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Reproductive Toxicity of Monocrotophos to Bobwhite Quail
Reproductive Toxicity of Monocrotophos to Bobwhite Quail K. L. STROMBORG US Fish and Wildlife Service, Division of Ecological Services, University of Wisconsin-Green Bay, Green Bay, Wisconsin 54302 (Received for publication December 28, 1984)
1986 Poultry Science 6 5 : 5 1 - 5 7
INTRODUCTION
feeding design was used to determine whether monocrotophos affected reproduction by a mechanism other than inhibition of food consumption. The response to progressively decreasing dietary concentrations was also compared to the response to constant dietary concentrations (Stromborg, 1981; Kenaga et al, 1979).
Pesticide registration requirements often mandate testing new pesticides for reproductive effects on one or more avian species (US Environmental Protection Agency, 1978). Unfortunately, the results of such tests are rarely published because the data are considered proprietary. Consequently, published data on the reproductive effects of the widely used organophosphate and carbamate pesticides are limited (Stromborg, 1981). In earlier studies, I have found that both northern bobwhite (Colinus virginianus) and ring-necked pheasants (Phasianus colchicus) are useful subjects for such tests (Stromborg, 1977, 1981). Both species were tested with an organophosphate of intermediate toxicity, diazinon. The present study was conducted to examine the response of bobwhites to another organophosphate, monocrotophos (Azodrin®), that has an extremely high toxicity to birds (Hill et al, 1975). Although the avian reproductive toxicity of monocrotophos had been tested before (Schom et al, 1979), the present study was designed to investigate several aspects of toxicity not evaluated in that study. In addition to describing dose-response relationships between monocrotophos and bobwhite reproductive parameters, a pair-
MATERIALS AND METHODS
Commercially obtained 16-week-old bobwhites were randomly paired, and individual pairs were housed in 51 x 30 x 27 cm breeding cages. The birds were provided with food and water ad libitum and, to stimulate egg production, a constant 15L:9D photoperiod was maintained. When the birds were 28 weeks of age, three groups of 30 pairs each were chosen from those pairs laying at that time. The groups were designated constant (to be exposed to the same pesticide concentration for the entire treatment period), pair-fed (to be given control food for the treatment period), and decreasing (to be exposed to steadily decreasing pesticide concentrations during the treatment period). After the 15-day treatment period, all birds were returned to the basic control diet for an additional 14-day posttreatment period.
51
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ABSTRACT Pairs of 1st-year breeding bobwhites were fed constant or decreasing concentrations of monocrotophos for 15 days. In addition, a control diet was used in a pair-fed group matched with the pairs in the constant group. Dietary concentrations for the constant group were logarithmically spaced at .100, .178, .316, .562, 1.000 ppm of actual insecticide and also at 0 ppm (control) for five pairs at each concentration. The beginning concentrations for the decreasing pairs were identical to the constant group but regularly decreased to reach 25% of the starting concentrations by Day 13. Food consumption, egg production, hatchability of eggs under artificial incubation, and survival of hatched chicks for 2 weeks were recorded pairwise during 15-day treatment and 14-day posttreatment periods. Mortality was high at the greatest constant concentration and in the associated pair-fed group. Food consumption and egg production rates were negatively dose-related during the treatment period in the constant and decreasing groups. The laying rate of pair-fed hens was reduced to the same extent as in the constant group. Reproductive inhibition was not permanent, and pairs resumed laying after a dose-related recovery interval. No dose-related effects on hatchability or chick survival were detected. There was no evidence of a pesticide effect on reproduction other than that exerted through pesticide-induced anorexia. (Key words: bobwhite, reproduction, organophosphate, monocrotophos, toxicity)
52
STROMBORG ductive effects (Stromborg, 1977). All birds were weighed 14 days before the treatment period began to provide weights for matching pairs. Constant and decreasing birds were weighed at the end of the 15-day treatment period and at the end of the experiment; pair-fed birds were weighed at the end of the experiment. Eggs were collected daily and incubated in weekly groups at 37.6 C and approximately 60% relative humidity. On Day 21 of incubation, eggs were transferred to individual hatching compartments. Within 24 hr of hatching, chicks were leg-banded to identify parentage, beak-trimmed, and placed in heated brooder units for 2 weeks to monitor survival. Data were analyzed by fitting simple linear regression models to various dose ranges and selecting the best fit by using the criterion of the highest coefficient of determination Stromborg, 1977, 1981). Dosage levels were transformed to log (dosage + 1.000). Doseresponse slopes were compared with Student's t-test. When birds died, they were not replaced, and data are reported as calculated hen-day egg production. RESULTS Although this experiment was designed to investigate only the sublethal effects of monocrotophos, at the highest treatment levels, many of the birds died. In general, birds died after an extended period (approximately 2 weeks) of depressed food consumption and accompanying severe weight loss of 50 to 60% (Table 1). One exception to this pattern was a pair-fed female that died on the 1st day of the experiment. This bird had no evident abnormalities, was not receiving a toxicant, but exhibited catastrophic weight loss from the pretreatment weighing. With this exception, weight losses and times to death in all three groups were similar. More birds died in the constant group than in the pair-fed group, suggesting that monocrotophos contributed directly to these deaths. The severe anorexia induced by monocrotophos was potentially lethal as reflected by the deaths of some pair-fed birds from starvation. Only one of the decreasing group died. Constant Exposure Test. Food consumption of pairs at the highest four treatment levels was depressed during the treatment period (P<.001), but there was no dose-related change (P>.50) in food consumption during the post-
