Interactions of Soil-Derived Dissolved Organic Matter with Phenol in

The influence of dissolved soil organic matter (DSOM) derived from three geosorbents of different chemical composition and diagenetic history on the ...
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Environ. Sci. Technol. 2004, 38, 338-344

Interactions of Soil-Derived Dissolved Organic Matter with Phenol in Peroxidase-Catalyzed Oxidative Coupling Reactions QINGGUO HUANG AND WALTER J. WEBER, JR.* Energy and Environment Group, Department of Chemical Engineering, 4103 ERB, The University of Michigan, Ann Arbor, Michigan 48109-2099

The influence of dissolved soil organic matter (DSOM) derived from three geosorbents of different chemical composition and diagenetic history on the horseradish peroxidase (HRP) catalyzed oxidative coupling reactions of phenol was investigated. Phenol conversion and precipitate-product formation were measured, respectively, by HPLC and radiolabeled species analysis. Fourier transform infrared (FTIR) spectroscopy and capillary electrophoresis (CE) were used to characterize the products of enzymatic coupling, and the acute toxicities of the soluble products were determined by Microtox assay. Phenol conversion and precipitate formation were both significantly influenced by cross-coupling of phenol with dissolved organic matter, particularly in the cases of the more reactive and soluble DSOMs derived from two diagenetically “young” humic-type geosorbents. FTIR and CE characterizations indicate that enzymatic crosscoupling in these two cases leads to incorporation of phenol in DSOM macromolecules, yielding nontoxic soluble products. Conversely, cross-coupling appears to proceed in parallel with self-coupling in the presence of the relatively inert and more hydrophobic DSOM derived from a diagenetically “old” kerogen-type shale material. The products formed in this system have lower solubility and precipitate more readily, although their soluble forms tend to be more toxic than those formed by dominant crosscoupling reactions in the humic-type DSOM solutions. Several of the findings reported may be critically important with respect to feasibility evaluations and the engineering design of associated remediation schemes.

Introduction Phenolic contaminants span a wide spectrum of compounds, including phenol, chlorophenols, alkylphenols, and hydoxylated polychlorinated biphenyls and polycyclic aromatics. These types of contaminants are introduced to the environment through a variety of agricultural and industrial activities and as a result of partial degradation of certain other widespread aromatic organic contaminants (1-5). The multiple toxicity effects and relatively high solubility and environmental mobility of such phenolic contaminants make them targets of major environmental concern. An important * Corresponding author phone: (734) 763-2274; fax:(313) 9364391;e-mail: [email protected]. 338

