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Toxicological Studies with Mirex in Bobwhite Quail1
Toxicological Studies with Mirex in Bobwhite Quail1 RONALD J. KENDALL, RAYMOND NOBLET, L. H. SENN, and J. R. HOLMAN Department of Entomology and Economic Zoology, Clemson University, Clemson, South Carolina 29631 (Received for publication April 27, 1978) ABSTRACT Bobwhite quail (Colinus virginianus) received dietary mirex in concentrations of 1, 20, and 40 ppm to investigate reproductive effects of long term exposure to this chemical. Residue analyses of F 0 generation breeders indicated that male adipose tissue contained approximately 10 times the mirex level in the diet. Elimination of mirex in females probably was facilitated by egg laying, which reduced mirex buildup in adipose tissue to five times the dietary level. Both sexes were noted to concentrate mirex in fat and breast tissue in direct proportion to the intake of dietary mirex. Eggs collected from F„ generation breeders were not affected deleteriously by mirex as measured by embryo survival to 3 weeks, and the number of eggs failing to hatch. Indeed, increased rates of egg fertility and hatchability were associated with higher dietary concentrations. Chick survival data was obtained in F 0 and F[ generation hatchlings from hatching through 2 weeks. No chick mortality attributable to pesticide stress was detected in either group of birds. Eggs collected from F, generation breeders that received 1 ppm were not affected harmfully as measured by embryonation, embryo survival, and hatchability rates. Comparison of residues in wild bobwhites with residues in our experimental findings indicates mirex is apparently not affecting deleteriously reproductive success of wild quail. INTRODUCTION The organochlorine pesticide mirex (dodecachlorooctahydro-1, 3, 4,-metheno-2H-cyclobuta [cd] pentalene) has been used extensively in the Southeastern United States for control of the imported fire ant (Solenopsis invicta). Although mirex provides effective control of fire ants at very low rates of application (1.7 gm mirex/acre in 1.25 lb of corncob grits-soybean oil bait) it is an extremely stable compound which allows it to become incorporated into the food webb (Mirex Report, 1972). Recent monitoring studies (Collins et al, 1974, Markin et al, 1974, and Bevenue et al, 1975) following mirex bait applications reported widespread residues in terrestrial and aquatic biota. Borthwick et al. (1973) reported biological concentration of mirex particularly in predacious birds. A few studies have been conducted to determine effects of mirex in experimental birds. Heath et al (1972) reported that mirex was less than one-fourth as toxic as DDT to 2-week-old bobwhites. In pen studies with Japanese quail (Coturnix coturnix japonica) receiving up to 80 ppm dietary mirex, Davison et al. (1975) found
'Technical contribution no. 1430 from South Carolina Agricultural Experiment Station. 1978 Poultry Sci 57:1539-1545
no reproductive effects. However, Hyde et al (1973) determined mallard duckling {Anas platyrhynchos) survival to be reduced in a 100 ppm mirex treatment. Heath and Spann (1973) concluded that short term mirex dosages of 40 ppm in the feed of bobwhite quail induced no perceptible reproductive effects, but mortality of dosed males averaged 75% compared to 17% of control males. When chickens were given a diet containing 600 ppm mirex, hatching rate and chick survival were significantly reduced compared to controls (Naber and Ware, 1964). The literature as a whole is variable as to conclusive evidence of the effects of mirex in birds. Since the chlorinated hydrocarbon pesticides are such an integral part of the discussion of reproductive inhibition in many avian species (Stickel and Rhodes, 1970), further studies with low dietary levels of mirex in birds are needed. Bobwhite quail are important game animals and their food habits (Rosene, 1969) would allow frequent contact with mirex; therefore, pen studies with bobwhites over two generations were conducted to investigate reproductive effects of long term exposure to dietary mirex. At 1 ppm mirex treatment was designed to test the effects of a mirex dosage that bobwhites might contact in the field (Kendall et al, 1977). Dietary levels of 20 and 40 ppm were used to determine effects of high levels of
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KENDALL ET AL.
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mirex on reproductive success; these levels were considered to be sublethal to adults.
