green et al 2006 diclofenac residues in livestock

Journal of Applied Ecology

2006

43

949ndash956

copy 2006 The Authors Journal compilation copy 2006 British Ecological Society

Blackwell Publishing Ltd

PRIORITY CONTRIBUTION

Collapse of Asian vulture populations risk of mortality from residues of the veterinary drug diclofenac in carcasses of treated cattle

RHYS E GREENdagger MARK A TAGGARTDagger DEVOJIT DASsect DEBORAH J PAIN C SASHI KUMARpara ANDREW A CUNNINGHAM and RICHARD CUTHBERT

Royal Society for the Protection of Birds The Lodge Sandy Bedfordshire SG19 2 DL UK

dagger

Conservation Science Group Department of Zoology University of Cambridge Downing Street Cambridge CB2 3EJ UK

Dagger

School of Biological Sciences Department of Plant and Soil Science University of Aberdeen St Machar Drive Aberdeen AB24 3UU UK

sect

Bombay Natural History Society Hornbill House S B Singh Road Mumbai 400 023 India

para

Sreenilayam Pattanur PO Edayannur Kannur District Kerala 670 595 India and

Institute of Zoology Zoological Society of London Regentrsquos Park London NW1 4RY UK

Summary

1

The populations of three species of South Asian vultures (

Gyps bengalensis

Gyps indicus

and

Gyps tenuirostris

) have declined rapidly within the last decade and all are now criticallyendangered Veterinary use of the non-steroidal anti-inflammatory drug diclofenacappears to be a major cause of the declines Vultures are likely to be exposed to the drugwhen they feed on carcasses of livestock that were treated with diclofenac before death

2

We measured the concentration of diclofenac in the tissues of treated Indian humpedand European cattle (

Bos indicus

and

Bos taurus

) in relation to the interval betweendosing and death We used a dosendashresponse model to assess the risk posed to wildvultures if they feed on carcasses of treated livestock

3

Diclofenac concentrations in fat intestine kidney and liver were considerably higher thanthose in muscle but concentrations in the first four tissues initially depleted more rapidly(half-life 6ndash8 h) with time since the last injection of the drug compared with muscle (half-life 15 h) Depletion rates became much slower in all tissues 25ndash98 h after the last injection

4

Diclofenac concentration averaged across the carcass was enough to causeappreciable mortality (gt 10 of birds per meal) if oriental white-backed vultures

G bengalensis

were to take a large meal from the carcass of an animal that was given itslast dose of the drug within a day or two before death Vultures that feed selectively ontissues with high concentrations of the drug such as kidney liver and intestine wouldbe exposed to a higher risk and for longer after dosing

5

Synthesis and applications

The tissues of cattle treated with diclofenac are a hazard towild vultures that feed on an animal that dies within a few days after treatment Intestinekidney and liver have the highest diclofenac concentrations but the concentrationaveraged across all the edible tissues of the carcass is also hazardous Withdrawal ofdiclofenac from veterinary use on animals whose carcasses may become available toscavenging vultures is recommended In

ex situ

and

in situ

conservation projectsvultures should be fed on carcasses of animals that are known not to have been treatedwith diclofenac in the week before death

Key-words

bird population decline dosendashresponse relationship endangered speciesnon-steroidal anti-inflammatory drug pharmacokinetics renal failure visceral gout


(2006)

43

949ndash956doi 101111j1365-2664200601225x

Correspondence Dr R E Green Department of Zoology Downing Street Cambridge CB2 3EJ UK (e-mail reg29hermescamacuk)

950

R E Green

et al



43

949ndash956

Introduction

Three species of vultures endemic to South Asia theoriental white-backed vulture

Gyps bengalensis

(Gmelin) long-billed vulture

Gyps indicus

(Scopoli)and slender-billed vulture

Gyps tenuirostris

Gray are athigh risk of global extinction and are listed as criticallyendangered because of rapid population declines withinthe last decade in the Indian subcontinent (Prakash

et al

2003 Green

et al

2004 IUCN 2004) There isstrong evidence that veterinary use of the non-steroidalanti-inflammatory drug (NSAID) diclofenac is a majorcause of the population declines (Oaks

et al

2004Green

et al

2004 Shultz

et al

2004) Diclofenac iswidely available as a veterinary drug in the Indiansubcontinent where it is used to treat inflammationfever and pain associated with disease and injury indomestic livestock Vultures are believed to be exposedto the drug when they feed on carcasses of livestock thatwere treated with diclofenac before death Followingexperimental exposure to tissues of ungulates treatedwith a veterinary dose of diclofenac

G bengalensis

died within a few days from kidney failure Extensivevisceral gout was evident at post-mortem examination(Oaks

et al

2004) Rapid death associated with visceralgout has also been observed in two other vulturespecies African white-backed vulture

Gyps africanus

Salvadori and Eurasian griffon vulture

Gyps fulvus

(Hablitzl) treated experimentally with diclofenac (Swan

et al

2006a) Hence susceptibility to diclofenac poi-soning seems to be widespread in the genus so

