Anticoagulant Rodenticides Research Articles

Fast and simultaneous analysis of carbamate pesticides and anticoagulant rodenticides used in suspected cases of animal poisoning

Carbamate pesticides (CBs) are reported as one of the main causes of intentional or accidental poisoning of animals. Anticoagulant rodenticides (ARs) form the main class of poisons implicated in analyzed poisoned baits. These two groups of pesticide compounds include multiple substances, and thus, the development of a simple and rapid multiclass/multiresidue analytical method for simultaneous identification of both toxicant classes should be a useful strategy for analytical laboratories to reduce analysis time and cost. The present study aimed to elaborate and validate a rapid method to simultaneously determine 11 CBs and 8 ARs in samples of real matrices (bait, stomach content, and liver) from suspected animal poisoning cases. QuEChERS sample treatment and liquid chromatography coupled to hybrid high resolution mass spectrometry were used. The method resulted in good linearity (R2 ≥ 0.98) for all compounds, recovery was between 70% and 120% for CBs and 40–90% for ARs, and precision was ≤ 20% for all compounds. The method was successfully applied to the analysis of 871 real samples originating from suspected cases of animal poisoning, collected from April 2019 to October 2020. Furthermore, full scan dependent data acquisition allowed qualitative retrospective data analysis of an additional 15 compounds outside the scope of the method to be performed; these compounds could potentially be involved in unresolved poisoning cases.

Risk of Anticoagulant Rodenticide Exposure for Mammals and Birds in Parc National des Pyrénées, France.

The extensive use of anticoagulant rodenticides (ARs) to control rodent populations poses intoxication risks for wildlife: persistence of ARs in rodents can cause secondary exposure and poisoning of predators or scavengers. We investigated risk factors for wildlife exposure to ARs in the Parc National des Pyrénées (PNP), France, using a multivariable logistic regression analysis. A total of 157 liver samples were collected from carcasses of 10 mammal and three bird species found in the PNP between 2010 and 2018 and screened for presence of AR residues. First- and second-generation ARs were detected in more than 60% of red fox (Vulpes vulpes) and stone marten (Martes foina) samples and in around 40% of wild cat (Felis silvestris), European pine marten (Martes martes), American mink (Neovison vison), and Eurasian Buzzard (Buteo buteo) samples. Wildlife exposure to ARs was significantly associated with species having a regular consumption of small mammals (odds ratio [OR]: 2.5, 95% confidence interval [CI]: 1.1-5.8) being collected in the Ossau valley (OR: 2.5, 95% CI: 1.1-6.1) and between 2013 and 2015 (OR: 4.8, 95% CI: 2.0-11.7). We identified wild species that could be targeted for risk-based surveillance program for AR secondary exposure and determined high risk areas in which alternative measures should be applied for rodent control.

Potential impact of diphacinone application strategies on secondary exposure risk in a common rodent pest: implications for management of California ground squirrels

Anticoagulant rodenticides are a common tool used to manage rodents in agricultural systems, but they have received increased scrutiny given concerns about secondary exposure in non-target wildlife. Rodenticide application strategy is one factor that influences exposure risk. To understand the impact of application strategy, we tested residues of a first-generation anticoagulant (diphacinone) in liver tissue of radiotransmittered California ground squirrels (Otospermophilus beecheyi) following spot treatments, broadcast applications, and bait station applications in rangelands in central California during summer and autumn 2018–2019. We also documented the amount of bait applied, the mean time from bait application until death, and the proportion of ground squirrels that died belowground. We documented the greatest amount of bait applied via bait stations and the least by broadcast applications. We did not document a difference in diphacinone residues across any application strategy, although survivors had an order of magnitude lower concentration of diphacinone than mortalities, potentially lowering secondary exposure risk. We did not observe any difference among bait delivery methods in time from bait application to death, nor did we identify any impact of seasonality on any of the factors we tested. The vast majority of mortalities occurred belowground (82–91%), likely reducing secondary exposure. Secondary exposure could be further reduced by daily carcass searches. Results from this study better define risk associated with first-generation anticoagulant rodenticide applications, ultimately assisting in development of management programs that minimize non-target exposure.

