Blue mussels, Mytilus edulis, were collected in Osaka Port, Kyoto Maizuru Bay and Hokkaido, Japan, and analyzed with a fluorescence spectrophotometer followed by fluorescence-HPLC on a C-18 reversed-phase column to examine environmental contaminants in the samples from each location. The fluorescence (Em λ_max=ca. 350nm) and excitation spectra (Ex λ_max=ca. 310nm) of samples obtained from different areas were similar. The ratios of fluorescence intensity of the samples from Osaka Port and Kyoto Maizuru Bay to that of samples from the environmentally clean coast of Hokkaido were ten and five to one, respectively. The levels of individual polycyclic aromatic hydrocarbons (PAHs) ranged from 66ppb for pyrene to 1.3ppb for benzo (a) pyrene in mussels from Osaka Port as determined by HPLC. On the other hand, the maximum concentrations in mussels of unknown fluorescent substances in the samples from Osaka Port and Hokkaido were 1600ppb and 20ppb, respectively, when picene was adopted as standard. These results suggest that the developed method can be used to monitor changes of the marine environment by the use of fluorescent substances as indicators.

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In the determination of hydrogen peroxide in semi-dried sardine by the oxygen electrode method, hydrogen peroxide added at the level of 1-100ppm was completely decomposed. However, sometimes trace amounts of hydrogen peroxide were actually detected in the samples.
The cause of the variability was investigated. The added hydrogen peroxide was primarily decomposed enzymatically. On the other hand, hydrogen peroxide was formed by the oxidation products of lipid during extraction, and it was not decomposed because of the inactivation of the enzymes in the extract.
The oxygen electrode method was modified to measure the residual hydrogen peroxide in semi-dried sardine after treatment with hydrogen peroxide. Acidic extractant (sulfuric acid containing zinc sulfate and potassium bromate) was suitable for the extraction, and catalase treatment of the sample was necessary. This modified method enabled us to determine the residual hydrogen peroxide without interference from enzymes and the oxidation products of lipid.

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A high-performance liquid chromatographic method (HPLC method) for the quantitative determination of destomycin A (DM) and fradiomycin (FM) in beef was developed. The procedures involve protein precipitation with trichloroacetic acid (TCA) and column chromatography using ion-exchange resin (Amberlite CG-50 Li⁺ type). The eluate was analyzed by reversed-phase, ion-pair chromatography using post-column on-line derivatization with o-phthalaldehyde.
The HPLC determination of DM and FM was carried out on a Shim-pack CLC-ODS column by measuring relative fluorescence (excitation maximum 340nm and emission maximum 450nm).
Recoveries of DM and FM added to beef at 10ppm were 81.9 and 71.6%, respectively. Consequently DM and FM could be determined simultaneously. The limits of detection were 0.1ppm for DM and 0.2ppm for FM.

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Enteropathogenicity of diarrhetic shellfish toxins (DST) was evaluated in rabbit intestinal loops, mouse intestinal loops and suckling mice, by using the methods established for the detection of bacterial enterotoxins. Crude toxin of DST (cr-DST) was prepared by acetone extraction from scallops (Patinopecten yessoensis) implicated in the poisoning outbreaks in Osaka in 1978 and 1982. Individual DSTs such as dinophysistoxin-1 and -3 (DTX-1 and DTX-3), pectenotoxin-1 (PTX-1) and okadaic acid were also tested on suckling mice for diarrheagenicity.
The rabbit loop injected with 8 MU of cr-DST showed significant fluid accumulation in 18 hours. The mouse loop injected with 0.8 MU of cr-DST also showed significant fluid accumulation in 6 hours. In the suckling mouse test, positive results were observed with 0.1 MU of cr-DST in 4 hours. The small intestines with fluid accumulation in the loop tests were sectioned and stained with hematoxylin-eosin for histopathological studies. Destruction of epithelium and edema of the lamina propria were observed. In the suckling mouse test, DTX-1, DTX-3 and okadaic acid showed positive results, whereas PTX-1 did not.
From these data, we concluded that DST shows enterotoxic activities in these animal models and that okadaic acid, DTX-1 and DTX-3 are diarrheagenic.

