[Examination of Analytical Method for Tris(2,3-dibromopropyl)phosphate and Bis(2,3-dibromopropyl)phosphate to Revise the Official Methods Based on "Act on the Control of Household Products Containing Harmful Substances"].

In Japan, the use of frame retardants [tris(2,3-dibromopropyl)phosphate: TDBPP and bis(2,3-dibromopropyl)phosphate: BDBPP] in several household textile products is banned under the "Act on the Control of Household Products Containing Harmful Substances." As the official analytical methods for testing these substances have not been revised for over 42 years, several issues such as the using of harmful reagents, have been pointed out. Therefore, we developed a new method to revise the official method in our previous study. In this study, the validity of the developed test method is evaluated at six laboratories using two types of textile samples spiked with TDBPP and BDBPP at three concentrations (4, 8, and 20 µg/g). TDBPP and BDBPP are extracted under reflux using methanol containing hydrochloric acid. TDBPP is analyzed using GC-MS, and BDBPP is also analyzed using GC-MS after methylation with trimethylsilyl diazomethane. Although the accuracy (70-120%), repeatability (<10%), and reproducibility (<15%) of a few samples, mainly low concentration samples, are out of range, overall, the concentration level of detection limits of TDBPP and BDBPP (8 and 10 µg/g) in official analytical methods are quantifiable with sufficient precision using the proposed method. Furthermore, harmful reagents are not used in this method. Thus, the method validated in this study is effective as a revised method for the testing of TDBPP and BDBPP in household textile products.

Development of a safer and improved analytical method for polycyclic aromatic hydrocarbons in creosote products.

Polycyclic aromatic hydrocarbons (PAHs) in creosote products used for wood preservation are regulated in Japan. Although the analytical method for this regulation has been stipulated by law, two main problems have been highlighted, namely the use of dichloromethane, a potential carcinogen, as a solvent and inadequate purification. Therefore, an analytical method to solve these problems was developed in this study. Actual creosote-treated wood samples were examined, and it was found that acetone could be used as an alternative solvent. Purification methods using centrifugation, silica gel cartridges, and strong anion exchange (SAX) cartridges were also developed. It was found that the SAX cartridges strongly retained PAHs, and an effective purification method was developed using this phenomenon, in which contaminants were removed by washing with diethyl ether/hexane (1/9 v/v), which could not be achieved with a silica gel cartridge. This strong retention was attributed to cation-π interactions. The analytical method developed in this study yielded good recoveries (81.4-113.0%) with low relative standard deviations (<6.8%), and the limit of quantification (0.02-0.29 µg/g) was significantly lower than the current creosote product regulation. Therefore, this method can safely and effectively extract and purify PAHs from creosote products.

Characterization of synthetic turf rubber granule infill in Japan: Polyaromatic hydrocarbons and related compounds.

Although the health effects of artificial turf fillings have been investigated in Europe and the United States, the actual situation in Japan is unclear. To address this issue, the concentrations of 46 polyaromatic hydrocarbons (PAHs) and related compounds in rubber infills were analyzed prior to their use in synthetic turf fields in Japan. Based on information obtained from the sample suppliers, the investigated samples were divided into five categories: discarded tires, industrial rubber, combinations of these products or unidentified components (mixture/unknown), synthetic rubber specifically manufactured for synthetic turf, and special-purpose thermoplastic elastomers (TPEs). The industrial rubber samples were mixtures of styrene butadiene rubber, natural rubber, and ethylene propylene diene rubber (EPDM). The synthetic rubber samples consisted only of EPDM. A few or none of the PAHs were detected in the synthetic rubber and TPE samples. However, in the discarded tire and industrial rubber samples, benzo[a]pyrene, cyclopenta[cd]pyrene, and 30 other compounds were detected. A comparison between these two categories indicated that the discarded tire samples exhibited higher concentrations of the target compounds than the industrial rubber samples. This finding can be attributed to the presence of EPDM in almost all of the industrial rubber samples, which were not present in the discarded tire samples. The maximum PAH concentrations obtained in the present study were equivalent to or lower than the previously reported PAH concentrations. The total concentrations of the eight PAHs included in the European Chemical Agency (ECHA) assessment of health risks were lower in the present study than those reported by the ECHA. Furthermore, elution testing was performed with four simulated biofluids (gastric and intestinal juices, saliva, and perspiration). The actual elution amounts of all compounds were less than the limits. This report provides basic data for the risk assessment of PAHs in rubber infills.

