Grantee Research Project Results
Final Report: Oxidative Coupling as a Cause of Substituent Release from Aromatic Pollutants
EPA Grant Number: R823847Title: Oxidative Coupling as a Cause of Substituent Release from Aromatic Pollutants
Investigators: Bollag, Jean-Marc , Dec, Jerzy
Institution: Pennsylvania State University
EPA Project Officer: Packard, Benjamin H
Project Period: September 1, 1995 through October 1, 1998 (Extended to September 30, 1999)
Project Amount: $425,000
RFA: Exploratory Research - Chemistry and Physics of Water (1995) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Safer Chemicals
Objective:
Oxidative coupling is an important part of many significant environmental and industrial processes, such as humification, delignification, paper bleaching, and binding of xenobiotics to soil and their polymerization in aqueous environments. The objectives of this project were to study nondegradative reactions, such as binding and polymerization, in which anthropogenic chemicals react with various humic materials and result in the formation of oligomeric products or stable complexes with soil organic matter. The investigatory approach in this project relied on monitoring the rates of substituent release and substrate transformation during the oxidative coupling processes, and on the use of molecular spectroscopy (MS, IR, NMR) to elucidate the reaction pathways (Dec and Bollag, 1997).Summary/Accomplishments (Outputs/Outcomes):
Both binding and polymerization rely largely on the activity of certain enzymes from the class of oxidoreductases, which are classified as monophenol monooxygenases (tyrosinase), polyphenol oxidases (laccase), and peroxidases (Rugierro, et al., 1996). As a group, these enzymes are often referred to as phenoloxidases; their occurrence in soil is well documented (Gianfreda and Bollag, 1996; Dec and Bollag, 2000). Mineral colloids that are omnipresent in soil environments contribute greatly to the incorporation of xenobiotics into the soil organic matter (Bollag, et al., 1998; Najdja, et al., 2000). The influence of minerals is partly due to the fact that they possess catalytic properties and are able to mediate xenobiotic transformations (Huang and Bollag, 1998). Frequently, it is difficult to determine whether a pollutant is transformed in soil abiotically or enzymatically. Incorporation that results from oxidative coupling is believed to have a detoxification effect and has been long suggested as a means of decontamination (Bollag, et al., 1998). Obtaining information on the structure of the complexes formed through oxidative coupling reactions is thus of great environmental importance.Dehalogenation of Chlorinated Phenols During Oxidative Coupling. Decontamination processes involving oxidative coupling (polymerization and binding to humic acid) were accompanied by the dehalogenation of chlorinated substrates, such as 2-, 3-, and 4-chlorophenol (2-CP, 3-CP, and 4-CP), 2,4,5-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP), and analogously chlorinated anilines (Dec and Bollag, 1995; Park, et al., 2000b). The transformation was mediated by both oxidoreductive enzymes and inorganic catalysts. For horseradish peroxidase, the greatest substrate transformation (percent Tr) and dehalogenation (percent Cl) were observed for DCP (94 percent Tr and 24 percent Cl), followed by TCP, 4-CP, and 2-CP. The lowest rates were observed for 3-CP (10 percent Tr and 0.2 percent Cl), because the impaired electron rarely occupies the meta position. Similar patterns were observed for the Trametes laccase and birnessite ( -MnO2).
The transformation was preceded by oxidation of the substrates to free radicals. Once the free radicals were generated, coupling was completed without further involvement of the catalyst. Initially, various dimeric products were formed depending on the resonance form of the free radical; these dimeric products were subsequently involved in the formation of bigger oligomers (up to tetramers and pentamers) that precipitated out of the water solution. Dehalogenation patterns indicated that chlorine atoms were released (in the form of ions), if they were attached to carbon atoms at the ortho or para positions that hosted an impaired electron and were thus directly engaged in coupling.
The transformation patterns were reversed with tyrosinase, with the greatest rate of removal and dehalogenation observed for monochlorophenols, including the meta chlorinated 3-CP (98 percent Tr and 50.3 percent Cl) and limited dehalogenation of DCP (0.8 percent) and TCP (0.2 percent), because the tyrosinase-mediated oxidation of chlorophenols led to the formation of o-quinones and phenoxide anions, not the generation of free radicals, and oxidative coupling occurred by a different mechanism. The dehalogenation patterns revealed that the polymerization of chlorophenols in the presence of tyrosinase resulted from nucleophilic attack of the o-quinone by phenoxide anion that was formed during the first step of the tyrosinase-mediated oxidation of chlorophenols to o-quinones. In the case of 2-CP, for instance, there were two routes of nucleophilic substitution of chlorinated o-quinones by these ionized species: one with and one without chloride release. The rate of dehalogenation depended on the pH of the reaction mixture, enzyme activity, and incubation time. Reduced chloride release was observed at the nonoptimal pH of the reaction mixture, limited amount of catalysts, and short incubation times as a result of insufficient generation of free radicals under the unfavorable conditions.
