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Adult and Embryo Responses to Organophosphate Pesticide: Azodrin
Adult and Embryo Responses to Organophosphate Pesticide: Azodrin c. B. SCHOM Department of Biological Sciences, Brock University, St. Catharines, Ontario, L2S 3A1 U. K. ABBOTT and N. E. WALKER Department of Avian Sciences, University of California, Davis, California 95616 (Received for publication April 17, 1978)
INTRODUCTION Both embryo and adult birds are affected by toxic compounds, in particular by the organophosphate pesticides (Abbott et al., 1964, Schom et al, 1972). Dursban, an organophosphate pesticide, interferes with normal embryogenesis in chickens, Chukar Partridge, and Bobwhite quail (Schom et al, 1973), as does Azodrin (Schom et al., 1972 and Schom and Abbott, 1977) and Malathion (Roger, 1967 and Walker, 1971). Azodrin has also been reported toxic to adult birds; however, there appeared to be a species difference in maximum acceptable toxicant concentrations (MATC) (Schom et al., 1972). The relationship of the adult and embryonic responses has yet to be determined, as does the relationship of one species response to that of another. In this study the effect of Azodrin on the adult and the embryo as well as the relationship between adult and embryonic responses have been examined. MATERIALS AND METHODS Feeding Trials. Females with known production records from the University of California White Leghorn Chicken, Chukar Partridge, and Bobwhite Quail flocks were chosen. In general all birds were two years old. The Chukar Partridge, one male to two females (three birds to a cage), and the Bobwhite Quail, one male to two females (nine birds to a cage), were housed with both sexes exposed to Azodrin in the feed. As gamebird males died they were replaced so that males were always present. The males, in 1979 Poultry Sci 58:60-66
60
separate individual cages and untreated, were milked immediately before the biweekly artificial insemination of the females. The Azodrin fed to the birds was mixed to the desired concentration in a rolling mill—ppm in the species standard breeder feed. Only chickens had the level of Azodrin fed changed after 14 days on experiment. All other trials were held at the same level until the experiment was terminated. For replications and levels of Azodrin fed see Table 1. Eggs from the feeding trials were collected daily and stored at 12.8 C no more than seven days before being set in a Jamesway 252 incubator. The eggs were candled at 3 days of incubation with infertile eggs and those with dead embryos removed, opened, and examined, as were all eggs from which chicks did not hatch. During the experiment all adult birds were weighed once every 14 days, and at the end of the experiment all surviving birds were checked before disposal. A few eggs were removed at random and frozen (about a dozen from each trial). They were stored at —17.8 C until residue analysis could be done by the Residue Laboratory, Department of Toxicology, Davis, California. The standard flame photometric analysis was done on a pooled sample of eggs. Egg Injection. The University of California, Davis, gamebird flocks were used to supply the fertile eggs for the yolk injection trials. The flocks were managed as described by Schom and Abbott (1974). Chicken eggs were shipped in weekly from the Donsing Hatchery, Reo
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ABSTRACT Azodrin was applied to adult and embryo chickens, Chukar Partridge, and Bobwhite Quail. Chronic exposure of adult birds to Azodrin mixed in their feed indicated that no a priori predictions could be made about one species based on the results of another; each had a different no effect (MACT) level. The chickens were between 25 and 100 ppm, the Chukar Partridge 5 and 25 ppm, and the Bobwhite quail less than 1.25 ppm. The chicken adults were most resistant, and the quail were least resistant to chronic exposure to Azodrin. Yolk-injected Azodrin caused the embryos of all three species to develop abnormally. The chicken and Chukar embryos developed a generalized achondroplasia, the quail were amuscular, only. In general, the 3 day quail embryos were most resistant to injected Azodrin and the chicken embryo least resistant. The relationship between adult and embryo response was negative.
AVIAN RESPONSE TO ORGANOPHOSPHATE
61
TABLE 1. The levels, number of birds, and length of time Azodrin fed to the different species Chicken PPM fed 100 25 5
Chukar
Bobwhite
Number of females
Number of days
Number of females
Number of days
Number of females
Number of days
9 9
first 14 days remaining 98 days
10 10 10
42 70 112
12 12 12 12 12 12 12
14 98 112 112 42 105 112
25 1.25
Egg Spraying. E m b r y o n a t e d eggs, candled at either 3 days or 10 days of i n c u b a t i o n t o elimin a t e infertile and dead e m b r y o s , were t a k e n t o an isolated location and sprayed with pesticide (2.1 fig/cm2 of egg surface) before being placed in a separate forced draft i n c u b a t o r . All egg were h a t c h e d in t h e incubator a n d t h e chicks checked for general condition, configuration, and behavior prior t o disposal. All u n h a t c h e d eggs were o p e n e d a n d t h e e m b r y o classified. All statistical analyses followed p r o c e d u r e s outlined in Snedecor and Cochrane ( 1 9 6 7 ) or Duncan (1965).
