Trends in Analytical Chemistry 110 (2019) 150e159

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Methods for sampling and detection of microplastics in water and

sediment: A critical review
Joana Correia Prata*, Joa
~o P. da Costa, Armando C. Duarte, Teresa Rocha-Santos
Centre for Environmental and Marine Studies (CESAM), Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Microplastics are widespread contaminants, virtually present in all environmental compartments.
Available online 9 November 2018 However, knowledge on sources, fate and environmental concentration over time and space still is
limited due to the laborious and varied analytical procedures currently used. In this work we critically
Keywords: review the methods currently used for sampling and detection of microplastics, identifying flaws in
Microplastics study design and suggesting promising alternatives. This work provides insights on bulk sample
Sampling of microplastics
collection, separation, digestion, identification and quantification, and mitigation of cross-contamination.
Analytical procedures
The sampling of microplastics will improve in representativeness and reproducibility through the
FTIR
Raman spectroscopy
determination of bulk sample volume, filter's pore size, density separation and digestion solutions, but
Staining dyes also through use of novel methods, such as the enhancement of visual identification by staining dyes, and
the generalized use of chemical characterization.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction methods of sampling microplastics from sediment and water,

providing criticism and perspectives of future developments. A
Microplastics, small plastic pieces <5 mm intentionally pro- search using the terms “sampling/extraction/determination/
duced to be used in consumer products (e.g. as exfoliants in methods þ microplastics” was performed on Web of Science in May
cosmetics) and in activities as abrasives (e.g. air blasting) (pri- 2018, selecting 49 works only directly related to the development
mary microplastics) or resulting from the fragmentation of larger of sampling procedures for microplastics. Google Scholar was also
objects (secondary microplastics), are highly persistent con- used to gather data on the methods used in sampling studies in
taminants potentially harmful to organisms or ecosystems [14]. water (N ¼ 20) [3,13,17,24,35,39,40,42,47,62,65,69,76,90e92,
From the Artic [20] to the Antarctic seawater [13], to sediments 102,103,108,112,113] and Maes et al. 2017b [67] sediment (N ¼ 20)
[8,34], rivers [69,71,83], soil [110] and even the air we breathe [5,7,12,23,41,46,55,60,61,67,68,76,77,91,96,98,100,102,106,115], and
[29], microplastics are currently present in all environmental summarized on Fig. 1 through the use of Microsoft Excel 2016. In-
matrixes. Concerns over these particles have led to a growing formation has been organized into sampling steps: bulk sample
literature attempting to quantify microplastics in the environ- collection, separation, digestion, and identification. At last, we
ment and their effects on organisms [4]. However, the lack of suggest five measures to reduce cross-contamination due to its
universal and validated methods led to a wide range of analytical importance in the validation of results.
approaches, compromising a large-scale interpretation of current
results. 2. Collection of water and sediment samples
Currently, research groups may struggle with selecting methods
for sampling microplastics due to the large range of available op- Collection of water and sediment samples is the first step of
tions. In this review, we attempt to summarize the most relevant microplastics sampling methodologies (Table 1). The choice be-
tween sampled medium is dependent on available equipment but
also the objective of the work. For instance, sampling the water
column may be the most adequate medium if the objective is to
* Corresponding author.
determine the exposure of pelagic organisms. However, micro-
E-mail addresses: [email protected] (J.C. Prata), [email protected] (J.P. da Costa), plastic distribution is largely influenced by meteorological, tem-
[email protected] (A.C. Duarte), [email protected] (T. Rocha-Santos). poral and geographical factors that may compromise

https://doi.org/10.1016/j.trac.2018.10.029
0165-9936/© 2018 Elsevier B.V. All rights reserved.
 J.C. Prata et al. / Trends in Analytical Chemistry 110 (2019) 150e159 151

Collection Density separation Digestion Identification

Dry weight Wet weight NaCl NaI ZnCl2 Water H2O2 (30%) Fenton's reagent No Visual FTIR Raman GC-M S
Sediment

25%
35%
45%
20%
55% 60%
65%
60%

Nets Pumps Sieves NaCl ZnCl2 Seawater No H2O2 (30%) Enzymes HCl No Visual FTIR Raman SEM Dyes

16% 15%
25%
Water

40%
26% 58% 10%
60%
75% 40%

Fig. 1. Details from sampling methods reviewed from the literature for microplastics in sediment (top row, N ¼ 20) and water (bottom row, N ¼ 20) regarding collection, density
separation, digestion, and identification.

Table 1
Methods of sample collection in water and sediment.

