Anal. Chem. 2003, 75, 6696-6700
Determination of Polybrominated Diphenyl Ethers in Environmental Standard Reference Materials Lingyan Y. Zhu and Ronald A. Hites*
School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana 47405
The total concentrations (∑PBDE) of 15 polybrominated diphenyl ethers, BDEs 17, 28, 47, 49, 66, 71, 85, 99, 100, 138, 153, 154, 183, 190, and 209, and a predominant polybrominated biphenyl (PBB) congener, 2,2′,4,4′,5,5′-polybrominated biphenyl (PBB-153) were determined in six natural matrix Standard Reference Materials (SRM) provided by the National Institute of Standards and Technology (NIST). The ∑PBDE concentrations (( one standard deviation) in SRM 1945 (whale blubber), SRM 1946 (fish tissue), SRM 1947 (fish tissue), and SRM 1974b (mussel tissue) were 126 ( 7, 79 ( 3, 134 ( 5, and 7.9 ( 0.2 ng/g of wet wt, respectively. The ∑PBDE concentrations were 54 ( 2 and 163 ( 4 ng/g of dry wt for SRM 2977 (mussel tissue) and SRM 2978 (mussel tissue), respectively. Among the three kinds of marine animals, mussel tissue had the lowest total concentration of PBDEs. These values can provide an informal reference or benchmark to laboratories who measure these compounds. Polybrominated diphenyl ethers (PBDEs) are used extensively as flame retardants in various consumer products, including polyurethane foam cushions, high-use textiles, electronic appliances, and printed circuit boards.1,2 In 1992, the total annual global production of PBDE was 40 000 metric tons,3 and by 1999, it was 67 000 metric tons.4 The three major commercial products available in the market are called the deca, octa, and penta products, the latter two of which consist of several BDE congeners. The estimated global consumption of the deca, octa, and penta products was 55 000, 3800, and 8500 metric tons, respectively, in 1999.5 PBDEs are mostly resistant to acidic and basic attack and to biological degradation.6,7 They have relatively low vapor pressures * Corresponding author. E-mail:
[email protected]. (1) Hedelmalm, P.; Carlsson, P.; Paml, V. Waste from electrical and electronic productssa survey of the contents of materials and hazardous substances in electric and electronic components, In Tema Nord, 554, Nordic Council of Ministers, 1995, Copenhagen, Denmark. (2) Meironyte´, D.; Nore´n, K.; Bergman, A° . J. Toxicol. Environ. Health, Part A 1999, 58, 329-341. (3) WHO/ICPS, Environmental Health Criteria 152: Polybrominated Biphenyls, Geneva, Switzerland, 1994. (4) Bromine Science and Environmental Forum, http://www.bsef.com. (5) Watanabe, I.; Sakai, S. I. Environ. Int. 2003, 29, 665-682. (6) Pijnenburg, A. M. C. M.; Everts, J. W.; de Boer, J.; Boon, J. P. Polybrominated Biphenyl and Diphenyl Ether Flame Retardants: Analysis, Toxicity, and Environmental Occurrence, Reviews of Environmental Contamination and Toxicology; Springer-Verlag: New York, 1995. (7) Allchin, C. R.; Law, R. J.; Morris, S. Environ. Pollut. 1999, 105, 197-207.
