- Email: [email protected]
Residues in Egg Yolks and Raw and Cooked Tissues from Laying Hens Administered Selected Chlorinated Hydrocarbon Insecticides
564
K. W. WASHBURN
at a faster rate than the minor component normally found. Inheritance studies using Fi, F2 and backcross progeny demonstrated that the differences in the minor components were due to a set of allelic, codominant genes. Individuals homozygous for either allele had a single minor component either fast or slow, in addition to the normal major hemoglobin component. The gene frequency in this population was .94 for the normal and .06 for the mutant alleles. All other populations tested were homozygous for the normal allele. REFERENCES D'Amelio, V., and A. M. Salvo, 1961. Further studies on the embryonic chick hemoglobin. An electrophoretic and immunoelectrophoretic analysis. Acta. Embryol. Morphol. Exper. 4:250. Dunlap, J. S., V. L. Johnson and D. S. Farner, 1956. Multiple hemoglobin in birds. Experientia, 12: 352-353. Fraser, R. C , 1964. Electrophoretic characteristics and cell content of the hemoglobins of developing chick embryos. J. Exp. Zool. 156:185-196. Fruton, J. S., and S. Simmonds, 1961. General Biochemistry, Second Edition. John Wiley and Sons, Inc. New York, N. Y.
Helm, H. J. van der, and T. H. J. Huisman, 1958. The two hemoglobin components of the chicken. Science, 127: 762. Hess, C. W., 1962. Randombred populations of the southern regional poultry breeding project. World's Poultry Science J. 18: 147-152. Huisman, T. H. J., and J. M. Schillhorn Van Veen, 1964. Studies on animal hemoglobin. III. The possible role of intercellular inorganic phosphate on the oxygen equilibrium of the hemoglobin in the developing chicken. Biochem. Acta. 88: 367374. Huisman, T. H. J., J. M. S. Van Veen, A. M. Doxy and C. M. Nechtman, 1964. Studies of animal hemoglobins. II. The influence of inorganic phosphate on the physico-chemical and physiological properties of the hemoglobin of the adult chicken. Biochem. Biophys. Acta. 88: 352-366. Manwell, C , C. M. A. Baker, J. D. Rosansky and M. Foght, 1963. Molecular genetics of avian proteins, II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. Natl. Acad. Sci. 49:496-503. Rodan, G. P., and F. G. Ebaugh, 1957. Paper electrophoresis of animal hemoglobins. Proc. Soc. Exp. Biol. Med. 95:397-401. Saha, A., R. Dutta and J. Ghoh, 1957. Paper electrophoresis of avian and mammalian hemoglobins. Science, 125: 447^48. Wilt, F. H., 1962. The ontogeny of chick embryo hemoglobin. Proc. Natl. Acad. Sci. 48: 15821590.
Residues in Egg Yolks and Raw and Cooked Tissues from Laying Hens Administered Selected Chlorinated Hydrocarbon Insecticides T. A. MCCASKEY 1 , A. R. STEMP 2 , B. J. LISKA AND W. J. STADELMAN Department of Animal Sciences, Purdue University, Lafayette, Indiana 47907 (Received for publication August 3, 1967)
INTRODUCTION
T
HE persistence of insecticide residues in animal products such as meat, milk and eggs, is of considerable 1
Present address: Department of Dairy Science, Auburn University, Auburn, Alabama. 2 Present address: National Dairy Products Corporation, Glenview, Illinois.
importance. The slow depletion from animal tissues and the toxicity of many insecticides are two areas of research endeavor that have commanded considerable attention in recent years. Insecticide residues appear in animal tissues through exposure of animals to contaminated environments or through the consumption
INSECTICIDE RESIDUES
565
of feed contaminated with residues. nical kelthane, 94% technical telodrin or Insecticide residues found in animal 90% technical ovex (Entomological Sotissues appear in highest concentration in ciety of America Reference Standards the adipose tissue. Presumably due to a from City Chemical Corporation, N. Y. smaller percentage of body fat, a higher or Nutritional Biochemicals Corp. Cleveconcentration of insecticide residues on a land, Ohio). An amount of insecticide fat basis has been reported to occur in equal to 10-15 p. p. m. of the average adipose tissue of chickens than in the weight of daily feed consumed was adadipose tissue of cattle, hogs, or lambs ministered in a gelatin capsule on each of (Gannon et al., 1959; and Terriere et al., 5 consecutive days. 1959). Hens were caged individually and Although there have been numerous furnished water and a commercial stanreports concerned with insecticide residue dard laying mash ad libitum. Eggs were contents of chicken tissues and eggs, more collected from each group of hens 2 days information is needed in respect to the after the insecticide administration comamounts of residues in cooked tissues from menced. The yolks were pooled and poultry contaminated with insecticides. frozen in polyethylene bags until anaLiska et al. (1967) reported that cooking lyzed. The hens were sacrificed 3 days hen carcasses containing DDT, lindane after the last dose of insecticide, and the or dieldrin for 3 hours at 121°C. rendered carcasses were cut into halves. One-half essentially all of the insecticides from the of each carcass was frozen in a polyethylbody tissues of the hen carcasses. The ene bag and later was