Journal of Hazardous Materials 300 (2015) 114–120

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Diastereoisomer- and species-specific distribution of hexabromocyclododecane (HBCD) in fish and marine invertebrates Min-Hui Son, Jongchul Kim, Eun-Su Shin, Sung-hee Seo, Yoon-Seok Chang ∗ School of Environmental Science and Engineering, POSTECH, San 31, Hyojadong, Namgu, Pohang 790-784, Republic of Korea

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• This study shows the levels of HBCD •

• • •

diastereoisomers in fish and marine invertebrates. Fish and marine invertebrates with different metabolic capacities may affect the distribution patterns of HBCD diastereoisomers. ␣-HBCD was the dominant diastereoisomer in almost all species. There are no significant differences in levels of 2 HBCD between nektonic and benthic organisms. Feeding habits and feeding modes of marine organisms also have a similar effect on the diastereoisomeric fraction of ␣-HBCD.

a r t i c l e

i n f o

Article history: Received 9 April 2015 Received in revised form 4 June 2015 Accepted 9 June 2015 Available online 19 June 2015 Keywords: HBCD Marine organisms Nekton Benthos Feeding habits Feeding modes

a b s t r a c t The levels and distributional characteristics of hexabromocyclododecane (HBCD) diastereoisomers have been largely reported for various fish and select shellfish. In this study, we reclassified a number and variety of marine invertebrates, including shellfish, to further contribute to the comprehensive understanding of the effects and assessment of human exposure to HBCD. Overall, 30 marine invertebrate species (n = 188) were investigated and the following order of 2 HBCD (␣- and ␥-HBCD) was observed: fish > chordata > cephalopoda > echinodermata > bivalve > crustacea. The marine invertebrates that were  reclassified into nektonic and benthic organisms showed similar concentration of 2 HBCD. The feeding habits and modes of the marine organisms were considered to compare the degree of bioaccumulation and diastereoisomer-specific distribution of HBCD due to the effects of the environment in and around pollution sources, as well as the organisms’ metabolic capacities. To the best of our knowledge, this is the first study to examine the species-specific distribution patterns of HBCD for both fish and marine invertebrates. We expect to significantly expand the understanding of the environmental fate of HBCD for marine organisms. © 2015 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. Fax: +82 54 279 8299. E-mail address: [email protected] (Y.-S. Chang). http://dx.doi.org/10.1016/j.jhazmat.2015.06.023 0304-3894/© 2015 Elsevier B.V. All rights reserved.

Fish have widely been used as typical environmental indicators of marine pollution to show the bioaccumulation of pollutants