Sample Type Advantages Disadvantage

Water Neuston and Manta nets Easy to use; Expensive equipment;

Sample large volumes of water; Requires boat;
Largely used (good to compare between locations); Time-consuming;
Produces large numbers of microplastics for further testing. Potential contamination by vessel and tow ropes;
Lower limit of detection is 333 mm.
Plankton net Easy to use; Expensive equipment;
Lowest limit of detection 100 mm; Requires boat;
Quick to use; Static sampling requires water flow;
Samples medium volumes of water. May become clogged or break;
Sampling of lower volumes of water than Manta trawl.
Sieving Does not require specialized equipment nor boat; Laborious and time consuming;
Easy to collect samples. Samples medium volumes;
Manual transfer of water with buckets
Pumps Samples large volumes of water; Requires equipment;
Effortless; Requires energy to work;
Allows choice of mesh size. Potential contamination by the apparatus;
May be difficult to carry between sampling locations.
Filtration or Sieving Easy to collect samples; Sampling of low volumes;
ex situ Known volume of water; Transportation of water samples to the lab;
Allows choice of mesh size. Potential contamination by the apparatus;
Time consuming depending on mesh size.
Sediment Beach sediment collection Easy to implement; Variation with sampled area and depth.
Rapid sampling;
Allows collection of large volumes of sample or replicates.
Seabed collection (Grab sampler, Easy to use; Expensive equipment;
box corer, gravity core) May allow replicates. Requires boat;
Variation with sampled area and depth;
Sampling may disturb sediment surface.

reproducibility of the results. On the other hand, methodology available, expressing their results in both total microplastics, but
and quantity of sampled material may influence representative- also in classes, which should be standardized. A uniform classi-
ness of results. Results are usually expressed as total microplastics fication of what is considered a microplastics should also be
per unit of sample (e.g. L1, in water), sometimes providing proposed, clarifying the inclusion (or not) of pieces such as fibers
detailed classifications of size classes, color and shape (e.g. fiber, and polymeric rubbers. For these reasons, most current research
particle, fragment). We suggest authors provide all the data on environmental concentrations of microplastics represent a
152 J.C. Prata et al. / Trends in Analytical Chemistry 110 (2019) 150e159

snapshot in time and space, limiting interpretation between Based on the 20 studies on water sampling, we found that the
studies. most used are nets (11), followed by pumps (5) and sieves (3)
(Fig. 1). Only one study used bottles and buckets to collect water
2.1. Water sampling samples. Only 8 of these studies stated the approximate volume of
sampling (10e2000 L). This information should always be present
Microplastics are distributed in the water column dependent on since it may determine the representativeness of the results. NOAA
their properties, such as density, shape, size, adsorption of chem- recommends the use of manta nets followed by sieving (0.3 mm)
icals and biofouling, and on environmental conditions such as and filtration (0.3 mm) [70].
water density, wind, currents and waves. Thus, quantity and quality
of microplastics recovered are highly dependent on sampling 2.2. Sediment sampling
location and depth. Sampling and processing methods are similar
for both fresh and saltwater samples, enabling a future standardi- The distribution of microplastics on sediments is uneven, largely
zation of methods. However, differences can be found in the dis- influenced by their properties and environmental factors, such as
tribution of microplastics in each system, influenced by winds and currents. Results will be largely dependent on the
environmental characteristics, such as hydrodynamic profiles, as sampling area (e.g. high tide line, intertidal areas, transects) and
well as density. The differences in density of fresh and saltwater, depth since some areas may contain higher concentrations of
respectively 1.00 g cm3 and 1.03 g cm3, may lead to distinct microplastics. For instance, collection of sediments on the tide-line,
distribution of microplastics in the water column in each system the high accumulation area for microplastics, may result in over-
(i.e. generally, microplastics will be deeper in the water column in estimation [38]. Collection of microplastics on beaches include
freshwater systems). Thus, depth and location may need to be direct sampling with forceps, sieving and collection of sediment
adjusted depending on the sampling location and salinity. Repre- samples. Collection of samples from the seabed requires a vessel
sentativeness may be dependent on the sampling of large volumes and the use of specialized equipment that is lowered to the seabed
of water, often achieved by reduction in situ through the use of nets, to collect the samples (e.g. grab sampler, box corer). An accurate
sieves or pumps. Towing of neuston nets or manta nets (333 mm) estimation of microplastic concentration in sediment samples may
allows the sampling of near-surface or surface water, respectively. require definition of sampling depth, since the top 1e5 cm presents
Bongo nets are paired nets used to gather replicate samples from higher concentrations than the top 10 cm, and number of replicates,
the water column. Plankton nets are usually hauled or towed at low since 11 samples are recommended per each 100 m of beach to
velocities since the small mesh size (~100 mm) can quickly become estimate microplastic concentration at a 90% confidence level [6].
clogged. Besides horizontal towing or hauling, these nets allow Sample weight (25e3000 g) or volume (0.05e1.2 L) largely varies
vertical or obliquely sampling. A flow meter should be attached to between studies, potentially affecting representativeness. Even
all these nets to allow estimation of water volume sampled and the though only 9 of 20 studies reported sediment concentrations in g,
expression of results by m3. An alternative to nets includes water kg or L of dry weight (Fig. 1), this method is recommended to
pumps that can be comprised by intakes from the vessel, deck eliminate variations related to humidity. NOAA recommends the
pumps or even used at coastal areas. use of 400 g (w.w.) per replicate, followed by drying and weighing
Mesh size may largely influence concentrations reported. For to adjust the results [70], while the MSFD technical subgroup [73]
instance, a nylon net (100 mm) revealed concentrations almost a recommends using at least five replicates of the top 5 cm of
hundred times higher than a manta net (333 mm), 0.1 and sediment.
0.00135 MP L1 respectively (Vermaire et al., 2016) [99]. None-
theless, manta nets allow the sampling of large volumes of water 3. Separation of microplastics from samples
and are widely used allowing some standardization of methods.
Plankton nets also have smaller mesh sizes (~100 mm), allowing Microplastics must be separated from water and sediment
sampling under a minute and recovering concentrations 30 times samples in order to be quantified and characterized. Samples may
higher than manta nets [28]. However, plankton nets must be be subjected to two separation steps: (1) a reduction step that al-
deployed for a short period of time due to clogging with organic lows to reduce sample volume, for instance, through the use of nets
and mineral material in suspension, limiting the volume of water during collection or bulk collection followed by sieving; (2) a sep-
sampled. Regarding fibers, an 80 mm mesh is able to filter 250 times aration step usually through filtration and/or density separation.
more fibers than a 330 mm net [30]. An alternative is to use a pump Density separation by the use of NaCl is recommended by both
to collect water on a vessel [24] or on shore [49]. For instance Lenz MSFD technical subgroup (2013) [73] and NOAA [70].
and Labrenz [59] have developed a large portable filtration device
based on pumps. On shore filtration or sieving using buckets is 3.1. Filtration or sieving
possible, but time consuming and demanding. Water samples can
also be collected in glass bottles and processed in the lab. Dubaish Filtering or sieving is the most frequent method in separation of
and Liebezeit [25] collected 100 mL of surface water, reporting high microplastics from water samples and for the supernatant con-
variability of the method. Thus, collection of higher volumes of taining plastics from density separation of sediment samples. Fil-
water may be required to achieve representativeness. Reduction of ter's pore size or sieve's mesh can vary greatly. Pore or mesh size
sample size can also be achieved in the lab by a pre-treatment using determine the lower size of microplastics detected. However, small
steel meshes of decreasing sizes [114]. Regarding contamination, pore or mesh sizes may also result in quick obstruction by organic
nylon nets and pumping systems may be a source of microplastic and mineral matter. In sediments, samples may be subjected to a
contamination whereas metal sieves and glass bottles avoid the use pre-treatment such as larger sieves (e.g. 4 works used sieving as a
of plastic materials. However, these plastic free materials are usu- pre-treatment to reduce sample volume), followed by density
ally associated with processing limited volumes of water. Thus, it separation and filtration of the supernatant through filters (16) or
may lead to a difficult choice between representativeness and sieves (4). In the reviewed works, pore or mesh size varies between
avoiding potential contamination. Defining a minimum volume of 0.3 and 200 mm, with 3 works lacking this information. In water, the
sample to achieve representativeness could facilitate this decision reduction step usually is related to sample collection (e.g. manta
and alleviate sampling efforts. nets) followed by filtration (16) or sieving (4) with pore or mesh
 J.C. Prata et al. / Trends in Analytical Chemistry 110 (2019) 150e159 153

