Environ. Sci. Technol. 2009, 43, 7536–7542
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Origin of Hydroxylated Brominated
Diphenyl Ethers: Natural Compounds
or Man-Made Flame Retardants?
Y I W A N , * ,† S T E V E W I S E M A N , †
HONG CHANG,† XIAOWEI ZHANG,†
P A U L D . J O N E S , † M A R K U S H E C K E R , †,‡
KURUNTHACHALAM KANNAN,§
SHINSUKE TANABE,| JIANYING HU,⊥
MICHAEL H. W. LAM,∇ AND
J O H N P . G I E S Y †,#,∇,O
Department of Biomedical Veterinary Sciences and Toxicology
Centre, University of Saskatchewan, Saskatoon,
Saskatchewan S7N 5B3, Canada, ENTRIX, Inc., Saskatoon,
Saskatchewan S7N 5B3, Canada, Wadsworth Center, New
York State Department of Health and Department of
Environmental Health Sciences, School of Public Health, State
University of New York, Empire State Plaza,
Albany, New York 12201-0509, Center for Marine
Environmental Studies, Ehime University, Matsuyama, Japan,
College of Urban and Environmental Sciences, Peking
University, Beijing, 100871 China, Department of Zoology and
Center for Integrative Toxicology, Michigan State University,
East Lansing, Michigan, Centre for Coastal Pollution and
Conservation and Department of Biology and Chemistry, City
University of Hong Kong, Kowloon, Hong Kong, SAR China,
and State Key Laboratory of Marine Environmental Science,
College of Oceanography and Environmental Science, Xiamen
University, Xiamen, P. R. China
Received May 6, 2009. Revised manuscript received August
5, 2009. Accepted August 6, 2009.
Polybrominated diphenyl ethers (PBDEs) have been widely
used as flame retardants. The structurally related hydroxylated
PBDEs (OH-PBDEs) and methoxylated PBDEs (MeO-PBDEs)
occur in precipitation, surface water, wildlife, and humans. The
formation of OH-PBDEs in wildlife and humans is of considerable
concern due to their greater toxicities relative to PBDEs
and MeO-PBDEs. Research to date suggests that OH-PBDEs
are formed by hydroxylation of PBDEs, and MeO-PBDEs are then
formed by methylation of the OH-PBDEs. Here we show
significant metabolic production of OH-PBDEs from MeOPBDEs while hydroxylation of synthetic PBDEs to OH-PBDEs
was negligible. Concentrations of PBDEs, OH-PBDEs, and MeOPBDEs were analyzed in tuna, albatross, and polar bears
collected from marine environments worldwide, and we found
a closer relationship between OH-PBDEs and MeO-PBDEs
than had been previously reported. Furthermore, for the first
* Corresponding author tel: (306) 966-4978; fax: (306) 966-4796;
e-mail: yi.wan@usask.ca.
†
University of Saskatchewan.
‡
ENTRIX, Inc.
§
Wadsworth Center, New York State Department of Health and
Department of Environmental Health Sciences, School of Public
Health, State University of New York.
|
Ehime University.
⊥
Peking University.
#
Michigan State University.
∇
City University of Hong Kong.
O
Xiamen University.
7536
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 19, 2009
time the metabolic relationships between PBDEs, OH-PBDEs,
and MeO-PBDEs were elucidated in vitro using rainbow trout,
chicken, and rat microsomes. We propose the production of OHPBDEs from naturally occurring MeO-PBDEs as a previously
unidentified mechanism that could be an important contributor
for the occurrence of OH-PBDEs found in wildlife from
remote areas. Our results suggest that risk assessment
paradigms for PBDEs and their metabolites need reevaluation
and that human exposure to MeO-PBDEs that occur naturally
in marine organisms should be considered.
Introduction
Brominated flame retardants (BFRs) have emerged as
contaminants of concern due to their widespread use,
ubiquitous environmental distribution, great bioaccumulation potential, and toxicity. Polybrominated diphenyl ethers
(PBDEs) are one of the most widely used BFRs with an annual
global consumption of 70,000 t in 1999 (1). Over the last 30
years concentrations of PBDEs in human blood, breast milk,
and other body tissues have been increasing with doubling
times of approximately 4-6 years (2, 3).
Hydroxylated (OH-) and methoxylated (MeO-) PBDEs,
which are analogous to PBDEs in structure, have been found
in wildlife tissues (4-8), and laboratory studies have shown
the formation of OH-PBDEs after exposure to PBDEs (9-13).
There is considerable interest in the origin of OH-PBDEs
and MeO-PBDEs in biota and abiotic environmental matrices.
The concern over OH-PBDEs is of particular interest since
they elicit a variety of effects on exposed organisms including
disruption of thyroid hormone homeostasis, oxidative phosphorylation disruption, altered estradiol synthesis, and
neurotoxic effects (14-20). The fact that OH-PBDEs were
found at greater concentrations than PBDEs in marine algae
led to the suggestion that OH-PBDEs can be formed naturally
in marine algae or by their associated microorganisms (4, 8).
It has also been shown that OH-PBDEs are biotransformation
products of PBDEs. This conversion has been reported in
fish, rat, and human cell cultures (9, 12, 13). However, the
exposure concentrations of PBDEs in these in vitro or in vivo
studies were generally great (µg/g level), and the resultant
products, OH-PBDEs, occurred at trace concentrations
(<0.01-1% of PBDEs) (9, 12, 13). In contrast, environmental
concentrations of PBDEs in marine organisms are in the pg/g
to ng/g range, which suggests that OH-PBDEs concentrations should be much smaller if they were the metabolic
products of PBDEs. However, relatively great concentrations
of OH-PBDEs have been found in marine organisms,
suggesting the existence of other sources of these compounds.
MeO-PBDEs have also been found in various animals at
concentrations sometimes greater than those of PBDE
congeners (21, 22), and two abundant congeners (6-MeOBDE-47 and 2′-MeO-BDE-68) have been found to be natural
products in marine organisms (21). Since bacterial methylation of phenols might be a significant alternative to
biodegradation in the environment (23), it has also been
suggested that some MeO-PBDEs are formed via methylation
of OH-PBDEs (5, 21).
Bromophenols (BRPs) are a group of compounds related
to PBDEs, which have been identified as key natural flavor
components of marine fish (24, 25). Some BRPs have been
reported to be metabolites of OH-PBDEs (26), while some
are widely used as flame retardants (2,4,6-triBRP) with a
worldwide production of 9500 t in 2001 (27). The metabolic
relationships among PBDEs, MeO-PBDEs, OH-PBDEs, and
10.1021/es901357u CCC: $40.75
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BRPs in organisms have not been studied in detail. The
current hypothesis on the origin of OH-PBDEs and MeOPBDEs has been based on metabolism studies with high
dosing concentrations (9, 12, 13) and on studies of chemicals
with similar structure, e.g., polychlorinated biphenyls (PCBs)
and their hydroxylated metabolites (28).
In this study, concentrations of PBDEs, OH-PBDEs, MeOPBDEs, and BRPs were quantified in livers of tuna (Katsuwonus pelamis), five albatross species (Thalassarche chlororhynchos, Phoebetria palpebrata, Thalassarche chrysostoma,
Thalassarche cauta, and Thalassarche melanophrys), and
polar bear (Ursus maritimus) collected from remote marine
locations. In addition, in vitro biotransformation of PBDEs,
MeO-PBDEs, and OH-PBDEs was investigated in microsomal
fractions of liver from rainbow trout (Oncorhynchus mykiss),
chicken (Gallus gallus), and rat (Rattus norvegicus). The aim
of this study was to investigate the relationships among
PBDEs, MeO-PBDEs, OH-PBDEs, and BRPs in marine wildlife
tissues and gain insight into sources and pathways of
transformation.
Materials and Methods
Tissue Collection. Livers from fifteen albatross, ten tuna,
and ten polar bear were used for PBDEs, OH-PBDEs, MeOPBDEs, and BRPs quantification. Albatross were collected
from the Indian and South Atlantic Oceans, polar bear were
collected in Northern and Western Alaska, and tuna were
collected from the North Pacific Ocean in 1992-2002 (Table
S1 in Supporting Information). All samples were kept frozen
at -20 °C until analysis.
