Analysis of Conventional and Biodegradable Microplastics in Solid Sample Matrices: A Contribution to Method Standardization


 Microplastics are the new emerging pollutants ubiquitously detectable in aquatic and terrestrial ecosystems. Fate and behavior as well as ecotoxicity are of increasing environmental concern particularly in sediments and soils as natural sinks. For a global environmental risk assessment reliable and easy to apply analytical methods are mandatory to obtain comparable data. This is based on the isolation of microplastics out of the solid sample matrices prior to instrumental detection. Thus, this study provides a validated approach for density separation, which emerged from a comparative study using different salt solutions to isolate conventional and for the first time biodegradable microplastics from different solid sample matrices, i.e., sand, artificial soil, and compost. Four solutions (water, sodium chloride, sodium hexametaphosphate and sodium bromide) of different densities were applied followed by oxidizing digestion. Finally, the impact of the procedures on size and surface properties of microplastics was tested. Dependent on the sample matrix, highest recovery rates of 87.3-100.3 % for conventional polymers and 38.2–78.2 % for biodegradable polymers were determined with sodium bromide. It could be shown that the type of solid sample matrix influences the recovery rates and has to be considered when choosing a sample preparation technique.


Introduction
Since the start of the large-scale production of plastic with about 2 Mt in the 1950s, production and use have rapidly grown due to bene ts like low weight, hygienic use and easy plasticity (Geyer et al. 2017). In 2019, production of plastics reached a maximum with 368 Mt worldwide (PlasticsEurope 2020). Generally, plastics are released deliberately and accidentally from different origins, through different pathways into the environment, which results in a ubiquitous plastic pollution (Schell et al. 2020). It is estimated that out of the generated 6300 Mt plastic waste up to 2015, nearly 80 % were accumulated in the environment (Geyer et al. 2017). Studies con rmed the persistence of plastics in nature. The main degradation process is assumed to be fragmentation into smaller plastic particles so called "microplastics" (MPs) which are polymers with a size between 1-1000 µm (Hartmann et al. 2019).
However, some de nitions contain sizes below 5 mm or 10 mm (Braun et al. 2018).
To overcome littering problems, biodegradable plastics have been developed with a current share of 0.6 % of the global plastic market (PlasticsEurope 2020; Bioplastics 2019). With 43.8 %, starch blends are produced the most, followed by polylactid (PLA) with 24.0 % and polybutylensuccinate (PBS) with 11.4 % of biopolymer production (Haider et al. 2019). Biodegradable alternatives for conventional plastics in mulching for agricultural industry have been developed like MaterBi (MB) by Novamont GmbH (Morra et al. 2016). Theoretically, biodegradable plastics will be converted to water and carbon dioxide by biological forces like microorganism metabolism and can become part of the carbon cycle. However, the degradation potential of plastics strongly depends on the chemical structure (e.g. molecular mass or crystallinity) as well as environmental conditions (e.g. temperature or UV radiation). Number and composition of microorganisms and enzymes present in the corresponding compartment also in uence the degradability of plastic (Thakur et al. 2018; Haider et al. 2019). In case of biodegradable polymers, studies suggest that additives will increase the persistency and might make them potentially undegradable under natural conditions (Lambert and Wagner 2017). Recent studies point out an even higher risk to the environment of some biodegradable plastics than conventional plastics due to faster fragmentation rates into MPs and incomplete degradation into water and carbon dioxide (Qin et al. 2021; Liao and Chen 2021). Still, formation and effects of biodegradable plastics and MPs to environment and human health are widely unknown.
Sampling strategies, sample preparation methods and measurement techniques for MP analysis differ strongly within the scienti c community (Thomas et al. 2020). Currently used techniques to measure MPs lack harmonization and show certain limits, especially for biodegradable MPs, ranging from limited size detection to limitation of detected polymer types ( A reliable method for MP analysis in solid sample matrices was not established until today, but is urgently needed to guarantee comparability between scienti c investigations. As there is only a low percentage of MPs in solid samples, MPs needs to be isolated from the soil matrix. Since MPs are also distributed heterogeneously, mixed samples with the highest possible sample mass will lead to more meaningful results regarding content of MPs in solid samples. This in turn leads to increased effort in sample preparation as organic and inorganic matrices have to be removed for analytics. Especially high organic contents within the sample matrix lead to interfering signals and have to be removed (Thomas et al. 2020 In this study we aim to generate a validated and easy to apply sample preparation method to detect conventional and biodegradable MPs in solid sample matrices. Therefore, a minimum standard has to be found, which enables a worldwide application to collect comparable data on MPs pollution worldwide. We compared the applicability of different solutions with increasing density for density separation as well as oxidative digestion under various conditions. The solutions used for density separation were water (H 2 O), sodium chloride (NaCl), sodium bromide (NaBr) and sodium hexametaphosphate (SHMP). These are considered to be harmless to humans and the environment according to the Globally Harmonized System of Classi cation, Labelling and Packaging of Chemicals (GHS). This is considered to be particularly important because the users have different chemical backgrounds. NaCl and NaBr solutions provided high recovery rates of MPs in other studies and are relatively cheap (Scheurer and

