1

Spectral characterization of two bioaccumulated methoxylated polybrominated 1

diphenyl ethers 2

Emma L. Teuten,1* Carl G. Johnson,1 Manolis Mandalakis,2 Lillemor Asplund,2 Örjan 3

Gustafsson,2 Maria Unger,2,3 Göran Marsh,3 and Christopher M. Reddy1 4

5

1Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods 6

Hole, MA 02543, USA 7 2Department of Applied Environmental Science (ITM), Stockholm University, 10691 Stockholm, Sweden 8

3Department of Environmental Chemistry, Stockholm University, 10691 Stockholm, Sweden 9

10

Abstract 11

Two methoxylated polybrominated diphenyl ethers (MeO-PBDEs) were isolated from a 12

True’s beaked whale (Mesoplodon mirus) and identified by NMR (1H, 1H,1H and 1H,13C) 13

and high resolution mass spectrometry as 2-(2’,4’-dibromophenoxy)-3,5-dibromoanisole 14

(6-MeO-BDE47) and 2-(2’,4’-dibromophenoxy)-4,6-dibromoanisole (2’-MeO-BDE68). 15

Previously the structures of these bioaccumulated compounds have been determined by 16

comparison of their mass spectra and gas chromatographic (GC) retention times with 17

those of authentic standards. While this method is accepted and generally successful, 18

NMR of the isolated compounds allows us to definitively identify the congeners. Our 19

characterizations are consistent with those made for MeO-PBDEs in other organisms, 20

identified by chromatographic methods. 21

22

* Corresponding author: eteuten@whoi.edu, fax 508-457-2164.

2

Keywords: Nuclear magnetic resonance (NMR); Mass spectrometry (MS); Isolation; 1

Compound specific analysis. 2

3

Introduction 4

Polybrominated diphenyl ethers (PBDEs), a common class of flame retardants, have been 5

found widely distributed throughout the environment (de Wit, 2002). A variety of 6

structurally related hydroxylated PBDE (HO-PBDE) and methoxylated PBDE (MeO-7

PBDE) marine natural products have been known for decades (Sharma and Vig, 1972; 8

Carté and Faulkner, 1981). Since initially being observed bioaccumulated in biota by 9

Haglund et al. (1997), MeO-PBDEs have been identified in an increasing number of 10

organisms at high trophic levels. To date, they have been detected in a range of species 11

including fish (Asplund et al., 1999; Letcher et al., 2003; Kierkegaard et al., 2004; 12

Sinkkonen et al., 2004), birds (Olsson et al., 2000; Sinkkonen et al., 2004), and marine 13

mammals (Haglund et al., 1997; Vetter et al., 2002; Pettersson et al., 2004). Their 14

dispersion appears ubiquitous throughout the World oceans, with their presence detected 15

from the Arctic (Sinkkonen et al., 2004) to the Antarctic (Vetter et al., 2002). 16

Bioaccumulation of these compounds is not limited to marine organisms; MeO-PBDEs 17

have also been observed in several species of freshwater fish (Letcher et al., 2003; 18

Kierkegaard et al., 2004). The two MeO-PBDEs most frequently observed 19

bioaccumulated in animal tissue are 2-(2’,4’- dibromophenoxy)-3,5-dibromoanisole (6-20

MeO-BDE47) and 2-(2’,4’-dibromophenoxy)-4,6-dibromoanisole (2’-MeO-BDE68) 21

(Figure 1) which have also been isolated from marine sponges (Anjaneyulu et al., 1996; 22

Utkina et al., 2002) and green algae (Kuniyoshi et al., 1985). The MeO-PBDE 23

3

abbreviations used herein are derived from their halogenation patterns, following the 1

International Union of Pure and Applied Chemistry (IUPAC) system for numbering 2

polychlorinated biphenyls (PCBs) (Marsh et al., 2004a). 3

To date, specific MeO-PBDE congeners have been identified by comparison of 4

their mass spectra and gas chromatographic (GC) retention times with those of authentic 5

standards (Asplund et al., 1999; Olsson et al., 2000; Vetter et al., 2002; Letcher et al., 6

