Grantee Research Project Results
Final Report: Ecotoxicity Risks Associated with the Land Treatment of Petrochemical Wastes
EPA Grant Number: R826242Title: Ecotoxicity Risks Associated with the Land Treatment of Petrochemical Wastes
Investigators: Janz, David M.
Institution: Oklahoma State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1997 through September 30, 2000
Project Amount: $406,229
RFA: Exploratory Research - Environmental Biology (1997) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
Petrochemical waste contains both organic and inorganic contaminants that pollute soil and may pose significant ecological risks to wildlife. Petrochemical waste typically is disposed in land treatment units; such units are widespread throughout Oklahoma and the United States. Land treatment is a waste management technology that involves controlled application of wastes onto or into soil for the purpose of biodegradation of organics, immobilization of inorganics, and avoiding the bioaccumulation of hazardous chemicals. Few studies have been conducted evaluating possible toxicity risks to terrestrial organisms residing on these units. Although many consider land treatment of petrochemical industrial wastes to be a viable and safe management practice, our previous field studies have raised environmental concerns regarding immunotoxicity and fluorosis risks to wild vertebrates. Our objective in this study was to examine these concerns by monitoring immunologic and pathologic responses of cotton rats (Sigmodon hispidus) in situ by seasonally collecting resident animals from replicated land treatment and reference sites throughout Oklahoma. Biomarker responses (immune system function, hematology, biotransformation enzyme activities) were compared to soil and tissue contaminant levels to identify potential causative agents. Cotton rat populations and rodent community assemblages also were assessed on landfarm sites.
Summary/Accomplishments (Outputs/Outcomes):
Over a 2-year period, cotton rats and soils were collected and analyzed from five land treatment units and five matched reference units. Land treatment units with soil metal contamination contained (number in parenthesis): Cr, Cu, Pb (5); Al, As, Ni, Sr, Zn (4); Ba (3); and Cd, V (2). Land treatment units with soil polycyclic aromatic hydrocarbons (PAH) contamination (number in parenthesis) were napthalene, phenanthrene, benzo (g,h,i) perylene (3); acenapthene, anthracene, pyrene, benzo (a) anthracene, chrysene, benzo (b) fluoranthene, benzo (a) pyrene, [B(a)P], indeno (1,2,3-cd) pyrene, dibenz (a,h) anthracene (2); and acenapthylene, fluorene, fluoranthene, benzo (k) fluoranthene (1). Total PAH and total petroleum hydrocarbons (TPH) were elevated on all five land treatment units. Mean sum of B(a)P equivalents (B(a)Pequiv) were slightly elevated on land treatment units as compared to reference units. Units 1 and 3 were significantly higher in levels of metals, total PAH, TPH, and B(a)Pequiv than the other units.
Pb and F bioaccumulated in bone and Pb bioaccumulated in the kidneys of cotton rats. F in bone of 496 to 2212 mg/kg was 3- to 15-fold greater than mean F in bone of cotton rats from reference units. Elevated levels of Pb in bone of 4.6 to 24.8 mg/kg was 460- to 2500-fold greater than mean Pb in bone of cotton rats from reference units. Elevated levels of Pb in the kidney of 0.31 to 1.08 mg/kg was 10- to 36-fold greater than mean Pb levels in the kidneys of cotton rats from reference units. Bone F was an accurate predictor of the severity of dental fluorosis (P = 0.0001, r = 0.78). Strong relationships were found between bone F and HCl-extractable F (P = 0.016, r = 0.73) and bone F and total F (P = 0.0001, r = 0.93) in soils of land treatment units. A strong relationship was discovered between Pb in bone and Pb in soil (P = 0.0001, r = 0.96). Land treatment appears to have been effective as a remediation technology in reducing levels of individual PAHs and the sum of B(a)P equivalents to background levels on units 2, 4, and 5, but not on units 1 and 3. Thus, certain contaminants such as Pb and F tend to bioaccumulate in cotton rats collected from land treatment units. Land treatment was ineffective in reducing levels of these contaminants to background levels that will not pose an increase in risk to terrestrial mammals. Therefore, we recommend that to avoid accumulation of these contaminants in cotton rats and their possible deleterious effects, these contaminants should be measured, and land application rates of petrochemical waste should be managed, to avoid excessive loading into soil systems.
