The Enigma of Norbormide, a Rattus-Selective Toxicant.

Norbormide (NRB) is a Rattus-selective toxicant, which was serendipitously discovered in 1964 and formerly marketed as an eco-friendly rodenticide that was deemed harmless to non-Rattus species. However, due to inconsistent efficacy and the emergence of second-generation anticoagulants, its usage declined, with registration lapsing in 2003. NRBs' lethal action in rats entails irreversible vasoconstriction of peripheral arteries, likely inducing cardiac damage: however, the precise chain of events leading to fatality and the target organs involved remain elusive. This unique contractile effect is exclusive to rat arteries and is induced solely by the endo isomers of NRB, hinting at a specific receptor involvement. Understanding NRB's mechanism of action is crucial for developing species-selective toxicants as alternatives to the broad-spectrum ones currently in use. Recent research efforts have focused on elucidating its cellular mechanisms and sites of action using novel NRB derivatives. The key findings are as follows: NRB selectively opens the rat mitochondrial permeability transition pore, which may be a factor that contributes to its lethal effect; it inhibits rat vascular KATP channels, which potentially controls its Rattus-selective vasoconstricting activity; and it possesses intracellular binding sites in both sensitive and insensitive cells, as revealed by fluorescent derivatives. These studies have led to the development of a prodrug with enhanced pharmacokinetic and toxicological profiles, which is currently undergoing registration as a novel efficacious eco-sustainable Rattus-selective toxicant. The NRB-fluorescent derivatives also show promise as non-toxic probes for intracellular organelle labelling. This review documents in more detail these developments and their implications.

Anticoagulant rodenticide toxicity in dogs: A retrospective study of 349 confirmed cases in Saskatchewan.

Objective: To evaluate the signalment and clinical, laboratory, treatment, and outcome features of dogs diagnosed with anticoagulant rodenticide (AR) intoxication in Saskatchewan. Animals: We studied 349 dogs. Procedure: Medical records from the Veterinary Medical Centre (Saskatoon, Saskatchewan) between 1999 and 2022 were reviewed. Cases were included if they met at least 1 of the following criteria: owner witnessed the dog ingesting an AR; AR was seen in the vomitus when emesis was induced; the dog had clinical signs of coagulopathy, with elevation of PT ± aPTT that normalized after vitamin K1 therapy, in the presence of appropriate clinical and paraclinical data and the absence of other causes of hypocoagulable state determined by the primary clinician. Results: Fifty-three percent of cases were seen between July and October. Most dogs (61%) came from an urban setting. Ninety-two percent of dogs ingested a 2nd-generation AR and the most frequent toxin was bromadiolone. Clinical signs were reported in 30% of AR intoxications and included lethargy (86%), dyspnea (55%), and evidence of external hemorrhage (44%). The most common site of hemorrhage was the pleural space, accounting for 43% of hemorrhage sites. Consumptive thrombocytopenia was reported in 24% of dogs with evidence of AR-induced hemorrhage, with moderate (platelet count < 60 K/µL) and marked (< 30 K/µL) thrombocytopenia in 7/12 and 2/12 dogs, respectively. Blood products were administered to 84% of dogs with AR-induced hemorrhage; the most common product administered was fresh frozen plasma (56% of cases). Among dogs with AR-induced hemorrhage, those that received blood products were more likely to survive to discharge (81%) compared to those that did not (19%) (P = 0.017). Eighty-six percent of dogs with AR-induced hemorrhage survived to discharge. Conclusion and clinical relevance: The pleural space was the most common site of hemorrhage. Moderate thrombocytopenia was a common finding. Eighty-six percent of dogs with AR-induced hemorrhage survived to discharge.

