Grantee Research Project Results

Final Report: Determination of Binding Interactions Between Xenobiotic Chemicals and Soil

EPA Grant Number: R826646
Title: 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 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 objectives of this research project were focused on: (1) immobilization of trinitrotoluene (TNT) in composted soil; (2) immobilization of chlorinated aromatics in various humic materials; (3) binding of trifluralin to soil; and (4) release of sequestered and irreversibly bound xenobiotics during incubation with fresh soil. Immobilization phenomena occurring in soil are of great environmental importance because they reduce the bioavailability and degradation of organic xenobiotics. This research integrated several experimental approaches that, in recent years, have shown great potential for investigating soil-bound chemicals. The compounds under investigation were labeled with ¹⁴C and ¹³C or ¹⁵N for analysis by radiocounting and ¹³C or ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. ¹⁹F also was used to evaluate xenobiotic binding by NMR spectroscopy.

Summary/Accomplishments (Outputs/Outcomes):

A clay loam soil from Pennsylvania, without a history of exposure to explosives, was incubated with 5 g kg^-1 of ¹⁵N -labeled 2,4,6-TNT and 200 Ci kg^-1 of ¹⁴C -TNT for 3 days, and then amended with compost at a 1:2 (mass/mass) soil to compost ratio. The compost was prepared by mixing 40 percent alfalfa hay, 40 percent grass hay, 10 percent spent mushroom compost, and 10 percent municipal biosolids. The mixture of soil and compost was inoculated with cattle manure, amended with glucose and starch, and incubated for 37 days under anoxic conditions. The anoxic incubation was followed by 26 days of forced aerobic incubation.

At the end of the aerobic growth phase, most of the radioactivity was associated with organic matter; only 8.7 percent could be extracted with water and methanol, but no TNT was present in the extracts as determined by high-performance liquid chromatography. The unextractable radioactivity was associated with humic acid (40.0±1.0 percent), fulvic acid (FA) (14.3±1.4 percent), and humin (28.2±0.5 percent). Radioactive materials associated with humic acid and humin were analyzed by solid-state ¹⁵N -NMR spectrometry. The NMR spectra indicated that nitro groups of TNT had been reduced to amino groups; these were subsequently involved in the formation associations, with soil organic matter consistent with covalent binding.

Chlorinated phenols and anilines are transformed and detoxified in soil through oxidative coupling reactions mediated by enzymes or metal oxides. The reactions may be influenced by humic constituents, such as syringaldehyde or catechol, which originate from lignin decomposition, and also are subject to oxidative coupling. In this study, the effect of humic constituents on xenobiotic transformation was evaluated in vitro based on the determination of unreacted chlorophenols and chloroanilines. In experiments with peroxidase, laccase, and birnessite (-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 can be explained by 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 seemed to undergo 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.

Incubations of chlorinated phenols and anilines with oxidoreductive catalysts (peroxidase, laccase, tyrosinase, and birnessite) in the presence of humic acid led to oligomerization of the substrates or their binding to organic matter. The effect of humic acid on the overall transformation depended on the substrate, type of catalyst, the concentration, and the source of humic acid. At low humic acid concentrations (less than 10 mg/L), the transformation of 4-chlorophenol (4-CP) was enhanced, but at higher concentrations of humic acid (more than 10 mM), no further enhancement occurred. The transformation of 4-chloroaniline (4-CA) was only slightly affected after the addition of humic acid. In experiments with ¹⁴C -labeled substrates, 4-CP mainly was bound to humic acid and formed few oligomers, whereas 4-CA largely was subject to oligomerization with less binding to humic acid. Binding and oligomerization of 4-CP did not change with increasing concentration of humic acid, but with 4-CA, binding increased and oligomerization decreased. It appears that nucleophilic binding of 4-CA depended largely on the availability of carbonyl and quinone groups in humic acid and, therefore, the distribution of the transformed substrate between oligomers and organic matter greatly depended on the source of humic acid.