Wildlife poisoning: a novel scoring system and review of analytical methods for anticoagulant rodenticide determination.

Anticoagulant rodenticides (ARs) are commonly used to control rodent populations and frequently involved in wildlife and domestic animal poisoning. These poisoning cases (especially for ARs) are a challenge for forensic toxicologists, and adequate post-mortem examination and toxicological analyses become essential for a proper diagnosis. Publications describing different analytical methods for AR analysis in biological samples are growing, and a clear compilation of the overall picture is needed to standardize methodologies in future research. This review aims to compile and compare the analytical procedures applied for AR determination in the literature. Using this information, a scoring system was developed for those techniques using liver and blood as matrices, and the techniques were ranked considering different criteria (i.e. sample amount required, recoveries, limits of quantification (LOQs), number of ARs analysed, points of the calibration curve and multi-class methods). This review shows an overview of the main methods used for AR analysis in forensic toxicology and will help to elucidate future directions to improve multi-residue techniques to detect the ARs involved in wildlife lethal poisoning.

An Easy Procedure to Quantify Anticoagulant Rodenticides and Pharmaceutical Active Compounds in Soils.

A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction was validated for the extraction of seven coumarin anticoagulant rodenticides (ARs) and 36 pharmaceutical active compounds (PhACs) residues in soil samples using liquid chromatography tandem mass spectrometry (LC-MS/MS). The aim of this work was to develop a method for the monitoring of these compounds in agricultural lands as well as in forensic applications for the determination of ARs poisoning. As far as is known, this is the first time that a QuEChERS-based method is used for the extraction of ARs in soil, as well as on such a quantity of PhACs. A matrix effect study was carried out on samples of soil devoted to agriculture in the midland area of the Canary Islands (clay loam type). It was in house validated (accuracy, precision, and linearity) at seven spiked levels between 0.5 and 50 ng g−1. The limits of quantification (LOQ) ranged between 0.5 and 50.0 ng g−1 and the limits of detection (LOD) ranged from 0.024 to 6.25 ng g−1. The method was then successfully used for both the determination of the target analytes in the soils from the agricultural plots that had been irrigated with regenerated water, and in the soil collected from underneath wild bird carcasses (which had been the subject of forensic investigation).

Anticoagulant rodenticide exposure and toxicosis in bald eagles (Haliaeetus leucocephalus) and golden eagles (Aquila chrysaetos) in the United States.

Raptors, including eagles, are geographically widespread and sit atop the food chain, thereby serving an important role in maintaining ecosystem balance. After facing population declines associated with exposure to organochlorine insecticides such as dichlorodiphenyltrichloroethane (DDT), bald eagles (Haliaeetus leucocephalus) have recovered from the brink of extinction. However, both bald and golden eagles (Aquila chrysaetos) are exposed to a variety of other toxic compounds in the environment that could have population impacts. Few studies have focused on anticoagulant rodenticide (AR) exposure in eagles. Therefore, the purpose of this study was to determine the types of ARs that eagles are exposed to in the USA and better define the extent of toxicosis (i.e., fatal illness due to compound exposure). Diagnostic case records from bald and golden eagles submitted to the Southeastern Cooperative Wildlife Disease Study (University of Georgia) 2014 through 2018 were reviewed. Overall, 303 eagles were examined, and the livers from 116 bald eagles and 17 golden eagles were tested for ARs. The percentage of AR exposure (i.e., detectable levels but not associated with mortality) in eagles was high; ARs were detected in 109 (82%) eagles, including 96 (83%) bald eagles and 13 (77%) golden eagles. Anticoagulant rodenticide toxicosis was determined to be the cause of mortality in 12 (4%) of the 303 eagles examined, including 11 bald eagles and 1 golden eagle. Six different AR compounds were detected in these eagles, with brodifacoum and bromadiolone most frequently detected (81% and 25% of eagles tested, respectively). These results suggest that some ARs, most notably brodifacoum, are widespread in the environment and are commonly consumed by eagles. This highlights the need for research to understand the pathways of AR exposure in eagles, which may help inform policy and regulatory actions to mitigate AR exposure risk.

