Environ. Sci. Technol. 2000, 34, 771-775
Effect of Environmental Factors on the Degradation of 2,6-Dichlorophenol in Soil PATRICK STEINLE,† PHILIPP THALMANN,‡ PATRICK HO ¨ HENER,§ KURT W. HANSELMANN,# AND G E R H A R D S T U C K I * ,† Ciba Specialty Chemicals Inc., WS-2090, CH-4133 Pratteln, Switzerland, Department of Environmental Science, Swiss Federal Institute of Technology, Ra¨mistrasse 101, CH-8000 Zu ¨ rich, Switzerland, Institute of Environmental Engineering, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland, and Institute of Plant Biology/Microbiology, University of Zurich, Zollikerstrasse 107, CH-8008 Zu ¨ rich, Switzerland.
Chlorinated phenols (CP) are frequently found as harmful soil contaminants. Depending on the environment, CP may persist for extended periods of time. The influence of environmental factors on the degradation of 2,6-dichlorophenol (2,6-DCP) in unsaturated soil was examined using Ralstonia basilensis RK1 as inoculum for bioaugmentation. The disappearance of 2,6-DCP in soil microcosms was caused by bacterial mineralization. This was proved using U-14Clabeled 2,6-DCP. After 5 days of incubation, 61% of the initial activity was detected as 14CO2, while only 20% of the radioactivity remained in the soil, and 2,6-DCP was not detected. The relative importance of individual factors and possible two-factor interactions was assessed using a fractional-factorial experimental design. The following individual factors were identified as important: 2,6-DCP concentration, temperature, inoculum size, and the presence of an additional substrate. The strongest factorial interaction was observed between bacterial inoculation and 2,6-DCP concentration. For practical reasons, the influence of oxygen, organic matter, and the age of the contamination were not included in the factorial design; however, these factors were analyzed separately and found to significantly affect the biodegradation of 2,6-DCP. The findings of this study are important for the design of bioremediation techniques as well as the prediction of natural attenuation.
Introduction Chlorophenols (CP) are harmful soil contaminants that are frequently released into the environment either directly as pesticides or due to improper handling of intermediary chemical products and wastes (1, 2). In the last years, there * Corresponding author phone: ++41 61 636 97 29; fax: ++41 61 636 93 29; e-mail:
[email protected]. † Ciba Specialty Chemicals Inc. ‡ Department of Environmental Science, Swiss Federal Institute of Technology. § Institute of Environmental Engineering, Swiss Federal Institute of Technology. # University of Zurich. 10.1021/es990587l CCC: $19.00 Published on Web 01/20/2000
2000 American Chemical Society
have been successful pilot- and field scale studies for on-site bioremediation of soils contaminated with CP (1, 3). Processes for the treatment of contaminated groundwater have also been developed and were applied successfully (4). There is, however, an important lack of reliable and comprehensive data concerning the influence of environmental factors on the degradation of chlorophenols in situ, in the unsaturated soil. Such data are needed to enhance the reliability of predictions on the fate of CP in soil, and the success of in situ bioremediation actions for sites where excavation and external treatment of the soil is not possible. Few studies have investigated the influence of more than one environmental factor on the degradation of xenobiotic compounds (5-9). Even fewer have attempted to weight such factors and to assess possible interactions (10). In the present study, the influence of environmental factors on the degradation of 2,6-DCP in unsaturated soil was examined. A statistical experimental design (11) was employed to weigh and quantify the relative importance of the examined factors and to assess possible interactions between them. The usefulness of such statistical experimental designs to assess interactions between factors influencing biodegradation of xenobiotic compounds has been demonstrated by Millette et al. (10). The environmental factors studied included soil humidity, oxygen concentration, age of contamination, temperature, organic matter content, contaminant concentration, presence of an additional substrate, and density of CP-degrading bacteria. The range of the factors was set such as to correspond to values prevalent in situ at a CP-polluted site near Amponville, France, or to values that could be attained by cost-efficient bioremediation measures. The response variable kR used to express the readiness of biological degradation was the ratio of the initial 2,6-DCP concentration C0 divided by the time t50 required for the removal of 50% of the initial 2,6-DCP. 2,6-DCP was looked at as strategically the most critical compound for the CP-congeners because it is present in concentrations up to several mmol × kg-1 at the Amponville-site, and because it has been shown in previous experiments (12) to be the least biodegradable of the three major pollutants 2,4-DCP, 2,6-DCP, and 2,4,6-TCP. The effect of bioaugmentation was studied using Ralstonia basilensis RK1 which is the only bacterial strain known to degrade 2,6-DCP aerobically in pure culture (12).
