Continuing Mortality of Vultures in India Associated with Illegal Veterinary Use of Diclofenac and a Potential Threat from Nimesulide

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Continuing mortality of vultures in India associated with illegal veterinary use of diclofenac and a potential threat from nimesulide

R ICHARD J. CUTHBERT,MARK A. TAGGART,MOHINI S AINI,ANIL S HARMA A SIT D AS,MANDAR D. KULKARNI,PARAG D EORI,SACHIN R ANADE R OHAN N. SHRINGARPURE,TOBY H. GALLIGAN and R HYS E. GREEN

Abstract The collapse of South Asia’s Gyps vulture popula- Veterinary use of nimesulide is a potential threat to the tions is attributable to the veterinary use of the non-steroidal recovery of vulture populations. anti-inflammatory drug (NSAID) diclofenac. Vultures died Keywords Diclofenac, environmental pollution, Gyps,me- after feeding on carcasses of recently-medicated animals. loxicam, nephrotoxicity, nimesulide, non-steroidal anti- The governments of India, Nepal and Pakistan banned the inflammatory drug, vulture veterinary use of diclofenac in . We analysed results of  necropsies and  NSAID assays of liver and/or kidney To view supplementary material for this article please visit for vultures of five species found dead in India between http://dx.doi.org/./SX  and . Visceral gout and diclofenac were detected in vultures from nine states and three species: Gyps bengalensis, Gyps indicus and Gyps himalayensis. Visceral gout Introduction was found in every vulture carcass in which a measurable level of diclofenac was detected. Meloxicam, an NSAID of opulations of three species of Gyps vultures endemic to low toxicity to vultures, was found in two vultures and ni- PSouth Asia fell by more than % between the early mesulide in five vultures. Nimesulide at elevated tissue s and  (Prakash et al., , , ), leading concentrations was associated with visceral gout in four of to their being categorized as Critically Endangered in the these cases, always without diclofenac, suggesting that nime- IUCN Red List (BirdLife International, ). Research has sulide may have similar toxic effects to those of diclofenac. established that veterinary use of diclofenac, a non-steroidal Residues of meloxicam on its own were never associated anti-inflammatory drug (NSAID), was the principal cause with visceral gout. The proportion of Gyps vultures found of this collapse in vulture numbers (Oaks et al., ;Green dead in the wild in India with measurable levels of diclofe- et al., , ;Shultzetal.,). Diclofenac is nephro- nac in their tissues showed a modest and non-significant toxic at low doses to all species of Gyps vultures tested decline since the ban on the veterinary use of diclofenac. (Oaks et al., ;Swanetal.,a). Residues of the drug The prevalence of visceral gout declined less, probably be- were found in carcasses of domesticated ungulates available cause some cases of visceral gout from  onwards were to vultures in India (Green et al., ; Taggart et al., , associated with nimesulide rather than diclofenac. ) and vultures were exposed when they consumed carcasses of ungulates treated shortly before death (Oaks et al., ; Green et al., ). Post-mortem examination of Gyps vultures killed by diclofenac poisoning in experiments showed RICHARD J. CUTHBERT* and TOBY H. GALLIGAN RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, Sandy, Bedfordshire, UK extensive visceral gout and necrosis of kidney tissues similar to

MARK A. TAGGART, Environmental Research Institute, University of the that seen in a high proportion of vultures found dead in the Highlands and Islands, Castle St, Thurso, UK wild (Oaks et al., ;Shultzetal.,; Meteyer et al.,

MOHINI SAINI,ANIL SHARMA and ASIT DAS Centre for Wildlife Conservation, ;Swanetal.,a). Bans on the veterinary use of diclo- Management & Disease Surveillance, Indian Veterinary Research Institute, fenac have been in force since  in India, Pakistan and Izatnagar, Uttar Pradesh, India Nepal, and since  in Bangladesh. The safety to Gyps vul- MANDAR D. KULKARNI,PARAG DEORI,SACHIN RANADE and ROHAN N. SHRINGARPURE Bombay Natural History Society Mumbai, India tures of meloxicam, an alternative veterinary NSAID, has  † been established experimentally (Swan et al., b; Swarup RHYS E. GREEN (Corresponding author) Conservation Science Group,  Department of Zoology, University of Cambridge, Downing Street, et al., ). In India the proportion of ungulate carcasses con- Cambridge, CB2 3EJ, UK. E-mail [email protected] taminated with diclofenac fell by about half within  years of *Also at: Wildlife Conservation Society, Goroka, Eastern Highlands Province, the introduction of the ban (Cuthbert et al., ). In asso- Papua New Guinea ciation with this decrease in diclofenac prevalence, the rate †Also at: RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, Sandy, Bedfordshire, UK of decline of Gyps vulture populations in India, Nepal and  Received  January . Revision requested  February . Pakistan has slowed (Jamshed et al., ; Prakash et al., Accepted  March . First published online  May . ), as has the decline in two other vulture species,

