Salinomycin Concentration in Eggs and Tissues of Laying Hens

Šinigoj-gačnik K., Zorman Rojs o: Salinomycin Concentration in Eggs and Tissues of Laying Hens. acta Vet. Brno 2008, 77: 423-429. The objective of our study was to monitor the presence of salinomycin in eggs and tissues of laying hens fed with rations containing 60 mg·kg-1 salinomycin sodium for five days. Residues of salinomycin were determined on the day of withdrawal from salinomycin treatment in the breast and thigh muscle, liver, abdominal fat and ovarian yolk. in eggs, residues of salinomycin were monitored in both yolk and albumen daily from the beginning of treatment until the tenth day after withdrawal. Salinomycin was first found in yolk 1 day after starting the treatment and persisted for 8 days after withdrawal. The highest average level, 480 g·kg-1, was present on the third day after withdrawal. in albumen, levels of salinomycin residues were significantly lower, with a maximum level of 15 g·kg-1 reached on the fifth day of the experiment; and they were only found up to one day after withdrawal. Salinomycin was found in all ovarian yolks present in the body of layer hens in a concentration range between 237 and 553 g·kg-1. it was also found in the abdominal fat (concentration range from 62 to 237 g·kg-1) and the liver (concentration ≤ 10 g·kg-1), but not in breast or thigh muscle tissues. No changes in salinomycin residues were observed after cooking and frying the eggs. Salinomycin, poultry tissues, eggs, residues, cooking, residue stability Salinomycin (SaL) is a monovalent polyether antibiotic with ionophoric properties which is used worldwide as a feed additive for the prevention of coccidiosis in poultry broilers, turkeys and replacement chickens. Several reports have been made on the toxic effects of SaL during the past decades. Toxic syndromes may result from its misuse which has occurred when SaL was administered to species for which it was not intended such as horses (Rollinson et al. 1987; nicpon et al. 1997) and turkeys (Stuart 1983; Franz et al. 1992; andreasen and Schleifer 1995; Van assen 2006) or from adverse interactions with a simultaneously administered drug (Miller et al. 1986; Wendt et al. 1997). Salinomycin is not intended for layer hens. nonetheless, residues of SaL in poultry products for human consumption, especially eggs, may occur if laying hens receive a feed contaminated with broiler feed that contains SaL. as reported by Hoop (1998), increased mortality, cannibalism, reduced food intake, decreased egg production and a significant reduction in hatchability was observed when a feed containing 77 mg·kg-1 SAL was accidentally administered to layer breeders. The occurrence of a significant drop in hatchability caused by SaL was reported by jones et al. (1990) because of the foetotoxic effect of SaL (atef et al. 1989). Kan et al. (1990) reported the carry-over of low levels of coccidiostats from layer feed to eggs, and Kennedy et al. (1996) presented the results of monitoring the presence of ionophore antibiotics in eggs from northern ireland. akhtar et al. (1996) studied the concentration of SaL in eggs after feeding the birds with a feed containing between 30 and 150 mg·kg-1 SaL; they reported that in yolk the concentration of SaL three days after withdrawal ranged between 57 and 300 g·kg-1 and depended on the concentration of SaL in the feed. Similarly high concentrations of SaL in yolk were reported by Hoop (1998). Address for correspondence: Dr. K. Šinigoj-gačnik Institute for Food Hygiene and Bromatology Veterinary Faculty, University of Ljubljana gerbičeva 60, 1000 Ljubljana, Slovenia Phone: +386 1 4779 187 Fax: +386 1 4779 174 E-mail: Ksenija.gacnik@vf.uni-lj.si http://www.vfu.cz/acta-vet/actavet.htm ACTA VET. BRNO 2008, 77: 423–429; doi:10.2754/avb200877030423

