Nimesulide poisoning in white-rumped vulture Gyps bengalensis in Gujarat, India

Population of white-rumped vulture has not recovered in India to a desired level even after diclofenac was banned in 2006. During 2019, there were two known separate incidents of white-rumped vulture mortality involving four white-rumped vultures in Gujarat. After post-mortem examinations, tissues of all four vultures were received for toxicological investigation at the National Centre for Avian Ecotoxicology, SACON. Tissues were screened for a set of toxic pesticides, and none of them was at detectable level. Subsequently, the tissues were analysed for thirteen NSAIDs and paracetamol. Of all the drugs tested, only nimesulide was detected in all the tissues (17–1395 ng/g) indicative of exposure. Visceral gout was also observed in all the four vultures during post-mortem. Residues of nimesulide in tissues with symptoms of gout indicated that the vultures died due to nimesulide poisoning. Although, other than diclofenac, many NSAIDs are suspected to be toxic to white-rumped vultures, only nimesulide is reported in the recent past with clear symptom of gout in wild dead white-rumped vultures similar to diclofenac. Since, nimesulide appears to act similar to diclofenac in exerting toxic effects, if veterinary use of nimesulide continues, white-rumped vulture are bound to suffer. Hence, it is recommended that nimesulide should be banned by the government to conserve white-rumped vulture in the Indian subcontinent. Further, an effective system is recommended to be put in place to collect the tissues of dead vultures for toxicological investigations and eventual conservation of the critically endangered species.


Introduction
Catastrophic population decline in the white-rumped vulture Gyps bengalensis in India was reported as early as 1990 . While diclofenac was reported to have caused the decline (Oaks et al. 2004;Shultz et al. 2004;Green et al. 2006;Cuthbert et al. 2014), the population of whiterumped vulture has not recovered even after the drug was banned in 2006 for veterinary use in India. The species is still "at high risk of global extinction", listed as critically endangered since 2000 (BirdLife International 2020), and categorised under Schedule I of Indian Wildlife Protection Act 1972;2002 (Amended). According to Prakash et al. (2019), the population of the white-rumped vulture in India as of 2015 was about 6000. Despite efforts, population of the species is yet to reach a healthy level. Moreover, while mortality of white-rumped vulture due to diclofenac was continued to be reported in India (Muralidharan and Dhananjayan 2010;Cuthbert et al. 2016;Nambirajan et al. 2018b), pesticide poisoning also made its contribution (Muralidharan S, Unpublished data).
Despite the ban on diclofenac, it was reported to have accounted for 10-46% of all NSAIDs available in the Indian market for veterinary use as on 2017 (Galligan et al. 2020). Studies revealed that in addition to diclofenac, other NSAIDs, namely, aceclofenac, ketoprofen, nimesulide, flunixin and carprofen are also to be toxic to Gyps vultures (Naidoo et al. 2010Sharma 2012;Fourie et al. 2015;Zorrilla et al. 2015;Cuthbert et al. 2016;Galligan et al. 2016;Eleni et al. 2019), and the same are among the 11 NSAIDs available in India for veterinary use as on 2017 (Galligan et al. 2020). Hence, an investigation was carried out to assess whether any of these NSAIDs was responsible for the death of four white-rumped vultures received dead at the National Centre for Avian Ecotoxicology (NCAE), Sálim Ali Centre for Ornithology and Natural History (SACON), Coimbatore for toxicological investigation during 2019. Since, veterinarians' choice to treat inflammation and related ailments in cattle is NSAIDs, and not all NSAIDs are safe to vultures, conservation initiatives to revive the population of white-backed vulture have become an ongoing affair in India. It also became imperative to check if there was misuse of diclofenac in India and also any other chemical was contributing to the population decline in white-rumped vulture.

Sample collection
Four white-rumped vultures were located dead accidentally on two separate incidents in Gujarat during 2019. While two were found dead below a roosting site in Sanand, two other vultures were also located dead below a roosting site in Wild Ass Sanctuary, Dhrangadhra. Gujarat forest officials located the dead vultures, and one of the co-authors of the study (Aditya Roy) coordinated transporting the dead vultures to veterinary hospital in Ahmedabad. While vultures from Sanand were taken to Jivdaya Charitable Trust, vultures from Wild Ass Sanctuary were brought to Wildlife Care Centre, Ahmedabad. Post-mortem examination of all the four birds in both the hospitals was performed by an experienced veterinarian (Dr Shashikant Jadhav), who is also a co-author of the study. Details pertaining to sample collection are given Table 1.
In all the four vultures, the crops were empty. Chalky white crystal/powder was found as a layer on all the four vultures' internal organs, namely, liver heart, kidney and intestinal serosa although intensity was different (Figs. 1, 2 and 3). In two birds which came from Sanand, air sacs, subcutaneous tissues in the neck region had signs of gout. One of the two birds from Wild Ass Sanctuary had chalky white deposits in the hock joint suggestive of articular gout (Fig. 4). While putrefaction had set in both birds from Wild Ass Sanctuary, one vulture had blood clots on its pectoral muscle and coelomic cavity. After post-mortem examination, organs, namely, kidney, liver, intestine and gut contents were dissected out, wrapped in aluminium foil, labelled, frozen immediately and transported subsequently through cold chain to the National Centre for Avian Ecotoxicology, SACON, Coimbatore. Samples on receipt at the laboratory were stored at − 20°C until chemical analyses were performed.

