In recent taxonomical studies concerning psychrotrophic coliform organisms several new species such as Rahnella aquatilis, Serratia fonticola, Enterobacter amnigenus, E. intermedium, etc. were proposed and approved. Based on the new taxonomy, various coliform isolates from mountain soils were identified in this study. During the period from October through December, 59 soil samples were collected from various places in and around Mt. Mito, located at the Tokyo-Yamanashi prefectural border. Enumeration and isolation of coliform organisms were done by using desoxycholate agar and incubating at 30°C. Fairly low coliform counts, less than 10³ per g, were obtained from most samples. Among 182 coliform isolates, 61% were identified as Rahnella aquatilis, 17.6% Citrobacter freundii, 5.5% Escherichia coli, 3.8% Serratia fonticola, 2.7% Klebsiella pneumoniae, 1.6% Enterobacter cloacae, 0.5% Enterobacter aerogenes and 7.1% were not determined. Almost all strains of R. aquatilis failed to grow at 37°C, the majority of them showed negative arginine dihydrolase reaction, and approximately half of them produced gas from glycerol. Rahnella aquatilis organisms appear to account for a large proportion of the coliform flora in unpolluted mountain soils.

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In order to elucidate the mechanism of food poisoning outbreaks caused by Vibrio parahaemolyticus, the viability of this bacterium was examined after a one-hour incubation in acidic media (pH 5.0, 5.5 and 6.0) with several organic acids and sodium chloride at 37°C.
The viability hardly decreased even at pH 5.0 in the medium without any organic acid in the presence of 0.5% sodium chloride solution. In the medium with 0.1M acetate, lactate or succinate the viability decreased markedly, but citrate, malate, tartrate or oxalate had almost no effect. The effects of acetate and lactate decreased with increasing sodium chloride concentration (3%). Among Kanagawa-phenomenon-positive strains, negative strains of V. parahaemolyticus and V. alginolyticus, almost no difference was observed in the behavior in acidic media with the various organic acids mentioned above.

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A sensitive and simple method for determination of monensin in chicken tissues by high performance liquid chromatography (HPLC) with a fluorescence detector was developed.
Monensin was extracted with a mixture of 0.5% metaphosphoric acid and methanol (1:1) from samples and reextracted with chloroform. The chloroform extract was evaporated to dryness. Monensin in the residue was converted into the corresponding ADAM-monensin by reaction with 9-anthryldiazomethane (ADAM). After clean-up with a Sep-pak silica cartridge, ADAM-monensin derivative was determined by HPLC with a fluorescence detector (excitation at 365nm, emission at 412nm).
The calibration graph was rectilinear from 0.1μg/ml to 1.0μg/ml. The average recoveries of monensin from chicken meat and liver fortified at the level of 0.5ppm were 82.1% for meat and 67.8% for liver.

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An analytical method was developed for monitoring environmental pollutants by using blue mussels or gray mullets as a biological indicator. The method involves extraction with acetonitrile, followed by liquid-liquid partitioning, the use of a Florisil dry column for defatting, and a final silica gel column clean-up to yield five fractions. The fractions from blue mussels or gray mullets were analyzed by using FPD-GC, FID-GC, ECD-GC, GC-MS and fluorescence spectrophotometry. Various kinds of pollutants were present in them, and clear quantitative and qualitative differences were observed among the substances from samples collected in regions of relatively clean sea and polluted sea.
The results so far obtained indicate that this analytical method is applicable to a monitoring system for environmental pollutants and that blue mussels are a more appropriate biological indicator than gray mullets for monitoring various kinds of compounds in marine samples.

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A method for distinguishing aloe material added to candy supplements by thin layer chromatography-densitometry has been developed. Candy containing aloe is blended, and extracted with water. The extract is cleaned up on a Sep-pak C₁₈ disposable cartridge by using methanol as the eluant. The eluate is spotted on a silica gel plate, and developed with ethyl acetate-methanol-water (100:16.5:13.5). The biological activity of aloe in candy on the developed plate is determined by densitometry with ultraviolet detection.
TLC patterns obtained from candy extracts could be classified into two categories, which may reflect the use of Aloe arborescens Miller and aloe powder (Japanese Pharmacopoeia) (or reagent aloin) as aloe material.