analyzed to repreinsecticide residues were in highest con- sent raw tissues and the other one-half centration in the abdominal fat, less in was cooked for 3 hours at 121°C. Fat drippings were collected from chicken dark meat and least in white meat. tissue at 1 | and 3 hours of cooking. SamThe investigation reported here was conducted to determine the effect of cook- ples of white and dark tissues, raw and ing on the insecticide residue, lipid, and cooked, were taken from the thigh, leg, moisture contents of tissues from laying and breast of each hen and were analyzed hens administered selected insecticides in for insecticide residue, fat, and moisture their diets. Eggs collected from the hens content. during the investigation were also anaThe insecticide residue contents of the lyzed for residues. These insecticides were samples were determined using the colchosen as representative of insecticides umn cleanup method reported by Stemp that could appear in feeds in addition to et al. (1964) and electron-capture gas those previously reported on (Liska et chromatography (ECGC). A Wilkens al., 1967). Pestilizer gas chromatograph, Model 680, and a Leeds and Northrup, Model H, METHODS recorder with a disc integrator were used Thirty-six Leghorn hens were ran- for the insecticide analyses. The columns domly allotted to 6 treatments groups of used for the analyses of the insecticides 6 hens. One group served as a control. were as follows: 5% D - l l (silicone DowEach of the remaining groups was as- 11) was used for ovex, methoxychlor and signed to one of the following insecticide telodrin, and 5% QF-1 (Dow Corning treatments: 89.5% technical methoxy- silicone) for chlordane and kelthane. Colchlor, 60% technical chlordane, 85% tech- umns prepared with 5% D-ll or 5%
566
T. A. MCCASKEY, A. R. STEMP, B. J. LISKA AND W. J. STADELMAN
QF-1 were on 60-80 mesh HMDS (hexamethyldisilazane) treated Chromosorb W solid support and were packed in 5-feet by 1/8-inch OD coiled pyrex glass columns. The columns were operated at 187°C. with a nitrogen flow rate of 80 ml. per minute. A 2.0 g. sample of egg yolk, or a 1.0 g. sample of poultry tissue or fat, was used for analysis. Samples taken from each of 6 hens were separately analyzed in duplicate. The following amounts of eluant (20% methylene chloride in petroleum ether) were used during the cleanup of the samples containing the various insecticides: 800 ml. for ovex and methoxychlor, 400 ml. for telodrin and kelthane, and 600 ml. for chlordane. The moisture content of the chicken tissues was determined by loss of weight during lyophilization of the samples. Lipid content of the tissues was determined by weight difference of the tissues before and after fat extraction with a Soxhlet fat extraction apparatus employing ethyl ether.
tissue of chicken and from milk (Stemp and Liska, 1966). Insecticide residues were recovered from all chicken adipose samples in significant amounts (Table 2). Kelthane was found in highest concentrations of all insecticides recovered from abdominal fat and methoxychlor in the smallest quantities. In view of the fact that DDT (Liska et al., 1964), dieldrin (Gannon el al., 1959), and endrin (Terriere et al., 1959) have been reported to accumulate in the depot fat of hens when fed a diet containing the insecticides, the values reported for kelthane are not unreasonable. Methoxychlor did not accumulate to any appreciable extent in the adipose tissue of the hens fed the insecticide at the 10-15 p. p. m. level for 5 consecutive days. This finding is in accord with that reported by Olney et al. (1962). Ovex, chlordane, and telodrin were found in about the same relative concentrations in the raw abdominal fat of hens fed the insecticides.
The association of the insecticides with lipid was evidenced by the following obRecovery values for ovex, methoxy- servations: (1) the highest amount of inchlor, chlordane, telodrin, and kelthane secticide was found in adipose tissue, in egg yolk reported in 200 ml. fractions (2) the insecticides in rendered-out fat of eluant used to reclaim the insecticides were in about the same concentration as during the sample clean-up procedure are presented in Table 1. The values are TABLE 2.—Residue levels of raw and processed* samples from carcasses of hens similar to the recovery values for the administered insecticides same insecticides recovered from adipose RESULTS AND DISCUSSION
Sample b
TABLE 1.—Percent recovery'- of ovex, methoxychlor, chlordane, telodrin, and kelthane from egg yolk
Insecticide
Percent recovery in 200 milliliter portions of eluant 0-200
Ovex Methoxychlor Chlordane Telodrin Kelthane
5b 0 68 95 100
2-400
4-600
4 59 13 0 0
28 22 2 0 0
Insecticide 1 Ovex Methoxychlor Chlordane Telodrin Kelthane
2
3
13.3° 11.6 13.4 0.9 0.5 0.4 11.0 11.0 10.9 10.6 8.9 13.3 28.4 22.7 22.4
4 0.3 —d 2.1 0.2 2.1
5
6
7
0.4 — 2.3 0.5 4.2
0.2
0.4
0.8
0.8
1.2
3.9
6-800 8-1,000 Total 61 7 2 0 0
0° 3 0 0 0
98 91 85 95 100
a Recovery was determined by the method of Langlois et al. (1964) at the 1 p.p.m. level of insecticide added to egg yolk. b Average of two samples to nearest 1%. 0 Less than 1%.
