Environ. Sci. Technol. 2009, 43, 6058–6063
Identification of Hydroxylated Metabolites of Hexabromocyclododecane in Wildlife and 28-days Exposed Wistar Rats S I C C O H . B R A N D S M A , * ,†,‡ L E O T . M . V A N D E R V E N , § J A C O B D E B O E R , †,‡ A N D P I M E . G . L E O N A R D S †,‡ Institute for Environmental Studies, VU University, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands, Netherlands Institute for Fisheries Research, PO Box 68, 1970 AB IJmuiden, The Netherlands, and Laboratory for Health Protection Research, National Institute of Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven, The Netherlands
Received March 24, 2009. Revised manuscript received May 31, 2009. Accepted June 3, 2009.
We studied the presence of hydroxylated metabolites of hexabromocyclododecane (HBCD) in three wildlife species (tern egg, seal, and flounder) and in Wistar rats exposed to 30 and 100 mg HBCD/kg bw/day for 28 days. A nondestructive extraction, fractionation, and cleanup method was developed to separate the hydroxylated HBCD metabolites from the biotic sample matrix. Four different groups of hydroxylated HBCD metabolites were identified in rat adipose, liver, lung, and muscle tissues by liquid and gas chromatography (LC and GC) combined with mass spectrometry (MS): monohydroxy metabolites of HBCD, pentabromocyclododecene (PBCDe), tetrabromocyclododecene (TBCDe), and dihydroxy-HBCD. Dihydroxy-PBCDe was identified by GC-MS but could not be confirmed by LCMS. Debromination of HBCD to PBCDe was another metabolic pathway observed. In tern eggs from the Western Scheldt the monohydroxy-HBCD was found and in the blubber of harbor seal (Wadden Sea) the monohydroxy metabolites of HBCD and PBCDe were found. No hydroxylated metabolites were detected in the tissue of flounder (Wadden Sea). To our knowledge, this is the first study to identify different hydroxylated metabolite groups of HBCD in rat and wildlife samples.
Introduction HBCD belongs to the large family of brominated flame retardants (BFRs) and is primarily used in thermal insulation material (polystyrene roof isolation) in the building industry. It is also the principal flame retardant in upholstered textiles (1). HBCD is the most widely used cycloaliphatic BFR, and is the BFR with the third highest production volume after tetrabromobisphenol-A (TBBP-A) and decabromodiphenyl ether (BDE209) (1). Its use has substantially increased over the last twenty years. The worldwide usage of HBCD in 2001 was about 16,700 tons, most of which was destined for the European market (9500 tons) (1). HBCD is persistent, and * Corresponding author phone: +31 20 5989566; fax: +31 20 5989553; e-mail:
[email protected]. † VU University. ‡ Netherlands Institute for Fisheries Research. § National Institute of Public Health and the Environment (RIVM). 6058
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bioaccumulates. It is not yet officially considered as a potential persistent organic pollutant (POP), but it is a candidate POP (2). Commercial mixtures of HBCD consist of ca. 80-90% γ-, 8-9% R-, and 6% β-HBCD. The thermal stability of HBCD is influenced by the percentage of the γ-diastereoisomer. Impurities found in the technical mixture of HBCD are e.g. five isomers of tetrabromocyclododecene (TBCDe), isobutanol, and some unidentified compounds (3-5). In environmental analyses, most attention is paid to R-, β-, and γ-diastereoisomers of HBCD. Like polybrominated diphenylethers (PBDEs), HBCD is an additive BFR and thus not covalently bound to the polymeric materials. During use or disposal of the products HBCD can be released to the environment. HBCD has been detected globally in the environment, including remote areas such as the Arctic (6-9). The diastereoisomers of HBCD show differences in structural and physical-chemical properties, which may result in differences in biological uptake and metabolism (10, 11). Data are available on the different isomers present in biota and sediment (e.g., 6-9). In sediment the pattern is normally similar to that of the technical mixture, with γ-HBCD being the most dominant diastereoisomer followed by the R- and β-diastereoisomers. In biota R-HBCD is often the dominant diastereoisomer followed by the γ- and β-diastereoisomers. The following three factors could be responsible for this observation. First, bioisomerization of β-, and γ-HBCD to R-HBCD as observed in fish (12); second, R-HBCD is more water-soluble than β- and γ-HBCD and therefore, more readily available for uptake (5).; third, in vitro experiments with rat and harbor seal microsomes showed that the biotransformation of β- and γ-HBCD was faster than that of R-HBCD (13). A small number of other