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Aflatoxicol and Aflatoxins B1 and M1 in Eggs and Tissues of Laying Hens Consuming Aflatoxin-Contaminated Feed
Aflatoxicol and Aflatoxins Bi and M t in Eggs and Tissues of Laying Hens Consuming Aflatoxin-Contaminated Feed M. W. TRUCKSESS, L. STOLOFF, 1 and K. YOUNG Division of Chemistry and Physics, Food and Drug Administration,
Washington, DC 20204
R. D. WYATT and B. L. MILLER Department of Poultry Science, University of Georgia, Athens, Georgia 30602
ABSTRACT This study was undertaken to relate quantitatively the aflatoxin residue found in eggs and tissues to the aflatoxin intake via feed. Eighteen hens were fed an aflatoxin Bl (B[)contaminated feed (8 Mg/g) for 7 days, after which half the group was sacrificed; the remainder were sacrificed after an additional 7 days on an aflatoxin-free diet. Eggs were collected over the entire 14-day period. Aflatoxicol (R 0 ), B„ or both were found in eggs and tissues (kidneys, liver, muscle, blood, and ova). Aflatoxin M, (M : ) (.04 to .1 ng/g) was found only in the kidneys. Levels of R0 and B t were approximately the same in eggs, ova, kidneys, and liver. In eggs, the levels of R 0 and Bj (.02 to .2 ng/g) increased steadily for 4 or 5 days, after which time the levels plateaued and then decreased after B, withdrawal at the same rate as they had increased. At 7 days after withdrawal, only trace amounts of R 0 (.01 ng/g) remained in eggs. All tissues, except blood, from hens sacrificed immediately before aflatoxin withdrawal contained R 0 (.04 to .4 ng/g) or R 0 and Bj (.04 to .8 ng/g). The R 0 (.03 to .11 ng/g) was the only aflatoxin detected in muscle, and Bj (.05 to .07 ng/g) was the only aflatoxin in blood. Seven days after aflatoxin withdrawal, B, (.08 ng/g) was found in one of nine livers and R 0 (.01 to .04 ng/g) in eight of nine muscles analyzed, but no aflatoxins were found in any other tissues. (Key words: aflatoxicol, aflatoxin Bit aflatoxin M,, laying hens, eggs, tissues). 1983 Poultry Science 62:2176-2182 INTRODUCTION Residues of aflatoxin B1 (Bi) have been found in eggs from hens and in tissues from hens and broilers fed an aflatoxin-contaminated ration by m e t h o d s capable of detecting aflatoxins M i ( M ^ and Bi (Rodricks and Stoloff, 1977: J a c o b s o n and Wiseman, 1 9 7 4 ; Trucksess et al, 1 9 7 7 ) . However, t h e p o t e n t i a l for finding aflatoxicol ( R 0 ) , t h e m o s t t o x i c of t h e k n o w n aflatoxin metabolites (Campbell and Hayes, 1 9 7 6 ) , in either eggs or m e a t from laying hens had n o t been investigated, because a d e q u a t e analytical m e t h o d o l o g y has n o t been available. T w o m e t h o d s recently developed t o d e t e r m i n e R o , B1, and M i in animal tissues and eggs (Trucksess, 1 9 8 2 ; Trucksess and Stoloff, 1 9 8 1 , 1 9 8 3 ; Trucksess et al, 1982) have n o w been used t o d e t e r m i n e this p o t e n t i a l . MATERIALS AND METHODS Aflatoxin was p r o d u c e d by growing Aspergillus parasiticus ( N R R L 2 9 9 9 ) on rice 1
Retired.
(Shotwell et al., 1 9 6 6 ; West et al., 1 9 7 3 ) . T h e m o l d y rice was autoclaved and then dried and g r o u n d t o a fine p o w d e r . T h e rice p o w d e r was analyzed for aflatoxin c o n t e n t by t h e CB m e t h o d (Association of Official Analytical Chemists, 1 9 8 0 ) and was f o u n d t o contain t h e following p r o p o r t i o n s : 80% B 1 ( 20% G j , and less t h a n 1% B 2 and G 2 . A weighed q u a n t i t y of rice p o w d e r was incorporated into a basal layer ration t o furnish Bi a t a level of 8 Mg/g feed. Twenty-four Single C o m b White Leghorn hens were selected on t h e basis of good rate of egg p r o d u c t i o n . T h e hens were r a n d o m l y divided into a control g r o u p of 6 hens and a treated g r o u p of 18 hens. T h e control h e n s were given an aflatoxin-free diet, while t h e treated hens were given the diet containing t h e m o l d y rice p o w d e r . T h e hens were housed at 22 C in individual wire cages u n d e r 17 hr lighting with feed and w a t e r available ad lib. F o o d c o n s u m p t i o n was recorded daily; b o d y weights were d e t e r m i n e d at t h e beginning of t h e e x p e r i m e n t and on Days 7 and 14; eggs were collected daily, weighed, shelled, and stored at —20 C until analyzed. On Day 7 of
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(Received for publication April 6, 1983)
AFLATOXINS IN EGGS AND TISSUES
2177
TABLE 1. Average body weight, food intake, egg production, egg weight, and relative weights of kidneys, liver, and ovaries for laying hens after 7 days on aflatoxin-contaminated diet (B1 level, 8 iig/g) and after an additional 7 days on control diet
No. of hens
Body weight
Food intake
(%)
2 9 3 9
(g) 1685 1850 1843 1741
(g/day)
Day Day Day Day
124 124 111 144
64 79 86 79
1
7 control 7 treated 14 control 14 treated
Relative weight1 Egg weight
Kidneys
Liver
Ovaries
(g) 67 58 61 59
.68 .72 .59 .72
2.55 2.84 2.46 3.29
2.75 2.02 2.78 1.98
Grams organ weight per 100 g body weight.
