Date Full Report Received11/03/2011
Date Abstract Report Received11/03/2011
InvestigationInstitution: Kansas State University
Primary Investigator: DACVCP, Mike Apley DVM, PhD
Co-Investigators: Locke Karriker, Gary Griffith
Funded ByNational Pork Board
The objective of this study was to describe tissue residues of chlortetracycline, oxytetracycline, and tetracycline in slaughter hogs and also bacitracin in cull sows. For the tetracyclines, the residues were monitored out to 28 days after ending the treatments. All analysis was carried out on a mass spectrometer representative of advanced equipment used for residue detection around the world. The methods used were developed in the analytical lab conducting this study, and are not necessarily equivalent to regulatory methods in any country. However, tissue concentration levels of detection and quantitation are similar to the most sensitive methods in use. In addition to mass spectrometry analysis, Charm II analysis was performed on muscle for all 5 treatment groups in the tetracycline portion of the study.
In the tetracycline category study, 5 groups of pigs received the following treatments.
1. Chlortetracycline in the water calculated to deliver 10 mg/lb body weight daily for 5 days
2. Oxytetracycline in the water calculated to deliver 10 mg/lb body weight daily for 5 days
3. Tetracycline in the water calculated to deliver 10 mg/lb body weight daily for 5 days
4. Chlortetracycline in the feed calculated to deliver 10 mg/lb body weight daily for 14 days
5. Oxytetracycline in the feed calculated to deliver 10 mg/lb body weight daily for 14 days
Within each treatment, 5 pigs were euthanized and samples collected at the time that drug administration was stopped (day 0), and at 7, 14, 21, and 28 days following the end of drug administration. The timing of drug administration resulted in the first samples being collected 28 days prior to the projected finish date of the pigs. Weights on the first sample date were approximately 200 lbs, and were approximately 270 lbs on day 28.
Samples collected included muscle, liver, kidney, fat, colon contents (at 21 and 28 days only), bone (21 and 28 days only), plasma, and urine. Detailed results are reported for each tissue in the final report. For the purposes of this summary, emphasis will be placed on muscle. For all of the tetracyclines in the study, all tissues displayed a dramatic drop in concentrations from day 0 to day 7. The discussion here focuses on days 7 through 28. Results are discussed in parts per billion (ppb) which is equivalent to nanograms per gram (ng/g). A nanogram is 1 billionith of a gram.
Results are discussed in light of the level of detection (the concentration at which we are sure the compound is there, but cannot reliably quantify it) and level of quantitation (the concentration at which we can reliably say how much is present). These two levels are abbreviated as LOD and LOQ, respectively. Half-lives will also be discussed, which is the time required for the concentration of a drug in a tissue to decrease by one half.
Chlortetracycline results: (LOD 1 ppb, LOQ 4 ppb in muscle) At day 7, muscle residues ranged from 11.8 to 29.0 ppb in the feed group and from 5.8 to 13.0 ppb in the water group. At 28 days withdrawal time, the feed group residues ranged from 4.7 to 12.0 ppb. At 28 days, the pigs in the water group displayed detectable residues only in 2 pigs, and concentrations ranging from 4.9 to 6.0 ppb in the other 3 pigs. The elimination half-life calculated in muscle after feed administration was 18.0 days, explaining the similar concentrations in the muscle at 7 and 28 days. Due to the number of detected but non-quantifiable concentrations in the water group, a tissue withdrawal for muscle could not be calculated, but calculated withdrawal times for liver and kidney were very similar to the feed group, suggesting muscle would also be similar.
Very low plasma concentrations were detected at 28 days in 3 of 5 pigs in the feed group, but none of the pigs in the water group. Significant concentrations could still be detected in the urine at 28 days in all 4 pigs from which a urine sample could be collected in the feed group, and 3 pigs in the water group (28 to 397 ppb).
Concentrations in bone suggest a significant reservoir of chlortetracycline contributing to these persistent low concentrations in other tissues. At 28 days, chlortetracycline concentrations in bone ranged from 375 to 1120 ppb in the feed group (100 to 766 ppb in the water group), which were very similar to the concentrations found at 21 days for both groups. No chlortetracycline could be detected in colon contents at 28 days in either the feed or water groups. Pen floor manure samples taken on the day that drug administration ceased for both groups were found to contain chlortetracycline ranging from 163,103 to 274,903 ppb on a dry matter basis.
