Vol. 4 8, No. 6
June, 1999
Norihide Nakada, Tomohiko Isobe, Hajime Nishiyama, Keiji Okuda, Shinobu Tsutsumi, Junya Yamada, Hidetoshi Kumata and Hideshige Takada
*Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-5809
(Received 18 January 1999, accepted 31 March 1999)A comprehensive analytical method of endocrine disruptors (i.e., alkylphenols, phthalic acid esters, bisphenol A, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, DDTs) in environmental samples was developed. The method employs Soxhlet-extraction with dichloromethane and two-step-silica-gel and activated-charcoal column chromatography, which provides eleven fractions, followed by determination on capillary GC equipped with MSD or ECD. The present method was applied to Tokyo Bay sediments. Most of the target compounds were identified and quantified clearly, demonstrating the applicability to various environmental samples. Their concentrations are as follows: 4-t-octylphenol: 10.0 ng/g; 4-t-nonylphenol: 184 ng/g; benzyl n-butyl phthalate: 6.12 ng/g; dicyclohexyl phthalate: 2.56 ng/g; bisphenol A: 6.58 ng/g; benz[a]anthracene: 58.1 ng/g; benzo[a]pyrene: 93.7 ng/g; IUPAC#77 coplanar PCB: 0.82 ng/g; DDT: 0.42 ng/g; DDD: 1.00 ng/g; DDE: 1.37 ng/g. This method provides a powerful analytical tool to investigate a wide range of endocrine disruptors in samples of limited quantity.
Keywords: alkylphenols; phthalic acid esters; bisphenol A; polycyclic aromatic hydrocarbons; coplanar PCBs.
Shinkichi Yamada, Toshihiro Asano, Sen-ichi Aizawa and Motoshi Nakamura*
*Faculty of Engineering, Shizuoka University, 3-5-1, Johoku, Hamamatsu-shi, Shizuoka 432-8561
(Received 12 January 1999, Accepted 5 February 1999)The kinetics and thermodynamics of the complexation reactions of monophenyltin (MPT), dimethyltin (DMT), dibutyltin (DBT), diphenyltin (DPT), trimethyltin (TMT), tributyltin (TBT) and triphenyltin (TPT) with 3-hydroxyflavone have been studied, and the results were applied to the fluorometric determination of organotins. The observed rate constant for the reactions hardly depended on the differences in the number and kind of organic substituent at tin. However, because the stability of the complexes was incresed in the order MPT>DPT>>TPT for phenyltins and DPT>DBT>DMT for di-substituted organotins, these differences were applied to the fluorometric determination of organotins. In the determination of the respective mono- and di-substituted organotins, MPT up to 5 µM, DMT up to 3 µM, DBT up to 4 µM and DPT up to 3 µM could be determined with a detection limit of 10, 20, 20 and 30 nM, respectively, and the tolerance level of tri-substituted organotins was relatively high. Noticeably, the presence of more than a 100-fold amount (by molarity) of TMT was successfully tolerated in the determination of 0.5 µM of any mono- or di-substituted organotin.
Keywords: complexation of organotins with 3-hydroxyflavone; fluorometric determination of organotins.Tomomi Iwamura, Kiwao Kadokami, Daisuke Jin-ya, Yoshifumi Hanada and Manabu Suzuki*
*Kitakyushu City Institute of Environmental Sciences, 1-2-1, Shin-ike, Tobata-ku, Kitakyusyu-shi, Fukuoka 804-0082
(Received 25 January 1999, Accepted 5 April 1999)A method for the simple and rapid determination of organotins (monobutyl-, dibutyl-, tributyl-, monophenyl-, diphenyl- and triphenyltin), in water and sediment has been developed by isotope dilution GC/MS. After deuterated organotins as internal standards were added in a water sample, orgatotins were derivatized with NaBEt4 at pH 5; then, ethylated organotins were extracted with hexane and cleaned up by using a florisil minicolumn. In the case of a sediment sample, organotins were firstly extracted with 1 M HCl-methanol/ethyl acetate (1:1), and the extract was concentrated. Then, the concentrate was added to a buffer solution; the following procedures were performed in the same way as that for water. Instrument measurement was carried out by GC/MS-SIM. The results concerning the overall recovery tests at ng/l levels for water and µg/kg levels for sediments showed that the mean recoveries were 104 and 98.1%, respectively. Their mean relative standard deviations were 3.7 and 9.8%, respectively. The detection limits of the method for target chemicals ranged from 0.16 to 3.5 ng/l for water, which were about one order lower than that by the conventional methods, and from 0.48 to 11 µg/kg for sediment. The method was applied to real samples; di- and trisubstituted organotins were detected in seawater and sediment, but only dibutyltin was found in river water.
