A method for the determination of dibutyl phthalate (DBP) and diethyl phthalate (DEP) in water as their nitro derivatives was studied by ECD-gas chromatography. DBP and DEP were extracted from sample water with hexane and the extract was treated with conc. H₂SO₄-conc. HNO₃ mixture to form nitro derivatives. The nitro derivatives of DBP were confirmed to be 3-nitro- and 4-nitro-DBP by gas chromatography-mass spectrometry. The recoveries of DEP and DBP in respective levels of 2 ppb and 1 ppm was 67-88% for DEP and 78-105% for DBP throughout the present method.

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Effect of various heavy metals was examined by experimental purification with activated sludge process using an artificial waste water. Presence of some heavy metals in the drainage water showed distinct obstacle on progress in the treatment. Especially, the chemical oxygen demand removal rate was decreased by the addition of Hg²⁺, Cu²⁺, and Ni²⁺, but Zn²⁺ and Pb²⁺ did not show a marked effect at the tested concentration of 1 to 40 ppm. Effect of heavy metals on the reduction activity of triphenyltetrazolium chloride (TTC) on activated sludge was similar to the above results, and Pb²⁺ showed little or no inhibition at 40 ppm. It was found that TTC reduction with dehydrogenase activity of Zoogloea and Sphaerotilus was largely inhibited by 2×10^-5M of Hg²⁺ (4.01 ppm) or Cu²⁺ (1.27 ppm). From these results, measurement of TTC reduction rate by dehydrogenase activity of activated sludge or Zoogloea was considered to be valuable as an index of purification-inhibition by influent of inhibitors such as heavy metals.

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A column chromatographic method for separation and concentration of polychlorinated biphenyls (PCBs) and terphenyls (PCTs), and dichlorodiphenyldichloroethylene (DDE) in water was investigated using dodecylbenzenesulfonic acid-bindig polyamide as an adsorbent. One to three liters of tap water doped with PCBs or PCTs at the ppb level was passed through a column and compounds adsorbed were eluted with ethanol. After dilution of the eluate with water, they were extracted with hexane. When determined by the electron-capture gas chromatography, all the componds tested were quantitatively recovered. This method will be applicable to the separation of other non-polar substances in water and waste water.

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Dry collecting method for mercury in ambient air by gold-amalgamation process was studied and gold-quartz wool collector was newly designed. The collector consists of a quartz tube of 4 mm in inner diameter, 6 mm in outer diameter, and 150 mm in length, in which the collecting agent made of gold wire and quartz wool are placed. The most effective collection was made with 300-1000 mg, of gold wire. For example, the use of a mixture of 500 mg of gold wire of 25 μm diameter and 50 mg of quartz wool with absorptive flow rate of 2 l/min showed collecting capability in the range of 4.5-18 μg of mercury. The collection of mercury could be carried out by one collector 20 times under above conditions without any loss of mercury. The absorbed mercury sample was stable for 1 week at room temperature. Interference of mercury collection by the fume of vegetable oil, exhaust gas of automobiles, and active gases such as SO₂ and Cl₂, was also examined, and the interference was excluded by the attachment of combustion furnace and NaOH filter to the collector.

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A method is described for the determination of benzo[a]pyrene in a sediment sample based on column chromatographic purification and spectrofluorometric analysis. Benzo[a]pyrene was extracted from sediment samples with n-hexane after refluxing with 10% KOH-90% EtOH. It was then separated from interfering materials by passing the n-hexane extract through silica gel and acetylated cellulose columns. The eluate from the latter column with ethanol-ether-water (4 : 2 : 3) mixture gave the same fluorescence and excitation spectrum as benzo[a]pyrene, and was found to be free from the contaminants originally present, by high-pressure liquid chromatography with UV detection at 254 nm. This procedure for the determination of benzo[a]pyrene was applied to the sediment samples from Hamana Lake, Tagonoura harbor, and paddy field in Shizuoka-city. Benzo[a]pyrene was found at levels varying from 0.0 to 255.2 ppb, with average recovery of 82.4 to 85.1%.