Use of rodenticide bait stations by commensal rodents at the urban-wildland interface: Insights for management to reduce nontarget exposure.

Pest management professionals use anticoagulant rodenticides, usually placed in tamper-resistant bait stations, to control commensal rodents, but significant concerns remain about exposure of nontarget species, especially at the urban-wildland interface. We deployed digital cameras to monitor use of bait stations placed in 90 residential yards across Orange County, California, USA. Two bait stations, supplied with nontoxic bait, were monitored in each yard for approximately 30 consecutive days during two camera-trapping sessions between December 2017 and March 2019. One station was placed on the ground, while the other was elevated 1-1.5m to determine if elevating stations could reduce nontarget exposure. Black rats (Rattus rattus L.) were present at 80% of sites, with mean activity ranging from 0 to 9.6h each night. There were no significant differences between elevated and ground stations in the time to discovery, time to bait station entry, or nightly activity of rats. Rats discovered bait stations more quickly, and mean nightly activity was greater, in yards where rats were detected more frequently. Although native rodents visited and entered bait stations occasionally, they were relatively rare among our sites (13.3%), and were detected five times less often at elevated stations compared to those on the ground. Yards visited by these rodents were significantly nearer to areas of green open space and natural vegetation, and tended to have no significant barriers to entry, e.g. solid fences or walls. By elevating bait stations and avoiding placing rodenticides in yards that are likely to be visited by wildlife, pest management professionals may be able to reduce the risk of nontarget exposure, including secondary poisoning of predators and scavengers, while still providing effective control of commensal pests. © 2021 Society of Chemical Industry.

Annual abundance of common Kestrels (Falco tinnunculus) is negatively associated with second generation anticoagulant rodenticides

Rats and mice can damage food and agricultural products as well as transmit diseases, thereby requiring control of their numbers. Application of Second Generation Anticoagulant Rodenticides (SGARs) often reduces rodent numbers locally. However, predators eating rodents, including non-target species, that have consumed SGARs may be secondarily exposed and potentially lethally poisoned. Here we study whether SGARs may have contributed to the widespread population declines of a rodent-eating raptor, the Common Kestrel (Falco tinnunculus) in the UK. We show that 161 (66.8%) of the 241 Kestrels submitted for ecotoxicology tests between 1997 and 2012 had detectable levels of at least one SGAR in their livers. Adult Kestrels had significantly higher prevalence of SGARs than juveniles, suggesting accumulation of SGARs through time. The prevalence and concentrations of individual SGARs in Kestrels were significantly higher in England than in Scotland. SGAR prevalence in Kestrels were positively associated with some land cover types, primarily arable cereals and broad-leaved woodland, and negatively associated with mainly mean elevation, probably reflecting variation in SGAR usage across land cover types. By using volunteer-collected data on national Kestrel abundance 1997–2012, we show that there is a negative correlation between the Kestrel population index in a specific year and the concentration of bromadialone as well as the total SGAR concentration in the same year. Although correlative, this is the first study to provide evidence for a potential population-limiting effect of SGARs on a raptor.

Monitoring the hepatic residues of cis- and trans-diastereoisomers of second generation anticoagulant rodenticides reveals a different bioaccumulation of diastereoisomers in the food chain of the Réunion harrier (Circus maillardi).

