Journal of Toxicology and Environmental Health
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Embryotoxicity of irradiated and nonirradiated catalytic converter‐treated automotive exhaust David J. Hoffman & Kirby I. Campbell To cite this article: David J. Hoffman & Kirby I. Campbell (1977) Embryotoxicity of irradiated and nonirradiated catalytic converter‐treated automotive exhaust, Journal of Toxicology and Environmental Health, 3:4, 705-712, DOI: 10.1080/15287397709529605 To link to this article: http://dx.doi.org/10.1080/15287397709529605
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Date: 05 November 2015, At: 15:09
EMBRYOTOXICITY OF IRRADIATED AND NONIRRADIATED CATALYTIC CONVERTERTREATED AUTOMOTIVE EXHAUST David J. Hoffman, Kirby I. Campbell
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Health Effects Research Laboratory, Environmental Research Center, U.S. Environmental Protection Agency, Cincinnati, Ohio
This study was undertaken to examine the relative embryotoxicity in chick embryos of photochemically reacted and unreacted diluted automotive exhaust emissions from a system equipped with a catalytic converter. Clean air controls and H2SO4 aerosol controls equivalent in concentration to those found in the catalytic exhaust atmosphere were also studied. From day 1 through day 14 of development, continuous exposure to nonirradiated exhaust resulted in decreased survival, lowered embryonic weight, a small increase in heartlbody weight ratio, and altered hematocrit and serum enzyme activities (LDH and GOT). Irradiated exhaust had little effect on survival or on embryonic weight but resulted in a higher liver/body weight ratio as well as altered hematocrit and serum enzyme activities. Interactions or cumulative effects of different compositions of exhaust atmospheres may play a role in differing biological responses between unreacted and irradiated exhaust. Sulfuric acid aerosol had a minimal effect on survival and resulted in only a slight decrease in embryonic weight and serum LDH activity, with no other apparent effects. In previous studies where the catalytic converter was not used, more pronounced effects on survival, increased heart/body weight ratio, elevated serum GPT activity, and liver discoloration were observed. Thus, the introduction of an oxidizing catalytic converter appeared to alleviate some but not all of the embryotoxic effects of automotive exhaust.
INTRODUCTION The developing avian embryo has been shown to be sensitive to both qualitative and quantitative manipulations of its gaseous environment. Such studies have involved exposures to pollutant gases including automotive exhaust emissions (Hoffman and Campbell, 1977), carbon monoxide (Baker and Tumasonis, 1972; McGrath and Moffa, 1972; Hoffman and Campbell, 1977), methane (Dugnat, 1965), and nitrous oxide (Rector and Eastwood, 1964) as well as to variations in oxygen and carbon dioxide (Cruz and Romanoff, 1944; Grabowski and Paar, 1958; Romanoff, 1972; Hoffman, 1975). The authors thank Joan Mattox, Isaac Washington, and Suzanne Hoffman for their excellent laboratory assistance and You-Yen Yang for statistical analyses. We also thank personnel of the Engineering and Analytical Chemistry Sections for atmosphere generation and aerometry. Requests for reprints should be sent to David J. Hoffman or Kirby I. Campbell, Health Effects Research Laboratory, Environmental Protection Agency, 26 West St. Clair Street, Cincinnati, Ohio 45268.
705 Journal of Toxicology and Environmental Health, 3:705-712,1977 Copyright © 1977 by Hemisphere Publishing Corporation
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There is evidence that a number of substances including sulfuric acid and other related sulfur compounds produced by automotive engines equipped with a catalytic converter are toxic. Amdur (1969), in a review of the literature, pointed out a number of these aspects, indicating, for example, that H 2 SO 4 is a greater pulmonary irritant than SO2 and that sensitive individuals may show a response to levels of SO2 as low as 1 ppm. Previous studies in this laboratory have dealt with the effects of carbon monoxide and automotive exhaust from engines that were not equipped with the catalytic converter (Hoffman and Campbell, 1977). The present study was initiated to examine the relative embryotoxicity of irradiated and nonirradiated exhaust emissions from the catalyst-equipped system in developing chick embryos. In addition, an atmosphere of H 2 SO 4 aerosol at a level equivalent to that found in the catalytic exhaust was used as an added control. Evaluations were made with respect to embryonic and organ development as well as to a number of serum enzymes. These enzymes have demonstrated activity level elevations after hypoxia, carbon monoxide, and irradiation exposure in developing chick embryos (Lindy and Rajasalmi, 1966; Roberts, 1968; Baker and Tumasonis, 1972; Wendt and Just, 1975; Hoffman and Campbell, 1977).