Oryx, 2016, 50(1), 104–112 © 2015 Fauna & Flora International doi:10.1017/S003060531500037X Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 02 Oct 2021 at 01:53:50, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S003060531500037X Mortality of vultures 105

red-headed vulture Sarcogyps calvus and Egyptian vulture carcasses collected in India for which data were reported by Neophron percnopterus, which may also be affected by Shultz et al. (). diclofenac (Galligan et al., ). Here we report the findings of necropsies of dead vultures collected in India during –, which includes Necropsies of vulture carcasses and assessment periods before and after the diclofenac ban in .We of visceral gout re-evaluate the previously observed perfect association of Detailed necropsy examinations were undertaken by trained visceral gout with diclofenac and other NSAIDs by compar- veterinarians and field biologists who followed a standard ing necropsy results with measurements of the concentra- protocol (Cunningham et al., ). This included external tions of nine NSAIDs in liver and/or kidney. We evaluate and internal visual examination for gross abnormalities, and changes over time in the prevalence of visceral gout and the collection of liver and/or kidney tissues for subsequent NSAID contamination in carcasses of vultures found dead NSAID residue analysis. Where possible, morbid tissues in the wild. fixed in % NBF were processed by a conventional technique to obtain  μm thick paraffin embedded sections (Luna, ). The sections stained with routine hematoxylin and eosin stain Methods were examined microscopically for pathological changes associated with nephrotoxicity. De Galantha stain was employed Collection of vulture carcasses for demonstration of urate crystals. Images of representative changes were documented. Samples of kidney and/or liver Carcasses of vultures were collected from July  to were removed and frozen for NSAID assays. April  in nine states of India (Assam, Gujarat, In some cases, when carcasses were found in remote lo- Haryana, Himachal Pradesh, Jharkhand, Madhya Pradesh, cations or where trained personnel were not available, full Maharashtra, Rajasthan and Uttarakhand), extending necropsies could not be performed and less detailed exam- from the western to the eastern extremes of the country. inations were conducted in the field. We consider these We report here only results for specimens for which the examinations, together with the detailed post mortems, suf- year of collection was known and results of necropsy, ficient for the detection of the presence or absence of vis- NSAID assay or both were available. A total of  vultures ceral gout based upon observation of white crystals of uric were examined at necropsy for visceral gout (one cinereous acid deposited on the surfaces of organs such as the liver (see vulture Aegypius monachus, one Eurasian griffon vulture Oaks et al.,  and Swan et al., a for illustrations). In Gyps fulvus, three Himalayan griffon vultures Gyps hima- reviewing the post-mortem reports, we found two cases of layensis,  long-billed vultures Gyps indicus and  oriental white deposits on internal organs, like those seen in gout, white-backed vultures Gyps bengalensis). Liver and/or kid- in which this was considered at the time to be possibly ney samples from  vultures were assayed for NSAIDs due to an unidentified fungal infection in one case and can- (one A. monachus, one G. fulvus, three G. himalayensis, didiasis (a specific fungal infection) in another. In both  G. indicus and  G. bengalensis). Carcasses of  vultures cases, fungal infection was not confirmed by microscopy. ( A. monachus,  G. fulvus,  G. himalayensis,  G. indicus and In the case of the bird with suggested candidiasis, subse-  G. bengalensis) were assessed for both gout and NSAIDs. quent histopathological examination of the kidney revealed No NSAID assays were available for seven G. indicus severe gout nephrosis. This suggested that white deposits and eight G. bengalensis with necropsy results, and one observed on macroscopic examination had been misidenti- G. bengalensis with an NSAID assay did not have a necropsy fied as fungal infection. We therefore judged that gout had result. Vulture carcasses were collected by staff of the Bombay been mistakenly identified as fungal disease in both cases Natural History Society and volunteers, local conservation and reassigned both as having visceral gout. NGOs and individuals. Two carcasses of G. bengalensis were obtained as a result of special efforts to collect and treat the large numbers of birds killed and injured by collisions with Measurement of NSAID residues kite strings during the annual kite-flying festival in the city of Ahmedabad (Gujarat), but the others were found Samples of frozen liver and kidney (c. . g) were thawed opportunistically, dead or dying in the field. After their and weighed (to ± mg) into new glass test tubes and homo- population decline became apparent, vultures in India were genized with  ml of HPLC grade acetonitrile. This mixture strictly protected under Schedule I of the Wildlife was then centrifuged at  g for  minutes and the super- Protection Act (), which requires permits before live or natant filtered using disposable PTFE syringe filter units of dead specimens can be collected. This restriction influenced . micron. The filtered extract was stored in LC vials at both the number of carcasses obtained and the distribution −°C until analysis. Samples collected up until June of collection localities. Our study included all  vulture  were analysed for diclofenac only by LC–ESI/MS

