ENVIRONMEN
91. RESE+RCFI
Health
14, 223 - 231 ( 1977)
Effects of Short-Term Inhalation of Nitrogen Dioxide and Ozone Mixtures
RICHARD -i; Lijb Sciences + Biomrdicul
EHRLICH,” J. C. FINDLAY,* J. D. FENTERS,* AND D. E. GARDNER+ Cliicujio, illitfois Di\,ision, II T Rr.search Itl.sritiltr, Resenrd~ Brunch, U.S. Envirannwnfirl Prorrcfion Research Trianglr Pork. North C’rrrolino 2771 I
60616, rrrrd Agency.
Received October 27. 1976 The effects of single and multiple daily 3-hour exposures to nitrogen dioxide (NO,) and ozone (0,) mixtures on the resistance to streptococcal pneumonia were investigated. The concentrations of NO, ranged from 1.5 to 5.0 ppm. and those of 0,. from 0.05 to 0.5 ppm. The effect of a single exposure to the mixture was additive, whereby the excess mortality rates were equivalent to those induced by the inhalation of each individual pollutant. The ability to clear inhaled bacteria from the lungs was diminished in mice exposed to the NO, -0, mixtures for 3 hours. This impairment was manifested by the increased frequency of isolation of Srreptococcrrs from the lungs for up to 6 days after the respiratory challenge. Excess mortalities observed after 20 daily 3-hour exposures suggested that a synergistic et’iect might be present upon repeated inhalation of pollutant mixtures, that made them more effective in reducing resistance to respiratory infection. The results emphasize the need for the establishment of primary air quality standards for short-term NO, exposures.
INTRODUCTION
Results of experimental studies clearly demonstrate that inhalation of either ozone (Miller and Ehrlich, 1958: Coffin and Gardner, 1971) or nitrogen dioxide (,Ehrlich, 1966) significantly enhances the susceptibility to bacterial pneumonias. However, only sparse data are available on the effects of exposures to mixtures of these two air pollutants on the resistance to respiratory infections. To elucidate such effects, studies were conducted in mice which were exposed to ozone, nitrogen dioxide, or a mixture of the two and then challenged with St~eptococcr/.s aerosol. This experimental model reflects the overall toxic response of the respiratory system, such as edema, inflammation, cellular necrosis, reduced macrophage function, and ciliostasis. Thus, it indicates the impairment of the basic defense mechanisms in the lung by the combined exposure to ambient concentrations of air pollutants and the superimposed infectious challenge. MATERIALS Animr~ls. Female
AND METHODS
CF-1 (ARS, Madison, Wis.). and CD,F, mice (Murphy Laboratory, Plainfield, Ind.) were used in the experiments. The 5- to &week-old mice were quarantined for 7 - 15 days before being used in the studies. During the quarantine and throughout the experiments, the mice were housed in groups of eight in stainless steel shoe-box cages, and food and water were provided ad libitum. For the 3-hour exposure to the pollutants and during the infectious challenge, the mice were housed individually in separate compartments of specially designed stainless steel wire cages.
224
EHRLICH
ETAL
Exposure chambers. Four identical 120 x 60 x 60-cm (432 liter) Plexiglas chambers were used for exposure to air pollutants or filtered air. The compressed air carrying the pollutants was dried and purified by passage through an Alemite filter, a disposable air purifier, and a flow equalizer. The air stream entered the chambers from the top of one side at a rate of 60 k 5 liters/minute and was exhausted at the top of the opposite side. Homogeneous distribution of the pollutants was further assured by continuous operation of a small blower during the animal exposures. To prevent a buildup of ammonia, deotized cage boards were placed on the floor of each chamber. Temperature in the chambers was maintained at 24 t 2°C at ambient (~40% RH) humidity. Nitrogen dioxide (NO,). A 1% NO, gas mixture in balanced air, 99.5% pure (Matheson, Joliet, Ill.), was diluted with the filtered air in a glass mixing chamber and then was passed into the animal exposure chamber. The NO, concentration was monitored continuously by a NO -NO, -NO, chemiluminescent analyzer (Model 8101 B, Bendix Corp., Ronceverte, W. Va.) and was expressed in parts per million (parts per million x 1.88 = milligrams per cubic meter). Ozorze (03). A high-voltage generator (IITRI) was used to convert filtered air to 0,. To provide the desired concentration, 0, was diluted with filtered air in a glass mixing chamber and then was passed into the animal exposure chamber. The O9 concentration was monitored continuously with an 0, chemiluminescent analyzer (Model OA 310, Meloy Laboratories, Springfield, VA.) and was expressed in parts per million (parts per million x 1.96 = milligrams per cubic meter). NO, -0, mixture. Each gas was introduced into separate glass vessels and then was combined in a glass mixing chamber and was passed into the animal exposure chamber. The concentration of gases was monitored continuously with the NO, and 0, chemiluminescent analyzers. Infectious challenge. Streptococcus pyogenes (Group C), originally isolated from a pharyngeal abcess of a guinea pig, was used as the infectious agent. To maintain the stock culture, mice were injected intraperitoneally with a suspension of Streptococcrrs and were killed 24 hours later. Blood obtained from the heart was incubated on blood agar for 48 hours at 37”C, and isolated Streptococcus colonies were inoculated in Todd Hewitt broth (BBL). After 18 hours of incubation at 37°C the growth was harvested, and l-ml aliquots were frozen and stored at -70°C. For aerosol dissemination the thawed bacteria were regrown in Todd Hewitt broth for 18 hours at 37”C, and the optical density of the suspension was adjusted in 0.1% peptone water to approximately 65% density, as measured at 440 pm in a Spectronic 20 densitometer. Mice were challenged with the infectious agent in a 400-liter Plexiglas aerosol chamber (71 x 61 x 92 cm) contained within a microbiological safety cabinet. A continuous-flow glass DeVilbiss nebulizer (Model 84) was used to produce the bacterial aerosol by supplying filtered air at a flow rate of approximately 8 liters/ minute. For the infectious respiratory challenge, mice were housed in individual compartments of stainless steel wire cages and were exposed to the aerosol for 10 minutes at 24 _t 2°C and 65 ? 5% RH. After the challenge, the mice were removed from the chamber and were held in a clean-air isolated, animal room. During the 1Cday observation period deaths were recorded daily.
