Toxicity of Diclofenac to Gyps Vultures

Toxicity of diclofenac to Gyps vultures
Gerry E. Swan1, Richard Cuthbert2, Miguel Quevedo3, Rhys E. Green2&4, Deborah J.
Pain2&*, Paul Bartels5, Andrew A. Cunningham6, Neil Duncan1, Andrew A. Meharg7,
J. Lindsay Oaks8, Jemima Parry-Jones9, Susanne Shultz2&10, Mark A. Taggart7,
Gerhard Verdoorn11, Kerri Wolter12
1Department of Paraclinical Sciences, Faculty of Veterinary Science , University of
Pretoria, Onderstepoort, 0110, South Africa
2Royal Society for the Protection of Birds, The Lodge, Sandy, Bedfordshire SG19 2DL,
UK
3Veterinary Department, ZooBotánico Jerez, c/ Taxdirt s/n, Jerez 11404, Cádiz, Spain
4Conservation Biology Group, Department of Zoology, University of Cambridge CB2
3EJ, UK
5Wildlife Biological Resource Centre, National Zoological Gardens of SA, Pretoria,
South Africa
6Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1
4RY
7School of Biological Sciences, Department of Plant and Soil Science, University of
Aberdeen, St Machar Drive, Aberdeen, AB24 3UU.
8Department of Veterinary Microbiology and Pathology, Washington State
University, Pullman, WA 99164-7040
9International Center for Birds of Prey, 4872 Sewee Rd, Awendaw, Charleston,
SC29429 USA
10Current address: School of Biological Sciences, University of Liverpool, Liverpool
L69 7ZB.
11Plot 22, De Wildt Endangered Wildlife Trust, South Africa and currently Birdlife
South Africa, PO Box 515, Randburg2125, South Africa
12Vulture Unit, DeWildt Cheetah and Wildlife Trust, PO Box 1756, Hartbeespoort
0216
*Corresponding author: debbie.pain@rspb.org.uk
Summary
Three endemic vulture species Gyps bengalensis, G. indicus and G. tenuirostris are
Critically Endangered following dramatic declines in South Asia resulting from
exposure to diclofenac, a veterinary drug present in the livestock carcasses that they
scavenge. Diclofenac is widely used globally and could present a risk to Gyps species
from other regions. In this study, we test the toxicity of diclofenac to a Eurasian (Gyps
fulvus) and an African (G. africanus) species, neither of which is threatened. A dose
of 0.8mg kg-1 of diclofenac was highly toxic to both species, indicating that they are at
least as sensitive to diclofenac as G. bengalensis, for which we estimate an LD50 of
0.1-0.2 mg kg-1. We suggest that diclofenac is likely to be toxic to all eight Gyps
species, and that G. africanus, which is phylogenetically close to G. bengalensis,
would be a suitable surrogate for the safety testing of alternative drugs to diclofenac.
Keywords: diclofenac, vultures, toxicity, Gyps, NSAID
Short Title: Diclofenac toxicity to Gyps
Word count: 2,519 words (including summary, acknowledgements, references and
figure legends)
1. INTRODUCTION
Three species of vultures endemic to South Asia are in grave danger of extinction
and are now listed as Critically Endangered (IUCN 2004). Populations of Oriental
white-backed (Gyps bengalensis), long-billed (G. indicus) and slender-billed vultures
(G. tenuirostris) have declined by more than 95% since the early 1990s (Prakash et al.
2003, Green et al. 2004). Mortality caused by diclofenac, a non-steroidal antiinflammatory
drug (NSAID), is the main cause of the observed population declines
(Oaks et al. 2004; Shultz et al. 2004; Green et al. 2004). Diclofenac is a widely available
veterinary drug in south Asia, where it is used to treat domestic livestock. Vultures
are exposed to the drug when they consume carcasses of animals that were treated
with diclofenac shortly before death. Vultures die from kidney failure within days of
exposure to diclofenac-contaminated tissues, with post-mortem findings of extensive
visceral gout (Oaks et al. 2004). Identical findings have been recorded in carcasses of
wild G. bengalensis and G. indicus across the Indian sub-continent (Oaks et al. 2004;
Shultz et al. 2004). To date, diclofenac has been identified as a risk for three species of
vultures in the Indian sub-continent, but diclofenac, as well as other NSAIDs, may
pose a danger to five other Gyps vultures found in Asia, Europe and Africa.
