Environ. Sci. Technol. 2010, 44, 6068–6073
Contribution of Synthetic and
Naturally Occurring Organobromine
Compounds to Bromine Mass in
Marine Organisms
YI WAN,† PAUL D. JONES,‡
STEVE WISEMAN,† HONG CHANG,†
DAVE CHORNEY,§
KURUNTHACHALAM KANNAN,|
KUN ZHANG,⊥ JIAN-YING HU,
JONG SEONG KHIM,#
SHINSUKE TANABE,∇
MICHAEL H. W. LAM,O AND
J O H N P . G I E S Y * ,†,O,[,¶,+
Toxicology Centre, University of Saskatchewan, Saskatoon,
Saskatchewan S7N 5B3, Canada, School of Environment and
Sustainability and Toxicology Centre, University of
Saskatchewan, Saskatoon, Saskatchewan, S7N 5C8B3, Canada,
Radiochemistry & SLOWPOKE Reactor, SRC Analytical
Laboratories, Saskatoon, Saskatchewan, 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, Laboratory for Earth Surface Processes,
College of Urban and Environmental Sciences, Peking University,
Beijing 100871, China, Division of Environmental Science and
Ecological Engineering, Korea University, Seoul 136-713, Korea,
Center for Marine Environmental Studies, Ehime University,
Matsuyama, Japan, Department of Biomedical Veterinary
Sciences, University of Saskatchewan, Saskatoon, Saskatchewan
S7N 5B3, Canada, Department of Biology and Chemistry, City
University of Hong Kong, Kowloon, Hong Kong, SAR China,
School of Biological Sciences, University of Hong Kong, Hong
Kong, SAR, PR China, and Department of Zoology and Center
for Integrative Toxicology, Michigan State University,
East Lansing, Michigan
Received March 22, 2010. Revised manuscript received
June 17, 2010. Accepted July 8, 2010.
An extraction, separation, and purification method was
developed for the identification and quantification of total
* Corresponding author address: Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N
5B3, Canada; tel (direct): 306-966-2096; tel (secretary): 306-966-4680; fax:
306-966-4796; mobile: 517-614-6123; e-mail: jgiesy@aol.com.
†
Toxicology Centre, University of Saskatchewan.
‡
School of Environment and Sustainability and Toxicology Centre,
University of Saskatchewan.
§
Radiochemistry & SLOWPOKE Reactor, SRC Analytical
Laboratories.
|
Wadsworth Center, New York State Department of Health and
Department of Environmental Health Sciences.
⊥
Laboratory for Earth Surface Processes, College of Urban and
Environmental Sciences, Peking University.
#
Division of Environmental Science and Ecological Engineering,
Korea University.
∇
Center for Marine Environmental Studies, Ehime University.
O
Department of Biomedical Veterinary Sciences University of
Saskatchewan.
[
Department of Biology and Chemistry, City University of Hong
Kong.
¶
School of Biological Sciences, University of Hong Kong.
+
Department of Zoology and Center for Integrative Toxicology,
Michigan State University.
6068
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 16, 2010
bromine (TBr), extractable organobromine (EOBr), and five
classes of identified EOBrs. Instrumental neutron activation
analysis (INAA) was utilized to quantify EOBr and TBr. The
method was then applied to liver samples of tuna, albatross, and
polar bear collected from remote marine locations. Polybrominated biphenyls (PBBs), polybrominated diphenyl ethers
(PBDEs), bromophenols (BRPs), hydroxylated (OH-) and
methoxylated (MeO-) PBDEs were analyzed as identified
EOBr. The majority of the bromine in these marine organisms
was nonextractable or inorganic, with EOBr accounting for
10-28% of the TBr. Of the identified EOBr, in tuna and albatross,
naturally occurring compounds, including MeO-PBDEs, OHPBDEs, and BPRs, were prevalent. However, the identifiable
EOBr in polar bears consisted primarily of synthetic compounds,
including PBDEs and PBBs. Overall, 0.08-0.11% and
0.008-0.012% of EOBr and TBr, respectively, were identified.
The proportion of EOBr that was identified in marine organisms
was relatively small compared to the proportions for
organofluorine and organochlorine compounds. This could be
related to the great diversity of naturally occurring organobromine compounds in the environment. Naturally occurring
brominated fatty acids were estimated to be the predominant
compounds in the EOBr fraction.
Introduction
Due to their persistence, bioaccumulation, and toxicity,
organohalogen compounds are of concern as contaminants
in aquatic and terrestrial ecosystems. Some well-known
synthetic organohalogens such as perfluorooctanesulfonate
(PFOS), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) have become ubiquitous
environmental pollutants (1-3). In addition to the known
synthetic organohalogens there are a number of unidentified
organohalogens in the environment (4, 5). Identification and
quantification of these novel organohalogen compounds is
essential for the assessment of potential risks to exposed
organisms.
Synoptic quantification of individual organohalogen
compounds along with quantification of total halogens is an
effective method to estimate the mass balance of identified
and unidentified organohalogen compounds in the environment. Several studies have examined the contributions of
known organohalogens to extractable organohalogens and
total organohalogens in abiotic and biological matrices (4-8).
Mass balance studies of chlorine in samples collected near
a former chloralkali facility indicated that the identified
organochlorines accounted for 48% and 1-35% of the
extractable organic chlorine (EOCl) in sediment and biota,
respectively (4). Methods for the mass balance analysis of
fluorine have been developed and applied to environmental
samples (5, 8). In these studies, most of the fluorine (>50%)
was in the nonextractable fraction and the identified perfluorinated compounds (PFCs) contributed 30-85% of
extractable organic fluorine (EOF) in human blood and
wildlife tissues (5, 8).