HEALTH
EFFECTS
OF NOp-03
225
MIXTURES
The inhaled dose of bacteria was determined by killing three mice immediately after the infectious challenge. The lungs were removed, weighed, and homogenized in sterile 0.1% peptone water. The suspension was diluted and plated on blood agar, and the colonies were counted after 48 hours of incubation at 37°C. The inhaled dose, expressed as the number of viable bacteria per gram of lung tissue, ranged from 10 to 30 x lo3 organism/g. Experimental protocol. In all experiments the mice were exposed for 3 hours to the individual pollutants and corresponding pollutant mixtures. For repeatedexposure studies mice were exposed to the pollutants daily for 3 hours, five times/week for 1, 2, or 4 weeks. Control mice were treated identically, with the exception of being exposed to filtered air instead of air containing the pollutants. Within 1 hour after termination of exposure to the pollutants, groups of mice representing all experimental and control conditions were simultaneously infected by the respiratory route with airborne Streptococcus. RESULTS
Previous reports from our laboratories (Ehrlich, 1966) have indicated that a single 2-hour exposure of mice to 3.5 ppm of NO, significantly enhanced the mortality resulting from a superimposed bacterial pneumonia initiated by inhalation of airborne Klebsiella pneumoniae. Coffin and Gardner (1972) reported a similar enhancement in mortalities after a 3-hour exposure to 0.08 ppm of 0, and challenge with Streptococcus aerosol. To provide more unified data in terms of duration of exposure and the infectious agent, mice were exposed for 3 hours to various concentrations of either NO, or 0, and then were challenged with Streptococcus aerosol. The death rates obtained during numerous replicate exposures to the pollutants are summarized in Fig. 1. The mortalities of infected mice not exposed to the pollutants represent the total number of control mice used in all experiments, This mortality rate (26.6%) was
Slgnltmmt (
)
Number
0
Mortality
Change
(pSO.05)
Of Mice
15
2.0 Not
(ppm)
3 5
5.0 03 (pm)
FK. 1. Mortality rates in mice exposedfor 3 hours to various concentrations of nitrogen dioxide or ozone and challenged with .Srrc+m~occ~r~s aerosol.
226
ET AL.
EHRLICH
used as the basis of estimation of the significance of the changes in mortality rates induced by exposures to the pollutants. The statistical significance of the differences was determined by a Chi-square test with a 2 x 2 contingency table. A significant increase in mortality rates over those observed among control animals was seen upon the 3-hour exposure to 0.1 ppm of 0, or 2.0 ppm of NO,. Moreover, a linear relationship was present between the concentration of the pollutants and the mortality rates, with a correlation coefficient of 0.969 for NO, and 0.996 for 0,. TO determine the effects of inhalation of air pollutant mixtures, groups of mice were exposed to selected concentrations of NO,, 03, NO, -OS mixture, or filtered air. The four groups, usually consisting of 24 mice each, were then simultaneously challenged with Streptococcus aerosol. Thus, the mortality rates could be initially compared on the basis of individual exposure experiments, and results of replicate experiments were then pooled for statistical analysis. The differences between mortality rates among mice exposed to the pollutants and challenged with Stwptococc~s and the corresponding control mice challenged with the infectious aerosol only during each exposure were calculated. The excess mortalities based on a minimum of four replicate experiments at each concentration of pollutants are summarized in Table 1. The data indicate that the effect of the 3-hour inhalation of NO,-0, mixtures was additive. In most instances the differences in mortality rates were equivalent to the sum of those induced by inhalation of each individual pollutant. The mean survival time of mice was also affected by the exposures to the individual pollutants as well as to the pollutant mixtures. All noninfected mice exposed to the pollutants only survived the observation period of 14 days, while the mean survival time of infected control mice not exposed to the pollutants was 12 days. Significant shortening of survival time was observed upon exposure to 3.5 or 5 ppm of NO, or 0.5 ppm of 0,. Thus, although exposures to 2.0 ppm of NO2 or 0.1 ppm of O3 significantly enhanced the mortality rates, they did not influence or accelerate the disease so that earlier deaths would occur. Decreases in mean survival time were also observed upon exposure to the N02-O3 mixtures. Significantly reduced survival occurred upon exposure to mixtures containing 0.5 TABLE Exctss
MORTIIIJIY
IS
OZOSF: AND
Concentration NWA 0 1.5 2.0 3.5 5.0
MIW
E~POS~;D
CHALLENGED
FOR WI
1 3 HOURS
TO NITROGES
H STREPTOC.OCCC:J
DIOXIDE
WD
AEROSOL
Excess mortality (“r) (pollutant - control)
(ppm) 0
0.05
0.1
0.5
0 -1.7 14.3” 28.T’ 35.7”
5.4 4.6 22.w -
7.2 4.2 38.5”
I 6” ‘8 23.9’ 56.2’ 68.7” 65.3”
” Significant change in mortality from corresponding
infected controls (P s 0.05).
