Effect of Environmental Factors onthe Degradation of2,6-Dichlorophenol in SoilP A T R I C K S T E I N L E , †

P H I L I P P T H A L M A N N , ‡

P A T R I C K H O H E N E R , §

K U R T W . H A N S E L M A N N , # A N DG E R H A R D S T U C K I * , †

Ciba Specialty Chemicals Inc., WS-2090,CH-4133 Pratteln, Switzerland, Department of EnvironmentalScience, Swiss Federal Institute of Technology,Ramistrasse 101, CH-8000 Zurich, Switzerland, Institute ofEnvironmental Engineering, Swiss Federal Institute ofTechnology, CH-1015 Lausanne, Switzerland, and Institute ofPlant Biology/Microbiology, University of Zurich,Zollikerstrasse 107, CH-8008 Zurich, Switzerland.

Chlorinated phenols (CP) are frequently found as harmfulsoil contaminants. Depending on the environment, CP maypersist for extended periods of time. The influence ofenvironmental factors on the degradation of 2,6-dichlorophenol(2,6-DCP) in unsaturated soil was examined using Ralstoniabasilensis RK1 as inoculum for bioaugmentation. Thedisappearance of 2,6-DCP in soil microcosms was causedby bacterial mineralization. This was proved using U-14C-labeled 2,6-DCP. After 5 days of incubation, 61% of the initialactivity was detected as 14CO2, while only 20% of theradioactivity remained in the soil, and 2,6-DCP was notdetected. The relative importance of individual factors andpossible two-factor interactions was assessed using afractional-factorial experimental design. The followingindividual factors were identified as important: 2,6-DCPconcentration, temperature, inoculum size, and the presenceof an additional substrate. The strongest factorialinteraction was observed between bacterial inoculationand 2,6-DCP concentration. For practical reasons, theinfluence of oxygen, organic matter, and the age of thecontamination were not included in the factorial design;however, these factors were analyzed separately and foundto significantly affect the biodegradation of 2,6-DCP. Thefindings of this study are important for the design ofbioremediation techniques as well as the prediction ofnatural attenuation.

IntroductionChlorophenols (CP) are harmful soil contaminants that arefrequently released into the environment either directly aspesticides or due to improper handling of intermediarychemical products and wastes (1, 2). In the last years, there

have been successful pilot- and field scale studies for on-sitebioremediation of soils contaminated with CP (1, 3). Processesfor the treatment of contaminated groundwater have alsobeen developed and were applied successfully (4). There is,however, an important lack of reliable and comprehensivedata concerning the influence of environmental factors onthe degradation of chlorophenols in situ, in the unsaturatedsoil. Such data are needed to enhance the reliability ofpredictions on the fate of CP in soil, and the success of insitu bioremediation actions for sites where excavation andexternal treatment of the soil is not possible.

Few studies have investigated the influence of more thanone environmental factor on the degradation of xenobioticcompounds (5-9). Even fewer have attempted to weight suchfactors and to assess possible interactions (10).

In the present study, the influence of environmentalfactors on the degradation of 2,6-DCP in unsaturated soilwas examined. A statistical experimental design (11) wasemployed to weigh and quantify the relative importance ofthe examined factors and to assess possible interactionsbetween them. The usefulness of such statistical experimentaldesigns to assess interactions between factors influencingbiodegradation of xenobiotic compounds has been dem-onstrated by Millette et al. (10).

The environmental factors studied included soil humidity,oxygen concentration, age of contamination, temperature,organic matter content, contaminant concentration, presenceof an additional substrate, and density of CP-degradingbacteria. The range of the factors was set such as tocorrespond to values prevalent in situ at a CP-polluted sitenear Amponville, France, or to values that could be attainedby cost-efficient bioremediation measures. The responsevariable kR used to express the readiness of biologicaldegradation was the ratio of the initial 2,6-DCP concentrationC0 divided by the time t50 required for the removal of 50%of the initial 2,6-DCP. 2,6-DCP was looked at as strategicallythe most critical compound for the CP-congeners becauseit is present in concentrations up to several mmol × kg-1 atthe Amponville-site, and because it has been shown inprevious experiments (12) to be the least biodegradable ofthe three major pollutants 2,4-DCP, 2,6-DCP, and 2,4,6-TCP.The effect of bioaugmentation was studied using Ralstoniabasilensis RK1 which is the only bacterial strain known todegrade 2,6-DCP aerobically in pure culture (12).

