Metabolism of aceclofenac in cattle to vulture-killing diclofenac.

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac is highly toxic to Gyps vultures, and its recent widespread use in South Asia caused catastrophic declines in at least 3 scavenging raptors. The manufacture of veterinary formulations of diclofenac has since been banned across the region with mixed success. However, at least 12 other NSAIDs are available for veterinary use in South Asia. Aceclofenac is one of these compounds, and it is known to metabolize into diclofenac in some mammal species. The metabolic pathway of aceclofenac in cattle, the primary food of vultures in South Asia, is unknown. We gave 6 cattle the recommended dose of aceclofenac (2 mg/kg), collected blood thereafter at intervals for up to 12 h, and used liquid chromatography with mass spectrometry in a pharmacokinetic analysis of aceclofenac and diclofenac in the plasma. Nearly all the aceclofenac administered to the cattle was very rapidly metabolized into diclofenac. At 2 h, half the aceclofenac had been converted into diclofenac, and at 12 h four-fifths of the aceclofenac had been converted into diclofenac. Therefore, administering aceclofenac to livestock poses the same risk to vultures as administering diclofenac to livestock. This, coupled with the risk that aceclofenac may replace diclofenac in the veterinary market, points to the need for an immediate ban on all aceclofenac formulations that can be used to treat livestock. Without such a ban, the recovery of vultures across South Asia will not be successful.

Persistence of brodifacoum in cockroach and woodlice: implications for secondary poisoning during rodent eradications.

When planning rodent eradications, that normally involve the use of the anticoagulant poison brodifacoum, it is imperative to minimise impacts on other "non-target" species that dwell alongside the targeted rodents and may indeed be the intended beneficiaries of the eradication. Such impacts can arise either from primary poisoning when the non-target species ingest bait pellets containing toxicant or by secondary poisoning when the non-target species eats prey that has itself eaten brodifacoum. Cockroaches and woodlice, likely to scavenge bait pellets, are widely distributed on tropical and sub-tropical islands where they are eaten by ground-dwelling birds. Combining work on Henderson Island, South Pacific, site of a recent rat eradication project, and UK laboratory experiments, our study first measured brodifacoum concentrations in cockroaches given temporary ad lib access to poison bait pellets, approximately mimicking the aftermath of bait distribution for a rodent eradication. In two separate experiments using different species/exposure times, the mean brodifacoum concentrations among cockroaches immediately after bait exposure was 262±s.e. 131 and 477±168µgkg(-1) wet weight. Values decreased quickly in the following 2 weeks, and then continued to decline at a slower rate over the following 4 weeks in the more prolonged laboratory experiment. A supplementary experiment with woodlice recorded a similar brodifacoum concentration in the animals at the end of the exposure period, 223±66µgkg(-1), and a similar time course for the post-exposure decline. In the context of rails (Rallidae), a group of birds known to be particularly susceptible to primary brodifacoum poisoning, these results suggested that, in terms of acute exposure, individual birds would need to eat a minimum of their own body weight (and more commonly 2-5 times that) of live cockroaches before facing a 50% risk of death. Therefore, we conclude that in eradication scenarios, acute secondary poisoning is of lower concern for these birds than primary poisoning.

Transfer of metals to plants and red deer in an old lead mining area in Spain.

Lead mining in the Sierra Madrona mountains and the valley of Alcudia in Southern Spain began in the 1st millennium B.C., and the area was intermittently exploited up until the end of the 20th century. The degree of contamination by Pb, Zn, Cd, Cu, As and Se of soil, water and sediment, and the transfer to 13 species of plants, and then to red deer (Cervus elaphus) have been studied. Mined areas had higher concentrations in stream sediments than control areas. The highest concentrations were observed for Pb (1481 microg g(-1) d.w.) and As (1880 microg g(-1)) in the sediment of a stream flowing beside the spoil dump at Mina de Horcajo. Plants from mining sites contained consistently higher levels of Pb and As, and their concentrations in plants were correlated. The highest concentrations of Pb were in Gramineae (Pb: 97.5, As: 2.4 microg g(-1) d.w.), and the lowest in elmleaf blackberry (Rubus ulmifolius). The highest mean liver concentrations were found in red deer from the mining sites for Pb (0.805 microg g(-1) d. w.), Cd (0.554 microg g(-1)), Se (0.327 microg g(-1)), and As (0.061 microg g(-1)), although these were well below the levels associated with clinical poisoning.