Extraction and Cleanup of Liver Tissue. Samples (approximately 5-10 g wet weight (ww)) were first freeze-dried,
spiked with a mixture of 13C-labeled PBDE and BRP surrogates, and extracted by accelerated solvent extraction
(Dionex ASE-200, Sunnyvale, CA). Extraction was conducted
with n-hexane/dichloromethane (DCM) (1:1) as the first
extraction solvent at a temperature of 100 °C and pressure
of 1500 psi, and then the samples were extracted with
n-hexane/methyl tert-butyl ether (MTBE) (1:1) as the second
extraction solvent at a temperature of 60 °C and pressure of
1000 psi. Two cycles (10 min) were performed for each solvent
per sample, and the two extraction fractions were combined
for subsequent cleanup. Extracts were rotary evaporated to
near dryness at 35 °C. Lipid content of each extract was
determined gravimetrically by evaporating the entire extract
to constant weight. Extracts were then dissolved in 8 mL of
hexane, and 4 mL of 0.5 M potassium hydroxide (KOH) in
50% ethanol was added. Phenolic compounds were separated
from the neutrals by partitioning with KOH (29). The aqueous
layer (KOH) was extracted with 8 mL of n-hexane three times
(neutral fraction), followed by acidification with 1.5 mL of
2 M hydrochloric acid. Then phenolic compounds were
extracted with n-hexane/MTBE (9:1; v/v) three times (phenolic fraction).
The neutral fraction was concentrated to approximately
2 mL and sequentially subjected to acidified silica gel and
neutral alumina column chromatography. The acidified silica
gel column was packed with 2 g of sodium sulfate and 8 g
of acidified silica (50 g of silica gel mixed with 27 mL of
concentrated sulfuric acid). After application of the sample,
the column was eluted with 15 mL of n-hexane and 10 mL
of DCM. The eluate was concentrated and passed through
a neutral alumina column (4 g of sodium sulfate, 4 g of neutral
alumina, 4 g of sodium sulfate), eluted with 20 mL of n-hexane
and then with 25 mL of 60% DCM in n-hexane. The second
fraction was concentrated and fortified with 13C-PBDE 138
for analysis of PBDEs and MeO-PBDEs.
The phenolic fraction was evaporated to dryness under
a gentle stream of nitrogen. A 480 µL aliquot of the
derivatization solvent (acetonitrile/methanol/water/pyridine
(5:2:2:1; v/v/v/v)) was added, and then 40 µL of methyl
chloroformate (MCF) was added. The reaction mixture was
shaken on a vortex at room temperature for 1 h before it
was diluted with 1.2 mL of pure water. The aqueous solution
was extracted with 6 mL of n-hexane three times, and the
extracts were subjected to acidified silica gel chromatography
as described above. The column was eluted with 30 mL of
n-hexane and 30 mL of DCM, and the eluate was concentrated
to 40 µL for OH-PBDE and BRP analysis. In this study the
MCF derivatization products of OH-PBDEs and BRPs were
designated as MCFO-PBDEs and MCFO-BRPs.
Identification and quantification of all target compounds
was performed using a Hewlett-Packard 5890 series II highresolution gas chromatograph interfaced to a Micromass
Autospec high-resolution mass spectrometer (HRGC-HRMS)
(Micromass, Beverly, MD). The chemicals and instrument
condition are provided in the Supporting Information.
Extraction and Cleanup of Microsome Reaction Mixtures. Prior to extraction, each of the microsomal reaction
mixture samples was spiked with a mixture of 13C-labeled
PBDE and BRP surrogates followed by addition of 2 mL of
pure water, 0.25 mL of concentrated hydrochloric acid, and
3 mL of 2-propanol. The aqueous layer was extracted with
5 mL of n-hexane/MTBE (1:1; v/v) two times. The extracts
were washed with 4 mL of pure water four times, followed
by addition of 4 mL of 0.5 M KOH in 50% ethanol. The
separation of phenolic and neutral compounds closely
mirrored that of the tissue samples except that the neutral
fraction was only subjected to an acidified silica gel column
eluted with 15 mL of n-hexane and 10 mL of DCM. The
phenolic fraction was dried and derivatized with MCF as
described above however the acidified silica gel column
following the derivatization was packed with 2 g of sodium
sulfate and 4 g of acidified silica and samples were eluted
with 15 mL of n-hexane and 15 mL of DCM.
In Vitro Microsomal Incubations. To investigate whether
MeO-PBDEs are more readily metabolized to OH-PBDEs than
synthetic PBDEs, we used microsomes isolated from several
surrogate species, namely rainbow trout, chicken, and rat,
that represent the different classes of species used for tissue
chemistry analysis. Rat S9 fraction was purchased from MP
Biomedicals (Solon, OH) and was isolated from Aroclor 1254
exposed individuals. Microsomes were isolated from rainbow
trout exposed to PCB-126 and microsomes were isolated from
chicken exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). These microsomes were previously isolated as part
of other studies in our laboratory. Previous studies have
clearly demonstrated that exposure to either Aroclor 1254,
PCB126, or TCDD induces the microsomal activity of the
same CYP450 1A homologues in exposed organisms (30).
Rainbow trout and chicken microsomes were prepared
according to the method of Kennedy and Jones (31), and the
details are provided in the Supporting Information. All
reactions were performed in 0.1 M NaH2PO4 buffer (pH 7.4)
containing 1 mM ethylenediaminetetraacetic acid (EDTA),
10 mM dithiothreitol (DTT), and 100 µM NADPH. The final
reaction volume was 100 µL and contained either 50 µL
(rainbow trout and chicken) or 25 µL (rat) of the microsomal
preparation and 3 uL of exposure chemicals. Individual
congeners (BDE-99, 6-MeO-BDE-47, and 6-OH-BDE-47) and
mixtures of compounds (PBDEs mix, MeO-PBDEs mix, and
OH-PBDEs mix) were used, and the concentrations in the
incubation mixture were 1.5 × 103 and 1.2 × 102 to 1.5 × 102
ng/mL for individual congeners and mixtures of compounds,
respectively (Table 1). The protein concentrations in the
reaction vial were 5.5, 6.4, and 9.0 mg/mL for rainbow trout,
chicken, and rat, respectively. Reactions were performed at
37 °C for 20 h with constant agitation. Incubations without
chemicals and without microsomes were used as negative
controls to assess background contaminants and the posVOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7537
TABLE 1. Percentages of Brominated Compounds Relative to the Dosing Concentration after Metabolism with Chicken, Rainbow
Trout, and Rat Microsomes Exposed to PBDEs, MeO-PBDEs, and OH-PBDEs (%)a
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exposed group (chemicals)
6-OH-BDE-47
4′-OH-BDE-49
6-OH-BDE-90
2-OH-BDE123
4′-OH-BDE103
24-DiBRP
246-TriBRP
245-TriBRP
2′-MeO-BDE-68
6-MeO-BDE-47
5-MeO-BDE-47
4′-MeO-BDE-49
5′-MeO-BDE-100
4′-MeO-BDE-103
4′-MeO-BDE-99
4′-MeO-BDE-101
BDE-28
BDE-49
BDE-47
BDE-66
BDE-100
BDE-119
BDE-99
BDE-85
BDE-154
BDE-153
BDE-183
BDE-99b
PBDEs mixb
6-MeO-BDE-47b
MeO-PBDEs mixb
6-OH-BDE-47b
OH-PBDEs mixb
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.24 ( 0.06
0.05 ( 0.01
N.D.
N.D.
N.D.
37.6 ( 8.6
N.D.
N.D.
N.D.-0.01
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
17.8 ( 1.9
N.D.-22.1
27.0 ( 7.7
N.D.-31.9
32.8 ( 13.3
0.58 ( 0.28
31.2 ( 10.6
25.4 ( 19.2
20.1 ( 6.9
28.7 ( 9.7
28.5 ( 16.2
6.2 ( 3.2
N.D.
N.D.
N.D.
N.D.
0.6 ( 0.5
N.D.
N.D.-0.1
N.D.
15.5 ( 5.3
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
4.8 ( 5.1
27.3 ( 4.8
N.D.-53.3
N.D.
48.1 ( 19.6
N.D.-1.0
N.D.-3.0
0.4 ( 0.3
17.8 ( 5.8
23.1 ( 10.6
36.9 ( 14.8
21.7 ( 8.6
3.0 ( 1.2
19.8 ( 7.4
18.2 ( 6.5
26.1 ( 9.2
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
49.4 ( 46.6
N.D.
N.D.
N.D.
N.D.
3.2 ( 3.4
N.D.-0.25
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
53.8 ( 47.9
196 ( 108
46.8 ( 22.7
89.8 ( 21.7
N.D.
