Environ. Sci. Technol. 1996, 30, 1253-1260
Pathway and Rate of Chlorophenol Transformation in Anaerobic Estuarine Sediment SHIGEKI MASUNAGA,† S R I D H A R S U S A R L A , * ,† J E N N I F E R L . G U N D E R S E N , ‡,§ A N D YOSHITAKA YONEZAWA† National Institute for Resources and Environment, 16-3 Onogawa, Tsukuba, Ibaraki, 305 Japan, and College of William and Mary, School of Marine Science, Virginia Institute of Marine Science, Department of Environmental Sciences, Gloucester Point, Virginia 23062
The dechlorination of chlorophenols (CPs) in anaerobic estuarine sediment was studied. The sediment used was fairly contaminated with various anthropogenic chemicals from the surrounding industries. The sulfate content of the interstitial and overlying waters was approximately 20 mmol/L, and the sediment was apparently sulfidogenic. All 13 CPs tested transformed following a first-order reaction kinetics. The rate constants for CP disappearance ranged between 0.010 and 0.38 day-1 or had half-lives between 1.8 and 70 days. The intermediates detected in the experiment indicated that the ortho-dechlorination was the preferred pathway. The disappearance of the parent compound and the accumulation and disappearance of dechlorinated intermediates were simulated using a branched chain first-order irreversible reaction kinetics, and the contributions of different pathways were estimated. A comparison of the estimated rate constants with the redox potential of the reaction showed that the hypothesis of microbial selection of most thermodynamically favorable pathways observed for chlorobenzene dechlorination was not applicable for CPs.
Introduction Chlorophenols (CPs) are an important class of chemical compounds and intermediates in chemical manufacturing industries, such as pesticides and dyes. Contamination of soil and sediment by CPs has often been reported (1, 2). The microbial degradation of CPs has been reported extensively in the last few years in aerobic and anaerobic * Author to whom all correspondence should be addressed; telephone: 81-209-58-8311; fax: 81-298-58-8309; e-mail address: pl682@ nire.go.jp. † National Institute for Resources and Environment. ‡ College of William and Mary. § Present address: U.S. EPA, National Health and Environmental Effect Laboratory, Atlantic Ecology Division, 27 Tarzwell Dr., Narragansett, RI 02882.
0013-936X/96/0930-1253$12.00/0
1996 American Chemical Society
conditions (3, 4). The transformation of CPs, especially dechlorination under anaerobic conditions, has become of great interest. The dechlorination of a number of CPs were reported recently in soils, sediments, and sewage sludges under various conditions (5-7). For example, fresh sewage sludge was found to dechlorinate monochlorophenol (MCP) isomers with transformation rates in the order of ortho > meta > para (6). The microbial communities from two different pond sediments were found to dechlorinate CPs in the order: ortho > meta or para (7). The transformation of trichlorophenols (TCPs) to dichlorophenols (DCPs) and MCPs by a methanogenic enrichment culture from a sewage sludge also shows a similar preference for dechlorination (8, 9). Mikesell and Boyd (10) reported that anaerobically digested sewage sludges that were acclimated to three MCP isomers degraded pentachlorophenol (PCP) by ortho-, meta-, and para-dechlorination, respectively, but accumulated less chlorinated phenols. Transformations under sulfidogenic conditions were observed (11), although the addition of sulfate often inhibited the transformation of CPs. Ha¨ggblom and Young (12) found that sulfidogenic culture from an anaerobic reactor dechlorinated DCPs to MCP and phenol. As indicated by the above studies, under the anaerobic conditions (both methanogenic and sulfidogenic), the first step of CP transformation appears to be dechlorination. However, quantitative studies dealing with relative contribution of different dechlorination pathways are limited. The specific objectives of this study are (i) to investigate the transformation of CPs and to determine rate constants and (ii) to identify and determine the pathway of transformation reactions in anaerobic sediment.
