An assessment of the ability to ingest and excrete microplastics by filter-feeders: A case study with the Mediterranean mussel

Accepted Manuscript An assessment of the ability to ingest and excrete microplastics by filter-feeders: A case study with the Mediterranean mussel Cátia Gonçalves, Marta Martins, Paula Sobral, Pedro M. Costa, Maria H. Costa PII:

S0269-7491(18)32559-4

DOI:

https://doi.org/10.1016/j.envpol.2018.11.038

Reference:

ENPO 11865

To appear in:

Environmental Pollution

Received Date: 8 June 2018 Revised Date:

24 October 2018

Accepted Date: 12 November 2018

Please cite this article as: Gonçalves, Cá., Martins, M., Sobral, P., Costa, P.M., Costa, M.H., An assessment of the ability to ingest and excrete microplastics by filter-feeders: A case study with the Mediterranean mussel, Environmental Pollution (2018), doi: https://doi.org/10.1016/ j.envpol.2018.11.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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An assessment of the ability to ingest and excrete microplastics by filter-feeders: A case study

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with the Mediterranean mussel

3 Cátia Gonçalves1, , Marta Martins1,2, , Paula Sobral1, Pedro M. Costa3, Maria H. Costa1

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do Ambiente, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516

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Caparica, Portugal.

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Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.

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MARE – Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia

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UCIBIO, REQUIMTE – Departamento de Química, Faculdade de Ciências e Tecnologia,

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Vida, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516 Caparica,

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Portugal.

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UCIBIO – Research Unit on Applied Molecular Biosciences, Departamento de Ciências da

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Corresponding author Tel.: +351 212 948 300 Ext. 10114 E-mail address: [email protected] (Cátia Gonçalves); [email protected] (Marta Martins)

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Abstract

21 Plastic debris has been recognized as a growing threat to marine biota due to its widespread

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distribution and possible interactions with marine species. Concerns over the effects of plastic

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polymers in marine ecosystems is reflected in the high number of toxicological studies,

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regarding microplastics (<5 mm) and marine fauna. Although several studies reported that

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organisms ingest and subsequently eliminate microplastics (MP), the potential effects at organ

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and tissue level remain unclear, especially considering exposure to different microplastic sizes

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and concentrations. The present study aimed at investigating potential pathophysiological

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effects of the ingestion of MP by marine filter-feeders. For the purpose, Mediterranean mussel

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(Mytilus galloprovincialis) was exposed to spherical polystyrene MP (2 and 10 µm Ø) over

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short- and medium-term exposure periods, under single and combined concentrations that

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represent high, yet realistic doses (10 and 1000 MP·mL-1). Overall, results suggest rapid MP’

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clearance from water column by filtering, regardless of MP size. Ingestion occurred, identified

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by MP in the lumen of the gut (mostly in midgut region), followed by excretion through faeces.

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However, no MP were found in gills or digestive gland diverticula. Biochemical indicators for

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oxidative stress were generally irresponsive regardless of organ and time of exposure. Small

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foci of haemocytic infiltration in gastric epithelia were found, albeit not clearly related to MP

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ingestion. Globally, no evident histopathological damage was recorded in whole-body sections

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of exposed animals. The present findings highlight the adaptative ability of filter-feeding

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bivalves to cope with filtration of suspended MP, resulting in rapid elimination and reduced

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internal damage following ingestion of spherical MP. Nevertheless, the fact that the animals are

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able to translocate MP to the gut reveals that filter feeding organisms may indeed became a

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target of concern for fragmented materials with smaller, mixed sizes and sharper edges.

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Capsule

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Effects of microplastic ingestion by filter-feeding bivalves.

