Article pubs.acs.org/est
Brominated Flame Retardants in Matched Serum Samples from Swedish First-Time Mothers and Their Toddlers Leena M. O. Sahlström,*,† Ulla Sellström,† Cynthia A. de Wit,† Sanna Lignell,‡ and Per Ola Darnerud‡ †
Department of Applied Environmental Science (ITM), Stockholm University, SE-106 91 Stockholm, Sweden Science Department, National Food Agency, Box 622, SE-751 26 Uppsala, Sweden
‡
S Supporting Information *
ABSTRACT: Tri-decabrominated diphenyl ethers and 21 other flame retardants were determined in matched serum samples from 24 Swedish mothers (Uppsala county) and their toddlers (11−15 months of age). The median concentrations of individual polybrominated diphenyl ethers (PBDEs) ranged from 0.036 to 0.95 ng/g lipid in mothers and from 0.057 to 1.5 ng/g lipid in toddlers. BDE-209 was detected in all but one sample. BDE-153 was the predominant congener in the mothers while in toddlers, BDE-209 was found in the highest concentrations. The levels of BDE-47, -100, -207, -208, and -209 in toddlers were significantly higher (p < 0.05) than those in their mothers. Dechlorane Plus (anti- and syn-) and α- and β-tetrabromoethylcyclohexane were detected in a few (2−4) serum samples from both mothers and toddlers. This study also reports concentrations of α-HBCD and eight emerging brominated flame retardants (EBFRs) in the standard reference material serum (SRM 1958, NIST). Lack of correlations between the matched serum samples indicate different exposure routes for octa-decaBDEs in mothers versus toddlers. Congener-to-congener correlations within the mother or toddler cohorts suggest diet as an important exposure pathway for tetra-nonaBDEs for mothers, breastfeeding as a predominant exposure pathway for tetra-hexaBDEs, and dust for octa-decaBDEs for toddlers.
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INTRODUCTION Brominated flame retardant (BFR) chemicals are used in high volumes globally to produce consumer products with reduced flammability. The use of these often bioaccumulative and persistent chemicals has been ongoing for decades, and has resulted in exposure of the environment, wildlife, and humans.1 One group of BFRs, the polybrominated diphenyl ethers (PBDEs), has been banned (penta- and octaBDE technical mixtures) or phased out (decaBDE mixture) in several countries.2,3 However, they are still present in older consumer products and production of decaBDE may still occur in countries lacking restrictions for their use.4 Hexabromocyclododecane (HBCD), a BFR used in textiles and building insulation is currently being phased-out.5 BFRs have been detected in indoor air and dust,6 dietary items,7 and also in humans.8 A number of emerging BFRs (EBFRs) are replacing the banned or restricted PBDEs and HBCD.9 These include 2ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB), bis(2-ethylhexyl) tetrabromophthalate (BEH-TEBP), bis(2,4,6-tribromophenoxy) ethane (BTBPE), decabromodiphenyl ethane (DBDPE), Dechlorane Plus (DDC-CO), tetrabromoethylcyclohexane (DBE-DBCH), 2,3,5,6-tetrabromo-p-xylene (TBX), 2-bromoallyl 2,4,6-tribromophenyl ether (BATE), 1,2,3,4,5pentabromobenzene (PBBz), tetrabromo-o-chlorotoluene (TBCT), pentabromotoluene (PBT), pentabromoethylbenzene (PBEB), 2,3-dibromopropyl-2,4,6-tribromophenyl ether (TBP© 2014 American Chemical Society
DBPE), hexabromobenzene (HBBz), hexachlorocyclopentenyldibromocyclooctane (DBHCTD), and octabromotrimethylphenylindane (OBTMPI). Biological effects of PBDEs and HBCD are mainly observed on circulating thyroid hormone levels and neurobehavioral development.10,11 Effects on neurobehavioral development in children have been associated with exposures to lower brominated BDEs.12−14 Effects of EBFRs on humans have not been studied. However, a recent study in rats indicates that exposure to Firemaster 550 (FM 550), a FR mixture containing EH-TBB and BEH-TEBP, causes endocrine disrupting effects.15 The very limited toxicological information existing on EBFRs has been summarized by The European Food Safety Authority (EFSA).16 A few studies have reported higher tetra-hexaBDE levels in serum from children compared to adults.17−19 In most of these studies, the children were over 3 years of age (and no longer breast-feeding) or the samples were pools representing many individuals with a wide age range. In one study, higher tetrahexaBDE and BDE-209 levels were found in two children (5 years and 18 months of age) compared to their parents.20 Received: Revised: Accepted: Published: 7584
March 6, 2014 June 9, 2014 June 13, 2014 June 13, 2014 dx.doi.org/10.1021/es501139d | Environ. Sci. Technol. 2014, 48, 7584−7592
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Article