RESULTS Feeding Trials. M a x i m u m acceptable t o x i cant c o n c e n t r a t i o n s (MATC), n o effects levels,
TABLE 2. Level of Azodrin fed and corresponding percent hen day production and percent mortality^
Chicken (9 females) PPM fed
Prod
100 to day 14 100 day 0 to end experiment 25 day 15 to day 112 25 day 0 to end experiment 5 2.5 1.25 .0
17
Mort
Chukar (10 females) Mort
Prod
Quail (12 females) Prod
Mort
(%> 60 32
65
No statistically significant differences.
100
11
11
23 32
50 0
30
11
0.1 12 29 18 41
75 91 66 50 8
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Linda, California. All eggs were held for n o m o r e t h a n seven days before being incubated three days, removed and candled, infertile and dead e m b r y o s removed, and y o l k injected. T h e doses used were 1.5 m g / c h i c k e n egg; .75 m g / Chukar Partridge egg; and .5 m g / B o b w h i t e Quail egg. All injections were m a d e using .9% saline as t h e vehicle with .5 ml for chicken and .25 ml for game birds. All solutions were sterilized for filtration, . 4 4 N millipore filter. T h e eggs were removed at 8, 10, 12, 16, and 18 days of i n c u b a t i o n for all species plus 21 days for chickens and 2 4 days for t h e game birds. T h e eggs were o p e n e d and t h e e m b r y o s were weighed and classified before being preserved in Bouin's fixative. Tissues were sect i o n e d and stained with H a e m o t o x y l i n and Eosin for histological studies.
SCHOM ET AL.
62
Chukar Control Azodrin
f
Quail Control A z o d r i n (5 ppm) Death X
(5 ppm)
Chicken Control A z o d r i n 1 0 0 ppm - d a y 0 t o 28 D e a t h X 2 5 p p . - flay 29 t o 112 •
5
600
56 Days on
Days o n
Experiment
84
112
Experiment
D a y s on
Experiment
differed for the three species. The chicken hens could survive at least 14 days on 100 ppm Azodrin, although with low hen day production (Table 2) following a switch to 25 ppm on the 15 th day they recovered both losses in body weight (Fig. 1) and hen day production (Table 2, Fig. 2). The Chukar Partridge and Bobwhite Quail levels of mortality and hen day production were unacceptable at 25 ppm; therefore the trials were terminated early. The Chukar functioned normally when fed five ppm, giving a MATC between 5 and 25 ppm chronic level for 112 days. The quail were still above their MATC at 1.25 ppm (Table 2, day died marked as an x on Figure 1). The Chukar body weights were unaffected by 5 ppm Azodrin and the chicken by 25 ppm, but quail were depressed by 1.25 ppm (Fig. 1). This quail body weight depression can be accounted for in part by a reduction in food consumed (Table 3). Similar measurements were not available for chicken and Chukar because the feeder design permitted too much wastage.
Chukar Control Azodrin Chicken Control Azodrin
0
28
This also meant that the total amount of pesticide consumed by the birds was not known, just the amount per unit feed presented to the birds. All weights reported for females only. Hen day production of control and 5 ppm treated Chukar did not differ. Chicken hen day production was depressed by 100 ppm in feed but recovered on 25 ppm reaching control levels by the 84th day on trial. Quail levels were never near the control at 5 ppm (Fig. 2). Chicken and Chukar embryogenesis was unaffected by Azodrin in the feed. In general, however, quail percentage hatch was lower for all levels fed (Table 4). In fact, fertility fell as did egg production (Fig. 2, Table 4), probably due to male sterility (Fig. 3). The one quail trial with high overall fertility was probably due to males inseminating the female before the Azodrin was fed, i.e., while the males were still fertile, as high fertilities were recorded early in the trial but fell by the end (Fig. 3). This is an acceptable explanation as quail females remain fertile for several weeks after
Quail Control Azodrin
( 5 ppm) •
(5 ppm)
— 100 ppm d a y 0 t o 28 25 ppm d a y 29 t o 1 1 2
56 Days o n
84 Experiment
112
28
56 Days o n
84
112
Experiment
FIG. 2. The hen day production (eggs/hen/day) for control and Azodrin-treated birds.
