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Published in final edited form as:
Chemosphere. 2009 June ; 76(2): 239–249. doi:10.1016/j.chemosphere.2009.03.018.

PCBs contamination in seafood species at the Eastern Coast of
Thailand
Chate Jaikanlayaa,b, Daam Settachana,c, Michael S. Denisond, Mathuros Ruchirawata,c,e,and Martin van den Bergb,*
aInter-University Program on Environmental Toxicology, Technology and Management of

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Chulabhorn Research Institute, Asian Institute of Technology and Mahidol University, Center for
Environmental Health, Toxicology and Management of Chemicals, 272 Rama 6 Road, Rajathevee,
Bangkok 10400, Thailand bInstitute for Risk Assessment Sciences, Utrecht University, Yalelaan 2,
P.O. Box 80176, 3508 TD Utrecht, The Netherlands cLaboratory of Environmental Toxicology,
Chulabhorn Research Institute, 54 Moo 4 Vibhavadee-Rangsit Highway, Laksi, Bangkok 10210,
Thailand dDepartment of Environmental Toxicology, Meyer Hall, University of California Davis, One
Shields Avenue, Davis, California 95616, United States eDepartment of Pharmacology, Faculty of
Science, Mahidol University, 272 Rama 6 Road, Rajathevee, Bangkok 10400, Thailand

Abstract

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Polychlorinated biphenyls (PCBs) are a large group of persistent organic substances spreadthroughout the world. The most toxic PCBs are those that are dioxin-like (dl-PCBs). Environmental
studies on PCBs in Thailand are limited, especially with regards to dl-PCBs. This study is one of thefirst in this country that demonstrates contamination of seafood with PCBs and determines the levelsof PCBs and total dioxin like activity in mussels, oysters and shrimp, from the Eastern Coast of
Thailand. Sixty pooled samples of mussels and twenty-seven pooled samples of oysters werecollected from cultivation farms and twenty-one pooled samples of shrimp were collected fromfisherman piers. Qualitative and quantitative measurements of 49 PCB congeners was obtained by
HRGC-ECD analysis and total dioxin-like activity using the CAFLUX bioassay. Total PCB
concentrations varied between three species, ranging between 19 and 1100 ng g−1 lipid adjusted
weight, and the levels of PCBs in shrimp was three time higher than that in mussels and oysters. With
respected to the pattern of PCB congeners, it implied that the source of PCBs exposure in this area
could be from the regional contamination. The calculated CAFLUX bioanalytical equivalents(BEQs) values ranged between 0.8 and 18 pg BEQ g−1 lipid adjusted weight, and showed a good
relationship with the chemical-derived TEQs. Therefore, the CAFLUX bioassay can be used for
effective screening of dioxin-like activity in marine species effectively.

Keywords
Dioxin; CAFLUX; BEQ; Mussels; Oysters; Shrimp

1. Introduction
Polychlorinated biphenyls (PCBs) are one of the largest groups of persistent organic pollutants(POPs). They comprise a total of 209 possible chlorine-substituted biphenyl congeners, but© 2009 Elsevier Ltd. All rights reserved.
*Corresponding author. Tel.: +31 30 2535265; fax: +31 30 2535077. M.vandenBerg@uu.nl, martin@martin-resi.eu (M. van den Berg).

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PCBs in samples analyzed for environmental and regulatory purposes usually consist ofcomplex mixtures of about 130 congeners originating from technical mixtures (e.g. Aroclors,
Clophens, Kanechlors etc.) (Safe, 1990). Many PCBs are considered to be resistant tobiological or chemical degradation and are persistent in the environment (IPCS, 1993). PCBs
were produced and used as lubricants in transformers and capacitors, stabilizers in paints,
polymers and as adhesives (Kimbrough and Jensen, 1989). At present, PCBs are still being
released into the environment from hazardous waste sites, illegal or improper disposal ofindustrial wastes, old consumer products, leaking electrical transformers and incineration ofsome chemical waste. In addition, other sources of PCBs exposure appear to result fromredistribution of PCBs previously introduced into the environment (IPCS, 1993). As a result,these compounds are still environmentally ubiquitous and bioaccumulate in the lipids of
exposed organisms. Furthermore, biomagnification of PCBs through the (human) food chain
has also been well documented in literature (Voogt et al., 1990; IPCS, 1993; de Boer et al.,
2001; Borga et al., 2001).

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Exposure of PCBs is still of concern due to their potential adverse health effects to humans
and the environment at relatively low dose levels. These compounds have been linked toreproductive effects, immunotoxicity and carcinogenicity (Fernandez et al., 2004). In spite of
the fact that levels have decreased over the last several decades, there are still concerns that
PCBs continue to have an impact on normal endocrine function and reproduction in fish andwildlife (Okumura et al., 2004; Sapozhnikova et al., 2004). Additionally, transfer of PCBs toinfants, either in utero or postnatally via human milk still occurs and may result in growth
delay, developmental defects and neurocognitive deficits (Wang et al., 2004).