(liquid chromatography–electrospray ionization mass G. indicus when the species are compared during the same spectrometry) using an Agilent  LC and D MS, fol- period (see above), ignoring the change over time in species lowing methods in Taggart et al. (). The limit of composition in the regression analysis would bias the esti- quantification (LOQ) for this technique (back-calculated mated trend to be more positive (less negative) than it − to wet tissue concentration) was . mg kg . Samples should be. collected after June  were analysed for nine different The proportion of adult vultures in the sample examined veterinary NSAIDs (carprofen, diclofenac, flunixin, ibupro- was slightly higher after  than before (Table ), and this fen, indometacin, ketoprofen, meloxicam, naproxen and would tend to bias the trend in the opposite direction. To nimesulide) that were selected based on their potential allow for these differences we fitted multiple logistic regression risk to avian species, presence within pharmacies in India models with the main effects of species (G. himalayensis, and likelihood of entering the veterinary sector in the region G. indicus and G. bengalensis) and age class (nestling, imma- (Taggart et al., ; Cuthbert et al., ). They were ture [i.e. juvenile and sub-adult] and adult) each included as analysed by LC–ESI/MS in negative ion mode (utilizing a three-level factors in addition to time (collection year) as a C column) following methods described in Taggart et al. continuous variable. (). LOQ values for these nine NSAIDs were .–. We excluded from the logistic regression models the re- − mg kg (see Supplementary Table S in Taggart et al., sults for the single specimens of A. monachus and G. fulvus − ). The LOQ for diclofenac was the same (. mg kg ) because the small sample size for these species did not allow in the analyses conducted before and after June .Ofthe us to fit reliable statistical models of the effects of species dif-  carcasses for which diclofenac measurements were made, ferences. We also excluded results for two Gyps bengalensis values were available for both liver and kidney for  carcasses, carcasses whose recovery was associated with a kite festival liver only for eight carcasses and kidney only for three (see above). Carcasses whose age class was not recorded carcasses. were also excluded (six carcasses from the gout dataset and two carcasses from the diclofenac dataset). Statistical analysis We expected that the prevalence of diclofenac contamination and visceral gout would decline with increasing time The statistical significance of associations between the pres- during this period, based upon independent information on ence of gout and the detection of diclofenac residues in liver the downward trend in diclofenac contamination of ungu- and/or kidney was assessed using the Fisher exact test for a lates (Cuthbert et al., ). Hence, we used one-tailed  ×  contingency table (Siegel & Castellan, ). The two- t-tests in these analyses to assess the statistical significance tailed exact probability was calculated for the observed out- of trends over time. We plotted the expected proportions come under a null hypothesis of no association. from the models against time for adult G. bengalensis and We estimated trends over time in the prevalence of diclo- adult G. indicus. fenac residues in the liver and/or kidney and the prevalence of visceral gout in vultures by logistic regression analysis, Comparison of the decline in diclofenac prevalence in  (Crawley, ) with the presence/absence of diclofenac vulture carcasses with the decline expected from surveys contamination or the presence/absence of gout as binary of diclofenac in ungulate carcasses dependent variables and calendar year of collection as an independent variable. It was necessary to allow for species We calculated the expected probability of death caused by and age class in these analyses because previous studies diclofenac per vulture meal (C)inmid and the annual have indicated that the prevalence of both gout and diclofe- survival rate of adults in the absence of diclofenac using the nac contamination of Gyps vultures in the Indian subcon- simulation model of a vulture population described by tinent vary with those variables. Both prevalences tend Green et al. (). We obtained values of C and survival to be higher in G. bengalensis than in G. indicus (Green that gave a value for the annual rate of population decline et al., ; Shultz et al., ) and higher in adults than for G. bengalensis in India equal to the value observed at immatures (Gilbert et al., ; Green et al., ). For ex- that time (population multiplication rate λ = .; Green ample, in a large sample of G. bengalensis found dead in et al., ) and a value for the proportion of dead adult Pakistan, the prevalence of visceral gout increased progres- G. bengalensis with diclofenac in mid  equal to that sively with age, being % in nestlings, % in fledglings, % from the multiple logistic regression fitted to post-mortem in subadults and % in adults (Gilbert et al., ). data (see preceding section), which was .%. We then es- The proportions of carcasses of different Gyps species timated C for mid  by reducing the mid  estimate changed over time. For example, after  a larger propor- by %, which is the change in this parameter estimated for tion of the carcasses examined were of G. bengalensis rather the  years between mid  and mid  from the sur- than G. indicus (Table ). Given that the prevalence of gout veys of diclofenac concentrations in liver samples of ungu- and diclofenac tended to be higher in G. bengalensis than in late carcasses available to vultures in India by Cuthbert et al.