Keywords: organotin; tributyltin; isotope dilution GC/MS; ethylation.Masahiko Takino*, Shigeki Daishima and Kenji Yamaguchi**
*Kansai branch office, Yokogawa Analytical Systems Inc., 3-3-11, Niitaka, Yodogawa-ku, Osaka 532-0033
**Yokogawa Analytical Systems Inc., 2-11-13, Nakacho, Musashino-shi, Tokyo 180-8543
An analytical method for the determination of bisphenol A and alkylphenols with liquid chromatography/mass spectrometry (LC/MS) has been developed. Under reversed-phase liquid-chromatographic conditions, bisphenol A and seven alkylphenols have been analyzed using atmospheric-pressure chemical ionization(APCI) and electrospray ionization (ESI) methods. The effects of post-column modification of the liquid chromatographic mobile phase, the fragmentor voltage with ESI methods and the temperature of the vaporizer with APCI methods on the sensitivity for the detection of bisphenol A and alkylphenols were examined. The sensitivities of the APCI methods were higher than those of the ESI methods. Large volumes of river water samples were directly injected into the precolumn, and then bisphenol A and alkylphenols were separated by an analytical column with a column-switching technique. The limit of detection (LOD) of these phenols ranged from 5 ppt to 20 ppt. The overall recoveries of phenols were from 93.0 to 112.4% {RSD (n=5) from 2.1 to 5.3%} for river water.
Keywords: LC/ESI-MS; LC/APCI-MS; column switching; bisphenol A; alkylphenols; large volume injection; endcrine disruptor.
Jun Yonekubo, Syunya Sasaki, Makiko Ichiki, Michiko Kanai and Hideki Sasaki*
*Nihon Waters K.K., No.5 Koike Bldg., 1-3-12, Kita-Shinagawa, Shinagawa-ku, Tokyo 140-0001
(Received 18 January 1999, Accepted 6 April 1999)Concentration by solid phase extraction (SPE) and LC/MS direct analysis methods has been established for bisphenol A (BSA) and nonylphenol (NP) as endocrine disruptors (ED) samples. In 1) concentration by SPE, used for reverse-phase cartridge, on average for 5-times analysis of the % recovery with % the RSD is higher than 80% and less than 17%, respectively. 2) Of the three ionization methods (EI, APCI and ESI) for LC/MS detection, ESI is considered because of better ionization concerning sensitivity. The limit of detection (LOD) according to a method based on the standard deviation (SD) of the measurement is 0.07 ng ml-1 (BSA) and 0.73 ng ml-1 (NP), respectively, from this LC/MS method with ESI detection. Considering the result from a blank test, the detection limit of this method was chosen to be 0.1 ng ml-1 (BSA) and 7.0 ng ml-1 for a real-sample analysis. This method also provides good reproducibility (%RSD<3.2) and linearity (r2>0.99) from each LOD mentioned above to the 1000 ng ml-1 range.
Keywords: bisphenol A; nonylphenol; endocrine disruptors; LC/MS.
