Proceedings of the International Symposium on
Lowland Technology, Saga University, September, 2002
THE CONCENTRATION LEVEL OF ORGANIC POLLUTANTS AND ITS CELL LINE ASSAYS
FROM SEDIMENT SAMPLES IN CREEKS ENTERING TO THE LAKE SHIHWA, KOREA
C.H., KOH, J.S., KHIM
School of Earth and Environmental Sciences (Oceanography), Seoul National University, Seoul 151-742, Korea
K., KANNAN, J.P., GIESY
National Food Safety and Toxicology Center, Department of Zoology, and Institute for Environmental Toxicology, Michigan
State University, East Lansing, MI, 48824-1311, USA
ABSTRACT: Lake Shihwa is an artificial lake, located on the west coast of Korea, which has experienced environmental
deterioration since it was formed by construction of a sea-dike in 1994. This study used instrumental analysis and in vitro
bioassays to characterize organic contaminants in sediment collected from 8 locations in creeks entering to the Lake Shihwa,
Korea. The relative abundance of organic pollutants in Lake Shihwa was in the order of nonylphenol, PAHs, bisphenol A,
octylphenol, PCBs, and organochlorine pesticides. Nonylphenol was predominant in landward regions of the Lake Shihwa.
Initial screening of raw sediment extracts showed significant dioxin-like and estrogenic activities in H4IIE-luc and MVLN cell
bioassay, respectively. Most activities associated with Florisil column fraction samples indicated that compounds in mid-polar
and polar fractions were responsible for the significant responses observed. Overall, most of the in vitro bioassay responses
appear to have been caused by unidentified and/or undetectable, relatively polar, compounds associated with Lake Shihwa
sediment.
Lake Shihwa, located on the west of Korea, was a channel
area in a bay composed of a huge tidal flat (~ 100 km2). It turned to a
lake after separation from the sea by a 12 km-long dike in 1994.
Reclamation was the purpose of the dike construction and a water
reservoir was produced in the channel area and named as Lake
Shihwa. Stopping the tidal currents by the dike and continuous input
of municipal and industrial wastes via several creeks from
landward region has resulted in serious environmental deterioration
since 1996. Despite the potential for direct and accidental release of
organic contaminants, only one study evaluated the concentration
and distribution of organic contaminants in this region (Khim et
al. 1999a). This study is focused on persistent organic pollutants
(POPs) such as polychlorinated biphenyls (PCBs), organochlorine
(OC) pesticides (HCB, HCHs, CHLs, DDTs), polycylclic
aromatic hydrocarbons (PAHs), nonylphenol (NP), octylphenol
(OP), and bisphenol A (BPA) and their biological effects in
sediments of Lake Shihwa.
Due to the complex nature of contaminants in sediments,
sample extracts were fractionated according to polarity to
isolate and identify target contaminants. Both instrumental
analyses and in vitro recombinant cell bioassays using
H4IIE-luc cells for dioxin-like activity and MVLN cells for
estrogenic activity were performed to quantify target
contaminants and to evaluate dioxin-like and estrogenic
potencies (Khim et al. 1999a, Hilscherova et al. 2000).
Assessment based solely on instrumental analysis may over
or underestimate potential hazards of sediment
contamination. Thus, a combination of instrumental analysis
and in vitro bioassay were used to assess sediment
contamination. It may contribute to explain environmental
deterioration of this area in various aspects and guide remediation
effort.
MATERIALS AND METHOD
Sampling
Study Area
KOREA
Shinkilcreek
KyeonggiBay
S1
Shiheung
S2
City S 3
S5 S4
S7
LakeShihwa
S6 S8
sea-dike
Taebu Is.
Ansan
City
37 o 18' N
INTRODUCTION
Hwasung
County
2 km
126 o 42' E
Figure 1 Map of the Lake Shihwa study area
355
Proceedings of the International Symposium on
Lowland Technology, Saga University, September, 2002
Whole sediment
procedure are presented elsewhere (Khim et al. 1999b).