TABLE 1 Co-occurrence of visceral gout and residues of NSAIDs in carcasses of wild Gyps vultures collected in India during –. Results are shown separately for a period when assays were only performed for diclofenac (–) and a later period (–) when carprofen, flunixin, ibuprofen, indometacin, ketoprofen, meloxicam, naproxen and nimesulide were also assayed. For these additional drugs, only residues of nimesulide and meloxicam were detected. Numbers of nestlings, immatures, adults and birds of unknown age, respectively, are shown in brackets.

Nimesulide Species Gout Diclofenac Nimesulide Meloxicam & meloxicam No NSAID 2000–2004 G. fulvus Yes 0 0 No 0 1 (0,1,0,0) G. himalayensis Yes 0 0 No 0 1 (0,1,0,0) G. indicus Yes 6 (0,5,1,0) 0 No 0 3 (0,2,1,0) G. bengalensis Yes 7 (0,3,4,0) 0 No 0 4 (1,1,2,0) All species Yes 13 (0,8,5,0) 0 No 0 9 (1,5,3,0) 2005–2011 G. fulvus Yes 0 0 0 0 0 No 0 0 0 0 0 G. himalayensis Yes 1 (0,1,0,0) 0 0 0 0 No 0 0 0 0 1 (0,1,0,0) G. indicus Yes 0 0 0 0 0 No 0 0 0 0 1 (0,1,0,0) G. bengalensis Yes 11 (2,1,8,0) 3 (0,1,1,1) 0 1 (0,0,1,0) 0 No 0 1 (0,0,1,0) 1 (0,0,0,1) 0 4 (1,3,0,0) All species Yes 12 (2,2,8,0) 3 (0,1,1,1) 0 1 (0,0,1,0) 0 No 0 1 (0,0,1,0) 1 (0,0,0,1) 0 6 (1,5,0,0)

− TABLE 2 Concentrations (mg kg ) of nimesulide and meloxicam and the presence of visceral gout in all six carcasses (of G. bengalensis)in which either or both of these drugs was detected. Other NSAIDs (diclofenac, carprofen, flunixin, ibuprofen, indometacin, ketoprofen and naproxen) were assayed in all these birds but none was detected.

Collection Nimesulide* Meloxicam* Visceral Age class Stateyear Liver Kidney Liver Kidney gout? Adult Gujarat 2008 0.309 2.753 ,LOQ ,LOQ Yes Juvenile Gujarat 2010 0.297 0.014 ,LOQ ,LOQ Yes Adult Jharkhand 2010 0.010 0.014 ,LOQ ,LOQ No Unknown Gujarat 2010 ,LOQ ,LOQ 0.187 0.684 No Unknown Gujarat 2011 0.156 0.689 ,LOQ ,LOQ Yes Adult Gujarat 2011 0.573 1.459 0.019 0.028 Yes

*,LOQ, below the limit of quantification of the assay

(). We used the method given by Green et al. ()to the same quantity derived from the surveys of diclofenac in calculate the expected proportion of deaths of adult G. ben- ungulate livers. To do this we generated lists of , boot- galensis caused by diclofenac in mid  from this reduced strap estimates of the proportions of vultures with diclofenac mid  value of C. We obtained confidence limits for this for each of the two methods. We aligned the two expected proportion from the bootstrap confidence limits randomly-ordered lists, calculated the absolute difference for the change in expected death rate per meal given by between values for each pair of bootstrap replicates and took Cuthbert et al. (; see their Table ). We performed a sig- the proportion of replicates in which the difference had the nificance test of the difference between the expected propor- opposite sign to that observed in the real data to be the prob- tion of deaths of adult G. bengalensis caused by diclofenac in ability of observing by chance a difference as large as or larger mid  based upon vulture necropsies and the estimate of than that obtained from the original calculations.