Brominated flame retardants (BFRs) have recently
emerged as contaminants of concern. Among BFRs, PBDEs
and polybrominated biphenyls (PBBs) are of primary interest
due to their high production volume, widespread use, and
ubiquitous occurrence in the environment (9, 10). The
occurrence of hydroxylated (OH-) and methoxylated (MeO-)
PBDEs has been investigated in aquatic ecosystems (11), and
concentrations of MeO-PBDEs were greater than those of
10.1021/es100914r
 2010 American Chemical Society
Published on Web 07/22/2010
TABLE 1. Concentrations of Bromine in Five Groups of Identified Organic Brominated Compounds, Identified EOBr, EOBr, and TBr
Extracted from Liver Samples Collected from Species Inhabiting Remote Marine Locationsa
species
name
water (%) lipid (%)
67 ( 3.7
(58-72)
albatross 60 ( 2.2
(55-64)
polar bear 60 ( 5.2
(54-71)
tuna
a
5.0 ( 2.7
(1.9-9.0)
10 ( 3.5
(4.3-15)
7.1 ( 2.2
(4.7-12)
MeO-PBDEs
OH-PBDEs
BRPs
PBDEs
PBBs
identified
EOBr
EOBr
TBr
0.31 ( 0.10
(0.17-0.47)
0.65 ( 0.85
(0.08-2.8)
0.014 ( 0.01
(0.003-0.03)
0.014 ( 0.005
(0.008-0.03)
0.34 ( 0.24
(0.11-0.90)
0.004 ( 0.005
(0.001-0.02)
0.45 ( 0.21
(0.14-0.67)
0.021 ( 0.033
(N.D.-0.10)
0.11 ( 0.12
(N.D.-0.39)
0.13 ( 0.05
(0.063-0.22)
0.19 ( 0.21
(0.05-0.85)
0.49 ( 0.16
(0.19-0.68)
0.003 ( 0.006
(N.D.-0.014)
0.049 ( 0.057
(N.D.-0.21)
0.34 ( 0.29
(0.064-1.0)
0.9 ( 0.3
(0.5-1.3)
1.3 ( 1.1
(0.2-3.8)
1.0 ( 0.5
(0.5-1.9)
1100 ( 540
(600-2000)
1700 ( 950
(810-3500)
2800 ( 2300
(310-6100)
10700 ( 3400
(8300-17200)
11300 ( 2600
(7000-14400)
12400 ( 6100
(6200-21800)
ng/g ww, mean ( SD (range in parentheses). N.D. Not detected.
PBDEs in some samples (12). Bromophenols (BRPs), which
are structurally similar to PBDEs, are key natural flavor
components of some marine fish. However, synthetic BRPs
have also been produced and widely used as flame retardants
(2,4,6-triBRP) with a worldwide production of 9500 t in 2001
(13).
Extractable organic bromine (EOBr) has been investigated
in a variety of environmental samples (4, 6, 14-16). However,
few studies have determined the proportion of EOBr that
could be identified, and the contribution of natural and
synthetic compounds to EOBr concentrations in environmental samples is unknown. In this study, the relative
contribution of natural and synthetic organobrominated
compounds to EOBr and total bromine (TBr) was determined.
Both EOBr and TBr were determined by instrumental neutron
activation analysis (INAA) of either crude samples or organic
solvent extracts of samples. PBBs, PBDEs, and related
brominated compounds were analyzed as identified EOBr
(9, 11, 17, 18). An extraction and cleanup method for TBr,
EOBr, and five classes of identified EOBr was developed. The
method was applied to determine concentrations of EOBr
and TBr as well as the concentrations of individual congeners
of PBBs, PBDEs, OH-PBDEs, MeO-PBDEs, and BRPs in livers
of tuna (Katsuwonus pelamis), five species of albatrosses
(Thalassarche chlororhynchos, Phoebetria palpebrata, Thalassarche chrysostoma, Thalassarche cauta, and Thalassarche
melanophrys) and polar bear (Ursus maritimus) collected
from remote marine locations. Absolute and relative contributions of identified EOBr to EOBr and TBr were determined, and the predominant forms of brominated compounds in the marine organisms are suggested.
Materials and Methods
Tissue Collection. Livers from fifteen albatross, ten tuna,
and ten polar bear were used for quantification of TBr, total
EOBr, and identified EOBr. Albatross were collected from
the Indian and South Atlantic Oceans, polar bear were
collected from Northern and Western Alaska, and tuna were
collected from the North Pacific Ocean in 1992-2002 (Table
1) (11). All samples were stored at -20 °C from the time of
collection until analysis.
Sample Preparation. An efficient fractionation method,
requiring a single extraction of 5 g ww of liver tissue that
allows for the simultaneous analysis of TBr, EOBr, and five
groups of identified EOBr was developed (Figure 1). The
identified EOBr included 10 PBBs, 21 PBDEs, 12 MeO-PBDEs,
10 OH-PBDEs, and 16 BRPs. Details of chemicals and
standards are provided in Supporting Information. To
estimate the concentrations of organobromines in the
environment, EOBr has been quantified in various matrixes,
such as sediment, atmosphere, and aquatic biota (4, 6, 15).
The current study is the first to compare TBr, EOBr, and
identified EOBr in tissues (Figure 1) (4). Due to the limited
sample mass available for the analysis of multiple target
compounds, direct quantifications of TBr in tissues were not
attempted. In contrast to the method used to quantify total
FIGURE 1. Flow diagram for the procedure used to fractionate
total bromine, extractable organic bromine, and known extractable organic bromines in liver tissue samples.
chlorine (TCl) and total fluorine (TF), TBr was quantified by
summing EOBr and nonextractable bromine (NEBr), and the
concentrations of NEBr were determined by analyzing the
residues from solvent extracted samples and normalized to
lipid and water contents. The calculation is based on an
assumption that negligible amounts of bromine (Br) were
lost during extraction.
