Determination of volatile and semivolatile mutagens in air using solid

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Anal. Chem. 1991, 63,1644-1650

1644

Determination of Volatile and Semivolatile Mutagens in Air Using Solid Adsorbents and Supercritical Fluid Extraction J. M. Wong, N. Y. Kado, P. A. Kuzmicky, H.-S. Ning, J. E. Woodrow, D. P. H. Hsieh, and J. N. Seiber* Department of Environmental Toxicology, University of California, Davis, California 95616

Volatlk toxicants may be present In emkrlons from moMk andstatknsryroucesaswdlaslnInacrk. Methodsfor cdkctlng and concentrating volatlles from alr samples have been developad. solkcphase adsorbents were compared In thelr trapplng efflclencles for dlchloromethano (DCM), ethylene dlbtomlde (EDB), 4-nltroMphenyl (4-NB), 2-nltrofluorene (2-NF), and ftuoranthene (FI). Charcoal and CarW e v e were the mort effklenl medla for retainlng DCM, whUe XAD-4 was the bert adsorbent tor EDB and the aromatk compounds. Extrclctlon of dkeci spikes of compounds from adsorbents urlng wpercrltlcal carbon dbxlde resulted In >90% recovery of EDB and 6 0 4 2 % recovery of the aromatlcs. Inlegratlon of trapplng and desorptlon methods wlth the SabnoneWa mkroruspendon bloaesay was demonstrated wlth EDB and 4-NB recoveries from ah; chemical malysla and Massay gave “parable remutts (wHhln 10%).

INTRODUCTION Due to increasing concern over air pollution from trace organics, it is becoming important to assess the nature and quantities of the species present in air and their impact on human health and the environment. Some vapor-phase organics, such as PAHs polynuclear aromatic hydrocarbons, halogenated hydrocarbons, and some pesticides, are known to be hazardous to humans and other biota in the environment (1). Once air samples have been chemically analyzed, rapid screening by relatively inexpensive short-term bioassays can be used to determine toxicity, mutagenicity, and the other potential hazards of the analytes. More expensive long-term studies with experimental animals can then be carried out to determine health effects of the suspect compounds. Few studies examining the toxicity of highly volatile vapor-phase organic mixtures have been conducted because of the lack of adequate methodology to quantitatively analyze volatile samples and to assay their toxicities. The major problem in assessing volatile toxicants is that conventional collection and desorption techniques result in sample loss. Most studies of air pollutants focus on the less volatile organics trapped on solid adsorbents or as particulates trapped on filters (2-4). Both XAD adsorbents and Tenax have been routinely used in many studies to collect airborne compounds such as PAHs, hydrocarbons, PCBs (polychlorinated biphenyls), N- and S-containing compounds, halogenated compounds, and pesticides (5-11 ) . Other adsorbents used to sample organics in air include the Chromosorb polymers, polyurethane foam (PUF), and carbonaceous adsorbents (12-14). Analytes are usually recovered from the adsorbents by Soxhlet extraction or sonication, both of which may require long extraction periods and large volumes of solvent and result in incomplete recovery or sample decomposition. Extracts are then concentrated by rotary vacuum, by the Kuderna-Danish technique, or under a stream of nitrogen prior to trace analysis. Before testing in a bioassay, the solvent must then be exchanged to a bioassay-compatible solvent such as dimethyl sulfoxide (2). An alternative method of sample recovery is by thermal desorption of the adsorbent 0003-2700/01/0383-1644$02.50/0

(e.g. Tenax) and collection of anal* in a cryogenic trap (15, 16). This technique is limited by analyte and adsorbent stability, and it too may result in incomplete recovery. Supercritical fluid extraction (SFE) of organics from adsorbents represents a powerful alternative to traditional methods of sample preparation. When compared to conventional extraction methods, SFE can provide a more rapid and efficient extraction, increased selectivity, and potential sample fractionation. These advantages can be attributed to properties of a solvent at temperatures and pressures above its critical point. Supercritical fluids exhibit densities similar to those of a liquid, yet with solute diffusivities and viscosities closer to those of a gas; these properties facilitate mass transfer of solutes resulting in a rapid and efficient extraction. The solvent strength of the fluid, which depends on density, may be varied by changes in the pressure or by using solvent modifiers (17). In addition, fluids with low critical temperatures allow extractions at relatively mild conditions, thus minimizing chemical changes. Typically, carbon dioxide, with its critical temperature of 304.2 K, critical pressure of 72 atm (7295Wa), and critical density of 0.468g / d , has been a fluid of choice (18). The critical point is accessible, it is a good solvent for organics, and it is nontoxic, nonflammable, and inexpensive. Several studies have shown that SFE with COz can yield rapid and quantitative recoveries of pesticides from soil and vegetation (19, 20), natural products from foods (21), and PAHs, PCBs, and dioxins from solid matrices (22-26). Applications of SFE for air samples include extracting PAHs from Tenax, alumina and charcoal, cigarette smoke pollutants and toxic organics from Tenax, PAHs from XAD-2, and woodsmoke effluent from PUF (13,27-30). Extracting with supercritical fluids can pose a major advantage for isolating volatile species, since it is easy to remove the extracting fluid, such as COz, as a gas from the analyte. There is no need for a solvent evaporation step or a step to exchange solvents for bioassay testing. By producing concentrated extra&, analyte recovery can be drastically simplified and potential sample loss can be minimized, since the extracts can be directly analyzed without additional sample preparation. In the present study, the model volatile mutagenic compounds dichloromethane, ethylene dibromide, Cnitrobiphenyl, 2-nitrofluorene, and fluoranthene were used to optimize collection and extraction methods for air samples. These compounds represented different classes of chemicals with volatiles ranging from 349 mmHg (dichloromethane)to 0.01 mmHg (fluoranthene). Adsorbents tested included charcoal, Carbosieve SIII, XAD-4, Tenax TA, and Chromosorb 102. Analyte recovery was investigated by using SFE with COz as the solvent. Upon volatilization of the COz, concentrated extracts were analyzed directly without additional concentration steps. Integration of trapping and desorption methods with the Salmonella bioassay was demonstrated with ethylene dibromide and 4-nitrobiphenyl.