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The oral acute toxicity and 12-month chronic toxicity of basic cupric carbonate, regarded as the principal ingredient of green patina (Roku-sho in Japanese), were tested in Slc: Wistar rats.
In the acute toxicity test, seven groups consisting of 10 five-week-old rats of each sex were orally given basic cupric carbonate in doses of 764-2281mg/kg. Three or 4 days after administration, hematuria was noted in some male and female rats that received doses of 1, 100mg/kg or more. The necropsy of dead animals revealed hemorrhage of the glandular stomach mucosa and hematuria in the urinary bladder in the same groups. The calculated LD₅₀ values were 1350 mg/kg in males and 1495mg/kg in females.
In the chronic toxicity test, five groups consisting of 42 five-week-old rats of each sex were fed on diets containing basic cupric carbonate at levels of 0 (control), 70, 220, 670 and 2, 000ppm for 12 months. Depression of body weight gain was noted in the 2, 000ppm group of both sexes, but no dead animal was observed throughout the test period. There were marked increases of serum GOT, GPT and LDH activities in the male 2, 000ppm group at 1, 3, 6 and 12 months. Histologically, the incidences of single cell necrosis in the liver was increased significantly in the 2, 000ppm group of both sexes. The results suggest that the diet containing basic cupric carbonate at a level of 2, 000ppm was toxic to rats, and that 670ppm is the maximum no-effect dose under the conditions used.

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Green patina, a kind of rust which forms on the surface of copper, has traditionally been suspected of being virulently poisonous, so the Food Sanitation Law of Japan prescribes that kitchen utensils and tableware made of copper must not have any green patina on the surface. However, little is known about the toxicity. In order to gain more insight into the toxicity, we have initiated a study into the toxicological effect of basic cupric carbonate in the diet on rats. Rats were fed a diet containing basic cupric carbonate, which is the major component of the patina, at doses of 0, 70, 220, 670 and 2, 000ppm as Cu(OH)₂·CuCO₃·H₂O. After feeding for 1, 3, 6 and 12 months, the levels of copper in the blood and tissues were determined by atomic absorption analysis. At the same time, fluctuations in the levels of iron and zinc, which are essential elements, were determined in the tissues.
Levels of copper, iron and zinc in blood were practically unchanged after feeding for 12 months at the highest dose, 2000ppm. Copper in the liver increased markedly at doses of 670 and 2000ppm from 1 month, and the level reached about 50 times that of the control group. The tendency was more marked in females than in males. The level of copper in the kidney significantly increased at larger doses than 670ppm.
Iron concentration in the kidney did not change at smaller doses than 220ppm, but significantly decreased at higher doses than 670ppm. The level of copper in the kidney increased only at the highest dose.
In conclusion, basic cupric carbonate, is not as toxic as formerly believed, and the levels of essential metals in tissues were not affected by feeding at smaller doses than 670ppm in the diet for 12 months.

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The activities of δ-aminolevulinic acid (ALA) synthase, ALA dehydratase and porphobilinogen (PBG) deaminase among heme biosynthetic enzymes were determined in bone marrow cells and peripheral blood of rats administered basic cupric carbonate at doses of 0, 70, 220, 670 and 2, 000ppm in the diet for 12 months. The activities were all significantly increased in male rats at doses of 670 and 2, 000ppm, but not in female rats. The result shows that there are sex differences in the toxicological effects of basic cupric carbonate on the heme biosynthetic pathway in erythropoietic tissues.

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A method for the separative determination of EDTA-2Na and EDTA-metals from various foods was developed. Food samples were dialyzed against 0.02M Tris-hydrochloric acid buffer (pH 8.5) and the dialyzed sample solution was loaded on an anion-exchange column (cartridge). EDTA-metals except EDTA-Fe flowed through the column, but EDTA-2Na was quantitatively adsorbed on the column, and could be eluted with 0.2N hydrochloric acid. EDTA-metals and EDTA-2Na were reacted with cupric sulfate solution at pH 2.5 and converted to EDTA-Cu. EDTA-Cu from EDTA-metals was concentrated and purified on a Dowex column, and EDTA-Cu from EDTA-2Na was purified on a Unicellex column. Then EDTA-Cu in each solution were separately determined by high performance liquid chromatography. Recoveries of EDTA-Ca (250ppm) from various foods were more than 92.4% by the proposed method. Most of the EDTA-2Na added to samples was recovered as EDTA-metals caused by minerals in the foods, but the recoveries of total EDTA were more than 91.2%.

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A practical determination of diarrhetic shellfish poison (DSP) was established by measuring the 50% acid phosphatase activity inhibitory concentration (APIC₅₀) with Tetrahymena pyriformis, in place of the mouse lethal assay. That is, DSP was extracted with acetone and ether from the toxic hepatopancreas of scallops, and applied to a silicic acid column. The column was washed with ether-methanol (40:1), and DSP was eluted with ether-methanol (1:1). A suitable concentration of DSP was added to 20ml of the medium for growth of T. pyriformis in an Erlenmeyer flask. This flask was inoculated with 0.2ml (approximately 4, 000 cells) of preculture of T. pyriformis and incubated at 28°C for 24 hours. The cultured cells were washed twice with 0.5% saline, and destroyed by sonication for 5min. Then the acid phosphatase activity in T. pyriformis was determined.
The APIC₅₀ and the 50% growth inhibitory concentration (GIC₅₀) of T. pyriformis were equivalent to 1 Mouse Unit (0.05MU/g of sample). The presence of DSP in samples can be judged qualitatively by using the naked eye when the toxin content is above the GIC₅₀ of T. pyriformis. For the analysis of a few samples, it is convenient to apply GIC₅₀ for quantitation of DSP, but the application of APIC₅₀ is preferable for the treatment of many samples.

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In the previous paper, the preparation of three kinds of iodoresorcinols and the separation and identification of the reaction products of iodine with resorcinol were reported. In this work, an analytical method for the intermediate and side reaction products of Food Red No. 3 (Erythrosine, Colour Index No. 45430, FD & C Red No. 3) and the reaction products of iodine with 2-(2′, 4′-dihydroxybenzoyl)benzoic acid (2′, 4′-diOHBBA) was developed.
The intermediate resorcinol and the side reaction products, 2, 4, 6-triiodoresorcinol, 2, 4-diiodoresorcinol and 2′, 4′-diOHBBA, were determined by a combination of thin layer chromatography (TLC) and densitometry (J. Food Hyg. Soc. Japan 26, 243-252 (1985)). The relationship between the integrated value on the densitogram and the concentration of each compound was found to be linear in the range of 25-150ppm (0.05-0.3μg/spot) for resorcinol, 500-10, 000ppm (1-20μg/spot) for 2, 4, 6-triiodoresorcinol, 100-400ppm (0.2-0.8μg/spot) for 2, 4-diiodoresorcinol, 200-2, 000ppm(0.4-4μg/spot)for 4, 6-diiodoresorcinol and 250-1500ppm (0.5-3μg/spot) for 2′, 4′-diOHBBA (Fig. 1). The detection limit was 0.008μg for resorcinol, 0.8μg for 2, 4, 6-triiodoresorcinol, 0.08μg for 2, 4-diiodoresorcinol, 0.16μg for 4, 6-diiodoresorcinol and 0.06μg for 2′, 4′-diOHBBA (Table 1).
Reaction of iodine (3 g-atom) with 2′, 4′-diOHBBA (1mol) yielded three compounds, which could be separated by TLC (Fig. 2). Spots A (Rf 0.73) and B (Rf 0.52) are thought to be 2-(3′, 5′-diiodo-2′, 4′-diOHB)BA and 2-(3′-iodo-2′, 4′-diOHB)BA, respectively (Table 2).
The intermediate and the side reaction products in Food Red No. 3 were extracted with a mixture of 10% hydrochloric acid-ethanol-water (1:1:1) from this color and filtered. The filtrate was extracted with ethyl ether. The ethyl ether extract was analyzed by TLC-densitometry. The average recoveries for resorcinol and 2′, 4′-diOHBBA added to Erythrosine standard (free from the intermediate and side reaction products) at the level of 0.2% were 82.0 and 74.8%, respectively, but 2, 4, 6-triiodoresorcinol was not recovered. The poor recovery results from degradation of 2, 4, 6-triiodoresorcinol itself (Fig. 4). The degradation products were 2, 4-di, 2- and 4-mono-iodoresorcinols and two other unidentified compounds (Figs. 5 and 6).
Ten commercial samples of Food Red No. 3 were analyzed by this method. The intermediate resorcinol and above side reaction products were not found in any of the samples. One of the side reaction products of Food Red No. 3, 2, 4, 6-triiodoresorcinol might coexist with the degradation products, di- and mono-iodoresorcinols in this color.

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The binding potential of ochratoxin A (Oct. A), a carcinogenic mycotoxin, to protein was investigated by using bovine serum albumin (BSA) and proteinous food. An interaction between Oct. A and BSA was suggested in the aqueous system, where fluorescence with high intensity was observed. This was also confirmed by thin-layer chromatography on a microporous cellulose acetate membrane. Measurements of the extractability of Oct. A with chloroform showed that the binding ability was high in the range of pH 4.0 to 6.0. The major binding type was presumed to be ionic, based on a comparison with the extractability of ochratoxin A methyl ester. The binding parameters determined by an equilibrium dialysis method were n=2.38±0.17 for the number of binding sites and K=7.67±0.39 (×10⁵/M) for the affinity constant in phosphate buffer at pH 7.4. The binding was weakened with increasing ionic strength of the solution and with increasing content of methanol. On the basic of these results, the first extraction procedure for Oct. A by the A. O. A. C. method, which gave a low extractability of 29% for Katsuobushi, dried bonito meat rich in protein, was modified to obtain a high recovery. The addition of 2M sodium chloride and further acidification by addition of 4 volumes of 0.5M phosphoric acid resulted in an increase of the extractability to 85%.

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A method for the determination of dephosphate-bromophenophos (TBB) in cow's milk by gas-liquid chromatography using an electron capture detector is described. TBB was extracted with acetonitrile and re-extracted into ethyl acetate-n-hexane (2:1). The crude extract was purified by partition into n-hexane under alkaline and acidic conditions and was then converted into the trimethylsilyl derivative. This derivative was separated on an OV-1 column, exhibiting a single peak on the chromatogram without any interference. A calibration graph was constructed by plotting the peak height ratio of TBB to the internal standard against the amount of TBB, and satisfactory linearity was observed in the range of 25 to 350pg. TBB added to cow's milk specimens was recovered at the rate of 100.1±1.18%. The proposed method was applied to quantitation of TBB in cow's milk after oral administration of bromophenophos (12mg/kg). TBB in cow's milk reached a maximum level (0.12-0.18ppm) at 24-48hr and then disappeared within 84hr.

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Studies on a fractionation procedure using droplet counter-current chromatography (DCC) for food contamination monitoring have been carried out. The elution profile of components could be predicted from the partition coefficients by making use of simple Gaussian curves by the least-squares method using a microcomputer. Volumes of stationary phase, V_S, and mobile phase, V_M, could be measured directly in the case of DCC. The partition coefficient, K, of various compounds between the stationary phase and the mobile phase could also be obtained easily. Elution profiles, including retention volume, V_R, and peak width, W_i, from DCC could therefore be estimated when the partition coefficient alone is known. The following equations are proposed for fractionation of various chemical contaminants. V_R=KV_S+d/2, F_i=m(V_M+V_i)/√n+d, F_i+1=(F_i-d)(2√n+m)/(2/√n-m)+d: where d is the injection volume, F_i is the volume of the i-th fraction, V_i is the center of the i-th fraction, n is the theoretical plate number, and m is an integer for mσ separation.

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The residues of organochlorine compounds in yearly pooled human milk samples, which had been collected in the Tokyo Metropolitan area in 1979-1983, were evaluated by capillary gas chromatography using an electron capture detector (ECD/GC) and gas chromatography/mass spectrometry with packed columns.
The concentrations of these compounds determined by selected ion monitoring (SIM) were identical to those determined by capillary ECD/GC.
The concentrations on a whole milk basis (fat basis) by SIM were as follows: α-HCH, 0.10-0.31ppb (6-9ppb); β-HCH, 14.9-39.2ppb (837-1140ppb); γ-HCH, 0.10-0.16ppb (3-7ppb); o, p′-DDE, 0.06-0.11ppb (3ppb); p, p′-DDE, 18.2-56.4ppb (1020-1630ppb); o, p′-DDT, 0.11-0.41ppb (6-12ppb); p, p′-DDT, 2.22-7.86ppb (125-228ppb); p, p′-DDD, 0.03-0.10ppb (2-3ppb); HCB, 1.30-3.03ppb (73-93ppb), S-421 (a synergist for pyrethroid insecticides), 0.24-0.80ppb (10-23ppb); dieldrin, 0.36-1.55ppb (20-45ppb). However, no residues of δ-HCH and o, p′-DDD were detected (<0.01ppb on a whole milk basis). In addition, residues of chlordane-related compounds on a whole milk basis (fat basis) were determined to be as follows by SIM: oxychlordane, 0.36-0.60ppb (16-20ppb); heptachlor epoxide, 0.39-1.12ppb (22-33ppb); trans-nonachlor, 0.80-0.91ppb (24-45ppb); cis-nonachlor 0.13-0.16ppb (4-7ppb); trans-chlordane, 0.01-0.02ppb (1ppb); cis-chlordane, 0.02-0.03ppb (1ppb); compound 2 (dihydroheptachlor isomer), 0.03-0.04ppb (1-2ppb); compound 5 (chlordane isomer), 0.02-0.03ppb (1-2ppb); compound 6 (nonachlor isomer) 0.14-0.17ppb (5-8ppb).

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[in Japanese]

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