Decarboxylation, Demethoxylation, and Demethylation Reactions. Evidence obtained during this project suggested that, in addition to halogens, the carboxyl and methoxyl groups can be released during oxidative coupling reactions. The release of the nonhalogen substituents was studied using 14C-labeled catechol, p-hydroxybenzoic acid (HBA), vanillic acid (VA), p-coumaric acid (CA), and ferulic acid (FA), which were incubated with various catalysts; the gaseous products were collected by entrapment. Radioactivity measurements showed that decarboxylation ranged from 17.8 to 64.7 percent of the initial radioactivity. The evolution of 14CO2 was observed in either case, whether the 14C-labeled carboxyl group was attached directly to the aromatic ring (HBA, VA) or to the aliphatic side chain (CA and FA). Practically no 14CO2 evolution was observed when the label was located in the aromatic ring or in the aliphatic side chains. Significant amounts of 14CO2 were released when uniformly labeled catechol was incubated with birnessite (10?25 percent) (Majcher, et al., 1999). Release of 14CO2 in this case represented another category of catalytic reaction leading to the cleavage of aromatic ring, and it was specific to catechol.
In demethoxylation experiments, phenolic substrates (2-, 3-, and 4-methoxyphenol, 2,6-dimethoxyphenol, syringic acid, and vanillic acid) were incubated at a concentration of 1.5 mM or 10 mM with horseradish peroxidase and laccase from Trametes villosa. The extent of demethoxylation ranged from 1.1 to 13.9 percent depending on the substrate, its concentration, and the type of enzyme. Unlike the electron-withdrawing carboxyl group, the methoxy moiety is electron-donating, and thus more strongly attached to the aromatic ring; therefore, the percentage of demethoxylation was considerably less than that of decarboxylation. In experiments involving 2-, 3-, and 4-methylphenol incubated with horseradish peroxidase, no demethylation occurred. As a result of their highly electron-donating nature, methyl groups are too strongly attached to aromatic rings to be released during oxidative coupling reactions.
Transformation of Xenobiotic Substrates in the Presence of Humic Materials. Both the aqueous and soil environments contain large amounts of natural phenols (e.g., ferulic acid, syringaldehyde, pyrogallol, hydroxybenzoic acid, or catechol) that originate from lignin decomposition and are major substrates for oxidative coupling reactions leading to the formation of humus. These humic constituents influenced the transformation of xenobiotics during oxidative coupling by competing for active sites on enzyme molecules or mineral surfaces, or by crosscoupling with the xenobiotic molecules (Roper, et al., 1995; Park, et al., 2000a). In experiments with peroxidase, laccase, and birnessite, the transformation of chlorophenols was considerably enhanced by the addition of syringaldehyde, which proved to be very reactive. Less enhancement was observed if 4-hydroxybenzoic acid was used, 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 resulted from different transformation mechanisms involving either o-quinone coupling (with tyrosinase) or free radical coupling (with peroxidase, laccase, or birnessite). Regardless of the catalyst used to mediate the reactions, chloroanilines underwent nucleophilic addition to quinones and quinone oligomers, which resulted from coupling of the humic constituents. Catechol, which readily forms quinones and quinone oligomers, was most efficient in enhancing these reactions. The study demonstrated that the outcome of the two-substrate reaction could be predicted if the mechanism of transformation was understood.
The enhancement of chloroaniline and chlorophenol transformation by humic constituents was only possible if the mechanism involving nucleophilic addition or free radical coupling was common to both substrates. Oxidative coupling reactions (mediated both by enzymes and abiotic catalysts) were evaluated in detail during this project (Naidja, et al., 1997, 1998, 1999). Additionally, using laccases of white rot fungi Trametes versicolor and Pycnoporus cinnabarinus, it was possible to precipitate the toxic pollutant 2-hydroxydibenzofuran in the form of nontoxic polymers that were identified by spectroscopic methods as dimeric and trimeric products (Jonas, et al., 1998). Laccase activity also was found to play an important role in the biodegradation of pentachlorophenol by Trametes versicolor (Ricotta, et al., 1996).