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pathway for environmental transformation of such compounds is oxidative coupling facilitated by natural catalysts (6-15). Peroxidases comprise an important class of enzymes able to catalyze the oxidative coupling reactions of a broad range of phenolic compounds (11-15). Phenolic substrates are converted catalytically in these reactions to phenoxy radicals in the presence of hydrogen peroxide (16, 17). The phenoxy radicals produced in the enzymatic reaction step can then, in post-enzymatic reactions, couple with each other (17, 18) or with other reactive substances present in any given system (19, 20). Self-coupling of phenol molecules with each other dominates in systems that lack appropriate substrates to participate in cross-coupling reactions with phenol, leading to formation of precipitated polymeric products that can be readily removed from water (21-26). Significant crosscoupling of phenol with other reactive substances usually occurs in aquatic systems in contact with soils and sediments, generally resulting in their incorporation in soil organic matter (11, 19). Cross-coupling reactions between phenolic contaminants and various types of organic substances have been demonstrated in a number of earlier investigations (20, 27-34). Phenolic contaminants have been found, for example, to bind with humic substances through C-C or aryl ether linkages via cross-coupling (34). The reactivity of humic constituents with respect to cross-coupling reactions has been reported to correlate with their structural characteristics and functionalities (20, 28). Studies involving bulk soils have shown that the resistances of phenolic compounds to water and solvent extractions are increased as a result of covalent binding to solid-phase soil organic matter (SOM) through cross-coupling reactions (1, 4, 35). In an earlier study (36) we linked the cross-coupling reactivity of SOM to the particular diagenetic history of the soil. Specifically, we found diagenetically “young” humic-type SOMs to be more reactive than “old” kerogen materials in terms of cross-coupling with phenol. Because cross-coupling reactions are expected to decrease the environmental mobility and ecotoxicity of phenolic contaminants by binding to solid-phase SOMs, the enhancement of enzymatic coupling reactions has been suggested as a potential means for their immobilization in soil/sediment remediation scenarios (11, 19, 37). While it is clear that cross-coupling of phenolic contaminants to solid-phase soil organic matter will lead to contaminant immobilization, the effects of cross-coupling with dissolved soil organic matter (DSOM) derived from various types of soil and sediment solid phases have not been systematically investigated. The study of phenol/DSOM cross-coupling reactions reported here was performed with a particular emphasis on clarifying issues of potential products and their respective precipitancy and toxicities. Precipitancy represents the tendency of a substance to precipitate, and thus has a strong relationship to substrate mobility. The results of this study therefore have important implications with respect to the environmental mobilities and ecotoxicities of coupling products that might be formed in different types of soil solutions. Such information is important for an integral assessment of the environmental fate and ecological impacts of phenolic contaminants, and thus critical for evaluating the feasibility of applying enzymatic coupling reactions for soil/sediment remediation. Horseradish peroxidase (HRP) mediated oxidative coupling of phenol was investigated in the current study in the presence of DSOMs derived from three geosorbents having different chemical compositions and diagenetic histories, and the specific effects of each type of DSOM on the coupling 10.1021/es0304289 CCC: $27.50

 2004 American Chemical Society Published on Web 11/22/2003

TABLE 1. Elemental Compositions of Geosorbent SOMs geosorbent materials

SOM type

Canadian peat Chelsea soil Lachine shale

humic humic kerogen

a

constituents (wt %) carbon oxygen hydrogen 49.7 5.6 8.3

42.5 9.1 n.d.a

5.7 0.9 n.d.a

Not determined.

reactions and products were examined. The extent of phenol conversion and the formation of precipitated products were quantified, respectively, by HPLC and radiolabeled species analyses, and the toxicities of the reaction mixtures were assayed using Microtox procedures. The experimental results reveal that the DSOMs studied have profound impacts on phenol transformations in terms of reaction extent, product precipitancy, and potential ecological effects. These impacts were found to correlate well with the characteristics and properties of the parent geosorbents from which the DSOMs were derived. FTIR and capillary electrophoresis (CE) characterizations of the parent geosorbents, the derivative DSOMs, and the reaction products were made to facilitate interpretation of the means by which phenol coupling is affected.

