Development of specific enzyme-linked immunosorbent assays for multiple vitellogenins in marbled sole, Pleuronectes yokohamae.

Non-competitive, enzyme-linked immunosorbent assays (ELISAs) for three distinct sole vitellogenins (VtgAa, VtgAb and VtgC) were designed using their purified lipovitellin (Lv) products and corresponding digoxigenin-labeled, anti-Lv polyclonal antibodies, primarily for employment in monitoring estrogenic pollution of the environment. The working range of the ELISAs was from 0.97 to 1,000 ng/mL for all Vtg subtypes. Each ELISA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed ELISAs were lower than 10%. Three Vtg subtypes were induced in serum of immature fish by estradiol-17ß (E2) injection (0.5 mg/kg body weight). All Vtg subtypes were induced one day after the injection, reaching peak levels (Lv equivalents) within three days, as follows: 39.1 ±â€¯28.9 µg/mL (VtgAa), 57.9 ±â€¯30.7 µg/mL (VtgAb) and 12.6 ±â€¯4.8 µg/mL (VtgC). In wild-caught males, VtgAa, VtgAb and VtgC were detected in ranges from 0.26 to 1.21, 0.19 to 8.69, and 0.17 to 53.50 µg/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 ±â€¯733.93 µg/mL) was significantly higher than for VtgAa (150.33 ±â€¯22.35 µg/mL) or VtgC (57.08 ±â€¯6.00 µg/mL); thus VtgAb appeared to be the most dominant Vtg subtype. The present study entails the first report on development of subtype-specific Vtg ELISAs in marbled sole, which empowers us to detect and monitor estrogenic contamination in aquatic environments inhabited by this species.

Acute Toxicity of Water-Accommodated Fraction and Chemically Enhanced WAF of Bunker C Oil and Dispersant to a Microalga Tetraselmis tetrathele.

This study assessed the toxicity of water-accommodated fraction (WAF) and chemically enhanced WAF (CEWAF) of bunker C oil and dispersant (DISP) to a microalga, Tetraselmis tetrathele. The 72-h median effective concentration (72-h EC50) of CEWAF and DISP were determined at 3.30% and 2.40%, respectively. The no observed effect concentration (NOEC) of CEWAF to T. tetrathele was at 2.0% and lowest observed effect concentration (LOEC) was at 3.0% while NOEC and LOEC of DISP to T. tetrathele were determined at 1.0% and 2.0%, respectively. The addition of dispersant to oil increased the amount of total PAH present in the CEWAF test solutions. DISP alone was highly toxic, and the toxicity of CEWAF was primarily caused by the presence of dispersant.

Reproductive toxicity of 1-nitronaphthalene and 1-nitropyrene exposure in the mummichog, Fundulus heteroclitus.

After pregnant mummichog were implanted with 1-nitronaphthalene or 1-nitropyrene via cholesterol pellet, we investigated the effects of the chemicals on embryo normality, hatchability and days to hatch of normal embryos, and growth and survival of hatched larvae from normal embryos of the implanted mummichog. Hatchability was the parameter most sensitive to the effects of both 1-nitronaphthalene and 1-nitropyrene. The 4-week lowest-observed-effect concentration (LOEC) of 1-nitronaphthalene, based on the actual concentrations in the eggs in the test, was 447 ng g(-1) wet wt.; and the LOEC and no-observed-effect concentration (NOEC) of 1-nitropyrene were 958 and 344 ng g(-1) wet wt., respectively. The 4-week LOEC of 1-nitronaphthalene, based on the concentration in the water, was estimated at 4.8 µg L(-1) by using the reported bioconcentration factor; and the LOEC and NOEC of 1-nitropyrene, based on the concentration in the water, were estimated at 3.1 and 8.6 µg L(-1), respectively. The reported environmental concentrations of 1-nitropyrene and 1-nitronaphthalene are over three magnitudes lower than the toxicity values we obtained. Therefore, the effects of environmental levels of 1-nitropyrene and 1-nitronaphthalene on fish reproduction, not including genomic effects on embryos, appear to be almost negligible. However, DNA damage has been detected in marine organisms exposed to 1-nitropyrene. Further studies of the genotoxicity of nitrated polycyclic aromatic hydrocarbons at environmental levels are therefore needed to evaluate their ecotoxicological risks.