The pollutants and catalysts also were incubated with natural humic acid to further approximate the conditions in soil and aquatic environments (Park, et al., 2000a). The effect of humic acid on the transformation of chlorinated phenols and anilines differed considerably from the effect of the monomeric humic constituents, such as syringaldehyde, catechol, and 4-hydroxybenzoic acid. Humic acid combines a variety of components in its structure that can either enhance, reduce, or have no effect on the transformation of xenobiotic substrates; therefore, it was not surprising that the effect of humic acid on the transformation of chlorinated phenols and anilines was less pronounced than the effect of monomer humic constituents. Strong enhancements in substrate transformation observed for monomeric humic constituents often were diminished considerably in reactions with humic acid.
On the other hand, humic acid considerably enhanced many transformation reactions that were either unaffected, slowed down, or only slightly enhanced by humic monomers. Incubations of chlorinated phenols and anilines with oxidoreductive catalysts in the presence of humic acid led to polymerization 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, and the concentration and source of humic acid. At low humic acid concentrations (<10 mg/L), the transformation of 4-CP was enhanced, but at higher concentrations of humic acid (>10 mg/L), 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 was mainly bound to humic acid and formed few oligomers, whereas 4-CA was largely 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 was determined that nucleophilic binding of 4-CA largely depended on the availability of carbonyl and quinone groups in humic acid; therefore, the distribution of the transformed substrate between oligomers and organic matter greatly depended on the source of humic acid.
The results indicated that in soil environments treated with oxidoreductive catalysts, chlorinated phenols should be transformed mainly through binding to humic acid. Binding also may be a major transformation pathway in aquatic systems containing sufficient concentrations of dissolved humus (e.g., 50 mg/L, as in this project). According to the data obtained, chloroanilines should mostly undergo oligomerization with less binding, but chloroaniline binding may greatly increase at high concentrations of humic acid due to covalent binding through nucleophilic addition to carbonyl and quinone components of the organic matter. It can be expected that with long incubation times (several days, rather than 24 hours as in this study) considerable amounts of chloroanilines may be subject to further binding controlled by nucleophilic addition.
To elucidate the involvement of oxidative coupling in the binding of other chemicals, the herbicide bentazon was incubated with laccase from Trametes villosa and catechol (Kim, et al., 1997, 1998). Binding of bentazone to catechol was not a free radical reaction. 1H nuclear magnetic resonance (NMR) and 13C NMR analyses and mass spectrometry of the reaction products showed that catechol was oxidized to a semiquinone radical that was further oxidized to o-quinone, to which bentazone was bound via the protonated nitrogen of the heterocyclic ring. This strongly indicated that the xenobiotic thiodiazinones are incorporated into soil organic matter by nucleophilic addition to quinone-like substances during the humification processes.
Dehalogenation of Chlorinated Phenols and Anilines in the Presence of Humic Materials. The measurement of chloride ions in the reaction mixtures revealed that phenolic products of lignin decomposition can be involved indirectly in dehalogenation through crosscoupling with chlorinated substrates (Park, et al., 2000b). Dehalogenation also may result from oxidative coupling of chlorinated phenols and anilines to humic acid. The effect of humic substances on dehalogenation depended on the mechanism of oxidative coupling. In a free-radical reaction mediated by peroxidase, laccase, or birnessite ( -MnO2), syringaldehyde enhanced the dehalogenation of chlorinated phenols, but it did not enhance the dehalogenation of 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 chlorophenols, whereas syringaldehyde had little effect. Humic acid in most cases enhanced the dehalogenation of the chlorophenols, but 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 as a result of multiple crosscouplings to one molecule of the substrate. In view of this research, it is clear that humic substances may modify the dehalogenation patterns of chlorinated phenols and anilines in soil and aqueous environments. The effects of humic substances on chloride release resulted from their participation in oxidative coupling; therefore, the underlying mechanisms of dehalogenation did not change compared to reactions involving only the chlorinated substrates.