Masakazu Horie, Terumitu Yoshida, Rie Ishii, Susumu Kobayashi* and Hiroyuki Nakazawa**
*Saitama Prefectural Institute of Public Health, 639-1, Kamiokubo, Urawa, Saitama 338-0824
**Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-0063
A simple and rapid method using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) has been developed for the determination of bisphenol A in canned drinks. LC separation was carried out on a Zorbax XDB-C18 column (150×2.1 mm) using 0.01% acetic acid-acetonitrile (60:40) as the mobile phase at a flow rate of 0.2 ml/min. The negative ionization produced a typical (M-H)- molecular ion of bisphenol A (m/z 227). The calibration graph for bisphenol A was rectilinear from 10 to 1000 pg with a detection limit by selected ion monitoring (SIM) of about 5 pg. Samples were cleaned up on a Isolute Multimode cartridge (500 mg). The recovery of bisphenol A from beverages fortified at levels 5 and 50 ng/ml was 85.4~96.3%, with high precision. Bisphenol A concentrations of canned drinks were N.D. ~212.1 ng/ml. The limit of quantification of bisphenol A in canned drinks was 0.5 ng/ml, except for coffee drinks.
Keywords: bisphenol A; LC/MS; epoxy resin; migration; canned drinks.
Yuji Takao, Ho Chul Lee* and Koji Arizono**
*Faculty of Environmental Studies, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521
**Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502
A combination solid-phase microextraction (SPME) and on-column silylation was applied for a rapid and highly sensitive determination of environmental endocrine disruptors in water samples. In this study, bisphenol A {BPA, 2,2-bis(4-hydroxyphenyl) propane} was selected. BPA was extracted by absorption over the surface of SPME fiber which was directly exposed to the sample. The fiber was introduced into the injection port of GC/MS, where BPA was thermally desorbed followed by injecting bis(trimethylsilyl)trifluoroacetamide (BSTFA) as a derivative with a syringe in the gas phase under He flow. In a comparison without silylation, the peak area of on-column silylated BPA was increased to approximately 20 times. The detection limit was about 1 ppb, lower than expected for originating in a epoxy-resin adhesive from a SPME fiber.
Keywords: SPME; bisphenol A; on-column silylation; endocrine disruptors.
Junko Oda*
*Okayama Prefectural Institute for Environmental Science and Public Health, 739-1, Uchio, Okayama 701-0212
(Received 13 January 1999, Accepted 26 February 1999)An extraction and separation method using supercritical fluid extraction (SFE) has been investigated for the pretreatment of PAHs as well as benzo[a]pyrene (BaP) in airborne particulates, which is estimated to be one of the so-called endocrine disruptors. The instrumental parameters of SFE were considered for the quantitative SFE of 30 PAHs spiked in airborne particulates. The extraction efficiency of SFE with the optimized operating conditions was compared to that of the classical method using sonication extraction to be applied to real sample analysis. The extraction efficiencies for 30 representative PAHs were influenced by such factors as the extraction temperature, density of the extraction fluid, volume of the extraction fluid, the extraction time, elute solvent from an analyte trap and the extraction size of samples. It was shown that the combination of three different conditions of SFE and the use of 5% methanol-modified supercritical carbon dioxide lead to more efficient recoveries of PAHs from airborne particulates. The SFE recoveries for higher molecular-weight PAHs (>178 amu) than phenanthrene ranged from 70.3 to 103%, while those with sonication extraction ranged from 67.8 to 99.5%. The SFE results obtained from 31 samples of native particulates in air were also comparable to the sonication values in terms of the BaP concentrations and its correlation coefficient. The average of the SFE recoveries for the BaP concentrations was 120%, and the correlation coefficient was 1.00 versus to their values certified, respectively, with sonication procedure. The results of this work indicate that the proposed method with SFE is useful for the rapid and effective pretreatment of airborne particulates. The SFE condition optimized from the experimental results was as follows. Step-1: extraction fluid, pure CO2; extraction temperature, 60°C; density, 0.65 g/ml; static/dynamic extraction time, 2/10 min; extract trap temperature, -5°C; rinse trap temperature, 30°C; trap rinse, 1.6 ml of acetone. Step-2: extraction fluid, 5% methanol-modified CO2; extraction temperature, 80°C; density, 0.65 g/ml; static/dynamic extraction time, 2/20 min; extract trap temperature, 80°C. Step-3: extraction fluid, pure CO2; extraction temperature, 80°C; density, 0.35 g/ml; static/dynamic extraction time, 2/5 min; extract trap temperature, 80°C; rinse trap temperature, 40°C; trap rinse, 1.6 ml of acetone.
