* Toxicology Centre, 44 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada; and
Syncrude Canada Ltd., Edmonton Research Centre, 9421 17th Avenue, Edmonton, Alberta T6N 1H4, Canada
Received August 16, 2001; accepted December 26, 2001
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ABSTRACT |
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Key Words: naphthenic acids; oil sands; mammalian; acute and subchronic toxicity.
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INTRODUCTION |
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The Athabasca oil sands (AOS) deposit covers about 42,000 km2 surrounding Fort McMurray (57° 3.07` N, 111° 36.02` W), approximately 440 km northeast of Edmonton, Alberta. It is the largest of 4 major oil sands deposits in the province and contains over 200 billion cubic meters of petroleum, making it the world's largest single oil deposit (AOSTRA, 1990). Oil sands are a mixture of bitumen (crude petroleum), sand, and water. Separation of bitumen from other components is accomplished by the Clarke Hot Water Extraction process, where mined oil sands are mixed with hot (7993°C) water and caustic soda (sodium hydroxide). After the bitumen is removed, residual sand, clay, and water, along with other inorganic and organic contaminants, are diverted to settling ponds. By 2025, an estimated 1 billion m3 of tailings pond water (TPW) will have accumulated as a result of mining the AOS (Herman et al., 1994
).
A consequence of the hot water extraction process is that the alkalinity (pH = 8) promotes solubilization of naphthenic acids (pKa 5), thereby concentrating them as mixtures of sodium salts in aqueous tailings. The actual amounts of naphthenic acids in the holding ponds of the two major companies operating in the AOS, Syncrude Canada Ltd. (Syncrude) and Suncor Energy Inc. (Suncor), are typically between 80 and 110 mg/l (FTFC, 1995
). The bioavailability and persistence of naphthenic acids in contaminated waters are believed to be high. These compounds are highly soluble and have an extremely low volatility (Henry's constant = 8.56 x 10-6 atm x m3/mol). Sorption to dissolved and particulate organic matter is limited by the polarity of dissolved naphthenates. The settling of fines in tailings ponds could decrease naphthenic acid bioavailability to some extent since naphthenic acids are amphipathic compounds that have surfactant-like properties and will accumulate at aqueous/nonaqueous interfaces, such as between water and fines. Physical biodegradation mechanisms for naphthenic acids have not been well investigated, but microbial biodegradation does occur, although primarily for naphthenic acids with carbon numbers
21 (Holowenko et al., in press
).
Although there are numerous contaminants in TPW, evidence suggests that naphthenic acids are the component primarily responsible for toxicity to aquatic organisms. For example, acute lethality of TPW from Mildred Lake Settling Basin (the primary holding pond of Syncrude) to rainbow trout (Onchorhynchus mykiss) and water fleas (Daphnia magna) was significantly reduced when tailings were treated to remove naphthenic acids. The treatment did not significantly influence the concentrations of other potentially important contaminants in the TPW (MacKinnon and Boerger, 1986). Another aquatic toxicity study with freshwater fish used commercial sodium salts of naphthenic acids and produced 96-h LC50 values of 50 mg/l for adult kutum (Rutulis frisii kutum) and sturgeon (Acipenser gueldenstaedi), and 75 mg/l for roach (Rutulis rutulis caspicus). Sixty-day LC50 values for these species were 2, 11, and 14 mg/l, respectively (Dokholyan and Magomedov, 1983
). Significant nonlethal effects observed included decreased blood glucose levels, reduced leukocyte count, and increased muscle glycogen. It should be emphasized that the chemical composition of naphthenic acid mixtures is source-specific, so extrapolation of toxicity test results to different sources should be done cautiously.
