- Email: [email protected]
Formaldehyde Levels on and in Chicken Eggs Following Preincubation Fumigation
552
T. L. GOODWIN, L. D. ANDREWS AND J. E.
G. S. Linton, 1957. Relationships among diet composition, fleshing, fatness, and edible quality of female roasting turkeys. 1 Fish products in starters and growers. Poultry Sci. 36: 635-646. Rand, N. T., F. A. Kummerow and H. M. Scott, 1957. The relationship of dietary protein, fat, and energy on the amount, composition and origin of chick carcass fat. Poultry Sci. 36: 1151. Shrimpton, D. H., and W. S. Miller, 1960. Some causes of toughness in broilers (young roasting chickens) II. Effects of breed, management, and sex. British Poultry Sci. 12: 111-112. Summers, J. D., S. J. Slinger and G. C. Ashton, 1965. The effect of dietary energy and protein on carcass composition with a note on a method for estimating carcass composition. Poultry Sci. 44: 501-509. Wise, R. G., and W. J. Stadelman, 1957. Effect of beating by mechanical pickers on the tenderness of poultry meat. Poultry Sci. 36: 1169-1170. Wise, R. G., and W. J. Stadelman, 1959. Tenderness at various muscle depths associated with poultry processing techniques. Food Technol. 13: 689691.
Formaldehyde Levels on and in Chicken Eggs Following Preincubation Fumigation J. E. WILLIAMS 1 AND H. S. SIEGEL 2 Southeast Poultry Research Laboratory, A gricultural Research Service, United Stales Department of A griculture, Athens, Georgia 30601 (Received for publication August 29, 1968)
INTRODUCTION
T
HE preincubation fumigation of chicken and turkey hatching eggs with formaldehyde (HCHO) gas for the early destruction of salmonella shell surface contamination, following the work of Wilson (1951), Lancaster el al. (1954), and Stover (1960), has become a rather common practice in the United States. Formalin (40% HCHO) is mixed with the oxidizing agent potassium permanganate (KMnO^ to generate the gas to which 1 2
Animal Disease and Parasite Research Division. Animal Husbandry Research Division.
eggs are exposed in a tightly closed cabinet or room. Paraformaldehyde is also available as a powder from which the gas can be released by heating. It is recommended that fumigation be carried out as soon as possible after the eggs are laid and dry cleaned. In using the gas at a high concentration, temperature is not considered to be a critical factor, but is generally kept at least 21°-24°C. (70° — 75°F.). The presence of moisture is important, and the relative humidity should be maintained at or near 70%. Harry (1954) reported that the union of HCHO with the shell surface during
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 13, 2015
White turkeys fed different cereal grains. Poultry Sci. 35: 1162. Harkin, A. M., C. Kitzmiller, G. L. Gilpin and S. J. Marsden, 1958. Quality of cooked meat of turkeys fed animal or vegetable protein diets with vitamin and fat supplements. Poultry Sci. 37: 1328-1339. Harshaw, H. M., 1936. Effect of diet, range, and fattening on the physical and chemical composition of cockerels. J. Agri Res. 53: 357-368. Klose, A. A., M. F. Pool, M. B. Wiele, H. L. Hanson and H. Lineweaver, 1959. Poultry tenderness. I. Influence of processing on tenderness of turkeys. FoodTechnol. 13: 20-24. Klose, A. A., A. A. Campbell, H. L. Hanson and H. Lineweaver, 1961. Effects of duration and type of chilling and thawing on tenderness of frozen turkeys. Poultry Sci. 40: 683-688. Klose, A. A., M. F. Pool, D. de Fremery, A. A. Campbell and H. Lineweaver, 1960. Effect of laboratory scale agitated chilling on poultry quality. Poultry Sci. 39: 1193-1198. Marion, W. W., 1967. Meat tenderness in the avian species. J. World's Poultry Sci. 23: 6-19. Marsden, S. J., L. M. Alexander, J. C. Lamb and
WEBB
FORMALDEHYDE ON E G G A F T E R FUMIGATION
fumigation is not chemical since it could be removed by soaking fumigated eggs in water. T h e need for quantitative information of the levels and persistence of H C H O on the surface, within the sub-shell membrane structures, and in the albumen of fumigated eggs prompted the work reported here.