Results arithmetic means of the concentrations of diclofenac in liver and kidney in this subset of individuals were similar − Prevalence of visceral gout and not significantly different (liver, mean . mg kg , − − range .–. mg kg ; kidney, mean . mg kg , − Visceral gout was not present in either of the single range .–. mg kg ; Wilcoxon signed ranks test, A. monachus or G. fulvus carcasses examined. In the other P=.).  three species the overall proportions with gout were % for In the five vulture carcasses in which nimesulide was       G. himalayensis ( / ), % for G. indicus ( / ) and % for detected, levels were above the limit of quantification in   G. bengalensis ( / ). both liver and kidney (Table ). There was a weak and non- significant positive correlation between the nimesulide concentrations in the liver and kidney (r = ., t = ., Co-occurrence of visceral gout and NSAID residues in P=., Supplementary Fig. S). Four of the five carcasses the liver and/or kidney with measurable residues of nimesulide had visceral gout. The During the period –, when diclofenac was the only carcass with nimesulide and no gout had low concentrations NSAID being measured, diclofenac residues were present in of nimesulide near to the limit of quantification in both tis- liver and/or kidney tissue in all Gyps vulture carcasses in sues, whilst the four birds with gout had considerably higher  which visceral gout was identified, and in none of the car- concentrations in one or both tissues (Table ). In the two casses with no gout (Table ). This perfect association be- vulture carcasses in which meloxicam was detected, it was tween diclofenac and visceral gout is highly significant above the limit of quantification in both liver and kidney. In (Fisher exact test P , .). In the later period, – one of these carcasses there was visceral gout, but the concen- , in which other NSAIDs were also assayed in addition tration of meloxicam was very low in both liver and kidney to diclofenac, all Gyps vulture carcasses with diclofenac re- whereas the concentration of nimesulide in both tissues was sidues also had visceral gout and no carcasses without gout very high. The other carcass with meloxicam had high con- had diclofenac. However, four of the  carcasses with gout centrations of the drug in both liver and kidney but no sign  did not have measurable levels of diclofenac, but did have of visceral gout (Table ). Meloxicam was the only NSAID residues of other NSAIDs (Table ). All four of these detected in this bird. carcasses had nimesulide in both the liver and kidney and one of them had meloxicam, as well as nimesulide, in both tissues. Of eight carcasses without gout, one had very Changes over time in the prevalence of diclofenac and low residues of nimesulide and one had residues of melox- visceral gout icam (Table ). Hence, during – the perfect association between diclofenac and gout found in the earlier Logistic regression analysis of the trend in the prevalence of period became weaker, though it remained statistically diclofenac in liver and/or kidneys of vultures indicated a de-  significant (Fisher exact test P = .). A logistic regres- cline that was close to statistical significance (Fig. a). In a sion analysis of the  vulture carcasses with gout, in multiple regression model with effects of species and age ac- which the presence/absence of diclofenac was the binary de- counted for, the logarithm of the odds of a vulture carcass   pendent variable and year of collection was the independent having a measurable level of diclofenac declined by .        variable, suggested a tendency for the proportion of vulture per year ( SE = . , t = . , one-tailed P = . ). carcasses with gout that had no diclofenac residues to in- The proportion of adult G. bengalensis expected from the re- crease over time, but this trend was not statistically signifi- gression model to have a measurable level of diclofenac fell from % in the middle of , just before the diclofenac cant (t = .,P=.). ban, to % four years later in mid  (Fig. a). For G. indicus, the equivalent change was from  to %. Concentrations of NSAIDs in liver and kidney of vultures Logistic regression analysis of the trend in the prevalence of visceral gout indicated a non-significant decline at a In the  vulture carcasses for which diclofenac assays slower rate than that for diclofenac (Fig. b). In a multiple were performed for both liver and kidney, diclofenac was regression model with effects of species and age accounted detected above the limit of quantification in both tissues for, the logarithm of the odds of a vulture carcass having in all  cases in which it was detected in either tissue, and gout declined by . per year ( SE = ., t = ., was below the limit of quantification in both tissues in the one-tailed P = .). The proportion of adult G. bengalensis remaining  cases. In the  cases with diclofenac above expected from the regression model to have gout fell from the LOQ in both tissues, there was a significant positive cor- % in the middle of  to % four years later in mid relation between the concentrations in liver and kidney (r =  (Fig. b). For G. indicus the equivalent change was ., t = .,P=., Supplementary Fig. S). The from  to %.

