Active monitoring of long-eared owl (Asio otus) nestlings reveals widespread exposure to anticoagulant rodenticides across different agricultural landscapes.

The widespread use of anticoagulant rodenticides (ARs) poses a worldwide threat to farmland wildlife. These compounds accumulate in tissues of both target and non-target species, potentially endangering both direct consumers and their predators. However, investigations on ARs in blood of free-ranging predatory birds are rare. Here, the long-eared owl (Asio otus) has been used as a model predator to assess AR exposure in different agricultural landscapes from a Mediterranean semiarid region. A total of 69 owlets from 38 nests were blood-sampled over 2021 and 2022, aiming to detect AR residues and explore factors that determine their exposure, such as land uses. In addition, prothrombin time (PT) test was conducted to assess potential effects of AR contamination. Overall, nearly all the samples (98.6 %) tested positive for at least one compound and multiple ARs were found in most of the individuals (82.6 %). Among the ARs detected, flocoumafen was the most common compound (88.4 % of the samples). AR total concentration (ΣARs) in blood ranged from 0.06 to 34.18 ng mL-1, detecting the highest levels in the most intensively cultivated area. The analysis of owl pellets from 19 breeding territories showed relevant among-site differences in the contribution of rodents and birds into the diet of long-eared owls, supporting its high dietary plasticity and indicating AR presence at multiple trophic levels. Moreover, a positive and significant correlation was found between ΣARs and PT (Rho = 0.547, p < 0.001), which demonstrates the direct effect of ARs on free-living nestlings. Our results provide a preliminary overview of AR exposure in a little-studied owl species inhabiting agricultural and rural landscapes. Despite the low detected levels, these findings indicate widespread exposure -often to multiple compounds- from early life stages, which raises concern and draws attention to an ongoing and unresolved contamination issue.

Mercury Exposure in Birds of Prey from Norway: Relation to Stable Carbon and Nitrogen Isotope Signatures in Body Feathers.

Mercury (Hg) and stable carbon and nitrogen isotope ratios were analysed in body feathers from nestlings of white-tailed eagles (Haliaeetus albicilla) (WTE; n = 13) and Northern goshawks (Accipiter gentilis) (NG; n = 8) and in red blood cells (RBC) from NG (n = 11) from Norway. According to linear mixed model, species factor was significant in explaining the Hg concentration in feathers (LMM; p < 0.001, estimate (WTE) = 2.51, 95% CI = 1.26, 3.76), with concentrations higher in WTE (3.01 ± 1.34 µg g-1 dry weight) than in NG (0.51 ± 0.34 µg g-1 dry weight). This difference and the isotopic patterns for each species, likely reflect their diet, as WTE predominantly feed on a marine and higher trophic-chain diet compared to the terrestrial NG. In addition, Hg concentrations in RBCs of NG nestlings were positively correlated with feather Hg concentrations (Rho = 0.77, p = 0.03), supporting the potential usefulness of nestling body feathers to biomonitor and estimate Hg exposure. Hg levels in both species were generally below the commonly applied toxicity threshold of 5 µg g-1 in feathers, although exceeded in two WTE (6.08 and 5.19 µg g-1 dry weight).

Feather mercury levels in beached Magellanic penguin (Spheniscus magellanicus) in northern Argentina during the non-breeding season.

Mercury (Hg) is a great concern for marine environments. Bird feathers have been widely used to assess Hg pollution. In this study, we determine mercury concentrations in body feathers of juvenile Magellanic penguins (Spheniscus magellanicus) from the southeastern sector of Buenos Aires province, Argentina, during a non-breeding season, considering both sexes. Hg levels, considering both females and males together, ranged between 265.5 and 1515.52 ng/g. These levels are well below the concentrations in feathers suggested for taking actions focused on the protection of seabirds. Non-significant differences between sexes regarding Hg levels were found, probably because juveniles were sexually immature and females did not excrete Hg by egg laying yet. Hg concentrations found in this study were an order of magnitude higher than those reported 10 years ago for the species in breeding areas on the Argentine coast. Thus, the present study provides relevant information indicating a possible increase of Hg pollution in the southwestern region of the Atlantic Ocean and thus trigger for the development of monitoring programs and regional strategies to improve the conservation status of this species.

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.

