Effects of Humic Substances on the Pattern of Oxidation Products of

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Environ. Sci. Technol. 2003, 37, 386-394

Effects of Humic Substances on the Pattern of Oxidation Products of Pentachlorophenol Induced by a Biomimetic Catalytic System Using Tetra(p-sulfophenyl)porphineiron(III) and KHSO5 MASAMI FUKUSHIMA,* HIROYASU ICHIKAWA, MIKIO KAWASAKI, AKIRA SAWADA, KENGO MORIMOTO, AND KENJI TATSUMI National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba-West, Tsukuba 305-8569, Japan

In the presence of humic substances (HSs), the oxidative conversion of pentachlorophenol (PCP) was found to be efficiently catalyzed by tetra(p-sulfophenyl)porphineiron(III) (Fe(III)-TPPS) using KHSO5 as an oxygen donor. Orthotetrachloroquinone (o-TeCQ), 2-hydroxyl-nonachlorodiphenyl ether (2H-NCDE), 4-hydroxyl-nonachlorodiphenyl ether (4HNCDE), and octachlorodibenzo-p-dioxin (OCDD) were identified as the major byproducts of the reaction. Decreased amounts of these byproducts were produced in the presence of HS. In particular, the addition of HSs with a lower degree of humification resulted in a large decrease in the formation of dimers, such as 2H-NCDE, 4H-NCDE, and OCDD. More than 60% of the chlorine, which was released from PCP, was found in the HS fractions after the reaction. This suggests that chlorinated intermediates from PCP were incorporated into the HS. Pyrolysis-GC/MS and 13C NMR studies confirmed that the binding of the chlorinated intermediates was covalent in nature and that the intermediates were copolymerized with HS via oxidative coupling reactions. A Microtox test demonstrated that the toxicity of the HS fraction containing PCPderived intermediates was much lower than that of the mixture of PCP and HS in the absence of a catalytic reaction.

Introduction Biomimetic catalytic systems using iron(III)-porphyrin complexes (Por-Fe(III)) have the potential to serve as clean processes for the removal of organic pollutants in soils. To understand remedial processes for organic pollutants via enzymatic reactions, catalytic systems using Por-Fe(III) were examined with respect to the oxidation of organic pollutants, such as chlorophenols (1). Considering their application to soil remediation, information concerning the effects of soil organic matter, such as humic substances (HSs), would be desirable. Therefore, we previously investigated the effects of HSs on the removal of pentachlorophenol (PCP) via a biomimetic catalytic reaction using a combination of tetra(p-sulfophenyl)porphineiron(III) (Fe(III)-TPPS) and KHSO5 * Corresponding author phone: +81-298-61-8328; fax: +81-29861-8326; e-mail: [email protected]. 386

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(2). In that study, HSs with a lower degree of humification were found to be more useful in enhancing the removal of PCP in the Fe(III)-TPPS/KHSO5 system. However, the patterns of byproducts, which are derived from PCP, have not been elucidated quantitatively. Chlorophenols are oxidized by peroxidase and ligninase that contain Por-Fe(III) as an active center, and this gives rise to the formation of more toxic dimers, such as chlorinated dioxins (3-14). However, it has been reported that the addition of humic precursors, such as phenolic acids, can lead to a reduction in the formation of dioxins from chlorophenols because of coupling reactions that occur between the precursors and reaction intermediates (13, 14). Bollag concluded that such coupling between humic precursors and organic pollutants may be useful in decontaminating soils (15). However, the detoxification of pollutants via their incorporation into HSs from a biological point of view is yet to be clearly demonstrated. Moreover, it has been reported that, in biomimetic systems using water-soluble Por-Fe(III), 2,4,6-trichlorophenol (2,4,6TrCP) is efficiently converted to 2,6-dichlorobenzoquinone (16, 17), but the formation of dimers has not been examined. In other oxidation processes, such as UV-light, Fenton, and MnO2 oxidations, the formation of dioxins has been observed during the oxidation of chlorophenols (18-21). The formation of dimers from chlorophenols has not been studied in the case of biomimetic catalytic systems using Por-Fe(III). The primary objective of the present study was to elucidate the patterns of byproducts by which PCP is oxidized in the Fe(III)-TPPS/KHSO5 system in the absence and presence of HS. Second, to evaluate the toxicity of chlorinated intermediates produced in the reaction and incorporated into HS, a Microtox test was examined.