studies of HBCD metabolism exist. In one study four metabolites of HBCD of unknown structure were found in rats (14). In-vitro biotransformation studies with R-, β-, and γ-HBCD showed changes in the diastereoisomer pattern of HBCD, and three HBCD metabolites were detected with a liquid chromatograph (LC) combined with a mass spectrometer (MS) (13). Two metabolites were identified as monohydroxy-HBCD. The third metabolite could not be identified (13). Hydroxylated metabolites of β- and γ-HBCD were found, however no significant decrease in R-HBCD concentration was observed during the incubation with rat and harbor seal microsomes. However, in vitro experiments with microsomes of dab and flounder showed that R-HBCD was also biotransformed resulting in two monohydroxy metabolites of HBCD (13). Huthala et al. (15) confirmed this result by the finding of monohydroxy-HBCD after an in vitro study in rainbow trout liver microsomes. Other degradation/metabolite products have been observed by Abdallah et al. (16) and Hiebl and Vetter (17). In office dust samples four isomers of pentabromocyclododecene (PBCDe) and two isomers of TBCDe were identified with LC and gas chromatography (GC) (16). PBCDe was detected in chicken eggs and whitefish (Coregonus sp.) with GC (17). The focus of the current study was to screen and identify different hydroxylated metabolites groups in HBCD-exposed rats and wildlife samples. Also the debromination of the HBCD in the exposed rats was studied. At the Dutch National Institute for Public Health and the Environment (RIVM) Wistar rats were exposed for 28 days to HBCD. This took place within the framework of the EU-project Flame retardants Integrated Risk assessment for Endocrine effects (FIRE) 10.1021/es900879k CCC: $40.75
2009 American Chemical Society
Published on Web 07/01/2009
FIGURE 1. (A) Full scan GC-MS ECNI spectrum of HBCD standard. (B) Full scan GC-MS spectrum of monohydroxy-HBCD (fraction 12) at GC retention time of 26.84 min from the adipose tissue of the male rat (100 mg HBCD/kg bw/day). (18). The current study focused on liver as a biotransforming organ, lungs, muscles, and adipose tissues of these rats that were exposed to 30 and 100 mg HBCD/kg bw/day. GC-MS and LC-MS were used for the identification of the HBCD metabolites.
Materials and Methods HBCD Exposed Rats. Briefly, Wistar rats (5 female and 5 male animals per dosing group) were dosed orally with the technical mixture of HBCD spiked to their feed (18). The dosing groups included 0, 0.3, 1, 3, 10, 30, 100, and 200 mg HBCD/kg bw/day. After 28 days the rats were sacrificed, and multiple organs were sampled (18). For the current study the liver, lung, muscle, and adipose tissue of individual rats from the control, 30 mg, and 100 mg HBCD/kg bw/day groups (males and females) were used for the identification of metabolites of HBCD. More details of the exposure study can be found in Van der Ven et al. (18). Wildlife. Samples of tern eggs from the Western Scheldt, The Netherlands (Terneuzen colony), and muscle of flounder and blubber of harbor seal from the Wadden Sea, The Netherlands were screened for the presence of HBCD metabolites. Sample treatment was according to the protocol for rats, and sample intakes were equivalent to approximately 1 g of lipid. Purity Check of HBCD Dosing Solution. To identify possible brominated impurities present in the technical mixture of HBCD used to dose rats in an in vivo toxicity test (see below), a solution of 20 mg/L was prepared. This mixture was fractionated using normal phase-high performance liquid chromatography (NP-HPLC) (procedure see below) and analyzed by LC-MS and GC-MS. The peaks of brominated compounds found in the technical mixture were identified and compared to the peaks found in the exposed rats. Extraction, Fractionation, and Cleanup. For the extraction of HBCD and metabolites a method developed by Jensen et al. (19) was used and slightly modified by the addition of a NP-HPLC fractionation step. Approximately 0.2 g of rat adipose tissue or liver or ca. 2 g of lung or muscle were homogenized in hexane/acetone (1:3.5) and twice extracted with hexane/methyl tert-butyl ether (MTBE) (9:1) using a Vortex mixer. The extraction solvents covered a wide polarity
range to extract not only parent compounds but also the more polar metabolites. A small number of the extracted polar metabolites (e.g., glucuronide conjugates) remained outside the scope of this study due to matrix interferences. The extract was further purified with GPC, and fractionated into 17 fractions using NP-HPLC. All fractions were analyzed by LCQ-MS and GC-MS. For further details on NP-HPLC, GPC, LCQ-MS, and GC-MS settings, see Supporting Information.