MATERIALS AND METHODS
Pen Studies. Fifty 1-day-old bobwhite chicks ( F 0 generation) were placed in each of four brooders (200 chicks in 3 mirex treatments plus control). Treatements consisted of mirex added to the diet as follows: 0 , 1 , 20, and 40 ppm (jug of mirex/g of diet). The mirex was dissolved in 25 ml of hexane, poured into the diet, and the feed was thoroughly mixed following evaporation of the hexane. The chicks received the mirex diets ad libitum beginning at 1-day of age and were continued on the diets through the grow-out and egg-laying periods. There was some early mortality in F 0 chicks; however, this appeared to be natural die-off and no antibiotics were administered. The 35-day-old F 0 quail were transferred from brooders into two quail battery breeding pens (GQF Manufacturing Company, Savannah, GA) for grow-out. The breeding pens were housed in a 2.1 x 4.3 m room maintained under constant photoperiod (12L:12D) and temperature (24 C). At 13 weeks F 0 birds were paired off and egg-laying was induced by increasing the light regimen 30 min/week until a 17 hr day was reached. A constant light duration (17L:7D) was maintained in the study room during the egg production period. There were at least five replicates of pairs (1 male + 1 female/pen section) in each mirex treatment plus controls (control, 7 pairs; 1 ppm, 5 pairs; 20 ppm, 6 pairs; 40 ppm, 6 pairs). Test birds began laying at 21 weeks and eggs were collected daily, coded and stored at approximately 19 C and 70% relative humidity. At four biweekly intervals of laying, eggs were incubated and candled after 1 and 3 weeks of incubation to measure embryonation and embryo survival rates. The eggs were incubated for 24 days, and we determined hatchability as well as survival of the hatchlings to 2 weeks. After (36 weeks) the experiment terminated, adult birds ( F 0 generation) were sacrificed with chloroform vapor for residue analysis. One replicate (3 mirex treatments plus control) of Fi generation hatchlings receiving dietary mirex was grown out for investigation of reproductive potential of second generation treatment birds. Ten-week-old quail were transferred'according to mirex treatments (0, 1, 20, and 40 ppm) into four (.6 x 1.5 m) wire pens
and placed in a poultry house in the necessity of reducing maintenance costs. Ulcerative enteritis was detected in Fj birds and treated with i H j O soluble quail antibiotic (Tylan, Elanco Chemicals). Birds were allowed to colony breed with sex ratios of five males to five females per pen. Two treatments (20 and 40 ppm) and the controls were lost at 16 weeks due to predation by dogs. One ppm treatment birds began laying at 21 weeks and the eggs were handled as previously described. Analytical Procedures. Fat (50-200 mg) and breast muscle (100-400 mg) were excised from two pairs of sacrificed F„ generation breeders/ treatment. The samples were weighed, freeze dried, reweighed, and ground with mortar and pestle using granular sodium sulfate as an abrasive. Mirex was extracted from breast muscles with hexane and from fat samples using petroleum ether. After grinding, the samples were collected in 125 ml flasks and fat samples were lightly boiled on a steam bath for 1 min. All samples were then shaken on a wrist-action shaker for 1 hr.. All the extracts were cleaned using standardized florisil column chromatography. Ten centimeters of 60/100 mesh PR-grade florisil topped with 2 cm of anhydrous sodium sulfate was placed into a 1 cm ID glass column fitted with a fritted glass disc. The florisil had been activated by oven heating at 150 C for 3 hr. After the extract was passed through Whatman-40 filter paper for removal of tissue debris, it was placed on the column and allowed to percolate into the florisil, at which time the mirex was eluted with 40 ml of 5% ethyl ether in petroleum ether. The columns were stripped with 40 ml of a 15% ethyl ether in petroleum ether solution before introduction of the next sample. The eluants were collected in 80 ml glass beakers, evaporated to dryness on a steam bath, and transferred into aluminum-lined-cap sample vials that contained 5 ml of nanograde hexane. Stock solutions were diluted when residues exceeded the linear response range of the electron-capture detector. Mirex residues were detected with a MicroTek 220 gas chromatograph equipped with a 63 Ni electron-capture detector. Separations and confirmations were carried out on 6 ft x .25 in OD U-shaped glass columns packed with the following materials: 10% DC-200 on Gas Chrom Q (80/100) and 1.5% OV-17, 1.95% QF1 on Gas Chrom Q (80/100). Nitrogen carrier gas flow rate was set at 100 cc/min and purge
MIREX IN BOBWHITE QUAIL flow at 10 cc/min. Injection port, column, and detector temperatures were 220, 210, and 300 C, respectively. The limit of detection of mirex in tissue samples was set at .01 ppm. Recovery rate for samples spiked with mirex was approximately 70%; residues are not corrected for recovery rates. All concentrations of mirex are reported on a dry weight basis. Factors for converting mirex residue data from dry-weight to wet-weight concentrations are fat, .77 and breast, .29. Data were analyzed using analysis of variance with the computer-based statistical analysis system (SAS) (Barr and Goodnight, 1971). The experiment was set up as a completely random design (CRD) in assigning mirex treatments to the quail pairs. Mirex residue data for breast and fat tissue of F 0 generation birds were analyzed as a 2 x 4 factorial. A regression analysis was performed to test the correlation between mirex levels in breast and adipose tissue versus dietary levels. Analysis of egg productivity data of F 0 breeder quail was accomplished with a one-way analysis of variance.
RESULTS Residues. Mirex residues were much higher in adipose tissue than breast muscle (Table 1), and males (P<.05) contained more mirex in fatty tissue (Fig. 1) and breast muscle (Fig. 2). There were positive correlations (P<.05) be-
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MIREX (PPM) IN THE DIET
FIG. 1. Mirex residues in F 0 generation breeder quail (fat ppm, dry weight basis). All Revalues are significant (P<.05). Estimating equations (Y = residues in ppm (Fat), Z = mirex concentration in the diet)— Males, Y = 7.82 + 8.54Z-Females, Y = .14 + 4.84Z.
tween dietary levels of mirex and residues of mirex deposited in fat (Fig. 1) and breast muscle (Fig. 2). A significant interaction (P<.05) was found between dietary levels of mirex X sex X mirex levels in fat, while the interaction was not significant (P>.05) using mirex residues in breast muscle. At the highest treatment level
TABLE 1. Mirex residues (ppm dry weight) in bobwbite quail F0 generation breeders Mirex
Fat ppm
Breast ppm
Sex
.257 .266 .311 .561
.000 .000 .000 .000
F M M F
6.027 6.528 13.767 20.209
.701 .094 .436 .143
F F M M
20 20 20 20
88.168 99.578 183.739 201.317
2.726 1.707 2.832 12.190
F F M M
40 40 40 40
143.773 246.640 305.318 379.871
2.863 2.330
F F M M
8.124 10.902
KENDALL ET AL.