Days on
Experiment
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FIG. 1. The body weights of control and Azodrin treated birds, 9 chicken, 10 Chukar, and 12 quail females. No males included.
AVIAN RESPONSE TO ORGANOPHOSPHATE
63
TABLE 3. Food consumption (quail) at different levels ofAzodrin in the diet Quail
Number birds
g Food/bird/day
Az 0 ppm Az 5 ppm Az 2.5 ppm Az 1.25 ppm
5 15 15 15
19.9 16.3 18.7 17.2
112
FIG. 3. Bobwhite quail fertility recorded for trials with the indicated level of Azodrin in the diet.
(Fig. 4). There were histological differences between treated and control chicken embryos but not for quail embryos. Only quail and chicken embryos were examined. The muscle histology was normal in both species, the cartilage appear normal in quail. The chicken embryo limb cartilage was abnormal, giving an appearance of being disorganized with fewer trabiculae; in fact, the cartilage was very similar to cartilage described by Hall (1977) following treatment with thallium sulphate. The skin of treated chicken embryos was very much thinner than the control;
TABLE 4. Embryonic response of a random sample from bens fed Azodrin3-
Number females
Dead
Number eggs laid Inf.
1-7
8-14
143 125
34.5 32.0
5.6 2.4
2.8 1.6
7.0 6.4
1 5 - 20 2 1 - 2 4
-
pip
Hatched
(%)
Chicken control (0 ppm) 100 ppm to day 14 25 ppm to day 112
9 9
Chukar control (0 ppm)
10
177
17.5
5.6
2.0
4.5
5 ppm Azodrin
10
239
20.1
3.8
1.7
4.6
Quail control (0 ppm)
12
581
29.8
1
1.4
6.5
2.6
7.7
50.9
5 ppm 2.5 ppm 1.25 ppm
12 12 12
114 198 100
21.0 70.1 61
2.6 1 1
0.5
8.0 4 4
8.8 2.5 8
12.2 2.5
47.4 18.7 26
No statistically significant differences.
126
1.4 2.4
46.2 55.2
5.6
15.3
49.7
2.1
9.2
58.2
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initial insemination (Schom and Abbott, 1974), and leads to the conclusion that low fertility in quail trials is probably due to the males response to Azodrin treatment rather than an effect on females' reproduction or the embryos. This conclusion is a possibility as no detectable residues were found in any eggs tested from birds in the feeding trials (Table 5); therefore, none should be present to act on the developing embryo. Yolk Infection. The chicken and Chukar embryos responded by developing a generalized micromelia, parrot beak syndromes, and achondroplasia. The quail, however, were amuscular only, that is they seemed to resist Azodrin effects on cartilage. The symptoms were all visible by the eighth day and continued through the 18th day with a higher dose causing more severe response. In general, the chicken embryo was severely affected, with Chukar somewhat less, and quail showing the least morphologic change as previously reported by Schom et al. (1972) and Schom and Abbott (1977). This was true of the growth rate of the embryos also
SCHOM ET AL.
64
TABLE 5. Residues in eggs collected from birds fed pesticide (Azodrin) Species
Treatment
Level fed
Level in egg
Chicken
Azodrin Control
25 ppm 0
>.01 ppm >.01 ppm
Chukar
Azodrin Control
5 ppm 0
>.01 ppm >.01 ppm
Quail
Azodrin Control
5 ppm 0
>.01 ppm >.01 ppm
DISCUSSION The three species adult females have different MACT's based on their hen day production, weight loss and mortality. The chicken's was between 25 and 100 ppm in feed, the Chukar's 5 and 25 ppm, and the quail's less than 1.25 ppm. The embryos from birds fed with the Azodrin showed no abnormalities. This was not surprising as no Azodrin residues were found in
7.0
7.0
5.0
5.0 3.0
2.0
2.0
s t;
3.0
10
4V-
11
U
16
Days of Ir.cubation
18
20
11
14
16
18
Days of I n c u b a t i o n
Days of
Incubation
FIG. 4. The log growth curves for embryos from eggs of chicken, chukar, and quail treated with 1.5 Mg> -75 Mg, and .5 jug Azodrin per egg, respectively.