Results and Discussion Technical HBCD Mixture, Blank, and Control Rat. The highest response of HBCD standard was found in NP-HPLC fraction 7. The full scan GC-MS spectrum showed that the highest ion clusters are m/z 79 and 160, followed by m/z 561 [M - Br]- (Figure 1A). At high concentrations, bromine adduct formation on the HBCD molecule results in an ion cluster at m/z 720 [M + Br - H]- (Figure 1A). At lower concentrations the ion clusters of m/z 641 and 720 corresponding with [M - H]- and [M + Br - H]- respectively, are no longer found; the bromine cluster at m/z 561 [M - Br]- may then be used for identification of HBCD. An advantage of LCQ-MS compared to GC-MS, in addition to avoiding thermal degradation in the GC injector or column, is the separation of the different diastereoisomers of HBCD. The LCQ-MS chromatograms and the full scan spectra of the three R-, β-, and γ-HBCD diastereoisomer standards are shown in Figures 2A and 3A, B, C. All isomers show bromine clusters at m/z values 677, 687, and 702. Mass 677 is an adduct of chlorine (m/z 35) to the molecular ion (m/z 642), and m/z 687 an adduct of two sodium molecules to the molecular ion (m/z 642) minus hydrogen. The bromine clusters at m/z 702 (m/z 60 higher than the molecular ion m/z 642) could not be explained. These diastereoisomers eluted in different NPHPLC fractions: R- and γ-HBCD in fraction 6 and 7, and β-HBCD in fraction 9. It is known that five isomers of TBCDe are present as byproduct of the HBCD synthesis (4). In the current study we found several peaks of brominated compounds in the technical HBCD mixture using GC-MS, including two isomers of PBCDe and two isomers of TBCDe (in fractions 3-7). The presence of PBCDe and TBCDe is related to impurities in the VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. (A) Selected ion LCQ-chromatogram of r-, β-, and γ-HBCD standard extract. Selected ion LCQ-chromatograms of the hydroxylated metabolites in adipose tissue of the male rat exposed to 100 mg HBCD/kg bw/day: (B) PBCDe in fraction 6, (C) monohydroxy-HBCD in fraction 12, (D) monohydroxy-PBCDe in fraction 15, (E) monohydroxy-TBCDe fraction 15, (F) dihydroxy-HBCD in fraction 16. technical mixture or to thermal degradation of HBCD on the GC column or in the GC injector. To confirm the identity of these compounds a high concentration (20 mg/L) of the technical mixture of HBCD was also analyzed with LCQ-MS; 0.025% (w/w) of impurities in the technical HBCD mixture could be detected, assuming similar response factors. PBCDe and TBCDe were not detected with LCQ-MS, which suggested that PBCDe and TBCDe observed with GC-MS in fractions 3-7 are thermal degradation products of HBCD or impurities present in quantities lower than 0.025% of the technical HBCD mixture. No other brominated (e.g., hydroxylated) impurities were observed in the technical HBCD mixture. The control rats (dosing level 0) and procedural blanks also revealed no hydroxylated metabolites or PBCDe and TBCDe. Identification of HBCD Metabolites in Rat Tissues. Differences in distribution of the metabolites over different tissues are known to occur, e.g., hydroxylated-polychlorinated biphenyls (OH-PCBs) are mainly found in blood and not in adipose tissue of the polar bears (20). Therefore, metabolites of HBCD were studied in adipose tissue, liver, muscle, and lung