EXPERIMENTAL SECTION Chemicals. Solvents such as methanol, acetone, ethyl acetate, toluene, and hexane were ‘resi-analyzed” grade (J. T. Baker), and

anhydrous diethyl ether was supplied by Fisher Scientific. 0 1001 American Chemical Society

ANALYTICAL CHEMISTRY, VOL.

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Table I. Adsorbents for Trapping Study

adsorbent

mesh size

charcoal Carbosieve SI11 Chromosorb 102 Tenax-TA XAD-4

60/80 60180 60/80 20150

adsorbent characteristics surface area, pore diam, A m2/g 500-2000 800

15-25 35 750

Ethylene dibromide (EDB), 99.4% purity, was supplied by Chem-Services,and dichloromethane (DCM),HPLC grade, 99.9% purity, 4-nitrobiphenyl (4-NB), analytical grade, 2-nitrofluorene (2-NF), 98% purity, and fluoranthene (Fl), 98% purity, were purchased from Aldrich Chemical Co. Adsorbents. On the basis of adsorbent stability, commercial availability, and desorption techniques, several adsorbents were selected for trapping characterization (Table I). XAD-4 was selected rather than XAD-2 because of our prior experience in the laboratory of its trapping efficienciesand its higher surface area and smaller pore diameter might prove better for volatile compounds. Adsorbents were packed into disposable pipets (10 cm X 5 mm id.) and held in place by glass wool plugs precleaned by solvent extraction with ethyl acetate. The amounts of adsorbent in the sampling tubes varied somewhat but were approximately 1-2 mL (200-900 mg), depending on adsorbent density and cost. Adsorbents were prepared for air sampling by the following procedures. XAD-4 resin was washed with successive volumes of 0.5 N HCl, 0.5 N NaOH, and distilled deionized water for approximately 30 min each wash (31). The resin was then cleaned by Soxhlet extraction with acetone, ethyl acetate, and diethyl ether, each for 24 h. The resin was dried for 48 h under vacuum at 50 "C. Charcoal was deactivated by heating for 24 h at 500 "C. Tenax, Chromosorb 102, and Carbosieve were heated to 230 "C and purged with helium. Sampling Apparatus. Two systems were used to deliver model compounds to a test airstream. The amount of chemical delivered to the airstream was largely determined by the sensitivity of the bioassay to the compound. The volatile compounds (DCM and EDB) were metered into a prefiltered airstream by diffusion from a reservoir through capillary tubes. Diffusion rates were controlled by the capillary diameter and diffusion pathlength, and rates were verified by determining the weight loss of the compound from the reservoir prior to and after sampling. Semivolatilecompounds (aromatics)were coated onto the inner surface of a glass tube upstream of the sampling tubes. The glass tube was heated to 60 "C with heating tape to increase volatilization of the compounds from the glass surface. A sampling period of 24 h was required to volatilize approximately 50 pg of each compound. After sampling, the glass tube was rinsed with 3 mL of toluene and analyzed to determine the unvolatilized chemical. The amount volatilized was calculated as the difference between the initial amount and that remaining after sampling. Adsorbent Trapping Efficiency. Recovery of 1mg of DCM by charcoal, Carbosieve SIII, and XAD-4 was evaluated by extracting the adsorbent samples with 10 mL of toluene in septum-sealed crimp-top vials with agitation on a rotary shaker for 20 min. Toluene was used as the extracting solvent since it could be chromatographically separated from DCM. Recovery of 100 pg of EDB by all of the adsorbents was evaluated by extraction with 3 mL of ethyl acetate in screw-cap septum-sealed vials, shaken for 20 min on a rotary shaker. For charcoal, 10 mL of ethyl acetate was injected in septum-sealed crimp-top vials due to excessive pressures that built up in the screw-cap vial. 4-NB, 2-NF, and F1 were extracted from Chromosorb 102, Tenax TA, and XAD-4 with 3 mL of ethyl acetate in triplicate in screw-cap teat tubes with 20-min agitation on a rotary shaker. The extracts were combined and analyzed by GC. All air samples included backup tubes to determine chemical breakthrough and blanks for background interferences. Supercritical Fluid Extraction. A dynamic flowthrough supercriticalextractor was constructed with a syringe pump (Isco SFC-500 microflow pump) to pump supercritical carbon dioxide

