Doi:10.1016/j.aquatox.2003.12.005
Aquatic Toxicology 67 (2004) 167–179
Effects of triclosan on the early life stages and reproduction of
medaka
Oryzias latipes and induction of hepatic vitellogenin
Hiroshi Ishibashi , Naomi Matsumura , Masashi Hirano , Munekazu Matsuoka ,
Hideki Shiratsuchi , Yasuhiro Ishibashi , Yuji Takao , Koji Arizono
a
Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan
b
Environmental Protection Center, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan
c
Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan
Received 11 September 2003; received in revised form 5 December 2003; accepted 8 December 2003
Triclosan (2,4,4-trichloro-2-hydroxydiphenyl ether) is widely used as antibacterial agent in various industrial products, such
as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. In this study, theeffects of TCS on the early life stages and reproduction of medaka (
Oryzias latipes) were investigated. The 96-h median lethalconcentration value of TCS for 24-h-old larvae was 602 g/l. The hatchability and time to hatching in fertilized eggs exposed to313 g/l TCS for 14 days were significantly decreased and delayed, respectively. An assessment of the effects of a TCS 21-dayexposure period on the reproduction of paired medaka showed no significant differences in the number of eggs produced andfertility among the control and 20, 100 and 200 g/l TCS treatment groups. However, concentrations of hepatic vitellogenin wereincreased significantly in males treated with TCS at 20 and 100 g/l. In the F1 generations, although the hatching of embryos inthe 20 g/l treatment showed adverse effects, there was no dose–response relationship between hatchability and TCS treatmentlevels. These results suggest that TCS has high toxicity on the early life stages of medaka, and that the metabolite of TCS may bea weak estrogenic compound with the potential to induce vitellogenin in male medaka but with no adverse effect on reproductivesuccess and offspring.
2003 Elsevier B.V. All rights reserved.
Keywords: Triclosan;
Oryzias latipes; Early life stage; Reproduction; Vitellogenin
ment and functioning of various systems in animalsand humans
Recently, a number of studies have been performed
Altered hormone status or
worldwide that examine endocrine-disrupting chem-
gonad histology has been reported in fish inhabiting
icals (EDCs) and their interactions with the develop-
water released from wastewater treatment plants andthe induction of plasma vitellogenin (VTG, egg yolk
protein precursor) has been recorded in male rain-
Corresponding author. Tel.: +81-96-383-2929x486;
bow trout (
Oncorhynchus mykiss) caged in a polluted
E-mail address: [email protected] (K. Arizono).
0166-445X/$ – see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2003.12.005
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
These alterations in the physiology of fish may be
estrogens (e.g. diethylstilbestrol and bisphenol A).
linked to declines in fish populations; therefore, vari-
Recent studies have demonstrated changes in fin
ous screening and testing systems for EDCs have been
length and non-significant trends in the sex ratio of
medaka exposed to TCS, and suggested that it was
a weakly androgenic contaminant
Some laboratory assays report a
reduction in fecundity and/or fertility of fish exposed
7-benzyloxyresorufin
O-debenzylase (BROD) and
to estrogenic compounds, such as 17-estradiol (
7-pentoxyresorufin
O-depentylase (PROD) activities,
), 4-nonylphenol (),
which are associated with CYP2B1 activity, were re-
and bisphenol A (Many of these
markably induced by all doses of TCS in rats. These
chemicals have been detected in aquatic environ-
results suggested that TCS induces the P450 isoforms
ments (and can adversely affect
of the CYP2B subfamily in the rat liver, and that the
the reproductive health of freshwater and marine fish
induced P450 isozymes were closely related to the
populations. Therefore, there is a need to evaluate the
toxicity of TCS or its chlorinated derivatives. How-
reproductive biology of fishes exposed to EDCs.
ever, the potential for TCS to act as an EDC has not
been studied in depth nor has it potential to disrupt
ether) is widely used as an antibacterial agent in liq-
reproductive function in fish.
uid toothpaste, soap, shampoo, and cosmetics (
Medaka (
Oryzias latipes) is widely used in ecotox-
and is frequency found in
icology and has been proposed to be a suitable fish
wastewater effluent. Water samples collected near the
for evaluating EDCs. Medaka is also a suitable test or-
outfall of a wastewater treatment plant in Rhode Is-
ganism for assessing reproductive effects because its
land, USA, showed 10–20 g/l of TCS in the effluent
large eggs with clear chorions can easily be observed.