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in general, the dermis and epidermis were reduced (Fig. 5). Quail skin was unaffected. Egg Spraying. There was no apparent difference between embryos hatched from eggs sprayed with Azodrin and control eggs for either chicken or quail chicks. The large number of quail embryos which did not hatch (DNH) suggests that something in the handling procedure was faulty, therefore making interpretation difficult (Table 6).
the eggs of birds fed with the drug. So exposure of the female to the pesticide in feed represents no danger to humans eating the eggs, unlike Dursban (Schom et al, 1973). The yolk injected chicken embryos were more severely affected than the yolk injected quail embryos, the reverse of the adult response to chronic levels of Azodrin (Table 7). Therefore, the most resistant adults have the least resistant embryos, and visa versa, indicating a negative relationship between adult response to chronic levels and embryo response to laboratory injected doses. Predictions, therefore, cannot be made about the magnitude of adults response based on Azodrin injected into the yolk of developing embryos. The yolk injected chicken embryo skin and cartilage was abnormal as was the histology. The quail were amuscular only with normal histology. These symptoms correspond to what Landauer (1975) called cholinomimetic and non-cholinomimetic responses. The only problem here is that Azodrin as an anti-acetylcholinesterase must be a cholinomematic agent as defined by Landauer (1975). To cause the development of two different sets of responses the two species must have at least one very different enzyme that does not always respond the same. This difference in response is not limited to just Azodrin but is common to at least 11 other organophosphate pesticides (Abbott, 1972). It, therefore, may be possible to test the (Moscioni et al., 1977) hypothesis proposing Kynurenine Formamidase as a mechanism for cartilage abnormalities (achondroplasia) by using chicken and quail preparations.
AVIAN RESPONSE TO ORGANOPHOSPHATE
65
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 10, 2015 FIG. 5. 16-day old chicken embryos. Top: metatarsal cartilage. Bottom: back skin, lumbar region. Left: control. Right: treated with 1.5 mg Azodrin at three days of incubation. Note the smaller more closely packed cells in the hyaline cartilage (A), and the irregular shape trabeculae with fewer crossbridges (B). Note also, the reduction in number of cells between the trabeculae in the treated embryos, the more areolar appearance of the back dermis and the thinner epidermis. Hematoxylin and Eosin X100.
66
SCHOM ET AL. TABLE 6. Embryogenic response to Azodrin sprayed on chicken and quail eggs*
Species
Days incubation
Level ng/cm 2
Chicken
10
0 2
1 3
0 2 0 2
86 77 13 14
Quail
3 10
Did not hatch
Hatched, weak 3
Normal
0
29 31
4 7 4 1
90 105 63 80
TABLE 7. Comparison of adult and embryonic response to Azodrin Species
Adult response
Embryonic response
Chicken Chukar Quail
+a ++ +++
+ ++ ++ +
+ severity of response.
REFERENCES Abbott, U. K., 1972. Effect of pesticide and related compounds on several avian species. Sum. Rep. Food Prot. Toxicol. Cent., University of California Davis, CA. Abbott, U. K., R. M. Craig, and K. O. Keith, 1964. Effects of malathion spray on embryonated chicken eggs exposed under field conditions at Tuolume Meadow, Yosemite National Park. Poultry Sci. 4 3 : 1297. Duncan, A. J., 1965. Quality control and industrial statistics. 3rd ed. Richard D. Irwin, Homewood, IL. Hall, B. K., 1977. Thallium induced achondroplasia in chicken embryos and the concept of critical peri-
ods during development. Teratology 15:1—15. Landauer, 1975. Cholinomimetic teratogens: Studies with chicken embryos. Teratoloty 12:125—145. Moscioni, A. D., J. L. Engel, and J. E. Casida, 1977. Kynurenine Formamidase inhibition as a possible mechanism for certain teratogenic effects of organophosphorous and methylcarbamate insecticides in chicken embryos. Biochem. Pharmacol. 26:2251-2258. Roger, J. C , 1967. Mode of teratogenic action of organophosphate esters in hen eggs. Ph.D. dissertation, University of California, Berkeley, CA. Schom, C. B., and U. K. Abbott, 1974. Studies with Bobwhite quail: Reproductive characteristics. Poultry Sci. 53:1860-1865. Schom, C. B., and U. K. Abbott, 1977. Temporal, morphological and genetic responses of avian embryos to Azodrin, an organophosphate insecticide. Teratology 15:81-87. Schom, C. B., U. K. Abbott, and N. E. Walker, 1973. Organophosphate pesticide effects on domestic and game bird species: Dursban. Poultry Sci. 52:2083. Schom, C. B., N. E. Walker, and U. K. Abbott, 1972. Azodrin and its effect on four avian species. Poultry Sci. 51:1860. Snedecor, G. W., and W. G. Cochrane, 1967. Statistical methods. Iowa State University Press, Ames, IA. Walker, N. E., 1971. The effect of Malathion and Mamaoxon on esterase and gross development of chick embryos. Toxic. Appl. Pharmacol. 19:590— 601.
