The development of this protocol was founded by IPA Adriatic Cross-border Cooperation Programme 2007-2013, within the DeFishGear project (1&#176;str/00010).

1. Sampling of microplastics on the sea surface
<ol>
	<li>Deploy the manta net from the side of the vessel using a spinnaker boom or &#187;A-frame&#171; using lines and karabiners.</li>
	<li>Deploy the manta net out of the wake zone (approx. 3 - 4 m distance from the boat) in order to prevent collecting water affected by turbulence inside the wake zone.</li>
	<li>Write down the initial GPS coordinates and initial time in the data sheet.</li>
	<li>Start to move in one straight direction with a speed of approx. 2 - 3 knots for 30 min and begin the time measurement.</li>
	<li>After 30 min stop the boat and write down final GPS coordinates, the length of the route (the most correct way is to calculate the length from the GPS coordinates) and the average boat speed into the data sheet provided and lift the manta net out of the water.</li>
	<li>Rinse the manta net thoroughly from the outside of the net with seawater using a submersible pump or water from the boat water reservoir. Rinse in the direction from the manta mouth to the cod end in order to concentrate all particles adhered to the net into the cod end. 
	Note: Never rinse the sample through the opening of the net in order to prevent contamination.</li>
	<li>Safely remove the cod end and sieve the sample in the cod end through a 300 &#181;m mesh size sieve or less.</li>
	<li>Rinse the cod end thoroughly from the outside and pour the rest of the sample through the sieve. Repeat this step until there are no longer any particles inside the cod end.</li>
	<li>Concentrate all material on the sieve in one part of the sieve.</li>
	<li>With the use of a funnel, rinse the sieve into a glass jar or plastic bottle using 70 % ethanol.</li>
	<li>Close the bottle,&#160;wipe it with paper towels and label the lid and outside of the jar with the sample name and date with waterproof marker (you should also put a second label written with a pencil on velum paper in a jar to avoid the possible loss of the sample name due to the erased label on the jar). Transfer labeled plastic bottle into the cool box. 
	Note to general sampling conditions: The wind speed should not be more than 2 Beaufort, since the waves are too high and the net is not stable on the sea surface. It is important to maintain a steady linear course at a constant speed during the trawls. Half of the manta net opening should be submersed during sampling. Duration of sampling should be 30 min (in cases where there is a large amount of natural material, e.g. plankton bloom, the duration of sampling can be shorter). Avoid the use of plastic tools and containers. Avoid synthetic clothing (e.g. fleece), ropes and contact of manta net with vessel to prevent contamination of the sample. Be very careful not to damage the manta net or the boat hull while deploying and capturing the net.</li>
</ol>
2. Separation of microplastics from the sea surface samples
<ol>
	<li>If the sample does not contain any items larger than 25 mm and appears to be clean, continue directly with step 3.</li>
	<li>Pour sample through the sieve (&#8804;300 &#181;m mesh size) and remove all natural or artificial litter objects of a size &#62;5 mm (macro and mezzo litter) from the sample, using visual identification and tweezers. Be careful to rinse each removed object carefully with distilled water in order to remove any microplastic litter adhered to it. Store all natural and artificial litter objects in separate containers. Dry all natural and artificial litter objects in a desiccator (or in the open air, but in a closed dish) and weigh them. Identify all litter objects &#62;25 mm (macro litter) according to the Master List of Categories of Litter Items16.</li>
	<li>After removing all larger objects, concentrate all remaining pieces in one part of the sieve using squirt bottles or tap water. Pour the sample into a glass container using a minimum amount of 70 % ethanol with the help of a funnel. 
	Note: In this step the use of 70 % ethanol is crucial to preserve the sample. Also in the step of visual inspection of the sample, ethanol helps to discolor the organisms and colorful plastics therefore become easier to find.</li>
	<li>Take a small amount of the sample (subsample) and pour it into a glass Petri dish. Analyze the sample with the use of a stereomicroscope (20 - 80x zoom) and search for microplastic particles.</li>
	<li>Each microplastic particle should be categorized into one of the categories listed in Table 1 and put into a Petri dish or other glass vials, marked with a category name. The Petri dish needs to be closed at all times. 
	Note: When separating microplastics from your sample be conservative and select more rather than less particles for the analysis. The real chemical structure of particles will still be determined later on. Be sure to analyze larger objects from all sides as microplastics may be stuck and therefore hidden under larger items. It may also be helpful to move already analyzed objects to one side of the Petri dish.</li>
	<li>Put the Petri dish under the microscope with measuring equipment (ocular ruler calibrated by the micrometer slide or image analysis software) and measure the size of each particle (measure the longest diagonal), except filaments, and note its color. Each subsample should be reviewed by another person. Be careful to rinse the glass container containing the sample so that all particles adhering to the glass walls are washed into the Petri dish.</li>
	<li>Weigh the microplastic particles of each category separately by the use of analytical scale. Microplastic particles need to be dried prior to weighing. The closed Petri dish can be put in a desiccator or the samples can be left to dry in a closed dish till particles became dry (the weight of closed petri dish with particles is constant).</li>
	<li>Identify micro litter. When analyzing a sample in search of microplastics, please consider that some particles will be easily visible (color, shape, size) while others may be trickier to find. Below are a few features that identify microplastic particles in the sample: For example, no cell structure, uneven, sharp, crooked edges, uniform thickness, distinctive colors (blue, green, yellow, etc.).</li>
</ol>
3. Chemical identification of microplastics
<ol>
	<li>ATR-FTIR spectroscopy
	<ol>
		<li>Prior to the analysis clean the detection system with alcohol and a lint free cloth.</li>
		<li>Record a background spectrum. Place the sample on the sample holder and collect the spectra. Identify the obtained ATR- FTIR spectra using an automated comparison of the obtained spectrum with spectra in a database.</li>
	</ol>
	</li>
	<li>Micro ATR-FTIR spectroscopy
	<ol>
		<li>Prior to the analysis clean the detection system with alcohol and a lint free cloth.</li>
		<li>Place the sample on a glass filter. Note: Other filters can be used but their polymer nature can interfere with the characterization.</li>
		<li>Place the filter with the sample on the automatic scanning table and use the joystick to locate the sample.</li>
		<li>Record an optical image and mark an area (e.g. 20 by 20 &#181;m) where the sample will be characterized.</li>
		<li>Record a background spectrum.</li>
		<li>Place the sample on the sample holder and collect the spectra at the predefined location.</li>
		<li>Identify the obtained micro ATR-FTIR spectra using an automated comparison of the obtained spectrum with spectra in a database.</li>
	</ol>
	</li>
</ol>