Fumigation and Storage of Eggs. Brownshell eggs from adult White P l y m o u t h Rock and Athens Random Bred (ARB) flocks maintained at the laboratory were used. T h e eggs were fumigated on the day of collection, or one d a y following. Some which were slightly stained were dry cleaned prior to delivery to the laboratory. An automatic egg fumigation cabinet (U.S. P a t e n t 3,326,114) operated at room temperature was used for enclosing the eggs during fumigation. This cabinet, designed for application of paraformaldehyde fumigation, was modified b y removal of the electric heating pan and V-shaped metal sheeting below the egg t r a y supports. An auxiliary electric 115v, 60Hz circulating fan was mounted a t the b o t t o m of the cabinet to provide strong, constant air circulation during the period of fumigation. M e t a l cross bars were installed to provide support of eight separated layers of eggs placed on 48-capacity plastic trays. These were spaced with three trays of eggs per layer providing a full-load capacity of 1152 eggs for the 30 cubic foot cabinet. Test eggs in groups of eight were routinely placed on the front section of a plastic tray four rows from the top on the right side of the cabinet. This tray, as well as t h e rest of the cabinet, was filled t o capacity with other eggs. Unfumigated eggs (blanks) in groups of two were placed on a clean plastic rack at room temperature in an area separated from the fumigation room and were stored there
for the same period as required to fumigate groups of test eggs. While only five test eggs and one blank were included in each experimental group, the additional eggs were available in the event of egg breakage during collection procedures. T h e level of fumigant used in these studies was t h a t generated by the addition of 1.2 ml. of formalin (40% H C H O ) to 0.6 g. of potassium permanganate ( K M n 0 4 ) per cubic foot of cabinet space. The period of fumigation was 20 minutes followed by either immediate removal of the eggs or a 15-minute exhaust period before opening the cabinet. Gas masks were utilized when eggs were removed from the cabinet without exhaust. During periods when not in use, the cabinet doors remained open to allow complete depletion of the gas between daily fumigations. H e a t and water are generated in the fumigation cabinet by the chemical reaction during release of the H C H O gas. T h e mean of 75 dry-bulb readings in the cabinet during the 20-minute fumigation interval over a 10-month period was 26°C. (78.8°F.), wet-bulb 22°C. (71.6°F.) and a relative humidity of 7 4 % . An immediate rise in both dry- and wet-bulb readings was registered inside the cabinet as soon as the fumigant was released. Readings were made on a psychrometer through a glass plate in the cabinet door or automatically recorded through thermocouples and humidity detectors installed inside the cabinet. Eggs were stored on clean plastic racks either at room temperature (24°C.) or in egg coolers at 10°C. (50°F.) after fumigation. Those eggs which were washed after fumigation were submerged in cold, running t a p water for 10 minutes. Collection of Outer Structures and Albumen. Shells. Test eggs were individually removed from t h e cabinet a n d deposited
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 13, 2015
MATERIALS AND METHODS
553
554
J.
E.
W I L L I A M S AND H.
A small opening was drilled through the shell only, just adjacent to the air cell area using a hand drill. T h e pointed end of the egg was broken away, and the contents were discarded. Applying weak air pressure (9 p.s.i.) over the opening in the shell, the membranes were blown down so t h a t they protruded from the pointed end of the egg and were pulled from the shell with a clean forceps and discarded (Williams and Whittemore, 1967). T h e shell now free of contents and membranes was returned to the beaker from which it had originally come. The beaker, sampling fluid, and shell were weighed on a triple-beam balance and the weights recorded. Using a clean glass rod the shell was broken into fine fragments. Shell membranes. The inner and outer membranes from each egg were separated from the shell, using the above technique and removed with a clean forceps. T h e membranes from 3 eggs were pooled into a single semimicro, Monel blendor cup
SIKGEL
containing 50 ml. of distilled water to which were added 3-4 drops of H 3 P 0 4 . T h e membranes were ground for 3 minutes in the blendor set a t medium speed to form a shell membrane homogenate. Albumen. A small opening was drilled through the shell only, a t the equator of the egg. T h e opening was lightly brushed with a cotton swab moistened with distilled water to remove any shell debris. T h e point of a hot, flamed forceps was then used to burn an opening through the shell membranes. Using a chemically clean 30-ml. syringe with a 13-gauge cannula, about 25 ml. of albumen were removed from the egg with care being taken to hold the cannula as close to the inner surface of the inner membrane as possible to avoid rupturing the yolk. An earlier technique tried and abandoned because of excessive yolk rupture was to make collections through the inner shell membrane at the air cell. The collected albumen was discharged from the syringe after cannula removal into chemically clean 50-ml. screw-capped test tubes. If a sample was accidentally contaminated with yolk it was discarded. T h e net weight of albumen from each egg was determined. Chemical Analyses for HCHO. Shells. T h e shell fragments and sampling fluid were poured from each beaker into a separate 250-ml. graduated cylinder after which the beaker was well rinsed into the cylinder with a jet of distilled water. Ten ml. of Tergitol No. 7 (Union Carbide Co., Los Angeles, California) were added to the cylinder along with 14-18 drops of H3PO4 to lower the p H of the final solution to 4.0. Sufficient distilled water was then added to bring the volume in the cylinder to 250 ml. T h e contents were transferred to a 1000-ml. flat-bottomed boiling flask. Each boiling flask was sprayed inside
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directly into a chemically clean 250-ml. beaker containing approximately 100 ml. of distilled water to which were added 3 5 drops of concentrated ortho-phosphoric acid (H3PO4). Eggs stored at room temperature or 10°C. were similarly collected a t selected periods after fumigation. All egg blanks were collected in the same manner. T h e test and egg blanks were left in the sampling fluid in the collection beakers for 10 minutes with frequent swirling to assure t h a t the egg surface was well washed. Using a clean stainless-steel wire loop, each egg was removed from the sampling liquid and its surface rinsed well into the beaker with a jet of distilled water. T h e eggs were placed on a plastic rack to drain. Each beaker and its sampling liquid was carefully weighed on a triplebeam balance and weights recorded to the nearest 0.1 g.
S.