M.-H. Son et al. / Journal of Hazardous Materials 300 (2015) 114–120

according to trophic levels. In addition, marine invertebrates such as bivalve and crustacea have reflected sediment contamination levels [1,2]. Particularly, the distributional characteristics of organic xenobiotics in marine organisms have substantiated viscerogenic transformation and compound-specific uptake, and their enantiomers have shown enantiomer-specific accumulation in their bodies [3,4]. Furthermore, diverse biotransformation enzymes (e.g., cytochrome P450, benzo(a) pyrene-hydroxylase, flavoprotein monooxygenase, uridine diphosphate (UDP)-glucuronyl transferase, and glutathione S-transferase) are involved in the xenobiotic metabolism of fish and marine invertebrates [5] and the unique feeding modes (e.g., deposit- and filter-feeding) [6] of marine invertebrates may induce different pollutant levels and explain endemic distributional features. As discussed in previous studies, the detoxification process of halogenated organic compounds (cycloaliphatic brominated flame retardants, HBCD) in juvenile rainbow trout was demonstrated by xenobiotic-metabolizing enzymes (i.e., cytochrome P4501A (CYP1A) enzymes) [3]. The phase I cytochrome P450 enzyme activities (i.e., functionalization) induced a specific diastereoisomer-dominated profile of HBCD in biotic matrices [7]. However, few studies have examined the fate, distribution, and biotransformation of HBCD diastereoisomers in various environmental biota. Hexabromocyclododecane (HBCD) is a representative brominated flame retardant, following tetrabromobisphenol A (TBBPA) [8] and polybrominated diphenyl ethers (PBDEs). It is extensively used worldwide for expanded and extruded polystyrene (EPS/XPS) insulation applications in buildings and construction industries, despite its tendency to cause neurodevelopmental toxicity, endocrine disruption, and thyroid toxicity [9]. HBCD was recently included as a persistent organic pollutant (POP) on the Stockholm Convention POPs list, which is why studies regarding the fate, distribution, and toxicity in the environment have become important. Like many other POPs, HBCD also has many features, such as long-range transport, bioaccumulation, and persistence in environmental matrices [10]. HBCD is an additive flame retardants, and it is not covalently bonded to the materials. Thus, it is released more easily into the natural environment than other reactive organic chemicals [11]. HBCD consists mainly of ␣-, ␤-, and ␥-HBCD diastereoisomers in technical mixtures and each diastereoisomer has different water solubilities and 1octanol/water partitioning coefficients (Sw ; 48.8, 14.7, and 2.1 ␮g/L, log Kow values; 5.65, 6.05, and 6.34 for ␣-, ␤-, and ␥-HBCD, respectively). To date, three diastereoisomers of HBCD have been reported in abiotic and biotic samples, including the atmosphere, soil, foods, wild marine species, breast milk, and human serum [12–16]. However, as indicated by Marvin et al. [17], the fate and enzymatic metabolism of HBCD in many marine invertebrates with different lifestyles have not been comprehensively investigated, and it is necessary to thoroughly understand the aqueous environmental fate of HBCD. Distribution of the ␣-HBCD dominant in marine organisms contrasts with the actual composition of those isomers in technical mixtures. It is reasonable to assume that these differences result from the vulnerability to thermal stress, biological stability and persistence, and potential bioaccumulation [18,19]. These results were especially pronounced among biota, such as marine organisms, that were naturally exposed to a contaminated marine environment. Recently, Du et al. [20] has reported the bioisomerization of HBCD from ␥- to ␣-HBCD by unknown enzymes in zebrafish and proved the likelihood of an abundance of ␣-HBCD diastereoisomer in biota. As previously explained, the hydrophobicity and high proportion of ␥-HBCD in technical mixtures may substantially increase its bioaccumulation and uptake into the body. However, despite these conceivable explanations regarding how these isomers can be overwhelmingly distributed in marine biota, the levels of ␥-HBCD

