Environ. Sci. Technol. 2000, 34, 3568-3573
Vertical Profiles of Dioxin-like and
Estrogenic Activities Associated
with a Sediment Core from Tokyo
Bay, Japan
K U R U N T H A C H A L A M K A N N A N , * ,†
DANIEL L. VILLENEUVE,†
NOBUYOSHI YAMASHITA,‡
TAKASHI IMAGAWA,‡
SHINYA HASHIMOTO,§
AKIRA MIYAZAKI,‡ AND JOHN P. GIESY†
National Food Safety and Toxicology Center, Department of
Zoology, Institute for Environmental Toxicology, Michigan
State University, East Lansing, Michigan 48824, National
Institute for Resources and Environment, 16-3 Onogawa,
Tsukuba 305-8569, Japan, and Tokyo University of Fisheries,
4-5-7 Konan, Minato-ku, Tokyo 108, Japan
In vitro bioassays were used to measure dioxin-like and
estrogenic activities associated with florisil fractions
of extracts from a sediment core collected from Tokyo
Bay, Japan. Florisil fractions 2 (F2) and 3 (F3) elicited significant
dioxin-like responses in vitro. Dioxin-like activities of F2
samples were correlated with the vertical profile of PAH
concentrations (R 2 ) 0.85). Contribution of PAHs to Ah
receptor-mediated activities in sediments was greater than
those by PCDDs/DFs, PCBs, and PCNs. The dioxin-like
activity of F3 samples suggests the presence of relatively
polar, Ah receptor-active compounds in the Tokyo Bay
sediment core. Significant estrogenic activities, which may
be related to the presence of certain estrogenic PAHs,
were observed for F2 samples. Estrogen equivalents (E2EQs) calculated from the concentrations and relative potencies
of known estrogenic compounds in F2 were greater than
bioassay-derived E2-EQs. This suggests that complex
interactions between estrogenic and antiestrogenic
compounds (PAHs, PCDD/DFs, and PCNs) may have
modulated the activity. F3 samples were toxic to MVLN
cells; therefore, their estrogenic activities could not
be estimated.
Introduction
Environmental matrixes such as sediments contain complex
mixtures of residues of organic compounds of both natural
and anthropogenic origin. The concentrations and toxic
potencies of compounds present in such complex mixtures
can range over several orders of magnitude and can be
modulated by interactions (synergism, antagonism, etc.)
among chemicals. This complicates hazard evaluation for
complex mixtures of xenobiotics present in environmental
matrixes. Instrumental analytical techniques are available
to identify and quantify some compounds in complex
* Corresponding author phone: (517)432-6321; fax: (517)432-2310;
e-mail: kuruntha@pilot.msu.edu.
† Michigan State University.
‡ National Institute for Resources and Environment.
§ Tokyo University of Fisheries.
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 17, 2000
mixtures, but there are many compounds for which neither
methods nor standards are available. Furthermore, instrumental analyses provide little information on the biological
effects of complex mixtures and do not account for possible
interactions among individual chemicals. In vitro bioassays
are useful tools for characterizing complex mixtures of
contaminants, which act through a known mechanism of
action. In vitro bioassays provide a biologically relevant,
integrated measure of the combined potency of all compounds in a sample (1, 2). When combined with instrumental
analysis, sample fractionation techniques, and mass-balance
analysis, in vitro bioassays can be used to identify specific
compounds or classes of compounds associated with observed biological activity (3-6).
In this study, florisil fractions of extracts from a sediment
core collected from Tokyo Bay, Japan, were analyzed using
in vitro bioassays in order to evaluate vertical profiles of
dioxin-like and estrogenic activities. Two in vitro bioassays
were used. In vitro luciferase assay with recombinant rat
hepatoma cells (H4IIE-luc; 7) was used to screen for
compounds capable of modulating aryl hydrocarbon receptor
(AhR)-mediated gene expression. In vitro luciferase assay
with recombinant MCF-7 human breast carcinoma cells
(MVLN; 8) was used to screen extracts for compounds that
can modulate gene expression through an estrogen receptor
(ER)-mediated mechanism. Where possible, bioassay-derived
potencies, relative to a 2,3,7,8-tetrachlorodibenzo-p-dioxin
or 17-β-estradiol standard (TCDD-EQ or E2-EQ), were
compared to relative potency estimates calculated by multiplying the concentrations of known dioxin-like or estrogenic
compounds (reported elsewhere: 9) by assay-specific relative
potencies and summing the total (TEQ or EEQ). This type of
mass-balance analysis (3) was used to evaluate whether the
known composition of the extracts could account for the
magnitude of dioxin-like or estrogenic activity observed.