Materials and Methods Estuarine Sediment. The sediment was collected from the mouth of the Tsurumi River, which flows into Tokyo Bay. A large number of factories discharge their treated wastewaters into the river. Contamination of the sediment with various industrial chemicals, such as toluene, xylene, ethylbenzene, aniline, trichlorobenzene, hexachlorobenzene, and decabromodiphenyl ether has been reported (13). The concentrations of some the background pollutants are shown in Table 1. The sediment was taken from the river bed in 1992 using an Ekman-Barge sediment sampler. The sediment was black in color, indicating a possible sulfate-reducing activity. Macrobenthic organisms such as small crabs, small fish, and shellfish were present in the collected sediment. The sediment was passed through a 2-mm sieve to remove debris. The concentrations of sodium chloride (NaCl), sulfate, and nitrate in the sediment interstitial water were 1.7%, 1.98 × 103 mg/L (20.6 mmol/L), and below the detection limit, respectively. River water (24 °C) was also sampled just above the bottom of the sampling point. The NaCl, sulfate, and nitrate concentrations were 1.5%, 1.84 × 103 mg/L (19.2 mmol/L), and 4.0 mg/L, respectively. The oxygen concentration of the river water was approximately 7 mg/L. The sieved sediment sample was mixed with the river water under nitrogen gas bubbling to make a slurry. The pH of the sediment slurry was 5.6. The total solid concentration and
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TABLE 1
Percentage of Recovery of Chlorophenols and Threshold Concentration Values threshold concentrationb ×10-7 (mol/L)
% recoverya
spiked
2-MCP 3-MCP 4-MCP 2,3-DCP 2,4-DCP 2,5-DCP 2,6-DCP 3,4-DCP 3,5-DCP 2,3,4-TCP
100 100 100 88 99 90 97 95 100 79
1.0 2.1 NAd NA NA 2.5 NA 4.0 1.4 NA
3,4,5-TCP
100
0.8
70 49
NA NA
chlorophenol
2,3,4,5-TeCP PCP
intermediate
pollutantsc originally present (× 10-7 mol/L) NDj 0.3k
NA (3-CP) NA (2-CP, 4-CP) NA (2-CP, 3-CP) NA (2-CP) 0.5 (3-CP + 4-CP)e 1.7 (3-CP)f 4.0 (3,4-DCP)g NA (3-CP + 4-CP, 2,3-DCP) 0.6 (3,4-DCP)g NA (3-CP + 4-CP, 3,5-DCP) NA (all intermediates)h 0.4 (2,4,5-TCP)i NA (all other intermediates)h
0.2 ND ND ND 1.2 ND ND ND ND ND
a Percentage recovery is calculated based on the initial spiked amount. b Threshold concentration is the concentration of CP isomer remaining after 300-day incubation. c Concentration of phenol and 1,4-DCB in the sediment were 1.2 × 10-4 and 1.9 × 10-7 mol/L, respectively. d NA threshold value if any was below the detection limit (1 × 10-8 mol/L). e Threshold value of sum of 3-CP and 4-CP as metabolites of 3,4-DCP. f Threshold value of 3-CP as a metabolite of 3,5-DCP. g Threshold value of 3,4-DCP as a metabolite of 3,4,5-TCP. h List of intermediates are shown in Table 3. i Threshold value of 2,4,5-TCP as a metabolite of PCP; the threshold concentration for the rest of the isomers was below the detection limit. j ND, below detection limit (1 × 10-8 mol/L). k Sum of 3-CP and 4-CP.