2003; Kierkegaard et al., 2004; Marsh et al., 2004a). This method is convenient and 7

generally acceptable for identifying bioaccumulated compounds. However, since 8

polyhalogenated aromatic compounds can have very similar retention times (Mullin et 9

al., 1984; Eganhouse and Gossett, 1991), the most definitive method for identifying the 10

exact structures of the bioaccumulated MeO-PBDEs is to isolate the individual 11

compounds, elucidate their structures, and compare them with authentic standards. In the 12

present work, the specific congeners of two MeO-PDBEs isolated from a North Atlantic 13

True’s beaked whale (Mesoplodon mirus), were identified as 6-MeO-BDE47 and 2’-14

MeO-BDE68 using proton NMR, 1H,1H correlated spectroscopy (COSY) and 1H,13C 15

heteronuclear chemical shift correlation (HETCOR). The structural assignments are in 16

agreement with those made using relative GC retention times and mass spectra. 17

18

Methods 19

Instrumentation 20

High resolution mass spectrometry (HRMS) data were acquired at 20,000 resolution by 21

direct exposure ionization (DEI) on an Autospec-Q mass spectrometer, using an 22

acceleration voltage of 6 kV and ionization energy of 20 eV. Continuum data were 23

4

acquired in constant voltage scanning mode, and perfluorokerosene used as an internal 1

calibration standard. Proton and 2-dimensional (1H,1H and 1H,13C) NMR spectra (400 2

MHz) were recorded on an Avance 400 DPX (Bruker) in CDCl3, setting the residual 3

CHCl3 signal to 7.24 ppm relative to trimethylsilane. Gas chromatography mass 4

spectrometry (GC-MS) employed an Aglient 6890 series GC system connected to a 5973 5

network mass selective detector (70 eV ionization energy), using a J & W Scientific DB-6

XLB column (60 m × 0.25 mm i.d. 0.25 μm film thickness). Electron capture negative 7

ionization (ECNI) spectra were obtained using methane as a reagent gas. Experiments 8

involving the flame ionization detector (FID) used an HP 5890 Series II GC, with a CP-9

Sil 5CB column (60 m × 0.25 mm i.d. 0.25 μm film thickness). 10

11

2-Dimensional NMR techniques 12

The 2-dimensional NMR techniques used in this identification offered additional 13

information to the 1-dimensional experiments, particularly with regard to connectivity 14

between atoms. COSY 1H,1H experiments, which show coupling between nearby protons, 15

aided in the identification of proton locations. In aromatic systems, such as those studied 16

here, coupling is observed over longer distances, due to electron delocalization. The 17

NMR samples were too dilute to allow for direct measurement of 13C chemical shifts. 18

However, short-range 1H,13C HETCOR experiments which investigate through-bond 19

coupling of adjacent carbon and hydrogen atoms allowed the chemical shift of a carbon 20

bonded to a particular proton to be determined. Long-range HETCOR experiments were 21

used to probe the coupling between atoms two to three bonds away from each other. 22

23

5

Sample acquisition and preparation 1

Blubber (10 kg) from a dead, stranded M. mirus was obtained from the Virginia Marine 2

Science Museum in January 2004. The homogenized blubber was filtered, dried over 3

Na2SO4 and designated the total lipid extract (TLE). 4

5

Isolation of MeO-PBDEs 6

Preliminary tests showed the recoveries of the MeO-PBDEs from 2 g of TLE were 7

comparable when the lipids were removed by i) gel permeation chromatography (GPC) 8

and ii) reaction with H2SO4. Consequently, for the large scale extraction portions of TLE 9