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Keywords

51 Microplastic ingestion, polystyrene, Mytilus galloprovincialis, histopathology, digestive tract

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Introduction

56 The enormous plastic-dependence that characterizes our global society has led to an increase in

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demand and production of these materials over the last decades. The wide range of unique

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properties (endurance and price, in particular) and the endless applications has driven

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worldwide plastic production to reach 335 million tonnes in 2016, with tendency to increase

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(Plastics Europe, 2018). The high consumption patterns of these polymers along with the lack of

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efficient management policies regarding plastic’s recovery or recycling in many parts of the

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world, were responsible for the ubiquity of plastic debris in several ecosystems. Once in the

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environment, plastics are subjected to several degradation processes, such as photo-degradation

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by UV light, mechanical and microbial degradation, eventually leading to fragmentation. Due to

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the role of aquatic waterbodies, especially marine, as ultimate fate of pollutants, the occurrence

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of microplastics (MP), i.e., plastic fragments with < 5 mm, has already been reported in areas as

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distinct as oceanic beaches, open ocean, deep-sea sediments, remote island shorelines,

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wastewater effluents and even in Artic polar waters (Cózar et al., 2014; Frias et al., 2014;

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Galgani et al., 1996; Ivar do Sul et al., 2013; Lusher et al., 2015; Mason et al., 2016; Van

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Cauwenberghe et al., 2013; Woodall et al., 2014).

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Over the last years there have been several indications that plastic debris, in their different

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dimensions, pose a serious threat to terrestrial and aquatic environments because of possible

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interactions with the biota (Avio et al., 2017; Duis and Coors, 2016; Lusher et al., 2017).

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Microplastics, in particular, due to their reduced dimensions, can be easily transported by water

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and become bioavailable for both producers and consumers (Auta et al., 2017). In fact, studies

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with wild marine fauna from different phyla (e.g. molluscs, arthropods and chordates) reported

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the presence of MP in the organisms’ gastrointestinal tracts (Braid et al., 2012; Lusher et al.,

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2013; Murray and Cowie, 2011; Neves et al., 2015; Van Cauwenberghe and Janssen, 2014).

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Nonetheless, the potential consequences of MP to these animals are far from being fully

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understood (Avio et al., 2017).

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It has been suggested that macroplastics, once ingested, tend to accumulate within the gut,

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causing physical damage, internal abrasion and blockages of the digestive tract (Laist, 1987;

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Derraik et al., 2002; Gregory et al., 2009). Wright (2013) suggested that such adverse effects

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may be also applied to invertebrates after MP ingestion, albeit lacking empirical validation. On

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the other hand, laboratory experiments performed by von Moos et al. (2012) reported that

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exposure to high-density polyethylene (HDPE) MP, of irregular shape and ranging from 0 − 80

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µm, may be responsible for the increase of inflammatory response and lysosomal membrane

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destabilization in the bivalve M. edulis. Nevertheless, this study was performed with unusual

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studies with concentration expressed as number of items or particles per volume. Additionally,

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manifold laboratorial experiments are often designed with MP concentrations several orders of

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magnitude above the highest values of those found in marine environment (Auta et al., 2017;

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Wright et al., 2013). For instance, mussels were exposed to concentrations of about 1.0 × 106

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items·mL-1 (Farrel and Nelson, 2013), whereas the highest concentration reported from marine

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environment is 0.009 MP·mL-1 in NE Pacific Ocean (Desforges et al., 2014). Moreover, Norén

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(2007) identified “microplastics hotspots” with concentrations of approximately 0.1 MP·mL-1

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(diam. ~0.5 – 2 mm) in a Swedish harbour area adjacent to a polyethylene (PE) production

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plant. Recently, Lebreton et al. (2018) reported loads of MP (0.05 – 0.5 cm) higher than 1.1×107

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items·km-2 in the Great Pacific Garbage Patch, located in North Pacific Subtropical Gyre. The

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discrepancy between concentrations and even units is a result of the lack of standardization in

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plastic quantification methods. This is more obvious for the very small size ranges, which may

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entail some difficulties in the process of risk assessment about the real effects of MP towards

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marine biota in the low levels of the food web. Still, it has been pointed out that future research

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should consider that overall abundance of MP in oceans should increase, due to the continuous

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fragmentation of larger plastic items (Duis and Coors, 2016; Wright et al., 2013).