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From a toxicological point of view, most attention is being paid to those PCB congeners that
produce a similar toxicity to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and they arecommonly known as dioxin-like PCBs (dl-PCBs). Given that these specific congeners lack
multiple ortho-chlorines, but contain adjacent meta- and para-substituted chlorine atoms, they
have also been called “coplanar” PCBs. Some of these PCBs show particularly high “dioxinlike” toxicity, especially the PCBs with IUPAC Nos. 77, 126, and 169 (Safe, 1990; van den
Berg et al., 1998). In addition, considerable dioxin-like toxicity is also attributed to somemono–ortho-chlorinated PCBs (MO-PCBs) (e.g. PCBs 105, 114, 118, 156 and 189). Based on
similarities of their toxic and biochemical potencies to that of TCDD, the relative toxicity of
PCB congeners is directly compared with TCDD and expressed as toxic equivalency factors(TEFs) (van den Berg et al., 2006). Given the relatively high concentrations of MO-PCBs invarious food and wild-life samples, their contribution to the total dioxin-like activity is likely
significant compared to other dioxin-like compounds e.g. PCB Nos. 77, 126, and 169, thechlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) (Jiang et al., 2007;
Durand et al., 2008; Zhang et al., 2008).
To measure the concentration of dioxin-like compounds, high resolution gas chromatography
combined with high resolution mass spectrometry (HRGC/HRMS) is the preferred technique.
However, this analytical method needs highly skilled operators, expensive instrumentation, istime consuming, very costly and is not suitable for high throughput screening. Therefore,
alternative bioanalytical screening methods have been developed (Denison et al., 1993; Nagyet al., 2002; Denison et al., 2004). The chemically activated luciferase expression (CALUX)
assay and chemically activated fluorescence expression (CAFLUX) assay are recently
developed highly sensitive bioanalytical techniques to detect and measure dioxin-likecompounds. These techniques take advantage of the specific mechanism of action of dioxinlike compounds, namely binding to the Ah-receptor, nuclear translocation of the ligand-bound
AhR and its dimerization with the Ah-receptor nuclear translator (ARNT) protein, followedby binding of the ligand:Ah-receptor:ARNT complex its specific DNA recognition site (dioxin
response elements (DREs) resulting in stimulation of transcription of adjacent genes, including

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luciferase or green fluorescent protein genes in the CALUX or CAFLUX recombinant celllines (Nagy et al., 2002; Denison et al. 2004)). Results are expressed as dioxin bioanalyticalequivalents (BEQs), i.e. the ability of the mixture of dioxin-like compounds to induce luciferase
or fluorescent protein expression, relative to TCDD (Eljarrat et al., 2002; Gizzi et al., 2005;
Jeong et al., 2005). BEQs are directly related to the overall toxic equivalents (TEQs) determined
in in vivo toxicity studies (van den Berg et al., 2006). Use of these bioassays allows the detection
and relative quantification of dioxin like compounds in various extracts of environmental or
food matrices at a sensitivity that approaches that of instrumental analysis methods.

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The concentrations of PCBs and dioxin-like compounds are usually higher in food samples
with higher lipid content and generally associated with food of animal origin. Thus, lipid richfood consumption is an important pathway for human exposure to PCBs and it has been
estimated that the major route of exposure to these compounds is still from ingestion ofcontaminated food (Safe, 1990; Schecter et al., 1997; Bocio and Domingo, 2005; Charnley andDoull, 2005; Huwe and Larsen, 2005; Llobet et al., 2008; Zhang et al., 2008). Several studies
of persistent organochlorine compounds, including PCBs, have by been reported from South
East Asia (Monirith et al., 2003). However, specific studies with PCBs from the Gulf ofThailand are much more limited (Monirith et al., 2003) and only two studies have been
conducted to date that to assess the distribution of PCBs in this region (Thao et al., 1993; Kanatireklap et al., 1997). Moreover, information about dioxin-like compounds from this area iseven more limited. This is surprising given that the Eastern area of Thailand is one of the mainindustrialized areas along the Gulf of Thailand. In addition, the coast of this region has also avery rich marine and estuarine ecosystem, with seafood being the main source of food for many
people. Thus, given the high amount of seafood (e.g. shellfish and shrimp) consumption, it isessential to have adequate knowledge about the levels of PCBs and other dioxin likecompounds in these materials. With-out this information the possible risks for humans of PCBs
and related compounds via consumption of local seafood can not be determined.
The present study was focused on assessing contamination levels of PCBs and total dioxin likeactivity in mussels, oysters and shrimp from the Eastern Coast of Thailand. Total dioxin-like
activity was determined by using an AhR-CAFLUX bioassay (Nagy et al., 2002) and HRGCECD was used to obtain qualitative and quantitative PCB congener specific information inthese three types of human food sources. The TEQ results of the chemical analyses of werecompared with the BEQ values obtained with the AhR-CAFLUX assay.