and 80–100 g/g of TCS in the sediment (
Early life stage toxicity tests using medaka are also
TCS and its chlorinated deriva-
considered to be sensitive biosensors. In this study, we
tives are readily converted into various chlorinated
used medaka as a test organism, and investigated the
dibenzo-
p-dioxins by heat and ultraviolet irradiation
effect of TCS on the early life stage and reproduction
of medaka. Furthermore, we evaluated the induction
ve also detected methylated TCS in fishes
of hepatic VTG in male medaka, 7-ethoxyresorufin
and shellfishes from Tokyo Bay and the Tama River
O-deethylase (EROD) and PROD activities in female
of Japan. The U.S. Geological Survey used five newly
liver microsomes exposed to TCS, and also measured
developed analytical methods to measure the concen-
the estrogenic activity of TCS using a yeast two-hybrid
trations of 95 chemical compounds (such as phar-
assay in vitro.
maceuticals, hormones, and other organic wastewatercontaminants) in water samples from a network of139 streams across 30 states during 1999 and 2000
2. Materials and methods
to provide the first nationwide reconnaissance of theoccurrence of organic wastewater contaminants in
2.1. Test chemical
water resources and reported that TCS was one of themost frequently detected compounds (maximum con-
TCS (>98.0% purity) was obtained from Wako Pure
taminant levels; 2.3 g/l)
Chemical Industries Ltd., Tokyo, Japan and dissolved
studied the aquatic toxicity of TCS in
in dimethyl sulfoxide (DMSO, Wako Pure Chemical
activated sludge microorganisms, algae, invertebrates,
Industries) to prepare test solutions.
and fish and reported a median effective concen-tration for
Daphnia magna of 390 g/l at 48 h and
2.2. Test organisms
median lethal concentrations for
Pimephales prome-las and
Lepomis macrochirus of 260 and 370 g/l,
Sexually mature medaka were purchased from
respectively, at 96 h. Additionally, the chemical struc-
a local fish farm in Kumamoto, Japan, and were
ture of TCS closely resembles known non-steroidal
maintained in glass tanks in our laboratory. In the
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
pre-exposure period, 40 mating pairs were selected
riod cycle, and were not fed during the toxicity tests.
and each pair was placed in a 1-l glass beaker un-
Each group of 24-h-old larvae were exposed to nom-
der a 16:8 light:dark photoperiod at 25 ± 1 ◦C. The
inal TCS concentrations of 78, 156, 313, 625, 1250
fish were fed a diet of
Artemia nauplii (
<24 h after
and 2500 g/l diluted in dechlorinated tap water at
hatching) once daily and a commercial diet (Kyorin,
25 ± 1 ◦C for 96 h. The controls in the 24-h-old lar-
Himeji, Japan) three times daily for 21 days. During
vae testing were conducted in dechlorinated tap water,
the last 10 days of the acclimatization period, the
and the larvae in the solvent control were exposed to
spawned eggs from the females were counted, and
solvent carrier only (0.1% DMSO). The test solutions
the fertility of the females was assessed a few hours
in the tanks were not changed during the experimental
after oviposition. Based on the number and viability
period. The larvae were observed daily under a stereo-
of the spawned eggs, we chose 25 acclimatized pairs
scopic microscope, and dead larvae, characterized by
of medaka to use for the exposure test. The selected
no heart activity were removed daily. The 96-h LC50
pairs were allocated into treatment groups using a
values for the 24-h-old larvae were calculated by pro-
stratified sampling method. Eggs were generated in
bit analysis.
this pre-trial also used in the study as well.
2.4. Exposure conditions for mature medaka of
2.3. Exposure conditions for embryos and larvae
Embryos less than 24 h post-fertilization and
Five male/female pairs of adult mature medaka
24-h-old larvae were used in exposure experiments.