Experimental Section Soil. The loamy-sand soil used for this study was chosen to correspond to the subsoil of a CP-polluted landfill at Amponville (France). It had no history of previous chemical contamination, which allowed controlled spiking with chemicals and bacteria. For experiments with low organic matter content and low buffer capacity, sand with a similar size distribution was used. Soil properties were previously determined (13). Bacterial Strain and Culture Conditions. Ralstonia basilensis RK1 (DSM 11853) for soil inoculation was pregrown at room temperature (20 ( 3 °C) in shake-flask cultures containing 300 µM 2,6-DCP dissolved in a phosphate-buffered mineral medium (12). CFUs of the preculture were determined by direct plating on nutrient agar prior to inoculation of the soil. Chemicals. 2,6-DCP (98-99% pure) was purchased from EGA-Chemie, Steinheim, Germany. U-14C-labeled 2,6-DCP (97.5% pure, 1.9 MBq × mg-1) was provided by Anawa Trading, Du ¨ bendorf, Switzerland. VOL. 34, NO. 5, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Chemical analysis. For 2,6-DCP analysis, soil samples (5 g dw) were put in a centrifuge tube and mixed with 5 mL of an acetonitrile/H2O/H3PO4 mixture (900:100:1). The tube was shaken at 250 rpm for 30 min and then centrifuged at 2500 × g for 15 min. The supernatant was diluted 1:1 with deionized H2O prior to analysis by reversed-phase HPLC and UVdetection. Headspace-O2 and -CO2 were measured by GCthermal conductivity detector (TCD). Chloride was extracted from the soil with water and quantified by ion-chromatography. Soil Microcosms. Soil was dried overnight at 60 °C, put in a dough mixer (Hobart, Zu ¨ rich, Switzerland), and then combined under constant stirring with 2,6-DCP, bacterial culture solution, mineral medium (12), and phenol to reach the desired incubation conditions. Phenol and 2,6-DCP were added from 15 mM stock solutions at pH 8. Stirring was continued for 30 min. One hundred grams dry weight (dw) of soil were filled into 100 mL Schott flasks closed with Teflonlined rubber septa. The volume of the headspace amounted to 75 ( 6 mL. The effect of individual factors on 2,6-DCP degradation was studied using quadruplet samples, whereas for the factorial design, duplicate samples were set up for each incubation grade. Abiotic controls were prepared by adding 0.2 g of NaN3 to the microcosms. Uncontaminated controls without 2,6-DCP were prepared for the investigation of individual factors. To prepare an aged 2,6-DCP contamination in the soil, 500 g of soil was filled in 1 L Schott flasks and autoclaved three times for 30 min in 24 h intervals to prevent microbial degradation. Sterile 2,6-DCP and water were added. The flasks were tightly closed, vigorously shaken for 30 min, and stored at room temperature in the dark. Sampling was carried out under sterile conditions after 182 and 365 days. Partitioning of 2,6-DCP in Soil. For toxicity considerations, it was assumed that only the fraction of 2,6-DCP present in the water phase was relevant. This fraction was estimated according to the following assumptions (cf. Supporting Information): (i) the deprotonated fraction of 2,6-DCP is completely dissolved, and (ii) the protonated fraction is distributed among the gas-, liquid-, and solidphases within the microcosms according to the Henry and sorption constants of 2,6-DCP. Sampling of Microcosms. For headspace measurements of O2 and CO2, the GC-TCD was equipped with a needle, which was directly introduced through the flask septa for sampling. Then, the microcosms were opened, the soil was mixed manually to obtain a representative sample, and a 5 g of portion of soil was removed for 2,6-DCP analysis. Microoxic samples were flushed with N2 for 20 min after sampling. Mineralization Experiments. A mass balance for 2,6-DCP degradation was established in inoculated and abiotic microcosms, using U-14C-labeled 2,6-DCP. These experimental conditions were those of combination no. 4 of the factorial design (Table 1). One kilogram (dw) of soil was conditioned in a dough mixer as described above, except that 155 kBq U-14C-labeled 2,6-DCP dissolved in 30 µL of ethanol was added along with the unlabeled 2,6-DCP. Twenty 250 mL Schott flasks were each filled with 50 g dw soil. Each Schott flask was equipped with a Falcon tube in the headspace containing 5 mL of 2 M NaOH. For sampling, duplicate flasks were sacrificed. Mineralization was determined by acidifying the soil with 10 mL of 5 M H3PO4 under shaking for 30 min thereby trapping the 14CO2 in NaOH. NaOH was removed and mixed to 15 mL of scintillation cocktail (SC). Remaining activity in the soil was quantified by burning of 1 g of soil in a furnace (Thermicon P, Heraeus, Zu ¨ rich, Switzerland), alimented with 8 L O2 × h-1. The resulting 14CO2 in the offgas was captured in 5 mL of NaOH (2 M), which was mixed in 15 mL of SC. Counting was performed with a liquid scintillation analyzer. 772