<ol>
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C., Thiel, M. <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:+a+review+of+the+methods+used+for+identification+and+quantification.">Microplastics in the marine environment: a review of the methods used for identification and quantification.</a> Environmental science &amp; technology. 46 (6), 3060-3075 (2012).</li><li>L&#246;der, M. G. J., Kuczera, M., Mintenig, S., Lorenz, C., Gerdts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+plane+array+detector-based+micro-Fourier-transform+infrared+imaging+for+the+analysis+of+microplastics+in+environmental+samples.">Focal plane array detector-based micro-Fourier-transform infrared imaging for the analysis of microplastics in environmental samples.</a> Environmental Chemistry. 12 (5), 563-581 (2009).</li><li>Ioakeimidis, C., 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=The+degradation+potential+of+PET+bottles+in+the+marine+environment:+An+ATR-FTIR+based+approach.">The degradation potential of PET bottles in the marine environment: An ATR-FTIR based approach.</a> Scientific reports. 6, 23501 (2016).</li><li>McDermid, K. J., McMullen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+small-plastic+debris+on+beaches+in+the+Hawaiian+archipelago.">Quantitative analysis of small-plastic debris on beaches in the Hawaiian archipelago.</a> Marine pollution bulletin. 48 (7), 790-794 (2004).</li><li>Eriksen, 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=Microplastic+pollution+in+the+surface+waters+of+the+Laurentian+Great+Lakes.">Microplastic pollution in the surface waters of the Laurentian Great Lakes.</a> Marine pollution bulletin. 77 (1-2), 177-182 (2013).</li></ol>

The authors have nothing to disclose.