Experimental Section Materials. Fe(III)-TPPS was prepared according to procedures described in a previous paper (2). 2-Hydroxyl (2H-NCDE) and 4-hydroxyl (4H-NCDE) nonachlorodiphenyl ethers were prepared by the reduction of 2,3,5,6-pentachloro-4-pentachlorophenoxy-2,5-cyclohexadieneone (PPC) with NaI and NaBH4, respectively (22, 23). PPC was prepared by the oxidation of PCP with HNO3 in a mixture of trifluoroacetic acid and trifluoroacetic anhydride at -20 °C (24). Stock solutions of 2H- and 4H-NCDEs were prepared by dissolution in toluene. PCP was purchased from Nacalai Tesque (99.0% purity). Tetrachlorohydroquinone (TeCHQ), tetrachlorocatechol (TeCC), and o-tetrachloroquinone (o-TeCQ) were obtained from Tokyo Chemical Industry (98% purity) and Aldrich (98% purity), respectively. Other chlorophenols were used as standard materials for py-GC/MS: 2,3,5-trichlorophenol (2,3,5-TrCP), 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP) (Tokyo Chemical Industry, 98% purity), 2,3,4,5-TeCP (Kanto Chemicals, 10 ppm in cyclohexane). 13C6-labeled PCP (ISOTEC INC, 99 at. % of 13C) was used for the 13C NMR study. A standard sample of octachlorodibenzo-p-dioxin (OCDD) (50 µg mL-1 in toluene) was purchased from GL Sciences. Fourteen types of humic (HA) and fulvic (FA) acids were used in the present study as follows: peat soil (BHA1, BHA2, SHA), compost soil (CHA), wheat straw-applying soil (WHA), tropical peat (GOHA, BBHA), commercial (AHA), brown forest soil (DHA, DFA), and ando soil (THA, AIHA, IHA, IFA). The origins and elemental compositions of these preparations have been summarized in a previous paper (2). Oxidation of PCP. A 25 mL aliquot of 0.02 M NaH2PO4/ Na2HPO4/citrate buffer at pH 3-7, which contained 0-100 mg L-1 of HS, was placed in a 100-mL Erlenmeyer flask. A 10.1021/es020747k CCC: $25.00

 2003 American Chemical Society Published on Web 12/06/2002

125 µL aliquot of 0.01 M PCP in acetonitrile and a 625 µL aliquot of aqueous Fe(III)-TPPS (200 µM) was added to the buffer solution. Subsequently, 313 µL of aqueous 0.01 M KHSO5 was added, and the flask was then shaken in a thermostatic shaking water bath at 25 ( 0.1 °C. After a 60 min reaction period, 2.5 mL of 1 M ascorbic acid aqueous was added, and pH of the solution was adjusted to 11-11.5 by the addition of aqueous K2CO3. Subsequently, 5 mL of acetic anhydride was added dropwise to the solution, and 0.6 mL of a 1 mM anthracene (ATC) hexane solution was added as an internal standard for the GC/MS analyses. This mixture was triply extracted with 30 mL of n-hexane, and the extract was dehydrated with Na2SO4 anhydride. After filtration, the extract was concentrated to 500 µL under a stream of dry N2. A 1 µL aliquot of the extract was introduced into an HP5971/HP5890 series II (Hewlett-Packard) GC/MS system. A Quadrex methyl silicon capillary column (0.25 mm i.d. × 25 m) was employed in the separation. The temperature ramp was as follows: 65 °C for 1.5 min, 65-120 °C at 35 °C min-1, 120 -300 °C at 7 °C min-1, and a 300 °C hold for 5 min. The recoveries of extractions (n ) 3) for 1 µM of TeCC, 2H-NCDE, 4H-NCDE, and OCDD were 87 ( 9%, 95 ( 8%, 90 ( 11%, and 95 ( 9%, respectively. The detection limits of TeCC, 2H-NCDE, 4H-NCDE, and OCDD for the GC/MS analysis after extraction with hexane were 39 nM, 25 nM, 38 nM, and 22 nM, respectively. The concentrations of PCP and Cl- in the test solution before and after the reaction were also determined by HPLC and ion chromatography, respectively. All oxidation runs were conducted in duplicate. Preparation of the HS Fractions. The oxidation of PCP by HS was conducted at pH 6, resulting in the incorporation of intermediates into HS fraction. The volume of initial buffer solution was scaled up from 25 mL to 100 mL. In this experiment, SHA and BHA1 were used at concentrations of 100 mg L-1. After the oxidation, 50 mL of 2-propanol was added to the test solution. The HS fraction was then concentrated by ultrafiltration through a Millipore YM1 ultrafiltration cellulose membrane (nominal cutoff 1000 daltons). The concentrated HS fraction was washed twice with 50 mL of 2-propanol and then twice with 100 mL of water. The pH of the concentrated fraction was adjusted to 1 via the addition of H2SO4, followed by stirring for 4 h. This mixture was then centrifuged (10 000 rpm, 10 min), and the resulting precipitate was dialyzed against water using a Spectra/Por cellulose ester membrane (nominal cutoff 500 daltons). The HS fraction (Ca. 10 mg) was obtained in powder form by freeze-drying. We applied two types of control for the HS fractions: (1) HS was reacted with the Fe(III)-TPPS/ KHSO5 system in the absence of PCP (reaction blank), and (2) HS was mixed with PCP but without the catalytic reaction (unreacted). The concentrations of PCP and Cl- in the test solution before and after the reaction were determined by HPLC and ion chromatography, respectively. Adsorptive Organic Halogen (AOX). To check the mass balance of the chlorinated species in the test solution before and after the oxidation, the AOX value was measured. For the measurement of AOX in the test solution before and after the reaction, a 200 µL aliquot of the test solution was introduced onto a column, packed with 40 mg of activated carbon (AC). Organic chlorine is adsorbed to the AC, but inorganic chlorine, such as Cl-, is not. After washing the AC column with aqueous KNO3 (8.2 g L-1), the AC was plated on a quartz boat. This was then introduced into a TOX-10Σtype halogen analyzer (Mitsubishi Kasei). For measurement of the amount of chlorine incorporated into HS, 1 mg of the HS fraction after reaction with PCP was dissolved in 1 mL of aqueous 0.01 M NaOH. After dilution with a pH 6 buffer to 100 mg L-1, a 200 µL aliquot of this solution was passed through the AC column. The AOX value was then measured using the halogen analyzer as described above. The detection

