Environ. Sci. Technol. 1904, 18, 333-337
Table IV. Maximum Safe Sampling Volumes ( V S ,m 3 ) at Designated Collection Efficiencies' 90% fluorene phenanthrene anthracene pyrene
95%
1.0 x l o 2 6.6 X l o 2 9.2 X 10' 8.2 x 103
'Single plug, 7.8 c m diameter
X
7.7 x 5.0 X 6.9 X 6.2 x
Registry No. FL, 86-73-7; PH, 85-01-8; AN, 120-12-7;PY, 129-00-0.
Literature Cited Cautreels, W.; Cauwenberghe, K. V. Atmos. Environ. 1978, 12, 1133-1141. De Wiest, F.; Rondia, D. Atmos. Enuiron. 1976,10,487-489. Yamasaki, H.; Kuwata, K.; Miyamoto, H. Bumeki Kagaku 1978,27, 317-321. Pupp, C.; Lao, R. C.; Murray, J. J.; Pottie, R. F. Atmos. Environ. 1974, 8, 915-925. Yamasaki, K.; Kuwata, K.; Miyamoto, H. Environ. Sci. Technol. 1982, 16, 189-194. Thrane, K. E.; Mikalsen, A. Atmos. Environ. 1981, 15, 909-9 18. Keller, C. D.; Bidleman, T. F. Atmos. Environ., in press. Burdick, N. F.; Bidleman, T. F. Anal. Chem. 1981, 53, 1926-1929. Simon, C. G.; Bidleman, T. F. Anal. Chem. 1979, 51, 1110-1 113. Sonnefeld, W. J.; Zoller, W. H.; May, W. E. Anal. Chem. 1983.55. 275-280. Billings,'W. N.; Bidleman, T. F. Atmos. Environ. 1983, 17, 383-391. MacKay, D.; Bobra, A,; Chan, D. W.; Shiu, W. Y. Environ. Sci. Technol. 1982, 16 645-649. Banerjee, S.; Samuel, H.; Yalkowsky, S.; Valvani, S. C. Environ. Sci. Technol. 1980, 14, 1227-1229. Chiou, C. T.; Schmedding, D. W. Environ. Sci. Technol. 1982, 16, 4-9. MacKay, D. Environ. Sci. Technol. 1982, 16, 274-278. Reilley, C. N.; Hildebrand, G. P.; Ashley, J. W., Jr. Anal. Chem. 1962, 34, 1198-1213. Senum, G. L. Enuiron. Sci. Technol. 1981,15,1073-1075.
10' 10'
10' 103
7.5 cm thick ( N = 7.5).
defined by the front midpoint, the two chromatographic terms are equivalent (16). When VB and N are known, the collection efficiency of the PUF bed at different air volumes can be predicted from Figure 7. Also, this figure can be applied to predict the maximum air volume for a required collection efficiency. For field sampling we use two 7.8 cm diameter X 7.5 cm thick plugs, so the front plug has N = 7.5. From Figure 7, when N = 7.5 and the required collection efficiency = 95%, vs/ V, = 0.62, or the maximum air volume should be no greater than 62% of VB in order to guarantee that 95% of the vapor has been collected on the first plug of the sampling train. By use of VBvalues from Table 11, maximum safe Vs values have been calculated for the four PAH at 90% (Vs/ V, = 0.82) and 95% (Vs/VB = 0.62) collection efficiencies (Table IV). From these results we can conclude that PH, AN, and PY will be quantitatively collected by two of our field PUF plugs in a 24-h sampling period (-700 m3 air) for ambient temperatures not exceeding 20 OC, in good agreement with field results (7). For sampling PAH vapors at temperatures other than 20 "C, one could calculate Psfrom published vapor pressure-temperature relationships (IO),estimate PLfrom eq 1, determine VBby using Figure 5b, and then use Figure 7 to estimate Vs. Because of its higher volatility, FL is not quantitatively collected for VS = 700 m3. A reduced Vs or a longer trap would be needed to retain FL.
