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TECHNICAL REPORTS
Bioremediation and Biodegradation

Transport and Biodegradation of Perchlorate in Soils

University of California Davis, Department of Land, Air, and Water Resources, Davis, CA 95616

* Corresponding author (d_tipton{at}hotmail.com)

Received for publication July 31, 2001.

	ABSTRACT

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Perchlorate (ClO^-₄) contamination of ground water and surface water is a widespread problem, particularly in the western United States. This study examined the effect of biodegradation on perchlorate fate and transport in soils. Solute transport experiments were conducted on two surface soils. Pulses of solution containing perchlorate and Br^- were applied to saturated soil columns at steady state water flow. Perchlorate behaved like a nonreactive tracer in Columbia loam (coarse-loamy, mixed, superactive, nonacid, thermic Oxyaquic Xerofluvent) but was degraded in Yolo loam (fine-silty, mixed, superactive, nonacid, thermic Mollic Xerofluvent). Batch experiments demonstrated that perchlorate removal from solution in Yolo loam was caused by biodegradation. Other batch experiments with Yolo loam surface and subsurface soils, Columbia loam surface soil, and dredge tailings demonstrated that perchlorate biodegradation required anaerobic conditions, an adequate carbon source, and an active perchlorate-degrading microbial population. The sequential reduction of perchlorate and NO^-₃ by an indigenous soil microbial community in Yolo loam batch systems was also studied. Nitrate reduction occurred much sooner than perchlorate reduction in soils that had not been previously exposed to perchlorate, but NO^-₃ and perchlorate were simultaneously reduced in soils previously exposed to perchlorate. The results of this study have implications for in situ remediation schemes and for agricultural soils that have been contaminated by perchlorate-tainted irrigation water.

	INTRODUCTION

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THE LARGE-SCALE DISPOSAL of explosives containing ammonium perchlorate salts has resulted in perchlorate contamination of both ground water and surface water, particularly in the western United States. Perchlorate has been found in water supplies of more than 15 million people in California, Nevada, and Arizona (USEPA, 1999). It is also known to contaminate the Colorado River, a major source of irrigation water in the southwestern United States, which could result in the absorption of perchlorate into crops (Urbansky et al., 2000). The potential health concern associated with perchlorate is that it interferes with the uptake of iodide by the pituitary gland, inhibiting the production of thyroid hormones required for normal metabolism (Urbansky, 1998).

Perchlorate is very soluble and extremely slow to react with other chemicals (Urbansky, 1998). These characteristics make perchlorate-contaminated water very difficult to remediate with standard methods of water treatment (Urbansky, 1998; Logan, 2001). Because of this, most research on perchlorate to date has focused on developing methods to remove perchlorate from drinking water (Giblin et al., 2000a,b; Herman and Frankenberger, 1999; Miller and Logan, 2000; Wallace et al., 1998; Kim and Logan, 2000, 2001). Factors that affect the fate and transport of perchlorate in the environment have received less attention. Several strains of perchlorate-reducing bacteria, all facultative anaerobes, have been isolated from environmental samples (Coates et al., 1999) and significant numbers of perchlorate-reducing bacteria have been found in water and soil samples (Coates et al., 1999; Wu et al., 2001). Perchlorate has been observed to adsorb slightly to variable charge soils in low pH environments (Ji and Kong, 1992). The capacity of biodegradation or adsorption to affect the fate and transport of perchlorate in soils, however, has not been studied extensively. Such information is essential to assess the risk of human exposure to perchlorate and predict the potential for contamination of water supplies and irrigated crops.

The purpose of this research was to determine if biodegradation and/or sorption affects the fate and transport of perchlorate in soils and to investigate the conditions under which these processes may occur. Because of the potential for agricultural soils to be contaminated by perchlorate through irrigation, agricultural soils were used in this study. First, solute displacement experiments were conducted in saturated soil columns to determine if biodegradation and/or sorption could affect perchlorate fate and transport in two surface soils. Then, a series of batch experiments were conducted in surface and subsurface soils to measure the potential of native microbial communities to transform perchlorate under different conditions.

