ENVIRONMENTAL
RESEARCH
15,
100-107
Embryotoxicity Automotive DAVID
(1978)
of Irradiated and Nonirradiated Exhaust and Carbon Monoxide J.
HOFFMAN
Received
AND
KIRBY
February
I.
CAMPBELL
19, 1977
This study was undertaken to compare the effects on chick embryogenesis of continuous exposure to diluted unreacted and photochemically reacted automotive exhaust with exposure to comparable levels of carbon monoxide. From Day 1 through 14 days of development. survival decreased almost equally in both exhaust groups but no gross anomalies were apparent in surviving embryos. An equivalent level of carbon monoxide had considerably less effect on survival than did exhaust and did not result in any gross anomalies. Exhaust and carbon monoxide exposure groups exhibited decreased embryonic weight, hematocrit. and feather formation and increased heart/body weight ratio, liver discoloration. and increased serum GPT activity. Carbon monoxide exposure resulted in increased spleen weight. Irradiated exhaust had a less pronounced effect than unreacted exhaust with respect to several parameters including serum enzymes. Differing effects of exposure to nonirradiated versus irradiated exhaust appear to be associated with components other than carbon monoxide. Cumulative effects of these components could explain the variations in biological response between the two exhaust atmospheres.
INTRODUCTION
It has become increasingly apparent that there is a need to evaluate the manner in which gaseous atmospheric pollutants may influence and interfere with the development of vertebrate embryos. In mammalian systems, variables are sometimes encountered due to various physiological and metabolic measures of protection and interaction afforded by the maternal organism to the developing fetus. This is not the case in oviparous vertebrates where development is extramaternal. For this reason, the developing avian embryo has repeatedly demonstrated its sensitivity to both qualitative and quantitative manipulations of its gaseous environment. Such studies have involved exposures to pollutant gases including carbon monoxide (Baker and Tumasonis, 1972: McGrath and Moffa, 1972), methane (Dugnat. 1963, and nitrous oxide (Rector and Eastwood, 1964), as well as variations in oxygen and carbon dioxide (Cruz and Romanoff, 1944; Grabowski and Paar, 1958; Romanoff, 1972; Hoffman, 1975). In the present study investigations were undertaken to compare the effects on avian development of unreacted and photochemically reacted automotive exhaust emissions with those effects due to comparable levels of carbon monoxide alone. Evaluations were made with respect to embryonic and organ development as well as to a number of serum enzymes. Elevations in the activity of these enzymes in chick embryos have been demonstrated in hypoxia, carbon monoxide, and irradiation studies (Lindy and Rajasalmi, 1966; Roberts, 1968: Baker and Tumasonis, 1972; Wendt and Just, 1975). 100 0013-9351/78/0151-0100$02.00/0 Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
EMBRYOTOXICITY
OF
MATERIALS
AND
101
EXHAUS-i
METHODS
The exhaust emission generation system used in these studies consisted of a 1975 Ford’ 400 CID production model engine with the catalyst removed. The engine was cycled continuously (24 hours/day) on an engine dynamometer using the seven-mode modified California cycle. The fuel used was Indolene, an unleaded motor fuel compounded by the American Oil Company as a reference fuel and commonly used for research since it is highly consistent in composition. Thiophene was added to the fuel to give a sulfur content of 0.1% by weight which is an enhanced level but still within the sulfur range of market fuels. Emissions were diluted with filtered conditioned air at a dilution ratio of 10.4 to 1 exhaust (Table 1). A portion of the diluted exhaust was distributed directly to exposure chambers (nonirradiated). Another portion was passed through uvvisible light irradiation chambers, providing photochemically reacted exhaust for the “irradiated-exhaust” chambers. The complete facility has been described in detail elsewhere (Hinners rt crl., 1968: Burkart et al.. 1974). Carbon monoxide (Union Carbide CP grade) from a gas cylinder was mixed with clean air to give a concentration equivalent to that in the exhaust atmopheres and was metered into the carbon monoxide exposure chamber. Diluted auto exhaust, carbon monoxide, or clean-air atmospheres were supplied to exposure chambers continuously at the rate of 15 air changes per hour. Nitrogen dioxide was calculated as the difference between NO, and nitric oxide (NO). Gaseous emission components in exposure chamber atmospheres were measured either automatically or on a periodic basis using the following methods: carbon E,~posure
system.
Test Pollutant Exhaust
dilution
Untreated
ratio
CO (ppm) THC (ppm as methane) NO (ppm) NO, Hydrocarbon ( ppm) Methane Nonmethane aliphatics Aldehydes (ppm) SO, dilution tube 3.88 (ppm) Ammonia (ppm) Particulate (mgim3) Total. dilution tube 0.48 Sulfate. dilution tube 0.21 Sulfuric acid
’ Mention vironmental
of commercial products Protection Agency.
exhaust
chamber Irradiated
exhaust
CO control
10.411 228 59 7.7 4.1
10.411 218 52 8.9 5.2
4.48 5.06 0.84 2.86 0.014
3.43 5.15 0.89 2.76 0.016
-
0.66 0. I9
0.72 0.09 -
-
-
does
atmosphere
not necessarily
constitute
endorsement
230 -
-
by the U.S.