Received for review May 26,1983. Revised manuscript received September 16,1983. Accepted October 5,1983. This work was supported by the US.Department of Energy under the National Environmental Research Park (NERP)Program. Contribution No. 514 of the Belle W. Baruch Institute.
Fractionation, Isolation, and Characterization of Ames Mutagenic Compounds in Kraft Chlorination Effluents Bjarne Holmbom," Ronald H. Voss, Richard D. Mortlmer, and Alfred Wong Pulp and Paper Research Institute of Canada, Pointe Claire, P.Q., Canada H9R 3J9
Mutagenic extracts from kraft pulp chlorination-stage effluents were fractionated, and the distribution of mutagenicity was determined by the Ames test with tester strain TA 100. Most of the mutagenicity was caused by nonvolatile compounds extractable with ethyl acetate. Strong acids accounted for the principal part of the mutagenicity. The mutagenic components could be concentrated to a narrow band by silica thin-layer chromatography and reverse-phase high-pressure liquid chromatography. The results provide further support for the conclusion that the previously identified hydroxyfuranone (C5H303C1,)is a major TA 100 mutagen in chlorinationstage effluents. The isolation of this compound, its Ames mutagenicity, and some of its chemical properties are described.
Introduction Short-term genetic bioassays are becoming increasingly *Towhom correspondenceshould be addressed at the Laboratory of Forest Products Chemistry, Abo Akademi, SF-20500 Turku 50,
Finland. 0013-936X/84/0918-0333$01.50/0
important tests for the identification of individual compounds or complex environmental mixtures which may pose a potential health hazard (1-3). The best known and most widely used short-term bioassay is the Salmonella microsome assay or Ames test (4) which measures the ability of test samples to induce genetic alterations (mutations) in specially developed strains of Salmonella bacteria. The limitations of this test notwithstanding (e.g., false negatives and positives), it is generally recognized as an important tool for the preliminary screening of potentially mutagenic and possibly carcinogenic chemical substances. Since early 1977, Ames mutagenicity of spent wash liquors from the first (chlorination) stage of kraft pulp bleaching has been the subject of extensive research in several laboratories in Scandinavia (5-11) and North America (12-18). These studies have shown that the Ames mutagenic substances present in chlorination-(C-) stage effluent are primarily direct-acting mutagens causing base substitutions in DNA molecules. They are also fairly unstable at alkaline pH and can be destroyed by reaction with SO2. Whereas kraft C-stage effluents exhibit sig-
0 1984 American Chemical Society
Environ. Sci. Technol., Vol. 18, No. 5, 1984 333
nificant Ames mutagenicity, untreated whole mill effluents, through dilution and/or chemical degradation, have been observed to be nonmutagenic or, less frequently, weakly mutagenic only after concentration (9, 13). Nonetheless, information about the identity of the Ames mutagenic compounds and the availability of suitable methods for analyzing such compounds are necessary for firmly establishing their fate and stability. Several compounds, viz., chloroacetones and 2-chloropropenal, have been cited to be important mutagens present in kraft chlorination effluents (6-8, 14, 18). We recently reported (19) the isolation and identification of a novel Ames mutagenic compound with the molecular formula C5H303C13having a hydroxyfuranone structure and tentatively identified as 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). CHC12
HO
CI
kSo MX
In this paper we~reportand discuss test data on the distribution of Ames mutagenicity in C-stage effluent through various fractionation and isolation procedures. Some chemical and mutagenic properties of the isolated MX mutagen are also reported.
