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Critical Review
Quality Criteria for the Analysis of Microplastic in Biota Samples. Critical review Enya Hermsen, Svenja Mintenig, Ellen Besseling, and Albert A. Koelmans Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01611 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018
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Environmental Science & Technology
Quality criteria for the analysis of microplastic in biota samples. Critical review
1 2 3 4 5
Enya Hermsen†,a, Svenja M. Mintenig‡,§,a, Ellen Besseling†,ǂ, Albert A. Koelmans†,ǂ,*
6 7 8 9 10 11 12 13 14 15 16 17 18
†
Aquatic Ecology and Water Quality Management Group, Wageningen University, The Netherlands. Copernicus Institute of Sustainable Development, Utrecht University, The Netherlands. § KWR Watercycle Research Institute, Nieuwegein, The Netherlands. ǂ Wageningen Marine Research, IJmuiden, The Netherlands. ‡
Total number of words
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Abstract: 168
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Abstract + text + acknowledgements: 6386
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Number of tables: 2
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Number of figures: 1
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Number of references: 88
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Supporting information: Yes
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a
contributed equally * corresponding author:
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TOC art
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Abstract
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Data on ingestion of microplastics by marine biota are quintessential for monitoring and risk
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assessment of microplastics in the environment. Current studies, however, portray a wide spread in
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results on the occurrence of microplastic ingestion, highlighting a lack of comparability of results
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which might be attributed to a lack of standardisation of methods. We critically review and evaluate
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recent microplastic ingestion studies in aquatic biota, propose a quality assessment method for such
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studies, and apply the assessment method to the reviewed studies. The quality assessment method
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uses ten criteria: Sampling method and strategy, Sample size, Sample processing and storage,
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Laboratory preparation, Clean air conditions, Negative controls, Positive controls, Target component,
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Sample (pre-)treatment, and Polymer identification. The results of this quality assessment show a
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dire need for stricter quality assurance in microplastic ingestion studies. On average studies score 8.0
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out of 20 points for ‘completeness of information’, and ‘zero’ for ‘reliability’. Alongside the
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assessment method, a standardised protocol for detecting microplastic in biota samples
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incorporating these criteria is provided.
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Keywords: marine plastic debris, microplastic, ingestion, analytical methods, quality assurance
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Introduction
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The ubiquity of microplastic (plastic particles < 5 mm1), combined with associated effects, has raised
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concerns regarding marine species, ecosystems, and the impact it may have on human health.
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Microplastic have been detected in a wide variety of habitats in the ocean, from shallow coasts to
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the deep sea.2-4 Increasing numbers of studies report the ingestion of microplastic by marine biota
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across multiple trophic levels, including animals often targeted by fisheries (Table 1).5-9 The ingestion
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of microplastics seemingly concerns a wider range of species than the ingestion of meso- and
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macroplastics; indeed, it is considered the most frequent interaction between plastic debris and
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marine organisms.10
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Ingested microplastic particles are thought able to evoke a biological response through both physical
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and chemical mechanisms, although many of these effects have yet to be studied. Ingestion of
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microplastics is thought to cause physical damage in small organisms2, and has been speculated to
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provide a pathway for some associated chemicals to enter and spread in the food web all the way up
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to humans, with microplastic particles as vectors.11-13 Additionally, ingestion by biota is considered a
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possible sink for microplastics.14 Therefore, measuring quantities of ingested plastic is of high priority
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in order to properly assess the risk of such hazards.
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Physical impacts for small organisms like internal abrasions and blockages have been reported.2
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Moreover, microplastic particles were shown to cause damage leading to cellular necrosis,
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inflammation, and lacerations of tissues in gastrointestinal tracts according to a review of plastic
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impact on biota.15 In bigger organisms, ingestion of larger objects (i.e. macroplastics) has been
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demonstrated too.16, 17
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In addition to the impact of ingested microplastics proper, persistent organic pollutants (POPs) may
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concentrate on the particles. It is suggested this could pose a possible new route for POPs to enter
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the food chain11,
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Contrarily, evidence in Northern Fulmars (Fulmarus glacialis) suggests a transfer of POPs from the
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lipids in the animal to the plastic, rather than the other way around.18
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The concerns for the impacts of microplastic are reinforced by the hypothesis that microplastics may
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be able to spread through the food web by means of trophic transfer, a phenomenon that has been
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observed in a few instances.21, 22 This is cause for concern especially in commercially valuable species,
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as it possibly poses a threat to human food-safety.23 To what extent this transfer occurs in the food
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web remains to be studied further.
