Styrofoam Debris as a Source of Hazardous Additives for Marine

Apr 21, 2016 - We hypothesized that if styrofoam debris acts as a source of the additives in the marine environment, organisms inhabiting such debris ...
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Styrofoam Debris as a Source of Hazardous Additives for Marine Organisms Mi Jang,†,‡ Won Joon Shim,†,‡ Gi Myung Han,† Manviri Rani,† Young Kyoung Song,†,‡ and Sang Hee Hong*,†,‡ †

Oil and POPs Research Laboratory, Korea Institute of Ocean Science and Technology, Jangmok-myon 391, Geoje 656-834, Republic of Korea ‡ Korea University of Science and Technology, Daejeon 305-320, South Korea S Supporting Information *

ABSTRACT: There is growing concern over plastic debris and their fragments as a carrier for hazardous substances in marine ecosystem. The present study was conducted to provide field evidence for the transfer of plastic-associated chemicals to marine organisms. Hexabromocyclododecanes (HBCDs), brominated flame retardants, were recently detected in expanded polystyrene (styrofoam) marine debris. We hypothesized that if styrofoam debris acts as a source of the additives in the marine environment, organisms inhabiting such debris might be directly influenced by them. Here we investigated the characteristics of HBCD accumulation by mussels inhabiting styrofoam. For comparison, mussels inhabiting different substrates, such as high-density polyethylene (HDPE), metal, and rock, were also studied. The high HBCD levels up to 5160 ng/g lipid weight and the γ-HBCD dominated isomeric profiles in mussels inhabiting styrofoam strongly supports the transfer of HBCDs from styrofoam substrate to mussels. Furthermore, microsized styrofoam particles were identified inside mussels, probably originating from their substrates.



INTRODUCTION Global plastic production increased from 1.7 million tons in the 1950s to approximately 311 million tons in 2014.1 Due to its mass production and persistency, plastic becomes a prevalent component of marine litter.2−4 The accumulation of plastic debris in the marine environment has various adverse effects on marine ecosystems, navigational safety, and the aesthetic quality of beaches. The financial damage to marine ecosystems caused by plastics is estimated to be $13 billion each year,5 from losses incurred by fisheries, tourism, and time spent cleaning up beaches. At least 395 species, including marine mammals, birds, sea turtles, crustaceans, and bivalves, are known to have suffered from entanglement or the ingestion of marine debris.6 The role of marine debris as a carrier of hazardous chemicals in the marine environment is an emerging issue. There are two types of chemicals: additives and absorbed chemicals. Additives are chemicals that are added during manufacturing processes to enhance the performance of plastics and include antioxidants, plasticizers, etc. Some of them are known to be endocrinedisrupting chemicals (e.g., phthalate and bisphenol A), which can disturb the reproduction and development of marine organisms.7 Plastics contain large amounts of additive chemicals that can leach into the surrounding environment.8,9 For example, polyvinyl chloride products contain up to 50% phthalates by weight.10 The hydrophobic surfaces of plastics are liable to accumulate hydrophobic pollutants (e.g., PCBs, © XXXX American Chemical Society

PAHs, and also additives leached out of other plastics such as PBDEs) in seawater. Therefore, plastics can concentrate persistent pollutants up to 6 orders of magnitude more than in ambient seawater.11 The chemicals present in plastics can be transported from contaminated to remote uncontaminated areas along with the movement of the plastics themselves.12,13 If marine organisms ingest plastic debris, the plastics may act as a mode of exposure to contaminants for marine organisms.14 In the 1980s, Ryan et al.15 first raised the possibility that seabirds could assimilate chemicals from plastics in their stomachs. Many controlled experiments have demonstrated that hazardous chemicals can be transferred to marine organisms from microplastics,16,17 resulting in the deterioration of the organisms’ physiological functions and health.18−20 The opposite view is that the role of plastics in the bioaccumulation of hazardous chemicals is negligible and could even reduce the uptake of such chemicals.21,22 Scientists have also attempted to obtain field evidence on the effects of marine microplastic pollution. They compared the levels and profiles of chemicals in organisms (fish, whales, and birds) with the abundance and chemical profiles of microplastics in surrounding waters (or gut Received: November 7, 2015 Revised: March 3, 2016 Accepted: April 21, 2016

