Generation Anticoagulant Rodenticides Research Articles

Anticoagulant rodenticide exposure in common buzzards: Impact of new rules for rodenticide use

Rodenticides are a key component of rodent management strategies, but birds of prey are susceptible to non-target exposure. New rules on sale and use of rodenticide products were introduced across the UK in 2016 in an industry-led stewardship scheme, with the aim of reducing this risk. To determine if this intervention has achieved its aim, exposure to second generation anticoagulant rodenticides (SGARs) was measured in buzzards. Liver samples from 790 buzzards collected between 2005 and 2022 (excluding 2016 and 2017 samples) were analyzed and the percentage presence and concentrations of SGARs from pre-stewardship and post-stewardship samples were compared.There was no statistically significant decrease in the percentage of buzzards exposed to bromadiolone, difenacoum or combined SGAR residues after the introduction of stewardship. The percentage of buzzards exposed to brodifacoum increased significantly post-stewardship, from 8 % to 27 %. There were no significant decreases in the concentrations of individual SGARs post-stewardship but concentration of combined SGARs increased significantly post-stewardship. Individual buzzards were significantly more likely to be exposed to multiple SGARs post-stewardship.Rodenticide poisoning was recorded as the cause of death for 5 % of pre- and post-stewardship buzzards with detectable levels of SGARs, and 90 % of these had combined SGAR residues >0.1 mg/kg.These findings suggest that the industry-led stewardship scheme has not yet had the intended impact of reducing SGAR exposure in non-target wildlife. The study highlights a substantial increase in exposure of buzzards to brodifacoum and to multiple SGARs post-stewardship, indicating that further changes to the stewardship scheme may be necessary.

Greater predisposition to second generation anticoagulant rodenticide exposure in red foxes (Vulpes vulpes) weakened by suspected infectious disease

Anticoagulant rodenticide (AR) exposure in wild carnivores is a current and global concern due to continuous and widespread use worldwide. We studied the prevalence of ARs in liver samples of 25 red foxes (Vulpes vulpes), 3 European badgers (Meles meles) and 2 genets (Genneta genneta) from Alicante (Spanish Levante region) obtained in 2021 and 2022. In addition to trauma, poisoning by pesticides is the most frequent cause of death in wild carnivores in this region. The present research aims to explain a possible association between the fact of suffering from an infectious disease and the increase in ARs concentrations in the affected animals.Both first- and second-generation ARs were analysed by HPLC/MS/TOF in liver samples. Apart from the cause of death, the influence of other variables such as age, sex and body weight were also assessed on AR liver concentrations. Potential health risks for individuals and populations in the study area have also been studied. Our research detected higher AR concentrations in the group of red foxes clinically diagnosed with infectious disease compared to the group of apparently healthy red foxes, mostly killed by trauma. Furthermore, our results lead us to suggest that red fox could be considered a good sentinel species for the risk of exposure to ARs in other wild mammals. All the livers analysed contained ARs and the most detected compounds in red fox were difenacoum, bromadiolone, brodifacoum, present in all the samples analysed, and flocoumafen (in 96 %). Additionally, 53 % of the animals had at least one second generation anticoagulant rodenticide (SGAR) above the threshold value reported as triggering adverse health effects (200 ng/g). Regarding this, the level of risk in red fox in this area might be classified as high and worrying. Moreover, we suggest that individuals and populations with weakened health due to other diseases (for example, infectious or parasitic diseases) might be more prone to high exposure to anticoagulant rodenticides and, very probably, would be more sensitive to suffering serious effects at lower doses of ARs.

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.