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| 3.01
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22
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MIREX (PPM) IN THE DIET
FIG. 2. Mirex residues in F 0 generation breeder quail (breast ppm—dry weight basis). All R* values are significant (P<.05). Estimating equations (Y = residues L \ ppm (breast), Z = mirex-concentration in the diet)— Males, Y .53 + .25Z-Females, Y = .32 + .06Z.
(40 ppm) the relative difference in residues in adipose tissue and breast muscle of male vs. females was larger than at the lower levels of dietary mirex (Fig. 1 and 2). Productivity. Mirex did not significantly (P>.05) affect the percentage of quail embryos surviving to 3 weeks nor the percentage of eggs (P>.05) failing to hatch (Table 2). A difference was detected (P<.05) in the percentage of eggs which failed to embryonate. Eggs of 1 ppm treatment birds had a mean infertility of 30% compared to 17.5% for eggs of the 20 ppm treatment (Table 2). The controls had a 28% mean infertility of eggs which was approximately midway between the 1 and 20 ppm treatments; therefore, no fertility problems attributable to pesticide stress were indicated. In comparison to a 42% hatchability rate in controls, the 20 and 40 ppm mirex treatment birds produced eggs with higher hatchability rates of 58% and 52%, respectively (Table 2). Chick Survival. A similar pattern of chick survival occurred during the first two weeks of life in the mirex treatments as compared to the control group. The similarity in the slopes of the lines on the graph from days 1 to 6 indicated that mirex was not a factor in early chick
die-off. Mortality problems involving dietary mirex were not encountered through grow-out and egg laying in F 0 generation quail. A 2-week survival check (hatching to 2 weeks) was accomplished with Fi generation birds (Fig. 4). A plot of the means of two replicates of Fj chick data (Fig. 4) revealed a similar rate as the F 0 generation (Fig. 3). Fj generation treatment chicks showed approximately the same mortality pattern as controls between 1 to 6 days of age as noted by the slopes of the lines on the graph. The rate of die-off, however, was somewhat sharper in the Fj than in the F 0 generation birds. Ulcerative enteritis was detected in the F t quail, and this factor probably influenced the livability of the chicks. The number of Fj quail chicks surviving stabilized at day 7 and remained stable through the rest of the two-week check (Fig. 4). No mortality problems occurred through grow-out in Fj generation quail. Only the 1 ppm Fj generation quail were available for the egg production phase of the experiment. Eggs collected from Fi generation 1 ppm breeders were incubated, and no problems in embryonation, embryo survival, and hatchability rates were detected (Table 2). Higher percentages of eggs failing to hatch and eggs embryonating were detected in 1 ppm Ft vs. F 0 breeders. Due to the small number of eggs as well as only one treatment available, the authors suggest that extrapolation from the data be limited. However, it was particularly noted that 1 ppm Fj breeders had the ability to lay fertile eggs.
DISCUSSION Previous work on the toxicity of mirex in birds has demonstrated that this compound is of low subacute toxicity and at best of moderate chronic toxicity (Galbreath 1965; Heath et al, 1972; Hyde et al, 1973; Stickel et al, 1973). Data presented in this paper concerning the effects of dietary mirex on bobwhite quail do not alter these conclusions. Davison et al. (1975) in studies with Japanese quail reported that residues of mirex from whole body homogenate samples were approximately five times the mirex concentration in the diet. These results were similar to the work reported here; however care must be taken in interpretation because only fat and breast tissue were analyzed. Our results indicate that fatty tissue of female quail contained approxi-
MIREX IN BOBWHITE QUAIL
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TABLE 2. Productivity data of F0 and -F, generation bobwhite quail Treatment
Na
Eggsb
% Fail c
% Died d
% Hatch e
% F Hatch f
F 0 generation C 1 20 40
4 4 4 4
50.8 39.3 44.5 47.5
28.4 38.9 17.5 22.2
6.6 1.3 5.3 8.7
42.0 39.9 58.1 52.7
22.5 19.8 19.1 16.3
F, generation 1
2
28.0g
11.5
6.1
48.3
34.1
N = total number of replicates per treatment. Eggs = mean number of eggs collected for incubation over the 4 replicates of each treatment—pairs of quail/treatment = C-7, 1 ppm-5, 20 ppm-6, 40 ppm-6. Mean percentage of eggs which failed to embryonate when checked at 1 week. Mean percentage of embryos which had died when checked at 3 weeks. Mean percentage of eggs hatching. Mean percentage of eggs failing to hatch. *Eggs collected from 1 ppm Fl generation breeders housed in a colony breeding pen with a sex ratio of five males to five females.
mately five times the dietary mirex concentration. However, fatty tissue of males had up to 10 times the level of mirex found in the diet. This difference of mirex levels in male vs.female quail agree with data reported by Heath and Spann (1973); they noted that the egg can serve as a major elimination route for mirex in females. The ability of the female birds in eliminating mirex also showed up when residues in
breast muscle were determined. A trend of higher mirex levels in breast muscle of males was obvious; however, differences were not clearly defined except in the 40 ppm treatment. Control birds showed low mirex residues in fat
30
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|
55 o X
3 o I o
u < o
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-I
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a: UJ m
45
CONTROL
I
25
s => z
20ppm 20
15
Ippm 40ppm
10
C0NTR0L
X
40 10 F„ QUAIL AGE IN DAYS
FIG. 3. Two week survival check on F 0 generation birds (beginning on hatching date).
5 10 15 F, QUAIL AGE IN DAYS FIG. 4. Two week survival check on F , generation birds (beginning on hatching date) — (X of 2 replicates used for plot of data).