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Marine filter-feeders, such as Mytilus sp., have already been considered an efficient indicator

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species in studies towards the potential effects of MP ingestion (Avio et al., 2017). In addition

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to much previous work within the domains of ecotoxicology and environmental monitoring,

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mussels also benefit from wide geographical distribution plus their sedentary disposition. As

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filter feeders, these animals rise as ideal targets for studying the uptake and accumulation of

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MP, which may thus be transferred through ingestion by organisms from the lower to higher

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levels of the food web. In spite of the existence of several studies concerning MP ingestion by

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marine mussels, there is still some considerable concern about the pathways of MP within these

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organisms and what are the consequences of the ingestion of MP at tissue and organ level. Thus,

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the present study aims at investigating MP ingestion and progression along the digestive tract of

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a model filter-feeder, the Mediterranean mussel (M. galloprovincialis) and to assess potential

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adverse effects. It is aimed at interpreting responses under the scope of time of exposure and

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microplastic size, in order to better understand risk and value for biomonitoring.

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Materials and methods

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Mussel collection

128 Mediterranean mussels (length: 4.35 ± 0.24 cm; width: 2.08 ± 0.19 cm) were randomly

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collected at a clean area in the West coast of Portugal, between April and September 2016. All

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individuals were transported to the laboratory and maintained in controlled conditions (16°C,

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34‰ salinity). Immediately before the acclimatization period, all mussels were scrubbed to

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remove epiphytes and other fouling organisms from valves.

134 MP characterization and test concentrations

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The experiments were conducted with spherical polystyrene MP (PS–MP), one of the most

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frequently polymers found in marine waters (Duis and Coors, 2016). Three different diameters

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of MP were employed, bearing in mind the typical food particles size for mussels (Fernández-

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Reiriz et al., 2015): 2 µm (Alfa-Aesar®), 6 µm (yellow-green fluorescent, Alfa-Aesar®), 10 µm

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(Sigma-aldrich®), 5 µm (red fluorescent, Magsphere), 10 µm (red fluorescent, Magsphere). The

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maximum excitation-emission spectra of fluorescent MP are 441 – 486 nm and 545 – 630 nm

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for yellow-green MP and red fluorescent MP, respectively.

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Regardless of MP size, two MP concentrations were selected, 10 MP·mL-1 and 1000 MP·mL-1.

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Animals were exposed to single and combined concentrations of differently-sized MP (1:1 or

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1:1:1). Test solutions were prepared from an intermediate dilution with a final concentration of

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1.0×106 MP·mL-1 and the appropriate nominal MP concentration was then spiked into each

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beaker.

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Preliminary assays confirmed neutral buoyancy of these MP in natural seawater, as stated by

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manufacturer and available literature (Karami, 2017). Dispersion of MPs was verified using a

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Coulter Counter® Multisizer™ 3 (Beckman Coulter™), with an aperture tube of 100 µm.

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Analysis did not show the presence of microparticles with sizes other than 6 or 10 µm,

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confirming no significant aggregation.

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MP exposure experiments

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A series of short- and medium-term bioassays were performed sequentially, meaning that each

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experiment was planned in accordance with the results obtained in the previous experiment. All

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bioassays were held in 2-L glass beakers, with 1 L of ultra- filtered and aerated natural seawater,

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with one individual per beaker and a minimum of three mussels per experimental condition and 6

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sampling time. All experiments included a control treatment which was prepared similarly

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although without MP. The experiments were carried out at constant water temperature of 16°C

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and 34‰ of salinity. For the medium-term bioassay, the photoperiod was set at 10:14 hours

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light:dark. Experimental setups and mussels’ sampling are detailed below:

166 Exp. 1 – To estimate the uptake of MP by mussels. Mussels were exposed to 6 µm and 10 µm

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MP (single and combined) at concentrations of 1000 MP⋅mL-1, making a total of three

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treatments plus the control. These experiments had the duration of 90-min (n = 3 per condition).

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Water samples were collected from each treatment every 10 min and immediately screened for

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the presence of MP using a Coulter Counter.. At the end of the experiment, all mussels were

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placed in clean filtered seawater (one mussel per glass beaker). Afterwards, mussels’ faeces

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were collected with a pipette, from the bottom of the glass beaker, for subsequent microplastic

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detection.

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Exp. 2 – To trace MP within mussels’ whole body. Once being demonstrated that mussels were

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able to filter PS microplastics in relatively few minutes, a 20-min bioassay was performed with

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the same experimental conditions as Exp. 1: MP diameter: 6 µm and 10 µm; MP concentration

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1000 MP⋅mL-1; n = 3 per experimental condition and sampling time (T0, T5, T10, T15, T20).