the ratio of ignition loss/total solid of the sediment slurry were 272 ((2.8) g/kg (or 311 g/L) and 12.1% ((0.033), respectively. These were an average of four replicate measurements. Chemicals. Chlorophenols were purchased from Tokyo Kasei Kogyo Co. Ltd. (TCI), Tokyo, Japan, while 2,5dichlorophenol was obtained from Wako Pure Chemicals Industries, Ltd., Osaka, Japan. Ten out of the 13 isomers had purity >99.9%, while 2,3,4-TCP, 3,4,5-TCP, and PCP had monochlorophenol impurity in trace concentration. The CPs were used without further purification for all experiments. Stock solutions for the 13 CP isomers at a concentration of 2 × 10-3 mol/L were prepared in methanol. Preparation of Test Tubes. For the incubation experiment, 5 mL of the sediment slurry, stirred by bubbling N2 gas through the mixture, was placed in a screw-top test tube and sealed under N2 atmosphere using a Teflon-coated cap. Forty-three test tubes with sediment slurry were prepared for each CP isomer. They were kept sealed at room temperature for 1 week to ensure that the system was anaerobic. Eight out of the 43 test tubes were then autoclaved at 120 °C for 20 min to obtain sterile controls. Due to the large number of test tubes to be handled during incubation, only the addition of the test compound was done in the anaerobic chamber. Transformation Experiments. All the test tubes were placed in an anaerobic chamber (10% hydrogen, 10% carbon dioxide, and 80% nitrogen), and 10 µL of stock solution was spiked by opening the screw caps. The concentration of the test compounds in the slurry was 4 × 10-6 mol/L, which was 1-2 orders of magnitude higher than the background pollutants. Just after spiking, time zero samples were frozen at -20 °C. The remaining test tubes were placed in a constant temperature room at 25 °C during the experiment. The test tubes were mixed by turning upside down by hand for a few times on every Monday, Wednesday, and Friday. The test tubes were sampled once a day at the beginning of the incubation and then at intervals as the experiment
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progressed. The test tubes were stored in a -20 °C freezer until further analysis. The incubation lasted for 1 year. All experiments were carried out in duplicate. For analysis, one set of samples were used, while the other set was kept as a backup. Analysis. To quantify the loss of parent compound and the appearance of its intermediate metabolites, the entire sample (5 mL of sediment slurry) was used for analysis. After thawing the test tubes, 1 N HCl (200 µL) followed by 1 mL of acetonitrile and 1 mL of benzene were added and then mixed for 1 h on a reciprocal shaker. The test tubes were then left overnight in the refrigerator. The following day, the mixtures were spiked with 10 µL (250 mg/L naphthalene-d8 solution served as an internal standard), shaken for 15 min, and centrifuged for 15-20 min at 2000 rpm. The supernatant benzene extract layer was placed in an autosampler vial for analysis. The extracted samples were analyzed by GC/MS (HP5971 MSD) with a 25-m capillary column (Hewlett Packard HP5). CPs were quantified without derivatization using a selected-ion monitoring method. Molecular ion (M+) and the (M + 2)+ ion were monitored for each isomer. The final results were corrected by the recovery rate of each isomer based on the surrogate standard. The recovery rates of all the CP isomers were found to be in the range of 50100% (Table 1), which indicates the percentage of CP extracted from the sediment to the initial spiked concentration. Sulfate Monitoring. The monitoring of sulfate depletion during the course of the experiment was carried out with and without the presence of chlorophenol. The incubation experiment was similar to the procedure described above for CPs. The sulfate concentration was analyzed using an ion chromatograph (Dionex 4000i).
Results Effect of Methanol. As described before, the stock solutions were prepared in methanol. This introduces an extra
FIGURE 1. SO42- reduction in the presence and in the absence of chlorophenol (3,4,5-TCP).
carbon source. The addition of methanol or other forms of organic matter normally present in river water can serve as electron donors to support transformation reactions or to accelerate the rate of metabolism (9, 14). Madsen and Aamand (9) and others reported that the addition of an extra carbon source could accelerate the rate of dehalogenation reaction. However, in this study, no attempts were made to examine the influence of methanol on the overall rate of dechlorination reaction. Sulfate Reduction. The depletion of SO42- during the course of experiment is shown in Figure 1 in the absence as well as in the presence of chlorophenol. The sulfate reduction followed a first-order reaction with a rate constant of 0.0026 and 0.0033 day-1 with and without the presence of chlorophenol, respectively. Between days 100 and 200, SO42- concentration nearly reached a plateau (Figure 1). Similarly, the concentration of 3,4,5-TCP also reached a plateau. This supports the dechlorination of chlorophenol occurring under SO42- reduction (15). Transformation Rates. The transformation of 13 CPs was investigated in a sediment slurry at an initial pH of 5.6. The sampling was done at frequent intervals during initial degradation to determine the lag period, which is a characteristic of biodegradation (16). However, the existence of a definite lag time was not confirmed. This was partly due to a scatter in the observed data. Also, the preexposure of the sediment to various chemicals including some CP isomers may be the cause of the nonexistence of a lag time. The decrease in concentration of the spiked CP and the increase of possible dechlorinated intermediates were analyzed. Results of typical experimental runs are shown in Figures 2-5. The depletion of CP could be approximated to a first-order reaction against the parent compound concentration. The first-order rate constants (k) and 95% confidence intervals for all CPs are listed in Table 2. The fit of the data to first-order reaction was very good and all the rate constants obtained were significantly different from zero at 1% significance level. The half-lives in test sediment ranged from 1.8 to 70 days. In a similar study using the same sediment, the authors have found that the half-life periods for chlorobenzenes were between 17 and 46 days, except for 1,4-dichlorobenzene and 1,3-dichlorobenzene which were 385 and 433 days, respectively (17). The formation of phenol, the ultimate dehalogenated product, could not be quantified because phenol was
FIGURE 2. Transformation of some dichlorophenol isomers in estuarine sediment: (a) 2,4-DCP and (b) 2,6-DCP. Solid and dashed lines represent the fit of the first-order reaction kinetics for the spiked compound in nonsterile sediment data and autoclaved sediment data, respectively. Simulation was done using a branched first-order kinetics for the intermediates.
