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

MOHINI SAINI,ANIL SHARMA and ASIT DAS Centre for Wildlife Conservation, ; Green et al., ). Post-mortem examination of Gyps Management & Disease Surveillance, Indian Veterinary Research Institute, vultures killed by diclofenac poisoning in experiments showed Izatnagar, Uttar Pradesh, India extensive visceral gout and necrosis of kidney tissues similar to MANDAR D. KULKARNI,PARAG DEORI,SACHIN RANADE and ROHAN N. SHRINGARPURE Bombay Natural History Society Mumbai, India that seen in a high proportion of vultures found dead in the   † wild (Oaks et al., ;Shultzetal., ; Meteyer et al., RHYS E. GREEN (Corresponding author) Conservation Science Group,   Department of Zoology, University of Cambridge, Downing Street, ;Swanetal., a). Bans on the veterinary use of diclo- Cambridge, CB2 3EJ, UK. E-mail [email protected] fenac have been in force since  in India, Pakistan and *Also at: Wildlife Conservation Society, Goroka, Eastern Highlands Province, Nepal, and since  in Bangladesh. The safety to Gyps vul- Papua New Guinea † tures of meloxicam, an alternative veterinary NSAID, has Also at: RSPB Centre for Conservation Science, Royal Society for the Protection  of Birds, Sandy, Bedfordshire, UK been established experimentally (Swan et al., b; Swarup  Received  January . Revision requested  February . et al., ). In India the proportion of ungulate carcasses con- Accepted  March . taminated with diclofenac fell by about half within  years of

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

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. micron. The filtered extract was stored in LC vials at than G. indicus (Table ). Given that the prevalence of gout −°C until analysis. Samples collected up until June and diclofenac tended to be higher in G. bengalensis than in  were analysed for diclofenac only by LC–ESI/MS G. indicus when the species are compared during the same (liquid chromatography–electrospray ionization mass period (see above), ignoring the change over time in species spectrometry) using an Agilent  LC and D MS, fol- composition in the regression analysis would bias the esti- lowing methods in Taggart et al. (). The limit of quan- mated trend to be more positive (less negative) than it tification (LOQ) for this technique (back-calculated to wet should be. − tissue concentration) was . mg kg . Samples collected The proportion of adult vultures in the sample examined after June  were analysed for nine different veterinary was slightly higher after  than before (Table ), and this NSAIDs (carprofen, diclofenac, flunixin, ibuprofen, indo- would tend to bias the trend in the opposite direction. To metacin, ketoprofen, meloxicam, naproxen and nimesulide) allow for these differences we fitted multiple logistic regression that were selected based on their potential risk to avian spe- models with the main effects of species (G. himalayensis, cies, presence within pharmacies in India and likelihood of G. indicus and G. bengalensis) and age class (nestling, imma- entering the veterinary sector in the region (Taggart et al., ture [i.e. juvenile and sub-adult] and adult) each included as ; Cuthbert et al., ). They were analysed by LC– three-level factors in addition to time (collection year) as a ESI/MS in negative ion mode (utilizing a C column) follow- continuous variable. ing methods described in Taggart et al. (). LOQ values We excluded from the logistic regression models the re- − for these nine NSAIDs were .–. mg kg (see sults for the single specimens of A. monachus and G. fulvus Supplementary Table S in Taggart et al., ). The LOQ because the small sample size for these species did not allow − for diclofenac was the same (. mg kg ) in the analyses con- us to fit reliable statistical models of the effects of species dif- ducted before and after June .Ofthe carcasses for ferences. We also excluded results for two Gyps bengalensis which diclofenac measurements were made, values were avail- carcasses whose recovery was associated with a kite festival able for both liver and kidney for  carcasses, liver only for (see above). Carcasses whose age class was not recorded eight carcasses and kidney only for three 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

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

surveys of diclofenac concentrations in liver samples of un- expected proportion of deaths of adult G. bengalensis caused gulate carcasses available to vultures in India by Cuthbert by diclofenac in mid  based upon vulture necropsies and et al. (). We used the method given by Green et al. the estimate of the same quantity derived from the surveys of () to calculate the expected proportion of deaths of diclofenac in ungulate livers. To do this we generated lists of adult G. bengalensis caused by diclofenac in mid  , bootstrap estimates of the proportions of vultures with from this reduced mid  value of C. We obtained confi- diclofenac for each of the two methods. We aligned the two dence limits for this expected proportion from the bootstrap randomly-ordered lists, calculated the absolute difference confidence limits for the change in expected death rate per between values for each pair of bootstrap replicates and took meal given by Cuthbert et al. (; see their Table ). We the proportion of replicates in which the difference had the performed a significance test of the difference between the opposite sign to that observed in the real data to be the

