这项工作由研究项目资助:"NanoBioTech ambiente e salute。Progetto 2:Ambiente。Strumenti e metodi per il monitoraggio ecotossicologico delle Nanoparticelle"（ "Nano-BioTechnology:environment and health。Project 2:Environment。Tools and methods for ecotoxicological监测纳米粒子" ）授予LM来自Regione Lazio-Consorzio Hypatia的LM。 AR在以前引用的项目的框架下由Tor Vergata / Regione Lazio-Consorzio Hypatia大学的博士后资助。与ISPRA-Tor Vergata大学（N. 2015/52857）的协议允许相互使用设施和研究人员的交流。 作者感谢Maria Cristina Thaller教授担任所有微生物活动的守护天使，以提高对微生物世界的兴趣，并大力帮助改善我们对该问题的研究。作者衷心感谢AndreaTornambè和Erika Magaletti与浮游植物生态和生态毒理学ISPRA实验室的宝贵协作。

1.试剂/材料的制备<ol><li>准备（约300）无菌1.5 mL管，用于连续稀释细菌悬浮液，以及15 mL无菌管作为测试浓度标记的测试容器。 </li><li>准备2％NaCl溶液作为曝光介质并灭菌。或者，使用灭菌的合成或天然海水，盐度范围为5至40。 </li><li>根据标签方向准备具有2％NaCl的胰蛋白酶大豆琼脂（TSA）生长培养基，并考虑培养基中已经存在的NaCl量。 </li><li>将TSA（冷却但仍然是液体的）倒入以前用测试浓度和暴露时间，重复数量和稀释因子标记的90-mm培养皿中; 19 mL是适当的体积。 </li><li>根据标签方向制备胰蛋白胨大豆肉汤（TSB）生长培养基。加入适量的NaCl以获得与曝光介质相同的盐度。 </li><li>准备具有双蒸水的CuSO 4储备溶液，并使用0.22μm注射器过滤器消毒（必需）等分试样。在环境样品的情况下，准备适当的样品稀释间隔，并使用0.22μm注射器过滤器进行灭菌。 </li><li>在测试浓度标记的15 mL管中制备测试溶液。用5 mL曝光介质（2％NaCl）填充阴性对照。用适量的暴露介质和CuSO 4储备液填充其他管，以获得最终体积为5 mL的测试浓度。 </li></ol>细菌接种物制备<ol><li>试验前12-18 h，制备鳗弧菌液体新鲜培养物。使用无菌环，从固体培养基（TSA）上的过夜培养中选择一个单独的，分离良好的菌落。接种填充有10mL TSB的管，并将细菌培养物在25℃下孵育12-18小时。 </li&gt; <li> 12-18小时后，用分光光度法估计接种物的细菌浓度。涡旋接种物并测量600 nm波长的光密度，使用TSB作为空白。 </li><li>为了获得已知的细菌浓度，通过加入通过以下公式计算的TSB量稀释2mL的涡旋接种物: TSB mL = [（OD / 0.14）* 2] -2。 </li><li>验证稀释接种物的光密度是0.140（±0.005），这对应于麦克法兰德比浊法标准的0.5分。 </li><li>将稀释的接种物以3,000g离心10分钟。消除上清液并将微生物沉淀物重新悬浮在1mL的2％NaCl溶液（曝光介质）中。 </li></ol> 3.测试曝光<ol><li>加入150μL重悬细菌接种物到每个管，包括对照。在黑暗和连续条件下，在25℃下孵育鳗弧菌悬浮液6小时摇晃以避免沉淀。 </li><li>在暴露时间的开始（T 0 ）和结束（T 6 ），使用菌落形成单位（CFU）计数法在所有暴露的细菌悬浮液中进行细菌计数。 </li><li>准备连续稀释每种暴露的细菌悬浮液一式三份，应用十倍稀释因子（最多10 -5 ）。将100μL每个细菌悬浮液加入到已经填充有900μL曝光介质（2％NaCl）的相应管中。继续进行连续稀释，每个步骤涡旋以重新悬浮细菌。 </li><li>在对应的片段中的TSA培养皿上10μL的10 -4和10 -5稀释液。通过旋转盘子快速让液滴滑入一个小圆圈。将板在25℃孵育48小时。 </li></ol> 4. CFU计数<ol><li> 48小时后，计数在培养皿上生长的菌落;藏有b的盘子5和50个菌落是准确计数的最佳选择。 </li><li>通过应用以下公式计算每毫升暴露的每个细菌悬浮液的活细菌数: 10 -4 →CFU / mL = n°CFU×100×10,000 10 -5 →CFU / mL = n°CFU×100×100,000 </li><li>使用并行重复获得的计数的平均值来评估与对照相比的死亡率。以下列公式计算死亡率百分比: M％= 100 - [（N / C）* 100] 注意:其中N =暴露于毒物后生长的CFU / mL数; C =在对照培养基中生长的CFU的数量。 </li><li>使用适当的统计软件（参见材料表），使用非线性回归分析计算LC 50 （ 即，将活性复制细菌数量减少50％/ mL的CFU / mL）。运行单向方差分析（ANOVA）通过事后成对t检验来评估治疗之间的显着差异。 </li></ol>