Experimental SectionSoil. The loamy-sand soil used for this study was chosen tocorrespond to the subsoil of a CP-polluted landfill atAmponville (France). It had no history of previous chemicalcontamination, which allowed controlled spiking with chemi-cals and bacteria. For experiments with low organic mattercontent and low buffer capacity, sand with a similar sizedistribution was used. Soil properties were previouslydetermined (13).

Bacterial Strain and Culture Conditions. Ralstoniabasilensis RK1 (DSM 11853) for soil inoculation was pregrownat room temperature (20 ( 3 °C) in shake-flask culturescontaining 300 µM 2,6-DCP dissolved in a phosphate-bufferedmineral medium (12). CFUs of the preculture were deter-mined by direct plating on nutrient agar prior to inoculationof the soil.

Chemicals. 2,6-DCP (98-99% pure) was purchased fromEGA-Chemie, Steinheim, Germany. U-14C-labeled 2,6-DCP(97.5% pure, 1.9 MBq × mg-1) was provided by AnawaTrading, Dubendorf, Switzerland.

* Corresponding author phone: ++41 61 636 97 29; fax: ++4161 636 93 29; e-mail: gerhard.stucki@cibasc.com.

† 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.

Environ. Sci. Technol. 2000, 34, 771-775

10.1021/es990587l CCC: $19.00 2000 American Chemical Society VOL. 34, NO. 5, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 771Published on Web 01/20/2000

Chemical analysis. For 2,6-DCP analysis, soil samples (5g dw) were put in a centrifuge tube and mixed with 5 mL ofan acetonitrile/H2O/H3PO4 mixture (900:100:1). The tube wasshaken at 250 rpm for 30 min and then centrifuged at 2500× g for 15 min. The supernatant was diluted 1:1 with deionizedH2O prior to analysis by reversed-phase HPLC and UV-detection. Headspace-O2 and -CO2 were measured by GC-thermal conductivity detector (TCD). Chloride was extractedfrom the soil with water and quantified by ion-chromatog-raphy.

Soil Microcosms. Soil was dried overnight at 60 °C, putin a dough mixer (Hobart, Zurich, Switzerland), and thencombined under constant stirring with 2,6-DCP, bacterialculture solution, mineral medium (12), and phenol to reachthe desired incubation conditions. Phenol and 2,6-DCP wereadded from 15 mM stock solutions at pH 8. Stirring wascontinued for 30 min. One hundred grams dry weight (dw)of soil were filled into 100 mL Schott flasks closed with Teflon-lined rubber septa. The volume of the headspace amountedto 75 ( 6 mL. The effect of individual factors on 2,6-DCPdegradation was studied using quadruplet samples, whereasfor the factorial design, duplicate samples were set up foreach incubation grade. Abiotic controls were prepared byadding 0.2 g of NaN3 to the microcosms. Uncontaminatedcontrols without 2,6-DCP were prepared for the investigationof individual factors. To prepare an aged 2,6-DCP contami-nation in the soil, 500 g of soil was filled in 1 L Schott flasksand autoclaved three times for 30 min in 24 h intervals toprevent microbial degradation. Sterile 2,6-DCP and waterwere added. The flasks were tightly closed, vigorously shakenfor 30 min, and stored at room temperature in the dark.Sampling was carried out under sterile conditions after 182and 365 days.

Partitioning of 2,6-DCP in Soil. For toxicity consider-ations, it was assumed that only the fraction of 2,6-DCPpresent in the water phase was relevant. This fraction wasestimated according to the following assumptions (cf.Supporting Information): (i) the deprotonated fraction of2,6-DCP is completely dissolved, and (ii) the protonatedfraction is distributed among the gas-, liquid-, and solid-phases within the microcosms according to the Henry andsorption constants of 2,6-DCP.

Sampling of Microcosms. For headspace measurementsof O2 and CO2, the GC-TCD was equipped with a needle,which was directly introduced through the flask septa forsampling. Then, the microcosms were opened, the soil wasmixed manually to obtain a representative sample, and a 5g of portion of soil was removed for 2,6-DCP analysis. Micro-oxic samples were flushed with N2 for 20 min after sampling.