TCDD-EQ or E2-EQ estimates significantly greater than TEQ
or EEQ estimates would suggest either the presence of
unidentified dioxin-like or estrogenic compounds or the
synergistic interactions between components of the extract.
TCDD-EQ or E2-EQ estimates significantly less than TEQ or
EEQ estimates would suggest the presence of antagonists or
interfering compounds in the extracts. This type of massbalance analysis has been applied in the identification and
characterization of dioxin-like and estrogenic compounds
in both surficial sediment and surface waters (6, 10). This
study applied the same mass-balance principles to help
characterize the historical profile of dioxin-like and estrogenic
activities associated with a dated sediment core.
Materials and Methods
Samples and Fractionation. A sediment core was collected
in May 1995 from the northern part of Tokyo Bay (35′35′′ N
and 139′55′′ E) using an acrylic tube (120 cm long and 11 cm
i.d.). The core was sliced at 2-cm intervals for up to 20 cm
and then at 5-cm intervals for up to 93 cm using a clean
stainless steel slicer. Each section was freeze-dried and stored
in a refrigerator until analysis. A detailed description of the
sample collection, extraction, and fractionation procedure
has been provided elsewhere (9). Briefly, sediments were
Soxhlet extracted using dichloromethane (DCM) and hexane
(3:1, 400 mL). Extracts were treated with acid-activated copper
granules to remove sulfur. Concentrated extracts were passed
through 10 g of activated Florisil packed in a glass column
(10 mm i.d.) for fractionation. The first fraction (F1; nonpolar),
eluted with 100 mL of hexane, contained PCBs. PAHs and
certain organochlorine pesticides were eluted in the second
10.1021/es001044a CCC: $19.00
 2000 American Chemical Society
Published on Web 07/27/2000
FIGURE 1. Luciferase induction in H4IIE-luc (dioxin-responsive)
cell bioassay elicited by Tokyo Bay sediment core extract fractions
1 (F1), 2 (F2), and 3 (F3) and procedural blank. Response magnitude
presented as percentage of the maximum response observed for
a 3130 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin (% TCDD max).
Horizontal line equals 3 SD above the mean solvent control response
(set to 0% TCDD max).
fraction (F2; midpolar) with 100 mL of 20% DCM in hexane.
The APs such as nonylphenol (NP) and octylphenol (OP)
were eluted in the third fraction (F3; polar) with 100 mL of
50% DCM in methanol. PCNs were eluted in F1 and F2, while
PCDDs/DFs were eluted in all the three fractions, with a
predominant proportion in F2.
Cell Culture and Bioassay. H4IIE-luc cells are rat
hepatoma cells, which were stably transfected with a luciferase gene under control of dioxin-responsive elements
(DREs) (7). MVLN cells are human breast carcinoma cells
stably transfected with a luciferase reporter gene under
control of estrogen-responsive elements (EREs) of the
Xenopus vitellogenin A2 gene (8, 11). Culturing conditions
for both cell lines have been described previously (6). MVLN
and H4IIE-luc cells were cultured in 100-mm disposable Petri
plates and incubated at 37 °C in a humidified 95:5 air:CO2
atmosphere. Cells for bioassay were plated into the 60 interior
wells of 96-well culture plates (250 µL/well) at a density of
approximately 18 000 cells/well. Cells were incubated overnight prior to dosing. Test wells were dosed with 2.5 µL of
the appropriate florisil fraction. Samples were tested using
three replicate wells. Control wells received appropriate
solvents. Sample responses, expressed as mean relative
luminescence units (RLU), from three replicate wells were
converted to a percentage of the mean maximum response
observed for standard curves generated on the same day (%
E2 max and % TCDD max for 17-β-estradiol and TCDD
standards, respectively). 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). Dose-response relationships were examined for sediment core extracts from
selected depths to estimate potencies relative to 17-βestradiol (E2) and TCDD. Dose-responses consisted of six
concentrations prepared by 3-fold dilution from the final
extract. Details regarding the derivation of relative potency
estimates from bioassay results have been described elsewhere (1, 5, 6, 12, 13).