	MATERIALS AND METHODS

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Solute Transport Experiments
Columbia loam and Yolo silt loam surface soils were collected in 1997 and 1993, respectively, from agricultural fields in Yolo County, California. Soils were air-dried, crushed, and stored at room temperature. Both soils were sieved through a 1-mm sieve. Characteristics of both soils are given in Table 1.

After all experiments had been conducted on a single column, the column was dismantled. The soil was weighed wet and then dried in an oven at 110°C for 24 h. The soil was then weighed again to determine the volumetric water content. All three of the Columbia loam experiments were conducted on the same column. The average porewater velocities of these experiments ranged from 0.017 to 0.018 cm min^-1, and the pulse lengths ranged from 300 to 306 min. Yolo loam Experiment 1 was performed on a column that had not been previously exposed to perchlorate, and its average measured porewater velocity was 0.009 cm min^-1 and pulse length was 480 min. Yolo loam Experiments 2 and 3 (average porewater velocity for both experiments was 0.005 cm min^-1) were performed on the same column, which had been previously exposed to perchlorate. The pulse lengths for Experiments 2 and 3 were 650 and 495 min, respectively.

Using the CXTFIT solute transport model (Toride et al., 1995), an analytical solution of the convection–dispersion equation was fit to the Br^- breakthrough data. In the Columbia loam experiments, the dispersion coefficient was fit to the data. In the Yolo loam experiments, both the pore water velocity and the dispersion coefficient were fit, because model estimates of porewater velocities were consistently 12 to 14% larger than the measured velocities for these experiments. This was probably caused by anion exclusion of the Br^- and perchlorate or by the presence of immobile water in the Yolo loam.

Batch Experiments
The Yolo loam and Columbia loam soil samples used in the column experiments were also used for the flooded batch experiments. Fresh Yolo loam and Columbia loam soils (collected in July and August 2000, respectively) were used for subsequent batch experiments. Both soils were collected from the top 20 cm of agricultural fields located in Yolo County, California and were crushed and sieved through a 2-mm sieve. The Yolo subsoil was collected in August 2000 from an agricultural field on the University of California, Davis campus at a depth of approximately 5 m. This soil was not sieved, but was mixed thoroughly. The dredge tailings were collected in October 2000 at White River Aggregates, a sand and gravel plant located in Rancho Cordova, CA, at a depth of approximately 9 m. Dredge tailings are materials that were deposited in the 1800s as a result of gold dredging activities. The tailings collected were similar to those on an adjacent site where perchlorate contamination of ground water had occurred, but these particular samples had not been exposed to perchlorate. The dredge tailings were sieved through a 2-mm sieve and mixed before use.

All soils were stored at 4°C until they were used in experiments. The Yolo subsurface soil was stored at field moisture, and the rest of the soils were stored air-dry. Table 1 gives selected characteristics of each soil.

Flooded Batch Experiments
In the Yolo loam surface soil flooded batch experiments, 50 g of soil was placed into 250-mL bottles. Sterilized water (15 mL) was added to the soil in the bottles to induce microbial activity the day before the experiment started, and the bottles were sealed with screw caps. The following day, 100 mL of 5, 10, 50, or 100 mg L^-1 perchlorate solution was added to the bottles. Three replicate bottles containing nonsterilized soil and two replicate bottles containing sterilized soil were used for each soil at each concentration. The bottles were incubated at room temperature (approximately 25°C) and were not shaken. At every sampling period, the bottles were shaken slightly and a 5-mL sample of the slurry was taken from each bottle. In subsequent experiments, 100 g of air-dry soil was added to 200 mL of 50 or 100 mg L^-1 perchlorate solution in 500-mL Erlenmeyer flasks. All samples were filtered through a 0.22-µm filter and frozen until they were analyzed.

