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.

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.

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.

Silent killers? The widespread exposure of predatory nocturnal birds to anticoagulant rodenticides.

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

Anthropogenic threats to owls: Insights from rehabilitation admittance data and rodenticide screening in Texas.

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

Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry.

A specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the anticoagulant rodenticide diphacinone (DPN) in mouse and rat liver. Tissue samples were extracted with a mixture of water and acetonitrile containing ammonium hydroxide. The extracted sample was cleaned up with a combination of liquid-liquid partitioning and dispersive solid phase extraction. Chromatographic separation was achieved using a Waters X-Bridge BEH C-18 LC column (50 mm, 2.1 mm ID, 2.5 µm particle size) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for DPN was m/z 339.0 → 167.0 for quantitation and 339.0 → 172.0 and 339.0 → 116.0 for confirmation. The linear range was 0.5 to 375 ng/mL. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97% - 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). The recovery of DPN fortified at 3 different levels averaged 106% in rat liver and 101% in mouse liver. The established method was successfully used to determine DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. The observed residue levels were consistent with values observed in other rodent studies. However, the amount of bait consumed was lower for the novel baits evaluated in this study.

Comprehensive Evaluation of an HPLC-MS-MS Method for Quantitation of Seven Anti-Coagulant Rodenticides and Dicoumarol in Animal Serum.

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

The effects of brodifacoum cereal bait pellets on early life stages of the rice coral Montipora capitata.

Midway Atoll in the Northwestern Hawaiian Islands is home to ground nesting birds that are threatened by invasive mice. Planned rodent eradication efforts for the island involve aerial application of cereal bait pellets containing the chemical rodenticide brodifacoum. Given the nature of the application method, drift of cereal bait pellets into the coastal waters surrounding Midway Atoll is unavoidable. To understand whether cereal bait pellets impact marine invertebrates, gametes and larvae of the reef-building coral Montipora capitata were exposed to brodifacoum, cereal bait pellets containing brodifacoum, and inert cereal bait pellets without the rodenticide. Fertilization success and larval survival were assessed at nominal brodifacoum concentrations of 1, 10, and 100 ppb. Fertilization success decreased by 15% after exposure to 100 ppb brodifacoum solutions. Larval survival was not reduced by exposure to brodifacoum solutions. Cereal bait pellets containing brodifacoum reduced fertilization success at 10 ppb brodifacoum in 0.4 g per L pellet solutions by 34.84%, and inhibited fertilization at 100 ppb brodifacoum in 4 g of pellet per L solution. Inert cereal bait pellets had similar effects, reducing fertilization success at 0.4 g of pellet per L by 40.50%, and inhibiting fertilization at 4 g per L pellet solutions. Larval survival was reduced by >43% after prolonged exposure to 4 g per L pellet solutions. The highest concentration used in this study was meant to represent an extreme and unlikely condition resulting from an accidental spill. Our findings indicate large amounts of cereal bait pellets entering the coastal environment of Midway Atoll, if occurring during a coral spawning event, would reduce coral reproduction by decreasing fertilization success. It is difficult to know the ecologically relevant concentrations of cereal bait pellets in coastal environments due to unavoidable bait drift after land applications, but results indicate small amounts of pellet drifting into coastal environments would not severely reduce coral reproductive capacity. Best management practices should consider known coral reproductive periods when scheduling applications of pellets on tropical islands to reduce the risk of negative impacts of large-scale accidents on corals.

Anticoagulant Rodenticide Contamination of Terrestrial Birds of Prey from Western Canada: Patterns and Trends, 1988-2018.

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

Enantiomer fraction evaluation of the four stereoisomers of second-generation anticoagulant rodenticides in biological matrices with polysaccharide-based chiral selectors and liquid chromatography tandem mass spectrometry.