2. Materials and methods
2.1. Chemicals and materials

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The analytical grade solvents n-pentane, dichloromethane, n-hexane and isooctane werepurchased from Merck (Darmstadt, Germany). Acetone and toluene used for rinsing glassware
were also purchased from Merck, Germany. Aluminium oxide 60 active basic (Activity I) andanhydrous sodium sulfate (Merck, Darmstadt, Germany) were activated at 550 °C for 5 h prior
to use. Silica gel 60 (0.063–0.200 nm) for column chromatography (70–230 mesh ASTM)(Merck, Darmstadt, Germany) was activated at 220 °C8h prior to use. All chemical standards
were purchased from Accu-Standard Inc. (New Haven, CT USA) with following purity; 99.1%
for TCDD, 100% for PCB 29, 99.8% for PCB 112. PCBs congeners used for identification by
HRGC-ECD and their purity are presented in Table 1.
2.2. Study area and sample collection
The provinces Chonburi and Rayong that cover the Eastern Coast of Thailand were selected
for this study. The selection of green mussels (Perna viridis Linneaus), oysters (Saccostrea
commercialis) and shrimp (Penaeus merguiensis) was based upon their availability at the

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sampling locations, their common use for human consumption, their high economic value andimportance in the local ecosystem. These marine benthic organisms are very suitable as sentinel
species for local contamination, since they tend not to migrate. All samples were collectedduring January to February of 2006 and 2007. The locations of mussel sampling (M1, M2, M3
and M4) are shown in Fig. 1 and are still existing mussel farms. Oysters were collected atcommercial farms as well and these farms (O1 and O2) are connected to the open sea andtherefore are representative for ecosystem exposure. Since there were no commercial shrimp
farms in the mussel and oyster collection areas, shrimp samples were collected at the fisherman
piers along the coast of these 2 provinces at sites located close to mussels and oysters farms.
The number of individual animals in each pooled sample varied from 20 to 30 for mussels, 30–
40 for oysters and 5–10 for shrimp. After collection, all biotic samples were transported in ice
to the laboratory and stored at −80 °C until use. All samples were freeze dried before analysis.
2.3. Sample preparation

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Prior to analysis, the samples were thawed and pooled samples of each species were composed.
The soft edible part was homogenized with a vertical blender. Sample extraction was performedby using a Milestone (ETHOS SEL) Microwave Solvent Extraction Lab-station according to
the following procedure. Five gram sub-samples were accurately weighed into microwave
extraction vessels. One extraction set included 5 samples and 1 blank. Approximately 2 g ofanhydrous sodium sulfate was mixed with the samples and the surrogate standard (PCB 29;
0.08 μg g−1) was added into all vessels. This was followed by the addition of a 30 mL of npentane and dichloromethane (1:1, v/v) solvent mixture. The extraction vessels were assembled
according to the manufacturer’s instructions and the extraction was conducted at 120 °C for
15 min at 70% power. The clean-up procedure was adapted from the method described earlier(de Boer, 1988). After the extraction, vessels were cooled to room temperature and thesupernatant from the extraction vessels was carefully transferred to the top of a 15 g Al2O3 ·8%H2O column (basic Al2O3 activity I, i.d. 2 cm). Elution took place with 120 mL of hexane(the extraction vessels rinsed twice with 20 mL and final elution with 80 mL hexane). Thiselute was concentrated to 2 mL by rotary evaporation after adding 2 mL isooctane as a keeper.
The extract was then transferred to the top of a 2.2 g of SiO2 · 2%H2O column (silica gel 60,
0.063–0.200 mm, i.d. 0.8 cm) and eluted with 10 mL of isooctane and the fraction containing
PCBs was collected and concentrated to final solution at 1mL by N2 gas. This final solution
was divided into 2 parts; 0.5 mL of the internal standard (PCB 112; 0.04 μg g−1) was added to
the first part for HRGC-ECD analyses, while the second part (0.5 mL) was stored at −20 °C
prior to CAFLUX analysis. All samples were analyzed in duplicate. It should be noted that thesurrogate standard (PCB 29) does not contribute to the BEQ or TEQ concentrations for PCBs.
However, after this clean-up procedure each sample could still have included PCDDs and
PCDFs or other halogenated compounds, which were not separated by this clean-up procedure.

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2.4. Chemical analysis
The PCB congeners presented in Table 1 were targeted for qualitative and quantitative HRGCECD analysis. The quantification of these 49 PCB congeners was performed by GC-μECD(Hewlett-Packard 6890). Two capillary columns, Factorfour VF5 ms and VF1701 ms, 50 m
length × 0.15 mm i.d. × 0.25 lm film thickness (Varians, Palo Alto, CA USA), were used toenhance identification and confirmation of PCB congeners in the GC chromatograms. Two
microlitre of final elute sample was injected in splitless mode. Injector and detector
temperatures were set at 270 °C and 300 °C, respectively. The temperature program for bothcolumns was similar (initially set at 90 °C for 3 min, raised by 20 °C/min to 215 °C and heldfor 30 min, then raised again by 5 °C/min to 270 °C, and held for 23 min. Helium was used as
carrier gas with velocity at 4.6 mL/min. Nitrogen was used as make up gas for the detector
with a velocity at 60 mL/min in the constant flow mode.

