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An efficient and gentle enzymatic digestion protocol for the extraction of microplastics from bivalve tissue
Marine Pollution Bulletin 142 (2019) 129–134
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Note
An efficient and gentle enzymatic digestion protocol for the extraction of microplastics from bivalve tissue
T
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Lisa W. von Friesena, , Maria E. Granberga, Martin Hassellövb, Geir W. Gabrielsenc, Kerstin Magnussona a
IVL, Swedish Environmental Research Institute, Kristineberg 566, SE-45178 Fiskebäckskil, Sweden University of Gothenburg, Department of Marine Sciences, Kristineberg 566, SE-45178 Fiskebäckskil, Sweden c Norwegian Polar Institute, Hjalmar Johansensgate 14, N-9296 Tromsø, Norway b
A R T I C LE I N FO
A B S T R A C T
Keywords: Microplastics Enzymatic digestion protocol KOH Pancreatic enzymes Tissue digestion Emerging pollutants
Standardized methods for the digestion of biota for microplastic analysis are currently lacking. Chemical methods can be effective, but can also cause damage to some polymers. Enzymatic methods are known to be gentler, but often laborious, expensive and time consuming. A novel tissue digestion method with pancreatic enzymes and a pH buffer (Tris) is here presented in a comparison to a commonly applied digestion protocol with potassium hydroxide. The novel protocol demonstrates a highly efficient removal of bivalve tissue (97.7 ± 0.2% dry weight loss) already over-night. Furthermore, it induces no impairment in terms of ability to correctly identify four pre-weathered plastic polymers and six textile fiber polymers by Fourier transform infrared spectroscopy after exposure. The high-throughput protocol requires minimal handling, is of low cost and does not pose risk to the performer or the environment. It is therefore suggested as a candidate for a standardized digestion protocol, enabling successful analysis of microplastics ingested by bivalves.
1. Introduction A rapid increase in the number of studies investigating environmental occurrence of microplastics is currently being seen. As the number of studies grows, so does the global record of microplastic pollution; from urban coastal surface waters to remote deep ocean floors (Bergmann et al., 2017a; Li et al., 2016). Improved knowledge on the occurrence and fate of microplastics in the marine environment is essential to assess bioavailability and potential effects in marine ecosystems. This knowledge is also needed for implementation of proper measures and determination of environmental quality standards. Several analytical challenges currently hamper investigations of microplastic pollution in environmental matrices, affecting both accurate quantification and comparability between studies (Lusher et al., 2017; Wesch et al., 2016). Ultimately, the sought microplastic particle shall be successfully and representatively separated from the sample matrix without risk of harming or misplacing the particle, or contaminating the sample (Fig. 1). In order to achieve a best practice method, factors such as work intensity, time efficiency, cost, as well as both human and environmental health in terms of exposure to chemicals also need to be considered. The latter aspect is particularly important when developing
methods suitable for large-scale monitoring purposes and routine analysis. In the relatively new research field of microplastics, there is an urgent need for standardization of sampling, quantification and interpretation of results to obtain comparable data across studies (OSPAR, 2015; Vandermeersch et al., 2015). When analyzing the occurrence of microplastics in organisms, digestion of tissue is a commonly applied method and has often been achieved through the use of strong oxidizing acids (e.g. HNO3; Claessens et al., 2013), strong bases hydrolyzing or saponification of fat tissues (e.g. potassium hydroxide (KOH); Foekema et al., 2013) or combinations of hydrolyzing and oxidizing chemicals such as KOH and NaClO (Enders et al., 2017). The use of KOH has been recommended as sufficient for removing biological material (Dehaut et al., 2016; Kühn et al., 2017), but the trade-off of the protocol is between the required exposure time and induced impact on polymers. A time-efficient removal of tissue is obtained by KOH at 60 °C over-night (Rochman et al., 2015), however this has been found to have deleterious impact on some polymers (Dehaut et al., 2016; Lusher et al., 2017). KOH in room temperature has also been verified efficient in removing tissue and presenting no visible impact on tested polymers by Foekema et al. (2013), but both polyethylene terephthalate (PET) and polycarbonate is
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Corresponding author at: IVL, Swedish Environmental Research Institute, Nordenskiöldsgatan 24, SE-21119 Malmö, Sweden. E-mail addresses: [email protected] (L.W. von Friesen), [email protected] (M.E. Granberg), [email protected] (M. Hassellöv), [email protected] (G.W. Gabrielsen), [email protected] (K. Magnusson). https://doi.org/10.1016/j.marpolbul.2019.03.016 Received 30 January 2019; Received in revised form 8 March 2019; Accepted 8 March 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.
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Eliminating the matrix
No impact on sought particles
No contamination of unwanted particles
Large-scale applicability
Successful identification of all microplastic particles
Time efficiency
Environmental health
Human health
Cost efficiency
Fig. 1. Requirements for a microplastic extraction protocol. Suggested requirements of a standardized protocol for the extraction of microplastics from environmental matrices applicable on a wider scale.
1
classified as non-resistant to this treatment as compiled from tabulated data by Lusher et al. (2017). When specifically focusing on impact on textile microfibres, substantial visual (morphological) change has been reported for PET, polylactic acid and modacrylic after four days of exposure to KOH (personal communication: Piarulli, S.). Considering the high amount of microfibers commonly being reported in environmental samples (e.g. Barrows et al., 2018; Railo et al., 2018; Salvador Cesa et al., 2017), the KOH protocol may not be advisable. The exposure time of four days up to three weeks may not be practically feasible for large scale routine-studies. Several enzymatic digestion protocols have been proposed to provide efficient digestion of tissue as well as low impact on exposed polymers (Catarino et al., 2017; Cole et al., 2014; Courtene-Jones et al., 2017; Löder et al., 2017; Railo et al., 2018; Rist et al., 2018). However, a frequently occurring drawback is the labor intensity associated with the many working steps included (e.g. Cole et al., 2014; Löder et al., 2017). This could markedly increase the risk of contamination as well as loss of particles (Lusher et al., 2017), subsequently decreasing applicability of the protocol. Some industrial enzymes are very expensive (e.g. Proteinase-K; Cole et al., 2014), several of the mentioned enzymatic protocols have been evaluated solely on smaller sized organisms (e.g. 0.15–0.33 g; Railo et al., 2018) and some require long exposure times (e.g. up to 15 days; Löder et al., 2017). Additionally, already weathered reference plastic particles have seldom been used when investigating the potential impact of digestion protocols on polymers. Considering that weathered microplastics are the ones present in environmental samples and ultimately the targeted particles, exclusion of these bring risk of underestimating potential impact induced by the digestion protocol in question. The main aim of this study was to evaluate the applicability of commercially isolated pancreatic enzymes (PEz) as a novel digestion method for the extraction of microplastics from bivalve tissues. The aim was also to experimentally compare the digestion efficiency of the PEz protocol to the commonly used digestion protocol of KOH (e.g. Foekema et al., 2013), both in terms of removal of biological material and required exposure time. Furthermore, the study was aimed at evaluating potential impact by the novel PEz protocol on both weathered polymers collected in the field and on different textile fibers, to further ensure the protocol's suitability of treating environmental samples and successfully identify the microplastic particles present.
2
3
a
c
b
4
a
b
Fig. 2. Level of coverage. The four different levels of coverage on filters after digestion treatment and filtration. 1) Serripes groenlandicus before any digestion treatment (scale in cm), 2) Level 1 = all/most tissue digested, whole mesh of filter possible to see, very easy to analyze, 3) a: Level 2 = low load of undigested tissue on the filter, easy to analyze, b: Level 3 = filter partly covered by undigested tissue, hard but possible to analyze, c: Level 4 = filter fully covered by undigested tissue, not possible to analyze. 4) A comparison of digestion results over-night, a: KOH (potassium hydroxide), an example of level 4 coverage where the whole organism is still present after treatment, b: pancreatic enzymes with Tris, an example of level 1 coverage.
