Name: extraction of organochlorine pesticides from plastic pellets and plastic type analysis
Uploaded: 2017-07-01T13:00:00
Description: Watch this Scientific Journal Video about Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis at JoVE.com

This work was funded by IPA Adriatic Cross-border Cooperation Program 2007-2013, within the DeFishGear project (1&#176;str/00010).

1. Plastic Pellet Sampling

<ol>
	<li>Before going to the field, triple rinse all required sampling materials (e.g., tweezers and aluminum foil) with acetone or ethanol (99%). In case the material cannot be solvent-rinsed, heat it at 450 &#176;C overnight in an oven (e.g., glassware). 
	NOTE: In tourist areas, obtain information about possible beach cleaning activities that would remove most of the marine litter including microplastics. If possible, plan the sampling ahead of this operatio...

<ol>
	<li>Plastic Europe. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics - the Facts 2015. An analysis of European plastics production, demand and waste data. , (2017).</li><li>Wang, J., Tan, Z., Peng, J., Qiu, Q., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviors+of+microplastics+in+the+marine+environment.">The behaviors of microplastics in the marine environment.</a> Mar Environ Res. 113, 7-17 (2016).</li><li>UNEP. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Marine plastic debris and microplastics - Global lessons and research to inspire action and guide policy change. , (2016).</li><li>Ogata, Y., 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=International+Pellet+Watch:+Global+monitoring+of+persistent+organic+pollutants+(POPs)+in+coastal+waters.+1.+Initial+phase+data+on+PCBs,+DDTs,+and+HCHs.">International Pellet Watch: Global monitoring of persistent organic pollutants (POPs) in coastal waters. 1. Initial phase data on PCBs, DDTs, and HCHs.</a> Mar Pollut Bull. 58 (10), 1437-1446 (2009).</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> Mar Pollut Bull. 62 (8), 1596-1605 (2011).</li><li>Antunes, J. C., Frias, J. G. L., Micaelo, A. C., Sobral, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resin+pellets+from+beaches+of+the+Portuguese+coast+and+adsorbed+persistent+organic+pollutants.">Resin pellets from beaches of the Portuguese coast and adsorbed persistent organic pollutants.</a> Estuarine Coastal Shelf Sci. 130, 62-69 (2013).</li><li>Cole, M., Lindeque, P., Halsband, C., Galloway, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+as+contaminants+in+the+marine+environment:+A+review.">Microplastics as contaminants in the marine environment: A review.</a> Mar Pollut Bull. 62 (12), 2588-2597 (2011).</li><li>Takada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Call+for+pellets!+International+Pellet+Watch+Global+Monitoring+of+POPs+using+beached+plastic+resin+pellets.">Call for pellets! International Pellet Watch Global Monitoring of POPs using beached plastic resin pellets.</a> Mar Pollut Bull. 52 (12), 1547-1548 (2006).</li><li>Teuten, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+and+release+of+chemicals+from+plastics+to+the+environment+and+to+wildlife.">Transport and release of chemicals from plastics to the environment and to wildlife.</a> Phil Trans R Soc B. 364, 2027-2045 (2009).</li><li>Heskett, 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=Measurement+of+persistent+organic+pollutants+(POPs)+in+plastic+resin+pellets+from+remote+islands:+Toward+establishment+of+background+concentrations+for+International+Pellet+Watch.">Measurement of persistent organic pollutants (POPs) in plastic resin pellets from remote islands: Toward establishment of background concentrations for International Pellet Watch.</a> Mar Pollut Bull. 64 (2), 445-448 (2012).</li><li>Besseling, E., Wegner, A., Foekema, E., Van Den Heuvel-Greve, M., Koelmans, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+microplastic+on+fitness+and+PCB+bioaccumulation+by+the+lugworm+Arenicola+marina+(L.).">Effects of microplastic on fitness and PCB bioaccumulation by the lugworm Arenicola marina (L.).