Keywords: polycyclic aromatic hydrocarbon; supercritical fluid extraction; airborne particulate; extraction and separation; extraction efficiency.
Tsuyoshi Ibaraki, Kuniaki Kawata, Noriko Utagawa, Masaaki Sakai, Ikuei Kifune* and Masatoshi Morita**
*Niigata Prefectural Research Laboratory for Health and Environment, 314-1, Sowa, Niigata 950-2144
**The National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba, Ibaraki 305-0053
A GC/MS method has been developed for endocrine disrupting aromatic compounds in water and sediment. The target compounds were benzophenone, 4-nitrotoluene, styrene dimmers (i.e. 2,4-diphenyl-1-butene, cis-1,2-diphenylcyclobutane, trans-1,2-diphenylcyclobutane and 1,3-diphenylpropane) and styrene trimmers (i.e. 1a-phenyl-4a-(1'-phenylethyl)tetralin, 1a-phenyl-4e-(1'-phenylethyl)tetralin, 1e-phenyl-4a-(1'-phenylethyl)tetralin, 1e-phenyl-4e-(1'-phenylethyl)tetralin and 2,4,6-triphenyl-1-hexene) together with 2-nitrotoluene and 3-nitrotoluene. A water sample of 350 ml or a sediment sample of 20 g with 350 ml of purified water in a 500 ml flask was added to 0.2 µg of surrogate compounds (benzophenone-d10, 1,2-diphenylethane-d14 and nitrobenzene-d5) and 5 ml of hexane. The flask was connected to an improved essential oil distillator and the mixture was distilled for 90 min. The hexane extract was condensed to 1 ml and fractionated by column chromatography using a silica-gel cartridge; the target compounds and the surrogate compounds were eluted from the column using 4 ml of acetone-hexane (20:80, v/v) after a fraction with 4 ml of hexane. The eluate was condensed to 1 ml and then added with internal standards for GC/MS determination. The recoveries of the target compounds were 88 to 111% for water samples and 80 to 113 % for sediment samples. The minimum detectable concentrations were 0.005 to 0.01 mg l-1 in water and 0.3 to 0.5 mg g-1 in sediment.
Keywords: endocrine disruptors; styrene dimmers, styrene trimmers, benzophenone and nitrotoluenes; essential oil distillator; GC/MS; water and sediment.
Ryoichi Kizu, Shoko Kato, Osamu Usui and Kazuichi Hayakawa*
*Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1, Takara-machi, Kanazawa 920-0934
(Received 5 February 1999, Accepted 3 April 1999)The authors have proposed a new in vitro assay method for estrogenicity. The cellular progesterone receptor (PgR) level in the human breast cancer cell line MCF-7 was measured because PgR has been known to be upregulated by 17β-estradiol (E2). The cellular PgR level was measured by a western blotting technique. While an E2 treatment increased the PgR level, tamoxyfen, a typical E2 antagonist decreased the PgR level elevated by the E2 treatment. These results revealed that the cellular PgR level is a good indication to evaluate the estrogenicity. Next, the estrogenic activities of Nakhodka heavy oil and a commercial C-heavy oil were examined. Heavy oil crude extracts prepared with ethanol were subjected to the assay. While the extracts increased the PgR level under the E2-free condition, the extracts decreased the PgR level elevated by the E2 treatment. These results indicate that the heavy oil contains compounds which show weak estrogenic activity and act as E2 antagonists. Dead cells were observed after the oil extract treatment, and their number increased with an increase of oil extract concentration in the cell-culture medium. Environmental samples such as oil-extract, river water, and air particulate extract comprise a wide variety of compounds, and are thought to have a cell-killing effect. A cellular PgR expression method is considered to be more appropriate for assessing the estrogenic activities of environmental samples, compared with the existing assay methods, such as the cell-proliferation method (E-Screen assay) and the reporter-gene methods.