Numerous cases of wildlife exposure to five second-generation anticoagulant rodenticides have been reported worldwide, and residues of these chiral pesticides in biological matrices are still quantified by achiral liquid chromatography methods. However, they are a mixture of cis- and trans-diastereomers, thus a mixture of four stereoisomers. Their persistence must be evaluated in a differentiated way in the food chain of concerned predator species in order to reduce the environmental impact. This article presents an evaluation of the chiral selectivity of five polysaccharide-based chiral selectors for the four stereoisomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone. Different chromatographic parameters, influencing the chiral separation, such as organic modifier (acetonitrile, methanol), percentage of formic acid and water content in the mobile phase are systematically tested for all columns. It was shown that little amount of water added to the acetonitrile mobile phase may influence the retention behaviors between reversed phase and HILIC-like modes, and consequently the enantiomer elution order of the four stereoisomers. On the contrary, reversed phase is always the observed mode for the methanol water mobile phase. A suitable combination of all these parameters is presented for each second-generation anticoagulant rodenticide with a description of the enantioresolution, the enantiomer elution order and the retention times of the respective stereoisomers. A method is validated for all stereoisomers of each second-generation anticoagulant rodenticide with chicken liver and according to an official bioanalytical guideline. As an example, the enantiomer fraction is evaluated in the liver of a raptor species (rodent predator) exposed to bromadiolone and difenacoum. The results showed that only one enantiomer of trans-bromadiolone and one enantiomer of cis-difenacoum is present in hepatic residues, although all four stereoisomers are present in bromadiolone and difenacoum rodenticide baits.

Methods in food defence: a simple and robust screening for 16 rodenticides by LC-DAD/FLD following QuEChERS-based extraction.

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

Spatial variation of rodenticides and emerging contaminants in blood of raptor nestlings from Germany.

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

Widespread exposure of powerful owls to second-generation anticoagulant rodenticides in Australia spans an urban to agricultural and forest landscape.

The powerful owl (Ninox strenua) is a threatened apex predator that consumes mainly arboreal marsupial prey. Low density populations reside in urban landscapes where their viability is tenuous. The catalyst for this research was the reported death of eight powerful owls around Melbourne, Australia, in less than one year (2020/2021). Eighteen deceased owls were toxicologically screened. We assessed toxic metals (Mercury Hg, Lead Pb, Cadmium Cd and Arsenic As) and anticoagulant rodenticides (ARs) in liver (n = 18 owls) and an extensive range of agricultural chemicals in muscle (n = 14). Almost all agricultural chemicals were below detection limits except for p,p-DDE, which was detected in 71% of birds at relatively low levels. Toxic metals detected in some individuals were generally at low levels. However, ARs were detected in 83.3% of powerful owls. The most common second-generation anticoagulant rodenticide (SGAR) detected was brodifacoum, which was present in every bird in which a rodenticide was detected. Brodifacoum was often present at toxic levels and in some instances at potentially lethal levels. Presence of brodifacoum was detected across the complete urban-forest/agriculture gradient, suggesting widespread exposure. Powerful owls do not scavenge but prey upon arboreal marsupials, and generally not rodents, suggesting that brodifacoum is entering the powerful owl food web via accidental or deliberate poisoning of non-target species (possums). We highlight a critical need to investigate SGARs in food webs globally, and not just in species directly targeted for poisoning or their predators.

Anticoagulant rodenticide exposure in raptors from Ontario, Canada.

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

Antemortem and postmortem rodenticide analysis in forensic toxicology as a part of an LC-MS/MS-based multi-target screening strategy.

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

Exposure of predatory and scavenging birds to anticoagulant rodenticides in France: Exploration of data from French surveillance programs.

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

Development and Validation of a Quantitative UHPLC-MS-MS Method for the Determination of Alpha-Chloralose in Feline Blood and Application on Blood Samples Collected from Cats with Symptoms of Alpha-Chloralose Poisoning.

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

Rapid method for the detection of rodenticides in contaminated foods.

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