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Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens H. Yoshimura
a b
a
, N. Osawa
a c
a
, F. S. C. Rasa , D. a
a
Hermawati , S. Werdiningsih , N. M. R. Isriyanthi & T. Sugimori
a d
a
Balai Pengujian Mutu dan Sertifikasi Obat Hewan (Veterinary Drug Assay Laboratory), Gunung Sindur, Bogor, 16340, Indonesia b
National Veterinary Assay Laboratory, 1–15–1 Tokura, Kokubunji‐shi, Tokyo 185, Japan c
Japan Food Research Laboratories, 52–1 Motoyoyogi‐cho, Shibuya‐ku, Tokyo 151, Japan d
Japan International Cooperation Agency, Shinjuku Mitsui Bldg., 2–1–2 Nishi‐shinjuku, Shinjuku‐ku, Tokyo 163, Japan Published online: 10 Jan 2009.
To cite this article: H. Yoshimura , N. Osawa , F. S. C. Rasa , D. Hermawati , S. Werdiningsih , N. M. R. Isriyanthi & T. Sugimori (1991) Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens, Food Additives & Contaminants, 8:1, 65-69, DOI: 10.1080/02652039109373956 To link to this article: http://dx.doi.org/10.1080/02652039109373956
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views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 1, 6 5 - 6 9
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Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens H. YOSHIMURA†, N. OSAWA‡, F. S. C. RASA, D. HERMAWATI, S. WERDININGSIH, N. M. R. ISRIYANTHI and T. SUGIMORI§ Balai Pengujian Mutu dan Sertifikasi Obat Hewan (Veterinary Drug Assay Laboratory), Gunung Sindur, Bogor 16340 Indonesia (Received 3 April 1990; revised 2 July 1990; accepted 5 July 1990) Doxycycline (DOTC) and oxytetracycline (OTC) were dissolved in drinking water (0.5 g/l) and supplied to laying hens for 7 consecutive days. Eggs laid were collected daily during and after medication, and the antibiotic concentrations in the yolk and albumin were determined by the cup-plate method with Bacillus cereus var. mycoides ATCC 11778. The concentrations of both antibiotics were increased in yolk day by day with the advance in medication, reached peaks 2 days after withdrawal and then declined gradually. Mean peak concentrations in the yolk were 6.70 µg/g for DOTC and 1.42 µg/g for OTC. Peak concentrations in the albumin occurred in the middle stage of medication, where the mean values were 12.24 µg/g for DOTC and 1.03 µg/g for OTC. DOTC was detected in albumin until 24 days after withdrawal and for 2 days more in yolk than in albumin. OTC was detected in yolk until 9 days after withdrawal. The depletion period of OTC was shorter for the albumin, where the residue disappeared in all eggs 6 days after withdrawal. In spite of similarities between DOTC and OTC in structure, DOTC was deposited in higher concentrations and lasted for a longer period in eggs. This characteristic was considered due to its greater lipophilicity, closely correlated with oral absorption and tissue penetration. Keywords: doxycycline, oxytetracycline, residues, eggs.
Introduction
Tetracyclines are effective against various Gram-positive and Gram-negative bacteria, rickettsia and some protozoa. Among them, oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) have been utilized widely to control poultry diseases such as chronic respiratory disease, infectious synovitis, infectious coryza, and fowl typhoid. It has been reported, however, that these tetracyclines are deposited in eggs when given to laying hens via the drinking water or feed. According to Nogawa etal. (1981), TC was deposited in higher concentrations, followed by OTC, after medication via the drinking water. The concentration in the yolk and albumin was 3-fold lower for CTC than for TC (Nogawa etal. 1981). Frieser etal. (1986) and Roudaut et al. (1987, 1989) also found similar observations after medication via the drinking water and feed. All of these tetracyclines seem to persist longer in yolk Present addresses: †National Veterinary Assay Laboratory, 1-15-1 Tokura, Kokubunji-shi, Tokyo 185, Japan--to whom correspondence should be addressed; ‡Japan Food Research Laboratories, 52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo 151, Japan; §Japan International Cooperation Agency, Shinjuku Mitsui Bldg., 2-1-2 Nishi-shinjuku, Shinjuku-ku, Tokyo 163, Japan 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
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H. Yoshimura et al.