En-
102
HOFFMAN
AND
CAMPBELL
monoxide (CO), NDIR spectroscopy; total hydrocarbons (THC), flame ionization detection; nitrogen oxides NO,, chemiluminescence. The following components were measured periodically: C, -C, hydrocarbons, gas chromatography; total aldehydes, calorimetry; sulfur dioxide (SO,), chemiluminescence and calorimetry amonia; calorimetry; ozone, chemiluminescence. Particulates were determined daily using the following methods: total mass, filtration gravimetry; sulfates, colorimetry; acidity, pH measurement. Particle size was determined periodically but less frequently by use of the Andersen multistage particle-fractioning sampler. E.uperimental design. Fertile White Leghorn eggs obtained from a commercial hatchery were placed randomly in incubators within each of four exposure chambers on the same day of procurement. The exposures included (1) purified air (controls), (2) nonirradiated exhaust, (3) irradiated exhaust, and (4) carbon monoxide. The incubators were maintained at 38”C, 60% relative humidity, and chamber atmospheres were circulated through them 24 hours/day. Eggs were exposed from Day 1 of development and were candled on Day 3 at which time infertile eggs were removed and 40 viable ones per exposure group were permitted to remain. On Day 7 eggs were candled again and dead embryos were removed and examined. 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 recorded. Hematocrits were obtained by centrifuging blood samples in capillary tubes under standardized conditions. After centrifugation, the serum was removed and glucose levels and enzyme activities were measured. Serum glucose was determined using the o-toluidine method of Feteris (1965). Serum lactic dehydrogenase (LDH) activity was measured by increased absorbance of NADH at 340 nm (Amador et al., 1963), serum glutamic oxalacetic transaminase (,SGOT), by the rate of disappearance of NADH at 340 nm (Henry et al., 1960), and serum glutamic pyruvic transaminase (SGPT), by a modified Karmen method of Henry et al. (1960). Weight and biochemical data were statistically analyzed using one-way analysis of variance and Duncan’s multiple-range test, and survival rates were examined using the chi-square test. RESULTS
By 14 days of development survival rates were lower in the nonirradiatedexhaust, irradiated-exhaust, and carbon monoxide groups compared to the cleanair group (Table 2). Candling revealed survival in these groups to be affected from Days 7 to 14. The survival rate was approximately 50% in the groups exposed to untreated exhaust and irradiated exhaust. However, equivalent levels of carbon monoxide alone had a much less pronounced effect on survival, decreasing it by 30%. Statistical examination (chi square) of the survival data revealed the effects of carbon monoxide to be of borderline significance (0.10 < P < 0.15) but those of
EMBRYOTOXICITY
EFFECTS
Treatment Clean air Nonirradiated exhaust Irradiated exhaust Carbon monoxide
Number exposed
OF
TABLE 2 OF UNTREATED Exkr.~us~. ~RRADIATLD EXHAUST, CARBON MONOXIDE ON AVIAN DEVELOPMENT Survival rate (7C)
Organ: Embryonic weight
40
86
8.7
i
40
45*
6.2**
40
48*
40
70””
” Values are expressed :* P < 0.05. **P < 0.01.
103
EXHAUSI
as the mean
body
weight
Heart 0.97
AND
ratio
(c/o body
Liver
weight) Spleen
0.95
?z 0.12
1.89
2 0.24
0,045
2 0.007
+ 1.07
1.36”*
t 0.15
2.32**
2 0.15
0.048
? 0.011
7.4**
-t 1.23
1.21**
+ 0.12
2.11*
k 0.24
0.050
k 0.005
6.7””
+ 0.97
1.28”*
+ 0.16
2.13**
z!z 0.21 0.063”
2 0.008
+ SD.