Gas-Sparged Batch Experiments
A 125-g sample of Yolo loam or Yolo subsoil was added to 250 mL of perchlorate solution in 500-mL Erlenmeyer flasks. Each flask was capped with a rubber stopper and placed on a shaker in a 25°C room. At every sampling time, a 5-mL sample of each slurry was taken from the flasks within a laminar flow hood. The samples were centrifuged, and the supernatant was filtered through a 0.22-µm filter and frozen until analysis. After sampling, the flasks were sparged with nitrogen (N₂), oxygen (O₂), or air for 5 min. Three replicate bottles containing nonsterilized soil and two replicate bottles containing sterilized soil were used for each soil.

During the incubation of the Yolo subsoil experiments, the 25°C room malfunctioned and reached temperatures as high as 40°C on Days 7 and 12 of the experiment.

Dredge Tailings Inoculation
A sample of 15 g of Yolo loam soil or 15 g of sterilized Yolo loam soil was added to 125 g of dredge tailings. The soil mixture was used in N₂–sparged batch experiments as described above with a 10 mg L^-1 perchlorate and 500 mg L^-1 acetate solution. One set of flasks contained dredge tailings that were not inoculated with Yolo loam soil.

During the incubation of the dredge tailings inoculation experiments, the 25°C room malfunctioned and reached temperatures as high as 40°C on Day 26 of the experiment.

Redox Experiments
Yolo loam soil (125 g) was added to 500-mL Erlenmeyer flasks filled with 250 mL of 10 mg L^-1 perchlorate solution. The apparatus was the same as the one described for the gas-sparged batch experiments. The flasks were placed on a shaker in a 25°C room between sampling times. At the beginning of the experiment, the flasks were sparged for 10 min with N₂ after the initial sampling. Subsequently, the headspace was sparged with N₂ when the flasks were opened during sampling and measurement of redox and pH.

During sampling, three platinum redox electrodes (calibrated with pH buffers and quinhydrone), a combination pH electrode, and a calomel reference electrode were placed in the flasks under a N₂ stream in a laminar flow hood. The electrodes were allowed to equilibrate for at least 5 min before the readings were taken. The electrodes were rinsed with distilled water and ethyl alcohol before placement into each flask.

When both the NO^-₃ and the perchlorate concentrations had dropped to zero, the flasks were respiked with 10 mL of a filter-sterilized, N₂–sparged solution containing 2000 mg L^-1 NO^-₃, 4000 mg L^-1 acetate, and 2000 mg L^-1 perchlorate solution, bringing the overall perchlorate and NO^-₃ concentrations of the slurry to approximately 80 mg L^-1. Samples were taken over time as previously described.

Sterilization
All glassware, apparatus materials, and pipette tips used in the batch experiments were autoclaved prior to use. Sterile controls were autoclaved for 1 to 1.5 h, allowed to cool for 12 to 16 h, and then autoclaved again for 1 to 1.5 h.

Chemical Analysis
Reagent-grade ammonium perchlorate (NH₄ClO₄), potassium bromide (KBr), sodium acetate (NaC₂H₃O₂), and sodium chloride (NaCl) were used for standards and solutions. Reagant-grade sodium perchlorate (NaClO₄), rather than NH₄ClO₄, was used in the experiments in which NO^-₃ was measured.

Perchlorate concentrations were analyzed with an ion chromatograph (IC) with an Ionpac AS16 column (Dionex, Sunnyvale, CA). Flooded batch experiments with initial perchlorate concentrations greater than 10 mg L^-1 were analyzed with a perchlorate electrode and double junction reference electrode (Orion Research, Beverley, MA) until the perchlorate concentrations dropped below 5 mg L^-1.

Chloride was analyzed using a chloridometer (American Instrument Company, Silver Spring, MD). Bromide analysis was conducted with a combination Br^- electrode (Orion Research). Nitrate was analyzed with an ion chromatograph and Ionpac AS4 column (Dionex).