Gindicus

and

G tenuirostris

are also likely to be susceptibleVisceral gout and diclofenac residues in tissues havealso been found in most carcasses of wild

Gyps

vulturesfrom across India Pakistan and Nepal examined sincethe decline began (Oaks

et al

2004 Shultz

et al

2004)The proportion of carcasses of

G bengalensis

and

Gindicus

found dead in the wild with signs of diclofenacpoisoning is consistent with this being the main andpossibly the only cause of the vulture decline (Green

et al

2004)No detailed studies have been made previously of

the exposure of vultures to diclofenac and the risk ofdeath posed by each exposure In this paper wereport on the concentrations of diclofenac in thetissues of experimentally treated cattle and assessthe risk to wild vultures if they were to feed on them inrelation to the time between treatment and the deathof the treated animal For this we used data fromthree experiments One was carried out to estimate theexposure of wild vultures to diclofenac administeredto cattle using the standard daily dose of the drugrecommended for veterinary use in India (experi-ment 1) The other experiments (experiments 2 and3) were undertaken by a pharmaceutical manu-facturing company to establish maximum residuelimits as required by the European Agency for theEvaluation of Medicinal Products (EMEA LondonUK)

Methods

1

Ten female Indian humped cattle

Bos indicus

L 1ndash7 yearsold (mean 3middot8 years) with an average mass of 202 kg(range 30ndash300 kg) were housed on a farm in KeralaIndia After keeping the animals for at least 2 weeks toensure that they were free of any previous NSAID theymight have received each was given one injection of adiclofenac sodium formulation (25000 mg L

minus

1

3-D VetIntas India) into the neck muscle at a dose of 1 mg kg

minus

1

live weight At 21 h (20middot7ndash22middot5 h) 46 h (45middot8ndash46middot8 h) 71 h(71middot0ndash71middot3 h) 167 h (166middot4ndash168middot8 h) and 334 h (333middot1ndash334middot3 h) after the injection respectively two animalswere slaughtered Samples of tissue weighing 25ndash30 gwere taken from intestine kidney and liver and fromintercostal or gluteal muscle from the opposite side ofthe body from the injection site Samples were frozenat

minus

20

deg

C

2

Sixteen young European cattle

Bos taurus

L (eight malesand eight females) 0middot6ndash2middot0 years old (mean 1middot1 years)and weighing 140ndash280 kg (mean 187middot5 kg) were housedat a facility in France Each animal received an inject-able proprietary veterinary formulation of diclofenacsodium (50 000 mg L

minus

1

) injected into the neck muscle ata dose of 2middot5 mg kg

minus

1

live weight on each of 6 consecutivedays At 2ndash4 h 12 h 24 h and 144 h after the last injectionrespectively four animals were slaughtered by exsan-guination after stunning Samples of tissue weighing 2ndash5 g were taken from perirenal fat kidney liver andtriceps muscle A summary of this experiment has beenreported previously in item 18 of EMEA (2004)

3

Eight mature dairy cows

B taurus

aged 2middot5ndash10middot0 yearsold (mean 5middot3 years) and weighing 555ndash715 kg (mean638middot8 kg) were housed at a facility in France Each animalreceived an injectable proprietary veterinary formulationof diclofenac sodium (50 000 mg L

minus

1

) injected into theneck muscle at a dose of 2middot5 mg kg

minus

1

live weight on eachof 6 consecutive days Four animals were slaughtered byexsanguination after stunning at 96 h and another fourat 176 h after the last injection Samples of perirenalfat (10 g) kidney (20 g) liver (20 g) and triceps muscle(20 g) were taken A summary of this experiment hasbeen reported previously in item 18 of EMEA (2004)

Diclofenac concentrations in tissues were measuredusing high-performance liquid chromatography (HPLC)methods calibrated against a known standard concen-tration of the drug In experiment 1 diclofenac wasextracted from 0middot5 g of tissue by homogenization with

951

Vulture mortality from veterinary diclofenac



43

949ndash956

2 mL of HPLC-grade acetonitrile which was thencentrifuged at 1000

g

for 5 min Analysis of diclofenactissue concentrations was undertaken using a validatedHPLC method with electrospray ionization mass spec-trometry (ESIMS) detection following the methods ofOaks

et al

(2004) The Agilent 1100 series instrument(1946D) (Agilent Technologies UK Cheadle RoyalBusiness Park Stockport SK8 3GR UK) was calibratedusing six diclofenac sodium salt (Sigma-Aldrich D6899New Road Gillingham Dorset SP8 4XT UK) standardsranging from 0middot005 to 1 mg L

minus

1

The calibration waslinear across this range with an

r

2

value of gt 0middot999 Thelimit of quantification (LOQ) was 0middot01 mg kg

minus

1

In experiments 2 and 3 diclofenac was extracted using

a multistep process For fat aliquots of 0middot5ndash1middot0 g oftissue were homogenized in 8 mL of dichloromethaneFor kidney liver and muscle aliquots of 0middot5ndash1middot0 g oftissue were homogenized in 4 mL of ethanol Quantita-tive determination of diclofenac was performed using aHewlett Packard HP 1050 series system Chromato-graphy was carried out on a column packed with a C18stationary phase The mobile phase was a mixture ofacetonitrilesodium acetate 0middot1