METHOD Exposure System The exhaust emission generation system used in these studies consisted of a 1975 Ford 1 400 CID production model engine equipped with an Engelhard/American Lava substrate monolith catalyst in the exhaust pipe system and was operated continuously (24 hr/day) on an engine dynamometer using the seven-mode modified California cycle and high-sulfur fuel (Indolene, with added thiophene to result in a sulfur content of 1,000 ppm). Emissions were diluted with filtered conditioned air at a dilution ratio of 10.9 parts air to 1 part exhaust (Table 1). A portion of the diluted exhaust was distributed directly to exposure chambers (nonirradiated). Another portion was passed through ultraviolet-visible light irradiation chambers, providing photochemically reacted exhaust in the "irradiated exhaust" chambers. The complete facility has been described in detail elsewhere (Hinners et al., 1968; Burkhart et al., 1974). Sulfuric acid aerosol control chambers were used to differentiate the effects due to H 2 SO 4 alone from the effects due to the total exhaust emissions. A Retec X-70/N nebulizer was used to aerosolize an aqueous solution of the acid. The droplet size was reduced, before the aerosol was introduced into the exposure chamber, by passing the fresh aerosol through a heated glass 1 Mention of commercial products does not necessarily constitute endorsement by the U.S. Environmental Protection Agency.
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TABLE 1. Average Concentration of Exposure Atmosphere Components
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Test chamber atmosphere Pollutant
Untreated exhaust
Irradiated exhaust
HjSO4 control
Exhaust dilution ratio CO, ppm THC, ppm (as methane) NO, ppm NO2, ppm Hydrocarbon, ppm: Methane Nonmethane, aliphatics Aldehydes, ppm SO2,ppm (dilution tube, 2.04) Ammonia, ppm Paniculate, mg/m3: Total (dilution tube, 15.37) Sulfate (dilution tube, 7.61) Sulfuric acid (dilution tube, 7.50)
10.9/1 29.4 16.3 12.73 0.00
10.9/1 25.1 13.6 11.08 0.42
-
5.20 1.28 0.24 1.45
3.85 1.45 0.19 1.40
— -
0.03
0.02
-
15.00
13.94
14.37
6.58
6.09
6.44
5.21
5.54
6.06
tube. The final acid concentration (mg/m 3 ) and particle size (/urn) in the chamber atmosphere were controlled by varying (1) the solution concentration in the nebulizer, (2) the aerosol generating pressure, (3) the heat input into the heating tube, and (4) the volume flow rate of diluting air carrying the aerosol mist into the exposure chamber. Diluted auto exhaust, H 2 SO 4 aerosol, or clean air atmospheres were supplied to exposure chambers continuously at the rate of 15 air changes per hour. A few important gaseous emission components in exposure chamber atmospheres were measured automatically and on a frequent periodic basis using the following methods: carbon monoxide (CO), NDIR spectroscopy; total hydrocarbons (THC), flame ionization detection; and nitrogen oxides (NO X ), chemiluminescence. Also measured periodically were Q - Q hydrocarbons, gas chromatography; total aldehydes, colorimetry; SO 2 , chemiluminescence and colorimetry; ammonia, colorimetry; and ozone, chemiluminescence. Nitrogen dioxide (NO2) was calculated as the difference between total NOX and nitric oxide (NO). Particulates were determined daily by using the following methods: total mass, filtration gravimetry; sulfates, colorimetry; and acidity, pH measurement. Particle size was determined periodically but less frequently by use of the Anderson multistage particle fractioning sampler. The size range of H 2 SO 4 particles in the exhaust and H 2 SO 4 control chambers was 0.20-0.30 //m mass median aerodynamic diameter (MMAD).
