(7) Lindstrom, F., Diehl, H., Ibid., 32, 1123 (1960). (8) MacNevin, W., “The Analytical Balance,” p. 38, Handbook Publishers, Inc. Sandusky, Ohio, 1951. (9) Mylius, F., Funk, R., Chem. Ber. 30, 1716 (1897). (10) Powell, J., Fritz, J., James, D., ANAL.CHEM.32, 954 (1960).
(11) Rammelsberg, C., Chem. Zentr. 9,
730 (1838). (12) Ringbom, A., “Treatise on Analytical Chemistry,” Part I, Vol. I, p. 543, I. Kolthoff, P. Elving, eds., The Interscience Encvclooedia. “ . , Inc.. New York. 1959. (13) Schwarxenbach, G., “Die komplexometrische Titration,” Ferdinand Enke Verlag, Stuttgart, 1956.
(14) Simon, V., Zqka, J., Collection Czechoslov. Chem. Comniuns. 21, 327 (1956). (15) Willard, H., Ralston, R., Trans. Electrochem. SOC.62, 239 (1932). RECEIVEDfor review July 14, 1961. Accepted April 20, 1963. From the M.S. thesis of B. G. Stephens. Presented a t the South Carolina Academy of Sciences Meeting, Spartanburg, March 1961.
Analysis of Air Pollution Mixtures: A Study of Biologically Effective Components FRANCES L. ESTESl Biochemistry Department, Baylor University-College o f Medicine, Houston, Tex.
b The biological effectiveness of an oxidant-type air pollution mixture was determined from the inhibition in growth of E. coli following exposure to the pollution mixture. Absorption in phosphate buffer and adsorption on a column of C-22 Firebrick coated with 2 0 7 0 (w./w.) disodium phosphate appeared to remove completely the effective components. Both agents removed a part of the oxidant material as determined b y the phenolphthalin method. The material absorbed in the phosphate buffer markedly inhibited the oxygen consumption of leucocytes exposed to it. The buffer completely removed the as yet unidentified 302-mp absorbing material. However, the apparent concentration in the buffer did not correspond to the apparent concentration in the gas. The data suggested that the 302-mp absorbing material contained a biologically effective agent, which might be peroxyacetyl nitrate.
E
studies have been made on the analysis of air pollutant mixtures and automobile exhaust fumes. Air pollutant mixtures have been shown to damage plants and bacteria. The voluminous literature on the analysis of the pollutant mixture indicates the complexity of the mixture and some of the problems encountered in chemical analyses. I n a n effort to examine specific biochemical effects of a pollutant mixture, the question of analysis becomes a most pressing one. Are the observed biochemical effects evoked by a single or multiple component(s) of the mixture? Assuming that the effective agent is a single component, the XTENSIVE
1 Present address, Department of Surgery, University of Texas-Medical Branch, Galveston, Tex.
998
ANALYTICAL CHEMISTRY
probability remains that other components have a synergistic effect on the biochemical system or are themselves converted to more reactive compounds. These possibilities are emphasized by the requirement of irradiation for the formation of pollutant mixture. To explore the in vitro biochemical effects of a n oxidant-type air pollution mixture, two general problems are involved: Determination of Specific Effects. Because of t h e nature of biological systems, direct effect on t h e system must be distinguished from effects on t h e substrate or environment of t h e system, or on the method of determination. Determination of Specific Cause. Since we are dealing with a complex mixture, direct evidence for indicating particular biochemically reactive agents requires either separation of these agents from t h e rest of the mixture or the ultimate in specificity from the method of analysis. Inherent in each of these is a third problem, namely, the possibility that the means of separation as well as the method of determination can of themselves be involved in the cause and in the effect. Our previous work (13) and that of Goetx and Tsuneishi (8, 9) shom-ed that an oxidant-type air pollution mixture inhibited the rate of subsequent growth of E. coli. The development of equipment for uniform exposure of cells and the measurement of subsequent growth led to a reproducible method for determining inhibition ( I S ) . For known compounds, an index of effectiveness of gaseous agents on microorganisms involving concentration of the agent and time was indicated ( I S ) . For the pollutant mixture the use of such an index was not feasible since the particular inhibitory compound(s) had not been identified. Further examination of the effects of the pollutant mixture on E. coli in-