the experiment, 3 of the control hens and 9 of the treated hens were sacrificed. The kidneys, liver, breast muscle, blood, and ovaries were removed from each hen, weighed, and stored at —20 C. Starting on Day 8, both the control and the treated group were fed the aflatoxin-free diet for another 7 days. On Day 14 the remaining hens were sacrificed, and the same tissues were removed as on Day 7. The liver, muscle, kidneys, and blood were analyzed with the method for R 0 , B j , and JV^ in tissues (Trucksess, 1982; Trucksess and Stoloff, 1981; Trucksess et al., 1982). The method was scaled down for the samples of kidneys and blood because of the small quantities available. The method consists of extraction with chloroform aqueous methanol, and hexane and column chromatography with silica gel. Fluorescence measurement after separation on a Cjg reverse phase high performance liquid chromatographic (HPLC) column determines
R 0 . Fluorescence densitometry after separation on silica gel thin layer chromatographic (TLC) plates determines Bx and M j . Eggs and ova (developing eggs inside the ovaries) were analyzed with the method for R 0 , B], and Mi in eggs (Trucksess and Stoloff, 1983). The method consists of extraction with aqueous acetone, liquid-liquid partition with petroleum ether, and column chromatography with silica gel. Fluorescence measurement as in the method for tissues determine R 0 , B i , and M!. In most cases, two sequential eggs from the same hen were pooled for each assay to obtain a large enough sample for the lowest determination limit of the method. Single egg analyses were performed on four eggs laid on Day 1, one egg laid on Day 8, and one egg laid on Day 14. Ova larger than ca. .5 cm in diameter were separated from the ovarian connective tissue and pooled for each hen for assay. RESULTS AND DISCUSSION
TABLE 2. Approximate limits of determination (ng/g)' for aflatoxicol (R„) and aflatoxins B1 and M, in hen tissues Aflatoxin Eggs Ro B, M,
.01 .02 .04
Ova
Kidneys
Blood
Liver
Muscle
.03 .06 .04
.04 .20 .04
.02 .05 .06
.01 .02 .04
.01 .02 .04
'These limits are based on the combination of sample size used for HPLC and the TLC resolution of the aflatoxins from interferences and are not necessarily the limits of these methods. Recovery studies were not conducted for all of these levels.
The effects of Bj at a level of 8 jug/g feed on various biological parameters in laying hens are shown in Table 1. There were no significant differences except for relative weights of liver and ovaries in these parameters between the control and experimental groups after either the 7-day aflatoxin feeding period or the 7-day withdrawal period. One control hen that produced no eggs during the test period was considered an anomaly and discounted as a control. The weights of livers relative to body weights of hens exposed to aflatoxin for 7 days increased over the relative liver weights of the control hens and continued to increase after withdrawal of the aflatoxin-containing feed, as
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Group
Egg production
1
1.0
Single egg analysis.
.3
.7 .8
.9
.5
1.2
Avg
1.1 1.7 1.0
1.5
1.2
.21 .5'
.2'
.3'
1.0
1 2 3 4 6 7 8 9 10 11 12 14 17 19 20 25 27 30
Hen
.8
.8
2.0
1.1
.8 1.4 1.5 .7
1.3
.7
2.5
1.7
2.1
1.4 1.5
1.7
1.9 1.4 2.1 •8
2.3
3.5
1.9
2.0 2.7
1.9
1.6 1.2 1.7
2.4
4.5
.8
.8
5.0
1.9
1.9
5.5
2.4
2.9 1.9
6.0
2.0
2.7
1.3 1.5 1.8 1.2 2.0
1.4 1.6 2.0
2.3
2.7 3.8
6.5
2.1
2.7
1.4
7.5
Day
1.5
1.1
1.9
1
8.0
.8
.8
8.5
.8
1.2
.5 1.3
.7
.5
9.5
T A B L E 3 . B ! residues (ng/g) X ICT1 in eggs from laying hens that consumed feed containing B1 at 8 iig/gfor 7 day consecutive eggs and the time interval is the average of the 2 days on which those
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1
.95
Single egg analysis.
.23
.87 .62
1.1
.56
.99
.86
.93 1.3 .63
1.5
Avg
.23' .27'
.20'
.231
1.0
1 2 3 4 6 7 8 9 10 11 12 14 17 19 20 25 27 30
Hen no.