Results and Discussion
Dioxin-like Activity. None of the F1 extracts elicited a
significant increase in luciferase expression in H4IIE-luc cells
(Figure 1). On the basis of the detection limit for TCDD in
the H4IIE-luc bioassay, approximately 5.0 pg of TEQ/g dry
wt would be needed to produce a significant response. Thus,
these results suggest that the total concentration of TEQs in
F1 samples was less than 5.0 pg/g dry wt. Tri- and tetrachloronaphthalenes elute in F1, but they have not been shown
to elicit significant activity in H4IIE-luc cells (12). PCBs were
the only AhR agonists known to be present in F1. PCB
congeners that elicit dioxin-like activity, including non- and
mono-ortho-PCBs, were detected in the order of, on average,
CB118 (0.006-8.4 ng/g) > CB105 (0.004-3.8 ng/g) > CB77
(0.003-3.2) > CB156 <0.0005-0.68 ng/g) > CB126 (0.00010.05 ng/g) > CB169 (<0.0001-0.006 ng/g) (Table 1). Congener-specific PCB concentrations were multiplied by their
H4IIE-specific potencies relative to TCDD in order to estimate
the concentration of TEQs in F1 (14). The greatest concentration of TEQs contributed by PCBs was 1.2 pg/g, dry wt,
which was observed at a depth of 12-14 cm and corresponded
to the maximum total PCB concentration among the sediment core sections (Table 1; Figure 2). Despite being fifth in
order of abundance, pentachlorobiphenyl congener 126
(3,3′,4,4′,5-P5CB) accounted for 80-90% of the total TEQs
contributed by PCBs throughout the sediment core (Figure
3).
The vertical profile of TEQs contributed by PCBs (PCBTEQs) was similar to their concentration profile, which
increased from the 1940s, peaked in the 1980s, and then
gradually declined (Figure 2). PCB-TEQ concentrations in
surface sediments corresponding to the 1990s were 3-fold
less than the maximum value observed for sediment deposited in the 1980s. Interestingly, instrumental analysis also
suggested the presence of non-ortho-coplanar congener 77
(3,3′,4,4′-T4CB) at concentrations of 4 pg/g, dry wt, at 85-90
cm depth. Concentrations of other non-ortho-congeners CB
126 (3,3′,4,4′,5-P5CB) and CB 169 (3,3′,4,4′,5,5′-H6CB) were
0.1 and <0.1 pg/g, respectively, at 85-90 cm depth. These
results suggest the occurrence of certain coplanar PCB
congeners in sediment deposited in the early 1900s, prior to
widespread industrial production and use. Overall, the TEQ
concentration contributed by PCBs was less than that which
would be expected to elicit luciferase induction in H4IIE cells.
Thus, the lack of significant H4IIE-luc response to F1 supports
the results of instrumental analysis (9).
F2 samples for sediment from depths of 0-70 cm of the
sediment core exhibited significant dioxin-like activity (Figure
1). Magnitudes of induction as great as 90% TCDD max were
observed. F2 samples for sediment from depths greater than
70 cm were not active (Figure 2). Penta- through octachloronaphthalenes, PCDDs/DFs, and PAHs were the target analytes
present in F2 (9). Several PCN congeners that have been
shown to elicit luciferase activity in H4IIE-luc cells were found
in sediment core extracts (Figure 3; 12). TEQs contributed
by PCNs (PCN-TEQs) were generally less than those contributed by PCBs. However, at depths of 30-60 cm, PCNTEQs were similar to or greater than those of PCB-TEQs (Table
1). The greatest PCN-TEQ concentration, 0.44 pg/g, dry wt,
was observed at a depth of 12-14 cm, and the vertical profile
of PCN-TEQs resembled the concentration profile of PCNs
(Figure 2). TEQs contributed by PCNs in surface sediments
were 4-fold less than the greatest PCN-TEQ concentration,
observed at 12-14 cm depth (Table 1). PCN congeners 66/
67 (1,2,3,4,6,7-/1,2,3,5,6,7-H6CN) accounted for 51-86% of
the total PCN-TEQs (Figure 3). PCN-TEQ concentrations were
less than the 5.0 pg/g, dry wt, concentration required to yield
a significant response in the H4IIE-luc assay. This suggests
that PCNs alone were not responsible for the AhR-mediated
activity associated with F2 of sediment core extracts.