Samples (approximately 5 g wet weight (ww)) were
homogenized and freeze-dried. Water content was measured
gravimetrically. Sample extraction was conducted using an
accelerated solvent extractor (Dionex ASE-200, Sunnyvale,
CA). Two kinds of solvents were used for the extraction: (1)
n-hexane/dichloromethane (DCM) (1:1) at 100 °C and 1500
psi, and (2) n-hexane/methyl tert-butyl ether (MTBE) (1:1)
at 60 °C and 1000 psi. Two extraction cycles (10 min each)
were performed for each solvent per sample (about 50 mL
for each solvent), and both extraction fractions were combined. The residual materials after solvent extraction were
air-dried and used for quantification of NEBr. The solvent
extract was reduced to 10 mL by rotary evaporation and 2
mL was used for the determination of EOBr.
Lipid content of each extract was determined gravimetrically by evaporating the remaining extract to constant weight.
Extracts were then spiked with a mixture of 13C-labeled PBB,
PBDE, and BRP surrogates for analysis of PBBs, PBDEs, MeOPBDEs, OH-PBDEs, and BRPs. Thus, the spiking solutions
did not influence quantification of Br EOBr, and the details
of the analysis of PBDEs, MeO-PBDEs, OH-PBDEs, and BRPs
have been reported previously (11). The method was modified
to accommodate simultaneous identification and quantification of PBBs (Figure 1). The neutral and phenolic fractions
in extracts were separated with 0.5 M potassium hydroxide
(KOH) in 50% ethanol. The methods for purification of PBDEs,
VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6069
MeO-PBDE, BRPs, and OH-PBDEs have been reported
previously (11). For analysis of PBBs, the extract was treated
with sulfuric acid because PBBs, unlike MeO-PBDEs and
PBDEs, are not degraded by concentrated sulfuric acid.
Extracts were passed through a column 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. Eluates were concentrated to 40 µL for quantification of PBBs. Instrument analyses
of identified EOBr are in Supporting Information.
Calculations of Bromine Concentrations. Concentrations of EOBr (ng Br/g ww) were determined (eq 1)
CEOBr )
CBr in extract × 10
× 1000
Wwet
(1)
where CBr in extract is the concentration of bromine in the extract
(µg/mL), 10 is the final volume of extract (mL), and Wwet is
the wet weight of sample (g). The residual materials after
solvent extraction were used for quantification of Br to
determine the nonextractable Br content (NEBr). Concentrations of NEBr (ng Br/g ww) were calculated (eq 2)
CNEBr ) CBr in RSP × (1 - Wat%) × (1 - Lipid%) × 1000
(2)
where CBr in RSP is the concentration of bromine in the residual
sample powders (RSP) (µg/g), Wat% is the water content of the
samples (%), and Lipid% is the lipid content of the samples (%).
Thus, the concentration of TBr, expressed as ng Br/g ww, was
the sum of concentrations of EOBr and NEBr (eq 3).
CTBr ) CEOBr + CNEBr
(3)
Identifiable EOBr, including PBBs, PBDEs, MeO-PBDEs,
OH-PBDEs, and BRPs, were quantified in liver samples.
Masses of Br contributing to total EOBr were determined by
multiplying the concentration of each compound identified
by its mole fraction of Br. Concentrations of identified EOBr
were the sum Br concentrations of all detected compounds
(eq 4).
∑(
m
Cknown EOBr )
i)1
)
80 × Ni
× Ci
MWi
(4)
where Ci is the concentration of organic brominated compound i (ng/g ww), Ni is the number of Br atoms contained
in the compound i, MWi is the molecular weight of compound
i, 80 is the molecular weight of Br, and m is the number of
detected organobromine compounds.
Bromine Analysis. Concentrations of Br were determined by INAA. Samples were activated for 15 min in 1.5 mL
NAA-grade polyvials at a neutron flux of 5.0 × 1011 (n/cm2)/s
in the SLOWPOKE 2 nuclear reactor (Saskatchewan Research
Council Analytical Laboratories, Saskatoon, SK, Canada). After
activation, γ-rays from 80Br were measured by γ-ray spectrometry by use of an Ortec model GMX20P4 HPGe solidstate detector and Ortec DSPEC Pro 8192 channel digital
gamma ray spectrometer for peak area calculations. Quantification was based on γ-peaks from 80Br (t1/2 ) 17.6 min,
Eγ ) 616 keV). The count time was 15 min. Known
concentrations of bromoform (CHBr3) dissolved in toluene
were used as standards for the quantification of EOBr and
TBr using INAA.
Quality Assurance and Quality Control (QA/QC). Counting efficiency of INAA was determined for EOBr and TBr for
each type of sample geometry. This was done by analyzing
a set of four standards and calculating counts per second per
microgram of bromine (cps/µg). The average efficiency factor
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 16, 2010
was used to calculate the EOBr and TBr concentrations. To
smooth out minor fluctuations that occur in individual
standards or batches, each time a new set of calibration
standards was quantified, a new average was calculated from
all standards run and maintained as a running average.