Levels of heavy metals and metalloids in critically endangered Iberian lynx and other wild carnivores from Southern Spain.

The Iberian lynx (Lynx pardinus) is the most endangered felid in the world with a wild population which probably stands at less than 200 individuals inhabiting two areas in Southern Spain (Doñana and Sierra Morena) that are known to have been contaminated by heavy metals and metalloids due to a long history of mining activities. This contamination may pose a threat to long term conservation efforts and hence, the concentrations of seven elements (As, Se, Cd, Zn, Cu, Pb, Hg) were determined in the liver, muscle and bone of 9 lynx, as well as 17 red foxes (Vulpes vulpes), 11 Egyptian mongooses (Herpestes ichneumon), 4 common genets (Genetta genetta) and 1 Eurasian badger (Meles meles). The mean concentrations found were below the threshold levels indicative of chronic intoxication in all the species studied. In general, genet and red fox were species with the highest concentrations of several elements in Doñana, whilst Iberian lynx had the lowest levels of most of them. Lynx from Sierra Morena had significantly higher concentrations of bone Pb (2.05 microg/g d.w.) than those from Doñana (0.13 microg/g d.w.), probably due to the mineralised underlying geology and/or the abandoned mine workings in Sierra Morena. Egyptian mongoose presented liver concentrations of Hg up to 9.7 microg/g d.w. A strong relationship between Hg and Se levels was found in liver and muscle samples of all the studied species, especially in mongoose. In conclusion, levels of the studied elements do not appear to represent a significant threat for the lynx or for the other carnivores studied. However, given the critical status of the Iberian lynx, a continuous monitoring scheme remains necessary.

The pharmacokinetics of meloxicam in vultures.

Vulture populations across the Asian subcontinent have declined dramatically in the last 15 years and are now on the verge of extinction. Although the cause of the population decline was initially unknown, the decrease has recently been conclusively linked to the use of the nonsteroidal anti-inflammatory drug diclofenac in cattle that inadvertently ended up in the vulture food chain. With the vulture numbers continuing to decline by up to 48% a year, the Indian, Nepali and Pakistan governments have recently banned the manufacture and importation of veterinary diclofenac. They have also suggested meloxicam as an alternate anti-inflammatory for use in cattle. This recommendation was based on extensive acute safety studies in the African White-backed vulture (Gyps africanus), which evaluated worst case scenarios of maximum intake based on a once in three day feeding pattern. However, the possible cumulative pharmacokinetic and pharmacodynamic effects in vultures receiving multiple daily doses of meloxicam over time were not assessed. At present very little pharmacokinetic or pharmacodynamic information is available to add further support for the safety of meloxicam in this animal species. This article discusses the oral and intramuscular pharmacokinetics of meloxicam in Cape Griffon vultures (Gyps coprotheres). Therapeutic drug monitoring was also undertaken in White-backed, Egyptian (Neophron pernopterus) and one Lappet Faced vulture (Torgos tracheliotos). In all these species, meloxicam was characterized by a short half-life of elimination. The rapid metabolism of meloxicam in combination with a short duration of effect in the studied species Gyps vultures shown in this study makes it unlikely that the drug could accumulate. This confirms the safety of repeated exposure to meloxicam in vultures of this genus.

The use of toxicokinetics and exposure studies to show that carprofen in cattle tissue could lead to secondary toxicity and death in wild vultures.

Veterinary medicines can be extremely damaging to the environment, as seen with the catastrophic declines in Gyps vulture in South Asia due to their secondary exposure to diclofenac in their primary food source. Not surprisingly, concern has been raised over other similar drugs. In this study, we evaluate the toxicity of carprofen to the Gyps vulture clade through plasma pharmacokinetics evaluations in Bos taurus cattle (their food source) and Gyps africanus (a validated model species); tissue residues in cattle; and the effect of carprofen as a secondary toxicant as both tissue-bound residue or pure drug at levels expected in cattle tissues. Carprofen residues were highest in cattle kidney (7.72 ± 2.38 mg/kg) and injection site muscle (289.05 ± 98.96 mg/kg of dimension of 5 × 5 × 5 cm). Vultures exposed to carprofen as residues in the kidney tissue or pure drug equivalents showed no toxic signs. When exposed to average injection site concentrations (64 mg/kg) one of two birds died with evidence of severe renal and liver damage. Toxicokinetic analysis revealed a prolonged drug half-life of 37.75 h in the dead bird as opposed to 13.99 ± 5.61 h from healthy birds dosed intravenously at 5 mg/kg. While carprofen may generally be harmless to Gyps vultures, its high levels at the injection site in treated cattle can result in lethal exposure in foraging vultures, due to relative small area of tissue it is found therein. We thus suggest that carprofen not be used in domesticated ungulates in areas where carcasses are accessible or provided to vultures at supplementary feeding sites.

