Envlron. Scl. Technol. 1986, 2 0 , 1144-1150
Nitrogen-Containing Aromatic Compounds in Sediments from a Polluted Harbor in Puget Sound Cheryl A. Krone,* Douglas G. Burrows, Donald W. Brown, Paul A. Roblsch, Andrew J. Friedman, and Donald C. Mallns
Environmental Conservation Division, Northwest and Alaska Fisheries Center, Seattle, Washington 98 112 Creosote oil and organic extracts of marine sediments were subjected to silica/alumina column chromatography to obtain fractions greatly enriched in nitrogen-containing aromatic compounds (NCAC), which were then characterized by gas chromatography (GC) with nitrogen-specific detection and GC/mass spectrometry. A large number of NCAC were identified in sediments from creosote-contaminated Eagle Harbor, Puget Sound, WA, as well as in the sample of commercially available creosote oil. No NCAC were detected in sediments from a pristine reference area (detection limit 10 ng/g). The total NCAC concentration in the Eagle Harbor sediments ranged from about 200 to 1200 pg/g of sediment (dry weight). Because many NCAC are known mutagens/carcinogens/teratogens, their presence in high concentrations in sediments may pose various health risks for marine biota. W
Introduction The occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment has been widely studied. Much less attention has been focused on the types and amounts of heterocyclic analogues of PAH, particularly nitrogencontaining aromatic compounds (NCAC). Since many of the sources of NCAC and PAH are the same (e.g., combustion processes and petroleum products), these heterocycles would be expected to be widespread in areas of concentrated human activities. NCAC have been found in such varied materials as cigarette smoke, auto and diesel exhaust, urban air particulate matter, petroleum, shale oil, coal tar, and synthetic fuels (1-8). The presence of NCAC in the environment is of concern because many of these compounds are known mutagens and/or carcinogens (9). For example, quinoline and all of its monomethyl isomers were found to be mutagens in the Ames Salmonella assay (4). Quinoline, 4-methylquinoline, and 8-methylquinoline also initiated skin tumors in SENCAR mice (IO). Carbazole has been shown to induce neoplastic lesions in the livers and forestomachs of mice when included in the diet (11). 7H-Dibenzo[cg]carbazole exhibits strong heptatocarcinogenic activity in XVIInc/z mice (12). Numerous isomers of benzacridine and their alkylated forms possess carcinogenic properties toward laboratory animals (9). Probably the most well-studied materials with regard to NCAC are petroleum and related products, including crude oils, shale oils, and synthetic fuels. Eleven different &ylbenzo[h]quinolines were found in one crude oil sample (6), and about 150 NCAC were detected in an Arabian crude oil (7). However, only a few of these NCAC were identified by comparison to standard compounds. Concentrations of NCAC in these crude oils ranged from 0.2 to 0.8 mg/kg compared to 2.7 mg/kg for benzo[a]pyrene. Dozens of NCAC including indoles, quinolines, carbazoles, and acridines were found in coal-derived synthetic fuels (8). The presence of a complex series of NCAC in marine sediments from Buzzards Bay, MA, was reported, but individual compounds were not identified (13). Approximately a dozen NCAC were identified by gas chromatog1144
Environ. Sci. Technol., Vol. 20, No. 11, 1986
