Kuehl et al. 1
'etrachloro-9H-Carbazole, a Previously Unrecognized Contaminant in Sediments
of the Buffalo River
Douglas W. Kuehl
Environmental Research Laboratory-Duluth
U.S. Environmental Protection Agency
6201 Congdon Boulevard
Duluth, Minnesota 55804
and
Elizabeth Durban, Brian C. Butterxrorth, and Darcy Linn
Center for Lake Superior Studies
University of Wisconsin-Superior
Superior, Wisconsin 54880
Running Head: Tetrachloro-9H-Carbazole in Sediments
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Kuehl et al. 2
ABSTRACT
Three sediment samples taken from the Buffalo River, New York, have been
analyzed for polychlorinated planar molecules similar in structure to the
highly toxic 2 ,3,7,8-TCDD. Although no TCDD was found, residues of
chlorinated dibenzofuran, fluorene, biphenylene, phenanthrene (anthracene),
naphthalene, and 9H-carbazole were identified. 1,3,6,8-tetrachloro-9H-
carbazole was identified as the major chemical of this type in the sediments,
and quantified at levels as high as 25 ng/g at one site.
Additional Index Words: Toxic substances, dioxin, chlorinated hydrocarbons,
GC/MS
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Kuehl et aL. 3
INTRODUCTION
The Buffalo River in New York, which receives both industrial and
municipal sewage discharges, is recognized as one of the most heavily
polluted river systems in the United States. Fishes in the Buffalo River.
were found to exhibit a high incidence of tissue lesions, which led
investigators to correlate high levels of mutagenicity (Ames Test) to
polynuclear aromatic hydrocarbons in proximity to a dye manufacturing
plant (Black et al. 1980). Other studies showed that sediments from this
section of the river also contained high levels of aromatic amines (Diachenko
1979, Nelson and Kites 1980). Many textile dyes containing araino groups have
been shown to be mutagenic by the Ames Test (Friedman et al. 1980).
For the past several years, our lab has been involved in the analysis of
Great Lakes fish and sediment samples for unusual polychlorinated chemicals
that are not routinely monitored, such as polychlorinated styrenes (Kuehl et
al. 1981). These studies are an essential part of hazard assessment for the
environmental regulation of industrial activity, and can be used for
establishing priorities in regulatory and health related research. This work
has continued with exploratory studies similar to those we have previously
published (Kuehl et_ aK 1980).
This paper reports GC/MS analysis of a previously unrecognized
environmental contaminant identified in a sample extract that contains a very
select class of chemicals, polychlorinated olanar aromatic compounds. This
class of chemicals contains the very toxic chlorinated dibenzo-_p-dioxins and
dibenzofurans, which have previously been shown to cause edemas in fish
(Helder 1981). This study examines sediments from three sites in the Buffalo
River near Lake Erie, and is a result of a continued concern over high levels
of potentially hazardous chemicals in sediments and fish from this river.
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Kuehl et al. 4
MATERIALS AND METHODS
Sample Collection
Core samples were collected by field crews of the U.S. Environmental
Protection Agency, Great Lakes National Program Office, Chicago, Illinois,
and the top 6 in. were shipped frozen in glass jars to the Environmental
Research Laboratory-Duluth (ERL-D), where they remained frozen at -20°C.
Sample Preparation
Two sets of samples were prepared for GC/MS analysis, A) a composite of
each of three sediments was used for qualitative analysis, and B) a set of
individual sediments was used for quantitative analysis.
Each jar of sediment was allowed to thaw, stirred 5 minutes and
transferred to a funnel where the excess water was allowed to drain off (5%
moisture). Subsamples (10 g) of each sediment were combined and blended to
yield the composite sample used for qualitative GC/MS analysis. In addition,
a 10 g subsample to be used for quantitative analysis of each sediment was
spiked with 1.4 ng 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) as a
surrogate spike used to evaluate the efficiency of the sample preparation
method. The samples were then thoroughly blended with an equivalent weight
of glass beads (140 mesh), and soxhlet extracted for 24 hours with
hexane/acetone (1:1) (Burdick and Jackson). The soxhlet extraction round
bottom flask contained 1-g freshly cleaned copper for sulfur removal.