Microplastics sampling on the sea surface by manta net is a widely used method for the sampling of microplastics on the sea surface, but to date there has been no unified methodology. A large volume of water can be filtered through the manta net, thus the possibility of trapping a relevant number of microplastics is high and the results are perceived to be reliable. Comparability of results among different samples is assured by normalization. In our case, the concentrations were related to the sampled area by multiplying trawl distance by the horizontal width of the net opening. Another option is to use a flow meter, fixed at the net opening. The use of a flow meter is possible since the manta net with its lateral wings is very stable on the sea surface and therefore hopping on the waves is minimal. A flow meter records the volume of filtered water and thus enables the normalization of results per volume of sampled water16.
The most frequently used manta nets have around 300 &#181;m mesh size and are 3 - 4.5 m long. These dimensions were optimized to avoid clogging of the net and to allow the sampling a volume of water as large as possible. Trawling speed is recommended to be between 2 - 3 knots, but it is dependent on wave height, wind speed and sea currents. It is very important that the manta net is under supervision the whole time during sampling and if it starts hopping, the trawling speed must be reduced. The trawling time is recommended to be around 30 min, but depends on seston concentrations. It can happen that seston sometimes clogs the manta net. In this case the trawling has to be stopped immediately, otherwise the microplastic particles can be lost and the net can get damaged. Manta net is the most often fixed from the side of the vessel. This is also the most suitable option, while the manta net is surely out of the wake zone. In some surveys manta net was fixed from the stern of the vessel17, 18, but in that case you have to be sure that the net is out of the wake zone. The distance, on which the trawl is set for sampling, should be determined individually, since the zone of turbulences caused by the vessel varies from the size of the vessel and from the speed of the boat19, 20.
Separation of microplastic particles from the sea surface samples is most often done just by visual identification21. Particles bigger than 1 mm can be identified easily by the naked eye, while particles smaller than 1 mm require the use of a stereomicroscope. To reduce the possibility of confusing the non-plastic particles with plastic ones, using the polarization light on stereomicroscopes is recommended. The possibility of misidentification of plastic particles gets higher with smaller particles. Thus particles &#62;0.5 mm can only be identified visually21, by the use of stereomicroscope. For particles smaller than 0.5 mm an additional, more accurate method is required e.g. micro ATR-FTIR spectroscopy21.
During the process of microplastics separation from the sample the possibility of sample contamination with the airborne filaments is very high. For this reason, control Petri dishes left open on the working table are strongly recommended for the identification of potential contaminant airborne particles. Namely, the quality of the data strongly depends on: 1) the precision of the person working with the sample, 2) the quality and magnification of the stereomicroscope, and 3) the quantity of organic matter in the sample16. After visual identification it is strongly recommended to analyze the sorted particles with one of the available techniques for chemical identification of the material8.
Several methods exist for polymer identification, among which the FTIR spectroscopy and Raman spectroscopy are the most frequently used22. FTIR and Raman spectroscopy are complementary techniques and their accuracy is similar. In our protocol, the FTIR and micro FTIR spectroscopy with &#34;attenuated total reflectance&#34; (ATR) are presented. They are simple to use and they enable fast and accurate results. Plastic polymers possess highly specific infrared (IR) spectra with distinct band patterns, thus making IR spectroscopy an optimal technique for the identification of microplastics21. The energy of IR radiation excites a specific molecular vibration when interacting with a sample, which enables the measurement of characteristic IR spectra22. FTIR spectroscopy can also provide additional information on particles, such as intensity of oxidation23 and level of degradation24. While ATR-FTIR is suitable for chemical identification of larger particles (&#62;0.5 mm), micro ATR-FTIR spectroscopy can provide information on the chemical structure of particles &#60;0.5 mm, as it combines the function of a microscope and an infrared spectrometer.
Before using FTIR and micro FTIR spectroscopy, microplastic particles have to be previously dried, since water strongly absorbs IR radiation22, and purified, in case they are covered with biofilms and/or other organic and inorganic adherents, which can influence the IR spectra. The most non-invasive way to purify samples is by stirring and rinsing with fresh water25. If this is not enough, then the use of 30 % hydrogen peroxide is recommended. All other methods can have negative effects on the microplastic particles (e.g. ultrasonic cleaning can further break particles, strong acidic or alkaline solutions can damage several plastic polymers, etc.) and therefore their use is not recommended. More promising is the use of a sequential enzymatic digestion as a plastic friendly purification step. Purification using different technical enzymes (e.g. lipase, amylase, proteinase, chitinase, cellulase, proteinase-K) has been successfully applied to reducing a biological matrix of plankton and thus proved to be a valuable technique to minimize matrix artifacts during FTIR spectroscopy measurements22.
Separation of microplastics by visual identification and chemical identification of selected particles are both extremely time-consuming processes. This work has to be done by an accurate and patient person who has experience with stereomicroscopes, not only in recognizing the plastic particles, but also in recognizing biological matter. Even an experienced person cannot discriminate all potential microplastic particles unambiguously from chitin or diatom fragments22. Therefore, the error rate of visual sorting ranges from 20 %26 to 70 %21 and increases with decreasing particle size.

