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The global impact of anticoagulant rodenticides (ARs) on non-target species is well-recognized. Birds of prey, as apex predators, are highly vulnerable to AR exposure and are widely used as biomonitors for priority pollutants in Europe. This study investigates differential SGAR exposure in raptors from insular versus continental regions, hypothesizing greater exposure in insular areas due to ecological factors like reduced prey diversity, intensive rodenticide use, and resistant rodent populations. We analyzed the livers of 190 common kestrels (Falco tinnunculus) and 104 common buzzards (Buteo buteo) across the Iberian Peninsula and its archipelagos using LC-MS/MS to assess their role as AR sentinels and the differences between insular and continental areas. Results revealed a high prevalence (>80%) of second-generation anticoagulant rodenticides (SGARs), with brodifacoum and bromadiolone, being the most frequent. Multiple SGAR detections were also common (≈50%). A binomial logistic regression showed that species and region significantly influence the likelihood of SGAR exposure. Kestrels had a greater probability of exceeding 100 ng/g wet weight (ww) compared to buzzards. Raptors from insular territories were ten times more likely to have higher SGAR concentrations than those from continental areas. However, the legal restriction on SGAR bait concentrations that came into effect in 2018 did not significantly impact exposure levels. This study highlights the need for targeted conservation efforts to mitigate AR exposure risk in vulnerable island ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Anticoagulant rodenticides (ARs) are potent pesticides acting as vitamin K epoxide reductase inhibitors causing haemorrhaging or external bleeding from orifices and/or skin lesions in intoxicated rodents. However, their non-selective mode of action makes them particularly harmful for non-target wildlife, which may be exposed to ARs via ingestion of AR-containing baits (primary exposure), feeding on AR-intoxicated rodents and carrions (secondary exposure), consuming AR-contaminated necrophagous species (tertiary exposure), and exposure to surface waters receiving baited sewer systems and ARs from outdoor-placed traps after heavy rain events. In the present study, we assessed the suitability of hairs as a non-invasive matrix for monitoring the possible exposure of mammals to ARs with a focus on the first-generation anticoagulant rodenticides (FGARs) warfarin, coumatetralyl, and chlorophacinone and the second-generation anticoagulant rodenticides (SGARs) brodifacoum, bromadiolone, difenacoum, flocoumafen, and difethialone. The Red fox (n = 24) was selected as the species representing the potentially exposed non-target wildlife in a littoral area of Northern Italy along the Adriatic coast (Cavallino-Treporti municipality). Half (n = 12) of the analysed hair samples were positive for at least one of the targeted ARs, with a higher prevalence of SGARs (n = 11; 46%) compared to FGARs (n = 1; 4%). The most frequently quantified ARs were brodifacoum (25%), difethialone (13%), and flocoumafen (13%), with concentrations ranging from 0.08 ng g -1 (difethialone) to 0.96 ng g -1 (brodifacoum). These data documented that a relevant part of the Red foxes living in the study area were exposed to ARs and, most importantly, provided the first evidence that hair residues can be used as a non-invasive matrix for assessing the possible exposure of mammals to ARs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pesticide contamination poses a significant threat to non-target wildlife, including amphibians, many of which are already highly threatened. This study assessed the extent of pesticide exposure in dead frogs collected during a mass mortality event across eastern New South Wales, Australia between July 2021 and March 2022. Liver tissue from 77 individual frogs of six species were analysed for >600 legacy and contemporary pesticides, including rodenticides. More than a third (36 %) of the liver samples contained at least one of the following pesticides: brodifacoum, dieldrin, DDE, heptachlor/heptachlor epoxide, fipronil sulfone, and 2-methyl-4-chlorophenoxyacetic acid (MCPA). Brodifacoum, a second-generation anticoagulant rodenticide, was found in four of the six frog species analysed: the eastern banjo frog (Limnodynastes dumerilii), cane toad (Rhinella marina), green tree frog (Litoria caerulea) and Peron's tree frog (Litoria peronii). This is the first report of anticoagulant rodenticide detected in wild amphibians, raising concerns about potential impacts on frogs and extending the list of taxa shown to accumulate rodenticides. Dieldrin, a banned legacy pesticide, was also detected in two species: striped marsh frog (Limnodynastes peronii) and green tree frog (Litoria caerulea). The toxicological effects of these pesticides on frogs are difficult to infer due to limited comparable studies; however, due to the low frequency of detection the presence of these pesticides was not considered a major contributing factor to the mass mortality event. Additional research is needed to investigate the effects of pesticide exposure on amphibians, particularly regarding the impacts of second-generation anticoagulant rodenticides. There is also need for continued monitoring and improved conservation management strategies for the mitigation of the potential threat of pesticide exposure and accumulation in amphibian populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Second-generation anticoagulant rodenticides (SGARs) are widely used to control rodent populations, resulting in the serious secondary exposure of predators to these contaminants. In the United Kingdom (UK), professional use and purchase of SGARs were revised in the 2010s. Certain highly toxic SGARs have been authorized since then to be used outdoors around buildings as resistance-breaking chemicals under risk mitigation procedures. However, it is still uncertain whether and how these regulatory changes have influenced the secondary exposure of birds of prey to SGARs. Based on biomonitoring of the UK Common Buzzard ( Buteo buteo ) collected from 2001 to 2019, we assessed the temporal trend of exposure to SGARs and statistically determined potential turning points. The magnitude of difenacoum decreased over time with a seasonal fluctuation, while the magnitude and prevalence of more toxic brodifacoum, authorized to be used outdoors around buildings after the regulatory changes, increased. The summer of 2016 was statistically identified as a turning point for exposure to brodifacoum and summed SGARs that increased after this point. This time point coincided with the aforementioned regulatory changes. Our findings suggest a possible shift in SGAR use to brodifacoum from difenacoum over the decades, which may pose higher risks of impacts on wildlife.

