STABILITY

OF

ANTIBIOTICS

IN

MEAT

DURING

SIMULATED

HIGH

TEMPERATURE DESTRUCTION PROCESS

H.J. van Egmond, J.F.M. Nouws, R. Schilt, W.D.M. van Lankveld-Driessen, E.P.M. Streutjens-van Neer, F.G.H. Simons State Institute for Quality Control of Agricultural Products (RIKILT), Bornsesteeg 45, NL6708 PD Wageningen, The Netherlands

Abstract With a simulation model at laboratory-scale, the stability of sixteen antibiotics during the destruction process of animal and offal's was investigated. The antibiotics were added to a mixture of pork-meat, pork-kidney, and pork-liver (90/5/5) (w/w/w), subsequently pasteurised at 80C (15 min.), sterilised at 134C (3 bar, 20 min.) and dried at 100C (4 hours). During the different stages of this process, samples were taken and analysed for antimicrobial activity by bioassay. The remaining activity after the full destruction process was for lincomycin 80%, flumequine 69%, enrofloxacin 68%, neomycin 46%, tylosin 44%, sulfamethazine 38% and spiramycin 15%. Penicillin, amoxicillin, ampicillin, cloxacillin, oxytetracycline, doxycycline, colistin, dihydro-streptomycin and sulfamethoxazole were fully degraded (less than 10% remaining activity) after the sterilisation step (134C). It is concluded that the high temperature destruction process does not guarantee a full break-down of residues of veterinary drugs present in condemned animals.

Introduction Condemned carcasses of animals and remains from slaughter are processed in a high temperature destruction process to obtain bone meal and fat suitable for the production of animal feed. In these materials, residues of veterinary drugs (e.g., antibiotics) and other substances can be present. In case condemned animals are processed to which antibiotics are administred for treatment of severe diseases without applying the normal withdrawal period, the residue levels may exceed the MRL levels. The risk is even larger when the pharmacokinetics are significantly influenced by the impaired health of the animal that may result in a decrease in drug absorption, metabolism and excretion. Consequently, the levels of veterinary drugs present in the animals may be relatively high. In general it is assumed that residues of veterinary drugs will be degraded by the high temperature in the destruction

process. The high temperature destruction process can be globally devided in the following steps: 1) crushing of the carcasses 2) pasteurisation at 80C, to inhibit the formation of stench 3) sterilisation at 134C, 3 bar, during 20 min 4) drying at approximately 100C during several (4 h) using fat to improve the transport through the heating units. In this study, the stability of sixteen antibiotics in a pork meat-kidney-liver mixture during the destruction process in a laboratory-scale simulation model was investigated with microbiological methods which can also detect possibly formed microbiological active transformation products. The applied concentrations were based on normally found antibiotic concentrations at injection sites or organs of diseased animals during antibiotic treatment [5,6].

Methods and materials

Pure drug substances. Stock solutions of the pure drug substances (corrected for potency) were prepared in appropriate solvents. The stock solution and working solutions (buffer calibrators) (table 2) were freshly prepared on the day of analysis. Test material. Lean pig meat, kidney and liver were obtained from a local butcher and homogenised. Subsequently, a mixture was prepared with meat, kidney and liver (90/5/5 (w/w/w)). The mixture was stored at 18C and portions were thawed one day prior to analysis at 0-5C. Bioassay Composition and preparation of test plates. For each antibiotic or group a specific test plate was used (Table1). Autoclaved (15', 121C) agar was cooled to 50C and the pH was adjusted. Subsequently the micro-organisms (spores or cells) ( 105-106 colony forming units /ml agar) were added and the agar was poured out in bioassay plates (245*245*20 mm) with a thickness of 2.2 mm. After solidification, holes of 14 mm were punched out of the agar. Bioassay. To each hole, 250 l extract or standard solution was added in duplicate. After a prediffusion of 30 min at room temperature, the plate was incubated for 8-12 hours or during the night at the appropriate temperature. Finally, the inhibition zones were accurately measured with a vernier caliper (0.1 mm accuracy).