Toxicité des rodenticides anticoagulants chez les chiens : étude rétrospective de 349 cas confirmés en Saskatchewan. Objectif: Évaluer le signalement et les caractéristiques cliniques, de laboratoire, de traitement et de résultats des chiens diagnostiqués avec une intoxication par un rodenticide anticoagulant (AR) en Saskatchewan. Animaux: Nous avons étudié 349 chiens. Procédure: Les dossiers médicaux du Veterinary Medical Centre (Saskatoon, Saskatchewan) entre 1999 et 2022 ont été examinés. Les cas ont été inclus s'ils répondaient à au moins 1 des critères suivants : le propriétaire a vu le chien ingérer un AR; de l'AR a été observée dans les vomissures lorsque des vomissements ont été provoqués; le chien présentait des signes cliniques de coagulopathie, avec une élévation du PT ± aPTT qui s'est normalisée après un traitement par la vitamine K1, en présence de données cliniques et paracliniques appropriées et en l'absence d'autres causes d'état hypocoagulable déterminées par le clinicien initial. Résultats: Cinquante-trois pour cent des cas ont été observés entre juillet et octobre. La plupart des chiens (61 %) venaient d'un milieu urbain. Quatre-vingt-douze pour cent des chiens ont ingéré un AR de 2e génération et la toxine la plus fréquente était la bromadiolone. Des signes cliniques ont été rapportés dans 30 % des intoxications par AR et incluaient de la léthargie (86 %), de la dyspnée (55 %) et des signes d'hémorragie externe (44 %). Le site d'hémorragie le plus fréquent était l'espace pleural, représentant 43 % des sites d'hémorragie. Une thrombocytopénie de consommation a été rapportée chez 24 % des chiens présentant des signes d'hémorragie induite par l'AR, avec une thrombocytopénie modérée (nombre de plaquettes < 60 K/µL) et marquée (< 30 K/µL) chez 7 chiens sur 12 et 2 chiens sur 12, respectivement. Des produits sanguins ont été administrés à 84 % des chiens présentant une hémorragie induite par l'AR; le produit le plus fréquemment administré était le plasma frais congelé (56 % des cas). Parmi les chiens présentant une hémorragie induite par l'AR, ceux qui ont reçu des produits sanguins étaient plus susceptibles de survivre jusqu'à leur congé (81 %) que ceux qui n'en ont pas reçu (19 %) (P = 0,017). Quatre-vingt-six pour cent des chiens présentant une hémorragie induite par l'AR ont survécu jusqu'à leur sortie. Conclusion et pertinence clinique: L'espace pleural était le site d'hémorragie le plus fréquent. Une thrombocytopénie modérée était fréquente. Quatre-vingt-six pour cent des chiens présentant une hémorragie induite par l'AR ont survécu jusqu'à leur sortie.(Traduit par Dr Serge Messier).

Broad-scale pesticide screening finds anticoagulant rodenticide and legacy pesticides in Australian frogs.

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.

Significant Turning Point: Common Buzzard (Buteo buteo) Exposure to Second-Generation Anticoagulant Rodenticides in the United Kingdom.

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.

Anticoagulant rodenticide poisoning in farmed Patagonian mara (Dolichotis patagonum).

BACKGROUND: Anticoagulant rodenticide (AR) poisoning was diagnosed in 3 Patagonian maras (Dolichotis patagonum) raised in the mara farm in Thailand. To date, there have been no reports of maras with diagnosed AR poisoning. CASE PRESENTATION: The first clinical sign of the sickening maras was anorexia. Fifteen from 50 maras were dead over a 3-5 day period after the clinical signs had occurred. Positive results to AR were detected in all of the maras' liver specimens by screening test using thin layer chromatography and spectrophotometry methods. Supportive therapy was selected for the treatment of the 35 surviving maras. During the follow - up observation period of 12 months, all of the surviving maras were healthy and no reproductive loss. CONCLUSIONS: This is the first report on suspected AR poisoning in maras in Thailand based on history taking, clinical signs, gross pathology lesions and chemical analysis. AR poisoning in the present report is possibly from contaminated animal food. Therefore, quality control of food should be fastidious when feeding maras.