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See below for the detailed histological procedure.

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Exp. 3 – To investigate oxidative stress responses to MP exposure. Mussels (n = 3 per

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experimental condition and sampling time: T12, T24 and T48) were exposed to 10 and 1000

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MP⋅mL-1 of 2 µm, 6 µm and 10 µm PS microplastics (single and combined).After 12h, 24h and

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48h of exposure, mussels were collected and the organs related to filtration and digestion, such

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as gills and digestive gland, were excised and stored at -20°C for biochemical analyses.

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Exp. 4 – To determine the occurrence of potential histopathological effects in whole digestive

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tract of mussels after medium-term MP exposure (21 days), followed by a depuration period (7

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days). Mussels (n = 6 per experimental condition and sampling time) were exposed to 5 µm and

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10 µm MP (single and combined) at a concentration of 1000 MP⋅mL-1. Mussels were fed daily

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with the microalgae Tetraselmis suecica and the seawater was renewed 50% every 2 days and

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100% weekly. Recontamination was done after each renewal. At the end of both exposure and

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depuration periods, the six mussels per treatment were prepared for whole-organism

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histopathological analysis. The mussels’ digestive tract was divided in three main regions as

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described by Morton (1983): conjoined style sac and midgut (R2), separated midgut (R3) and

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rectum (R4). The oesophagus region (R1) was not surveyed in this work due to the specific

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ventro-dorsal approach conducted in all organisms. For the histopathological analyses were

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accounted: i) the number of sections with MP, per region and ii) the number of inflammatory

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foci in gastric epithelium, per focal plane and per region.

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Microscopy and histopathological analyses

203 Freshly-collected faeces (kept on ice upon collection to minimise protozoa activity) were

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mounted in ultrapure water and immediately inspected for the presence of MP under the

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microscope. Histopathological analyses were done in whole-body sections, after being

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previously preserved in Davidson’s fixative (Costa, 2018). In summary, after fixation, animals

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were divided ventro-dorsally, which produced two parts: anterior and posterior. Histological

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sections were obtained in both parts to gather a representative image of the digestive tract. One

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slide per part (per individual) was analysed, containing, on average, three sections per slide. To

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ensure preservation of PS–MP external form, the histological procedure was followed by

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mandatory modifications developed by Gonçalves et al. (2018), in order to make the process

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compatible with PS–MP. In brief: samples were dehydrated in a progressive series of

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isopropanol and embedded in paraffin. Samples were then sectioned at 5 µm thickness using a

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Jung RM 2035 BioCut Microtome (Leica Microsystems). Sections were stained with

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Haematoxylin and Eosin (H&E). Deparaffination was done by alternately heating and

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immersing the slides in 100% isopropanol until complete removal of paraffin. The slides were

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mounted with 50% glycerol and sealed with nail polish.

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Biochemical biomarkers

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In order to determine the effects of MP ingestion, a toxicological screening based on non-

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specific biomarkers was performed. Biochemical analyses were performed in the mussels’

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digestive gland and gills. Lipid peroxides were determined from the thiobarbituric acid-reactive

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substances (TBARS) protocol developed by Uchiyama and Mihara (1978) and adapted to a

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microplate reader by Costa et al. (2011). Glutathione S-transferase (GST) activity was analysed

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by measuring the increase in absorbance at 340 nm during 6 min, using chloro-2,4-

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dinitrobenzene (CDNB) as substrate. Both biomarkers were performed in Biochrom ASYS

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UVM 340 microplate reader.

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Statistical analysis

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Normality and homoscedasticity of data were assessed through the Kolmogoroff-Smirnoff’s and

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Levene’s test, respectively. After the invalidation of at least one of these assumptions, non-

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parametric tests were employed. The Kruskal-Wallis ANOVA-by-ranks H test was used for

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multiple comparisons. Friedman’s test was used for post-hoc comparisons. Spearman rank-order

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correlation R statistic was used to search for individual links between the number of

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inflammatory foci and the presence of microplastics in all regions. A significance level α = 0.05

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was set for all analyses. All statistics were performed using Statistica (StatSoft®).