[Examination of Analytical Method for Mothproofing Agents to Revise the Official Methods Based on "Act on the Control of Household Products Containing Harmful Substances"].

In Japan, the use of mothproofing agents [dieldrin and 4,6-dichloro-7-(2,4,5-trichlorophenoxy)-2-trifluoromethylbenzimidazole; DTTB] in textiles is regulated by the Act on the Control of Household Products Containing Harmful Substances. Since official analytical methods for these agents have been in place for approximately 40 years, we developed an improved method in a previous study. In the present study, we validated this method. Accordingly, six laboratories analyzed the sample prepared at 3 µg/g (1/10 of the regulation value) and 30 µg/g (the regulation value). The high accuracy of the results for these samples in almost all the cases (accuracy: 70-120%, repeatability: <10%, reproducibility: <15%), confirmed the validity of the method. In addition, we examined three samples that were distributed before the introduction of the regulation. The analysis results for these samples showed little variation between laboratiories, indicating that our method is also applicable to actual samples. Meanwhile, the quantitative value was clearly lower in one laboratory than in the others. We presumed that the enhanced effect of the sample matrix (matrix effect) on the internal standards in GC-MS analysis was the main cause for this trend. Therefore, we examined the analytical method using polyethylene glycol 300 (PEG) as an analyte protectant. As PEG minimized the GC-MS response difference between the standard solution and the matrix-containing solution, GC-MS analysis with PEG would be useful for matrix effect measurements in this method.

[Examination of Analytical Methods for Methanol, Trichloroethylene, and Tetrachloroethylene to Revise the Official Methods Based on the Act on the Control of Household Products Containing Harmful Substances].

In Japan, the use of methanol, trichloroethylene, and tetrachloroethylene in aerosol household products is banned under the Act on the Control of Household Products Containing Harmful Substances. As the official analytical methods for testing for these substances have not been revised for over 35 years, several issues have been pointed out. Thus, we developed a new method to revise the official method in our previous study. In this study, validation of the proposed method for detecting the target substances was conducted using two aerosol-product samples (A and B), which contained methanol, trichloroethylene, and tetrachloroethylene. Sample A comprised regulated values of these compounds, while sample B comprised one-tenth of the regulated amounts. They also contained several volatile compounds that served as interfering substances. Subsequently, the samples were analyzed using head space/gas chromatography-mass spectrometry, and it was confirmed that the three target substances were separated from the other chemicals on chromatograms. Validation tests were conducted at seven laboratories to evaluate the proposed method using the prepared samples. In one laboratory, the recovery of trichloroethylene and tetrachloroethylene in sample B was slightly higher at 120%, while the recoveries obtained from the other tests were between 70% and 120%. Relative standard deviation at each laboratory was less than 10%. Furthermore, the relative standard deviations between the validation tests with respect to each chemical were less than 15%. Therefore, the method validated in this study was considered to be effective as a revised method for testing for methanol, trichloroethylene, and trichloroethylene in household aerosol products.

[Analysis of Dieldrin and DTTB in Textiles].

Standard analytical methods for the detection of dieldrin and 4,6-dichloro-7-(2,4,5-trichlorophenoxy)-2-trifluoromethylbenzimidazole (DTTB) in textiles, which are regulated by Japanese law ("Act on the Control of Household Products Containing Harmful Substances"), have been in place for more than 30 years. In this study, we developed an improved analytical method, based on GC-MS, that uses safe reagents and can simultaneously detect dieldrin and DTTB analytes. In the standard (existing) analytical method, dimethyl sulfate, which is a potential carcinogen, is used to derivatize DTTB. In the developed method, phenyltrimethylammonium hydroxide, as an alternative reagent, was used to derivatize DTTB in good results. Dieldrin and the derivatized DTTBs gave highly linear calibration curves when analyzed by GC-MS. Moreover, we found that both analytes are adequately extracted from textiles by refluxing in hydrochloric acid and methanol. Furthermore, we established a purification method using the Bond Elut PRS column that effectively removed interfering substances in woolen products. Finally, we developed an improved analysis method by combining the above-mentioned techniques; the developed method exhibited a recovery rate of 94-104% and a relative standard deviation of less than 7% for both analytes. In addition, the limits of quantitation (dieldrin: 1.3 µg/g, DTTB: 0.72 µg/g) were sufficiently lower than the Japanese regulatory value of 30 µg/g.