The Réunion harrier is an endangered raptor and endemic species to the Réunion Island. Second generation anticoagulant rodenticides (SGARs) are widely used pesticides on the island in order to control rodent populations. The latter are responsible for the transmission of leptospirosis to humans, the damage of sugarcane crops, and the decline of endemic endangered birds. SGARs are very persistent chiral pesticides and consequent secondary exposure or poisoning of the Réunion harrier has been observed (73% of prevalence in a group of 58 harriers). Commercial formulations of SGARs are a mixture of trans- and cis-diastereoisomers. Both diastereoisomers of all SGARs have been shown to inhibit coagulation function with the same potency. On the other hand, they have been shown to have a significant difference in terms of tissue-persistence. This difference has led to residue levels in rats with a significantly lower proportion of one of the isomers compared to the bait composition. In this study, residue levels of the diastereoisomers of all SGARs were evaluated in the livers of 58 harrier carcasses. The respective concentrations and proportions of cis- and trans- diastereoisomers of all SGARs are presented. cis-Brodifacoum and trans-bromadiolone had the highest concentrations (up to 438 and 573ng/g ww respectively), while trans-brodifacoum was less than 46ng/g and cis-bromadiolone was barely detected. cis-Difenacoum showed the highest prevalence and the highest concentration was 82ng/g ww, while trans-difenacoum was never detected. This study demonstrated that only cis-brodifacoum and trans-bromadiolone (and cis-difethialone, but with a low prevalence) had hepatic concentrations above a toxic threshold. The cis- and trans-diastereoisomers of SGARs had differential bioaccumulation in the food chain of the Réunion harrier compared to commercial baits. This suggests that a change of the proportions of SGARs diastereoisomers in baits could reduce the risk of secondary poisoning of predators, but maintain primary toxicity.

Brodifacoum pharmacokinetics in acute human poisoning: implications for estimating duration of vitamin K therapy

Standard of care follow-up therapy for patients poisoned by long-acting anticoagulant rodenticides (LAARs) is daily high-dose (up to 100 mg per day) oral vitamin K1 (VK1) for weeks to months to over a year. The availability of CLIA-certified quantitative testing for plasma LAAR concentrations can now assist health care providers in determining when to safely discontinue VK1 therapy. We present estimates of treatment duration required to reach safe concentrations (< =10ng/ml) using serial measurements of plasma brodifacoum (BDF, a potent LAAR) concentrations obtained from patients poisoned after inhaling synthetic cannabinoids containing BDF. We fit the data to zero-order (linear) and first-order (exponential) curves, the latter to account for enterohepatic circulation of BDF. The results show that estimates of therapy duration are significantly longer when exponential clearance is assumed. Accordingly, we recommend that plasma BDF concentrations be monitored simultaneously with international normalization ratio (INR) during follow-up of poisoned patients, and that concentrations be determined after VK1 therapy is discontinued to document persistence of safe concentrations.

Sensitivity of turtles to anticoagulant rodenticides: Risk assessment for green sea turtles (Chelonia mydas) in the Ogasawara Islands and comparison of warfarin sensitivity among turtle species

Although anticoagulant rodenticides (ARs) are effectively used for the control of invasive rodents, nontarget species are also frequently exposed to ARs and secondary poisonings occur widely. However, little data is available on the effects of ARs, especially on marine organisms. To evaluate the effects of ARs on marine wildlife, we chose green sea turtles (Chelonia mydas), which are one of the most common marine organisms around the Ogasawara islands, as our primary study species. The sensitivity of these turtles to ARs was assessed using both in vivo and in vitro approaches. We administered 4 mg/kg of warfarin sodium either orally or intravenously to juvenile green sea turtles. The turtles exhibited slow pharmacokinetics, and prolongation of prothrombin time (PT) was observed only with intravenous warfarin administration. We also conducted an in vitro investigation using liver microsomes from green sea turtles, and two other turtle species (softshell turtle and red-eared slider) and rats. The cytochrome P450 metabolic activity in the liver of green sea turtles was lower than in rats. Additionally, vitamin K epoxide reductase (VKOR), which is the target enzyme of ARs, was inhibited by warfarin in the turtles at lower concentration levels than in rats. These data indicate that turtles may be more sensitive to ARs than rats. We expect that these findings will be helpful for sea turtle conservation following accidental AR-broadcast incidents.

Epidemiological Study of Pesticide Poisoning in Domestic Animals and Wildlife in Portugal: 2014-2020.