2. Methods The study consisted of two parts; I) an evaluation of the relative tissue digestion efficiency and microplastic recovery rate of the pancreatic enzyme (PEz) and potassium hydroxide (KOH) digestion protocols, and II) an evaluation of the potential impact on different plastic polymers caused by the PEz protocol. The chosen test organism was the 130
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digestion protocols: digestion efficiency in terms of DW loss (DE, %), level of coverage (LC) which refers to the remaining level of undigested material on filter after treatment (scale 1–4; Fig. 2), and recovery rate of the 40 spiked microplastic particles (RR, %). The DE was calculated as the percentage of DW loss during treatment (Eq. 1, adjusted from Courtene-Jones et al., 2017 to be based on DW), based on the established WW/DW ratio. The total DW remaining on both filters (300 and 20 μm) was used for calculations.
Greenland Smoothcockle (Serripes groenlandicus) (Bruguiere, 1789), which is a relatively large Arctic bivalve species (Fig. 2). Individuals included in this study ranged between 6 and 35 g wet weight (WW) without shell. 2.1. Part I: comparison between the two digestion protocols Individuals of S. groenlandicus were collected via scuba diving at the island Prins Karls Forland, Svalbard (78.7946667°, 011.0013333°), and frozen (−20 °C) until further analysis. Upon analysis, the mussels were thawed, shells removed and WW recorded. The mussels were individually placed in clean glass jars. To establish the WW/dry weight (DW) ratio, mussels were oven dried at 105 °C until constant weight was reached (n = 2). For reference plastic polymers, floating pieces of macroplastics were collected by hand from the sea surface north of the island Nordaustlandet, Svalbard (80.9000000°, 022.1253333°), and stored dark and cold until further analysis. These ocean collected plastics have naturally been subjected to weathering, photo-degradation and biofouling, factors that influence the properties of plastics (Lobelle and Cunliffe, 2011). Thus, a higher similarity to actual microplastics in the environment is ensured, compared to when using virgin plastics. Two different types of low-density polyethylene (LDPE) particles were created out of the collected macroplastics for part I of the study; from multifilament fishing rope (LDPE-F) and plastic packaging (LDPE-P). LDPE-F was cut into 300–600 μm pieces and LDPE-P was cut into 20–300 μm pieces using a metal scalpel under a stereomicroscope (Leica, M205C). The two sizes were chosen in order to set up the quantification of recovery rates as environmentally realistic as possible. All samples were spiked with 20 LDPE-F and 20 LDPE-P particles respectively, which were mixed into the sample prior to digestion treatment. The two protocols (PEz and KOH) were evaluated at four exposure times; over-night, two, four and eight days (n = 3). Samples were weight-matched between the two protocols and time exposures, i.e. the average weight for protocol*time was kept within ± 2 g WW difference. The pancreatic enzyme used was the commercially available Creon® 40,000 (Abbott Laboratories GmbH, Germany, Mylan), originating from porcine pancreas, containing lipase (40,000 Ph.Eur), amylase (25,000 Ph.Eur) and protease (1600 Ph.Eur) as active substances. The content of the enzyme capsules was retrieved and added to the tissue samples together with Tris (tris(hydroxymethyl)aminomethane)/Tris hydrochloride solution (Trizma®, pH 8.0, 1 M, 0.2 μm filtered, Sigma-Aldrich, T3038, USA). PEz was added to a concentration of 0.05 g PEz per g WW of tissue (cost of Creon®: ~0.05 € per g WW to digest). The amount of Tris was added to reach and maintain a sample pH of 8.0 ± 0.1, which is the optimal performing range of pancreatic enzymes (Berdutina et al., 2000) (pH-Fix 7.0–14.0, Macherey-Nagel) (roughly: 0–15 g WW: 10 ml, 15–20 g WW: 15 ml, 20–35 g WW: 20 ml), and to ensure that the solution covered the organism. In the KOH treatment (1 M, Eka Nobel, Sweden), an equivalent ratio between tissue WW and volume of KOH solution as for the PEz protocol was applied. Control samples (n = 3) of PEz and Tris without any bivalve tissue were included to validate potential influence of PEz addition on the final filter weight. Samples treated with PEz were incubated at 37.5 °C on a shaking table at 126 rpm (Innova 40, Incubator Shaker Series, New Brunswick Scientific), whereas KOH treated samples were kept in room temperature on a shaking table at 126 rpm. Upon reaching the respective experimental exposure time, samples were vacuum filtered in sequence through 300 and 20 μm nylon filters (Sefar Nitax) and thoroughly rinsed in slightly warmed Milli Q-water (~40 °C, 18.2 MΩcm TC, 0.22 μm, Millipore). The filters were pre-weighed (accuracy 0.1 mg, Mettler Toledo XP205) after being dried for 24 h at 50 °C, and after filtering they were again dried at 50 °C until constant weight was reached. Three variables were measured to quantify the efficiency of the two
DE (%) =
DWB − DWA × 100 DWB
(1)
where DE is digestion efficiency (%), DWB represents the calculated DW before digestion (g) and DWA the measured DW after digestion (g). The resulting LC following digestion was graded into four levels; 1) all/most tissue digested, whole mesh of filter possible to see, very easy to analyze, 2) low load of undigested tissue on the filter, easy to analyze, 3) filter partly covered by undigested tissue, hard but possible to analyze, 4) filter fully covered by undigested tissue, not possible to analyze (Fig. 2). LC up to level three was considered acceptable for a reliable analysis of microplastics on the filters. The RR was calculated as the percentage of the 40 spiked microplastic particles recovered after treatment. Identification of the particles was performed under a stereomicroscope (Leica, M205C). 2.2. Part II: evaluation of potential impact on polymers induced by pancreatic enzymes An exposure test was carried out to investigate whether PEz altered the chemical composition of microplastic polymers to an extent that could hamper correct identification. Ten particles types in total were exposed to PEz and Tris over-night under the same conditions as in part I, except the presence of bivalve tissue. Weathered fragments of LDPE, expanded polystyrene (ePS) and polypropylene (PP) were created from collected marine debris. The LDPE is represented by the two types of spiking particles in part I of the study (LDPE-F and LDPE-P). In addition to weathered fragments, textile fibers (non-weathered) of polylactic acid (PLA), polyethylene terephthalate (PET), polyester (also PET), nomex, PP and modacrylic were also exposed to the PEz protocol. The quantified variable was the correlative matching rate of Fourier-Transform infrared (FTIR, ThermoScientific, Nicolet iN10) spectroscopy absorbance spectra between the respective particle type before and after exposure to PEz and Tris over-night. This evaluation is comparable to Catarino et al. (2017) who performed a similar before and after comparison of FTIR-spectra of four different polymers. However, here we further quantified the correlative matching rate of spectra between original and exposed particle type. A library with absorbance spectra (n = 5) was created for each type of plastic particle before PEz exposure. The FTIR was run in reflectance mode with 256 scans (resolution 4 cm−1, spectral range 4000–675 cm−1, detector cooled by liquid nitrogen) corrected against 256 background scans. After over-night exposure, the absorbance spectra of particles (n = 3) were matched through library search (OMNIC Picta) to the respective created library, and the correlative matching rate (%) to the initial library of spectra was noted. An acceptable matching rate was set to > 90%, which is markedly higher than limits commonly applied in microplastic identification studies (e.g. Bergmann et al., 2017b; Obbard et al., 2014). This is to take height for the fact that actual environmental microplastics can be expected to be even more degraded within organisms' bodies, further complicating correct identification. Particles were pre-washed in ethanol (96%) and air dried before FTIR analysis to remove excess water and potential remains from the exposure to PEz. 2.3. Data analysis Initially, descriptive statistics were generated for all data to test 131
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Table 1 Resulting digestion efficiencies, level of coverage and recovery rates. Digestion efficiency (% dry weight loss during treatment), level of coverage (scale 1–4, 300 μm/ 20 μm filters) and recovery rate (% recovery of spiked microplastic particles) at four different exposure times for the two evaluated protocols; pancreatic enzymes with Tris (pH 8.0, 1 M) and potassium hydroxide (KOH) solution (1 M).
