Contents

Preface

This report represents the results of the project "Microplastics in marine bivalves from the Nordic environment". The project was managed by the Norwegian Institiute for Water Research (NIVA) by contract from the Norwegian Environment Agency (Miljødirektoratet) and funded by the Nordic Council of Ministers (Nordisk ministerråd). Coordinator at the Norwegian Environment Agency is Runar Mathisen and the project manager at NIVA is Norman W. Green.

Acknowledgments

Thanks are due to the many listed below (divided by contribution):

• Collection of samples or retrieving stored samples
  • Norway: Gunhild Borgersen, Bjørnar Beylich and their colleagues at NIVA, Claudia Halsband and her colleagues at Akvaplan-niva, and Peter Leopold (freelance biologist).
  • Denmark: Mathias Kjeldgaard and Emilie Kallenbach at NIVA-Denmark, Grete Dinesen, Ciaran McLaverty and Lene Friis Møller and their colleagues at Technical University of Denmark (DTU) National Institute of Aquatic Resources.
  • Sweden: Matthew MacLeod and Anna Sobek and their colleagues at Department of Environmental Science and Analytical Chemistry (ACES).
  • Finland: Outi Setälä and Maiju Lehtiniemi and their colleagues at Finnish Environment Institute (SYKE).
  • Faroe Islands: Maria Dam and Katrin Hoydal and their colleagues at Faroes Islands Environment Agency (Umhvørvisstovan).
  • Iceland: Halldór Pálmar Halldórsson and Hermann Dreki Guls and their colleagues at University of Iceland Sudurnes Research Centre (SRC), and Anne de Vries at University Centre in the Westfjords.
  • Greenland: Anna Maria Roos and her colleagues at Greenland Institute of Natural Resources.
• Analyes and analytical quality assurance: Nina Tuscano Buenaventura, Maria Therese Hultman, Elisabeth Rødland, Inger Lise Nerland Bråte, Saer Samanipour and Rachel Hurley at NIVA, Joakim Skovly at Eurofins.
• Written assessment: Lise Nerland Bråte, Nina Tuscano Buenaventura, Maria Therese Hultman, Rachel Hurley, Saer Samanipour, Amy Lusher and Norman W. Green at NIVA, Claudia Halsband at Akvaplan-niva.
• Quality assurance: Bert van Bavel and Marianne Olsen at NIVA.

Oslo, 29 November 2019.

Norman W. Green

Project Manager

NIVA

Summary

The starting point of this study was to investigate microplastics in biota across the entire Nordic marine environment. Microplastics are found in all compartments of the marine environment, and there is a call from both the scientific community and decision makers to monitor the abundance and composition of these microscopic plastic particles to understand any potential impacts upon the marine ecosystem. Previous studies of microplastics in Nordic biota have mainly been conducted in the North Sea and the Baltic Sea, and very few studies were from Skagerrak and Kattegat, as well as from the northern areas and the western areas near the Faroe Islands, Iceland and Greenland. In a pre-study conducted in 2016–2017 bivalves were suggested as suitable bioindicators for monitoring of small microplastic fraction (< 1mm). Bivalves tend to be sessile, they filter large volumes of seawater, they are relatively abundant and they are already used to monitor contaminants. Furthermore, the seafloor is considered an accumulation site for microplastics and many species of bivalves live on or near the seafloor.

ln this large-scale survey of microplastics in marine bivalves from a total of 100 Nordic coastal sites were studied covering much of the Nordic marine environment ranging from Svalbard in the north, Greenland in the west, Baltic Sea in the east and the North Sea in the south. Microplastic abundance and composition were studied in five selected bivalve species that have some connection to the seafloor; the hard-bottom species of the blue mussel and closely related species (Mytilus spp) and the arctic Hiatella (Hiatella arctica), the soft bottom species of the Baltic clam (Limecola balthica), Abra nitida and Thyasira spp. Three different methods were applied; visual identification following point mode transmission Fourier transform infrared spectroscopy (µFT-IR), image scanning using automated attenuated total reflectance FT-IR (µATR-FT-IR) and pyrolysis gas chromatography mass spectrometry (Py-GCMS) of selected particles.

This study found that four out of five bivalve species contained microplastics. Hiatella arctica was not found to contain microplastics, however this is based on a very limited number of sites (n=3) covering a relatively small area. Most microplastics were detected in Mytilus spp. from highly urbanised areas, but also at some sites located close to harbours at stations on the west side of Iceland and in Bodø harbour in the northern part of Norway. For Mytilus spp. the two most affected sampling sites were from the Oslofjord (North Sea); Akershuskaia and Færder relative to other sites. This suggests that the Oslofjord is highly impacted by microplastic input. For the remaining bivalves, the trend was not as clear cut as it was for Mytilus spp., but it did point towards specific areas that showed higher levels of microplastics. This includes the North Sea along the west coast of Denmark and the southern part of Norway, as well as Skagerrak and Kattegat, in addition to coast off Stockholm in the Baltic Sea.

Microplastics were not found above the limit of detection (LOD) in bivalves from Svalbard, Faroe Islands or Greenland at the sites investigated. Despite not finding microplastics in bivalves from these locations, it cannot be ruled out that there are specific point sources in these areas that are emitting microplastics. For this to be further studied, one could target possible point sources such as wastewater treatment plants that are known to release microplastics.

A combination of methods was used to point towards potential sources or the main contributors to the marine microplastics load. Visual examination provided the morphology of particles down to around 70 µm (shape, size and appearance such as colour, lack of cellular structures and so on), FT-IR gave polymeric composition, whilst pyrolysis gave information on the mass of different compounds and thereby their identity. All these methods combined can be used for inferring potential sources, at least to some extent. Microplastics derived from road-associated activity have been suggested as one of the largest sources of microplastics into the Nordic marine environment. However, not much empirical data has been available to support these estimations until now. In this study, 16 sites were dominated by rubbery fragments that are hypothesised as originating from road run-off or harbour activity, or a combination. This was based on the visual assessment (black colour, rubbery behaviour, appropriate size and sausage shape), the finding of markers for rubber using pyrolysis (indications of butadiene and isoprene), carbon black interference for the FT-IR spectrum analysed, and the similarity in appearance from the different far reaching sites suggesting a common source or pathway.

Most microplastics detected in bivalves from this study were fragments, representing 87% of the overall count, whilst fibres accounted for the remaining 13%. This contradicts previous Mytilus spp. data from the Norwegian environment. This could represent a qualitative difference in the type of microplastics released into the sea, or methodological reasons such as; higher FT-IR coverage of particles, the strict and continuously improved procedures for contamination prevention; the exclusion of sites with microplastic levels below LOD or a generally lower fibre recovery rate in this study compared to fragments.

Most microplastics found in bivalves were below 130 µm in their longest dimension, with an average of 158 µm when including only microplastics below 1000 µm. There were significant differences between particle sizes at different sites. The reasons for the significant differences in microplastic size across sites are not yet understood. This could be related to large proportions of small black particles at certain sites. All the five sites with the highest levels of black rubbery fragments (M-19, M-14, M-16, M-22 and M-15) were the sites with the smallest sized particles on average. The two sites with the highest proportion of fibres were M-10 and M-11, which were two out of the five the sites with significantly larger microplastics. Fibres tend to be longer than fragments when measured in their longest dimension. The microplastics detected in Abra nitida and Limecola balthica were smaller than the microplastics detected in Mytilus spp., which probably reflects the fact that they are smaller sized organisms.

Besides the dominant black rubbery fragments, it was also evident that marine bivalves from the Nordic environment were exposed to a wide variety of polymeric materials. Overall, 11 other polymers were detected in bivalves from the Nordic marine environment:

Based on the visual ID and point µFT-IR (Mytilus spp., Limecola balthica and Abra nitida)

• Polyethylene (PE)
• Polypropylene (PP)
• Semi-synthetic biobased plastics (modified cellulose)
• Epoxy plastics (e.g. paint fragments)
• Polyvinyl chloride (PVC)
   

Based on scanning µFT-IR (Abra nitida and Thyasira spp.)

• Polyacrylate
• Polyethylene (PE)
• Polydimethylsiloxane (silicone)
• Calcium stearate (a plastic additive)
• Semi-synthetic biobased plastics
   

Based Py-GCMS (Mytilus spp.)

• Polyhydroxybutyrate (PHB)
• Polylactic acid (PLA)
• Polycaprolactone (PCL)
• Polyethylene naphthalate (PEN)
   

Based on this extensive study as well as previous national and intersessional work, three species of bivalves living on or in the sediment could be used to monitor microplastics (> 63–1000 µm) in the Nordic environment: the hard-bottom species the common blue mussel and closely related species (Mytilus spp.) for most of the Nordic coast, the soft bottom Baltic clam (Limecola balthica) for the Baltic Sea and Abra nitida for the Norwegian coast and some parts of the North Sea. It seems that Thyasira spp. did not contain microplastics larger than 63 µm. Both Thyasira spp. and Abra nitida contained microplastics smaller than 63 µm. These species could be used for monitor microplastics smaller than 63 µm, but further method development and sampling are required.

Sammendrag

Utgangspunktet for denne studien var å undersøke forekomst av mikroplast i biota i marint miljø på tvers av Norden. Mikroplast har gjennom ulike studier blitt påvist i alle i deler av det marine miljøet. Det er et ønske fra både forskere og myndigheter å kunne overvåke mengde og type av de mikroskopiske plastpartiklene i havmiljøet for å forstå hvilken påvirkning de kan ha på det marine økosystemet. Tidligere studier av mikroplast i nordisk biota har i hovedsak foregått i Nordsjøen og i Østersjøen mens veldig få studier er utført i Skagerrak og Kattegat eller i de nordligste og vestlige områdene rundt Færøyene, Island og Grønland. I en tidligere studie fra 2016–2017 ble muslinger foreslått som egnede bioindikatorer for overvåkning av små partikler av mikroplast (< 1 mm). Dette er blant annet begrunnet med at muslinger er stedbundne, de filtrerer store mengder sjøvann, de er ganske tallrike og vidt utbredt, og de er allerede etablert som overvåkningsorganisme for miljøgifter. Dessuten lever mange av muslingene på eller i nærheten av havbunnen, som er ansett som et akkumuleringssted for mikroplast.

