Environ. Sci. Technol. 1985, 19, 249-255
The Mutagenic Activity of Irradiated Toluene/NO,/H,O/Air
Mixtures
Paul B. Shepson," Tadeusz E. Klelndlenst, Edward 0. Edney, George R. Namle, and James H. Pittman
Northrop Services, 1nc.-Atmospheric
Sciences, Research Triangle Park, North Carolina 27709
Larry 1. Cupltt
Environmental Sciences Research Laboratory, U S . Environmental Protection Agency, Research Triangle Park, North Carolina 277 11 Larry. D. Claxton
Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 277 11 Irradiated mixtures of toluene/NO,/H,O/air were brought to a steady-state distribution of reactants and products in a 22.7 m3 flow-mode smog chamber, and the effluent was tested for mutagenic activity by exposing Salmonella typhimurium strains TAlOO and TA98 to it. Two different product distributions were examined, with that corresponding to a longer extent of reaction yielding a greater mutagenic response. Vapor-phase concentrations for several aldehydes, cresols, peroxyacetyl nitrate, toluene, nitrogen oxides, ozone, and particulate matter were obtained for the reaction chamber and exposure chamber air masses. Calculations were made as to the total quantity of each product species that deposited into the biotesting medium. This information was used to speculate on what may have caused the mutagenic response.
Introduction In the past several years there has been an increased level of concern that exposure to polluted urban atmospheres may pose a significant human health threat, specifically with regard to the incidence of cancer. Recently, measurements have been made af the mutagenic poteptial of actual urban air masses a t a number of sites (I), indicating a significant increased level of mutagenic activity at heavily industrialized locations. In addition, several volatile hydrocarbons [e.g., acrolein and vinyl chloride (2, 3)], and some photooxidation products of hydrocarbons [e.g., formaldehyde, epoxides, and nitro compounds ( 4 , 5 ) ] have been found to be mutagenic or tumor-producing agents. Because a large percentage of those chemicals found to be mutagenic are oxygenated or nitrogenated species (5),it would seem plausible that it may not be the volatile organic compounds emitted into the atmosphere themselves that pose the greatest risk, but their photooxidation products. We have, therefore, conducted this work with the goal of attempting to determine the identity and, in general, the structural types of photooxidation products of various atmospheric pollutants that exhibit the greatest mutagenic activity. We chose as a model compound toluene, since many of its photooxidation products (e.g., formaldehyde and peroxyacetyl nitrate) are common to other hydrocarbons such as propylene (6),its photooxidation chemistry is fairly well understood ( 3 ,and products such as aromatic nitrates will be present and because it also represents an important reactive component of the ambient hydrocarbon mix (8). Toluene/NO,/H,O/air masses were prepared for this experiment by irradiating approximately 1ppm of toluene, 0.5 ppm of NO,, and 50% relative humidity air in a 22.7-m3 Teflon smog chamber. The smog chamber is operated in a flow mode, where reactants are continuously added a t constant concentra0013-936X/85/0919-0249$01.50/0
tion, and behaves as a continuously stirred tank reactor (CSTR) in which the reactant and product distributions can be frozen at a particular level for a given constant flow rate. If, for example, the flow rate through the chamber is such that the residence time 7 (where 7 = volume/flow rate) is 3 h, the product distribution will be roughly the same as that for a 3-h static-mode irradiation. With this method, then, a bioassay of the reactor effluent can be performed for fairly long periods of time with a constant composition mixture of pollutants. The bioassay chosen for this work was the Salmonella/mammalian microsome test (9). This test was chosen because of its speed, relative simplicity, sensitivity (10,12), the good degree of correlation of positive results with known carcinogens, and lack of false positives and negatives (11). This test uses a mutant histidine-auxotrophic strain of Salmonella typhimurium. Mutations are detected as the number of bacterial colonies that grow in a histidine-free medium. The exposures were conducted by passing the chamber effluent through 190-L biochambers for a period of roughly 18 h. In this paper we discuss the results for four exposures at two different product distributions in the toluene/NO, system.
Experimental Section A schematic diagram of the reaction chamber and biochamber system is shown in Figure 1. All reactants are mixed in a 150-L stainless steel inlet manifold and then transferred to the chamber through the end plate. Toluene (Fisher Scientific, HPLC grade) was added to the inlet manifold by bubbling N2 (MG Scientific, prepurified grade) through an impinger bottle containing 100 mL of toluene maintained at -0 "C using a Forma Scientific Model 2800 cooling bath. Flow through the impinger was controlled with a Tylan Model FC260 (0-20 cm3/min) mass flow controller. NO was added by flowing a mixture of 0.5% NO in N2 (MG Scientific) into the inlet manifold with the aid of a Tylan Model FC260 mass flow controller. Clean air is produced by using an AADCO clean air generator supplied with compressed air from a Quincey Model 325-15 air compressor. The dilution air from the AADCO was controlled in the 0-5 ft3/min range with a Teledyne Hasting-Raydist Model NAHL-5P mass flow controller. This air was humidified by using a Sonimist Model 600 L ultrasonic spray nozzle. The reactants were flowed into the chamber at 2-5 ft3/min and mixed by using a 60.5-cm diameter three-blade impeller powered with a 0.25-hp motor. The chamber is constructed of a 7.5 m long cylindrical Teflon bag connected on each end to 1.96-m diameter aluminum end plates which are coated with fluorocarbon paint. Irradiation of the reactor is provided by a total of 180 GE F-40 blacklight bulbs and 36 sunlamps.
