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Dieldrin, Ca and P Balance, and Characteristics of the Egg in the Quail (Coturnix coturnix japonica)
Dieldrin, Ca and P Balance, and Characteristics of the Egg in the Quail (Coturnix coturnix japonica) M«M. ANDUJAR, G. VARELA and M«P. NAVARRO Instituto de Nutrition1, Consejo Superior de Investigaciones Cientificas, Facultad de Farmacia, Madrid, Espana (Received for publication July 26, 1977)
INTRODUCTION F o o d c o n t a m i n a t i o n by pesticides affects birds since their diet foodstuffs m a y be poll u t e d . Lindane, D D T , and dieldrin molecules are c o m m o n l y found in foods (Velle, 1 9 7 1 ; Hill et al., 1 9 7 3 ) . O n c e these substances have been ingested by t h e bird they are easily a c c u m u lated in t h e fatty tissues, and t h e y have been detected in p o u l t r y p r o d u c t s (Rousseau et al., 1971). Dramatic situations have been described concerning t h e levels of environmental c o n t a m i nation d u e t o chlorinated h y d r o c a r b o n pesticides, highly d e t r i m e n t a l t o t h e p o p u l a t i o n s of s o m e species of birds of prey in t h e United States and western E u r o p e , where there is a high level of D D T and dieldrin c o n t a m i n a t i o n (Ratcliffe, 1 9 6 7 ; Hickey and A n d e r s o n , 1 9 6 8 ) . T h e deficiencies t h a t were observed in reproduction in b o t h birds which develop in such ecosystems and in birds which were e x p e r i m e n tally treated with chlorinated h y d r o c a r b o n s pesticides, were associated, a m o n g o t h e r manifestations, with an impaired egg-laying (Neill et al., 1 9 6 9 ) with alterations in t h e characteristics of t h e egg shell, decrease in its weight ( A d a m e c et al., 1972) or thickness (Bitman et al, 1 9 7 0 ) , as well as an increased e m b r y o n i c m o r t a l i t y (Muller and L o c k m a n , 1972). Nevertheless, t h e
1 Address: Instituto de Nutricion, C.S.I.C. Facultad de Farmacia Ciudad Universitaria, Madrid-3 (Espana).
1978 Poultry Sci 57:596-602
findings are highly variable and we find t h a t Davison and Sell ( 1 9 7 2 ) as well as other workers did n o t observe any variations in t h e n u m b e r or weight of t h e eggs of hens t h a t had been treated with D D T or dieldrin. Alterations m a y be different due t o t h e chlorinated h y d r o c a r b o n s in t h e metabolism of calcium (Andujar, 1 9 7 6 ) which would be closely linked t o t h e r e p r o d u c t i v e physiology in t h e case of an egg-laying, b u t t h e exact m e c h a n i s m of its interference has n o t been described. Peakall ( 1 9 7 0 ) observed t h a t high levels of DDT decreased t h e deposition of m a r r o w b o n e in ringdoves, which t h e a u t h o r considered to be related t o t h e decrease in circulation levels of estradiol which was also found t o exist (Conney, 1 9 6 7 ) . However, Bitman et al. ( 1 9 6 9 ) did n o t find any modifications in t h e b o n e mineral c o n t e n t s of quail treated with t h e same dose of t h e same pesticide. Neither were levels of 1 0 m g of dieldrin/kg of b o d y weight in chicks capable of depleting t h e calcium c o n t e n t s of their tibias (Muller and L o c k m a n , 1 9 7 1 ) . Perhaps t h e m o s t intense effects t h a t are a t t r i b u t e d t o t h e chlorinated h y d r o c a r b o n s are the alterations in t h e characteristics of t h e egg shell as a result of faulty calcification. Bitman et al. ( 1 9 7 0 ) and Pocker et al. ( 1 9 7 1 ) a t t r i b u t e d these anomalies t o a decrease in t h e activity of the carbonic a n h y d r a s e in the uterine shell gland. On t h e basis of t h e f o r e m e n t i o n e d findings on t h e widespread use of dieldrin and t h e high sensitivity of birds to this pesticide, we
596
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ABSTRACT A study was made of the Ca and P balance, Ca and P content in the femur, physical characteristics of the egg, mineral structure of the shell, and the number of eggs in quails treated with dieldrin (20 mg/kg of diet) for 48 days. The diet contained 3.24% Ca and 0.72% P. The Ca and P balance, the bone contents of Ca and P and the calcemia in the males were not changed by the pesticide. In the females, the pesticide decreased the amount of excreted Ca and Ca in the egg, for which reason the coefficient of nutritive utilization (CNU), the coefficient of corporal retention (CCR), and the Ca level in the femur were greater in treated laying quails. The calcemia remained stable, and the balance of P was not significantly modified by the dieldrin. The study of weight, size, and strength of the egg did not reveal any influence of the dieldrin, and egg production fluctuated throughout the test period.
