Brodifacoum Research Articles

Validation and Uncertainty Calculation of Rodenticide Analysis Methods in a Simulated Gastric Content Matrix – Uncertainty of Rodenticide Analysis Methods

Warfarin (WAR), brodifacoum (BDF) and bromadiolone (BDL) are compounds present in rodenticides, highly toxic to rats, humans and other animals. These compounds can be detected in complex matrices, such as stomach contents, by liquid chromatography techniques (HPLC) with mass spectrometry (MS) or fluorescence detection (FLD). However, no validated method showed determination of uncertainty in the quantification of these compounds. In this study, we compare the validation parameters of two analytical methods, HPLC FLD and ultra high performance liquid chromatography (UHPLC – MS), with uncertainty estimation for the three cited compounds. The results showed that UHPLC-MS outperformed HPLC FLD, however both methods were considered adequate for detection of WAR, BDF or BDL in samples of simulated human stomach contents, especially in cases of suspected contamination.

Developmental toxicity induced by brodifacoum in zebrafish (Danio rerio) early life stages.

The present study mainly focused on the assessment of developmental toxicity induced by exposure to brodifacoum (BDF) in zebrafish at early life stages. Zebrafish embryos were exposed to 0.2, 0.4, and 0.8mg/L of BDF from 6 to 96 hr post-fertilization (hpf), and the toxic effects of BDF on early embryonic development were investigated in terms of morphological changes, oxidative stress, and alterations in heart development-related genes. The experimental results showed that BDF significantly decreased the heart rate, survival rate, body length, and spontaneous movements of zebrafish embryos at 0.8mg/L, and the morphological developmental abnormalities were also observed at 96 hpf. In addition, exposure to BDF significantly increased oxidative stress levels in zebrafish embryos by increasing the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels, and decreased glutathione (GSH) levels. Furthermore, BDF treatment-induced alterations in the expression levels of the heart development-related genes (gata4, sox9b, tbx2b, and nppa). Results from this study indicated that exposure to BDF could lead to marked growth inhibition and significantly alter the activities of antioxidant enzymes in zebrafish embryos. Moreover, BDF exposure exhibited severe cardiotoxicity and significantly disrupted heart development-related genes. The results indicated that BDF could induce developmental and cardiac toxicity in zebrafish embryos.

Effects of anticoagulant rodenticide poisoning on spatial behavior of farm dwelling Norway rats

Commensal rodent species cause damage to crops and stored products, they transmit pathogens to people, livestock and pets and threaten native flora and fauna. To minimize such adverse effects, commensal rodents are predominantly managed with anticoagulant rodenticides (AR) that can be transferred along the food chain. We tested the effect of the uptake of the AR brodifacoum (BR) by Norway rats (Rattus norvegicus) on spatial behavior because this helps to assess the availability of dead rats and residual BR to predators and scavengers. BR was delivered by oral gavage or free-fed bait presented in bait stations. Rats were radio-collared to monitor spatial behavior. BR residues in rat liver tissue were analyzed using liquid chromatography coupled with tandem mass spectrometry. Norway rats that had consumed BR decreased distances moved and had reduced home range size. Treatment effects on spatial behavior seemed to set in rapidly. However, there was no effect on habitat preference. Ninety-two percent of rats that succumbed to BR died in well-hidden locations, where removal by scavenging birds and large mammalian scavengers is unlikely. Rats that ingested bait from bait stations had 65% higher residue concentrations than rats that died from dosing with two-fold LD50. This suggests an overdosing in rats that are managed with 0.0025% BR. None of the 70 BR-loaded rats was caught/removed by wild predators/scavengers before collection of carcasses within 5–29 h. Therefore, and because almost all dead rats died in well-hidden locations, they do not seem to pose a significant risk of AR exposure to large predators/scavengers at livestock farms. Exposure of large predators may originate from AR-poisoned non-target small mammals. The few rats that died in the open are accessible and should be removed in routine searches during and after the application of AR bait to minimize transfer of AR into the wider environment.