Methods
The experimental design to separate MPs from solid sample matrices includes preparation of three different sample matrices and addition of arti cial MPs. Separation steps of density separation and digestion of organic matter were performed. Four different solutions were applied according to their density and toxicity. Recovery rates as well as changes in size and surface structure of MPs were analyzed with stereo microscope and ATR-FTIR (Fig. 2). A chemical identi cation of potential MPs is associated with the cost of an analyzer, but assumed to be mandatory, because of the high error rates of 20-70 % through visual sorting (Bläsing and Amelung 2018). An ATR-FTIR analyzer is relatively cheap e.g. compared to an automated µFPA-FTIR microscope. Therefore, 500 µm was chosen as the lowest size limit as it represents the optical resolution maximum of this technique.
To meet the standards of analytical quality assurance, every combination of plastic mixture, extraction solution and substrate was tested in triplicates with a blank for every series of tests to investigate contamination. Beyond, sources of MPs contamination were excluded (see 2.6).

Preparation Of Mps
Different types of conventional and biodegradable plastic polymers were produced from mainly postconsumer products (Table 1). Conventional polymers were chosen according to their relevance and the probability of appearance as MPs in the environment. With PE/PP ( ) as light density polymers and PVC/PET ( ) as high-density polymers, it can be estimated if the method is able to recover a variety of plastics with different densities. Densities were measured due displacement of volume with a pycnometer (50 ml) and ethanol (99 %; TH. Geyer).
Furthermore, three biodegradable polymers consisting of a range of newly produced polymers (PLA, PBS, MB) were applied in density separation validation tests from solid samples for the rst time.
Reference MPs were prepared in four steps: rst, products were cut. Second, pieces were frozen in liquid nitrogen and ground (Table 1) Besides the densities of used MPs, the color, chemical spectra as well as the sizes of particles before and after sample preparation were determined (see 2.7).

Preparation Of Solid Sample Matrices
Three solid sample substrates (sand, OECD arti cial soil and compost) were examined, representing different environmental compartments (beach/sediment, terrestrial soil and natural sample with high content of organic matter).

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An amount of 250 g was applied per sample. This is together with Möller et al. 2021 the largest sample mass that was investigated compared to previously published studies that measure MPs concentrations in soil with density separation.

a) Sand substrates
To determine the in uence of different particle sizes in the matrix, sands in different size classes ranging between 60-300 µm, 400-800 µm and 700-1200 µm (Scherf Quarzsand) as well as silicon dioxide in a size range of 53-74 µm (Eijelkamp synthetic sand) were purchased. These were combined to sand sample substrates according to sand fractions in OECD arti cial soil (Table 2). In relation to OECD 222, the size fraction of 0.05-0.2 mm has to make up at least 50 % which was evenly divided between the two obtained fractions in this section. The composition of the other 50 % followed the formula of 100 µm 5 % of total substrate composition. Therefore, sand was dried at 200°C over night and sieved with a vibratory sieve shaker (Retsch AS 200) for 10 min, amplitude 1.2.
Afterwards, the sand sample substrates were placed in a mu e furnace at 550°C for at least 6 h to remove potential MP contamination. Finally, the composite plastic samples were added to the sand sample substrates. The mixture was stirred manually for 30 s. minerals which belong to the kaolin group. The proportion of kaolinite (70 %) was selected to be higher than of halloysite nanoclay/kaolin clay (30 %) as it is the most widespread clay mineral in soils (Dixon 1989).
As there was no further de nition of nely ground, the size of sphagnum peat (Floragard Floratorf) was oriented to the descriptions of OECD 232: Collembolan Reproduction Test in Soil which speci es nely ground to a particle size of 2 1 mm. Therefore, sphagnum peat was sieved to 1.00-3.15 mm due to available mesh size.
All proportions were dried to constant weight.
A mixture of sand size classes with same proportions as the sand substrate was prepared ( Table 2) and placed in a mu e furnace at 550°C for at least 6 h. Afterwards, 35.00 g kaolinite and 15.00 g halloysite nanoclay/kaolin clay were added as well as 25.00 g of sphagnum peat. Additionally, 1.25 g of CaCO 3 were added which corresponds to 0.5 % of soil mass for obtaining pH 6.0 0.5. The pH was determined according to OECD 222 by mixing 20 g of arti cial soil with 100 mL KCl (1M) for 5 min thoroughly. The mixture was left to settle for 2-4 h and then measured with a pH-meter (pH 526, WTW), calibrated at pH 4.0 and 7.0.
The formation of soil aggregates was accelerated by wetting and drying soil. The applied amount of water was oriented to 60 % of water holding capacity as demanded in OECD 222 which was assessed with HYPROP 2 (Meter Group). Two samples of OECD arti cial soil (250 g) were put in a sample ring and placed into water for complete saturation for three days. Afterwards, measurements were conducted over 13 days. The water holding capacity at pF 1.8 was calculated to 32.7 %. Therefore, 45 mL of distilled water and the composite plastic sample were stirred manually together with the substrate for two minutes. Afterwards, 5-10 mL of distilled water were used to rinse the spoon. The sample was let stand for four days to create agglomerates with components and dried at 60°C for 24 h.