Experimental Section Chemicals. Sigma Chemical Co. (St. Louis, MO) was the source of extracellular horseradish peroxidase (HRP, type-I, RZ ) 1.3), catalase from bovine liver, hydrogen peroxide (30.8%, ACS reagent), 2,2′-azino-bis(3-ethylbenz-thiazoline6-sulfonic acid) (ABTS, 98%, in diammonium salt form), and phenol-UL-14C (51.4 mCi/mmol). The phenol (99+%, biochemical grade) was from Acros Organics (Belgium, NJ), and ScintiSafe Plus 50% liquid scintillation cocktail was from Fisher Scientific (Fairlawn, NJ). DSOM Solutions. DSOM solutions were derived from three different types of geological materials employed in previous studies in our laboratories (38-41): Canadian peat (CP), Chelsea soil (CS), and Lachine shale (LS). Elemental compositions of the organic matter components of the three geosorbents are summarized in Table 1. Stock DSOM solutions were prepared by first mixing 50 g of each geosorbent with 1 L of 5-mM phosphate buffer (pH ) 7.0), this buffer system having been found in preliminary tests to have an enhancement effect on SOM dissolution. This solution also served to maintain a constant ionic strength and pH across different DSOM solutions. The soil/buffer mixtures were equilibrated for 1 week at room temperature under dark conditions and agitated manually several times a day. At the end of this “dissolution” period, the solution phase was separated from the soil and passed through a pasteurized 0.2-µm filter (Nalgen Nunc International, Rochester, NY). The DSOM stock solutions were then stored in amber bottles at 4 °C until used in the experiments described below. The total organic carbon (TOC) concentration of each DSOM stock solution was measured using a Shimadzu TOC500 Total Organic Carbon Analyzer (Shimadzu Corp.). The respective values of TOC for the CP, CS, and LS stock solutions were 196.5, 42.9, and 6.4 mg/L. A preliminary test showed that centrifugation (1300g, 35 min) of the DSOM stock solutions and their acidified samples (pH ) 1) did not result in significant TOC reduction in the supernatants. This test indicates that the organic matter contained in the DSOM solutions was comprised primarily of dissolved substances, likely fulvic acids. The DSOM experimental working solutions were prepared by diluting the stock solutions with the 5-mM phosphate buffer to predetermined levels immediately prior to each enzymatic-coupling experiment.