and mid  (see Table  of Cuthbert et al., ). This change in expected death rate per meal, when used in the vulture population model of Green et al. (; see Methods), gave an expected proportion of deaths of adult G. bengalensis caused by diclofenac in mid  of .% (% confidence limits .–.%). The estimated proportion of adult G. bengalensis carcasses with diclofenac in mid  derived from the logistic regression model of vulture necropsy results was .%(% confidence limits .– .%) whereas the same proportion derived from the surveys of diclofenac in ungulate carcasses was .%, if it is assumed in both cases that the proportion of vulture carcasses with diclofenac in mid  was .%(Fig. a). The decline in the proportion of vulture carcasses with diclofenac derived from vulture necropsies was smaller than that derived from diclofenac surveys of ungulate carcasses (.–.%cf..–.%, respectively). However, the difference between the estimates derived from the two methods does not approach statistical significance (bootstrap P = .).

Discussion

Our results indicate that diclofenac remained a significant cause of mortality for India’s vultures and that the drug has continued to kill birds long after the  regulations to pre- vent its veterinary use. The proportion of Gyps vultures found dead in the wild in India that had measurable levels of diclofenac in their tissues showed only a small and non-significant decline in the  years following the ban on the veterinary use of

FIG. 1 (a) Proportions of carcasses of wild adult Gyps bengalensis diclofenac covered by our study. The estimated size of the de- and G. indicus contaminated with diclofenac, in relation to the cline was broadly consistent with an independent estimate year of collection. Curves represent expected values from a based upon measurements of the change in the prevalence logistic regression model that included the main effects of and concentration of diclofenac in carcasses of domesticated species, age class and year. Symbols show the expected ungulates available to vultures (Cuthbert et al., ). proportions of adult G. bengalensis contaminated with diclofenac Continued mortality of vultures in India caused by diclofenac in mid  (circle) and mid  (triangle) from the data from vulture carcasses, and the expected proportion of adult after the ban is consistent with the continued availability of the G. bengalensis deaths caused by diclofenac in mid  (square) drug in pharmacies. Based upon surveys conducted between based upon the results of surveys of diclofenac contamination of November  and June  in eleven Indian states, carcasses of domesticated ungulates. Vertical lines are % Cuthbert et al. () reported that diclofenac formulated confidence limits. (b) Proportions of carcasses of wild adult for non-veterinary use was offered for sale for veterinary use G. bengalensis and G. indicus with visceral gout, in relation to in % of pharmacies that sold any type of NSAID. the year of collection. Curves represent expected values from a All vultures with measurable diclofenac in liver and/or kid- logistic regression model that included the main effects of ney had visceral gout and this association was highly statistic- species, age class and year. ally significant. Wild Asian Gyps vultures that died with visceral gout and with measurable diclofenac in their tissues in our study had similar concentrations of diclofenac in the kidney Comparison of the decline in diclofenac prevalence in to those that died in similar circumstances in the study of  vulture carcasses with the decline expected from surveys Oaks et al. ( ). Means and ranges were similar in our study of wild G. bengalensis and G. indicus carcasses from India of diclofenac in ungulate carcasses − − (mean = . mg kg , range = .–. mg kg ), The expected vulture death rate per meal estimated from the study of Oaks et al. () of wild G. bengalensis − surveys of diclofenac residues in liver samples from ungulate carcasses from Pakistan (mean = . mg kg , range = − carcasses declined by % in the  years between mid  .–. mg kg ) and the study of Oaks et al. ()