A

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Figure 1. Map showing mussel sampling stations according to substrate type: (a) styrofoam buoy •, (b) HDPE buoy Δ, (c) metal buoy ▲, and (d) rock ■. Sections of styrofoam buoys were also collected along with the mussel samples.

contents).23−26 However, there is still limited information available regarding the issue of whether plastic marine debris is a medium that transports hazardous compounds to marine organisms. There is thus a need for more data on this issue, including evidence from the field. Hexabromocyclododecanes (HBCDs, brominated flame retardants) have been detected in expanded polystyrene (commonly known as styrofoam) buoys and their marine debris, as well as in other polystyrene (PS) consumer products.8,27 HBCDs were also enriched in marine sediments near to oyster aquaculture farms, where a large number of styrofoam buoys were deployed. 27 HBCDs have been categorized as persistent organic pollutants (POPs) in the Stockholm Convention since 2013 due to their potential toxicity, environmental persistence, bioaccumulative tendencies, and long-range transportability.28 Styrofoam is a widely used polymer material for applications on land and at sea. Its use on land includes its role as insulation material for construction and packaging purposes, while its use at sea includes its role as a floating device in the fishery industry. Styrofoam buoys of 40− 70 L in size are commonly used as floats in the aquaculture industry for oyster, mussel, and sea squirt production (Figure S1a), while styrofoam buoys of more than 200 L in size are often used as barges for fishing and transportation decks (Figure S1b). Approximately 2 million styrofoam buoys are produced for the aquaculture industry every year in South Korea.29 However, the rate of retrieval of used buoys is around 28%.30 Jang et al.31 estimated that about 990,000 styrofoam buoys are lost or disposed of as waste every year making styrofoam the dominant type of beach marine debris, including micro-, meso-, and macrodebris, in South Korea.32,33 Most plastic marine debris is buoyant and can host rafting organisms in the ocean.34 Similarly, floating styrofoam buoys, both lost and in active use, have become a habitat for marine organisms.35 We hypothesized that if styrofoam debris acts as a source of hazardous additives in the marine environment, inhabiting organisms should be most directly influenced by

them. To verify this hypothesis, this study investigated mussels inhabiting styrofoam buoys. For comparison, mussels attached to different substrates, such as high-density polyethylene (HDPE), metal, and rock, were also studied. The body burdens and stereoisomeric profiles of HBCDs in mussels from the four substrates were compared. In addition, the presence of small styrofoam particles in mussels from styrofoam buoy was identified by analyzing their feces and soft tissues. This study provides field evidence supporting the potential of marine plastic debris as a source and carrier of hazardous substances in marine ecosystems.



MATERIALS AND METHODS

Experimental Design. Mussel Sampling According to Substrate Type. Mussels (Mytilus galloprovincialis) were collected at Geoje Island (Stns. 1−15) and the east coast (Stns. 16−22) of South Korea in September and October 2013 (Figure 1). The samples were categorized according to their substrate type as follows: styrofoam buoys (25 composites from 10 styrofoam buoys), HDPE buoys (18 composites from 5 HDPE buoys), metal buoys (7 composites from 2 metal buoys), and rocks (13 composites from 13 stations) (Table S1). The styrofoam and HDPE buoys from Stns. 1−8 were approximately 62 L (diameter 35 cm × height 45 cm) in size and were floating on the sea surface as marine debris or floating markers for fishing. The styrofoam buoys from Stns. 9−11 were more than 200 L (diameter 50 cm × height 90 cm) in size and were used as floating aids for small transport barges. At Stn. 5, mussels were collected from two metal buoys that were used as navigational aids. Mussels were also collected from 6 natural rocky stations in Geoje (Stns. 2, 3, 12−15). To identify regional differences due to the level of styrofoam pollution, mussels living on natural rock were also collected along the east coast (Stns. 16−22). The mussels collected from substrates were carefully washed with filtered tap water before the removal of soft tissue so that no styrofoam particles remained on the shell. After opening B

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Figure 2. (a) Concentration and (b) stereoisomeric ratio (α/γ) of HBCDs in styrofoam buoys and mussels from four substrates: styrofoam, HDPE, metal, and rock. a: Geoje (Stns. 2, 3, 12−15), b: East coast (Stns. 16−22).