0 1985 American Chemical Society
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Environ. Sci. Technol., Vol. 19, No. 3, 1985 249
Clean
B
Generator
Mass Flow Controller
Mass Flow ct Controller
1
c--- Aluminum End Plates
chamber
_____)
F
f
M:n"g
' Samples and Exhaust
Reaction Chamber 22.7m3 Biochamber
Humidifier -t
--7 I
Lights
U
f - h-
Teflon
Heated Lines
lFNO in N2
4Samples and 4
Exhaust Flgure 1. Experimental schematic.
Four 190-L biochambers were used for exposure to Salmonella: the clean air biochamber (BCA), reactants biochamber (BR), filtered effluent biochamber (BF), and unfiltered effluent biochamber (BU), situated as shown in Figure 1. The gases were transferred to each of the biochambers through 3/8-in. Teflon tubing a t a flow rate of 14 L/min maintained and measured with a needle valve and rotameter. The effluent going into BF was filtered by using a heated quartz fiber filter (Gelman 142 mm AE). The lines going into BF and BU are heated to -50 "C by using heating tape. All lines were made as short as possible to maximize transfer efficiency. Glass Petri dishes containing Salmonella typhimurium were provided by the Health Effects Research Laboratory (HERL) of U S . EPA. The Salmonella was plated according to procedures developed a t HERL (22). Tester strains TAlOO and TA98 were used with and without S9 metabolic activation (rat liver homogenates). Strain TAlOO operates on a base-pair substitution mechanism, and TA98 operates on a frameshift mutation mechanism. Approximately 12 plates of each strain with and without S9 mix were exposed (for 18.5 h) in each biochamber. The number of revertant colonies per plate were counted a t HERL, after a 48-h incubation a t 37 "C. Toluene was measured on a Varian Model 1400 gas chromatograph (GC) containing a glass column packed with 0.1% SPl0oO on Carbopack C, operated at a He flow rate of 20 cm3/min and a temperature of 200 OC. Injection was performed every 5 min by using a solenoid-actuated Seizcor six-port valve that was switched on and off with a Chrontrol Model CD timer. Calibrations were performed by preparing 1ppm of toluene in 200-L Teflon bags by injection of microliter quantities of pure toluene into the air stream used to fill the bag. NO and NO, were mea-
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Environ. Scl. Technol., Vol. 19, No. 3, 1985
sured by using a CSI Model 1600 oxides of nitrogen analyzer calibrated with a certified standard of NO in N2 obtained from MG Scientific. Ozone was measured on a Bendix Model 8002 ozone analyzer calibrated by using a Dasibi Environmental Corp. UV ozone monitor. Relative humidity in the reactor was measured with an EG+G Model 880 dew point hygrometer calibrated by using saturated salt solutions. Particle concentrations were measured with a TSI Model 3020 condensation nuclei counter. Peroxyacetyl nitrate (PAN) was measured with a Varian electron capture detector after separation on a column packed with 10% Carbowax 400 on SO/lOO Supelcoport at 25 "C. The instrument was calibrated according to the procedure described by Lonneman et al. (13). Formaldehyde, acetaldehyde, glyoxal, methylglyoxal, and benzaldehyde were measured by using the DNPH/high-performance liquid chromatography (HPLC) technique (14). The reacted hydrazones were separated in a column packed with Zorbax ODS and detected by using a Varian Model 5000 HPLC. Calibrations were performed by using dilute solutions of twice-recrystallizedhydrazone samples for each aldehyde. Benzaldehyde and 0-,m-,and p-cresol analyses were also performed with a HP5985 gas chromatograph/mass spectrometer (GC/MS). Five-liter samples were pumped through an open (5 mm i.d., -20 mL volume) Pyrex U-tube, which was fitted with 0-5-mm high-vacuum stopcocks and cooled to liquid oxygen temperature (90 K). Ten milliliters of a 2 ppm sample of perfluorobenzene (Scott Environmental) was added to each sample as an internal standard. The Pyrex tube was then connected into the carrier gas line of the GC/MS. The sample trapped in the U-tube was then desorbed with heat and transferred to a second, smaller (2 mL) Pyrex trap (at
Table I. Bioassay Results, Average Revertants per Plate (SD)" exposureb
S BCA BR BF BU
TAlOO 119 (14) 117 (16) 162 (23) 263 (39) 181 (26) 222 (40) 242 (23) 308 (50) 211 (30) 285 (41)
11/17 and 11/30, 7 = 3.0 h TA100+S9 TA98 117 (5) 117 (12) 160 (35) 280 (47) 171 (26) 250 (45) 209 (37) 286 (31) 189 (24) 274 (37)
25 (5) 48 (4) 39 (6) 81 (15) 30 (4) 28 (6) 75 (11) 31 (5) 67 (7)
TA98+S9
TAlOO
31 (5) 50 (4) 52 (5) 88 (13) 51 (6) 72 (12) 44 (9) 81 (10) 45 (6) 78 (12)
124 (19) 154 (15) 213 (23) 158 (23) 321 (40) 242 (44) 689 (27) 751 (109) 705 (44) 863 (112)
10/11 and 10/26, 7 = 6.7 h TA100+S9 TA98 119 (16) 146 (19) 219 (44) 179 (18) 380 (24) 249 (31) 639 (106) 548 (177) 583 (52) 636 (81)
TA98+S9
31 (5)
40
21 (9)
16 (4)
24 (10)
49 (7)
63 (22)
91 (10)
76 (8)
80 (11)
OFirst and second lines show 11/17 (or 10/11) and 11/30 (or 10/26) results, respectively. b S = spontaneous; BCA = clean air; BR = reactants; BF = filtered; BU = unfiltered.
90 K) in the carrier gas line situated just above the column head. Injection was carried out by rapidly heating this secondary trap. Separation was achieved on a 6 f t long 2 mm i.d. glass column packed with 0.1% SPlOOO on 80/100 Carbopack C at a He flow rate of 20 cm3/min. The column was held a t 40 "C for 2 min and then heated to 225 "C a t 30 "C/min. Quantitation was performed by measuring peak areas relative to the perfluorobenzene peak area and multiplying by the previously determined relative response factors. Nitric acid was measured by drawing air through a 25-mm nylon filter (1-pm pore size), extraction of the filter with M perchloric acid solution, and subsequent analysis for nitrate ion with a Dionex System 12 ion chromatograph using a Dionex Model 60361 anion separator column. As a model for the quantity of gas-phase component that solubilized into the biotest medium, 50 mL of distilled buffered water was added to five Petri dishes in each chamber. Acid and base/neutral fractions were extracted with methylene chloride after each exposure (15). The extracts were then analyzed for benzaldehyde and the cresols by GC/MS. The distilled water was also analyzed for formaldehyde by the chromotropic acid technique (16) and for nitrite and nitrate ion by ion chromatography. Because of interference from the buffer, these latter two were measured by using plates with unbuffered distilled water. Results In order to choose the appropriate conditions for a dynamic-mode exposure, static toluene/NO, irradiations were performed to determine the product distributions as a function of irradiation time. Figure 2 shows the results for a static &e., no flow) irradiation of 970 ppb of toluene and 390 ppb of NO, along with the time profiles for several major components of interest. As can be seen in this figure, there is a sharp burst of particle formation a t or near the ozone maximum. We have found the overall reaction rate to be heavily dependent on the chamber humidity, possibly due to heterogeneous production (17) of nitrous acid (HONO),which can photolyze to produce OH radicals. We therefore attempted to operate the chamber a t -50% relative humidity (at 22 "C) in all experiments. The relative humidity for the static experiment was 57% a t 19
"C. For the dynamic runs, an attempt was made to maximize the compositional differences between the two sets of exposures. We were, however, limited on the fast flow (i.e., small T ) end by the maximum output of the clean air generator (5 ft3/min or T = 2.7 h) and on the slow flow (large T ) end by the required sampling and biochamber feed rates (2 ft3/min or T = 6.7 h). For the dynamic ex-
0 Toluene
0
NO
0 NO,-NO A 0% X
PAN CNC
8000
7000 6000 0
5000 0 0
*
4000
%
3000
2000 1000
0 11/16
1
T1ME.h
10'26
Flgure 2. Static-mode toluene/NO, /H,O/air irradiation.
