Environ. Sci. Technol. 1988, 20, 1008-1013
Acetaldehyde: The Mutagenic Activity of Its Photooxidation Products Paul B. Shepson," Tadeusz E. Klelndlenst, Edward 0. Edney, and Chris M. Nero Northrop Services, 1nc.-Environmental
Sciences, Research Triangle Park, North Carolina 27709
Larry T. Cupitt Atmospheric Sciences Research Laboratory, U S . Environmental Protection Agency, Research Triangle Park, North Carolina 277 11
Larry D. Claxton Health Effects Research Laboratory, US. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
We exposed Salmonella typhimurium strain TAlOO to the gas-phase products of the photooxidation of acetaldehyde by using the effluent from a flow-mode smog chamber. The total mutagenic activity of the photooxidation products under simulated atmospheric reaction conditions was measured. Significant mutagenic activity was observed when the reaction time was long and considerable concentrations of ozone and organic peroxy-type products were present and when the reaction time was short and the major products were formaldehyde and peroxyacetyl nitrate (PAN). The photooxidation of acetaldehyde is shown to account for a significant fraction of the mutagenic activity observed from the photooxidation of propylene. It was found that most of the observed response can be accounted for by the presence of PAN in the reaction chamber. The apparent mutagenic activity of PAN in these experiments was found to be considerably larger than in previous laboratory measurements.
Introduction As part of a long-term research effort aimed at assessing the extent to which chemical mutagens can be produced from the atmospheric oxidation of hydrocarbons, we recently reported (1) that propylene (C3H6),a component of automobile exhaust, is converted to bacterial mutagens in the course of its photooxidation. However, we have also shown that the major organic products of C3H6reaction with OH [formaldehyde (HCHO) and acetaldehyde (CH,CHO)], with NO3 ( I ) , and with O3(2),cannot account for the mutagenic activity observed in that system. In addition, the principal inorganic species (NO, NOz, 03, HNO,, and HzOz) present in these irradiated HC/NO, systems are not significantly miitagenic in tests with Salmonella typhimurium strain TA100. However, peroxyacetyl nitrate (PAN), a product of the photooxidation of CH3CH0 and an important component of urban atmospheres, has been reported to be a weak mutagen with this strain (3). This fact tends to support speculation that other photooxidation products of CH3CH0 are mutagenic and could account for the response observed in the irradiated C3H6/N0, system. Observation of the production of mutagenic products from CH,CHO photooxidation would have a significant impact on the assessment of the total mutagenic activity of urban air, because CH&HO is a major reactive component of urban air, present at levels as high as -10-20 ppb ( 4 ) . In this paper we report the results of exposures of Salmonella typhimurium strain TAlOO (without metabolic activation) to the species present in irradiated CH3CHO/NO/N02/air mixtures. The experiments were conducted in a 22.7-m3flow-mode Teflon smog chamber at reactant concentrations in the high parts-per-billion (ppb) range. The results indicate a significant mutagenic activity for products under reaction conditions that favor 1008
Environ. Sci. Technol., Vol. 20, No. 10, 1986
HCHO and PAN as major products. The results were examined with the aim of determining the nature of the chemical species that could have caused the response and determining to what extent these products can account for the observed response in the irradiated propylene/NO, system. Several surrogate acetaldehyde photooxidation product mixtures (containing PAN) are shown to exhibit a large enough response to account for that observed in the irradiated CH3CHO/N0, mixtures.
