Aromatic Amines in and near the Buffalo River Charles R. Nelson and Ronald A. Hites" School of Public and Environmental Affairs and Department of Chemistry, Indiana University, 400 East Seventh Street, Bloomington, Indiana 47405
Three sediment samples taken from the Buffalo River and two soil samplles taken near its bank have been analyzed for 2-propanol-exi;ractable, basic organic compounds by using GC/MS. Eleven aromatic amines related to the commercial production of inalachite green and crystal violet were identified in both the sediment and soil samples. Apparently a dye manufacturing plant used this part of the river bank as a dump, and several of its waste chemicals were leached into the river. It is possible that the compounds reported here are, a t least partially, responsible for tumors observed in fish taken from this river. The Buffalo River in Buffalo, NY, has had many pollution problems resu'lting, at least in part, from the discharge of wastes by several industries located on the river. As a consequence of both chemical and domestic sewage pollution, this river has been classified as one of the most heavily polluted bodies of water in the United States ( 2 ) . Recently, it was reported that several bottom-feeding species of fish obtained from the Buffalo River exhibited a high incidence of proliferative tissue lesions (2) and that sediments from the Buffalo River contained highly mutagenic compounds (2, 3). These latter data, which were the result of Ames bacterial mutagenesis assays, revealed a strong correlation between the level of mutagenic activity of sediment extracts and the proximity of the samplin;: sites to a dye manufacturing plant ( 2 , 3 ) . In addition, several aromatic amines (including l-naphthylamine) which were formerly produced by this dyestuff manufacturer have been detected in fish obtained from the Buffalo River ( 4 ) . Although these data suggest a possible link between the activities of the dyestuff manufacturer and the mutagenic materials present in the river sediment, the specific identities and origins of ithe mutagenic compounds in the sediment remain points of considerable uncertainty and interest. This paper describes the identification of the 2-propanol-extractable, basic organic compounds present in soil and river sediment obtained1 near this dye manufacturing plant and discusses the enviironmental implications of the results. E x p e r i m e n t a l Section
Sampling Procedure and Storage. Sediment samples were collected in midchannel by using an Eckman dredge during June 1979 a t the sampling locations shown in Figure 1.One-quart, precleaned, glass jars were used for the sediment
samples. The jars were covered with aluminum foil before replacement of the screwcap lids and were refrigerated until analyzed. Soil samples were collected in 1978 and were kept frozen until analyzed. Extraction. The sediment samples (140-150 g) and defrosted soil samples (15-20 g) were Soxhlet extracted with 2-propanol (500 mL) for 24 h. The extracting solvent was removed under vacuum, and the residue was redissolved in methylene chloride, Preliminary studies revealed that the sediment extracts were heavily contaminated with aliphatic hydrocarbons. Consequently, the basic compounds were separated by extracting the methylene chloride solution (150 mL) with 1.0 N HzS04 (100 mL). The acidic extract was made basic (pH 12) with aqueous NaOH and extracted with ethyl acetate (2 X 100 mL). The combined ethyl acetate extracts were dried over anhydrous MgS04 and concentrated under reduced pressure. All solvents were of nanograde quality purchased from Mallinckrodt. Instrumentation and Analysis. Preliminary gas-chromatographic analysis of the sample extracts was performed on a 2-mm i.d., 0.25-in. o.d., 6-ft glass column packed with 3% OV-17 on 80/100 mesh Supelcoport. The column was temperature programed from 70 to 310 O C a t 8 OC/min in a Hewlett-Packard 5720A gas chromatograph equipped with a flame ionization detector. Gas-chromatographic mass spectrometry was performed on a Hewlett-Packard 5982A quadrupole mass spectrometer operated under computer control. Electronimpact mass spectra were obtained at 70 eV ionizing energy by continuously scanning a mass range of 50-600 amu every 4 s. The GC conditions described above were also used for packed column GC/MS. A lO-m, high-resolution, glass capillary column coated with SP 2100 (0.25-mm i.d.) and directly interfaced to the mass spectrometer was also utilized in the analyses. The capillary column was temperature programmed from 70 to 260 OC a t 4 OC/min. Results and Discussion
Since it is very difficult to show all of the chromatographic and mass spectral data on which the identifications are based, example data only will be presented here. Figure 2 is the mass spectrum of the most abundant of the identified compounds, bis(4-dimethylaminopheny1)phenylmethane (see Figure 3, compound 8 for its structure). This spectrum is typical of most compounds seen in this study. The major mass spectral features are (a) intense singly and doubly charged molecular ions a t m / e 330 and 165, (b) ions due to the loss of the unsubsti-
TO LAKE ERIE
\
\
\ RR
SOUTH PARK ST.