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

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

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites.

Anticoagulant rodenticides (ARs) are used worldwide for rodent population control to protect human health and biodiversity, and to prevent agricultural and economic losses. Rodents may develop a metabolic resistance to ARs. In order to help understand such metabolic resistance, mass spectrometry was used to position the hydroxylated group of hydroxyl metabolites of second-generation ARs (SGARs). Most AR pesticides are derived from the 4-hydroxycoumarin/thiocoumarin family. We used low-resolution and high-resolution mass spectrometry to understand the fragmentation pathways of the ARs and their respective metabolites, and to better define the structure of their tandem mass spectrometry product ions. Seven specific product ions were evidenced for five ARs, with their respective chemical structures. Those ions were obtained as well from the mass spectra of the hydroxyl metabolites of four SGARs, difenacoum (DFM), brodifacoum (BFM), difethialone (DFTL) and flocoumafen (FLO), with different positions of the hydroxyl group. The differences in chemical structure between DFM on the one hand and BFM, FLO and DFTL on the other could explain the differences in bioavailability between these two groups of molecules. The defined product ions will be used to investigate the part played by the metabolic issue in the field resistance of SGARs.

Baiting location affects anticoagulant rodenticide exposure of non-target small mammals on farms.

Commensal rodents such as Norway rats (Rattus norvegicus Berk.), black rats (R. rattus L.) and house mice (Mus musculus L.) damage stored produce and infrastructure, cause hygienic problems and transmit zoonotic pathogens to humans. The management of commensal rodents relies mainly on the use of anticoagulant rodenticides (ARs). ARs are persistent and bio-accumulative, which can cause exposure of non-target species. We compared the baiting strategies to use brodifacoum (BR) in bait boxes indoors only versus in and around buildings in replicated field trials at livestock farms to assess resulting BR residues in non-target small mammals. When bait was used indoors only, the percentage of trapped non-target small mammals with BR residues as well as BR concentration in liver tissue was about 50% lower in comparison to bait application in and around buildings. These effects occurred in murid rodents and shrews but not in voles that were generally only mildly exposed. During the baiting period, BR concentration in murids was stable but decreased by about 50% in shrews. Restricting the application of BR bait to indoors only can reduce exposure of non-target species. The positive effect of this baiting strategy on non-target species needs to be balanced with the need for an effective pest rodent management within a reasonable time. More research is needed to clarify which management approaches strike this balance best.

Acute toxicity and clotting times of anticoagulant rodenticides to red-toothed (Odonus niger) and black (Melichthys niger) triggerfish, fathead minnow (Pimephales promelas), and largemouth bass (Micropterus salmoides).

Anticoagulant rodenticides (ARs) are used in rat eradication efforts on island wildlife refuges. AR bait pellets can get into coral reef areas during broadcasting and lead to exposure of non-target organisms, such as marine fishes. The objective of this study was to determine the sensitivity of representative saltwater fishes, Red-toothed triggerfish (Odonus niger) and Black triggerfish (Melichthys niger), and common freshwater fishes, fathead minnow (Pimephales promelas), and largemouth bass (Micropterus salmoides) to first generation ARs, diphacinone (DPN) and chlorophacinone (CPN), as well as a second-generation AR, brodifacoum (BROD). Acute toxicity of ARs was evaluated by single dose, intraperitoneal injections. The median lethal dose (LD50) ranges were 137−175 μg DPN/g, 155−182 μg CPN/g, and 36−48 μg BROD/g for Red-toothed triggerfish and 90−122 μg DPN/g, 125−164 μg CPN/g, and 50−75 μg BROD/g for black triggerfish. Laboratory surrogate test fish species fathead minnow and largemouth bass were of similar sensitivity toward AR-induced toxicity compared to triggerfish based on LD50 values. Sublethal effects on elevated clotting time occurred in dose-dependent fashion in all fish tested. Fish appear to have low sensitivity to AR chemicals as compared to other taxa, in particular mammals and birds, based on across-taxa comparisons of species sensitivity distributions of whole body, single dose acute lethality (LD50 values). The sensitivity of fish to waterborne exposures of ARs has yet to be fully evaluated and indeed may prove more hazardous to fish.

