Second-generation anticoagulant rodenticides in the blood of obligate and facultative European avian scavengers.

The widespread use of second-generation anticoagulant rodenticides (SGARs) and their high persistence in animal tissues has led to these compounds becoming ubiquitous in rodent-predator-scavenger food webs. Exposure to SGARs has usually been investigated in wildlife species found dead, and despite growing evidence of the potential risk of secondary poisoning of predators and scavengers, the current worldwide exposure of free-living scavenging birds to SGARs remains scarcely investigated. We present the first active monitoring of blood SGAR concentrations and prevalence in the four European obligate (i.e., vultures) and facultative (red and black kites) avian scavengers in NE Spain. We analysed 261 free-living birds and detected SGARs in 39.1% (n = 102) of individuals. Both SGAR prevalence and concentrations (ΣSGARs) were related to the age and foraging behaviour of the species studied. Black kites showed the highest prevalence (100%), followed by red kites (66.7%), Egyptian (64.2%), bearded (20.9%), griffon (16.9%) and cinereous (6.3%) vultures. Overall, both the prevalence and average ΣSGARs were higher in non-nestlings than nestlings, and in species such as kites and Egyptian vultures foraging in anthropic landscapes (e.g., landfill sites and livestock farms) and exploiting small/medium-sized carrions. Brodifacoum was most prevalent (28.8%), followed by difenacoum (16.1%), flocoumafen (12.3%) and bromadiolone (7.3%). In SGAR-positive birds, the ΣSGAR (mean ± SE) was 7.52 ± 0.95 ng mL-1; the highest level detected being 53.50 ng mL-1. The most abundant diastereomer forms were trans-bromadiolone and flocoumafen, and cis-brodifacoum and difenacoum, showing that lower impact formulations could reduce secondary exposures of non-target species. Our findings suggest that SGARs can bioaccumulate in scavenging birds, showing the potential risk to avian scavenging guilds in Europe and elsewhere. We highlight the need for further studies on the potential adverse effects associated with concentrations of SGARSs in the blood to better interpret active monitoring studies of free-living birds.

Determinants of the exposure of Eurasian griffon vultures (Gyps fulvus) to fluoroquinolones used in livestock: The role of supplementary feeding stations.

Veterinary pharmaceuticals, including antibiotics, are emerging contaminants of concern worldwide. Avian scavengers are exposed to pharmaceuticals through consumption of livestock carcasses used for feeding wildlife for conservation purposes at supplementary feeding stations. Here we tested the hypothesis that griffon vultures (Gyps fulvus) would be more exposed to antibiotics (i.e., quinolones) when feeding on livestock carcasses from intensive farming than when they rely on carcasses from extensive farming or wild animals. We sampled 657 adult griffon vultures captured between 2008 and 2012. In addition, we sampled tissues from domestic livestock supplied at feeding stations in the study area between 2009 and 2019; pig (n = 114), sheep (n = 28), cow (n = 1) and goat (n = 2). Samples were analysed by liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS). Quinolones were detected in plasma from 12.9% of the griffon vultures analysed. Quinolone prevalence in griffon vultures varied significantly among feeding stations but was also affected by the total amount of carcasses supplemented, especially the mass of pig carcasses. These results aligned with a 21.1% quinolone prevalence in pig carcasses sampled at feeding stations, wherein enrofloxacin and ciprofloxacin levels of up to 3359 ng/g and 1550 ng/g, respectively, were found. Given enrofloxacin pharmacokinetics in pig tissues, 5.3% of the analysed pigs may have died no more than one day after treatment. Quinolone presence in vultures was negatively associated with blood lead levels, which mostly originates from lead ammunition and indicates a higher consumption of game animal carcasses. Carcass disposal for feeding avian scavengers must always assess and manage the risks posed by veterinary pharmaceuticals, especially when livestock provided may have died soon after treatment.

Increasing incidence of barbiturate intoxication in avian scavengers and mammals in Spain.

