Name: sampling, sorting, and characterizing microplastics in aquatic environments with high suspended sediment loads and large floating debris
Uploaded: 2018-07-28T14:00:00
Description: Watch this Scientific Journal Video about Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris at JoVE.com

The project for which this protocol was established was funded by the National Oceanic and Atmospheric Administration (NOAA) Marine Debris Program (# NA16NO29990029). We thank Miles Corcoran at the National Great Rivers Research and Education Center (NGRREC) in Alton, Illinois, for help with site selection and boat operation. Field and lab work was completed with the help of Camille Buckley, Michael Abegg, Josiah Wray, and Rebecca Wagner.

1. Water Sample Collection
<ol>
	<li>Collect water samples and water quality data of interest by boat where the river is well-mixed, ideally at locations where river stage or discharge is known (e.g., United States Geological Survey (USGS) gauging stations).20 To assure that the water is well-mixed, guide the boat using a handheld meter immersed in the river to where conductivity stays relatively constant.</li>
	<li>At the sampling sites, record location coordinates and depth. To find t...

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+pollution.+Plastic+waste+inputs+from+land+into+the+ocean.">Marine pollution. Plastic waste inputs from land into the ocean.</a> Science. 347 (6223), 768-771 (2015).</li><li>Andrady, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+the+marine+environment.">Microplastics in the marine environment.</a> Marine Pollution Bulletin. 62 (8), 1596-1605 (2011).</li><li>Cole, M., Lindeque, P., Halsband, C., Galloway, T. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastic+is+an+abundant+and+distinct+microbial+habitat+in+an+urban+river.">Microplastic is an abundant and distinct microbial habitat in an urban river.</a> Environmental Science &amp; Technology. 48 (20), 11863-11871 (2014).</li><li>Farrell, P., Nelson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+level+transfer+of+microplastic:+Mytilus+edulis+(L.)+to+Carcinus+maenas+(L.).">Trophic level transfer of microplastic: Mytilus edulis (L.) to Carcinus maenas (L.).</a> Environmental Pollution. 177, 1-3 (2013).</li><li>Rochman, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+evidence+supports+a+ban+on+microbeads.">Scientific evidence supports a ban on microbeads.</a> Environmental Science &amp; Technology. 49 (18), 10759-10761 (2015).</li><li>Taylor, M. L., Gwinnett, C., Robinson, L. F., Woodall, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+microfibre+ingestion+by+deep-sea+organisms.">Plastic microfibre ingestion by deep-sea organisms.</a> Scientific Reports. 6, 33997 (2016).</li><li>Mani, T., Hauk, A., Walter, U., Burkhardt-Holm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+profile+along+the+Rhine+River.">Microplastics profile along the Rhine River.</a> Scientific Reports. 5, 17988 (2015).</li><li>Morritt, D., Stefanoudis, P. V., Pearce, D., Crimmen, O. A., Clark, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+in+the+Thames:+a+river+runs+through+it.">Plastic in the Thames: a river runs through it.</a> Marine Pollution Bulletin. 78 (1-2), 196-200 (2014).</li><li>. National Park Servies Available from: <a target="_blank" href="https://www.nps.gov/miss/riverfacts.htm">https://www.nps.gov/miss/riverfacts.htm</a> (2017)</li><li>. United States Census Bureau Available from: <a target="_blank" href="https://www.census.gov/geo/maps-data/data/tiger-data.html">https://www.census.gov/geo/maps-data/data/tiger-data.html</a> (2010)</li><li>. United States Geological Survey (USGS) Available from: <a target="_blank" href="https://waterdata.usgs.gov/nwis/rt">https://waterdata.usgs.gov/nwis/rt</a> (2016)</li><li>Grimes, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishery+Production+and+the+Mississippi+River.">Fishery Production and the Mississippi River.</a> Fisheries. 28 (8), 17-26 (2001).</li><li>Talvitie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+wastewater+treatment+plants+act+as+a+potential+point+source+of+microplastics?+Preliminary+study+in+the+coastal+Gulf+of+Finland,+Baltic+Sea.">Do wastewater treatment plants act as a potential point source of microplastics? Preliminary study in the coastal Gulf of Finland, Baltic Sea.</a> Water Science and Technology. 72 (9), 1495-1504 (2015).</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=.">.</a> United States Environmental Protection Agency (USEPA) Method 160.2: Residue, Non-filtereable (Gravimetric, Dried at 103-105C). , (1971).</li><li>Nor, N. H., Obbard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+Singapore's+coastal+mangrove+ecosystems.">Microplastics in Singapore's coastal mangrove ecosystems.</a> Marine Pollution Bulletin. 79 (1-2), 278-283 (2014).</li><li>Woodall, L. C., Gwinnett, C., Packer, M., Thompson, R. C., Robinson, L. F., Paterson, G. 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Microplastic collection using drift nets is the conventional method in environments like the ocean where both sediment and plastic concentrations are low, thus requiring large sample volumes. However, drift nets are not always practical or safe in rivers with high sediment loads and large floating or submerged debris. Additionally, it is not feasible to use a drift net when attempting to thoroughly capture and quantify microplastic materials, particularly fibers, as most nets used for plastic surveys have mesh sizes &#88...