Long-Term Trends of Second Generation Anticoagulant Rodenticides (Sgars) Show Widespread Contamination of a Bird-Eating Predator, the Eurasian Sparrowhawk (Accipiter Nisus) in Britain

Long-Term Trends of Second Generation Anticoagulant Rodenticides (Sgars) Show Widespread Contamination of a Bird-Eating Predator, the Eurasian Sparrowhawk (Accipiter Nisus) in Britain

Conservation Letter: Raptors and Anticoagulant Rodenticides

Conservation Letter: Raptors and Anticoagulant Rodenticides

Annual abundance of common Kestrels (Falco tinnunculus) is negatively associated with second generation anticoagulant rodenticides

Rats and mice can damage food and agricultural products as well as transmit diseases, thereby requiring control of their numbers. Application of Second Generation Anticoagulant Rodenticides (SGARs) often reduces rodent numbers locally. However, predators eating rodents, including non-target species, that have consumed SGARs may be secondarily exposed and potentially lethally poisoned. Here we study whether SGARs may have contributed to the widespread population declines of a rodent-eating raptor, the Common Kestrel (Falco tinnunculus) in the UK. We show that 161 (66.8%) of the 241 Kestrels submitted for ecotoxicology tests between 1997 and 2012 had detectable levels of at least one SGAR in their livers. Adult Kestrels had significantly higher prevalence of SGARs than juveniles, suggesting accumulation of SGARs through time. The prevalence and concentrations of individual SGARs in Kestrels were significantly higher in England than in Scotland. SGAR prevalence in Kestrels were positively associated with some land cover types, primarily arable cereals and broad-leaved woodland, and negatively associated with mainly mean elevation, probably reflecting variation in SGAR usage across land cover types. By using volunteer-collected data on national Kestrel abundance 1997–2012, we show that there is a negative correlation between the Kestrel population index in a specific year and the concentration of bromadialone as well as the total SGAR concentration in the same year. Although correlative, this is the first study to provide evidence for a potential population-limiting effect of SGARs on a raptor.

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 573ng/g ww respectively), while trans-brodifacoum was less than 46ng/g and cis-bromadiolone was barely detected. cis-Difenacoum showed the highest prevalence and the highest concentration was 82ng/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.

Chiral liquid chromatography-tandem mass spectrometry analysis of superwarfarin rodenticide stereoisomers – Bromadiolone, difenacoum and brodifacoum – In human plasma

Superwarfarins are second-generation long-acting anticoagulant rodenticides that can cause unintended human and wildlife toxicity due, in part, to their prolonged half-lives. Commercially available superwarfarin rodenticides are synthesized as racemates with two asymmetric carbons, producing four stereoisomers. To support studies of human plasma half-lives of individual superwarfarin stereoisomers, a method was developed based on LC-MS/MS to separate and quantify stereoisomers of the commercially important superwarfarins bromadiolone, difenacoum and brodifacoum. Human plasma samples were prepared using protein precipitation and centrifugation. Chiral-phase HPLC separation was carried out on-line with tandem mass spectrometric quantitative analysis of the eluting stereoisomers using selected-reaction monitoring with positive ion electrospray on a triple quadrupole mass spectrometer. All four stereoisomers of each superwarfarin were resolved within 12.5 min with calibration curves spanning 2–3 orders of magnitude and lower limits of quantitation between 0.87 and 2.55 ng/mL. This method was used to determine the half-lives of superwarfarin stereoisomers in plasma from patients who had inhaled synthetic cannabinoid products contaminated with superwarfarins. These data may be used to guide the development of safer next generation anticoagulant rodenticides stereoisomers.

Temporal Persistence of Bromadiolone in Decomposing Bodies of Common Kestrel (Falco tinnunculus).

Bromadiolone is a second generation anticoagulant rodenticide (SGAR) used to control pest rodents worldwide. SGARs are frequently involved in secondary poisoning in rodent predators due to their persistence and toxicity. This study aims to evaluate the persistence of bromadiolone in liver at different stages of carcass decomposition in experimentally-dosed common kestrels (Falco tinnunculus) to understand the possibility of detecting bromadiolone in cases of wildlife poisoning and the potential risk of tertiary poisoning. Twelve individuals were divided into the bromadiolone-dose group (dosed with 55 mg/kg b.w) and the control group. Hepatic bromadiolone concentrations found in each stage of decomposition were: 3000, 2891, 4804, 4245, 8848, and 756 ng/g dry weight at 1–2 h (fresh carcass), 24 h (moderate decomposition), 72 h, 96 h (advanced decomposition), seven days (very advanced decomposition), and 15 days (initial skeletal reduction) after death, respectively. Liver bromadiolone concentrations in carcasses remained relatively stable over the first four days and raised on day 7 of decomposition under the specific conditions of this experiment, presenting a risk of causing tertiary poisoning. However, at the initial skeletal reduction stage, liver bromadiolone concentration declined, which should be considered to interpret toxicological analyses and for proper diagnosis. This experimental study provides for the first time some light to better understand the degradation of SGARs in carcasses in the wild.

