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.

Active metabolite of the neurotoxic rodenticide bromethalin along with anticoagulant rodenticides detected in birds of prey in the northeastern United States.

Little is known about the ecologic fate of the neurotoxic rodenticide bromethalin, which is currently registered for use in the United States, Canada, and other countries including Australia. There is minimal research on bromethalin's potential to cause secondary toxicosis in nontarget wildlife. The aim of this study was to evaluate adipose tissue in four species of birds of prey presented to a wildlife clinic in Massachusetts, USA, for desmethylbromethalin (DMB), the active metabolite of bromethalin. Birds were also screened for anticoagulant rodenticides (ARs) in liver tissue to present a more complete picture of rodenticide exposures in this geographic area and to evaluate the impact of current mitigation measures in place during the time of sampling, 2021-2022. A total of 44 hawks and owls were included; DMB was found in 29.5% of birds and ARs were present in 95.5%. All birds with DMB detections also had residues of ARs. Among birds positive for ARs, 81% had two or more compounds. To the authors' knowledge the data presented here represent the first published monitoring study to document bromethalin/DMB bioaccumulation in obligate carnivores. As DMB is a more potent neurotoxicant than its parent compound, these results are cause for concern and an indication that further monitoring and study of the potential risk of bromethalin to wildlife species is needed. These findings have global implications as increasing concern regarding exposure to and toxicosis from ARs in nontarget wildlife worldwide leads to a search for alternatives and effective mitigation approaches.

Anticoagulant rodenticides are associated with increased stress and reduced body condition of avian scavengers in the Pacific Northwest.

Anticoagulant rodenticides (AR) have been used globally to manage commensal rodents for decades. However their application has also resulted in primary, secondary, and tertiary poisoning in wildlife. Widespread exposure to ARs (primarily second generation ARs; SGARs) in raptors and avian scavengers has triggered considerable conservation concern over their potential effects on populations. To identify risk to extant raptor and avian scavenger populations in Oregon and potential future risk to the California condor (Gymnogyps californianus) flock recently established in northern California, we assessed AR exposure and physiological responses in two avian scavenger species (common ravens [Corvus corax] and turkey vultures [Cathartes aura]) throughout Oregon between 2013 and 2019. AR exposure was widespread with 51% (35/68) of common ravens and 86% (63/73) of turkey vultures containing AR residues. The more acutely toxic SGAR brodifacoum was present in 83% and 90% of AR exposed common ravens and turkey vultures. The odds of AR exposure in common ravens were 4.7-fold higher along the coastal region compared to interior Oregon. For common ravens and turkey vultures that were exposed to ARs, respectively, 54% and 56% had concentrations that exceeded the 5% probability of toxicosis (>20 ng/g ww; Thomas et al., 2011), and 20% and 5% exceeded the 20% probability of toxicosis (>80 ng/g ww; Thomas et al., 2011). Common ravens exhibited a physiological response to AR exposure with fecal corticosterone metabolites increasing with sum ARs (ΣAR) concentrations. Both female common raven and turkey vultures' body condition was negatively correlated with increasing ΣAR concentrations. Our results suggest avian scavengers in Oregon are experiencing extensive AR exposure and the newly established population of California condors in northern California may experience similar AR exposure if they feed in southern Oregon. Understanding the sources of ARs across the landscape is an important first step in reducing or eliminating AR exposure in avian scavengers.

Long-term trends of second generation anticoagulant rodenticides (SGARs) show widespread contamination of a bird-eating predator, the Eurasian Sparrowhawk (Accipiter nisus) in Britain.