methanol (23860vv) and was used at a flow rate of 0middot7 mL min

minus

1

Acoulometric detection method was used The LOQ was0middot005 mg kg

minus

1

in experiments 2 and 3 in all tissues exceptfat in experiment 3 for which the LOQ was 0middot01 mg kg

minus

1

Inspection of the data indicated that the concentrationof diclofenac in tissue declined approximately expo-nentially after the last injection but that the rate ofdecline slowed abruptly after a certain time which wecalled the transition time (Fig 1) We decided to modelthe diclofenac concentration

d

as a function of timesince the last injection

t

using the piece-wise relationship

d

= exp(

a

ij

+

b

i

1

t

) for

t

lt

t

i

and

d

= exp(

a

ij

+

b

i

1

t

i

+

b

i

2

(

t ndash t

i

)) for

t

gt

t

i

where

a

ij

is a constant specific tothe

i

th tissue and

j

th experiment

b

i

1

is the exponentialrate of decline of diclofenac concentration in the

i

thtissue between the last injection and transition time

t

i

after which the decline rate changes to

b

i

2

Deviationsfrom expected values were assumed to be log-normallydistributed with residual variance s

i

2

specific to each tissueThere were insufficient data to estimate decline rates

and transition times for a given tissue separately for eachof three experiments so only the

a

parameters were treatedas specific to both tissue and experiment The

b

and

t

parameters were assumed to differ among tissues butto be the same for a given tissue in each experiment Inpractice this gave a reasonable fit to the data (Fig 1)

Parameter values were estimated by a quasi-Newtonmaximum-likelihood (M-L) method using the

module of

503 A M-L procedure was preferred toordinary linear least-squares regression after log trans-formation of

d

because some observed concentrations

were below the LOQ If least-squares linear regressionhad been used these observations would have had to beassigned an arbitrary concentration lower than theLOQ so that logarithms could be taken The choice ofthis value would affect the resulting parameter estimatesOmitting the lt LOQ observations would also be unsatis-factory as it would bias the parameter estimates There-fore the presence of observations lt LOQ was handledin the M-L procedure by incorporating into the modelleft-censoring of

d

at the LOQ value appropriate to agiven experiment and tissue (Kalbfleisch 1979)

As a measure of the goodness-of-fit of the models wecalculated expected values from them and then obtainedthe Pearson correlation coefficient

r

between observedand expected diclofenac concentrations for each tissueData for which the observed value was lt LOQ wereexcluded from this calculation

We wished to calculate the concentration of diclofenacaveraged over all parts of the carcass of a cow or domes-ticated water buffalo

Bubalus bubalis

(L) available tovultures as food To do this we needed to know theproportion of the edible mass of the entire carcass thatis made up of each tissue The composition of typicalcarcasses of

Bos taurus

Bos indicus

and

Bubalus bubalis

was determined from the scientific literature Ideally wewould have determined composition separately foreach species from samples of animals representative ofthose available dead as food for vultures in India Inpractice the information available did not allow thisand it was necessary to assume that the same meanvalues could be used for the three species

Initially we expressed the mass of each body componentas a percentage of the animalrsquos live weight The mass of theliver is 1middot3ndash1middot5 of live weight for

Bos taurus

(Budras ampHabel 2003) and 1middot5 for

Bos indicus

(Kumar Ghosh1998) We used 1middot5 as the typical value The mass of thekidneys is 0middot25 of live weight in

Bos taurus

accordingto Budras amp Habel (2003) and 0middot55 in

Bos indicus

Kumar Ghosh (1998) Using the relative mass of thekidneys and liver from table 14 of Grubh (1974) andthe value for liver given above we estimated that themass of the kidneys is 0middot41 of live weight in Bubalusbubalis The mean of these three estimates is 0middot40

The mass of all internal organs (offal) of Bos taurusis given as 14 of live weight by the Competition Com-mission (1985) After subtracting the estimates of themass of the liver and kidneys given above this leaves 12middot1of live weight comprised by the other internal organs

The percentage of muscle present in carcasses dressedfor meat preparation (ie with the head feet hide bloodalimentary tract and contents removed) was taken tobe 33 of live weight for Bos taurus (Callow 1962) and37middot1 for Bubalus bubalis (Charles amp Johnson 1972)giving a mean of 35middot0 We estimated the mean mass ofedible tissue assumed to be mostly muscle on heads of

952R E Green et al

copy 2006 The Authors Journal compilation copy 2006 British Ecological Society Journal of Applied Ecology 43 949ndash956

Bos indicus and Bubalus bubalis from data in table 14 ofGrubh (1974) as 2middot0 of live weight Combining these twoestimates gave total muscle mass as 37middot1 of live weight

The percentage of fat present in carcasses dressedfor meat preparation averaged across Bos taurusBos indicus and Bubalus bubalis is 6middot4 of live weight(Charles amp Johnson 1972) For Bos taurus the mass offat other than that in the dressed carcass is 8 of liveweight (Competition Commission 1985) giving a totalfat content of 14middot4 of live weight The remaining bodycomponents the hide and the blood were taken to be7 and 3 of live weight respectively (CompetitionCommission 1985)

