Literature Cited (1) Rook, J. J., Water Treatment Exam., 23,234 (1974). (2) Jolley, R. L., J . Water Pollut. Control Fed., 47,601 (1975). (3) Glaze. W. H.. Henderson, J. E., IV. J. Water Pollut. Control.Fed., 47,2411 (1975). (4) Lindstrom, K., Nordin, J., J Chromatogr., 128,13 (1976). ( 5 ) Rook. J. J.. J Am Water Works Assoc.. 68.168 (1976). (6) Dence, C., Sarkanen, K., Tappi, 43,87 (1960). (7) Das. B. S., Reid, S. G., Betts, J. L., Patrick, K., J . Fish.Res. Board Can.,’26,3055 (1969). (8) Sarkanen, K. V., Dence, C. W., J . Org, Chem., 25,715 (1960). (9) Slates, H. L., Taub, D., Kuo, C. H., Wendler, N. L., J. Org. Chem., 29,1424 (1964). (10) Shimizu, Y., Hsu, R. Y., Chem. Pharm. Bull., 23,2179 (1975). (11) Pearl, I. A., J . Org. Chem., 12,85 (1947). (12) Sohma, T., Konishi, K., Takeda Kenkyusho Nempo, 26,138 (1967); Chem. Abstr., 68,95447s (1968). (13) Nicholson, A. A,, Meresz, O., Lemyk’,B., Anal. Chem., 49, 814 (1977). (14) Aue, W. A,, Hastings, C. R., Berhardt, K. O., Pierce, J. O., 11,Hill, H. H., Moseman. R. F., J . Chromatogr., 72,259 (1972). (15) Larson, R. A., Weston, J. C., Howell, S. M., J. Chromatogr., 111, 43 (1975). (16) Rockwell, A. L., Larson, R. A,, in “Water Chlorination: Envi-
ronmental Impact and Health Effects”, Jolley, R. L., Gorchev, H., Hamilton, D. H., Eds., p 67, Ann Arbor Science Publishers, Ann Arbor, Mich., 1978. (17) “Standard Methods for the Examination of Water and Wastewater”, p 342, American Public Health Association, Washington, D.C., 1976. (18) Rook, J. J., Enuiron. Sci. Technol., 11,478 (1977). (19) Butterworth, J., Walker, T. K., Biochem. J., 23,926 (1929).
(20) Bjork, R. G., Anal. Biochem., 63,80 (1975). (21) Kempf, T.,Pribyl, J., Gas- Wasserfach., Wasser-Abwasser, 116, 278 (1975). (22) Afghan, B. K., Leung, R., Ryan, J. F., Water Res., 8, 789 (1974). (23) Tiffin, L. O., Plant Physiol., 41, 510, 515 (1966). (24) Christman, R. F., Oglesby, R. T., in “Lignins: Occurrence, Formation, Structure and Reactions”, Sarkanen, K. V., Ludwig, C. H., Eds., p 769, Wiley-Interscience, New York, N.Y., 1971. (25) Anderson, H. A,, Russell, J. D., Nature (London), 260, 597 (1976). (26) Carlson, R. M., Carlson, R. E., Kopperman, H. L., Caple, R., Enuiron. Sci. Technol., 9,674 (1975). (27) Smith, J. G., Lee, S.-F., Netzer, A., Water Res., 10, 985 (1976). (28) Whitehead, D. C., Nature (London),202,417 (1964). (29) Wang, T. S.C., Yang, T.-K.,Chuang, T. T., Soil Sei., 103,239 (1967). (30) Lodhi, M. A. K., Am. J . Bot., 6 3 , l (1976). (31) Hunter, J. V., in “Organic Compounds in Aquatic Environments”. Faust. s.D.. Hunter, J. V., Eds., Q 51, Marcel Dekker, New York, N.Y., 1971. (32) Degens, E. T.. Reuter, J. H., Shaw, K. N. F., Geochim. Cosmochim.&ta, 28,45 (1964). (33) Matsumoto, G., Ishiwatari, R., Hanya, T., Water Res., 11,693 (1977). (34) Larson, R. A,, Freshuater Biol., 8,91 (1978).
