[Validation Study on an Improved Quantitative Method for Aflatoxins in Foods].

As an analytical method for aflatoxins in foods, the analytical method based on the notification by the director of the Food Safety Department, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare (August 16, 2011) has been established. In order to improve the operability and analytical performance of the conventional method, this study aimed to construct an improved method that optimized selection of immunoaffinity column (IAC) and purifying condition, and omitted evaporation after the purification with IAC. In the recovery test performed by adding 2.5 ng/g of aflatoxin B1, B2, G1 and G2 standard solutions into 9 kinds of food samples, the improved method achieved the established target values: 77.0-99.7% of recovery, 1.7-5.6% of intra-assay coefficient of validation, and 0.9-3.6% of inter-assay of coefficient of variation, respectively. The improved method also achieved 4.3-10.5% greater recovery and 1.5 hours shorter preparation time than the conventional one. These results indicate applicability of the improved method for 9 kinds of foods and its efficacy as an analytical method for aflatoxins in foods.

[Development of an LC-MS/MS-Based Rapid and Simple Analytical Method for Six Fungicides].

In this study, we developed an LC-MS/MS-based rapid and simple analytical method for six fungicides; imazalil, o-phenylphenol, thiabendazole, fludioxonil, azoxystrobin and pyrimethanil, the latter three were newly approved for use after 2011. For expediting and simplification, we merged the extraction method with that of the pesticide analysis. For purification step, loading of 1 mL of sample extracts to 500 mg Oasis HLB column and elution with 8 mL of acetonitrile gave satisfactory results. The performance of the present method was confirmed for orange, grapefruit, and lemon samples fortified with the six fungicides. The results showed that the average recovery ranged from 89.7 to 100.0%, intra- and inter-assay CV% ranged from 1.5 to 5.0% and from 0.5 to 4.9%, respectively, achieving the target values of the Japanese official guideline for residual pesticide analysis. The limits of quantification of this method were determined to be 1 mg/kg for o-phenylphenol, and 0.2 mg/kg for the other five fungicides. These values were lower than their corresponding regulation values. In addition, we confirmed the usability of the present method for fungicide inspection of commercially available citrus fruits. During 2017-2019, there was no conflict between the food labeling and the fungicides detected and no fungicide with the concentration exceeding maximum residue level was detected.

Concentrations of total mercury and methylmercury in red snow crabs (Chionoecetes japonicus) caught off the coast of Japan.

The total mercury (T-Hg) and methylmercury (MeHg) concentrations in red snow crabs (Chionoecetes japonicus) caught off the coast of Japan were analyzed. The T-Hg concentration ranged from 0.03 to 0.56 mg/kg (mean: 0.21 mg/kg) in the raw muscle, and 0.02 to 0.74 mg/kg (mean: 0.27 mg/kg) in the boiled muscle. The MeHg concentration ranged from 0.04 to 0.54 mg/kg (mean: 0.20 mg/kg) in the raw muscle. The mean ratio of MeHg to T-Hg was 0.88. The crab body weight was found to significantly correlate with the concentrations of T-Hg (r = 0.488) and MeHg (r = 0.490) (p ≤ 0.01). For the general population in Japan, the intake of MeHg from eating red snow crab was estimated to be lower than 0.013 mg/week, which was less than one-sixth of the tolerable MeHg intake (0.08 mg/week).

[Enhancement of Pesticide Peak Response in GC-MS in the Presence of Multiple Co-Existing Reference Pesticides].

We carried out a collaborative study in six laboratories to confirm the universality of the enhancing effect of co-existing reference pesticides on the GC-MS peak response to a target pesticide (malathion, procymidone, or flucythrinate). First, we confirmed the response enhancement of the target pesticides with increasing numbers of co-existing reference pesticides in solution. Then, using diluted green soybean matrix, we analyzed the target pesticides with two types of matrix-matched calibration, containing the target pesticides or 166 other pesticides. In both cases, the response-enhancing effect of co-existing pesticides was confirmed in all laboratories. The enhancement was reduced by addition of green soybean matrix to the sample and calibration solutions. Our results show that it is necessary to estimate the peak response-enhancing effect of co-existing pesticides in the calibration solution to obtain accurate results with GC-MS determination. The enhancing effect could be reduced by addition of food matrix to the sample and calibration solutions.