The surrogate/reference standards used were purchased from Wellington Laboratories Inc. (Guelph, Canada), Cambridge Isotope Laboratories Inc. (Andover, USA), and AccuStandard Inc. (New Haven, USA); for details see Tables S-1 and S-2, Supporting Information (SI). Sampling. Study participants were recruited among 60 firsttime mothers who delivered one healthy child in Uppsala University Hospital in 2009−2010 and were included in the POPUP study (Persistent Organic Pollutants in Uppsala Primiparas)30 soon after delivery. When their children were about 11 months old, they were recontacted and asked to participate in a follow-up study. Thirty-five women agreed, and blood samples from 24 of the mothers and their toddlers (11− 15 months of age) (matched mother-child samples with sufficient blood volumes for analysis) were collected on the same visit in the participants’ homes by registered nurses. The sampling was organized by the Swedish National Food Agency in Uppsala, Sweden. Blood samples were collected in Vacuette tubes containing clot activator and centrifuged at 2800 rpm for 15 min to obtain serum and stored in −25 C until sample preparation and analysis. The mothers filled in a questionnaire about nursing, introduction of food/dietary habits of the child, daycare, etc. Information about the age of the study participants, length of breastfeeding, and the length of time toddlers were eating food and the education level of the mothers are shown in Table S-3, Supporting Information. The mothers were born in the Nordic countries. This study was approved by the Regional Ethics Committee in Uppsala, Sweden (Permit 2004:M-177). Extraction and Cleanup. Sample extraction was performed according to Hovander et al.31 with some modifications. In short, 0.5−5 g serum was weighed into a glass test tube, 13Clabeled surrogate standards (BDE-155, 127 pg; BDE-197, 120 pg; BDE-209, 124 pg; BTBPE, 238 pg; anti-DDC−CO, 129 pg; syn-DDC−CO, 127 pg; α-HBCD, 111 pg; β-HBCD, 111 pg; γHBCD, 110 pg) were added, and the sample was diluted 1:1 with 2-propanol, mixed, and then further diluted 2:1 with MilliQ water. The samples were extracted twice with 1:1 cHx:MTBE. The extracts were combined and washed with 1% KCl water solution. The solvent was evaporated and the lipid content was determined gravimetrically. Then the lipids were dissolved in n-Hx, and the sample was fractionated into three fractions (see Supporting Information, Table S-1 for specific analytes in each fraction) on a SiO2 SPE column according to Sahlström et al.32 Fractions I and III were further cleaned-up by treatment with concentrated sulfuric acid, while fraction II, containing acid sensitive analytes, was further cleaned-up on an aminopropyl column. Prior to the instrumental analyses the fractions were transferred to appropriate vials containing a recovery standard (13C−CB-180, 525 pg for fractions I and II, and d18-β-HBCD, 50 pg for fraction III), and the final volumes were adjusted to 25 μL. Instrumental Analyses. Fractions I and II were injected into a gas chromatograph (GC) (Trace GC Ultra) coupled to a mass spectrometer (MS) (DSQ II MS, both Thermo Scientific, Waltham, USA). The GC was equipped with a programmable temperature vaporizer injector and a HT 5 fused silica column (Thermo Scientific, Waltham USA, 0.25 mm inner diameter, 0.1 μm film thickness). Two column lengths were used, a shorter (15 m) for the analysis of octa-decaBDEs, OBTMPI, DBDPE, EH-TBB, BEH-TEBP, BTBPE, anti- and syn-DDCCO, and a longer one (30 m) for tri-heptaBDEs and other BFRs (α- and β-DBE-DBCH, TBX, BATE, PBBz, TBCT, PBT,
There is thus a lack of data on PBDE concentrations, in particular the higher brominated congeners, in children below the age of 3 in comparison with adults. The major routes of exposure to lower brominated BDEs and HBCD are generally considered to be from dietary intake and dust ingestion.8,21 Dust ingestion is estimated to be more important as an exposure pathway for congeners in the pentaBDE mixture in North America,22 whereas diet may be more important for European exposure to these congeners.8,23 For infants, intake via breast feeding is considered to be a major exposure route for the lower brominated BDEs,24 but dust ingestion may also contribute.25 HBCD concentrations in serum of adults have been shown to correlate with dust ingestion, but not with diet26 There is little information on the importance of dietary versus dust ingestion as exposure routes in children or adults for the higher brominated BDEs, and virtually no such information is available for EBFRs. The findings of higher PBDE concentrations in children compared to adults have raised questions about possible differences in exposure between children and adults,17−19 for example, that dust may be a more important pathway than diet for children’s exposure to BDE-209.20 In this study, matched blood serum samples from 24 primapara mothers and their toddlers (11−15 months of age) were analyzed for tri-decaBDEs, isomer-specific HBCDs and EBFRs. Our objectives were to compare concentrations and study associations between individual BDE congeners, including BDE-209, and other BFRs in mothers and their toddlers and relate these to possible exposure pathways for these compounds. At 11−15 months of age, possible exposure routes for these children were breastfeeding, food and dust primarily in their home environment as they had not yet started daycare. The frequency of mouthing behavior is the highest in children ≤24 months of age,27 and thus the children in this study were expected to have high exposure to dust ingestion in their homes. During maternity leave, possible exposure routes for the mothers were diet and indoor dust from their home and other environments. This study also presents the first results of octa-decaBDE and EBFR concentrations in children of this young age group, filling an important data gap.28
