Name: Sampling and Identification of Microplastics in Groundwater (Video) | JoVE Journal
Uploaded: 2025-11-07T14:10:00.000+00:00
Duration: 8 min 27 s
Description: Learn standardized groundwater microplastics sampling using borehole filtration and ATR-FTIR spectroscopy for accurate identification. This protocol enables reliable environmental monitoring and research.

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5 minutes

The development of this protocol was funded by the Slovenian Research and Innovation Agency under the research project &#34;Improved methods for determination of transport processes of MPs in groundwater resources&#34; (GWMicroPlast) (J1-50030) and the research program Groundwater and Geochemistry (P1-0020), as well as by the Research Science Funds project &#34;Microplastic in the geosphere&#34; (Manca).

1. Preparing the borehole for sampling
NOTE: To prevent contamination in the field, keep the filtering system closed except when inserting the filters or taking samples. Avoid the use of plastic tools and containers. Avoid synthetic clothing (e.g., fleece); wear a white cotton lab coat.
<ol>
	<li>Open the borehole and remove any samplers, if present. Measure the groundwater level using a water level meter.</li>
	<li>Record the GPS coordinates of the borehole, the date of sampling, the water level, and other sampling details on the data sheet (Table 1).</li>
</ol>
2. Setting up the sampling equipment
<ol>
	<li>Assemble and carefully lower the submersible pump into the well to the desired depth. Ensure that the pump intake is positioned approximately 1 m above the top of the filter screen to prevent sediment intake and maintain optimal water flow. 
	NOTE: During installation, handle the pump with care to prevent damage to its wiring or mechanical components.</li>
	<li>Set up the filtering system without the filter support screens and filters and place it horizontally.</li>
	<li>Close the main valve and open the bypass valve.</li>
	<li>Connect the water supply hose.</li>
</ol>
3. Cleaning the borehole
<ol>
	<li>Start the submersible pump and let the water flow through the bypass to clean the borehole. Pump at least 3x the volume of water present in the borehole or continue pumping until physicochemical parameters stabilize, to ensure fresh groundwater is sampled.</li>
</ol>
4. Cleaning the filtering system before sampling
<ol>
	<li>Open the valves of the sampling branches and the main valve of the filtering system. Afterwards, close the bypass valve. 
	NOTE: Allow water to flow through the filtering system for a sufficient duration to ensure effective flushing, which may depend on the pumping rate. During the cleaning process, the filter support screens and filters must be removed.</li>
</ol>
&#160;5. Inserting the filters
<ol>
	<li>First open the bypass valve and then close the main valve.</li>
	<li>Open the filter chamber and check that the filter chambers are clean. Rinse with ultrapure water, if necessary.</li>
	<li>Insert the filter support screen.</li>
	<li>Rinse the filter of the desired pore size with ultrapure water and place it on the filter support screen.</li>
	<li>Close the filter chamber.</li>
	<li>Repeat the process for all branches. 
	&#8203;NOTE: Several filters with decreasing pore sizes can be inserted sequentially for cascade filtration. Additional filter chambers with filters of the same pore sizes as the sampling filters can be subsequently installed. This is required by the Annex of Commission Delegated Decision (EU) 2024/1441 of 11 March 2024, supplementing Directive (EU) 2020/2184 of the European Parliament and of the Council for sampling MPs in drinking water, which mandates the use of 100 &#181;m and 20 &#181;m filters followed by another set of 100 &#181;m and 20 &#181;m filters (as blanks for quality control purposes).</li>
</ol>
6. Sample collection
<ol>
	<li>Read and record the water meter or reset it to zero.</li>
	<li>Open the main valve, close the bypass valve, and mark the start time of the sampling.</li>
	<li>Monitor the pressure gauge during sampling to ensure the pressure does not exceed four bars to avoid damaging the equipment and minimizing MP fragmentation.</li>
	<li>Stop sampling when the planned water volume is filtered or when the filters start to clog, indicated by increased pressure or significantly decreased flow.</li>
	<li>To stop sampling, first open the bypass valve and then close the main valve and the valves on the filtering branches.</li>
	<li>Turn off the pump.</li>
	<li>Record the time and final readings from the water meter. 
	NOTE: To obtain a representative groundwater sample at the sampling point, it is recommended to sample larger volumes of water, e.g., at least 1 m3/replicate. 
	&#8203;It is imperative to continuously monitor the entire sampling system, particularly the water meter and pressure gauge, throughout the sampling procedure. An increase in pressure accompanied by a decrease in water flow indicates that the filter is beginning to clog. When this occurs, it is advisable to stop the system and replace the used filters with new ones. By collecting the sample across multiple filters in this manner, the desired volume of filtered water can still be achieved efficiently.</li>
</ol>
7. Collecting the filters
<ol>
	<li>Rinse a glass Petri dish with ultrapure water.</li>
	<li>Open the filter chamber and carefully transfer the filter maintained in a horizontal position, into a clean Petri dish.</li>
	<li>Seal the Petri dish with a sealing film and label it with the sample name and the date of sampling.</li>
	<li>Repeat this procedure for all filter chambers (Figure 2).</li>
	<li>After the sample collection, disassemble the system. Always rinse the system with freshwater and dry it before storage. 
	&#8203;NOTE: Alternatively, store the entire filter chamber component of the filtering system for further processing in the laboratory.</li>
</ol>
8. Separation of microplastics from samples
NOTE: To prevent contamination in the laboratory, before starting the analysis of the samples, make sure the laboratory is cleaned of dust, close the window, and use air conditioning with a HEPA filter. Avoid using plastic tools and containers. Glassware must be rinsed with ultrapure water and checked under the microscope before use. Use non-synthetic clothing. Wear a white cotton lab coat to minimize contamination.
NOTE: The stereomicroscope must be equipped with a camera and image analysis software that enables accurate particle size measurement.
<ol>
	<li>Remove the sealing film and open the Petri dish.</li>
	<li>Transfer the Petri dish under the stereomicroscope with at least 30x magnification and search for potentially plastic particles, focusing on characteristics such as color, shape, and other visible features.</li>
	<li>Transfer each particle that seems to be plastic, take a photo, and measure its size. Evaluate the following properties for each MP particle: size (particles: feretmax or area-equivalent diameter; fibers: width and length), shape (particles: fragment, film, foam, pellet, granule; fibers), color, and chemical composition (methodology is described below) (Figure 3). 
	NOTE: Keep in mind that some MPs will be easily identifiable by their color and shape, while others may be more challenging. 
	&#8203;The main parameters for MP identification are described in the protocol for MP sampling on the sea surface and sample analysis26. Polarizing light can be very helpful in separating MP from sediment and organic particles. When isolating potential MPs from the samples, opt for a conservative approach by selecting more rather than fewer particles for detailed analysis.</li>
</ol>
9. Chemical identification of microplastics
NOTE: Chemical analysis of potential MP can be conducted for large MPs (1-5 mm) using ATR-FTIR and for small MP (&#60;1 mm) using micro-FTIR. Alternative methods, such as Raman spectroscopy, are also possible. 
The FTIR instrument software should support precise control of measurement parameters and real-time spectral data acquisition, along with advanced processing tools such as background correction and smoothing. It must include a comprehensive polymer library and enable reliable identification of substances through spectral library comparison to support accurate analysis of microplastics in environmental samples, particularly in complex matrices such as groundwater.
<ol>
	<li>ATR-FTIR spectroscopy
	<ol>
		<li>Before beginning the analysis, thoroughly clean the ATR crystal and sample pressor using 70% alcohol and a lint-free cloth.</li>
		<li>Configure the measurement settings to typically 16 scans with a wavenumber ranging from 4000 to 450 cm -1 and a resolution 4 cm -1. Then, collect the background spectrum. Place the particles one by one onto the ATR crystal, apply pressure, and initiate the measurements.</li>
		<li>Match the acquired infrared spectra with those in reference libraries to confirm the particles as MP. Typically, a 70% correlation with library spectra is considered sufficient for positive identification (Figure 4).</li>
		<li>Export the obtained data for further analysis and reporting.</li>
	</ol>
	</li>
	<li>Micro-FTIR spectroscopy
	<ol>
		<li>Ensure all relevant parts of the instrument, such as the stage, are cleaned with alcohol and a lint-free cloth, prior to analysis.</li>
		<li>If sampling has not been conducted directly on a surface suitable for the selected measurement mode, such as ATR or reflection, place the potential plastic particles on an appropriate reflective surface, such as gold- or aluminum-coated membranes or microscope slides.</li>
		<li>Select the measurement settings for the session, including the number of scans, spectral range, resolution, and session name. Locate the sample and capture a mosaic image of the area where all particles are situated.</li>
		<li>For reflection measurements, first measure the background before measuring the infrared spectra of the potential plastic particles.</li>
		<li>Identify and mark the points where the infrared spectra of the selected particles will be measured. If necessary, select multiple points on each particle. After selecting all the points, start the measurement. 
		&#8203;NOTE: It is also feasible to obtain infrared images of larger areas of interest, particularly if the sample is collected directly on a suitable infrared measurement surface.</li>
		<li>Compare the collected infrared spectra with those from reference libraries. Choose a threshold value for establishing the presence of MPs. Typically, a 70% match is sufficient for positive identification.</li>
		<li>Export the obtained data for further analysis and reporting.</li>
	</ol>
	</li>
</ol>