TEQ concentrations contributed by various 2,3,7,8substituted PCDD/DF congeners (PCDD/DF-TEQs) ranged
from 4.2 to 336 pg/g, dry wt (Table 1). These concentrations
were 2-3 orders of magnitude greater than those contributed
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TABLE 1. Concentration (pg/g, dry wt) Profiles of TEQs Contributed by PCDDs, PCDFs, PCNs, PCBs, and PAHs at Selected Sections
of a Sediment Core from Tokyo Bay, Japana
PCDDs
PCDFs
PCDD/DFs
PCNs
PCBs
PAHs
a
2-4
6-8
10-12
12-14
14-16
16-18
20-25
25-30
35-40
45-50
50-55
60-65
70-75
85-90
24.7
97.0
122
0.21
0.38
600
30.2
113
143
0.24
nab
950
45
149
194
0.27
0.42
1290
75.5
226
301
0.44
1.19
1800
75.1
261
336
0.31
0.62
1450
32.5
93.1
126
0.19
0.50
1070
12.3
56.1
68.4
0.13
0.22
1230
5.05
27.8
32.8
0.11
0.12
900
2.51
14.4
16.9
0.03
0.04
550
1.66
11.2
12.8
0.012
0.020
620
1.37
7.79
9.16
0.010
0.007
540
1.07
7.22
8.29
0.005
0.007
365
1.27
4.42
5.68
0.003
0.003
38
1.39
2.84
4.23
0.002
0.004
30
H4IIE relative potencies were used for PCDDs, PCDFs, PCBs, PAHs, and PCNs (12, 14, 16).
b
na, not analyzed.
FIGURE 2. Vertical profile of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) contributed by PCDD/DFs, PCBs, PCNs, and PAHs in
a sediment core collected from Tokyo Bay, Japan. All the TEFs were based on H4IIE rat hepatoma cell bioassays. PAH-TEFs were from
ref 16, whereas those of PCBs and PCDD/DF TEFs were from ref 14. PCN TEFs were from ref 12.
by PCBs and PCNs (Table 1) and were great enough to elicit
significant responses in the H4IIE-luc bioassay. PCDD/DFTEQ concentrations were correlated with bioassay response
magnitudes (% TCDD max), but the correlation was weak
(see Figure 5 in Supporting Information). Despite the fact
that PCDF concentrations were less than those of PCDDs,
PCDFs accounted for 70-87% of the total PCDD/DF-TEQs
throughout the core. The PCDF congener 1,2,3,4,6,7,8-H7CDF
accounted for 50-70% of the total PCDD/DF-TEQs at depths
of 0-50 cm. In deeper sections (50-90 cm), PCDF congeners
1,2,3,6,7,8-H6CDF, 1,2,3,7,8-P5CDF, and 2,3,7,8-TCDF accounted for greater than 10% of the total TEQs (Table 1;
Figure 3). Concentrations of PCDD/DF-TEQs in surface
sediments were 3-fold less than those observed at a depth
of 14-16 cm. The great contribution of 1,2,3,4,6,7,8-H7CDF
to PCDD/DF-TEQs could be due its great relative potency in
H4IIE bioassays as compared to the reported values of
consensus TEF (14, 15). Overall, the results suggest that
PCDDs/DFs may have contributed significantly to the dioxinlike activity observed for F2 samples.
Several PAHs, including benzo[k]fluoranthene (BkF),
benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), chrysene
(Chr), benz[a]anthracene (BaA), indeno[1,2,3,-cd]pyrene (IP),
dibenz[a,h]anthracene (DaA), and anthracene (Ant) have
been shown to elicit dioxin-like responses or induce cytochrome P4501A1 activity in vitro (16-18). H4IIE-specific
potencies, relative to TCDD, have been reported for a number
of the PAHs quantified in this study (16). These were used
to estimate TEQs contributed by PAHs (PAH-TEQs). Relative
potencies of Bkf, BaP, BbF, Chr, BaA, IP, and DaA were
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0.00478, 0.000354, 0.00253, 0.0002, 0.000025, 0.0011, and
0.00203, respectively (16). H4IIE-luc responses elicited by F2
extracts were significantly correlated with sediment PAH
concentrations, PAH-TEQs, and PAH profile (see Figure 6 in
Supporting Information). The correlation between PAH-TEQs
and H4IIE-luc response magnitudes (% TCDD max) was
greater than that for PCDD/DF-TEQs. Calculated PAH-TEQs
were 5-50 times greater than PCDD/DFs-TEQs (Table 1).
