Environ. Sci. Technol. 1990, 24, 1261-1264
(21) Abott, W. S. J . Econ. Entomol. 1925, 18, 265. (22) Sprague, J. B.; Fogels, A. Watch the y in Bioassay. En-
vironmental Protection Service Technical Report No. EPS-5-AR-77-1,107-118; Halifax, Canada, 1977. (23) Price, E. E.; Swift, M. C. Can. J . Fish. Aquat. Sci. 1985, 42, 1749. (24) Fryer, G. Freshwater Biol. 1985, 15, 347. (25) Yan,N. D.; Scheider, W. A.; Dillon, P. J. Water Pollut. Res. Can. 1977, 12, 213.
(26) Keller, W.; Pitblado, J. R. J. Biogeogr. 1989, 16, 249. (27) Neary, B. P.; Dillon, P. J.; Munro, J. R.; Clark, B. J. The Acidification of Ontario Lakes: An Assessment of their Sensitivity and Current Status with Respect to Biological Damage. Ontario Ministry of the Environment Report, Dorset, Canada, 1990. Received for review August 28, 1989. Accepted March 8, 1990.
Nonaqueous Ion-Exchange Separation Technique for Use in Bioassay-Directed Fractionation of Complex Mixtures: Application to Wood Smoke Particle Extracts Douglas A. Bell,*nt Hani Karam,t and Richard M. Kamens Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27514
We have explored the feasibility of an alternative method for acid/base/neutral separation of atmospheric samples such as wood smoke particle extracts that contain highly polar or acidic organic species. Ion-exchange resins, Amberlyst 15 and Amberlyst 26, were used with organic solvents for fractionating a standard mixture of organic acid, base, and neutral compounds, and a dichloromethane extract from pine wood smoke particles. Total recovery of individual standard compounds was 85-124% and qualitative separation between chemical classes was good. The recovery of wood soot extract through the fractionation system was 107 f 13%, with 15% in the basic fraction, 55% in the neutral fraction, and 20% and 17% in two acidic fractions. The majority of bacterial mutagenicity (TA98+S9,49%; TA98-S9,61%) appeared in the neutral fraction but significant direct-acting mutagenicity (39 % ) was found in the acidic fractions.
Table I. Recovery of Fractionation Standard Compounds through the Ion-Exchange Separation
Introduction Isolation of biologically active chemical fractions and the identification of specific toxic agents from complex environmental mixtures is a difficult technical challenge (I). This process, often referred to as bioassay-directed fractionation, requires chemical separation techniques that allow high recovery of total sample mass and individual toxic species (2). Application of bioassay-directed fractionation to the polar fractions of wood smoke, which contain 75-9090 of the mutagenic activity, has been particularly difficult (3). Common chemical separation methods for combustion samples such as normal-phase silica gel HPLC and aqueous-phase acid/base/neutral liquid/liquid partitioning have resulted in low mass recovery of polar compounds and loss of mutagenicity in polar fractions. Bell et al. (4) used normal-phase silica gel HPLC to separate dichloromethane extracts from freshly emitted and sunlight-reacted pine wood smoke particles. This method resulted in respective mass recoveries of 76% and 50%, and mutagenicity (TA98+S9) recoveries of 67% and 46%. Nishioka et al. (5) performed acid, base, and
"Aldrich, >99%. bChem Service, >99%. 'Kodak, >99%. Mean of four runs unless otherwise noted. Mean f SEM. Total mass of standard applied to columns was 12, 24, 30, or 30 mg. This represents a range of -0.05-0.1 mequiv (-1% of the column capacity). eSquareroot of the sum of the variance for the four fractions converted to Dercent of the mean.
