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Tetracycline residues in bones of slaughtered animals
Food Control 11 (2000) 175±180
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Tetracycline residues in bones of slaughtered animals M. K uhne a,*, S. Wegmann a, A. Kobe b, R. Fries b b
a School of Veterinary Medicine, D-30173 Hannover, Germany Faculty of Agriculture, University of Bonn, D-53115 Bonn, Germany
Received 19 July 1999; received in revised form 16 September 1999; accepted 20 September 1999
Abstract The incidence of tetracycline (TC) residues in bones of slaughtered animals (pigs, turkeys, chickens, ducks and calves) was investigated using a screening ¯uorescence test and HPLC analysis. Fluorescence ®ndings vary from 18.8% to 100% depending on species. The concentrations of TCs in the bones were 0.14±50.0 mg kgÿ1 . Additionally, an experimental evaluation of the speci®ty and sensitivity of the ¯uorescence screening test was performed. Chickens were fed low dosages of oxytetracycline (OTC) and subsequently analysed for detectable residues after a withdrawal period of 1, 5, 15 and 25 days. The results are discussed with regard to the possible toxicological signi®cance of TC residues in bones. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Tetracyclines; Bones; Residues
1. Introduction Tetracyclines have been widely used as growth promoters and therapeutics in animal husbandry. Despite early warnings about increasing resistances of microorganisms to tetracyclines and the banning of tetracyclines as growth promoters, actually more than 65% of the antibiotics prescribed for veterinary therapeutic use within the European Community (2294 of 3494 tons) are tetracyclines (FEDESA, 1998). After Milch, Rall and Tobic (1957) having given ®rst indications on the persistence of tetracyclines in bones, Buyske, Eisner and Kelly (1960) described residues of tetracycline and chlortetracycline in bones of treated animals and the speci®c ¯uorescence of bound tetracyclines in bones. Since then, this property of tetracyclines has intensively been used in medical sciences to study either their pharmacocinetics (Blomquist & Hanngren, 1966) or special physiological (Ballanti, Coen, Taggi, Mazzaferro, Perruza & Bonucci, 1995), embryological (van de Velde, Vermeiden & Bloot, 1985), and surgical issues (Aoyagi, Sasaki, Ramamurthy & Golub, 1996).
*
Corresponding author. Fax: +49-511-856-7694. E-mail address: [email protected] KuÈhne).
(M.
In 1993, we started to detect ¯uorescence in bones in order to screen for tetracyclines in slaughtered animals (K uhne & Ebrecht, 1993). The aim was to get information about: · Can ¯uorescence be used as a suitable screening for tetracycline residues in animals? · To what extent do tetracyclines occur in bones of slaughtered animals? It was the aim of this study, to assess the potential risk from mechanical deboning of meat, that contains signi®cant amounts of bone meal and bone splinters (Varnam & Sutherland, 1995), and the use of meat and bone meal in animal feeding.
2. Materials and methods 2.1. Experiments on the detection of residues of oxytetracycline (OCT) in bones after application to chickens Material. Thirty chickens were fed an oxytetracycline (124 mg kgÿ1 ) supplemented standard diet (medical premix bioptivet G-Bä from the 6th to 16th day of fattening. They were humanely killed the 1st, 5th, 15th and 25th day after withdrawal of the medicated feed. Additionally, 5 animals served as a control. Method. The skeleton was thoroughly prepared (separation of fat and periosteum) and examined
0956-7135/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 6 - 7 1 3 5 ( 9 9 ) 0 0 0 9 2 - 4
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M. K uhne et al. / Food Control 11 (2000) 175±180
visually for yellow ¯uorescence using an ultraviolet (UV)-lamp (366 nm, 4 W; Merck Nr. 13203). The intensity of ¯uorescence was de®ned as follows: Positive : Positive : Positive :
extent of ¯uoresceing areas <20% extent of ¯uoresceing areas 20±80% extent of ¯uoresceing areas >80%
Furthermore the oxytetracycline concentration in the femur was assessed using high performance liquid chromatography (HPLC). Extraction, clean-up procedure and HPLC. The whole os femoris was cleaned, sawed to pieces of 1±2 mm and incubated with 10 ml of 1 M hydrochloric acid for 10 h. The sediment was extracted once more after centrifugation. The combined supernatant was ®ltered and immediately loaded on a previously conditioned XAD-2 Amberlite column. The conditioning and elution procedure was published elsewhere (K uhne, Kobe, Ebrecht & Fries, 1992). HPLC was performed on a 250 mm ´ 4 mm cartridge column packed with LiChrospher RP 18 (Merck 1.50833). The mobile phase was 25% acetonitrile and 75% aqua bidest. (with 1% phosphoric acid) and maintained at 0.7 mlÿ1 min. Detection was carried out by a UV-detector at 360 nm (PU 4020, Philips). The con®rmation of positive results was performed with a diode array detector (SPD-M6A, Shimadzu). OTC stock standard solution: OTC dihydrate (Sigma O-5750) 1 mg in 1 ml water, working standards diluted in mobile phase according to demand. Calculation of results. HPLC was performed using 20 ll extract. A calibration curve was constructed of peak area against concentration for a range from 4 to 100 ng. The quantity of the OTC in each sample was then calculated from the measured peak area. This ®gure was corrected for injection volume and mean percentage recovery (calculated from spiked samples). 