of rat exposed to 30 and 100 mg HBCD/kg bw/day. All HBCD metabolites observed in the adipose tissue, reported in Table 1 and 2, were also found in muscle, lung, and liver of the exposed rats. There was no indication of differences in the metabolic profile among these three types of tissues. As quantification of the metabolites was not performed, no information on the relative concentrations among the tissue types can be provided. However, the highest response of hydroxylated metabolites were observed in the adipose tissue, e.g., 25 times higher response of monohydroxy-HBCD in adipose tissue than in muscle on wet weigh basis, which suggests that they accumulate in lipid-rich tissue. Szabo et al. (21) exposed mice orally to [14C] γ-HBCD to measure the tissue distribution. The highest concentration was observed in the liver followed by blood, fat, and brain. Szabo et al. (21) suggested that the biological persistency of HBCD in mice may be limited. No information on metabolites was given in this study. Extracts of the male adipose tissues (exposure level: 100 mg HBCD/kg/bw/day) were used for the identification of other metabolites of HBCD since the metabolite profile was the same as in female adipose tissue. 6060
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An overview of the major hydroxylated metabolites in the adipose tissue of the 100 mg HBCD/kg bw/day dose group identified by LCQ-MS is given in Table 1. The hydroxylated metabolites are found in NP-HPLC fractions 12-16, and elute later then the parent compound HBCD (fractions 6, 7, and 9). All metabolites that produced LCQ-MS full scan spectra that allowed for metabolite identification are shown in Table 1. Identification of the metabolites was based on a comparison between the LCQ-MS spectra of the NP-HPLC fractions of the exposed rats and spectra of the three isomers, R-, β-, and γ-HBCD, and retention times (Figures 2A and 3A, B, C). For the monohydroxy-HBCD the same bromine clusters as for HBCD were observed, apart from the difference of 16 m/z corresponding to the oxygen atom. The LCQ-MS chromatogram of fraction 12 shows two peaks at LC retention time of 4.11 and 5.76 min with a bromine cluster at m/z 693 (Figure 2C). Adduct formation of chlorine (m/z 35) to the molecular ion of the monohydroxy-HBCD (m/z 658) results in a bromine cluster with m/z 658 + 35 ) 693. Two other bromine clusters are visible in Figure 3E and F with m/z values of 703 and 718. These are m/z 45 and 60 higher than the molecular ion of monohydroxy-HBCD (m/z 658). The formation of the cluster with m/z 703 can be explained by adduct formation of two sodium molecules to the molecular ion of monohydroxy-HBCD and the loss of one hydrogen (m/z 658 + 23 + 23-1 ) 703). The formation of the bromine clusters with m/z 718 could not be explained. The two metabolites found in fraction 12 with LC retention times of 4.11 and 5.76 min were therefore identified as monohydroxy metabolites of HBCD. The hydroxy metabolites of HBCD found in rats confirm the in vitro results reported by Zegers et al. (13). They suggested that the metabolites formed during in vitro rat microsomes experiments (m/z 79, 81 and [M + 16]) were due to two monohydroxy metabolites of HBCD. In our study a larger number of hydroxylated metabolites were observed. In total four different types of metabolites