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Chemosphere 221 (2019) 107e114

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Chemosphere

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The uptake of microfibers by freshwater Asian clams (Corbiculafluminea) varies based upon physicochemical properties

Lingyun Li a, Lei Su a, Huiwen Cai a, Chelsea M. Rochman b, Qipei Li a,Prabhu Kolandhasamy a, Jinping Peng c, Huahong Shi a, *

a State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, 200241, Chinab Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, Canadac Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, 510006, China

h i g h l i g h t s

* Corresponding author.E-mail address: hhshi@des.ecnu.edu.cn (H. Shi).

https://doi.org/10.1016/j.chemosphere.2019.01.0240045-6535/© 2019 Elsevier Ltd. All rights reserved.

g r a p h i c a l a b s t r a c t

� The uptake of microfibers by clamsvaries by polymer type and size.

� The uptake of fibers was greater in1000 items/L than that in 100 items/Lgroups.

� Clams were more likely to uptakepolyester fibers in the smaller sizerange.

� Microplastics with “environmentallyrelevant properties” should be usedin future.

a r t i c l e i n f o

Article history:Received 12 September 2018Received in revised form12 December 2018Accepted 3 January 2019Available online 4 January 2019

Handling Editor: Tamara S. Galloway

Keywords:MicroplasticMicrofiberPhysicochemical propertiesUptakeAsian clam

a b s t r a c t

Microplastic is an umbrella term that covers particles with various physical and chemical properties.However, microplastics with a consistent shape, polymer type and size are generally used in exposurestudies (e.g., spherical polyethylene or polystyrene beads 1e100 mm in size). In the present study, weexposed freshwater Asian clams (Corbicula fluminea) to microfibers with different physicochemicalproperties at concentrations of 100 and 1000 fibers/L. The first experiment in this study exposed clams tomicrofibers made from six different polymers, demonstrating that Asian clams uptake more polyester(PET) (4.1 items/g) relevant to other polymers. The next experiment exposed clams to PET fibers ofdifferent size classes, demonstrating that uptake in the size range 100e250 mm (1.7 items/g) was greaterthan other size classes. These results suggest that physicochemical properties such as polymer and sizeplay important roles in the uptake of microfibers by organisms. Thus, we strongly suggest that theproperties of microplastics used in future laboratory exposure experiments be considered, with the aimof being “environmentally relevant”, i.e., similar to what is found in nature.

© 2019 Elsevier Ltd. All rights reserved.

1. Introduction

The term “microplastics” refers to plastic particles or fragments

less than 5mm in size (Arthur et al., 2009). Microplastic hasbecome a global environmental problem because of its ubiquitousdistribution and high abundance in aquatic environments (Law andThompson, 2014). The uptake of microplastics has been demon-strated in a large variety of aquatic organisms, includingzooplankton, bivalves and fish (Desforges et al., 2015; Li et al., 2015;Jabeen et al., 2017). The persistence nature and ubiquitous presence

L. Li et al. / Chemosphere 221 (2019) 107e114108

of microplastics make the investigation of their potential risks toaquatic organisms important and urgent (Rochman et al., 2016).

Because of the great complexity of the aquatic environment, it isdifficult to observe the impacts of microplastics in the field. As such,laboratory studies are critical to increase our understanding aboutpotential effects in nature. To date, many laboratory exposure ex-periments have been conducted to evaluate the potential impactsof microplastics on aquatic organisms (Von Moos et al., 2012;Grigorakis et al., 2017; Kolandhasamy et al., 2018). In most labo-ratory studies, commercialized microplastics with a sphericalshape, single polymer and precise size are used (Browne et al.,2008; Cole and Galloway, 2015; Hu et al., 2016). This tends to bebased upon what is available commercially for experimentation.

However, microplastics are made up of different physical (e.g.,shape and size) and chemical (e.g., polymer and additive) proper-ties (Eriksen et al., 2013; Koelmans et al., 2014; Fok and Cheung,2015; Hu et al., 2018). In addition, microplastics in nature areconstantly changing via various physical, chemical and biologicalmechanisms. For example, weathering and biofouling (Andrady,2017; Galloway et al., 2017). As a consequence, the heterogeneityof physical and chemical properties of microplastics is overlookedin most laboratory studies, and the toxicological effects of micro-plastics observed in laboratory may not reflect toxicological con-ditions in nature (Phuong et al., 2016; Burton, 2017).