Monitoring the concentrations of nonsteroidal anti-inflammatory drugs and cyclooxygenase-inhibiting activities in the surface waters of the Tone Canal and Edo River Basin.

Environmental pollution by pharmaceuticals has become a major problem in many countries worldwide. However, little is known about the concentrations of pharmaceuticals in water sources in Japan. The objective of this study was to clarify variations in the concentrations of seven nonsteroidal anti-inflammatory drugs (NSAIDs) and in cyclooxygenase(COX)-inhibiting activities in river water and domestic wastewater collected from the Tone Canal and the Edo River Basin in Japan. Total NSAID concentrations were higher in the Tone Canal than in the Edo River, and the highest concentration was observed at the domestic wastewater inflow point located in the Tone Canal (concentration averages of salicylic acid, ibuprofen, felbinac, naproxen, mefenamic acid, diclofenac, and ketoprofen in wastewater samples were 55.3, 162.9, 39.7, 11.8, 30.8, 259.7, and 48.3 ng L(-1), respectively). Gas chromatography-tandem mass spectrometry showed that wastewater samples collected during cooler seasons contained higher levels of COX-inhibiting activity. COX-inhibiting activities were highly correlated with NSAID concentrations (particularly for ketoprofen and diclofenac); however, other COX inhibitors, such as NSAIDs that were not examined in this study and/or other chemicals with COX-inhibiting activity, could exist in the water samples because the concentrations of NSAIDs obtained from the water samples did not account for the total COX-inhibiting activities observed. Therefore, COX inhibition assays may be helpful for evaluating the aquatic toxicity of COX inhibitors. In this study, we demonstrated that COX inhibitors in surface water may influence aquatic organisms more than was expected based on NSAID concentrations. Thus, further studies examining other COX inhibitors in the aquatic environment are necessary.

Mutagenic potentials of Amberlite XAD-2-resin extracts obtained from river and drinking waters in the Northwest district of Chiba, Japan.

Amberlite XAD-2 resin extracts of river and drinking water sampled from the Northwest district of Chiba Prefecture in each month during the period from January to December 2008 were investigated to characterize and determine their mutagenic potentials and polycyclic aromatic hydrocarbon (PAH) levels. The extracts from the river water were shown to be mutagenic in Salmonella typhimurium TA98 (a flameshift mutagen) without S9 mix, with higher mutagenic responses in summer and early fall seasons. While the drinking water extracts exhibited weak mutagenicity in both the TA98 and TA100 strains (a base-pair substitution mutagen) without S9 mix, with high mutagenic responses in fall and early winter seasons. GC/MS determinations of the water concentrates showed some seasonal scatter in PAH levels in river water. In contrast, comparatively high concentrations of PAHs were observed for drinking water samples collected during warmer seasons. Statistical studies revealed that there is a lower correlation between the levels of flameshift mutagenicity and the concentrations of PAH in the river water concentrations, but a higher correlation between them in the drinking water samples.

In vitro cyclooxygenase inhibition assay for evaluating ecotoxicity of the surface water and domestic wastewater in the Tone Canal, Japan.