2.2. Occurrence of TCs in bones of slaughtered animals 2.2.1. Pigs Material. 17 150 fattening pigs, 3275 sows and 345 piglets, slaughtered in commercial meat plants in Northern Germany and having passed the meat inspection. Method. Visual detection of ¯uorescence at the vertebrae and ribs of carcasses in not illuminated chilling rooms using a UV-lamp (for details see Section 2.1). De®nition of intensity was according to Section 2.1. 2.2.2. Poultry 2.2.2.1. Turkeys Material. Random samples from 85 ¯ocks of turkeys (3000±6500 animals), representing a total number of 357 000, were taken during the routine meat inspection
in several poultry meat plants in Northern Germany. These animals represented about 40% of the total number of slaughtered animals during the sampling period. Method. For the visual detection of ¯uorescence the os femoris of the animals was prepared according to the procedure described in Section 2.1. For the detection of tetracyclines and their 4-epimers in turkey bones with HPLC, the procedure described in Section 2.1 was slightly modi®ed. Samples: dierent long bones. Separation was performed on a 125 mm ´ 4 mm cartridge column (Merck 1.50943-0001), detection at 360 and 370 nm with a UV detector (SPD 10 A, Shimadzu) and photodiode array detector (SPD-M6A, Shimadzu), mobile phase was 60% 0.01 M oxalic acid and 40% acetonitrile and pumped at 0.4 ml minÿ1 at ambient temperature. Tetracycline stock standard solutions: OTC dihydrate (Sigma O-5750), tetracycline (TC) (Sigma T-3258), chlortetracycline (CTC) hydrochloride (Sigma C-4881), 4-epi-tetracycline (Acros Chimica 23312-1000), 4-epichlortetracycline (Acros Chimica 26823-1000), each 1 mg in 1 ml water, working standards diluted in mobile phase according to demand. Calculation of results was done according to Section 2.1. 2.2.2.2. Chickens Material. A total of 152 samples of frozen chickens, representing 76 dierent lots, were collected in food shops in various cities in Northern Germany. 30 samples displayed the producers' label with very detail production speci®cations. This particular producer guarantees to restrain from use of TCs and from use of meat and bone meal. All samples positive with the visual detection were analysed with HPLC. Method. Analysis as described in Section 2.2.2.1. 2.2.2.3. Ducks Material. A total of 86 samples of frozen ducks of European origin, representing 51 dierent lots, were collected in food shops in dierent cities in Northern Germany. All samples identi®ed as ¯uorescence positive during the visual detection, were chosen for HPLC analysis of TC concentrations. Method. Analysis as described in Section 2.2.2.1. 2.2.3. Calves Material. 1560 veal calves, slaughtered in commercial meat plants in Southern Germany and having passed the meat inspection. Samples: rib bones of 45 animals, identi®ed as ¯uorescence positive during the visual detection, were chosen for HPLC analysis of TC concentrations. Method. Analysis as described in Section 2.2.2.1.
M. K uhne et al. / Food Control 11 (2000) 175±180
177
Fig. 1. Decrease of ¯uorescening areas on bones of chicken after withdrawal of OTC supplemented feed. (a) 1 day after withdrawal. (b) 5 days after withdrawl. (c) 15 days after withdrawl.
3. Results 3.1. OTC in bones of chickens after withdrawal After application of OTC, signi®cant concentrations were found in the bones. UV radiation of bones led to yellow ¯uorescence of the bones. One day after withdrawal of the medicated feed a complete and regular ¯uorescence of the bones could be observed (Fig. 1a). On the 5th day after withdrawal the ¯uoresceing areas had decreased considerably (example: Fig. 1b). Individual variations within the group were observed. Samples collected 15 days after withdrawal showed a further reduction of ¯uorescence. Only small spots, especially on the tubular bones, could be found (Fig. 1c). 25 days after withdrawal, there was no more ¯uorescence detectable. Table 1 shows the results of the HPLC determination of OTC concentrations in the femur. The total amount of OTC in the femur decreased from 26.1 (in 1.3 g) to 1.1 lg (in 3.9 g). Samples collected 25 days after withdrawal of the medicated feed contained no detectable concentrations of OTC. Sensitivity and selectivity of the method. The lowest OTC concentration giving a positive result with the ¯uorescence screening test was 0.05 mg kgÿ1 . In all samples with positive ¯uorescence OTC could be identi®ed using HPLC.
Table 1 OTC concentrations in the femur and ¯uorescence on the surface after application of a feed medicated with 124 mg kgÿ1 of OTC to chickensa Day after withdrawal
Concentration (lg OTC kgÿ1 ) mean/S.D.
Fluorescence on the surface
Total weight of the femur (g) mean/ S.D.
1 n 10 5 n 3 15 n 3 25 n 3
20.1/4.3 5.4/2.2 0.28/0.3 n.d.
Negative
1.3/0.2 1.8/0.2 3.9/0.4 6.8/0.7
a n: number of samples; S.D.: standard deviation; g: gram; n.d.: not detectable; de®nition of positive results according to Section 2.
