polymers

Article
Extraction and Identification of a Wide Range of Microplastic
Polymers in Soil and Compost
Franja Prosenc 1, * , Pia Leban 2 , Urška Šunta 1 and Mojca Bavcon Kralj 2

1 Research Institute, Faculty of Health Sciences, University of Ljubljana, 1000 Ljubljana, Slovenia;
[email protected]
2 Department for Sanitary Engineering, Faculty of Health Sciences, University of Ljubljana,
1000 Ljubljana, Slovenia; [email protected] (P.L.); [email protected] (M.B.K.)
* Correspondence: [email protected]

Abstract: Microplastic pollution is globally widespread; however, the presence of microplastics

in soil systems is poorly understood, due to the complexity of soils and a lack of standardised
extraction methods. Two commonly used extraction methods were optimised and compared for the
extraction of low-density (polyethylene (PE)) and high-density microplastics (polyethylene (PET)),
olive-oil-based extraction, and density separation with zinc chloride (ZnCl2). Comparable recoveries
in a low-organic-matter matrix (soil; most >98%) were observed, but in a high-organic-matter matrix
(compost), density separation yielded higher recoveries (98 ± 4% vs. 80 ± 11%). Density separation
was further tested for the extraction of five microplastic polymers spiked at different concentrations.
Recoveries were >93% for both soil and compost, with no differences between matrices and individual
polymers. Reduction in levels of organic matter in compost was tested before and after extraction,
as well as combined. Double oxidation (Fenton’s reagent and 1 M NaOH) exhibited the highest





 reduction in organic matter. Extracted microplastic polymers were further identified via headspace
Citation: Prosenc, F.; Leban, P.;
solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME–GC–MS). This
Šunta, U.; Bavcon Kralj, M. Extraction method has shown the potential for descriptive quantification of microplastic polymers. A linear
and Identification of a Wide Range of relationship between the number of particles and the signal response was demonstrated for PET,
Microplastic Polymers in Soil and polystyrene (PS), polyvinyl chloride (PVC), and PE (R2 > 0.98 in alluvial soil, and R2 > 0.80 in
Compost. Polymers 2021, 13, 4069. compost). The extraction and identification methods were demonstrated on an environmental sample
https://doi.org/10.3390/ of municipal biowaste compost, with the recovery of 36 ± 9 microplastic particles per 10 g of compost,
polym13234069 and the detection of PS and PP.

Academic Editor: Jacopo La Nasa Keywords: microplastic extraction; oil extraction; density separation; GC–MS; mass spectrometry
identification; plastic polymers; polyethylene terephthalate; polyethylene; terrestrial
Received: 28 October 2021
Accepted: 19 November 2021
Published: 23 November 2021

1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Microplastic (MP) pollution is widespread across all ecosystems, and has been widely
published maps and institutional affil- studied in marine systems, whereas other environmental compartments—such as soil—
iations. have only started to emerge as a field of research in the past few years [1,2]. Soil is a
very versatile and complex matrix with a broad range of organic matter content (from
0.02% in desert soils to up to 100% in bog soils) [2]. Specifically, the high organic matter
content of soils hampers the extraction of MPs due their having similar densities to most
Copyright: © 2021 by the authors.
common plastic polymers and, as such, poses a challenge in the determination of MPs
Licensee MDPI, Basel, Switzerland.
in soils. Recently, standards for the collection and preparation of water samples for the
This article is an open access article
identification and quantification of MP particles and fibres have been developed (ASTM
distributed under the terms and D8332-20 and D8333-20) [3,4]. However, to date, there are no such standards for soil and
conditions of the Creative Commons soil-like samples, even though various methods for the detection of MPs in soil have been
Attribution (CC BY) license (https:// proposed.
creativecommons.org/licenses/by/ MPs enter soil via the addition of soil amendments—such as biowaste compost
4.0/). and waste sewage sludge/biosolids—the use of agricultural plastics, irrigation with (re-

Polymers 2021, 13, 4069. https://doi.org/10.3390/polym13234069 https://www.mdpi.com/journal/polymers

Polymers 2021, 13, 4069 2 of 16

claimed) wastewater, flooding, and atmospheric deposition, as well as littering and street
runoff [2,5–7]. Studies reporting on primary sources of MPs in soil consider tire wear,
fibres from synthetic clothing, artificial turf, and agricultural plastics (plastic mulch films,
greenhouses, fruit protection foams, etc.) as notable sources [8–10]. Agricultural soils are
particularly prone to accumulating MPs [11]. A study quantifying MPs in soil reported
between 7100 and 42,960 plastic particles per kilogram of soil in southwestern China, 95%
of which were <1 mm [12]. A study in northwestern China found 40 ± 126 light-density
polyethylene (LDPE) MPs per kilogram of soil in the top 10 cm of agricultural soil, and
100 ± 141 LDPE MPs per kilogram of soil in the 10–30 cm layer [13]. In east China, a
study found 40.2 ± 15.6 MPs per kilogram of unamended agricultural soil, while in soil
amended with sludge, between 68.6 ± 21.5 and 149.2 ± 52.5 MPs per kilogram of soil
were found [14]. In Germany, 0.34 ± 0.36 MPs were found per kilogram of dry weight
of soil that had never been fertilised with MP-containing fertilisers [15]. Organic waste
from households that is composted, or anaerobically digested with further composting
of digestate, is usually applied to soil as a fertiliser. This is a common practice to return
nutrients, trace elements, and humus to the soil; however, most municipal organic waste
is contaminated with plastics—either with non-biodegradable plastic bags and/or other
plastic items [16]. A study by Weithmann et al. reported between 20 and 24 MP particles
per kilogram of dry weight biowaste compost, and between 14 and 146 MPs per kilogram
of dry weight biowaste digestate [17].
MPs in soil have been shown to have deleterious effects on soil organisms; however,
most studies were carried out at (currently) environmentally irrelevant concentrations.
These high concentrations, however, represent future, presumably higher levels, as plastic
production, use, and consequent pollution increase [18]. MPs have been shown to alter
the microbial activity in soil and sediments, and to increase root and shoot mass [19,20],
affect the mortality and growth rate of earthworms (Lumbricus terrestris) [21], affect the
reproduction of nematodes (Caenorhabditis elegans) [22], and alter the immune response
in crustaceans (Porcellio scaber) [23]. In addition to the biotic effects of MPs, they have
also been shown to influence soils’ biophysical properties, such as pH, content and size
distribution of water-stable aggregates, water-holding capacity, soil bulk density, and
saturated hydraulic conductivity [19,24–26].
To precisely evaluate the risks of MP pollution in agroecosystems and terrestrial en-
vironments, reliable methods for the identification and quantification of MPs are needed.
Extraction of MP particles from these complex matrices is a paramount step in their analysis.
To date, various methods for the extraction of MPs have been developed, including density
separation, oil extraction, electrostatic separation, magnetic-field separation, solvent ex-
traction, and circular separation [27–31]. Density separation using various saturated saline
solutions—such as sodium iodide (NaI), sodium chloride (NaCl), sodium bromide (NaBr2 ),
calcium chloride (CaCl2 ), zinc bromide (ZnBr2 ), and zinc chloride (ZnCl2 )—is the most
frequently applied extraction method for solid samples. In this method, MP particles are
separated from the solid matrix based on the difference between the density of MPs and the
density of the saturated saline solution. Various technical solutions have been used to sepa-
rate MP-containing supernatants without disturbing the settled sediment. Most commonly,
the supernatant is decanted onto a filter after a settling period, then withdrawn with a
pipette or sucked with the aid of a pump [13,32]. Imhof et al. [33] devised the “Munich
Plastic Sediment Separator”—a custom-made stainless steel apparatus that separates the
settled sediment from the MP-containing supernatant with a valve. Konechnaya et al. [34]
separated MP-containing supernatant from various sediments by slowly overflowing the
samples with saline solution and collecting the MP-containing overflow. Recently, Grause
et al. [35] proposed using a centrifuge to speed up the separation step and decanting the
supernatant with MPs onto a filter. Many of these methods, however, were not tested on
soils or organic-rich matrices, such as compost.
Oil-based separation has recently been developed, with promising applications. In
this method, MPs are extracted based on the oleophilic properties of most plastic polymers,
Polymers 2021, 13, 4069 3 of 16