115

was found to be considerably lower due to the bioisomerization and preferential biodegradation [7,21]. To overcome the flaws of previous studies (e.g., restricted range and number of species), we conducted a full investigation regarding the transport mechanism of HBCD in marine organisms and systematically approached the issue. The primary aims of this study were to (1) scrutinize the distribution characteristics and levels of HBCD diastereoisomers in 30 different species of marine invertebrates, seven marine species and one freshwater fish, (2) compare the differences of HBCD exposure in different habitats and the swimming abilities for nektonic and benthic organisms, (3) track the fate of HBCD diastereoisomers according to feeding habits and modes, and (4) alert the general population to the bioaccumulation of HBCD in the human body that results from increased fish and shellfish intake. 2. Materials and methods 2.1. Chemicals Unlabeled and 13 C12 -labeled ␣-, ␤-, and ␥-HBCD were acquired from Wellington Laboratories (Andover, MA, USA). All solvents for sample preparation and cleanup (acetone, dichloromethane, n-hexane) were HPLC grade (Burdick & Jackson, NJ, USA). Silica and alumina were obtained from Merck (Darmstadt, Germany) and Sigma–Aldrich (Milwaukee, WI, USA), respectively, and anhydrous sodium sulfate (Na2 SO4 , purity 98%) was purchased from Kando Chemical Co., Inc. 2.2. Sample preparation and cleanup Fish and marine invertebrate species were selected for sampling based on the National Health and Nutrition Examination Survey (NHANES). The sampling sites for this study are shown in Fig. S1. Eight fish species and 30 marine invertebrate species were purchased from conventional fish markets at five and six locations in 2012 and 2013, respectively. All samples were captured in their natural environment with diverse geographical origins and stored in a freezer at −20 ◦ C until extraction. Approximately, 4.5 g of the fish (marine vertebrates) and 10 g of marine invertebrate samples were weighed and transferred into precleaned thimbles. With the exception of several small-sized species, the entrails of the fish and marine invertebrate species were removed (Table S1). All samples were homogenized with anhydrous sodium sulfate and extracted with hexane/dichloromethane (1:1, 250 mL) for 20 h in a Soxhlet apparatus. The extract was concentrated to measure their lipid contents using gravimetry and were subsequently cleaned using a multilayer silica gel/alumina column that was packed with anhydrous sodium sulfate (5 g), neutral silica (4 g, 3% deactivated), acidic silica (5 g, 44% sulfuric acid), neutral silica (4 g, 3% deactivated), neutral alumina (5 g, 3% organic-free reagent water w/w), and anhydrous sodium sulfate (5 g) from top to bottom. First fraction was discarded with 80 mL of hexane, and second fraction was eluted with 150 mL of hexane/dichloromethane (1:1, v/v). 2.3. HPLC/ESI(−)MS/MS analysis Seven calibration standards (1, 5, 20, 50, 100, 200, and 500 ng/mL) were prepared in methanol, and concentration for 13 C -labeled ␣-, ␤-, and ␥-HBCD was fixed at 50, 50, and 20 ng/mL 12 (r2 > 0.999). The HBCD diastereoisomers were identified by using an Agilent 1100 series HPLC binary pump (Agilent Technologies, Palo Alto, CA, USA) and an Agilent ZORBAX Eclipse XDB-C18 (4.6 × 75 mm, 3.5 ␮m) coupled with an API 2000 triple quadrupole mass spectrometer from Applied Biosystems/MDS Sciex (Foster

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City, CA). The gradient mobile phase consisted of methanol, acetonitrile, and water at a constant flow rate of 320 ␮L/min. The initial solvent composition was 60% water/30% methanol/10% acetonitrile. The mixture was changed linearly over 4 min to reach a final composition of 50% methanol/50% acetonitrile using two solvent mixtures of 50% methanol/50% acetonitrile (A) and 75% water/25% methanol (B). In addition, the initial flow composition of 20% A/80% B was linearly increased over 4 min to reach 100% A. The final composition was held for 10 min, and a linear ramp of 4 min was used to return the mobile phase to the initial composition. An electrospray source (ESI) in negative-ion mode with multiple reaction monitoring (MRM) was used for the mass spectrometry measurements. The MS detection of native and 13 C12 -labeled HBCD isomers was based on the [M − H]− → [Br]− transition at m/z 640.6 → 79, 640.6 → 81 and m/z 652.6 → 79, 652.6 → 81, respectively. 2.4. Quality assurance/quality control (QA/QC) Procedural blank samples were analyzed to characterize the interference or contamination for every batch of the 15 samples, and the obtained results were corrected for blank values. Next, 10 ␮L of the internal standard 13 C12 -labeled ␣- and ␤-HBCD at 2.5 ng/␮L were spiked into the sample before extraction. Furthermore, 10 ␮L of the syringe standard 13 C12 -labeled ␥-HBCD was spiked at 1.0 ng/␮L to determine the recovery before the instrumental analysis. The recoveries of HBCD were 71.7, 67.7, and 69.0% for ␣-, ␤-, and ␥-HBCD, and it ranged from 47.5 to 125.0%. The limits of detection (LODs), which were defined as the minimum amount of analyte that produced a peak with a signal-to-noise ratio of 3:1, and the limits of quantification (LOQs), which were defined as the minimum amount of analyte that produced a peak with a signalto-noise ratio 10:1, were determined. LODs of ␣-, ␤-, and ␥-HBCD were 2.9, 2.2, and 1.1 pg/g-ww, respectively, and LOQs were 8.8, 6.8, and 3.5 pg/g-ww, respectively. Statistical analyses were performed using SPSS Statistics 20 and the Mann–Whitney U-test was used to determine statistical significance (p < 0.05). To have a better statistical analysis, levels of HBCD in muscle of marine organisms were considered. 3. Results and discussion 3.1. Diastereoisomeric patterns of HBCD Some of the most persistent organic pollutants (POPs) have hydrophobic properties and are usually expressed on a lipid-weight (lw) basis. However, the concentrations based on lipid weight did not demonstrate any differences in the trophic levels (TLs) and were not linearly correlated with the lipid contents (%) [22]. Therefore, the wet-weight (ww)-based concentration was applied to understand the diastereoisomeric patterns of HBCD in marine organisms. All fish and marine invertebrate species were categorized by division or class, swimming ability, feeding habits, and feeding modes. Generally, ␣- and ␥-HBCD diastereoisomers were observed in almost all species. In contrast, the ␤-HBCD diastereoisomer occurred at irregularly low levels (detailed information is shown in Table S2). In analyzing the fate and diastereoisomer-specific distribution of HBCD in aquatic organisms, only ␣- and ␥-HBCD were considered. 3.2. Levels and distribution of HBCD diastereoisomers in fish as marine vertebrates Bioisomerization from ␥- to ␣-HBCD and preferential biodegradation of specific isomer (i.e., ␥- or ␤-HBCD) has been observed in zebrafish and mice [21,23]. In addition, the total cytochrome