Concentrations of PAH-TEQs ranged from 11.5 pg/g at 9093 cm depth to 1800 pg/g at 12-14 cm depth (Figure 2). The
concentration of PAH-TEQs in surface sediments was 333
pg/g, dry wt, which was 5.4-fold less than the highest observed
at 12-14 cm depth. Averaged across all depths, BbF accounted for approximately 43% of the total PAH-TEQs,
followed by BkF and IP at 33% and 20%, respectively (Figure
3). These results suggest that, although PCDDs/DFs may have
significantly contributed to the responses elicited by F2
samples, AhR-active PAHs in F2 samples probably account
for the majority of the response observed.
Dose-response curves were obtained for selected F2
samples by analyzing them at six different dilutions (3-fold
serial dilutions). Bioassay-derived TCDD-EQs were then
estimated from the dose-response relationships (13). TCDDEQ concentrations, at selected depths, ranged from 27 to
992 pg/g, dry wt. TCDD-EQ estimates were 2-12-fold less
than those predicted from instrumental TEQs (Table 2).
Nevertheless, instrumental TEQs and bioassay-derived TCDDEQs were correlated. These results suggest that interactions
among less active or inactive compounds present in the
mixture may have modulated the activity. These results are
FIGURE 3. Contribution (%) of dioxin-like congeners to 2,3,7,8tetrachlorodibenzo-p-dioxin equivalents of PCDDs, PCDFs, PCBs,
PCNs, and PAHs in a sediment core at 12-14 cm depth from Tokyo
Bay, Japan. The depth of the 12-14-cm core was selected because
this section had the greatest concentration of most of the compounds
analyzed.
TABLE 2. Predicted and Estimated Dioxin-like Activities (pg/g,
dry wt) in Fraction 2 (F2) of Tokyo Bay Sediment Core
Extracts at Selected Depths
depth (cm)
F2 bioassay
TCDD-EQsa
F2 (instrumental
TEQs)b
0-2
8-10
12-14
20-25
35-40
50-55
65-70
34
475
992
74
52
100
27
1950
4330
8330
4860
2450
2070
750
a Bioassay-derived TCDD-EQs were calculated as EC - X
TCDD/EC Xsample where X is the maximum response magnitude observed for the
sample. Uncertainty in the TCDD-EQ estimates, due to deviation from
parallelism to the standard curve, was less than 3-fold (13). b Instrumental TEQ values for F2 represent those from PAHs. TEQs from PCDDs/
DFs were not included.
similar to that obtained for riverine sediments from the Czech
Republic (19) in which concentrations of TEQs were greater
concentrations of TCDD-EQs. Certain alkylated PAHs have
been shown to interact with AhR agonists and elicit antagonistic activity in H4IIE bioassays (20). The results of this study
are in accordance with the earlier studies reporting great
contribution of PAHs to dioxin-like activity in sediments (5,
19, 21).
Several F3 extracts also elicited significant luciferase
activity in H4IIE-luc cells (Figure 1), although the magnitude
of induction was generally less than that elicited by F2
samples. A small portion of PCDDs/DFs (<10%) eluted in F3
(<10% of the total concentration). Assuming 10% of the total
PCDD/DF-TEQs eluted in F3, PCDD/DF-TEQ concentrations
in F3 samples associated with the upper 18 cm of the core
would have ranged from 12 to 33 pg/g dry wt (Table 1). On
the basis of regression against the TCDD standard curve,
such concentrations of TEQs would be expected to yield
bioassay responses of approximately 25-45% TCDD max.
Observed responses were similar to or less than this predicted
magnitude of response (Figure 1). This suggests that PCDD/
DFs eluted in F3 may be able to account for at least a portion
FIGURE 4. Luciferase induction in MVLN (estrogen responsive)
cell bioassay elicited by Tokyo Bay sediment core extract fractions
1 (F1), 2 (F2), and 3 (F3) and procedural blank. Response magnitude
presented as percentage of the maximum response observed for
a 1000 pM 17-β-estradiol (% E2 max). Horizontal line equals 3 SD
above the mean solvent control response (set to 0% E2 max).
