Anionic surfactant enhanced bacterial degradation of tributyltin in soil

International Biodeterioration & Biodegradation 75 (2012) 7e14

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Anionic surfactant enhanced bacterial degradation of tributyltin in soil Lada Mathurasa a, b, Chantra Tongcumpou b, c, David A. Sabatini d, Ekawan Luepromchai b, e, * a

International Postgraduate Programs in Environmental Management, Graduate School, Chulalongkorn University, Bangkok, Thailand National Center of Excellence for Environmental and Hazardous Waste Management (NCE-EHWM), Chulalongkorn University, Bangkok, Thailand c Environmental Research Institute, Chulalongkorn University, Bangkok, Thailand d School of Civil Engineering and Environmental Science, The University of Oklahoma, OK, USA e Bioremediation Research Unit, Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 October 2011 Received in revised form 14 June 2012 Accepted 25 June 2012 Available online 29 August 2012

The effect of an anionic surfactant, sodium dihexylsulfosuccinate (SDHS) addition on remediation of tributyltin (TBT) contaminated soil was investigated. Aerobic soil-slurry batch experiments were used to determine the adsorption, desorption, and bacterial degradation of TBT as well as its metabolites such as dibutyltin (DBT) and monobutyltin (MBT). The extent of butyltin adsorption over the concentration range of 1e100 mg Sn l
1 was in the order of MBT > DBT > TBT. The sub-critical micelle concentration (CMC) of SDHS (i.e., 10 mM) did not increase the amounts of desorbed TBT at the beginning of experiment, however, it enhanced the bacterial degradation of 100 mg Sn kg
1 TBT as seen from the increasing amounts of DBT and MBT in the liquid phase after 2 weeks. At the same time, the desorbed TBT was readsorbed to the soil in the experiment without SDHS. The complex of TBT and SDHS monomers was expected to enhance the interaction of TBT with indigenous soil bacteria. At supra-CMC SDHS (i.e., 70 mM), the desorbed TBT was not degraded because high concentrations of TBT and SDHS posed synergistic toxic effects to the bacteria. Consequently, the presence of anionic surfactant at sub-CMCs will be beneficial for the cleanup of TBT contaminated sites. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Anionic surfactant Tributyltin Soil Biodegradation Desorption Adsorption

1. Introduction Tributyltin (TBT) and other organotins have been found in soil at concentrations ranging from 1.1 to 800 mg kg
1, where the anthropogenic sources of these compounds are sewage sludge, pesticides, insecticides, biocides, timber treatment, industrial waste, and mining (De Carvalho Oliveira and Santelli, 2010). TBT is a persistent organic pollutant with a half-life of 78e89 days in sandy soil, while its monosubstituted species i.e., monobutyltin (MBT) has two to four times longer half-life (Heroult et al., 2008). In general, the persistence of organotins is governed by moderate to fast aerobic biotic degradation processes, slow anaerobic biotic degradation, slow abiotic degradation by photolysis, and fast, but reversible, adsorption/desorption processes (Rüdel, 2003). The adsorption of organotins has been attributed to hydrophobic interaction and complexation with organic matter in soils (Huang and Matzner, 2004). This process was also known to reduce * Corresponding author. Bioremediation Research Unit, Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Tel.: þ66 2 218 5087; fax: þ66 2 252 7576. E-mail address: [email protected] (E. Luepromchai). 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2012.06.027

bioavailability of TBT and thereby prevent its biodegradation (Sakultantimetha et al., 2011a). Tributyltin biodegradation involves the sequential removal of organic groups from the tin atom by microorganisms and generally occurs at low TBT concentrations since high concentration of triorganotins exerts toxic effects through interactions with membrane lipids of the microorganisms (Gadd, 2000). In sediment, TBT biodegradation can be enhanced by providing aeration and adjusting salinity but not by nutrient addition or inoculation of TBT-degrading bacteria (Sakultantimetha et al., 2011a). On the other hand, the presence of low concentration of supplemental substrates such as acetic acid can increase biodegradation of TBT in activated sludge (Stasinakis et al., 2005). Only limited studies have evaluated TBT biodegradation in soil. For example, Heroult et al. (2008) reported that initial concentration, temperature, and soil organic matter are responsible for the differences in organotin biodegradation under natural conditions. To enhance TBT biodegradation, this study was interested to use sodium dihexylsulfosuccinate (SDHS, Aerosol MAþ80) to desorb butyltins from soil and then monitored the degradation of desorbed TBT to its metabolites (i.e., dibutyltin (DBT) and MBT) by indigenous soil bacteria. SDHS was selected as a model anionic surfactant because