Bromadiolone and brodifacoum, the most frequently used anticoagulant rodenticides, are highly toxic and pose a threat to public health by causing food poisoning incidents. Here, we developed a fluorescence microplate immunoassay for facile and sensitive detection of bromadiolone and brodifacoum by introducing three commercial chemicals (p-phenylenediamine, polyethyleneimine, H 2 O 2 ) as a new substrate of horseradish peroxidase and then generating fluorescence signals based on an in situ fluorogenic reaction (detection time within 75 min). This assay exhibited higher efficiency in generating fluorescence signals, thereby exhibiting a 6-fold improvement in sensitivity compared with colorimetric ELISA. The limit of detection was 0.23-0.28 ng/mL (ng/g) for bromadiolone and 0.34-0.61 ng/mL (ng/g) for brodifacoum in corn and human serum, with recovery ratios higher than 82.3 %. These satisfactory results illustrated our proposed assay was a potential tool for food analysis and poisoning diagnosis caused by bromadiolone and brodifacoum., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The study deals with the environmental residues of anticoagulant rodenticides (ARs) in Slovenia to evaluate the toxicological risk of secondary poisoning of red foxes (Vulpes vulpes) as representatives of non-target wildlife, and in relation to the investigated use patterns of ARs and specific local parameters in Slovenia. From 2019 to 2022, 148 liver tissue samples of adult red foxes were collected from almost all state geographical regions. The samples were extracted with methanol/water (2:1, v/v), cleaned-up using a solid supported liquid-liquid extraction, and measured by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) with reporting limits of 0.5 to 5.0 ng/g. Residues of at least one rodenticide were detected in 77.7 % of the samples. The second generation ARs of bromadiolone, brodifacoum and difenacoum were the most frequently found, appearing in 75.0, 51.4, and 18.9 % of the samples, respectively. Concentrations of pooled ARs ranged from 1.5 to 2866.5 ng/g with mean and median values of 601.4 and 350.2 ng/g, respectively. We determined bromadiolone and brodifacoum at concentrations of ≥800 ng/g in 10.8 and 10.1 % of the samples, and 1.4 and 0.7 % of the samples contained residues >2000 ng/g, respectively. These concentrations are much higher than those found in comparable studies in Europe and elsewhere in the world. Residues of ARs were detected in all monitored statistical regions of Slovenia, with higher concentrations in the eastern parts of the country. First generation ARs were found in only 9.5 % of samples, and residues were below 10 ng/g with one exception (coumatetralyl with 55 ng/g). The results of the study indicate a serious toxicological risk for red foxes in Slovenia as part of the Western Balkans, and will contribute to the growing body of knowledge about the protection of European ecosystems, as wildlife is not limited by national borders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Second-generation anticoagulant rodenticides (SGARs) are persistent chiral pesticides used to control rodent populations. Raptors are protected species and may be exposed through the ingestion of rodents contaminated with SGARs. Commercial formulations of SGARs are a mixture of four stereoisomers (E1, E2, E3, E4): the cis- and trans-diastereoisomers are each a racemic mixture of two enantiomers. In this study, the residue levels of all SGARs (bromadiolone, difenacoum, brodifacoum, difethialone, flocoumafen) were evaluated in the liver of 529 raptor carcasses. All species (n = 18) and 75 % of individuals (n = 396) were SGAR positive and 29 % (n = 154) had summed hepatic concentrations above 100 ng/g ww. Concentrations were higher for predators with facultative scavenging behaviors than for predators and obligate scavengers. Bromadiolone, brodifacoum and difenacoum had equivalent hepatic prevalence (between 48.9 and 49.9 %), and difethialone was detected less frequently (31.7 %). Concentrations and enantiomeric fractions of the four stereoisomers of all SGARs are described in to demonstrate the biological enantioselectivity of these chiral pesticides in the food chain. A difference was observed between the proportions of SGARs diastereoisomers and stereoisomers in the liver of all raptor species and in commercial baits. The enantioselective bioaccumulation of E1-trans-bromadiolone, E3-cis-brodifacoum, E1-cis-difenacoum and E3-cis-difethialone was characterized and represented 96.8 % of total SGARs hepatic residues. While hepatic concentrations were heterogeneous, the proportions of stereoisomers and diastereoisomers were homogeneous with no inter-individual or inter-species differences (only E1-trans-bromadiolone is present in hepatic residues). However, proportions of brodifacoum stereoisomers and diastereoisomers were more scattered, probably due to their slower elimination. This could provide an opportunity to date the exposure of individuals to brodifacoum. We highlight the need to consider each SGAR as four molecular entities (four stereoisomers) rather than one. These findings suggest new commercial formulations with the less persistent stereoisomers could reduce secondary exposure of non-target species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs), particularly second-generation compounds (SGARs), are extensively used in pest management, impacting non-target wildlife. The California kingsnake (Lampropeltis californiae), an invasive species in Gran Canaria, is under a control plan involving capture and euthanasia. This research aimed to detect 10 different ARs in these snakes, explore geographical and biometrical factors influencing AR exposure, and assess their potential as sentinel species for raptors, sharing similar foraging habits. Liver samples from 360 snakes, euthanized between 2021 and 2022, were analysed for ARs using LC-MS/MS. Results showed all detected rodenticides were SGARs, except for one instance of diphacinone. Remarkably, 90 % of the snakes tested positive for ARs, with over half exposed to multiple compounds. Brodifacoum was predominant, found in over 90 % of AR-positive snakes, while bromadiolone and difenacoum were also frequently detected but at lower levels. The study revealed that larger snakes and those in certain geographic areas had higher AR concentrations. Snakes in less central or more peripheral areas showed lower levels of these compounds. This suggests a correlation between the snakes' size and distribution with the concentration of ARs in their bodies. The findings indicate that the types and prevalence of ARs in California kingsnakes on Gran Canaria mirror those in the island's raptors. This similarity suggests that the kingsnake could serve as a potential sentinel species for monitoring ARs in the ecosystem. However, further research is necessary to confirm their effectiveness in this role., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This study assesses the impact of second-generation anticoagulant rodenticides (SGARs) on the common kestrel (Falco tinnunculus canariensis) in Tenerife, Canary Islands. The analysis of 390 liver samples over 19 years using HPLC-MS/MS showed that 93.1 % of kestrels were exposed to SGARs in this island. A notable shift in SGAR profiles was observed, with bromadiolone and flocoumafen decreasing, while brodifacoum levels increased sharply from 2018 onwards. Comparatively, Tenerife kestrels had a higher detection frequency of SGARs (93.1 %) than those in the rest of the islands of the archipelago (68.2 %), with median concentrations nearly double (∑AR = 180.9 vs 102.4 ng/g liver, P < 0.0001). Furthermore, on average, kestrels from Tenerife were found to have a higher number of different rodenticide compounds per individual. A Generalized Linear Model (GLM) analysis revealed that several factors contribute to the likelihood of SGAR exposure: being an adult kestrel, the enactment of legal restrictions on SGAR bait concentrations in 2018, higher livestock density, and greater human population density. These findings suggest that both bioaccumulation over the birds' lifespans and environmental factors related to human and agricultural activity are influencing the levels of SGARs detected. Alarmingly, 44.7 % of kestrels had SGAR levels above the toxicity threshold established for other raptor species (200 ng/g liver), signaling a high poisoning risk. This is despite EU regulations to protect wildlife, with our findings indicating an increase in both exposure rates and SGAR concentrations since these laws were enacted. The data highlight a critical environmental threat to endemic species on islands like Tenerife. The common kestrel, not considered globally endangered, is nonetheless facing regional threats from SGAR contamination. These results emphasize the urgent need for effective regulations to address the persistent and growing impact of SGARs on island biodiversity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Rodent control strategies are primarily based on the use of anticoagulant rodenticides (ARs), making them widely used worldwide. However, due to their high toxicity and availability, ARs are among the leading causes of animal poisoning in Europe. They are the primary agents involved in intoxication in cats and the second in dogs. Additionally, their long persistence in the body can lead to secondary exposure, particularly in wild predators. The laboratory findings and clinical signs of intoxication can range from increased clotting time (prolonged prothrombin time and activated partial thromboplastin time) to severe bleeding and death. Despite the prevalence and severity of this intoxication, only a few methods are available for the identification and quantification of ARs in animals, and most of them are suitable only for post-mortem diagnosis. In this study, we present the validation of a rapid and sensitive method for the identification and quantification of ARs in animal whole blood, using a small sample volume. The developed LC-MS/MS method demonstrated high accuracy and precision at the limit of quantification (LOQ), as well as at low, medium, and high concentrations. It exhibited higher sensitivity (LOQ 0.1 - 0.3 ng/mL) compared to previously published methods. After validation, the method was successfully applied to real cases of suspected poisoning events, resulting in the identification of several positive samples. The examples presented in this study highlight the utility of this method for diagnosis and follow-up, emphasizing the importance of method sensitivity in order to avoid misclassifying truly positive samples as negative., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs) influence predator populations and threaten the stability of ecosystems. Understanding the prevalence and impact of rodenticides in predators is crucial to inform conservation planning and policy. We collected dead birds of four nocturnal predatory species across differing landscapes: forests, agricultural, urban. Liver samples were analysed for eight ARs: three First Generation ARs (FGARs) and five SGARs (Second Generation ARs). We investigated interspecific differences in liver concentrations and whether landscape composition influenced this. FGARs were rarely detected, except pindone at low concentrations in powerful owls Ninox strenua. SGARs, however, were detected in every species and 92 % of birds analysed. Concentrations of SGARs were at levels where potential toxicological or lethal impacts would have occurred in 33 % of powerful owls, 68 % of tawny frogmouths Podargus strigoides, 42 % of southern boobooks N. bookbook and 80 % of barn owls Tyto javanica. When multiple SGARs were detected, the likelihood of potentially lethal concentrations of rodenticides increased. There was no association between landscape composition and SGAR exposure, or the presence of multiple SGARs, suggesting rodenticide poisoning is ubiquitous across all landscapes sampled. This widespread human-driven contamination in wildlife is a major threat to wildlife health. Given the high prevalence and concentrations of SGARs in these birds across all landscape types, we support the formal consideration of SGARs as a threatening process. Furthermore, given species that do not primarily eat rodents (tawny frogmouths, powerful owls) have comparable liver rodenticide concentrations to rodent predators (southern boobook, eastern barn owl), it appears there is broader contamination of the food-web than anticipated. We provide evidence that SGARs have the potential to pose a threat to the survival of avian predator populations. Given the functional importance of predators in ecosystems, combined with the animal welfare impacts of these chemicals, we propose governments should regulate the use of SGARs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs) are used to control rodent populations. Poisoning of non-target species can occur by accidental consumption of commercial formulations used for rodent control. A robust method for determining ARs in animal tissues is important for animal postmortem diagnostic and forensic purposes. We evaluated an ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) method to quantify 8 ARs (brodifacoum, bromadiolone, chlorophacinone, coumachlor, dicoumarol, difethialone, diphacinone, warfarin) in a wide range of animal (bovine, canine, chicken, equine, porcine) liver samples, including incurred samples. We further evaluated UPLC-MS in 2 interlaboratory comparison (ILC) studies; one an ILC exercise (ICE), the other a proficiency test (PT). The limits of detection of UPLC-MS were 0.3-3.1 ng/g, and the limits of quantification were 0.8-9.4 ng/g. The recoveries obtained using UPLC-MS were 90-115%, and relative SDs were 1.2-13% for each of the 8 ARs for the 50, 500, and 2,000 ng/g spiked liver samples. The overall accuracy from the laboratories participating in the 2 ILC studies (4 and 11 laboratories for ICE and PT studies, respectively) were 86-118%, with relative repeatability SDs of 3.7-11%, relative reproducibility SDs of 7.8-31.2%, and Horwitz ratio values of 0.5-1.5. Via the ILC studies, we verified the accuracy of UPLC-MS for AR analysis in liver matrices and demonstrated that ILC can be utilized to evaluate performance characteristics of analytical methods.

A specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the anticoagulant rodenticide diphacinone (DPN) in mouse and rat liver. Tissue samples were extracted with a mixture of water and acetonitrile containing ammonium hydroxide. The extracted sample was cleaned up with a combination of liquid-liquid partitioning and dispersive solid phase extraction. Chromatographic separation was achieved using a Waters X-Bridge BEH C-18 LC column (50 mm, 2.1 mm ID, 2.5 μm particle size) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for DPN was m/z 339.0 → 167.0 for quantitation and 339.0 → 172.0 and 339.0 → 116.0 for confirmation. The linear range was 0.5 to 375 ng/mL. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97% - 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). The recovery of DPN fortified at 3 different levels averaged 106% in rat liver and 101% in mouse liver. The established method was successfully used to determine DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. The observed residue levels were consistent with values observed in other rodent studies. However, the amount of bait consumed was lower for the novel baits evaluated in this study., Competing Interests: The authors have no competing interests to the publication of this work., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Owls (Strigiformes) provide myriad ecosystem services and are sentinels for ecosystem health. However, they are at continued risk from anthropogenic threats such as vehicle collisions, entanglement with human-made materials, and exposure to anticoagulant rodenticides (ARs), a widespread pesticide known to affect owls. Texas is an important region for numerous migratory and non-migratory owl species in the United States (US), yet assessments of threats owls face here are lacking preventing the development of informed conservation strategies. This study coupled assessment of admittance data from two wildlife rehabilitation centers in Texas with AR liver screening to (1) identify which species of owls are commonly admitted, (2) evaluate seasonality of admittance, and (3) assess causes of admittance for owls in Texas. Between 2010 and 2021, 1,620 owls were admitted into rehabilitation, representing eight species of which the Great-horned Owl (Bubo virginianus) was the most common. For all owls combined admittance rates were highest in the spring, driven by an influx of juveniles (n = 703, 43.40%). The leading cause of admittance amongst species was 'no apparent injury' (n = 567, 34.94%). Where clear diagnoses could be made, the leading causes of admittances were 'entrapment in human infrastructure' (n = 100, 6.11%) and 'collision with vehicles' (n = 74, 4.56%). While the admittance data did not reveal any cases of AR poisoning, liver screening demonstrated high incidences of AR exposure; of 53 owls screened for ARs, 50.94% (n = 27) tested positive with 18 showing exposure to multiple ARs. Brodifacoum was the most frequently detected AR (n = 19, 43.18%) and seven owls (25.93%) tested positive within lethal ranges. Our results suggest that owls in Texas are at risk from myriad anthropogenic threats and face high exposure rates to ARs. In doing so, our results can inform conservation strategies that mitigate anthropogenic threats faced by owls in Texas and beyond., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gomez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Anti-coagulant rodenticides (ARs) are commonly utilized for controlling rodent populations; however, non-target companion and wildlife animals are also exposed. A method was developed for quantitation of seven ARs (chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone and warfarin) and dicoumarol (a naturally occurring anti-coagulant) in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (negative mode) combined with multiple reaction monitoring. In-house method validation in the originating laboratory using non-blinded samples revealed method limits of quantitation at 2.5 ng/mL for all analytes. The inter-assay accuracy ranged from 99% to 104%, and the relative standard deviation ranged from 3.5% to 20.5%. Method performance was then verified in the originating laboratory during an exercise organized by an independent party using blinded samples. The method was successfully transferred to two naïve laboratories and further evaluated for reproducibility among three laboratories by means of Horwitz ratio (HorRat(R)) values. Such extensive validation provides a high degree of confidence that the method is rugged, robust, and will perform as expected if used by others in the future., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Midway Atoll in the Northwestern Hawaiian Islands is home to ground nesting birds that are threatened by invasive mice. Planned rodent eradication efforts for the island involve aerial application of cereal bait pellets containing the chemical rodenticide brodifacoum. Given the nature of the application method, drift of cereal bait pellets into the coastal waters surrounding Midway Atoll is unavoidable. To understand whether cereal bait pellets impact marine invertebrates, gametes and larvae of the reef-building coral Montipora capitata were exposed to brodifacoum, cereal bait pellets containing brodifacoum, and inert cereal bait pellets without the rodenticide. Fertilization success and larval survival were assessed at nominal brodifacoum concentrations of 1, 10, and 100 ppb. Fertilization success decreased by 15% after exposure to 100 ppb brodifacoum solutions. Larval survival was not reduced by exposure to brodifacoum solutions. Cereal bait pellets containing brodifacoum reduced fertilization success at 10 ppb brodifacoum in 0.4 g per L pellet solutions by 34.84%, and inhibited fertilization at 100 ppb brodifacoum in 4 g of pellet per L solution. Inert cereal bait pellets had similar effects, reducing fertilization success at 0.4 g of pellet per L by 40.50%, and inhibiting fertilization at 4 g per L pellet solutions. Larval survival was reduced by >43% after prolonged exposure to 4 g per L pellet solutions. The highest concentration used in this study was meant to represent an extreme and unlikely condition resulting from an accidental spill. Our findings indicate large amounts of cereal bait pellets entering the coastal environment of Midway Atoll, if occurring during a coral spawning event, would reduce coral reproduction by decreasing fertilization success. It is difficult to know the ecologically relevant concentrations of cereal bait pellets in coastal environments due to unavoidable bait drift after land applications, but results indicate small amounts of pellet drifting into coastal environments would not severely reduce coral reproductive capacity. Best management practices should consider known coral reproductive periods when scheduling applications of pellets on tropical islands to reduce the risk of negative impacts of large-scale accidents on corals., Competing Interests: The authors declare that they have no competing interests., (© 2022 Barkman and Richmond.)

