Article pubs.acs.org/est
Stimulation of Hexachlorocyclohexane (HCH) Biodegradation in a Full Scale In Situ Bioscreen Alette A. M. Langenhoff,*,†,‡ Sjef J. M. Staps,†,∥ Charles Pijls,§ and Huub H. M. Rijnaarts†,‡ †
Deltares, P.O. Box 85467, 3508 AL Utrecht, The Netherlands Sub-department of Environmental Technology, Wageningen University, P.O. Box 17, 6700 AA Wageningen, The Netherlands § Tauw bv, P.O. Box 133, 7400 AC Deventer, The Netherlands ‡
ABSTRACT: The feasibility of a bioscreen for the in situ biodegradation of HCH and its intermediates is demonstrated at a contaminated site in The Netherlands, via the discontinuous addition of methanol as electron donor. An infiltration system was installed and operated at the site over a length of 150 m and a depth of 8 m, to create an anaerobic infiltration zone in which HCH is converted. The construction of the infiltration system was combined with the redevelopment of the site. During passage through the bioscreen, the concentration of HCH in the groundwater decreased from 600 μg/ L to the detection limit of the individual HCH isomers (0.01 μg/L) after one year of operation. The concentration of the intermediate biodegradation products benzene and chlorobenzene increased and achieved steady state values of respectively 800 and 2700 μg/L. Benzene and chlorobenzene were treated aerobically on site in an existing wastewater treatment plant. By changing the infiltration regime, it is conclusively shown that HCH removal is the result of the biological degradation and stimulated by the addition of methanol as electron donor. To our knowledge, this is the first successful field demonstration of the stimulated transformation of HCH to intermediates in a full scale anaerobic in situ bioscreen, combined with an aerobic on site treatment to harmless end products.
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INTRODUCTION
When studying biodegradation of hydrophobic organic compounds the concept of bioavailability needs to be taken into account, as compounds tend to partition among solid, liquid, and gas phases in the subsurface. For example, HCH can be found as a solid, dissolved in the aqueous phase, sorbed to soil and sediment materials, and/or associated with colloids and other large organic molecules (e.g., natural organic matter) in water, decreasing its bioavailability.4 Bioavailability of hydrophobic organic contaminants in the subsurface is affected by sorption/desorption in two important ways. First, sorption diminishes the organic concentration in the water phase and only this fraction can be utilized as substrates by microorganisms.5 The separation of organic compounds by sorption from the aqueous phase is likely to reduce the rate and extent of biotransformation in the subsurface. Second, because desorption and source zone diffusion must occur before biodegradation can proceed, the overall rate of bioremediation can be limited or even controlled by these mass transfer processes, and not by the activity of the degrading microorganisms.6 The microbial degradation of HCH was extensively reviewed by Phillips et al.7 In general, the α-, γ-, and δ-HCH isomers can
Hexachlorocyclohexane (HCH) is one of the most widely produced and used pesticide. As a result, the pollution of soil and groundwater with HCH has caused serious environmental problems. HCH exists of eight isomers, with lindane (γ-HCH) as the best known and effective insecticide component of HCH. Only 8−15% of technical HCH consists of the γ-isomer, and technical HCH contains 60−70% α-HCH, 5−12% ß-HCH, 10−12% δ-HCH and 3−4% ε-HCH,1 which do not have insecticide activity. Originally, the technical mixture was used as insecticide, until it was discovered that only the γ-isomer was an effective insecticide. As a result, the other isomers were separated from γ-HCH, and dumped at waste sites resulting in polluted soils and groundwater.2 Nowadays, the use of HCH is forbidden in most countries or has at least decreased, but the relatively high resistance of HCH to degradation continues to lead to environmental problems. Current treatment technologies of HCH contaminated sites focus on landfilling (usually a long intensive and incomplete process, and transfer instead of removal pollution), total destruction of the contaminated soil by thermal or chemical ex situ treatment, and pump and treat for the contaminated groundwater.3 The high costs that are associated with these treatments are hardly applied, despite that final pollution elimination is mandated by the Stockholm Convention. © 2013 American Chemical Society
Received: Revised: Accepted: Published: 11182
June 6, 2013 August 23, 2013 August 26, 2013 August 26, 2013 dx.doi.org/10.1021/es4024833 | Environ. Sci. Technol. 2013, 47, 11182−11188
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Article
conditions.20−22 The degradation of chlorobenzene under anaerobic conditions has been demonstrated,23 but degradation rates are very slow, making it an unattractive process for field bioremediation. We have previously demonstrated in batch experiments that the α-, ß-, γ-, and δ-isomers of HCH can be completely degraded to harmless compounds via sequential anaerobic−aerobic conditions.24 Thus, a complete mineralization of the most important HCH isomers can be expected when an anaerobic zone is followed by an aerobic phase. These findings led to the development of an in situ bioscreen, which was tested and demonstrated in the field at full scale operation. This paper discusses the feasibility of this system, focusing on the anaerobic zone. The concept of a combined intrinsic and stimulated in situ bioremediation is evaluated for HCH contaminated sites, using data from this field study in The Netherlands.