Experimental Methods and Results The experimental apparatus and methods employed for the gas-phase exposure of S. typhimurium to the products of various photochemical reaction mixtures have been described in detail previously ( 1 , 2 , 5 )and are only briefly described here. The method for the dynamic exposures relies on continuously adding the photochemical reactants (CH3CHO/NO/NO2) at low ppb to ppm levels into a 22.7-m3Teflon smog chamber irradiated with a combination of sunlamps and blacklights. If the flow of reactants into the chamber, effluent out of the chamber, and the light intensity are held constant, a steady-state reactant and product distribution can be maintained for extended periods of time, allowing for adequate exposure of the bioassay medium. The product distribution depends in part on the residence time, r , of gases in the chamber, where r = (chamber volume/total flow rate). The exposures are conducted by bringing the reaction chamber reactant and product concentrations to steady-state values and allowing the chamber effluent to flow through 190-L exposure chambers loaded with -50 test plates that contain the bacteria Salmonella typhimurium, strain TA100. The exposure relies on the deposition of the soluble product species into the test medium as they flow through the exposure chambers. The extent to which this occurs for many of the products of CHBCHOphotooxidation has been previously reported (1, 2). The bioassay test procedures employed were essentially those of Ames et al. (6). The test plates were prepared by adding 0.1 mL of the S. typhimurium culture to 3 mL of an agar overlay at 45 "C. This mixture was then poured onto 45 mL of minimal histidine concentration agar in a Pyrex Petri plate. Colony counting was done with an Artec 880 automatic colony counter using previously published guidelines (7). The reactants (CH,CHO, NO, and NO2) were metered into the chamber by using 0.1-1.0% mixtures of the individual chemicals in N2 (obtained from MG Scientific), as previously described (5). The sampling and analysis for the measurement of NO, NO,, 03,PAN, and HCHO were conducted as described in that work. Acetaldehyde was measured with a Varian Model 1200 gas chromatograph (GC)/flame ionization detector employing a 6.4 mm by 2 m stainless steel column packed with 8 O / l O O Porapak QS
0013-936X/86/0920-1008$01.50/0
0 1986 American Chemical Society
0 CH3CH0+2
Table I. Average Reactant and Product Concentrations (ppb) for Irradiated CH,CHO/NO, Exposures
0 PAN HCHO
0 NO
A CH,0N02 x 2 0
A NO,- NO
7
= 2.6 h effluent concn
compound
input concn
CHSCHO NO, NO
1237 f 124 427 f 26 406 f 35
1064 f 113 388 f 22 40 f 4 40 f 7 102 f 7 72 f 6
0 3
500
T
input concn
PAN HCHO
= 4.0 h effluent concn
1190 f 26 977 f 58 212 f 13 174 f 10 31f3 5f2 311 f 12 171 f 11 101 f 16
400 600
300
0 0
q
550
200
7= i=
2.6h idisplaced 0.2h to the right) 4.0h
T
475
v I
100
/
/
1
/
0 1
2
3
4
5
6
7
Time, hours
Figure 1. CH3CHO/NO/N02 static-mode irradiation.
operated isothermally a t 145 OC. Sample injection was achieved with 10-mL glass and Teflon syringes. Methyl nitrate (CH,0N02) was measured by using the same GC that was used for the PAN measurements (5). The GC was calibrated with pure samples synthesized according to the procedure of Johnson (8). To determine the temporal behavior of the reactants and products in the irradiated CH3CHO/N0, system, a static-mode irradiation was conducted. For this experiment the reactor was operated as a conventional smog chamber; that is, the reactants were added through the mixing manifold to the desired initial concentrations, the lights were turned on, and dilution air at 10 L/min was added to account for an equivalent sampling rate at the effluent end of the chamber. The initial reactant concentrations were 1440,418, and 436 ppb for CH3CH0, NO, and NO,, respectively. Figure 1shows the time profiles for CH3CHO, PAN, HCHO, CH30N02,NO,-NO (which is associated with PAN + NO2), NO, and 03. We report here the results of two dynamic-mode exposures under conditions where the product distributions should have been considerably different. One was conducted with input concentrations of 1237,406,and 427 ppb of CH,CHO, NO, and NO,, respectively. This exposure was conducted at T = 2.6 h. The product distribution was similar to that at -1.5 h in Figure 1. Under these conditions, where considerable NO is present, the product distribution is simplified because peroxy radical-radical reactions will not occur (see Discussion). The other exposure was conducted at inlet concentrations of 1190,31, and 212 ppb of CH3CH0, NO, and NO,, respectively. The product distribution at steady state was similar to that at -6 h in Figure 1. [Only NO, is substantially higher in the static experiment at 6 h. NO2 is the major component of difference since the PAN concentrations are similar for the static and dynamic (7 = 4.0 h) experiments.] Under these conditions organic peroxides can be produced, and the product mixture is considerably more complex. The average chamber inlet and effluent reactant and product concentrations for these two exposures are presented in Table I.