so that the MPs, when in contact with oil, move to the oil layer independent of their density.
Water and a small volume of oil are added to the solid sample, stirred, and left to settle,
and then the top oil layer, containing MPs, is separated from the rest of the sample. In
several studies, a separation funnel was used to drain the sediment and aqueous fraction
from the sample and, in this way, retain the MP-containing oil layer [27,36,37]. Several
types of oil—such as canola, castor, and olive oils—were used for the separation of MPs
from sediments, soil, and sludge. It has been reported, however, that separation funnels
could easily become obstructed when extracting solid samples [36]. The same issue was
encountered in our preliminary experiments. Scopetani et al. [38], therefore, designed a
system to freeze the samples after the settling period and cut off the frozen oil layer.
After extraction, MPs can be identified by employing advanced instrumentation, us-
ing non-destructive (A) and destructive methods (B): (A) scanning electron microscopy
(SEM) combined with spectroscopic techniques, such as micro-Fourier-transform infrared
(µFTIR) and Raman spectroscopies; (B) thermogravimetric analysis (TGA), thermal desorp-
tion gas chromatography coupled with mass spectrometry (TED-GC–MS), and pyrolysis
employing gas chromatography coupled with mass spectrometry (Py-GC–MS) [39–43].
Even though the well-established methods can offer enhanced identification of polymers,
the field strives towards developing low-cost and simple techniques to facilitate analysis
in most laboratories with basic analytical equipment. For this reason, solid-phase mi-
croextraction (SPME)—as a simple, inexpensive, and easy-to-handle technique—combined
with gas chromatography coupled with mass spectrometry (GC–MS) analysis was used
to develop an efficient method for the identification of MPs in environmental matrices,
termed headspace solid-phase microextraction–gas chromatography–mass spectrometry
(HS-SPME–GC–MS) [44,45].
This study aimed to determine the most suitable method for the extraction of various
MP polymers from soil and compost, based on recovery, ease of handling, the potential for
operator error, and time efficiency. Two methods—an olive-oil-based method, and density
separation with saturated ZnCl2 solution, never compared before—were evaluated for
the extraction of MPs. Moreover, the effects of different vessels on MP recovery with an
oil-based method were evaluated for the first time. Additionally, a simpler identification
method based on HS-SPME–GC–MS is presented in this study as a viable alternative to
traditionally used identification methods, and shows the potential for descriptive quan-
tification of MPs. This method allowed for the simultaneous identification of various
polymers in a mixture, and showed good linearity by increasing the number of spiked MPs
in real matrices (both alluvial soil and compost), which is a significant advantage over the
traditionally used methods.

2. Materials and Methods

2.1. Preparation of Microplastics
Two types of MP polymers were used in the development and optimisation of the
extraction methods—polyethylene terephthalate (PET), as a polymer with high density (ρ
= 1.33–1.48 g cm−3 ), and low-density polyethylene (LDPE), as a polymer with low density
(ρ = 0.91–0.94 g cm−3 ) (Figure 1a). PET particles were prepared by cutting a plastic bottle
(Radenska, Radenci, Slovenia) into <5 mm pieces. LDPE particles were prepared by melting
plastic pellets (Tera Tolmin, Ltd., Tolmin, Slovenia), and then grating the melted plastic
mass using a metal cheese grater and sieving the particles with a 2 mm sieve (Retsch,
Dusseldorf, Germany).
After the method optimisation, the method was tested on five MP polymers—namely,
polypropylene (PP), polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), and
PET (Table 1, Figure 1b). The same particles were then identified via HS-SPME–GC–MS.
The MP particles were prepared with cryo-milling according to an adapted protocol [44].
In short, 5–10 g of plastic material (pellets or small pieces) was added to a stainless-steel
grinding jar, together with a stainless steel 25 mm grinding ball. The jar was submerged
in liquid nitrogen for 6 min, after which the grinding was carried out in a MillMix 20 ball
 Table 1. Plastic polymers used, their source, and their properties.

Polymer Type Melting

Source Density (g·
cm−3)
(1–2 mm) Temperature (°C)
Polymers 2021, 13, 4069 PP Pellets (Golias Ltd.) 0.85–0.88 179 4 of 16
PE Bottle cap (Radenska) 0.91–0.96 108–141
PS Pellets (Golias Ltd.) 1.04–1.10 242–276
PVC Blister pack 1.38–1.40 220–305
mill (Tehtnica, Železniki, Slovenia) at between 25 and 30 Hz for 1.5 min, depending on the
PET Plastic bottle (Radenska) 1.33–1.48 264
polymer. For PET and PE, the procedure of cooling and grinding was repeated more than
PP: polypropylene; PE: polyethylene; PS: polystyrene; PVC: polyvinyl chloride; PET: polyethylene
terephthalate.
once; the fine fraction was then sieved through 2, 1, and 0.5 mm sieves (Retsch, Germany)
to obtain a fine fraction of 1–2 mm.