than in albumin (Nogawa et al. 1981, Roudaut et al. 1989). Doxycycline (DOTC), classified as a tetracycline, has the abilities to be absorbed well from the intestinal tract and concentrated in tissues to a greater extent (Aronson 1980). Its structure is similar to OTC. Although DOTC was shown to pass into eggs (Archimbault et al. 1983), no data have been presented concerning its concentration in the yolk and albumin of eggs, compared with any of the other tetracyclines. Administration of antimicrobials to poultry via the drinking water is carried out widely in tropical countries, where the amount of drinking water consumed is thought to be considerably larger than in the temperate zone. This paper deals with the concentration accumulated and residue period in eggs of DOTC administered via the drinking water to laying hens in Indonesia. OTC was used for comparison.
Materials and methods
Drugs used DOTC (a specially prescribed product containing 800 mg doxycycline hydrochloride/g, Fujita Pharm. Co., Ltd., Shinagawa-ku, Tokyo) and OTC (Terramycin water-soluble containing 800 mg oxytetracycline hydrochloride/g, Pfizer Ltd., Shinjuku-ku, Tokyo) were used. Test animals Forty laying hens (Indian River) weighing approx. 1 • 5 kg were maintained individually in cages in a room exposed to open air. Antimicrobial-free feed specially prescribed in the present laboratory were supplied ad libitum throughout the experiment, including 2 weeks before medication. Experiments DOTC and OTC were dissolved in the drinking water (0-5 g/1) and supplied continuously for 7 days to 15 hens each. The remaining hens served as controls to obtain antimicrobial-free eggs; they received non-medicated drinking water. Eggs laid were collected daily during and after medication. All eggs were kept at -20°C until assayed. Consumption of the medicated drinking water was recorded daily for each hen. Assay method After thawing the eggs to be assayed, the yolk and albumin were separated, weighed and made 3-fold homogenates with 0-1 M monopotassium phosphate buffer (pH 4-5 ± 0-1) in a glass mortar. The antibiotic concentrations were determined for all eggs by the cup-plate method with a single layer of agar (1 • 1 mm in depth), inoculated with spores of Bacillus cereus var. mycoides ATCC 11778, as described by Nogawa et al. (1981). The agar plate was made with no. 8 medium (pH 5-8 ±0-1) proposed by Kramer et al. (1968), by the addition of 0-135% KH2PO4. The number of spores inoculated was 3 x 103/ml agar. Standard curves were prepared with known amounts of added DOTC and OTC in pooled yolk and albumin of the eggs collected from control hens. The threshold sensitivities in yolk and albumin were 0-15 and 0-04 /*g/g for DOTC, and 0-3 and 0-07 /tg/g for OTC.
Residues of doxycycline and oxytetracycline in eggs
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Results The intake of medicated drinking water was smaller in DOTC-medicated hens than in OTC-medicated hens; the mean daily intake of drinking water was 211 ± 83 ml in the DOTC group, and 327 ± 154 ml in the OTC group. Intakes of DOTC and OTC were 85 ± 35 and 116 ± 53 mg/kg per day, respectively (data not shown). Table 1. Concentrations (fig/g) of doxycycline and oxytetracycline in yolk and albumin during and after medication to laying hens via the drinking water (0-5 g/1 for 7 days). Doxycycline Days
Oxytetracycline
Yolk
Albumin
Yolk
Albumin
0 10 0-45 10-14 0-91 10-22 2-401 1-03 3-33 1 1-57 3-78 1 0 3-63 10-04
0 10 8-6112-07 12-2412-98 11-3612-47 10-83 14-31 11-1010 8-9714-07
0 10 0 10 0 10 0-2410-24 0-6010-49 0-75 10-23 1-19 + 0-61
0 10 0-3210- 15 0-3010- 11 0-37 1 0 - 22 1-03 1 0 - 54 0-83 + 0-24 0-57 1 0 - 36
—
1-1110-20 1-4210-64 1-33 + 0-51 1-18 + 0-33 0-6610-24 0-21 10-28 0-31 +0-35 0-15 10-27 0-20 + 0-40 0-1010-24 0 1 0 + 0 1 0 1
0-67 1 0 - 39 0-71 1 0 - 29 0-4910- 43 0-53 1 0 - 17 0-4410- 19 0-18 1 0 - 21 0 +0 0 10 0 10 0 10 0 +0 0 10 0 10 0 10
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
Medication 1 2 3 4 5 6 7
After withdrawal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
—
6-7012-23 6-3413-62 3-60 + 2-03 3-27 + 0-74 3-3911-78 1-49 12-07 2-07 1 1-82 1-20 + 0-85 0-8010-77 0-85 10-59 0-63 1 0-62 0-4610-20 0-58 10-48 0-5010-37 0-57 + 0-22 0-69 + 0-16 0-38 + 0-36 0-3010-24 0-4610-24 0-25 10-31 0-1410-13 0-5010 0-13 10-23 0-13 + 0-22 0 10 0-0910-18 0 1 0 + 0 1 Values are mean 1 SD. —: No eggs collected. ND: Not determined.