of captive G. bengalensis that died after experimental admin- of nimesulide in cattle is unlikely to be much more rapid istration of meat from water buffalo or goat given a veterinary than for diclofenac. To date, the liver has been the only − dose of diclofenac shortly before death (mean = . mg kg , organ routinely sampled in surveys of NSAIDs in ungulate − range = .–. mg kg ). We therefore consider that carcasses in India (Taggart et al., , ; Cuthbert et al., diclofenac was likely to have been the cause of death of all ). Nimesulide concentrations in ungulate livers may be of the vultures reported in the present study in which the unusually low relative to other tissues. Alternatively, the ex- drug was detected at above the limit of quantification. posure pathway for vultures may involve sources of carrion The estimated decline in the prevalence of visceral gout that have not been surveyed, such as poultry waste. These in Gyps vultures found dead in India was smaller than that questions should be addressed through further field sam- for diclofenac prevalence, and did not approach statistical pling and experiments to study tissue distribution of nime- significance. This may be because cases of visceral gout sulide in ungulates. that were not associated with the presence of diclofenac in Another NSAID found in vulture carcasses was meloxi- liver and/or kidney tissues began to occur from  on- cam, which is the only drug established by experiment to be wards. Four G. bengalensis with gout collected during of low toxicity to Gyps vultures and other scavenging birds – had no measurable diclofenac, but all had high (Swan et al., b; Cuthbert et al., ; Swarup et al., concentrations of nimesulide in the liver and/or kidney. ). One vulture carcass with visceral gout had a low One of these carcasses also contained meloxicam residues concentration of meloxicam and a high concentration of but at concentrations so low that involvement of meloxicam nimesulide in liver and kidney tissues, as described above. in the death of the bird is unlikely. Another G. bengalensis Another vulture carcass had a high concentration of melox- carcass had a very low concentration (near LOQ) of nime- icam in liver and kidney and no trace of any other NSAID, sulide in both liver and kidney and no visceral gout. Hence, and it had no evidence of gout. The cause of death of this wild vultures in India are being exposed to nimesulide. bird was recorded to be diphtheroid enteritis. Therefore Exposure at a high level is associated with visceral gout our findings are consistent with the safety to vultures of vet- and death. Reddy et al. () suggested that nimesulide erinary use of meloxicam indicated by experimental studies is likely to be safe for vultures, based upon a comparison on captive birds. However, carcasses of wild vultures should of the toxicity of nimesulide and diclofenac to domestic continue to be monitored to check that this is the case under fowl Gallus domesticus. However, there are large differences field conditions. in the toxicity of NSAIDs among bird species (Cuthbert The NSAIDs carprofen, flunixin, ibuprofen, indometa- et al., ), so the safety of nimesulide in domestic fowl cin, ketoprofen and naproxen were not detected in any of cannot be taken to indicate its safety to distantly-related the  vulture carcasses assayed for them. In experiments vultures. on the African vultures Gyps coprotheres and Gyps afri- Nimesulide is legally approved for veterinary use in India canus, ketoprofen has been shown to be nephrotoxic at and was offered for sale for this purpose in % of pharma- doses that are likely to be encountered by wild vultures cies that sold any type of NSAID in surveys conducted (Naidoo et al., ). A wild G. fulvus was recently found during – in eleven Indian states (Cuthbert et al., dead in Spain with visceral gout associated with high levels ). The prevalence of nimesulide in pharmacies was par- of flunixin residues in liver and kidney tissues (Zorrilla et al., ticularly high in Gujarat, where it was offered for sale for ). This augments previous evidence from surveys of the veterinary use in % of shops visited ( out of  shops; therapeutic use of NSAIDs on captive vultures in zoos, re- R.J. Cuthbert, unpubl. data). It is therefore notable that all habilitation centres and other collections (Cuthbert et al., four vultures with both nimesulide residues and visceral ) that flunixin may be nephrotoxic to Gyps vultures. gout were collected from Gujarat. However, there is little evi- Cuthbert et al. () also found evidence of nephrotoxicity dence of nimesulide in carcasses of domesticated ungulates in of carprofen in one Gyps vulture. Experiments on captive India. Taggart et al. () did not find any nimesulide resi- vultures to measure the toxicity of flunixin and carprofen dues in liver samples from , ungulate carcasses collected have not yet been conducted. between April and December . After including further Taggart et al. () found that some liver samples from samples collected up until July  (total n = ,), only domesticated ungulates available to vultures in India during − three ungulate carcasses with low (,. mg kg ) levels of April–December  contained residues of ibuprofen and − nimesulide were found (. mg kg ; M.A. Taggart, unpubl. ketoprofen, but flunixin was not detected (Table  in data). Taggart et al., ). In surveys conducted during – Comparison of the time to maximum plasma concentra-  in  Indian states by Cuthbert et al. (), flunixin  tion (tmax) and elimination half-life (T/) of nimesulide in was being offered for sale for veterinary use in % of phar- cattle following intramuscular injection shows that its macies that sold any type of NSAID, ibuprofen in % and pharmacokinetics are broadly similar to those of diclofenac ketoprofen in %. The fact that we did not find residues of (EMEA, ; Mahapatra et al., ). Hence, the elimination these drugs in the sample of  vulture carcasses assayed for