Freeze drying
Soxhlet extraction
Cu treatment
Florisil column
Raw extract (RE)
In vitro bioassay
Non-polar (F1)
PCBs, HCB,
p,p’-DDE
Mid-polar (F2)
PAHs, DDTs,
HCHs, CHLs
Polar (F3)
NP, OP,
BPA
GC/MS & ECD
In vitro bioassay
GC/MS & ECD
In vitro bioassay
GC/MS & HPLC
In vitro bioassay
Figure 2 Extraction and fractionation sequence
for Lake Shihwa sediment samples
Surface sediments (0-5 cm) samples were collected from 8
locations on Lake Shihwa including Shinkil creek in October
2000 (Figure 1). Sampling was designed to determine
potential sources of contaminants from inland regions such
as Shinkil creek (location S1-6) and sewage treatment plant
(location S7-8). A global positioning system was employed
to identify each location precisely. All samples were
collected using a shovel. After collection, pebbles and
twigs were removed, then samples were freeze dried and
ground with a mortar and pestle. Samples were stored in
pre-cleaned HDPE (high density polyethylene) bottles at
-20 °C, until extraction. Total organic carbon (TOC) was
analyzed for the sediment samples, allowing concentrations
of target analytes to be normalized to TOC.
Extraction and fractionation
Sediment samples (~40 g + ~ 100 g Na2SO4) or
procedural blanks (~120 g Na2SO4) were Soxhlet extracted
for 20 h using 400 mL high purity dichloromethane (DCM).
Extracts were treated with acid-activated copper granules to
remove sulfur, concentrated to approximately to 5 mL by
rotary evaporation (39oC), and then to 1 mL under a gentle
stream of nitrogen.
Extracts were passed through 10 g of activated Florisil
(60-100 mesh size; Sigma, St. Louis, MO, USA) packed in a
glass column (10 mm i.d.) for clean up and fractionation.
The first fraction (F1) eluted with 100 mL of high purity
hexane contained PCBs, hexachlorobenzene (HCB) and
p,p’-DDE. Remaining OC pesticides and PAHs were eluted
in the second fraction (F2) using 100 mL 20 % DCM in
hexane. NP, OP and BPA were eluted in the third fraction
(F3) with 100 mL 50 % DCM in high purity methanol.
Florisil separation was confirmed using a spike recovery test
(n=3). Recoveries of target analytes through all the analytical
steps are 80-100 %. Further details of the fractionation
356
Instrumental analysis
OC pesticides and PCBs were quantified using a gas
chromatograph (Perkin Elmer series 600) equipped with 63Ni
electron capture detector (GC-ECD). A fused silica capillary
column coated with DB-5MS [(5%-phenyl)-methylpolysiloxane, 30m × 0.25mm i.d.; J&W Scientific, Folsom,
CA, USA] having a film thickness of 0.25 µm was used. An
equivalent mixture of 98 individual PCB congeners
(AccuStandard) and a mixture of OC pesticides (CLP-023R,
CLP-024R, AccuStandard) were used as a standard.
Concentrations of 98 individually resolved peaks were
summed to obtain total PCB concentrations. PCB congeners
have been referred by Ballschmiter and Zell numbers. OC
pesticides were quantified from individually resolved peak
areas based on the peak areas of standards. Detection limits
of OC pesticides and PCBs for this method were 0.01 and
1.00 ng/g, dry weight (dry wt), respectively.
PAHs were quantified using a Hewlett Packard 5890
series II gas chromatograph equipped with a 5972 series
mass spectrometer detector. A fused silica capillary
column (30 m × 0.25 mm i.d.) coated with DB-17 [(50%
phenyl)-methyl polysiloxane; J&W Scientific, Folsom, CA,
USA] at 0.25 µm film thickness was used. The PAH
standard (AccuStandard, New Haven, CT, USA) consisted
of 16 priority pollutant PAHs identified by the U.S.
Environmental Protection Agency (U.S. EPA; method 8310).
The mass spectrometer was operated under the SIM mode
using the molecular ions selective for individual PAHs.
Concentrations based on individually resolved peaks were
summed to obtain the total PAH concentrations. The
detection limit for PAHs was 10.0 ng/g, dry wt.