Digestion efficiency (%)
a
Level of coverage (scale 1–4)a,b (300 μm/20 μm)
Exposure time
Pancreatic enzymes + Tris
KOH
Over-night 2 days 4 days 8 days
97.7 97.3 94.8 96.5
33.1 40.2 52.7 59.7
Over-night 2 days 4 days 8 days
1.5 1.0 2.0 2.0
Recovery rate (%)a,c
± ± ± ±
± ± ± ±
0.2 1.6 3.1 1.3
0.7/2.5 0.0/2.0 1.0/2.3 1.4/3.0
87.0 ± 5.9
± ± ± ±
0.7 0.0 0.6 0.0
4.0 3.7 4.0 4.0
± ± ± ±
± ± ± ±
5.5 13.4 9.2 5.3
0.0/2.3 0.6/2.7 0.0/2.0 0.0/2.3
± ± ± ±
0.6 0.6 0.0 0.6
75.0 ± 11.5
a
Data is presented as average ± 1 standard deviation (n = 3). Level 1 = all/most tissue digested, whole mesh of filter possible to see, very easy to analyze, Level 2 = low load of undigested tissue on the filter, easy to analyze, Level 3 = filter partly covered by undigested tissue, hard but possible to analyze, Level 4 = filter fully covered by undigested tissue, not possible to analyze. c Based on the best performing exposure time for the respective protocol: over-night for the pancreatic enzyme treatment and eight days for the KOH treatment. b
with protocol PEz over-night. The following material in a wide variety of particle shapes and colors were successfully identified with FTIR: wool, cotton, cellophane, polyurethane (PU), acrylic, polystyrene, polyamide, polypropylene, polyethylene terephthalate, LDPE, highdensity PE and paint (unpublished data). To minimize potential contamination, all work was performed in a clean air cabinet (Clean Air Techniek B.V.) with equipment rinsed two times in Milli-Q water and covered in aluminum foil at all times possible. 100% white cotton laboratory coats were used at all times. Procedural contamination controls (n = 5) were performed to enable quantification of contamination, with a resulting average number of 1 ± 0.6 microlitter particles (synthetic and non-synthetic) per sample. The following materials were detected in the procedural contamination controls: cotton, cellophane, PU and acrylic.
normality (Shapiro Wilk) and evaluate whether parametric or nonparametric tests were suitable. If normality and homogenous variances (Levene's test) were met, one-way analysis of variance was performed. If non-normality was discovered, Mann-Whitney U Test or KruskalWallis tests for independent samples with subsequent Bonferroni corrections of pairwise comparisons were applied. The statistical analyses were performed in SPSS (IBM, 24) and statistical significance was attributed to p < 0.05.
3. Results 3.1. Part I: comparison between the two digestion protocols The digestion efficiency (DE) of the pancreatic enzyme protocol (PEz) was very high, 97.7 ± 0.2%, already after an over-night exposure, whereas the DE of the KOH (potassium hydroxide) protocol reached its maximum DE of 59.7 ± 5.3% after 8 days of exposure (Table 1). The DE of protocol PEz was higher than the DE of protocol KOH for all exposure times tested (Mann-Whitney U, p = 0.000). The addition of PEz did not affect the final weight of the filters (MannWhitney U, p = 0.937). The level of coverage (LC) on 300 μm filters (LC300) for protocol KOH was never classified as acceptable (i.e. LC300 > 3 at all times) whereas for protocol PEz, it was acceptable already after an over-night exposure (1.5 ± 0.7) (Table 1). The LC of 20 μm filters (LC20) for both protocols were acceptable after an over-night exposure (protocol PEz = 2.5 ± 0.7, protocol KOH = 2.3 ± 0.6). LC300 was higher in protocol KOH for all exposure times compared to protocol PEz (Kruskal Wallis, p = 0.000) and to the control (Kruskal Wallis, p = 0.010), but no difference was found between protocol PEz and the control (Kruskal Wallis, p = 1.000). LC20 was higher than the control for all exposure times in both protocol KOH (Kruskal Wallis, p = 0.012) and protocol PEz (Kruskal Wallis, p = 0.008), and no difference was found between the two protocols (Kruskal Wallis, p = 1.000). There was no difference in the total recovery rate (low-density polyethylene from fishing rope and plastic packaging: LDPE-F + LDPEP) between protocol KOH, protocol PEz and the control (One-way analysis of variance (ANOVA), F2;13 = 3.504, p = 0.061) (Table 1). Similarly, no differences were found between protocol KOH, PEz or the control in the recovery rate of LDPE-P or LDPE-F individually (LDPE-P: One-way ANOVA, F2;13 = 1.877, p = 0.192; LDPE-F: Kruskal-Wallis, independent samples, p = 0.108). Additional analysis of actual anthropogenic microlitter particles present within Serripes groenlandicus (not the same individuals as used for the protocol evaluation), was successfully performed after digestion
3.2. Part II: evaluation of potential impact on polymers induced by pancreatic enzymes The novel digestion protocol here presented did not alter any of the exposed polymers to an extent where the identifiability was impaired, i.e. the average correlative matching rate of exposed particles to the library of original spectra was > 90% for all particle types (Figs. 3, 4). Furthermore, no visual (morphological) change in shape or color was observed on exposed LDPE-F or LDPE-P plastic particles (Supplemental material 1). 4. Discussion This study clearly showed that pancreatic enzymes (PEz) in combination with Tris were effective in digesting tissue of Serripes groenlandicus for microplastic analysis. The novel protocol was superior to potassium hydroxide (KOH) digestion both in terms of removal of organic matter and time requirement. Since the KOH protocol never reached an acceptable digestion efficiency (DE) or level of coverage on 300 μm filters (LC300) during the exposure times used in this study, it is concluded to not be suitable for extraction on these premises. The KOH protocol resulted in equally high recovery rates (RR) of the spiked microplastics as the PEz protocol and the control. This result may, however, be somewhat misleading since the spiked particles were added to the sample but not incorporated in the organism itself as natural microplastic particles would have been. Any naturally incorporated (non-spiked) microplastic particles within the organism would not have been extracted with protocol KOH since the tissues frequently remained entirely undigested throughout the treatment (Fig. 2). The lack of digestion also explains the low level of coverage on 132
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Average matching rate to original (%)
100
used in this study is suggested for analysis of microplastic particles > 20 μm. For smaller fractions, further evaluation and optimization of the protocol could be needed. The fact that some residues of organic matter still remain after treatment is not considered to interfere with the ability of a correct visual quantification of microplastics. The here presented PEz protocol can most likely be optimized to fit other types of tissues and larger samples than those evaluated in the present study. However, since bivalves are commonly used within marine monitoring (OSPAR, 2015), the presented configuration is suggested to be widely applicable. The RR of pre-weathered spiked microplastics averaged 87.0 ± 5.9%, which is in line with, or slightly below, previously reported RR of microplastic particles originating from virgin plastics or plastics from everyday items, e.g. (93 ± 10%: Catarino et al., 2017; 100%: Dehaut et al., 2016; 84.5 ± 3.3%: Löder et al., 2017). However, particles used in the current study (low-density polyethylene from fishing rope and plastic packaging, LDPE-F: 300–600 μm, LDPE-P: 20–300 μm) were smaller than in previous studies and it is acknowledged that smaller sized microplastics are easier to misplace. Since the here measured RR did not differ between the two protocols or the control treatment, it indicates that handling may lead to misplacement of particles rather than remaining undigested tissue hampering detection. Consequently, there is room for some improvement regarding handling routines and the equipment used in the proposed method. The degree of underestimation of microplastic concentrations in environmental samples is seldom addressed in reports or scientific articles but could easily be implemented based on RR in the applied systems, and should further take care for potential size- and polymer-dependent underestimations. Even though weathered plastics and some textile fibers known to be more susceptible to degradation were used in this investigation (e.g. polyethylene terephthalate (PET): Lusher et al., 2017; PET, polylactic acid & modacrylic: personal communication Piarulli, S.), we did not observe any detectable effects induced by the PEz protocol on the plastic polymers that could impair correct identification or quantification. Nevertheless, it is acknowledged that the degradation state of real microplastics within biota in the environment could be higher or different than the ones tested. To increase the environmental realism, future studies investigating the impact on plastic polymers by various digestion protocols should include field collected particles. Considering their high presence when reported, future investigations should additionally include non-synthetic anthropogenic materials that so far have been largely overlooked in studies of microlitter (Barrows et al., 2018; Ladewig et al., 2015). Because anthropogenic materials (also non-plastics) contain e.g. chemical additives and/or pigments, it is important to be able to quantify all types of anthropogenic microlitter in the environment (Barrows et al., 2018; Ladewig et al., 2015). Without this information it is not possible to obtain an accurate exposure scenario for risk assessment of anthropogenic microlitter, which has been acknowledged by the European Commission (2010/477/EU) and further addressed by Railo et al. (2018). The additional types of microlitter that were successfully identified in S. groenlandicus also included non-synthetic materials (unpublished data), which further supports the applicability of the presented PEz digestion method. Beyond the present study, the developed PEz protocol has successfully been applied to several other species for the analysis of microplastics (other bivalve species, gastropods, amphipods, gastrointestinal tracts of fish) (unpublished data). When analyzing crustaceans, parts of the exoskeleton can remain undigested, although they are easily distinguishable and will likely not hamper visual identification. Slight modifications of the protocol configuration have been made for the different species, as expected and previously observed by Löder et al. (2017) when applying an enzymatic digestion protocol on various matrices and species. Sample specific properties as well as the scientific question to be answered will ultimately determine the most suitable