raphy (GC) in lake and marine sediments (24,15). Their presence was attributed primarily to street dust and/or fallout of atmospheric particulate matter (14, 15). Recently, contaminated sediments from Eagle Harbor, Puget Sound, WA, were found to contain high levels of PAH (16). Bordering the shoreline of Eagle Harbor are the small community of Winslow (popuIation 2500), a Washington State Ferry Terminal, a wood-treatment (creosote) plant, and several marinas. The distribution of PAH in sediment samples from this area was indicative of creosote oil pollution (16). Moreover, Eagle Harbor has recently been placed on the EPA Superfund list and designated for clean-up on the basis of creosote pollution (17). Creosote oil is a complex mixture of chemicals obtained during the distillation of coal tar (18). Substantial amounts of both PAH and NCAC are found in coal tar, up to 30% and 2% by weight, respectively (19). In addition to the high levels of PAH in Eagle Harbor sediments, the NCAC carbazole was present in levels ranging from about 79 to 1300 ng/g of sediment (dry weight) in 13 samples from two sites (16). Several other NCAC were also detected in these samples. Thus, a more extensive study of the identity and concentrations of NCAC in sediments from Eagle Harbor and in a commercial creosote oil was initiated, and the results are reported here. Materials and Methods Reference compounds were obtained from commercial sources (Aldrich, Milwaukee, WI; Pfaltz and Baur, Waterbury, CT; Lancaster Synthesis, Windham, NH; Sigma, St. Louis, MO) with the exception of l-methyl-, 2-methyl-, 3-methyl-, and 4-methylcarbazoles, which were provided by Dr. E. LaVoie, Naylor Dana Institute for Diseases Prevention, and benzo[a]carbazole, which was synthesized (20,21).Sediments were collected from three contaminated areas in Eagle Harbor and from a relatively rural reference site (West Beach, Deception Pass, WA). Extracts of the sediments (representing between 2 and 10 kg of each sediment) and a sample of commercially available creosote (American Tar Co., Seattle, WA) were analyzed for nitrogen-containing compounds by capillary gas chromatography (GC) with nitrogen-phosphorus (NPD), flame ionization (FID), and mass spectrometry (MS) detectors (22). Sediment extracts were prepared by tumbling 100-g portions with CHzClz(500 mL) and 500 g of NaZSO4in l-L glass bottles (2 times for 18 h and once for 6 h). The CH2C12extract was placed in an Erlenmeyer flask, fitted with a three-ball Snyder column, and concentrated to 5 mL, and the CH2Clzwas exchanged with hexane, The organic sediment extracts and the commercial creosote oil were fractionated by silica/alumina column chromatography. The columns were prepared by adding a slurry of 10 g of alumina (activated at 170 "C for 2 h) in CH,Cl2 to a 19 mm i.d. glass column having a 250-mL solvent reservoir. Twenty grams of silica gel [Davison type 923,100-200 mesh (nominal), activated by heating to 700 "C for 18 h and stored at 170 "C]was added to the column as a CHzClzslurry. The nitrogen-enriched (polar) fractions
Not subject to U.S. Copyright. Published 1986 by the American Chemical Society
Table I. Numbers of Nitrogen-Containing Aromatic Compound Isomers in Sediment and Creosote Identified by GC/MS compd class methylpyridines (C2-alky1)pyridines (C, + Cz-alkyl)benzonitriles (C1-alkyl)quinolines,etc.a (C2-alkyl)quinolines,etc. (C3-alkyl)quinolines,etc. (C1-alky1)naphthalenenitriles benzoquinolines, etch
no. of isomers sediment creosote 0 0 0 0
3 4
6 4
2 3 3 10 9 4 7 5
compd class (Cl-alkyl)benzoquinolines, etc. ((2,-alky1)carbazoles (Cz-alkyl)benzoquinolines, etc. (Cz-alkyl)carbazoles benzocarbazoles benzacridines, etcc (C1-alky1)benzacridines
no. of isomers sediment creosote 9
10
4 14 11 4 2 3
4 13 11
4 2 3
a Includes isomers of quinoline and isoquinoline. *Includes all isomers of M , 179, N1 heterocycles, i.e., acridine, phenanthridine, and benzoquinolines. Includes all isomers of M , 229, N1 heterocycles, is., benzacridines, benzophenanthridines, and dibenzoquinolines.
