This paper reviews the literatures on toxicity bioassay for sea water and chemicals by using sea urchin eggs. The test methodologies pertaining to various stages of the life cycle, e.g. gametes (egg and sperm), fertilization and development (embryo and larva) are described. Especially, the latest method to enhance the sensitivity, by improving the previous methods of marine pollution bioassay using sea urchin eggs presented by the author (Kobayashi, 1971, 1974, 1977 and 1985 etc.), is introduced. The effects of various waters and chemicals upon the eggs and embryos in respective developmental stages at first cleavage and pluteus are observed. Rates of first cleavage, pluteus stages and some anomalies of development in the test water are examined. On the basis of these experimental results, a new ranking of the sea water pollution (Ranking IV, 1990) is proposed. The latest method is more sensitive, and simple than those in the cases of ranking I and III (1972 and 1985).
Both the results of laboratory experiments and of field assays for environmental assessment are included. With regard to the choice of the test organisms for pollution bioassay, the international use of urchins as test species is to be encouraged, because a wide and abundant distribution should be required for candidate species.

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In environmental water has been reported the occurrence of thousands of chemicals, in which there are many toxic substances including carcinogens. These hazardous micropollutants may be not completely removed by conventional water purification process. To secure the safety of drinking water, it is highly necessary to monitor these toxic substances. For this purpose, simple bio-assay systems such as Ames test have been widely developed to monitor mutagenic micropollutants in water. In the present paper, the principle and limitation of bio-assay system were discussed. Based on the discussion, we examined whether cytotoxicity test is available as bioassay of water micropollutants. Cytotoxicity test using cultured HL-60 cells, liposomal damage test, and Ames mutagenicity test were applied to the substances recovered from tap and raw waters. No correlation was observed between the results of mutagenicity and cytotoxicity, while the results of cytotoxicity related to those of liposomal membrane damage. These results indicate that cytotoxicity tests will be effective to estimate the safety of drinking water and that a concept of cytotoxicity matrix may be available for identification of toxic micropollutants.

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The runoff loadings and characteristics of pesticides applied to paddy fields after transplanting of rice plant were estimated by means of analysis of pesticides in drainage river water in weekly and at the rainfall events.
The largest loadings of herbicides were found between one and two weeks after transplanting of rice plant. On the other hand, the largest loadings of insecticides were found at about ten days later than that of herbicides.
The runoff loadings of herbicides during one rainfall event ranged 20 to 25% of total runoff amounts. Therefore, the runoff investigation of pesticides in river water at and after the rainfall are very important for estimation of pesticides runoff from agricultural fields to drainage river.
The runoff characteristics of insecticides and herbicides were differed, and it was recognized that the runoff loadings of insecticides were decreased more rapidly than herbicides at the rainfall event.
The runoff rates of pesticides were ranged from 25% for fenobucarb to 0.02% for fenitrothion.
From this results concerned in rainfall event, the runoff rates of pesticides from agricultural fields to river were more higher than that of the previous literature.

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Environmental behavior and fate of butyltin compounds in a water-sediment system has been studied.
Following results on butyltin behavior have been obtained experimentally :
1) Sea water-sediment soil partitioning coefficient is 1,500-3,000 (l·kg^-1) both for dibutyltin (DBT) and tributyltin (TBT) when the sorption is reversible ;
2) Degradation of TBT in anaerobic sediment seemed to be abiotic and its half life is 460 days ;
4) Flux acorss the water-sediment interface can be represented by the film model for butyltin compounds, and its transport coefficient is order of 10^-6(m·s^-1) and affected by turbulence of the overlaying water ; and
5) TBT desorbs from sediment soil more easily than DBT : spontaneous desorption ratio are 0.16 and 0.015 (-), and first order desorption kinetic coefficient are 2.3×10^-4 and 4.2×10^-5 (s^-1) respectively.
Fate analysis of TBT in a sea water-sediment system shows ;
1) TBT from paint chips has severer impact (raising concentrations and high persistence) than dissoluted TBT ; and
2) Resuspension of highly contaminated sediment causes recontamination of the overlaying water : a few centimeters of erosion of TBT contaminated sediment will raise the concentration in the overlaying water up to the level of the pore water.