Effects of vitamin K1 treatment on plasma concentrations of long-acting anticoagulant rodenticide enantiomers following inhalation of contaminated synthetic cannabinoids

Background: An outbreak of synthetic cannabinoid (SC)-associated coagulopathy and bleeding in Illinois, USA was determined to be due to inhalation of SC contaminated with brodifacoum (BDF), difenacoum (DiF), and bromadiolone (BDL), highly potent long-acting anticoagulant rodenticides (LAARs). Treatment with high-dose vitamin K1 (VK1) prevented mortality; however, plasma LAAR levels were not measured risking recurrence of coagulopathy and bleeding due to premature discontinuation. The goal of this study was to determine if plasma LAAR levels were reduced following standard of care treatment to normalize coagulopathy.Methods: Blood samples were collected from a cohort of 32 patients, and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis used to quantify plasma LAAR levels including enantiomers.Results: BDF was detected in 31 samples; 30 also contained DiF and 18 contained BDL. Initial plasma levels were 581 ± 87, 11.0 ± 1.9, and 14.9 ± 5.9 ng/mL for BDF, DiF, and BDL, respectively (mean ± SE). At discharge plasma, BDF levels remained elevated at 453 ± 68 ng/mL. Plasma half-lives for BDF, DiF, and BDL were 7.5 ± 1.3, 7.2 ± 1.9, and 1.8 ± 0.3 days, respectively. The half-life for trans-BDF enantiomers (5.7 ± 0.8 days) was shorter than for cis-enantiomers (7.6 ± 1.9 days). BDF half-lives were shorter, and coagulopathy normalized faster in patients receiving intravenous VK1 as compared to oral VK1. Patients prescribed VK1 at discharge had fewer re-admittances.Conclusions: These results demonstrate that plasma LAAR levels at discharge were elevated in poisoned patients despite normal coagulation, and that the route of VK1 administration affected LAAR pharmacokinetics and INR normalization. We propose plasma LAAR levels and coagulation be monitored concomitantly during follow-up of patients with LAAR poisoning.KEY POINTSIn patients treated with high-dose vitamin K1 for LAAR poisoning, plasma levels remained 40-fold above safe levels upon discharge from hospital.LAAR half-lives, normalization of coagulopathy, and readmittances were reduced by treatment with intravenous vitamin K1.

Brodifacoum does not modulate human cannabinoid receptor-mediated hyperpolarization of AtT20 cells or inhibition of adenylyl cyclase in HEK 293 cells.

BackgroundSynthetic cannabinoids are a commonly used class of recreational drugs that can have significant adverse effects. There have been sporadic reports of co-consumption of illicit drugs with rodenticides such as warfarin and brodifacoum (BFC) over the past 20 years but recently, hundreds of people have been reported to have been poisoned with a mixture of synthetic cannabinoids and BFC. We have sought to establish whether BFC directly affects cannabinoid receptors, or their activation by the synthetic cannabinoid CP55940 or the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC).MethodsThe effects of BFC on the hyperpolarization of wild type AtT20 cells, or AtT20 cells stably expressing human CB1- or CB2- receptors, were studied using a fluorescent assay of membrane potential. The effect of BFC on CB1- and CB2-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) activation was measured using a BRET assay of cAMP levels in HEK 293 cells stably expressing human CB1 or CB2.ResultsBFC did not activate CB1 or CB2 receptors, or affect the hyperpolarization of wild type AtT20 cells produced by somatostatin. BFC (1 µM) did not affect the hyperpolarization of AtT20-CB1 or AtT20-CB2 cells produced by CP55940 or Δ9-THC. BFC (1 µM) did not affect the inhibition of forskolin-stimulated AC activity by CP55940 in HEK 293 cells expressing CB1 or CB2. BFC (1 µM) also failed to affect the desensitization of CB1 and CB2 signaling produced by prolonged (30 min) application of CP55940 or Δ9-THC to AtT20 cells.DiscussionBFC is not a cannabinoid receptor agonist, and appeared not to affect cannabinoid receptor activation. Our data suggests there is no pharmacodynamic rationale for mixing BFC with synthetic cannabinoids; however, it does not speak to whether BFC may affect synthetic cannabinoid metabolism or biodistribution. The reasons underlying the mixing of BFC with synthetic cannabinoids are unknown, and it remains to be established whether the “contamination” was deliberate or accidental. However, the consequences for people who ingested the mixture were often serious, and sometimes fatal, but this seems unlikely to be due to BFC action at cannabinoid receptors.