I denne omfattende kartleggingen er det undersøkt for mikroplast i marine muslinger fra totalt 100 ulike steder over praktisk talt hele Norden, fra Svalbard i nord til Nordsjøen i sør, og fra Grønland i vest til Østersjøen i øst. Forekomst og sammensetning av mikroplast ble undersøkt i fem forskjellige muslingarter med tilknytning til havbunnen: hardbunnsartene blåskjell og nær beslektede arter (Mytilus spp.) og den arktiske Hiatella arctica, og bløtbunnsartene Østersjømusling (Limecola balthica), Abra nitida og Thyasira spp. Tre forskjellige metoder ble benyttet for identifisering av mikroplast i muslingene; visuell identifikasjon ved hjelp av mikroskop, partikkelspesifikk analyse med Fourier transform infrarød spektroskopi (µFT-IR) og automatisert bildeskanning ved bruk av µATR-FT-IR, og pyrolysegasskromatografi massespektrometri (Py-GCMS) av utvalgte partikler.

Av de fem undersøkte artene inneholdt fire arter mikroplast over deteksjonsgrensen (LOD). Hiatella arctica inneholdt ikke mikroplast, men dette var basert på et lite antall studiesteder (n = 3) innenfor et begrenset område. Mest mikroplast ble påvist i Mytilus spp. fra urbaniserte områder, i tillegg til noen steder i nærheten av mindre urbane havneområder på vestkysten av Island og ved Bodø havn i nord-Norge. I blåskjell ble det funnet flest mikroplastpartikler i Oslofjorden (Nordsjøen), og nærmere bestemt ved Akershuskaia og ved Færder sammenlignet med de andre stedene. Dette antyder at Oslofjorden i stor grad er påvirket av tilførsler av mikroplastpartikler. For de andre muslingartene var ikke tendensen like tydelig som for blåskjell, men resultatene indikerte høyere nivåer av mikroplast. Dette inkluderte Nordsjøen representert ved vestkysten av Danmark og Sør-Norge, i tillegg til Skagerrak og Kattegat samt området utenfor Stockholm i Østersjøen.

Mikroplast ble ikke funnet over deteksjonsgrensen i de undersøkte blåskjellene fra Svalbard, Færøyene eller Grønland. Dette utelukker imidlertid ikke at det finnes punktutslipp av mikroplast i disse områdene. Det anbefales å gjøre målrettede undersøkelser av muslinger i nærheten av mulige punktutslipp slik som for eksempel renseanlegg.

I denne studien ble det benyttet en kombinasjon av ulike metoder for å vurdere mulige kilder til mikroplast i marine miljø. Visuell undersøkelse ga partiklenes morfologi ned til ca. 70 µm (form, størrelse, farge, mangel på cellulære strukturer mm), FT-IR ga partiklenes polymersammensetning, mens pyrolyse-GCMS ga informasjon om massen til ulike stoff og dermed informasjon om polymertype. Samlet kan disse metodene til en viss grad gi indikasjon på mulig opphav til partiklene. Mikroplast fra veiavrenning har blitt foreslått som en av de største kildene til mikroplastutslipp i det nordiske miljøet. Fram til nå har det imidlertid vært svært begrenset med empiriske data som kan støtte disse estimatene. Ved 16 av de 100 undersøkte stedene var dominert av gummiaktigepartikler. Disse partiklene antas å stamme fra veiavrenning eller havneaktivitet, eller en kombinasjon. Denne antagelsen er basert på den visuelle vurderingen (svart farge, gummiaktig fremtoning, passende størrelse og pølseaktig form), funn av kjemiske markører for gummi ved bruk av py-GCMS (butadien og isopren), «carbon black» interferens for FT-IR-spekteret. Stor likhet mellom partikler fra svært ulike lokaliteter antyder at opphavet er en type kilde som ikke er lokalspesifikk.

Flesteparten av mikroplastpartiklene som ble identifisert i blåskjell (87%) var fragmenter, mens fibre utgjorde de resterende 13%. Dette skiller seg fra tidligere undersøkelser av blåskjell i Norge der fibre har vært dominerende. Dette kan skyldes faktiske forskjeller i type mikroplastpartikler som er tilført havmiljøet, men kan også ha metodiske årsaker. Eksempler på dette kan være at en høyere andel av de identifiserte partiklene er sjekket ved hjelp av FT-IR i denne studien enn i tidligere studier, prosedyrer for å forebygge forurensning av prøver under innsamling og analyse forbedres kontinuerlig, prøver med mikroplastnivåer under deteksjonsgrensen er ekskludert i denne studien eller det kan være ulik grad av fibergjenvinning i forhold til fragmenter mellom forskjellige studier.

Flesteparten av mikroplastpartiklene var mindre enn 130 µm i sin lengste dimensjon, med et gjennomsnitt på 158 µm når partikler over 1000 µm ble ekskludert. Det var signifikante forskjeller mellom partikkelstørrelser for de forskjellige stasjonene. Årsakene til dette er ikke fullt ut forstått, men det kan til dels være relatert til de høye nivåene av gummipartikler for enkelte stasjoner. De fem innsamlingsstedene der det ble funnet flest svarte gummipartikler (M-19, M-14, M-16, M-22 og M-15) hadde også de laveste gjennomsnittlige partikkelstørrelsene. De to stasjonene der det ble funnet flest fibre var blant de fem stasjonene der de største mikroplast-partiklene ble påvist. Fibre er ofte lange og den lengste dimensjonen av partiklene har derfor en tendens til å være lengre enn fragmenter. Mikroplastpartiklene som ble funnet i Abra nitida og Limecola balthica var mindre enn partiklene i Mytilus spp., noe som trolig reflekterer at disse artene er mindre i størrelse enn blåskjell.

Foruten de dominerende svarte gummipartiklene var det tydelig at muslinger i nordisk havmiljø blir eksponert for en lang rekke andre polymer-materialer. Totalt ble 11 andre polymer-typer påvist:

Basert på visuell ID og punkt μFT-IR (Mytilus spp., Limecola balthica og Abra nitida)

• Polyetylen (PE)
• Polypropylen (PP)
• Semi-syntetisk materiale (modifisert cellulose)
• Epoksyplast (f. eks. malingsfragmenter)
• Polyvinylklorid (PVC)
  
   

Basert på automatisert skanning av µFT-IR (Abra nitida og Thyasira spp.)

• Polyakrylat
• Polyetylen (PE)
• Polydimetylsiloksan (silikon)
• Kalsiumstearat (et plasttilsetningsstoff) 
• Semi-syntetisk materiale
  
   

Basert på Py-GCMS (Mytilus spp.)

• Polyhydroksybutyrat (PHB)
• Polymelkesyre (PLA)
• Polykaprolakton (PCL)
• Polyetylen naftalat (PEN)

Basert på denne omfattende studien samt tidligere nasjonalt og internasjonalt arbeid, ser det ut til at tre muslingarter kan være egnet for å overvåke mikroplast (63–1000 µm) i det nordiske havmiljøet; blåskjell og nær beslektede arter (Mytilus spp.) i mesteparten av kystområdene i Norden, Østersjømusling (Limecola balthica) i Østersjøen og Abra nitida langs deler av norskekysten og Nordsjøen. Våre funn antyder at Thyasira spp. ikke tar opp mikroplast større enn 63 µm, men i både Thyasira spp. og Abra nitida ble det påvist mikroplast mindre enn 63 µm. Disse artene kan derfor være egnet til å overvåke små mikroplastpartikler < 63 µm, men mer metodeutvikling og flere datapunkter er nødvendig for å vurdere dette nærmere.

Abbreviations

1. Background for report

Microplastics (MPs) are found in marine environments worldwide. Marine organisms can interact with microplastics through adhesion, absorption, ventilation and ingestion (Lusher, 2015). Ingestion has been described as the primary mode of interaction between organisms and microplastics as a form of environmental contamination, however, the consequences of this interaction is still not clear. Laboratory experiments have found negative impact on feeding, growth, energy levels, fecundity and reproduction, as well as sublethal effects within immune systems (Wright et al., 2013). Unfortunately, many laboratory exposures to date use unrealistic exposure regimes, such as very high concentrations, and further research is still required to build a clear picture of the consequences of microplastic exposure.

A very recent report from the Norwegian Scientific Committee for Food and Environment (VKM), concluded that it is challenging to perform environmental risk assessment of microplastics due to data gaps and constraints within the scientific literature such as those mentioned above (VKM 2019). The microplastic levels in different environmental matrices as well as extent across large geographical regions is not fully understood, which is also hindering the development of risk assessments.

As many species have been shown to contain microplastics, biota can be used to monitor microplastics levels whilst simultaneously providing important interaction data. Organisms which live in the water column or in surface waters can provide information on buoyant plastics, as well as transitory plastics which are on route to deeper sediments, following changes related to buoyancy and density (Andrady 2017). Considering reports suggesting the sediments can be the end point for as much as 90% of microplastics (Booth et al., 2017), microplastics in organisms that live in, on or near the sediment should be investigated. Benthic organisms, or those associated with the benthic community, may therefore be suitable as a sentinel species for monitoring microplastics in the environment.

The VKM report concluded that further information is required to evaluate microplastics in the Norwegian and the Nordic environments to understand microplastic abundance and potential sources. This echoes the conclusion of a Nordic Council of Ministers (NMR) scoping project from 2017 on the status of microplastic knowledge from the Nordic marine environment (Bråte et al., 2017), where the Nordic marine environment was defined as: the Norwegian Sea, Greenland Sea, the Norwegian and Danish sector of the North Sea, Skagerrak and Kattegat, as well as the Baltic Sea. It did also include all sea areas close to Greenland (south, east and north, but not west), sea areas north and north-east of Svalbard, and coastal sea areas north-east of Varangerhalvøya (Figure 1) (Bråte et al., 2017).