20-75

15-40 850 200 50

source

amt used, mg

SKC-West, Inc. Supelco Supelco Alltech Rohm & Haas

625 340 210 720

900

Liquid C 0 2 under 1900 psi helium headspace

I

I

mice-acetone

bath

Syringe Pump

Flgurs 1. Supercritical fluM extraction (SFE) system.

through an extraction cell containing adsorbent (Figure 1). Supercritical fluid chromatography grade liquid carbon dioxide under a helium head space of 1500 psi was used for all extractions. The cell volume was approximately 2 mL. A length of 25 pm i.d. (375 pm 0.d.) fused-silica capillary (Lee Scientific) was used as a depressurizing flow restrictor. The effluent from the outlet of the capillary was directed into a volume of methanol to trap and concentrate volatile chemicals for subsequent analysis. Methanol was selected as the trapping solvent due to its miscibility with water and compatibility with the Salmonella bioassay. Different volumes of methanol were used depending on the mutagenicity of the compound and therefore the concentration needed for response in the bioassay. For the extraction of DCM and EDB, a 40-cm length of restrictor was used to maintain a fluid flow rate of approximately 175 pL/min. The extraction cell temperature was maintained at 50 OC. DCM was extracted from charcoal at pressures ranging from 2000 to 6OOO psi. EDB was extracted from XAD-4 with CO, at 3000 psi at a flow rate of 200 pL/min. To prevent plugging of the restrictor, a 10-cm length of 0.53 mm i.d. deactivated fused-silica tubing was connected to the restrictor by using a press-fit column connector and warmed with heating tape to prevent freezing within the connector. The carbon dioxide extracted DCM was bubbled into 2 mL of toluene chilled to -50 "C in a dry iceacetone bath. For EDB, the effluent was bubbled into 100 pL of methanol in a hematocrit tube chilled to -50 OC in a dry ice-acetone bath. For the aromatic compounds, a 20-cm length of restrictor was used to maintain a fluid flow rate of 300 pL/min. The extraction cell was heated to 50 "C, and the extraction pressure was 6000 psi. 4-NB, 2-NF, and Fl were trapped in 2 mL of methanol, which was cooled to below room temperature by the expansion of the carbon dioxide. The carbon dioxide modifier was added to the pump by filling a reservoir in-line between the carbon dioxide tank and the pump. Gas Chromatography. Both DCM and EDB were analyzed by using a Model 5730A gas chromatograph equipped with a asNi electron capture detector (ECD) and either a 30 m X 0.53 mm i.d. J & W DB-5 (1.5-pm film thickness) megabore column or a 30 m X 0.53 mm i.d. J & W DB-17 (1.5-pm film thickness) megabore column under the following conditions: column flow rate was 6.8 mL/min 10% methane/argon; makeup gas flow was 40.2 mL/min 10% methane/argon; column temperature was 65 "C; detector temperature was 300 "C; injector temperature was 250 "C. dNB, 2-NF, and Fl were analyzed on an HP 5890 GC equipped with a asNiECD and a 30 m X 0.53 mm i.d. J & W DB-5 (1.5-pm

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Table 11. Adsorbent Collection Efficiencies for Dichloromethane

adsorbent charcoal

XAD-4 Carbosieve SIX1

flow rate, L/min

vap dens, PPm

first trap

0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 2.0

270 58 3.7 149 31 3.7 130 56 7

84.1 f 16.6 61.1 f 8.0 54.4 f 9.5 29.7 f 5.0 12.0 f 1.5 b 76.1 f 11.7 91.2 f 9.7 73.7 f 8.4

collection efficiencv. second trap b 16.1 f 1.4 b 21.9 f 5.9 10.5 f 2.9 b b b b

total 84.1 f 16.6 77.2 f 8.9 54.4 f 9.5 51.6 f 10.8 22.5 f 3.6 b 76.1 f 11.7 91.2 f 9.7 73.7 f 8.4