The QA/QC for the analysis of PBDEs, MeO-PBDEs, OHPBDEs, and BRPs has been reported previously (11). A
laboratory blank and a matrix spike were analyzed for every
batch of 15 samples. Beef liver was used for matrix spike
samples, and the spiking solutions were added before
accelerated solvent extraction. Recoveries of spiked materials
from samples were 80-127%, 81-126%, 87-128%, 81-123%,
and 65-126% for PBBs, MeO-PBDEs, PBDEs, OH-PBDEs,
and BRPs, respectively, in the entire analytical procedure.
Concentrations quantified in spiked samples were within
20% of the spiked concentrations. Thus, both accuracy and
precision of the analysis were deemed to be acceptable.
Quantification of PBBs, PBDEs, and BRPs was performed
with 13C-PBBs, 13C-PBDEs, and 13C-BRPs as surrogates.
Concentrations of OH-PBDEs were quantified relative to
2,3,4,6-13C-TeBRPs, and MeO-PBDEs were quantified relative
to 13C-PBDEs with the same number of Br atoms.
The instrumental limits of quantification for Br analysis
were 2 µg Br/g dw for solid samples and 0.08-0.5 µg Br/mL
for liquid samples. Method detection limits (MDL) were
defined to be mean plus three times the standard deviation
of concentrations in the blank. MDLs for compounds that
were not detected in the blank were set to be the instrumental
minimum detectable amounts. The MDLs were 0.4 pg/g ww
for MeO-PBDEs, 0.2-10.1 pg/g ww for PBDEs, 2-4 pg/g ww
for OH-PBDEs, 2.0-10 pg/g ww for BRPs, and 2-50 pg/g
ww for PBBs. For those results that were less than the MDL,
a sensitivity analysis was conducted and no statistically
significant differences could be found by using different
values ranging from 0 to the MDL. Thus, a value of 0 was
assigned to avoid missing values in statistical analyses.
Results and Discussion
Bromine Fractions. Concentrations of TBr, EOBr, and
identified EOBr in liver of tuna, albatross, and polar bear are
presented (Table 1). Mean ((SD) concentrations of TBr were
10 700 ( 3400, 11 200 ( 2600, and 12 400 ( 6100 ng/g ww
in tuna, albatross, and polar bear, respectively. Concentrations of EOBr were approximately 3- to 9-fold less than those
of NEBr, which ranged from 1100 ( 540 (tuna) to 2800 (
2300 ng/g ww (polar bear). Among the species analyzed, polar
bear liver contained the greatest concentrations of both TBr
and EOBr. However, concentrations of identified EOBr were
relatively small, ranging from 0.9 ( 0.3 (tuna) to 1.3 ( 1.1
ng/g ww (albatross) and accounted for only a small proportion
of the TBr.
Few studies have investigated concentrations of EOBr in
marine biota. The mean concentration of EOBr in harbor
porpoise (1700 ng/g ww) from the Baltic Sea (16) was
comparable to concentrations in tuna and albatross observed
in this study. However, concentrations of EOBr in marine
organisms in the present study were greater than those in
Atlantic herring from the Baltic Sea (120-240 ng/g ww) (16)
and northern pink shrimp from the North Atlantic Ocean
(60-1030 ng/g ww) (19). Overall, the reported concentrations
of EOBr in the organisms analyzed in this study ranged from
of 60 to 6100 ng/g ww. Similar variation in EOBr concentrations has been reported for tissues of terrapins, birds, and
fish collected from coastal waters of Georgia, U.S., which
ranged from 110 to 3700 ng/g ww. Although the underlying
reasons for this variation are unknown, it has been suggested
that concentrations of EOBr are species- and location-specific (4).
Concentrations of bromine associated with identified
EOBr compounds are presented (Figure 2 and Table 1). The
FIGURE 3. Relative contribution (%) of identified EOBr, EOBr,
and NEBr to TBr concentration in livers of polar bear, albatross, and tuna from remote marine locations.
FIGURE 2. Concentrations of major organic brominated compounds detected in liver samples collected from species inhabiting remote marine locations.
proportion of Br contributed by MeO-PBDEs was relatively
large in tuna (0.31 ( 0.10 ng/g ww) and albatross (0.65 ( 0.85
ng/g ww). The greatest contributions of OH-PBDEs to
identified EOBr were found in albatross (0.34 ( 0.24 ng/g
ww), and the greatest bromine contributions from BRPs were
observed in tuna (0.48 ( 0.21 ng/g ww). In these two species,
the predominant congeners of MeO-PBDEs, OH-PBDEs, and
BRPs were 6-MeO-BDE-47, 6-OH-BDE-47, and BRP-24,
respectively. Previous studies have demonstrated that these
compounds originate primarily from natural synthesis by
marine algae (11, 12, 20, 21). Thus, most of the identified
compounds contributing to concentrations of identified EOBr
in tuna and albatross are from naturally occurring compounds. Contributions of Br from the naturally occurring
MeO-PBDEs (0.014 ( 0.01 ng/g ww) and OH-PBDEs (0.004
( 0.005 ng/g ww) were small in polar bear, relative to those
in tuna and albatross. The relative contributions of organobromine compounds of human origin in polar bear liver
(PBDEs: 0.49 ( 0.16 ng/g ww and PBBs: 0.34 ( 0.29 ng/g ww)
were greater than those in tuna or albatross. Profiles of relative
proportions of identified compounds including PBDEs and
PBBs in polar bears were dominated by BDE-47 and PBB153, respectively. Similar profiles of PBDEs and PBBs have
been reported for other marine organisms (17, 22). Because
point sources play only a minor role in regional contamination of the Arctic marine ecosystem (23), the relatively great
concentrations of PBDEs and PBBs in polar bear are likely
due to atmospheric transport and deposition of these
anthropogenic compounds (24).