Several criteria for defining bioindicators were suggested when monitoring for microplastics in the Nordic marine environment (Bråte et al., 2017) including:

• Species should be abundant in the environment and easy to sample
• Ethical considerations (e.g. not use species that are threatened or protected)
• Cost of sampling/analysing the biota – sampling simultaneously for other pollutants, rapid sample process to increase the number of samples that can be analysed
• Species should be commercially and/or ecologically important
• Can be comparable to global studies (global range)
• Should be abundant in the Nordic area
• Known to contain microplastics

In the absence of agreed target species or tissues to monitor, most microplastic studies in the Nordic area so far have used fish stomachs; however, several issues have been raised with using fish stomachs for monitoring purposes based on the abovementioned criteria (among others). These include the complexity of the sample matrix, the stage of gut clearance (retention time) when sampled, their motile behaviour and the potential for ingestion of plastics in the trawl during sampling. Marine bivalves, such as Mytilus spp., were suggested to be more suitable than fish when it comes to standardisation of sampling and analysis (Bråte et al., 2017). Mytilus spp. have been identified as a suitable species for monitoring microplastics in the environment (Dehaut et al., 2016; Beyer et al., 2017; Lusher et al., 2017a; Bråte et al., 2018; Li et al., 2019), particularly suited to studying the finer (<1mm) waterborne faction of microplastics (Lusher et al., 2017a, Bråte et al., 2018). Due to the size preferences of bivalves, they should, however, not be used as the only bioindicator for marine plastic pollution. Other species, such as the Northern Fulmar sea bird (Fulmarus glacialisor) or larger benthic species, might be better suited for plastics larger than 1 mm.

In the 2017 scoping project, it was also found that most microplastic studies were conducted in species from the North Sea and the Baltic Sea, and very few studies had been performed using biota from Skagerrak, Kattegat, north in the Nordic area, west and north of the Faroe Islands, Iceland and Greenland (Bråte et al., 2017).

1.1 Aims and deliverables

 The overall aims of this study were to:

• primarily investigate spatial trends across the Nordic marine environment in microplastic abundance and composition using bivalves as bioindicators;
• assess the use of multiple bivalve species to monitor microplastics in the marine environment; and
• infer potential sources of microplastics found in indicator organisms.

The results from this investigation will be used to assess the differences in abundance and composition of microplastics across the entire Nordic water regions, as well as provide insights into gradients of presumed sources or transport vectors (e.g. ocean currents). The focus for the selection of species were those that live on, in or in the vicinity of the bottom sediments. The aim of the investigation was also to help guide authorities as to how microplastics might be routinely monitored. The investigation was also meant to provide a basis for presentation of results in more scientific fora. The programme was carried out between June 2018 and November 2019, with the final report ready at the end of November.

2. Methods

2.1 Bivalve species, their distribution and ecology

Based on ecological criteria, see for instance Beyer et al., (2018), marine bivalves were chosen as the taxonomic target group, with a special focus on Mytilus spp. In total, NIVA identified five bivalves that live either in or in close proximity to sediments: Mytilus spp., Limecola balthica (formerly called Macoma balthica), Abra nitida, Thyasira spp., and Hiatella arctica. In addition to criteria relating to the feasibility of microplastics analyses and avoidance of contamination, the species chosen for this study have a geographical distribution throughout the Nordic study area and are comparable in their general biology and ecology, while they represent different life strategies and feeding modes. They also occupy different habitats, which in turn affect their exposure to microplastics. By studying these five species it allowed comparisons of microplastic contamination between species living in, on and above benthic sediments (Table 1). Furthermore, these species have been also routinely collected for scientific purposes, leading to good knowledge of where to find them and knowledge regarding their biology. Bivalves are easy to collect, process and analyse, and many laboratory studies of microplastic have been conducted using these organisms. The five selected species occupy different habitats, which may influence their exposure to microplastics, enabling comparisons of microplastic contamination of specimens living in, on and above marine sediments.

Table 1: Bivalve species included in the current study and description of their ecology.

Blue mussel (Mytilus edulis), a very common cosmopolitan bivalve, is distributed throughout the North Atlantic and along the coast of the White Sea. The more boreal M. edulis may be confused with the Mediterranean M. galloprovincialis, or the Pacific M. trossulus, and in some locations along northern European coasts two or all three species co-occur and/or produce hybrids (Oliver et al., 2010). Collected individuals along the Norwegian coast has also been seen not to be entirely restricted to M. edulis (Brooks & Farmen 2013), and may include M. trossulus, and M. galloprovincialis. Hence, in this report, these species are referred to collectively as Mytilus spp (Figure 2). Along the west coast of Svalbard Mytilus spp. have been observed since the early 2000s and have spread in the region, with circumstantial evidence for reproductive activity and recruitment of young individuals in recent years (Mathiesen et al., 2017; Leopold et al., 2019). The shell is inequilateral and roughly triangular in shape, but shell shape varies considerably with environmental conditions. The shell colour varies from purple or blue to brown. Mytilus spp. are variable in size, from populations not exceeding 20–30 mm in length to the largest specimens measuring up to 20 cm (Marlin, 2019). Mytilus spp. live in intertidal areas attached to rocks and other hard substrates with a strong and slightly elastic fibrous structure located in the foot, the so-called byssal threads, which are secreted by byssal glands. Mytilus spp. are suspension filter feeders and ingest phytoplankton (dinoflagellates, small diatoms, flagellates, various unicellular algae), zoospores, other protozoans, and detritus, filtered from the surrounding water. They also play a vital role in the removal of bacteria, and also toxins from the water column. Indigestible materials can be rejected as pseudofaeces (Kiørboe et al., 1980).

Limecola balthica (Figure 3), commonly called the Baltic clam or Baltic tellin, is a small infaunal clam in the family Tellinidae. Limecola balthica lives in the northern parts of both the Atlantic and Pacific oceans, and also extends to the Subarctic both in North America and in Europe. The European distribution ranges from southern France north to the White Sea and Pechora Sea and also includes the inner brackish parts of the Baltic Sea (Strelkov et al., 2007). Limecola balthica is an euryhaline species, i.e. it is adapted to a wide range of salinities, down to 3–4‰ (10% of ocean salinity). It usually lives in the intertidal or shallow subtidal zone, in estuaries and on tidal flats (Oliver et al., 2010). In the brackish Baltic Sea, it lives submerged down to water depths of >100m (Strelkov et al., 2007). The shells are smooth, relatively flat, oval or somewhat trigonal in shape, and less than 30 mm long (Denisenko et al., 2003). The shell colour is polymorphic, varying between individuals and localities and can vary between white, pink, yellow and orange. Concentric growth rings indicating the age of the specimen are often clearly visible. Living buried in the mud or silt, they extend two narrow siphons to the surface of the seafloor. Through the siphons, they feed on organic matter on the sediment surface or the overlaying water.

Abra nitida, (Figure 4), is a marine clam in the family Semelidae, distributed all along the Norwegian coast. The specimens are small (approximately 20 mm in length and <15 mm in height), and have thin asymmetrical shells in a glossy, pearly-white colour, sometimes translucent and scattered with small specks. Abra nitida inhabits self-made burrows in mud, sandy mud, silty sand and muddy gravel in the sublittoral zone (down to 183 m depth) (Marlin 2019). where it mainly feeds on detritus. They are considered an important food source for flat fish (Harbo 2001).

Thyasira spp. (Figure 5), is a genus of small globular bivalves with thin and fragile shells. Several species co-occur in Nordic waters, e.g. T. flexuosa, T. dunbari and T. gouldi,I and are very similar in morphology. T. gouldi has a pan-arctic distribution and are found along the north coast of Norway, and around the coast of Greenland. They inhabit a small chamber in the top few centimetres of soft mud or sand-mud sediments rich in organic matter (Marlin 2019). The family Thyasiridae contains symbiotic and asymbiotic species that live beneath the seabed surface. Feeding strategies may vary from suspension feeding, deposit feeding to bacteria farming. Sulfur-oxidizing symbiotic bacteria are 'farmed' along burrow linings and then collected with the bivalve foot (a variation of bivalve deposit-feeding called pedal feeding) (Zanzerl et al., 2019).

Hiatella arctica, (Figure 6), also called the 'wrinkled rock borer’, has a thick shell with an irregular, but generally almost rectangular, shape. It is cosmopolitan species found from pole to pole and from the intertidal zone down to 800 m depth. The bivalves attach to hard substrates such as rocks, kelp hapterons (holdfasts) or crevices, but are also able to bore themselves into soft rock (Rees & Dare, 1993). Their development involves a relatively long pelagic phase in the larval stage. Hitaella arctica is a suspension feeder (Denisenko et al., 2003).

2.2 Location and sampling of bivalves

In total, 100 sites were sampled; collected from the east coast of Greenland and coastal waters of Iceland, Faroe Islands, Norway (including Svalbard), Denmark, Sweden and Finland. The samples were partly from new sampling (2018–2019) and partly from stored samples (2013–2018). The selection of sample locations was largely driven by the availability of bivalve samples and field sampling schedules from the summer of 2018 and early spring of 2019 of the participating research entities. The target species was to have a wide geographical distribution with some overlap in the distributions of other target bivalve species. The overlap was important to provide a possible means to compensate for different feeding strategies in order to make a pan regional assessment. The choice of samples and locations was based on:

• geographical distribution in Nordic marine environment
• availability of archived samples and on-going field work
• choice of indicator organisms (bivalves)
• transects to investigate supposed pollution gradient
• some stations with same bivalves to investigate differences in uptake of microplastic and provide a possible way to “normalize” data across a large region.

In total, 100 samples (i.e. sites) were used (Table 2, from Figure 7 to Figure 11). Except for Mytilus spp. collected in shallow water (<2m), samples were collected from soft bottom sediment.

As mentioned above, the natural distributions of these target species are not entirely overlapping which complicated a pan-regional assessment. To address this issue, at some sites two target species were analysed in order to compare the composition of microplastics in the attempt to make a broader regional assessment. Details concerning the samples can be found in Table 16 in Appendix 7.1.

Possible differences in contaminant uptake between Mytilus spp. were assumed to be small and they were not taken into account. This species was collected from shallow water (0–2 depth) by hand during the period August to October 2018, except for three samples of small individuals (<5mm) derived from stored grab samples taken during the years 2014–2015 at sampling depths that ranged from 50 to 61 m.

All but three samples of Limecola balthica were collected during field work conducted in September 2018 and January 2019. Only three samples were collected from stored grab samples taken during the years 2015 and 2016. The sampling depths ranged from 16 to 32 m.

All but three samples for Abra nitida were derived from stored grab samples taken during the years 2013–2017, and for a few of these samples, Abra spp. and A. longicallis were also included. The three samples were collected using a Van Veen grab during field work in August–September 2018 and were classified as Abra nitida. The sampling depths ranged from 27 to 426 m.