» Three hours at 121° C. 1—Raw abdominal fat. 2—Fat drippings obtained after 1$ hours of cooking. 3—Pat drippings obtained after 3 hours of cooking. 4—Raw white meat. 5—Raw dark meat. 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. 0 Average of six samples each replicated two times to nearest 0.1d p.p.m. Less than 0.1 p.p.m. b
567
INSECTICIDE RESIDUES TABLE 3.—Lipid content of samples of raw and processed* tissues from carcasses of hens administered insecticides
TABLE 4.—Percent moisture content of raw and processed* tissues from carcasses of hens administered insecticides
Sample b
Sample b — 5 6
Insecticide Ovex Methoxychlor Chlordane Telodrin Kelthane
4
5
6
7
6.8" 8.7 7.8 5.9 10.3
10.7 12.2 10.6 11.5 12.1
5.7 6.5 5.8 4.8 6.8
10.5 10.8 9.6 10.7 11.6
* Three hours at 121°C. 4—Raw white meat. 5—Raw dark meat. 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. 0 Average of six samples to nearest 0.1%.
b
in the raw abdominal fat, and (3) tissues with the least fat contained the least insecticide residue. The insecticide residue content of raw dark chicken tissue (leg and thigh) was higher than the residue content found in the raw white meat (breast) of the same birds. Methoxychlor was not found in the raw or cooked meat samples of the birds receiving the insecticide in their diets. The small amount found in raw adipose tissue indicates that methoxychlor is not readily stored in the hen carcass. White and dark meat which have considerably less lipid than adipose tissue contained very little methoxychlor residue. After cooking, the methoxychlor content of white and dark meat would be reduced further by rendering out residue associated with lipid. The percent of fat in the raw and cooked white and dark chicken tissues from carcasses of hens administered insecticides is shown in Table 3. The average lipid content of the raw white meat was 7.9% and for the raw dark meat it was 11.4%. After 3 hours of cooking at 121°C, the white meat had an average of 5.9% lipid and the dark meat had an average of 10.6%. The moisture content of the raw and processed, white and dark tissues from
4 Ovex Methoxychlor Chlordane Telodrin Kelthane
72.4' 72.9 73.3 72.7 72.7
74.7 74.7 75.3 74.1 74.8
59.2 59.2 59.3 60.0 59.4
7 57.3 55.3 56.2 53.8 56.8
» Three hours at 121°C. 4—Raw white meat. 5—Raw dark meat. 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. c Average of six samples to nearest 0.1%.
b
hens fed 10-15 p. p. m. of various insecticides in their diets is shown in Table 4. The average moisture content was 74.7% for raw dark tissue and 72.8% for raw white tissue. After cooking the tissues, the average moisture content was 55.9% for dark meat and 59.4% for white meat. The results indicate that moisture was more easily removed from the dark tissue than from the white tissue. The data presented in Table 5 show the insecticide content per gram of fat of raw and processed, white and dark TABLE 5.—Residue content of fat* of raw and processed^ tissues from carcasses of hens administered insecticides Sample" Insecticide 5
6
7
d
3.7
3.5
3.8
e
—
—
—
4 Ovex Methoxychlor Chlordane Telodrin Kelthane
4.4
26.9 3.4 20.4
21.7 4.3 34.7
13.8
—
17.6
8.3
—
33.6
* Parts per million of insecticide in tissue times 100 divided by percent lipid of tissue. b Three hours at 121°C. • 4—Raw white meat 5—Raw dark meat 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. d Average of six samples to nearest 0.1 p.p.m. e Residue level in tissue could not be determined accurately below 0.1 p.p.m.
568
T. A. MCCASKEY, A. R. STEMP, B. J. LISKA AND W. J. STADELMAN TABLE 6.—Insecticide residue levels in yolks of eggs from hens administered insecticides Days of egg collection8 Insecticide Ovex Methoxychlor Chlordane Telodrin Kelthane
2
3 h
1.7
—
2.2
. . <0.1 3.6 <0.1 4.1
4 1.2' <0.1 3.5 0.1 9.9
5 1.1 0.3 3.4 0.4 12.6
6 1.1 0.2 3.8
—
9.3
7 2.4 0.3 3.1 0.5 14.2
8 0.4 2.0 0.7
—
a
Hens were fed 10-15 p.p.m. of insecticide on Day 1 through Day 5. Eggs were collected on Day 2 through Day 8. b No eggs were laid by the hens. ° Parts per million of insecticide to the nearest 0.1 p.p.m. Each sample represents the level of insecticide in pooled yolks of eggs collected from hens comprising each test group.