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Despite these worries concerning microplastic ingestion, the effects in the natural environment and
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the implications for the food web remain poorly understood. Due to the absence of suitable
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standardized methods, data are too often incomparable, are not representative, and lack quality
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; however, it has not been irrefutably shown that this actually happens.18-20
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assurance.24-28 Hence, our knowledge on the fate and impacts of microplastics remains incomplete.
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The microplastic research field is young, and as research performed now lays down the foundations
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for later studies, there is a dire need for a standardised protocol for carrying out studies on the
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ingestion of microplastics by marine biota in order to mitigate this issue.27 Although first steps
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towards standardisation of methodologies in environmental samples are being made27,
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comparability of current data is being impeded by the wide variety of methodologies, which has led
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to data of different quality.24, 29 In order to deal with the wide spread in quality of the data produced
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by studies, an example can be taken from the field of toxicology. In toxicology, it is common practise
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to assess the reliability of studies with consensus criteria, like the so-called Klimisch score30, or the
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recently proposed CRED (Criteria for Reporting and Evaluating Ecotoxicity Data).31 These methods
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both offer scoring systems with different reliability categories, generating standardised
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documentation of validity evaluation. They were developed to guide risk assessors in performing
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unbiased, transparent, and detailed evaluations, while guiding researchers in performing and
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reporting studies in a manner deemed appropriate.31 We argue that research and risk assessment
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with respect to the impacts of plastic debris are in urgent need for the development and use of such
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criteria.32
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The aim of the present study is to critically review the literature on ingestion of microplastic by
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marine biota. Based on this review, we develop a scoring method for ecological studies and the
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analytical methodologies employed to detect plastic debris in aquatic biota samples. The scoring
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method is subsequently applied retrospectively to the reviewed studies. This assessment does not
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result in an absolute judgement, but is an indicator of the usefulness of these studies for risk
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assessment and monitoring purposes of microplastic ingestion in natural populations. We also
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provide average scores per evaluation criterion, illustrating which methodological aspects need
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improvements most. Finally, our synthesis provides the basis for a quality assurance protocol for the
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analysis of microplastic debris in biota samples.
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, the
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Methods
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An extensive literature review was undertaken by accessing the Web of Science, ScienceDirect, and
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Scopus databases for studies of microplastic ingestion in marine biota in natural populations,
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including studies from all years up until those published in June 2017. Queries included the following
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search terms: “microplastic AND ingestion AND marine”, “microplastic AND uptake AND marine”,
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“microplastic AND marine biota”, “microplastic AND biota AND monitor*”. Reference lists of the
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found articles, reviews, and ‘reversed searches’ were consulted as well, resulting in a representative
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collection of 35 currently available studies. Laboratory exposure experiments were excluded from 5 ACS Paragon Plus Environment
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the collection. Furthermore, studies were only included if they provided data on the ingestion of
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microplastic. For these studies, the ingestion incidence was calculated as the fraction of sampled
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individuals containing microplastic. The 95% confidence intervals for these binominal proportions
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were assessed using the Wilson method.33 Subsequently, studies were scored according to method
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quality criteria discussed in the next section. All studies were assessed by two separate authors
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independently, after which differences in scoring were discussed, and tuned until the assessment
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was done consistently across all studies. To maximize transparency and traceability, the scoring
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explanations, scoring criteria and scorings for all papers are provided as Supporting Information
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(Table S1, Table S2 and Table S3, respectively). The eventual assessments do not express the value of
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studies. They only reflect the compliance of studies to reliability criteria as perceived by the authors
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of the present paper, in hindsight. Although we maximized our effort to be complete and thorough in
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this process, misinterpretations or misjudgements cannot be completely excluded.