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it has low degree of biodegradability and is widely used for soil and groundwater remediation (Franzetti et al., 2006). To our knowledge, the application of anionic surfactant for removal of TBT and other butyltins from soil has not been studied. On the other hand, nonionic surfactant i.e., Tween 80 has been used to increase TBT bioavailability in sediment, however the biodegradation of TBT is inhibited because the sediment microorganisms prefer the surfactant as substrate over TBT (Sakultantimetha et al., 2011b). TBT and its degrading metabolites have both organic and metal properties thus it is difficult to predict their fate after adding anionic surfactant. Butyltins were expected to be desorbed from soil by anionic surfactant similar to the mobilization of metals (i.e., Al, Fe, Cd, Cu, Mn, Ni, Pb, Sn, and Zn) from sediment via complexation with anionic amphiphiles and denudation of hydrophobic host phases (Singh and Turner, 2009). On the other hand, these compounds may be desorbed and biodegraded similar to other organic compounds during surfactant enhanced bioremediation of soil and groundwater (Yu et al., 2007). Consequently, this research aims to investigate the effects of SDHS addition on improving TBT desorption as well as bacterial degradation in a soil sample. An aerobic soil-slurry batch experiment was applied to allow homogeneous mixing between TBT, SDHS, and soil bacteria. Since the amounts of remaining surfactant in contaminated soil during remediation can be varied, the study also compared the effects of SDHS at sub-CMC, where surfactant is in monomer form, and supra-CMC, where surfactant appears in micelle forming unit.

Merck. (NH4)2SO4, KH2PO4, K2HPO4, MgSO4, CaCl2, and trace element solution (FeSO4, CuSO4, CoCl2, MnCl2, NaMoO4, and ZnSO4) for preparing mineral salt medium (MSM) were purchased from Merck. For R2A medium broth, proteose peptone, casamino acids, and yeast extract were purchased from Difco, while dextrose, soluble starch, K2HPO4, MgSO4$7H2O, and sodium pyruvate were purchased from Merck. Solid phase microextraction (SPME) device that contained a fiber coated with polydimethylsiloxane (PDMS, 100 mm) were purchased from Supelco. Organotin stock solutions containing 100 g l
1 as tin were prepared in methanol and stored at 4  C in the dark. The working solutions were obtained by dilution with studied medium and were not buffered. Sodium dihexylsulfosuccinate (SDHS) was purchased from Fluka. SDHS stock solution of 500 mM was sterilized by pass through a 0.2 mm membrane filter. From the preliminary study, the critical micelle concentration (CMC) of SDHS determined, according to the method of Millioli et al. (2002), was 49 mM in a soilewater system (1:10 ratio of soil and DI). Consequently, the study selected the SDHS concentrations at 10 and 70 mM to represent the sub-CMC and supra-CMC, respectively. All labware were cleaned by rinsing 3 times with hexane, soaking in 10% nitric acid overnight, and rinsing again with deionized water (DI). Polycarbonate plasticware was used since they have been found to minimally adsorb butyltins (Huang, 2004).

2. Materials and methods

A batch technique was used to determine the equilibrium partitioning of butyltin species i.e., TBT, DBT, and MBT between soil and water. A 2-g aliquot of unamended soil was suspended in 20 ml DI containing each butyltin species at a concentration range of 1e 100 mg Sn l
1 in a 50 ml polycarbonate tube. The experiments were conducted in triplicate. The sample tubes were slanted on a rotary shaker and shaken in the dark at 250 rpm for 24 h at 25  1  C to ensure that the equilibrium was reached (Burton et al., 2006). To separate the liquid phase, the sample was centrifuged at 1077  g, for 15 min, then the supernatant was filtered through a No. 41 Whatman filter paper. The filtrate was measured for pH and analyzed for the amount of each butyltin, as tin, using microwave assisted digestion with aqua regia (ISO 11466, 1995) and inductively coupled plasma atomic emission spectroscopy (ICPeAES). The concentration of butyltin in solid phase was calculated by subtracting the remaining mass in liquid phase from the total mass.

2.1. Soil sample A sandy clay loam soil with low organic matter content was used to highlight the effect of anionic surfactant on TBT sorption and biodegradation. The soil was collected from an uncontaminated residential area in Pattalung province, Thailand. The sample was taken from a depth of 3to 10 cm as a bulk and sieved at 2 mm to remove large debris and ensure homogeneous mixing. The sieve soil was stored at 4  C until used. The soil sample was sent to the Department of Soil Science, Kasetsart University, Thailand for analysis of soil texture by the hydrometer method, organic matter (OM) by the Walkley-Black titration method, and cation exchange capacity (CEC) by the ammonium acetate method. Proportions of sand, silt, and clay content were 51.8%, 15.8%, and 32.4%, respectively. The values of OM and CEC were low at 1.06% and 5.2 cmol kg
1, respectively. The soil pH was measured after mixing the soil with deionized water (DI) at 1:10 ratio for 24 h and the value at equilibrium was 6.2  0.1. The pHzpc (pH of zero point of charge) of the soil measured by a zeta meter was about 4. The amounts of soil used in all experiments were calculated based on dry weight. Background butyltins in the soil sample were under the detection limit of 0.002 mg Sn kg
1. Butyltin-contaminated soil was prepared by spiking butyltin stock solution at a specific concentration into the soil sample and allowing it to dry under a hood to remove the remaining solvent. The spiked soil was aged for 2 weeks in the dark prior to the experiment. 2.2. Chemicals and labware Monobutyltin trichloride (MBT, 95%), dibutyltin dichloride (DBT, 96%), tributyltin chloride (TBT, 96%), tetrabutyltin (TeBuT, 93%), tripropyltin chloride (TPrT, 98%), sodium tetraethylborate (NaBEt4), and amphotericin B were purchased from Sigmae Aldrich. CaCl2$2H2O, Na2SO4, NaHCO3, and MgCl2$6H2O for preparing synthetic groundwater medium were purchased from