</a> Environ Sci Technol. 47 (1), 593-600 (2013).</li><li>Rochman, C. M., Hoh, E., Kurobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SJ+Ingested+plastic+transfers+hazardous+chemicals+to+fish+and+induces+hepatic+stress.">The SJ Ingested plastic transfers hazardous chemicals to fish and induces hepatic stress.</a> Sci Rep. 3, 3263 (2013).</li><li>Endo, S., 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=Concentration+of+polychlorinated+biphenyls+(PCBs)+in+beached+resin+pellets:+Variability+among+individual+particles+and+regional+differences.">Concentration of polychlorinated biphenyls (PCBs) in beached resin pellets: Variability among individual particles and regional differences.</a> Mar Pollut Bull. 50 (10), 1103-1114 (2005).</li><li>Frias, J. P. G. L., Sobral, P., Ferreira, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+pollutants+in+microplastics+from+two+beaches+of+the+Portuguese+coast.">Organic pollutants in microplastics from two beaches of the Portuguese coast.</a> Mar Pollut Bull. 60 (11), 1988-1992 (2010).</li><li>Karapanagioti, H. K., Endo, S., Ogata, Y., Takada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffuse+pollution+by+persistent+organic+pollutants+as+measured+in+plastic+pellets+sampled+from+various+beaches+in+Greece.">Diffuse pollution by persistent organic pollutants as measured in plastic pellets sampled from various beaches in Greece.</a> Mar Pollut Bull. 62 (2), 312-317 (2011).</li><li>Mizukawa, K., 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=Monitoring+of+a+wide+range+of+organic+micropollutants+on+the+Portuguese+coast+using+plastic+resin+pellets.">Monitoring of a wide range of organic micropollutants on the Portuguese coast using plastic resin pellets.</a> Mar Pollut Bull. 70 (1-2), 296-302 (2013).</li><li>Gauquie, J., Devriese, L., Robbens, J., De Witte, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+qualitative+screening+and+quantitative+measurement+of+organic+contaminants+on+different+types+of+marine+plastic+debris.">A qualitative screening and quantitative measurement of organic contaminants on different types of marine plastic debris.</a> Chemosphere. 138, 348-356 (2015).</li><li>Yeo, B. G., 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=POPs+monitoring+in+Australia+and+New-Zealand+using+plastic+resin+pellets,+and+International+Pellet+Watch+as+a+tool+for+education+and+raising+public+awareness+on+plastic+debris+and+POPs.">POPs monitoring in Australia and New-Zealand using plastic resin pellets, and International Pellet Watch as a tool for education and raising public awareness on plastic debris and POPs.</a> Mar Pollut Bull. 101 (1), 137-145 (2015).</li><li>Kova&#269; Vir&#353;ek, M., Palatinus, A., Koren, &. #. 3. 5. 2. ;., Peterlin, M., Horvat, P., Kr&#382;an, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+microplastics+sampling+on+the+sea+surface+and+sample+analysis.">Protocol for microplastics sampling on the sea surface and sample analysis.</a> J Vis Exp. (118), e55161 (2016).</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> Environ Chem. 12 (5), 563-581 (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> Stockholm Convention on Persistent Organic Pollutants (POPs) as amended in 2009 . , (2017).</li><li>EPA - Environmental protection Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Method 3620C: Florisil Cleanup, part of Test Methods for Evaluating Solid Waste, Physical/Chemical Methods (2014). , (2017).</li><li>Hirai, H., 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=Organic+micropollutants+in+marine+plastics+debris+from+the+open+ocean+and+remote+and+urban+beaches.">Organic micropollutants in marine plastics debris from the open ocean and remote and urban beaches.</a> Mar Pollut Bull. 62 (8), 1683-1692 (2011).</li></ol>