3.2. TC residues in pig bones There were signi®cant dierences between fattening pigs, sows and piglets (Table 2). Highest incidence was found in piglets. 3.3. TC residues in turkey bones All samples (85 of 85) showed ¯uorescence on the surface of the os femoris (Table 2). During preparation of samples for HPLC, some samples with only slight positive ¯uorescence on the surface (positive ) showed
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Table 2 TC ®ndings in bones of slaughtered animals (¯uorescence on bone surfaces)a Species
(n)
Negative (%)
Positive (%)
Positive (%)
Positive (%)
Pigs Fattening pigs Sows Piglets Turkeys Chickens Ducks Veal calves
(17 150) (3275) (345) (85) (152) (86) (1560)
30 89.6 27.2 ± 65.1 4.7 81.2
29.5 6.1 12.8 48.8 1.3 ± 4.4
25.1 3.4 16.5 24.4 3.3 26.7 3.9
15.4 0.9 43.5 26.8 30.3 68.6 10.5
a
n: number of animals/samples; de®nition of positive results according to Section 2.
intensive ¯uorescence in deeper layers of the bone tissue, forming ¯uoresceing rings under UV light. Colour pictures of these ®ndings have been published elsewhere (K uhne, 1998). HPLC. 83 of 85 samples contained detectable TC residues in the bones. 75 samples contained TC in concentrations of 0.14±18.27 mg kgÿ1 (sum of TC and its 4epimer), 4 contained OTC in concentrations of 0.36± 32.15 mg kgÿ1 , 3 had CTC residues of 0.5±4.67 mg kgÿ1 (CTC and its 4-epimer), and one had doxycycline in an amount of 11.29 mg kgÿ1 . Compared with the intensity of ¯uorescence on bone surfaces (Fig. 2), the concentrations of TCs increased with intensity of ¯uorescence. Some statistical parameters of the grouped results are given in Table 3. 3.4. TC residues in chicken bones 34.9% of the samples (53 of 152) showed ¯uorescence on the surface of the os femoris (Table 2). No sample with the label described in Section 2.2.2.2 showed ¯uorescence.
Table 3 TC concentrations in turkey bones (mg OTC/TC/CTC kgÿ1 bone)a
Range Mean S.D.
Positive n 39
Positive n 20
Positive n 23
0.19±6.5 1.72 1.16
1.6±8.9 4.66 2.13
2.01±32.15 10.49 7.0
a
n: number of samples; range: minimum and maximum concentration; S.D.: standard deviation; de®nition of positive results according to Section 2.
HPLC. 51 of 53 samples contained detectable TC residues in the bones. Two samples contained TC in concentrations between 0.9 and 15.1 mg kgÿ1 (sum of TC and its 4-epimer), the remaining 49 samples contained OTC in concentrations of 0.15±42.3 mg kgÿ1 . As the number of samples with slight positive ¯uorescence was limited, the correlation between intensity of ¯uorescence and TC concentrations was not examined. 3.5. TC residues in duck bones 95.3% of the samples (82 of 86) showed ¯uorescence on the surface of the os femoris (Table 2). HPLC. 82 of 82 samples contained detectable tetracycline residues in the bones. 1 sample contained 70 mg kg ÿ1 doxycycline, 40 samples contained either OTC or TC (and its 4-epimer) in concentrations of 1.2±36.2 mg kgÿ1 , the remaining 41 samples contained chlortetracycline in concentrations of 1.6±45 mg kgÿ1 . As the number of samples with slight positive ¯uorescence was limited, the correlation between intensity of ¯uorescence and TC concentrations was not examined. 3.6. TC residues in bones of veal calves
Fig. 2. TC residues in turkey bones (intensity of ¯uorescence on bone surfaces vs. concentrations).
18.8% (293 of 1560) showed ¯uorescence on the surface of the os femoris (Table 2). As already noticed (turkey bones), some samples with only slight positive ¯uorescence on the surface (positive
M. K uhne et al. / Food Control 11 (2000) 175±180
) showed intensive ¯uorescence in deeper layers of the bone tissue, forming ¯uoresceing rings under UV light. HPLC. 45 samples of ¯uoresceing rib bones were analysed for TC residues (15 samples positive , 15 samples positive , 15 samples positive ). In all samples residues of TCs were detected. There was no predominant TC: Either TC with its 4epimer, CTC with its 4-epimer and OTC was found. 10 samples contained more than one TC. The concentrations are given in Fig. 3. There was not found a correlation between intensity of ¯uorescence and TC concentrations.
4. Discussion 1. Screening for TCs in bones using the ¯uorescence test. The therapeutical dosage of OTC when administered orally to chickens is 1000±1500 mg OTC kgÿ1 feed. To study the sensitivity of the visual detection we used the smallest recommended subtherapeutical dosage. This dosage (124 mg kgÿ1 ) gave consistently positive results. As studied earlier by Br uggemann, L osch, Merkenschlager and Oterdinger (1966), even smaller dosages (10 mg kgÿ1 ) would lead to detectable residues in the bones, when they were used for longer periods. The visual detection using the UV-lamp gave positive results at concentrations of 50 lg OTC kgÿ1 bone tissue. Nevertheless, as found during the analysis of turkey bones, ¯uoresceing areas in deeper layers of the bone resulting from earlier applications could hardly be seen, if only bone surfaces are examined. 2. TC residues in slaughtered animals. The results of random samples from dierent species of animals showed a high incidence of TC residues (TC, CTC, OTC and doxycycline) in the bones of slaughtered animals.
Fig. 3. TC residues in bones of veal calves (intensity of ¯uorescence on bone surfaces vs. concentrations).
