Brodifacoum Toxicity in American Kestrels (Falco sparverius) with Evidence of Increased Hazard on Subsequent Anticoagulant Rodenticide Exposure.

A seminal question in ecotoxicology is the extent to which contaminant exposure evokes prolonged effects on physiological function and fitness. A series of studies were undertaken with American kestrels ingesting environmentally realistic concentrations of the second-generation anticoagulant rodenticide (SGAR) brodifacoum. Kestrels fed brodifacoum at 0.3, 1.0, or 3.0 µg/g diet wet weight for 7 d exhibited dose-dependent hemorrhage, histopathological lesions, and coagulopathy (prolonged prothrombin and Russell's viper venom times). Following termination of a 7-d exposure to 0.5 µg brodifacoum/g diet, prolonged blood clotting time returned to baseline values within 1 wk, but brodifacoum residues in liver and kidney persisted during the 28-d recovery period (terminal half-life estimates >50 d). To examine the hazard of sequential anticoagulant rodenticide (AR) exposure, kestrels were exposed to either the first-generation AR chlorophacinone (1.5 µg/g diet) or the SGAR brodifacoum (0.5 µg/g diet) for 7 d and, following a recovery period, challenged with a low dose of chlorophacinone (0.75 µg/g diet) for 7 d. In brodifacoum-exposed kestrels, the challenge exposure clearly prolonged prothrombin time compared to naive controls and kestrels previously exposed to chlorophacinone. These data provide evidence that the SGAR brodifacoum may have prolonged effects that increase the toxicity of subsequent AR exposure. Because free-ranging predatory and scavenging wildlife are often repeatedly exposed to ARs, such protracted toxicological effects need to be considered in hazard and risk assessments. Environ Toxicol Chem 2020;39:468-481. © 2020 SETAC.

Placebo Oral Rabies Vaccine Bait Uptake by Small Indian Mongooses (Herpestes auropunctatus) in Southwestern Puerto Rico.

The small Indian mongoose (Herpestes auropunctatus) is a rabies reservoir in areas of the Caribbean including Puerto Rico, but no rabies vaccination program targeting this host exists. We used two derivatives of iophenoxic acid (IPA) to evaluate placebo oral rabies vaccine bait uptake by mongooses in southwestern Puerto Rico. We hand-distributed baits at an application rate of 200 baits/km2 at three, 400 ha, sites during autumn 2016 and spring 2017. Each site contained 90-100 cage traps in a 100 ha central trapping area. We used ethyl-IPA as a biological marker during the autumn and methyl-IPA during the spring. We live captured mongooses for 10 consecutive days, beginning 1 wk following bait application. We obtained a serum sample from captured mongooses and analyzed the sera for ethyl- and methyl-IPA by liquid chromatography-mass spectrometry. During autumn 2016, 63% (55/87) mongooses sampled were positive for ethyl-IPA. In spring 2017, 69% (85/123) of mongooses were positive for methyl-IPA. Pooling seasons, accounting for recaptures between years, and disregarding marker type, 74% (133/179) unique mongooses were positive for IPA biomarker, indicating bait consumption during either the autumn, spring, or both trials. We conclude that distributing baits at an application rate of 200 baits/km2 is sufficient to reach over 60% of the target mongoose population in dry forest habitats of Puerto Rico.

Volatile metabolomic signatures of rabies immunization in two mesocarnivore species.

Rabies is a zoonotic disease caused by infection with rabies virus, which circulates naturally in several wild carnivore and bat reservoirs in the United States (US). The most important reservoir in the US from an animal and public health perspective is the raccoon (Procyon lotor). To prevent the westward expansion of a significant raccoon rabies epizootic along the eastern seaboard, an operational control program implementing oral rabies vaccination (ORV) has existed in the US since the 1990s. Recently, two vaccine efficacy studies conducted with raccoons and striped skunks (Mephitis mephitis) provided the opportunity to determine if volatile fecal metabolites might be used to non-invasively monitor ORV programs and/or predict virus protection for these species. The volatile metabolome is a rich source of information that may significantly contribute to our understanding of disease and infection. Fecal samples were collected at multiple time points from raccoons and striped skunks subjected to oral treatment with rabies vaccine (or sham). Intramuscular challenge with a lethal dose of rabies virus was used to determine protection status at six (raccoons) and 11 (skunks) months post-vaccination. In addition to fecal samples, blood was collected at various time points to permit quantitative assessment of rabies antibody responses arising from immunization. Feces were analyzed by headspace gas chromatography with mass spectrometric detection and the chromatographic responses were grouped according to cluster analysis. Cluster scores were subjected to multivariate analyses of variance (MANOVA) to determine if fecal volatiles may hold a signal of immunization status. Multiple regression was then used to build models of the measured immune responses based on the metabolomic data. MANOVA results identified one cluster associated with protective status of skunks and one cluster associated with protective status of raccoons. Regression models demonstrated considerably greater success in predicting rabies antibody responses in both species. This is the first study to link volatile compounds with measures of adaptive immunity and provides further evidence that the volatile metabolome holds great promise for contributing to our understanding of disease and infections. The volatile metabolome may be an important resource for monitoring rabies immunization in raccoons and striped skunks.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Use of faecal volatile organic compound analysis for ante-mortem discrimination between CWD-positive, -negative exposed, and -known negative white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is a naturally occurring infectious, fatal, transmissible spongiform encephalopathy of cervids. Currently, disease confirmation relies on post-mortem detection of infectious prions in the medial retropharyngeal lymph nodes or obex in the brain via immunohistochemistry (IHC). Detection of CWD in living animals using this method is impractical, and IHC and other experimental assays are not reliable in detecting low concentrations of prion present in biofluids or faeces. Here, we evaluate the capability of faecal volatile organic compound analysis to discriminate between CWD-positive and -exposed white-tailed deer located at two positive cervid farms, and two groups of CWD-negative deer from two separate disease-free farms.