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To examine autoxidation of unsaturated fatty acids and the effect of antioxidants or synergists, especially on the interaction between them, experiments were carried out on methyl oleate, by its oxidation under the same condition as for methyl lionoleate, i.e., oxygen was passed through the samples at 90°. The increase in peroxide value (POV) and carbonyl value (COV or DNPV) of methyl oleate was about one-tenth of that of methyl linoleale. Results of thiobarbituric acid value (TBA) test and changes of infrared spectra through oxidation were also compared with those of methyl linoleate. Nonanal, octanal, methyl octanoate, methyl heptanoate, methyl formyloctanoate, and a substance estimated as methyl formylheptanoate were detected as volatile carbonyl compounds and esters formed. The antioxidant effects of α-tocopherol and butylhydroxyanisole, or synergistic effect of ascorbic acid was observed similarly as in the case of methyl linoleate.

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For planning the cleanup of the eutrophic moat water, nutrient taken in algae and seasonal changes of influent and effluent of nutrient were investigated. Samples of moat water, effluent water and influent water such as ground water and storm water, were collected 4 times a year, algae were separated by a filter paper, and nutrients in the filtrate water and non-filtrate water were determined. In summer (August), organic nitrogen in algae increased markedly compared with those in other seasons. Inorganic nitrogen dissolved in water, such as nitrite-nitrogen and nitrate-nitrogen, was not determined. It was considerd that nitrogen in moat water was supplied not only from the ground water but also from the bottom sediment. Phosphate in algae increased in summer and was found a little in other seasons. It was suggested that phosphate in the moat water was supplied only from the ground water.

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For lactate determination in milk and milk powder, application of enzymic method using lactic dehydrogenase (LDH) was studied. After clarification of milk (or reconstituted milk) with zinc sulfate and potassium ferrocyanide, the test solution thus obtained was mixed with pH 9.5 buffer composed of 1.0 M glycine and 0.4 M hydrazine. NAD, and L- and D-LDH were added, mixed, and incubated for 30 min at 37°. The lactic acid/lactate content was estimated from NADH formed which was determined by spectrophotometry at 340 nm. Although the results from contemporary international standard method (colorimetry) and the present method were not significantly different, the enzymic method was found to be better than the former in reproducibility. Comparative results on 16 kinds of commercial milk and milk powder are shown for reference.

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Colorimetric determination of hesperidin in cold cure preparations was examined. Hesperidin reacted with diethyl-p-phenylenediamine in slight alkalinity, with the use of potassium ferricyanide as the oxidation agent. The reaction product was extracted with isobutanol and this extract solution, colored blue, had an absorption maximum at 650 nm. This followed the Lambert-Beer's law in the concentration range of 30-210 μg/3 ml of hesperidin. Several chemicals contained in the preparations interfered in this measurement but they were removed by extraction with acetone or water.

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Rats were injected subcutaneously with cadmium (3 mg/kg) ; 5 ml of the supernatant of the liver homogenate from these animals was incubated with cadmium, and the reaction mixture was gel filtered over Sephadex G-75 column. Cadmium in vitro was mainly bound to metallothionein fraction and the amount of zinc, approximately equal to the increased amount of cadmium in metallothionein fraction, decreased. Rats pretreated subcutaneously with cadmium (3 mg/kg) were injected 2 days later intravenously with labeled cadmium (0-3 mg/kg). Almost all of the radioactivity was already bound to metallothionein fraction after 30 min. The ratio of cadmium to zinc both bound to two different types of metallothionein fractionated by gel filtration and ion exchange chromatography increased in proportion to the amount of cadmium additionally injected. These findings indicate that the zinc bound to metallothionein is substituted with cadmium both in vivo and in vitro.