179
Same samples contained even more than one TC. The concentrations ranged from 0.14 to 50.0 mg kgÿ1 . The intensity of ¯uorescence in bones of dierent species had a large variety. A number of samples showed intensive ¯uorescence on the surface. Following the conclusions of Buyske et al. (1960) and our own results with OTC and chicken, intensive ¯uorescence indicates, that the last administration of TCs to the animals was only days (or a few weeks) before slaughtering. Fluorescence in deeper layers of the bone, as found in turkey and calf bones, might be a reference to use in earlier stages of rearing. The ®ndings in bones of slaughtered pigs were inhomogenous and dependent on the age of the animals with highest incidence in piglets and lowest in sows. The highest incidence of all species was found in turkey bones. Predominantly, we found residues of TC with its main metabolite 4-epi-tetracycline. Rearing of turkeys is usually performed in large ¯ocks with a high infectious pressure. During the rearing period of 18±24 weeks, respiratory diseases may occur in almost every farm und might be a trigger for antibiotic treatments of the ¯ocks. The dierent residue levels found in this study might depend on the time after withdrawal of the medicated feed or water and the individual dosage. In chickens and ducks also a high rate of contamination with TCs was found. The percentage of slight positive results with the ¯uorescence test was small compared with turkey samples. Chickens are only kept for 4±5 weeks, so in most cases the period between administration of TCs and slaughtering will be shorter than in turkey ¯ocks. In our study with calves the rate of contamination was medium. In 22% of the samples multiple TC residues were found. An important question arising from this results is the correlation with residues in edible tissues. K uhne (1998) examined TC concentrations of muscle, kidney and liver of turkey carcasses with ¯uorescence in bones and found increasing percentage of positive results in kidney and liver with increasing ¯uorescence on the surfaces of bones. However, in all samples the concentrations of TCs were below the maximum residue levels set by the European Community (Commission of the European Community, 1999). 3. Toxicological signi®cance of TC residues in bones. A toxicological signi®cance of TC residues emerges from the possible contamination of mechanically deboned meat and the contamination of meat and bone meal. Mechanically deboned meat. In 1996, 350 000 tons of poultry separator meat and 250 000 tons of pig separator meat has been produced within the European Community (BgVV, 1998), containing bone meal and bone splinters up to 2.5% (Anhalt, 1980) or even 5% (Varnam and Sutherland, 1995). Assuming the concentrations of TCs in our study as possible contamination
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M. K uhne et al. / Food Control 11 (2000) 175±180
source for mechanically deboned meat (i.e. 50 mg kgÿ1 and 2.5% bone splinters in meat), concentrations of up to 1250 lg kgÿ1 may be possible. The temperatures used for the preparation of poultry meat products like poultry surimi, pie ®llings and sausages, are not sucient to destroy tetracycline residues (Rose, Bygrave, Farrington & Shearer, 1996). Meat and bone meal. Meat and bone meal is a costeective and often used protein source in poultry and pig fattening. According to European legislation meat and bone meal is manufactured in special factories, that must use eective methods for the inactivation of hazardous microorganisms and agents. In rendering plants the applied heat is a minimum of 133°C (Celsius) at 3 bar for 20 min. Particle size prior to processing of meat und bone meal must be not more than 5 cm (Commission of the European Community, 1997). The technique provides a suitable inactivation of hazards of microbiological origin. Nevertheless, Honikel, Schmidt, Woltersdorf and Leistner (1978) stated, that bones must be at least heated to 140°C to get a considerable decrease of TC residues in bones. A calculation of presumptive concentrations of TC in raw material for the production of meat and bone meal will in most cases exceed the ppm level (i.e. 50 mg kgÿ1 , average percentage of bones in a steer destined for slaughter: 10% (Gracey & Collins, 1992).
5. Conclusions The occurence of TC residues in bones of slaughtered animals is more likely a normal than an exceptional ®nding. This fact has severe consequences for the potential toxicological risk of products that contain bone portions like mechanically deboned meat and meat and bone meal. Screening methods, like the detection of ¯uorescence, are able to detect suspicious carcasses in meat plants. References Anhalt, G. (1980). Maschinelle Fleisch-Knochen-Separation, Technologie und Anforderungen. Arch. Lebensmittelhyg., 31, 12±16. Aoyagi, M., Sasaki, T., Ramamurthy, N. S., & Golub, L. M. (1996). Tetracycline/Flurbiprofen combination therapy modulates bone remodeling in ovariectomized rats: preliminary observations. Bone, 19, 629±635.