Marine fish

8

∑ 2HBCD (ng/g-ww)

116

Freshwater fish

6

4

2

*

0 ho anc

vy

l t t y cod saur mulle ckere alibu erring h h a y fic a m i r c g pa

fish cat



Fig. 1. 2 HBCD in marine and freshwater fish. The TL for each species was 2.51, 4.00, 3.70, 2.10, 3.09, 4.35, 3.15, and 4.50 in the order named. Asterisk shows the species containing entrails and the bars indicate the standard deviations.

P450 content (pmol mg−1 ) that was fundamentally involved in the metabolism of organic xenobiotics varied in fish and different marine invertebrate. Tomy et al. [24] explained the discrepancy in the distribution profile of HBCD diastereoisomers with TL (the TL for eight fish species was given in the caption of Fig. 1). Marine vertebrates, including all of the fish reported in this study, have been reported as higher TL organisms with greater metabolic capacities. Therefore, a higher concentration and predominance of ␣-HBCD in fish is expected than in marine invertebrates such as benthic organisms [7].  The total concentrations ( 2 HBCDs) and diastereoisomer profiles of HBCD in seven marine fish, one freshwater fish, and four marine invertebrate groups, comprising 30 species in total, were monitored to determine the species-specific characteristics. As shown in Fig. S2, the bioaccumulated  2 HBCDs (ng/g-ww) in the fish were greater than in most of the marineinvertebrate groups in the lower-TL organisms. The highest 2 HBCDs were found in the fish followed by chordata > cephalopoda > echinodermata > bivalve > crustacea.  2 HBCDs differed among the 8 fish species (Fig. 1) and more prominent fraction was observed  in fish (Fig. S3). In addition, several fish with high levels of 2 HBCD, including catfish, mackerel, and halibut may be affected by their feed (e.g., fish meat and fish oil) [25] and the pollutant levels in nearby seas [14]. The total concentrations varied between different species, but the concentrations in the marine (1.78 ng g−1 , ww) and freshwater fish (1.02 ng g−1 , ww) were equivocally dissimilar (p = 0.072). The results did not describe the positive relationship between concentration and TL. Although the lipid contents (%) in the fish varied from 0.4 to 18.6% (Table S2), whereas the lipid-weight-based concentrations (ng/g-lw) did not adequately show the species-specific distribution characteristics of the pollutants compared to the marine invertebrate species with usually low lipid contents (generally, less than 1%). Given the proportion of ␥-HBCD in technical mixtures and the stereoisomer-specific biotransformation based on the metabolic capacity of marine organisms, the dominance of ␣-HBCD should be considered among other factors such as the decomposition, rearrangement, and biodegradation of ␥-HBCD in the environment. Moreover, the increase of ␣-HBCD level has a positive relationship with the distance from urban and industrial areas, such that high levels of ␥-HBCD may indicate continuous emission of fresh HBCD from the source [12,26]. Therefore, marine and freshwater fish can be spontaneously exposed to varied levels of HBCD

