Transgenerational effects of triazole fungicides on gene expression and egg compounds in non-exposed offspring: A case study using Red-Legged Partridges (Alectoris rufa).

Triazole fungicides are widely used to treat cereal seeds before sowing. Granivorous birds like the Red-legged Partridge (Alectoris rufa) have high exposure risk because they ingest treated seeds that remain on the field surface. As triazole fungicides can act as endocrine disruptors, affecting sterol synthesis and reproduction in birds several months after exposure, we hypothesized that these effects could also impact subsequent generations of exposed birds. To test this hypothesis, we exposed adult partridges (F0) to seeds treated at commercial doses with four different formulations containing triazoles as active ingredients (flutriafol, prothioconazole, tebuconazole, and a mixture of the latter two), simulating field exposure during late autumn sowing. During the subsequent reproductive season, two to four months after exposure, we examined compound allocation of steroid hormones, cholesterol, vitamins, and carotenoids in eggs laid by exposed birds (F1), as well as the expression of genes encoding enzymes involved in sterol biosynthesis in one-day-old chicks of this F1. One year later, F1 animals were paired again to investigate the expression of the same genes in the F2 chicks. We found changes in the expression of some genes for all treatments and both generations. Additionally, we observed an increase in estrone levels in eggs from partridges treated with flutriafol compared to controls, a decrease in tocopherol levels in partridges exposed to the mixture of tebuconazole and prothioconazole, and an increase in retinol levels in partridges exposed to prothioconazole. Despite sample size limitations, this study provides novel insights into the mechanisms of action of the previously observed effects of triazole fungicide-treated seeds on avian reproduction with evidence that the effects can persist beyond the exposure windows, affecting unexposed offspring of partridges fed with treated seeds. The results highlight the importance of considering long-term chronic effects when assessing pesticide risks to wild birds.

A heavy burden: Metal exposure across the land-ocean continuum in an adaptable carnivore.

Urbanisation and associated anthropogenic activities release large quantities of toxic metals and metalloids into the environment, where they may bioaccumulate and threaten both wildlife and human health. In highly transformed landscapes, terrestrial carnivores may be at increased risk of exposure through biomagnification. We quantified metallic element and metalloid exposure in blood of caracals (Caracal caracal), an adaptable felid inhabiting the rapidly urbanising, coastal metropole of Cape Town, South Africa. Using redundancy analysis and mixed-effect models, we explored the influence of demography, landscape use, and diet on the concentration of 11 metals and metalloids. Although species-specific toxic thresholds are lacking, arsenic (As) and chromium (Cr) were present at potentially sublethal levels in several individuals. Increased use of human-transformed landscapes, particularly urban areas, roads, and vineyards, was significantly associated with increased exposure to aluminium (Al), cobalt (Co) and lead (Pb). Foraging closer to the coast and within aquatic food webs was associated with increased levels of mercury (Hg), selenium (Se) and arsenic, where regular predation on seabirds and waterbirds likely facilitates transfer of metals from aquatic to terrestrial food webs. Further, several elements were linked to lower haemoglobin levels (chromium, mercury, manganese, and zinc) and elevated levels of infection-fighting cells (mercury and selenium). Our results highlight the importance of anthropogenic activities as major environmental sources of metal contamination in terrestrial wildlife, including exposure across the land-ocean continuum. These findings contribute towards the growing evidence suggesting cities are particularly toxic areas for wildlife. Co-exposure to a suite of metal pollutants may threaten the long-term health and persistence of Cape Town's caracal population in unexpected ways, particularly when interacting with additional known pollutant and pathogen exposure. The caracal is a valuable sentinel for assessing metal exposure and can be used in pollution monitoring programmes to mitigate exposure and promote biodiversity conservation in human-dominated landscapes.

A non-invasive method to monitor farmland bird exposure to triazole fungicides.

