For In Vitro bioassay system to evaluate water environment, it may be important to correlate In Vitro data closely to whole body toxicity. In order to establish the evaluation system for In Vitro bioassay, we focused on oxidative stress and investigated toxicities of organic halogenated compound using both In Vitro micronucleus test and in vivo hepatic injuries test.
Micronuclei formation of HeLa cells was induced by chlorophenols and the induction was enhanced with addition of S9mix. The enhancement was inhibited by scavengers of hydrogen peroxide, hydroxyl radical or superoxide radical, indicating that micronuclei formation of HeLa cells was induced by oxidative stress which was caused with metabolites of chlorophenols.
Oral treatment of 2,4,6-trichlorophenol or carbon tetrachloride increased the amount of serum GOT. in vivo free radical reactions which were estimated with in vivo ESR technique were also enhanced specifically in upper abdomen, indicating that metabolites of these compounds caused liver injuries through oxidative stress.
The present study demonstrated the evaluation system of In Vitro and in vivo bioassays concerning oxidative stress caused with organic halogens. In Vitro bioassay system will be more valuable and reliable, comparing In Vitro data with in vivo one using indices of same mechanism of toxicity.

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Most drinking water contains suspected carcinogens such as trihalomethanes and other hazardous by-products. These are considered to be produced by chlorination of organic substances.
To screen carcinogenicity, bioassay methods using mammalian cells as well as microorganisms will be inevitably necessary. Thus, we examined the mutagenic activities of the drinking water by Ames test using S. typhimurium (TA100 and TA98) and chromosomal aberration test using cultured Chinese hamster lung cells (CHL/IU).
Drinking water from the Yodo River showed almost as high mutagenicity as chlorinated raw water and sewage effluent, and also the cell nuclei stained by fluorescent dye (Hoechst No. 33258) showed the chromatin condensation evoking DNA fragmentations, and eventually leading to chromosomal aberrations. These indicate apoptosis of the cells but not necrosis.
From the results revealed by the bioassay, the water quality was evaluated from the viewpoint of genotoxicity of drinking water for human health.

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Microcosm system was applied to assess effect of anionic surfactant (LAS) on aquatic ecosystem. Anionic surfactant such as LAS was added to an flask microcosm consisting of four species of bacteria as decomposer, one species of ciliate protozoa (Cyclidium glaucoma), two rotifers (Philodina sp. and Lepadella sp.) and one aquatic oligochaete (Aeolosoma hemprichi) as predator, and a green alga (Chlorella sp.) and a filamentous blue-green alga (Tolypothrix sp.) as producer, comparing with that of an natural lake model ecosystem derived from natural lake water. In the flask microcosm system and the natural lake model ecosystem, biodegradation rates of LAS were almost same and NOECs (no observed effect concentration) of LAS were also below 1.5 mg·l^-1. It was found that flask microcosm test could provide precise ecological effect assessment of LAS on number of microorganisms because the system showed higher reproducibility and stability than natural lake model ecosystem. It was suggested that flask microcosm test was useful ecological effect assessment method which can reflect natural aquatic ecosystem.

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Genotoxicity and cytotoxicity of Tama river water was determined with micronucleus in vitro test and colony formation inhibition test, using a human cultured cell line. River water samples were taken from 5 stations of Tama River ; Hamuraseki, Hinobashi, Sekidobashi, Toumeishita and Marukobashi, and 1 station of its main tributary, Asakawa ; Araibashi. The organic substances in water were once recovered with solid phase adsorbent and then eluted with dichloromethane and methanol in this order. After evaporating, the residues were dissolved in DMSO and used for the assays. The dichloromethane fractions from Sekidobashi and Marukobashi strongly inhibited colony formation but micronucleus formation was not increased by any of them except Araibashi station, suggesting the occurrence of cytotoxic substances in river water from these stations. The methanol fractions from Toumeishita and Marukobashi strongly increased micronucleus formation but colony formation inhibition was not observed with the fractions, indicating the occurrence of genotoxic substances. The results suggest that Tama river water is polluted with genotoxic and cytotoxic substances and that the genotoxic substances have more polarity than cytotoxic ones.
in vitro micronucleus test and colony formation inhibition test using cultured cells are simple and effective methods for evaluating both genotoxicity and cytotoxicity of environmental water.