Present address: National Research Council, Genetic Toxicology Division, US.EPA, Research Triangle Park, NC 27711. 4 Present address: Triangle Laboratories, Research Triangle Park, NC 27709. 0013-936X/90/0924-1261$02.50/0
% recovery
compound benzoic acid" phthalic acid" (n = 2) dibenzofuran"
1-naphthol"
base
neutral
acid
polar acid
totale
1 6 f 2 7 2 f 2 1 8 f 5 106f3 31 f 3 33 f 7 50 f 8 115 f 11
102 f 2 2051 7 6 i 4 102 f 3
102 f 2 7 f 3 103i5 102 f 3
9-fluorenone" 7,8-benzoquinoline" 85 f 3 85 f 3 (n = 2) 4-nitrophenolb 2 f l 83f7 11f4 96f8 pyrene" 104 f 3 104 f 3 naphthalic acid 8 8 f 4 11f4 8 f 3 1 0 7 f 6 anhydrideb benz [a ]anthracene124 f 6 124 f 6 7J2-dione'
neutral fractionation of pine smoke extracts by aqueousphase liquid/liquid partitioning and observed -40% mass loss and 3370mutagenicity loss. Clearly, there is a need for an alternative to these techniques when confronted with highly polar, acidic organic mixtures. The goal of this work was to explore the feasibility of a simple, alternative method for chemically separating acidic, basic, and neutral fractions found in pine wood smoke extract while achieving high mass and mutagenicity recovery required for bioassay-directed fractionation. We report here preliminary data on the use of a nonaqueous ion-exchange technique for fractionating a standard mixture of organic acid, base, and neutral compounds, and a dichloromethane extract from pine smoke particles. We have evaluated this scheme with respect to quantitative recovery of authentic chemical standards, mass of wood smoke extract, and mutagenicity. We are currently modifying and refining this technique for use on source emissions from wood stoves, ambient air samples impacted by residential wood stoves and mobile sources, and stack
0 1990 American Chemical Society
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Environ. Sci. Technol., Vol. 24, No. 8, 1990 1261
NON-AQUEOUS
samples from municipal waste incinerators.
Materials and Methods Reagents. High purity grade chloroform, dichloromethane (DCM), tetrahydrofuran (THF), and methanol were purchased from Burdick and Jackson (Muskegon, MI). The macroreticular ion-exchange resins (Amberlyst 15 and Amberlyst 26) were manufactured by Rohm and Hass and obtained from Alfa Products (Danvers, MA). Trifluoroacetic acid and isopropylamine were obtained from Fluka (Ronkonkoma, NY). Source and purity of standard compounds are listed in Table I. Conditioning of Ion-Exchange Resins. Preparation was similar to that of Bell et al. (6) and Jewell et al. (7). Amberlyst 15, a sulfonic acid cation-exchange resin (capacity 4.7 mequiv/g), was washed with 100 mL of a methanolic potassium hydroxide solution (5% by weight) and then rinsed with 100 mL of methanol. It was then converted to the acid state by slowly adding 100 mL of 5% volume HCl in methanol and stirring for 30 min. The resin was washed with distilled deionized water until the pH of the washing water was neutral. The final step was to Soxhlet extract the resin for 24 h each with methanol, dichloromethane, and a chloroform/ 5% tetrahydrofuran mixture. The cation-exchange resin was stored in chloroform in the dark in a glass container with a Teflon-lined cap until used. The anion-exchange resin, Amberlyst 26 (quaternary amine, capacity 4.4 mequiv/g), was washed with 100 mL of 5% HCl in methanol and rinsed with 100 mL of methanol. After being rinsed, the resin was activated by adding 100 mL of 5% methanolic potassium hydroxide solution. The resin was washed with deionized water, Soxhlet extracted sequentially with methanol, DCM, methanol, and DCM, and stored in DCM. Resins should be used within 30 days of conditioning. Approximately 6 g (dry weight) of resin was slurry packed in DCM into a 25-mL buret plugged with glass wool and fitted with a Teflon stopcock. Columns were rinsed with 50 mL of the initial solvent prior to addition of the sample. The quantity of resin used in these separations was estimated to provide greater than 90% excess anion-exchange capacity relative to the sample. Separation Procedure. A high concentration standard was prepared containing each of the compounds listed in Table I at a concentration of -6 mg/mL in 10% methanol/DCM and aliquots of this mixture were used for the qualitative and quantitative analysis. The relative amounts of total acidic (40%), basic (lo%), and neutral (50%) components reflect the approximate distribution of these chemical classes in wood smoke. Many of these compounds have been identified in wood smoke by Kamens et al. (8) and Ramdahl and Becher (9). The method reported here is modified from that reported by Jewell et al. (7) and Bell et al. (6). The ionexchange separation procedure is shown schematically in Figure 1. Fractionation standard (12-30 mg) or wood smoke extract (69.1 mg dissolved in 10% methanol/DCM) was placed on the Amberlyst 15 column. Acids and neutrals were eluted with 50 mL of 20% methanol/DCM. Previous trials suggested that the presence of methanol in the elution solvent was needed to provide sufficient mobile-phase polarity to maintain the polar solutes in solution during the separation. The retained bases were removed from the resin with 50 mL of 20% volume isopropylamine in methanol. The acid/neutral mixture was concentrated by rotary evaporation to -1 mL and placed on the anion-exchange column. Neutrals were eluted in 50 mL of 100% DCM. 1282 Environ. Sci. Technol., Vol. 24, No. 8, 1990
ION EXCHANGE
Sample
Cation Exchange Resin A15
I
Bases Retained
m
NeutralsiAcids 20% MeOHlDCM
1.