The treatment of seeds with pesticides is an extended practice in current agriculture. There is a high risk of exposure in granivorous birds, such as the red-legged partridge (Alectoris rufa), that can consume those seeds remaining on the surface during sowing. Fungicide exposure could in turn affect bird reproductive capacity. To better understand to what extent triazole fungicides are a threat to granivorous birds, we need an easy and reliable method to quantify field exposure. In this study, we tested a novel non-invasive method to detect the presence of triazole fungicide residues in farmland bird faeces. We experimentally exposed captive red-legged partridges to validate the method, and then applied it in a real scenario to assess exposure of wild partridges. We exposed adult partridges to seeds treated with two formulations containing triazole fungicides as active ingredients: Vincit®Minima (flutriafol 2.5%) and Raxil®Plus (prothioconazole 25% and tebuconazole 15%). We collected two types of faeces (caecal and rectal samples) immediately after exposure and 7 days later and quantified the concentrations of the three triazoles and their common metabolite (1,2,4-triazole). The three active ingredients and 1,2,4-triazole were only detected in faeces collected immediately after exposure. Triazole fungicide detection rates in rectal stool were 28.6%, 73.3% and 80% for flutriafol, prothioconazole and tebuconazole, respectively. In caecal samples, detection rates were 40%, 93.3% and 33.3%, respectively. 1,2,4-triazole was detected in 53% of rectal samples. For an applied use of the method in the field, we collected 43 faecal samples from wild red-legged partridges during autumn cereal seed sowing and found detectable levels of tebuconazole in 18.6% of the analysed wild partridges. The results of the experiment were then used to estimate actual exposure levels from this prevalence value found in wild birds. Our study shows that faecal analysis can be a useful tool to assess farmland bird exposure to triazole fungicides, when samples are fresh and the method has been validated for the detection of target molecules.

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.

Weasel exposure to the anticoagulant rodenticide bromadiolone in agrarian landscapes of southwestern Europe.

Bromadiolone is an anticoagulant rodenticide (AR) commonly used as a plant protection product (PPP) against rodent pests in agricultural lands. ARs can be transferred trophically to predators/scavengers when they consume intoxicated live or dead rodents. ARs exposure in weasels Mustela nivalis, small mustelids specialized on rodent predation, is poorly known in southern Europe. Moreover, in this species there is no information on bioaccumulation of AR diastereomers e.g., cis- and trans-bromadiolone. Trans-bromadiolone is more persistent in the rodent liver and thus, is expected to have a greater probability of trophic transfer to predators. Here, we report on bromadiolone occurrence, total concentrations and diastereomers proportions (trans- and cis-bromadiolone) in weasels from Castilla y León (north-western Spain) collected in 2010-2017, where bromadiolone was irregularly applied to control outbreaks of common voles Microtus arvalis mainly with cereal grain bait distributed by the regional government. We also tested variables possibly associated with bromadiolone occurrence and concentration, such as individual features (e.g., sex), spatio-temporal variables (e.g., year), and exposure risk (e.g., vole outbreaks). Overall bromadiolone occurrence in weasels was 22% (n = 32, arithmetic mean of concentration of bromadiolone positives = 0.072 mg/kg). An individual showed signs of bromadiolone intoxication (i.e., evidence of macroscopic hemorrhages or hyperaemia and hepatic bromadiolone concentration > 0.1 mg/kg). All the exposed weasels (n = 7) showed only trans-bromadiolone diastereomer in liver, whilst a single analyzed bait from those applied in Castilla y León contained trans- and cis-bromadiolone at 65/35%. Bromadiolone occurrence and concentration in weasels varied yearly. Occurrence was higher in 2012 (100% of weasels), when bromadiolone was widely distributed, compared to 2016-2017 (2016: 20%; 2017: 8.33%) when bromadiolone was exceptionally permitted. The highest concentrations happened in 2014 and 2017, both years with vole outbreaks. Our findings indicate that specialist rodent predators could be exposed to bromadiolone in areas and periods with bromadiolone treatments against vole outbreaks.

Poisoned chalice: Use of transformed landscapes associated with increased persistent organic pollutant concentrations and potential immune effects for an adaptable carnivore.

