is slower and less extensive a t pHs 9.65 and 2.25 than a t p H 8.0. Part of this effect is probably due to the changes in metal-NTA complexation. In a solution containing only Ca2+, ultimate degradation was slower than in solutions containing several transition metals. The rate of ultimate degradation decreases as the products of the ozonation process increase in concentration. Natural Systems. In Ohio River water, ozonation with an average of 2 mg of ozone/L for 900 s results in conversion of a t least 75% of NTA carbon to COz. Simultaneous analysis of total organic carbon during ozonation suggests that NTA competes effectively with the large fraction of organic carbon normally found in the river. In cistern water ultimate degradation was not significant even after 2700 s. This result was ascribed to a significant drop in p H during ozonation. Literature Cited (1) Rice, R. G., Water Pollut. Control, 77,51-5 (1978). (2) Osheroff, B., in “Ozone/Chlorine Dioxide Oxidation Products of Organic Materials”, Rice, R. G., and Cotruvo, J. A., Eds., International Ozone Institute, Inc., Cleveland, Ohio, 1978, p p 7-20. (3) Stumm, W., Helu. Chim. Acta, 37,773 (1954). (4) Kilpatrick, M. L., Herrich, C. D., Kilpatrick, M., J . Am. Chem. Soc., 78,1784 (1965). (5) Hoigne, J., Bader, H., Water Res., 10,377 (1976).
(6) Hoigne, J., Bader, H., Prog. Water Technol., 10,657 (1978). (7) Hoigne, J., Bader, H., Ozone Scz. Eng., I, 73 (1979). (8) Shambaugh, R. L., Melnyk, P. B., Water Pollut. Control, 50,113 (1978). (9) Stolzberg, R. S., Hume, D. N., Enuiron. Sci. Technol., 9, 654 (1975). (10) Stolzberg, R. J., Hume, D. N., Anal. Chem., 49,374 (1977). (11) Woodiwiss, C. R., Walker, R. D., Brownridge, F. A,, Water Res., 13,599 (1979). (12) Nebel, C., Gottschling, R. D., Unangst, P. C., O’Neill, D. J., Zintel, G. V., Water Sewage Works, 123,76 (1976). (13) McDuff, R. E., Morel, F. M., Technical Report EQ-73-02, W. M. Keck Laboratory of Environmental Engineering Science, California Institute of Technology, Pasadena, Calif. (14) Hoover, T . B., “Polarographic Determination of NTA”, US. EPA Report EPA-R2-73-254. (15) “Standard Methods for the Examination of Water and Wastes”, 14th ed., American Public Health Association, Washington, D.C., 1976, Method 423A. (16) Hoigne, J., Bader, H., Enuiron Sci. Technol., 12,79 (1978). (17) Gilbert, E., in ref 2, pp 227-42. (18) Kuo, P. P. K., Chian, E. S. K., Chang, B. J., in ref 2, pp 22742. (19) Spanggord, R. J., McClurg, V. J., in ref 2, pp 115-25. (20) Ishizaki, N., Dobbs, R. A,, Cohen, J. M., in ref 2, pp 210-26.
Received for reuiew October 25, 1979. Accepted February 4, 1980. Presented at the Environmental Chemistry Diuision, 178th National Meeting of the American Chemical Society, Washington,D.C., Sept 10-14,1979.
Identification of Organic Compounds in a Mutagenic Extract of a Surface Drinking Water by a Computerized Gas Chromatography/ Mass Spectrometry System (GC/MS/COM) W. Emile Coleman*, Robert G. Melton, Frederick C. Kopfler, Karen A. Barone, Theresa A. Aurand, and Mark G. Jellison
U.S.Environmental Protection Agency, Health Effects Research Laboratory, 26 W. St. Clair Street, Cincinnati, Ohio 45268 The organics in a Cincinnati, Ohio drinking water sample were concentrated by a reverse osmosis (RO) process. The diethyl ether soluble extract of the RO concentrate, which proved to be mutagenic in studies using the Ames test, was partitioned into acid and baseheutral fractions. The unpartitioned ethyl ether concentrate, an acid and methylated acid fraction, the unpartitioned baseheutral extract, and five baseheutral eluants from a silica gel microcolumn were analyzed for the presence of organics using a computerized gas chromatography/mass spectrometry system (GC/MS/COM) equipped with glass capillary columns. Analysis of individual fractions indicated a predominance of polychlorinated biphenyls (PCBs) and chlorinated aromatics in the second baseheutral partition and many polynuclear aromatics (PNA) in the fourth baseheutral partition. Approximately 460 compounds were identified in this tap water extract, including 41 PNAs, 15 PCBs, and a number of amines, amides, and other halogenated mecies. The presence of traces of organic chemicals in drinking water has caused concern, since it is thought that they might contribute to the induction of cancer or other illnesses in consumers. The potential health effects of these compounds can be studied only after they are isolated from the water and identified. But since many are of high molecular weight, they cannot be identified by available techniques. Conventional techniques used for isolating organic compounds from water 576
Environmental Science & Technology
for analytical purposes are not practical for isolating sufficient material for bioassay procedures. Consequently, a technique using reverse osmosis and lyophilization to reduce large amounts of water to volumes that could be extracted in the laboratory while retaining most of the organic carbon was developed ( I 1. In 1974,400-galsamples of drinking water from the distribution system of Cincinnati, Ohio, were concentrated and extracted by this procedure each month. Eighty percent of the organic matter obtained in each sample was used for biological tests and 20% was retained for chemical analysis if the biological tests were positive. Two of these samples (March and June) were tested for mutagenic activity in the Ames Salmonella system (2).Both of the concentrates were mutagenic when tested in strains TAlOO and TA98 in the presence and absence of an Arochlor-1254-stimulated rat liver homogenate metabolic activation system. Both samples were fractionated by sequential extraction with petroleum ether, diethyl ether, and acetone. When these fractions were assayed, most of the mutagenic activity was in the diethyl ether fraction ( 3 ) . The sample collected in March was chosen for chemical analysis to attempt to determine the components responsible for the mutagenic activity. The portion of that sample retained for chemical analysis was fractionated in a manner similar to that used to fractionate the bioassay portion. The diethyl ether fraction was then analyzed by glass capillary column gas chromatography/mass spectrometry. This paper reports the results.