95
90
85
80
Fig. 3. Evaluation of identifiability after treatment. Average matching rates (%) between original Fourier transform infrared spectroscopy spectrum and after an over-night exposure to the pancreatic enzyme digestion protocol for the respective particle types tested. Data is presented as average ± 1 standard deviation (n = 3). Note that the y-axis is broken. Dashed line marks the acceptable matching rate of 90%. Weathered polymers are indicated with (w), those without a marking within parenthesis are non-weathered textile fibers. PET: polyethylene terephthalate, PLA: polylactic acid, PP: polypropylene, ePS: expanded polystyrene, LDPE: low-density polyethylene (F: fishing rope, P: plastic packaging).
1.2
0.8 After
0.6
Before
0.4
Absorbance
1.0
0.2 0.0 4,000
3,500
3,000
2,500 2,000 1,500 Wavenumber cm-1
1,000
500
Fig. 4. Fourier transform infrared spectroscopy (FTIR) evaluation. An example of FTIR spectra before and after exposure of low-density polyethylene from fishing rope (LDPE-F) (pre-weathered) to the developed pancreatic enzyme digestion protocol. The FTIR spectra are baseline corrected at the following wavenumbers; 3959, 3738, 3522, 3229, 2445, 2100, 1847, 839 and 677.
20 μm filters (LC20). The application of the PEz digestion protocol was successful without any pre-treatment such as separation of tissue into smaller pieces and resulted in a low LC which allowed successful visual detection of anthropogenic microlitter. The DE was satisfactory already after over-night exposure (97.7 ± 0.2% dry weight (DW) loss). In comparison to previously evaluated enzymatic digestion protocols, the DE of PEz was higher than the use of trypsin (88 ± 2.5% wet weight (WW) loss: Courtene-Jones et al., 2017) and similar to the use of Proteinase-K (> 97% DW loss: Cole et al., 2014). However, an important difference and advantage of the PEz protocol is the comparatively few working-steps required, which is both time efficient and reduces potential contamination. The LC is essential since it ultimately determines the possibility to visually detect sought microplastic particles. LC of the PEz protocol was classified as acceptable for both filter sizes already after over-night exposure. But since LC20 (PEz) was higher than LC300 (PEz) and also higher than LC20 of the control, the configuration of the PEz protocol 133
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extraction protocol as also emphasized by Karlsson et al. (2017).
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The use of potassium hydroxide (KOH) solution as a suitable approach to isolate plastics ingested by marine organisms. Mar. Pollut. Bull. 115, 86–90. https://doi.org/10.1016/j.marpolbul.2016.11.034. Ladewig, S.M., Bao, S., Chow, A.T., 2015. Natural fibers: a missing link to chemical pollution dispersion in aquatic environments. Environ. Sci. Technol. 49, 12609–12610. https://doi.org/10.1021/acs.est.5b04754. Li, W.C., Tse, H.F., Fok, L., 2016. Plastic waste in the marine environment: a review of sources, occurrence and effects. Sci. Total Environ. 566–567, 333–349. https://doi. org/10.1016/j.scitotenv.2016.05.084. Lobelle, D., Cunliffe, M., 2011. Early microbial biofilm formation on marine plastic debris. Mar. Pollut. Bull. 62, 197–200. https://doi.org/10.1016/j.marpolbul.2010. 10.013. Löder, M.G.J., Imhof, H.K., Ladehoff, M., Löschel, L.A., Lorenz, C., Mintenig, S., Piehl, S., Primpke, S., Schrank, I., Laforsch, C., Gerdts, G., 2017. Enzymatic purification of microplastics in environmental samples. Environ. Sci. Technol. 51, 14283–14292. https://doi.org/10.1021/acs.est.7b03055. Lusher, A.L., Welden, N.A., Sobral, P., Cole, M., 2017. Sampling, isolating and identifying microplastics ingested by fish and invertebrates. Anal. Methods 9, 1346–1360. https://doi.org/10.1039/C6AY02415G. Obbard, R.W., Sadri, S., Wong, Y.Q., Khitun, A.A., Baker, I., Thompson, R.C., 2014. Global warming releases microplastic legacy frozen in Arctic Sea ice. Earth's Future 2, 315–320. https://doi.org/10.1002/2014EF000240. OSPAR, 2015. International Council for the Exploration of the Sea, L., 2015. OSPAR Request on Development of a Common Monitoring Protocol for Plastic Particles in Fish Stomachs and Selected Shell Fish on the Basis of Existing Fish Disease Surveys. ICES Advice (Book 1, 1.6.6.1). Railo, S., Talvitie, J., Setälä, O., Koistinen, A., Lehtiniemi, M., 2018. Application of an enzyme digestion method reveals microlitter in Mytilus trossulus at a wastewater discharge area. Mar. Pollut. Bull. 130, 206–214. https://doi.org/10.1016/j. marpolbul.2018.03.022. Rist, S., Steensgaard, I.M., Guven, O., Nielsen, T.G., Jensen, L.H., Møller, L.F., Hartmann, N.B., 2018. The fate of microplastics during uptake and depuration phases in a blue mussel exposure system. Environ. Toxicol. Chem. 1–24. https://doi.org/10.1002/etc. 4285. Rochman, C.M., Tahir, A., Williams, S.L., Baxa, D.V., Lam, R., Miller, J.T., Teh, F.-C., Werorilangi, S., Teh, S.J., 2015. Anthropogenic debris in seafood: plastic debris and fibers from textiles in fish and bivalves sold for human consumption. Sci. Rep. 5, 14340. https://doi.org/10.1038/srep14340. Salvador Cesa, F., Turra, A., Baruque-Ramos, J., 2017. Synthetic fibers as microplastics in the marine environment: a review from textile perspective with a focus on domestic washings. Sci. 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5. Conclusion and recommendations An efficient and gentle enzymatic digestion protocol, applying pancreatic enzymes and Tris, was successfully developed and applied for the extraction of microplastics from the bivalve Serripes groenlandicus. Considering the promising results of the protocol in terms of i) simplicity to perform, ii) low cost, iii) time efficiency, iv) efficient digestion of tissue, v) no impact on pre-weathered polymers and textile fibers vi) low toxicity in terms of human and environmental health, it is suggested as a promising candidate to the highly needed standardized method for investigating occurrence of microplastics in bivalves. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2019.03.016. Declarations of interest None. Role of financial sources in the process None. Data behind this research note is available upon request. CRediT authorship contribution statement Lisa W. von Friesen: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing - original draft, Writing - review & editing. Maria E. Granberg: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing - original draft, Writing - review & editing. Martin Hassellöv: Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Writing - review & editing. Geir W. Gabrielsen: Funding acquisition, Investigation, Methodology, Supervision, Writing - review & editing. Kerstin Magnusson: Conceptualization, Data curation, Funding acquisition, Methodology, Supervision, Validation, Writing - original draft, Writing - review & editing. Acknowledgements We thank Haakon Hop, Mikko Vihtakari and Piotr Kuklinski at the Norwegian Polar Institute for their assistance with bivalve collection, as well as the crew of the MOSJ expedition with R/V Lance 2017 for their support. We would also like to thank Lars Ljungqvist at the Sven Lovén Centre for Marine Infrastructure for support regarding equipment and instruments. We are grateful for advice during FTIR-analyses from Therese Karlsson at the University of Gothenburg and Sinja Rist at the Technical University of Denmark. Thanks are also directed to France Collard at the Norwegian Polar Institute for helpful comments on the manuscript, and to the constructive feedback received from reviewers. Funding This work was supported by JPI-Oceans Baseman [grant number 942-2015-1863], JPI-Oceans Plastox [grant number 942-2015-1862], BONUS Micropoll [grant number 2015-122], VINNOVA [grant number 2017-00001] and the ecotoxicology section of the Norwegian Polar Institute, Tromsø, Norway. References Barrows, A.P.W., Cathey, S.E., Petersen, C.W., 2018. Marine environment microfiber contamination: global patterns and the diversity of microparticle origins. Environ.