exhaust to be significant (P < 0.05). Embryonic weight was significantly lower (15-290/o) than controls by 14 days of development in nonirradiated-exhaust, irradiated-exhaust, and carbon monoxide exposure groups, with untreated exhaust resulting in the most severe decrease. Reduced growth rate and feather formation were apparent in all three exposure groups but were the most evident in the nonirradiated exhaust group (Fig. 1). Surviving embryos did not exhibit any gross anatomic defects. Alterations in organ weights were apparent after all three exposures (Table 2). Exhaust exposure and carbon monoxide resulted in significant increases (27-43%) in grams of heart/l00 g body weight ratios, with the greatest increase occurring in the nonirradiated-exhaust exposure group. When absolute values of heart weights (g) were compared, differences approached the 0.05 level of significance (P = 0.057). Significantly higher (12-23%) 1iver:body weight ratios occurred and liver discoloration was apparent in all three exposure groups; when absolute liver weight values (grams) were compared, there were no significant differences. A significantly higher spleen:body weight ratio was found in the carbon monoxide exposure group but not in either exhaust exposure group; this difference remained significant when absolute spleen weights were compared. During Days lo- 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 reflective of stage-retarded growth of the entire embryo, but could be due to either organ hypertrophy or selective stunting of the embryo. Hematocrit and serum enzyme activities in exhaust and carbon monoxide groups also differed significantly from controls (Table 3). Nonirradiated-exhaust, irradiated-exhaust, and carbon monoxide exposure groups exhibited hematocrits which were 19 to 23% lower than those of the clean-air group. Serum glucose concentrations appeared slightly elevated in the exposed groups but did not differ significantly from controls. Serum LDH activities were slightly but not significantly higher in the nonirradiated-exhaust and carbon monoxide exposure groups
HOFFMAN
FIG. B ottom ff :ather
1. Fourteen-day chick embryos. Top-row row chicks are from the untreated-exhaust formation. x 1.3.
AND
CAMPBELL
chicks are from the clean-air exposure chamber. exposure chamber, showing decreased growth and
EhlBRYOTOXICITY
OF
TABLE EFFPCTS
OF US.I-REATED CAKBOS
Number exposed
Treatment Clean air Nonirradiated
Irradiated exhaust Carbon monoxide * Values are expressed -:rfJ < 0.01.
ESHAI.S~, DLVELOPM.~ I
IRRADIATED
OS AVIAS
Serum Glucose Hematocrit
mg/lOO
.\sn
GOT (1.U.)
LDH (1.U.)
ml
GPT 1I.U.)
26 ?I*’
t 3.2’ + 3.4
186 193
2 t
19 I5
263 286
231 k ,4
68 91“‘:
I
11 1-o
24
40
+
14
zj’fs’
-’ 6
40 40
TJO’“” * 2.5 10”~; 2 3.0
187 192
t t
37 17
‘22’‘83
+ 35 240
78 64
21021 214
28’
r6 23
40
exhaust
3
EXH~L~ST,
MONOXIDE
105
EXHAUST
as the mean
k SD.
and were significantly lower (16%) in the irradiated-exhaust group. Serum GOT activities were significantly higher (35%) in the nonirradiated exhaust group and were somewhat elevated but not significantly so in the irradiated-exhaust group. Serum GPT activities were significantly higher in the nonirradiated-exhaust and carbon monoxide exposure groups (25 and 40%, respectively). DISCUSSION
Nonirradiated-exhaust. irradiated-exhaust. and carbon monoxide exposures were not equivalent in magnitude of embryotoxicity with respect to a number of parameters. Carbon monoxide exposure had considerably less effect on embryonic survival than did untreated- and irradiated-exhaust exposures and did not increase serum GOT activity. However, carbon monoxide exposure did result in decreased embryonic weight and feather formation. increased heart weight, liver discoloration, decreased hematocrit, and elevated serum GPT activity, which were also evident in exhaust-exposed groups. A significant increase in spleen weight occurred which was not apparent after either exhaust exposure. Irradiated exhaust was found to affect survival rate and hematocrit to almost the same extent as untreated exhaust. but decreased embryonic weight, increased heart weight, and increased serum enzyme activities (GOT and GPT) were less pronounced. Irradiated-exhaust exposure resulted in serum LDH activity that was significantly lower than the control level. Cumulative effects of overall composition of the exhaust with respect to different pollutants such as hydrocarbon concentration and composition, SO, levels, and other constituents including unknown ones may account for minor variations seen between irradiated- and nonirradiated-exhaust groups (Table 1). However, ozone and oxidants, measured periodically but not shown in this table, did not appear within the limits of detection and therefore were probably not a determining factor. Additional study would be required to resolve these questions. All living embryos were free of gross teratogenic anomalies after carbon monoxide and exhaust exposures. Baker and Tumasonis (1972) reported that carbon monoxide above 425 ppm was required to produce developmental defects, and McGrath and Moffa (1972) reported that 200 ppm of carbon monoxide interfered with survival, which is in agreement with the present study. 111rrtero exposure to carbon monoxide at 180 ppm has been reported to cause decreased birth
106
HOFFMAN
AND
CAMPBELL
weight and decreased neonatal survival in rabbits (Astrup et crl., 1972), and similar results have been noted at 230 ppm in rats (Hoffman, unpublished data). A number of the embryotoxic effects observed in this study have been reported to occur following hypoxia in chick embryos. It has been established that chick embryos of less than 15 days of development are unable to increase their erythrocyte count and have exhibited decreased hematocrit in response to hypoxic stress (Jalavisto et al.. 1965: Grabowski and Schroeder, 1968; Ackerman, 1970). Enlarged hearts and intraembryonic blood vessels have been reported following hypoxia in chick embryos (Grabowski and Paar, 1958; Grabowski and Schroeder. 1968). Increased spleen weight has been reported in various species of adult mammals exposed to carbon monoxide (Van Liere and Stickney, 1963). Elevated serum activities of intracellular enzymes characteristic of certain organs have indicated cellular leakage associated with probable cellular injury following such stresses as irradiation, hypoxia, and carbon monoxide exposure during avian embryogenesis (Lindy and Rajasalmi. 1966; Roberts, 1968: Baker and Tumasonis, 1972; Wendt and Just, 1975). The observed increases in serum GOT and GPT activities and heart and liver weight, along with hepatic discoloration, are consistent with the possibility of cellular damage in these organs. 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 low LDH level in the irradiated-exhaust group reflects an exhaust-induced delay in normal development . ACKNOWLEDGMENTS The authors thank Joan Mattox. laboratory assistance and You-Yen Engineering and Analytical Chemistry
Isaac Washington, and Suzanne Hoffman for their excellent Yang for statistical analyses. We also thank personnel of the Sections for atmosphere generation and aerometry.