Sensitivity assessment of diphacinone by pharmacokinetic analysis in invasive black rats in the Bonin (Ogasawara) Archipelago, Japan.

The Bonin Archipelago is a United Nations Educational, Scientific and Cultural Organization's World Natural Heritage Site in Japan with a unique ecosystem; however, the invasive rodents preying on endemic species have been a significant concern. The anticoagulant rodenticide, diphacinone, sprayed by the Ministry of the Environment, has succeeded; however, its repeated use leads to rodenticide resistance. This study evaluated the sensitivity by in vivo pharmacokinetics/pharmacodynamics (PK/PD) analysis and physiologically-based pharmacokinetic modeling to diphacinone in black rats (Rattus rattus) captured on the Bonin Archipelago in February 2022. The Bonin rats exhibited prolonged coagulation time after diphacinone administration. They recovered earlier than susceptible black rats, indicating that Bonin rats were less susceptible, though there were no genetic mutations in Vkorc1, the target enzyme of diphacinone. After the administration of diphacinone, hepatic expression levels of Fsp1, identified as the vitamin K reductase, was decreased, however, the Bonin rats exhibited the most minor suppression. The PK analysis showed that the excretion capacity of the Bonin rats was lower than that of the resistant black rats. In the PBPK modeling, the resistant black rats showed higher clearance than the Bonin and susceptible black rats due to high hepatic metabolic capacity. The Bonin rats demonstrated slow absorption and relatively low clearance. This study highlighted the reduced rodenticide-sensitive tendency of wild black rats in the Bonin Archipelago at an in vivo phenotype level. At the same time, they do not have known rodenticide resistance mechanisms, such as hepatic metabolic enhancement or Vkorc1 mutations. It is crucial to monitor the biological levels to evaluate rodenticide sensitivity accurately.

Anticoagulant Rodenticide Toxicity in Terrestrial Raptors: Tools to Estimate the Impact on Populations in North America and Globally.

Anticoagulant rodenticides (ARs) have caused widespread contamination and poisoning of predators and scavengers. The diagnosis of toxicity proceeds from evidence of hemorrhage, and subsequent detection of residues in liver. Many factors confound the assessment of AR poisoning, particularly exposure dose, timing and frequency of exposure, and individual and taxon-specific variables. There is a need, therefore, for better AR toxicity criteria. To respond, we compiled a database of second-generation anticoagulant rodenticide (SGAR) residues in liver and postmortem evaluations of 951 terrestrial raptor carcasses from Canada and the United States, 1989 to 2021. We developed mixed-effects logistic regression models to produce specific probability curves of the toxicity of ∑SGARs at the taxonomic level of the family, and separately for three SGARs registered in North America, brodifacoum, bromadiolone, and difethialone. The ∑SGAR threshold concentrations for diagnosis of coagulopathy at 0.20 probability of risk were highest for strigid owls (15 ng g-1) lower and relatively similar for accipitrid hawks and eagles (8.2 ng g-1) and falcons (7.9 ng g-1), and much lower for tytonid barn owls (0.32 ng g-1). These values are lower than those we found previously, due to compilation and use of a larger database with a mix of species and source locations, and also to refinements in the statistical methods. Our presentation of results on the family taxonomic level should aid in the global applicability of the numbers. We also collated a subset of 440 single-compound exposure events and determined the probability of SGAR-poisoning symptoms as a function of SGAR concentration, which we then used to estimate relative SGAR toxicity and toxic equivalence factors: difethialone, 1, brodifacoum, 0.8, and bromadiolone, 0.5. Environ Toxicol Chem 2024;43:988-998. © 2024 His Majesty the King in Right of Canada and The Authors. 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.

Developing a GC-EI-MS/MS method for quantifying warfarin and five hydroxylated metabolites generated by the Fenton reaction.