Due to their extensive filter-feeding activities, bivalves are likelyto take up and accumulate large quantities of microplastics fromtheir living environment. In recent years, bivalves have receivedconsiderable attention inmicroplastics studies (Van Cauwenbergheand Janssen, 2014; Li et al., 2016; Xu et al., 2016). Freshwater clamsare one representative type of filter-feeders. The Asian clam(Corbicula fluminea) originates fromAsia and can be found in awiderange of freshwater habitats across the world (Sousa et al., 2008).The Asian clam has been proposed as a bioindicator to trace themicroplastic pollution, and as a toxicological test animal in thelaboratory. Su et al. (2018) has proved that clams would reflect thevariability of microplastic pollution in the freshwater environ-ments. Asian clam has also been used in toxicity testing. One studyfound that the ingestion of microplastics induced tubular dilationin the gut tissues (Rochman et al., 2017).

In the present study, we purchased multiple plastic productsfrom the market and made them into microfibers with differentpolymers and sizes. We focused on fibers because they are thedominant microplastic shape in the freshwater, like lake and river,and the fiber polymers varied (Su et al., 2016; Miller et al., 2017;Sighicelli et al., 2018). We exposed Asian clams to these microfiberswith diverse properties. The uptake of microfibers was measuredand compared among the clams. Our objective was to determinehow uptake of microfibers varies based upon the physical andchemical properties.

2. Materials and methods

2.1. Origin and maintenance of Asian clams

Asian clams were collected from Dianshan Lake in China andsubsequently transferred to glass tanks that were part of a semi-static freshwater system with de-chlorinated and filtered tap wa-ter (1 mm pore size, 47mmGF/B glass microfiber filters, Whatman)in the laboratory. Dianshan Lake is an important water resource forShanghai and a natural habitat for clams (Liu et al., 2014). The clamsweremaintained at 20± 1 �C and under a 12 h light-dark cycle withconstant aeration (Cid et al., 2015). The water was changed every24 h, and the clams were fed with dried powder of Spirulina spp. ata concentration of 0.25mg/L at the same time every 24 h. Clamswere kept under laboratory conditions for one week prior to use.

2.2. Preparation of microfibers

Plastic products were purchased from the market, and micro-fibers from six different polymers were prepared for further poly-mer experiments: black polyester-amide (PEA), red polyester (PET),black acrylic (AC), blue polyamide (PA), red rayon (RA) and whitepolyvinyl alcohol (PVA). The raw material was cut with dissectingscissors into tiny pieces that were as small as possible (Gutow et al.,2016). Fibers were collected with ultrapure Milli-Q water, passedthrough a 5mm mesh sieve and then transferred onto a 5 mm poresize nylon membrane filter. The main properties of the fibers weremeasured (Supplementary Table 1). The size distributions of thefibers were provided in Supplementary Fig. 1. The polymers wereidentified using a micro-Fourier transform infrared spectroscopymicroscope (LUMOS m-FT-IR, Bruker) in attenuated total reflectance(ATR) mode (Dris et al., 2016). The fibers were also examined with ascanning electron microscope (SEM, Hitachi S-4800, Japan) basedon the methodology of Li et al. (2016) (Fig. 1). The SEM was con-ducted for better comparing the diameters and observing thesmooth or rough surfaces of fibers, this could provide evidence forthe subsequent analysis that if the different diameters and surfacestructures have effect on the uptake by clams. The densities of themicroplastics were measured using the density gradient solutions,in which the floating and sinking statuses of the tested micro-plastics were observed (Li et al., 2018). All the fibers were heavierthan water and could sink to the bottom of tanks within 2min(Supplementary Table 1). A softer material would have a lowerelastic modulus, thus the softness of fibers are usually indicated bytheir elastic modulus. The elastic modulus of fibers were deter-mined using atomic force microscopy (AFM) with a BrukerDimension Icon under Peak Force Quantitative NanomechanicalMapping (PF-QNM) mode. The data was analyzed via NanoScopeAnalysis 1.8. Six aqueous stocks of approximately 3500 fibers/mLwere prepared for each of the different polymers.

PET fibers were chosen for further size experiments. Five sizeclasses of PET microfibers were prepared (Supplementary Fig. 2).The five classes, classified based on the length of their fibers, were I(5e100 mm), II (100e250 mm), III (250e500 mm), IV (500e1000 mm)and V (1000e5000 mm). Fibers in the range of 5e100 mm werecollected using nylon membrane filters with 5 mm and 100 mm poresizes; fibers >100 mm were collected using a series of sieves with250 mm, 500 mm,1000 mm and 5000 mmmesh sizes. Aqueous stocksof approximately 1000 fibers/mL were prepared for the five sizecategories. The concentrations of microfibers in the solutions weredetermined by counting with a Sedgewick rafter counting cell un-der an Carl Zeiss Discovery V8 Stereo microscope (MicroImagingGmbH, G€ottingen, Germany), and the sizes were subsequentlymeasured with an AxioCam digital camera.