all be degraded metabolically under both aerobic and anaerobic conditions,8−11 and especially studies on the aerobic degradation of γ-HCH are becoming available.12−14 The microbial degradation of γ-HCH has even been demonstrated with compound-specific stable isotope analyses.15 In contrast to these three isomers, ß-HCH was for a long time thought as recalcitrant toward biodegradation under anaerobic and aerobic conditions.9,16 However, the last 15 years of research showed that ß-HCH can be microbiologically degraded to the intermediates benzene and chlorobenzene under anaerobic conditions.17,18 An anaerobic coculture was enriched from these experiments and one bacterium was identified as a Dehalobacter sp., and is responsible for the dechlorination of ß-HCH. The other bacterium was isolated and characterized as being a Sedimentibacter sp. This strain is not able to dechlorinate ßHCH but its presence is needed by Dehalobacter sp. for its growth and dechlorination.19 The anaerobic degradation of HCH occurs most likely via a dehalogenation to tetrachlorocyclohexane and dichlorocyclohexane.17,18 Further degradation gives benzene, but a dehydrohalogenation to chlorobenzene is also possible (Figure 1).
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MATERIALS AND METHODS Site Description. The contaminated site is an industrial facility where HCH was produced by a former site owner, and dissolved and nondissolved HCH isomers have formed a contaminated area of 300 × 100 m up to a depth of 18 m (Figure 2).24 Typical HCH concentrations in the groundwater range from 100 to 2000 μg/L (0.4 to 6.9 μM). At this site, three sandy aquifers can be defined down to a depth of 25 m. They are separated by peat-clay layers. The groundwater velocities in the first, second and third aquifer are 7.5, 15 and 30−60 m/yr, respectively. The groundwater flow in the aquifers is in the direction north−northeast and toward a freshwater system (a canal), which forms a natural boundary at one side of the site. The canal is considered as the surface water receptor to be protected by plume interception measures from HCH contamination. Benzene and chlorobenzene were found in the core of the plume in concentrations of 140 μg/L (1.6 μM) and 50 μg/L (0.9 μM), respectively. This indicates that intrinsic anaerobic
Figure 1. Anaerobic biodegradation pathway of HCH (TeCCH = tetrachlorocyclohexene; DCCH = dichlorocyclohexene, CB = chlorobenzene).
The intermediates benzene and chlorobenzene can be biodegraded; benzene is degradable under both aerobic and anaerobic conditions, and chlorobenzene mainly under aerobic
Figure 2. Side view of the field site, including the design of the bioremediation system CB = chlorobenzene, B = benzene. 11183
dx.doi.org/10.1021/es4024833 | Environ. Sci. Technol. 2013, 47, 11182−11188
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Figure 3. Schematic top view of the HCH contaminated site (150 × 50 m) in The Netherlands.
