The Science of the Total Environment, 123/124 (1992) 205-217 Elsevier Science Publishers B.V., Amsterdam
205
Biological and chemical interactions of pesticides with soil organic matter J e a n - M a r c Bollag a, C a r l a J. M y e r s a a n d R o b e r t D. M i n a r d b
aLaboratory of Soil Biochemistry, The Pennsylvania State University, University Park, PA 16802, USA bDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA ABSTRACT There is little doubt that organic matter plays a major role in the binding of pesticides in soil, and that this phenomenon is usually the most important cause for interaction of pesticides in the soil environment. Fulvic or humic acids are the chemicals most commonly involved in the binding interactions. Binding can occur with the original pesticide or a transformation product, the reaction being caused by abiotic agents or biotic agents (microbial or plant enzymes). The reactions or processes involved appear to be the same as those responsible for the formation of humic substances, i.e. for the humification process. Binding of pesticides to organic matter can occur by sorption (Van der Waal's forces, hydrogen bonding, hydrophobic bonding), electrostatic interactions (charge transfer, ion exchange or ligand exchange), covalent bonding or combinations of these reactions. Our investigation focused primarily on the binding of substituted phenols and aromatic amines to humus monomers and humic substances. In model reactions, we demonstrated the formation of covalent linkages between pesticides and humus constituents and fulvic or humic acids in the presence of phenol oxidases or clay minerals. With chlorinated phenols and carboxylic acids, it was possible to isolate and identify cross-coupling products and to elucidate the site and type of binding. The binding of chlorinated phenols to humic substances was determined by using 14C-labelled chemicals and by measuring the uptake of radioactivity by the humic material. These experiments provide a base for explaining the formation of bound residues in certain cases and for assuming the toxic potential of the immobilized pollutants.
Key words: pesticides; humic substances; bound residues; chlorophenols; covalent binding
INTRODUCTION As a result o f c u r r e n t agricultural a n d industrial practices, the environm e n t has b e c o m e c o n t a m i n a t e d with excessive quantities o f m a n - m a d e chemicals. M a n y o f these x e n o b i o t i c s are highly resistant to n a t u r a l degradation processes a n d t e n d to a c c u m u l a t e in soil, g r o u n d w a t e r a n d waste water. T h e r e is great c o n c e r n o v e r the p o t e n t i a l toxic, carcinogenic a n d t e r a t o g e n i c
206
J.-M. BOLLAG ETAL.
nature of these compounds and extensive efforts are now being taken towards cleaning up polluted sites. However, many of the physical-chemical methods presently available for decontamination purposes involve largescale excavation or long-term treatment, either of which may be prohibitively costly and/or ineffective. Consequently, there is an immediate need to develop alternative methods for the in situ decontamination of soil and aquatic environments. One promising approach involves the binding of pollutants to soil organic matter. Since many pesticides and their degradation products resemble humic precursors, these compounds are often incorporated into soil organic matter during humification. It has been suggested that this naturally occurring process can be exploited as a means of neutralizing toxic compounds. Binding of xenobiotics to humus has three important consequences: (1) the amount of material available to interact with the biota is reduced; (2) the toxicity of the compound is diminished; and (3) xenobiotics can be incorporated into insoluble precipitates, therefore reducing the leaching and transportability of these compounds. This review focuses on the use of oxidative coupling to bind pesticides to humus. Information will be presented on the binding mechanism, the chemical nature of bound substances and the ultimate fate of these chemicals in the environment. INTERACTION BETWEEN PESTICIDES A N D SOIL HUMIC SUBSTANCES
A significant portion of pesticides applied in agriculture have been found to remain associated with the soil over long periods of time. Thus, it is important to ascertain the toxicity and stability of these chemicals in the soil environment. Soil itself plays a major role in determining the fate of chemical pollutants. In the soil, xenobiotics may be transformed by biotic or abiotic processes. Ideally, xenobiotics are mineralized to release carbon dioxide, water, and mineral elements. However, varying intermediate products may also form and often these intermediates are toxic pollutants in their own right. Additionally, pesticides and their metabolites can be transported through the soil by the processes of leaching, bioconcentration and volatilization. Xenobiotics directly interact with soil through the processes of adsorption and covalent bond formation. Adsorption occurs via several mechanisms including Van der Waals forces, ion exchange, hydrogen bonding, chargetransfer complexation, and hydrophobic interactions (Khan, 1978; Pignatello, 1989). The nature and strength of adsorption depend on the chemical and structural characteristics of the xenobiotic and this interaction is particularly sensitive to changes in the environment. For example, residues
BIOLOGICAL AND CHEMICAL INTERACTIONS OF PESTICIDES WITH SOIL ORGANIC MA'I'rER
207