(400 mL) were treated with concentrated H2SO4 (150 mL) to degrade the lipids. The 10

resulting mixture was extracted into hexane (3 × 350 mL) and the hexane layers washed 11

with 10 mL H2SO4. Hexane extracts of three of the above extractions were combined and 12

reduced in volume to 300 mL. The concentrated hexane extract was brought to neutral 13

pH by washing with water and the hexane removed by rotatory evaporation, yielding 15 - 14

30 g of extract. The non-polar compounds were isolated using silica gel column (50 g) 15

eluting with 2% dichloromethane (DCM) in hexane (1.1 L). Remaining lipids were 16

removed by GPC, utilizing Bio-beads SX-8 (Biorad Laboratories) as the stationary phase 17

and 60% DCM in hexane as the mobile phase in a 50 cm × 2.5 cm i.d. glass column. 18

Extracts from 8 kg of TLE were combined and compounds separated using a column 19

containing 4 g Al2O3 above 8 g deactivated silica. Fractions were eluted with an 20

increasing gradient of DCM in hexane. MeO-PBDEs were eluted with 50% DCM in 21

hexane. 6-MeO-BDE47 (0.7 mg) and 2’-MeO-BDE68 (1.2 mg) were isolated by 22

preparative capillary gas chromatography (PCGC) (Eglinton et al., 1996) using a CP-Sil 23

6

5CB column (60 m × 0.25 mm i.d. 0.25 μm film thickness). After ~200 injections the 1

MeO-PBDEs were rinsed from the U-tubes using DCM. Column bleed was removed 2

using a silica gel (~1 g) column and 10 % DCM in hexane as the eluant, and the purity of 3

the recovered MeO-PBDEs was determined by GC-FID (>99%). 4

5

Results and Discussion 6

The studied MeO-PBDEs are two of many halogenated organic compounds (HOCs) 7

observed in the True’s beaked whale. They are relatively abundant at concentrations of 8

~1 μg g-1 of lipid, with only 2,2-bis(p-chlorophenyl)-1,1-dichloroethane (DDE) and three 9

polychlorinated biphenyls (PCBs) present at higher concentrations. The concentrations 10

(μg g-1 of lipid) of the more abundant compounds were accurately determined relative to 11

authentic standards as follows: DDE, 4.2 μg g-1; PCB-138, 1.2 μg g-1; PCB-153, 1.0 μg g-12

1; PCB-180, 1.0 μg g-1 (Teuten et al., 2005). 13

The original impetus of this work was the isolation of sufficient quantities of the 14

MeO-PBDEs from an environmental matrix for determination of their origin using 15

natural abundance radiocarbon analysis. Radiocarbon content has been proven an 16

excellent tool for determining whether HOCs are of a natural or industrial origin (Reddy 17

et al., 2002, Reddy et al., 2004). Extraction of 10 kg of blubber generated sufficient 18

material to conduct a detailed spectral analysis in addition to making the radiocarbon 19

measurement, which showed that the two isolated MeO-PBDEs were naturally produced 20

(Teuten et al., 2005). Both 6-MeO-BDE47 and 2’-MeO-BDE68 are known marine 21

natural products, and it is possible that natural sources account for a substantial portion of 22

these MeO-BDEs accumulated in marine organisms. However, no fresh water sources of 23

7

these compounds are known. Since they have been found bioaccumulated in freshwater 1

fish in highly industrialized areas (Letcher et al., 2003; Kierkegaard et al., 2004), their 2

formation from anthropogenic PBDEs is also possible. Indeed, HO-PBDEs are known to 3

form from PBDEs in vivo (Hakk and Letcher, 2003), and are also proposed intermediates 4

in the pyrolytic formation of polybrominated dibenzodioxins from PBDEs (Weber and 5

Kuch, 2003). Microorganisms have been shown to methylate the hydroxyl group of 6

Triclosan, a polychlorinated diphenyl ether (Hundt et al., 2000); similar pathways are 7

likely available for conversion of HO-PBDEs to MeO-PBDEs. Hence formation of MeO-8