8-60+ 1-68 5-9912-00 5-19 + 3-82 2-021 1-11 6-05 15-88 1-13 + 0-58 1-921 1-53 0-89 + 0-42 1-1410-72 1-75 1 1-52 0-9210-95 1-561 1-05 0-9910-39 1-07 10-48 1-06 + 0-48 1-54 + 0-09 0-55 + 0-51 0-62 + 0-45 0-53 10-46 0-27 10-19 0-13 + 0-08 0-81 1 0 0-13 + 0-22 0-15 10-25 0 10 0 +0 0 10 0 10 0 10
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The concentrations of DOTC and OTC in the yolk and albumin during and after medication are shown in table 1. DOTC was detected in both yolk and albumin the next day after commencing medication, its concentrations increased day by day with the advance in medication. The concentrations of DOTC were much higher in albumin than in yolk, especially in the middle stage of medication, where the mean peak values amounted to 10-80-12-24 jtg/g in albumin vs. 3-78 jtg/g in yolk. The concentration in yolk reached a plateau (6-34-6-7O/tg/g) 1-2 days after withdrawal and then declined gradually. The residue lasted in albumin for 24 days after withdrawal and longer by 2 days in yolk than in albumin. OTC appeared in yolk 3 days after commencing medication. The concentration was increased day by day with the advance in medication and reached a peak (l-42jtg/g on the average) the next day after withdrawal. Compared to yolk, albumin contained OTC in lower concentration; the mean peak value measured the day after withdrawal was 0-71 jtg/g. The antibiotic was detected in yolk for 9 days after withdrawal. The depletion period was shorter for the albumin where the residue disappeared in all eggs 6 days after withdrawal. Discussion The oral absorption and tissue penetration of tetracyclines are largely influenced by their lipophilicity. In human beings, absorption rates of CTC, OTC and TC were 30%, 58% and 77%, respectively, while more lipophilic DOTC was absorbed more completely (90-100%) after oral dosage (Barza et al. 1975). In tissues and body fluids of dogs the concentration of DOTC was several times higher than that of OTC (Barza et al. 1975). In fact, DOTC has been applied to human beings and dogs at the recommended oral dosage approximately one-tenth that of three other tetracyclines (Aronson 1980). The recommended dosages via the drinking water in poultry are 0-05-0-2 g/1 for DOTC and 0-033-0-5 g/1 for OTC. In the present experiment, conducted to compare the concentrations of DOTC and OTC in eggs after the same dosage levels, the mean peak values were 5 times higher in yolk and more than 10 times higher in albumin for DOTC than for OTC. This characteristic of DOTC is considered to be due to its greater lipophilicity, causing more efficient absorption and tissue penetration. Nogawa et al. (1981) compared TC, OTC and CTC with regard to their residues in eggs after medication to laying hens via the drinking water (0-5 g/1) for 7 consecutive days. According to them, the mean peak concentration of OTC in the yolk was 0-77 /*g/g, which occurred 2 days after withdrawal. Roudaut et al. (1987) also noted a similar increase in the concentration of OTC in yolk during medication of 0-5 g/1 for 5 days. The present experiment, however, showed that mean peak concentration of OTC in the yolk, reached 2 days after withdrawal, was approximately double that reported by Nogawa et al. (1981). As with the depletion period, Nogawa et al. (1981) and Roudaut et al. (1987) noted that OTC was detected in the yolk for 4 days after withdrawal, a much shorter period than in the present experiment. The depletion period in the albumin also lasted longer in the present experiment than in others (Nogawa et al. 1981, Roudaut et al. 1987). These differences in the antibiotic concentration and depletion period in eggs may be due to differences in the intake of drinking water between different countries; in the experiment of Nogawa et al. (1981) a hen (approximately 1-7 kg) consumed 100 mg daily via the drinking water corresponding to 59 mg/kg per day, while in the