Reverse phase high performance liquid chromatography
(HPLC) with fluorescence detection was used to quantify NP,
OP, and BPA. High purity p-nonylphenol and p-tertoctylphenol standards (Schenectady International, Freeport,
TX, USA) and BPA (4,4’-isopropylidenediphenol; Sigma
Chemical Co., St. Louis, MO) were prepared in high purity
acetonitrile (ACN). Samples and standards were injected (10
µl) by a Perkin Elmer Series 200 autosampler (Perkin Elmer,
Norwalk, CT, USA) onto an analytical column, Prodigy
ODS (3), 250 × 4.6 mm column (Phenomenex, Torrance,
CA, USA), which was connected to a guard column
(Prodigy ODS (3), 30 × 4.6 mm), and eluted with a flow
of ACN and water at a gradient from 50 % ACN in water to
98 % ACN in water delivered by Perkin Elmer Series 200
pump for 20 min. Detection was accomplished using a
Hewlett
Packard
1046A
fluorescence
detector
(Hewlett-Packard, Wilmington, DE, USA) with an
excitation wavelength of 229 nm and an emission
wavelength of 310 nm. NP, OP, and BPA detection limits for
the analytical method were 1 ng/g, dry wt.
Proceedings of the International Symposium on
Lowland Technology, Saga University, September, 2002
In vitro bioassay
Each sample was tested as both an raw extracts (REs)
and fractionated extracts (FEs) in the in vitro bioassays.
Luciferase and protein assays were conducted after 72 h
incubations (Sanderson et al. 1996). Sample responses,
expressed as mean relative luminescence units (RLU) over
three replicate wells, were converted to relative response
units, expressed as a percentage of the maximum response
observed
for
2,3,7,8tetrachlorodibenzo-p-dioxin
(TCDD; %-TCDD-max.) or 17-β-estradiol (E2, %-E2
-max.) standard curves generated on the same day. This was
done to normalize responses for day-to-day variability in
response magnitude. The mean solvent control response
(RLU) was subtracted from both sample and standard
responses (RLU) on a plate-by-plate basis, prior to
conversion to a percentage, in order to scale values from 0 to
100 %-standard-max. Significant responses were defined as
those outside the range defined by three times the standard
deviation (expressed in %-standard-max.) of the mean
solvent control response (0 %-standard-max.). Total protein
in the wells was used as an index of cell number to detect
outliers that were not apparent by simple visual inspection.
Mass balance analysis (or potency balance analysis) was
used to examine whether or not the known concentration
and/or composition of a sample (identified by instrumental
analyses) could account for the magnitude or potency of
biological response observed. Further details of in vitro
bioassay and mass balance analysis have been described
elsewhere (Khim et al 1999a, Villeneuve et al. 2000).
RESULT AND DISCUSSION
Concentrations of trace organic pollutants
The relative abundance of target organic contaminants
measured in sediment was, NP > PAHs > BPA > OP > PCBs
> OC pesticides (Table 1). PCBs were detected in the all
locations at concentrations ranging from 9.05 to 126 (mean:
30.9) ng/g dry wt (Table 1). Total PCB concentrations in
sediment were generally one or two orders magnitude less
than those of NP and PAHs. Locations of S3 and S4 in the
Shinkil creek contained relatively great concentrations of
PCBs (126 and 47.2 ng/g, dry wt, respectively). The spatial
gradient of PCB concentrations in sediments suggests the
presence of sources along Shinkil Creek.
Lesser
chlorinated congeners such as tetra- and penta-CBs were the
most prevalent homologs in Lake Shihwa sediments.
Previous studies have also reported the presence of lower
chlorinated PCB congeners in sediments collected from
Masan, Ulsan, and Onsan Bays (Khim et al. 1999a, Khim et
al. 2001a). Among different OC pesticides analyzed,
concentrations of HCHs (sum of α−, β−, γ− hexachlorocyclohexanes) were the greatest, ranging from 0.55 to 10.7
ng/g, dry wt.
The sedimentary PAH concentrations ranged from 12.8
to 643 (mean: 226) ng/g, dry wt (Table 1). PAH
concentrations were as great as 643 ng/g, dry wt, in sediment
from locations S3 in middle of the Shinkil creek. Mean PAH
concentration in sediments from the Lake Shihwa (mean:
224 ng/g, dry wt) was approximately 2-fold greater than
outer Kyeonggi Bay (Kim et al. 1999). There was a gradient
in concentrations of PAHs in Shinkil creek, which suggested
the input of PAHs from the upper region of the Shinkil creek.
