In addition to the previous finding that aliphatic amines reacted with the nitrite ion to form a cyanide ion, aromatic amines, such as p-nitroaniline, aniline, p-toluidine, and ethyl p-aminobenzoate, also reacted with the nitrite ion to form a cyanide ion, the concentration of which was markedly higher than that from the reaction with aliphatic amines. The formation of a cyanide ion from aromatic amines suggests the cleavage of the benzene ring by the nitrite ion.

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Gas chromatographic method for the determination of a trace of beryllium in natural and treated water was investigated. The water sample of 500 ml was extracted with acetylacetone and benzene in the presence of ethylenediaminetetraacetic acid (EDTA) at pH 5.5-6.0, and then it was reverse extracted with 6 N hydrochloric acid. Recovery of beryllium at this stage was more than 95%. A sample solution for a gas chromatographic analysis was prepared by extraction with trifluoroacetylacetone and benzene from the solution concentrated with acetylacetone as above. The extract containing beryllium trifluoroacetylacetonate was washed with 0.1 N sodium hydroxide and then subjected to gas chromatography. The analytical limit is approximately 1×10^-9 g/liter of beryllium. Beryllium content in the natural water at three water supplies, lake water, and river water ranged from 2.5×10^-9 to 8.6×10^-9 g/liter. Beryllium content in the treated water at each of the water supplies decreased from one quarter to one-third compared with natural water.

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Among damages to fisheries by water pollution, appearance of fish with offensive odor has created the social problems by the loss of their commercial value. Since 1962, fish with offensive odor have appeared in Nagara River. Such foulsmelling fish are caught along about 20 km of this river in Hashima area from Chusetsu Bridge, where Arata River and Sakai River flow into Nagara River, and about 8 km from Ise Bridge to Nagashima area. Such offensive odor was found in the fish inhabiting the bottom of the river and those living in the river for a long period. These fish were found to live in the polluted basin and ingested a kind of substance with an offensive odor, which was considered to be one of the factors for the appearance of fish with offensive odor.

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Binding of cadmium with the supernatant components of the liver, kidney, and small intestinal mucosa of rats was examined in vitro using the supernatants of these three organs from (a) untreated control rats and from (b) rats given subcutaneous injection of 3 mg/kg of cadmium and (c) those given 10 mg/kg of cadmium orally. A mixture of 5.5 ml of each supernatant fraction and 50 μl of cadmium chloride solution (0-80 μg Cd/5 ml supernatant) was incubated at 37° for 30 min and each of the mixtures was submitted to gel filtration to collect high molecular and metallothionein fractions. Cadmium bound to these fractions was determined with the following results. 1) Cadmium was present bound to these two fractions in vitro, as in the case of in vivo. 2) Thionein was small in amount in the liver supernatant from control rats, similar to kidney and small intestinal mucosa, and binding of cadmium to high molecuiar fraction became greater than that to metallothionein fraction from addition of 5 μg of cadmium. 3) In spite of a larger amount of endogenous metallothionein (6.9 μg Cd/ml supernatant) in the supernatant of the liver after subcutaneous injection of cadmium, binding of cadmium to thionein became greater up to the addition of 40 μg of cadmium in vitro. Addition of larger amount than that resulted in the increased binding of cadmium with high molecular fraction. 4) In the kidney supernatant after subcutaneous injection, endogenous metallothionein was present in the amount of 2.4 μg Cd/ml supernatant. In the small intestinal mucosa, endogenous metallothionein was present in the amount of 0.1 μg (b) or 1.6 μg (c). In the supernatant fraction of these organs, the amount of cadmium bound to thionein in vitro was practically the same as that in the organs from untreated controls.

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In the determination of fluorine, recovery of fluorine was significantly raised by steam-distillation of silicate-containing sulfuric acid. Accurate estimation of low fluorine content in the sample became possible by the use of this method and was found that the steam-distillation using sulfuric acid is much better the determination of fluorine than that using perchloric acid.