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Note
An efficient and gentle enzymatic digestion protocol for the extraction of microplastics from bivalve tissue
T
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Lisa W. von Friesena, , Maria E. Granberga, Martin Hassellövb, Geir W. Gabrielsenc, Kerstin Magnussona a
IVL, Swedish Environmental Research Institute, Kristineberg 566, SE-45178 Fiskebäckskil, Sweden University of Gothenburg, Department of Marine Sciences, Kristineberg 566, SE-45178 Fiskebäckskil, Sweden c Norwegian Polar Institute, Hjalmar Johansensgate 14, N-9296 Tromsø, Norway b
A R T I C LE I N FO
A B S T R A C T
Keywords: Microplastics Enzymatic digestion protocol KOH Pancreatic enzymes Tissue digestion Emerging pollutants
Standardized methods for the digestion of biota for microplastic analysis are currently lacking. Chemical methods can be effective, but can also cause damage to some polymers. Enzymatic methods are known to be gentler, but often laborious, expensive and time consuming. A novel tissue digestion method with pancreatic enzymes and a pH buffer (Tris) is here presented in a comparison to a commonly applied digestion protocol with potassium hydroxide. The novel protocol demonstrates a highly efficient removal of bivalve tissue (97.7 ± 0.2% dry weight loss) already over-night. Furthermore, it induces no impairment in terms of ability to correctly identify four pre-weathered plastic polymers and six textile fiber polymers by Fourier transform infrared spectroscopy after exposure. The high-throughput protocol requires minimal handling, is of low cost and does not pose risk to the performer or the environment. It is therefore suggested as a candidate for a standardized digestion protocol, enabling successful analysis of microplastics ingested by bivalves.
1. Introduction A rapid increase in the number of studies investigating environmental occurrence of microplastics is currently being seen. As the number of studies grows, so does the global record of microplastic pollution; from urban coastal surface waters to remote deep ocean floors (Bergmann et al., 2017a; Li et al., 2016). Improved knowledge on the occurrence and fate of microplastics in the marine environment is essential to assess bioavailability and potential effects in marine ecosystems. This knowledge is also needed for implementation of proper measures and determination of environmental quality standards. Several analytical challenges currently hamper investigations of microplastic pollution in environmental matrices, affecting both accurate quantification and comparability between studies (Lusher et al., 2017; Wesch et al., 2016). Ultimately, the sought microplastic particle shall be successfully and representatively separated from the sample matrix without risk of harming or misplacing the particle, or contaminating the sample (Fig. 1). In order to achieve a best practice method, factors such as work intensity, time efficiency, cost, as well as both human and environmental health in terms of exposure to chemicals also need to be considered. The latter aspect is particularly important when developing
methods suitable for large-scale monitoring purposes and routine analysis. In the relatively new research field of microplastics, there is an urgent need for standardization of sampling, quantification and interpretation of results to obtain comparable data across studies (OSPAR, 2015; Vandermeersch et al., 2015). When analyzing the occurrence of microplastics in organisms, digestion of tissue is a commonly applied method and has often been achieved through the use of strong oxidizing acids (e.g. HNO3; Claessens et al., 2013), strong bases hydrolyzing or saponification of fat tissues (e.g. potassium hydroxide (KOH); Foekema et al., 2013) or combinations of hydrolyzing and oxidizing chemicals such as KOH and NaClO (Enders et al., 2017). The use of KOH has been recommended as sufficient for removing biological material (Dehaut et al., 2016; Kühn et al., 2017), but the trade-off of the protocol is between the required exposure time and induced impact on polymers. A time-efficient removal of tissue is obtained by KOH at 60 °C over-night (Rochman et al., 2015), however this has been found to have deleterious impact on some polymers (Dehaut et al., 2016; Lusher et al., 2017). KOH in room temperature has also been verified efficient in removing tissue and presenting no visible impact on tested polymers by Foekema et al. (2013), but both polyethylene terephthalate (PET) and polycarbonate is
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Corresponding author at: IVL, Swedish Environmental Research Institute, Nordenskiöldsgatan 24, SE-21119 Malmö, Sweden. E-mail addresses: [email protected] (L.W. von Friesen), [email protected] (M.E. Granberg), [email protected] (M. Hassellöv), [email protected] (G.W. Gabrielsen), [email protected] (K. Magnusson). https://doi.org/10.1016/j.marpolbul.2019.03.016 Received 30 January 2019; Received in revised form 8 March 2019; Accepted 8 March 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.
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Eliminating the matrix
No impact on sought particles
No contamination of unwanted particles
Large-scale applicability
Successful identification of all microplastic particles
Time efficiency
Environmental health
Human health
Cost efficiency
Fig. 1. Requirements for a microplastic extraction protocol. Suggested requirements of a standardized protocol for the extraction of microplastics from environmental matrices applicable on a wider scale.
1
classified as non-resistant to this treatment as compiled from tabulated data by Lusher et al. (2017). When specifically focusing on impact on textile microfibres, substantial visual (morphological) change has been reported for PET, polylactic acid and modacrylic after four days of exposure to KOH (personal communication: Piarulli, S.). Considering the high amount of microfibers commonly being reported in environmental samples (e.g. Barrows et al., 2018; Railo et al., 2018; Salvador Cesa et al., 2017), the KOH protocol may not be advisable. The exposure time of four days up to three weeks may not be practically feasible for large scale routine-studies. Several enzymatic digestion protocols have been proposed to provide efficient digestion of tissue as well as low impact on exposed polymers (Catarino et al., 2017; Cole et al., 2014; Courtene-Jones et al., 2017; Löder et al., 2017; Railo et al., 2018; Rist et al., 2018). However, a frequently occurring drawback is the labor intensity associated with the many working steps included (e.g. Cole et al., 2014; Löder et al., 2017). This could markedly increase the risk of contamination as well as loss of particles (Lusher et al., 2017), subsequently decreasing applicability of the protocol. Some industrial enzymes are very expensive (e.g. Proteinase-K; Cole et al., 2014), several of the mentioned enzymatic protocols have been evaluated solely on smaller sized organisms (e.g. 0.15–0.33 g; Railo et al., 2018) and some require long exposure times (e.g. up to 15 days; Löder et al., 2017). Additionally, already weathered reference plastic particles have seldom been used when investigating the potential impact of digestion protocols on polymers. Considering that weathered microplastics are the ones present in environmental samples and ultimately the targeted particles, exclusion of these bring risk of underestimating potential impact induced by the digestion protocol in question. The main aim of this study was to evaluate the applicability of commercially isolated pancreatic enzymes (PEz) as a novel digestion method for the extraction of microplastics from bivalve tissues. The aim was also to experimentally compare the digestion efficiency of the PEz protocol to the commonly used digestion protocol of KOH (e.g. Foekema et al., 2013), both in terms of removal of biological material and required exposure time. Furthermore, the study was aimed at evaluating potential impact by the novel PEz protocol on both weathered polymers collected in the field and on different textile fibers, to further ensure the protocol's suitability of treating environmental samples and successfully identify the microplastic particles present.