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All extracts were analyzed in batches that also included blank samples and identification of
the congener specific standard calibration curves. Multi-level calibration curves in the linear
response interval of the detector were created for the quantification of 49 PCB congeners anda good correlation (r2 > 0.99) was achieved. The identification of 49 congeners was based on
their retention times relative to the internal standard (PCB 112) used for quantification on GCμECD. The limit of detection (LOD) for these PCBs was approximately 4 pg g−1. Results arereported as ‘not detected’ (ND) when the peak area of a specific congener was lesser than thepeak area of the LOD. TEQ concentrations of the mono–ortho PCBs 105, 114, 118, 156 and
189 were calculated using the WHO 2005 TEF values (van den Berg et al., 2006).
2.5. CAFLUX bioassay

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The AhR-CAFLUX cells were cultured in phosphate-buffered saline medium with l-glutamineand supplemented by 10% fetal calf serum and 1% of penicillin/streptomycin (45.5/45.5 unitsmL−1) at 37 °C and in a 5% CO2 atmosphere saturated with water. After culturing, cells were
harvested by trypsinization and 50 μL cell suspension was removed for cell counting. The othercells were seeded in 96-wells plates with 200 μL of cell suspension at a density of 1 × 104 cells/
well and placed in the incubator for 24 h. Prior to dosing on the plate, the second half of sample
final solutions (0.5 mL) were transferred in 4 μL of DMSO using a centrifuge under vacuum,and 400 μL of medium was added to each extract in DMSO. After incubation, the medium wasremoved and the cells were seeded with 200 μL medium containing standard TCDD, DMSO,
or a sample extract dissolved in DMSO. A standard solution series for TCDD was added toeach plate at concentrations of 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.0025, 0.001, and 0.0005 nM.
The limit of detection of the CAFLUX bioassay was about 1 pM. On each plate, nine DMSO
solutions of TCDD, DMSO alone, and sample extract were analyzed in 2 wells (2 μL/well).
The mean of these results was used for calculation of TCDD bioanalytical equivalents. DMSO
only was added to the outer wells of the plate. After 48 h of incubation, the medium was replaced
by PBS and EGFP fluorescence measured in a Microplate Reader (Spectra MAX Genuini XS)
as previously described (Nagy et al., 2002). All tissue culture media were purchased from
Gibco/Innovation (Grand Island, NY, USA).
2.6. MTT measurement
The MTT assay was performed on the wells with sample extracts and the three highest
concentrations of TCDD to determine possible cytotoxicity. After removal of the medium, thecells were incubated for 30 min with 200 μL serum-free medium containing 1 mg MTT
mL−1. Medium was then removed and replaced by 200 μL iso-propanol per well, followed by
a 15 min incubation and measurement of absorbance at 595 nm in a spectrophotometer(SPECTRAmaxPLUS384).

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2.7. Lipid analysis
The lipid content of the samples was determined by the method developed by Bligh and Dyer
(Honeycutt et al., 1995; Smedes and Askland, 1999). Briefly, one gram of freeze dried sample
was mixed for 30 s with 20 mL methanol, 10 mL chloroform, and 80 μL deionized water.
Another 10 mL chloroform and 10 mL deionized water were added and the solution mixed.
The mixture was centrifuged for 10 min at 1000 rpm, followed by the removal of the upper
methanol layer. A 10 mL aliquot of the lower chloroform layer was collected, weighted, andevaporated, after which the weight of the remaining fat residue was determined.
2.8. Statistical analysis
The quantification of unknown samples was calculated from using the fitted concentration–
response curve obtained using a sigmoidal dose–response nonlinear regression curve fit(GraphPad Prism 3.0, GraphPad Software Inc., San Diego, CA). Chemo-analytical results were

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processed using Principle Components Analysis (PCA). Spearman’s Rank correlation
coefficient was using for chemo-analytical and bio-analytical dioxin equivalents (BEQ) values.
All statistical calculations were processed by SPSS version 12.0 for Windows, SPSS Inc.
(Chicago, IL USA).

3. Results
3.1. General sample characteristics
General sample information (size, weight and lipid content) is presented in Table 2. The sizeof mussels from location M2 and M4 was larger than that from location M1 and M3 and this
could be due to differences in age of the mussels. The mussels from M2 and M4 were about 1
year old, while those from M1 and M3 were about 6–8 months old. Lipid content of the shrimp,
mussels and oysters varied between species (see Table 2).
3.2. Total PCB concentrations

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The average total concentrations of 49 PCBs (ΣPCB) in mussels, oysters and shrimp samples
are presented in Table 2 and were expressed either on a dry weight, wet weight or lipid adjusted
weight. Total PCB concentrations varied between species and ranged between 19 and 1100 ng
g−1 lipid adjusted weight (lw). Among the three seafood species studied, the highest
concentrations of ΣPCB were observed in shrimp with average concentrations of 775 ± 230
ng g−1 lw. For mussels and oysters, the highest concentrations were found at the 3rd location(M3) and the 2nd location (O2) being respectively 454 ± 125 and 304 ± 65 ng g−1 lw. In Fig.
2 the quantitative differences between mussels, oysters and shrimp are expressed either on a
dry or wet weight basis or lipid adjusted weight. From these results it can be concluded thatbased on dry or wet weight there is a clear overlap in PCB concentrations for the mussels andoysters. However, when adjusted for lipid, PCB concentrations in the shrimp wereapproximately three times higher than those in the mussels and oysters.
3.3. Congener specific profiles