Since the 1950s, Warfarin has been used globally as both a prescription drug and a rodenticide. Research has shown that warfarin and other rodenticides are present in the environment and food chain. However, emerging contaminants are subject to degradation by biotic and abiotic processes and advanced oxidation processes. In some cases, detecting the parent compound may not be possible due to the formation of structurally changed species. This approach aims to identify hydroxylated transformation products of warfarin in a laboratory setting, even after the parent compound has undergone degradation. Therefore, the Fenton reaction is utilized to insert hydroxylation into the parent compound, warfarin, by hydroxyl and hydroperoxyl radicals generated by Fe2+/Fe3+ redox reaction with hydrogen peroxide. Using multiple reaction monitoring, a GC-MS/MS method, incorporating isotopically labeled reference compounds, is used to quantify the expected derivatized species. The analytes are derivatized using trimethyl-3-trifluoromethyl phenyl ammonium hydroxide, and the derivatization yield of warfarin is determined by using isotopically labeled reference compounds. The method has a linear working range of 30 to 1800 ng/mL, with detection limits ranging from 18.7 to 67.0 ng/mL. The analytes are enriched using a C18-SPE step, and the recovery for each compound is calculated. The Fenton reaction generates all preselected hydroxylated transformation products of warfarin. The method successfully identifies that 4'-Me-O-WAR forms preferentially under the specified experimental conditions. By further optimizing the SPE clean-up procedures, this GC-MS-based method will be suitable for detecting transformation products in more complex matrices, such as environmental water samples. Overall, this study provides a better understanding of warfarin's degradation and offers a robust analytical tool for investigating its transformation products.

[Anticoagulant rodenticide poisoning in dogs: Retrospective analysis of clinical and laboratory diagnostic data]. / Vergiftung mit Cumarinderivaten beim Hund: Retrospektive Analyse klinischer und labordiagnostischer Daten.

OBJECTIVE: In this retrospective study, patient records of dogs suffering from poisoning with coumarin derivatives were evaluated to characterize the clinical appearance more precisely. MATERIAL UND METHODS: Retrospective data analysis included 52 dogs with hemostaseologically proven anticoagulant rodenticide poisoning which were treated as inpatients at the Clinic for Small Animals between September 2011 and October 2018. RESULTS: In only 2 dogs (4%) the intake of poison could be observed with certainty. The most common clinical signs observed were reduced general behavior (79%), pallor of the mucosa (79%), anorexia (60%), and dyspnea/tachypnea (60%). In contrast, macroscopically visible internal and external bleedings occurred less frequently. Initially, all cases showed a highly altered prothrombin time and most patients a considerably prolonged activated partial thromboplastin time. Anemia was present in 75% of patients. All dogs included in the study received initially an intravenous treatment with 10 mg/kg vitamin K1. Pretreatment with 1 mg/kg prednisolone was given for prophylaxis of possible incompatibility reactions. No patient showed signs of anaphylactic reaction. Transfusions of whole blood or concentrated red cells were given to only 10 of the 52 animals; only one received 2 transfusions of erythrocytes. 94% of the animals could be discharged home for outpatient therapy after a median length of hospitalization of 3 days (1-9 days) with physiological or almost physiological coagulation test results. CONCLUSION: Anticoagulant rodenticide poisoning is often associated with non-specific symptoms and good prognosis if treated adequately. CLINICAL RELEVANCE: Coagulation diagnostics is always indicated in cases with unclear disorders. In life-threatening emergencies, immediate intravenous infusion of high-dose vitamin K1 is a very effective treatment and results in a rapid increase in coagulation factor activity.

Rodenticide poisoning leading to cerebral hemorrhage: A case report.

