- Email: [email protected]
Some Physiological Responses of New Hampshire Chickens to a Mixture of Penta- and Hexachloronaphthalenes1
Some Physiological Responses of New Hampshire Chickens to a Mixture of Penta- and Hexachloronaphthalenes 1 W . J . PUDELKIEWICZ,2 R . V. BOUCHER, E . W . CALLENBACH AND R . C. MlLLEE Departments of Agricultural and Biological Chemistry and Poultry Husbandry, The Pennsylvania State University, University Park, Pennsylvania (Received for publication August 5, 1958)
T
HE use of chlorinated naphthalenes in electrical insulation and as additives to some lubricating greases used in feed processing and farm machinery has exposed farm animals to the inadvertent ingestion of these compounds. One causative agent of hyperkeratosis (X-disease) in catlie has been reported to be chlorinated naphthalene (Bel, 1952; Sikes etal., 1952). Cotter (1944) reported several deaths among workers who had been in prolonged contact with these compounds. Schwartz (1936) was the first to report that chlorinated naphthalenes caused acute yellow atrophy of the liver. Pudelkiewicz et al. (1958) in a study of physiological responses of Broad Breasted Bronze poults to a mixture of penta- and hexachloronaphthalenes reported that 5 p.p.m. was definitely toxic while 100 p.p.m. was fatal to all poults within 33 days. They also reported that octachloronaphthalene showed no toxic effects at a dietary level of 125 p.p.m. No other reports could be found in the literature dealing with the effect of chlorinated naphthalenes upon poultry, although several investigators reported on the toxic effects of related chlorinated molecules, such as, chlordane (Rosenberg et al., 1950), dieldrin (Eden, 1951) and aldrin, (Arant, 1952). The former reported that 'Authorized for publication on July 29, 1957, as paper No. 2180, in the Journal series of the Pennsylvania Agricultural Experiment Station. 2 Present address: Department of Poultry Science, University of Connecticut, Storrs, Connecticut.
0.50% chlordane in the diet stopped egg production within 5 days, while no adverse effect was noted in egg production or hatchability at the 0.05% level. Post mortem inspection showed an excessive quantity of straw-colored pericardial fluid. Eden stated that 3 and 6-week old New Hampshire chicks which received 50 and 100 p.p.m. of dieldrin in the diet died before the end of the 90-day experimental period while Arant reported that the upper limit of safety for aldrin in the diet of 3 and 6-week old chicks was below 25 p.p.m. The objective of this investigation was to study the pathological and physiological responses of chickens through the life cycle to a mixture of penta- and hexachloronaphthalenes in the diet. EXPERIMENTAL
Day-old New Hampshire chicks were reared in battery-type brooders equipped with thermostatically controlled electrical heating units, 0.5 inch mesh hardware cloth floors and automatic waterers. Practical starter and breeder diets were fed which satisfied all the known nutrient requirements of chicks, as recommended by the National Research Council (1950). The composition of the diets is presented in Table 1. The chlorinated naphthalene, Halowax 1014 (a mixture of penta- and hexachloronaphthalenes), was dissolved in cottonseed oil and the solution hand-mixed into the starter diet. When the breeder diet was used, a premix was prepared by dissolving
424
CHLORONAPHTHALENE TOXICITY TABLE 1.—Percentage composition of the starter and breeder diets—all experiments Ingredients
Starter Breeder
Ground yellow corn Ground wheat 61.80 Wheat middlings, standard Ground oats 5.00 Alfalfa meal, dehydrated 3.00 Soybean oil meal, 5 0 % protein 10.00 Soybean oil meal, 4 4 % protein Fish meal, menhaden, 6 0 % protein 5.00 M e a t scrap, 5 0 % protein Dried whey 5.00 Riboflavin supplement, 8 mg. per gram Brewers' dried yeast 5.00 1.80 Ground limestone Steamed bone meal * 1.00 Iodized salt 0.25 D-activated animal sterol, 3,0001.C.U. per gram 0.02 Fish oil, 300 I.C.U. vitamin D per gram 1,500 I.U. vitamin A per gram Manganese sulfate, technical 0.03 0.10 Choline chloride, 2 5 % Cottonseed oil 2.00
60.44
—
—
10.00 5.00 2.50
— — —
5.00 2.50 5.00 2.00 0.03 2.40 3.50 1.00 0.40 0.01
—
0.20 0.02
—
— —
Halowax in a minimum of cottonseed oil and hand-mixing with a known quantity of the diet. Weighed amounts of this premix were then hand-mixed into the diet to give the desired final concentrations. Increasing amounts of Halowax 1014 were added to the diet, each level being 5 times that of the preceding one, as described in detail under experiments one and three. The amount of oil added to the diet by the premix varied from 0.0016% in the 4 p.p.m. diet to 0.04% in the 100 p.p.m. diet. Feed and water were supplied ad libitum. Vitamin A in the liver was determined by the Carr-Price colorimetric method as described in "Methods of Vitamin Assay of the Association of Vitamin Chemists" (1947) and vitamin A in blood plasma according to the method of Kimble (1939). The cholesterol was determined by the micro method of Zlatkis et al. (1953). The Folin isolation method for serum uric acid and the Folin and Wu method for serum creatinine were taken from Hawk et al. (1947). Serum sodium and serum potassium determinations were carried out essentially by the method described by Toth et al. (1948) with the use of a flame photometer. Chlorinated naphthalene was determined by a method developed by Shank (1954) of this department which was sensi-
425