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Most PCB congeners studied could be detected in the three seafood species. However, somecongeners present in the reference mixture could not be detected in any species, these include
PCBs 194, 195, 205, 206, 208 and 209. In addition, no attempt was made to identify andquantify the potent dioxin like PCBs 77, 126 and 169, as concentrations are generally much
lower than the more common non dioxin-like congeners like PCBs 28, 52, 101 and 138. For
reliable identification and quantification of PCBs 77, 126 and 169 in environmental samples
HRGC/HRMS is needed for analysis. Differences in pattern distribution of the PCB congeners
that were studied in mussels, oysters and shrimp are visualized in Fig. 3. A relatively large
variability in individual concentrations can be observed for all congeners. The major difference
between the three species is the fact that the shrimp was found to contain a much higher
proportion of lower chlorinated PCBs than oysters and mussels. In addition, it was found thatneither of the three species were retaining octa- and nona-chlorinated PCBs at concentrations
higher than the established limit of detection (4 pg g−1). In mussels and oysters penta- andhexachlorinated biphenyls represented the major portion of ΣPCB, with PCB 153 being thequantitatively the most significant congener. In contrast, the tri- and tetrachlorinated congeners
were the most abundant PCBs in shrimp with PCB 28 being the most dominant congener froma quantitative point of view.
In Fig. 3(D), the isomer distributions of PCBs relative to ΣPCB in the oysters, mussels andshrimps are shown. Furthermore, it can be observed that the distributions of isomer groups
from tri- to octa-chlorobiphenyls are relatively similar for oysters and mussels. The highcontribution of Cl3 and Cl4 PCBs, almost 60% of the total, in shrimp is apparent and clearly adownward trend in PCB retention can be observed with increasing number of chlorine atoms.
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Page 73.4. Principal component analysis

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Principal component analysis (PCA) has been used extensively for explaining the polluted
situation of PCBs in the environmental metrics (Howel, 2007). In this study the total varianceof the fist two components were used to explain the original variables. PCA was applied to thehomologous congener profiles of all samples. The first two components of mussels, oysters,
and shrimp accounted for 52.29%, 55.83% and 64.63% of total variation, respectively.
Therefore, the majority of variables were attributable to the first two components. The scoreplot of principle component PC1 and PC2 for three species (Fig. 4) had shown that the highest
values of the 1st principal component (PC1) in mussels sample (39.42% of variation) were
PCB nos. 138, 179 and 180, the highest values of PC1 in oysters sample (32.48% of variation)
were PCB Nos. 101, 149 and 158, and the highest values of PC1in shrimp (50.96% of variation)
were PCB nos. 8, 31, 66 and 60. Therefore, the PCA results demonstrated the similar dominantcongeners as observed with the congener specific profiles in Fig. 3.
3.5. Bioassay derived dioxin bioanalytical equivalencies (BEQs)

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The AhR-CAFLUX cell bioassay was used to screen for the presence of dioxin-like chemicals
in mussels, oysters and shrimp and BEQs determined. BEQ values are directly related to
instrumental TEQs as they are based on the same Ah-receptor mechanism of action. These
results are shown in Table 2. As with the chemical analysis, the results in BEQs have been
expressed either on dry or wet weight and lipid adjusted. The highest concentrations in BEQs
(11.5 ± 4.1 pg BEQ g−1 lw) were detected in mussels from the 3rd location (M3), followed bythose from the 2nd location of mussels (M2) (8.0 ± 2.7 pg BEQ g−1 lw). The lowest BEQ
concentrations were measured in oysters (5.8 ± 4.3 pg BEQ g−1 lw).
3.6. Relationship between CAFLUX-derived BEQ and chemically-derived TEQ values for MOPCBs
The average AhR CAFLUX-derived BEQ values of mussels, oysters and shrimp were 7.8 ±
3.8, 6.4 ± 3.7 and 7.6 ± 1.9 pg BEQ g−1 lw, respectively. The trend observed between the three
species was found to be similar to the average ΣPCB levels determined by chemical analysis.
Therefore, the results of CAFLUX bioassay were directly compared to TEQ levels of MOPCBs as determined by chemical analysis. These chemically-derived values were calculated
by using only the MO-PCBs detected in this study (PCB 105, 114, 118, 156 and 189) multipliedby their most recent TEF value which was 0.00003 for all of congeners (van den Berg et al.,2006)(Table 2). The chemically-derived TEQ values of mussels, oysters and shrimp were 0.9± 0.9, 0.5 ± 0.3, and 1.5 ± 0.7 pg TEQ g−1 lw. These comparisons reveal that all AhR CAFLUX
BEQ values were consistently higher than the chemically-derived TEQ values with the same
sample.

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However, a highly significant linear relationship between both BEQ and chemically-derived
TEQ values was obtained (Fig. 5) for mussels (R2 = 0.72, p < 0.0001), oysters (R2 = 0.71, p <0.0001), and shrimp (R2 = 0.70, p < 0.0001). A relationship between CAFLUX-derived BEQs
and total PCBs was also found in mussels, oysters and shrimp with R2 = 0.6 (p < 0.001), R2 =
0.4 (p < 0.001) and R2 = 0.4 (p < 0.001), respectively. Based on the relationships presented inFig. 5, it can be seen that the AhR CAFLUX had a good predictive value. With respect to thedifferences observed between both methods it should be kept in mind that the chemicallydetermined TEQs in this study were only based on MO-PCBs and did not include otherpersistent and more potent dioxin like compounds (PCDDs, PCDFs and PCBs), which could
not be analyzed by HRGC-ECD. Including these additional chemicals would result in asignificantly higher potency value for the chemically determined TEQs.