Chemicals and reagents
Certified standards of the fourteen individual drugs with purity higher than 98% were obtained from Sigma-Aldrich, USA and certified standards of thirty-two individual pesticides with purity more than 97% were purchased from Agilent Technologies, USA. While chromatography grade acetonitrile and sodium chloride were purchased from Merck, India, gradient grade acetonitrile for LC and hypergrade acetonitrile for LC-MS were of Merck KGaA, Germany, and anhydrous magnesium sulphate was of Himedia. Clean-up reagents, namely, Primary Secondary Amine (PSA), C18 bulk sorbent and graphitized carbon block (GCB) were from Agilent Technologies, USA. Ultrapure water was produced with a water purification system (Purelab classic, Elga, UK) in the laboratory at NCAE, SACON.

Residue extraction
Residues of NSAIDs and pesticides were extracted from tissues such as liver, kidney and gut contents of the vultures using multi-residue extraction method as adopted by Anastassiades et al. (2003) for vegetables and optimised by Nambirajan et al. (2018a) for bird tissues. Tissues were ground well using mortar and pestle to make it homogenised. About 2 g of homogenised samples were accurately weighed and placed in a 50-mL centrifuge tube. Five millilitres of purified water was added, hand shaken to homogenise well and kept for 30 min. Subsequently, 10 mL of acetonitrile was added to it and shaken again vigorously for a minute using Spinix Vortex shaker (Tarsons). Two grams of anhydrous magnesium sulphate (MgSO 4 ) and 0.5 g of sodium chloride were added to the centrifuge tube and shaken vigorously again for a minute, and centrifuged at 2000 g for 5 minutes (Remi, R-24 Research Centrifuge). After centrifugation, 2 mL of the organic layer of centrifugate was transferred into a 15-mL centrifuge tube which contained 100 mg of Primary Secondary Amine (PSA), 100 mg of C18 bulk sorbent and 250 mg of MgSO4, and shaken well. Then it was centrifuged at 1000 g for 5 min, and 1 mL of the centrifugate was transferred into separate vials for instrumental analyses.

Instrumental analyses
Residues of pesticides and drugs were quantified using Shimadzu LCMS-8045 Ultra-Fast Triple Quadrupole Mass Spectrometer with Nexera X2 Ultra Performance Liquid Chromatography as a frontend. Instrument conditions for estimation of drugs were as follows: Shim-pack GISS C18 (3 μm, 2.1 × 150 mm) column (Shimadzu, Japan) was used for separation. Five microlitres of sample was subjected to a binary gradient elution profile, which consisted 0.1% acetic acid in water (solution A) and 100% acetonitrile (solution B), as follows: starting conditions 70% A/30% B for 1.0 min, a 7min linear gradient from 70% A/30% B to 10% A/90% B, followed by a 3-min re-equilibration step with 70% A/30% B before the next injection. Flow rate was 0.4 mL min −1 and the column temperature was 40°C. ESI interface was used with 4 kV interface voltage. Interface, heating block and dissolvation line temperatures were 300, 400 and 250°C, respectively. Nebulizing, heating and drying gas flows were 3.0, 10.0 and 10.0 L min −1 , respectively. Instrument conditions for pesticide residue estimation were as follows: Shim-pack GISS C18 (3 μm, 2.1 × 150 mm) column (Shimadzu, Japan) was used for separation. Five microlitres of sample was subjected to a binary gradient elution profile, which consisted 0.1% acetic acid in water (solution A) and 100% acetonitrile (solution B) as follows: starting conditions 65% A/35% B for 1.0 min, a 5-min linear gradient from 65% A/35% B to 10% A/90% B, followed by a 6-min column-wash step in 10% A/90% B, and a 6-min re-equilibration step with 10% A/90% B before the next injection. Flow rate was 0.3 mL min −1 and the column temperature was 40°C. The MS conditions were the same as adopted for drug analysis.
Quality assurance/quality control Both the types of contaminants were analysed in multiple reaction monitoring (MRM) mode to obtain the maximum sensitivity for the detection of the target molecules. For confirmation, two MRM transitions and an appropriate ratio between the abundances of the two optimised MRM transitions (MRM2/MRM1) were used along with retention time matching. MRM transition details are given in supplementary table S1 & S2. A matrix-matched calibration curve was constructed for each compound at eight calibration levels using 1, 2.5, 5, 10, 25, 50, 100 and 200 ng/mL of mixed pesticide and NSAID standards. Tissue of an Indian peafowl which died due to kite string injury without any of the target compounds was used for matrix match standard preparation and recovery study. The peafowl tissue was confirmed for absence of all the target pesticides and drugs before use. The criterion for the acceptance of the linearity (R 2 ≥ 0.98) was fulfilled for all analytes in the method, and the estimated values are expressed in ng/g (wet weight basis). Limit of quantification (LOQ) and recoveries (25 ng/g, n = 6) of only the reported compound, nimesulide, are 10 ng/g and 96.5%, respectively. LOQ and recoveries from fortified samples of all other compounds are presented in supplementary table S1 & S2. Results were not corrected for percent recovery. Analyses were done in batches of 10 samples plus three quality controls, namely, blank, method blank and mid-range standard.