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Residues of doxycycline and oxytetracycline in eggs
69
present experiment mean OTC intake was 116 mg/kg per day. OTC intake by a hen via the drinking water (0-5 g/1) conducted by Roudaut et al. (1987) was 50 mg/kg per day, which is close to that of Nogawa et al. (1981). The Joint FAO-WHO Expert Committee on Food Additives (1969) established acceptable levels of antibiotic residues in eggs. In some counties the withdrawal periods have been proposed for laying hens after medication of antibiotics (Berger 1971, Milhaut 1978). The results of this experiment, however, reveal that withdrawal periods become unexpectedly longer when antibiotics are given via the drinking water in tropical countries. Acknowledgements
We would like to thank Drh. Syamsul Bahri Siregar MSc, Director of the Veterinary Drug Assay Laboratory, who afforded us every facility for this experiment. The technical assistance of I. Wayan Sutha and Komarudin is also gratefully acknowledged. References ARCHIMBAULT, Ph., AMBROGGI, G., and JOINEAUD, J., 1983, La doxycycline chez la volaille:
biodisponibilité et passage dans les œufs. Revue de Médecine Vétérinaire,134, 291-295. ARONSON, A. L., 1980, Pharmacotherapeutics of the newer tetracyclines. Journal of the American Veterinary Medical Association, 176, 1061-1068. BARZA, M., BROWN, R. B., SHANKS, C., GAMBLE, C., and WEISTEIN, L., 1975, Relation between
lipophilicity and pharmacological behaviour of minicycline, doxycycline, tetracycline, and oxytetracycline in dogs. Antimicrobial Agents and Chemotherapy, 8, 713-720. BERGER, R., 1971, The use of antibiotics in animal production in Finland. Bulletin de l'Office international des Epizooties, 75, 849-850. FRIESER, J., GEDEK, W., and DORN, P., 1986, Zum Nachweis und zur Bedeutung von Tetracyclin-
Rückständen im Ei. Deutsche Tierärztliche Wochenschrift, 93, 17-20. JOINT FAO-WHO EXPERT COMMITTEE ON FOOD ADDITIVES, 1969, Specifications for the identity and
purity of food additives and their toxicological evaluation: some antibiotics. World Health Organization Technical Report Series, 430, 15-44. KRAMER, J., CARTER, G. G., ARRET, B., WILNER, J . , WRIGHT, W. W., and KIRSHBAUM, A., 1968,
Antibiotic Residues in Milk, Dairy Products, and Animal Tissues: methods, reports, and protocols. Food and Drug Administration, Department of Health, Education, and Welfare, Washington, DC, pp. 1-11 MILHAUT, G., 1978, L'utilisation rationnelle des médicaments vétérinaires et le temps d'attente. Recueil de Médecine Vétérinaire,154, 177-185. NOGAWA, H., NAGURA, S., TSUCHIYA, M., and YONEZAWA, S., 1981, Residues of tetracycline anti-
biotics in eggs laid by hens given drinking water medicated. Annual Report of the National Veterinary Assay Laboratory, 18, 25-30. ROUDAUT, B., MORETAIN, J. P., and BOISSEAU, J., 1987, Excretion of oxytetracycline in eggs after
medication of laying hens. Food Additives and Contaminants, 4, 297-307. ROUDAUT, B., MORETAIN, J. P., and BOISSEAU, J., 1989, Excretion of tetracycline and chlortetracycline
in eggs after oral medication of laying hens. Food Additives and Contaminants, 6, 71-78.