Each determination is the mean and standard deviation of triplicate analyses. Less than detection limit of 0.5%. film thickness)megabore column. The operating conditions were as follows: column flow rate was 15 mL/min helium; makeup gas flow rate was 60 mL/min nitrogen; column temperature was 220 OC; injector temperature was 250 OC; detector temperature was 300 "C. Fluoranthene was separated from interferences on an HP 5730A gas chromatograph equipped with a "Ni ECD and a 30 m X 0.53 mm i.d. J & W DB-17(1.5-rm film thickness) megabore column under the following conditions: column flow rate was 8 mL/min 10% methane/argon; makeup gas flow was 56 mL/min 10% methane/argon; column temperature was 230 OC; detector temperature was 300 OC; injector temperature was 250 OC. Mutagenicity Testing. A microsuspension procedure previously reported by Kado et al. (32,33),which is a simple modification of the Salmonella/micro"e test of Ames et al. (34) was used to test samples for mutagenic activity. The assay is approximately 10 times more sensitive than the Amea Salmonella procedure, based on a b l u t e amounta of materialadded per tube. The procedure was adapted for volatile compounds by using a closable test tube. Tester strains TA98 and TAl00 were provided by Dr. B. N. Ames, Berkeley, CA. For the modification, bacteria were grown overnight in Oxoid Nutrient Broth No. 2 (Oxoid Ltd., Hanta, England) to approximately (1-2) X loa cells/mL and harvested by centrifugation (5ooo X g, 4 OC, 10 min). W were reauspended in ice-cold phosphate-buffered saline (0.15 M PBS, pH 7.4) to a concentration of approximately 1 x 1O'O cells/mL. The S9 (metabolic enzymes added to the assay tubes) and S9 mix were prepared according to the procedure of Ames et al. (34). The S9 from Aroclor 1254 pretreated male Sprague-Dawley rata contained 40.3 mg of protein/&, as determined by the modified Biuret method of Ohnishi and Barr (35). For the microsuspension assay, the following ingredients were added, in order, to 13 X 100 mm sterile glass screw-cap culture t u b kept on ice: 0.1 mL of S9 mix, 0.005 mL of sample in DMSO or methanol, and 0.1 mL of concentrated bacteria in PBS (10I0/mL of PBS). The tubes were incubated in the dark at 37 "C with rapid shaking, and after 90 min, the tubes were placed in an ice bath and 2 mL of molten top agar containing 90 nmol of histidine and biotin were added (34).The solutions were poured onto minimal glucose plates, incubated at 37 OC in the dark for 48 h, and counted by an automatic plate counter (Biotran counter, New Brunswick Scientific,Princeton, NJ). Strain markers were routinely determined for each experiment. RESULTS AND DISCUSSION Evaluation of Adsorbents. The trapping of DCM was evaluated with charcoal, Carbosieve, and XAD-4. Tenax and Chromosorb 102 were not tested because they have been previously shown to be inefficient trapping agents for highly volatile compounds such as DCM (36,37). To determine recovery of DCM from the adsorbents by toluene extraction, 1 mg of DCM was directly spiked onto each adsorbent. Recoveries from Carbosieve, XAD-4, and charcoal were 32.1 i 5.8%, 48.4 f 7.2%, and 97.9 f 9.9%, respectively. Since low extraction efficiencies were observed with Carbosieve and XAD-4, trapping efficiencieswere corrected for the extraction

Table 111. Solvent Extraction Recoveries for Model Compounds

EDB adsorbent tube spikes Chromosorb

(100 fig)

% recovery 4-nitro2-nitr0biphenyl fluoranthene fluorene (50 rg) (50 rg) (50 rg)

92.3 f 5.5 3.1 80.1 f 7.5

95.6

68.5 f 4.7 80.6 f 5.3

91.3 f 1.7 87.0 f 5.9

59.2 f 5.3 b b 62.9 f 5.3

69.5 b

102

Tenax-TA charcoal Carbosieve XAD-4

103.8 f 4.6 74.0 f 1.8 78.1 f 1.8 92.5 3.2

*

71.6 f 10.6 b b 72.7 4.2

b 73.4

7.4

* 1.2

a Mean and standard deviation of triplicate samples. Extracting solvent ww ethyl acetate for all compounds. Aromatic compounds were recovered from tube spikes by using toluene. Not detected.

recovery for comparison of adsorbents on the basis of co&ction ability alone. Charcoal collection efficiencies were not corrected since extractions were quantitative. DCM was collected at 0.1, 0.5, and 3.0 L/min for all the adsorbents tested. Table I1 is a summary of collection efficiencies for both"primary and backup traps. Collection efficiencies for DCM were calculated by using the following equation:

9% collection efficiency = amt on adsorbent x 100 wt loss of chemical reservoir

# In general, collection efficiency of charcoal was related to vapor density and inversely related to flaw rate. In this study, at low flow rates and high vapor densities both charcoal and Carbosieve SI11 proved suitable for DCM due to their high collection efficiencies and low breakthrough. Collection efficiencies of Carbosieve for DCM did not vary with flow rate or vapor density. A recent study of carbon-containing adsorbents determined breakthrough volumes for 0.25 g of Carbosieve and charcoal to be 66 and 39 L, respectively, for DCM at 20 OC (38). Major drawbacks to using Carbosieve in the present study included ita frne mesh size, which limited the flow rate through the adsorbent, and its high cost. Since charcoal is relatively inexpensive, larger amounts may be used to compensate for its lower breakthrough volume; fwthermore ita larger mesh size does not restrict flow through the sampling tubes. XAD-4 performed poorly as a trapping agent for DCM at all vapor concentrations and flow rates despite its large surface area and small pore size. At the lowest vapor density, the amounts of DCM trapped by XAD-4 were below the detection limit of the method. Collection efficiencies of the adsorbents for EDB were examined at 0.1, 0.5, and 3.0 L/min. Solvent extraction effi-

