA assay kit</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">K1391</td>
			<td style="width:313px;">Kit includes GR division arrested (DA) cells and LiveBLAzer FRET loading kit.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">PrestoBlue cell viability reagent&#160;</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">A-13261</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Trypan blue, 0.4% in PBS</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">T8154</td>
			<td>Also available at ThermoFisher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Corning 96 well black wall, clear-bottom plate</td>
			<td style="width:123px;">Corning</td>
			<td style="width:115px;">3603</td>
			<td style="width:313px;">Individually wrapped, sterile with lid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Whatman glass fiber filters, GF/A, 1.6 &#181;M</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">WHA1820025</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Microplate aluminum sealing film</td>
			<td style="width:123px;">E&#38;K Scientific</td>
			<td style="width:115px;">T592100</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Oasis HLB 6 cc cartridge, 200 mg sorbent</td>
			<td style="width:123px;">Waters</td>
			<td style="width:115px;">WAT106202</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">17&#946; Estradiol</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">E2758</td>
			<td style="width:313px;">CAS #50-28-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Ascorbic acid</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:115px;">A61-100</td>
			<td style="width:313px;">Also available at Sigma-Aldrich</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Dexamethasone&#160;</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">D4902</td>
			<td style="width:313px;">CAS #50-02-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">D8418</td>
			<td>Molecular grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Solvents (acetone, hexane, methanol)</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">HPLC grade</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:275px;">Sodium azide</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">S2002</td>
			<td style="width:313px;">Chemical is highly toxic and must be handled with caution. Use protective clothing and weigh under a fumehood. Also available at EMD Millipore.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:275px;">Automated cell counter or hemocytometer</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">Suppliers include Bio-Rad, Fisher Scientific, Sigma-Aldrich and ThermoFisher.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Class II biological safety cabinet</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">CO2 incubator</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">Cryogenic freezer&#160;</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">Liquid nitrogen storage dewar is recommended.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">Fluorescence microplate reader</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">&#160;The reader must have bottom read capabilities.</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">* No recommended source, the choice of this equipment depends on budget, frequency of use, and lab space.</td>
		</tr>
	</tbody>
</table>

screening for endocrine activity in water using commercially-available in vitro transactivation bioassays

体外转录生物测定表明希望作为水质监测工具，但他们采用和广泛应用已部分阻碍由于缺乏规范的方法和强大的，用户友好的技术可用性。在这项研究中，使用可商购的，分裂停滞的细胞系，以定量筛选本感兴趣的环境质量的专业人员水样中的化学物质的内分泌活性。其中包括全面的质量保证/质量控制一个单一的，标准化的协议（QA / QC）检查是为雌激素和糖皮质激素受体的活性（ER和GR分别）使用基于细胞的荧光共振能量转移（FRET）分析开发。在加利福尼亚州（美国）处理城市废水和地表水从淡水系统的样品，采用固相萃取提取和使用标准化原为内分泌活动分析山坳。背景和特定端点引用的化学物质的剂量 - 反应满足必要的可靠的测量QA / QC准则。地表水样品的生物测定筛选反应，主要是检测不到的。与此相反，从二级处理厂流出物样品具有最高的可测量的活性，与估计的生物测定当量浓度（BEQs）到392纳克地塞米松/升的GR和17纳克17β雌二醇/升的ER。为三级污水样品的生物测定的反应比对二级污水测量低，说明经过深度处理一个较低的残余内分泌活性化学物质。这个协议表明 ，利用市售的体外转录生物测定，除法被捕细胞"试剂盒"，可适于筛选在水中内分泌活动。