Compared to aquatic organisms, there is limited information on the mammalian toxicity of naphthenic acids. The po LD50 of male mice was found to be 3.55 g/kg body weight for commercial sodium salts of naphthenic acids. Symptoms observed included central nervous system depression, convulsions, and respiratory arrest, leading to death (CEATAG, 1998). For rats, the po LD50 was 3.0 g/kg for commercial naphthenic acids (U.S. EPA, 1987
), while a mixture of isomers of dimethylcyclohexane (a specific naphthenic acid) had a po LD50 of 1.75 g/kg (Uzhdavini and Glukharev, 1984
). Intramuscular administration of cyclopentane naphthenic acid to rats at 0.15 g/kg/day for 10 days resulted in increased vascular permeability of capillaries (Maizelis et al., 1980
). To the authors' knowledge, results of mammalian toxicity testing involving repeated po exposure to naphthenic acids have not been reported previously.
The objective of the current study was to determine whether AOS naphthenic acids posed a health risk to indigenous mammals by means of exposure through their drinking water. Naphthenic acids were isolated from TPW collected from the upper (03 m), clarified zone of Mildred Lake settling basin. Wistar rats, serving as surrogate mammals, were used to test the acute po toxicity as well as the po subchronic toxicity of the naphthenic acids. Animals were gavaged with either single (acute study) or repeated (subchronic study) doses of the purified naphthenic acids, reconstituted in distilled water. Dosage levels in each study were chosen to bracket worst-case environmental exposures that small mammals could achieve if their drinking water was contaminated with naphthenic acids at concentrations comparable to TPW (i.e., 100 mg/l). Effects on body weight, water and food consumption, and other clinical signs of toxicosis were monitored, followed by histopathological examination of major organs. In subchronic testing, plasma biochemical and hematological effects were also evaluated.
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MATERIALS AND METHODS |
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Animals and animal husbandry.
All animals used in this study were outbred Wistar rats, purchased from Charles River Canada (St. Constant, Ontario, Canada) and were 9 weeks old at the onset of dosing. Rats were housed individually in polycarbonate cages lined with heat-treated hardwood bedding, with ad libitum access to Prolab Lab DietTM 5P00 rat chow (PMI® Nutrition International Inc., St. Louis, MO) and tap water. Animals were maintained under a light:dark schedule of 12h:12h at a temperature of 20°C. In the acute study, 40 female rats were randomly assigned to 4 groups of 10 animals each, designated control, low-dose, medium-dose, and high-dose. Twenty male rats were randomly assigned to 2 groups of 10 animals each, designated control and high-dose. In the subchronic study, 48 female rats were randomly assigned to 4 groups of 12 animals each (control, low, medium, and high dose). Animals were allowed a 2-week acclimatization period prior to toxicity testing during which body weight and food and water consumption were monitored. All aspects of this study were conducted with approval from the University of Saskatchewan Committee on Animal Care and Supply, in compliance with the Principles of Animal Care of the Canadian Federation of Biological Societies.
Acute toxicity test.
Animals were fasted for 12 h prior to dosing on day 0. Treatment females were dosed by po gavage, using a stainless steel feeding needle, with 5.45 ml/kg body weight of aqueous solutions containing either 55,080, 5508, or 550.8 mg/l naphthenic acids. The dosages delivered were therefore 300 (high dose), 30 (medium dose), or 3 (low dose) mg/kg body weight naphthenic acids. These dosages reflected 50, 5, and 0.5 times, respectively, a worst-case, single day exposure for wild mammals. This is based on a naphthenic acid concentration in environmental drinking water of 100 mg/l, typical of relatively fresh TPW (FTFC, 1995), and a water consumption rate of 60 ml/kg/day. Treatment males received the high dose in the same volume (per unit body weight) as females. Female and male controls were given 5.45 ml/kg tap water by gavage. Animals were returned to an ad libitum diet immediately after dosing.
All rats were monitored continuously for 12 h after dosing for signs of toxicosis. For the remainder of the 14-day study period, animals were monitored daily for changes in body weight, food and water consumption, and any additional behavioral or clinical signs of toxicosis. Rats were euthanized on day 14 by CO2 inhalation, and selected organs removed, weighed, and fixed in 10% neutral buffered formalin. Fixed tissues were sectioned, stained with hematoxylin and eosin, and examined microscopically for toxicant-induced changes.