Validation of Multi-Residue Method for Quantification of Antibiotics and NSAIDs in Avian Scavengers by Using Small Amounts of Plasma in HPLC-MS-TOF.

Pharmaceuticals are still considered emerging pollutants affecting both aquatic and terrestrial ecosystems. Scavenging bird species may be exposed to veterinary drugs when they feed on livestock carcasses provided at supplementary feeding stations, as these are often stocked with ailing and/or recently medicated animals. Because those animals may be a source of several different pharmaceutical compounds, analytical methods to evaluate residue levels and exposure potential should enable detection and quantification of as many different compounds as possible, preferably from small sample volumes. Four different extraction methods were tested to conduct HPLC-MS-TOF analysis of some of the most common veterinary drugs used in livestock in Spain. The method deemed most viable was a simple extraction, using methanol and 100 µL of plasma, that allowed quantification of seven antibiotics (tetracycline, oxytetracycline, ciprofloxacin, enrofloxacin, nalidixic acid, trimethoprim, sulfadiazine) and five nonsteroidal anti-inflammatory drugs (NSAIDs) (meloxicam, flunixin, carprofen, tolfenamic acid, phenylbutazone). The method was then applied to analysis of 29 Eurasian griffon vulture (Gyps fulvus) nestling samples, wherein enrofloxacin and tolfenamic acid were most commonly detected (69% and 20%, respectively). To our knowledge, this is the first study including NSAIDs in the exposure assessment of different classes of veterinary pharmaceuticals in live avian scavengers.

Temporal changes in metal concentrations in Andean condor feathers: a potential influence of volcanic activity.

Geothermal activities (e.g., volcanic eruptions) represent one of the most important natural sources of metal emissions (heavy metals and metalloids). They can be one of the main risks for the ecosystems in regions like North of Argentiniean Patagonia, a sparsely populated area, close to an extensive network of active volcanoes on the Andes Range. The 2011 eruption of the Puyehue-Cordon Caulle volcanic complex (PCCVC) has been the largest volcanic event of the last decades. The effects of exposure to ashes on wildlife and humans have been sparsely studied, and only one biomonitoring study has used higher trophic species. The exposure to metals of the species in Patagonia has been poorly studied. The main objectives of our study were to assess metal screening and to evaluate a possible relation between the levels of metals in the Andean condor (Vultur gryphus) population and the volcanic activity of the area. We investigated the effects of the eruption of the PCCVC in 2011, using samples of molt primary feathers of the Andean condor, collected in nine roosts around Bariloche, Argentina (maximum distance 85 km). Data available suggest the molt of the primary feathers of the Andean condor has a duration of 6 years. We carried out sampling before (2007, 2009) and after (2017) the volcanic eruption (2011). The feathers sampled in 2017 should have been developed in 2011-2012, reflecting the environmental situation of the period immediately following the eruption of the PCCVC. For the first time, we have screened metals in 48 molted primary feathers of Andean condor, showing the levels of 9 metals and metalloids (Si, Cr, Cu, Zn, As, Se, Cd, Pb, Hg). Si, Zn, As, and Cd showed higher levels in the feathers sampled after the eruption. The levels of Cr and Pb (although apparently not related to the volcanic eruption) in some samples are compatible with potential adverse effects in living organisms. The screening results represent an important database (the first for this species) that can be used in in future studies for comparative purposes. HIGHLIGHTS: •We present the first database of metals in condor feathers, which may be useful for future studies. •Volcanic eruptions might represent an important source of metal and metalloid emissions in this area. •The Andean condor from Argentine Patagonia could be affected by volcanic activity in the area. •Andean condor feathers may be good biomonitoring units of this contamination. •Pb pollution does not seem to be related to the volcano's eruption.

Organochlorine pesticides in feathers of three raptor species in southern Brazil.

In this study, we determined the presence of organochlorine pesticides (OCPs) in back feathers from three raptor species, Phalcoboenus chimango, Milvago chimachima and Caracara plancus. Samples were obtained from live animals and ten OCPs were detected: α-HCH, ß-HCH and γ-HCH (lindane), heptachlor, heptachlor epoxide, aldrin, endosulfan I, endosulfan II, endosulfan sulfate and p,p'-DDE. The concentrations found were higher than those reported in other raptor species, and C. plancus showed greater values than P. chimango and M. chimachima for all the substances. These differences may be justified by the species feeding habits and the characteristics of the sampling area, since C. plancus were sampled in an area with a higher concentration of agricultural crops and urbanized environments. In relation to the possible negative effects, four individuals of C. plancus presented DDE values related to sublethal effects for other species. This is the first study reporting OCPs in back feathers of these species in Brazil, and further studies evaluating OCP exposure and related effects are important for a better understanding of the consequences of different OCP concentrations on the health of these species.