Spontaneous Hemorrhage Associated With Synthetic Cannabinoid Use

Synthetic cannabinoids (SCBs) are a popular new class of designer drugs. In 2018, the Centers for Disease Control and Prevention advised that illicit products containing SCB compounds were laced with brodifacoum (BDF), a highly potent superwarfarin easily purchased as a rodenticide. Multiple outbreaks of bleeding from BDF exposure have since been reported. Patients with BDF toxicity present with bleeding ranging from mild epistaxis to life-threatening hemorrhage. Given the increasing popularity of SCBs as recreational drugs, outbreaks from BDF exposure are likely to continue, presenting a public health risk. The nurse practitioner has a vital role in the complex, ongoing management of this patient population.

The relative toxicity of brodifacoum enantiomers

Brodifacoum (BDF) is a potent, long-acting anticoagulant rodenticide that can cause fatal poisoning in humans. The chemical structure of BDF includes 2 chiral carbons, resulting in 2 pairs of diastereomers, BDF-cis (R/S and S/R) and BDF-trans (R/R and S/S). However, the relative potency of these molecules is not known. The purpose of this study was to compare the in vitro and in vivo toxic effects of the 2 BDF diastereomer pairs. In adult Sprague-Dawley rats BDF-cis was significantly more toxic than BDF-trans (LD50 values of 219 versus 316 μg/kg, respectively) while racemic BDF had intermediate potency (266 μg/kg). In adult New Zealand white rabbits, BDF-cis had a longer half-life than BDF-trans which could contribute to its observed increased toxicity. Lastly, BDF-cis (10 μM), but not BDF-trans, damaged cultured SH-SY5Y human neuroblastoma cells by attenuating mitochondrial reductive capacity. Taken together, these data suggest that different toxic manifestations of BDF poisoning in mammals could be attributed, in part, to differences in relative enantiomer concentrations present in racemic formulations of this commercially-available toxicant.

The Bile Sequestrant Cholestyramine Increases Survival in a Rabbit Model of Brodifacoum Poisoning.

Patients exposed to long acting anticoagulant rodenticides (LAARs) are typically administered large amounts of oral vitamin K1 (VK1) to counteract life-threatening anticoagulant effects. Although VK1 treatment effectively prevents mortality, additional methods are needed to reduce the long duration of VK1 treatment which can last for months at high expense. We developed a model of brodifacoum (BDF) poisoning, one of the most potent LAARs, in adult male New Zealand White (NZW) rabbits. The LD50 for oral BDF was determined to be 192 μg/kg, similar to that calculated for adult rats. However, in contrast to rats, NZW rabbits exhibited severe internal hemorrhage including in the brain, symptoms which mimic what occurs in cases of human poisoning. Similar to warfarin, BDF and other LAARs undergo enterohepatic recirculation which contributes to their long half-lives. We therefore tested effects of cholestyramine (CSA), an FDA-approved bile sequestrant, on BDF-induced mortality. When given daily (0.67 g/kg, oral) starting the day of BDF administration, CSA reduced mortality from 67% to 11%. At the same CSA prevented the increase in clotting time, and reduced the decrease in core body temperature due to BDF. Given its excellent safety record and that it is approved for children older than 6 years, these findings suggest CSA could be considered as an adjunct to VK1 for treatment of LAAR poisoning.