M.-H. Son et al. / Journal of Hazardous Materials 300 (2015) 114–120

(A)

(B)

0.16

117

*

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∑ 2HBCD (ng/g-ww)

∑ 2HBCD (ng/g-ww)

0.8 0.12 0.10 0.08 0.06

0.6

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0.04

0.2 0.02 0.00 c ow sn

rab

e blu

cra

b

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(C)

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sea

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(D) 1.2

∑ 2HBCD (ng/g-ww)

∑ 2HBCD (ng/g-ww)

2.0

1.5

1.0

1.0 0.8 0.6 0.4

0.5 0.2 0.0

0.0 l l ter elk nch hel one ails lam lam kle lam llop se wh co en s abal r sn ite c ila c coc ite c sca mus oys e h n h p v w ri n w ma ia as st a e

us uid sq top oc tra mi

s s h uid uid pu pu fis sq sq cto cto ttle ka to cu do o e be o g bf eg we g-l lon



Fig. 2. Comparison of 2 HBCD (ng/g-ww) in marine invertebrates: (A) crustacea, (B) echinonodermata and chordata, (C) bivalve, and (D) cephalopoda. Asterisks show the marine invertebrate species containing entrails.

diastereoisomers, thereby leading to the preferential uptake of dominant isomers in the area. Assuming that the eight fish species have similar enzymatic capacities, the larger variation of 2 HBCD might be related to species-specific processes. Recently, Ichihara et al. [27] reported the distribution of HBCD in sewage treatment plants and river water from Japan. It was found that the ␥-HBCD isomer was predominant in the aquatic environment and that its exposure of ␥-HBCD to marine organisms was high regardless of the solubility of each diastereoisomer. The distribution of HBCD in fish may be attributable to the previously mentioned phenomena with the biotransformation enzyme activity and bioisomerization from ␥- to ␣-HBCD. 3.3. Levels and distribution of HBCD diastereoisomers in marine invertebrates In marine invertebrates with lower metabolic competence and biotransformation rates than fish, the uptake of each diastereoisomer may have been affected by several factors in the environmental matrix (e.g., sediments and suspended particles). The approximate fraction of ␣-HBCD would be 0.11 if we assumed approximately 10% of ␣- and 80% of ␥-HBCD in technical mixtures. Only a moderate increase was confirmed in clams as discussed by Tomy et al. [24]. The total cytochrome P450 content of crustacea was analogous to that observed in fish. Fish may have faster biotransformation rates for organic xenobiotics than mollusk. Several studies supported lower levels of HBCD in crustacea than fish [14,28]. Our results also showed the crustacea had low levels of HBCD diastereoisomers (range: 0.033–0.071 ng/g-ww) (Fig. 2A). If all marine organisms are assumed to be captured in the same areas of the sea, the dominance of ␣-HBCD may depend exclusively on the organism’s metabolic capacity and trophic level [24]. However, the archetypal distribu-