2
3
a
c
b
4
a
b
Fig. 2. Level of coverage. The four different levels of coverage on filters after digestion treatment and filtration. 1) Serripes groenlandicus before any digestion treatment (scale in cm), 2) Level 1 = all/most tissue digested, whole mesh of filter possible to see, very easy to analyze, 3) a: Level 2 = low load of undigested tissue on the filter, easy to analyze, b: Level 3 = filter partly covered by undigested tissue, hard but possible to analyze, c: Level 4 = filter fully covered by undigested tissue, not possible to analyze. 4) A comparison of digestion results over-night, a: KOH (potassium hydroxide), an example of level 4 coverage where the whole organism is still present after treatment, b: pancreatic enzymes with Tris, an example of level 1 coverage.
2. Methods The study consisted of two parts; I) an evaluation of the relative tissue digestion efficiency and microplastic recovery rate of the pancreatic enzyme (PEz) and potassium hydroxide (KOH) digestion protocols, and II) an evaluation of the potential impact on different plastic polymers caused by the PEz protocol. The chosen test organism was the 130
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digestion protocols: digestion efficiency in terms of DW loss (DE, %), level of coverage (LC) which refers to the remaining level of undigested material on filter after treatment (scale 1–4; Fig. 2), and recovery rate of the 40 spiked microplastic particles (RR, %). The DE was calculated as the percentage of DW loss during treatment (Eq. 1, adjusted from Courtene-Jones et al., 2017 to be based on DW), based on the established WW/DW ratio. The total DW remaining on both filters (300 and 20 μm) was used for calculations.
Greenland Smoothcockle (Serripes groenlandicus) (Bruguiere, 1789), which is a relatively large Arctic bivalve species (Fig. 2). Individuals included in this study ranged between 6 and 35 g wet weight (WW) without shell. 2.1. Part I: comparison between the two digestion protocols Individuals of S. groenlandicus were collected via scuba diving at the island Prins Karls Forland, Svalbard (78.7946667°, 011.0013333°), and frozen (−20 °C) until further analysis. Upon analysis, the mussels were thawed, shells removed and WW recorded. The mussels were individually placed in clean glass jars. To establish the WW/dry weight (DW) ratio, mussels were oven dried at 105 °C until constant weight was reached (n = 2). For reference plastic polymers, floating pieces of macroplastics were collected by hand from the sea surface north of the island Nordaustlandet, Svalbard (80.9000000°, 022.1253333°), and stored dark and cold until further analysis. These ocean collected plastics have naturally been subjected to weathering, photo-degradation and biofouling, factors that influence the properties of plastics (Lobelle and Cunliffe, 2011). Thus, a higher similarity to actual microplastics in the environment is ensured, compared to when using virgin plastics. Two different types of low-density polyethylene (LDPE) particles were created out of the collected macroplastics for part I of the study; from multifilament fishing rope (LDPE-F) and plastic packaging (LDPE-P). LDPE-F was cut into 300–600 μm pieces and LDPE-P was cut into 20–300 μm pieces using a metal scalpel under a stereomicroscope (Leica, M205C). The two sizes were chosen in order to set up the quantification of recovery rates as environmentally realistic as possible. All samples were spiked with 20 LDPE-F and 20 LDPE-P particles respectively, which were mixed into the sample prior to digestion treatment. The two protocols (PEz and KOH) were evaluated at four exposure times; over-night, two, four and eight days (n = 3). Samples were weight-matched between the two protocols and time exposures, i.e. the average weight for protocol*time was kept within ± 2 g WW difference. The pancreatic enzyme used was the commercially available Creon® 40,000 (Abbott Laboratories GmbH, Germany, Mylan), originating from porcine pancreas, containing lipase (40,000 Ph.Eur), amylase (25,000 Ph.Eur) and protease (1600 Ph.Eur) as active substances. The content of the enzyme capsules was retrieved and added to the tissue samples together with Tris (tris(hydroxymethyl)aminomethane)/Tris hydrochloride solution (Trizma®, pH 8.0, 1 M, 0.2 μm filtered, Sigma-Aldrich, T3038, USA). PEz was added to a concentration of 0.05 g PEz per g WW of tissue (cost of Creon®: ~0.05 € per g WW to digest). The amount of Tris was added to reach and maintain a sample pH of 8.0 ± 0.1, which is the optimal performing range of pancreatic enzymes (Berdutina et al., 2000) (pH-Fix 7.0–14.0, Macherey-Nagel) (roughly: 0–15 g WW: 10 ml, 15–20 g WW: 15 ml, 20–35 g WW: 20 ml), and to ensure that the solution covered the organism. In the KOH treatment (1 M, Eka Nobel, Sweden), an equivalent ratio between tissue WW and volume of KOH solution as for the PEz protocol was applied. Control samples (n = 3) of PEz and Tris without any bivalve tissue were included to validate potential influence of PEz addition on the final filter weight. Samples treated with PEz were incubated at 37.5 °C on a shaking table at 126 rpm (Innova 40, Incubator Shaker Series, New Brunswick Scientific), whereas KOH treated samples were kept in room temperature on a shaking table at 126 rpm. Upon reaching the respective experimental exposure time, samples were vacuum filtered in sequence through 300 and 20 μm nylon filters (Sefar Nitax) and thoroughly rinsed in slightly warmed Milli Q-water (~40 °C, 18.2 MΩcm TC, 0.22 μm, Millipore). The filters were pre-weighed (accuracy 0.1 mg, Mettler Toledo XP205) after being dried for 24 h at 50 °C, and after filtering they were again dried at 50 °C until constant weight was reached. Three variables were measured to quantify the efficiency of the two
DE (%) =
DWB − DWA × 100 DWB
(1)
where DE is digestion efficiency (%), DWB represents the calculated DW before digestion (g) and DWA the measured DW after digestion (g). The resulting LC following digestion was graded into four levels; 1) all/most tissue digested, whole mesh of filter possible to see, very easy to analyze, 2) low load of undigested tissue on the filter, easy to analyze, 3) filter partly covered by undigested tissue, hard but possible to analyze, 4) filter fully covered by undigested tissue, not possible to analyze (Fig. 2). LC up to level three was considered acceptable for a reliable analysis of microplastics on the filters. The RR was calculated as the percentage of the 40 spiked microplastic particles recovered after treatment. Identification of the particles was performed under a stereomicroscope (Leica, M205C). 2.2. Part II: evaluation of potential impact on polymers induced by pancreatic enzymes An exposure test was carried out to investigate whether PEz altered the chemical composition of microplastic polymers to an extent that could hamper correct identification. Ten particles types in total were exposed to PEz and Tris over-night under the same conditions as in part I, except the presence of bivalve tissue. Weathered fragments of LDPE, expanded polystyrene (ePS) and polypropylene (PP) were created from collected marine debris. The LDPE is represented by the two types of spiking particles in part I of the study (LDPE-F and LDPE-P). In addition to weathered fragments, textile fibers (non-weathered) of polylactic acid (PLA), polyethylene terephthalate (PET), polyester (also PET), nomex, PP and modacrylic were also exposed to the PEz protocol. The quantified variable was the correlative matching rate of Fourier-Transform infrared (FTIR, ThermoScientific, Nicolet iN10) spectroscopy absorbance spectra between the respective particle type before and after exposure to PEz and Tris over-night. This evaluation is comparable to Catarino et al. (2017) who performed a similar before and after comparison of FTIR-spectra of four different polymers. However, here we further quantified the correlative matching rate of spectra between original and exposed particle type. A library with absorbance spectra (n = 5) was created for each type of plastic particle before PEz exposure. The FTIR was run in reflectance mode with 256 scans (resolution 4 cm−1, spectral range 4000–675 cm−1, detector cooled by liquid nitrogen) corrected against 256 background scans. After over-night exposure, the absorbance spectra of particles (n = 3) were matched through library search (OMNIC Picta) to the respective created library, and the correlative matching rate (%) to the initial library of spectra was noted. An acceptable matching rate was set to > 90%, which is markedly higher than limits commonly applied in microplastic identification studies (e.g. Bergmann et al., 2017b; Obbard et al., 2014). This is to take height for the fact that actual environmental microplastics can be expected to be even more degraded within organisms' bodies, further complicating correct identification. Particles were pre-washed in ethanol (96%) and air dried before FTIR analysis to remove excess water and potential remains from the exposure to PEz. 2.3. Data analysis Initially, descriptive statistics were generated for all data to test 131
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Table 1 Resulting digestion efficiencies, level of coverage and recovery rates. Digestion efficiency (% dry weight loss during treatment), level of coverage (scale 1–4, 300 μm/ 20 μm filters) and recovery rate (% recovery of spiked microplastic particles) at four different exposure times for the two evaluated protocols; pancreatic enzymes with Tris (pH 8.0, 1 M) and potassium hydroxide (KOH) solution (1 M).