tive to approximately 0.1 mg. The recovery obtained with this method was approximately 75%. Thus, the method was limited in its use as a quantitative measurement but was satisfactory as a qualitative test. Experiment 1. Reports in the literature showed that plasma vitamin A was greatly decreased in cattle afflicted with hyperkeratosis (Olafson, 1947). In a review article on bovine hyperkeratosis by Lee (1952) it was reported that administration of vitamin A caused temporary improvement in the condition but did not prevent its fatal termination regardless of the amount of vitamin A given. To test the hypothesis that some of the toxic effects of Halowax 1014 might be due to a vitamin A deficiency, a relatively large amount of the vitamin was added to the diet of one group after the day-old chicks were first placed on a vitamin A-low diet. The chicks were depleted of vitamin A stores in order that a more uniform response might take place when they were placed on a vitamin A supplemented diet. Six groups of chicks, after receiving the vitamin A-low diet from one to 12 days of age, were transferred to a diet that contained 2,000 I.U. of vitamin A per pound and 0, 4, 20, 100, 500, or 2,500 p.p.m. of Halowax 1014 for a period of 40 days. Each group contained 10 males and 10 females. A second 500 p.p.m. group was included which received an additional 8,000 I.U. of vitamin A per pound of diet. At the end of the experiment, the chicks were sacrificed and blood and liver samples taken for analysis of vitamin A. Experiment 2. One group of 10 unsexed day-old chicks was fed a control diet and another group of 12 was fed a diet that contained 100 p.p.m. of Halowax 1014. After 35 days, the chicks were sacrificed and individual blood samples were taken and stored at — 25°C. until analyzed. Blood constituents such as vitamin A, sodium, potassium, uric acid, creatinine and
426
W. J. PUDELKIEWICZ, R. V. BOUCHER, E. W. CALLENBACH AND R. C. MILLER
cholesterol as well as the hematocrit were determined in order to measure responses which might follow the addition of Halowax 1014 to the diet. Experiment 3. Experiment 3 was designed to study the effect of Halowax 1014 upon the egg production, hatchability and blood constituents of New Hampshire pullets. Diets which contained 0, 4, 20 or 100 p.p.m. of Halowax 1014 were fed to chickens from day-old to 28 weeks of age. The same starter diet was fed as in experiments 1 and 2. The pullets and cockerels were transferred to individual cages at sexual maturity and fed the supplemented Pennsylvania State University breeder diet for the remainder of the experiment. After allowing one week for the chickens to become accustomed to the new environment, egg production was recorded. Semen was collected from cockerels once each week and used immediately to inseminate pullets on the same treatment. Onetenth milliliter of undiluted pooled semen from two cockerels was used for each insemination. Eggs were collected daily and stored at SS°F. Settings were made biweekly, all in the same incubator. Fertility
of eggs was determined by candling on the nineteenth day of incubation. RESULTS AND DISCUSSION Experiment 1. The results of the vitamin A analyses of blood and liver along with the mortality, average gain in weight and percent difference in gain from the control appear in Table 2. Chicks generally tolerated 4 and 20 p.p.m. of Halowax 1014 without showing toxic symptoms. The first indication of toxicity was observed at the 100 p.p.m. level where the average gain was retarded about 8%. The diet that contained 2S00 p.p.m. was fatal to all chicks within two weeks. Pathological symptoms observed were an enlarged fibrotic liver and the presence of an "ascites-type" straw-colored fluid in the body cavities. The latter condition generally began by an accumulation of the fluid in the pericardial sac. In advanced stages, the peritoneal cavity of the chick was filled with this fluid which was first observed between the 4th and 5th week in chicks that ingested a diet which contained 100 or more parts per million of Halowax 1014. In some instances, the fluid contributed up to 25%
TABLE 2.—Effect of increasing amounts of Halowax 1014 on average gain in weight, mortality and vitamin A concentration in blood plasma and liver of 52-day old New Hampshire chicks1— Experiment 1
M p.p.m. 0 4 20 100 500 5002 2,500
%
5 0 5 15 65 25 100
F
Vitamin A content of
Difference in gain from control
Average gain
Halowax3 Mortality 1014
Av.
M
gm. gm. gm. 597 498 547 582 473 528 615 532 574 515 491 503 230 298 264 393 325 359
% -
3 3 -14 -61 -34
F
Av.
% —
% —
-
5 7 - 1 -40 -35
-
3 5 - 8 -52 -34
Liver
Plasma M
F
Av.
I.U./100 ml. 235 185 210 208 220 214 280 248 264 188 205 196 50 155 102 308 420 364
M
F
I.U./gm. 16 14 10 11 10 12 6 6 2 4 67 80
Av. 15 10 11 6 3 73
1 Chicks were fed a vitamin A-low diet for the first 12 days followed by supplementation with 2,000 I.Uof vitamin A per pound and with the indicated amounts of Halowax for the next 40 days. 2 This group received an additional 8,000 I.U. of vitamin A per pound of diet. 3 Halowax 1014 is a mixture of penta- and hexachloronaphthalenes.
427
CHLORONAPHTHALENE TOXICITY
TABLE 3.—Hematocrit and serum sodium, potassium, uric acid, creatinine and cholesterol of New Hampshire chicks that received a normal diet and one that contained 100 p.p.m. of Halowax 1014 from day-old to 5 weeks of age—Experiment 2 Control M (6) Sodium, mg./lOO ml. Potassium, mg./lOO ml. Uric Acid, mg./lOO ml. Creatinine, mg./lOO ml. Cholesterol, mg./lOO ml. Hematocrit, %
392 28 1.8 0.72 154 27
Treated
F (4) 340 29 1.7 0.82 • 131 26
Av. (10)
M (7)
F (5)
Av. (12)
366 28 1.8 0.76 145 27
335 26 2.4* 0.91 139 23**
347 25 2.5* 0.88 124 23**
341 25 2.4* 0.90 131 23**
Figures in parentheses indicate number of birds per group. : Significant at P=0.05. * Significant at P=0.01.