330 ( M I )
M - ( C H 3 )N.phenyl
I
l2 RR -SOIL
1.2 150
SED. 3
W
SE6. 2
Figure 1. Map of the Buffalo River (New York, USA) showing the sampling sites. The lclcations of the Ohio and South Park Street bridges and three railroad bridges are marked. 0013-936X/80/0914-1147$01.00/0
I, ,
194
, , 200 l , I! ,
,-L: , i , 250
286
,
:-I.,
,
300
, L u ,
lb
,
Figure 2. Mass spectrum of a major component of the 2-propanolextractable, basic compounds isolated from a sediment sample. It was subsequently identified as that of bis(4-dimethylaminophenyl)phenylmethane (see compound 8, Figure 3 for its structure).
@ 1980 American Chemical Society
Volume 14,Number 9,September 1980
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falo River is dredged annually (the previous dredging took place during the fall of 1978),the concentration data in Table I reflect only the accumulation of these chemicals during the 7-8-month period following the dredging. Most of the compounds found in these soil and river sediment samples are either starting materials, intermediates, byproducts, or products associated with the commerical synthesis of two dyes: malachite green and crystal violet. Compound 9 is the leuco base of crystal violet, and compound 8 is the leuco base of malachite green. Both of these leuco bases are produced by the condensation, under acidic conditions, of N,N-dimethylaniline with compounds of the form
OH
R = H or (CH3)2N Figure 3. Structures of some of the compounds found in and near the Buffalo River. Compound numbers refer to Table I.
tuted phenyl ring (M - 77 at mle 253) and the N,N-dimethylamino substituted phenyl ring (M - 120 at rnle 210), and (c) ions due to the loss of 16 amu (presumably CH3 and H) from the M - 77 and M - 120 ions to give ions at mle 237 and 194. The positions of substituents on ring systems cannot always be assigned on the basis of mass spectrometry alone. In this case, we have assigned the position of the N,N-dimethylamino groups to the 4 position in all structures (see Table I). We have based this assignment not on mass spectral features but rather on the known synthetic chemistry of the dye-manufacturing industry. The compounds identified in the sediment and soil samples are listed in Table I along with their approximate concentrations which are not corrected for extraction efficiencies and, thus, represent minimum values. All compound identifications were confirmed by comparison of the mass spectra and exact gas-chromatographic retention times with those of authentic reference compounds. The structures of several of the compounds listed in Table I are given in Figure 3. Since the Buf-
These are, in turn, formed from the reaction of N,N-dimethylaniline with the appropriately substituted benzaldehyde. Although these diphenyl carbinols were not observed in the soil or sediment samples, compounds formed by both their reduction (compounds 6 and 7) and their oxidation (compound 5 ) were identified. Of the other compounds, clearly compounds 1,2,4, and 12 are starting materials for this synthesis. The remaining compounds are either other products of the dyestuff plant (1-naphthylamine and N-ethyl-Nphenylbenzylamine) or common industrial chemicals (compounds 13 to 16). The high concentrations and close structural similarity of the various aromatic amines present in the soil samples suggest that this particular site may have been used as a chemical dump by the dyestuff manufacturer. In fact, production information indicates that a variety of triarylmethane dyes and leuco dyes bases were produced by this plant ( 5 ) .The close proximity of the soil sampling sites to the Buffalo River and the similarity in structure and concentration ratios of several of the aromatic amines present in both the soil and sediment samples suggest that these compounds may have entered the water and sediment of the Buffalo River via leaching from the chemical dump. We should point out that both 1-naphthylamine and N-ethyl-N-phenylbenzylamine
Table I. Compounds Found in Soil and Sediment from Buffalo River, New York concn, compd no.