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No statistically significant differences.
INTRODUCTION Both embryo and adult birds are affected by toxic compounds, in particular by the organophosphate pesticides (Abbott et al., 1964, Schom et al, 1972). Dursban, an organophosphate pesticide, interferes with normal embryogenesis in chickens, Chukar Partridge, and Bobwhite quail (Schom et al, 1973), as does Azodrin (Schom et al., 1972 and Schom and Abbott, 1977) and Malathion (Roger, 1967 and Walker, 1971). Azodrin has also been reported toxic to adult birds; however, there appeared to be a species difference in maximum acceptable toxicant concentrations (MATC) (Schom et al., 1972). The relationship of the adult and embryonic responses has yet to be determined, as does the relationship of one species response to that of another. In this study the effect of Azodrin on the adult and the embryo as well as the relationship between adult and embryonic responses have been examined. MATERIALS AND METHODS Feeding Trials. Females with known production records from the University of California White Leghorn Chicken, Chukar Partridge, and Bobwhite Quail flocks were chosen. In general all birds were two years old. The Chukar Partridge, one male to two females (three birds to a cage), and the Bobwhite Quail, one male to two females (nine birds to a cage), were housed with both sexes exposed to Azodrin in the feed. As gamebird males died they were replaced so that males were always present. The males, in 1979 Poultry Sci 58:60-66
60
separate individual cages and untreated, were milked immediately before the biweekly artificial insemination of the females. The Azodrin fed to the birds was mixed to the desired concentration in a rolling mill—ppm in the species standard breeder feed. Only chickens had the level of Azodrin fed changed after 14 days on experiment. All other trials were held at the same level until the experiment was terminated. For replications and levels of Azodrin fed see Table 1. Eggs from the feeding trials were collected daily and stored at 12.8 C no more than seven days before being set in a Jamesway 252 incubator. The eggs were candled at 3 days of incubation with infertile eggs and those with dead embryos removed, opened, and examined, as were all eggs from which chicks did not hatch. During the experiment all adult birds were weighed once every 14 days, and at the end of the experiment all surviving birds were checked before disposal. A few eggs were removed at random and frozen (about a dozen from each trial). They were stored at —17.8 C until residue analysis could be done by the Residue Laboratory, Department of Toxicology, Davis, California. The standard flame photometric analysis was done on a pooled sample of eggs. Egg Injection. The University of California, Davis, gamebird flocks were used to supply the fertile eggs for the yolk injection trials. The flocks were managed as described by Schom and Abbott (1974). Chicken eggs were shipped in weekly from the Donsing Hatchery, Reo
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 10, 2015
ABSTRACT Azodrin was applied to adult and embryo chickens, Chukar Partridge, and Bobwhite Quail. Chronic exposure of adult birds to Azodrin mixed in their feed indicated that no a priori predictions could be made about one species based on the results of another; each had a different no effect (MACT) level. The chickens were between 25 and 100 ppm, the Chukar Partridge 5 and 25 ppm, and the Bobwhite quail less than 1.25 ppm. The chicken adults were most resistant, and the quail were least resistant to chronic exposure to Azodrin. Yolk-injected Azodrin caused the embryos of all three species to develop abnormally. The chicken and Chukar embryos developed a generalized achondroplasia, the quail were amuscular, only. In general, the 3 day quail embryos were most resistant to injected Azodrin and the chicken embryo least resistant. The relationship between adult and embryo response was negative.
AVIAN RESPONSE TO ORGANOPHOSPHATE
61
TABLE 1. The levels, number of birds, and length of time Azodrin fed to the different species Chicken PPM fed 100 25 5
Chukar
Bobwhite
Number of females
Number of days
Number of females
Number of days
Number of females
Number of days
9 9
first 14 days remaining 98 days
10 10 10
42 70 112
12 12 12 12 12 12 12
14 98 112 112 42 105 112
25 1.25
Egg Spraying. E m b r y o n a t e d eggs, candled at either 3 days or 10 days of i n c u b a t i o n t o elimin a t e infertile and dead e m b r y o s , were t a k e n t o an isolated location and sprayed with pesticide (2.1 fig/cm2 of egg surface) before being placed in a separate forced draft i n c u b a t o r . All egg were h a t c h e d in t h e incubator a n d t h e chicks checked for general condition, configuration, and behavior prior t o disposal. All u n h a t c h e d eggs were o p e n e d a n d t h e e m b r y o classified. All statistical analyses followed p r o c e d u r e s outlined in Snedecor and Cochrane ( 1 9 6 7 ) or Duncan (1965).