posures, residence times ( 7 ) of 3.0 and 6.7 h were chosen. Because of the dependence of the reaction rate on humidity and apparent wall radical sources, the extent of reaction at these times was difficult to duplicate exactly. Figure 2 shows, by experiment date, the extent of reaction (by the vertical lines) a t which the bioassays were performed on the basis of the observed distribution of toluene, NO, NO,-NO, and ozone. The 11/16 and 11/30 and the 10/11 and 10/26 experiments were meant to be duplicates of each other. Each of the four experiments were conducted a t the same initial reactant concentrations, as indicated in Figure 2. The results of the biotesting for these four experiments are presented in Table I. The "spontaneous" samples are a group of plates prepared and counted in the same way as the exposed plates, but which remain in the biotesting laboratory throughout the exposure. The spontaneous plates thus measure the natural reversion rate observed under sterile conditions. Although the data for the clean air biochamber are, on the average, larger than the spontaneous, we do not interpret this as being a positive response to clean air. Rather, we attribute this to exposure of the clean air plates to a number of environmental factors (such as a brief exposure to sunlight) which the spontaneous plates do not experience. For the purposes of interpreting the data for the other biochambers, it is reaEnviron. Scl. Technol., Vol. 19, No. 3, 1985
251
+
0 Effluent Toluene CNC @ Input Toluene V BR Toluene 0 NO BF Toluene A NO,-NO BU Toluene
I
I
I
I
6000
1200 1100 1000
5000
900 n
4000
800 2
0
I-
Pa
g 700
2
3000
600
I-
$ 500
5 u
m r
, 3
2000
400 300
200
1000
100
0 Open
Closed
TIME, h
Flgure 3. Reaction profile: 10/26,7 = 6.7 h.
Table 11. Average (*20% ) Product Concentrations test plates,' nmol/plate
effluent, ppb
= 3.0 14 48 48 5 16 20 7 11 3.5 3.5 62 169 0
7
PAN 0 3
HCHO CHaCHO CHOCHO CHSC(0)CHO CBH&HO o-cresol m-cresol p-cresol HN03
co
particles/cm3 a Calculated
= 6.7 132 280 82 8 24 34 12 5 1.0 1.5 84 490 400
7
7
= 3.0 91
7
= 6.7 198
367
933
34 125 38 111 37 37
103 333 66 30 6 9
from eq 1.
sonable to compare the numbers to those for the clean air biochamber. In addition, two clean air irradiations and exposures to the irradiated clean air were performed at T = 3.0 and 7 = 6.7 h. In these experiments, no significant increase in the revertant level for the irradiated clean air, relative to the nonirradiated air, was observed for either residence time. Therefore, it can be concluded that irradiation of the clean air does not produce any gas-phase mutagens. The following products were measured in these experiments: PAN, ozone, formaldehyde, acetaldehyde, glyoxal, methylglyoxal, benzaldehyde, the three cresol isomers, nitric acid, CO, and total particulate matter. The average effluent concentrations for each species are presented in Table 11. These concentrations agreed for the duplicate experiments to within f20%. In Figure 3 we present the reactant profiles for the 10/26 (T = 6.7 h) experiment, which demonstrate the approach to steady state and the degree to which it was maintained in these experiments. Once the effluent reactant and product distributions reached steady state, the four biochambers were loaded with the covered Petri plates. The 252
Environ. Sci. Technoi., Vol. 19, No. 3, 1985
biochamber product concentrations were then allowed to come back up to their steady-state values. At this point the various product concentrations were measured in the biochambers with the plates covered. The values obtained thus represented the transfer efficiencies between the chamber effluent and the biochambers. These measurements required an -2-h period before the plates could be uncovered. At the end of this 2-h period the plates were uncovered and the exposure was begun. During the 18.5-h exposures the product concentrations were measured in the chamber effluent and in the biochambers. From the differences in biochamber concentrations before and after the plates were uncovered, we can determine the relative amounts of exposure to each chemical (see Discussion). It was found from these measurements that, for all but glyoxal, the transfer efficiencies of the various products to the biochambers were at least 50%. This type of measurement could not be done for acetaldehyde, since it evaporates from the plates when they are opened, possibly as a metabolic product from the bacteria. (The biochamber data are not presented, since the transfer efficiencies are high, and for polar compounds such as formaldehyde, glyoxal, and methylglyoxal, the majority of the product mass passing through the biochambers is deposited into the plates.) As a model for determining the amount of vapor-phase material deposited into the plates, four plates containing buffered water (phosphate/diphosphate buffer, pH 7.4) and one with pure deionized water were placed in each biochamber during the exposure period. The buffered water is used as the best model of the agar that can also be easily analyzed. Deionized water is used to circumvent ion chromatographic analysis problems. The concentrations found in the water plates can then be compared with the calculated deposition into the bioassay medium (see Discussion). This was done for the 10/26, T = 6.7 h run for the cresols, benzaldehyde, formaldehyde, nitrite, and nitrate. The cresols, benzaldehyde, and formaldehyde were measured in the buffered water plates, and nitrite and nitrate were measured in the deionized water plates. The results are presented in Table 111.
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Table 111. Water Plate Concentrations (Nanomoles per Plate) (10/26,7 = 6.7 h)
mpexpo- CBHS- 0sure CHO cresol cresol cresol HCHO NO, BCA BR BU BF