Whatman, Maidstone, UK) with five replicates and was depurated for 2 days. After depuration, the feces (in seawater) and tissues of mussels were collected and used for particle identification. To check whether mussels reingested the egested styrofoam particles during the depuration periods in the closed depuration system, an additional depuration test was performed using a continuous flow-through chamber system, and similar results (size and number of styrofoam particles remaining in mussels after depuration) were obtained (see the Supporting Information). Sample Preparation. HBCD Analysis in Styrofoam and Mussels. The analytical protocols for HBCD analysis in styrofoam were as described previously.8,37 Briefly, styrofoam samples were dissolved with dichloromethane (DCM). The dissolved samples were solvent-exchanged with acetonitrile and used for instrumental analysis. Homogenized mussel samples were extracted using a Soxhlet apparatus with DCM and hexane (4:1, v/v). The extracts were treated with concentrated sulfuric acid to remove bulk lipids. The hexane layers were collected and solvent-exchanged with acetonitrile for instrumental analysis. A mixture of 13C-α,β,γ-HBCDs as surrogate standards was spiked into both styrofoam and mussel samples before extraction to determine the performance of the analytical procedures. To calculate the recovery of the surrogate standards, d18-γ-HBCD was spiked as an internal standard before the instrumental analysis. The details of the HBCD analysis in styrofoam and mussels are described in the Supporting Information.

each shell, no styrofoam particles were found on the surface of tissue following a visual inspection. Biometric data, including length, width, height, and wet tissue weight, were recorded (Table S2). Sections of the styrofoam buoys were also sampled using a stainless steel knife (Stns. 1−4 and 6−11) and subjected to chemical analysis. For each styrofoam buoy, quadruplicate analyses were performed. Depuration Experiment: Field Mussels from Styrofoam Buoys. To identify whether mussels from styrofoam buoys contained styrofoam particles and, if so, the extent to which the particles influenced the HBCD levels, a depuration test was performed using mussels additionally taken from styrofoam buoys in May 2014. A schematic diagram of the experiment is shown in Figure S2. There were two test batches: a chemical analysis batch and a particle identification batch. In the chemical analysis batch, mussels were taken from two buoys with different HBCD content such as 4001 and 13 μg/g (namely, high- and low-HBCD buoys, respectively). Mussels from each buoy were divided into a depuration group and a nondepuration group. For the nondepuration group, mussels (7 individuals/sample × 3 replicates) were frozen immediately after carefully washing the outer part of the shell. For the depuration group, mussels were placed in beakers containing 3 L of filtered seawater as triplicate samples for 2 days. The typical gut depuration time is known to vary by up to 15 h in mussels (Mytilus edulis).36 Tissues from the two groups were sampled from the shell for HBCD analysis. In particle identification batch, mussels were taken from the high-HBCD buoy. One individual was placed in a beaker containing 400 mL of filtered seawater (GF/F 0.75 μm; C