sizes varying from 0.45 to 55500 mm, with 3 works not expressing been used with ZnCl2 (1.5 g cm3) with an efficiency of 95.8% [18],
this information (Fig. 1). Thus, a standardized pore or mesh size and could be used with other salt saturated solutions such as NaI. In
should be defined to allow comparison between works, even the reviewed works, all sediments were subjected to density sepa-
though this is sometimes dependent on protocol constrains (which ration, using NaCl (13), ZnCl2 (4), NaI (2) and deionized water (1),
could be overcome by creating a universal sampling protocol), and while only 5 works on water samples used density separation using
this information should always be clearly expressed. NaCl (3), ZnCl2 (1), and seawater (1) (Fig. 1).
In elutriation, usually a liquid such as water is injected at the
bottom of a column, allowing the separation of buoyant micro-
3.2. Density separation: flotation and elutriation
plastics from the settling organic matter and sediment [53]. Micro-
plastics are collected in a mesh in the column and then separated
Differences in density can be used to separate plastics
using dense solutions (e.g. NaI) [11,54]. The advantage of elutriation
(0.8e1.6 g cm3) (Table 2) from sediment (2.7 g cm3), usually by
is the cheap and efficient separation of microplastics from large
carefully mixing the sediment with salt saturated solutions and col-
volumes of sediments, allowing higher environmental representa-
lecting the supernatant containing microplastics for further filtration
tiveness, and reduction of sample volume undergoing density sep-
[82]. It is accepted that solutions >1.4 g cm3 are required to separate
aration [54]. However, this method takes at least 1 h per sample
microplastics from sediments, since their density is dependent on
(comprised of 3 subsamples) and requires previous sieving by size
polymer type, additive concentration, and even adsorbed substances
range [54]. The Munich Plastic Sediment Separator (MPSS) uses a
and organisms [80]. A simple density separation using water is able
similar system, where a dense solution of ZnCl (1.6e1.7 kg L1) is
to recover some types of plastics, such as PE (polyethylene) and PP
injected at the bottom of the column, allowing microplastics to
(polypropylene), from soil samples [110] or fibers from sediments,
ascend and be collected in the supernatant or by direct vacuum
due to their shape and large surface [80]. NaCl is one of the most used
filtration, but at greater time expense (e.g. settling phase may take
salts for density separation, as it is highly available, cheap and eco-
1e2 h) [45]. Nuelle et al. [78] also used a pre-step of fluidization
friendly [78]. Reagent grade NaCl is recommend since it achieves
using NaCl and air-induced overflow to reduce sample size up to 80%.
slightly higher densities and thus has a higher extraction efficiency
Oil has also been tested as a separation method due to the hy-
for slightly heavier polymers, such as HDPE (high density poly-
drophobic properties of plastics. Imhof et al., [45] used pine oil along
ethylene) [84]. However, Quinn et al. [80] found that NaCl
with froth conditioner to improve wetting, reduce surface tension
(1.2 g cm3) had low recovery rates (<90%) and larger error bars, as
and mediate the detachment of plastics from sediment in deionized
well as NaBr (1.4 g cm3), while both NaI (1.6 g cm3) and ZnBr2
water, with low recovery rates (55.0%) particularly of high density
(1.7 g cm3) were able to separate heavier polymers with good re-
plastics. Canola oil has also been used, with recovery rates of 96.1%,
covery rates (99%) and tight error bars. Furthermore, separation using
shorter sampling time (~2 h) compared to salt saturated solutions
NaI and ZnBr2 requires a single washing of the sediment, while NaCl
and little retention of organic matter [21]. A drop of olive oil has also
requires three. However, NaI reacts with cellulose filters, turning
been added to salt saturated solutions to help gather plastic particles
them black and complicating visual identification, while ZnBr is
in the supernatant, improving recovery rates from 64% to 82% [51].
hazardous to the environment and expensive, which can be over-
Even though oil seems to have limitations in separating plastics and
come by continuous reuse through careful filtration and density
requires a cleaning step with a detergent, it can be combined with
adjustment. NaI is also able to recover oleophobic fibers (93.3%),
saturated solutions to improve recovery rates.
better than CaCl2 (69%) [21], and with MeOH is able to recover most
NaCl is a highly available, cheap and innocuous separation so-
microplastics from marine snow (90e98%) [111]. Following Kedzier-
lution, advantages that led to its recommendation by both the
ski et al. [52] protocol using an elutriation column, NaI can be recy-
MSFD technical subgroup (2013) [73] and NOAA [70]. However,
cled up to 10 cycles through rising and evaporation steps, having
NaCl limitations, namely the low recovery of higher density poly-
similar costs to NaCl. Thus, the use of NaI is the recommended since it
mers Table 2, classify this solution as unsuitable for microplastic
is environmentally safe and can be recycled for several cycles, as long
separation, since it leads to underestimation of environmental
as it is not used with a cellulose filter. The Sediment-Microplastic
concentrations. Thus, recovery of microplastics, especially of
Isolation, an apparatus comprised of two tubes connected by a
heavier polymers, can be improved through the use of higher
valve that allows separation of the supernatant and sediment, has
density solutions, in which we propose NaI due to its high density,
Table 2 safety and possibility of reuse, and possibly in combination with
Separation of polymer types by solutions used in density separation. separation columns or the use of oil to improve recovery rates.
Polymer Density (g cm3) Water NaCl NaI ZnBr2