Effects of chlorpyrifos on the metabolome of the freshwater carp, Cyprinus carpio.

This study investigated the effects of waterborne chlorpyrifos with concentrations of 1 and 100 µg/L for L and H-groups, respectively, on metabolome profiles of carp plasma using (1)H-NMR. Principal component analysis suggests that chlorpyrifos exposure firstly affected in L and H-groups on day 2 or 4, and followed a second effect in both exposure groups on day 14. Levels of metabolites related to the energy production in the body, such as glucose, glycerol, valine, leucine, isoleucine, lactate, alanine, 3-D-hydroxybutyrates and acetoacetate, significantly changed by exposures of chlorpyrifos. Those results suggest that energy production was severely affected in carp. The exposure could also be highly elevated ammonia levels especially in H-group due to severe convulsion in muscle caused by the inhibition of acetylcholinesterase activity.

Accumulation of endocrine disrupting chemicals (EDCs) in the polychaete Paraprionospio sp. from the Yodo River mouth, Osaka Bay, Japan.

This study presents the levels of endocrine disrupting chemicals (EDCs) accumulated by Paraprionospio sp. from the Yodo River mouth, Osaka Bay. Since high concentrations of nonylphenol (NP), bisphenol A (BP), octylphenol (OP), 17ß-estradiol (E2), and estrone (E1) have been measured in sediment from Osaka Bay, some bioaccumulation could be expected particularly in benthic animals. EDCs were analysed in Paraprionospio sp., a dominant benthic species in Osaka Bay. The results showed that Paraprionospio sp. had accumulated varying concentrations (wet weight; w.w.) of NP at 1,460-4,410 ng/g; BP at 22.5-39.6 ng/g; OP at 18.9-45.4 ng/g; E2 at 0.89-4.35 ng/g; and E1 at 0.06-2.50 ng/g. Accumulation of NP and OP were highest among the samples gathered in summer (July 2008), while concentrations of BP, E2, and E1 did not much differs within 3 years. EDC levels in Paraprionospio sp. were apparently greater than those in sediments showing bioaccumulation.

Effect of seasonal variation in seawater dissolved mercury concentrations on mercury accumulation in the muscle of red sea bream (Pagrus major) held in Minamata Bay, Japan.

Japanese stingfish (Sebastiscus marmoratus) and Bambooleaf wrasse (Pseudolabrus japonicas) are monitored annually for mercury pollution in Minamata Bay, Japan. The average total mercury concentration in the muscle of these two species in Minamata Bay was 0.36 mg kg(-1) wet weight and 0.20 kg(-1) wet weigh, respectively, between 2008 and 2010. This is higher than levels elsewhere in Japan (0.125 mg kg(-1) wet weight and 0.038 mg kg(-1) wet weight, respectively). The FDA (2001) and EPA (2004) suggested that a proportion of mercury accumulated in fish is derived from seawater. We reared young red sea bream (Pagrus major) over a 2-year period in Minamata Bay and Nagashima (control) to evaluate the uptake of mercury from seawater and dietary sources. Fish were fed a synthesized diet that did not contain mercury. There was no difference in mercury accumulation in the muscle of red sea bream between Minamata Bay and Nagashima. Thus, our results suggest that the majority of mercury accumulated in fish muscle is not from seawater.

Bioaccumulation of dietary endocrine disrupting chemicals (EDCs) by the polychaete, Perinereis nuntia.

To investigate the biomagnification factor (BMF) of EDCs by the polychaete, Perinereis nuntia, organisms were exposed to EDCs through their diet. BMF values ranged from 0.001 to 0.028 indicating that EDCs were not biomagnified. Elimination rates were (0.20-0.25 day(-1)) and were higher than uptake rate (0.0003-0.003 day(-1)) verifying that EDCs were not biomagnified by P. nuntia due to their fast elimination. The calculated half-life of each EDC in this study varies from 2.76 to 3.45 days. Overall, the findings from this study demonstrated that the studied EDCs are not biomagnified in P. nuntia but exposure does occur from the diet.