ionically bound to soil organic matter are frequently released due to fluctuations in the pH or cation concentration of the soil. When compounds are bound via adsorption processes, a portion of the material remains available to interact with the biota, suggesting that the process of adsorption is reversible. However, abundant evidence indicates that with time adsorbed residues become more stable and more resistant to extraction and microbial degradation (Calderbank, 1989). This may be due to a redistribution of the xenobiotic from a weaker to a stronger site and/or to covalent bond formation between xenobiotics and soil. The most persistent complexes result from the direct covalent binding of xenobiotics to soil humic matter or clay. The pesticides which are most likely to bind covalently to soil have chemical functionalities similar to the components of humus. Humic material is derived from the remains of decomposing plants, animals and microorganisms and is composed primarily of humic and fulvic acids. These molecules are polymeric and consist of an aromatic core containing mono-, di- and polyphenolic subunits. In fact, phenolic compounds account for up to 30% by weight of the humic polymer. It is important to note that humic substances occur not only in the major fraction of soil, but also in sewage effluents, peat, coal and lignite and in river, marine and lake waters and sediments. Thus, pesticides that structurally resemble phenolic compounds can covalently bind to humus and these complexes can be found in all of the above environments. Covalently bound residues are extremely stable and are characterized by their resistance to acid or base hydrolysis, thermal treatment and microbial degradation (Hsu and Bartha, 1974; Bollag et al., 1978; Helling and Krivonak, 1978; Saxena and Bartha, 1983). OXIDATIVE COUPLING REACTIONS
The constituents of humus are linked together through various reactions such as an oxidative coupling reaction in a process referred to as humification. It is now believed that oxidative coupling reactions are also responsible for the incorporation of organic pesticides and their degradation products (e.g. anilines and phenols) into humus. Oxidative coupling is the process by which phenols, anilines and other compounds are linked together after oxidation by an enzyme or chemical agent. In the first step of the reaction, susceptible organic compounds are oxidized to form unstable free radicals. The free radicals go on to react with other molecules yielding free radical intermediates that can further react, ultimately leading to the formation of polymers. The reaction results in the formation of C - C and C - O bonds between phenolic species and C - N and N - N bonds between aromatic amines (Sjoblad and Bollag, 1981). With
208
J.-M. BOLLAG ET AL.
respect to phenolic cross-coupling, the initial step usually requires the removal of an electron and a hydrogen ion from the hydroxyl group, which generates a phenoxy radical. The free radical intermediate then couples at positions ortho and para to the hydroxyl group (the meta position is nonreactive) to yield a dimer. Phenolic dimers can be further oxidized resulting in the formation of oligomeric and polymeric products. Conversely, if the oxidation potential of the enzyme or chemical is high enough, C - C coupled dimers can be further oxidized to form extended quinones. Oxidative coupling reactions are mediated by a number of biological and abiotic catalysts, including plant and microbial enzymes, inorganic chemicals, clay and soil extracts (Bollag, 1983; Shindo and Huang, 1984; Wang et al., 1986). Coupling may also occur spontaneously in the presence of oxygen at neutral and alkaline pH. The enzymes which catalyze oxidative coupling reactions have been divided into two classes, the peroxidases and the monophenol monooxygenases. All peroxidases contain an iron porphyrin ring and require either hydrogen peroxide or alkyl peroxide as a coenzyme. The monophenol monooxygenases are further subdivided into two distinct groups, tyrosinases and laccases. These two enzyme groups require molecular oxygen, but no coenzyme, for activity. Two types of reactions are catalyzed by tyrosinase: (1) the hydroxylation of monophenol and (2) the oxidation of o-diphenols to o-quinones (Sjoblad and Bollag, 1981). Free radicals are not generated during reactions catalyzed by tyrosinase. However, in alkaline environments, polymeric molecules may form if quinone products react with one another via autooxidation. The laccases have a broad substrate specificity and are able to oxidize mono-, di-, and polyphenols, as well as aromatic amines. Laccases readily transform phenolic substrates to yield the corresponding anionic free radical which may go on to couple with other molecules and generate polymers. For general polymerization purposes, the laccases may prove to be the most beneficial because, unlike the peroxidases, they do not require an expensive coenzyme, and because they yield highly reactive intermediates. OXIDATIVE COUPLING OF PHENOLS AND ANILINES The reaction products generated from the coupling of xenobiotics to humic matter are highly heterogeneous and complex and therefore quite difficult to analyze. In order to elucidate the mechanism of coupling, model in vitro systems were designed in which aromatic substrates were incubated with extracellular microbial enzymes. The reaction products were separated by thin-layer chromatography (TLC) or by high-performance liquid chromatography (HPLC) and then characterized by mass spectroscopy (MS). In this way, we examined the enzymatic polymerization of a naturally
(CIRHIsQ9)
OCH3
OCH3
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OC~3i OH
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0
m/z 290 (C15H1406)
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m/z 306 (CI6HI806)
I
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.OCH3
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CI13 ~ _•~ 7 o