Salinomycin (SaL) is a monovalent polyether antibiotic with ionophoric properties which is used worldwide as a feed additive for the prevention of coccidiosis in poultrybroilers, turkeys and replacement chickens.Several reports have been made on the toxic effects of SaL during the past decades.Toxic syndromes may result from its misuse which has occurred when SaL was administered to species for which it was not intended such as horses (Rollinson et al. 1987;nicpon et al. 1997) and turkeys (Stuart 1983;Franz et al. 1992;andreasen and Schleifer 1995;Van assen 2006) or from adverse interactions with a simultaneously administered drug (Miller et al. 1986;Wendt et al. 1997).Salinomycin is not intended for layer hens.nonetheless, residues of SaL in poultry products for human consumption, especially eggs, may occur if laying hens receive a feed contaminated with broiler feed that contains SaL. as reported by Hoop (1998), increased mortality, cannibalism, reduced food intake, decreased egg production and a significant reduction in hatchability was observed when a feed containing 77 mg•kg -1 SAL was accidentally administered to layer breeders.The occurrence of a significant drop in hatchability caused by SaL was reported by jones et al . (1990) because of the foetotoxic effect of SaL (atef et al. 1989).Kan et al. (1990) reported the carry-over of low levels of coccidiostats from layer feed to eggs, and Kennedy et al. (1996) presented the results of monitoring the presence of ionophore antibiotics in eggs from northern ireland. akhtar et al. (1996) studied the concentration of SaL in eggs after feeding the birds with a feed containing between 30 and 150 mg•kg -1 SaL; they reported that in yolk the concentration of SaL three days after withdrawal ranged between 57 and 300 g•kg -1 and depended on the concentration of SaL in the feed.Similarly high concentrations of SaL in yolk were reported by Hoop (1998).
The objective of the present study was to trace the changes of SaL concentration in eggs from laying hens that had been receiving a feed containing 60 mg•kg -1 SaL for five days.We determined SaL in yolk and albumen from the first day of SaL administration and followed its disappearance after the withdrawal of the feed containing SaL.We also compared the SaL contents in tissues and ovarian yolks in layers sacrificed after 4 days of SaL administration.Food, especially that of animal origin, is usually cooked before consumption; to better understand the behaviour of SaL residues in eggs and to estimate consumer exposure to the residues, we also studied the effect of heat treatment on SaL present in eggs.

Management of birds and experimental design
56-week-old layers, breeding line Hisex, and 35-week-old layers, breeding line Rahmann, were included in the experiment.Hens were allocated in litter floor pens in a controlled environment with a constant access to food and water.There was one group of 10 and two groups of 5 animals, each in a separate pen.
one group of layers with five animals, breeding line Rahmann (group a), received a medicated feed for 4 days, and one group of the breeding line Hisex with ten animals (group B), received a medicated feed for 5 days.SaL was included in the diet as premix, supplied as a 6% powder Kokcisan®, at the concentration of 60 mg•kg -1 .On the 5 th day three hens from group a were sacrificed and on the 6 th day chickens from group B were switched over to the feed free of SaL. one group of 5 layer chickens of the breeding line Hisex (group C) served as a control and received non-medicated feed throughout the experiment.