¹H NMR-based metabolomics approach to assess toxicity of bunker a heavy oil to freshwater carp, Cyprinus carpio.

Using a ¹H NMR metabolomics approach, the effects of dietary exposure of bunker A heavy oil (0.01, 0.1, 1, and 5% in diet) on freshwater carp, Cyprinus carpio, were examined. Statistical analysis by PCA score plots showed that the amount of metabolites in exposure groups 0.1, 1, and 5% differed from those in the control group. Although no discernible effects on metabolites were noted in the 0.1% exposure group as well as in the lowest concentration (0.01%) group, several metabolites such as amino acid (e.g., leucine, isoleucine, valine, glutamine, histidine, proline, and methionine), 3-D-hydroxybutyrate, and glycerol were elevated, while another metabolite such as formate was reduced in 1 and 5% groups. These changes in the metabolites associated with the tri-carboxylic-acid (TCA) cycle suggest that oil exposure resulted in the disturbance of the TCA cycle in the liver of the carp. Isobutyrate, a marker of anoxia, was also increased in 1 and 5% exposures groups and was directly related to low hemoglobin concentrations leading to reduced oxygen transport by blood. In addition, significant elevation of creatinine in the plasma of carps exposed to 5% heavy oil suggests disturbance in kidney function. Thus, metabolomics approach can detect toxic effects of hazardous pollutants on fish.

Histopathological alterations in gill, liver and kidney of common carp exposed to chlorpyrifos.

Histopathological alterations in gill, liver and kidney of common carp, Cyprinus carpio, intoxicated with sub-lethal concentrations of chlorpyrifos (O,O,-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothioate) pesticide (1 and 100 µg/L) for a period of 14 days were analyzed under light microscope. Gill exhibited hyperplasia and hypertrophy of gill epithelium, blood congestion, dilation of marginal channel, epithelial lifting, lamellar fusion, lamellar disorganization, lamellar aneurysm, rupture of the lamellar epithelium, rupture of pillar cells and necrosis. Alterations in hepatocytes were more pronounced, including nuclear and cellular hypertrophy, cellular atrophy, irregular contour of cells and nucleus, cytoplasmic vacuolation, cytoplasmic and nuclear degeneration, cellular rupture, pyknotic nucleus, necrosis and melanomacrophages aggregations. Histopathological lesions in kidney were cellular and nuclear hypertrophy, narrowing of tubular lumen, cytoplasmic vacuolation, hyaline droplet degeneration, nuclear degeneration, occlusion of tubular lumen, tubular regeneration, dilation of glomerular capillaries, degeneration of glomerulus and hemorrhage in Bowman's space. The most significant conclusion drawn from this study was that with the increased concentration and duration the toxicosis of chlorpyrifos would be enhanced as shown through the analysis of mean assessment value (MAV) and degree of tissue changes (DTC) also.

Histopathological effects and EROD induction in common carp exposed to dietary heavy oil.

Common carp, Cyprinus carpio, was exposed to 1% and 5% dietary heavy oil for 2 weeks. Higher polyaromatic hydrocarbons (PAHs) accumulation induced higher hepatic EROD activity in exposure groups compared with the control group. Significant histological alternations were observed in the liver such as nuclear and cellular hypertrophy, accumulation of eosinophilic granules in cytoplasm, nuclear degeneration and fibrillar inclusion in cytoplasm. Severe damage of the kidney was indicated by nuclear and cellular degeneration, and ultimately necrosis in the tubular epithelium cells. In addition, dilated glomerulus with hemorrhage was found in the renal capsule. The mean assessment value (MAV) and degree of tissue changes (DTC), as semi-quantitative analyses, were significantly increased with concentration and duration of exposure. Likewise, frequencies of pathological lesions in both liver and kidney were also increased with concentration and duration of exposure.

Interspecies differences in the accumulation of tributyltin and its metabolites under dietary exposure in sea perch, Lateolabrax japonicus, and red sea bream, Pagrus major.