Problems and solutions of polyethylene glycol co-injection method in multiresidue pesticide analysis by gas chromatography-mass spectrometry: evaluation of instability phenomenon in type II pyrethroids and its suppression by novel analyte protectants.

Polyethylene glycol 300 is commonly used as a base material for "analyte protection" in multiresidue pesticide analysis via gas chromatography-mass spectrometry. However, the disadvantage of the co-injection method using polyethylene glycol 300 is that it causes peak instability in α-cyano pyrethroids (type II pyrethroids) such as fluvalinate. In this study, we confirmed the instability phenomenon in type II pyrethroids and developed novel analyte protectants for acetone/n-hexane mixture solution to suppress the phenomenon. Our findings revealed that among the examined additive compounds, three lipophilic ascorbic acid derivatives, 3-O-ethyl-L-ascorbic acid, 6-O-palmitoyl-L-ascorbic acid, and 6-O-stearoyl-L-ascorbic acid, could effectively stabilize the type II pyrethroids in the presence of polyethylene glycol 300. A mixture of the three ascorbic acid derivatives and polyethylene glycol 300 proved to be an effective analyte protectant for multiresidue pesticide analysis. Further, we designed and evaluated a new combination of analyte protectant compounds without using polyethylene glycol or the troublesome hydrophilic compounds. Consequently, we obtained a set of 10 medium- and long-chain saturated fatty acids as an effective analyte protectant suitable for acetone/n-hexane solution that did not cause peak instability in type II pyrethroids. These analyte protectants will be useful in multiresidue pesticide analysis by gas chromatography-mass spectrometry in terms of ruggedness and reliable quantitativeness. Graphical abstract Comparison of effectiveness of the addition of lipophilic derivatives of ascorbic acid in controlling the instability phenomenon of fluvalinate with polyethylene glycol 300.

Persistent organic pollutants in red-crowned cranes (Grus japonensis) from Hokkaido, Japan.

The red-crowned crane (Grus japonensis) from eastern Hokkaido is classified as a Special Natural Monument in Japan. In this study, we determined the concentrations of persistent organic pollutants (POPs) in red-crowned crane muscle tissues (n = 47). Polychlorinated biphenyls (PCBs) had the highest median concentration (240ng/g lipid weight), followed by dichlorodiphenyltrichloroethane and its metabolites (DDTs) (150ng/g lipid weight), chlordane-related compounds (CHLs) (36ng/g lipid weight), hexachlorobenzene (HCB) (16ng/g lipid weight), hexachlorocyclohexanes (HCHs) (4.4ng/g lipid weight), polybrominated diphenyl ethers (PBDEs) (1.8ng/g lipid weight), and finally, Mirex (1.5ng/g lipid weight). Additionally, a positive correlation was found among POP concentrations. No sex differences beyond body parameters were observed. Additionally, red-crowned cranes exhibited a high enantiomeric excess of (+)-alpha-HCH, with enantiomer fractions varying from 0.51 to 0.87 (average: 0.69).

Evidence of exposure to chemicals and heavy metals during pregnancy in Japanese women.

Purpose: Prenatal exposure to environmental chemicals is a growing concern, because such exposures have been shown to be associated with various diseases. The levels of chemicals and heavy metals in maternal blood, cord blood, maternal urine and amniotic fluid in Japanese pregnant women were investigated. Methods: A total of 145 women, including 14 fetal growth restriction cases, were included in the present study. The levels of phthalates (di[2-ethylhexyl]phthalate and mono[2-ethylhexyl]phthalate), perfluorinated compounds (perfluorooctane sulfonate, perfluorohexanoic acid, perfluorooctanoic acid, and perfluorononanoic acid), pesticides (dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, 3-phenoxybenzoic acid, and octachlorodipropyl ether), bisphenol A, nicotine (nicotine, nornicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine), polybrominated diphenyl ethers, and heavy metals were measured. The relationship between fetal growth and the levels of chemicals and heavy metals were investigated. Results: Phthalates, perfluorinated compounds, pesticides, polybrominated diphenyl ethers, and heavy metals were detected in high frequency, whereas nicotine and bisphenol A were almost negative. Phthalates, perfluorinated compounds, and several heavy metals were transferred to the fetus. High perfluorononanoic acid levels in the maternal blood and cord blood, and low perfluorooctanoic acid level in the cord blood were significantly and negatively associated with fetal growth. Conclusions: The present study showed that pregnant women in Japan and their fetuses are exposed to a variety of chemicals and heavy metals.