ANALYTICAL CHEMISTRY, VOL. 63,NO. 15, AUGUST 1, 1991

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Table IV. Adsorbent Collection Efficiencies for Ethylene Dibromide and Aromatic Compounds

compd EDB

adsorbent charcoal Chromosorb 102 XAD-4

Tenax-TA Carbosieve 4-NB

XAD-4

Tenax-TA Chromosorb 102 F1

XAD-4

Tenax-TA Chromosorb 102 2-NF

XAD-4

Tenax-TA Chromosorb 102

flow rate, L/min

vap dens, PPb

0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 2.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0 0.1 0.5 3.0

760 127 27 760 151 17 793 156 24 868 168 35 926 174 78

collection efficiency, 5%" second first trap trap 69.6 f 6.8 79.7 f 7.5 89.0 f 3.4 72.6 f 3.3 51.1 f 1.9 11.7 f 1.6 100.6 f 7.9 86.1 f 1.3 102.5 f 7.0 51.8 f 3.0 49.3 f 2.3 11.6 f 1.2 48.5 f 2.4 49.1 f 4.1 47.1 f 2.5 68.2 f 5.3 92.8 f 6.7 93.9 f 3.5 57.3 f 3.4 72.6 f 11.7 74.2 f 5.0 64.5 f 8.3 78.1 f 12.4 87.5 f 6.8 85.9 f 7.5 62.6 f 6.8 92.4 f 6.5 72.6 f 14.3 70.0 f 7.6 73.0 f 10.9 72.8 f 4.2 73.4 f 13.9 76.8 f 5.8 75.7 f 15.7 96.4 f 9.1 90.2 f 13.6 55.2 f 8.6 75.5 f 9.2 59.4 f 6.8 53.2 f 6.2 65.8 f 4.5 72.7 f 8.2

b b b 1.5 f 2.6 13.7 f 2.1 20.5 f 2.1 b b 4.7 f 2.7 b 3.0 f 1.9 8.2 f 0.7 0.3 f 0.01 0.3 f 0.04 0.1 i 0.02 C C C C C C

C C

C C C

C C C C C C

C C C C C

C C C

C C

total

69.6 f 6.8 79.7 f 7.5 89.0 f 3.4 74.1 5.8 64.8 f 2.4 32.2 f 1.5 100.6 f 7.9 86.1 f 1.3 107.1 f 8.9 51.8 f 3.0 52.3 f 0.7 19.8 f 1.7 48.4 f 1.8 49.4 f 4.1 47.2 f 2.4 68.2 f 5.3 92.8 f 6.7 93.9 f 3.5 57.3 f 3.4 72.6 f 11.7 74.2 f 5.0 64.5 f 8.3 78.1 f 12.4 87.5 f 6.8 85.9 f 7.5 62.6 f 6.8 92.4 f 6.5 72.6 f 14.3 70.0 f 7.6 73.0 f 10.9 72.8 4.2 73.4 f 13.9 76.8 f 5.8 75.7 f 15.7 96.4 f 9.1 90.2 f 13.6 55.2 f 8.6 75.5 f 9.2 59.4 f 6.8 53.2 f 6.2 65.8 f 4.5 72.7 f 8.2

*

"Each determination is the mean and standard deviation of triplicate samples. *Less than detection limit of 0.1%. eNo aromatic compounds detected in second trap in all analyses. ciencies of EDB from the adsorbents are shown in Table 111. Collection efficiencies of the various adsorbents tested with EDB (Table IV)were calculated by using the same equation as for DCM. Charcoal and XAD-4 trapped EDB nearly quantitatively, probably due to their high surface area and porosity. There was detectable breakthrough of EDB by XAD-4 at the highest flow rate. Carbosieve trapped EDB poorly, with some breakthrough detected at all flow rates. Although chemically similar to XAD-4,Chromosorb 102 exhibited increasingly higher breakthrough of EDB with increasing flow rate and an overall lower collection efficiency, likely due to ita smaller surface area and large pore diameters. Tenax also trapped EDB ineffectively, with much breakthrough occurring to the backup tubes. A previous study has shown that for Tenax, a safe sampling volume of 30 L was observed for EDB at 20 OC, demonstrating the limited collection ability of this adsorbent for volatile organic vapors (39). Trapping of 4-NB, 2-W,and F'l was evaluated with Tenax TA, XAD-4, and Chromosorb 102. Solvent extraction recoveries are shown in Table m. Charcoal and Carhieve were not evaluated for trapping since direct spiking of the compounds onto the adsorbents multed in irreversible adsorption and thus no recovery by solvent extraction.