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<li>Long, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interactions+between+microplastics+and+phytoplankton+aggregates%3A+impact+on+their+respective+fates%5BTitle%5D)+AND+%22Mar+Chem%22%5BJournal%5D)">Interactions between microplastics and phytoplankton aggregates: impact on their respective fates.</a> Mar Chem. 175, (2015).</li>
<li>Viaroli, S., Lancia, M., Re, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplastics+contamination+of+groundwater%3A+current+evidence+and+future+perspectives%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">Microplastics contamination of groundwater: current evidence and future perspectives.</a> Sci Total Environ. 824, (2022).</li>
<li>Li, H., Lu, X., Wang, S., Zheng, B., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vertical+migration+of+microplastics+along+soil+profile+under+different+crop+root+systems%5BTitle%5D)+AND+%22Environ+Pollut%22%5BJournal%5D)">Vertical migration of microplastics along soil profile under different crop root systems.</a> Environ Pollut. 278, (2021).</li>
<li>Ren, Z., Gui, X., Xu, X., Zhao, L., Qiu, H., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplastics+in+the+soilgroundwater+environment%3A+aging%2C+migration%2C+and+cotransport+of+contaminants+-+a+critical+review%5BTitle%5D)+AND+%22J+Hazard+Mater%22%5BJournal%5D)">Microplastics in the soilgroundwater environment: aging, migration, and cotransport of contaminants - a critical review.</a> J Hazard Mater. 419, (2021).</li>
<li>Lwanga, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+microplastic+sources%2C+transport+pathways+and+correlations+with+other+soil+stressors%3A+a+journey+from+agricultural+sites+into+the+environment%5BTitle%5D)+AND+%22Chem+Biol+Technol+Agric%22%5BJournal%5D)">Review of microplastic sources, transport pathways and correlations with other soil stressors: a journey from agricultural sites into the environment.</a> Chem Biol Technol Agric. 9 (1), (2022).</li>
<li>Maaß, S., Daphi, D., Lehmann, A., Rillig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transport+of+microplastics+by+two+collembolan+species%5BTitle%5D)+AND+%22Environ+Pollut%22%5BJournal%5D)">Transport of microplastics by two collembolan species.</a> Environ Pollut. 225, (2017).</li>
<li>Rillig, M. C., Ziersch, L., Hempel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplastic+transport+in+soil+by+earthworms%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Microplastic transport in soil by earthworms.</a> Sci Rep. 7 (1), (2017).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Microplastics+in+drinking+water%22">Microplastics in drinking water</a>. , World Health Organization. Geneva. Licence: CC BYNCSA (2019).</li>
<li>Chia, R. W., Lee, J. Y., Jang, J., Cha, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Errors+and+recommended+practices+that+should+be+identified+to+reduce+suspected+concentrations+of+microplastics+in+soil+and+groundwater%3A+a+review%5BTitle%5D)+AND+%22Environ+Technol+Innov%22%5BJournal%5D)">Errors and recommended practices that should be identified to reduce suspected concentrations of microplastics in soil and groundwater: a review.</a> Environ Technol Innov. 28, (2022).</li>
<li>Drummond, J. D., Nel, H. A., Packman, A. I., Krause, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Significance+of+hyporheic+exchange+for+predicting+microplastic+fate+in+rivers%5BTitle%5D)+AND+%22Environ+Sci+Tech+Let%22%5BJournal%5D)">Significance of hyporheic exchange for predicting microplastic fate in rivers.</a> Environ Sci Tech Let. 7 (10), (2020).</li>
<li>Pittroff, M., Munz, M., Valenti, B., Loui, C., Lensing, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fate+of+microplastics+in+deep+gravel+riverbeds%3A+evidence+for+direct+transfer+from+river+water+to+groundwater%5BTitle%5D)+AND+%22Microplastics%22%5BJournal%5D)">Fate of microplastics in deep gravel riverbeds: evidence for direct transfer from river water to groundwater.</a> Microplastics. 4 (2), 26(2025).</li>
<li>Matjašič, T., Mori, N., Hostnik, I., Bajt, O., Viršek, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplastic+pollution+in+small+rivers+along+rural-urban+gradients%3A+variations+across+catchments+and+between+water+column+and+sediments%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">Microplastic pollution in small rivers along rural-urban gradients: variations across catchments and between water column and sediments.</a> Sci Total Environ. 858, (2023).</li>
<li>Samandra, S., Johnston, J. M., Jaeger, J. E., Symons, B., Xie, S., Currell, M., Ellis, A. V., Clarke, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplastic+contamination+of+an+unconfined+groundwater+aquifer+in+Victoria%2C+Australia%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">Microplastic contamination of an unconfined groundwater aquifer in Victoria, Australia.</a> Sci Total Environ. 802, (2022).</li>
<li>Panno, S. V., Kelly, W. R., Scott, J., Zheng, W., McNeish, R. E., Holm, N., Hoellein, T. J., Baranski, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplastic+contamination+in+karst+groundwater+systems%5BTitle%5D)+AND+%22Groundwater%22%5BJournal%5D)">Microplastic contamination in karst groundwater systems.</a> Groundwater. 57 (2), 189-196 (2019).</li>
<li>Shi, J., Dong, Y., Shi, Y., Yin, T., He, W., An, T., Tang, Y., Hou, X., Chong, S., Chen, D., Qin, K., Lin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Groundwater+antibiotics+and+microplastics+in+a+drinkingwater+source+area%2C+northern+China%3A+occurrence%2C+spatial+distribution%2C+risk+assessment%2C+and+correlation%5BTitle%5D)+AND+%22Environ+Res%22%5BJournal%5D)">Groundwater antibiotics and microplastics in a drinkingwater source area, northern China: occurrence, spatial distribution, risk assessment, and correlation.</a> Environ Res. 210, (2022).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Guidance+on+the+monitoring+of+marine+litter+in+European+seas%22">Guidance on the monitoring of marine litter in European seas</a>. , MSFD Technical Group on Marine Litter. (2023).</li>
<li>Kovač Viršek, M., Palatinus, A., Koren, Š, Peterlin, M., Horvat, P., Kržan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+microplastics+sampling+on+the+sea+surface+and+sample+analysis%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">Protocol for microplastics sampling on the sea surface and sample analysis.</a> JoVE. (118), (2016).</li>
<li>Lee, J. Y., Cha, J., Ha, K., Viaroli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplastic+pollution+in+groundwater%3A+a+systematic+review%5BTitle%5D)+AND+%22Environ+Pollut+Bioavailab%22%5BJournal%5D)">Microplastic pollution in groundwater: a systematic review.</a> Environ Pollut Bioavailab. 36 (1), 2299545(2024).</li>
<li>Cha, J., Lee, J. Y., Chia, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comment+on+the+paper+%26%2334%3BMicroplastic+contamination+of+an+unconfined+groundwater+aquifer+in+Victoria%2C+Australia%26%2334%3B%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">Comment on the paper &#34;Microplastic contamination of an unconfined groundwater aquifer in Victoria, Australia&#34;.</a> Sci Total Environ. 820, (2022).</li>
<li>Weber, F., Kerpen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Underestimating+microplastics%3F+Quantification+of+the+recovery+rate+of+microplastic+particles+including+sampling%2C+sample+preparation%2C+subsampling%2C+and+detection+using+%C2%B5-Raman+spectroscopy%5BTitle%5D)+AND+%22Anal+Bioanal+Chem%22%5BJournal%5D)">Underestimating microplastics? Quantification of the recovery rate of microplastic particles including sampling, sample preparation, subsampling, and detection using µ-Raman spectroscopy.</a> Anal Bioanal Chem. 415 (15), 2963-2973 (2023).</li>
</ol>