tion patterns of HBCD diastereoisomers could not be considered in our study because the marine organism samples were captured in different areas of the sea. Echinoderms and chordates with a microsomal cytochrome P450 monooxygenase system may substantially reflect the distributional property of pollutants, as distinct from that of the  habitat [29,30]. The mean 2 HBCD of diastereoisomer in echinoderms (i.e., sea cucumber, jellyfish, sea urchin, and spoon worm) was 0.20 ng/g-ww (range: 0.090–0.32 ng/g-ww) which was lower than the concentration in chordate (0.34 ng/g-ww). Jellyfish was less affected by contaminated  sediments than other echinoderms, showing similar levels of 2 HBCD (0.13 ± 0.12 ng/g-ww) those of cephalopod.  In bivalves, the 2 HBCDs values among the 12 species varied significantly (range: 0.044–1.61 ng/g-ww) (Fig. 2C), and the fraction of ␣-HBCD to ␥-HBCD diastereoisomer increased slightly based on the amount of lipids (Fig. S3, low-CYP groups). Generally, bivalves live at the bottom of the sea or rivers and consume seaweeds (with the exception of a few species) and intriguing results were observed for pen shell, whelk, abalone,  and conch, after the removal of the entrails (Fig. 2C). The 2 HBCDs were 0.12 ng/g-ww and 0.66 ng/g-ww in these bivalves without entrails and those still containing entrails, respectively, showing significant differences (p < 0.01). Additionally, the lipid content between bivalve with and without entrails varied from 1.38% to 0.30%, which suggests enhanced bioaccumulation of lipophilic compounds in reservoir-like entrails (p < 0.01). Among cephalopoda, the high concentration of HBCD diastereoisomers was observed in beka squid (0.81 ± 0.41 ng/gww), and it might be related with the entrails and lipid content (1.49%) of those species. Other cephalopod species showed low  2 HBCDs (range: 0.12–0.31 ng/g-ww) and mean lipid content

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M.-H. Son et al. / Journal of Hazardous Materials 300 (2015) 114–120 1.8

1.0

1.2 0.6 1.0 0.8 0.4 0.6 0.4

0.2

0.2

Concentration (ng/g-ww)

Concentration (ng/g-ww)

0.8

1.4

Diastereoisomeric fraction of α-HBCD

1.6

α-HBCD diastereoisomer β-HBCD diastereoisomer

0.6

0.6

0.4

0.4

0.2

0.2

0.0 0.0

0.0 Fish

Nektonic

Benthic

Fig. 3. Comparison of the mean HBCD concentrations (ng/g-ww) between nektonic and benthic organisms (p > 0.05).

(0.75%) (Fig. 2D). In contrast to bivalve and cephalopoda, echinodermata and chordata had low lipid contents. However, their  2 HBCDs and the variations of HBCD diastereoisomer fractions varied. We suggest four possible responsible factors: (1) the lipid contents in each species, (2) the aquaculture feeds used by fisheries, (3) the long-distance fishing from pollutant sources, and (4) the intimate contact with sediments. These multiple factors may be closely associated with the uptake, accumulation, and distribution of HBCD diastereoisomers in their bodies. 3.4. Distributional patterns of HBCD diastereoisomers in nektonic and benthic organisms Although the estimated bioaccumulation factors (BAFs) of the nektonic organisms were considerably higher than those of the benthic organisms, the total concentrations in the benthic organisms that were directly exposed to polluted sediments were similar to or greater than those in the nektonic organisms  [31]. Nevertheless, some benthic organisms contained low 2 HBCDs despite their prolonged exposure to contaminants in depositional habitats. Nektonic organisms are usually high TL species (i.e., fish) and have tremendously higher BAFs than benthic invertebrates. Therefore, fish were excluded from the comparison of HBCD distributional patterns between  the nektonic and benthic organisms. As shown in Fig. 3, the 2 HBCDs (ng/g-ww) of each group showed no differences between active and passive HBCD uptake (p > 0.05). The  2 HBCDs and the mean proportions of ␥-HBCD were 1.68, 0.23, and 0.16 ng/g-ww and 9.89, 52.79, and 48.29% for the fish, nektons, and benthos, respectively. The disparity of 2 HBCDs and diastereoisomeric fraction of ␣-HBCD between the nektonic and benthic marine invertebrates could have been caused by partial deposition into sediments, sorption to organic matters in the sea or river, and solubility of ␣-HBCD in the water. With regard to the fate of HBCD diastereoisomers, sorption mechanisms with dissolved and particulate organic matter must be analyzed in further studies. As stated above, this study indicated that typical benthic organisms, such as clams, cockles, oysters, and mussels, have high levels of HBCD diastereoisomers, and it is considered due to the entrails. However, nektonic organisms, such as squid, cuttlefish, octopus, and long-legged octopus, have relatively low levels of HBCD diastereoisomers. Similarly, the high ␥-HBCD levels in blue mussel (Mytilus edulis) have been explained by the adsorption of sedimentary particles of HBCD and their assimilation in their intestinal system [26]. Accordingly, the wide variations in total concentrations and fractions of HBCD diastereoisomers in bivalve