limit for AOX was 0.6 mg L-1. The AOX analyses were conducted in triplicate. Pyrolysis-GC/MS (Py-GC/MS). A JPH-3 type Curie-point pyrolyzer (Japan Analytical Industry) was interfaced with the GC/MS system. The powdered HS fraction (1 mg), as prepared above, was pyrolyzed at 500 °C for 4.0 s with 0.5 mg of a methylation reagent, tetramethylammonium hydroxide (TMAH). The temperature program for the GC/MS was as follows: 50 °C for 1 min, 50-300 °C at 5.0 °C min-1, and a 300 °C hold for 4 min. 13C NMR. After the catalytic reaction with 13C-labeled PCP in the presence of BHA1 or SHA, a powder of the HS fraction was prepared according to the method described above. The resulting powder (50 mg) was dissolved in 1.2 mL of DMSOd6 (ISOTEC INC). The 13C NMR spectrum was sampled in the inverse-gated decoupling mode using a JEOL Lambda FTNMR spectrometer (Nippon Denshi) with a resonance frequency for 13C of 125 MHz. The spectra were recorded using a pulse angle of 45° and a 2.0-s pulse delay, with 52 000 accumulated scans being obtained. Microtox Toxicity Test. The toxicities of the test solutions were evaluated on the basis of the reduction of light emitted by a microorganism (vibrio fischer NRRL B-1117) when exposed to a toxic sample from which the toxicity value is then calculated (25, 26). The toxicity of the test solutions was performed by using a Microtox kit equipped with a model500 type bioluminescence spectrophotometer (AZUR Environmental). In this test, the original test solution was diluted with 0.02 M citrate/phosphate buffer (pH 6) up to 0.05-0.44 mg L-1 of AOX, and the diluted solution was then used in the tests. After a 500 µL aliquot of aqueous 2% NaCl was pipetted into a glass cuvette, a 10 µL aliquot of an aqueous suspension of the bacteria was added. After incubating at 15 °C for 15 min, the luminescence of the solution (I0s) was measured. Subsequently, a 500 µL aliquot of the diluted test solution was added, and the luminescence (Its) was then measured after 5, 15, and 30 min of incubation. A control measurement was performed using aqueous 2% NaCl in the absence of toxic compounds, in which the luminescence intensity was measured before (I0) and after (It) incubation. The degree of reduction of the light emitted by the bacteria (%E) can be represented as

%E )

RtI0s - Its It + RtI0s - Its

× 100

where Rt represents the correction factor (Rt ) It/I0). In this experiment, the concentration of toxic compound in the test solution was normalized to the organic halogen concentration, which was based on the AOX values, because PCP could be oxidatively converted to a variety of chlorinated compounds. The AOX value at which %E is equal to 50% corresponds to the EC50 value, indicating the concentration of toxic compound required to reduce the activity by 50%.