them may reflect the small size of our sample rather than their CUTHBERT, R., PARRY-JONES, J., GREEN, R.E. & PAIN, D.J. () true absence from vulture carcasses. There is a % probability NSAIDs and scavenging birds: potential impacts beyond Asia’s  – of our survey finding no contamination with any of these critically endangered vultures. Biology Letters, , . CUTHBERT, R.J., TAGGART, M.A., PRAKASH, V., CHAKRABORTY, S.S., drugs, even if the true prevalence of the drugs in vulture   DEORI, P., GALLIGAN,T.etal.( ) Avian scavengers and the carcasses had been as high as %((–.) = .). threat from veterinary pharmaceuticals. Philosophical Transactions Our study highlights the continuing threat to Asia’s vul- of the Royal Society B, , http://dx.doi.org/./rstb.. tures from veterinary use of diclofenac and identifies a new EMEA (THE EUROPEAN AGENCY FOR THE EVALUATION OF  potential threat from nimesulide. Toxicity testing of nime- MEDICINAL PRODUCTS)( ) Committee for Veterinary Medicinal Products, Diclofenac Summary Report EMEA/MRL// sulide on Gyps vultures is needed to establish whether or not -FINAL September . EMEA, London, UK. the compound is nephrotoxic. Illegal misuse in the veterin- GALLIGAN, T.H., AMANO, T., PRAKASH, V.M., KULKARNI, M., ary sector of diclofenac products labelled ‘for human use SHRINGARPURE, R., PRAKASH, N. et al. () Have population only’ is the cause of much of the ongoing threat from that declines in Egyptian vulture and red-headed vulture in India slowed  drug and further action to eliminate this has been recom- since the ban on veterinary diclofenac? Bird Conservation International, , –. mended (Cuthbert et al., ). Identification of NSAIDs GILBERT, M., VIRANI, M.Z., WATSON, R.T., OAKS, J.L., BENSON, P.C., that are effective on cattle and also safe for vultures at the KHAN, A.A. et al. () Breeding and mortality of oriental maximum likelihood exposure level would be valuable for white-backed vulture Gyps bengalensis in Punjab Province, Pakistan. vulture conservation, but so far the only known example Bird Conservation International, , –. of such a drug is meloxicam. GREEN, R.E., NEWTON, I., SHULTZ, S., CUNNINGHAM, A.A., GILBERT, M., PAIN, D.J. & PRAKASH,V.() Diclofenac poisoning as a cause of vulture population declines across the Indian subcontinent. Journal of Applied Ecology, , –. Acknowledgements GREEN, R.E., TAGGART, M.A., DAS, D., PAIN, D.J., SASHI KUMAR, C., CUNNINGHAM, A.A. & CUTHBERT,R.() Collapse of Asian We thank the Ministry of Environment and Forests and the vulture populations: risk of mortality from residues of the veterinary drug diclofenac in carcasses of treated cattle. Journal of Applied Chief Wildlife Wardens for the states of Assam, Gujarat, Ecology, , –. Haryana, Himachal Pradesh, Jharkhand, Madhya Pradesh, GREEN, R.E., TAGGART, M.A., SENACHA, K.R., RAGHAVAN, B., Maharashtra, Rajasthan and Uttarakhand for permissions, PAIN, D.J., JHALA,Y.&CUTHBERT,R.() Rate of decline of and Kartik Shastri, Ruchi Dave, S. Saravaran, Satya Prakash, the oriental white-backed vulture population in India estimated Sumantha Ghosh, Puja Basu, Bhrigu Neog and Vibhu Pra- from a survey of diclofenac residues in carcasses of ungulates. PLoS ONE, (), e. kash for collecting vulture carcasses. Andrew Cunningham, JAMSHED, M., CHAUDHRY, I., OGADA, D.L., MALIK, R.N., Romain Pizzi, Yedra Feltrer, Devojit Das, Percy Avari, VIRANI, M.Z. & GIOVANNI, M.D. () First evidence that Jeherul Islam and Devendra Podadhe performed the nec- populations of the Critically Endangered long-billed vulture Gyps ropsies. Ian Newton provided useful comments. We grate- indicus in Pakistan have increased following the ban of the toxic fully acknowledge financial support and assistance for the veterinary drug diclofenac in South Asia. Bird Conservation International, , –. project from the Director, Indian Veterinary Research  ’ LUNA, L.G. ( ) Manual of Histologic Staining Methods of the Armed Institute, the UK Government s Darwin Initiative and the Forces Institute of Pathology, rd edition. W.B. Saunders, Royal Society for the Protection of Birds. Philadelphia, USA. MAHAPATRA, L., SAHOO, G.R., PANDA, M.K. & PARIJA,S.() Pharmacokinetic profile of nimesulide in bovine calves. Journal of Bioequivalence & Bioavailability, , –. References METEYER, C.U., RIDEOUT, B.A., GILBERT, M., SHIVAPRASAD, H.L. & OAKS, J.L. () Pathology and proposed pathophysiology of BIRDLIFE INTERNATIONAL () Gyps bengalensis. In The IUCN Red diclofenac poisoning in free-living and experimentally exposed List of Threatened Species v. .. 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