Mineralization Experiments. A mass balance for 2,6-DCPdegradation was established in inoculated and abioticmicrocosms, using U-14C-labeled 2,6-DCP. These experi-mental conditions were those of combination no. 4 of thefactorial design (Table 1). One kilogram (dw) of soil wasconditioned in a dough mixer as described above, exceptthat 155 kBq U-14C-labeled 2,6-DCP dissolved in 30 µL ofethanol was added along with the unlabeled 2,6-DCP. Twenty250 mL Schott flasks were each filled with 50 g dw soil. EachSchott flask was equipped with a Falcon tube in the headspacecontaining 5 mL of 2 M NaOH. For sampling, duplicate flaskswere sacrificed. Mineralization was determined by acidifyingthe soil with 10 mL of 5 M H3PO4 under shaking for 30 minthereby trapping the 14CO2 in NaOH. NaOH was removedand mixed to 15 mL of scintillation cocktail (SC). Remainingactivity in the soil was quantified by burning of 1 g of soilin a furnace (Thermicon P, Heraeus, Zurich, Switzerland),alimented with 8 L O2 × h-1. The resulting 14CO2 in the off-gas was captured in 5 mL of NaOH (2 M), which was mixedin 15 mL of SC. Counting was performed with a liquidscintillation analyzer.

Experimental SetupIndividual Factors. To analyze the effect of individualenvironmental factors, standard conditions were defined asfollows: 617 µmol 2,6-DCP × kg-1 soil, 20 °C incubationtemperature, no aging of the contamination, 2.1% organicmatter 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 factorswere changed to values indicated on the x-axes in Figure 1.Abiotic and uncontaminated controls were included for eachfactor.

Fractional-Factorial Experimental Design. Table 1 liststhe different incubation conditions of the fractional-factorialdesign, by which the relative importance of the factors wasweighted and the effect of interactions was examined. Wechose 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 allowsstatements on the effect of the individual factors as well ason two-factor interactions (11). Design and evaluation ofthese experiments were supported by statgraphics-software(Statistical Graphics Corp., Princeton, N. J.).

Statistical Evaluation of Data. For the evaluation of theraw data, the time t50 required for the removal of 50% of theinitial 2,6-DCP concentration C0 was determined by nonlinearregression. Removal of 2,6-DCP as a function of time wasdescribed by one of three different functions (Table 2). Theselected functions fulfilled the following criteria: (i) close fitto the data over the entire time range (R 2 > 90%) and thusgood estimation of t50, (ii) broad applicability (function fitsall 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 theMarquardt method, using statgraphics software. Three fittingruns were performed, each using the results of the previousrun as starting values. For the first run, the parameters forthe logistic equation were estimated by a linear regressionof -ln(S(t)/S0) versus time. For the logarithmic equation, kand B were replaced by µmax and X0 given in ref 12. For thesecond-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 of2,6-DCP biodegradation. It was employed as a responsevariable to evaluate the fractional factorial design. To increase

TABLE 1: Microcosm Incubation Conditions for the FractionalFactorial Experimental Design

combinationno. temp

RK1inoculation

[CFU× g-1 dw]

soil moisture[% WHCmax]

phenolconcn[µmol

× kg-1 dw]

2,6-DCPconcn[µmol

× kg-1 dw]

high value 20 106 90 106 617low value 8 103 30 0 621 8 103 90 0 622 20 103 30 0 623 20 106 30 0 6174 20 106 90 0 625 8 103 30 0 6176 20 106 30 106 627 8 106 90 106 628 20 103 90 0 6179 20 103 90 106 6210 8 103 90 106 61711 12 105 60 53 33912 8 103 30 106 6213 20 106 90 106 61714 8 106 30 0 6215 8 106 90 0 61716 8 106 30 106 61717 20 103 30 106 617

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the degrees of freedom of the evaluation, and thus to increasethe statistical significance of the results, only major effectsand interactions between two factors were taken into account.Interactions with insignificant effects (p > 0.3, determinedby analysis of variance) were eliminated from further analysis.

Results and DiscussionProof of 2,6-DCP Mineralization in Unsaturated Soils. Inall experiments, 2,6-DCP disappeared faster in living than inabiotic controls. The average (linear) rate of 2,6-DCP disap-pearance in the controls was 0.11 ( 0.02 d-1 (t50 455 ( 74 d),whereas t50 in the samples varied greatly from 2 d to > 300d, depending on the incubation conditions. The differencein rate between the abiotic controls and the samples can beattributed to microbial degradation.

