Article
pubs.acs.org/est
Polyhalogenated Carbazoles in Sediments of Lake Michigan: A New
Discovery
Jiehong Guo,† Da Chen,*,‡ Dave Potter,§ Karl J. Rockne,∥ Neil C. Sturchio,⊥ John P. Giesy,# and An Li*,†
†
School of Public Health, University of Illinois at Chicago, Chicago, Illinois 60612, United States
Cooperative Wildlife Research Laboratory and Department of Zoology, Southern Illinois University, Carbondale, Illinois 62901,
United States
§
Research Division, Wellington Laboratories, Guelph, Ontario N1G 3M5, Canada
∥
Department of Civil and Materials Engineering, University of Illinois at Chicago, Chicago, Illinois 60607, United States
⊥
Department of Earth and Environmental Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, United States
#
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N
5B3, Canada
‡
S Supporting Information
*
ABSTRACT: Previously unknown halogenated compounds were detected during the analysis
of halogenated flame retardants in two sediment cores collected from Lake Michigan. Gas
chromatography coupled with high- or low-resolution mass spectrometry (MS) was used to
determine the chemical structures for a total of 15 novel polyhalogenated carbazoles (PHCs)
with the general molecular formula C12H9-x-y-zNClxBryIz. On the basis of the mass spectra
generated by electron impact (EI) and electron capture negative ionization (ECNI) MS, eight
PHCs were tentatively identified as polybrominated carbazoles, while the others were mixed
halogenated carbazoles containing, in addition to bromine, either chlorine or iodine or both.
Patterns of halogen substitution of PHCs included Br2 to Br5, ClBr2, ClBr3, ClBr4, ClBr3I, Br4I,
and Br3I2. 3,6-Dibromocarbazole and 1,3,6,8-tetrabromocarbazole were also found among the
PHCs. Profiles of the concentration versus depth of sediment at the two sites showed various
patterns among polybrominated carbazoles. The abundance of mixed halogenated carbazoles
peaked at depths of 12−16 cm, remained at relatively constant levels in deeper sediment, but
declined markedly in more recently deposited sediments. This is the first study discovering the
seven mixed halogenated carbazoles in the environment. Detailed methods for their detection and identification are provided.
■
other aquatic organisms.3 For example, the soil fungus
Penicillium sp. produces 2,4-dichlorophenol, and grasshoppers
secrete 2,5-dichlorophenol.6 Methoxylated and hydroxylated
polybrominated diphenyl ethers (PBDEs) as well as polybrominated phenols found in some marine organisms were
proven to be of natural origin.7−10 Some naturally produced
organohalogens have molecular structures similar to those of
synthetic PBT chemicals, warranting more research on their
environmental behavior and impact.
A group of halogenated organic compounds, which could be
of natural and/or anthropogenic origins, are polyhalogenated
carbazoles (PHCs).11−14 Chemical structures of PHCs
resemble those of polyhalogenated dibenzofurans, with a NH
group replacing the oxygen. A number of studies have identified
either chlorinated or brominated carbazoles in riverine or
marine sediments as well as soils.11,14−18 In the Great Lake
region, 1,3,6,8-tetrachlorocarbazole was discovered in the
INTRODUCTION
Numerous anthropogenic organic chemicals contain halogens.
According to the 2014 Chemical Data Reporting (CDR)
information submitted to the United States Environmental
Protection Agency (U.S. EPA), annual production of bromine
(Br), chlorine (Cl), and iodine (I) in the United States was 2.1
× 105, 9.9 × 106, and 6.1 × 103 metric tonnes, respectively.1 In
North America, 610 chemicals were prioritized for environmental monitoring, about 62% of which are halogenated
organic compounds.2 These chemicals have caused global
environmental and human health concerns, because of their
persistent, bioaccumulative, and potentially toxic (PBT) nature.
In addition, more than 3800 naturally occurring organohalogens have been identified from both biogenic and abiogenic
sources.3 Natural processes, such as volcanic eruptions and
forest fires, are known to produce various organohalogens,
including congeners of polychlorinated biphenyls (PCBs).4
Chlorophenols are naturally produced but are also manufactured and used on a large scale in industry and are listed as
priority pollutants by the U.S. EPA.5 Natural biogenic
organohalogens are usually produced by organisms, such as
bacteria, fungi, plants, marine and freshwater algae, and some
© 2014 American Chemical Society
Received:
Revised:
Accepted:
Published:
12807
August 12, 2014
September 24, 2014
October 1, 2014
October 1, 2014
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sediment of the Buffalo River in 1984.18 In the sediment of
Lake Michigan, a group of tri- to pentabrominated carbazoles
were discovered and the most abundant congener was
identified as 1,3,6,8-tetrabromocarbazole.16 Recently, a carbazole with both chlorine and bromine substituents was detected
in sediments of streams across southern Ontario, Canada.19 In
the present study, a number of carbazole-like compounds with a
variety of halogen substitution patterns were discovered in
sediments from Lake Michigan. The objectives of the study
were to (1) identify and characterize structural information on
these compounds and (2) reveal their temporal trends in
sediments of Lake Michigan. To our knowledge, this is the first
report on the presence of this suite of PHCs in the
environment.
time of 10 min per cycle. The extract was concentrated by a
rotary evaporator and solvent-exchanged into hexane. A glass
column (11 mm inner diameter × 40 cm long) was prefilled
with dichloromethane (DCM). The column was then filled
from the bottom to the top with 1 g of granular anhydrous
Na2SO4, 16 g of alumina, 4 g of silica gel, and 4 g of granular
anhydrous Na2SO4. After the column was packed, DCM was
completely replaced by hexane. Concentrated extract was added
to the prepared columns and eluted with 100 mL of hexane (F1), 200 mL of 4:1 hexane/DCM mixture (F-2), and finally, 100
mL of DCM (F-3). F-2 and F-3 containing the target analytes
were concentrated and placed in 200 μL glass inserts of GC
vials, prior to analysis by gas chromatography (GC)−lowresolution mass spectrometry (LRMS) in electron impact (EI)
or electron capture negative ionization (ECNI) mode.