FIGURE 3. Transformation of 2,3,4-TCP and its metabolites in estuarine sediment. The solid and dashed lines for 2,3,4-TCP show the firstorder fit to the nonsterile and autoclaved sediment data, respectively.
already present in the sediment prior to the beginning of the experiment. A large peak was observed on the GC/SIM chromatogram at the retention time of phenol and had an identical mass spectrum to that of phenol. The loss of CPs in autoclaved sediment was not significant as shown in Figures 2-5. The data from autoclaved sediment were also fitted to a first-order model, and the rate constants are shown in Table 2. The k values obtained in autoclaved
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TABLE 2
First-Order Rate Constants for Chlorophenol Depletion in Sediment and Sterile Sediment test sediment
a
sterile sediment
chlorophenol
ka (day-1)
t1/2 b (day)
r2c
k (day-1)
r2
2-MCP 3-MCP 4-MCP 2,3-DCP 2,4-DCP 2,5-DCP 2,6-DCP 3,4-DCP 3,5-DCP 2,3,4-TCP 3,4,5-TCP 2,3,4,5-TeCP PCP
0.101 ( 0.033** 0.024 ( 0.007** 0.059 ( 0.017** 0.053 ( 0.008** 0.045 ( 0.012** 0.015 ( 0.003** 0.139 ( 0.022** 0.001 ( 0.001** 0.040 ( 0.009** 0.377 ( 0.153** 0.032 ( 0.006** 0.107 ( 0.017** 0.327 ( 0.093**
6.9 29.2 11.7 13.2 15.3 46.3 5.0 69.9 17.3 1.8 21.7 6.5 2.1
0.88 0.83 0.91 0.96 0.89 0.91 0.98 0.95 0.90 0.89 0.93 0.97 0.91
0.0029 ( 0.0020* 0.0004 ( 0.0006 0.0002 ( 0.0010 0.0006 ( 0.0012 0.0007 ( 0.0004** 0.0016 ( 0.0007** 0.0027 ( 0.0027 -0.0005 ( 0.0013 0.0002 ( 0.0007 0.0005 ( 0.0009 -0.0002 ( 0.0007 -0.0002 ( 0.0008 -0.0006 ( 0.0017
0.69 0.35 0.03 0.24 0.75 0.88 0.56 0.15 0.06 0.21 0.07 0.06 0.10
k is the first-order rate constant; *, significant at 5% level. **, significant at 1% level. b t1/2 is the half-life period. c r2 is the coefficient of determination.
FIGURE 4. Transformation of 2,3,4,5-TeCP and its metabolites in estuarine sediment. The solid and dashed lines for 2,3,4,5-TeCP show the first-order fit to the nonsterile and autoclaved sediment data, respectively.