FORMALDEHYDE ON E G G A F T E R
T h e shells of egg blanks were collected and chemically analyzed as outlined above for test eggs. These eggs were at no time exposed to H C H O gas. I n calculating values for the H C H O levels on the shells of fumigated eggs, the results obtained in the chemical analysis of distillates from blank eggs were used as blank values and were subtracted from the former values. Shell membranes. T h e shell membrane homogenates were transferred from the Monel blendor cups to individual 250-ml. volumetric flasks. T h e procedures for the dilution, distillation and chemical analysis of the level of H C H O in the shell membranes were identical to those given above for shells. Controls were also included and results again used as blanks in calculating the H C H O concentration in shell membrane distillates. Albumen. T h e albumen samples were deposited in individual 250-ml. volumetric flasks. T h e procedures for the dilution, distillation and chemical analysis of the level of H C H O in the albumen were the same as those outlined above for shells. Albumen samples from control eggs were also run and used as described above. Five treated samples and one blank
200
stored at rm. temp.(24°C) SEMEAN
180 Y
160 -
> 3J140 a i
<
Il20h E 100 Q.
a 80
60 40 0 30 60 90 120 MINUTES AFTER FUMIGATION FIG. 1. Adsorption and persistence of HCHO on the shells of eggs removed immediately after fumigation and following cabinet venting for 15 minutes. Each point on the graph represents a mean of 10
sample constituted a run, and for the purposes of statistical analysis each run was treated as a replicate. D a t a were analysed by analysis of variance in various models according to the experimental design and, where applicable, means were separated b y K r a m e r ' s (1956) modification of the D u n c a n multiple range test. RESULTS
A dsorption and persistence of HCHO on the shells of fumigated eggs. Figure 1 graphically compares the concentrations of H C H O on t h e shells of eggs immediately after fumigation and following the 15-
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with a silicone defoamer (Anti-foam A Spray) and to each was added 20-30 inert granules (Boileezers) to prevent excessive bumping. A small plug of glass wool was placed in the neck of each boiling flask to guard against overflow if the liquid bumped occasionally. The flasks were connected to Allyn condensers through all-ground-glass joints and boiled slowly until all of the liquid was distilled. T h e distillate was collected in a 250-ml. Erlenmeyer flask containing 10 ml. distilled water mixed with 3 drops of H 3 P 0 4 . Duplicate aliquots of the distillate were chemically analyzed for H C H O (in p.p.m.) as described by the Association of Official Agricultural Chemists (1960).
555
FUMIGATION
556 160 r
J. E. WILLIAMS AND H. S. SIEGEL
140
120
£l00 x UJ
Q < 80
o "-. 6 0
£ a 40
160 r
20
140
i^M 0
60 MINUTES
120 7
14 DAYS
T
SE MEAN
unwashed
120
21
TIME AFTER REMOVAL FROM F U M I G A T I O N FIG. 2. Effect of post-fumigation storage conditions on the concentration of HCHO on shells. Each point on the graph represents a mean of 10 eggs.
minute cabinet exhaust. Eggs were held at room temperature (24°C.) for periods ranging from 0 to 120 minutes after removal from the cabinet before HCHO determinations were made. The analysis of variance indicated no significant differences in the HCHO level between eggs taken immediately after fumigation or after the 15-minute exhaust period. There was a highly significant reduction in HCHO levels in both groups by 30 minutes after removal from the cabinet (p<0.01). Some variation existed in the amount
>100 x LU
Q < 80 a: O
£ Q. a
cabinet vented for 15min. stored at rm. temp.(24°C)
60
40 ^washed 20
0
30 60 MINUTES TIME AFTER REMOVAL FROM FUMIGATION FIG. 3. The effect of washing on the level and persistence of HCHO*on*the shells of fumigated eggs. Each point on_thej;raph represents a mean of 10 eggs.
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of HCHO adsorbed by different eggs as illustrated by the standard errors shown. This variation was also noted by Harry (1954) and ascribed to differences naturally existent in the shell texture. Effect of post-fumigation storage temperature on the concentration of HCHO on shells. Although the levels of HCHO on the shells of fumigated eggs stored at 10°C. were consistently lower than those stored at room temperature (24°C.) (Figure 2), the differences between the two groups were not significant except at 120 minutes after fumigation (p<0.05). Determinations made at 7, 14, and 21 days indicated very low levels on eggs
c a b i n e t v e n t e d f o r 15 m i n .