1.2 1.3 2.1 .5
1.4
.80
2.5
.68 1.2
.68
2.0
1.6
2.1
1.5 1.3
1.9
1.8 1.9 1.8 .49
2.1
3.5
2.0
2.2 2.8
2.0
1.6 1.0 1.9
2.6
4.5
.55
.55
5.0
2.2
2.2
5.5
2.0
1.4 2.5
6.0
.78 1.4
1.2
.79
9.5
1.2
1.5 .99
.99
8.5
1.1
.53
1.91
8.0
2.1 2.5
3.1
1.8
7.5
2.7
1.6 1.9 1.8 1.7 2.0
1.9 1.6 2.3
1.8
2.3 3.3
6.5
Day
1
1
1
TABLE 4. Aflatoxicol (R0) residues (ng/g) X Iff1 in eggs from laying hens that consumed feed containing B, at 8 Ug pool of two consecutive eggs and the time interval is the average of the 2 days on which t
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TRUCKSESS ET AL. days on an aflatoxin-free diet. No Mx was detected in any of the eggs laid during the treatment period or the recovery period. No Ro, Bi, or Mj was found in any eggs from control hens during both periods. The biological half-life retention times (Ty2 in hr) of R 0 and B1 in eggs were 19 and 20, respectively; these values were calculated from the peak at 6.5 days to the end of the experiment at 14 days by a first order equation. The R 0 (.13 to .42 ng/g) and Bj (.17 to .37 ng/g) levels found in the ova of the hens sacrificed on Day 7 are given in Table 5. The levels of R 0 and B^ in these samples were about the same as the levels of R 0 and Bj in eggs collected on Days 6 or 7. No M t was found in any of the ova. No aflatoxins were found in the ova from the control hens or the hens sacrificed on Day 14. Aflatoxin apparently can enter the egg at any stage of its development. It takes 7 to 8 days for each oocyte to develop into a mature ovum (yolk). The ovum then descends into the oviduct, where the egg white is secreted, and is enveloped by the eggshell, a process that takes slightly more than 24 hr. R 0 and B : detection in eggs collected on Day 1 after feed with aflatoxin was offered indicates that B] and Ro were transmitted into the egg through the egg white because the egg yolk was fully developed before Day 1. That ova and eggs show approximately the same levels of Ro and Bi contamination on Day 7 indicates that the toxins were also able to diffuse directly into the egg yolk. Although the distribution of each toxin between egg white and egg yolk to
TABLE 5. Aflatoxicol (R 0) and aflatoxin B1 and Mx residues (ng/g) in tissues from laying hens that consumed feed containing B, at a level of 8 Vg/gfor 7 days to day of sacrifice Ova
Muscle
Blood
B,
Ro
B.
.83 .66 .81 .27 .67 .19 .28 .11 .28
.08 .10 .10 .11 .07 .03 .07 .10 .06
ND .05 .05 .07 .07 .05 .05 .07 .06
Liver
Kidneys
Hen no.
Ro
B,
Ro
B,
M,
Ro
1 2 3 4 6 7 8 9 10
.31 .42 .24 .29 .24 .14 .13 .23 .24
.37 .33 .24 .21 .18 .20 .21 .17 .23
.15 .10 ND .12 .12 .08 .11 .15 .07
ND ND .56 ND .20 .20 .47 .62 .20
.10 .05 .07 .04 .04 .04 .04 ND .04
.32 .25 .22 .08 .30 .12 .17 .09 .26
1 No M, was detected in ova, liver, muscle, or blood; no B, in muscle; no R 0 in blood; see Table 2 for limit of determination.
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seen when the treated birds were sacrificed 7 days after exposure to Bj-free feed. The enlargement of the livers was probably due to infiltration of fat, as noted from their color and texture. The relative ovary weights decreased in the treated hens over the same periods. This decrease adversely affected the size and number of the ova, a phenomenon detrimental to future egg production, a finding in agreement with a previous study evaluating the effect of aflatoxin on egg production (Huff et al., 1975). The approximate limits of determination of aflatoxin residues in the various tissues are given in Table 2. In some cases (kidneys, blood, ova), the limiting factor was the small amount of sample available; in others (liver, muscle, eggs), it was the presence of interferences. Bx and R 0 but no Mj were found in eggs. Analytical results for Bj in eggs are given in Table 3 and for R 0 in Table 4. The transfer of aflatoxin into eggs appears to occur rapidly after initial administration. R 0 (.02 ng/g) and B t (.03 ng/g) were found in eggs laid only one day after the hens had received aflatoxincontaminated feed. In each egg or pooled eggs analyzed, the levels of the two toxins, when detected, were about the same throughout the entire experimental period. The toxin concentration in eggs increased with time and reached a maximum by Day 4 or 5 (Ro ca. .2 ng/g; Bi ca. .2 ng/g) and remained at that level until aflatoxin withdrawal. After the aflatoxincontaminated feed was discontinued on Day 8, aflatoxin residues in the eggs decreased rapidly. Low levels of R 0 (.01 ng/g) and no detectable Bj were found in eggs from treated hens after 7
Blood weight = .1 body weight.
'Percent of B; intake.
2
10
16 11 10 3 11 11 8 8 10
Ro
Liver
Muscle weight = .65 body weight.
46
43
Avg
1
61 79 58 33 56 8 37 33 45
53 72 38 44 46 5 44 32 49
1 2 3 4 6 7 8 9 10
B,
Ro
Hen no.
Eggs
21
38 29 37 12 25 17 13 9 11
B,
2 1 1 1 2 1
2 1
Ro
B,
Kidneys
M,
66
67 87 74 97 42 33 53 96 42
Ro
Muscle1
11 15 8 12 8 8 5 8 7
Ro
Ova
15 12 8 8 6 12 8 6 7
B,
11
10 10 11 14 7 13 9 15 10
Ro
Blo
TABLE 6. Extractable aflatoxins in tissues from laying hens fed Bi for 7 days in their feed. Residues are prese from each hen and the total residues as percent of the total B, consum
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TRUCKSESS ET AL.