t
Anion Exchange Resin A26
Open Column System
2. Bases Eluted 20% IPMMeOH
Concentrate to 1 ml
n
I
Acids /Retained
II
U
f
3. Neutrals DCM
4. Acids COZIMeOH
5. Polar Acids 10%TFAlMeOH
Figure 1. Acid, base, and neutral separation scheme utilizing ionexchange resins.
Retained acids were then eluted by adding 50 mL of C02/methanol solution. This fraction is referred to as the acid fraction. The C02/methanol solution was created by bubbling gaseous C02through methanol for -30 min. The polar acid fraction was collected by eluting with a 10% trifluoroacetic acid/methanol solution. Each elution was carried out at a rate of -1 mL/min. The volume of solvent used for the wood smoke separation was 75 mL/ fraction. Basic and acidic fradions were rotary evaporated just to dryness to remove any remaining isopropylamine, C02, or trifluoroacetic acid. They were then redissolved in fresh solvent. Samples to be bioassayed were solvent exchanged into DMSO. Quantitation of compounds in the fractions was by gas chromatography utilizing a 30-m fused-silica J&W, DB 1701 (phenyl(cyanopropy1)methyl) column with hydrogen as carrier gas and a flame ionization detector as described in Kamens et al. (10). Mutagenicity Assay. Samples were tested by the plate incorporation test with Salmonella typhimurium strain TA98 with and without a 10% rat liver homogenate mixture (S9) prepared from Aroclor-induced rats (11,12). Due to a limitation of mass for most fractions, fractions were tested with duplicate plates at four doses that were chosen by estimating the probable linear dose-response range based on previous testing. Mutagenicity slope values were calculated by simple linear regression using all dose levels.
Results and Discussion Recovery of Authentic Compounds. Table I summarizes the recovery of authentic compounds through the ion-exchange separation method (four trials). Total recovery of individual compounds in the fractionation standard was high, and qualitative separation between chemical classes was good. There was no overlap of acid or neutral compounds into the base fraction, and the base, benzoquinoline, showed 85% recovery. Good qualitative separation of neutrals was also achieved. With the exception of naphthalic acid anhydride, no neutral compounds appeared in the acid or base fractions. The acidic compound, 4-nitrophenol, appeared almost entirely in the acidic fractions and showed 96% recovery. These data
Table 11. Mass Distribution and Recovery for Wood Smoke Particle Extract Sample mass, mg
9i rec
whole
69.1
base neutral acid polar acid
10.2 38.0 14.2 11.9
sum
74.3
100.0 14.8 55.0 20.5 17.2 107.5O
sample
The mass measurement error (SD)for individual samples was typically A770 based on replicate analysis and A13% for the sum of four samples (square root of the sum of the variances of four samoles). (I
suggest that ion-exchange separation may be useful in studies such as Nishioka et al. (14), which focused on recovery and identification hydroxynitroarenes in atmospherically reacted samples. An area of concern in these data is the presence of significant quantities of weak acid species, specifically benzoic acid, 1-naphthol, and phthalic acid in the neutral fraction (16%, 20%, and 31%, respectively). Given that the total milliequivalents of acid in these four separations varied from -0.05 to 0.1 mequiv (see Table I) and that the total ion-exchange capacity of the column was -26 mequiv, it is unlikely that breakthrough, or overloading of the column, caused the elution of the weak acids in the neutral fraction. In additional trials, we explored the hypothesis that the presence of methanol could reduce the selectivity of the anion-exchange resin for weak organic acids. We found that increasing the methanol concentration of the neutral elution solvent to 10% in step 3 (Figure 1)resulted in a dramatic increase (from 16% to 46%) in the quantity of benzoic acid in the neutral fraction. Because many of the acidic components could not be maintained in solution in 100% DCM, elution of the neutrals/acids from the cation resin in step 1 required 20% methanol/DCM. Therefore, we infer from the above results that the presence of residual methanol from step 1, concentrated during the rotory evaporation step, is the reason for the observed lack of selectivity of this ion-exchange resin for weak acids. Ongoing work suggests that a related anion-exchangeresin, AG-MP1 (Bio-Rad, Richmond, CA), has better selectivity in the presence of methanol, particularly if used in a solid-phase extraction protocol (15). Wood Smoke Mass Recovery. A wood smoke extract sample was chemically separated, and mass distribution and recovery results are shown in Table 11. The recovery of mass through the separation scheme was 107.5 f 13%. The base, neutral, and acid fractions contained 14.8%, 5570, and 37.7%, respectively, of the fractionated mass. For pine smoke stack sample, Nishioka et al. (5)recovered 59% of the mass fractionated using an aqueous-phase liquid/liquid partitioning procedure. Of the total mass fractionated, bases comprised 1.9%, neutrals 29.390, and acids 27.8%. Mutagenicity Recovery. The TA98 mutagenic potency, percent distribution, and recovery are shown in Table 111. The whole unfractionated pine wood smoke extract was slightly more potent than other samples tested in this laboratory (typical values for TA98: -S9, 0.3 revertants/pg; +S9,1.2 revertants/pg). The neutral fraction contained the most mutagenicity with or without S9 activation. The acid fractions also contained significant direct-acting (-S9) mutagenicity (11% and 28%). The base fraction had a high S9-dependent mutagenic potency
Table 111. Mutagenic Potency, Percent Distribution and Mutagenicity Recovery for Wood Smoke Whole Extract and Fractions in TA98
sample whole extract base neutral acid polar acid sum of fractions
TA98-S9 TA98tS9 rev/pg rev/ % mutag rev/pg rev/ % mutag extrt" fractnb distribn' extrta fractn* distribnC 0.47 0.08 0.52 0.24 0.76
32280 820 19800 3440 9060
100 3 61 11 28 103
1.77 1.35 1.60 0.31 0.72
122530 13730 60650 4400 8550
100 11 49 4 7 71
Revertants per microgram of extract indicates relative potency of the samples. Mean (n = 3) revertant counts for controls: spontaneous 4 9 , 26; spontaneous +S9, 31; 3 kg of 2-nitrofluorene, 268; 0.5 pg of 2-aminoanthracene,477. Calculated slope error for most samples was less than 5%; however, only a single teat was performed and typical historical error between repeat tests is *-15%. bRevertants per fraction is (revertants per microgram of extract)(fraction m a d [ (potency)(mass)]. Percent mutagenicity distribution is (potency)(mass) as a percent of the unfractionated or whole sample.