6696 Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
at room temperature (∼3 × 10-4 Pa for tetra-BDEs to ∼2 × 10-7 Pa for octa-BDEs) and high octanol-water partition coefficients (∼106 for tetra-BDEs to ∼1010 for deca-BDEs).8-11 As a result, PBDEs are environmentally persistent pollutants, just like the structurally similar PCBs.12-15 Unlike PCBs and organochlorine pesticides, however, PBDEs are still widely produced and used throughout the world. They enter the environment during their production, delivery, use, and disposal and through the recycling of materials containing PBDEs.16 Once released into the environment, PBDEs accumulate in biota, such as fish, meat, dairy products, and people.17 Toxicological studies indicate that hydroxylated metabolites of PBDEs may be potent competitors of the thyroid hormone, thyroxine, and could disrupt normal thyroid hormone function in wildlife and in people.18-20 PBDEs were first detected in the environment in 1979, when several PBDE congeners, including decabromo diphenyl ether (BDE-209), were detected in the soil and sediment around a PBDE manufacturing plant in the United States.21 In 1981, PBDEs were found in Swedish fish samples taken from the River Viskan.22 Eventually, the presence of PBDEs in the environment was confirmed by other researchers.12,23-26 Even though the concentrations of PBDE are still lower than those of PCBs and DDT, PBDE levels in the environment and in people have greatly increased in the last 10-15 years.27-29 (8) Watanabe, I.; Kashimoto, T.; Ryo, T. Chemosphere 1987, 16, 2389-2396. (9) Darnerud, P. O.; Eriksen, G. S.; Jo´hannesson, T.; Larsen, P. B.; Viluksela, M. Environ. Health Perspect. 2001, 109, 49-68. (10) Rahman, F.; Langford, K. H.; Scrimshaw, M. D.; Lester, J. N. Sci. Total Environ. 2001, 275, 1-17. (11) de Wit, C. A. Chemosphere 2002, 46, 583-624. (12) Watanabe, I.; Kashimoto, T.; Ryo, T. Chemosphere 1987, 16, 2389-2396. (13) de Boer, J.; Wester, P. G.; Klamer, H. J. C.; Lewis, W. E.; Boon, J. P. Nature 1998, 394, 28-29. (14) Sellstro ¨m, U.; Kierkegaard, A.; de Wit, C.; Jansson, B. Environ. Toxicol. Chem. 1998, 17, 1065-1072. (15) Renner, R. Anal. Chem. 2000, 34, 223A-226A. (16) Sjo ¨din, A.; Patterson, D. G., Jr.; Bergman, A° . Environ. Sci. Technol. 2001, 35, 3830-3833. (17) Booij K.; Zegers, B. N.; Boon, J. P. Organohalogen Compd. 2000, 47, 89-92. (18) Meerts, I. A. T. M.; van Zanden, J. J.; Luijks, E. A. C.; van Leeuwen-Bol, I.; Marsh, G.; Jakobsson, E.; Bergman, A° .; Brouwer, A. Toxicol. Sci. 2000, 56, 95-104. (19) Meerts, I. A. T. M.; Marsh, G.; van Leeuwen-Bol, I.; Luijks, E. A. C.; Jakobsson, E.; Bergman, A° . Organohalogen Compd. 1998, 37, 309-312. (20) Meerts, I. A. T. M.; Luijks, E. A. C.; Marsh, G.; Jakobsson, E.; Bergman, A.; Brouwer, A. Organohalogen Compd. 1998a, 37, 147-150. (21) DeCarlo, V. J. N. Y. Acad. Sci., Ann. 1979, 320, 678-681. (22) Andersson, O ¨ .; Blomkvist, G. Chemosphere 1981, 10, 1051-1060. (23) Jansson, B.; Asplund, L.; Olsson, M. Chemosphere 1987, 16, 2343-2349. (24) de Boer, J. Chemosphere 1989, 18, 231-2140. (25) Kuehl, D. W.; Haebler, R.; Potter, C. Chemosphere 1991, 22, 1071-1084. (26) Loganathan, B. G.; Kannan, K.; Watanabe, I.; Kawano, M.; Irvine, K.; Kumar, S.; Sikka, H. C. Environ. Sci. Technol. 1995, 29, 1832-1838. 10.1021/ac034643j CCC: $25.00
© 2003 American Chemical Society Published on Web 10/24/2003
Table 1. SRM Sample Information SRM ID
species
sampling location
sampling year
description
1945 1946 1947 1974b 2977 2978
whale blubber fish tissue fish tissue mussel tissue mussel tissue mussel tissue
Cape Cod, MA Apostle Island, Lake Superior Lake Michigan Dorchester Bay, Boston Harbor Guanabara Bay, Brazil Raritan Bay, NJ
Sept 1991 Oct 1997 Oct 1997 Oct 1999 1996 1991
adult female pilot whale lake trout lake trout Mytilus edulis freeze-dried, Perna Perna freeze-dried, unknown species