Clinical characteristics and treatments of anti-coagulant rodenticide poisoning in children lacking of poisoning history

Objective To summarize the clinical characteristics, diagnosis and treatments of 50 children with no history of anti-coagulation rodenticide poisoning. Methods Fifty children lacking of poisoning history were admitted to our hospital from Feb 2011 to Dec 2015, who were due to hemorrhage and finally diagnosed as rodenticide poisoning.The clinical manifestation, characteristics of blood coagulation dysfunction, toxicology screening results and treatments of 50 cases were retrospectively analyzed. Results All 50 cases were from rural area, and denied the history of taking rodenticide by mistake.The time from the onset to admission to the hospital was from 1 d to 120 d, with an average of 11.66 d. Major clinical manifestations included: 34 cases(68%)of skin ecchymosis, 26 cases(52%)of epistaxis, 10 cases(20%)of bleeding gums and 4 cases(8%)of hematoma at the injection site, with PT 20 to over 200 s and APTT 31 to over 200 s(maximal detection value). The children were diagnosed as rodenticide poisoning when one of brodifacoum, bromadiolone, and diphacinone-sodium or combination of brodifacoum and bromadiolone were detected from their blood specimens, with an concentration of 5 to 3 270 μg/L.The diagnosed children were intravenous dripped with specific antagonist vitamin K1 and the critically ill children were added fresh frozen plasma.All children recovered from hemorrhage within 24 hours after treatment.The level of PT, APTT was close to normal after 3 to 7 days of treatment, and the using of vitamin K1 was accumulated about 30 to 70 mg.The children were given maintenance treatment of vitamin K1 for three months after discharge until the condition was stable. Conclusion Commonly used rodenticide has the characteristics of fat soluble, easy to accumulate, long incubation period, and long treatment period and so on.Children with no bleeding performance have the clinical tendency of acute bleeding, prolonged PT and APTT should be considered the possibility of rodenticide poisoning.Blood or urine samples should be screened for toxicology as soon as possible, and meanwhile children should be given antagonist treatment with special antidote vitamin K1 until the coagulation function is stable in normal range to avoid recurrence of the illness. Key words: Anti-coagulant; Rodenticide; Poisoning; Vitamin K1

Recovery of brodifacoum in vomitus following induction of emesis in dogs that had ingested rodenticide bait

AIM: To assess the benefit of inducing emesis in dogs that have ingested rodenticide bait containing brodifacoum (BDF), by determining the amount of BDF in bait recovered from the vomitus relative to the estimated amount consumed.METHODS: Between 2014 and 2015 samples of vomitus from seven dogs that ingested rodenticide baits containing BDF were submitted by veterinarians in New Zealand. All seven dogs had been given apomorphine by the veterinarian and vomited within 1 hour of ingesting the bait. Some or all of the bait particles were retrieved from each sample and were analysed for concentrations of BDF using HPLC. Based on estimations of the mass of bait consumed, the concentration of BDF stated on the product label, and the estimated mass of bait in the vomitus of each dog, the amount of BDF in the vomited bait was calculated as a percentage of the amount ingested.RESULTS: For five dogs an estimation of the mass of bait ingested was provided by the submitting veterinarian. For these dogs the estimated percentage of BDF in the bait retrieved from the vomitus was between 10–77%. All dogs were well after discharge but only one dog returned for further testing. This dog had a normal prothrombin time 3 days after ingestion.CONCLUSIONS AND CLINICAL RELEVANCE: The induction of emesis within 1 hour of ingestion can be a useful tool in reducing the exposure of dogs to a toxic dose of BDF. The BDF was not fully absorbed within 1 hour of ingestion suggesting that the early induction of emesis can remove bait containing BDF before it can be fully absorbed.