of the body weight of the chicks. Anemic conditions often prevailed which were manifested by pale tissue in the breast and leg muscle. The vitamin A content of blood plasma was found to be quite variable; however, a decrease occurred with the 500 p.p.m. treatment. The group that received the added vitamin A in the diet showed a corresponding increase in the blood plasma vitamin A content. Liver storage of vitamin A was generally increasingly depressed as the amount of chlorinated naphthalene in the diet increased. This seemed reasonable to expect in view of the liver injury that occurred in the treated birds. Increased dietary intake of vitamin A was reflected by a large increase in liver storage. A lack of normal feather pigmentation was observed in chicks that received dietary Halowax 1014. The change in pigmentation varied directly with the dietary intake of Halowax 1014 and at the 500 p.p.m. level, chicks that survived 5 weeks of the treatment had feathers that were light buff in color. This condition with New Hampshire chicks appeared to be possibly analogous to the depigmentation of the skin of Negroes who had come into prolonged contact with chlorinated naphthalenes in industry (Cotter, 1944). (In a later experiment with dark crosses, New Hampshire
males X Barred Rock females, no change was observed in feather pigmentation.) In contrast to the above results, Broad Breasted Bronze poults had shown a 33% decrease in average gain upon ingesting a diet that contained 5 p.p.m. of Halowax 1014 for 40 days (Pudelkiewicz et al., 1958). At levels up to 100 p.p.m., neither a lack of feather pigmentation nor the presence of excessive amounts of fluids were observed in body cavities of poults. Experiment 2. The results of experiment 2 are presented in Table 3. Uric acid in the serum of chickens that received 100 p.p.m. of dietary Halowax 1014 was significantly increased while the hematocrit was highly significantly decreased. No significant differences were observed in serum sodium, potassium, cholesterol or creatinine. Experiment 3. The results of experiment 3 are presented in Tables 4 and 5. Although no apparent differences in the rate of egg production occurred between the TABLE 4.—Egg production and halchability of New Hampshire pullets that received Halowax 1014 for 28 weeks from day-old-Experimenl 3 Halowax
, lets
p
,ets
1014 p.p.m. No. 0 5 4 5 20 7 100 2
Egg E j . P ro duc- * * f ^'f/ set me tion % No. 84 122 % 80 103 20 14 74 126 39 0 0
Dead embryos and pips
Hatchability All
Fertile
%
%
%
11 18 84
71 71 10
89 82 16
428
W. J. PUDELKIEWICZ, R. V. BOUCHER, E. W. CAIXENBACH AND R. C. MILLER
groups that received the 0 to 20 p.p.m. treatments, there was a marked decrease in the hatchability of the eggs from pullets that received the diet that contained 20 p.p.m. of Halowax 1014. No eggs were produced by the pullets that received the 100 p.p.m. diet. Approximately 84% of the embryos of eggs produced by pullets that received 20 p.p.m. of dietary Halowax. failed to hatch or died during the incubation period. Thus, although the pullets of this group appeared to be similar to the controls in egg production and general appearance, the presence of 20 p.p.m. of Halowax 1014 resulted in a decrease in hatchability. The question arises whether or not the death of these embryos was due to a disturbance in the function of some biological system of the embryo by Halowax 1014 or to interference with some biological function of the dam which caused production of eggs which was deficient in some essential nutrient necessary to the normal development of the embryo. A sensitive quantitative method for the determination of micro quantities of chlorinated naphthalene in eggs and body tissues would aid in supplying part of this information. Performance of pullets that received 4 p.p.m. generally appeared to be equal in all respects to that of pullets that received the control diet.
On the basis of a limited number of values, the hematocrit decreased in pullets that received diets which contained 100 p.p.m. of Halowax 1014 from an average of 28% in the controls to 19% in the treated pullets (Table 5). The combined averages of both sexes for serum sodium and serum potassium in chickens that received 0, 20 or 100 p.p.m. of Halowax 1014 were 367 and 26, 314 and 21, and 280 and 18 mg. per 100 ml., respectively. Both serum sodium and serum potassium were found to be significantly lower ( 1 % level) in chickens that received the 100 p.p.m. treatment. The values for the 20 p.p.m. treatment were consistently intermediate between the other two treatments for both constituents. The combined averages of both sexes for serum cholesterol in chickens that received 0, 20 or 100 p.p.m. of Halowax 1014 were 110, 141 and 161 mg. per 100 ml., respectively. Serum cholesterol increased significantly at the 5% level (Snedecor, 1946). Serum uric acid in chickens that received the treated diet appeared to increase. Several samples were too concentrated to be read in the colorimeter and were recorded as being greater than 10 mg. per 100 ml. of serum. A study was undertaken in order to determine whether or not the progeny of the
TABLE 5.—Hematocrit and serum sodium, potassium, uric acid and cholesterol of New Hampshire chickens that received a normal diet and one that contained Halowax 1014 from day-old to 28 •weeks of age—Experiment 3 20 p.p.m.
Control
Sodium, mg./lOO ml. Potassium, mg./lOO ml. Uric acid, mg./lOO ml. Cholesterol, mg./lOO ml. Hematocrit, %
M (2)
F (4)
394 25 S.6 118
340 26 4.9 103 28
—
100 p.p.m.
Av. (6)
F (5)
M (3)
367 26 5.2 110
314 21
288 19 10+* 1 160 24
—
—
141 23
F (2) 272 16 8.2* 163 19**
Av. (5) 280** 18**
—
161* 22
Figures in parentheses indicate number of birds per group. * Significant at P = 0.05. ** Significant at P = 0 . 0 1 . 1 There was such a high concentration of uric acid in some samples that they could not be read in the colorimeter and were recorded as being greater than 10 p.p.m.