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The protective effect of six selenium compounds (sodium selenite, sodium selenate, selenocystine, selenocysteamine, selenocystamine, and selenomethionine) on the toxicity of mercuric chloride to rats was compared on the basis of survival rate, body weight change, and renal damage. Twenty-four hours after one of six selenium compounds was injected with mercuric chloride in rats, distribution of mercury and selenium in the tissues was determined by nondestructive neutron activation analysis. The protective effect of six selenium compounds on the toxicity of mercuric chloride decreased in the order of sodium selenite, sodium selenate, selenocystine, selenocysteamine, selenocystamine, and selenomethionine. Injection of the six selenium compounds characteristically altered the distribution of mercury in the rats and markedly increased the whole-body retention of mercury. The molar ratio of selenium to mercury in the tissues of animals simultaneously injected with mercuric chloride and selenium compound was nearly 1 : 1, except in a few cases. The result of this experiment demonstrated that mercury reacted peculiarly with selenium at an equimolar ratio in vivo and was transformed to a compound with lower toxicity. The difference in the protective effect among sodium selenite, sodium selenate, selenocystine, and selenomethionine on the toxicity of mercuric chloride was found to be explicable from the distributions of mercury and selenium, and the ratio of selenium to mercury in the tissues.

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The distribution of mercury and selenium in the tissues 15 and 30 min, and 1, 4, and 24 hr after the injection of mercuric chloride (10 μmol/kg, i.p.) or sodium selenite (10 μmol/kg, s.c.), and after simultaneous injection of mercuric chloride and sodium selenite was determined by non-destructive neutron activation analysis. The molar ratio of selenium to mercury in the tissues of animals simultaneously injected with mercuric chloride and sodium selenite was calculated from the distributions of mercury and selenium in the tissues. In the case of simultaneous injection of mercuric chloride and sodium selenite, the reaction of mercury with selenium was found to occur mainly in the blood (red cells and plasma) at an equimolar ratio. When injection of mercuric chloride preceded the injection of sodium selenite by 4 hr, the reaction of mercury with selenium was found to occur mainly in the kidneys rather than in blood at an equimolar ratio. The results of this experiment demonstrated that the reaction of mercury with selenium occured at an equimolar ratio in vivo, regardless of dose conditions and reaction sites. The reaction product of mercury with selenium in the plasma was suggested to move gradually from the plasma to the liver, kidneys, and spleen.

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The reaction mechanism of mercury with selenium in the plasma of rats simultaneously injected HgCl₂ and Na₂SeO₃ was investigated. Sera collected after independent or simultaneous injection of HgCl₂ (10 μmol/kg, i.p. or i.v.) and Na₂SeO₃ (10 μmol/kg, s.c.) were chromatographed on Sephadex G-200 column. Distribution of mercury and selenium in the serum protein fractions of each injection group was determined by atomic absorption analysis and neutron activation analysis. On the other hand, bovine serum albumin, reduced glutathione, Na₂SeO₃, and HgCl₂ were reacted as the model reaction of mercury with selenium in the plasma. The reaction product of mercury with selenium in this reaction was chromatographed on Sephadex G-200 column and compared with in vivo experimental results. These results suggested that the final reaction product of mercury with selenium in the plasma was a colloidal HgSe, formed by the following two reactions : (1) Mercury from the Hg-binding protein transfers to the Se-binding protein and forms an intermediate such as protein-S-Se-Hg⁺. (2) The Hg-binding protein reacts with the Se-binding protein and forms an intermediate such as protein-S-Hg-Se-S-protein, or selenium from the Se-binding protein transfers to the Hg-binding protein and forms an intermediate such as protein-S-Hg-Se^-. Finally, these intermediates decompose to HgSe and then it converts to colloidal HgSe by association. The latter reaction was proved to occur in the plasma after simultaneous injection of HgCl₂ (i.v.) and Na₂SeO₃ (s.c.).

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The acid phosphatase and β-N-acetylglucosaminidase activities of femur were examined in rats intraperitoneally administered cadmium acetate, lead acetate, zinc acetate, or combined metals (0.89 μmol as a metal ion/100 g body weight) 5 times at 2-day intervals. Acid phosphatase activity in the diaphysis was significantly decreased by the administration of cadmium and increased by the injection of lead or zinc compared with that of normal rats, but this enzyme activity in the epiphysis was significantly elevated by cadmium alone. Also, β-N-acetylglucosaminidase activity in the diaphysis was significantly reduced by the administration of cadmium or zinc and enhanced by the treatment with lead compared with that of normal rats, but this enzyme activity in the epiphysis was significantly increased by cadmium alone. On the other hand, these enzyme activities in both diaphysis and epiphysis after injection of combined metals did not show a significant alteration. The present results indicate that the specific action of each metal was modified by the administration of these metals in combination.

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