Interspecies differences in the accumulation of dietary tributyltin (TBT) between sea perch, Lateolabrax japonicus, and red sea bream, Pagrus major, were studied. Although TBTs in both species reached steady-state condition in low-concentration group (L-group, 200 ng/g nominal concentration) by 1 week, it increased up to the end of exposure in high-concentration group (H-group, 3000 ng/g nominal concentration). In H-group, the accumulation rate of TBT in sea perch from 1 to 2 weeks was much higher than in red sea bream, which were 2.4-fold for sea perch and 1.7-fold for red sea bream, although TBT concentrations were similar between sea perch and red sea bream in L-group. Furthermore, in the H-group, the concentrations of TBT at 1 and 2 weeks were about 1.3- and 1.9-fold, respectively, higher in the sea perch than in the red sea bream. On the other hand, DBT residue in red sea bream was about 1.4-fold higher in sea perch for the L-group but concentrations were similar in both fishes for the H-group. These results suggest that red sea bream could metabolize faster the TBT to DBT than sea perch. This study also reveals that fish probably could absorb TBT through the food chain. The uptake of TBT by fish should be regarded in the real environment, because TBT concentration in seawater has been decreasing and now already at significantly low level.

Pesticide residues in coastal waters affected by rice paddy effluents temporarily stored in a wastewater reservoir in southern Japan.

This paper presents the occurrence, distribution, and environmental impact of pesticide residues in coastal water in southern Japan that receives effluents from a wastewater reservoir temporarily storing surface runoffs of rice paddy fields located near the coastline during the 2005 rice planting season. Concentrations of 14 target pesticides were measured by GC-MS and the hazards posed by the most important pesticides detected were evaluated by acute toxicity tests using a marine diatom, Chaetoceros sp., and a marine amphipod, Hyale barbicornis. Six pesticides (fenobucarb, flutolanil, iprobenfos, mefenacet, phthalide, pyriproxyfen) were detected in the coastal water, with three pesticides (fenobucarb, iprobenfos, and mefenacet) having 100% frequencies of detection. The maximum concentration of mefenacet, at 4.22 microg/L, was at least one magnitude higher than that of fenobucarb and iprobenfos, at 0.27 and 0.19 microg/L, respectively, while the three remaining pesticides had concentrations just around the detection limit of 0.01 microg/L. Consequently, detected concentrations of mefenacet were highly correlated with salinity levels, confirming that the wastewater reservoir is a major source of rice pesticide residues in this particular coastal environment. Hence, the spatial distribution of mefenacet was simulated using their relationship and the results indicate that mefenacet has a tendency to enter and spread to a relatively wide portion of the coastal area during the rice planting season. There is also a possibility that the other pesticides used in rice farming such as fenobucarb and iprobenfos may show similar distribution patterns in coastal waters when they are present in the wastewater reservoir at higher concentrations. These pesticides, however, manifested low acute toxicities to both Chaetoceros sp. and H. barbicornis, suggesting little impact to marine organisms.

Assessment of pesticide residues in freshwater areas affected by rice paddy effluents in Southern Japan.

Pesticide residues in five freshwater areas that are directly affected by rice paddy effluents in southern Japan were measured to determine their maximum concentrations and temporal variations. Water samples were collected every week during the 2005 rice planting season in Kagoshima Prefecture and stations were established in Amori River, Sudo River, Nagaida River (that drains into the bigger Kotsuki River), rice paddy drainage canal, and wastewater reservoir (that collects effluents from rice paddy fields). Of the 14 target pesticides examined, a total of 11 were detected in all stations. Mefenacet, fenobucarb, and flutolanil were the three pesticides with the highest maximum concentrations and were also detected frequently. Analysis of temporal variations of pesticides showed that herbicides had relatively higher concentrations in the earlier stages of the rice planting season, while insecticides and fungicides had relatively higher concentrations at the later stages. There was no significant difference among stations with regards to the temporal patterns of the top three pesticides. The calculated toxic units were less than 1 in all stations, implying low or negligible environmental risk of pesticides detected to freshwater organisms.

