In Ireland, the European Communities (Control of Animal Remedies and their Residues) Regulations 2009 implement the provisions of Council Directives 96/22/EC and 96/23/EC. They prohibit the import, manufacture, sale, supply, administration, or possession of substances having oestrogenic, androgenic, gestagenic, or thyrostatic action and beta-agonists. Limited exceptions are outlined in 96/22/EC for animal remedies that may contain these substances.
The National Residue Control Plan (NRCP), drawn up in accordance with Council Directive 96/23/EC on measures to monitor certain substances and residues in live animals and animal products, is approved by the European Commission and implemented by the Food Safety Authority of Ireland. The scope of testing under the NRCP, for which the Irish Equine Centre is an official laboratory for part of these analyses, is very comprehensive, covering all 11 animal/food areas and 18 distinct residue groups that fall into four broad categories:
- Banned substances, such as growth promoting hormones;
- Approved veterinary medicines;
- Approved animal feed additives; and
- Environmental contaminants.
The extensive testing carried out under this Plan and the low incidence (less than 0.2 percent) of non-compliant samples over the last few years indicate that the controls in place are ensuring that the administration of banned growth promoting hormones and banned substances to food-producing animals in Ireland remains low or those illegally using these substances have evaded detection.
Growth promoters, which are tested for under the NRCP, are hormonal and antibiotic substances that may be used in food producing animals for growth promotion in livestock animals, thus increasing the production of muscle meat and the reduction of fat. The type of growth promoter used is dependent on the animal species and mode of rearing—with steroid growth promoters used for beef cattle and antibiotic growth promoters, which are usually added to feedstuffs, such as the coccidiostats used in the poultry industry and chlorotetracycline used in the porcine industry.
It is not clear what the potential human health impacts of growth promoters are, particularly when it is possible that exposure may be extended over long periods of time. Of the hormonal growth promoters (anabolic steroids) the use of three natural steroids (17β-estradiol, progesterone, and testosterone) and three synthetic hormones (zeranol, trenbolone acetate, and progestin melengestrol acetate) have been banned in the EU since 1988. For all of these six hormones, endocrine, developmental, immunological, neurobiological, immunotoxic, genotoxic, and carcinogenic effects could be possible. Of the various susceptible risk groups, prepubertal children is the group that was reported to be of greatest concern by a report on the Opinion of the Scientific Committee on Veterinary Measures Relating to Public Health (SCVPH)—April 10, 2002 (on review of previous SCVPH opinions of April 30, 1999 and May 3, 2000 on the potential risks to human health from hormone residues in bovine meat and meat products). Therefore, from a consumer health perspective, it is essential that testing is carried out for these substances.
Of the antibiotic growth promoters, prophylactic use is common practice in animal feed and can be vital for animal health, particularly with intensified animal rearing. Suppression of disease causing organisms by prophylactic use of antibiotics may reduce the incidence of clinical and subclinical disease, resulting in better animal health and growth. The same classes of antibiotics used to treat humans are given to animals. A report by the U.S. FDA in 2013 revealed that the meat industry accounts for nearly four fifths of all antibiotics used. The issue of antibiotic resistance is of paramount concern. If antibiotics are administered at a therapeutic level (in the absence of prophylactic use), the consumer may be exposed to the antibiotic particularly if it’s not used judiciously with observation of withdrawal times so that maximum residue limits (MRLs) are not breached. This in turn could potentiate the issue of antibiotic resistance, which will affect all species.
Due to the economic benefits to be gained from the use of illegal growth promoters, they will continue to be used in animal production. As analytical methods of detection become more sensitive, methods of evasion have become more sophisticated. The ban on the use of steroid implants (which were easily detected upon ante-mortem inspection) resulted in the production and distribution of liquid-based steroid formulations. The use of low-dose multi-compound cocktails, which are below detectable levels but have a synergistic effect when used together, and natural hormone administrations such as 17β-estradiol, make detection and confirmation of these substances difficult. This in turn presents an enormous challenge to regulatory authorities tasked with enforcing their ban.
The Special Investigation Unit (Ireland) was set up to provide specialist inspectors within the Department of Agriculture to investigate and deal with practices relating to the use of illegal substances in animal production, such as was the case with the use of angel dust (clenbuterol) in the 1980s. Surveillance for the presence of residues of veterinary substances in food-producing animals is regulated by the Directive 86/469/EEC, which offers specific guidelines for sampling procedures on farms and in slaughterhouses. The category of compounds and species will determine the level and frequency of sampling and this is outlined in the NRCP each year. Sampling intensity increases in response to the incidence of non-compliant samples. This was evident during the bute horsemeat scandal in 2013 with intensified sampling for phenylbutazone in horses, which is a group A prohibited substance under Directive 96/23/EC (Annex I). This also occurred for anthelmintics in sheep/goats and dioxins in all species and food commodities.
Analytical methods of detection have become more sensitive over time. This is due to the requirement to meet detection levels or MRLs outlined in legislation. For some substances, these have been lowered and methods must be sensitive enough to be able to detect very low concentrations of substances in multiple matrices. There are several analytical methods used for detection of drug residues in a sample. Enzyme-linked immunosorbent assay (ELISA) methods employed for this analysis usually target one substance and their cross reactants. This methodology works well as a screening tool for targeted screening. The disadvantage of using ELISA-based methods is in the event that a single sample needs to be screened for multiple drug residues since each drug residue has to be tested using a specific kit. The Siemens Immulite, which is an automated chemiluminescent immunoassay, is used for hormone detection such as progesterone and estradiol in serum samples. Again, as for the ELISA, an individual kit is required for detection of each individual hormone.
Randox Food developed another method of biochemical analysis using the Evidence Investigator. The Evidence Investigator biochip array technology (BAT) is used to perform simultaneous quantitative detection of multiple analytes from a single sample. The core technology is the Randox biochip, a device containing an array of discrete test regions containing immobilized antibodies specific to the drug residues under test—according to the kit type. The Evidence Investigator itself is a benchtop analyzer housing digital imaging instrumentation that captures the chemiluminescent signal emitted from drug residue conjugates labeled with HRP.
All of these methods are rapid, reliable, and sensitive. Drug residues or hormones may be detected in very small concentrations. The results obtained from analysis must be reproducible and all methods must be validated in accordance with the requirements of Commission Decision 2002/657/EC, which sets down standards for the performance of analytical methods and the interpretation of results.
An important area of focus in relation to residue testing is the dairy industry. Because milk and milk products, such as baby milk powder, are a huge export market for Ireland, it is important to screen these products for growth promoters and antibiotics. It is a legal requirement that raw milk not contain residues, including antibiotics. Each farmer is required to keep a record of all medicines purchased and administered to animals. Milk from dairy cows that are on antibiotic treatment must be discarded and kept out of the food chain until the withdrawal period has been observed. Milk testing programs employed by the dairy co-ops are tested at the farm level and have limitations. If antibiotics are detected, the milk must be discarded and not used for human consumption. Severe commercial penalties ensue for the farmer.
Randox InfiniPlex for milk BAT method using the Evidence Investigator is a possible option that can provide a single analysis to screen for all currently monitored residues covered under legislation. This method screens for in excess of 120 drug residues, including antibiotics and growth promoters. Technologies like this are essential in an emerging and demanding market where consumer health is of paramount importance.
Treacy is the senior laboratory analyst in the Forensics Laboratory at the Irish Equine Centre. Reach her at ATreacy@irishequinecentre.ie.
Professor Tom Buckley, MSc., FIBMS, FAMLS (firstname.lastname@example.org), the department head for Microbiology and Forensics at the Irish Equine Centre, contributed to this article.