Food Additives & Contaminants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac19
Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens H. Yoshimura
a b
a
, N. Osawa
a c
a
, F. S. C. Rasa , D. a
a
Hermawati , S. Werdiningsih , N. M. R. Isriyanthi & T. Sugimori
a d
a
Balai Pengujian Mutu dan Sertifikasi Obat Hewan (Veterinary Drug Assay Laboratory), Gunung Sindur, Bogor, 16340, Indonesia b
National Veterinary Assay Laboratory, 1–15–1 Tokura, Kokubunji‐shi, Tokyo 185, Japan c
Japan Food Research Laboratories, 52–1 Motoyoyogi‐cho, Shibuya‐ku, Tokyo 151, Japan d
Japan International Cooperation Agency, Shinjuku Mitsui Bldg., 2–1–2 Nishi‐shinjuku, Shinjuku‐ku, Tokyo 163, Japan Published online: 10 Jan 2009.
To cite this article: H. Yoshimura , N. Osawa , F. S. C. Rasa , D. Hermawati , S. Werdiningsih , N. M. R. Isriyanthi & T. Sugimori (1991) Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens, Food Additives & Contaminants, 8:1, 65-69, DOI: 10.1080/02652039109373956 To link to this article: http://dx.doi.org/10.1080/02652039109373956
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views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 1, 6 5 - 6 9
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Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens H. YOSHIMURA†, N. OSAWA‡, F. S. C. RASA, D. HERMAWATI, S. WERDININGSIH, N. M. R. ISRIYANTHI and T. SUGIMORI§ Balai Pengujian Mutu dan Sertifikasi Obat Hewan (Veterinary Drug Assay Laboratory), Gunung Sindur, Bogor 16340 Indonesia (Received 3 April 1990; revised 2 July 1990; accepted 5 July 1990) Doxycycline (DOTC) and oxytetracycline (OTC) were dissolved in drinking water (0.5 g/l) and supplied to laying hens for 7 consecutive days. Eggs laid were collected daily during and after medication, and the antibiotic concentrations in the yolk and albumin were determined by the cup-plate method with Bacillus cereus var. mycoides ATCC 11778. The concentrations of both antibiotics were increased in yolk day by day with the advance in medication, reached peaks 2 days after withdrawal and then declined gradually. Mean peak concentrations in the yolk were 6.70 µg/g for DOTC and 1.42 µg/g for OTC. Peak concentrations in the albumin occurred in the middle stage of medication, where the mean values were 12.24 µg/g for DOTC and 1.03 µg/g for OTC. DOTC was detected in albumin until 24 days after withdrawal and for 2 days more in yolk than in albumin. OTC was detected in yolk until 9 days after withdrawal. The depletion period of OTC was shorter for the albumin, where the residue disappeared in all eggs 6 days after withdrawal. In spite of similarities between DOTC and OTC in structure, DOTC was deposited in higher concentrations and lasted for a longer period in eggs. This characteristic was considered due to its greater lipophilicity, closely correlated with oral absorption and tissue penetration. Keywords: doxycycline, oxytetracycline, residues, eggs.
Introduction
Tetracyclines are effective against various Gram-positive and Gram-negative bacteria, rickettsia and some protozoa. Among them, oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) have been utilized widely to control poultry diseases such as chronic respiratory disease, infectious synovitis, infectious coryza, and fowl typhoid. It has been reported, however, that these tetracyclines are deposited in eggs when given to laying hens via the drinking water or feed. According to Nogawa etal. (1981), TC was deposited in higher concentrations, followed by OTC, after medication via the drinking water. The concentration in the yolk and albumin was 3-fold lower for CTC than for TC (Nogawa etal. 1981). Frieser etal. (1986) and Roudaut et al. (1987, 1989) also found similar observations after medication via the drinking water and feed. All of these tetracyclines seem to persist longer in yolk Present addresses: †National Veterinary Assay Laboratory, 1-15-1 Tokura, Kokubunji-shi, Tokyo 185, Japan--to whom correspondence should be addressed; ‡Japan Food Research Laboratories, 52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo 151, Japan; §Japan International Cooperation Agency, Shinjuku Mitsui Bldg., 2-1-2 Nishi-shinjuku, Shinjuku-ku, Tokyo 163, Japan 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
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H. Yoshimura et al.