RESULTS Feeding Trials. M a x i m u m acceptable t o x i cant c o n c e n t r a t i o n s (MATC), n o effects levels,
TABLE 2. Level of Azodrin fed and corresponding percent hen day production and percent mortality^
Chicken (9 females) PPM fed
Prod
100 to day 14 100 day 0 to end experiment 25 day 15 to day 112 25 day 0 to end experiment 5 2.5 1.25 .0
17
Mort
Chukar (10 females) Mort
Prod
Quail (12 females) Prod
Mort
(%> 60 32
65
No statistically significant differences.
100
11
11
23 32
50 0
30
11
0.1 12 29 18 41
75 91 66 50 8
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Linda, California. All eggs were held for n o m o r e t h a n seven days before being incubated three days, removed and candled, infertile and dead e m b r y o s removed, and y o l k injected. T h e doses used were 1.5 m g / c h i c k e n egg; .75 m g / Chukar Partridge egg; and .5 m g / B o b w h i t e Quail egg. All injections were m a d e using .9% saline as t h e vehicle with .5 ml for chicken and .25 ml for game birds. All solutions were sterilized for filtration, . 4 4 N millipore filter. T h e eggs were removed at 8, 10, 12, 16, and 18 days of i n c u b a t i o n for all species plus 21 days for chickens and 2 4 days for t h e game birds. T h e eggs were o p e n e d and t h e e m b r y o s were weighed and classified before being preserved in Bouin's fixative. Tissues were sect i o n e d and stained with H a e m o t o x y l i n and Eosin for histological studies.
SCHOM ET AL.
62
Chukar Control Azodrin
f
Quail Control A z o d r i n (5 ppm) Death X
(5 ppm)
Chicken Control A z o d r i n 1 0 0 ppm - d a y 0 t o 28 D e a t h X 2 5 p p . - flay 29 t o 112 •
5
600
56 Days on
Days o n
Experiment
84
112
Experiment
D a y s on
Experiment
differed for the three species. The chicken hens could survive at least 14 days on 100 ppm Azodrin, although with low hen day production (Table 2) following a switch to 25 ppm on the 15 th day they recovered both losses in body weight (Fig. 1) and hen day production (Table 2, Fig. 2). The Chukar Partridge and Bobwhite Quail levels of mortality and hen day production were unacceptable at 25 ppm; therefore the trials were terminated early. The Chukar functioned normally when fed five ppm, giving a MATC between 5 and 25 ppm chronic level for 112 days. The quail were still above their MATC at 1.25 ppm (Table 2, day died marked as an x on Figure 1). The Chukar body weights were unaffected by 5 ppm Azodrin and the chicken by 25 ppm, but quail were depressed by 1.25 ppm (Fig. 1). This quail body weight depression can be accounted for in part by a reduction in food consumed (Table 3). Similar measurements were not available for chicken and Chukar because the feeder design permitted too much wastage.
Chukar Control Azodrin Chicken Control Azodrin
0
28
This also meant that the total amount of pesticide consumed by the birds was not known, just the amount per unit feed presented to the birds. All weights reported for females only. Hen day production of control and 5 ppm treated Chukar did not differ. Chicken hen day production was depressed by 100 ppm in feed but recovered on 25 ppm reaching control levels by the 84th day on trial. Quail levels were never near the control at 5 ppm (Fig. 2). Chicken and Chukar embryogenesis was unaffected by Azodrin in the feed. In general, however, quail percentage hatch was lower for all levels fed (Table 4). In fact, fertility fell as did egg production (Fig. 2, Table 4), probably due to male sterility (Fig. 3). The one quail trial with high overall fertility was probably due to males inseminating the female before the Azodrin was fed, i.e., while the males were still fertile, as high fertilities were recorded early in the trial but fell by the end (Fig. 3). This is an acceptable explanation as quail females remain fertile for several weeks after
Quail Control Azodrin
( 5 ppm) •
(5 ppm)
— 100 ppm d a y 0 t o 28 25 ppm d a y 29 t o 1 1 2
56 Days o n
84 Experiment
112
28
56 Days o n
84
112
Experiment
FIG. 2. The hen day production (eggs/hen/day) for control and Azodrin-treated birds.
Days on
Experiment
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FIG. 1. The body weights of control and Azodrin treated birds, 9 chicken, 10 Chukar, and 12 quail females. No males included.