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Water and ethanol extracts from leaf and stem-rhizome of Acanthopanax senticosus were administered to rats, and their effects on serum biochemical parameters, organ weights and histochemistry were investigated. The mutagenicity was also examined. The results obtained may be summarized as follows:
1) The extracts were not mutagenic to Salmonella typhimurium TA100 and TA98.
2) The micronucleus test in mice was negative.
3) No significant difference in body weight was observed between the treated groups and the control group.
4) The serum biochemical examination in rats showed the following significant differences from the control (p>0.01): ALP, γ-GTP, Ch-E, T. G, T. P, Cl and IP for the group given leaf extract with ethanol (40mg/kg), T. G and Na for the group given stem-rhizome extract with ethanol (40mg/kg), T. G, ALB and IP for the group given stem-rhizome extract with ethanol (400mg/kg), T-BIL, T. G, CREAT, BUN, K and Cl for the group given stem-rhizome extract with water (40mg/kg) and T. G, ALB, Cl and IP for the group given stem-rhizome extract with water (400mg/kg).
5) The liver and kidney weights in the group given leaf extract with ethanol were different from the control values (p<0.01).
6) The histological findings after administration of leaf extract with ethanol showed no pathological change.

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There have been two poisoning cases which were suggested to have been caused by eating seaweed belonging to the Gracilariaceae family in Japan, one in Yamagata Prefecture in 1980 and the other in Ehime Prefecture in 1982. We therefore examined the biological activities of Gracilaria verrucosa gathered in Ehime Prefecture by using guinea pigs and mice as test animals. The seaweed was extracted with ether, with chloroform-methanol (1:1) and then with methanol. On i. p. administration to guinea pigs, the chloroform-methanol extract and the methanol extract induced dilatation of ear blood vessels, sneezing, urination, evacuation, loss of righting reflex, etc. The methanol extract showed a laxative action and fatal toxicity in mice on p. o. administration. The ether extract stimulated the central nervous system when given p. o., i. p. or i. v. These effects were, however, obtained only at very high doses of the samples. Accordingly, it is not clear whether the seaweed gathered was responsible for the human acute toxic symptoms.

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Raw material of Gracilaria verrucosa was divided into three portions. One was untreated, the second was soaked in water and the last was soaked in limewater. Methanol extracts were prepared from them and administered to guinea pigs intraperitoneally, and the acute toxicity and symptoms were observed. Differences in chemical components among them were also investigated. The results indicated that the method applied to prepare the commercial seaweed product is efficaceous.

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Food Green No. 3 (Fast Green FCF, Colour Index No. 42053, FD & C Green No. 3) is prepeared by condensation of 1mol of p-hydroxy-o-sulfobenzaldehyde (HSBA) with 2mol of N-ethyl-N-benzylaniline sulfonic acid (EBASA). In this work, the isolation of two major lower-sulfonated subsidiary colors in Food Green No. 3, the structures of the subsidiary colors, and the separation and identification of the subsidiary colors by high performance liquid chromatography were investigated.
Two subsidiary colors were isolated from Food Green No. 3 by thin layer chromatography. One of the subsidiary colors was identified as 3-[N-ethyl-N-[4-[(4-ethylaminophenyl) (4-hydroxy-2-sulfophenyl) methylene]-2, 5-cyclohexadienylidene] ammoniomethyl] benzenesulfonate (EA-subsidiary color) from the infrared (IR), nuclear magnetic resonance and FAB mass spectra (Figs. 2, 5 and 6). The other appear to be the color (EBA-subsidiary color) obtained by condensation of 1mol of N-ethyl-N-benzylaniline (EBA), 1mol of HSBA and 1mol of EBASA as judged from the IR and absorption spectra. The absorption maxima of EA- and EBA-subsidiary colors in 30v/v% methanol were at 610nm and 624nm, respectively (Table 1 and Fig. 7).
Food Green No. 3 and EA- and EBA-subsidiary colors could be simultaneously separated by reversed phase high performance liquid chromatography on a Zorbax ODS column with 0.5% ammonium carbonate solution-methanol as the eluant (J. Food Hyg. Soc. Japan 27, 30 (1986)), with detection at 624nm for Food Green No. 3 and EBA-subsidiary color and 610nm for EA-subsidiary color. The retention times of Food Green No. 3, and EA- and EBA-subsidiary colors were about 6min, 8min and 10min, respectively (Fig. 8). Linear calibration curves were obtained in the range of 10-100ppm (50-500ng) for Food Green No. 3, and EA- and EBA-subsidiary colors (Figs. 9 and 10). The minimum detectable amount was 5ng (1ppm, 5μl) for Food Green No. 3 and EBA-subsidiary color, and 25ng (5ppm, 5μl) for EA-subsidiary color. The recoveries of EA- and EBA-subsidiary colors added to pure Fast Green FCF (free of subsidiary colors) at levels of 1-10% were 98.4-101. 8% and 98.4-102.1%, respectively (Table 2).
EA- and EBA-subsidiary colors in six commercial samples of Food Green No. 3 were analyzed by this method. EA-subsidiary color content was determined to be 0.4-5.7%, and EBA-subsidiary color was detected at levels of 0.2-3.5% (Table 3 and Fig. 11). This method is considered to be suitable for the analysis of EA- and EBA-subsidiary colors in Food Green No. 3.