DIELDRIN, Ca AND P BALANCE IN COTURNIX EGGS
attempted to make a study of its possible interaction with the nutritional utilization of Ca and P, with egg-laying, and with some of the physical and chemical characteristics of the egg.
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Forty quail (twenty adult males and twenty laying females) were divided into four groups of homogeneous weight, composed of ten animals each. One group of males and another of females acted as controls against the other two groups which ingested the same diet with an addition of dieldrin at the rate of 20 mg/kg of diet. The treatment period lasted 48 days. During that time the birds drank deionized water and ate food on an ad lib. basis. The experiment was carried out in a heat controlled chamber (21 ± 2 C) lit 24 hours a day in which the birds were accomodated in individual cells with a common drinking trough, an external feeding-trough, and individual systems for the collection of excreta and eggs. Throughout 48 days of the experiment, the egg-laying and the weight of the egg was recorded. The size and the resistance of the egg shell were determined on the middle days of the experimental period (20 to 28) and on the last eight days (40 to 48), in which Ca and P balance was also carried out. The experimental diet contained in dry matter 23.1% protein, 6.5 ether extract, 4.5% crude fiber, 55.2% nitrogen free extract materials, 10.72% minerals (3.24% Ca, 0.72% P and Ca/P: 4.5), and 1.7 mg/kg of vitamin D 3 . Calcium was determined by atomic absorption spectrophotometry using a solution of strontiun chloride to avoid the possible interference of other ions. The phosphorus was determined photocolorimetrically. The size of the egg was determined by measuring its two diameters (large diameter = D, and smaller diameter = d) with the help of a calipher. Resistance of the eggshell was expressed by the minimum weight that produces its breakage, but not its fragmentation. In order to study the structure of the shell, we followed the petrographic technique adopted by Navarro and Murillo (1976). On the basis of the analytic data of Ca or P that was ingested (I), excreted (E x ), and transferred to the egg (E), the nutritional utilization of these two minerals was determined by calculating the following index, according to Navarro and Murillo (1976).
598
M«M. ANDUJAR, G. VARELA AND M«P. NAVARRO
Dietary calcium utilized: U = I~E X Calcium retained as body calcium: R = U-E Coefficient of nutritive utilization:
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We have also estimated the percentage of calcium retained (R) or transferred to the egg (E) in relation to that utilized (U):
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CIE= —X 100 I S
Percentage of calcium retained = —x 100 U Percentage of calcium transferred to the egg
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= — X 100 U In male quails, the utilized and retained Ca or P are the same. The results were analyzed by analysis of variance. RESULTS AND DISCUSSION Although dieldrin, when added to the diet of egg-laying quail in a concentration of 20 mg per kg of diet, caused a slight decrease in the amount of Ca ingested (Table 1), this decrease was not statistically significant. The larger changes took place in the elimination of Ca, which decreased in all of its routes: egg-laying, calcium content of the egg (P<.05), and excretion (P<.01) (Table 1); however, the decrease was not large for any of these routes. Nevertheless, it should be pointed out that excretion was the route that was most affected in quantitative terms. Peakall (1969) and Nowicki et al. (1972) have suggested that the action of certain chlorinated hydrocarbons, and specifically that of DDT, is not associated with an effect on gastrointestinal absorption of Ca, but rather, exerted through the Ca that is already absorbed, due to anomalies in its storage and mobilization. Since in birds the urine and feces are excreted together, it is impossible at this time for us to determine whether the forementioned effects occur at a digestive level because the intestinal absorption increases, or on the
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other hand, it is the result of a metabolic phenomenon which would lead to a decreased urinary excretion, or perhaps, a combined action of both effects. In fact since Ca elimination decreased while ingested Ca did not in the same proportions, both responses led to an increase in CNU value (Table 1), utilization, and body Ca retention. These effects were demonstrated in relative terms with respect to the ingesta (CCR) well as in absolute terms expressed by the rise in calcium content of the femur in quails that ingested the pesticide (Table 2). The lower elimination of egg Ca did not result in a significant decrease relative to the amount ingested, since the differences between the two lots were small and also because the amount of Ca ingested by the treated birds was also lower (Table 1). The fraction of absorbed Ca that was retained by the body increased (P<.01) with pesticide ingestion, while the portion that was incorporated to the egg underwent a significant decrease ( P < 0 1 ) (Table 1). The forementioned decrease of Ca in the egg due to dieldrin resulted in a diminishing of the Ca in the egg shell, (Table 4). This is in agreement with observations of Lehner and Egbert (1969) in ducks, although it is in disagreement with the observations of Davison and Sell (1972) in hens. The P of the whole egg remained unaltered. We do not think that this decrease in Ca content of the shell can be attributed to a real lack of this cation in the organism, since
contrary to the bone depletion observed by Peakall (1970) with DDT, our results pointed to an increase of the Ca retained in the skeleton, because the quails fed with dieldrin showed a slight increase (P<.05) in the amount of Ca and P in the femur, but the Ca/P relationship was not altered (Table 2). In other studies with DDT (Bitman et ai, 1969) and dieldrin (Muller et ai, 1971), no changes were observed in the bone either. Furthermore, the plasma calcium values in our tests, as those
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FIG. 2. Fluctuations of the egg-laying in quails that received an experimental diet (o) and the same diet with the addition of 20 mg of dieldrin/kg (•). The ordinates indicate the average number of eggs laid per quail/day; the abscissas indicate the experimental days divided into 4 day intervals; the calculation of the daily egg-laying average is carried out in each of the forementioned intervals of time.