2.3. Exposure experiments

Intact and active clams were selected from the glass tanks forthe exposure experiments. The average length of the clams was2.2± 0.1 cm, and the average total body wet weight was 4.1± 0.4 g.Two concentrations of microfibers (100 and 1000 items/L) wereused in all exposure experiments. Ten clams were put in each tankwith 2 L of water, the experiments were carried out in triplicate.Aliquots for the exposure experiments were prepared by pipettingmicrofibers from stocks after the process of ultrasonication. Controlclams were fed with algae only. During the exposure period, thewater was changed daily, and both food and microfibers were re-dosed at nominal concentrations every day. Two experimentswere run chronologically.

In experiment 1, we studied the uptake of different polymers of

Fig. 1. Polymers and surface structures of different fibers (A1-F1) identified using m-FT-IR (A2-F2) and SEM (A3-F3). Scale bar: A1-F1¼200 mm, A3-F3¼ 5 mm. Abbreviations: PET,polyester; PEA, polyester-amide; AC, acrylic; PA, polyamide; RA, rayon; PVA, polyvinyl alcohol.

L. Li et al. / Chemosphere 221 (2019) 107e114 109

L. Li et al. / Chemosphere 221 (2019) 107e114110

fibers. The exposure period of experiment 1 lasted two days sincethe abundance of fibers reached a high level in the clams at thattime point in the preliminary test. Each polymer was regarded as aseparate treatment group. There were 12 experimental treatmentgroups (6 polymers and 2 concentrations) and 2 control groupswith three replicate tanks in each group, i.e., 42 tanks in total. Theclams were collected after 2 days of exposure.

In experiment 2, we studied the uptake of different PET mi-crofiber sizes. According to the results of experiment 1, PET fiberswere present at the highest levels in clams, so they were used inexperiment 2. Moreover, PET is a common polymer type in somefield investigations (Wang et al., 2017; Lahens et al., 2018; Su et al.,2018). Each size category of fibers was regarded as one treatment.There were 10 experimental treatment groups (5 size classes and 2concentrations) and 2 control groups with three replicate tanks ineach group, i.e., 36 tanks in total. The clamswere collected after twodays of exposure.

2.4. Digestion and filtration treatment

Clams were collected and wrapped in clean aluminum foil afterthe exposure period and kept at �20 �C for further analysis. Tapwater was pre-filtered through 1 mm pore size, 47mmGF/B glassmicrofiber filters (Whatman) to prevent airborne contamination.Conical flasks, glass dishes and a dissecting knife were rinsed threetimes with the filtered water. The shell length and total weight ofeach clam were recorded first. Next, the clam shells were opened,and their inner contents were rinsed gently with ultrapure Milli-Qwater to remove their intervalvular water. Then, the soft tissues ofthe clamswere separated andweighted. The soft tissues of 10 clamsin each replicate were pooled into a 250-mL conical flask, andapproximately 200mL of 10% KOHwas added to each flask to digestthe soft tissues (Dehaut et al., 2016). The conical flasks werecovered with glass lids and set in an incubator. The incubatortemperature was 65 �C, and the rotation speed was 80 rpm. After12 h, the solution in each conical flask was immediately transferredonto a 5 mm pore size nylon membrane filter using a vacuum sys-tem. The membrane filters were kept in petri dishes for furtherobservation.

2.5. Observation and quantification of microfibers

The observation and quantification of the microfibers followedour previous methods (Yang et al., 2015). In brief, the fibers wereobserved with a Carl Zeiss Discovery V8 Stereo microscope(MicroImaging GmbH, G€ottingen, Germany), and images of the fi-bers were taken with an AxioCam digital camera.

2.6. Statistical analysis

Differences in the abundance of microfibers among treatmentgroups were determined using the Kruskal-Wallis non-parametrictest with multiple comparisons. A significance level of 0.05 waschosen.

3. Results

3.1. Uptake of microfibers of different polymers

No microfibers were observed in control groups. Only PET andPEA fibers were found ingested by clams in the 100 items/L mi-crofiber treatment group, and PET, PEA, AC and PA were foundingested by clams in the 1000 items/L microfiber treatment group(Fig. 2). PET fibers showed various curved forms and even bent into

loops (Fig. 2A); PEA fibers mostly had arched forms (Fig. 2B); ACand PA fibers only presented in straight line forms (Fig. 2C and D)after ingestion. The colors of the PET, PEA and AC fibers did notchange significantly after digestion, but the color of the PA fiberschanged from dark blue to light blue. The oxidation of KOH causedthe PA fibers to fade slightly without destroying their structures.The abundance of PET fibers was higher (0.5 items/g) than that ofPEA fibers (0.3 items/g) in the 100 items/L microfiber treatmentgroup (Fig. 2E). PET fibers showed the highest abundance (4.1items/g), and PA fibers showed the lowest abundance (0.1 items/g),in the 1000 items/L microfiber treatment group (Fig. 2F,Supplementary Table 2). The abundances were also provided for allpolymers as items/individual in Supplementary Fig. 3.