Determination of residue levels of the avicide 3-chloro-4-methylaniline hydrochloride in red-winged blackbirds (Agelaius phoeniceus) by gas chromatography-tandem mass spectrometry.

The avicide 3­chloro­4­methylanaline hydrochloride (chloro­p­toluidine hydrochloride, CPTH, DRC-1339) is used to control pest bird species that damage agricultural crops. A specific and sensitive gas chromatography-tandem mass spectrometry method was developed and validated for the determination of CPTH in avian breast muscle, GI tract, kidney, and liver. Tissue samples were extracted with a solution of acidified water and acetonitrile. The sample was made basic and cleaned up with a combination of liquid-liquid partitioning and solid phase extraction. Separation was achieved using a HP-5 ultra-inert GC column (15 M, 0.25 µm film) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for CPTH was m/z 140.9 → 106.2 for quantitation and 139.9 → 105.2 and 139.9 → 77.2 for confirmation. The linear range was 5 to 5000 ng/mL. The precision for the determination of CPTH in all tissues averaged 7.2% and the accuracy averaged 6.7%. The recovery of CPTH fortified at 5 different levels averaged 101% in liver, 98.8% in GI tract, 92.9% in breast muscle, and 87.9% in kidney. The established method was successfully used to determine CPTH residue levels in red-winged blackbirds exposed to three different doses of CPTH.

Effects of Low-level Brodifacoum Exposure on the Feline Immune Response.

Anticoagulant rodenticides have been implicated as a potential inciting factor in the development of mange in wild felids, but a causative association between anticoagulant rodenticide exposure and immune suppression has not been established. Specific-pathogen-free domestic cats were exposed to brodifacoum over a 6-week period to determine whether chronic, low-level exposure altered the feline immune response. Cats were vaccinated with irrelevant antigens at different points during the course of the experiment to assess recall and direct immune responses. Measures of immune response included delayed-type hypersensitivity tests and cell proliferation assays. IgE and antigen-specific antibodies were quantified via ELISA assays, and cytokine induction following exposure to vaccine antigens was also analyzed. While cats had marked levels of brodifacoum present in blood during the study, no cats developed coagulopathies or hematologic abnormalities. Brodifacoum-exposed cats had transient, statistically significant decreases in the production of certain cytokines, but all other measures of immune function remained unaffected throughout the study period. This study indicates that cats may be more resistant to clinical effects of brodifacoum exposure than other species and suggests that the gross impacts of environmentally realistic brodifacoum exposure on humoral and cell-mediated immunity against foreign antigen exposures in domestic cats are minimal.

Grass is not always greener: rodenticide exposure of a threatened species near marijuana growing operations.

OBJECTIVE: Marijuana (Cannabis spp.) growing operations (MGO) in California have increased substantially since the mid-1990s. One environmental side-effect of MGOs is the extensive use of anticoagulant rodenticides (AR) to prevent damage to marijuana plants caused by wild rodents. In association with a long-term demographic study, we report on an observation of brodifacoum AR exposure in a threatened species, the northern spotted owl (Strix occidentalis caurina), found freshly dead within 669-1347 m of at least seven active MGOs. RESULTS: Liver and blood samples from the dead northern spotted owl were tested for 12 rodenticides. Brodifacoum was the only rodenticide detected in the liver (33.3-36.3 ng/g) and blood (0.48-0.54 ng/ml). Based on necropsy results, it was unclear what role brodifacoum had in the death of this bird. However, fatal AR poisoning has been previously reported in owls with relatively low levels of brodifacoum residues in the liver. One likely mechanism of AR transmission from MGOs to northern spotted owls in California is through ingestion of AR contaminated prey that frequent MGOs. The proliferation of MGOs with their use of ARs in forested landscapes used by northern spotted owls may pose an additional stressor for this threatened species.

Toxicity reference values for chlorophacinone and their application for assessing anticoagulant rodenticide risk to raptors.