1 7 5 r , 0 1
I
I
I
,
,
2
3
4
5
6
, , , , , , , 7
8
9
I
I
I
,
,
, ,
10 11 12 13 14 15 16 17 18 19 20
Exposure Time. hours
Figure 2. Dose-response curves for irradiated CH,CHO/NO, sures.
expo-
The exposures were conducted by loading the exposure chambers with 45 test plates and 5 survivor plates. For the survivor plates, the bacterium concentration was diluted by roughly lo4,and additional histidine was added to the plates. The magnitude of the dilution is such that, in the absence of toxicity, -500 colonies per plate will be produced. Therefore, these plates can be used to indicate the possibility of toxicity effects for the test plates. Throughout the exposure the effluent was allowed to flow through the exposure chambers at 14 L/min. Once steady-state concentrations were achieved in the exposure chambers, all the plates were uncovered. Then after 2.5, 5.0, 10.0, 15.0 (for the 7 = 2.6-h experiment only), and 20.0 h, the appropriate fraction of the total number of plates was covered. Covering the plates effectively stopped the exposure, thereby establishing five and four different exposure levels in the 7 = 2.6-h and T = 4.0-h exposures, respectively. At the end of the 20-h exposure period, the covered plates were removed from the exposure chambers and incubated at 37 OC for 48 h, and the final revertant and survivor colonies were counted. For both experiments a reactant exposure chamber, which drew its sample from the inlet manifold of the reaction chamber, was used to obtain the zero product dose level. The plates from this exposure chamber were all exposed for 20 h. The results of both exposures are presented as dose-response curves in Figure 2. For these experiments the numbers at each exposure period are averages for -25 plates. Throughout the T = 2.6-h exposure there was no discernible change in the survivor levels. For the 7 = 4.0-h exposure the survivor counts were 675,742, 552, and 0 for 2.5, 5.0, 10.0, and 20.0 h, respectively, indicating the potential for toxicity effects in the latter part of the exposure. Although there appears to be some curvature for both dose-response curves, we Environ. Sci. Technol., Vol. 20, No. 10, 1986
1009
440 420
Table 11. Initial Component Concentrations (ppb) for Acetaldehyde Photooxidation Product Exposures
0 Effluent (displaced 0 2h to the right)
400
compound
380
340
320
108 383 1897 180 57 34
200
0 3
CHBONOZ
300 280
330 320 310 300 290 -
exposure
40
350
260
0 Clean Air
340
240
220 200 180
3
! 280
160
120
PAN only
PAN NO2 CHBCHO HCHO
360
140
mixture exposure
$
-
-
100
0
P
2 I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
260 250 240
9 1 0 1 1 1 2 1 3 1 4
230
I
I
I
I
Effective ExposureTime, h
1 . :
7
270
I
I
0 Effluent (displaced 0 2h t o the right)
220 210
Figure 3. Dose-response curve for CH,CHO photooxidation product mixture exposure.
