Adrian C. E. Fesser

Long-term trends and sources of organochlorine contamination in Canadian tundra peregrine falcons, Falco peregrinus tundrius

Levels of eggshell thinning, and organochlorine residues in egg contents, blood plasma of adults and juveniles, tissue samples, and prey species were determined for a population of migratory Peregrine Falcons (Falco peregrinus tundrius) breeding in the Canadian Arctic. Temporal trends were assessed by comparing data collected during 1991-1994, with data from 1982-1986, for the same population. Shells (n=54) from 1991-1994 averaged 15% thinner than eggs produced prior to the introduction of DDT. No improvement in shell thickness was detected between decades. Mean DDE residue levels in eggs showed a decline from 7.6 mg kg (1982-1986) to 4.5 mg kg (1991-1994), but there was no significant change in SigmaPCB residues. Moreover, the proportion of clutches with eggs exceeding critical SigmaPCB, DDE, and dieldrin residue levels (10%) did not change between decades. Relative to Greenland and Alaskan populations, F. p. tundrius at Rankin Inlet show high levels of organochlorine contamination and little reduction in residues over the last decade. These Tundra Peregrines continue to be exposed to organochlorines in Latin America; however, results also link relatively high levels in the study population with waterfowl species that do not leave Canada in winter.

Quantitative screening of stilbenes and zeranol and its related residues and natural precursors in veal liver by gas chromatography-mass spectrometry.

An existing gas chromatography-mass spectrometry-based quantitative screening method for the regulatory analysis of the resorcylic acid lactones zeranol, taleranol, and zearalanone and the stilbene anabolic steroids diethylstilbestrol and dienestrol was extended to include natural precursors of zeranol (zearalenone, alpha-zearalenol, and beta-zearalenol) in veal liver. No changes in sample preparation were required; the instrumental conditions were selected to effect a suitable chromatographic separation and detection of the analytes. Validation experiments were performed to verify the performance and applicability of the extended method for the quantitative screening of the original and additional analytes in veal liver in the concentration range from 0.5 to 2.0 microg/kg. The limits of detection were 0.08-0.19 microg/kg. The limits of quantitation were 0.27-0.64 microg/kg. Recoveries were 29-67%. Combined relative measurement uncertainty estimates were 6-21%.

Identification of hormone esters in injection site in muscle tissues by LC/MS/MS.

The detection of hormone abuse for growth promotion in food animal production is a global concern. Initial testing for hormones in Canada was directed at the compounds approved for use in beef cattle, melengestrol acetate, trenbolone acetate and zeranol, and the banned compound diethylstilbestrol (DES). No hormonal growth promoters are approved for use in veal production in Canada. However, instances of use of trenbolone and clenbuterol were detected in Canada in the 1990s. During the development of a new analytical method for testosterone and progesterone, there were reports of suspicious injection sites being found in veal calves. Upon implementation of the method, analysis of investigative samples revealed significant residues of testosterone in some injection sites. To prove that the source of these residues was exogenous, a fully validated method for hormone esters was developed to confirm the presence of exogenous hormones in these injection sites. The QUECHERS model was employed in methods development and resulted in a simple, effective extraction technique that consisted of sample pre-homogenization, liquid/liquid partitioning, extract dilution, filtration and use of LC/MS/MS to provide detection selectivity. The result was an adaptable MS/MS confirmation technique that meets the needs of Canadian regulatory authorities to confirm the misuse of injectable testosterone, and potentially other hormones, in food animal production.

Investigation of Charm Test II receptor assays for the detection of antimicrobial residues in suspect meat samples

Charm Test II receptor assays for beta-lactams, sulfonamides, (dihydro)streptomycin and erythromycin were applied to 257 bovine muscle and kidney samples, and 215 porcine muscle and kidney samples collected from animals suspected to contain antimicrobial residues. The assays were run in conjunction with Agriculture and Agri-food Canadas routine diagnostic confirmation analyses for suspect samples collected at federally inspected packing plants. All samples were subjected to the Charm Test II receptor assays and thin layer chromatography-bioautography (TLC-BA). Selected samples were quantitatively analysed using a liquid chromatographic method for penicillin G and a thin layer chromatography-fluorescence densitometry (TLC-FD) method for sulfonamides. The Charm Test II assays for beta-lactams, (dihydro)streptomycin and erythromycin were an acceptable alternative to the TLC-BA screen for laboratory confirmation of the presence of these compounds, with enhanced sensitivity for (dihydro)streptomycin and erythromycin. In addition, the Charm Test II provided a sensitive screen for sulfonamides as confirmed by the standard TLC-FD procedure. The analysis time, laboratory space and analyst time required to complete the Charm Test II assays is less than that for TLC-BA. Operating costs are similar for both analyses, but the Charm Test II does require capital expenditure for a scintillation counter.