Pharmaceuticals are considered emerging contaminants in terms of impacts on wildlife. One chemical group of concern is euthanasia agents used in veterinary medicine. Here we present data on the occurrence of barbiturate intoxication using samples collected from 2004 to 2020 of suspected wildlife and domestic animal poisoning cases in Spain (n = 3210). Barbiturate intoxication was seen in 3.4% (45/1334) of the total number of confirmed intoxicated animals. Barbiturates were detected in 0.2% (1/448) of baits containing detectable poisons. The most frequently detected barbiturate was pentobarbital (42/45, 93.3%), but we also detected phenobarbital, barbital, and thiopental (2.2% prevalence for each). Avian scavengers were most frequently affected by barbiturate intoxication (n = 36), especially Eurasian griffon vultures (Gyps fulvus) (n = 28). Median pentobarbital concentrations detected in intoxicated griffon vultures was 27.3 mg kg-1 in gastric content and 38.1 mg kg-1 in liver, which highlights the acute effect of the chemical soon after ingestion. At least two large intoxication events affecting griffon vultures were related to the consumption of carcasses from euthanized livestock. We also found phenobarbital in a prepared bait linked to the intoxication of one Eurasian buzzard (Buteo buteo). This study highlights the need for stronger regulation of barbiturates to avoid secondary intoxications due to improper disposal of euthanized livestock.

Integrating active and passive monitoring to assess sublethal effects and mortality from lead poisoning in birds of prey.

The ingestion of lead (Pb) ammunition is the most important exposure pathway to this metal in birds and involve negative consequences to their health. We have performed a passive monitoring of Pb poisoning in birds of prey by measuring liver (n = 727) and blood (n = 32) Pb levels in individuals of 16 species found dead or sick in Spain between 2004 and 2020. We also performed an active monitoring by measuring blood Pb levels and biomarkers of haem biosynthesis, phosphorus (P) and calcium (Ca) metabolism, oxidative stress and immune function in individuals (n = 194) of 9 species trapped alive in the field between 2016 and 2017. Passive monitoring results revealed some species with liver Pb levels associated with severe clinical poisoning (>30 µg/g d.w. of Pb): Eurasian griffon vulture (27/257, 10.5%), red kite (1/132, 0.8%), golden eagle (4/38, 10.5%), and Northern goshawk (1/8, 12.5%). The active monitoring results showed that individuals of bearded vulture (1/3, 33.3%), Eurasian griffon vulture (87/118, 73.7%), Spanish imperial eagle (1/6, 16.7%) and red kite (1/18, 5.6%) had abnormal blood Pb levels (>20 µg/dL). Blood Pb levels increased with age, and both monitoring methods showed seasonality in Pb exposure associated with a delayed effect of the hunting season. In Eurasian griffon, blood Pb concentration was associated with lower δ-ALAD activity in blood and P levels in plasma, and with higher blood lipid peroxidation and plasma carotenoid levels in agreement with other experimental and field studies in Pb-exposed birds. The study reveals that Pb poisoning is a significant cause of death and sublethal effects on haem biosynthesis, P metabolism and oxidative stress in birds of prey in Spain.

Accumulation of diastereomers of anticoagulant rodenticides in wild boar from suburban areas: Implications for human consumers.

We studied the prevalence of anticoagulant rodenticides (ARs) in liver and muscle tissues of wild boar captured in the urban area of Barcelona, the suburban area of Collserola Natural Park and the rural area of Santa Quiteria, next to Cabañeros National Park, in Spain. The objective was to assess the influence of both urbanisation and wild boar (Sus scrofa) trophic opportunism on the accumulation of these compounds. We have also evaluated the risk for human consumers of this game meat. Wild boars from Barcelona city showed the highest prevalence of ARs detection (60.8%), followed by the adjoining suburban area of Collserola N.P. (40%) and the rural distant area of Santa Quiteria (7.7%). Liver bioaccumulated ARs (45.2%) more frequently than muscle (11.9%). A significant proportion (13.7%) of wild boar captured in Barcelona city exceeded 200 ng/g of total ARs in liver, a threshold for adverse effects on blood clotting. For difenacoum, there was a predominance of cis isomer, while for brodifacoum and bromadiolone cis and trans isomers appeared in a similar proportion. According to the scarce available information on ARs toxicity in humans, the risk of acute poisoning from game meat consumption seems to be low. However, repeated exposure through liver consumption should be considered in further risk assessments because of the high concentration detected in some samples (up to 0.68 mg/kg).