PBDEs from industrial PBDEs, particularly in polluted freshwater areas, cannot be 9

excluded. 10

Extraction of individual compounds from such a large sample required extensive 11

clean-up, involving treatment with concentrated sulfuric acid followed by a variety of 12

chromatographic techniques. The progress of the clean-up was monitored by GC. Figure 13

2 shows GC-FID traces for A) the extracted HOCs after removal of the lipids, B) the 14

MeO-PBDE fraction collected from a silica gel-Al2O3 column and C) 2’-MeO-BDE68 15

isolated in >99% purity by PCGC. Spectral analysis showed the peaks with retention 16

times of 28.1 min and 28.4 min corresponded to 2’-MeO-BDE68 and 6-MeO-BDE47, 17

respectively (relative to BDE-138 at 36.7 min). Half of each isolated MeO-PBDE was 18

prepared for NMR and 10 μg was submitted for HRMS. 19

Mass spectra of both compounds exhibit isotopic distributions characteristic of 20

tetrabrominated compounds. Accurate mass data for the molecular ions of 6-MeO-21

BDE47 (511.7260 ± 0.0005) and 2’-MeO-BDE68 (511.7258 ± 0.0006) match those 22

calculated for C13H879Br4O2, with errors of 0.4 ppm and 0.0 ppm, respectively. Similar 23

8

precisions and errors were observed for the 81Br containing ions of the molecular ion 1

cluster (Table 1). A search of other possible molecular formulae within ± 5 ppm of the 2

measured molecular ion for the two isolated compounds, revealed almost 200 3

possibilities with the following atomic restrictions: C (0-20), H (0-40), O (0-4), N (0-4), S 4

(0-4), Br (0-6), Cl (0-6). After elimination of those that did not contain the correct 5

number of halogens to give the observed isotopic pattern, eight possibilities remained. Of 6

these, three can be discarded as they have an odd number of nitrogen atoms, and hence 7

cannot give stable compounds with an even (rounded) molecular weight. A further three 8

can be eliminated due to their high degree of saturation, which is inconsistent with the 1H 9

NMR results showing more aromatic than aliphatic protons. At this point, two possible 10

molecular structures remain: C13H8Br4O2, whose calculated mass matches the molecular 11

ions of the two isolated compounds within 0.4 ppm as discussed above, and 12

C11H9Br4ON4P. The calculated mass of the latter provides a less close match to the 13

experimental data (1.6 ppm and 2.0 ppm for 6-MeO-BDE47 and 2’-MeO-BDE68 14

respectively). A literature search did not reveal any compounds with this molecular 15

formula, and it is difficult to conceive a structure with this elemental composition that 16

could withstand the harsh sulfuric acid treatment used in the isolation of these 17

compounds. Hence, C13H8Br4O2 is the most feasible molecular formula, which is 18

consistent with tetrabrominated MeO-PBDEs. 19

Low resolution EI-MS of the two isolated compounds showed both to have a 20

cluster of peaks at 354, corresponding to the [M-2Br] ion, a commonly observed 21

fragment ion for polybrominated diphenyl ethers (Marsh et al., 1999a, Cooper et al., 22

2003). Also observed for each of the isolated compounds was an ion resulting from the 23

9

loss of BrCH3 (M-94), which is characteristic of MeO-PBDEs with the methoxy group in 1

the ortho position (Marsh et al., 2003). This fragment ion is not observed for MeO-2

PBDEs with the methoxy substituent in the meta and para positions (Marsh et al., 2003). 3

The mass spectral data provides strong support for the two compounds isolated 4

being ortho substituted methoxylated tetrabrominated diphenyl ethers. This assignment is 5

further supported by the 1H NMR, 1H,1H COSY and 1H,13C HETCOR experiments. The 6

analyte concentrations were too low for a direct 13C NMR spectrum to be obtained, but 7

information about the chemical environments of many carbon atoms was obtained using 8