2.3. Adsorption of butyltins

2.4. Desorption of butyltins by SDHS The desorption experiment was conducted in a 50 ml polycarbonate tube containing 2 g of 100 mg Sn kg
1 butyltin amended soil and 20 ml of 0e70 mM SDHS solution. To investigate the effect of liquid medium on desorption of butyltins, SDHS was diluted in either DI, MSM prepared following Wong et al. (2004), or synthetic groundwater (GW) prepared following Dang et al. (2008). The components of synthetic groundwater were as follows (in mg l
1): CaCl2$2H2O, 230, Na2SO4, 1200, NaHCO3, 370, and MgCl2$6H2O, 135. The recipe is usually used to simulate typical groundwater (Dang et al., 2008). The experiments were conducted in triplicate. The sample tubes were shaken in the dark at 250 rpm, 25  1  C for 24 h. The amounts of desorbed butyltin were determined after separating the liquid phase from soil as same as in the adsorption experiment. Total butyltins were quantified from the microwave digested liquid sample by ICPeAES. The percent desorption was calculated based on the mass of butyltin in the initial soil sample and liquid phase.

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2.5. Potential biodegradation of butyltins and SDHS The potential biodegradabilities of butyltin and SDHS were examined through the concentrations of soil bacteria that could utilize either butyltin or SDHS as sole carbon source. A three-tube most-probable-number (MPN) technique was conducted in microtiter plates containing MSM with either 0.01e5 mg Sn l
1 butyltin or 0.26e70 mM SDHS. These concentration ranges covered the amounts of desorbed butyltin from soil as well as the amounts of SDHS used in the study. In addition, 0.26 mM SDHS was selected since this concentration was previously found to support bacterial growth in soil (Chmelárová et al., 2000). The effect of SDHS on bacterial dispersion during MPN analysis was preliminary found to be negligible. The control set containing only MSM was also conducted in parallel. The experiment was conducted in triplicates. Bacterial concentrations were determined after incubating the samples for 2 weeks at room temperature (25  1  C). To facilitate the detection of bacterial growth, a respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate} and a mixture of glucose, succinate, and pyruvate (16.6 mM each in tris buffer, pH 6.8) as electron donor were added to the microtiter plate wells. The addition of these electron donors can increase the detection sensitivity by 3e40 folds (Johnsen et al., 2002). The bacterial respiration of WST-1 was measured immediately using microplate autoreader at 450 nm with a reference wavelength of 630 nm and again after 5 h. 2.6. Bacterial degradation of TBT in soil containing SDHS Aerobic soil-slurry batch experiment was used to study TBT bacterial degradation and conducted in the same way as in adsorption and desorption experiments. However, the liquid medium in this experiment was MSM to prevent the soil bacteria from nutrient deficiency. In general, 2 g of soil amended with 100 mg Sn kg
1 TBT was mixed with 20 ml of MSM containing 0e70 mM SDHS in a 50 ml polycarbonate tube. The samples were prepared in triplicates. The mixtures were shaken at 25  C, 250 rpm for 2 months, in the dark. To check for the presence of oxygen in the test tubes, methylene blue reduction method was conducted where 10 g l
1 methylene blue was applied to an extra tube of each treatment following Winn and Koneman (2006). It was found that the condition in the test tubes remained aerobic throughout the 2-mo period. During incubation, the samples were sacrificed weekly from the 2nd to 8th week for further analysis. Tributyltin bacterial degradation was monitored from the changes in concentrations of TBT and its degrading metabolites during the experiment and compared between control and test sets. For the test set, 2.5 mg l
1 amphotericin B was applied to the liquid medium to reduce the activity of indigenous soil fungi and thereby highlight the effects of bacterial activity on TBT degradation. Ingham and Coleman (1984) showed that amphotericin B (Fungizone) was one of the most effective fungicides, however bacterial populations in soil usually increased following fungal reduction. Consequently, the control set was prepared by adding both 2.5 mg l
1 amphotericin B and 1% (w/v) sodium azide to the liquid medium. Although, sodium azide can inhibit soil respiration, it cannot sterilize the soil (Trevors, 1996). The control set was therefore used to represent a system with much lower microbial activity than that of the test set. To monitor the changes in bacterial concentration, 100 ml of soil slurry from each tube was analyzed by a three-tube MPN technique in microtiter plate. An R2A broth medium was used to determine the total bacterial concentration because the medium was known to favor the cultivation of most soil bacteria (Maier et al., 2009). For TBT-degrading bacteria, MSM containing 0.1 mg Sn l
1 of TBT was