Table 1. Composition of test plates

Analyte Penicillin, Amoxicillin, Ampicillin, Cloxacillin Oxytetracycline Doxycycline Tylosin, Lincomycin Spiramycin Neomycin DH-streptomycin Enrofloxacin Flumequine Colistin Sulfamethazine, Sulfamethoxazole Indicator micro-organism B. calidolactis C953 B. cereus ATCC 11778 M. luteus ATCC 9341 B. subtilis BGA Entero RIK 144 Entero RIK 14 B. broncheseptica ATCC 4617 B. subtilis BGA Composition of plates Plate count agar (PCA) Standard II Nhr-agar with supplements Mueller Hinton agar Plate count agar (PCA) Plate count agar (PCA) several constituents Tryptone soya broth + agar New Dutch Kidney test medium pH 7.0 6.0 8.0 8.0 8.0 6.0 6.0 7.0 Incubation temperature (C) 55 (8-12 hours) 30 30 30 30 30 30 37

Experimental Simulation of the destruction process. Ten portions of 10 grams of the meat-kidney-liver mixture were weighed in glass bottles. To each bottle, 0.5 ml of a standard solution was added. Two portions were directly analysed (portions 1 and 2). The remaining eight portions were heated 15 min at 80C in a water bath. Portion 3 and 4 were cooled and stored. Portions 5-8 were heated for 20 min at 134 1C (3 bar) in an autoclave. Portion 5 and 6 were cooled and stored. Portion 7-10 were heated in a oven at 100 C. After 2 hours portion 7 and 8 were cooled and after 4 hours portion 9 and 10. All portions were weighed and analysed. The addition of fat to the simulated process has been omitted due to practical reasons. All antibiotics were tested at two levels (high and low, see table 2). Extraction. To 10 grams of sample, an appropriate amount of extraction solvent (table 2) was added to yield a weight of 50 or 100 g (corrected for loss of fluid during the destruction). The sample was shaken for 15 min on a horizontal shaker and homogenised for 2 min in a stomacher device. After centrifugation the pH was adjusted and appropriate dilutions for bioassay were made with buffers. With each series, a blank meat-kidney-liver sample was analysed.

Table 2. Levels, extraction solvent, solvent and calibration curves

Analyte Penicillin Amoxicillin Ampicillin Cloxacillin Oxytetracycline Doxycycline Tylosin Lincomycin Spiramycin Neomycin

Level (mg/kg) 25;2.5 25;2.5 25;2.5 5;50 12.5;125 6.25;62.5 16;160 24;240 16;160 35;350

Extraction solvent acetone/phosphate buffer pH 6 mixture (1/4) (v/v/) methanol/hydrochloric acid (12 N) mixture (98/2)(v/v) methanol/phosphate buffer pH 8 mixture (6/4) (v/v) 0.2 M CaCl2 solution with 20% NaCl (w/v) 0.5 M acetic acid solution methanol/phosphate buffer pH 8 mixture (6/4) (v/v/) methanol/phosphate buffer pH 8 mixture (6/4) (v/v) methanol 50%/ trichloroacetic acid 25% mixture (4/1) (v/v) methanol 50%/ trichloroacetic acid% mixture (4/1) (v/v)

Dilution in phosphate buffer pH 6

Calibration range (g/ml) 0.04-0.0025 0.04-0.0025 0.04-0.0025 0.40-0.025 2.0-0.125 012-0.008 3.20-0.20 3.20-0.20 3.20-0.20 1.60-0.10

phosphate buffer pH 5.5 phosphate buffer pH 8 NaCl/CaCl2/ Tris buffer pH 8 mixture triethanol-amine buffer pH 8 phosphate buffer pH 8 phosphate buffer pH 6 phosphate buffer pH 5.5 phosphate buffer pH 7