Figure 1. Microplastic particles prepared for (a) extraction method optimisation and (b) extraction of
Figure 1. Microplastic particles prepared for (a) extraction method optimisation and (b) extraction
a wide rangepolymers
of a wide range of microplastic of microplastic polymers (PP: polypropylene;
(PP: polypropylene; PE: polyethylene;PE:PS:
polyethylene; PS: polystyrene; PVC:
polystyrene;
polyvinyl chloride; PET: polyethylene
PVC: polyvinyl chloride; PET: polyethylene terephthalate). terephthalate).

2.2. Optimisation ofTable 1. PlasticExtraction

Microplastic polymers used, their source, and their properties.
Two methods for the extraction
Polymer Type of MPs from soil were adapted from the literature
Density Melting
and optimised: olive-oil-based extraction, and Source
density separation with saturated
(1–2 mm) (g cm−ZnCl
3) 2 Temperature (◦ C)

solution [34,38]. Both methodsPP

werePellets
tested(Golias
on two types of MP polymers: LDPE,
Ltd., Ljubljana, Slovenia)
repre-
0.85–0.88 179
senting MPs with low density; PE and PET, representing MPs with high density.
Bottle cap (Radenska) In both
0.91–0.96 108–141
methods, 10 and 20 MP particles
PS of each composition were Ltd.)
Pellets (Golias spiked into 10 g of 1.04–1.10
alluvial soil 242–276
or biowaste compost. AlluvialPVC soil used in this experiment
Blister packwas collected on the banks of
1.38–1.40 220–305
the Sava River basin (15° PET
31′ 47.65″ E, 45°55′Plastic bottle
27.21″ (Radenska)
N), in the immediate vicinity1.33–1.48
of ag- 264
ricultural fields, atPP:
a polypropylene;
depth of 0–10 PE:cm.
polyethylene; PS: polystyrene;
The compost was from PVC:apolyvinyl
biowaste chloride; PET: polyethylene terephthalate.
composting
plant that collects and processes separately collected organic waste (JP VOKA Snaga Ltd.,
Ljubljana, Slovenia),2.2.with
Optimisation
a reportedof organic
Microplastic Extraction
matter content of 24–44%.
Two methods for the extraction of MPs from soil were adapted from the literature and
optimised: olive-oil-based extraction, and density separation with saturated ZnCl2 solu-
tion [34,38]. Both methods were tested on two types of MP polymers: LDPE, representing
MPs with low density; and PET, representing MPs with high density. In both methods,
10 and 20 MP particles of each composition were spiked into 10 g of alluvial soil or biowaste
compost. Alluvial soil used in this experiment was collected on the banks of the Sava River
basin (15◦ 310 47.6500 E, 45◦ 550 27.2100 N), in the immediate vicinity of agricultural fields, at a
depth of 0–10 cm. The compost was from a biowaste composting plant that collects and
processes separately collected organic waste (JP VOKA Snaga Ltd., Ljubljana, Slovenia),
with a reported organic matter content of 24–44%.

2.2.1. Oil-Based Method

This method was carried out as described by Scopetani et al. [38], with some modifi-
cations, as shown in Figure 2a. As a vessel for the sample, three technical configurations
were used: a polytetrafluoroethylene (PTFE or Teflon) cylinder (Dastaflon Ltd., Medvode,
Slovenia), a glass cylinder (Promal, Logatec, Slovenia), and a modified plastic syringe with
the hub removed to create an open, flat aperture (Soft-Ject® , Henke-Sass, Wolf GmbH,
Tuttlingen, Germany). Ten grams of alluvial soil was spiked with either 10 or 20 LDPE or
 2.2.1. Oil-Based Method
This method was carried out as described by Scopetani et al. [38], with some modi-
fications, as shown in Figure 2a. As a vessel for the sample, three technical configurations
were used: a polytetrafluoroethylene (PTFE or Teflon) cylinder (Dastaflon Ltd.,
Polymers 2021, 13, 4069 Medvode, Slovenia), a glass cylinder (Promal, Logatec, Slovenia), and a modified plastic 5 of 16
syringe with the hub removed to create an open, flat aperture (Soft-Ject ® , Henke-Sass,
Wolf GmbH, Tuttlingen, Germany). Ten grams of alluvial soil was spiked with either 10
or 20 LDPE or PET MPs added to each sample vessel and closed on one end with a cap.
PET MPs
Then, 30 mL added to 2each
of dH sample
O was mixed vessel
withand 3 mL closed on one
of olive oil.end with a cap.
Afterwards, theThen,
vessels30 mL
were of
dH O was mixed with 3 mL of olive oil. Afterwards, the vessels
capped on the other end and shaken to homogenise the sample. Samples were left to
2 were capped on the other
end and
stand for shaken to homogenise
2 h to sediment, the sample.
and then Samples were
frozen overnight at −18left°C.toAfter
standfreezing,
for 2 h tothe
sediment,
frozen
and then frozen overnight at − 18 ◦ C. After freezing, the frozen samples were pushed out
samples were pushed out of the vessels with a piston. The frozen oil layer containing
of the
MPs vessels
was with melted
removed, a piston.at The
room frozen oil layer and
temperature, containing
filtered MPs wasaremoved,
through 47 mm GF/C melted at
filter
room temperature,
(Whatman, Maidstone,and UK).
filtered through
The MPs and a 47remaining
mm GF/C debris filter (Whatman,
were rinsed Maidstone,
with hexane UK).
The MPs and remaining debris were rinsed with hexane (CARLO
(CARLO ERBA Reagents S.A.S., Val-de-Reuil, France) and dH2O to remove any remain- ERBA Reagents S.A.S.,
Val-de-Reuil,
ing oil. In the France)
case of and dH2 Othe
compost, to remove
procedure anywas
remaining
similaroil. In the
to that forcase of compost,
alluvial the
soil, with
procedure was similar to that for alluvial soil, with slight modifications; the method was
slight modifications; the method was optimised using 10 PET MPs in a plastic syringe,
optimised using 10 PET MPs in a plastic syringe, and the oxidation step was added after
and the oxidation step was added after extraction to reduce organic matter content. Ox-
extraction to reduce organic matter content. Oxidation was achieved by submerging the
idation was achieved by submerging the GF/C filter with the sample remaining on the
GF/C filter with the sample remaining on the filter in 60 mL of Fenton’s reagent for 2 h
filter in 60 mL of Fenton’s reagent for 2 h under constant stirring [46]. The oxidised sam-
under constant stirring [46]. The oxidised sample was again filtered through the GF/C filter.
ple was again filtered through the GF/C filter. Extraction of MPs in each treatment (ves-
Extraction of MPs in each treatment (vessel, MP type, and concentration) was repeated six
sel, MP type, and concentration) was repeated six times. Recovery of MPs was calculated
times. Recovery of MPs was calculated using the following equation:
using the following equation:
N𝑁extracted
𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑× 100
%) =
R𝑅 ((%) = N × 100 (1)
(1)
𝑁spiked
𝑠𝑝𝑖𝑘𝑒𝑑