CHLORONAPHTHALENE TOXICITY
dams that received Halowax 1014 would grow normally and might have increased tolerance to this compound. The average 28-day gain of the progeny of New Hampshire chickens that received the control, the 4 and the 20 p.p.m. Halowax containing diet was 172, 137 and 91 gm., respectively. These groups contained 6, 6 and 4 chicks, respectively. It would appear from these limited data that the progeny of treated dams did not grow at the normal rate when compared with the progeny of controls. This may indicate a carry-over of Halowax 1014 from the dam to the progeny. Chlorinated naphthalene was detected in liver and kidney tissue; however, no accurate quantitative determination could be made. In tissue distribution studies of aldrin fed to rats, Borgmann et al. (1952) reported that the largest concentration was found in liver and kidney tissue. This appears to be generally true in studies of toxic material since these organs play a key role in detoxification and elimination of toxic substances. The external pathological symptoms of 28-week old chickens at 100 p.p.m. of Halowax 1014 were characterized by pale and underdeveloped combs and wattles in both sexes. These characteristics were especially striking in some males which were mistaken for females until the time of autopsy. The abnormalities most frequently observed at autopsy were the enlarged livers and the presence of clear straw-colored fluid in the pericardial sac and the peritoneal cavity. SUMMARY
Halowax 1014 (a mixture of pentaand hexachloronaphthalenes) was fed to New Hampshire chickens through the life cycle in order to study its influence as measured by certain physiological responses. A dietary intake of 2,500 p.p.m. resulted
429
in 100% mortality in 2 weeks, an intake of 100 p.p.m. to 28 weeks of age prevented egg production, 20 p.p.m. was responsible for poor hatchability while a 4 p.p.m. appeared to be well tolerated by the chickens of all ages. Thus, the upper limit of tolerance for Halowax 1014 would appear to be between 4 and 20 p.p.m. The vitamin A content of the liver varied inversely with the Halowax content of the diet. The hematocrit of chicks that received 100 p.p.m. of the chlorinated naphthalene for only 35 days was significantly lower ( 1 % level) than the hematocrit of controls. In 28-week old chickens that were fed the 100 p.p.m. diet, the sodium and potassium content of the serum was significantly lower, while the cholesterol and uric acid content of the serum was significantly higher (5% level). The most common pathological symptoms observed were the enlarged and fibrotic liver, the presence of a straw-colored fluid in the pericardial sac and peritoneal cavity and the lack of normal feather pigmentation in New Hampshire chicks. ACKNOWLEDGMENTS
The authors are grateful to Dr. L. V. Curtin, Buckeye Cotton Oil Company, Ivorydale, Ohio for the cottonseed oil, to the Bakelite Company, New York for the Halowax 1014 and to Dr. S. Gordeuk, Jr., The Pennsylvania State University, who performed many of the autopsies. REFERENCES Arant, F. S., 1952. Toxicity of aldrin to chickens. J. Econ. Ent. 45: 121. Association of Vitamin Chemists, Inc. 1947. Methods of Vitamin Assay. Interscience Publishers, Inc., New York, N.Y. Bell, W. B., 1952. Further studies on the production of bovine hyperkeratosis by the administration of a lubricant. Virginia J. Sci. 3: 169-177. Borgmann, A. R., P. A. Dahm and C. H. Kitselman, 1952. Toxicological studies of aldrin to
430
W. J. PUDELKIEWICZ, R. V. BOUCHER, E. W. CALLENBACH AND R. C. MILLER
laboratory animals. Food and Drug Administration Hearings, Washington, D.C. Exhibit 1218. Committee on Animal Nutrition, 1950. Recommended nutrient allowances for poultry. National Research Council, Washington, D.C. Cotter, L. N., 1944. Pentachlorinated naphthalenes in industry. J. Am. Med. Assoc. 125: 273-274. Eden, W. C , 1951. Toxicity of dieldrin to chickens. J. Econ. Ent. 44: 1013. Hawk, P. B., B. L. Oser and W. H. Summerson, 1949. Practical Physiological Chemistry. 12th edition. The Blakiston Company, Philadelphia, Pa. Kimble, N. A., 1939. The photocolorimetric determination of vitamin A and carotene in human plasma. J. Lab. Clin. Med. 24: 1055-1065. Lee, A. M., 1952. Our newer knowledge of bovine hyperkeratosis (X-disease). Reprint from Proceedings United States Livestock Sanitary Association. Fifty-sixth annual meeting. Pp. 175194. Olafson, P., 1947. Hyperkeratosis (X-disease) of cattle. Cornell Vet. 37: 279-291. Pudelkiewicz, W. J., R. V. Boucher, E. W. Callenbach and R. C. Miller, 1958. Some physiological responses of Broad Breasted Bronze poults to
chlorinated naphthalenes. Poultry Sci. 37: 185187. Rosenberg, M. M., T. Tanaka and H. E. Adler, 1950. Toxicity of chlordane to laying pullets. Am. J. Vet. Research, 11: 236-239. Schwartz, L., 1936. Dermatitis from synthetic resins and waxes. Am. J. Pub. Health, 26: 586592. Shank, M. T., 1954. Personal communication. Sikes, D., and M. E. Bridges, 1952. Experimental production of hyperkeratosis ("X-disease") of cattle with a chlorinated naphthalene. Science, 116: 506-507. Snedecor, W. W., 1946. Statistical Methods Applied to Experiments in Agriculture and Biology. The Iowa State College Press, Ames, Iowa. Toth, S. J., A. L. Prince, A. Wallace and D. S. Mikkelsen, 1948. Rapid quantitative determination of eight mineral elements in plant tissue by a systematic procedure involving use of a flame photometer. Soil Science, 66: 459-466. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.
The Interrelationship of Reproductive State and Seasonal Acclimatization on the Hen's Resistance to Lethal High Temperature H A R O L D S.
Department
WEISS
of Poultry Science, Rutgers, the State University, New Brunswick, New Jersey (Received for publication August 5, 1958)
N
ON-LAYING hens should withstand heat stress better than layers on the theoretical grounds that their metabolism and heat production are lower (Hutchinson, 19S4). Robinson and Lee (1947) showed that decreased feed intake, such as might be expected in non-layers, reduces some of the undesirable effects of hot environments Paper of the Journal Series, New Jersey Agricultural Experiment Station, New Brunswick, N.J. Supported in part by a grant from CIBA Pharmaceutical Products, Inc., Summit, N.J.
on hens. The lower body temperature and respiration rate found by Hillerman and Wilson (1955) in non-layers compared to layers at high air temperatures implies further that the non-layer is more capable of acclimatization. On the other hand, Hutt (1939) was unable to find any relationship between production and susceptibility to high temperature in a flock exposed to a naturally occurring heat wave intense enough to cause mortality from heat prostration.