Ballanti, P., Coen, G., Taggi, F., Mazzaferro, S., Perruza, I., & Bonucci, E. (1995). Extent of alkaline phosphatase cytochemistry vs. extent of tetracycline ¯uorescence in the evaluation of histodynamic variables of bone formation. Bone, 16, 493±498. Blomquist, L., & Hanngren, A. (1966). Fluorescence technique applied to whole body sections for distribution studies of tetracyclines. Biochem. Pharmacol., 15, 215±219. Br uggemann, J., L osch, U., Merkenschlager, M., & Oterdinger, I. (1966). Ablagerung von Tetracyclin im Knochengewebe von Tieren bei dem Zusatz von Tetracyclin zum Futter. Zbl. Vet. A., 13, 59±74. BgVV. (1998). T atigkeitsbericht 1997. Arbeitsergebnisse 1.2.24 (p. 141). M unchen: M unchner Medizin. Buyske, D. A., Eisner, H. J., & Kelly, R. G. (1960). Concentration and persistence of tetracycline and chlortetracycline in bone. J. Pharmacol. Exp. Therap., 130, 150±156. Commission of the European Community. (1997). Commission decision of 25th March 1997 laying down the animal health requirements and the veterinary certi®cation for the import of processed animal protein for certain third countries which use alternative heat treatment systems and amending decision 94/344/ EC. O. J., L 84, pp. 36±43. Commission of the European Community. (1999). Commission regulation (EC) No 508/1999 of 4th March 1999 amending annexes I to IV to council regulation (EEC) No 2377/90 laying down a community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstus of animal origin. O. J., L 60, pp. 16±52. European Federation of Animal Health. (1998). Press release, 6th Sept. 1998, to be found on the internet: http://www.fedesa.be. Gracey, J. F., & Collins, D. S. (1992). Meat hygiene (p. 111). London: Bailliere Tindall. Honikel, K. A., Schmidt, U., Woltersdorf, W., & Leistner, L. (1978). Eect of storage and processing on tetracycline residues in meat and bones. J. Assoc. O. Anal. Chem., 61, 1222±1227. K uhne, M., Kobe, A., Ebrecht, A., & Fries, R. (1992). R uckst ande von Oxytetracyclin und Furazolidon bei Jungmasth uhnern nach Verabreichung subtherapeutischer Dosen. Lebensm. ± Wiss. u. Technol., 25, 484±486. K uhne, M., & Ebrecht, A. (1993). The detection of ¯uorescence in bones-a suitable screening for tetracyclines. In N. Haagsma, A. Ruiter, & P.B. Czedik-Eysenberg, Proceedings of EuroResidue II, Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, 3±5 May 1993 (pp. 429±432). Utrecht: University of Utrecht. K uhne, M. (1998). Untersuchungen zum Vorkommen von TetracyclinR uckst anden bei Puten. Fleischwirtsch., 78 (4), 369±370. Milch, R. A., Rall, D. P., & Tobic, J. E. (1957). Bone localisation of the tetracyclines. J. Nat. Can. Inst., 19, 87±91. Rose, M. D., Bygrave, J., Farrington, W. H. H., & Shearer, G. (1996). The eect of cooking on veterinary drug residues in food: 4. Oxytetracycline. Food Addit. Contam., 13, 275±286. Van de Velde, J. P., Vermeiden, J. P. W., & Bloot, A. M. (1985). Medullary bone matrix formation, mineralization, and remodeling related to the daily egg-laying cycle of Japanese quail: a histological and radiological study. Bone, 6, 321±327. Varnam, A. H., Sutherland, J. P. (1995). Meat and meat products ± technology, chemistry and microbiology (pp. 125, 159, 245). London: Chapman & Hall.
www.elsevier.com/locate/foodcont
Tetracycline residues in bones of slaughtered animals M. K uhne a,*, S. Wegmann a, A. Kobe b, R. Fries b b
a School of Veterinary Medicine, D-30173 Hannover, Germany Faculty of Agriculture, University of Bonn, D-53115 Bonn, Germany
Received 19 July 1999; received in revised form 16 September 1999; accepted 20 September 1999
Abstract The incidence of tetracycline (TC) residues in bones of slaughtered animals (pigs, turkeys, chickens, ducks and calves) was investigated using a screening ¯uorescence test and HPLC analysis. Fluorescence ®ndings vary from 18.8% to 100% depending on species. The concentrations of TCs in the bones were 0.14±50.0 mg kgÿ1 . Additionally, an experimental evaluation of the speci®ty and sensitivity of the ¯uorescence screening test was performed. Chickens were fed low dosages of oxytetracycline (OTC) and subsequently analysed for detectable residues after a withdrawal period of 1, 5, 15 and 25 days. The results are discussed with regard to the possible toxicological signi®cance of TC residues in bones. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Tetracyclines; Bones; Residues
1. Introduction Tetracyclines have been widely used as growth promoters and therapeutics in animal husbandry. Despite early warnings about increasing resistances of microorganisms to tetracyclines and the banning of tetracyclines as growth promoters, actually more than 65% of the antibiotics prescribed for veterinary therapeutic use within the European Community (2294 of 3494 tons) are tetracyclines (FEDESA, 1998). After Milch, Rall and Tobic (1957) having given ®rst indications on the persistence of tetracyclines in bones, Buyske, Eisner and Kelly (1960) described residues of tetracycline and chlortetracycline in bones of treated animals and the speci®c ¯uorescence of bound tetracyclines in bones. Since then, this property of tetracyclines has intensively been used in medical sciences to study either their pharmacocinetics (Blomquist & Hanngren, 1966) or special physiological (Ballanti, Coen, Taggi, Mazzaferro, Perruza & Bonucci, 1995), embryological (van de Velde, Vermeiden & Bloot, 1985), and surgical issues (Aoyagi, Sasaki, Ramamurthy & Golub, 1996).
*
Corresponding author. Fax: +49-511-856-7694. E-mail address: [email protected] KuÈhne).
(M.
In 1993, we started to detect ¯uorescence in bones in order to screen for tetracyclines in slaughtered animals (K uhne & Ebrecht, 1993). The aim was to get information about: · Can ¯uorescence be used as a suitable screening for tetracycline residues in animals? · To what extent do tetracyclines occur in bones of slaughtered animals? It was the aim of this study, to assess the potential risk from mechanical deboning of meat, that contains signi®cant amounts of bone meal and bone splinters (Varnam & Sutherland, 1995), and the use of meat and bone meal in animal feeding.