AVIAN RESPONSE TO ORGANOPHOSPHATE
63
TABLE 3. Food consumption (quail) at different levels ofAzodrin in the diet Quail
Number birds
g Food/bird/day
Az 0 ppm Az 5 ppm Az 2.5 ppm Az 1.25 ppm
5 15 15 15
19.9 16.3 18.7 17.2
112
FIG. 3. Bobwhite quail fertility recorded for trials with the indicated level of Azodrin in the diet.
(Fig. 4). There were histological differences between treated and control chicken embryos but not for quail embryos. Only quail and chicken embryos were examined. The muscle histology was normal in both species, the cartilage appear normal in quail. The chicken embryo limb cartilage was abnormal, giving an appearance of being disorganized with fewer trabiculae; in fact, the cartilage was very similar to cartilage described by Hall (1977) following treatment with thallium sulphate. The skin of treated chicken embryos was very much thinner than the control;
TABLE 4. Embryonic response of a random sample from bens fed Azodrin3-
Number females
Dead
Number eggs laid Inf.
1-7
8-14
143 125
34.5 32.0
5.6 2.4
2.8 1.6
7.0 6.4
1 5 - 20 2 1 - 2 4
-
pip
Hatched
(%)
Chicken control (0 ppm) 100 ppm to day 14 25 ppm to day 112
9 9
Chukar control (0 ppm)
10
177
17.5
5.6
2.0
4.5
5 ppm Azodrin
10
239
20.1
3.8
1.7
4.6
Quail control (0 ppm)
12
581
29.8
1
1.4
6.5
2.6
7.7
50.9
5 ppm 2.5 ppm 1.25 ppm
12 12 12
114 198 100
21.0 70.1 61
2.6 1 1
0.5
8.0 4 4
8.8 2.5 8
12.2 2.5
47.4 18.7 26
No statistically significant differences.
126
1.4 2.4
46.2 55.2
5.6
15.3
49.7
2.1
9.2
58.2
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initial insemination (Schom and Abbott, 1974), and leads to the conclusion that low fertility in quail trials is probably due to the males response to Azodrin treatment rather than an effect on females' reproduction or the embryos. This conclusion is a possibility as no detectable residues were found in any eggs tested from birds in the feeding trials (Table 5); therefore, none should be present to act on the developing embryo. Yolk Infection. The chicken and Chukar embryos responded by developing a generalized micromelia, parrot beak syndromes, and achondroplasia. The quail, however, were amuscular only, that is they seemed to resist Azodrin effects on cartilage. The symptoms were all visible by the eighth day and continued through the 18th day with a higher dose causing more severe response. In general, the chicken embryo was severely affected, with Chukar somewhat less, and quail showing the least morphologic change as previously reported by Schom et al. (1972) and Schom and Abbott (1977). This was true of the growth rate of the embryos also
SCHOM ET AL.
64
TABLE 5. Residues in eggs collected from birds fed pesticide (Azodrin) Species
Treatment
Level fed
Level in egg
Chicken
Azodrin Control
25 ppm 0
>.01 ppm >.01 ppm
Chukar
Azodrin Control
5 ppm 0
>.01 ppm >.01 ppm
Quail
Azodrin Control
5 ppm 0
>.01 ppm >.01 ppm
DISCUSSION The three species adult females have different MACT's based on their hen day production, weight loss and mortality. The chicken's was between 25 and 100 ppm in feed, the Chukar's 5 and 25 ppm, and the quail's less than 1.25 ppm. The embryos from birds fed with the Azodrin showed no abnormalities. This was not surprising as no Azodrin residues were found in
7.0
7.0
5.0
5.0 3.0
2.0
2.0
s t;
3.0
10
4V-
11
U
16
Days of Ir.cubation
18
20
11
14
16
18
Days of I n c u b a t i o n
Days of
Incubation
FIG. 4. The log growth curves for embryos from eggs of chicken, chukar, and quail treated with 1.5 Mg> -75 Mg, and .5 jug Azodrin per egg, respectively.