REFERENCES Ackerman. N. R. (1970). The physiological effects of hypoxia on the erythrocytes of the chick embryo. De\,elop. Bid. 23, 310-323. Amador. E., Dorfman. L. E.. and Wacker. W. E. C. (1963). Serum lactic dehydrogenase activity: An analytical assessment of current assays. C/in. Chetn. 9, 391-9. Astrup, P., Olsen. H. M., Trolle. D.. and Kjeldsen, K. (1972). Effect of moderate carbon-monoxide exposure on fetal development. LNpt(,et 2, l220- 1222. Baker. F. D., and Tumasonis. C. F. (1972). Carbon monoxide and avian embryogenesis. Arch. En~,i~-WI. Health 24, 53-61. Burkart, J. K.. Hinners, R. G.. and Malanchuk. M. (1974). “Catalytic Converter Exhaust Emissions.” Proceedings, Air Pollution Control Association. 67th Annual Meeting. Denver. Cola.. 74-129. Cruz, S. R., and Romanoff. A. L. (1944). Effect of oxygen concentration on the development of the chick embryo. Physiol. Zoo/. 17, 1844 187. Dugnat. J. M. (1965). Action du gaz d‘e’clairage et du gaz de lacq sur le systeme nerveux de I’embryon d’oiseau. C. R. Sot. Bid. 159, 1545-1547. Feteris. W. A. (1965). A serum glucose method without protein precipitation. Amer. J. Med. Tec~hnol. 31, 17-21. Grabowski. C. T., and Paar. J. A. (1958). The teratogenic effects of graded doses of hypoxia on the chick embryo. Amer. J. Anal. 103, 313-348. Grabowski, C. T.. and Schroeder. R. L. (1968). Time lapse photographic study of chick embryos exposed to teratogenic doses of hypoxia. J. Emlqd. Exp. Morphol. 19, 347-362. Henry, R.. Chiamori, N.. Golub, 0. J.. and Berkman. S. (1960). Revised spectrophotometric methods
EMBRYOTOXICITY
OF
107
EXHACST
for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase. lactic acid dehydrogenase. Amer. J. C/in. Pn~hrd. 34, 381-398. Hinners. R. G.. Burkart, J. K.. and Punte. C. L. (1968). Animal inhalation exposure chambers. fi'u~~ir-or~.
Hoffman.
Hrtrlth
16,
D. J. (197.5).
T~wto/o~~
12,
and AK/I.
19-206.
Physiological
effects
of hypoxia
and trypan
blue
in 17.day
chick
embryos.
57-60.
Jalavisto. E.. Kuurinka I., and Kyllastinen, M. (1965). Responsiveness of the erythron to variations of oxygen tension in the chick embryo and young chicken. Plr~.siol. Sctrjrd. 63, 4799486. Lindy. S.. and Rajasalmi. (1966). Lactate dehydrogenase isozymes of chick embryo: Response to variations of ambient oxygen tension. Scirrtc-cl 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 P~~//ut. Co~rv. ALS. 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. A~r.sthr.sio/ogy 25, 109. Roberts, C. M. ( 1968). The response of the early chick embryo heart to anoxia. J. Cc//. P/I~.~;~I/. 68, 263 -268. Romanoff. A. L. (1967). Chemistry of the embryonic organ tissues. 1,1 “Biochemistry of the Avian Embryo” (A. L. Romanoff and A. J. Romanoff, Eds.). pp. 53- 117. Wiley. New York. Romanoff. A. L. (1977). Atmospheric changes. I/r “Pathogenesis of the Avian Embryo” (A. L. Romanoff and A. J. Romanoff. Eds.), pp. 90-102. Wiley. New York. Van Liere. E. J., and Stickney. J. C. (1963). Acclimitization to hypoxia. In “Hypoxia.” pp. 199-701. University of Chicago Press. Chicago, III. Wendt. T. E.. and Just. H. (1975). Untersuchungen der Glutamat-Oxalazetat-Transaminase bei bestrahlten and unbestrahlten Huhnerembryonen. Sf~cc/z/enthc,rnpil, 149, 333-337.