Four-ring aromatic hydrocarbons, such as fluoranthene and
pyrene, were the predominant PAHs in the Lake Shihwa
sediment. Molecular ratios of specific PAH compounds,
such as fluoranthene to pyrene (Fluo/Py) ratio and
indeno[1,2,3-cd]pyrene to benzo[ghi]perylene (IP/BP) ratio,
were calculated to evaluate the potential sources of PAHs
(Baumard et al. 1998). The Fluo/Py ratios for Lake Shihwa
sediment samples varied depending on the locations with an
overall mean value of 0.842. The ratio of Fluo/Py in all the
locations except for S1 and S6 locations (range: 0.62-0.91)
were less than 1.0. The ratios of IP/BP were less than 1.0 for
S2-5 locations (range: 0.56-0.92). These results suggest that
the sources of PAHs to Lake Shihwa were both petrogenic
and pyrolytic. The locations of S2-5 along the Shinkil creek,
which are proximal to petrochemical industries, may receive
more petrogenic inputs whereas the upper region and lake
may be influenced by pyrolytic inputs.
NP was predominant contaminant in Lake Shihwa
Table 1 Concentrations (ng/g, dry wt) of PCBs and organochlorine pesticides (HCB, HCHs, CHLs, and DDTs), PAHs ,
nonylphenol (NP), octylphenol (OP), and bisphenol A (BPA) in sediment samples from Lake Shihwa, Korea
Location
S1
S2
S3
S4
S5
S6
S7
S8
Mean
PCBs
HCB
HCHs
CHLs
DDTs
PAHs
NP
OP
BPA
15.6
9.30
126
47.2
19.9
10.9
9.40
9.05
30.9
0.69
0.75
3.49
2.80
0.94
0.10
0.09
0.18
1.13
2.45
1.46
10.7
0.97
2.76
0.55
2.17
0.88
2.74
0.12
0.06
0.22
0.04
0.03
<0.01
<0.01
0.02
0.08
0.35
0.17
2.14
0.71
0.40
0.22
0.15
0.28
0.55
610
121
643
292
80.2
25.8
12.8
24.9
224
1640
254
4930
3640
949
668
367
622
1630
42.8
6.10
94.2
99.7
42.6
20.9
5.17
8.59
40.0
47.5
26.2
74.3
115
<1.00
13.2
17.1
31.0
46.3
357
Proceedings of the International Symposium on
Lowland Technology, Saga University, September, 2002
sediment at the mean concentrations of 1630 ng/g, dry wt
(Table 1). Maximum concentrations of NP, OP, and BPA in
sediment were 4930, 99.7, and 115 ng/g, dry wt. NP and
BPA concentrations in Shinkil creek showed distinct
concentration gradient from upstream to downstream sites.
The greater concentrations of APs at locations S3 and S4 can
be explained by its proximity to sewage waste input near
these locations.
Comparison to other study
Several studies have examined the occurrence and
distribution of POPs such as PCBs, OC pesticides, and
PAHs in Korean coastal areas (Kim et al. 1999, Khim et al.
1999a, b, 2001a, Lee et al. 2001a, b). Sedimentary PCBs and
PAHs in Korea have been reported to range from a few ng/g
to several µg/g, dry wt. Concentrations of PCBs and PAHs
in Lake Shihwa were similar to those in Masan, Ulsan, and
Yeongil Bay. (Koh et al. 2002) Where as concentrations of
APs and BPA in sediment from Lake Shihwa were 2-3- fold
greater than those in other bay areas in Korea. Greater
concentrations of APs and BPA in Lake Shihwa are
consistent with greater input from heavily populated cities
such as Shiheung and Ansan city around the lake. The data
presented here establish the baseline for future monitoring of
these compounds in Lake Shihwa areas.
Potential for biological and ecological effects
Sixteen priority PAH compounds including aryl
hydrocarbon receptor (AhR) active compounds such as
benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene,
and dibenz[a,h]anthracene were detected in sediments. Nonortho-coplanar PCB congeners 77 (3,3’,4,4’-tetraCB), 126
(3,3’,4,4’,5-pentaCB), and 169 (3,3’,4,4’,5,5’-hexaCB) were
also detected in a few samples. Based on the concentrations
of dioxin-like compounds, 2,3,7,8-TCDD equivalents
(TEQs) were estimated using relative potency factors (RPs)
specific to the H4IIE-luc cell for selected PAHs and non- and
mono-ortho-PCB congeners. Concentrations of total TEQs
ranged from 2.36 to 45.8 pg/g, dry wt. Contributions of
PCBs and PAHs concentrations to total TEQs varied among
locations. This result suggested that both dioxin-like PCBs
and PAHs were responsible for the H4IIE-luc responses
observed in sediment RE samples. The TEQs estimated for
selected PCBs and PAHs in Lake Shihwa sediment were at
the lower end of the sediment quality guideline (SQG) of
0.014-210 pg/g, dry wt, reported for dioxin equivalents
(Iannuzzi et al. 1995). SQGs such as effect range low (ERL),
threshold, median, and extreme effects concentrations (TEC,
MEC, EEC) for PAHs and PCBs were applied to further
evaluate the quality of sediment in Lake Shihwa (Long et al.