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KENDALL ET AL.
tissue but none was detected in breast muscle. This agreed with Putnam et al. (1974) who showed that poultry house dust on walls and ground litter can serve as a source of contamination of controls in pesticide studies. Contrary to the results of Heath and Spann (1973), no mortality problems were encountered with dosed male bobwhites even at the 40 ppm mirex level. Conflicting results may be due to differences in environmental conditions experienced by quail in the two experiments. Our birds were placed under little or no stress except from the pesticide whereas Heath and Spann (1973) conducted an outdoor colony breeding experiment. Reproductive studies reported here with bobwhite quail indicated that survival of chick embryos as well as the number of eggs failing to hatch were not deleteriously affected by mirex. These data agreed with results reported by Heath and Spann (1973) and Davison et al. (1975). In addition, our results show a lower percentage of infertile eggs and increased hatchability rates in the higher mirex treatments. We have no explanation for this other than the observation of large fat deposits in all of the experimental birds, which possibly enabled partitioning of ingested mirex into adipose tissue, thus allowing treatment birds to compete reproductively with controls even with high levels of mirex exposure. Some problems with egg fertility were noted, and we suggest that because of the small number of quail pairs available, extrapolation from the data should be limited. Fertility, survival of embryos to 3 weeks, and hatchability rates were determined for eggs collected from 1 ppm F t generation breeders. No reproductive problems attributable to pesticide stress were noted in these Fi breeder quail. Due to the fact that only one treatment (1 ppm) of Fj breeders was available, conclusions were limited in this phase of the experiment. However, the data show that Vx generation mirex treatment birds laid fertile eggs. Beginning on the day of hatching, 2-week chick survival checks were made on F 0 and Fj generation quail. During this early critical period (Rosene, 1969), mortality of bobwhite chicks was not related to exposure to mirex. Heath et al. (1972) also showed bobwhite chicks were not unusually sensitive to mirex; they listed an extremely high LC 5 0 of 2511 ppm mirex in 5-day diets of 2-week-old birds. In relating the research reported here to bobwhite quail found in mirex treated areas, Ken-
dall et al. (1977) found mirex residues in wild quail that were similar to those receiving 1 ppm mirex in this study. Baetcke et al. (1972) reported that adipose tissue of bobwhite quail contained as much as 3.148 ppm mirex 1 year after mirex bait application. Collins et al. (1974) reported that mirex residues in bobwhites collected from treated areas were similar to the concentrations discussed here. Comparison of mirex residues in our experimental quail, that exhibited no treatment effects in regard to mortality or reproductivity, to residues in wild bobwhites indicates no demonstrable threat of mirex to wild bobwhite quail populations. ACKNOWLEDGMENTS The authors express their gratitude to Lynn Luszcz who assisted in the pesticide analyses and Jay D. Hair for helpful suggestions during the course of the experiment. This research was supported by a grant from the South Carolina Plant Pest Regulatory Service.
REFERENCES Baetcke, K. P., J. D. Cain, and W. E. Poe, 1972. Mirex and DDT residues in wildlife and miscellaneous samples in Mississippi—1970. Pestic. Monit. J. 6: 14-22. Barr, A. J., and J. H. Goodnight, 1971. Statistical analysis system. North Carolina State University Press, Raleigh, NC. Bevenue, A., J. N. Ogata, L. S. Tengan, and J. W. Hylin, 1975. Mirex residues in wildlife and soils, Hawaiian Pineapple—Growing Areas—1972—1974. Pestic. Monit. J. 9:141-149. Borthwick, P. W., T. W. Duke, A. J. Wilson, Jr., J. I. Lowe, J. M. Patrick, Jr. and J. C. Oberheu, 1973. Accumulation and movement of mirex in selected estuaries of South Carolina, 1 9 6 9 - 7 1 . Pestic. Monit. J. 7 : 6 - 2 6 . Collins, H. L., G. P. Markin, and J. Davis, 1974. Residue accumulation in selected vertebrates following a single aerial application of mirex bait, Louisiana-1971-72. Pestic. Monit. J. 8:125-130. Davison, K. L., J. H. Cox, and C. K. Graham, 1975. The effect of mirex on reproduction of Japanese quail and on characteristics of eggs from Japanese quail and chickens. Arch. Environ. Contam. Toxicol. 3:84-95. Galbreath, E. H., 1965. Toxicity of mirex bait to certain species of wildlife. M.S. Thesis, University of Georgia. Heath, R. G., E. F. Hill, and J. F. Kreitzer, 1972. Comparative dietary toxicities of pesticides to birds. No. 152. U. S. Bur. Sport Fish, and Wildl. Heath, R. G., and J. W. Spann, 1973. Reproduction and related residues in birds fed mirex. Page 421— 434 in Pestic. Symp., 8th Inter-Amer. Conf. Toxicol. Occup. Med.
