Analysis of Nine NSAIDs in Ungulate Tissues Available to Critically Endangered Vultures in India MARK A. TAGGART, †,‡ KALU R. SENACHA, § RHYS E. GREEN, | , ⊥ RICHARD CUTHBERT, ⊥ , * YADVENDRADEV V. JHALA, # ANDREW A. MEHARG, † RAFAEL MATEO, ‡ AND DEBORAH J. PAIN ∇ School of Biological Sciences, University of Aberdeen, United Kingdom, Instituto de Investigacio ´n en Recursos Cinege ´ticos, Ciudad Real, Spain, Bombay Natural History Society, Mumbai, India, Conservation Science Group, University of Cambridge, United Kingdom, Royal Society for the Protection of Birds, Bedfordshire, United Kingdom, Wildlife Institute of India, Dehradun, India, and Wildfowl and Wetlands Trust, Slimbridge, United Kingdom Received January 22, 2009. Revised manuscript received April 16, 2009. Accepted April 29, 2009. In 2006, India, Pakistan, and Nepal banned the manufacture of veterinary formulations of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac. This action was taken to halt the unprecedented decline of three Gyps vulture species that were being poisoned by diclofenac residues commonly present in carcasses of domestic livestock upon which they scavenged. To assess the affect of this ban and evaluate residue prevelances of other NSAIDs, we present a method to detect diclofenac and eight more NSAIDs by liquid chromatography-mass spectrometry and apply this to 1488 liver samples from carcasses of livestock taken across seven Indian states. Diclofenac was present in 11.1% of samples taken between April and December 2006, and meloxicam (4%), ibuprofen (0.6%), and ketoprofen (0.5%) were also detected. Although meloxicam is safe for a range of avian scavengers, including Gyps vultures, data regarding the safety of other NSAIDs is currently limited. If wild Gyps on the Indian subcontinent are to survive, diclofenac bans must be completely effective, and NSAIDs that replace it within the veterinary drug market must be of low toxicity toward Gyps and other scavenging birds. Introduction In 2004, it emerged that the nonsteroidal anti-inflammatory drug (NSAID) diclofenac was causing poisoning and mortality of Gyps vultures across the Indian subcontinent (1, 2). Residues of this NSAID, widely used to treat pain, fever, and inflammation in livestock, were being ingested by vultures scavenging on carcasses of ungulates that had died soon after treatment. Residues were detectable in 10.1% of carcasses in India in 2004-2005 (3), and the LD 50 (dose that is lethal to 50% of test subjects) for the Oriental white-backed vulture (Gyps bengalensis) was estimated at 98-225 μg kg -1 (1, 4). These LD 50 values are an order of magnitude less than, for example, the highly toxic carbamate insecticide aldicarb to red-winged blackbirds (Agelaius phoeniceus) and mallard ducks (Anas platyrhynchos) and are half that of carbofuran for the same species (5). As a class I “highly toxic” compound to Gyps vultures (LD 50 < 1000 μg kg -1 )(6), diclofenac poisoning has caused resident Gyps populations across the Indian subcontinent to collapse over the last 17 years. G. bengalensis, long-billed vulture (G. indicus), and slender-billed vulture (G. tenuirostris) are now listed as Critically Endangered (2000, G. bengalensis; 2002, G. indicus and G. tenuirostris), having previously been listed as Least Concern [International Union for Conservation of Nature (IUCN)] (7). In the 1980s, G. bengalensis was probably the commonest large bird of prey in the world (8), and at the beginning of the 1990s, Gyps in India were estimated to number 60 million individuals. Indian populations began to crash in the early to middle 1990s (9, 10), with G. bengalensis falling 99.9% between 1992 and 2007 at a mean annual decline rate of 43.9% during 2000-2007 (11). Similar trends for G. indicus and G. tenuirostris are reported, and these majestic species now sit on the brink of extinction. This is the first time that a common veterinary pharma- ceutical, thought to be of low toxicity toward numerous species, has had such a widespread, devastating effect at the population level on nontarget wildlife. Modeling suggests diclofenac alone is likely to be the major cause of the observed population collapse (12, 13), and two other species, Egyptian vulture (Neophron percnopterus, Endangered) and red- headed vulture (Sarcogyps calvus, Critically Endangered) are undergoing similar declines in the region due to an as yet unidentified cause (14). Also, despite the recognized toxicity of diclofenac toward the Gyps genus, its use is now increasing across Africa (15). This may endanger four more species, i.e, the cape vulture (G. coprotheres, vulnerable), Rueppell’s griffon vulture (G. rueppellii, Near Threatened), African white- backed vulture (G. africanus; Near Threatened), and griffon vulture (G. fulvus; Least Concern), whose populations are already under pressure from persecution, hunting, habitat loss, a decrease in wild ungulate food availability, and power line collisions (7, 16). In May 2006, the Drug Controller General (India) withdrew all licenses to manufacture diclofenac for veterinary use (17), and similar action was taken in Nepal and Pakistan. If wild Gyps populations are to recover or captive bred birds released successfully these bans must be highly effective. Further, there is currently a lack of data regarding the toxicity of many common NSAIDs toward avian scavengers, and concerns exist that NSAIDs other than diclofenac may pose comparable risks (18). It is therefore critical that as diclofenac is phased out, it is replaced with well-studied alternatives known to be of low toxicity. One such alternative, meloxicam, has been clearly identified (19, 20) but the risk remains that less well- examined NSAIDs could still emerge (21). Data regarding livestock carcass residues of NSAIDs other than diclofenac have not to date been presented in the literature. Many analytical procedures are available to detect single NSAIDs in differing matrices and to simultaneously analyze multiple NSAIDs in pure mixtures, surface and wastewater, serum, and urine (refs 22-26 represent ex- amples). There are however only two reports to our knowl- edge describing the analysis of multiple NSAID residues in tissues (27, 28). Although these describe method development * Corresponding author phone: +44 (0)1767 680551; fax: +44 (0)1767 685079; e-mail: Richard.Cuthbert@rspb.org.uk. † University of Aberdeen. ‡ Instituto de Investigacio ´n en Recursos Cinege ´ticos. § Bombay Natural History Society. | University of Cambridge. ⊥ Royal Society for the Protection of Birds. # Wildlife Institute of India. ∇ Wildfowl and Wetlands Trust. Environ. Sci. Technol. 2009, 43, 4561–4566 10.1021/es9002026 CCC: $40.75  2009 American Chemical Society VOL. 43, NO. 12, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4561 Published on Web 05/15/2009