11.0 ( 7.4
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
a
Values are means of three exposures, one with each of the three microsomal sources. N.D.: concentrations less than
the detection limit; concentrations with three times greater than the background were reported as detected. b BDE-99
contained BDE-99 with dosing concentrations of 1.5 × 103 ( 75 ng/mL; PBDEs mix contained BDE-28, BDE-49, BDE-47,
BDE-66, BDE-100, BDE-119, BDE-99, BDE-85, BDE-154, BDE-153, and BDE-183 with dosing concentrations of 1.2 × 102 ( 10
ng/mL for each congener; 6-MeO-BDE-47 contained 6-MeO-BDE-47 with dosing concentrations of 1.5 × 103 ( 75 ng/mL;
MeO-PBDE mix contained 2′-MeO-BDE-68, 6-MeO-BDE-47, 5-MeO-BDE-47, 4′-MeO-BDE-49, 5′-MeO-BDE-100, 4′-MeO-BDE103, 4′-MeO-BDE-99, and 4′-MeO-BDE-101 with dosing concentrations of 1.5 × 102 ( 10 ng/mL for each congener;
6-OH-BDE-47 contained 6-OH-BDE-47 with dosing concentrations of 1.5 × 103 ( 75 ng/mL; and OH-PBDE mix contained
OH-BDE-47, 4′-OH-BDE-49, 6-OH-BDE-90, and 2-OH-BDE123 with dosing concentrations of 1.5 × 102 ( 10 ng/mL for each
congener.
sibility of nonenzyme mediated changes in chemical structure. After the incubation, the samples were extracted
immediately for chemical analysis.
Quality Assurance and Quality Control (QA/QC). Concentrations of all congeners were quantified by the internal
standard isotope-dilution method using mean relative response factors determined from standard calibration runs.
All equipment rinses were carried out with acetone and
hexane to avoid sample contamination. The procedure
described above was validated by analyzing spiked beef liver
(matrix spike samples). The spiking concentrations were at
least three times the original basal concentrations in the
matrix medium. During the sample analysis, a laboratory
blank and a matrix spike were incorporated in the analytical
procedures for every batch of 15 samples. Recoveries for
spiked samples were 81-126%, 87-128%, 81-123%, and
65-126% for MeO-PBDEs, PBDEs, OH-PBDEs, and BRPs
(except DiBRPs) respectively. The recoveries of DiBRPs in
the spiked samples were slightly high (71-213%) possibly
due to matrix-induced ionization enhancement. The concentrations of DiBRPs in matrix spike samples corrected by
the surrogates were within the acceptable range. Concentrations of all target compounds corrected by surrogates in
matrix spike samples are shown in Table S3 in the SI.
Concentrations quantified in the spiked beef liver varied
within 20% of the spiked concentrations, showing the
accuracy and precision of the tissue analysis. PBDEs and
BRPs were quantified in sample extracts relative to 13C-PBDEs
7538
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 19, 2009
and 13C-BRPs, respectively. OH-PBDEs were quantified
relative to 2,3,4,6-13C-TeBRPs, and MeO-PBDEs were quantified relative to 13C-PBDEs with same number of bromines.
Recoveries of 13C-PBDEs and 13C-BRPs (except 2,4-13C-DiBRP:
70-300%) were in the range of 50-130% in all samples. The
method detection limits (MDL) were set to be the mean of
the concentration plus three times the standard deviation in
the blank samples, in which BDE-28, BDE-49, BDE-47, BDE66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153, 2,4-DiBRP,
and 2,4,6-TriBRP were detected. The MDLs for the other
compounds, which were not detected in blank samples, were
set to the instrumental minimum detectable amounts. The
detection limits were 0.4 pg/g ww for MeO-PBDEs; 0.2 pg/g
ww for PBDEs except for BDE-28 (0.7 pg/g ww), BDE-49 (0.6
pg/g ww), BDE-47 (10.1 pg/g ww), BDE-66 (0.6 pg/g ww),
BDE-100 (3.5 pg/g ww), BDE-99 (5.6 pg/g ww), BDE-85 (0.6
pg/g ww), BDE-154 (2.5 pg/g ww), BDE-153 (1.4 pg/g ww);
2.0 pg/g ww for 2′-OH-6′-Cl-BDE-7 and 6′-OH-BDE-17, and
4.0 pg/g ww for 3-OH-BDE-47, 5-OH-BDE-47, 2’OH-BDE68, 6-OH-BDE-47, 4′-OH-BDE-49, 2′-OH-6′-Cl-BDE-68, 6-OHBDE-90, 2-OH-BDE-123; 2.0 pg/g ww for DiBRPs except for
2,4-DiBRP (2.5 pg/g ww), 4.0 pg/g ww for TriBRPs except for
2,4,6-BRP (6.4 pg/g ww), 8 pg/g ww for TeBRPs, and 10 pg/g
ww for PeBRP. For those results less than the MDL, half of
the MDL was assigned to avoid missing values in statistical
analyses.
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FIGURE
1. Concentrations
of
ΣPBDEs,
ΣMeO-PBDEs,
ΣOH-PBDEs, and ΣBRPs in livers of tuna, albatross, and polar
bear collected from remote marine locations worldwide. Data
are presented in box-and-whisker plots; 50% of the cases have
values within the boxes, and the edges of the box mark the
25th and 75th percentiles. *: significantly greater than in the
other species.
Results and Discussion
Levels of PBDEs, MeO-PBDEs, OH-PBDEs, and BRPs in
Marine Organisms. Marine species at higher trophic concentrations have been reported to accumulate relatively great
concentrations of PBDEs and related compounds (32, 33).
Three top marine predator species (tuna, albatross, and polar
bear) collected from the Pacific, Atlantic, Indian, and Arctic
Oceans were selected for this study, and were analyzed for
PBDEs, MeO-PBDEs, OH-PBDEs, and BRPs. Besides the
generally analyzed PBDEs, MeO-PBDEs, and OH-PBDEs, 2′OH-6′-Cl-BDE-47 and 2′-OH-6′-Cl-BDE-68 were selected as
target compounds due to their potential steroidogenic effects
(34) and identification in previous investigations (6). BRPs
were included because of the previous detection of these
compounds in PBDE metabolism studies (26).
All four groups of brominated chemicals were found in
liver tissues of each marine species, including polar bears
from the Arctic Ocean (Figure 1). The greatest concentrations
of ΣOBRPs were found in livers of tuna (0.67 ( 0.32 ng/g wet
weight (ww)). This may be due to the fact that BRPs are the
key natural flavor components of marine fish (24), and/or
fish may have potential for metabolic conversion of OHPBDEs to BRPs (26) or accumulating the commercial flame
retardants (27). The greatest concentrations of ΣOH-PBDEs
were detected in livers of albatross (0.54 ( 0.38 ng/g, ww),
followed by tuna (0.025 ( 0.008 ng/g ww) and polar bear
(0.012 ( 0.009 ng/g ww). OH-PBDE concentrations in
albatross livers were much greater than those previously
reported in whales, gulls, and polar bears (7, 35, 36). This
could be due to differences among species, and/or the
analytical methods used. In this study, samples were extracted
using an accelerated solvent extraction method with two
solvents, and OH-PBDEs were then derivatized with methyl
chloroformate (MCF), which showed excellent reproducibility
and fewer background interferences compared to diazomethane (37).
Concentrations of ΣPBDEs in the marine organisms
studied were not related to those of ΣOH-PBDEs (Figure 1).
Concentrations of ΣOH-PBDEs in tuna and polar bear were
significantly lower than those in albatross (p < 0.05, one-way
analysis of variance (ANOVA)), although large variation in
the concentrations of ΣPBDEs was found in those animals
(0.19 ( 0.067 to 0.74 ( 0.23 ng/g ww); ΣOH-PBDEs concentrations were the greatest in albatrosses (p < 0.05), which
had moderate concentrations of ΣPBDEs (0.27 ( 0.30 ng/g
ww). In contrast, concentrations of ΣMeO-PBDEs were related
to those of ΣOH-PBDEs in polar bears and albatross, but not
in tuna. In tuna, relatively high concentrations of ΣMeOPBDEs coincided with elevated concentrations of ΣBRPs (p
< 0.05), possible metabolites of OH-PBDEs (26). A significant
correlation between precursors and metabolites is indicative
of metabolic transformation, but not definitive. No significant
relationships were found between concentrations of ΣPBDEs
and ΣOH-PBDEs, which suggests the existence of other
natural sources for OH-PBDEs. But significant correlations
were found between the concentrations of ΣMeO-PBDEs and
ΣOH-PBDEs, and more significant correlations were obtained
between the concentrations of ΣMeO-PBDEs and ΣOHPBDEs+ΣBRPs (Figure 2 and Table S1 in the SI). Since the
samples were collected in remote marine locations worldwide
between 1992 and 2002, the relationships between MeOPBDEs and OH-PBDEs were independent of time and
FIGURE 2. Relationships between concentrations of ΣOH-PBDEs, ΣOH-PBDEs + BRPs, and ΣMeO-PBDEs: (a) log (ΣOH-PBDEs) )
0.6632log(ΣMeO-PBDEs) + 0.2915, r2 ) 0.4065, p < 0.001; (b) log (ΣOH-PBDEs + ΣBRPs) ) 0.4339log(ΣMeO-PBDEs) + 1.521, r2 )
0.4819, p < 0.001.