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used. The microtiter plates were incubated at room temperature (25  1  C) for 2 weeks and the bacterial growth was detected by WST-1 assay as described earlier. For chemical analysis, the soil slurry samples were centrifuged at 1077  g for 15 min to separate liquid and soil phases. Butyltins in soil phase were quantified as total tin by microwave digestion followed by ICPeAES, while the amounts of TBT and its degrading metabolites in liquid phase were analyzed by a method below. The concentration of SDHS was determined following Liu et al. (2004). 2.7. Quantification of butyltin species The presence of butyltin and its degrading intermediates in liquid phase was simultaneously determined by a one-step ethylationeextraction using NaBEt4 derivatization followed by gas chromatography with flame photometric detection (GCeFPD). The technique was modified from Bancon-Montigny et al. (2000). Briefly, a 15 ml supernatant from TBT bacterial degradation experiment was introduced into 30 ml derivatization reactor containing 5 ml of sodium acetateeacetic buffer, pH 4.8. Tripropyltin and tetrabutyltin were added as internal standards to check for the derivatization and SPMEs extraction efficiencies, respectively. The derivatization by ethylation was carried out using 0.1 ml of 2% NaBEt4. To extract the derivatized compounds, a 100 mm PDMS coating SPME fiber was put into headspace of the sample (Le Gac et al., 2003). The solution was mixed by stirring for 50 min to achieve the maximum extraction of TBT, DBT, MBT, and inorganic tin. Then, the SPME fiber was placed directly into the injection port of Agilent Model 6890 gas chromatograph fitted with a flame photometric detector. The injection mode was splitless and the separation was carried out on a capillary column HP-5 (25 mm  0.32 mm  0.17 nm). The operating condition was set following the method of Hsia et al. (2004). Since the presence of SDHS in the sample reduced the peak area of butyltin, the calibration curves were established for each concentration of SDHS. 3. Results 3.1. Adsorption of butyltins Adsorption of each butyltin species at 1e100 mg Sn l
1 was studied with unamended soil. The equilibrium adsorption (qe) of DBT was higher than TBT and their adsorption curves were fitted with linear isotherm (Fig. 1). The Kd values of TBT and DBT were 41 l kg
1 (r2 ¼ 0.96) and 148 l kg
1 (r2 ¼ 0.96), respectively. At the same time, MBT was completely adsorbed at 1e80 mg Sn l
1

Fig. 1. Adsorption isotherm of TBT (A), DBT (-) and MBT (:) where Ce is equilibrium concentration of butyltin in aqueous phase and qe is concentration of butyltin in solid phase.

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(Fig. 1). Butyltin in the environment can be in nonionic and cationic forms, however; the pH ranges of the soil slurries were 5.85e6.23 and less than pKa of TBT (w6.25, Arnold et al., 1997) and of DBT (<7.00, Hoch et al., 2003), thus TBT and DBT cations were the predominant forms. The degree of butyltins adsorption was MBT > DBT > TBT and the pHzpc of the studied soil was about 4. Consequently, the exchange of butyltin cations on negatively charge surface of soil particles was a major mechanism of butyltin adsorption. 3.2. Desorption of butyltins by SDHS When using DI only (0 mM SDHS), the equilibrium desorption of all butyltins at the concentration of 100 mg Sn kg
1of soil ranged from 4 to 24% and in the order of TBT > DBT > MBT (Table 1). These results are similar to other tested media (i.e., MSM and GW). When SDHS was added to the medium, the results indicated that SDHS could not completely desorb TBT, DBT, and MBT from soil even at supra-CMC SDHS (70 mM, Table 1). Nevertheless, butyltin desorption especially for TBT and DBT by 70 mM SDHS was significantly higher than by 0 mM and 10 mM SDHS in all medium (Table 1). The sub-CMC SDHS (10 mM) did not increase the amounts of desorbed TBT as seen from the similar percent desorption at 0 mM and 10 mM SDHS. When compared with each butyltin species, SDHS diluted in different media had different desorption abilities. However, there was no apparent trend. At equilibrium, the concentrations of desorbed TBT, DBT, and MBT at 0e10 mM SDHS were 2.09  0.50, 1.03  0.41, and 0.48  0.21 mg Sn l
1, respectively while at 70 mM SDHS were 4.52  0.90, 2.44  0.25, and 0.91  0.47 mg Sn l
1, respectively. 3.3. Potential biodegradation of butyltins and SDHS Potential biodegradation of butyltins and SDHS in soil was determined from their ability to support growth of soil bacteria. When using MSM without butyltins, the concentration of soil bacteria was under the detection limit (5.0log MPN kg
1 soil). The concentration of butyltin degraders was increased from 5 to 7log MPN kg
1 soil when the concentration of butyltin was increased from 0.01 to 1 mg Sn l
1 (Fig. 2a). The results indicated that TBT, DBT, and MBT at these concentrations could be utilized by soil bacteria as sole carbon source. When compared between

