Grantee Research Project Results
Final Report: Bioavailability & Risk Assessment of Complex Mixtures
EPA Grant Number: R825408Title: Bioavailability & Risk Assessment of Complex Mixtures
Investigators: Donnelly, Kirby C. , Safe, Stephen H. , Autenrieth, R. L. , McDonald, T. J. , Reeves, William , Huebner, Henry J.
Institution: Texas A & M University
EPA Project Officer: Hahn, Intaek
Project Period: November 15, 1996 through November 14, 1999 (Extended to April 15, 2000)
Project Amount: $443,997
RFA: Environmental Fate and Treatment of Toxics and Hazardous Wastes (1996) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management , Safer Chemicals
Objective:
The objectives of this research project were to: (1) evaluate the toxicity of isolated fractions and model compounds from several complex mixtures; and (2) study the bioavailability of soil-bound polycyclic aromatic hydrocarbon (PAH) mixtures.
Summary/Accomplishments (Outputs/Outcomes):
Risk assessments of PAH mixtures are hindered by a lack of reliable information on the potency of both mixtures and their individual components. In addition, minimal information is available to define the rate of sorption of PAHs onto soils or sediments. Several studies have suggested that soil-bound PAHs are unavailable for leaching or uptake by a biological system, whereas others have suggested that the solubility of PAHs increases as the oxidized metabolites of biodegradation are generated. The major objectives of this research were to investigate the bioavailability and genotoxic interactions of complex mixtures. In the initial stages of research, two complex mixtures (a wood preserving waste (WPW) and a manufactured gas plant residue (MGP)) were fractionated. Mixtures were separated into aliphatic, aromatic, and PAH fractions using column chromatography. The PAH fraction was further separated into fractions based on the number of aromatic rings. Initial steps have been completed in the separation of the WPW and MGP mixtures.
Preliminary testing with a fractionated MGP sample indicates that the 2- and 3-ring fractions have limited activity in the Salmonella/microsome mutagenicity or ethoxyresorufin-O-deethylase (EROD) enzyme induction assays, whereas the 4-, 5- and more-than-5-ring fractions induce a strong genotoxic response and a significant increase in EROD induction. The mutagenic response observed for the MGP fractions ranged from 41 net revertants for the 2-ring fraction, to 159 net revertants for the 5-ring fraction. The crude extract induced 89 net revertants per mg.
This preliminary separation of MGP fractions demonstrated that genotoxic activity could be isolated in the higher molecular weight components. Chemical analysis, however, indicates that the initial separations enriched specific 2- and 3-ring compounds, but also resulted in a great deal of "smearing" between fractions. Because the degree of separation obtained with the initial fractionation procedure was less than planned, a revised protocol using a larger column and different packing materials was employed.
Using a preparatory-scale column, PAHs were isolated from a coal tar and then separated by ring number using an HPLC with a photodiode array detector (PDA). Five fractions (A-E) were generated, each possessing a unique composition and expected potency. The toxicity of each fraction was measured in the Salmonella/mutagenicity assay and the chick embryo screening test (CHEST). Their abilities to induce ethoxyresorufin-O-deethylase and inhibit gap junction intercellular communication in rat liver Clone 9 cells also were measured. In the Salmonella/mutagenicity assay, fractions were found to induce a genotoxic response in the order C > D > E > B > A. Toxic equivalency factors (TEFs) for fractions A-E were in the order E D > C > B > A. TEF values were 20652, 20929, 441, 306 and 74.1 µg BaP equivalents/g, respectively. A lack of agreement between assay-predicted potencies and chemical analysis-predicted potencies was observed with other assays and other methods of calculation. The results demonstrate the limitations of using a single method to predict the toxicity of a complex PAH mixture. The analysis of crude extracts, isolated fractions, model chemical and reconstituted mixtures provides information that could be used to validate the TEF approach for risk assessment.
Estimating the bioavailability of soil-bound contaminants has become the subject of much research and discussion in recent years. Much of the debate focuses on single chemicals and simple mixtures. Although mechanistically useful, these studies do not address contamination in the form of complex mixtures as often seen with PAHs. In this study, a Weswood silt loam soil was amended with three model chemicals (benzo(a)pyrene, 2,4,6-trinitrotoluene, and pentachlorophenol), or one of the two complex mixtures (the WPW and MGP). Sufficient soil was spiked to allow extractions on day 0 and day 360. Soil was extracted in triplicate using each of six extracting solutions: pH 2 water with pepsin, pH 7 water with pancreatin and bile salts, a model system adjusted from pH 2 to pH 7, water, methanol:water (1:1), and methanol:water (1:3), as well as the standard U.S. Environmental Protection Agency (EPA) method using hexane:acetone. The results obtained with the model compounds and complex mixtures indicated that sequestration was similar in both treatment groups. In addition, similar levels of PAHs were detected on day 0 and day 360 for each extraction procedure from the soil amended with the complex PAH mixture, indicating that under the conditions of the study, aging was minimal.