RATIONALE: Anticoagulant rodenticides (ARs) are a substantial fraction of murine types. AR poisoning causes bleeding from the skin, mucous membranes, and multiple organs. However, reports of AR-induced cerebral hemorrhage are scarce. PATIENT CONCERNS: A 40-year-old male presented with dizziness, headache, and limb weakness for 5 days and with coagulopathy. Two days prior to the onset of these symptoms, the patient was exposed to dead mice. DIAGNOSES: Rodenticide intoxication-induced cerebral hemorrhage. INTERVENTIONS: Vitamin K1 infusion, administration of dehydrating agents to reduce intracranial pressure, and correction of acid-base and electrolyte imbalances. OUTCOMES: After 9 days of treatment, the patient's symptoms were relieved, and reexamination revealed that coagulation parameters returned to normal levels. The patient was eventually discharged for observation with oral vitamin K1. CONCLUSIONS: Rodenticide poisoning can lead to intracerebral hemorrhage, and treatment with vitamin K1 infusion is effective. LESSON: Rodenticide poisoning-induced cerebral hemorrhage is rarely reported. Because its symptoms are nonspecific, it is easy to miss the diagnosis or misdiagnose. When patients present with direct and indirect symptoms such as dizziness, headache, and limb weakness, rodenticide poisoning should be considered. Coagulation function and head computed tomography or magnetic resonance imaging examination should be performed at the earliest to confirm the diagnosis and provide timely treatment.

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

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

Exposure assessment of anticoagulant rodenticides in the liver of red foxes (Vulpes vulpes) in Slovenia.

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.

Stereoselective bioaccumulation of chiral anticoagulant rodenticides in the liver of predatory and scavenging raptors.

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.

Significance of additives to enhance the acceptance of poison bait in poultry rodents of Haripur, Khyber Pakhtunkhwa, Pakistan.

Rodent infestation on poultry farms incurs heavy economic losses to this industry by causing feed loss and disease introduction. Development and continuous improvement of rodents control techniques are vital to minimize and control the damages caused by rodents. Here, we test the feed preference of rodents for locally available and palatable food grains viz. millet (whole), wheat (cracked) and rice (broken) and taste additives namely whole egg (5%), eggshell (5%), peanut cracked (5%) and yeast (2%) that were offered mixed in millet-wheat (50:50 by wt.) bait. We tested the preferences of different food additives through a process of feed choice mechanism. We applied two different techniques to compare the preference of mixed feed baits, these techniques included no-choice with multiple choice feeding tests and paired choice with multiple choices feeding tests. The results indicated that consumption of bait with added whole egg was significantly higher (p > 0.05). Further test for its effectiveness as a carrier for rodenticides revealed 56%, 82% and 92%, reduction in rodent activities with zinc phosphide (2%), coumatetralyl (0.0375%) and Brodifacoum (0.005%) respectively. Our results point to a need on continuous improvement of feed baits by using different combinations to effectively control the rodent infestation.

First evidence of widespread positivity to anticoagulant rodenticides in grey wolves (Canis lupus).

Second-generation Anticoagulant Rodenticides (ARs) can be critical for carnivores, due to their widespread use and impacts. However, although many studies explored the impacts of ARs on small and mesocarnivores, none assessed the extent to which they could contaminate large carnivores in anthropized landscapes. We filled this gap by exploring spatiotemporal trends in grey wolf (Canis lupus) exposure to ARs in central and northern Italy, by subjecting a large sample of dead wolves (n = 186) to the LC-MS/MS method. Most wolves (n = 115/186, 61.8 %) tested positive for ARs (1 compound, n = 36; 2 compounds, n = 47; 3 compounds, n = 16; 4 or more compounds, n = 16). Bromadiolone, brodifacoum and difenacoum, were the most common compounds, with brodifacoum and bromadiolone being the ARs that co-occurred the most (n = 61). Both the probability of testing positive for multiple ARs and the concentration of brodifacoum, and bromadiolone in the liver, systematically increased in wolves that were found at more anthropized sites. Moreover, wolves became more likely to test positive for ARs through time, particularly after 2020. Our results underline that rodent control, based on ARs, increases the risks of unintentional poisoning of non-target wildlife. However, this risk does not only involve small and mesocarnivores, but also large carnivores at the top of the food chain, such as wolves. Therefore, rodent control is adding one further conservation threat to endangered large carnivores in anthropized landscapes of Europe, whose severity could increase over time and be far higher than previously thought. Large-scale monitoring schemes for ARs in European large carnivores should be devised as soon as possible.