2. Materials and methods 2.1. Experiments on the detection of residues of oxytetracycline (OCT) in bones after application to chickens Material. Thirty chickens were fed an oxytetracycline (124 mg kgÿ1 ) supplemented standard diet (medical premix bioptivet G-Bä from the 6th to 16th day of fattening. They were humanely killed the 1st, 5th, 15th and 25th day after withdrawal of the medicated feed. Additionally, 5 animals served as a control. Method. The skeleton was thoroughly prepared (separation of fat and periosteum) and examined
0956-7135/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 6 - 7 1 3 5 ( 9 9 ) 0 0 0 9 2 - 4
176
M. K uhne et al. / Food Control 11 (2000) 175±180
visually for yellow ¯uorescence using an ultraviolet (UV)-lamp (366 nm, 4 W; Merck Nr. 13203). The intensity of ¯uorescence was de®ned as follows: Positive : Positive : Positive :
extent of ¯uoresceing areas <20% extent of ¯uoresceing areas 20±80% extent of ¯uoresceing areas >80%
Furthermore the oxytetracycline concentration in the femur was assessed using high performance liquid chromatography (HPLC). Extraction, clean-up procedure and HPLC. The whole os femoris was cleaned, sawed to pieces of 1±2 mm and incubated with 10 ml of 1 M hydrochloric acid for 10 h. The sediment was extracted once more after centrifugation. The combined supernatant was ®ltered and immediately loaded on a previously conditioned XAD-2 Amberlite column. The conditioning and elution procedure was published elsewhere (K uhne, Kobe, Ebrecht & Fries, 1992). HPLC was performed on a 250 mm ´ 4 mm cartridge column packed with LiChrospher RP 18 (Merck 1.50833). The mobile phase was 25% acetonitrile and 75% aqua bidest. (with 1% phosphoric acid) and maintained at 0.7 mlÿ1 min. Detection was carried out by a UV-detector at 360 nm (PU 4020, Philips). The con®rmation of positive results was performed with a diode array detector (SPD-M6A, Shimadzu). OTC stock standard solution: OTC dihydrate (Sigma O-5750) 1 mg in 1 ml water, working standards diluted in mobile phase according to demand. Calculation of results. HPLC was performed using 20 ll extract. A calibration curve was constructed of peak area against concentration for a range from 4 to 100 ng. The quantity of the OTC in each sample was then calculated from the measured peak area. This ®gure was corrected for injection volume and mean percentage recovery (calculated from spiked samples). 2.2. Occurrence of TCs in bones of slaughtered animals 2.2.1. Pigs Material. 17 150 fattening pigs, 3275 sows and 345 piglets, slaughtered in commercial meat plants in Northern Germany and having passed the meat inspection. Method. Visual detection of ¯uorescence at the vertebrae and ribs of carcasses in not illuminated chilling rooms using a UV-lamp (for details see Section 2.1). De®nition of intensity was according to Section 2.1. 2.2.2. Poultry 2.2.2.1. Turkeys Material. Random samples from 85 ¯ocks of turkeys (3000±6500 animals), representing a total number of 357 000, were taken during the routine meat inspection
in several poultry meat plants in Northern Germany. These animals represented about 40% of the total number of slaughtered animals during the sampling period. Method. For the visual detection of ¯uorescence the os femoris of the animals was prepared according to the procedure described in Section 2.1. For the detection of tetracyclines and their 4-epimers in turkey bones with HPLC, the procedure described in Section 2.1 was slightly modi®ed. Samples: dierent long bones. Separation was performed on a 125 mm ´ 4 mm cartridge column (Merck 1.50943-0001), detection at 360 and 370 nm with a UV detector (SPD 10 A, Shimadzu) and photodiode array detector (SPD-M6A, Shimadzu), mobile phase was 60% 0.01 M oxalic acid and 40% acetonitrile and pumped at 0.4 ml minÿ1 at ambient temperature. Tetracycline stock standard solutions: OTC dihydrate (Sigma O-5750), tetracycline (TC) (Sigma T-3258), chlortetracycline (CTC) hydrochloride (Sigma C-4881), 4-epi-tetracycline (Acros Chimica 23312-1000), 4-epichlortetracycline (Acros Chimica 26823-1000), each 1 mg in 1 ml water, working standards diluted in mobile phase according to demand. Calculation of results was done according to Section 2.1. 2.2.2.2. Chickens Material. A total of 152 samples of frozen chickens, representing 76 dierent lots, were collected in food shops in various cities in Northern Germany. 30 samples displayed the producers' label with very detail production speci®cations. This particular producer guarantees to restrain from use of TCs and from use of meat and bone meal. All samples positive with the visual detection were analysed with HPLC. Method. Analysis as described in Section 2.2.2.1. 2.2.2.3. Ducks Material. A total of 86 samples of frozen ducks of European origin, representing 51 dierent lots, were collected in food shops in dierent cities in Northern Germany. All samples identi®ed as ¯uorescence positive during the visual detection, were chosen for HPLC analysis of TC concentrations. Method. Analysis as described in Section 2.2.2.1. 2.2.3. Calves Material. 1560 veal calves, slaughtered in commercial meat plants in Southern Germany and having passed the meat inspection. Samples: rib bones of 45 animals, identi®ed as ¯uorescence positive during the visual detection, were chosen for HPLC analysis of TC concentrations. Method. Analysis as described in Section 2.2.2.1.
M. K uhne et al. / Food Control 11 (2000) 175±180
177
Fig. 1. Decrease of ¯uorescening areas on bones of chicken after withdrawal of OTC supplemented feed. (a) 1 day after withdrawal. (b) 5 days after withdrawl. (c) 15 days after withdrawl.
3. Results 3.1. OTC in bones of chickens after withdrawal After application of OTC, signi®cant concentrations were found in the bones. UV radiation of bones led to yellow ¯uorescence of the bones. One day after withdrawal of the medicated feed a complete and regular ¯uorescence of the bones could be observed (Fig. 1a). On the 5th day after withdrawal the ¯uoresceing areas had decreased considerably (example: Fig. 1b). Individual variations within the group were observed. Samples collected 15 days after withdrawal showed a further reduction of ¯uorescence. Only small spots, especially on the tubular bones, could be found (Fig. 1c). 25 days after withdrawal, there was no more ¯uorescence detectable. Table 1 shows the results of the HPLC determination of OTC concentrations in the femur. The total amount of OTC in the femur decreased from 26.1 (in 1.3 g) to 1.1 lg (in 3.9 g). Samples collected 25 days after withdrawal of the medicated feed contained no detectable concentrations of OTC. Sensitivity and selectivity of the method. The lowest OTC concentration giving a positive result with the ¯uorescence screening test was 0.05 mg kgÿ1 . In all samples with positive ¯uorescence OTC could be identi®ed using HPLC.