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The here presented scoring method was designed to assess current studies on reliability of their data
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on microplastic ingestion in marine field biota, and is based on several aspects that define a
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reproducible and controlled study. The method evaluates the inherent adequacy of the employed
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methods for monitoring and risk assessment purposes, relating to a standardised methodology, and
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the description of the procedure and results. By scoring high in all categories, a study can be defined
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as “reliable”, providing reproducibility, clarity, and plausibility of its findings.
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Quality Assessment System
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Previous scoring systems that have been proposed for assessing the reliability of ecotoxicology
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studies are the Klimisch30, and the more recent CRED scoring systems.31 The Klimisch criteria have
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received critique for being unspecific and for lacking essential criteria and guidance, leaving too
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much room for interpretation.31 The CRED evaluation method gives extensive guidance on how to
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use the set criteria, and gives recommendations for reporting.31 Following the example set by the
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CRED method, the present evaluation method for microplastic ingestion studies provides several
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criteria which must be assessed, including guidance on how to assess each criterion. The quality
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assessment method is made up of ten criteria: (1) Sampling method and strategy, (2) Sample size, (3)
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Sample processing and storage, (4) Laboratory preparation, (5) Clean air conditions, (6) Negative
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controls, (7) Positive controls, (8) Target component, (9) Sample (pre-)treatment, and (10) Polymer
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identification (Table 1). For each criterion, a score of 0, 1, or 2 can be assigned to the publication
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under review. Scores signify the following: 2= reliable without restrictions, 1= somewhat reliable, but
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with restrictions, 0= not reliable. If information is lacking on certain aspects in the publication this is
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considered unreliable, leading to a lower score. After each criterion is scored, an overall reliability 6 ACS Paragon Plus Environment
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score is calculated by taking the product of all criteria scores, resulting in a maximum attainable
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overall theoretical reliability score of 1024 points, indicating a high reliability of a publication. This
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contrasts with both the CRED and Klimisch method: these methods assign a category of reliability to
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each criterion, but do not quantify it with a score.30, 31 In the evaluation method presented here, the
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quantification through scoring is deemed important, because each criterion is considered crucial and
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equally important to the reliability of the results of a study. This means when a study scores zero
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points on a criterion, too much uncertainty still surrounds the results of the study, marking the
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results unreliable. This also means that when only one criterion is evaluated as “not reliable” (zero
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points) the overall reliability score of the study will be zero. Besides this overall reliability score, we
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provide an accumulated score; calculated as the sum of the individual scores. This score has a
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maximum of 20 points and can be seen as a combination of the reliability and the completeness of
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information in a publication.
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In the following ten paragraphs, argumentation is provided on each of the ten scoring categories,
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including explanation based on the currently reviewed studies, and specification of scoring criteria. A
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supporting, more detailed overview of the scoring criteria is provided as Supporting Information
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(Table S1, Table S2).
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Sampling methods and strategy - Several factors related to sampling method and strategy affect the
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results of microplastic detection in biota samples. For instance, due to differences in density, and
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sinking as a result of biofouling, plastic is found at different depths of the water column.10,
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Microplastics are also known to accumulate in the sediment28, 35, 36, with deep sea bottoms likely to
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make up a sink for the particles.34, 37, 38 It is plausible that feeding strategy has an influence on the
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type and amount of microplastic ingested39, 40, with planktivorous and filter feeders expected to be
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more susceptible to ingestion of low density particles floating in the top layers of the water column,
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while demersal and bottom dwelling species are more likely to encounter high density microplastics.