dicated that glutamic dehydrogenase was probably one of the enzymes involved (4). The characteristics of this enzyme ( I d ) suggested that the inhibition resulted from oxidation of the sulfhydryl groups. This hypothesis could not be directly demonstrated since, using either per cent bacteria killed or increase in lag time as an index of biological effect, there was no simple relationship between the biological effect and the usual chemical analysis of the pollutant mixture for oxidants. Methods for determining oxidants are not specific; the phenolphthalin method, for example, includes as oxidants ozone, nitrogen oxides, alkyl nitrites, peroxyalkylnitrites, and a variety of peroxides (IO). Since there is no specific method for differentiation of the oxidants, separation of components in the pollution mixture was attempted. Three general approaches to separation of the gas mixture were used: cold trapping, absorption in liquids, and adsorption on solids. PROCEDURE
The general criterion of biological effectiveness used was the inhibition in growth of E. coli folloning exposure to the pollutant mixture or fraction. The apparatus and techniques required have been described in detail ( I S ) . I n brief, a thin layer of organisms, collected on a Rlillipore filter, was exposed to t h e gas stream, transferred to a growth chamber, and the degree of inhibition was determined as a function of time, from the change in absorbance of t h e medium. The aldolase studies were made on cell-free extracts of E. coli prepared from a 24-hour broth culture. The washed cells \yere suspended in sterile water and the cells were ruptured in a sonic oscillator. The ruptured cell suspension was centrifuged a t 25,000 X g for 30 minutes a t 0” C., and the supernatant
fluid was kept frozen. For aldolase determination, working concentrations were prepared by dilution of the cell-free extract. Aldolase activity mas determined by the method of Sibley and Lehninger (17). Relative activity has been expressed on the basis of the diluted preparations. Leucocytes obtained from intraperitoneal exudates of guinea pigs ( b ) were suspended in phosphate buffer p H 7.0 through which the pollutant mixture had been bubbled. After 20 minutes the cells mere recovered and resuspended in unexposed phosphate buffer. Oxygen consumption, determined on a Warburg apparatus, was compared with cells u hich had been twice suspended in unexposed phosphate buffer. Oxidants u ere determined by the phenolphthalin method (10); and nitrogen dioxide by that of Saltzman (15). ;icidity n as determmed by a titration of standard alkali solution exposed to the pollutant mixture. The 302-mp absorbance of a 4y0 sodium hydroxide solution exposed t o the pollutant mixture has been evaluated in terms of the absorbance of a sodium nitrate solution. I t must not be assumed that the numerical values represent more than an index of nitrogencontaining materials; in the presence of a base, primary aliphatic nitro compounds absorb more strongly than does sodium nitrate ( 7 ) . The production of oxidant type pollutant mixture for these studies was based on the earlier work of HaagenSmit ( 1 1 ) . I n an all-glass system, nitrogen oxides were supplied by bubbling air through nitric acid a t 30 to 50 ml. per minute; 10 p l . per hour of a hydrocarbon mixture was intermittently introduced and the reactants were carried by a stream of compressed air into a 12-liter and a 5-liter flask in series. These flasks vere irradiated by two cylindrically arranged banks of BL type fluorescent lights providing 480 and 360 ~ a t t sillumination. Mean residence time m s about 70 minutes for the 17-liter volume maintained a t 80" C. n-ith total air input of 200 cc. per minute.