All but five samples for Thyrasira spp. were derived from stored grab samples taken during the years 2013–2017. These samples included T. sarsii, T. obsulata, T. equalis and T. gouldi. The five samples were collected using a Van Veen grab during field work in August–September 2018 and were classified as Thyrasira spp. The sampling depths ranged from 27 to 423 m.

The domination of Norwegian sites regarding Abra nitida and Thyasira spp. was mainly due to the costs of sampling of new specimens from a broader range of sites. The samples used in current study were already sampled but not yet analysed. The use of historical samples was also to test if such samples, which were preserved in ethanol, are suitable for studying microplastics occurrence.

The three Hiatella arctica samples were derived from store grab samples taken in 2014 with a depth range of 148–167 m.

Table 2: Number of bivalves sites and distribution (see also from Figure 7 to Figure 11 and more detailed description in Table 16 in Appendix 7.1.).

Figure 7: Sites where blue mussel and closely related species (Mytilus spp.) were sampled during the period 2014–2018. Detailed maps show east coast of Greenland (M.1), southwest coast of Iceland (M.2) and the Faroe Islands (M.3). Further information is available in Table 16 in Appendix 7.1.

Figure 8: Sites where the bivalve Abra nitida were sampled during the period 2013–2018. Detailed map shows the Oslofjord (A.1). Further information is available in Table 16 in Appendix 7.1.

Figure 9: Sites where the bivalve Thyrasira spp. were sampled during the period 2014–2018. For further information is available in Table 16 in Appendix 7.1.

Figure 10: Sites where the bivalve Limecola balthica were sampled during the period 2015–2019. Detailed maps show east coast of Sweden near Stockholm (L.1) and the south coast of Finland (L.2). Further information is available in Table 16 in Appendix 7.1.

Figure 11: Sites where the bivalve Hiatella arctica were sampled in 2014. Detailed map shows the west coast of Norway, north of Rørvik (H.1). Further information is available in Table 16 in Appendix 7.1.

2.3 Sample preparation

Different sizes of individuals were samples, which had to be prepared for analysis in slightly different ways. Large individuals (Mytilus spp.) followed validated methods with minor modifications (Bråte et al., 2018). Smaller individuals, which included Mytilus spp. (<15 mm), Thyasira spp., Abra nitida, Limecola balthica and Hiatella arctica were processed in two fractions: Fraction A (> 63 µm) and Fraction B (< 63 µm).

2.3.1 Large bivalves

Mytilus spp. were the only bivalve species to be processed using the modified standard procedure (n= 29 sites, 545 individuals^[1]). Between 14 and 20 individuals were processed per site. In addition, three Mytilus spp. sites were analysed together with the other four taxonomic groups and termed ‘small bivalves’ (see Section 2.3.2 and Table 16 in Appendix 7.1). All samples which were stored frozen, were then defrosted and their lengths were measured (cm) with callipers before opening. Soft tissue was dissected out before being weighed (g, w.w.) and placed in a pre-rinsed, clean glass beaker. A premade, filtered solution of 10% KOH was added to each beaker with a ration of 1:10 (biota: KOH, v/v). Beakers were sealed with aluminium foil and placed in an incubator for 24 h at 60 °C with continuous agitation (120 rpm). Samples were removed from the incubator and allowed to cool before being filtered under vacuum onto glass microfibre filter papers (GF/A, pore size, 1.6μm) (Figure 12). Filter papers were dried before being visually inspected for the presence of suspected microplastics plastics following the steps described in Section 2.4.1.

Mytilus spp. dry weight was calculated from additional individuals not previously analysed for microplastics an additional 1–3 individuals (see Table 19 in Appendix 7.3 for exact numbers). Four sites did not have a enough surplus individuals (M-5, M-8, M-17 and M-22). For these sites, the overall mean of all sites was calculated (84.35% w. w) and used to convert the wet weight of the individuals to dry weight, as presented in Table 20 in Appendix 7.3.

Figure 12: Flow of sample preparation of Mytilus spp. (figure reproduced from Bråte et al., 2018).

2.3.2 Small bivalves

Four other bivalve species, Thyasira spp., Abra nitida, Limecola balthica, Hiatella arctica, in addition to three sites with very small Mytilus spp., were processed for microplastic analysis (Table 3).

The species were too small for tissue dissection (Figure 13) and had to be incubated with KOH as whole organisms, followed by a 5% acetic acid (CH₃COOH) treatment to fully dissolve whole and/or debris of shell as well as other calcium carbonate (CaCO₃) based structures (e.g. shell remains) in the sample. Digestion of bivalve shells, mainly consisting of CaCO₃, occurs at pH <6, with increased solubility when exposed to lower pH. Removal of CaCO₃ debris is crucial as presence of shell and/or pearl formation may interfere with the FT-IR scanning of the sample in the later stages of analysis. A few large specimens of Limecola balthica which in some cases were large in size (between 3–10 individuals, ranging from 0.004 g w.w. up to 6.051 g w.w. – see in Table 21 in Appendix 7.6.1). The larger Limecola balthica had a thick shell, and therefore the shell was removed from the solution as soon as the soft tissue had detached.

Table 3: Number of individuals of Thyasira spp., Abra nitida, Limecola balthica, Hiatella arctica and Mytilus spp. from the Nordic environment processed as small bivalves, therefore splitting them in to Fraction A and Fraction B for analysis.

Figure 13: Selection of images taken during sample processing. A: Thyasira spp. in a glass sample container, preserved in ethanol. B: Abra nitida in a glass sample container preserved in ethanol and stained with rose bengal. Individuals selected for sample processing; C: Thyasira spp., D: Abra nitida, E and F: Limecola balthica and G: Hiatella arctica.

The stored samples of Thyasira spp., Abra nitida and Hiatella arctica, and to a lesser degree, Limecola balthica were preserved in formalin and then stored in ethanol. No evaporation of the excess ethanol was performed prior to weighing the sample. In some cases, this led to difficulties in determining the wet weight of some samples due to the ethanol and small tissue content. To avoid any external sample contamination during processing of the samples, the bivalve species were weighed in glass beakers prior to the addition of KOH. The same sample preparation was also performed for smaller Mytilus spp. from three sites (M-25, M-31 and M-32). Since Limecola balthica were larger, the shells were removed manually after KOH treatment in a Laminar Flow, Class II cabinet to avoid any external contamination, see Table 16 to Table 19 in Appendix 7.1.

Digestion of the soft tissue using KOH was performed for the bivalves as previously described in Section 2.4.1. The processing of the small bivalves is described in Figure 14. In short, soft tissue was digested after incubation of the whole individual in 10% KOH at 40^oC on a shaking table at 125 rpm, leaving only shell debris, sediments, and potential microplastics remaining after <24h. The pH of the KOH solution was 13.5–14.5 after 24h of incubation. To further dissolve all shell debris and other calcium carbonate structures, the pH was adjusted to 4.3–5.0, by first adding 1:1 (v/v) of 10% acetic acid (a novel method that was developed for this project), followed by addition of a 1:1 – 5:7 ratio of 5% acetic acid to increase the solubility of calcium carbonate. The samples where then incubated at 40ºC (and sometimes 60ºC depending on the amount of organic matter), at 125 rpm from 1 up to 12 hours. Some polymers can be affected at 60ºC (Dehaut et al., 2016), therefore it was aimed to keep the temperature at 40 ºC whenever possible. Only one sample had to be incubated at 60ºC.

Following digestion, the samples were filtered, as described in Figure 14, and were separated into two fractions. The samples were split across two different filters papers during filtering to improve both the visual inspection and µFT-IR steps (single point mode for Fraction A: >63 µm and µATR imaging for Fraction B: <63 µm). In short, the samples for Fraction B were first passed through a 63 µm stainless steel mesh and filtered onto a Cellulose Nitrate (CN) filter (25 mm, 8 µm), using a glass vacuum filtering system (Ø17 mm; Sterlitech, USA). All remaining debris on the 63 µm steel mesh were then filtered onto a Whatman GF/A filter (pore size 1.6 µm; Ø 45 mm), using a Nalgene filtering system (such as for Fraction A). The two filters (GF/A and CN) represent particle sizes above 63 µm (Fraction A) and below 63µm (Fraction B), respectively. The pre-filtration of Fraction B was performed to limit the presence of sand and/or silt particles on the CN filter. Such particles may damage the germanium crystal used by the FT-IR instrument during µATR imaging.

Figure 14: Sample preparation of small bivalves Thyasira spp., Abra nitida, Limecola balthica, Hiatella arctica and Mytilus spp.

2.4 Microplastic analysis

Figure 15: Flow of analysis method for Fraction B (<63 µm). A: T2_rep1. Visible image taken with the software of the approximate site of analysis. Size: 500x500 µm. B: Chemical image (spectral image) after atmospheric correction for T2_rep1. The red and green edge is the edge of the germanium crystal. C: Combined chemical image (spectral image) after PCA for T2_rep1.D: Single chemical image (spectral image) of structure 2 after PCA for T2_rep1. E: Representing spectrum obtain from structure 2 after PCA exemplified for T2_rep1. The spectrum does match with calcium stearate.

2.5 Pyrolysis gas chromatography mass spectrometry of selected Mytilus spp. samples

2.6 Quality assurance and quality control (QA/QC)

Table 4: Procedural blanks with corresponding LOD and LOQ for this current study for Mytilus spp.
All but two microplastics (MPs) were cellulosic fibres (n=54).

2.6.2 Visual ID

To reduce the subjectivity by different researchers performing the visual ID, one person performed all visual analyses. Furthermore, all samples were blind labelled to prevent the observer being biased by sample locations. To ensure for contamination control, petri dish lids were only open when necessary for analysis.

2.6.3 Recovery tests

Recovery tests can be applied to understand how much, and what types, of microplastics are obtained from environmental samples with the method used. This gives an idea of how much and what kind of microplastics are likely to be extracted from environmental matrices. However, it is important to note that recovery tests are often based on virgin reference materials that have not been out in the environment and therefore tend to behave differently. This can result in the recovery tests showing an underestimation in particle recovery. On the other hand, virgin particles are easier to identify chemically, such as by FT-IR, than weathered particles since they do not have a coating of organic material, are oxidized, or so on, which can lead to an overestimation in the recovery test. All together it is therefore likely that the recovery test is not giving the full picture of the recovery, but it can be used as an indicator of the method applied and its accuracy for different types of microplastics.

