Katherine E. Horak

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, Russells 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 Russells 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.

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 Russells 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.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Development of methods for avian oil toxicity studies using the double crested cormorant (Phalacrocorax auritus)

Oral and external dosing methods replicating field exposure were developed using the double crested cormorant (DCCO) to test the toxicity of artificially weathered Deepwater Horizon Mississippi Canyon 252 oil. The majority of previous oil dosing studies conducted on wild-caught birds used gavage methods to dose birds with oil and determine toxicity. However, rapid gut transit time of gavaged oil likely reduces oil absorption. In the present studies, dosing relied on injection of oil into live feeder fish for oral dosing of these piscivorous birds, or applying oil to body contour feathers resulting in transdermal oil exposure and oral exposure through preening. Both oral and external oil dosing studies identified oil-related toxicity endpoints associated with oxidative stress such as hemolytic anemia, liver and kidney damage, and immuno-modulation or compromise. External oil application allowed for controlled study of thermoregulatory stress as well. Infrared thermal images indicated significantly greater surface temperatures and heat loss in treated birds following external oil applications; however, measurements collected by coelomically implanted temperature transmitters showed that internal body temperatures were stable over the course of the study period. Birds exposed to oil externally consumed more fish than control birds, indicating metabolic compensation for thermal stress. Conversely, birds orally dosed with oil experienced hypothermia and consumed less fish compared to control birds.

Changes in white cell estimates and plasma chemistry measurements following oral or external dosing of double-crested cormorants, Phalacocorax auritus , with artificially weathered MC252 oil

Scoping studies were designed whereby double-crested cormorants (Phalacocorax auritus) were dosed with artificially weathered Deepwater Horizon (DWH) oil either daily through oil injected feeder fish, or by application of oil directly to feathers every three days. Preening results in oil ingestion, and may be an effective means of orally dosing birds with toxicant to improve our understanding of the full range of physiological effects of oral oil ingestion on birds. Blood samples collected every 5-6 days were analyzed for a number of clinical endpoints including white blood cell (WBC) estimates and differential cell counts. Plasma biochemical evaluations were performed for changes associated with oil toxicity. Oral dosing and application of oil to feathers resulted in clinical signs and statistically significant changes in a number of biochemical endpoints consistent with petroleum exposure. In orally dosed birds there were statistically significant decreases in aspartate amino transferase (AST) and gamma glutamyl transferase (GGT) activities, calcium, chloride, cholesterol, glucose, and total protein concentrations, and increases in plasma urea, uric acid, and phosphorus concentrations. Plasma electrophoresis endpoints (pre-albumin, albumin, alpha-2 globulin, beta globulin, and gamma globulin concentrations and albumin: globulin ratios) were decreased in orally dosed birds. Birds with external oil had increases in urea, creatinine, uric acid, creatine kinase (CK), glutamate dehydrogenase (GLDH), phosphorus, calcium, chloride, potassium, albumin, alpha-1 globulin and alpha-2 globulin. Decreases were observed in AST, beta globulin and glucose. WBC also differed between treatments; however, this was in part driven by monocytosis present in the externally oiled birds prior to oil treatment.

Reprint of: CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to deepwater Horizon Mississippi Canyon 252 oil

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Organ weights and histopathology of double-crested cormorants (Phalacrocorax auritus) dosed orally or dermally with artificially weathered Mississippi Canyon 252 crude oil

A series of toxicity tests were conducted to assess the effects of low to moderate exposure to artificially weathered Deepwater Horizon Mississippi Canyon 252 crude oil on representative avian species as part of the Natural Resource Damage Assessment. The present report summarizes effects of oral exposure (n=26) of double-crested cormorants (DCCO; Phalacrocorax auritus) to 5 or 10ml oil kg-1 day-1 for up to 21 days or dermal application (n=25) of 13ml oil to breast and back feathers every three days totaling 6 applications in 21 days on organ weights and histopathology. Absolute and relative kidney and liver weights were increased in birds exposed to oil. Additionally, gross and/or histopathologic lesions occurred in the kidney, heart, pancreas and thyroid. Clinically significant renal lesions in the orally dosed birds included squamous metaplasia and increased epithelial hypertrophy of the collecting ducts and renal tubules and mineralization in comparison to controls. Gross cardiac lesions including thin walls and flaccid musculature were documented in both orally and dermally dosed birds and myocardial fibrosis was found in low numbers of dermally dosed birds only. Cytoplasmic vacuolation of the exocrine pancreas was noted in orally dosed birds only. Thyroid follicular hyperplasia was increased in dermally dosed birds only possibly due to increased metabolism required to compensate damaged feather integrity and thermoregulate. Gastrointestinal ulceration was found in orally dosed birds only. There were no significant hepatic histopathologic lesions induced by either exposure route. Therefore, hepatic histopathology is likely not a good representation of oil-induced damage. Taken together, the results suggest that oral or dermal exposure of DCCOs to artificially weathered MC252 crude oil induced organ damage that could potentially affect survivability.