was detected in the absence than in the presence of the glass wool; yet no more oxidant material was detected in the trap. Evidence that biologically effective terial was involved is shown in Figure 1. For testing the effect on E. coli, .P 7-mm. i.d. borosilicate tube containing a 5-em. column of glass wool mas inserted into the effluent line. I n these experiments, the negative controls exposed directly to the pollutant mixture failed to groir., whereas those organisms exposed behind the glass wool showed considerable recovery of growth rate. The probability that a t least a part of the biologically active components was associated with water was indicated by the observation that a Ringersphosphate solution, through which the pollutant mixture had been bubbled, n ould inhibit the oxygen consumption of isolated leucocytes (Table 11). -4-11though the inhibition was not so great as that observed by direct exposure of the cells to the gas, this technique demonstrated that for these cells the inhibitory effect of the pollutant mixture was not caused by change in p H or ionic strength of the suspending medium. These observations have been interpreted as indicating that either a portion of the active material was water-soluble, or the aerosols retained their innate characteristics and part of their activity, even in aqueous SUEpensions (3). Drying the pollutant mixture n-ith Ascarite or magnesium perchlorate, as has frequently been done for gas chromatographic analysis of pollutant mixtures ( 6 , 1 9 ) ,resulted in complete loss of biological activity of the effluent. ilnalysis of random samples of the pollutant mixture by gas chromatography on a Carbowax 6000 column indicated that there were about eight components in the mixture more rap-
0--0
0
-
Eaposed w i l h Column
Table I.
Oxidant Passed through Trap at 5-Min. Intervals 3 Total 1 2
~l~~~ Oxidant Wool Trapped 3 . 9 5.0 5 . 0 1 3 . 9 Absent 2.4 Present 2.2 1 . 9 4 . 4 4.5 1 0 . 8 Reported as Kg. 03/3 liters of pollutant mixture, collection a t 200 cc./min. for 15 minutes. 0
Table II. Effect of Buffer Soluble Pollutants on Oxygen Consumption of Leucocytes
Decrease in 02 Analysis of Gas t o JVhich Uptake Buffer Was E x p o s e d Compared Oxidant, 302 mp.mg. with NOa-/liter Control, % p.p.m. O3 2.5 2.8 2.6 2.8 1.9 1.9
/
O E x p o r e d , No C o l u m n
RESULTS AND DISCUSSION
Attempts to isolate components of the gas in a cold trap, containing glass wool to increase surface area, resulted in observations that suggested the glass wool destroyed rather than trapped the oxidant material. Table I shows a comparison of attempts to cold trap the oxidants in a Sheppard trap enclosed in a dry ice-ethanol bath with and without glass wool in the trap. During a 15minute flow (3 liters) of the synthetic pollutant mixture, three oxidant tests were made on the effluent beyond the trap. For each interval more oxidant
0
1
2 3 4 5 G R O W T H TIME IHOURS)
Figure 1. Effect of glass wool. 60 minutes Analysis of line:
5.8 Oxidant, pg. O&, NOz, p.p.m./l., 0.36 Acid, meq./l., 0.030 302 m p as mg. NO&, 2.79
6
Trapped Oxidant"
7
Exposure
2.3 2.3
3.5
3.3 2.3 3.2
48 20 11 40 19 19
idly eluted than water. The trailing of the water did not permit separation of any components which might be associated with it. It was not possible to apply the technique of indirect exposure used for the leucocytes to the examination of the effects of the pollutant mixture on the growth of E. coli. Hence, aliquots of the cells were exposed to the pollutant mixture before and after a bubbler containing 10 ml. of 0.25M phosphate buffer. For another aliquot of cells, 1 ml. of the t q o s e d buffer was substituted for fresh buffer in preparing the medium for subsequent gron-th. Results are presented in Figure 2. For these studies the growth of the negative control (exposed n ithout bubbler) showed little inhibition. A comparison of the analysis of the eRuent for this and the following experiments indicated that the recovery of the negatiye control was not the result of lower concentration of any component for which chemical analysis was made. This has been regarded as further indication that more definitive analytical methods are required. With the bubbler containing phosphate buffer in the line, there was little or no inhibition of the growth of the organisms. One milliliter of the exposed buffer added to 7 ml. of the growth medium had no effect on the growth of the cells. It was possible that dilution of the pollutant(s) was involved. For other experiments analysis of the effluent, before and after the bubbler, is summarized in Table 111. A portion of all components had been removed. The buffer appeared to be most efficient for VOL 34, NO. 8, JULY 1962
0
999
0
0
Control Wilh Buffer
E
0
2-
v,
I
0
I
1 2 GROWTH
I
3 TIME
I
J
4 5 (HOURS)
6
I
Figure 2 . Effect of the pollutant mixture, after bubbling through phosphate buffer, on growth of E. coli. Exposure time 60 minutes Analysis of line: Oxidant, pg. o&, 6.6 NOn, p.p.m./l., 0.23 Acid, meq./l., 0.025 302 m p as mg. N&/l., 3.06
rem01 ing the as yet unidentified 302mp absorbing material (see analytical section). To determine the concentration of these materials in the absorbing solutions, the method of analysis for oxidant was modified, since the presence of phosphate interfered. Sodium chloride solution of comparable ionic strength and pH was substituted. Coniparison of the analysis of the gas and the solution is given in Table IT. I n all experiments the analysis of the absorbing solution indicated higher oxidant con-
Table 111.