1 g cm3 1.2 g cm3 1.6 g cm3 1.7 g cm3

3.3. Unusual methods for separation and quantification of
microplastics
PP 0.9e0.91 þ þ þ þ
PE 0.92e0.97 þ þ þ þ
PA 1.02e1.05 e þ þ þ Other unusual methods have been used in the separation and
PS 1.04e1.1 e þ þ þ quantification of microplastics. For instance, Felsing et al. [33]
Acrylic 1.09e1.20 e þ þ þ separated microplastics (63e5000 mm) using a Korona-Walzen-
PMA 1.17e1.20 e þ þ þ Scheider electrostatic bell separator, reducing sample volume by
PU 1.2 e þ þ þ
PVC 1.16e1.58 e ± þ þ
99%. Shimizu et al., [89] propose using the frequency of impact of
PVA 1.19e1.31 e ± þ þ microplastics in solution with an electrode, caused by Brownian
Alkyd 1.24e2.10 e e þ þ motion, to infer concentration. Flow cytometry with visual sto-
Polyester 1.24e2.3 e e þ þ chastic network embedding (viSNE) also allows quantification of
PET 1.37e1.45 e e þ þ
small microplastics (1e20 mm) in water samples [85]. However,
POM 1.41e1.61 e e ± þ
limitations of these methods include availability of specialized
Label: þ: separation, ±: possible separation, e: not separated. Polymers: PE: poly- equipment, time spent in each sample, incapability of character-
ethylene, PP: polypropylene, PS: polystyrene; PA: polyamide (nylon), POM: poly-
oxymethylene, PVA: polyvinyl alcohol, PVC: polyvinylchloride, PMA: poly methyl
ization of polymer types, potential saturation of measuring equip-
acrylate, PET: polyethylene terephthalate, PU: polyurethane (polymer density ment and changes in surface charge of plastics due to weathering
adapted from Ref. [44]). and biofouling.
154 J.C. Prata et al. / Trends in Analytical Chemistry 110 (2019) 150e159

