This work was supported by the US Army Medical Research and Materiel Command and by the Small Business Innovation Research and Small Business Technology Transfer program; Contract No. W81XWH-13-C-0093. We would like to thank Dr. Lucy Lee at the University of Fraser for being our RTgill-W1 cell culture mentor, and to acknowledge Dr. Niels Bols of Waterloo University for the development of the RTgill-W1cell line.

1.测试材料的制备注意:为了制备生物芯片进行测试，RTgill-W1细胞几种汇合烧瓶需要准备。需要瓶的数量的一个很好的估计一个合流T175烧瓶中接种16生物芯片。 <ol><li>执行使用无菌技术II级生物安全柜（biohood）以下步骤。用70％乙醇消毒biohood并放置在罩的任何材料。 <ol><li>通过解冻纤连蛋白的一个为1mg小瓶中并在100毫升无菌的L-15培养基稀释为10微克/ ml的浓度制备为流体生物芯片纤连蛋白底物。冻结在-20℃下在无菌的50ml锥形聚丙烯管40,42毫升分装。在解冻到播种前生物芯片室温下几个小时。一旦解冻，不要再次冷冻。 </li><li>通过加入50ml胎牛血清，5毫升的200mM的L-丙氨酰-L-谷氨酰胺SUP制备细胞培养基plement和5ml青霉素/链霉素溶液（10,000单位青霉素/毫升和10000微克链霉素/ ml）的库存至500ml的L-15介质。这将产生560毫升含9％的血清的细胞培养介质。冷藏。 注意:这将是用于培养瓶和生物芯片的完整的细胞培养基。 </li></ol></li><li>粉状媒体样​​品瓶<ol><li>提前准备用自动粉末分配器含有60毫克±L-15ex粉0.5％，0.1 DRAM单元口小瓶。标签的小瓶粉末配发的日期和冷藏瓶（以50只），在再封金属化塑料袋;各含有了三片1克硅胶干燥包。 注释:L-15ex粉状介质小瓶可制成并存储了预先测试的长达9个月。 </li></ol></li><li>通过稀释家用漂白用去离子（DI）水达到100ml 20％的漂白剂的溶液中。估计有5毫升漂白溶液将需要对每个双向ochip。 </li><li>通过切割高压灭菌生物相容性管（待接种2个部分的每个生物芯片）为27毫米的部分使生物芯片管组件和适合与聚碳酸酯滑移Luer配件的管的两端。放置管组件中的耐高压加热的袋和高压釜在134℃8分钟。此外釜生物芯片插头（2％生物芯片）在一个单独的包在相同的设置。 </li><li>高压釜DI水30分钟at121℃。 注意:此水将用于播种后漂洗生物芯片。估计采用10ml将需要对每个生物芯片。 注:各生物芯片通道的实际体积为2毫升，但溶于5ml无菌水通过除去漂白溶液后的各信道漂洗。 </li><li>通过切割生物相容性管27毫米的部分和附接阳滑鲁尔装配件到管道的两端使注射器注入管道组件。发生在纸热封袋消毒和高压灭菌管组件8分钟，134＃176℃。 </li></ol> 2.生物芯片射流播种过程注意:请那里的生物芯片或媒体使用无菌技术II级生物安全柜进行处理的所有程序。 <ol><li>原定播种前二十四小时，从biohood制造商的包装和无菌塑料仪表到位的情况下删除该生物芯片。 </li><li>使用20％的漂白剂溶液如下消毒生物芯片 注意:从历史上看，此漂白过程已可防止在生物芯片真菌生长长期储存过程中的事件由生产商所做的生物芯片的等离子体灭菌是无效的。 <ol><li>用20毫升无菌注射器用注射器注射管组件连接并在biohood工作，注入将2ml 20％的漂白剂溶液的成生物芯片的各通道。让生物芯片坐1小时的漂白粉溶液。 </li><li>一小时真空asp的后愤怒的从两个通道使用连接到真空抽吸管路中的无菌雄性滑路厄组件的漂白溶液中。漂洗该生物芯片时，使用生物芯片管组件中的一个为漏极。 </li><li>使用附连到无菌注射器注射组件无菌20毫升注射器，冲洗芯片的每个通道用5ml无菌水，使多余的水排入在biohood的容器。然后真空吸掉作为漂白刚才描述和放置生物芯片放回塑料仪表案件，并在biohood离开，直到与细胞接种第二天的水。 </li></ol></li><li>六十分钟，接种生物芯片，注入将2ml10μg/ ml的纤连蛋白溶液加入到生物芯片的各通道之前。留在biohood 60分钟的生物芯片，然后用细胞接种在生物芯片之前真空抽吸掉纤连蛋白（如在步骤2.2.3描述）。放置无菌生物芯片管组件的两个部分（参照在生物芯片的端口1.4节）。 </li><li> Trypsinize使用在美国典型培养物保藏中心（ATCC）的产品说明片16所概述的程序每16生物芯片1汇合RTgill-W1 T175烧瓶中。 <ol><li>吸掉来自细胞的汇合烧瓶（多个）媒体。 </li><li>冲洗用15ml PBS的细胞层，然后吸关闭。 </li><li>加6 ml胰蛋白酶/ EDTA至每个T175烧瓶中的细胞层和允许细胞trypsinize〜5分钟。 </li><li> 15 ml完全L-15细胞培养基添加到每个烧瓶中以停止胰蛋白酶消化。 </li><li>结合细胞悬液在无菌的一次性容器。 注:容器尺寸可从150-500毫升，取决于被接种的生物芯片的数目而变化。有5 ml的细胞悬浮液将每生物芯片需要估计。 </li></ol></li><li>除去约1毫升细胞悬浮液，并放入用于计数一个离心管中。使用显微镜明用10X objective和血球，计数细胞的10微升等分试样，并计算达到2.5×10 5个细胞/ ml的细胞悬浮液所需要的完整的L-15细胞培养基的体积。 注意:如果使用的细胞悬浮液的种子烧瓶继续培养，这将是这样做的点。调整用完整的L-15细胞培养基的细胞悬浮液的浓度。 </li><li>使用无菌20毫升syringeattached到无菌注射器注入管道组件（见1.6节），注入2.5细胞悬液到生物芯片的各通道的外部端口（ 即 ，不具有连接管的端口），允许一些额外的细胞悬浮液的流出管到废物容器中的biohood。 注意:这将保证整个通道和连接的管道将充满的细胞悬浮液。 <ol><li>通过与路厄件和泵系列插入软管的自由端为每个生物芯片通道的封闭环纳克成用于每个通道的外端口。 </li><li>擦去多余的介质了与用70％乙醇浸湿的纸巾闭环，并放置在生物芯片放回塑料盒在20℃的孵化器。给每个生物芯片的唯一标识号。 </li></ol></li><li>天4和7，请从20℃培养箱生物芯片和补充媒体在所有温度平衡完整的L-15细胞培养基的生物芯片。按照相同的步骤，如步骤2.6除刚刚使用L-15细胞培养基代替（无细胞悬液）。将生物芯片早在20℃培养箱中4天进食后。 <ol><li>当天7喂奶后，删除和丢弃从芯片的软管，并在生物芯片插入蒸压排水塞。 </li><li>放置生物芯片中有6℃培养箱一个框，直到用于测试。 注意:该芯片可以储存在冷藏温度下长达9个月，但仍存活的测试我n个ECIS的读者。 </li></ol></li></ol> 3. ECIS测试与生物芯片<ol><li>如果使用准备测试的化学物质。 （参见布伦南等人于2012年7制备的试验化学品）。 </li><li>从手提箱中取出ECIS阅读器和测试设备。