Despite widespread use and benefit, there are growing concerns regarding hazards of second-generation anticoagulant rodenticides to non-target wildlife which may result in expanded use of first-generation compounds, including chlorophacinone (CPN). The toxicity of CPN over a 7-day exposure period was investigated in American kestrels (Falco sparverius) fed either rat tissue mechanically-amended with CPN, tissue from rats fed Rozol(®) bait (biologically-incorporated CPN), or control diets (tissue from untreated rats or commercial bird of prey diet) ad libitum. Nominal CPN concentrations in the formulated diets were 0.15, 0.75 and 1.5 µg/g food wet weight, and measured concentrations averaged 94 % of target values. Kestrel food consumption was similar among groups and body weight varied by less than 6 %. Overt signs of intoxication, liver CPN residues, and changes in prothrombin time (PT), Russell's viper venom time (RVVT) and hematocrit, were generally dose-dependent. Histological evidence of hemorrhage was present at all CPN dose levels, and most frequently observed in pectoral muscle and heart. There were no apparent differences in toxicity between mechanically-amended and biologically-incorporated CPN diet formulations. Dietary-based toxicity reference values at which clotting times were prolonged in 50 % of the kestrels were 79.2 µg CPN consumed/kg body weight-day for PT and 39.1 µg/kg body weight-day for RVVT. Based upon daily food consumption of kestrels and previously reported CPN concentrations found in small mammals following field baiting trials, these toxicity reference values might be exceeded by free-ranging raptors consuming such exposed prey. Tissue-based toxicity reference values for coagulopathy in 50 % of exposed birds were 0.107 µg CPN/g liver wet weight for PT and 0.076 µg/g liver for RVVT, and are below the range of residue levels reported in raptor mortality incidents attributed to CPN exposure. Sublethal responses associated with exposure to environmentally realistic concentrations of CPN could compromise survival of free-ranging raptors, and should be considered in weighing the costs and benefits of anticoagulant rodenticide use in pest control and eradication programs.

Assessment of toxicity and potential risk of the anticoagulant rodenticide diphacinone using Eastern screech-owls (Megascops asio).

In the United States, new regulatory restrictions have been placed on the use of some second-generation anticoagulant rodenticides. This action may be offset by expanded use of first-generation compounds (e.g., diphacinone; DPN). Single-day acute oral exposure of adult Eastern screech-owls (Megascops asio) to DPN evoked overt signs of intoxication, coagulopathy, histopathological lesions (e.g., hemorrhage, hepatocellular vacuolation), and/or lethality at doses as low as 130 mg/kg body weight, although there was no dose-response relation. However, this single-day exposure protocol does not mimic the multiple-day field exposures required to cause mortality in rodent pest species and non-target birds and mammals. In 7-day feeding trials, similar toxic effects were observed in owls fed diets containing 2.15, 9.55 or 22.6 ppm DPN, but at a small fraction (<5%) of the acute oral dose. In the dietary trial, the average lowest-observed-adverse-effect-level for prolonged clotting time was 1.68 mg DPN/kg owl/week (0.24 mg/kg owl/day; 0.049 mg/owl/day) and the lowest lethal dose was 5.75 mg DPN/kg owl/week (0.82 mg/kg owl/day). In this feeding trial, DPN concentration in liver ranged from 0.473 to 2.21 µg/g wet weight, and was directly related to the daily and cumulative dose consumed by each owl. A probabilistic risk assessment indicated that daily exposure to as little as 3-5 g of liver from DPN-poisoned rodents for 7 days could result in prolonged clotting time in the endangered Hawaiian short-eared owl (Asio flammeus sandwichensis) and Hawaiian hawk (Buteo solitarius), and daily exposure to greater quantities (9-13 g of liver) could result in low-level mortality. These findings can assist natural resource managers in weighing the costs and benefits of anticoagulant rodenticide use in pest control and eradication programs.

Acute toxicity, histopathology, and coagulopathy in American kestrels (Falco sparverius) following administration of the rodenticide diphacinone.

The acute oral toxicity of the anticoagulant rodenticide diphacinone was found to be over 20 times greater in American kestrels (Falco sparverius; median lethal dose 96.8 mg/kg body weight) compared with Northern bobwhite (Colinus virginianus) and mallards (Anas platyrhynchos). Modest evidence of internal bleeding was observed at necropsy, although histological examination of heart, liver, kidney, lung, intestine, and skeletal muscle revealed hemorrhage over a wide range of doses (35.1-675 mg/kg). Residue analysis suggests that the half-life of diphacinone in the liver of kestrels that survived was relatively short, with the majority of the dose cleared within 7 d of exposure. Several precise and sensitive clotting assays (prothrombin time, Russell's viper venom time, thrombin clotting time) were adapted for use in this species, and oral administration of diphacinone at 50 mg/kg increased prothrombin time and Russell's viper venom time at 48 and 96 h postdose compared with controls. Prolongation of in vitro clotting time reflects impaired coagulation complex activity, and generally corresponded with the onset of overt signs of toxicity and lethality. In view of the toxicity and risk evaluation data derived from American kestrels, the involvement of diphacinone in some raptor mortality events, and the paucity of threshold effects data following short-term dietary exposure for birds of prey, additional feeding trials with captive raptors are warranted to characterize more fully the risk of secondary poisoning.