Received f o r reuieu August 7, 1978. Accepted October 6, 1978. Presented in part at the Conference on Water Chlorination (Enuironmental Impact and Health Effects), Gatlinburg, Tenn., Nou 1977. Supported by the Enuironmental Associates, Academy of Natural Sciences of Philadelphia.
Determination of Several Industrial Aromatic Amines in Fish Gregory W. Diachenko Division of Chemical Technology, Food and Drug Administration, Washington, D.C. 20204
w A procedure is described for the determination of selected industrial aromatic amines in fish. Ground fish tissue is digested with aqueous sodium hydroxide and extracted with benzene. The extract is washed with dilute acid and cleaned up using gel permeation chromatography. The amines are separated and quantitated using nitrogen-selective gas-liquid chromatography. Recoveries of N-ethyl-N-phenylbenzylamine, N-ethyl-N-(m -tolyl)benzylamine, N,N-dibenzylmethylamine, diphenylamine, and N-phenyl-l-naphthylamine from fish tissue fortified a t levels of 20-100 ppb (pglkg) averaged a t least 80%. Recoveries of 1-naphthylamine and 3,3’-dichlorobenzidine were somewhat lower. Fish samples obtained from rivers near nine textile and dyestuff manufacturers known to use certain aromatic amines have been analyzed. 1-Naphthylamine was detected in fish from the Buffalo and Delaware Rivers downstream from two dyestuff manufacturers. N-Ethyl-N-phenylbenzylamine and N ethyl-N-(m-toly1)benzylamine were also detected in the Buffalo River fish. The carcinogenicity of several industrial aromatic amines has stimulated interest in investigating possible environmental contamination by this class of compounds. In 1974, the Occupational Safety and Health Administration issued regulations on 14 chemical compounds that were either known or suspected human carcinogens ( I ) , including aromatic amines such as 1- and 2-naphthylamine and 3,3’-dichlorobenzidine. These carcinogenic compounds could create a potentially serious problem should there be widespread con-
tamination of the human food chain. The Food and Drug Administration became concerned with the possible contamination of human foods by industrial aromatic amines because of their known or suspected carcinogenicity, relatively large production volumes, and potential for biomagnification. Quantities produced vary from approximately 584 X 106 Ib/year for aniline to several million pounddyear for numerous other compounds ( 2 ) . Many aromatic amines are used as intermediates for dyes and pigments, and as antioxidants and antiozonants in rubber products. These varied applications suggest many possible modes of entry into the environment, ranging from direct discharge to conversion of azo dyes t o the precursor amines by bacteria. Many workers have published detection schemes for various aromatic amines, using gas-liquid, high performance liquid, or thin-layer chromatography, sometimes coupled with ancillary techniques such as ultraviolet or fluorescence spectrometry (3-12). Most investigations into amines in fish or foods have centered around naturally derived volatile amines such as di- and trimethylamine, or more recently the N-nitrosamines (13-16). T o this author’s knowledge, however, there are no published methodologies or findings of unhalogenated industrial aromatic amines in aquatic organisms such as fish. This study reports the presence of microgram/kilogram (parts per billion) quantities of selected industrial aromatic amines in fish tissue and a n analytical technique for their detection and quantitation. The technique is aimed a t detecting the less water-soluble aromatic amines which are expected to biomagnify or concentrate t o higher levels in fish tissue than are found in the aqueous environment. The pres-
This article not subject to U.S. Copyright. Published 1979 American Chemical Society
Volume 13, Number 3, March 1979
329
Table 1. Retention Time of Industrial Aromatic Amines Relative to Parathion a compound
N,Ndiethyl-mtoluidine 3,4-dichloroaniline 1-naphthylamine 2-naphthylamine diphenylamine 1-ethyl-1-naphthylamine N,Ndibenzylmethylamine Kphenylbenzylamine Methyl-Kphenylbenzylamine Kethyl-K(mtolyl)benzylamine Knitrosodiphenylamine 2-nitrodiphenylamine 1-nitro-2-naphthylamine benzidine Kphenyl-1-naphthylamine
re1 retentlon tlme
0.13 0.20 0.26 0.28
0.33 0.36 0.42
0.43 0.49 0.66 0.88 0.95 0.95 1.4
1.6
N,N'-bis(dimethylpenty1)-pphenylenediamine
2.5
Kphenyldibenzylamine 3,3'-dichlorobenzidine
4.1
2.6
Data were obtained using a 6 ft X 2 mm i.d. 10% OV-101 column at 200 with 20 mL/min carrier flow using Hall detector, nitrogen mode. Retention time of parathion is about 8 min. a
OC
ence of 1-naphthylamine,N-ethyl-N-phenylbenzylamine, and N-ethyl-N-(m-toly1)benzylamine in fish collected near dye manufacturing plants is reported.