Collection efficiencies for the aromatic compounds were calculated by the following equation: 90 ' collection efficiency = amt on adsorbent/(extn recovery) X [initial 100 pg - (amt on glass tube after sampling)]

100 Table IV summarizes the aromatic compound collection efficiencies adjusted for extraction recoveries from the adsorbents. Analysis of backup traps of all samples revealed no detectable breakthrough of any of these analytes on any adsorbent. All three adsorbenta trapped 4NB effectively, while Tenax TA and Chromosorb trapped 2-NF leas effectively than did XAD-4. Chromoeorb 102 and Tenax were similar in their abilities to trap the three compounds from an airstream while XAD-4 exhibited slightly better collection efficiencies. On the basis of collection performances of the adsorbenta for the model compounds, a sampling train consisting of XAD-4 followed by charcoal or Carbosieve could be used to collect a range of volatile chemicals present in a complex mixture. Highly volatile compounds, such as DCM, that would break through the XAD-4, would be retained by either charcoal or Carbosieve, while XAD-4 would trap the less

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Table V. Aromatics Recovery from XAD-4 by C02 Extraction

extraction time, min 15 30

60 90 120 180 remaining on XAD-4O 15 30 60 90 120 180 remaining on XAD-4'

no modifier b 0.1 0.4 8.7 49.6 83.9 1.5

d 0.1 0.1 0.2 0.5 11.5 52.5

6% methanol

6% acetone

C02 modifier 6% ethyl acetate

6% hexane

12% hexane

% 4-Nitrobiphenyl" 0.9 b b 1.5 59.6 25.1 78.6 74.2 84.5 70.6 84.6 70.2 b 0.9

5.5 15.1 36.0 76.4 55.7 78.4 b

12.5 37.3 78.6 81.5 92.1 73.2 0.2

11.9 37.3 74.5 76.4 74.7 56.1 b

% 2-Nitrofluorene" 0.4 d 0.6 0.2 3.8 40.4 25.2 59.4 65.3 62.6 79.7 63.9 0.6 d

4.0 7.6 7.5 41.7 48.1 64.6 0.9

d 1.1 18.0 42.7 70.9 65.0 1.0

1.2 20.2 40.3 67.2 71.2 59.3 0.1

% Fluoranthene"

15 30 60 90 120 180 210 remaining on XAD-4'

e e e

e e

e e

e e

e

e

e

e

e

e

e

e

e 4.7 31.7 49.1 48.6 6.3

e 3.1 20.7 34.8 42.3 29

e 12.7 27.0 63.0 NU 11.3

e

e e

e e e e

e

25.9 66.2 61.1

ND e

"50-pg spike. bLesa than detection limit of 0.09%. CSolventextraction with ethyl acetate. dLess than detection limit of 0.06%. #Less than detection limit of 4.5%. fND = not determined.

volatile components, ranging from EDB through the PAHs. On the basis of this study, future trapping studies in this laboratory for complex mixtures will utilize multiadsorbent samplers consisting of XAD-4 and charcoal. Supercritical Fluid Extraction. Supercritical fluid extraction (SFE) was examined for desorbing model compounds from the adsorbents. Due to its high volatility, DCM presented the "worst case" situation using SFE to recover compounds from air sampling. Its volatility posed problems in both recovery from charcoal adsorbent and transfer to a solvent carrier trap for subsequent analysis. In experiments, 1mg of DCM was directly spiked onto charcoal, which was then extracted with C02 under pressures ranging from 2000 to 6000 psi and trapped into 2 mL of toluene at -50 "C. Although toluene is not a bioassay-compatible solvent, it was used in these tests for determination of extraction recoveries. Aliquots of toluene were analyzed at l b m i n extraction intervals, and afterward, the CO,-extracted charcoal was again extracted with toluene to determine DCM remaining on the charcoal. Pressures greater than 3500 psi were required to completely extract DCM within 75-90 min, but under these conditions, the cold toluene did not completely trap all of the desorbed DCM. In experiments where no detectable DCM remained on the charcoal, less than 20% was detected in the cold toluene trap. Evidently, DCM had been lost during transfer from charcoal to the toluene trap perhaps due to volatilization during COSdecompression. Typical methods of determining levels of DCM and other volatile chemicals in air have used a carbon-based adsorbent, with thermal desorption into a gas chromatograph to avoid potential sample loss (16,401. According to the results from this study using SFE, compounds of volatility similar to that of DCM will require a trapping-desorption technique that employs a means of recovering the compound from the extracting solvent or gas stream which does not involve simple evaporation of the solvent carrier.

For moderately volatile chemicals such as EDB, SFE proved to be an excellent method for desorption and recovery. Pressures of 3000 psi for 60 min were required to extract EDB from XAD-4. Recoveries of 500- and 5-pg spikes to XAD-4 were 89.9 f 5.0% and 95.3 f 12.7%, respectively. A slight loss of methanol (510%) was observed during the extraction, which may account for small losses of EDB due to covolatilization. Carbon dioxide at 3000 psi pumped through clean XAD-4 and then bubbled into 500 pg of EDBspiked methanol for 60 min did show some loss (87.0f 9.2% recovery) from the methanol. This covolatilization may further explain the low recoveries of DCM, since it is much more volatile than EDB. The aromatic compounds were relatively difficult to desorb from XAD-4. Extraction experiments were monitored by analyzing methanol aliquots during the extraction period. 4-NB recoveries from XAD-4 were significantly affected by the temperature of extraction, especially comparing subcritical (25 "C) to supercriticalconditions (50 "C),and by the preeence of methanol modifier (Table V), which resulted in high recoveries of 4NB within 120 min. Switching t~ modifiers, such as acetone, ethyl acetate, and hexane (6% and 12% v/v), also resulted in high recoveries within 90 min. Of the three compounds, 4-NB was most easily extracted from XAD-4,although the volume of cold methanol in the trap had to be carefully maintained during the extraction to avoid covolatilization of 4-NB. 2-NF was not as easily extracted from XAD-4, although temperature and modifier notably affected recoveries (Table V). Acceptable recoveries of 2-NF required extraction periods of at least 180 min using methanol modifier and an extraction temperature of 50 "C. Acetone (6%) increased the rate of extraction, and hexane (6%) modifier further enhanced the rate of extraction. Increasing the modifier composition to 12% hexane did not significantly increase the extraction rate. Of the model compounds extracted, F1 was the most dif-

ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1, 1991

Table VI. Recovery of PAHs from XAD-4 Using SFE (6000 psi, 180 min, 12% Hexane-Modified Carbon Dioxide) spiking level, pg

4-NB

50 5

76.7 f 8.8 88.5 12.7

% recove@ 2-NF

87.0 i 2.5 92.3 8.3

F1 70.8 i 3.3 60.6 5.7

Mean and standard deviation of triplicate samples.

ficult to recover from XAD-4 using SFE. With methanol as the modifier, there was no measurable recovery of F1. Using acetone and ethyl acetate as modifiers, it was possible to recover almost 50% of F1, while 6% hexane even further increased the recovery of F1 (Table V). Using 12% hexane resulted in acceptable recoveries in 180 min with no detectable F1 remaining on the XAD-4. Although use of methanol, acetone, and ethyl acetate modifiers resulted in acceptable recoveries for 4-NB and 2-NF, hexane (12%) produced the greatest recoveries for all three compounds within a 180-min extraction period. Table VI shows the recoveries of the three compounds at two spiking levels (using 12% hexane). Recoveries of both 4-NBand 2-NF were >75% at both spiking levels, while the recovery of F1 was slightly lower. Compared to previous studies using 24-h Soxhlet extraction for desorption of PAHs from adsorbents or particulates (5, 6), SFE was more rapid, needed very little solvent, and did not require removal of the extracting solvent. Previous SFE studies have shown quantitative recoveries of high molecular weight polynuclear aromatic hydrocarbons from Tenax and XAD-2 within 10-15 min at pressures of 3000-5000 psi (24, 27). Several reasons may account for the differences in extraction in this study: First, the highly microporous structure of the XAD-4 resin (50-A pore diameter) may permit more efficient penetration of adsorbed chemicals into the adsorbent. Second, the higher surface area of XAD-4 (750 m2/g) compared to Tenax (375 m2/g) and XAD-2 (300 m2/g) may result in increased binding of analytes to the XAD-4 surface. Third, the apolar composition of XAD-4 (styrene divinylbenzene) compared to Tenax (diphenyl phenylene oxide) could result in strong adsorptive interactions with nonpolar, aromatic analytes. The nature of the matrix seems to have a significant effect on the extraction process. Using modifiers with C02 may increase the solubilities of the solutes in the supercritical fluid by polarization of the solvent and dielectric constant, by acid-base interactions, and the modifier may physically displace the solutes by competing for adsorptive sites on the matrix (41). Finally, the flow rate of C02 through the matrix Table VII. Analysis of EDB and 4-NBby

may also affect the rate of extraction, especially if partitioning of the analyte between the matrix and fluid is the rate-limiting step. Higher flow rates of C02 were not used in this study since vigorous bubbling of COz in the solvent traps was observed with the flow rates teated. It was felt that higher flow rates might result in compound loss. Integration of 4-NB and EDB Air Samples with Bioassay Using SFE. These experiments examined the possible integration of air sampling and bioassay using SFE. The diffusion vapor system was used to deliver approximately 500 pg of EDB in an airstream over a period of 4 h to tubes containing XAD-4 resin. Resin samples and blanks were extracted with supercritical C02 at 3000 psi for 60 min. For all samples and blanks, a 5-pL aliquot was removed for GC analysis (Table VII) and the remainder of the extract was teated for mutagenicity. Our results indicate that an average of 77.3 f 3.5% of EDB in the air was recovered, with less than 1% remaining on the resin. Using the microsuspension bioassay, EDB standards were used to generate dose-response curve8 and on the basis of these curves, the concentration of EDB in the test samples was estimated. The amount of EDB estimated by bioassay in the supercritical fluid extract was compared to the quantities measured by GC analysis (Table VII). Quantitation of EDB by the two methods agreed to within 8%. Experiments were performed in which 4-NB vapors were trapped by XAD-4 and the samples were recovered for analysis by SFE (12% hexane-mdied C02, 180 min). The methanol extract (5pL) was analyzed by GC, and the remaining extract was analyzed by the bioassay (Table VII). An average of 50 f 12.8% of 4-NB spiked into the airstream was recovered by SFE. Comparison of the results from chemical analysis and Ames bioassay for 4-NB again showed close agreement (6%). CONCLUSIONS Volatile pollutants are a potentially major class of toxic chemicals present in both the indoor and outdoor air. Pollutants may differ considerably in physical properties and in concentrations in air. It is possible that toxicants may be present in the air which have yet to be identified or tested for hazardous properties. In thia study, commercially available solid adsorbents were evaluated for trapping volatile compounds. Adsorbents with high surface areas (charcoal, Carbosieve, and XAD-4) trapped volatile compounds such as DCM and EDB quantitatively. All of the adsorbents tested trapped the less volatile aromatic compounds efficiently. With SFE, extracts can be obtained from air-sampling adsorbents without additional sample concentration steps or solvent exchange into a bioassay-compatible solvent. Sample loss is minimized, and the concentrated samples can be di-