从6℃培养箱中取出事先准备好的生物芯片。放置在生物芯片上的纸巾。打开ECIS读者。 </li><li>用10ml注射器，分配10毫升控制水的成标记0.5盎司透明塑料控制罐和10ml试验样品放入一个标记0.5盎司透明塑料试验瓶。 注意事项:1）一定要去除气泡的精确测量。 2）注射器和罐子颜色编码;蓝色用于控制和红色测试。 </li><li>从铝箔袋删除两个粉状媒体小瓶。粉状媒体瓶打开其中一个（使用多功能的工具，如果需要的话）和一小瓶的全部内容倒入与控制水的罐子，下探整个小瓶成解决方案也是如此。重复与测试罐子此过程。盖上盖子并摇晃罐子，以确保粉末溶解。 </li><li>用9ml要么控制（蓝色注射器）或测试（红色注射器）从各自的小瓶解决方案填充每个彩色10毫升注射器。从注射器去除气泡。 </li><li>从生物芯片端口​​取下插头连接的流失。 </li><li>将生物芯片到ECIS的读者可移动塑料托盘，并盖上盖子。 </li><li>附加充式注射器外生物芯片的端口，并附加注射器活塞。 </li><li>从主屏幕上，选择"下一步"开始前测试。 注:读者软件会检查阻抗。如果阻抗范围内由用户（通常在1000和3000欧姆）的屏幕将寄存器设定为"墨盒合格"， 如图3所示，并指示用户选择"下一步"，该软件将进行到步骤3.10）。如果阻抗不内因为有缺陷的生物芯片或读者（通常是由于电极的错位）生物芯片的连接故障的设定范围，然后屏幕将注册"墨盒故障"，用户将不得不采取"取消"的选项或"验证"测试。 <ol><li>选择"取消"回到步骤3.9）。选择"确认"继续接收"柱体"消息，然后选择"下一步"以后的步骤3.10）。 注意:最好是把生物芯片并目视检查其是否有缺陷或泄漏，如果一个"墨盒故障"以新生物芯片在继续之前收到的消息。 </li></ol></li></ol><img alt="图3" src="/files/ftp_upload/53555/53555fig3.jpg" /> 图3:用可接受的阻抗读数生物芯片的ECIS阅读器截图的截图显示了在每个4个共同的欧姆阻抗初始读数ntrol电极（CE）和流体生物芯片内的4试样电极（SE）。 <a href="https://www.jove.com/files/ftp_upload/53555/53555fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="10"><li>当读者提示，使用软键盘输入样品信息，然后选择"接受"完成时。 注意:ECIS阅读器软件将自动加盖与日期，时间和由用户输入的其他信息的各数据集;例如生物芯片号码，以及化学和浓度的类型。阻抗数据的两个分将从插入生物芯片被收集和一个屏幕上的定时器将倒计时时间。 </li><li>当被一个红色框内闪动屏幕上的说明提示2分钟后，点击"下一步"。当闪烁的绿框"现在注入样品"提示，同时注入从所附的注射器的控制和测试介质装入生物芯片陈荫罴平。完成后离开注射器在生物芯片的地方。 注意:阻抗值将为60分钟收集每分钟一次。如果ECIS阅读器软件确定该处理通道是从控制信道在试验开始后的10分钟和60分钟之间的任何点统计学差异，则在屏幕上显示将表明，样品"污染"。如果治疗不从控制通道不同，那么屏幕上会显示"没有检测到污染"在试运行的结束。 </li><li>记录的测试结果被污染或不污染每个样品。 </li><li>在试运行结束后，删除和丢弃的生物芯片。冲洗并空气干燥该注射器，测试小瓶和可拆卸的塑料托盘其中容纳在测试期间的生物芯片。 注:ECIS读者有4 GB用于操作软件的板载存储，运行测试产生的控制模型和测试文件。这使得若干个ousand测试文件被存储在阅读器。文件可以用USB跳跃驱动器进行检索，并且如果需要传送到计算机，以便进一步分析用于研究目的。 </li></ol>