Initial analyses were performed on Agilent 6890/5973 GC−
MS (Agilent Technologies, Santa Clara, CA) in ECNI mode. It
was equipped with a Gerstel programmable temperature
vaporization (PTV) injection port, operated in solvent vent
mode. The inlet temperature started at 40 °C, held for 2 min,
and then increased to 300 °C at 600 °C/min. The injection
volume was 60 μL (20 μL × 3) for each run. The carrier gas
was helium. The vent flow was 100 mL/min, and the purge
flow was 100 mL/min, at 2.75 min. GC was equipped with a
Restek Rtx1614 capillary column (15 m × 0.25 mm inner
diameter × 0.10 μm film thickness). The initial oven
temperature was set at 50 °C, held for 3 min, then increased
to 300 °C at 10 °C/min, and kept for 10 min until the run was
completed. The carrier gas flow was kept constant at 1.5 mL/
min. To achieve baseline separation of all peaks and ensure the
quality of mass spectra of targeted analytes, a 30 m Agilent DB5MS capillary column (0.25 mm inner diameter × 0.25 μm film
thickness) was also used. The initial oven temperature was 50
°C, held for 3 min, and then increased to 150 °C at 10 °C/min
and further to 300 °C at 5 °C/min. The flow of carrier gas was
kept constant at 1.2 mL/min. For both columns, the
temperature of the GC−MS interface was kept at 300 °C.
Full scan mass spectra were obtained in LRMS with ECNI
(m/z 35−800) or EI (m/z 50−800) ionization. The temperature of the ECNI ion source was set at 200 °C, and methane
was used as reagent gas. The temperature of the EI ion source
was 230 °C. The quadrupole analyzer was maintained at 150
°C during ECNI and EI analyses.
To achieve adequate responses from the accurate mass
determination using GC−high-resolution MS (HRMS), 16 g of
dry composite sediment from Lake Michigan was extracted.
The extract was fractionated in the same silica gel/alumina
column but with slightly different eluting solvents, i.e., 100 mL
of hexane (F-1), 60 mL of hexane/DCM mixture (4:1, v/v) (F2), and 240 mL of hexane/DCM mixture (4:1, v/v) (F-3). F-3
contained target analytes, was concentrated using a rotary
evaporator, and was placed in 200 μL glass inserts of GC vials
prior to analysis.
Accurate mass determination was performed using Agilent
6890 GC coupled to Waters Autospec Ultima MS. The fraction
containing target analytes was injected into the GC−HRMS
system and analyzed in full-scan mode (m/z 50−1000). Voltage
scan experiments were created to scan narrow mass ranges that
encompassed molecular fragments of interest. These narrow
mass ranges also included one to three reference peaks from the
mass calibrant, perfluorokerosene (PFK). The instrument was
tuned to a resolution of at least 10 000. A spectrum calibration
curve was constructed using the PFK reference mass peaks in a
■
EXPERIMENTAL SECTION
Sampling. Two sediment cores were collected in Lake
Michigan onboard the U.S. EPA Research Vessel (R/V) Lake
Guardian. One was collected using a box corer in September
2010 at site M018 (latitude, 42.7338°; longitude, −86.9995°)
in the southern basin. The other was collected by a multi-corer
in June 2011 from M041 (latitude, 44.7367°; longitude,
−86.7215°) in the deep Chippewa basin. At each site, four
subcores (10 cm in diameter) were obtained and sectioned
using hydraulic extruders with 1 cm intervals until 10 cm and
then 2 cm intervals to a depth of 30 cm. To minimize potential
“smearing”, sediment within 2 mm of the wall of the coring
tube was trimmed off and discarded. After each segment was
cut, all sectioning gear was thoroughly cleaned using tap water,
acetone, and deionized water. At each site, the segments at
corresponding depths were combined and mixed with stainlesssteel spoons in glass bowls. Well-mixed composite samples
were distributed into pre-cleaned 125 mL amber glass jars with
Teflon-liner screw caps. The samples were immediately frozen.
They were transported in coolers to the laboratory, where they
were stored at −20 °C until further processing and analysis.
Chemicals and Reagents. Chemical standards 3,6dibromocarbazole (3,6-DiBC) (97%) and 1,3,6,8-tetrabromocarbazole (1,3,6,8-TeBC) were purchased from Sigma-Aldrich
(St. Louis, MO) and the Florida Center for Heterocyclic
Compounds of the University of Florida (Gainesville, FL),
respectively. Surrogates 4′-fluoro-2,3′,4,6-tetrabromodiphenyl
ether (F-BDE69) and injection standard decabromobiphenyl
(BB209) were purchased from AccuStandard (New Haven,
CT). All solvents were high-performance liquid chromatography (HPLC)- or Optima-grade and purchased from Fisher
Scientific (Pittsburgh, PA). Silica gel (100−200 mesh, 75−150
μm, grade 644), alumina (neutral, Brockmann I, 50−200 μm by
Acros Organics), and granular anhydrous sodium sulfate
(Na2SO4) were also from Fisher Scientific. The sorbents silica
gel and alumina as well as Na2SO4 were activated at 500 °C for
8 h, stored at 160 °C, and cooled to room temperature in
desiccators before use.