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4. Discussion and conclusions
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In this study, edible mussels, oysters and shrimps from the east coast of Thailand have been
analyzed for PCBs. The concentrations of total PCBs in these seafood products from this area
were rather similar with those observed from other Asiatic areas (e.g. China, Hong Kong, Japan,
Malaysia etc (Tsutsumi et al., 2001; Monirith et al., 2003; Jiang et al. 2007)). Expressed on
wet or dry weight, the variation of total PCBs within each seafood species was rather low. For
mussels and oysters this can be explained by the fact that both species have similar feeding
behavior. In addition, the mussels and oysters used in this study were from the cultivation farms
and equally exposed to seawater, in which PCBs and other organochlorine compounds have abetter mobility compared to soil or sediment. Based on wet or dry weight shrimps contained
significantly less PCBs than mussels and oysters. However, when expressed on a lipid weightbasis, shrimp contain significantly higher levels of PCBs than mussels. The much lower lipid
content in shrimp might contribute to this observation. Our results are in agreement with studies
from Western Europe (Roose et al., 1998; Voorspoels et al., 2004). Many countries have useddifferent approaches to determine the limits of exposure of non dioxin-like PCBs, i.e. Germany
is using congeners specific limits for PCB Nos. 28, 52, 101, 138, 153 and 180 in food of animal
origin at 8 ng g−1 wet weight or 600 ng g−1 fat, Belgium is using the sum of seven PCBs
congeners; PCB Nos. 28, 52, 101, 118, 138, 153 and 180 in bovine, pork, poultry, animal fats
and oils, eggs and derived products, if >2% fat, at 200 ng g−1 fat, or the Netherlands are alsousing the seven PCBs for foods of animal origin at 20 ng g−1 wet weight or 2000 ng g−1 fat(European Commission, 2000). In our study, the sum of these indicators PCB were 101 ± 61,
142 ± 54, and 446 ± 148 ng g−1 fat and 1.0 ± 0.6, 2.3 ± 0.9, and 1.1 ± 0.4 ng g−1 wet weight,
in mussels, oysters and shrimp, respectively. It should be noticed that these seven PCBs
together represented the major proportion, approximately 50%, of total PCB concentrations inthis study. If we use these seven indicator PCBs as limit values for non dioxin-like activity our
results indicated levels in all three food species below those limit values.
The congener patterns in our study show that the penta- and hexachlorinated isomers comprised
the major fraction of the ΣPCB in mussels and oysters, more than 50% of the total content,
followed by the tri- and tetrachlorinated isomers. For the shrimps the pattern is clearly different
with a dominance of the lower chlorinated PCBs, especially those with three or four chlorines.
This difference can most likely be explained by a possible difference in rate of metabolism of
PCBs among these three species (mussels, oysters and shrimp), as well as differences in diet,behavior and trophic level. In view of the similarities in PCB pattern observed in oysters,mussels and shrimp between different locations, our results might indicate that the PCB
contamination in this marine environment is more likely to be a result from diffuse long rangeor regional transport than directly from local point sources.

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A good statistical relationship was found between chemically-derived TEQs and bio-assay
derived BEQ values. However, the CAFLUX-derived BEQs were significantly higher than
TEQs calculated on the basis of analytical data using the WHO 2005 TEFs (van den Berg etal., 2006). The AhR activity measured in this bioassay does not permit to identify the specificdioxin like chemicals. The most likely explanation for this difference is the presence ofadditional dioxin-like compounds in the mussels, oysters and shrimp. In our study, the only
PCBs with dioxin like properties were the MO-PCBs. In many biological samples it has been
observed that these congeners can contribute significantly (e.g. 50–60%) to the dioxin like
content of a sample extract (Schoeters et al., 2004; So et al., 2005; Jiang et al., 2007). However,chlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like non-ortho
PCBs (PCB 77, 126 and 169) can be major contributors to the total BEQs/TEQs found in
biological samples (Baars et al., 2004; Schoeters et al., 2004; Jiang et al., 2007). Thus, it not
surprising that there is a consistent difference between the bioassay BEQs and chemicallyderived TEQs in our study.

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The CALUX and CAFLUX bioassays are increasingly being used for quantitative screeningof dioxin-like compounds in various marine samples, including fishes, mussels, oysters,
starfishes, sea birds, and marine mammals (Schoeters et al., 2004; Gizzi et al., 2005; Jeong etal., 2005; So et al., 2005). The results of our study show that the AhR CAFLUX bioassay canbe used effectively for initial screening of dioxin like activity in marine food samples. In spiteof the fact that we did no chemical analysis of all known dioxin like compounds, the bioassay
derived TEQs showed good correlations with either ΣPCBs or MO-PCBs. The fact that thechemically-derived TEQs based on MO-PCBs consistently underestimated the AhR CAFLUX
BEQs can easily be explained by the presence of other dioxin-like compounds that were notmeasured. Obvious candidates for these compounds include the chlorinated dibenzofurans andnon-ortho PCBs 77, 126 and 169, which have significantly higher potencies than the MO-PCBs(van den Berg et al., 2006). The AhR CAFLUX bioassay applied here, like that of the CALUX
assay, can be utilized for screening large numbers of food and environmental samples. It canalso be used for estimation of the human daily intake of potential dioxin-like compounds andassessment of the potential health risks for human consumers due to food consumption.
However, it should be emphasized that at levels above a regulatory cutoff level (e.g. a Tolerable
Daily Intake (TDI) or Maximum Residue Limit), final confirmation of the identity of the
responsible chemical(s) should come from congener specific chemical analytical techniques.