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Biodegradation of organic substances by attached bacteria on biological activated carbon (BAC) was studied to clarify the advantages of granular activated carbon (GAC) as a support medium over conventional medium without adsorption capacity with regard to biodegradation activity and community structure of attached bacteria. Anthracite (AN) was used as a reference support medium without adsorbability. Low molecular organic substances with different biodegradability and adsorbability (phenol, benzoic acid, glucose and m-aminobenzoic acid) were fed into completely mixed BAC and AN reactors. The rates of biodegradation by BAC reactors fed with the biodegradable organic substances were approximately 3 times as high as those by AN reactors. The difference in adsorbability of the organic substances onto GAC had little effect on the rate of biodegradation. m-Aminobenzoic acid with low biodegradability was degraded only by the BAC reactor. The structure of GAC with micro and macro pores did not provide better habitat for attached bacteria with regard to the size of population in comparison with AN without pores. Bacterial floras on GAC was probably little difference from those on anthracite. Therefore GAC as a bacterial support medium stimulated the biodegradation activity of each bacterium without increase in the population and probably with little change in the species composition.

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It has been commonly known that mercury is one of the most toxic metals and that organic mercury is more toxic than inorganic mercury. This paper treats relationship between bioaccumulation and toxicity of various chemical forms of mercury on cell growth of the yeast Saccharomyces cerevisiae, DNA damage by mercury and effect of chelate agents on mercury toxicity.
Four kinds of strains of S. cerevisiae were used. They are a wild type (AOY1) and three mutants. CA11-mutation repair-deficient mutant (rad18), CA13-exicion repair-deficient mutant (rad2) and CA15-recombination repair-deficient mutant (rad52) for DNA damage test.
In terms of EC₅₀ toward AOY1, the order of toxicity was Hg(NO₃)₂ > HgSO₄ > HgCl₂. For the same nitrate mercury, monovalent mercury(HgNO₃) was more toxic than divalent mercury(Hg(NO₃)₂). Toxicity of organic mercury(CH₃HgCl) on cell growth was two order higher than inorganic mercury(HgCl₂). Comparing with the survival rate after 8 hours of inoculation and the content of both chemical forms of mercury in cells, the survival rate for CH₃HgCl was about one hundredth of HgCl₂, but the concentration of CH₃HgCl in cells was about 170 times higher than that of HgCl₂. Addition of chelate agents, EDTA and methyl penicillamine to the medium did not modify the toxicity by mercury. The mutant which is deficient in recombination repair system was most sensitive for mercury among three mutants mentioned above.

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In resent years, some of dioxin congeners have been detected in landfill leachate and drainage of paper mill and causing serious social problems. Appropriate methods for treating these congeners are urgently needed.
This study was undertaken to develope the method to biodegrade the dioxin congeners using immobilized Pseudomonas aeruginosa which is able to utilize specifically dibenzofuran (DBF), fundamental element of dioxin. As an immobilizing chemical, polyethyleneglycol, polyacrylamide and sodium alginate were tested. The pellets immobilized with sodium alginate were unsuitable for using as the immobilizing agent because they dissolved extensively in a growth medium. The optimum temperature for degradation of DBF was found to be 30°C and the optimum pH was about 7. The biodegradation of DBF was significantly enhanced in the presence of landfill leachate.
In a long-term batch cultivation system, it was made clear that the physical strength of the pellet was maintained and the degradation activities of the immobilized microorganisms remained high. In a continuous cultivation system, DBF was degraded as efficiently as in the batch cultivation system.