Subchronic toxicity test.
For the repeated dose study, animals were exposed to naphthenic acids by po gavage 5 days per week (MondayFriday), for a 90-day period (68 total doses). Treatment animals (all females) were dosed by po gavage, using a stainless steel feeding needle, with 7.0 ml/kg of aqueous solutions containing either 8549, 854.9, or 85.5 mg/l naphthenic acids. The dosages delivered were therefore 60 (high dose), 6 (medium dose), or 0.6 (low dose) mg/kg body weight naphthenic acids per day. Control females were gavaged daily with 7.0 ml/kg tap water. These dosages reflected 10, 1, and 0.1 times, respectively, a worst-case, daily exposure for wild mammals, based on daily water consumption rates and environmental naphthenic acid concentrations discussed above. Animals were maintained on an ad libitum diet of rat chow and tap water throughout the test, with body weight, food and water consumption, and behavioral and clinical signs monitored daily.
Blood samples were collected from the ventral tail vein of each rat on day 45 of dosing. Samples were submitted to Prairie Diagnostic Services, Western College of Veterinary Medicine, Saskatoon, SK, Canada, for comprehensive plasma biochemical and hematological analyses. At the completion of the repeated dose study on day 91, rats were anesthetized with isofluorane, and blood samples collected by cardiac puncture were immediately submitted for plasma biochemical and hematological analyses as above. All plasma biochemical analyses were performed using an automated chemistry analyzer (Abbott Spectrum IITM, Chicago, IL). Sorbitol dehydrogenase activity was measured in place of alanine aminotransferase and aspartate aminotransferase in the standard clinical chemistry panel, since the former plasma enzyme is considered more hepatospecific and sensitive in the detection of hepatocellular injury in rats (Loeb, 1997). Following cardiac puncture, rats were euthanized by exsanguination. Selected organs were removed, fixed, sectioned, and stained for histopathological examination as previously described. To evaluate the presence of glycogen in hepatocytes, duplicate sections of liver were stained with Periodic Acid-Schiff, with and without prior treatment with diastase. Positive staining that was lost after diastase treatment was considered to be due to the presence of glycogen (modified from Luna, 1968
).
Statistical analyses.
Statistical analysis was performed using SigmaStatTM statistical software, version 2.0. In the acute study, a one-way ANOVA was used to compare means of female dose and control groups (4) with respect to consumption and body weight at regular timepoints, as well as the postmortem organ weights. A pairwise multiple comparison test was then used in cases where statistical significance was reached. For the male dose and control groups (2), a Student's t-test was used to compare group means. In the subchronic study, one-way ANOVA was again used to compare group means for consumption, plasma biochemical/hematological parameters, and organ weights, while a one-way repeated measures ANOVA was used to compare body weight trends. An ANOVA on ranks was performed on histological scoring of tissue lesions for both studies. Probability values of p 0.05 were considered statistically significant.
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RESULTS |
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Consumption and body weight trends.
Mean water and food consumption and body weight trends are illustrated in Figures 1 and 2 for female and male rats, respectively. Water consumption of dosed and control groups was similar for each sex. High-dose females and males exhibited a significant decrease in food consumption for 4 days following dosing, but all groups were similar thereafter. A small but significant posttreatment drop in body weight was observed in males, but not females. The sudden drop in body weight from day 1 to day 0 for all groups resulted from the predosing fast. Gorging on day 0 (after food was reintroduced) was evidenced by the spike in food consumption, but was less marked in dosed animals, particularly the high-dose groups of both sexes.
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Consumption and body weight trends.
Mean water and food consumption and body weight trends are illustrated in Figure 4. Compared with controls, water consumption by high-dose rats was significantly elevated throughout most of the experiment, but was depressed for the low-dose group. Food consumption was significantly less in high-dose rats for the first 11 days of dosing. However, from day 11 to the end of the study, food consumption of all groups was similar. Mean body weight of animals in the high-dose group was significantly lower than the weight of other groups throughout the exposure period.