We took the total mass of edible tissue available tovultures on a typical carcass to be the sum of thesecomponents The hide of cattle and buffaloes is oftenremoved for leather production soon after death so

we assumed arbitrarily that the hide was only availableto vultures on half of carcasses After conversion topercentages of total edible tissue the masses of thedifferent body components are liver 2middot1 kidneys0middot6 alimentary tract and other offal 16middot8 muscle51middot5 fat 20middot0 hide 4middot9 and blood 4middot2 We tookthe diclofenac concentration measured in samples ofintestine to apply to the whole alimentary tract and tooffal other than liver and kidney and the concentrationin samples of muscle to apply also to hide and blood

The average diclofenac concentration in the edibleparts of the entire carcass was calculated separately for

Fig 1 Concentration of diclofenac in tissues of cattleslaughtered at different times after administration of the finalinjection of a course of the drug Symbols and lines on eachpanel represent results and models from experiment 1 withBos indicus (diamonds solid line) experiment 2 with youngBos taurus (squares dashed line) and experiment 3 withmature dairy cows Bos taurus (triangles dotted line) Eachpoint is the datum for a single animal Fitted piece-wise modelsare shown in which concentration declines exponentially withtime For each tissue the initial rapid rate of decline changesto a slower rate after a certain interval The timing of thischange and the decline rates were modelled as being the samefor a given tissue for all trials but the intercepts were allowedto vary among trials (Table 1)

953Vulture mortality from veterinary diclofenac


experiments 1 and 2 We considered it inappropriateto perform this calculation for experiment 3 becausethe first animals were slaughtered after the transitiontime For each experiment and each hour after the lastinjection we multiplied the diclofenac concentrationfor each tissue obtained from the fitted model by theproportion of the edible mass of the entire carcass thatis made up of each tissue (see above) We then summedthese products to obtain the weighted mean diclofenacconcentration for the entire carcass We did not havemeasurements of diclofenac concentration for fat inexperiment 1 or for intestine in experiment 2 soexperiment-specific a values were not available to use inthe calculations for these tissue experiment combina-tions To overcome this we calculated the means foreach of the two experiments of the a values for the threetissues kidney liver and muscle that were measured inboth To estimate the a value for fat in experiment 1 wetook the a value for fat in experiment 2 added the meanfor the three tissues from experiment 1 and subtractedthe mean from experiment 2 To estimate the a value forintestine in experiment 2 we took the a value for intestine inexperiment 1 added the mean for the three tissues fromexperiment 2 and subtracted the mean from experiment 1

G B E N G A L E N S I S -

Following Swan et al (2006b) we assumed that a wildvulture would ingest 1middot023 kg of various tissues from thecarcass of a treated animal in a short time This quantityof food would sustain a free-living G bengalensis for3 days We assumed that the average concentration ofdiclofenac in the food was the same as the average forall edible parts of the carcass obtained from the fittedmodel as described above This concentration wasmultiplied by 1middot023 and the product was divided by4middot75 (the average mass in kg of G bengalensis del Hoyo

Elliott amp Sargatal 1994) to give the ingested dose in mgkgminus1 vulture body weight We then used dosendashresponsemodels fitted by probit analysis by Swan et al (2006a)to experimental data for captive G bengalensis fromOaks et al (2004) which estimated the relationshipbetween the rate of diclofenac-induced mortality andthe dose of diclofenac ingested We used two versionsof the model which were fitted with and without datafrom an outlier a vulture that died with visceral goutafter apparently receiving a very low dose of diclofenac(Swan et al 2006a)

Results

Diclofenac concentration declined rapidly (half-life 6ndash8 h) in fat intestine kidney and liver up to the transitiontime (25ndash46 h) after which it declined more slowly (half-life 35ndash57 h Fig 1 and Table 1) The concentration inmuscle declined more slowly than in other tissues (half-life 15 h) and the transition occurred later (98 h) Themodels fitted the data reasonably well for all five tissues(Fig 1 and Table 1) There was a non-significant tendency(Pearson r3 = 0middot687 P = 0middot20) for those tissues with along half-life in the period before the transition to alsohave a relatively long half-life after the transition

In all three experiments diclofenac concentrations inkidney and liver were considerably higher than those inmuscle (Fig 1) Concentrations in intestine approachedthose in kidney and liver and were higher than those inmuscle Concentrations in fat were intermediate Fora given tissue diclofenac concentrations tended to behigher in experiments 2 and 3 than in experiment 1(Fig 1) Hence the estimated average diclofenacconcentration in the entire carcass was considerably

Table 1 Estimates of the parameters of piece-wise models relating tissue concentrations of diclofenac in mg kgminus1 wet weight tothe time in hours since the last injection Details of the models are given in the Methods aij is an intercept specific to the ith tissueand jth experiment bi1 is the exponential rate of decline of diclofenac concentration in the ith tissue between the last injection andthe transition time ti in hours bi2 is the rate of decline after the transition time and si is the residual standard deviation Half-lifein hours was calculated as loge(0middot5)b for the periods before and after the transition Values of aij in italics are for experimentndashtissuecombinations for which observations were not available and were calculated using the mean of a values for kidney liver andmuscle (see Methods) Also shown is the Pearson correlation coefficient r between observed and modelled concentrations