Results and Discussion Identification of Byproducts. To identify and determine the byproducts, hexane extracts of the reaction mixtures were examined by GC/MS. The PCP used in the present work contained approximately 1% impurities (mainly 2,3,5,6TeCP). Therefore, we first checked the impurities in the PCP by GC/MS analysis of a hexane extract of an aqueous mixture of PCP and BHA1 (Figure 1a). However, in this chromatogram, no chlorinated compounds, except for PCP, were detected. Figure 1b,c shows chromatograms for the hexane extracts of the reaction mixtures. In the absence of HS (Figure 1b), PCPderived dimers, such as 2H-NCDE (28.2 min), 4H-NCDE (28.8 min), and OCDD (29.1 min), were detected. However, in the presence of BHA1, the peaks corresponding to these dimers VOL. 37, NO. 2, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. GC/MS chromatograms of a hexane extract of reaction mixtures. [PCP]0: 50 µM, [Fe(III)-TPPS]: 5 µM, [KHSO5]: 125 µM, pH 6, reaction time: 60 min, ATC: anthracene (internal standard). were reduced. In both the absence and presence of BHA1, TeCC (14.4 min) was detected. The formation of o- and/or 388

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p-quinones has been reported in the catalytic oxidation of phenols using Por-Fe(III) (16, 17, 27). In the present work,

FIGURE 2. Reaction kinetics for PCP disappearance and oxidation in the absence of HS. [PCP]0: 50 µM, [Fe(III)-TPPS]: 5 µM, [KHSO5]: 125 µM, pH 6. (a): PCP disappearance and Cl- release during the oxidation; sum of products: [PCP] + [o-TeCQ] + 2([2H-NCDE] + [4H-NCDE] + [OCDD]), (b): formation of o-TeCQ, 2H-NCDE and OCDD, (c): formation of 4H-NCDE. because the reaction mixture was reduced by the addition of ascorbic acid in the pretreatment step of the extraction, the quinones may have been reduced to hydroquinones. Therefore, the TeCC detected herein likely corresponds to o-TeCQ. Characteristics of Disappearance and Oxidation of PCP. Figure 2a shows the reaction kinetics for PCP disappearance and dechlorination in the absence of HS. The “Sum” in Figure 2a represents the summation of the residual PCP and all byproducts. If o-TeCQ, 2H-NCDE, 4H-NCDE, and OCDD shown in Figure 2b,c would be all of the byproducts, the “Sum” might be equal to the initial concentration of PCP ([PCP]0). In the absence of HS (Figure 2a), the values for the summation were equal to the [PCP]0 for up to a 30 min reaction period but were slightly decreased at 60 and 90 min. This implies that, with time, the byproducts may react to form other products. Figure 3 shows the kinetics of disappearance and oxidation of PCP in the presence of BHA1. The disappearance and dechlorination of PCP were enhanced in the presence of BHA1 (Figure 3a), and the formation of dimers, such as 2HNCDE, 4H-NCDE, and OCDD, was largely reduced (Figure 3b,c). A tendency existed, in the kinetics of oxidative

FIGURE 3. Reaction kinetics for PCP disappearance and oxidation in the presence of BHA1 (50 mg L-1). Conditions and (a)-(c) are similar to Figure 2. conversion, for small amounts of byproducts to be formed in the initial period of the reaction (3 min), which then gradually decreased with increasing reaction time. Moreover, in Figure 3a, the “Sum” is not equal to [PCP]0 for each reaction period and decreased with increasing reaction time. These results may be attributed to the fact that (1) other byproducts are formed and (2) PCP and byproducts are incorporated into BHA1. Because organic acids, such as chloromaleic and chloromuconic acids, represent expected PCP-derived byproducts (28), we determined such compounds by ion chromatography. However, these were not detected in the reaction mixtures in the absence and presence of BHA1. Thus, further oxidation to organic acids does not appear to be involved in the PCP oxidation via the Fe(III)-TPPS/KHSO5 system. Moreover, PCP-derived trimers or tetramers can be expected as additional byproducts. However, these also were not detected in the hexane extract in either the absence or presence of BHA1. Therefore, in the presence of BHA1, the majority of PCP and PCP-derived byproducts may be incorporated into BHA1 with increasing reaction times. Effects of pH and BHA1 Concentration. Figure 4a,b shows the effect of pH on the oxidative conversions of PCP in the absence and presence of BHA1, respectively. In both cases, the percentage of PCP disappearance increased with inVOL. 37, NO. 2, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Effect of BHA1 concentration on PCP disappearance and on oxidative conversion. [PCP]0: 50 µM, [Fe(III)-TPPS]: 5 µM, [KHSO5]: 125 µM, reaction time: 60 min.