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Results

242 The findings from Exp. 1 indicate that mussels are able to rapidly remove both PS–MP sizes (6

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and 10 µm Ø) from the water column (Fig. 1A). After 20 min of exposure, about 40% and 60%

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of 6 and 10 µm Ø MP were removed, respectively, in both single or combined treatments.

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Additionally, the results from the first experiment showed that, after being ingested, PS–MP

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passed through the entire digestive tract and were expelled together with organism’ faeces. This

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fact was verified by the presence of MP in mussels’ faeces (Fig.1B) after exposure to all

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treatments.

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The histological observations from Exp. 2 revealed that the larger MP (i.e. 10 µm Ø) could be

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found in digestive tract just after 5 min of exposure, whereas the smaller required about 15 min.

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In either case, and in the combined exposure, MP were only found in the lumen of gut and not

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in the gills or the digestive gland. No traces of MP were found in the remaining visceral mass.

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Despite small foci of haemocytic infiltration in the gastric epithelium (Fig.2A), no severe

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histopathological alterations were found in the remaining digestive tract, in this bioassay.

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The experiment was repeated (Exp. 3) to determine biochemical alterations, with emphasis on

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oxidative stress. No significant differences were found either in lipid peroxidation or in the

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activity of Glutathione S-transferase (Kruskal-Wallis H, p > 0.05), regardless of treatments and

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time of exposure (see Fig.S1 in supplementary material).

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After 21 days of exposure (Exp. 4), mussels from each experimental condition revealed MP in

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their digestive tracts, namely in stomach’s and digestive gland´s diverticula. After seven days of

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depuration, animals still exhibited MP inside the stomach. No MP were found in the other

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organs, such as gills or gonads, regardless of MP size and exposure time.

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In general, the number of sections with MP was higher during exposure, independently of

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experimental condition or gut region, in comparison with the depuration period, as shown in

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Fig. 4A. The midgut (R3) was the region with the largest number of sections with MP, during

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both exposure and depuration periods (Fig.3A and 4B). No significant differences were found

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between treatments within each region of the gut (Kruskal-Wallis H, p > 0.05). All treatments

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presented significant differences (Friedman’s test, p < 0.05) among the three regions, except for

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the mixture treatment (21 days) and the MP5 treatment (depuration period).

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Regardless of MP size, singly or combined, no severe internal lesions were found, including

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abrasion of digestive epithelia, even during the depuration period. However, the findings from

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Exp. 4 revealed that all individuals exhibited inflammatory foci in these epithelia, but without a

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clear relation with the presence of MP. Only one individual, from the MP 10 µm treatment, was

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found with evident inflammation around MP lodged within the epithelium of the stomach

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(Fig.3B). Accordingly, no significant correlations were found between the average number of

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inflammatory foci and the presence of MP in any region.

283 Among all regions, the separated midgut (R3) was the one with the highest average number of

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inflammatory foci per focal plane (Fig.4C) but no significant differences were found regarding

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the number of inflammatory foci among treatments, per region of gut (Kruskal-Wallis H, p >

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0.05), as shown in Fig. 4D. Additionally, there were no significant differences between all

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treatments from exposure time and its homologous from depuration period for this alteration

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(Kruskal-Wallis H, p > 0.05).

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Discussion

294 The present results revealed that, at the tested concentrations (10 and 1000 MP·mL-1),

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commercial polystyrene spherical MP (2 – 10 µm Ø) were promptly removed from the water by

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filter-feeding mussels, passed through the whole digestive tract and were released through

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faeces without significant damage to gut epithelia. Accordant with reduced histopathological

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effects, lipid peroxidation and Glutathione S-transferase activity indicated that exposure and

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ingestion of polystyrene MP did not cause significant oxidative stress in medium-term

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exposures.

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As filter-feeders, mussels are well-adapted to feed on suspended particles, whether composed of

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organic or inorganic materials (Ward and Shumway, 2004). The results from the present

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research revealed that mussels indeed filtered microplastics with no clear preference for any of

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the tested MP size nor for single or combined exposure, in accordance with other studies with

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filter-feeders, even with different MP concentrations and diameters (Browne et al., 2008; Farrel

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and Nelson, 2013; Van Cauwenberghe, 2015; Duis and Coors, 2016).