Wildlife around cities bioaccumulate multiple harmful environmental pollutants associated with human activities. Exposure severity can vary based on foraging behaviour and habitat use, which can be examined to elucidate exposure pathways. Carnivores can play vital roles in ecosystem stability but are particularly vulnerable to bioaccumulation of pollutants. Understanding the spatial and dietary predictors of these contaminants can inform pollutant control, and carnivores, at the top of food webs, can act as useful indicator species. We test for exposure to toxic organochlorines (OCs), including dichloro-diphenyl-trichloroethane (DDT) and polychlorinated biphenyls (PCBs), in a medium-sized felid, the caracal (Caracal caracal), across the peri-urban and agricultural landscapes of the city of Cape Town, South Africa. Concentrations in both blood (n = 69) and adipose tissue (n = 25) were analysed along with detailed spatial, dietary, demographic, and physiological data to assess OC sources and exposure risk. The analysis revealed widespread exposure of Cape Town's caracals to organochlorines: detection rate was 100% for PCBs and 83% for DDTs in blood, and 100% for both compounds in adipose. Caracals using human-transformed areas, such as vineyards and areas with higher human population and electrical transformer density, as well as wetland areas, had higher organochlorine burdens. These landscapes were also highly selected foraging areas, suggesting caracals are drawn into areas that co-incidentally increase their risk of exposure to these pollutants. Further, biomagnification potential was higher in individuals feeding on higher trophic level prey and on exotic prey. These findings point to bioaccumulation of OC toxicants and widespread exposure across local food webs. Additionally, we report possible physiological effects of exposure, including elevated white blood cell and platelet count, suggesting a degree of immunological response that may increase disease susceptibility. Cape Town's urban fringes likely represent a source of toxic chemicals for wildlife and require focused attention and action to ensure persistence of this adaptable mesocarnivore.

Determination of glyphosate exposure in the Iberian hare: A potential focal species associated to agrosystems.

Glyphosate is the most used herbicide worldwide. It is a small and highly polar pesticide whose physicochemical properties makes its analytical determination difficult. Here, a procedure based on liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed for glyphosate determination in samples of gastric content from wildlife. Iberian hare (Lepus granatensis), a herbivorous mammal species, strongly associated to agrosystems was selected as model species. The procedure involves direct analysis of sample without derivatization or instead of neither further cleaning steps. The procedure was validated by inter-day accuracy and precision studies with gastric content of hare spiked with glyphosate at ecologically relevant concentrations for the species (0.1-6 µg/g), and with 1 µg/g of isotopically labelled internal standard (glyphosate-2-13C,15N). Finally, glyphosate residues in hunted animals from pesticide-treated and pesticide-free areas (n = 75 and 28, respectively), as well as from hares found dead in the field (n = 11) were analysed. The linearity of both standards in extraction solutions and procedural calibration curves with spiked samples was similar, both with determination coefficients (r2) higher than 0.99. Satisfactory recoveries in spiked samples were achieved within the range of 95% to 118% (CV ≤ 20%). The limit of detection of glyphosate in hare gastric content was 0.03 µg/g. Prevalence of glyphosate in hunted animals from pesticide-treated areas ranged between 9 and 22%, increasing to 45% in animals found dead. The glyphosate concentrations detected in the gastric content of hares ranged from 0.11 to 16 µg/g. No residues were detected in animals from pesticide-free areas. In practice, the developed methodology may be particularly useful in the context of research and other work on the exposure in wildlife of one of the most used pesticides nowadays.

Bird exposure to fungicides through the consumption of treated seeds: A study of wild red-legged partridges in central Spain.

Sown seeds are a key component of many farmland birds' diets due to natural food shortages in autumn and winter. Because these seeds are often treated with pesticides, their ingestion by birds can result in toxic effects. For risk assessment, data on treated seed toxicity should be combined with information about exposure risk for wild birds and the factors that modulate it. We characterized the exposure of red-legged partridges to pesticide-treated seeds through the analysis of digestive contents of birds shot by hunters (n = 194) in an agricultural region in central Spain. We measured the contribution of sown seeds to the partridges' diet and how it related to pesticide exposure. Moreover, we evaluated the influence of landscape composition on the intake of sown seeds and pesticides by partridges. During peak sowing time, seeds constituted half (50.7%) of the fresh biomass ingested by partridges, which consumed mostly winter cereal seeds (42.3% of biomass). Residues of seven fungicides and one insecticide (active ingredients) were detected in 33.0% of birds. The presence of pesticides in digestive contents was linked to the ingestion of cereal sown seeds. Moreover, dietary exposure of birds to pesticides was modulated by landscape characteristics, being lower in areas with heterogeneous landscapes, greater habitat mosaic and more natural vegetation. The estimated dietary intake of pesticides resulting from our field observations, in combination with experimental data on pesticide toxicity, raise concerns about the risks that pesticide-treated cereal seeds pose to granivorous bird populations. Our results highlight the importance of farming landscape composition and diversification, which should be considered as a priority in the agricultural policy to mitigate pesticide risks to farmland birds through the consumption of treated seeds.