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The constant and pair-fed groups contained pairs that were matched between groups to minimize differences in nutritional requirements between matched pairs. The total weights of the pairs being matched were within 10 g of each other, and males differed by less than 5 g (approximately 3% in each instance). Each of the 30 constant pairs was then randomly assigned to one of six treatment levels of monocrotophos (five pairs per level). Based on a preliminary range-finding trial, concentrations, in addition to control, were logarithmically spaced at .100, .178, .316, .562, and 1.000 ppm in an attempt to confine treatments to a nonlethal range including at least one noeffect concentration. Food consumption of the constant pairs was monitored over successive 3-day intervals, and the amount of food eaten by a constant pair was fed to the matched pair-fed control. Control food was used for the pair-fed pairs, and the total 3-day constant consumption was equally divided over 3 days. Thus, the difference between pairs in a match was the presence or absence of pesticide during the treatment period and a 3-day lag between constant and pair-fed trials. The decreasing pairs were randomly selected from the remaining laying pairs. The beginning monocrotophos concentrations for these pairs were the same as those used for the constant group. However, every 3 days, the pesticidetreated food was diluted with control food to simulate an environmental decline (Stromborg, 1981; Kenaga et al, 1979). Based on estimated 50% disappearance times of 2 to 16 days (Beynon and Wright, 1972; Westlake et al, 1970; Lindquist and Bull, 1967; Young and Bowman, 1967), 7 days was selected as an intermediate value. To simulate this exponential decline, 707 g of treated food were diluted with 293 g of control food every 3 days; thus, on Day 7, the concentrations were 50% of the starting concentrations and, on Day 14, they were 25%. The basic diet was a commercial game bird breeder mash supplemented with 1% corn oil by weight that served as a pesticide vehicle. Technical monocrotophos was dissolved in a minimal volume of acetone and then thoroughly mixed into corn oil to make a final mixture of the required weight. All calculations were based on 8.16% actual insecticide in the technical material. Acetone at the concentrations used has been found to be without repro-
PESTICIDE TOXICITY IN BOBWHITE QUAILS
53
TABLE 1. Time (days) after the beginning of treatment to mortality and associated weight losses at death of bobwhites given constant dosages of monocrotophos, restricted control diets (pair-fed), or decreasing dosages of monocrotophos Days tci death Dosage
Constant d
Pair-fed d
9
9
Weight loss at death Decreasing
Constant
Pair-fed
d
d
d
9
9
(ppm) .316
Decreasing d
9
("") 16
10 15
11
13 15 15 15 16
14 16
11 11 11
12
treatment period (Table 2). At the highest level, treatment period food consumption was 17% of the control level. Anorexia was reflected in a decrease in rate or total cessation of egg production (P<.001) at all but the lowest (.100 ppm) dosage. The doseresponse relationship was truncated at the .316 ppm concentration, because all pairs at the highest three concentrations stopped laying at the beginning of the treatment period. Recovery of reproductive function after cessation of pesticide feeding was measured in two ways. First, the number of days following the last treatment until the first posttreatment egg was laid was used as a measure. This recovery interval was related to dose (P<.001) over the same total range of concentrations as the egglaying inhibition occurred (Table 2). In other words, even though the tabulated limits of the relationships were different, the upper limit for recovery included all of the severely affected groups with complete cessation of egg laying. Even those birds eventually began laying again; the duration of effect was related to concentration. The other measure of recovery was the posttreatment laying rate. This was also related to concentration (P<.001) and reflected the length of time for birds to resume laying, because birds laid consistently once they resumed egg laying. Therefore the dose-related nature of this measure reflected the time for recovery rather than performance after recovery. The eggs that were laid were apparently unaffected because there were no treatmentrelated effects on fertility, hatchability, or
52 68 26 52
41 58
48 53
58
56 56 56 58 58
67 51
55 59 59
48
chick survival (P>.10) in any of the experimental groups. Overall, 92.6% of the total 1222 eggs laid were fertile, 77.8% of the fertile eggs hatched, and 90.1% of the chicks survived for 2 weeks. Both males and females lost weight in a dose-related fashion (P<.001) during the treatment period and regained most of the loss (.001.50) to the slope of the constant group. Recovery was also affected (P<.001) over the same range as the constant group. Slopes of the recovery dose-response relationships were different (P<.05) between the pair-fed and constant groups. The pair-fed group took longer to
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14 14 15 15
52
1
16
.562
1.000
9
STROMBORG
54
TABLE 2. Response of bobwhite pairs to monocrotophos during and after a 15-day treatment period
in the diet
Parameter (Y)
Constant dosage
Food consumption, g/day
Y = 34 - (99 ± 9) a log(X b + 1) .100
Y = 33 - ( 2 3 ± 1 0 ) l o g ( X c + 1) .100
Egg production, eggs/day
Y = . 9 - (6.4±1.2)log(X + 1) .100
Y = . 9 - ( 3 . 7 + .8)Iog(X + 1) .100
Change in weight, % of pretreatment, males
Y = 3 - (74+17)log(X + 1) 0
Y= 3 - ( 2 2 + 6)log(X + 1) .100
Change in weight, % of pretreatment, females
Y = 1 - (79±17)log(X+ 1) 0
Y = 5 - ( 3 4 + 16)log(X + 1) .100
Food consumption, g/day
Y = 42 + (4 + 6)log(X + 1) 0
Y = 42 + ( 1 0 + l l ) l o g ( X + 1) 0
Egg production, eggs/day
Y = . 9 - (2.6 + .5)log(X + 1) .100
Y = 1 . 0 - (2.0 + .8)Iog(X + 1) .316
Recovery, days to first egg
Y = .4 + (40.1 + 6.0)log(X + 1) 0
Y = - 2 . 1 + (29.3 + 4.8)log(X + 1) .100
Change in weight, % of pretreatment, males
Y = - 4 + (46 + 13)log(X + 1) 0
Y = - 5 + (13 + 5)log(X+ 1) 0
Change in weight, % of pretreatment, females
Y = - 2 + (57±16)log(X + 1) 0
Y = - 4 + (35 + 10)log(X + 1) 0
Decreasing dosage Treatment period
b ± Standard error. X = Dietary concentration in ppm of monocrotophos. X = Initial dietary concentration which was decreased exponentially every three days to simulate field decay. *P<.05. **P<.01. ***P<.001.