Plastic was first observed in the ocean as early as the 1930s1. Recent estimates of marine plastic debris range from over 243,000 metric tons (MT) of plastic on the ocean&#39;s surface to 4.8-12.7 million MT of plastic entering the ocean from terrestrial sources annually2,3. Early studies on marine plastic debris focused on macroplastics (&#62;5 mm diameter) as they are easily visible and quantifiable. However, it was recently discovered that macroplastics represent &#60;10% of plastic debris, by count, in the ocean, indicating that the overwhelming majority of plastic debris is microplastic (&#60;5 mm diameter)2.
Microplastics are categorized into two groups: primary and secondary microplastics. Primary microplastics consist of plastics that are manufactured at a diameter &#60;5 mm and include nurdles, the raw pellets used to make consumer products, microbeads used as exfoliants in personal care products (e.g., facial wash, body scrub, toothpaste), and abrasives or lubricants in industry. Secondary microplastics are created within the environment as larger plastic debris is fragmented by photolysis, abrasion, and microbial decomposition4,5. Synthetic fibers are also secondary microplastics and are a growing concern. A single garment can release &#62;1,900 fibers per wash in a domestic washing machine6. These microfibers, as well as microbeads from personal care products, are washed down drains and into the sewer system before entering wastewater treatments plants. Murphy (2016) found that a wastewater treatment plant serving a population of 650,000 reduced the microplastic concentration by 98.4% from influent to effluent, yet 65 million microplastics remained in effluent and sludge each day7. Even with high percentages of microplastics being removed during the treatment processes, millions, possibly billions, of microplastics pass through wastewater treatment plants daily and enter surface waters in effluent6,8,9,10,11.
Due to their environmental release, microplastics have been found in the digestive and respiratory tissues of marine organisms across all trophic levels12,13,14,15. Their impact after uptake is variable, with some studies not observing harm, while others demonstrate numerous effects such as physical and chemical tissue damage4,6,14,15. Due to these discoveries, interest in this field has increased over the past five decades. However, only recently have studies begun to quantify plastic debris, particularly microplastics, in freshwater systems, such as rivers and lakes, or assess the effect on organisms dwelling in these habitats12,16,17,18. Rivers are a major source of plastic debris found in the ocean as they receive wastewater effluent and surface water runoff that contain microplastics and macroplastics.
The protocol detailed here can be used to collect microplastic samples where drift nets are not feasible; specifically, in aquatic environments with high concentrations of suspended sediments and large floating debris like the Mississippi River. The Mississippi River watershed is one of the world&#39;s largest and has a population of &#62;90 million people, likely making it one of the largest sources of plastic debris to the ocean19,20. Each year, the Mississippi River discharges an average of 735 km3 of freshwater into the Gulf of Mexico, along with high concentrations of suspended sediments (~60 to &#62;800 mg/L) and large debris13,21. Water samples were collected at two depths (i.e., surface and 0.6-depth) at various locations along the Mississippi River and its tributaries in translucent 1 L low-density polyethylene (PE) containers using a peristaltic pump. In the lab, samples were filtered using nylon mesh sieves and mixed cellulose ester membrane filters simultaneously with a custom-made 63.5 mm (2.5 in) polyvinyl chloride (PVC) cylinder with union joints to insert the sieves and filters22. The inclusion of PVC unions in the filtration device allows for filtration by as many or as few particle size classes as desired. Additionally, it can be used to capture microplastic debris down to sub-micron sizes using membrane filters when studying synthetic fibers. Once filtered, samples were dried and suspected plastics were identified and sorted from the mesh sieves and membrane filters under a stereomicroscope. Suspected plastics were then examined using micro-attenuated total reflectance Fourier transform infrared spectroscopy (micro ATR-FTIR) to eliminate non-synthetic materials or determine polymer type. Considering the size of microplastic particulates and fibers, contamination is commonplace. Sources of contamination include atmospheric deposition, clothing, field and lab equipment, as well as deionized (DI) water sources. Multiple steps are included throughout the protocol to reduce contamination from various sources while conducting all stages of the study.