Results and discussion
Residues of all the thirty-two, most commonly used pesticides as listed in methodology, in tissues, namely, kidney, liver and gut contents of all the four white-rumped vultures collected from Sanand and Dhrangadhra were below detection limits. A standard and sample chromatogram are given in supplementary figure S1 and S2, respectively. It was confirmed that the vultures did not die due to any of the tested toxic pesticides. Subsequently, the tissues were analysed for fourteen drugs as listed in methodology. Of all the fourteen dugs analysed, only nimesulide was detected in all the tissues considered for the investigations ( Table 2). Levels of other drugs were below detection limit in all the tissue samples analysed. Standard (figure S3) and sample chromatogram (figure S4) are given in supplementary information. Calibration curve (figure S5), standard ( Figure S6) and sample ( Figure S7) peaks of nimesulide are also given as supplementary information. Of all the four vultures investigated, the one received from Sanand had higher concentration of nimesulide residues ranging from 792.1 to 1395.4 ng/g. Among the three tissues, kidney had the highest level of nimesulide (1395.4 ng/g) followed by gut content (1340.7 ng/g), while liver had the lowest. Tissues of the vultures received from Wild Ass Sanctuary comparatively had low concentration of nimesulide. This may be because the carcases were partially decayed at the time of collection. N i m e s u l i d e ( N -( 4 -n i t r o -2 -p h e n o x y p h e n y l ) methanesulfonamide) being a nonsteroidal anti-inflammatory drug has analgesic, antipyretic and anti-inflammatory properties. Data from a case study on drug-induced liver injury of drugs, namely, nimesulide, ketoprofen, ibuprofen, diclofenac and paracetamol in Italy revealed nimesulide to be associated with higher, and ibuprofen and ketoprofen with moderate risk of hepatotoxicity in human (Donati et al. 2016). Because of the death of a six-year-old child due to nimesulide-induced concomitant hepatic and renal failure in India (Gupta et al. 2012), controversy exists in the safety of the drug to human.
Nevertheless it is being widely used in India (banned for use in under 12) and many other developing countries on human beings. However, in many developed countries, such as USA, UK, Canada, Australia, New Zealand and Japan, nimesulide is not permitted for human use due to its high risk of liver damage. Also the International Society of Drug Bulletins (ISDB) in a press release dated December 2007 recommended withdrawal of nimesulide worldwide due to its potential to cause serious liver damage in humans (ISDB 2007). However, there is no such guideline available on its use in cattle.
While nimesulide is legally approved for veterinary use in India, it was the most available NSAID (48%) next to meloxicam for sale for veterinary use in eleven states in India between November 2007 and June 2010 (Cuthbert et al. 2011). A recent covert survey revealed that eleven NSAIDs were available for sale in pharmacies for veterinary use between 2012 and 2018 including nimesulide and diclofenac (Galligan et al. 2020). However, residues of nimesulide were recorded in none of the liver tissues of ungulates (n = 1488) collected across seven states in India including Gujarat during April and December 2006 (Taggart et al. 2009). There is no recent study in India that reported NSAIDs in the carcases available for wild white-rumped vultures to feed. However, residues of nimesulide were detected in the tissues of five white-rumped vultures which were found dead in the wild in Gujarat after 2008, and also were associated with gout as observed in vultures which died due to diclofenac poisoning . Cuthbert et al. (2016) recorded elevated levels of nimesulide in kidney (14-2753 ng/g) and liver (156-573 ng/ g) in four white-rumped vultures collected dead from Gujarat between 2008 and 2011. All the four white-rumped vultures had visceral gout and not diclofenac residues. Hence, it was concluded that nimesulide may have toxic effect similar to diclofenac and these four white-rumped vultures might have died due to renal failure caused by nimesulide. Levels of nimesulide recorded in the current investigation in the kidney (61-1395 ng/g) and liver (17-792 ng/g) tissues of whiterumped vultures received from Sanand and Wild Ass Sanctuary were similar to the levels reported by Cuthbert et al. (2016). Moreover, the high levels of nimesulide in gut contents (196-1341 ng/g) of white-rumped vultures from both the incidents clearly show that all the white-rumped