Nowadays the intentional poisoning of domestic and wild animals is a crime in the European Union (EU), but as in the past the poison is still used in rural areas of a number of European countries to kill animals that were considered harmful for human activities. From January 2014 up until October 2020, the Laboratory of Pharmacology and Toxicology of the Faculty of Veterinary Medicine (LFT-FMV) has done the analytical detection of poisoning substances in 503 samples of wildlife and domestic animals and pesticides residues were found in 239 of the samples analyzed. In this retrospective study, toxicology results from domestic species (dog, cat, sheep, cows, and horses), wildlife species (red foxes, birds of prey, lynx, and wild boar), and food baits, are presented. During this period the samples analyzed at the LFT-FMV, were received from all over the country. Analytical detections were performed via solvent extraction followed by thin layer chromatography. Molluscicides (47%, n = 109) and Carbamates (24%, n = 57) were found to be the first category of pesticides involved in intoxications, in both domestic and wild animals, followed by rodenticides (13%, n = 30)—in this group second and third generation, were the most represented; Strychnine is the third (11%, n = 26) even though this pesticide has been banned in Portugal since 1988 and in the European Union since 2006 and finally Organophosphates (5%, n = 11) in the small number. This study allowed to realize that a great number of positive samples involved banned pesticides (i.e., Aldicarb and Strychnine) but, at the same time, many positives cases were due to the exposure to commercially available products (i.e., Methiocarb and Anticoagulant rodenticides). Also, it's possible to identify the areas where domestic species are the most affected (i.e., Setubal and Lisboa) and the areas where the wild animals are the mainly affected species (i.e., Faro, Castelo Branco, and Bragança).

Chiral liquid chromatography-tandem mass spectrometry analysis of superwarfarin rodenticide stereoisomers – Bromadiolone, difenacoum and brodifacoum – In human plasma

Superwarfarins are second-generation long-acting anticoagulant rodenticides that can cause unintended human and wildlife toxicity due, in part, to their prolonged half-lives. Commercially available superwarfarin rodenticides are synthesized as racemates with two asymmetric carbons, producing four stereoisomers. To support studies of human plasma half-lives of individual superwarfarin stereoisomers, a method was developed based on LC-MS/MS to separate and quantify stereoisomers of the commercially important superwarfarins bromadiolone, difenacoum and brodifacoum. Human plasma samples were prepared using protein precipitation and centrifugation. Chiral-phase HPLC separation was carried out on-line with tandem mass spectrometric quantitative analysis of the eluting stereoisomers using selected-reaction monitoring with positive ion electrospray on a triple quadrupole mass spectrometer. All four stereoisomers of each superwarfarin were resolved within 12.5 min with calibration curves spanning 2–3 orders of magnitude and lower limits of quantitation between 0.87 and 2.55 ng/mL. This method was used to determine the half-lives of superwarfarin stereoisomers in plasma from patients who had inhaled synthetic cannabinoid products contaminated with superwarfarins. These data may be used to guide the development of safer next generation anticoagulant rodenticides stereoisomers.

Anticoagulant Rodenticides in Red-Tailed Hawks (Buteo jamaicensis) from New York City, New York, USA, 2012–18

Anticoagulant rodenticides (AR), principally difethialone, brodifacoum, and bromadiolone, were detected in the livers of 89% of 72 Red-tailed Hawks (Buteo jamaicensis) from New York City, New York, US examined for cause of death over a 7-yr period (January 2012-December 2018). Fatal hemorrhage likely attributable to AR exposure was diagnosed in 41% (30/74) of cases, and 46% (18/39) of the cases analyzed with no gross evidence of AR-mediated hemorrhage had liver concentrations of AR that overlapped those with an AR-poisoning diagnosis. Although urban areas like New York City can support surprisingly dense populations of Red-tailed Hawks, the threat posed by extensive use of AR can be large.

Another look at reactions of 4-hydroxycoumarin with hydroxyl radical in the environment: deprotonation and diffusion effects

Quantum chemistry calculations suggest that chemical fate of the anticoagulant rodenticide 4-hydroxycoumarin in the environment is crucially dependent on deprotonation in aqueous medium.