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作者宣称他们没有竞争的经济利益。

这项工作描述了一种新的生物测定与海洋细菌V. anguillarum成功应用于评估CuSO 4 （一种参考毒物）的毒性作用，显示出明确的剂量反应关系。海洋细菌V. anguillarum被选为模式生物，因为它是无害的，无所不在的，代表海洋生态系统。 测试可以在广泛的盐度值（5-40）下进行，并且可以使用盐水溶液和合成或天然海水作为曝光介质，只要微生物能够在整个测试期间容易地存活。这样可以分析不同种类的样品，包括咸水和海洋环境。 在暴露阶段不需要生长培养基，避免干扰污染物8及其对生物反应的可能影响。钍e协议可靠，快速，成本有效，而且比较容易。液 - 板微计数9的过程具有使用小（样品）体积的优点，尽管这意味着高度的精度和鲁棒性。三项独立试验的结果和每次治疗的重复显示了该方法的高重复性。使用细菌作为生物学模型，以及该技术的适应性，有利于此过程的生态和环境相关性。其他关键的技术问题是在细菌接种物的制备中的准确性和在该步骤的一些步骤中所需的不育性。 提出的测试比其他海洋生态毒理学测定（24-96小时）更快（6小时），并且不会引起使用高等生物所带来的伦理问题。此外，参考毒物的数据显示LC 50值与用急性t获得的值相当<html>对其他海洋物种的遗传10,11表现出良好的敏感性。在细菌生物测定中， V. fischeri发光抑制测试是最常见和标准化的12 。该生物测定非常快速（15-30分钟），并且对于固相样品的测试是有效的，但它可能受到着色和混浊样品的影响，这些样品干扰了发光测量。盐度是使用上述试验的限制因素，2％NaCl需要13 。相反，这里提出的具有V. illar illar the the um um um um um um um um um um um um um um。。。。。。。。。。。。。。。。。。。。。。。。。。。我们的研究结果与V. fisheri的文献中的比较 14 ，ss ="xref"&gt; 15,16表示可比较的EC 50值，进一步支持此生物测定的有效性。 该生物测定评估了在目前可用于微生物的测试中使用的细菌培养性（通常称为死亡率）的降低，而不是群体生长速率或酶活性抑制。 LC 50计算允许与通常应用于通常以生存/死亡率为终点的海洋环境的生态毒理学评估的其他生物测定法进行比较。为了评估/确认该测试的可靠性和可重复性，并且支持其在规范协议中的标准化和使用，迫切需要进行相互校验。 纳米材料的越来越多的使用及其在环境中的潜在释放意味着需要进行风险评估17 。但是，克拉斯这些新兴污染物的sical（eco）毒理学方法似乎没有提供可承受的结果，可能需要一些适应性18 。这种新的生物测定的特征使其易于有效的应用于纳米颗粒的毒性评估。事实上，在不同盐度下进行测定的可能性将会说明不同离子强度下的纳米粒子行为，一个可以显着影响毒性的环境参数变量。此外，在纳米颗粒的生态毒性评估中，特别推荐使用生长培养基和营养素，因为有机物质可以通过增加毒性作用促进它们的吸收，或者可能引起聚集，降低生物利用度，从而降低其毒性21 。 总之，鳗弧菌的生物测定是ap古典和新兴污染物风险评估的浪漫工具，以及海洋和咸水环境状况的评估。