T
HE use of chlorinated naphthalenes in electrical insulation and as additives to some lubricating greases used in feed processing and farm machinery has exposed farm animals to the inadvertent ingestion of these compounds. One causative agent of hyperkeratosis (X-disease) in catlie has been reported to be chlorinated naphthalene (Bel, 1952; Sikes etal., 1952). Cotter (1944) reported several deaths among workers who had been in prolonged contact with these compounds. Schwartz (1936) was the first to report that chlorinated naphthalenes caused acute yellow atrophy of the liver. Pudelkiewicz et al. (1958) in a study of physiological responses of Broad Breasted Bronze poults to a mixture of penta- and hexachloronaphthalenes reported that 5 p.p.m. was definitely toxic while 100 p.p.m. was fatal to all poults within 33 days. They also reported that octachloronaphthalene showed no toxic effects at a dietary level of 125 p.p.m. No other reports could be found in the literature dealing with the effect of chlorinated naphthalenes upon poultry, although several investigators reported on the toxic effects of related chlorinated molecules, such as, chlordane (Rosenberg et al., 1950), dieldrin (Eden, 1951) and aldrin, (Arant, 1952). The former reported that 'Authorized for publication on July 29, 1957, as paper No. 2180, in the Journal series of the Pennsylvania Agricultural Experiment Station. 2 Present address: Department of Poultry Science, University of Connecticut, Storrs, Connecticut.
0.50% chlordane in the diet stopped egg production within 5 days, while no adverse effect was noted in egg production or hatchability at the 0.05% level. Post mortem inspection showed an excessive quantity of straw-colored pericardial fluid. Eden stated that 3 and 6-week old New Hampshire chicks which received 50 and 100 p.p.m. of dieldrin in the diet died before the end of the 90-day experimental period while Arant reported that the upper limit of safety for aldrin in the diet of 3 and 6-week old chicks was below 25 p.p.m. The objective of this investigation was to study the pathological and physiological responses of chickens through the life cycle to a mixture of penta- and hexachloronaphthalenes in the diet. EXPERIMENTAL
Day-old New Hampshire chicks were reared in battery-type brooders equipped with thermostatically controlled electrical heating units, 0.5 inch mesh hardware cloth floors and automatic waterers. Practical starter and breeder diets were fed which satisfied all the known nutrient requirements of chicks, as recommended by the National Research Council (1950). The composition of the diets is presented in Table 1. The chlorinated naphthalene, Halowax 1014 (a mixture of penta- and hexachloronaphthalenes), was dissolved in cottonseed oil and the solution hand-mixed into the starter diet. When the breeder diet was used, a premix was prepared by dissolving
424
CHLORONAPHTHALENE TOXICITY TABLE 1.—Percentage composition of the starter and breeder diets—all experiments Ingredients
Starter Breeder
Ground yellow corn Ground wheat 61.80 Wheat middlings, standard Ground oats 5.00 Alfalfa meal, dehydrated 3.00 Soybean oil meal, 5 0 % protein 10.00 Soybean oil meal, 4 4 % protein Fish meal, menhaden, 6 0 % protein 5.00 M e a t scrap, 5 0 % protein Dried whey 5.00 Riboflavin supplement, 8 mg. per gram Brewers' dried yeast 5.00 1.80 Ground limestone Steamed bone meal * 1.00 Iodized salt 0.25 D-activated animal sterol, 3,0001.C.U. per gram 0.02 Fish oil, 300 I.C.U. vitamin D per gram 1,500 I.U. vitamin A per gram Manganese sulfate, technical 0.03 0.10 Choline chloride, 2 5 % Cottonseed oil 2.00
60.44
—
—
10.00 5.00 2.50
— — —
5.00 2.50 5.00 2.00 0.03 2.40 3.50 1.00 0.40 0.01
—
0.20 0.02
—
— —
Halowax in a minimum of cottonseed oil and hand-mixing with a known quantity of the diet. Weighed amounts of this premix were then hand-mixed into the diet to give the desired final concentrations. Increasing amounts of Halowax 1014 were added to the diet, each level being 5 times that of the preceding one, as described in detail under experiments one and three. The amount of oil added to the diet by the premix varied from 0.0016% in the 4 p.p.m. diet to 0.04% in the 100 p.p.m. diet. Feed and water were supplied ad libitum. Vitamin A in the liver was determined by the Carr-Price colorimetric method as described in "Methods of Vitamin Assay of the Association of Vitamin Chemists" (1947) and vitamin A in blood plasma according to the method of Kimble (1939). The cholesterol was determined by the micro method of Zlatkis et al. (1953). The Folin isolation method for serum uric acid and the Folin and Wu method for serum creatinine were taken from Hawk et al. (1947). Serum sodium and serum potassium determinations were carried out essentially by the method described by Toth et al. (1948) with the use of a flame photometer. Chlorinated naphthalene was determined by a method developed by Shank (1954) of this department which was sensi-
425