Cyclooxygenase (COX) plays an important role in eicosanoid metabolism. Nonsteroidal anti-inflammatory drugs (NSAIDs) function as COX inhibitors and are frequently detected in the aquatic environment. Here, we measured the in vitro COX-inhibiting activity of the surface water and domestic wastewater in the Tone Canal, Japan. The concentrations of several NSAIDs in the some samples were also determined using gas chromatography-tandem mass spectrometry for confirming the validity of the assay. The target compounds were extracted from the samples using a solid-phase extraction cartridge. A dose-response relationship between the inhibiting activity and sample volume were observed in the wastewater sample. The higher COX-inhibiting activities were observed in the wastewater sample, as compared with the samples of the surface water in the canal. These inhibiting activities reflected the trends of NSAIDs distribution in the canal. However, the inhibiting activities of the water samples could not be entirely explained by the NSAIDs that were selected for instrumental analysis in this study. Other compounds that were not measured by instrumental analysis in this study might contribute to the inhibiting activities. Therefore, the COX-inhibiting assay would be effective for evaluating inclusive ecotoxicity in the aquatic environment.

Formation of polybrominated and polychlorinated ethylphenoxyethylphenols (PXEPEPs) during aqueous chlorination of 4-ethylphenol solutions in the presence of bromide ions.

This investigation was undertaken to determine the effect of the bromide concentration on the formation of polyhalogenated ethylphenoxyethylphenols (PXEPEPs), including predioxins, during the chlorination of 4-ethylphenol in solution. An aqueous solution of 4-ethylphenol was treated with hypochlorite in the presence of various concentrations of bromide ions. The changes in the compositions of the halogenated products in hexane extracts of the chlorinated solution were analyzed by gas chromatograph (GC) and a flame ionization detector (FID) and mass spectrometry (MS). 4-ethylphenol was shown to from several halogenated compounds, including PXEPEPs, as by-products of chlorination. The number of substituted chlorine or bromine atoms ranged from 0 to 4. The formation of bromine-substituted PXEPEPs was observed in the presence of 0.1 equivalents of bromide ions per mole of 4-ethylphenol. The number of substituted bromine atoms increased with the amount of co-existing bromide ions. In the presence of more than one equivalent of bromide ions per mole of 4-ethylphenol, the number of bromine atoms substituted in the PXEPEPs increased, whereas the number of chlorine atoms substituted in the PXEPEPs decreased. GC-MS total ion chromatograms confirmed the formation of polybrominated and polychlorinated predioxins during the aqueous chlorination of 4-ethylphenol in the presence of bromide ions. However, at ten equivalents of bromide ions per mole of 4-ethylphenol, no predioxins were observed in the hexane extract obtained from the aqueous 4-ethylphenol solution after being treated with chlorine. The formation of PXEPEPs during the chlorination of 4-ethylphenol in the presence of bromide ions was also influenced by the reaction pH.

Monitoring of cholinesterase-inhibiting activity in water from the Tone canal, Japan, as a biomarker of ecotoxicity.

The cholinesterase (ChE)-inhibiting activity of water and the concentrations of representative inhibitors were monitored in the Tone canal, Japan, during April to December 2006. The ChE-inhibiting activity, measured by using horse serum as enzyme source, increased from late April to early June, and from September to October. Although the trends in the ChE-inhibiting activity of the samples were consistent with concentration changes of organophosphorus pesticides, ChE-inhibiting activity was not observed in samples replicated on the basis of the chemical concentrations detected. The water samples were treated with chlorine to enhance the ChE-inhibiting activity by conversion of thiophosphate pesticides to phosphate pesticides. The ChE-inhibiting activity increased in almost all the chlorine-treated samples, although organophosphorus pesticides were either not detected or detected in traces in the samples by gas chromatographic-mass spectrometric analysis. These results suggested that assay of ChE-inhibiting activity is important for evaluating the ecotoxicity of environmental water, because toxicological investigations based solely on inhibitor concentrations may underestimate the contamination. Furthermore, the combined method of oxidation by chlorination and the ChE assay is very effective for screening and monitoring of organophosphorus pesticides in environmental water.

Monitoring of triclosan in the surface water of the Tone Canal, Japan.

Triclosan, which is used widely as an antibacterial agent, was ubiquitously found in the water samples collected from the Tone Canal, Chiba, Japan. The concentrations of triclosan ranged from 11 to 31 ng/L. Higher concentrations of triclosan were observed in water samples collected from downstream, as compared to in the water samples collected upstream. The daily monitoring of a selected point from where the domestic wastewater inflow occurs showed that the triclosan levels in the water samples ranged from 55 to 134 ng/L; the levels of the loading amounts peaked from 10:00 to 12:00.