Consequently, we conducted toxicity testing on Gyps africanus and G. fulvus; two
species of Least Concern (IUCN 2004).
2. MATERIALS AND METHODS
(a) Median lethal dose
Experimental results from a previous study (Oaks et al. 2004) were used to estimate the median lethal dose (LD50)
of diclofenac to G. bengalensis. In these experiments, vultures were either administered diclofenac orally (at doses
of 2.5 mg kg–1 and 0.25 mg kg–1) or fed tissues from goats (Capra aegagrus hircus) or buffaloes (Bubalus bubalis)
treated with diclofenac a few hours before slaughter. The LD50 was estimated by probit analysis; the probability of
death during the experiment being modelled in relation to the logarithm (base 10) of the dose of diclofenac
administered (mg kg–1 vulture body weight), as a cumulative normal distribution function with parameters m and s,
the mean and standard deviation of the logarithm of the lethal dose respectively. Estimation was by a quasi-newton
maximum-likelihood method using SYSTAT 5.01. The estimate of m and its asymptotic 95% confidence limits
were back-transformed to give the LD50 and its confidence interval. Inspection of the results indicated an outlier
(Electronic Appendix); a bird that apparently received a very low dose of diclofenac, but died of gout.
Consequently, we present results both with and without this bird.
(b) Toxicity testing
To minimise the number of birds needed for toxicity testing, the fitted probit model was used to
determine the probability of killing a vulture at differing doses. Results from the model (including the
outlier) indicate that the probability of killing an individual G. bengalensis given 0.8 mg kg–1 diclofenac is
0.8676. If two birds are dosed, the binomial probability that neither of them will die is (1-0.8676)2 =
0.0175. Excluding the outlier, the probability of death from this dose is 0.9284, and the probability that
neither of the two vultures would be killed is 0.0051. Consequently, treating two vultures is sufficient to
determine whether the toxicity of diclofenac to another species is similar to that for G. bengalensis.
Injured or non-releasable captive vultures, in relatively good body condition, were used for the
toxicity trials (Electronic Appendix). G. africanus and G. fulvus were provided by the de Wildt Cheetah
and Wildlife Trust (South Africa) and Zoobotánico Jerez (Spain) respectively. Two G. africanus were
randomly allocated to each of diclofenac-treated and control groups; three G. fulvus were allocated to
each of diclofenac-treated and control groups. Diclofenac sodium was administered by oral gavage at a
dose of 0.8 mg kg–1. Control birds were sham-treated with sterilized water (i.e. administered sterilized
water by oral gavage). Body mass, feeding behaviour, signs of toxicity, and time to first signs of toxicity
and mortality was recorded for all birds. 2.5 ml of blood was taken to quantify uric acid, albumin, and
diclofenac concentrations, and creatinine kinase, and serum alanine transferase (ALT) activity 24 h prior
to or immediately before dosing (for G. africanus and G. fulvus respectively), and at 4, 12, and 24 h after
dosing. Only one blood sample was taken from sham-treated G. fulvus. A post-mortem examination was
carried out on each vulture that died or was euthanased (including one sham-treated G. africanus),
recording all gross external and internal abnormalities. Histopathological examinations were conducted
on major tissues. Liver and kidney samples were collected from all dead birds and stored at -70°C.
Analysis of diclofenac tissue residues and plasma concentrations was undertaken at the University of
Aberdeen using a validated LC-MS method following the methods of Oaks et al. (2004). The studies
were approved by the University of Pretoria Animal Use and Care Committee (V026/04), South Africa,
and the Consejería de Medio Ambiente, Junta de Andalucía (Regional Environmental Administration
Office), Spain.