Charm II analysis of muscle samples for chlortetracycline (LOD reported as 100 ppb) identified all 0 withdrawal time samples in both the feed and water groups as suspect (161.0 to 545.0 ppb). No other residues at withdrawal times 7 to 28 days were identified in muscle (maximum residue of 29.0 ppb).
Oxytetracycline results: (LOD 1 ppb, LOQ 4 ppb in muscle) At day 7, muscle residues ranged from 10.0 to 15.7 ppb in the feed group and from 4.5 to 18.8 ppb in the water group. At 28 days withdrawal time, the feed group residues ranged from 4.7 to 9.9 ppb. At 28 days, the pigs in the water group displayed detectable residues only in 4 pigs, and a concentration of 5.1 ppb in the fifth pig. The elimination half-life calculated in muscle after feed administration was 31.6 days. Due to the number of detected but non-quantifiable concentrations in the water group, a tissue withdrawal for muscle could not be calculated.
Low plasma concentrations were detected at 28 days in 4 of 4 pigs in the feed group (one pig died during the study), and all 5 pigs in the water group. Significant concentrations could still be detected in the urine at 28 days in 2 pigs from which a urine sample could be collected in the feed group and 4 pigs from which urine could be collected in the water group (8.9 to 526 ppb).
Bone samples retained significant residues at 28 days for all pigs in both the feed and water groups (12.7 to 117 ppb). In the feed group, colon contents at 28 days were not detected in one pig and ranged from 404 to 713 ppb in 3 other pigs. Colon contents at 28 days in the water group displayed nondetectable (2 pigs), detectable (2 pigs) and quantifiable (276 ppb) residues.
Pen floor manure samples taken on the day that drug administration ceased ranged from 593,857 to 1,400,000 ppb on a dry matter basis.
Charm II analysis of muscle samples for oxytetracycline (LOD reported at 100 ppb) identified no residues in the feed and water groups as suspect (maximum residue of 223.0 ppb).
Tetracycline Results: (LOD 0.5 ppb, LOQ 4 ppb in muscle) There was only a water administration group for tetracycline. At day 7, muscle concentrations ranged from 16.6 to 24.1 ppb. At day 28, muscle concentrations ranged from 12.5 to 26.9 ppb. The muscle elimination half-life for tetracycline was calculated at 118.5 days, with kidney being similar at 92.7 days. Plasma concentrations were detectable in all 5 pigs at day 28, as were concentrations in the urine. Concentrations in colon contents were only detected in 1 of the 5 pigs at day 28. Bone concentrations ranged from 44.6 to 75.0 ppb at 28 days, which were very similar concentrations to the 5 pigs on day 21.
Charm II analysis of muscle samples for tetracycline (LOD reported at 25 ppb) identified all 0 withdrawal time samples as suspect (161.0 to 899.0 ppb). Four other samples were identified as suspect on days 7-21 (18.0 to 36.2 ppb), with all other samples on these days determined as negative (9.8 to 26.9 ppb).
Results summary for the tetracyclines: Muscle tissue analyzed from chlortetracycline, oxytetracycline, and tetracycline treatment groups maintained concentrations well below the U.S. tolerance (1000 ppb) and some foreign export maximum residue limits (MRLs, 50 ppb) out to 28 days. The concentrations were high enough that they could be detected and quantified by advanced analytical techniques, which could pose trade issues in markets where detected, or very low, quantifiable residues are used as a regulatory standard. Bone samples displayed prolonged residues, which may be a major component of the persistent, low-level residues demonstrated in this study. Colon contents and urine displayed quantifiable residues for all treatment groups in at least some pigs at 21 and 28 days withdrawal depending on the group. The high initial manure concentrations at 0 withdrawal time, and the persistent manure and urine contributions to the pen floor environment, could contribute to recycling of drug in environments where manure consumption is possible.
In the hands of this laboratory, and in incurred samples (residues in the muscle from drug given to the animal rather than added in the lab), the chlortetracycline and tetracycline CHARM II assays detected residues at or near the stated level of detection. The oxytetracycline CHARM II assay failed to detect oxytetracycline residues up to 223 ppb with a stated level of detection of 100 ppb.
Bacitracin results: Bacitracin was fed to sows at a rate of 750 g/ton in 5 lbs of feed daily for 2 weeks. Residues were detected (LOD 38 ppb) in one muscle sample at 0 days withdrawal and two samples at 7 days withdrawal. Multiple samples had detected or quantifiable concentrations in the liver at 0 and 7 days withdrawal (LOD 97 ppb, LOQ 292 ppb).
Contact information: Mike Apley, Kansas State University, 785-410-5643, firstname.lastname@example.org