A suite of Phase I (oxidation) and Phase II (conjugation) biotransformation enzymes were determined in resident cotton rats. Male and female cotton rats were collected in summer, fall, and winter from four landfarm sites and four ecologically similar reference sites. Hepatic methoxyresorufin O-deethylase (MROD)-a specific catalytic marker of cytochrome P450 CYP1A2-activity was significantly induced (2-3 fold) in male and female rats collected from landfarms, compared to rats collected from reference sites. In contrast, effects on hepatic ethoxyresorufin O-deethylase (EROD)-a specific catalytic marker of CYP1A1-activity were inconsistent due to season, sex, and treatment variation. A significant decrease in EROD and MROD activity was found in cotton rats held for 48 hours prior to sacrifice, compared to rats sacrificed on the day of capture. These results indicate that when using EROD and MROD activities as biochemical markers of exposure to aryl hydrocarbon receptor (AhR) agonists present in petrochemical wastes, animals should be sacrificed as quickly as possible after capture. The cotton rats collected from one landfarm unit exhibited consistent elevation of EROD, MROD, and pentoxyresorufin O-deethylase (PROD) activity. Rats from this unit also exhibited significantly elevated CYP1A2 protein expression determined by Western blotting. There were no consistent effects at any of the landfarm sites of contaminant exposure on hepatic glutathione S-transferase (GST) activity, glutathione levels, or CYP1A1 protein expression. Thus, AhR agonists, such as PAHs at certain landfarm units, were bioavailable to rats, as indicated by induction of hepatic CYP1A protein expression and CYP1A-dependent biotransformation enzyme activities.
We also investigated effects of contaminants on the immune system and hematology of cotton rats (Sigmodon hispidus) living on five abandoned petrochemical landfarms. Cotton rats were sampled seasonally (summer and winter) from each landfarm and from five ecologically matched reference sites for 2 years (1998-2000), and returned to the laboratory for immunological and hematological assays. Overall analysis indicated that rats inhabiting landfarms exhibited decreased relative spleen size compared to rats collected from reference sites, with one landfarm showing the greatest reduction. Cotton rats collected from landfarms also had increased hemoglobin, hematocrit and platelet levels, and decreased blood leukocytes during summer. During winter, an increase in the number of popliteal node white blood cells was observed from rats collected on landfarms. No difference was detected for lymphocyte proliferation in response to concanavalin A, pokeweed, or interleukin-2. Lymphokine-activated killer cell lytic ability showed a seasonal pattern, but no treatment differences. No differences between landfarm and reference sites were detected in the hypersensitivity reaction of rats given an intradermal injection of phytohemagluttinin (PHA-P). Comparisons within individual sites indicated that two sites had the greatest effects on immune function and hematology of cotton rats. These results suggest that residual petrochemical waste affects the immune system and hematology of cotton rats living on abandoned landfarms, particularly during summer, and is complicated by variation in the contaminants found on individual petroleum sites.
We evaluated the effectiveness of tumor necrosis factor-alpha (TNF- alpha) produced by splenocytes harvested from cotton rats collected from petrochemical landfarms and reference sites. Additional actinomycin d to test samples increased lysis of test cells (WEHI 164 murine-fibrosarcoma cells) by 100 times, making the assay unusable. A range of lipopolysaccharide (LPS) concentrations (0.0, 0.05, 0.5, 5.0, and 50.0 µg/ml) was tested to determine the appropriate concentration to stimulate TNF- alpha production in cotton rat splenocytes. Fifty µg/ml of LPS was found to be the most effective concentration for stimulating TNF- alpha production. Cotton rats collected from petrochemical landfarms had a 12.6 percent increase in lytic activity associated with TNF- alpha production over rats from reference sites. By stimulating splenocytes with an LPS concentration greater than 50.0 µg/ml, TNF- alpha can be used effectively to detect changes in the immune system of mammals inhabiting contaminated sites.