Qualitative Analysis. Extensive sample preparation and
cleanup were conducted to separate target analytes (PHCs)
from potentially interfering compounds (mainly PBDEs) and
produce high-quality mass spectra of PHCs. In brief, 8 g of
freeze-dried, composite sediment from Lake Michigan was
extracted with an accelerated solvent extraction system (Dionex
ASE350, Thermo Fisher Scientific, Inc.), using a procedure
similar to that described in the U.S. EPA Method 3545A. The
sediment was extracted with a hexane and acetone mixture (1:1,
v/v) at 100 °C for 3 cycles, with heat time of 5 min and static
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Table 1. Elemental Compositions of Halogenated Carbazoles Found in Lake Michigan Sediments and Their Characteristic Ions
during the EI and ECNI Mass Spectral Analyses
accurate mass (Da)
name
formula
UNC-1
UNC-2
UNC-3
3,6-DiBC
UNC-4
UNC-5
UNC-6
UNC-7
UNC-8
UNC-9
1,3,6,8-TeBC
UNC-10
UNC-11
UNC-12
UNC-13
UNC-14
UNC-15
C12H6NClBr2
C12H6NBr3
C12H7NBr2
C12H7NBr2
C12H6NBr3
C12H5NClBr3
C12H6NBr3
C12H5NClBr3
C12H6NBr3
C12H5NBr4
C12H5NBr4
C12H6NBr3
C12H4NClBr4
C12H4NBr5
C12H4NClBr3I
C12H4NBr4I
C12H4NBr3I2
measured
434.7661
434.7661
478.7155
478.7155
512.6766
556.6261
560.6603
604.6122
calculateda
356.855575
400.805068
322.894545
322.894545
400.805068
434.766098
400.805068
434.766098
400.805068
478.715591
478.715591
400.805068
512.676621
556.626114
560.662625
604.612118
652.598122
EI ions
differenceb
−0.000002
−0.000002
0.000091
0.000091
0.000021
0.000014
0.002325
−0.000082
CI ions
molecular
main fragment
Qc
q1d
q2d
spectrum figure
358.8
404.8
324.8
324.8
404.8
438.7
404.8
438.7
404.8
482.7
482.7
404.8
516.6
560.6
562.6
608.6
654.5
279.9
323.8
245.9
245.9
323.8
357.8
323.8
357.8
323.8
401.7
403.6
323.8
438.6
481.6
438.7
482.7
529.8
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
79/81
358.9
402.8
324.9
324.9
402.8
438.8
402.8
438.8
402.8
482.7
482.7
402.8
516.7
560.6
562.7
608.7
654.5
280.9
324.9
S17
S7
S8
S3 and S5
S9
S18
S10
2
S11
S12
S4 and S6
S13
S19
S14
3
S20
S21
324.9
358.8
324.9
358.8
324.9
402.7
402.7
324.9
436.7
480.7
438.7
482.8
527.7
a
Calculations are based on the following exact masses: C, 12.000000; H, 1.007825; Br, 78.918348; Cl, 34.968855; F, 18.998405; N, 14.003074; and
I, 126.904352.20 bDifference between calculated and measured masses. cQuantifier ion. dQualifier ion.
Figure 1. Partial GC−ECNI−LRMS chromatograms (30 m DB-5MS column, full scan, m/z 35−800) of deep sediment (26−30 cm) extract from
sampling site M041 (A, F-2; B, F-3) and (C) standard mixture of halogenated flame retardants. In panel A, UNC-15 is zoomed in.
by use of the PFK reference peaks. The corrected data file was
then opened, and the background was subtracted from the
region of interest to generate the final mass spectrum. The
portion of the total ion current (TIC) where no peaks eluted. A
secondary reference correction (Masslynx 4.1) was applied to
the acquired data file to autocorrect each mass in the spectrum
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Figure 2. (Top) ECNI (scan range m/z 35−800) and (bottom) EI (scan range m/z 50−800) mass spectra (GC−LRMS) of UNC-7 in Lake
Michigan sediment samples.
Quantifications of 3,6-DiBC and 1,3,6,8-TeBC were based on
calibration curves developed from their corresponding
reference standards. Calibration curves were generated from
the bromine response ( 79 Br − or 81 Br − ) ratios versus
concentration ratios of 3,6-DiBC and 1,3,6,8-TeBC to injection
standard BB209. Because of the lack of commercially available
reference standards for the 15 unknown compounds (UNCs)
(Figure 1), semi-quantification was achieved via SIM for 79Br−
and 81Br−, and based on the calibration curves for 3,6-DiBC or
1,3,6,8-TeBC. Specifically, UNC-1, UNC-3, UNC-5, and UNC7 were semi-quantified using the calibration curve of 3,6-DiBC
because of the nearness in GC retention times. The calibration
curve of 1,3,6,8-TeBC was used to semi-quantify other UNCs,
including all tribromocarbazoles, one of which (UNC-8)
matches with the impurity in the chemical standard of
1,3,6,8-TeBC.
Quality Control. Two laboratory procedural blanks
(Na2SO4) were analyzed along with sediment samples from
each of the two cores (M018 and M041). Blanks were analyzed
using the same laboratory procedures and instrument as used
for sediments in quantitative analysis. Concentrations of PHCs
in the procedural blanks ranged from no detection (nd) to 0.1
ng/g of dry weight (dw). Surrogate F-BDE69 was added to
each sample before extraction, and its average recovery was 121
± 15% for M018 samples and 80 ± 11% for M041 samples.
Two samples (one for each core) were analyzed in duplicate,
and the average relative percentage differences (RPDs) of the
duplicate analyses for individual PHCs ranged from 0.4 to 26%.
In addition, duplicate blanks (Na2SO4) were spiked with
1,3,6,8-TeBC (8 ng), and its average recovery was 95.4 ± 8%.
Instrument detection limits (on the basis of 3 times the signal-
Masslynx 4.1 elemental composition program was used to
determine the best fitting formula for each isotope signal in the
mass spectrum of interest. All injections were made in the
splitless mode at a temperature of 280 °C. The oven
temperature program was the same as that for GC−LRMS
analysis.
Quantitative Analysis. Quantitative or semi-quantitative
analyses of PHCs were conducted using a procedure similar to
that used for qualitative analyses. Approximately 5 g of freezedried sediments were spiked with surrogates F-BDE69 (4 ng),
stabilized overnight, and then extracted with accelerated solvent
extraction (ASE). The cleanup column was packed with 8 g of
alumina and 4 g of silica gel. Concentrated extract was loaded
on the column and eluted with 100 mL of hexane (F-1), 100
mL of 4:1 hexane/DCM mixture (F-2), and 100 mL of DCM
mixture (F-3). The latter two fractions contained PHCs. They
were concentrated to 2 mL, and aliquots were placed in 200 μL
glass inserts of GC vials. Injection standard BB209 (0.525 ng)
was added to the insets prior to instrumental analysis.
Separation and quantification of PHCs were performed on
the Agilent 6890/5973 GC−ECNI−MS system with the use of
the 15 m Restek Rtx1614 column described above, and MS was
operated in the selective ion monitoring (SIM) mode. Other
instrumental conditions were the same as those used in
qualitative analyses. Table 1 lists the ions used for
quantification (79Br− and 81Br−) for each PHC as well as
their respective qualifier ions for confirmation during ENCI−
LRMS analyses. The molecular ion and an additional fragment
ion for each analyte under EI full scan are also included in
Table 1.