Figure 2. Schematic representation of microplastic extraction methods: (a) oil-based method, and (b)
Figure 2. Schematic representation of microplastic extraction methods: (a) oil-based method, and
density
(b) separation.
density separation.
2.2.2. Density Separation
2.2.2. Density Separation
This method was carried out as described by Konechnaya et al. [34], with some
modifications (Figure 2b). Saturated ZnCl2 solution with a density of 1.6 g cm−3 was
prepared by dissolving 1 kg of ZnCl2 (anhydrous RE, CARLO ERBA Reagents S.A.S.,
Val-de-Reuil, France) in 751.4 mL of dH2 O. The solution was adjusted to a pH of 3 with 5 M
KOH, and then filtered through a 47 mm GF/C filter (Whatman, Maidstone, UK). Then,
10 g of alluvial soil was spiked with either 10 or 20 LDPE or PET MPs, and added to a 50 mL
centrifuge tube. ZnCl2 solution was added to the 50 mL mark in the tube, and the sample
was shaken for 30 s. The separation was carried out by centrifuging the sample at 9000 rpm
for 15 min (Hettich Universal 320 centrifuge, Andreas Hettich GmbH & Co. KG, Tuttlingen,
Germany). The supernatant, together with floated MP particles, was filtered through a
Polymers 2021, 13, 4069 6 of 16

GF/C filter. The filtrate—used ZnCl2 solution—was reused up to 20 times, since its density
did not change. In the case of compost, the procedure was similar to that for alluvial soil,
with slight modifications; the method was optimised using 10 PET MPs, and an oxidation
step was added after extraction to reduce organic matter content. Oxidation was achieved
by submerging the GF/C filter with the sample remaining on the filter in 60 mL of Fenton’s
reagent for 2 h under constant stirring [46]. The oxidised sample was again filtered through
the GF/C filter. Extraction of MPs in each treatment (MP type and concentration; matrix)
was repeated six times. Recovery of MPs was calculated using Equation (1).

2.3. Extraction of a Wide Range of Microplastic Polymers

The more optimal of the two extraction methods (i.e., better recovery, easier to handle,
less time consuming) was tested on MPs of five different polymer compositions—namely,
PE, PP, PS, PVC, and PET (Table 1)—spiked into alluvial soil and compost. MPs were
spiked into 10 g of matrix in different concentrations—namely, 10 MPs (2 particles of each
polymer composition), 25 MPs (5 particles of each composition), and 50 MPs (10 particles of
each composition). The extraction procedure was carried out as described in Section 2.2.2.
Treatment of compost samples included double oxidation—one before extraction, and
another after extraction. Before extraction, the whole sample (10 g of compost with MPs)
was added to 60 mL of Fenton’s reagent and left for 2 h under constant stirring. Afterwards,
the sample was filtered through the GF/C filter, and the sample remaining on the filter was
oxidised again, this time using 50 mL of 1 M NaOH under constant stirring overnight at
50 ◦ C. After the oxidation, the sample was filtered through a 100 µm stainless steel mesh
(Fipis, Ribnica, Slovenia). The experiment was conducted in four replicates. Recoveries
were calculated using Equation (1), and identification of cumulative extracted MPs was
done using the HS-SPME–GC–MS method, as described in Section 2.4.

2.4. Identification of Microplastic Polymers Using Headspace Solid-Phase Microextraction

with GC–MS
MP polymers from alluvial soil and compost samples were identified via the HS-
SPME–GC–MS method described by Šunta et al. [44], with some modifications. The HS-
SPME–GC–MS method is based on the adsorption of volatile compounds emitted during
melting of plastic particles onto SPME fibre, followed by GC–MS analysis and detection
of characteristic fragment ions for individual polymers (PET—m/z 163; PS—m/z 104;
PVC—m/z 91; PP—m/z 142; and PE—m/z 85). Due to observed difficulties in the melting
of PET in polymer mixtures, the method was optimised in the steps of sample preparation
and thermal decomposition. In the sample preparation step, PET MPs were separated from
other MPs and analysed separately. In the thermal decomposition step, headspace vials
were placed in a sand bath with a temperature of 260 ◦ C (probe thermometer, Amarell
GmbH & Co. KG, Kreuzwertheim, Germany) for 15 min and 3 min for melting of PET
MPs and other MP polymers, respectively. With this improved protocol, identification of
MP polymers extracted from spiked samples of alluvial soil and compost was carried out
in cumulative samples composed of three replicates. A linear relationship between the
number of MP particles and the signal response, with the area under the chromatographic
peak being the measure, was determined using the coefficient of determination (R2 ).

3. Results and Discussion

3.1. Comparison of Microplastic Extraction Methods
The two most widely reported methods for the extraction of MPs from soil and soil-like
matrices—oil-based extraction, and density separation—were optimised for MP polymers
of high (PET) and low density (LDPE) and compared. As per the recommendations of
Scopetani et al. [38], olive oil was used for the separation of oleophilic MPs from the matrix
in the oil-based method. Olive oil supposedly has the strongest affinity for a wide range
of plastic polymers [38]. Different solutions to separate MPs from the matrix with the
oil-based method are reported in the literature—from a custom-made PTFE cylinder with a
Polymers 2021, 13, 4069 7 of 16