FIGURE 4. Effect of pH on PCP disappearance and on oxidative conversion in the Fe(III)-TPPS/KHSO5 system. (a) [PCP]0: 50 µM, [Fe(III)-TPPS]: 5 µM, [KHSO5]: 125 µM, (b) (a) + 50 mg L-1 of BHA1, reaction time: 60 min. creasing pH. In the absence of BHA1 (Figure 4 a), OCDD and 4H-NCDE were the major byproducts at pH 3. The percentage of 4H-NCDE remained constant over the pH range of 3-7. Moreover, the formation of OCDD decreased at pH 4, and the percentage of 2H-NCDE increased above pH 4. However, in the presence of BHA1 (Figure 4b), the percentage of all byproducts decreased with increasing pH. In particular, at pH 6 and 7, no OCDD and 2H-NCDE were detected. In the absence of both Fe(III)-TPPS and KHSO5, PCP and byproducts thereof may be incorporated into HS via hydrophobic interactions (29, 30). Because the pKa value of PCP is known to be 4.75, the majority of PCP is present as an anionic species above pH 5. Moreover, the amounts of anionic species for 2H- and 4H-NCDEs may appear to increase with increasing pH. Thus, because of electrostatic repulsion, the binding of PCP, 2H- and 4H-NCDEs to BHA1, which has a large negative electrostatic field, may be inhibited. To examine whether PCP binds with BHA1 at variety of pH values in the absence of a catalytic reaction, the HPLC data for PCP in a mixture of PCP and BHA1 were compared with those in the presence of PCP alone. Even in the presence of BHA1 (50 mg L-1), the retention time and peak area of PCP remained constant in the pH range of 3-7. Thus, the PCP disappearance detected in Figure 4b is not due to the hydrophobic binding of PCP to BHA1. Figure 5 shows the effect of BHA1 concentration on the percentage of PCP disappearance and byproduct formation. The percentage of PCP disappearance increased with BHA1 concentration and the percentages of byproduct conversion decreased with increasing BHA1 concentration. In a control experiment without a catalytic reaction, the HPLC peak area and retention time of PCP in the presence of 50 mg L-1 of BHA1 were similar to those with 100 mg L-1 of BHA1. These 390

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results also support the view that no binding between PCP and BHA1 occurred without the catalytic reaction. Byproducts in a Variety of HSs. Table 1 shows the percentages of PCP-derived byproducts in the presence of a variety of HSs. The ∆[PCP] term represents the concentration of PCP oxidized in the Fe(III)-TPPS/KHSO5 system. The [Cl-]/∆[PCP] values (1.3-1.7) indicate that 1-2 chlorine atoms are released from PCP during the oxidation. The byproducts detected, such as TeCC, 2H-NCDE, 4H-NCDE, and OCDD, may be appropriate, with respect to the [Cl-]/ ∆[PCP] ratios. In the photo-Fenton reaction, the [Cl-]/∆[PCP] values were reported to be above 3 (21). In this case, 30-40% of the PCP was oxidized to CO2. However, in the Fe(III)TPPS/KHSO5 system, further oxidation products, such as aromatic ring cleavage organic acids, were not detected in the reaction mixtures. Therefore, the further oxidation of PCP to organic acids and CO2 may not occur in the Fe(III)TPPS/KHSO5 system. In a previous study (2), the presence of HSs with a lower degree of humification leads to an enhancement in PCP disappearance. The origins of such types of HSs are peat and compost soils (BHA1, BHA2, SHA, and CHA in Table 1). In the absence of HS, the majority of the PCP was converted to 2H-NCDE, 4H-NCDE, and OCDD. In the presence of HS, the percentages of such dimers decreased and the percentages of unknown species increased, compared to the corresponding percentages in the absence of HS. In the presence of HSs with a lower degree of humification (BHA1, BHA2, SHA, and CHA), the percentages of residual 2H-NCDE, 4HNCDE, and OCDD decreased to below 2.1%, 10%, and 0.9%, respectively. The percentages of unknown species for these HSs were relatively larger (80% f 4.9 5.8 9.9 10 9.8 8.1 11 10 11 7.1 8.4 5.2 5.5

12 DL>f 1.0 0.9 8.0 2.5 5.5 1.7 1.7 2.1 4.1 1.9 4.8 2.1 1.4

63 2.7 2.5 7.0 64 24 50 17 14 17 41 18 44 10 18

7.8 0.3 0.7 0.7 5.7 3.3 3.2 2.5 1.5 1.6 3.4 1.9 3.6 0.9 1.6

13 97 91 86 12 60 32 71 72 70 41 71 39 82 74

a [PCP] - [PCP] b [Cl-] after 60 min of reaction period. c [o-TeCQ]/∆[PCP] × 100. d (2 × C)/∆[PCP] × 100 (C ) [2H-NCDE], 0 60 min, [PCP]0 ) 53 ( 2 µM. [4H-NCDE], and [OCDD]). e Unextractable species with n-hexane: 100 - %(o-TeCQ + 2H-NCDE + 4H-NCDE + OCDD). f Below detection limit.