http://journals.cambridge.org Downloaded: 27 May 2015 IP address: 194.35.219.98 Mortality of vultures 5 probability of observing by chance a difference as large as or correlation between the concentrations in liver and kidney larger than that obtained from the original calculations. (r = ., t = .,P=., Supplementary Fig. S). The arithmetic means of the concentrations of diclofenac in liver and kidney in this subset of individuals were similar − Results and not significantly different (liver, mean . mg kg , − − range .–. mg kg ; kidney, mean . mg kg , − Prevalence of visceral gout range .–. mg kg ; Wilcoxon signed ranks test,   Visceral gout was not present in either of the single P= . ). A. monachus or G. fulvus carcasses examined. In the other In the five vulture carcasses in which nimesulide was three species the overall proportions with gout were % for detected, levels were above the limit of quantification in  G. himalayensis (/), % for G. indicus (/) and % for both liver and kidney (Table ). There was a weak and non- G. bengalensis (/). significant positive correlation between the nimesulide concentrations in the liver and kidney (r = ., t = ., P=., Supplementary Fig. S). Four of the five carcasses Co-occurrence of visceral gout and NSAID residues in with measurable residues of nimesulide had visceral gout. The the liver and/or kidney carcass with nimesulide and no gout had low concentrations of nimesulide near to the limit of quantification in both tis- – During the period , when diclofenac was the only sues, whilst the four birds with gout had considerably higher NSAID being measured, diclofenac residues were present in concentrations in one or both tissues (Table ). In the two liver and/or kidney tissue in all Gyps vulture carcasses in vulture carcasses in which meloxicam was detected, it was which visceral gout was identified, and in none of the car- above the limit of quantification in both liver and kidney. In  casses with no gout (Table ). This perfect association be- one of these carcasses there was visceral gout, but the concen- tween diclofenac and visceral gout is highly significant tration of meloxicam was very low in both liver and kidney ,   – (Fisher exact test P . ). In the later period, whereas the concentration of nimesulide in both tissues was  , in which other NSAIDs were also assayed in addition very high. The other carcass with meloxicam had high con- to diclofenac, all Gyps vulture carcasses with diclofenac re- centrations of the drug in both liver and kidney but no sign sidues also had visceral gout and no carcasses without gout of visceral gout (Table ). Meloxicam was the only NSAID  had diclofenac. However, four of the carcasses with gout detected in this bird. did not have measurable levels of diclofenac, but did have residues of other NSAIDs (Table ). All four of these carcasses had nimesulide in both the liver and kidney and Changes over time in the prevalence of diclofenac and one of them had meloxicam, as well as nimesulide, in both visceral gout tissues. Of eight carcasses without gout, one had very low Logistic regression analysis of the trend in the prevalence of residues of nimesulide and one had residues of meloxicam diclofenac in liver and/or kidneys of vultures indicated a de-  – (Table ). Hence, during the perfect association cline that was close to statistical significance (Fig. a). In a between diclofenac and gout found in the earlier period be- multiple regression model with effects of species and age ac- came weaker, though it remained statistically significant counted for, the logarithm of the odds of a vulture carcass   (Fisher exact test P = . ). A logistic regression analysis having a measurable level of diclofenac declined by . of the  vulture carcasses with gout, in which the presence/ per year ( SE = ., t = ., one-tailed P = .). absence of diclofenac was the binary dependent variable and The proportion of adult G. bengalensis expected from the re- year of collection was the independent variable, suggested a gression model to have a measurable level of diclofenac fell tendency for the proportion of vulture carcasses with gout from % in the middle of , just before the diclofenac that had no diclofenac residues to increase over time, but ban, to % four years later in mid  (Fig. a). For     this trend was not statistically significant (t = . ,P= . ). G. indicus, the equivalent change was from  to %. Logistic regression analysis of the trend in the prevalence of Concentrations of NSAIDs in liver and kidney of vultures visceral gout indicated a non-significant decline at a slower rate than that for diclofenac (Fig. b). In a multiple regression In the  vulture carcasses for which diclofenac assays model with effects of species and age accounted for, the loga- were performed for both liver and kidney, diclofenac was rithm of the odds of a vulture carcass having gout declined by detected above the limit of quantification in both tissues . per year ( SE = ., t = ., one-tailed P = .). in all  cases in which it was detected in either tissue, and The proportion of adult G. bengalensis expected from the was below the limit of quantification in both tissues in the regression model to have gout fell from %inthemiddle remaining  cases. In the  cases with diclofenac above of  to % four years later in mid  (Fig. b). For the LOQ in both tissues, there was a significant positive G. indicus the equivalent change was 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. ()

http://journals.cambridge.org Downloaded: 27 May 2015 IP address: 194.35.219.98 Mortality of vultures 7 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. .. Http://www.iucnredlist.org oriental white-backed vultures (Gyps bengalensis). Journal of [accessed  November ]. Wildlife Diseases, , –. CRAWLEY, M.J. () The R Book. John Wiley & Sons, Chichester, NAIDOO, V., WOLTER, K., CROMARTY, D., DIEKMANN, M., UK. DUNCAN, N., MEHARG, A.A. et al. () Toxicity of non-steroidal CUNNINGHAM, A.A., PRAKASH, V., PAIN, D., GHALSASI, G.R., WELLS, anti-inflammatory drugs to Gyps vultures: a new threat from G.A.H., KOLTE, G.N. et al. () Indian vultures: victims of an ketoprofen. Biology Letters, , –. infectious disease epidemic? Animal Conservation, , –. OAKS, J.L., GILBERT, M., VIRANI, M.Z., WATSON, R.T., METEYER, C.U., CUTHBERT, R.J., DAVE, R., CHAKRABORTY, S.S., KUMAR, S., PRAKASH, RIDEOUT, B.A. et al. () Diclofenac residues as the cause of S., RANADE, S.P. & PRAKASH,V.() Assessing the ongoing threat vulture population decline in Pakistan. Nature, , –. from veterinary non-steroidal anti-inflammatory drugs to Critically PRAKASH, V., BISHWAKARMA, M.C., CHAUDHARY, A., CUTHBERT, R., Endangered Gyps vultures in India. Oryx, , –. DAVE, R., KULKARNI, M. et al. () The population decline of CUTHBERT, R., PAIN, D.J., GREEN, R.E., SWAN,G.&SWARUP,D. Gyps vultures in India and Nepal has slowed since veterinary use of () Comparative toxicity studies of NSAIDs in birds: a criticism diclofenac was banned. PLoS ONE, (), e. of Reddy et al. Environmental Toxicology and Pharmacology, , PRAKASH, V., GREEN, R.E., PAIN, D.J., RANADE, S.P., SARAVANAN, S., –. PRAKASH, N. et al. () Recent changes in populations of resident

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