RESEARCH
15,
100-107
Embryotoxicity Automotive DAVID
(1978)
of Irradiated and Nonirradiated Exhaust and Carbon Monoxide J.
HOFFMAN
Received
AND
KIRBY
February
I.
CAMPBELL
19, 1977
This study was undertaken to compare the effects on chick embryogenesis of continuous exposure to diluted unreacted and photochemically reacted automotive exhaust with exposure to comparable levels of carbon monoxide. From Day 1 through 14 days of development. survival decreased almost equally in both exhaust groups but no gross anomalies were apparent in surviving embryos. An equivalent level of carbon monoxide had considerably less effect on survival than did exhaust and did not result in any gross anomalies. Exhaust and carbon monoxide exposure groups exhibited decreased embryonic weight, hematocrit. and feather formation and increased heart/body weight ratio, liver discoloration. and increased serum GPT activity. Carbon monoxide exposure resulted in increased spleen weight. Irradiated exhaust had a less pronounced effect than unreacted exhaust with respect to several parameters including serum enzymes. Differing effects of exposure to nonirradiated versus irradiated exhaust appear to be associated with components other than carbon monoxide. Cumulative effects of these components could explain the variations in biological response between the two exhaust atmospheres.
INTRODUCTION
It has become increasingly apparent that there is a need to evaluate the manner in which gaseous atmospheric pollutants may influence and interfere with the development of vertebrate embryos. In mammalian systems, variables are sometimes encountered due to various physiological and metabolic measures of protection and interaction afforded by the maternal organism to the developing fetus. This is not the case in oviparous vertebrates where development is extramaternal. For this reason, the developing avian embryo has repeatedly demonstrated its sensitivity to both qualitative and quantitative manipulations of its gaseous environment. Such studies have involved exposures to pollutant gases including carbon monoxide (Baker and Tumasonis, 1972: McGrath and Moffa, 1972), methane (Dugnat. 1963, and nitrous oxide (Rector and Eastwood, 1964), as well as variations in oxygen and carbon dioxide (Cruz and Romanoff, 1944; Grabowski and Paar, 1958; Romanoff, 1972; Hoffman, 1975). In the present study investigations were undertaken to compare the effects on avian development of unreacted and photochemically reacted automotive exhaust emissions with those effects due to comparable levels of carbon monoxide alone. Evaluations were made with respect to embryonic and organ development as well as to a number of serum enzymes. Elevations in the activity of these enzymes in chick embryos have been demonstrated in hypoxia, carbon monoxide, and irradiation studies (Lindy and Rajasalmi, 1966; Roberts, 1968: Baker and Tumasonis, 1972; Wendt and Just, 1975). 100 0013-9351/78/0151-0100$02.00/0 Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
EMBRYOTOXICITY
OF
MATERIALS
AND
101
EXHAUS-i
METHODS
The exhaust emission generation system used in these studies consisted of a 1975 Ford’ 400 CID production model engine with the catalyst removed. The engine was cycled continuously (24 hours/day) on an engine dynamometer using the seven-mode modified California cycle. The fuel used was Indolene, an unleaded motor fuel compounded by the American Oil Company as a reference fuel and commonly used for research since it is highly consistent in composition. Thiophene was added to the fuel to give a sulfur content of 0.1% by weight which is an enhanced level but still within the sulfur range of market fuels. Emissions were diluted with filtered conditioned air at a dilution ratio of 10.4 to 1 exhaust (Table 1). A portion of the diluted exhaust was distributed directly to exposure chambers (nonirradiated). Another portion was passed through uvvisible light irradiation chambers, providing photochemically reacted exhaust for the “irradiated-exhaust” chambers. The complete facility has been described in detail elsewhere (Hinners rt crl., 1968: Burkart et al.. 1974). Carbon monoxide (Union Carbide CP grade) from a gas cylinder was mixed with clean air to give a concentration equivalent to that in the exhaust atmopheres and was metered into the carbon monoxide exposure chamber. Diluted auto exhaust, carbon monoxide, or clean-air atmospheres were supplied to exposure chambers continuously at the rate of 15 air changes per hour. Nitrogen dioxide was calculated as the difference between NO, and nitric oxide (NO). Gaseous emission components in exposure chamber atmospheres were measured either automatically or on a periodic basis using the following methods: carbon E,~posure
system.
Test Pollutant Exhaust
dilution
Untreated
ratio
CO (ppm) THC (ppm as methane) NO (ppm) NO, Hydrocarbon ( ppm) Methane Nonmethane aliphatics Aldehydes (ppm) SO, dilution tube 3.88 (ppm) Ammonia (ppm) Particulate (mgim3) Total. dilution tube 0.48 Sulfate. dilution tube 0.21 Sulfuric acid
’ Mention vironmental
of commercial products Protection Agency.
exhaust
chamber Irradiated
exhaust
CO control
10.411 228 59 7.7 4.1
10.411 218 52 8.9 5.2
4.48 5.06 0.84 2.86 0.014
3.43 5.15 0.89 2.76 0.016
-
0.66 0. I9
0.72 0.09 -
-
-
does
atmosphere
not necessarily
constitute
endorsement
230 -
-
by the U.S.
