The concentration of estrogenic substances in water and sediment was surveyed in Ago Bay, which is a typical enclosed sea area. Eight water samples and seven sediment samples were collected from the mouth of the Maekawa River at the entrance of the Ago Bay. To examine estrogenic substances, a yeast screen assay developed by Prof. J. P. Sumpter was used. The highest concentrations of estrogenic substances in water and sediment were noted in the Maekawa River (16.6ng·l^-1 and 24.3ng·g^-1). The concentrations gradually decreased towards the entrance of Ago Bay from the mouth of the Maekawa River. It was suggested that this decrease may be the result of dilution, adsorption to the seabed and biodegradation of the estrogenic substances. The water and sediment assay samples were fractionated by high pressure liquid chromatography and assayed to determine the chemical species of estrogenic substances (high pressure liquid chromatography-bioassay detection method). The estrogenic substances detected corresponded to E1 and/or E2, and were noted in all samples. These are typical natural female hormones, and their origin is presumably domestic sewage and other wastewater containing feces and urine.

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The objectives of the present research were (i) to report the mass balance of chlorine during pentachlorophenol (PCP) photodegradation and (ii) to reveal the photodegradation pathway experimentally with a theoretical proof based on the density functional theory (DFT). The chlorine of PCP was completely mineralized to produce chloride ions after 24 hours of UV irradiation. As intermediates, 2,3,5,6-tetrachlorophenol, 2,3,4,6-tetrachlorophenol and 2,5-dichlorophenol were identified. At least 80% of the chlorine balance during PCP photodegradation was accounted by PCP, these intermediates, and chloride ions. A DFT calculation showed differences in the C-Cl bond dissociation energy level and the positions of respective PCP molecular and the PCP intermediates. The dechlorination intermediates predicted using the calculated C-Cl bond dissociation energy were consistent with those experimentally confirmed, indicating the feasibility of this theoretical method in predicting the dechlorination pathway.

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The runoff characteristics of pesticides were observed every three days for four months in the Yodo River and its two tributaries, and monthly for their traverse distributions in the Yodo River while flowing down from the junction of the upper three big rivers to the Torikai Big Bridge. Simultaneous rice transplanting and subsequent application of pesticides in catchments caused high concentrations and loadings of pesticides. When the first-half stage of storm runoff events after pesticide application was observed, the concentrations and loadings of pesticides in the river were very high. The concentration peaks of pesticides were lower and broader in the Yodo River than in its tributaries due to the time lag in local pesticide application in its big catchment area. The peak times and heights of many pesticide concentrations on the right and left sides, and in the center in the traverse section of the Yodo River differed from one another owing to their differences among the three big upstream rivers and small midstream tributaries. The frequency distributions of pesticide concentrations in the fixed observation period became the logarithmic normal distributions due to many unditected low values. The runoff risks of pesticides in the rivers could be evaluated by the maximum concentration, ΣC·ΣT, cumulative loading per catchment area, and cumulative ditected concentration.

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In this study, we investigated the effects of an endocrine disruptor, 17β-estradiol, on the reproduction of freshwater shrimp, Paratya compressa improvisa (Natantia, Decapoda, Crustacea). Female shrimps with fertilized eggs were exposed to 0.1, 1.0, and 10μg·l^-1 17β-estradiol. Larval mortality after 14 days of hatching for these female shrimps significantly increased from 2.0% in the control group to 66-80% in the exposed groups. On the other hand, when larvae of this species, obtained within 24 h after hatching, were exposed to 0.1, 1.0, and 10μg·l^-1 17β-estradiol, larval mortality after 14 days of hatching slightly increased from 2.0% in the control group to 3.9-9.2% in the exposed groups. The sex ratio (female/male) of adults exposed to 17β-estradiol changed markedly from 1.1 in the control group to 0.5 in the exposed groups at 1.0 and 10μg·l^-1. Female shrimps with fertilized eggs were observed after 150 days of hatching in the control group and the exposed groups at 0.1 and 1.0μg·l^-1. The larval mortality of the next generation after 14 days of hatching gradually increased from 0.8-3.4% in the control group to 8.1-16% in the exposed group at 0.1μg·l^-1 and 64-84% in the exposed group at 1.0μg·l^-1. These results suggest that this compound affects the reproduction of this species.

