2002 Progress Report: The Use of Mussel Gene Expression Profiles to Determine the Pollutant Decontamination Efficacy of Solvated Electron Reduction Technology - An Interdisciplinary Collaboration

EPA Grant Number: R829421E01
Title: The Use of Mussel Gene Expression Profiles to Determine the Pollutant Decontamination Efficacy of Solvated Electron Reduction Technology - An Interdisciplinary Collaboration
Investigators: Wang, Shiao Y. , Biesiot, Patricia M. , Pittman, Charles U.
Institution: University of Southern Mississippi , Mississippi State University - Main Campus
EPA Project Officer: Chung, Serena
Project Period: May 8, 2002 through May 7, 2004 (Extended to May 7, 2006)
Project Period Covered by this Report: May 8, 2002 through May 7, 2003
Project Amount: $228,750
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (2001) RFA Text |  Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)


The objectives of this research project are to: (1) improve and better understand the use of solvated electron reduction chemistry using (Na/NH3) to remediate polynuclear aromatic hydrocarbon (PAH)-contaminated soils and sludge; and (2) develop a suite of molecular biomarkers of chemical stress response using freshwater mussels as the model organism. These are complimentary objectives because solvated electron reductions in Na/NH3 produce a complex variety of reduced products from PAH mixtures. The PAHs themselves are totally converted to these products and are absent from the remediated soils. The question becomes: is it acceptable to leave these byproducts in the soil? Because specific genes are activated to meet metabolic needs, the expression pattern of genes in mussels in a healthy environment differs from that of genes in mussels in a polluted environment. By comparing the gene expression profile of mussels exposed to treated and untreated PAHs to that of control mussels, it will be possible to determine whether the toxicity of PAHs has been reduced or eliminated as a result of solvated electron reduction. Thus, gene expression is being studied as an early warning system to evaluate the efficacy of the Na/NH3 remediation of PAHs.

Progress Summary:

The reductions of eight individual PAHs have been carried out in Na/NH3 by Dr. Pittman's laboratory at the Mississippi State University as planned. These compounds include naphthalene, anthracene, phenanthrene, acenaphthylene, fluoranthene, pyrene, chrysene, and 1,2-benzanthracene. The products were identified in the case of naphthalene and anthracene, but the product distributions quickly become very complex as more complicated PAHs, or mixtures of them, are used. Dimeric products were formed by dimerization of radical anion intermediates when the concentration of proton donors was low and this increases product complexity. The key observation is that the PAHs disappear entirely (down to gas chromatography detection limits), while a plethora of other products are produced when PAH mixtures are treated with Na/NH3. Both untreated and reduced PAHs—including naphthalene, anthracene, phenanthrene, fluoranthene, pyrene, and chrysene—have been shipped to Dr. Wang's laboratory at the University of Southern Mississippi for exposure to mussels and molecular biology studies.

Soils, purposely contaminated with 10,000 ppm, 5,000 ppm, 1,000 ppm, and 100 ppm amounts of equimolar mixtures of the PAHs listed above, were treated with excess Na/NH3, and conditions were developed where no residual PAHs were detected after soil extractions with toluene. Other conditions were developed where the residual contents of individual PAHs were below 5 ppm. Within these samples, the complex mixture of reduction products was not specifically identified. Can this complex reduction product mixture be left in the remediated soil without harmful effects on the environment? This becomes the central focus of our research. Results to date indicate that solvated electron reduction technology can be an effective method to destroy PAHs.

A second thrust has been to use Na/NH3 solutions to reduce polychlorinated biphenyls (PCBs), pentachlorophenols (PCPs), and chlorinated solvents such as tetrachloroethylene. This has been performed on neat samples and on soil samples contaminated with these pollutants. Soils, contaminated with PCBs, PCPs, or halogenated pesticides (such as Mirex™), will be remediated by Na/NH3, and the remediated soils will be provided, on demand, to Dr. Wang's group for his use in investigating the exposure of fish and mussels to water leaching through these remediated soils.