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Additionally, some species are known for diurnal vertical migration and are subjected to a wide
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variety of microplastic encountered, possibly affecting their ingestion rates. Non-ecological factors
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such as mesh size will have influence on life stage of the caught individuals in the sample, whereas a
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small mesh size could lead to cod-end feeding.41 Sampling methods can greatly influence the
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outcome of a study; therefore, it is important that such characteristics of the sampling are recorded,
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in order to create a reproducible study.28,
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interpret the outcome, and account for possible contamination in the results.
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In this section, studies are scored on reportage, and therefore reproducibility, of the sampling, but
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also on choice of sampling methods itself. Studies scoring high in this section reported extensively on
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their methods (e.g. type of gear, sampling location and depth) and controlled their own sampling, or 7
42
34
Also, by reporting such details it could be easier to
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were fully aware of what had happened to the specimens during sampling. Articles with low scores
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either failed to report on (parts of) their sampling (Table 2), or used, for instance, store bought
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individuals when making inferences on natural populations.43, 44 The use of store or market bought
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individuals is not inherently wrong, as long as the interest of the study lies on contamination of sea
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food, not on natural populations. Scores of 1 indicate that for part of the sample, sampling was not
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performed correctly, while for another part of the sample it was: the aim of the study should be
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correctly matched to the sampling method. For example, Vandermeersch et al. (2015)27 partially used
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store bought individuals, while using self-sampled ones for a different part of the study. The
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microplastic uptake in mussels from different estuaries was compared with the uptake by
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commercial mussels. The commercial mussels were bought in stores, leading to uncertainty about
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the treatment of these mussels prior to the analysis: microplastic found in these mussels could have
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originated from contamination during handling in the production chain, rather than from
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microplastic ingestion by the mussels themselves. Would the aim of this study have been to check
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microplastic content in store bought individuals (i.e. checking on general contamination, not
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ingestion) this would not have been an issue. This study scored 1 in this section, since part of the
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study can be considered reliable with sampling method correctly matched to the aim of the specific
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part of the study.
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Sample size - Both the International Council for the Exploration of the Sea ICES (2015)42 and the
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European Strategy Framework Directive’s Technical Subgroup on Marine Litter (MSFD-TSGML)
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(2013)45 recommend a sample size of at least 50 individuals. This sample size of 50 is arbitrarily
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chosen, since, due to the wide variety in microplastic ingestion reported by different studies, no clear
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indication of the true ingestion incidence of microplastic by biota can be estimated. When more
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clarity can be given in the future, this recommended sample size should be adjusted accordingly. If
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ingestion incidence appears to be low, higher sample sizes will be needed to give reliable results; if
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populations show high incidence of microplastic ingestion, lower sample sizes will suffice.
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The scoring in this category is fairly straightforward, using the recommended 50 individuals as a
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threshold until it is possible to perform a reliable power analysis in order to calculate a more
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appropriate sample size for ingestion studies. Too low a sample size may provide interesting data,
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but no conclusions should be drawn, as the statistical power of such a study simply would be too low
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to infer any trends. A larger sample size is always advisable, since it will lead to more reliable results,
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i.e. narrower confidence intervals (Figure 1). Studies with a sample size over 50 specimens taken
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from a food web or ecoregion scored 2. A score of 0 was ascribed to studies using less than 50
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specimens. Studies with > 50 specimens in total and > 25 specimens per research unit (e.g. a species,
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a food web or an ecoregion) received a score of 1. For now, we also applied these criteria to a study 8 ACS Paragon Plus Environment
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that reported the presence of microplastic in a single stranded whale,16 leading to a very wide
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confidence interval (Figure 1). However, for whales or for rare and protected species the n=50
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criterion is difficult or even unethical to achieve in a sampling effort meant to assess trends in
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microplastic ingestion. For such big or protected organisms, retrospective data obtained from
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stranded animals and from bycatch through different reports need to be combined in order to reach
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a sample size with sufficient rigour.17 This would require harmonisation of protocols in order to
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increase comparability of studies, for which guidance is beyond scope of the current review.
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We further advise provision of the confidence interval in the reported count (e.g.,5, 46); however, this
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was not yet included as criterion in the current scoring. Based on the total number of animals and
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the number of animals that ingested microplastics, we calculated the confidence intervals and
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provide an overview in Figure 1.