Dihydro-streptomycin Enrofloxacin

32;320 10;100

3.20-0.20 0.08-0.005

Flumequine

20;200

1.60-0.10

Colistin

16;160

1.60-0.10

Sulfamethazine Sulfamethoxazole

10;100 10;100

2.0-0.125 0.50-0.031

Results

For each antibiotic the decrease in antimicrobial activity was calculated and related to the original activity in portions 1 and 2 (see Figures 1 and 2). In the samples showing antimicrobial activity, the identity of the substance(s) was investigated using agar gel high voltage electrophoresis. In all samples, the antibiotic added was identified and no indications of the presence of breakdown products showing anti-microbial activity were found. In the blank meat-kidney-liver samples, no growth inhibition was observed. The results of the high level and low level experiments showed good comparision with the exception of neomycine and enrofloxacin. These antibiotics showed at the high level 20% more degradation after the full proces, compared to the lower level. This may be due to analytical inaccuracy at the lower level. In Figures 1 and 2 the results of the high level experiments are presented.

Lincomycin was rather stable during the destruction proces (Fig.1). A gradually decrease in remaining antimicrobial activity during the various steps in the destruction process was observed for flumequine, enrofloxacin, sulfamethazine, spiramycin, neomycin and tylosin (Fig.1 and 2). The mean (high and low level) remaining activity after the full destruction process was for lincomycin 80%, flumequine 69%, enrofloxacin 68%, neomycin 46%, tylosin 44%, sulfamethazine 38% and spiramycin 15%. Penicillin, amoxicillin, ampicillin, cloxacillin, oxytetracycline, doxycycline, colistin, streptomycin and sulfamethoxazole were degraded (less than 10% remaining activity) after the sterilisation step (134C). The degree of degradation did not depend on the concentration. Moreover the degradation was not group related, but drug related.

Discussion and conclusion

Heat stability of (residues of) veterinary drugs during the processing of food, e.g. meat, liver, etc has been studied by a variety of methods. These varied in testmatrices, duration and heigth of temperature treatments. Although the studies are not easily to compare and to perform, the results indicate that the information obtained is important, with respect to unintential exposure of food producing animals to drug remains in their feeds. In Table 3 a summary is given.

Table 3. Stability of some veterinary drugs during processing of food (meat) Substance Clenbuterol Levamisol Sulfamethazine Oxytetracycline Chloortetracycline Oxfenbendazol Neomycin Streptomycin Lasalocide Ivermectine Benzylpenicilline (penicilline G) Dimetridazol and ronidazol Stability during processing (cooking, baking, frying, etc.) Stable Stable Stable at 122C Relatively stable, unstable at 100C Relatively stable, unstable at 100C (in bones relatively stable at 130C) Relatively stable Stable Stable Stable below 100 C Stable Not stable Stable Reference [9] [8] [1][7] [2][10] [2] [11] [4] [4] [12] [14] [3][4][13] [15]

For sulfamethazine, oxytetracyclin, neomycin and penicillin the results were in accordance with the literature. Streptomycin was fully degraded after the heating step (134C), in contrast with other studies [4]. As a result of this study it is concluded that the high temperature druing the destruction process does not give a guarantee for the breakdown of veterinary drugs possibly present in the source material used. Especially the stable substances which can be orally absorbed and have a biological half-life > 3 hours may reveal tissue residues exceeding MRL (spiramycin, tylosin, sulfamethazine, enrofloxacin, flumequine). Whether, for the poorly or non-absorbable antibiotics a potential risk for increase of resistance among gastro-intestinal flora exists, is unknown. Additional research of the breakdown of antibiotics and other veterinary drugs is necessary to obtain a better insight. Additionally, survey research can reveal the current presence of the stable antibiotics in bone meal obtained from the destruction process.

Acknowledgements

The authors thank the Ministry of Agriculture, Nature Management and Fisheries, The Department of the Environment, Quality and Health (VVM) for the financial support. The authors also are indebted to Rendac Son for their kind co-operation.

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