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TABLE 1: Microcosm Incubation Conditions for the Fractional Factorial Experimental Design RK1 phenol 2,6-DCP inoculation concn concn combination [CFU soil moisture [µmol [µmol no. temp × g-1 dw] [% WHCmax] × kg-1 dw] × kg-1 dw] high value low value 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
20 8 8 20 20 20 8 20 8 20 20 8 12 8 20 8 8 8 20
106 103 103 103 106 106 103 106 106 103 103 103 105 103 106 106 106 106 103
90 30 90 30 30 90 30 30 90 90 90 90 60 30 90 30 90 30 30
106 0 0 0 0 0 0 106 106 0 106 106 53 106 106 0 0 106 106
617 62 62 62 617 62 617 62 62 617 62 617 339 62 617 62 617 617 617
Experimental Setup Individual Factors. To analyze the effect of individual environmental factors, standard conditions were defined as follows: 617 µmol 2,6-DCP × kg-1 soil, 20 °C incubation temperature, no aging of the contamination, 2.1% organic matter content, 90% maximum water holding capacity (WHCmax), 0.2 atm O2, 106 CFU Ralstonia basilensis RK1 × g-1 soil. Keeping all other factors constant, the individual factors were changed to values indicated on the x-axes in Figure 1. Abiotic and uncontaminated controls were included for each factor. Fractional-Factorial Experimental Design. Table 1 lists the different incubation conditions of the fractional-factorial design, by which the relative importance of the factors was weighted and the effect of interactions was examined. We chose a 2n-1-fractional-factorial design with one center point, where n is the number of factors examined at two levels each (high and low, Table 1). This experimental set up allows statements on the effect of the individual factors as well as on two-factor interactions (11). Design and evaluation of these experiments were supported by statgraphics-software (Statistical Graphics Corp., Princeton, N. J.). Statistical Evaluation of Data. For the evaluation of the raw data, the time t50 required for the removal of 50% of the initial 2,6-DCP concentration C0 was determined by nonlinear regression. Removal of 2,6-DCP as a function of time was described by one of three different functions (Table 2). The selected functions fulfilled the following criteria: (i) close fit to the data over the entire time range (R 2 > 90%) and thus good estimation of t50, (ii) broad applicability (function fits all experimental runs exhibiting similar degradation patterns), and (iii) factors can be determined by the Marquardt-method (14). The functions (Table 2) were fitted to the data by the Marquardt method, using statgraphics software. Three fitting runs were performed, each using the results of the previous run as starting values. For the first run, the parameters for the logistic equation were estimated by a linear regression of -ln(S(t)/S0) versus time. For the logarithmic equation, k and B were replaced by µmax and X0 given in ref 12. For the second-order function, k1 and k2 were set at -0.1 and 0.1, respectively. The ratio kR ) C0/t50 was used to express the readiness of 2,6-DCP biodegradation. It was employed as a response variable to evaluate the fractional factorial design. To increase
FIGURE 2. Degradation of 2,6-DCP in unsaturated soil. Values represent the average from two independent runs at a standard deviations of 300 d, depending on the incubation conditions. The difference in rate between the abiotic controls and the samples can be attributed to microbial degradation. A representative mass balance of the microbial conversion of 2,6-DCP was determined in detail in the combination no. 4 of the factorial design (Table 1): 2,6-DCP (6.45 µmol) disappeared completely, and 9.6 µmol chloride ions or 75%