A representative mass balance of the microbial conversionof 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 for2,6-DCP mineralization since they were more than 10 timeshigher than those calculated (38.7 µmol) for complete 2,6-DCP oxidation. However, the O2/CO2-balance measured inaged microcosms agreed well with the biological 2,6-DCPremoval. Due to the sterilization procedure, there wasrespiration only from the bacteria inoculated after the agingperiod. The respiration due to 2,6-DCP degradation duringthe 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 no2,6-DCP. The O2 consumption amounted thus to 132 ( 30µmol, CO2 production was 167 ( 21 µmol, corresponding to61 ( 14% and 77 ( 10% of the values expected for themineralization of the removed 2,6-DCP (36 µmol).

Further evidence for the mineralization of 2,6-DCP wasobtained using U-14C-labeled 2,6-DCP (Figure 2). 61% of theinitial activity could be trapped as 14CO2 in NaOH within 5days. The remaining activity in the soil might have beenincorporated into biomass and/or be strongly absorbed. Fromday 7 onward, the 2,6-DCP concentration was below thedetection limit, whereas in the poisoned control, 88% of theinitial 2,6-DCP were still detected after 9 days (data notshown).

Effect of Individual Environmental Factors on theDegradation of 2,6-DCP. Figure 1 shows the effect of thevariation of individual factors on the 2,6-DCP biodegradationratio kR in soil microcosms. All other factors remained at thestandard conditions.

2,6-DCP Concentration. The initial concentration of 2,6-DCP had a strong effect on kR, which was highest at 247 µmol2,6-DCP × kg-1 and decreased strongly toward both lowerand higher initial concentrations (Figure 1A).

The lower kR at 62 than at 247 µmol 2,6-DCP × kg-1 dwmight be explained by a considerable amount of substratethat was difficult for the bacteria to access, which increasedt50 in the microcosm with low 2,6-DCP concentration, whereasat the higher concentration, the poorly accessible fractiondoes not have to be degraded before t50 is reached. Atconcentrations above 247 µmol × kg-1, the decrease of kR isdue to substrate inhibition: 2,6-DCP is known to be toxic tobacteria, acting as uncoupling agent on biological membranes(15). In microtox-assays, EC50 for Vibrio fisherii strain NRRLB 1117 was found to be 239 µM (13). In liquid cultures, thegrowth rate of R. basilensis RK1 decreases from a maximumat 309 µM 2,6-DCP toward no growth at 1200 µM and above

FIGURE 1. Effects of variation of individual factors in unsaturatedsoil on the biodegradation index kR (C0/t50; in µmol × kg-1 × d-1).All other factors were kept at standard conditions mentioned in thetext. Data points represent the average of four replicates, standarddeviations were below 10%.

TABLE 2: Degradation Patterns and Functions Used forNonlinear Regression, Depending on the IncubationConditionsa

incubation condition

degradationpattern

observedfunction used for

nonlinear regression

low 2,6-DCP concn logistic S(t) ) (S0/(1 + B × e(S0×k×t))high 2,6-DCP concn,

wet soillogarithmic S(t) ) S0 + B × (1 - e(k×t))

high 2,6-DCP concn,dry soil

second-orderdecline

S(t) ) S0 + k1× t + k2 × t2

a S(t): substrate concentration at time t; S0: initial substrateconcentrated; B: factor; k: rate.

FIGURE 2. Degradation of 2,6-DCP in unsaturated soil. Valuesrepresent the average from two independent runs at a standarddeviations of <10%. (A) Degradation of unlabeled 2,6-DCP. 2: %of 2,6-DCP remaining in soil at experimental conditions given forcombination no. 4 (Table 2). (B) Degradation of U-14C 2,6-DCP inunsaturated soil. 9: remaining radioactivity in soil after acidification;0: remaining radioactivity of the abiotic control; [: radioactivitytrapped as CO2. 14CO2 evolving from the abiotic control: <1%; ].

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(12). Considering partitioning of 2,6-DCP in soil, the actualconcentration in the pore water in microcosms spiked with62 µmol × kg-1 soil was estimated to be 233 µM. Inmicrocosms spiked with 617 µmol × kg-1 soil, a pore waterconcentration of 2329 µM 2,6-DCP was calculated. While thefirst value is close to what allows a maximum growth rate ofR. basilensis in liquid batch culture, the latter lies in the toxicrange where complete inhibition was observed. Apparently,the soil environment provides protection from 2,6-DCPtoxicity as evidenced by the slow degradation of 2,6-DCP(Figure 1A). The protection mechanism might be “buffer anddepot”, as described by (16) for activated carbon.