recover than the constant group; at the highest treatment level, the difference was approximately 4.8 days (50% greater than the constant group). The dose-response relationship for the final reproductive measurement, posttreatment laying rate, was also significant (P<.001) and similar in range and slope (.40
two groups to the treatment. The pair-fed birds returned to production slower than the monocrotophos-dosed birds, although the difference was relatively small. The ranges for the two measures of reversal of reproductive inhibition were also different. The pair-fed group exhibited no dose-related changes in weight over the course of the experiment (P>.05) (Table 3). When the net changes
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Posttreatment period
PESTICIDE TOXICITY IN BOBWHITE QUAILS
55
TABLE 3. Response ofintact matched pairs of bobwhites receiving ad libitum amounts of food with monocrotopbos added (constant dosage) or control food in amounts determined by the matched constant pair (pair-fed) Parameter (Y)
Constant dosage
Pair-fed
Egg production, eggs/day
Y = 1.0 - (6.9 + 1.3) a log(X b + 1 ) .100
Egg production, eggs/day
Y = 1 . 0 - (5.2 + l . l ) l o g ( X + 1) .100
Y = 1,1 - (6.2 + l . l ) l o g ( X + 1) .100
Recovery, days to first egg
Y = - . 5 + (45.2+7.6) log(X + 1) 0
Y = - . 8 + (70.5 + 9.2)log(X + 1) 0
Treatment period Y = 1.0 - (6.7 + 1.6)log(X c + 1) .100
Posttreatment period
Net change in weight, % of pretreatment, females
Y = 1 - (31 + 10)log(X + 1) .178
b ± Standard error. X = Dietary concentration of monocrotophos. X = Dietary concentration given to matched pair in the constant group. *No significant dose-related response; P>.05. **P<.05. ***P<001.
over this interval were analyzed for the matched pairs in the constant groups, a dose-related reduction in weight of less than 5% was found for 10 females in one limited dose range (.01.05) over any dose range. Decreasing Exposure Test. Only one male in the highest decreasing concentration group died during the experiment in contrast to the extensive mortality in the constant group (Table 1). The reduced toxicitv of the decreasing schedule was also reflected in the other parameters measured. Pairs receiving the decreasing concentrations reduced their food consumption in a dose-related manner (P<.01) during the treatment period, but there were no dose-related effects after pesticide administration ceased (P>.20). The reduction in food consumption during the treatment period was much less pronounced (P<.001) in the decreasing group than in the constant group (Table 2). Posttreatment food consumption was not related to treatment level in either group.
The treatment period egg-laying rate was dose-related (P<.001) in the decreasing group. The slope of this dose-response relationship cannot be compared directly with the constant group, because the ranges of effects were different in the two groups (Table 2). By inspection, it was apparent that the slope of the response was lower and the effect was distributed over a wider range in the decreasing group. Recovery from pesticide effects on egg laying was also related to treatment (P<.001) over a broader range of concentrations than in the constant group. Additionally, the posttreatment laying rate was related to treatment (P<.05) but only at the highest (>.316 ppm) concentrations. Both males and females in the decreasing group lost weight (P<.001 for males, P<.05 for females) during the treatment period and gained weight (P<.05 for males, P<.001 for females) after dosage was discontinued. Although the range of concentrations which these relationships describe were generally different in
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Net change in weight, % of pretreatment, males
STROMBORG
56
the constant and decreasing groups (Table 2), maximum weight losses were less in the decreasing group (—4% for males; —6% for females) than in the constant group (—19% for males; —14% for females). Consequently, maximum weight gains during the posttreatment period were also less in the decreasing group (0% for males; 6% for females) than in the constant group (10% for males; 9% for females). DISCUSSION
ACKNOWLEDGMENTS I thank C. Bratsch, K. Banes, and A. Shull
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The high toxicity of monocrotophos to quail reported by Hill et al. (1975), Tucker and Crabtree (1970), and Schom et al. (1972, 1979) was also observed in the present study. Only 1 of 10 quail exposed to 1.000 ppm monocrotophos survived for the entire 15-day treatment period. As was the case in earlier studies of diazinon (Stromborg, 1981), a primary effect of the organophosphate, monocrotophos, was to induce severe anorexia. In contrast to the diazinon study, there was little evidence that any of the other effects on surviving birds was related to physiological effects independent of anorexia; the pair-fed and constant groups, which differed only in whether or not they were fed monocrotophos, responded