The authors have no conflicts of interest to disclose.

The laboratory analysis of MPs in this protocol follows the Annex of Commission Delegated Decision (EU) 2024/1441 of 11 March 2024, supplementing Directive (EU) 2020/2184 of the European Parliament and of the Council by establishing a methodology to measure MPs in water intended for human consumption (Annex of Commission Delegated Decision (EU) 2024/1441) and Guidance on the Monitoring of Marine Litter in European Seas31 developed for the implementation of Marine Strategy Framework Directive (MSFD). Upon completion of this protocol, each MP particle or fiber with its own ID is documented with an image, spectra, and all acquired properties. Each MP particle is described in terms of its shape, size, color, transparency, and chemical composition (polymer type).
According to the Annex of Commission Delegated Decision (EU) 2024/1441, MPs are divided into two types of shapes: particles and fibers. Particles can be further classified into fragments, films, foams, pellets, and granules. This classification is used in monitoring MPs for the MSFD. The differentiation between different kinds of MPs shapes is described in MSFD guidelines31 and video protocol for sampling MP on the sea surface32. The size of each particle is measured as the maximum Feret diameter31 or area-equivalent diameter (Annex of Commission Delegated Decision (EU) 2024/1441), while the dimensions of fibers are recorded in terms of their length and width31. Although color and transparency are not included in the data acquired per the Annex of Commission Delegated Decision (EU) 2024/1441, they are included in the list of MPs properties collected for MSFD. The classifications are outlined in Tables 7.3 and 7.4 of the MSFD guidelines31, respectively.
Based on the result of the spectrum search in the spectrum library, MP particles are categorized into one of the priority polymer groups listed in the Annex of Commission Delegated Decision (EU) 2024/1441 Section 1, points (14) and (15), or classified as other materials under Section 1, point (15). The chemical composition of fibers should be analyzed only if their dimensions and the instrument capabilities allow for positive identification of polymer type; otherwise, they shall be indicated as unidentified fibers.
Monitoring MPs in groundwater is limited to a small number of research studies with differing methodologies that may affect the final results14. Key factors include: 1) flushing before sampling, 2) pump type, 3) filtration system, 4) filter material and pore size, 5) sampling volume, 6) sample treatment, 7) MPs detection and quantification, and 8) quality control. These factors and their characteristics are discussed in more detail below.
In most existing studies, prepumping (flushing) before sampling is not mentioned. But information about it should also be provided33, since purging has a significant impact on the concentration of MPs28. Our protocol includes prepumping, as this process is crucial for sampling fresh groundwater from the aquifer rather than stagnant water accumulated in the borehole. To effectively clean the borehole, it is necessary to pump out 2-3x the volume of the borehole or continue until the physicochemical parameters of water stabilize14. The water sample should be collected approximately 1 m above the top of the filter screen to prevent sediment intake and ensure the homogeneous sample.
Collecting groundwater samples from boreholes requires the use of a pump. The flow rate is strongly influenced by the sampling depth, the type of pump used, and the condition of the borehole, which is closely linked to the location-specific water characteristics-such as sediment content-as well as the prevailing and recent weather conditions at the time of sampling.
Typically, most pumps contain plastic parts, which can potentially contaminate samples, yet most studies do not specify the type of pump used. Pumps with all components made of stainless steel are recommended. If this is not possible, the materials should be specified in terms of their chemical composition using FTIR analysis. The spectra of materials used must be included in the library, and the particles from the samples must be compared against them to account for potential contamination.
For pumps equipped with impellers, larger MP particles may be damaged or even fragmented as they pass through the pump. To avoid overestimating the presence of MPs, it is essential to assess whether the sampling pump contributes to the fragmentation of plastic particles. This can be tested by introducing a known number of well-characterized MP particles into previously cleaned water and pumping it through the filtering system. After pumping, the particles should be re-characterized to determine if any physical changes-such as fragmentation or surface alteration-have occurred. This validation step helps ensure that the sampling process does not artificially increase MP counts due to mechanical degradation.
To provide a large volume of sampled water, a filtering system suitable for use in the field is necessary. In our protocol, to avoid contamination, the filtering system (the sampling system is patented under SLO-P-202300155 and is patent pending EPO-EP24217168.4) is made of stainless steel with all its components welded and does not include Teflon (PTFE) or any other insulating tape made of plastic materials. As filters can clog very quickly, cascade filtration is included as an option, which can also allow direct distribution of particles into desired size classes. Typically, filters with pore sizes of 100 &#181;m and 20 &#181;m or 10 &#181;m are used. Additional filters can be added to address possible clogging issues (e.g., an additional filter with pores between 100 &#181;m and 20 &#181;m). The Annex of Commission Delegated Decision (EU) 2024/1441 recommends cascade filtration with four stages: the first filter has pores of 100 &#181;m, the second 20 &#181;m, the third 100 &#181;m, and the fourth 20 &#181;m. The first two filters are used for MPs analysis, and third and fourth are used for the assessment of MPs contamination levels (sampling quality control) and to ensure that all particles have been filtered by the first two.
The flowmeter (particularly if it is a mechanical one) should be installed after the cascade of filters for sampling to accurately measure the volume of sampled water. During sampling, it is essential to monitor system pressure. If the pressure exceeds 4 bars, this indicates filter clogging, and the system must be shut down immediately to prevent damage to the filter or the system. To avoid additional fragmentation of MPs, sampling should be conducted at the lowest possible pressure.
As mentioned, the Commission Delegated Decision (EU) 2024/1441 proposes using 100 &#181;m and 20 &#181;m filters. In this study, nylon filters were used. However, in this case, it is necessary to collect the MP particles from the filters and transfer them directly to ATR-FTIR or reflective slides suitable for working in reflection mode with FTIR or Raman microscopy. Nylon net filters are effective for filtering through filters with pore sizes of 100 &#181;m, as particles of this size can be manually handled. However, when using 20 &#181;m filters, silicone or aluminum-coated filters are recommended to facilitate measurements in transmission or reflection mode using FTIR or Raman microscopy.
The literature suggests sampling at least 500 L of water to avoid underestimating the presence of MPs34. According to the Annex of Commission Delegated Decision (EU) 2024/1441, 1 m3 of water should be sampled. The recommended volume needs to be considered to avoid overestimating the MP concentration in the sample. This large volume of water increases the risk of filter clogging, making cascade filtration highly recommended.
The samples from the presented system are immediately prepared for MPs analysis using a stereomicroscope. This is a very time- and cost-efficient method. Many studies detail pretreatment steps to remove sand and organic matter; however, these procedures are time-consuming and may involve the use of toxic chemicals. Such pretreatment steps may also negatively affect the results as they lead to additional fragmentation or loss of particles, especially smaller ones (&#60; 100 &#181;m)35. Therefore, it is preferable to avoid these steps unless necessary. Such instructions are also described in the Commission Delegated Decision (EU) 2024/1441, where it is proposed that sample analysis by FTIR micro-spectroscopy may be done directly on the original collection filters, if they are compatible with the analytical method used, and a flotation step is used if necessary.
First, filter samples are analyzed with stereomicroscopes, whereby each particle is photo documented, ID-labeled, measured for size, and assessed for shape. The determination of shape is important as it can provide information about the source of the MPs. Fibers typically come from textile washing and originate from wastewater treatment plants, while fragments typically come from the degradation of large plastic particles in the environment. In the second part of the analysis, the chemical composition of each particle is determined using ATR-FTIR or FTIR/ Raman microscopy. Generally, all particles from the filter to reflective slides are transferred to perform analysis in reflective mode with FTIR microscopy. If the sample is collected on a 20 &#181;m silicone filter, direct analysis with the Raman or FTIR microscope can be performed, provided the filter is not excessively covered with sand particles. If the particles are embedded in sand, density separation and/or chemical or enzymatic treatments may be applied to reduce the presence of non-plastic materials such as minerals, metal oxides, and natural organic matter. MP particles must be dried before measurement, as water strongly absorbs IR radiation.
To ensure the credibility of the results, the following quality control elements must be considered: First, air pollution prevention: to prevent atmospheric contamination during sampling, a closed filter system is essential. To avoid airborne contamination in the process of analysis, a white cotton lab coat and air filtration through a HEPA filter are required. Second, prevention of contamination by the filter system: the only component of the system where polymers are unavoidable are the hoses. Silicone hoses are recommended and should be replaced in case of visible damage. It is necessary to know the components of the pump and test the pump for potential contamination of samples if polymer presence is established. Third, prevention of contamination during laboratory work: only laboratory equipment made of glass and washed with ultrapure water is suitable for MPs analysis. Fourth, parallel samples to ensure reproducibility and statistical significance of the results: this filtration system allows simultaneous sampling from four branches. A minimum of three parallel samples should be obtained to ensure the same conditions for all samples and to save time. Fifth, blank samples must be included in all steps of sampling and laboratory processes. During the field sampling, blank filters 100 &#181;m and 20 &#181;m are installed after 100 &#181;m and 20 &#181;m sample filters, as proposed in the Annex of Commission Delegated Decision (EU) 2024/1441. Blank samples obtained in the sampling procedure must be considered in all analytical steps by following the same procedure.
The protocol outlined in this paper meets all the requirements set forth by the Commission Delegated Decision (EU) 2024/1441 and, therefore, provides an excellent resource to all researchers studying or monitoring MPs in groundwater. The filtration system described can also be used for freshwater or marine waters, as it enables sampling of MPs at different depths. The sampling method is simple, user-friendly, and provides fast and accurate results in most cases without needing pretreatment of the sample.