FORMALDEHYDE
ON E G G A F T E R
HCHO level in the albumen of fumigated eggs. T h e concentration of H C H O in the albumen of eggs after fumigation was extremely low (Table 2). After storage for one hour and 24 hours at room temperature, the concentration did not change significantly ( p > 0.05). DISCUSSION
Preincubation fumigation of freshlylaid, brown-shell chicken eggs a t room temperature results in immediate deposition of a high level of H C H O on the shell TABLE 1.—Concentration {p.p.m.) of formaldehyde in shell membranes of brown eggs before and after washing No. of eggs per group
Unwashed, ppm HCHO (x + SE)
Washed, ppm HCHO (x±SE)
IS
3.70 + 0.6
2.60 + 0.3
TABLE 2.—Concentration (p.p.m.) of formaldehyde in albumen of brown eggs after storage Storage time (hrs.) 0 No. of eggs per group 15
1
24
p.p.m. HCHO p.p.m. HCHO p.p.m. HCHO (x±SE) (x+SE) (x"±SE) 0.12 + 0.02
0.08+0.02
0.16 + 0.02
surface. On clean eggs this t r e a t m e n t kills pathogenic, as well as m a n y other types of organisms, normally on the shell. T h e immediate kill is quite high; however, the initial level of H C H O on the shells of fumigated eggs does not persist and at room temperature has become quite low after about two hours. This study illustrates t h a t there is a wide variation in the amount of H C H O taken up on the surface of individual eggs during fumigation. This is apparently associated with the variations t h a t exist in the texture of the shell. A wide range of standard errors was encountered. The level of H C H O on fumigated eggs stored at 10° C. while consistently lower, was not significantly lower than on those stored at 24°C. Low levels of H C H O persisted on the surface of eggs up to as long as 21 days; however, no residual protective effect from this was demonstrated. Washing of freshly fumigated eggs in running, cold t a p water for 10 minutes removed most of the H C H O from the shell surface. This procedure might be used if there exists a need for the immediate removal of the fumigant from the shell; however, washing in water or water plus detergents or sanitizers is not recommended for eggs t h a t are to be incubated for hatching. Low levels of the fumigant were found in the shell membrane homogenates indicating t h a t the gas penetrated t h e shell only slightly. T h e H C H O level demonstrated in the shell membranes probably
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(10 p.p.m. or less) with no significant differences between temperature groups. The effect of washing on the level and persistence of HCHO on the shells of fumigated eggs. As shown in Figure 3, washing freshly fumigated eggs for 10 min. in running, cold t a p water reduced H C H O to very low levels on the shell surface. I t is of interest to note t h a t the standard errors with washed eggs were considerably less than were those with unwashed eggs. HCHO penetration into the shell membranes of fumigated eggs. T h e mean concentration of H C H O ( + SE) in shell membrane homogenates of unwashed eggs immediately after fumigation and exhaust of the gas was not significantly higher than in the membranes of those washed in cold running t a p water (Table 1). There were 15 eggs included in each group. T h e levels of H C H O demonstrated in the membranes were extremely low compared to levels on the shell itself. T h e gas apparently did not penetrate to any extent through the shell structure.
557
FUMIGATION
558
J. E. WILLIAMS AND H.
resulted from the direct contact with the fumigant of scattered larger openings in the shell surface. Very low concentrations of HCHO were found in the albumen of fumigated eggs indicating that there was little penetration of the gas into the albumen during fumigation. SUMMARY
ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of Messrs. Richard L. Dixon and Keith Wesley in this work. REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis, 9th edition, Washington, D. C. Harry, E. G., 1954. The influence of certain chemicophysical characteristics of formaldehyde on its use as a disinfectant. Proc. 10th World's Poultry Cong., p. 217-221. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Lancaster, J. E., R. F. Gordon and E. G. Harry, 1954. Studies on disinfection of eggs and incubators. III. The use of formaldehyde at room temperature for the fumigation of eggs prior to incubation. Brit. Vet. J. 110: 238-246. Stover, D. E., 1960. Fumigation of hatching eggs. The Bulletin, Dept. of Agr., State of California, 49: 30-33. Williams, J. E., and A. D. Whittemore, 1967. A method for studying microbial penetration through the outer structures of the avian egg. Avian Dis. 11: 467-490. Wilson, J. E., 1951. The control of salmonellosis in poultry with special reference to fumigation of incubators. Vet. Record, 63: 501-503.
NEWS AND
NOTES
(Continued from page 542) was Editor of Poultry Tribune from 1944 until 1951 and was elected a Vice-President of the company in 1965. A graduate of Ohio State University, he is Past President of the American Agricultural Editors Association, and is a member of the Poultry Science Association. He is the author of "ABC of Poultry Raising." In 1962, he received the Reuben Brigham Award from the American Association of Agricultural College Editors for meritorious service to agriculture. A.P.H.S. NOTES At the annual meeting of the American Poultry Historical Society held during the Fact Finding Conference in Kansas City, Missouri, the following officers were elected: President—O. August Hanke,
Mount Morris, Illinois; First Vice-President— E. M. Funk, University of Missouri, Columbia, Missouri; Second Vice-President—H. R. Bird, University of Wisconsin, Madison, Wisconsin; Secretary —Paul E. Sanford, Kansas State University, Manhattan, Kansas; and Treasurer—Robert L. Hogue, Purdue University, Lafayette, Indiana. Hanke, who succeeds C. S. Shaffner, University of Maryland, was Publishing Director of the Watt Publishing Company, retiring in 1967. He is now a Public Relations and Agribusiness Consultant. In 1926 he received a Bachelor of Science degree in Agriculture from the University of Wisconsin, and joined the Watt Publishing Company as Associate Editor of Poultry Tribune, and later became Editor, Editorial Director, Assistant Publisher, and Publishing Director. The title of his thesis in Agricultural
{Continued on page 589)
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Fresh brown-shell chicken eggs were exposed to fumigation with formaldehyde (HCHO) gas at a level of 1.2 ml. of formalin and 0.6 g. potassium permanganate per cu. ft. It was found that high and very bactericidal levels of the gas were demonstrable on the shells of the eggs immediately after fumigation as well as after a 15minute period of gas exhaust. The fumigant level was lower, but not significantly so, when the eggs were stored at 10°C. after fumigation. After about 2 hours' storage at room temperature (24°C.) the concentration of HCHO on the eggs had dropped radically and by 21 days it was extremely low. Penetration into the shell membranes or the albumen or fumigated eggs was also very low.