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l.w.).41 Mussels from HDPE buoys, metal buoys, and rocks collected from Geoje also had relatively high levels of HBCDs (median: 61.5 ng/g l.w.) in comparison to levels reported from other Asian countries (median: 3.3 ng/g l.w.),42 including Cambodia, China, Hong Kong, India, Indonesia, Malaysia, Philippines, and Vietnam. The elevated levels of HBCDs in mussels from this region could be due to the widespread use of styrofoam buoys in the aquaculture industry. Jinahe Bay, including the Geoje sector, is one of the major oysterproducing areas in South Korea, where numerous floating styrofoam buoys are used in oyster hanging-culture farms to support oyster strings (Figure S1a). Because HBCDs are additive-type flame retardants (physically combined with polymer materials), they have a marked tendency to leach out of polymer materials into the surrounding environment. Substantial leaching of HBCDs from styrofoam spherules into the seawater has been found together with their enrichment in coastal sediments near aquaculture farms using styrofoam buoys.27,43 Therefore, the abundant use of styrofoam buoys may elevate the background level of HBCDs in the water column of the bay, resulting in the elevation of HBCD concentrations in aquatic organisms inside the bay. The HBCD concentrations in mussels from rock along the east coast of Korea (Stns. 16−22) were in the range of 6.97−35.1 ng/g l.w. (18.6 ± 10.1, 15.9; Figure 2a). The overall HBCD concentration in mussels from the east coast was significantly lower than from Geoje (t-test, p < 0.001). Nationwide marine debris monitoring data (items above 25 cm in size) indicate that styrofoam debris is five times more abundant along the southeastern coast than the east coast.44 Therefore, the regional difference in HBCD levels in mussels from rock is likely to be related to their different styrofoam pollution load. However, the overall HBCD concentrations in mussels (0.52−98.3 ng/g wet weight; w.w.) in this study were lower than the environmental quality standard (EQS) for fish (167 ng/g w.w.) and fishery products (6100 ng/g w.w.) established by the European Commission to protect aquatic predators and human health.45 Isomeric signatures of contaminants can provide useful information about their origin and recent input. For example, Tanaka et al.26 presented the occurrence of higher-brominated PBDE congeners in adipose tissues of seabirds as evidence for the bioaccumulation of plastic-derived chemicals because the assimilation efficiency of high brominated congeners in biota is low due to the large molecular size, and, as a result, their biomagnification potential in a food web is low.46 We expected that the isomeric profile in styrofoam substrates would be reflected in the mussels inhabiting them, if the styrofoam substrate is the main source of HBCDs for the mussels. Commercial HBCD mixtures consist of three main stereoisomers, α-, β-, and γ-HBCDs, at rates of 10−13%, 1−12%, and 75−89%, respectively. In the environment, γ-HBCD is dominant in abiotic matrices such as air, water, and sediment, while α-HBCD is dominant in biota due to its high metabolic stability and the potential for β- and γ-HBCDs to biotransform into α-HBCD.47,48 The isomeric patterns of HBCDs in styrofoam buoys were rich in γ-HBCD, with a mean rate of 82 ± 11% of the total, which is close to that of commercial HBCDs. Compared with that of styrofoam buoys, the proportion of γ-HBCD was reduced and that of α-HBCD was increased in mussel tissues (Figure 2b). The α/γ ratios in mussels from styrofoam, HDPE, and metal buoys, and rocks were in the ranges of 0.12−3.97 (median: 0.78), 0.73−5.02 (2.21), 0.61−3.17 (1.20), and 1.72−32, (7.10), respectively. It

Identification of Styrofoam Particles in Mussels and Feces. The mussel feces in water were treated with 20 mL of hydrogen peroxide (H2O2, 34.5%) to remove organic matter and then left for 1 week at room temperature. The treated water samples were filtered over a 5-μm mesh-polycarbonate membrane filter (diameter 47 mm; SPI Supplies, West Chester, PA, USA). Filters were stored in covered glass Petri dishes to avoid atmospheric contamination and were dried at room temperature. The tissues of depurated mussels were digested using the enzymatic digestion technique proposed by Simonsen et al.38 and Catarino et al.39 Briefly, each mussel was placed in a 500 mL beaker with 10 mL of lipase (Lipex 100L; Novozymes, Bagsværd Denmark), 5 mL of protease (Savinase 16L; Novozymes), and 185 mL of distilled water. The temperature was kept at 60 ± 5 °C, and a stirring speed of 300 rotations per min was used. After digestion, the solution was subsequently filtered over a 20-μm mesh-polycarbonate membrane filter (diameter 47 mm; Sterlitech Corporation, Kent, WA, USA). Styrofoam particles on the filters were identified using a Fourier-transform infrared spectroscopy (FTIR) microscope (Thermo Nicolet 6700 and Continuum; Thermo Scientific, Waltham, MA, USA). For the styrofoam particles identified by microscopy, spectroscopic confirmation was performed using FTIR. Distinct FTIR spectra were obtained for all styrofoam particles of more than 100 μm in size. The minimum size of styrofoam particles from which distinct FTIR spectra could be obtained was 50 μm in cases where particles had an appropriate thickness; however, spectroscopic confirmation was difficult if the particles were too thin and porous. We counted the particles larger than 50 μm that had the typical morphological characteristics of styrofoam (i.e., a milky white color, and a shiny and amorphous surface) but could not be confirmed spectroscopically. Particles smaller than 50 μm were not counted. The quality control and quality assurance procedures used for HBCD analysis and particle identification are described in the Supporting Information.