Numerous cases of wildlife exposure to five second-generation anticoagulant rodenticides have been reported worldwide, and residues of these chiral pesticides in biological matrices are still quantified by achiral liquid chromatography methods. However, they are a mixture of cis- and trans-diastereomers, thus a mixture of four stereoisomers. Their persistence must be evaluated in a differentiated way in the food chain of concerned predator species in order to reduce the environmental impact. This article presents an evaluation of the chiral selectivity of five polysaccharide-based chiral selectors for the four stereoisomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone. Different chromatographic parameters, influencing the chiral separation, such as organic modifier (acetonitrile, methanol), percentage of formic acid and water content in the mobile phase are systematically tested for all columns. It was shown that little amount of water added to the acetonitrile mobile phase may influence the retention behaviors between reversed phase and HILIC-like modes, and consequently the enantiomer elution order of the four stereoisomers. On the contrary, reversed phase is always the observed mode for the methanol water mobile phase. A suitable combination of all these parameters is presented for each second-generation anticoagulant rodenticide with a description of the enantioresolution, the enantiomer elution order and the retention times of the respective stereoisomers. A method is validated for all stereoisomers of each second-generation anticoagulant rodenticide with chicken liver and according to an official bioanalytical guideline. As an example, the enantiomer fraction is evaluated in the liver of a raptor species (rodent predator) exposed to bromadiolone and difenacoum. The results showed that only one enantiomer of trans-bromadiolone and one enantiomer of cis-difenacoum is present in hepatic residues, although all four stereoisomers are present in bromadiolone and difenacoum rodenticide baits., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

As the dominant means for control of pest rodent populations globally, anticoagulant rodenticides (ARs), particularly the second-generation compounds (SGARs), have widely contaminated nontarget organisms. We present data on hepatic residues of ARs in 741 raptorial birds found dead or brought into rehabilitation centers in British Columbia, Canada, over a 30-year period from 1988 to 2018. Exposure varied by species, by proximity to residential areas, and over time, with at least one SGAR residue detected in 74% of individuals and multiple residues in 50% of individuals. By comparison, we detected first-generation compounds in <5% of the raptors. Highest rates of exposure were in barred owls (Strix varia), 96%, and great horned owls (Bubo virginianus), 81%, species with diverse diets, including rats (Rattus norvegicus and Rattus rattus), and inhabiting suburban and intensive agricultural habitats. Barn owls (Tyto alba), mainly a vole (Microtus) eater, had a lower incidence of exposure of 65%. Putatively, bird-eating raptors also had a relatively high incidence of exposure, with 75% of Cooper's hawks (Accipiter cooperii) and 60% of sharp-shinned hawks (Accipiter striatus) exposed. Concentrations of SGARs varied greatly, for example, in barred owls, the geometric mean ∑SGAR = 0.13, ranging from <0.005 to 1.81 μg/g wet weight (n = 208). Barred owls had significantly higher ∑SGAR concentrations than all other species, driven by significantly higher bromadiolone concentrations, which was predicted by the proportion of residential land within their home ranges. Preliminary indications that risk mitigation measures implemented in 2013 are having an influence on exposure include a decrease in mean concentrations of brodifacoum and difethialone in barred and great horned owls and an increase in bromodialone around that inflection point. Environ Toxicol Chem 2022;41:1903-1917. © 2022 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada., (© 2022 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.)

Wildlife exposures to pest controlling substances have resulted in population declines of many predatory species during the past decades. Many pesticides were subsequently classified as persistent, bioaccumulative, and toxic (PBT) and banned on national or global scales. However, despite their risks for non-target vertebrate wildlife, PBT substances such as anticoagulant rodenticides (ARs) are still permitted for use in Europe and have shown to threaten raptors. Whereas risks of ARs are known, much less information is available on emerging agrochemicals such as currently used PPPs and medicinal products (MPs) in higher trophic level species. We expect that currently used PPPs are relatively mobile (vs. lipophilic) as a consequence of the PBT criteria and thus more likely to be present in aqueous matrices. We therefore analyzed blood of 204 raptor nestlings of three terrestrial (red kite, common buzzard, Montagu's harrier) and two aquatic species (white-tailed sea eagle, osprey) from Germany. In total, we detected ARs in 22.6% of the red kites and 8.6% of the buzzards, whereas no Montagu's harriers or aquatic species were exposed prior to sampling. ΣAR concentration tended to be higher in North Rhine-Westphalia (vs. North-Eastern Germany) where population density is higher and intense livestock farming more frequent. Among the 90 targeted and currently used PPPs, we detected six substances from which bromoxynil (14.2%) was most frequent. Especially Montagu's harrier (31%) and red kites (22.6%) were exposed and concentrations were higher in North Rhine-Westphalia as well. Among seven MPs, we detected ciprofloxacin (3.4%), which indicates that risk mitigation measures may be needed as resistance genes were already detected in wildlife from Germany. Taken together, our study demonstrates that raptors are exposed to various chemicals during an early life stage depending on their sampling location and underpins that red kites are at particular risk for multiple pesticide exposures in Germany., (© 2022. The Author(s).)

Fortunately, the intentional contamination of food or water supplies out of criminal or terroristic motivation is a rather rare event. However, in the face of asymmetric warfare and as the consequences of such an event would be severe, food defence as a necessary supplement to food safety is gaining increased attention. While some progress has been made in developing non-target detection devices, the contamination of food or water supplies using readily available rodenticides may still be revealed only by complex analytical techniques. The presented study therefore aimed to develop a quick and easy screening method for the detection of sixteen globally common rodenticides in foodstuffs. Robust operation with limited personnel and analytical resources were one benchmark to be met by the method, which uses a slightly modified QuEChERS (quick, easy, cheap, effective, rugged, safe) protocol for dispersive solid-phase extraction and subsequent ion-pair chromatography with diode-array and fluorescence detection. Quantification limits were as low as 5 µg/kg with satisfying bias (recovery) and repeatability rates of 77 to 117% and 1.8 to 17.1%, respectively. The developed method provides reliable and robust detection of these deadly poisons at toxic concentrations, which was demonstrated impressively in an improvised assault scenario., (© 2022. The Author(s).)

Alpha-chloralose (AC) is used as a rodenticide as well as an anesthetic agent in laboratory animals. It was previously also used as an avicide. Detection of AC in blood samples or in body tissues collected postmortem is key for the diagnosis of clinical cases and a requirement for surveillance of secondary toxicosis, including potential cases in wild animals. Reports on poisoning of humans and non-laboratory animals confirmed by the detection of AC or its metabolites are available, however poisoning of domestic animals are rarely available. Furthermore, reports on clinical cases in domestic animals rarely report quantifications of AC in blood or body tissues. The present study describes the validation of a quantitative ultra high performance liquid chromatography--tandem mass spectrometry (UHPLC--MS-MS) method that can be used in cases of suspected AC poisoning in cats. The validation study showed the method to be fit for purpose. In serum, the limit of quantification was 100 ng/mL and the limit of detection was 30 ng/mL. The new analytical method was applied on blood samples collected from 20 individual cats with a preliminary clinical diagnosis of acute AC poisoning. AC was confirmed in all 20 feline blood samples, and the concentration range of AC was 538-17,500 ng/mL. The quantitative method developed in this study was found to be a fast and selective method for confirmation of AC poisoning using blood samples from cats., (© The Author(s) 2021. Published by Oxford University Press.)