生态毒理学生物测定评估化学品或环境样品与标准生物学模型的毒性，将物理，化学和生物应激物的作用整合到生态系统上。由于生态系统的复杂性，生态毒理学风险评估必须考虑一系列涉及不同营养水平的生物体的生物测定。实验室动物的毒性测定可能是昂贵的，耗时的，并且在道德上是有问题的。限制动物测试和开发替代方法（ 例如细菌和非脊椎动物）的动力现在是当前欧洲立法框架中所报告的关键问题，其中包括欧盟动物保护指令，第七修正案欧盟化妆品指令和REACH。 甲壳类动物，鱼类和藻类主要用于海洋环境中的毒性测量1 。细菌是重要的成分生态系统的变化以及对微生物群落的改变可能对生物地球化学循环和其他关键生态系统服务产生重大影响。基于微生物的毒性测试越来越受欢迎，因为它们相对快速，可重现和便宜，不会引起伦理问题2 。这项工作的目的是描述一种生态毒理学方案，以评估当暴露于环境污染物时海洋细菌鳗弧菌 （ 鳗 弧菌 ）的响应。 鳗弧菌是具有极性鞭毛的革兰氏阴性短曲线状细菌（0.5×1.5μm）。通常在咸水或盐水中发现，它是耐盐的，最佳盐度约为20，最佳温度在25至30℃之间。由于其普遍存在及其在海洋中的重要生态作用，被选为生物体模型全球4 。已知鳗弧菌的一些血清型可导致各种海洋或微咸鱼类物种5,6的弧菌病 。为此，实验的一些步骤需要标准的微生物学实践，但不需要特殊的安全设备或预防措施。拟议的毒性试验方案使用细菌培养性（ 即，复制和形成菌落的能力）作为终点，并允许LC 50的测定，导致细菌积极复制和形成菌落50％的浓度的浓度，之后6小时曝光。在弧菌中 ，与其他微生物一样，我们通常表示为死亡率的这种减少可部分归因于可行但不可培养（VBNC）阶段7中的个体。在本研究中，我们应用这种方法来测量硫酸铜（CuSO 4）的毒性作用），一种参考毒物。 该方法被开发用于为污染物/化合物的生态毒性评估提供适合的基于微生物的测试，包括新兴的污染物如纳米材料。与现有的用于微生物的方法相比，该方案的新颖性主要与暴露介质和终点有关。事实上，暴露在盐溶液中进行，避免生长培养基与被研究的污染物的任何可能的干扰，这可能影响生物反应8 。终点是细菌培养性的降低，这可以容易地与基于生存/死亡率的海洋/微咸水环境中用于生态毒理学筛选的其它急性终点相比较。此外，该方案使用已经在大肠杆菌 9上使用的液 - 板微量计数技术，减少体积，因此实验效率ort（有关详细信息，请参阅协议的步骤3.3和3.4）。

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			<td height="42" style="height:42px;width:317px;">Vibrio anguillarum (strain AL 102, serotype O1)</td>
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			<td style="width:167px;">Obtained from the laboratory collection of NOIFMA (Norway)</td>
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			<td height="21" style="height:21px;">Tryptic Soy Agar&#160;</td>
			<td style="width:128px;">Liofilchem</td>
			<td style="width:165px;">610052</td>
			<td>Dehydrated Culture Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptic Soy Broth growth medium</td>
			<td style="width:128px;">Liofilchem</td>
			<td style="width:165px;">610053</td>
			<td>Dehydrated Culture Media</td>
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			<td height="25" style="height:25px;">CuSO4 &#183;5H2O</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:165px;">209198</td>
			<td></td>
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			<td height="21" style="height:21px;">NaCl&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:165px;">S-3014</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Name of Equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:165px;">Catalog Number</td>
			<td>Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Biosafety Laminar Flow Hood&#160;</td>
			<td style="width:128px;">ESCO</td>
			<td style="width:165px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator&#160;</td>
			<td style="width:128px;">Fratelli Galli</td>
			<td style="width:165px;">Mod. 2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Name of Software</td>
			<td style="width:128px;">Company</td>
			<td style="width:165px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchmark Dose Software&#160;</td>
			<td style="width:128px;">US EPA</td>
			<td style="width:165px;">Benchmark Dose 2.4.0&#160;</td>
			<td align="right">2012</td>
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ecotoxicological method with marine bacteria vibrio anguillarum to evaluate the acute toxicity of environmental contaminants