Table 1. Overview of the Used Phases of Infiltration, and HCH, Benzene, and Chlorobenzene Concentrations at the End of the Various Phases of Infiltration in a Monitoring Well A, Located Behind the Second Infiltration Trencha phase phase phase phase phase a
I II III IV
infiltration solution
duration
HCH (μg/ L)
benzene (μg/ L)
chlorobenzene (μg/ L)
bromide (Br), infiltration at t = 0 methanol, N, Br, 4 intermitting infiltrations at t = 150, 272, 356, and 454 days no infiltration methanol, N, Br, infiltration at t = 797 days
150 days 310 days 340 days 40 days
500 0.13 98% purity) was from Fluka (Zwijndrecht, The Netherlands). All other chemicals were from Merck Nederland B.V. (Amsterdam, The Netherlands). Batch Experiments. Batch experiments were performed in 120 mL bottles with 40 mL anaerobic media and 20 g soil from the industrial site as previously described.24 In short, 1 mg/L HCH was added to the batches. The stimulated degradation of HCH was tested by the addition of acetate or methanol (10 mM), All batches were incubated statically in the dark at 14 °C, and the concentrations of HCH, benzene, and chlorobenzene were measured routinely. Analytical Procedures. Sampling. Groundwater samples were taken according to the EPA method, as described in Appendix A of the EPA Technical protocol for evaluating natural attenuation of chlorinated solvents in groundwater.25
All monitoring wells at the right-hand site of the site (Figure 3) were sampled every two or three months. Benzene and Chlorobenzene. Groundwater samples were analyzed on a GC (Varian Star 3600 CX) equipped with Solid phase Micro Extraction (SPME). Samples were injected onto a capillary column (Stabilwax DB; 30 m × 0.32 mm, 1 μm bead size) and analyzed with a photo ionization detector. Oven temperature conditions for the separation were: 50 °C for 5 min, 20 °C/min to 180 °C and 7 min at 180 °C, total run length was 18.5 min. Detection limits were 1 μg/L. HCH. Ten grams of soil were first dried, followed by extraction with hexane with 10% diethylether. 75 mL groundwater was extracted three times with 5 mL of hexane. The extracts were dried over anhydrous sodium sulfate, and the residual fraction was concentrated to 1 mL under a nitrogen flow. An injection standard (1,2,3,4-tetrachloronaphthalene) was added to the extract. The extracts were analyzed using a GC (HP6890 GC) connected to a mass-spectrometer (HP8593 MSD). Samples were injected on a HP5-MS column (30 m × 0.25 mm, film thickness 0.25 μm). Oven temperature conditions for the separation were: 60 °C for 1 min, 6 °C/ min to 220 °C, 20°/min to 280 °C and 1 min at 280 °C, total run length was 33 min. The mass-spectrometer was used in SIM-mode (molecular weight 181 and 219 for HCH). Detection limits were 0.01 μg/L for the individual isomers. Methane. Methane was analyzed in 8 mL samples on a Varian 3800 GC equipped with a flame ionizing detector (FID), and a Combi Pal auto sampler (CTC Analytics, Zwingen, Switzerland). The sample was shaken and heated for 20 min at 80 °C. Thereafter, the gas phase (100 μL) was injected splitless onto a Porabond-Q column (0.32 mm × 25 m, Varian). The injector temperature was 200 °C, the detector temperature was 300 °C and helium was used as a carrier gas at a flow rate of 2 mL/min. Oven temperature conditions for the separation were 3 min at 40 °C, 10 °C/min to 70 °C, and 15 °C/min to 250 °C for 7 min. Anions. Nitrate, sulfate, and bromide were analyzed by anion exchange chromatography (Dionex DX-120). Samples (50 μL) were injected onto an IONPAC AS9-SC column at 20 °C. The flow rate was 1.3 mL/min with an eluent of 2.0 mM Na2CO3 and 0.75 mM Na2CO3 in Milli-Q water. Hydrogen. Determination of the hydrogen concentration in the field is done according to the “bubble strip” method;26 sampling at the well head and analysis on site with a reducing gas detector. Hydrogen was analyzed on a RGA3 reduction gas analyzer equipped with a 60/80 Unibeads precolumn and a 60/ 80 molecular sieve 5A column. The detector and column temperature were 265 and 105 °C, respectively. The detection limit in the groundwater was approximately 0.015 nM H2.
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RESULTS AND DISCUSSION Previously, batch experiments were performed in duplicate with material from the site to demonstrate the complete degradation of HCH and the stimulation of the biodegradation. These batch experiments showed that the degradation of HCH was stimulated via the addition of a suitable electron donor, for example, compost percolate, or landfill leachate.24 In the batch experiments performed in this study, methanol was used as electron donor. Methanol was widely available near the contaminated site, thus making it very practical if methanol was a suitable electron donor. In addition, ε-HCH was also present at the site, and information was needed about the degradation of this isomer. In batch experiments with material 11185
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from the site the HCH degradation and production of benzene and chlorobenzene from β- and ε-HCH were compared with methanol as electron donor (Figure 4). ε-HCH was degradable under both anaerobic and aerobic conditions. The rate of anaerobic ε−HCH degradation and intermediate production was 3 times slower than for the anaerobic degradation of βHCH, and the aerobic degradation rate of ε−HCH was comparable to the anaerobic degradation of β-HCH. All batches showed a stoichiometric production of the intermediates benzene and chlorobenzene (results not shown). Autoclaved controls with or without methanol did not show any increase in benzene or chlorobenzene concentration. HCH was measured at the beginning and the end (t = 78 days) of the experiments by extracting the whole content of the batches for analyses, and confirmed that all added HCH was degraded.
Figure 5. Bromide profile at the site in monitoring well A (standard deviations in data shown were 5%).
After start-up of the infiltration system, methanol was not detected in the groundwater samples (