Enzymatic Coupling. Phenol coupling reactions in the DSOM and DSOM-free control solutions were conducted at room temperature in 13 × 100-mm glass centrifuge tubes operated as completely mixed batch reactors (CMBRs). Each reactor contained 7 mL of solution containing DSOM at different levels of TOC, a mixture of 14C-labeled (∼0.05 µCi) and unlabeled phenol at an initial concentration of 500 µM, and the HRP catalyst at 0.5 unit/mL as measured by the ABTS method (42). Each reaction condition was investigated in triplicate. Hydrogen peroxide in an amount sufficient to yield a resultant concentration of 1 mM was amended to each reactor to initiate the coupling reaction. After 2.5 h of incubation, the reactors were centrifuged at 1300g for 35 min to separate the solution and precipitate phases. A 0.5mL sample of supernatant was then taken for measurement of radioactivity and a 1.0-mL sample for HPLC analysis. These analyses were conducted immediately after sampling. The 0.5-mL sample for analysis of radioactivity was mixed with 3 mL of ScintiSafe Plus 50% liquid scintillation cocktail and analyzed for dissolved 14C using a Beckman LS6500 liquid scintillation counter (Beckman Instruments, Inc.). The concentration of precipitated 14C was calculated by mass balance differencing and expressed as an equivalent phenol concentration in solution. An Agilent 1100 Series HPLC system (Agilent Technologies) equipped with a phenomenex C18(2) column (150 × 3.0 mm, 5 µm particle size) was used to measure the concentration of residual phenol remaining in each sample. The mobile phase containing methanol and 1% acetic acid aqueous solution at a ratio of 70:30 was operated at a 0.25-mL/min rate. Phenol concentration was detected by UV absorbance at 270 nm, and the extent of phenol conversion calculated by mass balance differencing. Both radioactivity and HPLC measurements of the blank control samples indicated that phenol losses during the 2.5-h reaction time and subsequent analytical processes were negligible. Capillary Electrophoresis Analysis and Microtox Assay. Enzymatic coupling reactions were carried out with unlabeled phenol following the same procedure described above to prepare samples for capillary electrophoresis analysis and Microtox assay. Reactions were conducted in different DSOM solutions at a common TOC concentration (6.4 mg/L) and in a DSOM-free solution. After the reaction was completed and the phases separated by centrifugation, a 1-mL sample of supernatant was taken for CE characterization and 2.5mL sample for toxicity measurements. A P/ACE MDQ capillary electrophoresis system (Beckman Coulter Inc.) equipped with an uncoated fused-silica column (21-cm effective length, 75-µm i.d.) was used in the CE analyses. This technique separates dissolved electrolytes based on their respective charge-to-mass ratios. All neutral solution components move together along the length of the electrophoresis column at the rate of electroosmotic flow (EOF). Anionic components move more slowly than the EOF because of electric field effects; in general, the higher the charge-to-mass ratio of the anion, the greater the retardation and the longer the retention time. The conditions involved in these analyses include the following: (i) a running buffer of borate (100-mM, pH 8.3); (ii) a voltage of 15 kV; (iii) a temperature of 25 °C; (iv) a detector wavelength of 254 nm; and, (v) an injection period of 10 s. The aqueous samples for toxicity measurements were amended with 20 unit/mL of catalase to decompose any remaining H2O2. Microtox assay was performed using a model 500 Toxicity Analyzer (Azure Environmental) following the basic test procedure prescribed by the instrument manufacturer. Each sample was diluted to five serial levels, and reductions in light-emission of the photobacteria were measured after a 5-min contact period. The dilution level of each sample corresponding to a 20% decrease in light VOL. 38, NO. 1, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Coupling product distributions for enzymatic reactions in a DSOM-free solution and DSOM solutions having a common TOC level (6.4 mg/L). Initial phenol concentration ) 500 µM, initial H2O2 concentration ) 1 mM, HRP dosage ) 0.5 unit/mL, reaction time ) 2.5 h. FIGURE 1. Effects of DSOM concentration as TOC on phenol conversion (open symbols) and precipitate formation (solid symbols) resulting from enzymatic coupling in DSOM reaction solutions. Initial phenol concentration ) 500 µM, initial H2O2 concentration ) 1 mM, HRP dosage ) 0.5 unit/mL, reaction time ) 2.5 h. Data points are the means of triplicate experiments with 1 SD error bars. emission was reported as an EC20 value; EC20 values were used instead of EC50 in order to avoid extrapolations. FTIR Analysis. Precipitated products for FTIR analysis were prepared in 28 × 98-mm glass centrifuge tubes, each containing 30 mL of reaction solution. Enzymatic coupling reactions were conducted with unlabeled phenol in DSOM stock solutions and in a DSOM-free solution. The resultant reaction mixtures were acidified to pH ≈ 2.0 with 0.3 mL of 1.0 M hydrochloric acid to facilitate product precipitation. Preliminary tests indicated that this acidification procedure did not cause precipitation in the DSOM solutions in the absence of enzymatic coupling reactions. The precipitated products were collected, washed several times with Milli-Q water, and freeze-dried before FTIR characterization. A method commonly employed in SOM studies (43, 44) was used here for FTIR characterizations. Briefly, pellets for analysis were prepared by mixing, grinding, and pressing samples of the precipitated products with potassium bromide salt. A Perkin-Elmer Spectrum BX FTIR spectrophotometer was employed for the analysis. IR signals were acquired by averaging 256 scans ranging from 400 to 4000 cm-1 at a resolution of 4 cm-1. FTIR spectra were also obtained for the three geosorbents from which the DSOMs were derived.

Results and Discussion Enzymatic Phenol Coupling in DSOM Solutions. Figure 1 presents phenol conversion and precipitate formation resulting from reactions in different DSOM solutions of varying TOC levels. As described in the Experimental Section, phenol conversion represents the total transformed phenol as measured by HPLC, including both precipitated and soluble products. Precipitate formation, in turn, is only that fraction of phenol forming precipitated products, as analyzed by 14C radiolabeling. It is evident in Figure 1 that phenol conversion increases in all three DSOM solutions as TOC concentration increases above a level of approximately 1 mg/L. The degree of enhancement of phenol conversion varies among the different DSOM solutions, decreasing in the order Chelsea soil > Canadian peat > Lachine shale. Unlike phenol conversion, however, formation of precipitated products was significantly suppressed in the presence of the DSOMs derived from Chelsea soil and Canadian peat and significantly increased in the presence of that from Lachine shale. The commonalities among and differences between reactions in 340