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Sample processing and storage - After sampling, samples need to be stored until examination in the
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laboratory. Samples are often frozen5, 9, 47, 48, or whole specimens of smaller species are preserved in
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fixatives such as formalin, ethanol, or formaldehyde.49-53 ICES (2015)42 recommends to store biota
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samples on board using aluminium foil for freezing at -20°C or preservation in ethanol in glass
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containers. In the present study it was not considered necessary to wrap each individual in
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aluminium foil, as long as specimens were quickly frozen after capture at -20°C, and stored in a
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closed container. If this is combined with a pre-examination rinse of the specimens (see “Laboratory
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preparation”), it should suffice in mediating contamination of the exterior of the specimen. Under no
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circumstance should the specimen be opened on board. This is considered as a high, and difficult to
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assess risk, for contamination due to unregulated conditions on board. We further recommend to
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avoid dissecting individuals outside clean air conditions at all times (see “Clean air conditions”).
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High scores were assigned to studies freezing their samples shortly after capture at -20°C or storing
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them on ice, leaving any further handling till the laboratory. Alternative methods storing the samples
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in closed off containers with a fixative were also given highest scores in case potential effects of
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these chemicals on different plastics were studied before application. Recently, the resistance of
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microplastics to formaldehyde/ethanol has been confirmed.49 Studies scoring low in this section
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performed dissections, or otherwise opened the specimens, on board. Middle scores again indicate
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some aspects of the study do not comply, but do partially meet the standards (e.g. different
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processing for different subsamples).
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Laboratory preparation - Contamination is a prevalent issue in microplastic research, creating
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uncertainty around the results of many studies.27, 28, 54 This risk and uncertainty have been dealt with
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in different ways. Different forms of prevention have been applied, in varying degrees of success. 9 ACS Paragon Plus Environment
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Foekema et al. (2013)6 decided to exclude small fibres from analyses after finding a sharply
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decreased abundance when working under clean air conditions. ICES (2015)42 proposed in their
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preliminary protocol to exclude all fibres smaller than 5 mm in length from results. Although this may
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provide a way to reduce the issue of contamination in results, it is less than ideal; by excluding all
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small fibres from results, truly ingested fibres will be excluded from the results too. This could lead to
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an underestimation of ingestion rates and a potential knowledge gap in the ingestion of microplastic.
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Therefore, proper prevention is needed. In the laboratory, contaminations with synthetic polymers
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should be avoided as they may influence ingestion results.6, 27 Equipment, tools and work surfaces
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should be free of particles, in order to avoid easy contamination. To this end all materials used
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should be washed and rinsed thoroughly with high quality water (e.g. Milli-Q water) before use, and
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preferably kept in a clean air cabinet.
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Factors such as clothing should be considered. Often, contamination arises in the form of
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microfibers.27,
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avoided by solely wearing 100% natural fibre clothing, such as cotton. Only wearing a 100% cotton
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lab coat may not suffice; if one was to wear a polyester shirt underneath, it would not be
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unimaginable that some fibres could end up in the samples. For the current scoring in this study, if all
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other precautions were met, a 100% cotton lab coat was considered sufficient.
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In some studies, precautions were made by wiping surfaces and tools using alcohol.55 This method is
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probably not thorough enough to deal with contamination; merely wiping surfaces, be it with alcohol
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or water, could still leave particles. They could be missed, detach from the wipe during wiping, or the
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wipe itself could even prove to be a source of contamination (i.e. the material, or dust already
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collected on the wipe before use). Rigorously washing and rinsing of the equipment are considered
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to be the only proper option here.
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Additional to the preparation of surfaces and tools, the sample specimens itself require some
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preparation. The exterior of the animal should be rinsed6, 46, and checked for contamination. In case
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of small specimens such as zooplankton, this is not an easy feat. In the study performed by Desforges
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et al. (2015)50 this issue was overcome by individually checking each specimen under a microscope,
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and picking off any external contamination with a pair of tweezers.