The accurate measurement of trace levels of pollutants greatly benefits from standardization and from interlaboratory calibration. To facilitate this process, the National Institute of Standards and Technology (NIST) has issued several natural-matrix Standard Reference Materials (SRMs), with both certified and reference concentrations of individual PCB congeners.30-32 However, there is very little information from NIST or in the literature on PBDE levels in SRMs or in other natural-matrix certified reference materials. The one exception is a study by Alaee et al., who developed a GC/HRMS method and applied it to the determination of PBDE concentrations in certified reference materials of fish from Lake Ontario.33 Given the availability and homogeneity of the NIST SRMs, we suggest that these materials can be used for the interlaboratory calibration of PBDE concentrations. To this end, we describe here the analytical methods and quantitation results for PBDE concentrations in six SRM samples. They are SRM 1974b (a frozen mussel tissue homogenate), SRM 2977, SRM 2978 (freeze-dried mussel tissues), SRM 1945 (a frozen whale blubber homogenate), SRM 1946, and SRM 1947 (frozen fish tissue homogenates from the Great Lakes). EXPERIMENTAL SECTION Materials. The SRMs were donated to us by the NIST Standard Reference Materials Program. Information about each SRM sample is listed in Table 1. Detailed information on the collection and preparation of each SRM can be found in the SRM’s Certificates of Analysis.34-38 The 15 PBDE congeners investigated in this study were as follows: 2,2′,4-TrBDE (BDE-17), 2,4,4′(27) Schro ¨ter-Kermani, C.; Helm, D.; Herrmann T.; Paepke, O. Organohalogen Compd. 2000, 47, 49-52. (28) Thomsen, C.; Lundanes, E.; Becher, G. Environ. Sci. Technol. 2002, 36, 1414-1418. (29) Ryan, J. J.; Patry, B. Organohalogen Compd. 2000, 47, 57-60. (30) Schantz, M. M.; Benner, B. A.; Hays, M. J.; Kelley, W. R.; Vocke, R. D.; Demiralp, R.; Greenberg, R. R.; Schiller, S. B.; Lauenstein, G. G.; Wise, S. A. Fresenius J Anal. Chem. 1995, 352, 166-173. (31) Schantz, M. M.; Koster, B. J.; Oakley, L. M.; Schiller, S. B.; Wise, S. A. Anal. Chem. 1995, 67, 901-910. (32) Schantz, M. M.; Parris, R. M.; Wise, S. A. Chemosphere 1993, 27, 19151922. (33) Alaee, M.; Sergeant, D. B.; Ikonomou, M. G.; Luross, J. M. Chemosphere 2001, 44, 1489-1495. (34) Certificate of Analysis, Standard Reference Material (SRM) 1945, Organics in Whale Blubber, National Institute of Standards and Technology (NIST): Gaithersburg, MD, 1994. (35) Certificate of Analysis, Standard Reference Material (SRM) 1946, Lake Superior Fish Tissue, National Institute of Standards and Technology (NIST): Gaithersburg, MD, 2002. (36) Certificate of Analysis, Standard Reference Material (SRM) 1974b, Organics in Mussel Tissue (Mytilus edulis), National Institute of Standards and Technology (NIST): Gaithersburg, MD, 2003. (37) Certificate of Analysis, Standard Reference Material (SRM) 2977, Mussel Tissue (Organic Contaminants and Trace Elements), National Institute of Standards and Technology (NIST): Gaithersburg, MD, 2000.
TrBDE (BDE-28), 2,2′,4,4′-TeBDE (BDE-47), 2,2′,4,5′-TeBDE (BDE-49), 2,3′,4,4′-TeBDE (BDE-66), 2,3′,4′,6-TeBDE (BDE-71), 2,2′,3,4,4′-PeBDE (BDE-85), 2,2′,4,4′,5-PeBDE (BDE-99), 2,2′,4,4′,6PeBDE (BDE-100), 2,2′,3,4,4′,5′-HxBDE (BDE-138), 2,2′,4,4′,5,5′HxBDE (BDE-153), 2,2′,4,4′,5,6′-HxBDE (BDE-154); 2,2′,3,4,4′,5′,6HpBDE (BDE-183),2,3,3′,4,4′,5,6-HpBDE (BDE-190), and decabromodiphenyl ether (BDE-209). They were all purchased in nonane solution from Cambridge Isotope Laboratories (Cambridge, MA). In addition, we investigated a predominant polybrominated biphenyl (PBB) congener, 2,2′,4,4′,5,5′-polybrominated biphenyl (PBB-153), a standard of which was purchased from Ultra Scientific (North Kingstown, RI). The recovery standard, 2,3′,4,4′,5PeBDE (BDE-118), was donated to us by Cambridge Isotope Laboratories. All the solvents used for the extraction and cleanup procedures were of residue-analysis grade. Sample Preparation. Four to seven samples of each of the six SRMs were extracted and analyzed for PBDE congeners. The extraction, isolation, and analytical procedures were similar to those we have used previously for PBDEs.39 Generally, a portion of the SRM sample was ground with 60 g of precleaned anhydrous Na2SO4. After spiking with the internal standards [13C12]-2,3,3′,4,4′,5hexachlorodiphenyl ether (CDE-156) and [13C12]-2,2′,3,3′,4,4′,5,5′octachlorodiphenyl ether (CDE-194) (Cambridge Isotope Laboratories), the samples were Soxhlet extracted for 24 h with 50% acetone in hexane. Throughout the extraction and analysis procedure, the analytes were protected from light by wrapping the containers with aluminum foil or by using amber glassware. The lipid concentration was determined by gravimetric measurement. The majority of the lipids were removed by adding ∼4 mL of concentrated H2SO4 (EM Science, Gibbstown, NJ) to each extract