Second generation anticoagulant rodenticides (SGARs) are widely used to control rodents around the world. However, contamination by SGARs is detectable in many non-target species, particularly carnivorous mammals or birds-of-prey that hunt or scavenge on poisoned rodents. The SGAR trophic transfer pathway via rodents and their predators/scavengers appears widespread, but little is known of other pathways of SGAR contamination in non-target wildlife. This is despite the detection of SGARs in predators that do not eat rodents, such as specialist bird-eating hawks. We used a Bayesian modelling framework to examine the extent and spatio-temporal trends of SGAR contamination in the livers of 259 Eurasian Sparrowhawks, a specialist bird-eating raptor, in regions of Britain during 1995-2015. SGARs, predominantly difenacoum, were detected in 81% of birds, with highest concentrations in males and adults. SGAR concentrations in birds were lowest in Scotland and higher or increasing in other regions of Britain, which had a greater arable or urban land cover where SGARs may be widely deployed for rodent control. However, there was no overall trend for Britain, and 97% of SGAR residues in Eurasian Sparrowhawks were below 100 ng/g (wet weight), which is a potential threshold for lethal effects. The results have potential implications for the population decline of Eurasian Sparrowhawks in Britain. Fundamentally, the results indicate an extensive and persistent contamination of the avian trophic transfer pathway on a national scale, where bird-eating raptors and, by extension, their prey appear to be widely exposed to SGARs. Consequently, these findings have implications for wildlife contamination worldwide, wherever these common rodenticides are deployed, as widespread exposure of non-target species can apparently occur via multiple trophic transfer pathways involving birds as well as rodents.

In vitro assessment of the dermal penetration potential of sodium fluoroacetate using a formulated product.

This paper presents experimental data on the skin absorption of sodium fluoroacetate from a formulated product using an in vitro approach and human skin. Sodium fluoroacetate is a pesticide, typically applied in formulation (1080) for the control of unwanted vertebrate invasive species. It has been assigned a Skin Notation by the ACGIH, and other international workplace health regulatory bodies, due to its predicted ability to permeate intact and abraded human skin. However, there is a distinct lack of experimental data on the skin absorption of sodium fluoroacetate to support this assignment. This study found that sodium fluoroacetate, as a formulated product, permeated the human epidermis when in direct contact for greater than 10 hr. A steady-state flux (Jss) of 1.31 ± 0.043 µg/cm2/hr and a lag time of 6.1 hr was calculated from cumulative skin permeation data. This study provides important empirical evidence in support of the assignment of a Skin Notation.

New insights on in vitro biotransformation of anticoagulant rodenticides in fish.

The assessment of the bioaccumulation potential of chemicals is an essential and mandatory part of their regulatory environmental risk and hazard assessment. So far, in vitro data on fish metabolism is rarely available for biocidal active substances such as anticoagulant rodenticides. In this case study we present in vitro biotransformation rates of eight biocidal and one pharmaceutical anticoagulants in rainbow trout (Oncorhynchus mykiss) liver subcellular S9 fraction (RT-S9) determined following the Organisation for Economic Co-operation and Development test guideline 319B method at two different incubation temperatures (i.e., 12 ± 1 °C and 23 ± 2 °C). Furthermore, we address challenges associated with the usability and interpretation of in vitro data to support the decision making within the regulatory bioaccumulation assessment in bridging the gap between in silico methods and in vivo studies. According to our results, four of the tested substances (i.e., chlorophacinone, coumatetralyl, bromadiolone, and difenacoum) exhibited significant intrinsic clearance (p < .001) in the RT-S9 assay. Overall, the observed metabolism was (very) slow and clearance rates were temperature-dependent. Whether the determined in vitro biotransformation rate had a substantial influence on the predicted bioconcentration factor during extrapolation was subject to the lipophilicity of the test substance. Further improvements of existing concepts are needed to overcome uncertainties in the prediction of bioconcentration factors for chemicals such as anticoagulants.

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

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

Evaluating a Rapid Field Assessment System for Anticoagulant Rodenticide Exposure of Raptors.

Anticoagulant rodenticides (ARs) are commonly used to control rodent pests. However, worldwide, their use is associated with secondary and tertiary poisoning of nontarget species, especially predatory and scavenging birds. No medical device can rapidly test for AR exposure of avian wildlife. Prothrombin time (PT) is a useful biomarker for AR exposure, and multiple commercially available point-of-care (POC) devices measure PT of humans, and domestic and companion mammals. We evaluated the potential of one commercially available POC device, the Coag-Sense® PT/INR Monitoring System, to rapidly detect AR exposure of living birds of prey. The Coag-Sense device delivered repeatable PT measurements on avian blood samples collected from four species of raptors trapped during migration (Intraclass Correlation Coefficient > 0.9; overall intra-sample variation CV: 5.7%). However, PT measurements reported by the Coag-Sense system from 81 ferruginous hawk (Buteo regalis) nestlings were not correlated to those measured by a one-stage laboratory avian PT assay (r = - 0.017, p = 0.88). Although precise, the lack of agreement in PT estimates from the Coag-Sense device and the laboratory assay indicates that this device is not suitable for detecting potential AR exposure of birds of prey. The lack of suitability may be related to the use of a mammalian reagent in the clotting reaction, suggesting that the device may perform better in testing mammalian wildlife.