MIREX IN BOBWHITE QUAIL Hyde, K. M., J. B. Graves, A. B. Watts, and F. L. Bonner, 1973. Reproductive success of mallard ducks fed mirex. J. Wildl. Manage. 37:479-484. Kendall, R. J., R. Noblet, J. D. Hair and H. B. Jackson, 1977. Mirex residues in bobwhite quail after aerial application of bait for fire ant control, South Carolina-1975-76. Pestic. Monit. J. 11:64-68. Markin, G. P., H. L. Collins, and J. Davis, 1974. Residues of the insecticide mirex in terrestrial and aquatic invertebrates following a single aerial application of mirex bait, Louisiana—1971—1972. Pestic. Monit. J. 8:131-134. Mirex report, 1972. Report of the Mirex Advisory Committee, Environmental Protection Agency, Washington, DC. Naber, E. C , and G. W. Ware, 1965. Effects of kepone and mirex on reproductive performance in the lay-
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ing hen. Poultry Sci. 44:875-880. Putnam, E. M., R. N. Brewer, and G. J. Cottier, 1974. Low level pesticide contamination of soil and feed and its effect on broiler tissue residue. Poultry Sci. 53:1695-1698. Rosene, W., 1969. The bobwhite quail, its life and management. Rutgers University Press, New Brunswick, NJ. Stickel, L. F., and L. I. Rhodes, 1970. The thin eggshell problem. Page 31—35 in Proc. Symp., The Biological Impact of Pesticides in the Environmental Health Sciences Series No. 1, Oregon State University, Corvallis, OR. Stickel, W. H., J. A. Gaylen, R. A. Dyrland, and D. L. Hughes, 1973. Toxicity and persistence of mirex in birds. Page 437—466 in Pestic. Symp. 8th InterAmer. Conf. Toxicol. Occup. Med.
'Technical contribution no. 1430 from South Carolina Agricultural Experiment Station. 1978 Poultry Sci 57:1539-1545
no reproductive effects. However, Hyde et al (1973) determined mallard duckling {Anas platyrhynchos) survival to be reduced in a 100 ppm mirex treatment. Heath and Spann (1973) concluded that short term mirex dosages of 40 ppm in the feed of bobwhite quail induced no perceptible reproductive effects, but mortality of dosed males averaged 75% compared to 17% of control males. When chickens were given a diet containing 600 ppm mirex, hatching rate and chick survival were significantly reduced compared to controls (Naber and Ware, 1964). The literature as a whole is variable as to conclusive evidence of the effects of mirex in birds. Since the chlorinated hydrocarbon pesticides are such an integral part of the discussion of reproductive inhibition in many avian species (Stickel and Rhodes, 1970), further studies with low dietary levels of mirex in birds are needed. Bobwhite quail are important game animals and their food habits (Rosene, 1969) would allow frequent contact with mirex; therefore, pen studies with bobwhites over two generations were conducted to investigate reproductive effects of long term exposure to dietary mirex. At 1 ppm mirex treatment was designed to test the effects of a mirex dosage that bobwhites might contact in the field (Kendall et al, 1977). Dietary levels of 20 and 40 ppm were used to determine effects of high levels of
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KENDALL ET AL.
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mirex on reproductive success; these levels were considered to be sublethal to adults.
MATERIALS AND METHODS
Pen Studies. Fifty 1-day-old bobwhite chicks ( F 0 generation) were placed in each of four brooders (200 chicks in 3 mirex treatments plus control). Treatements consisted of mirex added to the diet as follows: 0 , 1 , 20, and 40 ppm (jug of mirex/g of diet). The mirex was dissolved in 25 ml of hexane, poured into the diet, and the feed was thoroughly mixed following evaporation of the hexane. The chicks received the mirex diets ad libitum beginning at 1-day of age and were continued on the diets through the grow-out and egg-laying periods. There was some early mortality in F 0 chicks; however, this appeared to be natural die-off and no antibiotics were administered. The 35-day-old F 0 quail were transferred from brooders into two quail battery breeding pens (GQF Manufacturing Company, Savannah, GA) for grow-out. The breeding pens were housed in a 2.1 x 4.3 m room maintained under constant photoperiod (12L:12D) and temperature (24 C). At 13 weeks F 0 birds were paired off and egg-laying was induced by increasing the light regimen 30 min/week until a 17 hr day was reached. A constant light duration (17L:7D) was maintained in the study room during the egg production period. There were at least five replicates of pairs (1 male + 1 female/pen section) in each mirex treatment plus controls (control, 7 pairs; 1 ppm, 5 pairs; 20 ppm, 6 pairs; 40 ppm, 6 pairs). Test birds began laying at 21 weeks and eggs were collected daily, coded and stored at approximately 19 C and 70% relative humidity. At four biweekly intervals of laying, eggs were incubated and candled after 1 and 3 weeks of incubation to measure embryonation and embryo survival rates. The eggs were incubated for 24 days, and we determined hatchability as well as survival of the hatchlings to 2 weeks. After (36 weeks) the experiment terminated, adult birds ( F 0 generation) were sacrificed with chloroform vapor for residue analysis. One replicate (3 mirex treatments plus control) of Fi generation hatchlings receiving dietary mirex was grown out for investigation of reproductive potential of second generation treatment birds. Ten-week-old quail were transferred'according to mirex treatments (0, 1, 20, and 40 ppm) into four (.6 x 1.5 m) wire pens