Acknowledgments
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The authors would like to thank Prof. Dr. Jacob de Boer and Dr. Pim Leonards for providing an analytical training at
the Netherlands Institute for Fisheries Research (The Netherlands) and Dr. Patrik Andersson from the University of
Umea (Sweden) for his constructive comments on Principal Component Analysis. The research was supported by theCenter for Environmental Health, Toxicology and Management of Chemicals (Thailand) and the Institute for Risk
Assessment Sciences of the Utrecht University (The Netherlands). Support for the development of the CAFLUX
bioassay was received from the United States National Institutes of Environmental Health Sciences (ES04699).

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Fig. 1.

Sampling locations along the east coast of the Gulf of Thailand.

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Fig. 2.

Box plots of total PCB concentrations in mussels, oysters and shrimp collected at various
locations from the Eastern Coast of the Gulf of Thailand, expressed either as dry weights (A),wet weights (B) or lipid adjusted weights (C). Each box represents the median and 25–75th
percentiles. The whiskers represent the 10th & 90th percentiles. Circles represent the outliers.

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Fig. 3.

Congener specific distributions and concentrations (mean ± SD) of individual PCB congeners
in mussels (A), oysters (B) and shrimp (C); and their isomer groups distribution (D). Isomergroups have been divided in lightly (Cl3 and Cl4), medium (Cl5 and Cl6) and heavily (Cl7 andCl8)) chlorinated.

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Fig. 4.

Score plot of principal component analysis of mussels (A), oysters (B) and shrimp (C) collected
at various locations from east coast of the Gulf of Thailand.

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Fig. 5.

Statistical relationship between HRGC-ECD-derived MO-PCB TEQs and CAFLUX-derived
BEQs of mussels (A), oyster (B) and shrimp (C) collected at various locations from east coastof the Gulf of Thailand.

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NIH-PA Author Manuscript
99.0

Chemosphere. Author manuscript; available in PMC 2010 June 1.
2,3,4,3′,4′
2,3,4,5,4′
2,4,5,3′,4′
3,4,5,3′,4′
2,3,4,2′,3′,4′

118

126

128

99.7

100.0

99.8

100.0

99.0

99.7

99.5

99.0

100.0

100.0

114

2,4,5,4′

74

105

2,5,3′,4′

70

99.0

99.0

99.0

2,4,5,3′

67

2,4,5,2′,5′

2,4,3′,4′

66

99.0

99.0

2,4,5,2′,4′

2,3,4,4′60

99

2,5,2′,5′

52

99.0

99.0

101

2,4,2′,5′

49

100.0

2,3,2′,5′

44

99.7

99.0

2,3,6,2′,5′3,4,4′

37

2,3,4,2′,5′3,4,2′

33

100.0

100.0

95

2,5,4′

31

87

2,4,4′

28

99.5

99.0

3,4,3′,4′2,5,2′

18

2,3,4, 2′,3′

2,4,2′

17

99.6

Purity %

82

2,4′

8

77

Chlorine substitution pattern

IUPAC No.

209

208

206

205

201

199

195

194

191

189

187

183

180

179

177

171

170

169

166

158

156

153

151

149

138

132

IUPAC No.

2,3,4,5,6,2′,3′,4′,5′,6′2,3,4,5,6,2′,3′,5′,6′2,3,4,5,6,2′,3′,4′,5′2,3,4,5,6,3′,4′,5′2,3,4,6,2′,3′,5′,6′2,3,4,5,6,2′,3′,6′

2,3,4,5,6,2′,3′,4′

2,3,4,5,2′,3′,4′,5′2,3,4,6,3′,4′,5′2,3,4,5,3′,4′,5′

2,3,5,6,2′,4′,5′2,3,4,6,2′,4′,5′2,3,4,5,2′,4′,5′

2,3,5,6,2′,3′,6′

2,3,5,6,2′,3′,4′

2,3,4,6,2′,3′,4′

2,3,4,5,2′,3′,4′

3,4,5,3′,4′,5′2,3,4,5,6,4′

2,3,4,6,3′,4′

2,3,4,5,3′,4′

2,4,5,2′,4′,5′

2,3,5,6,2′,5′

2,4,5,2′,3′,6′

2,3,4,2′,4′,5′2,3,4,2′,3′,6′Chlorine substitution pattern

100.0

99.8

100.0

99.6

100.0

100.0

99.8

100.0

99.8

100.0

99.0

99.0

99.7

100.0

100.0

99.6

99.0

100.0

99.0

100.0

99.0

99.2

100.0

98.9

99.8

99.8

Purity %NIH-PA Author Manuscript

PCB congeners used for this study and their purity

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Table 1
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82.08 ± 0.78
(80.62–83.50)
1.17 ±0.11(0.92–1.38)

Moisture content,%

Lipid content, %wet weight

1.68 ± 1.48
(0.19–4.96)
173.22 ± 153.20(19.18–510.56)

Wet weight, ng g−1
ww.