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MATERIALS AND METHODS Chemicals and Materials. Cyclo(c)-hexane (SupraSolv), 2-propanol, and n-hexane (both LiChrosolv) were obtained from Merck (Darmstadt, Germany); methyl-tert-butyl ether (MTBE, HPLC-grade) from Rathburn Chemicals Ltd. (Walkerburn, Scotland, UK), iso-octane (HPLC-grade) from LabScan (Gliwice, Poland), methanol (B&J Brand) from Honeywell (Seelze, Germany); and sulfuric acid (AnalaR, BDH) from VWR International (Pennsylvania, USA). Water was obtained from a Milli-Q water purification unit (Millipore AB, Solna, Sweden). Other materials used were silica gel 60 (0.063−0.200 mm) from Merck; potassium chloride and anhydrous Na2SO4 (both reagent grade) from Scharlau (Barcelona, Spain); ISOLUTE aminopropyl columns (0.5 g), empty reservoirs and frits from Biotage (Uppsala, Sweden). The standard reference material (SRM 1958, organic contaminants in fortified human serum)29 from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) was used as quality control (QC) sample in the analyses. 7585
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determine possible presence of these EBFRs in the standard reference material. The quantification of the above-mentioned “other BFRs” was performed for screening purposes and thus based on one-point calibration curves and the results are to be considered as semiquantitative. The method limits of quantification (mLOQ) were derived from low-level serum samples as the minimum amount of analyte present in the sample giving a signal-to-noise (S/N) ratio of 10, (Table S-6 in the Supporting Information). mLOQ was calculated individually for each sample with respect to the different sample intakes. The method limits of detection (mLODs) were estimated as one-third of the mLOQ. For analytes present in the laboratory blanks, the mLODs and mLOQs were set to the mean blank values plus 3 and 5 times the standard deviation of the blanks, respectively. For the nonaBDEs, mLODs and mLOQs were defined individually for each sample when degradation of BDE-209 occurred. The nonaBDE levels were considered quantifiable when the original peak area (after subtracting the area initiated from degradation of BDE-209 during cleanup/analysis) exceeded 20% of the total peak area measured. Method Recoveries and SRM 1958. The average method recoveries of 13C-labeled surrogate standards and spiked EBFRs ranged from 46−101% and 57−101%, respectively (Tables S-7 and S-8, Supporting Information). The PBDE concentrations in the SRM serum obtained in this study agreed well with the certified values33 (Table S-9) with one exception. The BDE206 concentration in this study was somewhat lower than the certified value and could possibly be due to our correction for the addition of BDE-206 due to degradation of BDE-209 during sample preparation. α-HBCD and eight EBFRs (α-DBEDBCH, TBX, PBBz, TBCT, PBEB, HBBz, BTBPE, DBDPE) were also detected in the SRM 1958 serum (Table S-9, Supporting Information). There are no certified values available for these compounds in SRM 1958. However, according to the NIST Certificate of Analysis,29 HBCD, BTBPE, HBBz, and DBDPE (500 pg/mL each) were additionally spiked into the material. The α-HBCD concentration in the SRM serum measured in this study was in agreement with a previously reported value.34 Statistics. The statistical analyses were performed using JMP 10, (SAS Institute Inc., Cary, NC, USA). The data were transformed to fit a normal distribution by taking the natural logarithm of the lipid weight-based concentrations in both mothers and toddlers. Only analytes with detection frequencies of 50% or higher were analyzed statistically. Data below mLOD and mLOQ were set as the mLOD or mLOQ divided by the square-root of 2, respectively. Student’s paired comparisons ttest was used to compare the concentrations of individual analytes in matched mother versus toddler serum samples. Student’s t-test was used to compare BDE concentrations between female and male toddlers. Pearson’s correlation analyses were computed to study associations of specific BDE concentrations between matched serum samples from mothers and their toddlers. Correlation analyses were also performed between the different BDE congeners in mothers’ and in toddlers’ serum, separately. Correlations were also studied between BDE concentrations and age, and length of breastfeeding and toddlers’ food eating period, respectively. The significance was set at α = 0.05 in all the statistical analyses.