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Figure 3. (Top) ECNI (scan range m/z 35−800) and (bottom) EI (scan range m/z 50−800) mass spectra (GC−LRMS) of UNC-13 in the extract
of Lake Michigan sediment samples.
the identities of UNC-1−UNC-15 as a suite of polyhalogenated
carbazoles, including eight polybrominated carbazoles and
seven mix-halogenated carbazoles (i.e., containing more than
one type of halogen atom) (Table 1). The identification and
characterization of these two groups are separately discussed
below.
Polybrominated Carbazoles. 3,6-DiBC and 1,3,6,8-TeBC
were among the initially targeted analytes of this work. The EI
and ECNI full-scan mass spectra of their standards are given in
Figures S3 and S4 of the Supporting Information, respectively.
Figures S5 and S6 of the Supporting Information display the EI
and ECNI full-scan spectra of these two compounds in a
sediment extract, respectively, which agree well with those of
the standards. In the EI−MS spectrum of 1,3,6,8-TeBC, the
molecular ion cluster is centered at m/z 482.7, representing the
[M+4]+ ion, and no ions were observed above this cluster to
the maximum measured m/z value of 800 by the GC−LRMS
analysis and 1000 by the GC−HRMS analysis. The major
fragment ions included [M−Br]+, [M−2Br]+, [M−3Br]+, and
[M−4Br]+ (see Figures S4 and S6 of the Supporting
Information). The GC−HRMS analysis of the sediment extract
further confirmed the elemental composition of C12H5NBr4 for
the peak corresponding to 1,3,6,8-TeBC in standard solution,
with the exact mass of molecular ion [M]+ being 478.7155,
which matches well with the calculated mass of 478.715591 Da.
In addition to 3,6-DiBC and 1,3,6,8-TeBC, eight other
brominated carbazoles substituted with different numbers of
bromine atoms were identified in the sediment extracts (Table
1, Figure 1, and Figure S1 of the Supporting Information).
Their MS spectra are presented in Figures S7−S14 of the
Supporting Information. These brominated UNCs included a
to-noise ratio) for 3,6-DiBC and 1,3,6,8-TeBC are 2.3 and 0.6
pg, respectively, when they were analyzed on GC−ECNI−
LRMS.
■
RESULTS AND DISCUSSION
The extensive cleanup and fractionation of sediment extracts
facilitated the acquisition of high-quality GC chromatograms
and mass spectra of UNCs. Figure 1 and Figure S1 of the
Supporting Information show the chromatograms of optimally
isolated fractions of a sediment extract obtained using GC−
LRMS with ECNI and EI ion sources, respectively. This sample
was obtained from the deepest segments of core M041, where
interference from emerging halogenated flame retardants is
negligible. A total of 15 halogenated UNCs as well as 3,6-DiBC
and 1,3,6,8-TeBC were observed in the sediment extracts. For
comparison, GC chromatograms of a mixture of halogenated
flame retardant standards generated under the same instrumental conditions are included in Figure 1 and Figure S1 of the
Supporting Information. Both the 15 m and 30 m GC columns
resulted in the same elution order of targeted analytes, which
were separated from each other as well as from most other
halogenated compounds in fractionated sample extracts.
The mass spectra of individual compounds are shown in
Figure 2, Figure 3, and Figures S2−S21 of the Supporting
Information. The UNCs differed from the known halogenated
analytes in mass spectra. Full-scan EI spectra revealed that the
molecular ion of the UNCs appeared at odd m/z values.
According to the nitrogen rule of MS, the odd number of
nominal mass implies an odd number of nitrogen atoms in the
molecule.20 In the absence of chemical reference standards,
evidence from both LRMS and HRMS spectra strongly support
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dibrominated (UNC-3), five tribrominated (UNC-2, UNC-4,
UNC-6, UNC-8, and UNC-10), a tetrabrominated (UNC-9),
and a pentabrominated carbazole (UNC-12). The identification
was mainly based on the accurate mass determination of
molecular ions (Table 1) and the interpretation of both EI−MS
and ECNI−MS spectra. The EI or ENCI mass spectra of these
UNCs consistently revealed fragmentation patterns of consecutive loss of bromines, similar to those of 3,6-DiBC or 1,3,6,8TeBC (see Figures S3 and S4 of the Supporting Information).
The EI ionization of all polybrominated carbazoles consistently
produced a fragment ion cluster centered around m/z 162,
which appears to represent the backbone of substituted
carbazoles. The GC−HRMS analyses further supported the
identity of these UNCs as polybrominated carbazoles. For
example, the accurate mass determination suggested an
elemental composition of C12H4NBr5 for UNC-12, which has
the exact mass of 556.6261 Da for its molecular ion. This agrees
well with its calculated mass 556.626114 Da (Table 1). On the
basis of the GC retention times and the MS spectra, UNC-8
and UNC-12 appear to be a tribromocarbazole (see Figure S15
of the Supporting Information) and a pentabromocarbazole
(see Figure S16 of the Supporting Information) present as
impurities in the 1,3,6,8-TeBC chemical standard. The
substitution positions of bromine atoms on the carbazole
backbone remain unknown. In laboratory syntheses, direct
bromination of carbazole appears to favor 3, 3,6, 1,3,6, and
1,3,6,8 substitution patterns for mono-, di-, tri-, and
tetrahomologues, respectively.21
Mixed Halogenated Carbazoles. The EI and ECNI fullscan mass spectra of seven mixed halogenated carbazoles
(UNC-1, UNC-5, UNC-7, UNC-11, UNC-13, UNC-14, and
UNC-15) are presented in Figures 2 and 3 as well as Figures
S17−S21 of the Supporting Information. The analyses of both
low- and high-resolution mass spectra suggest that these peaks
correspond to a suite of compounds structurally similar to
brominated carbazoles but containing more complicated
compositions of halogen substituents. Because UNC-7
exhibited a high response during the EI analysis, it is used as
an example to illustrate the structural identification strategy.