	RESULTS

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Solute Transport Experiments
In the Columbia loam experiments (data not shown), the perchlorate breakthrough curves were nearly identical to the Br^- breakthrough curves and the analytical solution of the convection–dispersion equation with the retardation coefficient equal to 1.0. Bromide and perchlorate recovery ranged from 93.8 to 99.6%

In the Yolo loam experiments, the measured Br^- breakthrough curves were similar to those calculated with the convection–dispersion equation with the retardation coefficient equal to 1.0. The measured perchlorate breakthrough curves, however, contained less mass than the Br^- breakthrough curves (Fig. 1) . Although nearly all of the Br^- was recovered from these experiments (99.7–103%), only 58.1 to 89.0% of the perchlorate was recovered. This indicates that perchlorate was removed from solution during transport through the soil.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Measured and predicted breakthrough curves in Yolo loam Experiments 1 (a), 2 (b), and 3 (c).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Perchlorate degradation and Cl- formation in Yolo loam flooded batch experiments. Each point represents the mean (± standard deviation) of three replicates. Perchlorate in sterile controls was not degraded.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Perchlorate concentrations over time in flooded batch experiments with Columbia loam collected in 1997 and fresh Columbia loam. Each point represents the mean (± standard deviation) of three replicates. Perchlorate in sterile controls was not degraded.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Perchlorate concentrations over time in unamended and acetate-amended fresh Columbia loam flooded batch experiments. Each point for the unamended flasks represents an average (± standard deviation) of three replicates. Each replicate is shown separately for the flasks to which acetate was added. Perchlorate in sterile controls was not degraded.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Perchlorate concentrations over time in flooded batch experiments with unamended Yolo subsoil, Yolo subsoil amended with glucose, and Yolo subsoil amended with acetate. Each point represents the mean (± standard deviation) of three replicates.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Perchlorate concentrations over time in Yolo loam gas-sparged batch experiments. Perchlorate in sterile controls was not degraded. Each point represents the average of three replicates (± standard deviation).

Dredge Tailings Inoculation
In preliminary experiments (data not shown), low microbial perchlorate-degrading activity in the dredge tailings was observed, even in the presence of a carbon source. To determine if this low perchlorate-degrading activity was caused by the absence of indigenous perchlorate-reducing microbes or environmental conditions insufficient for biodegradation, another experiment was conducted in which flasks containing dredge tailings were inoculated with Yolo loam soil known to contain perchlorate-degrading microbes.

In the dredge tailings inoculation experiments, perchlorate was degraded at a slower rate in the flasks amended with acetate alone or acetate and sterilized Yolo loam than flasks amended with acetate and nonsterilized Yolo loam (Fig. 7) . This indicates that the lower perchlorate-degrading activity in the dredge tailings than the other soils was probably due to the presence of fewer perchlorate-reducing microbes in this material, rather than insufficient conditions for biodegradation. Perchlorate in the flasks containing sterilized Yolo loam and sterilized dredge tailings also was degraded slightly (Fig. 7), suggesting that autoclaving was not able to completely inhibit the microbial activity in these flasks. The high temperatures to which the experiments were exposed during the malfunction of the constant temperature room may have slowed the rate of perchlorate degradation in these experiments, as most soil bacteria grow optimally at 15 to 35°C (Sylvia et al., 1998).

View larger version (33K): [in this window] [in a new window]   Fig. 7. Perchlorate concentrations over time in dredge tailings or sterilized dredge tailings amended with acetate, acetate and sterilized Yolo loam, or acetate and nonsterilized Yolo loam. Each point in represents the mean (± standard deviation) of three replicates.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Nitrate concentrations (a) and perchlorate concentrations (b) over time in Yolo loam N2–sparged batch experiments. Flasks were respiked with perchlorate and NO-3 on Day 15. Each point in (a) and (b) represents the average (± standard deviation) of three replicates.

