ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 67 (2007) 157–162
www.elsevier.com/locate/ecoenv
Determinations of dioxinlike activity in selected mollusks from the coast
of the Bohai Sea, China, using the H4IIE-luc bioassay
Maoyong Songa,b, Yan Xub, Qinting Jiangb, Paul K.S. Lamb, Desmond K. O’Tooleb,
John P. Giesyb,c, Guibin Jianga,
a
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
P.O. Box 2871, Beijing 100085, China
b
Research Centre for Coastal Pollution and Conservation, Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon,
Hong Kong, China
c
Department of Zoology, National Food Safety and Toxicology Center, Institute of Environmental Toxicology, Michigan State University, East Lansing,
MI 48824, USA
Received 20 October 2005; received in revised form 28 February 2006; accepted 4 March 2006
Available online 2 May 2006
Abstract
Extracts of mollusks collected from eight cities along the coast of the Bohai Sea, China, were used to determine dioxinlike activity
using an in vitro bioassay approach. Dioxinlike activity of total extracts was detected by measuring luciferase activity in a stable
transfected rat hepatoma cell line, H4IIE, containing an aryl hydrocarbon receptor (AhR)-responsive element linked to a luciferase
reporter gene. Luciferase activity was expressed as a percentage of the maximum response observed for 2,3,7,8-TCDD (% TCDDmax).
All mollusk samples elicited significant dioxinlike activity except those from Dalian (N1). The greatest magnitudes (7SD, data ex N1) of
activity observed for these extracts ranged from 4.8870.48% to 11.3871.43% TCDDmax. The mean activity was 7.3771.85%
TCDDmax. Concentrations of TCDD-EQs in mollusk extracts were from 227.4 to 547.5 pg g1 lipid. The cytotoxic effect of the mollusk
extracts on cells was assessed at the same time. Six of the mollusk extracts (N2, N3, N4, S1, S2, and S4) caused noticeable growth
inhibition over the exposure period at concentrations higher than 0.33% of the extracts concentrations (0.83 mL extract well1). All the
dilutions of sample N1 were cytotoxic to the H4IIE cells.
r 2006 Elsevier Inc. All rights reserved.
Keywords: Bioassay; H4IIE-luc; Dioxinlike activity; Bohai Sea coast; Mollusk
1. Introduction
Many xenobiotic pollutants, which are released into the
environment through industrial and municipal wastewaters, represent a potential threat to aquatic ecosystems.
Some substances are persistent while others are (bio)transformed into additional compounds, further increasing the
apparent number of chemicals released into the environment (Gagné and Blaise, 1998). A group of toxicants of
current interest is the dioxinlike compounds that elicit toxic
effects similar to those of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). These compounds are persistent and
Corresponding author. Fax: +8610 62923563.
E-mail address: gbjiang@mail.recees.ac.cn (G. Jiang).
0147-6513/$ - see front matter r 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2006.03.004
accumulate in the food chain (Ahlborg et al., 1994). Such
dioxinlike compounds include polychlorinated dibenzo-pdioxins
(PCDDs),
polychlorinated
dibenzofurans
(PCDFs), polychlorinated biphenyls (PCBs) (Van den Berg
et al., 1998), and other compounds that may elicit the aryl
hydrocarbon receptor (AhR) mediated biochemical and
toxic response (Behnisch et al., 2001). They are of great
concern due to their hepatotoxicity, embryotoxicity,
teratogenicity, immunotoxicity, dermal toxicity, lethality,
carcinogenicity, association with a wasting syndrome, and
tumorogenicity, found expressed in many different species
at low concentrations (Hilscherova et al., 2000; Peterson
et al., 1993).
High-resolution gas chromatography and mass spectrometry (HRGC-MS) are used for the detection and
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M. Song et al. / Ecotoxicology and Environmental Safety 67 (2007) 157–162
quantification of dioxinlike compounds and are considered
the ‘‘Gold Standard’’ technique (Vanderperren et al.,
2004), but they are quite expensive and time-consuming.