c) Compost
Compost was derived from a biowaste plant in Lower Saxony, Germany (ALBA, Watenbüttel). The compost consists of biowaste from private households as well as garden waste (66 %) and was fermented at > 55°C for 14 days according to the composting plant operator. Characteristics show a content of organic matter triple the amount of OECD arti cial soil. Furthermore, pollution with plastics is documented in the share of foreign matter with 0.08 % dry weight. The moisture content of 38.5 % was obtained by drying compost to a constant weight.
Compost was dried at 60°C and sieved. The composite plastic sample and 100 mL of distilled water were added to 250 g of compost dry weight (size class < 1 mm) and stirred manually for 1 min to create agglomerates containing MPs whereas the amount of added water corresponds to the moisture content before drying. Afterwards, samples were dried again (60°C, 24 h). As compost was not newly mixed from components in contrast to OECD arti cial soil, no standing time was applied.

Preparation Of Density Solutions
Density separation was performed with distilled water ( ) and brine solutions of different densities. Saturated solutions were made by dissolving the respective salt into a volume of 1 l distilled water. In case of NaCl (> 99%, z. A., Ph. Eur.; CHEMSOLUTE®) and NaBr (99%, p.a., ACS; Roth) the amount of salt per water volume (358 g NaCl /L H 2 O and 905 g NaBr/L H 2 O) was depending on the speci c solubility of each salt. The solutions were stirred in a beaker (2 l) on a magnetic stirring plate overnight resulting in solution densities of ( ) for NaCl and ( ) for NaBr. As there was no information available about solubility of SHMP (65-70% P 2 O 5 basis; SIGMA-ALDRICH), the substance was added to distilled water sequently and stirred until no further salt dissolved and an excess formed at the beaker bottom ( ).
To lower the amount of disposed chemicals as well as costs, separation solutions of SHMP and NaBr were recycled and reused throughout the test series of density separation. Solutions were decanted onto a pleated lter (Macherey-Nagel MN 615 1/4, diameter 385 mm). The salt was retrieved in a rotary evaporator (Rotavapor RE 111; Büchi Labortechnik AG). Nevertheless, solutions that were used for density separation with compost were not recycled as the dark color indicated pollution with humic substances from compost.

Density Separation, Digestion And Recovery
The recovery rates of MPs in solid sample matrices by density separation with distilled water and different salt solutions were determined. Therefore, spiking experiments were performed, where 250 g of solid sample matrix was spiked with the composite plastic sample. First, 500 mL of the respective separation solution was added into a 2 L glass beaker and stirred with a rectangular stirring bar at 200 rpm. Afterwards, each spiked solid sample was transferred together with another 500 mL separation solution into the glass beaker, resulting in a total volume of 1 L separation solution per sample. Glass beakers were also used in other studies (Chen et  Afterwards, MPs were sorted out with tweezers visually with the naked eye and under a stereo microscope. Then, a second density separation was applied. Residues were mixed with 500 mL of the respective density solution, stirred manually and let stand for 1-2 minutes. MPs were then sorted from the surface and washed in distilled water, dried at room temperature and stored in petri dishes for analysis.
Preliminary experiments showed that density separation eliminated most of the sample matrix compared to digestion. There was one exception in compost samples with NaBr as density solution. Thus, density separation was applied before digestion in all experiments except compost with NaBr.