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The adsorption and degradation of triphenyltin (TPT) by immobilized Pseudomonas chlororaphis CNR15 cells were investigated in a batch mode. The cells were entrapped in calcium alginate beads. Pyoverdine, which is a siderophore that participates in the degradation of TPT, was secreted from the immobilized P. chlororaphis CNR15 cells, and the concentration of pyoverdine increased for at least one month. The rate of TPT adsorption by the immobilized cells was significantly higher than that by the beads without cells, while the diphenyltin and monophenyltin produced by TPT degradation were largely soluble forms. The degradation activity was influenced by temperature and pH, and showed a maximum at 30°C and pH 8.8 These results suggest that the rate of TPT degradation by immobilized P. chlororaphis CNR15 depends on both the production and catalytic properties of pyoverdine.

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A new acute toxicity test method was developed using the larvae of Japanese Medaka (Oryzias latipes). The test was carried out in small glass petri dishes and was found to be feasible for simultaneously testing many chemicals. The most preferable larval stage and exposure period for the test were determined to be 48-72h posthatch with an exposure time of 48h. To obtain larvae for the test, adult pairs as many as 1/7 the number of larvae are required. The egg should be collected after 8 days of pairing, and the larvae hatched on the 9^th or 10^th day from commencement of embryo incubation at 25°C should be separated and cultured at a population density of less than 1.0g-weight·l^-1. The calculated coefficients of variation of the test were 15% for 48-h LC₅₀ and 22% for 48-h EC₅₀. Using this method, the acute toxicity levels of eight widely used pesticides were evaluated. High toxicity levels were detected for diazinon and isoprothiolane, which were reported as low-toxicity pesticides in an acute toxicity test using Medaka adults.

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A new quantitative evaluation technique called the fragmented method^* for the detection of testis-ova in male fish using a medaka (Oryzias latipes) S-rR strain has been developed in this study. A current histological method is still being applied to detect the testis-ova in male fish exposed to estrogens or suspected endocrine-disrupting chemicals, even though some disadvantages, such as oversight of the testis-ova due to the limited observation area of the testis, are apparently indicated. The method we developed here enables the accurate determination of the number and the developmental stages (size) of oocytes in a whole testis. The testis was removed from the specimen, fixed with 10% buffered formalin solution and broken into small fragments with a dissecting needle and aciform forceps in glycelin solution containing a small amount of methylene blue or toluidine blue on a slide glass. All developing oocytes of various sizes were clearly stained and thereby were observable under a dissecting microscope. Spermatozoa were also distinguishable in this method. In conclusion, this method is not only proven as a convenient and cost-effective method for quantitatively detecting testis-ova appearance in fish, but is also expected to contribute to the understanding of testis-ova appearance and its biological significances to future studies of endocrine disruption.
^*The fragmented method is a new technique for quantitatively detecting testis-ova in male fish using the medaka (Oryzias latipes) strain described in this paper. This technique was developed in the research that the National Institute of Advanced Industrial Science and Technology (AIST) entrusted to Sumika Technoservice Corporation.

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Bisphenol A (BPA) is used as the primary raw material for polycarbonate plastics and epoxy resins worldwide. Recently, BPA has been reported as a weakly estrogenic chemical and detected widely in aquatic environments in Japan. Various chemical oxidation processes were examined with the aim of reducing the estrogenic activity of BPA. The results of our examination showed that O₃ and O₃/H₂O₂ processes were very effective in reducing BPA and its estrogenic activity. On the other hand, TOC was not decreased markedly by O₃ and O₃/H₂O₂ processes. The structure of BPA after O₃ oxidation was examined by IR analysis. These results showed that aromatic ring of BPA was degraded and the resulting intermediate has no estrogenic activity.
It was also investigated whether the O₃ process was effective in reducing the estrogenic activity of BPA in river water. The Ames mutagenicity of the ozonation sample was also assayed. The test result showed that the Ames mutagenicity of BPA was not influenced by the O₃ process. These results indicated that the O₃ process was useful in reducing estrogenic activity of BPA in waste water and aquatic environments.

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The objective of this study was to optimize the yeast two-hybrid system for quantitative detection of the estrogenic activity of organics in sewage water. Most of the estrogenic activity of sewage water concentrates prepared by Amberlite XAD-2 resin column concentration method was due to that of 17β-estradiol (E₂). As well as E₂, sewage water concentrates showed higher estrogenic activity with the rise of assay pH than with the conventional pH (pH 4). When assay was performed at pH 7.6. the estrogenic activity of sewage water concentrates increased approximately 25 times as high as that in the case of the conventional pH. This increase was thought to be caused by the following reason: organic constituents of sewage water concentrates disturbed the detection of the activity of E₂ at the conventional pH. but humic acid as an organic constituent did not interfere with the detection of the activity of E₂ at pH 7.6. The estrogenic activity of sewage water concentrate assayed at pH 7.6 was almost the same as activity corresponding to E2 concentration that was measured by the ELISA method. This pH-optimized yeast two-hybrid system is useful for comprehensive evaluation of estrogenic substances in sewage water and environmental water.