In the environment, chemicals exist and occur as complex
mixtures including different congeners and isomers. Quantitative instrumental analysis of complex mixtures of these
chemicals is complicated by the fact that techniques and
standards for identifying many of the congeners and
isomers do not exist (Garrison et al., 1996). In addition,
instrumental analysis provides little information about the
additive, synergistic, or antagonistic interactions produced
by the chemical mixtures. In recent years, a variety of in
vitro assays using cultured cells have been developed as an
alternative to the use of animals in assessing the risk to fish
and wildlife posed by these dioxinlike compounds, which
may also reflect the risk to humans. In this study the stable
transfected rat hepatoma cell line, H4IIE, has been applied
in the H4IIE-luc assay, which is based on the AhRdependent mechanism for the detection of dioxinlike
compounds. Recombinant H4IIE cells (H4IIE-luc) exhibit
AhR-mediated luciferase expression (Aarts et al., 1995). It
has proved to be a rapid, sensitive, and relatively
inexpensive technique compared with some instrumental
analytical methods (Sakai and Takigami, 2003).
The Bohai Sea is a semienclosed sea of China and has a
coastline length of nearly 3800 km. There are many heavy
industries and a variety of fisheries located along the coast.
To investigate distribution of these xenobiotic substances
four kinds of mollusks distributed along the Bohai Coast
that are harvested for human consumption were chosen as
our test organisms. Most of the mollusk samples were
collected from the important seaports. The dioxinlike
activity in the mollusk extracts was studied using the
recombinant H4IIE-luc cell line.
2. Materials and methods
2.1. Reagents
The following standards and reagents were used in this study: 2,3,7,8TCDD (Sigma, Germany), n-hexane (99%) (Merck, Germany), dichloromethane (DCM, 99%) (TEDIA company, USA), silica gel 60, column
chromatography (Merck, Germany), acid-activated copper powder, 99%
(Fluka), and anhydrous sodium sulphate (Na2SO4) (Sigma, Germany). All
the solvents in this study were redistilled.
The following were used for cell culturing and testing: Dulbecco’s
Modified Eagle’s Medium (Sigma), the LIVE/DEADs Viability/Cytotoxicity Kit (L-3224) (Molecular Probes, Invitrogen, Eugene, OR), the
luminescence receptor gene assay system (LucLiteTM kit) (Packard
BioScience, Netherlands). Milli-Q purified water was used throughout.
The bioassay was conducted with the rat hepatoma cell line, H4IIE, that
has been stably transfected with a receptor gene to allow expression of the
luciferase enzyme. The cell line was provided by Department of Biology
and Chemistry, City University of Hong Kong.
2.2. Sampling
Fig. 1 shows the eight sampling sites, four of them are spread out along
the northern Bohai coast and the other four are along the southern coast.
The sampling was carried out in August 2003. Four species were selected
Fig. 1. Map of the locations along the coast of the Bohai Sea, China.
for this study: Rapana venosa collected from N1, S1, S3 and S4; Neverita
didyma collected from N2 and N4; a species of Amusium collected from
N3 and Neptunea cumingii collected from S2. The species of mollusks
(Table 1) were identified according to the catalog of marine mollusks
(Zhao et al., 1982). The collected mollusks were stored in an ice-cooled
box during the transportation and then kept at 20 1C in the laboratory.
Before analysis, stainless-steel scalpel blades were used to cut open the
mollusks and remove the soft tissues, which were then thoroughly rinsed
with Milli-Q water to remove extraneous impurities. Samples were
homogenized in a stainless-steel blender, and subsamples were freezedried for analysis.
2.3. Sample extraction and clean-up
Sample extraction was performed as follows. Five grams of the freezedried mollusk samples was mixed with 15 g anhydrous Na2SO4 to remove
residual moisture, 10 g of copper powder to remove any sulfur, and 10 g
silica gel. Then the mixture was extracted with 300 mL of a mixture (1:1) of
DCM and hexane for 16 h in a Soxhlet apparatus. The procedural blank
consisted of the same amount of Na2SO4, silica, and copper powder. After
the initial extraction steps the sample was rinsed three times with 15 mL
hexane to ensure total recovery of the extract. The extract was evaporated
(40 1C) and eventually transferred to 1 mL n-hexane. The freeze-dried
sample (1 g) was put into a glass column; the lipids were eluted with a
mixture of hexane and DCM. The lipids extract was evaporated under a
gentle N2 stream at 37 1C. Lipids were then gravimetrically determined.