The Long-Lasting Rodenticide Brodifacoum Induces Neuropathology in Adult Male Rats.

Superwarfarins are very long-lasting rodenticides effective in warfarin-resistant rodents at extremely low doses. The consequences of chronic superwarfarin levels in tissues, due to biological half-lives on the order of 20 days, have not been examined. We now characterized the neurological effects of brodifacoum (BDF), one of the most widely used superwarfarins, in adult male Sprague Dawley rats. Dosing curves established the acute oral lethal dose for BDF as 221 ± 14 μg/kg. Measurement of tissue BDF levels showed accumulation throughout the body, including the central nervous system, with levels diminishing over several days. Immunocytochemical staining showed that both astrocyte and microglial activation was increased 4 days after BDF administration, as were levels of carbonylated proteins, and neuronal damage assessed by fluorojade B staining. Direct toxic effects of BDF on neurons and glia were observed using enriched cultures of cerebellar neurons and cortical astrocytes. Proteomic analysis of cerebellar lysates revealed that BDF altered expression of 667 proteins in adult rats. Gene ontology and pathway analysis identified changes in several functional pathways including cell metabolism, mitochondria function, and RNA handling with ribosomal proteins comprising the largest group. In vitro studies using primary astrocytes showed that BDF suppressed de novo protein synthesis. These findings demonstrate that superwarfarin accumulation increases indices of neuroinflammation and neuropathology in adult rodents, suggesting that methods which minimize BDF toxicity may not address delayed neurological sequelae.

LC-MS-MS Analysis of Brodifacoum Isomers in Rat Tissue.

Brodifacoum (BDF) is a second-generation anticoagulant rodenticide structurally related to warfarin but containing two chiral centers. Highly stable, BDF can contaminate food and water supplies causing accidental poisoning of humans and nontarget animals. To determine the distribution of BDF isomers in serum and tissues, a quantitative method was developed and validated according to FDA guidelines based on high-performance liquid chromatography-tandem mass spectrometry. A single liquid-liquid extraction step provided recoveries exceeding 93%. Reversed-phase chromatographic separations required <6 min, and quantitative analysis utilized a triple-quadrupole mass spectrometer equipped with negative ion electrospray and selected reaction monitoring. The standard curve had a linear regression coefficient of 0.999 and intra- and inter-assay variations of <10%. The chromatographic method enabled the resolution and measurement of pairs of BDF diastereomers in commercial materials as well as in rat tissues. This method is suitable for measuring BDF exposure as well as basic science studies of the distribution and elimination of BDF diastereomers to various tissues.

Brodifacoum Induces Early Hemoglobinuria and Late Hematuria in Rats: Novel Rapid Biomarkers of Poisoning

Introduction: Brodifacoum (BDF) is a superwarfarin that is used primarily as a rodenticide. There have been increasing numbers of reports of human cases of accidental or intentional BDF ingestion with high mortality rate. Its broad availability and high lethality suggest that BDF should be considered a potential chemical threat. Currently, there is no biomarker for early detection of BDF ingestion in humans; patients typically present with severe coagulopathy. Since we demonstrated earlier that warfarin can induce acute kidney injury with hematuria, we tested whether BDF would also lead to change in urinary biomarkers. Material and Methods: BDF was administered to Sprague Dawley rats via oral gavage. N-acetylcysteine (NAC) was given per os in drinking water 24 h prior to BDF. Urinalysis was performed at different times after BDF administration. Anticoagulation and serum creatinine levels were analyzed in the blood. Results: We observed that within a few hours the animals developed BDF-dose-dependent transient hemoglobinuria, which ceased within 24 h. This was accompanied by a transient decrease in hematocrit, gross hemolysis and an increase in free hemoglobin in the serum. At later times, animals developed true hematuria with red blood cells in the urine, which was associated with BDF anticoagulation. NAC prevented early hemoglobinuria, but not late hematuria associated with BDF. Conclusions: We propose that transient early hemoglobinuria (associated with oxidative stress) with consecutive late hematuria (associated with anticoagulation) are novel biomarkers of BDF poisoning, and they can be used in clinical setting or in mass casualty with BDF to identify poisoned patients.