Aflatoxin residues in t h e tissues of t h e hens sacrificed 7 days after withdrawal of aflat o x i n - c o n t a m i n a t e d feed were close t o t h e limit of d e t e r m i n a t i o n . Bx (.08 ng/g) was found in o n e of t h e livers; R 0 (.01 t o .04 ng/g) was found in eight of nine muscle samples. No aflatoxins were found in kidneys or b l o o d ; n o R 0 or M.x in livers; and n o Bx o r M ! in muscle. For each hen sacrificed after 7 days o n feed containing aflatoxin, t h e t o t a l residues, t h e total Bi intake, and t h e sum of t h e aflatoxin residues as a percent of t h e Bi intake are listed in Table 6. The major part of t h e total aflatoxin residue was found in t h e eggs and muscle. T h e average for 9 hens of all aflatoxin residues in all tissues as a percentage of t o t a l Bj intake was 31 X 10~ 4 . This study d e m o n s t r a t e s t h a t Bi and its m e t a b o l i t e , R 0 , can be detected in eggs and edible tissues from hens given feed contaminated with Bj at a level of 8 A(g/g. The level of aflatoxin residues in eggs increased steadily for 4 or 5 days t o a plateau and decreased after aflatoxin withdrawal at a b o u t t h e same rate t h a t it increased. At 7 days after withdrawal, only trace a m o u n t s of Ro ( < . 0 1 ng/g) could be found in eggs. All tissues from hens sacrificed before aflatoxin withdrawal contained Bj or a m e t a b o l i t e . Liver and ova contained t h e highest levels of Bj and R 0 . Kidney was the only tissue in which Mj was found. Blood contained only low levels of B j , while muscle contained only
R 0 . Ro could still be d e t e c t e d in t h e muscle 7 days after aflatoxin withdrawal, at which t i m e all o t h e r tissues except one liver sample showed no d e t e c t a b l e aflatoxin residues. If t h e average residue t o intake ratio (219 ng/6.95 mg) observed in this s t u d y were t o hold for lower levels of aflatoxin in feed, t h e n a level 10% of t h a t used in this s t u d y (Bj at a level of .8jUg/g feed) would result in -aflatoxin residues at or below t h e limit of analytical d e t e c t i o n .
REFERENCES Association of Official Analytical Chemists, 1980. Official Methods of Analysis. 13th ed. Ch. 26. Assoc. Offic. Anal. Chera., Arlington, VA. Campbell, T. C , and J. R. Hayes, 1976. The role of aflatoxin metabolism in its toxic lesion. Toxicol. Appl. Pharmacol. 35:199-222. Huff. W. E., R. D. Wyatt, and P. B. Hamilton, 1975. Effects of dietary aflatoxin on certain egg yolk parameters. Poultry Sci. 54:2014-2018. Jacobson, W. C , and H. G. Wiseman, 1974. The transmission of aflatoxin Bj into eggs. Poultry Sci. 53:1743-1745. Rodricks, J. V., and L. Stoloff, 1977. Aflatoxin residues from contaminated feed in edible tissues of food producing animals. Pages 67—79 in Mycotoxins in Human and Animal Health. J. V. Rodricks, C. W. Hesseltine, and M. Mehlman, ed. Pathotox Publ., Park Forest South, IL. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425—428. Trucksess, M. W., 1982. Aflatoxicol in animal tissues, Ph.D. diss., Am. Univ., Washington, DC. Trucksess, M. W., and L. Stoloff, 1981. High performance liquid chromatographic determination of aflatoxicol in milk, blood, and liver. J. Assoc. Offic. Anal. Chem. 64:1083-1087. Trucksess, M. W., and L. Stoloff, 1983. Determination of aflatoxicol and aflatoxins Bj and M, in eggs. J. Assoc. Offic. Anal. Chem. (in press). Trucksess, M. W., L. Stoloff, W. C. Brumley, D. M. Wilson, O. M. Hale, L. T. Sangster, and D. M. Miller, 1982. Aflatoxicol and aflatoxins B1 and M, in the tissues of pigs receiving aflatoxin. J. Assoc. Offic. Anal. Chem. 65:884-887. Trucksess, M. W., L. Stoloff, W. A. Pons, Jr., A. F. Cucullu, L. S. Lee, and A. O. Franz, Jr., 1977. Thin layer chromatographic determination of aflatoxin Bj in eggs. J. Assoc. Offic. Anal. Chem. 60:795-798. West, S., R. D. Wyatt, and P. B. Hamilton, 1973. Improved yield of aflatoxin by incremental increases of temperature. Appl. Microbiol. 25:1018-1019.
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finding t h e same level in b o t h ova and complete eggs would indicate t h a t transmission is relatively constant t h r o u g h o u t t h e process of egg f o r m a t i o n . Analytical results for aflatoxins in t h e tissues of t h e hens sacrificed on Day 7 on t h e aflatoxin-containing feed are presented in Table 5. Aflatoxins were found in all tissues: Ro (-07 to .15 ng/g) and M1 (.04 t o .10 ng/g) in eight of nine kidneys; Bx (.20 t o .62 ng/g) in six kidneys; and R 0 (.08 to .32 ng/g) and B x (.11 to .83 ng/g) in all livers. M j was n o t found in any livers. Ro (.03 to .11 ng/g) was t h e only toxin found in muscle; and Bi (.05 to .07 ng/g) was t h e only toxin f o u n d in b l o o d . No aflatoxin was d e t e c t e d in any tissue from control hens.
