Grantee Research Project Results
2000 Progress Report: Determination of Binding Interactions Between Xenobiotic Chemicals and Soil
EPA Grant Number: R826646Title: Determination of Binding Interactions Between Xenobiotic Chemicals and Soil
Investigators: Bollag, Jean-Marc , Dec, Jerzy
Institution: Pennsylvania State University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1998 through September 30, 2001
Project Period Covered by this Report: October 1, 1999 through September 30, 2000
Project Amount: $408,775
RFA: Exploratory Research - Environmental Chemistry (1998) RFA Text | Recipients Lists
Research Category: Sustainable and Healthy Communities , Land and Waste Management , Air , Safer Chemicals
Objective:
The purpose of this project is to evaluate the mechanisms of pollutant immobilization in terrestrial systems. Immobilization phenomena occurring in soil are of great environmental importance because they reduce the bioavailability and degradation of organic xenobiotics. This research integrates several experimental approaches that, in recent years, have shown great potential for investigating soil-bound chemicals. The compounds under investigation are labeled with 14C and 13C or 15N for analysis by radiocounting and 13C or 15N NMR spectroscopy. In this project period, the research focused on three topics: (1) composting of 2,4,6-trinitrotoluene (TNT) polluted soil, (2) immobilization of chlorinated aromatics in various humic materials, and (3) release of sequestered and irreversibly bound xenobiotics during incubation with fresh soil.
Progress Summary:
A local clay loam soil without a history of exposure to explosives was mixed with 15N- and 14C-labeled TNT and amended with compost consisting of alfalfa hay, grass hay, spent mushroom compost, and municipal biosolids. The mixture was inoculated with methanogens from cattle manure, and incubated for 37 days under anaerobic conditions and then for 26 days under aerobic incubation. The incubation was monitored for gas production (H2, CO2, and CH4) and 14CO2 evolution. On the completion of the aerobic phase, the composted soil was processed by solvent extraction and fractionation, followed by the analysis of the fractions by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid scintillation counting (LSC), and liquid or solid state 15N NMR.
At the end of composting, most of the TNT was associated with organic matter; only 6.9 percent of the added radioactivity could be extracted with water (together with soluble organic matter) and another 1.8 percent with methanol; no unaltered TNT, however, was present in the extracts. Fractionation of the extracted solids revealed that unextractable radioactivity was associated with humic acid (40.0±1.0 percent), fulvic acid (14.3±1.4 percent), and humin (28.2±0.5 percent). The spectrum for the humic acid sample containing bound 15N TNT, in addition to the same background signals, showed a large peak in the region of 60-90 ppm that was attributed to the presence of protonated aromatic amines originating from TNT. Another peak at 130 ppm was attributed to amides and/or tertiary amines originating from the bound residue. The 15N-NMR spectrum of the humin showed broad resonances in the range 40-80 ppm indicating the presence of TNT residues incorporated in the form of primary and secondary aromatic amines.
Selected chlorinated phenols and anilines (some labeled with 14C) were incubated with various enzymes and birnessite (d-MnO2) in the presence or absence of different humic constituents or humic acids and the reaction mixtures were analyzed by HPLC. The distribution of reaction products was determined by LSC following the acidification and centrifugation of the reaction mixtures.
In experiments with peroxidase, laccase, and birnessite (d-MnO2), the transformation of most chlorophenols was considerably enhanced by the addition of syringaldehyde. Less enhancement was observed using 4-hydroxybenzoic acid, and the addition of catechol resulted in a reduction of most transformations. The opposite was observed in experiments with tyrosinase, in which case catechol caused considerable enhancement of chlorophenol transformation. The varying effect of catechol was due to different transformation mechanisms involving either o-quinone coupling (with tyrosinase) or free radical coupling (with peroxidase, laccase, or birnessite). Regardless of the agent used to mediate the reactions, chloroanilines underwent nucleophilic addition to quinone oligomers that resulted from coupling of the humic constituents. Catechol, which readily forms quinones and quinone oligomers, was most efficient in enhancing these reactions.
Incubation of chloroaromatics with humic acid in the presence of various oxidoreductases or inorganic reagents led to binding of the pollutants to organic matter. At the same time, some pollutants were subject to oligomerization. At low humic acid concentrations, chlorophenol transformation was enhanced, but stopped to increase at higher concentrations of humic acid. Chloroanilines were largely oligomerized with less binding, but binding increased at higher concentrations of humic acid. The distribution of the transformed substrate between oligomers and organic matter greatly depended on the source of humic acid.
Concerns exist that pollutants incorporated into soil organic matter may be released upon exposure to microbial activity, hydrolytic conditions, or other environmental factors that may disrupt the bonds formed. To test this possibility, various soil materials (whole soil, whole soil extracted with methanol, humic acid, or humin) containing free and/or bound residues of 14C-labeled xenobiotics (2,4-dichlorophenol, TNT, or cyprodinil) were mixed with fresh uncontaminated soils and incubated for 141 days under forced aeration. The incubations were monitored for evolution of 14CO2 and volatile xenobiotics. Soil samples taken at specific times were extracted with methanol, and fractionated into fulvic acid, humic acid, and humin. The distribution of radioactivity in specific fractions was determined by LSC.
The experiments showed some decrease in bound radiocarbon with time due to release. Small amounts of this radioactivity were found in methanol extracts. The release coincided with the evolution of 14CO2 indicating the occurrence of microbial degradation. The amounts of radioactivity present in methanol extracts (2 to 25 percent) and 14CO2 (5 to 40 percent) differed considerably depending on the compound under investigation. The results indicate that after incorporation into humic materials, the pollutant is practically indistinguishable from soil organic matter. It can be assumed that mineralization of the bound residue would occur at a rate similar to that of mineralization of natural humus. Even if some covalently bound molecules are released and become bioavailable, it will not occur in mass quantities to cause toxic effects.
Future Activities:
Future work will include the derivatization of xenobiotic/organic matter complexes by silylation and methylation followed by 13C/15N-NMR spectroscopy and high-performance size exclusion chromatography. Xenobiotics sequestered in the remote site or soil organic matter will be released upon derivatization and become available for the determination of their chemical nature. Both methylation and silylation will result in fragmentation of humic materials to make them soluble in organic solvents. This, in turn, will improve the resolution HPLC chromatograms and NMR spectra. The methanol- and NaOH-extracted soil samples generated in all experiments are stored in the freezer for tests on the bioavailability of bound chemicals to be conducted in an aerobic respirometer system.
Journal Articles:
No journal articles submitted with this report: View all 5 publications for this projectSupplemental Keywords:
risk assessment, ecological effects, chemicals, toxic substances, organics, bound residues, aging in soil, terrestrial system, soil organic matter, pollution prevention, cleanup, detoxification, environmental chemistry, analytical methods., RFA, Scientific Discipline, Toxics, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Environmental Chemistry, Ecosystem/Assessment/Indicators, Chemical Mixtures - Environmental Exposure & Risk, Contaminated Sediments, Remediation, pesticides, Ecological Effects - Environmental Exposure & Risk, Fate & Transport, Ecological Risk Assessment, sediment treatment, degradation of organic pollutants, fate and transport, risk assessment, hydrocarbon, xenobiotics, contaminant transport, sediment transport, sorbed contaminants, contaminated sediment, chemical contaminants, PAH, chemical transport, chemical kinetics, ecological assessment, assessment methodsRelevant Websites:
http://www.personal.psu.edu/jxb10/
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http://myprofile.cos.com/jdec10
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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.