Intensive livestock farming as a major determinant of the exposure to anticoagulant rodenticides in raptors of the Canary Islands (Spain)

The Canary Islands (Spain) is a biodiversity hotspot, with more than 4500 registered endemic species. However, it is subject to high anthropogenic pressure that threatens its wildlife in various ways. In the context of forensic toxicological surveys, the presence of anticoagulant rodenticides (AR) has been investigated in the liver of 831 animal carcasses with georeferenced data from 2011 to May 2020. The high concentrations of toxic pesticides in carcasses and in baits found close to the corpses indicated that all the reptiles and most of the mammals tested positive for AR were intentionally poisoned, although mainly by other substances. The frequency of detection of AR in non-raptor birds (n = 343) was only 4.1%, being the Canary raven the most frequently affected species (7/97, 7.2%). On the contrary, in raptors (n = 308) the detection frequency was almost 60%, with an average of more than 2 ARs per animal. The highest concentrations were found in the common kestrel. We present for the first-time results of AR contamination in two species of raptors that are very rare in Europe, Eleonora's falcon (n = 4) and Barbary falcon (n = 13). The temporal trend of positive cases remains stable, but since the entry into force of the restriction to the concentration of the active ingredient in baits (<30 ppm), a decrease in the concentrations of these compounds in the raptors' liver has been detected. Conversely, we registered an increase in the number of ARs per animal. From the study of the geographic information system (GIS) it can be deduced that intensive livestock farms are an important determinant in the exposure of raptors to ARs. Those birds that have their territory near intensive production farms have higher levels of exposure than those of birds that live far from such facilities.

Biomarkers Potency to Monitor Non-target Fauna Poisoning by Anticoagulant Rodenticides.

The widespread use of pesticides to control agricultural pests is a hot topic on the public scene of environmental health. Selective pest control for minimum environmental impact is a major goal of the environmental toxicology field, notably to avoid unintended poisoning in different organisms. Anticoagulant rodenticides cause abnormal blood coagulation process; they have been widely used to control rodents, allowing inadvertent primary and secondary exposure in domestic animals and non-target predatory wildlife species through direct ingestion of rodenticide-containing bait or by consumption of poisoned prey. To report toxic effect, the most common approach is the measurement of liver or plasma residues of anticoagulant rodenticides in dead or intoxicated animals showing clinical symptoms. However, one major challenge is that literature currently lacks a hepatic or plasma concentration threshold value for the differentiation of exposure from toxicity. Regarding the variation in pharmacology properties of anticoagulant rodenticides inter- and intra-species, the dose-response relationship must be defined for each species to prejudge the relative risk of poisoning. Beyond that, biomarkers are a key solution widely used for ecological risk assessment of contaminants. Since anticoagulant rodenticides (AR) have toxic effects at the biochemical level, biomarkers can serve as indicators of toxic exposure. In this sense, toxicological knowledge of anticoagulant rodenticides within organisms is an important tool for defining sensitive, specific, and suitable biomarkers. In this review, we provide an overview of the toxicodynamic and toxicokinetic parameters of anticoagulant rodenticides in different animal species. We examine different types of biomarkers used to characterize and differentiate the exposure and toxic effects of anticoagulant rodenticide, showing the strengths and weaknesses of the assays. Finally, we describe possible new biomarkers and highlight their capabilities.

Exposure of passerine birds to brodifacoum during management of Norway rats on farms