Parameter

Parameter estimates

Fat Intestine Kidney Liver Muscle

ai1 ndash0middot7077 1middot4120 1middot1095 0middot5490 minus2middot2125ai2 0middot1192 2middot2389 1middot4048 1middot2441 minus0middot7222ai3 0middot5202 ndash 2middot1658 1middot6468 minus0middot5331bi1 minus0middot09916 minus0middot11538 minus0middot08775 minus0middot08347 minus0middot04684bi2 minus0middot01982 minus0middot01208 minus0middot01554 minus0middot01435 minus0middot01021ti 25middot19 41middot76 45middot83 42middot73 98middot34si 0middot6760 0middot3256 0middot4551 0middot3858 0middot7326Half-life1 6middot99 6middot01 7middot90 8middot30 14middot80Half-life2 34middot97 57middot37 44middot60 48middot30 67middot89r 0middot646 0middot997 0middot895 0middot979 0middot698

954R E Green et al


higher at all times after the last injection for experiment2 than experiment 1 (Fig 2)


The average diclofenac concentrations in tissues of thecattle experimentally treated with the drug were suffi-cient to cause appreciable mortality in vultures that fedon the carcass of an animal that died within 1ndash4 daysof the last treatment (Fig 3) Vultures that ingested theaverage concentration across all tissues would receive asufficient dose to kill gt 10 of birds if a treated cow haddied within 33 h (experiment 1) or 59 h (experiment 2)of the last injection These results are for the dosendashresponse model that includes the datum from an outlier(see the Methods) If the version of the model thatexcludes this datum is used then the equivalent timesare 9 h (experiment 1) and 19 h (experiment 2)

Vultures that fed exclusively on tissues with higherthan average concentrations of diclofenac would beat risk over a much longer interval after treatment Forexample if birds were to feed only on liver they wouldreceive a sufficient dose to kill gt 10 of them if the treated

cow had died within 56 h (experiment 1) or 105 h(experiment 2) of the last injection (model includingthe outlier) If the dosendashresponse model that excludesthe outlying datum is used the equivalent times are19 h (experiment 1) and 28 h (experiment 2)

Discussion

Our study demonstrates that diclofenac concentrationsin the tissues of treated cattle decline rapidly with timeafter the last injection However enough diclofenacremains to cause appreciable mortality (gt 10) if birdswere to take a large meal from the carcass of an animalthat was given its last dose of the drug within a few daysof death although the relationship of mortality to thetime since injection was affected by the version of thedosendashresponse model used Lower mortality rates werecalculated if a vulture that died with visceral gout afterapparently ingesting a very small dose of diclofenacwas excluded when fitting the model However forreasons given by Swan et al (2006a) it is unclearwhether the results for this bird should be included inthe calculation or not Hence we present calculationsbased upon both versions

Fig 2 Concentration of diclofenac in various cattle tissues inrelation to the time between administration of the final injectionof a course of the drug and slaughter Models are the same asthose presented in Fig 1 The thin lines on each panel representresults for different tissues The thick line shows the estimateddiclofenac concentration averaged over all parts of a carcassthat are available to vultures (see text) The upper panel showsresults from experiment 1 with Bos indicus and the lower panelshows results from experiment 2 with young Bos taurus

Fig 3 Proportion of Gyps bengalensis estimated to be killedby diclofenac in relation to time between administration of thefinal injection of a course of the drug and the death of thetreated cow (see text) Results are shown separately for a dosendashresponse model fitted to all data from the experiments of Oakset al (2004) (solid line) and after excluding an outlier (dottedline) The upper panel shows results from experiment 1 withBos indicus and the lower panel shows results from experiment2 with young Bos taurus



The treatment regimes used in our experiments arelikely to be broadly comparable with practice in the Indiansubcontinent In India the recommended veterinarycourse of diclofenac for cattle and buffaloes is 1middot0 mg kgminus1

on each of 3 consecutive days This is the dose we used inexperiment 1 although only one injection was administeredrather than three Given the short half-life of diclofenacin cattle tissue giving a course of three injections at 24-hintervals would probably have elevated tissue concentra-tions by a modest amount In Pakistan the recommendedcourse is 2middot5 mg kgminus1 on each of 3 consecutive days Thisdaily dose level was used in experiments 2 and 3 althoughinjections were given for 6 rather than 3 days It seemslikely that larger doses of diclofenac than recommendedare sometimes used in practice in India The highestconcentrations of diclofenac found in liver from live-stock carcasses sampled in the field in India were up tothree times the maximum we found in liver in theseexperiments (M Taggart manuscript in preparation)