For this current study two different recovery tests were performed, one for KOH method used for Mytilus spp. and one for KOH plus acetic acid used for the small bivalves.

Mytilus spp. -KOH

Reference particles were directly spiked into the samples to ensure efficient transfer of material. The test was done with or without mussel tissue, at different temperatures (60 or 40ºC) and for different incubation times (24, 48 or 72 hours). The mussels used were commercially available mussels that had been depurated (gut cleared). The reference microplastics were counted onto a spatula under a Nikon SMZ 745T stereomicroscope at 20x magnification. They were then placed directly into each control sample. The spatula was inspected for any residual particles, which were washed into the control sample if present. All beakers were spiked with four types of reference particles available in-house at NIVA, in the same beaker with ten particles of each type (Figure 16 and Figure 17):

1. PET fibres – Orange polyester fibres (101–2194 µm in length; IQR: 493–992 µm) were produced by washing blankets (‘Skogsklocka’, IKEA, Norway) in a clean washing machine system (Candy Smart, model no. CS 1692D3-S). Fibres were filtered from the laundry effluent and dried before use. Density: ~0.96 to 1.45 g/cm3 (Zhao et al., 2018)
2. Tyre particles between 250–500 µm were obtained from Genan, Denmark. This material is generated during the recycling of end-of-life passenger car tyre. The material was sieved to obtain the fraction 250–500 µm and purified to remove residual metal and fibre contamination from the tyre recycling process. Approximate density: 1.16 g/cm3
3. PVC fragments were obtained from Goodfellow, UK. These were sieved to isolate particles between 150–250 µm. Approximate density: 1.38 g/cm3
4. PET fragments were also obtained from Goodfellow, UK. These were sieved to obtain the 250–300 µm fraction. Approximate density: 1.38 g/cm3

In general, the highest recovery rate for the four polymers tested were obtained in presence of biota, with an average recovery of 74 ± 10% (ranging between 58 to 92%), when removing the polyester (PET) fibres (see appendix). The test also displayed that tyre particles had the highest recovery rate (81 ± 15%) across all treatments tested (w./wo biota, temperature and incubation time). The highest loss of reference materials was identified for the PET fibres, displaying a high variability of recovery across the different treatments tested. The inconsistent number of fibres recovered amongst the different treatments suggests other factors such as precision during polymer spike-in, polymers stuck to the incubation vessel, insufficient rinsing and/or filtering of the samples may introduce the source of variability. Fibres have previously been suggested to be more challenging to recovery in both lab-based recovery tests (Thiele et al., 2019). For the two other fragments tested, PVC and PET, the particles were recovered at 71 ± 7% and 67 ± 8%, respectively across all biota treatments.

The recovery rate of polymers was comparable to previously published studies (Karami et al., 2017; Thiele et al., 2019).

Figure 16: Illustration from recovery test with Mytilus spp. tissue present. Ten particles of PVC fragments, tyre particles and PET fragments. One PET fibre is also present in the lower right corner.

Figure 17: Illustration from recovery test with Mytilus spp. tissue present and seven PET fibres.

Other bivalves – KOH + acetic acid

A qualitative recovery test (non-numerical) was applied to test effects of sample treatment (method used for the smaller bivalves) on spiked reference materials. In this test, however, no biotic material was present. This was to see the ‘worst case scenario’ of the treatment with no ‘protection’ by biotic material. Acetic acid was chosen as a digestion agent as it has previously shown in various ‘chemical resistant tables’ to have little/no corrosion on polymers when tested at a maximum concentration of 5% at 40 degrees, incubated for up to 30 days. Both KOH and acetic acid can be corrosive on polymers, but concentrations of 10% KOH and 5% acetic acid had no visual qualitative effect (inspection of FT-IR spectra) on seven reference polymers Table 5 (see also Table 18 in Appendix 7.2). Since no effects were anticipated, and due to the previous KOH test applied for KOH, the current recovery test was not based on numerical particle recovery, but rather spiked based on mg (and sometimes particle numbers were given), and the results were investigated by inspecting the FT-IR data. Based on this recovery test, no qualitative effects were found on the tested polymers.
The particles tested were^{[1]Majority of densities from Zhao et al., 2018, the rest from technical information following the reference materials.} :

1. PP pellets, 3 mm – 0.153 gram (5 particles). Approximate density: 0.83 to 0.85 g/cm3
2. PA-66 (nylon) pellets – 0.07 gram (5 particles). Density: 1.13 g/cm3 (Zhao et al., 2018)
3. LDPE – 0.142 gram (5 particles). Approximate density: ~ 917 to 0.93 g/cm3
4. PET fibres – see bivalve recovery test for info of the particles. 0.0067 gram (unknown particle number)
5. PET fragments between 150 – 250 µm; 0.014 gram (unknown particle number). Approximate density: 1.38 g/cm3
6. PVC fragments between 150 and 250 µm; 0.029 gram (unknown particle number). Approximate density: 1.38 g/cm3³
7. Tyre particles see bivalve recovery test for info of the particles. 0.0175 gram (unknown particle number). Approximate density: 1.16 g/cm3

Table 5: Polymers tested for impact of KOH and acetic acid.

3. Results

3.1 Mussels (Mytilus spp.)

Table 6: Results from a Tukey-Kramer HSD test on log transformed data of the number of microplastics (MPs) at the different sites. Levels not connected by same letters are significantly different. For example, site M-19 is significantly different from M-16, M.22, M-4, M-21, M-9 and M-18.

Figure 18: Average number of microplastics (MPs) per individual of Mytilus spp. Blue colour indicates Mytilus spp. individuals at sites with values above the LOD (2.77 MPs per individual) and the size is relative to the number of MPs. Red sites were studied, but values were below the LOD. For more information of sites see appendix. Asterisk * refers to upper quantification limit for particles at site M-19

Table 7: Microplastics (MPs) per individual above the LOD. Yellow colour indicates values above the LOD (2.77 MPs per individual) and green above the LOQ (8.31 MPs per individual). All values below the LOD were set to 0. The values are blank corrected.

3.1.2 Influence of Mytilus spp. size and number of microplastics

Mytilus spp. size, in terms of both length and weight, varied between the sites (Figure 19). It is an advantage when performing monitoring to aim for sampling mussels that are approximately the same size. However, due to large variation in Mytilus spp. size between different Nordic sample areas, it was difficult to define a size for sample collection. The largest mussels processed were mussels from one site in the Faroe Islands (M-20), while M- 14, M-15 and M-19 also were relatively large. All of these sites had mussels with microplastic levels above the LOD, except from M-20. The smallest mussels were from the Baltic Sea (M-22, 23 and 24), where it is challenging to sample mussels suitable for monitoring since they are small and not highly abundant in number. For the five sites that contained the highest number of microplastics (more than 1 MPs per individual), i.e. M-19, M-14, M-16, M-22 and M-15, the number of microplastics per individual was plotted against the size of the mussels (gram w.w and length in mm) to see if there were any impact of size of the individual on microplastic content. Based on this analysis, Figure 20 A and B, it was evident that there was no impact of Mytilus spp. size (weight nor length) on the microplastic levels. This suggests that Mytilus spp. size is not the driving factor for microplastic intake, rather the level of exposure is likely to be a more important factor.

Figure 19: Box and whisker plots of Mytilus spp. Data displaying, A: dry weight, and B: length of individuals. Outliers are represented as dots. Most of the sites are based on n=20 individuals, for full information see Table 20 and Table 21 in Appendix 7.3.

Figure 20: Microplastics (MPs) presented as log-MPs in mussels from the five sites (M-19, M-14, M-16, M-22 and M-15) above LOD that exceeded 1 MPs per individual plotted against A: log wet weight (g) and B: log length (mm). R²: R-squared - Coefficient of determination. See site locations in Figure 7.

3.1.3 Microplastic morphology (size, shape and polymeric composition)

Another important aspect of microplastic studies is to understand what type of microplastics are found, their characteristics; their shape (round, irregular, fibres), their size and what material they are made of. In this way it may be possible to come closer to understand the pollution source. Most of the microplastics found in this study were small black fragments or sausage-shaped particles, probably composed of rubber and suggested to be derived from road-run off or harbour activities. As for most other MP studies, more MP were found in the smallest analysed size classes.

Most microplastics detected in this study were fragments, accounting for 87% of the overall number, whilst fibres accounted for the remining 13% (Table 8 and Figure 21). Only two sites were dominated by fibres: site M-11 in the Outer Trondheimsfjord (Norway) and M-10 from Øster Hurup (Denmark). This contradicts previous Mytilus spp. data from the Norwegian environment (Lusher et al., 2017a; Bråte et al., 2018) and other international studies that have also found that fibres dominate in biota samples (Rezania et al., 2018). There might be several reasons for this difference. This could represent a qualitative difference in the type of microplastics released into the sea, such as fibre release from WWTPs. This is, however, not likely to be the case since so many sites had a domination of fragments and, to our knowledge, no specific large-scale measures for a reduction in fibre release have been implemented. More credible reasons are therefore:

• A higher FT-IR coverage with 100% of the particles subjected to FT-IR analysis^{[1]Excluding rubbery particles.} leading to improved identification (and exclusion from the dataset) of more natural fibres such as cotton or wool which typically visually resemble plastic fibres during visual analysis
• This could be a reflection of the strict, and continuously improved procedures for contamination prevention during analysis
• The exclusion of sites with microplastic levels below the LOD. A general observation was that fibres dominated in sites with microplastic values lower than the LOD
• A generally lower recovery rate in this study, as seen from section 2.6.3, for fibres than for fragments

The size of microplastics detected in mussels from the Nordic environment ranged from 34 µm to 7386 µm (average: 300 µm), Table 9. Most microplastics found in mussels were below 130 µm in their longest dimension (Figure 22) with an average of 158 µm when including only microplastics below 1000 µm (n_particles=571) and excluding particles above 1000 µm (n_particles=33). We believe that 158 µm as an average represents a superficial assessment of microplastics found in this study based upon the significant skew in the particle size distribution A left skewed size distribution is in accordance with other microplastic studies, including the previous studies of microplastics in mussels from the Norwegian environment (Lusher et al., 2017a and Bråte et al., 2018). One study carried out in England saw that there was a significant difference between the fragment size in mussels and surrounding sea water, compared to the surrounding sediment, where sediments contained larger fragments. However, there was no significant difference between the size of fibres found in mussels compared to sediment (Scott et al., 2019). The microplastic size in an organism may not always directly proportional to what is in their surrounding environment, which is an issue that needs to be further investigated.