This article not subject to U.S. Copyright. Published 1980 American Chemical Society
Experimental
Sampling and Concentration: Reverse Osmosis Method (RO). The organic pollutants in 400 gal of Cincinnati drinking water, direct from the tap in our laboratory, were concentrated by the RO method of Kopfler ( I ) . The theory and practice of RO have been reviewed by others (4, 5 ) . Kopfler (1, 6 ) , in earlier papers, showed the schematic diagrams of the major components of the dual RO concentrator and of the extraction of the concentrates. The same extraction scheme was used for the subject sample. The pentane and methylene chloride extracts as shown in ref 3 and 6 were combined, dried, and evaporated down to constant weight. The diethyl ether soluble portion of this combined concentrate, which proved to be mutagenic, was the origin of the organic compounds identified in this paper. A detailed procedure for obtaining the ether fraction for GC/MS analysis is described by Melton and Barone (7). The rationale for using these methods and the study for which similar samples have been collected have been discussed by Tardiff et al. ( 8 ) . Fractionation and Partitioning. Burdick and Jackson Distilled in Glass solvents were used for all extractions. The RO concentrate (5-20 mg) was dissolved in 10 mL of ethyl ether and placed in a 10-mL separatory funnel. The organic layer was extracted with four 1-mL aliquots of 5% NaOH and washed with two 1-mL aliquots of distilled water. After separation, the aqueous layer was acidified to pH 2 with 6 M H2S04 solution, saturated with NaC1, and reextracted with four 2-mL aliquots of methylene chloride. The methylene chloride layers were combined and then dried with 0.5 g of sodium sulfate (previously muffled a t 400 "C for 1h). Twenty percent of the dried methylene chloride was set aside for direct GC/MS analysis for acids on glass capillary columns. The remaining solution (80%)was derivatized with diazomethane to form methyl esters from the acids extracted with 5% NaOH (9).
The original diethyl ether solution, which contained neutral and basic organics at this point, was reextracted with 1mL of 5% NaOH, and the aqueous layer was combined with the original aqueous fraction as shown in Figure 1.The ether layer was dried with 0.5 g of sodium sulfate and evaporated in a conical graduated glass tube to 0.5 mL with an Organomation N-Evaporator a t 45 "C. (The N-Evaporator allowed one to adjust and maintain a variety of bath temperatures while evaporating the solvent under a stream of nitrogen.) Two milliliters of hexane was added to the tube, and the sample was again evaporated a t 60 "C to 0.1 mL. After adding 0.5 mL of hot (60 "C) hexane to the concentration tube, the solution was transferred to the top of a 14 cm X 5 mm i.d. microchromatographic column (disposable transfer pipet) (10) packed with 0.2 g of 5% deactivated silica gel (11,12).Organics were partitioned by eluting from the column with the following aliquots of eluants: (1)0.5 mL of hexane, (2) 1 mL of hexane, (3) 4 mL of hexane, (4)4 mL of hexane/benzene (1:1), (5) 4 mL of benzene, (6) 4 mL of methylene chloride, and ( 7 ) 4 mL of methanol. Each of the fractions was collected in a premuffled 4-mL graduated glass-stoppered chromaflex sample tube (Kontes, No. K-422560). Eluants 1to 5 and eluants 6 and 7 were typically evaporated to a volume of 10 to 20 pL a t temperatures of 60 and 40 "C, respectively, prior to GC/MS analysis. When the sample volume had to be adjusted, methylene chloride was the solvent of choice. Words of caution: Logsdon ( 1 3 ) extracted aqueous .solutions containing from 2 up to 900 mg/L residual chlorine with methylene chloride and the subsequent GC/MS analysis of the concentrated extracts showed numerous isomers of mono- and dichlorocyclohexanes. If the residual chlorine in the water sample was removed by the addition of sodium sulfite before solvent extraction, then no chlorinated cyclohexanes were observed. The source of these
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