similarly in body weight changes and egg production. In addition, mortality occurred in both groups, although it was somewhat more rapid and severe in the pesticide-fed groups. This mortality appeared to be associated with anorexiainduced catastrophic weight losses. Comparisons of the present results with those reported by Schom et al. (1972, 1979) are complicated by dissimilar procedures, but the qualitative patterns of results are similar. In the latter studies, some bobwhites survived at dietary concentrations as high as 25 ppm for 98 days; in the current study, only one bobwhite survived 1.000 ppm for 15 days. At 1.25 ppm, Schom et al. (1979) found 50% survival for 112 days. Clearly, bobwhites in that study survived higher reported dietary concentrations than in the present study. Possible explanations include their use of older (generally >2 years), proven breeders, whereas, I used young, firsttime breeders. They also apparently began to feed monocrotophos before the females began laying, and I used birds that were already laying regularly. This might have introduced differences in metabolic requirements and therefore, total toxicant ingestion. In addition, the formulation and diluent used by Schom et al. (1979)
were not specified, and such differences might have been responsible for some of the difference with the results of the present study. As was the case in prior studies of diazinon (Stromborg, 1981), a decreasing dietary concentration schedule ameliorated the effects of monocrotophos on bobwhites. The implication is that the quail were responding primarily to current conditions rather than to strongly cumulative toxic effects of either chemical. The reversibility of egg laying inhibition, reduced food consumption, and weight losses also supports this interpretation. Although Tucker and Crabtree (1970) reported a high degree of cumulative toxicity, in the present study, only a slight indication of cumulative toxicity was provided by the greater mortality in the constant as opposed to the pair-fed groups. This might have been related to the same avoidance of lethal doses of organophosphates reported by Hill (1972). Because the pair-fed quail were beginning to die from starvation by the end of the treatment period, it is reasonable to believe that the constant birds that had unlimited access to pesticide-treated food began to eat sufficient quantities of toxic food to cause mortality despite the avoidance exhibited early in the treatment period. Thus, toxicity of monocrotophos might not have been cumulative, but rather, reflected a starvation-induced reversal of the avoidance reaction. In contrast to the diazinon experiment (Stromborg, 1981), the dose-response relationships were not so clearly defined in this experiment. Unplanned mortality reduced sample size at the upper limit of the dosage range and, for several parameters, the upper two or three treatment levels caused identical responses. This reduced the sample sizes available for describing the dose-response and resulted from the greater relative range of concentrations used in the present monocrotophos experiment. The range tested in the diazinon experiment was 4.3-fold; in the monocrotophos experiment, it was 10fold. For experiments of this type, preliminary range-finding trials can be utilized to good advantage to narrow the treatment range and avoid collecting data above or below the effective dose-response range. In the present experiment, limited range-finding was conducted; additional effort in this area might have improved the efficiency of the final experiment.
PESTICIDE TOXICITY IN BOBWHITE QUAILS
for assistance with bird care, and M. Holmes for typing the manuscript. T. Custer and B. Rattner reviewed the manuscript. REFERENCES
Schom, C. B„ U. K. Abbott, and N. E. Walker, 1979. Adult and embryo responses to organophosphate pesticide: Azodrin. Poultry Sci. 58:60—66. Schom, C. B., N. E. Walker, and U. K. Abbott, 1972. Azodrin and its effects on four avian species. Poultry Sci. 51:1860. (Abstr.) Stromborg, K. L., 1977. Seed treatment pesticide effects on pheasant reproduction at sublethal doses. J. Wildl. Manage. 41:632-642. Stromborg, K. L., 1981. Reproductive tests of diazinon on bobwhite quail. Pages 19—30 in Avian and Mammalian Wildlife Toxicology: Second Conference, ASTM STP 757. D. W. Lamb and E. E. Kenaga, ed. Am. Soc. Testing and Materials, Philadelphia, PA. Tucker, R. K., and D. G. Crabtree, 1970. Handbook of toxicity of pesticides to wildlife. Resource Publ. No. 84, US Fish and Wildl. Service, Washington, DC. US Environmental Protection Agency, 1978. Federal Register 43 (132):29696-29741. Westlake, W. E., F. A. Gunther, and L. R. Jeppson, 1970. Persistence of Azodrin residues on and in Valencia oranges and in laboratory-processed citrus pulp cattle feed. Agric. Food Chem. 18: 864-865. Young, J. R., and M. C. Bowman, 1967. Azodrin for corn earworm and fall armyworm control and its persistence in sweet corn. J. Econ. Entomol. 60: 1282-1284.