Since rodenticides represent a substance group relevant in toxicological analyses, the aim of this work was the development of a complex multi-target screening strategy for the identification with liquid chromatography-tandem mass spectrometry. A simple protein precipitation was used as the sample preparation strategy. Further, a Luna 5 μm C18 (2) 100 Å, 150 × 2 mm analytical column was applied for the separation of relevant analytes with a Shimadzu HPLC. Signal detection was performed with a SCIEX API 5500 QTrap MS/MS system. The rodenticides investigated (α-chloralose, brodifacoum, bromadiolone, coumatetralyl, difenacoum, and warfarin) could be incorporated effectively into a multi-target screening strategy covering about 250 substances representing different groups with a limit of detection appropriate for substance identification. The strategy can easily be modified to perform semi-quantitative measurements for this substance group and could be supplemented by quantification based on standard addition., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The powerful owl (Ninox strenua) is a threatened apex predator that consumes mainly arboreal marsupial prey. Low density populations reside in urban landscapes where their viability is tenuous. The catalyst for this research was the reported death of eight powerful owls around Melbourne, Australia, in less than one year (2020/2021). Eighteen deceased owls were toxicologically screened. We assessed toxic metals (Mercury Hg, Lead Pb, Cadmium Cd and Arsenic As) and anticoagulant rodenticides (ARs) in liver (n = 18 owls) and an extensive range of agricultural chemicals in muscle (n = 14). Almost all agricultural chemicals were below detection limits except for p,p-DDE, which was detected in 71% of birds at relatively low levels. Toxic metals detected in some individuals were generally at low levels. However, ARs were detected in 83.3% of powerful owls. The most common second-generation anticoagulant rodenticide (SGAR) detected was brodifacoum, which was present in every bird in which a rodenticide was detected. Brodifacoum was often present at toxic levels and in some instances at potentially lethal levels. Presence of brodifacoum was detected across the complete urban-forest/agriculture gradient, suggesting widespread exposure. Powerful owls do not scavenge but prey upon arboreal marsupials, and generally not rodents, suggesting that brodifacoum is entering the powerful owl food web via accidental or deliberate poisoning of non-target species (possums). We highlight a critical need to investigate SGARs in food webs globally, and not just in species directly targeted for poisoning or their predators., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs) are used globally to control rodent pest infestations in both urban and agricultural settings. It is well documented that non-target wildlife, including predatory birds, are at risk for secondary anticoagulant exposure and toxicosis through the prey they consume. However, there have been no large-scale studies of AR exposure in raptors in Ontario, Canada since new Health Canada legislation was implemented in 2013 in an attempt to limit exposure in non-target wildlife. Our objective was to measure levels of ARs in wild raptors in southern Ontario to assess their exposure. We collected liver samples from 133 raptors representing 17 species submitted to the Canadian Wildlife Health Cooperative (CWHC) in Ontario, Canada, between 2017 and 2019. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively assess the level of exposure to 14 first- and second-generation ARs. Detectable levels of one or more ARs were found in 82 of 133 (62%) tested raptors, representing 12 species. The most commonly detected ARs were bromadiolone (54/133), difethialone (40/133), and brodifacoum (33/133). Of AR-positive birds, 34/82 (42%) contained residues of multiple (> 1) anticoagulant compounds. Our results indicate that AR exposure is common in raptors living in southern Ontario, Canada. Our finding that brodifacoum, difethialone, and bromadiolone were observed alone or in combination with one another in the majority of our sampled raptors indicates that legislative changes in Canada may not be protecting non-target wildlife as intended., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Wild raptors are widely used to assess exposure to different environmental contaminants, including anticoagulant rodenticides (ARs). ARs are used on a global scale for rodent control, and act by disruption of the vitamin K cycle that results in haemorrhage usually accompanied by death within days. Some ARs are highly persistent and bioaccumulative, which can cause significant exposure of non-target species. We characterized AR exposure in a heterogeneous sample of dead raptors collected over 12 years (2008-2019) in south-eastern France. Residue analysis of 156 liver samples through LC-MS/MS revealed that 50% (78/156) were positive for ARs, with 13.5% (21/156) having summed second-generation AR (SGAR) concentrations >100 ng/g ww. While SGARs were commonly detected (97.4% of positive samples), first-generation ARs were rarely found (7.7% of positive samples). ARs were more frequently detected and at greater concentration in predators (prevalence: 82.5%) than in scavengers (38.8%). Exposure to multiple ARs was common (64.1% of positive samples). While chlorophacinone exposure decreased over time, an increasing exposure trend was observed for the SGAR brodifacoum, suggesting that public policies may not be efficient at mitigating risk of exposure for non-target species. Haemorrhage was observed in 88 birds, but AR toxicosis was suspected in only 2 of these individuals, and no difference in frequency of haemorrhage was apparent in birds displaying summed SGAR levels above or below 100 ng/g ww. As for other contaminants, 17.2% of liver samples (11/64) exhibited Pb levels compatible with sub-clinical poisoning (>6 μg/g dw), with 6.3% (4/64) above the threshold for severe/lethal poisoning (>30 μg/g dw). Nine individuals with Pb levels >6 μg/g dw also had AR residues, demonstrating exposure to multiple contaminants. Broad toxicological screening for other contaminants was positive for 18 of 126 individuals, with carbofuran and mevinphos exposure being the suspected cause of death of 17 birds. Our findings demonstrate lower but still substantial AR exposure of scavenging birds compared to predatory birds, and also illustrate the complexity of diagnosing AR toxicosis through forensic investigations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rodenticides are toxic chemicals used to control rodent populations and are among the most common household toxicants. Ingestion of foods contaminated with rodenticides may cause severe illness or death in humans and animals. A rapid analytical method was developed for the identification of nine common rodenticides in foods using solid-liquid extraction followed by dispersive-solid phase extraction prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and UV detection. The method validation on a variety of food matrices including cornmeal, peanut, whole wheat flour and pork liver produced average recoveries between 91.2 and 107% with relative standard deviations between 2.6 and 14% for all studied rodenticides. The method detection limits ranged from 2.7 to 8.2 μg/kg (ppb) for eight rodenticides analyzed by LC-MS/MS and between 0.10 and 0.21 mg/kg (ppm) for bromethalin which was analyzed by LC with UV detection. This method could be useful in preparedness for emergency response situations involving widespread food contamination, terrorist acts or for forensic studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Anticoagulant rodenticides (ARs) used to control mammalian pest populations cause secondary exposure of predatory species throughout much of the world. It is important to understand the drivers of non-target AR exposure patterns as context for assessing long-term effects and developing effective mitigation for these toxicants. In Australia, however, little is known about exposure and effects of ARs on predators. We detected AR residues in 74% of 50 opportunistically collected carcasses of the Tasmanian wedge-tailed eagle (Aquila audax fleayi), an endangered apex predator. In 22% of birds tested, or 31% of those exposed, liver concentrations of second generation ARs (SGARs) were >0.1 mg/kg ww. Eagles were exposed to flocoumafen, a toxicant only available from agricultural suppliers, at an exceptionally high rate (40% of birds tested). Liver SGAR concentrations were positively associated with the proportion of agricultural habitat and human population density in the area around where each eagle died. The high exposure rate in a species not known to regularly prey upon synanthropic rodents supports the hypothesis that apex predators are vulnerable to SGARs. Our results indicate that AR exposure constitutes a previously unrecognized threat to Tasmanian wedge-tailed eagles and highlight the importance of efforts to address non-target AR exposure in Australia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Prior studies (2006-2016) in birds of prey admitted to a wildlife clinic in Massachusetts, USA, revealed widespread exposure to second-generation anticoagulant rodenticides (SGARs) among red-tailed hawks (Buteo jamaicensis, RTHAs). Continued monitoring of species for which historic data are available can reveal trends in exposure that aid in evaluating the effectiveness of risk-mitigation measures. While the majority of exposure-monitoring studies utilize liver tissue collected postmortem, antemortem modalities, such as serum analysis, may be desirable for risk assessments in certain populations. However, the sensitivity of serum for detecting anticoagulant rodenticides (ARs) is not well studied. Paired liver and serum samples from 43 RTHAs were evaluated from 2017 to 2019. In liver tissue, 100% of birds were positive for ARs, with the SGARs brodifacoum, bromadiolone, and difethialone identified most frequently; 91% of birds had liver residues of 2 to 4 ARs. These findings represent the highest exposure both to ARs overall and to multiple ARs in RTHAs compared to previous studies. All birds diagnosed with AR toxicosis (n = 14) were positive for ARs in serum; however, all subclinically exposed birds (n = 29) were negative in serum. These data show that exposure to SGARs remains widespread in RTHAs in this geographic area. In addition, although serum analysis is not sensitive for detecting sublethal exposures in RTHAs, it can potentially support a diagnosis of AR toxicosis in conjunction with other consistent signs. Environ Toxicol Chem 2020;39:2325-2335. © 2020 SETAC., (© 2020 SETAC.)