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A high performance liquid chromatographic (HPLC) method using an ion-pair partition system was established for simultaneous determination of sodium saccharin (SA) and glycyrrhizin (GL) in foods. SA and GL in foods such as soy sauce, soy paste and pickled herring with malt were extracted with ammonium hydroxide solution and the extracts were treated with a Sep-pak C₁₈ cartridge prior to HPLC analysis. SA and GL could be separated within 20min on a reversed-phase column, “LiChrosorb RP-18”, with ethanol. 0.05M sodium dihydrogenphosphate (2:3) containing 0.02M cetyltrimethylammonium chloride, adjusted to pH 3.0, as the eluent. The eluate was monitored at 245nm. Recoveries of SA and GL from foods were 97.8-99.5% and 93.8-99.1%, respectively. The detection limits were 0.005g/kg. Among higher-grade soy sauce samples, an inverse correlation seemed to exist between SA and GL contents.

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The utility of the substrate-labeled fluorescent immunoassay technique (SLFIA method) was investigated for the determination of kanamycin in pork. The sample was blended with 0.5% metaphosphoric acid-phosphate buffer (1:1), and centrifuged. After filtration of the supernatant, the filtrate was analyzed by the SLFIA method. The calibration curve was rectilinear from 0.025μg/ml to 0.25μg/ml. Recoveries of kanamycin from pork spiked at the levels of 0.1 and 0.5μg/g were 85.3 and 78.7%, respectively. The accuracy and reproducibility were high, and an excellent correlation between the results of the SLFIA method and the bioassay method was obtained (r=0.98). This method is applicable to levels as low as 0.1μg/g kanamycin in pork with high accuracy and reproducibility.

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An analytical method was developed for the determination of copper chlorophyll and copper chlorophyllin in food. Firstly, copper chlorophyll was extracted from foods with ethyl acetate under mild alkaline conditions, and then copper chlorophyllin was extracted with n-butanol under mild acidic conditions. The ethyl acetate layer was washed with water, dried with anhydrous sodium sulfate and evaporated to dryness. The residue was dissolved in hexane-chloroform (9:1) and applied to a silica gel column. Copper chlorophyll was eluted with methyl ethyl ketone-methanol (3:2). The eluate was evaporated to dryness, and the residue was dissolved in acetone. The n-butanol layer was washed with water and evaporated to dryness. The residue was dissolved in methanol.
Thin layer chromatography was used for the qualitative determination of copper chlorophyll and copper chlorophyllin. Atomic absorption spectrometry was used for quantitative determination of copper.
The recoveries of copper chlorophyll and copper chlorophyllin added to foods at levels of 10ppm were in the range from 80% to 97% and the detection limit of copper was 0.1ppm. By using this method, 41 commercial food samples were analyzed. Copper chlorophyll was detected in 8 samples of processed bracken.

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[in Japanese]

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[in Japanese]

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