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the three DSOM solutions are further evident in Figure 2, which compares product distributions between dissolved and precipitated forms in solutions of DSOM having a common TOC concentration (6.4 mg/L) and in a DSOM-free control solution. The observed effects of DSOM on the enzymatic coupling reactions of phenol are indicative of cross-coupling reactions proceeding to different extents and resulting in products of different solubility. Spectroscopic examinations described in following sections provide further insights to the DSOM effects on phenol coupling reactions, and attempts are made to relate the observed effects to the properties and diagenetic stages of the geosorbents from which the DSOMs were derived. Such relationships are of important practical significance in identifying and optimizing potential engineering applications of enzymatic coupling reactions in soil/ subsurface remediation. Characteristics of the Geosorbents and DSOMs. The three geosorbent materials used to prepare the DSOM solutions differ significantly in chemical composition and diagenetic history. These materials have been well characterized and extensively tested in previous studies in our laboratories, and details of these characterizations and tests are readily available in the literature (38-41). The materials can be described briefly as follows: (i) Canadian peat, originating from recently deposited plant material, is in the very beginning stage of early diagenesis; (ii) Chelsea soil, a humiccontaining surface geosorbent, is also a young geological material but diagenetically more mature than Canadian peat; and (iii) Lachine shale is a kerogen-containing ancient sediment that has undergone extensive diagenesis. The FTIR spectra shown in Figure 3 reflect differences in the chemical functionalities of the geosorbent organic matters. Canadian peat contains carbonyl (1734, 1679 cm-1), hydroxyl (3432 cm-1), aromatic (1615 cm-1), and aliphatic (1324, 2904 cm-1) groups. For Chelsea soil, the broad band (1624 cm-1) ranging from 1500 to 1800 cm-1 may result from hybridization of the responses of various carbonyl and aromatic moieties. This broad band, in contrast to the multiple peaks in the same wavenumber region for Canadian peat, may be indicative of the greater diagenetic maturity of the Chelsea soil, which also contains hydroxyl (3390 cm-1) and aliphatic (1380 cm-1) functionalities in addition to carbonyl and aromatic groups. Aromatic (1620, 1525 cm-1) and aliphatic (1289 cm-1) groups are most evident in the Lachine shale SOM. This chemically reduced kerogen-type geological material shows no carbonyl band characteristics (1640-1780 cm-1), indicating the lack of oxidized functionalities (i.e., carboxylic, aldehyde, and ketone groups). The relatively weak band at 3608 cm-1 can be attributed to

FIGURE 3. FTIR spectra for the parent geosorbents. hydroxyl groups. All three geosorbent materials exhibit a strong band around 1020 cm-1, which can be attributed to either ether bonds or Si-O bonds with inorganic components. The order of the TOC concentrations of the DSOM stock solutions prepared, respectively, from Canadian peat (196.5 mg/L), Chelsea soil (42.9 mg/L), and Lachine shale (6.4 mg/ L) may be taken as an indication of the relative “solubility” of the organic contents comprised in these geosorbents and the relative “hydrophilicity” of the three DSOMs. The order appears to be consistent with the nature of the materials and their relative abundances of hydrophilic functionalities as reflected by the FTIR spectra (Figure 3). Characterizations of Coupling Products. Capillary electrophoresis (CE) was employed to characterize the organic components of each DSOM solution and the soluble products resulting from enzymatic coupling reactions in that solution. Humic substances are rich in carboxylic and phenolic functionalities and anionic in character, and capillary electrophoresis has thus proven to be a useful tool for characterizing DSOM (45, 46). The soluble polyphenol products resulting from phenol self-coupling are also ionic and thus amenable to CE characterization. Electropherograms for the DSOM solutions and the DSOM-free background solution (5 mM phosphate buffer) are shown in Figure 4 as light gray lines and those for each product mixture after enzymatic coupling as bold black lines. The enhanced EOF (neutral component) responses caused by residual phenol and H2O2 can be observed in each after-reaction electropherogram. The electropherogram fingerprints shown in Figure 4 provide a unique perspective on the phenol coupling reactions occurring in each solution. A multispiked shape is observed in the after-reaction electropherogram given in part A of Figure 4 for the DSOM-free solution. It is known that the self-coupling reactions of phenol alone result in polyphenol products composed of various numbers of phenol monomers linked by either C-C or C-O-C bonds (18, 47). The charge-to-mass ratios of the ionized polyphenol products are thus diversified, depending as they do on polymerization levels and types of bond linkages. This characteristic of selfcoupling reaction products is reflected in the multispiked shape of the after-reaction electropherogram for the DSOM-