In recent years, microplastics (MPs) have been identified as an important environmental pollutant. Due to their potential to enter the atmosphere, MPs are part of the water cycle1. Atmospheric deposition and surface runoff are the main pathways through which MPs enter surface water2. MPs originate from various terrestrial and aquatic sources, with terrestrial sources contributing to 80% of their total quantity3. Upon entering aquatic ecosystems, the majority of terrestrial MPs are transported to oceans via rivers. The remaining MPs persist in freshwater environments, with surface waters in densely populated and urbanized areas-characterized by long water residence times and significant anthropogenic influence-exhibiting higher levels of MP pollution. Surface waters can also connect to groundwater in riverbeds, floodplains, wetlands, and springs4.
Although research conducted in freshwater habitats made up less than 4% of the available literature in 20185, growing recognition of the need to better understand MP sources in aquatic environments has since driven a notable increase in studies focused on freshwater systems6. While the number of studies on groundwater remains limited, evidence of the presence of MPs in groundwater has been well-documented. As groundwater is the most important source of freshwater in the world, providing more than two billion people with access to safe drinking water, water for domestic, agricultural, and industrial use7, the presence of MPs in groundwater raises new questions about groundwater safety in the 21st century8.
Terrestrial sources of MPs can be very diverse, with fibrous MPs from the washing of textiles accounting for approximately 35% of the MPs detected in aquatic systems9. Other important sources of different types of MPs in the environment include personal care and cosmetic products, tires, agricultural plastic films, artificial turf and road coatings, landfills, improperly disposed plastics, packaging, and construction industry pollutants9,10.
Due to their many different sources, MPs can differ substantially in terms of their chemical composition, color, shape, density, size, and other characteristics11. International standard ISO 24187:2023 classifies MPs into two categories: 1) &#34;large microplastics&#34;: solid, water-insoluble plastic particles with sizes ranging between 1 mm and 5 mm, and 2) &#34;microplastics&#34;: any solid plastic particles insoluble in water with sizes ranging between 1 &#181;m and 1 mm. Particles smaller than 1 &#181;m are considered nanoparticles12. The polymer materials most frequently found in the form of MPs are polyethylene (PE) and polypropylene (PP), as the most widely produced polymer materials13.
MPs can be transported into groundwater from soils and sediments, via interactions with surface water and seawater, and through recharge/discharge processes. During these processes, MPs can be transported into and from the groundwater14. The hyporheic zone (HZ) serves as an important interface for the exchange between the river and the shallow groundwater system15. The transport of MPs through the HZ is influenced by various particle properties (size, shape, material composition), as well as certain hydrological and geochemical factors, including riverbed morphology and flow turbulence15. The relationship between pore diameter and MP size is another crucial factor in these processes16, as MPs with smaller dimensions move more easily through the pore space from the surface into the subsurface layers15,16.
MPs can enter groundwater through the unsaturated zone17. The unsaturated zone represents an important connection between the land surface and the groundwater18. The processes of transport and retention of MPs in the unsaturated zone depend on particle properties, soil properties, and environmental factors17,19,20. Soil organisms such as earthworms, collembolans, and mites can influence the transport of MPs from the soil surface to deeper layers through various mechanisms such as displacement, ingestion, engulfment, and adherence21,22. Invertebrates can indirectly affect the transport of microplastics by forming macropores in the soil, which act as pathways for microplastic movement through leaching21.
The primary concern regarding the presence of MPs in groundwater is their persistence related to the large surface area, less environmentally friendly degradation processes (leading to the formation of micro- and even nano-sized particles), and strong hydrophobicity17. Their persistence causes a potential risk of affecting groundwater quality from a chemical and biological perspective. MPs can chemically pollute groundwater by leaching unbound monomers and additives, as well as sorbed chemicals from the environment (e.g., hydrophobic persistent organic pollutants)23. MPs can also serve as a substrate for biofilm formation and influence the microbiology of groundwater. Biofilms on MPs may also contain free-living microorganisms and pathogens23. If ingested, the particles themselves represent a physical hazard. The smaller the particle size, the greater the possibility that they will be absorbed into cells or cross biological barriers of organisms23.
Research into MPs in groundwater is increasingly recognized as critical due to the potential risk that MPs pose to human health. Consequently, the revised Drinking Water Directive for measuring MPs entered into force in January 2021. EU Member States were obliged to transpose the Directive into national law and ensure compliance with its provisions by 12 January 2023. Nevertheless, the number of studies on MPs conducted to date remains limited. There is currently no standard procedure for sampling and analyzing MPs in groundwater. Studies assessing the occurrence of MPs in groundwater are difficult to compare as they use different sampling and analytical approaches. Therefore, recent studies have highlighted the urgent need to standardize the protocols for MPs sampling and analysis to ensure the collection of high-quality samples and obtain comparable results12,17,24,25.
Filtration-based sampling and grab sampling are two commonly used methods for collecting groundwater samples in MP studies to date. Filtration involves passing water through mesh filters, either stainless steel cartridges26 or sieves27, in the field to capture MP particles. A major drawback of using cartridges and sieves is the difficulty in thoroughly cleaning them of residual particles, which compromises the ability to ensure complete sample analysis and increases the risk of cross-contamination. Grab sampling, frequently used in many studies28,29,30, is a simpler approach where water is collected directly using bottles or containers without pretreatment. While suitable for exploratory studies, grab sampling does not accurately reflect MP concentrations due to small sample volumes.
This study introduces a newly developed system for sampling MPs in groundwater (Figure 1), based on field filtration using commercially available filters of customizable pore sizes. The system enables simultaneous filtration of multiple samples and supports cascade filtration. Designed as a fully enclosed setup, it effectively prevents environmental contamination of samples. A detailed sampling protocol is provided, accompanied by video guidelines, along with procedures for subsequent analysis of the chemical composition and other characteristics of the detected MPs. The system aims to enhance the quality, consistency, and comparability of future research in this field.

