Article pubs.acs.org/crt
Levels of Tetrabromobisphenol A, Tribromobisphenol A, Dibromobisphenol A, Monobromobisphenol A, and Bisphenol A in Japanese Breast Milk Teruyuki Nakao,† Ema Akiyama,† Hideki Kakutani,† Ayami Mizuno,† Osamu Aozasa,‡ Yukiko Akai,§ and Souichi Ohta*,† †
Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan Faculty of Science and Engineering, Setsunan University, 17-8, Ikedanaka-machi, Neyagawa, Osaka 572-8508, Japan § Faculty of Nursing, Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan ‡
ABSTRACT: The levels of bisphenol A (BPA) and tetrabromobisphenol A (TeBBPA) were determined in breast milk samples from 19 Japanese mothers. BPA and TeBBPA levels were 36 ng/g lipid (range: 1.4−380 ng/g lipid) and 1.9 ng/g lipid (range: N.D. − 8.7 ng/g lipid), respectively. Tribromobisphenol A (TriBBPA) was similarly detected in all samples (mean: 5.5 ng/g lipid). We investigated the alteration of BPA-related compounds in breast milk over a period of three months. No trend could be observed for time-dependent changes in TeBBPA levels. High levels of TriBBPA were detected in breast milk samples with a high concentration of TeBBPA. We further examined concentration changes in BPA-related compounds in the breast milk of two donors over a period of 24 h. The results suggested that TriBBPA was a debrominated metabolite of TeBBPA, which had been ingested via food consumption and immediately transferred to the breast milk. On the basis of the present results, we estimated and compared the daily intake of BPA, TriBBPA, and TeBBPA from breast milk for infants. The estimated average intake of TriBBPA was 4 times higher than TeBBPA, at 48 and 12 ng/kg/day, respectively. The level of TeBBPA in breast milk was low, suggesting a low risk of causing adverse health effects. In conclusion, the concentration of both TriBBPA and TeBBPA must be determined in breast milk to accurately clarify the exposure of these compounds to infants.
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chain.11−13 Because introduction of TeBBPA into humans occurs via the ingestion of contaminated food, TeBBPA has also been detected in human blood,14−16adipose tissue,13,16 and breast milk.16−20 Furthermore, in vitro studies have shown that TeBBPA disrupts thyroid hormone,21,22 estrogen,23,24 and immunosuppressive25,26 homeostasis, and in vivo studies have demonstrated its effects on endocrine signaling6 and neurobehavioral7 activity. However, there are few reports on the in vivo metabolism of TeBBPA, and in particular, little is known of the average contamination levels in biological samples and toxicity of each debrominated TeBBPA metabolite, such as tribromobisphenol A (TriBBPA), dibromobisphenol A (DiBBPA), and monobromobisphenol A (MoBBPA).27 TriBBPA is a known ligand for peroxisome proliferator-activated receptor (PPARγ), which is involved in human lipid and glucose metabolic processes.28 Furthermore, because the fully debrominated TeBBPA product is bisphenol A (BPA), the contribution of secondary toxic effects due to BPA needs to be considered in addition to the health effects of TeBBPA during such studies. BPA is a component of polycarbonate plastics and epoxy resins, and is globally one of the most produced chemicals by volume. In Japan, BPA production exceeded 450,000 tons in
INTRODUCTION Flame retardants are used to confer flame resistance to polymer materials, such as plastic, rubber, wood, and textiles, and comprise both inorganic and organic compounds. Of these, brominated flame retardants (BFRs) are used in various industrial products because of their high economic and flameretardant efficiencies. However, several recent reports have demonstrated environmental pollution,1,2 homeostasis disruption,3−5 and toxic effects6,7 due to BFRs such as tetra- and pentabromodiphenyl ethers (TeBDEs and PeBDEs), hexabromobiphenyl (HxBB), and hexabromocyclododecane (HBCD). In 2009 and 2013, these compounds were designated as persistent organic pollutants (POPs) under the Stockholm Convention. Tetrabromobisphenol A (TeBBPA) is one of the most commonly used BFRs in the world as a relatively safe flame retardant without marked toxicity. In Japan, TeBBPA accounted for approximately 34% (18,000 tons) of the annual demand (54,000 tons) of BFRs in 2010.8 TeBBPA, in which the hydrogens of BPA are substituted with bromine, is a common reactive BFR for ABS and epoxy resins due to its low toxicity and cost. However, TeBBPA is likely to contaminate the environment during its production, use, and disposal. In fact, TeBBPA has been detected in river sediment9 and sludge from wastewater treatment plants.10 In addition, there have been reports of TeBBPA bioaccumulation in aquatic organisms and marine mammals throughout the food © 2015 American Chemical Society