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FIG. 1. View under the petrographic microscope (500X; parallel nicols) of sections parallel to the surface of the egg shell of quail that received for 48 days: a control diet (A) and the same diet with the addition of dieldrin (20 mg/kg of diet) (B) The prismatic nodules, which correspond to the column of calcite microcrystals (A), can be seen apparently fragmented after the treatment with the pesticide (B).
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It is true that dieldrin did not alter significantly the nutritive utilization of P in our tests. Nevertheless the results for P, judged by the
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reported by Call and Call (1974) on the same species, did not undergo any variations (Table 2). Our results cannot be explained by the hypothesis that alterations in Ca metabolism are mediated by estrogenic degradation caused by chlorinated hydrocarbons (Peakall, 1970). Since calcemia was not altered and the content of Ca and P in the femur did not decrease, it is difficult for us in our experimental conditions to consider the possibility of a mechanism based on the decrease of estrogens concentration in blood. As it is generally known (Taylor, 1966; Navarro and Murillo, 1976), this concentration is one of the main factors that take part in the maintenance of the calcemia and bone Ca. Neither the size nor the total weight of the egg were significantly affected by the treatment at the periods (20 to 28 days and 40 to 48 days) when the determinations were carried out (Table 4). This observation concurs with Davison and Sell (1972) regarding to DDT. The breaking strength by weight of the shell was not affected by the pesticide (Table 4). Cecil et al. (1971) reported the same observations on quails treated with DDT. The structure of the shell of the quails egg, which is similar to that described by Taylor (1970) in hens and Navarro and Murillo (1976) in quails, showed some differences due to the dieldrin, including an apparent decrease in the size of the calcite microcrystal nodules with a parallel increase in number and a decrease of the pores per surface unit (Figure 1). These results are similar to those given for birds treated with DDT (Peakall et al., 1973). The frequency of egg-laying (Figure 2) showed fluctuations throughout the experimental period. It was identical in the two groups of quail at the beginning of the test period, but in quail fed with dieldrin it decreased slightly and progressively as the treatment continued, until it finally became significantly lower (P<.05) towards the end of the first month (middle days of the experimental period). Nevertheless, these large differences gradually disappeared. The decrease observed in the number of eggs laid per treated quail was not statistically significant. Similar egg-laying fluctuations, which are difficult to explain, were observed by Bitman et al. (1969) and Cecil et al. (1971) for the same species treated with DDT.
601
DIELDRIN, Ca AND P BALANCE IN COTURNIX EGGS
animal (Sell and Davison, 1973), and dose (Varela et al., 1975). Furthermore, feed consumption by the male quail was much smaller than that of females, and the amount of pesticide ingested by the males was, in consequence, much lower.
parameters and coefficients controlled (ingested, excreted, transferred to the egg, CNU, CCR, CIE, etc. (Table 3)) showed the same trends as the more distinct effects noted in Ca. The treatment with dieldrin did not significantly modify the amount of Ca and P ingested by the male quail. The overall excretion of the two minerals and body retention for both minerals did not undergo any marked alterations either (Tables 1 and 3). The contents of Ca and P in the femur did not reveal any detrimental effect in the treated birds, as would be expected, since body retention for both minerals was not affected, and in addition, the Ca/P bone relationship remained constant with a value of 2.1. Neither did the pesticide alter the calcemia (Table 2). In short, Ca and P balances in the male quail did not seem to be affected by dieldrin. Pesticide action depends to a large extent on a series of factors such as species, sex, age of the
REFERENCES
TABLE A.—Effect of dieldrin (20 ppm in the diet) on egg production of the egg in quails
characteristics
Diet a C + dieldrin (20 ppm)
C
Means Average number of eggs per quail and days Days 1-48 Days 2 0 - 2 8 Days 4 0 - 4 8 Average weight (g) Days 2 0 - 2 8 Days 4 0 - 4 8
Means
SE
SE
.96 .94 .98
.01 .03 .02
.91 .80 .92
.03'
9.89 10.02
.21 .20
9.51 9.94
.19 .17
3.11 2.44
.03 .04
3.03 2.42
.03 .02
3.13 2.43
.02 .02
3.09 2.42
.02 .01
.03 .03
Average size (cm) Days 2 0 - 2 8 DO
db Days 4 0 - 4 8 D d Breaking strength (g) Days 2 0 - 2 8 Days 4 0 - 4 8 Ca in shell (%) P in egg (mg)
1,190 1,218 26
16.6
Diet C is a commercial diet which contains 3.24% Ca and .72% P. " D " is longer diameter and "d" is shorter diameter. *P<.05 comparing with untreated quails.