Digestion efficiency (%)
a
Level of coverage (scale 1–4)a,b (300 μm/20 μm)
Exposure time
Pancreatic enzymes + Tris
KOH
Over-night 2 days 4 days 8 days
97.7 97.3 94.8 96.5
33.1 40.2 52.7 59.7
Over-night 2 days 4 days 8 days
1.5 1.0 2.0 2.0
Recovery rate (%)a,c
± ± ± ±
± ± ± ±
0.2 1.6 3.1 1.3
0.7/2.5 0.0/2.0 1.0/2.3 1.4/3.0
87.0 ± 5.9
± ± ± ±
0.7 0.0 0.6 0.0
4.0 3.7 4.0 4.0
± ± ± ±
± ± ± ±
5.5 13.4 9.2 5.3
0.0/2.3 0.6/2.7 0.0/2.0 0.0/2.3
± ± ± ±
0.6 0.6 0.0 0.6
75.0 ± 11.5
a
Data is presented as average ± 1 standard deviation (n = 3). Level 1 = all/most tissue digested, whole mesh of filter possible to see, very easy to analyze, Level 2 = low load of undigested tissue on the filter, easy to analyze, Level 3 = filter partly covered by undigested tissue, hard but possible to analyze, Level 4 = filter fully covered by undigested tissue, not possible to analyze. c Based on the best performing exposure time for the respective protocol: over-night for the pancreatic enzyme treatment and eight days for the KOH treatment. b
with protocol PEz over-night. The following material in a wide variety of particle shapes and colors were successfully identified with FTIR: wool, cotton, cellophane, polyurethane (PU), acrylic, polystyrene, polyamide, polypropylene, polyethylene terephthalate, LDPE, highdensity PE and paint (unpublished data). To minimize potential contamination, all work was performed in a clean air cabinet (Clean Air Techniek B.V.) with equipment rinsed two times in Milli-Q water and covered in aluminum foil at all times possible. 100% white cotton laboratory coats were used at all times. Procedural contamination controls (n = 5) were performed to enable quantification of contamination, with a resulting average number of 1 ± 0.6 microlitter particles (synthetic and non-synthetic) per sample. The following materials were detected in the procedural contamination controls: cotton, cellophane, PU and acrylic.
normality (Shapiro Wilk) and evaluate whether parametric or nonparametric tests were suitable. If normality and homogenous variances (Levene's test) were met, one-way analysis of variance was performed. If non-normality was discovered, Mann-Whitney U Test or KruskalWallis tests for independent samples with subsequent Bonferroni corrections of pairwise comparisons were applied. The statistical analyses were performed in SPSS (IBM, 24) and statistical significance was attributed to p < 0.05.
3. Results 3.1. Part I: comparison between the two digestion protocols The digestion efficiency (DE) of the pancreatic enzyme protocol (PEz) was very high, 97.7 ± 0.2%, already after an over-night exposure, whereas the DE of the KOH (potassium hydroxide) protocol reached its maximum DE of 59.7 ± 5.3% after 8 days of exposure (Table 1). The DE of protocol PEz was higher than the DE of protocol KOH for all exposure times tested (Mann-Whitney U, p = 0.000). The addition of PEz did not affect the final weight of the filters (MannWhitney U, p = 0.937). The level of coverage (LC) on 300 μm filters (LC300) for protocol KOH was never classified as acceptable (i.e. LC300 > 3 at all times) whereas for protocol PEz, it was acceptable already after an over-night exposure (1.5 ± 0.7) (Table 1). The LC of 20 μm filters (LC20) for both protocols were acceptable after an over-night exposure (protocol PEz = 2.5 ± 0.7, protocol KOH = 2.3 ± 0.6). LC300 was higher in protocol KOH for all exposure times compared to protocol PEz (Kruskal Wallis, p = 0.000) and to the control (Kruskal Wallis, p = 0.010), but no difference was found between protocol PEz and the control (Kruskal Wallis, p = 1.000). LC20 was higher than the control for all exposure times in both protocol KOH (Kruskal Wallis, p = 0.012) and protocol PEz (Kruskal Wallis, p = 0.008), and no difference was found between the two protocols (Kruskal Wallis, p = 1.000). There was no difference in the total recovery rate (low-density polyethylene from fishing rope and plastic packaging: LDPE-F + LDPEP) between protocol KOH, protocol PEz and the control (One-way analysis of variance (ANOVA), F2;13 = 3.504, p = 0.061) (Table 1). Similarly, no differences were found between protocol KOH, PEz or the control in the recovery rate of LDPE-P or LDPE-F individually (LDPE-P: One-way ANOVA, F2;13 = 1.877, p = 0.192; LDPE-F: Kruskal-Wallis, independent samples, p = 0.108). Additional analysis of actual anthropogenic microlitter particles present within Serripes groenlandicus (not the same individuals as used for the protocol evaluation), was successfully performed after digestion
3.2. Part II: evaluation of potential impact on polymers induced by pancreatic enzymes The novel digestion protocol here presented did not alter any of the exposed polymers to an extent where the identifiability was impaired, i.e. the average correlative matching rate of exposed particles to the library of original spectra was > 90% for all particle types (Figs. 3, 4). Furthermore, no visual (morphological) change in shape or color was observed on exposed LDPE-F or LDPE-P plastic particles (Supplemental material 1). 4. Discussion This study clearly showed that pancreatic enzymes (PEz) in combination with Tris were effective in digesting tissue of Serripes groenlandicus for microplastic analysis. The novel protocol was superior to potassium hydroxide (KOH) digestion both in terms of removal of organic matter and time requirement. Since the KOH protocol never reached an acceptable digestion efficiency (DE) or level of coverage on 300 μm filters (LC300) during the exposure times used in this study, it is concluded to not be suitable for extraction on these premises. The KOH protocol resulted in equally high recovery rates (RR) of the spiked microplastics as the PEz protocol and the control. This result may, however, be somewhat misleading since the spiked particles were added to the sample but not incorporated in the organism itself as natural microplastic particles would have been. Any naturally incorporated (non-spiked) microplastic particles within the organism would not have been extracted with protocol KOH since the tissues frequently remained entirely undigested throughout the treatment (Fig. 2). The lack of digestion also explains the low level of coverage on 132
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used in this study is suggested for analysis of microplastic particles > 20 μm. For smaller fractions, further evaluation and optimization of the protocol could be needed. The fact that some residues of organic matter still remain after treatment is not considered to interfere with the ability of a correct visual quantification of microplastics. The here presented PEz protocol can most likely be optimized to fit other types of tissues and larger samples than those evaluated in the present study. However, since bivalves are commonly used within marine monitoring (OSPAR, 2015), the presented configuration is suggested to be widely applicable. The RR of pre-weathered spiked microplastics averaged 87.0 ± 5.9%, which is in line with, or slightly below, previously reported RR of microplastic particles originating from virgin plastics or plastics from everyday items, e.g. (93 ± 10%: Catarino et al., 2017; 100%: Dehaut et al., 2016; 84.5 ± 3.3%: Löder et al., 2017). However, particles used in the current study (low-density polyethylene from fishing rope and plastic packaging, LDPE-F: 300–600 μm, LDPE-P: 20–300 μm) were smaller than in previous studies and it is acknowledged that smaller sized microplastics are easier to misplace. Since the here measured RR did not differ between the two protocols or the control treatment, it indicates that handling may lead to misplacement of particles rather than remaining undigested tissue hampering detection. Consequently, there is room for some improvement regarding handling routines and the equipment used in the proposed method. The degree of underestimation of microplastic concentrations in