have drawn straight lines representing the initial slopes to facilitate comparison of the mutagenic activities of the two mixtures. In addition, several exposures were conducted by preparing, in the reaction chamber, surrogate mixtures of various combinations of the photooxidation products of acetaldehyde. In this paper we present the results from two such exposures, one of which was conducted with a mixture of CH3CH0, HCHO, 03,NO2, CH30N02,and PAN and the other with pure PAN only. The CH,CHO, HCHO, and NO2used were pure commercial samples. The O3 was provided by using a Welsbach Model T-408 O3 generator supplied with zero grade O2 (MG Scientific). The PAN was synthesized in Teflon bags using irradiated C12/N02/CH3CH0mixtures and was distilled as previously described (3). These experiments were conducted by adding the pure products into the reaction chamber (in the dark) until the initial desired exposure concentration levels were reached. The exposure was conducted by flowing the reaction chamber air through the exposure chamber at 14 L/mm, while continuously adding make-up air to the reaction chamber at 14 L/min. Therefore, the component concentrations were continuously diluted during the exposure. In order to avoid reactions of O3with NO2 (to yield NO3, which will subsequently react with CH3CHO),the O3 was added to the transfer line between the reaction chamber and exposure chamber by using a dilute 03/air mixture. Also, for the PAN only exposure, a small amount of NO2was added to the chamber in order to stabilize the PAN (see Discussion, reactions 2, -2). Dose-response curves were obtained as with the irradiated CH3CHO/N0, exposures. The effective exposure times for these curves are obtained by using expression I:
where t,, is the exposure time in real time and k is the dilution rate constant. The initial concentrations used for these two experiments are presented in Table 11. The results of the exposures are presented in Figure 3 1010
Environ. Sci. Technol., Vol. 20, No. IO, 1986
200
190 180 1
3
2
5
4
6
a
7
9
io
Effective Exposure Time, h
Flgure 4. Dose-response curve for pure PAN exposure.
(CH3CHO photooxidation products mixture) and Figure 4 (PAN only), as dose-response curves. For these two experiments the numbers at each exposure period are averages for eight plates. The survivor plates for both experiments showed no evidence for toxicity.
Discussion The photooxidation of CH3CH0 in the presence of oxides of nitrogen occurs by direct photolysis of CH3CH0 or through an OH radical chain mechanism. In the presence of NO and air, the reaction proceeds (9) as shown: CH3CH0
+ OH -% CH3C(0)02+ H 2 0
CH&(O)O2 + NO2
+ + + + + + -
CH,C(0)02
CH3C(0)02N02 (PAN)
+ NO
CH&(O)O
Oa
(1) (2, -2)
CH,C(O)O + NO2
(3)
CH302+ C02
(4)
+
+
CH30z CO HO2 CH3CHO hv CH302 NO CH30 + NO2 CH30 O2 HCHO H 0 2 CH30 NOz CH3ONO2 CH30 NO CH30N0 CH30 + NO CH30N0 hv HO2 + NO OH + NO2 -+
+
+
(5) (6)
(7) (8) (9)
(10)
(11)
Because the photolysis of methyl nitrite is fast (9),it is not likely that the concentration of methyl nitrite reaches significant levels. Thus, the only organic products measured in the presence of NO are PAN, HCHO, and CH3ON02. At long reaction times, when all the NO is converted to NO2,the CH302,H02, and CH3C(0)02radicals can react with NO2 or with each other, leading to various
peroxy-type compounds and a more complex product distribution. In evaluating the relative mutagenic activities of the two (7= 2.6 h and 7 = 4.0 h) irradiated CH,CHO/NO, product mixtures, it would seem reasonable to compare them on the basis of the number of ppm of CH,CHO consumed. For the 7 = 2.6-h exposure ACH3CH0 = 0.173 ppm, and for the 7 = 4.0-h exposure ACH3CH0 = 0.213 ppm. From the initial slopes in Figure 2, we calculate mutagenic activities for these mixtures of 70 and 94 revertants plate-l h-l ppm-l for the 7 = 2.6-h and 4.0-h exposures, respectively. Since the responses in the two systems are comparable, and the product distribution is much simpler in the short residence time experiment, we will restrict our attempts at identifying those species responsible to the short residence time experiment. We have previously reported (1)that CH30N02and the inorganic products expected to be present under these conditions (O,,NO2,and "OB) are all nonmutagenic with TA100. This leaves HCHO and PAN as known products that need to be considered. As discussed in a previous publication ( 0 , the amount of each chemical that deposits into the plates can be calculated from