The exposure of non-target wildlife to anticoagulant compounds used for rodent control is a well-known phenomenon. Exposure can be primary when non-target species consume bait or secondary via uptake of poisoned animals by mammalian and avian predators. However, nothing is known about the exposure patterns in passerine birds that are commonly present on farms where rodent control is conducted.We used liquid chromatography coupled with tandem mass spectrometry to screen for residues of anticoagulant rodenticides (ARs) in liver tissue of passerine birds that were present during rodent control with a product containing brodifacoum (BR). The 222 birds of 13 species were bycatch of rodent snap trapping in 2011–2013 on 11 livestock farms run synchronously with baiting.During baiting, ARs were detected in about 30% of birds; 28% carried BR. In liver tissue of 54 birds that carried BR, concentrations ranged from 4 to 7809 ng/g (mean 490 ± 169 ng/g). Among common bird species with AR residues, BR was most prevalent in robins (Erithacus rubecula) (44%) and dunnocks (Prunella modularis) (41%). Mean BR concentration was highest in great tits (Parus major) (902 ± 405 ng/g). The occurrence and concentrations of BR residues were about 30% higher in birds collected close to bait stations compared to birds collected further away.The results demonstrate that several ground feeding songbird species are exposed to ARs used on farms. If BR was present in liver tissue, concentrations were variable, which may imply a combination of primary and secondary exposure of songbirds. Exposure was mostly restricted to the immediate surroundings of farms where bait was used, which might limit the transfer to the wider environment. Efforts should be made to reduce the access for birds to AR bait to prevent high exposure.

Linking landscape composition and biological factors with exposure levels of rodenticides and agrochemicals in avian apex predators from Germany

Intensification of agricultural practices has resulted in a substantial decline of Europe's farmland bird populations. Together with increasing urbanisation, chemical pollution arising from these land uses is a recognised threat to wildlife. Raptors are known to be particularly sensitive to pollutants that biomagnify and are thus frequently used sentinels for pollution in food webs. The current study focussed on anticoagulant rodenticides (ARs) but also considered selected medicinal products (MPs) and frequently used plant protection products (PPPs). We analysed livers of raptor species from agricultural and urban habitats in Germany, namely red kites (MIML; Milvus milvus), northern goshawks (ACGE; Accipiter gentilis) and Eurasian sparrowhawks (ACNI; Accipiter nisus) as well as white-tailed sea eagles (HAAL; Haliaeetus albicilla) and ospreys (PAHA; Pandion haliaetus) to account for potential aquatic exposures. Landscape composition was quantified using geographic information systems. The highest detection of ARs occurred in ACGE (81.3%; n = 48), closely followed by MIML (80.5%; n = 41), HAAL (38.3%; n = 60) and ACNI (13%; n = 23), whereas no ARs were found in PAHA (n = 13). Generalized linear models demonstrated (1) an increased probability for adults to be exposed to ARs with increasing urbanisation, and (2) that species-specific traits were responsible for the extent of exposure. For MPs, we found ibuprofen in 14.9% and fluoroquinolones in 2.3% in individuals that were found dead. Among 30 investigated PPPs, dimethoate (and its metabolite omethoate) and thiacloprid were detected in two MIML each. We assumed that the levels of dimethoate were a consequence of deliberate poisoning. AR and insecticide poisoning were considered to represent a threat to red kites and may ultimately contribute to reported decreased survival rates. Overall, our study suggests that urban raptors are at greatest risk for AR exposure and that exposures may not be limited to terrestrial food webs.

Sublethal dose of warfarin induction promotes the accumulation of warfarin resistance in susceptible Norway rats

Resistance to anticoagulant rodenticides in Norway rats has been primarily explained by selection on the mutations in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene. Whether warfarin resistance can be induced independent of Vkorc1 mutations and its role in the acquirement of warfarin resistance is unclear. In this study, we screened susceptible Norway rats with a sublethal dose of warfarin from the generation F0–F5 and found the warfarin tolerance level of the screened rats was significantly improved. Further, lethal feeding period (LFP) tests indicated 5.7–7.1% of screened rats were resistant, while all unscreened rats were susceptible. Analysis of the Vkorc1 polymorphisms suggested none of the screened F5 rats carried any potential warfarin resistance Vkorc1 mutations. As a fitness cost, the litter size of the screened rats tended to be smaller over generations, implicating the reproductive success could be impacted by warfarin induction. Our findings reveal that susceptible Norway rats can actively acquire inheritable warfarin resistance under constant warfarin induction without changing the Vkorc1 genotype. Further exploration of epigenetic and cryptic genetic mechanisms of warfarin resistance in those resistant rats is essential to fully understand the 'missing' heritability of warfarin resistance.