The immune selection hypothesis states that individuals exposed to immunosuppressive contaminants will be negatively affected with respect to survival and population density. We tested the immune selection hypothesis by comparing population size, monthly survival, and reproduction of cotton rats inhabiting the petrochemical landfarms and uncontaminated reference sites. Populations were monitored during summer and winter for 2 years using mark-recapture techniques in association with program MARK. Toxicity indices were calculated for metal and PAH soil contamination. After every season of trapping, a sample of cotton rats was returned to the laboratory to measure male and female reproductive organ weight, placental scars per female, and number of embryos per female. Cotton rat populations inhabiting landfarms experienced reduced population densities and lower monthly survival. Populations inhabiting landfarms reached maximum densities of half that of reference populations. Survival was lowest in populations from landfarms during summer months. Toxicity indices indicated that both metal and PAH concentrations were affecting survival and population density. Cotton rat populations had more variable sex ratios and lower proportion of juveniles than populations from reference sites. No differences were observed in the weight of epididymes, testes, uteri, or ovaries. The results of this study support the predictions made by the immune selection hypothesis.
Toxicologists have become increasingly aware of the importance of apoptosis (programmed cell death) as a molecular control point in toxicity, and toxicant-stimulated apoptosis may represent an early cellular indicator of toxicity in vertebrates. We determined the extent of apoptosis in ovary and thymus collected from female cotton rats inhabiting the five landfarm and five reference sites in this study. There was a significantly elevated extent of ovarian cell apoptosis in cotton rats collected from landfarm sites. These female rats also exhibited a lower number of uterine scars, a measure of successful gestation, in comparison to rats collected from reference sites. Although thymic cell apoptosis was elevated at one of the five landfarm sites, there was no significant overall increase in this parameter using individual sites as replicates. In a subsequent study, we collected rats from one landfarm and reference site, and determined the extent of apoptosis in bone marrow and spleen cells. Although bone marrow and spleen cell apoptosis was elevated at the landfarm site, compared to reference site (approximately 2-fold), these findings were not statistically significant due to the small number of rats available at the time of analysis. Overall, data suggest that the extent of apoptotic cell death in reproductive and immune organs represent an early cellular indicator of toxicity in wildlife exposed to complex mixtures of environmental contaminants.
Studies on the effects of contamination on wildlife have been largely focused at the individual level. Biomarkers have been used to detect changes in the health of individuals from acute exposure; however, little attention has been given to the effects of chronic exposure at the population or community level. Rodent assemblages from uncontaminated reference sites and petrochemical landfarms were examined to identify potential alterations in structure and composition. Reference sites showed typical rodent assemblage structure dominated by hispid cotton rats (Sigmodon hispidus) and fulvous harvest mice (Reithrodontomys fulvescens). Rodent assemblages inhabiting landfarms also were dominated by cotton rats; however, harvest mice were replaced by deer mice (Peromyscus maniculatus). Contaminated sites also were characterized by an increase in house mice (Mus musculus) and an absence of voles (Microtus spp.). Cotton rat abundance had a negative impact on rodent diversity on both sites, but was more intense on reference sites. Evenness was indirectly affected by the abundance of cotton rats. Diversity varied temporally on both landfarms and reference sites, with peaks in diversity associated with periods of low cotton rat abundance. Thus, rodent assemblages were impacted by landfarms, either directly through effects of contaminants on the health of individuals, or indirectly by changes in the habitat.