Proportions of Identified Compounds in EOBr and
TBr. A mass balance analysis of Br composition showed that
NEBr accounted for a major proportion of TBr (72 ( 24% to
90 ( 4%) in all samples (Figure 3). Similar to the results
reported here, the major proportions of total fluorine (TF)
found in human blood and dolphin livers were nonextractable
organic fluorine (NEOF) and inorganic fluoride (IF) (5, 8).
The EOBr accounted for a relatively small fraction of TBr in
each species, ranging from 10 ( 4% in tuna to 28 ( 24% in
polar bear. Although the contributions of EOBr to TBr in
polar bear liver were variable (2-53%), the large percentages
are comparable to the contributions of extractable organic
FIGURE 4. Relationship between carbon-halogen bond energies and contributions of identified anthropogenic organohalogens to extractable organic halogens. Bond energies were
referenced from 26.
fluorine (EOF) to TF measured in livers of marine mammals
in Hong Kong such as the Indo-pacific humpback dolphin:
58%, and finless porpoise: 27% (8).
Identified EOBr accounted for only 0.08-0.11% and
0.008-0.012% of EOBr and TBr, respectively (Figure 3) in the
three species, all of which are at the top of the food chain.
Contributions of identified EOBr to the total concentrations
of EOBr were small compared to the ratios of identified EOF
to total EOF (Indo-pacific humpback dolphin: 30%, finless
porpoise: 30%, and human blood: higher than 60%; refs 5,
8) and identified EOCl to total EOCl (fish: 5-25%; blue crab:
35%, birds: 1-14%, and terrapin: 4.2%; refs 4, 14). The small
proportion of identified EOBr contributing to total EOBr
concentration is probably due to the diversity of naturally
occurring organobromine compounds. Previous studies have
shown the existence of nearly 3200 known naturally occurring
organohalogen compounds in the environment, with more
than 1600 containing bromine (25). The predominance of
naturally occurring organobrominated compounds is likely
a result of the relatively low energy requirement for the
formation of the C-Br bond. The stronger halogen-carbon
bonds are less likely to be derived from naturally biological
processes. The relationship between identifiable synthetic
and unidentified organohalognes is given in Figure 4. Since
the C-F bond is relatively strong, it is much less likely to be
formed naturally and there are likely to be fewer F atoms in
naturally occurring organic molecules. Therefore, most of
the organically bound fluorine is attributable to identifiable,
synthetic, per- and polyfluorinated compounds.
VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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6071
TABLE 2. Concentrations of Organobromine Compounds Reported in Previous Studiesa
brominated flame retardants
sample
ref
glaucous gull
polar bear
eel
cormorant
predatory birds
deep sea fish
deep sea fish
food web
wild tuna
whale oil
sediment
fish and
mussels
18
18
27
9
17
28
28
29
30
31
32
tuna
albatross
polar bear
PBDEs
PBBs
TBBPA
HBCD
1325
21.9
N.D.
533
24.6
0.1 N.D.
57.5
1419
14.4
63.0 N.D.-0.26
1753
16.9
<1.5
5.1
<1.5
1.0-3.0
6.4-115.4
58-74
originated from both sources
PBDD/Fs
BRPs
naturally occurring brominated compound
OH-PBDEs MeO-PBDEs
26.5
N.D.
TBA
MHC-1
0.1
28.9
6.5
0.7-110.1
134-167
0.1
0.3
530
<0.2
7040
4.7-146
11.7-30.1
3580-5241 3.1-4.2
9.7
0.6
0.1-1.6
26-30
0.008
0.4-118
13.3-360.8
<5-1140
0.2
0.6
0.8
BRI
54.3
N.D.
33
b 5.7
b 2.6
b 11.0
PBHDs
18.8
0.4
2.1
0.6
6.7
0.6
<1-3
13.4
12.3
0.4
a
(ng/g lw (lipid weight). Number underlined indicates the predominant brominated compounds detected. The
concentrations of EOBr in tuna, albatross, and polar bear in current study were 23 000, 17 000, and 40 000 ng/g lw,
respectively. TBBPA: tetrabromobisphenol A; HBCD: hexabromocyclododecane; PBDD/Fs: polybrominated dibenzop-dioxins and dibenzofurans; PBHDs: polybrominated hexahydroxanthene derivatives; TBA: tribrominated anisole; BRI:
brominated indole. Locations of references: (18), Norwegian Arctic; (27), Irish waters; (9), Japan; (17), Norway; (28, 30), and
(33), Mediterranean Sea; (29), Sydney Harbour; (31), historical sample; (32), North and Baltic Sea. b Current study.
Estimating the Major Organobromine Compounds in
EOBr. There is a variety of synthetic and natural organobrominated compounds that were not quantified in this study
that could have contributed to the EOBr of these samples
(Table 2). Since the masses of samples available for this study
were limited and standards are not available for many of the
classes of brominated compounds that could have contributed to the EOBr, an assessment of the potential for those
unquantified compounds to contribute to the EOBr was made
from information available in the literature (Table 2). PBDEs
were generally the predominant compounds among brominated flame retardants, which is also consistent with the
fact that they are used in the greatest quantities globally.