The ECNI mass spectrum of UNC-7 reveals high relative
abundances of the [Br]− ions at m/z 79 and 81 (top of Figure
2). It also exhibits successive losses of two bromine atoms,
resembling the mass spectrum of 1,3,6,8-TeBC. In EI scan
(bottom of Figure 2), an ion cluster centered at m/z 438.7
dominates the abundances of fragment ions and appears to be
the molecular ion cluster because essentially no ions were
observed above the m/z 438.7 cluster to the maximum m/z
800. This molecular ion cluster exhibits an isotopic signature
corresponding to the presence of one chlorine and three
bromine atoms.20 On the basis of the calculated isotope pattern
of ClBr3, [M+4]+ should be the base peak and the calculated
relative abundance of [M]+, [M+2]+, [M+6]+, and [M+8]+ to
[M+4]+ would be 26.2, 85.3, 48.7, and 7.88%, respectively. For
UNC-7, the measured ratios were at 25.5, 84.9, 48.6, and
6.94%, respectively, agreeing well with the calculated values.
The GC−HRMS analysis revealed the elemental composition
of UNC-7 to be C12H5NClBr3 because the accurate mass [M]+
(12C121H514N35Cl79Br3) was 434.7661 Da and is essentially the
same as the calculated mass of 434.766097 (Table 1). Major EI
fragment ions include [M−Br]+, [M−2Br]+, and [M−3Br]+,
resembling the fragmentation pattern of 1,3,6,8-TeBC.
Cleavage of Cl was also observed in the mass spectrum,
producing minor fragment ions, such as [M−Cl]+, [M−Cl−
Br]+, [M−Cl−2Br]+, and [M−Cl−3Br]+ (Figure 2). Cleavage
of all halogen substituents produces a fragment ion cluster
centered at m/z 162.0 (i.e., [M−Cl−3Br]+), which corresponds
to a carbazole ion fragment. Using the same strategy, we
identified three additional halogenated carbazole congeners that
contain both bromine and chlorine atoms, including UNC-1
(C 12 H 6 NClBr2 ), UNC-5 (C 12 H 5 NClBr 3 ), and UNC-11
(C12H4NClBr4).
In addition to bromine and chlorine, iodine was also present
in some halogenated carbazoles found in this work. The ECNI
spectrum of UNC-13 as well as those of UNC-14 and UNC-15
exhibits a major fragment ion cluster centered at m/z 126.9
(Figure 3), which appears to represent the ion of [I]−.22 HRMS
analyses suggest molecular compositions of C12H4NClBr3I for
UNC-13 (centered at m/z 562.6) and C12H4NBr4I for UNC-14
(centered at m/z 608.6). The EI−LRMS ionization of UNC-13
results in a suite of dehalogenated fragment ions, including
[M−Br]+, [M−I]+, [M−Cl−I]+, [M−Br−I]+, [M−Cl−Br−I]+,
[M−2Br−I]+, [M−Cl−2Br−I]+, and [M−Cl−3Br−I]+ (Figure
3). The low concentration of UNC-15 in sediment made it
difficult to determine the actual mass with HRMS and resulted
in a less clear EI−LRMS spectrum compared to those of other
UNCs (see Figure S21 of the Supporting Information).
Nonetheless, its ECNI−LRMS and high m/z portion of its
EI−LRMS spectra (see Figure S21 of the Supporting
Information) indicate an ionization fragment pattern similar
to those of other halogenated carbazoles. Therefore, UNC-15 is
tentatively identified as a mixed halogenated carbazole with the
molecular formula of C12H4NBr3I2.
A close examination of the EI mass spectra revealed the
possible presence of a doubly charged ion of [M]2+ with m/z
value of M/2, in some PHCs. For example, in the EI spectra of
UNC-7 (Figure 2) and UNC-13 (Figure 3), the ion clusters
centered at m/z 219 and 282 may represent their respective
doubly charged molecular ion [M]2+. Doubly charged ions were
also found in other PHCs, such as tribromocarbazole UNC-2
(see Figure S7 of the Supporting Information), UNC-11 (see
Figure S19 of the Supporting Information), pentabromocarbazole UNC-12 (see Figure S14 of the Supporting Information),
and UNC-14 (see Figure S20 of the Supporting Information)
with m/z values of 202.3, 258.3, 280.1, and 303.2, respectively.
For dibromocarbazoles (see Figures S3, S5, and S8 of the
Supporting Information) and tetrabromocarbazole (see Figures
S4, S6, and S12 of the Supporting Information), the ion clusters
of m/z 163−165 and 240−242 may represent doubly charged
ions and/or dehalogenated fragment ions. Some PHCs could
also produce doubly charged ions after losing two halogens. For
example, UNC-12 and UNC-14 both exhibit an EI fragment
ion cluster centered at m/z 200, which might represent
[M−2Br]2+ and [M−I−Br]2+, respectively. Carbazole itself also
exhibits a doubly charged ion with m/z 84 (see Figure S2 of the
Supporting Information). Doubly charged ions are typical for
polycyclic aromatic hydrocarbons (PAHs)20 and dibenzofuran.23 Characteristic doubly charged ions have been observed in
mass spectra of polybrominated diphenoxybenzenes.24 PBDE
congeners can also produce doubly charged ions of [M−2Br]2+
upon EI ionization, particularly for those with no ortho bromine
or having 2,3- substitution on one ring and no ortho bromines
on the other.25 The formation of a dibenzofuran-like fragment
ion during EI ionization of PBDEs was proposed.26 In the
present study, the doubly charged ion [M]2+ observed in the EI
spectra of halogenated carbazoles may reflect their structural
similarity to halogenated dibenzofurans.
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Article
Figure 4. Depth profiles of PHCs and BDE209 in Lake Michigan sediment cores M018 (red) and M041 (blue).
Concentrations and Depth Profile. Concentrations of
3,6-DiBC and 1,3,6,8-TeBC ranged from 1 to 41 ng/g of dw
and from 7 to 65 ng/g of dw, respectively. The latter is similar
to the concentration range (i.e., from nd to 54 ng/g) reported
by Zhu and Hites in their sediment samples from Lake
Michigan.16 On the basis of semi-quantified results, in both
cores, the most abundant PHCs were UNC-7 and 1,3,6,8TeBC, followed by UNC-12 and UNC-14. UNC-4, UNC-6,
UNC-8, and UNC-15 had the lowest levels (<4 ng/g of dw).