Of all six polymers of fibers, the value of elastic modulus was thelowest for PET fibers, followed by PEA and AC (Fig. 3AeC). Theuptake of fibers in clams were negatively correlated with the valueof elastic modulus (p< 0.05) (Fig. 3D).

3.2. Uptake of PET fibers of different sizes

No fibers were found in the control groups (Fig. 4A), and fibersfrom all five size classes were found in the clams of the treatmentgroups (Fig. 4BeF). Morphologically, the extraction procedurecaused curliness in the fibers. The fibers extracted from the clamswere more likely to be curved than fibers before exposure(Fig. 4BeF; Supplementary Fig. 2). The abundance of fibers in eachsize class was low (0.1e0.2 items/g) and did not significantly differamong the five classes in the 100 items/L microfiber treatmentgroup (Fig. 4G). The abundance of fibers in the Size II (100e250 mm)class was the highest (1.7 items/g), and the abundance of fibers inthe Size V (1000e5000 mm) class was the lowest (0.2 items/g) in the1000 items/L microfiber treatment group (Fig. 4H, SupplementaryTable 3). The uptake of fibers belonging to any size class wasgreater in the 1000 items/L exposure treatments than that in the100 items/L exposure treatments. The abundance were also pro-vided for all size classes of PET fibers as items/individual inSupplementary Fig. 3.

4. Discussion

4.1. The uptake of microfibers with different properties

Microplastic concentrations used in previous laboratory studiesusually tend to be several orders of magnitude above the levels innature (Lenz et al., 2016). To overcome this issue, we used micro-plastics levels of 100 items/L and 1000 items/L to simulate “envi-ronmentally relevant concentrations” in our laboratory exposures.The highest concentrations of microplastics found in nature are thesame order of magnitude as the 1000 items/L treatment (Mooreet al., 2011). This order of concentration was also be used as envi-ronmentally relevant data in some exposure studies (Xu et al., 2016;Gray and Weinstein, 2017). Our results showed that the amount ofuptake dependent upon the availability of microplastics in theenvironment, which is in accordance with Scherer et al. (2017).

Since fibers were usually the dominant category found in somefield studies, we specifically chose fibers of six different polymersfor further exposure experiments (Zhang et al., 2018). In addition,the risk assessment studies based on plastic microfibers are limited(Jemec et al., 2016).We found great variations in the uptake of thesefibers. Generally, plastics of different polymers show a wide varietyof density, surface structure, chemical ingredients and hardness.These properties govern the distribution and sinking of micro-plastics in thewater column and subsequently affect the interactionof microplastics with biota (Wang et al., 2016; Bagaev et al., 2017).In the present study, all the fibers were heavier thanwater and sank

Fig. 2. Uptake of fibers of different polymers in Asian clams after 2 days of exposure. Each value represents the mean ± standard deviation of the three replicates (n¼ 3). A-D, showimages of the fibers extracted from the tissues of clams after the exposure period. The arrows indicate the observed microplastics. E-F, show the abundance of fibers in the 100items/L (E) and 1000 items/L (F) exposure treatments. The letters above the bars indicate significant differences (p< 0.05). If two arbitrary groups have the same letter, then they arenot significantly different. Abbreviations: Ctrl., control group; PET, polyester; PEA, polyester-amide; AC, acrylic; PA, polyamide; RA, rayon; PVA, polyvinyl alcohol. Scalebar¼ 200 mm.

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to the bottom of the tanks within 2min, which provided a relativelyfair opportunity for the fibers to be encountered by clams. In termsof surface structure, all fibers had smooth surfaces. Although thePET and RA fibers were both red, the RA fibers were not ingested byclams. The PET and PVA fibers had similar length distributions anddiameters, but the PVA fibers were not ingested by clams. It isreasonable to deduce that color, length distribution, diameter,density, settling velocity and surface structure were not key factorsleading to differences in the uptake of different fibers in the presentstudy.

The value of elastic modulus is an index to indicate the softnessof fibers. Our results suggested that PET were the softest fibers, andthe uptake of fibers was positively related to their softness.Therefore, our results indicated that the ingestion of fibers by clamswas greatly determined by the property of softness of fibers. To ourbest knowledge, this is the first time to point out the relation be-tween softness of fibers and their bioavailability to the organisms.Still, it is possible that the differences were also driven by differ-ences in chemical mixtures among polymers.