Collecting samples and analysis
Eggs from groups B and C were collected every morning from the beginning of the experiment -during treatment and for 10 days after withdrawal -and stored at + 4 °C.on each day the presence of SaL in eggs was determined in five eggs.on the day of analysis, each egg was broken, albumen was separated from yolk and each was analysed separately.
From the three layer chicken from group a sacrificed on day 5 of the experiment, thigh and breast muscle, liver, abdominal fat and ovarian yolks were removed.Tissues from each bird were homogenised, packed in separate bags and stored frozen at below -18 °C until the day of analysis.ovarian yolks were weighed and stored frozen.The control tissues were obtained and prepared in the same way from one randomly selected bird from group C.
For the determination of SaL in egg yolk and albumen we used the method described by Šinigoj-gačnik (2001).Extraction was achieved with acetonitrile, followed by partitioning between saturated salt and carbon tetrachloride, the organic layer was then further purified by solid phase extraction (SPE) on silica columns.Determination was done with high-performance thin-layer chromatography (HPTLC) with chemical detection using p-anisaldehyde as a derivative reagent.The intensity of coloured spots was measured by densitometry at 510 nm.The recoveries with standard deviation (± SD) were 85.1% ± 17.4% and 70.3% ± 10.4% for egg yolk and albumen, with the detection limit being 10 g•kg -1 and 7.5 g•kg -1 , respectively.The calibration curves in the matrix showed good correlation with correlation coefficients 0.9973 and 0.9982 for egg yolk and albumen, respectively.HPTLC with bioautographic detection (Dimenna et al. 1986a) was also performed simultaneously with a detection limit of 7.5 g•kg -1 .
For the determination of SaL in muscle and liver tissue the procedure described by Dimenna et al. (1986a) was chosen with some minor modifications as there was no need for purification of the extracts with carbon tetrachloride.SaL was extracted from the muscle and liver tissue by isooctane and SPE followed on silica column.Extraction of SaL from the fat was carried out with a methanol-water mixture, followed by liquid-liquid purification with n-hexane and carbon tetrachloride (Dimenna et al. 1986b).a final cleaning by SPE followed.Thin-layer chromatography (TLC) by bioautographic identification with the test organism B. subtilis (Dimenna et al. 1986a) was used for identification and quantification in order to achieve low detection limits.For muscle and liver tissues ethyl acetate-water (97 + 3) was used as the mobile-phase, while for the fat, chloroform-methanolammonium hydroxide (95 + 5 + 0.5) was preferred.Quantitative evaluation was done by measuring the size of inhibitory zones of B. subtilis growth.The recoveries of the methods with standard deviation (± SD) were 72.9% ± 8.6%, 49.9% ± 9.2% and 75.3% ± 12.4% for muscle tissue, liver and fat, respectively.The limits of detection were 7.5 g•kg -1 , 10 g•kg -1 and 20 g•kg -1 for the muscle tissue, liver and fat, respectively.The calibration curves in the matrix showed a good correlation using a logarithmical relationship, Y = m ln X + b, where Y was the area of inhibitory zones of B. subtilis growth and X was the concentration of SaL in the matrix.The correlation coefficients were 0.9928, 0.9924 and 0.9923 for the muscle tissue, liver and fat, respectively.all quantitative results were achieved using the method of matrix match calibration curve.
Cooking experiment of eggs Three eggs with incurred SaL were cooked for 5 min.no fat or water was added.Each incurred egg was broken and melange was made.From each egg, half of the prepared melange was analysed in order to determine the SaL content without cooking and the other half (ca 20 g) was exposed to heat treatment when the egg melange was fried.During frying the melange was stirred all the time and the temperature was monitored with a contact thermometer niCr-ni typK, reaching 80 °C in 2 min and remaining between 80 to 90 °C for 3 min.Fried melange was cooled and analyses of the SaL content followed.The same procedure was performed with blank eggs with a standard addition of SAL.

