A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

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.

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 bird's life-history.

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.

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.

Toxicological and thermoregulatory effects of feather contamination with artificially weathered MC 252 oil in western sandpipers (Calidris mauri).

The external contamination of bird feathers with crude oil might have effects on feather structure and thus on thermoregulation. We tested the thermoregulatory ability of western sandpipers (Calidris mauri) in a respirometry chamber with oil applied either immediately prior, or three days before the experiment. The birds were then exposed to a sliding cold temperature challenge between 27°C and -3°C to calculate thermal conductance. After the experiment, a large blood sample was taken and the liver extracted to measure a range of parameters linked to toxicology and oxidative stress. No differences in thermal conductance were observed among groups, but birds exposed to oil for three days had reduced body temperatures and lost more body mass during that period. At necropsy, oiled birds showed a decrease in plasma albumin and sodium, and an increase in urea. This is reflective of dysfunction in the kidney at the loop of Henle. Birds, especially when exposed to the oil for three days, showed signs of oxidative stress and oxidative damage. These results show that the ingestion of externally applied oil through preening or drinking can cause toxic effects even in low doses, while we did not detect a direct effect of the external oil on thermoregulation over the temperature range tested.

Effect of oral exposure to artificially weathered Deepwater Horizon crude oil on blood chemistries, hepatic antioxidant enzyme activities, organ weights and histopathology in western sandpipers (Calidris mauri).

Shorebirds were among birds exposed to Mississippi Canyon 252 (MC252) crude oil during the 2010 Deep Water Horizon (DWH) oil spill in the Gulf of Mexico. The western sandpiper (Calidris mauri) was chosen as one of four species for initial oral dosing studies conducted under Phase 2 of the avian toxicity studies for the DWH Natural Resource Damage Assessment (NRDA). Thirty western sandpipers were assigned to one of three treatment groups, 10 birds per group. The control group was sham gavaged and the treatment groups were gavaged with 1 or 5mL oil kg bw-1 daily for 20 days. Periodic blood samples for hemoglobin measurements were collected during the trial. A final blood sample used to determine hemoglobin concentration in addition to complete blood counts, plasma clinical chemistries, haptoglobin concentration and plasma electrophoresis was collected when birds were euthanized and necropsied on day 21. Tissues were removed, weighed and processed for subsequent histopathological evaluation. There were numerical decreases in hemoglobin concentrations in oil-dosed birds over the 21-day trial, but values were not significantly different compared to controls on day 21. There were no significant differences between controls and oiled birds in complete blood counts, plasma chemistries, haptoglobin concentration, and plasma electrophoresis endpoints. Of the hepatic oxidative stress endpoints assessed, the total antioxidant capacity assessment (Trolox equivalents) for the control group was lower compared to the 1mL oil kg bw-1 group. Absolute liver weights in the 5mL oil kg bw-1 group were significantly greater compared to controls. While not conclusive, the numerical decrease in hemoglobin concentration and significant increase in absolute liver weight are consistent with exposure to oil. Histological changes in the adrenal gland could be considered a non-specific indicator of stress resulting from exposure to oil. It is possible that the quantity of oil absorbed was not sufficient to induce clearly evident hemolytic anemia or that the western sandpiper is relatively insensitive to ingested oil.

Assay validation of the cardiac isoform of troponin I in double crested cormorant (Phalacrocorax auritus) plasma for diagnosis of cardiac damage.

Cardiac abnormalities, initially found in Deepwater Horizon weathered MC252 crude oil exposed Double Crested Cormorants (DCCOs) upon gross necropsy, were further investigated using echocardiography. Clinical and statistically significant changes including decreased ventricular myocardial contractility and arrhythmia were elucidated by echocardiography and interpreted by boarded cardiologists as potentially life threatening. The objective of this investigation was to initiate development of an antemortem, sensitive blood screening test for cardiac damage due to oil exposure of avian species. An assay for the cardiac isoform of troponin I (cTnI) which is known to be highly cross-reactive across mammalian species was chosen and analytically validated in DCCO. This is the first time this test has been analytically validated in avian species. All plasma samples from birds assessed as healthy had trace concentrations (<0.016ng/ml). The assays was precise and accurate revealing a coefficient of variation <3% and an R2>0.99. Diagnostic investigation revealed that the test appears to have diagnostic potential for the diagnosis of cardiomyocyte damage. Diagnostic sensitivity and specificity were 91% and 73% in this laboratory population. Due to an equivocal sample population in which health could not be proven, further investigation is needed to diagnostically validate troponin I in the assessment of oil exposure in DCCO.