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m/z 444 (C23H2409)
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205
Biological and chemical interactions of pesticides with soil organic matter J e a n - M a r c Bollag a, C a r l a J. M y e r s a a n d R o b e r t D. M i n a r d b
aLaboratory of Soil Biochemistry, The Pennsylvania State University, University Park, PA 16802, USA bDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA ABSTRACT There is little doubt that organic matter plays a major role in the binding of pesticides in soil, and that this phenomenon is usually the most important cause for interaction of pesticides in the soil environment. Fulvic or humic acids are the chemicals most commonly involved in the binding interactions. Binding can occur with the original pesticide or a transformation product, the reaction being caused by abiotic agents or biotic agents (microbial or plant enzymes). The reactions or processes involved appear to be the same as those responsible for the formation of humic substances, i.e. for the humification process. Binding of pesticides to organic matter can occur by sorption (Van der Waal's forces, hydrogen bonding, hydrophobic bonding), electrostatic interactions (charge transfer, ion exchange or ligand exchange), covalent bonding or combinations of these reactions. Our investigation focused primarily on the binding of substituted phenols and aromatic amines to humus monomers and humic substances. In model reactions, we demonstrated the formation of covalent linkages between pesticides and humus constituents and fulvic or humic acids in the presence of phenol oxidases or clay minerals. With chlorinated phenols and carboxylic acids, it was possible to isolate and identify cross-coupling products and to elucidate the site and type of binding. The binding of chlorinated phenols to humic substances was determined by using 14C-labelled chemicals and by measuring the uptake of radioactivity by the humic material. These experiments provide a base for explaining the formation of bound residues in certain cases and for assuming the toxic potential of the immobilized pollutants.
Key words: pesticides; humic substances; bound residues; chlorophenols; covalent binding
INTRODUCTION As a result o f c u r r e n t agricultural a n d industrial practices, the environm e n t has b e c o m e c o n t a m i n a t e d with excessive quantities o f m a n - m a d e chemicals. M a n y o f these x e n o b i o t i c s are highly resistant to n a t u r a l degradation processes a n d t e n d to a c c u m u l a t e in soil, g r o u n d w a t e r a n d waste water. T h e r e is great c o n c e r n o v e r the p o t e n t i a l toxic, carcinogenic a n d t e r a t o g e n i c
206
J.-M. BOLLAG ETAL.
nature of these compounds and extensive efforts are now being taken towards cleaning up polluted sites. However, many of the physical-chemical methods presently available for decontamination purposes involve largescale excavation or long-term treatment, either of which may be prohibitively costly and/or ineffective. Consequently, there is an immediate need to develop alternative methods for the in situ decontamination of soil and aquatic environments. One promising approach involves the binding of pollutants to soil organic matter. Since many pesticides and their degradation products resemble humic precursors, these compounds are often incorporated into soil organic matter during humification. It has been suggested that this naturally occurring process can be exploited as a means of neutralizing toxic compounds. Binding of xenobiotics to humus has three important consequences: (1) the amount of material available to interact with the biota is reduced; (2) the toxicity of the compound is diminished; and (3) xenobiotics can be incorporated into insoluble precipitates, therefore reducing the leaching and transportability of these compounds. This review focuses on the use of oxidative coupling to bind pesticides to humus. Information will be presented on the binding mechanism, the chemical nature of bound substances and the ultimate fate of these chemicals in the environment. INTERACTION BETWEEN PESTICIDES A N D SOIL HUMIC SUBSTANCES
A significant portion of pesticides applied in agriculture have been found to remain associated with the soil over long periods of time. Thus, it is important to ascertain the toxicity and stability of these chemicals in the soil environment. Soil itself plays a major role in determining the fate of chemical pollutants. In the soil, xenobiotics may be transformed by biotic or abiotic processes. Ideally, xenobiotics are mineralized to release carbon dioxide, water, and mineral elements. However, varying intermediate products may also form and often these intermediates are toxic pollutants in their own right. Additionally, pesticides and their metabolites can be transported through the soil by the processes of leaching, bioconcentration and volatilization. Xenobiotics directly interact with soil through the processes of adsorption and covalent bond formation. Adsorption occurs via several mechanisms including Van der Waals forces, ion exchange, hydrogen bonding, chargetransfer complexation, and hydrophobic interactions (Khan, 1978; Pignatello, 1989). The nature and strength of adsorption depend on the chemical and structural characteristics of the xenobiotic and this interaction is particularly sensitive to changes in the environment. For example, residues
BIOLOGICAL AND CHEMICAL INTERACTIONS OF PESTICIDES WITH SOIL ORGANIC MA'I'rER
207