(approximate body weight 300 mg; approximate total
Eggs spawned from each female fish were carefully
length 30 mm) were exposed to 1 l of TCS at nomi-
collected within a few hours after fertilization, washed
nal concentrations of 20, 100 and 200 g/l in glass
in a petri dish containing about 30 ml of 10% artifi-
beakers for 21 days at 25 ± 1 ◦C. The control group
cial seawater (Senju Pharmaceutical Co. Ltd., Osaka,
was only exposed to dechlorinated tap water, and the
Japan), checked for fertilization and developmental
solvent control group was exposed only to solvent car-
stage under a stereoscopic microscope, and then sub-
rier (DMSO 0.1 ml/l). Test solutions were exchanged
jected to chemical exposure. The 60 embryos used
every 24 h, and the fish were subjected to a 16:8
in each treatment were separated into two groups of
light:dark photoperiod. The fish were fed the same
30 eggs each to provide duplication. Each group of
diet as during the pre-exposure period. Eggs spawned
embryos was exposed to nominal TCS concentrations
from each female fish were carefully collected within
of 78, 156, 313, 625, 1250 and 2500 g/l diluted in
a few hours after fertilization, pooled in a petri dish
dechlorinated tap water for 14 days. The embryos in
containing about 5 ml of 10% artificial seawater, and
the control treatment were exposed to dechlorinated
checked for fertilization and developmental stage
tap water, and those in the solvent control group were
under a stereoscopic microscope. During the 21-day
exposed to solvent carrier only (0.1% DMSO). The
testing period, the number of eggs spawned in each
eggs in each group were placed in a petri dish con-
treatment group was counted daily, and the ratio
taining 30 ml of each test solution and incubated on a
of fertilized eggs was calculated. Each embryo was
16:8-h light:dark photoperiod cycle at 25 ± 1 ◦C. The
maintained in dechlorinated tap water until hatching
test solution in the wells was changed every 24 h. The
(see above), and the hatchability and time to hatching
development of the embryos was observed daily under
was calculated. During the last 24 h of TCS exposure,
a stereoscopic microscope, and dead embryos were
all the fertilized eggs from each pair in each treat-
removed during the observation. The median lethal
ment group were collected, the hatched larvae were
concentration (LC50) values for the embryos were
maintained for 90 days in dechlorinated tap water
calculated by probit analysis. Hatchability and time to
alone and observed for cumulative mortality, total
hatching was calculated using data from all embryos.
length, body weight, and sex ratio with the appear-
Fifteen 24-h-old larvae from each treatment were
ance of secondary sex characteristics. At the end of
placed in glass beakers containing 100 ml of each test
the F0 generation exposure, livers and gonads were
solution at 25 ± 1 ◦C with 16:8 light:dark photope-
sampled, and the body weight and the total length of
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
each fish were measured. The gonadosomatic (GSI,
2.6. Measurement of hepatic vitellogenin
%) and hepatosomatic indices (HSI, %) were alsocalculated as a ratio of gonad or liver weight to body
Hepatic VTG levels in male medaka were mea-
sured using enzyme linked immunosorbent assay ina VTG assay kit specifically for medaka (TransGenic
2.5. Determination of TCS concentrations in test
Inc., Kumamoto, Japan). The measurement of hepatic
VTG was performed according to the manufacturesprocedure. Purified medaka VTG (1, 4, 26, 64 and
The TCS concentrations in the test water of each
256 ng/ml) was used as a standard, and VTG in di-
treatment groups were measured once a week dur-
luted samples was measured in duplicate. The assays
ing the exposure period. One liter of the water was
were performed at room temperature. Concentrations
taken from each of the five beakers of each treatment
of VTG in hepatic samples were calculated from the
group. These samples were added 1 ml of 1 M HCl and
linear part of the log-transformed medaka VTG stan-
1 g of nonylphenol-d4 (Kanto Chemical Co., INC.,
dard curve. The detection limit of VTG in the present
Tokyo, Japan) as an internal standard, respectively.
study was 1 ng/ml.
After the mixing, the water was extracted with 10 mlof dichloromethane shaking for 30 min and this op-
2.7. Measurement of hepatic EROD and PROD
eration was repeated. The collected dichloromethane
solution was mixed with anhydrous sodium sulfate(Kanto Chemical Co. Inc., Tokyo, Japan) for dehydra-
EROD and PROD activities in liver microsomes
tion. The solution was concentrated to approximately
from female medaka were measured by dealkylation
0.5 ml by high-purity nitrogen gas flowing, and
of ethoxyresorufin and pentoxyresorufin, respec-
200 l of
N,O-bis(trimethylsilyl)trifluoroacetamide
tively, and detection of the resulting resorufin by
(BSTFA; SUPELCO Inc., Bellefonte, PA, USA) was
high-performance liquid chromatography (HPLC)
added to the solution for the silylation of TCS and
with fluorescent detection as described by
nonylphenol-d4. The resultant sample was sealed and
were produced by preincubat-
left to stand at room temperature for 1 h to allow the
ing 500 nM substrates ethoxyresorufin or pentoxyre-
sorufin in 0.05% v/v methanol, liver microsomal
An ion-trap mass detector and gas chromatogram
proteins and 0.5 mM NADPH in a final volume of
(GC3800/Saturn200, Varian Inc., USA) with a capil-
400 l of 50 mM phosphate buffer (pH 7.7) at 22 ◦C
lary column of cross-linked 5%-phenylmethylsiloxane
for 10 min. The reaction was started by the addition of
(DB-5MS, J&W Scientific Inc., USA) was used for
100 mM glucose-6-phosphate, incubated at 22 ◦C for
TCS analysis. Separation was performed in splitless
20 min, and stopped by placing the samples in hot wa-
mode under the following conditions: injector pres-
ter for 5 min (90 ◦C). After cooling on ice for 5 min,
sure 10.0 psi, split ratio = 30, split vent opening
the samples were centrifuged at 1800 ×
g for 10 min.