Washington, DC 20204
R. D. WYATT and B. L. MILLER Department of Poultry Science, University of Georgia, Athens, Georgia 30602
ABSTRACT This study was undertaken to relate quantitatively the aflatoxin residue found in eggs and tissues to the aflatoxin intake via feed. Eighteen hens were fed an aflatoxin Bl (B[)contaminated feed (8 Mg/g) for 7 days, after which half the group was sacrificed; the remainder were sacrificed after an additional 7 days on an aflatoxin-free diet. Eggs were collected over the entire 14-day period. Aflatoxicol (R 0 ), B„ or both were found in eggs and tissues (kidneys, liver, muscle, blood, and ova). Aflatoxin M, (M : ) (.04 to .1 ng/g) was found only in the kidneys. Levels of R0 and B t were approximately the same in eggs, ova, kidneys, and liver. In eggs, the levels of R 0 and Bj (.02 to .2 ng/g) increased steadily for 4 or 5 days, after which time the levels plateaued and then decreased after B, withdrawal at the same rate as they had increased. At 7 days after withdrawal, only trace amounts of R 0 (.01 ng/g) remained in eggs. All tissues, except blood, from hens sacrificed immediately before aflatoxin withdrawal contained R 0 (.04 to .4 ng/g) or R 0 and Bj (.04 to .8 ng/g). The R 0 (.03 to .11 ng/g) was the only aflatoxin detected in muscle, and Bj (.05 to .07 ng/g) was the only aflatoxin in blood. Seven days after aflatoxin withdrawal, B, (.08 ng/g) was found in one of nine livers and R 0 (.01 to .04 ng/g) in eight of nine muscles analyzed, but no aflatoxins were found in any other tissues. (Key words: aflatoxicol, aflatoxin Bit aflatoxin M,, laying hens, eggs, tissues). 1983 Poultry Science 62:2176-2182 INTRODUCTION Residues of aflatoxin B1 (Bi) have been found in eggs from hens and in tissues from hens and broilers fed an aflatoxin-contaminated ration by m e t h o d s capable of detecting aflatoxins M i ( M ^ and Bi (Rodricks and Stoloff, 1977: J a c o b s o n and Wiseman, 1 9 7 4 ; Trucksess et al, 1 9 7 7 ) . However, t h e p o t e n t i a l for finding aflatoxicol ( R 0 ) , t h e m o s t t o x i c of t h e k n o w n aflatoxin metabolites (Campbell and Hayes, 1 9 7 6 ) , in either eggs or m e a t from laying hens had n o t been investigated, because a d e q u a t e analytical m e t h o d o l o g y has n o t been available. T w o m e t h o d s recently developed t o d e t e r m i n e R o , B1, and M i in animal tissues and eggs (Trucksess, 1 9 8 2 ; Trucksess and Stoloff, 1 9 8 1 , 1 9 8 3 ; Trucksess et al, 1982) have n o w been used t o d e t e r m i n e this p o t e n t i a l . MATERIALS AND METHODS Aflatoxin was p r o d u c e d by growing Aspergillus parasiticus ( N R R L 2 9 9 9 ) on rice 1
Retired.
(Shotwell et al., 1 9 6 6 ; West et al., 1 9 7 3 ) . T h e m o l d y rice was autoclaved and then dried and g r o u n d t o a fine p o w d e r . T h e rice p o w d e r was analyzed for aflatoxin c o n t e n t by t h e CB m e t h o d (Association of Official Analytical Chemists, 1 9 8 0 ) and was f o u n d t o contain t h e following p r o p o r t i o n s : 80% B 1 ( 20% G j , and less t h a n 1% B 2 and G 2 . A weighed q u a n t i t y of rice p o w d e r was incorporated into a basal layer ration t o furnish Bi a t a level of 8 Mg/g feed. Twenty-four Single C o m b White Leghorn hens were selected on t h e basis of good rate of egg p r o d u c t i o n . T h e hens were r a n d o m l y divided into a control g r o u p of 6 hens and a treated g r o u p of 18 hens. T h e control h e n s were given an aflatoxin-free diet, while t h e treated hens were given the diet containing t h e m o l d y rice p o w d e r . T h e hens were housed at 22 C in individual wire cages u n d e r 17 hr lighting with feed and w a t e r available ad lib. F o o d c o n s u m p t i o n was recorded daily; b o d y weights were d e t e r m i n e d at t h e beginning of t h e e x p e r i m e n t and on Days 7 and 14; eggs were collected daily, weighed, shelled, and stored at —20 C until analyzed. On Day 7 of
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Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 9, 2015
(Received for publication April 6, 1983)
AFLATOXINS IN EGGS AND TISSUES
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TABLE 1. Average body weight, food intake, egg production, egg weight, and relative weights of kidneys, liver, and ovaries for laying hens after 7 days on aflatoxin-contaminated diet (B1 level, 8 iig/g) and after an additional 7 days on control diet
No. of hens
Body weight
Food intake
(%)
2 9 3 9
(g) 1685 1850 1843 1741
(g/day)
Day Day Day Day
124 124 111 144
64 79 86 79
1
7 control 7 treated 14 control 14 treated
Relative weight1 Egg weight
Kidneys
Liver
Ovaries
(g) 67 58 61 59
.68 .72 .59 .72
2.55 2.84 2.46 3.29
2.75 2.02 2.78 1.98
Grams organ weight per 100 g body weight.