of the amount expected were found. The amount of O2 consumed and CO2 produced did not serve as evidence for 2,6-DCP mineralization since they were more than 10 times higher than those calculated (38.7 µmol) for complete 2,6DCP oxidation. However, the O2/CO2-balance measured in aged microcosms agreed well with the biological 2,6-DCP removal. Due to the sterilization procedure, there was respiration only from the bacteria inoculated after the aging period. The respiration due to 2,6-DCP degradation during the first 5 days was calculated for the spiked samples, subtracting the respiration from controls (115 ( 20 µmol O2 consumed and 108 ( 13 µmol CO2 produced) receiving no 2,6-DCP. The O2 consumption amounted thus to 132 ( 30 µmol, CO2 production was 167 ( 21 µmol, corresponding to 61 ( 14% and 77 ( 10% of the values expected for the mineralization of the removed 2,6-DCP (36 µmol). Further evidence for the mineralization of 2,6-DCP was obtained using U-14C-labeled 2,6-DCP (Figure 2). 61% of the initial activity could be trapped as 14CO2 in NaOH within 5 days. The remaining activity in the soil might have been incorporated into biomass and/or be strongly absorbed. From day 7 onward, the 2,6-DCP concentration was below the detection limit, whereas in the poisoned control, 88% of the initial 2,6-DCP were still detected after 9 days (data not shown). Effect of Individual Environmental Factors on the Degradation of 2,6-DCP. Figure 1 shows the effect of the variation of individual factors on the 2,6-DCP biodegradation ratio kR in soil microcosms. All other factors remained at the standard conditions. 2,6-DCP Concentration. The initial concentration of 2,6DCP had a strong effect on kR, which was highest at 247 µmol 2,6-DCP × kg-1 and decreased strongly toward both lower and higher initial concentrations (Figure 1A). The lower kR at 62 than at 247 µmol 2,6-DCP × kg-1 dw might be explained by a considerable amount of substrate that was difficult for the bacteria to access, which increased t50 in the microcosm with low 2,6-DCP concentration, whereas at the higher concentration, the poorly accessible fraction does not have to be degraded before t50 is reached. At concentrations above 247 µmol × kg-1, the decrease of kR is due to substrate inhibition: 2,6-DCP is known to be toxic to bacteria, acting as uncoupling agent on biological membranes (15). In microtox-assays, EC50 for Vibrio fisherii strain NRRL B 1117 was found to be 239 µM (13). In liquid cultures, the growth rate of R. basilensis RK1 decreases from a maximum at 309 µM 2,6-DCP toward no growth at 1200 µM and above VOL. 34, NO. 5, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(12). Considering partitioning of 2,6-DCP in soil, the actual concentration in the pore water in microcosms spiked with 62 µmol × kg-1 soil was estimated to be 233 µM. In microcosms spiked with 617 µmol × kg-1 soil, a pore water concentration of 2329 µM 2,6-DCP was calculated. While the first value is close to what allows a maximum growth rate of R. basilensis in liquid batch culture, the latter lies in the toxic range where complete inhibition was observed. Apparently, the soil environment provides protection from 2,6-DCP toxicity as evidenced by the slow degradation of 2,6-DCP (Figure 1A). The protection mechanism might be “buffer and depot”, as described by (16) for activated carbon. Temperature. Lowering the temperature led to a decrease in the degradation activity, with a four times lower kR at 8 °C than at 20 °C (Figure 1B). The effect of temperature on 2,6-DCP-degradation could be fitted to the Arrhenius equation. With the linear regression of ln k (Table 2, function 2) versus T-1, the activation energy Ea was estimated at 65.16 kJ × mol-1 and the frequency factor A at 24.69 M-1 × d-1 (R 2 ) 95.8%). The activation energy obtained is in the range of activation energies found for liquid batch cultures (17). Studies on pentachlorophenol degradation (18, 19) revealed significantly higher activation energies, reaching approximately seven times higher degradation rates with a temperature increase of 10 °C. Age of Contamination. Most studies on the effect of the age of contamination on biodegradation in soil report slower degradation rates and lower extent of degradation with longer incubation times of the chemicals in soil (cf. refs 20 and 21). In our experiments, with 2,6-DCP aged in sterilized soil for 6 or 12 months prior to inoculation of strain RK1, the opposite was observed: kR was much higher in aged than in freshly spiked soil (Figure 1C). There are several possible explanations for this phenomenon: (i) Due to abiotic processes, such as oxidation, polymerization (22, 23), or diffusion into microfissures of the solids (24), the extractable 2,6-DCP concentration at the beginning of the experiment was lower in the aged samples (344 ( 24 µmol × kg-1 and 473 ( 9 µmol×kg-1 in the soils aged for 12 and 6 months, respectively) than in the freshly contaminated control (617 ( 10 µmol × kg-1). Lower initial extractable 2,6-DCP led to increased kR. The decrease of extractable 2,6-DCP from aged soil was concomitant with an even stronger decrease in