	DISCUSSION

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Because the hydraulic conductivity of the Yolo loam soil is less than that of the Columbia loam soil, the flow rates of the Yolo loam solute transport experiments were much slower than those of the Columbia loam experiments, which may have given more time for biodegradation to occur in the Yolo loam. The flooded flask batch experiments, however, showed that perchlorate was not degraded readily in the Columbia loam soil used in the transport experiments (Fig. 3). Therefore, even if the flow rates of the Columbia loam transport experiments had been as slow as those used in the Yolo loam experiments, it is unlikely that biodegradation would have occurred.

Perchlorate was not degraded in the Columbia loam transport experiments probably because the soil was collected 3 yr before the experiments were conducted so that it no longer contained a large perchlorate-degrading microbial population. The high nitrate in the Columbia loam used in the transport experiments (Table 1) due to the fertilizers used in this soil was probably leached away during the initial flow of the column experiments, and therefore probably did not influence the experiments significantly. The microbial populations appeared to be better preserved in the Yolo loam even though this soil also had been stored for several years. This greater microbial activity may be partly due to the higher organic carbon content in the Yolo loam than the Columbia loam soil (Table 1). Columbia loam soil that was freshly collected did have the capacity to biodegrade perchlorate (Fig. 3). The microbes in this soil were probably carbon-limited, as the addition of acetate made the rate of degradation much faster (Fig. 4).

The batch experiments also indicated that in subsurface soils that contain low amounts of organic carbon, such as the Yolo subsoil (Table 1), perchlorate-reducing microbes may not be active at all unless carbon is made available to them (Fig. 5). This result could have implications for subsurface in situ remediation schemes.

In other materials, such as the dredge tailings, indigenous perchlorate-reducing bacteria required a longer lag time to become active than the other soils studied, even in the presence of a carbon source (Fig. 7). Perchlorate concentrations in the acetate-amended flasks did not decrease in the first 40 d of the experiment. After this time, the perchlorate concentrations began to decrease, but only slightly. Carbon limitations might explain why perchlorate contamination is often a problem when perchlorate disposal or releases occur on gravelly or stony material. Perchlorate concentrations decreased to an average of 3.86 mg L^-1 in the flasks containing sterilized Yolo loam (Fig. 7). This may indicate that the Yolo loam soil provided nutrients that were limiting the growth of the indigenous microbial population in the dredge tailings. Only the addition of acetate and nonsterilized Yolo loam (which contained a perchlorate-degrading microbial population) was able to induce perchlorate degradation in the dredge tailings to low concentrations (Fig. 7).

The gas-sparged batch experiments (Fig. 6) showed that perchlorate biodegradation in soils occurs under anaerobic conditions, confirming the results of previous studies (Rikken et al., 1996; Attaway and Smith, 1993). Although the Yolo loam soil columns were not manipulated to make them anaerobic before the start of the experiments, it is probable that after the columns were saturated, microbial activity quickly consumed all of the available O₂, causing anaerobic conditions in areas of the columns. Similarly, in the flooded batch experiments, which also were not made anaerobic at the onset of the experiment, microbial O₂ consumption probably created conditions sufficiently anoxic for perchlorate degradation to occur.

Perchlorate and NO^-₃ reduction (Fig. 8) occurred under the same range of average redox conditions (about 180–308 mV). These values are similar to redox conditions previously observed for NO^-₃ reduction. In previous studies, NO^-₃ reduction in soils has been observed in the range of 200 to 300 mV (Patrick and Jugsujinda, 1992; Kralova et al., 1992; Bailey and Beauchamp, 1971).

According to thermodynamic theory, the order of terminal electron acceptors used by microbes under anaerobic conditions is determined by the energy-yielding capacity of the electron acceptors. Usually in the absence of O₂, NO^-₃ is used first, followed by Mn⁴⁺, Fe³⁺, and SO^2-₄ (Patrick and Jugsujinda, 1992). Because perchlorate reduction is slightly more thermodynamically favorable than NO^-₃ reduction (Coates et al., 2000), perchlorate should in theory be reduced before NO^-₃.