HETCOR and long range HETCOR spectroscopy. The NMR results are summarized in 9

Figure 3. 10

Proton and 1H,13C correlated NMR spectra indicate that the only aliphatic carbon 11

has a chemical shift consistent with a methoxy group (δH = 3.8 - 3.9 ppm, δ13C = 60 - 62 12

ppm). All of the other carbon atoms observed by HETCOR and long-range HETCOR 13

have chemical shifts of 115 to 155 ppm, supporting their aromatic nature. Most 14

significantly, details of the bromine substitution pattern, hence the specific congeners, are 15

revealed by studying the 1H NMR and COSY. From the 1H coupling it is evident that 16

both compounds have three protons on one aromatic ring (A) and two on the other (B). In 17

both cases, ring A is substituted in the ortho and para position as evidenced by the 18

coupling constants for H-3’ with H-5’ (2 Hz) and H-5’ with H-6’ (9 Hz). Coupling 19

constants (2 Hz) for the protons on ring B show that the substituents are on alternate 20

carbons. Observation of COSY coupling between H-6 and the methyl protons indicates 21

the MeO-PBDE with a retention time of 28.4 min as 6-MeO-BDE47. For the MeO-PBDE 22

with retention time = 28.1 min, no coupling between the methyl and aromatic protons 23

10

was observed, hence we have identified this compound as 2’-MeO-BDE68. The 1H NMR 1

chemical shifts obtained (shown in Table 2) match those reported by Marsh et al. (2003) 2

for synthesized standards to within 0.03 ppm. 3

Previous identification of MeO-PBDEs in animal tissue has relied on comparative 4

mass spectra and relative GC retention times (Asplund et al., 1999; Olsson et al., 2000; 5

Vetter et al., 2002; Letcher et al., 2003; Kierkegaard et al., 2004; Marsh et al., 2004a). 6

Although this technique is commonly used for identifying bioaccumulated HOCs, it is 7

possible for different congeners with the same degree of halogenation to coelute. In a 8

thorough study of relative retention times of PCBs, five pairs of isomers exhibited 9

identical relative retention times (Mullin et al., 1984). Here, we have definitively 10

identified the specific congeners of bioaccumulated MeO-PBDEs based on their NMR 11

spectra. Our detailed spectral identification of 6-MeO-BDE47 and 2’-MeO-BDE68 as the 12

predominant bioaccumulated MeO-PBDEs in True’s beaked whale blubber is consistent 13

with data for Swedish fish (Kierkegaard et al., 2004). High abundances of 6-MeO-14

BDE47 and 2’-MeO-BDE68 have also been reported in several marine mammals from 15

Australia (Vetter et al., 2002; Melcher et al., 2004) and Japan (Marsh et al., 2004b). Mass 16

spectra for the isolated MeO-PBDEs are consistent with spectra for 6-MeO-BDE47 and 17

2’-MeO-BDE68 obtained using both electron ionization (EI) (Marsh et al., 1999b; 18

Athanasiadou, 2003) and electron capture negative ionization (ECNI) (Athanasiadou, 19

2003). To check the agreement of our structural assignments with those determined with 20

the traditional approach used in other studies, the GC properties of the above MeO-21

PBDEs were compared with those isolated from a long-finned pilot whale (Globicephala 22

melas) from the northern North Atlantic Ocean, which had been identified as 6-MeO-23

11

BDE47 and 2’-MeO-BDE68 by chromatographic comparison to authentic standards 1

(Mandalakis et al.). The relative GC retention times and mass spectra for both pairs of 2

compounds were identical. 3

4

Summary 5

Two abundant MeO-PBDEs isolated from the blubber of a True’s beaked whale have 6

been identified as 6-MeO-BDE47 and 2’-MeO-BDE68, using NMR and mass spectral 7

data. These structural assignments are consistent with those made previously using 8

chromatographic methods, and unequivocally identify the specific congeners of these 9

compounds. 10

11

Acknowledgements 12

We thank the Virginia Marine Science Museum for providing us with the M. mirus 13

blubber and Bob Nelson for his assistance in the laboratory. We gratefully acknowledge 14

financial support from the National Science Foundation (OCE-0221181), the Postdoctoral 15