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Publication Date (Web): August 21, 2009 | doi: 10.1021/es901357u
FIGURE 3. Patterns of relative concentrations of the major
congeners of PBDEs, MeO-PBDEs, and OH-PBDEs in the liver
tissues of tuna (T), albatross (A), and polar bear (PB) from
remote marine locations. Sum concentrations of each group of
chemicals are listed for each species at the bottom of the
figure (pg/g ww).
location, which indicates that MeO-PBDEs and OH-PBDEs
share a common source or metabolic pathway in marine
animals.
Profiles of PBDEs, MeO-PBDEs, and OH-PBDEs in
Marine Organisms. The profiles of the relative concentrations
of major congeners among the detected PBDEs, MeO-PBDEs,
and OH-PBDEs are shown in Figure 3. Consistent with the
previous studies, BDE-47 was the predominant compound
in all three marine predators of the 21 PBDE congeners,
followed by BDE-99, BDE 154, and BDE-153. But the patterns
of relative concentrations of PBDE and OH-PBDE congeners
varied among species (Figure 3). Of the 10 OH-PBDEs
analyzed, 6-OH-BDE-47, 4′-OH-BDE-49, and 2′-OH-BDE-68
were detected with detection frequency of 88%, 43, and 3%,
respectively. Similar profiles of predominant congeners were
reported in previous investigations (7, 35). Six MeO-PBDEs
(6-MeO-BDE-47, 2′-MeO-BDE-68, 5′-MeO-BDE-100, 5′-MeO-
BDE-99, 6-MeO-BDE-85, and 6-MeO-BDE-90) were found
in the samples, with the greatest concentrations and detection
frequencies were observed for 6-MeO-BDE-47, 2′ -MeO-BDE68, and 5′-MeO-BDE-100. It should be noted that the
structures of two major MeO-PBDE congeners (6-MeO-BDE47 and 2′-MeO-BDE-68) are similar to those of the two major
OH-PBDEs (6-OH-BDE-47 and 2′-OH-BDE-68). The variations in patterns among species were also similar for MeOPBDEs and OH-PBDEs. Similar profiles of OH-PBDEs and
MeO-PBDEs have been reported previously in whales,
glaucous gulls, and polar bears (7, 35). The similarity in
congener profiles further supports a direct relationship
between MeO-PBDEs and OH-PBDEs. Significant correlations were also found between concentrations of 6-OH-BDE47 and 6-MeO-BDE-47 (Figure S1 in the SI). Significant
correlations for compounds with similarity in structures
could suggest methylation of OH-PBDEs to MeO-PBDEs
(5, 21). Of the 16 BRP congeners, 2,4-DiBRP and 2,4,6-TriBRP
were the two major compounds in all samples, and 2,4,5TriBRP was only detected in polar bear with a detection
frequency of 80%. Similar profiles were also reported in ocean
fishes, and the “ocean-like” flavors in seafood have been
attributed to these compounds (24).
In vitro Metabolism of PBDEs, MeO-PBDEs, and OHPBDEs. To further elucidate the relationships among the
brominated substances analyzed, in vitro metabolism of
PBDEs, MeO-PBDEs, and OH-PBDEs by microsomal fractions
of rainbow trout, chicken, and rat were conducted (Table 1,
Figure 4, and Tables S4-S6 in the SI). Profiles of the analyzed
compounds were similar among the three exposed species.
Relatively low concentrations of 6-OH-BDE-47 were observed
in 6-MeO-BDE-47 exposed rat microsomes, and concentrations of 6-OH-BDE-47 in 6-OH-BDE-47 and OH-PBDE
mixture exposed rainbow trout microsomes were comparable
to the dosing concentrations (Tables S4-S6). This could
be due to the metabolic differences among species, since the
experimental conditions for the three species were similar
(e.g., protein concentration, dosing concentrations).
FIGURE 4. Proposed metabolic relationships among PBDEs, MeO-PBDEs, OH-PBDEs, BRPs, and PBDDs. Solid arrow: mechanism
demonstrated in this study; thin arrow: reported pathways; dashed arrow: minor pathway. (1) microsomes exposed to 6-MeO-BDE-47;
(2) microsomes exposed to MeO-PBDE mixtures; (3) microsomes exposed to OH-PBDE mixtures; (4) microsomes exposed to BDE-99
and PBDE mixtures, and ref by (9, 12, 13); (5) microsomes exposed to 6-OH-BDE-47 and OH-PBDE mixtures, and ref by (26); (6) ref by
(39); (7) ref by (39).
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Concentrations of OH-PBDEs were all less than the
method detection limit in the PBDE exposure groups, which
may be due to the relatively small concentrations compared
with other studies. Small proportions of OH-PBDEs have
been reported previously in in vitro and in vivo studies
(9, 12, 13), and a recent study using salmon microsomes
did not detect OH-PBDEs after exposure to BDE-99 (38).
In contrast, significant amounts of 6-OH-BDE-47 were
generated from 6-MeO-BDE-47, and more OH-PBDE
congeners were detected when additional MeO-PBDE
congeners were incubated with microsomes, even at lesser
concentrations (100 ppb, Figure 4). Based on radiocarbon
measurements, 6-MeO-BDE-47 has been reported to be a
natural product, and MeO-PBDEs were considered to be
metabolites of OH-PBDEs (5, 21). Our results are the first
to demonstrate the demethoxylation of MeO-PBDEs to
OH-PBDEs at environmentally relevant concentrations
(Figure 4). The biotransformation ratio for the conversion
of MeO-PBDEs to OH-PBDEs (about 10% of parent
material) was 100-1000 times greater than those between
PBDEs and OH-PBDEs reported elsewhere (<0.01-1%)
(9, 12, 13). Thus, a significant amount of the toxic OHPBDEs (2′-OH-BDE-68 and 6-OH-BDE-47) found in wildlife
and humans could be derived from naturally occurring
MeO-PBDEs. No MeO-PBDEs or PBDEs were detected
when OH-PBDEs were incubated with microsomes, which
indicates a lack of methylation of OH-PBDEs to MeOPBDEs, as has been suggested previously (5, 21). 2,4-DiBRP
was the major BRP congener after microsomal metabolism
of OH-PBDEs and MeO-PBDEs, and previous studies also
suggested that polybrominated dibenzo-p-dioxins (PBDDs)
could be formed through condensation of BRPs with OHPBDEs as intermediate products (39). Thus, BRPs, OHPBDEs, and PBDDs are related as shown in Figure 4. At the
end of the exposure, concentrations of 4′-OH-BDE-49 were
greater than the original exposure concentrations. This
suggested that 4′-OH-BDE-49 may be formed via the
metabolism of OH-PentaBDE congeners, which in themselves may be produced via demethoxylation of MeOPentaBDE congers (Figure 4). These results demonstrate
that MeO-PBDEs can be transformed in vitro to OH-PBDEs
(Figure 4), which is also consistent with the relationships
found in the livers of wild marine animals. Radiocarbon
content analysis is a direct tool to determine the origins
of compounds, and this technology has been used successfully in determining MeO-PBDEs origins (21). However,
concentrations of OH-PBDEs are much lower than those
of MeO-PBDEs in the environment, and it is hard to isolate
sufficient amounts of pure compounds from environmental
samples. In this study, measurements of PBDEs, OHPBDEs, and MeO-PBDEs in tissues of wildlife and controlled in vitro metabolism studies provide sufficient
evidence that demethoxylation of MeO-PBDEs contributes
to OH-PBDEs found in wildlife.
OH-PBDEs, which have been reported in higher trophic
level organisms including humans, are known to be toxic
(14-20). Research to date suggests that there are two sources
of OH-PBDEs in the environment: natural production and
anthropogenic formation via the metabolism of PBDEs
(9-13, 21). The results of our study demonstrate that the
primary source of OH-PBDEs in marine animals could be
from the demethylation of MeO-PBDEs, which have been
shown to be of natural origin (21). In addition, concentrations
of MeO-PBDEs in wild animals are generally greater (as much
as 10-fold) than those of PBDEs (21, 22). Some investigations
have shown that the human daily intake of MeO-PBDEs from
fish oil dietary supplements is 3-fold greater than that of
PBDEs (22), which suggests that humans may be at greater
risk of exposure to OH-PBDEs via metabolism of MeO-PBDEs.
Recent studies have reported the detection of several OH-
PBDE congeners in human blood samples (40, 41), and the
concentrations of OH-PBDEs were higher in people consuming large amounts of fish (40). Because MeO-PBDEs are
found at relatively high concentrations in marine organisms
(21, 22), future studies on human exposure to OH-PBDEs
should include the analysis of MeO-PBDEs. Since OH-PBDEs
can have relatively great toxic potency, we suggest that risk
assessments of PBDEs and related compounds should be
reevaluated based on our discovery of this additional pathway
of OH-PBDEs production in biota.