In the redox experiments in this study, the lag time preceding NO^-₃ reduction was much shorter than that preceding perchlorate reduction in soils that had not been previously exposed to perchlorate. Once the soil was exposed to perchlorate, however, perchlorate and NO^-₃ were reduced simultaneously (Fig. 8). Thus, the sequence in which perchlorate and NO^-₃ are reduced in the environment may be more dependent on the history of the microbial population than on the energy yield of the terminal electron acceptors. Because NO^-₃ is naturally occurring in soils, microbial populations are poised to degrade it as soon as appropriate redox conditions are present (Fig. 8a).

It is possible that NO^-₃ and perchlorate were reduced simultaneously instead of sequentially in this study because the difference in reduction potential between NO^-₃ and perchlorate is so slight. A redox-controlled system might be needed to observe the sequential reduction of perchlorate and NO^-₃ in a system where both perchlorate- and NO^-₃–reducing microbial populations are established. In another soil slurry study, the reduction of NO^-₃ and Mn⁴⁺ was observed to occur sequentially in a redox-controlled system, but had been observed to occur simultaneously in a previous soil slurry study that was not redox-controlled (Patrick and Jugsujinda, 1992). This phenomenon was attributed to the fact that the reduction potentials of NO^-₃ and Mn⁴⁺ are so close that the soil became reducing enough to support Mn⁴⁺ reduction before all of the NO^-₃ was reduced.

The finding that microbes in some agricultural soils degrade perchlorate without carbon amendments or inoculation is significant. Perchlorate-contaminated irrigation water has the potential to contaminate agricultural soils, and possibly crops (Urbansky et al., 2000). This research, however, indicates that there is potential for perchlorate to be reduced in surface soils before it is taken up by crops or migrates to shallow ground water. Denitrification rates are significant in many agricultural soils (Barton et al., 1999), and although denitrification is known to occur under anaerobic conditions, it can also occur in well-drained aerobic soils because of anaerobic microsites caused by heterogeneities in soil. (Parkin, 1987; Christensen et al., 1990, Højberg et al., 1994). Because perchlorate reduction and nitrate reduction occur under similar conditions, perchlorate reduction may also be significant in anaerobic microsites of relatively well-drained soils.

Adsorption did not appear to be significant in the soils under the conditions studied. The breakthrough data from the solute transport experiments did not indicate that the transport of perchlorate was retarded relative to bromide, and there was no indication of adsorption in the batch experiment sterile controls. Other soils, however, especially those with a high anion exchange capacity, may adsorb perchlorate under low pH conditions. More study is needed to determine if adsorption of perchlorate can affect its transport under these conditions.

This research shows that biodegradation can significantly affect the transport of perchlorate in different soils. Conditions needed for biodegradation in soils include anaerobic conditions and the presence of sufficient carbon. The presence of perchlorate-degrading bacteria also is an important factor, since in some materials, such as the dredge tailings used in this study, indigenous perchlorate-degrading microbes take a long time to develop a population large enough to reduce perchlorate, even when a carbon source is available.

Redox conditions required for perchlorate degradation are similar to those for NO^-₃ reduction. The lag time for microbial perchlorate reduction was much longer than the lag time for NO^-₃ reduction in soils not previously exposed to perchlorate. Growth of microbial populations able to degrade perchlorate and/or the induction of enzymes able to degrade perchlorate may strongly affect whether perchlorate or NO^-₃ is used first as a terminal electron acceptor. The results of this research have implications for in situ remediation schemes for perchlorate-contaminated ground water and for agricultural fields contaminated by perchlorate-tainted irrigation water.

	ACKNOWLEDGMENTS

 
This research was supported by Grant 5P42ES04699 from the National Institute of Environmental Health Sciences, NIH. Although the information in this document has been funded wholly or in part by the USEPA and NIH, no official endorsement should be inferred.

	REFERENCES

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