tive to approximately 0.1 mg. The recovery obtained with this method was approximately 75%. Thus, the method was limited in its use as a quantitative measurement but was satisfactory as a qualitative test. Experiment 1. Reports in the literature showed that plasma vitamin A was greatly decreased in cattle afflicted with hyperkeratosis (Olafson, 1947). In a review article on bovine hyperkeratosis by Lee (1952) it was reported that administration of vitamin A caused temporary improvement in the condition but did not prevent its fatal termination regardless of the amount of vitamin A given. To test the hypothesis that some of the toxic effects of Halowax 1014 might be due to a vitamin A deficiency, a relatively large amount of the vitamin was added to the diet of one group after the day-old chicks were first placed on a vitamin A-low diet. The chicks were depleted of vitamin A stores in order that a more uniform response might take place when they were placed on a vitamin A supplemented diet. Six groups of chicks, after receiving the vitamin A-low diet from one to 12 days of age, were transferred to a diet that contained 2,000 I.U. of vitamin A per pound and 0, 4, 20, 100, 500, or 2,500 p.p.m. of Halowax 1014 for a period of 40 days. Each group contained 10 males and 10 females. A second 500 p.p.m. group was included which received an additional 8,000 I.U. of vitamin A per pound of diet. At the end of the experiment, the chicks were sacrificed and blood and liver samples taken for analysis of vitamin A. Experiment 2. One group of 10 unsexed day-old chicks was fed a control diet and another group of 12 was fed a diet that contained 100 p.p.m. of Halowax 1014. After 35 days, the chicks were sacrificed and individual blood samples were taken and stored at — 25°C. until analyzed. Blood constituents such as vitamin A, sodium, potassium, uric acid, creatinine and
426
W. J. PUDELKIEWICZ, R. V. BOUCHER, E. W. CALLENBACH AND R. C. MILLER
cholesterol as well as the hematocrit were determined in order to measure responses which might follow the addition of Halowax 1014 to the diet. Experiment 3. Experiment 3 was designed to study the effect of Halowax 1014 upon the egg production, hatchability and blood constituents of New Hampshire pullets. Diets which contained 0, 4, 20 or 100 p.p.m. of Halowax 1014 were fed to chickens from day-old to 28 weeks of age. The same starter diet was fed as in experiments 1 and 2. The pullets and cockerels were transferred to individual cages at sexual maturity and fed the supplemented Pennsylvania State University breeder diet for the remainder of the experiment. After allowing one week for the chickens to become accustomed to the new environment, egg production was recorded. Semen was collected from cockerels once each week and used immediately to inseminate pullets on the same treatment. Onetenth milliliter of undiluted pooled semen from two cockerels was used for each insemination. Eggs were collected daily and stored at SS°F. Settings were made biweekly, all in the same incubator. Fertility
of eggs was determined by candling on the nineteenth day of incubation. RESULTS AND DISCUSSION Experiment 1. The results of the vitamin A analyses of blood and liver along with the mortality, average gain in weight and percent difference in gain from the control appear in Table 2. Chicks generally tolerated 4 and 20 p.p.m. of Halowax 1014 without showing toxic symptoms. The first indication of toxicity was observed at the 100 p.p.m. level where the average gain was retarded about 8%. The diet that contained 2S00 p.p.m. was fatal to all chicks within two weeks. Pathological symptoms observed were an enlarged fibrotic liver and the presence of an "ascites-type" straw-colored fluid in the body cavities. The latter condition generally began by an accumulation of the fluid in the pericardial sac. In advanced stages, the peritoneal cavity of the chick was filled with this fluid which was first observed between the 4th and 5th week in chicks that ingested a diet which contained 100 or more parts per million of Halowax 1014. In some instances, the fluid contributed up to 25%
TABLE 2.—Effect of increasing amounts of Halowax 1014 on average gain in weight, mortality and vitamin A concentration in blood plasma and liver of 52-day old New Hampshire chicks1— Experiment 1
M p.p.m. 0 4 20 100 500 5002 2,500
%
5 0 5 15 65 25 100
F
Vitamin A content of
Difference in gain from control
Average gain
Halowax3 Mortality 1014
Av.
M
gm. gm. gm. 597 498 547 582 473 528 615 532 574 515 491 503 230 298 264 393 325 359
% -
3 3 -14 -61 -34
F
Av.
% —
% —
-
5 7 - 1 -40 -35
-
3 5 - 8 -52 -34
Liver
Plasma M
F
Av.
I.U./100 ml. 235 185 210 208 220 214 280 248 264 188 205 196 50 155 102 308 420 364
M
F
I.U./gm. 16 14 10 11 10 12 6 6 2 4 67 80
Av. 15 10 11 6 3 73
1 Chicks were fed a vitamin A-low diet for the first 12 days followed by supplementation with 2,000 I.Uof vitamin A per pound and with the indicated amounts of Halowax for the next 40 days. 2 This group received an additional 8,000 I.U. of vitamin A per pound of diet. 3 Halowax 1014 is a mixture of penta- and hexachloronaphthalenes.
427
CHLORONAPHTHALENE TOXICITY
TABLE 3.—Hematocrit and serum sodium, potassium, uric acid, creatinine and cholesterol of New Hampshire chicks that received a normal diet and one that contained 100 p.p.m. of Halowax 1014 from day-old to 5 weeks of age—Experiment 2 Control M (6) Sodium, mg./lOO ml. Potassium, mg./lOO ml. Uric Acid, mg./lOO ml. Creatinine, mg./lOO ml. Cholesterol, mg./lOO ml. Hematocrit, %
392 28 1.8 0.72 154 27
Treated
F (4) 340 29 1.7 0.82 • 131 26
Av. (10)
M (7)
F (5)
Av. (12)
366 28 1.8 0.76 145 27
335 26 2.4* 0.91 139 23**
347 25 2.5* 0.88 124 23**
341 25 2.4* 0.90 131 23**
Figures in parentheses indicate number of birds per group. : Significant at P=0.05. * Significant at P=0.01.