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Evaluation of genotoxicity of organic extracts from the Yodo River system was examined with umu test and Ames test both using an O-acetyltransferase-overexpressing strain. Organic extracts in river water were collected by the XAD-2 resin column method and the blue rayon hanging method at 4 locations for one year.
The same level of genotoxic activity was observed among the presence and the absence of S9 mix in umu test using S. typhimurium NM2009. But, higher activities were observed in the presence of S9 mix in Ames test using S. typhimurium YG1024. These results shows that S. typhimurium YG1024 in Ames test is highly sensitive to aromatic amines in water environment. There were a significant correlation between genotoxic activity induced by NM2009 in umu test and one by YG1024 in Ames test in the presence of S9 mix. No significant correlation was found between genotoxicities and water pollution indicators. The present results shows that umu test or Ames test using an O-acetyltransferase-overexpressing strain are simple, sensitive and useful bioassay for monitoring the safety of river water.

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Various bioassays have been applied to evaluate safety of environmental water and treated water.
The umu test is a method for the detection of genotoxicity by using SOS function of Salmonella typhimurium.
The protocol of the nmu test is divided into 5 phases, (1) pre-incubation of test strain, (2) addition of samples, (3) induction of umuC gene, (4) β-galactosidase enzymatic reaction, and (5) measurement of absorption in a spectrophotometry and calculation of the activity.
In usual procedure, all phases are performed in batch-type reactors, using test tubes.
In this study, we tried to develop the device of flow-injection style to perform the umu test automatically. The culture was flowed through a tube and added samples by using a rotary pump. The various conditions were investigated and modified for detection of genotoxicity of AF-2.
Even after 24hr of incubation under control, test strain showed activity. Within 1.5hr, the test strain was most active at 36°C and longer than 2.0hr, it was so at 37°C.
At each phases some more conditions have been investigated, and we suggest that new device of flow-injection style could be effective to screen or monitor the genotoxicity of environmental water.

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Evaluation of genotoxicity related to nitrated polycyclic aromatic hydrocarbons (NO₂-PAHs) was investigated on sediment contaminated with road runoff by using test system named umu-test which has highly sensitive potential with nitroarenes. The trace of genotoxicity on NO₂-PAHs could be detected in the effluent from a sewage treatment plant where domestic sewage and rain water in urban area were introduced. And also, we obtained nitrated genotoxicity in the river water where the effluent was discharged. A source of the genotoxicity related nitro-derivatives would originate the sediment contaminated with road runoff, because mobile exhaust gas include many toxicants with nitro derivatives of PAHs. Road runoff water, especially, extracts of particulate matters showed strong genotoxicity on NO₂-PAHs. Genotoxic potency on nitrated hydrocarbons could be obtained in the extract of particulate parts of water of a treatment plant of exhaust gas of motor cars with gasoline or diesel engines. It is possible to detect the trace of NO₂-PAHs by this test system easily. The source and the potency for unit weight of each sample can be evaluated.

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In the previous works, we performed experimental evaluations of cytotoxicity of the Tama-river water and found that the daily cytotoxicity variation of this river water was so large that the water may cause serious effects on human body in a short period of time. From these findings, we confirmed that the rapid and simple bioassay for detecting toxicity should be developed in very near future. As a rapid detection method, LDL (low density lipoprotein)-uptaking activity of mammalian cell is considered to be one of the most rapid endpoints for the cells exposed to toxicic substances. In this work, a novel bioassay based on LDL-uptaking activity was proposed and developed, which successfully detected rive water cytotoxicity very quickly as compared with other methods using cultured cell. The methodology developed in this work is considered to be one of the promising canditates of bioassay sensors for water quality management.