the experiment, 3 of the control hens and 9 of the treated hens were sacrificed. The kidneys, liver, breast muscle, blood, and ovaries were removed from each hen, weighed, and stored at —20 C. Starting on Day 8, both the control and the treated group were fed the aflatoxin-free diet for another 7 days. On Day 14 the remaining hens were sacrificed, and the same tissues were removed as on Day 7. The liver, muscle, kidneys, and blood were analyzed with the method for R 0 , B j , and JV^ in tissues (Trucksess, 1982; Trucksess and Stoloff, 1981; Trucksess et al., 1982). The method was scaled down for the samples of kidneys and blood because of the small quantities available. The method consists of extraction with chloroform aqueous methanol, and hexane and column chromatography with silica gel. Fluorescence measurement after separation on a Cjg reverse phase high performance liquid chromatographic (HPLC) column determines
R 0 . Fluorescence densitometry after separation on silica gel thin layer chromatographic (TLC) plates determines Bx and M j . Eggs and ova (developing eggs inside the ovaries) were analyzed with the method for R 0 , B], and Mi in eggs (Trucksess and Stoloff, 1983). The method consists of extraction with aqueous acetone, liquid-liquid partition with petroleum ether, and column chromatography with silica gel. Fluorescence measurement as in the method for tissues determine R 0 , B i , and M!. In most cases, two sequential eggs from the same hen were pooled for each assay to obtain a large enough sample for the lowest determination limit of the method. Single egg analyses were performed on four eggs laid on Day 1, one egg laid on Day 8, and one egg laid on Day 14. Ova larger than ca. .5 cm in diameter were separated from the ovarian connective tissue and pooled for each hen for assay. RESULTS AND DISCUSSION
TABLE 2. Approximate limits of determination (ng/g)' for aflatoxicol (R„) and aflatoxins B1 and M, in hen tissues Aflatoxin Eggs Ro B, M,
.01 .02 .04
Ova
Kidneys
Blood
Liver
Muscle
.03 .06 .04
.04 .20 .04
.02 .05 .06
.01 .02 .04
.01 .02 .04
'These limits are based on the combination of sample size used for HPLC and the TLC resolution of the aflatoxins from interferences and are not necessarily the limits of these methods. Recovery studies were not conducted for all of these levels.
The effects of Bj at a level of 8 jug/g feed on various biological parameters in laying hens are shown in Table 1. There were no significant differences except for relative weights of liver and ovaries in these parameters between the control and experimental groups after either the 7-day aflatoxin feeding period or the 7-day withdrawal period. One control hen that produced no eggs during the test period was considered an anomaly and discounted as a control. The weights of livers relative to body weights of hens exposed to aflatoxin for 7 days increased over the relative liver weights of the control hens and continued to increase after withdrawal of the aflatoxin-containing feed, as
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Group
Egg production
1
1.0
Single egg analysis.
.3
.7 .8
.9
.5
1.2
Avg
1.1 1.7 1.0
1.5
1.2
.21 .5'
.2'
.3'
1.0
1 2 3 4 6 7 8 9 10 11 12 14 17 19 20 25 27 30
Hen
.8
.8
2.0
1.1
.8 1.4 1.5 .7
1.3
.7
2.5
1.7
2.1
1.4 1.5
1.7
1.9 1.4 2.1 •8
2.3
3.5
1.9
2.0 2.7
1.9
1.6 1.2 1.7
2.4
4.5
.8
.8
5.0
1.9
1.9
5.5
2.4
2.9 1.9
6.0
2.0
2.7
1.3 1.5 1.8 1.2 2.0
1.4 1.6 2.0
2.3
2.7 3.8
6.5
2.1
2.7
1.4
7.5
Day
1.5
1.1
1.9
1
8.0
.8
.8
8.5
.8
1.2
.5 1.3
.7
.5
9.5
T A B L E 3 . B ! residues (ng/g) X ICT1 in eggs from laying hens that consumed feed containing B1 at 8 iig/gfor 7 day consecutive eggs and the time interval is the average of the 2 days on which those
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1
.95
Single egg analysis.
.23
.87 .62
1.1
.56
.99
.86
.93 1.3 .63
1.5
Avg
.23' .27'
.20'
.231
1.0
1 2 3 4 6 7 8 9 10 11 12 14 17 19 20 25 27 30
Hen no.