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Umu test for detecting the SOS-inducing activity (genotoxicity) using Salmonella typhimurium TA1535/pSK1002 has been applied for studies of genotoxicity of chlorine-treated amino acids. Five amino acids tested (glycine, lysine, methionine, proline, serine) showed the SOS-inducing activity after the chlorine treatment. The SOS-inducing activity of the chlorine-treated amino acid solution was relatively stable at freezing condition, though the activity decreased rapidly at room temperature. Formation of the SOS-inducing substancs were effected to the concentration of amino acids in the reaction mixture. The suitable concentration of amino acids for formation of SOS-inducing substaces was different from each amino acids.
SOS-inducing activity of some halogenated organic chemicals such as halo alcohol and halo acetonitrile which were detected in the amino acids and chlorine reaction mixture were observed. However, 11 wellknown halogenated chemicals (mono-, di-, tri- chloroacetic acid, mono-, di-, tri- chloroacetonitrile, mono-, di- chloroacetone, 2,2'-dichloro-ethyl ether, chloral hydrate, nitromethane) did not show SOS-induction.
Present studies indicate that harmless amino acids could be converted into genotoxic substances by chlorine treatment. To clarify the structure and formation mechanisms of genotoxic products, more detailed studies are concidered to be necessory.

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First of all, 1,1,1-trichloroethane (MCF), tetrachloroethylene (PCE) and trichloroethylene (TCE) concentrations of 51 well waters in Nagaoka City of Japan were measured in 1989. Maximum, minimum and average concentrations of MCF were 20.0, below 0.1 and 1.8μg·l^-1. Those for PCE were 2.27, below 0.02 and 1.26μg·l^-1, and for TCE were 2.29, below 0.01 and 1.17, respectively. Many wells at Sekihara area, which is in the west of the city, were contaminated exclusively with MCF.
Next, detailed study of the groundwater quality at Sekihara area was carried out both in 1989 and 1991. The number of the wells was 17 in 1989 and 24 in 1991. The maximum, minimum and average concentrations of MCF were 18.6, below 0.1 and 2.9 in 1989, and 173.4, 0.1 and 9.2 in 1991. No decrease in the MCF concentration between 1989 and 1991 was observed at all. The source of the MCF was suspected to be an electric factory in this area.
Last, halogenated organic chemicals in the wastewater discharged from the factory were analyzed in two drain ditches. Sampling was carried out at the interval of 3 hours for a day. Considerable amount of MCF and total organic halogens (TOX) was detected.The TOX amount in a ditch, of which water is originally coming from groundwater, was 9.3g in a day and in another one for rainwater drain was 144g during the run-off of 6 hours' rainfall. The ratio of MCF to TOX was very small in the both case.

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Soil gas was collected and analyzed for tetrachloroethylene to examine the possibility of soil gas monitoring as a tool for identifying pollutant source and delineating plume boundary of pollution in specified groundwater. Field surveys in a shallow groundwater region show that tetrachloroethylene concentration in soil gas detected around pollutant source seems to be affected by gaseous diffusion from highly contaminated soil. With distance away from pollutant source, it is becoming in a possitive relationship to the tetrachloroethylene concentration in shallow groundwater. In particular, soil gas monitoring is recognized to be an effective technique for identifying pollutant source, as tetrachloroethylene concentration contours displayed around pollutant source are extending like concentric circles.

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The change of microbial phase in the course of acclimation to hard-biodegradable chemicals such as dimethylformamide (DMF), which is widely used as solvent and discharged from chemical industries, was analyzed on the basis of quinone profile in the aerobic biofilter to assure the optimal operating condition for the further improvement of their removal from wastewater. The microbial film cultivated by feeding the artificial wastewater containing glucose and peptone (GP wastewater, hereafter) was acclimated to DMF within 3 weeks after the onset of continuous feeding of DMF, and a remarkable change in the quinone profile was observed along with the acclimation. The predominant quinone of the microbial film cultivated on the GP wastewater was featured ubiquinone (Q)-8, and those on the mixture of GP wastewater and DMF and on DMF as a sole carbon source were MK-9 (H₂) and Q-10, respectively. The predominant bacteria in the film could be changed from the β-group of Proteobacteria, generally inhabiting the activated sludge, to the facultative C₁-using bacteria such as Mycobacterium and to the obligate C₁-using bacteria such as Methylobacterium along with the acclimation. Relationship between the removal efficiency of DMF and the change in quinone profile was clarified in the aerobic biofilter.