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As part of validation processes of in vitro toxicity assays for the risk assessment of environmental hazards, we applied an in vitro toxicity test using two human cell lines, neuroblastoma NB-1 cells and glioblastoma U-87 MG cells, to the assessment of organochlorine compounds detected in the water environment. The in vitro toxicity assay using NB-1 cells was calibrated by testing reference chemicals proposed by MEIC (Multicenter Evaluation of In Vitro Cytotoxicity), an international program for the validation of in vitro cytotoxicity assays.
Beforehand, an assay using cells in frozen stock without subcultivation was examined by comparing IC₅₀ values with the ordinary assay using subcultured cells. IC₅₀ values for MEIC reference chemicals from the former assay showed good correlation with those from the latter assay, suggesting that the assay using cells in frozen stock can he used at least for the assessment of basal cytotoxicity.
IC₅₀ values for ten organochlorine compounds frequently detected in the sediment samples from contaminated rivers, p-chloroaniline, 3,4-dichloroaniline, p-dichlorohenzene, o-dichlorobenzene, Tris (2-chloroethyl)-phosphate, 2,5-dichlorophenol, 2,5-dichloroanisol, Triclosan and Triclocarban, were obtained with the in vitro assays and compared with their LD₅₀ values in rats. No significant correlation, however, was seen between the IC₅₀ and LD₅₀ values, indicating that further improvement of in vitro toxicity assays is necessary for the application to the risk assessment of environmental hazards.

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Interactions of binary mixutues among organochlorine compounds detected in the water and sediment of urban rivers were analysed in cultured limb bud cells using Toxic Unit analysis and isobole diagram.
Type and mode of action of mixtures vary depedns on the mixture ratio and end points (chondrogenesis, cell proliferation) which resulted in varied potential of developmental toxicity among mixtures.
As limb bud cell culture (LBC) assesses cell differentiation and cell proliferation simultaneously, LBC combined with Toxic Unit analysis and isobole diagram provides useful tool for analysis of interation of mixture.

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Seasonal and basinal variations of direct mutagenicity of Nakdong river in Korea, were investigated by Ames test. Nakdong river is 512.5 km from middle of Korea to Pusan and the river water is the source of water supply of Taegu and Pusan cities. Semiconductor, dye and food plants and domestic wastewater arc inflowed to upper and middle stream of the river. Dissolved substances in water were recovered with XAD-2 resin adsorption method.
The adsorbed substances were eluted with hexane, dichloromethane and methanol in this order. The mutagenicity of each fraction was estimated by Ames test without S9 mix. The samples in May, 1991 showed high mutagenicity with TA98 strain while no mutagenicity with TA100 one, suggesting that Nakdong river is polluted by frame-shift type direct mutagens. To estimate the origin of mutagenicity in this river basin, river waters were taken from 5 stations of Nakdong river, 1 station of Kumho river and 1 station of Jinchun stream, tributaries of Nakdong river, for one year from May, 1995. Similar results to May, 1991 were observed showing pollution with frame-shift type direct mutagen. The present results suggest that wastewater from dye and food plants located in the tributary may contribute to the pollution of direct mutagenicity in Nakdong river.

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As sewerage has spread in Japan, evaluation of the effects of sewage and treated sewage on aquatic organisms has been becoming important. In this paper, applicability of two bioassays to sewage samples was discussed. One is an algal growth inhibition test using the green alga, Selenastrum capricornutum, proposed by Environment Canada that is conducted in a microplate for simplicity of handling. Because sewage samples include high concentration of nutrients, the test medium was modified here. The other is teratogenicity test with embryos of the frog, Xenopus laevis proposed by ASTM. Application of both the assays to sewage samples has been little reported. Therefore, we applied these bioassays to sewage, treated sewage and its receiving water at three sewage treatment plants (STPs). MICROTOX acute toxicity test was also conducted for comparison with the above bioassays. As a result from all the bioassays, the following findings were observed : i) significant reduction of the toxicity of sewage during biological treatment in all the STPs, ii) no significant toxicity of disinfection with ozone and low-chlorine-dose, iii) high toxicity due to disinfection with high-chlorine dose from MICROTOX test, iv) toxicity due to high-chlorine-dose from algal test even after dechlorination, v) no significant toxicity of high-chlorine-dose from frog embryo test. Considering the above results, these bioassays are useful for evaluating the toxicity of sewage samples to each organism.

