For long, veterinary use of diclofenac has been found to be the culprit behind wiping off vultures from the Indian subcontinent. Even though the centre banned veterinary use of diclofenac in 2006 following an outcry on vulture deaths, nobody knows whether the drug is still being used in the hinterland as there is hardly any reliable field-level diclofenac diagnostic.
A team of German researchers has now offered help. The team has come out with a diclofenac detection method, which would be ideal for the vulture breeding centre at Pinjore to regularly test meat supply for the trace amount of the drug.
Even though the technique was originally developed for testing the presence of pharmaceutical in waste water, it is now being extended to India for vulture conservation efforts.
First study in 2006-07
The first pilot study was done in 2006-07 involving researchers from Technical University, Munchen, Royal Society for the Protection of Birds in the UK, Bombay Natural History Society and the Centre for Wildlife at the Indian Veterinary Research Institute, Izaatnagar. It proved the suitability of the test for detecting diclofenac in animal tissue.
“After showing the principal applicability of the test, another study with more than 1000 samples has started to screen diclofenac in different tissues, including that of vultures. So far the results demonstrate the usefulness of the assay, being a cost-efficient alternative to more expensive and time-consuming HPLC-MS (high-performance liquid chromatography with mass spectrometric detection) method,” Dietmar Knopp, team leader at TU Munchen told Deccan Herald.
In 1990s, diclofenac was a popular medicine for livestock in India, Pakistan and Nepal. When vultures gorge on cattle carcasses, they too ingest the drug in large quantity and fall prey to the drug. The populations of three species of these birds of prey – the Indian vulture, the Oriental white-backed vulture and the slender-billed vulture – have shrunk to a mere three percent of their original number.
The diclofenac ban came in 2006, followed by the creation of the centre for breeding, which plans to reintroduce the vultures into the wild after 10 years. But raising the bird offspring with diclofenac-free food, necessitates testing meat for possible traces of the drug. Also the test has to be simple enough to be carried out in field conditions. The scientists started by producing a specific diclofenac antibody that they could use to develop a highly sensitive immunological test to detect the drug. The test uses miniaturised plastic microtiter plates and has the advantage of not requiring complex sample treatment, which translates into quick and low-cost analyses.
“The assay is a cheap and user-friendly test which can be used on-site. It does not need very experienced personnel and highly sophisticated instrumentation. More importantly, the test does not give false negative results. It does not indicate that a sample is free of diclofenac when it actually contains the drug,” Knopp said.
Diclofenac binds strongly with protein molecules and the conjugate can be immobilised in the wells of the microtiter plate. When the sample and the antibody are added, the antibody can either react with the drug molecules of the sample or with the molecules previously bonded to the fixed diclofenac-protein conjugate. The higher the diclofenac concentration in the sample, the fewer antibodies remain to bind with the diclofen
ac-bonded protein in the well. The concentration of bound antibodies can then be determined in a colour reaction.
Fears over use of diclofenac
Notwithstanding the ban on veterinary use, conservationists believe diclofenac may still be in illegal use. Analysing tissues from dead vultures for diclofenac might give a rough idea about the actual compliance with the regulation. The test is useful for analysing the meat used to feed the vultures in the sanctuaries.
“As it takes roughly 10 years to raise vultures before they can be released into the wild, nobody knows how long diclofenac will be illegally available and used for animal medication,” Knopp said.
This method is also suitable for many other fields of application, as shown for instance in studies on diclofenac contamination of waste water in Bavaria and Austria. With more than 80 tonnes of diclofenac being sold in Germany every year, the test may soon be required in Europe as well as it is among the pharmaceutical substances most frequently found in surface water samples.
“Studies have shown that diclofenac leads to kidney damage in trout,” says Reinhard Niessner, head of the Institute of Hydrochemistry at the TUM. “As this substance degrades only very slowly, there may be need for action soon. As new waste water treatments are developed and implemented in the future, simple methods for monitoring their efficiency will be required,” he said.