Tests On In uence Of Sample Preparation
To test the in uence of sample preparation on MPs, several potential impact factors were examined individually and also in combination. The individually tested impact factors were dry storage at 60°C, applied separation solutions for density separation, H 2 O 2 (30 %) at room temperature and H 2 O 2 (30 %) at 60°C. Three particles per polymer type in a size > 1 mm were put into a petri dish and tested for eight days for each impact factor except with H 2 O 2 at 60°C which was tested for 3 d. The duration was based on the time periods of the separation process and corresponds to at least twice the time for each condition in the separation process described above. The combination was a sequence of dry storage at 60°C, the separation solution NaBr that extracts the most MP contents and H 2 O 2 at room temperature and at 60°C with same durations as for individual factors, respectively. Afterwards, particles were washed in distilled water, dried and analyzed.

Contamination Mitigation
To prevent contamination of the samples with MPs during the ongoing experiments, cotton lab coats and butyronitrile gloves were worn in the laboratory and lab surfaces cleaned with distilled water (Torres and De-la-Torre 2021). Equipment was cleaned in a dishwasher and dried in a drying oven. Furthermore, only ltered distilled water (20 m) was applied in test series and for cleaning. All tests of density separation and removal of organic matter were performed under a ow bench and a fume hood. The time a sample was exposed to air was limited as much as possible to prevent atmospheric contamination. Additionally, beakers were covered with aluminum foil during stirring and settling. Furthermore, the air in the lab was ltered (Philips air puri er 2000 AC 2887; Koninklijke Philips N.V.; Amsterdam; Netherlands) as well as the supplied air from outside. Isolated MPs were stored in glass petri dishes until analysis. Besides, a blank sample was performed for each test series.

Microscopy And Ftir Polymer Identi cation
MP composite samples and MPs for in uence tests were manually sorted onto glass petri dishes and photographed under a stereo microscope (Zeiss Stemi SV8 with Olympus SC30, f = 100, 80x, Oberkochen, Germany) with analysisPRO before and after application of the separation protocol. Special attention was paid to lay all particles the same way to ensure comparability for tests of in uence of sample preparation. For other samples of recovery tests, this could not be ensured due to extent of particles.
Then, the photographs were evaluated by automatic computer image analysis to determine the damaging effects on the size of the MPs due to the impact factors. Image analysis was done using the open source computer vision library OpenCV (opencv.org): Contours were localized with the openCV function ndContours, and the particle area was calculated from the contour. Maximum particle length was identi ed by automatic stepwise contour rotation and the openCV function boundingRect. All pictures had identical scale (84 pixels/mm), and contours with an area smaller than 500 pixels (0,07 mm 2 ) were regarded as noise and excluded from the processing. The particles were fragmented and not round which is more likely to re ect their occurrence in the environment. Some particles were also frayed. The fabricated MPs are multidimensional so a two-dimensional picture can only represent a certain aspect of the particles. Thus, two parameters of area and maximum length were considered to reduce effects of observational error.
Besides, MPs were characterized chemically with ATR-FTIR (Bruker Tensor 27 with MVP-Pro, resolution 4 cm − 1 , 16 Scans, area 4000 − 380 cm − 1 ). Several recovered MPs from the MP composite samples were measured with ATR-FTIR to ensure su ciency of visual classi cation. In addition, each polymer for the in uence tests was determined by measuring three particles on two sides. The following six spectra were accumulated to reference spectra.

Statistical Analysis
For recovery tests, means, standard deviation and recovery rates of MPs were calculated for each sample and compared for every combination of substrate and solution. Besides, numbers of recovered MPs for the rst and second removal from the solution surface were compared. For tests of in uence, an analysis of changes in surface structure was performed with ATR-FTIR. Likewise to reference spectra, six spectra per polymer and impact factor were accumulated and afterwards compared with reference spectra. The similarity of spectra was tested with Pearson's correlation coe cient likewise to Vermeiren et al. 2020.