time 1 min after injection, injection port tempera-
The supernatant was filtered with a PTFE membrane
ture 300 ◦C. Then oven temperature program was as
filter of 0.45 m pore size (Millipore, Bedford, MA,
follows: initial temperature 35 ◦C, hold for 1 min, in-
USA) and immediately analyzed by HPLC. Resorufin
crease at 10 ◦C/min to 250 ◦C, hold for 7 min. Mass
standards at concentrations of 12.5, 25, 50, 100 and
spectra were measured in full scan mode.
200 nM for were prepared from stock standard solu-
The total peak area of the
m/z = 345–348 peaks
tions. Blank samples contained all components expect
was used for quantification of trimethylsilylated TCS
for the NADPH, which was added after termination
(TCS-TMS), and the 183 peak was used for quantifi-
of the reaction.
cation of nonylphenol-d4-TMS. The amount of TCSin the water was calculated by the ratio of these 2 val-
2.8. Yeast two-hybrid assay
ues. The detectable limit in this work was estimatedto be about 0.01 g/l based on a linear calibration line
The estrogenic activity of TCS was measured by
and blank tests.
a yeast two-hybrid assay as described by
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
with and without possible metabolic
using Stat View J 5.0 (SAS Institute Inc., Cary, NC,
activation by rat liver S9 preparations (Kikkoman
Company, Noda, Japan). Briefly, yeast cells (
Sac-charomyces cervisiae Y190) for the assay were pre-pared by incorporating the human estrogen receptor
3. Results
␣, expression plasmid of the coactivator TIF2 and
-galactosidase expression reporter
3.1. Effect on the early life stages of medaka
Aliquots of TCS (10 mM in DMSO, 20 l)with or without a 1-h incubation with rat liver S9
The acute toxicity tests were carried out using
mix at 37 ◦C were incubated (30 ◦C, 4 h) with yeast
24-h-old larvae medaka and embryos less than 24-h
cells in a 96-wells microplate (SUMILON, Sumit-
post-fertilization. As calculated by probit analysis,
omo Bakelite, Tokyo, Japan). A solution was added
the 96-h LC50 values of TCS were 602 g/l for
to induce chemiluminesence and enzymatic digestion
24-h-old larvae and 399 g/l for embryos. Embry-
(Zymolyase 20T), followed by a light emission ac-
onic development, hatchability, and time to hatching
celerator solution. The chemiluminesence produced
of medaka eggs were affected by TCS treatment
by the released -galactosidase was measured on
(Hatchability of fertilized eggs exposed to
a 96-well plate luminometer (Luminescencer-JNR
TCS for 14 days was significantly decreased relative
AB2100, ATTO Bio-Instrument, Tokyo, Japan). The
to the controls (
P < 0
.05) in the treatment groups
estrogenic activity was calculated based on the chemi-
with concentrations over 313 g/l. Time to hatching
luminescent signal intensity.
was also significantly delayed relative to the controls(
P < 0
.05) in the treatment groups with concentra-
2.9. Statistical analysis
tions over 313 g/l. All embryos died within 3 dayspost-fertilization in the 1250 and 2500 g/l TCS treat-
The data from the control and solvent control
ments, and died prior to 10 days post-fertilization in
groups by a chi-square test, a Student's
t-test, or a
the 625 g/l treatment (data not shown).
Bonferroni's
U-test to determine whether differencesexisted before data analysis. When no differenceswere found, these groups were pooled for subsequent
analysis. If differences were found, the control group
Time to hatching (days)
without the solvent control was excluded from sub-
sequent analyses. The experimental data, except for
the HSI values of female medaka and sex ratios of
offspring, were checked for assumptions of homo-
geneity of variance across treatments using a Bartlett
test. When the assumptions were met, data were an-
alyzed by one-way analysis of variance followed by
Dunnett's multiple comparison tests
Time to hatching (days)
When no homogeneity was observed in the data,
non-parametric a Kruskal–Wallis test was used, fol-
lowed by a Mann–Whitney
U-test with Bonferroni's
156 313 625 1250 2500
adjustment The data on sex ratios,
Nominal TCS concentration (µg/l)
which were determined by the secondary sex charac-teristics, were tested by chi-square analysis for dif-
Fig. 1. Effect on hatchability (solid bar) and time to hatching
ferences between treatment and control groups. The
(open symbol) in fertilized eggs of TCS 14-day exposure. Values
data on HSI values of female medaka were assessed
are shown as the mean hatchability and time to hatching of theembryos, respectively. Error bars represent the standard deviation
by the Mann–Whitney
U-test with Bonferroni's ad-
of the mean. The asterisk symbol (*) denotes significant differences
justment. Differences were considered significant at
were compared to the control group embryos (
P < 0
.05). Cont.:
P < 0
.05. All statistical analyses were performed
control; S.C.: solvent control.