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Beynon, K. I., and A. N. Wright, 1972. The breakdown of [ I4 C]monocrotophos insecticide on maize, cabbage and apple. Pestic. Sci. 3:277— 292. Hill, E. F., 1972. Avoidance of lethal dietary concentrations of insecticide by house sparrows. J. Wildl. Manage. 36:635-639. Hill, E. F., R. G. Heath, J. W. Spann, and J. D. Williams, 1975. Lethal dietary toxicities of environmental pollutants to birds. Special Scientific Report-Wildlife, No. 191, US Fish and Wildl. Service, Washington, DC. Kenaga, E. E„ R. J. Fink, and J. B. Beavers, 1979. Dietary toxicity tests with mallards, simulating residue decline of chlorphyrifos and avoidance of treated food. Pages 36—44 in Avian and Mammalian Wildlife Toxicology, ASTM STP 693. E. E. Kenaga, ed. Am. Soc. Testing and Materials, Philadelphia, PA. Lindquist, D. A„ and D. L. Bull, 1967. Fate of 3hydroxy-ZV-methyl-cz's crotonamide dimethyl phosphate in cotton plants. J. Agric. Food Chem. 15:267-272.
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1986 Poultry Science 6 5 : 5 1 - 5 7
INTRODUCTION
feeding design was used to determine whether monocrotophos affected reproduction by a mechanism other than inhibition of food consumption. The response to progressively decreasing dietary concentrations was also compared to the response to constant dietary concentrations (Stromborg, 1981; Kenaga et al, 1979).
Pesticide registration requirements often mandate testing new pesticides for reproductive effects on one or more avian species (US Environmental Protection Agency, 1978). Unfortunately, the results of such tests are rarely published because the data are considered proprietary. Consequently, published data on the reproductive effects of the widely used organophosphate and carbamate pesticides are limited (Stromborg, 1981). In earlier studies, I have found that both northern bobwhite (Colinus virginianus) and ring-necked pheasants (Phasianus colchicus) are useful subjects for such tests (Stromborg, 1977, 1981). Both species were tested with an organophosphate of intermediate toxicity, diazinon. The present study was conducted to examine the response of bobwhites to another organophosphate, monocrotophos (Azodrin®), that has an extremely high toxicity to birds (Hill et al, 1975). Although the avian reproductive toxicity of monocrotophos had been tested before (Schom et al, 1979), the present study was designed to investigate several aspects of toxicity not evaluated in that study. In addition to describing dose-response relationships between monocrotophos and bobwhite reproductive parameters, a pair-
MATERIALS AND METHODS
Commercially obtained 16-week-old bobwhites were randomly paired, and individual pairs were housed in 51 x 30 x 27 cm breeding cages. The birds were provided with food and water ad libitum and, to stimulate egg production, a constant 15L:9D photoperiod was maintained. When the birds were 28 weeks of age, three groups of 30 pairs each were chosen from those pairs laying at that time. The groups were designated constant (to be exposed to the same pesticide concentration for the entire treatment period), pair-fed (to be given control food for the treatment period), and decreasing (to be exposed to steadily decreasing pesticide concentrations during the treatment period). After the 15-day treatment period, all birds were returned to the basic control diet for an additional 14-day posttreatment period.
51
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ABSTRACT Pairs of 1st-year breeding bobwhites were fed constant or decreasing concentrations of monocrotophos for 15 days. In addition, a control diet was used in a pair-fed group matched with the pairs in the constant group. Dietary concentrations for the constant group were logarithmically spaced at .100, .178, .316, .562, 1.000 ppm of actual insecticide and also at 0 ppm (control) for five pairs at each concentration. The beginning concentrations for the decreasing pairs were identical to the constant group but regularly decreased to reach 25% of the starting concentrations by Day 13. Food consumption, egg production, hatchability of eggs under artificial incubation, and survival of hatched chicks for 2 weeks were recorded pairwise during 15-day treatment and 14-day posttreatment periods. Mortality was high at the greatest constant concentration and in the associated pair-fed group. Food consumption and egg production rates were negatively dose-related during the treatment period in the constant and decreasing groups. The laying rate of pair-fed hens was reduced to the same extent as in the constant group. Reproductive inhibition was not permanent, and pairs resumed laying after a dose-related recovery interval. No dose-related effects on hatchability or chick survival were detected. There was no evidence of a pesticide effect on reproduction other than that exerted through pesticide-induced anorexia. (Key words: bobwhite, reproduction, organophosphate, monocrotophos, toxicity)
52
STROMBORG ductive effects (Stromborg, 1977). All birds were weighed 14 days before the treatment period began to provide weights for matching pairs. Constant and decreasing birds were weighed at the end of the 15-day treatment period and at the end of the experiment; pair-fed birds were weighed at the end of the experiment. Eggs were collected daily and incubated in weekly groups at 37.6 C and approximately 60% relative humidity. On Day 21 of incubation, eggs were transferred to individual hatching compartments. Within 24 hr of hatching, chicks were leg-banded to identify parentage, beak-trimmed, and placed in heated brooder units for 2 weeks to monitor survival. Data were analyzed by fitting simple linear regression models to various dose ranges and selecting the best fit by using the criterion of the highest coefficient of determination Stromborg, 1977, 1981). Dosage levels were transformed to log (dosage + 1.000). Doseresponse slopes were compared with Student's t-test. When birds died, they were not replaced, and data are reported as calculated hen-day egg production. RESULTS Although this experiment was designed to investigate only the sublethal effects of monocrotophos, at the highest treatment levels, many of the birds died. In general, birds died after an extended period (approximately 2 weeks) of depressed food consumption and accompanying severe weight loss of 50 to 60% (Table 1). One exception to this pattern was a pair-fed female that died on the 1st day of the experiment. This bird had no evident abnormalities, was not receiving a toxicant, but exhibited catastrophic weight loss from the pretreatment weighing. With this exception, weight losses and times to death in all three groups were similar. More birds died in the constant group than in the pair-fed group, suggesting that monocrotophos contributed directly to these deaths. The severe anorexia induced by monocrotophos was potentially lethal as reflected by the deaths of some pair-fed birds from starvation. Only one of the decreasing group died. Constant Exposure Test. Food consumption of pairs at the highest four treatment levels was depressed during the treatment period (P<.001), but there was no dose-related change (P>.50) in food consumption during the post-