The average concentration of diclofenac in the entireedible carcass is likely to be affected by the number of dosesand particularly the daily dose level The concentration washigher in experiments 2 and 3 in which 2middot5 mg kgminus1 wasused than in experiment 1 in which the dose was 1middot0 mgkgminus1 In experiment 2 the ratio of the diclofenac concen-tration in the entire carcass calculated over the first 24 hafter the last injection was 2middot58 times that for experiment1 a very similar ratio to that of the dose levels used Theestimated concentration of diclofenac in the entire ediblecarcass immediately after the last injection (calculated asfor the intercepts in Fig 2) was about two-thirds of theamount of diclofenac injected into the cattle per kilogramof edible tissue in both experiments (experiment 1 post-injection carcass concentration 0middot91 mg kgminus1 cf 1middot39 mgkgminus1 drug injected experiment 2 carcass concentration2middot19 mg kgminus1 cf 3middot47 mg kgminus1 drug injected given that 72of the live weight consists of edible tissues) However itshould be noted that several other things differed betweenthe experiments such as the species of cattle the numberof injections given and the laboratory where the diclofenacassays were performed so the difference in tissue con-centration cannot be attributed unambiguously to thedifference in the amount given per dose

The concentrations of diclofenac we measured intissues of cattle in our experiments are broadly similarto results reported by Oaks et al (2004) for a Bubalusbubalis that was slaughtered 4 h after receiving the lastof three daily injections of diclofenac at 2middot5 mg kgminus1Diclofenac concentrations in the kidney liver and muscleof this animal were 5middot7 1middot5 and 0middot76 mg kgminus1 respectivelycompared with values of 2middot87 2middot48 and 0middot40 mg kgminus1 forthe same tissues of young Bos taurus estimated 4 h afterthe last treatment from the results of experiment 2When expressed as normal standard deviates using theresidual standard deviation from the model the valuesfor Bubalus bubalis are +1middot51 minus1middot31 and +0middot87 relativeto the modelled values for young Bos taurus Hence thedifferences are well within the expected range Howeverthe values observed for the Bubalus bubalis are high

relative to the modelled values for Bos indicus fromexperiment 1 (2middot14 1middot24 and 0middot09 mg kgminus1 or +2middot16+0middot49 and +2middot90 as standard normal deviates) Thebetter agreement of the Bubalus bubalis data with theexpected values from experiment 2 than experiment 1may be because the Bubalus bubalis and the Bos taurusin experiment 2 both received the same daily dose ofdiclofenac (2middot5 mg kgminus1) whereas the Bos indicus inexperiment 1 received a much lower dose (1middot0 mg kgminus1)

Our findings are of practical value for efforts toprevent the extinction of critically endangered vulturespecies in two main ways First they extend the experi-mental results of Oaks et al (2004) by measuring theperiod after treatment for which contamination of cattletissues causes vulture mortality This new informationis being used to develop a more realistic version of themodel described by Green et al (2004) of the relation-ship between the prevalence of diclofenac in ungulatecarcasses and vulture population trends From this itshould be possible to ascertain more accurately fromfield surveys of diclofenac residues in cattle and buffalocarcasses whether a sufficient proportion is contaminatedwith diclofenac to account for the observed vulturepopulation declines Better means of interpreting suchsurveys will be essential for monitoring the effectivenessof future action taken to remove diclofenac from thefood supply of vultures such as government measuresto restrict veterinary use of diclofenac and encourageits replacement by meloxicam (Swan et al 2006b)Secondly the findings are useful for the husbandry ofcaptive vultures being held for captive breeding andfuture reintroduction projects and the supplementaryfeeding of wild vultures at lsquovulture restaurantsrsquo which isintended to divert them from feeding on contaminatedtissue Our results indicate that these birds are likely tobe safe from diclofenac poisoning if they are fed oncarcasses of cattle that are known not to have been treatedwith diclofenac within about a week before death

Acknowledgements

We gratefully acknowledge Fatro SpA (Italy) forpermission to use data from the experiments they con-ducted to establish maximum residue limits for diclofenac(experiments 2 and 3) and thank Beata Truszkowska forproviding the raw data and details of the experimentalmethods We thank Vibhu Prakash and Susanne Shultzfor help in planning and setting up experiment 1 Weare grateful to Ian Newton Simon Thirgood and twoanonymous referees for useful criticisms of a previousversion The research is part of a programme co-fundedby the UK governmentrsquos Darwin Initiative for theSurvival of Species the Royal Society for the Protec-tion of Birds and the Zoological Society of London

References

Budras K-D amp Habel RE (2003) Bovine Anatomy AnIllustrated Text Schluumltersche Hanover Germany

956R E Green et al


Callow EH (1962) The relationship between the weight of atissue in a single joint and the total weight of the tissue in aside of beef Animal Production 4 37ndash46

Charles DD amp Johnson ER (1972) Carcass composition ofthe water buffalo (Bubalus bubalis) Australian Journal ofAgricultural Research 23 905ndash911

Competition Commission (1985) Animal Waste A Report onthe Supply of Animal Waste in Great Britain CompetitionCommission London UK

EMEA (2004) The European Agency for the Evaluation ofMedicinal Products Committee for Veterinary MedicinalProducts Diclofenac Summary Report EMEAMRL88503-FINAL EMEA London UK