Acknowledgments
This research was supported by a Discovery Grant from the
National Science and Engineering Research Council of
Canada (Project 326415-07) and a grant from Western
Economic Diversification Canada (Projects 6578 and 6807).
J.P.G. was supported by the Canada Research Chair program
and an at large Chair Professorship at the Department of
Biology and Chemistry and Research Centre for Coastal
Pollution and Conservation, City University of Hong Kong.
We acknowledge the support of an instrumentation grant
from the Canada Foundation for Infrastructure. We thank
Thomas Evans, U.S. Fish and Wildlife Service, Anchorage,
Alaska, for providing polar bear liver tissues.
Supporting Information Available
This material is available free of charge via the Internet at
http://pubs.acs.org.
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ES901357U
1
Supporting Information for
2
Origin of Hydroxylated Brominated Diphenyl Ethers: Natural Compounds or
3
Man-made Flame Retardants?
4
Yi Wan1*, Steve Wiseman1, Hong Chang1, Xiaowei Zhang1, Paul D. Jones1, Markus Hecker1,2,
5
Kurunthachalam Kannan 3, Shinsuke Tanabe4, Jianying Hu5,
6
Michael H. W. Lam7, John P. Giesy1,6,7,8
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1
Dept. Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan,
Saskatoon, Saskatchewan S7N 5B3, Canada
2
ENTRIX, Inc., Saskatoon, Saskatchewan S7N 5B3, Canada
3
Wadsworth Center, New York, NY, State Department of Health and Department of
Environmental Health Sciences, School of Public Health, State University of New York,
Empire State Plaza, Albany, NY 12201-0509, USA
4
Center for Marine Environmental Studies, Ehime University, Matsuyama, Japan
5
College of Urban and Environmental Sciences, Peking University, Beijing, 100871 China
6
Department of Zoology and Center for Integrative Toxicology, Michigan State University,
East Lansing, MI, USA
7
Centre for Coastal Pollution and Conservation and Department of Biology and Chemistry,
City University of Hong Kong, Kowloon, Hong Kong, SAR China
8
State Key Laboratory of Marine Environmental Science, College of Oceanography and
Environmental Science, Xiamen University, Xiamen, P. R. China
*To whom correspondence should be addressed. E-mail: yi.wan@usask.ca
23
24
25
26
27
28
29
30
31
32
33
34
35
36
This file includes:
Chemicals
Microsomal Preparation
Instrument Condition
Statistical Analysis
Fig. S1
Table S1
Table S2
Table S3
Table S4
Table S5
Table S6
1
1
Chemicals.
2
Twenty-one PBDEs (BDE-7, BDE-15, BDE-30, BDE-17, BDE-28, BDE-49, BDE-71,
3
BDE-47, BDE-66, BDE-77, BDE-100, BDE-119, BDE-99, BDE-85, BDE-126, BDE-154,
4
BDE-153,
5
(6-MeO-BDE-17, 4-MeO-BDE-17, 2’ -MeO-BDE-68, 6-MeO-BDE-47, 5-MeO-BDE-47, 4’
6
-MeO-BDE-49, 5’-MeO-BDE-100, 4’-MeO-BDE-103, 5’-MeO-BDE-99, 4’-MeO-BDE-101,
7
6-MeO-BDE-90,
and
8
6’-OH-BDE-17,
6-OH-BDE-47,
9
4’-OH-BDE-49,
BDE-139,
BDE-140,
BDE-138,
6-MeO-BDE-85),
and
ten
OH-PBDEs
3-OH-BDE-47,
2’-OH-6’-Cl-BDE-68,
BDE-183),
twelve
(2’-OH-6’-Cl-BDE-7,
5-OH-BDE-47,
6-OH-BDE-90,
MeO-PBDEs
2’-OH-BDE-68,
2-OH-BDE-123)
and
sixteen
10
bromophenols (BRPs, including 2,6-DiBRP, 2,5-DiBRP, 2,4-DiBRP, 2,3-DiBRP, 3,5-DiBRP,
11
3,4-DiBRP,
12
3,4,5-TriBRP, 2,3,5,6-TeBRP, 2,3,4,6-TeBRP, 2,3,4,5-TeBRP, and 2,3,4,5,6-PeBRP) were
13
selected
14
2,3,4,6-13C-TeBRPs and 2,3,4,5,6-13C-PeBRPs) were used as surrogate standards for
15
OH-PBDEs and BRPs, and
16
13
17
for PBDEs and MeO-PBDEs.
18
2,4,6-TriBRP,
as
target
2,3,6-TriBRP,
compounds.
13
13
2,3,5-TriBRP,
C-BRPs
2,4,5-TriBRP,
(2,4-13C-DiBRP,
C-PBDEs (13C-BDE-28,
13
2,3,4-TriBRP,
2,4,6-13C-TriBRPs,
C-BDE-47,
13
C-BDE-100,
C-BDE-99, 13C-BDE-154, 13C-BDE-153 and 13C-BDE-183) were used as surrogate standard
PBDEs,
13
C-PBDEs, BRPs,
Wellington
Laboratories
13
C-BRPs and eight MeO-PBDEs standards were obtained
19
from
Inc.
(Guelph,
Ontario,
Canada).
3-OH-BDE-47,
20
5-OH-BDE-47 and 2’-OH-BDE-68 were obtained from AccuStandard (New Haven,
21
Connecticut, USA).
22
and the remaining seven OH-PBDEs were synthesized in the Department of Biology and
6-MeO-BDE-17, 4-MeO-BDE-17, 6-MeO-BDE-90, 6-MeO-BDE-85
2
1
Chemistry, City University of Hong Kong, and purities of all metabolites were >98% (1).
2
Dichloromethane (DCM), n-hexane, methyl tert-butyl ether (MTBE), acetonitrile and
3
methanol were pesticide residue grade obtained from OmniSolv (EM Science, Lawrence, KS,
4
USA).
5
size), pyridine (anhydrous, 99.8%), methyl chloroformate (MCF), hydrochloric acid (37%,
6
A.C.S. reagent), 2-propanol, and potassium hydroxide (KOH) were purchased from
7
Sigma-Aldrich (St. Louis, MO, USA).
8
was obtained from Molecular Probes (Eugene, OR,USA), sodium phosphate dibasic
9
(Na2HPO4), sodium phosphate monobasic (NaH2PO4) and potassium phosphate monobasic
10
(KH2PO4), resorufin, ethylenediaminetetraacetic acid (EDTA), and dithiothreitol (DTT) were
11
obtained from Sigma-Aldrich (St. Louis, MO, USA).
12
including NADPH, were obtained from Sigma-Aldrich and were reagent grade or better
13
unless stated otherwise.
Sodium sulfate, silica gel (60-100 mesh size), aluminum oxide (neutral, 150 mesh
For biochemical analyses, 7-ethoxyresorufin (7-ER)
All other biochemical reagents,
14
15
Microsomal Preparation.
16
Approximately 200 mg of tissue was homogenized in cold phosphate buffer (200 mL 0.1
17
M Na2HPO4 mixed with 800 mL 0.1 M KH2PO4 with pH adjusted to 7.4) and samples were
18
centrifuged for 15 min at 9000 g.
19
removed and then centrifuged at 100,000 g for 60 min. The resulting pellet was dissolved in
20
phosphate buffer and stored at -80 °C until analysis.
21
out at 4 °C and samples were kept on ice throughout the procedure.
22
concentrations were determined using the bichinchoninic acid (BCA) method using bovine
Following centrifugation the supernatant (S9 fraction) was
3
All centrifugation steps were carried
Sample protein
1
serum albumin (BSA) as a standard and according to the manufacturers protocol
2
(Sigma-Aldrich Corp., St Louis, MO). Prior to PBDE and MeO-PBDE exposure studies all
3
microsomal fractions were assayed for ethoxyresorufin O-deethylase (EROD) activity (31).
4
Briefly, resorufin and BSA standards were added to the first 12 wells of the plate.
5
wells did not contain microsomes and were used to establish resorufin and protein standard
6
curves.
7
concentration 5 µM) and sodium phosphate buffer to a final volume of 175 µL.
8
5 min incubation at 37 °C, the enzymatic reaction was started by adding 25 µL of NADPH
9
(1.2 mg/mL in sodium phosphate buffer) to each well.