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Experimental Design Forty fertile White Leghorn eggs obtained from a commercial hatchery were assigned randomly to incubators within each of four exposure chambers on the day of procurement. Exposures included (1) purified air (controls), (2) nonirradiated exhaust, (3) irradiated exhaust, and (4) H 2 SO 4 aerosol at a level approximately equivalent to that in the exhaust atmospheres (6.5 mg/m 3 ). The incubators were maintained at 38°C and 60% relative humidity, and chamber atmospheres circulated through them. Eggs were exposed from day 1 of development and were candled on days 2 and 7, and infertile ones were discarded on day 2, at which time 40 viable eggs per treatment group were permitted to remain. All remaining eggs were opened on day 14 of exposure. A small section of the chorioallantoic membrane was cut and carefully hooked over the edge of the shell opening. An exposed chorioallantoic artery was nicked to permit filling a nonheparinized capillary tube. Care was taken not to contaminate the blood with other embryonic fluids by adequately draining the area before taking the sample. Embryos were excised from the fetal membranes, blotted, and then weighed. An examination for gross developmental anomalies was made and heart, liver, and spleen weights were measured. Hematocrits were obtained by centrifuging blood samples in capillary tubes under standardized conditions. After centrifugation and separation of serum, glucose levels, serum lactic dehydrogenase (LDH), serum glutamic oxalacetic transaminase (GOT), and serum glutamic pyruvic transaminase (GPT) activities were measured. Serum glucose was determined using the o-toluidine method of Feteris (1965). Serum LDH activity was measured by increased absorbancc of NADH at 340 nm (Amador et al., 1963), serum GOT by the rate of disappearance of NADH at 340 nm (Henry et al., 1960), and serum GPT by a modified Karmen method of Henry et al. (1960). Data were compared statistically using one-way analysis of variance and Duncan's multiple range tests. Survival rates were compared by the chi-square test. RESULTS By day 14 of development the survival rate in the nonirradiated exhaust exposure group was 28% lower than that in the clean air group (Table 2). Irradiated exhaust did not have any effect on survival and equivalent levels of H 2 SO 4 aerosol resulted in survival that was only 11% below the clean air level. Chi-square analysis of these survival rates indicated that only the effect of untreated exhaust was statistically significant. Candling showed survival to be affected from day 7 to day 14. Embryonic weight was 35% lower in the nonirradiated exhaust exposure group and 12% lower in the H 2 SO 4 aerosol exposure group, but not
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TABLE 2. Effects of Nonirradiated and Irradiated Catalytic Exhaust and of H 2 SO 4 Aerosol on Avian Development
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Treatment Clean air Nonirradiated exhaust Irradiated exhaust H 2 SO 4 aerosol
Organ/body weight ratio" (g/100g)
Survival rate
Embryonic weight 0
(%)
(g)
40
85
8.6 ± 0.88
0.97 ± 0.10
2.03 ±0.14
0.051 ± 0.008
40
61C
5.6 ± 0.48 6
1.09 ± 0.14 C
1.93 ± 0.11
0.053 ± 0.005
40 40
88 76
8.3 ± 1.5 7.6 ± 0.75 6
0.97 ±0.16 0.96 ± 0 . 1 8
2.23 ± 0.22 c 2.01 ± 0.21
0.052 ± 0.007 0.056 ± 0.006
No. exposed
Heart
Liver
Spleen
"Values are means ± SD. "Significant a t p < 0.01. c Significant at p < 0.05.
significantly lower in the irradiated exhaust group. Reduced growth and feather formation was apparent in the nonirradiated exhaust group. Surviving embryos did not exhibit any gross abnormalities in any of the experimental groups. A small but significant increase (12%) in the heart/body weight ratio was apparent in the nonirradiated exhaust exposure group. When absolute values of heart weights (g) were compared, this difference was not significant. A significantly higher (10%) liver/body weight ratio occurred in the irradiated exhaust exposure group. When absolute values of liver weights (g) were compared, this difference was not significant. During days 10-15 of chick embryogenesis the heart/body weight ratio remains somewhat constant and the liver/body weight ratio is increasing very slightly (Romanoff, 1967). Therefore an increase in heart or liver/ body weight ratio is probably not indicative of stage-retarded growth of the entire embryo, but could be due to either hypertrophy of these organs or selective stunting of the embryo. Hematocrit values in both nonirradiated and irradiated exhaust groups were significantly lower than those in controls by 21 and 7%, respectively (Table 3). Serum glucose concentrations in the nonirradiated exhaust group were somewhat although not significantly lower than in controls. Serum LDH activity levels were significantly lower in the untreated exhaust, irradiated exhaust, and H 2 SO 4 aerosol exposure groups (36, 12, and 20%, respectively). Serum GOT activities were significantly higher in the untreated and irradiated exhaust groups by 55 and 35%, respectively, and were slightly but not significantly higher in the H 2 SO 4 exposure group. Serum GPT activities appeared slightly but not significantly higher in all three exposure groups.