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The uptake and ingestion of MP have been investigated in several marine invertebrates exposed

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under laboratory conditions (Avio et al., 2017; Wright et al., 2013). These studies demonstrated

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that sea urchins (Tripneustes gratilla), amphipods (Orchestia gammarellus), polychaetes

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(Arenicola marina) and mussels (Mytilus sp.) have the ability to ingest microplastics, which

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may be partly released through faeces (Kaposi et al., 2014; Thompson, et al., 2004; Van

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Cauwenberghe et al., 2015; Wegner et al., 2012). In the present study, the presence of MP in the

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mussels’ faeces demonstrated not only that ingestion indeed occurs, but also that mussels’

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digestive tract may be adapted for particle selection according to their organic content. Based in

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several experiments with bivalves and phytoplankton-silt suspensions, Ward and Shumway

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(2004) suggested that mussels are able to selectively retain food items in the pallial cavity

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whereas inorganic material is rejected via pseudofaeces. This process can be described by the

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postingestive phase, where particles are circulated by the action of the rotating crystalline style

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(in the R2 region) and cilia on the stomach wall. This mechanism allows the settlement of

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heavier particles into the rejection grooves of the stomach, which are transported to the midgut

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(R3 region) and posteriorly incorporated into faecal pellets (Morton, 1983; Ward and Shumway,

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2004). This sorting mechanism, may explain why larger MP appeared within the stomach just

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after 5 minutes of exposure, while smaller MP required more time (Exp. 2). In addition, the

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movements of the style should rapidly clear MP from the R2 region, which explains the low

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incidence of MP observed and the subsequent increase in R3 region. Furthermore, the lack of

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physical abrasion in the digestive epithelium may be also associated with the fact that the action

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of this crystalline style binds food items into a slurry, avoiding possible adverse interactions

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with this structure.

332 The histopathological observations performed on the whole digestive tract of mussels revealed

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that, for both short- and medium-term exposure periods, the digestive epithelia exhibited

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intraepithelial inflammatory foci, albeit with an elusive relation to MP ingestion. According to

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De Vico and Carella (2012), this inflammatory response is classified as being of nodular-type

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and is commonly associated to factors as diverse as bacterial challenge or exposure to toxicants.

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In fact, these and other histopathological alterations in the mussel digestive gland (as in other

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bivalves) are markedly unspecific, i.e. can result from many factors, ranging from pollutants to

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harmful dinoflagellates and parasites (e.g. Carella et al., 2015; Cuevas et al., 2015; Galimany et

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al., 2008). The present findings suggest that nodular inflammatory responses in gut epithelia

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may be related to cleaning and recycling processes that occur during digestion. The absence of

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compelling evidence of immediate overwhelming of the mussels’ anti-oxidant defences is

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consistent with reduced pathological traits associated with MP ingestion, following short-term

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exposure. Nonetheless, at this stage any potential impairment of the anti-oxidative response at

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longer exposures (enzymatic and non-enzymatic) cannot be fully ascertained and needs further

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research.

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Altogether, the ingestion of polystyrene MP after a medium-term exposure of 10 and 1000

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MP·mL-1, may not exert significant deleterious effects in adult Mytilus galloprovencialis. In

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accordance, no significant effects at cellular level or filter-feeding activity were observed in M.

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edulis, after exposure to 43 items fluorescent polystyrene microspheres (3.0 and 9.6 µm) per

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millilitre (Browne et al., 2008). On the other hand, recent studies had reported several

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histological changes and strong inflammatory responses, up to the formation of granulocytomas

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and lysosomal membrane destabilization in M. edulis, after exposure to 27 000 items·mL-1 of 0

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– 80 µm of high-density polyethylene particles (von Moos et al., 2012). The lack of agreement

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about what “environmentally-relevant” concentrations mean leads to the suspicion that the

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reported effects of MP ingestion that may not correspond to reality, if compared with the

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highest reported concentration in marine environment. In relation, Phuong et al. (2016) stated

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that one of the main issues is the difficulty to establish standardised protocols and units for

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quantification of MP in the environment. In addition to the problem of “ecologically-relevant”

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MP concentrations, it remains to be asserted how irregularly-shaped and -sized MP, i.e.