Table 1 Effect of SDHS addition and different medium on desorption of TBT, DBT, and MBT. Specie of butyltin SDHS (mM) Desorption (%)a DIb

MSMb

GWb

TBT

0 10 70

23.6  2.4A,a 14.8  0.5A,b 24.6  0.6A,a 23.9  2.7A,a 14.7  4.1A,b 23.8  3.3A,a 48.9  10.4B,a 37.3  2.8B,a 49.4  8.0B,a

DBT

0 10 70

13.1  2.0A,a 6.4  0.6B,a 26.3  1.3C,a

MBT

0 10 70

4.2  0.7A,a 4.5  1.6A,a 6.7  1.4A,a

14.2  7.1A,a 11.0  0.9A,a 10.2  2.9A,b 6.9  0.6B,ab 21.7  1.7B,b 25.2  2.1C,a 8.1  2.2A,b 3.9  1.5A,a 14.0  5.1B,b

4.0  1.6A,a 4.4  1.9A,a 6.5  2.7A,a

a The initial concentration of each butyltin species in soil was 100 mg Sn kg
1. The effect of SDHS concentrations on desorption of the same butyltin specie from the same medium was significantly different (p < 0.05, ANOVA with Duncan’s test) when indicated by different capital letters of the same column. The effect of medium on desorption of the same butyltin specie at the same SDHS concentration was significantly different (p < 0.05, ANOVA with Duncan’s test) when indicated by different lower-case letter of the same row. b The media used for SDHS preparation were DI; deionized water 18 U, MSM; mineral salt medium, and GW; synthetic groundwater.

butyltin species, the concentrations of TBT-degrading bacteria were higher than DBT- and MBT-degrading bacteria especially at the concentrations of 0.1e1 mg Sn l
1. These indicated that TBT was better in terms of carbon source for soil bacteria which may be due to its highest molecular carbon content. However, no butyltin degrader was observed at 5 mg Sn l
1. The results indicated that high concentrations of butyltins (>1 mg Sn l
1) were toxic to soil bacteria. For SDHS, only low concentration of SDHS (i.e., 0.26 mM) could be utilized by soil bacteria as sole carbon source (Fig. 2b). No bacteria were detected at 10 mM (sub-AMC) and 70 mM SDHS (supra-CMC). These indicated that SDHS at very low concentrations was potentially biodegradable; however, they might not be sufficient to support bacterial growth. In fact, the results from the TBT bacterial degradation experiment showed that the concentrations of total and TBT-degrading bacteria were almost constant in the soil slurry containing 100 mg Sn kg
1 TBT and 10 mM SDHS throughout the 8-week incubation period (Fig. 5a and c). 3.4. Bacterial degradation of TBT in soil containing SDHS The bacterial degradation of TBT was studied in soil slurry containing 100 mg Sn kg
1 TBT and varied concentrations of SDHS. The concentrations of SDHS remained unchanged in all experiments over the 2-mo period. At 0 time, more than 90 mg Sn kg
1 TBT was present in the soil phase with 0 and 10 mM SDHS, while only 47 mg Sn kg
1 soil TBT was found with 70 mM SDHS (Fig. 3). The data corresponded to 10% and 50% TBT desorption at 0e10 and 70 mM SDHS, respectively. These values were in the range of percent TBT desorption from soil with SDHS diluted in MSM from the previous experiment (Table 1). The amounts of remaining TBT in soil from both control and test sets were similar when 0 or 70 mM SDHS was applied throughout the 2-mo experiment (Fig. 3). However, butyltins in the control soil containing 10 mM SDHS decreased during the first few weeks while their concentration increased to the initial concentration at week 6th (Fig. 3a). On the other hand, the amount of total butyltin in the test soil with 10 mM SDHS was gradually decreased and remained at 50e55 mg Sn kg
1 TBT after week 6th (Fig. 3b). Thus, TBT was probably degraded by soil bacteria. To confirm its bacterial degradation, TBT-degrading metabolites including DBT, MBT, and inorganic tin were monitored in the liquid phase; however, the inorganic tin was not detectable in any experiment. When using 10 mM SDHS, the concentration of DBT in the liquid phase from the test soil was increased up to 3.26 mg Sn l
1 at the 2nd week, which was much higher than that of the control soil (0.45 mg Sn l
1) (Fig. 4). In addition, the amount of DBT in the test set with 10 mM SDHS was higher than TBT throughout the experiment (Fig. 4b). The MBT appeared to slightly increase in the test soil toward the end of study, while only small amounts of MBT were found in control soil (Fig. 4). The presence of DBT and MBT in the control set with 10 mM SDHS was probably due to the degradation of TBT by bacteria that grew after incubation. In fact, the concentration of bacteria in this soil was around 5e 6log MPN kg
1. The reduction of TBT in soil along with the increase of DBT and MBT in aqueous concentrations indicated that TBT bacterial degradation was occurred in the soil slurry containing 10 mM SDHS by sequential debutylation. On the other hand, the concentrations of all butyltin species in both control and test sets with 70 mM SDHS remained almost constant throughout the study period (Fig. 4c, d). The results indicated that TBT was not degraded under this condition. The control and test sets with 70 mM SDHS had similar concentrations of DBT and MBT at the beginning of study (Fig. 4c, d). These metabolites were probably formed during the aging of TBT-amended soil.