HEALTH
EFFECTS
OF
SO~-03
227
MIXl-LIKES
ppm of 0,, irrespective of the concentrations of NO, present and to the mixture containing 3.5 ppm of NO2 and 0.1 ppm of 0, In studies of the effects of multiple exposures, mice were exposed to the pollutants daily for 3 hours, five days per week for 1. 2, and 4 weeks. Within 1 hour after termination of the final exposure, the mice, along with control mice exposed to filtered air, were challenged with Streptococcrrs aerosol. Two mixtures of the pollutants were included: 2.0 ppm of NO, and 0.05 ppm of 0,: and 1.5 ppm of NO, and 0.1 ppm of OZ1. Each mixture contained a concentration of one of the two pollutants, exposure to which had previously resulted in excess mortalities. The results of two replicate experiments for each exposure regimen are summarized in Fig. 2. The excess mortalities are based on exposure of 48 mice at each experimental point to the 1.5 ppm of NO, and 0.1 ppm of 0, mixture, and 104 mice to the 2.0 ppm of NO, and 0.05 ppm of 0, mixture. Repeated daily exposure to the mixture consisting of 2.0 ppm of NO, and 0.05 ppm of 0,, followed by the infectious challenge, resulted in significant excess deaths over those observed in control mice. The excess mortalities were present
* pso.05 **ps 01
.\” ; Z F
I5 IO
r” 2
5
z 6
0 -
1.5 PP~
NOI
+ 0.1 ppm
0,
-5 t 5
IO Number
Flc.. mixtures
2. Excess and
mortality
challenged
in mice with
after
Of
multiple
.S~e~~toc~oc~rr
3-hr
daily
5 aerosol.
20 Exposures
3-hour
exposures
to nitrogen
dioxide
and
ozone
228
EHRLICH
ET AL
irrespective of the number of daily 3-hour exposures. Death rates resulting from exposures to 2.0 ppm of NO* alone increased somewhat after five daily exposures, were significantly higher after 10 exposures, but did not differ from control mice after 20 exposures. Only small changes in mortality rates were seen after 5, lo, or 20 daily exposures to 0.05 ppm of 0, alone. The results indicate that daily 3-hour exposures to either NO, or O3 had no major effect on the mortality rates. On the other hand, daily exposures to the NOz-0, mixture containing the same concentration of each pollutant resulted in significant excess mortalities. This could suggest the presence of a synergistic relation between the two pollutants that makes them more effective in reducing resistance to respiratory infection. Results of daily 3-hour exposures to a mixture consisting of 1.5 ppm of NO, and 0.1 ppm of O3 indicate that 0, appears to be the primary contributing factor in inducting excess mortalities. There were no remarkable differences in mortalities at any of the exposure conditions after five daily exposures. However. after 10 or 20 exposures to 0, per se or to the NO* -0, mixture, marked excess deaths were present. Inasmuch as excess mortalities seen upon inhalation of the mixture were approximately the same as those observed in mice exposed to 0, only, it can be assumed that they were due primarily to the presence of O3 in the mixture. Lung Clearance
of Inhaled
Bacteria
The effects of inhalation of pollutants on clearance of viable Streptococcus from lungs were investigated in mice exposed for 3 hours to mixtures consisting of 3.5 ppm of NO, and 0.1 ppm of O,, or 2.0 ppm of NO, and 0.05 of ppm OS. Within 1 hour after termination of exposure, mice were challenged with airborne Streptococcus, and, immediately, live mice were killed and the lungs were removed. weighed, homogenized, and cultured quantitatively. These initial counts (0 hours), expressed as the number of viable bacteria per gram of lung, were considered as 100%. Thereafter, groups of five mice, either exposed to filtered air or to the NO,-0, mixture, were killed at 1, 2, 3, 4, and 5 hours, and at 1, 2, 3, and 6 days after the respiratory challenge. Lungs of these mice were assayed in an identical manner. The hourly counts were calculated as the percentage of recovery of those present at the zero hour. The clearance rates of viable bacteria, determined by the least-squares method, showed a marked delay after exposure to the 3.5 ppm of NO, and 0.1 ppm of O3 mixture. The time required to clear 50% of inhaled bacteria in control mice was approximately 81 minutes, and in those exposed to the pollutant mixture, 131 minutes. No differences were observed in bacterial clearance from the lungs upon exposure to the 2 ppm of NO, and 0.05 ppm of 0, mixture. The daily clearance rates were calculated as the number of mice out of the total number of mice killed on a given day having viable Streptococcus present in their lungs. The effects of exposure to both mixtures of the pollutants were much more pronounced over this extended assay period (Table 2). Among the mice exposed to the 3.5 ppm of NO, and 0.1 ppm of 0, mixture, 15119 (79%) and, of those exposed to 2.0 ppm of NO, and 0.05 ppm of O3 mixture, 16/18 (89%‘) showed viable Streptococcus in the lungs, whereas, among the corresponding controls, 7120 (35%) and 8/20 (40%‘) were positive. Thus, it appears that the capacity to clear inhaled bacteria is markedly impaired by inhalation of mixtures of the pollutants.
HEALTH
RETEKTION
OF INHALED
3
HOERS
EFFECTS
OF NO*-O3
229
MIXTURES
TABLE 2 STREPTOCOCCIIN Lu~cs ok
VIABLE TO NITROGEN
DIOXIDE
AND
OZONE
MICE
FOR
Mixture II”
Mixture I” Day of lung assay
Control
Expt
1 2 3 6
215’ 315 o/5 215
515 315 314 415
” 3.5 ppm of NO, and 0.1 ppm of 03. ’ 2 ppm of NO2 and 0.05 ppm of 0,. ‘. Number of mice showing viable Streptoc-occrrs
EXPOSED
MIXTCRES
Control O/5 ?I5 415 215
Expt 414 415 314 515
out of total number of mice assayed.