The concentrated extract was further cleaned up through a column that
contained 10 g of activated florisil (Hilscherova et al., 2000). The
extraction aliquot was added to the column. A quatity of 100 mL hexane
was eluted to obtain Fraction 1, which contained the PCBs and a portion
of the PCDD/PCDFs. A mixture of hexane containing 20% DCM was
next used to elute organochlorine (OC) compounds, PAHs, alkylphenolethoxylates and the remaining PCDD/PCDFs to obtain Fraction 2. The
H4IIE-luc bioassay was performed on each fraction to calculate the
contribution that each of these fractions made to the overall AhRmediated activity.
2.4. Assessment of cytotoxicity of mollusk extract
Prior to testing in the H4IIE-luc assay the extracts were tested for
cytotoxicity towards the H4IIE cells. Only a dose range without an obvious
cytotoxic effect was applied to the subsequent bioassay. Cytotoxocity
testing was conducted with H4IIE cells using the procedure in the LIVE/
DEADViability/Cytotoxicity Kit. Live cells retain the polyanionic dye AM
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159
Table 1
Characteristics and number of mollusk samples collected from sites along the coast of the Chinese Bohai Sea
Sample
Sites
Species
Length mm7SD
No.
N1
N2
N3
N4
S1
S2
S3
S4
Dalian
Yingkou
Jinzhou
Huludao
Weihai
Yantai
Laizhou
Yangkou
Rapana venosa
Neverita didyma
Amusium sp.
Neverita didyma
Rapana venosa
Neptunea cumingii
Rapana venosa
Rapana venosa
7173
6174
8576
6073
8776
4976
5673
2875
5
10
3
4
4
29
8
12
2.5. Cell culture and bioassay
Culture conditions and the assay procedure for the recombinant H4IIE
rat hepatoma cell line, containing a luciferase reporter gene under control
of dioxin-responsive enhancers, have been described previously (Khim et
al., 1999a, b). In brief, 60 interior wells of a 96-well culture plate were
seeded with 250 mL of a cell suspension to give a density of about 15,000
cells well1, and 250 mL phosphate-buffered saline (PBS) was added to the
outer wells. Cells were incubated overnight prior to dosing with 2.5 mL of
standard, solvent, and test extract dissolved in hexane. Standard plates
were dosed with 2,3,7,8-TCDD at 30, 10, 3, 1, 0.3, and 0.1 pg well1,
prepared by one-in-two dilutions. Test plates were dosed with mollusk
extract at 2.5, 0.83, 0.28, 0.09, 0.03, and 0.01 mL well1, also prepared by
one-in-two dilutions. All additions were done in triplicate. Cells were
cultured under aseptic conditions in a humidified CO2 incubator at 37 1C,
5% CO2. The luciferase assay was carried out after 72 h exposure. Briefly,
H4IIE cells were washed three times with PBS after removal of culture
medium. Cells were lysed with 75 mL PBS containing Ca2+ (1 g L1 CaCl2)
and Mg2+(1 g L1 MgCl2). Then 75 mL luciferase assay reagent was added
to each well. Plates were incubated for 20 min at room temperature in the
dark. Luciferase activities were measured with an automate luminometer
(Dynatech ML 3000 luminometer; Chantilly, VA). The detailed method
for in vitro assays with the H4IIE-luc cell line has been described elsewhere
(Sanderson et al., 1996). Responses of extracts, expressed as the mean
relative luminescence units (RLUs), were converted to relative response
units, expressed as a percentage of the maximum response observed for
2,3,7,8-TCDD (% TCDDmax). Comparing to the TCDD standard,
bioassay-derived dioxin equivalents (TCDD-EQ) were determined directly
from the dose–response relationship generated by the testing sample at
multiple levels of dilution.
3. Results and discussion
As shown in Fig. 2, the semilogarithmic dose curve of
TCDD used as standard in the H4IIE-luc assay has a
sigmoidal appearance. Each point of the curve represents
the ratio of mean luciferase response to TCDDmax
response. The detection limit was calculated separately
for each multiwell plate and was shown to be very
reproducible. The limit of detection (LOD) and EC50 for
120
100
80
% TCDDmax
calcein which produces an intense uniform green fluorescence in the live
cells. Dead cells retain the dye ethD-1 which produces a bright red
fluorescence in the dead cells. Graph the average calcein fluorescence and
standard deviation for the data visually. If the blank has greater viability
decrease than the other treatment, the solvent may be toxic to the cells. If
viability decreases with increasing concentration of the test substance, the
test substance may be toxic to the cells. In either of these cases, the luciferase
data must be regarded with great suspicion.