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Biol.- Animal. 45, 127-134 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RT-gill-W1+(ATCC+CRL-2523)+Culture+Method.">RT-gill-W1 (ATCC CRL-2523) Culture Method.</a> American Type Culture Collection (ATCC). , (2015).</li></ol>

Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Citations of commercial organizations or trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations. Authors Linda M. Brennan and Mark W. Widder, both of the US Army Center for Environmental Health Research, declare that they have no competing financial interests. Michael Mayo, Michael McAleer and Alex Greis are employees of Nanohmics that produces the disposable biochips and instruments used in this Article.10251980

ECIS的技术在实验室环境下表现良好，并能在浓度是相关的人体健康检测潜在的水污染物。该技术的便携和包装使得它有利于现场使用。 在该协议的技术成功的关键步骤如下:1）培养，播种期间保持无菌条件下，与生物芯片的馈送，2）保持种子生物芯片在冷藏条件下，直到准备用于自RTgill-W1细胞测试很长，一旦它们进行温度高于25℃时，将无法生存，3）准确称L形15ex在粉状介质小瓶和精确地测量水样品，以避免产生假阳性，其可通过在移位引起的媒体而不是样品毒性渗透压，4）按照ECIS屏幕上的用户说明运行测试。在阅读器软件将提醒如果双向用户ochip是用于测试（基于初始阻抗读数）不可接受时生物芯片首先插入读卡器。如果阻抗水平测试不能接受的，该软件将不允许用户与测试继续进行，直到一个新的生物芯片时使用。为不可接受的阻抗读数的原因通常是由于与ECIS读者销或沿着所述生物芯片的胶合边缘之一流体泄漏的生物芯片的电极的轻微未对准。 有一些限制这种技术，因为ECIS传感器只被用饮用水测试和不与表面水。所述RTgill-W1细胞是在生物芯片上不能较长时间（时间帧可以是从数小时至数天取决于温度耐受冷冻或温度大大高于25℃。在生物芯片起作用的温度范围最好从冷冻以室温7，他们准备立即使用，然而，作为后R权从冷库emoved。便携式冷藏容器目前在该领域使用的军队人员对温度敏感的用品。这些相同的容器可以用于种子生物芯片运输。 这种技术的另一个限制是，即使它是一个宽波段毒性传感器，它不，如果在所有反应良好，以抑制胆碱酯酶的化合物，如某些杀虫剂。为了填补这一能力差距，ECIS的传感器被设计以提供具有更广泛的毒性测试的用户测试水样时，在结合使用市售的快速农药试验测定。该试剂盒是一种快速酶法测定设计为在30分钟内检测有机磷和氨基甲酸酯杀虫剂。 ECIS的传感器补充WQAS-PM（水质分析系统 - 预防医学）域水测试系统，目前使用的军事预防医学人员发现，阿尔森IC，铅，氰化物或饮用水样本。虽然ECIS传感器将不能识别污染物是什么，这将表明，如果某些金属或有机化合物的存在，这表明水可能不适合人食用。 ECIS的测试结果可用一个小时之内。然后将水样可发出用于进一步分析的污染物的鉴定是否有阳性测试结果。 如上所述，ECIS的读者被设计为，其包括为了覆盖污染物检测的广谱单独酶ACE试剂盒的系统的一部分。这两个读者都被包装在运输领域为士兵野外使用一个坚固的情况下。