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Leachate from a landfill site of old landfill-age are containing refractory organics and high concentration of ammonium nitrogen. It is not very clear how refractory organics from advanced treatment of landfill leachate behaves in natural environment. In this study, four treatment processes were employed, including biological activated carbon fluidized bed (BACFB) process, pre-ozone Ireatuent-BACFB process (ozone-BACFB), internediate ozone treatment-BACFB process (BACFB-ozone-BACFB) and post-ozone treatment-BACFB process (BACFB-ozone).Then landfill leachate and process treated water from the treatment processes were evaluated for assessment of the performance of the above processes from biological oxygen consumption point of view. The intermediate ozone treatment-BACFB process removed 77% of dissolved organic carbon (DOC). The biological oxygen consumption over a period of 5 days of a landfill leachate was 20 mg·l^-1, but the value of that a period of 100 days was over 300 mg·l^-1. Therefore, refractory organics in landfill Ieacate can become source of organic pollution. On the other hand, the biological oxygen consumption over a period of 100 days of effluent from intermediate ozone treatment-BACFB process was 15 mg·L^-1, much lower than that of the landfill leacate. Intermediate ozone treatment-BACFB process was thus efficient advanced treatment system from biological oxygen consumption point of view.

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The toxicity of monochloramine (NH₂Cl), which is the main by-product of sewage effluent chlorination, was evaluated from growth inhibition in different condition of nutrients by a green alga Selenastrum capricornutum. The effect of NH₂C1 on algal growth was depended on the nutrients condition, and nutrients limited algal cells were more sensitive than the non-limited cells. When using the nutrient-limited algal cells for the test organisms, lowest-observed-effect concentration (LOEC) and 50% effective concentration (EC₅₀) after 96 hours exposed period were 0.001 and 0.002 mg-Cl₂·l^-1, respectively.
The obtained LOEC and EC₅₀ were lower than the water quality criteria for residual oxidants and the detection limit of oxidant determination. It is clear that chemical analysis is insufficient to monitor the various effluents. From these results, an algal growth test using the nutrient-limited algal cells is very sensitive and useful for the toxicity evaluation of disinfected effluents.

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Chlorinated organic compounds has high priority for toxicity screening among environmental hazardous chemicals. In the present study, we used immortalized rat hepatocytes as a liver model in vitro to evaluate the toxicity of nine chlorinated organic compounds. Toxicity of nine chlorinated organic compounds were evaluated to cellular viability of immortalized rat hapatocytes. The potency of the toxicity based on 50% inhibitory concentration (IC₅₀) value was in the following order : triclocalban>triclosan>3,4-dichloroaniline>2,5-diclorophenol>2,5-dichloroanisole>p-dichlorobenzene>p-chloroaniline>o-dichlorobenzene=tris (2-chloroethyl) phosphate. The rank order of cytotoxic potency of nine chemicals was compared with toxicity information using animals. The rank order of cytotoxic potency did not relative to the order referenced mean lethal dose (LD₅₀) as an index of acute toxicity of rats or mice. However, the rank order of cytotoxic potency relatively correlated non-observed adverse effect level (NOAEL) under the exposure duration adjusted for chronic toxicity in vivo. These data suggests that the origin of testing cell had better to make match target organ of toxic chemicals for extrapolation from data of bioassay in vitro to in vivo.

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We investigated the possibility of a method to monitor pesticides in water by assaying effects of organophosphorus and carbamate pesticides upon acetylcholinesterase (AChE) activity. The procedure is very simple as follows ; the human red blood cell suspension was added to sample water and then AChE activity was detrmined by autoanalyzer.
Using this method, we assayed 33 kinds of organophosphorus pesticide, 8 kinds of its oxon, and 12 kinds of carbamate pesticide.
Nineteen out of the 33 organophosphorus pesticides, 7 out of the 8 oxons of organophosphorus pesticide, and all of the 12 carbamate pesticides tested were detected at the concentration of less than 1mg·l^-1.
Even at the concentration 100ng·l^-1, EDDP, Benfracarb, Chlorpyrifos oxon, Isoxathion oxon and Diazinon oxon were detected.
In addition, 13 pesticides were detected at the concentration of 100ng·l^-1 in the water containing 1mg·l^-1 chlorine, which is equivalent to the chlorine level in water disinfection process.
This analytical method, although the detectable concentration levels are varied widely according to the type of pesticides, is capable of detecting many of organophosphorus and carbamate pesticides in water.

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