FIGURE 4. Electropherograms for DSOM reaction solutions before and after enzymatic coupling. Initial phenol concentration ) 500 µM, initial H2O2 concentration ) 1 mM, HRP dosage ) 0.5 unit/mL, DSOM concentration ) 6.4 mg/L as TOC, reaction time ) 2.5 h. free solution in part A of Figure 4. Conversely, no such multispiked shapes appear in the postreaction electropherograms for the Chelsea soil or Canadian peat DSOM reaction mixtures shown in parts C and D of Figure 4. Instead, the electropherograms for these two DSOM reaction mixtures after coupling are magnified in scale but similar in shape to their electropherograms before the reaction. Additional electrophoresis tests were performed involving the addition of Chelsea soil and Canadian peat DSOMs to enzymedepleted product mixtures resulting from phenol selfcoupling reactions in DSOM-free solutions, and in the electropherograms thus obtained the multiple peaks corresponding to polyphenol products were still evident. It is unlikely that this behavior can be attributable to association or aggregation of polyphenol products with DSOMs and thus reasonable to conclude that (i) formation of polyphenol products via phenol self-coupling is largely suppressed in these two reaction mixtures containing DSOMs derived from Chelsea soil and Canadian peat, respectively, and (ii) the dominant cross-coupling reactions in these DSOM systems lead to incorporation of phenol molecules in the DSOMs. Multiple peaks can be observed in part B of Figure 4 in the postreaction electropherogram for the reaction mixture containing Lachine shale DSOM, but these multiple peaks differ in pattern from those shown in part A for self-coupling products formed in the DSOM-free solution. This indicates that both cross-coupling and self-coupling reactions may occur simultaneously in the case of the DSOM derived from Lachine shale. As noted earlier, the kerogen-type SOM of VOL. 38, NO. 1, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. FTIR spectra for products formed by enzymatic coupling in a DSOM-free solution and stock DSOM solutions. Enzymatic reaction conditions: initial phenol concentration ) 500 µM, initial H2O2 concentration ) 1 mM, HRP dosage ) 0.5 unit/mL, reaction time ) 2.5 h. Lachine shale is distinctly different than the SOMs of the Chelsea soil and Canadian peat, being chemically more reduced and inert and having fewer soluble oxygen-containing functionalities. Because of the relatively inert nature of the shale DSOM, cross-coupling may not dominate in that reaction mixture as it did in those involving the soil and peat DSOMs. Phenol self-coupling may therefore become relatively stronger and occur in parallel with cross-coupling reactions in the shale reaction mixture. This would explain why the multispiked fingerprint corresponding to the presence of polyphenols can still be observed in the postreaction electropherogram for the shale DSOM solution but in a pattern that is different from those of products formed solely by self-coupling reactions in DSOM-free solution. The FTIR spectra for the precipitated products shown in Figure 5 confirm cross-coupling between phenol and the DSOMs. The spectrum for self-coupling products formed in the DSOM-free solution gives clear evidence of polyphenol structures having aromatic moieties (1632, 1580, 1488, and 1455 cm-1) bonded to each other by aryl ether bonds (1209 cm-1) and containing hydroxyl groups (3420 cm-1). No band is evident in the characteristic region of carbonyl functionality (1640-1780 cm-1). Conversely, such bands can be easily discerned in the spectra for the products formed in the Canadian peat (1757 cm-1) and Chelsea soil (1721 cm-1) DSOM mixtures. Tests involving addition of self-coupling products to DSOM solutions followed by collection, washing, and FTIR analysis of the precipitates yielded no discernible evidence of carbonyl peaks, thus precluding the possibility that the carbonyl signals can be caused by sorption of DSOMs to precipitated products. The presence of carbonyl groups in the products formed in Canadian peat and Chelsea soil DSOM solutions therefore indicates that carbonyl-containing DSOM moieties have been incorporated in the products through reactions, confirming cross-coupling between phenol and the DSOMs. Again, in contrast to the products formed in the soil and peat DSOM reaction mixtures, no characteristic carbonyl peaks are evident in the spectrum given in Figure 5 for the 342