and placed in a poultry house in the necessity of reducing maintenance costs. Ulcerative enteritis was detected in Fj birds and treated with i H j O soluble quail antibiotic (Tylan, Elanco Chemicals). Birds were allowed to colony breed with sex ratios of five males to five females per pen. Two treatments (20 and 40 ppm) and the controls were lost at 16 weeks due to predation by dogs. One ppm treatment birds began laying at 21 weeks and the eggs were handled as previously described. Analytical Procedures. Fat (50-200 mg) and breast muscle (100-400 mg) were excised from two pairs of sacrificed F„ generation breeders/ treatment. The samples were weighed, freeze dried, reweighed, and ground with mortar and pestle using granular sodium sulfate as an abrasive. Mirex was extracted from breast muscles with hexane and from fat samples using petroleum ether. After grinding, the samples were collected in 125 ml flasks and fat samples were lightly boiled on a steam bath for 1 min. All samples were then shaken on a wrist-action shaker for 1 hr.. All the extracts were cleaned using standardized florisil column chromatography. Ten centimeters of 60/100 mesh PR-grade florisil topped with 2 cm of anhydrous sodium sulfate was placed into a 1 cm ID glass column fitted with a fritted glass disc. The florisil had been activated by oven heating at 150 C for 3 hr. After the extract was passed through Whatman-40 filter paper for removal of tissue debris, it was placed on the column and allowed to percolate into the florisil, at which time the mirex was eluted with 40 ml of 5% ethyl ether in petroleum ether. The columns were stripped with 40 ml of a 15% ethyl ether in petroleum ether solution before introduction of the next sample. The eluants were collected in 80 ml glass beakers, evaporated to dryness on a steam bath, and transferred into aluminum-lined-cap sample vials that contained 5 ml of nanograde hexane. Stock solutions were diluted when residues exceeded the linear response range of the electron-capture detector. Mirex residues were detected with a MicroTek 220 gas chromatograph equipped with a 63 Ni electron-capture detector. Separations and confirmations were carried out on 6 ft x .25 in OD U-shaped glass columns packed with the following materials: 10% DC-200 on Gas Chrom Q (80/100) and 1.5% OV-17, 1.95% QF1 on Gas Chrom Q (80/100). Nitrogen carrier gas flow rate was set at 100 cc/min and purge
MIREX IN BOBWHITE QUAIL flow at 10 cc/min. Injection port, column, and detector temperatures were 220, 210, and 300 C, respectively. The limit of detection of mirex in tissue samples was set at .01 ppm. Recovery rate for samples spiked with mirex was approximately 70%; residues are not corrected for recovery rates. All concentrations of mirex are reported on a dry weight basis. Factors for converting mirex residue data from dry-weight to wet-weight concentrations are fat, .77 and breast, .29. Data were analyzed using analysis of variance with the computer-based statistical analysis system (SAS) (Barr and Goodnight, 1971). The experiment was set up as a completely random design (CRD) in assigning mirex treatments to the quail pairs. Mirex residue data for breast and fat tissue of F 0 generation birds were analyzed as a 2 x 4 factorial. A regression analysis was performed to test the correlation between mirex levels in breast and adipose tissue versus dietary levels. Analysis of egg productivity data of F 0 breeder quail was accomplished with a one-way analysis of variance.
RESULTS Residues. Mirex residues were much higher in adipose tissue than breast muscle (Table 1), and males (P<.05) contained more mirex in fatty tissue (Fig. 1) and breast muscle (Fig. 2). There were positive correlations (P<.05) be-
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MIREX (PPM) IN THE DIET
FIG. 1. Mirex residues in F 0 generation breeder quail (fat ppm, dry weight basis). All Revalues are significant (P<.05). Estimating equations (Y = residues in ppm (Fat), Z = mirex concentration in the diet)— Males, Y = 7.82 + 8.54Z-Females, Y = .14 + 4.84Z.
tween dietary levels of mirex and residues of mirex deposited in fat (Fig. 1) and breast muscle (Fig. 2). A significant interaction (P<.05) was found between dietary levels of mirex X sex X mirex levels in fat, while the interaction was not significant (P>.05) using mirex residues in breast muscle. At the highest treatment level
TABLE 1. Mirex residues (ppm dry weight) in bobwbite quail F0 generation breeders Mirex
Fat ppm
Breast ppm
Sex
.257 .266 .311 .561
.000 .000 .000 .000
F M M F
6.027 6.528 13.767 20.209
.701 .094 .436 .143
F F M M
20 20 20 20
88.168 99.578 183.739 201.317
2.726 1.707 2.832 12.190
F F M M
40 40 40 40
143.773 246.640 305.318 379.871
2.863 2.330
F F M M
8.124 10.902
KENDALL ET AL.
1542 13.0
10.5 • •FEMALE QUAIL x-MALE QUAIL
;a.o
_ 5.5
| 3.01
-2.0
6
14
22
30
38
MIREX (PPM) IN THE DIET
FIG. 2. Mirex residues in F 0 generation breeder quail (breast ppm—dry weight basis). All R* values are significant (P<.05). Estimating equations (Y = residues L \ ppm (breast), Z = mirex-concentration in the diet)— Males, Y .53 + .25Z-Females, Y = .32 + .06Z.