The peak concentrations of 1,3,6,8-TeBC and UNC-7 were
comparable to the level of decabromodiphenyl ether (BDE209)
in the surface sediments of the cores. In comparison to the
literature values, 1,3,6,8-tetrachlorocarbazole was found at 25
ng/g in the sediment of the Buffalo River, New York.18 A
dibromocarbazole was found with an estimated concentration
of 93 ng/g in one of the marine sediment samples, along with
3-chlorocarbazole and a dichlorocarbazole in soil, river, and
marine sediments near the city of Kavala, Greece.14 Chlorinated
carbazoles were also found in soils and sediments from both
polluted and unpolluted sites.11,15,17
Profiles of concentrations of 3,6-DiBC and 1,3,6,8-TeBC as a
function of depth were different (Figure 4). Concentrations of
3,6-DiBC tended to be higher in more recently deposited
sediments than in the deep sediments. Similar trends were
observed for UNC-3, UNC-6, UNC-8, and UNC-10, which are
dibromo- or tribromocarbazoles. In contrast, 1,3,6,8-TeBC as
well as most other UNCs, including all of the mixed
halogenated UNCs, reached maxima at a depth of 12−16 cm
and remained relatively high and constant in deeper sediments
at both locations; however, the concentrations have dropped
considerably in more recently deposited sediments with depth
of <10 cm. All of these trends are distinctly different from that
of BDE209, as shown in Figure 4.
Collectively, a total of 17 polyhalogenated carbazoles were
found in sediments of Lake Michigan. Halogen substitution
patterns include Br2−Br5, ClBr2, ClBr3, ClBr4, ClBr3I, Br4I, and
Br3I2. This is the first study discovering a suite of mixed
halogenated carbazoles in the environment. Their sources
remain unclear. Chlorinated and brominated carbazoles may be
formed enzymatically by some natural biogeochemical
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Environmental Science & Technology
processes.12,27 However, given the high levels found near
potential pollution sources, they may also result from
anthropogenic processes, such as dye manufacturing and
chloralkali production of chlorine.18,28 Iodine-containing
natural products are relatively rare,29 and no other study has
reported iodinated carbazoles in the environment. Carbazole
and its derivatives were found to be carcinogenic and
mutagenic,30−32 and 3,6-dichlorocarbazole has dioxin-like
toxicity17 in some animal studies. Therefore, the occurrence,
spatial distribution, temporal trends, sources and emissions,
environmental transport and fate, and ecotoxicity and impact
on human health of halogenated carbazoles warrant future
research.
■
ACKNOWLEDGMENTS
■
REFERENCES
This research was part of the Great Lakes Sediment
Surveillance Program (GLSSP) funded by a Cooperative
Agreement from the U.S. EPA Great Lakes Restoration
Initiative with Assistance #GL-00E00538 (U.S. EPA Program
Officer Todd Nettesheim). Jiehong Guo was also supported by
the Predoctoral Fellowship provided by the Institute for
Environmental Science and Policy at the University of Illinois at
Chicago. John P. Giesy acknowledges the support to his work
provided by the Department of Biology and Chemistry and
State Key Laboratory in Marine Pollution, City University of
Hong Kong.
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S Supporting Information
*
Full name, GC retention time, and cleanup fraction of UNCs
and standards (Table S1), partial GC−EI−LRMS chromatograms of deep sediment (26−30 cm) extract from sampling site
M041 (A, F-2; B, F-3) and (C) standard mixture of XFRs
(Figure S1), EI−MS mass spectrum of carbazole in NIST2010
Mass Spectra Library (Figure S2), ECNI and EI mass spectra of
3,6-dibromocarbazole chemical standard (Figure S3), ECNI
and EI mass spectra of 1,3,6,8-tetrabromocarbazole chemical
standard (Figure S4), ECNI and EI mass spectra of 3,6dibromocarbazole in Lake Michigan sediments (Figure S5),
ECNI and EI mass spectra of 1,3,6,8-tetrabromocarbazole in
Lake Michigan sediment (Figure S6), ECNI and EI mass
spectra of UNC-2 in Lake Michigan sediment (Figure S7),
ECNI and EI mass spectra of UNC-3 in Lake Michigan
sediment (Figure S8), ECNI and EI mass spectra of UNC-4 in
Lake Michigan sediment (Figure S9), ECNI and EI mass
spectra of UNC-6 in Lake Michigan sediment (Figure S10),
ECNI and EI mass spectra of UNC-8 in Lake Michigan
sediment (Figure S11), ECNI and EI mass spectra of UNC-9 in
Lake Michigan sediment (Figure S12), ECNI and EI mass
spectra of UNC-10 in Lake Michigan sediment (Figure S13),
ECNI and EI mass spectra of UNC-12 in Lake Michigan
sediment (Figure S14), ECNI and EI mass spectra of an
impurity tribromocarbazole in the chemical standard of 1,3,6,8tetrabromocarbazole (Figure S15), ECNI mass spectrum of an
impurity pentabromocarbazole in the chemical standard of
1,3,6,8-tetrabromocarbazole (Figure S16), ECNI and EI mass
spectra of UNC-1 in Lake Michigan sediment (Figure S17),
ECNI and EI mass spectra of UNC-5 in Lake Michigan
sediment (Figure S18), ECNI and EI mass spectra of UNC-11
in Lake Michigan sediment (Figure S19), ECNI and EI mass
spectra of UNC-14 in Lake Michigan sediment (Figure S20),
and ECNI and EI mass spectra of UNC-15 in Lake Michigan
sediment (Figure S21). This material is available free of charge
via the Internet at http://pubs.acs.org.
■
■
Article
AUTHOR INFORMATION
Corresponding Authors
*Telephone: 1-618-453-6946. Fax: 1-618-453-6944. E-mail:
dachen@siu.edu.
*Telephone: 1-312-996-9597. Fax: 1-312-413-9898. E-mail:
anli@uic.edu.
Notes
The authors declare no competing financial interest.
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Article
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K.-W. Degradative fate of 3-chlorocarbazole and 3,6-dichlorocarbazole
in soil. Environ. Sci. Pollut. Res. 2011, 18 (4), 547−555.
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Tetrachloro-9H-carbazole, a previously unrecognized contaminant in
sediments of the Buffalo River. J. Great Lakes Res. 1984, 10 (2), 210−
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analysis. Environ. Sci. Technol. 2014, 48 (16), 9591−9599.