<table><tbody><tr><td>ATR-FTIR spectrometer</td><td>PerkinElmer</td><td>L160000F</td><td>SpectrumTwo</td></tr><tr><td>FTIR microscope</td><td>ThermoFischer Scientific</td><td>IQLAADGAAGFARMMBMN</td><td>RaptIR</td></tr><tr><td>Glass Petri dishes</td><td>Brand</td><td>455717</td><td>60 mm in diameter</td></tr><tr><td>Nylon Net Membranes</td><td>Merck</td><td>NY1H04700</td><td>Hydrophilic, 100&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, 100</td></tr><tr><td>Nylon Net Membranes</td><td>Merck</td><td>NY2004700</td><td>Hydrophilic, 20&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, 100</td></tr><tr><td>Precise forceps</td><td>BraunB</td><td>BD335R</td><td>Micro forcep</td></tr><tr><td>Sealing film</td><td>Amcor Flexibles North</td><td>PM992</td><td>Parafilm M</td></tr><tr><td>Stereomicroscope&amp;nbsp;</td><td>Zeiss</td><td>495015-9880-010</td><td>StereoDiscovery V8</td></tr><tr><td>Submersible water pump</td><td>Grundfoss</td><td>96510217</td><td>SQ 5-70</td></tr><tr><td>Water level meter</td><td>Hydrotechnik</td><td>/</td><td>Type 010</td></tr></tbody></table>

sampling and identification of microplastics in groundwater

Sampling and Identification of Microplastics in Groundwater

Microplastics pollution in groundwater remains significantly underreported within scientific literature. This paper presents a comprehensive protocol outlining the methodology for the sampling of groundwater from boreholes, as well as the steps of microplastics separation and analysis. It provides an extensive description of a filtration sampling system designed specifically for this purpose, along with the detailed sampling procedure. In addition, it presents the laboratory analysis of microplastic particles, including their characterization based on size, shape, color, transparency, and chemical structure using attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and micro-FTIR spectroscopy. Factors that can influence results are discussed, and special attention is paid to preventing contamination of samples. The methodology described also considers the requirements of the Annex of Commission Delegated Decision (EU) 2024/1441 of 11 March 2024, supplementing Directive (EU) 2020/2184 of the European Parliament and of the Council. This comprehensive written protocol, accompanied by video guidance, is intended to support the development of a synchronized methodology for monitoring microplastics in groundwater or drinking water. This resource will be of interest to researchers in the field of microplastics worldwide.