These
All wells containing micosomes received 50 µL of 7-ER working solution (final
Following a
Exactly 10 min later, the reaction was
10
stopped with the addition of 150 µL of cold acetonitrile (0.15 mg/mL). Cells were incubated
11
for 10 min at room temperature, after which fluorescence values for resorufin were read on a
12
fluorescence plate reader (CytoFluor 2350, Millipore, Bedford, MA, USA). Resorufin was
13
read with a 530-nm excitation filter and a 630-nm emission filter.
14
15
Instrumental Conditions.
16
Identification and quantification of all target compounds was performed using a
17
Hewlett-Packard 5890 series II high-resolution gas chromatograph interfaced to a
18
Micromass® Autospec® high-resolution mass spectrometer (HRGC-HRMS) (Micromass®,
19
Beverly, MD).
20
capillary column for all target compounds (30 m length, 0.25 mm ID, 0.1 µm film thickness,
21
Agilent, Carlsbad, CA), helium was used as carrier gas.
22
ions monitored for PBDEs, MeO-PBDEs, OH-PBDEs and BRPs are shown in Table S2 in
Chromatographic separation was achieved on a DB-5MS fused silica
4
The GC temperature program and
1
Supporting Information.
The mass spectrometer was operated in a Selected Ion-Monitoring
2
(SIM) mode.
3
7,000.
4
The electron ionization energy was 37 eV and the ion current was 750 µA.
The resolution for all reference gas peaks in all time windows was more than
The injector temperature was held at 285 °C and the ion source was kept at 285 °C.
5
6
Statistical Analysis.
7
Correlations between the target compounds were examined by Pearson’s rank correlation test,
8
and when the p value was less than 0.05, the linear regression was regarded as significant.
9
Differences in concentrations of target compounds among species were compared using
10
one-way analysis of variance (ANOVA) (2).
11
variances.
12
equality of variances could not be assumed, Welch’s and Brown-Forsythe's robust tests were
13
used to perform one-way ANOVA analysis.
14
determine which means differed from one another.
15
Differences (HSD) was used where variances were presumed to be equal, and the
16
Games-Howell test was used where equality of variances could not be assumed (SPSS 11,
17
SPSS Inc., Chicago, IL).
18
USA).
19
20
21
22
23
24
25
26
Levene’s test was used to check the equality of
Where variances were equal, data were analyzed by the F test.
Where the
Multiple paired comparisons were used to
Tukey’s Honestly Significant
The software used was SPSS 11.0 (SPSS Inc., Chicago, IL,
Reference
1.
He, Y.H.; Murphy, M.B.; Yu, R.M.K.; Lam, M.H.W.; Hecker, M.; Giesy, J.P.; Wu, R.S.S.
5
1
2
3
4
5
6
7
2.
Effects of 20 PBDE metabolites on steroidogenesis in the H295R cell line. Toxicol. Lett.
2008, 176, 230-238.
Wan, Y.; Hu, J.Y.; An, W.; Zhang, Z.B.; An, L.H.; Hattori, T.; Itoh, M.; Masunaga, S.
Congener-specific tissue distribution and hepatic sequestration of PCDD/Fs in wild
herring gulls from Bohai Bay, North China: Comparison to coplanar PCBs. Environ. Sci.
Technol. 2006, 36, 1462-1468.
6
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
10000
Conc. of 6-OH-BDE-47 (pg/g ww)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1000
100
10
1
0.1
1
10
100
1000
10000
Conc. of 6-MeO-BDE-47 (pg/g ww)
Figure S1. Relationship between concentrations of 6-OH-BDE-47 and 6-MeO-BDE-47. log
(6-OH-BDE-47) = 0.5861log(6-MeO-BDE-47) + 0.5984, r2 = 0.5011, p<0.001
7
Table S1. Details and concentrations target compounds in organisms used in this study.
Species name
25
Pacific tuna
1999
North Pacific Ocean
M
-
23
106
4.8
-16.5
11.1
192
493
25.9
798
34
Pacific tuna
1999
North Pacific Ocean
M
-
30
115
7.8
-17.0
10.9
114
344
33.9
1000
49
Pacific tuna
1999
North Pacific Ocean
M
-
33
115
7.6
-17.2
11.5
153
548
44.0
726
54
Pacific tuna
1999
North Pacific Ocean
M
-
31
117
3.7
-17.3
10.8
326
669
20.3
255
58
Pacific tuna
1999
North Pacific Ocean
M
-
25
105
7.0
-16.3
9.3
171
761
24.3
1000
63
Pacific tuna
1999
North Pacific Ocean
M
-
36
124
1.9
-16.8
11.2
199
459
22.6
918
66
Pacific tuna
1999
North Pacific Ocean
M
-
19
101
2.0
-18.9
10.9
209
610
19.9
827
84
Pacific tuna
1999
North Pacific Ocean
M
-
31
115
2.6
-16.5
10.7
254
375
26.8
722
88
Pacific tuna
1999
North Pacific Ocean
M
-
41
130
3.3
-17.1
11.8
201
267
15.9
200
90
Pacific tuna
1999
North Pacific Ocean
M
-
23
104
9.0
-17.3
11.3
93.7
447
17.8
272
EB02470
Yellow-nosed
albatross
1995
Indian Ocean
M
-
2.95
83.9
14.7
-18.3
13.6
407 3900
1400
42.2
EB04184
Light-mantled
1995
sooty albatross
South Atlantic Ocean M
-
3.2
91.4
4.7
-22.7
12.3
EB04307
Grey-headed
albatross
1996
Indian Ocean
M
-
3.94
87.1
4.3
-20.5
12.0
EB04314
Grey-headed
albatross
1996
Indian Ocean
M
-
3.55
80.4
9.1
-18.3
13.7
EB04714 Shy albatross
1995
South Atlantic Ocean M
-
4.9
96.2
10.9
-16.9
EB04719 Shy albatross
1996
Indian Ocean
M
-
5.48
97.3
11.8
Black-browed
albatross
1992
Indian Ocean
M
-
5.1
88.9
EB05097 Black-browed
1994
Indian Ocean
M
-
4.85
89.6
EB05088
Date of
collection
Location
Age
Length Lipid
Sex (year) Wt (kg) (cm) (%)
δ13C (‰, δ15N (‰, ΣPBDEs ΣMeO-P ΣOH-PB ΣBRPs
V-PDB)
AIR)
BDEs
DEs
Sample
code
8
97.2
161
490
140
74.0
213
692
35.4
75.0
127
168
<DL
16.7
86.5
187
229
<DL
-17.4
15.0
115
709
445
<DL
10.4
-18.2
14.6
90.8
160
323
<DL
14.4
-18.1
15.4
431
224
259
<DL
albatross
EB05103
Black-browed
albatross
1995
Indian Ocean
F
-
3.45
81.5
7.4
-19.1
13.1
EB05118
Black-browed
albatross
1996
South Pacific Ocean
M
-
4.55
88.5
5.5
-18.3
13.5
EB05121
Black-browed
albatross
1996
Indian Ocean
M
-
4.75
88.2
8.8
-18.0
13.9
EB05122
Black-browed
albatross
1996
Indian Ocean
F
-
3.7
84.4
9.6
-17.7
14.3
EB05881
Yellow-nosed
albatross
1996
Indian Ocean
F
-
2.49
78.2
11.4
-18.5
11.6
EB05884
Yellow-nosed
albatross
1996
Indian Ocean
M
-
2.55
78.8
14.3
-18.2
14.8
EB05922
Grey-headed
albatross
1994
Indian Ocean
-
-
-
-
14.0
-19.5
12.4
980565LB Polar bear
1993-2002 Arctic Ocean
F
3
-
-
11.9
-18.2
990083LD Polar bear
1993-2002 Arctic Ocean
M
5
-
-
6.2
990112LB Polar bear
1993-2002 Arctic Ocean
M
5
-
-
990127LB Polar bear
1993-2002 Arctic Ocean
M
6
-
990592LA Polar bear
1993-2002 Arctic Ocean
F
11
990594LB Polar bear
1993-2002 Arctic Ocean
M
990600LB Polar bear
1993-2002 Arctic Ocean
990652LC Polar bear
140
254
768
<DL
229 4500
665
32.9
424 1500
312
<DL
167 1600
219
141
1200 1500
505
28.8
396
342
1300
48.4
92.3
472
311
29.6
21.0
1000
31.8
9.9
621
-18.8
20.3
1000
11.3
<DL
129
4.7
-17.9
20.2
743
39.5
32.8
45.7
-
5.7
-17.8
20.3
848
56.0
<DL
64.3
-
-
8.4
-17.6
20.7
820
9.9
<DL
335
19
-
-
6.6
-16.2
20.5
288
14.8
8.9
123
M
5
-
-
7.6
-18.7
20.6
818
25.5
<DL
12.6
1993-2002 Arctic Ocean
M
3
-
-
8.6
-17.1
20.1
427
5.7
12.9
6.5
990658LB Polar bear
1993-2002 Arctic Ocean
M
10
-
-
7.0
-17.6
20.0
625
22.8
20.7
134
990671LC Polar bear
1993-2002 Arctic Ocean
F
14
-
-
4.8
-19.2
21.2
755
16.6
8.9
76.7
The scientific names were Katsuwonus pelamis, Thalassarche chlororhynchos, Phoebetria palpebrata, Thalassarche chrysostoma, Thalassarche cauta,
Thalassarche melanophrys and Ursus maritimus for pacific tuna, yellow-nosed albatross, light-mantled sooty albatross, grey-headed albatross, shy albatross,
9
black-browed albatross and polar bear, respectively.