in 10 mL of hexane and shaking the mixture for 5 min. After centrifugation for 10 min, the upper hexane layer was recovered and combined with 5 mL of hexane used to wash the sulfuric acid residue. After reducing the volume to ∼1 mL under N2 flow, the samples were loaded on to a 3% water-deactivated silica (Grace Davison, Columbia, MD) column (20 cm × 1.9 cm i.d.). One fraction of 150 mL of dichloromethane was collected. The solvent was blown down with N2 to 500 µL, and the samples were fractioned on an alumina (ICN Biomedicals GmbH, Eschwege, Germany) column (6 cm × 0.6 cm i.d.). The column was first eluted by 8 mL of hexane, followed by 8 mL of 40% dichloromethane in hexane. The PBDEs were in the second fraction. After solvent exchange to hexane, BDE-118 was added as a recovery standard. The samples were analyzed by GC/MS. (38) Certificate of Analysis, Standard Reference Material (SRM) 2978, Mussel Tissue (Organic Contaminants and Trace Elements), National Institute of Standards and Technology (NIST): Gaithersburg, MD, 2000. (39) Dodder, N. G.; Strandberg, B.; Hites, R. A. Environ. Sci. Technol. 2002, 36, 146-151.
Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
6697
Instrumental Parameters. All these SRM samples were analyzed for the 15 PBDE congeners and PBB-153 using an Agilent 6890 series gas chromatograph coupled to an Agilent 5973 mass spectrometer with helium as the carrier gas. The 2-µL injections were made in the pulse splitless mode, with a purge time of 2.0 min. The injection port was held at 285 °C. For the determination of all PBDEs and PBB except for BDE-209, the GC column was a 60 m × 250 µm (i.d.) fused-silica capillary tube coated with DB-5-MS (0.25-µm film thickness; J&W Scientific, Folsom, CA). A 60-m column was used to ensure separation of BDE-154 from PBB-153. The GC oven temperature program was as follows: isothermal at 110 °C for 1.90 min, 15 °C/min to 180 °C, 1.85 °C/min to 300 °C, and held at 300 °C for 45 min. The GC-to-MS transfer line was held at 285 °C. The mass spectrometer was operated in electron capture negative ionization mode (ECNI) using methane as the reagent gas, and the ion source temperature was 150 °C. Selected ion monitoring of the two bromide ions at m/z 79 and 81 was used to detect the PBDEs. The ions at m/z 351.9 and 349.9 due to (M - HCl + 2)- and (M - HCl)- were used to detect CDE-156, and ions at m/z 457.8 and 455.8 due to (M + 4)- and (M + 2)- were used to detect CDE-194. The response factors for all compounds were determined using quantitation standards with known amounts of all the target compounds, internal standards, and recovery standards. The internal standard [13C12]-CDE-156 was used as a retention time reference, and [13C12]-CDE-194 was used as the internal standard to quantitate all the PBDE congeners. BDE-118 was used as a recovery standard. [13C12]-CDE-194 eluted in the middle of the retention range of all the congeners; thus, it provides satisfactory response factors for all congeners investigated. ECNI was also used for the determination of BDE-209. It was analyzed on a shorter DB-5-MS column (15-m × 250-µm i.d.; 0.25µm film thickness; J&W Scientific) with the following temperature program: 110 °C for 1 min, 15 °C/min to 300 °C, and held at 300 °C for 17 min. Selected ion monitoring of the two bromide ions at m/z 79 and 81 was also used to detect BDE-209. [13C12]-CDE194 was used as the internal standard to quantitate it. Quality Control. Three quality control criteria were used to ensure the correct identification of the target compounds: (a) The GC retention times matched those of the standard compounds. (b) The signal-to-noise ratio was greater than 3:1. At this S/N, the detection limits were 0.0086-0.15 ng/g of wet wt, depending on the individual congener. (c) The isotopic ratio between the quantitative ion and confirmation ion was within (15% of the theoretical value. The recoveries of the internal standard were higher than 70% for all the SRM samples, except for SRM 1945 (whale blubber), where the average recovery was 50 ( 5% due to the high load of lipids in this sample. Of course, the measurements were all automatically corrected for recovery by using the chlorinated diphenyl ether internal standard, which had been added to the samples before extraction. Each batch of samples included one method blank. The method blank consisted of 60 g of precleaned Na2SO4, which was spiked with the same amount of internal standard as the samples. Method blank results were all below the detection limits except for BDE-47 (0.27 ( 0.027 ng), BDE-99 (0.18 ( 0.032 ng), and BDE-209 (1.6 ( 0.48 ng). The levels of BDE-47 and BDE-99 in the method blank were well below 1% of the levels found in the SRM samples. 6698
Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
All SRM samples were analyzed in two batches. The time between the two batches was one month. In each batch, at least two replicates were included. The results of these two batches were equivalent, demonstrating that our analysis method is stable and reliable with good repeatability between different batches. The final results are calculated as the average of all the measurements of the same sample. For most of the congeners, the relative standard deviations (RSD) were ∼10%, illustrating the good reproducibility of this analysis method. A worldwide interlaboratory study on PBDEs was conducted by de Boer et al., and our laboratory was involved in this study.40 This study showed RSDs between 17 and 77%, depending on the individual congeners, suggesting that there are large variations between different laboratories, which may be using different analysis methods. RESULTS AND DISCUSSION PBDE Concentrations. The concentrations of PBDEs are listed by congener in Tables 2 and 3. BDE-71, 138. and 190 were not found in any of the six SRM samples, presumably due to their low concentrations in the commercial mixtures. Table 2 lists the PBDE concentrations in four of the SRMs based on the wet weight of the sample. Because SRM 2977 and 2978 came as freeze-dried powders, the PBDE concentrations in these two mussel samples had to be normalized to the sample’s dry weight (see Table 3). The concentrations in SRM 1974b are also reported as nanograms per gram of dry weight as a comparison between the three mussel samples. The moisture content in mussel tissue SRM 1974b was determined by weight lost on heating at 110 °C for 4 h. Our value (90.83%) was similar to the value (89.87%) reported in the NIST Certificate document for this material.36 BDE-47, 99, and 100 were usually the most abundant congeners in all of the SRM samples. The amounts of BDE-209 in the samples (1.6-2.8 ng) were about the same as in the blank samples (1.2-1.9 ng); thus, we cannot conclude that BDE-209 is present in these SRMs. Even though BDE-209, which is the main congener (97%) in the deca product, is the most abundant congener produced, it is rarely found in biota.13,39,41 BDE-209 was found at low levels in mussels collected in Osaka Bay, Japan;12 in human blood serum from Sweden,42 and in peregrine falcon eggs in Sweden.43 Presumably BDE-209 has a low bioaccumulation potential because of its large molecular size and very high Kow value, suggesting it is predominantly bound to particles.5 We can compare these concentrations to those reported in the literature for similar types of samples. Lake Michigan salmonids were reported to have a ∑PBDE concentration of 80 ng/g of wet wt (sum of BDE-47, 66, 99, 100, 153, and 154).44 The PBDE concentrations we found in the lake trout tissue SRM 1946 and 1947 (79 and 134 ng/g of wet wt, respectively) are in the same range as these samples, although as expected, the fish (SRM 1947) from Lake Michigan are more contaminated than those from Lake (40) de Boer, J. Chemosphere 2002, 46, 625-633. (41) de Boer, J.; van der Horst, A.; Wester, PG. Organohalogen Compd. 2000, 47, 85-88. (42) Sjo ¨din, A.; Hagmar, L.; Klasson-Wehler, E.; Kronholm-Diab, K.; Jakobsson, E.; Bergman, A° . Environ. Health Perspect. 1999, 107, 643-648. (43) Sellstro ¨m, U.; Lindberg, P.; Haggerberg, L.; de Wit, C. Proceedings of the Second International Workshop on brominated flame retardants, Stockholm, Sweden; The Swedish Chemical Society: Stockholm, 14-16 May, 2001; pp 159-162. (44) Manchester-Neesvig, J. B.; Valters, K.; Sonzogni, W. C. Environ. Sci. Technol. 2001, 35, 1072-1077.
Table 2. PDBE Concentrations (ng/g of wet wt) ( One Standard Deviation in SRM Fish, Whale Blubber, and Mussel Tissuea SRM 1945 SRM 1946 SRM 1947 SRM 1974b whale blubber lake trout lake trout mussel Cape Cod Lake Superior Lake Michigan Boston Harbor no. of replicates
a
6
4
4
5
BDE-17 BDE-28 BDE-47 BDE-49 BDE-66 BDE-71 BDE-85 BDE-99 BDE-100 BDE-138 BDE-153 BDE-154 BDE-183 BDE-190
0.21 ( 0.06 1.53 ( 0.30 46.6 ( 2.0 2.57 ( 0.11 1.53 ( 0.13