1,250 1,200
112 83 .8 .6
23*
15.7
94 100 1.0 .7
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Adamec, O., M. Ledec, J. Kosutzky, and E. Bobakova, 1972. Disorders of egg shell formation in hens and ducks exposed to technical DDT. Zivozisma Vyroba, 17:835-840. Andujar, M.a M., 1976. Algunos aspectos de la utilizacion nutritiva de una dieta en la codorniz; influencia sobre la misma de los contaminantes organoclorados. Tesis Doctoral. Univ. Granada, EspaiTa. Bitman, J., H. C. Cecil, and G. F. Fries, 1970. DDTinduced inhibition of avian shell gland carbonic anhydrase: a mechanism for thin eggshells. Science, 168:594-596. Bitman, J., H. C. Cecil, S. J. Harris, and G. F. Fries, 1969. DDT induces a decrease in eggshell calcium. Nature, 244:44-46.
602
M<*M. ANDUJAR, G. VARELA AND MaP. NAVARRO Norman, 1972. Inhibition of biological activity of cholecalciferol (vitamin D 3 ) by o,p'-DDT or p,p'DDT in rachitic cockerel. J. Agr. Food Chem. 20:376-380. Peakall, D. B., 1969. Effect of DDT on calcium uptake and vitamin D metabolism in birds. Nature, 224:1219-1220. Peakall, D. B., 1970. p,p'-DDT: effect on calcium metabolism and concentration of estradiol in the blood. Science, 168:592-594. Peakall, D. B., J. L. Linger, R. W. Risebrough, J. B. Pritchard, and W. B. Kinter, 1973. DDE-induced egg-shell thinning: structural and physiological effects in three species. Com. Gen. Pharmacol. 4:305-313. Pocker, T., M. Beug, N. Watamori, and C. Guilbert, 1971. Enzyme inhibition by DDT questioned. Chem. Eng. News, 49:20-27. Ratcliffe, D. A., 1967. Decrease in eggshell weight in certain birds of prey. Nature, 215:208—210. Rousseau, M., J. Bourtallier, et Y. Taliercio, 1971. Pollution des oeufs par des residus de pesticides organochlores (frequence, importance, origine). Bull. Acad. Vet., France, XLIV: 2 7 - 3 2 . Sell, J. L., and K. L. Davison, 1973. Changes in the activities of hepatic microsomal enzymes caused by DDT and dieldrin. Fed. Proc. 32:2003-2009. Taylor, T. G., 1966. The endocrine control of calcium metabolism in the fowl. In Physiology of domestic fowl. Edited by C. Horton-Smidi and E. C. Amoroso, ed. Oliver, and Boyd, Edimburgh and London: pp. 199-202. Taylor, T. G., 1970. How an eggshell is made. Sci. Am. 222:89-95. Varela, G., A. Torralba, and J. Escriva, 1975. Influence of prolonged ingestion of some organochlorated pesticides on digestibility and nutritive value of a diet in rats. Proc. Nutr. Soc. 34.-93A. Velle, W., 1971. How the egg is formed and the effect of insecticides on reproduction in birds. Nor. Vet. Tidsskr. 83:448-453.