The size distribution of microplastics is another key property

Fig. 3. The elastic modulus of fibers and their relationship with the uptake of fibers in clams. A, show representative topography map of PET fiber obtained by atomic force mi-croscopy (AFM); B, show the elasticity modulus distribution of PET fiber; C, show the average elasticity modulus of six different types of fibers; D, show the correlation betweenelastic modulus and the abundances of fibers in clams. Abbreviations: PET, polyester; PEA, polyester-amide; AC, acrylic; PA, polyamide; RA, rayon; PVA, polyvinyl alcohol.

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affecting the bioavailability of microplastics in aquatic organisms(Wright et al., 2013). In the present study, we used the full size-spectrum of microfibers, 5 mm-5000 mm in the laboratory expo-sure experiments. In the field, microplastics ranging from 5 to5000 mm can be found in the tissue of Asian clams, and the range of100e1000 mm is the dominant size class (Su et al., 2016, 2018). Thegreater uptake of fibers in the middle size class in the present studyis consistent with the optimum size of microplastics found in filter-feeders in previous field studies (Li et al., 2016; Qu et al., 2018).Siphons are the primary feeding apparatus of clams. The averagediameter of clam siphons was 620 mm in this study. It was hard forthe clams to take up fibers >1000 mm due to the limitation of theirfeeding apparatus (Rosenkranz et al., 2009). Our results suggestedthat clams could uptakemore soft fibers (e.g., PET), which indicatedthat bigger fibers still might bend and go through the narrow si-phons of clams. In addition to ingestion, adherence is anothermicroplastics uptake route and has been observed in mussels(Kolandhasamy et al., 2018). It is possible that the fibers mayadhered to clams.

4.2. The importance of microplastics with environmentally relevantproperties

Our results strongly suggest that the physicochemical propertiesof microplastics significantly affect their bioavailability to clams. Inour previous studies, fibers were usually the dominant microplasticshape not only in the water but also in the clams (Su et al., 2016,2018). In addition, this study mainly focused on properties ofpolymer and size of microfibers, which neglects the complexity of

the microplastics in real environments. For instance, crystallinity,surface chemistry and weathering status are also important prop-erties affecting the behaviors of microplastics (Andrady, 2017;Galloway et al., 2017).

Since laboratory conditions are not always consistent withenvironmental conditions, the uptake and accumulation of micro-plastics in the laboratory cannot reflect the reality observed in thefield (Burton, 2017). Several researchers have emphasized that“environmentally relevant concentrations” of microplastics shouldbe considered in microplastic studies in the laboratory (Lenz et al.,2016; Rochman, 2016). The idea of “environmentally relevantconcentrations” is very useful to the design of exposure experi-ments for common chemicals as well as microplastics (Besselinget al., 2017; Ziajahromi et al., 2018). However, microplastics are acocktail with dynamic chemicals and properties. It is still notenough to just consider “environmentally relevant concentrations”in the exposure experiments (Karami, 2017; Lambert et al., 2017;Pottoff et al., 2017). Therefore, we propose that the concept of“environmentally relevant properties” should be used in the labo-ratory to better simulate microplastics in field conditions. “Envi-ronmentally relevant properties” refers to the properties ofmicroplastics present in real environments, including not only thelevels of microplastics but also other physicochemical propertiessuch as shape, polymer and size.

To simulate microplastics with “environmentally relevantproperties”, first, the properties of microplastics such as abun-dance, color, shape, polymer and size distribution in natural envi-ronments should be described as precisely and comprehensively aspossible. Currently, we describe these properties separately and

Fig. 4. Uptake of PET fibers of different size classes in Asian clams after 2 days of exposure. Each value represents the mean± standard deviation of three replicates (n¼ 3). A-F,show images of the control (A) and microplastics extracted from the treatment clams after exposure (BeF). The arrows indicate the observed microplastics. G-H, show theabundance of fibers in the 100 items/L (G) and 1000 items/L (H) exposure treatments. The letters above the bars indicate significant differences (p< 0.05). If two arbitrary groupshave the same letter, then they are not significantly different. Abbreviations: Ctrl., control group; I, Size I (5e100 mm); II, Size II (100e250 mm); III, Size III (250e500 mm); IV, Size IV(500e1000 mm); V, Size V (1000e5000 mm). Scale bar¼ 200 mm.

L. Li et al. / Chemosphere 221 (2019) 107e114 113

rely too much on the total abundance for the comparison ofmicroplastic contaminant levels across different studies. In subse-quent studies, cocktails of microplastics with diverse properties canbe made from available plastic products based on the microplasticpollution profile in real environments. All the chosen microplasticscan be combined with different property distributions for furtherexposure experiments. Since microplastics are an emergingcontaminant with complex and unique properties, it is necessary todevelop effective methods for studying how microplastics mayaffect wildlife under laboratory conditions.