Most studies focusing on organic contaminants associated to plastic pellets have relied on classical extraction methods of the adsorbed chemicals. The Soxhlet apparatus is the most widely used technique with typical extraction times ranging from 12 to 24 h and with high consumption of organic solvents (i.e., from 100 to 250 mL per extraction)23. Maceration extractions require a long contact time between the sample and the organic solvent (e.g., 6 days)4 an...

Global production of plastics is continuously rising since the 1950&#39;s to reach 311 million tons in 2014 with about 40% used in packaging1. In parallel, increasing quantities of these materials are accumulating in the environment, which might pose a serious threat to the ecosystems2. Although already reported in the 1970&#39;s, the occurrence of plastic debris in the marine environment has only received a greater attention in the past decade. Especially microplastics, plastic fragments with a diameter of &#8804; 5 mm, are now recognized as one of the main marine water quality issues3.
Plastic resin pellets are small granules generally in the shape of a cylinder or a disk and with a diameter of a few mm (e.g., 2 to 5 mm)4,5. They fall in the category of microplastics. These plastic granules are industrial raw material from which final plastic products are manufactured through re-melting and molding at high temperature6. They can be unintentionally released to the environment during manufacturing and transport. For instance, they can be directly introduced to the ocean through accidental spills during shipping4,7,8. They can be carried from land to oceans by surface run-off, streams and rivers. Because of their environmental persistence, plastic pellets are widely distributed in the oceans and found on beaches all over the world4. They can negatively affect marine organisms and can enter the food chain, where their effects are unpredictable6,7. Furthermore, several studies have revealed the presence of environmental contaminants adsorbed onto plastic pellets collected in a coastal environment, which act as vector of these potentially toxic chemicals4,9,10. In fact, there is laboratory evidence suggesting that these chemicals can bioaccumulate in tissues of organisms after being released from ingested plastic fragments11,12.
In order to better assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that can determine sorbed organic contaminants. An important step is the extraction of the chemicals from the plastic matrices, which can present heterogeneous physical-chemical characteristics depending on the polymer type, its degradation stage, and pre-treatments. Most of the investigations reported in the literature use maceration or Soxhlet techniques4,5,6,9,13,14,15,16,17,18, which are solvent and/or time consuming. Regarding the growing interest for this issue, alternatives should be developed, for a faster evaluation of organic contaminants adsorbed on plastic pieces. In addition, plastic chemical analysis provides information about the chemical structure of the microplastics. As a result, the predominant types of polymers and copolymers present in the environment can be evaluated. Although plastic fragments are usually made of polyethylene (PE) and polypropylene (PP)5, some sampling locations can present a particular profile where other categories are significantly represented (e.g., ethylene/vinyl acetate copolymer and polystyrene (PS)). FT-IR spectroscopy is a reliable and user-friendly technique for polymer identification commonly used to identify microplastics19,20.
The main aim of the present work is to offer a rapid and simple option for extracting OCPs and related compounds from plastic pellets by means of a PFE. However, the design of the protocol includes all steps leading to the determination of sorbed OCPs, from the sampling of the resin pellets to the analysis of the compounds. The method of identifying the plastic type is also described. The developed methodology focuses on 11 OCPs and related compounds: i) DDT (2,4&#39;- and 4,4&#39;-dichlorodiphenyltrichloroethane) and its two main metabolites DDE (2,4&#39;- and 4,4&#39;-dichlorodiphenyldichloroethylene) and DDD (2,4&#39;- and 4,4&#39;-dichlorodiphenyldichloroethane); ii) the isomer gamma-hexachlorocyclohexane (&#947;-HCH) as the main ingredient of the pesticide lindane and the two isomers &#945;-HCH and &#946;-HCH released during its production15; iii) and the two biologically active isomers endosulfan I (Endo I) and II (Endo II) present in the technical endosulfan. The studied pesticides are broad-spectrum insecticides, chemically stable, hydrophobic, and classified as persistent organic pollutants (POPs) by the Stockholm Convention21.