of the activity induced by F3 samples associated with the
upper 18 cm of the core. Assuming 10% of the total PCDD/
DF-TEQs eluted in F3, PCDD/DF-TEQ concentrations in F3
samples associated with core sections deeper than 18 cm
would have ranged from 0.4 to 6.8 pg/g dry wt (Table 1). On
the basis of regression against the TCDD standard curve, 6.8
pg/g TEQ would be expected to yield a response of approximately 17% TCDD max, while most F3 samples associated with sections deeper than 18 cm would not be
expected to yield a significant response. Responses for F3
samples associated with depths greater than 18 cm were
markedly higher than predicted (Figure 1). This suggests that
PCDD/DFs probably do not account for all the activity
associated with F3, particularly for greater depths in the
sediment core. Thus, although PCDD/DFs may contribute
to the activity observed, the results suggest the presence of
additional, unidentified, relatively polar AhR agonists in
Tokyo Bay sediments. This agrees with earlier studies, which
reported the presence of acid labile, polar AhR agonists in
surficial sediment from Korea (5, 6). A recent study has
suggested that the AhR may be capable of binding a wider
range of structures than previously suspected (22). Metabolic
products of marine biota, such as brevitoxin-6 (produced by
the dinoflagellate Ptychodiscus brevis), are one potential
source of polar AhR-active compounds in marine environment (22). Similarly, natural products, such as harmane and
topolone, derived from wood pulp, and tryptophan derivatives have also been shown to induce cytochrome P4501A1
(22). Thus, the cause of the dioxin-like activity associated
with F3 samples remains a topic for further investigation.
Estrogenic Activity. Florisil fractions F1, F2, and F3 from
each of the 25 sediment extracts were screened for their ability
to promote ERE-mediated gene expression in MVLN cells
(Figure 4). Only one of the 25 F1 samples, the sample
associated with the 35-40-cm section, elicited a significant
estrogenic response in the MVLN bioassay (Figure 4). The
magnitude of response was less than 10% E2 max. On the
basis of regression against the E2 standard curve, this
response would be associated with approximately 2.0 pg E2EQ/g, dry wt, while most F1 samples would have contributed
less than 1.6 pg E2-EQ/g, dry wt, to the total E2-EQ
concentration of the associated core section. The lack of
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estrogenic response for most F1 samples was expected and
consistent with the polarity of known estrogen agonists.
F2 samples were significantly more estrogenic than F1
samples. Estrogenic activity was observed in sediment as
deep as 85-90 cm (Figure 4). Response magnitudes as great
as 22% E2 max were observed. On the basis of regression
against the standard E2 curve, approximately 25 pg of E2EQ/g, dry wt, would be required to yield a response of 20%
E2 max. If present, organochlorine pesticides such as
toxaphene, chlordecone, endosulfan, and p,p′-DDT would
elute in F2. Such pesticides have been shown to elicit weak
estrogenic responses in vitro (23). However, such responses
have been shown to occur at concentrations greater than 1
µg/g. Concentrations of organochlorine pesticides in Tokyo
Bay sediments were not expected to be great enough to
contribute to the estrogenic activity observed for F2 samples.
A recent study reported DDT concentrations of <2 ng/g, dry
wt, in Tokyo Bay sediment (24). Similarly, concentrations of
organochlorine pesticides in sediment from Masan Bay,
Korea, were insufficient to account for estrogenic activity
associated with F2 samples (5). Thus, organochlorine pesticides do not appear to be a likely cause of the estrogenic
activity observed.
MVLN responses in this study were significantly correlated
with the concentration of PAHs detected in the sediment
core (see Figure 7 in Supporting Information). Some PAHs
have been shown to elicit estrogenic responses in vitro (17).
Estrogenic potencies of chrysene, benz[a]anthracene, and
benzo[a]pyrene, relative to E2, have been reported to be
0.0005, 0.0001, and 0.001 (17). On the basis of these relative
potency values, EEQs contributed by PAHs were in the range
of 0.7-50 pg/g, dry wt, at depths of 25-93 cm and 20-160
pg/g, dry wt, at depths of 0-25 cm. Predicted response
magnitudes, based on PAH-EEQ estimates, were consistently
greater than the MVLN response magnitudes observed.
Furthermore, PAH-EEQs (up to 160 pg/g) were greater than
bioassay-derived E2-EQ estimates for F2 samples (up to 25
pg/g). These results suggest that interactions with antiestrogens or other interfering compounds may have modulated the activity of estrogenic PAHs. Dioxin-like compounds
such as PCDD/DFs, which have been shown to be antiestrogenic both in vitro and in vivo, were known to be present
in F2 samples (25, 26). Thus, interactions between compounds in the F2 samples could account for the discrepancies.