En-
102
HOFFMAN
AND
CAMPBELL
monoxide (CO), NDIR spectroscopy; total hydrocarbons (THC), flame ionization detection; nitrogen oxides NO,, chemiluminescence. The following components were measured periodically: C, -C, hydrocarbons, gas chromatography; total aldehydes, calorimetry; sulfur dioxide (SO,), chemiluminescence and calorimetry amonia; calorimetry; ozone, chemiluminescence. Particulates were determined daily using the following methods: total mass, filtration gravimetry; sulfates, colorimetry; acidity, pH measurement. Particle size was determined periodically but less frequently by use of the Andersen multistage particle-fractioning sampler. E.uperimental design. Fertile White Leghorn eggs obtained from a commercial hatchery were placed randomly in incubators within each of four exposure chambers on the same day of procurement. The exposures included (1) purified air (controls), (2) nonirradiated exhaust, (3) irradiated exhaust, and (4) carbon monoxide. The incubators were maintained at 38”C, 60% relative humidity, and chamber atmospheres were circulated through them 24 hours/day. Eggs were exposed from Day 1 of development and were candled on Day 3 at which time infertile eggs were removed and 40 viable ones per exposure group were permitted to remain. On Day 7 eggs were candled again and dead embryos were removed and examined. 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 recorded. Hematocrits were obtained by centrifuging blood samples in capillary tubes under standardized conditions. After centrifugation, the serum was removed and glucose levels and enzyme activities were measured. Serum glucose was determined using the o-toluidine method of Feteris (1965). Serum lactic dehydrogenase (LDH) activity was measured by increased absorbance of NADH at 340 nm (Amador et al., 1963), serum glutamic oxalacetic transaminase (,SGOT), by the rate of disappearance of NADH at 340 nm (Henry et al., 1960), and serum glutamic pyruvic transaminase (SGPT), by a modified Karmen method of Henry et al. (1960). Weight and biochemical data were statistically analyzed using one-way analysis of variance and Duncan’s multiple-range test, and survival rates were examined using the chi-square test. RESULTS
By 14 days of development survival rates were lower in the nonirradiatedexhaust, irradiated-exhaust, and carbon monoxide groups compared to the cleanair group (Table 2). Candling revealed survival in these groups to be affected from Days 7 to 14. The survival rate was approximately 50% in the groups exposed to untreated exhaust and irradiated exhaust. However, equivalent levels of carbon monoxide alone had a much less pronounced effect on survival, decreasing it by 30%. Statistical examination (chi square) of the survival data revealed the effects of carbon monoxide to be of borderline significance (0.10 < P < 0.15) but those of
EMBRYOTOXICITY
EFFECTS
Treatment Clean air Nonirradiated exhaust Irradiated exhaust Carbon monoxide
Number exposed
OF
TABLE 2 OF UNTREATED Exkr.~us~. ~RRADIATLD EXHAUST, CARBON MONOXIDE ON AVIAN DEVELOPMENT Survival rate (7C)
Organ: Embryonic weight
40
86
8.7
i
40
45*
6.2**
40
48*
40
70””
” Values are expressed :* P < 0.05. **P < 0.01.
103
EXHAUSI
as the mean
body
weight
Heart 0.97
AND
ratio
(c/o body
Liver
weight) Spleen
0.95
?z 0.12
1.89
2 0.24
0,045
2 0.007
+ 1.07
1.36”*
t 0.15
2.32**
2 0.15
0.048
? 0.011
7.4**
-t 1.23
1.21**
+ 0.12
2.11*
k 0.24
0.050
k 0.005
6.7””
+ 0.97
1.28”*
+ 0.16
2.13**
z!z 0.21 0.063”
2 0.008
+ SD.