The first results of this protocol is the database of all MPs found in each sample (Table 2), which can be used for further analysis of the quantity of MP and its properties (color, size, shape in material composition).
The primary objective of MP sampling and sample analysis is to determine the quantity of MP particles per sample (Figure 5). These data can subsequently be normalized per cubic meter (m3). The normalization formula is as follows: 
MP particles per m3 per sample = N / V
Where: 
N = sum of MP particles per sample 
V = sample volume (m3)
<img alt="Vacuum system diagram; pressure indicator, valve symbols; gas flow control process." class="xfigimg" src="/files/ftp_upload/68652/68652fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Schematic of the sampling system used in the protocol. The sampling system consists of an inlet pipe with three legs, wherein one leg is arranged for connection to a pump, a second leg is arranged for connection to a distribution unit, and a third leg is arranged to ensure a bypass of the water past the sampling units. The distribution unit has four symmetrically arranged branches for connection to the corresponding sampling unit, wherein in the center of the distribution unit, a pressure measuring device is installed. Each sampling unit is provided with a valve, three filter holders, and a flow meter installed downstream of the filter holder to prevent sample contamination. <a href="https://www.jove.com/files/ftp_upload/68652/68652fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Soil particle analysis, two petri dishes, sediment sorting, laboratory experiment." class="xfigimg" src="/files/ftp_upload/68652/68652fig2v3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Example of filters after 1 m3 of sampled groundwater. Left: nylon net filter with a pore size of 100 µm, right: nylon net filter with a pore size of 20 µm. Filters can differ in the amount of sediment and organic particles, depending on the sampling location. <a href="https://www.jove.com/files/ftp_upload/68652/68652fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy analysis of microplastic fibers and fragments; magnification, polymer identification, particle size." class="xfigimg" src="/files/ftp_upload/68652/68652fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Examples of representative particles of various shapes. (A) fragments; (B) fibers. Scale bars are shown in the images. Particles can be of many different colors, shapes, and sizes. The size of fragments is measured as Feret diameter or area-equivalent diameter, while fibers are measured in their length and width. <a href="https://www.jove.com/files/ftp_upload/68652/68652fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="FTIR spectroscopy result; IR spectrum of polystyrene-butadiene copolymer; spectral analysis chart." class="xfigimg" src="/files/ftp_upload/68652/68652fig4.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Example spectra measured on a selected particle with marked peaks and their wavelengths [cm-1], compared with a spectral library. The sample spectrum should show at least 70% correlation with the reference spectra in the library. <a href="https://www.jove.com/files/ftp_upload/68652/68652fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar chart of microplastic concentration across various sampling locations; environmental data analysis." class="xfigimg" src="/files/ftp_upload/68652/68652fig5.jpg" data-altupdatedat="1783396841000" data-alt="Figure 5"> 
Figure 5: Example results of the number of microplastics per m3 per sampling location. <a href="https://www.jove.com/files/ftp_upload/68652/68652fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Location: Borehole 1</td>
			<td colspan="2">Date: April 1</td>
			<td colspan="5">Researcher(s): Researcher 1, researcher 2, researcher 3</td>
		</tr>
		<tr>
			<td colspan="9">GPS coordinates: 46.056946 N 14.505751 E</td>
		</tr>
		<tr>
			<td colspan="9">Project: Groundwater sampling</td>
		</tr>
		<tr>
			<td colspan="9">Weather conditions: Sunny</td>
		</tr>
		<tr>
			<td colspan="9">Temperature (°C): 20</td>
		</tr>
		<tr>
			<td colspan="9">Samples</td>
		</tr>
		<tr>
			<td>Sample ID</td>
			<td colspan="2">GW1.1</td>
			<td colspan="2">GW1.2</td>
			<td colspan="2">GW1.3</td>
			<td colspan="2">GW1.4</td>
		</tr>
		<tr>
			<td>Filtration position</td>
			<td colspan="2">1</td>
			<td colspan="2">2</td>
			<td colspan="2">3</td>
			<td colspan="2">4</td>
		</tr>
		<tr>
			<td>Filter type</td>
			<td colspan="2">Nylon - 100 µm, 20 µm</td>
			<td colspan="2">Nylon - 100 µm, 20 µm</td>
			<td colspan="2">Nylon - 100 µm, 20 µm</td>
			<td colspan="2">Nylon - 100 µm, 20 µm</td>
		</tr>
		<tr>
			<td>Sampling depth (m)</td>
			<td colspan="2">25</td>
			<td colspan="2">25</td>
			<td colspan="2">25</td>
			<td colspan="2">25</td>
		</tr>
		<tr>
			<td>Start time</td>
			<td colspan="2">10.00</td>
			<td colspan="2">10.00</td>
			<td colspan="2">10.00</td>
			<td colspan="2">10.00</td>
		</tr>
		<tr>
			<td>Water meter START (m3)</td>
			<td colspan="2">437.4199</td>
			<td colspan="2"></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td>End time</td>
			<td colspan="2">10.45</td>
			<td colspan="2"></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td>Water meter END (m3)</td>
			<td colspan="2">438.421</td>
			<td colspan="2"></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td>Sampled water volume (m3)</td>
			<td colspan="2">1.001</td>
			<td colspan="2"></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td>Other</td>
			<td colspan="2"></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
	</tbody>
</table>
Table 1: Example sampling data sheet, including parameters such as location, date, environmental conditions, and data related to water filtration.
<table border="1" fo:font-size="7px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style="table-layout: auto; width: 1px; border-collapse: collapse;">
	<tbody>
		<tr>
			<td>Location</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Sample</td>
			<td style="white-space: nowrap; min-width: 0;">Particle ID</td>
			<td style="white-space: nowrap; min-width: 0;">Shape</td>
			<td style="white-space: nowrap; min-width: 0;">Size (mm)</td>
