Determining an approximate minimum toxic dosage of diphacinone in horses and corresponding serum, blood, and liver diphacinone concentrations: a pilot study.

Poisoning of nontarget species is a major concern with the use of anticoagulant rodenticides (ARs). At postmortem examination, differentiating toxicosis from incidental exposure is sometimes difficult. Clotting profiles cannot be performed on postmortem samples, and clinically significant serum, blood, and liver AR concentrations are not well-established in most species. We chose diphacinone for our study because, at the time, it was the publicly available AR most commonly detected in samples analyzed at the University of Kentucky Veterinary Diagnostic Laboratory. We determined an approximate minimum toxic dosage (MTD) of oral diphacinone in 3 horses and measured corresponding serum, blood, and liver diphacinone concentrations. Diphacinone was administered orally to healthy horses. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and serum and blood diphacinone concentrations were measured daily. At the study endpoint, the horses were euthanized, and diphacinone concentration was measured in each liver lobe. The horse that received 0.2 mg/kg diphacinone developed prolonged (>1.5× baseline) PT and aPTT; the horse that received 0.1 mg/kg did not. This suggests an approximate oral MTD in horses of 0.2 mg/kg diphacinone. Median liver diphacinone concentration at this dosage was 1,780 (range: 1,590-2,000) ppb wet weight. Marginal (model-adjusted) mean diphacinone concentrations of liver lobes were not significantly different from one another (p = NS). Diphacinone was present in similar concentrations in both serum and blood at each time after administration, indicating that both matrices are suitable for detection of diphacinone exposure in horses.

Cluster of cases of massive hemorrhage associated with anticoagulant detection in race horses.

Five horses originating from 4 different California race tracks were submitted to the California Animal Health and Food Safety Laboratory for necropsy and diagnostic workup. The 5 horses had a history of sudden collapse and death during exercise. In all of them, massive hemoperitoneum and hemorrhages in other cavities or organs were observed. The liver from these 5 animals and from 27 horses that had been euthanized due to catastrophic leg injuries (controls) were subjected to a rodenticide anticoagulant screen. Traces of brodifacoum, diphacinone, or bromadiolone were detected in the 5 horses with massive bleeding (5/5), and no traces of rodenticides were detected in control horses (0/27). Other frequent causes of massive hemorrhages in horses were ruled out in 4 of the cases; one of the horses had a pelvic fracture. Although only traces of anticoagulants were found in the livers of these horses and the role of these substances in the massive bleeding remains uncertain, it is speculated that exercise-related increases in blood pressure may have reduced the threshold for toxicity of these anticoagulants.

Nontarget mortality of New Zealand lesser short-tailed bats (Mystacina tuberculata) caused by diphacinone.

Primary and secondary poisoning of nontarget wildlife with second-generation anticoagulant rodenticides has led to restrictions on their use and to increased use of first-generation anticoagulants, including diphacinone. Although first-generation anticoagulants are less potent and less persistent than second-generation compounds, their use is not without risks to nontarget species. We report the first known mortalities of threatened New Zealand lesser short-tailed bats (Mystacina tuberculata) caused by diphacinone intoxication. The mortalities occurred during a rodent control operation in Pureora Forest Park, New Zealand, during the 2008-09 Austral summer. We observed 115 lesser short-tailed bat deaths between 9 January and 6 February 2009, and it is likely that many deaths were undetected. At necropsy, adult bats showed gross and histologic hemorrhages consistent with coagulopathy, and diphacinone residues were confirmed in 10 of 12 liver samples tested. The cause of mortality of pups was diagnosed as a combination of the effects of diphacinone toxicity, exposure, and starvation. Diphacinone was also detected in two of 11 milk samples extracted from the stomachs of dead pups. Eight adults and 20 pups were moribund when found. Two adults and five pups survived to admission to a veterinary hospital. Three pups responded to treatment and were released at the roost site on 17 March 2009. The route of diphacinone ingestion by adult bats is uncertain. Direct consumption of toxic bait or consumption of poisoned arthropod prey could have occurred. We suggest that the omnivorous diet and terrestrial feeding habits of lesser short-tailed bats make them susceptible to poisoning with the bait matrix and the method of bait delivery used. We recommend the use of alternative vertebrate pesticides, bait matrices, and delivery methods in bat habitat.

Effectiveness of rodenticides for managing invasive roof rats and native deer mice in orchards.

Roof rats (Rattus rattus) and deer mice (Peromyscus maniculatus) are occasional pests of nut and tree fruit orchards throughout California and in many other parts of the USA and beyond. In general, the most practical and cost-effective control method for rodents in many agricultural environments is the use of rodenticides (toxic baits), but little or no information exists on the efficacy of current rodenticides in controlling roof rats and deer mice in orchards. Therefore, our goals were to develop an index of rodent activity to monitor efficacy of rodenticides and to subsequently test the efficacy of three California Department of Food and Agriculture rodenticide baits (0.005 % chlorophacinone treated oats, 0.005 % diphacinone treated oats, and 0.005 % diphacinone wax block) to determine their utility for controlling roof rats and deer mice in agricultural orchards. We determined that a general index using the number of roof rat photos taken at a minimum of a 5-min interval was strongly correlated to the minimum number known estimate of roof rats; this approach was used to monitor roof rat and deer mouse populations pre- and post-treatment. Of the baits tested, the 0.005 % diphacinone treated oats was most effective for both species; 0.005 % chlorophacinone grain was completely ineffective against roof rats. Our use of elevated bait stations proved effective at providing bait to target species and should substantially limit access to rodenticides by many non-target species.