The 4-hydroxycoumarin derivatives are the most used rodenticides and act as classical anticoagulants, interfering with the production of clotting factors in liver by antagonizing the action of vitamin K reductase, thereby inhibiting recycling of vitamin K1, involved in activation of blood clotting factors, resulting in massive bleeding. In this paper, we present the case of a 72-year old man providing abnormal coagulation parameters (PT-INR between 16.1 and 19.1) after hospitalization. Blood samples tested positive for flocoumafen and difenacoum, two superwarfarin rodenticides. Patient's hair specimens, sampled 19 days after his hospitalization, showed that traces of both difenacoum and flocoumafen were detected in the first 1 cm; in the intermediate segments (1-2 and 2-3 cm), both difenacoum and flocoumafen were absent, while in the distal segment (3-4.5 cm), only difenacoum was found, but in significant amounts (140 pg/mg). Exposure to difenacoum in the past months, at least 4-5 before hospitalization, was confirmed by the presence of the rodenticide in the distal segment. Moreover, among the seized material, two specimens resulted compatible with the two rodenticides., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Superwarfarins, second-generation long-acting anticoagulant rodenticides, are 4-hydroxycoumarin analogues of warfarin that contain a large hydrophobic side chain. These compounds contain two chiral centers and are synthesized for commercial use as two pairs of diastereomer., Objective: To support studies of superwarfarin pharmacokinetics and other efforts to improve clinical care for poisoning victims, a quantitative assay was developed for the measurement of diastereomer of bromadiolone, difenacoum, flocoumafen, brodifacoum, and difethialone in human plasma., Method: Based on ultrahigh-pressure liquid chromatography-tandem mass-spectrometry (UHPLC-MS/MS), this method was validated according to U.S. Food and Drug Administration (FDA) guidelines. Sample preparation involved simple protein precipitation followed by reversed phase UHPLC, which resolved all five pairs of cis/trans diastereomer in less than 10 min. Superwarfarins were measured using negative ion electrospray followed by selected-reaction monitoring on a triple quadrupole mass spectrometer., Results: Calibration curves covered 3-4 orders of magnitude with linear regression coefficients of >0.999. The lower limits of quantitation were from 0.013 to 2.41 ng/mL, and intra-day and inter-day accuracy and precision coefficients of variation were <12%., Conclusions: A 10-min UHPLC-MS/MS assay was developed and validated for the separation and quantitative analysis of the pairs of diastereomer of five superwarfarins in human plasma., Highlights: This method was used to identify and measure superwarfarins and their cis/trans diastereomers in plasma obtained from patients treated for coagulopathy following consumption of contaminated synthetic cannabinoid products., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A seminal question in ecotoxicology is the extent to which contaminant exposure evokes prolonged effects on physiological function and fitness. A series of studies were undertaken with American kestrels ingesting environmentally realistic concentrations of the second-generation anticoagulant rodenticide (SGAR) brodifacoum. Kestrels fed brodifacoum at 0.3, 1.0, or 3.0 µg/g diet wet weight for 7 d exhibited dose-dependent hemorrhage, histopathological lesions, and coagulopathy (prolonged prothrombin and Russell's viper venom times). Following termination of a 7-d exposure to 0.5 µg brodifacoum/g diet, prolonged blood clotting time returned to baseline values within 1 wk, but brodifacoum residues in liver and kidney persisted during the 28-d recovery period (terminal half-life estimates >50 d). To examine the hazard of sequential anticoagulant rodenticide (AR) exposure, kestrels were exposed to either the first-generation AR chlorophacinone (1.5 µg/g diet) or the SGAR brodifacoum (0.5 µg/g diet) for 7 d and, following a recovery period, challenged with a low dose of chlorophacinone (0.75 µg/g diet) for 7 d. In brodifacoum-exposed kestrels, the challenge exposure clearly prolonged prothrombin time compared to naive controls and kestrels previously exposed to chlorophacinone. These data provide evidence that the SGAR brodifacoum may have prolonged effects that increase the toxicity of subsequent AR exposure. Because free-ranging predatory and scavenging wildlife are often repeatedly exposed to ARs, such protracted toxicological effects need to be considered in hazard and risk assessments. Environ Toxicol Chem 2020;39:468-481. © 2020 SETAC., (© 2019 SETAC.)

Rodents infest urban environments, causing damage and acting as vectors for disease transmission. Currently, anticoagulants are the most widely used chemical rodenticides, and their extensive and widespread use can contaminate the environment. To ensure effectiveness and avoid accumulation of rodent baits in the environment, it is important to evaluate how long rodent baits maintain their palatability and efficacy. In rodent control programs, rodent baits are placed in locations such as sewers, but after a few days, baits appear altered, causing doubts about the control efficacy. For this reason, baits are replaced periodically, which increases costs and generation of chemical waste. The objective of this study was to evaluate the palatability and efficacy of commercial paraffin-type rodent bait blocks placed in sewers in São Paulo City over a period of 90 days. Bait blocks were placed in sewers and collected after 30, 60, and 90 days. Additionally, in a laboratory two-choice test, wild-caught urban Norway rats were offered 40-60 g of bait and an equal volume of standard rat pellets. The amount of bait and rat pellet consumed was registered, the palatability was calculated, and the efficacy was measured as the percentage mortality over 14 days. The results showed that, even when they had an altered appearance, bait blocks remained palatable to the rats and were effective after at least 90 days. Leaving bait blocks for longer periods could be an effective strategy for reducing costs and could help to ensure the control of urban rodents in an environmentally sustainable way.

Anticoagulant rodenticides (ARs) are known to cause extensive secondary exposure in top predators in Europe and North America, but there remains a paucity of data in Asia. In this study, we collected 221 liver samples from 21 raptor species in Taiwan between 2010 and 2018. Most birds were recovered from rescue organizations, but some free-ranging individuals were obtained from bird-strike prevention measures at airports. ARs were detected in 10 species and more than half of the total samples. Common rodent-eating Black-winged Kites (Elanus caeruleus) had the highest prevalence (89.2%) and highest average sum concentration (0.211 ± 0.219 mg/kg), which was similar between free-ranging birds at airports and injured birds from rescue organizations. Scavenging Black Kites (Milvus migrans) and snake-eating Crested Serpent-eagles (Spilornis cheela) had the second highest prevalence or sum concentration, respectively. Seven different AR compounds were detected, of which brodifacoum was the most common and had the highest average concentration, followed by flocoumafen and bromadiolone. The frequency of occurrence in the three most numerous species (Black-winged Kite, Crested Goshawk [Accipiter trivirgatus], and Collared Scops-owl [Otus lettia]) was significantly higher in autumn than summer, which is consistent with the timing of the Taiwanese government's supply of free ARs to farmers. Regional differences in the detection of individual compounds also tended to reflect differences in human population density and use patterns (in agriculture or urban-dominated environments). Clinical poisoning was confirmed in Black Kites with sum concentrations as low as 0.026 mg/kg; however, further study of interspecific differences in AR sensitivity and potential population effects are needed. In addition, continued monitoring remains important given the Taiwanese government has modified their farmland rodent control policy to gradually reduce free AR supplies since 2015., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC 50 ) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 μg/L, 0.32 μg/L, and 0.55 μg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis. Graphical abstract.

In recent months, there has been increased reporting of seized drug and toxicology cases containing rodenticides, the active ingredient in rat poisons. Seeing as rodenticides are not scheduled substances, they are not commonly screened for in seized drug analysis. This work investigates the use of TD-DART-MS for the simultaneous detection of rodenticides and drugs. Six rodenticides were evaluated, an optimal method was established, and limits of detection in the tens of nanograms were calculated. Additional studies highlight that detection at less than 1% by weight in mixtures with AB-FUBINACA, cocaine, heroin, or methamphetamine was possible. This work presents an optimized method for detection of these compounds, allowing for the simultaneous detection of drugs and rodenticides, providing drug chemists with a tool for rapid identification of these compounds for forensic or public health purposes., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Non-target wildlife, particularly birds of prey, are widely exposed to and acutely poisoned by anticoagulant rodenticides (ARs). An unresolved issue surrounding such exposure, however, is the potential for sublethal effects. In particular, the consequences of AR exposure and resulting coagulopathy on health and survival of unintentionally exposed animals, which often encounter a multitude of anthropogenic stressors, are understudied. In a wildlife rehabilitation setting, AR intoxication may be masked by more obvious injuries related to collision with vehicles or electrocution, thereby obfuscating proximate from ultimate cause of mortality. An assessment of coagulation function of admitted wildlife may provide a means of identifying animals exhibiting sublethal coagulopathy, and ultimately ensuring provision of appropriate and swift treatment. In conjunction with routine diagnostics for injury and disease, we performed two blood clotting assays (prothrombin time, Russell's viper venom time) affected by vitamin K-dependent coagulopathy of samples from six species of live raptors admitted to a rehabilitation facility. We also measured clotting time in pre-fledgling barn owl chicks (Tyto furcata) from 10 nest sites in Lower Mainland Canada. Prolonged clotting time or failure to form a clot altogether was observed in 23.0% of 61 sampled raptors admitted to the rehabilitation facility. This is a biologically significant proportion of individuals given the fortuitous and likely biased nature by which raptors are found and admitted to rehabilitation facilities. In contrast, there was little evidence of coagulopathy in 19 pre-fledgling barn owl chicks. The utility of avian coagulation tests for diagnosing AR exposure is promising, yet there remains a need to establish species specific reference values and standardize assay methodologies among testing facilities., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