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
Table 1Actual concentrations of TCS in water in the experiments to determine persistence in aquaria (0–24 h)
Nominal concentration (g/l)
Mean measured concentration (g/l)
a Average of 0 and 24 h values.
b Data represent as the mean ± S.D.
c nd: not determined at 0.01 g/l.
3.2. Effects on the reproduction of medaka
the controls (
P < 0
.05) when exposed to 200 g/lTCS. However, there were no significant differences
3.2.1. TCS concentrations in the test water
in total length among the five groups of males and
The means and standard deviation of measured TCS
body weight among females or males in the five TCS
concentrations in the test water at 0 h were 17
.3 ± 3
.3,
treatment groups after exposure.
75
.2 ± 18
.5, and 162
.1 ± 18
.2 g/l, and those at 24 hwere 8
.3 ± 1
.4, 46
.3 ± 18
.5, and 111
.7 ± 5
.1 g/l as
3.2.3. Fecundity and fertility
shown in TCS concentration in the con-
No significant differences in the total number of
trol and solvent control treatment was less than the
eggs collected during the TCS-exposure period were
determination limit in all analyses. The actual expo-
observed among the control, solvent control and all
sure concentrations of the test water were determined
TCS treatment groups (Fertility in all TCS
by the averages of 0 h and 24 h values.
treatment groups, the control and solvent control ex-ceeded 90%, and no significant differences were ob-
3.2.2. Total length and body weight
In this study, we investigated the effects of TCS on
the reproduction of medaka. The mean total lengths
3.2.4. HSI and GSI
and body weights of the five adult mature male/female
Levels of HSI and GSI in adult male and female
pairs of medaka following exposure to nominal TCS
medaka exposed to TCS concentrations of 20, 100 and
concentrations of 20, 100 and 200 g/l for 21 days
200 g/l for 21 days are showed in The HSI
are shown in The total length of the fe-
of male medaka exposed to 200 g/l TCS was sig-
male medaka was significantly decreased relative to
nificantly higher than that of the control group fish
Table 2Total length and body weight of medaka at the end of TCS exposure for 21 days
a Nominal concentrations of TCS during the exposure period.
b Data represent the mean ± S.D. (
n = 5).
c Significantly different when compared to the control groups (
P < 0
.05).
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
3.2.6. EROD and PROD activities in liver
microsomes in female medaka
No significant differences were observed in PROD
or EROD activity in the liver microsome from female
medaka at the end of TCS exposure for 21 days in the
control, solvent control and all TCS treatment groups(
3.3. Hatchability, time to hatching and sex ratio of
Total number of eggs / mating pair
1 generations, although embryonic hatch-
ing in those exposed to 20 g/l showed adverse ef-
fects (P < 0.05), there was no linear dose–responserelationship between hatching and TCS treatments
(TCS did not affect cumulative mortality
of offspring maintained in clean water for 90 daysafter hatching, and body weight and total length did
Mean fertility (%)
not differ significantly among treatment and control
groups (data not shown). The sex ratio of males tofemales was approximately 1:1 for all groups; no
significant differences were observed between the
control and treatment groups.
Nominal TCS concentration (µg/l)
Fig. 2. Weekly changes (first, second, and third weeks) of fecundity
3.4. Estrogenic activity in vitro assay
(A) and fertility (B) of eggs from paired mature medaka during21-day TCS exposure period (n = 5 per mating pair). Error bars
The estrogenic activity of TCS was assessed using
represent the standard deviation of the mean. Cont.: control; S.C.:
a yeast two-hybrid assay in vitro, both with and with-
solvent control.
out possible metabolic activation by the rat liver S9preparation (Estrogenic activity of TCS was
(P < 0.05). The HSI of female medaka exposed to
analyzed with metabolic activation by rat S9 liver as
20 g/l TCS was significantly higher than that of the
TCS showed only weak estrogenic activity in the ab-
control fish (P < 0.05). The GSI of male medakaexposed to 100 and 200 g/l TCS was significantly
higher than that of the control fish (P < 0.01 and
Hatchability, time to hatching, and sex ratio in the F
0.05, respectively). The GSI of female medaka ex-
of fertilized eggs from paired F0 generation medaka exposed to
posed to 20 and 200 g/l TCS was significantly higher
than that of the control fish (P < 0.05 and <0.01,
3.2.5. Hepatic vitellogenin concentrations in male
Levels of hepatic VTG were significantly higher for
male medaka exposed to TCS at 20 and 100 g/l for
21 days compared to the control group (P < 0.05)
a Nominal concentration of TCS during the exposure period.