Residue depletion in tissues and fluids from swine fed sulfamethazine, chlortetracycline and penicillin G in combination

Twenty-four hogs were fed a ration for 14 days containing three times the recommended label dose of a combination drug which included sulfamethazine, chlortetracycline and penicillin G. Groups of six hogs were slaughtered 0, 2, 4, or 8 days after withdrawal. Six untreated control hogs were slaughtered 5 days before the first group, of six treated hogs, were slaughtered. Residue concentrations were determined in kidney, liver, muscle, serum and urine. At zero withdrawal the kidney from one hog contained 0.018 mg penicillin G per kg and the serum from the same hog contained 0.016 mg penicillin G per litre. Penicillin G was not detected in any other samples that were analysed. Chlortetracycline concentrations in tissues at zero withdrawal time were below accepted Canadian Maximum Residue Limits (MRL) for chlortetracycline of 1 mg/kg in muscle, 2 mg/kg in liver and 4 mg/kg in kidney and were below the limit of quantitation in all tissues 4 days after withdrawal. Sulfamethazine persisted in the tissues longer than penicillin G or chlortetracycline. Sulfamethazine concentrations were above the Canadian MRL of 0.1 mg/kg at zero withdrawal time and did not decrease to below the MRL until 8 days after withdrawal. Our results suggest that, if the label withdrawal period of 10 days is observed, an increase in the dosage of up to three times the recommended rate is unlikely to increase significantly the risk that residues would occur in the tissues of treated hogs at concentrations which exceed MRLs. Sulfamethazine concentrations in all matrices decreased after storage at -76 degrees C for 6 months.

Laboratory Evaluation of the Charm Farm Test for Antimicrobial Residues in Meat

Charm Farm Tests were applied to 54 muscle and 44 kidney bovine samples, and 95 muscle and 90 kidney porcine samples collected for Canadas national meat inspection program from animals suspected of containing antimicrobial residues. The assays were run in conjunction with Agriculture and Agri-food Canadas routine confirmation analyses for suspect samples collected at federally inspected packing plants. When the bovine and porcine results were combined, 19% of the kidney and 8% of the muscle results were false negatives. Using the Charm Farm Test to screen only the kidney samples would have resulted in 100% identification of the muscle samples that were found to contain violative levels of drug residues. One percent of the kidney and 16% of the muscle results were false positives, on the basis of the results of the confirmatory tests. Minimum detectable levels found in fortified (i.e., artificially contaminated) muscle and kidney with the Charm Farm Test for ceftiofur, chlortetracycline, oxytetracycline, tetracycline, erythromycin, gentamycin, neomycin, penicillin G, streptomycin, sulfamethazine, sulfadimethoxine, tylosin, tilmicosin, and trimethoprim were similar to those claimed by the manufacturer. Minimum detectable levels for the Charm Test II (dihydro) streptomycin and erythromycin receptor assays are reported for fortified muscle and kidney to provide an indication of the relative sensitivities of the Charm Farm Test and the Charm II Tests for these compounds. To further assess the Charm Farm Tests potential as a replacement for existing tests in packing plants, parallel testing needs to be conducted on fresh tissues in a plant environment.

Laboratory Testing of the Charm Test II Receptor Assays and the Charm Farm Test with Tissues and Fluids from Hogs Fed Sulfamethazine, Chlortetracycline, and Penicillin G

The potentials of the Charm Test II receptor assays for the detection of residues of sulfonamides, tetracyclines, and β-lactams and the Charm Farm Test for screening for antimicrobial residues were tested. Market hogs were fed rations containing three times the label level of sulfamethazine, chlortetracycline, and penicillin G for 2 weeks. Groups were killed after 0, 2, 4 and 8 days of withdrawal. Quantitative chemical methods were used to determine residue levels in fluids and tissues. Results were compared with those obtained using the qualitative Charm Test II receptor assays and the Charm Farm Test antimicrobial inhibition assay. For the Charm Test II assays for sulfonamides, tetracyclines and β-lactams, respectively, 1.6, 5, and 6% of the results were false positive and 7, 5, and 0% were false negative, based on the limits of detection for the test kits and the quantitative results. On a similar basis for the Charm Farm Test, 16% of the results were false positive (6 kidney, 4 muscle, and 6 urine samples) and 1% were false negative (sulfamethazine in one urine sample).

Evaluation and Testing of Charm Test II Receptor Assays for the Detection of Antimicrobial Residues in Meat

Charm Test II receptor assays for β-lactams, sulfonamides, streptomycin and erythromycin were tested with standard solutions of targeted antimicrobials and with fortified kidney, liver and muscle samples. The degree of response obtained with the β-lactam assay varied among equimolar amounts of 8 β-lactams. The sulfa assay was positive, with varying response, to equimolar amounts of four different sulfonamides and negative for trimethoprim. The streptomycin assay also detected dihydrostreptomycin, but was negative for gentamicin and neomycin. The erythromycin assay was negative for oleandomycin but positive for tilmicosin and tylosin. No evidence of cross reactivity was detected among the compounds targeted by the four assays.