S. SIEGEL
T. L. GOODWIN, L. D. ANDREWS AND J. E.
G. S. Linton, 1957. Relationships among diet composition, fleshing, fatness, and edible quality of female roasting turkeys. 1 Fish products in starters and growers. Poultry Sci. 36: 635-646. Rand, N. T., F. A. Kummerow and H. M. Scott, 1957. The relationship of dietary protein, fat, and energy on the amount, composition and origin of chick carcass fat. Poultry Sci. 36: 1151. Shrimpton, D. H., and W. S. Miller, 1960. Some causes of toughness in broilers (young roasting chickens) II. Effects of breed, management, and sex. British Poultry Sci. 12: 111-112. Summers, J. D., S. J. Slinger and G. C. Ashton, 1965. The effect of dietary energy and protein on carcass composition with a note on a method for estimating carcass composition. Poultry Sci. 44: 501-509. Wise, R. G., and W. J. Stadelman, 1957. Effect of beating by mechanical pickers on the tenderness of poultry meat. Poultry Sci. 36: 1169-1170. Wise, R. G., and W. J. Stadelman, 1959. Tenderness at various muscle depths associated with poultry processing techniques. Food Technol. 13: 689691.
Formaldehyde Levels on and in Chicken Eggs Following Preincubation Fumigation J. E. WILLIAMS 1 AND H. S. SIEGEL 2 Southeast Poultry Research Laboratory, A gricultural Research Service, United Stales Department of A griculture, Athens, Georgia 30601 (Received for publication August 29, 1968)
INTRODUCTION
T
HE preincubation fumigation of chicken and turkey hatching eggs with formaldehyde (HCHO) gas for the early destruction of salmonella shell surface contamination, following the work of Wilson (1951), Lancaster el al. (1954), and Stover (1960), has become a rather common practice in the United States. Formalin (40% HCHO) is mixed with the oxidizing agent potassium permanganate (KMnO^ to generate the gas to which 1 2
Animal Disease and Parasite Research Division. Animal Husbandry Research Division.
eggs are exposed in a tightly closed cabinet or room. Paraformaldehyde is also available as a powder from which the gas can be released by heating. It is recommended that fumigation be carried out as soon as possible after the eggs are laid and dry cleaned. In using the gas at a high concentration, temperature is not considered to be a critical factor, but is generally kept at least 21°-24°C. (70° — 75°F.). The presence of moisture is important, and the relative humidity should be maintained at or near 70%. Harry (1954) reported that the union of HCHO with the shell surface during
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 13, 2015
White turkeys fed different cereal grains. Poultry Sci. 35: 1162. Harkin, A. M., C. Kitzmiller, G. L. Gilpin and S. J. Marsden, 1958. Quality of cooked meat of turkeys fed animal or vegetable protein diets with vitamin and fat supplements. Poultry Sci. 37: 1328-1339. Harshaw, H. M., 1936. Effect of diet, range, and fattening on the physical and chemical composition of cockerels. J. Agri Res. 53: 357-368. Klose, A. A., M. F. Pool, M. B. Wiele, H. L. Hanson and H. Lineweaver, 1959. Poultry tenderness. I. Influence of processing on tenderness of turkeys. FoodTechnol. 13: 20-24. Klose, A. A., A. A. Campbell, H. L. Hanson and H. Lineweaver, 1961. Effects of duration and type of chilling and thawing on tenderness of frozen turkeys. Poultry Sci. 40: 683-688. Klose, A. A., M. F. Pool, D. de Fremery, A. A. Campbell and H. Lineweaver, 1960. Effect of laboratory scale agitated chilling on poultry quality. Poultry Sci. 39: 1193-1198. Marion, W. W., 1967. Meat tenderness in the avian species. J. World's Poultry Sci. 23: 6-19. Marsden, S. J., L. M. Alexander, J. C. Lamb and
WEBB
FORMALDEHYDE ON E G G A F T E R FUMIGATION
fumigation is not chemical since it could be removed by soaking fumigated eggs in water. T h e need for quantitative information of the levels and persistence of H C H O on the surface, within the sub-shell membrane structures, and in the albumen of fumigated eggs prompted the work reported here.