tissues from hens administered the insecticides orally. The residue levels of ovex, chlordane and telodrin were distributed about 1:1 in the fat of raw white and dark tissues. Kelthane was found in higher concentration in the fat of the dark meat. The data indicate that cooking did not greatly affect the ovex or kelthane content of white or dark tissue. Cooking chicken tissue reduced the chlordane content more in dark than in white meat. Cooking white and dark tissue also reduced the content of telodrin in the tissues, but the amount could not be determined accurately below 0.1 p. p. m. Insecticide residues were found in the yolks of eggs collected from the 5 groups of hens administered the insecticides (Table 6). Kelthane accumulated more in the egg yolks than any of the other insecticides. This phenomenon is in accord with the higher content of kelthane found in the tissues of the hens than the content of the other insecticides in the corresponding tissues (Table 2). The level of kelthane in the yolks steadily increased during the feeding trial. Although chlordane did not accumulate in the egg yolks to the same extent as kelthane, a similar trend was noted in the chlordane content of egg yolks from hens administered the insecticide. The accumulation of ovex in egg yolks during this investigation was
about 1 to 2 p.p.m. Methoxychlor was found in smaller concentration than any of the other insecticides in egg yolks. A slow accumulation and a relatively low content of methoxychlor in the egg yolks from hens administered methoxychlor have been observed also by Olney et al. (1962) and Thompson et al. (1967). SUMMARY
Leghorn laying hens were administered 10-15 p.p.m. of ovex, methoxychlor, chlordane, telodrin, or kelthane, calculated from weight of their average daily feed consumption, in gelatin capsules for 5 consecutive days. Eggs were collected from the hens and the insecticide contents of the yolks were determined. The insecticide content of the abdominal fat and white and dark chicken meat from each hen was determined and the residue content of the corresponding samples was determined after cooking for 3 hours at 121°C. The insecticides were found in the abdominal fat of the hens in significant amounts. Kelthane was recovered in largest amount and methoxychlor in smallest amount. The amounts of insecticide residues in the rendered fat samples were in the same relative concentrations as the amounts in raw abdominal fat. The insecticide content of raw dark
569
INSECTICIDE RESIDUES
meat was higher than that found in raw white meat. After cooking, the proportion of insecticide was still greater in the dark meat. Methoxychlor was not recovered from raw or cooked chicken tissues in excess of 0.1 p.p.m. Telodrin was recovered from raw tissue but not from cooked tissue. Ovex, chlordane and telodrin were distributed about equally in the fat of raw white and dark meat while kelthane was distributed more in the fat of raw dark meat. Cooking did not greatly affect the content of ovex or kelthane in the fat of white or dark tissue. The cooking process reduced the telodrin and chlordane contents of the chicken tissues. Insecticides were found in the yolks of eggs collected from hens administered insecticides. Kelthane accumulated more in the yolks than the other insecticides studied and methoxychlor accumulated the least. ACKNOWLEDGMENTS
This investigation was supported in part by PHS Research Grant EF-0004905 from the Division of Environmental Engineering and Food Protection, Public Health Service and North Central Regional Project NCM-37. This is Journal Paper No. 3125 of the Purdue University Agricultural Experiment Station.
REFERENCES Gannon, N., R. P. Link and G. C. Decker. 1959. Storage of dieldrin in tissues of steers, hogs, lambs, and poultry fed dieldrin in their diets. J. Agr. Food Chem. 7:826-828. Langlois, B. E., A. R. Stemp and B. J. Liska. 1964. Rapid cleanup of dairy products for analysis of chlorinated insecticide residue by electron capture gas chromatography. J. Agr. Food Chem. 12: 243-245. Liska, B. J., B. E. Langlois, G. C. Mostert and W. J. Stadelman. 1964. Residues in eggs and tissues of chickens on rations containing low levels of DDT. Poultry Sci. 43: 982-984. Liska, B. J., A. R. Stemp and W. J. Stadelman. 1967. Effect of method of cooking on chlorinated insecticide residue content in edible chicken tissues. J. Food Technol. 21: 117-120. Olney, C. E., W. E. Donaldson and T. W. Kerr. 1962. Methoxychlor in eggs and chicken tissues. J. Econ. Entomol. 55:477-479. Stemp, A. R., B. J. Liska, B. E. Langlois and W. J. Stadelman. 1964. Analysis of egg yolk and poultry tissues for chlorinated insecticide residues. Poultry Sci. 43: 273-275. Stemp, A. R., and B. J. Liska. 1966. One-step florisil clean-up and electron-capture gas chromatographic procedure for detection of additional insecticide residues in milk. J. Dairy Sci. 49: 1270-1272. Terriere, L. C , G. H. Arscott and U. Kiigemagi. 1959. The endrin content of eggs and body tissue of poultry receiving endrin in their daily diet. J. Agr. Food Chem. 7: 502-504. Thompson, E. M., G. J. Mountey and G. W. Ware. 1967. Methoxychlor residues in chicken eggs. J. Econ. Entomol. 60:235-237.