5. Conclusion

Our results strongly suggest that the physicochemical properties

of microfibers significantly affect their bioavailability in aquaticorganisms. Considering the great differences between the micro-plastics used in the present exposure experiments and those in thereal environmental conditions, we propose that diverse types,shapes and sizes of microplastics with “environmentally relevantproperties” should be considered and used in future laboratoryexposure experiments.

Acknowledgements

This work was supported by the Natural Science Foundation ofChina (21507031, 41571467) and grants from the National KeyResearch and Development Programs (2016YFC1402204).

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Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.chemosphere.2019.01.024.

References

Andrady, A.L., 2017. The plastic in microplastics: a review. Mar. Pollut. Bull. 119 (1),12e22.

Arthur, C., Baker, J., Bamford, H., 2009. In: Proceedings of the International ResearchWorkshop on the Occurrence, Effects and Fate of Microplastic Marine Debris.September 9e11, 2008. University of Washington Tacoma, Tacoma, WA. Group.530.

Bagaev, A., Mizyuk, A., Khatmullina, L., Isachenko, I., Chubarenko, I., 2017. Anthro-pogenic fibers in the Baltic Sea water column: field data, laboratory and nu-merical testing of their motion. Sci. Total Environ. 599e600, 560e571.

Besseling, E., Foekema, E.M., van den Heuvel-Greve, M.J., Koelmans, A.A., 2017. Theeffect of microplastic on the uptake of chemicals by the lugworm Arenicolamarina (L.) under environmentally relevant exposure conditions. Environ. Sci.Technol. 51 (15), 8795e8804.

Browne, M.A., Dissanayake, A., Galloway, T.S., Lowe, D.M., Thompson, R.C., 2008.Ingested microscopic plastic translocates to the circulatory system of themussel, Mytilus edulis (L.). Environ. Sci. Technol. 42 (13), 5026e5031.

Burton, G.A., 2017. Stressor exposures determine risk: so, why do fellow scientistscontinue to focus on superficial microplastics risk? Environ. Sci. Technol. 51(23), 13515e13516.

Cid, A., Picado, A., Correia, J.B., Chaves, R., Silva, H., Caldeira, J., de Matos, A.P.,Diniz, M.S., 2015. Oxidative stress and histological changes following exposureto diamond nanoparticles in the freshwater Asian clam Corbicula fluminea(Müller, 1774). J. Hazard Mater. 284, 27e34.

Cole, M., Galloway, T.S., 2015. Ingestion of nanoplastics and microplastics by Pacificoyster larvae. Environ. Sci. Technol. 49 (24), 14625e14632.

Dehaut, A., Cassone, A.-L., Fr�ere, L., Hermabessiere, L., Himber, C., Rinnert, E.,Rivi�ere, G., Lambert, C., Soudant, P., Huvet, A., Duflos, G., Paul-Pont, I., 2016.Microplastics in seafood: benchmark protocol for their extraction and charac-terization. Environ. Pollut. 215, 223e233.

Desforges, J.P., Galbraith, M., Ross, P.S., 2015. Ingestion of microplastics byzooplankton in the northeast Pacific ocean. Arch. Environ. Contam. Toxicol. 69(3), 320e330.

Dris, R., Gasperi, J., Saad, M., Mirande, C., Tassin, B., 2016. Synthetic fibers in at-mospheric fallout: a source of microplastics in the environment? Mar. Pollut.Bull. 104 (1e2), 290e293.

Eriksen, M., Mason, S., Wilson, S., Box, C., Zellers, A., Edwards, W., Farley, H.,Amato, S., 2013. Microplastic pollution in the surface waters of the Laurentiangreat lakes. Mar. Pollut. Bull. 77 (1e2), 177e182.

Fok, L., Cheung, P.K., 2015. Hong Kong at the Pearl River Estuary: a hotspot ofmicroplastic pollution. Mar. Pollut. Bull. 99 (1e2), 112e118.

Galloway, T.S., Cole, M., Lewis, C., 2017. Interactions of microplastic debristhroughout the marine ecosystem. Nat. Ecol. Evol. 1 (5), 116.

Gray, A.D., Weinstein, J.E., 2017. Size and shape dependent effects on microplasticparticles on adult daggerblade grass shrimp (Palaemonetes pugio). Environ.Toxicol. Chem. 36 (11), 3074e3080.