ionically bound to soil organic matter are frequently released due to fluctuations in the pH or cation concentration of the soil. When compounds are bound via adsorption processes, a portion of the material remains available to interact with the biota, suggesting that the process of adsorption is reversible. However, abundant evidence indicates that with time adsorbed residues become more stable and more resistant to extraction and microbial degradation (Calderbank, 1989). This may be due to a redistribution of the xenobiotic from a weaker to a stronger site and/or to covalent bond formation between xenobiotics and soil. The most persistent complexes result from the direct covalent binding of xenobiotics to soil humic matter or clay. The pesticides which are most likely to bind covalently to soil have chemical functionalities similar to the components of humus. Humic material is derived from the remains of decomposing plants, animals and microorganisms and is composed primarily of humic and fulvic acids. These molecules are polymeric and consist of an aromatic core containing mono-, di- and polyphenolic subunits. In fact, phenolic compounds account for up to 30% by weight of the humic polymer. It is important to note that humic substances occur not only in the major fraction of soil, but also in sewage effluents, peat, coal and lignite and in river, marine and lake waters and sediments. Thus, pesticides that structurally resemble phenolic compounds can covalently bind to humus and these complexes can be found in all of the above environments. Covalently bound residues are extremely stable and are characterized by their resistance to acid or base hydrolysis, thermal treatment and microbial degradation (Hsu and Bartha, 1974; Bollag et al., 1978; Helling and Krivonak, 1978; Saxena and Bartha, 1983). OXIDATIVE COUPLING REACTIONS
The constituents of humus are linked together through various reactions such as an oxidative coupling reaction in a process referred to as humification. It is now believed that oxidative coupling reactions are also responsible for the incorporation of organic pesticides and their degradation products (e.g. anilines and phenols) into humus. Oxidative coupling is the process by which phenols, anilines and other compounds are linked together after oxidation by an enzyme or chemical agent. In the first step of the reaction, susceptible organic compounds are oxidized to form unstable free radicals. The free radicals go on to react with other molecules yielding free radical intermediates that can further react, ultimately leading to the formation of polymers. The reaction results in the formation of C - C and C - O bonds between phenolic species and C - N and N - N bonds between aromatic amines (Sjoblad and Bollag, 1981). With
208
J.-M. BOLLAG ET AL.
respect to phenolic cross-coupling, the initial step usually requires the removal of an electron and a hydrogen ion from the hydroxyl group, which generates a phenoxy radical. The free radical intermediate then couples at positions ortho and para to the hydroxyl group (the meta position is nonreactive) to yield a dimer. Phenolic dimers can be further oxidized resulting in the formation of oligomeric and polymeric products. Conversely, if the oxidation potential of the enzyme or chemical is high enough, C - C coupled dimers can be further oxidized to form extended quinones. Oxidative coupling reactions are mediated by a number of biological and abiotic catalysts, including plant and microbial enzymes, inorganic chemicals, clay and soil extracts (Bollag, 1983; Shindo and Huang, 1984; Wang et al., 1986). Coupling may also occur spontaneously in the presence of oxygen at neutral and alkaline pH. The enzymes which catalyze oxidative coupling reactions have been divided into two classes, the peroxidases and the monophenol monooxygenases. All peroxidases contain an iron porphyrin ring and require either hydrogen peroxide or alkyl peroxide as a coenzyme. The monophenol monooxygenases are further subdivided into two distinct groups, tyrosinases and laccases. These two enzyme groups require molecular oxygen, but no coenzyme, for activity. Two types of reactions are catalyzed by tyrosinase: (1) the hydroxylation of monophenol and (2) the oxidation of o-diphenols to o-quinones (Sjoblad and Bollag, 1981). Free radicals are not generated during reactions catalyzed by tyrosinase. However, in alkaline environments, polymeric molecules may form if quinone products react with one another via autooxidation. The laccases have a broad substrate specificity and are able to oxidize mono-, di-, and polyphenols, as well as aromatic amines. Laccases readily transform phenolic substrates to yield the corresponding anionic free radical which may go on to couple with other molecules and generate polymers. For general polymerization purposes, the laccases may prove to be the most beneficial because, unlike the peroxidases, they do not require an expensive coenzyme, and because they yield highly reactive intermediates. OXIDATIVE COUPLING OF PHENOLS AND ANILINES The reaction products generated from the coupling of xenobiotics to humic matter are highly heterogeneous and complex and therefore quite difficult to analyze. In order to elucidate the mechanism of coupling, model in vitro systems were designed in which aromatic substrates were incubated with extracellular microbial enzymes. The reaction products were separated by thin-layer chromatography (TLC) or by high-performance liquid chromatography (HPLC) and then characterized by mass spectroscopy (MS). In this way, we examined the enzymatic polymerization of a naturally
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OCH3
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OC~3i OH
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CI13 ~ _•~ 7 o
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DIMER
OCH3 OH
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m/z 444 (C23H2409)
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