(40 ppm) the relative difference in residues in adipose tissue and breast muscle of male vs. females was larger than at the lower levels of dietary mirex (Fig. 1 and 2). Productivity. Mirex did not significantly (P>.05) affect the percentage of quail embryos surviving to 3 weeks nor the percentage of eggs (P>.05) failing to hatch (Table 2). A difference was detected (P<.05) in the percentage of eggs which failed to embryonate. Eggs of 1 ppm treatment birds had a mean infertility of 30% compared to 17.5% for eggs of the 20 ppm treatment (Table 2). The controls had a 28% mean infertility of eggs which was approximately midway between the 1 and 20 ppm treatments; therefore, no fertility problems attributable to pesticide stress were indicated. In comparison to a 42% hatchability rate in controls, the 20 and 40 ppm mirex treatment birds produced eggs with higher hatchability rates of 58% and 52%, respectively (Table 2). Chick Survival. A similar pattern of chick survival occurred during the first two weeks of life in the mirex treatments as compared to the control group. The similarity in the slopes of the lines on the graph from days 1 to 6 indicated that mirex was not a factor in early chick
die-off. Mortality problems involving dietary mirex were not encountered through grow-out and egg laying in F 0 generation quail. A 2-week survival check (hatching to 2 weeks) was accomplished with Fi generation birds (Fig. 4). A plot of the means of two replicates of Fj chick data (Fig. 4) revealed a similar rate as the F 0 generation (Fig. 3). Fj generation treatment chicks showed approximately the same mortality pattern as controls between 1 to 6 days of age as noted by the slopes of the lines on the graph. The rate of die-off, however, was somewhat sharper in the Fj than in the F 0 generation birds. Ulcerative enteritis was detected in the F t quail, and this factor probably influenced the livability of the chicks. The number of Fj quail chicks surviving stabilized at day 7 and remained stable through the rest of the two-week check (Fig. 4). No mortality problems occurred through grow-out in Fj generation quail. Only the 1 ppm Fj generation quail were available for the egg production phase of the experiment. Eggs collected from Fi generation 1 ppm breeders were incubated, and no problems in embryonation, embryo survival, and hatchability rates were detected (Table 2). Higher percentages of eggs failing to hatch and eggs embryonating were detected in 1 ppm Ft vs. F 0 breeders. Due to the small number of eggs as well as only one treatment available, the authors suggest that extrapolation from the data be limited. However, it was particularly noted that 1 ppm Fj breeders had the ability to lay fertile eggs.
DISCUSSION Previous work on the toxicity of mirex in birds has demonstrated that this compound is of low subacute toxicity and at best of moderate chronic toxicity (Galbreath 1965; Heath et al, 1972; Hyde et al, 1973; Stickel et al, 1973). Data presented in this paper concerning the effects of dietary mirex on bobwhite quail do not alter these conclusions. Davison et al. (1975) in studies with Japanese quail reported that residues of mirex from whole body homogenate samples were approximately five times the mirex concentration in the diet. These results were similar to the work reported here; however care must be taken in interpretation because only fat and breast tissue were analyzed. Our results indicate that fatty tissue of female quail contained approxi-
MIREX IN BOBWHITE QUAIL
1543
TABLE 2. Productivity data of F0 and -F, generation bobwhite quail Treatment
Na
Eggsb
% Fail c
% Died d
% Hatch e
% F Hatch f
F 0 generation C 1 20 40
4 4 4 4
50.8 39.3 44.5 47.5
28.4 38.9 17.5 22.2
6.6 1.3 5.3 8.7
42.0 39.9 58.1 52.7
22.5 19.8 19.1 16.3
F, generation 1
2
28.0g
11.5
6.1
48.3
34.1
N = total number of replicates per treatment. Eggs = mean number of eggs collected for incubation over the 4 replicates of each treatment—pairs of quail/treatment = C-7, 1 ppm-5, 20 ppm-6, 40 ppm-6. Mean percentage of eggs which failed to embryonate when checked at 1 week. Mean percentage of embryos which had died when checked at 3 weeks. Mean percentage of eggs hatching. Mean percentage of eggs failing to hatch. *Eggs collected from 1 ppm Fl generation breeders housed in a colony breeding pen with a sex ratio of five males to five females.
mately five times the dietary mirex concentration. However, fatty tissue of males had up to 10 times the level of mirex found in the diet. This difference of mirex levels in male vs.female quail agree with data reported by Heath and Spann (1973); they noted that the egg can serve as a major elimination route for mirex in females. The ability of the female birds in eliminating mirex also showed up when residues in
breast muscle were determined. A trend of higher mirex levels in breast muscle of males was obvious; however, differences were not clearly defined except in the 40 ppm treatment. Control birds showed low mirex residues in fat
30
> to
|
55 o X
3 o I o
u < o
50
-I
z> O
a: UJ m
45
CONTROL
I
25
s => z
20ppm 20
15
Ippm 40ppm
10
C0NTR0L
X
40 10 F„ QUAIL AGE IN DAYS
FIG. 3. Two week survival check on F 0 generation birds (beginning on hatching date).
5 10 15 F, QUAIL AGE IN DAYS FIG. 4. Two week survival check on F , generation birds (beginning on hatching date) — (X of 2 replicates used for plot of data).
1544
KENDALL ET AL.