Fumigation and Storage of Eggs. Brownshell eggs from adult White P l y m o u t h Rock and Athens Random Bred (ARB) flocks maintained at the laboratory were used. T h e eggs were fumigated on the day of collection, or one d a y following. Some which were slightly stained were dry cleaned prior to delivery to the laboratory. An automatic egg fumigation cabinet (U.S. P a t e n t 3,326,114) operated at room temperature was used for enclosing the eggs during fumigation. This cabinet, designed for application of paraformaldehyde fumigation, was modified b y removal of the electric heating pan and V-shaped metal sheeting below the egg t r a y supports. An auxiliary electric 115v, 60Hz circulating fan was mounted a t the b o t t o m of the cabinet to provide strong, constant air circulation during the period of fumigation. M e t a l cross bars were installed to provide support of eight separated layers of eggs placed on 48-capacity plastic trays. These were spaced with three trays of eggs per layer providing a full-load capacity of 1152 eggs for the 30 cubic foot cabinet. Test eggs in groups of eight were routinely placed on the front section of a plastic tray four rows from the top on the right side of the cabinet. This tray, as well as t h e rest of the cabinet, was filled t o capacity with other eggs. Unfumigated eggs (blanks) in groups of two were placed on a clean plastic rack at room temperature in an area separated from the fumigation room and were stored there
for the same period as required to fumigate groups of test eggs. While only five test eggs and one blank were included in each experimental group, the additional eggs were available in the event of egg breakage during collection procedures. T h e level of fumigant used in these studies was t h a t generated by the addition of 1.2 ml. of formalin (40% H C H O ) to 0.6 g. of potassium permanganate ( K M n 0 4 ) per cubic foot of cabinet space. The period of fumigation was 20 minutes followed by either immediate removal of the eggs or a 15-minute exhaust period before opening the cabinet. Gas masks were utilized when eggs were removed from the cabinet without exhaust. During periods when not in use, the cabinet doors remained open to allow complete depletion of the gas between daily fumigations. H e a t and water are generated in the fumigation cabinet by the chemical reaction during release of the H C H O gas. T h e mean of 75 dry-bulb readings in the cabinet during the 20-minute fumigation interval over a 10-month period was 26°C. (78.8°F.), wet-bulb 22°C. (71.6°F.) and a relative humidity of 7 4 % . An immediate rise in both dry- and wet-bulb readings was registered inside the cabinet as soon as the fumigant was released. Readings were made on a psychrometer through a glass plate in the cabinet door or automatically recorded through thermocouples and humidity detectors installed inside the cabinet. Eggs were stored on clean plastic racks either at room temperature (24°C.) or in egg coolers at 10°C. (50°F.) after fumigation. Those eggs which were washed after fumigation were submerged in cold, running t a p water for 10 minutes. Collection of Outer Structures and Albumen. Shells. Test eggs were individually removed from t h e cabinet a n d deposited
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 13, 2015
MATERIALS AND METHODS
553
554
J.
E.
W I L L I A M S AND H.
A small opening was drilled through the shell only, just adjacent to the air cell area using a hand drill. T h e pointed end of the egg was broken away, and the contents were discarded. Applying weak air pressure (9 p.s.i.) over the opening in the shell, the membranes were blown down so t h a t they protruded from the pointed end of the egg and were pulled from the shell with a clean forceps and discarded (Williams and Whittemore, 1967). T h e shell now free of contents and membranes was returned to the beaker from which it had originally come. The beaker, sampling fluid, and shell were weighed on a triple-beam balance and the weights recorded. Using a clean glass rod the shell was broken into fine fragments. Shell membranes. The inner and outer membranes from each egg were separated from the shell, using the above technique and removed with a clean forceps. T h e membranes from 3 eggs were pooled into a single semimicro, Monel blendor cup
SIKGEL
containing 50 ml. of distilled water to which were added 3-4 drops of H 3 P 0 4 . T h e membranes were ground for 3 minutes in the blendor set a t medium speed to form a shell membrane homogenate. Albumen. A small opening was drilled through the shell only, a t the equator of the egg. T h e opening was lightly brushed with a cotton swab moistened with distilled water to remove any shell debris. T h e point of a hot, flamed forceps was then used to burn an opening through the shell membranes. Using a chemically clean 30-ml. syringe with a 13-gauge cannula, about 25 ml. of albumen were removed from the egg with care being taken to hold the cannula as close to the inner surface of the inner membrane as possible to avoid rupturing the yolk. An earlier technique tried and abandoned because of excessive yolk rupture was to make collections through the inner shell membrane at the air cell. The collected albumen was discharged from the syringe after cannula removal into chemically clean 50-ml. screw-capped test tubes. If a sample was accidentally contaminated with yolk it was discarded. T h e net weight of albumen from each egg was determined. Chemical Analyses for HCHO. Shells. T h e shell fragments and sampling fluid were poured from each beaker into a separate 250-ml. graduated cylinder after which the beaker was well rinsed into the cylinder with a jet of distilled water. Ten ml. of Tergitol No. 7 (Union Carbide Co., Los Angeles, California) were added to the cylinder along with 14-18 drops of H3PO4 to lower the p H of the final solution to 4.0. Sufficient distilled water was then added to bring the volume in the cylinder to 250 ml. T h e contents were transferred to a 1000-ml. flat-bottomed boiling flask. Each boiling flask was sprayed inside
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directly into a chemically clean 250-ml. beaker containing approximately 100 ml. of distilled water to which were added 3 5 drops of concentrated ortho-phosphoric acid (H3PO4). Eggs stored at room temperature or 10°C. were similarly collected a t selected periods after fumigation. All egg blanks were collected in the same manner. T h e test and egg blanks were left in the sampling fluid in the collection beakers for 10 minutes with frequent swirling to assure t h a t the egg surface was well washed. Using a clean stainless-steel wire loop, each egg was removed from the sampling liquid and its surface rinsed well into the beaker with a jet of distilled water. T h e eggs were placed on a plastic rack to drain. Each beaker and its sampling liquid was carefully weighed on a triplebeam balance and weights recorded to the nearest 0.1 g.
S.