<table border="0" cellpadding="0" cellspacing="0" style="width:1076px;" width="1075">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">1L Cubitainer Containers, Low-Density Polyethylene</td>
			<td style="width:127px;">VWR</td>
			<td style="width:119px;">89094-140</td>
			<td style="width:612px;">Containers used to collect and store samples.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:219px;">2-1/2&#34; Clear Schedule 40 Rigid PVC Pipe</td>
			<td style="width:127px;">United States Plastic Corporation</td>
			<td style="width:119px;">34138</td>
			<td style="width:612px;">The PVC pipe used to make the device comes as an 2.43 m pipe. The pipe was then cut to the desired lengths for each section seperated by union joints. Section lengths were decided by predicting smaller pore sizes would clogg the device quicker. Longer sections were placed above the smaller pore sizes to collect and hold water to prevent needing to disassemble the device to change a filter while a sample remained in the device. For one filtration device one 18 in, one 12 in, and two 6 in peices are needed.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">2-1/2&#34; PVC SCH 40 Socket Union&#160;</td>
			<td style="width:127px;">Supply House</td>
			<td style="width:119px;">457-025</td>
			<td style="width:612px;">Union joints were glued to PVC pipe to house nylon sieves and mixed cellulose membranes.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:219px;">Nylon 6 Woven Mesh Sheet, Opaque Off-White, 12&#34; Width, 12&#34; Length, 500 microns Mesh Size, 38% Open Area (Pack of 5)</td>
			<td style="width:127px;">Small Parts via Amazon</td>
			<td style="width:119px;">CMN-0500-C/5PK-05</td>
			<td style="width:612px;">Mesh sheets were cut into circles to match the diameter of the outer diameter of the PVC pipe. The edges were glued to esure no fraying would occur. The glue &#39;s diamter should not extend into the inner diameter of the PVC so that it will not be affected during filtration.&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:219px;">Nylon 6 Woven Mesh Sheet, Opaque White, 12&#34; Width, 12&#34; Length, 100 microns Mesh Size, 44% Open Area (Pack of 5)</td>
			<td style="width:127px;">Small Parts via Amazon</td>
			<td style="width:119px;">B0043D1TB4</td>
			<td style="width:612px;">Mesh sheets were cut into circles to match the diameter of the outer diameter of the PVC pipe. The edges were glued to esure no fraying would occur. The glue &#39;s diamter should not extend into the inner diameter of the PVC so that it will not be affected during filtration.&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:219px;">Nylon 6 Woven Mesh Sheet, Opaque White, 12&#34; Width, 12&#34; Length, 50 microns Mesh Size, 37% Open Area (Pack of 5)</td>
			<td style="width:127px;">Small Parts via Amazon</td>
			<td style="width:119px;">B0043D1SGA</td>
			<td style="width:612px;">Mesh sheets were cut into circles to match the diameter of the outer diameter of the PVC pipe. The edges were glued to esure no fraying would occur. The glue &#39;s diamter should not extend into the inner diameter of the PVC so that it will not be affected during filtration.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">Mixed Cellulose Ester Membrane, 0.45um, 142mm, 25/pk</td>
			<td style="width:127px;">VWR</td>
			<td style="width:119px;">10034-914</td>
			<td style="width:612px;">Mixed cellulose membrane filter with 0.45 um was used as the last filter. A large diameter was used to allow the filter to be folded into a cone to increase surface area of the filter to prevent clogging.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">Metal Mesh Basket Tea Leaves Strainer Teapot Filter 76mm Dia 3pcs</td>
			<td style="width:127px;">Uxcell via Amazon</td>
			<td style="width:119px;">a15071600ux0260</td>
			<td style="width:612px;">The mesh basket used to provide extra support for the membrane filter to prevent tearing when pressure was applied by a vacuum pump.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">1/2&#34; PVC Barbed Insert Male Adapter</td>
			<td style="width:127px;">Supply House</td>
			<td style="width:119px;">1436-005</td>
			<td style="width:612px;">A vacuum adapter was added to allow vacuum filtration in the case of slow filtration due to high sediment concentration.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">1/2 in. O.D. x 3/8 in. I.D. x 10 ft. PVC Clear Vinyl Tube</td>
			<td style="width:127px;">Home Depot</td>
			<td style="width:119px;">702229</td>
			<td style="width:612px;">Tubing used to connect the vacuum pump to the filtration device.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">YSI Professional Plus Multiparameter Instrument with Quatro Cable</td>
			<td style="width:127px;">YSI</td>
			<td>6050000</td>
			<td style="width:612px;">Handheld meter used to measure additional water quality parameters parameters (e.g., turbidity, temperature, conductivity, pH, and dissolved oxygen (DO)).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">2100P Portable Turbidimeter</td>
			<td style="width:127px;">Hach</td>
			<td style="width:119px;">4650000</td>
			<td style="width:612px;">Handheld meter used to measure turbidity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FEP-lined PE tubing</td>
			<td style="width:127px;">Geotech</td>
			<td style="width:119px;">87050529</td>
			<td style="width:612px;">Tubing used with perestaltic pump to collect water samples from desired depths.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Geopump Peristaltic Pump Series II</td>
			<td style="width:127px;">Geotech</td>
			<td style="width:119px;">91350123</td>
			<td style="width:612px;">Pump used to collected water samples.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">MeiJi Techno EMZ-8TR Microscope</td>
			<td style="width:127px;">Microscope.com</td>
			<td style="width:119px;">EMZ8TR-PLS2</td>
			<td style="width:612px;">Microscope used analyze mesh sieves and membrane filters to quanitfy suspect microsplastics.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Nicolet iS10 FTIR Spectrometer</td>
			<td style="width:127px;">Thermo Electron North America</td>
			<td style="width:119px;">912A0607</td>
			<td style="width:612px;">FTIR used to analyze suspect microplastics.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Nicolet iN5 FTIR microscope</td>
			<td style="width:127px;">Thermo Electron North America</td>
			<td style="width:119px;">912A0895</td>
			<td style="width:612px;">FTIR microscope used to analyze suspect microplastics.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Germanium (Ge) ATR</td>
			<td style="width:127px;">Thermo Electron North America</td>
			<td style="width:119px;">869-174400</td>
			<td style="width:612px;">Geranium ATR accessory used along with the Nicolet iN5 FTIR microscope to analyze suspect microplastic.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Aluminum EZ-Spot Micro Mounts (Pkg of 5)</td>
			<td style="width:127px;">Thermo Electron North America</td>
			<td style="width:119px;">0042-545</td>
			<td style="width:612px;">Microscope slides used along with the Nicolet iN5 FTIR microscope to analyze suspect microplastic.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Aluminum Coated Glass Sample Slides</td>
			<td style="width:127px;">Thermo Electron North America</td>
			<td style="width:119px;">0042-544</td>
			<td style="width:612px;">Microscope slides used along with the Nicolet iN5 FTIR microscope to analyze suspect microplastic.</td>
		</tr>
	</tbody>
</table>