where Xi is the concentration of species i (micromolesper plate), aiis the fraction of material that deposits into the plates once all plates are uncovered, P@) is the partial pressure (yatm) of species i in the exposure chamber before the plates are open, R is the universal gas constant, T is the absolute temperature, VE is the volume of effluent passing through the exposure chamber (L), and Npis the total number of plates. Results using eq I1 have previously been shown to agree well with concentrations measured in surrogate water plates used as models for test plates (1, 3). We have also demonstrated that the deposition is linear within experimental uncertainty for HCHO as the plates are covered during an exposure (2). Recent measurements in our laboratory indicate that, at an exposure chamber flow rate of 14 L/min and for 50 plates per exposure chamber, LY is approximately equal to 0.15 for PAN and 0.85 for HCHO. Assuming 100% transfer efficiency of PAN and HCHO to the exposure chambers, we calculate 0.21 and 0.84 pmol per plate at 20 h of total exposure time for PAN and HCHO, respectively. We have previously measured the mutagenic activities for PAN and HCHO with TAlOO and found them to be approximately 34 and 12 revertants pmol-l per plate, respectively (1,3). The total number of revertants per plate due to the presence of PAN and HCHO is thus calculated to be 7 and 10 revertants per plate at 20 h. The sum of these two therefore can account for only -7% of the total observed response for the 7 = 2.6-h exposure. Since, at this point, it appeared that none of the known acetaldehyde photooxidation products could individually account for the observed response, it became reasonable to suspect that the response could be the result of some type of cooperative effect between or among reaction products. As perhaps the simplest way to test for this possibility, we conducted an exposure of TAlOO to a mixture of acetaldehyde and its major photooxidation products, i.e., CH3CH0, HCHO, CH30N02, 03,NOz, and PAN. The concentrations chosen for this exposure were similar in magnitude to those present in the r = 2.6 h irradiated CH3CHO/N0, exposure. As can be seen in Figure 3, the observed mutagenic activity (- 160 excess revertants at a 10-h effective expo-
sure time) is much larger than anticipated on the basis of the known mutagenic activities of each of the species present. To determine which combination or combinations of these products caused the response, we began conducting exposures with one of the products removed (in the order in which they are listed above) in each successive experiment. In each case, a similarly (relatively) large mutagenic activity was observed, until finally an exposure of TAlOO to pure PAN (with -40 ppb of NO2) was conducted. Figure 4 indicates that the exposure to 200 ppb of PAN yielded the equivalent response of 110 excess revertants at 10 h, corresponding to a significant fraction of the total observed response with the irradiated CH,CHO/NO, mixtures. This observed mutagenic activity for PAN is much larger than that previously observed in laboratory experiments. Using our previously measured value for the fraction (15%) of PAN that deposits into the plates under these exposure conditions, we calculate 0.21 pmol of PAN per plate at 10 h, which yields a mutagenic acticity for PAN of -520 revertants/ ymol. The magnitude of the discrepancy between this experiment and our laboratory measurements for the mutagenic activity of PAN, which were conducted three separate times and found to be reproducible, led us to believe that our PAN samples might be contaminated with an unknown potent mutagen. Therefore, as an independent check on these results, we prepared PAN from 03/N02/ CH,CHO mixtures, distilled it as before, and repeated the experiment. This experiment was conducted with 430 ppb of PAN and yielded 103 excess revertants per plate at 10 h, corresponding to a PAN mutagenic activity of -230 revertants/pmol. This experiment tended to confirm the result that the PAN mutagenic activity, when measured with PAN in the reaction chamber (in the dark), was considerably higher (although there is