Blood Toxic Elements and Effects on Plasma Vitamins and Carotenoids in Two Wild Bird Species: Turdus merula and Columba livia.

Birds have historically suffered adverse effects by toxic elements, such as As, Pb, Hg, and Cd. However, reports on exposure to a wide range of elements, including rare earth elements and other minor elements of emerging concern, and the potential consequences for wildlife are still scarce. This study evaluates blood concentrations of 50 elements and their related effects on lutein and vitamin levels in the Eurasian blackbird (Turdus merula) and wild rock pigeon (Columba livia), inhabiting different scenarios of contaminant exposure. Blood concentrations of As, Cd, and Pb (and Mn in T. merula) were increased in both species captured in the mining area, compared to the control site. T. merula also showed increased As, Cd, and Pb concentrations in blood in the agricultural-urban area, as compared to the control area, together with the highest Hg levels, which could be related to agricultural practices and industrial activities. Decreases of 33 and 38% in the plasma retinol levels in T. merula inhabiting the mining and the agricultural-urban areas, respectively, as compared to the control site, were associated with increased Pb, As, and Cd exposure. This could be due to a metal-driven suppressive effect in retinol metabolism and/or its over-use for coping with metal-related oxidative stress.

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.

Birds feeding on tebuconazole treated seeds have reduced breeding output.

Drilled seeds are an important food resource for many farmland birds but may pose a serious risk when treated with pesticides. Most compounds currently used as seed treatment in the EU have low acute toxicity but may still affect birds in a sub-chronic or chronic way, especially considering that the sowing season lasts several weeks or months, resulting in a long exposure period for birds. Tebuconazole is a triazole fungicide widely used in agriculture but its toxicity to birds remains largely unknown. Our aim was to test if a realistic scenario of exposure to tebuconazole treated seeds affected the survival and subsequent reproduction of the red-legged partridge (Alectoris rufa). We fed captive partridges with wheat seeds treated with 0%, 20% or 100% of tebuconazole application rate during 25 days in late winter (i.e. tebuconazole dietary doses were approximately 0.2 and 1.1 mg/kg bw/day). We studied treatment effects on the physiology (i.e. body weight, biochemistry, immunology, oxidative stress, coloration) and reproduction of partridges. Exposed birds did not reduce food consumption but presented reduced plasmatic concentrations of lipids (triglycerides at both exposure doses, cholesterol at high dose) and proteins (high dose). The coloration of the eye ring was also reduced in the low dose group. Exposure ended 60 days before the first egg was laid, but still affected reproductive output: hatching rate was reduced by 23% and brood size was 1.5 times smaller in the high dose group compared with controls. No significant reproductive effects were found in the low dose group. Our results point to the need to study the potential endocrine disruption mechanism of this fungicide with lagged effects on reproduction. Risk assessments for tebuconazole use as seed treatment should be revised in light of these reported effects on bird reproduction.

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.

Egg Overspray with Herbicides and Fungicides Reduces Survival of Red-Legged Partridge Chicks.

Within the environmental risk assessment conducted for pesticide registration in the European Union (EU), avian reproductive toxicity is characterized after exposing adults. However, eggs of ground-nesting species can be exposed when pesticide applications occur during laying or incubation. We simulated environmentally realistic exposure of red-legged partridge (Alectoris rufa) eggs to an herbicide (2,4-D) and a fungicide (tebuconazole) applied to winter cereal crops during the breeding season of most farmland birds. We analyzed the effects on hatching success, offspring survival, and physiology. Exposure by overspray led to greater pesticide accumulation in the eggshell or content than exposure through contact with treated soil (3.1-13.7 times higher, depending on the pesticide and target sample). Egg overspray with tebuconazole significantly increased chick mortality, which was 26% higher than that of controls. 2,4-D caused a similar but a close to significant increase (chick mortality 24% higher than controls). Exposure to either pesticide through contact with treated soils did not affect chick survival but altered some biochemical parameters posthatching. Our experiment shows that egg spraying with pesticides should be considered as a relevant exposure scenario in risk assessment procedures, given its potential to affect the reproductive success of ground-nesting farmland birds.