Urbanization and cattle density are determinants in the exposure to anticoagulant rodenticides of non-target wildlife.

The persistence and toxicity of second generation anticoagulant rodenticides (SGARs) in animal tissues make these compounds dangerous by biomagnification in predatory species. Here we studied the levels of SGARs in non-target species of wildlife and the environmental factors that influence such exposure. Liver samples of terrestrial vertebrates (n = 244) found dead between 2007 and 2016 in the region of Aragón (NE Spain) were analysed. The presence of SGARs was statistically analysed with binary or ordinal logistic models to study the effect of habitat characteristics including human population density, percentage of urban surface, livestock densities and surface of different types of crops. SGARs residues were detected in 83 (34%) of the animals and levels >200 ng/g were found in common raven (67%), red fox (50%), red kite (38%), Eurasian eagle-owl (25%), stone marten (23%), Eurasian buzzard (17%), northern marsh harrier (17%), and Eurasian badger (14%). The spatial analysis revealed that the presence of SGARs residues in wildlife was more associated with the use of these products as biocides in urban areas and cattle farms rather than as plant protection products in agricultural fields. This information permits to identify potential habitats where SGARs may pose a risk for predatory birds and mammals.

Mapping the spatio-temporal risk of lead exposure in apex species for more effective mitigation.

Effective mitigation of the risks posed by environmental contaminants for ecosystem integrity and human health requires knowing their sources and spatio-temporal distribution. We analysed the exposure to lead (Pb) in griffon vulture Gyps fulvus-an apex species valuable as biomonitoring sentinel. We determined vultures' lead exposure and its main sources by combining isotope signatures and modelling analyses of 691 bird blood samples collected over 5 years. We made yearlong spatially explicit predictions of the species risk of lead exposure. Our results highlight elevated lead exposure of griffon vultures (i.e. 44.9% of the studied population, approximately 15% of the European, showed lead blood levels more than 200 ng ml(-1)) partly owing to environmental lead (e.g. geological sources). These exposures to environmental lead of geological sources increased in those vultures exposed to point sources (e.g. lead-based ammunition). These spatial models and pollutant risk maps are powerful tools that identify areas of wildlife exposure to potentially harmful sources of lead that could affect ecosystem and human health.

Risk assessment of bearded vulture (Gypaetus barbatus) exposure to topical antiparasitics used in livestock within an ecotoxicovigilance framework.

Between 2004 and 2013, 486 suspected scavenger poisoning cases, including 24 bearded vultures (Gypaetus barbatus), were investigated in the Pyrenees and surrounding areas in Spain as part of a monitoring programme regarding accidental and intentional poisoning of wildlife. Poisoning was confirmed in 36% of all analysed cases where scavenger species were found dead within the distribution range of bearded vultures. Organophosphates and carbamates were the most frequently detected poisons. Four of the bearded vulture cases were positive for the presence of topical antiparasitics (3 with diazinon and 1 with permethrin). These likely represented accidental exposure due to the legal use of these veterinary pharmaceuticals. In order to confirm the risk of exposure to topical antiparasitics in bearded vultures, pig feet (n=24) and lamb feet (n=24) were analysed as these are one of the main food resources provided to bearded vultures at supplementary feeding stations. Pig feet had no detectable residues of topical antiparasitics. In contrast, 71.4% of lamb feet showed residues of antiparasitics including diazinon (64.3%), pirimiphos-methyl (25.4%), chlorpyrifos (7.1%), fenthion (1.6%), permethrin (0.8%) and cypermethrin (27.8%). Washing the feet with water significantly reduced levels of these topical antiparasitics, as such, this should be a recommended practice for lamb feet supplied at feeding stations for bearded vultures. Although the detected levels of antiparasitics were relatively low (≤1 µg/g), a risk assessment suggests that observed diazinon levels may affect brain acetylcholinesterase and thermoregulation in bearded vultures subject to chronic exposure.