3. RESULTS
(a) Median lethal dose
The maximum-likelihood estimate of the logarithm of the LD50 (m) for G. bengalensis
was -1.011, which, after back-transformation, is equivalent to an LD50 of 0.098 mg kg-1
vulture body weight (95% CI 0.027–0.351 mg kg-1). The maximum-likelihood estimate
of parameter s was 0.820 (with asymptotic 95% CI of 0.238–0.1401). Excluding the
outlier, the maximum-likelihood estimate of m is –0.6486, equivalent to an LD50 of
0.225 mg kg-1 vulture bw (95% CI 0.117–0.432 mg kg-1) and the estimate of s was 0.377
(95% CI 0.135–0.618). There was no indication of a difference in the dose-response
relationship between the two methods of administration (likelihood ratio test, χ2(2) =
0.52, P > 0.75), although small sample sizes limited the statistical power of this test.
(b) Toxicity of diclofenac
All diclofenac-treated G. africanus (n=2) and G. fulvus (n=3) died within 2 days of
treatment. All controls (n=5) survived the experimental period and exhibited no
abnormal behaviour. At 24 hours post-treatment, four diclofenac-treated birds
showed lethargy and different degrees of neck-drooping. Subsequently, these signs
of toxicity increased in intensity. Death occurred 39 and 42 hours post-treatment in
G. africanus and after 28 and 35 hours in two G. fulvus. The third treated G fulvus
exhibited no signs of toxicity until it was found dead 48 hours after treatment. Postmortem
examination revealed extensive visceral gout in all diclofenac-treated birds
(Electronic Appendix). Histological examination revealed significant lesions in the
kidneys, liver and spleen with extensive uric acid crystal deposition. No control birds
showed any signs of toxicity during the experiment. A post-mortem conducted on
one control G. africanus revealed no gout.
There were large increases after treatment in plasma concentrations of uric
acid and alanine transferase (ALT) in both diclofenac-treated G. africanus (figure 1)
and significant differences between the response to treatment of diclofenac-treated
and sham-treated control birds (treatment*sampling time interaction, uric acid F3,6 =
11.82, P = 0.006, ALT F3,6 = 25.43, P = 0.001). There were no statistically significant
differences (P> 0.30) between diclofenac-treated and control G. africanus for any of
the other blood parameters measured (Electronic Appendix). All three diclofenactreated
Gyps fulvus showed elevation of plasma uric acid concentration, but there
was no clear pattern of response for ALT (figure 1). Plasma uric acid levels at 24
hours after diclofenac treatment were correlated with the dose administered for G.
bengalensis alone (figure 2; Spearman correlation coefficient, rS = 0.940, one-tailed P =
0.011) and also when data for G. bengalensis, G. fulvus and G. africanus were combined
(rS = 0.656, one-tailed P = 0.025). However, three G. bengalensis that received low doses
of diclofenac (<0.03 mg kg-1) had concentrations within the normal range for wild G.
africanus and untreated captive G. africanus and G. fulvus (figure 2), even though one
of these died with gout. At necropsy, all diclofenac-treated G. africanus and G. fulvus
had detectable diclofenac residues in liver (mean = 0.278 mg kg-1; range 0.108-0.752)
and kidney (mean = 0.319 mg kg-1; range 0.149-0.813). No diclofenac was found in the
euthanased sham-treated G. africanus. When data for G. africanus, G. fulvus and G.
bengalensis were combined, there was a significant relationship between diclofenac
dose and kidney diclofenac concentrations at post-mortem examination (Electronic
Appendix, correlation coefficient, rS = 0.503, one-tailed P = 0.030, n = 8), with no
statistically significant variation in the relationship between species or between
routes of diclofenac administration.
4. DISCUSSION
This study demonstrates that diclofenac was highly toxic to G. africanus and G. fulvus
at 0.8 mg kg-1. Hence, these species are likely to be at least as sensitive to diclofenac
poisoning as G. bengalensis, which has an estimated LD50 of 0.098 or 0.225 mg kg-1.