exhaust to be significant (P < 0.05). Embryonic weight was significantly lower (15-290/o) than controls by 14 days of development in nonirradiated-exhaust, irradiated-exhaust, and carbon monoxide exposure groups, with untreated exhaust resulting in the most severe decrease. Reduced growth rate and feather formation were apparent in all three exposure groups but were the most evident in the nonirradiated exhaust group (Fig. 1). Surviving embryos did not exhibit any gross anatomic defects. Alterations in organ weights were apparent after all three exposures (Table 2). Exhaust exposure and carbon monoxide resulted in significant increases (27-43%) in grams of heart/l00 g body weight ratios, with the greatest increase occurring in the nonirradiated-exhaust exposure group. When absolute values of heart weights (g) were compared, differences approached the 0.05 level of significance (P = 0.057). Significantly higher (12-23%) 1iver:body weight ratios occurred and liver discoloration was apparent in all three exposure groups; when absolute liver weight values (grams) were compared, there were no significant differences. A significantly higher spleen:body weight ratio was found in the carbon monoxide exposure group but not in either exhaust exposure group; this difference remained significant when absolute spleen weights were compared. During Days lo- 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 reflective of stage-retarded growth of the entire embryo, but could be due to either organ hypertrophy or selective stunting of the embryo. Hematocrit and serum enzyme activities in exhaust and carbon monoxide groups also differed significantly from controls (Table 3). Nonirradiated-exhaust, irradiated-exhaust, and carbon monoxide exposure groups exhibited hematocrits which were 19 to 23% lower than those of the clean-air group. Serum glucose concentrations appeared slightly elevated in the exposed groups but did not differ significantly from controls. Serum LDH activities were slightly but not significantly higher in the nonirradiated-exhaust and carbon monoxide exposure groups
HOFFMAN
FIG. B ottom ff :ather
1. Fourteen-day chick embryos. Top-row row chicks are from the untreated-exhaust formation. x 1.3.
AND
CAMPBELL
chicks are from the clean-air exposure chamber. exposure chamber, showing decreased growth and
EhlBRYOTOXICITY
OF
TABLE EFFPCTS
OF US.I-REATED CAKBOS
Number exposed
Treatment Clean air Nonirradiated
Irradiated exhaust Carbon monoxide * Values are expressed -:rfJ < 0.01.
ESHAI.S~, DLVELOPM.~ I
IRRADIATED
OS AVIAS
Serum Glucose Hematocrit
mg/lOO
.\sn
GOT (1.U.)
LDH (1.U.)
ml
GPT 1I.U.)
26 ?I*’
t 3.2’ + 3.4
186 193
2 t
19 I5
263 286
231 k ,4
68 91“‘:
I
11 1-o
24
40
+
14
zj’fs’
-’ 6
40 40
TJO’“” * 2.5 10”~; 2 3.0
187 192
t t
37 17
‘22’‘83
+ 35 240
78 64
21021 214
28’
r6 23
40
exhaust
3
EXH~L~ST,
MONOXIDE
105
EXHAUST
as the mean
k SD.
and were significantly lower (16%) in the irradiated-exhaust group. Serum GOT activities were significantly higher (35%) in the nonirradiated exhaust group and were somewhat elevated but not significantly so in the irradiated-exhaust group. Serum GPT activities were significantly higher in the nonirradiated-exhaust and carbon monoxide exposure groups (25 and 40%, respectively). DISCUSSION
Nonirradiated-exhaust. irradiated-exhaust. and carbon monoxide exposures were not equivalent in magnitude of embryotoxicity with respect to a number of parameters. Carbon monoxide exposure had considerably less effect on embryonic survival than did untreated- and irradiated-exhaust exposures and did not increase serum GOT activity. However, carbon monoxide exposure did result in decreased embryonic weight and feather formation. increased heart weight, liver discoloration, decreased hematocrit, and elevated serum GPT activity, which were also evident in exhaust-exposed groups. A significant increase in spleen weight occurred which was not apparent after either exhaust exposure. Irradiated exhaust was found to affect survival rate and hematocrit to almost the same extent as untreated exhaust. but decreased embryonic weight, increased heart weight, and increased serum enzyme activities (GOT and GPT) were less pronounced. Irradiated-exhaust exposure resulted in serum LDH activity that was significantly lower than the control level. Cumulative effects of overall composition of the exhaust with respect to different pollutants such as hydrocarbon concentration and composition, SO, levels, and other constituents including unknown ones may account for minor variations seen between irradiated- and nonirradiated-exhaust groups (Table 1). However, ozone and oxidants, measured periodically but not shown in this table, did not appear within the limits of detection and therefore were probably not a determining factor. Additional study would be required to resolve these questions. All living embryos were free of gross teratogenic anomalies after carbon monoxide and exhaust exposures. Baker and Tumasonis (1972) reported that carbon monoxide above 425 ppm was required to produce developmental defects, and McGrath and Moffa (1972) reported that 200 ppm of carbon monoxide interfered with survival, which is in agreement with the present study. 111rrtero exposure to carbon monoxide at 180 ppm has been reported to cause decreased birth
106
HOFFMAN
AND
CAMPBELL
weight and decreased neonatal survival in rabbits (Astrup et crl., 1972), and similar results have been noted at 230 ppm in rats (Hoffman, unpublished data). A number of the embryotoxic effects observed in this study have been reported to occur following hypoxia in chick embryos. It has been established that chick embryos of less than 15 days of development are unable to increase their erythrocyte count and have exhibited decreased hematocrit in response to hypoxic stress (Jalavisto et al.. 1965: Grabowski and Schroeder, 1968; Ackerman, 1970). Enlarged hearts and intraembryonic blood vessels have been reported following hypoxia in chick embryos (Grabowski and Paar, 1958; Grabowski and Schroeder. 1968). Increased spleen weight has been reported in various species of adult mammals exposed to carbon monoxide (Van Liere and Stickney, 1963). Elevated serum activities of intracellular enzymes characteristic of certain organs have indicated cellular leakage associated with probable cellular injury following such stresses as irradiation, hypoxia, and carbon monoxide exposure during avian embryogenesis (Lindy and Rajasalmi. 1966; Roberts, 1968: Baker and Tumasonis, 1972; Wendt and Just, 1975). The observed increases in serum GOT and GPT activities and heart and liver weight, along with hepatic discoloration, are consistent with the possibility of cellular damage in these organs. 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 low LDH level in the irradiated-exhaust group reflects an exhaust-induced delay in normal development . ACKNOWLEDGMENTS The authors thank Joan Mattox. laboratory assistance and You-Yen Engineering and Analytical Chemistry
Isaac Washington, and Suzanne Hoffman for their excellent Yang for statistical analyses. We also thank personnel of the Sections for atmosphere generation and aerometry.