Temperature. Lowering the temperature led to a decreasein the degradation activity, with a four times lower kR at 8°C than at 20 °C (Figure 1B). The effect of temperature on2,6-DCP-degradation could be fitted to the Arrhenius equa-tion. With the linear regression of ln k (Table 2, function 2)versus T-1, the activation energy Ea was estimated at 65.16kJ × 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 onpentachlorophenol degradation (18, 19) revealed significantlyhigher activation energies, reaching approximately seventimes higher degradation rates with a temperature increaseof 10 °C.

Age of Contamination. Most studies on the effect of theage of contamination on biodegradation in soil report slowerdegradation rates and lower extent of degradation with longerincubation times of the chemicals in soil (cf. refs 20 and 21).In our experiments, with 2,6-DCP aged in sterilized soil for6 or 12 months prior to inoculation of strain RK1, the oppositewas observed: kR was much higher in aged than in freshlyspiked soil (Figure 1C).

There are several possible explanations for this phenom-enon: (i) Due to abiotic processes, such as oxidation,polymerization (22, 23), or diffusion into microfissures ofthe solids (24), the extractable 2,6-DCP concentration at thebeginning of the experiment was lower in the aged samples(344 ( 24 µmol × kg-1 and 473 ( 9 µmol×kg-1 in the soilsaged for 12 and 6 months, respectively) than in the freshlycontaminated control (617 ( 10 µmol × kg-1). Lower initialextractable 2,6-DCP led to increased kR. The decrease ofextractable 2,6-DCP from aged soil was concomitant with aneven stronger decrease in soil toxicity, as measured by amicrotox-assay (according to ref 13, data not shown). (ii) Toprevent microbial degradation of 2,6-DCP during the agingprocess, the soil had been autoclaved, which is known toalter its physicochemical properties (25). (iii) The bacteriainoculated into the sterilized soil were not exposed tocompetition and predation, which may have contributed tothe 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 fromthe 2,6-DCP concentration in the sand pore water. Theconcentration calculation revealed that the bacteria wereexposed to about 3914 µM 2,6-DCP. At this concentrationlevel, growth of R. basilensis RK1 is inhibited.

Soil Moisture. Low soil moisture decreased kR (Figure1E). Again, there might be a masked effect of concentration,as a low water content leads to a high 2,6-DCP concentrationin the pore water (from 2329 µM at 90% WHCmax to 6781 µMat 30% WHCmax). In liquid culture, concentrations in thisrange do not allow growth of R. basilensis RK1. In soilcontaining organic matter, there is some protection againstthe toxicity; however, kR is still clearly lowered.

Oxygen Level. The oxygen concentration is crucial forthe degradation of 2,6-DCP by the aerobic bacterium

Ralstonia basilensis RK1 (12). In the micro-oxic microcosms,with oxygen concentrations below 0.01 atm O2, the degrada-tion proceeded very slowly (Figure 1F).

Bioaugmentation. Strain RK1 was able to contribute toa faster degradation of 2,6-DCP under the incubationconditions studied (Figure 1G). The linear correlation of thenumber of inoculated Ralstonia basilensis RK1 and kR

amounted to + 0.7 only, mainly because a higher kR wasobtained 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 cor-relation between bioaugmentation and kR.

Relevance of Individual Environmental Factors and ofInteractions between Them. The aim of the fractional-factorial experimental design was to find the relative im-portance of individual environmental factors on biodegra-dation of 2,6-DCP in soil and to identify importantinteractions between pairs of factors. Interactions of higherorder were not taken into consideration.

Three factors were excluded from these experiments:oxygen concentration, as O2 has been shown to be absolutelyneeded for efficient 2,6-DCP degradation; organic mattercontent; and the age of the contamination, as they cannotbe varied independently. All other factors were examinedonly at two levels each (high and low, Table 1). As anadditional factor, the presence of another less toxic substrate(106 µmol phenol × kg-1 dw) was investigated. Phenol is anexcellent growth substrate for R. basilensis RK1 (12), and itwas expected that its addition might lead to faster degradationof 2,6-DCP, possibly by raising the RK1 cell number.