Keywords: estrogenic activity; PgR expression assay; MCF-7; heavy oil.
Koichi Moriguchi, Takeshi Kamata, Takashi Harimoto, Yonezo Matsumoto and Hisako Nakashima*
*Sumika Chemical Analysis Service, Ltd., 3-1-135, Kasugade-naka, Konohana-ku, Osaka 554-0022
(Received 18 January 1999, Accepted 14 April 1999)Conformational analysis of stereoisomers of 1-phenyl-4-(1'-phenylethyl)-tetralin (1,4-PPET) and 1,3,5-triphenylcyclohexane (1,3,5-TPCH) were investigated using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, among several styrene trimers that remained in polystyrene products for food containers. Four stereoisomers of 1,4-PPET were isolated from a mixture of styrene oligomers formed during the thermal polymerization of styrene by employing a recycling method of preparative high-performance liquid chromatography (preparative HPLC). As for 1,3,5-TPCH, two kinds of stereoisomers were obtained by means of two kinds of synthetic methods with different steroselectivities. We confirmed that the four stereoisomers of 1,4-PPET are (1-eq, 4-eq), (1-ax, 4-eq), (1-ax, 4-ax) and (1-eq, 4-ax) conformers (ax: axial, eq: equtorial), in order of their gas-chromatographic (GC) elutions, respectively, based on the relationships of the vicinal coupling constants of tetralin protons. This result was also supported by the 1H and 13C NMR chemical shifts (δH, δC) of 1', 2'(CH3) protons and 2'(CH3) carbon. As for 1,3,5-TPCH, we obtained (trans, cis) and (cis, cis) isomers as two kinds of stereoisomers in order of their GC elutions using two kinds of synthetic methods with different stereoselectivities, and concluded that they were (1-ax, 3-eq, 5-eq) and (1-eq, 3-eq, 5-eq) conformers, respectively, based on the relationship among the typical coupling constants of cyclohexane protons and the corresponding 1H and 13C NMR chemical shifts of the cyclohexane rings. The characterization of the conformation of styrene trimers seems to be significant for future investigations of endocrine-disrupting mechanisms.
Keywords: conformational analysis; stereoisomers; styrene trimers; enodcrine disrupting chemicals; 1H and 13C-NMR.
Takashi Korenaga, Toshiyuki Kajihara and Yoko Tsukiyama*
*Faculty of Integrated Arts and Sciences, University of Tokushima, 1-1, Minami-jousanjima-cho Tokushima 770-8502
(Received 18 January 1999, Accepted 5 April 1999)Polycyclic aromatic hydrocarbons (PAHs) are hazardous environmental contaminants. Benzo[a]pyrene, a typical example of PAHs, is suspected to be an endocrine disruptor. Reported here are the results of the determination of PAHs in soil from several locations in Tokushima and plant samples, such as rice straw, polished rice and unpolished rice. Samples were extracted for 16 h with 120 ml of dichloromethane using a soxhlet thimble. The extract portion was then cleaned by passing it through activated silica gel with aceton/hexane (5/95, v/v). The PAHs were analyzed by HPLC and GC/MS. This method was successfully applied to the determination of PAHs in real environmental samples. The determination limits for PAHs were in the range of 0.1~0.5 µg/kg for soils and plants. Replicate analyses gave errors ranging between ±10~30%for soils and plants. According to this determination, some PAHs, including benzo[a]pyrene, can be detected in soil samples and plants. For instance, the total concentration of detected PAHs was 250 µg/kg in soil samples of University of Tokushima.
Keywords: polycyclic aromatic hydrocarbons; benzo[a]pyrene; endocrine disruptor; soils; plants.