RESULTS AND DISCUSSION Transfer of Additive Chemicals from the Plastic Substrate to Its Inhabitants. Accumulation Characteristics According to Substrate Type. The overall concentration of HBCDs in mussels inhabiting styrofoam buoys in Geoje (Stns. 1−4 and 6−11) was in the range of 23.1−5160 ng/g lipid weight (l.w.), with mean and median concentrations of 523 ± 1091 and 129 ng/g l.w., respectively (Figure 2a). The HBCD concentrations in mussels from other substrates in Geoje were 37.6−96.6 ng/g l.w. (mean and median: 60.0 ± 17.3, 61.3) from HDPE buoys, 34.5−90.7 ng/g l.w. (55.9 ± 21.4, 61.5) from metal buoys, and 20.1−75.4 ng/g l.w. (57.1 ± 20.9, 62.1) from rocks. The mussels inhabiting the styrofoam substrate accumulated more HBCDs than the mussels from the other substrates (Kruskal−Wallis, p < 0.005), which implies that the styrofoam substrate is a potential source of hazardous substances for attached organisms. On the other hand, there was no significant difference in HBCD levels in mussels from HDPE, metal, and rock in Geoje (one-way ANOVA, p = 0.8). The HBCD concentrations in mussels from styrofoam were among the highest measured in mussels from coastal areas worldwide. The levels were comparable to (or higher than) those reported in industrialized areas in South Korea (140−550 ng/g l.w.; Onsan and Masan bays)40 and Japan (32−5200 ng/g D

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Figure 3. A comparison of HBCD concentrations and stereoisomeric ratios (α/γ) between styrofoam buoys and the mussels that inhabit them.

is notable that the isomeric composition in mussels from styrofoam buoys was closer to that in styrofoam substrates (α/γ ratios: 0.04−0.59, median: 0.12) than in the other substrates. This implies that the chemical signature in a styrofoam substrate is reflected in its inhabitants. In summary, the concentrations as well as the isomeric profiles identified in this study support the assertion that HBCDs in mussels mainly originate from the styrofoam

substrate. This is in accordance with our previous observations in coastal sediments, where the HBCD concentration and γHBCD composition were high in the proximity of aquaculture farms that used styrofoam buoys extensively.27 These observations support the hypothesis that plastic debris is a source of hazardous substances in the marine environment. Relationship between Styrofoam Substrate and Mussels Inhabiting It. HBCDs were detected in all styrofoam samples E