TABLE 2. Chlorine Mass Balance before and after Reaction via the Fe(III)-TPPS/KHSO5 System in the Presence of HS (100 mg L-1) at pH 6 concentration of Cl/mg L-1 chlorine species reactiona

organic Cl in PCP before the AOX measured before the reaction inorganic Cl- after the reactionb AOX measured after the reaction calculated AOX after the reactionc organic Cl in PCP after the reactiona AOX in HS measured after the reactiond

SHA

BHA1

9.2 ( 0.2 8.8 ( 0.9 2.1 ( 0.1 7.5 ( 0.6 7.1 ( 0.2 1.0 ( 0.04 5.6 ( 0.7

9.0 ( 0.3 9.1 ( 0.8 1.9 ( 0.1 7.5 ( 0.8 7.1 ( 0.3 0.8 ( 0.03 6.1 ( 0.7

a The concentrations of PCP before and after the reaction were measured by HPLC. b The concentrations of Cl- after the reaction were measured by ion chromatograph. c (Organic Cl in PCP before the reaction) - (inorganic Cl- after the reaction). d AOX in 100 mg L-1 of HS fraction after the reaction with PCP.

solution of the HS fractions are shown in Table 2 (5.6 mg L-1 for SHA and 6.1 mg L-1 for BHA1). No chlorine was detected in aqueous solutions of unreacted HSs. Thus, it can be assumed that the AOX values in the HS fractions represent bound chlorinated species. To estimate the percentages of chlorine in the HS fractions, the AOX values in the test solutions were analyzed before and after the reaction. The mass actions of the chlorinated species in the test solutions are also summarized in Table 2. The chlorine contents (9.2 ( 0.2 and 9.0 ( 0.3 mg L-1 for SHA and BHA1), which were calculated from [PCP]0, were in good agreement with the AOX values in the test solutions prior to the reaction (8.8 ( 0.9 and 9.1 ( 0.3 mg L-1). These results indicate that PCP in the test solution can be detected as AOX species. In addition, the AOX values after the reaction can be calculated by subtracting the concentrations of inorganic chlorine, as measured by ion chromatograph, from those of the chlorine content of the PCP before the reaction, which were calculated from the [PCP]0. The AOX values after the reaction (7.5 ( 0.6 and 7.5 ( 0.8 mg L-1 for SHA and BHA1) were the same within experimental uncertainty as the calculated values (7.1 ( 0.2 and 7.1 ( 0.3 mg L-1). Therefore, all of organic chlorinated species in the test solution can be adsorbed on the AC and can be detected as AOX.