10
Table S2. GC temperature program and ions monitored for HRMS of PBDEs, MeO-PBDEs, MCFO-PBDEs and MCFO-BRPs
Function and
bromine level
m/z
m/z type
Substance
Function and
bromine level
m/z
m/z type
Substance
PBDEs
Temperature program: 110 ℃ (10 min) - 250 ℃ @ 25 ℃/min, 250 ℃ - 260 ℃ @ 1.5 ℃/min, 260 ℃ - 323 ℃ (15 min) @ 25 ℃/min
Fn-1; Br-2: 14.54-16.00 min
Fn-2; Br-3: 16.00-18.00 min
325.8941
M
DiBDEs
403.8047
M
TriBDEs
327.8923
M+2
DiBDEs
405.8028
M+2
TriBDEs
329.8905
M+4
DiBDEs
407.8010
M+4
TriBDEs
254.9856
-
PFK Lock Mass
430.9792
-
PFK Lock Mass
254.9856
-
Lock Mass Check
430.9792
-
Lock Mass Check
M
13
415.8452
M
13
M+2
13
M+2
13
M+4
13
M+4
13
337.9345
339.9325
341.9305
C-DiBDEs
C-DiBDEs
C-DiBDEs
Fn-3; Br-4: 18.00-21.00 min
417.8431
419.8411
C-TriBDEs
C-TriBDEs
C-TriBDEs
Fn-4; Br-5: 21:00-23.48 min
483.7133
M+2
TeBDEs
561.6237
M+2
PeBDEs
485.7114
M+4
TeBDEs
563.6219
M+4
PeBDEs
487.7096
M+6
TeBDEs
565.6201
M+6
PeBDEs
11
492.9697
-
PFK Lock Mass
566.9665
-
PFK Lock Mass
492.9697
-
Lock Mass Check
566.9665
-
Lock Mass Check
M+2
13
573.6639
M+2
13
M+4
13
M+4
13
M+6
13
M+6
13
495.7536
497.7517
499.7497
C-TeBDEs
C-TeBDEs
575.6622
C-TeBDEs
Fn-5; Br-6: 23.48-25.42 min
577.6602
C-PeBDEs
C-PeBDEs
C-PeBDEs
Fn-6; Br-7: 25.42-39.00 min
481.6977
M-2Br+2
HxBDEs
561.6060
M-2Br+2
HpBDEs
483.6958
M-2Br+4
HxBDEs
563.6442
M-2Br+4
HpBDEs
485.6936
M-2Br+6
HxBDEs
554.9665
-
504.9697
-
PFK Lock Mass
554.9665
-
504.9697
493.7379
495.7360
497.7337
-
573.6462
Lock Mass Check
M-2Br+2
13
M-2Br+4
13
M-2Br+6
13
C-HxBDEs
575.6442
PFK Lock Mass
Lock Mass Check
M-2Br+2
13
M-2Br+4
13
M+2
13
M+4
13
C-HpBDEs
C-HpBDEs
C-HxBDEs
C-HxBDEs
MeO-PBDEs
Temperature program: 150 ℃ (2 min) - 245 ℃ (2 min) @ 2 ℃/min, 245 ℃ - 320 ℃ (2 min) @ 30 ℃/min
Fn-1; Br-3: 24.00-35.18 min
417.8431
419.8411
Fn-2; Br-4: 35.18-48.00 min
M+2
13
M+4
13
C-TriBDEs
C-TriBDEs
12
495.7536
497.7517
C-TeBDEs
C-TeBDEs
454.9728
-
PFK Lock Mass
513.7237
M+2
MeO-TeBDEs
454.9728
-
Lock Mass Check
515.7217
M+4
MeO-TeBDEs
435.8136
M+2
MeO-TriBDEs
517.7197
M+6
MeO-TeBDEs
437.8116
M+4
MeO-TriBDEs
542.9665
-
PFK Lock Mass
542.9665
-
Lock Mass Check
575.6622
M+4
13
M+6
13
M+4
13
13
577.6602
Fn-3; Br-5: 24.00-35.18 min
593.6323
M+4
M+6
642.9601
-
642.9601
-
655.5703
C-PeBDEs
Fn-4; Br-6: 48.00-53.54 min
595.6303
653.5723
C-PeBDEs
MeO-PeBDEs
653.5723
C-HxBDEs
MeO-PeBDEs
655.5703
M+6
C-HxBDEs
PFK Lock Mass
642.9601
-
PFK Lock Mass
Lock Mass Check
Lock Mass Check
642.9601
-
M+4
13
673.5408
M+6
MeO-HxBDEs
M+6
13
675.5338
M+8
MeO-HxBDEs
C-HxBDEs
C-HxBDEs
MCFO-PBDEs
Temperature program: 150 ℃ (2 min) - 320 ℃ (2 min) @ 10 ℃/min
Fn-1; Br-2: 12.00-13.13 min
429.7155
431.7135
Fn-2; Br-3: 13.13-14.00 min
M-CO2+4
13
479.8034
M+2
MCFO-TriBDEs
M-CO2+6
13
481.8014
M+4
MCFO-TriBDEs
C-MCFO-TeBRPs
C-MCFO-TeBRPs
13
430.7929
-
PFK Lock Mass
483.7994
M+6
MCFO-TriBDEs
430.7929
-
Lock Mass Check
480.9697
-
PFK Lock Mass
435.8544
M+2
MCFO-chloroDiBDEs
480.9697
-
Lock Mass Check
437.8524
M+4
MCFO-chloroDiBDEs
439.8504
M+6
MCFO-chloroDiBDEs
Fn-3; Br-4: 14.00-17.00 min
Fn-4; Br-5: 17.00-21.00 min
559.7115
M+4
MCFO-TeBDEs
637.6221
M+4
MCFO-PeBDEs
561.7095
M+6
MCFO-TeBDEs
639.6201
M+6
MCFO-PeBDEs
563.7075
M+8
MCFO-TeBDEs
641.6181
M+8
MCFO-PeBDEs
554.9665
-
PFK Lock Mass
642.9601
-
PFK Lock Mass
554.9665
-
Lock Mass Check
642.9601
-
Lock Mass Check
593.6726
M+4
MCFO-chloroTeBDEs
595.6706
M+6
MCFO-chloroTeBDEs
597.6686
M+8
MCFO-chloroTeBDEs
MCFO-BRPs
Temperature program: 100 ℃ (5 min) - 180 ℃ @ 3 ℃/min, 180 ℃ - 300 ℃ (2 min) @ 25 ℃/min
Fn-1; Br-2: 17.00-24.00 min
Fn-2; Br-3: 24.00-33.00 min
248.8550
M-CO2CH3
MCFO-DiBRPs
328.7635 M-CO2CH3 MCFO-TriBRPs
250.8530
M-CO2CH3+2
MCFO-DiBRPs
330.7615 M-CO2CH3+2 MCFO-TriBRPs
14
263.8785
M-CO2
MCFO-DiBRPs
330.9792
-
PFK Lock Mass
265.8765
M-CO2+2
MCFO-DiBRPs
330.9792
-
Lock Mass Check
M-CO2
13
343.7870
M-CO2+2
MCFO-TriBRPs
271.8966
M-CO2+2
13
345.7850
M-CO2+4
MCFO-TriBRPs
280.9824
-
PFK Lock Mass
349.8071
M-CO2+2
13C-MCFO-TriBRPs
280.9824
-
Lock Mass Check
351.8051
M-CO2+4
13C-MCFO-TriBRPs
269.8986
C-MCFO-DiBRPs
C-MCFO-DiBRPs
Fn-3; Br-4: 33.00-36.00 min
Fn-4; Br-5: 36.00-38.30 min
408.6719
M-CO2CH3+4
MCFO-TeBRPs
486.5824 M-CO2CH3+4 MCFO-PeBRPs
410.6699
M-CO2CH3+6
MCFO-TeBRPs
488.5804 M-CO2CH3+6 MCFO-PeBRPs
423.6954
M-CO2+4
MCFO-TeBRPs
492.9697
-
PFK Lock Mass
425.6934
M-CO2+6
MCFO-TeBRPs
492.9697
-
Lock Mass Check
M-CO2+4
13
503.6039
M-CO2+6
MCFO-PeBRPs
431.7135
M-CO2+6
13
505.6019
M-CO2+8
MCFO-PeBRPs
430.9729
-
PFK Lock Mass
509.6240
M-CO2+6
13
M-CO2+8
13
429.7155
430.9729
-
C-MCFO-TeBRPs
C-MCFO-TeBRPs
Lock Mass Check
15
511.6220
C-MCFO-PeBRPs
C-MCFO-PeBRPs
Table S3. Concentrations of target compounds in matrix spike samples (ng/ml).