There were significant differences between microplastic sizes at different sites. Site M-7, M-21, M-11, M-4, M-10 and M-9 had significantly larger microplastics than the other sites (with the exception of M-18). Site M-19 (Akershuskaia) and M-22 (Outside Stockholm) had significantly smaller sized particles than the rest (with the exception of site M-18) (Table 10). The reasons for the significant differences in microplastic size across sites are not understood. This could be related to large proportions of small black particles at certain sites. All the five sites with the highest levels of black rubbery fragments (M-19, M-14, M-16, M-22 and M-15) are the sites with the smallest sized particles. The two sites with the largest proportion of fibres were M-10 and M-11, which were two out of five sites with the significantly larger microplastics. Fibres are often long and tend to be longer than fragments in their longest dimension.

Table 8: Shape of microplastics (MPs) across the different sites exceeding the LOD (n_particles=601).

Figure 21: Morphology of microplastics (MPs) found in Mytilus spp. from the Nordic environment. Dark blue colour illustrates fragments, while lighter blue indicates fibres. Asterisk * refers to upper quantification limit for particles at site M-19.

Figure 22: Size distribution of microplastics from sites above the LOD in Mytilus spp. from the Nordic environment. Microplastics below 1000 µm are included (n=571), while particles above 1000 µm up to 7386 µm (n=33) are excluded. The average size of microplastics was 158 µm. For the full data set, see appendix.

Table 9: Size of the microplastics (MPs) found in mussels at the sites exceeding the LOD.

Table 10: Results from a Tukey-Kramer HSD test on log transformed data of the microplastic sizes at the different sites. Sites not connected by same letters are significantly different. For example, sites M-21, M-11, M-4, M-10 and M-9 are significantly different from sites M-15, M-16, M-14, M-22 and M-19, but not from each other.

3.1.4 Pyrolysis gas chromatography mass spectrometry (Py-GCMS)

Targeted approach

Since many methods are needed to find out what material microplastics are made of, mass estimations were also applied to some mussel samples. This showed us that for three of the sites where rubber particles were found, Mytilus spp. had measurable levels of two compound that are found in rubber. This increases the confidence that rubber is being detected in mussels from these sites. For other common plastics, no measurable levels were detected with this method.

Spectroscopic methods such as Fourier transform infrared (FT-IR) scanning of particles has its limitations, e.g. not providing mass information. Pyrolysis gas chromatography mass spectrometry (Py-GCMS) can provide such mass information (Löder & Gerdts 2015) and mass spectrometry methods can therefore be a good supplement, or addition, to FT-IR analysis. Mass spectrometry methods will also remove the need for manual hand-picking of particles, as well as the visual pre-identification step which can introduces human bias. However, the methods are not yet fully developed for the heterogenous group of microplastics, and they tend to have quite high detection limits for many polymer types. One will also lack the morphological characteristics of microplastics that can be useful in a number of applications, e.g. a toxicological perspective or for source tracing. Black rubbery fragments are hard to analyse by FT-IR due to their high content of carbon black. Some selected Mytilus spp. samples were sent to Eurofins for Py-GCMS. The ten samples (ten GF/A filers representing one individual each) were chosen since they contained rubbery fragments and gave a good overall geographic distribution amongst the Nordic Mytilus spp. samples; M-19 (Akershuskaia; two individuals), Bodø harbour (M-16; two individuals), Færder (M-14; one individual), Bolungarvik harbour (M-9; one individual), Hanstholm (M-15; two individuals) and outside Stockholm (M-22; two individuals).

Isoprene and/or butadiene, substituents in rubber, were detected above the LOQ in mussels from three sites; Akershuskaia (isoprene and butadiene), Færder in the Oslofjord (butadiene) and Bolungarvik harbour at Iceland (butadiene) (Table 11). No other polymers above 100 µg/kg were detected with the current pyrolysis method. However, the results might have been interfered by organic material that in some samples had (based on Eurofins comments) strong peaks that could potentially mask plastic polymeric materials.

Table 11: Results from the targeted pyrolysis gas chromatography mass spectrometry (Py-GCMS) of ten mussels individuals for rubbers plus eight polymers; polyethylene (PE); polypropylene (PP); Polystyrene (PS); Polyvinylchloride (PVC); Polyethylene terephthalate (PET); Polyamide6/nylon (PA6); poly(methyl methacrylate) (PMMA); Polycarbonate (PC). The highlighted indicates that markers of rubbers were detected.

Non-target approach

Four additional polymers were detected using the non-targeted approach when processing the pyrolysis data: polyhydroxybutyrate (PHB), polylactic acid (PLA), polycaprolactone (PCL) and polyethylene naphthalate (PEN) (Table 12). All of these are types of polyester. Polyester refers to a broad category of polymer types, of which PET is most common (used to produce plastic bottles and most polyester clothing). PHB, PLA and PCL were found in almost all samples except the second replicate of individual 7. PEN was found in seven of the individuals.

PHB is a type of biodegradable polyester which is used in both biodegradable/compostable consumer products and some medical applications. PLA is another type of polyester and is often produced based on fermented plant starch such as from corn, cassava, sugarcane or sugar beet pulp. PLA has been used to produce a range of bio-based consumer products. PCL is also a biodegradable polyester and was the earliest available synthetic polymer, while PEN polyester is similar to PET but with a higher temperature resistance. To our knowledge, these polymers have not been detected in environmental samples. Their specific source in this case is not known, but they must be abundant in environmental samples since they occur in all of the ‘hot-spots’ that were analysed in this study. However, results from the non-target sample analysis are relatively uncertain and only are an indication of the presence of PHB, PLA, PCL and PEN. Further confirmatory analyses are required to verify the occurrence of these polymer types in mussels from the Nordic environment.

Table 12: Non-targeted data treatment of pyrolysis gas chromatography mass spectrometry (Py-GCMS). +: ++: +++: means the levels of signal therefore indirectly the quantity of the plastics in the samples. Coloured field highlights polymers that were detected above the LOD.

3.1.5 Black rubbery fragments in the Nordic environment 

Most of the identified particles found in mussels from this study were black rubbery fragments, most of which were found in the Oslofjord at Akershuskaia M-19 and Færder M-14 (Table 13), illustrated in Figure 25. The change in the composition of microplastics (more fragments than fibres which is a result of these black small rubbery fragments) found in mussels in current study compared to previously studies from the Norwegian environment, is discussed under section 3.1.3. The black rubbery fragments were detected at eight different sites in the Nordic coastal environment; namely, Norway (Oslofjord and Norwegian Sea; M-19, M-14 and M-16), Sweden (Baltic Sea; M-22), Denmark (Skagerrak and Kattegat; M-15, M-4 and M-10) and one site at Iceland (M-9). Mussels from less urbanised site did not contain these rubbery fragments, with exception of a harbour area in west Iceland. The black particles found at these sites resemble each other, as illustrated from Figure 26 to Figure 32 and is therefore likely to be from the same type of source or release pathway.

Table 13: Sites with Mytilus spp. containing black rubbery fragments in descending order of abundance.

Figure 23: Occurrence of black rubbery fragments in Mytilus spp. from the Nordic environment. Asterisk * refers to upper quantification limit for particles at site M-19.

Figure 24: Examples of rubbery fragments found in two Mytilus spp. individuals from Akerhuskaia (M‑19).

Figure 25: Examples of rubbery fragments found in one Mytilus spp. individual from 36A, Færder (M‑14).

Figure 26: Examples of rubbery fragments found in two Mytilus spp. individuals from 97A2, Bodø harbour (M-16).

Figure 27: Examples of rubbery fragments found in two Mytilus spp. individuals from V4-ACES (M‑22).

Figure 28: Examples of rubbery fragments found in one Mytilus spp. individual from DKW3, Hanstholm (M-15).

Figure 29: Examples of rubbery fragments found in one Mytilus spp. individual from DKW4 – Hirtshals (M-4).

Figure 30: Examples of rubbery fragments found in one Mytilus spp. individual from DKE1, Øster Hurup novo strand (M-10).

Figure 31: Examples of rubbery fragments found in one Mytilus spp. individual from St. IC7, Bolungarvik harbour, Iceland (M-9).

Small unidentifiable black particles were identified in water samples from the Nordic environment as early as 2009, they were considered to be of anthropogenic origin (Norén et al., 2009). This was from a study conducted in Swedish waters (Baltic Sea and Kattegat) on behalf of the Swedish Environmental Protection Agency. The major component of anthropogenic particles found in the study were black rubbery fragments, illustrated in Figure 32, suggested to be from road and rubber wear. Similar particles were also detected in water samples from another study focusing on samples from Skagerrak, with a maximum load of 779 black particles per litre (Norén & Naustvoll, 2011). These studies illustrated that black rubbery fragments, of unknown origin and source, are abundant in water samples from at least some parts of the Nordic environment. Norén and Naustvoll (2011) also highlighted that the oil component in asphalt roads (bitumen) contains toxic hydrocarbons such as PAHs. This component can also be seen as having acute toxicity to the freshwater species Daphnia magna (Wik & Dave, 2006).

Additionally, in 2014, MEPEX published a report on behalf of the Norwegian Environment Agency that estimated that so-called road dust – microplastics derived from road associated activity – were the largest source of microplastics to the Norwegian environment (Sundt et al., 2014). However, empirical data on black particles from the Norwegian environment is still lacking. Therefore, further analyses such as those performed in our study are required to fill knowledge gaps.

Figure 32: Picture of black particles in water samples from the Baltic Sea and Kattegat (from Noren et al., 2009).