4. Sample processing: removal of organic matter plastics [79], leave oily residues and bone fragments [22,66] or re-
deposit tissue residues on plastic surfaces, complicating charac-
Environmental samples contain biologic material. For instance, terization by vibrational spectroscopy [101]. KOH has good diges-
Crichton et al. [21] reported that sediment from beaches contained tion of organic matter and recovery of plastics [10,74]. Protocols
0.5e7.0% of biologic material. Biological material is often confused using KOH (10%) at 60 C overnight [66] or 60 C for 24 h [22]
with plastics (e.g. darker algae fragments), leading to overestimation showed to be one of the most effective digestive treatments, as
of environmental concentrations and increasing the number of par- well as NaOH [15]. Nonetheless, KOH may cause discoloration of
ticles subjected to further analysis. Thus, there is a need to create a nylon, PE and uPVC (unplasticized PVC), degradation of nylon,
simple method of digestion capable of reducing organic matter polyester, PE, PC (polycarbonate), PET, PVC, LDPE, CA (cellulose
without affecting the structural or chemical integrity of polymers acetate) [15,50,56,66,74]. NaOH also may cause CA, PA, PET degra-
[33,72]. Nonetheless, the need for digestion varies depending on the dation and color change in PVC and PET [22]. Regarding digestion
quantity of organic matter in each sample. For instance, not all the efficiency, Kühn et al. [56] tested several samples of organic matter
reviewed studies performed a digestion step: in sediment seven used often found in beaches (i.e. seaweed, squid beaks, Polychaeta beaks,
H2O2 (30%) and one Fenton's reagent (H2O2 with ferrous iron catalyst) sheep wool, seal whiskers, fish otoliths, bird feathers, manila rope,
and in water five used H2O2 (30%), two enzymatic digestion and one metal fish hook, paraffin and palm fat) revealing that fish otoliths,
HCl (5e10%) (Fig. 1). NOOA recommends the use of H2O2 (30%) with squid beaks, paraffin and palm fat survived the digestion process
Fe(II) solution (0.05 M)(sulfate (Fenton's reagent) heated at 75 C to a with KOH (1 M) for 2 days at room temperature. Thus, hard parts
glass beaker containing the microplastics fraction for both water and and fats seem to not be fully digested by alkali. Acid and alkali
sediment samples. The use of a digestion step is highly recommended digestion may also be used sequentially (e.g. NaOH and HNO3) with
when identification is mainly based on visual inspection. However, good digestion of biologic material and recovery rates [81].
most works did not remove organic matter from their samples,
possibly because authors deemed them low in organic matter. The 4.3. Oxidizing agents
NOOA oxidizing method has yet to be widely used, with a single study
mentioning its use e an example of the difficulties in standardization. Hydrogen peroxide (H2O2, 30e35%) is an oxidizing agent able to
Digestion protocols, and their uniformization, are even more impor- digest organic matter more efficiently than NaOH and HCl, with
tant when processing biota samples (e.g. fish). Besides oxidizing little to no degradation of polymers [78,79,111]. Nuelle et al. [78]
methods, digestion can also be acidic, alkaline or enzymatic (Table 3). reported resistance of PVC, PET, nylon, ABS (acrylonitrile buta-
diene styrene), PC, PUR (polyurethane), PP, LDPE, LLDPE (linear
4.1. Acid digestion LDPE), HPDE to H2O2, with some discoloration, while Karami et al.
[50] reported degradation of nylon and color change of PET
Acid digestion may be used to degrade organic matter. However, following H2O2 (35%) treatment at 50 C for 96 h. Digestion may also
some polymers (e.g. nylon, PET e polyethylene terephthalate) have cause the production of foam that may lead to the reduction of
low resistance to acids and may also be degraded, especially in high microplastics retrieved [66]. Temperature of incubation seems to be
concentration and high temperatures [79]. Yet, there must be an op- a determinant factor on the efficiency of H2O2. For instance Cole et
timum of concentration and temperature used to efficiently remove al. [15] reported that incubation with H2O2 (35%) at room tem-
biologic material in a reasonable period of time. For instance, Naidoo perature for 7 days only degraded 25% of organic matter, whereas
et al. [75] found that heating nitric acid (HNO3, 55%) to 80 C allows to Avio et al. [2] reported to have used H2O2 (15%) at 50 C overnight to
digest fish tissues 26 times faster. Nonetheless, caution is advised efficiently remove organic matter. Zhao et al. [111] reports that 15%
when heating digestion solutions above 60 C, since these tempera- is preferred to 20% H2O2, and both these treatments have better
tures may destroy microplastics [74]. Hydrochloric acid (HCl) seems to results than HCl. Thus, H2O2 treatments may be able to efficiently
be the least effective treatment in treating large quantities of biologic remove organic matter with little effect on microplastic integrity.
material [15,66,111]. Nonetheless, Karami et al. [50] reported that HCl
(37%) at 25 C had a digestion efficiency >95% but with melting of PET. 4.4. Enzymatic digestion
This difference may reflect different protocols, with variations in
concentration and temperature that affect digestion efficiency. Nitric Enzymes have been used as alternative digestion methods.
acid (HNO3) is widely used in acid digestion. Yet, nitric acid may leave Enzymatic digestion is less hazardous (e.g. can be used without a
oily residues or tissue debris, cause loss of nylon and melting of PS fume hood) and less likely to induce damage to microplastics [66].
(polystyrene), LDPE (low density polyethylene), PET and HDPE, or However, enzyme efficiency will vary with type of organic material
yellowing of polymers, including Claessens et al. 2013 [11] PP (poly- present in the sample [19]. Enzyme protocols include the pre-
propylene), PVC (polyvinyl chloride) and PET [10,11,22,50,66]. Naidoo digestion of sediments with an industrial enzyme blend (2.5%) at
et al. [75] reports PE, HDPE, PS, polyester and PVC survived HNO3 45 C for 60 min followed by H2O2 (30%) removal of debris [21]. To
(55%) at room temperature for a month, only with degradation of digest fish tissues Karlsson et al. [51] used proteinase K (500 mg mL1)
nylon and whitening of PVC. Once again, resistance of polymers to with CaCl2 incubated at 50 C for 2 h, followed by shaking (20 min)
digestion is dependent on various factors, such as the presence of and further incubations (60 C, 20 min), then treated with H2O2 (30%)
organic matter in the sample (leading to lower degradation of poly- with recoveries of 97% but with calcium deposition over particles that
mers) [11] and temperature of the solution. In this case, it is more may complicate further characterization. Protein K has also been used
realistic to think that HNO3 will have some effects on the integrity of in seawater to digest biologic material retained in samples collected
plastics, since heating is needed to achieve digestion in a timely by plankton net, allowing a digestion efficiency up to 97% at 50 C
manner. Thus, acid digestion may be used with caution since it may [15]. Courtene-Jones et al. [19] tested Tripsin, Collagenase and Papain
lead to underestimation of microplastics in environmental samples. with digestion efficiencies of 72e88% and no effect on polymers
€ der et al. [64] proposed the use of a basic enzymatic puri-
tested. Lo
4.2. Alkali digestion fication protocol with 98.3% efficiency, based on the use of detergent
(5% w/w sodium dodecyl sulfate), a sequential use of enzymes
Alkali digestion is an alternative to acid digestion with great (protease, cellulase, chitinase) and two hydrogen peroxide treat-
potential. However, alkali digestion may also damage or discolor ments (one between enzyme treatments and one in the end), adding
 J.C. Prata et al. / Trends in Analytical Chemistry 110 (2019) 150e159 155