(The higher hepatic VTG concentrations of
b Data represent the mean ± S.D.
the 200 g/l treatment groups compared to the control
c Significantly different when compared to the control groups
group were not significantly different.
(P < 0.05).
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
Fig. 3. Hepatosomatic (HSI) and gonadosomatic (GSI) indices of male (A and C) and female (B and D) medaka at the end of 21 days ofTCS exposure. Values are shown as the mean HSI and GSI of male and female fish, respectively (n = 5). Error bars represent the standarddeviation of the mean. The asterisk symbols (* and **) denote significantly different when compared to the control group at P < 0.05and P < 0.01, respectively. Cont.: control; S.C.: solvent control.
sence of rat S9 liver treatment. The estrogenic activity
of the effects of a TCS 21-day exposure period on the
of TCS was enhanced about two-fold by exposure to
reproduction of paired medaka showed no significant
S9 metabolic activation.
differences in the number of eggs produced and amongthe control and 20, 100 and 200 g/l TCS treatmentgroups. However, concentrations of hepatic VTG were
increased significantly in males treated with TCS at20 and 100 g/l. In the F1 generations, although the
In this study, the effects of TCS on the early life
hatching of embryos in the 20 g/l treatment showed
stages and reproduction of medaka (O. latipes) were
adverse effects, there was no dose–response relation-
investigated. The 96-h median lethal concentration
ship between hatchability and TCS treatment levels.
value of TCS for 24-h-old larvae was 602 g/l. The
These results suggest that TCS has high toxicity on
hatchability and time to hatching in fertilized eggs ex-
the early life stages of medaka, and that the metabo-
posed to 313 g/l TCS for 14 days were significantly
lite of TCS may be a weak estrogenic compound
decreased and delayed, respectively. An assessment
with the potential to induce VTG in male medaka but
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
Hepatic VTG levels (ng/mg liver) 100
Chemiluminescent intensity (CLN)
Nominal TCS concentration (µg/l)
Nominal TCS concentration (µg/l)
Fig. 4. Hepatic vitellogenin (VTG) concentrations of male medaka
Fig. 6. Dose–response curves for TCS with (䊉) and without (䊊)
after exposure to TCS for 21 days. Values are shown as the mean
metabolic activation by rat liver S9 preparation using the agonist
VTG concentration of male fish (n = 5). Error bars represent the
test for the yeast two-hybrid assay for estrogen receptor. The
standard deviation of the mean. The asterisk symbol (*) denotes
values were represented as the chemiluminescence intensity of
significantly different when compared to the control group fish
-galactosidase. Symbols represent the mean (n = 4) and bars
(P < 0.05). Cont.: control; S.C.: solvent control.
represent the standard deviation.
with no adverse effect on reproductive success andoffspring.
The acute toxicity (96-h LC50 values) of TSC for
medaka in early life stages, 24-h-old medaka larvae
and embryos less than 24 h post-fertilization were 602and 399 g/l, respectively. These toxicity profiles for
medaka larvae were similar to those found in a pre-
EROD activity (pmol/mg/min)
vious paper (However, the toxicsensitivity of embryos to TCS was higher than for
24-h-old larvae. reported on the
factors affecting the sensitivity of embryonic-larvalmedaka to diazinon, reported that 96-h LC50 val-
ues changed significantly from 111 M in stage34 embryos (8 days post-fertilization) to 31.5 Min 24-h-old larvae. Embryos are surrounded by a
chorion, a protective yet semi-permeable barrier. Ourresults suggest that TCS was the most lethal on the
PROD activity (pmol/mg/min)
early life stages of medaka, and may that differenttoxic mechanisms may be observed between embryos
and larvae. Embryonic development, hatching, and
time to hatching for medaka eggs is affected by TCS
Nominal TCS concentration (µg/l)
treatment. The hatchability of fertilized eggs exposedto TCS for 14 days was significantly decreased rela-
Fig. 5. EROD (A) and PROD (B) activities in liver microsomes
tive to the control groups in the treatment groups with
from female medaka at the end of TCS exposure for 21 days.
