In water treatment, ultraviolet irradiation, ozonation, and chlorination are believed to produce various active oxygen species, which seem to participate in reactions with fumic acid, pollutants and bacteria. It was, however, very difficult to determine the actual kinds and amount of active oxygen during the processes because of deficiencies of method. In order to determine the kinds and amounts of active oxygen species which may participate in water treatment process, the spin-trapping ESR (electron spin resonance) technique was applied to measuring unstable free radicals generated during chlorination and ozonation. As spin-trapping reagent, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was dissolved in phosphate-buffered water, and the solution was treated by either chlorination or ozonation. Immediately after the treatment, ESR spectrum was measured at room temperature. Typical ESR spectra due to DMPO-OH were observed from both treatments, indicating the generation of hydroxy radical in chlorination and ozonation. In addition to DMPO-OH, several different signals were observed and their appearance depended on the chlorine or ozone concentration.

In order to examine whether cytotoxicity test is available as bioassay of water micropollutants, cytotoxicities of 40 chemicals, which have been reported to occur in drinking water, were investigated using 3 different types of cultured cells including HL-60 cells, mouse peritoneal macrophages and rat liver hepatocytes, and the results were compared with their mutagenicity and carcinogenicity. Cancer promotion activity was assayed by use of morphological change of HL-60 cells. Lethality of chemicals was measured with trypan-blue exclusion test of HL-60 cells or lactose dehydrogenase released from rat hepatocytes. Influence of chemicals on cell functions was examined with phagocytic activity of macrophages and glycogenolytic activity of hepatocytes. Some of the chemicals showed strong cytotoxicities which depended on the structure of chemicals and the nature of cells, while cancer promotion activity was not found in any sample. Among the 3 tests, viability test using HL-60 cells was the most susceptible to the chemicals. No correlation was observed between the results of cytotoxicities and their mutagenicity. The results indicate that the combination of some cytotoxicity tests should become effective both to estimate the safety of drinking water and to identify the hazardous micropollutants.

In order to establish the method of bio-assay for the safety of drinking water, mutagenic activity, promotion activity for cancer, cytotoxicity and liposomal membrane damage of micropollutants in water were studied. The dissolved substances in water were recovered from raw, chlorinated, and rapid-filtrated waters by adsorption with XAD-2. Mutagenicity of the recovered substances was estimated with Ames Salmonella mutagenicity test. Cancer promotion activity was assayed by use of morphological change of HL-60 cell. Cytotoxicities of the recovered substances were evaluated with the cultured cells, HL-60 and mouse peritoneal macrophages. Some of the samples showed strong direct mutagenicity and cytotoxicities, while cancer promotion activity was not found in any sample. No correlation was observed between the results of mutagenicity and cytotoxicities, while the results of cytotoxicities related to those of liposomal membrane damage. The findings indicate that the combination of mutagenicity test with the other bio-assay such as cytotoxicity should be necessary to secure the safety of drinking water.

Identification of the species of active oxygen generated in water by chlorination or ozonation was carried out by the spin trapping method using DMPO as the spin trap agent with ESR spectrometry. On chlorinating with NaOCl solution (15 mg/litre), typical ESR signals for DMPO-OH adduct were observed. Increasing the concentration of NaOCl, however, caused complex spectra to appear. The addition of thiourea or D-mannitol, as a radical scavenger, did not significantly affect on the intensity of the signals. On ozonation, two radicals, DMPO-OH and an unknown radical, were always observed. These results indicate the generation of several radicals in the solution treated with NaOCl and ozone. An interaction of the polio virus with ♦OH was also studied. The preliminary test showed that the ♦OH generated by Fenton's reaction slightly disinfected the virus. Precise tests changing the concentration of active oxygen and the reaction condition are currently in progress.