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Fig. 2. Concentrations of bacteria in soil that able to utilize butyltins (a) or SDHS (b) as sole carbon source. Bars represent the standard deviation of mean from triplicate samples. Note: The concentrations of TBT-, DBT-, and MBT-degrading bacteria from the media with similar butyltin concentrations were significantly different (p < 0.05, ANOVA with Duncan’s test) when indicated by different letter.

3.5. Changes in soil bacterial concentrations during TBT desorption and bacterial degradation Bacterial concentrations in the soil slurry were monitored for 2-mo period and compared between soil with and without TBT to determine the effects of TBT and its metabolites of degradation on soil bacteria. The concentrations of total bacteria in TBT amended soil with 10 mM SDHS were around 11.2e9.6log MPN kg
1 soil at the beginning of the study and were similar to that of unamended soil regardless of SDHS concentrations (Fig. 5a, b). After incubation, the total bacterial concentrations in these soil samples were slightly decreased and might have been caused by a lack of carbon source. Among these soil samples, the TBT amended soil containing 70 mM SDHS had the lowest concentrations of total bacteria especially after the 2nd week (Fig. 5a). The results indicated that the combination of desorbed TBT (i.e., 3.38e 4.49 mg Sn l
1, Fig. 4d) and 70 mM SDHS had more toxic effect on the soil bacteria than either compound alone. The synergistic toxic effect of TBT and SDHS was more apparent considering the concentrations of TBT-degrading bacteria in the soil samples with 70 mM SDHS. TBT-degrading bacteria in TBT amended soil containing 70 mM SDHS were not found after the 1st week, while they were detected in the unamended soil until the 7th week (Fig. 5c, d). TBT-degrading bacteria were the minor populations in all soil samples, where their concentrations were about 3 log scales lower than the concentrations of total bacteria (Fig. 5). After incubation, the concentrations of TBT-degrading bacteria as well as total bacteria in TBT amended soil containing 10 mM SDHS declined and

were slightly lower than that in the unamended soil (Fig. 5). The results were directly correlated with an increasing amount of DBT and MBT in the liquid phase of soil slurry (Fig. 4b) and the relevant toxicity of these butyltin species to soil bacteria (Fig. 2a). The changes in concentrations of soil bacteria confirmed that TBT at low concentrations was biodegraded after sub-CMC SDHS addition. In addition, the high concentration of SDHS inhibited TBT degradation because of the synergistic toxic effects of SDHS and desorbed TBT on soil bacteria. 4. Discussion The fate of TBT in soil after addition of SDHS could be explained through adsorption, desorption, and bacterial degradation processes. In adsorption, TBT and its metabolites (i.e., DBT and MBT) had linear sorption isotherms over the concentration range of 1e 100 mg Sn l
1 and with the order of adsorption of MBT > DBT > TBT in the sandy clay loam soil. The results were consistent with Huang and Matzner (2004) who studied the adsorption of 10e100 mg Sn l
1 butyltin diluted in artificial rain water in organic soils (wetland and upper layer of podzol soil) and mineral soils (Bw-C horizon of podzol soil). The Kd values of butyltins in organic and mineral soils were 1320e38,400 l kg1 and 21.4e10,700 l kg
1, respectively (Huang and Matzner, 2004). The butyltin adsorption coefficients from our study were in the range of mineral soil in their study. In this study, the major sorption mechanism of butyltins was ion exchange between butyltin cations and negatively charged soil particles. This TBT adsorption

Fig. 3. Time difference in changing amounts of total butyltins in soil slurry with the initial TBT concentration of 100 mg Sn kg
1; (a) control and (b) test sets. The concentrations of applied SDHS were varied from 0 (A), 10 (-) and 70 (:) mM. Bars represent the standard deviation of mean from triplicate samples.