of the sediment extract and creosote oil were obtained by collecting compounds that eluted from the silica/alumina column with 20% methanol in CH,C12, the saturated and aromatic hydrocarbons having been previously eluted with solvents of lesser polarity (22). GC/NPD analysis was carried out on a Varian Model 3700 gas chromatograph equipped with a thermionicspecific (nitrogen-phosphorus) detector, a fused silica capillary column (bonded SE-54, ca. 30 m X 0.25 mm i.d.), and a Varian CDS I11 data system. Initial column oven temperature of 60 "C was held for 1 min and then increased to 290 "C at 4 OC/min. Split injection was employed. The GC/MS system (Hewlett Packard 5840 GC with a 30 m X 0.25 mm i.d. bonded-phase SE-54 fused silica capillary column interfaced to a Finnigan 3200 MS with an INCOS 2300 data system) was operated with a splitless injection for 0.3 min. The initial column temperature of 40 "C was held for 3 min and then increased to 320 "C at 4 OC/min. Compounds were identified by comparison with spectra obtained from standards analyzed under the same conditions, by comparison to the spectra in the NBS/NIH mass spectral library in the data system, and by interpretation.
Results The total PAH and NCAC concentrations in three Eagle Harbor sediment samples were 29 000,1300, and 1100 pg of PAH/g of sediment (dry weight) and 1250,200, and 240 pg of NCAC/g of sediment, respectively. The creosote oil contained 175000 pg of PAH/g and 93 000 pg of NCAC/g. The chromatographic profiles of NCAC in the three Eagle Harbor sediment samples and in the sediment extracts from Eagle Harbor analyzed by Malins et al. (16) were all very similar. Over 200 distinct NCAC were detected by capillary gas chromatography with the nitrogen-phosphorus-specific detector (NPD). Figure 1shows the complex suite of NCAC contained in the nitrogenenriched fractions of a representative sediment from Eagle Harbor chosen for detailed analysis and in the creosote oil. Inspection of the chromatogram (Figure 1)from the commercially available creosote oil reveals a similarly complex pattern of peaks, with many additional peaks representing compounds more volatile than l-naphthalenenitrile (peak 7). NCAC were not detected in the nitrogen-enriahed fraction from a sediment collected in a pristine area (West Beach) (detection limit 10 ng/g of dry weight sediment). PAH in this sediment were below detection limits (4 ng/g of dry weight). The three largest individual peaks in the chromatogram of the Eagle Harbor sediment NCAC fraction were carbazole (peak 12), acridine (peak lo), and l-naphthalenenitrile (peak 7). The concentrations of these compounds
were 18, 6.8, and 7.1 bg/g of sediment (dry weight), respectively, Numerous alkylated derivatives of these compounds were also found including 7 methylnaphthalenenitriles, 4 methylcarbazoles, 11 (C2-alkyl)carbazoles,and S.methylacridines/methylphenanthridines/methylbenzoquinolines (Table I). Total concentrations for each of these four groups of isomers were about 7.7, 18, 13, and 20 pg/g of sediment (dry weight), respectively (Table 11). Creosote oil was found to contain these same substances as well as significant amounts of the more water-soluble, lower molecular weight NCAC. These included pyridine (170 pg/g), benzonitrile (1200 pg/g), quinoline (18 000 pg/g), and isoquinoline (8100 pglg). As above, a variety of alkylated homologues of these compounds were detected (Table I) ranging in individual concentrations from 25 to 3800 pg/g of creosote oil. Phenol and alkylated phenols, naphthol, dibenzofuranol, and several ketones (indanone, acridinone, and benzanthraquinone) were also present in the polar fraction from Eagle Harbor sediment (Table 11). No pentachlorophenol was seen in the polar fractions from either the sediments or creosote oil. There was also little evidence for the presence of significant levels of compounds containing two nitrogen atoms (e.g., diazaarenes), All NCAC identified by GC/MS and their concentrations in the sediment and creosote oil sample are listed in Table 11.