DISCUSSION
It is well recognized that effects of air pollutants may be additive or synergistic with each other as well as with other environmental stresses. Such combined effects can be ascribed to a variety of factors such as: one pollutant affecting the site of deposition of another; one pollutant affecting the lung clearance mechanisms so that the second one cannot be removed; or one pollutant producing an effect in the lung which makes it more vulnerable to the effects of the second pollutant. Occasionally, the type of interaction can be predicted on the basis of the chemical composition of each component. More often, however, it is not possible to forecast the effect of pollutant mixtures, and, thus, empirical studies are required. Only sparse experimental data are available on the effects of exposure to NO, -0, mixtures on the resistance to respiratory infections. Coffin and Bloomer ( 1967) reported increased mortality rates in mice which were exposed for 4 hours to light-irradiated automobile exhaust and then infected with airborne StreptococC’US. The NO, concentration in the exhaust gas was 0.3 ppm, approximately tenfold below that reported to enhance the susceptibility to respiratory infection (Ehrlich, 1966). However, concentrations of oxidants were within the effective range of ozone, where threshold values of 0.1 ppm have been reported (Coffin and Gardner, 1972). Thus, the authors ascribed the changes in resistance to infection primarily to the presence of oxidants. Goldstein et al. (1974) studied the pulmonary defense mechanisms in mice exposed to NO,-0, mixtures and infected with airborne Staphylococcus aureus. Mice were exposed to the gas mixtures either for 17 hours before or for 4 hours after the infectious challenge. The authors concluded that the effects, expressed as bactericidal dysfunction in the lungs, were present when the concentration of either pollutant approximated its individual threshold value; 7.0 ppm of NO, or 0.4 ppm of O3 for the 4-hour exposure. Our studies clearly demonstrate the additive effects of NO, and 0, during a single 3-hour exposure when superimposed with an infectious challenge. Moreover, results of 20 daily 3-hour exposures suggest that a synergistic effect might be present upon repeated inhalation of a mixture of the two pollutants. TO assess further the interaction between the two pollutants, regression analysis
230
EHRtiCH -
100
r
-.-
-
r
01” 0
ET AL
NO, I5 PP~ NO2 20PPm NO2 35PPm
” 0.1
” 0.2
0.3 03 (w-4
”
--
0, 0,
-.-
0,
005ppm 0.1 ppm 0.5 PPnl
1.0
2.0
‘1 0.4
0.5
0
NO,
FIG. 3. Expected excess deaths from streptococcal pneumonia nitrogen dioxide and ozone mixtures.
3.0 (ppd
4.0
5.0
in mice exposed for 3 hours to
was applied to the excess mortality data obtained during exposures to the NO,-OZS mixtures. The least-squares lines plotted in Fig. 3 make it possible to estimate the increase in deaths which can be expected upon exposure to 1.5, 2.0. or 3.5 ppm of NO1 in the presence of various concentrations of O,, or conversely, upon exposure to 0.05,O. 1, or 0.5 ppm of Og, in conjunction with various concentrations of N02. The data show that a marked increase in mortality rates among infected mice can be expected upon a 3-hour exposure to NOs--Os mixtures in concentrations frequently encountered in ambient urban pollution. Results of our studies re-emphasize the necessity for the establishment of primary air quality standards for short-term exposures to NO,. The data provide estimates concerning the health effects of single and multiple short-term exposures to NO, alone or in the presence of 0,. The current NO, air quality standard of 0.05 ppm (0.94 mg/m”) represents an arithmetic mean averaged on an annual basis. The experimental evidence presented in this paper further supports the contention that short-term exposures to NO, are indeed biologically harmful. The results also suggest that, in the establishment of air quality standards, consideration should be given to the presence of mixtures of pollutants. It appears. therefore, that the primary air quality standards should consider the concomitant control of NO, and 0, at any one time, e.g., NO, should not exceed X concentration when 0, is present in Y concentration. ACKNOWLEDGMENTS The authors are indebted to Ms. S. Daseler and Mr. J. Hingeveld for their technical assistance. The studies were supported by funds provided by the U.S. Environmental Protection Agency under Contract Nos. 68-O?- 1267 and 68-02-2274. Portions of the paper were presented at the International Conference on Photochemical Oxidant Pollution and Its Control. September 12-17, 1976, Raleigh. North Carolina.
HEALTH
EFFECTS
OF
NO?-O3
MIXTURES
231
REFERENCES Coffin, D. L.. and Blommer, E. J. (1967). Acute toxicity of irradiated auto exhaust indicated by mortality from streptococcal pneumonia. Arch. Etwiron. Hec~ltlz 15, 36-38. Coffin. D. L.. and Gardner, D. E. t 1972). Interaction of biological agents and chemical pollutants. Am. Omcp.
Hyg.
15, 219-234.
Ehrlich, R. t 1966). Effect of nitrogen dioxide on resistance to respiratory infection. Eucferiol. Rr\,. 30, 604-614. Goldstein, E., Warshauer. D.. Lippert, W.. and Tarkington. B. (1974). Ozone and nitrogen dioxide exposure. Arch. .!CmGw~. Hdrh 28, 85 -90. Miller, S., and Ehrlich. R. (1958). Susceptibility to respiratory infections of animals exposed to ozone. .I. Infi~c.
Dis.