<table border="0" cellpadding="0" cellspacing="0" width="1097">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Alpha&#8211;HCH</td>
			<td style="width:300px;">Dr. Ehrenstorfer, Augsburg, Germany</td>
			<td style="width:136px;">DRE-C14071000</td>
			<td style="width:316px;">H301, H351, H400, H410, H312</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta&#8211;HCH</td>
			<td>Fluka, Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:136px;">33376-100MG</td>
			<td>H301, H312, H351, H410</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Lindane</td>
			<td>Fluka, Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:136px;">45548-250MG</td>
			<td>H301, H312, H332, H362, H410</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endosufan I</td>
			<td>Supleco, Sigma-Aldrich Bellefonte, PA, USA</td>
			<td style="width:136px;">48576-25MG</td>
			<td>H301, H410</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endosulfan II</td>
			<td>Supleco, Sigma-Aldrich, Bellefonte, PA, USA</td>
			<td style="width:136px;">48578-25MG</td>
			<td>H301, H410</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,4&#39;&#8211;DDD</td>
			<td>Fluka, Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:136px;">35485-250MG</td>
			<td>H351</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4,4&#8217;&#8211;DDD</td>
			<td>Dr. Ehrenstorfer, Augsburg, Germany</td>
			<td style="width:136px;">DRE-C12031000</td>
			<td>H301, H351, H400, H410, H312</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,4&#8217;&#8211;DDE</td>
			<td>Dr. Ehrenstorfer, Augsburg, Germany</td>
			<td style="width:136px;">DRE-C12040000</td>
			<td>H351, H400, H410, H302</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4,4&#8217;-DDE</td>
			<td>Fluka , Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:136px;">35487-250MG</td>
			<td>H302, H351, H410</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,4&#8217;&#8211;DDT</td>
			<td>Dr. Ehrenstorfer, Augsburg, Germany</td>
			<td style="width:136px;">DRE-C12081000</td>
			<td>H301, H311, H330, H351, H400, H410</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4,4&#8217;&#8211;DDT</td>
			<td>National Institute of Standards and Technology, Gaithersburg, USA</td>
			<td style="width:136px;">RM8469-4,4&#39;-DDT</td>
			<td>H301, H311, H351, H372, H410</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">n-Hexane&#160;</td>
			<td style="width:300px;">VWR International GmbH, Graumanngasse, Viena, Austria</td>
			<td style="width:136px;">83992.320</td>
			<td>H225, H315, H336, H373, H304, H411</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acetone for HPLC</td>
			<td style="width:300px;">J.T.Baker, Avantor performance Materials B.V., Teugseweg, Netherlands</td>
			<td style="width:136px;">8142</td>
			<td>H225, H319, H 336</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">FL-PR Florisil 1000mg/6mL</td>
			<td style="width:300px;">Phenomenex, Torrance, CA, USA</td>
			<td style="width:136px;">8B-S013-JCH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fat free quartz sand 0.3-0.9 mm</td>
			<td>Buchi, Flawil, Switzerland</td>
			<td style="width:136px;">37689</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:345px;">Gas chromatograph Hawlett Packard HP 6890 Series gas chromatograph with GERSTEL MultiPurpose Sampler MPS 2XL with ECD and FID detector</td>
			<td>Agilent technologies, Santa Clara USA</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Presure fluid extractor, Speed Extractor E-916</td>
			<td>Buchi, Flawil, Switzerland</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solid phase extractor</td>
			<td>Supleco, Sigma-Aldrich Bellefonte, PA, USA</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Concentrator miVac DUO</td>
			<td>Genevac SP Scientific, Suffolk UK</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GC capillary column Zebron ZB-XLB (30 x 0.25 x 0.25)</td>
			<td>Phenomenex, Torrance, CA, USA</td>
			<td style="width:136px;">122-1232</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">ATR FT-IR Spectrometer, Spectrum-Two</td>
			<td style="width:300px;">Perkin Elmer</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Plastic pellets are usually found along the high and low tide lines of sandy beaches (Figure 1A). They can also stick to seagrass freshly stranded on beaches, after a storm for instance. They can occasionally be found on pebble and stony beaches in accumulation areas of stranded material.
Plastic pellets are usually easily recognizable by their shape, size and color as shown in ...

Watch this Scientific Journal Video about Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis at JoVE.com

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

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 ...

