This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.

注意:下面的协议说明了SPE 100毫升的水样品与C18的吸附剂和洗脱用50％体积/体积甲醇进行。富集的样品稀释分析用酶联免疫吸附试验（ELISA）试剂盒的酶之前达到10％体积/体积甲醇。 1.准备试剂<ol><li>制备水样<ol><li>之前的任何其它步骤，筛选各100毫升的水样品与0.2μm孔径的过滤器。 </li><li>通过稀释的参考溶液适当体积入水体积穗与期望浓度的样品。例如，制备100毫升的水样品与E2的100纳克/升的稀释3.3微升的E2参考溶液在300微克/升的浓度。稀释此溶液十次以获得一个样品掺入10纳克/升的E2。稀释后者另一个十倍，得到100ml的样品与1纳克/升的E2。 </li><li>放置过滤后的样品（未修饰的或加标）在玻璃瓶中以GL45线程。使用示例在同一天。取一小部分，以与ELISA法进行分析，来估计初始浓度。 </li></ol></li><li>通过在去离子（二 - ）水在紧管稀释甲醇至50％体积/体积制备300μl的洗脱液。 </li></ol> 2.制备固相萃取柱的<ol><li>通过加入第一1600微升甲醇中，然后加入400μl的二 - 水至40mg反相吸附剂的制备20毫克/毫升悬浮十八烷基二氧化硅吸附剂颗粒。盖紧盖子，搅拌用旋涡。 </li><li>安装在塔底部的膜<ol><li>选择一个尼龙膜孔径为11微米，将其放置在防尘组织构成的双层。用3毫米直径冲头，切断在膜2的小零件。 </li><li>抓住小膜用平头镊子过滤器之一，并将其​​放置在列的一侧。 </li><li>拧平底接头与管并拧紧。该膜为n流到位和吸着能够可靠地加入。上朝向的最后一列指示的体绘制的箭头，其中膜置于。 </li></ol></li><li>准备填充吸附柱<ol><li>固定柱向底部的箭头指向在支架上。列必须是垂直地设置。 </li><li>附加空10毫升一次性注射器到列的管的端部，通过使用鲁尔锁定连接​​器。 </li><li>准备微量移液器用20-200微升小费，定容至100微升。此枪头格式适于吸附剂柱的要制备的大小。该程序将具有较大的枪头，如用1毫升吸管中使用的那些更加困难。 </li><li>搅动吸附剂悬浮液用涡旋，并迅速吸管100微升在中心上的列。同时注入，吸轻轻所有移液溶液通过使用注射器用另一只手槽膜。在这届年龄，溶液灌装注射器必须明确微粒，粒子床最终可以在列中观察到。 </li><li>通过搅拌所有移液步骤之间的纸浆悬浮液，以确保在溶液中的颗粒的均匀悬浮液重复此过程2次。由此产生的列包含吸附剂6毫克。 </li><li>当将300μl悬浮液已被加载并通过用注射器抽吸干燥，保持注射器在适当位置，并放置在顶部的第二尼龙膜通过使用钳子用另一只手。 </li><li>拧紧第二个连接器管和处置注射器。该固相萃取柱就可以使用了。 </li></ol></li></ol> 3.准备系统<ol><li>通过使用鲁尔锁接头拧紧设备上的固相萃取柱。箭头必须是指向相同的方向在设备上显示的。 </li><li>通过拧紧连接含有样品到装置中的瓶它提供了GL45安全帽。 </li><li>加载200微升洗脱液中的"淋洗液"水库。 </li><li>负载800微升二水的"稀释"水库。 </li><li>将一个瓶在排污口在提取步骤收集处理过的水。格式并不重要，但体积需要足够大的方面的样品体积。 </li><li>将小瓶在1.5毫升体积最小容量的传感器出口。 1 mL的最大容积将过小，收集丰富洗脱液和稀释液时气泡会形成在该出口。 </li><li>验证压力调节器处于关闭位置时通过手动转动它，反向旋转，直至进一步的移动是不可能的。 </li></ol> 4. SPE与原型<ol><li>通过按下背面的按钮开关上的原型。 </li><li>制备用户界面和选择程序<ol><li>打开用户界面。选择计算机的通讯端口来进行设备从列表连接，然后点击按钮"下一步"。 </li><li>输入值580 PSET和30 dpset。泵将调整运行时，以维持在加压系统和储层压力，在580±30毫巴。 </li><li>选择自动模式。 </li><li>在自动模式下的角落，装入箱子"配置文件路径"程序文件。 </li></ol></li><li>调节压力调节器<ol><li>启动泵。 </li><li>手动转动压力调节器，直到读取预浸料值小于但接近320毫巴。 </li><li>停止泵。 </li></ol></li><li>启动SPE程序<ol><li>按在自动模式下的角落"开始"。泵将接通和提取，洗脱和稀释步骤将自动跟随另一个。在50分钟中进行，整个过程在100毫升样品。 </li><li>验证预浸的值。它必须是在范围320 - 350毫巴在提取步骤以确保最佳的流速。 </li><li>关闭小瓶并储存在4-5°C黑暗直至分析。在下面的30小时进行分析，以防止分析物的降解。 </li><li>处置处理过的水。 </li></ol></li><li>清洁系统 注意:在待清洁每次萃取步骤的系统需要防止交叉污染。 <ol><li>制备甲醇70％体积/体积的在一个GL 45玻璃瓶10ml的溶液中。 </li><li>拔下固相萃取柱和插头与连接器的管道。 </li><li>在自动模式下，选择"清洗"文件和相同的压力设置启动。 </li></ol></li></ol> 5.检测与ELISA雌二醇浓度<ol><li>制备校准样品浓度在设有所使用的ELISA试剂盒的协议表示。准备一张CALIBRA中置具有相同的矩阵作为样品，和一个校准设置用甲醇10％体积/体积。 </li><li>分配在盘上的孔中的样品的所需量，根据制造商的说明。使用校准样品，过滤但未处理与SPE，和丰富的样品甲醇10％V不变并加标水样/ V。使用每样本3个孔，以减少与测定相关的误差。 </li><li>遵循即随试剂盒提供的反应物的加入，培养时间，洗涤和进一步反应与酶的协议的指示。 </li><li>读出的光信号在每个孔中根据试剂盒制造商的用酶标仪仪器指令和收集数据。 </li><li>使用平板读取器仪器的软件，以适合校准曲线，并确定与相同矩阵的初始样品中的E2浓度，并且在富集样品用甲醇10％体积/体积。 </li></ol>