Diclofenac residues in carcasses of domestic ungulates available to vultures in India.

Gyps vulture populations across the Indian subcontinent are declining rapidly and evidence indicates that veterinary use of the non-steroidal anti-inflammatory drug (NSAID) diclofenac is the major cause. Exposure of vultures to diclofenac is likely to arise from the consumption of livestock carcasses that have been treated shortly before death, however, detailed information regarding the prevalence and residual levels of diclofenac in carcasses available to vultures in India remains unreported. Here, we present data on diclofenac residues in 1848 liver samples taken from carcasses of dead livestock sampled at 67 sites in 12 states within India, between May 2004 and July 2005. Diclofenac residues were detected in carcasses in all states except Orisa, where only one site was sampled. The overall prevalence of detectable diclofenac (>10 microg kg(-1)) across all states was 10.1% and varied significantly among states, with up to 22.3% prevalence determined in Bihar. The geometric mean concentration of diclofenac found in samples in which the drug was detected was 352 microg kg(-1). The prevalence of carcasses containing diclofenac is similar to that previously proposed to be required to have caused the observed Gyps vulture declines in India. On the 11th of May 2006, the Drug Controller General (India) ordered the withdrawal of all licenses granted for the manufacture of diclofenac for veterinary use within India. However, if Gyps vultures are to be protected, potentially substantial existing stocks now need to be quickly and effectively removed from the Indian veterinary market.

Lead contamination and associated disease in captive and reintroduced red kites Milvus milvus in England.

Since 1989, a red kite Milvus milvus reintroduction programme has been underway in the United Kingdom, with 4-6 week old nestlings brought into captivity and held for 6-8 weeks before reintroduction. As scavengers, red kites may consume unretrieved game, and ingest shot or lead (Pb) fragments in their prey's flesh. We evaluated exposure to Pb in captive and wild red kites by taking blood samples from 125 captive young red kites prior to release, through analysing 264 pellets (regurgitated by wild birds) collected from under a roost site, and analysing Pb concentrations in livers and/or bones of 87 red kites found dead between 1995 and 2003. Lead isotope analyses of livers were also conducted in an effort to identify Pb exposure routes. Forty-six (36.8%) kites sampled prior to release had elevated blood Pb concentrations (201-3340 microg l(-1)). The source of this Pb was probably small fragments of lead ammunition in the carcasses of birds or mammals either fed to the nestlings by their parents or, more likely, subsequently whilst in captivity. Once released, kites were also exposed to lead shot in their food, and a minimum of 1.5-2.3% of regurgitated pellets contained Pb gunshot. Seven of 44 red kites found dead or that were captured sick and died within a few days had elevated (>6 mg kg(-1) dry weight [d.w.]) liver Pb concentrations, and six of these (14%) had concentrations of >15 mg kg(-1) d.w., compatible with fatal Pb poisoning. Post-mortem analyses indicated that two of these birds had died of other causes (poisoning by rodenticide and a banned agricultural pesticide); the remaining four (9%) probably died of Pb poisoning. Bone samples from 86 red kites showed a skewed distribution of Pb concentration, and 18 samples (21%) had Pb concentrations >20 mg kg(-1) d.w., indicating elevated exposure to Pb at some stage in the birds' life. Lead isotopic signatures (Pb (208/206); Pb (206/207)) in liver samples of the majority of kites were compatible with those found in lead shot extracted from regurgitated pellets. Lead isotope ratios found in the livers of kites with very low Pb concentrations were distinct from UK petrol Pb isotopic signatures, indicating that birds were exposed to little residual petrol Pb. We conclude that the primary source of Pb to which red kites are exposed is lead ammunition (shotgun pellets or rifle bullets), or fragments thereof, in their food sources; in some cases exposure appears sufficient to be fatal. We make recommendations to reduce Pb poisoning in both captive and wild red kites and other scavenging species.