Journal Articles on this Report : 8 Displayed | Download in RIS Format
Other project views: | All 12 publications | 12 publications in selected types | All 10 journal articles |
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Type | Citation | ||
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Carlson RI, Wilson JA, Lochmiller RL, Janz DM, Schroder JL, Basta NT. Ecotoxicological risks associated with the land treatment of petrochemical wastes. II. Effects of hepatic Phase I and Phase II detoxification enzymes in cotton rats. Journal of Toxicology and Environmental Health-Part A 2003;66(4):327-343. |
R826242 (Final) |
not available |
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Kim S, Stair EL, Lochmiller RL, Lish JW, Qualls CW. Evaluation of myelotoxicity in cotton rats (Sigmodon hispidus) exposed to environmental contaminants. I. In vitro bone marrow progenitor culture. Journal of Toxicology and Environmental Health 2001;62(2):83-96. |
R826242 (Final) |
not available |
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Kim S, Stair EL, Lish JW, Lochmiller RL, Rafferty DP, Qualls CW. Evaluation of myelotoxicity in cotton rats (Sigmodon hispidus) exposed to environmental contaminants. II. Myelotoxicity associated with petroleum industrial wastes. Journal of Toxicology and Environmental Health 2001;62(2):97-105. |
R826242 (Final) |
not available |
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Kim S, Stair EL, Lochmiller RL, Rafferty DP, Schroder JL, Basta NT, Lish JW, Qualls CW. Widespread risks of dental fluorosis in cotton rats (Sigmodon hispidus) residing on petrochemical waste sites. Journal of Toxicology and Environmental Health 2001;62(2):107-125. |
R826242 (Final) |
not available |
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Rafferty DP, Lochmiller RL, Kim S, Qualls CW, Schroder JL, Basta N, McBee K. Fluorosis risks to resident hispid cotton rats on land-treatment facilities for petrochemical wastes. Journal of Wildlife Diseases 2000;36(4):636-645. |
R826242 (2000) R826242 (Final) |
Exit |
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Savabieasfahani M, Lochmiller RL, Janz DM. Elevated ovarian and thymic cell apoptosis in wild cotton rats inhabiting petrochemical-contaminated terrestrial ecosystems. Journal of Toxicology and Environmental Health-Part A 1999;57(8):521-527. |
R826242 (1999) R826242 (Final) |
not available |
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Schroder JL, Basta NT, Payton M, Wilson JA, Carlson RI, Janz DM, Lochmiller RL. Ecotoxicological risks associated with the land treatment of petrochemical waste. I. Residual soil contamination and bioaccumulation by cotton rats.Journal of Toxicology and Environmental Health-Part A 2003;66(4):305-325. |
R826242 (Final) |
not available |
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Wilson JA, Carlson RI, Janz DM, Lochmiller RL, Schroder JL, Basta NT. Ecotoxicological risks associated with land treatment of petrochemical waste. III. Immune function and hematology of cotton rats. Journal of Toxicology Environmental Health-Part A 2003;66(4):345-363. |
R826242 (Final) |
not available |
Supplemental Keywords:
terrestrial, ecosystem, indicators, assessment, southcentral, landfarm, petrochemical pollution, wildlife toxicology, ecotoxicity, oil refinery pollution, immunotoxicity, metabolism, soil pollution, zoology, heavy metals, PAHs., RFA, Scientific Discipline, Toxics, Waste, Ecosystem Protection/Environmental Exposure & Risk, National Recommended Water Quality, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Environmental Chemistry, Ecological Effects - Environmental Exposure & Risk, chemical mixtures, Ecological Effects - Human Health, Biochemistry, Environmental Monitoring, Hazardous Waste, Ecology and Ecosystems, Ecological Risk Assessment, Hazardous, Ecological Indicators, ecological exposure, adverse impacts, biomarkers, wildlife, dose-response, immunotoxicity, petrochemical wastes, lead, petrochemical waste, biomonitoring, ecological assessment, oil refinery pollution, oil spills, assessment methods, biotransformation, ecotoxicological studies, water quality, arsenicRelevant Websites:
http://www.soilchem.okstate.edu/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.