Microplastic pollution in the sea represents a growing concern to contemporary society, due to the constant increase in plastic production and its subsequent disposal and accumulation in the marine environment1. Even if plastic macro litter would no longer enter the seas, microplastic pollution would continue to grow due to fragmentation of already existing plastic litter in the sea2. The majority of microplastic pollution studies were carried out in marine and fresh water ecosystems and mainly addressed sea surface pollution3.
The term microplastic refers to plastic particles smaller than 5 mm in size4. This term describes a heterogeneous mixture of particles, which can differ in size (from a few microns to several millimeters), color and shape (from very different shapes of fragments to long fibers). Microplastic particles can be of a primary or secondary origin5. Microplastic of primary origin is manufactured as small particles used in the cosmetics industry (pilling cr&#232;me etc.) or chemical industry as precursor for other plastic products (e.g. plastic pellets used in plastic industry). Microplastic of secondary origin arise via the degradation of larger plastic pieces in the environment due to physical and chemical processes, induced by light, heat, oxygen, water and organisms6. In 2015, four types of microplastic sources were defined: larger plastic litter, cleaning products, medicines and textiles6. The main source (80 %) of larger plastic litter is assumed to be land based7. Microplastic from cosmetic products, medicines and textile enters water ecosystems through sewage and storm waters6. Microplastic particles most frequently found in water ecosystems are fragments from larger plastic litter and textile fibers8. 
Microplastics have several negative effects on the environment. Their small size allows them to enter the food web through ingestion by marine organisms9, 10. Ingested particles can cause physical damage or block the digestive system of animals11. Particles can also be carriers of persistent organic pollutants (POPs). Their hydrophobic surface and favorable ratio of large surface area to small volume, enables POPs to adsorb onto the microplastics12. In the environment or digestive systems of animals who ingest them, POPs and other plastic additives can be leached from microplastic particles13. 
Previous studies reported the ubiquitous presence of microplastics in the marine environment3, from the water column to the bottom sediments. The threat of microplastic pollution was already identified by the Marine Strategy Framework Directive in the EU and, consequently, mandatory monitoring of microplastics was advised14. Accordingly, the EU Technical Subgroup on Marine Litter (TSG-ML) prepared recommendations for monitoring of microplastics in the European seas15. Thus, the video guidelines for microplastics sampling are of high importance, as they support comparative monitoring and a coherent management process all over the world.
This protocol was developed within the DeFishGear project for the first monitoring of microplastic pollution in the Adriatic Sea. Recommendations from the document "Guidance on Monitoring of Marine Litter in European Seas" by TSG-ML15 were taken into account. This protocol describes the methodology for microplastics sampling on the sea surface, separation of microplastics from the samples, and chemical analysis of microplastic particles to confirm that particles are from plastic material and to identify the type of plastic. Sampling was done by the use of a manta net, which is the most suitable equipment for sampling in calm waters16. Separation of microplastics from the samples was carried out by visual identification using a stereomicroscope. Isolated particles were later chemically identified using Fourier transform infrared (FTIR) spectroscopy and micro FTIR spectroscopy.