Chlorinated phenols and anilines were transformed by oxidoreductive catalysts with release of chloride ions in both the absence and presence of humic substances (syringaldehyde, catechol, and humic acid). Dehalogenation of these xenobiotics resulted from oxidative coupling reactions occurring at the chlorinated sites of the substrates. The effect of humic substances on dehalogenation depended on the mechanism of oxidative coupling. In a free-radical reaction mediated by peroxidase, laccase, or -MnO₂, syringaldehyde enhanced the dehalogenation of most of the chlorinated phenols, but it did not enhance the dehalogenation of the chloroanilines. With catechol, which does not form free radicals, dehalogenation was reduced or remained the same for both the chlorophenols and the chloroanilines. However, in tyrosinase-mediated reactions controlled by nucleophilic addition, catechol enhanced the dehalogenation of most of the chlorophenols, whereas syringaldehyde had little effect. Humic acid, in most cases, enhanced the dehalogenation of the chlorophenols, but it had little effect on the dehalogenation of the chloroanilines. On a molar basis, changes in dehalogenation caused by humic substances were proportional to the respective changes in substrate transformation. Only syringaldehyde was capable of releasing disproportionately high amounts of chloride ions from chlorophenols, apparently as a result of multiple cross-couplings to one molecule of the substrate.

Trifluralin is a widely used herbicide for the control of broad leaf weeds in cotton, alfalfa, and soybeans. Previous research indicated that in wet soils of high organic matter content, trifluralin is likely to undergo binding and thus sustain significant losses in herbicidal activity. Bound residue formation may, at the same time, result in a delayed pollution problem. To evaluate trifluralin binding, experiments were undertaken in which the ¹⁴C -labeled herbicide was incubated for 7 weeks with four soils of different organic matter contents under anaerobic metabolic conditions enhanced by flushing with N2 gas. The presence of fluorine (¹⁹F) in the trifluralin molecule was used to obtain structural information by ¹⁹F NMR spectroscopy. As determined by radiocounting, trifluralin binding ranged between 10 and 53 percent of the initial ¹⁴C depending on soil tested. NMR analyses of the methanol-extractable and unextractable ¹⁹F suggests that soil binding largely may involve reduced metabolites of the herbicide. Incubation of trifluralin with zero valent iron produced a product (Fe-TR) that tentatively was identified by mass spectrometry as 2,6-diaminotrifluralin. This product, and one of the standard metabolites (1,2-diaminotrifluralin or TR6), spontaneously formed covalent bonds with FA, as indicated by the appearance of new resonances in the ¹⁹F NMR spectra taken periodically over a 3-week contact time. No unaltered Fe-TR or TR6 could be recovered by chloroform extraction of these complexes. At short contact times, TR6 and Fe-TR formed weak physical bonds with FA, as the respective spin-spin relaxation times (T1) decreased from 1300-1831 msec for TR6 or Fe-TR analyzed, in the absence of FA to 150-410 msec, for TR6/FA or Fe-TR/FA mixtures. In general, the results indicated that trifluralin immobilization involved a variety of mechanisms (covalent binding, adsorption, sequestration) and with time became increasingly stable.

Concerns exist about pollutants incorporated into soil organic matter being released on 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 (DCP), 2,4,6- TNT, or cyprodinil] were mixed with fresh uncontaminated soils and incubated for 141 days under forced aeration. The incubations were monitored for evolution of ¹⁴C O₂ and volatile xenobiotics. Soil samples taken at specific times were extracted with methanol, and fractionated into FA, humic acid, and humin. The distribution of radioactivity in specific fractions was determined by liquid scintillation counting (LSC). The experiments showed some decrease in bound radiocarbon with time because of release. Small amounts of this radioactivity were found in methanol extracts. The release coincided with the evolution of ¹⁴C O₂, indicating the occurrence of microbial degradation. The amounts of radioactivity present in methanol extracts (2 to 25 percent) and ¹⁴C O₂ (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, this will not occur in mass quantities to cause toxic effects.

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

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Other project views:	All 5 publications	5 publications in selected types	All 2 journal articles

Publications

Type	Citation	Project	Document Sources
Journal Article	Dec J, Bollag J-M. Phenoloxidase-mediated interactions of phenols and anilines with humic materials. Journal of Environmental Quality 2000;29(3):665-676.	R826646 (1999) R826646 (Final) R823847 (Final)	Abstract: Access DL-Abstract Exit

Supplemental Keywords:

soil, risk assessment, effects, bioavailability, metabolism, organism, enzymes, chemicals, toxics, bacteria, intermediates, ecosystem, terrestrial, remediation, bioremediation, cleanup, environmental chemistry, analytical, measurement methods, agriculture, industry., 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 methods

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Progress and Final Reports:

Original Abstract

1999 Progress Report

2000 Progress Report