This protocol describes the extraction of organochlorine pesticides absorbed on plastic pellets sampled from beaches, as well as the chemical analysis of the plastics. Plastic pellet sampling. Before going in the field, prepare your sampling materials.Carefully clean the stainless steel tweezers and the containers, and you will also need gloves, zip bags, the identity beach form, and eventually an icebox. Once onsite, investigate the area to find the pellets. Did you see them?Wearing gloves, collect the plastic pellets from the beach with the tweezers. In order to have representative results, it is advised to sample 50 to 100 pellets per location. However, if the required number of pellets cannot be reached, collect the maximum pellets possible and specify it in the identity beach form.Plastic pellets are usually found along the high and low tide lines of sandy beaches. Also, check any potential accumulation areas of stranded material. Sometimes, the pellets can be found sticking to fresh seagrass on beaches, after a storm for instance.At the end of the sampling, wrap the collected pellets in aluminum foil. Glass bottles can be used as an alternative or eventually paper bags. Do not forget to fill in the identity beach form.Once in the laboratory, gently wipe off any particles, dry the pellets, if necessary, prior to storage, and store them in dark at low temperature. Extraction of organochlorine pesticides from plastic pellets. Using tweezers, sort the pellets by color.Gather 10 pellets of similar color, which will constitute one replicate. To this end, create visual references, white/transparent, whitish/yellowish, yellow/orange, amber/brown, and pigmented. Weigh the sample on an analytical balance.Switch on the pressurized fluid extractor. Download the extraction method, and warm up the instrument to 60 degrees centigrade. While the instrument is warming up, prepare the extraction cell.First, place the bottom filter and a frit in the extraction cell, and close it. Fill approximately half the cell with quartz sand, using a funnel. Since it is exposed to the same extraction conditions as the samples, special care must be taken to use ultra-clean sand.Add the 10 weighed pellets. Add quartz sand up to one centimeter to the top of the cell. Insert the top filter in the cell, and place the cell in the instrument.Place the collecting vessels in the instrument, and start the extraction method. Once the sequence is completed, empty the extraction cell in a cleaned glass Petri dish. Then, retrieve the 10 pellets in the sand.Store them until further analysis for plastic identification. Concentration and clean-up of the extract. Transfer the obtained extract from the collecting vessel to a glass tube.Place the tube in a rotating concentrator for 20 minutes at 35 degrees Celsius. In this way, you will concentrate the extract to one milliliter. In the meantime, prepare the solid-phase extraction by placing a waste tube in the rack and a Florisil cartridge on the manifold in the close d-valve position.Turn the vacuum on at the source, and add four milliliters of hexane in the cartridge to activate the sorbent. Open the valve, and let the solvent pass through the entire sorbent bed. Then, close the valve, and allow the sorbent to soak in hexane for five minutes.Open the value, and let the sorbent pass through, but close the valve before the sorbent dries off. When the sample is concentrated, transfer it to the cartridge with a glass Pasteur pipette. Gently open the valve and let it pass through slowly.One to two drops per second is the appropriate speed. Rinse the glass tube containing the extract with half a milliliter of hexane, and add it to the cartridge when the extract has passed through. When the entire solvent has passed through, close the valve, and turn off the vacuum.Replace the waste tube by a collecting tube, and use a clean solvent guide needle. Add nine millimeters of solvent composed of 10%acetone and 90%hexane. Turn on vacuum at the source, allow the sorbent to soak in the solvent for one minute.Open the value, and collect the entire eluate and the collecting tube. Place the collecting tube in the concentrator, and evaporate the solvent for nine minutes at 35 degrees in order to reach one milliliter of eluate. Transfer the concentrated eluate in an amber vial, using a glass Pasteur pipette.Analysis of the cleaned and concentrated extract. Download the analytical method on the control software of the gas chromatograph equipped with a micro electron capture detector. The injection is done in splitless mode.Place the vial containing the cleaned and concentrated extract in the autosampler rack, and run the method. Inject two microliters of samples. Plastic type identification.In order to identify the plastic polymer type, transfer the pellets in a glass Petri dish, and place the dish in a plastic bag. Hold one pellet, and cut it with a scalpel. The plastic bag prevents the loss of pellets.Cutting the pellets allows an easier identification of the polymer than processing uncut pellets. The measurement is carried out on a Fourier transform infrared spectrometer. Place the fragment on the attenuated total reflectance crystal, and screw the sample holder.Scan the sample, and record its spectrum. Identify the plastic type by comparing the obtained spectrum to a spectral library. In this example, the sample was made of polyethylene.In black, the spectrum of the sample, and in red, you can see the reference spectrum. Polyethylene, as shown in this example, is the most common polymer type of plastic pellets found. The second most spread category is polypropylene.Ethylene/vinyl acetate copolymer is the third most common. Polystyrene is also occasionally found. After the analysis, identify the different compounds on the chromatogram by their retention types, and write down the corresponding peak areas.As can be seen from this example of spiked pellets, all 11 pesticides have been extracted. To calculate the concentrations of the pesticides absorbed on pellets, you will need to determine the calibration equations and the recoveries from the solid-phase extraction and concentration steps by using standard solutions. Here is an example of a chromatogram of plastic pellets collected on a beach in Greece.Eight pesticides out of 11 were detected. For example, lindane has a retention time of about 9.92 minutes and a recovery of 96%according to standard solutions. On this chromatogram, lindane was detected at the retention time 9.915 minute with a peak area of 3, 270 arbitrary units.The area is then recalculated according to the recovery. Then, the concentration of the one milliliter extract can be calculated based on the calibration equation. Finally, taking into account the mass of the 10 pellets, the concentration of lindane in this sample was 5.33 nanograms per gram of pellets.The median concentrations of several replicates are then determined, and results can be plotted by compound categories, HCHs, DDTs, and endosulfans. Here is an example of plastic type identification for a pool of 10 pellets. The issue of microplastics and associated contaminants is a growing concern, and this simple protocol allows a rapid determination of 11 organochlorine pesticides and their related degradation products on plastic pellets found on beaches.

extraction-organochlorine-pesticides-from-plastic-pellets-plastic

Research

JoVE Journal

Environment

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

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

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 Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na2CO3) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca2+). From the six extraction methods used, only the Na2CO3 extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca2+. The Ca2+-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.

The protocol provides a methodology to solubilize aerobic granular sludge in order to extract alginate-like extracellular polymers (ALE).

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) ...

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 ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

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

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

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

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform ...