The degradation of aflatoxins (AF) by Aspergillus niger or Aspergillus flavus (AF non-producing strain) from peanuts naturally contaminated and rice artificially contaminated with AF were studied.
Conidia of the two strains were separately inoculated into contaminated peanut and rice cultures. The cultures were analyzed at 5, 10, 15, 20 and 25 days after inoculation. At the same time, AF-contaminated peanut and rice cultures without conidia were analyzed as controls. In the culture with A. niger, loss of AF B₁, B₂, G₁ and G₂ was faster and more extensive than in the culture with AF non-producing A. flavus. After 5 days, low levels of AF remained in the culture with A. niger (80-90% was lost), and no AF were detected after 10 to 15 days. In contrast, after 15 days, the loss of AF in the culture with AF non-producing A. flavus ranged from about 40 to 90%, and low levels of AF were still detected after 25 days.
The degradation of AF by these two Aspergilli was confirmed by means of the chicken embryo test.

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The chemical form of arsenic in Laminaria japonica Aresh. (Japanese name: “makonbu”, Laminariaceae) was investigated and the following results were obtained.
1) The arsenic in “makonbu” is easily extracted with water-methanol (4:1) mixture (the recovery was more than 90%).
2) Ultrafiltration indicated that the water-methanol soluble arsenic compounds have low molecular weight (MW 500-1, 000).
3) In activated charcoal column chromatography, the arsenic compounds in “makonbu” were eluted with water-ethanol (4:1) mixture, but not with water (column recovery 99.2%). In QAE-Sephadex A-25 (Cl type) column chromatography, the arsenic compounds in “makonbu” were eluted with acetic acid-methanol (1:25) mixture (column recovery 80.1%). After both procedures, the content of arsenic in the eluate reached 2.78%.
4) The arsenic compounds gave negative reactions with ninhydrin, dithizon and phenol-sulfuric acid reagent.

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Toxins of the gonyautoxin group (GTXs), the major components of paralytic shellfish poison, were isolated from the digestive glands of toxic scallop. No adverse effect of GTXs was observed in male ddY mice pretreated intraperitoneally with GTXs at 2/5 to 3/5 of LD₅₀ for 10 days. This pretreatment did not produce tolerance to a subsequent challenge dose of GTXs as the intraperitoneal LD₅₀ values were 0.760MU/20g for the GTXs-pretreated group and 0.774MU/20g for the control group pretreated with 0.01N hydrochloric acid. Residual toxic efficiency of GTXs was determined by measuring the LD₅₀ for GTXs at intervals of 1, 2, 3 and 4 hours after pretreatment with a single dose of 0.4MU/20g. This efficiency gradually decreased with time and was essentially lost 4 hours after pretreatment.

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Analytical methods for macrolide antibiotics (Macs.) in the muscle and liver of swine were examined.
Tylosin (TS), erythromycin (EM), spiramycin (SP), oleandomycin (OM) and kitasamycin (KT) were well extracted with methyl alcohol after deproteinization by means of salt solutions, then adsorbed on an Amberlite XAD-2 resin column and eluted with methyl alcohol. The eluate was concentrated to dryness and dissolved in phosphate buffer (pH 8.0) to prepare a test solution. The cup plating method with Micrococcus luteus as the test organism was employed for the assay, and the average recoveries were more than 93.2% and 92.5% from muscle and liver, respectively. As little as 0.05μg/g (TS, SP, OM, KT) or 0.01μg/g (EM) was detectable.
The extraction and the column chromatography were carried out on samples to which other antibiotics had been added in order to check the specificity of the present method. Chlortetracycline (5.0μg/g), fradiomycin (5.0μg/g), kanamycin (5.0μg/g), monensin (5.0μg/g) and colistin (5.0μg/g) showed no inhibition zone in the cup plating method, but bacitracin (5.0μg/g), ampicillin (5.0μg/g) and oxytetracycline (5.0μg/g) showed an inhibition zone.
Bioautography was suitable for both separation and confirmation of the identity of Macs.

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A low temperature ashing (LTA) pretreatment method was developed for the analysis of heavy metals in foods, and the results were compared with those obtained by the conventional wet and dry ashing methods.
1) The LTA method requires few reagents, and utilizes oxygen plasma. Ashing can thus be done easily and simply, with little risk of contamination of samples by metals.
2) Before LTA, treatments may be necessary to remove moisture from samples and to prevent losses from samples with high contents of oils.
3) There was no significant loss of metals in the LTA method except in the case of arsenic. The recoveries of added cadmium, zinc, manganese, lead, chromium, nickel and copper were nearly 100%.
4) Loss of arsenic could be avoided in LTA by using a radio frequency power (R. F. power) of less than 100W.
5) Analysis of standard samples made available by the joint FAO/WHO investigation group showed that the present method gives accurate results.

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Antimicrobial action of esters of p-hydroxybenzoic acid (PHBAE) was tested on Escherichia coli JE1011 and its NS mutants (outer membrane mutants). JE1011 was resistant to more hydrophobic PHBAE (n-propyl, isopropyl, n-butyl and isobutyl esters), while the NS mutants were sensitive to them. On treatment with EDTA, the cells of JE1011 released protein, lipopolysaccharide and phospholipids. It was shown that the outer membrane of these cells was damaged by EDTA-treatment. The more hydrophobic PHBAE had a stronger antimicrobial action on the cells of JE1011 treated with EDTA. These results suggest that the wild-type outer membrane of gram-negative bacteria functions as a permeability barrier towards the more hydrophobic PHBAE.

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A method was developed for the simultaneous determination of pyrethroid insecticides in grains by means of ECD gas chromatography.
This method consisted of extraction with acetonitrile, clean-up by Florisil column chromatography and determination by ECD-GC. Pyrethroids on the column were eluted with 100ml each of 10% ether in n-hexane and 10% ethyl acetate in n-hexane. Permethrin was eluted in the first fraction, and allethrin, phthalthrin and pyrethrins were eluted in the second fraction. Pyrethroids were determined by ECD-GC with a column of 2% XE-60+2% OV-17 and a column of 3% SE-30. No overlap of pyrethroids and organochlorine pesticides on gas chromatograms was observed. Recoveries of pyrethroids added to grains were in the range of 78-96%.

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Exponentially grown cells of Escherichia coli JE 1011 were heated at 45°C, 48°C or 50°C in phosphate buffer or nutrient broth at pH 7.0. Heating at 45°C did not affect the growth of cells, while heating at 48°C and 50°C elongated the lag time as a function of heating time. During heating for 30 min, protein, lipopolysaccharide, phospholipid, magnesium ion (Mg²⁺), and 260nm absorbing materials were released from the heated cells. The heating at 45°C caused the release of lipopolysaccharide and outer membrane proteins, indicating structural damage to the outer membrane. On heating at 48°C and 50°C, 260nm absorbing materials and surface enzymes leaked from the cells extensively. It was shown that heating at these temperatures resulted in structural and functional damage to the cytoplasmic membrane in addition to structural damage to the outer membrane. Hewever, even the heating at 50°C did not cause the decomposition of DNA. More hydrophobic esters of p-hydroxybenzoic acid had stronger antimicrobial action on these heated cells, and their activity was greater towards cells heated at higher temperature. The more hydrophobic PHBAE (propyl and butyl esters) had bactericidal action on Escherichia coli JE 1101 at 50°C.

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Hexobarbital sleeping time (HST) and zoxazolamine paralysis time (ZPT) were measured in male mice after oral administration of 14 food additives. HST and ZPT were significantly (P<0.05) prolonged when hexobarbital or zoxazolamine was given intraperitoneally after pretreatment with diphenyl, o-phenylphenol, piperonyl butoxide or thiabendazole. One hour after the pretreatment, the maximum prolongation of HST and ZPT was observed.
To prolong HST or ZPT in mice, it was necessary to administer 50mg/kg of diphenyl, o-phenylphenol or thiabendazole to them. However, a dose of only 5mg/kg of piperonyl butoxide prolonged HST and ZPT.

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Propyl gallate (100mg per rat) was given by stomach tube to fed or fasted rats, and the urinary metabolites were determined by the iatroscan method. Most metabolites were excreted within 3 days after the administration.
The urine of fed rats contained the following metabolites (% of dose; mean of 5 rats): glucuronide of propyl gallate 5%, free gallic acid 7% and its conjugate 22%, free 4-O-methyl gallate 28% and its conjugate 10%. Free propyl gallate and resorcinol were not detectable in the urine, but pyrogallol was observed in 3 of 5 rats.
The amounts of total gallate, total 4-O-methyl gallate and glucuronide of propyl gallate excreted in the fasted rats were similar to those in the fed rats, but the formation of gallic acid conjugate was depressed to about 2/5 by fasting. Pyrogallol and resorcinol were observed in the urine of 4 of 5 fed rats and 1 of 5 fasted rats.

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The metabolism of propyl gallate by isolated hepatocytes of the rat and the effect of alcohol on the metabolism were investigated. When the hepatocytes (5×10⁶ cells) were incubated with 10^-3M propyl gallate for 1hr, the cells hydrolyzed about 65% of the substrate, and produced large amount of free gallic acid and its conjugate and a small amount of 4-O-methyl gallate. However, the addition of methionine caused an increase in 4-O-methyl gallate formation from propyl gallate.
The hepatocytes also produced an unidentified substance in addition to the normal products in the presence of ethanol (5×10^-3M). The maximum amount of the unidentified substance in an incubation mixture containing 0.1M ethanol was observed after the incubation for 1hr, and the amount then gradually decreased. The unidentified substance was identified as ethyl gallate by TLC, GC and GC-MS. Ethyl gallate might be produced by the transfer reaction of gallic acid produced by the action of esterase. The ester transfer reaction also occurred with other alcohols such as methanol, iso-butyl alcohol and iso-amyl alcohol, but ethanol was the most effective.

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An improved analytical method for toxaphene was developed.
The presence of PCBs, DDTs and certain other organochlorine insecticides interferes with the ECD-GC method for toxaphene determination. Therefore, interfering pesticides and PCBs were removed by acid treatment, and then continuous distillation was carried out. Operational conditions were as follows: the test solution was evaporated to dryness in a flask and cold sulfuric acid and fuming nitric acid were added to the residue. The mixture was allowed to stand for 15 minutes at 0°C, and then 100ml of cold water was added. The improved oil distillation apparatus was connected to the flask, and 4ml of n-heptane was placed in the apparatus. The flask was heated for 1 hour, then the n-heptane layers was used for ECD-GC analysis.
By this method, DDTs, Drins and PCBs were converted to compounds which did not register on ECD-GC. Toxaphene was scarcely affected, and was recovered in a good yield. This method is more convenient than a column chromatographic method.

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CNP (chlornitrofen), widely used as a paddy herbicide in Japan, is known to be converted readily to the corresponding amino derivative by reduction of the nitro group in the environment. An unusually high level of CNP residue concentration was reported in fresh-water fish and shellfish during the application period. Therefore, the occurrence of CNP reduction products in samples with high CNP residue levels was investigated and two unknown compounds were detected which were not identical with any standard pesticides on GLC. These two compounds, obtained in preparative scale experiments, showed high sensitivity in ECD-GC and NP-FID GC and gave CNP-amino compound on alkaline hydrolysis. From the GC-MS analysis and the above mentioned chemical property, the structures of these compounds were elucidated as CNP-acetamide (CNP-a) and CNP-formamide (CNP-f); these structures were confirmed by comparison of the products with authentic samples prepared from CNP.
Analytical methods for residual CNP-a and CNP-f were established and the residual concentrations were investigated in fresh-water fish and shellfish at regular intervals through a year at a fixed location in Miyagi prefecture. The maximum concentrations of CNP derivatives were 0.58ppm (CNP-a) and 0.24ppm (CNP-f) in freshwater clam, 0.04 (CNP-a) and 0.05ppm (CNP-f) in loach, and 0.06 (CNP-a) and 0.13ppm (CNP-f) in crucian carp during May and June, and these levels decreased rapidly after the end of the herbicide application period.

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The LD₅₀ of cadmium chloride to rats was studied after oral administration with or without L-cysteine, and the accumulation and excretion of cadmium were also examined during continuous oral administration.
The LD₅₀ of cadmium chloride by single oral administration to rats was 204.2mg/kg. However, in the presence of L-cysteine (1500mg/kg), the LD₅₀ decreased to 58.3mg/kg.
Compared with the rats given continuous oral administration of cadmium chloride (10mg/kg/day) alone for 8 weeks, the simultaneous oral dosing of cadmium chloride (10mg/kg/day) and L-cysteine (1500mg/kg/day) increased the amount of cadmium retained in the organs. Cadmium retained in the organs reached a maximum level after 6 weeks. In the rats given cadmium chloride, the urinary excretion of cadmium was about 0.1μg/day through 8 weeks. In the rats given L-cysteine with cadmium chloride, the daily excretion of cadmium was similar (0.1μg) until cadmium retained in the organs reached the maximum level. Thereafter, the urinary excretion of cadmium increased markedly to about 1.0μg/day.

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A method was developed for the determination of propineb and propylenethiourea in agricultural products. Propineb was measured as propylenediamine after hydrolysis with acidic stannous chloride. The propylenediamine was reacted with 1-dimethylaminonaph-thalene-5-sulfonyl chloride, and after standing for 3 hours, the product was separated by thin-layer chromatography and quantitated by dual-wavelength densitometry. Propylenethiourea was quantitated by FPD-GC of its N-methanesulfonylated S-(m-trifluoromethyl-benzyl) derivative. The recoveries of propineb added to crops were in the range of 41 to 74%. Those of Propylenethiourea added to crops were in the range of 77 to 107%.
In the analysis of commercial products, propylenethiourea was detected in 50% of beer samples in the range of 0.001 to 0.007ppm.

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A gas liquid chromatography (GLC) method was developed for the determination of tetrakis-[methylene-3-(3′, 5′-di-tert-butyl-4′-hydroxyphenyl) propionate] methane, known as Irganox 1010, a high-molecular phenolic antioxidant in polyethylene food packaging films.
Direct analysis by GLC is inappropriate for Irganox 1010 because of the high molecular weight. Irganox 1010 was therefore converted to methyl (3, 5-di-tert-butyl-4-hydroxyphenyl) propionate by saponification with potassium hydroxide and then determined by GLC. The product was identified by GLC-MS.
The results of the analysis of Irganox 1010 in polyethylene food packaging films by the present method were compared with the results of high performance liquid chromatography (HPLC) for the same samples. The results of the two methods agreed well, and the coefficients of variation in the GLC and HPLC methods were found to be 2.9% and 3.2%, respectively.

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Residual α-methylstyrene (α-MSt) in products made of styrene (St)-based polymers, such as polystyrene (PS), St-acrylonitrile copolymers (SAN) and acrylonitrile-butadiene-St resins (ABS), was determined by gas liquid chromatography. St-based polymers were dissolved in dichloromethane and analyzed for α-MSt by direct injection of the polymer solutions. Further, α-MSt migrations from ABS sheet to water, 4% aceteic acid, 20% ethanol and n-heptane were measured by mass fragmentography, using selected ion recording at m/e 118, 117 and 103.
In commercial PS and SAN products, α-MSt was not detected at a quantitation limit of 20ppm, but in ABS products such as ladles, graters, sheets and lunch trays, residual α-MSt amounted to 72-2496ppm, α-MSt migration from 2 commercial ABS sheets into 3 food simulants (water, 4% acetic acid and 20% ethanol) was not detected. However, some migration into n-heptane was detected, so α-MSt appears to have some tendency to migrate into oils and fats and fatty foods.

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A method was developed for the determination of ethylenediaminetetraacetic acid (EDTA) in foods such as canned clams and vegetables, salad dressings and mayonnaises by high performance liquid chromatography (HPLC).
1, 2-Diaminopropane-N, N, N′, N′-tetraacetic acid (DPTA) was added to foods as an internal standard before extraction. EDTA and DPTA were extracted with 0.5% cupric sulfate solution and were analyzed by HPLC using a Zorbax ODS column with 0.75M sodium sulfate solution as the eluent. The ultraviolet absorption detector was set at 245nm. EDTA was quantitated by the internal standard method. Recoveries of EDTA (as disodium salt 0.5mg/10g) ranged from 98.6 to 103.8% and the coefficient of variation ranged from 0.3 to 3.7%.
By this method, American commercial salad dressing and mayonnaise were found to contain 36-42ppm EDTA. No EDTA (less than 5ppm) was detected in Japanese commercial foods.

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[in Japanese]

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