0.8

Diastereoisomeric fraction of α-HBCD

0.8

α-HBCD diastereoisomer β-HBCD diastereoisomer

0.0 CR

CD

HF

HB

Fig. 4. Comparison of the mean HBCD concentration (ng/g-ww) considering the feeding habits and feeding modes (left axis, stacked vertical bar) and diastereoisomeric fraction of ␣-HBCD diastereoisomer (right axis, colored circle) (p > 0.05 for CR vs CD and HF vs HB).

should be interpreted with various approaches. Therefore, concrete explanation for the results found for each species are still lacking. In king and tiger prawns, which are nektonic crustacea, the HBCD levels were well below the average (0.059 ng/g-ww) of the other nektonic species. Considering the low concentrations of HBCD in benthic crustacea, such as lobster, blue crab, and snow crab, the results were plausible. The mobility of the organisms, the effects of the contaminated sediments, the organisms’ proximity to pollution sources, and the organisms’ enzymatic capacity could cause the low levels of HBCD  concentration in crustacea. According to Haukås et al. [32], the HBCD in shore crabs may be related to habitat and feeding patterns because shore crabs are predators and scavengers. Overall, our results showed no conspicuous differences in terms of HBCD exposure between vagile and sedentary marine organisms (p > 0.05). These results suggest wide geographical and local contamination levels.

3.5. Diastereoisomer-specific distribution of HBCD in consideration of feeding habits and feeding modes To further identify new trends for the diastereoisomer-specific bioaccumulation and biotransformation of HBCD, we categorized the benthic organisms into two groups based on their feeding habits (herbivores, H; carnivores, C; omnivores, O) and feeding modes (filter feeder, F; deposit feeder, D; raptorial feeder, R; and browser, B), regardless of TL. This information is presented in Table S3. As shown in Fig. 4, this classification system was applied to reveal the potential bioaccumulation of HBCD. Fish were excluded from this comparative study because they are carnivores and raptorial feeder, and have a high TL (>2.5). Except for the long-legged octopus, the other cephalopoda and crustacea had identical feeding habits and modes. Under these considerations,  the variation in the HBCD levels was entirely reasonable. The 2 HBCDs and diastereoisomeric fraction of ␣-HBCD decreased as follows:  CR > CD > HF > HB and HF > CR > HB > CD, respectively. The high 2 HBCDs for carnivorous raptorial feeders (CR) confirmed the effects of feeding habits and feeding modes relative to herbivorous browser (HB) feeders (0.63 ng/g-ww and 0.14 ng/g-ww, respectively, for CR and HB), but the fraction of ␣-HBCD was similar. The increased concentrations in these organisms (CR) were probably related to the biomagnification of HBCD and the fraction of ␣-HBCD may be related to the HBCD patterns in the aquatic enzymatic capacities and distributed  environment. HF had a lower 2 HBCDs than CR and CD, but the diastereoisomeric fraction of ␣-HBCD were high (0.58 and 0.43

M.-H. Son et al. / Journal of Hazardous Materials 300 (2015) 114–120

High CYP450 groups

(A)

1.0 Fish Crustacea

Diastereoisomeric fraction of γ-HBCD

Diastereoisomeric fraction of γ-HBCD

Low CYP450 groups

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1.0

119

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0.6

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Diastereoisomeric fraction of α-HBCD

Diastereoisomeric fraction of α-HBCD

Fig. 5. Diastereoisomeric fraction of ␣- and ␥-HBCD diastereoisomers in the (A) high CYP450 groups and (B) low CYP450 groups.