compd name
1
sediment 2
* ppm SO11
3
1
2
Aromatic Amines
1 2 3 4
5 6 7 8 9 10 11 12 13 14
15 16 a
aniline N,Ndimethylaniline 4,4'-bis(dimethylamino)biphenyl 4-(dimethylamino)benzaldehyde 4-(dimethylamino)benzophenone 4-(dimethylamino)diphenylmethane bis(4-dimethylaminophenyl)methane bis-(4-dimethylaminophenyl)phenylmethane tris(4-dimethylaminophenyl)methane 1-naphthylamine Methyl-Mphenylbenzylamine benzaldehyde nitrobenzene 2,4-dinitrotoluene 2,6-dinitrotoluene trichlorobenzenes
ND = not detected.
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Environmental Science & Technology
ND ND 0.004 ND ND ND 0.1 0.1 ND 0.08 0.07
Other Compounds ND ND ND ND ND
ND 0.007 ND
10 200 ND 300 20 ND 1000 ND ND ND
ND 40 400 20 300 20 20 2000 2000 ND ND
ND ND ND ND ND
4 8 ND ND 10
ND ND 50 50 40
ND ND ND ND ND ND 0.2 0.1 ND 0.06 ND
ND ND 0.01 ND 0.02 ND 0.1
ND ND ND ND ND
0.1
5
Table II. Dose-Dependent Mutagenicity of Two Compounds Found in the Buffalo River concn, Ccg/mL
4-(dimethylamino)benzophenone
4-(dimethylamino)benzaldehyde
background benzo [ a] pyrene
25 50 75
100 100 300 1000 20
105
x
IMF
*
18 22
41 49 31 40 79
13f 1 200 f 50
induced mutant fraction: see ref 6 for details. All compounds were assayed in the presence of Aroclor-induced postmitochondriai supernatant. a
IMF =
turing plant used the Buffalo river bank as a dump for experimental or failed dye production batches. Leaching and runoff from this dump have transferred many of the dye-related chemicals into the Buffalo River and its sediment. In the river, fish have been exposed to these compounds by direct contact with contaminated sediment. Since some of these compounds could be mutagenic or carcinogenic, it is not unreasonable to assume that the chemicals originally derived from the dyestuff manufacturer’s dump are at least partially responsible for the tumors observed in fish taken from this river. Acknowledgment We thank W. Zapisek (Canisius College) for providing the soil samples and J. Spagnoli (New York State Department of Environmental Conservation) for help in obtaining the sediment samples. We also thank W. G. Thilly and B. M. Andon for the bioassay experiments. Literature Cited
are present in substantial amounts in the sediment (see Table I) and that bothi have previously been detected in fish obtained from the Buffalo River ( 4 ) . Compounds 3,4,5, and 8 were bioassayed to determine their individual mutagenic activity by using a quantitative forward bacterial mutagenesis assay described elsewhere (6, 7). Benzo[a]pyrene was run as a positive control in each mutation assay. Only compounds producing an induced mutant fraction at least twice background were considered to give a positive mutagenic test. Both 4-(dimethy1amino)benzophenone and 4-(dimethylamino)benzaldehydeinduced significant mutation to 8-azaquanine resistance in S. typhimurium (see Table 11). These two compounds have up to 5% of the activity of benzo[a]pyrene on a weight basis. The following scenario emerges from these chemical and biological data. Over several years, this dyestuff manufac-
(1) Kraybill, H. F. Prog. Exp. Tumor Res. 1976,20, 34. (2) Black, J. J.; Holmes, M.; Dymerski, P. P.; Zapisek, W. F. In “Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment”, Afghan, B. K., Mackay, D., Eds.; Plenum Press: New York, 1980; p 559. (3) Paigen, B.; Braun, M.; Steenland, K.; Holmes, E.; Cohen, H., Roswell Park Memorial Institute, Buffalo, NY, private communication, 1980. (4) Diachenko, G. W. Enuiron. Sci. Technol. 1979,23, 329. (5) “1974 Directory of Chemical Producers, United States”; Stanford Research Institute: Menlo Park, CA, 1974. (6) Skopek, T. R.; Liber, H. L.; Kaden, D. A.; Thilly, W. G. Proc. Natl. Acad. Sci. U.S.A. 1978. 75. 4465. (7) Krishnan, S.; Kaden,.D. A,;Thilly, W. G.; Hites, R. A. Enuiron. Sci. Technol. 1979, 23, 1532.