Although the cell bioassay used by Clemons et al. (17)
was analogous to the MVLN bioassay, there may be cell-line
specific differences in the relative estrogenic potencies of
PAHs. Overall, the results suggest that estrogenic PAHs
account for at least a portion of the estrogenic activity of F2.
This agrees with earlier reports of PAHs contributing to
estrogenic activity in sediments (5, 20).
F3 samples were toxic to MVLN cells, therefore their
estrogenic potency could not be determined. MVLN cells are
relatively sensitive to the presence of toxic components in
extracts (6,20). Nonlyphenol (NP) and octylphenol (OP) were
the target analytes present in F3. NP and OP are weak
estrogenic compounds and their MVLN-specific potencies,
relative to E2, have been reported to be 0.0000125 and
0.000019, respectively (27). On the basis of the measured
concentrations of NP, E2-EQs contributed by NP were
estimated to be 50-70 pg/g, dry wt, at the top 10 cm. Such
concentrations of E2-EQs would be expected to elicit a
significant response in the MVLN bioassay. However, any
estrogenic activity attributable to NP and OP were obscured
by the toxic effects of the F3 samples.
Overall, these results suggest the utility of in vitro bioassays
in assessing dioxin-like and estrogenic potential of sediments.
Mass balance calculations suggest that PAHs account for a
considerable portion of both the dioxin-like and estrogenic
activity of the Tokyo Bay sediment core (Table 3). Bioassay3572
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 17, 2000
TABLE 3. Ranges of Concentrations (ng/g, dry wt), TEQs (pg/g,
dry wt), and E2-EQs (pg/g, dry wt) of Target Analytes in a
Sediment Core from Tokyo Baya
PCDDs
PCDFs
PCNs
PCBs
PAHs
nonylphenol
concn
TEQs
0.39-29.4
0.034-5.53
0.2-4.43
1.1-150
38-2010
<10-5540
1.0-76
2.8-260
0.002-0.44
0.003-1.2
66-8330
E2-EQs
0.8-160
0.7-70
a Relative potencies were used for PCDDs, PCDFs, PCBs, PAHs, and
PCNs (12, 14, 16, 17).
derived TCDD-EQs were significantly correlated with instrumental TEQs, although instrumental values were 2-12fold greater. These results suggested the existence of
antagonistic interactions among various compounds present
in F2. Similarly, potential antagonistic interactions between
estrogenic PAHs and antiestrogenic compounds such as
TCDD, PCNs, and hydroxylated PCBs in F2 were suggested
by the discrepancy between instrumental PAH-EEQs and
bioassay responses. Next to PAHs, PCDDs/DFs were the
greatest contributors to TEQs in the Tokyo Bay sediment
core. While PAHs can be toxic to benthic organisms, they
would not be expected to biomagnify like PCDD/DFs. On
this basis, PCDD/DFs are compounds of concern in the
marine food chain in Tokyo Bay. The mass-balance profile
of dioxin-like activity in F3 suggests the presence of unidentified, relatively polar compound that can act through
AhR-mediated mechanism. This adds to a growing body of
evidence for the presence of unidentified dioxin-like compounds in sediments. Although the mass-balance analyses
used in this study have limitation, as discussed earlier (5),
risk assessment based solely on instrumental analyses may
not accurately reflect actual hazards. Although in vitro
bioassays cannot be directly extrapolated to determine the
risk for adverse effects, they point out additional sources of
uncertainty, which should be considered.
Acknowledgments
This work was supported by grants from the Chlorine
Chemistry Council of the Chemical Manufacturers Association (United States) and National Institute for Resources and
Environment (NIRE, Japan).
Supporting Information Available
Figures showing relationships between PCDD/DF-TEQs
measured from instrumental analysis and H4IIE-luc bioassay
responses (Figure 5), PAH-TEQs measured from instrumental
analysis and H4IIE-luc bioassay responses (Figure 6), and
PAH concentrations and MVLN bioassay responses (Figure
7) (4 pages). This material is available free of charge via the
Internet at http://pubs.acs.org.
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Received for review February 25, 2000. Revised manuscript
received June 16, 2000. Accepted June 21, 2000.
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