Mus musculus populations in Western Australia lack VKORC1 mutations conferring resistance to first generation anticoagulant rodenticides: Implications for conservation and biosecurity.

Humans routinely attempt to manage pest rodent populations with anticoagulant rodenticides (ARs). We require information on resistance to ARs within rodent populations to have effective eradication programs that minimise exposure in non-target species. Mutations to the VKORC1 gene have been shown to confer resistance in rodents with high proportions of resistance in mice found in all European populations tested. We screened mutations in Mus musculus within Western Australia, by sampling populations from the capital city (Perth) and a remote island (Browse Island). These are the first Australian mouse populations screened for resistance using this method. Additionally, the mitochondrial D-loop of house mice was sequenced to explore population genetic structure, identify the origin of Western Australian mice, and to elucidate whether resistance was linked to certain haplotypes. No resistance-related VKORC1 mutations were detected in either house mouse population. A genetic introgression in the intronic sequence of the VKORC1 gene of Browse Island house mouse was detected which is thought to have originated through hybridisation with the Algerian mouse (Mus spretus). Analysis of the mitochondrial D-loop reported two haplotypes in the house mouse population of Perth, and two haplotypes in the population of Browse Island. Both house mouse populations exhibited no genetic resistance to ARs, in spite of free use of ARs in Western Australia. Therefore weaker anticoagulant rodenticides can be employed in pest control and eradication attempts, which will result in reduced negative impacts on non-target species. Biosecurity measures must be in place to avoid introduction of resistant house mice, and new house mouse subspecies to Western Australia.

Enantiomeric fraction evaluation of the four stereoisomers of difethialone in biological matrices of rat by two enantioselective liquid chromatography tandem mass spectrometry methods: Chiral stationary phase or derivatization

The need for the control of rodent populations with anticoagulant rodenticides remains actual, and enantioselective analytical methods are mandatory to understand ecotoxicity issues of those chiral pesticides. This study presents two enantioselective methods to achieve the residue levels and differentiated persistence of the four stereoisomers of difethialone (called in this work E1-trans, E2-cis, E3-cis and E4-trans), which is one of the most toxic second generation anticoagulant rodenticide. Their enantiomeric fraction evaluation in biological matrices of rats was determined by two LC-MS/MS methods. The first one (chiral-LC-MS/MS) combined a chiral column employed in reversed-phase mode (with acetonitrile-water mobile phase) to be compatible with mass spectrometry detection. The second one was also a LC-MS/MS method but with a reversed phase column after a derivatization step with (1S)-(−)-camphanic chloride. Extraction process combined Solid-Liquid extraction and sorbent cartridges. The methods were fully validated. The chiral column was chosen as a reference method for our laboratory because it was quicker and cheaper, and enantioresolution and sensitivity were better. This chiral-LC-MS/MS method was used to measure the enantiomeric fraction of the four stereoisomers of difethialone in rodent biological matrices (liver, plasma, blood and feces) of female rats treated with 3.5 mg/kg of difethialone. The results showed that metabolism is not the same for all the stereoisomers: cis-E3-difethialone was the most persistent, and E4-trans-difethialone was the most quickly eliminated.This chiral-LC-MS/MS method will be used to study the pharmacokinetics of the four stereoisomers of difethialone, and for ecotoxicological surveillance to evaluate the specific persistence of each stereoisomer of difethialone in case of secondary exposure of wildlife non-target species.