PBEB, TBP-DBPE, HBBz, DBHCTD). The GC methods are described in detail in the Supporting Information (Table S-4). Helium (purity 4.6, Aga, Lidingö, Sweden) was used as the carrier gas (1.5 mL/min). Electron capture negative ionization (ECNI) with ammonia (purity 5.0, Aga) as moderating gas (5.0 mL/min) was used, and the MS was operated in selected ion monitoring mode recording the bromide ions (m/z of −79 and −81) and heavier mass fragments for the different analytes (Supporting Information, Table S-4). The quantification was performed with XCalibur 2.0.7 (Thermo Finnigan, San Jose, CA, USA). Fraction III was injected into an ultra performance LC (ACQUITY UPLC) coupled to a tandem-quadrupole MS (Xevo TQ-S) to determine the three major HBCD stereoisomers (α-, β-, and γ-HBCD). The separation was performed on a UPLC column (ACQUITY UPLC HSS C18, 1.8 μm; 2.1 mm × 100 mm), with a precolumn (ACQUITY UPLC HSS C18; 1.8 μm VanGuardTM; 2.1 × 5 mm) coupled. The UPLC/MS instrument and columns used were from Waters (Milford, USA). A mobile phase linear gradient from 78:22 to 93:7 methanol/water was applied. Electrospray ionization (ESI) in negative mode was applied for the ionization of the analytes, and the MS was run in multiple reaction monitoring mode, measuring the quasi-molecular ions [M-H]− as parent ions and bromide ions as daughter ions. The instrumental parameters and method are described in the Supporting Information, Table S-5. The quantification was performed with MassLynx V4.1 (Waters, Milford, USA). Quality Control. All glassware was heated to 470 °C (4 h) and rinsed with acetone before use. UV-filters were mounted on windows and light fixtures in the laboratory; amber glassware was used when possible, and the samples were covered with aluminum foil throughout the sample preparation to prevent photodegradation and/or possible contamination from the laboratory facilities. Ten laboratory (solvent) blanks (BL) and 10 quality control (QC) samples (SRM 1958) were processed together with the serum samples (2 BL and 2 QC per batch of 10 samples). The data were blank-corrected by subtracting the mean amount detected in the blanks. Only four compounds, BDE-197, -209 and α- and β-DBE-DBCH were detected in the laboratory blanks (detection frequencies 80, 100, 80, and 90%, respectively). Three empty Vacuette tubes were tested by rinsing with n-Hx. No BFRs were detected in these “field blanks”. The formation of octa- and nonaBDEs from degradation of BDE-209 during sample extraction/cleanup, was corrected for by measuring the 13C-octa- and −nonaBDEs formed from the surrogate 13C-BDE-209 standard in each individual sample assuming that 13C- and native BDE-209 are degraded equally. Quantification was performed using surrogate standards and 4− 12 point linear calibration curves. For the analytes lacking 13Clabeled equivalents (e.g., EH-TBB, BEH-TEBP, and DBDPE), the relative recovery versus the surrogate standard was used to correct the results. The method recoveries of the abovementioned three BFRs and of α- and β-DBE-DBCH, TBX, BATE, PBBz, TBCT, PBT, PBEB, TBP-DBPE, HBBz, DBHCTD, OBTMPI (here referred to as “other BFRs”) were determined by fortifying five SRM 1958 aliquots at one (130 pg) level (270 pg of OBTMPI and also 1100 pg of DBDPE). The average recovery obtained for each analyte was used to correct the quantified amounts in the samples. Five nonfortified SRM 1958 aliquots were also analyzed to 7586
dx.doi.org/10.1021/es501139d | Environ. Sci. Technol. 2014, 48, 7584−7592
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Table 1. PBDE Concentrations (ng/g Lipid) in Matched Serum Samples from Mothers and Toddlers from Uppsala, Sweden mothers (N = 24) lipid content (%) BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-196 BDE-197 BDE-203 BDE-206 BDE-207 BDE-208 BDE-209 ΣBDE12c
toddlers (N = 24)
DF %a
GMb
median
range
DF %a
GMb
median
range
50 100 46 50 100 4 100 21 13 100 96 100 100
0.51 0.047 0.58 0.098 0.11 0.95 0.046 0.43 0.51 0.078 0.37 0.081 0.86 4.7
0.48 0.036 0.56 0.078 0.10 0.95 0.043 0.47 0.42 0.066 0.33 0.074 0.68 4.5
0.39−0.76