FIGURE 5. Transformation of PCP and its metabolites in estuarine sediment. The solid and dashed lines for PCP show the first-order fit to the nonsterile sediment and autoclaved sediment data, respectively.
sediment were not significantly different from zero for most of the CP isomers. The order of transformation rates among MCPs was ortho > para > meta. This order was different to some of the previous reports in which the orders of ortho > meta > para and para > meta > ortho were observed in sewage sludge and sulfidogenic conditions, respectively (5, 12). Chlorophenols with ortho-chlorine(s) had shorter half-lives than those without ortho-chlorines (2-CP vs. 3-CP and 4-CP; 2,3-DCP, 2,4-DCP, and 2,6-DCP vs. 3,4-DCP and 3,5-DCP; 2,3,4-TCP vs. 3,4,5-TCP). However, ortho-substituted CP with another chlorine at the diagonal position of the orthochlorine had a longer half-life, e.g., 2,5-DCP (Table 2). However, Smith and Woods (18) in a study of reductive dechlorination of CPs by vitamin B12s reported that chlorines ortho to the phenol group were particularly resistant to dechlorination. Transformation Pathways. In all the CPs tested, dehalogenated intermediates were detected with the exception of MCPs. The formation of phenol and the dechlorinated intermediate of MCP, could not be certified due to the presence of phenol in the collected sediment. The detected intermediates are shown in Table 3. Accumulated intermediates from 2,3-DCP, 2,4-DCP, and 2,5-
DCP indicated that ortho-chlorine was preferably removed compared to the meta- and para-chlorine. This was also true with 2,3,4-TCP, 2,3,4,5-tetrachlorophenol (2,3,4,5TeCP), and PCP. The preferential ortho-dehalogenation was also reported by other researchers (6, 7, 11). In the case of 2,3-DCP, 2,3,4-TCP, and PCP, the accumulation of dehalogenated intermediates indicated that the first transformation step was primarily dehalogenation. On the other hand, the mass balance of the accumulation of dechlorinated intermediates for 2,4-DCP, 2,5-DCP, and 3,4-DCP did not account for the total loss of the parent compound, thereby indicating that dehalogenation may not be the only transformation pathway.
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Estimating Contribution of Each Transformation Pathway. The CPs, especially higher chlorinated ones, were shown to dechlorinate by more than one pathway. The concentration profiles of intermediate metabolites in Figures 2-5 indicate that the substrates were transformed independently in the presence of each other. To estimate the contribution of each dechlorination step to the overall reaction, a branched chain first-order reaction kinetic model was used (19). Since no interactions of the substrates were observed, they were not accounted for in
TABLE 3
Intermediates Detected in Chlorophenol Transformation Experiments substrate
intermediatesa
2-MCP 3-MCP 4-MCP 2,3-DCP 2,4-DCP 2,5-DCP 2,6-DCP 3,4-DCP 3,5-DCP 2,3,4-TCP 3,4,5-TCP 2,3,4,5-TeCP
no intermediates detected no intermediates detected no intermediates detected 3-MCP 4-MCP > 2-MCP 3-MCP > 2-MCP 2-MCP 3-/4-MCPb 3-MCP 3,4-DCP > 2,3-DCP; 3-/4-MCPb 3,4-DCP > 3,5-DCP; 3-/4-MCPb 3,4,5-TCP ) 2,4,5-TCP > 2,3,5-TCP ) 2,3,4-TCP; 3,4-DCP > 3,5-DCP > 2,4-/2,5-DCPc; 3-/4-MCPb 2,3,4,5-TeCP; 2,4,5-TCP ) 3,4,5-TCP > 2,3,5-TCP; 3,4-DCP > 2,4-/2,5-DCPc ) 3,5-DCP; 3-/4-MCPb
PCP
Phenol could not be quantified. > and ) indicate the relative maximum concentration among isomers. b GC peaks for 3-MCP and 4-MCP could not be separated. c GC peaks for 2,4-DCP and 2,5-DCP could not be separated. a
the model. The model consists of three mass balance relationships, the first two accounting for the dechlorination reaction, for example, 2,4-DCP dechlorinating to 2-CP (intermediate -1) and 4-CP (intermediate -2), and the third one accounting for the possible nondechlorination pathways. The model assumes a first-order reaction kinetics for the parent compound and also for intermediate metabolites. The equations are represented as dechlorinated intermediate –1 [D1] uka
Parent CP [PC]
vka wka
dechlorinated intermediate –2 [D2]
kb1 kb2
nondechlorinated intermediates [NX]
kbx
where [PC] is the concentration of parent CP; [D1] is the concentration of dechlorinated intermediate -1; [D2] is the concentration of dechlorinated intermediate -2; [NX] is the total concentration of non-dechlorination intermediates; ka, kb1, kb2, and kbx are the first-order transformation rate constants for parent CP, dechlorinated intermediates -1 and -2, and the total of non-dechlorinated intermediates, respectively; u, v, and w are the contribution factor of each pathway (u + v + w ) 1). f is the pathway by a single type of reaction; - f is the pathway by a single or possibly multiple types of reaction. The differential equations describing the observed dechlorination and nondechlorination reactions are as follows:
d[PC] ) -ka[PC] dt
(1)
d[D1] ) uka[PC] - kb1[D1] dt
(2)
d[D2] ) vka[PC] - kb2[D2] dt
(3)
d[NX] ) wka[PC] - kbx[NX] dt
(4)
FIGURE 6. Simulation of 2,3,4,5-TeCP transformation using branched chain first-order irreversible reaction kinetics.