Tsuyoshi Ibaraki, Chikako Oguma, Akiko Tanabe, Kuniaki Kawata, Masaaki Sakai and Ikuei Kifune*
*Niigata Prefectural Research Laboratory for Health and Environment, 314-1, Sowa, Niigata 950-2144
(Received 18 January 1999, Accepted 2 March 1999)A solid-phase extraction-gas chromatography/mass spectrometric method was applied to determine endocrine disrupting pesticides in river-water samples. The target compounds were seven pesticides (cypermethrin, ethylparathion, fenvalerate, metribuzin, nitrofen, permethrin and vinclozolin). A water sample of 500 ml was passed through a Sep-Pak PS-2 cartridge. The pesticides were eluted with 4 ml of acetone and 4 ml of hexane, and then 4ml of ethyl acetate. The eluate was concentrated to 1ml and determined by GC/MS. The overall recoveries from ground water were 80% to 107%, except for pyrethroids (cypermethrin, fenvalerate and permethrin). A membrane-extraction disk (SDB-XC) was also applied to determine these pesticides and another six pesticides (alachlor, atrazine, carbaryl, malathion, simazine and trifluralin). Pesticides in a water sample of 500 ml were extracted using a membrane disk, and then eluted by 20 ml of ethyl acetate. After dehydration, the eluate was concentrated and determined by GC/MS. Pyrethroids can be extracted relatively well by this method. The overall recoveries of all the target compounds from ground water and river water were 49% to 99% and 56% to 112%, respectively. The limits of quantification in water were 0.1 µg l-1 to 0.2 µg l-1.
Keywords: endocrine disruptor; pesticide; membrane extraction disk; solid phase extraction; GC/MS.
Shunya Sasaki, Jun Yonekubo*, Michael S. Young, Dorothy J. Phillips and Uwe D. Neue**
*Nihon Waters K. K., No. 5 Koike Bldg., 1-3-12, Kita-Shinagawa, Shinagawa-ku, Tokyo 140-0001
**Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, U.S.A.
A simple and rugged solid-phase extraction (SPE) method for the determination of acidic herbicides, including phenoxyacetic acids, which are considered to be endocrine disruptors (EDs), was developed. Spiked tap-water and well-water samples were alkaline hydrolyzed with NaOH, followed by a simple SPE procedure using a hydrophilic-lipophilic balanced (HLB) copolymer. The hydrophilic character of a HLB copolymer allows packing a sufficient amount of hydrophilic property to prevent the wettability problems encountered with ODS and polystyrene packings. High and reproducible recoveries were obtained for both tap-water and well-water samples at a 0.4 µg/l spike level. For five replicate analyses using 75 ml of spiked water, the method detection limit (MDL) of UV-HPLC analysis was 0.04~0.18 µg/l.
Keywords: endocrine disruptors; solid-phase extraction; hydrophilic-lipophilic balanced copolymer; acidic herbicides; phenoxyacetic acid.
Takao Katase and Yunsoku Kim
College of Bioresource Sciences, Nihon University, 1866, Kameino, Fujisawa-shi, Kanagawa 252-0813
(Received 18 January 1999, Accepted 14 April 1999)Bis(2-ethylhexyl)adipate (DEHA) has been found to reduce the binding of tritiated 17β-estradiol to its receptor by screening an inhibitory effect on that to the rainbow-trout estrogen receptor (Jobling et al. 1995). Adipic acid esters (AAEs) are utilizable to a plasticized polyvinyl chloride and are also migratory from plastics. We studied the identification of AAEs potentially migrated from plastic films for food wrapping, and estimated their migration. Approximately fifty pieces of used wrapping films for foods were collected for about one month. DEHA was not found in those samples but di-n-hexyl, -n-heptyl, di-n-octyladipate, -n-nonyl and -n-decyladipates were identified by gas-liquid chromatography and mass spectrometry. Additionally, an unidentified adipate (AAEX) was found in 12 films. Approximately fourteen mg of total amounts of AAEs was migrated into n-heptane solvent from one side of the surface of 9 cm×9 cm films, equivalent to ca. 0.2 g. The daily intake of AAE from food-wrapping film was estimated as 0.04 mg kg-1 day-1. A portion of thirteen percent of the film weight was migratory to natural and human environments. Approximately one tenth of a million tons of the films is annualy produced by factories in Japan. Further studies on endocrine disruption by plastic materials in our daily lives will be needed.
Keywords: adipates; plastic-films; endocrine disruptor; mass spectrometry.
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