roughly a factor of 2 difference in the two results) than the 34 revertants/ymol previously measured in laboratory experiments. Assuming that the response observed with the surrogate mixtures was due entirely to PAN, these experiments were not very reproducible, contrary to-our previous experienceswith this test (the exposures showed a poor correlation between PAN concentration and mutagenic activity of the mixture; the mixtures containing PAN yielded 150 f 75 excess revertants at 10 h). It therefore seemed reasonable to question whether the sensitivity of the test had changed during the course of our studies with PAN and systems that produce PAN (e.g., irradiated C,H,/NO,). To test this possibility, we repeated C3H6/N0, exposures under conditions identical with those conducted roughly 11/2 years previously (I). A repeat of the 7 = 7.5 h irradiated C,HG/NO, exposure yielded -500 excess revertants at 20 h, which compares well with the value of -600 excess revertants reported in that work. In addition, no significant change in the laboratory Ames test positive controls (NaN,) was observed over this period. These observations therefore enhance our confidence in the stability of the test. The question therefore remains as to why there is such a large difference between our laboratory and reaction chamber measurements of the mutagenic activity of PAN, where the PAN is prepared and tested in the same way. The major difference in the two cases is the residence time of PAN in the two systems. In the laboratory measurements (3) the time required to transfer the PAN from the vacuum-line cold trap to the exposure chamber was roughly 1-2 min. In comparison, for the PAN exposures conducted in the reaction chamber, the residence time of PAN in the chamber is on the order of several hours. This
-
Environ. Sci. Technol., Vol. 20, No. 10, 1986
1011
then raises the question of whether PAN might be decomposing (either homogeneously or heterogeneously) in the reaction chamber to yield more mutagenic products. In the absence of NO2, the peroxyacetyl radicals formed in reaction -2 can react with themselves to produce a variety of products, including organic peroxides. However, in the presence of 40 ppb of NO2, the equilibrium concentration of peroxyacetyl radicals with 200 ppb of PAN is calculated to be -70 parts per trillion (ppt) (using k2 = 1.0 X cm3 molecule-l s-l and = 4.0 X s-l from ref 10). If the rate constant (which as far as we know is unknown) for the peroxyacetyl radical self-reaction were as high as 1 X lo-'' cm3 molecule-I s-l, this would lead to an initial reaction rate of peroxyacetal radicals of only 0.8 ppb h-l. Therefore, if there are homogeneous reaction products, these concentrations would necessarily be small. In addition, the total PAN decay in the chamber, after correction for dilution, appears to be no more than 2% h-l (4ppb h-* for the pure PAN exposure). Therefore, the total concentration of heterogeneous decomposition products would also be small. An attempt was made to test for the possibility that PAN decomposition products were the cause of the response by conducting several dynamic-mode exposures with the CH3CH0/O3/NOZsystem. This system should yield PAN (almost exclusively) as the major product, along with some HNO,, according to reactions 12 and 13, followed by reaction 2. Several exposures were conducted 0, + NOz NO, O2 (12)
-
+ NO, + CH3CH0 2HNO, + CH,C(0)02
-
(13)
with steady-state PAN concentrations of 170 ppb. These experiments were conducted with reaction chamber residence times of either 2.7 or 7.5 h to determine if the residence time of PAN in the chamber influences the observed mutagenic activity. The observed mutagenic activities of these mixtures were all on the order of 150 excess revertants after 10 h of exposure. Unfortunately, the variability in the observed response (nearly a factor of 2) precluded observing an effect based on the somewhat small change in residence time. One might conceivably argue that if the source of the mutagenic activity were a PAN decomposition product, the dose-response curves (e.g., Figure 4) would curve upward, since the mutagen concentration in the chamber would be continuously increasing. This, however, did not seem to be the case. It is apparent from these experiments that the observed response with PAN is large