<table><tbody><tr><td>In this protocol no specific equipment or reagents were used.</td><td></td><td></td><td></td></tr></tbody></table>

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 first result of the described protocol are microplastic particles categorized into six categories according to their visual features (Table 1). The first category, and usually the most abundant one, are fragments (Figure 1). They are rigid, thick, with sharp crooked edges and an irregular shape. They can be in a variety of different colors. The second category are films (Figure 2). They also appear in irregular shapes, but in comparison with fragments, they are thin and flexible and usually transparent. The third category are pellets (Figure 3), usually originating from the plastics industry. They are irregular, round shapes, and normally bigger in size, around 5 mm in diameter. They are usually flat on one side and can be of various colors. The fourth category are granules (Figure 4). In comparison with pellets, they have a regular round shape and usually a smaller size, around 1 mm in diameter. They appear in natural colors (white, beige, brown). The fifth category are filaments (Figure 5). They are, next to fragments, the most abundant type of microplastic particles. They can be short or long, with different thicknesses and colors. The last category are foams (Figure 6). They most often come from large particles of styrofoam. They are a soft, irregular shape and white to yellow in color.
The main result of microplastics sampling and sample analysis is the number of microplastic particles per sample. These data can be further normalized per km2. The formula used for normalization is:
microplastic particles per sample / sampling area,
where sampling area is calculated by multiplying sampling distance by the width of the opening of the manta net (Tables 2, 3; Figure 7). In addition, particles can be analyzed with image analysis software. The results include maximum length and area of each particle (Table 4). Figure 8a show particles before image analysis and Figure 8b is after image analysis, where each particle is measured and numbered. Lastly, a chemical analysis of the total or highest possible number of particles per sample is recommended. Using Fourier transform infrared spectroscopy a spectrum of the selected particle is acquired, as shown on Figure 9. This spectrum is then compared with the spectra from the software library (Figure 10). The final result will show if a given particle is plastic or not and indicate the type of plastic from the chemical structure.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>1</td>
			<td>Fragments</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Films</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Pellets</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Granules</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Filaments</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Foams</td>
		</tr>
	</tbody>
</table>
Table 1: Categories of microplastic particles.
<img alt="Figure 1" src="/files/ftp_upload/55161/55161fig1.jpg" /> 
Figure 1: Example of particles from category: Fragments.&#160; <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55161/55161fig2.jpg" /> 
Figure 2: Example of particles from category: Films.&#160; <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55161/55161fig3.jpg" /> 
Figure 3: Example of particles from category: Pellets.&#160; <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55161/55161fig4.jpg" /> 
Figure 4: Example of particles from category: Granules. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55161/55161fig5.jpg" /> 
Figure 5: Example of particles from category: Filaments.&#160; <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55161/55161fig6.jpg" /> 
Figure 6: Example of particles from category: Foams. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Sampling distance [km]</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Manta width [km]</td>
			<td>0.0006</td>
		</tr>
		<tr>
			<td>Sampling area [km2]</td>
			<td>0.0012</td>
		</tr>
	</tbody>
</table>
Table 2: Example of data from survey, used for calculation of microplastic particles per km2.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>No</td>
			<td>No/km2</td>
		</tr>
		<tr>
			<td>fragments</td>
			<td>301</td>
			<td>250833</td>
		</tr>
		<tr>
			<td>films</td>
			<td>45</td>
			<td>37500</td>
		</tr>
		<tr>
			<td>pellets</td>
			<td>15</td>
			<td>12500</td>
		</tr>
		<tr>
			<td>granules</td>
			<td>8</td>
			<td>6667</td>
		</tr>
		<tr>
			<td>foams</td>
			<td>33</td>
			<td>27500</td>
		</tr>
		<tr>
			<td>filaments</td>
			<td>223</td>
			<td>185833</td>
		</tr>
	</tbody>
</table>
Table 3: Example of results from survey, where the categorized data into 6 groups are counted and normalized per km2 (No - number of particles).
<img alt="Figure 7" src="/files/ftp_upload/55161/55161fig7.jpg" /> 
Figure 7: Example of representative results after visual categorization of particles (No -&#160;number of particles). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Index Region</td>
			<td>Area [mm&#178;]</td>
			<td>Maximum length [mm]</td>
		</tr>
		<tr>
			<td>1</td>
			<td>8.010</td>
			<td>5.506</td>
		</tr>
		<tr>
			<td>2</td>
			<td>10.517</td>
			<td>5.628</td>
		</tr>
		<tr>
			<td>3</td>
			<td>12.185</td>
			<td>5.429</td>
		</tr>
		<tr>
			<td>4</td>
			<td>3.367</td>
			<td>3.367</td>
		</tr>
		<tr>
			<td>5</td>
			<td>2.475</td>
			<td>2.155</td>
		</tr>
		<tr>
			<td>6</td>
			<td>1.809</td>
			<td>2.943</td>
		</tr>
		<tr>
			<td>7</td>
			<td>6.604</td>
			<td>5.238</td>
		</tr>
		<tr>
			<td>8</td>
			<td>5.779</td>
			<td>4.037</td>
		</tr>
		<tr>
			<td>9</td>
			<td>4.472</td>
			<td>3.791</td>
		</tr>
		<tr>
			<td>10</td>
			<td>16.907</td>
			<td>5.355</td>
		</tr>
		<tr>
			<td>11</td>
			<td>7.246</td>
			<td>3.733</td>
		</tr>
		<tr>
			<td>12</td>
			<td>7.867</td>
			<td>4.622</td>
		</tr>
		<tr>
			<td>13</td>
			<td>6.411</td>
			<td>5.056</td>
		</tr>
		<tr>
			<td>14</td>
			<td>3.281</td>
			<td>3.070</td>
		</tr>
		<tr>
			<td>15</td>
			<td>12.937</td>
			<td>5.554</td>
		</tr>
		<tr>
			<td>16</td>
			<td>6.709</td>
			<td>3.716</td>
		</tr>
	</tbody>
</table>
Table 4: Example of image analysis results where area [mm2] and maximum length [mm] of each particle are measured.
<img alt="Figure 8" src="/files/ftp_upload/55161/55161fig8.jpg" /> 
Figure 8: Example of image acquired a) before and b) after image analysis of particles with image analysis software. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/55161/55161fig9.jpg" /> 
Figure 9: Example of a spectra measured on a selected particle with marked peaks and their wavelengths [cm-1]. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/55161/55161fig10.jpg" /> 
Figure 10: Example of comparison of acquired spectra from selected particle to best match from the ATR-FTIR spectra library.&#160; <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55161/55161fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis at JoVE.com