Effect of Bubbling Pollutant Mixture through Phosphate Buffer
Oxidant,
pg. Or;/Liter
of Gas
Direct After phosphate buffer Table IV.
centrations than ~ I direct I analysis of the gas. Does this mean that there was poterit'ial oxidant material in the gas n-liich became actual oxidant material in solution? I n exploring column packing materials for gas chromatographic analysis, vie observed that for a variety of columns, mineral acids could he quantitatively eluted from the columns and that the retention properties of the column appeared to rhange gradually n-hen avid-containing samples were analyzed. This observation led to exploring suitable caolumn parking materials which would effectively rcmove acids from the gas samples without removing ot'her polar compounds which might be present. Studies on C-22 Firebrick, 30- to BO-mesh, coated with 207, ( W . ~ ' W . ) disodium phosphate packed in 5-ern. columns (7-nini. i.d.) indicated quantitative removal from an air stream of organic, as well as inorganic, acids as represented by hydrochloric, phosphoric, nitric, acetic, propionic, and butyric acids. The disodium phosphate-Firebrick column also permitted only seniiquantit'at'ive recovery of representative alcohols, ketones, and nitro-hydrocarbons having boiling points of 132" C. or less. Thus, each of these compounds is held in part by bhe column. With a column of 20% ( ~ J i v . ) disodium phosphate on Firebrick in the effluent line, good recovery of the growth rat'e m-as exhibited by the organisms exposed to t'he pollutant mixture. -1s seen in Figure 3 the growth rate of the exposed. organisms
6.3 4.6
sor,
P.P.M. 0.33 0.12
NO?,
0 3 !
p.p.m. 0 33 0 51 0 36 0 36 0 38
Table V.
Acid, meq./liter 0 028 0 030 0 028 0 036 0 028
302 mp as mg. XOs/liter 2.83 2.57 2.52 2.89 2.78
Analysis of t,he Solution Calculated to Gas 037 302 mp a s pg./liter mg. NO3/hter of gas of gas 6.1 1.75 7.5 1.99 6.3 1.75 9.1 2.46 10.5 2.69
Analysis o f Pollutant Mixture after a Disodium Phosphate Column
Without column With column Without column With column
1000
302 RIM, as Rlg. S03-/Liter 2.35 0.00
Concentration of Pollutant Materials in Gas and Absorbing Solution
.4nalysis of the Gas pg./liter 49 5 4 5 3 5 2 4 7
Acid, Meq. /Liter 0 028 0 002
Oxidant, pg. 03/Liter 69 0 2
6 5 1 2
ANALYTICAL CHEMISTRY
KO?, P.P.M. 0 28 0 13 0 26 0 25
Acid, i\leq./Liter 0 029 0 004 0 028 0 007
302 RIp as Mg.
SOo/Liter 2 27 0 13 1 91 0 89
60r
.5Ot
P
c-->
Control Exposed
e--*
E x p o s e d , No C o l u m n
H
+ Column
*
40u
i 0
0
-
3
0
p
'
l
,
,
20
./r
m
-=
IO
0
_ --- e - - 1
_-
e---e---e---*
2 3 4 5 G R O W T H TIME ( H O U R S )
6
Figure 3. The protective effect of a disodium phosphate-Firebrick column on the growth of E. co!i Analysis of the line:
Oxidant, pg. 03/L NO?, p.p m. Acid, meq./l.
