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 operation. If sampling during the clean-up season, specify the details of this activity in the identity form (e.g., dates, clean-up method used, etc.)</li>
	<li>Wearing gloves, collect plastic pellets from the beach with solvent-rinsed stainless steel tweezers.</li>
	<li>Sample 50 to 100 pellets per location, which corresponds to 5 to 10 replicates per location (10 pellets per replicate). If the required number of pellets cannot be obtained, collect the maximum pellets possible and specify it in the identity beach form.</li>
	<li>At the end of the sampling, wrap the collected pellets in solvent-rinsed aluminum foil. Glass bottles can be used as an alternative or even paper bags.</li>
	<li>Fill in the identity form of the selected beach with the missing information (i.e., beach location, weather conditions, details on pellets, etc.).</li>
	<li>Transport the samples to the laboratory in an icebox if the ambient temperature exceeds 25 &#176;C. This step can be skipped in the case of short trips (e.g., &#60;1 h).</li>
	<li>Once in the laboratory, gently wipe off removable particles (e.g., sand) of the pellets. Dry the samples if necessary in a desiccator prior to storage (darkness, T &#60;25 &#176;C). Avoid rooms where OCPs might be in use (e.g., storage of standard solutions).</li>
	<li>Store the pellets in the fridge (4 &#176;C) for short periods (i.e., few days) or in the freezer (-18 &#176;C) for longer periods in solvent-rinsed aluminum foil.</li>
	<li>Avoid exposure of samples to artificial light or sunlight. Handle the samples as little as possible before analysis to decrease the risk of contamination.</li>
</ol>
2. Extraction of OCPs from Plastic Pellets
<ol>
	<li>To reduce the risk of contamination, work in a clean laboratory using carefully washed glassware as follows: 2 rinses with analytical-grade acetone, dichloromethane and n-hexane. Dry the glassware under nitrogen flow and protect from contact with ambient air (e.g., cover with cleaned aluminum foil). Apply this cleaning procedure in the further steps of the protocol (i.e., sections 3 and 4).</li>
	<li>Using solvent-rinsed tweezers, sort the pellets by color in the following categories: white/transparent, whitish/yellowish, yellow/orange, amber/brown, and pigmented (e.g., red, green, blue, etc.)</li>
	<li>Gather 10 pellets of similar color randomly (i.e., plastic type not considered), which will constitute one replicate.</li>
	<li>Weigh the sample on an analytical balance and record the mass. At this stage, the samples can be put back in the fridge or freezer.</li>
	<li>To take into account the background contamination, perform a blank sample with each set of replicates (e.g., 1 blank for 5 replicates). To this end, apply the same protocol as described above, but do not add plastic pellets in the extraction cell. This blank sample will undergo the further steps of the protocol and be analyzed together with the samples.</li>
	<li>Switch on the PFE. Download the extraction method and warm-up the instrument to 60 &#176;C. The details of the method are as follows:
	<ol>
		<li>Set the temperature to 60 &#176;C and the pressure to 100 bar.</li>
		<li>Select one cycle with heat-up time of 1 min, a hold time of 25 min, and a discharge time of 2 min.</li>
		<li>Set the solvent and gas (N2) flush times to 3 min each.</li>
		<li>Select n-hexane as the extraction solvent.</li>
	</ol>
	</li>
	<li>While the instrument is warming up, prepare the extraction cell as described below. If necessary, adapt the protocol to the supplier&#39;s instructions of your instrument:
	<ol>
		<li>Place the bottom filter and the frit in the extraction cell. Close it and turn it over.</li>
		<li>Fill approximately half the cell with cleaned quartz sand using a funnel.</li>
		<li>Add the weighed sample (i.e., one replicate of 10 pellets). Frozen plastic pellets should be placed in the fridge overnight prior to extraction.</li>
		<li>Add quartz sand up to 1 cm from the top of the cell. Take special care to use ultra clean quartz sand (or alternatively glass beads) since it is exposed to the same extraction conditions as the samples. To clean the sand, successively extract it in the PFE in analytical-grade dichloromethane and n-hexane, applying 2 or more cycles per solvent (e.g., 30 min at 100 &#176;C under 100 bar). Alternatively, use an ultrasonic bath and/or rotating evaporator. Repeat the cleaning procedure, if necessary.</li>
		<li>Insert the top filter in the cell and place the cell in the instrument.</li>
	</ol>
	</li>
	<li>Place the collecting vessels in the instrument and start the extraction method (total run of about 35 min).</li>
	<li>When the method is completed, empty the extraction cell in a cleaned glass vessel (e.g., beaker, glass cell-culture dish) and retrieve the 10 pellets in the sand. Store them in a container until further analysis for plastic identification (e.g., zip bag or glass vial).</li>
</ol>
3. Concentration and Clean-up of the Extract
<ol>