piston, to separation funnels [27,37,38]. Separation funnels were tested, and were found to
be impractical for the extraction of MPs from soil and compost, due to frequent clogging.
This problem was also encountered by other researchers [36]. Three vessels were tested for
the oil-based method: a PTFE cylinder, similar to the one used by Scopetani et al. [38]; a
glass cylinder; and a modified plastic syringe. The glass cylinder was used as a cheaper
alternative to the PTFE cylinder (i.e., EUR 15 vs. EUR 48 per piece), while the plastic syringe
is a cheap and widely available laboratory consumable. Recoveries obtained in alluvial soil
for all three vessels were high (>97%), and comparable for all spiked MPs (10 and 20 PET
MPs, as well as 10 and 20 LDPE MPs), Table 2. The glass cylinder, however, was found
to break frequently—not in the stages of freezing the sample, but rather when pushing
the sample out after the freezing process. In compost, the recovery obtained for 10 PET
MPs in a modified syringe was lower, at only 80%. It should be noted that in compost, only
marginal conditions were tested—that is, using the lowest quantity of spiked MPs (10 MPs
per 10 g of matrix) as well as the MPs with the highest density (PET), which are usually
more challenging to separate. The recoveries achieved in this study for high-density MPs
in soil were comparable to those obtained by Scopetani et al. [38], who used the extraction
system most similar to ours (98% vs. 95%, respectively); for low-density MPs, however,
the recoveries achieved were higher in this study (98% vs. 90%). In compost, Scopetani
et al. [38] observed lower recovery of low-density MPs (PE and polyurethane (PU)) (80%),
although the recovery of medium- and high-density MPs did not seem to be affected by
the matrix. In this study, only high-density MPs (PET) were tested in the compost matrix,
and their recovery was lower as compared to recovery in soil (80% vs. 98%, respectively).
Other studies reporting on the use of the oil-based method used different solutions for
extraction systems, e.g., separation funnels. Mani et al. [27] used separation funnels and
castor oil, and achieved an average recovery of 99% of four polymers spiked into fluvial
suspended surface solids, marine suspended surface solids, marine beach sediments, and
agricultural soil; the matrix did not significantly influence MP recovery; however, the tested
matrices were low in organic matter content and, therefore, less challenging. Crichton
et al. [37] mixed samples with water and oil in an Erlenmeyer flask, and transferred the
liquid fraction into a separation funnel during the sedimentation period. They achieved an
average recovery of 96% of five polymers spiked into sediment beach samples.

Table 2. Comparison of recoveries of PET and LDPE microplastic particles, achieved in two extraction methods: oil-based
extraction in three different vessels, and density separation. n = 6; results are presented as mean ± standard deviation (SD).

Oil-Based Method (%)

No. and Type of Density
Type of Soil PTFE Glass Plastic
MPs Separation (%)
Cylinder Cylinder Syringe
10 PET 96.7 ± 8.2 100.0 ± 0.0 96.7 ± 5.2 93.3 ± 5.2
20 PET 97.5 ± 2.7 97.5 ± 2.7 97.5 ± 4.2 99.2 ± 2.0
Alluvial soil
10 LDPE 98.3 ± 4.1 98.3 ± 4.1 98.3 ± 4.1 98.3 ± 7.5
20 LDPE 96.7 ± 8.2 96.7 ± 4.1 97.5 ± 2.7 100.0 ± 0.0
Compost 10 PET N.A. 1 N.A. 1 80.0 ± 11.0 98.3 ± 4.1
1 Not applicable.

The oil-based method did not prove to be straightforward in this experiment. MPs
were often found to be stuck to the inner walls of vessels, as well as to the surface of the
frozen samples. This was due to the formation of a thin layer of oil on the walls of the
vessels, which the MPs were attracted to. To combat this, the inner walls were rinsed
with hexane; after the sample was removed, the surface of the frozen sample was scraped,
and these fractions were further processed to isolate MPs. This method was also time-
consuming (2 h for settling, overnight for freezing, and up to 45 min for melting the cut-off
sample containing MPs for further processing, e.g., filtration).
The second method tested was density separation with saturated ZnCl2 solution. This
method followed previously published protocols, with further optimisation [32–34]. Sedi-
Polymers 2021, 13, 4069 8 of 16

mentation (usually overnight) was sped up with centrifugation (15 min). This significantly
improved time efficiency, and improved decanting of the supernatant due to more compact
sediment. Recoveries obtained in alluvial soil and compost were high (>93%, most >98%,
respectively), and were comparable for all spiked MPs (different compositions and quanti-
ties) (Table 2). Additionally, the ZnCl2 solution was reused up to 20 times without losing
the desired density, which significantly reduced the cost and the environmental footprint of
the method. Density separation achieved better recoveries in compost as compared to the
oil-based method (98% vs. 80%), with an oxidation step after filtration in both methods (see
Sections 2.2.1 and 2.2.2). For this method, oxidation with Fenton’s reagent before extraction
was also tested, and the recovery obtained was 100% (n = 6). In the study by Konechnaya
et al. [34], the authors used ZnCl2 solution and separated MPs from sandy matrices by
overflow, and the obtained recoveries were comparable to those obtained in this study in
alluvial soil (between 94 and 104% vs. 98%, respectively). Very recently, Grause et al. [35]
used a similar approach to the one used in this study—density separation sped up via
centrifugation using CaCl2 solution to extract MPs from agricultural soil. They optimised
the centrifugation protocol on PET MPs spiked into soil, and obtained 95% recoveries,
which is consistent with the results of our study; their method, however, was not tested
on samples rich in organic matter; likewise, CaCl2 solution with a density of 1.4 g cm−3
could be unsuitable for the extraction of some MP polymers, e.g., some PET, chlorinated
polyvinyl chloride (CPVC), polyvinylidene chloride (PVDC), etc. [35].
In addition to better recoveries in compost, the significant advantage of the density
separation method over the oil-based method was in better time efficiency (approximately
30 min to obtain MPs on the filter vs. approximately 1 day, respectively), as well as in
the ease of handling. The potential for scaling up—e.g., analysing multiple-kilogram
samples—could be feasible with the use of a large-volume centrifuge. For this reason,
density separation was further used in testing the method on a wide range of plastic
polymers at various concentrations, as described in Section 3.2.

3.2. Extraction of a Wide Range of Microplastic Polymers

The extraction method that proved to be more optimal—density separation—was
used for the extraction of MP particles of different polymer compositions (Table 1) spiked
into alluvial soil and compost, at various concentrations. The five chosen MP poly-
mers (PE, PP, PET, PVC, and PS) were chosen based on their frequent occurrence in the
environment [15,17,47]. Cumulative recoveries were high (>93%) for all spiked MP poly-
mers, especially at spiking quantities of 25 and 50 MPs (>98%) per 10 g of matrix (Figure 3c).
Moreover, there were no significant differences in the recoveries of individual MP polymers
from soil (Figure 3a) and compost (Figure 3b), nor were there any significant differences in
recoveries from the two matrices. Blank samples of non-spiked alluvial soil were treated
in the same way as the spiked samples to check for potential cross-contamination from
aerial deposition, clothing, and/or lab equipment. Blank alluvial soil resulted in zero
recovered MPs (n = 3). Blank compost samples were also treated in the same way; however,
compost originating from the industrially composted organic fraction of household waste
inherently contains plastic contamination from plastic bags and other sources [17]. A total
of 32 ± 2 native MPs (n = 3) were recovered from the compost blank sample; however, their
authenticity was not additionally checked with identification methods or the hot needle
test. Native MPs did not hinder the determination of recovery of spiked MPs, since the
spiked MPs were easily visually distinguishable. This method was additionally tested by
inexperienced operators—primary school pupils, 14 years of age, with no prior laboratory
experience; the recovery achieved ranged from 75% to 95%, which shows that the method
is simple for handling.
 and other sources [17]. A total of 32 ± 2 native MPs (n = 3) were recovered from the
compost blank sample; however, their authenticity was not additionally checked with
identification methods or the hot needle test. Native MPs did not hinder the determina-
tion of recovery of spiked MPs, since the spiked MPs were easily visually distinguishable.
This method was additionally tested by inexperienced operators—primary school pupils,
Polymers 2021, 13, 4069 9 of 16
14 years of age, with no prior laboratory experience; the recovery achieved ranged from
75% to 95%, which shows that the method is simple for handling.