Spatial and temporal exposure patterns in non-target small mammals during brodifacoum rat control

Worldwide pest rodents on livestock farms are often regulated using anticoagulant rodenticides (ARs). Second generation ARs in particular can cause poisoning in non-target species due to their high toxicity and persistence. However, research on exposure of small mammals is rare. We systematically investigated spatial and temporal exposure patterns of non-target small mammals in a large-scale replicated study. Small mammals were trapped at different distances to bait stations on ten farms before, during and after brodifacoum (BR) bait application, and liver samples of 1178 non-target small mammals were analyzed for residues of eight ARs using liquid chromatography coupled with tandem mass spectrometry. BR residues were present in 23% out of 742 samples collected during and after baiting. We found clear spatial and temporal exposure patterns. High BR residue concentrations mainly occurred within 15m from bait stations. Occurrence and concentrations of residues significantly decreased with increasing distance. This pattern was found in almost all investigated taxa. After baiting, significantly more individuals contained residues than during baiting but concentrations were considerably lower. Residue occurrence and concentrations differed significantly among taxa, with the highest maximal residue concentrations in Apodemus species, which are protected in Germany. Although Sorex species are known to be insectivorous we regularly found residues in this genus. Residues of active agents other than brodifacoum were rare in all samples. The confirmation of substantial primary exposure in non-target small mammals close to the baiting area indicates considerable risk of secondary poisoning of predators, a pathway that was possibly underestimated until now. Our results will help to develop risk mitigation strategies to reduce risk for non-target small mammals, as well as their predators, in relation to biocidal AR usage.

Inheritance of Low Grade Brodifacoum Resistance in the Norway Rat

We conducted a study to ascertain if an observed reduction of susceptibility to brodifacoum in Norway rats (Rattus norvegicus) present on at least 3 farms in Berkshire, United Kingdom, was heritable. This trait was demonstrated in individual rats by their survival after consumption of 0.0005% (m/m) bro difacoum in the diet for 7 days (Gill and MacNicoll 1991) and is termed «low grade» resistance. It is inherited through at least 5 generations and is not associated with vitamin K deficiency. Our data also suggest a different explanation for the inheritance of difenacoum resistance compared with that reported by Greaves and Cullen-Ayres (1988)

Coagulopathic Effects and Therapy of Brodifacoum Toxicosis in Dogs

The clinical signs and laboratory changes of brodifacoum (BDF) intoxicated dogs and their response to vitamin K1 treatment were examined. Brodifacoum, a second-generation anticoagulant rodenticide, was fed to four dogs for 3 consecutive days producing a cumulative dose of 1.1 mg BDF/kg body weight. Clinical observations of the animals were made daily throughout the study. Monitored laboratory parameters included: one-stage prothrombin time (OSPT), activated partial thromboplastin time (APTT), activated coagulation time (ACT), complete blood counts, thrombocyte counts, and serum chemistry values. Response to vitamin K1 therapy was evaluated clinically and by laboratory tests. Serum BDF concentrations were monitored. Inappetence and hemorrhagic tendencies were exhibited by day 5 postrodenticide exposure. One-stage prothrombin time, APTT, and ACT were 25% greater than time zero values at 24, 24, and 72 hours postdosing, respectively. All laboratory parameters returned to normal within 48 hours of initiating vitamin K1 therapy (0.83 mg/kg orally, TID for 5 days). Serum brodifacoum concentrations were highest (1065-1215 ng/mL) during the 3 days after BDF dosing and were detectable (3.0-7.5 ng/mL) until day 24 postexposure. A mean BDF elimination half-life of 6 +/- 4 days was observed.