Scholar Program at Woods Hole Oceanographic Institution (WHOI) (with funding 16

provided by the Henry and Camille Dreyfus Foundation Inc. and the J. Seward Johnson 17

Fund to E.L.T.), the WHOI Ocean Life Institute and the Swedish Foundation for 18

Strategic Environmental Research (MISTRA Idestöd, contract no. 2002-057). This is 19

WHOI contribution # ______. 20

21

22

23

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Figure 1. Bioaccumulated methoxylated diphenyl ethers isolated from M. mirus. 1

OOCH3

BrBr

Br

Br

2

3 5

61

1'2'

3'

4'

5'6'

4

OOCH3Br

Br

2

3

11'

2'3'

4'

5'6'

Br

Br

54

6

2'-MeO-BDE686'-MeO-BDE47

A B A B

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

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Figure 2. GC-FID of HOCs isolated from M. mirus at various stages during the isolation 1

of 2’-MeO-BDE68. (A) HOCs separated by GPC, (B) MeO-PBDE fraction collected 2

using Al2O3 column, (C) 2’-MeO-BDE68 collected by PCGC. 3

A

B

C

4

5

6

7

8

9

10

11

12

13

19

Figure 3. NMR results for 6-MeO-BDE47 and 2’-MeO-BDE68. The dotted lines 1

illustrate 1H,1H coupling observed between protons in the COSY experiments. Chemical 2

shifts (δ, ppm) are in italics. δH are underlined. δ13C were inferred from the HETCOR 3

experiments. Not all of the 13C chemical shifts for 6-MeO-BDE47 could be assigned due 4

to low analyte concentration. 5

60

131

153 116

129116116

7.1

7.8

7.2

7.4

3.8

6.3

3.8

MeO-BDE-47

62

148 120

131

117122118

137121

150

152115

3.9

7.5

6.97.4

7.8

6.8132

OOCH3

Br

Br

Br

Br

H

HH H H

MeO-BDE-68

136

114

120

141OOCH3

Br

Br

Br

H

HH Br

H

H

6

7

8

9

20

Table 1. A comparison of calculated and measured HRMS values for the molecular ion 1

clusters of 6-MeO-BDE47 and 2’-MeO-BDE68 2

6-MeO-BDE47 2’-MeO-BDE68 Calculated

mass Measured massa difference/

ppm

Measured massa difference/

ppm

511.7258 511.7260 ± 0.0005 0.4 511.7258 ± 0.0006 0.0

513.7238 513.7240 ± 0.0004 0.4 513.7237 ± 0.0006 -0.2

515.7218 515.7214 ± 0.0006 -0.7 515.7214 ± 0.0006 -0.6

517.7198 517.7198 ± 0.0004 0.0 517.7195 ± 0.0006 -0.6

519.7180 519.7172 ± 0.0004 -1.5 519.7175 ± 0.0009 -1.0

a Measured masses are an average of a minimum of 4 injections. 3

4

5

6

7

8

9

10

11

12

13

14

15

21

Table 2. Proton NMR chemical shifts and coupling constants 1

Proton chemical shift /ppm

CH3 H-3 H-4 H-5 H-6 H-3’ H-5’ H-6’

Coupling

constants /Hz

6-MeO-

BDE47

3.79 --- 7.10 --- 7.42 7.76 7.24 6.33 4JH-4, H-6 = 2.1

4JH-3’, H-5’ = 2.4

3JH-5’, H-6’ = 8.8

2’-MeO-

BDE68

3.93 6.94 --- 7.53 --- 7.81 7.41 6.77 4JH-3, H-5 = 2.3

4JH-3’, H-5’ = 2.3

3JH-5’, H-6’ = 8.7

2 3 4 5 6 7 8