FORMALDEHYDE ON E G G A F T E R
T h e shells of egg blanks were collected and chemically analyzed as outlined above for test eggs. These eggs were at no time exposed to H C H O gas. I n calculating values for the H C H O levels on the shells of fumigated eggs, the results obtained in the chemical analysis of distillates from blank eggs were used as blank values and were subtracted from the former values. Shell membranes. T h e shell membrane homogenates were transferred from the Monel blendor cups to individual 250-ml. volumetric flasks. T h e procedures for the dilution, distillation and chemical analysis of the level of H C H O in the shell membranes were identical to those given above for shells. Controls were also included and results again used as blanks in calculating the H C H O concentration in shell membrane distillates. Albumen. T h e albumen samples were deposited in individual 250-ml. volumetric flasks. T h e procedures for the dilution, distillation and chemical analysis of the level of H C H O in the albumen were the same as those outlined above for shells. Albumen samples from control eggs were also run and used as described above. Five treated samples and one blank
200
stored at rm. temp.(24°C) SEMEAN
180 Y
160 -
> 3J140 a i
<
Il20h E 100 Q.
a 80
60 40 0 30 60 90 120 MINUTES AFTER FUMIGATION FIG. 1. Adsorption and persistence of HCHO on the shells of eggs removed immediately after fumigation and following cabinet venting for 15 minutes. Each point on the graph represents a mean of 10
sample constituted a run, and for the purposes of statistical analysis each run was treated as a replicate. D a t a were analysed by analysis of variance in various models according to the experimental design and, where applicable, means were separated b y K r a m e r ' s (1956) modification of the D u n c a n multiple range test. RESULTS
A dsorption and persistence of HCHO on the shells of fumigated eggs. Figure 1 graphically compares the concentrations of H C H O on t h e shells of eggs immediately after fumigation and following the 15-
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with a silicone defoamer (Anti-foam A Spray) and to each was added 20-30 inert granules (Boileezers) to prevent excessive bumping. A small plug of glass wool was placed in the neck of each boiling flask to guard against overflow if the liquid bumped occasionally. The flasks were connected to Allyn condensers through all-ground-glass joints and boiled slowly until all of the liquid was distilled. T h e distillate was collected in a 250-ml. Erlenmeyer flask containing 10 ml. distilled water mixed with 3 drops of H 3 P 0 4 . Duplicate aliquots of the distillate were chemically analyzed for H C H O (in p.p.m.) as described by the Association of Official Agricultural Chemists (1960).
555
FUMIGATION
556 160 r
J. E. WILLIAMS AND H. S. SIEGEL
140
120
£l00 x UJ
Q < 80
o "-. 6 0
£ a 40
160 r
20
140
i^M 0
60 MINUTES
120 7
14 DAYS
T
SE MEAN
unwashed
120
21
TIME AFTER REMOVAL FROM F U M I G A T I O N FIG. 2. Effect of post-fumigation storage conditions on the concentration of HCHO on shells. Each point on the graph represents a mean of 10 eggs.
minute cabinet exhaust. Eggs were held at room temperature (24°C.) for periods ranging from 0 to 120 minutes after removal from the cabinet before HCHO determinations were made. The analysis of variance indicated no significant differences in the HCHO level between eggs taken immediately after fumigation or after the 15-minute exhaust period. There was a highly significant reduction in HCHO levels in both groups by 30 minutes after removal from the cabinet (p<0.01). Some variation existed in the amount
>100 x LU
Q < 80 a: O
£ Q. a
cabinet vented for 15min. stored at rm. temp.(24°C)
60
40 ^washed 20
0
30 60 MINUTES TIME AFTER REMOVAL FROM FUMIGATION FIG. 3. The effect of washing on the level and persistence of HCHO*on*the shells of fumigated eggs. Each point on_thej;raph represents a mean of 10 eggs.
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of HCHO adsorbed by different eggs as illustrated by the standard errors shown. This variation was also noted by Harry (1954) and ascribed to differences naturally existent in the shell texture. Effect of post-fumigation storage temperature on the concentration of HCHO on shells. Although the levels of HCHO on the shells of fumigated eggs stored at 10°C. were consistently lower than those stored at room temperature (24°C.) (Figure 2), the differences between the two groups were not significant except at 120 minutes after fumigation (p<0.05). Determinations made at 7, 14, and 21 days indicated very low levels on eggs
c a b i n e t v e n t e d f o r 15 m i n .