A California Without Rodenticides: Challenges for Commensal Rodent Management in the Future

Author(s): Quinn, Niamh; Kenmuir, Sylvia; Krueger, Laura | Abstract: Rodenticides are an essential tool in the integrated pest management (IPM) of infestations of commensal rodents. With the introduction of Assembly Bill 2422 California Natural Predator Protection Act, the State of California is potentially facing a future with serious restrictions on the use of anticoagulant rodenticides to manage commensal rodents in urban areas. Assembly Bill 2422 has been proposed to protect predators from rodenticide poisoning and seeks to significantly restrict the application of first and second generation anticoagulant rodenticides for use in many urban and no-urban areas of California. Exclusion and cultural practices, such as landscape management and sanitation, are important and successful tools for managing rodent populations. However, quick and efficient control of commensal rodent infestations often necessitates the use of rodenticides. While rodenticide is an important tool, exposure of wildlife to anticoagulant rodenticides has been evident for many years in the state of California. When rodents are consumed by predators, second generation anticoagulant rodenticides can be detected as residues in the livers of predators. Many species of animals are documented as having succumbed to rodenticide toxicosis, however the effects of chronic, sub-lethal exposure to predators are not well understood. This paper will discuss the current and proposed changes to rodenticide legislation in California, impacts of the legislation on communities across California, and gaps in research preventing the adoption of evidence-based best management strategies for rodent control. In order to improve the success of commensal rodent control programs in California, effective strategies for rodent management must be developed.

Urbanization and cattle density are determinants in the exposure to anticoagulant rodenticides of non-target wildlife

The persistence and toxicity of second generation anticoagulant rodenticides (SGARs) in animal tissues make these compounds dangerous by biomagnification in predatory species. Here we studied the levels of SGARs in non-target species of wildlife and the environmental factors that influence such exposure. Liver samples of terrestrial vertebrates (n = 244) found dead between 2007 and 2016 in the region of Aragón (NE Spain) were analysed. The presence of SGARs was statistically analysed with binary or ordinal logistic models to study the effect of habitat characteristics including human population density, percentage of urban surface, livestock densities and surface of different types of crops. SGARs residues were detected in 83 (34%) of the animals and levels >200 ng/g were found in common raven (67%), red fox (50%), red kite (38%), Eurasian eagle-owl (25%), stone marten (23%), Eurasian buzzard (17%), northern marsh harrier (17%), and Eurasian badger (14%). The spatial analysis revealed that the presence of SGARs residues in wildlife was more associated with the use of these products as biocides in urban areas and cattle farms rather than as plant protection products in agricultural fields. This information permits to identify potential habitats where SGARs may pose a risk for predatory birds and mammals.

The stereoisomerism of second generation anticoagulant rodenticides: a way to improve this class of molecules to meet the requirements of society?

Second generation anticoagulant rodenticides (SGAR) are generally highly efficient for rodent management even towards warfarin-resistant rodents. Nevertheless, because of their long tissue-persistence, they are very associated with non-target exposure of wildlife and have been identified as 'Candidates for Substitution' by the European Union's competent authority. A promising way to reduce ecotoxicity issues associated to SGAR could be the improvement of SGAR based on their stereoisomery, and due to this improvement, positioning about SGAR might be reconsidered. © 2018 Society of Chemical Industry.

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.

A California without Rodenticides: Challenges for Commensal Rodent Management in the Future

