The discovery of the ozone hole over the Antarctica and various research works to find the cause of such hole revealed that the present regulation of ozone depleting substances may not be sufficient for the adequate protection of the ozone layer. On the basis of the latest findings indicated by the world scientists in the four assessment panel reports pursuant to the Article 6 of the Montreal Protocol (science, environmental effects, technical and economic aspects) and other information, the Parties to the Montreal Protocol adopted the amendment and adjustment of the Protocol in June 1990, at the Second Meeting of the Parties to the Montreal Protocol. This document briefly reviews the latest scientific and technical information for the amendment and adjustment of the Protocol, and the major thrusts of the amendment and adjustment, including elimination of the production and consumption of controlled CFCs by the year 2000, and the problems which are still left before us to be solved.

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Biomethylation of selenium (Se) by freshwater green algae, Ankistrodesmus sp., Chlorella vulgaris and Selenastrum sp., which had been isolated from the Tone River and Lake Kasumigaura in Japan, were investigated. All of the three algae produced methylated Se compounds from the corresponding inorganic compounds. The formation of trimethylselenonium ion (TMSe) by algae reached a plateau 2-4 d after culturing and the amount of Se an TMSe was less than about 0.001% of the added Se. TMSe was also found in the algae and was in the range from 0.04 to 0.3% of total-Se accumulated in the algae. Dimethylselenide (DMSe) was found in the headspace gases of the cultured flask and was in the range from 0.001 to 0.016% of the added Se. Total-Se accumulated in the algae was in the range from 0.002 to 0.2% of added Se. The presence of arsenic in the medium inhibited DMSe formation in the algae. Green algae in the freshwater ecosystems might be related to methylation of Se, and biomethylation of Se by green algae is thought to be one of the detoxification processes.

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In HPLC analysis of foods to which sorbic acid is added as preservative, a peak of unknown substance is often observed before that of sorbic acid. Especially, the peak was observed very often in fish-paste products, Japanese pickles, foods boiled down in soy, pickles in vinegar and others. The rate of appearance exceeded 83% in the above foods. The structural analysis of this unknown substance was carried out with GC/MS, GC/FTIR, NMR. The mass spectrum of the unknown substance showed fragments ions at m/z 112 (M⁺), 97 (M⁺-Me), 67 (M⁺-CHO₂), 45 (CHO₂), 41 (CH₃-CH=CH-), the fragmentation pattern being nearly identical with that of sorbic acid (M⁺, 112). The ¹³C-NMR of the unknown substance showed one methyl carbon at δ14.120 (q), four olefin carbons at δ120.22 (d), δ127.32 (d), δ137.07 (d), δ141.47 (d) and one carboxyl carbon at δ172.62 (s). The ¹H-NMR showed one methyl proton at δ1.91, four olefin protons at δ5.87, δ6.00, δ6.19 and δ7.73. The following coupling constants due to olefilic protons were observed : J_{2, 3}=15.23 Hz (trans bond), J_{3, 4}=11.0 Hz and J_{4, 5}=11.0 Hz (cis bond), and J_{5, 6}=7.0 Hz and J_{4, 6}=1.5 Hz. From the above spectral data, the structure of the unknown substance was identified to be trans-2, cis-4-hexadienoic acid, a geometrical isomer of sorbic acid.

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A simple and rapid method for the determination of acesulfame K (AK), saccharin (SA) and aspartame (APM) by high performance liquid chromatography (HPLC) using an ion-pair partition system was described. Samples were dialyzed with 1% phosphoric acid for 24 h. For APM, the dialyzate was passed through a Bond Elut SCX column. The column was washed with water, and APM was eluted with a mixture of methanol-15% sodium chloride (1 : 1). For AK and SA, tetra-n-propyl ammonium bromide was added to the dialyzate. The sample solution was passed through a Bond Elut C₁₈ column, and AK and SA were eluted with a mixture of methanol-water (4 : 6). The eluate was passed through a Bond Elut SAX column. The column was washed with 0.5% phosphoric acid and water. Then AK and SA were eluted with 0.3 N hydrochloric acid. Three sweeteners were separated on a Finepak C₁₈S column with a mixture of methanol-water (2 : 8) adjusted to pH 4.0 containing 0.1 M tetra-n-propylammonium hydroxide and detected at 210 nm. The recoveries of AK, SA and APM added to various kinds of foods at levels of 50 and 200 μg/g were 90-104%, 90-103%, 92-102%, respectively. The detection limits of AK and APM were 10 μg/g and of SA was 5 μg/g.