GC and Bioassay from XAD-4 Air Samples Using Supercritical Fluid Extraction

amt of EDB recovered (GC), fig

bioassay amt of TA100, pg +s9

bioassay amt of TA100, pg -s9

sample no.

amt of EDB delivered, pg

1 2 3 4 (blank)

500 540 480 0

404 399 368 0

250 200 240

400 319 360

81 74 77

0

0

0

amt of 4-NB

amt of 4-NB recovered (GC),

bioassay amt of TA98, pg -s9

bioassay amt of TA100, pg -s9

54 41 58

45 38 30 0

sample no.

delivered, pg

1 2 3 4 (blank)

76.6 69.1 75.7 0

1649

44.1 33.5 30.8 0

No detectable EDB remaining on XAD-4 resin after COz extraction. traction.

0

% EDB recovered (CC)"

% 4-NB recoveredb(GC)

57.6 48.4 40.6 0

No detectable 4-NB remaining on XAD-4 resin after COz ex-

1650

ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1, 1991

rectly analyzed by the bioassay. This study demonstrated the extraction of EDB, 4-NB, 2-NF, and F1 from an adsorbent used for air sampling by using supercritical carbon dioxide. For more volatile compounds, such as DCM, alternative techniques are necessary to quantitatively recover extracted analytes using SFE due to their volatilization in the expanding COz stream. Experiments demonstrating the integration of sampling and extraction methods with the bioassay were conducted with EDB and 4-NB, which further validated the use of SFE. Analysis of samples showed recoveries of 77% for EDB with good agreement between GC and bioassay. Recoveries from air for 4-NB were 50%, while GC and bioassay results agreed to within 6%. Due to differences in extraction conditions for EDB and the aromatic compounds, partial fractionation of the sample may be accomplished during extraction. The potential for class-selective extraction of organics from diesel exhaust particles and coal tar has been demonstrated (24, 42, 43). Studies have separated alkanes from PAHs (24) and separated PAHs by ring size and degree of alkylation (42,43). In most cases, selectivity of fractionation has been limited to classes of compounds. On the basis of the results of our experiments, extraction with COz for 60 min at 3000 psi, followed by extraction at 6000 psi with 12% hexane in COz could be used to recover the components of the sample for chemical analysis and to fractionate the components by either volatility or chemical class to simplify analyses. In this manner, a bioassay-directed analysis of fractions can be used to pinpoint those mutagens present in air samples. Further development of SFE for extraction, fractionation, and recovery of airborne compounds in air samples is warranted for the identification and assessment of hazardous chemicals in the atmosphere. LITERATURE CITED (1) Fishkin, L. I n chendoel h4utagms, Frhc@k and h48for W ~ t s c t l o nMlaender, : A,, Ed.: Plenum: New York, 1970 Vol. 4. (2) Egeback. K. E.; Tejle, G.; Stenberg, U.; Westholm, R.; Akberg, T.; Ranrmg, U.; Sundval, U. I n pdLnucker Arcrmetlc h)&oarbms; Cook. M., Ed.; BatteUe publishers: cdumbus, OH, 1982. (3) Scheuble, D.; Lewtas, J. Anal. Chem. 1988, 58, 1060A-1075A. (4) Westholm, R. N.; Almen, J.; U, H.; Rannug, J.; Egeback, K.X.; eagg, K. En-. Scl. Techno/. 1991. 25. 332-338. (5) AHhekn, I.: Becher, 0.;Hongda, J. K.; Ramdehl, T. Enbhw. Mutegen. 1984, 6, 91-102. (6)Doh, L. D.; &gley, S. T.; Leddy, 0. G.; Johnson, J. H. Envkon. Scl. Te~hnol.1987, 21. 757-785. (7) Otson, R.; Leach, J. M.; Chung, L. T. K. Anal. Chem. 1987, 59. 1701- 1705. ( 8 ) Krost, K. J.; Pelllzzarl, E. D.: Walburn, S. G.; Hubbard, S. A. Anal. chem. 1982, 54, 810-817. (9) Kawata, K.; Uehara, T.; Kltune, I.; Tomlnaga. Y.; Okawa, K. Bun&/ 1982, 31, 453-457.

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RECEIVED for review January 2,1991. Accepted May 6,1991. This study was supported by the California Air Resources Board (Contract No. A6-174-321, by the National Institute of Environmental Health Sciences (NIEHS Superfund Grant No. ES04699), and by the NIEHS training grant (No. ES07059) to J.M.W.