sampling, sorting, and characterizing microplastics in aquatic environments with high suspended sediment loads and large floating debris

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only recently have microplastics in freshwater systems, such as rivers and lakes, been quantified. Microplastic sampling at the surface usually consists of deploying drift nets behind either a stationary or moving boat, which limits the sampling to environments with low levels of suspended sediments and floating or submerged debris. Previous studies that employed drift nets to collect microplastic debris typically used nets with &#8805;300 &#181;m mesh size, allowing plastic debris (particles and fibers) below this size to pass through the net and elude quantification. The protocol detailed here enables: 1) sample collection in environments with high suspended loads and floating or submerged debris and 2) the capture and quantification of microplastic particles and fibers &lt;300 &#181;m. Water samples were collected using a peristaltic pump in low-density polyethylene (PE) containers to be stored before filtering and analysis in the lab. Filtration was done with a custom-made microplastic filtration device containing detachable union joints that housed nylon mesh sieves and mixed cellulose ester membrane filters. Mesh sieves and membrane filters were examined with a stereomicroscope to quantify and separate microplastic particulates and fibers. These materials were then examined using a micro-attenuated total reflectance Fourier transform infrared spectrometer (micro ATR-FTIR) to determine microplastic polymer type. Recovery was measured by spiking samples using blue PE particulates and green nylon fibers; percent recovery was determined to be 100% for particulates and 92% for fibers. This protocol will guide similar studies on microplastics in high velocity rivers with high concentrations of sediment. With simple modifications to the peristaltic pump and filtration device, users can collect and analyze various sample volumes and particulate sizes.

To validate the recovery rates of this protocol, three samples (V1-V3) from Oso Bay, Corpus Christi, Texas (adjacent to the Texas A&#38;M University Corpus Christi Campus), were spiked with 10 blue PE particulates (ranging from 50-100 &#181;m in diameter) and 50 green nylon fibers of various lengths (Figure 3). Sample TSS was calculated (Section 2) and then the samples were filtered using the methods outlined in Sections 3-5. The blue PE...