总体目标是开发用于细胞接种，储存和流体生物芯片的测试中的ECIS生物传感器的方法。对于该生物传感器的开发目标是，以满足美国陆军规格，可以检测饮用水供应的可能的污染正在由士兵一个字段便携式装置。对于毒性传感器的要求是，它可以在有关对人类健康浓度迅速检测有毒工业化合物的广谱（一个小时内），该装置是现场便携式，和生物组分将具有一个货架寿命至少九个月。冷藏，但不冻结，易腐成分是可以接受的。 从历史上看，与生物成分将它们现场便携式水检测技术（如抗体，酶，或核酸）已经被分析物特异性1-3。这些类型的生物传感器的缺点在于，它们将ONLŸ同时检测一种类型的化学品。如果怀疑该多个化学是本需要多个传感器。如果特定的传感器是不是在测试曲目，在水中化学污染物很容易被忽视。 广泛的毒性传感器，另一方面，必须填写此项技术差距的潜力。这些通常具有细胞成分对他们4-8。广泛的毒性生物传感器的优点是它们可以检测各种各样的化学污染物，其中包括它们的混合物和未知数，在时间5,9,10相对较短的存在。 使用细胞单层的电阻抗作为一个可能的毒性传感器，它也被称为电细胞-基底阻抗感测（ECIS）的测量的概念，首先由Giaever和Keese酒店11所述。在过去的二十年中，已经证明是细胞VIAB的敏感指标ility和细胞毒性。基本上，即附着在电极上的生物芯片的细胞单层暴露于高频率和低的电压和电流强度交流电流信号。细胞的汇合单层阻碍电子的流动。当细胞单层的完整性受到损害（当被引入一种有毒化学物质，如），ECIS的传感器记录在电阻抗的变化11-14。 图1示出ECIS的原则关系到细胞单层上的生物芯片。 <img alt="图1" src="/files/ftp_upload/53555/53555fig1.jpg" /> 图1:关于与简化ECIS读者电气原理生物芯片细胞单层ECIS插图原理 <a href="https://www.jove.com/files/ftp_upload/53555/53555fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 12所述的ECIS传感器技术被使用。这些细胞不适合现场使用实际的，然而，因为它们需要频繁更换培养基，只在有限的保质期，并且需要人工的 CO 2的环境和37℃温育温度。它被发现从虹鳟鱼鳃上皮细胞（RTgill W-1细胞）衍生的市售的细胞系可以在在环境CO 2室温进行测试，形成在生物芯片融合单层，可以存储在冷藏温度，和不得不在有关对人类健康的12个浓度一快速反应（1小时或更少）的化学品的广谱。 RTgill-W1细胞毒理学应用，以及在基础研究，是由Lee 等人 15审查。 对于播种，存储和容纳流体生物芯片检测方法就在ECIS传感器流体生物芯片RTgill-W1细胞单层如下所述。流体生物芯片可以保存长达9个月在冷藏状态，并且可以在一个冷藏集装箱运，对于饮用水supplies.The伴随ECIS阅读器，或测试单元的测试，被单独提供。生物芯片有两个组成部分的人;上部的聚碳酸酯层，用两个分开的流体通道，以及包含每第四频道电极焊盘用于阻抗传感低级电子层。有每个垫10工作电极;每个电极是250微米的直径。组装的生物芯片具有用于当插入ECIS测试单元获取阻抗读数金电极连接。每两个封闭流体的U形通道将持有2毫升RTgill-W1细胞悬浮液; 图2示出了在ECIS读者汇合的细胞上的单个感测电极的倍率的流体的生物芯片。 图2:在ECIS读者射流生物芯片的放大区域显示RTgill-W1细胞的单一感应电极覆盖单层<a href="https://www.jove.com/files/ftp_upload/53555/53555fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies, Inc. www.lifetechnologies.com</td>
			<td>16000-085</td>
			<td>Store @ -20 &#176;C. Thaw @ room temperature before use. Ingredient for complete L-15 cell culture media (10%).</td>
		</tr>
		<tr>
			<td>Fibronectin, bovine plasma</td>
			<td>EMD Millipore Corp.&#160;&#160; www.emdmillipore.com&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>341631-1 mg</td>
			<td>Store @ -20 &#176;C. Thaw @ room temperature before use. Mix with L-15 media for a concentration of 10 ug/mL and freeze @ -20 &#176;C in aliquots. Use as substrate for biochips.</td>
		</tr>
		<tr>
			<td>L-15 media without L-glutamine</td>
			<td>Lonza&#160; www.lonzabioscience.com</td>
			<td>12-700F</td>
			<td>Basal media for cell culture and feeding biochips. Store at 6 &#176;C.</td>
		</tr>
		
		<tr>
			<td>L-15ex powdered media with phenol red</td>
			<td>US Biological&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.usbio.net</td>
			<td>L1501</td>
			<td>Media is weighed out in 60 mg aliquots in 0.1 dram vials and stored at 6 &#176;C in foil pouches with dessicant packs. Nine month shelf-life. Mixed with 10 mL of water sample for testing in biochips.</td>
		</tr>
	
		<tr>
			<td>PBS, w/o Ca++ or Mg++</td>
			<td>Lonza&#160; www.lonzabioscience.com</td>
			<td>17-516F</td>
			<td>Store at room temperature. Used for rinsing media when trypsinizing cell culture flasks.&#160;&#160;</td>
		</tr>
		
		<tr>
			<td>Trypsin, EDTA</td>
			<td>Lonza&#160; www.lonzabioscience.com</td>
			<td>CC-5012</td>
			<td>Store @ -20 &#176;C. Thaw at room temperature and use to trypsinize cell culture flasks.&#160;</td>
		</tr>
	
		<tr>
			<td >T175 culture flasks</td>
			<td >Fisher Scientific 
			www.fishersci.com</td>
			<td >12-565-30</td>
			<td >Used for culturing RTgill-W1 cells.</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Chlorox&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.chlorox.com</td>
			<td></td>
			<td>Diluted to 20% with millique or distilled water for cleaning ECIS chips. Any household bleach is acceptable.</td>
		</tr>
		