Journal Articles on this Report : 15 Displayed | Download in RIS Format
Other project views: | All 20 publications | 19 publications in selected types | All 15 journal articles |
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Type | Citation | ||
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Bollag JM, Dec J, Huang PM. Formation mechanisms of complex organic structures in soil habitats. Advances in Agronomy 1998;63:237-266. |
R823847 (1998) R823847 (Final) |
not available |
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Dec J, Bollag JM. Effect of various factors on dehalogenation of chlorinated phenols and anilines during oxidative coupling. Environmental Science & Technology 1995;29(3):657-663. |
R823847 (1998) R823847 (Final) |
not available |
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Dec J, Bollag JM. Determination of covalent and noncovalent binding interactions between xenobiotic chemicals and soil. Soil Science 1997;162(12):858-874. |
R823847 (1998) R823847 (Final) |
not available |
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Dec J, Bollag J-M. Phenoloxidase-mediated interactions of phenols and anilines with humic materials. Journal of Environmental Quality 2000;29(3):665-676. |
R823847 (Final) R826646 (1999) R826646 (Final) |
Exit |
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Jonas U, Hammer E, Schauer F, Bollag JM. Transformation of 2-hydroxydibenzofuran by laccases of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus and characterization of oligomerization products. Biodegradation 1997;8(5):321-328. |
R823847 (1998) R823847 (Final) |
not available |
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Kim JE, Fernandes E, Bollag JM. Enzymatic coupling of the herbicide bentazon with humus monomers and characterization of reaction products. Environmental Science & Technology 1997;31(8):2392-2398. |
R823847 (1998) R823847 (Final) |
not available |
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Kim JE, Wang CJ, Bollag JM. Interaction of reactive and inert chemicals in the presence of oxidoreductases: reaction of the herbicide bentazon and its metabolites with humic monomers. Biodegradation 1997;8(6):387-392. |
R823847 (1998) R823847 (Final) |
not available |
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Majcher EH, Chorover J, Bollag JM, Huang PM. Evolution of CO2 during birnessite-induced oxidation of C-14-labeled catechol. Soil Science Society of America Journal 2000;64(1):157-163. |
R823847 (Final) |
not available |
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Naidja A, Huang PM, Bollag JM. Activity of tyrosinase immobilized on hydroxyaluminum-montmorillonite complexes. Journal of Molecular Catalysis A-Chemical 1997;115(2):305-316. |
R823847 (1998) R823847 (Final) |
not available |
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Naidja A, Huang PM, Bollag JM. Comparison of reaction products from the transformation of catechol catalyzed by birnessite or tyrosinase. Soil Science Society of America Journal 1998;62(1):188-195. |
R823847 (1998) R823847 (Final) |
not available |
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Park JW, Dec J, Kime JE, Bollag JM. Effect of humic constituents on the transformation of chlorinated phenols and anilines in the presence of oxidoreductive enzymes or birnessite. Environmental Science & Technology 1999;33(12):2028-2034. |
R823847 (1998) R823847 (Final) |
not available |
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Park J-W, Dec J, Kim J-E, Bollag J-M. Dehalogenation of xenobiotics as a consequence of binding to humic materials. Archives of Environmental Contamination and Toxicology 2000;38:405-410. |
R823847 (Final) |
Exit |
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Park JW, Dec J, Kim JE, Bollag JM. Transformation of chlorinated phenols and anilines in the presence of humic acid. Journal of Environmental Quality 2000;29(1):214-220. |
R823847 (Final) |
not available |
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Ricotta A, Unz RF, Bollag JM. Role of laccase in the degradation of pentachlorophenol. Bulletin of Environmental Contamination and Toxicology 1996;57(4):560-567. |
R823847 (1998) R823847 (Final) |
not available |
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Roper JC, Sarkar JM, Dec J, Bollag JM. Enhanced enzymatic removal of chlorophenols in the presence of co-substrates. Water Research, December 1995;29(12):2720-2724. |
R823847 (1998) R823847 (Final) |
not available |
Supplemental Keywords:
water, soil, risk assessment, enzymes, enzymatic treatment, soil minerals, detoxification, bioremediation, cleanup, terrestrial systems, wastewaters, environmental chemistry, agriculture, industry, food processing., Scientific Discipline, Water, Physics, Environmental Chemistry, Chemistry, Engineering, Chemistry, & Physics, immunoassay, aromatic pollutants, oxidative coupling, anilinic compounds, dehalogenation, stochiometry, humification, phenolic compoundsRelevant Websites:
http://www.cos.comProgress 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.