Grigorakis, S., Mason, S.A., Drouillard, K.G., 2017. Determination of the gut retentionof plastic microbeads and microfibers in goldfish (Carassius auratus). Chemo-sphere 169, 233e238.

Gutow, L., Eckerlebe, A., Gim�enez, L., Saborowski, R., 2016. Experimental evaluationof seaweeds as a vector for microplastics into marine food webs. Environ. Sci.Technol. 50 (2), 915e923.

Hu, L., Su, L., Xue, Y., Mu, J., Zhu, J., Xu, J., 2016. Uptake, accumulation and elimi-nation of polystyrene microspheres in tadpoles of Xenopus tropicalis. Chemo-sphere 164, 611e617.

Hu, L., Chernick, M., Hinton, D.E., Shi, H., 2018. Microplastics in small waterbodiesand tadpoles from Yangtze river Delta, China. Environ. Sci. Technol. 52 (15),8885e8893.

Jabeen, K., Su, L., Li, J., Yang, D., Tong, C., Mu, J., Shi, H., 2017. Microplastics andmesoplastics in fish from coastal and fresh waters of China. Environ. Pollut. 221,141e149.

Jemec, A., Horvat, P., Kunej, U., Bele, M., Kr�zan, A., 2016. Uptake and effects ofmicroplastic textile fibers of freshwater crustacean Daphnia magna. Environ.Pollut. 219, 201e209.

Karami, A., 2017. Gaps in aquatic toxicological studies of microplastics. Chemo-sphere 184, 841e848.

Koelmans, A.A., Besseling, E., Foekema, E.M., 2014. Leaching of plastic additives tomarine organisms. Environ. Pollut. 187, 49e54.

Kolandhasamy, P., Su, L., Li, J., Qu, X., Jabeen, K., Shi, H., 2018. Adherence ofmicroplastics to soft tissue of mussels: a novel way to uptake microplasticsbeyond ingestion. Sci. Total Environ. 610e611, 635e640.

Lahens, L., Strady, E., Kieu-Le, T.C., Dris, R., Boukerma, K., Rinnert, E., Gasperi, J.,Tassin, B., 2018. Macroplastic and microplastic contamination assessment of atropical river (Saigon River, Vietnam) transversed by a developing megacity.Environ. Pollut. 236, 661e671.

Lambert, S., Scherer, C., Wagner, M., 2017. Ecotoxicity testing of microplastics:considering the heterogeneity of physicochemical properties. Integr. Environ.

Assess. Manag. 13 (3), 470e475.Law, K.L., Thompson, R.C., 2014. Microplastic in the seas. Science 345 (6193),

144e145.Lenz, R., Enders, K., Nielsen, T.G., 2016. Microplastic exposure studies should be

environmentally realistic. Proc. Natl. Acad. Sci. U. S. A. 113 (29), E4121eE4122.Li, J., Yang, D., Li, L., Jabeen, K., Shi, H., 2015. Microplastics in commercial bivalves

from China. Environ. Pollut. 207, 190e195.Li, J., Qu, X., Su, L., Zhang, W., Yang, D., Kolandhasamy, P., Li, D., Shi, H., 2016.

Microplastics in mussels along the coastal waters of China. Environ. Pollut. 214,177e184.

Li, L., Li, M., Deng, H., Cai, L., Cai, H., Yan, B., Hu, J., Shi, H., 2018. A straightforwardmethod for measuring the range of apparent density of microplastics. Sci. TotalEnviron. 639, 367e373.

Liu, X., Wu, Z., Xu, H., Zhu, H., Wang, X., Liu, Z., 2014. Assessment of pollution statusof Dalianhu water sources in Shanghai, China and its pollution biologicalcharacteristics. Environ. Earth Sci. 71 (10), 4543e4552.

Miller, R.Z., Watts, A.J.R., Winslow, B.O., Galloway, T.S., Barrows, A.P.W., 2017.Mountains to the sea: river study of plastic and non-plastic microfiber pollutionin the northeast USA. Mar. Pollut. Bull. 124 (1), 245e251.

Moore, C.J., Lattin, G.L., Zellers, A.F., 2011. Quantity and type of plastic debris flowingfrom two urban rivers to coastal waters and beaches of Southern California.J. Integr. Coast. Zone Manag. 11 (1), 65e73.

Phuong, N.N., Zalouk-Vergnoux, A., Poirier, L., Kamari, A., Chatel, A., Mouneyrac, C.,Lagarde, F., 2016. Is there any consistency between the microplastics found inthe field and those used in laboratory experiments? Environ. Pollut. 211,111e123.

Pottoff, A., Oelschl€agel, K., Schmitt-Jansen, M., Rummel, C.D., Kühnel, D., 2017. Fromthe sea to the laboratory: characterization of microplastic as prerequisite for theassessment of ecotoxicological impact. Integr. Environ. Assess. Manag. 13 (3),500e504.