Development of monoclonal antibody-based ELISA for the quantification of orange allergen Cit s 2 in fresh and processed oranges.

The aim of this study was to develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) for the quantification of a major allergen (Cit s 2) in fresh and processed oranges. Purified recombinant Cit s 2 (rCit s 2)-small ubiquitin-like modifier (SUMO) was used for the production of mAbs. In the optimized ELISA, the recovery of rCit s 2 from Navel oranges or orange juice was 107-132%, and the intra- and inter-assay coefficients of variation were 3.1-8.8% and 4.4-11%, respectively. The Cit s 2 content in fresh oranges was determined to be 1,800±430ng/g, while this content was much lower in the processed foods. The developed ELISA demonstrated high reproducibility, sensitivity, and accuracy, and this assay may help individuals with orange allergy by determining Cit s 2 quantities in food products and controlling their Cit s 2 intake.

Evaluation of the matrix-like effect in multiresidue pesticide analysis by gas chromatography with tandem mass spectrometry.

In multiresidue pesticide analysis using gas chromatography, it has long been recognized that an increase in the number of pesticides present in a standard solution can result in an enhancement of the peak responses of certain pesticides. Despite being widely acknowledged, this phenomenon has been rarely studied and is poorly understood. In this study, the authors have tentatively called this phenomenon the "matrix-like effect" and demonstrated it clearly using gas chromatography with tandem mass spectrometry. Five selected pesticides, namely, omethoate, terbufos, malathion, procymidone, and permethrin, and four internal standard candidates, namely, triphenyl phosphate, naphthalene-d8 , phenanthrene-d10 , and fluoranthene-d10 , were used to evaluate the matrix-like effect following the addition of 58, 108, and 166 other pesticides. With the exception of naphthalene-d8 , the responses of all evaluated pesticides and internal standard candidates were dramatically enhanced by the addition of up to 166 coexisting pesticides. The relative response factors of the five pesticides to each internal standard candidate were not constant under the conditions studied, meaning that these internal standard candidates did not adequately compensate for the matrix-like effect, at least for the five evaluated pesticides. The results revealed that the presence of various mixtures of pesticides in standard solutions might act as an unintentional analyte protectant, that is, some sort of troublesome "quasi-matrix."

[A Survey of School Lunch Provision for Elementary School Students with Food Allergy in Seven Cities of Osaka Prefecture].

The number of students with food allergy is currently increasing. Moreover, the unintentional mixing or accidental ingestion of allergy-causing food materials in school lunches has attracted great attention. The aim of this study was to verify the current status of elementary school lunch provision for students with food allergy. We investigated the elementary school lunch services in seven cities in Osaka prefecture. The egg elimination diet was provided in five of the seven cities. In four of these five cities, we did not detect the presence of egg residue either on the surface of various cookware used to prepare the egg elimination diet or in the food itself. In this investigation, the egg elimination diet was provided properly, but we observed differences among the cities in the manual preparation of foods for food allergy diets. To step up these efforts, our results suggest the necessity of preparing a manual to consider individual conditions of school lunch services.

Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells.

The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an "epimutagen combination" that is effective at low concentrations as detected in maternal peripheral and cord blood.

Dechlorane Plus and decabromodiphenyl ether in atmospheric particles of northeast Asian cities.