Received f o r review January 4, 2980. Accepted June 16 1980. This work has been supported by the U.S. Environmental Protection Agency (Grant No. 806350).
CORRESPONDENCE
SIR: The work of Rollins and Homolya ( 1 ) has been noted with great interest. The levels of chlorides that were found come as no surprise, as I had personally been involved in the early studies ol’ Carotti and Kaiser ( 2 ) . Rollins and Homolya have estimated ambient levels of HC1 aerosol and suggest a secondary transformation possibility in which some of the HC1 might be transformed into ammonium chloride. The presence of the latter species has been previously reported by Cunningham et al. ( 3 ) and Hindman et al. ( 4 ) . Each of these studies found the presence of ammonium chloride concurrent with ammonium sulfate. Hindman et al. ( 4 ) found relatively few particles that were only sodium chloride; most of the particles consisted of many elements and compounds, including sulfur forms. For the most part, the role of HC1 in the atmosphere has all but been ignored. Most investigators discount the direct emission of HC1 associated with the burning of fossil fuels, although in oil the amount of available chloride for emission can be linked not only to that inherent in the fuel but also to the amount of excess water in the fuel. The latter is related to sea water, most probably the residual of tanker ballast or wash water, and can be highly variable. The creation of HC1 in the atmosphere is generally assumed to be a breakdown of sea salt (NaCl) by photooxidation with nitrogen dioxide (5, 6) along with aldehydes (12) or through sulfate reactioins (6, 7 ) .A more complex relationship is pro-
jected by Yue and Mohnen (8) for the production of HC1 in clouds. They show that the amount of HC1 released from an aqueous system depends, in part, on the ambient concentraand H2S04, as well as the liquid water tions of S02, ”3, content available in the cloud. The production of HC1 increases with the amount of SO2 available but is limited by the This suggests a competition betyeen HC1 and increase of “3. sulfate for the available NH3 as is also implied by Cunningham et al. ( 3 ) and Brosset et al. ( 7 ) . The production of HC1 in an aqueous acidic system is based on the fact that SO2 can be absorbed and converted to S042-. As the concentration of S04*- increases either through production or evaporation, the solution vapor pressure will approach that of sulfuric acid. Since the vapor pressure of sulfuric acid is less than that of hydrochloric acid, as the reduction proceeds past the equilibrium point between the two, HC1 will be driven from the system (followed by nitric acids, if present). Whether or not the system is driven to total HC1 depletion is not now relevant. However, the important question to be answered is what happens to the HC1 aerosol so produced in accordance with the scenario or by direct emission from combustion sources. Rollins and Homoloya (1)have suggested a transformation of HC1 into ammonium chloride. This suggests a complex role for HC1 that relates directly to the creation of strong acid aerosols and aqueous sites suitable for the conversion of SO2
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