363

resulting from plastic disintegration and erosion, can provoke different effects in filter-feeders at

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a long run (see Duis and Coors, 2016). Nonetheless, the present results demonstrated that the

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effects of MP ingestion by mussels may not be as evident as it could be suspected.

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Conclusions

369 Taken together, these results suggest that filter-feeding bivalves are well-adapted to cope with

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the filtration of suspended microplastics, at least with respect to commercial pellets with smooth

372

edges, as with other undigestible small-sized organic and inorganic particles present in the

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aquatic environment. The interactions between MP and mussels under potentially ecologically-

374

relevant concentrations may be restricted to MP ingestion and posterior rejection through

375

faeces. The results highlight the need to investigate the effects caused by filtering rougher,

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fragmented MP. Indeed, higher environmental relevance should be applied to laboratory

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experiments, namely considering i) potentially adsorbed contaminants, ii) biofouling associated

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with the MP in the aquatic environment and also, iii) longer exposure times.

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Acknowledgments

382 383

This study was supported by the project PLASTOX (JPIOCEANS/0003/2015), which is also

384

acknowledged for the fellowship to C. Gonçalves. This work had also the financial support of

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Fundação

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UID/MAR/04292/2013 granted to MARE. Marta Martins was supported by FCT through the

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post-doctoral grant ref: SFRH/BPD/109734/2015. Pedro M. Costa also acknowledges FCT for

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the grant IF/00265/2015. The authors also acknowledge Aquário Vasco da Gama for providing

389

the microalgae.

a

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Ethics and conflicting interest statement

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The authors declare that there are no pending issues regarding ethics, conflicts of interest or

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animal testing to be declared.

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Figure captions

587 Fig. 1 Results from the mussels’ exposure to PS-MP (6 and 10 µm Ø) during 90-min. The

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mixture treatment corresponds to both MP sizes combined (1:1). A) Removal of polystyrene

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MP by filtration over 90 min, expressed as percentage of MP in water, relatively to the initial

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load. B) Photomicrograph representing the PS–MP in the faeces of mussels subjected to the

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same experiment. Scale bar: 25 µm.

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Fig. 2 Toxicopathological effects in mussels exposed to MP. A) Photomicrograph representing

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small foci of haemocytic infiltration in the gastric epithelium of a mussel exposed to PS–MP 10

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µm Ø (arrowheads), for 20 min. Scale bar: 25 µm. B) Lipid peroxides (given by TBARS) in

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digestive glands of mussels exposed to different diameters of MP, for 48 h.

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Fig. 3 Photomicrographs representing PS–MP within different regions of the digestive tract of a

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mussel exposed to MP (5 µm and 10 µm), for 21 days. Scale bars: 25 µm. A) Section of the

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separated midgut region (R3) containing 5 µm PS microplastics (arrowheads). B) Section of

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conjoined style sac and midgut region (R2) with 10 µm PS microplastics within the stomach

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epithelium. Note that both microplastics are surrounded by haemocytic cells.

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Fig. 4 Mean results from histopathological analyses of whole digestive tract of mussels from

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medium-term (21-day) bioassay. The Mix-treatment correspond to both MP sizes combined

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(1:1). Error bars indicate 95% confidence intervals. A) Mean results from all regions (R2, R3,

611

R4) of average number of sections with microplastics. B) Average number of sections with

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microplastics. C) Average number of inflammatory foci per focal plane. D) Mean results from

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all regions (R2, R3, R4) of the average number of inflammatory foci per focal plane. Horizontal

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bars indicate significant differences between the three regions under the same treatment

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(Friedman’s test p < 0.05). ∗ Indicates significant differences between the same treatment in

616

both exposure and depuration periods (Kruskal-Wallis H, p < 0.05). Controls are omitted from

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A) and B), as no MP were hitherto found.

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Highlights

2 Mussels are able to ingest and excrete polystyrene microplastics

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MP are rapidly processed through the mussels’ digestive tract

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No significant pathological effects occurred even during longer-term exposure

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No clear evidence of oxidative stress caused by ingested polystyrene MP

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