1.2 1.3 2.1 .5
1.4
.80
2.5
.68 1.2
.68
2.0
1.6
2.1
1.5 1.3
1.9
1.8 1.9 1.8 .49
2.1
3.5
2.0
2.2 2.8
2.0
1.6 1.0 1.9
2.6
4.5
.55
.55
5.0
2.2
2.2
5.5
2.0
1.4 2.5
6.0
.78 1.4
1.2
.79
9.5
1.2
1.5 .99
.99
8.5
1.1
.53
1.91
8.0
2.1 2.5
3.1
1.8
7.5
2.7
1.6 1.9 1.8 1.7 2.0
1.9 1.6 2.3
1.8
2.3 3.3
6.5
Day
1
1
1
TABLE 4. Aflatoxicol (R0) residues (ng/g) X Iff1 in eggs from laying hens that consumed feed containing B, at 8 Ug pool of two consecutive eggs and the time interval is the average of the 2 days on which t
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TRUCKSESS ET AL. days on an aflatoxin-free diet. No Mx was detected in any of the eggs laid during the treatment period or the recovery period. No Ro, Bi, or Mj was found in any eggs from control hens during both periods. The biological half-life retention times (Ty2 in hr) of R 0 and B1 in eggs were 19 and 20, respectively; these values were calculated from the peak at 6.5 days to the end of the experiment at 14 days by a first order equation. The R 0 (.13 to .42 ng/g) and Bj (.17 to .37 ng/g) levels found in the ova of the hens sacrificed on Day 7 are given in Table 5. The levels of R 0 and B^ in these samples were about the same as the levels of R 0 and Bj in eggs collected on Days 6 or 7. No M t was found in any of the ova. No aflatoxins were found in the ova from the control hens or the hens sacrificed on Day 14. Aflatoxin apparently can enter the egg at any stage of its development. It takes 7 to 8 days for each oocyte to develop into a mature ovum (yolk). The ovum then descends into the oviduct, where the egg white is secreted, and is enveloped by the eggshell, a process that takes slightly more than 24 hr. R 0 and B : detection in eggs collected on Day 1 after feed with aflatoxin was offered indicates that B] and Ro were transmitted into the egg through the egg white because the egg yolk was fully developed before Day 1. That ova and eggs show approximately the same levels of Ro and Bi contamination on Day 7 indicates that the toxins were also able to diffuse directly into the egg yolk. Although the distribution of each toxin between egg white and egg yolk to
TABLE 5. Aflatoxicol (R 0) and aflatoxin B1 and Mx residues (ng/g) in tissues from laying hens that consumed feed containing B, at a level of 8 Vg/gfor 7 days to day of sacrifice Ova
Muscle
Blood
B,
Ro
B.
.83 .66 .81 .27 .67 .19 .28 .11 .28
.08 .10 .10 .11 .07 .03 .07 .10 .06
ND .05 .05 .07 .07 .05 .05 .07 .06
Liver
Kidneys
Hen no.
Ro
B,
Ro
B,
M,
Ro
1 2 3 4 6 7 8 9 10
.31 .42 .24 .29 .24 .14 .13 .23 .24
.37 .33 .24 .21 .18 .20 .21 .17 .23
.15 .10 ND .12 .12 .08 .11 .15 .07
ND ND .56 ND .20 .20 .47 .62 .20
.10 .05 .07 .04 .04 .04 .04 ND .04
.32 .25 .22 .08 .30 .12 .17 .09 .26
1 No M, was detected in ova, liver, muscle, or blood; no B, in muscle; no R 0 in blood; see Table 2 for limit of determination.
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seen when the treated birds were sacrificed 7 days after exposure to Bj-free feed. The enlargement of the livers was probably due to infiltration of fat, as noted from their color and texture. The relative ovary weights decreased in the treated hens over the same periods. This decrease adversely affected the size and number of the ova, a phenomenon detrimental to future egg production, a finding in agreement with a previous study evaluating the effect of aflatoxin on egg production (Huff et al., 1975). The approximate limits of determination of aflatoxin residues in the various tissues are given in Table 2. In some cases (kidneys, blood, ova), the limiting factor was the small amount of sample available; in others (liver, muscle, eggs), it was the presence of interferences. Bx and R 0 but no Mj were found in eggs. Analytical results for Bj in eggs are given in Table 3 and for R 0 in Table 4. The transfer of aflatoxin into eggs appears to occur rapidly after initial administration. R 0 (.02 ng/g) and B t (.03 ng/g) were found in eggs laid only one day after the hens had received aflatoxincontaminated feed. In each egg or pooled eggs analyzed, the levels of the two toxins, when detected, were about the same throughout the entire experimental period. The toxin concentration in eggs increased with time and reached a maximum by Day 4 or 5 (Ro ca. .2 ng/g; Bi ca. .2 ng/g) and remained at that level until aflatoxin withdrawal. After the aflatoxincontaminated feed was discontinued on Day 8, aflatoxin residues in the eggs decreased rapidly. Low levels of R 0 (.01 ng/g) and no detectable Bj were found in eggs from treated hens after 7
Blood weight = .1 body weight.
'Percent of B; intake.
2
10
16 11 10 3 11 11 8 8 10
Ro
Liver
Muscle weight = .65 body weight.
46
43
Avg
1
61 79 58 33 56 8 37 33 45
53 72 38 44 46 5 44 32 49
1 2 3 4 6 7 8 9 10
B,
Ro
Hen no.
Eggs
21
38 29 37 12 25 17 13 9 11
B,
2 1 1 1 2 1
2 1
Ro
B,
Kidneys
M,
66
67 87 74 97 42 33 53 96 42
Ro
Muscle1
11 15 8 12 8 8 5 8 7
Ro
Ova
15 12 8 8 6 12 8 6 7
B,
11
10 10 11 14 7 13 9 15 10
Ro
Blo
TABLE 6. Extractable aflatoxins in tissues from laying hens fed Bi for 7 days in their feed. Residues are prese from each hen and the total residues as percent of the total B, consum
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TRUCKSESS ET AL.