Green RE Newton I Shultz S Cunningham AAGilbert M Pain DJ amp Prakash V (2004) Diclofenacpoisoning as a cause of vulture population declines acrossthe Indian subcontinent Journal of Applied Ecology 41793ndash800

Grubh RB (1974) The birds of Gir Forest (the ecology andbehaviour of vultures in Gir Forest) PhD Thesis Universityof Bombay Bombay India

del Hoyo J Elliott A amp Sargatal J (1994) Handbook of theBirds of the World 2 New World Vultures to GuineafowlLynx Edicions Barcelona Spain

IUCN (2004) IUCN Red list of Threatened Species httpwwwiucnredlistorg accessed 5 December 2005 IUCNGland Switzerland

Kalbfleisch JG (1979) Probability and Statistical InferenceVol II Springer-Verlag New York NY

Kumar Ghosh R (1998) Primary Veterinary Anatomy 2ndedn Current Books International Calcutta India

Oaks JL Gilbert M Virani MZ Watson RT

Meteyer CU Rideout BA Shivasprasad HLAhmed S Chaudry MJI Arshad M Mahmood SAli A amp Khan AA (2004) Diclofenac residues as thecause of vulture population declines in Pakistan Nature427 630ndash633

Prakash V Pain DJ Cunningham AA Donald PFPrakash N Verma A Gargi R Sivakumar S ampRahmani AR (2003) Catastrophic collapse of Indianwhite-backed Gyps bengalensis and long-billed Gyps indicusvulture populations Biological Conservation 109 381ndash390

Shultz S Baral HS Charman S Cunningham AADas D Ghalsasi GR Goudar MS Green RE Jones ANighot P Pain DJ amp Prakash V (2004) Diclofenacpoisoning is widespread in declining vulture populationsacross the Indian subcontinent Proceedings of the RoyalSociety of London B (Supplement) 271 S458ndashS460 DOI101098Rsbl20040223

Swan GE Cuthbert R Quevedo M Green RE Pain DJBartels P Cunningham AA Duncan N Meharg AAOaks JL Parry-Jones J Shultz S Taggart MAVerdoorn G amp Wolter K (2006a) Toxicity of diclofenac toGyps vultures Biology Letters 2 279ndash282

Swan G Naidoo V Cuthbert R Green RE Pain DJSwarup D Prakash V Taggart M Bekker L Das DDiekmann J Diekmann M Killian E Meharg APatra RC Saini M amp Wolter K (2006b) Removing thethreat of diclofenac to critically endangered Asian vultures2006 Public Library of Science Biology 4 396ndash402

Received 18 May 2006 final copy received 21 June 2006 Editor Simon Thirgood

950

R E Green

et al



43

949ndash956

Introduction

Three species of vultures endemic to South Asia theoriental white-backed vulture

Gyps bengalensis

(Gmelin) long-billed vulture

Gyps indicus

(Scopoli)and slender-billed vulture

Gyps tenuirostris

Gray are athigh risk of global extinction and are listed as criticallyendangered because of rapid population declines withinthe last decade in the Indian subcontinent (Prakash

et al

2003 Green

et al

2004 IUCN 2004) There isstrong evidence that veterinary use of the non-steroidalanti-inflammatory drug (NSAID) diclofenac is a majorcause of the population declines (Oaks

et al

2004Green

et al

2004 Shultz

et al

2004) Diclofenac iswidely available as a veterinary drug in the Indiansubcontinent where it is used to treat inflammationfever and pain associated with disease and injury indomestic livestock Vultures are believed to be exposedto the drug when they feed on carcasses of livestock thatwere treated with diclofenac before death Followingexperimental exposure to tissues of ungulates treatedwith a veterinary dose of diclofenac

G bengalensis

died within a few days from kidney failure Extensivevisceral gout was evident at post-mortem examination(Oaks

et al

2004) Rapid death associated with visceralgout has also been observed in two other vulturespecies African white-backed vulture

Gyps africanus

Salvadori and Eurasian griffon vulture

Gyps fulvus

(Hablitzl) treated experimentally with diclofenac (Swan

et al

2006a) Hence susceptibility to diclofenac poi-soning seems to be widespread in the genus so

Gindicus

and

G tenuirostris

are also likely to be susceptibleVisceral gout and diclofenac residues in tissues havealso been found in most carcasses of wild

Gyps

vulturesfrom across India Pakistan and Nepal examined sincethe decline began (Oaks

et al

2004 Shultz

et al

2004)The proportion of carcasses of

G bengalensis

and

Gindicus

found dead in the wild with signs of diclofenacpoisoning is consistent with this being the main andpossibly the only cause of the vulture decline (Green

et al

2004)No detailed studies have been made previously of

the exposure of vultures to diclofenac and the risk ofdeath posed by each exposure In this paper wereport on the concentrations of diclofenac in thetissues of experimentally treated cattle and assessthe risk to wild vultures if they were to feed on them inrelation to the time between treatment and the deathof the treated animal For this we used data fromthree experiments One was carried out to estimate theexposure of wild vultures to diclofenac administeredto cattle using the standard daily dose of the drugrecommended for veterinary use in India (experi-ment 1) The other experiments (experiments 2 and3) were undertaken by a pharmaceutical manu-facturing company to establish maximum residuelimits as required by the European Agency for theEvaluation of Medicinal Products (EMEA LondonUK)