of the body weight of the chicks. Anemic conditions often prevailed which were manifested by pale tissue in the breast and leg muscle. The vitamin A content of blood plasma was found to be quite variable; however, a decrease occurred with the 500 p.p.m. treatment. The group that received the added vitamin A in the diet showed a corresponding increase in the blood plasma vitamin A content. Liver storage of vitamin A was generally increasingly depressed as the amount of chlorinated naphthalene in the diet increased. This seemed reasonable to expect in view of the liver injury that occurred in the treated birds. Increased dietary intake of vitamin A was reflected by a large increase in liver storage. A lack of normal feather pigmentation was observed in chicks that received dietary Halowax 1014. The change in pigmentation varied directly with the dietary intake of Halowax 1014 and at the 500 p.p.m. level, chicks that survived 5 weeks of the treatment had feathers that were light buff in color. This condition with New Hampshire chicks appeared to be possibly analogous to the depigmentation of the skin of Negroes who had come into prolonged contact with chlorinated naphthalenes in industry (Cotter, 1944). (In a later experiment with dark crosses, New Hampshire
males X Barred Rock females, no change was observed in feather pigmentation.) In contrast to the above results, Broad Breasted Bronze poults had shown a 33% decrease in average gain upon ingesting a diet that contained 5 p.p.m. of Halowax 1014 for 40 days (Pudelkiewicz et al., 1958). At levels up to 100 p.p.m., neither a lack of feather pigmentation nor the presence of excessive amounts of fluids were observed in body cavities of poults. Experiment 2. The results of experiment 2 are presented in Table 3. Uric acid in the serum of chickens that received 100 p.p.m. of dietary Halowax 1014 was significantly increased while the hematocrit was highly significantly decreased. No significant differences were observed in serum sodium, potassium, cholesterol or creatinine. Experiment 3. The results of experiment 3 are presented in Tables 4 and 5. Although no apparent differences in the rate of egg production occurred between the TABLE 4.—Egg production and halchability of New Hampshire pullets that received Halowax 1014 for 28 weeks from day-old-Experimenl 3 Halowax
, lets
p
,ets
1014 p.p.m. No. 0 5 4 5 20 7 100 2
Egg E j . P ro duc- * * f ^'f/ set me tion % No. 84 122 % 80 103 20 14 74 126 39 0 0
Dead embryos and pips
Hatchability All
Fertile
%
%
%
11 18 84
71 71 10
89 82 16
428
W. J. PUDELKIEWICZ, R. V. BOUCHER, E. W. CAIXENBACH AND R. C. MILLER
groups that received the 0 to 20 p.p.m. treatments, there was a marked decrease in the hatchability of the eggs from pullets that received the diet that contained 20 p.p.m. of Halowax 1014. No eggs were produced by the pullets that received the 100 p.p.m. diet. Approximately 84% of the embryos of eggs produced by pullets that received 20 p.p.m. of dietary Halowax. failed to hatch or died during the incubation period. Thus, although the pullets of this group appeared to be similar to the controls in egg production and general appearance, the presence of 20 p.p.m. of Halowax 1014 resulted in a decrease in hatchability. The question arises whether or not the death of these embryos was due to a disturbance in the function of some biological system of the embryo by Halowax 1014 or to interference with some biological function of the dam which caused production of eggs which was deficient in some essential nutrient necessary to the normal development of the embryo. A sensitive quantitative method for the determination of micro quantities of chlorinated naphthalene in eggs and body tissues would aid in supplying part of this information. Performance of pullets that received 4 p.p.m. generally appeared to be equal in all respects to that of pullets that received the control diet.
On the basis of a limited number of values, the hematocrit decreased in pullets that received diets which contained 100 p.p.m. of Halowax 1014 from an average of 28% in the controls to 19% in the treated pullets (Table 5). The combined averages of both sexes for serum sodium and serum potassium in chickens that received 0, 20 or 100 p.p.m. of Halowax 1014 were 367 and 26, 314 and 21, and 280 and 18 mg. per 100 ml., respectively. Both serum sodium and serum potassium were found to be significantly lower ( 1 % level) in chickens that received the 100 p.p.m. treatment. The values for the 20 p.p.m. treatment were consistently intermediate between the other two treatments for both constituents. The combined averages of both sexes for serum cholesterol in chickens that received 0, 20 or 100 p.p.m. of Halowax 1014 were 110, 141 and 161 mg. per 100 ml., respectively. Serum cholesterol increased significantly at the 5% level (Snedecor, 1946). Serum uric acid in chickens that received the treated diet appeared to increase. Several samples were too concentrated to be read in the colorimeter and were recorded as being greater than 10 mg. per 100 ml. of serum. A study was undertaken in order to determine whether or not the progeny of the
TABLE 5.—Hematocrit and serum sodium, potassium, uric acid and cholesterol of New Hampshire chickens that received a normal diet and one that contained Halowax 1014 from day-old to 28 •weeks of age—Experiment 3 20 p.p.m.
Control
Sodium, mg./lOO ml. Potassium, mg./lOO ml. Uric acid, mg./lOO ml. Cholesterol, mg./lOO ml. Hematocrit, %
M (2)
F (4)
394 25 S.6 118
340 26 4.9 103 28
—
100 p.p.m.
Av. (6)
F (5)
M (3)
367 26 5.2 110
314 21
288 19 10+* 1 160 24
—
—
141 23
F (2) 272 16 8.2* 163 19**
Av. (5) 280** 18**
—
161* 22
Figures in parentheses indicate number of birds per group. * Significant at P = 0.05. ** Significant at P = 0 . 0 1 . 1 There was such a high concentration of uric acid in some samples that they could not be read in the colorimeter and were recorded as being greater than 10 p.p.m.