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

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 ...

protocol-for-microplastics-sampling-on-sea-surface-sample

Universidad San Sebastian

Research

JoVE Journal

Environment

49.3K Views.  Institute for water of the Republic of Slovenia. 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.

Video: Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

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.

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

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.

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

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

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.

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 ...

Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status

The following protocol is intended to respond to the requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: &#34;There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region&#34;, where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: &#34;There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region&#34;.

The protocol focuses on the collection of sea turtle samples, describing all the steps from the animal recovery and necropsy to the classification and quantification of ingested marine litter. Moreover, the representative results show how to use the collected data to elaborate the possible thresholds for Good Environmental Status.

The following protocol is intended to respond to the requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the ...

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Biology

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use

Microplastics (MPs) are becoming a global concern due to the potential risk to human health. Case studies of plastic products (i.e., plastic single-use cups and kettles) indicate that MP release during daily use can be extremely high. Precisely determining the MP release level is a crucial step to identify and quantify the exposure source and assess/control the corresponding risks stemming from this exposure. Though protocols for measuring MP levels in marine or freshwater has been well developed, the conditions experienced by household plastic products can vary widely. Many plastic products are exposed to frequent high temperatures (up to 100 &#176;C) and are cooled back to room temperature during daily use. It is therefore crucial to develop a sampling protocol that mimics the actual daily-use scenario for each particular product. This study focused on widely used polypropylene-based baby feeding bottles to develop a cost-effective protocol for MP release studies of many plastic products. The protocol developed here enables: 1) prevention of the potential contamination during sampling and detection; 2) realistic implementation of daily-use scenarios and accurate collection of the MPs released from baby feeding bottles based on WHO guidelines; and 3) cost-effective chemical determination and physical topography mapping of MPs released from baby feeding bottles. Based on this protocol, the recovery percentage using standard polystyrene MP (diameter of 2 &#181;m) was 92.4-101.2% while the detected size was around 102.2% of the designed size. The protocol detailed here provides a reliable and cost-effective method for MP sample preparation and detection, which can substantially benefit future studies of MP release from plastic products.

This study detailed a reliable and cost-effective protocol for microplastics collection and detection from the daily use of plastic products.

Microplastics (MPs) are becoming a global concern due to the potential risk to human health. Case studies of plastic products (i.e., plastic single-use ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Summary

Explore More Videos

Chapters in this video

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

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

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

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

Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status

Seawater Sampling and Collection

Small Volume (1-3L) Filtration of Coastal Seawater Samples

Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use