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Environmental Science & Technology (10 buoys × 4 replicates) in the concentration range of 0.15 to 5220 μg/g dry weight (d.w.; median: 17.2 μg/g d.w.) (Figures 2a and 3a). As observed in our previous study,27 the HBCD levels were not consistent among styrofoam buoys. Large buoys (>200 L) tended to contain higher levels of HBCDs than smaller ones (40−60 L) (Figure 3a). The highest concentrations were detected in two large styrofoam buoys (Stns. 9 and 11), with the concentrations of 4630 ± 521 (4240−5220) μg/g d.w. and 3330 ± 865 (2090−4020) μg/g d.w., respectively, which is comparable to the levels detected in construction foam boards.8 HBCDs are added to construction materials as fire-retardants, but their use in styrofoam buoys that float on the sea surface is not essential. The wide variation in HBCD levels among buoys implies unintentional addition during the manufacturing process. Producers mainly use nonflame retardant PS beads for buoy production because flame retardant beads are more expensive. However, owing to stock issues, producers occasionally use flame retardant beads (personal communication). In addition, we found that commercial nonflame retardant PS beads also contain small amounts of HBCDs (unpublished). As a result, HBCDs can be detected in various PS consumer products including ice boxes, disposable trays, and packing materials.8 These findings imply that improper control of the production and use of plastic raw materials could lead to the dispersion of hazardous substances in the environment. Comparing each pair of buoy and mussel samples, it was found that the HBCD levels in styrofoam buoys were reflected in the mussels inhabiting them (Figure 3a). In other words, HBCD concentrations were high in mussels from styrofoam buoys with high HBCD content (Stns. 9 and 11), with the exception of those from Stn. 3. The HBCD levels in styrofoam from Stn. 3 were 100 times lower than in styrofoam from Stn. 11; however, mussels from both stations had similar concentrations of HBCDs. The unexpected enrichment of HBCDs in mussels from Stn. 3 could be related to the environmental conditions at this location. Unlike the other stations, Stn. 3 was located in a semienclosed area with limited water circulation. The buoy was collected alongside a floating pier where many styrofoam fragments and small particles had accumulated (Figure S1c). During sampling, we observed that many small styrofoam particles were dispersed on the sea surface around the buoy, and particles were also trapped between the byssal threads of mussels. For this reason, we suspected that the surrounding styrofoam debris could be an additional source of HBCDs. To check the HBCD levels in the water column at Stn. 3, surface seawater (4 L) was collected, and its dissolved phase was used for HBCD analysis. The total HBCD concentration in dissolved seawater (n = 2) was 3.6 ± 2.4 μg/L, which was higher than the HBCD levels commonly detected in seawater from Geoje (0.82 ± 0.21 μg/L, unpublished data) and even higher than those inside aquaculture farms (1.5 ± 0.91 μg/L, unpublished data). The enhanced HBCD levels in the water column might cause an increase in the HBCD uptake by mussels via the gills. Additionally, styrofoam particles surrounding the buoy could be taken up by mussels, enhancing their HBCD body residue. As previously mentioned, γ-HBCD was the predominant stereoisomer in all styrofoam buoys. However, there were slight differences in the isomeric composition between styrofoam buoys (Figure 3b), which may be related to the isomeric composition of the raw material (PS beads) and the processing temperature during manufacture. The isomeric profiles of the

mussels tended to reflect those of their styrofoam substrates. Each set of styrofoam and mussel samples showed a positive relationship in their α/γ ratios, except for samples from Stns. 7 and 8. α-HBCD was relatively abundant in the sample sets from Stns. 1 and 2, while γ-HBCD was relatively abundant in those from Stns. 3, 9, and 11. In the mussels, there was a good relationship between the HBCD concentration and the α/γ ratio (r = 0.78, p < 0.001, Figure 3c). The proportion of γHBCD was high in mussels with a high HBCD body burden (Stns. 3, 9, and 11). Even in the samples with a low concentration (red box in Figure 3c), the HBCD levels and α/γ ratio were linearly related (r = 0.61, p < 0.01). Considering the high metabolic stability of α-HBCD and potential of β- and γ-HBCDs to biotransform into α-HBCD,46 the relatively strong γ-HBCD signal in mussels could imply high-level exposure. Therefore, the isomeric profiles of HBCDs in mussels seemed to be affected by both the substrate characteristics and the contaminant load. Identification of Styrofoam Particles in Mussels Inhabiting Styrofoam. During sample treatment, we carefully washed and visually inspected the shells and tissues so as not to include any particles. If mussels contained styrofoam particles in their digestive tract and the particles were also extracted, they could affect the HBCD concentration and isomeric profile of mussel samples. Therefore, to identify whether mussels from styrofoam buoys contained styrofoam particles and, if so, the extent to which the particles influenced the HBCD levels and isomeric profiles in the mussels, a depuration experiment was performed using mussels additionally taken from styrofoam buoys. The experimental design is described in detail in the Materials and Methods and is shown in Figure S2. There were two batches: a chemical analysis batch and a particle identification batch. To check the changes in HBCD levels and profiles in mussels according to depuration, mussels were taken from two buoys having different HBCD content, namely, 4001 and 13 μg/g (high- and low-HBCD buoys, respectively). In mussels from the high-HBCD buoy, the HBCD concentration was reduced from 10.5 ± 1.7 to 7.5 ± 0.6 ng/g w.w. after depuration, constituting a decrease of 28%. In contrast, only a 3% decrease (1.69 ± 0.3 to 1.64 ± 0.2 ng/g w.w.) was measured in mussels from the low-HBCD buoy (Table 1). This implies that Table 1. Results of the Chemical Analysis Batch in Depuration Experiment: The Changes in HBCD Concentrations in Mussels from High- and Low-HBCD Buoys According to Depurationa HBCD concentration in mussel (ng/g w.w.)