60
40
20
0
0.1
1
10
100
-1
2,3,7,8-TCDD pg well
Fig. 2. Dose–response curve of 2,3,7,8-TCDD for the induction of
luciferase activity in the H4IIE-luc assay. Luciferase induction is expressed
in terms of the percentage of luciferase activity relative to that of
30 pg well1 2,3,7,8-TCDD (TCDDmax). Values represent the mean7SD;
n ¼ 3.
luciferase induction by 2,3,7,8-TCDD were 0.15 and
2.2170.13 pg well1, respectively. Significant response
was defined as those outside the range defined by three
times the standard deviation (expressed as % TCDDmax) of
the mean blank response (0% TCDDmax). For the testing
samples, the extract dilutions (mL well1) were used as a
measure of fluorescence induction instead of the concentration of 2,3,7,8-TCDD.
Cytotoxicity is a significant influencing factor when
evaluating the dioxinlike activity of complex extracts. In
our study, cytotoxicity was assessed as a viability index. If
viability decreased with increasing concentration of the test
substance, the test substance may be toxic to the cells and
any morphological damage to cells can be observed by
microscopic examination. All the dilutions of sample N1
were cytotoxic to the H4IIE cells, and only one sample (S3)
was not cytotoxic at any test extract concentrations. The
other six of mollusk extracts (N2, N3, N4, S1, S2, and S4)
caused noticeable growth inhibition over the exposure
period at concentrations of 0.33% extracts (0.83 mL extract
well1) and 0.1% extracts (2.5 mL extract well1). As shown
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Total ectract
Fraction 1
Fraction 2
20
Luciferase activity (% TCDDmax )
18
16
14
12
10
8
6
4
Significant
Response
2
0
Fig. 3. Viability and dose–response curve of H4IIE exposed to different
concentrations of test sample (N4). Viability is expressed as the percentage
of AM calcein luciferase activity of the control well (cell with no solvent
and test sample added); Luciferase induction is expressed in terms of the
percentage of the maximum response of TCDD. Values presented are the
average of three parallel test wells; Bars represent standard deviations.
as Fig. 3, there was still a significant dose-dependent
increase of dioxinlike activity in test wells despite the
decrease in the viability index.
Results of the in vitro H4IIE-luc assay on the extract and
fractions of mollusks collected from different sampling
locations are summarized (Fig. 4). Seven of the eight
extracts induced significant luciferase activity, except the
sample from N1. The greatest magnitudes (7SD) of
activity observed for these extracts ranged from
4.8870.48 to 11.3871.43% TCDDmax. The mean activity
(7SD, data ex N1) was 7.3771.85% TCDDmax. For F1,
no significant induction was observed even for the greatest
concentration to this fraction except N2 and N4. Of the
two fractions, only F1 caused significant cytotoxicity to
H4IIE cells. The range of cytotoxic concentrations was
0.83 mL extract well1 and 2.5 mL extract well1 (all
concentrations for N1). This means that the cytotoxic
compounds were concentrated in F1. The greatest induction of luciferase activity was caused by compounds in F2
for most samples, except N3. This fraction contained
PAHs, their derivatives, OC pesticides and a portion of the
PCDD/PCDFs. Some PAHs, such as benzo(a)pyrene
(BAP), dibenzo(a,h)anthracene (DBA), benzo(a)anthracene (BA), benzo(k)fluoranthene (BkF), benzo(b)fluoranthene (BbF), chrysene (Chr), and indeno(1,2,3c,d)pyrene (IdP), are a class of chemicals that can
significantly contribute to the induction TEQs. In study
these seven PAHs accounted for more than 85% of the
calculated total induction equivalents in mollusks (Gardinal and Wade, 1998). Anderson et al. (1999) have described
how small mussels deployed in the Naval Station exhibited
very high bioaccumulation of PAHs, in addition to very
strong induction of CYP1A1 measured by reporter gene
system (RGS) response.