Results and Discussion
The results of the studies on SaL concentration in eggs after administration of a medicated feed containing 60 mg•kg -1 SaL to laying hens are summarized in Table 1 and presented in Fig. 1. in egg yolks SaL was first found on the second day of exposure to SaL in feed.its concentration grew slowly and reached the maximum value (849 g•kg -1 ) on the third day after withdrawal.afterwards, the concentration slowly diminished until some egg yolks became SaL-free on the eighth day after withdrawal.However, some egg yolks still contained minute amounts of SaL (up to 11 g•kg -1 ) until the tenth day after withdrawal. in contrast, the concentrations of SaL in albumen were extremely low (5 -20 g•kg -1 ), and SaL was present only on the days of receiving the feed that contained it and two days after withdrawal.as can be seen from Table 1, the contents of SaL in egg yolks varied greatly between eggs laid by different animals on the same day.For example, in five analysed eggs laid on the 6 th day after withdrawal, the concentrations of SaL in yolk were 13, 51, 80, 152 and 161 g•kg -1 .These findings can be explained by the physiology of egg production.Medicating laying hens leads to accumulation of drugs in egg yolks present in the oviduct during the phase of fast follicular growth and thus high drug concentrations are still to be expected in eggs 7 to 11 days after withdrawal.The drug residues in albumen rapidly diminish since the oviduct only contains water soluble proteins sufficient for the production of two eggs.our results are comparable to the results published by aerts (1990) and akhtar et al. (1996), leading to the conclusion that SaL accumulates in egg yolks progressively, depending on the time of medication and the concentration of SaL in the feed. in our study we also followed the changes of SaL contents in the egg yolk and albumen from the 425 Table 1.Concentrations of salinomycin (in g•kg -1 ) in egg yolk and albumen during the five-day feeding trial of laying hens with medicated feed (60 mg•kg -1 ) and 10 days post withdrawal beginning of medication and observed its disappearance after withdrawal.The maximum average amounts of SaL in egg yolk were found in eggs from the first to the third day after withdrawal of the medicated feed.With the exception of one egg yolk sample from day 3 after withdrawal which contained 849 g•kg -1 SaL, almost the same maximum amounts were found from the fifth day of medication until the fourth day after withdrawal and ranged between 400 and 500 g•kg -1 .The level of SaL in yolk rapidly diminished between the fifth and eighth day after withdrawal, but the rate of disappearance became slower on the ninth and tenth day after withdrawal.The contents of SaL residues in tissues and ovarian yolks of hens fed for four days with medicated feed are presented in Table 2 and Table 3. no residues of SaL were detected in breast and thigh muscle. in the liver, SaL was present at concentrations ≤ 10 g•kg -1 , and the concentrations in the abdominal fat ranged from 62 to 237 g•kg -1 .With the exception of abdominal fat, residue levels of SaL in tissues were similar to those found in 37-day fed broilers (Korsrud et al. 1996;Šinigoj-gačnik et al. 1997).The amounts of SaL in the abdominal fat of hens on the day of withdrawal ranged from 63 to 237 g•kg -1 as shown in Table 2, and in broilers the average level was 95 ± 19 g•kg -1 as reported by Korsrud et al. (1996), and from 31 to 152 g•kg -1 as reported by Šinigoj-gačnik et al. (1997).This difference can be attributed to different fat compositions in hens and broilers.From these results we can conclude that the time of feeding SaL has no influence on the SaL contents in tissues other than eggs.Contents of SaL found in ovarian yolks of one hen confirmed the accumulation of SaL in ovarian yolk (Table 3).The highest quantity was found in the heaviest ovarian yolk.The findings confirm the theory of drug accumulation in the follicles during egg development.assuming that one egg is laid every day, we understand why the highest concentrations of SaL in yolk were found only after withdrawal.if hens do not 426 Fig. 1.Concentration of salinomycin (in μg•kg -1 ) in egg yolk and albumen of eggs laid by hens receiving feed containing salinomycin (60 mg•kg -1 ) for 5 days .lay eggs daily, then prolonged high concentrations of SaL in yolks were observed over a 5-day period.
Results from the cooking experiment are presented in Fig. 2 and show that frying eggs for 5 min had no influence on the content of SaL.
Since SaL possesses cardiovascular properties (Fahim et al. 1986), it could have adverse effects on human health when consumed orally with eggs and other food of animal origin.The exact toxic effects for humans after oral consumption have not yet been clarified, but from a recently published case of accidental human poisoning with SaL in a feed mill (Story and Doube 2004) it is evident that we must not underestimate even the residues of SaL present in eggs, and a careful control of the feed for laying hens and of possible residues of veterinary drugs, especially coccidiostats, in eggs is needed.Fig. 2. Concentration of salinomycin (in μg•kg -1 ) before and after 5 min-heat treatment of eggs with standard addition of salinomycin at a concentration of 100 μg•kg -1 (rec) and in eggs with incurred salinomycin at a concentration of 123 μg•kg -1 (egg 1), 338 μg•kg -1 ) (egg 2) and 113 μg•kg -1 ) (egg 3) .

Table 2 .
Contents of salinomycin (in g•kg -1 ) in tissues of hens receiving feed containing salinomycin (60 mg•kg -1 ) for 4 days and sacrificed on the 5 th day

Table 3 .
Contents of salinomycin in ovarian yolks of laying hen, receiving feed containing salinomycin (60 mg•kg -1 ) for 4 days and sacrificed on the 5 th day