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Call, D. J., and J. K. J. Call, 1974. Blood chemistry of Japanese quail fed dieldrin. Poultry Sci. 53:54—56. Cecil, H. C, J. Bitman, and S. J. Harris, 1971. Effects of dietary p,p'-DDT and p,p'-DDE on egg production and egg shell characteristics of Japanese quail receiving an adequate calcium diet. Poultry Sci. 50:657-659. Conney, A. H., R. M. Welch, R. Kuntzman, and J. J. Burns, 1967. Effects of pesticides on drug and steroid metabolism. Clin. Pharmacol. Ther. 8:2-10. Davison, K. L., and J. L. Sell, 1972. Dieldrin and p,p'-DDT effects on egg production and egg shell thickness of chickens. Bull. Environ. Contam. Toxicol. 7 : 9 - 1 8 . Hickey, J. J., and D. W. Anderson, 1968. Chlorinated hydrocarbons and eggshell changes in raptorial and fish-eating birds. Science, 162:271-273. Hill, E. G., F. B. Fishwick, and R. M. Thompson, 1973. Pesticide residues in foodstuffs in Great Britain: organochlorine residues in imported cereals, nuts, pulses, and animal foodstuffs. Pestc. Sci. 4:33-39. Lehner, P. N., and A. Egbert, 1969. Dieldrin and eggshell t h i c k n e s s in d u c k s . N a t u r e , 224:1218-1219. Muller, H. D., and D. C. Lockman, 1971. The influence of dieldrin on bone calcium deposition in domestic chicks. Poultry. Sci. 1611 (Abstr.). Muller, H. D., and D. C. Lockman, 1972. Fecundity and progeny growth following subacute insecticide ingestion by the mallard. Poultry Sci. 51:239—241. Navarro, M.a P., and A. Murillo, 1976. Calcium balance in the quail (Coturnix coturnix japonica). 1. Influence of sex and diethylstilbestrol. Poultry Sci. 55:2201-2209. Neill, D. D., J. V. Shutze, and H. D. Muller, 1969. The influence of feeding dieldrin and parathion to the Hungarian partridge on reproduction. 58th. Ann. Mtng. Poultry Sci. Ass., Inc. Poultry Sci. 48:1850. Nowicki, H. G., R. G. Wong, J. F. Myrtle, and A. W.
INTRODUCTION F o o d c o n t a m i n a t i o n by pesticides affects birds since their diet foodstuffs m a y be poll u t e d . Lindane, D D T , and dieldrin molecules are c o m m o n l y found in foods (Velle, 1 9 7 1 ; Hill et al., 1 9 7 3 ) . O n c e these substances have been ingested by t h e bird they are easily a c c u m u lated in t h e fatty tissues, and t h e y have been detected in p o u l t r y p r o d u c t s (Rousseau et al., 1971). Dramatic situations have been described concerning t h e levels of environmental c o n t a m i nation d u e t o chlorinated h y d r o c a r b o n pesticides, highly d e t r i m e n t a l t o t h e p o p u l a t i o n s of s o m e species of birds of prey in t h e United States and western E u r o p e , where there is a high level of D D T and dieldrin c o n t a m i n a t i o n (Ratcliffe, 1 9 6 7 ; Hickey and A n d e r s o n , 1 9 6 8 ) . T h e deficiencies t h a t were observed in reproduction in b o t h birds which develop in such ecosystems and in birds which were e x p e r i m e n tally treated with chlorinated h y d r o c a r b o n s pesticides, were associated, a m o n g o t h e r manifestations, with an impaired egg-laying (Neill et al., 1 9 6 9 ) with alterations in t h e characteristics of t h e egg shell, decrease in its weight ( A d a m e c et al., 1972) or thickness (Bitman et al, 1 9 7 0 ) , as well as an increased e m b r y o n i c m o r t a l i t y (Muller and L o c k m a n , 1972). Nevertheless, t h e
1 Address: Instituto de Nutricion, C.S.I.C. Facultad de Farmacia Ciudad Universitaria, Madrid-3 (Espana).
1978 Poultry Sci 57:596-602
findings are highly variable and we find t h a t Davison and Sell ( 1 9 7 2 ) as well as other workers did n o t observe any variations in t h e n u m b e r or weight of t h e eggs of hens t h a t had been treated with D D T or dieldrin. Alterations m a y be different due t o t h e chlorinated h y d r o c a r b o n s in t h e metabolism of calcium (Andujar, 1 9 7 6 ) which would be closely linked t o t h e r e p r o d u c t i v e physiology in t h e case of an egg-laying, b u t t h e exact m e c h a n i s m of its interference has n o t been described. Peakall ( 1 9 7 0 ) observed t h a t high levels of DDT decreased t h e deposition of m a r r o w b o n e in ringdoves, which t h e a u t h o r considered to be related t o t h e decrease in circulation levels of estradiol which was also found t o exist (Conney, 1 9 6 7 ) . However, Bitman et al. ( 1 9 6 9 ) did n o t find any modifications in t h e b o n e mineral c o n t e n t s of quail treated with t h e same dose of t h e same pesticide. Neither were levels of 1 0 m g of dieldrin/kg of b o d y weight in chicks capable of depleting t h e calcium c o n t e n t s of their tibias (Muller and L o c k m a n , 1 9 7 1 ) . Perhaps t h e m o s t intense effects t h a t are a t t r i b u t e d t o t h e chlorinated h y d r o c a r b o n s are the alterations in t h e characteristics of t h e egg shell as a result of faulty calcification. Bitman et al. ( 1 9 7 0 ) and Pocker et al. ( 1 9 7 1 ) a t t r i b u t e d these anomalies t o a decrease in t h e activity of the carbonic a n h y d r a s e in the uterine shell gland. On t h e basis of t h e f o r e m e n t i o n e d findings on t h e widespread use of dieldrin and t h e high sensitivity of birds to this pesticide, we
596
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on June 18, 2015
ABSTRACT A study was made of the Ca and P balance, Ca and P content in the femur, physical characteristics of the egg, mineral structure of the shell, and the number of eggs in quails treated with dieldrin (20 mg/kg of diet) for 48 days. The diet contained 3.24% Ca and 0.72% P. The Ca and P balance, the bone contents of Ca and P and the calcemia in the males were not changed by the pesticide. In the females, the pesticide decreased the amount of excreted Ca and Ca in the egg, for which reason the coefficient of nutritive utilization (CNU), the coefficient of corporal retention (CCR), and the Ca level in the femur were greater in treated laying quails. The calcemia remained stable, and the balance of P was not significantly modified by the dieldrin. The study of weight, size, and strength of the egg did not reveal any influence of the dieldrin, and egg production fluctuated throughout the test period.