Toxicology of chemical biocides: Anticoagulant rodenticides - Beyond hemostasis disturbance.

The use of anticoagulant rodenticides (ARs) is one of the most commonly employed management methods for pest rodents. ARs compete with vitamin K (VK) required for the synthesis of blood clotting factors in the liver, resulting in inhibition of blood coagulation and often animal death due to hemorrhage. Besides rodents (target species), ARs may affect non-target animal species and humans. Out of hemostasis disturbance, the effects of ARs may be related to the inhibition of proteins that require VK for their synthesis but are not involved in the coagulation process, to their direct cytotoxicity, and their pro-oxidant/proinflammatory activity. A survey of the cellular and molecular mechanisms of these sublethal/asymptomatic AR effects is given in this review. Data from field, clinical, and experimental studies are presented. Knowledge of these mechanisms might improve hazard characterization and identification of potential ecotoxicological risks associated with ARs, contributing to a safer use of these chemicals.

Evaluation of anticoagulant rodenticide sensitivity by examining in vivo and in vitro responses in avian species, focusing on raptors.

Anticoagulant rodenticides (ARs) are used to control pest rodent species but can result in secondary poisoning of non-target animals, especially raptors. In the present study, differences in AR sensitivity among avian species were evaluated by comparing in vivo warfarin pharmacokinetics and effects, measuring cytochrome P450s (CYPs) expression involved in AR metabolism, and conducting in vitro inhibition assays of the AR target enzyme Vitamin K 2,3-epoxide reductase (VKOR). Oral administration of warfarin at 4 mg/kg body weight did not prolong prothrombin time in chickens (Gallus gallus), rock pigeons (Columba livia), or Eastern buzzards (Buteo japonicus). Rock pigeons and buzzards exhibited shorter plasma half-life of warfarin compared to chickens. For the metabolite analysis, 4'-hydroxywarfarin was predominantly detected in all birds, while 10-hydroxywarfarin was only found in pigeons and raptors, indicating interspecific differences in AR metabolism among birds likely due to differential expression of CYP enzymes involved in the metabolism of ARs and variation of VKOR activities among these avian species. The present findings, and results of our earlier investigations, demonstrate pronounced differences in AR sensitivity and pharmacokinetics among bird species, and in particular raptors. While ecological risk assessment and mitigation efforts for ARs have been extensive, AR exposure and adverse effects in predatory and scavenging wildlife continues. Toxicokinetic and toxicodynamic data will assist in such risk assessments and mitigation efforts.

The combined effect of bromadiolone and ivermectin (iBr) in controlling both rodents and their fleas.

Rodent pests not only cause severe agricultural loss but also spread zoonotic pathogens to human beings. Anticoagulant rodenticides are widely used to decrease the population densities of rodents but often lead to the spillover of ectoparasites because fleas and ticks may gather on surviving rodents. Therefore, it is necessary to kill fleas and ticks before culling rodents to minimize the risk of pathogen transmission. In this study, we used a mixture of ivermectin (an antiparasitic drug) and bromadiolone (an anticoagulant rodenticide) to control both rodent and flea/tick abundances. We found that in a laboratory test, 0.01% ivermectin bait was not lethal for greater long-tailed hamsters after 7 days of treatment, while 0.1% ivermectin bait was lethal for approximately 33% of treated rodents. In a field test, bait containing 0.001%, 0.005%, 0.01%, and 0.05% ivermectin decreased the number of fleas per vole of Brandt's voles to 0.42, 0.22, 0.12, and 0.2, respectively, compared with 0.77 in the control group, indicating that 0.01% ivermectin bait performed best in removing fleas. In another laboratory test, bait containing a 0.01% ivermectin and 0.005% bromadiolone mixture caused the death of all voles within 6-14 days after the intake of the bait. In the field test, the bait containing 0.01% ivermectin and 0.005% bromadiolone reduced the average number of fleas per vole to 0.35, which was significantly lower than the 0.77 of the control group. Our results indicate that a 0.01% ivermectin and 0.005% bromadiolone mixture could be used to control both rodents and fleas to minimize the spillover risk of disease transmission when using traditional rodenticides.