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In order to clarify the substances responsible for the offensive odor in fish, volatile fatty acids, which play an important role as a biochemical oxygen demand (BOD) substance, were examined by gas chromatography. It was proved that the concentration of volatile fatty acids was high in the river water and in the bottom mud where the fish with offensive odor lived. Such acids were also found in a high concentration in the muscles and digestive organs of the fish with offensive odor, but was not detected in their liver, or in the fish in breeding ponds and in river ayu living in Nagara Bridge basin. Therefore, fish were kept in a tank with constant concentration of volatile fatty acids and the taste of fish was examined at regular intervals. At the same time, concentration of volatile fatty acids in the internal organs of the fish was analyzed. Accumulation of volatile fatty acids was recognized in internal organs except in the liver, and the offensive odor appeared on the 37 th day. At that time, a total of 73.7 ppm of total fatty acids was detected in the muscles. This value agreed with the concentration of fatty acids in the fish with offensive odor. These results suggested that volatile fatty acids were responsible as a substance giving the offensive odor to fishes.

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Tibia from 9-day chick embryo was cultivated at 38° for 5 days by watch-glass method, with and without (control) cadmium chloride. Growth of tibia was inhibited completely in the presence of 10 ppm cadmium chloride in the medium. In the tibia cultivated in the presence of 20 ppm CdCl₂, content of calcium, inorganic phosphate (iP), hydroxyproline (OH-Pro), uronic acid, and deoxynucleic acid (DNA) were determined. Transfer of cadmium from the medium into tibia was apparent, as none was detected in the control. It was observed that when CdCl₂ was added the increase of each content in the tibia after 5 days' cultivation was inhibited as follows : Ca 29%, iP 61%, OH-Pro 86%, uronic acid 103%, and DNA 154%. Histopathological examination of the tibia cultivated in the presence of CdCl₂ showed that (1) all of the cartilage cells revealed various regressive changes such as pyknosis, karyorrhexis, and coagulative necrosis, (2) a very faint reaction appeared in both PAS-stained matrix and cells of the cartilage, and (3) ossification remained nearly in the state before the cultivation, showing necrosis of osteoblastic cells.

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Effect of bear, ox, pig, and wild boar galls, in which bile acid components had been analyzed quantitatively, and various bile acids on growth of intestinal bacteria was investigated. 1) Free deoxycholic, chenodeoxycholic, and ursodeoxycholic acids showed strong antibacterial activities, whereas the antibacterial activities of their conjugated acids decreased distinctively. 2) Chenodeoxycholic acid has antibacterial potency as strong as that of deoxycholic acid on anaerobic Clostridium butyricum and Bacteroides fragilis. 3) Salmonella enteritidis and Escherichia coli were not inhibited in the medium containing 1% (w/v) of any of the bile acids and animal galls tested. 4) Streptococcus faecalis was affected by free deoxycholic and chenodeoxycholic acids and a certain sample of bear gall. Growth of Bacteroides fragilis was inhibited by every free bile acid tested, and that of Clostridium butyricum was inhibited by all the free bile acids except cholic acid. 5) The results obtained from the tests of ox and pig gall suggest that these galls contain a component responsible for overcoming the antibacterial activities of bile acids on anaerobic bacteria.

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Quantitative analysis of metal dithizonates was carried out by extraction with chloroform, thin-layer chromatography, elution with chloroform, and colorimetry. The metal ions Hg²⁺, Cu²⁺, Pd²⁺ and Bi³⁺ were extracted from the sample solution (200 ml) with 0.1% dithizone-chloroform solution (5 ml) at pH 1.8-2.0, and Ni²⁺, Co²⁺, Zn²⁺, and Pb²⁺ at pH 7.0-8.0. The extracted dithizonates of Bi³⁺, Pb²⁺ and Zn²⁺ were unstable compared to other metal dithizonates. The extract solution (100 μl) was applied on MN-silica gel S-HR layer by chromatocharger. The layer was developed with benzene or a solution of carbon tetrachloride-methylene chloride-benzene (1 : 7 : 4). After the thin-layer chromatographic separation, zones of each dithizonate were scraped off from the thin-layer plate by a vacuum extractor and eluted from the adsorbent with chloroform (4 ml). The eluate was made up to 5 ml with chloroform and amount of metals determined by colorimetry. The determination ranges of metal ions obtained by this method were as follows : Cu²⁺ and Ni²⁺, 0.125-1.0 ppm ; Pd²⁺, 0.125-2.125 ppm ; Zn²⁺, 0.125-0.625 ppm ; Co²⁺, 0.125-1.0 ppm ; Hg²⁺, 0.25-2.5 ppm ; Bi³⁺, 0.5-2.5 ppm, and Pb²⁺, 0.5-3.0 ppm. The coefficient of variation of these determined metal values was about 4%.

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