细菌是生态系统的重要组成部分，微生物群落的改变可以对生物地球化学循环和食物网产生重大影响。基于微生物的毒性测试被广泛使用，因为它们相对快速，可重复，便宜，并且与伦理问题无关。在这里，我们描述了一种生态毒理学方法来评估海洋细菌鳗弧菌的生物反应。该方法评估化学化合物的急性毒性，包括新型污染物如纳米颗粒以及环境样品。终点是由于暴露于有毒物质而导致细菌培养性降低（ 即复制和形成菌落的能力）。这种减少通常可以称为死亡率。该测试允许测定LC 50 ，导致细菌积极复制和形成菌落的50％减少的浓度，之后6小时曝光。以菌落形成单位（CFU）计数培养的细菌，并评估"死亡率"并与对照进行比较。在这项工作中，评估了硫酸铜（CuSO 4 ）的毒性。观察到明确的剂量反应关系，三次独立试验后平均LC 50为1.13 mg / L。该方案与现有的微生物方法相比，适用于更宽的盐度范围，对彩色/混浊样品没有限制。它使用盐水溶液作为曝光介质，避免生长培养基与被研究污染物的任何可能的干扰。 LC 50计算有助于与通常用于海洋环境生态毒理学评估的其他生物测定法进行比较。

将鳗弧菌暴露于四种浓度的CuSO 4的三个独立试验的结果显示出明显的剂量 - 反应关系和显着减少的细菌积极地复制和形成集落，随着参考毒物浓度的增加（ 图1 ; ANOVA，F = 20.28，p &lt;0.001）。与对照（CNTR）相比，CFU / mL的数量以1.25mg / L（事后Tukey检验，p &lt;0.05）显着降低。在最高测试浓度下检测到任何可培养的细菌。在暴露介质的宽盐度范围（ 即 5,20或35g / L NaCl，数据未显示）中，没有发现CuSO 4毒性的差异。 补充图1中报告了显示非线性回归的统计分析的代表性输出。 LC 50值计算为三项独立试验（&lt;strong&gt;表1）突出了该方法的可行性和可重复性。 <img alt="图1" src="/files/ftp_upload/55211/55211fig1.jpg" /> 图1:暴露于CuSO的鳗弧菌 4.暴露于不同浓度的CuSO 4 6小时的CFU / mL（CFU =菌落形成单位）的平均数。这些值代表三次独立试验的平均值（±SD）。相对于对照（CNTR）积极复制和形成菌落的细菌的减少报告在黄色框中（以百分比表示）。与基于事后Tukey测试的对照的显着差异用星号表示（* = p &lt;0.05; ** = p &lt;0.01）。 <a href="http://ecsource.jove.com/files/ftp_upload/55211/55211fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 表1:暴露于CuSO 4的 鳗弧菌的致死浓度（LC50）值。报告了三项独立试验和手段（±SD）的结果。 补充图1:非线性回归分析。对测试结果进行非线性回归分析（Logit-Hill模型）的代表性输出。 <a href="http://ecsource.jove.com/files/ftp_upload/55211/suppl_fig_1.tif" target="_blank">请点击这里下载此图。</a>

Watch this Scientific Journal Video about Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants at JoVE.com

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

这项工作描述了一种新的方案来评估污染物的生态毒性，包括新兴的污染物，如纳米材料，使用海洋细菌鳗弧菌 。该方法允许在暴露6小时后，确定导致细菌培养性降低50％的浓度的LC 50或死亡率。

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

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Environment

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

10.9K 次观看.  Institute for Environmental Protection and Research (ISPRA), Rome (Italy). 这项工作描述了一种新的方案来评估污染物的生态毒性，包括新兴的污染物，如纳米材料，使用海洋细菌鳗弧菌 。该方法允许在暴露6小时后，确定导致细菌培养性降低50％的浓度的LC 50或死亡率。 

视频: Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

科研

环境科学

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

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

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

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

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

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

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

一个铁（II）的实验室模拟的前寒武纪富海洋上升流系统探索光合细菌生长

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

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 ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

藤壶Balanus improvisus作为海洋模型的培养和基因表达

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.