soil toxicity, as measured by a microtox-assay (according to ref 13, data not shown). (ii) To prevent microbial degradation of 2,6-DCP during the aging process, the soil had been autoclaved, which is known to alter its physicochemical properties (25). (iii) The bacteria inoculated into the sterilized soil were not exposed to competition and predation, which may have contributed to the survival of a high number of active 2,6-DCP degraders. Organic Matter Content. In microcosms containing sand (0.1% (w/w) organic matter), t50 increased to about 5 years, and kR was below 0.5 µmol 2,6-DCP × kg-1 × d-1 (Figure 1D). An explanation for this strong effect might again come from the 2,6-DCP concentration in the sand pore water. The concentration calculation revealed that the bacteria were exposed to about 3914 µM 2,6-DCP. At this concentration level, growth of R. basilensis RK1 is inhibited. Soil Moisture. Low soil moisture decreased kR (Figure 1E). Again, there might be a masked effect of concentration, as a low water content leads to a high 2,6-DCP concentration in the pore water (from 2329 µM at 90% WHCmax to 6781 µM at 30% WHCmax). In liquid culture, concentrations in this range do not allow growth of R. basilensis RK1. In soil containing organic matter, there is some protection against the toxicity; however, kR is still clearly lowered. Oxygen Level. The oxygen concentration is crucial for the degradation of 2,6-DCP by the aerobic bacterium 774
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FIGURE 3. Graphical presentation of the statistical evaluation of the effects of individual environmental factors (A) and combined effects of two factors (B) on kR of 2,6-DCP in unsaturated soil. All experiments were run in duplicate; kR-values did not defer more than 10%. For details refer to the text. Ralstonia basilensis RK1 (12). In the micro-oxic microcosms, with oxygen concentrations below 0.01 atm O2, the degradation proceeded very slowly (Figure 1F). Bioaugmentation. Strain RK1 was able to contribute to a faster degradation of 2,6-DCP under the incubation conditions studied (Figure 1G). The linear correlation of the number of inoculated Ralstonia basilensis RK1 and kR amounted to + 0.7 only, mainly because a higher kR was obtained with 103 cells × g-1 soil dw than with 105 cells × g-1 dw due to unknown reasons. A further statistical test (test for any trend) confirmed, however, the positive correlation between bioaugmentation and kR. Relevance of Individual Environmental Factors and of Interactions between Them. The aim of the fractionalfactorial experimental design was to find the relative importance of individual environmental factors on biodegradation of 2,6-DCP in soil and to identify important interactions between pairs of factors. Interactions of higher order were not taken into consideration. Three factors were excluded from these experiments: oxygen concentration, as O2 has been shown to be absolutely needed for efficient 2,6-DCP degradation; organic matter content; and the age of the contamination, as they cannot be varied independently. All other factors were examined only at two levels each (high and low, Table 1). As an additional factor, the presence of another less toxic substrate (106 µmol phenol × kg-1 dw) was investigated. Phenol is an excellent growth substrate for R. basilensis RK1 (12), and it was expected that its addition might lead to faster degradation of 2,6-DCP, possibly by raising the RK1 cell number. The results of the statistical evaluation of the five individual factors and their interactions are graphically shown in Figure 3. The factors (temperature, inoculation density of strain RK1, soil moisture, addition of phenol, and 2,6-DCP concentration) were plotted against kR as response variable for 2,6-DCP degradation (Figure 3A). The slopes for soil temperature, inoculation density of strain RK1, and addition of phenol were positive, thus indicating a faster degradation at the higher level, whereas the contrary was true for soil moisture and 2,6-DCP concentration, showing a negative slope. The steepness of the slope is an indicator for the quantitative importance of the respective factor. The combined effects of couples of factors (two factor interactions) are shown in Figure 3B. The effect of an increase (from left to right) of the first mentioned factor of each couple of factors is shown for the low level of the second factor (line marked with -) and for the high level of the second factor
obtaining a more detailed picture of the influence of the respective factor over the chosen range.