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The constant and pair-fed groups contained pairs that were matched between groups to minimize differences in nutritional requirements between matched pairs. The total weights of the pairs being matched were within 10 g of each other, and males differed by less than 5 g (approximately 3% in each instance). Each of the 30 constant pairs was then randomly assigned to one of six treatment levels of monocrotophos (five pairs per level). Based on a preliminary range-finding trial, concentrations, in addition to control, were logarithmically spaced at .100, .178, .316, .562, and 1.000 ppm in an attempt to confine treatments to a nonlethal range including at least one noeffect concentration. Food consumption of the constant pairs was monitored over successive 3-day intervals, and the amount of food eaten by a constant pair was fed to the matched pair-fed control. Control food was used for the pair-fed pairs, and the total 3-day constant consumption was equally divided over 3 days. Thus, the difference between pairs in a match was the presence or absence of pesticide during the treatment period and a 3-day lag between constant and pair-fed trials. The decreasing pairs were randomly selected from the remaining laying pairs. The beginning monocrotophos concentrations for these pairs were the same as those used for the constant group. However, every 3 days, the pesticidetreated food was diluted with control food to simulate an environmental decline (Stromborg, 1981; Kenaga et al, 1979). Based on estimated 50% disappearance times of 2 to 16 days (Beynon and Wright, 1972; Westlake et al, 1970; Lindquist and Bull, 1967; Young and Bowman, 1967), 7 days was selected as an intermediate value. To simulate this exponential decline, 707 g of treated food were diluted with 293 g of control food every 3 days; thus, on Day 7, the concentrations were 50% of the starting concentrations and, on Day 14, they were 25%. The basic diet was a commercial game bird breeder mash supplemented with 1% corn oil by weight that served as a pesticide vehicle. Technical monocrotophos was dissolved in a minimal volume of acetone and then thoroughly mixed into corn oil to make a final mixture of the required weight. All calculations were based on 8.16% actual insecticide in the technical material. Acetone at the concentrations used has been found to be without repro-
PESTICIDE TOXICITY IN BOBWHITE QUAILS
53
TABLE 1. Time (days) after the beginning of treatment to mortality and associated weight losses at death of bobwhites given constant dosages of monocrotophos, restricted control diets (pair-fed), or decreasing dosages of monocrotophos Days tci death Dosage
Constant d
Pair-fed d
9
9
Weight loss at death Decreasing
Constant
Pair-fed
d
d
d
9
9
(ppm) .316
Decreasing d
9
("") 16
10 15
11
13 15 15 15 16
14 16
11 11 11
12
treatment period (Table 2). At the highest level, treatment period food consumption was 17% of the control level. Anorexia was reflected in a decrease in rate or total cessation of egg production (P<.001) at all but the lowest (.100 ppm) dosage. The doseresponse relationship was truncated at the .316 ppm concentration, because all pairs at the highest three concentrations stopped laying at the beginning of the treatment period. Recovery of reproductive function after cessation of pesticide feeding was measured in two ways. First, the number of days following the last treatment until the first posttreatment egg was laid was used as a measure. This recovery interval was related to dose (P<.001) over the same total range of concentrations as the egglaying inhibition occurred (Table 2). In other words, even though the tabulated limits of the relationships were different, the upper limit for recovery included all of the severely affected groups with complete cessation of egg laying. Even those birds eventually began laying again; the duration of effect was related to concentration. The other measure of recovery was the posttreatment laying rate. This was also related to concentration (P<.001) and reflected the length of time for birds to resume laying, because birds laid consistently once they resumed egg laying. Therefore the dose-related nature of this measure reflected the time for recovery rather than performance after recovery. The eggs that were laid were apparently unaffected because there were no treatmentrelated effects on fertility, hatchability, or
52 68 26 52
41 58
48 53
58
56 56 56 58 58
67 51
55 59 59
48
chick survival (P>.10) in any of the experimental groups. Overall, 92.6% of the total 1222 eggs laid were fertile, 77.8% of the fertile eggs hatched, and 90.1% of the chicks survived for 2 weeks. Both males and females lost weight in a dose-related fashion (P<.001) during the treatment period and regained most of the loss (.001
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14 14 15 15
52
1
16
.562
1.000
9
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TABLE 2. Response of bobwhite pairs to monocrotophos during and after a 15-day treatment period
in the diet
Parameter (Y)
Constant dosage
Food consumption, g/day
Y = 34 - (99 ± 9) a log(X b + 1) .100
Y = 33 - ( 2 3 ± 1 0 ) l o g ( X c + 1) .100
Egg production, eggs/day
Y = . 9 - (6.4±1.2)log(X + 1) .100
Y = . 9 - ( 3 . 7 + .8)Iog(X + 1) .100
Change in weight, % of pretreatment, males
Y = 3 - (74+17)log(X + 1) 0
Y= 3 - ( 2 2 + 6)log(X + 1) .100
Change in weight, % of pretreatment, females
Y = 1 - (79±17)log(X+ 1) 0
Y = 5 - ( 3 4 + 16)log(X + 1) .100
Food consumption, g/day
Y = 42 + (4 + 6)log(X + 1) 0
Y = 42 + ( 1 0 + l l ) l o g ( X + 1) 0
Egg production, eggs/day
Y = . 9 - (2.6 + .5)log(X + 1) .100
Y = 1 . 0 - (2.0 + .8)Iog(X + 1) .316
Recovery, days to first egg
Y = .4 + (40.1 + 6.0)log(X + 1) 0
Y = - 2 . 1 + (29.3 + 4.8)log(X + 1) .100
Change in weight, % of pretreatment, males
Y = - 4 + (46 + 13)log(X + 1) 0
Y = - 5 + (13 + 5)log(X+ 1) 0
Change in weight, % of pretreatment, females
Y = - 2 + (57±16)log(X + 1) 0
Y = - 4 + (35 + 10)log(X + 1) 0
Decreasing dosage Treatment period
b ± Standard error. X = Dietary concentration in ppm of monocrotophos. X = Initial dietary concentration which was decreased exponentially every three days to simulate field decay. *P<.05. **P<.01. ***P<.001.