Experimental Reagents. Burdick and Jackson "Distilled in Glass" solvents were used for all extractions. Inorganic reagents were ACS or analytical grade. Aromatic amine reference materials were obtained from Aldrich Chemical Co. (Milwaukee, Wis.); purity was usually greater from 95%. Caution: Benzene is presently considered to be a carcinogen. Use adequate ventilation. Procedures:Sample Preparation and Storage. Fish near dye or textile manufacturing plants were collected by various state agencies either by netting or electroshock techniques, and immediately placed on ice or dry ice. Frozen fish were filleted, doubly- ground, and thoroughly mixed. Fish were stored frozen if immediate analysis was not possible. Digestion and Extraction, Prepared fish tissue (50 g) was digested in a 1-L beaker containing 200 mL of 1 N NaOH for 2 h on a steam bath with occasional stirring. The cooled digest was transferred to a 1-L separatory funnel containing 15 mL of saturated NaCl solution. One hundred milliliters of benzene was added and the funnel shaken for 30 s. Four hundred milliliters of distilled water was then added and the mixture shaken for an additional 60 s. After allowing the layers to separate, the water layer was drained into a 1-L beaker and the benzene layer was transferred to a 500-mL separatory funnel and reserved. The extraction of the aqueous layer was repeated in the original separatory funnel with a second 100-mL portion of benzene, with vigorous shaking for 90 s. After allowing the layers to separate, the aqueous layer was discarded and the benzene extracts were combined in the 500-mL separatory funnel. Acid Wash. One hundred fifty milliliters of 2 X M H2S04 was added to the combined benzene extracts and the mixture shaken for 60 s. After allowing the emulsion to partially separate, the separatory funnel was placed in a clean 1-L beaker and stored in a freezer until both layers were frozen (usually overnight). Centrifugation may also be used to break 330
Environmental Science & Technology
the emulsion. After the layers were allowed to thaw, the aqueous layer was discarded; the clear benzene extract was passed through a drying column containing about 40 g of anhydrous Na2S04 and collected in a 500-mL Kuderna-Danish concentrator equipped with a 10-mL collector. The dried benzene extract was concentrated on a steam bath to about 10 mL, using a 3-ball Snyder column (Kontes Glass Co.). Gel Permeation Chromatographic (GPC) Cleanup. The benzene extract was diluted to 12.0 mL and 10.0 mL was injected (via loop) onto a GPC column. The GPC apparatus was a DuPont 820 liquid chromatograph (or any system capable of pressurizing the column) equipped with a 2.5 in. 0.d. X 19 in. stainless steel column containing 270 g of Bio-Beads S-X2 (Bio-Rad Laboratories, Richmond, Calif.) and a Model C906 sample introduction valve with a 10-mL sample loop (Waters Associates, Framingham, Mass.). Methylene chloride (CH2C12) mobile phase a t a flow rate of 8 mL/min was used to elute the sample. The first 500 mL of CHZClz eluate (containing the bulk of the lipid) was discarded and the next 440 mL (containing the aromatic amines) collected in a 500-mL Kuderna-Danish concentrator with a 10-mL graduated collector. The solution was then concentrated on a steam bath to less than 10 mL in the Kuderna-Danish concentrator equipped with a 3-ball Snyder column. Petroleum ether (150 mL) was added and reconcentrated to less than 10 mL to remove the bulk of the CH2C12. The extract was further concentrated to 0.5 mL or less, using a gentle stream of nitrogen a t room temperature. Gas Chromatographic (GC) Separation and Quantitation. The cleaned up fish extracts were analyzed on a Hewlett-Packard Model 5710 gas