The results of the statistical evaluation of the five individualfactors and their interactions are graphically shown in Figure3. The factors (temperature, inoculation density of strainRK1, soil moisture, addition of phenol, and 2,6-DCP con-centration) were plotted against kR as response variable for2,6-DCP degradation (Figure 3A). The slopes for soil tem-perature, inoculation density of strain RK1, and addition ofphenol were positive, thus indicating a faster degradation atthe higher level, whereas the contrary was true for soilmoisture and 2,6-DCP concentration, showing a negativeslope. The steepness of the slope is an indicator for thequantitative importance of the respective factor.

The combined effects of couples of factors (two factorinteractions) are shown in Figure 3B. The effect of an increase(from left to right) of the first mentioned factor of each coupleof factors is shown for the low level of the second factor (linemarked with -) and for the high level of the second factor

FIGURE 3. Graphical presentation of the statistical evaluation ofthe effects of individual environmental factors (A) and combinedeffects of two factors (B) on kR of 2,6-DCP in unsaturated soil. Allexperiments were run in duplicate; kR-values did not defer morethan 10%. For details refer to the text.

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(line marked with +). Any discrepancy between the two linescan be attributed to an interaction between the respectivefactors. Increasing temperature, cf., has only a minor effecton kR at high 2,6-DCP concentrations. At low 2,6-DCPconcentration, however, the same increase in temperatureleads to a several times higher kR. The strongest interactionswere observed between the 2,6-DCP concentration on oneside and temperature and inoculation on the other side(Figure 3B).

The effects of all individual factors and of two-factorinteractions were normalized by dividing their absolute effectthrough the mean effect of all factors and interactions andwere statistically analyzed regarding their significance on kR

(Figure 4). The concentration of 2,6-DCP was the mostimportant factor, followed by temperature, the inoculationof strain RK1, and phenol addition. Soil moisture and alltwo-factor interactions except the one between 2,6-DCPconcentration and inoculation of strain RK1 had no significanteffect on kR (p > 0.1).

The results obtained form the statistical analysis dem-onstrate the importance of 2,6-DCP concentration on kR. Ofcourse, this factor is, at the same time, the crucial issue andthe very reason for every remediation measures to take placeat chlorophenol-polluted sites. As the toxicity toward po-tential degrading microorganisms is an important reasonfor the persistence of CP in soil (presuming the availabilityof sufficient oxygen), the degradation can be expected totake place wherever the concentrations are low due tosorption and/or dispersion processes which are typicallyoccurring downgradient of heavily polluted sites. Downgra-dient remediation measures such as bioscreens (26) providedwith bacteria able to degrade CP could support such a spatialconfinement.

Both the beneficial influence of high temperature and ofbioaugmentation on 2,6-DCP degradation could be con-firmed. Assessing the interactions between individual factorsled to important findings (Figure 3B): The inhibition by high2,6-DCP concentrations attenuates the influence of bothtemperature, addition of strain RK1 and of phenol on 2,6-DCP degradation. Although not significant on the 90%confidence level, inoculation of strain RK1 seems to be moreeffective in dry than in wet soil, and the success ofbioaugmentation is enhanced when phenol as additionalsubstrate is available.

The effects of the variation of individual environmentalfactors on kR corresponded well with those found by thefactorial design. The factorial design allowed for determiningthe relative importance of the factors and in assessinginteractions. The variation of individual factors allowed for

obtaining a more detailed picture of the influence of therespective factor over the chosen range.

AcknowledgmentsThis work was partially supported by Grant 980033 from theSafety, Health and Environment Department of Novartis Inc.and Ciba Specialty Chemicals Inc. to P. Steinle. The authorsthank K. Eigenmann for his support and P. Ackermann andA. Fredenhagen for their amiable help with the radiolabeledexperiments.

Supporting Information AvailableCalculation of the 2,6-DCP concentration in the pore waterof unsaturated soil and tables of Cw calculated for differentmicrocosms and symbols and values used for the calculationof Cw. This material is available free of charge via the Internetat http://pubs.acs.org.

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Received for review May 24, 1999. Revised manuscript re-ceived December 1, 1999. Accepted December 2, 1999.

ES990587L

FIGURE 4. Standardized effects of individual environmental factorsand combined effects of two factors (interactions) on kR of 2,6-DCPin unsaturated soil. The vertical line represents the 90% confidenceinterval for a significant effect. - and + signs indicate negativeand positive effects, respectively.

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