Methods

1

Ten female Indian humped cattle

Bos indicus

L 1ndash7 yearsold (mean 3middot8 years) with an average mass of 202 kg(range 30ndash300 kg) were housed on a farm in KeralaIndia After keeping the animals for at least 2 weeks toensure that they were free of any previous NSAID theymight have received each was given one injection of adiclofenac sodium formulation (25000 mg L

minus

1

3-D VetIntas India) into the neck muscle at a dose of 1 mg kg

minus

1

live weight At 21 h (20middot7ndash22middot5 h) 46 h (45middot8ndash46middot8 h) 71 h(71middot0ndash71middot3 h) 167 h (166middot4ndash168middot8 h) and 334 h (333middot1ndash334middot3 h) after the injection respectively two animalswere slaughtered Samples of tissue weighing 25ndash30 gwere taken from intestine kidney and liver and fromintercostal or gluteal muscle from the opposite side ofthe body from the injection site Samples were frozenat

minus

20

deg

C

2

Sixteen young European cattle

Bos taurus

L (eight malesand eight females) 0middot6ndash2middot0 years old (mean 1middot1 years)and weighing 140ndash280 kg (mean 187middot5 kg) were housedat a facility in France Each animal received an inject-able proprietary veterinary formulation of diclofenacsodium (50 000 mg L

minus

1

) injected into the neck muscle ata dose of 2middot5 mg kg

minus

1

live weight on each of 6 consecutivedays At 2ndash4 h 12 h 24 h and 144 h after the last injectionrespectively four animals were slaughtered by exsan-guination after stunning Samples of tissue weighing 2ndash5 g were taken from perirenal fat kidney liver andtriceps muscle A summary of this experiment has beenreported previously in item 18 of EMEA (2004)

3

Eight mature dairy cows

B taurus

aged 2middot5ndash10middot0 yearsold (mean 5middot3 years) and weighing 555ndash715 kg (mean638middot8 kg) were housed at a facility in France Each animalreceived an injectable proprietary veterinary formulationof diclofenac sodium (50 000 mg L

minus

1

) injected into theneck muscle at a dose of 2middot5 mg kg

minus

1

live weight on eachof 6 consecutive days Four animals were slaughtered byexsanguination after stunning at 96 h and another fourat 176 h after the last injection Samples of perirenalfat (10 g) kidney (20 g) liver (20 g) and triceps muscle(20 g) were taken A summary of this experiment hasbeen reported previously in item 18 of EMEA (2004)

Diclofenac concentrations in tissues were measuredusing high-performance liquid chromatography (HPLC)methods calibrated against a known standard concen-tration of the drug In experiment 1 diclofenac wasextracted from 0middot5 g of tissue by homogenization with

951




43

949ndash956


g


et al


minus

1


r

2


minus

1




minus

1


minus

1


minus

1


d


t


d

= exp(

a

ij

+

b

i

1

t

) for

t

lt

t

i

and

d

= exp(

a

ij

+

b

i

1

t

i

+

b

i

2

(

t ndash t

i

)) for

t

gt

t

i

where

a

ij


i

th tissue and

j

th experiment

b

i

1


i


t

i


b

i

2


i

2



a


b

and

t



module of


d



d



r



Bubalus bubalis


Bos taurus

Bos indicus

and

Bubalus bubalis



Bos taurus


Bos indicus


Bos taurus


Bos indicus




952R E Green et al














Results




Parameter

Parameter estimates



954R E Green et al







Discussion












Acknowledgements


References


956R E Green et al



















951




43

949ndash956


g


et al


minus

1


r

2


minus

1




minus

1


minus

1


minus

1


d


t


d

= exp(

a

ij

+

b

i

1

t

) for

t

lt

t

i

and

d

= exp(

a

ij

+

b

i

1

t

i

+

b

i

2

(

t ndash t

i

)) for

t

gt

t

i

where

a

ij


i

th tissue and

j

th experiment

b

i

1


i


t

i


b

i

2


i

2



a


b

and

t



module of


d



d



r



Bubalus bubalis


Bos taurus

Bos indicus

and

Bubalus bubalis



Bos taurus


Bos indicus


Bos taurus


Bos indicus




952R E Green et al














Results




Parameter

Parameter estimates



954R E Green et al







Discussion












Acknowledgements


References


956R E Green et al



















952R E Green et al














Results




Parameter

Parameter estimates



954R E Green et al







Discussion












Acknowledgements


References


956R E Green et al

























Results




Parameter

Parameter estimates



954R E Green et al







Discussion












Acknowledgements


References


956R E Green et al



















954R E Green et al







Discussion












Acknowledgements


References


956R E Green et al



























Acknowledgements


References


956R E Green et al



















956R E Green et al



















green et al 2006 diclofenac residues in livestock

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