Figure
Figure 3.
3. Recovery
Recovery of of MPs
MPs (polymer
(polymer types:
types: PE,
PE,PP,
PP, PET,
PET, PVC, and PS) spiked into alluvial soil and
compost,
compost, extracted via the
extracted via thedensity
densityseparation
separationmethod.
method.Recovery
Recoveryofofindividual
individual polymer
polymer types
types from
from (a)
(a) alluvial
alluvial soilsoil
andand (b) compost,
(b) compost, spiked
spiked at a concentration
at a concentration of 5ofMPs
of 5 MPs eachofpolymer
each polymer
type. (c)type. (c) Cu-
Cumulative
mulative
recovery ofrecovery
all MPs,ofspiked
all MPs, spiked at
at different different concentrations.
concentrations. n = 4; resultsnare
= 4; results are
presented presented
as mean ± SD.as
mean ± SD.
There are numerous reports on using density separation to extract various MPs
fromThere
river are
andnumerous reportshowever,
sea sediments; on using fewer
densitystudies
separation
havetodealt
extract
with various MPs from
extraction from
river and seasoil
agricultural sediments; however,
and organic-rich fewer studies
matrices, such as have dealtwhich
compost, with areextraction from more
considerably agri-
cultural soilprocess.
difficult to and organic-rich
Hurley et al.matrices,
[46] testedsuch as compost,
a density which
separation are considerably
protocol more
with NaI solution
difficult to process.oxidation
and an additional Hurley etstepal. [46] tested
on soil andasludge
densitysamples
separation protocol
spiked with PE with NaI solu-
microbeads
tion and an additional oxidation step on soil and sludge
and PET fibres. Recoveries obtained in the reported study ranged between 92% samples spiked with PE and
mi-
crobeads and PET fibres. Recoveries obtained in the reported study
100% for PE microbeads, and between 79% and 86% for PET fibres. The recoveries of PE ranged between 92%
and 100% forresembled
microbeads PE microbeads, and between
the recoveries obtained79%inandthis86%
studyfor(>92%
PET fibres.
vs. >93%,The recoveries
respectively);of
PE microbeads
however, resembled
PET fibres were the recoveries
recovered to a obtained
lesser extentin this
thanstudy (> 92%
the PET vs. > 93%,
particles usedrespec-
in this
tively); however,
study (>79% PET fibres
vs. 100%, were recovered
respectively). Hurley toeta lesser
al. alsoextent
testedthan
the the PET
effect of particles used
an additional
in this study
oxidation step(> on
79% vs. 100%, and
recoveries, respectively).
found thatHurley
oxidationet al.with
alsoFenton’s
tested the effect of
reagent an MP
after ad-
ditional oxidation step on recoveries, and found that oxidation with
extraction led to lower recoveries than oxidation before MP extraction, but this result Fenton’s reagent af-
ter
wasMP notextraction
statisticallyledsignificant.
to lower recoveries than oxidation
No such effect was observed beforein MP
this extraction, but this
study; instead, we
result wasanot
observed statistically
difference in the significant.
quantity of Noorganic
such effect
matterwas observed
removed in this4).study;
(Figure Liu etinstead,
al. [31]
we observed
devised a difference
a circulation in the device
separation quantity andoftested
organic matter
various removed
saline (Figure
solutions 4). Liua et
to extract al.
wide
range
[31] of MPapolymers
devised circulation (polyamide
separation(PA), polycarbonate
device (PC), PP,
and tested various acrylonitrile
saline solutions butadiene
to extract
astyrene
wide (ABS),
range of PE,MPPS, poly(methyl methacrylate)
polymers (polyamide (PA),(PMMA), polyoxymethylene
polycarbonate (POM), PET,
(PC), PP, acrylonitrile
and PVC) from soil; with the use of NaBr and CaCl2 , they achieved recoveries from 95%
to 100%, similar to the recoveries reported herein. Similarly, Li et al. [48] constructed a
circulation system for the extraction of MPs from soil via density separation using NaBr
solution; the system was tested on common MP polymers (LDPE, PS, PP, and PVC) as
well as biodegradable MPs (polybutylene succinate (PBS), poly(adipic acid), butylene
terephthalate (PBAT), and polylactic acid (PLA)); recovery rates ranged from 92% to 99.6%,
resembling the recoveries reported herein. Grause et al. [35], using density separation with
centrifugation and CaCl2 solution, achieved recoveries between 97% and 98% for LDPE,
PP, PS, and flexible PVC MPs, comparable to the recoveries obtained in this study; PET
had lower recoveries as compared to the other tested MPs, at around 95%, whereas in this
study no differences in recoveries of the five tested MP polymers were observed.
 oxidation reduced organic matter content to the largest extent (Figure 4), and was there-
fore used in processing all compost samples used in the recovery experiments. Hurley et
al. [46] tested the impacts of different oxidation protocols on MP integrity; Fenton’s rea-
gent proved to reduce organic matter to the highest degree (106%) without damaging the
Polymers 2021, 13, 4069
MPs; alkaline digestion with 1 M NaOH, on the other hand, provided a lesser reduction 10 of 16
in organic matter (68%), but it also did not cause significant changes in the mass or size of
MP particles [46].

Reduction in organic content in compost samples with oxidation

Figure 4. Reduction
Figure oxidation at various stages of the
MP
MP extraction
extraction procedure.
procedure. The
The initial amount of all samples was 10 g of compost.

