In the United States, several proposed approaches for using bioassays for the risk assessment of complex hazardous mixtures require that selected mixtures be "sufficiently similar" to each other. The goal of this research was to evaluate the utility of a protocol using in vitro bioassays and chemical analysis as a basis for assessing mixture similarity. Two wood preserving wastes (WPWs) containing polycyclic aromatic hydrocarbons and pentachlorophenol were extracted and fractionated to generate potentially similar mixtures. Chemical analysis was conducted using gas chromatography/mass spectrometry. Genotoxicity was evaluated using the Salmonella/microsome and Escherichia coli prophage induction assays. The crude extract of one WPW was also tested in the chick embryotoxicity screening test (CHEST) assay. The CHEST assay provided the most sensitive measurement of toxicity. Overall, the biological potency of the samples was not well correlated with predicted potency based on chemical analysis. Although several mixtures appeared similar based on chemical analysis, the magnitude of the response in bioassays was often dissimilar. Fractionation was required to detect the genotoxicity of mixture components in vitro. The results confirm the need for an integrated protocol, combining chemical analysis, fractionation, and biological testing to characterize the risk associated with complex mixtures.
A large number of hazardous waste sites in the United States have undergone the initial stages of remediation or containment. At many of the remaining sites, the potential for exposure to ecological receptors is a primary concern. This manuscript reports on studies to investigate the impact on ecological receptors exposed to complex mixtures at a former creosote facility. Currently there are isolated areas on-site that were not addressed in the initial removal action that appear to be releasing polycyclic aromatic hydrocarbons (PAHs) to the surrounding environment. The U.S. EPA collected environmental samples and performed ex situ sediment bioassays to measure chronic toxicity; whereas, this study describes an in situ study to measure biomarkers of effect in two ecological receptors. Mosquitofish (Gambusia affinis) and cricket frogs (Acris crepitans) were collected from a small intermittent creek adjacent to the site, and reference stations. A weight-of-evidence ecological risk assessment was completed for the amphibian and fish communities. The ecological risk assessment was developed using analysis of media chemistry, body burden of specific PAHs, bioassay results, community surveys, and cellular genome size variation as a biomarker of genotoxicity. Flow cytometric estimates of chromosomal damage were significantly elevated for both mosquitofish and cricket frogs inhabiting the contaminated site, relative to at least one reference site. Surface water screening values for fish and amphibians exceeded screening values for PAHs by more than one order of magnitude in the on-site creek, and sediment PAH concentrations were extremely high (up to 1,549 μg/dry g). Tissue concentrations of PAHs were below screening values. Media chemistry, bioassay and genotoxicity data all support the same conclusion that on-site PAHs continue to impact aquatic receptors. The genotoxicity findings are consistent with and contribute to results of the weight-of-evidence ecological risk assessment. The results support continuing efforts to incorporate biomarkers as valuable lines of evidence within ecological risk assessment.
Bioremediation represents one of the most cost-effective technologies for treatment of petroleum hydrocarbons in contaminated surface soils. A major concern for regulatory agencies when evaluating bioremediation is how to determine acceptable levels for residual organics in soil. Although guidelines have been developed for some organics in soil, limited information is available to define acceptable levels of the metabolites of biological degradation. The products of oxidative degradation are likely to be more water soluble and may also be more toxic. The purpose of the current study was to monitor changes in compound concentration and genotoxicity in soils undergoing bioremediation. The site selected for this study was a former wood-preserving site in the northwestern United States. Soil samples were collected over a 4-year period from two 6075-m 2 land treatment units. Conditions for biodegradation were enhanced by the addition of water and nutrients, as well as by frequent tilling to add oxygen. Due to the location of the facility, the temperature was conducive to a more rapid rate of biodegradation for approximately 6 months per year. Soil samples were collected using a grid system and solvent extracted. Polycyclic aromatic hydrocarbons were quantified in soil extracts using gas chromatography-mass spectrometry (GC/MS), and genotoxicity measured using the Salmonella/microsome assay. After 2 years of treatment, concentrations of total and carcinogenic polycyclic aromatic hydrocarbons (PAHs) were reduced to approximately 10% the concentration in the untreated soil. However, the mean weighted activity of the untreated soil was 293 net revertants per g soil, whereas the extracts of soil collected after 2 years induced a mean weighted activity of 325 net revertants per g soil. Thus, although biodegradation clearly reduced the concentration of total and carcinogenic PAHs in the surface soils, the results from the genotoxicity bioassay indicate that there was a lag in the reduction of mutagenicity in treated soils.
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