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DISCUSSION |
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The results of the acute toxicity test suggested exposure to AOS naphthenic acids at levels of 300 mg/kg in rats had both cardiovascular and hepatic effects. A single po dose of 300 mg/kg produced significant cerebral hemorrhage in male rats. Vasoactive effects of naphthenic acids were also noted in a previous study by Maizelis et al. (1980). In that study, rats injected intramuscularly with 150 mg/kg cyclopentane naphthenic acid for 10 days demonstrated increased vascular permeability of cerebral capillaries. A similar effect on vascular permeability was not directly investigated in our study, but such an effect could be linked to the cerebral hemorrhaging or periarteriolar necrosis/fibrosis in the heart that was apparent here following acute exposure to naphthenic acids. It is unclear why the cerebral hemorrhage observed in the present study was more prevalent in male than in female rats.
The effects of acute naphthenic acid dosing on cardiac tissue have not previously been documented. Whether the observed periarteriolar necrosis/fibrosis was attributable to parent naphthenic acids or their metabolites is unknown at this time.
The clearest demonstration of a target organ in the acute toxicity test was the liver, where the occurrence of pericholangitis was consistent between sexes and highly dose-dependent. However, the pathogenesis of the observed pericholangitis remains to be determined.
Although sex differences may exist with respect to naphthenic acid toxicity, the subchronic toxicity test was restricted to female Wistar rats for logistical reasons (e.g., there was a limited supply of purified naphthenic acids). In addition, for these initial investigations of naphthenic acid toxicity, female responses to subchronic exposure were of greater interest since the data were required for future reproductive toxicity tests.
Subchronic exposure of female rats to naphthenic acids affected both food and water consumption. Although the suppression in food consumption was short-term, the elevation in water consumption in high-dose rats persisted throughout most of the 90-day exposure period. It is possible that the higher water consumption was simply due to elevated sodium intakes in the high-dose animals. Naphthenic acids were administered as sodium salt solutions, with the high-dose solution having a Na+ concentration of 0.1 molar. Since the gavage volume was 7 ml/kg body weight, naphthenic acid administration resulted in a daily sodium dose of 16.1 mg/kg body weight. This was over and above the 50 mg/kg body weight per day normally derived from the diet (Koolhaas, 1999), such that treatment increased the sodium intake of high-dose rats by 32%. Medium- and low-dose rats received dilutions of the high-dose solution, and therefore incremental sodium intake was negligible by comparison.
Although food consumption of all dose groups and controls were similar beyond day 11 in the subchronic study, the average body weight of the high-dose rats was significantly lower than that of other groups. The difference was consistent until the end of dosing, with high-dose animals being 89% lighter than controls. The effect on body weight was not extreme, but suggested that the 60 mg/kg naphthenic acid treatment had a sustained, suppressive influence on growth. It was not determined whether the loss in body mass was predominantly in the form of muscle or adipose tissue.
Subchronic dosing produced a marked increase in relative liver weight, which was 35% above control values for the high-dose group. This effect did not appear to be attributable to glycogen accumulation, since the relative liver weight of rats exhibiting high hepatic glycogen stores were similar to liver weights of rats not having high glycogen. A possible explanation for the increased liver weights of high-dose rats could be the induction of hepatic detoxification enzymes such as cytochrome P450 (Woodman, 1996), a mechanism currently being investigated. The relative weights of brain and kidneys in the high-dose group were also statistically greater than in control rats (11.5 and 9.1% higher, respectively). However, these differences are modest, and may simply reflect the slightly lower body weights of animals in the high-dose group. The elevations in terminal organ weights were not likely due to sodium and water retention. No pathological lesions were observed in the kidneys, rats had free access to drinking water, and plasma levels of sodium were normal both at the midpoint and end of the subchronic study.