REFERENCES Ackerman. N. R. (1970). The physiological effects of hypoxia on the erythrocytes of the chick embryo. De\,elop. Bid. 23, 310-323. Amador. E., Dorfman. L. E.. and Wacker. W. E. C. (1963). Serum lactic dehydrogenase activity: An analytical assessment of current assays. C/in. Chetn. 9, 391-9. Astrup, P., Olsen. H. M., Trolle. D.. and Kjeldsen, K. (1972). Effect of moderate carbon-monoxide exposure on fetal development. LNpt(,et 2, l220- 1222. Baker. F. D., and Tumasonis. C. F. (1972). Carbon monoxide and avian embryogenesis. Arch. En~,i~-WI. Health 24, 53-61. Burkart, J. K.. Hinners, R. G.. and Malanchuk. M. (1974). “Catalytic Converter Exhaust Emissions.” Proceedings, Air Pollution Control Association. 67th Annual Meeting. Denver. Cola.. 74-129. Cruz, S. R., and Romanoff. A. L. (1944). Effect of oxygen concentration on the development of the chick embryo. Physiol. Zoo/. 17, 1844 187. Dugnat. J. M. (1965). Action du gaz d‘e’clairage et du gaz de lacq sur le systeme nerveux de I’embryon d’oiseau. C. R. Sot. Bid. 159, 1545-1547. Feteris. W. A. (1965). A serum glucose method without protein precipitation. Amer. J. Med. Tec~hnol. 31, 17-21. Grabowski. C. T., and Paar. J. A. (1958). The teratogenic effects of graded doses of hypoxia on the chick embryo. Amer. J. Anal. 103, 313-348. Grabowski, C. T.. and Schroeder. R. L. (1968). Time lapse photographic study of chick embryos exposed to teratogenic doses of hypoxia. J. Emlqd. Exp. Morphol. 19, 347-362. Henry, R.. Chiamori, N.. Golub, 0. J.. and Berkman. S. (1960). Revised spectrophotometric methods
EMBRYOTOXICITY
OF
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EXHACST
for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase. lactic acid dehydrogenase. Amer. J. C/in. Pn~hrd. 34, 381-398. Hinners. R. G.. Burkart, J. K.. and Punte. C. L. (1968). Animal inhalation exposure chambers. fi'u~~ir-or~.
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T~wto/o~~
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and AK/I.
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effects
of hypoxia
and trypan
blue
in 17.day
chick
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57-60.
Jalavisto. E.. Kuurinka I., and Kyllastinen, M. (1965). Responsiveness of the erythron to variations of oxygen tension in the chick embryo and young chicken. Plr~.siol. Sctrjrd. 63, 4799486. Lindy. S.. and Rajasalmi. (1966). Lactate dehydrogenase isozymes of chick embryo: Response to variations of ambient oxygen tension. Scirrtc-cl 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 P~~//ut. Co~rv. ALS. 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. A~r.sthr.sio/ogy 25, 109. Roberts, C. M. ( 1968). The response of the early chick embryo heart to anoxia. J. Cc//. P/I~.~;~I/. 68, 263 -268. Romanoff. A. L. (1967). Chemistry of the embryonic organ tissues. 1,1 “Biochemistry of the Avian Embryo” (A. L. Romanoff and A. J. Romanoff, Eds.). pp. 53- 117. Wiley. New York. Romanoff. A. L. (1977). Atmospheric changes. I/r “Pathogenesis of the Avian Embryo” (A. L. Romanoff and A. J. Romanoff. Eds.), pp. 90-102. Wiley. New York. Van Liere. E. J., and Stickney. J. C. (1963). Acclimitization to hypoxia. In “Hypoxia.” pp. 199-701. University of Chicago Press. Chicago, III. Wendt. T. E.. and Just. H. (1975). Untersuchungen der Glutamat-Oxalazetat-Transaminase bei bestrahlten and unbestrahlten Huhnerembryonen. Sf~cc/z/enthc,rnpil, 149, 333-337.