		<tr>
			<td>70 % ethyl alcohol</td>
			<td></td>
			<td></td>
			<td>For disinfecting biohood surfaces and any materials being placed in biohood.</td>
		</tr>
		<tr>
			<td>Rainbow trout gill cells (RTgill-W1)&#160;</td>
			<td>American Type Tissue Culture Collection&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.atcc.org</td>
			<td>CRL-2523</td>
			<td>Cells cultured and used for biosensor (seeding biochips).</td>
		</tr>
	
		<tr>
			<td>GlutaMAX-1 Supplement, 200 mM</td>
			<td>Lonza&#160; www.lonzabioscience.com</td>
			<td>35050-061</td>
			<td>Store at room temperature.&#160; Ingredient for complete L-15 cell culture media (1%).</td>
		</tr>
	
		<tr>
			<td>Penn/Strep Stock 10K/10K</td>
			<td>Lonza&#160; www.lonzabioscience.com</td>
			<td>17-602E</td>
			<td>Store @ -20 &#176;C. Thaw @ room temperature before use. Ingredient for complete L-15 cell culture media (1%).</td>
		</tr>
		<tr>
			<td>Pharmed BPT tubing</td>
			<td>U.S. Plastic Corp.&#160;&#160;&#160;&#160;&#160; www.usplastic.com</td>
			<td>57317</td>
			<td>Cut in 27 mm sections and autoclaved. Used for seeding biochips with cells and as a closed loop between media changes.</td>
		</tr>
		
		<tr>
			<td>Polycarbonate luer fittings for Pharmed tubing assemblies</td>
			<td>Value Plastics&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>MTLS210-9</td>
			<td>Secured to each end of cut Pharmed tubing for insertion into bichips.</td>
		</tr>
	
		<tr>
			<td>20 mL syringes, slip-tip</td>
			<td>VWR Scientific&#160;&#160;&#160;&#160;&#160; us.vwr.com</td>
			<td>BD302831</td>
			<td>Used for injection of cell suspension for seeding ECIS chips, as well as for feeding chips.</td>
		</tr>
		
		<tr>
			<td>0.1 dram snap-cap polypropylene microvials</td>
			<td>Bottles Jars and Tubes, Inc.&#160;&#160; www.bottlesjarsandtubes.com</td>
			<td>30600</td>
			<td>Used to store 60 mg aliquots of L-15ex powdered media.</td>
		</tr>
		
		<tr>
			<td>60 mil Lexan fluidic ECIS biochips</td>
			<td>Nanohmics, Inc.&#160;&#160;&#160; www.nanohmics.com</td>
			<td></td>
			<td>Custom-made by Nanohmics, Inc. RTgill-W1 cells will be injected into the biochips and seeded chips will be placed in ECIS reader for testing.</td>
		</tr>
	
		<tr>
			<td>Autoclavable Plastic Instrument Box 
			17 1/2&#34; x 7 3/4&#34; x 2 3/8&#34;</td>
			<td >Medi-Dose EPS&#160;&#160;&#160;&#160; medidose.com</td>
			<td >IB701</td>
			<td >Used to store the following; autoclaved plugs, biochips that have been cleaned, seeded biochips.&#160;</td>
		</tr>
		<tr>
			<td>Paper heat-seal sterilization pouches, 7 &#189;&#8221; x 13&#8221;</td>
			<td>CardinalHealth&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.cardinalhealth.com</td>
			<td>90713</td>
			<td>Used for autoclaving tubing and fittings and plugs.</td>
		</tr>
	
		<tr>
			<td>Quantos automated powder dispenser</td>
			<td>Mettler Toledo&#160;&#160;&#160;&#160;&#160;&#160; www.mt.com</td>
			<td>QB5</td>
			<td>Automated dispension of 60 mg aliquots of powdered L-15ex into 0.1 dram vials.</td>
		</tr>
		
		<tr>
			<td>ECIS reader</td>
			<td>Nanohmics, Inc.&#160;&#160;&#160; www.nanohmics.com</td>
			<td></td>
			<td>Custom-made by Nanohmics, Inc. Seeded biochip is inserted into the reader for conducting water toxicity testing.</td>
		</tr>
	