high-HBCD buoy low-HBCD buoy

before depuration

after depuration

reduction rate (%)

10.5 ± 1.7 1.69 ± 0.3

7.50 ± 0.6 1.64 ± 0.2

28 3

HBCD concentrations in high- and low-HBCD buoys were 4001 ± 605 μg/g and 13 ± 22 μg/g, respectively. a

styrofoam particles were present in the digestive tract of mussels and that the ingested styrofoam particles mainly originated from their substrates. Assuming that the mussels collected from Stns. 1−11 contained a similar amount of styrofoam particles internally and the particles had mainly originated from the buoy that they inhabited, the HBCD levels F

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Figure 4. Pictures (a) of styrofoam particles from mussels and (b) their Fourier transform infrared (FT-IR) spectrum (red: styrofoam particles from mussel, blue: styrofoam buoy).

detected in mussels could be reduced in proportion to HBCD levels in their styrofoam substrates. Despite the 28% and 3% reduction in HBCD concentration for the mussels from highHBCD (Stns. 9 and 11) and low-HBCD (Stns. 1, 2, 4, 6, and 10) buoys, respectively, the overall concentration gradient among the mussels (Figure 3a) remained unchanged. Additionally, the overall isomeric profiles of HBCDs were consistent after depuration in mussels from both high- and low-HBCD buoys (27:3:70% to 34:3:63% and 74:5:21% to 70:7:23% for α:β:γ, respectively), although the proportion of γ-HBCD was slightly reduced in the former. This implies that the isomeric signatures in mussels could represent the profile formed through long-term accumulation. In the particle identification batch, several types of microplastic, mostly styrofoam and some other polymers (polypropylene, polyethylene, and epoxy resin), were detected in mussels. This paper mainly deals with styrofoam so as to discuss the transfer of additive HBCDs from styrofoam to the mussels that inhabit it. Field studies have reported the number, size, and shape of microplastics ingested by mussels; however, their polymer type has rarely been studied.49−51 Polymer-type information would also be useful for understanding their origins and related additives (e.g., HBCDs in styrofoam, phthalate in polyvinyl chloride, perfluorinated compounds in Teflon). For styrofoam particles, the polymer type was confirmed using an FTIR microscope; the spectra from mussels and styrofoam buoys are presented in Figure 4. Styrofoam particles were detected in all mussels (tissues + feces) with a mean abundance of 2.03 ± 0.98 particles/g w.w. (9.0 ± 5.1 particles per individual), at 1.66 ± 0.8 particles/g w.w. (7.4 ± 4.3 particles per individual) for feces, and 0.37 ± 0.2 particles/g w.w. (1.6 ± 0.9 particles per individual) for tissues (Table 2). Although similarly sized mussels (longest dimension: 40−50 mm) were used for this experiment, intraindividual variation was observed. Interestingly, the number and size of styrofoam particles inside mussels decreased after depuration. After depuration, the size ranges of styrofoam particles in tissues and feces were 58−83 μm (n = 8, median: 74 μm) and 52−715 μm (n = 40, median: 149 μm), respectively. This indicates that the larger styrofoam

Table 2. Results of the Particle Identification Batch in Depuration Experiment: The Abundance of Styrofoam Particles Recovered from Tissue and Feces of Mussels from High-HBCD Buoy after Depuration and the Estimated HBCD Concentrations in the Particles abundance of styrofoam particle (particle/g w.w.) estimated HBCD concentration (ng/g w.w.)a

tissue

feces

0.37 ± 0.2

1.66 ± 0.8

0.007 ± 0.005

2.306 ± 2.53

a

The HBCD concentrations in the particles from mussel tissues and feces were estimated based on the measurement data for styrofoam particles and styrofoam buoy (density of styrofoam buoy: 0.021 mg/ mm3; HBCDs in styrofoam buoy: 4,001 μg/g styrofoam, 86.52 ng/ mm3 styrofoam). The details of the calculation are given in Table S3.