vultures were exposed to nimesulide through food they consumed before death. Uric acid deposits, as crystals or powder, were observed in the internal organs of all four vultures although intensity was different (Figs. 1, 2, 3 and 4). Hence, it could be concluded that uric acid deposition observed in the viscera in all the four vultures during post-mortem and elevated levels of nimesulide residues in the organs and gut content were responsible for the mortality of all the white-rumped vultures in Gujarat during 2019. Reddy et al. (2006) suggested that nimesulide was likely to be safe for vultures based on a study on domestic fowl Gallus domesticus. However, Hassan et al. (2018) reported that three species of birds, namely, Japanese quail Coturnix japonica, Muscovy duck Cairina moschata and domestic pigeon Columba livia to be not suitable as surrogates for NSAID toxicity test in vultures, and the NSAID toxicity to Gyps vultures to be peculiar. Since, there are large differences in the toxicity of NSAIDs among different species of birds , safety of nimesulide to domestic fowl need not indicate safety to vultures. Further, due to variation in toxicity of nimesulide to various species of birds, Cuthbert et al. (2006) suggested that safety of nimesulide to other species of birds cannot be considered as safe to vultures too. This is further substantiated by the findings of the present study and Cuthbert et al. (2016).
It is now evident that nimesulide has the potential to harm vultures similar to diclofenac. Ban on the use of diclofenac for veterinary use in India as early as 2006, and the reduction in volume of diclofenac formulation for human use to restrict misuse for veterinary purpose in 2015, surely has slowed down the population decline of white-rumped vulture. But the population is still at low levels (5729) in India unlike Nepal and Pakistan where the population recovered subsequent to the ban on diclofenac (Chaudhry et al. 2012;Galligan et al. 2019;Prakash et al. 2019). This may be mainly because of illegal use of diclofenac and use of other toxic NSAIDs such as nimesulide, aceclofenac, ketoprofen, flunixin and carprofen on animals in India. Although, other than diclofenac, many NSAIDs are toxic or suspected to be toxic to white-rumped vulture, only nimesulide is reported with clear symptoms of gout in wild dead white-rumped vultures similar to diclofenac consistently in the recent past .
Further, aceclofenac administered to cattle also rapidly metabolises into diclofenac and harm white-rumped vulture . Diclofenac accounted for 10-46% of all NSAIDs available in the market for veterinary use as on 2017 (Galligan et al. 2020) and also diclofenac residues were recorded in 68.7% of white-rumped vultures received dead after the ban (between 2011 and 2014) in India (Nambirajan et al. 2018b). It appears at this junction that the ban on diclofenac alone will not help save vulture in the Indian subcontinent if we go by the studies in Gujarat. In the light of available facts, in addition to diclofenac, at least nimesulide and aceclofenac should be banned for veterinary use to conserve white-rumped vulture in Indian subcontinent. The volume of human formulations of these drugs also should be regulated like diclofenac to avoid misuse. As of now, only meloxicam is experimentally proved to be a safe NSAID to vultures Swarup et al. 2007). Fortunately, the safe drug, meloxicam is available in Indian market adequately for veterinary use (Cuthbert et al. 2011;Galligan et al. 2020). Veterinarians should be encouraged to continue use meloxicam, the only proven safe alternate NSAID, to treat domestic cattle at least in the entire distribution range of Gyps species.
Between March and April 2020, we know of at least two other incidents of mortality of white-rumped vulture in Assam and West Bengal. While 13 vultures died after eating carcass of pig in Jalpaiguri district, in West Bengal (Business Standard 2020), 19 vultures died in Sibsagar, Assam possibly due to poisoning (Hindustan Times 2020). Unfortunately, we could not collect tissue samples from both the locations for toxicological investigation. However, the circumstantial evidences suggested that both the cases to be poisoning. Since, mortality of vultures appears to an ongoing affair in India, an effective system is recommended to collect and submit tissues of dead vultures to identify contaminants, so as to take further action to conserve the endangered species. Further, toxicity testing of other NSAIDs on vultures is also suggested to be continued to save the Gyps vultures in Indian subcontinent.