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In summary, a score of 2 was assigned when non-synthetic clothing and a lab coat were used, and
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equipment and organism exterior were rinsed. A score of 1 was assigned for solely wiping laboratory
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surfaces and equipment or not wearing a lab coat, as long as negative control samples were run in
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parallel and examined for contamination. A score of zero was assigned when no precautions were
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met.
28 28
Additional contamination originating from researchers’ clothing can easily be
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Clean air conditions - Problems with airborne contamination are unavoidable, unless work is
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performed in clean air conditions.6, 27, 28 To this end, sample handling should be done in a laminar
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flow cabinet42,
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during sample handling and analysis.28,
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research; any handling of samples outside clean air conditions creates a high risk of airborne
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contamination.57
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Other studies placed their samples in a fume hood in order to minimise the risk of contamination.56
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However, since a fume hood draws air from the room into the hood (contrarily to a positive pressure
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laminar flow cabinet, which blows filtered air through the cabinet into the room), the risk of airborne
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contamination remains.57
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A few studies were seen that mitigated contamination by closing off samples as much as possible,
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and handling them as fast as possible.44, 53 These methods are not fool proof, and should not be relied
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upon without further indication on results of negative samples treated in parallel to actual samples.
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The proper use of clean air conditions was given a score of 2. A score of 0 was assigned to studies
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taking no regard for airborne contamination. Studies mitigating contamination by carefully keeping
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samples in a closed off situation as much possible scored 1 in this category, provided that negative
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controls were run in parallel and examined for contamination.
46, 56
or in a “clean room”, which is designed to minimise airborne contamination 57
The use of such facilities is a necessity in microplastic
316 317
Negative controls - Although increasing in recent studies, the use of controls in microplastic research
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is not standard practise. During sample handling the chances of contamination by microplastic
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particles and fibres are high, thus, the use of controls, treated and analysed in parallel to actual
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samples, is crucial.
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For a study to score 2, proper blanks should be included for each batch of samples, with at least
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three replicate blanks per batch. These controls should be performed without tissue, or with tissue
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that was confirmed to be devoid of microplastic, in parallel with samples containing the target
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component.42,
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specimens. Controls should be run regularly and with special attention to moments of high risk of
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contamination, such as moving specimens in and out of the laminar flow cabinet.29 Also the visual
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examination of samples forms a moment of high risk, which is why additionally placed and examined
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petri dishes next to the sample might be advisable.46
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Scores of 1 indicate a blank analysis of some form, nevertheless deemed insufficient here. This
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includes, for instance, solely open petri dishes or soaked paper that were placed next to the work
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surface and checked for contamination,46,
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contamination deriving from used chemicals or equipment. Studies scored 0 when no form of
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negative control was included in the study.
58
By doing so, the controls are given the same full treatment as the studied
48
or the filtration of air. These do not account for
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Positive controls - It is generally difficult to assess whether all microplastics present in a sample are
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effectively recovered from that sample. Especially small particles may be overlooked or missed, and
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losses may occur during all steps of sample preparation, processing and analysis. Therefore, it is
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considered crucial to include controls (triplicate) with added microplastic particles that are treated in
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parallel to the samples, in order to determine the recovery rate (score of 2 points). Ideally, positive
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controls should be included also for the smallest targeted size class, and the limit in the detected size
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should be reported. We are aware of only three studies that included reliable positive controls.41, 46, 59
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Davison and Asch, for instance41, blindly added random numbers of spherical beads from two size
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classes into fish stomach contents, so that the researcher would not know this number, and were
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able to trace back all added particles to achieve 100% recovery. A score of 1 was assigned to studies
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with some form of a positive control (e.g. testing only a part of the protocol) and a score of zero was
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assigned when no positive controls were included.