Author(s): Quinn, Niamh; Kenmuir, Sylvia; Krueger, Laura | Abstract: Rodenticides are an essential tool in the integrated pest management (IPM) of infestations of commensal rodents. With the introduction of Assembly Bill 2422 California Natural Predator Protection Act, the State of California is potentially facing a future with serious restrictions on the use of anticoagulant rodenticides to manage commensal rodents in urban areas. Assembly Bill 2422 has been proposed to protect predators from rodenticide poisoning and seeks to significantly restrict the application of first and second generation anticoagulant rodenticides for use in many urban and no-urban areas of California. Exclusion and cultural practices, such as landscape management and sanitation, are important and successful tools for managing rodent populations. However, quick and efficient control of commensal rodent infestations often necessitates the use of rodenticides. While rodenticide is an important tool, exposure of wildlife to anticoagulant rodenticides has been evident for many years in the state of California. When rodents are consumed by predators, second generation anticoagulant rodenticides can be detected as residues in the livers of predators. Many species of animals are documented as having succumbed to rodenticide toxicosis, however the effects of chronic, sub-lethal exposure to predators are not well understood. This paper will discuss the current and proposed changes to rodenticide legislation in California, impacts of the legislation on communities across California, and gaps in research preventing the adoption of evidence-based best management strategies for rodent control. In order to improve the success of commensal rodent control programs in California, effective strategies for rodent management must be developed.

Liver and fecal samples suggest differential exposure of red fox (Vulpes vulpes) to trans- and cis-bromadiolone in areas from France treated with plant protection products

Bromadiolone, a second generation anticoagulant rodenticide (SGARs), is authorized in France to control water voles (Arvicola scherman) outbreaks. SGARs enter the food chain and their persistence in rodents is responsible for secondary exposure or poisoning of predators and scavengers. Bromadiolone commercial formulations are a mixture of two diastereoisomers of bromadiolone: 70–90% is trans-bromadiolone and 10–30% is cis-bromadiolone. Both diastereoisomers were reported to inhibit coagulation function with similar potency. On the other hand, cis-bromadiolone has been shown to be less tissue-persistent than trans-bromadiolone in rats. Furthermore, cis-bromadiolone was not found in liver of red kites after bromadiolone poisonings of water voles.In this study, a multi-residue LC-MS/MS method for the quantification of the diastereoisomers of SGARs was used to investigate their proportions in field samples of another vole's predator, the red fox. Red fox livers (n=48) and scats (n=160) were collected in a pesticide use zone within a few months of bromadiolone application. We reported the concentrations of bromadiolone diastereoisomers in the livers and scats. Accumulation of bromadiolone was apparent in 81% (n=39) of the livers with mean and max concentrations of 355 and 2060ng/g, and in 23% of the faeces with mean and max concentrations of 78.5 and 593ng/g. However, cis-bromadiolone was not detected in the liver of 35 of 39 exposed red foxes and was present at very low concentrations (below 24.6ng/g) in 4 of 39 exposed red foxes. It was not detected in 11 of the positive scats and represented only 4.2% of the bromadiolone residues in scats. This demonstrated differential persistence of trans- and cis-bromadiolone in the food chain. The results suggest that a change of the proportions of bromadiolone diastereoisomers in baits could reduce the risk of secondary poisoning of predators, but retain primary toxicity for control water voles outbreaks.

The rapid determination of bromadiolone in liver and blood plasma by in-injector pyrolysis gas chromatography - Ion trap tandem mass spectrometry.

The unintentional poisoning of off-target animals by bromadiolone, a second generation anticoagulant rodenticide, is an undesirable outcome requiring sensitive analytical methods. In this study, a rapid and sensitive method for the determination of bromadiolone in liver and blood plasma by means of gas chromatography coupled with tandem mass spectrometry without need for derivatization was developed. The method is based on the in-injector pyrolysis of bromadiolone and subsequent gas chromatography coupled with ion trap tandem mass spectrometry with electron ionization. Sample preparation includes extraction with methanol, evaporation under nitrogen stream, and dissolution in toluene. The pyrolysis of bromadiolone was carried out in an injector at 390°C. Chromatographic separation of the pyrolytical fragment of bromadiolone was achieved using a VF-5ms column with helium as the mobile phase. Tandem in-time mass spectrometry of the separated pyrolytical fragment of bromadiolone was carried out using an ion trap mass spectrometer after electron ionization. Recovery ranged from 94 to 98%. The method showed good linearity up to 1000μgkg-1 for liver and 1000μgL-1 for plasma. The limit of detection was 0.38μgkg-1 for liver and 0.26μgL-1 for plasma. The developed method was used successfully in several animal poisoning cases.