			<td style="white-space: nowrap; min-width: 0;">Color</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Chemical composition</td>
			<td style="white-space: nowrap; min-width: 0;">% match</td>
			<td style="white-space: nowrap; min-width: 0;">Priority polymers</td>
			<td style="white-space: nowrap; min-width: 0;">Instrument</td>
			<td style="white-space: nowrap; min-width: 0;">Spectrum</td>
			<td style="white-space: nowrap; min-width: 0;">Other</td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_1_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">1</td>
			<td style="white-space: nowrap; min-width: 0;">particle</td>
			<td style="white-space: nowrap; min-width: 0;">0.54</td>
			<td style="white-space: nowrap; min-width: 0;">black</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polytetrafluoroethylene</td>
			<td style="white-space: nowrap; min-width: 0;">80.2</td>
			<td style="white-space: nowrap; min-width: 0;">PTFE</td>
			<td style="white-space: nowrap; min-width: 0;">Spectrum two</td>
			<td style="white-space: nowrap; min-width: 0;">abc1111</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_1_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">2</td>
			<td style="white-space: nowrap; min-width: 0;">fiber</td>
			<td style="white-space: nowrap; min-width: 0;">0.98</td>
			<td style="white-space: nowrap; min-width: 0;">blue</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyethylene teraphalate</td>
			<td style="white-space: nowrap; min-width: 0;">91.9</td>
			<td style="white-space: nowrap; min-width: 0;">PET</td>
			<td style="white-space: nowrap; min-width: 0;">RaptIR</td>
			<td style="white-space: nowrap; min-width: 0;">abc1112</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_2_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">1</td>
			<td style="white-space: nowrap; min-width: 0;">fiber</td>
			<td style="white-space: nowrap; min-width: 0;">1.54</td>
			<td style="white-space: nowrap; min-width: 0;">blue</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyamide</td>
			<td style="white-space: nowrap; min-width: 0;">75.0</td>
			<td style="white-space: nowrap; min-width: 0;">PA</td>
			<td style="white-space: nowrap; min-width: 0;">RaptIR</td>
			<td style="white-space: nowrap; min-width: 0;">abc1113</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_2_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">2</td>
			<td style="white-space: nowrap; min-width: 0;">fiber</td>
			<td style="white-space: nowrap; min-width: 0;">2.87</td>
			<td style="white-space: nowrap; min-width: 0;">red</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyethylene teraphalate</td>
			<td style="white-space: nowrap; min-width: 0;">98.0</td>
			<td style="white-space: nowrap; min-width: 0;">PET</td>
			<td style="white-space: nowrap; min-width: 0;">RaptIR</td>
			<td style="white-space: nowrap; min-width: 0;">abc1114</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_2_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">3</td>
			<td style="white-space: nowrap; min-width: 0;">fiber</td>
			<td style="white-space: nowrap; min-width: 0;">3.04</td>
			<td style="white-space: nowrap; min-width: 0;">red</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyethylene teraphalate</td>
			<td style="white-space: nowrap; min-width: 0;">71.3</td>
			<td style="white-space: nowrap; min-width: 0;">PET</td>
			<td style="white-space: nowrap; min-width: 0;">RaptIR</td>
			<td style="white-space: nowrap; min-width: 0;">abc1115</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_2_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">4</td>
			<td style="white-space: nowrap; min-width: 0;">fiber</td>
			<td style="white-space: nowrap; min-width: 0;">1.27</td>
			<td style="white-space: nowrap; min-width: 0;">blue</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyethylene teraphalate</td>
			<td style="white-space: nowrap; min-width: 0;">83.6</td>
			<td style="white-space: nowrap; min-width: 0;">PET</td>
			<td style="white-space: nowrap; min-width: 0;">RaptIR</td>
			<td style="white-space: nowrap; min-width: 0;">abc1116</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_3_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">1</td>
			<td style="white-space: nowrap; min-width: 0;">particle</td>
			<td style="white-space: nowrap; min-width: 0;">1.93</td>
			<td style="white-space: nowrap; min-width: 0;">white</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyethylene </td>
			<td style="white-space: nowrap; min-width: 0;">85.3</td>
			<td style="white-space: nowrap; min-width: 0;">PE</td>
			<td style="white-space: nowrap; min-width: 0;">Spectrum two</td>
			<td style="white-space: nowrap; min-width: 0;">abc1117</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_3_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">2</td>
			<td style="white-space: nowrap; min-width: 0;">particle</td>
			<td style="white-space: nowrap; min-width: 0;">0.96</td>
			<td style="white-space: nowrap; min-width: 0;">blue</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyethylene </td>
			<td style="white-space: nowrap; min-width: 0;">89.4</td>
			<td style="white-space: nowrap; min-width: 0;">PE</td>
			<td style="white-space: nowrap; min-width: 0;">Spectrum two</td>
			<td style="white-space: nowrap; min-width: 0;">abc1118</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
		<tr>
			<td style="white-space: nowrap; min-width: 0;">Borehole 1</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Borehole1_3_20242204</td>
			<td style="white-space: nowrap; min-width: 0;">3</td>
			<td style="white-space: nowrap; min-width: 0;">fiber</td>
			<td style="white-space: nowrap; min-width: 0;">0.54</td>
			<td style="white-space: nowrap; min-width: 0;">blue</td>
			<td colspan="2" style="white-space: nowrap; min-width: 0;">Polyamide</td>
			<td style="white-space: nowrap; min-width: 0;">73.2</td>
			<td style="white-space: nowrap; min-width: 0;">PA</td>
			<td style="white-space: nowrap; min-width: 0;">RaptIR</td>
			<td style="white-space: nowrap; min-width: 0;">abc1119</td>
			<td style="white-space: nowrap; min-width: 0;"></td>
		</tr>
	</tbody>
</table>
Table 2: Example database of all isolated microplastic particles per sampling, including parameters such as shape, size, color, and material for each particle.