but, due to low mass, contributed relatively little (11%) to the whole sample. Mutagenicity recovery was calculated from the sum of the percent distribution values for the fractions. These values were 103% and 71 7%. Variation between recovery of direct-acting and S9-dependent mutagenicity was observed in previous work with a similar separation scheme (6) and also during HPLC fractionation of oak combustion particles reacted with NOz and O3(8). Variation in mutagenicity recovery may reflect the combined error in the mutagenicity assay and the mass analysis (estimated at 15-2590 for mass and mutagenic potency combined). This suggests that recoveries are within the range of the experimental error. Mutagenicity testing of method blanks produced no significant response above the spontaneous mutation level, suggesting little effect of the method on the mutagenicity of the fractions. Chromatographic analysis of the method blanks detected a single trace contaminant peak. In addition, small quantities of isopropylamine and trifluoroacetic acid were found in the base and polar acid fractions. The chromatographic contaminant may be related to residual unpolymerized divinylbenzene monomers eluting from the ion-exchange resin. Isopropylamine and trifluoroacetic acid were tested for mutagenicity and did not produce a mutagenic response. Overall, these data suggest adequate recovery of mass and mutagenicity through this separation scheme and demonstrate the compatibility of the technique with the mutagenicity bioassay. This method appears to be feasible as a primary step in bioassay-directed fractionation, particularly for separating highly polar organic extracts such as wood smoke. Current research is aimed a t improving qualitative separation of weak acids by optimizing flow and solvent parameters and exploring additional resin types in a solid-phase extraction protocol. We are also evaluating this method with a variety of highly acidic samples such as particles from a municiple waste incinerator. Acknowledgments We thank Drs. L. Claxton and J. Lewtas, US. EPA, for their continued support of this project. We also acknowledge the useful suggestions made by M. Nishioka, Battelle Columbus Laboratory, and the excellent technical support of J. Fulcher and C. Braun, University of North Carolina. Environ. Sci. Technol., Vol. 24, No. 8, 1990
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Registry No. Amberlyst 15, 9037-24-5; Amberlyst 26, 69865-42-5; benzoic acid, 65-85-0; phthalic acid, 88-99-3; dibenzofuran, 132-64-9;1-naphthol,90-15-3; 9-fluorenone, 486-25-9; 7,8-benzoquinoline, 230-27-3; 4-nitrophenol, 100-02-7; pyrene, 129-00-0; naphthalic acid anhydride, 81-84-5; benz[a]anthracene-7,12-dione, 2498-66-0.
Literature Cited Scheutzle, D.; Lewtas, J. Anal. Chem. 1986,58,1060-1067. Lewtas, J. Fundam. Appl. Toricol. 1988, 10, 571-589. Nishioka, M.; Chuang, C. C.; Peterson, B. A.; Austin A.; Lewtas, J. Enuiron. Int. 1985, 11, 137-146. Bell, D. A.; Kamens, R. M.; Claxton, L. D.; Lewtas, J. Presented at 79th Annual Meeting, Air Pollution Control Association, Minneapolis, MN, 1986; APCA 86-77.4. Nishioka, M.; Strup, P.; Chuang, C. C.; Cooke, M. Draft final report prepared by Battelle Columbus Laboratories for ORD-USEPA, Contract No. 68-02-2686,Task Directive 134, 1986. Bell, D. A., Karam, H.; Kamens, R. M. Proceedings of the 1987 EPAIAPCA Symposium on Measurement of Toxic Air Pollutants; EPA600/9-87-010; Research Triangle Park, NC, 1987; APCA V1P-8, pp 411-415. Jewell, D. M.; Weber, J. H.; Benger, J. W.; Plancher, H.;
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Latham, D. R. Anal. Chem. 1972,44, 1391. (8) Kamens, R. M.; Bell, D. A.; Dietrich, A.; Perry, J.; Goodman, R.; Claxton, L. D.; Tejada, S. Environ. sci. Technol. 1985, 19, 63-69. (9) Ramdahl, T.; Becher, G. Anal. Chim. Acta 1982,144,83-91. (10) Kamens, R. M.; Karam, H.; Guo, J.; Perry, J. M.; Stockburger, L. Enuiron. Sci. Technol. 1989,23, 801-806. (11) Maron. D.: Ames. B. N. Mutat. Res. 1983. 113. 173-215. (12) Claxton, L.; Austin, A.; Kohan, M.; Evans, C.’Heakh Effects Research Laboratory, U.S. EPA, EPA-HERL-0323; 1982. (13) Kamens, R. M.; Rives, G.; Perry, J.; Bell, D. A.; Paylor, R. E., Jr.; Goodman, R. G.; Claxton, L. D. Environ. Sci. Technol. 1984, 18, 523-530. (14) Nishioka, M.; Howard, C.; Contos, D.; Ball, L.; Lewtas, J. Environ. Sci. Technol. 1988, 22, 908-915. (15) Bell, D. A.; Williams, R.; Brooks, L.; Thompson, D.; Zwiedinger, R.; Lewtas, J. Presented a t the International Conference on the Genetic Toxicology of Complex Mixtures, Washington, DC, 1989; abstract.
Received for review February 1, 1989. Revised manuscript received February 23,1990. Accepted March 12,1990. This work was funded wholely by cooperative agreement (CR 812514) and grant (R812256)from the U S . EPA.