1995, Swartz et al. 1999, MacDonald et al. 2000). TOC
normalized concentrations of 16 individual PAHs, total
358
PAHs, total PCBs were calculated. None of the locations
exceeded the ERLs for 16 PAHs and PCBs. When TEC
reported for total PCBs were compared two locations (S3
and S4) exceeded the limit of 35 ng/g, dry wt.
Dioxin-like activity in vitro
Extracts of sediment were screened for their ability to
induce aryl hydrocarbon receptor (AhR) mediated gene
expression in vitro using H4IIE-luc cells (Sanderson et al.
1996). Based on the initial screening of REs, all the sediment
samples showed significant dioxin-like activity in H4IIE-luc
bioassay (Figure 3). Response magnitudes ranged from
35.8 %- to 82.6 %-TCDD-max. In order to examine
potential cause-effect relationships between the target
AhR-agonists quantified in this study and the AhR-mediated
bioassay responses observed, sediment extracts were divided
into three fractions and each fraction was analyzed in the
H4IIE-luc assay.
F1 of Lake Shihwa sediment extracts did not induce or
depress AhR-mediated gene expression in H4IIE-luc cells.
Based on TEQPCB concentrations present in the samples four
(S3, 4, 6, and 7) of the 8 F1 samples analyzed should have
yielded a significant H4IIE-luc response. Each of those
significant responses was predicted to be greater than
80 %-TCDD-max. However, this predicted profile of
responses was not observed. Thus the lack of H4IIE-luc
response to F-1 suggested that antagonistic effect of the
compounds in F1 has been occurred.
F2 and F3 sediment extracts from all locations caused
significant induction of luciferase expression in H4IIE-luc
cells (Figure 3). The magnitude of induction elicited was as
great as 113 and 69.3 %- TCDD-max. in F2 and F3,
respectively. PAHs, some of which have been shown to
induce dioxin-like activity in vitro (Villeneuve et al. 2002)
partitioned to F2 and were detected in some samples. Based
on magnitude of induction expressed as %-TCDD-max.
there was no clear correlation between PAH concentrations
detected and in vitro luciferase induction. Based on TEQPAH
concentrations present in the samples only two (S1 and 3) of
the F2 samples should have yielded a significant H4IIE-luc
response. This suggests that unknown dioxin-like
compounds such as PCDDs, PCDFs, and PCNs other than
PAHs can be possible agonists for the dioxin-like activity in
F2 samples.
No known AhR-agonists were expected to partition into
F3. Despite this, all the F3 samples analyzed produced a
significant response in the H4IIE-luc bioassay (Figure 3).
The prevalence and magnitude of AhR activity in F3
suggests the presence of unidentified, relatively polar,
AhR-agonists in sediment from the Lake Shihwa area. The
results are consistent with previous studies, which examined
sediments from other Korean coastal areas (Khim et al.
1999a, 2001b, Koh et al. 2002). Thus, the unidentified
Proceedings of the International Symposium on
Lowland Technology, Saga University, September, 2002
120
F1
F2
F3
250
200
%-E2-Max.
%-TCDD-Max.
100
RE
80
60
40
RE
F1
F2
F3
S7
S8
150
100
50
20
0
0
S1
S2
S3
S4
S5
S6
S7
S1
S8
S2
S3
S4
S5
S6
Location
Location
Figure 3 Luciferase induction in the H4IIE-luc cell bioassay elicited
Figure 4 Luciferase induction in the MVLN cell bioassay elicited
by Lake Shihwa sediment raw extracts (REs) and Florisil fraction
by Lake Shihwa sediment raw extracts (REs) and Florisil fraction
1, 2, 3 (F1, F2, F3). Response magnitude presented as the maximum
1, 2, 3 (F1, F2, F3). Response magnitude presented as the maximum
response observed for 2000 pM TCDD standard (%-TCDD-max.).
response observed for 1000 pM E2 standard (%-E2-max.).