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Endocrine disruptors such as bisphenol A (BPA) and nonylphenol have been frequently detected in environmental water. The objectives of this study were to evaluate the effects of chlorination on the estrogenic activity of BPA and its chlorination by-products. To quantify the estrogenic activity, a yeast two-hybrid assay was conducted. For the measurements of the concentrations BPA and its chlorination by-products, high performance liquid chromatography/mass spectrometry (LC/MS) analysis was used. As a result of BPA chlorination, monochloro-, dichloro-, trichloro- and tetrachloro-BPAs were detected in addition to the original BPA. To evaluate the contribution of each chlorinated BPA to the total estrogenic activity, the detected chlorinated BPAs were fractionated and the estrogenic activity of each chlorinated BPA was examined. Monochloro-, dichloro- and trichloro-BPAs showed higher estrogenic activities than BPA itself, but these activities decreased and were finally eliminated with reaction time. From these results, it is concluded that BPA chlorination is effective in eliminating estrogenic activity although chlorinated BPAs, which have stronger estrogenic activities than BPA, are formed temporarily.

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The ecotoxic effects of leachate from waste disposal landfill sites were studied using Japanese medaka (Oryzias latipes) based on acute lethal toxicity, and the inductions of CYP1A and vitellogenin (Vtg). The leachate exhibited highly lethal toxicity to medaka, however the toxicity was reduced in the leachate treated by biological and chemical processes. These processes seem to decrease the amount of lethally toxic substances from the leachate. CYP1A and Vtg were induced by the leachate and treated water. We also investigated which of the contaminant chemicals detected in the leachate induces CYP1A or Vtg. Multiple regression analysis showed that benzo (g,h,i) perylene causes a significant CYP1A induction among the detected chemicals, but not Vtg induction. The monitoring applications of biomarkers are occasionally more practical for environmental assessment rather than for chemical monitoring, because it is technically impossible to survey all contaminant chemicals, and toxicity data for all chemicals are not available. In our study, not only untreated leachate but also treated leachate is contaminated with estrogenic endocrine disruptors and/or CYP1A inducers, although the lethal toxicity of the treated leachate decreased. This study demonstrated that aquatic environments, where leachate effluents are discharged into, are in danger of endocrine disruptor contamination from waste disposal landfill sites.

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If it is possible to develop an efficient process of removing estrogens from wastewater during sewage treatment, the load of endocrine disrupting chemicals in the water environment could be greatly reduced. In this study, the removal of estrogens from municipal wastewater by coagulation and foam separation using coagulant and milk casein was examined. The ELISA method was used to measure the efficiency of estrogen removal. In the conventional foam separation method, the removal efficiency of suspended solids was very high. However, most of the estrogen remained in the treated water since approximately 95% of the total estrogen in wastewater was in the dissolved fraction, while the amount of estrogens in the solid fraction was extremely small. Therefore, the removal of dissolved estrogen was difficult using the conventional foam separation process for removing solids. The addition of powdered activated carbon was examined as a pretreatment process. The adsorption efficiency of estrogen to activated carbon was markedly higher than that of other soluble fractions such as E260 (ultraviolet absorbance at 260 nm), chromaticity, and dissolved organic carbon in wastewater. With the addition of powdered activated carbon, both suspended solids and estrogen could be removed by coagulation and foam separation. The removal efficiencies of turbidity and estrogen were 99% and 94%, respectively, under the following dosage conditions: activated carbon, 50mg·l^-1 ; ferric coagulant, 30mg-Fe·l^-1 ; and casein, 50mg·l^-1.

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To evaluate the total hazard existing in effluent water, three different bioassays, namely, the in vitro basal cytotoxicity test using a human cell line, bioluminescence inhibition test using luminescent bacteria and algal growth inhibition test, were applied to landfill leachate samples. To simplify the algal growth inhibition test, a new method using 96-well microplates and a microplate fluorometer was developed. Based on the results obtained using the bioassays, scoring of the total hazard existing in the samples was attempted. While the scores obtained with the basal cytotoxicity and bioluminescence inhibition tests showed similar patterns, those obtained with the algal growth inhibition test showed a different pattern. Nevertheless, the total scores of the samples indicate that the total hazard existing in raw leachate was significantly reduced by a wastewater treatment process. Thus, this type of scoring of the total hazard existing in landfill leachate may be useful for priority setting for the risk management of landfill sites.