DIELDRIN, Ca AND P BALANCE IN COTURNIX EGGS
attempted to make a study of its possible interaction with the nutritional utilization of Ca and P, with egg-laying, and with some of the physical and chemical characteristics of the egg.
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T3 B
Forty quail (twenty adult males and twenty laying females) were divided into four groups of homogeneous weight, composed of ten animals each. One group of males and another of females acted as controls against the other two groups which ingested the same diet with an addition of dieldrin at the rate of 20 mg/kg of diet. The treatment period lasted 48 days. During that time the birds drank deionized water and ate food on an ad lib. basis. The experiment was carried out in a heat controlled chamber (21 ± 2 C) lit 24 hours a day in which the birds were accomodated in individual cells with a common drinking trough, an external feeding-trough, and individual systems for the collection of excreta and eggs. Throughout 48 days of the experiment, the egg-laying and the weight of the egg was recorded. The size and the resistance of the egg shell were determined on the middle days of the experimental period (20 to 28) and on the last eight days (40 to 48), in which Ca and P balance was also carried out. The experimental diet contained in dry matter 23.1% protein, 6.5 ether extract, 4.5% crude fiber, 55.2% nitrogen free extract materials, 10.72% minerals (3.24% Ca, 0.72% P and Ca/P: 4.5), and 1.7 mg/kg of vitamin D 3 . Calcium was determined by atomic absorption spectrophotometry using a solution of strontiun chloride to avoid the possible interference of other ions. The phosphorus was determined photocolorimetrically. The size of the egg was determined by measuring its two diameters (large diameter = D, and smaller diameter = d) with the help of a calipher. Resistance of the eggshell was expressed by the minimum weight that produces its breakage, but not its fragmentation. In order to study the structure of the shell, we followed the petrographic technique adopted by Navarro and Murillo (1976). On the basis of the analytic data of Ca or P that was ingested (I), excreted (E x ), and transferred to the egg (E), the nutritional utilization of these two minerals was determined by calculating the following index, according to Navarro and Murillo (1976).