for CR and CD, respectively). The fraction was calculated by the following equation: Diastereoisomeric fraction of ˛-HBCD =

C␣ C␣ + C␥

(1)

where C␣ is the concentration of ␣-HBCD (ng/g-ww) and C␥ is the concentration of ␥-HBCD (ng/g-ww). Abalone and conch were labeled as HB with low concentrations and fraction of ␣-HBCD, suggesting low metabolic capacities only carnivorous deposit and rates. The sea cucumber was the  feeder (CD) among the sample, and its 2 HBCDs was 0.39 ng/gww. Compared with other echinodermata, it was hypothesized that the feeding habits and feeding modes of the sea cucumber did not result in the removal of superfluous particles or pollutants. This theory is based on the assumption that the small samples (n = 2) did not lead to biased  results. The chordata sea squirt (HF) showed similar levels of 2 HBCDs with spoon worm (0.33 ng/g-ww and 0.32 ng/g-ww), but the fraction of ␣-HBCD was a little lower than that (0.70 and 0.80). Considering that the feeding habits of marine organisms affect the bioaccumulation of HBCD, the remarkable difference was found between carnivores and herbivores (p < 0.05, Mann–Whitey U-test), which indicated that  food for marine organisms had a positive relationship with 2 HBCD (0.62 ng/g-ww and 0.25 ng/gww, respectively). The exposure of HBCD between carnivores and herbivores showed the difference based on the feeding modes (0.63 ng/g-ww and 0.36 ng/g-ww for CR and CD, respectively, and 0.25 ng/g-ww and 0.14 ng/g-ww for HF and HB, respectively). 3.6. HBCD metabolites in fish and marine invertebrates Enzymatic metabolites of HBCD have been concretely identified using in vitro rat liver microsomes. However, only a few studies have shown the monohydroxy-HBCD and dihydroxy-HBCD in wildlife [7,19,33]. Because of the absence of reference standards for HBCD metabolites, these compounds should be determined by matching their molecular weights using a mass spectrometer. Coincidentally, the molecular weights of 13 C-labeled HBCDs and hydroxylated metabolites with six bromines partially overlap each other due to their multiple isotopes of bromine. Chromatographic separation of hydroxylated metabolites could be performed when determining their retention time. However, the coelution of metabolites should also be excluded, and the initial concentrations of each diastereoisomer were important for observing the enzymatic metabolites in the biota [35]. For the marine organisms with

low HBCD concentrations, the enzymatic metabolites of HBCD were not observed. Fig. 5 depicts the differences of diastereoisomeric fraction of ␣- and ␥-HBCD between the high- and the low cytochrome-P450 groups. Given the distributional patterns of HBCD apart from the environmental conditions, the increased values of ␣-HBCD in fish (Fig. 5A) might represent the enzymatic metabolism of high-TL organisms. Meanwhile, the low-TL organisms showed relatively high proportions of ␥-HBCD because of their less effective biotransformation to eliminate those diastereoisomer (Fig. 5B).

4. Conclusions We investigated the levels and distributional characteristics of HBCD diastereoisomers in  fish and marine invertebrates. All the fish species showed high 2 HBCDs and had dominant fraction of ␣-HBCD, compared to the other  nektonic and benthic organisms. In addition, high levels of 2 HBCDs were observed in bivalve containing entrails. In other words, further investigation on distribution of HBCD between muscle and organs in bivalve is necessary to understand and elucidate the fate of those pollutants in marine environment. Feeding habits and modes of marine invertebrates also showed the effects to total concentrations of HBCD and diastereoisomeric fraction of ␣-HBCD. Therefore, our results demonstrated that the fate and distributional patterns of HBCD diastereoisomers in marine organisms was quite different based on species, metabolic capacity, and habitat. Further study on diastereoisomer-specific and species-specific distribution of HBCD diastereoisomers in aquatic environment is needed to clarify the fate and bioavailability of HBCD.

Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0028723) and the Korea Ministry of Environment (MOE) as “the Environmental Health Action Program.”

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2015.06. 023

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