Table 3
Digestion methods for the removal of organic matter to improve the identification of microplastics, their efficiency and effects on synthetic polymers.

Digestion Treatment Recovery rate Polymer degradation Organic matter Reference

degradation

Acid HNO3 (35%), 60 C 1 h n.a. Fusion of PET and HDPE; 100% [10]
destruction of PA
HNO3 (65%), RT overnight, 60 C 2 h, n.a. PA degradation; yellowing n.a. [22]
dilution 80 C distilled water
HNO3 (65%) and HClO4 (65%) 4:1 overnight, n.a. PA degradation, yellowing n.a. [22]
boiled 10 min, dilution 80 C distilled water
HNO3 (5e69%), RT 96 h <95% Melted LDPE and PP; color n.a. [50]
change in PP, PVC, PET;
decrease Raman peaks
HNO3 (55%) RT 1 month n.a. Whitening of PVC, degradation of PA n.a. [75]
HCl (5e37%), 25-60 C 96 h n.a. Changes in PET and PVC >95% [50]
Alkali NaOH, 60 C 1 h 94% No 100% [10]
NaOH (10 M), 60 C 24 h n.a. CA degradation n.a. [22]
K2S2O8 (0.27 M) and NaOH n.a. CA degradation; unpredictable n.a. [22]
(0.24 M), 65 C 24 h weight increase
KOH (10%), RT 3 weeks n.a. No n.a. [22]
KOH (10%), 60 C 24 h n.a. CA degradation n.a. [22]
KOH (10%), 50 C 96 h n.a. Loss of PET and PVC n.a. [50]
KOH (10%), 40 C 96 h n.a. Loss of PET; yellowing of PA n.a. [50]
KOH (1 M), RT 2 days n.a. Degradation of LDPE, CA, Most, except otoliths, [56]
Cradonyl and PA. squid beaks, paraffin,
palm fat
NaOH (1 mol L1), 17.5 mL of 65% HNO3 95% Degradation of PA, PET, EPS, n.a. [81]
and 2.5 mL UP and drying LDPE, PVC; color change
in PVC and PET
Oxidative H2O2 (30%), 60 C for 1 h, 100 C for 7 h n.a. n.a. n.a. [32]
H2O2 (35%), RT, 40 C 96 h n.a. Decrease in Raman peaks n.a. [50]
of PVC and PA.
H2O2 (35%), RT, 50 C 96 h n.a. Degradation of PA; n.a. [50]
color change of PET;
foam and oxidization
H2O2 (6%) 70 C for 24 h 78% (PE) n.a. n.a. [93]
H2O2 (30%), 60 C until evaporation n.a. n.a. n.a. [114]
Enzymatic Corolase 7086, 60 C 1 h 93% No n.a. [10]
Tripsin, 38-42 C 30 min n.a. No 88% [19]
Collagenase, 38-42 C 30 min n.a. No 76% [19]
Papain, 38-42 C 30 min n.a. No 72% [19]
Pepsin (0.5%) and HCL (0.063 M), 35 C 2 h n.a. No Incomplete [22]
15 mL Tris-HCl 60 C 60 min, proteinase K (500 mg/mL) 97% Calcium layer n.a. [51]
and CaCl2 50 C 2 h, shaken 20 min, incubated
60ºc 2 h, 30 mL H2O2 (30%) overnight