598
M«M. ANDUJAR, G. VARELA AND M«P. NAVARRO
Dietary calcium utilized: U = I~E X Calcium retained as body calcium: R = U-E Coefficient of nutritive utilization:
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Coefficient of corporal calcium retention: q q C C R = y X 100 Coefficient of calcium incorporation to the e gg :
We have also estimated the percentage of calcium retained (R) or transferred to the egg (E) in relation to that utilized (U):
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CIE= —X 100 I S
Percentage of calcium retained = —x 100 U Percentage of calcium transferred to the egg
IA
TH
rH
X
00
= — X 100 U In male quails, the utilized and retained Ca or P are the same. The results were analyzed by analysis of variance. RESULTS AND DISCUSSION Although dieldrin, when added to the diet of egg-laying quail in a concentration of 20 mg per kg of diet, caused a slight decrease in the amount of Ca ingested (Table 1), this decrease was not statistically significant. The larger changes took place in the elimination of Ca, which decreased in all of its routes: egg-laying, calcium content of the egg (P<.05), and excretion (P<.01) (Table 1); however, the decrease was not large for any of these routes. Nevertheless, it should be pointed out that excretion was the route that was most affected in quantitative terms. Peakall (1969) and Nowicki et al. (1972) have suggested that the action of certain chlorinated hydrocarbons, and specifically that of DDT, is not associated with an effect on gastrointestinal absorption of Ca, but rather, exerted through the Ca that is already absorbed, due to anomalies in its storage and mobilization. Since in birds the urine and feces are excreted together, it is impossible at this time for us to determine whether the forementioned effects occur at a digestive level because the intestinal absorption increases, or on the
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599
DIELDRIN, Ca AND P BALANCE IN COTURNIX EGGS
other hand, it is the result of a metabolic phenomenon which would lead to a decreased urinary excretion, or perhaps, a combined action of both effects. In fact since Ca elimination decreased while ingested Ca did not in the same proportions, both responses led to an increase in CNU value (Table 1), utilization, and body Ca retention. These effects were demonstrated in relative terms with respect to the ingesta (CCR) well as in absolute terms expressed by the rise in calcium content of the femur in quails that ingested the pesticide (Table 2). The lower elimination of egg Ca did not result in a significant decrease relative to the amount ingested, since the differences between the two lots were small and also because the amount of Ca ingested by the treated birds was also lower (Table 1). The fraction of absorbed Ca that was retained by the body increased (P<.01) with pesticide ingestion, while the portion that was incorporated to the egg underwent a significant decrease ( P < 0 1 ) (Table 1). The forementioned decrease of Ca in the egg due to dieldrin resulted in a diminishing of the Ca in the egg shell, (Table 4). This is in agreement with observations of Lehner and Egbert (1969) in ducks, although it is in disagreement with the observations of Davison and Sell (1972) in hens. The P of the whole egg remained unaltered. We do not think that this decrease in Ca content of the shell can be attributed to a real lack of this cation in the organism, since
contrary to the bone depletion observed by Peakall (1970) with DDT, our results pointed to an increase of the Ca retained in the skeleton, because the quails fed with dieldrin showed a slight increase (P<.05) in the amount of Ca and P in the femur, but the Ca/P relationship was not altered (Table 2). In other studies with DDT (Bitman et ai, 1969) and dieldrin (Muller et ai, 1971), no changes were observed in the bone either. Furthermore, the plasma calcium values in our tests, as those
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EXPERIMENTAL DAYS
FIG. 2. Fluctuations of the egg-laying in quails that received an experimental diet (o) and the same diet with the addition of 20 mg of dieldrin/kg (•). The ordinates indicate the average number of eggs laid per quail/day; the abscissas indicate the experimental days divided into 4 day intervals; the calculation of the daily egg-laying average is carried out in each of the forementioned intervals of time.
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FIG. 1. View under the petrographic microscope (500X; parallel nicols) of sections parallel to the surface of the egg shell of quail that received for 48 days: a control diet (A) and the same diet with the addition of dieldrin (20 mg/kg of diet) (B) The prismatic nodules, which correspond to the column of calcite microcrystals (A), can be seen apparently fragmented after the treatment with the pesticide (B).
600
iWM. ANDUJAR, G. VARELA AND M«P. NAVARRO
It is true that dieldrin did not alter significantly the nutritive utilization of P in our tests. Nevertheless the results for P, judged by the
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reported by Call and Call (1974) on the same species, did not undergo any variations (Table 2). Our results cannot be explained by the hypothesis that alterations in Ca metabolism are mediated by estrogenic degradation caused by chlorinated hydrocarbons (Peakall, 1970). Since calcemia was not altered and the content of Ca and P in the femur did not decrease, it is difficult for us in our experimental conditions to consider the possibility of a mechanism based on the decrease of estrogens concentration in blood. As it is generally known (Taylor, 1966; Navarro and Murillo, 1976), this concentration is one of the main factors that take part in the maintenance of the calcemia and bone Ca. Neither the size nor the total weight of the egg were significantly affected by the treatment at the periods (20 to 28 days and 40 to 48 days) when the determinations were carried out (Table 4). This observation concurs with Davison and Sell (1972) regarding to DDT. The breaking strength by weight of the shell was not affected by the pesticide (Table 4). Cecil et al. (1971) reported the same observations on quails treated with DDT. The structure of the shell of the quails egg, which is similar to that described by Taylor (1970) in hens and Navarro and Murillo (1976) in quails, showed some differences due to the dieldrin, including an apparent decrease in the size of the calcite microcrystal nodules with a parallel increase in number and a decrease of the pores per surface unit (Figure 1). These results are similar to those given for birds treated with DDT (Peakall et al., 1973). The frequency of egg-laying (Figure 2) showed fluctuations throughout the experimental period. It was identical in the two groups of quail at the beginning of the test period, but in quail fed with dieldrin it decreased slightly and progressively as the treatment continued, until it finally became significantly lower (P<.05) towards the end of the first month (middle days of the experimental period). Nevertheless, these large differences gradually disappeared. The decrease observed in the number of eggs laid per treated quail was not statistically significant. Similar egg-laying fluctuations, which are difficult to explain, were observed by Bitman et al. (1969) and Cecil et al. (1971) for the same species treated with DDT.