Dietary intake of Deepwater Horizon oil-injected live food fish by double-crested cormorants resulted in oxidative stress

The Deepwater Horizon oil spill released 134 million gallons of crude oil into the Gulf of Mexico making it the largest oil spill in US history and exposing fish, birds, and marine mammals throughout the Gulf of Mexico to its toxicity. Fish eating waterbirds such as the double-crested cormorant (Phalacrocorax auritus) were exposed to the oil both by direct contact with the oil and orally through preening and the ingestion of contaminated fish. This study investigated the effects of orally ingestedMC252 oil-contaminated live fish food by double-crested cormorants on oxidative stress. Total, reduced, and oxidized glutathione levels, superoxide dismutase and glutathione peroxidase activities, total antioxidant capacity and lipid peroxidation were assessed in the liver tissues of control and treated cormorants. The results suggest that ingestion of the oil-contaminated fish resulted in significant increase in oxidative stress in the liver tissues of these birds. The oil-induced increase in oxidative stress could have detrimental impacts on the birds life-history.

Overview of avian toxicity studies for the Deepwater Horizon Natural Resource Damage Assessment

The Oil Pollution Act of 1990 establishes liability for injuries to natural resources because of the release or threat of release of oil. Assessment of injury to natural resources resulting from an oil spill and development and implementation of a plan for the restoration, rehabilitation, replacement or acquisition of natural resources to compensate for those injuries is accomplished through the Natural Resource Damage Assessment (NRDA) process. The NRDA process began within a week of the Deepwater Horizon oil spill, which occurred on April 20, 2010. During the spill, more than 8500 dead and impaired birds representing at least 93 avian species were collected. In addition, there were more than 3500 birds observed to be visibly oiled. While information in the literature at the time helped to identify some of the effects of oil on birds, it was not sufficient to fully characterize the nature and extent of the injuries to the thousands of live oiled birds, or to quantify those injuries in terms of effects on bird viability. As a result, the US Fish and Wildlife Service proposed various assessment activities to inform NRDA injury determination and quantification analyses associated with the Deepwater Horizon oil spill, including avian toxicity studies. The goal of these studies was to evaluate the effects of oral exposure to 1-20ml of artificially weathered Mississippi Canyon 252 oil kg bw-1 day-1 from one to 28 days or one to five applications of oil to 20% of the birds surface area. It was thought that these exposure levels would not result in immediate or short-term mortality but might result in physiological effects that ultimately could affect avian survival, reproduction and health. These studies included oral dosing studies, an external dosing study, metabolic and flight performance studies and field-based flight studies. Results of these studies indicated changes in hematologic endpoints including formation of Heinz bodies and changes in cell counts. There were also effects on multiple organ systems, cardiac function and oxidative status. External oiling affected flight patterns and time spent during flight tasks indicating that migration may be affected by short-term repeated exposure to oil. Feather damage also resulted in increased heat loss and energetic demands. The papers in this special issue indicate that the combined effects of oil toxicity and feather effects in avian species, even in the case of relatively light oiling, can significantly affect the overall health of birds.

Development of methods for avian oil toxicity studies using the Double-crested Cormorant

Oral and external dosing methods replicating field exposure were developed using the double crested cormorant (DCCO) to test the toxicity of artificially weathered Deepwater Horizon Mississippi Canyon 252 oil. The majority of previous oil dosing studies conducted on wild-caught birds used gavage methods to dose birds with oil and determine toxicity. However, rapid gut transit time of gavaged oil likely reduces oil absorption. In the present studies, dosing relied on injection of oil into live feeder fish for oral dosing of these piscivorous birds, or applying oil to body contour feathers resulting in transdermal oil exposure and oral exposure through preening. Both oral and external oil dosing studies identified oil-related toxicity endpoints associated with oxidative stress such as hemolytic anemia, liver and kidney damage, and immuno-modulation or compromise. External oil application allowed for controlled study of thermoregulatory stress as well. Infrared thermal images indicated significantly greater surface temperatures and heat loss in treated birds following external oil applications; however, measurements collected by coelomically implanted temperature transmitters showed that internal body temperatures were stable over the course of the study period. Birds exposed to oil externally consumed more fish than control birds, indicating metabolic compensation for thermal stress. Conversely, birds orally dosed with oil experienced hypothermia and consumed less fish compared to control birds.