Table 1 OTC concentrations in the femur and ¯uorescence on the surface after application of a feed medicated with 124 mg kgÿ1 of OTC to chickensa Day after withdrawal
Concentration (lg OTC kgÿ1 ) mean/S.D.
Fluorescence on the surface
Total weight of the femur (g) mean/ S.D.
1 n 10 5 n 3 15 n 3 25 n 3
20.1/4.3 5.4/2.2 0.28/0.3 n.d.
Negative
1.3/0.2 1.8/0.2 3.9/0.4 6.8/0.7
a n: number of samples; S.D.: standard deviation; g: gram; n.d.: not detectable; de®nition of positive results according to Section 2.
3.2. TC residues in pig bones There were signi®cant dierences between fattening pigs, sows and piglets (Table 2). Highest incidence was found in piglets. 3.3. TC residues in turkey bones All samples (85 of 85) showed ¯uorescence on the surface of the os femoris (Table 2). During preparation of samples for HPLC, some samples with only slight positive ¯uorescence on the surface (positive ) showed
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Table 2 TC ®ndings in bones of slaughtered animals (¯uorescence on bone surfaces)a Species
(n)
Negative (%)
Positive (%)
Positive (%)
Positive (%)
Pigs Fattening pigs Sows Piglets Turkeys Chickens Ducks Veal calves
(17 150) (3275) (345) (85) (152) (86) (1560)
30 89.6 27.2 ± 65.1 4.7 81.2
29.5 6.1 12.8 48.8 1.3 ± 4.4
25.1 3.4 16.5 24.4 3.3 26.7 3.9
15.4 0.9 43.5 26.8 30.3 68.6 10.5
a
n: number of animals/samples; de®nition of positive results according to Section 2.
intensive ¯uorescence in deeper layers of the bone tissue, forming ¯uoresceing rings under UV light. Colour pictures of these ®ndings have been published elsewhere (K uhne, 1998). HPLC. 83 of 85 samples contained detectable TC residues in the bones. 75 samples contained TC in concentrations of 0.14±18.27 mg kgÿ1 (sum of TC and its 4epimer), 4 contained OTC in concentrations of 0.36± 32.15 mg kgÿ1 , 3 had CTC residues of 0.5±4.67 mg kgÿ1 (CTC and its 4-epimer), and one had doxycycline in an amount of 11.29 mg kgÿ1 . Compared with the intensity of ¯uorescence on bone surfaces (Fig. 2), the concentrations of TCs increased with intensity of ¯uorescence. Some statistical parameters of the grouped results are given in Table 3. 3.4. TC residues in chicken bones 34.9% of the samples (53 of 152) showed ¯uorescence on the surface of the os femoris (Table 2). No sample with the label described in Section 2.2.2.2 showed ¯uorescence.
Table 3 TC concentrations in turkey bones (mg OTC/TC/CTC kgÿ1 bone)a
Range Mean S.D.
Positive n 39
Positive n 20
Positive n 23
0.19±6.5 1.72 1.16
1.6±8.9 4.66 2.13
2.01±32.15 10.49 7.0
a
n: number of samples; range: minimum and maximum concentration; S.D.: standard deviation; de®nition of positive results according to Section 2.
HPLC. 51 of 53 samples contained detectable TC residues in the bones. Two samples contained TC in concentrations between 0.9 and 15.1 mg kgÿ1 (sum of TC and its 4-epimer), the remaining 49 samples contained OTC in concentrations of 0.15±42.3 mg kgÿ1 . As the number of samples with slight positive ¯uorescence was limited, the correlation between intensity of ¯uorescence and TC concentrations was not examined. 3.5. TC residues in duck bones 95.3% of the samples (82 of 86) showed ¯uorescence on the surface of the os femoris (Table 2). HPLC. 82 of 82 samples contained detectable tetracycline residues in the bones. 1 sample contained 70 mg kg ÿ1 doxycycline, 40 samples contained either OTC or TC (and its 4-epimer) in concentrations of 1.2±36.2 mg kgÿ1 , the remaining 41 samples contained chlortetracycline in concentrations of 1.6±45 mg kgÿ1 . As the number of samples with slight positive ¯uorescence was limited, the correlation between intensity of ¯uorescence and TC concentrations was not examined. 3.6. TC residues in bones of veal calves
Fig. 2. TC residues in turkey bones (intensity of ¯uorescence on bone surfaces vs. concentrations).
18.8% (293 of 1560) showed ¯uorescence on the surface of the os femoris (Table 2). As already noticed (turkey bones), some samples with only slight positive ¯uorescence on the surface (positive
M. K uhne et al. / Food Control 11 (2000) 175±180
) showed intensive ¯uorescence in deeper layers of the bone tissue, forming ¯uoresceing rings under UV light. HPLC. 45 samples of ¯uoresceing rib bones were analysed for TC residues (15 samples positive , 15 samples positive , 15 samples positive ). In all samples residues of TCs were detected. There was no predominant TC: Either TC with its 4epimer, CTC with its 4-epimer and OTC was found. 10 samples contained more than one TC. The concentrations are given in Fig. 3. There was not found a correlation between intensity of ¯uorescence and TC concentrations.