Conditioned food aversion in domestic dogs induced by thiram.

BACKGROUND: The conflict between predators and humans for resources such as game species or livestock is an ancient issue, and it is especially sharp in the case of medium-large wild canids. In order to manage this conflict, lethal control methods are often used, which can sometimes be illegal, such as poisoning. As an alternative, conditioned food aversion (CFA) is a non-lethal method to reduce predation in which animals learn to avoid a given food due to the adverse effects caused by the ingestion of an undetectable chemical compound added to this food. The present study aimed to test thiram as a CFA agent in penned dogs as a first approach to use this substance for reducing the predation conflict associated with wild canids. RESULTS: Thiram, with or without an additional odor cue, produced CFA in penned dogs for more than 2 months. Moreover, thiram seemed to be undetectable and safe after the third ingestion of a 40-60 mg kg-1 dose. Desirable adverse effects, such as vomits, appeared around 1 h after exposure. These characteristics make thiram optimal for its use in predation reduction through CFA. However, individual variability could prevent CFA acquisition by some animals. CONCLUSIONS: Thiram has the potential to be used as a CFA agent in wildlife management and conservation to reduce predation by wild canids. Since thiram produced CFA without the problems of detectability and toxicity caused by other substances, it may be an alternative to lethal control methods used to reduce predation on game, livestock and endangered species. © 2019 Society of Chemical Industry.

Selection of new chemicals to be used in conditioned aversion for non-lethal predation control.

Globally, native predators and scavengers are threatened through the incidence of illegal poisoning due to increasing human-wildlife conflicts. The use of conditioned taste aversion (CTA) may mitigate such conflicts. CTA is a robust learning paradigm that occurs when animals associate a food with a discomfort induced by a chemical, thereby avoiding that food in subsequent encounters. We reviewed the potential of 167 chemical compounds to be used in CTA, considering effects, margin of safety, accessibility, and detectability. After the review, 15 compounds fulfilled the required characteristics, but only five (thiabendazole, thiram, levamisole, fluconazole and fluralaner) were finally selected to be tested in CTA assays with dogs. Of the tested compounds, thiabendazole, thiram and levamisole caused target food rejection by dogs and reduced the time spent eating during post-conditioning. However, despite being microencapsulated, levamisole appeared to be detectable by dogs, whereas thiram and thiabendazole were not. Fluconazole and fluralaner did not produce any CTA effect. Thiabendazole, thiram and levamisole can therefore induce CTA, and thus are potential candidates as aversive compounds for wildlife management. Thiram is an undetectable, relatively safe and accessible compound that can induce CTA in canids, and opens new possibilities to develop methods of non-lethal predation control.

Spatiotemporal variations of organochlorine pesticides in an apex predator: Influence of government regulations and farming practices.

BACKGROUND: Intensification of agricultural practices has caused several negative effects to the environment. The use of fertilizers and pesticides may alter geochemical cycles or cause direct wildlife intoxication. Detrimental effects of organochlorine pesticides (OCPs) have forced the authorities to ban or restrict its use. This study evaluates the variation in levels of OCPs in a sentinel species in relation to changes in government regulations and the spatial configuration of agricultural practices around the nests. METHODS: Between 2003 and 2007, we analysed OCP levels in 256 blood samples of Eurasian Eagle-owl (Bubo bubo) chicks nesting in area of intensive commercial agriculture with historical frequent use of pesticides, in South-eastern Spain. We studied year-to-year variations in OCP concentrations and their relation with land use configuration around raptor nests by Generalized Linear Mixed Models (GLMM). RESULTS: OCPs were detected in 100% samples surveyed in 2003 and 2004, while dropped to 27% in 2005, 6.8% in 2006 and 6.3% in 2007, coinciding with the ban of OCPs. The presence of the main OCPs was related to agricultural practices. In particular, endosulfan and lindane were related to irrigated crops and urban areas, while DDT-related compounds and dieldrin were associated with dry land farming. CONCLUSIONS: OCP concentrations in blood samples of Eurasian Eagle-owls may respond quickly to the implementations of new regulations about the use of agricultural products. This raptor was confirmed as a good sentinel species allowing rapid detection of changes in pesticides use.