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The effects of the short-term inhalation of hydrogen sulfide were studied on rats that were exposed to hydrogen sulfide gas (75 ppm) for 20 to 60 min. The levels of hydrogen sulfide were higher in most of the tissues than that of the blood shortly after the exposure to the gas. The highest level of hydrogen sulfide was obtained in the heart, followed by kidney, brain, spleen, liver and lung. Biochemical parameters in the serum increased the level of GOT but those of alkaline phosphatase, blood urea nitrogen, sodium ion, potassium ion and creatinine remained unchanged. Physiological examination revealed a reduction in the heart beat and delayed P-Q intervals on electrocardiograms during the exposure for 60 min. Histopathological examination demonstrated a slight congestion in the lungs but no abnormal lesions in other tissues. The results indicate that the short-term inhalation of hydrogen sulfide at a concentration of 75 ppm could evoke overall acute toxicity, mainly in cardiovascular system.

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The production of aflatoxin (AF) groups (B₁, B₂, G₁, G₂, M₁, M₂, P₁ and Q₁) and aflatoxicol (AFL) groups (A and B) by AF-producing Aspergillus flavus and A. parasiticus isolated from various foods, and the metabolism of AF B₁ to AF M₁, AF P₁, AF Q₁, AFL-A and AFL-B by AF-non-producing A. flavus also isolated from various foods, were studied. A simple and rapid method for the analysis of the 10 kinds of toxins was developed. Toxins produced in culture were extracted with chloroform, separated into AF and AFL fractions by a silica gel cartridge column, then assayed by high performance liquid chromatography (HPLC). AF M₁, AF M₂, AFL-A and AFL-B were detected in addition to AF B₁ and AF B₂ in nearly all of the cultures inoculated with AF-producing A. flavus. The same groups were detected in addition to AF B₁, AF B₂, AF G₁ and AF G₂ in the cultures inoculated with A. parasiticus. The time courses of AF M₁ and AF M₂ production were almost the same as those of AF B₁, AF B₂, AF G₁ and AF G₂. AFLs tended to appear after AFs had accumulated in the cultures. AF P₁ and AF Q₁ were not detected at all. AF B₁ added to the cultures was metabolized by all strains of non-AF-producing A. flavus. AFL-A and AFL-B were the common metabolites in all of the cultures. However, no strains which converted AF B₁ to AF M₁, AF P₁ and AF Q₁ were observed.

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Gas chromatographic (GC) separating conditions for 2- and 3- series of prostanoids were investigated by using prostaglandin F_2α (PGF_2α) and PGF_3α as model compounds. The combination of the dimethylisopropylsily (DMIPS) ether derivatives and the capillary column coated with a polar liquid stationary phase of 50%-phenylmethylsilicone enabled to separate them with the difference of about 0.3 methylene unit values. This condition was applied for GC/mass spectrometric analysis of the metabolites of eicosapentaenoic acid (EPA), demonstrating the obvious separation of Δ¹⁷-6-keto-PGF_1α and 11-dehydrothromboxane B₃ from the corresponding 2-series metabolites originated from arachidonic acid.