The samples were then reduced to a volume of 5 mL in the round bottom
flask fitted with a three-ball Snyder column. Each sample was transferred to
a 2 cm x 50 cm glass column containing 20 g Celite 545 (Baker Chemical Co.)
coated with 10 mL sulfuric acid (Ultrex, Baker Chemical Co.) and eluted with
200 mL hexane. The eluent continued to flow through a second column, 9 mm x
25 cm, containing 1 g cesium silicate (Stalling et al. 1981), and was
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Kuehl et al. 5
collected in a Kuderna-Danish (K-D) apparatus. The samples were again
reduced in volume to 5 mL for Chromatography on the carbon-glass system.
Carbon-Glass Column Chromatography
The carbon-glass column (CGC) was prepared by blending (Waring blender)
AMOCO PX-21 carbon (50 mg) with a shredded glass filter pad (600 rag) in -
methylene chloride (100 mL) for two min. The carbon coated glass fibers were
slurry packed into a 1 cm x 4 cm glass column fitted with glass fiber pads
and teflon end fittings.
The sample was pumped (FMI Corp., G-50) through an all teflon 8-way
Figure valve (Rainin) onto the column at a rate of 4 mL/min (Figure 1). Non-planar
1 near
here molecules were eluted with methylene chloride (50 mL) . Loosely bound planar
molecules were eluted with methylene chloride/benzene (1:1, 50 mL) while
tightly bound planar molecules were eluted with toluene (50 mL) flowing in
the reverse direction to the previous fractions. Both the benzene and
toluene fractions were reduced in volume on a rotary vacuum evaporator to
^0.5 mL. Samples were concentrated to 100 yL with dry ^(g) prior to
GC/MS analysis.
Gas Chromatography - Mass Spectrometry
Identification of polychlorinated planar chemical residues in the
composite sample was done by computer library search and manual
interpretation of GC/MS data. Spectra were generated on a Finnigan-MAT
4500-INCOS GC/MS/computer system in the electron impact ionization mode at 70
eV, by scanning m/z 50-550 every second. Samples were chromatographed on a
30 m x 0.25 mm SE-54 fused silica capillary column temperature programmed
from 80° to 250°C at 4°/min. Helium was used as the carrier gas at a linear
velocity of 35 cm/sec.
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Kuehl et al. 6
Quantification of 1,3,6,8-tetrachloro-9H-carbazole (1,3,6,8-TCC), and
determination of the percent recovery of the surrogate spike, 1,2,3,4-TCDD
were done on the same system using the multiple ion detection (MID) mode with
dibromobiphenyl as an internal standard, and using a set of external
standards of 1,2,3,4-TCDD and 1,3,6,8-TCC. Because of potential interfer-
ences in the analysis of 1,3,6,8-TCC from the molecular ions of isomers of
tetrachlorodibenzofurans (TCDF), the molecular ions of TCDF were also
monitored. The following nominal masses were monitored during a 2 sec. scan
period at the appropriate retention time window: dibromobiphenyl, 310;
1,2,3,4-TCDD, 320, 322; 1,3,6,8-TCC, 303, 305; and TCDF, 304, 306. Quantifi-
cation of 1,2,3,4-TCDD was based upon the response of mv/z 320 and
quantification of 1,3,6,8-TCC was based upon the response of m/z 303.
Synthesis of 1,3,6,8-Tetrachloro-9H-Carbazole
9H-carbazole (100 mg) (Chemical Service, Inc.) was added to a 500 mL
round bottom flask and dissolved in methylene chloride (100 mL). To this,
methylene chloride saturated with chlorine (100 mL) was slowly added over 30
minutes. The reaction was allowed to stir for 24 hours at room temperature.
The solution was transferred to a 500 mL beaker and the solvent was allowed
to evaporate off to yield light brown crystals. The product was transferred
to a filter funnel and washed with cold iso-octane to yield 165 mg white
crystals, m.p. 221-222°C uncorrected. GC/MS analysis showed the product to
be 95% tetrachloro-9H-carbazole, 3% trichloro-9H-carbazole and 2%
pentachloro-9H-carbazole. Nuclear magnetic resonance (NMR) showed protons at
H^> 8.25; Kg, 7.60; and HJJ , 1.95 ppm down field from tetramethyl silane
and integrating 2:2:1, respectively.
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Kuehl et al. 7
RESULTS AND DISCUSSION
A number of polychlorinated chemicals frequently reported in
environmental sample monitoring studies were identified by mass spectral
library search (INCOS FIT >850 of 1,000) in the planar molecular fraction of
the Buffalo River composite sediment extract. These chemicals include
pentachloroohenol, pentachlorotoluene, and various isomers of dichloro-
through hexachlorobenzene, dichloro- through octachloronaphthalene,
trichloro- through pentachlorodibenzofuran, and octachlorodibenzofuran. No
isomers of TCDD were found by plotting mass chromatograms of the molecular
ions m/z 320, 322, 324 and evaluating for a tetrachloro isotope pattern.