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Handbook of GC/MS; Wiley-VCH Verlag GmbH and Co. KGaA:
Weinheim, Germany, 2008; pp 293−420.
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Compounds, Indole and Carbazole Systems; John Wiley and Sons:
Hoboken, NJ, 2009; Vol. 8.
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polybrominated diphenyl ethers (PBDEs) in dust from personal
automobiles in conjunction with studies on the photochemical
degradation of decabromodiphenyl ether (BDE-209). Environ. Int.
2011, 37 (5), 899−906.
(23) Pring, B. G.; Stjernst, N. E. Complex dibenzofurans. XII. Use of
oxygen-18 labelling in interpretation of mass spectra of some biphenyl
and dibenzofuran derivatives. Acta Chem. Scand. 1968, 22 (2), 549−
561.
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Potter, D. Novel methoxylated polybrominated diphenoxybenzene
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2011, 45 (22), 9523−9530.
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G. Dependence of mass spectrometric fragmentation on the bromine
substitution pattern of polybrominated diphenyl ethers. J. Am. Soc.
Mass Spectrom. 2014, 25 (6), 1058−1067.
(26) Hites, R. A. Electron impact and electron capture negative
ionization mass spectra of polybrominated diphenyl ethers and
methoxylated polybrominated diphenyl ethers. Environ. Sci. Technol.
2008, 42 (7), 2243−2252.
(27) Gribble, G. W. Natural organohalogens: A new frontier for
medicinal agents? J. Chem. Educ. 2004, 81 (10), 1441.
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Sasaki, Y.; Weber, R. The fingerprint of chlorinated aromatic
compounds in contaminated sites from chloralkali process and a
historic chlorine production using GC-HR-TOF-MS screening.
Organohalogen Compd. 2009, 71, 002224−002229.
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A Comprehensive Update; Springer: New York, 2009; Vol. 91.
(30) Tsuda, H.; Hagiwara, A.; Shibata, M.; Ohshima, M.; Ito, N.
Carcinogenic effect of carbazole in the liver of (C57BL/6N × C3H/
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Supporting Information
(es-2014-03936u)
Polyhalogenated Carbazoles in Sediments of Lake Michigan – A New Discovery
Jiehong Guo, Da Chen*, Dave Potter,
Karl J. Rockne, Neil C. Sturchio, John P. Giesy, An Li*
* Corresponding authors: Dr. An Li, phone: 1-312-996-9597, email: anli@uic.edu
and Dr. Da Chen, phone: 1-618-453-6946, email: dachen@siu.edu
Table of Content
Table S1.
Figure S1.
Figure S2.
Figure S3.
Figure S4.
Figure S5.
Figure S6.
Figure S7.
Figure S8.
Figure S9.
Figure S10.
Figure S11.
Figure S12.
Figure S13.
Figure S14.
Figure S15.
Figure S16.
Figure S17.
Figure S18.
Figure S19.
Figure S20.
Figure S21.
Full name, GC retention time and cleanup fraction of UNCs and standards
Partial GC-EI-LRMS chromatograms of deep sediment (26-30cm) extract from
sampling site M041 (A: F-2; B: F-3), and standard mixture of XFRs (C).
EI-MS mass spectrum of carbazole in NIST2010 Mass Spectra Library
ECNI and EI mass spectra of 3,6-dibromocarbazole chemical standard
ECNI and EI mass spectra of 1,3,6,8-tetrabromocarbazole chemical standard
ECNI and EI mass spectra of 3,6-dibromocarbazole in Lake Michigan sediments
ECNI and EI mass spectra of 1,3,6,8-tetrabromocarbazole in Lake Michigan
sediment
ECNI and EI mass spectra of UNC-2 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-3 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-4 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-6 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-8 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-9 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-10 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-12 in Lake Michigan sediment
ECNI and EI mass spectra of an impurity tribromocarbazole in the chemical
standard of 1,3,6,8-tetrabromocarbazole
ECNI mass spectrum of an impurity pentabromocarbazole in the chemical
standard of 1,3,6,8-tetrabromocarbazole
ECNI and EI mass spectra of UNC-1 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-5 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-11 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-14 in Lake Michigan sediment
ECNI and EI mass spectra of UNC-15 in Lake Michigan sediment
Table S1. Full name, GC retention time and cleanup fraction of UNCs and standards
Peak
UNC-1
F-BDE69
HBB
BDE49
BDE47
UNC-2
UNC-3
mirex
PBBB
3,6-DiBC
BB101
PBBA
UNC-4
UNC-5
UNC-6
BDE100
TrBC
UNC-7
UNC-8
Dec602
BDE99
HCDBCO
EHTBB
UNC-9
1,3,6,8-TeBC
BB153
BDE154
UNC-10
BDE153
UNC-11
HBCDS
UNC-12
UNC-13
Dec603
Dec604
BDE183
UNC-14
BTBPE
TBPH
UNC-15
syn-DP
Full Name
4’-fluoro-2,3,4,6-tetrabromodiphenyl ether
hexabromobenzene
2,2´,4,5´-tetrabromodiphenyl ether
2,2',4,4'-tetrabromodiphenyl ether
mirex
pentabromobenzyl bromide
3,6-dibromocarbazole
2,2’4,5,5’-pentabromobiphenyl
pentabromobenzyl acrylate
2,2,4,4,6-pentabromodiphenyl ether
tribromocarbazole, impurity in 1,3,6,8-TeBC standard
dechlorane 602
2,2,4,4,5-pentabromodiphenyl ether
hexachlorocyclopentadienyl-dibromocyclooctane
2-ethylhexyl2,3,4,5-tetrabromobenzoate
1,3,6,8-tetrabromocarbazole
2,2',4,4',5,5'-hexabromodiphenyl
2,2',4,4',5,6'-hexabromodiphenyl ether
2,2,4,4,5,5-hexabromodiphenyl ether
hexabromocyclododecane
dechlorane 603
dechlorane 604 component A
2,2,3,4,4,5,6-heptabromodiphenyl ether
1,2-bis(2,4,6-tribromophenoxy)ethane
bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate
dechlorane plus (syn)
GC RT#
31.04
31.14
31.94
32.30
32.93
33.06
33.95
33.97
34.09
34.25
34.70
35.36
35.54
35.78
35.83
35.87
36.02
36.05
36.06
36.66
36.83
36.92
37.20
37.49
37.82
39.11
39.16
39.75
40.36
40.55
41.22
42.17
42.68
42.69
43.53
43.64
44.31
44.69
46.43
46.68
47.47
#
The GC retention times on a 30- m DB-5MS column.