chromatograph equipped with a temperature programmer and nitrogen-phosphorus detector Model 18789A or Model 310 Hall electrolytic conductivity detector in the catalytic reductive nitrogen mode. The nitrogen-phosphorus detector operating conditions were: 250 "C; 14 V; 3 mL/min hydrogen; 60 mL/min air; 30 mL/min nitrogen carrier gas; 6 ng of 1-naphthylamine gave 50% full scale deflection (FSD) a t 1 X 4 attenuation and a retention time of 2.7 min, using the 10% OV-101 column described below. The Hall detector operating conditions were: l/4 in. quartz combustion tube with nickel catalyst operated at 900 "C; 50% NaOH on 80/100 mesh Chromosorb W (HP) scrubber; 60 s solvent vent; 40-60 mL/min hydrogen carrier gas; 0.4 mL/min water conductivity solvent; 10 ng of 1-naphthylamine gave 50% FSD at 2 X 1attenuation and a retention time of 3.4 min, using the 10% OV-101 column described below. Three different 6 ft X 2 mm i.d. coiled glass columns were used for GC analyses. They were packed with 10%OV-101,6% OV-17, and 5% OV-225, respectively, each on 80/100 mesh Chromosorb W (HP). The injection port temperature was 250 O C for all columns. Temperature programming conditions for the 10% OV-101 and 6% OV-17 columns were: initial 160 or 180 "C for 8 min, program to 220 "C a t 4 "C/min, hold a t 220 "C for 16 min. The 5% OV-225column was operated at 150 "C for 8 min, programmed to 180 "C at 4 "C/min, and held a t 180 "C for 16 min. The carrier gas was adjusted to give a retention time of 3-4.5 min for 1-naphthylamine (about 20-40 mL/ rnin). A 5-30-pL sample aliquot (equivalent to about 400-2500 mg of fish) was injected onto the 1OOh OV-101 GC column with a 20-mL/min carrier flow and 200 "C isothermal column temperature to determine retention times relative to parathion. Table I lists the values for several industrial aromatic amines. Identification and quantitation can be accomplished on any of the three GC columns. Quantitation is accomplished by comparing sample peak heights to those of standards. Bracketing of sample injections with standards is desirable
Table II. Recovery of Aromatic Amines from Spiked a Ocean Perch Tissue compound
recovery, %
av. recovery, %
av dev from mean
std dev
1-naphthylamine diphenylamine Methyl-Kphenylbenzylamine Kethyl-K(mtolyl)benzylamine N,Ndibenzylmethylamine Kphenyl-1-naphthylamine 3,3‘-dichlorobenzidine
16, 26, 30, 32, 34, 35, 39 98, 98, 102, 102, 104, 105, 107 85,a5,a6,92,95 88, 97, 98, 98, 102 78, 79, 80, 81, 86, 86, 91 80, 80, 86, 88, 97, 98, 99 50, 54, 54, 60, 61, 64, 75, 80, 88
30 102 89 97 83 90 65
5.4 2.6 4.0 3.2 4.0 7.1 11.9
7.5 3.4 4.6 5.2 4.8 8.4 13.0
a
Spiking levels ranged from 20 to 100 ppb (edible portion basis).
due to occasional sensitivity variations of 10-30% over the course of the day. The identity of aromatic amines in fish was confirmed by retention times on all three GC columns, and also by comparison of major sample fragments with those of standards by GC-mass spectrometry (MS). A Finnigan 1015 C quadrupole mass spectrometer coupled to a Varian 1700 gas chromatograph through an all-glass jet separator was used for GC-MS identification. Acid Partitioning Cleanup for GC-MS Identification. In some samples excessive background necessitated additional cleanup to allow positive GC-MS identification of aromatic amines. Additional cleanup was performed by diluting the sample extract to 10 mL with petroleum ether in a 60-mL separatory funnel and extracting the amines twice with 25-mL portions of 0.2 M HzS04. The aqueous extract was made basic by the addition of 3.0 g of NaOH, and extracted twice with 50-mL portions of benzene in a 125-mL separatory funnel. The combined benzene extracts were passed through an Na2S04 drying column and concentrated to