Here, we provide a detailed description of sampling groundwater from a borehole for microplastics analysis using a patented sampling system developed for this purpose. The protocol details the methods for sampling microplastics from boreholes, as well as the separation and chemical identification of microplastics.

Microplastics pollution in groundwater remains significantly underreported within scientific literature. This paper presents a comprehensive protocol ...

Protocol for the Sampling and Identification of Microplastics from Groundwater. Preparing the borehole for sampling. Open the borehole and remove any samplers if present.Measure the groundwater level using the water level meter. Record the GPS coordinates of the borehole, the date of sampling, water level, and other sampling details on the data sheet. Setting up the sampling equipment.Assemble and carefully lower the submersible pump into the well to the desired depth. Set up the filtering system without the filter support screens and filters, and place it horizontally. Close the main valve and open the bypass valve.Connect the water supply hose. Cleaning the borehole. Start the submersible pump and let the water flow through the bypass to clean the borehole.Cleaning the filtering system before sampling. Open the valves of the sampling branches and the main valve of the filtering system. Afterwards, close the bypass valve.Inserting the filters. First, open the bypass valve and then close the main valve. Open the filter chamber and check that the filter chambers are clean.Rinse with ultrapure water if necessary. Insert the filter support screen. Rinse the filter with the desired pore size with ultrapure water and place it on the filter support screen.Close the filter chamber. Repeat the process for all branches. Several filters with decreasing pore sizes can be inserted sequentially for cascade filtration.Sample collection. Read and record the water meter or reset it to zero. Open the main valve, close the bypass valve, and mark the start time of the sampling.Monitor the pressure gauge during sampling to ensure the pressure does not exceed four bars to avoid damaging the equipment and minimizing microplastic fragmentation. Stop sampling when the planned volume of water is filtered or when the filters start to clog, that can be observed by the increased pressure or significantly decreased flow. To stop sampling, first open the bypass valve and then close the main valve and the valves on the filtering branches.Turn off the pump. Record the time and final readings from the water meter. Collecting the filters.Rinse a Petri dish with ultrapure water. Open the filter chamber and carefully transfer the filter maintained in a horizontal position into a clean Petri dish. Seal the Petri dish with a sealing film and label it with the sample name and date of sampling.Repeat the procedure for all filter chambers. After the sample collection, disassemble the system. Always rinse the system with fresh water and dry it before storage.Separation of microplastics from samples. Remove the sealing film and open the Petri dish. Transfer the Petri dish under the stereo microscope with at least 30 times magnification and search for potentially plastic particles, focusing on characteristics such as color, shape, and other visible features.With the transfer of each particle that seems to be plastic, take a photo and measure its size. The following properties have to be determined for each microplastic particle:size, shape, color, and chemical composition. Keep in mind that some microplastics will be easily identifiable by their color and shape, while others may be more challenging.Chemical identification of microplastics. The chemical analysis of potential microplastic can be done for large microplastics by ATR-FTIR, and for small microplastics by micro-FTIR. Alternative methods, such as Raman, are also possible ATR-FTIR spectroscopy.Before beginning the analysis, thoroughly clean the ATR crystal and sample pressor using alcohol and a lint-free cloth. Configure the measurement settings, typically 16 scans with a wave number ranging from 4, 000 to 450 reciprocal centimeter and resolution 4 reciprocal centimeter. Then collect the background spectrum.Place the particles one by one onto the ATR crystal, apply pressure and initiate the measurements. Match the acquired infrared spectra with those in reference libraries to confirm the particles as microplastic. Typically, a 70%correlation with library spectra is considered sufficient for positive identification.Export the obtained data for further analysis and reporting. Micro-FTIR spectroscopy. Ensure all relevant parts of the instruments, such as the stage, are cleaned with alcohol and a lint-free cloth prior to analysis.If sampling has not been done directly on a surface suitable for the selected measurement mode, such as ATR or reflection, place the potential plastic particles on an appropriate reflective surface, such as gold or aluminum-coated membranes or microscope slides. Select the measurement settings for the session, including the number of scans, spectral range, resolution, and session name. Locate your sample and capture a mosaic image of the area where all particles are situated.For reflection measurements, first measure the background before measuring the infrared spectra of potential plastic particles. Identify and mark the points where infrared spectra of the selected particles will be measured. If necessary, select multiple points on each particle.After selecting all the points, start the measurement. It's also feasible to obtain infrared images of larger areas of interest, particularly if the sample is collected directly on a suitable infrared measurement surface. Compare the collected infrared spectra with those from reference libraries.Choose a threshold value for establishing the presence of microplastics. Typically, a 70%match is sufficient for positive identification. Export the obtained data for further analysis and reporting.Representative results. The main result of this protocol is the database with all isolated microplastic particles, each characterized by shape, size, color, and material composition. The concentration of microplastic per sample and sampling location can be calculated.Conclusion.This comprehensive protocol ensures accurate identification of microplastics for research or monitoring purposes, considering the recommendations from the annex of Commission Delegated Decision EU 2024/1441.

sampling-and-identification-of-microplastics-in-groundwater

Research

JoVE Journal

Environment

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.

Protocol for Microplastics Sampling on the Sea Surface and Sample 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.

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

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.

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

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

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

Presented here is a method for extracting microplastics from soil and identifying their polymer types. The method has been optimized for execution, applicability, and cost-effectiveness. It lays a scientific foundation for standardizing the analytical method to identify microplastics in soils.

Separation and Identification of Conventional Microplastics from Farmland Soils

Here, we present a protocol showcasing an optically transparent and flat substrate for the streamlined capture and analysis of contaminating particles in drinking water. Presented here is the Silicon Nanomembrane Analysis Pipeline (SNAP): a flexible pipeline for the capture, quantification, and identification of particles in liquid media.

Multimodal Analysis of Microplastics in Drinking Water using a Silicon Nanomembrane Analysis Pipeline

Wild animals are commonly parasitized by a wide range of helminths.  The four major types of helminths are “roundworms” (nematodes), “thorny-headed worms” (acanthocephalans), “flukes” (trematodes), and “tapeworms” (cestodes).  Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically...

Helminth Collection and Identification from Wildlife

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects.  We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

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.

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

Learn standardized groundwater microplastics sampling using borehole filtration and ATR-FTIR spectroscopy for accurate identification. This protocol enables reliable environmental monitoring and research.

Sampling and Identification of Microplastics in Groundwater

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