347 348
Target component - Among the reviewed studies, different target components were described, that
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are mainly (parts of) the digestive tracts for larger biota like fish5, 6, 9, 46, 52, or whole specimens for
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smaller species, like bivalves27, 50, 60 Choosing a suitable target component is an important part of the
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study setup. For accurate estimation of microplastic ingestion, it is important to examine the entire
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gastrointestinal tract (GIT) (oesophagus to vent). By only examining the stomach, particles in the gut
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would be missed, leading to an underestimation of ingestion rate. When small animals such as
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bivalves and zooplankton are being studied, the entire specimen should be used.
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Studies examining full specimens or entire GITs received the highest score. Examination of parts of
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the GIT were scored lowest. In case a study examined a part of the GIT for a subsample, yet full GITs
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for the rest of the sample, it was scored 1.
358 359
Sample (pre-)treatment - To extract and characterise microplastics in biological samples, a digestion
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step is a crucial component, namely dissolving organic matter without degrading plastic polymers.
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Detection of microplastic in a biological sample without getting rid of the organic matter makes for
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an unreliable method; the chance of missing particles is high, especially small particles that are not
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visually detectable.27 Therefore, it is advised to make use of a digestion pre-treatment.42, 61
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Dehaut et al. (2016)62 performed a study testing six existing methods (including enzymatic, alkaline,
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and acidic digestion), comparing their effects on 15 different plastic polymers, as well as their
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efficiency in biological samples. Their tests showed that out of the six protocols, an adapted protocol
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of Foekema et al. (2013)6 was most successful. The original protocol involves the samples being left
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for digestion in 10% KOH solution and kept at room temperature for 3 weeks. The adapted protocol 12 ACS Paragon Plus Environment
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used 10% KOH solution, with 24 hours of incubation at 60°C.62 This adaptation was made in order to
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shorten the incubation time. The heating of samples during digestion pre-treatments to speed up the
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process is fairly common, and especially with acidic digestion methods this is often part of the
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protocol. However, this practise may be ill advised, since the heating of the samples could cause
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some microplastic particles to deform, or clump together.63 Therefore, it is advised to apply the
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original protocol of Foekema et al. (2013)6. The adequacy of the 10% KOH protocol has recently been
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confirmed by Kühn et al. (2017) 64 and Munno et al. (2018).63 However, for smaller organisms, like the
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soft tissue of mussels or plankton species, also enzymatic methods have been shown to provide high
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digestion rates with no damage to microplastic.65, 66
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Based on these findings, studies using a 10% KOH solution-based digestion, or an enzymatic digestion
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received the highest score of 2. Studies not incorporating a digestion step received no points. Studies
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using other digestion methods were scored 1. A score of 1 was also assigned to studies that did not
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need a digestion step because the size of particles was large enough, which can be achieved by
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sieving the samples over 300 µm. This mesh size allows adequate particle sorting as it is done
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frequently for e.g. water samples.67-69
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Polymer identification - Accurate identification of polymer types in environmental samples can be
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laborious. Hence, two aspects are relevant when assessing the polymer identities of a microplastic
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sample: (1) the quality of the method used for the identification (efficiency, sensitivity, accuracy,
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reproducibility) and (2) the quality of the selection of the subsample (representativeness).
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Polymer identity - Visual inspection (i.e. characterising microplastic by eye under a dissection or
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stereomicroscope) was found to be a frequently used identification method.8, 9, 47, 50, 56, 58, 70 However,
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visual examination cannot be used to identify the (polymer) identity of a particle. Without formal
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evidence of polymer identity, a particle cannot be reported as being a microplastic particle. The
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quality of visual examination is influenced by the observer, properties of the plastic, the targeted
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microplastic size, the magnification of the microscope, and the sample type.28 In a case study on
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microplastics in North Sea sediments, the usage of focal plane array (FPA) micro-Fourier transform
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infrared (micro-FTIR) spectroscopy revealed that only 1.4% of the particles visually sorted as
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microplastic actually were synthetic polymers.29 Fibres with a size over 500µm were found to be of
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natural origin after an initial selection as microplastic.28, 71 This uncertainty of visual identification
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further increases as particle size decreases which illustrates the importance of verifying the chemical
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origin of potential microplastics.