Significant level was 3.45 %-TCDD-max.
agonists appear to have a fairly ubiquitous distribution
proximal to Korea. Future studies should employ extensive
bioassay directed fractionation and chemical analysis in
effort to identify the causative agents in F3 samples from this
study and other locations in Korea.
Estrogenic activity in vitro
Lake Shihwa sediment extracts were also screened for
their ability to induce estrogen receptor (ER) mediated gene
expression in vitro using MVLN cells (Demirpence et al.
1993). All of the RE samples yielded a significant response
in the MVLN bioassay. Based on MVLN-specific relative
potencies previously determined for NP, OP, BPA, and two
weakly estrogenic PAHs (benzo[a]anthracene, dibenz[a,h]anthracene), concentrations of E2 equivalents (EEQs)
based on these residues in the extracts were predicted to
elicit a response in the MVLN bioassay (Villeneuve et al.
1998). Predicted EEQs for Lake Shihwa sediment samples
were as great as 101 %-E2-max. in location S3. Based on
EEQs concentrations present in the samples six (S1, 3-6, 8)
of the 8 F2 and F3 samples analyzed should have yielded a
significant MVLN response. Thus, estrogenic responses
observed in F2 and F3 samples could be explained partly by
the known concentrations of APs, BPA, and estrogenic
PAHs. Additional bioassay-directed fractionation and
chemical analysis would be necessary to identify the
causative agents, however. Overall, in vitro bioassay applied
in conjunction with instrumental analysis provides a
powerful tool for characterizing mechanism- specific
agonists present in the environment.
Role of Bioassays
The results of this study support the utility of in vitro
Significant level was 2.28 %-TCDD-max.
bioassays in characterizing the occurrence and distribution of
potentially adverse compounds in the environment.
Empirical bioassay results and mass balance analyses
suggested that the target compounds quantitated by
instrumental analysis accounted for only a portion of the
mechanism specific biological activity of Lake Shihwa
sediment extracts. Risk assessment based solely on the
the instrumental results may underestimate the potential
hazard of Lake Shihwa sediment contamination. Although in
vitro bioassay results cannot be directly extrapolated to
determine the risk for adverse effects on Lake Shihwa biota,
they pointed out additional sources of uncertainty which
should be considered.
SUMMARY
The relative abundance of measured POPs in Shinkil
creek from Lake Shihwa was in the order of NP, PAHs, BPA,
OP, PCBs, and OC pesticides. Nonylphenol was predominant contaminants in Lake Shihwa landward regions.
Concentrations of APs and PAHs in Lake Shihwa were
similar to those in Masan, Ulsan bays, Korea. Based on the
initial screening of REs, significant dioxin-like and
estrogenic activities were observed in H4IIE-luc and MVLN
bioassay. Most activities associated with Florisil column
fractionated samples showed that F2and F3 were responsible
for the significant responses. Although, there were poor
relationships between sedimentary concentrations and
bioassay activities, a combination use of instrumental
analysis and cell bioassay was useful tool to characterize
and/or assess the sediment quality of Lake Shihwa area.
359
Proceedings of the International Symposium on
Lowland Technology, Saga University, September, 2002
REFERENCES
Baumard, P., Budzinski, H. and Garrigues, P. (1998).
Polycyclic aromatic hydrocarbons in sediments and
mussels of the western Mediterranean Sea.
Environmental Toxicology and Chemistry 17: 765-776.
Demirpence, E., Duchesne, M.J., Badia, E., Gagne, D. and
Pons, M. (1993). MVLN cells: a bioluminescent
MCF-7- derived cell line to study the modulation of
estrogenic activity. Journal of Steroid Biochemical
Molecular Biology 46: 355-364.
Hilscherova, K., Machala, M., Kannan, K., Blankenship,
A.L. and Giesy, J.P. (2000). Cell bioassays for detection
of aryl hydrocarbon receptor (AhR) and estrogen
receptor (ER) mediated activity in environmental
samples. Environmental Science and Pollution
Research 7: 159-171.
Iannuzzi, T.J., Bonnevie, N.L. and Wenning, R.J. (1995). An
evaluation of current methods for developing sediment
quality guidelines for 2,3,7,8-tetrachlorodibenzo-pdioxin. Archives of Environmental Contamination and
Toxicology 28: 366-377.