Testing the shared-pathway hypothesis in the carotenoid-based coloration of red crossbills.

The mechanisms involved in the production of red carotenoid-based ornaments of vertebrates are still poorly understood. These colorations often depend on enzymatic transformations (ketolation) of dietary yellow carotenoids, which could occur in the inner mitochondrial membrane (IMM). Thus, carotenoid ketolation and cell respiration could share biochemical pathways, favoring the evolution of ketocarotenoid-based ornaments as reliable indices of individual quality under sexual selection. Captive male red crossbills (Loxia curvirostra Linnaeus) were exposed to redox-active compounds designed to penetrate and act in the IMM: an ubiquinone (mitoQ) or a superoxide dismutase mimetic (mitoTEMPO). MitoQ can act as an antioxidant but also distort the IMM structure, increasing mitochondrial free radical production. MitoQ decreased yellow carotenoids and tocopherol levels in blood, perhaps by being consumed as antioxidants. Contrarily, mitoTEMPO-treated birds rose circulating levels of the second most abundant ketocarotenoid in crossbills (i.e., canthaxanthin). It also increased feather total red ketocarotenoid concentration and redness, but only among those birds exhibiting a redder plumage at the start of the study, that is, supposedly high-quality individuals. The fact that mitoTEMPO effects depended on original plumage color suggests that the red-ketocarotenoid-based ornaments indicate individual quality as mitochondrial function efficiency. The findings would thus support the shared pathway hypothesis.

NSAIDs detected in Iberian avian scavengers and carrion after diclofenac registration for veterinary use in Spain.

Despite the now well recognised impact of diclofenac on vultures across the Indian subcontinent, this non-steroidal anti-inflammatory drug (NSAID) was registered in 2013 for livestock treatment in Spain, Europe's main vulture stronghold. We assessed the risk of exposure to diclofenac and nine other NSAIDs in avian scavengers in the Iberian Peninsula (Spain and Portugal) after the onset of diclofenac commercialization. We sampled 228 livestock carcasses from vulture feeding sites, primarily pig (n = 156) and sheep (n = 45). We also sampled tissues of 389 avian scavenger carcasses (306 Eurasian griffon vultures, 15 cinereous vultures, 11 Egyptian vultures, 12 bearded vultures and 45 other facultative scavengers). Samples were analysed by liquid chromatography with mass spectrometry (LCMS). Seven livestock carcasses (3.07%) contained NSAID residues: flunixin (1.75%), ketoprofen, diclofenac and meloxicam (0.44% each). NSAID residues were only detected in sheep (4.44%) and pig (3.21%) carcasses. Fourteen dead avian scavengers (3.60%) had NSAID residues in kidney and liver, specifically flunixin (1.03%) and meloxicam (2.57%). Flunixin was associated with visceral gout and/or kidney damage in three (0.98%) dead Eurasian griffons. To date, diclofenac poisoning has not been observed in Spain and Portugal, however, flunixin would appear to pose an immediate and clear risk. This work supports the need for well managed carrion disposal, alongside appropriate risk labelling on veterinary NSAIDs and other pharmaceuticals potentially toxic to avian scavengers.

Dust and bullets: Stable isotopes and GPS tracking disentangle lead sources for a large avian scavenger.