The lethargy and neck-drooping behaviour of diclofenac-dosed birds are consistent
with clinical studies in which birds showed lethargy for hours or days during the
deposition of renal urate, prior to the onset of visceral gout (Lierz 2003). At postmortem
examination, all treated birds showed extensive visceral gout. Microscopic
lesions and necrosis of proximal convoluted tubules seen in G. africanus were
consistent with the lesions described following experimental exposure of G.
bengalensis to diclofenac (Oaks et al. 2004). The results of toxicity testing in three Gyps
species (bengalensis, africanus and fulvus) and the finding of diclofenac residues and
visceral gout in wild G. bengalensis and G. indicus (Oaks et al., 2004, Schulz et al.,
2004), suggest that diclofenac is probably toxic to all eight vulture species within the
Gyps genus. As many G. fulvus and G. himalayensis winter in areas of India, Pakistan
and Nepal (del Hoyo et al. 1994) known to use veterinary diclofenac, ascertaining the
breeding areas and conservation status of populations of these species is a priority.
Determining the toxicity of diclofenac and other NSAIDs to vultures and
other scavenging birds is an urgent priority to ascertain the wider threat that these
drugs may pose. NSAIDs are widely used in veterinary medicine so vultures
(accipitrid and cathartid) and other scavenging birds (e.g. raptors, scavenging storks
and corvids) in many areas are likely to consume NSAID-treated animals (Anderson
et al. 2005).
Preliminary results from questionnaires on the clinical use of NSAIDs at
zoological institutions and raptor rehabilitation centres have found that renal disease
and/or acute visceral gout have been associated with the use of a range of NSAIDs
(RSPB and NBPC unpublished data). A better understanding of the exposure of
scavenging birds to NSAIDs and knowledge on the mechanism of the NSAID
toxicity is urgently needed to enable a better assessment of the likely current and
future impact of NSAIDs on the environment, and to determine which NSAIDs can
be safely used to replace diclofenac. In the short term, the demonstration in this
paper of the toxicity of diclofenac to G. africanus, the favourable conservation status
of this species and its close phylogenetic relationship to G. bengalensis (Seibold &
Helbig 1995) indicate that it could be used as a surrogate to establish the safety of
other NSAIDs to threatened Gyps vultures.
Acknowledgements
We would like to thank the following individuals and organisations: Dr Andrea
Raab (Aberdeen University, Chemistry Department) for help with analyses; The
Endangered Wildlife Trust for project development assistance; The Vulture Unit at
de Wildt for supply and housing of vultures; David Gibbons for comments; The
Faculty of Veterinary Science, Pretoria University, South Africa and the Consejería de
Medio ambiente , Junta de Andalucía, Spain, for their support.
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Figure 1. Uric acid and alanine transferase (ALT) concentrations in plasma measured
before and after oral treatment of vultures with 0.8 mg kg-1 of diclofenac. Lines connect
data for the same bird. Results are shown for two diclofenac-dosed (filled squares) and
two sham-treated (open squares) Gyps africanus, and for three diclofenac-dosed G. fulvus
(filled diamonds).


Figure 2. Uric acid concentration in the plasma of Gyps vultures at 24 h after
treatment with diclofenac in relation to dose. Open symbols represent Gyps
bengalensis from the experiments of Oaks et al. (2004), black symbols G. africanus and
grey symbols G.fulvus. Results for zero dose are for two untreated captive G. fulvus,
three treated G. fulvus sampled before treatment, two untreated control G. africanus,
two treated G. africanus sampled before treatment and the geometric mean for 14
wild G. africanus (filled square with vertical line showing the range from the 2.5th to
97.5th percentiles of a lognormal distribution from Gatome 2002, Table 5). Diamonds
are data for birds that died with gout after treatment. Squares represent untreated
birds (zero dose) and survivors of treatment. The outlying G. bengalensis that died
with gout after receiving a very low dose of diclofenac (see text) is labelled Gb11.