To apply the yeast two-hybrid test for the evaluation of river water, an effective method of concentrating estrogenic substances and a method of removing inhibitors were developed. When 250 ml of river water was supplied to a Sep-Pak Plus PS-2 cartridge and then 10 ml of acetone was used for desorption, typical estrogenic substances were recovered by as much as 93-101% and were concentrated quickly to 500-fold. Moreover, when the concentrated water sample was supplied to a column containing 1.5g of silica gel and was eluted with 8 ml of 30% acetone in toluene, inhibitors of estrogenic activity were removed. The validity of these methods was confirmed using actual river water samples obtained from 19 sites of the Sagami River and 7 sites of the Ayase River.

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In recent years, pharmaceuticals in the aquatic environment have attracted much attention in Europe and North America due to their potential to increase antibiotic resistance in microorganisms and their ecological effects on aquatic organisms. Little information, however, has been reported from Japan. In this study, we report on the levels of human and veterinary pharmaceuticals in aquatic environments in Japan. A liquid chromatograph coupled with a tandem mass spectrometer was used to detect these compounds. The results showed that certain pharmaceuticals were detected at ng·l^-1 level. The concentrations of human pharmaceuticals in rivers located in urban areas were higher than those in suburban areas, while pharmaceuticals mainly used for veterinary medicine were found of higher concentration in suburban areas. Human pharmaceuticals were also detected the sewage treatment plant effluent. In agricultural wastewater, some antibiotics for veterinary use were detected at high concentrations of up to μg·l^-1 level, which indicated possible ecological effects in some limited areas.

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Antibiotics are used extensively in medicine, livestock raising and aqua culture. The spread of antibiotic resistant bacteria from hospitals and livestock farms into the environment has become a public health concern. The information on the occurrence of resistant bacteria in water bodies, however, is very scarce. In this study, about 40 E. coli strains were isolated from river waters collected in the Kaname, Tama and Tsurumi Rivers. The Kaname River receives a large amount of agricultural effluent, while the Tama and Tsurumi Rivers contain a large percentage of sewage treatment plant effluent. Antibiotic resistance patterns were determined by detecting resistance against nine types of antibiotic. The pattern of the Kaname River water was different from those of Tsurumi and Tama River waters. E. coli strains isolated from the Kaname River were determined to originate from livestock feces, and E. coli strains isolated from the Tsurumi and Tama Rivers were of human origin.

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Most criteria of contaminants in aquatic ecosystems are based on their acute or chronic toxicity examined during short periods. However, it is important to evaluate the effects of long-term, low-level chronic exposure of populations to multiple stressors and to identify causative stressors for the conservation of the aquatic ecosystem. In this study, we present a novel approach to identifying multiple stressors to Psedorasbora parva by amplified fragment length polymorphism (AFLP) analysis. Pseudorasbora parva was captured in habitats contaminated mainly with sewage, treated sewage or agricultural effluent. We also analyzed 14 polycyclic aromatic hydrocarbons (PAHs) and 7 heavy metals in the sampling sites. The genetic diversity of the population calculated by AFLP analysis decreased with increasing contamination of their habitats by treated sewage or sewage. Genetic diversity was significantly correlated with concentration of dissolved benzo[k]fluoranthene, dissolved lead, and dissolved manganese. Nickel in sediments was also significantly correlated with the genetic diversity. Some of these contaminants might be acting as strong selective agents that decrease the genetic diversity of the population. Using fluorescence-based AFLP data, we could classify amplified fragment length patterns into several groups and they were found to correspond with the contamination properties of the sampling sites. These genotypes distributions in rivers and lakes may show the direction of selective forces to Psedorasbora parva.

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In recent years, antibiotics resident in sewage and in the water environment have become an emerging public concern in many developed countries. However, limited knowledge is available on the occurrence of antibiotics in sewage and discharge from wastewater treatment plants (WWTPs) in Japan. Moreover, little is known on the significance of their occurrence in the water environment from the viewpoints of biological adverse effects. The objectives of this research were, therefore, to determine the occurrence of selected antibiotics, namely levofloxacin (LVFX) and clarithromycin (CAM). That are commonly used in Japan, in discharge from WWTPs and then to evaluate their possible effects on algal growth. Therefore, we developed a novel analysis method for LVFX and CAM in wastewater by LC/MS/MS whose detection limits and recovery ratios are 2-3ng·l^-1 and 53-87%, respectively. We also conducted algal growth inhibition tests using Pseudokirchneriella subcapitata, and results showed that the EC₅₀s of LVFX and CAM are 1200μg·l^-1 and 11μg·l^-1, LOECs are 630μg·l^-1 and 6.3μg·l^-1, and NOECs are 310μg·l^-1 and 3.1μg·l^-1, respectively, LVFX and CAM concentrations in secondary effluent of five WWTPs that use the activated sludge process ranged from 152-323ng·l^-1 and 303-567ng·l^-1, respectively, which indicates that the PEC/PNEC ratio of LVFX is less than one but that of CAM exceeds two at the maximum secondary effluents if a safety factor of ten is considered. This suggests a possibility of algal growth inhibition due to CAM in WWTP discharge in the case of insufficient dilution of the receiving waters.