*Fractions collected from the silica gel / alumina chromatographic column in quantitative analysis
Fraction*
F-2
F-2
F-2
F-2
F-2
F-2
F-3
F-2
F-2
F-3
F-2
F-3
F-3
F-2
F-3
F-2
F-3
F-2
F-3
F-2
F-2
F-2
F-2 and F-3
F-2
F-2
F-2
F-2
F-3
F-2
F-2
F-3
F-2
F-2
F-2
F-2
F-2
F-2
F-2
F-3
F-2
F-2
Figure S1. Partial GC-EI-LRMS chromatograms (30m DB-5MS column, full scan m/z 35 -800, EIC ions: 325, 359, 403, 439, 481,
517, 561, 563, 608 and 655) of deep sediment (26-30cm) extract from sampling site M041 (A: F-2; B: F-3), and standard mixture of
XFRs (C).
167
100
50
NH
0
28 32
20
30
(mainlib) Carbazole
39
40
50
50
57
63
60
70 74
70
139
84
98
80
90
100
113
110
127
120
130
140
150
160
170
Figure S2. EI-MS mass spectrum of carbazole in the NIST2010 Mass Spectra Library (National Institute of Standards and
Technology, Gaithersburg, MD).
180
Figure S3. Mass spectra of 3,6-dibromocarbazole standard, by ECNI-MS with scan range m/z 35 -800 (top) and EI-MS with scan
range m/z 40 - 800 (bottom).
Figure S4. Mass spectra of 1,3,6,8-tetrabromocarbazole chemical standard, by ECNI-MS with scan range m/z 35 -800 (top) and EIMS with scan range m/z 40 - 800 (bottom).
x10 5 + Scan (20.56-20.59 min, 6 Scans) 2011M041MC-20 F-3 SCAN.D Subtract (2)
5
324.9
4.5
4
3.5
3
2.5
2
1.5
79.0
1
0.5
0
x10 6 +EI Scan (34.28-34.32 min, 7 Scans) 2011M041MC-1920 F-3 SCAN.D Subtract
6
324.7
5
4
3
82.8
2
163.8
1
106.8
119.2
243.8
137.9
0
70
80
90
100 110
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340
Counts vs. Mass-to-Charge (m/z)
Figure S5. Mass spectra of 3,6-dibromocarbazole in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top)
and EI-MS with scan range m/z 50-800 (bottom).
Figure S6. Mass spectra of 1,3,6,8-tetrabromocarbazole in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800
(top) and EI-MS with scan range m/z 50-800 (bottom).
Figure S7. Mass spectra of UNC-2 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
x10 5 -CI Scan (33.92-33.97 min, 14 Scans) 2011M041MC-20 F-3 30MSCAN.D
5.5
5
325.2
79.1
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
x10 7 +EI Scan (33.97-34.02 min, 10 Scans) 2011M041MC-1920 F-3 SCAN.D Subtract (2)
1.1
324.7
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
241.8
163.7
82.7
0.2
119.2
0.1
137.9
272.0
0
80
90
100
110
120
130
140
150
160
170
180 190 200 210 220 230
Counts vs. Mass-to-Charge (m/z)
240
250
260
270
280
290
300
310
320
Figure S8. Mass spectra of UNC-3 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
330
x10 4 + Scan (20.33-20.40 min, 9 Scans) 2011M041MC F-3 SCAN.D Subtract (3)
3.5
402.8
3
2.5
81.0
2
1.5
324.9
1
258.1
0.5
286.2
0
x10 6 +EI Scan (35.58-35.60 min, 4 Scans) 2011M041MC-1920 F-3 SCAN.D Subtract
1.1
402.7
1
0.9
0.8
0.7
182.9
168.9
0.6
0.5
0.4
253.8
154.9
121.0
82.0
0.3
196.9
224.9
140.9
96.0
261.8
244.8
323.7
110.0
0.2
285.8
0.1
304.8
0
80
100
120
140
160
180
200
220
240
260
280
Counts vs. Mass-to-Charge (m/z)
300
320
340
360
380
400
Figure S9. Mass spectra of UNC-4 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
420
x10 4 + Scan (20.47-20.53 min, 8 Scans) 2011M041MC F-3 SCAN.D Subtract (3)
3.5
404.8
3
2.5
2
1.5
79.0
1
0.5
325.9
0
x10 6 +EI Scan (35.86-35.91 min, 9 Scans) 2011M041MC-1920 F-3 SCAN.D Subtract
1.4
404.7
1.2
1
0.8
0.6
97.9
71.0
111.0
82.0
119.8
0.4
136.9
0.2
163.9
242.8
202.1
176.9
261.8
218.9
290.8
323.7
363.9
0
80
100
120
140
160
180
200
220
240
260
280
Counts vs. Mass-to-Charge (m/z)
300
320
340
360
380
400
Figure S10. Mass spectra of UNC-6 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S11. Mass spectra of UNC-8 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S12. Mass spectra of UNC-9 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S13. Mass spectra of UNC-10 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S14. Mass spectra of UNC-12 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S15. Mass spectraof an impurity tribromocarbazole in thechemical standard of 1,3,6,8-tetrabromocarbazole, by ECNI-MS with
scan range m/z 35 -800 (top) and EI-MS with scan range m/z 40 - 800 (bottom).
Figure S16. Mass spectraof an impurity pentabromocarbazole in the chemical standard of 1,3,6,8-tetrabromocarbazole, by ECNI-MS
with scan range m/z 35 – 800.
Figure S17. Mass spectra of UNC-1 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S18. Mass spectra of UNC-5 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800(top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S19. Mass spectra of UNC-11 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
303.2
Figure S20. Mass spectra of UNC-14 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).
Figure S21. Mass spectra of UNC-15 in sediments from Lake Michigan, by ECNI-MS with scan range m/z 35-800 (top) and EI-MS
with scan range m/z 50-800 (bottom).