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The MCF-7 human breast cancer cell line has been widely used in studies in the fields of cancer biology, steroid hormone biochemistry and toxicology. MCF-7 cells were demonstrated to be enhanced cell proliferation by estrogens. The estrogen-dependent growth of MCF-7 cells is often called E-Screen, which is one of the most sensitive assays for estrogenicity assessment. On the contrary, there is a potential for this assay to identify false positives by cell mitogens and false negatives by cytotoxic or general growth inhibitors. Therefore, when we subject environmental samples to an E-screen test, we have to examine the interaction of contaminant substances in the cells. In this study, we evaluated the endocrine-disrupting activity of multiply contaminated model samples using the E-screen test. Through this study, we demonstrated that some chemicals prevent the estrogenicity of endocrine-disrupting chemicals, and furthermore, this estrogenicity is induced by other chemicals. This result indicates that the fractionation of chemicals in environmental samples is essential for evaluating their estrogenicity using a bioassay technique.

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A bacterial strain capable of degrading triphenyltin (TPT) was isolated using a screening technique based on pyrocatechol violet colorimetry for the detection of TPT metabolites, diphenyltin, monophenyltin, and inorganic tin. The isolated strain, CNR22, was identified as Burkholderia cepacia. Strain CNR22 was also found to degrade tributyltin (TBT). After 240-h incubation, a strain CNR22 culture containing additional carbon sources, glucose and L-valine, showed the accumulation of 42 μmol·l^-1 inorganic tin and 17 μmol·l^-1 monobutyltin as the main metabolite from 100 μmol·l^-1 each of TPT and TBT, respectively. The organotin degradation activity was observed in the culture supernatant of strain CNR22 grown without organotin; the reaction was catalyzed by low-molecular-mass iron chelators of microbial origin. These substances degraded TPT and TBT to the corresponding monoorganotin. The monophenyltin (or monophenyltin-chelator complex) produced could be further converted to an inorganic tin (or an inorganic tin-chelator complex) in a sterile medium.

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A medaka (Oryzias latipes) vitellogenin bioassay is a powerful and useful tool for the screening of estrogenic compounds. In order to establish a rapid and simple method for the screening of estrogenic contaminants in an aquatic environment, we studied the effects of several test conditions on the induction of plasma vitellogenin in male medaka. During the holding period, treatments with animal drugs or NaCl for the prevention of an infective disease did not affect the induction of vitellogenin in male medaka. However, plasma vitellogenin in some male medaka was induced when they were kept with female or in water from a fish-breeding tank. During the testing period, vitellogenin induction in male medaka was not affected by the photoperiod cycle and temperature. However, this induction was affected by the frequency of water changing. In the present paper, we propose a rapid and simple method for the screening of estrogenic contaminants in an aquatic environment using a medaka vitellogenin assay. We also present the exposure test results of 4-nonylphenol and bisphenol A obtained using our simple method.

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Alkylphenols and bisphenol A (BPA) in rivers in Chiba City were determined by gas chromatography-mass spectrometry (GC-MS) after extractive derivatization with ethyl sulfate. The relevance of the use of areas and their concentrations was investigated. Nonylphenol (NP) concentrations in the rivers in the surveyed areas ranged from 0.2 to 24μg·l^-1, exceeding the No-Observed-Effect Concentration (NOEC) value (6.08μg·l^-1). 5800μg·kg^-1 NP and 170μg·kg^-1 BPA were observed in bottom sediments from Hanamigawa and Yoshikawa, respectively. These values suggested NP and BPA accumulation in these rivers.

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Very few investigations of the concentrations of estrogens in the effluents of urban drainage systems and in the receiving river water in areas with few constructed sewerage systems have been conducted. In this study, the concentrations of estrogens in the effluents of an urban drainage system and an onsite wastewater treatment plant, and in the receiving river were monitored for six months. The concentrations of estrogens and 17β-estradiol (E2) were determined by enzyme-linked immunosorbent assay (ELISA). Most estrogens were present in the dissolved fraction of the effluents and the river water. E2, which exists in natural waters according to many reports, accounted for 24% of the estrogens and has concentrations of 4.7±3.0 ng·l^-1 (n=6) and 8.6±5.8ng·l^-1 (n=6) in the effluents of the urban drainage system and the onsite wastewater treatment plant, respectively. The concentrations of estrogens and E2 in the receiving river water were two to three times higher than upstream levels. In addition, the concentrations of ammonium and estrogens showed a very high correlation (r=0.986) in all samples, suggesting that the increase in the concentrations of estrogens in the receiving river water was due to the presence of human excrement in the urban drainage system.

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