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Linear alkylbenzenesulfonates (LAS) and alcohol derived surfactants are common in commercial household detergent applications. These surfactants are sometimes found in river water, and in few cases foam is observed on river water.
The foam properties of these surfactants in water were studied and compared with 0.5 mg·l^-1 of alkylbenzenesulfonates (ABS) at which foam was well known to be limited. The surfactant solutions in test tubes were shaken for 5 min by mechanical shaker at about 3 times a second, allowed to stand and foaming recorded immediately and after 30 s, 1 min and 2 min. Foaming depended on a kind of surfactant and alkyl chain length of a surfactant molecule. The concentrations at which foaming is equal to ABS 0.5 mg·l^-1 are 0.3-0.4 mg·l^-1 for LAS, 0.2-0.4 mg·l^-1 for alkylpoly(oxyethylene) sulfates (AES) and alkyl poly(oxyethylene) ethers (AE) and 0.5-1.1 mg·l^-1 for α-olefin sulfonates (AOS). LAS, AES, or AE which generate much foam as compared with ABS are dominant in river water. AOS which are less reamers are little in river water. Then in river water the concentration at which foaming is equal to ABS 0.5 mg·l^-1 is about 0.3 mg·l^-1 for anionic surfactants only, and with nonionic surfactants it is less than 0.3 mg·l^-1 as anionic surfactants.

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Runoff loadings of linear alkylbenzensulfonates (LAS) were observed weekly from June 1990 to May 1991 in eight inflowing rivers to Lake Kasumigaura.
Annual maximums and means of LAS concentration ranged from 0.047mg·l^-1 in a rural river to 1.45mg·l^-1 in an urban river, and from 0.012mg·l^-1 in the rural river to 0.472mg·l^-1 in other urban river, respectively. Seasonal means of LAS concentrations became maximum in winter and minimum in spring or summer due to the biodegradation depended on water temperature during flowing-down. Because LAS loadings were influenced by flow, both maximums and minimums among their seasonal means of loadings were dispersed in three seasons. The loadings of LAS homologue with long alkyl chain length between 13 and 14 became less in rivers of bigger catchment areas, and the isomer loadings with external phenyl position became similar to that. Annual LAS loading per capita, ranged from 24 to 528g·y^-1·cap.^-1, showed almost log-log linear relationship to river catchment area. Annual runoff rate of LAS loading, ranged from 0.07 to 1.45, could be expressed as a function of logarithm of river catchment area. The annual loading per capita and its runoff rate were low level in large rural rivers and high in small urban rivers.

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Colony formation assay using L-929 cells was applied to 40 chemicals which were reported to occur in drinking water. In the 40 chemicals including 4 of specified chemical substances, 7 of pesticides, 19 of industrial chemicals and 10 of halogenated hydrocarbons, some of pesticides and halogenated hydrocarbons inhibited strongly the colony formation of L-929 cells, and the number and position of halogen in the chemicals influenced their cytotoxicities. The results were compared with those by the other cytotoxicity tests involving viability of HL-60 cells, phagocytic activity of mouse peritoneal macrophages, and LDH release or glycogenolytic activity of rat hepatocytes, and cytotoxicity matrix were obtained from the comparison of these four cytotoxicity tests. Availability of the cytotoxicity matrix is discussed to estimate the micropollutants in water.

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Determination method of organic carbon in suspended solid (SSOC) in urban surface water was investigated with the use of elemental analyzer.
Detection limit of SSOC on filter resulted in 3.4μg·cm^-2 by using no-binder glass fiber filter for water sample substrate and elemental analyzer for quantitative analysis. On the analysis, the temperature of the combustion furnace of elemental analyzer was 550°C. Influence of inorganic carbon in sample was negligible. This method proved to be useful with the reasons that precise SSOC data were available, and that filter sample was obtained from the process of suspended solid analysis and that data of elements such as hydrogen, nitrogen were obtained at the same time by elemental analyzer.

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We conducted an investigation with the levels of organic chlorinated compounds such as trichloroethylene (TCE) and dichloroethylene (DCE) in groundwater of wells at 62 poists in Kawasaki city. From this investigation, cis-1, 2-DCE was detected at the higher percentage than other chlorinated compounds.
High concentrations of TCE, tetrachloroetylene (PCE) and DCE were found in the Takatsu-ku area, where it was presumed that the PCE pollution of groundwater was came from soil polluted by any source before then.

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