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A simple and rapid method was developed for the determination of zinc, cadmium, copper and lead at the mg·kg^-1 level in soil samples by anodic stripping voltammetry using a mercury film microelectrode (surface area, 240μm²). Pre-electrolysis was performed at -1.3V vs. Ag/AgCl for 2min with stirring in a 0.1mol·l^-1 acetic acid-sodium acetate buffer solution (pH 4.2). After the stirring was stopped, anodic stripping voltammograms for deposits were recorded in the potential range of -1.3 to 0.15V vs. Ag/AgCl at a scan rate of 50mV·s^-1 in a differential pulse mode. Calibration curves (peak height vs. concentration) were linear (correlation coefficient >0.999) over the 0.2 to 10μg·l^-1 concentration range with a relative standard deviation of approximately 5% for 1μg·l^-1 (n=5). The detection limits (3σ) calculated from the repeated determination of 0.1μg·l^-1 metal ions for a deposition time of 2min ranged from 0.04 to 0.07μg·l^-1. Possible interferences were evaluated. This method was successfully applied to the simultaneous determination of zinc, cadmium, copper and lead in several soil samples without any separation steps of matrix elements. The time required for an analysis was within 3min.

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A fluorescence analysis method, which focuses on the fluorescence intensity of fulvic-like organic matter measured at λ_ex320nm/λ_em430nm, was applied to evaluate the characteristics of dissolved organic matter (DOM) in environmental waters. The duplicated analyses of a clean river water sample showed that this method was more sensitive by one order of magnitude than TOC and UV₂₆₀ methods in the DOC measurement. From the results of its application to spring waters in Saitama prefecture, the method was proved to be effective for the evaluation of water purity in terms of DOC. Moreover, fluorescence excitation-emission matrix spectroscopy (EEMs) was also proved to be effective for the discrimination of spring waters which had unusual DOM compositions. From the results of its application to water samples obtained from various rivers in Japan, it was concluded that the fluorescence excitation spectrometric analysis, which is a modification of our original method with three wavelengths, is effective for the removal of the influence of the fluorescence emitted by fluorescent whitening agents (FWAs) in treated domestic wastewater and gray water. Moreover, the fluorescence intensity at λ_ex320nm/λ_em430nm showed a very strong positive correlation with DOC.

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Methodology development for the population-level ecological risk assessment of chemicals with potential endocrine disruption has become a research focus in recent years. A multigeneration full-life-cycle experiment on 4-nonylphenol (4-NP) using S-rR strain medaka was designed and conducted with this focus. This paper provides an overview of the experiment and presents some of the important results of the experiment. The experiment was designed for three generations (approximately 70 days per generation) to determine 1) the effectiveness and biological significance of each of the orthodox endpoints for endocrine disruption from the standpoint of population-level impact, 2) the correlations between the respective endpoints, 3) the possibility of intergenerational sensitivity, and 4) the incidence between the males and females, S-rR strain medaka was employed due to its biological merit of hereditary difference in body color for male and female, which is useful for the detection of a slight endocrine disruption effect on secondary sexual characteristics and reproduction. Seventeen β-estradiol (E2) compounds were used as a positive control. Fertility, the frequency of testis-ovum appearance in the male gonad, and the secondary sexual characteristics were found to have good dose-effect relationship. None of strengthened effect expressed in most of the measure endpoints for 4-NP and E2 were observed to cross from one generation to another. Males were more sensitive than females.

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A rapid and simple method for the determination of nonionic surfactants is described. The method is based on the formation of an ion associate with a tetrabromophenolphthalein ethyl ester potassium salt and a potassium complex of Triton X-100 as an example of a nonionic surfactant. The ion associate is adsorbed to the inner wall of the vessel with vigorous shaking. After the complete removal of the solution in the tube, the ion associate was dissolved in ethanol and quantified spectrophotometrically. The proposed method is simple and rapid. The linear calibration curve for Triton X-100 was obtained at 1-5mg l^-1. The coefficient of variation for Triton X-100 at 5μg·l^-1 was 8.75% (n=9). The influence of the anionic surfactant used in this method was blocked by treatment with an anionic exchange resin.

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Many types and large quantities of detergents and hair care products are used in homes. If they are directly discharged into rivers, they may lead to serious pollution of river water, and have hazardous effects on aquatic organisms. Their toxicity was determined by the acute immobilization test using Daphnia magna as 48h-EC50 (concentration required to immobilize 50% of the daphnids at 48h exposure). The most toxic products are fabric softeners and laundry detergents. Shampoos and conditioners are less toxic. A significant difference in the 48h-EC50 values was observed among shampoos, but for the hand dishwashing detergents, less difference in the 48h-EC50 values was observed. The toxicities of detergents and hair care products in home wastewater were evaluated by concentration-48h-EC50 ratio in home wastewater. The laundry detergents group showed the largest concentration-48h-EC50 ratio of 5.9, and the conditioners or shampoos showed the smallest of 0.14 to 0.22. The concentration-48h-EC50 ratio was changed significantly by the selection of household detergents and hair care products with the most or least toxicity. The development of low-toxicity laundry detergent will lead to the reduction of the concentration-48h-EC50 ratio of home wastewater. The effect of popularization of dishwashing machines and conditioning shampoos on wastewater toxicity is also discussed.

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