Acknowledgments This work was partially supported by Grant 980033 from the Safety, Health and Environment Department of Novartis Inc. and Ciba Specialty Chemicals Inc. to P. Steinle. The authors thank K. Eigenmann for his support and P. Ackermann and A. Fredenhagen for their amiable help with the radiolabeled experiments. FIGURE 4. Standardized effects of individual environmental factors and combined effects of two factors (interactions) on kR of 2,6-DCP in unsaturated soil. The vertical line represents the 90% confidence interval for a significant effect. - and + signs indicate negative and positive effects, respectively. (line marked with +). Any discrepancy between the two lines can be attributed to an interaction between the respective factors. Increasing temperature, cf., has only a minor effect on kR at high 2,6-DCP concentrations. At low 2,6-DCP concentration, however, the same increase in temperature leads to a several times higher kR. The strongest interactions were observed between the 2,6-DCP concentration on one side and temperature and inoculation on the other side (Figure 3B). The effects of all individual factors and of two-factor interactions were normalized by dividing their absolute effect through the mean effect of all factors and interactions and were statistically analyzed regarding their significance on kR (Figure 4). The concentration of 2,6-DCP was the most important factor, followed by temperature, the inoculation of strain RK1, and phenol addition. Soil moisture and all two-factor interactions except the one between 2,6-DCP concentration and inoculation of strain RK1 had no significant effect on kR (p > 0.1). The results obtained form the statistical analysis demonstrate the importance of 2,6-DCP concentration on kR. Of course, this factor is, at the same time, the crucial issue and the very reason for every remediation measures to take place at chlorophenol-polluted sites. As the toxicity toward potential degrading microorganisms is an important reason for the persistence of CP in soil (presuming the availability of sufficient oxygen), the degradation can be expected to take place wherever the concentrations are low due to sorption and/or dispersion processes which are typically occurring downgradient of heavily polluted sites. Downgradient remediation measures such as bioscreens (26) provided with bacteria able to degrade CP could support such a spatial confinement. Both the beneficial influence of high temperature and of bioaugmentation on 2,6-DCP degradation could be confirmed. Assessing the interactions between individual factors led to important findings (Figure 3B): The inhibition by high 2,6-DCP concentrations attenuates the influence of both temperature, addition of strain RK1 and of phenol on 2,6DCP degradation. Although not significant on the 90% confidence level, inoculation of strain RK1 seems to be more effective in dry than in wet soil, and the success of bioaugmentation is enhanced when phenol as additional substrate is available. The effects of the variation of individual environmental factors on kR corresponded well with those found by the factorial design. The factorial design allowed for determining the relative importance of the factors and in assessing interactions. The variation of individual factors allowed for
Supporting Information Available Calculation of the 2,6-DCP concentration in the pore water of unsaturated soil and tables of Cw calculated for different microcosms and symbols and values used for the calculation of Cw. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review May 24, 1999. Revised manuscript received December 1, 1999. Accepted December 2, 1999. ES990587L
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