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in general, the dermis and epidermis were reduced (Fig. 5). Quail skin was unaffected. Egg Spraying. There was no apparent difference between embryos hatched from eggs sprayed with Azodrin and control eggs for either chicken or quail chicks. The large number of quail embryos which did not hatch (DNH) suggests that something in the handling procedure was faulty, therefore making interpretation difficult (Table 6).
the eggs of birds fed with the drug. So exposure of the female to the pesticide in feed represents no danger to humans eating the eggs, unlike Dursban (Schom et al, 1973). The yolk injected chicken embryos were more severely affected than the yolk injected quail embryos, the reverse of the adult response to chronic levels of Azodrin (Table 7). Therefore, the most resistant adults have the least resistant embryos, and visa versa, indicating a negative relationship between adult response to chronic levels and embryo response to laboratory injected doses. Predictions, therefore, cannot be made about the magnitude of adults response based on Azodrin injected into the yolk of developing embryos. The yolk injected chicken embryo skin and cartilage was abnormal as was the histology. The quail were amuscular only with normal histology. These symptoms correspond to what Landauer (1975) called cholinomimetic and non-cholinomimetic responses. The only problem here is that Azodrin as an anti-acetylcholinesterase must be a cholinomematic agent as defined by Landauer (1975). To cause the development of two different sets of responses the two species must have at least one very different enzyme that does not always respond the same. This difference in response is not limited to just Azodrin but is common to at least 11 other organophosphate pesticides (Abbott, 1972). It, therefore, may be possible to test the (Moscioni et al., 1977) hypothesis proposing Kynurenine Formamidase as a mechanism for cartilage abnormalities (achondroplasia) by using chicken and quail preparations.
AVIAN RESPONSE TO ORGANOPHOSPHATE
65
Downloaded from http://ps.oxfordjournals.org/ at Simon Fraser University on June 10, 2015 FIG. 5. 16-day old chicken embryos. Top: metatarsal cartilage. Bottom: back skin, lumbar region. Left: control. Right: treated with 1.5 mg Azodrin at three days of incubation. Note the smaller more closely packed cells in the hyaline cartilage (A), and the irregular shape trabeculae with fewer crossbridges (B). Note also, the reduction in number of cells between the trabeculae in the treated embryos, the more areolar appearance of the back dermis and the thinner epidermis. Hematoxylin and Eosin X100.
66
SCHOM ET AL. TABLE 6. Embryogenic response to Azodrin sprayed on chicken and quail eggs*
Species
Days incubation
Level ng/cm 2
Chicken
10
0 2
1 3
0 2 0 2
86 77 13 14
Quail
3 10
Did not hatch
Hatched, weak 3
Normal
0
29 31
4 7 4 1
90 105 63 80
TABLE 7. Comparison of adult and embryonic response to Azodrin Species
Adult response
Embryonic response
Chicken Chukar Quail
+a ++ +++
+ ++ ++ +
+ severity of response.
REFERENCES Abbott, U. K., 1972. Effect of pesticide and related compounds on several avian species. Sum. Rep. Food Prot. Toxicol. Cent., University of California Davis, CA. Abbott, U. K., R. M. Craig, and K. O. Keith, 1964. Effects of malathion spray on embryonated chicken eggs exposed under field conditions at Tuolume Meadow, Yosemite National Park. Poultry Sci. 4 3 : 1297. Duncan, A. J., 1965. Quality control and industrial statistics. 3rd ed. Richard D. Irwin, Homewood, IL. Hall, B. K., 1977. Thallium induced achondroplasia in chicken embryos and the concept of critical peri-
ods during development. Teratology 15:1—15. Landauer, 1975. Cholinomimetic teratogens: Studies with chicken embryos. Teratoloty 12:125—145. Moscioni, A. D., J. L. Engel, and J. E. Casida, 1977. Kynurenine Formamidase inhibition as a possible mechanism for certain teratogenic effects of organophosphorous and methylcarbamate insecticides in chicken embryos. Biochem. Pharmacol. 26:2251-2258. Roger, J. C , 1967. Mode of teratogenic action of organophosphate esters in hen eggs. Ph.D. dissertation, University of California, Berkeley, CA. Schom, C. B., and U. K. Abbott, 1974. Studies with Bobwhite quail: Reproductive characteristics. Poultry Sci. 53:1860-1865. Schom, C. B., and U. K. Abbott, 1977. Temporal, morphological and genetic responses of avian embryos to Azodrin, an organophosphate insecticide. Teratology 15:81-87. Schom, C. B., U. K. Abbott, and N. E. Walker, 1973. Organophosphate pesticide effects on domestic and game bird species: Dursban. Poultry Sci. 52:2083. Schom, C. B., N. E. Walker, and U. K. Abbott, 1972. Azodrin and its effect on four avian species. Poultry Sci. 51:1860. Snedecor, G. W., and W. G. Cochrane, 1967. Statistical methods. Iowa State University Press, Ames, IA. Walker, N. E., 1971. The effect of Malathion and Mamaoxon on esterase and gross development of chick embryos. Toxic. Appl. Pharmacol. 19:590— 601.
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No statistically significant differences.