103, 145
149.
91. RESE+RCFI
Health
14, 223 - 231 ( 1977)
Effects of Short-Term Inhalation of Nitrogen Dioxide and Ozone Mixtures
RICHARD -i; Lijb Sciences + Biomrdicul
EHRLICH,” J. C. FINDLAY,* J. D. FENTERS,* AND D. E. GARDNER+ Cliicujio, illitfois Di\,ision, II T Rr.search Itl.sritiltr, Resenrd~ Brunch, U.S. Envirannwnfirl Prorrcfion Research Trianglr Pork. North C’rrrolino 2771 I
60616, rrrrd Agency.
Received October 27. 1976 The effects of single and multiple daily 3-hour exposures to nitrogen dioxide (NO,) and ozone (0,) mixtures on the resistance to streptococcal pneumonia were investigated. The concentrations of NO, ranged from 1.5 to 5.0 ppm. and those of 0,. from 0.05 to 0.5 ppm. The effect of a single exposure to the mixture was additive, whereby the excess mortality rates were equivalent to those induced by the inhalation of each individual pollutant. The ability to clear inhaled bacteria from the lungs was diminished in mice exposed to the NO, -0, mixtures for 3 hours. This impairment was manifested by the increased frequency of isolation of Srreptococcrrs from the lungs for up to 6 days after the respiratory challenge. Excess mortalities observed after 20 daily 3-hour exposures suggested that a synergistic et’iect might be present upon repeated inhalation of pollutant mixtures, that made them more effective in reducing resistance to respiratory infection. The results emphasize the need for the establishment of primary air quality standards for short-term NO, exposures.
INTRODUCTION
Results of experimental studies clearly demonstrate that inhalation of either ozone (Miller and Ehrlich, 1958: Coffin and Gardner, 1971) or nitrogen dioxide (,Ehrlich, 1966) significantly enhances the susceptibility to bacterial pneumonias. However, only sparse data are available on the effects of exposures to mixtures of these two air pollutants on the resistance to respiratory infections. To elucidate such effects, studies were conducted in mice which were exposed to ozone, nitrogen dioxide, or a mixture of the two and then challenged with St~eptococcr/.s aerosol. This experimental model reflects the overall toxic response of the respiratory system, such as edema, inflammation, cellular necrosis, reduced macrophage function, and ciliostasis. Thus, it indicates the impairment of the basic defense mechanisms in the lung by the combined exposure to ambient concentrations of air pollutants and the superimposed infectious challenge. MATERIALS Animr~ls. Female
AND METHODS
CF-1 (ARS, Madison, Wis.). and CD,F, mice (Murphy Laboratory, Plainfield, Ind.) were used in the experiments. The 5- to &week-old mice were quarantined for 7 - 15 days before being used in the studies. During the quarantine and throughout the experiments, the mice were housed in groups of eight in stainless steel shoe-box cages, and food and water were provided ad libitum. For the 3-hour exposure to the pollutants and during the infectious challenge, the mice were housed individually in separate compartments of specially designed stainless steel wire cages.
224
EHRLICH
ETAL
Exposure chambers. Four identical 120 x 60 x 60-cm (432 liter) Plexiglas chambers were used for exposure to air pollutants or filtered air. The compressed air carrying the pollutants was dried and purified by passage through an Alemite filter, a disposable air purifier, and a flow equalizer. The air stream entered the chambers from the top of one side at a rate of 60 k 5 liters/minute and was exhausted at the top of the opposite side. Homogeneous distribution of the pollutants was further assured by continuous operation of a small blower during the animal exposures. To prevent a buildup of ammonia, deotized cage boards were placed on the floor of each chamber. Temperature in the chambers was maintained at 24 t 2°C at ambient (~40% RH) humidity. Nitrogen dioxide (NO,). A 1% NO, gas mixture in balanced air, 99.5% pure (Matheson, Joliet, Ill.), was diluted with the filtered air in a glass mixing chamber and then was passed into the animal exposure chamber. The NO, concentration was monitored continuously by a NO -NO, -NO, chemiluminescent analyzer (Model 8101 B, Bendix Corp., Ronceverte, W. Va.) and was expressed in parts per million (parts per million x 1.88 = milligrams per cubic meter). Ozorze (03). A high-voltage generator (IITRI) was used to convert filtered air to 0,. To provide the desired concentration, 0, was diluted with filtered air in a glass mixing chamber and then was passed into the animal exposure chamber. The O9 concentration was monitored continuously with an 0, chemiluminescent analyzer (Model OA 310, Meloy Laboratories, Springfield, VA.) and was expressed in parts per million (parts per million x 1.96 = milligrams per cubic meter). NO, -0, mixture. Each gas was introduced into separate glass vessels and then was combined in a glass mixing chamber and was passed into the animal exposure chamber. The concentration of gases was monitored continuously with the NO, and 0, chemiluminescent analyzers. Infectious challenge. Streptococcus pyogenes (Group C), originally isolated from a pharyngeal abcess of a guinea pig, was used as the infectious agent. To maintain the stock culture, mice were injected intraperitoneally with a suspension of Streptococcrrs and were killed 24 hours later. Blood obtained from the heart was incubated on blood agar for 48 hours at 37”C, and isolated Streptococcus colonies were inoculated in Todd Hewitt broth (BBL). After 18 hours of incubation at 37°C the growth was harvested, and l-ml aliquots were frozen and stored at -70°C. For aerosol dissemination the thawed bacteria were regrown in Todd Hewitt broth for 18 hours at 37”C, and the optical density of the suspension was adjusted in 0.1% peptone water to approximately 65% density, as measured at 440 pm in a Spectronic 20 densitometer. Mice were challenged with the infectious agent in a 400-liter Plexiglas aerosol chamber (71 x 61 x 92 cm) contained within a microbiological safety cabinet. A continuous-flow glass DeVilbiss nebulizer (Model 84) was used to produce the bacterial aerosol by supplying filtered air at a flow rate of approximately 8 liters/ minute. For the infectious respiratory challenge, mice were housed in individual compartments of stainless steel wire cages and were exposed to the aerosol for 10 minutes at 24 _t 2°C and 65 ? 5% RH. After the challenge, the mice were removed from the chamber and were held in a clean-air isolated, animal room. During the 1Cday observation period deaths were recorded daily.