Watch this Scientific Journal Video about Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris at JoVE.com

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

Most microplastic research to date has occurred in marine systems where suspended solid levels are relatively low. Focus is now shifting to freshwater systems, which may feature high sediment loads and floating debris. This protocol addresses collecting and analyzing microplastic samples from aquatic environments that contain high suspended solid loads.

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only ...

This method will help researchers quantify microplastic loads in rivers, which often contain high sediment loads, as they are a major source of plastic debris to the ocean. The main advantage of this technique is that it enables filtering and sorting of microplastics and water samples with high sediment loads at sizes not typically included in previous studies. To begin rinse the filtration device and nylon mesh sieves three times with deionized water.Then place the mesh sieves into each union joint, with the pour sizes decreasing from top to bottom. To prevent leaks seal each joint tightly. Next wet a mixed cellulose ester membrane filter with deionized water.While the filter is still damp fold it into a cone. Then place a stainless steel mesh basket into the last union joint. Carefully place the folded membrane filter into the basket.Fold the lip of the filter over the edge of the joint. Next place a mesh sieve on top of the membrane filter in the last union joint. Ensure that all of the union joints are sealed tightly.Then attach the hose form the top of the filtering flask to the base of the filtration device. Turn on the vacuum pump, ensuring that the pressure does not exceed 127 milliliters of mercury. Using a 500 milliliter graduated cylinder measure the total volume of the sample.Next record the volume of the sample and transfer it to the filtration device. To empty the filtering flask turn off the pump and detach the two hoses from the flask. Then empty the flask into a waste container.To continue the filtration cycle reattach the hoses and turn on the pump. Once the entire sample has been filtered use deionized water to rinse the sample container and graduated cylinder three times. After each rinse filter the deionized water used to rinse the container and graduated cylinder.Using deionized water rinse the walls of the filtration device three times. Then turn off the vacuum pump, and use a deionized water wash bottle to rinse the edges of the union joint. Turn the pump off and use forceps to remove the mesh sieve from the union joint.Place the sieve in a covered Petri Dish and dry it at 60 degrees celsius for 24 hours. Repeat this process for each union joint. Then turn the vacuum pump on and rinse the edges of a membrane filter using a deionized water wash bottle.Wash the particulates at the edges of the filter into the center, and ensure that all of the water passes through the filter. Next use forceps to remove and unfold the membrane filter. Place the filter in a foil envelope and dry at 60 degrees celsius for 24 hours.First examine the membrane filter under a stereo microscope. Suspected plastics will not have cellular structure. The fibers will have equal thickness throughout, and the particles will not appear shiny.Remove suspected plastics from the filter and place them into a collection vial containing 70%ethanol. Record the color and shape of each suspected plastic. For nylon mesh sieves stored in Petri Dishes after all suspected plastics have been removed from the filter examine the lid and bottom of the Petri Dish for additional suspected plastics.To validate this protocol three samples from Oso Bay were spiked with 10 blue polyethylene particles and 50 green nylon fibers. On average, 100%of the polyethylene particles and 92%of the nylon fibers were recovered from the samples. The loss of fibers may be due to a small amount of sample loss during filtration or incorrect identification.Once mastered multiple filtration apparatus can be run simultaneously with samples taking less than two hours each to filter. Sample sorting times under the microscope however are sample specific. While attempting this procedure it's important to remember to account for potential contamination using lab equipment and field blanks for each step in the process.Following this procedure other methods like Fourier Transform Infrared Spectroscopy can be used to verify material properties. With its development this technique enables researchers studying environmental contaminates to explore microplastic pollution and waterways with high suspended sediment loads, as well as floating and submerged debris.

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Research

JoVE Journal

Environment

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is built and can be transported to a temporary work space close to field sampling site(s) to facilitate rapid analysis. Cores are extruded into a portable glove bag, where they are sectioned and each 1-3 cm thick section (depending on core diameter) is sealed into 50 ml centrifuge tubes. Pore waters are separated with centrifugation outside of the glove bag and then returned to the glove bag for separation from the sediment. These extracted pore water samples can be analyzed immediately. Immediate analyses of redox sensitive species, such as sulfide, iron speciation, and arsenic speciation indicate that oxidation of pore waters is minimal; some samples show approximately 100% of the reduced species, e.g. 100% Fe(II) with no detectable Fe(III). Both sediment and pore water samples can be preserved to maintain chemical species for further analysis upon return to the laboratory.

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

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

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

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

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

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

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.

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.

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

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.