NEWS AND NOTES (continued from page 560) They must follow the rules for technical papers as set down in the C.S.I.R.O. Bulletin, "Instructions to Authors." Authors are to advise title and paper contents by the end of October, 1968. Authors will be advised of acceptance at an early date. Completed manuscripts must be submitted by February, 1969. Technical papers are invited under the following headings, or other areas of interest to any section of the poultry industry—nutrition, disease, genetics,
management and husbandry, marketmg and economics. The registration fee is $15.00 for W.P.S.A. members, $17.50 for non-members. This fee includes all technical sessions, the official dinner and cocktail party, morning and afternoon teas at technical sessions, official papers and one copy of the proceedings. Proceedings of the 1969 Australasian Poultry Science Convention will be published in a bound form. All delegates registering will receive a copy_
(continued on page 591)
K. W. WASHBURN
at a faster rate than the minor component normally found. Inheritance studies using Fi, F2 and backcross progeny demonstrated that the differences in the minor components were due to a set of allelic, codominant genes. Individuals homozygous for either allele had a single minor component either fast or slow, in addition to the normal major hemoglobin component. The gene frequency in this population was .94 for the normal and .06 for the mutant alleles. All other populations tested were homozygous for the normal allele. REFERENCES D'Amelio, V., and A. M. Salvo, 1961. Further studies on the embryonic chick hemoglobin. An electrophoretic and immunoelectrophoretic analysis. Acta. Embryol. Morphol. Exper. 4:250. Dunlap, J. S., V. L. Johnson and D. S. Farner, 1956. Multiple hemoglobin in birds. Experientia, 12: 352-353. Fraser, R. C , 1964. Electrophoretic characteristics and cell content of the hemoglobins of developing chick embryos. J. Exp. Zool. 156:185-196. Fruton, J. S., and S. Simmonds, 1961. General Biochemistry, Second Edition. John Wiley and Sons, Inc. New York, N. Y.
Helm, H. J. van der, and T. H. J. Huisman, 1958. The two hemoglobin components of the chicken. Science, 127: 762. Hess, C. W., 1962. Randombred populations of the southern regional poultry breeding project. World's Poultry Science J. 18: 147-152. Huisman, T. H. J., and J. M. Schillhorn Van Veen, 1964. Studies on animal hemoglobin. III. The possible role of intercellular inorganic phosphate on the oxygen equilibrium of the hemoglobin in the developing chicken. Biochem. Acta. 88: 367374. Huisman, T. H. J., J. M. S. Van Veen, A. M. Doxy and C. M. Nechtman, 1964. Studies of animal hemoglobins. II. The influence of inorganic phosphate on the physico-chemical and physiological properties of the hemoglobin of the adult chicken. Biochem. Biophys. Acta. 88: 352-366. Manwell, C , C. M. A. Baker, J. D. Rosansky and M. Foght, 1963. Molecular genetics of avian proteins, II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. Natl. Acad. Sci. 49:496-503. Rodan, G. P., and F. G. Ebaugh, 1957. Paper electrophoresis of animal hemoglobins. Proc. Soc. Exp. Biol. Med. 95:397-401. Saha, A., R. Dutta and J. Ghoh, 1957. Paper electrophoresis of avian and mammalian hemoglobins. Science, 125: 447^48. Wilt, F. H., 1962. The ontogeny of chick embryo hemoglobin. Proc. Natl. Acad. Sci. 48: 15821590.
Residues in Egg Yolks and Raw and Cooked Tissues from Laying Hens Administered Selected Chlorinated Hydrocarbon Insecticides T. A. MCCASKEY 1 , A. R. STEMP 2 , B. J. LISKA AND W. J. STADELMAN Department of Animal Sciences, Purdue University, Lafayette, Indiana 47907 (Received for publication August 3, 1967)
INTRODUCTION
T
HE persistence of insecticide residues in animal products such as meat, milk and eggs, is of considerable 1
Present address: Department of Dairy Science, Auburn University, Auburn, Alabama. 2 Present address: National Dairy Products Corporation, Glenview, Illinois.
importance. The slow depletion from animal tissues and the toxicity of many insecticides are two areas of research endeavor that have commanded considerable attention in recent years. Insecticide residues appear in animal tissues through exposure of animals to contaminated environments or through the consumption
INSECTICIDE RESIDUES
565
of feed contaminated with residues. nical kelthane, 94% technical telodrin or Insecticide residues found in animal 90% technical ovex (Entomological Sotissues appear in highest concentration in ciety of America Reference Standards the adipose tissue. Presumably due to a from City Chemical Corporation, N. Y. smaller percentage of body fat, a higher or Nutritional Biochemicals Corp. Cleveconcentration of insecticide residues on a land, Ohio). An amount of insecticide fat basis has been reported to occur in equal to 10-15 p. p. m. of the average adipose tissue of chickens than in the weight of daily feed consumed was adadipose tissue of cattle, hogs, or lambs ministered in a gelatin capsule on each of (Gannon et al., 1959; and Terriere et al., 5 consecutive days. 1959). Hens were caged individually and Although there have been numerous furnished water and a commercial stanreports concerned with insecticide residue dard laying mash ad libitum. Eggs were contents of chicken tissues and eggs, more collected from each group of hens 2 days information is needed in respect to the after the insecticide administration comamounts of residues in cooked tissues from menced. The yolks were pooled and poultry contaminated with insecticides. frozen in polyethylene