Figure 1. Urine Ingested and Soil Type
The final component of this research was an animal feeding study. Coal tar (CT) was spiked onto two aliquots of Weswood silty clay loam soil (pH 8.0, 1.7 percent organic matter, 12 percent sand, 49 percent silt, 39 percent clay) at a concentration of 5 percent (wt/wt). The first was aged 270 days at near-ambient temperatures in a greenhouse, and the second was spiked immediately before use. Both soils were incorporated into a lab rodent diet and administered to mail Fisher 344 rats. Additional feeds containing clean soil, no soil, and a CT control feed containing coal tar spiked onto an inert vehicle (3:1 SiO2:TiO2) at 5 percent (wt/wt) also were administered. The final concentration of CT in the prepared pellets was 0.35 percent by weight. Excretion of PAH metabolites in urine and PAH-DNA adducts were quantified. In tandem with the feeding study, the rate of desorption from soil to water of CT PAHs was measured in vitro for both soils. Urine for coal tar control animals from the first day in metabolism cages had an average of 29.7 ± 3.4 ppm hydroxypyrene in the urine/mg pyrene consumed. Freshly spiked and aged coal tar groups had 5.3 ± 1.4 and 4.4 ± 1.7 ppm hydroxypyrene in the urine/mg ingested, respectively (see Figure 1). Thus, while the PAHs were released at a relatively high rate from the coal tar amended feed, there were much lower levels of hydroxypyrene in urine of animals in the aged and freshly spiked coal tar groups.
Post-labeling analysis of hepatic DNA revealed a zone and three distinct adducts. The adduct closest to the origin co-migrated with benzo(j)fluoranthene-9,10-diol-11,12-epoxide-N-guanine (B(j)F-N,G) standard. RAL values for B(j)F-N,G adducts ranged from 16.54 ± 1.85 to 24.46 ± 6.4 nM adduct/M DNA. A significant difference (p<0.05) was observed in B(j)F-N,G adduct levels in the aged CT/soil group (16.54 ± 1.85 nM adduct/M DNA) compared to the control CT group (26.46 ± 6.4 nM/M DNA). Overall, rats receiving feed with coal tar on soil exhibited a significantly lower RAL/mg PAH ingested than those receiving feed with coal tar on the inert carrier. Additionally, there were no significant differences in the RAL/mg PAH ingested between the aged and freshly spiked soil groups.
In vitro desorption experiments also suggested that no significant changes in availability occurred over 270 days of aging as compared to freshly spiked soil. It was shown that aging had little or no effect on the availability of contaminants and that soil alone diminished availability substantially. In desorption studies, less than 65 percent of the pyrene was able to freely desorb from the soil that was aged for 270 days and the freshly spiked soil. An examination of carcinogenic PAHs (cPAHs) produced a similar result. Of the total amount of cPAHs, approximately 70 percent was present in the refractory phase in both soils.
Under the conditions of the current study, the availability of PAHs in coal tar amended soil did not decrease significantly with time. There are at least three possible explanations for the minimal degree of time-dependent sequestration observed in the study. First, the aliphatic and aromatic components of coal tar may affect the behavior of PAH mixtures in ways that are not accounted for by these experiments. Second, aging took place under ambient conditions in a greenhouse, conditions that may produce different sorbent/solute interactions than those observed at room temperature. Finally, the volume of coal tar added may have overwhelmed the capacity of the soil organic matter to adsorb and retain PAHs. Under this scenario, most of the sorption sites in the soil would be occupied by hydrophobic coal tar components, rendering the soil incapable of further sorption. In each of the above cases, it is apparent that complex PAH mixtures may behave differently from single compounds or simple mixtures.
This study has investigated the genotoxicity and bioavailability of complex mixtures. Fractionation studies suggest that chemical analysis does not accurately characterize risk. The available information suggests that compound interactions or unidentified compounds may contribute appreciably to the total risk. Bioavailability studies using in vitro and in vivo tests indicate that coal tar sequestration in soil was not affected by aging. Overall, the data indicate that current methods of risk assessment, which incorporate uncertainty factors of one to two orders of magnitude, should provide adequate representation of risk.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 8 publications | 4 publications in selected types | All 4 journal articles |
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Type | Citation | ||
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Reeves WR, Barhoumi R, Burghardt RC, Lemke SL, Mayura K, McDonald TJ, Phillips TD, Donnelly KC. Evaluation of methods for predicting the toxicity of polycyclic aromatic hydrocarbon mixtures. Environmental Science & Technology 2001;35(8):1630-1636. |
R825408 (Final) |
not available |
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Reeves WR, McDonald TJ, Bordelon NR, George SE, Donnelly KC. Impacts of aging on in vivo and in vitro measurements of soil-bound polycyclic aromatic hydrocarbon availability. Environmental Science & Technology 2001;35(8):1637-1643. |
R825408 (Final) |
not available |
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Reeves WR, Donnelly KC, McDonald TJ, Cizmas L. Partitioning and desorption behavior of polycyclic aromatic hydrocarbons from disparate sources. Science of the Total Environment 2004;332(1-3):183-192. |
R825408 (Final) |
not available |
Supplemental Keywords:
bioaccessability, complex mixtures, benzo(a)pyrene, pentachlorophenol, polycyclic aromatic hydrocarbon, genotoxicity., RFA, Health, Scientific Discipline, Toxics, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Contaminated Sediments, Remediation, Geochemistry, Environmental Chemistry, Health Risk Assessment, pesticides, Chemistry, Risk Assessments, chemical mixtures, Fate & Transport, complex mixtures, risk assessment, fate and transport, cumulative risk, fate, coal gasification sites, Superfund sites, effects assessment, risk assessment techniques, contaminant transport, soil sediment, acceptable residue levels, contaminated sediment, complex mitures, sediment transport, transport contaminants, geotoxicity, PAH metal mixtures, adverse human health affects, chemical contaminants, soils, contaminated soil, PAH, human exposure, toxicity, characterizing chemical mixtures, environmental transport and fate, public health, furans, environmental toxicant, Toxic Equivalency Factor, hazardous materials exposure, assessment methods, refinery sites, vadose zone, hazardous waste sites, dioxins, exposure assessment, environmental chemicals, toxicokineticsProgress 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.