Bioassay-derived dioxin equivalents (TCDD-EQs) were
estimated directly from the sample dose–response curve
*
*
N1
N2
N3
N4
S1
*
S2
S3
S4
Fig. 4. Maximal luciferase induction in the H4IIE-luc assay elicited by
crude extracts and fractions of the mollusks. Response magnitude is
expressed as percentage of TCDDmax. * The response of the test sample
below the mean solvent control response (set to 0% TCDDmax).
Table 2
The TCDD-EQs and lipid contents of samples from the sampling sites
measured by H4IIE-luc bioassay
N1
N2
N3
N4
S1
S2
S3
S4
Lipid (% dw)
TCDD-EQ-T
(pg TEQ g1 lipid)
TCDD-EQ-F2
(pg TEQ g1 lipid)
6.23
4.86
4.57
4.43
5.24
3.77
5.57
5.98
RLa
523.5
378.7
547.5
227.4
374.2
377.4
284.2
430.1
837.0
485.6
823.8
712.2
586.6
543.0
413.4
a
RL: All the test values have cytotoxicity in the H4IIE-luc assay and the
responses were lower than a significant response.
using methods described elsewhere (Sanderson et al., 1996;
Villeneuve et al., 2000). Concentrations of TCDD-EQs in
mollusk extracts were from 227.4 to 547.5 pg g1 lipid
(Table 2). Bioassay-derived TCDD-EQs were compared to
instrumentally derived TEQs in order to assess the
contamination level along the Bohai Sea coast. The
instrumental analyses were performed previously using
the same samples, except N3 and S3 (Zhao et al., 2005).
The results are shown in Table 3. The TEQ-values ranged
from 0.73 to 51.9 pg g1 lipid. Currently, the relative
toxicological potency of a complex mixture of dioxinlike
compounds is assessed by the toxic equivalent factor (TEF)
approach. Only seven dioxins (PCDDs), 10 furans
(PCDFs) and 12 dioxinlike PCBs have had TEF values
attributed to them and it is only these chemicals that are
analyzed for as part of the conventional targeted chemical
analysis TEQ determination (Hurst et al., 2004). There are
a number of other compounds and classes of compounds in
the extracts that can interact with AhR and potentially
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Table 3
Concentrations of PCBs and PCDD/Fs in mollusks from the Chinese Bohai Sea (pg TEQ g1 lipid wt)
PCB-WHO-TEQ
PCDD/Fs-WHO-TEQ
Total TEQ
N1
N2
N4
S1
S2
S4
7.9
44
51.9
0.78
14.4
15.18
0.53
16.4
16.93
0.68
0.05
0.73
16
24
40
1.4
6.1
7.5
Source: Data from Zhao et al. (2005).
cause dioxinlike activity. The H4IIE bioassay will responded to any AhR ligand if it is present in the matrix of
interest. It has been observed in other locations that the
extracts contained some chemicals that could exhibit the
luciferase activity (Koh et al., 2004). These unknown
compounds producing dioxinlike response in the H4IIE-luc
assay may account for the major differences between the
TCDDs with TEQs and TEQsH4IIE-luc. One recent study
reported that the luciferase induction in the H4IIE-luc cell
bioassay elicited by raw extracts of Zhoushan fish was
negative (Jiang et al., 2005). While another study reported
the concentrations of TEQs in the organisms collected
from the Bohai Sea coast ranged from 0.06 to 65 pg g1 wet
weight (Wan et al., 2005).
The Bohai Sea is an enclosed inner sea in north China,
and the coastal areas are highly developed economically.
With the economic development and population growth
the environmental impact is likely to be most severe along
the Bohai coastline. For example, 1 billion tons of
wastewater alone is discharged into the Bohai Sea from
Beijing, Tianjin, and Hebei province every year (Wan et al.,
2005). People in coastal cities have a greater chance of
exposure to toxic contaminants than inland populations
due to the consumption of seafood (Jiang et al., 2005).
Being highly lipophilic and resistant to metabolic breakdown, dioxin and dioxinlike compounds can accumulate in
predators at the top of food chain (Braune and Simon,
2003), resulting in potential risk to wildlife. Because dietary
uptake is the major route of dioxin exposure in humans,
many studies have used foods such of mollusks as a
suitable bioindicator for monitoring and determining the
dioxin contaminant levels in freshwater or marine environments (Abad et al., 2003; Monod et al., 1995; Wu et al.,
2001). Although the use of marine organisms for pollution
monitoring has some limitations, evaluation of contaminant profiles in mollusks can provide more information on
contaminant levels, bioaccumulation, and pollutant bioavailability to successive consumers in the food chain.