Among naturally occurring brominated compounds, concentrations of MeO-PBDEs, BRPs, and polybrominated
hexahydroxanthene derivatives (PBHDs) were generally
greater than other brominated compounds of natural origin
(28-30). In the current study, PBHDs were not analyzed, but
concentrations of PBHDs have been reported to be as great
as 5000 ng/g lipid weight (lw) in some deep-ocean fishes
(28). Since authentic PBHD standards were not commercially
available, PBHDs were identified and quantified based on
previously reported methods (28). While MeO-PBDEs in the
sample extracts can be detected by GC-EI-MS, no obvious
peaks that would have corresponded to masses of PBHDs
were observed (Supporting Information Figure S1). This result
suggests that concentrations of PBHDs in the samples studied
were not significant contributors to TBr. The presence of
PBHDs and other organobromine compounds were further
studied by GC-ECNI-MS, and no brominated compounds
with concentrations greater than approximately 1 ng/g ww
could be detected (Supporting Information Figure S2). In
addition, concentrations of the detected organobromine
compounds reported previously were very small compared
with those of EOBr (Table 2). Including the estimated
concentrations of these additional brominated compounds,
the identified proportion of the EOBr would not exceed 1%.
Brominated fatty acids (BFAs) could be the predominant
compounds in EOBr, since BFAs have been reported to occur
at concentrations ranging from 2.2 to 82 µg/g ww in marine
fishes and invertebrates (34). These concentrations are
comparable to those of EOBr determined in this study
(1.1-2.8 µg/g ww). However, reported concentrations of BFAs
6072
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 16, 2010
were based on the analysis of bromine content. As one of the
most interesting groups among the naturally occurring
halogen compounds, BFAs comprise a relatively large and
diverse class of compounds (35, 36). BFAs with different
structures have been identified in marine algae and invertebrates, and some compounds contain as many as 28 carbon
atoms (35, 36). However, none of the studies has attempted
to quantify BFAs. In the current study concentrations of
saturated BFAs (C2-C21) were estimated by use of GC-EIMS after derivatization of the extracts with N-methyl-N-(tertbutyldimethylsilyl) trifluoroacetamide with 1% t-BDMSchloride (MTBSTFA) (FigureS3 in Supporting Information).
However, the chain lengths of BFAs in marine organisms
were generally greater than 16 (35, 36), and standards for
BFAs of this chain length are not commercially available. A
number of possible peaks with relatively great concentrations
were observed (Figure S4 in Supporting Information), but
without authentic standards, it was difficult to accurately
identify or quantify the individual BFAs. Further studies
should focus on the analysis and potential effects of these
compounds.
Acknowledgments
The 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 6971 and 6807).
J.G. was supported by the Canada Research Chair program
and an at large Chair Professorship at the Department of
Biology and Chemistry and State Key Laboratory in Marine
Pollution, 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 livers.
Supporting Information Available
Detailed information on chemicals, standards, instrument
analysis, and analysis of saturated brominated fatty acids;
GC-EI-MS and GC-NCI-MS chromatography of sample
extracts. This material is available free of charge via the
Internet at http://pubs.acs.org.
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ES100914R
VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6073
1
SUPPORTING INFORMATION
2
Contribution of Anthropogenic and Naturally Occurring Organobromine Compounds
3
to Bromine Mass in Marine Organisms
4
5
Yi Wan, Paul D. Jones, Steve Wiseman, Hong Chang, Dave Chorney, Kurunthachalam
6
Kannan, Jong Seong Khim, Shinsuke Tanabe, Michael H. W. Lam, John P. Giesy*
7
8
9
10
11
12
13
14
15
16
17
18
19
* corresponding author
This supporting information includes:
Chemicals and Standards
Instrumental Analysis of Identified EOBr
Analysis of Saturated Brominated Fatty Acids
Table S1
Figure S1
Figure S2
Figure S3
Figure S4
20
21
Summary of the number of pages, figures, and tables
22
Number of pages: 13
23
Number of figures: 4
24
Number of tables: 1
S1
1
MATERIALS AND METHODS
2
Chemicals and Standards
3
Ten PBBs (PBB-3, PBB-15, PBB-18, PBB-52, PBB-101, PBB-153, PBB-180, PBB-194,
4
PBB-206, and PBB-209), twenty-one PBDEs (BDE-7, BDE-15, BDE-30, BDE-17, BDE-28,
5
BDE-49, BDE-71, BDE-47, BDE-66, BDE-77, BDE-100, BDE-119, BDE-99, BDE-85,
6
BDE-126, BDE-154, BDE-153, BDE-139, BDE-140, BDE-138, and BDE-183), twelve
7
MeO-PBDEs
8
5-MeO-BDE-47, 4’ -MeO-BDE-49, 5’-MeO-BDE-100, 4’-MeO-BDE-103, 5’-MeO-BDE-99,
9
4’-MeO-BDE-101,
(6-MeO-BDE-17,
4-MeO-BDE-17,
6-MeO-BDE-90,
and
2’-MeO-BDE-68,
6-MeO-BDE-85),
6-MeO-BDE-47,
ten
OH-PBDEs
10
(2’-OH-6’-Cl-BDE-7, 6’-OH-BDE-17, 6-OH-BDE-47, 3-OH-BDE-47, 5-OH-BDE-47,
11
2’-OH-BDE-68, 4’-OH-BDE-49, 2’-OH-6’-Cl-BDE-68, 6-OH-BDE-90, and 2-OH-BDE-123)
12
and sixteen bromophenols (2,6-DiBRP, 2,5-DiBRP, 2,4-DiBRP, 2,3-DiBRP, 3,5-DiBRP,
13
3,4-DiBRP,
14
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
15
selected as target compounds.
16
and
17
13
18
BRPs,
19
13
20
Laboratories Inc. (Guelph, Ontario, Canada).