Determination of anticoagulant rodenticides in faeces of exposed dogs and in a healthy dog population.

BACKGROUND: Exposure to anticoagulant rodenticides (ARs) in dogs is among the most common causes of poisoning in small animal practice, but information about toxicokinetic of these rodenticides in dogs is lacking. We analysed blood and faeces from five accidentally exposed dogs and 110 healthy dogs by reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry. The aim of the study was to estimate elimination of brodifacoum, bromadiolone and difenacoum after acute exposure, calculate the half-lives of these rodenticides in dogs, estimate faecal elimination in a litter of puppies born, and further to identify the extent of AR exposure in a healthy dog population. RESULTS: Three dogs were included after single ingestions of brodifacoum; two dogs ingested bromadiolone and one dog ingested difenacoum. Maximum concentrations in faeces were found after day 2-3 for all ARs. The distribution half-lives were 1-10 days for brodifacoum, 1-2 days for bromadiolone and 10 days for difenacoum. Brodifacoum and difenacoum had estimated terminal half-lives of 200-330 days and 190 days, respectively. In contrast, bromadiolone had an estimated terminal half-life of 30 days. No clinical signs of poisoning or coagulopathy were observed in terminal elimination period. In blood, the terminal half-life of brodifacoum was estimated to 8 days. Faeces from a litter of puppies born from one of the poisoned dogs were examined, and measurable concentrations of brodifacoum were detected in all samples for at least 28 days after parturition. A cross-sectional study of 110 healthy domestic dogs was performed to estimate ARs exposure in a dog population. Difenacoum was detected in faeces of one dog. Blood and faecal samples from the remaining dogs were negative for all ARs. CONCLUSIONS: Based on the limited pharmacokinetic data from these dogs, our results suggest that ARs have a biphasic elimination in faeces using a two-compartment elimination kinetics model. We have shown that faecal analysis is suitable and reliable for the assessment of ARs exposure in dogs and a tool for estimating the AR half-lives. Half-lives of ARs could be a valuable indicator in the exposed dogs and provides important information for veterinarians monitoring AR exposure and assessment of treatment length in dogs.

Comparison of anticoagulant rodenticide concentrations in liver and feces from apparently healthy red foxes.

Exposure of wildlife and domestic animals to anticoagulant rodenticides (ARs) is a worldwide concern, but few methods exist to determine residue levels in live animals. Traditional liver detection methods preclude determining exposure in live wildlife. To determine the value of assessing AR exposure by fecal analysis, we compared fecal and liver residues of ARs in the same animals. We collected liver and fecal samples from 40 apparently healthy red foxes (Vulpes vulpes) potentially exposed to ARs, and quantified brodifacoum, bromadiolone, coumatetralyl, difenacoum, difethialone, and flocoumafen residues by liquid chromatography-tandem mass spectrometry. Residues of ARs were detected in 53% of the fecal samples and 83% of the liver samples. We found good concordance between AR residues in feces and liver for coumatetralyl, difenacoum, and difethialone. Bromadiolone occurred in significantly greater frequency in livers compared to feces, but no significant difference in concentration between feces and liver in individual foxes could be detected. Brodifacoum displayed a significant difference in concentration and occurrence of positive samples between liver and feces. Our findings demonstrate that fecal analysis of ARs provides a feasible and valuable non-lethal means of determine AR exposure in live wildlife.

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites.

RATIONALE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Frequent detection of anticoagulant rodenticides in raptors sampled in Taiwan reflects government rodent control policy.

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 ±â€¯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.

Widespread anticoagulant poison exposure in predators in a rapidly growing South African city.