Acknowledgments
This study was supported by a Central Allocation Grant
(8730020) awarded by the Research Grants Council, Hong
Kong, and the Area of Excellence Scheme under the
University Grants Committee of the Hong Kong Special
Administration Region, China (Project AoE/P-04/2004).
References
Aarts, J.M.M.J.G., Dension, M.S., Cox, M.A., Schalk, M.A.C., Garrison,
P.A., Tullis, K., De Haan, L.H.J., Brouwer, A., 1995. Species-specific
antagonism of Ah receptor action by 2,20 ,5,50 -tetrachloro- and
2,20 ,3,30 ,4,40 -hexachlorobiphenyl. Eur. J. Phamacol. 293, 463–474.
Abad, E., Pérez, F., Lleena, J.J., Caixach, J., Rivera, J., 2003. Evidence for
a specific pattern of polychlorinates dibenzo-p-dioxin and dibenzofurans in bivalves. Environ. Sci. Technol. 37, 5090–5096.
Ahlborg, U.G., Becking, G.C., Birnbaum, L.S., Brouwer, A., Derks,
H.J.G.M., Feeley, M., Golor, G., Hanberg, A., Larsen, J.C., Liem,
A.K.D., Safe, S.H., Schlatter, C., Waern, F., Younes, M., Yränheikki,
R., 1994. Toxic equivalency factors for dioxin-like PCBs. Chemosphere 28, 1049–1067.
Anderson, J.W., Jones, J.M., Steinert, S., Sanders, B., Means, J.,
McMillin, D., Vu, T., Tukey, R., 1999. Correlation of CYP1A1
induction, as measured by the P450 RGS biomarker assay, with high
molecular weight PAHs in mussels deployed at various sites in San
Diego Bay in 1993 and 1995. Mar. Environ. Res. 48, 389–405.
Behnisch, P.A., Hosoe, K., Sakai, S-ichi., 2001. Combinatorial bio/
chemical analysis of dioxin and dioxin-like compounds in waste
recycling, feed/food, humans/wildlife and the environment. Environ.
Int. 27, 495–519.
Braune, B.M., Simon, M., 2003. Dioxins, furans, and non-ortho PCBs in
Canadian Arctic seabirds. Environ. Sci. Technol. 37, 3071–3077.
Gagné, F., Blaise, C., 1998. Differences in the measurement of cytotoxicity
of complex mixtures with rainbow trout hepatocytes and fibroblasts.
Chemosphere 37, 753–769.
Gardinal, P.R., Wade, T.L., 1998. Contribution of PAHs, PCBs, and
PCDD/PCDFs to the Total Induction Equivalents (SIEs) in mollusks.
Mar. Pollut. Bull. 37, 27–31.
Garrison, P.M., Tullis, K., Aarts, J.M.M.J.G., Brouwer, A., Giesy, J.P.,
Dension, M.S., 1996. Species-specific recombinant cell lines as bioassay
systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like
chemicals. Fund. Appl. Toxicol. 30, 194–203.
Hilscherova, K., Machala, M., Kannan, K., Blankenship, A.L., Giesy,
J.P., 2000. Cell bioassays for detection of Aryl Hydrocarbon (AhR)
and Estrogen Receptor (ER) mediated activity in environmental
samples. Environ. Sci. Pollut. Res. 7, 159–171.
Hurst, M.R., Balaam, J., Chan-Man, Y.L., Thain, J.E., Thomas, K.V.,
2004. Determination of dioxin and dioxin-like compounds in
sediments from UK estuaries using a bio-analytical approach:
chemical-activated luciferase expression (CALUX) assay. Mar. Pollut.
Bull. 49, 648–658.
Jiang, Q.T., Lee, T.K.M., Chen, K., Wong, H.L., Zheng, J.S., Giesy, J.P.,
Lo, K.K.W., Yamashitad, N., Lam, P.K.S., 2005. Human health risk
assessment of organochlorines associated with fish consumption in a
coastal city in China. Environ. Pollut. 136, 155–165.