Values are shown as the mean EROD and PROD activities of
TCS concentrations over 313 g/l. Time to hatching
female fish, respectively (n = 5). Error bars represent the standard
was also significantly delayed relative to the controls
deviation of the mean. Cont.: control; S.C.: solvent control.
in the treatment groups over 313 g/l TCS. All em-
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
bryos died prior to 3 days post-fertilization in the
animals and some hydroxylated
1250 and 2500 g/l TCS treatments, whereas they
PCBs show estrogenic activity
died prior to 10 days post-fertilization following ex-
p,p-DDT is metabolized to p,p-DDD and p,p-DDE
posure to 625 g/l. These results suggest that TCS
by reductive dechlorination and dehydrochlorination,
has the greatest lethal toxicity toward embryos, and
respectively p,p-DDD shows
also has the greatest inhibitory effect on egg hatching
estrogenic activity (These results
at the concentration of 313 g/l.
suggest that a metabolite of TCS can act as an es-
The chemical structure of TCS closely resembles
trogen receptor agonist. Recent studies have demon-
known non-steroidal estrogens. However, the potential
strated changes in fin length and non-significant trends
of TCS to act as an EDC has not been fully tests and
in sex ratio of medaka, and have suggested that TCS
thus we tested the possibility it may disrupt reproduc-
has potentially weak androgenic and antiestrogenic
tive function in fish. We assessed effects of TCS on
the reproductive biology of paired medaka during a
ported that methyltestosterone (an androgenic chem-
21-day exposure period. No significant differences in
ical) causes significant induction of plasma VTG in
the number of eggs produced or fertility was observed
both male and female fathead minnows (P. promelas).
among treatment groups. This indicates that TCS did
Therefore, our results reconfirm that TCS and/or its
not have adverse effects on the reproductive abilities of
metabolite may have a possibly weakly androgenic
male and female medaka at the concentrations tested in
and/or anti-estrogenic action that has the potential to
this study. During exposures of adult medaka to TCS,
induce hepatic VTG in male medaka.
the measured TCS concentrations in the test water at
0 h were 17.3±3.3 (mean±S.D., N = 4), 75.2±18.5,
the binding ability of nonylphenol (mixture) and
and 162.1±18.2 g/l, and those at 24 h were 8.3±1.4,
4-t-octylphenol with the medaka and human estrogen
46.3 ± 18.5, and 111.7 ± 5.1 g/l. Loss of estrogenic
receptor (␣) ligand-binding domain expressed through
compound such as nonylphenol from aquaria with
Escherichia coli by the competitive binding test
fish (was rapidly (half-life
with [3H] estradiol-17. Nonylphenol (mixture) and
8.0 h) than that without fish (half-life 36.5 h)
4-t-octylphenol showed a concentration-dependent
The loss of TCS concentration in our
affinity for the medaka estrogen receptor (␣). Their
study probably included uptake by the fish, although
relative affinities were approximately 1/10 and 1/5
this was not measured. Surface volatilization and ad-
of those of estradiol-17 respectively, and suggested
sorption to particulate matter and glass probably con-
that they have stronger affinity than for the human es-
tributed to the loss. In future studies we must clear the
trogen receptor (␣) (about 1/2000 to 1/3000 of those
uptake in medaka measuring the chemical concentra-
of estradiol-17). In a similar test for the  receptor,
tion of TCS. Using the static renewal system in our
nonylphenol showed the relative binding affinity of
study, the loss of TCS was kept minimum. Therefore,
approximately 1/110 against that of estradiol-17,
in a laboratory scale, we believe that the static renewal
which is approximately 30 times higher than that of
approach is also a suitable exposure tool for evaluat-
the human estrogen receptor. Therefore, the affinity
ing the estrogenic and reproductive effects of EDCs
of TCS and/or its metabolite for the intrinsic receptor
as well as flow-through conditions
may depend on the species. In this study, we used the
We evaluated the estrogenic activity of TCS as a
rat S9 liver fraction and human estrogen receptor ␣ to
biomarker of hepatic VTG synthesis in male medaka
test the estrogenic activity of TCS and/or its metabo-
exposed to TCS. Male medaka in groups treated with
lite. Unfortunately, we yet have not assay systems for
20 and 100 g/l of TCS for 21 days had significantly
the estrogenic activity based on medaka estrogen re-
higher hepatic VTG levels than the control group. Us-
ceptor. Moreover, we have not obtained medaka liver
ing in vitro yeast two-hybrid assays, no estrogenic ac-
S9 fractions treated with CYPs inducers, because PB
tivity was found for TCS without metabolic activation
induction of CYP has not been clarified in teleosts
by rat S9 liver but TCS showed weak estrogenic activ-
ity in the absence of the rat S9 liver. It is known that
Therefore, in a future study, we need
PCBs are converted into hydroxylated metabolites in
to develop a test method for estrogenic activity using
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
the medaka estrogen receptor. Moreover, using the in-
that teleost CYP2K1 pro-
strumental analysis, we must be characterized a major
tein levels and PROD activity were not induced by
active metabolite of TCS in a microsomal system of
exposure to PB in primary cultures of rainbow trout
medaka liver treated with novel CYPs inducers.