4. Discussion 1. Screening for TCs in bones using the ¯uorescence test. The therapeutical dosage of OTC when administered orally to chickens is 1000±1500 mg OTC kgÿ1 feed. To study the sensitivity of the visual detection we used the smallest recommended subtherapeutical dosage. This dosage (124 mg kgÿ1 ) gave consistently positive results. As studied earlier by Br uggemann, L osch, Merkenschlager and Oterdinger (1966), even smaller dosages (10 mg kgÿ1 ) would lead to detectable residues in the bones, when they were used for longer periods. The visual detection using the UV-lamp gave positive results at concentrations of 50 lg OTC kgÿ1 bone tissue. Nevertheless, as found during the analysis of turkey bones, ¯uoresceing areas in deeper layers of the bone resulting from earlier applications could hardly be seen, if only bone surfaces are examined. 2. TC residues in slaughtered animals. The results of random samples from dierent species of animals showed a high incidence of TC residues (TC, CTC, OTC and doxycycline) in the bones of slaughtered animals.
Fig. 3. TC residues in bones of veal calves (intensity of ¯uorescence on bone surfaces vs. concentrations).
179
Same samples contained even more than one TC. The concentrations ranged from 0.14 to 50.0 mg kgÿ1 . The intensity of ¯uorescence in bones of dierent species had a large variety. A number of samples showed intensive ¯uorescence on the surface. Following the conclusions of Buyske et al. (1960) and our own results with OTC and chicken, intensive ¯uorescence indicates, that the last administration of TCs to the animals was only days (or a few weeks) before slaughtering. Fluorescence in deeper layers of the bone, as found in turkey and calf bones, might be a reference to use in earlier stages of rearing. The ®ndings in bones of slaughtered pigs were inhomogenous and dependent on the age of the animals with highest incidence in piglets and lowest in sows. The highest incidence of all species was found in turkey bones. Predominantly, we found residues of TC with its main metabolite 4-epi-tetracycline. Rearing of turkeys is usually performed in large ¯ocks with a high infectious pressure. During the rearing period of 18±24 weeks, respiratory diseases may occur in almost every farm und might be a trigger for antibiotic treatments of the ¯ocks. The dierent residue levels found in this study might depend on the time after withdrawal of the medicated feed or water and the individual dosage. In chickens and ducks also a high rate of contamination with TCs was found. The percentage of slight positive results with the ¯uorescence test was small compared with turkey samples. Chickens are only kept for 4±5 weeks, so in most cases the period between administration of TCs and slaughtering will be shorter than in turkey ¯ocks. In our study with calves the rate of contamination was medium. In 22% of the samples multiple TC residues were found. An important question arising from this results is the correlation with residues in edible tissues. K uhne (1998) examined TC concentrations of muscle, kidney and liver of turkey carcasses with ¯uorescence in bones and found increasing percentage of positive results in kidney and liver with increasing ¯uorescence on the surfaces of bones. However, in all samples the concentrations of TCs were below the maximum residue levels set by the European Community (Commission of the European Community, 1999). 3. Toxicological signi®cance of TC residues in bones. A toxicological signi®cance of TC residues emerges from the possible contamination of mechanically deboned meat and the contamination of meat and bone meal. Mechanically deboned meat. In 1996, 350 000 tons of poultry separator meat and 250 000 tons of pig separator meat has been produced within the European Community (BgVV, 1998), containing bone meal and bone splinters up to 2.5% (Anhalt, 1980) or even 5% (Varnam and Sutherland, 1995). Assuming the concentrations of TCs in our study as possible contamination
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source for mechanically deboned meat (i.e. 50 mg kgÿ1 and 2.5% bone splinters in meat), concentrations of up to 1250 lg kgÿ1 may be possible. The temperatures used for the preparation of poultry meat products like poultry surimi, pie ®llings and sausages, are not sucient to destroy tetracycline residues (Rose, Bygrave, Farrington & Shearer, 1996). Meat and bone meal. Meat and bone meal is a costeective and often used protein source in poultry and pig fattening. According to European legislation meat and bone meal is manufactured in special factories, that must use eective methods for the inactivation of hazardous microorganisms and agents. In rendering plants the applied heat is a minimum of 133°C (Celsius) at 3 bar for 20 min. Particle size prior to processing of meat und bone meal must be not more than 5 cm (Commission of the European Community, 1997). The technique provides a suitable inactivation of hazards of microbiological origin. Nevertheless, Honikel, Schmidt, Woltersdorf and Leistner (1978) stated, that bones must be at least heated to 140°C to get a considerable decrease of TC residues in bones. A calculation of presumptive concentrations of TC in raw material for the production of meat and bone meal will in most cases exceed the ppm level (i.e. 50 mg kgÿ1 , average percentage of bones in a steer destined for slaughter: 10% (Gracey & Collins, 1992).
5. Conclusions The occurence of TC residues in bones of slaughtered animals is more likely a normal than an exceptional ®nding. This fact has severe consequences for the potential toxicological risk of products that contain bone portions like mechanically deboned meat and meat and bone meal. Screening methods, like the detection of ¯uorescence, are able to detect suspicious carcasses in meat plants. References Anhalt, G. (1980). Maschinelle Fleisch-Knochen-Separation, Technologie und Anforderungen. Arch. Lebensmittelhyg., 31, 12±16. Aoyagi, M., Sasaki, T., Ramamurthy, N. S., & Golub, L. M. (1996). Tetracycline/Flurbiprofen combination therapy modulates bone remodeling in ovariectomized rats: preliminary observations. Bone, 19, 629±635.
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