HEALTH
EFFECTS
OF NOp-03
225
MIXTURES
The inhaled dose of bacteria was determined by killing three mice immediately after the infectious challenge. The lungs were removed, weighed, and homogenized in sterile 0.1% peptone water. The suspension was diluted and plated on blood agar, and the colonies were counted after 48 hours of incubation at 37°C. The inhaled dose, expressed as the number of viable bacteria per gram of lung tissue, ranged from 10 to 30 x lo3 organism/g. Experimental protocol. In all experiments the mice were exposed for 3 hours to the individual pollutants and corresponding pollutant mixtures. For repeatedexposure studies mice were exposed to the pollutants daily for 3 hours, five times/week for 1, 2, or 4 weeks. Control mice were treated identically, with the exception of being exposed to filtered air instead of air containing the pollutants. Within 1 hour after termination of exposure to the pollutants, groups of mice representing all experimental and control conditions were simultaneously infected by the respiratory route with airborne Streptococcus. RESULTS
Previous reports from our laboratories (Ehrlich, 1966) have indicated that a single 2-hour exposure of mice to 3.5 ppm of NO, significantly enhanced the mortality resulting from a superimposed bacterial pneumonia initiated by inhalation of airborne Klebsiella pneumoniae. Coffin and Gardner (1972) reported a similar enhancement in mortalities after a 3-hour exposure to 0.08 ppm of 0, and challenge with Streptococcus aerosol. To provide more unified data in terms of duration of exposure and the infectious agent, mice were exposed for 3 hours to various concentrations of either NO, or 0, and then were challenged with Streptococcus aerosol. The death rates obtained during numerous replicate exposures to the pollutants are summarized in Fig. 1. The mortalities of infected mice not exposed to the pollutants represent the total number of control mice used in all experiments, This mortality rate (26.6%) was
Slgnltmmt (
)
Number
0
Mortality
Change
(pSO.05)
Of Mice
15
2.0 Not
(ppm)
3 5
5.0 03 (pm)
FK. 1. Mortality rates in mice exposedfor 3 hours to various concentrations of nitrogen dioxide or ozone and challenged with .Srrc+m~occ~r~s aerosol.
226
ET AL.
EHRLICH
used as the basis of estimation of the significance of the changes in mortality rates induced by exposures to the pollutants. The statistical significance of the differences was determined by a Chi-square test with a 2 x 2 contingency table. A significant increase in mortality rates over those observed among control animals was seen upon the 3-hour exposure to 0.1 ppm of 0, or 2.0 ppm of NO,. Moreover, a linear relationship was present between the concentration of the pollutants and the mortality rates, with a correlation coefficient of 0.969 for NO, and 0.996 for 0,. TO determine the effects of inhalation of air pollutant mixtures, groups of mice were exposed to selected concentrations of NO,, 03, NO, -OS mixture, or filtered air. The four groups, usually consisting of 24 mice each, were then simultaneously challenged with Streptococcus aerosol. Thus, the mortality rates could be initially compared on the basis of individual exposure experiments, and results of replicate experiments were then pooled for statistical analysis. The differences between mortality rates among mice exposed to the pollutants and challenged with Stwptococc~s and the corresponding control mice challenged with the infectious aerosol only during each exposure were calculated. The excess mortalities based on a minimum of four replicate experiments at each concentration of pollutants are summarized in Table 1. The data indicate that the effect of the 3-hour inhalation of NO,-0, mixtures was additive. In most instances the differences in mortality rates were equivalent to the sum of those induced by inhalation of each individual pollutant. The mean survival time of mice was also affected by the exposures to the individual pollutants as well as to the pollutant mixtures. All noninfected mice exposed to the pollutants only survived the observation period of 14 days, while the mean survival time of infected control mice not exposed to the pollutants was 12 days. Significant shortening of survival time was observed upon exposure to 3.5 or 5 ppm of NO, or 0.5 ppm of 0,. Thus, although exposures to 2.0 ppm of NO2 or 0.1 ppm of O3 significantly enhanced the mortality rates, they did not influence or accelerate the disease so that earlier deaths would occur. Decreases in mean survival time were also observed upon exposure to the N02-O3 mixtures. Significantly reduced survival occurred upon exposure to mixtures containing 0.5 TABLE Exctss
MORTIIIJIY
IS
OZOSF: AND
Concentration NWA 0 1.5 2.0 3.5 5.0
MIW
E~POS~;D
CHALLENGED
FOR WI
1 3 HOURS
TO NITROGES
H STREPTOC.OCCC:J
DIOXIDE
WD
AEROSOL
Excess mortality (“r) (pollutant - control)
(ppm) 0
0.05
0.1
0.5
0 -1.7 14.3” 28.T’ 35.7”
5.4 4.6 22.w -
7.2 4.2 38.5”
I 6” ‘8 23.9’ 56.2’ 68.7” 65.3”
” Significant change in mortality from corresponding
infected controls (P s 0.05).