Monitoring of PAHs and alkylated PAHs in aquatic organisms after 1 month from the Solar I oil spill off the coast of Guimaras Island, Philippines.

Following the oil spill accident of the Solar I tanker in 2006 off the coast of Guimaras Island in the Philippines, polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs in some aquatic organisms were investigated at Luzaran in Guimaras and Taklong Islands, which were heavily polluted with spilled oil, immediately and 1 month after the accident. The concentrations of total PAHs were 11.9-52.3 ng/g dry weight in fish. Meanwhile, total PAH concentrations in shellfish were 38.0-3,102 ng/g dry weight in Luzaran and 128-236 ng/g dry weight in Taklong. Pyrene, phenanthrene, and fluoranthene were dominant in most fish and chrysene in all shellfish. Significantly higher concentrations of all alkylated homologs were detected in shellfish than in fish. These differences had two possible causes, that is, the differences between fish and shellfish could be attributed to the uptake routes and/or their metabolizing abilities.

Potential mechanisms underlying embryonic developmental toxicity caused by benzo[a]pyrene in Japanese medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.

Influence of atrial fibrillation on cardiac prognosis in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD), a known risk factor for the development of congestive heart failure (CHF), was recently shown to predict the prevalence of atrial fibrillation (AF). Here, we explore the influence of AF on cardiac prognosis in COPD patients. METHODS: A total of 339 consecutive patients who underwent spirometry from 2010 to 2013 for various reasons were retrospectively examined. Based on the diagnostic criteria, patients were stratified into COPD and non-COPD groups, which were both further divided into those with AF (chronic AF or paroxysmal AF) or sinus rhythm (SR) based on previous electrocardiography results. Significances of differences in cardiac events were assessed by the chi-square test. Multivariate logistic regression analyses and Cox proportional hazard models were applied to evaluate the influence of AF on cardiac events. RESULTS: Of the 339 patients, 190 were diagnosed with COPD, with 42 of these were having AF. During the mean follow-up period of 7.4 ± 0.8 years, CHF developed more frequently in COPD patients with AF than in COPD patients without AF [50% vs 7%; odds ratio (OR) 12.4, 95% confidence interval (CI): 5.25-29.49, p < 0.05]. AF was an independent predictor of CHF development (OR 20.4, 95% CI: 6.55-79.80, p < 0.05) and cardiac mortality (OR 2.8, 95% CI: 1.79-4.72, p < 0.05). Moreover, positive correlations were found between the severity of pulmonary obstruction with AF and CHF development (R = 0.69, p < 0.05), as well as cardiac mortality (R = 0.78.p < 0.05). CONCLUSIONS: These results suggest that AF may be strongly associated with cardiac mortality and CHF in COPD patients.

Influence of cognitive impairment on cardiac mortality after percutaneous coronary intervention in very elderly patients: a retrospective observational study.

BACKGROUND: Cognitive impairment (CI) increases cardiac mortality among very elderly patients. Percutaneous coronary intervention (PCI) for ischemic heart disease (IHD) patients is considered a favorable strategy for decreasing cardiac mortality. Here, we investigated the influence of CI on cardiac mortality after PCI in very elderly patients. METHODS: We performed a retrospective observational analysis of patients who received PCI between 2012 and 2014 at the South Miyagi Medical Center, Japan. IHD patients over 80 years old who underwent the Mini-Mental State Examination for CI screening during hospitalization and/or who had been diagnosed with CI were included. Participants were divided into CI and non-CI groups, and cardiac mortality and incidence of adverse cardiac events in a 3-year follow-up period were compared between groups. Statistical analyses were performed using the t-test, χ2 test, and multivariable Cox regression analysis, with major comorbid illness and conventional cardiac risk factors as confounders. RESULTS: Of 565 patients, 95 were included (41 CI, 54 non-CI). Cardiac mortality during the follow-up period was significantly higher in the CI group (36%) compared with the non-CI group (13%) (OR = 4.3, 95% CI: 1.56-11.82, P < 0.05). CI was an independent cardiac prognostic factor after PCI and, for CI patients, living only with a CI partner was an independent predictor of cardiac death within three years. CONCLUSIONS: CI significantly affected cardiac prognosis after PCI in very elderly patients, particularly those living with a CI partner. To improve patients' prognoses, social background should be considered alongside conventional medical measures.