21
2’-OH-BDE-68
22
6-MeO-BDE-17, 4-MeO-BDE-17, 6-MeO-BDE-90, 6-MeO-BDE-85, 6’-OH-BDE-17 and the
13
2,4,6-TriBRP,
C-PBB-209),
C-BDE-85,
13
13
PBDEs,
C-BDE-99,
C-BRPs
2,3,6-TriBRP,
PBBs,
13
13
2,3,5-TriBRP,
2,4,5-TriBRP,
C-PBBs (13C-PBB-52,
(13C-BDE-28,
C-PBDEs
13
C-BDE-100,
(13C-2,4-DiBRP,
13
C-BDE-153,
13
13
13
C-PBB-153,
13
13
13
C-PBB-194,
13
C-BDE-47,
C-BDE-154, and
C-2,4,6-TriBRP,
2,3,4-TriBRP,
C-BDE-66,
13
C-BDE-183),
C-2,3,4,6-TeBRP,
and
C-2,3,4,5,6-PeBRP) and eight MeO-PBDEs standards were obtained from Wellington
were
obtained
from
3-OH-BDE-47, 5-OH-BDE-47 and
AccuStandard
S2
(New
Haven,
CT,
USA).
1
remaining seven OH-PBDEs were synthesized in the Department of Biology and Chemistry,
2
City University of Hong Kong.
3
high-resolution gas chromatograph interfaced to high-resolution mass spectrometer
4
(HRGC-HRMS).
5
acetonitrile and methanol were pesticide residue grade obtained from OmniSolv (EM Science,
6
Lawrence, KS, USA).
7
(neutral, 150 mesh size), pyridine (anhydrous, 99.8%), methyl chloroformate (MCF),
8
potassium hydroxide (KOH), hydrochloric acid (HCl), 2-bromohexanoic acid and
9
N-methyl-N-(tert-butyldimethylsilyl)
10
All standards were determined to be >98% pure by use of
Dichloromethane (DCM), n-hexane, methyl tert-butyl ether (MTBE),
Sodium sulfate, silica gel (60-100 mesh size), aluminum oxide
trifluoroacetamide
with
1%
t-BDMS-chloride
(MTBSTFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
11
12
Instrumental Analysis of Identified EOBr
13
Identification and quantification of PBBs, PBDEs, MeO-PBDEs, OH-PBDEs and BRPs
14
were performed with a Hewlett-Packard 5890 series II HRGC interfaced to a Micromass®
15
Autospec® HRMS (Micromass®, Beverly, MD).
16
on a DB-5MS fused silica capillary column for all target compounds (30 m length, 0.25 mm
17
ID, 0.1 µm film thickness, Agilent, Carlsbad, CA), helium was used as the carrier gas. The
18
mass spectrometer was operated in a selected ion-monitoring (SIM) mode.
19
spectrometer resolution for all reference gas peaks in all time windows was greater than 7,000.
20
The injector temperature was held at 285 °C and the ion source was kept at 285 °C. The
21
electron ionization energy was 37 eV and the ion current was 750 µA.
22
temperature program was from 110°C (10min) to 250 °C at a rate of 25 °C/min, then
23
increased to 260 °C at a rate of 1.5 °C/min, and then to 325 °C (15 min) at a rate of 25 °C/min.
S3
Chromatographic separation was achieved
The mass
For PBDEs, the
1
For OH-PBDEs, the temperature program was from 150 °C (2min) to 320 °C (2min) at a rate
2
of 10 °C/min.
3
°C (2 min) at a rate of 2 °C/min, and then increased to 320 °C (2 min) at a rate of 30°C/min.
4
For BRPs, the temperature program was from 100 °C (5 min) to 180°C at a rate of 3°C/min,
5
and then increased to 300 °C (2 min) at a rate of 25°C/min. For PBBs, the temperature
6
program was from 110 °C (3 min) to 250 °C at a rate of 25°C/min, then increase to 260 °C at
7
a rate of 1.5 °C/min, and increase to 320 °C (15 min) at a rate of 25 °C/min.
For MeO-PBDEs, the temperature program was from 150°C (2 min) to 245
8
A Hewlett-Packard 7890A gas chromatograph connected to a Hewlett-Packard 5975C
9
mass spectrometer was used to detect two predominant naturally occurring brominated
10
compounds (MeO-PBDEs and PBHDs) and to analyze brominated fatty acids in the samples.
11
The mass spectrometer was operated in the electron impact ionization mode (EI) with an
12
ionizing energy of 70 eV.
13
detector source temperature was kept at 280 °C.
14
splitless mode was used.
15
thickness of 0.25 µm) was used for separations.
16
the oven temperature was programmed to increase from 90 °C (1.5 min) to 230 °C at a rate of
17
15 °C /min and then to 300 °C at 5 °C /min, which was held for 15 min.
18
saturated brominated fatty acids, the temperature was programmed to increase from 100 °C (3
19
min) to 320 °C at a rate of 10 °C /min, which was held for 20 min.
The injector temperature was maintained at 300 °C, and the
The injection volume was 2 µL, and the
An HP-5MS capillary column (60 m × 0.25 mm i.d. with a film
For analysis of MeO-PBDEs and PBHDs,
For analysis of
20
To identify the existence of other possible brominated compounds in sample extracts,
21
samples were analyzed by GC-electron capture negative ionization mass spectrometry
22
(ENCI-MS) (Shimadzu QP 2010 plus, Kyoto, Japan). An HP-5MS capillary column (30 m
23
× 0.25 mm i.d. with a film thickness of 0.25 µm) was used for the separation.
24
temperature program was from 90 °C (1.5 min) to 230 °C at the rate of 15 °C /min, then
25
increase to 300 °C (15 min) at 5 °C /min. The interface and ion source temperatures were
S4
The
1
300 °C and 280 °C, respectively.
A splitless injector maintained at 250 °C was used.