However other planar compounds were tentatively identified based upon
Table 1 molecular ion data and retention time window data (Table 1) . These include
near here
isomers of trichloro- and tetrachlorophenanthrene (anthracene), pentachloro-
biphenylene, pentachlorofluorene, and pentachloro-9H-carbazole as minor
components. Based upon the GC/MS total ion current peak area of molecular
ions (m'+, m+2+), a tetrachloro-9H-carbazole (TCC) isomer was found at
the highest level of all of the chlorinated planar chemicals identified.
Because the major oeak in the chromatogram was tentatively identified as
a chemical analogous in structure (and in potential biological activity) to
2,3,7,8-TCDD and 2,3,7,8-TCDF, an attempt was made to confirm its structure.
This was accomplished by chlorinating 9H-carbazole in an organic solvent, and
Figure identifying the stable products by GC/MS (Figure 2) and NMR. It was observed
2 near
here that 9H-carbazole very easily added four chlorine to yield one major product.
Because of the ortho-para directing power of the nitrogen, one would predict
the structure to be 1,3,6,8-TCC, which was confirmed by NMR. Moreover, the
1,3,6,8-TCC standard and the major TCC peak in the sediment samples had
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Figure
3 near
here
Table 2
near here
Kuehl et al. 8
identical capillary column GC retention time (+2 sec). These findings
strongly indicate that the major TCC in the sediment appears to be
1,3,6,8-TCC. This cannot be completely established, however, until all 38
TCC congeners have been synthesized and characterized by GC retention time (a
task beyond the scope of this study).
Figure 3 is an example of the ion current profile for the analysis of
TCDFs and TCCs in one of the sediment samples. The peak at spectrum 703 is
the internal standard dibromobiphenyl. Major TCDF peaks are observed at
spectra 882 and 916, and 1,3,6,8-TCC is at spectrum 979. Minor peaks between
scan number 900 and 1200 appear to be a mixture of TCDFs and TCCs. Table 2
lists the sample identification, percent recovery of 1,2,3,4-TCDD, and
quantification of 1,3,6,8-TCC. The recovery of 1,3,6,8-TCC from each step
of the sample cleanup procedure was >95%. The recovery of the surrogate
spike, 1,2,3,4-TCDD, was =?80% for Casenovia Creek and Black Rock Canal
samples, however the determination of percent recovery of TCDD in the "dye
plant" sample was not possible because of a large interference at m/z 322.
The levels of 1,3,6,8-TCC were 0.30 ng/g at Casenovia Creek, 0.60 ng/g at
Black Rock Canal and 25 and 22 ng/g in duplicate analysis of the Dye Plant
site sample. No TCC was found in the procedural blank above 0.10 ng/g.
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Kuehl et al. 9
SUMMARY
Sediments from the Buffalo River have been found to contain a large
number of polychlorinated planar chemicals similar in structure to TCDD and
TCDF. These residues may be partially responsible for the unusual effects
upon Buffalo River fish. Also, in agreement with other published data,
levels of chemical residues found in the river sediment appear to be highest
near an area where a dye manufacturing plant has previously discharged into
the river (map included in reference 3).
Future studies should include the synthesis of all 38 TCC isomers to
verify isomer identification, an evaluation of acute and chronic toxicity of
these chemicals to freshwater fish, and a monitoring program to determine the
source and distribution of these chemicals.
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Kuehl et al. 10
ACKNOWLEDGMENT
The authors wish to thank Dr. Ronald Caple, University of Minnesota-
Duluth for assistance in obtaining NMR data.
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Kuehl et al. 11
REFERENCES
Black, J. J., Holmes, M., Dymerski, P. P., and Zapisek, W. F. 1980. Fish
tumor pathology and aromatic hydrocarbon pollution in a Great Lakes
estuary. In Hydrocarbons and halogenated hydrocarbons in the aquatic
environment, ed. B. K. Afghan, and D. McKay, pp. 559-565. New York:
Plenum 'Press.
Diachenko, G. W. 1979. Determination of several industrial aromatic amines
in fish. Environ. Sci. Technol. 13: 329-333.