A SNP in a Steroidogenic Enzyme Is Associated with Phenotypic Sex in Seriola Fishes.

Vertebrate sex development consists largely of two processes: "sex determination," the initial bifurcation of sexual identity, and "sex differentiation," which subsequently facilitates maleness or femaleness according to the sex determination signal. Steroid hormones promote multiple types of sexual dimorphism in eutherian mammals and avians [1-3], in which they are indispensable for proper sex differentiation. By contrast, in many poikilothermic vertebrates, steroid hormones have been proposed to be key players in sex determination as well as sex differentiation [4-8]. This hypothesis was introduced more than 50 years ago but has never been rigorously tested due to difficulties in discriminating the roles of steroids in sex determination and differentiation. We found that a missense SNP in the gene encoding the steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase 1 (Hsd17b1) was perfectly associated with ZZ/ZW sex determination in Seriola fishes. Biochemical analyses revealed that a glutamate residue present specifically in Z-type HSD17B1 attenuated interconversion between 17-keto and 17ß-hydroxy steroids relative to the allelic product from the W chromosome, which harbors glycine at that position, by disrupting the hydrogen bond network between the steroid and the enzyme's catalytic residues. Hsd17b1 mRNA is constitutively expressed in undifferentiated and differentiating gonads of both genotypic sexes, whereas W-type mRNA is expressed only in genotypic females. Meanwhile, Cyp19a1 is predominantly expressed in differentiating ovary. We conclude that the combination of Hsd17b1 alleles determines sex by modulating endogenous estrogen levels in Seriola species. These findings strongly support the long-standing hypothesis on steroids in sex determination.

Java medaka: a proposed new marine test fish for ecotoxicology.

Java medaka (Oryzias javanicus), which is distributed in estuarine waters of East to Southeast Asia, is proposed here for consideration as a new marine test fish. Semi-static acute toxicity tests were conducted at 26 degrees C for 1 week, 2 weeks, and 1 month old Java-medaka exposed to 4-chlorophenol and zinc, and 2 weeks olds exposed to potassium dichromate (hexavalent chromium) and sodium hypochlorite (residue chlorine). Ninety-six hours LC50s of 4-chlorophenol, zinc, hexavalent chromium, and residue chlorine were from 3.0 to 4.1, from 12.4 to 14.7, 7.4, and 0.05 mg/L, respectively. There were no large differences in sensitivity for 4-chlorophenol and zinc among the three growth stages. Compared with other fish species, these LC50s were relatively low suggesting that Java medaka has relatively high sensitivity to chemicals. This fish is quite easy to keep in the laboratory, with low mortalities at early stages and it takes about 3 months to mature from hatching, suggesting that this fish can be used not only for short-term toxicity tests but also for early life stage toxicity tests. From these results, Java-medaka is highly recommended as a marine test fish for ecotoxicity tests.

Effects of estrone on full life cycle of Java medaka (Oryzias javanicus), a new marine test fish.

Estrone is a natural estrogen detected in sewage treatment works effluents and in estuarine waters. However, there is little information on the effects of estrone on marine fish. This study investigated the effects of estrone on reproduction of the estuarine fish, Java medaka (Oryzias javanicus). Java medaka were exposed to concentrations of 39, 198, 484, 1,188, and 3,701 ng/L of estrone from embryonic stages up to adult stages for 239 d after hatching. The fertility and egg numbers of Java medaka exposed to 1,188 and 3,701 ng/L were significantly lower than that of control. The hepatic vitellogenin concentrations in male Java medaka exposed to estrone greater than 484 ng/L were significantly higher than that of control. Oocytes in testis (testis-ova) were not detected in the males in any of the exposure groups. The lowest-observed-effect concentration and no-observed-effect concentration for Java medaka were 484 and 198 ng/L of estrone. These results suggest that in relatively low estrone concentrations, 39 and 198 ng/L, Java medaka will not be affected by exposure to estrone.