Qu, X., Su, L., Li, H., Liang, M., Shi, H., 2018. Assessing the relationship between theabundance and properties of microplastics in water and in mussels. Sci. TotalEnviron. 621, 679e686.

Rochman, C.M., Browne, M.A., Underwood, A.J., Franeker, J.A., Thompson, R.C.,Amaral-Zettler, L.A., 2016. The ecological impacts of marine debris: unravelingthe demonstrated evidence from what is perceived. Ecology 97 (2), 302e312.

Rochman, C.M., 2016. Ecologically relevant data are policy-relevant data. Science352, 1172.

Rochman, C.M., Parnis, J.M., Browne, M.A., Serrato, S., Reiner, E.J., Robson, M.,Young, T., Diamond, M.L., Teh, S.J., 2017. Direct and indirect effects of differenttype of microplastics on freshwater prey (Corbicula fluminea) and their predator(Acipenser transmontanus). PLoS One 12 (11), e0187664.

Rosenkranz, P., Chaudhry, Q., Stone, V., Fernandes, T.F., 2009. A comparison ofnanoparticle and fine particle uptake by Daphnia magna. Environ. Toxicol.Chem. 28 (10), 2142e2149.

Scherer, C., Brennholt, N., Reifferscheid, G., Wagner, M., 2017. Feeding type anddevelopment drive the ingestion of microplastics by freshwater invertebrates.Sci. Rep. 7, 17006.

Sighicelli, M., Pietrelli, L., Lecce, F., Iannilli, V., Falconieri, M., Coscia, L., Di Vito, S.,Nuglio, S., Zampetti, G., 2018. Microplastic pollution in the surface waters ofItalian Subalpine Lakes. Environ. Pollut. 236, 645e651.

Sousa, R., Antunes, C., Guilhermino, L., 2008. Ecology of the invasive Asian clamCorbicula fluminea (Müller, 1774) in aquatic ecosystems: an overview. Ann.Limnol. -Int. J. Lim. 44 (2), 85e94.

Su, L., Xue, Y., Li, L., Yang, D., Kolandhasamy, P., Li, D., Shi, H., 2016. Microplastics inTaihu lake, China. Environ. Pollut. 216, 711e719.

Su, L., Cai, H., Kolandhasamy, P., Wu, C., Rochman, C.M., Shi, H., 2018. Using the Asianclam as an indicator of microplastic pollution in freshwater ecosystems. Envi-ron. Pollut. 234, 347e355.

Van Cauwenberghe, L., Janssen, C.R., 2014. Microplastics in bivalves cultured forhuman consumption. Environ. Pollut. 193, 65e70.

Von Moos, N., Burkhardt-Holm, P., K€ohler, A., 2012. Uptake and effects of micro-plastics on cell and tissue of the blue mussel Mytilus edulis L. after an experi-mental exposure. Environ. Sci. Technol. 46 (20), 11327e11335.

Wang, J., Tan, Z., Peng, J., Qiu, Q., Li, M., 2016. The behaviors of microplastics in themarine environment. Mar. Environ. Res. 113, 7e17.

Wang, W., Ndungu, A.W., Li, Z., Wang, J., 2017. Microplastics pollution in inlandfreshwaters of China: a case study in urban surface waters of Wuhan, China. Sci.Total Environ. 575, 1369e1374.

Wright, S.L., Thompson, R.C., Galloway, T.S., 2013. The physical impacts of micro-plastics on marine organisms: a review. Environ. Pollut. 178, 483e492.

Xu, X.-Y., Lee, W.T., Chan, A.K.Y., Lo, H.S., Shin, P.K.S., Cheung, S.G., 2016. Microplasticingestion reduces energy intake in the clam Atactodea striata. Mar. Pollut. Bull.124 (2), 798e802.

Yang, D., Shi, H., Li, L., Li, J., Jabeen, K., Kolandhasamy, P., 2015. Microplastic pollutionin table salts from China. Environ. Sci. Technol. 49 (22), 13622e13627.

Ziajahromi, S., Kumar, A., Neale, P.A., Leusch, F.D.L., 2018. Environmentally relevantconcentrations of polyethylene microplastics negatively impact the survival,growth and emergence of sediment-dwelling invertebrates. Environ. Pollut.236, 425e431.

Zhang, K., Shi, H., Peng, J., Wang, Y., Xiong, X., Wu, C., Lam, P.K.S., 2018. Microplasticspollution in China's inland water systems: a review of findings, methods,characteristics, effects, and management. Sci. Total Environ. 630, 1641e1653.