Anticoagulant rodenticides (ARs) are used worldwide to control rodent populations. ARs bioaccumulate across trophic levels and threaten non-target wildlife. We investigated the prevalence of AR exposure in seven predator species in the rapidly developing Greater Cape Town region of South Africa - a mosaic of natural, urban, and agricultural areas within a global biodiversity hotspot. We focused sampling on caracals (Caracal caracal, n = 28) as part of a larger caracal ecology study, but also opportunistically sampled Cape Clawless otters (Aonyx capensis, n = 9), large-spotted genets (Genetta tigrina, n = 4), honey badger (Mellivora capensis, n = 1), water mongoose (Atilax paludinosus, n = 1), small gray mongoose (Galerella pulverulenta, n = 1), and Cape Eagle owl (Bubo capensis, n = 1). We tested livers from all species, and blood from ten caracals, for eight AR compounds to assess prevalence and amount of exposure for each compound. We used generalized linear models to test spatial, demographic, and seasonal risk factors for ten measures of AR exposure in caracals. We detected at least one of the four most toxic AR compounds in six species. Exposure was high for caracals (92%) and all species combined (81%). For caracals, proximity to vineyards was the most important AR exposure risk factor. Vineyards in Cape Town do not use ARs to protect their vines but do host commercial hospitality structures where ARs are used. Vineyards may thus link caracals that forage within vineyards to the rat poisons used in and around their commercial structures. Residue levels were unexpected in large-spotted genets and Cape Clawless otters, suggesting invertebrate vectors. ARs may present a cryptic threat to populations already vulnerable to increasing habitat loss, vehicle collisions, poachers and fire. Targeted mitigation should include a mix of environmentally responsible policies that reduce AR use, particularly in areas near wildlife habitat.

Characterization of Bromethalin and its Degradation Products in Veterinary Toxicology Samples by GC-MS-MS.

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.

Anticoagulant rodenticide exposure in an Australian predatory bird increases with proximity to developed habitat.

Anticoagulant rodenticides (ARs) are commonly used worldwide to control commensal rodents. Second generation anticoagulant rodenticides (SGARs) are highly persistent and have the potential to cause secondary poisoning in wildlife. To date no comprehensive assessment has been conducted on AR residues in Australian wildlife. My aim was to measure AR exposure in a common widespread owl species, the Southern Boobook (Ninox boobook) using boobooks found dead or moribund in order to assess the spatial distribution of this potential threat. A high percentage of boobooks were exposed (72.6%) and many showed potentially dangerous levels of AR residue (>0.1 mg/kg) in liver tissue (50.7%). Multiple rodenticides were detected in the livers of 38.4% of boobooks tested. Total liver concentration of ARs correlated positively with the proportions of developed areas around points where dead boobooks were recovered and negatively with proportions of agricultural and native land covers. Total AR concentration in livers correlated more closely with land use type at the spatial scale of a boobook's home range than at smaller or larger spatial scales. Two rodenticides not used by the public (difethialone and flocoumafen) were detected in boobooks indicating that professional use of ARs contributed to secondary exposure. Multiple ARs were also detected in recent fledglings, indicating probable exposure prior to fledging. Taken together, these results suggest that AR exposure poses a serious threat to native predators in Australia, particularly in species using urban and peri-urban areas and species with large home ranges.

The prevalence and correlates of anticoagulant rodenticide exposure in non-target predators and scavengers in Finland.

The most common rodent control method worldwide is anticoagulant rodenticides (ARs), which cause death by internal bleeding. ARs can transfer to non-target predators via secondary exposure, i.e. by consuming contaminated rodents. Here we quantify the prevalence of seven AR substances in the liver tissues of altogether 17 mammalian or avian predator or scavenger species in Finland. In addition, we identify the environmental and biological factors potentially linked to secondary AR poisoning. No previous AR screenings have been conducted in the country, despite the widespread use of ARs and their potential impacts on the high levels of the ecosystem food chain. ARs were detected (≥0.3 µg/kg) in 82% of the 131 samples. The most prevalent and the AR with highest concentrations was bromadiolone (65% of samples). In 77% of the positive samples more than one (2-5) different ARs were detected. Of the environmental variables, we only found a weakly positive relationship between the coumatetralyl concentration and the livestock farm density. Conversely, overall AR concentration and number, as well as the concentration of three separate ARs (coumatetralyl, difenacoum and bromadiolone) differed among the three species groups tested, with the group "other mammals" (largely represented by red fox and raccoon dog) having higher values than the groups presented by mustelids or by birds. ARs are authorized only as biocides in Finland and a national strategy on risk management (e.g. for minimising secondary poisoning of non-target species) of ARs was adopted in 2011. Based on these results it appears that the risk mitigation measures (RMMs) either have not been followed or have not been effective in preventing wide scale secondary exposure. Continued monitoring of AR residues in non-target species is needed in order to evaluate the effectiveness of current RMMs and a need for new ones to reduce the risk of secondary poisoning.