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Coastal and marketing fishes in Japan were analyzed for polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar PCBs (Co-PCBs) at limits of determination of 0.01-0.1 ppt (pg/g) on wet weight basis in the selected ion monitoring method by using high resolution GC/MS (R=7000-10000). Highly toxic 2, 3, 7, 8-substituted PCDD and PCDF isomers (2, 3, 7, 8-PCDDs and 2, 3, 7, 8-PCDFs) were detected in all fish samples examined. The total concentrations of 2, 3, 7, 8-PCDDs and 2, 3, 7, 8-PCDFs were respectively 1.60-4.3 and 2.40-7.6 ppt in coastal fishes, and 0.04-1.50 and 0.01-3.7 ppt in marketing fishes. It was first clarified that all analyzed fish samples were also polluted by toxic Co-PCBs at levels of 290-1500 and 0.40-170 ppt in coastal and marketing fishes, respectively. On the other hand, 2, 3, 7, 8-T₄ CDD toxic equivalent (TEQ) levels of PCDDs and PCDFs were calculated by international toxicity equivalency factor (I-TEF), and that of Co-PCB by TEF reported by Hanberg et al. The total TEQ levels of PCDDs/PCDFs in coastal and marketing fishes were 0.63-1.41 ppt (mean±S.D. : 0.87±0.28 ppt) and <0.01-0.86 ppt (0.33±0.25 ppt), respectively. In addition, average TEQ levels of Co-PCBs in coastal and marketing fishes were 9.4±7.3 and 0.22±0.24 ppt, respectively. It is noteworthy that the TEQ level of Co-PCBs in coastal fish was greater than that of PCDDs or PCDFs.

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Isolation of the bacteria-free clone of Anabaena macrospora from unialgal culture was attemped by using such four methods as UV irradiation, capillary-pipette washing, akinete dilution and akinete washing methods. The axenic clones were obtained by the UV irradiation and capillary-pipette washing methods. The bacteria associated with A. macrospora were isolated and identified as Micrococcus sp.. Addition of these bacterial cells had no effects on the growth and the geosmin production ability of A. macrospora.

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Lipid peroxidation induced by copper chloride (Cu²⁺) was investigated in vitro in the rat erythrocytes. In the erythrocyte suspensions treated with Cu²⁺ hemolysis and formation of thiobarbituric acid reactive substances (TBA-RS) were induced, and catalase and glutathione peroxidase (GSH-Px) activities inhibited and reduced glutathione (GSH) content decreased. When the erythrocyte suspensions or erythrocyte membrane suspensions were treated with Cu²⁺, the amount of hydrogen peroxide in the supernatants of these suspensions increased and the amounts in the supernatants of erythrocyte suspensions were much higher than those of erythrocyte membrane suspensions. Chemiluminescence intensity as an index of lipid peroxidation in erythrocyte membranes and hemolysates treated with Cu²⁺ increased. The increase of chemiluminescence intensity induced by Cu²⁺ in hemolysates and erythrocyte membrane suspensions was inhibited by the addition of catalase. Since Cu²⁺ incorporated into erythrocyte decreased GSH content and inhibited activities of active oxygen scavenging enzyme, it is suggested that the Cu²⁺ produces hydrogen peroxide and subsequently causes lipid peroxidation of the constituent of erythrocytes.

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A main aqueduct of municipal sewage system at Tsukuba area in Ibaraki prefecture had been constructed by the Shield Method. When the construction of the main aqueduct was almost completed, several inhabitants who were residing in a zone along the route of construction with distances of 15-350 meters from the aqueduct had brought a suit that they have had attacks of diseases to be caused by contaminated well water with constituents of soil stabilisation chemicals applied in the construction. The accusers claimed that a large quantity of constituents of soil stabilisation chemicals was removed from applied location into underground water and spread to a wide area, and transferred to many different aquifers through gaps made by the construction of the aqueducts in impermeable layers, thus it resulted that well water in each well hole they were availed for drinking were contaminated with constituents of soil stabilisation chemicals, and this contamination orignated the diseases which attacked them. The points around connections of disease and the constituents of soil stabilisation chemicals, effects of constituents on the quality of well water and velocity of underground water movement were disputed in the court. The present auther gave an expert opinion that no evidence of contamination by stabilisation chemicals on the quality of well water. The judgement approved our opinion that the constituents of soil stabilisation chemicals did not cause health damages of accusers and delivered in favor of the defendant perfectly.

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