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system involving the shale DSOM. This, however, cannot be taken as evidence for the absence of cross-coupling reactions, because the shale organic matter itself lacks carbonyl groups, as indicated by the absence of characteristic carbonyl bands (1640-1780 cm-1) in its FTIR spectrum (Figure 3) In terms of linking the results of the FTIR and CE studies to a better understanding of mechanisms underlying the observed effects of DSOMs on phenol coupling, we first attribute the enhanced overall phenol conversion evident in Figures 1 and 2 to the occurrence of cross-coupling reactions of the phenol with DSOM. Enhancements of this nature have been reported earlier for peroxidase-catalyzed reaction systems containing “humic monomers” and humic acids as cosubstrates (19, 28, 31). The fact that enhancement of phenol conversion increases in the order Lachine shale < Canadian peat < Chelsea soil in the DSOM reaction mixtures is taken as indicative of the relative order of cross-coupling reactivity for these different DSOMs, an order consistent with previous reports of greater cross-coupling reactivity for Chelsea soil than for Lachine shale (36). CE analysis indicates that crosscoupling tends to dominate and suppress self-coupling of phenol, in the presence of DSOMs deriving from Chelsea soil and Canadian peat, i.e., DSOMs containing hydrophilic moieties and having abundant oxygen-containing functionalities. Cross-coupling reactions in such solutions should in fact result in products having relatively high solubility, explaining the reductions in precipitated product formation apparent for these two reaction mixtures in Figures 1 and 2. As suggested by CE studies, self-coupling and cross-coupling reactions appeared to occur in parallel in solutions of DSOM derived from the Lachine shale, solutions that contain less reactive and more hydrophobic DSOM components. Figures 1 and 2 indicate that products formed in this system tend to be less soluble and yield greater precipitate formation. This suggests that hydrophobic DSOM moieties may have been incorporated into the polyphenol products via parallel selfand cross-coupling processes, resulting in solubility reduction. Toxicity Evaluations. To evaluate the potential effects of the DSOMs studied on detoxification of the products of enzymatic coupling reactions, various reaction mixtures were assayed using the Microtox procedure referenced in the Experimental Section. Toxicity data for the product mixtures are compared in Table 2 to that for a control containing 500 µM unreacted phenol in DSOM-free buffer solution. The value of the toxicity indicator, EC20, corresponds to the number of dilutions required to effect a 20% decrease in the lightemission of photobacteria in a sample. For example, the EC20 value of 5.4 given in Table 2 for the control sample (500 µM phenol in buffer) means that photoluminance is reduced by 20% by a 5.4-fold dilution of that control sample (i.e., a dilution containing 92.6 µM phenol). The toxicities of the reaction mixtures are attributable to residual phenol and possibly to soluble coupling products. If it is assumed as a hypothetical that the toxicity of any reaction mixture is attributed solely to residual phenol, one can construct a correspondingly hypothetical “dummy” phenol concentration for that reaction mixture simply by multiplying its measured EC20 value by 92.6 µM. These dummy phenol concentrations are compared in Table 2 to the phenol transformation data acquired in separate experiments with radiolabeled phenol and depicted graphically in Figure 6. As shown in Figure 6, the product toxicities of the Chelsea soil and Canadian peat DSOM reaction mixtures can be accounted for entirely by residual phenol, indicating that the soluble coupling products formed in the presence of these DSOMs are not toxic. This indicates that enzymatic coupling reactions involving incorporation of phenol into these two DSOMs yield environmentally benign products. Conversely, the dummy phenol concentration for the reaction mixture