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To date, potential microplastics are identified mostly using spectroscopic29,
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degradation analyses.73-75 Particles sorted manually are mostly analysed using attenuated total
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reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy,49, 13 ACS Paragon Plus Environment
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69, 72
or thermal
but also pyrolysis GC-MS is
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applied.73 Both techniques result in a clear identification, but are restricted to bigger particles due to
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the manual particle handling. When aiming for microscopic particle determination, the coupling of a
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microscope to FTIR or Raman spectroscopy reveals the chemical identity of particles and allows
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particle sizes to be estimated. Both techniques are limited by a certain minimum particle size.72, 76, 77
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Alternatively, unsorted samples, i.e. where a polymer mixture might be present, can be analysed
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using thermal degradation techniques.74, 75 Since particles are not sorted manually, these techniques
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are not limited by a minimum particle size required, however they do not provide information on
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microplastic size either. Furthermore, they do provide information on ingested polymer masses
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instead of presenting the numbers of ingested microplastic particles. One of these techniques should
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be applied, and should always be favoured over the so-called ‘hot point-test’ applied by several
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studies.27,
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touched with a hot needle. However, this test does not allow polymer identification, is less suitable
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for thermoset and smaller plastics, and should therefore only be seen as a facilitation for visual
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sorting.
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Representative subsample of particles – Many studies report polymer identities for a small subset of
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sorted particles only.6, 53 This leaves considerable uncertainty with respect to the actual distribution
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of polymer types among samples. Based on practical experience using ATR-FTIR to determine
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polymer identities6, 16, 46, we advise that when numbers of pre-sorted particles are < 100, all particles
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should be analysed. For particle numbers > 100, analysis becomes more laborious but > 50 % should
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be identified for a representative subsample, with a minimum of 100 particles being analysed. The
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information given in the results section should contain the following: particle counts with confidence
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intervals, detection limits for the count and for minimum particle size, the polymer types
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determined, their percentages with regard to other polymer types and natural particles, and the
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microplastic size (classes).
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If a study identified polymer identities and applied the latter criteria, 2 points were assigned. For
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insufficient numbers of identified particles that could result in an unrepresentative subsample, 1
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point was assigned. Zero points were given if no polymer identification (i.e. purely visual sorting) was
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conducted.
42, 56 27
Plastic particles are ‘identified’ when a particle shows a sticky dark mark when
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Protocol for microplastic ingestion studies in biota
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In this article, as a synthesis of our review and method assessment, we propose a standardised
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protocol for the detection of ingested microplastic in (marine) biota, alongside the quality
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assessment method (Table 2). The protocol is adaptable for both vertebrates and invertebrates, as
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long as the components of the quality assessment system are upheld. The protocol was developed 14 ACS Paragon Plus Environment
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taking the recommended protocol by ICES (2015) 42 into account and amending with knowledge and
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evaluation of currently existing methodologies as outlined above. The protocol and the quality
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assessment system are such that, when following the protocol successfully, high reliability scores can
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be acquired. This protocol relies on the same literature analysis and argumentation as the
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assessment method, and follows the categories step-by-step.
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General Discussion
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Considerable uncertainty with respect to methodology was observed and quantified via the scoring
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system. Accumulated reliability scores ranged from 0 to 15, out of a maximum of 20, with an average
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of 8.0 (Table 1). As mentioned before, the results of such an assessment are not an absolute
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judgement, and the results should not be used as a ranking list of the value of studies. The scores are
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an indicator of the usefulness of these studies for risk assessment and monitoring purposes with
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respect to natural populations. The assessment evaluates common characteristics of a variety of
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studies. Not all decisions in a study can be captured in the scoring system; therefore, it is still
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important to critically look at a study and reflect upon its plausibility and comparability to other
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studies, not just upon its results.
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Often studies could not be assigned a high score due to missing information on certain
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characteristics, such as details of the sampling or analytical procedures. Average scores (n=35) per
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evaluation criterion were especially low (