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TABLE 3. Effects of Nonirradiated and Irradiated Catalytic Exhaust and of H2SO4 Aerosol on Avian Development
Treatment
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Clean air Nonirradiated exhaust Irradiated exhaust H2SO4 aerosol
No. exposed
Hematocrit0 (%)
Serum glucose0 (mg/100ml)
LDH0
(i.u.)
GOT0 (I.U.)
GPT° (i.u.)
58 ± 6
18+3
40
28 ± 2.5
193 ± 20
240+16
40
22 ± 3.4°
184 ± 23
144 ± 36°
106 ± 2 3 °
21 ± 5
40 40
26 + 3.0 c 27 + 3.9
194 ± 18 197 ± 16
211 ±26 C 181 ± 2 5 °
77 ± 8 ° 62 ± 9
22 + 4 20+4
"Values are means ± SD. Significant at p < 0.01. Significant at p < 0.05.
DISCUSSION Nonirradiated catalytic exhaust, irradiated catalytic exhaust, and H 2 SO 4 aerosol exposures differed in degree of embryotoxicity with respect to several parameters. Irradiated exhaust did not significantly affect survival rate or embryonic weight, both of which were significantly lower after exposure to untreated exhaust. Reduced growth and feather formation were apparent in the untreated exhaust exposure group. A small but significant increase in heart/body weight ratio occurred in the untreated exhaust exposure group, and a significantly higher liver/body weight ratio was apparent in the irradiated exhaust exposure group, but absolute organ weights did not differ significantly in either group. No organ or body weight effects were noted in the H 2 SO 4 aerosol group. Differences seen in hematocrit and serum enzyme activities (LDH and GOT) were more pronounced in the untreated exhaust exposure group than in the irradiated exhaust exposure group. Cumulative effects due to differences in exhaust composition with respect to certain measured, and perhaps unmeasured, constituents might account for variations seen between nonirradiated and irradiated exhaust groups. Total hydrocarbon was greater in the nonirradiated exhaust group, but NO2 was present in the irradiated exhaust group. Variations in individual hydrocarbons as well as possible synergistic interactions may be additional sources of differences between these two groups. Sulfuric acid aerosol had a minimal but not statistically significant effect on survival and resulted in a slight decrease in embryonic weight. Sulfuric acid aerosol did not affect hematocrit. Serum LDH was significantly lower but serum GOT was not affected by H 2 SO 4 aerosol exposure. Aerosol particles were determined to be in the size range 0.20-0.3 /im MMAD. This should present no problem of entry past the
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711
calcium carbonate layer of the shell, which is pierced by many pores 40-50 fim in diameter (Balinsky, 1970). It has been established that chick embryos at less than 15 days of development are unable to increase their erythrocyte count and have exhibited decreased hematocrit in response to hypoxic stress and CO exposure (jalavisto et al., 1965; Grabowski and Schroeder, 1968; Ackerman, 1970; Hoffman and Campbell, 1977). Hence the observed decreases in hematocrit found in the present study in 14-day-old embryos would be in keeping with these findings. Elevated serum activities of intracellular enzymes characteristic of certain organs have demonstrated cellular leakage, probably associated with cellular injury following such stresses as irradiation, hypoxia, and CO exposure during avian embryogenesis (Lindy and Rajasalmi, 1966; Roberts, 1968; Baker and Tumasonis, 1972; Wendt and Just, 1975; Hoffman and Campbell, 1977). The observed increase in serum GOT activity in the present study in combination with some increase in heart and liver weight ratios could be indicative of some cellular damage. Serum LDH activity levels normally increase rapidly over this interval of development (12-16 days) (Baker and Tumasonis, 1972). Thus, it is conceivable that the lower LDH levels seen in both catalytic exhaust exposure groups and the H 2 SO 4 aerosol group could reflect a delay in normal development. In previous studies, chick embryos were exposed to exhaust that was not modified by the catalytic converter (Hoffman and Campbell, 1977). More pronounced exhaust effects on embryonic survival, organ weights, liver discoloration, and elevated serum GPT activity levels were noted when the catalytic converter was not present. However, in the present study, even with the catalytic converter, decreased embryonic weight, decreased hematocrit, and elevated serum GOT activity levels were observed. Thus application of the catalytic converter appears to have alleviated some but not all of the embryotoxic effects of automotive exhaust. The incorporation of the oxidative catalyst into the exhaust system resulted in large reductions of CO, total hydrocarbon, and various individual organic compounds, but also resulted in a considerable increase in total particulate and H 2 SO 4 .
