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.
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