Received: November 29, 2014 Published: February 26, 2015 722
DOI: 10.1021/tx500495j Chem. Res. Toxicol. 2015, 28, 722−728
Article
Chemical Research in Toxicology
evaporation, the procedure adapted from Sala et al.36 was used for deisopropylation of the brominated intermediate. 2,6-DiBBPA was then purified by recrystallization. Structures of the synthesized chemicals were confirmed by ESI-MS and NMR. By analysis of each spectrum, each fractionated compound was identified as MoBBPA, 2,2′-DiBBPA, 2,6-DiBBPA, or TriBBPA. The purity of all compounds was higher than 99%. Analytical Methods. Breast milk samples (5.0 mL) were spiked with 5.0 ng each of 13C12-TeBBPA and 2H16-BPA in a glass flask. Next, 5.0 mL of 25% 2-propanol in formic acid was added, and the samples were sonicated for 5 min in an ultrasonic bath. The samples were then diluted with 5.0 mL of 50% 2-propanol in water, and, after another 5 min of sonication, were purified by solid-phase extraction (SPE). The SPE cartridges were first washed with eluent solution (70% dichloromethane in methanol) and then preconditioned with 5.0 mL of methanol, 5.0 mL of dichloromethane, and 7.0 mL of 70% dichloromethane in methanol. Cartridges were continuously conditioned with 5.0 mL of methanol and 5.0 mL of water. Treated samples were loaded onto the cartridge, and the flasks were rinsed with 5 mL of 25% methanol in water to remove any residual milk, which was also loaded onto the cartridge. These cartridges were then washed with 4.0 mL of 0.05% 2-propanol in water. After complete drying of the cartridges, the adsorbed matter in the cartridge was eluted with 7.0 mL of 70% dichloromethane in methanol, and the eluate was gently evaporated to dryness at 45 °C under a stream of nitrogen. The residue was added to 0.5 mL of 1 M KOH/ethanol and shaken for 30 min. Next, samples were derivatized by ethylation of the hydroxyl group using 0.5 mL of diethyl sulfate for 30 min. After the addition of 4.0 mL of 1 M KOH/ethanol, samples were left at 70 °C for 1 h. To the samples, 3 mL of water was added and then extracted twice with 2 mL of n-hexane. The organic phase was then dried over anhydrous Na2SO4. Purification was performed using a florisil column (1.0 g) and eluted with 10 mL of 4% diethyl ether in n-hexane. The purified extract was then concentrated to 50 μL of in n-nonane. Extractions and purifications were conducted under light shielding conditions. The final solution was analyzed for bisphenol A related compounds by GC/MS. The GC/MS setup consisted of an Agilent 6890N gas chromatograph coupled with a JEOL JMS-Q1000 MkII mass spectrometer. The ion source was operated in the electron ionization (EI, 70 eV, 200 μA, 250 °C) mode. The sample extract was analyzed on a J&W DB-17 column (30 m × 0.25 mm i.d., 0.15 μm film thickness) with an oven temperature program as follows: initial temperature 140 °C, held for 1.5 min, raised to 310 °C at 10 °C/min, and held for 3 min at 310 °C. The column was connected directly to the ion source of the mass spectrometer (interface temperature 250 °C). Sample introduction was performed by splitless injection (injection temperature 250 °C, 1 min splitless time) of a 1.0 μL aliquot of the sample extract. Helium was used as the carrier gas at 1.0 mL/min. Method of QA/QC for Analytical Data. TeBBPA and BPA were identified by comparing the retention times and mass spectra with those of the commercial standards, and MoBBPA, DiBBPA, and TriBBPA were identified using the synthesized standards. Furthermore, SIM chromatograms were used to determine the peak area ratios for [M-CH3]+/[M-C3H8]+. The acceptance criteria were set from −30% to 30% of ratios observed with the commercial and synthesized standards. Concentrations were corrected utilizing the recovery efficiency of their respective internal standards: 2H16-BPA for BPA, MoBBPA, and DiBBPA, and 13C12-TeBBPA for TriBBPA and TeBBPA. Samples which had high internal standard recoveries in the range of 60−120% were used for data collection. The limits of detections (LODs) and the limits of quantitations (LOQs) were defined as 3 (S/N = 3) and 10 times (S/N = 10) the noise level, respectively. LOQs of BPA, MoBBPA, DiBBPA, TriBBPA, and TeBBPA in breast milk were 0.002, 0.010, 0.010, 0.010, and 0.018 ng/g lipid, respectively. For the estimation of daily intake of BPArelated compounds through breast milk, any values between the LOD and LOQ were assumed to be 1/2 LOQ.
2012.29 Since the 1990s, BPA has been known as an ecosystem and endocrine disruptor. It has been suggested that BPA may have endocrine-30 and nerve-disrupting31 effects in infants, even at very low doses. Thus, in 2007, the European Food Safety Authority (EFSA) set its tolerable daily intake (TDI) at