Lipid adjusted
weight, ng g−1
lw.

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0.06 ± 0.03
(0.02–0.11)
5.93 ±2.90(1.62–11.54)
7.84 ±3.76(1.62–18.39)
0.94 ± 0.89
(ND–2.75)

Wet weight, pg
BEQ g−1 ww.

Lipid adjusted
weight, pg BEQ
g−1 lw.

CAFLUX-derived
BEQ, pg BEQ
g−1 lw.

Mono-PCBsderived TEQe,
pg TEQ g−1 lw.

ND = Not detectable.

0.32 ±0.16(0.09–0.62)

Dry weight, pg
BEQ g−1 dw.

Dioxin-likecompounds
concentrationsd

9.35 ±8.22(1.04–27.57)

Dry weight, ng g−1
dw.

Sum of 49 PCBs
concentrations

7.60 ± 0.80b(6.40–9.00)c

Mussels from 1st
location, namely
Sapan laung-jao (M1)(na = 15)

Seafood species

Size, cm.

Parameter

8.01 ±2.70(4.83–12.64)

0.08 ± 0.03
(0.05–0.12)

0.43 ±0.15(0.26–0.68)

390.94 ±48.18(329.59–492.53)

3.80 ± 0.47
(3.20–4.79)

21.11 ± 2.60
(17.80–26.60)

1.17 ± 0.07
(1.00–1.26)

82.13 ± 0.59
(81.00–82.91)

9.40 ± 0.70
(8.20–10.50)

Mussels from 2nd
location, namely
Koh loy (M2)(n = 15)

11.47 ±4.06(3.14–18.39)

0.11 ± 0.04
(0.01–0.06)

0.62 ± 0.22
(0.17–0.99)

453.82 ± 125.44(149.59–640.23)

4.41 ± 1.22
(1.45–6.22)

24.51 ± 6.77
(8.08–34.57)

1.17 ± 0.07
(1.06–1.32)

82.33 ± 0.82
(80.82–84.46)

5.80 ± 1.20
(5.50–7.20)

Mussels from 3rd
location, namely
Laem-chabang (M3)(n = 15)

5.95 ± 2.33
(3.78–10.24)

0.06 ± 0.02
(0.04–0.10)

0.32 ±0.13(0.20–0.55)

79.43 ± 35.77(33.28–145.74)

0.77 ± 0.35
(0.32–1.42)

4.29 ± 1.93
(1.80–7.87)

1.17 ± 0.09
(1.08–1.40)

82.08 ± 0.40
(81.28–82.85)

9.40 ± 0.70
(7.20–8.40)

Mussels from 4th
location, namely
Ban pae (M4)(n = 15)

0.54 ± 0.33
(ND–0.19)

6.42 ± 3.68
(0.84–15.31)

5.75 ± 4.34
(0.84–15.64)

0.09 ± 0.07
(0.01–0.26)

0.27 ± 0.20
(0.04–0.74)

192.61 ± 93.31(32.76–338.87)

3.17 ± 1.53
(0.54–5.57)

9.05 ±4.39(1.54 – 15.93)

2.50 ± 0.22
(2.06–2.72)

65.11 ± 0.48
(64.38–65.79)

3.60 ± 0.80
(2.10–5.20)

Oysters from 1st
location, namely
Au lungdam (O1)(n = 12)

7.57 ±3.69(3.82–15.31)

0.12 ± 0.06
(0.06–0.25)

0.36 ± 0.17
(0.18–0.72)

304.00 ± 64.86(207.57–457.59)

5.00 ± 1.07
(3.41–7.53)

14.29 ± 3.05
(9.76–21.51)

2.51 ±0.13(2.34–2.68)

65.18 ± 0.83
(63.75–66.84)

2.80 ± 0.90
(2.00–4.80)

Oysters from 2nd
location, namely
Pak prasae (O2)(n = 15)

1.47 ± 0.68
(ND–3.06)

7.57 ± 1.91
(3.13–10.45)

7.57 ± 1.91
(3.13–10.45)

0.02 ± 0.01
(0.01–0.03)

0.05 ± 0.01
(0.02–0.07)

774.61 ± 229.37(19.95–
1,100.57)

1.90 ± 0.56
(0.05–2.70)

5.42 ± 1.61
(0.14–7.70)

0.70 ± 0.40
(0.20–1.60)

64.64 ± 0.79
(61.81–66.12)

12.30 ± 0.84
(10.40–14.00)

Shrimp(n = 21)

PCB and dioxin-like compound concentrations in mussels, oysters and shrimp collected at various locations from the eastern coast of the Gulf of Thailand

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Table 2
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Dioxin-like compounds as determined with the AhR-CAFLUX assay.

eMono-PCBs as analyzed by HRGC-ECD are comprised with PCBs Nos. 105, 114, 118, 156 and 189.

dc
Range (min – max).

Mean value is expressed as mean ± standard deviation.

The number of pooled samples.

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