		<tr>
			<td>3 X 5 metalized 2.5 mil polypropylene reclosable bags</td>
			<td>Uline&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.uline.com</td>
			<td>S-16893</td>
			<td>Packaging and storage for both seeded biochips and powdered L-15ex media vials.</td>
		</tr>
		
		<tr>
			<td>Leatherman squirt ps4</td>
			<td>Amazon&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.Amazon.com</td>
			<td></td>
			<td>Used to open powdered media vials.</td>
		</tr>
		
		<tr>
			<td>1 gram silica gel desiccant packets&#160;</td>
			<td>Uline&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.uline.com</td>
			<td>S-3902</td>
			<td>Put in polypropylene bags with L-15ex powdered media vials to prevent the powder from picking up moisture.</td>
		</tr>
	
		<tr>
			<td>Sterile 250 or 500 mL Nalgene bottles</td>
			<td >Fisher Scientific 
			www.fishersci.com</td>
			<td >09-740-25C or E</td>
			<td >Hold cell suspensions for seeding ECIS chips in biohood.</td>
		</tr>
	
		<tr>
			<td>Plugs for biochips</td>
			<td>Nanohmics, Inc.&#160;&#160;&#160; www.nanohmics.com</td>
			<td></td>
			<td>Custom-made by Nanohmics, Inc. Used to seal ports on biochips before storage @ 6&#176;C.</td>
		</tr>
		<tr>
			<td>Drains for ECIS biochips</td>
			<td>Nanohmics, Inc.&#160;&#160;&#160; www.nanohmics.com</td>
			<td></td>
			<td>Custom-made by Nanohmics, Inc. Placed on 2 inner ports on biochips prior to insertion in ECIS reader.&#160; Allows for excess media to drain from channels during test injections.&#160;</td>
		</tr>
		
		<tr>
			<td>Hemocytometer</td>
			<td >Fisher Scientific 
			www.fishersci.com</td>
			<td >S17040</td>
			<td >Needed for counting cells prior to adjusting cell suspension for injection into biochips.</td>
		</tr>
		
		<tr>
			<td>Brightfield microscope w/ 10X objective</td>
			<td>Leitz Labovert</td>
			<td></td>
			<td>Any brightfield microscope is acceptable.</td>
		</tr>
		<tr>
			<td>Class II biological safety cabinet</td>
			<td></td>
			<td></td>
			<td>Any class II biological safety cabinet where cell culture can be performed under sterile conditions is acceptable.</td>
		</tr>
		
		<tr >
			<td >Microcentrifuge tubes, 0.6 mL</td>
			<td >Fisher Scientific 
			www.fishersci.com</td>
			<td >02-681-311</td>
			<td >Holds 1 mL of cell suspension prior to counting cells.</td>
		</tr>
	
		<tr >
			<td >Slip 10 cc red syringes</td>
			<td >Procedure Products, Inc.&#160;&#160;&#160;&#160;&#160;&#160; www.procedureproducts.com</td>
			<td >S/49S 30-R</td>
			<td >Withdraws 9 mL of test water sample and used to inject sample into biochip.</td>
		</tr>
		
		<tr >
			<td >Slip 10 cc blue syringes</td>
			<td >Procedure Products, Inc.&#160;&#160;&#160;&#160;&#160;&#160; www.procedureproducts.com</td>
			<td >S/49S 30-B</td>
			<td >Withdraws 9 mL of control water sample and used to inject sample into biochip.</td>
		</tr>
		
		<tr >
			<td >&#189; oz. clear pet plastic jar w/ white ribbed lined caps</td>
			<td >SKS Bottle &#38; Packaging, Inc.&#160;&#160;&#160;&#160;&#160;&#160;&#160; www.sks-bottle.com</td>
			<td >0605-30</td>
			<td >Sample vials used for mixing L-15ex powder and 10 mL of water sample for testing.</td>
		</tr>
		
		<tr >
			<td >50 mL sterile conical polypropylene centrifuge tubes</td>
			<td >Fisher Scientific&#160;&#160;&#160;&#160; www.fishersci.com</td>
			<td>12-565-269</td>
			<td >Used to hold 40 mL aliquots of 10 ug/mL fibronectin @ -20 &#176;C.</td>
		</tr>
	</tbody>
</table>

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.