Toxicokinetics and coagulopathy threshold of the rodenticide diphacinone in eastern screech-owls (Megascops asio).

In the United States, new regulations on second-generation anticoagulant rodenticides will likely be offset by expanded use of first-generation anticoagulant rodenticides. In the present study, eastern screech-owls (Megascops asio) were fed 10 µg diphacinone/g wet weight food for 7 d, and recovery was monitored over a 21-d postexposure period. By day 3 of exposure, diphacinone (DPN) was detected in liver (1.63 µg/g wet wt) and kidney (5.83 µg/g) and coagulopathy was apparent. By day 7, prothrombin time (PT) and Russell's viper venom time (RVVT) were prolonged, and some individuals were anemic. Upon termination of exposure, coagulopathy and anemia were resolved within 4 d, and residues decreased to <0.3 µg/g by day 7. Liver and kidney DPN elimination occurred in 2 phases (initial rapid loss, followed by slower loss rate), with overall half-lives of 11.7 d and 2.1 d, respectively. Prolonged PT and RVVT occurred in 10% of the exposed owls with liver DPN concentrations of 0.122 µg/g and 0.282 µg/g and in 90% of the owls with liver concentrations of 0.638 µg/g and 0.361 µg/g. These liver residue levels associated with coagulopathy fall in the range of values reported in raptor mortality incidents involving DPN. These tissue-based toxicity reference values for coagulopathy in adult screech-owls have application for interpreting nontarget mortality and assessing the hazard of DPN in rodent-control operations. Diphacinone exposure evokes toxicity in raptors within a matter of days; but once exposure is terminated, recovery of hemostasis occurs rapidly.

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.

Acute toxicity of diphacinone in Northern bobwhite: effects on survival and blood clotting.

The anticoagulant rodenticide diphacinone was slightly toxic (acute oral LD50 2014 mg/kg) to Northern bobwhite (Colinus virginianus) in a 14-day acute toxicity trial. Precise and sensitive assays of blood clotting (prothrombin time, Russell's Viper venom time, and thrombin clotting time) were adapted for use in quail, and this combination of assays is recommended to measure the effects of anticoagulant rodenticides. A single oral sublethal dose of diphacinone (434 mg/kg body weight) prolonged clotting time at 48 h post-dose compared to controls. At 783 mg/kg (approximate LD02), clotting time was prolonged at both 24 and 48 h post-dose. Prolongation of in vitro clotting time reflects impaired coagulation complex activity, and was detected before overt signs of toxicity were apparent at the greatest dosages (2868 and 3666 mg/kg) in the acute toxicity trial. These clotting time assays and toxicity data will assist in the development of a pharmacodynamic model to predict toxicity, and also facilitate rodenticide hazard and risk assessments in avian species.

The susceptibility of Bandicota bengalensis from Rangoon, Burma to several anticoagulant rodenticides.

The baseline susceptibility of the lesser bandicoot rat, Bandicota bengalensis, from Rangoon, Burma, to five anticoagulant rodenticides was established with no-choice feeding in the laboratory. The susceptibility of lesser bandicoots to the several poisons (brodifacoum, difenacoum, diphacinone, coumatetralyl, and warfarin) was such that they were offered at a 0.001% concentration. B. bengalensis was most susceptible to brodifacoum, and in descending order, difenacoum, coumatetralyl, diphacinone and warfarin. In comparison with Rattus norvegicus on warfarin at 0.005%, B. bengalensis proved more susceptible. Feeding tests at 0.005% concentration indicated that a 1-day feeding on brodifacoum and difenacoum would result in complete mortality, whereas coumatetralyl and warfarin would require 4 days feeding to a 100% kill. Brodifacoum and difenacoum are recommended at 0.002-0.005% bait concentrations and coumatetralyl at 0.005--0.01% concentrations for the control of B. bengalensis in the field in Rangoon. The use of any anticoagulant material in rat control should be alternated with acute toxicants to retard the possible development of anticoagulant resistance.

Vampire bat control by systemic treatment of livestock with an anticoagulant.

The blood of beef cattle given single doses (1 milligram per kilogram of body weight) of diphenadione (2-diphenylacetyl-1,3-indandione) became toxic to vampire bats (Desmodus rotundus) and remained toxic for 3 days without harming the cattle. Cattle at three ranches in Mexico treated with single intraruminal injections of diphenadione experienced a reduction in vampire bat bites of 93 percent. Bioassays of milk and liver from cattle treated orally with diphenadione in the laboratory indicated that there were no residue problems.