n.a. e not available; RT e room temperature; Polymers: PA e polyamide (nylon), PE e polyethylene, PET e polyethylene terephthalate, LDPE e low density polyethylene, HDPE
e high density polyethylene, PP e polypropylene, PS e polystyrene, PVC e polyvinyl chloride, CA e cellulose acetate, EPS e expanded polystyrene.

up to 13 days of sample processing. However, enzyme use is limited characterization. From the reviewed studies on water and sediment
by its high price. Industrial Corolase 7089, sold in liquid form, has (N ¼ 40), 50% used Fourier-transform infrared spectroscopy (FTIR)
been presented as a less expensive enzyme that could be used in based methods, 32.5% visual inspection, 10% Raman spectroscopy,
microplastic sampling with better results than chemical treatments whereas electron microscopy, staining dyes and gas
[10]. Nonetheless, enzymes are still being used in a small scale and chromatography-mass spectroscopy were each used in 2.5% (Fig. 1).
some protocols may require a following treatment with H2O2 to However, electron microscopy was used in three more studies also
remove undigested debris. subjected to FTIR.

4.5. Less used digestion methods 5.1. Visual inspection

Other methods include the use of microwaves, that seem to Visual inspection allows classification of particles as plastic
damage microplastics [51], ultrasonication, useful in combination based on physical characteristics, observed directly, or using a
with other methods (e.g. improved the digestion of sludge using stereoscope or microscope. This is one of the most used and widely
NaOH from 43.5% to 50.7%, around 7.2%) [48] and the use of sodium available methods of identification and quantification of plastic
hypochlorite (NaClO; 24.8 g L1) in distilled water (1:3 v/v) left particles, even used as pre-selection when chemical characteriza-
overnight as an efficient method in the digestion of fish stomach tion is performed. However, this method is subjective, may produce
contents, without affecting polymers and Raman spectra but wide variations between observers and is highly time consuming.
potentially causing discoloration [16]. For instance, visual detection of microplastics directly in beach
sediments by multiple observers had detections of 60e100%,
5. Identification, chemical characterization and varying with the individual, experience, fatigue and leading to
quantification overestimation (e.g. biologic material confused for black fragments)
or underestimation (e.g. white fragments) of certain types and
Identification and quantification of microplastics is almost al- colors of microplastics [57]. Misclassification of other material as
ways done by visual inspection, even if followed by chemical plastics, confirmed by following chemical analysis, is reported in
156 J.C. Prata et al. / Trends in Analytical Chemistry 110 (2019) 150e159