tissue but none was detected in breast muscle. This agreed with Putnam et al. (1974) who showed that poultry house dust on walls and ground litter can serve as a source of contamination of controls in pesticide studies. Contrary to the results of Heath and Spann (1973), no mortality problems were encountered with dosed male bobwhites even at the 40 ppm mirex level. Conflicting results may be due to differences in environmental conditions experienced by quail in the two experiments. Our birds were placed under little or no stress except from the pesticide whereas Heath and Spann (1973) conducted an outdoor colony breeding experiment. Reproductive studies reported here with bobwhite quail indicated that survival of chick embryos as well as the number of eggs failing to hatch were not deleteriously affected by mirex. These data agreed with results reported by Heath and Spann (1973) and Davison et al. (1975). In addition, our results show a lower percentage of infertile eggs and increased hatchability rates in the higher mirex treatments. We have no explanation for this other than the observation of large fat deposits in all of the experimental birds, which possibly enabled partitioning of ingested mirex into adipose tissue, thus allowing treatment birds to compete reproductively with controls even with high levels of mirex exposure. Some problems with egg fertility were noted, and we suggest that because of the small number of quail pairs available, extrapolation from the data should be limited. Fertility, survival of embryos to 3 weeks, and hatchability rates were determined for eggs collected from 1 ppm F t generation breeders. No reproductive problems attributable to pesticide stress were noted in these Fi breeder quail. Due to the fact that only one treatment (1 ppm) of Fj breeders was available, conclusions were limited in this phase of the experiment. However, the data show that Vx generation mirex treatment birds laid fertile eggs. Beginning on the day of hatching, 2-week chick survival checks were made on F 0 and Fj generation quail. During this early critical period (Rosene, 1969), mortality of bobwhite chicks was not related to exposure to mirex. Heath et al. (1972) also showed bobwhite chicks were not unusually sensitive to mirex; they listed an extremely high LC 5 0 of 2511 ppm mirex in 5-day diets of 2-week-old birds. In relating the research reported here to bobwhite quail found in mirex treated areas, Ken-
dall et al. (1977) found mirex residues in wild quail that were similar to those receiving 1 ppm mirex in this study. Baetcke et al. (1972) reported that adipose tissue of bobwhite quail contained as much as 3.148 ppm mirex 1 year after mirex bait application. Collins et al. (1974) reported that mirex residues in bobwhites collected from treated areas were similar to the concentrations discussed here. Comparison of mirex residues in our experimental quail, that exhibited no treatment effects in regard to mortality or reproductivity, to residues in wild bobwhites indicates no demonstrable threat of mirex to wild bobwhite quail populations. ACKNOWLEDGMENTS The authors express their gratitude to Lynn Luszcz who assisted in the pesticide analyses and Jay D. Hair for helpful suggestions during the course of the experiment. This research was supported by a grant from the South Carolina Plant Pest Regulatory Service.
REFERENCES Baetcke, K. P., J. D. Cain, and W. E. Poe, 1972. Mirex and DDT residues in wildlife and miscellaneous samples in Mississippi—1970. Pestic. Monit. J. 6: 14-22. Barr, A. J., and J. H. Goodnight, 1971. Statistical analysis system. North Carolina State University Press, Raleigh, NC. Bevenue, A., J. N. Ogata, L. S. Tengan, and J. W. Hylin, 1975. Mirex residues in wildlife and soils, Hawaiian Pineapple—Growing Areas—1972—1974. Pestic. Monit. J. 9:141-149. Borthwick, P. W., T. W. Duke, A. J. Wilson, Jr., J. I. Lowe, J. M. Patrick, Jr. and J. C. Oberheu, 1973. Accumulation and movement of mirex in selected estuaries of South Carolina, 1 9 6 9 - 7 1 . Pestic. Monit. J. 7 : 6 - 2 6 . Collins, H. L., G. P. Markin, and J. Davis, 1974. Residue accumulation in selected vertebrates following a single aerial application of mirex bait, Louisiana-1971-72. Pestic. Monit. J. 8:125-130. Davison, K. L., J. H. Cox, and C. K. Graham, 1975. The effect of mirex on reproduction of Japanese quail and on characteristics of eggs from Japanese quail and chickens. Arch. Environ. Contam. Toxicol. 3:84-95. Galbreath, E. H., 1965. Toxicity of mirex bait to certain species of wildlife. M.S. Thesis, University of Georgia. Heath, R. G., E. F. Hill, and J. F. Kreitzer, 1972. Comparative dietary toxicities of pesticides to birds. No. 152. U. S. Bur. Sport Fish, and Wildl. Heath, R. G., and J. W. Spann, 1973. Reproduction and related residues in birds fed mirex. Page 421— 434 in Pestic. Symp., 8th Inter-Amer. Conf. Toxicol. Occup. Med.
MIREX IN BOBWHITE QUAIL Hyde, K. M., J. B. Graves, A. B. Watts, and F. L. Bonner, 1973. Reproductive success of mallard ducks fed mirex. J. Wildl. Manage. 37:479-484. Kendall, R. J., R. Noblet, J. D. Hair and H. B. Jackson, 1977. Mirex residues in bobwhite quail after aerial application of bait for fire ant control, South Carolina-1975-76. Pestic. Monit. J. 11:64-68. Markin, G. P., H. L. Collins, and J. Davis, 1974. Residues of the insecticide mirex in terrestrial and aquatic invertebrates following a single aerial application of mirex bait, Louisiana—1971—1972. Pestic. Monit. J. 8:131-134. Mirex report, 1972. Report of the Mirex Advisory Committee, Environmental Protection Agency, Washington, DC. Naber, E. C , and G. W. Ware, 1965. Effects of kepone and mirex on reproductive performance in the lay-
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ing hen. Poultry Sci. 44:875-880. Putnam, E. M., R. N. Brewer, and G. J. Cottier, 1974. Low level pesticide contamination of soil and feed and its effect on broiler tissue residue. Poultry Sci. 53:1695-1698. Rosene, W., 1969. The bobwhite quail, its life and management. Rutgers University Press, New Brunswick, NJ. Stickel, L. F., and L. I. Rhodes, 1970. The thin eggshell problem. Page 31—35 in Proc. Symp., The Biological Impact of Pesticides in the Environmental Health Sciences Series No. 1, Oregon State University, Corvallis, OR. Stickel, W. H., J. A. Gaylen, R. A. Dyrland, and D. L. Hughes, 1973. Toxicity and persistence of mirex in birds. Page 437—466 in Pestic. Symp. 8th InterAmer. Conf. Toxicol. Occup. Med.