Fast and sensitive method for the diagnosis and follow-up of anticoagulant rodenticides poisoning in animal whole blood.

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.

An atypical presentation of anticoagulant rodenticide toxicosis in a dog.

A 5-month-old intact female Australian shepherd dog was referred to our clinic for neurologic signs including ataxia, a head tilt, and altered mentation following consumption of an unidentified rodenticide several days prior to developing clinical signs. A provisional diagnosis of bromethalin toxicosis had been made, given the neurologic signs seen and the general increased use of bromethalin-containing rodenticide products. However, on physical examination, the dog was noted to have scleral hemorrhage and bleeding at the venipuncture sites, which was inconsistent with bromethalin toxicosis. Coagulation testing was supportive of anticoagulant rodenticide toxicosis and the rodenticide was later identified as the first-generation anticoagulant rodenticide diphacinone. The neurologic signs seen were attributed to a coagulopathy causing multifocal hemorrhage into the central nervous system. Neurologic signs rapidly resolved following treatment with a frozen plasma transfusion and vitamin K1. This atypical presentation of an anticoagulant rodenticide toxicosis highlights the need for accurate product identification, if available, and thorough patient examination and laboratory testing. An atypical presentation of anticoagulant rodenticide toxicosis should be considered when neurologic signs are present with clinical bleeding, especially if the type of rodenticide is unknown, or even if it was not thought to have an anticoagulant as the active ingredient. Key clinical message: Given the change in commercially available rodenticide products, this case highlights the need for accurate product identification in cases of suspected toxicosis, and the variable clinical signs that can be seen following anticoagulant rodenticide toxicosis.

Présentation atypique d'une toxicose aux rodenticides anticoagulants chez un chien. Une chienne berger australien intacte âgée de 5 mois a été référée à notre clinique pour des signes neurologiques, notamment de l'ataxie, une inclinaison de la tête et une altération de l'état mental à la suite de la consommation d'un rodenticide non identifié plusieurs jours avant l'apparition des signes cliniques. Un diagnostic provisoire de toxicose à la brométhaline avait été posé, compte tenu des signes neurologiques observés et d'une utilisation historique accrue de produits rodenticides contenant de la brométhaline. Cependant, lors de l'examen physique, il a été constaté que le chien présentait une hémorragie sclérale et des saignements au niveau des sites de ponction veineuse, ce qui n'était pas cohérent avec une toxicose à la brométhaline. Les tests de coagulation ont confirmé la toxicose au rodenticide anticoagulant et le rodenticide a ensuite été identifié comme étant le rodenticide anticoagulant de première génération diphacinone. Les signes neurologiques observés ont été attribués à une coagulopathie provoquant une hémorragie multifocale du système nerveux central. Les signes neurologiques ont rapidement disparu après un traitement par transfusion de plasma congelé et de vitamine K1. Cette présentation atypique d'une toxicose aux rodenticides anticoagulants met en évidence la nécessité d'une identification précise du produit, si disponible, ainsi que d'un examen approfondi du patient et de tests de laboratoire. Une présentation atypique de toxicose des rodenticides anticoagulants doit être envisagée lorsque des signes neurologiques sont présents avec saignement clinique, en particulier si le type de rodenticide est inconnu, ou même si l'on ne pense pas qu'un anticoagulant soit l'ingrédient actif.Message clinique clé :Compte tenu de l'évolution des produits rodenticides disponibles dans le commerce, ce cas met en évidence la nécessité d'une identification précise du produit en cas de suspicion de toxicose et les signes cliniques variables qui peuvent être observés à la suite d'une toxicose au rodenticide anticoagulant.(Traduit par Dr Serge Messier).