bags until anaLiska et al. (1967) reported that cooking lyzed. The hens were sacrificed 3 days hen carcasses containing DDT, lindane after the last dose of insecticide, and the or dieldrin for 3 hours at 121°C. rendered carcasses were cut into halves. One-half essentially all of the insecticides from the of each carcass was frozen in a polyethylbody tissues of the hen carcasses. The ene bag and later was analyzed to repreinsecticide residues were in highest con- sent raw tissues and the other one-half centration in the abdominal fat, less in was cooked for 3 hours at 121°C. Fat drippings were collected from chicken dark meat and least in white meat. tissue at 1 | and 3 hours of cooking. SamThe investigation reported here was conducted to determine the effect of cook- ples of white and dark tissues, raw and ing on the insecticide residue, lipid, and cooked, were taken from the thigh, leg, moisture contents of tissues from laying and breast of each hen and were analyzed hens administered selected insecticides in for insecticide residue, fat, and moisture their diets. Eggs collected from the hens content. during the investigation were also anaThe insecticide residue contents of the lyzed for residues. These insecticides were samples were determined using the colchosen as representative of insecticides umn cleanup method reported by Stemp that could appear in feeds in addition to et al. (1964) and electron-capture gas those previously reported on (Liska et chromatography (ECGC). A Wilkens al., 1967). Pestilizer gas chromatograph, Model 680, and a Leeds and Northrup, Model H, METHODS recorder with a disc integrator were used Thirty-six Leghorn hens were ran- for the insecticide analyses. The columns domly allotted to 6 treatments groups of used for the analyses of the insecticides 6 hens. One group served as a control. were as follows: 5% D - l l (silicone DowEach of the remaining groups was as- 11) was used for ovex, methoxychlor and signed to one of the following insecticide telodrin, and 5% QF-1 (Dow Corning treatments: 89.5% technical methoxy- silicone) for chlordane and kelthane. Colchlor, 60% technical chlordane, 85% tech- umns prepared with 5% D-ll or 5%
566
T. A. MCCASKEY, A. R. STEMP, B. J. LISKA AND W. J. STADELMAN
QF-1 were on 60-80 mesh HMDS (hexamethyldisilazane) treated Chromosorb W solid support and were packed in 5-feet by 1/8-inch OD coiled pyrex glass columns. The columns were operated at 187°C. with a nitrogen flow rate of 80 ml. per minute. A 2.0 g. sample of egg yolk, or a 1.0 g. sample of poultry tissue or fat, was used for analysis. Samples taken from each of 6 hens were separately analyzed in duplicate. The following amounts of eluant (20% methylene chloride in petroleum ether) were used during the cleanup of the samples containing the various insecticides: 800 ml. for ovex and methoxychlor, 400 ml. for telodrin and kelthane, and 600 ml. for chlordane. The moisture content of the chicken tissues was determined by loss of weight during lyophilization of the samples. Lipid content of the tissues was determined by weight difference of the tissues before and after fat extraction with a Soxhlet fat extraction apparatus employing ethyl ether.
tissue of chicken and from milk (Stemp and Liska, 1966). Insecticide residues were recovered from all chicken adipose samples in significant amounts (Table 2). Kelthane was found in highest concentrations of all insecticides recovered from abdominal fat and methoxychlor in the smallest quantities. In view of the fact that DDT (Liska et al., 1964), dieldrin (Gannon el al., 1959), and endrin (Terriere et al., 1959) have been reported to accumulate in the depot fat of hens when fed a diet containing the insecticides, the values reported for kelthane are not unreasonable. Methoxychlor did not accumulate to any appreciable extent in the adipose tissue of the hens fed the insecticide at the 10-15 p. p. m. level for 5 consecutive days. This finding is in accord with that reported by Olney et al. (1962). Ovex, chlordane, and telodrin were found in about the same relative concentrations in the raw abdominal fat of hens fed the insecticides.
The association of the insecticides with lipid was evidenced by the following obRecovery values for ovex, methoxy- servations: (1) the highest amount of inchlor, chlordane, telodrin, and kelthane secticide was found in adipose tissue, in egg yolk reported in 200 ml. fractions (2) the insecticides in rendered-out fat of eluant used to reclaim the insecticides were in about the same concentration as during the sample clean-up procedure are presented in Table 1. The values are TABLE 2.—Residue levels of raw and processed* samples from carcasses of hens similar to the recovery values for the administered insecticides same insecticides recovered from adipose RESULTS AND DISCUSSION
Sample b
TABLE 1.—Percent recovery'- of ovex, methoxychlor, chlordane, telodrin, and kelthane from egg yolk
Insecticide
Percent recovery in 200 milliliter portions of eluant 0-200
Ovex Methoxychlor Chlordane Telodrin Kelthane
5b 0 68 95 100
2-400
4-600
4 59 13 0 0
28 22 2 0 0
Insecticide 1 Ovex Methoxychlor Chlordane Telodrin Kelthane
2
3
13.3° 11.6 13.4 0.9 0.5 0.4 11.0 11.0 10.9 10.6 8.9 13.3 28.4 22.7 22.4
4 0.3 —d 2.1 0.2 2.1
5
6
7
0.4 — 2.3 0.5 4.2
0.2
0.4
0.8
0.8
1.2
3.9
6-800 8-1,000 Total 61 7 2 0 0
0° 3 0 0 0
98 91 85 95 100
a Recovery was determined by the method of Langlois et al. (1964) at the 1 p.p.m. level of insecticide added to egg yolk. b Average of two samples to nearest 1%. 0 Less than 1%.