were found: the monohydroxy metabolites of TBCDe, PBCDe, and HBCD, and a dihydroxy-HBCD (Table 1). The LCQ-MS chromatograms and spectra of the monohydroxy-TBCDe and monohydroxy-PBCDe of fraction 15, and the dihydroxy-HBCD of fraction 16 are shown in Figures 2D, E, F and 3G, H, and I. The spectra of both the monohydroxy-PBCDe (C12H16Br5OH, Figure 3G) and monohydroxy-TBCDe (C12H17Br4OH, Figure 3H) show the molecular ion bromine clusters with a chlorine adduct, m/z 611, corresponding to C12H16Br5OH [M + Cl](576 + 35), and m/z 533, corresponding to [M + Cl]- (m/z 498 + 35), respectively. The double bond of PBCDe and TBCDe is due to the elimination of HBr. The spectrum of dihydroxy-HBCD (Figure 3I) can be explained by the molecular ion bromine cluster at m/z 709 and by the addition of a chlorine adduct (m/z 674 + 35), corresponding to C12H16Br6OHOH. To confirm the results of the LCQ-MS, the NP-HPLC fractions were also analyzed by GC-MS. The metabolites with GC-MS full scan spectra (e.g., Figure 1B) that allow identification of the metabolites are shown in Table 2. All the hydroxylated metabolites found with LCQ-MS were confirmed by GC-MS, but an additional metabolite was found with GC-MS, the dihydroxy-PBCDe. The concentrations of the two dihydroxy metabolites of PBCDe observed in NPHPLC fractions 15 and 16 are probably too low to be detected by LCQ-MS. However, the presence of dihydroxy-PBCDe in fraction 16 could also be a result of thermal debromination of the dihydroxy-HBCD on the GC column as shown for HBCD. This was not possible for the dihydroxy-PBCDe in fraction 15 because no dihydroxy-HBCD was found in this fraction. A full scan GC-MS spectrum of fraction 12 containing the monohydroxy-HBCD with GC retention time of 26.84 min is shown in Figure 1B. Comparing the GC-MS spectra of the technical mixture of HBCD (Figure 1A) with those of
FIGURE 3. LCQ-MS spectrum of the main Br-cluster of the major peaks shown in Figure 2: (A, B, C) r-, β-, γ-HBCD, (D) PBCDe, (E, F) monohydroxy-HBCD, (G) monohydroxy-PBCDe, (H) monohydroxy-TBCDe, (I) dihydroxy-HBCD.
TABLE 1. Metabolites in Adipose Tissue of Male Rats Dosed with 100 mg HBCD/kg bw/day Measured by LCQ-MS (Metabolite 1 Corresponds to Figure 2E, Metabolites 2 and 3 Correspond to Figure 2D, Metabolites 8 and 9 Correspond to Figure 2C, and Metabolite 10 Corresponds to Figure 2F) LCQ-MS
metabolite
retention time (min)
NP-HPLC fraction no.
1 2 3 4 5 6 7 8 9 10
monohydroxy-TBCDe monohydroxy-PBCDe monohydroxy-PBCDe monohydroxy-PBCDe monohydroxy-HBCD monohydroxy-HBCD monohydroxy-HBCD monohydroxy-HBCD monohydroxy-HBCD dihydroxy-HBCD R-HBCD β-HBCD γ-HBCD
5.35 3.15 4.50 4.77 3.08 3.55 4.13 4.11 5.76 1.99 7.45 8.44 12.21
15 15 15 12 15 15 15 12 12 16 6,7 9 6,7
a
Br-cluster 498 578 578 578 658 658 658 658 658 674 677 677 677
+ + + + + + + + + +
35 35 35 35 35 35 35 35 35 35
) ) ) ) ) ) ) ) ) )
533 613 613 613 693 693 693 693 693 709
molecular formula
molecular massa
[M + Cl]-
[M + 2Na - H]-
C12H17Br4OH C12H16Br5OH C12H16Br5OH C12H16Br5OH C12H17Br6OH C12H17Br6OH C12H17Br6OH C12H17Br6OH C12H17Br6OH C12H16Br6OHOH C12H18Br6 C12H18Br6 C12H18Br6
498 576 576 576 658 658 658 658 658 674 642 642 642
533 613 613 613 693 693 693 693 693 709 677 677 677
543 621 621 621 703 703 703 703 703 719 687 687 687
The highest peak of the bromine cluster is marked as molecular mass.