Plasma biochemical analysis at the midpoint (day 45) and end (day 91) of the subchronic study also suggested liver as a target organ for naphthenic acid toxicity, particularly the suppression in plasma cholesterol concentration. Hypocholesterolemia in the high-dose group was not likely due to a difference in diet, since food consumption patterns of all groups were similar beyond day 11, and because severe dietary restrictions are required to induce this effect in rats (Levin et al., 1993). Baseline plasma cholesterol levels are largely regulated via synthesis in the liver, and suppression with naphthenic acid exposure suggests interference with synthetic, feedback, or mobilization pathways associated with that organ. Some of these mechanisms are currently being explored. Changes in cholesterol or other plasma lipids are often preceded by more obvious changes in other diagnostic tests (Woodman, 1999), so the presence of hypocholesterolemia without other evidence of major hepatic dysfunction is surprising. Another possible mechanism for hypocholesterolemia that is being investigated is cytochrome P450 induction, since cholesterol is a substrate for the CYP 7 subfamily of this enzyme group (Okuda et al., 1993
). Changes in plasma amylase are usually associated with pancreatic disease in most species. However, in rats it may also be indicative of a hepatic effect, since all isozyme forms of amylase are also present in rat hepatocytes (Loeb, 1999
). In addition, there was no evidence of pathological lesions in the pancreas of treated rats. However, since organ-specific amylase isozymes were not quantified in this study, it is uncertain at this time whether dose-related changes in amylase reflect a liver and/or pancreatic effect.
The major histopathological observation associated with repeated naphthenic acid dosing was increased glycogen storage in the liver. Hepatic glycogen storage is a normal metabolic function, but abnormally high intracellular accumulation can be a signal of hepatotoxicity or perturbations of carbohydrate metabolism. Interestingly, increased muscle glycogen was reported by Dokholyan and Magomedov (1983) in fish exposed to sodium naphthenates. The possible pathogenic mechanism for these observations are unknown. Other histopathological lesions that were seen in the acute study were not observed to a significant degree in the subchronic study. For example, the prevalence of pericholangitis in high-dose females in the subchronic study was low. This may suggest that 60 mg/kg, even on a repeated-exposure basis, is below the threshold required to induce this type of change.
In conclusion, a number of significant physical, clinical, and pathological changes were associated with the po administration of AOS naphthenic acids to Wistar rats, either acutely (300 mg/kg) or subchronically (60 mg/kg/day). Several of these observations support the conclusion that the liver is a plausible target organ in both cases and for both sexes, although the mechanism of toxic injury appears distinct between short- and long-term exposures to naphthenic acids.
Based on the Wistar rat as a surrogate mammal, the results of this study suggest the risk of acute po toxicity to small, wild mammals exposed to AOS naphthenic acids in contaminated drinking water is low, since 300 mg/kg is approximately 50 times a likely worst case single-day exposure. This exposure estimate is based upon a typical tailings pond water concentration of 100 mg/l and an average water consumption rate of 60 ml/kg/day (60 ml/kg/day x 100 x 10-3 mg/ml = 6 mg/kg/day). However, adverse health effects from repeated low-dose exposure may be more likely, since 60 mg/kg is only 10 times the estimated worst-case daily exposure for small mammals. Naturally, the extrapolation of data from the laboratory to the field always involves uncertainty, and there exists the possibility that indigenous mammals could be more sensitive to naphthenic acids than Wistar rats. Age, season, diet, general health and contemporary interacting stressors, contaminant interaction, and interspecic differences in metabolism or excretion are just a few factors that could influence the response to naphthenic acid consumption. It should also be noted that the naphthenic acids used in the present toxicity studies were relatively "fresh" since they were isolated from recently produced tailings. Knowing that naphthenic acids will undergo some microbial biodegradation over time, mixture composition will change, and the overall concentration will decrease in the future. However, this study was a logical starting point for assessing the mammalian toxicity of these compounds, and will be a valuable reference for others researching the toxicity of naphthenic acids from the AOS or other sources.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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