enough to account for a significant fraction of that observed with the irradiated CH,CHO/NO, mixtures. However, the absolute magnitude of the response is small enough that small changes in the exposure conditions do not yield a conclusively observable effect, given the variability of the test results. It is therefore unclear to us what the cause of the apparent discrepancy in PAN mutagenic activity measurements may be. It is possible that an independent, perhaps spectroscopic, analysis of PAN decomposition pathways may reveal the formation of mutagenic products. One important objective of this study was to determine to what extent the photooxidation of CH&HO can account for the mutagenic activity observed in the C3H6/NOx system. In our experiments with that system ( I ) , a response of roughly 600 excess revertants per plate (relative to the clean air and reactants exposure chamber plates, assuming a linear response) at T = 7.5 h in a 20-h exposure was observed. Computer-modeling studies that we have conducted indicate that, at 7.5 h, -225 ppb of CH3CH0 (which is a product of OH and 0, reaction with C3HG)had 1012
Environ. Sci. Technol., Vol. 20,
No. 10, 1986
been consumed. This is essentially the same amount of CH3CH0 that was converted to products in the 7 = 4.0-h CH,CHO/NO, irradiation. This then indicates that CH3CH0 photooxidation products (presumably mainly PAN) may be able to account for -400 of the 600 excess revertants observed in the C3H6/N0, exposure. We also note that it is possible that PAN was the cause of some of the response observed in studies of irradiated C2H,/ DEHA/NO, mixtures (11). It is interesting to note that in the C,H,/NO, system a mutagenic activity of -0.8 revertants plate-l (ppb of C3H6 consumed)-l was observed (for a 20-h exposure), whereas -1.6 revertants plate-l (ppb of CH3CH0 consumed)-l was observed in the CH,CHO/NO, system. Therefore, on an absolute basis the CH,CHO/NO, mixtures seem to be at least as mutagenic as the C3H6/N0, mixtures. [We note, however, that under the conditions of the C,H,/NO, exposures there were C3H6 removal mechanisms (e.g., 0, reactions) that lead to nonmutagenic products.] Conclusions It is apparent from this study that PAN, or its decomposition products, may account for a significant fraction of the total observed response in the irradiated C,H6/N0, and CH,CHO/NO, systems. If, in fact, these results indicate that PAN, or its decomposition products, are significantly mutagenic, these are significant results, given that PAN is an important photochemical oxidant, present in urban atmospheres in the low-ppb range (12). Further research is clearly warranted to determine (a) the exact cause of the observed mutagenic activity in these experiments and (b) if PAN appears to be significantly mutagenic (or carcinogenic) in other short-term or long-term bioassay tests. This information is essential to an accurate assessment of the potential human health implications of exposure to PAN. Acknowledgments We thank E. Perry of Environmental Health Research and Testing, Inc., for her assistance with the bioassay work. Bruce Ames (University of California, Berkeley, CA) provided the S. typhimurium tester strain TA100. R e g i s t r y No. PAN, 2278-22-0; CH,CHO, 75-07-0; NO, 10102-43-9; NOz, 10102-44-0; NO,, 11104-93-1; O,, 10028-15-6; CHCl,, 67-66-3; CH3ONO2, 598-58-3; propylene, 115-07-1.
Literature Cited Kleindienst, T. E.; Shepson, P. B.; Edney, E. 0.;Cupitt, L. T.; Claxton, L. D. Environ. Sci. Technol. 1985, 19, 620-627. Shepson, P. B.; Kleindienst, T. E.; Edney, E. 0.;Cupitt, L. T.; Claxton, L. D. Enuiron. Sci. Technol. 1985, 19, 1094-1098. Kleindienst, T. E.; Shepson, P. B.; Edney, E. 0.;Claxton, L. D. Mutat. Res. 1985, 157, 123-128. Grosjean, D.; Swanson, R. D.; Ellis, C. Sci. Total Enuiron. 1983, 29, 65-85. Shepson, P. B.; Kleindienst, T. E.; Edney, E. 0.;Namie, G. R.; Pittman, J. H.; Cupitt, L. T.; Claxton, L. D. Environ. Sci. Technol. 1985, 19, 249-255. Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347-364. Claxton, L. D.; Toney, S.; Perry, E.; King, L. Environ. Mutat. 1984, 6, 331-342. Johnson, J. R., Ed. Organic Syntheses; Wiley: New York, 1939; Collect. Vol. XIX, p 64. Atkinson, R.; Lloyd, A. C. J. Phys. Chem. Ref. Data 1984, 13, 315-444. Hendry, D. G.; Kenley, R. A. J . Am. Chem. SOC.1977,99, 3198-3199.