than in albumin (Nogawa et al. 1981, Roudaut et al. 1989). Doxycycline (DOTC), classified as a tetracycline, has the abilities to be absorbed well from the intestinal tract and concentrated in tissues to a greater extent (Aronson 1980). Its structure is similar to OTC. Although DOTC was shown to pass into eggs (Archimbault et al. 1983), no data have been presented concerning its concentration in the yolk and albumin of eggs, compared with any of the other tetracyclines. Administration of antimicrobials to poultry via the drinking water is carried out widely in tropical countries, where the amount of drinking water consumed is thought to be considerably larger than in the temperate zone. This paper deals with the concentration accumulated and residue period in eggs of DOTC administered via the drinking water to laying hens in Indonesia. OTC was used for comparison.
Materials and methods
Drugs used DOTC (a specially prescribed product containing 800 mg doxycycline hydrochloride/g, Fujita Pharm. Co., Ltd., Shinagawa-ku, Tokyo) and OTC (Terramycin water-soluble containing 800 mg oxytetracycline hydrochloride/g, Pfizer Ltd., Shinjuku-ku, Tokyo) were used. Test animals Forty laying hens (Indian River) weighing approx. 1 • 5 kg were maintained individually in cages in a room exposed to open air. Antimicrobial-free feed specially prescribed in the present laboratory were supplied ad libitum throughout the experiment, including 2 weeks before medication. Experiments DOTC and OTC were dissolved in the drinking water (0-5 g/1) and supplied continuously for 7 days to 15 hens each. The remaining hens served as controls to obtain antimicrobial-free eggs; they received non-medicated drinking water. Eggs laid were collected daily during and after medication. All eggs were kept at -20°C until assayed. Consumption of the medicated drinking water was recorded daily for each hen. Assay method After thawing the eggs to be assayed, the yolk and albumin were separated, weighed and made 3-fold homogenates with 0-1 M monopotassium phosphate buffer (pH 4-5 ± 0-1) in a glass mortar. The antibiotic concentrations were determined for all eggs by the cup-plate method with a single layer of agar (1 • 1 mm in depth), inoculated with spores of Bacillus cereus var. mycoides ATCC 11778, as described by Nogawa et al. (1981). The agar plate was made with no. 8 medium (pH 5-8 ±0-1) proposed by Kramer et al. (1968), by the addition of 0-135% KH2PO4. The number of spores inoculated was 3 x 103/ml agar. Standard curves were prepared with known amounts of added DOTC and OTC in pooled yolk and albumin of the eggs collected from control hens. The threshold sensitivities in yolk and albumin were 0-15 and 0-04 /*g/g for DOTC, and 0-3 and 0-07 /tg/g for OTC.
Residues of doxycycline and oxytetracycline in eggs
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Results The intake of medicated drinking water was smaller in DOTC-medicated hens than in OTC-medicated hens; the mean daily intake of drinking water was 211 ± 83 ml in the DOTC group, and 327 ± 154 ml in the OTC group. Intakes of DOTC and OTC were 85 ± 35 and 116 ± 53 mg/kg per day, respectively (data not shown). Table 1. Concentrations (fig/g) of doxycycline and oxytetracycline in yolk and albumin during and after medication to laying hens via the drinking water (0-5 g/1 for 7 days). Doxycycline Days
Oxytetracycline
Yolk
Albumin
Yolk
Albumin
0 10 0-45 10-14 0-91 10-22 2-401 1-03 3-33 1 1-57 3-78 1 0 3-63 10-04
0 10 8-6112-07 12-2412-98 11-3612-47 10-83 14-31 11-1010 8-9714-07
0 10 0 10 0 10 0-2410-24 0-6010-49 0-75 10-23 1-19 + 0-61
0 10 0-3210- 15 0-3010- 11 0-37 1 0 - 22 1-03 1 0 - 54 0-83 + 0-24 0-57 1 0 - 36
—
1-1110-20 1-4210-64 1-33 + 0-51 1-18 + 0-33 0-6610-24 0-21 10-28 0-31 +0-35 0-15 10-27 0-20 + 0-40 0-1010-24 0 1 0 + 0 1 0 1
0-67 1 0 - 39 0-71 1 0 - 29 0-4910- 43 0-53 1 0 - 17 0-4410- 19 0-18 1 0 - 21 0 +0 0 10 0 10 0 10 0 +0 0 10 0 10 0 10
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
Medication 1 2 3 4 5 6 7
After withdrawal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
—
6-7012-23 6-3413-62 3-60 + 2-03 3-27 + 0-74 3-3911-78 1-49 12-07 2-07 1 1-82 1-20 + 0-85 0-8010-77 0-85 10-59 0-63 1 0-62 0-4610-20 0-58 10-48 0-5010-37 0-57 + 0-22 0-69 + 0-16 0-38 + 0-36 0-3010-24 0-4610-24 0-25 10-31 0-1410-13 0-5010 0-13 10-23 0-13 + 0-22 0 10 0-0910-18 0 1 0 + 0 1 Values are mean 1 SD. —: No eggs collected. ND: Not determined.