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Fig. 4. Time difference in changing concentrations of aqueous TBT (-) and its metabolites, DBT (:) and MBT (A) in soil slurry with the initial TBT concentration of 100 mg Sn kg
1 TBT; control set (a, c) and test set (b, d). The concentrations of applied SDHS were 10 (a, b) and 70 (c, d) mM. Bars represent the standard deviation of mean from triplicate samples. Note: Inorganic tin was under the detection limit of 0.01 mg Sn l
1.

mechanism has been confirmed in pure-phased clay mineral and in sand by Weidenhaupt et al. (1997) and Behra et al. (2003), respectively. Therefore, the addition of anionic surfactant is necessary for the increasing butyltin desorption and hence increasing their bioavailability through inducing the cationic

butyltins, especially TBT, by anionic head group of surfactant from the anionic surface of soil particles. For all tested concentrations, SDHS could not completely desorb butyltins at 1e100 mg Sn l
1 from sandy clay loam soil. However, the presence of supra-CMC SDHS significantly increased the

Fig. 5. Soil bacterial concentrations of total bacteria (a, b) and TBT degrading bacteria (c, d) in 100 mg Sn kg
1 TBT amended (a, c) and unamended soil (b, d). The concentrations of SDHS were 10 (-) and 70 (:) mM. Bars represent the standard deviation of mean from triplicate samples.

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percent desorption of butyltins. The order of desorption was TBT > DBT > MBT and correlated well with their adsorption which may be due to the irreversible adsorption of butyltins caused by strong cationic exchange on to clay minerals (Hermosin et al., 1993). This order of butyltin desorption is similar to that of mineral soil by Huang and Matzner (2004). There was no significant effect of different media on butyltin desorption even though there have been many studies reported that ions in aqueous phase (e.g. salinity) could increase sorption of TBT (Hoch et al., 2002; Burton et al., 2004). This may be due to the low ion concentrations of synthetic groundwater and mineral salt medium used in this study. Nevertheless, these tested media are representatives of water in soil pores in the real environment. TBT and SDHS at high concentrations were toxic to soil bacteria. However, butyltins up to 1 mg Sn l
1 (5.5 mM) could be utilized as the sole carbon source by bacteria in the soil sample. The concentrations of biodegradable butyltin were lower than in previous reports, for example Pseudomonas sp. and Aeromonas veronii Av27 isolated from contaminated estuarine, can utilize 2e3 mM TBT as the sole carbon source, respectively (Roy et al., 2004; Cruz et al., 2007). This study focused on TBT-degrading efficiency of the indigenous bacteria from uncontaminated soil, thus the low activity was acceptable. Nevertheless, the results indicated that butyltindegrading bacteria were available in the soil studied. SDHS at sub-CMC levels (10 mM) was not toxic to soil bacteria thus it could be used during biological treatment. Although, the addition of sub-CMC SDHS did not increase the amount of desorbed TBT, the TBT bacterial degradation was enhanced as seen from the reducing amounts of soil butyltins and the increasing concentrations of DBT and MBT in aqueous phase. Similarly, Aronstein et al. (1991) reported that the addition of nonionic surfactant at low concentration can increase the biodegradation of aromatic compound, even when the surfactant-induced desorption is not detected. Beaudette et al. (2000) also found that low concentration of Triton X-100 can aid intracellular solubilization of 2,4,5trichlorobiphenyl for complete oxidation by Trametes versicolor. They suggested that surfactants at below CMC remain as monomers and while they do not increase the solubility of hydrophobic compounds they can bind to surfaces within the medium such as microbial cells and pollutant particles. The complex of TBT and SDHS monomers was therefore expected to enhance the interaction of TBT with soil bacteria. Although, TBT was not completely transformed to inorganic tin in this study, its toxicity would be lower. The decreasing of organic substitute molecules in butyltins has been found to decrease their hydrophobicity for cell membrane association (Han and Cooney, 1995; Bertoli et al., 2001). The strong adsorption affinity of DBT and MBT to soil would also reduce their bioavailabilities and hence lead to lower toxicity than TBT. The addition of sub-CMC anionic surfactant was therefore suggested for bioremediation of soil contaminated with TBT. Since SDHS was difficult to degrade by soil bacteria, the competition between surfactant and TBT degradation as that found in Sakultantimetha et al. (2011b) could be avoided. This approach is also applicable as post treatment where excess SDHS is removed after soil flushing to prevent its accumulation in soil (Franzetti et al., 2006). In this study, the partition of butyltins between soil and water tended to be in equilibrium in the batch experiment system, thus TBT was not completely removed from the soil. Nevertheless, the continuous flow of groundwater in real sites could increase the amount of reversible desorbed TBT from the soil and thereby enhance TBT degradation. Further research focusing on how SDHS at various sub-CMCs increases the bacterial degradation of TBT in various soil samples is under way. The acquired knowledge would be beneficial for bioremediation of sites contaminated with TBT as well as other organometallic compounds.