The chlorine content of the species that do not bind to HS can be calculated by subtracting the summation of AOX in the HS fraction and inorganic chlorine from AOX before the reaction. Because the total chlorine contents in the system would be represented by the AOX value before the reaction, the rate of each chlorinated species in the reaction mixture can be calculated (Supporting Information, Figure SI-1). The percentages of chlorine, incorporated into the HS fractions, were calculated to be 67% for BHA1 and 64% for SHA, respectively. Moreover, the percentages of unbound organic chlorine, which remained in the reaction mixtures, were calculated to be 11% for BHA1 and 13% for SHA, respectively. From “organic Cl in PCP after the reaction” in Table 2, the percentages of organic chlorine for the residual PCP were calculated to be 9.0% for BHA1 and 12% for SHA. This indicates that the majority of organic chlorine, which was not incorporated into the HS fractions, is due to the residual PCP. Py-GC/MS Studies. To identify the PCP-derived byproducts that were incorporated into HS, py-GC/MS was examined to the HS fractions. Figure 6 shows chromatograms of the BHA1 fractions and the peak assignments, respectively. When Fe(III)-TPPS and KHSO5 were reacted with PCP in the presence of BHA1, the methylated forms of 2,3,5-TrCP (18.3 min), 2,3,4,5-TeCP (20.2 min), 2,3,5,6-TeCP (23.2 min), and PCP (24.8 min) were detected as new peaks in the py-GC/MS chromatogram, as compared to the chromatograms for unreacted BHA1 and reacted BHA1 without PCP (reaction blank). In the case of unreacted BHA1, a PCP peak was not observed. Thus, PCP can be removed by the separation processes of the HS fraction from the reaction mixture. Similar results were obtained with SHA. We investigated the degradation characteristics of 2H-NCDE, 4H-NCDE, and OCDD during pyrolysis. Although 2H- and 4H-NCDEs were degraded to PCP as a result of pyrolysis, OCDD was not. If OCDD were present in the HS fractions, the peaks with as retention time of 49.9 min would be expected. However, the peak corresponding to OCDD was not observed in the HS fraction after reaction with PCP. Therefore, OCDD is not present in the HS fractions. A possible interaction between OCDD and HS in the absence of a catalytic reaction may be hydrophobic binding, and OCDD is more hydrophobic than PCP, o-TeCQ, and 2H- and 4H-NCDEs. Thus, such byproducts, which are less hydrophobic than OCDD, may not be incorporated into the HS fractions via hydrophobic interactions. Chlorophenolderived intermediates, such as chlorophenoxy radicals, are covalently coupled to HSs or their precursors (14, 15, 19, 21, VOL. 37, NO. 2, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Pyrolysis-GC/MS chromatograms of the BHA1 fraction before and after the reaction and the assignments of the peaks. 31). Moreover, it has been reported that model-coupling dimers between PCP and HS moieties (e.g. 3-methoxy-4(pentachlorophenoxy)benzenealdehyde and 2,6-dimethoxy(pentachlorophenoxy)phenyl ether) are degraded to PCP and phenols as a result of pyrolysis (21). Therefore, the appearance of chlorophenols in Figure 6 can be attributed to the degradation of chlorinated compounds coupled to HS as a result of pyrolysis. 13C NMR Studies. The py-GC/MS studies indicated the existence of covalent binding between PCP-derived intermediates and HS fractions. To observe the nature of the binding between HS and PCP-derived intermediates, 13Clabeled PCP was reacted in the Fe(III)-TPPS/KHSO5 system containing HS, and the 13C NMR spectra of the HS fractions were then measured. Figure 7 shows the 13C NMR spectra of the BHA1 and SHA fractions. Except for peaks around 39.5 ppm corresponding to CH3 of DMSO, no signals were observed in a DMSO-d6 solution containing 5% HS under the same accumulation conditions. In Figure 6, the majority of chlorinated intermediates were detected as 2,3,4,5-TeCP (peaks 25 and 26) and PCP (peak 28). These may be degradation products as a result of the pyrolysis of PCP- and o-TeCQ-HS coupling compounds. Therefore, we also obtained 13C NMR spectra of PCP and o-TeCQ. As shown in Figure 7, peaks corresponding to PCP and o-TeCQ were not overlapped with chemical shifts in the HS fractions. These results indicate that many of the peaks shown in the spectra of HS fractions are reaction intermediates derived from 13Clabeled PCP, which are incorporated into the HSs. In the enzymatic oxidation of 13C-labeled 2,6-dichlorophenol with HS, peaks corresponding to ether C-O linkages appeared in the 139-141 ppm and 143-151 ppm ranges, respectively (32). Moreover, in the photo-Fenton oxidation of 13C-labeled PCP with HS, peaks corresponding to C-O ether linkages were observed at 143-153 ppm (21). Therefore, the peaks for the HS fractions, which appear in the ranges 392

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FIGURE 7. 13C-NMR spectra of PCP, o-TeCQ, and the HS fractions isolated after reaction with 13C-labeled PCP. of 142-146 ppm and 149-153 ppm, indicate the presence of ether C-O linkages. The peaks in the 128-133 ppm region can be attributed to shifts as the result of C-C and C-O binding (21). The peaks in the 161-171 ppm region for SHA can be assigned to carbonyl carbons, such as quinones (32). These results lead to the conclusion that reaction intermediates derived from 13C-labeled PCP are covalently incorporated into HS, mainly in the form of ether C-O linkages.

TABLE 3. EC50 for AOX in the Test Solutions before and after Reaction EC50/mg L-1 test solutions

5 min

15 min

30 min

before reaction without HS after reaction without HS before reaction with BHA1 after reaction with BHA1 before reaction with SHA after reaction with SHA

0.21 ( 0.03 0.13 ( 0.01 0.24 ( 0.04 NDa 0.22 ( 0.01 0.21 ( 0.01

0.23 ( 0.02 0.15 ( 0.01 0.26 ( 0.03 NDa 0.23 ( 0.00 0.22 ( 0.02

0.25 ( 0.01 0.15 ( 0.01 0.29 ( 0.01 NDa 0.24 ( 0.01 0.23 ( 0.02

a EC 50 after the reaction in the presence of BHA1 could not be determined.