The analytical solutions for these equations are
[PC] ) [PC]0e(-kat)
(5)
[D1] ) uka[PC]0(e-kat - ekb1t)/(kb1 - ka)
(6)
[D2] ) vka[PC]0(e-kat - ekb2t)/(kb2 - ka)
(7)
[NX] ) wka[PC]0(e-kat - e-kbxt)/(kbx - ka)
(8)
where t is the time from the start of the incubation, and [PC]0 is the initial concentration of parent chlorophenol. Using these solutions, the authors estimated the relative contribution of each transformation pathway. The estimation procedure will be explained using 2,4-DCP as an example. The identified dehalogenation intermediates for this substrate are 2-MCP and 4-MCP. The transformation rate constants for 2-MCP and 4-MCP determined in each spiked experiment were 0.10 and 0.06 day-1, respectively (Table 2). To determine the contribution of each pathway, eqs 5-7 were first calculated assuming that u ) v ) 0.5 (where transformation depends completely on dechlorination and the contribution of each pathway is the same). By comparing the observed and simulated maximum concentrations for each intermediate, the contribution factors u and v were adjusted. Next, the times when the observed and the simulated maximum intermediate concentration appeared were compared with each other by adjusting the first-order rate constants for the intermediates. In general, the rate constants observed for intermediate chlorophenols were 1-3 times larger than those for the spiked substrates. Lastly, the simulated maximum concentration was readjusted to the observed concentration by changing the contribution factors. By this method, the contribution of each pathway to the dechlorination reaction was estimated and the results are tabulated in Table 4. The results of the simulation are shown in Figures 2 and 6. The transformation of 2,3-DCP was estimated to be completely by ortho-dechlorination. The contribution of the 2-MCP pathway was estimated to be less than 4%. Similarly, 2,6-DCP and 3,5-DCP were degraded completely through ortho- and meta-dechlorination, respectively. However, for 2,5-DCP and 2,4-DCP, o-chlorine was preferentially removed over the m- or p-chlorine, but the summation of the dechlorination pathways did not explain the total loss of the parent compounds. Therefore, a
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TABLE 4
First-Order Rate Constant (k) and Redox Potential (E0′) for Each Chlorophenol Dechlorination Pathway overall k (day-1)
position of Cl removal
product
2-MCP 3-MCP 4-MCP 2,3-DCP
0.10 0.02 0.06 0.05
2,4-DCP
0.04
2,5-DCP
0.02
2,6-DCP 3,4-DCP
0.14 0.001
3,5-DCP 2,3,4-TCP
0.04 0.38
3,4,5-TCP
0.03
2,3,4,5-TeCP
0.11
PCP
0.33
ortho meta para ortho meta ortho para ortho meta ortho meta para meta ortho meta para meta para ortho meta para meta ortho meta para
phenolb phenolb phenolb 3-MCP 2-MCP 4-MCP 2-MCP 3-MCP 2-MCP 2-MCP 4-MCP 3-MCP 3-MCP 3,4-DCP 2,4-DCP 2,3-DCP 3,4-DCP 3,5-DCP 3,4,5-TCP 2,4,5-TCP 2,3,5-TCP 2,3,4-TCP 2,3,4,5-TeCP 2,3,4,6-TeCP 2,3,5,6-TeCP
parent
% contribution factor
100 NA 15 11 20 10 100 30c 100 96