8-60+ 1-68 5-9912-00 5-19 + 3-82 2-021 1-11 6-05 15-88 1-13 + 0-58 1-921 1-53 0-89 + 0-42 1-1410-72 1-75 1 1-52 0-9210-95 1-561 1-05 0-9910-39 1-07 10-48 1-06 + 0-48 1-54 + 0-09 0-55 + 0-51 0-62 + 0-45 0-53 10-46 0-27 10-19 0-13 + 0-08 0-81 1 0 0-13 + 0-22 0-15 10-25 0 10 0 +0 0 10 0 10 0 10
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H. Yoshimura et al.
The concentrations of DOTC and OTC in the yolk and albumin during and after medication are shown in table 1. DOTC was detected in both yolk and albumin the next day after commencing medication, its concentrations increased day by day with the advance in medication. The concentrations of DOTC were much higher in albumin than in yolk, especially in the middle stage of medication, where the mean peak values amounted to 10-80-12-24 jtg/g in albumin vs. 3-78 jtg/g in yolk. The concentration in yolk reached a plateau (6-34-6-7O/tg/g) 1-2 days after withdrawal and then declined gradually. The residue lasted in albumin for 24 days after withdrawal and longer by 2 days in yolk than in albumin. OTC appeared in yolk 3 days after commencing medication. The concentration was increased day by day with the advance in medication and reached a peak (l-42jtg/g on the average) the next day after withdrawal. Compared to yolk, albumin contained OTC in lower concentration; the mean peak value measured the day after withdrawal was 0-71 jtg/g. The antibiotic was detected in yolk for 9 days after withdrawal. The depletion period was shorter for the albumin where the residue disappeared in all eggs 6 days after withdrawal. Discussion The oral absorption and tissue penetration of tetracyclines are largely influenced by their lipophilicity. In human beings, absorption rates of CTC, OTC and TC were 30%, 58% and 77%, respectively, while more lipophilic DOTC was absorbed more completely (90-100%) after oral dosage (Barza et al. 1975). In tissues and body fluids of dogs the concentration of DOTC was several times higher than that of OTC (Barza et al. 1975). In fact, DOTC has been applied to human beings and dogs at the recommended oral dosage approximately one-tenth that of three other tetracyclines (Aronson 1980). The recommended dosages via the drinking water in poultry are 0-05-0-2 g/1 for DOTC and 0-033-0-5 g/1 for OTC. In the present experiment, conducted to compare the concentrations of DOTC and OTC in eggs after the same dosage levels, the mean peak values were 5 times higher in yolk and more than 10 times higher in albumin for DOTC than for OTC. This characteristic of DOTC is considered to be due to its greater lipophilicity, causing more efficient absorption and tissue penetration. Nogawa et al. (1981) compared TC, OTC and CTC with regard to their residues in eggs after medication to laying hens via the drinking water (0-5 g/1) for 7 consecutive days. According to them, the mean peak concentration of OTC in the yolk was 0-77 /*g/g, which occurred 2 days after withdrawal. Roudaut et al. (1987) also noted a similar increase in the concentration of OTC in yolk during medication of 0-5 g/1 for 5 days. The present experiment, however, showed that mean peak concentration of OTC in the yolk, reached 2 days after withdrawal, was approximately double that reported by Nogawa et al. (1981). As with the depletion period, Nogawa et al. (1981) and Roudaut et al. (1987) noted that OTC was detected in the yolk for 4 days after withdrawal, a much shorter period than in the present experiment. The depletion period in the albumin also lasted longer in the present experiment than in others (Nogawa et al. 1981, Roudaut et al. 1987). These differences in the antibiotic concentration and depletion period in eggs may be due to differences in the intake of drinking water between different countries; in the experiment of Nogawa et al. (1981) a hen (approximately 1-7 kg) consumed 100 mg daily via the drinking water corresponding to 59 mg/kg per day, while in the