During 2018, four free-ranging conures, from a naturalized flock in San Francisco, presented with a characteristic set of neurologic signs that had been reported in other individuals from this flock. The cause of morbidity or mortality in historic cases has not been identified. From these four subjects, fresh feces were collected during their initial days of hospitalization and submitted to the University of Georgia Infectious Diseases Laboratory and Center for Applied Isotope Studies for bromethalin and desmethyl-bromethalin quantitation. Using High Performance Liquid Chromatography, the laboratory detected bromethalin, a non-anticoagulant, single-dose rodenticide, in fecal samples from three subjects; half of these samples were also positive for desmethyl-bromethalin, bromethalin's active metabolite. In three subjects that died, the UGA laboratory screened brain and liver samples and found bromethalin in all samples; desmethyl-bromethalin was detected in all but one brain sample, which was below the detection limit. Our findings suggest the conures are more resistant to bromethalin than are other species in which bromethalin has been studied, and/or that the conures may be ingesting the toxin at a sublethal dose. More data is needed to better assess the long-term effects of bromethalin on animals exposed at the subacute/chronic levels, and also to better understand the compartmentalization of bromethalin and desmethyl-bromethalin in a wider variety of species., Competing Interests: The authors have declared that no competing interests exist.

Bromethalin is a neurotoxicant with unusual instability and chromatographic behavior that make it difficult to analyze by gas chromatography (GC) in forensic examination of non-target animal deaths. Physicochemical breakdown of bromethalin produced multiple unique products with discernible mass spectra. This paper describes an investigation of the GC electron impact-mass spectrometric properties of bromethalin and its capacity to generate up to twenty heat- or light-induced breakdown products. Two principal breakdown products are isomeric with one another and involve release of both fluorine and methyl groups to develop dehydrofluorodesmethylbromethalin products. These compounds have proven to be excellent surrogate markers in screening forensic samples for bromethalin exposure, particularly in veterinary samples in which the active metabolite desmethylbromethalin has not yet accumulated to any appreciable extent, such as baits and animal stomach contents. The compounds as well as their parent bromethalin were easily monitored by GC interfaced with a tandem-quadrupole mass spectrometer using multiple-reaction monitoring (MRM) modes. Unusual gas chromatographic properties of bromethalin included: (i) specific requirements for a maximum oven temperature; (ii) non-linear increases in detector response on increased injection volumes, hypothesized to result from variable diffusion coefficients. We report here the development of GC strategies that facilitate detection of bromethalin and its breakdown products, as well as their MRM analysis by tandem-quadrupole mass spectrometry. The developed approaches are applicable to feed, baits and stomach contents as well as extracted tissue samples such as liver and kidney., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Anticoagulant rodenticides (ARs) have been used for decades for rodent control worldwide. Research on the exposure of the environment and accumulation of these active substances in biota has been focused on terrestrial food webs, but few data are available on the impact of ARs on aquatic systems and water organisms. To fill this gap, we analyzed liver samples of bream (Abramis brama) and co-located suspended particulate matter (SPM) from the German Environmental Specimen Bank (ESB). An appropriate method was developed for the determination of eight different ARs, including first- and second-generation ARs, in fish liver and SPM. Applying this method to bream liver samples from 17 and 18 sampling locations of the years 2011 and 2015, respectively, five ARs were found at levels above limits of quantifications (LOQs, 0.2 to 2 μg kg -1 ). For 2015, brodifacoum was detected in 88% of the samples with a maximum concentration of 12.5 μg kg -1 . Moreover, difenacoum, bromadiolone, difethialone, and flocoumafen were detected in some samples above LOQ. In contrast, no first generation AR was detected in the ESB samples. In SPM, only bromadiolone could be detected in 56% of the samples at levels up to 9.24 μg kg -1 . A temporal trend analysis of bream liver from two sampling locations over a period of up to 23 years revealed a significant trend for brodifacoum at one of the sampling locations.

The extensive use of anticoagulant rodenticides (ARs) results in widespread unintentional exposure of non-target rodents and secondary poisoning of predators despite regulatory measures to manage and reduce exposure risk. To elucidate on the potential vectoring of ARs into surrounding habitats by non-target small mammals, we determined bromadiolone prevalence and concentrations in rodents and shrews near bait boxes during an experimental application of the poison for 2 weeks. Overall, bromadiolone was detected in 12.6% of all small rodents and insectivores. Less than 20 m from bait boxes, 48.6% of small mammals had detectable levels of bromadiolone. The prevalence of poisoned small mammals decreased with distance to bait boxes, but bromadiolone concentration in the rodenticide positive individuals did not. Poisoned small mammals were trapped up to 89 m from bait boxes. Bromadiolone concentrations in yellow-necked mice (Apodemus flavicollis) were higher than concentrations in bank vole (Myodes glareolus), field vole (Microtus agrestis), harvest mouse (Micromys minutus), and common shrew (Sorex araneus). Our field trials documents that chemical rodent control results in widespread exposure of non-target small mammals and that AR poisoned small mammals disperse away from bating sites to become available to predators and scavengers in large areas of the landscape. The results suggest that the unintentional secondary exposure of predators and scavengers is an unavoidable consequence of chemical rodent control outside buildings and infrastructures.

Raptor population growth is dynamic and trends vary across species and by location in the United States. For those species that are declining, it is important to identify potential causes including chemical contaminants. Sampling wild raptors is problematic due to their small population sizes and role as a top predator. Therefore, we obtained liver samples (n = 56) from carcasses of several raptor species, including common species like red-tailed hawks, red-shouldered hawks, barred owls, great horned owls, and osprey that arrived dead or were euthanized from a non-profit rehabilitation center in Charlotte, North Carolina. Raptors were found or collected in South Carolina, North Carolina, and Virginia, but most samples were located near the metropolitan region of Charlotte, NC. We analyzed livers for total mercury residue (mg/kg, dry weight) and five anti-coagulant rodenticides (μg/kg wet weight). Mercury was analyzed using a direct mercury analyzer approach and rodenticides were quantified by LC-MS. Mercury residues were high in piscivorous birds (15.09 mg/kg for osprey and 6.93 mg/kg for great blue herons, dry weight) and relatively high in red-shouldered hawks and one eastern screech owl tested. Six of our samples exceeded a health threshold of 1 mg/kg (wet weight) including three osprey and one each of great blue heron, red-shouldered hawk, and eastern screech owl. Brodifacoum was the only rodenticide consistently detected in our samples. Brodifacoum detections exceeded 75% in barred owls, great horned owls, and red-shouldered hawks. Sixty-nine percent of owl samples were within (or exceeded) a threshold of brodifacoum residue associated with a 10-20% risk of acute toxicity. Correlations between residues and human population density were not significant for either mercury or brodifacoum. Our data suggest that mercury residues for most raptors were not of significant concern with the exception of osprey and possibly red-shouldered hawks. Rodenticide exposures associated with a risk of acute toxicity appear to be common and warrant further investigation.