Recovery rates of MPs
Increasing densities of solutions were associated with increasing recovery rates of conventional and biodegradable MPs (Fig. 3) Looking at recovery rates of different polymers, more polymer types were retrieved with increasing density of the applied solution (Fig. 4). with H 2 O were also measured by (Hurley et al. 2018). However, recovery rates of PE were lower in other solutions (85-101 %). PE particles were hardest to identify of conventional polymers due to their color which matched the brown-yellow color of residues of organic matter the most. In addition, gas formation in second density separation hindered visual identi cation of particles on the solution surface in salt solutions. Except for PE for this reason, conventional polymers all showed highest recovery rates in NaBr with > 92 %.
Biodegradable MPs were also highest recovered on NaBr with 17-85 %. MB showed lowest recovery which can be again attributed to decreased visibility. In the residues after digestion, only MB lms with green color could be identi ed. An effect of color on recovery rate was also described by ( unsuitable. Similar effects could also be observed in these test series. Recovery rates in compost samples show a matching trend to OECD arti cial soil with increasing recovery rates in increasing density but they were lower in every solution in compost compared to OECD arti cial soil. The decreased recovery rates can be attributed to the content of organic matter which was also shown in other studies (Radford et al. 2021). Compost had a content of organic matter triple the amount of arti cial soil. In addition, with the increasing density of solutions, more organic matter of compost was surfacing. Thus, more organic matter had to be ltered and digested after density separation. In addition, there were residues of compost left after digestion. Removal of organic matter showed varying e ciency, depending on applied density solution but also depending on sample in general. This is attributed to varying temperatures of heating plates with 5°C and incomplete degradation of lters during digestion which hindered identi cation of polymers. For example, likewise to arti cial soil, MB particles could only be identi ed when having a green color, resulting in recovery rates < 30 %.
As mentioned before, the sequence of density separation and removal of organic matter was changed for compost samples with NaBr solutions as preliminary tests revealed surfacing of 80-90 % of the sample.
Based on research ndings by (Hurley et al. 2018), is was assumed that there is no considerable effect of the sequence on e ciency of separation of MPs. However, samples were then easier to handle. Residues of organic matter were also surfacing in density separation as not the whole sample was digested.
No MPs greater than 500 µm were detected in blanks of arti cial soil and sand and no intentionally added MPs in blanks of compost.
A disadvantage of the method is that it currently relies on visual sorting from solution surface as well as from lters after removal of organic matter and is therefore only applicable for identi cation of MPs > 500 µm. Separation of residues of organic matter from the sample has to be increased to make an automated analysis of smaller MPs possible.
Depending on sample and density solution, different amounts of organic matter were ooding. In arti cial soil, the whole amount of organic matter was surfacing in all test series while in compost it depended on density of solution. This depicts a different behavior of organic matter based on the type and composition. Thus, the application of different methods to remove organic matter can be helpful. To increase recovery rates and decrease visual dependency, conditions of digestion have to be improved to digest organic matter completely like increase heating to 24 h or using more reagent. Otherwise, a second digestion after density separation can be implemented.
Considering the recovery rates of the second removal from solution surface, 1-3 % of all recovered particles were recovered in second removal step with the same density solution, depending on the substrate (Table 3). Thus, this step is not concluded to be necessary for further studies. This data was not collected in other studies although several extraction steps were performed which can be timeconsuming (Chen et al. 2020;Huang et al. 2020). Samples with < 100 % recovery rate per polymer were not included as changes in size are then mainly in uenced by the size of recovered MPs. For this reason, no measurements of MB could be conducted and results of in uence tests not be proven. However, the evaluation method cannot exclude the possibility that there is also an effect on the other polymers.

Conclusion
There is a need of standardized procedures for MPs analysis to collect data for a true picture of the scale of MP contamination of the global environment. Thus, methods should be applicable around the globe.
The introduced protocol provides a simple and useful tool for separation of conventional and biodegradable MPs from solid sample matrices with different composition representing different environmental compartments (beach/sediment, terrestrial soil and natural sample with high content of organic matter), which are supposed to be sinks for MPs. It was shown that recovery rates of MPs increase with increasing density of separation solution and decrease with increasing content of organic matter within the sample matrix. To isolate a wide range of environmentally relevant polymers, solutions with a density > 1.5 g/cm 3 are recommended.
This study showed recovery rates of 94.7 % 10 % with NaBr as density solution for conventional polymers with a density of 0.86-1.37 g/cm 3 with an inexpensive, easy set up for density separation with a salt solution that does not harm the environment and has minimal effect on particle size and composition. Concentrations of 0.01 % w/w MPs in solid matrices were recovered which shows applicability for realistic concentrations in solid matrices.
Indeed, it could be shown that the method needs to be optimized for isolating biodegradable polymers.
However, this is the rst study to isolate biodegradable MPs from solid sample matrices. Although the share of bioplastics from globally produced plastics is currently < 1 %, it is expected to grow. Thus, it is important to further investigate formation and quanti cation of MPs from bioplastics in solid sample matrices.
With a size limit of 500 µm and the set up with beakers, the method applies as a common minimum consensus. To get a full picture of MP contamination with respect to particles < 500 µm, the procedure has to be specialized on the sample matrix. As the set up possibly leads a higher effort for sample isolation and causes misidenti cations, more specialized apparatus, such as separating funnels, are recommended. In addition, more elaborate and expensive analytical methods have to be applied.

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.