HEALTH
EFFECTS
OF
SO~-03
227
MIXl-LIKES
ppm of 0,, irrespective of the concentrations of NO, present and to the mixture containing 3.5 ppm of NO2 and 0.1 ppm of 0, In studies of the effects of multiple exposures, mice were exposed to the pollutants daily for 3 hours, five days per week for 1. 2, and 4 weeks. Within 1 hour after termination of the final exposure, the mice, along with control mice exposed to filtered air, were challenged with Streptococcrrs aerosol. Two mixtures of the pollutants were included: 2.0 ppm of NO, and 0.05 ppm of 0,: and 1.5 ppm of NO, and 0.1 ppm of OZ1. Each mixture contained a concentration of one of the two pollutants, exposure to which had previously resulted in excess mortalities. The results of two replicate experiments for each exposure regimen are summarized in Fig. 2. The excess mortalities are based on exposure of 48 mice at each experimental point to the 1.5 ppm of NO, and 0.1 ppm of 0, mixture, and 104 mice to the 2.0 ppm of NO, and 0.05 ppm of 0, mixture. Repeated daily exposure to the mixture consisting of 2.0 ppm of NO, and 0.05 ppm of 0,, followed by the infectious challenge, resulted in significant excess deaths over those observed in control mice. The excess mortalities were present
* pso.05 **ps 01
.\” ; Z F
I5 IO
r” 2
5
z 6
0 -
1.5 PP~
NOI
+ 0.1 ppm
0,
-5 t 5
IO Number
Flc.. mixtures
2. Excess and
mortality
challenged
in mice with
after
Of
multiple
.S~e~~toc~oc~rr
3-hr
daily
5 aerosol.
20 Exposures
3-hour
exposures
to nitrogen
dioxide
and
ozone
228
EHRLICH
ET AL
irrespective of the number of daily 3-hour exposures. Death rates resulting from exposures to 2.0 ppm of NO* alone increased somewhat after five daily exposures, were significantly higher after 10 exposures, but did not differ from control mice after 20 exposures. Only small changes in mortality rates were seen after 5, lo, or 20 daily exposures to 0.05 ppm of 0, alone. The results indicate that daily 3-hour exposures to either NO, or O3 had no major effect on the mortality rates. On the other hand, daily exposures to the NOz-0, mixture containing the same concentration of each pollutant resulted in significant excess mortalities. This could suggest the presence of a synergistic relation between the two pollutants that makes them more effective in reducing resistance to respiratory infection. Results of daily 3-hour exposures to a mixture consisting of 1.5 ppm of NO, and 0.1 ppm of O3 indicate that 0, appears to be the primary contributing factor in inducting excess mortalities. There were no remarkable differences in mortalities at any of the exposure conditions after five daily exposures. However. after 10 or 20 exposures to 0, per se or to the NO* -0, mixture, marked excess deaths were present. Inasmuch as excess mortalities seen upon inhalation of the mixture were approximately the same as those observed in mice exposed to 0, only, it can be assumed that they were due primarily to the presence of O3 in the mixture. Lung Clearance
of Inhaled
Bacteria
The effects of inhalation of pollutants on clearance of viable Streptococcus from lungs were investigated in mice exposed for 3 hours to mixtures consisting of 3.5 ppm of NO, and 0.1 ppm of O,, or 2.0 ppm of NO, and 0.05 of ppm OS. Within 1 hour after termination of exposure, mice were challenged with airborne Streptococcus, and, immediately, live mice were killed and the lungs were removed. weighed, homogenized, and cultured quantitatively. These initial counts (0 hours), expressed as the number of viable bacteria per gram of lung, were considered as 100%. Thereafter, groups of five mice, either exposed to filtered air or to the NO,-0, mixture, were killed at 1, 2, 3, 4, and 5 hours, and at 1, 2, 3, and 6 days after the respiratory challenge. Lungs of these mice were assayed in an identical manner. The hourly counts were calculated as the percentage of recovery of those present at the zero hour. The clearance rates of viable bacteria, determined by the least-squares method, showed a marked delay after exposure to the 3.5 ppm of NO, and 0.1 ppm of O3 mixture. The time required to clear 50% of inhaled bacteria in control mice was approximately 81 minutes, and in those exposed to the pollutant mixture, 131 minutes. No differences were observed in bacterial clearance from the lungs upon exposure to the 2 ppm of NO, and 0.05 ppm of 0, mixture. The daily clearance rates were calculated as the number of mice out of the total number of mice killed on a given day having viable Streptococcus present in their lungs. The effects of exposure to both mixtures of the pollutants were much more pronounced over this extended assay period (Table 2). Among the mice exposed to the 3.5 ppm of NO, and 0.1 ppm of 0, mixture, 15119 (79%) and, of those exposed to 2.0 ppm of NO, and 0.05 ppm of O3 mixture, 16/18 (89%‘) showed viable Streptococcus in the lungs, whereas, among the corresponding controls, 7120 (35%) and 8/20 (40%‘) were positive. Thus, it appears that the capacity to clear inhaled bacteria is markedly impaired by inhalation of mixtures of the pollutants.
HEALTH
RETEKTION
OF INHALED
3
HOERS
EFFECTS
OF NO*-O3
229
MIXTURES
TABLE 2 STREPTOCOCCIIN Lu~cs ok
VIABLE TO NITROGEN
DIOXIDE
AND
OZONE
MICE
FOR
Mixture II”
Mixture I” Day of lung assay
Control
Expt
1 2 3 6
215’ 315 o/5 215
515 315 314 415
” 3.5 ppm of NO, and 0.1 ppm of 03. ’ 2 ppm of NO2 and 0.05 ppm of 0,. ‘. Number of mice showing viable Streptoc-occrrs
EXPOSED
MIXTCRES
Control O/5 ?I5 415 215
Expt 414 415 314 515
out of total number of mice assayed.