In 2019, a report on Road dust-Associated Microplastic Particles (RAMP) on behalf of the Norwegian Environment Agency was published (Vogelsang et al., 2019). The main component of road dust was found to be rubber from tyre treads, polymers added to strengthen the bitumen used in road pavement and thermoplastic elastomers in road marking paints (Vogelsang et al., 2019). However, very limited field studies are available on the characterization of these microplastics that are released from road-related activities. It is challenging to analyse rubbery fragments using the ‘classical’ methods available for microplastic research such as FT-IR analysis or similar, as discussed in more detail in section 2.4.1. Therefore, it has been suggested to use markers for such particles and several markers for tyre particles have so far been suggested. These markers can be used as a proxy to calculate the concentration of tyre particles in a sample. For example, extractable organic zinc, different benzothiazoles and SBR/IR content have been suggested (Wagner et al., 2018; Vogelsang et al., 2019). Isoprene and butadiene, as found in mussels from the Oslofjord and Iceland, can also be examples of such markers. Carbon black is also a potential marker since 90% of the carbon black on a global scale is used in the rubber industry as a reinforcing filler in a variety of products. Though, it is very challenging to distinguish between engineered and carbon black (e.g. from combustion sources) (Vogelsang et al., 2019), and carbon black is also used as a reinforcing filler for certain other polymers.

Based on the limited literature on rubbery fragments, the main part of the tyre wear appears to be larger than 10 µm and up to 350 µm, with 85% ranging between 50 and 350 µm (Vogelsang et al., 2019). When rubber treads are present in road dust, they are typically sausage-shaped conglomerates with rough surfaces (Vogelsang et al., 2019). Many of the black particles found in this study were sausage- shaped. It would be beneficial to have a closer look at the shape of the particles found in this study with better magnification and resolution than a stereomicroscope, such as by using scanning electron microscopy.

Klöckner et al., (2019) highlights that the quantity and quality of particles emitted from road traffic is probably very variable depending on local circumstances such as traffic conditions, road material and surfaces; however, generally speaking, particles from road run-off will have a higher density than seawater (around 1 g/cm³) and will therefore sink. Tyre rubber have an approximate density of 1.2 g/cm³ (Degaffe & Turner 2011), and the particles collected from roads have been reported to range from 1.5 to 2.2 g/cm³ (Kayhanian et al., 2012) indicating that all types of particles emitted from road-runoff will sink towards the seafloor. Another potential pathway of road related particle, which is not mentioned a lot in the literature, is dumping of snow into the ocean. The M-19 site, Akershuskaia in the Oslofjord, has also previously been found to have the highest levels of microplastics in mussels dominating by similar rubbery fragments (Bråte et al., 2018). This could be due to snow dumped from a snow melting cleaning facility near Akershuskaia.^[1] 

Following the results obtained in this study and discussion above, it is not straight forward to determine the source of these rubbery fragments. However, based on an assembly of indications we hypothesize that the rubbery fragments in mussels from the eight urban sites in the Nordic area, could stem from either road runoff or harbour activity, or a combination.

This is based on the following indications:

• The visual assessment: black colour, rubber behaviour and often “sausage shaped” particles
• Carbon black indication from FT-IR analysis
• Py-GCMS analysis giving indications of rubbers for three sites (isoprene and/or butadiene)
• Size of the particles found in the mussels
• Previous findings of similar particles in water bodies and mussels from the Nordic environment

Lastly, since it was an apparent that there was a homogenic appearance of the particles across the Nordic sites, it indicates that the particles are from one ‘type’ of source or release pathway which must be common for the different sites. Road and/or harbour activity could be such a pathway. Which part of the harbour activity that might contribute to rubber release is not clear, but we cannot exclude that it is point sources in harbours that could result in this, such as boat traffic, dock protection, or as, mentioned above, snow dumping at specific sites.

Considerable effort is currently being put into solving these analytical issues, and this should help bridge the knowledge gap.

3.1.6 Other polymers

Rubbery fragments were by far the most dominant microplastic type observed in the mussels in this study; although other polymers were also found, including semi-synthetic biobased plastics and PE (Figure 33). For example, PE fragments were dominant in one site from Denmark (M-21; North Sea) and two other sites in Denmark, M-4 and M-10 (Skagerrak and Kattegat) (from Figure 34 to Figure 36). PE was also found in mussels from Bolungarvik harbour at Iceland (M-9). Semi-synthetic biobased plastics were found in mussels from five sites, but they were not found to be dominant. This confirms earlier findings (Bråte et al., 2018).

No individual mussels from Svalbard exceeded the LOD in regard to microplastic counts set in this study. However, when investigating the polymers found at this site, it is unlikely that the fragments found were a result of procedural contamination. In four out of fifteen Mytilus spp. individuals, epoxy fragments resembling paint fragments were present (Figure 37). If blank correction had performed for each morphology type (fibres, fragments), and not in the aggregated approach that was used in this study, these Svalbard results would have been eliminated. This is particularly the case when looking at all characteristics of the particles found in the blanks versus the samples (morphology, composition, etc.).

Figure 33: Polymeric composition of microplastics found in Mytilus spp. from the Nordic environment. Asterisk * refers to upper quantification limit for particles at site M-19. Only polymers representing >5% of the data for each site are shown, the rest are aggregated into ‘other’.

Figure 34: Microplastics in Mytilus spp. from DKW4, Hirtshals (M-4).

Figure 35: Polyethylene (PE) in two Mytilus spp. individuals from DKE1, Øster Hurup novo strand (M‑10).

Figure 36: Microplastics in Mytilus spp. from St. IC7, Bolungarvik harbour (M-9).

Figure 37: Microplastics in Mytilus spp. from Svalbard (M-1), values fell below LOD and were presumed not likely to be contamination. All from different Mytilus spp. individuals. A: Semi-synthetic biobased plastics and polyester (longest black) B-D: Epoxide polymer – a possible paint fragment(s) E: Semi-synthetic biobased plastics F: Epoxide polymer – a possible paint fragment.

3.2 Thyasira spp., Abra nitida, Limecola balthica and Hiatella arctica

Table ​14: Overview of analysed samples for Fraction A, * indicates small mussels (not to be confused with mussels from section 3.1). Colour indicates the presence of microplastics (MPs).

Figure 38: Microplastics in pooled samples of Thyasira spp., Abra nitida (A), Limecola balthica (L) and Hiatella arctica.

Figure 39: Microplastics in pooled samples and gram adjusted of Thyasira spp., Abranita nitida, Limecola balthica (T) and Hiatella arctica (H).

Figure 40: Polymeric composition of the microplastics found in Abra nitida (A) and Limecola balthica (L) based on pooled samples. Pink illustrates rubbery fragments, yellow illustrates semi-synthetic biobased plastics and blue indicates PVC.

3.2.2 Fraction B (<63µm)

Figure 41: Presence/absence and material composition of microplastics of Fraction B for Abra nitida (A) and Thyasira spp. (T) from the Nordic environment.

Table 15: Overview of microplastic (MP) polymers and associated contaminants detected in Thyasira spp. and Abra nitida, Fraction B (below 63µm) with ATR scanning FT-IR.

4. Discussion

4.1 Combined microplastic results for all bivalves from the Nordic environment

This large-scale microplastic study is a significant contribution to address the lack of empirical data for the Nordic marine environment as highlighted by Bråte et al., 2017. This study covers much of the sea areas in Nordic marine environment, ranging from Svalbard in the north, Greenland in the west, Baltic Sea in the east and the North Sea in the south. The amount of empirical data on marine bivalves from this area has dramatically increased with the 100 sites included in the survey. The results, especially the Mytilus spp., indicate that bivalves from urban sites in the Nordic marine environment are exposed to microplastics to a larger extent than less urbanised sites. This is not always the case for other microplastic studies that sometimes indicate, not local input, but rather less understood and studied sources/pathways. A case in point, were high levels detected in mussels from the Barents Sea that might be explained by accumulation zones driven by currents (Bråte et al., 2018).

In this current study, microplastics were identified at eleven sites for Mytilus spp., in six sites for Fraction A and two sites for Fraction B for Abra nitida, four sites of Limecola balthica from Fraction A, and two sites for Thyasira spp. from Fraction B (Figure 42).

The results indicate that the North Sea, Skagerrak, Kattegat and the Baltic Sea are the most impacted areas. The three northern sites that with microplastics above LODs, were sites associated to harbours; M-9 (Bolungarvik harbour), L-14 (near Tromsø) and M-16 (Bodø harbour). Microplastics above LOD were not detected in bivalves from the east coast of Greenland, the Faroe Islands and Svalbard. No bivalves were studied from the north-east part of Varangerhalvøya (northern Norway). Despite not finding microplastics in mussels from the coast of Greenland, Svalbard and the Faroe Islands, there may still be some specific point sources in these areas. To address this, one could focus on possible point sources of microplastics such as wastewater treatment plants (WWTPs) that are known to have especially synthetic, semi-synthetic biobased plastics and natural fibres in their effluent (Magnusson & Norén 2014). Such an approach should take into consideration not only the population and industry the WWTP serves but also how developed the WWTP is.

One of the main objectives with this study was to identify the most likely sources of microplastics found in the bivalve species throughout the Nordic marine environment. Three different methods were applied and compiled: visual identification following point mode transmission Fourier transform infrared spectroscopy (µFT-IR), image scanning using automated attenuated total reflectance FT-IR (µATR-FT-IR) and, for ten Mytilus spp samples, pyrolysis gas chromatography mass spectrometry (Py-GCMS). Overall, black rubbery fragments were the dominate microplastic type for three species; Mytilus spp., Abra nitida and Limecola balthica (Figure 43). This indicates that these rubbery fragments, possibly from road-runoff, are accumulating in bivalves from many sites in the Nordic marine environment, including North Sea, Skagerrak, Kattegat and the Baltic Sea. For a more in-dept discussion on these rubbery fragments, see section 3.1.5.

It was also evident from the three applied methods that these marine bivalves were exposed to a wide variety of polymeric materials, indicating numerous of sources or pathways. A total of 11 polymers were detected other than rubbery fragments:

Based on the visual ID and point µFT-IR (Mytilus spp., Limecola balthica and Abra nitida)

• Polyethylene (PE)
• Polypropylene (PP)
• Semi-synthetic biobased plastics (modified cellulose)
• Epoxy plastics (e.g. paint fragments)
• Polyvinyl chloride (PVC)

Based on scanning µFT-IR (Abra nitida and Thyasira spp.)