CHLORONAPHTHALENE TOXICITY
dams that received Halowax 1014 would grow normally and might have increased tolerance to this compound. The average 28-day gain of the progeny of New Hampshire chickens that received the control, the 4 and the 20 p.p.m. Halowax containing diet was 172, 137 and 91 gm., respectively. These groups contained 6, 6 and 4 chicks, respectively. It would appear from these limited data that the progeny of treated dams did not grow at the normal rate when compared with the progeny of controls. This may indicate a carry-over of Halowax 1014 from the dam to the progeny. Chlorinated naphthalene was detected in liver and kidney tissue; however, no accurate quantitative determination could be made. In tissue distribution studies of aldrin fed to rats, Borgmann et al. (1952) reported that the largest concentration was found in liver and kidney tissue. This appears to be generally true in studies of toxic material since these organs play a key role in detoxification and elimination of toxic substances. The external pathological symptoms of 28-week old chickens at 100 p.p.m. of Halowax 1014 were characterized by pale and underdeveloped combs and wattles in both sexes. These characteristics were especially striking in some males which were mistaken for females until the time of autopsy. The abnormalities most frequently observed at autopsy were the enlarged livers and the presence of clear straw-colored fluid in the pericardial sac and the peritoneal cavity. SUMMARY
Halowax 1014 (a mixture of pentaand hexachloronaphthalenes) was fed to New Hampshire chickens through the life cycle in order to study its influence as measured by certain physiological responses. A dietary intake of 2,500 p.p.m. resulted
429
in 100% mortality in 2 weeks, an intake of 100 p.p.m. to 28 weeks of age prevented egg production, 20 p.p.m. was responsible for poor hatchability while a 4 p.p.m. appeared to be well tolerated by the chickens of all ages. Thus, the upper limit of tolerance for Halowax 1014 would appear to be between 4 and 20 p.p.m. The vitamin A content of the liver varied inversely with the Halowax content of the diet. The hematocrit of chicks that received 100 p.p.m. of the chlorinated naphthalene for only 35 days was significantly lower ( 1 % level) than the hematocrit of controls. In 28-week old chickens that were fed the 100 p.p.m. diet, the sodium and potassium content of the serum was significantly lower, while the cholesterol and uric acid content of the serum was significantly higher (5% level). The most common pathological symptoms observed were the enlarged and fibrotic liver, the presence of a straw-colored fluid in the pericardial sac and peritoneal cavity and the lack of normal feather pigmentation in New Hampshire chicks. ACKNOWLEDGMENTS
The authors are grateful to Dr. L. V. Curtin, Buckeye Cotton Oil Company, Ivorydale, Ohio for the cottonseed oil, to the Bakelite Company, New York for the Halowax 1014 and to Dr. S. Gordeuk, Jr., The Pennsylvania State University, who performed many of the autopsies. REFERENCES Arant, F. S., 1952. Toxicity of aldrin to chickens. J. Econ. Ent. 45: 121. Association of Vitamin Chemists, Inc. 1947. Methods of Vitamin Assay. Interscience Publishers, Inc., New York, N.Y. Bell, W. B., 1952. Further studies on the production of bovine hyperkeratosis by the administration of a lubricant. Virginia J. Sci. 3: 169-177. Borgmann, A. R., P. A. Dahm and C. H. Kitselman, 1952. Toxicological studies of aldrin to
430
W. J. PUDELKIEWICZ, R. V. BOUCHER, E. W. CALLENBACH AND R. C. MILLER
laboratory animals. Food and Drug Administration Hearings, Washington, D.C. Exhibit 1218. Committee on Animal Nutrition, 1950. Recommended nutrient allowances for poultry. National Research Council, Washington, D.C. Cotter, L. N., 1944. Pentachlorinated naphthalenes in industry. J. Am. Med. Assoc. 125: 273-274. Eden, W. C , 1951. Toxicity of dieldrin to chickens. J. Econ. Ent. 44: 1013. Hawk, P. B., B. L. Oser and W. H. Summerson, 1949. Practical Physiological Chemistry. 12th edition. The Blakiston Company, Philadelphia, Pa. Kimble, N. A., 1939. The photocolorimetric determination of vitamin A and carotene in human plasma. J. Lab. Clin. Med. 24: 1055-1065. Lee, A. M., 1952. Our newer knowledge of bovine hyperkeratosis (X-disease). Reprint from Proceedings United States Livestock Sanitary Association. Fifty-sixth annual meeting. Pp. 175194. Olafson, P., 1947. Hyperkeratosis (X-disease) of cattle. Cornell Vet. 37: 279-291. Pudelkiewicz, W. J., R. V. Boucher, E. W. Callenbach and R. C. Miller, 1958. Some physiological responses of Broad Breasted Bronze poults to
chlorinated naphthalenes. Poultry Sci. 37: 185187. Rosenberg, M. M., T. Tanaka and H. E. Adler, 1950. Toxicity of chlordane to laying pullets. Am. J. Vet. Research, 11: 236-239. Schwartz, L., 1936. Dermatitis from synthetic resins and waxes. Am. J. Pub. Health, 26: 586592. Shank, M. T., 1954. Personal communication. Sikes, D., and M. E. Bridges, 1952. Experimental production of hyperkeratosis ("X-disease") of cattle with a chlorinated naphthalene. Science, 116: 506-507. Snedecor, W. W., 1946. Statistical Methods Applied to Experiments in Agriculture and Biology. The Iowa State College Press, Ames, Iowa. Toth, S. J., A. L. Prince, A. Wallace and D. S. Mikkelsen, 1948. Rapid quantitative determination of eight mineral elements in plant tissue by a systematic procedure involving use of a flame photometer. Soil Science, 66: 459-466. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.
The Interrelationship of Reproductive State and Seasonal Acclimatization on the Hen's Resistance to Lethal High Temperature H A R O L D S.
Department
WEISS
of Poultry Science, Rutgers, the State University, New Brunswick, New Jersey (Received for publication August 5, 1958)
N
ON-LAYING hens should withstand heat stress better than layers on the theoretical grounds that their metabolism and heat production are lower (Hutchinson, 19S4). Robinson and Lee (1947) showed that decreased feed intake, such as might be expected in non-layers, reduces some of the undesirable effects of hot environments Paper of the Journal Series, New Jersey Agricultural Experiment Station, New Brunswick, N.J. Supported in part by a grant from CIBA Pharmaceutical Products, Inc., Summit, N.J.
on hens. The lower body temperature and respiration rate found by Hillerman and Wilson (1955) in non-layers compared to layers at high air temperatures implies further that the non-layer is more capable of acclimatization. On the other hand, Hutt (1939) was unable to find any relationship between production and susceptibility to high temperature in a flock exposed to a naturally occurring heat wave intense enough to cause mortality from heat prostration.