在本研究中，选择了处理城市废水的4倍24小时的复合材料样品，从淡水系统在南加州地表水6抓取样品，包括超纯水的字段为空，说明该协议。 4流出物样品3是从以往的活性污泥的废水处理厂（"二次流出物"），并从与砂/碳过滤后加入生物处理（"叔流出物"）的先进废水处理厂的第四个。地表水样品从代表不同的土地用途（开，农业和城市）流域收集。 水提取物的稀释液没有表现出明显的细胞毒性，观察到小于20％的细胞死亡相比DMSO对照（ 表1）。无细胞（仅培养基）的背景低，以及C只有细胞和用DMSO响应单元之间omparison表明溶剂对荧光测量没有可测量的影响。测定特定的校准曲线参数，包括斜率和日志EC 50，分别的示范校准曲线随着时间的推移，重复性好可接受的历史值（ 表1）的范围内。对于ER测定可接受的校准曲线的一个例子示于图2。17β雌二醇和地塞米松的选择作为基准的化学品是基于测定的反应，发生处理过的废水和历史使用其他研究者在可能性的强度20- 22。在一式三份样品之间的反应的变异性小于20％的相对标准偏差。因此，所有的数据被认为是可接受的质量，并随后用废水和地表水样中估计的ER和GR生物活性（以ng / L表示为BEQ）。 图3）。然而，地表水提取物并没有表现出强烈的ER或GR活动。尽管10的REF，荧光反应往往接近或低于检测限。该领域的空白没有生物测定的反应证实了LOD以上提到的检测并不采样或程序实验室文物的结果。 在7检测ER和GR活性和10代表的水样，分别为（ 表2）4，用最高的17 BEQs纳克E2 / L的<html>与392纳克在二级污水发现地塞米松/ L。第三流出物的样品中的遗传活性的生物测定法的LOD以下。同样，大多数地表水样品展出上述LOD无GR活性，比二级污水ER活动的水平要低得多。 <img alt="图2" src="/files/ftp_upload/54725/54725fig2.jpg" /> 图2:校正曲线为对ER测定的剂量-反应曲线的实施例 ，绘制为平均荧光比率（±SEM）是S形并且用于确定EC 10和EC 50值。曲线的这一部分是用线性回归拟合，并用于派生生物测定当量浓度。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> LES / ftp_upload / 54725 / 54725fig3.jpg"/&gt; 图 3: 作图theRrelative富集因子生物测定当量浓度水样的平均蓝色/绿色荧光比率（±SEM）（BEQ）计算的样品的稀释系列显示出浓度依赖性的响应与上面的限至少两点检测（LOD）的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="2"> 雌激素受体（ER）测定 </td><td colspan="2"> 糖皮质激素受体（GR）测定 </td></tr><tr><td></td><td> 研究结果 </td><td> 验收标准 </td><td> 研究结果 </TD&gt; <td> 验收标准 </td></tr><tr><td> 校准参数 </td><td></td><td></td><td></td><td></td></tr><tr><td>平均log EC50（M） </td><td> -9.6 </td><td> -10.1至-9.1 </td><td> -8.5 </td><td> -9.0 -8.0到</td></tr><tr><td>平均坡度</td><td> 1.1 </td><td> 0.9〜1.5 </td><td> 2.0 </td><td> 1.8〜2.4 </td></tr><tr><td> R2 </td><td> 0.99 </td><td> &gt; 0.95 </td><td> 0.99 </td><td> &gt; 0.95 </td></tr><tr><td> QA / QC控制 </td><td></td><td></td><td></td><td></td></tr><tr><td>媒体背景</td><td>是</td><td>只有媒体&lt;单元+媒体</td><td>是</td><td>只有媒体&lt;单元+媒体</td></tr><tr><td>细胞毒性（％的死亡率） </td><td> 0％ </td><td> &lt;20％的死亡率</td><td> 0至5％ </td><td> &lt;20％的死亡率</td></tr><tr><td>样本精度（％RSD）&lt;/ TD&gt; <td> 2至18％的</td><td> &lt;20％ </td><td> 4至13％的</td><td> &lt;20％ </td></tr></tbody></table> 表1:QA / QC结果从污水处理厂出水出水生物分析筛选和地表水样品的总结。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 样品标识和说明 </td><td> ER-BEQ（NG E2 / L） </td><td> GR-BEQ（NG敏捷/ L） </td></tr><tr><td> A - 从全二级处理厂出水</td><td> 6.4 </td><td> 248 </td></tr><tr><td>乙 - 从全二级处理厂出水</td><td> 13 </td><td> 392 </td></tr><tr><td> c - 从中​​充分二级处理厂出水</td><td> 17 </td><td> 236 </td></tr><tr><td> ð - 从三级处理p最终出水兰特</td><td> 2.3 </td><td> &lt;22 </td></tr><tr><td> e - 来自农业区地表水</td><td> &lt;0.5 </td><td>三十</td></tr><tr><td>的F - 农业区地表水</td><td> &lt;0.5 </td><td> &lt;22 </td></tr><tr><td>摹 - 从市区地表水</td><td> 4 </td><td> &lt;22 </td></tr><tr><td> ^ h - 从市区地表水</td><td> 0.9 </td><td> &lt;22 </td></tr><tr><td>我 - 从露地地表水</td><td> 0.8 </td><td> &lt;22 </td></tr><tr><td>的J - 从露地地表水</td><td> &lt;0.5 </td><td> &lt;22 </td></tr><tr><td>的K - 领域的空白（超纯水） </td><td> &lt;0.5 </td><td> &lt;22 </td></tr></tbody></table> 表2:雌激素受体（ER-BEQ）和糖皮质激素受体生物测定当量浓度（GR-BEQ）交流。拉了一三种类型在加利福尼亚州（美国）收集的样本进行了分析:城市废水（AD），地表水（EJ）和字段为空（K）。

Watch this Scientific Journal Video about Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays at JoVE.com

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 ...

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

7.5K 次观看.  Southern California Coastal Water Research Project Authority. 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.

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

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EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

双工数字PCR方法的同时定量<em&gt;肠球菌。</em&gt;和人的粪便相关HF183标记的水域

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

表征和被动采样的应用在农药水监测

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

常淹发生的重构树简易造林技术

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

用原位曝光法测定淡水珍珠贻贝 Bioindication 的流环境适宜性

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

用小型化磷测定法测定膦酸在粒状氢氧化铁上吸附的优化方法

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied.

Fioriniae水和氯蓝莓和越橘花提取物的研究进展

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine laboratory measures of P bioavailability are based on chemical extractions performed on dried samples under oxidizing conditions. While useful, these tests are limited with respect to characterizing P release under prolonged water saturation. Labile orthophosphate bound to oxidized iron and other metals can rapidly desorb to solution in reducing environments, increasing P mobilization risk to surface runoff and groundwater. To better quantify P desorption potential and mobility during extended saturation, a laboratory microcosm method was developed based on repeated sampling of porewater and overlying floodwater over time. The method is useful for quantifying P release potential from soils and sediments varying in physicochemical properties and can improve site-specific P mitigation efforts by better characterizing P release risk in hydrologically active areas. Advantages of the method include its ability to simulate in situ dynamics, simplicity, low cost, and flexibility.

Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

测量实验室微量微生物中的磷释放,用于水质评估

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...