在本文中描述的技术ECIS在美国环保署（USEPA）进行测试赞助的技术测试和评估计划（TTEP）。被选定为测试作为有毒工业化合物，可能是饮用水的可能的污染物的广谱的代表十三化学品。在测试中，13 9被ECIS一个小时内的浓度是相关的人类健康检测8。 表1示出了这种污染物测试的结果; 图4是代表什么一个"被污染的"结果将类似于上ECIS的读者屏幕。在大多数情况下，蜂窝阻抗为被污染的样品与对照相比减少。偶尔，某些化合物可能引起的阻抗的增加。 也总结在表1中 </s仲&gt;是干净的水测试。第四清洁水的样品运行，并在任何样品中没有检测到污染（参见图5）为"无污染检测到"的代表性屏幕截图。 <img alt="图4" src="/files/ftp_upload/53555/53555fig4.jpg" /> 图4:ECIS读卡器截图一个"被污染的"水样的从被污染的水样归一化阻抗的图形和结果的一个例子。蓝线代表每个控制电极的归一化的阻抗;红线代表每个试样电极中的标准化阻抗。 <a href="https://www.jove.com/files/ftp_upload/53555/53555fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53555/53555fig5.jpg" /> 图5:ECIS读卡器截图一个"不污染检测到"水样的从已不被污染的水样归一化阻抗的图形和结果的一个例子。蓝线代表每个控制电极的归一化的阻抗;红线代表每个试样电极中的标准化阻抗。 <a href="https://www.jove.com/files/ftp_upload/53555/53555fig5large.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>类别</td><td>污染物</td><td colspan="3">浓度测试（毫克/升）1 </td><td colspan="2">检测≤1小时 N = 4/4芯片</td></tr><tr><td rowspan="8">农药</td><td>涕灭威</td><td colspan="3"> 0.17 </td><td colspan="2">没有</td></tr><tr><td>阿尔森<html> IC（亚砷酸钠） </td><td colspan="3"> 4.5 </td><td colspan="2">是</td></tr><tr><td>叠氮化物（叠氮化钠） </td><td colspan="3"> 46.​​7 </td><td colspan="2">是</td></tr><tr><td>苯线磷</td><td colspan="3"> 0.56 </td><td colspan="2">没有</td></tr><tr><td>甲胺磷</td><td colspan="3"> 1.4 </td><td colspan="2">没有</td></tr><tr><td>甲基对硫磷</td><td colspan="3"> 33.6 </td><td colspan="2">是</td></tr><tr><td>百草枯（二） </td><td colspan="3"> 4.6 </td><td colspan="2">没有</td></tr><tr><td>五氯酚（钠） </td><td colspan="3"> 71.9 </td><td colspan="2">是</td></tr><tr><td rowspan="5">工业化学品</td><td>氨</td><td colspan="3"> 924 </td><td colspan="2">是</td></tr><tr><td>铜（铜II硫酸盐） </td><td colspan="3"> 103 </td><td colspan="2">是</td></tr><tr><td>氰化物（钠） </td>3"&gt; 14 </td><td colspan="2">是</td></tr><tr><td>汞（氯化物） </td><td colspan="3"> 24.7 </td><td colspan="2">是</td></tr><tr><td>甲苯</td><td colspan="3"> 444 </td><td colspan="2">是</td></tr><tr><td>清洁水2 </td><td>没有</td><td colspan="3"> NA </td><td colspan="2">没有</td></tr><tr><td colspan="7"> 1浓度测试是相同的由的Widder 等人的手稿。 （2014年）。 </td></tr><tr><td colspan="7"> 2 40清洁水样品用没有污染运行。 </td></tr></tbody></table> 表1:通过检测ECIS水样污染物。

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

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 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.

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

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9.0K 次观看.  U.S. Army Center for Environmental Health Research. 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. 

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

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. ...

科研

环境科学

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

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 CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

一个外线<sub&gt; 2</sub&gt;浓度梯度用于测试CO<sub&gt; 2</sub&gt;浓度与土壤对草地生态系统功能的影响

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

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 ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

用于表征土壤微生物的脂质提取和分析方法在许多样品的实验中

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

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 ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...