Phenanthrene was used as the model PAH in exposure studies to isolate genes whose expressions are altered in response to PAH exposure. In addition to mussels, a fish (the Gulf killifish Fundulus grandis) was included in the study. Because of initial difficulties in obtaining mussels, research using the Gulf killifish is at a more advanced stage compared to work with mussels. Two different complementary DNA (cDNA) subtraction technologies were used to construct two subtracted cDNA libraries using fish messenger ribonucleic acid (mRNA). A subtraction library using bivalve mRNA currently is in progress. From the fish subtraction libraries, a total of 1,330 clones were screened using dot blots to identify cDNAs that were either up- or downregulated in response to phenanthrene exposure. Among the 41 cDNA clones of interest, the expression of 29 appeared elevated, while the remaining 12 appeared depressed. Nineteen of the upregulated cDNAs have been sequenced and PCR primers of 12 unique clones have been designed for real-time (RT)-polymerase chain reaction (PCR) expression analysis.

Among the 12 cDNAs, the expression of 5 has been shown by real-time reverse transcriptase-polymerase chain reaction to be altered in the liver of fish exposed to phenanthrene. The expression of 4 cDNAs in exposed fish increased by 4.6 to 8.9 fold over control fish, whereas the expression of one was reduced by 2.2 fold. One of the cDNAs has been identified using DNA sequence comparisons as vitellogenin, while four remain unidentified. The expression of vitellogenin was elevated by 4.6 fold in the liver of fish exposed to phenanthrene.

In conclusion, solvated electron reduction technology using Na/NH3 has been shown to be an effective method to destroy PAHs and may prove to be a cost-effective methodology to remediate contaminated soil. Research is in progress to determine whether remediated soil can be left onsite. Five cDNAs that appear useful as biomarkers of PAH exposure in fish have been isolated. Research is in progress to isolate biomarkers of exposure in mussels, as well as the isolation of additional biomarkers in fish.

Future Activities:

Future activities involve the Mississippi State University group preparing specific samples on demand for biological testing at the University of Southern Mississippi. This work will concentrate on the residues from individual PAHs and PAH mixtures, as well as making remediated soil samples for leaching so that effluent waters can be tested on freshwater bivalves and fish to study alterations in gene expression patterns. Work will continue as planned on the development of molecular biomarkers, and no change in project schedule is anticipated. A new subtracted cDNA library using bivalve mRNA is under construction and will be screened shortly using protocols developed for screening the fish subtracted libraries. The screening of a second subtracted fish cDNA library currently is in progress, and additional cDNA clones representing differentially expressed genes will be isolated and characterized. The expression patterns of these cDNAs in fish and mussels exposed to treated and untreated PAHs, as well as in fish and mussels exposed to effluents leached from PAH-contaminated soil before and after treatment, will be compared to determine efficacy of solvated electron reduction technology in eliminating the toxic nature of PAHs.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 6 publications 4 publications in selected types All 2 journal articles
Type Citation Project Document Sources
Journal Article Pittman Jr CU, He JB. Dechlorination of PCBs, CAHs, herbicides and pesticides neat and in soils at 25°C using Na/NH3. Journal of Hazardous Materials 2002;92(1):51-62. R829421E01 (2002)
R826180 (Final)
not available

Supplemental Keywords:

polynuclear aromatic hydrocarbon, PAH, polychlorinated biphenyl, PCB, fish, mussels, polymerase chain reaction, PCR, subtractive cloning, comparative DNA, cDNA, cDNA library, genetic biomarkers, environmental chemistry, remediation, solvated electron reduction, southeast, Gulf Coast, water, groundwater, soil, estuary, leachate, stressor., Scientific Discipline, Water, Waste, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Geochemistry, Contaminated Sediments, Analytical Chemistry, Fate & Transport, Ecology and Ecosystems, fate and transport, electrokinetics, contaminant transport, bioavailability, contaminated sediment, desorption kinetics, PAH, electron reduction technology, nitrate compounds, bioremediation of soils, chemical kinetics, munitions residues, mussel gene expression profiles, water quality, TNT, sorption experiments, munitions, contaminant transport models

Progress and Final Reports:

Original Abstract
  • 2003 Progress Report
  • 2004 Progress Report
  • Final Report