Mercury in the feathers of bird scavengers from two areas of Patagonia (Argentina) under the influence of different anthropogenic activities: a preliminary study.

Mercury (Hg) is a global pollutant that bioaccumulates and biomagnifies in food chains and is associated with adverse effects in both humans and wildlife. We used feather samples from bird scavengers to evaluate Hg concentrations in two different areas of Northern Patagonia. Hg concentrations were analyzed in feathers obtained from turkey vultures (Cathartes aura), Black Vultures (Coragyps atratus), and southern crested caracaras (Caracara plancus) from the two areas of Northern Patagonia (Argentina): Bariloche and El Valle. Hg was detected in all the samples analyzed, but the concentrations can be considered low for the three species in both sampling areas. The mean concentration of Hg in Bariloche was 0.22 ± 0.16 mg/kg dry weight (d.w.) in black vulture, 0.13 ± 0.06 mg/kg d.w. in turkey vulture, and 0.13 ± 0.09 mg/kg d.w. in southern crested caracara; in El Valle, the mean concentration of Hg was 1.02 ± 0.89 mg/kg d.w. in black vulture, 0.53 ± 0.82 mg/kg d.w. in turkey vulture, and 0.54 ± 0.74 mg/kg d.w. in southern crested caracara. Hg concentrations in feathers were explained by the sampling area but not by the species. The concentrations of Hg contamination were comparable to those obtained in other studies of terrestrial raptors and aquatic bioindicator raptors. The species of the present study occur throughout much of North and South America. Thus, they may be appropriate bioindicators across the species' range, which is particularly useful as a surrogate, especially in distribution areas shared with endangered scavengers such as the California condor (Gymnopsys californianus) and the Andean Condor (Vultur gryphus).

Estrogenic activity of zearalenone, α-zearalenol and ß-zearalenol assessed using the E-screen assay in MCF-7 cells.

Mycotoxins, including zearalenone (ZEA), can occur worldwide in cereals. They can enter the food chain and cause several health disorders. ZEA and its derivatives (α-zearalenol, α-ZOL and ß-zearalenol, ß-ZOL) have structural analogy to estrogen, thus they can bind to estrogen receptors (ERs). In order to characterize the estrogenic activity of ZEA, α-ZOL and ß-ZOL, the proliferation of ER-positive human breast cancer cells (MCF-7) exposed to these mycotoxins was measured. After exposure at levels ranging from 6.25 to 25 µM, cell proliferation was evaluated by using the E-Screen bioassay. In accordance with previous studies, our results show the estrogenic activity of ZEA, α-ZOL and ß-ZOL in MCF-7 cells. This effect is related to ZEA and its metabolites being flexible enough to bind to mammalian ERs. The relative proliferative effect (RPE) ranged from 10% to 91%. The α-ZOL induced the highest proliferative effect due to its higher affinity for the ERs compared to the other mycotoxins.

Oxidative damage and disturbance of antioxidant capacity by zearalenone and its metabolites in human cells.

Mycotoxin contamination of foods and feeds represent a serious problem worldwide. Zearalenone (ZEA) is a secondary metabolite produced by Fusarium species. This study explores oxidative cellular damage and intracellular defense mechanisms (enzymatic and non-enzymatic) in the hepatoma cell line HepG2 after exposure to ZEA and its metabolites (α-zearalenol, α-ZOL; ß-zearalenol, ß-ZOL). Our results demonstrated that HepG2 cells exposed to ZEA, α-ZOL or ß-ZOL at different concentrations (0, 6.25, 12.5 and 25µM) showed: (i) elevated ROS levels (1.5- to 7-fold) based on the formation of the highly fluorescent 2',7'-dichlorofluorescein (DCF), (ii) increased DNA damage measured by the comet assay (9-45% higher), (iii) decreased GSH levels and CAT activity (decreased by 54%-25% and by 62%-25% for GSH and CAT, respectively) and (iv) increased GPx and SOD activities (increased by 50%-90% and by 26%-70%, respectively), compared to untreated cells. Our results suggest that mycotoxin-induced oxidative stress and damage may play a major role in the cytotoxic effects of ZEA and its metabolites. GSH and endogenous enzymes function together in protecting cells from ROS and the consequent damage after mycotoxin exposure. ZEA has a lower capacity to induce oxidative stress and damage in HepG2 cells than its metabolites at the tested concentrations.