DISCUSSION
It is well recognized that effects of air pollutants may be additive or synergistic with each other as well as with other environmental stresses. Such combined effects can be ascribed to a variety of factors such as: one pollutant affecting the site of deposition of another; one pollutant affecting the lung clearance mechanisms so that the second one cannot be removed; or one pollutant producing an effect in the lung which makes it more vulnerable to the effects of the second pollutant. Occasionally, the type of interaction can be predicted on the basis of the chemical composition of each component. More often, however, it is not possible to forecast the effect of pollutant mixtures, and, thus, empirical studies are required. Only sparse experimental data are available on the effects of exposure to NO, -0, mixtures on the resistance to respiratory infections. Coffin and Bloomer ( 1967) reported increased mortality rates in mice which were exposed for 4 hours to light-irradiated automobile exhaust and then infected with airborne StreptococC’US. The NO, concentration in the exhaust gas was 0.3 ppm, approximately tenfold below that reported to enhance the susceptibility to respiratory infection (Ehrlich, 1966). However, concentrations of oxidants were within the effective range of ozone, where threshold values of 0.1 ppm have been reported (Coffin and Gardner, 1972). Thus, the authors ascribed the changes in resistance to infection primarily to the presence of oxidants. Goldstein et al. (1974) studied the pulmonary defense mechanisms in mice exposed to NO,-0, mixtures and infected with airborne Staphylococcus aureus. Mice were exposed to the gas mixtures either for 17 hours before or for 4 hours after the infectious challenge. The authors concluded that the effects, expressed as bactericidal dysfunction in the lungs, were present when the concentration of either pollutant approximated its individual threshold value; 7.0 ppm of NO, or 0.4 ppm of O3 for the 4-hour exposure. Our studies clearly demonstrate the additive effects of NO, and 0, during a single 3-hour exposure when superimposed with an infectious challenge. Moreover, results of 20 daily 3-hour exposures suggest that a synergistic effect might be present upon repeated inhalation of a mixture of the two pollutants. TO assess further the interaction between the two pollutants, regression analysis
230
EHRtiCH -
100
r
-.-
-
r
01” 0
ET AL
NO, I5 PP~ NO2 20PPm NO2 35PPm
” 0.1
” 0.2
0.3 03 (w-4
”
--
0, 0,
-.-
0,
005ppm 0.1 ppm 0.5 PPnl
1.0
2.0
‘1 0.4
0.5
0
NO,
FIG. 3. Expected excess deaths from streptococcal pneumonia nitrogen dioxide and ozone mixtures.
3.0 (ppd
4.0
5.0
in mice exposed for 3 hours to
was applied to the excess mortality data obtained during exposures to the NO,-OZS mixtures. The least-squares lines plotted in Fig. 3 make it possible to estimate the increase in deaths which can be expected upon exposure to 1.5, 2.0. or 3.5 ppm of NO1 in the presence of various concentrations of O,, or conversely, upon exposure to 0.05,O. 1, or 0.5 ppm of Og, in conjunction with various concentrations of N02. The data show that a marked increase in mortality rates among infected mice can be expected upon a 3-hour exposure to NOs--Os mixtures in concentrations frequently encountered in ambient urban pollution. Results of our studies re-emphasize the necessity for the establishment of primary air quality standards for short-term exposures to NO,. The data provide estimates concerning the health effects of single and multiple short-term exposures to NO, alone or in the presence of 0,. The current NO, air quality standard of 0.05 ppm (0.94 mg/m”) represents an arithmetic mean averaged on an annual basis. The experimental evidence presented in this paper further supports the contention that short-term exposures to NO, are indeed biologically harmful. The results also suggest that, in the establishment of air quality standards, consideration should be given to the presence of mixtures of pollutants. It appears. therefore, that the primary air quality standards should consider the concomitant control of NO, and 0, at any one time, e.g., NO, should not exceed X concentration when 0, is present in Y concentration. ACKNOWLEDGMENTS The authors are indebted to Ms. S. Daseler and Mr. J. Hingeveld for their technical assistance. The studies were supported by funds provided by the U.S. Environmental Protection Agency under Contract Nos. 68-O?- 1267 and 68-02-2274. Portions of the paper were presented at the International Conference on Photochemical Oxidant Pollution and Its Control. September 12-17, 1976, Raleigh. North Carolina.
HEALTH
EFFECTS
OF
NO?-O3
MIXTURES
231
REFERENCES Coffin, D. L.. and Blommer, E. J. (1967). Acute toxicity of irradiated auto exhaust indicated by mortality from streptococcal pneumonia. Arch. Etwiron. Hec~ltlz 15, 36-38. Coffin. D. L.. and Gardner, D. E. t 1972). Interaction of biological agents and chemical pollutants. Am. Omcp.
Hyg.
15, 219-234.
Ehrlich, R. t 1966). Effect of nitrogen dioxide on resistance to respiratory infection. Eucferiol. Rr\,. 30, 604-614. Goldstein, E., Warshauer. D.. Lippert, W.. and Tarkington. B. (1974). Ozone and nitrogen dioxide exposure. Arch. .!CmGw~. Hdrh 28, 85 -90. Miller, S., and Ehrlich. R. (1958). Susceptibility to respiratory infections of animals exposed to ozone. .I. Infi~c.
Dis.
103, 145
149.