70% of presumed microplastics [44], for 32% of particles and 25% of 6e7 times the price of the cellulose filters commonly used),
fibers [58]. Researchers have tried to improve visual inspection by limiting the wide application of this technique. Vibrational spec-
prodding particles with needles [87], testing plastics with a heated troscopy is limited by the high cost, availability of equipment, time
needle tip [9] and even melting plastics (130 C, 3e5 s) in the and effort required in analyzing and processing samples, complex
sample to ease detection [110]. Visual inspection also allows the data treatment, need for skilled personal and limited detection,
classification of plastic particles by size, color and shape, allowing especially in weathered or contaminated microplastics
to infer on their origin. [26,31,63,79,87,111]. Visual pre-selection of particles is often used
The use of staining dyes is a low-cost method to ease visual to lessen these practical problems but may induce bias [79].
identification. Unsatisfactory results have been reported for Oil red In pyrolysis-gas chromatography e mass spectrometry (Pyro-
EGN, Eosin B, Hostasol Yellow 3G and Rose Bengal [66]. Problems GC-MS), microplastics are thermally decomposed (pyrolyzed) un-
arise from the affinity of the dye for plastics and the confounding der inert conditions and the gas formed is cryo trapped and sepa-
effect of staining biogenic material in the sample, requiring a rated on a chromatographic column, identified by mass
thorough digestion step. For instance, Rose-Bengal allows a 92% spectrometry [27,87]. This method can provide the chemical char-
coloration of 25 mm microplastics, and following characterization acterization of a single microplastic or of a bulk sample, but it is
by FTIR, but from all the stained particles only 22e99% were destructive and provides no information regarding number, size or
actually plastics [114]. Presently, Nile Red seems to be the most shape. Thermoextraction and desorption coupled with gas
promising staining protocol for microplastics. Staining with Nile chromatography-mass spectroscopy (TED-GC-MS) combines a
Red requires a short term of incubation (10e30 min), provides high thermogravimetric analysis (TGA) for thermal degradation
recovery rates (96.6%) and allows vibrational spectroscopy with or (100e600 C) and solid phase extraction of plastic degradation
without a short cleaning step with bleach [32,66]. Results are seen products, further analyzed by thermal desorption in GC-MS (~3 h)
under an orange, red or green filter in the fluorescent microscope. [27,31]. The advantage of this method over Pyro-GC-MS is the use of
Even though biogenic material, such as algae, seaweed, wood, relatively high sample masses and enabling measurement of
feathers and mollusc shells are not stained [66] and weathering of complex heterogeneous matrices, allowing identification and
plastics does not seem to affect staining [88], some types of plastics quantification of polymers in environmental samples without pre-
such as PC, PUR, PET and PVC have weak signals [32] and fibers are selection [27]. Liquid chromatography uses an appropriate solvent
difficult to stain [94]. Since Nile Red is a solvatochromic dye, the for the polymer type and size exclusion chromatography in char-
fluorescence emission is dependent on the polarity of the solvent, acterization, requiring large amounts of sample [31]. These
possibly allowing classification of microplastics in large chemical methods present the advantage of analyzing relatively high masses,
groups based on fluorescent shift [66,88], especially through the improving representativeness, but they are destructive, and the
analysis of images with suitable software. Thus, validation of a information provided is limited to chemical composition.
staining protocol may provide researchers with a simple and time- Turner [97] used a portable X-ray fluorescence (XRF) spec-
effective tool to visually identifying plastics and improve the pre- trometer to characterize the elemental composition of polymers
selection method of particles to be submitted to chemical through the diffraction and reflection of radiation, with potential
characterization. use in the detection of some additives or adsorbed metals. Scanning
electron microscope (SEM) with an energy-dispersive X-ray
5.2. Chemical characterization microanalyser has also been used to collect information of
morphology and chemical composition of microplastics, requiring a
Chemical characterization of potential microplastics by Fourier previous pre-selection and mounting of the particles analyzed [36].
transform infrared spectroscopy (FTIR) and Raman spectroscopy is Fuller and Gautam [37] used a pressurized fluid extraction with
highly recommended. The MSFD technical subgroup [73] recom- solvents (methanol, hexane and dichloromethane), temperature
mends subjecting 10% of microplastics of sizes 100e5000 mm and and pressure conditions to separate the bulk microplastics fraction
all the suspected particles in the range of 20e100 mm to these from soil samples.
methods, but more may be required for the larger sizes due varying
accuracy in visual identification. Both vibrational spectroscopy 6. Mitigation of cross-contamination
methods are non-destructible, highly accurate, and complemen-
tary, producing a spectrum based on the interaction of light with Due to the wide contamination of the environment with
molecules: FTIR produces an infrared spectrum resulting from the microplastics, including air [29], measures should be taken during
change in dipole moment, whereas Raman provides a molecular sampling to reduce the contamination with these particles and fi-
fingerprint spectrum based on the polarizability of chemical bonds bers. The five rules to reduce cross-contamination of microplastic
[31,79]. Diverse FTIR techniques have been used in the character- samples are: (1) using glass and metal equipment instead of plas-
ization of microplastics, such as attenuated total reflection FTIR tics, which can introduce contamination; (2) avoiding the use of
(ATR-FTIR) that improves the information on irregular micro- synthetic textiles during sampling or sample handling, preferring
plastics, which in contrast to transmission FTIR is also applicable to the use of 100% cotton lab coat; (3) cleaning the surfaces with 70%
thick or opaque samples [19,87] and micro-FTIR that produces a ethanol and paper towels, washing the equipment with acid fol-
high-resolution map of the sample (down to 20 mm), without a pre- lowed by ultrapure water, using consumables directly from pack-
selection step [43,63,104]. Raman microscopy allows the charac- aging and filtering all working solutions; (4) using open petri
terization of microplastics <20 mm, but may be limited by weak dishes, procedural blanks and replicates to control for airborne
signals, overcome by increasing measurement duration, and fluo- contamination; (5) keeping samples covered as much as possible
rescence interference, dependent on material characteristics such and handling them in clean rooms with controlled air circulation,
as color, biofouling and degradation [1]. Stimulated Raman scat- limited access (e.g. doors and windows closed) and limited circu-
tering (SRS) has been used to identify microplastics on low Raman lation, preferentially in a fume hood or algae-culturing unit, or by
background filter membranes without pre-selection, scanning at covering the equipment during handling [15,26,95,105,107]. A fume
six wavenumber settings and characterizing 1 cm2 under 4.5 h, hood can reduce 50% of the contamination [105] while covering
compared to the 24 h needed in Raman mapping [109]. However, samples during filtration, digestion and visual identification can
the low Raman background filters used are highly expensive (about reduce more than 90% of contamination [95].
 J.C. Prata et al. / Trends in Analytical Chemistry 110 (2019) 150e159 157

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