Toxicity Test of Reaction Mixtures. To estimate the toxicities in the test solutions before and after oxidation with PCP, a Microtox test was performed. To check the activity of the bacteria, the EC50 values for phenol were determined prior to each measurement. These values were 22.5 ( 0.6 mg L-1 for 5 min and 24.1 ( 0.8 mg L-1 for 15 min and were in good agreement with values in the literature (20-29 mg L-1 for 5 min and 21-34 mg L-1 for 15 min (33)). However, the EC50 values determined for PCP (0.29 ( 0.03 mg L-1 for 5 min, 0.30 ( 0.01 mg L-1 for 15 min, and 0.31 ( 0.02 mg L-1 for 30 min) were lower than those found in the literature (0.9-1.3 mg L-1 for 5 min, 0.6-1.1 mg L-1 for 15 min, and 0.5-1.0 mg L-1 for 30 min (33)). The EC50 values for the test solutions are summarized in Table 3. In the absence of HS, the EC50 value was decreased from 0.21 to 0.25 mg L-1 to 0.13-0.15 mg L-1 after the reaction. The smaller EC50 value can be taken to indicate a higher toxicity. Therefore, in the absence of HS, the toxicity of the test solution is increased. It has been reported that the oxidation of chlorophenols by peroxidase results in a decrease in toxicity, i.e., the toxicity of quinones derived from chlorophenols are lower than that of the original compounds (6). Thus, the formation of o-TeCQ may not play a role in increasing the overall toxicity. However, more toxic byproducts, such as 2H-NCDE, 4H-NCDE, and OCDD, were produced in the present system. Thus, the smaller EC50 value after the reaction can be attributed to a small contribution by more toxic byproducts. However, the water solubility of these compounds was too low to permit an AOX measurement. Thus, it was not possible to evaluate the toxicities of these individual compounds using the Microtox test. In the presence of SHA, the EC50 values after the reaction were similar to those obtained before the reaction. However, the EC50 values for BHA1 after the reaction could not be precisely evaluated. For the case of BHA1, because the maximum %E value for the reaction mixture did not reach 50% (Supporting Information, Figure SI-2), it was not possible to determine the EC50 values. However, the maximum AOX level in the reaction mixture for BHA1 was the same as those levels in the absence of HS and in the presence of SHA. This implies that, in the presence of BHA1, the rate of formation of toxic compounds in the reaction mixture was smaller than those for the other cases. Because residual PCP can be regarded as the main toxic compound, the toxicity of the reaction mixture in the presence of HSs may be dependent mainly on residual PCP. Toxicity Test of HS Fractions. After the catalytic reaction, more than 60% of the chlorine species was contained in the HS fractions. Therefore, the issue of whether these chlorinated intermediates in the HS fractions are toxic is relevant. Figure 8 shows a comparison of the toxicity curves between the mixtures without the reaction (PCP + HS) and the HS fractions after reaction with PCP. The EC50 values for the HS fractions could not be determined, since maximum %E value was below 50%. However, in Figure 8, the %E values for the HS

FIGURE 8. Comparisons of toxicity curves for the mixture without the catalytic reaction (PCP + HS) and the HS fraction after a catalytic reaction with PCP. fractions were clearly smaller than those for the mixtures without the catalytic reaction. These results leads to the conclusion that the chlorinated intermediates present in the HS fraction are less toxic than the original mixture of PCP and HS. The polymerization of organic pollutants via oxidative coupling with HS has been reported in a variety of oxidative processes (13, 14, 21, 32). However, the toxicity of the HS fraction, which is coupled with PCP-derived intermediates, has not been evaluated directly. In the present study, the Microtox test demonstrated that the toxicity of the chlorinated intermediates derived from PCP could be significantly reduced as the result of their covalent binding to HS. These results support the view that, if the HS reacts with PCP in the biomimetic catalytic system, in-situ detoxification of contaminating soils with PCP may be an achievable goal.

Acknowledgments This work was supported by Grants-in-Aid for Scientific Research in Japan Society for the Promotion of Science (14380283).

Supporting Information Available Figures SI-1 and -2 of the rate of chlorinated species in the reaction mixture and the toxicity curves of the test solutions before and after reaction with PCP. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review May 22, 2002. Revised manuscript received October 25, 2002. Accepted November 4, 2002. ES020747K