Long-term increase in secondary exposure to anticoagulant rodenticides in European polecats Mustela putorius in Great Britain.

As a result of legal protection and population recovery, European polecats (Mustela putorius) in Great Britain are expanding into areas associated with greater usage of second-generation anticoagulant rodenticides (SGARs). We analysed polecat livers collected from road casualties from 2013 to 2016 for residues of five SGARs. We related variation in residues to polecat traits and potential exposure pathways, by analysing stable isotopes of carbon (δ13C) and nitrogen (δ15N) in their whiskers. 54 of 68 (79%) polecats had detectable residues of at least one SGAR. Bromadiolone (71%) was the most frequently detected compound, followed by difenacoum (53%) and brodifacoum (35%). Applying historical limits of detection to allow comparison between these new data and previous assessments, we show that in the 25 years from 1992 to 2016 inclusive, the rate of detection of SGARs in polecats in Britain has increased by a factor of 1.7. The probability of SGAR detection was positively related to increasing values of δ15N, suggesting that polecats feeding at a higher trophic level were more likely to be exposed. Total concentrations of SGARs in polecats with detectable residues were higher in polecats collected in arable compared to pastoral habitats, and in the west compared to the east of Britain. The number of compounds detected and total concentrations of SGARs increased with polecat age. There was no evidence of regional or seasonal variation in the probability of detecting SGARs, suggesting that the current risk of exposure to SGARs does not vary seasonally and has increased (from that in the 1990s) throughout the polecat's range. We recommend quantification of current practices in rodenticide usage, particularly in the light of recent regulatory changes, to enable assessment and mitigation of the risks of secondary exposure to rodenticides in non-target wildlife.

GABAA receptor subtype selectivity of the proconvulsant rodenticide TETS.

The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant (lethal dose in humans 7-10 mg) that is listed as a possible threat agent by the United States Department of Homeland Security. TETS has previously been studied in vivo for toxicity and in vitro in binding assays, with the latter demonstrating it to be a non-competitive antagonist on GABAA receptors. To determine whether TETS exhibits subtype selectivity for a particular GABAA receptor combination, we used whole-cell patch-clamp to determine the potency of TETS on the major synaptic and extrasynaptic GABAA receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABAA receptors, TETS exhibited the highest activity on α2ß3γ2 (IC50 480 nM, 95% CI 320-640 nM) and α6ß3γ2 (IC50 400 nM, 95% CI 290-510 nM). Introducing ß1 or ß2 subunits into these receptor combinations reduced or abolished TETS sensitivity, suggesting that TETS preferentially affects receptors with α2/ß3 or α6/ß3 composition. Since α2ß3γ2 receptors make up 15-20% of the GABAA receptors in the mammalian CNS, we suggest that α2ß3γ2 is probably the most important GABAA receptor for the seizure-inducing activity of TETS.

Elevated difenacoum metabolism is involved in the difenacoum-resistant phenotype observed in Berkshire rats homozygous for the L120Q mutation in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene.

BACKGROUND: Soon after difenacoum began to be used, resistance to this rodenticide was detected in rats in northeast Hampshire and northwest Berkshire in England. Resistance to difenacoum has been reported to be stronger in rats from Berkshire than in rats from Hampshire. Surprisingly, after the discovery of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene, rats from Berkshire and Hampshire were all shown to be homozygous for the L120Q mutation in Vkorc1. RESULTS: This study aimed to evaluate the resistance of Berkshire rats to confirm their extreme resistance and determine mechanisms supporting this resistance. For this purpose, we created a quasicongenic rat F7 strain by using a Berkshire rat as a donor to introduce the L120Q mutation in Vkorc1 into the genetic background of an anticoagulant-susceptible recipient strain. The use of F7 rats enabled demonstration of (i) the level of resistance to difenacoum conferred by the L120Q mutation, (ii) co-dominance of the L120 and Q120 alleles, (iii) the extreme resistance of Berkshire rats compared with Q120/Q120 rats as a consequence of additional resistance mechanisms, and (iv) the involvement of cytochrome P 450 (CYP450) enzymes in this extreme resistance. CONCLUSION: This study demonstrated that elevated CYP450 oxidative metabolism leading to accelerated difenacoum detoxification is involved in the Berkshire phenotype. © 2017 Society of Chemical Industry.