Effects of Agricultural Management Policies on the Exposure of Black-Winged Stilts (Himantopus himantopus) Chicks to Cholinesterase-Inhibiting Pesticides in Rice Fields.

Levels of exposure to pesticides in rice fields can be significant depending on the environmental policies practiced. The aim of European Union integrated management policy is to reduce pesticide use and impact on environment. Rice fields provide an alternative breeding habitat for many waterbirds that are exposed to the pesticides used and therefore can be valuable indicators of their risk for wildlife. To evaluate integrated management success we examined exposure of Black-winged Stilts (Himantopus himantopus) to cholinesterase-inhibiting pesticides in rice fields under different types of management by measuring plasma cholinesterase activity. Cholinesterase activity was lower in birds sampled in (a) 2008 after a period of intense pesticide application, than in (b) 2005-2007 and 2011 in rice fields subject to integrated management in Doñana (SW Spain) and (c) in control natural wetlands in Spain and Morocco. During 2009 and 2010, cholinesterase activity was lower in rice fields in Doñana than in rice fields in Larache and Sidi Allal Tazi (NW Morocco). Our results suggest that integrated management successfully reduced the exposure of Black-winged Stilts to pesticides in most of the years. Care should be taken to implement mosquito and pest crop controls on time and with environmentally friendly products in order to reduce its impact on wildlife.

Primary and secondary poisoning by anticoagulant rodenticides of non-target animals in Spain.

Anticoagulant rodenticide (AR) levels were studied in liver of 401 wild and domestic animals found dead in Spain with evidences of AR poisoning, including 2 species of reptiles (n=2), 42 species of birds (n=271) and 18 species of mammals (n=128). Baits (n=32) were also analyzed to detect the potential use of ARs in their intentional preparation to kill predators. AR residues were detected in 155 (38.7%) of the studied animals and 140 (34.9%) may have died by AR poisoning according to the clinical information, necropsy findings, residue levels and results of other toxicological analysis. Animals considered with sublethal AR exposure had total AR residues (geometric mean with 95% CI) in liver of 0.005 (0.003-0.007)µg/g wet weight (w.w.) and animals diagnosed as dead by AR poisoning had 0.706 (0.473-1.054)µg/g w.w. ARs were detected in 19% of baits illegally prepared to kill predators. In terms of the total incidents studied in our laboratory between 2005 and 2010 (n=1792 animals), confirmed poisonings represented 40.9% of the cases, and 21.1% of these were due to ARs (8.6% of the total sample). Nocturnal raptors (62%) and carnivorous mammals (38%) were amongst the secondary consumers with highest prevalence of AR exposure, especially to second generation ARs (SGARs). On the other hand, granivorous birds showed the highest prevalence of AR exposure (51%), especially to chlorophacinone in a region treated against a vole population peak in 2007. The presence of hemorrhages was significantly associated with AR levels in liver, but some animals (7.2%) with elevated residue levels (>0.2µg/g w.w.) showed no evidence of macroscopic bleeding. The use of accumulative SGARs and the application of baits on surface (i.e. treated grain by spreader machines) should be discontinued in future EU regulations on the use of rodenticides to prevent the poisoning of non-target wildlife species.

Possible cantharidin poisoning of a great bustard (Otis tarda).

A possible cantharidin intoxication of a great bustard (Otis tarda) was described. This wild bird died by a traumatism, but also presented diarrhoea, congestion of the gastrointestinal tract and kidneys and had ingested several blister beetles of the species Berberomeloe majalis. The analysis of the stomach content by GC-MS revealed the presence of cantharidin at a concentration of 1.37 µg/g of wet weight, a similar level than in other birds poisoned in captivity.