Aflatoxin residues in t h e tissues of t h e hens sacrificed 7 days after withdrawal of aflat o x i n - c o n t a m i n a t e d feed were close t o t h e limit of d e t e r m i n a t i o n . Bx (.08 ng/g) was found in o n e of t h e livers; R 0 (.01 t o .04 ng/g) was found in eight of nine muscle samples. No aflatoxins were found in kidneys or b l o o d ; n o R 0 or M.x in livers; and n o Bx o r M ! in muscle. For each hen sacrificed after 7 days o n feed containing aflatoxin, t h e t o t a l residues, t h e total Bi intake, and t h e sum of t h e aflatoxin residues as a percent of t h e Bi intake are listed in Table 6. The major part of t h e total aflatoxin residue was found in t h e eggs and muscle. T h e average for 9 hens of all aflatoxin residues in all tissues as a percentage of t o t a l Bj intake was 31 X 10~ 4 . This study d e m o n s t r a t e s t h a t Bi and its m e t a b o l i t e , R 0 , can be detected in eggs and edible tissues from hens given feed contaminated with Bj at a level of 8 A(g/g. The level of aflatoxin residues in eggs increased steadily for 4 or 5 days t o a plateau and decreased after aflatoxin withdrawal at a b o u t t h e same rate t h a t it increased. At 7 days after withdrawal, only trace a m o u n t s of Ro ( < . 0 1 ng/g) could be found in eggs. All tissues from hens sacrificed before aflatoxin withdrawal contained Bj or a m e t a b o l i t e . Liver and ova contained t h e highest levels of Bj and R 0 . Kidney was the only tissue in which Mj was found. Blood contained only low levels of B j , while muscle contained only
R 0 . Ro could still be d e t e c t e d in t h e muscle 7 days after aflatoxin withdrawal, at which t i m e all o t h e r tissues except one liver sample showed no d e t e c t a b l e aflatoxin residues. If t h e average residue t o intake ratio (219 ng/6.95 mg) observed in this s t u d y were t o hold for lower levels of aflatoxin in feed, t h e n a level 10% of t h a t used in this s t u d y (Bj at a level of .8jUg/g feed) would result in -aflatoxin residues at or below t h e limit of analytical d e t e c t i o n .
REFERENCES Association of Official Analytical Chemists, 1980. Official Methods of Analysis. 13th ed. Ch. 26. Assoc. Offic. Anal. Chera., Arlington, VA. Campbell, T. C , and J. R. Hayes, 1976. The role of aflatoxin metabolism in its toxic lesion. Toxicol. Appl. Pharmacol. 35:199-222. Huff. W. E., R. D. Wyatt, and P. B. Hamilton, 1975. Effects of dietary aflatoxin on certain egg yolk parameters. Poultry Sci. 54:2014-2018. Jacobson, W. C , and H. G. Wiseman, 1974. The transmission of aflatoxin Bj into eggs. Poultry Sci. 53:1743-1745. Rodricks, J. V., and L. Stoloff, 1977. Aflatoxin residues from contaminated feed in edible tissues of food producing animals. Pages 67—79 in Mycotoxins in Human and Animal Health. J. V. Rodricks, C. W. Hesseltine, and M. Mehlman, ed. Pathotox Publ., Park Forest South, IL. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425—428. Trucksess, M. W., 1982. Aflatoxicol in animal tissues, Ph.D. diss., Am. Univ., Washington, DC. Trucksess, M. W., and L. Stoloff, 1981. High performance liquid chromatographic determination of aflatoxicol in milk, blood, and liver. J. Assoc. Offic. Anal. Chem. 64:1083-1087. Trucksess, M. W., and L. Stoloff, 1983. Determination of aflatoxicol and aflatoxins Bj and M, in eggs. J. Assoc. Offic. Anal. Chem. (in press). Trucksess, M. W., L. Stoloff, W. C. Brumley, D. M. Wilson, O. M. Hale, L. T. Sangster, and D. M. Miller, 1982. Aflatoxicol and aflatoxins B1 and M, in the tissues of pigs receiving aflatoxin. J. Assoc. Offic. Anal. Chem. 65:884-887. Trucksess, M. W., L. Stoloff, W. A. Pons, Jr., A. F. Cucullu, L. S. Lee, and A. O. Franz, Jr., 1977. Thin layer chromatographic determination of aflatoxin Bj in eggs. J. Assoc. Offic. Anal. Chem. 60:795-798. West, S., R. D. Wyatt, and P. B. Hamilton, 1973. Improved yield of aflatoxin by incremental increases of temperature. Appl. Microbiol. 25:1018-1019.
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finding t h e same level in b o t h ova and complete eggs would indicate t h a t transmission is relatively constant t h r o u g h o u t t h e process of egg f o r m a t i o n . Analytical results for aflatoxins in t h e tissues of t h e hens sacrificed on Day 7 on t h e aflatoxin-containing feed are presented in Table 5. Aflatoxins were found in all tissues: Ro (-07 to .15 ng/g) and M1 (.04 t o .10 ng/g) in eight of nine kidneys; Bx (.20 t o .62 ng/g) in six kidneys; and R 0 (.08 to .32 ng/g) and B x (.11 to .83 ng/g) in all livers. M j was n o t found in any livers. Ro (.03 to .11 ng/g) was t h e only toxin found in muscle; and Bi (.05 to .07 ng/g) was t h e only toxin f o u n d in b l o o d . No aflatoxin was d e t e c t e d in any tissue from control hens.