2
3
Analysis of Saturated Brominated Fatty Acids
4
The analytical and identification method for brominated fatty acid is similar to that of
5
naphthenic acids reported previously (1). Due to large numbers of the brominated fatty acids,
6
only saturated acids with carbon numbers ranging from 2 to 20 were selected as target
7
compounds.
About 200 µl of ASE extracts were dried under nitrogen gas, and dissolved in
8
100 µl DCM.
The derivatizations were performed by adding 100 µl MTBSTFA reagent, and
9
then samples were heated at 60 °C for 30 min to generate the t-BDMS derivatives. The
10
derivatized extracts were analyzed by GC-EI-MS.
11
mass spectrum for t-BDMS derivatives of 2-bromohexanoic acid.
12
compounds were [M-tBDMS-C4H9]+, which is resulted from cleavage of the tert.-butyl group.
13
The ions of other saturated brominated fatty acids (C2-C21) were also calculated for
14
identification of the compounds (Table S1), since this ion can reflect the information of
15
molecular weight of the target compounds.
S5
Figure S3 shows the chromatography and
The base peak ion for the
1
Table S1, Monitoring ions for brominated fatty acids with number of carbons ranging from 2
2
to 21.
Number of C
Ion
2
195
197
3
209
211
4
223
225
5
237
239
6
251
253
7
265
267
8
279
281
9
293
295
10
307
309
11
321
323
12
335
337
13
349
351
14
363
365
15
377
379
16
391
393
17
405
407
18
419
421
19
433
435
20
447
449
21
461
463
3
4
5
S6
Albatross
2’-MeO-BDE68
6-MeO-BDE47
1
Tuna
2
Polar bear
3
4
Figure S1, GC-EI-MS chromatographic profiles of MeO-PBDEs and PBHDs in liver samples
5
collected from species inhabiting remote marine locations.
6
and 513.7; mass for TriBHD and TeBHD: 466, 464 and 387 referenced from 2.
7
S7
Mass for MeO-TriBDEs: 515.7
Albatross
a: 0-17.18.75 min
b: 18.5-33.25 min
CBDE183/BDE183
CBDE138
b
13
13
CBB153/BB153
CBDE154/BDE154
13
13
CBDE153/BDE153
6-MeO-BDE47
CBDE100/BDE100
13
CBDE99/BDE99
13
2’-MeO-BDE68
CBB52/BB52
C-BDE47/BDE47
13
13
C-BDE28/BDE28
13
13
a
1
c
d
Tuna
c: 0-17.18.75 min
d: 18.5-33.25 min
2
e
f
Polar bear
e: 0-17.18.75 min
f: 18.5-33.25 min
3
S8
1
Figure S2. GC-NCI-MS chromatographic profiles in liver samples collected from species inhabiting remote marine locations. Black line: TIC;
2
pink line: 79 (ion); blue line: 81 (ion).
3
ww in samples.
Concentrations of 13C-labelled surrogates were 100 ppb in the purified extracts, which is about 1 ng/g
4
S9
Abundance
1.6×106
Ion 251
Ion 253
1.4×106
1.2×106
1.0×106
0.8×106
0.6×106
0.4×106
0.2×106
13.00
1
14.00
15.00
16.00
17.00
18.00
19.00
20.00
Abundance
3.2×106
2.8×10
21.00
22.00
23.00
75
O
75
O
Si
6
Br
2.4×106
139
253
2.0×106
1.6×106
1.2×106
97 115 132
253
8×105
4×105
2
3
4
5
100
140
180
220
260
300
340
380
Figure S3, Chromatography (0.5 µg/ml) and mass spectrum for t-BDMS derivatives of
2-bromohexanoic acid.
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Abundance
1.5×105
Ion 349
Ion 351
1.3×105
a
1.1×105
9×104
7×104
5×104
3×104
1×104
17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00
1
Abundance
1.1×105
9×10
Ion 391
Ion 393
b
4
7×104
5×104
3×104
1×104
22.50
2
23.50
24.50
25.50
26.50
27.50
Abundance
8×104
Ion 405
Ion 407
c
7×104
6×104
5×104
4×104
3×104
2×104
1×104
3
29.00
31.00
33.00
35.00
S11
37.00
Abundance
2.3×104
Ion 461
Ion 463
d
1.9×104
1.5×104
1.1×104
7×103
3×103
1
2
3
4
5
6
7
8
26.00
28.00
30.00
32.00
34.00
Figure S4, GC-EI-MS chromatographic profiles of possible t-BDMS derivatives of
brominated fatty acids in liver samples collected from species inhabiting remote marine
locations. Ion of 75 and 73 were not shown. Peaks showed here were detected in all three
species. Possible detected compounds: a, t-BDMS derivatives of bromotridecanoic acid; b,
t-BDMS derivatives of bromohexadecanoic acid; c, t-BDMS derivatives of
bromohepadecanoic acid; d, t-BDMS derivatives of bromohenicosanoic acid. The estimated
concentrations of each peak were about 0.5 ng/g ww in samples.
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1
REFERENCES
2
3
4
1. St. John, W.P.; Rughani, J.; Green, S.A.; McGinnis, G.D. Analysis and characterization of
naphthenic acids by gas chromatography-electron impact mass spectrometry of
tert.-butyldimethylsilyl derivatives. J. Chromatogr. A 1998, 807, 241-251.
5
6
7
8
2. Hiebl, J.; Melcher, J.; Gundersen, H.; Schlabach, M.; Vetter, W. Identification and
Quantificaiton of polybrominated hexahydroxanthene derivatives and other halogenated
natural products in commercial fish and other marine samples. J. Agric. Food Chem. 2006,
54, 2652-2657.
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