recover than the constant group; at the highest treatment level, the difference was approximately 4.8 days (50% greater than the constant group). The dose-response relationship for the final reproductive measurement, posttreatment laying rate, was also significant (P<.001) and similar in range and slope (.40
two groups to the treatment. The pair-fed birds returned to production slower than the monocrotophos-dosed birds, although the difference was relatively small. The ranges for the two measures of reversal of reproductive inhibition were also different. The pair-fed group exhibited no dose-related changes in weight over the course of the experiment (P>.05) (Table 3). When the net changes
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Posttreatment period
PESTICIDE TOXICITY IN BOBWHITE QUAILS
55
TABLE 3. Response ofintact matched pairs of bobwhites receiving ad libitum amounts of food with monocrotopbos added (constant dosage) or control food in amounts determined by the matched constant pair (pair-fed) Parameter (Y)
Constant dosage
Pair-fed
Egg production, eggs/day
Y = 1.0 - (6.9 + 1.3) a log(X b + 1 ) .100
Egg production, eggs/day
Y = 1 . 0 - (5.2 + l . l ) l o g ( X + 1) .100
Y = 1,1 - (6.2 + l . l ) l o g ( X + 1) .100
Recovery, days to first egg
Y = - . 5 + (45.2+7.6) log(X + 1) 0
Y = - . 8 + (70.5 + 9.2)log(X + 1) 0
Treatment period Y = 1.0 - (6.7 + 1.6)log(X c + 1) .100
Posttreatment period
Net change in weight, % of pretreatment, females
Y = 1 - (31 + 10)log(X + 1) .178
b ± Standard error. X = Dietary concentration of monocrotophos. X = Dietary concentration given to matched pair in the constant group. *No significant dose-related response; P>.05. **P<.05. ***P<001.
over this interval were analyzed for the matched pairs in the constant groups, a dose-related reduction in weight of less than 5% was found for 10 females in one limited dose range (.01
The treatment period egg-laying rate was dose-related (P<.001) in the decreasing group. The slope of this dose-response relationship cannot be compared directly with the constant group, because the ranges of effects were different in the two groups (Table 2). By inspection, it was apparent that the slope of the response was lower and the effect was distributed over a wider range in the decreasing group. Recovery from pesticide effects on egg laying was also related to treatment (P<.001) over a broader range of concentrations than in the constant group. Additionally, the posttreatment laying rate was related to treatment (P<.05) but only at the highest (>.316 ppm) concentrations. Both males and females in the decreasing group lost weight (P<.001 for males, P<.05 for females) during the treatment period and gained weight (P<.05 for males, P<.001 for females) after dosage was discontinued. Although the range of concentrations which these relationships describe were generally different in
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Net change in weight, % of pretreatment, males
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56
the constant and decreasing groups (Table 2), maximum weight losses were less in the decreasing group (—4% for males; —6% for females) than in the constant group (—19% for males; —14% for females). Consequently, maximum weight gains during the posttreatment period were also less in the decreasing group (0% for males; 6% for females) than in the constant group (10% for males; 9% for females). DISCUSSION
ACKNOWLEDGMENTS I thank C. Bratsch, K. Banes, and A. Shull
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The high toxicity of monocrotophos to quail reported by Hill et al. (1975), Tucker and Crabtree (1970), and Schom et al. (1972, 1979) was also observed in the present study. Only 1 of 10 quail exposed to 1.000 ppm monocrotophos survived for the entire 15-day treatment period. As was the case in earlier studies of diazinon (Stromborg, 1981), a primary effect of the organophosphate, monocrotophos, was to induce severe anorexia. In contrast to the diazinon study, there was little evidence that any of the other effects on surviving birds was related to physiological effects independent of anorexia; the pair-fed and constant groups, which differed only in whether or not they were fed monocrotophos, responded similarly in body weight changes and egg production. In addition, mortality occurred in both groups, although it was somewhat more rapid and severe in the pesticide-fed groups. This mortality appeared to be associated with anorexiainduced catastrophic weight losses. Comparisons of the present results with those reported by Schom et al. (1972, 1979) are complicated by dissimilar procedures, but the qualitative patterns of results are similar. In the latter studies, some bobwhites survived at dietary concentrations as high as 25 ppm for 98 days; in the current study, only one bobwhite survived 1.000 ppm for 15 days. At 1.25 ppm, Schom et al. (1979) found 50% survival for 112 days. Clearly, bobwhites in that study survived higher reported dietary concentrations than in the present study. Possible explanations include their use of older (generally >2 years), proven breeders, whereas, I used young, firsttime breeders. They also apparently began to feed monocrotophos before the females began laying, and I used birds that were already laying regularly. This might have introduced differences in metabolic requirements and therefore, total toxicant ingestion. In addition, the formulation and diluent used by Schom et al. (1979)