Cis-bromadiolone diastereoisomer is not involved in bromadiolone Red Kite (Milvus milvus) poisoning

Anticoagulant rodenticides (ARs) are widely used pesticides to control rodent populations. Bromadiolone, a second generation anticoagulant rodenticide (SGARs), is authorized in France to control the population of water voles (Arvicola scherman). The persistence of SGARs in rodents is responsible for secondary exposure or poisoning of predators and scavengers, and is of ecological concern for the conservation of endangered species. Commercial formulations are a mixture of two diastereoisomers of bromadiolone: 70–90% is trans-bromadiolone and 10–30% is cis-bromadiolone. Both diastereoisomers have been shown to inhibit coagulation function with the same potency. On the other hand, cis-bromadiolone has been shown to be less tissue-persistent than trans-bromadiolone in rats. This difference led to residue levels in rats with substantially weakened proportion in cis-bromadiolone compared to the composition of baits.In this study, a multi-residue LC-MS/MS method for the quantification of the diastereoisomers of SGARs was used to investigate their proportions in field samples of predators. In 2011, 28 red kites (Milvus milvus) were found dead within a few months of bromadiolone application in grassland to control water vole outbreaks. In this study, we report the concentrations of the two diastereoisomers of bromadiolone measured in the livers of thirteen red kites. Exposure to bromadiolone was apparent in all the kites with hepatic concentrations of trans-bromadiolone ranging from 390 to 870ng/g (89 to 99% of summed SGARs). However, cis-bromadiolone was not detected in 5 of 13 red kites and was present at very low concentrations (below 2.2ng/g) in 8 of 13 kites, demonstrating that cis-bromadiolone is not involved in this red kite poisoning event. The results suggest that a change of the proportions of bromadiolone diastereoisomers in baits could reduce the risk of secondary poisoning of predators, but retain primary toxicity for control rodent outbreaks.

Core-shell LC–MS/MS method for quantification of second generation anticoagulant rodenticides diastereoisomers in rat liver in relationship with exposure of wild rats

Second generation anticoagulant rodenticides (SGARs), pesticides used worldwide to control rodent populations, exist in two diastereoisomer chemical species because they own two stereogenic centers. A core-shell LC-MS/MS multi-residue method for comprehensive quantitative analysis of the diastereoisomers of five SGARs as well as three first generation anticoagulant rodenticide molecules has been fully validated in liver of rats according to a bioanalytical guideline. A core-shell column (superficially porous particles) has been chosen for its ability to separate the diastereomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone and for its robustness to rat liver extracts. The highly selective chromatographic separation of the diastereoisomers contributes to good signal to noise ratios and then enhances the sensitivity of the method compared to the ones of fully porous columns. An elution gradient has been optimized with 10mM ammonium acetate and acetonitrile as aqueous/organic mobile phase respectively. Triple quadrupole mass detector has been used to achieve specifity and LLOQ from 0.92 to 2.2ng/g for each diastereoisomer, or first generation anticoagulant rodenticides. Then we evidenced diastereoisomeric ratios in liver of rats issued from not controlled exposure of wild rats (Rattus norvegicus) trapped in a French Parisian park through a campaign of rodent eradication. We compared them to diastereoisomeric ratios in SGARs commercial baits that contain both isomers, and showed that one of the two diastereoiomers had nearly disappeared in liver of rats. The proportions of cis-bromadiolone and trans-difenacoum were really lowered compared to the baits: 5/7 and 9/12 rats had only trans-bromadiolone and cis-difenacoum hepatic residues respectively. Liver persistence of the two diastereoisomers of bromadiolone and difenacoum was different due to differences in their pharmacokinetics in wild rats. The new core-shell LC-MS/MS method is particularly well adapted for further exploration of diastereoisomers ratios in rodent and predatory wildlife biological samples in order to evaluate ecological consequences of actual baits, to explore new formulated baits with a good balance between efficacity (ability to kill rodents) and diastereoisomers persistence, and hopefully to mitigate exposure of non-target species.