REFERENCES Ackerman, N. R. 1970. The physiological effects of hypoxia on the erythrocytes of the chick embryo. Dev. Biol. 23:310-323. Amador, E., Dorfman, L. E. and Wacker, W. E. C. 1963. Serum lactic dehydrogenase activity: An analytical assessment of current assays. Clin. Chem. 9:391-399. Amdur, M. O. 1969. Toxicologic appraisal of particulate matter, oxides of sulfur, and sulfuric acid. J. Air Pollut. Control Assoc. 19:638-644. Astrup, P., Olsen, H. M., Trolle, D. and Kjeldsen, K. 1972. Effect of moderate carbon-monoxide exposure on fetal development. Lancet 2:1220-1222.
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Baker, F. D. and Tumasonis, C. F. 1972. Carbon monoxide and avian embryogenesis. Arch. Environ. Health 24:53-61. Balinsky, B. I. 1970. Oogenesis. In An introduction to embryology, pp. 98-100. Philadelphia: Saunders. Burkhart, J. K., Hinners, R. G. and Malanchuk, M. 1974. Catalytic converter exhaust emissions. Proc. Air Pollut. Control Assoc. 67th Annu. Meet., Denver. 74-129. Cruz, S. R. and Romanoff, A. L. 1944. Effect of oxygen concentration on the development of the chick embryo. Physiol. Zool. 17:184-187. Dugnat, J. M. 1965. Action du gaz d'eclairage et du gaz de lacq sur le systeme nerveux de I'ambryon d'Oiseau. C. R. Seances Soc. Biol. Paris 159:1545-1547. Feteris, W. A. 1965. A serum glucose method without protein precipitation. Am. J. Med. Technol. 31:17-21. Grabowski, C. T. and Paar, J. A. 1958. The teratogenic effects of graded doses of hypoxia on the chick embryo. Am. J. Anat. 103:313-348. Grabowski, C. T. and Schroeder, R. O. 1968. Time lapse photographic study of chick embryos exposed to teratogenic doses of hypoxia. J. Embryol. Exp. Morphol. 19:347-362. Henry, R., Chiamori, N., Golub, O. J. and Berkman, S. 1960. Revised spectrophotometric methods for determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am. J. Clin. Pathol. 34:381-398. Hinners, R. G., Burkart, J. K. and Punte, C. L. 1968. Animal inhalation exposure chambers. Arch. Environ. Health 16:19-206. Hoffman, D. J. 1975. Physiological effects of hypoxia and trypan blue in 17-day chick embryos. Teratology 12:57-60. Hoffman, D. J. and Campbell, K. I. 1977. Embryotoxicity of irradiated and nonirradiated automotive exhaust and carbon monoxide. Environ. Res. In press. Jalavisto, E., Kuurinka, I. and Kyllastinen, M. 1965. Responsiveness of the erythron to variations of oxygen tension in the chick embryo and young chicken. Physiol. Scand. 63:479-486. Lindy, S. and Rajasalmi, M. 1966. Lactate dehydrogenase isozymes of chick embryo: Response to variations of ambient oxygen tension. Science 153:1401-1403. McGrath, J. J. and Moffa, J. V. 1972. System to evaluate the influence of chronic exposure to CO on the hatching eggs of the White Leghorn chicken. J. Air. Pollut. Control Assoc. 22:123. Rector, G. H. M. and Eastwood, D. W. 1964. The effects of an atmosphere of nitrous oxide and oxygen on the incubating chick. Anesthesiology 25:109. Roberts, C. M. 1968. The response of the early chick embryo heart to anoxia. J. Cell. Physiol. 68:263-268. Romanoff, A. L. 1967. Chemistry of the embryonic organ tissues. In Biochemistry of the avian embryo, pp. 53-117. New York: Wiley. Romanoff, A. L. 1972. Atmospheric changes. In Pathogenesis of the avian embryo, pp. 90-102. New York: Wiley. Van Liere, E. J. and Sickney, J. C. 1963. Acclimitization to hypoxia. In Hypoxia, pp. 199-201. Chicago: Univ. of Chicago Press. Wendt, T. E. and Just, H. 1975. Untersuchungen der glutamat-oxylazetat-transaminase bei bestrahlten und unbestrahlten huhnerembryonen. Strahlentherapie 149:333-337. Received February 11, 1977 Accepted June 24, 1977