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


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t
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                                      SAMPLE COLLECT

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 60
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                            20O         250
                        MASS  NUMBER
                                          300

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                                                   Kuehl et al.  18
                                                   (Fig. 3)
  lOO.On
   5O.O-
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I
                                         979
                                   916
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800      900       IOOO
 SPECTRUM NUMBER
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