• Polyacrylate
• Polyethylene (PE)
• Polydimethylsiloxane (silicone)
• Calcium stearate (plastic additive)
• Semi-synthetics biobased plastics

Based Py-GCMS (Mytilus spp.)

• Polyhydroxybutyrate (PHB)
• Polylactic acid (PLA)
• Polycaprolactone (PCL)
• Polyethylene naphthalate (PEN)

On the whole, the composition of these polymers varied considerably from site to site and did not point strongly towards a specific source or pathway. The polymers reflect a wide range of densities such as the natural buoyant polymers like polyethylene (PE) and the denser polymers such as polyvinyl choride (PVC). The polymers also represent a wide range of applications. Polyethylene is the most produced and used polymer worldwide (PlasticsEurope 2018), and hence, it is not possible to assign what is found in the bivalves to certain products or sources. Epoxy plastics, often used in paint, might be from boat paint but this can’t be confirmed. Semi-synthetic biobased fibres might come from WWTPs or be a result of airborne deposition, but again, this is speculation. The polyester-like polymers found with the Py-GCMS method (PHB, PLA, PCL and PEN) are, to our knowledge, not commonly detected in environmental samples. Possible sources for these are, unfortunately, not possible to confirm within the scope of this study. However, since these are present at all sites studied with Py-GCMS, it indicates that the source(s) are common to urban sites in the Nordic environment. More comprehensive and targeted studies would help bridge this gap in knowledge.

One purpose of the sampling design was to observe whether the accumulation of microplastics differed among the target species. This related to the different habitats and feeding modes of the selected species. There were 18 sites at which two or more species were sampled. This overlap mostly corresponded with the species: Abra nitida, Thyasira spp., Limecola balthica and Hiatella arctica. For the majority of these sites, no values above the LOD were detected for any of the species. Three sites were characterised by only one of the species recording confirmed microplastics but, for two of these sites, this referred to the Fraction B of Thyasira spp. This complicates efforts to make comparisons, as the Fraction B of one species is not comparable to Fraction A of another. Hence, it is not possible to draw justified conclusions regarding the difference in exposure or ecology of the 5 bivalves tested. Further research is necessary to better understand these dynamics in regard to the ingestion of microplastic particles.

Figure 42: Combined results for all polymers found in Mytilus spp. (M), Limecola balthica (L), Abra nitida (A) and Thyasira spp. (T). Asterisk * refers to upper quantification limit for particles at site M-19.

Figure 43: Combined results for the species containing black rubbery fragments from the Nordic environment; Abra nitida (A), Limecola balthica (L) and Mytilus spp. (M). Asterisk * refers to upper quantification limit for particles at site M-19.

4.2 Use of archived sampled for microplastic analysis

There are many factors to consider when collecting samples for microplastic analysis. For biota such as mussels, all containers for storage must be pre-rinsed with filtered (RO-)water before use to ensure there is no contamination. If the mussels are to be stored in a solution for preservation, the solution must also be pre-filtered to minimize any microplastic contamination. As atmospheric contamination cannot be accounted for except within lab facilities, fibres were excluded in samples stored in ethanol. Fibres, as large as 3000–8000 µm, were found in the pooled bivalve mussels. Also, fibres < 1000 µm long were detected in the several blanks from the NIVA lab. Fragments, however, can act as supporting material, if similarities between the data from the smaller mussels and the Mytilus spp. are observed such as the black rubbery fragments. Black rubbery fragments were never detected in the procedural blank samples. The Mytilus spp. were collected with the intent of analysing microplastics, and precautions were taken to minimize atmospheric and cross contamination from sampling areas to processing in the lab. They were also frozen and not fixed in ethanol, which is likely to be a better solution for microplastic studies. Due to the uncertainties of how the smaller mussels were collected, handled and stored, the data obtained from these samples should act as supporting material to the bivalve’s data.

4.3 Some considerations regarding microplastics in Nordic marine bivalves

The presence of microplastics in bivalves, as with other marine species, raises a concern regarding the consequences. These include consequences to marine life, to seafood supply and to human health. Several international reports published in the last three years have address these issues. Most recently, VKM published a report in October 2019 to address concerns regarding the implications of microplastics to the environment and human health. Earlier reports addressing the same issue have come from EFSA Panel on Contaminants in Food (Alexander et al., 2016), the Food and Agricultural organisation of the United Nations (Lusher et al., 2017b) and Science Advice for Policy by European Academics consortium (SAPEA, 2019). On a general marine ecosystem scale, evidence shows that organisms can intake microplastics of different morphologies in varying concentrations. The VKM report shows that the amount of data available for environmental exposure and laboratories studies investigating effects, are disjointed. Therefore, we are currently not able to perform an assessment of environmental risk.

Importantly, the exposure of microplastics to individuals in the wild will depend on the concentrations in the specific environmental compartment. Microplastics in the water column are generally seen as transitory, they are either sinking or floating, due to changes in buoyancy and density. Ultimately, microplastics will sink to the sediment, which may suggest that benthic organisms are more exposed than pelagic species. There is ample information that suggests no matter which environmental compartment an organism inhabits, they could interact with microplastic. Understanding the impact on biota in the wild, requires assessment in the laboratory. When an organism ingests microplastics, the consequences of ingestion can be assessed on different levels of biological organisation: molecular, cellular, organ, individual, population and community. By far most of the information available focuses on the individual level (VKM 2019). Impacts on individuals can include impacts on feeding, growth, reproduction, behaviour, and ultimately mortality. However, many of the studies use exposure concentrations, and exposure material, which lack environmental relevance. Therefore, interpretation of any generated data in terms of the impact on the health of individuals must be made with caution, and especially at a higher level of organisation such as on a population level.

Shellfish farming has been highlighted as a potential source of microplastics to the environment (Lusher et al., 2017b). There have been some studies which indicate mussels and clams collected from sites of aquaculture contained larger quantified of microplastics (Mathalon & Hill 2014; De witte et al., 2014) whereas others have shown no difference or pattern in observed levels (Davidson & Dudas 2016). Further research is required to understand microplastics generated through fisheries and aquaculture and their consequences for food products. The types of pre-processing, such as depuration before reaching the point of sale, could reduce the levels of microplastics in seafood. There is evidence that when mussels are depurated, they contained fewer microplastics (e.g., Birnstiel et al., 2019). This step is usually performed to prepare bivalves for human consumption.

There is sufficient data showing that many marine species that are consumed as food by humans does contain microplastics. However, further quantitative data is still required to build a picture of the risk for consumption. Alexander et al., (2016), Lusher et al., (2017b) and SAPEA (2019) all state that there is sufficient published evidence that microplastics are present in seafood. This is affirmed by VKM, although they go on to state that an exposure assessment is not yet possible.

There is a general lack of information on the toxicity of micro and nano-sized plastics to human health. The consequences, in terms of a human health perspective, as outlined by three reports state that:

• There are few relevant studies for human hazard assessment (Alexander et al., 2016)
• The toxicity of microplastics to humans is uncertain (SAPEA 2019)
• The available information does not provide sufficient basis to characterise potential toxicity to humans (VKM 2019)

5. Conclusions

ln this large-scale survey of microplastics in five Nordic marine bivalves (Mytilus spp., Limecola balthica, Abra nitida, Thyasira spp and Hiatella arctica) from a total of 100 sites, three different methods were applied to assess the microplastic occurrence and composition. This study found that four out of five bivalve species contained microplastics. Hiatella arctica did not contain microplastics, however, there were only three sites, close together, with this species. Most of the sites with microplastics were mussels from highly urbanised areas, or harbour areas. This was more evident for Mytilus spp. and less so for Abra nitida or Thryasira spp. The results indicate that the North Sea, as well as Skagerrak and Kattegat, in addition to the area near Stockholm in the Baltic Sea, higher accumulation of most microplastics. Not all particles are simple to analyse, and this is especially true for rubbery fragments. However, nearly all microplastics found in this study were rubbery fragments. It is speculated that these are derived from road run-off or harbour activity based on the combination of methods applied in this study. In addition, to the rubber fragments, 11 other polymers were also detected, indicating many different sources and pathways contributing to the microplastic load in the Nordic environment.

Based on the empirical data now available, three bivalves living near, on, or in the sediment could be used to monitor microplastics in the range from 63 to 1000 µm in the Nordic environment. The three bivalves are: the hard-bottom dwelling blue mussel and closely related species (Mytilus spp.) for most of the coastal Nordic marine environment, and the two soft bottom dwelling Baltic clam (Limecola balthica) from the Baltic Sea and Abra nitida from the Norwegian coast and parts of the North Sea. There is an indication that with more effort Thyasira spp. and Abra nitida could be used for monitoring microplastics smaller than 63 µm as well.

How microplastics are impacting the marine ecosystem is still not understood, but by combining environmental concentration data, such as those from this study, with experimental data, one can better understand which areas are likely to be most impacted. Even so, there remain many unanswered questions with regards to the impact that microplastics have on the environment. To address these issues, further method development is needed to increase the resolution and understanding of microplastic occurrence and composition in biota. With this knowledge the challenging task of confirming the sources of the variety of microplastics that occur in the marine environment may be advanced.

7. Appendix

7.1 Sample overview

Table 16: Sample overview. Location of sample through the site code was not known at the time to the those that did microplastic analyses. Method of sampling: VV = Van Veen 0.1m², HP = hand-picked.

7.2 Reference material treated with KOH + acetic acid

Table 17: Recovery test of reference material in the presence and absence of biota tissue after incubation at 40 or 60 degrees for 24–72h with 10% KOH at 100 rpm shaking. The results represent average value (± SD) expressed in percentage of the number of initially spiked in polymers.

Table 18 shows the quality score before after KOH + Acetic acid and Figure 44 represent FT-IR performed with Agilent Cary 360. Settings on the instrument were as following:

Sample Scans: 8
Background Scans:16
Resolution: 4
Range: 4000–650

Table 18: FT-IR before/after recovery test of the seven polymers tested for impact from KOH + acetic acid.

Figure 44: FT-IR spectra of the seven polymers tested for impact from KOH + acetic acid. See Table 18 for more information.

7.3 Weight and length of Mytilus spp.

Table 19: Wet weight and dry weight of analysed mussels.

Table 20: Estimated dry weight of Mytilus edulis based on Table 19.