Khim, J.S., Villeneuve, D.L., Kannan, K., Lee, K.T., Snyder, S.A., Koh,
C.H., Giesy, J.P., 1999a. Alkylphenols, polycyclic aromatic hydrocarbons, and organochlorines in sediment from Lake Shiwa, Korea:
instrumental and bioanalytical characterization. Environ. Toxicol.
Chem. 18, 2424–2432.
Khim, J.S., Villeneuve, D.L., Kannan, K., Koh, C.H., Giesy, J.P., 1999b.
Characterization and distribution of trace organic contaminants in
sediment from Masan Bay, Korea: 2. In vitro gene expression assays.
Environ. Sci. Technol. 33, 4206–4211.
ARTICLE IN PRESS
162
M. Song et al. / Ecotoxicology and Environmental Safety 67 (2007) 157–162
Koh, C.H., Khim, J.S., Kannan, K., Villeneuve, K.T., Senthilkumar, K.,
Giesy, J.P., 2004. Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-TCDD equivalents (TEQs) in sediment
from the Hyeongsan River, Korea. Environ. Pollut. 132, 489–501.
Monod, J.L., Arnaud, P.M., Armoua, A., 1995. PCB congeners in the
marine biota of Saint Paul and Amsterdam Islands, Southern Indian
Ocean. Mar. Pollut. Bull. 30, 272–274.
Peterson, R.E., Theobald, H.M., Kimmel, G.L., 1993. Developmental and
reproductive toxicity of dioxins and related compounds: cross-species
comparisons. Crit. Rev. Toxicol. 23, 283–335.
Sakai, S-ichi, Takigami, H., 2003. Integrated biomonitoring of dioxin-like
compounds for waste management and environment. Ind. Health 41,
205–214.
Sanderson, J.T., Aarts, J.M.M.J.G., Brouwer, A., Forese, K.L., Dension,
M.S., Giesy, J.P., 1996. Comparision of Ah receptor-mediated
luciferase and ethoxyresorufin o-deethylase induction in H4IIE cells:
implications for their use as buianalytical tools for the detection of
polyhalogenated aromatic hydrocarbons. Toxicol. Appl. Pharmacol.
137, 316–325.
Van den Berg, M., Birnbaum, L., Bosveld, A.T., Brunstrom, B., Cook, P.,
Feeley, M., Giesy, J.P., Hanberg, A., Hasegawa, R., Kennedy, S.W.,
Kubiak, T., Larsen, J.C., van Leeuwen, F.X., Liem, A.K., Nolt, C.,
Peterson, R.E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D.,
Tysklind, M., Younes, M., Waern, F., Zacharewski, T., 1998. Toxic
equivalency factors (TEFs) for PCBs, PCDDs for humans and wildlife.
Environ. Health Perspect. 106, 775–779.
Vanderperren, H., Van Wouwe, N., Behets, S., Windal, I.,
Van Overmerie, I., Fontaine, A., 2004. TEQ-value determination of
animal feed; emphasis on the CALUX bioassay validation. Talanta 63,
1277–1280.
Villeneuve, D.L., Blankenship, A.L., Giesy, J.P., 2000. Derivation and
application of relative potency estimates based on in vitro bioassay
results. Environ. Toxicol. Chem. 19, 2835–2843.
Wan, Y., Hu, J.Y., Yang, M., An, L.H., An, W., Jin, X.H., Hattori, T.,
Itoh, M., 2005. Characterization of trophic transfer for polychlorinated dibenzo-p-dioxins, dibenzofurans, non- and mono-ortho polychlorinated biphenyls in the marine food web of Bohai Bay, North
China. Environ. Sci. Technol. 39, 2417–2425.
Wu, W.Z., Schramm, K.W., Kettrup, A., 2001. Bioaccumulation of
polychlorinated dibenzo-p-dioxins and dibenzofurans in the foodweb
of Ya-Er lake area. China. Water Res. 35, 1141–1148.
Zhao, R.Y., Cheng, J.M., Zhao, D.D., 1982. Marine Mollusca Fauna of
Dalian. Oceanic Press, Beijing.
Zhao, X., Zheng, M., Liang, L., Zhang, Q., Wang, Y., Jiang, G., 2005.
Assessment of PCBs and PCDD/Fs along Chinese Bohai Sea Coastline
using mollusks as bioindicators. Arch. Environ. Contam. Toxicol. 49,
178–185.