TABLE 2. Acute Toxicities and Relevant Phenol Transformation Dataa for Product Mixtures Resulting from Enzymatic Coupling in DSOM Solutions reaction mixture

CS DSOM

CP DSOM

LS DSOM

controlb

EC20c dummy phenol concentrationd (µM) residual phenol concentratione (µM) soluble C-14 concentratione(µM)

2.0 185.2 184.4 494.8

2.2 203.7 216.3 484.2

4.7 435.2 281.5 311.9

5.4 500.0 500.0 500.0

a Enzymatic reactions conducted at 500 µM phenol and 0.5 unit/mL HRP. b No enzymatic reaction at 500 µM phenol in phosphate buffer. Expressed as number of dilutions. d Estimated concentrations assuming that only phenol contributes to toxicity. e Measured concentrations in separate experiments with radio-labeled phenol. c

results of the study reflect important factors that should be considered in applying the concepts of enzymatic coupling for remediation purposes. Some of the findings reported may in fact be critically important with respect to feasibility evaluations and the engineering design of appropriate remediation schemes.

Acknowledgments

FIGURE 6. Comparisons of toxicity data (dummy phenol concentrations) and relevant phenol transformation data for product mixtures resulting from enzymatic coupling in DSOM solutions. Initial phenol concentration ) 500 µM, initial H2O2 concentration ) 1 mM, HRP dosage ) 0.5 unit/mL, DSOM concentration ) 6.4 mg/L as TOC, reaction time ) 2.5 h. containing DSOM derived from Lachine shale is higher than the residual phenol concentration, indicating that products formed in this reaction system contribute to the observed toxicity. We concluded earlier that cross-coupling and selfcoupling occur in parallel in the Lachine shale solution, resulting in products having polyphenol structures incorporating DSOM moieties. Dissolved polyphenol products formed by phenol self-coupling have been reported to have greater acute toxicity than phenol (48, 49). Polyphenol-type products incorporating Lachine shale DSOM may therefore be toxic as well and thus contribute to the overall toxicity of the product mixture. It should be noted, however, that some parallel detoxification can still result in this reaction system because coupling products embedded in shale DSOMs may more readily precipitate from solution. In summary, this study reveals that DSOMs have profound impacts on the oxidative coupling of phenol in terms of reaction extent, product precipitancy, and potential ecological effects. Such impacts correlate with the properties and diagenetic stages of the geosorbents from which the DSOMs were derived. Cross-coupling reactions dominate in DSOM solutions derived from diagenetically “young” humic-type geosorbents. Such cross-coupling reactions lead to incorporation of phenol in DSOM macromolecules, yielding nontoxic soluble products. Conversely, cross-coupling appears to proceed in parallel with self-coupling in the presence of the relatively inert and more hydrophobic DSOM derived from a diagenetically “old” kerogen-type shale material. The products formed in this system have lower solubility and precipitate more readily, although their soluble forms tend to be more toxic than those formed by dominant crosscoupling reactions in humic-type DSOM solutions. The

We thank Deborah A. Ross and Carl W. Lenker for their diligent efforts and important contributions in performance of the experimental work reported and Thomas Yavaraski for invaluable assistance with the instrumental analyses. The research was supported in part by the Environmental Management Science Program of the United States Department of Energy (DOE) through Grant No. DE-FG0702ER63488 and in part by Research Grant P42ES04911-14 from the National Institutes for Environmental and Health Sciences. The content of this paper does not necessarily represent the views of either funding agency.

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Received for review April 7, 2003. Revised manuscript received July 16, 2003. Accepted October 8, 2003. ES0304289