Oxidative stress biomarkers in Eurasian eagle owls (Bubo bubo) in three different scenarios of heavy metal exposure.

The main aim of the present study is the assessment of oxidative stress related to metals in the Eurasian eagle owl (Bubo bubo) from three areas (agricultural and rural area, industrial area, and mining area) of Murcia, Southern Spain. Mean blood metal concentrations were Cd=0.07±0.21, Pb=3.27±5.21, Cu=10.62±4.77, Zn=311.47±67.14, Hg=2.32±3.83 µg/dl wet weight. Although individuals from the mining area had significant higher Pb and Hg concentrations, and significant lower glutathione-S-transferase (GST) and catalase (CAT) activities in red blood cells (RBC); the lack of differences in oxidative damage to membrane lipids (TBARS) among areas suggests that the antioxidant capacity of the different populations is able to deal with oxidant species and maintain TBARS levels in the same amount. Despite the low levels of metals, several oxidative stress biomarkers were correlated with metal concentrations. This study provides threshold concentrations at which metals cause effects on the antioxidant system in Eagle owls. Blood Cd concentrations greater than 0.3 µg/dl produced an inhibition in GPx (32%) and CAT (26%) activity in RBC. However, Cd concentrations higher than 0.02 µg/dl were enough to produce an inhibition of these enzymes. Regarding Pb levels, blood concentrations above 2 µg/dl produced an inhibition of 8% and 10.5% in GPx and CAT activities, respectively, in RBC. A depletion of 16% and 4% in tGSH levels was associated with Pb concentrations higher than 15 and 3 µg/dl, respectively, in individuals from the ancient mine site. In addition, Pb concentrations above 2 and 10 µg/dl produced a TBARS induction of 10% and 28%, respectively, in individuals from both the industrial and the mining area. Finally, Hg concentrations greater than 3 and 10 µg/dl resulted in a TBARS induction of 102% and 190%, respectively, in Eurasian eagle owls from the industrial area. Our findings show that Pb may produce effects on oxidative stress biomarkers in Strigiformes at lower concentrations than those typically accepted for considering physiological effects in Falconiformes (20 µg/dl in blood). In addition, we provide new data on Hg and Cd concentrations related to effects in the antioxidant system.

Factors that influence mercury concentrations in nestling Eagle Owls (Bubo bubo).

Mercury (Hg) is a global pollutant that bioaccumulates and biomagnifies in food chains, and is associated with adverse effects in both humans and wildlife. The Hg levels detected in blood obtained from Eagle Owl (Bubo bubo) chicks in Southeast Spain (Murcia) can be considered low (mean Hg concentration in blood from 2006 to 2012 was 36.83 ± 145.58 µg/l wet weigh, n=600), and it is therefore unlikely that Hg pollution can negatively affect their breeding. Positive correlation (r=0.339, p<0.001, n=229) was found to exist between the Hg concentrations in the blood and back feathers of the chicks. We provide a regression equation that could be helpful to estimate blood Hg levels when analyzing Hg concentrations in back feathers. Blood Hg concentrations in Eagle Owls have shown positive correlations with Hg levels in rabbit muscles, more evident in nests from the Northern area (r=0.600, p=0.014, n=16), where rabbits are the main prey of Eagle Owls. The best Linear Mixed Model to explain variations in blood Hg concentrations in nestling Eagle Owls includes year and location within the mining area as variables. The variable year is assigned the largest value of relative importance, followed by the location in the ancient mining sites and then the zone. Rainfalls may have an effect on the temporal differences in the blood Hg concentrations of nestling Eagle Owls. Although the studied region is not considered Hg polluted, the Hg levels were higher in Eagle Owls and European Rabbits from areas within the ancient mining sites as compared to those in the entire region. This result shows that spatial differences in Hg concentrations in Eagle Owls may be affected by local contamination, and that the role of diet composition may be less significant.