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The authors have nothing to disclose.

提出了水样然后分析使用免疫的制备新方法。该仪器能够以自动化和用户友好的方式进行固相萃取。 水样其注射之前到系统的过滤是非常关键的。溶液中存在的任何微粒仍然会潜在地引起流体网络的堵塞和阻碍SPE柱。另一个重要步骤是在SPE柱的制备。在列颗粒的量是至关重要的，以达到最佳的性能成为可能。制备吸附剂颗粒的悬浮液时，避免了粒子的附聚特必须小心​​。?...

样品制备是在任何分析过程中的重要一步。特别是，除去基体效应，干扰的缩减，且分析物的富集是必要的，以获得精确的结果，并达到检测的低限。内分泌干​​扰化合物（内分泌干扰物）是特别值得关注的，因为它们对活的生物体的动作，即使在环境中存在的非常低的水平。天然激素17β雌二醇存在于欧盟水污染的收藏和容易被加入到根据欧洲水框架指令调节优先物质清单。固相萃取（SPE）通常适用于小的污染物在水中的分析，与两个化学1-5（色谱，质谱）和免疫6-9检测方法。后者积累了环境监测领域的兴趣，如免疫测定法是在大量的各种格式提供，特定于たrget分析物，和达到低检测极限。6，7，10，11的各种酶联免疫吸附测定（ELISA）是可商购，并使得能够分析多个样品同时上的多孔板。该过程包括在连续的反应步骤，可能需要几个小时。反应的最终产物，可以检测光学来确定基于校准曲线对目标分子的浓度。 经典的SPE程序包括吸附剂预调节，样品萃取，清洗，洗脱，浓缩由洗脱液蒸发。用于稀释该提取物的溶剂选自根据检测方法。对于免疫学方法中，有机溶剂的影响强烈的方法的灵敏度的量。12 除了回收和预浓缩的表演中，该方法也需要简单和成本效益。在再修改的自动化Ë有助于减少人类相关的错误。在我们以前的工作13我们推出了样机的自动固相萃取，而我们的方法适用于天然激素17β雌二醇海水样品中的分析。在本视频中，我们想强调我们方法的技术优势相对于传统的离线和在线SPE，以及它与通过检测免疫反应的具体的兼容性。我们描述适用于水样品17β雌二醇的检测的协议。 SPE与十八烷基硅胶（C18）吸附剂相和洗脱用稀甲醇进行执行。

<table border="0" cellpadding="0" cellspacing="0" width="609">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter membrane 0.2 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">GNWP04700</td>
			<td style="width:167px;">For sample filtration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon membrane 11 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">NY1104700</td>
			<td>For SPE column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable biopsy punch 5 mm</td>
			<td style="width:107px;">Medical Budget</td>
			<td style="width:119px;">39302439</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nucleodur C18 ec&#160;</td>
			<td style="width:107px;">Macherey Nagel</td>
			<td style="width:119px;">713550.01</td>
			<td>50 &#956;m particle diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic sea water</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">SSWS500-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol standard</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;"></td>
			<td>300 ng/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol ELISA kit&#160;</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;">ADI-900-008</td>
			<td>96 wells, range 30 - 3000 ng/L</td>
		</tr>
	</tbody>
</table>

a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants

对环境水样的固相萃取（SPE）的新方法。所开发的原型是成本效益和用户友好的，并能进行快速，自动化和简单的SPE。预浓缩的溶液与通过免疫测定分析兼容，具有低的有机溶剂含量。一种方法被描述为天然激素17β雌二醇在100ml水样提取和预浓缩。反相SPE执行与十八烷基二氧化硅吸附剂和洗脱用200μl甲醇50％体积/体积的完成。洗脱剂是通过将二 - 水，以降低甲醇的量稀释。手动制备SPE柱后，整体过程1小时内自动执行。在该过程结束时，雌二醇浓度通过使用市售酶联免疫吸附测定（ELISA）进行测定。 100倍预浓缩得以实现，在只有10％体积/体积的甲醇含量。完全恢复的分子用1ng得以实现/升掺入去离子和合成海水样品。

吸附剂填料的再现性通过干燥评价和加权在玻璃小瓶的移液吸附剂，结果示于图1。重复性的喷射时100ml中的样品进行了测试，如在初始和预如图 2所示。浓度浓缩加标样品通过使用商业ELISA试剂盒17β雌二醇测定，并示于图3。 所提出的方法包括在制备吸附剂相（ <st...

Watch this Scientific Journal Video about A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants at JoVE.com

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

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 ...

a-simple-method-for-automated-solid-phase-extraction-water-samples

Research

JoVE Journal

Environment

13.6K 次观看.  Centre Suisse d'Electronique et de Microtechnique. A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

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

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

小体积的方法从水中病毒浓度

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

科研

环境科学

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. ...

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. ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

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 ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

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 ...

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 ...

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 ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.