Friedman, M., Diamond, M. J., and MacGregor, J. T. 1980. Mutagenicity of
textile dyes. Environ. Sci. Technol. 14: 1145-1146.
Helder, T. 1981. Effects of 2 ,3 , 7 ,8-tetrachlorodibenzo-_p-dioxin (TCDD) on
early life stages of rainbow trout (Salmo gairdneri Richardson).
Toxicology 19: 101-112.
Kuehl, D. W., Johnson, K. L., Butterworth, B. C., Leonard, E. N., and Veith,
G. D. 1981. Quantification of octachlorostyrene and related compounds
in Great Lakes fish by gas chroraatography - mass spectrometry. J^. Great
Lakes Res. 7(3): 330-335.
Kuehl, D. W. , Dougherty, R. C., Tondeur, Y.. Stalling, D. L. , Smith, L. M.,
and Rappe, C. 1980. Negative chemical ionization studies of
polychlorinated dibenzo-p-dioxins, dibenzofurans and naphthalenes in
environmental samples. In Environmental health chemistry - the
chemistry of environmental agents as potential human hazards, ed. J. D,
McKinney, pp. 245-261. Ann Arbor: Ann Arbor Science Publishers, Inc.
Nelson, C. R., and Hites, R. A. 1980. Aromatic amines in and near the
Buffalo River. Environ. Sci. Technol. 14: 1147-1149.
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Kuehl et al. 12
Stalling, D. L., Petty, J. D. , and Smith, L. M. 1981. Chromatographic
enrichment of acidic compounds from organic solvents using alkali metal
silicates. J. Chrqmatogr. Sci. 19: 18-26.
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Kuehl et al. 13
Table 1. GC Retention Time and Major Characteristic Ions of
Polychlorinated Planar Molecule Isomers in Buffalo River Sediment.
Chemical
Hexach lorobenzene
Pent achlorobiphenylene
Tetrachlorodibenzofuran
Tetrachlorodibenzofuran
Pentachlorofluorene
Trichloroanthracene/Phenanthrene
Tetrachloro-9H-Carbazole
Pent ach loro-9H-Carbazo le
Tetrachloroanthracene/Phenanthrene
Octachlorodibenzo furan
Relative
Retention Time* Major Ions
1
1
1
1
2
2
2
2
2
3
.00
.87
.88
.95
.00
.01
.08
.37
.41
.61
C16,
ci5,
C14,
C14,
C15,
C13,
C14,
C15,
C14,
Clg,
m/z
m/z
m/z
ra/z
m/z
m/ z
ml z
m/z
m/z
m/z
282
322
304
304
336
280
303
337
314
440
* Retention time relative to hexachlorobenzene.
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Kuehl et al. 14
Table 2. Buffalo River Sample Identification and Quantification Data
Sample
Code
NS01S55
NS01S79
NS03S66
Sample Latitude
Identification Longitude
Casenovia Creek 42°5ri3"
78C48'48"
Black Rock Canal 42e54'34"
78°54'08"
Dye Plant 42° 51 '43"
78°50'42"
Percent Recovery
1,2,3,4-TCDD 1,3,6,8-TCC
at 0.14 ng/g ng/g*
77 0.30
82 0.60
X** 25,22
* detection limit 0.10 ng/g
**not determined
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Kuehl et al. 15
FIGURE CAPTIONS
Figure 1. Carbon-glass chromatographic system.
Figure 2. Electron impact mass spectrum of 1,3,6,8-tetrachloro-9H-carbazole.
Figure 3. GC/MS total ion current chromatogram for sample NS03566, "Dye
Plant" site.
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Kuehl et al. 16
(Fig. 1)
SAMPLE - SOLVENT RESERVOIR
J1 ""I-* •»
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PUMP
i
CARBON ON GLASS
COLUMN
SOLVENT FLOW
FORWARD
REVERSE
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* \ Xv.
\^ 2^^ ^ \ V EIGHT WAY SLIDE INJECTOR
1 X\^\\XN\
1 \ > 5
[L X-. i/ /
L *»: .ci : .4
* *r- v / 3
\\ /
t
A
SAMPLE COLLECT
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60
lOO.Oi
H
C/>
Ul
— 50.0-
UJ
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UJ
cr
o.o-
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1
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100
^
150
Jk li
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20O 250
MASS NUMBER
300
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Kuehl et al. 18
(Fig. 3)
lOO.On
5O.O-
LU
I
979
916
7O3
600
TOO
882
800 900 IOOO
SPECTRUM NUMBER
noo
1200
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