Compound Conc.
Compound
PBDEs
Conc.
Compound
MeO-PBDEs
2
Conc.
Compound
OH-PBDEs
BRPs
BDE-28
1.1×10 ±6.0
6-MeO-BDE-17
0.90×10 ±4.2 2’-OH-6’-Cl-BDE-7
1.0×10 ±6.5
2,6-DiRP
0.81×102±11
BDE-47
1.1×102±4.9
4-MeO-BDE-17
0.89×102±6.4 6’-OH-BDE-17
1.1×102±0.8
2,5-DiRP
1.1×102±6.2
BDE-66
1.1×102±11
2’-MeO-BDE-68
0.97×102±5.4 6-OH-BDE-47
1.1×102±11
2,4-DiRP
1.1×102±4.1
BDE-100
1.1×102±3.4
6-MeO-BDE-47
0.96×102±3.9 4’-OH-BDE-49
1.2×102±8.3
3,5-DiRP
1.1×102±3.0
BDE-99
1.1×102±1.3
5-MeO-BDE-47
0.85×102±7.0 2’-OH-6’-Cl-BDE-68 0.80×102±3.2 2,3-DiRP
1.1×102±7.3
BDE-85
1.0×102±12
4’-MeO-BDE-49
0.90×102±7.5 6-OH-BDE-90
0.80×102±3.7 3,4-DiRP
1.1×102±5.9
BDE-154
0.95×102±12 5’-MeO-BDE-100 0.89×102±6.3 2-OH-BDE123
1.0×102±1.6
2,4,6-TriRP
1.1×102±0.8
BDE-153
1.1×102±2.4
6-MeO-BDE-90
0.90×102±5.0
2,3,6-TriRP
1.0×102±5.3
BDE-183
1.0×102±4.4
4’-MeO-BDE-103 0.93×102±1.6
2,3,5-TriRP
0.99×102±3.0
0.98×102±5.4
2,4,5-TriRP
1.1×102±2.9
2,3,4-TriRP
1.1×102±4.5
3,4,5-TriRP
1.2×102±3.5
2,3,5,6-TeRP
1.1×102±6.1
2,3,4,6-TeRP
1.1×102±5.9
2,3,4,5-TeRP
0.81×102±3.4
4’-MeO-BDE-99
2
Conc.
4’-MeO-BDE-101 1.1×102±10
6-MeO-BDE-85
0.90×102±2.6
2
2,3,4,5,6-PeRP 1.1×102±6.3
Approximately 10 g of beef liver was used for matrix spike samples. The spiking levels of all target compounds were 100 ng/ml. Recoveries of surrogates were
84.6-116% and 75.4-124.1% for 13C-BRPs (except 2,4-13C-DiBRP) and 13C-PBDEs, respectively.
16
Table S4. Concentrations of brominated compounds after metabolism with chicken microsomes exposed to PBDEs, MeO-PBDEs and
OH-PBDEs (ng/ml).
Dosing concentration
6-OH-BDE-47
4’-OH-BDE-49
6-OH-BDE-90
2-OH-BDE123
4'-OH-BDE103
24-DiBRP
246-TriBRP
245-TriBRP
2’-MeO-BDE-68
6-MeO-BDE-47
5-MeO-BDE-47
4’-MeO-BDE-49
5’-MeO-BDE-100
4’-MeO-BDE-103
4’-MeO-BDE-99
4’-MeO-BDE-101
BDE-28
BDE-49
BDE-47
BDE-66
BDE-100
BDE-119
BDE-99
BDE-85
BDE-154
BDE-153
BDE-183
BDE-99
1.5×103±75
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
PBDEs mix
1.2×102±10
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
3.0
0.7
<DL
<DL
<DL
6.2×102
<DL
<DL
<DL
<DL
24
<DL
42
38
58
1.1
52
53
34
48
56
Exposed group (Chemicals)
6-MeO-BDE-47 MeO-PBDEs mix 6-OH-BDE-47
1.5×103±75
1.5×102±15
1.5×103±75
2
1.3×10
16
60
<DL
39
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
40
<DL
17
1.6
22
<DL
<DL
<DL
1.6
0.3
<DL
<DL
17
<DL
1.7×102
19
<DL
<DL
32
<DL
<DL
19
<DL
<DL
3.2
<DL
<DL
18
<DL
<DL
16
<DL
<DL
24
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL: less than detection limit
17
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
OH-PBDEs mix
1.5×102±15
6.4
1.5×102
33
1.4×102
<DL
27
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
Table S5. Concentrations of brominated compounds after metabolism with rainbow trout microsomes exposed to PBDEs, MeO-PBDEs and
OH-PBDEs (ng/ml).
Dosing concentration
6-OH-BDE-47
4’-OH-BDE-49
6-OH-BDE-90
2-OH-BDE123
4'-OH-BDE103
24-DiBRP
246-TriBRP
245-TriBRP
2’-MeO-BDE-68
6-MeO-BDE-47
5-MeO-BDE-47
4’-MeO-BDE-49
5’-MeO-BDE-100
4’-MeO-BDE-103
4’-MeO-BDE-99
4’-MeO-BDE-101
BDE-28
BDE-49
BDE-47
BDE-66
BDE-100
BDE-119
BDE-99
BDE-85
BDE-154
BDE-153
BDE-183
BDE-99
1.5×103±75
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
PBDEs mix
1.2×102±10
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
4.6
0.7
<DL
0.1
<DL
4.2×102
<DL
<DL
0.2
<DL
19
<DL
32
34
32
0.6
33
7.5
20
27
25
Exposed group (Chemicals)
6-MeO-BDE-47 MeO-PBDEs mix 6-OH-BDE-47
1.5×103±75
1.5×102±15
1.5×103±75
2
1.1×10
3.0
1.5×103
<DL
35
<DL
<DL
80
<DL
<DL
<DL
<DL
<DL
79
<DL
5.2
<DL
16
<DL
3.3
<DL
0.7
0.3
<DL
<DL
31
<DL
3.2×102
50
<DL
<DL
76
<DL
<DL
45
<DL
<DL
3.8
<DL
<DL
40
<DL
<DL
34
<DL
<DL
52
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL: less than detection limit
18
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
OH-PBDEs mix
1.5×102±15
1.5×102
4.7×102
1.0×102
1.0×102
<DL
5.2
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
Table S6. Concentrations of brominated compounds after metabolism with rat microsomes exposed to PBDEs, MeO-PBDEs and OH-PBDEs
(ng/ml).
Dosing concentration
6-OH-BDE-47
4’-OH-BDE-49
6-OH-BDE-90
2-OH-BDE123
4'-OH-BDE103
24-DiBRP
246-TriBRP
245-TriBRP
2’-MeO-BDE-68
6-MeO-BDE-47
5-MeO-BDE-47
4’-MeO-BDE-49
5’-MeO-BDE-100
4’-MeO-BDE-103
4’-MeO-BDE-99
4’-MeO-BDE-101
BDE-28
BDE-49
BDE-47
BDE-66
BDE-100
BDE-119
BDE-99
BDE-85
BDE-154
BDE-153
BDE-183
BDE-99
1.5×103±75
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
PBDEs mix
1.2×102±10
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
3.4
0.8
<DL
<DL
<DL
6.5×102
<DL
<DL
<DL
<DL
21
27
23
<DL
29
0.4
28
30
19
29
21
Exposed group (Chemicals)
6-MeO-BDE-47 MeO-PBDEs mix 6-OH-BDE-47
1.5×103±75
1.5×102±15
1.5×103±75
40
2.7
7.1×102
<DL
49
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
98
<DL
4.5
<DL
1.1×102
<DL
4.4
3.8
<DL
1.2
<DL
<DL
32
<DL
2.1×102
34
<DL
<DL
57
<DL
<DL
34
<DL
<DL
6.6
<DL
<DL
30
<DL
<DL
32
<DL
<DL
41
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL: less than detection limit
19
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
OH-PBDEs mix
1.5×102±15
86
2.6×102
77
1.6×102
<DL
17
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL
<DL