The impacts of anticoagulant rodenticides (ARs) on non-target wildlife have been well documented in Europe and North America. While these studies are informative, patterns of non-target poisoning of wildlife elsewhere in the world may differ substantially from patterns occurring in Australia and other countries outside of cool temperate regions due to differences in the types of ARs used, patterns of use, legislation governing sales, and potential pathways of secondary exposure. Most of these differences suggest that the extent and severity of AR poisoning in wildlife may be greater in Australia than elsewhere in the world. While many anecdotal accounts of rodenticide toxicity were found - especially in conjunction with government control efforts and island eradications - no published studies have directly tested rodenticide exposure in non-target Australian wildlife in a comprehensive manner. The effects of private and agricultural use of rodenticides on wildlife have not been adequately assessed. Synthesis of reviewed literature suggests that anticoagulant rodenticides may pose previously unrecognised threats to wildlife and indigenous people in Australia and other nations with diverse and abundant reptile faunas relative to countries with cooler climates where most rodenticide ecotoxicology studies have been conducted. To address the identified knowledge gaps we suggest additional research into the role of reptiles as potential AR vectors, potential novel routes of human exposure, and comprehensive monitoring of rodenticide exposure in Australian wildlife, especially threatened and endangered omnivores and carnivores. Additionally, we recommend regulatory action to harmonise Australian management of ARs with existing and developing global norms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The anticoagulant rodenticides brodifacoum, chlorophacinone, and diphacinone have been proposed for broadcast application in some forested areas in Hawai'i to protect rare and endangered native bird species from introduced mice and rats. Groundwater resources in Hawai'i are prone to contamination due to the intrinsic aquifer vulnerability to leaching from the land surface. Because of the hydrogeologic complexity, Hawai'i uses a Tier-I leaching assessment tool, CLERS, to make registration decisions for new or existing chemicals. The CLERS tool uses soil and pesticide properties as well as water recharge through the soil profile in a GIS framework to estimate mass attenuation of the chemicals at a given depth and compares against this attenuation factor against those of a known leacher and a non-leacher. Disturbed soil samples were collected across the state of Hawai'i, including the islands of Hawai'i, Kaho'olawe, Kaua'i, Lana'i, Maui, Moloka'i, and O'ahu, with two sampling locations per island, except for Kaua'i which had three. As only limited information on chemical properties of these anticoagulants in soils is available, laboratory experiments were performed to determine the sorption capacity (K d ) and the degradation rate (T 1/2 ) of brodifacoum, chlorophacinone, and diphacinone to construct a proper chemical database. Depending on the soil type, T 1/2 values ranged between 37 and 248days for diphacinone, between 39 and 1000days for chlorophacinone, and between 72 and 462days for brodifacoum. These data were used in the CLERS tool to estimate leaching risks for these chemicals primarily in forested areas of the state where the chemicals are likely to be applied. The results from the CLERS tool indicate low risks of leaching of these three compounds into aquifers in five out of six major Hawaiian Islands. Diphacinone showed medium risk of leaching in a few remote areas in Maui., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Accidental poisoning with anticoagulant rodenticides is not uncommon in dogs, but few reports of the elimination kinetics and half-lives in this species have been published. Our objectives were to develop and validate a new method for the quantification of anticoagulant rodenticides in canine blood and faeces using reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and apply the method on a case of anticoagulant rodenticide intoxication., Results: Sample preparation was liquid-liquid extraction. Six anticoagulant rodenticides were separated using a UPLC ® BEH C 18 -column with a mobile phase consisting of 5 mM ammonium formate buffer pH 10.2 and methanol. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions. The limits of quantification were set at the levels of the lowest calibrator (1.5-2.7 ng/mL or ng/g). The method was successfully applied to a case from a dog accidentally poisoned with anticoagulant rodenticide. Coumatetralyl and brodifacoum concentrations were determined from serial blood and faecal samples. A terminal half-life of at least 81 days for coumatetralyl in blood was estimated, which is longer than previous reported in other species. A slow elimination of brodifacoum from the faeces was found, with traces still detectable in the faeces at day 513., Conclusions: This study offers a new method of detection and quantification of six frequently used anticoagulant rodenticides in canine faeces. Such drugs might cause serious health effects and it is important to be able to detect these drugs, to initiate proper treatment. The very long elimination half-lives detected in our study is important to be aware of in assessment of anticoagulant rodenticide burden to the environment.

Interest in the adverse effects of rodenticides on birds has focused primarily on raptors. However, non-raptor birds are also poisoned (rodenticide exposure resulting in adverse effects including mortality) by rodenticides through consumption of the rodenticide bait and contaminated prey. A literature search for rodenticide incidents (evidence of exposure to a rodenticide, adverse effects, or exposure to placebo baits) involving non-raptor birds returned 641 records spanning the years 1931 to 2016. The incidents included 17 orders, 58 families, and 190 non-raptor bird species. Nineteen anticoagulant and non-anticoagulant rodenticide active ingredients were associated with the incidents. The number of incidents and species detected were compared by surveillance method. An incident was considered to have been reported through passive surveillance if it was voluntarily reported to the authorities whereas the report of an incident found through field work that was conducted with the objective of documenting adverse effects on birds was determined to be from active surveillance. More incidents were reported from passive surveillance than with active surveillance but a significantly greater number of species were detected in proportion to the number of incidents found through active surveillance than with passive surveillance (z=7.61, p<0.01). Results suggest that reliance on only one surveillance method can underestimate the number of incidents that have occurred and the number of species that are affected. Although rodenticides are used worldwide, incident records were found from only 15 countries. Therefore, awareness of the breadth of species diversity of non-raptor bird poisonings from rodenticides may increase incident reportings and can strengthen the predictions of harm characterized by risk assessments., (Published by Elsevier B.V.)

In May 2016, thirteen dogs housed in backyards within a single neighborhood were reported to have developed convulsions and died within a 24 h period. An investigation of the scene by law enforcement resulted in submission of eight dogs for postmortem examination. It was suspected that a rapid acting toxin was the cause of death. A gas chromatography-mass spectrophotometry (GC-MS) protocol combined with thin-layer chromatography that allows screening for common convulsants failed to identify a toxin in either pooled gastric content or liver samples from select cases. After consultation with a veterinary toxicologist, sodium fluoroacetate poisoning was investigated. Sodium fluoroacetate, also known as 1080, is a pesticide that was available in the United States from the 1940's to the 1970's, but since 1972 has been banned or under EPA restricted use. When gastric content was re-tested using a GC-MS protocol with selective fluoroacetate ion monitoring and carbon 14 radiolabeling to facilitate quantification, 379 ppb sodium fluoroacetate was detected in a pooled gastric content sample. In spite of its banned status, sodium fluoroacetate remains a rarely reported cause of malicious poisoning in domestic dogs in the United Sates. This compound is highly toxic and is capable of causing death in dogs, humans, other mammals, and insects in ingested quantities as small as a few droplets. Even when geographic or historical proximity to a source is not evident, this intoxication should be considered in dogs exhibiting compatible clinical signs.

A method was developed for the screening and detection of food poisonings by ultra high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF MS). After extracted by acetonitrile and cleaned-up by QuEChERS, the extract was separated on a Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with the gradient elution of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile. TOF-MS scan-information dependent acquisition (IDA)-product ion scan was performed in positive electrospray ionization (ESI) mode to acquire high resolution MS and MS/MS spectra in one injection, and rapidly screen 581 target compounds by SCIEX OS software, including 546 pesticides, 24 mycotoxins, 11 rodenticides. The target compounds were qualitatively confirmed by mass accuracy of precursor, isotope distribution of precursor, fragment ions of precursor, and library search. Carbofuran was detected in 9 out of 11 samples with the proposed method. The retention time was further confirmed by the standard of carbofuran. The results showed the retention times were coincident between the samples and reference standard, and the deviations of accurate mass numbers were all less than 3.7×10 -6 . The scope of the relationship was good and the correlation coefficient was 0.998. The instrumental limit of detection ( S/N =3) was 0.3 μg/kg, and the limit of quantification ( S/N =10) was 1 μg/kg. The recoveries at 10, 50, 200 μg/kg levels were 75.6%-95.9%, and the RSDs ( n =6) were 3.6%-6.9%. The method is rapid, simple, accurate and sensitive. It is suitable for the rapid screening and detection of public safety incidents.