Analysis of Soil Samples from a Hazardous Waste Site Comparison

							Analysis of Soil Samples from a Hazardous Waste Site: Comparison of CALUX® Bioassay TEQ Determinations with High Resolution GC/MS
George C. Clark1, Michael S. Denison2, Richard W. Morris3, Michael Chu1, Andrew Chu1, and David J. Brown1
1 2

Xenobiotic Detection Systems, 1601 East Geer Street, Suite S, Durham, NC 27704, USA Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA 3 Analytical Sciences Inc., 2605 Meridian Parkway, Suite 200, Durham, NC 27713, USA

Introduction Remediation of hazardous material contaminated sites requires analysis of levels of dioxin-like chemicals that are potentially important contaminants of these areas. Traditionally highresolution gas chromatography/mass spectrometry (HRGC/MS) has been used to detect the presence of dioxin-like chemicals. This is a complex, expensive and time consuming method based on measuring the concentrations of 17 individual chlorinated dioxin and furan congeners that are considered toxic. To estimate a Toxic Equivalency (TEQ), the individual concentrations of each toxic congener is multiplied by a Toxic Equivalency Factor (TEF) and these determinations summed to produce a TEQ Determination. This TEQ determination provides an estimate of the potential toxicity of the sample for risk assessment purposes. A rapid, less expensive and more easily performed method of estimating TEQ determinations would aid in remediation efforts of hazardous waste sites. Xenobiotic Detection Systems, Inc. (XDS) has developed a cellular bioassay based on the mechanism of toxicity of dioxin for estimating TEQ contamination with dioxin-like chemicals. This system has been developed with a rapid method of sample extraction and processing and application of the extract to living cells that respond to dioxin-like chemicals. The cell bioassay is depicted in Figure1; it utilizes a recombinant cell line with a stably integrated AhR-responsive luciferase reporter gene. Exposure of this Chemically Activated Luciferase Expression (CALUX®) bioassay to extracts containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and/or related halogenated aromatic hydrocarbons produces the enzyme luciferase in a time, dose and chemical specific manner. Luciferase activity is determined by measuring light emitted and is directly proportional to the amount of dioxin-like chemicals within the test sample.

Figure 1. Schematic representation of the cellular processes involved in the CALUX® bioassay.

We participated in a double-blind study to compare the results of TEQ determinations for soil samples from a hazardous material remediation area measured by the CALUX® bioassay and HRGC/MS. Here we report the results of this double blind validation study.

Materials and Methods A corporation under contract from the US Environmental Protection Agency collected soil samples from a hazardous material remediation site. These samples were sent to an independent laboratory for HRGC/MS analysis of TEQ contamination. The laboratory sent an aliquot of each soil to XDS for CALUX® determination. Results of HRGC/MS analysis and CALUX® bioassay results were sent to an independent statistician (Richard W. Morris, Analytical Sciences, Inc.) so that a double-blind format was maintained. After all results were reported comparison of the results was performed.

HRGC/MS. Sediment and ash samples were spiked with 13C12-labeled PCDD/PCDF standards and analyzed for congener-specific PCDD/PCDFs at the corporation’s lab using EPA Method 8290. I-TEQs for PCDDs/PCDFs were calculated using TEF values from the World Health Organization1. CALUX® bioassay. XDS has a patented genetically engineered cell line (mouse hepatoma H1L6.1) that contains the gene for firefly luciferase under transactivational control of the aryl hydrocarbon receptor2. This cell line can be used for the detection and relative quantificatation of a sample’s total dioxin I-TEQ. Using a patent pending sample processing procedure it is also possible to use the CALUX® assay to estimate the I-TEQ contributions of PCDDs/PCDFs or the I-TEQ contributions of the coplanar PCBs3. The assay that uses this cell line is called the Chemically-Activated Luciferase Expression or CALUX® assay. The samples were extracted using a modification of the EPA 8290 extraction method4. Briefly, the dried samples were ground and one gram aliquots were placed in solvent cleaned glass vials with PTFE lined caps. The sample was extracted with a 20% solution of methanol in toluene then twice with toluene. During each extraction step the samples were incubated in an ultrasonic water bath. The three extracts from each sample were filtered, pooled and concentrated by vacuum centrifugation. The sample extract was suspended in hexane and prepared for the bioassay by a patent pending clean up method3. The eluate from the clean up method was concentrated under vacuum into dimethyl sulfoxide (DMSO). The DMSO solution was used to dose the genetically engineered cells in the CALUX® bioassay. Prior to dosing cells, the sample extracts in DMSO were suspended in cell culture medium. This medium was then used to expose monolayers of the H1L6.1 cell line grown in 96 well culture plates. In addition to the samples, a standard curve of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was assayed (500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.9, and 1.0 parts per trillion (ppt) TCDD). The plates were incubated for a time to produce optimal expression of the luciferase activity in a humidified CO2 incubator. Following incubation, the medium was removed and the cells were examined microscopically for viability. The induction of luciferase activity was quantified using the luciferase assay kit from Promega.

Results and Discussion A double-blind format comparison was made of TEQ determinations with dioxin-like chemicals in soil samples with the CALUX bioassay versus HRGC/MS. The two methods were highly correlated (R2 = 0.9815). Figure 2 depicts a dot plot comparing results from the two assays.

Figure 2. Correlation of CALUX® bioassay determination of TEQ versus HRGC/MS TEQ determinations in soil samples from a hazardous waste site. The study was performed with the corporation by contract with the US Environmental Protection Agency in a double blind format to compare measurements of TEQ contamination in soil samples by the CALUX® bioassay versus HRGC/MS. Results of both analytical procedures correlate highly (R2 = 0.9815).

These data demonstrate that the CALUX bioassay system provides a sensitive and less expensive system to rapidly evaluate remediation efforts of soils contaminated with dioxin-like chemicals. We are currently investigating if our analysis system can be modified into a kit format in which it could be used in the field to investigate contamination of remediation sites. This would be particularly valuable in that delays in receiving data can be a major cost factor in remediation of these hazardous sites.

References 1. Van den Berg, M., Birnbaum, L., Bosveld, A., Brunström, B., Cook, P., Feeley, M., Giesy, J., Hanberg, A., Hasegawa, R., Kennedy, S., Kubiak, T., Larsen, J., Van Leeuwen, R., Djien Liem, A., Nolt, C., Peterson, R., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M., Younes, M., Waern, F., and Zacharewski, T. (1998) Environ. Health Perspec. 106, 775. 2. Denison, M., Brouwer, A. and Clark, G. (1998). U.S. patent # 5,854,010. 3. Chu, M. and Clark, G. (2000). Patent application submitted. 4. US EPA Method 8290, September 1994. Acknowledgements The National Institutes of Environmental Health Sciences Grant (R44ES08372-02) supported portions of this research work. The authors would like to acknowledge the contributions of Bill Coakley of the US Environmental Protection Agencies Environmental Response Team in completing this research work.

CALUX® is a registered U.S. Trademark.