Atmospheric particles were collected in several cities in Japan (Sapporo, Sagamihara, Kanazawa, and Kitakyushu), Korea (Busan), and China (Beijing) using a high-volume air sampler equipped with a quartz fiber filter. The summer and winter samples were analyzed using gas chromatography-high-resolution mass spectrometry for Dechlorane Plus (DP). Decabromodiphenyl ether (BDE-209) was also analyzed for the samples from Kanazawa and Beijing. DP was detected in all samples. The mean total DP (ΣDP) concentration was highest (6.7 pg/m(3)) and lowest (0.87 pg/m(3)) in the winter samples from Sagamihara and Busan, respectively. The seasonal variation of DP concentrations varied by sampling site in this study. BDE-209 was detected in all the analyzed samples except for one of the Kanazawa winter samples. BDE-209 concentration was considerably higher in Beijing than in Kanazawa. Significant correlations were found between the concentrations of ΣDP and BDE-209 in the winter samples from Kanazawa and in both summer and winter samples from Beijing. This similarity in the atmospheric behavior of DP and BDE-209, especially in winter, is assumed to reflect a common end usage and release mechanism.

[Allergy due to probable contamination of bread used for school meals with milk].

BACKGROUND: In May 2012, two schoolchildren developed allergic symptoms after eating a school meal of fried bread in Osaka, Japan. One specific raw material, milk, should not be present in fried bread. However, we suspected the unintentional mixing of milk in the bread manufacturing process. Our aim was to verify the reason if this was so. METHODS: We first manufactured bread that contained milk as one of its components. We then thoroughly cleaned the manufacturing line except for the dough divider and, as per the revised instruction manual, continuously manufactured bread that did not contain milk. In this manner, we tried to simulate the conditions at the time of the incident. Casein is a major milk protein. We, therefore, determined casein concentration in the milk-free bread, using casein enzyme-linked immunosorbent assay. RESULTS: We observed casein levels higher than 1000 ppm in the initial lots of breads, but lower levels in the later lots. Casein levels also decreased upon frying of bread. CONCLUSION: High casein levels, which were observed in the earlier lots of breads, were lower in the later lots. In the manufacturing line, the remnants of the bread dough that contained milk presumably got mixed with the dough used in the subsequent manufacture of milk-free bread.

Inhalation and dietary exposure to Dechlorane Plus and polybrominated diphenyl ethers in Osaka, Japan.

This study estimated daily exposure to Dechlorane Plus (DP) and polybrominated diphenyl ethers (PBDE) via inhalation and diet. Samples of atmospheric particles and food (obtained by market basket method) from Osaka, Japan were analyzed for DP (syn-, anti-) and PBDE using gas chromatography-mass spectrometry. DP was detected in both atmospheric particles and food samples. Among the atmospheric particles, DP was detected in all samples. ΣDP concentration was 7.1-15.4 pg m(-3) and anti-DP was the dominant residue among DP isomers. PBDE was also detected in all the atmospheric particles. ΣPBDE concentration was 9.9-23.3 pg m(-3). In the market basket study, DP was detected in Groups Ш (sugar and confectionary), V (legumes and their products), X (fish, shellfish, and their products), and XI (meat and eggs) at concentrations of 3.3, 2.8, 1.9, and 1.5 pg g(-1) wet wt, respectively. PBDE was detected in Groups Ш, IV (oils and fats), V, X, XI, and XШ (seasonings and other processed foods) at concentrations of 153, 79.1, 74.6, 308, 94.8, and 186 pg g(-1) wet wt, respectively. The daily intake of ΣDP (750 pg day(-1)) via inhalation and diet was approximately one percent of that for ΣPBDE (62 ng day(-1)).

Detection of polybrominated diphenyl ethers in culture media and protein sources used for human in vitro fertilization.

The concentrations of 10 polybrominated diphenyl ethers (PBDEs) in commercial culture media and protein sources (PSs) for in vitro fertilization (IVF) of human ova were investigated. Samples of 15 IVF media (IVFM), nine sperm preparation media (SPM), and six PSs were analyzed. PBDEs were detected in 10 IVFM, seven SPM, and all PS samples in ranges of 0.6-35, 0.9-31, and 7.5-385pgg(-1), respectively. A dominant PBDE congener BDE-47 was detected in the PS and PS-supplemented samples. Our findings suggested that PS supplementation was the potential cause of PBDE-contamination of IVFM and SPM.