» Three hours at 121° C. 1—Raw abdominal fat. 2—Fat drippings obtained after 1$ hours of cooking. 3—Pat drippings obtained after 3 hours of cooking. 4—Raw white meat. 5—Raw dark meat. 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. 0 Average of six samples each replicated two times to nearest 0.1d p.p.m. Less than 0.1 p.p.m. b
567
INSECTICIDE RESIDUES TABLE 3.—Lipid content of samples of raw and processed* tissues from carcasses of hens administered insecticides
TABLE 4.—Percent moisture content of raw and processed* tissues from carcasses of hens administered insecticides
Sample b
Sample b — 5 6
Insecticide Ovex Methoxychlor Chlordane Telodrin Kelthane
4
5
6
7
6.8" 8.7 7.8 5.9 10.3
10.7 12.2 10.6 11.5 12.1
5.7 6.5 5.8 4.8 6.8
10.5 10.8 9.6 10.7 11.6
* Three hours at 121°C. 4—Raw white meat. 5—Raw dark meat. 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. 0 Average of six samples to nearest 0.1%.
b
in the raw abdominal fat, and (3) tissues with the least fat contained the least insecticide residue. The insecticide residue content of raw dark chicken tissue (leg and thigh) was higher than the residue content found in the raw white meat (breast) of the same birds. Methoxychlor was not found in the raw or cooked meat samples of the birds receiving the insecticide in their diets. The small amount found in raw adipose tissue indicates that methoxychlor is not readily stored in the hen carcass. White and dark meat which have considerably less lipid than adipose tissue contained very little methoxychlor residue. After cooking, the methoxychlor content of white and dark meat would be reduced further by rendering out residue associated with lipid. The percent of fat in the raw and cooked white and dark chicken tissues from carcasses of hens administered insecticides is shown in Table 3. The average lipid content of the raw white meat was 7.9% and for the raw dark meat it was 11.4%. After 3 hours of cooking at 121°C, the white meat had an average of 5.9% lipid and the dark meat had an average of 10.6%. The moisture content of the raw and processed, white and dark tissues from
4 Ovex Methoxychlor Chlordane Telodrin Kelthane
72.4' 72.9 73.3 72.7 72.7
74.7 74.7 75.3 74.1 74.8
59.2 59.2 59.3 60.0 59.4
7 57.3 55.3 56.2 53.8 56.8
» Three hours at 121°C. 4—Raw white meat. 5—Raw dark meat. 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. c Average of six samples to nearest 0.1%.
b
hens fed 10-15 p. p. m. of various insecticides in their diets is shown in Table 4. The average moisture content was 74.7% for raw dark tissue and 72.8% for raw white tissue. After cooking the tissues, the average moisture content was 55.9% for dark meat and 59.4% for white meat. The results indicate that moisture was more easily removed from the dark tissue than from the white tissue. The data presented in Table 5 show the insecticide content per gram of fat of raw and processed, white and dark TABLE 5.—Residue content of fat* of raw and processed^ tissues from carcasses of hens administered insecticides Sample" Insecticide 5
6
7
d
3.7
3.5
3.8
e
—
—
—
4 Ovex Methoxychlor Chlordane Telodrin Kelthane
4.4
26.9 3.4 20.4
21.7 4.3 34.7
13.8
—
17.6
8.3
—
33.6
* Parts per million of insecticide in tissue times 100 divided by percent lipid of tissue. b Three hours at 121°C. • 4—Raw white meat 5—Raw dark meat 6—White meat after 3 hours of cooking. 7—Dark meat after 3 hours of cooking. d Average of six samples to nearest 0.1 p.p.m. e Residue level in tissue could not be determined accurately below 0.1 p.p.m.
568
T. A. MCCASKEY, A. R. STEMP, B. J. LISKA AND W. J. STADELMAN TABLE 6.—Insecticide residue levels in yolks of eggs from hens administered insecticides Days of egg collection8 Insecticide Ovex Methoxychlor Chlordane Telodrin Kelthane
2
3 h
1.7
—
2.2
. . <0.1 3.6 <0.1 4.1
4 1.2' <0.1 3.5 0.1 9.9
5 1.1 0.3 3.4 0.4 12.6
6 1.1 0.2 3.8
—
9.3
7 2.4 0.3 3.1 0.5 14.2
8 0.4 2.0 0.7
—
a
Hens were fed 10-15 p.p.m. of insecticide on Day 1 through Day 5. Eggs were collected on Day 2 through Day 8. b No eggs were laid by the hens. ° Parts per million of insecticide to the nearest 0.1 p.p.m. Each sample represents the level of insecticide in pooled yolks of eggs collected from hens comprising each test group.