Lead intoxication is an important threat to human health and a large number of wildlife species. Animals are exposed to several sources of lead highlighting hunting ammunition and lead that is bioavailable in topsoil. Disentangling the role of each in lead exposure is an important conservation issue, particularly for species potentially affected by lead poisoning, such as vultures. The identification of lead sources in vultures and other species has been classically addressed by means of stable-isotope comparisons, but the extremely varied isotope signatures found in ammunition hinders this identification when it overlaps with topsoil signatures. In addition, assumptions related to the exposure of individual vultures to lead sources have been made without knowledge of the actual feeding grounds exploited by the birds. Here, we combine lead concentration analysis in blood, novel stable isotope approaches to assign the origin of the lead and GPS tracking data to investigate the main foraging grounds of two Iberian griffon vulture populations (N = 58) whose foraging ranges differ in terms of topsoil lead concentration and intensity of big game hunting activity. We found that the lead signature in vultures was closer to topsoil than to ammunition, but this similarity decreased significantly in the area with higher big game hunting activity. In addition, attending to the individual home ranges of the tracked birds, models accounting for the intensity of hunting activity better explained the higher blood lead concentration in vultures than topsoil exposure. In spite of that, our finding also show that lead exposure from topsoil is more important than previously thought.

Organochlorine pesticides and polychlorinated biphenyls in common kestrel eggs from the Canary Islands: Spatiotemporal variations and effects on eggshell and reproduction.

Organochlorine (OC) pesticides were widely used on the Canary Islands (Spain) for intensive crop production and against plagues of African locust. A previous study performed in 1988-1994 showed a high concentration of p,p'-DDE in the eggs of common kestrels (Falco tinnunculus) from the island of Tenerife. The present study shows OC pesticide and polychlorinated biphenyl (PCBs) levels in 40 unhatched common kestrel eggs collected from southern Tenerife between 2009 and 2016. The protoporphyrin IX in eggshells has also been analysed in order to explore the use of this pigment as a biomarker. Egg biometry, status of embryo development, eggshell thickness and mass of extractable lipids of each egg were recorded. Surrounding land use and reproductive parameters (hatching and fledging rates) were obtained for each nest. The most abundant compound was p,p'-DDE (15.0 µg/g d.w), followed by PCBs (0.46 µg/g d.w.). The decline in p,p'-DDE levels in southern Tenerife (with 23.6 µg/g d.w. in 1988-1994) was 36.4%. p,p'-DDE levels were positively associated with the surface of active and abandoned cropland in a 200 m-radius around the nest and with proximity to urban areas. PCB levels were associated with proximity to roads. Shell thickness was negatively affected by the p,p'-DDE concentration. The concentration of protoporphyrin IX in the eggshell was negatively associated with the concentration of hexachlorobenzene in the egg content. Despite the total ban on the use of p,p'-DDT in Spain since 1986, p,p'-DDE levels remain elevated in those areas in which that use was formerly intensive.

Blood concentrations of 50 elements in Eagle owl (Bubo bubo) at different contamination scenarios and related effects on plasma vitamin levels.

Some metals and metalloids (e.g. Pb, Hg, Cd and As) are well-known for their bioaccumulation capacity and their toxic effects on birds, but concerns on other minor elements and rare earth elements (ME and REE) are growing due to their intensive use in modern technology and potential toxicity. Vitamins and carotenoids play essential roles in nestling growth and proper development, and are known to be affected by the metals classically considered as toxic. However, we are unaware of any attempts to evaluate the exposure to 50 elements and related effects in plasma vitamins and carotenoids in raptor species. The main goals of this study are: (i) to assess the exposure to 50 elements (i.e. classic toxic elements, trace elements, REE and ME) in nestling Eagle owls (Bubo bubo) inhabiting three differently polluted environments (mining, industrial and control areas) in southeastern Spain, and (ii) to evaluate how element exposure affects plasma vitamin and carotenoid levels, hematocrit and body measurements (mass and wing length) of the individuals. Our results show that local contamination in the mining area contributes to increased blood concentrations of Pb, As and Tl in nestlings, while diet differences between control and mining/industrial areas may account for the different levels of Mn, Zn, and Sr in blood, and lutein in plasma. Plasma tocopherol levels were increased in the mining-impacted environment, which may be a mechanism of protection to prevent toxic element-related oxidative stress. Plasma α-tocopherol was enhanced by 20% at blood Pb concentrations ≥8 ng/ml, and nestlings exhibited up to 56% increase in α-tocopherol levels when blood Pb concentrations reached 170 ng/ml. Tocopherol seems to be a sensitive biomarker under an exposure to certain toxic elements (e.g. Pb, As, Tl).

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).