Column Present 6.6
0.31 0.029
Column Absent
1 .o 0.59 0.001
Column: 5 cm. of 7 mm. i.d. tubing containing C-22 firebrick, 30- to 60-mesh coated with 20y0 (w./w.) Na2HPOd
m s identical with that of tlie unexposed control. From the analysis of the effluent before and aEter the column, it would appear that oxidants were the most significant components removed in ternis of quantity. Further coni~iarisonsof t'he analyses of the pollutant mixture behind these columns are shovin in Table V. These experiments represent different columns of the same dimensions and packing. Of the coniponents tested, t'he nitrogen dioxide appeared to be the most variable. .Ittempts to elute possibly absorbed materials from the column were unsuccessful. Exposure of purified glutamic dehydrogenase to the pollutant mixture led to difficulties in the determination of enzymatic activity. Aldolase from E . coli is a,lso regarded as a sulfhydryl enzyme (1). Examinat,ion of tlie effectiveness of a disodium phosphateFirebrick column for the protection of aldolase activity has slio~vn t'hat the column absorbed acid materials (Table 1-1). Kere this the only rharacteristic of these columns, it' might be assunied that t'he more effect'ive inhibitor of aldolase activity was a decrease of pH. However, decreasing the p H alone, as with HC1, gives complete inhibition with recovery of activity after p H adjustment. This does not simulate tlie effect' of exposure to the pollutant misture. The data indicate t'hat tlie pollutant mixture has a twofold effect on aldolase activity. I n addition, the magnitude of the increased activity for aldolase exposed behind the column cannot be ignored. This apparent stimulation by the column requires further inwstigation.
From the data it \vas apparent that glass wool can partially remove the biologically effective components. The phosphate buffer and tlie disodium phosphate-Firebrick column appeared to remove completely tlie effective components. Both trapping agents removed a pait of the oxidant muterial. It 11-as not apparent' whether the ieiixiiniiig oxiclaiit material was biologically itieffertive or in too low a conceiitratiuii to be detected. Like\visc current methods n-ould n o t permit diff erwtiation among oxidant m:iterinls. The observation that the biologically effcctiT-e inciterial PKI water soluhlo eliminatc~tl the possibility that ozone i v w t,hc sole offending component. XI t,liougli considerablc cvidencc points to thi. destruct'ion of ozone by surfac*e :wtiori (I!+). ozone has aril>- slight solubility in water. It was obsericd t h a t the 302-mp atisorliiiig material n-as c*oinplc+ly rcnio\-cct hy the l~uffer and that. t'hc solution did not account' for the concimtrat,ion of the abwrbing material in Furt~liermorc, t h e ap1)arent oxitlalit inaterial 11:id increased in concentration. These obscrntions suggwted t,hat the 302-nip absorbing nintci,ial iwitnined a biologically effwtivv nitrogen containing agent nilp:irt>iitly not a c ~ c o u n t d for in the nitrogivi oxides as rlt+mnined by the Snltznian method. 'I'hc l~erosyacetyl nitrite, suggcsted by Scott et al. (It?), is less likely a compoileiit of the 302-mp absorbing niatcrial than thc recently suggested perosyacetyl nitrate ( 1 8 ) . Although iiitroniethaiie is a 1mdurt of the slow di~orn~iosition of the I)eroxyacctyl com-
pound, no evidence of nitro compounds v a s found in the effluent. The limitations of this method (8)in determining nitromethane do not completely eliminate its presence in the gas. Further examinations of the analysis of the pollutant mixture and exploration of its biochemical effects are in progress. ACKNOWLEDGMENT
The author appreciates the excellent technical assistance of Chao-Han Pan, John -L Cooper, and Pauline K. Baughman. LITERATURE CITED
(1) Bard, R. C., Gunsalus, I. C., J . Bacterzol. 5 9 , 387 (1950). (2) Estes, F. L., Baughman, P. I