Experimental Section Chlorination Effluent Samples. Samples of chlorination effluents from four mills in eastern, central, and western Canada were tested. All mills produced fully bleached softwood kraft pulp utilizing Clz (with about 10% of the active chlorine as chlorine dioxide) in the first bleaching stage. The Ames mutagenicity determined with the tester strain T A 100 was comparable for all effluent samples. Extraction of Mutagenic Materials. Solvent extractions were carried out as funnel extractions with distilled-in-glass solvents. Generally, 1 L of effluent was extracted with five 125-mL portions of solvent. Solvents were evaporated with a rotary evaporator at 40 "C under reduced pressure (-1.5 kPa). For effluent volumes larger than 2 L, Amberlite XAD-4 polymeric resin was used in place of solvent extraction to isolate the mutagenic substances from C-stage effluent. Adsorbed material was eluted with 4 bed volumes of methanol. Fractionation of Mutagenic Extracts. The various techniques which were used, separately or in tandem, to fractionate the C-stage extracts (obtained as per above) are described. (1) Separation of Strong Acids. Ethyl acetate extracts were fractionated by shaking with four portions of 2% aqueous NaHC03 solution. The aqueous phase was acidified to pH 2 and extracted with four portions of ethyl acetate. This ethyl acetate fraction contained the strong acids. (2) Extract Cleaning with Anion Exchanger. Some extracts were cleaned by elution through a column of DEAE-Sepharose CL-6B (Pharmacia Fine Chemicals) which is a weakly basic anion exchanger. The column was washed with several bed volumes of water, methanol, and finally ethyl acetate. Elution was carried out with 5 bed volumes of ethyl acetate. (3) Thin-LayerChromatography (TLC). TLC plates coated with silica gel containing UV-254 fluorescent indicator with layer thicknesses of 1.0 or 0.25 mm were used. Elutions were carried out either with hexane-diethyl 334
Environ. Sci. Technol., Vol. 18, No. 5, 1984
ether-acetic acid, 50:50:1 v/v (system A), or dichloromethane-acetic acid, 955 v/v (system B). Mucochloric acid (Aldrich Chemical Co.) was used as a reference compound. Separated zones were scraped off by using special sample recovery tubes (Chromaflex,Kontes Scientific Glassware). Adsorbed substances were eluted with ethyl acetate (2-3 mL). Evaporation of solvent was carried out under a stream of nitrogen. (4) Silica Column Chromatography. In the largescale isolation procedure the XADd/methanol extract was first repeatedly leached with hexane-diethyl ether-acetic acid (50:50:1 v/v) to give an extract free of highly polar material. This extract was then fractionated on a 45 cm/50 mm column of silica Gel 60 (Merck) by elution with the solvent system A described above. The first 700-mL portion of solvent eluted was essentially nonmutagenic and was discarded. Each subsequent 100-mL portion eluted was tested separately for mutagenicity. The three fractions between 800 and 1100 mL which were strongly mutagenic were combined and used in subsequent purification steps by TLC and high-pressure liquid chromatography in order to isolate the specific mutagen, i.e., MX. (5) High-pressure Liquid Chromatography (HPLC). A Hewlett-Packard 1084 B liquid chromatograph equipped with an automatic injector and a variable wavelength UV detector was used. The following columns were used: (a) RP-8, 25 cm/4.6 mm, 5 pm (code RP-8), (b) RP-8, 25 cm/9.4 mm, 10 pm (code RP-8 M9), and (c) RP-18, 25 cm/4.6 mm, 5 pm (code RP-18). The solvent system was water-acetonitrile programmed in different gradient modes. The pH of the water was adjusted to 3.0-3.2 with phosphoric acid. Collected fractions were worked up through repeated extractions with ethyl acetate after removal of most of the acetonitrile by evaporation in a rotary evaporator. Mutagenicity Testing. Mutagenicity tests were carried out at several contract laboratories specializing in mutagenicity testing. For shipment to the testing laboratories, test samples were dissolved in distilled water adjusted to pH 2.0 (=pH of C-stage effluents) with hydrochloric acid. Some samples also contained traces of ethyl acetate or acetonitrile. Blank tests confirmed that these solvents did not influence the mutagenic assays. Tests were made with Salmonella typhimurium strains according to the procedure developed by Ames and co-workers ( 4 ) . Most tests were made with the tester strain TA 100 without metabolic ( 4 - 9 ) activation. Results of the tests are given as average number of revertants (rev) from three to four plates. The standard deviation for revertant counts was typically