environmental samples is seldom addressed in reports or scientific articles but could easily be implemented based on RR in the applied systems, and should further take care for potential size- and polymer-dependent underestimations. Even though weathered plastics and some textile fibers known to be more susceptible to degradation were used in this investigation (e.g. polyethylene terephthalate (PET): Lusher et al., 2017; PET, polylactic acid & modacrylic: personal communication Piarulli, S.), we did not observe any detectable effects induced by the PEz protocol on the plastic polymers that could impair correct identification or quantification. Nevertheless, it is acknowledged that the degradation state of real microplastics within biota in the environment could be higher or different than the ones tested. To increase the environmental realism, future studies investigating the impact on plastic polymers by various digestion protocols should include field collected particles. Considering their high presence when reported, future investigations should additionally include non-synthetic anthropogenic materials that so far have been largely overlooked in studies of microlitter (Barrows et al., 2018; Ladewig et al., 2015). Because anthropogenic materials (also non-plastics) contain e.g. chemical additives and/or pigments, it is important to be able to quantify all types of anthropogenic microlitter in the environment (Barrows et al., 2018; Ladewig et al., 2015). Without this information it is not possible to obtain an accurate exposure scenario for risk assessment of anthropogenic microlitter, which has been acknowledged by the European Commission (2010/477/EU) and further addressed by Railo et al. (2018). The additional types of microlitter that were successfully identified in S. groenlandicus also included non-synthetic materials (unpublished data), which further supports the applicability of the presented PEz digestion method. Beyond the present study, the developed PEz protocol has successfully been applied to several other species for the analysis of microplastics (other bivalve species, gastropods, amphipods, gastrointestinal tracts of fish) (unpublished data). When analyzing crustaceans, parts of the exoskeleton can remain undigested, although they are easily distinguishable and will likely not hamper visual identification. Slight modifications of the protocol configuration have been made for the different species, as expected and previously observed by Löder et al. (2017) when applying an enzymatic digestion protocol on various matrices and species. Sample specific properties as well as the scientific question to be answered will ultimately determine the most suitable
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Fig. 3. Evaluation of identifiability after treatment. Average matching rates (%) between original Fourier transform infrared spectroscopy spectrum and after an over-night exposure to the pancreatic enzyme digestion protocol for the respective particle types tested. Data is presented as average ± 1 standard deviation (n = 3). Note that the y-axis is broken. Dashed line marks the acceptable matching rate of 90%. Weathered polymers are indicated with (w), those without a marking within parenthesis are non-weathered textile fibers. PET: polyethylene terephthalate, PLA: polylactic acid, PP: polypropylene, ePS: expanded polystyrene, LDPE: low-density polyethylene (F: fishing rope, P: plastic packaging).
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Fig. 4. Fourier transform infrared spectroscopy (FTIR) evaluation. An example of FTIR spectra before and after exposure of low-density polyethylene from fishing rope (LDPE-F) (pre-weathered) to the developed pancreatic enzyme digestion protocol. The FTIR spectra are baseline corrected at the following wavenumbers; 3959, 3738, 3522, 3229, 2445, 2100, 1847, 839 and 677.
20 μm filters (LC20). The application of the PEz digestion protocol was successful without any pre-treatment such as separation of tissue into smaller pieces and resulted in a low LC which allowed successful visual detection of anthropogenic microlitter. The DE was satisfactory already after over-night exposure (97.7 ± 0.2% dry weight (DW) loss). In comparison to previously evaluated enzymatic digestion protocols, the DE of PEz was higher than the use of trypsin (88 ± 2.5% wet weight (WW) loss: Courtene-Jones et al., 2017) and similar to the use of Proteinase-K (> 97% DW loss: Cole et al., 2014). However, an important difference and advantage of the PEz protocol is the comparatively few working-steps required, which is both time efficient and reduces potential contamination. The LC is essential since it ultimately determines the possibility to visually detect sought microplastic particles. LC of the PEz protocol was classified as acceptable for both filter sizes already after over-night exposure. But since LC20 (PEz) was higher than LC300 (PEz) and also higher than LC20 of the control, the configuration of the PEz protocol 133
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extraction protocol as also emphasized by Karlsson et al. (2017).
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5. Conclusion and recommendations An efficient and gentle enzymatic digestion protocol, applying pancreatic enzymes and Tris, was successfully developed and applied for the extraction of microplastics from the bivalve Serripes groenlandicus. Considering the promising results of the protocol in terms of i) simplicity to perform, ii) low cost, iii) time efficiency, iv) efficient digestion of tissue, v) no impact on pre-weathered polymers and textile fibers vi) low toxicity in terms of human and environmental health, it is suggested as a promising candidate to the highly needed standardized method for investigating occurrence of microplastics in bivalves. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2019.03.016. Declarations of interest None. Role of financial sources in the process None. Data behind this research note is available upon request. CRediT authorship contribution statement Lisa W. von Friesen: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing - original draft, Writing - review & editing. Maria E. Granberg: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing - original draft, Writing - review & editing. Martin Hassellöv: Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Writing - review & editing. Geir W. Gabrielsen: Funding acquisition, Investigation, Methodology, Supervision, Writing - review & editing. Kerstin Magnusson: Conceptualization, Data curation, Funding acquisition, Methodology, Supervision, Validation, Writing - original draft, Writing - review & editing. Acknowledgements We thank Haakon Hop, Mikko Vihtakari and Piotr Kuklinski at the Norwegian Polar Institute for their assistance with bivalve collection, as well as the crew of the MOSJ expedition with R/V Lance 2017 for their support. We would also like to thank Lars Ljungqvist at the Sven Lovén Centre for Marine Infrastructure for support regarding equipment and instruments. We are grateful for advice during FTIR-analyses from Therese Karlsson at the University of Gothenburg and Sinja Rist at the Technical University of Denmark. Thanks are also directed to France Collard at the Norwegian Polar Institute for helpful comments on the manuscript, and to the constructive feedback received from reviewers. Funding This work was supported by JPI-Oceans Baseman [grant number 942-2015-1863], JPI-Oceans Plastox [grant number 942-2015-1862], BONUS Micropoll [grant number 2015-122], VINNOVA [grant number 2017-00001] and the ecotoxicology section of the Norwegian Polar Institute, Tromsø, Norway. References Barrows, A.P.W., Cathey, S.E., Petersen, C.W., 2018. Marine environment microfiber contamination: global patterns and the diversity of microparticle origins. Environ.
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