Khim, J.S., Villeneuve, D.L., Kannan, K., Lee, K.T., Snyder,
S.A., Koh, C.H. and Giesy, J.P. (1999a). Alkylphenols,
polycyclic aromatic hydrocarbons (PAHs), and organochlorines in sediment from Lake Shihwa, Korea:
Instrumental and bioanalytical characterization. Environmental Toxicology and Chemistry 8: 2424 -2432.
Khim, J.S., Kannan, K., Villeneuve, D.L., Koh, C.H. and
Giesy JP. (1999b). Characterization and distribution of
trace organic contaminants in sediment from Masan Bay,
Korea: 1. Instrumental analysis. Environmental Science
and Technology 33: 4199-4205.
Khim, J.S., Lee, K.T., Kannan, K., Villeneuve, D.L., Giesy,
J.P. and Koh, C.H. (2001a). Trace organic contaminants
in sediment and water from Ulsan Bay and its vicinity,
Korea. Archives of Environmental Contamination and
Toxicology 40: 141-150.
Khim, J.S., Lee, K.T., Villeneuve, D.L., Kannan, K., Giesy,
J.P. and Koh, C.H. (2001b). In Vitro bioassay
determination of dioxin-like and estrogenic compounds
in environmental samples from Ulsan Bay and its
Vicinity, Korea. Archives of Environmental
Contamination and Toxicology 40: 151-160.
Kim, G.B., Maruya, K.A., Lee, R.F., Lee, J.H., Koh, C.H.
and Tanabe, S. (1999). Distribution and source of
polycyclic aromatic hydrocarbons in the vicinity of
Incheon Harbor, Korea. Marine Pollution Bulletin 38:
7-15.
360
Koh, C.H., Khim, J.S., Villeneuve, D.L., Kannan, K. and
Giesy, J.P. (2002). Analysis of trace organic
contaminants in sediment, pore water and water
samples from Onsan Bay, Korea: Instrumental analysis
and in vitro gene expression assay. Environmental
Toxicological and Chemistry (in press).
Lee, K.T., Tanabe, S. and Koh, C.H. (2001a). Contamination
of polychlorinated biphenyls (PCBs) in sediments from
Kyeonggi Bay and nearby areas, Korea. Marine
Pollution Bulletin 42: 273-279.
Lee, K.T., Tanabe, S. and Koh, C.H. (2001b). Distribution of
organochlorine pesticides in sediments from Kyeonggi
Bay and nearby areas, Korea. Environmental Pollution
114: 207-213.
Long, E.R., MacDonald, D.D., Smith, S.L. and Calder, F.D.
(1995). Incidence of adverse biological effects within
ranges of chemical concentrations in marine and
estuarine sediments. Environmental Management 19:
81-97.
MacDonald, D.D., Dipinto, L.M., Christopher, J.F., Ingersoll,
C.G., Long, E.R. and Swartz, R. (2000). Development
and evaluation of consensus-based sediment effect
concentrations for polychlorinated biphenyls. Environmental Toxicology and Chemistry 19: 1403-1413.
Sanderson, J.T., Aarts, J.M.M.J.G., Brouwer, A., Froese, K.L.,
Denison, M.S. and Giesy, J.P. (1996). Comparison of
Ah receptor-mediated luciferase and ethoxyresorufin-Odeethylase induction in H4IIE cells: implications for
their use as bioanalytical tools for the detection of
polyhalogenated aromatic hydrocarbons. Toxicological
and Applied Pharmacology 137: 316-325.
Swartz, R. (1999). Consensus sediment quality guidelines
for polycyclic aromatic hydrocarbon mixtures.
Environmental Toxicology and Chemistry 18: 780-787.
Villeneuve, D.L., Blankenship, A.L. and Giesy, J.P. (1998).
Interactions between environmental xenobiotics and
estrogen receptor-mediated responses. In: Denison
MS, Helferich WG, (eds), Toxicant-receptor interactions.
Taylor and Francis, Philadelphia, PA, USA, pp 69-99.
Villeneuve, D.L., Blankenship, A.L. and Giesy, J.P. (2000).
Derivation and application of relative potency estimates
based on in vitro bioassay results. Environmental
Toxicology and Chemistry 19: 2835-2843.
Villeneuve, D.L., Khim, J.S., Kannan, K. and Giesy, J.P.
(2002). Relative potencies of individual polycyclic
aromatic hydrocarbons to induce dioxin-like and
estrogenic responses in three different cell lines.
Environmental Toxicology 17: 128-137.