	<li>Transfer the obtained extract (about 40 mL) from the collecting vessel to a glass tube and evaporate it to 1 mL in a rotating concentrator set to 35 &#176;C for 20 min. Alternative methods could be used such as evaporation under nitrogen flow or rotating evaporator. The temperature and duration should be optimized accordingly.</li>
	<li>In the meantime, prepare the solid-phase extractor (SPE) by placing a waste tube in the rack and a cartridge filled with activated magnesium silicate sorbent (1 g) on the manifold in the close-valve position. The clean-up is based on the EPA method 3620C22 as follows:
	<ol>
		<li>Turn the vacuum on at the source and add 4 mL of hexane in the cartridge to activate the sorbent.</li>
		<li>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 5 min.</li>
		<li>Open the valve and let the solvent pass through, but close the valve before the sorbent dries off.</li>
		<li>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. 1-2 drops per second is an appropriate speed.</li>
		<li>Rinse the glass tube containing the extract with 0.5 mL of hexane and add it to the cartridge when the extract has passed through.</li>
		<li>When the entire solvent has passed through, close the valve and turn off the vacuum.</li>
		<li>Replace the waste tube with a collecting tube and use a clean solvent guide needle.</li>
		<li>Add 9 mL of acetone/hexane (10/90, v/v) to the cartridge and turn on the vacuum at the source. Allow the sorbent to soak in the solvent for 1 min.</li>
		<li>Open the valve and collect the entire eluate in the collecting tube.</li>
	</ol>
	</li>
	<li>Place the collecting tube in the concentrator and evaporate the solvent for 9 min at 35 &#176;C in order to reach 1 mL of eluate.</li>
	<li>Transfer the concentrated eluate into an amber autosampler vial with a glass Pasteur pipette. At this stage, the samples can be stored in the freezer prior to analysis.</li>
</ol>
4. Analysis of the Cleaned and Concentrated Extract
<ol>
	<li>Download the analytical method on the control software of the GC-&#956;ECD instrument (gas chromatograph equipped with a micro electron capture detector). The details of the method are as follows:
	<ol>
		<li>Set the injector to splitless mode, its temperature to 250 &#176;C, and the purge time to 1 min.</li>
		<li>Set the flow of the carrier gas (He) to 1.5 mL min-1.</li>
		<li>Program the column oven with the following temperature gradient: 60 &#176;C hold for 1 min, ramp of 30 &#176;C min-1 to 200 &#176;C, ramp of 5 &#176;C min-1 to 230 &#176;C, ramp of 3 &#176;C min-1 to reach 250 &#176;C, hold this temperature for 5 min.</li>
		<li>Set the detector temperature to 300 &#176;C and the back-up gas flow (N2) to 60 mL min-1.</li>
	</ol>
	</li>
	<li>Place the vial containing the sample (cleaned and concentrated) in the autosampler rack and run the method (run time of 23.3 min). Inject 2 &#181;L of sample.</li>
	<li>After the analysis, identify the different compounds on the chromatogram by their retention times and record the corresponding peak areas.</li>
	<li>Taking into account the recoveries (R) and the peak areas (A1), calculate the concentration (C1) of each OCP in the extract using the equations of the calibration curves as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/55531/55531eq1.jpg" /> 
	where b is the intercept at the origin and a is the slope of the calibration equation, 
	<img alt="Equation 2" src="/files/ftp_upload/55531/55531eq2.jpg" /></li>
	<li>Taking into account the mass (m) of the replicate (i.e., 10 pellets; see section 2.4) and the volume (V) of the final extract (i.e., 1 mL), calculate the concentration (C2) of each OCP adsorbed on the plastic pellets (i.e., ng of OCP per g of plastic pellet): 
	<img alt="Equation 3" src="/files/ftp_upload/55531/55531eq3.jpg" /></li>
</ol>
5. Plastic Type Identification
<ol>
	<li>Transfer the pellets in a glass Petri dish and place it in a plastic bag.</li>
	<li>Hold one pellet with tweezers and cut a slice of the pellet with a scalpel. The plastic bag prevents the loss of pellets during the cutting process.</li>
	<li>Clean the attenuated total reflectance (ATR) crystal of the FT-IR instrument with ethanol.</li>
	<li>Record a background spectrum.</li>
	<li>Place the fragment on the ATR crystal and screw the sample holder. The inner side of the piece must be in contact with the crystal.</li>
	<li>Scan the sample and record the spectrum.</li>
	<li>Identify the polymer constituting the plastic pellet by comparing the obtained spectrum to a spectra library. Although more time-consuming, the interpretation of the obtained spectra could be carried out manually as well, but most probably without reaching the degree of specificity achieved with a library search.</li>
</ol>

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The authors have nothing to disclose.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Environment

11.5K Views.  University of Nova Gorica. 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. 

Video: Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

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 ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

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 ...