the exposed rats shows that the bromine clusters observed at m/z 417, 497, 577, 657, and 736 all differ by 16 m/z units from the main clusters found in the technical mixture of HBCD, corresponding to one oxygen atom. The most important indication for a monohydroxy metabolite is the loss of m/z 18 corresponding to H2O between the clusters at m/z 577-559 and m/z 497-479, which confirms the presence of a hydroxy-group (Figure 1B). Due to the relatively high level of monohydroxy-HBCD in the extract [M - H]- (m/z 657) and [M + Br - H]- (m/z 736) are also visible. These were also observed in the technical mixture of HBCD (Figure 1A). The GC-MS spectra of the monohydroxy metabolites of PBCDe (C12H16Br5OH) and TBCDe (C12H17Br4OH) showed bromine clusters at m/z 497 and 417, respectively, ([M - Br]-). The intensity of [M - Br]decreases with decreasing numbers of bromines, which makes the identification of the lower brominated metabolites more difficult. However, in combination with the LCQ-MS spectra identification was still possible. The difference between the number of metabolites found with GC-MS and LC-MS can probably be explained by the greater sensitivity
of the GC-MS technique (LOD for the LCQ is 100 pg for each HBCD isomer, and 1 pg total HBCD for GC). For example, the identification of five of the nine metabolites of monohydroxy of TBCDe (fraction 12) was based on [M - Br]- of the GC-MS spectra (Table 2). Identification of those metabolites could not be confirmed by LCQ-MS. Therefore, the monohydroxy-TBCDe in fraction 12 could also be the result of thermal debromination of monohydroxy-PBCDe or HBCD on the GC column. Debromination of HBCD. Another metabolic pathway in the rat is debromination of HBCD to PBCDe and TBCDe. PBCDe was found in the adipose tissue of the rat (Figures 2B and 3D). PBCDe in the adipose tissue could be explained either by debromination of HBCD via metabolism in the rat or an accumulation of PBCDe impurities present in the technical mixture of HBCD used to dose the rats. However, the latter explanation would seem unlikely because the only impurities reported in the literature as byproduct of the HBCD synthesis are five isomers of tetrabromocyclododecene (4, 5). Furthermore, LCQ-MS analysis showed that the technical HBCD mixture used in our study contained no PBCDe or VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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527 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 79 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 160 417 417 417 417 401 479 479 479 479 b
The highest peak of the bromine cluster is marked as molecular mass. a
22 23
monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-TBCDe monohydroxy-PBCDe monohydroxy-PBCDe monohydroxy-PBCDe monohydroxy-PBCDe monohydroxy-PBCDe dihydroxy-PBCDe dihydroxy-PBCDe monohydroxy-HBCD monohydroxy-HBCD monohydroxy-HBCD monohydroxy-HBCD dihydroxy-HBCD HBCD unknown unknown 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Bold ) is most dominant bromine cluster.
415 415 415 415 415 431 431 497 497 497 497 481 479 479 479 479 479 559 559 559 559 417 417 417 417 417 417 417 417 417 497b 497 497 497 497 513 513 577 577 577 577 593 561 497 497 497 497 497 497 497 497 497 575 575 575 575 575 657 657 657 657 641 577 577 577 577 577 577 577 577 577 657 657 657 657 657 736 736 736 736 720 498 498 498 498 498 498 498 498 498 576 576 576 576 576 592 592 658 658 658 658 674 642 unknown unknown C12H17Br4OH C12H17Br4OH C12H17Br4OH C12H17Br4OH C12H17Br4OH C12H17Br4OH C12H17Br4OH C12H17Br4OH C12H17Br4OH C12H16Br5OH C12H16Br5OH C12H16Br5OH C12H16Br5OH C12H16Br5OH C12H15Br5OHOH C12H15Br5OHOH C12H17Br6OH C12H17Br6OH C12H17Br6OH C12H17Br6OH C12H16Br6OHOH C12H18Br6 unknown unknown 12 15 12 12 15 12 15 12 15 12 15 15 16 12 15 16 15 12 15 15 16 6,7,9 8 9
metabolite
18.41 18.50 18.79 18.98 19.02 19.30 19.35 19.73 19.81 22.25 22.63 22.90 23.52 23.74 24.14 25.98 26.50 26.84 27.30 27.69 29.16 24.98 22.45 23.09
molecular [M + [M [M [M massa Br - H]- [M - H]- [M - Br]- Br - H20]- 2Br - H]- 2Br - H20]- [M - 3Br]- [Br + Br]- [Br]- unknown 9
GC-MS
retention NP-HPLC time (min) fraction no.
molecular formula
TABLE 2. Metabolites in Adipose Tissue of Male Rats Dosed with 100 mg HBCD/kg bw/day Measured by GC-MS 6062
FIGURE 4. Tentative pathways for the metabolism of HBCD in rat.