Diclofenac disposition in Indian cow and goat with reference to Gyps vulture population declines.

Gyps vultures across India are declining rapidly and the NSAID diclofenac has been shown to be the major cause. Vultures scavenge livestock carcasses that have been treated with diclofenac within the days preceding death. We present data on diclofenac disposition in Indian cow and goat, and field data on the prevalence of diclofenac in carcases in the environment. In the disposition experiment, animals were treated with a single intramuscular injection of diclofenac at 1000 microg kg-1 bw. In cow, diclofenac was detectable in liver, kidney and intestine up to 71 h post-treatment; in plasma, half-life was 12.2 h. In goat, tissue residues were undetectable after 26 h. Prevalence of diclofenac in liver from 36 dead livestock collected in the field was 13.9%. Data suggest that diclofenac residues in Indian cow and goat are short-lived, but diclofenac prevalence in carcasses available to vultures may still be very high.

Arsenic levels in the soils and macrophytes of the 'Entremuros' after the Aznalcóllar mine spill.

In April 1998, a holding lagoon containing pyrite ore processing waste rich in arsenic, failed and released 5-6 million m(3) of highly polluting sludge and acidic water. Over 2700 ha of the internationally important Doñana National and Natural Parks were contaminated. The area of Natural Park to sustain the greatest impact was known as the Entremuros. This paper presents 0-5 cm soil monitoring data from the Entremuros, from sampling campaigns 6 and 18 months after the disaster; as well as macrophyte root, rhizome and stem data from samples taken 18 months after the spill. Results show a clear, decreasing, north-south arsenic soil pollution trend, both 6 and 18 months after the spill, and suggest a small reduction in total soil arsenic levels occurred over time; although a significant increase in extractable arsenic is also noted. The two macrophytes (Typha dominguensis and Scirpus maritimus) studied herein are not accumulating arsenic in stem parts, however, accumulation of arsenic on iron plaque on the roots of these plants may be occurring. Further work is recommended in order to determine the ecotoxicological significance of this process in relation to the avian food-chains of Doñana, and elsewhere.

The distribution of arsenic in soils affected by the Aznalcóllar mine spill, SW Spain.

In April 1998, a holding lagoon containing pyrite ore processing waste, failed and released an estimated 5-6 million m(3) of metal rich sludge and acidic waters into the Rio Guadiamar, SW Spain. Over 2700 ha of the internationally important Doñana National and Natural Parks were contaminated. The sludge component of the waste contained up to 0.6% arsenic. This paper presents an extensive set of 0-5 cm soil analyses results from samples taken 6-8 months after the spill. Data are presented on pseudo-total arsenic levels in these samples, and on arsenic removed by both single batch and sequential extraction techniques. Pseudo-total levels of arsenic in the sludge ranged from 1521 to 3510 mg kg(-1), and a mean 4.4% of this was found to be extractable using 2.5% acetic acid. Soils in the Guadiamar Valley and Entremuros areas (those worst affected by the spill) were found to contain 85.4-782 mg kg(-1) and 7.1-196 mg kg(-1) pseudo-total arsenic, respectively. The mean acetic acid extractable component in these areas was 2.5% and 4.9%, respectively. Background pseudo-total arsenic levels in the soils of the area have been found to be 4.2-13.6 mg kg(-1). Rapid input of acidic waters, and the acidic nature of the sludge itself, may have caused depletion of Mg, Na and K, and to a lesser extent Mn, Ca and PO(4) in the upper 5 cm of the worst affected soils. Sequential extraction results suggest clear As-Fe associations, and possible As-Mn associations within the more soluble phases.

Pb-Al relationships in waterfowl feces discriminate between sources of Pb exposure.

Hunted mallards (n = 56) were collected in the Ebro Delta (Spain) in 2007-08 to evaluate the use of feces as a non-invasive monitoring method to study lead shot ingestion. The prevalence of Pb shot ingestion in these birds was 28.6%, and similar to that reported before a ban on Pb shot use in 2003. Lead concentrations in terminal intestinal contents were higher in mallards with Pb shot in their gizzard. Feces Pb concentrations >34 microg/g d.w were indicative of Pb shot ingestion, and background Pb levels were <12.5 microg/g d.w. To discriminate between birds ingesting soil Pb, and those ingesting Pb shot, we correlated Al and Pb levels and showed that mallards with ingested Pb shot in the gizzard stood out as outliers within the regression. Feces Pb-Al relationships can be used as a simple non-invasive tool in monitoring programs regarding Pb shot ingestion in birds.