hepatocytes. However, CYP1A1 gene expression was
VTG production in male or juvenile fish has be-
strongly induced by PB based on the marked increases
come a useful biomarker for detecting estrogenic
in CYP1A1 mRNA, CYP1A1 protein, and EROD ac-
contamination in aquatic environments. However,
tivity. also demonstrated that
VTG induction in male fish exposed to environmen-
PB treatment did not affect PROD activity in imma-
tal estrogens has not yet been clearly correlated with
ture rainbow trout. Similar observations have been re-
reproductive impairment. re-
ported in medaka exposed to PB (
ported that VTG levels in the serum of male medaka
However, the levels of BROD and PROD activ-
were significantly inversely correlated with fertil-
ities were induced in liver microsomes from channel
ization after mature male medaka were exposed to
catfish (Ictalurus punctatus) by 3-methylcholanthrene
4-tert-octylphenol for 21 days. On the other hand,
(3-MC) (It is unclear whether
hepatic VTG production in adult male medaka ex-
our results are due to physiological differences in the
posed to ethinyl estradiol () and
inductions of BROD and/or PROD activities between
estradiol-17 ) for 21 days was
fish and mammals. We also attempted to measure
not correlated with fertility and/or egg production. In
PROD activity in male medaka, but it was impossible
the present study, although the production of VTG in
to collect sufficient liver microsome material in addi-
male medaka treated with 20 or 100 g/l of TCS for
tion to that needed for the VTG assay. These results
21 days was higher than that in the control groups, no
suggest that TCS might cause lethal and sublethal toxi-
reproductive impairment was observed.
city in male medaka of in the 200 g/l treatment group;
reported that although concentrations of
however, further experiments in the laboratory are re-
hepatic VTG were significantly increased in males
quire to evaluate CYP2B induction in male and female
exposed to bisphenol A at 3120 g/l, no reduction
medaka exposed to various chemicals such as 3-MC
of fecundity and fertility were observed. Therefore,
and/or PAHs.
these results suggest that increased VTG levels in
In the F1 generation, hatchability was adversely af-
male fish exposed to environmental estrogens may
fected in the 20 g/l treatment, but there was no lin-
not always correlate with reproductive impairment.
ear dose–response relationship. TCS did not affect cu-
After 21-day exposure to TCS, the HSI levels of fe-
mulative mortality, growth and sex ratio of offspring
male and male medaka were significantly higher than
maintained for 90 days after hatching. The sex ratio of
those of the control fish at 20 or 200 g/l TCS, re-
males to females was approximately 1:1 for all groups
spectively. In males, hepatic VTG concentrations were
with no significant differences observed between the
lower in the 200 g/l TCS treatment groups com-
control and treatment groups. Therefore, our results
pared to the 100 g/l TCS treatment groups.
suggest that TCS has high toxicity in the early life
that BROD and PROD activi-
stages of medaka, and that the metabolites of TCS
ties in rats, were induced by all doses of TCS; these
may be weak estrogenic compounds with the poten-
results suggested that TCS induced the hepatic P450
tial to induce elevated VTG in male medaka but pro-
isoforms of the CYP2B subfamily. The induced P450
ducing no adverse effects on reproductive success and
isozymes are closely related to the toxicity of TCS
or its chlorinated derivatives. In this study, we alsoassessed the induction of EROD and PROD activi-ties in hepatic microsomes from female medaka ex-
posed to TCS but found no difference between theEROD and PROD activities among the TCS treat-
This study was supported in part by funds from the
ment groups. In mammals, phenobarbital (PB) is an
Kurita Water and Environment Foundation, Japan. The
in vivo inducer of the CYP2B family, whereas in
authors would like to thank Dr. Louis Guillette (Uni-
teleosts, PB induction of CYP has not been clarified.
versity of Florida) for critical reading of manuscript.
H. Ishibashi et al. / Aquatic Toxicology 67 (2004) 167–179
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Source: http://www.pu-kumamoto.ac.jp/access/access01seika.html/01-12.html/011201.pdf
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