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FIGURE 5. Selected ion LCQ-MS chromatogram (m/z 693) and spectrum from fraction 12 of the tern egg (Western Scheldt). TBCDe impurities (at least lower than 0.025% of the technical HBCD mixture). In the rat adipose tissue a relatively high PBCDe was observed with LCQ-MS in NP-HPLC fractions 3, 5, and 6 (Figures 2B and 3D). This supports the hypothesis that the observed PBCDe comes from the biotransformation of HBCD. It is known that biotransformation from cyclododecane to cyclododecene by human and rat liver microsomes can occur as was reported for HCH (22). Recently, PBCDe was found in chicken egg and fish (17). However, in that study it was not clear if PBCDe was a metabolite of HBCD formed in the fish, chicken, or egg or accumulated from the food. Abdallah et al. (16) found four isomers of PBCDe and also two isomers of TBCDe in dust. In our study no TBCDe was found, however, the presence of monohydroxy-TBCDe indicates that further debromination of PBCDe to TBCDe may occur. A tentative pathway for HBCD metabolism is proposed in Figure 4. Metabolites of HBCD in Wildlife. Three wildlife samples were screened for the presence of HBCD metabolites (tern egg, flounder muscle, and harbor seal blubber). Metabolites of HBCD were found in the tern egg and seal blubber but not in the flounder sample. In the tern egg the monohydroxy of HBCD was found in fraction 12 with a LC retention time of 4.16 min (Figure 5). A metabolite with the same spectrum and retention time was also observed in the exposed rats (Figures 2C and 3E). In the seal blubber the monohydroxy metabolites of PBCDe and HBCD were identified in fraction 12 with GC retention times of 23.74 and 26.84 min, respectively. The concentrations were low and therefore only the monohydroxy-HBCD could be confirmed by LCQ-MS (LC retention time of 4.16 min). Identification of all metabolites was performed by GC-MS in the selected ion-monitoring mode measuring the m/z 79, 81, 493-501, and 573-583. The monohydroxy-PBCDe in fraction 12 could also be a result of the thermal debromination product of monohydroxy-HBCD on the GC column.
The metabolites in the two wildlife samples had the same GC retention times as the monohydroxy metabolites of PBCDe and HBCD (fraction 12) that were found in the adipose tissue of the exposed male rat (Table 1). That no hydroxylated metabolites were found in flounder is in contrast to the results of an in vitro study with flatfish (flounder and dab) microsomes, where evidence was found for the metabolism of HBCD (13). The metabolites formed in the liver of the flatfish might be excreted too rapidly to be accumulated in the muscle tissue. In an earlier study Zegers et al. (13) observed a peak in a marine mammal blubber extract with a retention time similar to the monohydroxy-HBCD peak detected in the in vitro experiment. Our results proved that this same hydroxylated metabolite is present in harbor seal (Wadden Sea) and tern eggs (Western Scheldt).
Acknowledgments We gratefully acknowledge financial support by the European Commission through the FIRE project (QLRT-2001-00596). We are solely responsible for the content of this paper, which does not necessarily represent the opinion of the European Community. Bart Zegers (NIOZ) and Ike van der Veen (IVM) are acknowledged for their support during the LC-MS measurements. We also thank Pim de Voogt (University of Amsterdam) for insightful input.
Supporting Information Available More analytical details and settings for the NP-HPLC, GPC, LCQ-MS, and GC-MS. Figure S1 shows the schematic illustration of the extraction, fractionation, and cleanup procedure to separate the HBCD from the metabolites. This material is available free of charge via the Internet at http:// pubs.acs.org.
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