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5. Conclusions TBT and its metabolites, DBT and MBT, acted like polar compounds in soil containing low organic carbon where ionic exchange was a major sorption mechanism. Supra-CMC SDHS can significantly improve desorption of butyltins; however, those high concentrations of applied SDHS and desorbed TBT caused synergistic toxic effects to soil bacteria. On the other hand, sub-CMC SDHS did not increase the amounts of desorbed TBT at the beginning of the experiment; however, it promoted TBT bacterial degradation and had low toxicity. The complex of TBT and SDHS monomers was expected to enhance the interaction of TBT with soil bacteria. Consequently, the presence of anionic surfactant at subCMCs will be beneficial for the cleanup of TBT contaminated sites. Acknowledgments The authors would like to thank the Office of the Higher Education Commission, Thailand; the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund); National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University; Graduate School, Chulalongkorn University; and DuPont Co. Ltd. for their financial support of this research. References Arnold, C.G., Weidenhaupt, A., David, M.M., Müller, S.R., Haderlein, S.B., Schwarzenbach, R.P., 1997. Aqueous speciation and 1-octanolewater partitioning of tributyl- and triphenyltin: effect of pH and ion composition. Environmental Science & Technology 31, 2596e2602. Aronstein, B.N., Calvillo, Y.M., Alexander, M., 1991. Effect of surfactants at low concentrations on the desorption and biodegradation of sorbed aromatic compounds in soil. Environmental Science & Technology 25, 1728e1731. Bancon-Montigny, C., Lespes, G., Potin-Gautier, M., 2000. Improved routine speciation of organotin compounds in environmental samples by pulsed flame photometric detection. Journal of Chromatography A 896, 149e158. Beaudette, L.A., Ward, O.P., Pickard, M.A., Fedorak, P.M., 2000. Low surfactant concentration increases fungal mineralization of a polychlorinated biphenyl congener but has no effect on overall metabolism. Letters in Applied Microbiology 30, 155e160. Behra, P., Lecarme-Theobald, E., Bueno, M., Ehrhardt, J.J., 2003. Sorption of tributyltin onto a natural quartz sand. Journal of Colloid and Interface Science 263, 4e12. Bertoli, E., Ambrosini, A., Zolese, G., Gabbianelli, R., Fedeli, D., Falcioni, G., 2001. Biomembrane perturbation induced by xenobiotics in model and living systems. Cellular & Molecular Biology Letters 6, 334e339. Burton, E.D., Phillips, I.R., Hawker, D.W., 2004. Sorption and desorption behavior of tributyltin with natural sediments. Environmental Science & Technology 38, 6694e6700. Burton, E.D., Phillips, I.R., Hawker, D.W., 2006. Tributyltin partitioning in sediments: effect of aging. Chemosphere 63, 73e81.  Závadská, I., Húska, J., Tóth, D., 2000. Dihexyl sulfosuccinate Chmelárová, Z, biodegradation by mixed cultures. Folia Microbiologica 45, 491e492. Cruz, A., Caetano, T., Suzuki, S., Mendo, S., 2007. Aeromonas veronii, a tributyltin (TBT)-degrading bacterium isolated from an estuarine environment, Ria de Aveiro in Portugal. Marine Environmental Research 64, 639e650. Dang, S.V., Kawasaki, J., Abella, L.C., Auresenia, J., Habaki, H., Gaspillo, P.D., Kosuge, H., 2008. Removal of arsenic from synthetic groundwater by adsorption using the combination of laterite and iron-modified activated carbon. Journal of Water and Environment Technology 6, 43e54. De Carvalho Oliveira, R., Santelli, R.E., 2010. Occurrence and chemical speciation analysis of organotin compounds in the environment: a review. Talanta 82, 9e24. Franzetti, A., Di Gennaro, P., Bevilacqua, A., Papacchini, M., Bestetti, G., 2006. Environmental features of two commercial surfactants widely used in soil remediation. Chemosphere 62, 1474e1480. Gadd, G.M., 2000. Microbial interactions with tributyltin compounds: detoxification, accumulation, and environmental fate. Science of the Total Environment 258, 119e127. Han, G., Cooney, J.J., 1995. Effects of butyltins and inorganic tin on chemotaxis of aquatic bacteria. Journal of Industrial Microbiology & Biotechnology 14, 293e299. Hermosin, M.C., Martin, P., Cornejo, J., 1993. Adsorption mechanisms of monobutyltin in clay minerals. Environmental Science & Technology 27, 2606e2611. Heroult, J., Nia, Y., Denaix, L., Bueno, M., Lespes, G., 2008. Kinetic degradation processes of butyl- and phenyltins in soils. Chemosphere 72, 940e946.

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