An oxidation
3.3. Identification step was employed
of Microplastic forCompost
Polymers in all compost samples
and Alluvial Soilin order to reduce the
organic
Identification of MPs extracted from spiked alluvial soil and compostOxidation
matter content and facilitate the identification of MP polymers. was
samples using
implemented at various stages of the MP extraction process to compare
density separation with ZnCl2 solution was possible via the HS-SPME–GC–MS method. the efficiency of
organic matter reduction. Oxidation with Fenton’s reagent after MP extraction reduced
As described in Section 2.4., the proposed method by Šunta et al. [44] was optimised for
the organic matter content to a lesser extent compared to oxidation with Fenton’s reagent
improved identification of PET. To improve the melting of PET, a thermal decomposition
before MP extraction, as shown in Figure 4. Additionally, double oxidation was trialled—
step was carried out in a sand bath, and PET particles were analysed separately from
with Fenton’s reagent before extraction, and 1 M NaOH after extraction. Double oxidation
other MP polymers. PS, PVC, and PP in alluvial soil and compost samples were identified
reduced organic matter content to the largest extent (Figure 4), and was therefore used in
processing all compost samples used in the recovery experiments. Hurley et al. [46] tested
the impacts of different oxidation protocols on MP integrity; Fenton’s reagent proved to
reduce organic matter to the highest degree (106%) without damaging the MPs; alkaline
digestion with 1 M NaOH, on the other hand, provided a lesser reduction in organic matter
(68%), but it also did not cause significant changes in the mass or size of MP particles [46].

3.3. Identification of Microplastic Polymers in Compost and Alluvial Soil

Identification of MPs extracted from spiked alluvial soil and compost samples using
density separation with ZnCl2 solution was possible via the HS-SPME–GC–MS method.
As described in Section 2.4, the proposed method by Šunta et al. [44] was optimised for
improved identification of PET. To improve the melting of PET, a thermal decomposition
step was carried out in a sand bath, and PET particles were analysed separately from other
MP polymers. PS, PVC, and PP in alluvial soil and compost samples were identified with
previously proposed characteristic compounds for the identification of MPs after their
thermal decomposition: styrene and dimer, chlorooctane, and 4,6-dimethyl 2-heptanone,
respectively [39,41,44,49]. In the chromatogram of analysed MP mixtures (PS, PVC, PP, and
PE), pentadecane—a characteristic compound proposed for the identification of PE—was
not observed; however, the series of higher alkanes (from tetradecane, C14 , up to eicosane,
C20 ) was observed, and the presence of PE was confirmed. PET extracted from compost
samples was identified using the previously proposed characteristic compound—dimethyl
terephthalate (DMTP) [41,44,49]—while DMTP from analysed PET particles extracted from
alluvial soil was not determined, and ethyl methyl terephthalate (signal 1a, Figure 5) at a
retention time of 16.994 min was used instead. Ethyl methyl terephthalate was chosen in
the case of alluvial soil samples because it is one of the terephthalate derivatives that are
produced upon thermal decomposition of PET, according to Yakovenko et al. [50].
 In the case of alluvial soil samples, a linear relationship between the number of par-
ticles and the signal response with R2 > 0.97 was observed for ethyl methyl terephthalate
(PET), styrene and the dimer — trans(cis)-1,2-diphenylcyclobutane (PS), chlorooctane
(PVC), and eicosane (PE) (Figure 5). For 4,6-dimethyl 2-heptanone (PP), the linear coeffi-
Polymers 2021, 13, 4069 cient was very high (R2 = 0.9997); nevertheless, the signal response (signal 6a in Figure
11 of 16
5)—or area under the chromatographic peak—was not increasing with the number of
MPs, as would be expected, but decreasing.

Headspace solid-phase
Figure 5. Headspace solid-phase microextraction–gas
microextraction–gas chromatography–mass
chromatography–mass spectrometry
spectrometry (HS-SPME–GC–MS)
(HS-SPME–GC–MS) chro- chro-
matograms
matograms of of identification
identification compounds
compounds from
from MP
MP particles
particles extracted
extracted from
from alluvial
alluvial soil
soil samples,
samples, spiked
spiked with
with three
three con-
con-
centrations (2 MPs, 5 MPs, and 10 MPs) of each polymer type (PET: dimethyl terephthalate (1a); PS: styrene
centrations (2 MPs, 5 MPs, and 10 MPs) of each polymer type (PET: dimethyl terephthalate (1a); PS: styrene (2a) and (2a) and di-
mer trans(cis)-1,2-diphenylcyclobutane (3a and 4a); PVC: chlorooctane (5a); PP: 4,6-dimethyl 2-heptanone (6a);
dimer trans(cis)-1,2-diphenylcyclobutane (3a and 4a); PVC: chlorooctane (5a); PP: 4,6-dimethyl 2-heptanone (6a); and PE: and PE:
eicosane
eicosane (7a)).
(7a)).

After optimisation of the HS-SPME–GC–MS method for the identification and analysis
of spiked samples of soil with different MP contents, the potential of this method for the
descriptive quantification of MPs was observed. In a detailed analysis of chromatographic
peaks of characteristic compounds for the identification of MPs, a relationship between
the number of analysed particles and the signal response (determined area under the
Polymers 2021, 13, x FOR PEER REVIEW 12 of 16
chromatographic peak) was observed (Figures 5 and 6). Cumulative samples of three
replicates were analysed (6, 15, and 30 MPs).

Figure
Figure 6. Headspace
Headspace solid-phase
solid-phase microextraction–gas
microextraction–gas chromatography–mass
chromatography–mass spectrometry
spectrometry (HS-SPME–GC–MS)
(HS-SPME–GC–MS) chro- chro-
matograms
matograms of of identification
identification compounds
compoundsfromfromMPMP particles
particles extracted
extracted from
from compost
compost samples,
samples,spiked
spikedwith
withthree
three concen-
concen-
trations
trations (2
(2 MPs,
MPs, 55 MPs,
MPs, and
and 10
10 MPs)
MPs) of
of each
each polymer
polymer type
type (PET:
(PET: ethyl
ethyl methyl
methyl terephthalate
terephthalate (1b);
(1b); PS:
PS: styrene
styrene (2b)
(2b) and
and
dimer
dimer trans(cis)-1,2-diphenylcyclobutane
trans(cis)-1,2-diphenylcyclobutane (3b(3b and
and 4b);
4b); PVC:
PVC: chlorooctane
chlorooctane (5b);
(5b); PP:
PP: 4,6-dimethyl
4,6-dimethyl 2-heptanone
2-heptanone(6b);
(6b);and
andPE:
PE:
eicosane (7b)).
eicosane (7b)).