FORMALDEHYDE
ON E G G A F T E R
HCHO level in the albumen of fumigated eggs. T h e concentration of H C H O in the albumen of eggs after fumigation was extremely low (Table 2). After storage for one hour and 24 hours at room temperature, the concentration did not change significantly ( p > 0.05). DISCUSSION
Preincubation fumigation of freshlylaid, brown-shell chicken eggs a t room temperature results in immediate deposition of a high level of H C H O on the shell TABLE 1.—Concentration {p.p.m.) of formaldehyde in shell membranes of brown eggs before and after washing No. of eggs per group
Unwashed, ppm HCHO (x + SE)
Washed, ppm HCHO (x±SE)
IS
3.70 + 0.6
2.60 + 0.3
TABLE 2.—Concentration (p.p.m.) of formaldehyde in albumen of brown eggs after storage Storage time (hrs.) 0 No. of eggs per group 15
1
24
p.p.m. HCHO p.p.m. HCHO p.p.m. HCHO (x±SE) (x+SE) (x"±SE) 0.12 + 0.02
0.08+0.02
0.16 + 0.02
surface. On clean eggs this t r e a t m e n t kills pathogenic, as well as m a n y other types of organisms, normally on the shell. T h e immediate kill is quite high; however, the initial level of H C H O on the shells of fumigated eggs does not persist and at room temperature has become quite low after about two hours. This study illustrates t h a t there is a wide variation in the amount of H C H O taken up on the surface of individual eggs during fumigation. This is apparently associated with the variations t h a t exist in the texture of the shell. A wide range of standard errors was encountered. The level of H C H O on fumigated eggs stored at 10° C. while consistently lower, was not significantly lower than on those stored at 24°C. Low levels of H C H O persisted on the surface of eggs up to as long as 21 days; however, no residual protective effect from this was demonstrated. Washing of freshly fumigated eggs in running, cold t a p water for 10 minutes removed most of the H C H O from the shell surface. This procedure might be used if there exists a need for the immediate removal of the fumigant from the shell; however, washing in water or water plus detergents or sanitizers is not recommended for eggs t h a t are to be incubated for hatching. Low levels of the fumigant were found in the shell membrane homogenates indicating t h a t the gas penetrated t h e shell only slightly. T h e H C H O level demonstrated in the shell membranes probably
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(10 p.p.m. or less) with no significant differences between temperature groups. The effect of washing on the level and persistence of HCHO on the shells of fumigated eggs. As shown in Figure 3, washing freshly fumigated eggs for 10 min. in running, cold t a p water reduced H C H O to very low levels on the shell surface. I t is of interest to note t h a t the standard errors with washed eggs were considerably less than were those with unwashed eggs. HCHO penetration into the shell membranes of fumigated eggs. T h e mean concentration of H C H O ( + SE) in shell membrane homogenates of unwashed eggs immediately after fumigation and exhaust of the gas was not significantly higher than in the membranes of those washed in cold running t a p water (Table 1). There were 15 eggs included in each group. T h e levels of H C H O demonstrated in the membranes were extremely low compared to levels on the shell itself. T h e gas apparently did not penetrate to any extent through the shell structure.
557
FUMIGATION
558
J. E. WILLIAMS AND H.
resulted from the direct contact with the fumigant of scattered larger openings in the shell surface. Very low concentrations of HCHO were found in the albumen of fumigated eggs indicating that there was little penetration of the gas into the albumen during fumigation. SUMMARY
ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of Messrs. Richard L. Dixon and Keith Wesley in this work. REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis, 9th edition, Washington, D. C. Harry, E. G., 1954. The influence of certain chemicophysical characteristics of formaldehyde on its use as a disinfectant. Proc. 10th World's Poultry Cong., p. 217-221. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Lancaster, J. E., R. F. Gordon and E. G. Harry, 1954. Studies on disinfection of eggs and incubators. III. The use of formaldehyde at room temperature for the fumigation of eggs prior to incubation. Brit. Vet. J. 110: 238-246. Stover, D. E., 1960. Fumigation of hatching eggs. The Bulletin, Dept. of Agr., State of California, 49: 30-33. Williams, J. E., and A. D. Whittemore, 1967. A method for studying microbial penetration through the outer structures of the avian egg. Avian Dis. 11: 467-490. Wilson, J. E., 1951. The control of salmonellosis in poultry with special reference to fumigation of incubators. Vet. Record, 63: 501-503.
NEWS AND
NOTES
(Continued from page 542) was Editor of Poultry Tribune from 1944 until 1951 and was elected a Vice-President of the company in 1965. A graduate of Ohio State University, he is Past President of the American Agricultural Editors Association, and is a member of the Poultry Science Association. He is the author of "ABC of Poultry Raising." In 1962, he received the Reuben Brigham Award from the American Association of Agricultural College Editors for meritorious service to agriculture. A.P.H.S. NOTES At the annual meeting of the American Poultry Historical Society held during the Fact Finding Conference in Kansas City, Missouri, the following officers were elected: President—O. August Hanke,
Mount Morris, Illinois; First Vice-President— E. M. Funk, University of Missouri, Columbia, Missouri; Second Vice-President—H. R. Bird, University of Wisconsin, Madison, Wisconsin; Secretary —Paul E. Sanford, Kansas State University, Manhattan, Kansas; and Treasurer—Robert L. Hogue, Purdue University, Lafayette, Indiana. Hanke, who succeeds C. S. Shaffner, University of Maryland, was Publishing Director of the Watt Publishing Company, retiring in 1967. He is now a Public Relations and Agribusiness Consultant. In 1926 he received a Bachelor of Science degree in Agriculture from the University of Wisconsin, and joined the Watt Publishing Company as Associate Editor of Poultry Tribune, and later became Editor, Editorial Director, Assistant Publisher, and Publishing Director. The title of his thesis in Agricultural
{Continued on page 589)
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Fresh brown-shell chicken eggs were exposed to fumigation with formaldehyde (HCHO) gas at a level of 1.2 ml. of formalin and 0.6 g. potassium permanganate per cu. ft. It was found that high and very bactericidal levels of the gas were demonstrable on the shells of the eggs immediately after fumigation as well as after a 15minute period of gas exhaust. The fumigant level was lower, but not significantly so, when the eggs were stored at 10°C. after fumigation. After about 2 hours' storage at room temperature (24°C.) the concentration of HCHO on the eggs had dropped radically and by 21 days it was extremely low. Penetration into the shell membranes or the albumen or fumigated eggs was also very low.
S. SIEGEL