particles were released from mussels to the surrounding water during depuration, while the smaller styrofoam particles were retained inside the mussels, which is in accordance with a previous study by Van Cauwenberghe et al. (tissue: 20−90 μm; feces: 15−500 μm).49 This size range of microplastic particles matches the particle size selected by bivalves during feeding (mainly 30−40 μm and up to 600 μm).52 A couple of styrofoam particles smaller than 50 μm were also observed during the microscopic analysis (but not confirmed spectroscopically), implying the potential existence of much smaller particles. The remaining particles in mussels could subsequently move up the food chain. The number of styrofoam particles found in mussels from styrofoam buoys was higher than that for microplastics in mussels from estuaries (0.13 ± 0.14 microplastic particles/g w.w.) and commercial markets (0.18 ± 0.15 microplastic particles/g w.w.) in Europe.53 We estimated the HBCD content in styrofoam particles detected from feces and tissues based on the volume of the styrofoam particles and the HBCD concentration in a styrofoam buoy (Table 2). The volume of styrofoam particles was calculated under the assumption that these particles were spherical and that the maximum lengths corresponded to their diameters. The HBCD concentration in the styrofoam buoy G

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Environmental Science & Technology was 4001 μg/g, corresponding to 86.52 ng/mm3. The details of this calculation are given in Table S3. The estimated HBCD content in styrofoam particles from the tissues of depurated mussels was 0.007 ± 0.005 ng/g w.w., which accounts for 0.09% of the HBCDs detected in mussel tissues (7.50 ± 0.6 ng/ g w.w.) after depuration in the chemical analysis batch; and the estimated HBCD content in styrofoam particles from the feces of depurated mussels was 2.306 ± 2.53 ng/g w.w., which is comparable with the HBCD decrement (2.96 ± 1.1 ng/g w.w.) in mussel tissues after depuration in the chemical analysis batch (Table 1). There are two ways in which HBCDs in styrofoam substrates could be transferred to the mussels inhabiting them: indirect uptake via the water column and direct uptake by styrofoam particle ingestion. For indirect uptake via the water column, HBCDs leached from styrofoam substrates into the water column could be rapidly adsorbed onto surrounding particulate organic matter and/or dissolved in the water, subsequently being taken up by mussels through ingestion of the particulates and across the gills, respectively. Direct intake of styrofoam particles formed by environmental weathering processes and biological activities could be an additional route, whereby HBCDs leach out of the styrofoam in the digestive tract and are subsequently absorbed by tissue. This study found an elevated concentration of hazardous additives in mussels attached to styrofoam substrates and the presence of styrofoam particles inside the mussels. The presence of styrofoam particles inside mussels implies that microplastic ingestion would be an important route of HBCD exposure for inhabiting mussels. Indirect uptake via the water column could be more substantial considering the high potential for HBCDs to be released from styrofoam into water.8,9 However, it is difficult to assert their relative contributions, because this study was not designed to examine them. The transport of styrofoam-associated contaminants to organisms could also be influenced by other factors, such as the leaching efficiency of HBCDs from styrofoam in water column and the digestive tract, their bioavailability, and the styrofoam particle concentration. Styrofoam is one of the major components of marine debris globally.54−57 After a tsunami struck Japan in 2011, tsunami debris, including styrofoam buoys and construction materials, drifted across the Pacific Ocean and washed ashore along the Alaskan and Hawaiian coasts. Owing to their buoyancy, styrofoam debris and fragments have great potential to travel long distances due to ocean currents and winds. The movement of styrofoam marine debris in the ocean not only is a problem of nuisance debris but also can result in the dispersion of hazardous substances.





particles from depurated mussels and the estimation of the HBCD concentrations in the particles (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: 82-55-639-8674. Fax: 82-55-639-8689. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS



REFERENCES

This study was supported by a research project titled “Environmental Risk Assessment of Microplastics in the Marine Environment” from the Ministry of Oceans and Fisheries, Korea.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b05485. Materials and methods including mussel sampling according to substrate type, chemicals and reagents, HBCD analysis in styrofoam and mussels; Validation of closed depuration system, quality control, and quality assurance; Photographs of aquaculture farms, barge for fishing and transportation, and sea surface of St. 3; Schematic diagrams of depuration experiment; Biometric data of mussel samples; Information on styrofoam H

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