were not specified, and such differences might have been responsible for some of the difference with the results of the present study. As was the case in prior studies of diazinon (Stromborg, 1981), a decreasing dietary concentration schedule ameliorated the effects of monocrotophos on bobwhites. The implication is that the quail were responding primarily to current conditions rather than to strongly cumulative toxic effects of either chemical. The reversibility of egg laying inhibition, reduced food consumption, and weight losses also supports this interpretation. Although Tucker and Crabtree (1970) reported a high degree of cumulative toxicity, in the present study, only a slight indication of cumulative toxicity was provided by the greater mortality in the constant as opposed to the pair-fed groups. This might have been related to the same avoidance of lethal doses of organophosphates reported by Hill (1972). Because the pair-fed quail were beginning to die from starvation by the end of the treatment period, it is reasonable to believe that the constant birds that had unlimited access to pesticide-treated food began to eat sufficient quantities of toxic food to cause mortality despite the avoidance exhibited early in the treatment period. Thus, toxicity of monocrotophos might not have been cumulative, but rather, reflected a starvation-induced reversal of the avoidance reaction. In contrast to the diazinon experiment (Stromborg, 1981), the dose-response relationships were not so clearly defined in this experiment. Unplanned mortality reduced sample size at the upper limit of the dosage range and, for several parameters, the upper two or three treatment levels caused identical responses. This reduced the sample sizes available for describing the dose-response and resulted from the greater relative range of concentrations used in the present monocrotophos experiment. The range tested in the diazinon experiment was 4.3-fold; in the monocrotophos experiment, it was 10fold. For experiments of this type, preliminary range-finding trials can be utilized to good advantage to narrow the treatment range and avoid collecting data above or below the effective dose-response range. In the present experiment, limited range-finding was conducted; additional effort in this area might have improved the efficiency of the final experiment.
PESTICIDE TOXICITY IN BOBWHITE QUAILS
for assistance with bird care, and M. Holmes for typing the manuscript. T. Custer and B. Rattner reviewed the manuscript. REFERENCES
Schom, C. B„ U. K. Abbott, and N. E. Walker, 1979. Adult and embryo responses to organophosphate pesticide: Azodrin. Poultry Sci. 58:60—66. Schom, C. B., N. E. Walker, and U. K. Abbott, 1972. Azodrin and its effects on four avian species. Poultry Sci. 51:1860. (Abstr.) Stromborg, K. L., 1977. Seed treatment pesticide effects on pheasant reproduction at sublethal doses. J. Wildl. Manage. 41:632-642. Stromborg, K. L., 1981. Reproductive tests of diazinon on bobwhite quail. Pages 19—30 in Avian and Mammalian Wildlife Toxicology: Second Conference, ASTM STP 757. D. W. Lamb and E. E. Kenaga, ed. Am. Soc. Testing and Materials, Philadelphia, PA. Tucker, R. K., and D. G. Crabtree, 1970. Handbook of toxicity of pesticides to wildlife. Resource Publ. No. 84, US Fish and Wildl. Service, Washington, DC. US Environmental Protection Agency, 1978. Federal Register 43 (132):29696-29741. Westlake, W. E., F. A. Gunther, and L. R. Jeppson, 1970. Persistence of Azodrin residues on and in Valencia oranges and in laboratory-processed citrus pulp cattle feed. Agric. Food Chem. 18: 864-865. Young, J. R., and M. C. Bowman, 1967. Azodrin for corn earworm and fall armyworm control and its persistence in sweet corn. J. Econ. Entomol. 60: 1282-1284.
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Beynon, K. I., and A. N. Wright, 1972. The breakdown of [ I4 C]monocrotophos insecticide on maize, cabbage and apple. Pestic. Sci. 3:277— 292. Hill, E. F., 1972. Avoidance of lethal dietary concentrations of insecticide by house sparrows. J. Wildl. Manage. 36:635-639. Hill, E. F., R. G. Heath, J. W. Spann, and J. D. Williams, 1975. Lethal dietary toxicities of environmental pollutants to birds. Special Scientific Report-Wildlife, No. 191, US Fish and Wildl. Service, Washington, DC. Kenaga, E. E„ R. J. Fink, and J. B. Beavers, 1979. Dietary toxicity tests with mallards, simulating residue decline of chlorphyrifos and avoidance of treated food. Pages 36—44 in Avian and Mammalian Wildlife Toxicology, ASTM STP 693. E. E. Kenaga, ed. Am. Soc. Testing and Materials, Philadelphia, PA. Lindquist, D. A„ and D. L. Bull, 1967. Fate of 3hydroxy-ZV-methyl-cz's crotonamide dimethyl phosphate in cotton plants. J. Agric. Food Chem. 15:267-272.
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