Arsenic rich iron plaque on macrophyte roots--an ecotoxicological risk?

Arsenic is known to accumulate with iron plaque on macrophyte roots. Three to four years after the Aznalcóllar mine spill (Spain), residual arsenic contamination left in seasonal wetland habitats has been identified in this form by scanning electron microscopy. Total digestion has determined arsenic concentrations in thoroughly washed 'root+plaque' material in excess of 1000 mg kg(-1), and further analysis using X-ray absorption spectroscopy suggests arsenic exists as both arsenate and arsenite. Certain herbivorous species feed on rhizomes and bulbs of macrophytes in a wide range of global environments, and the ecotoxicological impact of consuming arsenic rich iron plaque associated with such food items remains to be quantified. Here, greylag geese which feed on Scirpus maritimus rhizome and bulb material in areas affected by the Aznalcóllar spill are shown to have elevated levels of arsenic in their feces, which may originate from arsenic rich iron plaque.

After the Aznalcóllar mine spill: arsenic, zinc, selenium, lead and copper levels in the livers and bones of five waterfowl species.

In April 1998, a holding lagoon containing pyrite ore processing waste, failed and released 5-6 million m3 of highly polluting sludge and acidic water. Over 2650 ha of the internationally important Doñana Natural Park became contaminated, along with < 100 ha of the more pristine Doñana National Park. In order to assess the affect of the spill on waterfowl from Doñana, bone and liver samples from 124 individuals have been analysed for As, Pb, Cu, Zn and Se. Five species have been studied, from the Rallidae (rails), Anatini (dabbling ducks) and Aythyini (pochards) families. Geometric mean bone concentrations 2-3 months after the spill were in the order of Zn > Cu > Pb > Se > As, while liver concentrations were in the order of Zn > Cu > Se > Pb > As. Dry weight bone concentrations ranged from n.d-1.76 mg kg(-1) As, 109.4-247.6 mg kg(-1) Zn, 0.06-1.27 mg kg(-1) Se, n.d-134.11 mg kg(-1) Pb, and 2.18-8.92 mg kg(-1) Cu. Wet weight liver concentrations ranged from n.d-0.34 mg kg(-1) As, 29.8-220.1 mg kg(-1) Zn, 0.15-0.85 mg kg(-1) Se, n.d-3.80 mg kg(-1) Pb, and 7.30-742.96 mg kg(-1) Cu. The most important factor related to the accumulation of these metals was commonly species; however, location and sex also had important effects on liver As levels, location and age affected Cu levels, while Zn and Pb were affected by age, sex and location. Birds from Natural Park areas were found to have significantly higher levels of bone Zn, Pb and Cu, and liver As and Cu than birds from National Park areas. Female birds had higher liver As, Zn and Pb than males; whilst adults appeared to have lower bone As and Zn but higher liver Pb than chicks/juveniles. Although metal concentrations were elevated in certain individuals, in the majority of birds studied, they did not reach levels widely considered to be toxic. However, it would appear that As and Cu liver levels (which may be indicative of short-medium term pollutant exposure) were elevated in waterbirds which died in the spill contaminated Natural Park, 2-3 months after the disaster.

Nickel, cobalt, zinc and copper levels in brown trout (Salmo trutta) from the river Otra, southern Norway.

The Flåt Nickel mine at Evje in southern Norway was mined extensively from 1914 to 1945 with little regard for any potential environmental effect. Much of the ore extracted was smelted at a site adjacent to the river Otra south of Evje. Recent studies have revealed heavy metal pollution in the land surrounding the smelter and in water draining from the mine leading to concern for the aquatic ecosystem in the river Otra. Brown trout were sampled from an uncontaminated lake 9 km upstream from the smelter, from the base of the Oddebekken (a tributary draining the mine water into the Otra), from sites immediately upstream and down stream of the smelter and from a site 4 km down stream from the smelter. Fish from sites adjacent to the smelter and the base of the Oddebekken were smaller than those from the lake and down stream site. Concentrations of the metals were highest in fish sampled where the mine water entered the Otra and gradually decreased in fish further down the river. Fish from the uncontaminated lake had the lowest level of metals.