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Residues of doxycycline and oxytetracycline in eggs
69
present experiment mean OTC intake was 116 mg/kg per day. OTC intake by a hen via the drinking water (0-5 g/1) conducted by Roudaut et al. (1987) was 50 mg/kg per day, which is close to that of Nogawa et al. (1981). The Joint FAO-WHO Expert Committee on Food Additives (1969) established acceptable levels of antibiotic residues in eggs. In some counties the withdrawal periods have been proposed for laying hens after medication of antibiotics (Berger 1971, Milhaut 1978). The results of this experiment, however, reveal that withdrawal periods become unexpectedly longer when antibiotics are given via the drinking water in tropical countries. Acknowledgements
We would like to thank Drh. Syamsul Bahri Siregar MSc, Director of the Veterinary Drug Assay Laboratory, who afforded us every facility for this experiment. The technical assistance of I. Wayan Sutha and Komarudin is also gratefully acknowledged. References ARCHIMBAULT, Ph., AMBROGGI, G., and JOINEAUD, J., 1983, La doxycycline chez la volaille:
biodisponibilité et passage dans les œufs. Revue de Médecine Vétérinaire,134, 291-295. ARONSON, A. L., 1980, Pharmacotherapeutics of the newer tetracyclines. Journal of the American Veterinary Medical Association, 176, 1061-1068. BARZA, M., BROWN, R. B., SHANKS, C., GAMBLE, C., and WEISTEIN, L., 1975, Relation between
lipophilicity and pharmacological behaviour of minicycline, doxycycline, tetracycline, and oxytetracycline in dogs. Antimicrobial Agents and Chemotherapy, 8, 713-720. BERGER, R., 1971, The use of antibiotics in animal production in Finland. Bulletin de l'Office international des Epizooties, 75, 849-850. FRIESER, J., GEDEK, W., and DORN, P., 1986, Zum Nachweis und zur Bedeutung von Tetracyclin-
Rückständen im Ei. Deutsche Tierärztliche Wochenschrift, 93, 17-20. JOINT FAO-WHO EXPERT COMMITTEE ON FOOD ADDITIVES, 1969, Specifications for the identity and
purity of food additives and their toxicological evaluation: some antibiotics. World Health Organization Technical Report Series, 430, 15-44. KRAMER, J., CARTER, G. G., ARRET, B., WILNER, J . , WRIGHT, W. W., and KIRSHBAUM, A., 1968,
Antibiotic Residues in Milk, Dairy Products, and Animal Tissues: methods, reports, and protocols. Food and Drug Administration, Department of Health, Education, and Welfare, Washington, DC, pp. 1-11 MILHAUT, G., 1978, L'utilisation rationnelle des médicaments vétérinaires et le temps d'attente. Recueil de Médecine Vétérinaire,154, 177-185. NOGAWA, H., NAGURA, S., TSUCHIYA, M., and YONEZAWA, S., 1981, Residues of tetracycline anti-
biotics in eggs laid by hens given drinking water medicated. Annual Report of the National Veterinary Assay Laboratory, 18, 25-30. ROUDAUT, B., MORETAIN, J. P., and BOISSEAU, J., 1987, Excretion of oxytetracycline in eggs after
medication of laying hens. Food Additives and Contaminants, 4, 297-307. ROUDAUT, B., MORETAIN, J. P., and BOISSEAU, J., 1989, Excretion of tetracycline and chlortetracycline
in eggs after oral medication of laying hens. Food Additives and Contaminants, 6, 71-78.