601
DIELDRIN, Ca AND P BALANCE IN COTURNIX EGGS
animal (Sell and Davison, 1973), and dose (Varela et al., 1975). Furthermore, feed consumption by the male quail was much smaller than that of females, and the amount of pesticide ingested by the males was, in consequence, much lower.
parameters and coefficients controlled (ingested, excreted, transferred to the egg, CNU, CCR, CIE, etc. (Table 3)) showed the same trends as the more distinct effects noted in Ca. The treatment with dieldrin did not significantly modify the amount of Ca and P ingested by the male quail. The overall excretion of the two minerals and body retention for both minerals did not undergo any marked alterations either (Tables 1 and 3). The contents of Ca and P in the femur did not reveal any detrimental effect in the treated birds, as would be expected, since body retention for both minerals was not affected, and in addition, the Ca/P bone relationship remained constant with a value of 2.1. Neither did the pesticide alter the calcemia (Table 2). In short, Ca and P balances in the male quail did not seem to be affected by dieldrin. Pesticide action depends to a large extent on a series of factors such as species, sex, age of the
REFERENCES
TABLE A.—Effect of dieldrin (20 ppm in the diet) on egg production of the egg in quails
characteristics
Diet a C + dieldrin (20 ppm)
C
Means Average number of eggs per quail and days Days 1-48 Days 2 0 - 2 8 Days 4 0 - 4 8 Average weight (g) Days 2 0 - 2 8 Days 4 0 - 4 8
Means
SE
SE
.96 .94 .98
.01 .03 .02
.91 .80 .92
.03'
9.89 10.02
.21 .20
9.51 9.94
.19 .17
3.11 2.44
.03 .04
3.03 2.42
.03 .02
3.13 2.43
.02 .02
3.09 2.42
.02 .01
.03 .03
Average size (cm) Days 2 0 - 2 8 DO
db Days 4 0 - 4 8 D d Breaking strength (g) Days 2 0 - 2 8 Days 4 0 - 4 8 Ca in shell (%) P in egg (mg)
1,190 1,218 26
16.6
Diet C is a commercial diet which contains 3.24% Ca and .72% P. " D " is longer diameter and "d" is shorter diameter. *P<.05 comparing with untreated quails.
1,250 1,200
112 83 .8 .6
23*
15.7
94 100 1.0 .7
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602
M<*M. ANDUJAR, G. VARELA AND MaP. NAVARRO Norman, 1972. Inhibition of biological activity of cholecalciferol (vitamin D 3 ) by o,p'-DDT or p,p'DDT in rachitic cockerel. J. Agr. Food Chem. 20:376-380. Peakall, D. B., 1969. Effect of DDT on calcium uptake and vitamin D metabolism in birds. Nature, 224:1219-1220. Peakall, D. B., 1970. p,p'-DDT: effect on calcium metabolism and concentration of estradiol in the blood. Science, 168:592-594. Peakall, D. B., J. L. Linger, R. W. Risebrough, J. B. Pritchard, and W. B. Kinter, 1973. DDE-induced egg-shell thinning: structural and physiological effects in three species. Com. Gen. Pharmacol. 4:305-313. Pocker, T., M. Beug, N. Watamori, and C. Guilbert, 1971. Enzyme inhibition by DDT questioned. Chem. Eng. News, 49:20-27. Ratcliffe, D. A., 1967. Decrease in eggshell weight in certain birds of prey. Nature, 215:208—210. Rousseau, M., J. Bourtallier, et Y. Taliercio, 1971. Pollution des oeufs par des residus de pesticides organochlores (frequence, importance, origine). Bull. Acad. Vet., France, XLIV: 2 7 - 3 2 . Sell, J. L., and K. L. Davison, 1973. Changes in the activities of hepatic microsomal enzymes caused by DDT and dieldrin. Fed. Proc. 32:2003-2009. Taylor, T. G., 1966. The endocrine control of calcium metabolism in the fowl. In Physiology of domestic fowl. Edited by C. Horton-Smidi and E. C. Amoroso, ed. Oliver, and Boyd, Edimburgh and London: pp. 199-202. Taylor, T. G., 1970. How an eggshell is made. Sci. Am. 222:89-95. Varela, G., A. Torralba, and J. Escriva, 1975. Influence of prolonged ingestion of some organochlorated pesticides on digestibility and nutritive value of a diet in rats. Proc. Nutr. Soc. 34.-93A. Velle, W., 1971. How the egg is formed and the effect of insecticides on reproduction in birds. Nor. Vet. Tidsskr. 83:448-453.
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