Discussion Over 90 NCAC, many of which have been shown to produce toxic effects in marine organisms, have been identified in Eagle Harbor sediments. This is a far greater number of NCAC than had previously been identified in any marine sediment. The concentrations of NCAC in the Eagle Harbor sediments were 2 to 3 orders of magnitude greater than levels found in various marine sediments examined by other investigators. In general, information on the types and concentrations of NCAC in marine sediments is limited. This may be due in part to the fact that NCAC are reportedly present in the environment at concentrations 1-2 orders of magnitude lower than the more extensively studied PAH (13,14). The total weight of the "azaarene fraction" of marine sediments from Buzzards Bay, MA, and a nearby coastal marsh ranged from 0.14 to 0.21 pg/g of sediment (dry weight) (13). Direct probe fractional distillation of this material produced mass spectra that indicated the presence of NCAC containing three to eight rings and homologues with up to eight alkyl carbons. This analytical technique did not allow identification of individual compounds nor estimates of their concentration. Low levels of NCAC were also found in six sediment samples from diverse areas in Puget Sound (15),where total NCAC (based on the sums of the concentrations for 19 reference compounds) were 0.002-0.050 pg/g of sediEnviron. Sci. Technol., Vol. 20,No. 11, 1986
1145
Table 11. Concentrations of Nitrogen-Containing Aromatic Compounds Found in Eagle Harbor Sediment and Creosote Oil by GC/MS Analysis
EGH sediment ~~
compd pyridine methylpyridine methylpyridine (C2-alkyl)pyridine (Cz-alky1)pyridine (Cz-alky1)pyridine benzonitrile methylbenzonitrile methylbenzonitrile naphthalene (RS)d ((&-alkyl)benzonitrile quinoline (ST)e isoquinoline (ST) indole 2-methylquinoline (ST) 8-methylquinoline (ST) methyl(quinoline/isoquinoline) methyl(quinoline/isoquinoline) 7-methylquinoline (ST) 3-methylquinoline (ST) methyl(quinoline/isoquinoline) 4-methylquinoline (ST) 2,8-dimethylquinoline (ST) methyl(quinoline/isoquinoline) methylindole methyl(quinoline/isoquinoline) methylindole (Cz-alkyl)(quinoline/isoquinoline) 2,7-dimethylquinoline (ST) 2,6-dimethylquinoline (ST) (Cz-alkyl)(quinoline/isoquinoline) (Cz-alkyl)(quinoline/isoquinoline) 2,4-dimethylquinoline (ST) hexamethylbenzene (1S)f methylphenylpyridine (C2-alkyl)(quinoline/isoquinoline) ((&-alkyl)(quinoline/isoquinoline) 1-naphthalenenitrile (ST) (C3-alkyl)(quinoline/isoquinoIine) (C3-alkyl)(quinoline/isoquinoline) 2-naphthalenenitrile (ST) (C3-alkyl)(quinoline/isoquinoline) (C3-alkyl)(quinoline/isoquinoline) methylnaphthalenenitrile methylnaphthalenenitrile methylnaph thalenenitrile methylnaphthalenenitrile methylnaphthalenenitrile methylnaphthalenenitrile methylnaphthalenenitrile biphenylnitrile phenanthrene (RS) 7,8-benzo[h]quinoline (ST) acridine (ST) benzoquinoline phenanthridine 5,6-benzo[flquinoline (ST) carbazole (ST) methyl(acridine/etc.)g methyl(acridine/etc.) 2-methylbenzo[flquinoline (ST) methyl(acridine/etc.) azafluorene methylcarbazole methyl(acridine/etc.) methyl(acridine/etc.) methyl(acridine/etc.) methyl(acridine/etc.) methyl(acridine/etc.) 3-methylcarbazole (ST) 4-methylcarbazole (ST) (C,-alkyl) (acridineletc.)' methylcarbazole (Cz-alkyl)(acridine/etc.) 1146
Mr 79 93 93 107 107 107 103 117 117 128 131 129 129 117 143 143 143 143 143 143 143 143 157 143 131 143 131 157 157 157 157 157 157 162 155 157 157 153 171 171 153 171 171 167 167 167 167 167 167 167 179 178 179 179 179 179 167 193 193 193 193 167 181 193 193 193 193 193 181 181 207 181 207
Envlron. Sci. Technol., Vol. 20, No. 11, 1986
RI"
concn,bvcMg/g
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