tissues from hens administered the insecticides orally. The residue levels of ovex, chlordane and telodrin were distributed about 1:1 in the fat of raw white and dark tissues. Kelthane was found in higher concentration in the fat of the dark meat. The data indicate that cooking did not greatly affect the ovex or kelthane content of white or dark tissue. Cooking chicken tissue reduced the chlordane content more in dark than in white meat. Cooking white and dark tissue also reduced the content of telodrin in the tissues, but the amount could not be determined accurately below 0.1 p. p. m. Insecticide residues were found in the yolks of eggs collected from the 5 groups of hens administered the insecticides (Table 6). Kelthane accumulated more in the egg yolks than any of the other insecticides. This phenomenon is in accord with the higher content of kelthane found in the tissues of the hens than the content of the other insecticides in the corresponding tissues (Table 2). The level of kelthane in the yolks steadily increased during the feeding trial. Although chlordane did not accumulate in the egg yolks to the same extent as kelthane, a similar trend was noted in the chlordane content of egg yolks from hens administered the insecticide. The accumulation of ovex in egg yolks during this investigation was
about 1 to 2 p.p.m. Methoxychlor was found in smaller concentration than any of the other insecticides in egg yolks. A slow accumulation and a relatively low content of methoxychlor in the egg yolks from hens administered methoxychlor have been observed also by Olney et al. (1962) and Thompson et al. (1967). SUMMARY
Leghorn laying hens were administered 10-15 p.p.m. of ovex, methoxychlor, chlordane, telodrin, or kelthane, calculated from weight of their average daily feed consumption, in gelatin capsules for 5 consecutive days. Eggs were collected from the hens and the insecticide contents of the yolks were determined. The insecticide content of the abdominal fat and white and dark chicken meat from each hen was determined and the residue content of the corresponding samples was determined after cooking for 3 hours at 121°C. The insecticides were found in the abdominal fat of the hens in significant amounts. Kelthane was recovered in largest amount and methoxychlor in smallest amount. The amounts of insecticide residues in the rendered fat samples were in the same relative concentrations as the amounts in raw abdominal fat. The insecticide content of raw dark
569
INSECTICIDE RESIDUES
meat was higher than that found in raw white meat. After cooking, the proportion of insecticide was still greater in the dark meat. Methoxychlor was not recovered from raw or cooked chicken tissues in excess of 0.1 p.p.m. Telodrin was recovered from raw tissue but not from cooked tissue. Ovex, chlordane and telodrin were distributed about equally in the fat of raw white and dark meat while kelthane was distributed more in the fat of raw dark meat. Cooking did not greatly affect the content of ovex or kelthane in the fat of white or dark tissue. The cooking process reduced the telodrin and chlordane contents of the chicken tissues. Insecticides were found in the yolks of eggs collected from hens administered insecticides. Kelthane accumulated more in the yolks than the other insecticides studied and methoxychlor accumulated the least. ACKNOWLEDGMENTS
This investigation was supported in part by PHS Research Grant EF-0004905 from the Division of Environmental Engineering and Food Protection, Public Health Service and North Central Regional Project NCM-37. This is Journal Paper No. 3125 of the Purdue University Agricultural Experiment Station.
REFERENCES Gannon, N., R. P. Link and G. C. Decker. 1959. Storage of dieldrin in tissues of steers, hogs, lambs, and poultry fed dieldrin in their diets. J. Agr. Food Chem. 7:826-828. Langlois, B. E., A. R. Stemp and B. J. Liska. 1964. Rapid cleanup of dairy products for analysis of chlorinated insecticide residue by electron capture gas chromatography. J. Agr. Food Chem. 12: 243-245. Liska, B. J., B. E. Langlois, G. C. Mostert and W. J. Stadelman. 1964. Residues in eggs and tissues of chickens on rations containing low levels of DDT. Poultry Sci. 43: 982-984. Liska, B. J., A. R. Stemp and W. J. Stadelman. 1967. Effect of method of cooking on chlorinated insecticide residue content in edible chicken tissues. J. Food Technol. 21: 117-120. Olney, C. E., W. E. Donaldson and T. W. Kerr. 1962. Methoxychlor in eggs and chicken tissues. J. Econ. Entomol. 55:477-479. Stemp, A. R., B. J. Liska, B. E. Langlois and W. J. Stadelman. 1964. Analysis of egg yolk and poultry tissues for chlorinated insecticide residues. Poultry Sci. 43: 273-275. Stemp, A. R., and B. J. Liska. 1966. One-step florisil clean-up and electron-capture gas chromatographic procedure for detection of additional insecticide residues in milk. J. Dairy Sci. 49: 1270-1272. Terriere, L. C , G. H. Arscott and U. Kiigemagi. 1959. The endrin content of eggs and body tissue of poultry receiving endrin in their daily diet. J. Agr. Food Chem. 7: 502-504. Thompson, E. M., G. J. Mountey and G. W. Ware. 1967. Methoxychlor residues in chicken eggs. J. Econ. Entomol. 60:235-237.
NEWS AND NOTES (continued from page 560) They must follow the rules for technical papers as set down in the C.S.I.R.O. Bulletin, "Instructions to Authors." Authors are to advise title and paper contents by the end of October, 1968. Authors will be advised of acceptance at an early date. Completed manuscripts must be submitted by February, 1969. Technical papers are invited under the following headings, or other areas of interest to any section of the poultry industry—nutrition, disease, genetics,
management and husbandry, marketmg and economics. The registration fee is $15.00 for W.P.S.A. members, $17.50 for non-members. This fee includes all technical sessions, the official dinner and cocktail party, morning and afternoon teas at technical sessions, official papers and one copy of the proceedings. Proceedings of the 1969 Australasian Poultry Science Convention will be published in a bound form. All delegates registering will receive a copy_
(continued on page 591)