Environ. Sci. Technol. 1986, 20, 1013-1016 (11) Kleindienst, T. E.; Edney, E. 0.; Namie, G. R.; Claxton, L. D. Atmos. Enuiron. 1986,20, 971-978. (12) Singh, H. B.; Salas, L. J.; Smith, A.; Stiles, R.; Shigeshi,
H. U S . Environ. Prot. Agency 1981, EPA-600/S3-81-032. Received for review October 30, 1985. Revised manuscript received April 28, 1986. Accepted May 7, 1986. Although the
research described in this article has been funded wholly or in part by the U S . Environmental Protection Agency through Contract 68-02-4033 to Northrop Services, Inc.-Environmental Sciences, it has not been subjected to the Agency's required peer and policy review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.
Dissolution of Iron Sulfates from Pyritic Coal Waste Patrick J. Sullivan" Western Research Institute, University of Wyoming Research Corporation, Laramie, Wyoming 8207 1
Shas V. Mattigod Department of Soil and Environmental Sciences, University of California, Riverside, California 9252 1
Andrew A. Sobek Research and Development Center, B. F. Goodrich Company, Brecksville, Ohio 44141
N
A pyritic coal waste was leached under saturated con-
ditions for 6 months. Waste samples were removed after 3 and 6 months of leaching. These samples, in addition to a sample with no leaching, were used to make up equilibrium solutions. The leaching data showed a continuous release of iron and sulfate species and low acidity under anaerobic conditions. As leaching progressed, the relatively soluble phases were removed, leaving the control of activities to less soluble primary and secondary minerals. Data from the equilibrium solutions were used to calculate the saturation index for common secondary minerals that could form in the waste/water system. The saturation index data show that (1)the common iron mineral phases under anaerobic and acid conditions will continually dissolve, (2) halotrichite may be controlling the activities of Fe(II), Al, and SO4,and (3) illite may limit the activities of Mg, K, and Si. These data indicate that changes in the solid phase with leaching will change the long-term geochemistry of the wastelwater system.
Introduction When pyritic coal refuse and/or coal spoil is exposed at the earth's surface prior to burial, pyrite can be oxidized by gaseous oxygen, dissolved oxygen, and dissolved ferric iron. The stoichiometry of these reactions are given by Stumm and Morgan ( I ) :
+ 7/202 + HzO = Fe2+ + 2S042-+ 2H+ Fe2+ + 1 / 4 0 z + H+ = Fe3+ + 1/2H20 Fe3+ + 3H20 = Fe(OH),(s) + 3H+ FeSz + 14Fe3++ 8H20 = 15Fe2++ 2S04,- + 16H+ FeS,
(1) (2)
(3) (4)
After pyrite is oxidized (eq l), ferric iron is produced extremely slowly (eq 2). This second reaction, however, can be accelerated by microbial catalysis to increase the overall rate of ferrous iron oxidation. With the generation of ferric iron, insoluble ferric hydroxide can form under proper pH conditions (eq 3). Pyrite can also be oxidized by ferric iron resulting in the generation of more acidity and ferrous iron (eq 4). The continuation of this process requires that pyrite be oxidized (eq 1). With pyrite oxidation, Nordstrom (2) proposes the formation of secondary iron phases that include (1)ferrous 0013-936X/86/0920-1013$01.50/0
Table I. Mineral and Chemical Characteristics of Refuse (from Reference 5) mineral
percentage
quartz pyrite gypsum siderite jarosite total clay illite degraded illite kaolinite
10 25 5