Detection of Dechlorane Plus and brominated flame retardants in marketed fish in Japan.

Fish samples purchased from Japanese markets were analyzed for Dechlorane Plus (DP)(syn-, anti-), polybrominated diphenyl ether (PBDE), and hexabromocyclododecane (HBCD)(α, γ). Twenty fish were analyzed using gas chromatography-mass spectrometry for DP and PBDE, and using liquid chromatography-tandem mass spectrometry for HBCD. DP was detected in 18 samples and ∑DP concentrations were <0.2-14.2 pg g(-1)wet wt. Among the DP isomers, anti-DP was the dominant residue observed in this study. PBDE was detected in all samples. Concentrations of ∑PBDE ranged from 2.2 to 878 pg g(-1) wet wt. HBCD was detected in 18 samples, and ∑HBCD concentrations were <0.02-21.9 ng g(-1)wet wt. In fish landed near the East China Sea and the Sea of Japan, we detected relatively high concentrations of DP, PBDE, and HBCD. These results indicate that the seawaters around East Asia are contaminated with flame retardants. This study demonstrates the presence of DP in fish marketed in Japan for the first time.

Di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in media for in vitro fertilization.

In vitro fertilization (IVF) is one of the most important treatments of infertility to provide a chance of conceiving. In IVF treatment, sperm are washed and motile sperm are isolated with sperm washing media (SWM) for the purpose of fertilization; fertilized ova are then incubated for a maximum of 5 or 6d in media for IVF (IVFM). The exposure of fertilized ova to chemicals via such media has not been studied. We determined the concentrations of two contaminants; di(2-ethylhexyl)phthalate (DEHP) and its hydrolyzed product mono(2-ethylhexyl)phthalate (MEHP) in IVFM, SWM, and protein sources (PS: human serum albumin or serum substitute) for IVFM and SWM. The DEHP and MEHP in these media were extracted by a liquid-liquid extraction method and their concentrations determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Fifteen IVFM, nine SWM, and six PS obtained in Japan were examined. The concentrations of DEHP and MEHP in IVFM and SWM were <10-114 and <2.0-263 ng mL(-1), respectively. The concentrations of both DEHP and MEHP were higher in the media containing PS than in those without PS. Either MEHP alone or both DEHP and MEHP were detected in PS. The concentrations of DEHP and MEHP in PS were <10-982 and 47.0-1840 ng mL(-1), respectively. The DEHP and MEHP detected in these media were derived from PS. This is the first study on the chemical contamination of IVFM, SWM, and PS.

Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples.

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4¢,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.

Time trend of hexabromocyclododecane in the breast milk of Japanese women.

Hexabromocyclododecane (HBCD) is a type of brominated flame retardant. We investigated the time trend of the stereoisomer-specific concentrations of HBCD (alpha, beta, and gamma) in breast milk samples of Japanese women by using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pooled milk lipid samples collected in 1973, 1978, 1983, 1988, 1993, 1999, and from 2000 to 2006 were analysed. The concentration was below the detection limit in the samples collected between 1973 and 1983 and increased in those collected since 1988. Between 1988 and 2006, alpha-HBCD was detected in all 11 breast milk samples collected from 25 to 29-year-old Japanese women; gamma-HBCD, in 7; and beta-HBCD, in none of the samples. Time trend of summation operatorHBCD appeared to be related to that of the industrial HBCD consumption level in Japan.

Simultaneous analysis of 17 organochlorine pesticides in natural medicines by GC/MS with negative chemical ionization.

Many methods for the determination of pesticide residues in food have been reported. Although natural medicines should be confirmed to be as safe as food, few methods for the determination of pesticide residues in natural medicines have been reported. In this study, 17 organochlorine pesticides were detected in natural medicines using GC/MS with negative chemical ionization (NCI). GC/MS with NCI can detect halogenated pesticides selectively and thus is suitable for the detection of organochlorine pesticides. This study indicates that GC/MS with NCI is useful for analyzing organochlorine pesticides in natural medicines.