A high linear relationship (R2 > 0.99) between the signals of characteristic com-
pounds of individual polymer types of MPs extracted from compost samples on the one
hand, and the number of MPs on the other, was also observed in the case of DMTP (PET),
trans(cis)-1,2-diphenylcyclobutane (PS), chlorooctane (PVC), and eicosane (PE) (Figure
6). There was a low linear relationship observed for styrene (PS) with R2 = 0.8034, and
Polymers 2021, 13, 4069 12 of 16

In the case of alluvial soil samples, a linear relationship between the number of
particles and the signal response with R2 > 0.97 was observed for ethyl methyl terephthalate
(PET), styrene and the dimer—trans(cis)-1,2-diphenylcyclobutane (PS), chlorooctane (PVC),
and eicosane (PE) (Figure 5). For 4,6-dimethyl 2-heptanone (PP), the linear coefficient was
very high (R2 = 0.9997); nevertheless, the signal response (signal 6a in Figure 5)—or area
under the chromatographic peak—was not increasing with the number of MPs, as would
be expected, but decreasing.
A high linear relationship (R2 > 0.99) between the signals of characteristic compounds
of individual polymer types of MPs extracted from compost samples on the one hand, and
the number of MPs on the other, was also observed in the case of DMTP (PET), trans(cis)-
1,2-diphenylcyclobutane (PS), chlorooctane (PVC), and eicosane (PE) (Figure 6). There
was a low linear relationship observed for styrene (PS) with R2 = 0.8034, and none for
4,6-dimethyl 2-heptanone (PP).
Observed linearity (above R2 > 0.98 in alluvial soil, and above R2 > 0.80 in compost)
in a controlled laboratory environment with established particle size, number, average
particle mass, and polymer type demonstrated that the HS-SPME–GC–MS method could
be used for tentative descriptive quantification of PET, PS, PVC, and PE in analysed
matrices. However, it is a prerequisite that samples are prepared for the identification of
MPs using pretreatment methods for the elimination of organic matter and extraction of MP
polymers from the matrix. Quantitative measurements with the SPME headspace extraction
technique depend on the type of SPME coating, the partitioning coefficient between the
fibre coating and the analytes, the chemical properties of the analytes, the complexity of
the sample, and temperature, while particle size, mass, and chemical structure (additives)
also need to be considered in the identification of MPs [51,52].
The nonlinearity of 4,6-dimethyl 2-heptanone (PP) could be attributed to the possible
interactions in the headspace vial between volatile substances emitted from MP polymers
during the thermal decomposition step of the identification method. As the best signal
response can be observed at the lowest number of analysed PP particles, it is also possible
that competition for the active adsorption sites on the SPME fibre occurred between
4,6-dimethyl 2-heptanone and other volatile substances, as was also observed by Dutra
et al. [53] and Demets et al. [54]. Therefore, future studies on the quantification of MPs in
environmental matrices using HS-SPME–GC–MS would be of interest.

3.4. Applicability of the Developed Methods to Environmental Samples

Density separation was applied to real compost samples from municipal organic waste
to extract natively present MPs. The procedure was carried out as described in Section 2.3.
From 10 g of compost, 36 ± 9 MPs were extracted, with an average mass of 6.3 ± 1.8 mg
(n = 3). Extracted particles were placed in vials and identified via HS-SPME–GC–MS
as described in Section 2.4. The presence of two polymers was determined: PS and PP,
the latter being tentatively identified using methylated higher alkanes in the absence of
the characteristic compound for PP, i.e., 4,6-dimethyl 2-heptanone. It should be noted,
however, that the identification method has currently only been developed for the five
aforementioned plastic polymers (PET, PVC, PE, PS, and PE), while the determination of
characteristic compounds for other plastics is underway. Hence, the detection of other
potentially present MP polymers was not possible.
Biowaste compost is frequently contaminated with MPs, originating from incompletely
removed plastic bags, plastic packaging of food, incompletely degraded biodegradable
plastics, and other sources [16]. A study quantifying MPs in organic fertilisers found
between 20 and 24 MPs (>1 mm) per kg of compost [17], which is 150–180 times less than
the amount isolated in this study; the employed method for MP extraction, however, was
different (wet sieving), which could, in part, explain the differences in MP numbers. Other
studies reported up to 2800 MPs per kg of compost, which more closely aligns with the
abundance reported herein (2900–4600 MPs per kg) [55].
Polymers 2021, 13, 4069 13 of 16

The strengths of the methods reported in this study include high MP recoveries from
soil and compost via the density separation method, as well as the methods being easy to
handle and widely accessible. The identification method has the advantage of simultaneous
multi-polymer analysis and the use of common analytical equipment. There are, however,
some drawbacks: Neither method was optimised for large sample processing (e.g., >1 kg).
Additionally, the HS-SPME–GC–MS method is not yet developed for the identification
of other plastic polymers, e.g., PA and biodegradable plastic polymers. Furthermore,
the presence of organic matter in the MP isolate hinders polymer identification, as the
limit of detection (LOD) rises with increasing organic matter content; therefore, efforts to
significantly reduce organic matter should be made prior to sample analysis.

4. Conclusions
Two methods for the extraction of MP particles were tested and optimised: oil-based
extraction, and density separation. Both methods achieved high recoveries of spiked MP
polymers of low and high density in alluvial soil; however, in compost, density separation
with ZnCl2 solution proved to have better recoveries. In addition, considering other
factors—such as time efficiency, ease of handling, and the potential for scaling up—density
separation was shown to be the better option. Additionally, oxidation before and after
extraction was necessary in order to considerably reduce the organic matter content of
compost samples. Identification of MPs via the HS-SPME–GC–MS method was carried
out successfully via the detection of characteristic compounds for each polymer type.
The potential to use this method for descriptive quantification was proven by a linear
relationship between the number of particles and the signal response on chromatograms
in the case of PET, PS, PVC, and PE. Density separation was employed to isolate natively
present MPs from municipal biowaste compost (36 ± 9 MPs per 10 g). The presence of PS
and PP was confirmed with HS-SPME–GC–MS.
This study contributes towards the understanding of the suitability of available ex-
traction methods for soil and compost samples, and additionally presents the possibility of
using commonly available analytical equipment for MP identification.

Author Contributions: Conceptualisation, F.P. and M.B.K.; methodology, U.Š.; formal analysis, P.L.;
investigation, P.L.; resources, U.Š. and M.B.K.; data curation, P.L.; writing—original draft preparation,
F.P., P.L., U.Š., and M.B.K.; writing—review and editing, F.P., U.Š., and M.B.K.; visualisation, F.P.,
P.L., and U.Š.; supervision, F.P. and M.B.K.; project administration, F.P.; funding acquisition, F.P. and
M.B.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Slovenian Research Agency, grant number Z2-2643
(Agri-iMPact: Microplastic Prevalence and Impact in Agricultural Fields), grant number SP-0510/21
(Postgraduate Research Funding Programme: Young Researchers), and research core funding number
P3-0388 (Mechanisms of Health Maintenance). The APC was funded by the Slovenian Research
Agency, grant number Z2-2643.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the Zenodo digital
repository at https://doi.org/10.5281/zenodo.5607477 (accessed on 21 November 2021).
Acknowledgments: The authors acknowledge the Research Nature Institute, Ljubljana, Slovenia for
analytical support.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or
in the decision to publish the results.
Polymers 2021, 13, 4069 14 of 16

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